Pot-Honey Patricia Vit • Silvia R.M. Pedro Editors Pot-Honey A legacy of stingless bees • David W. Roubik Editors Patricia Vit Universidad de Los Andes Mérida, Venezuela The University of Sydney Lidcombe, NSW, Australia Silvia R.M. Pedro University of São Paulo Ribeirão Preto São Paulo, Brazil David W. Roubik Smithsonian Tropical Research Institute Ancon, Balboa Panama ISBN 978-1-4614-4959-1 ISBN 978-1-4614-4960-7 (eBook) DOI 10.1007/978-1-4614-4960-7 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2012952932 © Springer Science+Business Media New York 2013 This work is subject to copyright. 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Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com) This book is dedicated to our families, friends, colleagues—past, present, future− observers of stingless bee life, and stingless bee keepers Foreword The stingless bees are one of the most diverse, attractive, fascinating, conspicuous, and useful of all the insect groups of the tropical world. This is a formidable and contentious claim but I believe it can be backed up. They are 50 times more species rich than the honey bees, the other tribe of highly eusocial bees. They are ubiquitous in the tropics and thrive in tropical cities. In rural areas, they nest in a diversity of sites and are found on the flowers of a broad diversity of crop plants. Their role in natural systems is barely studied but they almost certainly deserve that hallowed title of keystone species. They are popular with the general public and are greatly appreciated in zoos and gardens. The chapters of this book provide abundant further evidence of the ecological and economic importance of stingless bees. Given their extreme interest, then it follows that this group must have been the subject of a huge body of scientific research. Unfortunately, this is not the case. Although the stingless bees contain 50 times as many species as the honey bees, the latter have been the subject of perhaps 50 times as much research effort, as estimated by published papers. We have squandered this precious natural heritage by our lack of attention, and in our failure we have limited our use of this resource. But this book starts to address that failure. The chapters of this book summarize much of the current knowledge of stingless bees and also provide new findings. The diversity of species, behaviors, and the wide geographic range is explored in the Part I. The close relationships between humans and stingless bees through history is the topic of the chapters of Part II. The importance of stingless bees in agricultural and natural ecosystems derives from their flower visitation behavior and resulting pollination; this is the focus of the third part. The final two parts provide reviews and original research on the use and properties of the products of the hives of stingless bees, in particular the honey. Stingless bees are an ancient source of sweetness and medicine for many indigenous people in the tropics, from the nomadic hunters and gatherers of northern Australia to the mighty Mayan empire of Central America. But modern commercial exploitation of this product has been hampered partially by a lack of information on its properties and composition. A strength of this book is the focus on “pot-honey,” honey derived from the pots of stingless bees, as opposed to the comb of honey vii viii Foreword bees. Perhaps now stingless bee honey will move from locally available and start to be seen in the global marketplace. Indigenous peoples may not have knowingly used stingless bees as pollinators of their crops, but certainly these industrious insects would have played an important role. Stingless bees also have an important role to play in education. These harmless and fascinating animals can be used in schools and universities, public gardens, and zoos, as case studies in ecological interactions. These bees may even have economical value as pets. Housing a colony of these bees in a city apartment provides an opportunity for urban dwellers to have some contact with nature. This book is one of the few specifically devoted to stingless bees. Let us hope that it stimulates a generation of further research so that the enormous potential of this group can be realized. Brisbane, Australia Tim A. Heard Foreword Yes, we can We live in a time when bees seem to become scarce in relation to their former numbers engaged in pollination and honey production. Our time is also one of competition and upset between different kinds of bees. First, in the nineteenth century, Apis mellifera invaded the Americas and Australia. That was large-scale invasion. And in the twentieth century and afterwards, we saw the invasion, in a larger scale, of the African A. mellifera scutellata in the tropical and subtropical Americas, and there was also a strong decline in the numbers of the meliponine bees. We, the friendly breeders of stingless bees, must in some way make them recover at least some parts of the areas already nearly lost. For doing so, we must improve and increase our breeding of stingless bees such as Scaptotrigona and Melipona, good for pollination. In other words we must as soon as possible improve MELIPONICULTURE and also increase the number of colonies engaged in different projects. We are not against any bee properly bred and cared for. However, we must also protect meliponiculture. For doing so, we must improve our breeding experience in MELIPONICULTURE. This is quite possible, since in Nature, in Africa, in some places A. mellifera and the native meliponines are present after millions of years of coexistence. However, now in parts of tropical America, A. mellifera scutellata seems to be still gaining ground, becoming generally the dominant bees. In such a situation it is important to publish papers about the best ways of helping the Meliponini to survive and also to let people know more about their life history and their potential in pollination and in other fields. I am glad to send my congratulations to the authors of the articles here published and for those who organized this initiative. Some efforts like this one are needed from time to time, for promoting the survival of stingless bees. I would say: yes, we can save them. We really can. São Paulo, Brazil Paulo Nogueira-Neto ix Introduction Just as variety is the spice of life, it is also the source of honey. It doesn’t matter which kind of honey. There is surely variety, and that explains many of honey’s attributes. An average honey taken from a bee colony living within tropical forest contains 50 plant products. Most are nectar or pollen, and some are from the storage containers or food pots, from which this volume takes its name. A few compounds, such as hydrogen peroxide, honey’s valuable antibiotic, form within the honey itself, while others derive from plants or the bees themselves. Now, what is there to explain about pot-honey? Here is a scholarly and lively collection of facts and important insights from people across the world to answer that question. It is explained, as it should be, by a journey across cultures, continents, scientific exploration, and time—a representative sample of knowledge, studies, and applications, some ancient and others nascent. For instance, as we develop analytical techniques both for sequencing honey-making bee genes and reliably defining and characterizing honey, we are exploring ways to market honey and protect the environment it comes from. This is only the beginning. Our human repertoire of honey uses and cultivation techniques can be matched with cultures from Australia to Argentina, from Mexico to Ivory Coast, and from India and Indonesia. This enterprise proffers revelations that few other culinary/linguistic/tribal/cultural/scientific studies can offer. To begin with, honey from insects is a novel feat. As humans, we have a fondness for this food (and drink—as explained herein) that is deep. At the peak of social evolution in insects there is honey. It seems curious that certain bees, wasps, and ants, truly social with long-lived colonies of a queen and workers, are the sole manufacturers of honey on the planet. Yet we take them for granted. There is not long to study some of these unique and natural honeys, before their makers waver on the edge of extinction, and then are no more. Why? Because they are denizens of the tropics and the world’s remaining wildlands. Most honey comes from bees, but not the bumble bees or the honey bees. The tropical and stingless honey-making bees, the Meliponini, are the original and still the predominant makers of honey. Those stingless bees are not a close relative of Apis, xi xii Introduction the stinging honey-bee of wide renown. Biology of the two kinds of honey-making bees diverged some 100 million years ago, now revealed in biogeographic and molecular information that provides conclusive evidence. The stingless bees invented honey. Not so many years ago, books on bee keeping would lay down the theme that there are only four honey bees on earth, then describe methods for bee keeping, and mead making, candlemaking and honey extraction, mostly in the temperate zone and since the Middle Ages. That pattern of presentation is now obsolete. We now contemplate there being a dozen living honey bee species. With the stingless bees, formerly “known” to contain about 200 species, we are surpassing 500 well-codified individual ways of being stingless bees—some actually larger than any honey bee—and many having powerful defense methods. With more exploration of tropical forests and other remote areas, such as the vast Australian “Outback,” the number will soon eclipse that figure. Stingless bee honey is unique not only for its origin in the rich vegetation of native environments but also for its unusual degree of sweetness, sourness, acidity, and a host of other qualities that we have studied. One of them is “medicinal value.” Another feature is the resin or “propolis” that is a part of the entire nesting home of a stingless bee colony. It is definitely an important ingredient in biology and food. Some stingless bees protect and, in turn, are fed and nurtured by bugs. The bugs feed on plant phloem and provide sugars and sustenance to a few species of meliponine bees. Another factor is the microbes. The rainy tropical forests in which stingless bees thrive, as well as some of the dry and hostile regions they can exist in, challenge the procurement and storage of concentrated sugar in a nest. If the predators do not locate this rich resource, the microbes and micro-predators most certainly will. Yet stingless bees survive. We find they are protected in multiple ways, by behavior and nesting habits, and their health in the environment has a long history of compatibility, if not co-option, with other organisms and many plant materials. How many kinds of honey exist in the world? Take the number of stingless bee species, multiply this by the number of seasons in the tropical or subtropical year (wet and dry, for the most basic), and then multiply this by a number including combinations of 20–50 pollen types. Of course, in an environment that has fewer flowering plant species, or where invasive honey bees are taking many of the flowers that the two bee groups compete for, that number is reduced. Indeed, a traditional scientific application of pollen study to the honey of bees has been in the identification of a single, predominant resource in a honey sample. Such “unifloral” honey is an economic standard, verified clearly by pollen identified in the honey, which permits commercialization and unquestionable legitimacy. Other kinds of honey are difficult to categorize in such a straightforward way. They are the flavor of the tropics. They come in too many varieties for superficial scrutiny, other than to state that they are diverse. A connoisseur would notice the difference. “Native honeys,” as we find them, are a remarkable kaleidoscope of bouquet, aroma, flavors, aftertaste, and even texture. Such sensorial adventure begins with both botanical and entomological Introduction xiii origin, often with an added benefit from their matrix of human cultural experience, in which they are embedded. From a human point of view, stingless bees in Asia (Indonesia and Malaysia) are “the bees that remove sticky substances form their legs,” the “galo galo”, or the “flute bees” with the long, tubular nest entrance, or the “beer bees,” whose fermenting honey encourages the production of alcohol, in a container of bee nests and water. Much the same is true for Africa, and the Australian stingless bees have a multitude of uses and metaphors attached to them. In the American tropics, they are frequently the garden bees—those kept close at hand for a case of sore throat, or a home remedy conferring stamina or at very least, well-being. A remarkable dose of needed sweetness, with which to surrender all pessimism and doubt. On the other hand, an astringent tang in the back of the throat and a near convulsion of shock with sweetness combined with something nearly its opposite is familiar to those of us who have consumed buckwheat honey. It is a monofloral honey that honey bees produce in Asia, where Apis cerana and Fagopyrum (Polygonaceae) are native. It is heavily laced with phenolic compounds. This general quality is perhaps the rule, rather than the exception, among the stingless bee honeys in our increasingly homogenized and monofloral world. However, the herbicide-treated and cleared plantations and orchards have given stingless bees, and other bees, a pasture that is more or less uniform, and it has flowers for only a part of the year. Its honey may be harvested, and appreciated, as something fairly novel. But it is far from natural. Still basically unknown, despite multicultural and multigeographic recognition, are the honey and other so-called “hive products” of most stingless bees. Like the perfumed essences emitted by orchids and many flowers, they may soon vanish forever. They are, first and foremost, the most biodiverse products that nature has to offer. What are they worth, both scientifically and culturally? Further, how much have we, and the myriad other species that interact with them lost, if they are neglected, abused, and consigned to extinction? These are essential and pressing questions that we hope the reader will pursue with us. Honey is a rare element of science and nature. What components or synergisms explain each mechanism of action? Is the greater water content of stingless bee honey a defect in quality, as would be recognized in A. mellifera honey, or an important medicinal factor? Sugar and water hold the invisible (and visible, with pollen grains) structure of honey—to arrange metals, secondary metabolites, microbes, chemical residues and final products, after processing by the bees in their nests. Genuine and false honey are simple comparisons, seen immediately by what is present and what is lacking. Honey is used as food, and as our cosmetics and medicines. The little bubbles in pot-honey suggest that ethanol is in the stingless bee storage pots, but in very low concentration. Modern technology has a wide range of applications to discern whether chemical compounds such as unique flavonoids, organic acids, or oxidative reactions in honey influence the immune system or interfere with cancer onset and progress. The Meliponini xiv Introduction introduce the reader to a fascinating world of the woodland bees and their cerumen pots, in which honey and pollen are kept. Our well-known 94-year-old mentor—admiring the first stingless bee he saw alive Trigona (Tetragonisca) angustula Latreille—said that this bee was special “because it is small, gentle, pretty, in Panama often nests in cavities in buildings in towns, makes excellent honey and does not visit filth.” Dr. Michener was correct. Biodiversity and similar admiration for the local species of meliponines are found in the following chapters describing stingless bees from Australia, Venezuela, French Guiana, Guatemala, Costa Rica, Argentina, and Mexico. Two chapters examine the possible roles of microorganisms living with stingless bees, and consider whether fermentation is a mutualistic interaction between yeasts and bees. Strategies in communication by stingless bees to locate, collect and process food in competitive niches are developed in two chapters. Historical views communicate the high valuation of stingless bees and their pot-honey, medicinal uses by Mayans, entomological descriptions in the oldest Brazilian report, and melittology and Melipona bee scientific heritage, which has a legacy of at least 4000 years. Afrotropical stingless bees are treated from a taxonomic perspective used by traditional healers, naturalists and systematists. Conservation of stingless bees is presented as a challenge in Africa and Mexico, where human disturbance and habitat fragmentation propel Meliponini and many organisms toward depletion or extinction. Pollen spectra and plant use by stingless bees for food and nesting are surveyed, with new details and analytical techniques. The sensory descriptions of pot-honey are accompanied with chapters on physicochemical analysis of pot-honey from bees in Australia, Bolivia, Brazil, Colombia, Guatemala, Mexico, and Venezuela— including microbial, nutritional, and metal composition—an electronic nose, nonaromatic organic acid profiles, and Nuclear Magnetic Resonance. The flavonoid studies show that meliponine pot-honey from Venezuela, Australia, Brazil, and Bolivia is richer in flavonoid glycosides than A. mellifera honey. Bioactivity of pot-honey considers antioxidant value, cancer prevention and therapy, and antibacterial properties of Latin American and Thai pot-honey, and a review on immunological properties of bee products. Propolis collected by stingless bees from Bolivia, Philippines, Thailand, and Venezuela also is characterized. A closing chapter on major initiatives of production, and marketing in some parts of Brazil, moves our attention toward sustainable economics and principles that would benefit with increased commercial availability and consumption of pot-honey. Human emotion and reaction to pot-honey indicate the evolution of natural contact between bees and our species. Sensory attributes of color, taste, texture, odor, and aroma are explored in detail. Pot-honey, as a healthy product, may someday follow millennia-old Traditional Chinese Medicine in the patterns of human response, ecology and cultural use. The inimitable Professor Camargo left a generous contribution placed here as a seminal chapter of this book. His authentic respect for the local names and cultural uses of the bees were instrumental in producing that which authors heard as a call to offer their insights and research findings. Introduction xv Future generations may have more ideas than time to further develop the science of pot-honey and decipher the messages carried, in monastic silence, by the bee chefs within their cerumen alchemist cauldrons. Mérida, Venezuela; Sydney, Australia Ribeirão Preto, Brazil Balboa, Panama Patricia Vit Silvia R.M. Pedro David W. Roubik Acknowledgments To the stingless bees and the stingless bee-keepers of the world, and for the pot-honey and meliponiculture that have evolved. In addition to contributing to inspiring several chapters, Charles D. Michener helped with additional editing and suggestions. Carlos Augusto Rosa and Paula São Thiago Calaça kindly contributed the list of microorganisms associated with bees. Various authors updated plants listed in their chapters. All botanical scientific names were checked and family names updated by Jorge Enrique Moreno Patiño in the lists of plants, according to the Missouri Botanical Garden (Tropics) database. The chapter reviewers provided timely and detailed comments and criticisms: Maria Lúcia Absy, Ingrid Aguilar, Ligia Almeida-Muradian, Monika O Barth, Alfred Botha, Susanna Buratti, José Camina, João Pedro Cappas e Sousa, José Ángel Cova, David De Jong, Rosires Deliza, Michael Engel, Wolf Engels, Miguel Ángel Fernández Muiño, Mabel Gil-Izquierdo, Cynthia FP Luz, Walter Farina, Daniela Freitas, Klaus Hartfelder, John-Erick Haugen, Tim Heard, Robert Kajobe, Gina Meccia, Charles D Michener, Gabriel AR Melo, Guiomar Nates-Parra, César Pérez, James Nieh, Auro Nomizo, Livia Persano Oddo, Silvia RM Pedro, Gabor Peter, Claus Rasmussen, Martyn Robinson, David W Roubik, Gianni Sacchetti, María Teresa Sancho Ortiz, Judith Slaa, Bruno A Souza, Marta Regina Verruma-Bernardi, Rogel Villanueva, Patricia Vit, and Alfredo Usubillaga. We acknowledge our institutions and authorities for the academic support. xvii Contents Part I Origin, Biodiversity and Behavior of the Stingless Bees (Meliponini) 1 The Meliponini ....................................................................................... Charles D. Michener 2 Historical Biogeography of the Meliponini (Hymenoptera, Apidae, Apinae) of the Neotropical Region ......................................... João Maria Franco de Camargo† 3 19 3 Australian Stingless Bees ....................................................................... Megan Halcroft, Robert Spooner-Hart, and Anne Dollin 35 4 Stingless Bees from Venezuela .............................................................. Silvia R.M. Pedro and João Maria Franco de Camargo 73 5 Stingless Bees (Hymenoptera: Apoidea: Meliponini) of French Guiana ................................................................................... Alain Pauly, Silvia R.M. Pedro, Claus Rasmussen, and David W. Roubik 87 6 Stingless Bees of Guatemala.................................................................. Carmen Lucía Yurrita Obiols and Mabel Vásquez 99 7 Stingless Bees of Costa Rica .................................................................. Ingrid Aguilar, Eduardo Herrera, and Gabriel Zamora 113 8 Stingless Bees in Argentina ................................................................... Arturo Roig-Alsina, Favio Gerardo Vossler, and Gerardo Pablo Gennari 125 9 Mexican Stingless Bees (Hymenoptera: Apidae): Diversity, Distribution, and Indigenous Knowledge ............................................ Ricardo Ayala, Victor H. Gonzalez, and Michael S. Engel 135 xix xx 10 Contents The Role of Useful Microorganisms to Stingless Bees and Stingless Beekeeping ....................................................................... Cristiano Menezes, Ayrton Vollet-Neto, Felipe Andrés Felipe León Contrera, Giorgio Cristino Venturieri, and Vera Lucia Imperatriz-Fonseca 153 11 Microorganisms Associated with Stingless Bees ................................. 173 Paula B. Morais, Paula S. São Thiago Calaça, and Carlos Augusto Rosa 12 Stingless Bee Food Location Communication: From the Flowers to the Honey Pots.................................................................................... Daniel Sánchez and Rémy Vandame 13 On the Diversity of Foraging-Related Traits in Stingless Bees .......... Michael Hrncir and Camila Maia-Silva Part II 187 201 Stingless Bees in Culture, Traditions and Environment 14 Stingless Bees: A Historical Perspective .............................................. Richard Jones 15 Medicinal Uses of Melipona beecheii Honey, by the Ancient Maya ......................................................................................... Genoveva R. Ocampo Rosales 229 Staden’s First Report in 1557 on the Collection of Stingless Bee Honey by Indians in Brazil.................................................................... Wolf Engels 241 Melipona Bees in the Scientific World: Western Cultural Views ........................................................................................ Raquel Barceló Quintal and David W. Roubik 247 Taxonomy as a Tool for Conservation of African Stingless Bees and Their Honey .................................................................................... Connal Eardley and Peter Kwapong 261 16 17 18 19 Effects of Human Disturbance and Habitat Fragmentation on Stingless Bees..................................................................................... Virginia Meléndez Ramírez, Laura Meneses Calvillo, and Peter G. Kevan 219 269 Part III What Plants Are Used by the Stingless Bees? 20 Palynology Serving the Stingless Bees.................................................. Ortrud Monika Barth 285 21 How to Be a Bee-Botanist Using Pollen Spectra.................................. David W. Roubik and Jorge Enrique Moreno Patiño 295 xxi Contents 22 Important Bee Plants for African and Other Stingless Bees ............. Robert Kajobe 23 Botanical Origin of Pot-Honey from Tetragonisca angustula Latreille in Colombia ............................................................................. Diana Obregón, Ángela Rodríguez-C, Fermín J. Chamorro, and Guiomar Nates-Parra Part IV 315 337 Sensory Attributes and Composition of Pot-Honey 24 Sensory Evaluation of Stingless Bee Pot-Honey .................................. Rosires Deliza and Patricia Vit 349 25 Melipona favosa Pot-Honey from Venezuela ....................................... Patricia Vit 363 26 Tetragonisca angustula Pot-Honey Compared to Apis mellifera Honey from Brazil.................................................................................. Ligia Bicudo de Almeida-Muradian 27 Honey of Colombian Stingless Bees: Nutritional Characteristics and Physicochemical Quality Indicators ............................................. Carlos Alberto Fuenmayor, Amanda Consuelo Díaz-Moreno, Carlos Mario Zuluaga-Domínguez, and Martha Cecilia Quicazán 28 The Pot-Honey of Guatemalan Bees..................................................... María José Dardón, Carlos Maldonado-Aguilera, and Eunice Enríquez 29 Pot-Honey of Six Meliponines from Amboró National Park, Bolivia...................................................................................................... Urbelinda Ferrufino and Patricia Vit 30 31 32 An Electronic Nose and Physicochemical Analysis to Differentiate Colombian Stingless Bee Pot-Honey .................................................... Carlos Mario Zuluaga-Domínguez, Amanda Consuelo Díaz-Moreno, Carlos Alberto Fuenmayor, and Martha Cecilia Quicazán Nuclear Magnetic Resonance as a Method to Predict the Geographical and Entomological Origin of Pot-Honey ............... Elisabetta Schievano, Stefano Mammi, and Ileana Menegazzo Nonaromatic Organic Acids of Honeys................................................ María Teresa Sancho, Inés Mato, José F. Huidobro, Miguel Angel Fernández-Muiño, and Ana Pascual-Maté 375 383 395 409 417 429 447 Part V Biological Properties 33 Flavonoids in Stingless-Bee and Honey-Bee Honeys .......................... Francisco A. Tomás-Barberán, Pilar Truchado, and Federico Ferreres 461 xxii Contents 34 Antioxidant Activity of Pot-Honey ....................................................... Antonio Jesús Rodríguez-Malaver 475 35 Use of Honey in Cancer Prevention and Therapy .............................. Patricia Vit, Jun Qing Yu, and Fazlul Huq 481 36 Bioactivity of Honey and Propolis of Tetragonula laeviceps in Thailand.............................................................................................. Chanpen Chanchao 37 Costa Rican Pot-Honey: Its Medicinal Use and Antibacterial Effect ........................................................................ Gabriel Zamora, María Laura Arias, Ingrid Aguilar, and Eduardo Umaña 495 507 38 Immunological Properties of Bee Products ......................................... José Angel Cova 513 39 Chemical Properties of Propolis Collected by Stingless Bees ............ Omur Gençay Çelemli 525 Part VI Marketing and Standards of Pot-Honey Production and Marketing of Pot-Honey ............................................ Rogério Marcos de Oliveira Alves 541 Appendix A Taxonomic Index of Bees ...................................................... 557 Appendix B List of Bee Taxa ...................................................................... 569 Appendix C Common Names of Stingless Bees ........................................ 581 Appendix D Taxonomic Index of Plant Families ...................................... 585 Appendix E List of Plant Taxa Used by Bees ........................................... 597 Appendix F Common Names of Plants Used for Nesting by Stingless Bees .................................................................... 615 Appendix G Common Names of Medicinal Plants Used with Honey by Mayas ............................................................ 617 Appendix H Microorganisms Associated to Stingless Bees or Used to Test Antimicrobial Activity ................................ 619 40 Appendix I Summary of Meliponine and Apis Honey Composition............................................................................ 623 Information of Collected Stingless Bees ............................... 627 Index ................................................................................................................ 629 Appendix J Contributors Ingrid Aguilar Centro de Investigaciones Apícolas Tropicales (CINAT), Universidad Nacional, Heredia, Costa Rica Ligia Bicudo de Almeida-Muradian Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, Brazil Rogério Marcos de Oliveira Alves Instituto Federal de Educação, Ciência e Tecnologia Baiano, Salvador, Bahia, Brazil María Laura Arias Centro de Investigaciones en Enfermedades Tropicales (CIET), Universidad de Costa Rica, San José, Costa Rica Ricardo Ayala Estación de Biología Chamela, Instituto de Biología, Universidad Nacional Autónoma de México (UNAM), San Patricio, Jalisco, Mexico Raquel Barceló Quintal History and Anthropology Area, Social Sciences and Human Studies Institute, Universidad Autónoma del Estado de Hidalgo, Pachuca, Mexico Ortrud Monika Barth Laboratório de Morfologia e Morfogênese Viral, Instituto Oswaldo Cruz, FIOCRUZ, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil João Maria Franco de Camargo† Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil Fermín J. Chamorro Laboratorio de Investigaciones en Abejas LABUN 128, Departamento de Biología, Universidad Nacional de Colombia, Bogotá, DC, Colombia Chanpen Chanchao Faculty of Science, Department of Biology, Chulalongkorn University, Bangkok, Thailand José Ángel Cova Clinical Immunology Institute, Faculty of Medicine, Universidad de Los Andes, Mérida, Venezuela xxiii xxiv Contributors María José Dardón Unidad de Conocimiento, Uso y Valoración de la Biodiversidad, Centro de Estudios Conservacionistas, Universidad de San Carlos de Guatemala, Ciudad de Guatemala, Guatemala Rosires Deliza Embrapa Agroindústria de Alimentos, Rio de Janeiro, RJ, Brazil Amanda Consuelo Díaz-Moreno Instituto de Ciencia y Tecnología de Alimentos ICTA, Universidad Nacional de Colombia, Bogotá, Colombia Anne Dollin Australian Native Bee Research Centre, North Richmond, Australia Connal Eardley School of Biological and Conservation Sciences, University of KwaZulu–Natal, Pietermaritzburg, South Africa Michael S. Engel Division of Entomology, Natural History Museum, University of Kansas, Lawrence, KS, USA Wolf Engels Zoological Institute, University of Tübingen, Tübingen, Germany Departamento de Genética, Universidade de São Paulo, Ribeirão Preto, Brazil Eunice Enríquez Unidad de Conocimiento, Uso y Valoración de la Biodiversidad, Centro de Estudios Conservacionistas, Universidad de San Carlos de Guatemala, Guatemala City, Guatemala Miguel Angel Fernández-Muiño Department of Biotechnology and Food Science, Faculty of Science, Universidad de Burgos, Burgos, Spain Federico Ferreres Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), Campus, Universitario Espinardo, Murcia, Spain Urbelinda Ferrufino Asociación Ecológica de Oriente, Santa Cruz, Bolivia Carlos Alberto Fuenmayor Instituto de Ciencia y Tecnología de Alimentos– ICTA, Universidad Nacional de Colombia, Bogotá, Colombia Gerardo Pablo Gennari INTA Estación Experimental Agropecuaria Famaillá, Instituto Nacional de Tecnología Agropecuaria, Famaillá, Tucumán, Argentina Ömür Gençay Çelemli Science Faculty, Department of Biology, Hacettepe University, Beytepe, Ankara, Turkey Victor H. Gonzalez Southwestern Oklahoma State University, Biological Sciences, USA Megan Halcroft School for Health and Science, Hawkesbury Campus, University of Western Sydney, Penrith, NSW, Australia Tim A. Heard CSIRO Ecosystem Science, Dutton Park, QLD, Australia Eduardo Herrera Centro de Investigaciones Apícolas Tropicales (CINAT), Universidad Nacional, Heredia, Costa Rica Contributors xxv Michael Hrncir Laboratório de Ecologia Comportamental Departamento de Ciências Animais, Universidade Federal do Semi-Árido, Mossoró, RN, Brazil José F. Huidobro Faculty of Pharmacy, Department of Analytical Chemistry, Nutrition and Food Science, University of Santiago de Compostela, Santiago de Compostela, Spain Fazlul Huq Cancer Research Group, School of Medical Sciences, The University of Sydney, Lidcombe, NSW, Australia Vera Lucia Imperatriz-Fonseca Universidade Federal Rural do Semiárido, Mossoro, RN, Brazil Richard Jones International Bee Research Association (IBRA), Cardiff, Wales, UK Robert Kajobe National Agricultural Research Organisation (NARO), Rwebitaba Zonal Agricultural Research and Development Institute (ZARDI), Fort Portal, Uganda Peter G. Kevan Canadian Pollination Initiative, School of Environmental Sciences, University of Guelph, Guelph, ON, Canada Peter Kwapong Department of Entomology & Wildlife, International Stingless Bee Centre, School of Biological Sciences, University of Cape Coast, Cape Coast, Ghana Felipe Andrés Felipe León-Contrera Universidade Federal do Pará, Belém, PA, Brazil Camila Maia-Silva Faculdade de Filosofia, Ciências e Letras, Universidade de São Paulo, Ribeirão Preto, SP, Brazil Carlos Maldonado-Aguilera Unidad de Conocimiento, Uso y Valoración de la Biodiversidad, Centro de Estudios Conservacionistas, Universidad de San Carlos de Guatemala, Guatemala City, Guatemala Stefano Mammi Department of Chemical Sciences, University of Padova, Padova, Italy Inés Mato Faculty of Pharmacy, Department of Analytical Chemistry, Nutrition and Food Science, University of Santiago de Compostela, Santiago de Compostela, Spain Virginia Meléndez Ramírez Departamento de Zoología, Campus de Ciencias Biológicas y Agropecuarias, Universidad Autónoma de Yucatán, Mérida, Yucatán, Mexico Ileana Menegazzo Department of Chemical Sciences, University of Padova, Padova, Italy Cristiano Menezes Embrapa Amazônia Oriental, Belém, PA, Brazil xxvi Contributors Laura Meneses Calvillo Departamento de Zoología, Campus de Ciencias Biológicas y Agropecuarias, Universidad Autónoma de Yucatán, Mérida, Yucatán, Mexico Charles D. Michener Division of Entomology, Natural History Museum, University of Kansas, Lawrence, KS, USA Paula B. Morais Laboratório de Microbiologia Ambiental e Biologia Six, Fundação Universidade Federal de Tocantins, Palmas, Tocantins, Brazil Jorge Enrique Moreno Patiño Smithsonian Tropical Research Institute, Balboa, Ancon, Republic of Panama Guiomar Nates-Parra Laboratorio de Investigaciones en Abejas LABUN 128, Departamento de Biología, Universidad Nacional de Colombia, Bogotá, DC, Colombia Paulo Nogueira-Neto Departamento de Ecologia Geral, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil Diana Obregón Laboratorio de Investigaciones en Abejas LABUN 128, Departamento de Biología, Universidad Nacional de Colombia, Bogotá, DC, Colombia Genoveva R. Ocampo Rosales Facultad de Filosofía y Letras, Universidad Nacional Autónoma de México, Del. Tlalpan, México, Mexico Ana Pascual-Maté Faculty of Sciences, Department of Biotechnology and Food Science, University of Burgos, Burgos, Spain Alain Pauly Department Entomology, Royal Belgian Institute of Natural Sciences, Brussels, Belgium Silvia R.M. Pedro Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil Martha Cecilia Quicazán Instituto de Ciencia y Tecnología de Alimentos—ICTA, Universidad Nacional de Colombia, Bogotá, Colombia Claus Rasmussen Department of Biological Sciences, Aarhus University, Aarhus C, Denmark Ángela Rodríguez-C Laboratorio de Investigaciones en Abejas LABUN 128, Departamento de Biología, Universidad Nacional de Colombia, Bogotá, DC, Colombia Antonio Jesús Rodríguez-Malaver Department of Biochemistry, Faculty of Medicine, Universidad de Los Andes, Mérida, Venezuela Arturo Roig-Alsina Museo Argentino de Ciencias Naturales, Buenos Aires, Argentina Contributors xxvii Carlos Augusto Rosa Departamento de Microbiologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil David W. Roubik Smithsonian Tropical Research Institute, Ancón, Balboa, Republic of Panamá Daniel Sánchez El Colegio de la Frontera Sur, Tapachula, Chiapas, Mexico María Teresa Sancho Cátedra de Nutrición y Bromatología, Departamento de Biotecnología y Ciencia de los Alimentos, Universidad de Burgos, Burgos (Castilla y León), Spain Paula S. São Thiago Calaça Fundação Ezequiel Dias (FUNED), Gameleira, Belo Horizonte, Brazil Elisabetta Schievano Department of Chemical Science, Università di Padova, Padova, Italy Robert Spooner-Hart School for Health and Science, Hawkesbury Campus, University of Western Sydney, Penrith, NSW, Australia Francisco A. Tomás-Barberán Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), Campus Universitario Espinardo, Murcia, Spain Pilar Truchado Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), Campus Universitario Espinardo, Murcia, Spain Eduardo Umaña Centro de Investigaciones Apícolas Tropicales (CINAT), Universidad Nacional, Heredia, Costa Rica Rémy Vandame El Colegio de la Frontera Sur, Tapachula, Chiapas, Mexico Mabel Vásquez Unidad de Conocimiento, Uso y Valoración de la Biodiversidad, Centro de Estudios Conservacionistas, Universidad de San Carlos de Guatemala, Guatemala City, Guatemala Giorgio Cristino Venturieri Embrapa Amazônia Oriental, Belém, PA, Brazil Patricia Vit Apitherapy and Bioactivity, Food Science Department, Faculty of Pharmacy and Bioanalysis, Universidad de Los Andes, Mérida, Venezuela Cancer Research Group, Discipline of Biomedical Science, The University of Sydney, NSW, Australia Ayrton Vollet-Neto Universidade de São Paulo, Ribeirão Preto, SP, Brazil Favio Gerardo Vossler CONICET, Laboratorio de Sistemática y Biología Evolutiva (LASBE), Museo de La Plata, La Plata, Argentina Jun Qing Yu Cancer Research Group, Discipline of Biomedical Science, The University of Sydney, Lidcombe, NSW, Australia xxviii Contributors Carmen Lucía Yurrita Obiols Unidad de Conocimiento, Uso y Valoración de la Biodiversidad, Centro de Estudios Conservacionistas, Universidad de San Carlos de Guatemala, Guatemala City, Guatemala Gabriel Zamora Centro de Investigaciones Apícolas Tropicales (CINAT), Universidad Nacional, Heredia, Costa Rica Carlos Mario Zuluaga-Domínguez Instituto de Ciencia y Tecnología de Alimentos—ICTA, Universidad Nacional de Colombia, Bogotá, Colombia Part I Origin, Biodiversity and Behavior of the Stingless Bees (Meliponini) Chapter 1 The Meliponini Charles D. Michener 1.1 Introduction The stingless bees are a primarily tropical group of over 500 species (and possibly 100 more as yet undescribed). The pot-honey that they produce is the main subject of this book. Given that bees are so well known for their stings, stinglessness among bees seems rather sensational. The term “stingless bee” requires some examination, however. First, all male bees are completely stingless; the sting is a modified ovipositor, a structure found only in females. Second, the parts of the sting of stingless bees are actually present, much reduced and modified and not functional for stinging. Third, there are various other groups of bees whose females have reduced and nonfunctional stings. For example, females of the common bee genus Andrena have stings that are too small to be used as stings, and the very different bee genus Dioxys and its relatives have the most reduced stings of all bees, smaller than those of the “stingless bees.” Nonetheless, the term stingless bees is well established for the tribe Meliponini and we will use it for this group of primarily tropical bees. The stingless bees, like the well-known honey bees (tribe Apini, genus Apis) and unlike the thousands of species of other bees, live in more or less permanent colonies made up of workers (modified females) and usually only one female reproductive, the queen, for each colony. Thus females appear in two castes, workers and queens. Of the many kinds of bees, the stingless bees are the only ones that have long-term (sometimes called permanent) colonies, morphologically different worker and queen castes, and also reduced stings (so cannot sting). To clarify the position of bees within the order Hymenoptera: there is a large group within that order in which the ovipositor no longer functions to place eggs, and is typically modified into a sting. Members of this group are called the aculeate C.D. Michener (*) Division of Entomology, Natural History Museum, University of Kansas, 1501 Crestline Drive, Room 140, Lawrence, KS 66049-4401, USA e-mail: michener@ku.edu P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_1, © Springer Science+Business Media New York 2013 3 4 C.D. Michener Hymenoptera or the Aculeata, which includes the bees, ants, and wasps. One major group of Aculeata consists of those with the pronotum short, not reaching the tegulae but forming a rounded lobe below each tegula. These were long called the superfamily Sphecoidea, the sphecoid wasps and the bees. More recently and correctly they are called the Apoidea, the apoid wasps and the bees. The bees, technically the Apiformes or Anthophila, are an apparently monophyletic group of the Apoidea. They differ from the apoid wasps in that they no longer sting prey to feed their larvae but depend instead on other foods, nearly always pollen, as their major protein source, and they have at least some branched or plumose hairs and commonly other structures that may facilitate pollen collecting as well as nectar gathering (Michener 2007; Engel 2011). The bees are divided into several families (seven according to Michener 2007), one of which is the Apidae, which includes the large subfamily Apinae, within which is the tribe Meliponini. Recognition of the Meliponini is usually easy, although a few other groups of bees resemble that tribe superficially. A bee collector in tropical America, who may be taking the common stingless bees from the collecting net with fingers, will occasionally be surprised by a sting from a similar looking bee of the tribe Tapinotaspidini, usually of the genus Paratetrapedia. The Meliponini belongs to a monophyletic group of four tribes (Apini, Meliponini, Bombini, and Euglossini) known as the corbiculate bees because their females have a corbicula (Fig. 1.1) on each hind tibia (except that queens of the first two tribes listed lack corbiculae, as do workers of a few species that live by taking carrion or by robbing nests of other stingless bees). The corbicula is a large smooth area, often concave, margined by fringes of long hairs. It is used to carry pollen or sometimes other substances into the nest. The Meliponini can be differentiated from all other bees by the lack or weakness (relative to other veins) of the submarginal crossveins and the second recurrent vein in the forewing (Fig. 1.2). As in the Apini, the hind tibial spurs are absent (Fig. 1.1). The beginner, seeking to recognize stingless bees, should know that while some Meliponini of the genus Melipona are as large as or even larger than the common honey bee (Apis mellifera Linnaeus), the great majority are much smaller. Perhaps the smallest is a Madagascar species of Liotrigona whose workers are as small as 1.8 mm in length. Many particulars about Meliponini can be learned from NogueiraNeto (1953, 1970, 1997), Roubik (1989, 2006), and Wille (1983). 1.2 Classification Some earlier authors (e.g., Lepeletier de Saint-Fargeau 1836; Dalla Torre 1896) placed all Meliponini in a single genus, Melipona. Others (e.g., Smith 1854; Michener 1944; Schwarz 1948) recognized two major genera, Melipona for the species now placed in that genus and Trigona for all the rest of the Meliponini except a few robber species commonly placed in a separate genus. Trigona in this broad sense is very diverse, not monophyletic, containing species with different relationships to Melipona, and it becomes evident that it should be broken up into smaller and more homogeneous units. 1 The Meliponini 5 Fig. 1.1 Outer side of hind tibia and basitarsus of worker of Trigona (Trigona) amalthea (Olivier) showing the corbicula and the lack of tibial spurs, as well as the lack of the auricle (and pollen press) found in Apis (prepared by Sara Taliaferro, based on Michener 2007) Several groups were named as subgenera of Trigona but in 1946 and thereafter Moure elevated subgeneric groups to the status of genera and described various new genera. The genus-group names, i.e., generic and subgeneric names, are listed below. The status of many names is unsettled; Moure’s followers consider nearly all the named supraspecific taxa as genera while others (Michener 1990, 2007; Sakagami 1975) place many, rather subjectively, as subgenera of a moderate number of genera. The authors of different chapters of this book show different opinions on some such matters. For example, Austroplebeia australis is the same species that in another chapter is called Trigona australis. While the Meliponini are found in all parts of the tropical zone except many Pacific islands, no genus occurs throughout that zone. For our purposes, there are three tropical regions in the world: the American tropics (= Neotropics), sub-Saharan African (= Afrotropical region), and the Indoaustralian (= Austroasian) region. For convenience the meliponine taxa are listed below, for each of these three regions. The number of 6 C.D. Michener Fig. 1.2 Wings of Melipona fasciata Latreille (above) and Euglossa cordata (Linnaeus) (below). The latter shows the wing venation of most bees, with arrows marking the vein segments that are weak or absent in the Meliponini (prepared by Sara Taliaferro, based on Michener 2007) species shown in parentheses after each taxon must be viewed with some caution because distinct new species must exist, and especially because in the Meliponini there appear to be numerous cryptic species not yet recognized. The number of species listed is derived, with some adjustments, from Camargo and Pedro (2007) for the Americas, from Eardley (2004) for Africa plus Pauly et al. (2001) for Madagascar, and from Rasmussen (2008) for the Indoaustralian region. Synonymous names shown in the lists below after equal (=) symbols are of two types. Some are absolute synonyms. Others are synonymized by judgment. An example of the latter is Celetrigona which can be used for a distinct group which is here included in Trigonisca. Regardless of possible deficiencies in the lists, they clearly show the great diversity of stingless bees in the American tropics (over 400 species) where, in many localities, they are the most abundant bees, hence presumably the most important pollinators. They also show the much smaller and less diverse meliponine fauna in Africa, with that of the Indoaustralian region intermediate. 1 The Meliponini 7 Neotropical Meliponini are found northward to Cuba and the states of Tamaulipas and Sonora in Mexico, and southward to Buenos Aires Province, Argentina. The species are listed by Camargo and Pedro (2007) and identification of species is facilitated by keys and descriptions in numerous revisional papers such as Schwarz (1948) and many excellent revisions by Camargo and his associates, such as Camargo and Pedro (2009), as well as by regional studies such as Schwarz (1938) for Guyana and Ayala (1999) for Mexico. The genus-group taxa are listed below; subgenera are indented, and as indicated above the number of species is shown in parentheses. Cephalotrigona Schwarz 1940 (5) Lestrimelitta Friese 1903 (20) Melipona Illiger 1806 (= Micheneria Kerr, Pisiani and Aily 1967, Michmelia Moure 1975, Melikerria Moure 1992, and Eomelipona Moure 1992) (72) Meliwillea Roubik, Lobo and Camargo 1997 (1) Nannotrigona Cockerell 1922 (10) Nogueirapis Moure 1953 (3) Oxytrigona Cockerell 1917 (11) Paratrigona Schwarz 1938 (= Aparatrigona Moure 1951) (34) Paratrigonoides Camargo and Roubik 2005 (1) Partamona Schwarz 1939 Parapartamona Schwarz 1948 (7) Partamona Schwarz 1939 s.str. (= Patera Schwarz 1938) (32) Plebeia Schwarz 1938 Plebeia Schwarz 1938 s.str. (= Mourella Schwarz 1946 and Friesella Moure 1946) (42) Scaura Schwarz 1938 (= Schwarzula Moure 1946) (7) Schwarziana Moure 1943 (2) Scaptotrigona Moure 1942 (= Sakagamilla Moure 1989) (22) Trichotrigona Camargo and Moure 1983 (= ?Frieseomelitta) (1) Trigona Jurine 1807 Duckeola Moure 1944 (2) Frieseomelitta Ihering 1912 (16) Geotrigona Moure 1943 (21) Tetragona Lepeletier and Serville 1828 (= Ptilotrigona Moure 1951 and Camargoia Moure 1989) (19) Tetragonisca Moure 1946 (4) Trigona Jurine 1807 s.str. (= Amalthea Rafinesque 1815, Aphaneura Gray 1832, and Alphaneura Gray 1832) (32) Trigonisca Moure 1950 (= Celetrigona Moure 1950, Dolichotrigona Moure 1950, and Leurotrigona Moure 1950) (43) Frieseomelitta, Duckeola, and Tetragonisca, along with the genus Trichotrigona, may constitute a genus Frieseomelitta, separate from Trigona; their separation from Trigona is indicated by the phylogenetic study of Rasmussen and Cameron (2010). The same study shows Lestrimelitta among the species of Plebeia, making the latter paraphyletic. These matters should be investigated further. Sub-Saharan or Afrotropical Meliponini are found from Senegal, Niger, and Eritrea on the north to KwaZulu-Natal Province, South Africa, and the whole of Madagascar on the south. The species are listed and revised by Eardley (2004). The genus-group taxa are listed below; subgenera are indented. 8 C.D. Michener Cleptotrigona Moure 1961 (1) Dactylurina Cockerell 1934 (2) Hypotrigona Cockerell 1934 (4) Liotrigona Moure 1961 (9) Meliponula Cockerell 1934 Axestotrigona Moure 1961 (2) Meliplebeia Moure 1961 (= Pebeiella Moure 1961 and Apotrigona Moure 1961) (7) Meliponula Cockerell 1934 s.str. (1) Plebeina Moure 1961 (1) Indoaustralian or Australasian Meliponini are found from India to Taiwan and the Caroline Islands (perhaps introduced) and from southeastern China to New South Wales, Australia. The species are listed by Rasmussen (2008). Identification to the genus and subgenus levels should be facilitated by the keys of Moure (1961) and Michener (2000, 2007). Identification to the species level is made possible by revisional works such as, for the Asian region, Schwarz (1937, 1939) and Sakagami (1975, 1978), and for Australia, Dollin et al. (1997). The genus-group taxa are listed below (with some advice from the late S.F. Sakagami). Austroplebeia Moure 1961 (9) Heterotrigona Schwarz 1939 Geniotrigona Moure 1961 (3) Heterotrigona Schwarz 1939 s.str. (3) Sundatrigona Inoue and Sakagami 1995 (= Trigonella Sakagami and Moure 1975) (2) Homotrigona Moure 1961 (4) Lepidotrigona Schwarz 1939 (12) Lisotrigona Moure 1961 (4) Lophotrigona Moure 1961 (1) Odontotrigona Moure 1961 Odontotrigona Moure 1961 s.str.(1) Tetrigona Moure 1961 (5) Papuatrigona Michener and Sakagami 1990 (1) Pariotrigona Moure 1961 (1) Platytrigona Moure 1961 (6) Tetragonula Moure 1961 Tetragonilla Moure 1961 (4) Tetragonula Moure 1961 s.str.(32) 1.3 Biology All stingless bees live in colonies, as already indicated, consisting of dozens to tens or hundreds of thousands of workers, and usually only one queen. At any one time a few to many males may or may not be present in such a colony. Contrary to honey bees (Apis), males are usually similar to workers in size and appearance and queens, quite different. 1 The Meliponini 9 Major works exist on the biology of stingless bees, including such matters as nest construction and resultant structures, defense, foraging, reproduction, caste, and sex determination, as well as culture (meliponiculture) by humans, uses of their honey and cerumen (a combination of plant resin with bee wax) importance as pollinators, etc. Schwarz (1948) undertook the great task of presenting and summarizing everything then known about meliponine biology. Other good book-length accounts of meliponine biology and importance to humans are by Nogueira-Neto (1953, 1970, 1997); the last in particular contains a very extensive list of publications on the biology of stingless bees. A review article covering the same fields is by Wille (1983). 1.3.1 Reproduction There is no solitary phase in meliponine life history; colony life is continuous. When a colony is dividing, workers from the parent colony fly to a new site and prepare it as a nest, carrying construction materials and food there in repeated trips. A nest entrance of the form characteristic of the species is often or always constructed first. Eventually a new, often unmated, young queen flies to the new nest from the parent colony. The queen soon mates, sometimes within the new nest. For some time (weeks or even months) workers continue to fly back and forth carrying materials from the parent nest to the new one, until eventually such contact ceases and the new colony becomes independent. Wille and Orozco (1975) described the events in the founding of a new colony of Partamona orizabaensis (Strand) (originally identified as Trigona cupira Smith) in which interchange continued for 6 months. During this process as well as at other times many males, often from other colonies, assemble nearby or hover near the nest entrances, presumably attracted by pheromones produced by young queens. 1.3.2 Foraging At a nest entrance workers can constantly be seen carrying pollen, nectar, or construction materials into the nest. The foods go into pots, usually made of rather soft cerumen. Pollen and honey (made from the nectar) are placed in separate pots, not mixed. Of course it is this honey, in pots, that is the main subject of this book. Communication for the collection of food by various species is summarized by Aguilar-Monge (2004) and in this book, in Chap. 12. The above is written as though all stingless bees, like most other bees, collect their foods (nectar and pollen) from flowers and carry the foods to the nest where the larvae are fed. A few stingless bees deviate from this pattern. Some are known to visit scale insects (Coccidae) and collect their wax and honeydew. Nests of Plebeia (Scaura) timida Silvestri are in cavities of living plants and contain scale insects that provide a domestic source of honeydew (Camargo and Pedro 2002; 10 C.D. Michener Camargo 2008); this bee collects only pollen, not nectar, from flowers. Species of Plebeia subgenus Scaura have enlarged hind basitarsi with which they collect pollen from leaves or other flat surfaces onto which they have drifted from flowers above (Camargo and Pedro 2002). Some and perhaps most meliponines will occasionally rob from damaged nests of the same or other species, carrying away honey, pollen, provisions from brood cells, and construction materials. Species of the genera Lestrimelitta in the Neotropics and Cleptotrigona in Africa carry such behavior to the extreme; they do not visit flowers but live by mass robbing of nests of other species of stingless bees, from which they carry food and nest-making materials to their own nests (Sakagami et al. 1993; Portugal-Araújo 1958). Trichotrigona, known from only one locality, may also live by robbing, apparently by individuals solitarily entering host nests (Camargo 2008). Trichotrigona nests contain no food storage pots, the host apparently providing for that need. Carrion is sometimes visited by stingless bees for the liquid or bits of solid material. Three species, however, the group of Trigona (Trigona) hypogea Silvestri, do not collect from flowers, have reduced corbiculae, and their protein source is carrion rather than pollen (Roubik 1982). Of course “honey” from such bees (or from those that use feces for construction materials) is not appropriate for human consumption. Many stingless bees, especially small species, are attracted to perspiration of humans and other animals. People in most tropical areas are well aware of these pestiferous insects. More should be learned about the very minute bees (1.8–3.3 mm in length), particularly of the genera Trigonisca, Hypotrigona, Liotrigona, Lisotrigona, and Pariotrigona. Some of these bees can be frequent pests on perspiring humans but, although they carry pollen, they are not very commonly seen on flowers. In Southeast Asia bees of the last two genera listed above are not commonly attracted to perspiration but are attracted to eyes and collect tears of mammals (including humans), birds, and reptiles (Bänziger et al. 2009). Tears are high in protein and appear to be a significant source of food for these bees. Behavior of the minute bees of other continents should be investigated further. 1.3.3 Nests Data on the nest structure of many species is provided by Wille and Michener (1973). An account of nest structures, their evolution and variability, as well as their functions in defense, temperature control, and the like is given by Roubik (2006). For nest construction, stingless bees secrete wax from the dorsal surface of the abdomen, and collect gum and resin or propolis from vegetation. Rich sources include secretions around cut or broken branches and gum secreted as a result of biting off bark and young shoots by the bees themselves. Such damage to citrus trees by Trigona (Trigona) is well known. Mixtures of these materials for nest construction are called cerumen. Certain species, and for certain parts of the nest, such cerumen is supplemented with mud, feces of vertebrates, probably bits of carrion, etc. 1 The Meliponini 11 Various combinations of these materials appear to be used to produce the hard and tough, hard and brittle, to soft and pliable cerumens used in construction of the various sheets, pillars, pots, cells, etc. of the nest. Nest sites vary widely. Many species use hollows, usually in tree trunks or large branches. Such hollows, usually caused by rot, are favored if they have small entrances that can be narrowed and if any extra openings can be closed by the bees’ construction activities. Some species appear to prefer cavities of other kinds, for example in limestone cliffs or in constructs by humans (Bänziger et al. 2009, 2011). Thus some species, especially small forms, are common in villages or towns where their nests are frequent in cavities between walls of buildings or in other sorts of man-made cavities. Examples are Trigona (Tetragonisca) angustula (Latreille) and Tetragonula fuscobalteata (Cameron). Such species may not have a preference for the types of cavities found in buildings; they may merely tolerate a wider variety of locations and cavity sizes and shapes than do most species. For the Meliponini as a whole, the cavities used vary from huge in the trunk of a forest tree for a large species with large colonies to the abandoned burrow of a cerambycid beetle for a small colony of a minute species of Trigonisca. Other species nest in the ground, perhaps in cavities resulting from rotting of large roots or from activities of rodents, ants, or other animals. Probably the bees enlarge and modify such cavities, but there is no evidence that the bees ever start at the surface and dig a nest cavity in the ground. Some species, however, do make their own nest cavities within exposed nests of ants or termites. Workers from a parent bee colony construct a typical nest entrance projecting from a termite or ant nest, and then dig to construct a cavity and nest, keeping it constantly lined to exclude the hosts from the growing bee nest inside the host’s nest. Such behavior seems to have originated independently in diverse groups of Meliponini. Arboreal termites (Nasutitermes) are the hosts for Plebeia (Scaura) latitarsis (Friese) in the Neotropical region (Wille and Michener 1973); arboreal leaf nests of ants (Camponotus) are hosts for Paratrigona peltata (Spinola) in Costa Rica while ants (Crematogaster) are the hosts for Heterotrigona (Sundatrigona) moorei (Schwarz) in Thailand and Sumatra (Sakagami et al. 1989). Some Meliponini do not nest in preformed cavities or in nests of other social insects, but they make their own “cavities” by constructing exposed walls surrounding a space in which they live. For example, some species of Partamona make nests against walls, cliffs, or tree trunks. Such a nest looks as though someone had thrown a large glob of mud against a vertical surface, but of course the bees constructed the nest by carrying mud, wax, cerumen, etc. Other species construct nests, sometimes very large, by building on or around small tree branches so that the nest is exposed on all sides. An excellent example is Trigona (Trigona) corvina Cockerell, whose thick, hard nest walls consist largely of bees’ feces full of pollen exines (Roubik and Moreno Patiño 2009). While the nests of stingless bees are rather diverse in structure, they all follow a basic pattern shown in Fig. 1.3. They are the most complex of bee nests. The heart of the nest, usually more or less in the center of the nesting cavity, is the brood chamber, containing the brood cells in each of which one bee is reared from egg to 12 C.D. Michener Fig. 1.3 Diagram of a stingless bee nest in a hollow tree trunk with parts labeled (modified from Nogueira-Neto 1970). The elongate food storage pots shown are unusual; they are more often irregularly spherical (prepared by Sara Taliaferro, based on Michener 2007) emergent adult. Thereafter the cell is destroyed. The cells, which open upward (or laterally in Dactylurina) are provisioned, an egg is laid in each (normally by the queen), after which the cell is closed; there is no progressive feeding of the larva. The cells are commonly arranged to form a stack of horizontal combs, sometimes joined to form a broad spiral. In Dactylurina, however, cells are in vertical combs arranged much as in Apis. And in scattered taxa among the Meliponini the comb arrangement is to varying degrees lost so that cells are in clusters. It is the species with cells in clusters that utilize small and irregular cavities, sometimes with the brood cells dispersed in different subcavities. Workers and males are reared in similar cells in the same cluster or comb; queens come from a few larger irregular brood cells, except in the genus Melipona in which queens are produced in ordinary brood cells among the cells producing other castes. In that genus the queens are unusually small; there is no evidence that they receive any special treatment during development and they are produced (and destroyed) in considerable numbers. This leads to the conclusion that the female castes are determined genetically in Melipona whereas in other Meliponini the larger amounts of food provided in their large cells appear to produce queens. 1 The Meliponini 13 Surrounding the brood chamber is the involucrum. It is frequently laminate, that is, made up of several layers with or without spaces between them in which bees can move about. The involucrum is absent in some species that have brood cells in clusters rather than combs. Outside the involucrum, in one or more clusters or even in a partial layer, are the food pots where honey and pollen are stored. Of course the honey pots and their contents are the main topic of this book. The pots vary among species in size and shape (unusually elongate in Fig. 1.3) but are always much larger than brood cells. Surrounding the whole nest, that is outside the storage pots, is a layer of batumen, which is hard gray, brown, or black material, often with a thin, brittle outermost layer that breaks if disturbed, allowing rapid exit of many bees for defense. In a cavity batumen may include a single lining layer often less than a millimeter thick that smooths irregularities in the wood or soil walls. To close off excess space the batumen may form a thick layer. For example in a long hollow in a tree trunk, strong batumen plates above and below the nest may close off the nest area from other parts of the hollow (Fig. 1.3). The strong and usually laminate outside walls of exposed nests are batumen; in part of the nest laminate batumen may grade into the laminate involucrum. An entrance tube, usually opening in the nest outside the involucrum, extends to the outside world by an entrance that varies widely among species and, except for exposed nests, is usually the only outside indication of the presence of a nest. It may be that scarcity of suitable nesting cavities has been a limiting factor for Meliponini. Since small and irregular cavities are more frequent than larger cavities that can be appropriately closed off, it is not surprising that minute size appears to have arisen repeatedly among stingless bees. Or perhaps small size characterized some ancestral Meliponini. Often small size is accompanied by brood cells in clusters, not surrounded by an involucrum. However, brood cells of A. australis (Friese) are in large clusters, with an involucrum, in rather large cavities (Michener 1961). 1.3.4 Defense Defense is a significant function of stings in many aculeate Hymenoptera, but of course not for stingless bees. Strong nest structure, difficult to penetrate, must be important. Attacks on intruders by worker stingless bees, however, cannot be ignored. Especially in species that construct exposed nests, workers can swarm out of the nest in large numbers. They get into the hair, eyes, ears, and sometimes under clothing. They crawl about, bite, are sticky, and some say they have offensive odors. Particularly objectionable are species of Oxytrigona (they do not have exposed nests). From enlarged mandibular glands they bite a liquid containing formic acid into the skin. The result is severe pain (hence the name fire bees) and long-lasting lesions. Defense against parasitic and predaceous arthropods must also be important for stingless bees. That the nests are completely sealed except for small and easily guarded entrances suggests that natural enemies have played a role in the evolution of meliponine nesting behavior. Of course foraging workers are subject to the usual predators of flying insects and floral visitors. 14 1.4 C.D. Michener History and Phylogeny The fossil record for bees is very incomplete. Nonetheless a few fossil Meliponini have been found. The oldest, and it may also be the oldest fossil bee, is the Late Cretaceous (about 70 million years ago, Mya) Cretotrigona prisca (Michener and Grimaldi) from New Jersey amber. This species is surprisingly similar to Trigona (Trigona) of the American tropics (Michener and Grimaldi 1988; Engel 2000). Two genera of stingless bees are known from the Eocene (44 Mya) Baltic amber. The species are Kelneriapis eocenica (Kelner-Pillault) and Liotrigonopsis rozeni Engel. Both species are minute (body length little over 3 mm) and have greatly reduced wing venation like the recent minute Meliponini. Engel (2001a, b) provided a detailed account of these species. More recent fossil Meliponini include the several species of the extinct genus Proplebeia Michener from Miocene (15–20 Mya) amber in the Dominican Republic and southern Mexico (Camargo et al. 2000). Except for Melipona which is perhaps introduced, Meliponini no longer exist in the Greater Antilles; perhaps they disappeared during a dry epoch or during subduction of portions of the various islands. It is noteworthy that Meliponini (Cretotrigona) are found at least as early as any fossil bees, yet they have striking derived features that unite the Meliponini and distinguish them from other bees. These characters such as reduced wing venation, reduced sting, etc. must have originated substantially after the bees originated from related wasps. For other synapomorphies of the Meliponini see Michener (2007). Engel (2004) suggests that bees differentiated from the related wasps in the later part of the Early Cretaceous, when flowering plants were becoming dominant, and that by Late Cretaceous the major lineages of bees, of which the Meliponini is one, had been established. Although bees in general probably arose in, and much of their early evolution probably occurred in, xeric areas, the stingless bees, to judge by their present distribution, probably evolved in forested zones. The fossils of stingless bees from New Jersey and the Baltic region indicate that in the Late Cretaceous and the Eocene Meliponini occurred well outside the modern tropical zone to which they are now almost completely restricted. The fact that the present meliponine faunas of South America and Africa have no genera in common indicates that these genera arose and differentiated after the origin of the South Atlantic Ocean in the Late Cretaceous. Rasmussen and Cameron (2010) estimated dates for various events in meliponine evolution. Earlier studies of phylogenetic relationships within the Meliponini were summarized by Michener (2007). Several of these studies, based primarily on morphological characters, suffered from utilizing too few characters; different studies gave quite different results. For example the genus Melipona is sometimes sister to all the other genera. Other studies place Melipona among the other genera. A study by Wille (1979) thoughtfully presented many characters but the basis for his phylogenetic tree is not very clear. Certain authors believed that the Meliponini originated in South America because of the great diversity of the group there now. Wille, however, believed that the tribe probably originated in Africa because of ancestral 1 The Meliponini 15 (plesiomorphic) characters such as a less reduced sting in all the African genera except Hypotrigona. Recent molecular work using sequences within gene fragments (Rasmussen and Cameron 2007, 2010) provides more satisfying results in that major clades make good sense geographically. The major division is between, I, the neotropical clade and the Old World clade, which is itself divisible into, II, the African clade and, III, the Indoaustralian clade. The two exceptions are the genera Austroplebeia and Lisotrigona which fall in clade II although geographically they belong with clade III. References Aguilar-Monge I. 2004. Communication and recruitment for the collection of food in stingless bees: a behavioral approach. 150 pp, thesis, University of Utrecht [Netherlands]. Ayala R. 1999. Revision de las abejas sin aguijon de México. Folia Entomologica Mexicana 106:1–123. Bänziger H, Boongird S, Sukumalanand P, Bänziger S. 2009. Bees that drink human tears. Journal of the Kansas Entomological Society 82:135–150. Bänziger H, Pumikong S, Kanokorn S. 2011. The remarkable nest entrance of tear drinking Pariotrigona klossi aand other stingless bees nesting in limestone cavities. Journal of the Kansas Entomological Society 84:22–35. Camargo JMF (2008) Biogeografía histórica dos Meliponini (Hymenoptera, Apidae, Apinae) da região neotropical, p 13–26. In: Vit P (ed) Abejas sin Aguijón y Valorización sensorial de su Miel. APIBA-FFB-DIGECEX-ULA, Mérida, Venezuela, 146 pp. Camargo JMF, Grimaldi DA, Pedro SRM. 2000. The extinct fauna of stingless bees (Hymenoptera: Apidae: Meliponini) in Dominican amber: Two new species and redescription of the male of Proplebeia dominicana (Wille and Chandler). American Museum Novitates 3293:1–24. Camargo JMF, Pedro SRM. 2002. Mutualistic association between a tiny Amazonian stingless bee and a wax-producing scale insect. Biotropica 34:1–6. Camargo JMF, Pedro SRM. 2007. Meliponini Lepeletier, 1836. pp. 272–578. In Moure JS, Urban D, Melo GAR, eds. Catalogue of Bees (Hymenoptera, Apoidea) in the Neotropical Region. Sociedade Brasileira de Entomologia; Curitiba, Brasil. xiv+1058 pp. Camargo JMF, Pedro SRM. 2009. Neotropical Meliponini: The genus Celetrigona Moure. Zootaxa 2135:37–54. de Dalla Torre CG. 1896. Catalogus Hymenopterorum, vol. X. Englemann; Lipsiae [Leipzig, Germany]. vii+643 pp. Dollin AE, Dollin LJ, Sakagami SF. 1997. Australian stingless bees of the genus Trigona. Invertebrate Taxonomy 12:861–896. Eardley CD. 2004. Taxonomic review of the African stingless bees. African Plant Protection 10:63–96. Engel MS. 2000. A new interpretation of the oldest fossil bee. American Museum Novitates 3296:1–11. Engel MS. 2001a. A monograph of the Baltic amber bees and evolution of the Apoidea. Bulletin of the American Museum of Natural History 259:1–192. Engel MS. 2001b. Monophyly and extensive extinction of advanced eusocial bees: Insights from an unexpected Eocene diversity. Proceedings of the National Academy of Science [USA] 98:1661–1664. Engel MS. 2004. Geological history of the bees. Revista Tecnologia e Ambiente [Criciúma, Brazil] 10:9–33. 16 C.D. Michener Engel MS. 2011. Systematic melittology: where from here? Systematic Entomology 36:2–15. Lepeletier de Saint-Fargeau A. 1836. Histoire naturelle des insectes, Hyménoptéres, vol. 1. Encycl. Rorat; Paris, France. 547 pp. Michener CD. 1944. Comparative external morphology, phylogeny, and a classification of the bees. Bulletin of the American Museum of Natural History 82:151–326. Michener CD. 1961. Observations on the nests and behavior of Trigona in Australia and New Guinea. American Museum Novitates 2026:1–46. Michener CD. 1990. Classification of the Apidae. University of Kansas Science Bulletin 54:75–164. Michener CD. 2000. The bees of the world. Johns Hopkins Univ. Press; Baltimore, United States. xiv + 913 pp. Michener, CD. 2007. The bees of the world. Second edition. Johns Hopkins Univ. Press; Baltimore, United States. xvi + 953 pp. Michener CD, Grimaldi DA. 1988. A Trigona from Late Cretaceous amber of New Jersey. American Museum Novitates 2917:1–10. Moure JS. 1946. Contribuição para o conhecimento dos Meliponinae. (Hym. Apoidea). Revista de Entomologia 17:437–443. Moure JS. 1961. A preliminary supraspecific classification of the Old World meliponine bees. Studia Entomologica [Brazil] 4:181–242. Nogueira-Neto P. 1953. A criação de abelhas indígenas sem ferrão. Chácaras e Quintais; São Paulo:iii + 280 pp. Nogueira-Neto P. 1970. A criação de abelhas indígenas sem ferrão. Second edition. Chácaras e Quintais; São Paulo:365 pp. Nogueira-Neto P. 1997. Vida e criação de abelhas indígenas sem ferrão. Nogueirapis; São Paulo:446 pp. Pauly A, Brooks RW, Nilsson LA, Pesenko YA, Eardley CD, Terzo M, Griswold T, Schwarz M, Patiny S, Muzinger J, Barbier Y. 2001. Hymenoptera Apoidea de Madagascar et des iles voisines. Annales Sciences Zoologiques (Musée Royal de l’Afrique Centrale, Tervuren) 286:1–390, pls. 1–16. Portugal-Araújo V. 1958. A contribution to the bionomics of Lestrimelitta cubiceps. Journal of the Kansas Entomological Society 31:203–211. Rasmussen C. 2008. Catalog of the Indo-Malayan / Australasian stingless bees. Zootaxa 1935:1–80. Rasmussen C, Cameron SA. 2007. A molecular phylogeny of the old world stingless bees (Hymenoptera: Apidae: Meliponini) and the non-monophyly of the large genus Trigona. Systematic Entomology 32:26–39. Rasmussen C, Cameron SA. 2010. Global stingless bee phylogeny supports ancient divergence, vicariance, and long distance dispersal. Biological Journal of the Linnean Society 99:206–232. Roubik DW. 1982. Obligate necrophagy in a social bee. Science 217:1059–1060. Roubik DW. 1989. Ecology and natural history of tropical bees. Cambridge Univ. Press; New York. x + 514 pp. Roubik DW. 2006. Stingless bee nesting biology. Apidologie 37:124–143. Roubik DW, Moreno Patiño JE. 2009. Trigona corvina: An ecological study based on an unusual nest structure and pollen analysis. Psyche 2009 (258756):1–7. Sakagami SF. 1975. Stingless bees (excl. Tetragonula) from the continental southeast Asia in the collection of Bernice P. Bishop Museum, Honolulu, Journal of the Faculty of Science, Hokkaido University, Series VI, Zoology 20:49–76. Sakagami SF. 1978. Tetragonula stingless bees of the continental southeast Asia and Sri Lanka. Journal of the Faculty of Science, Hokkaido University, Series VI, Zoology 21:165–247. Sakagami SF, Inoue T, Yamane S, Salmah S. 1989. Nests of the myrmecophilous stingless bee, Trigona moorei: How do the bees initiate their nest in an arboreal ant nest? Biotropica 21:265–274. 1 The Meliponini 17 Sakagami SF, Roubik DW, Zucchi R. 1993. Ethology of the robber stingless bee, Lestrimelitta limao. Sociobiology 21:237–277. Schwarz HF. 1937. Results of the Oxford University Sarawak (Borneo) expedition: Bornean stingless bees of the genus Trigona. Bulletin of the American Museum of Natural History 73:281– 328, pls.II-VII. Schwarz HF. 1938. The stingless bees (Meliponidae) of British Guiana and some related sforms, Bulletin of the American Museum of Natural History 74:437–508, pls. LII-LXII. Schwarz HF. 1939. The Indo-malayan species of Trigona. Bulletin of the American Museum of Natural History 76:83–141. Schwarz HF. 1948. Stingless bees (Meliponidae) of the western hemisphere. Bulletin of the American Museum of Natural History 90:xviii+546. Smith F. 1854. Catalogue of the hymenopterous insects in the collection of the part 2 British Museum, London, pp. 199–465, pls. vii-xii. Wille A. 1979. Phylogeny and relationships among the genera and subgenera of the stingless bees (Meliponinae) of the world. Revista de Biologia Tropical 27:241–277. Wille A. 1983. Biology of the stingless bees. Annual Review of Entomology 28:41–64. Wille A, Michener CD. 1973. The nest architecture of stingless bees with special reference to those of Costa Rica. Revista de Biologia Tropical 21 (supplemento 1):1–279. Wille A, Orozco E. 1975. Observations on the founding of a new colony by Trigona cupira (Hymenoptera:Apidae) in Costa Rica. Revista de Biologia Tropical 22:253–287. Chapter 2 Historical Biogeography of the Meliponini (Hymenoptera, Apidae, Apinae) of the Neotropical Region João Maria Franco de Camargo† Communicated by: David W. Roubik and Silvia R.M. Pedro Conference given at Universidad de Los Andes, Mérida, Venezuela, March 2008. Translation authorized by the Faculty of Pharmacy and Bioanalysis, Universidad de Los Andes. The Meliponini have a pantropical distribution (Indo-Australia, the Neotropics and Africa-Madagascar) which includes continental disjunctions unique among the Apidae, revealing a complex history of vicariance events of great antiquity. The trait of disjunction by vicariance permits the inference that Meliponini possibly had their origin on the ancient Gondwanan continent and possess a minimum age near 100 million years (Camargo and Pedro 1992). The oldest known fossil of Meliponini is Cretotrigona prisca, from upper Cretaceous New Jersey—USA, c.a. 65–96 Ma (Michener and Grimaldi 1988a, b; Engel 2000). From a few species (possibly only one that left descendants) which remained isolated in South America, after fragmentation of Gondwana, and final separation of that continent from Africa, came all existing diversity of the Neotropical region, J.M.F. Camargo † and S.R.M. Pedro Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, CEP 14040-901, Ribeirão Preto, SP, Brazil e-mail: dair.aily@hotmail.com D.W. Roubik Smithsonian Tropical Research Institute, Ancón, Balboa, Republic of Panamá MRC 0580-12, Unit 9100, Box 0948, DPO AA, 34002-9998, USA P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_2, © Springer Science+Business Media New York 2013 19 J.M.F. Camargo and P. Vit 20 Table 2.1 Genera and number of Meliponini species from the Neotropical region (in alphabetical order) Genus Number of species Aparatrigona Moure, 1951 Camargoia Moure, 1989 Celetrigona Moure, 1950 Cephalotrigona Schwarz, 1940 Dolichotrigona Moure, 1950 Duckeola Moure, 1944 Friesella Moure, 1946 Frieseomelitta Ihering, 1912 Geotrigona Moure, 1943 Lestrimelitta Friese, 1903 Leurotrigona Moure, 1950 Melipona Illiger, 1806 Meliwillea Roubik, Lobo and Camargo, 1997 Mourella Schwarz, 1946 Nannotrigona Cockerell, 1922 Nogueirapis Moure, 1953 Oxytrigona Cockerell, 1917 Parapartamona Schwarz, 1948 Paratrigona Schwarz, 1938 Paratrigonoides Camargo and Roubik, 2005 Partamona Schwarz, 1939 Plebeia Schwarz, 1938 Proplebeia Michener, 1982† Ptilotrigona Moure, 1951 Scaptotrigona Moure, 1942 Scaura Schwarz, 1938 Schwarziana Moure, 1943 Schwarzula Moure, 1946 Tetragona Lepeletier and Serville, 1828 Tetragonisca Moure, 1946 Trichotrigona Camargo and Moure, 1983 Trigona Jurine, 1807 Trigonisca Moure, 1950 2 3 1 5 10 2 1 16 20 19 2 69(+10 ssp.) 1 1 10 3 8 7 29 1 32 38 4 3 21 5 2 2 13 4 1 32 25 † extinct genus which comprises 33 genera, including one that is extinct, Proplebeia (Table 2.1), and 391 nominate taxa at the species-group level, following the recent catalog by Camargo and Pedro (2007b).1 Evolution of Neotropical Meliponini, in isolation since the upper Cretaceous, resulted not only in the abovementioned large taxonomic diversity, but also in a great variety in life histories, for example: species with obligate necrophagic habits, species The online version http://moure.cria.org.br/catalogue?id=27560, updated on 07 February 2012 by SRM Pedro, includes now 412 species (SRMP, personal note) 1 2 Historical Biogeography of the Meliponini (Hymenoptera, Apidae, Apinae)… 21 Fig. 2.1 Trigona hypogea, collecting meat at a dead lizard. Photo: provided by D. Wittmann that cultivate yeast associated with pollen, species having mutualistic relationships with scale insects, etc., in addition to a wide variety of methods used in nest construction. The obligate necrophagy habit (Fig. 2.1) is known in three species—Trigona necrophaga, T. hypogea, and T. crassipes (Roubik 1982; Camargo and Roubik 1991), the only bees which do not collect pollen (the corbicula is rudimentary in all of them) nor floral nectar; flesh of dead animals is their only protein source (and supply of lipids, carbohydrates and salts); sugars are obtained from ripe or rotting fruit on the ground, extrafloral nectaries, fallen flowers on the ground, etc. (and, possibly, the glycogen obtained from carcasses serves as a glucose source). Collected carrion is transported in the stomach, and regurgitated in storage pots, in the form of a yellowish or greenish jelly which is broken down (probably under the action of digestive enzymes) and mixed with “honey.” In the storage pots (Fig. 2.2), the proteinaceous paste mixed with honey undergoes the action of the bacteria. In the larval food of T. necrophaga, Gilliam et al. (1985) found five species of Bacillus with reducing enzymatic activity related to protein and lipid metabolism and hydrolysis of carbohydrates, likely involved in digestion of the animal remains, in addition to production of amino acids and antibiotics. In T. hypogea, the pots, after being filled with a proteinaceous substance, mixed with “honey,” are sealed and chemical reactions proceed inside them for 12–16 days (Noll et al. 1996). At the end of this maturation period, “honey” is obtained, free of reduced sugars, almost transparent, good tasting, and rich in free amino acids. The storage of pollen associated with yeast—Candida sp.—is only known in species of the genus Ptilotrigona, as reviewed by Camargo et al. (1982, sic =1992)2 and Camargo and Pedro (2004). Three species comprise the genus: Ptilotrigona lurida, 2 SRMP note. 22 J.M.F. Camargo and P. Vit Fig. 2.2 Necrophagous bee nest, Trigona hypogea (Itaituba, PA, Brazil); left, the storage pots with products derived from meat mixed with “honey”. Photo: J.M.F. Camargo of wide range in Amazonia, P. pereneae, endemic to western Amazonia, and P. occidentalis, which occurs from northwestern Ecuador to Darién and an isolated population in the area of the Osa Peninsula in Costa Rica (Camargo and Pedro 2004). The studies were made with P. lurida, for which dozens of nests were observed (Fig. 2.3). Pots containing “honey” or sweet liquids are rare or even absent in the nests, while pollen pots, associated with yeast (Fig. 2.4) are found in great number (in one of the three nests studied there was about 3.0 kg of pollen). The activity of yeast promotes the desiccation and stored life of the pollen; it makes pollen dry enough that it can produce a wrinkling and deformation of the pots. Another interesting aspect, still lacking complementary studies, is that utilization of resins (principally floral resins of the genus Clusia), collected by these bees and added to cerumen used for construction of storage pots and brood cells, is that it has bactericidal activity, but no fungicidal effect. The action of such resins can promote the growth of yeast free of bacteria (Lokvam and Braddock 1999; Camargo and Pedro 2004). It is only suggested but not proven, even now, that a part of the sugars, used by bees, may be derived from the metabolic activity of the yeast. Associations between certain species of Meliponini and free-living phytophagic hemipterans, which make sugar secretions (honeydew), are well known, but their mutualistic associations with sedentary hemipterans, coccids, are known only among species of the genus Schwarzula (Camargo and Pedro 2002). Silvestri (1902) 3 sic, = Schwarzula timida. Scaura timida was entered by error in the original text (Pedro SRM, personal communication). 2 Historical Biogeography of the Meliponini (Hymenoptera, Apidae, Apinae)… 23 Fig. 2.3 Nest of Ptilotrigona lurida (Camanaus, AM, Brazil); in the lower portion a large mass of pots can be seen, where the pollen associated with yeast is stored. Photo: J.M.F. Camargo was the first to suspect mutualism between Scaura timida3 and scale insects, but detailed observations only were made by Camargo and Pedro (2002), who observed dozens of nests of Schwarzula coccidophila, residing in galleries excavated by the larva of the moth Cossula sp. (Cossidae) in the branches of Campsiandra angustifolia (Caesalpiniaceae), on the banks of the Rio Negro, Amazonas state, Brazil. The scale insects (Cryptostigma sp.) are found attached to the gallery walls, in the nest interior, where they receive protection and care from the bees (Fig. 2.5), and, in exchange, offer sweet secretions and additional wax the bees use in nest construc- 24 J.M.F. Camargo and P. Vit Fig. 2.4 Ptilotrigona lurida, closeup of pollen covered with yeast. Photo: J.M.F. Camargo Fig. 2.5 Schwarzula coccidophila, closeup of the scale insects―Cryptostigma sp.―in the nest interior, being attended by a bee (Tapurucuara-Mirim, AM, Brazil). Photo: J.M.F. Camargo tion. The secretions are a subproduct of sap from the plant, on which the scale insects feed. When stimulated by attending bees, the scale insects liberate, through the anus, a small droplet of the sugary liquid, which is ingested by the attendant. These bees are the only known species which have, within their own nest, a permanent source of carbohydrates, in addition to additional wax for building. Only pollen is collected at flowers (Camargo and Pedro 2002). Another extraordinary behavior is found in Trichotrigona extranea (Fig. 2.6), a monotypic genus and until now only known from a single locality, in the middle 2 Historical Biogeography of the Meliponini (Hymenoptera, Apidae, Apinae)… 25 Fig. 2.6 Nest of Trichotrigona extranea, a bee that does not build storage pots and does not store any kind of food; closeup of brood cells (Samaúma, AM, Brazil). Photo: J.M.F. Camargo Rio Negro region of Amazonas, Brazil. The colonies are very small, with less than 200 adults, located in small cavities in dead branches (of Buchenavia suaveolens); they construct no storage pots and do not store food of any kind. It is likely these bees are cleptobiotic, but not in the manner of Lestrimelitta, which robs, during mass raids, the food stores of a host and transfers them to the storage pots of its own nest. Supposedly, the workers (and also possibly the males) of T. extranea, individually use and have free access to the food stores of the host species (perhaps of Frieseomelitta, very common in the region and sharing nest habits similar to those of Trichotrigona; Camargo and Pedro 2007a). There exists, also, a great diversity in nest architecture, ranging from subterranean, with complex structures for the control of humidity and air circulation, to 26 J.M.F. Camargo and P. Vit Fig. 2.7 Nest aggregation of Partamona batesi, in active termite nest (Nasutitermes acangussu); endemic in the Tefé region, central Amazonia, Brazil. Photo: J.M.F. Camargo nests in tree cavities, within the nests of other social insects, such as termites and ants, to exposed arboreal nests. Among these, species of the genus Partamona are noteworthy, which are among the most formidable nest builders known, primarily considering the nest entrance structures (Figs. 2.7 and 2.8), conspicuous and richly ornamented, which “facilitate” recognition of the nest and function as true flight targets (several of these species—like P. batesi, Figs. 2.7 and 2.8a—construct nests in large aggregations, with the nest entrances very close to each other). The nest of P. vicina, of Amazonas state, is one of the most sophisticated known (Fig. 2.9); the nest entrance structure (Fig. 2.9a) opens upon a wide chamber or vestibule, filled with a structure similar to intertwined roots, constructed with earth and resin (Fig. 2.9b), forming a large labyrinth, where workers stay and constitute 2 Historical Biogeography of the Meliponini (Hymenoptera, Apidae, Apinae)… 27 Fig. 2.8 Nest entrances of Partamona; (a) P. batesi (endemic in the Tefé region); (b) P. gregaria (endemic in the region of lower Tapajós); (c) P. pearsoni (endemic to north of the Amazon/Negro rivers); (d) P. chapadicola (endemic to Maranhão—eastern Pará); (e) P. vicina (of wide Amazonian distribution). Photo: J.M.F. Camargo the first force of nest defense; the vestibule is connected, through a small tunnel, to a second cavity or atrium (Fig. 2.9c), filled with waxy lamellae, cells and small pots, generally containing an acidic liquid, constituting a typical “false nest.” From this “false nest,” there is a small tunnel leading to the true nest, where the brood and food are located (Fig. 2.9d), and their various satellite chambers—containing honey pots. The entire assemblage of structures and chambers is important in nest defense, against invasions of other insects, primarily cleptobiotic social insects, such as Lestrimelitta spp., for example (cf. Camargo and Pedro 2003). There exists, also, a great diversity in form and size, from the robust Melipona fuliginosa, ca. 11.0–13.0 mm in length, to the minuscule Leurotrigona pusilla, ca. 2.0 mm in length (Fig. 2.10). Some species of Meliponini are exploited, economically, since pre-Colombian times. Some native peoples of South America, such as the Kayapós, from southern 28 J.M.F. Camargo and P. Vit Fig. 2.9 Nest of Partamona vicina, in active termite nest (Amitermes excellens); (a) entrance; (b) vestibule/labyrinth, where the defense force is located; (c) atrium/false nest; (d) true nest, with brood cells, food storage pots, etc. (Muçum, Tapajós, PA, Brazil). Photo: J.M.F. Camargo Pará, Brazil (Fig. 2.11), make varied use of the products from these bees, in food, medicine, ritual, tool making, etc., and also as a model for social organization for their own communities (cf. Posey and Camargo 1985; Camargo and Posey 1990). The causes of this diversification, especially taxonomic, in the Neotropical region, have been the subject of many speculations. Through the decades of 1960–1970 the postulate of ecological “refuges” emerged. This postulate attempted to associate the known pattern of endemism and speciation in Amazonia with climatic cycles (glacial and interglacial) in the recent quaternary. Although this attempt, a priori, can explain some of the current distribution patterns, it barely touches the problem of the history of the taxa; it only deals with regional fragments of recent history. 2 Historical Biogeography of the Meliponini (Hymenoptera, Apidae, Apinae)… 29 Fig. 2.10 Nest of Leurotrigona pusilla (Curicuriari, AM, Brazil), in a gallery made by a beetle. This is the smallest known meliponine (body length ca. 2.0 mm). The nest is of ca. 4 cm in length. Photo: J.M.F. Camargo Fig. 2.11 Kayapó Indians (Gorotire, PA, Brazil), on a trip to collect meliponine nests. These Indians are bee experts. Photo: J.M.F. Camargo Only recently, some work based on the methods conceived in phylogenetic systematics and vicariance biogeography, involving monophyletic taxa, with large ranges in the Neotropical region, permit access to some periods of evolutionary history/ 30 J.M.F. Camargo and P. Vit Fig. 2.12 Area and biological cladograms for the subgroups of Geotrigona (this is the first area cladogram proposed for Neotropical Meliponini), from Camargo and Moure (1996) biogeography with great significance in the Neotropical area, permitting, for the first time, integration of space, time and form. The first works on evolutionary biogeography of Meliponini through the viewpoint and protocol of vicariance biogeography were of Camargo and Moure (1996), Camargo (1996) and Camargo and Pedro (2003). The first biological and area cladograms were for the species of the genera Paratrigona and Geotrigona (Fig. 2.12), and more recently Partamona (Fig. 2.13). The results reveal that the species subgroups within each of these genera are notably congruent in terms of biogeographic compartmentalization, that is, when the taxa are placed on the biological cladograms by their respective areas of endemism, the results obtained for the subgroups of the first two genera are the same (particularly in relation to the species of Partamona), indicating the same relationships between areas or biogeographic compartments. These results, obviously, suggest a general pattern of biogeographic coevolution in the Neotropical region. The sequence of events in vicariance/cladogenesis provides, therefore, a definition of a hierarchy in the formation of biogeographic boundaries or geological compartmentalization and barriers, as in Figs. 2.14 and 2.15. The first great barrier is formed along the alignment of the Madeira/Amazonas Rivers (possibly epicontinental seas related to the Tapajonic transgressions, in the 2 Historical Biogeography of the Meliponini (Hymenoptera, Apidae, Apinae)… 31 Fig. 2.13 Areas of endemism and biogeographical components, inferred from the species of Partamona; Chocó-CA (from northwestern Peru to Mexico); SWAm (a component delimited, on the north, by the alignment of the Uaupés/Negro rivers, on the south, by the Madeira/Mamoré rivers, and on the west, by the Andean mountain range); NAm (north of the Negro/Amazonas rivers); SEAm (area to the south of the Madeira/Amazonas rivers to northwestern Argentina); Atl (Atlantic area, from Bahia to Paraná). See Fig. 1.15a (taken from Camargo and Pedro 2003) lower Miocene), dividing the Neotropical region into two large compartments: NW–SE (Fig. 2.14a). In the NW compartment a further break occurred (approximately along the line of the Caqueta/Japura rivers, possibly related to the transgression of the Maracaibo seas in the mid Miocene; Fig. 2.14b), separating North Amazonia (NAm) from all of southwestern Amazonia (SWAm) and the north Andean, Central American—Mexico block (Choco-AC). And, finally, a break separating SWAm from the Choco-AC component (Fig. 2.14c), related, possibly, with orogeny of the equatorial Andes, which attained heights greater than 3,000–4,000 m in the PlioPleistocene. In the SE component, there is a separation between the southeastern Atlantic region (Atl) and southeast Amazonia (SEAm). The breaks, giving rise to the crown (present) species, may be related to the climatic events of the Pleistocene, as postulated by the proponents of ecological “refuges.” The first image that arises from this biogeographic and geological compartmentalization of the Neotropical region is that Amazonia (Fig. 2.16) is not a single historical unit, and rather, it is composed of three great biogeographic compartments with distinct temporal and phylogenetic relationships (Fig. 2.14, area cladogram). 32 Fig. 2.14 Sequence of events of separation and vicariance in the Neotropical region. The shaded area is Amazonia, which, from the biogeographic perspective of vicariance, is not an historical unit, taken from Camargo (2006) Fig. 2.15 Principal biogeographic elements which unify the Neotropical region ; (a) the diverse area cladograms obtained; (b) those proposed by Amorim and Pires (1996); (c) those proposed by Camargo (1996) and Camargo and Moure (1996); (d) those proposed by Camargo and Pedro (2003) (taken from Camargo and Pedro 2003). See legend in Fig. 1.13 2 Historical Biogeography of the Meliponini (Hymenoptera, Apidae, Apinae)… 33 Fig. 2.16 The magnificent Amazonian forest (upper Rio Negro region), produced by millions of years of evolution, habitat of many Meliponini and a megadiverse biota, today at the mercy of an irresponsible and uncontrolled devastation. Photo: J.M.F. Camargo References Amorim DS, Pires MRS. 1996. Neotropical biogeography and a method for maximum biodiversity estimation. 183–219 pp. In: Bicudo EMC, Menezes NA, eds. Biodiversity in Brazil, a first approach. CNPq; São Paulo, Brasil. 326 p. Camargo JMF. 1996. Meliponini neotropicais (Apinae, Apidae, Hymenoptera): biogeografia histórica. pp 107–121. In: Garofalo CA et al., eds Anais do Encontro sobre Abelhas de Ribeirão Preto, SP, FFCLRP-USP; Ribeirão Preto, São Paulo, Brasil. xxii + 351 pp. Camargo JMF. 2006. A Amazônia não é uma unidade histórica. pp. 47–49. In: Santana WC, Lobo CH, Hartfelder KH et al., eds. Anais do VII Encontro sobre Abelhas. Ribeirão Preto, Brasil, FFCLRP-USP, FMRP-USP, publicação eletrônica em mídia digital (CDROM), p. 850. Camargo JMF, Moure JS (1996) Meliponini neotropicais: o genero Geotrigona Moure, 1943, (Apinae, Apidae, Hymenoptera), com especial referencia a filogenia e biogeografia. Arquivos de Zoologia 33:95–161 Camargo JMF, Pedro SRM. 1992. Systematics, phylogeny and biogeography of the Meliponinae (Hymenoptera, Apidae): a mini-review. Apidologie 23:509–522. Camargo JMF, Pedro SRM. 2002. Mutualistic association between a tiny Amazonian stingless bee and a wax-producing scale insect. Biotropica 34:446–451. Camargo JMF, Pedro SRM. 2003. Meliponini Neotropicais: o gênero Partamona Schwarz, 1939 (Hymenoptera, Apidae, Apinae) – bionomia e biogeografia. Revista Brasileira de Entomologia 47:311–372. Camargo JMF, Pedro SRM. 2004. Meliponini neotropicais: o gênero Ptilotrigona Moure (Hymenoptera, Apidae, Apinae). Revista Brasileira de Entomologia 48:353–377. Camargo JMF, Pedro SRM. 2007 a. Notas sobre a bionomia de Trichotrigona extranea Camargo & Moure (Hymenoptera, Apidae, Meliponini). Revista Brasileira de Entomologia 51:72–81. Camargo JMF, Pedro SRM. 2007 b. Meliponini Lepeletier, 1836. pp 272–578. In: Moure JS, Urban D, Melo GAR, eds. Catalogue of Bees (Hymenoptera, Apoidea) in the Neotropical Region. Sociedade Brasileira de Entomologia; Curitiba, Brasil. 1058 pp. 34 J.M.F. Camargo and P. Vit Camargo JMF, Posey DA. 1990. O conhecimento dos Kayapó sobre as abelhas sociais sem ferrão (Meliponidae, Apidae, Hymenoptera): notas adicionais. Boletim do Museu Paraense Emílio Goeldi, série Zoologia 6:17–42. Camargo JMF, Roubik DW. 1991. Systematics and bionomics of the apoid obligate necrophages: the Trigona hypogea group (Hymenoptera: Apidae; Meliponinae). Biological Journal of the Linnean Society 44:13–39. Camargo JMF, Garcia MVB, Júnior ERQ, Castrillon A. 1992. Notas prévias sobre a bionomia de Ptilotrigona lurida (Hymenoptera, Apidae, Meliponinae): associação de leveduras em pólen estocado. Boletim do Museu Paraense Emílio Goeldi, série Zoologia 8:391–395. Engel MS. 2000. A new interpretation of the oldest fossil bee (Hymenoptera: Apidae). American Museum Novitates 3296:1–11. Gilliam M, Buchmann SL, Lorenz BJ, Roubik DW. 1985. Microbiology of the larval provisions of the stingless bee, Trigona hypogea, an obligate necrophage. Biotropica 17:28–31. Lokvam J, Braddock JF. 1999. Anti-bacterial function in the sexually dimorphic pollinator rewards of Clusia grandiflora (Clusiaceae). Oecologia 119:534–540. Michener CD, Grimaldi DA. 1988 a. A Trigona from Late Creataceous amber of New Jersey (Hymenoptera: Apidae: Meliponinae). American Museum Novitates 2917:1–10. Michener CD, Grimaldi DA. 1988 b. The oldest fossil bee: apoid history, evolutionary stasis, and antiquity of social behavior. Proceedings of the National Academy of Science 85:6424–6426. Noll FB, Zucchi R, Jorge JA, Mateus S. 1996. Food collection and maturation in the necrophagous stingless bee, Trigona hypogea (Hymenoptera: Meliponinae). Journal of the Kansas Entomological Society 69:287–293. Posey DA, Camargo JMF. 1985. Additional notes on the classification and knowledge of stingless bees (Meliponinae, Apidae, Hymenoptera) by the Kayapó Indians of Gorotire, Pará, Brazil. Annals of Carnegie Museum 54:247–274. Roubik DW. 1982. Obligate necrophagy in a social bee. Science 217:1059–1060. Silvestri F. 1902. Contribuzione alla conoscenza dei meliponid del Bacino del Rio de la Plata. Rivista di Patologia Vegetale 10:121–174. Chapter 3 Australian Stingless Bees Megan Halcroft, Robert Spooner-Hart, and Lig Anne Dollin 3.1 Introduction Stingless bees have been an important part of indigenous Australian culture for centuries; however, modern meliponiculture in Australia is still very much in its infancy (Heard and Dollin 2000). A recent survey showed that interest in stingless bees is growing and Australians are becoming increasingly aware of and concerned about conservation of these species. More community members are keeping hives with this interest in mind (Halcroft, unpublished data). Beekeepers in the northern regions are able to produce honey in small quantities and some multiply hives for profit. Of the two stingless bee genera in Australia, Trigona (s.l.) is the most studied. The domestication of Trigona (Heterotrigona) carbonaria colonies began in the 1980s and Dr. Tim Heard conducted ground-breaking work in T. (Heterotrigona) carbonaria husbandry (Heard 1988a,b). As a result, most scientific research has been conducted on this species. Few studies have been conducted on Trigona (s.l.) pollination efficacy and have mainly used T. (Heterotrigona) carbonaria or T. (Heterotrigona) hockingsi in macadamia nut (Macadamia integrifolia) crops. Pollination studies on other horticultural crops are minimal and, as such, anecdotal reports pertaining to crop pollination are cited here. Austroplebeia have only recently become of interest to beekeepers and hobbyists. A small number of studies have been conducted on aspects of biology of A. australis and A. symei, as their brood structure and queen/worker interaction M. Halcroft (*) • R. Spooner-Hart School for Health and Science, Hawkesbury Campus, University of Western Sydney, Locked Bag 1797, Penrith, NSW 2751, Australia e-mail: megan@beesbusiness.com.au L.A. Dollin Australian Native Bee Research Centre, PO Box 74, North Richmond, NSW 2754, Australia P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_3, © Springer Science+Business Media New York 2013 35 36 M. Halcroft et al. is more easily observed than that of Trigona (s.l.), due to reduced nest structures. Recent doctoral research has been conducted (M. Halcroft) to better understand the development of the Australian stingless bee industry, phylogeny of Austroplebeia, the biology and behavior of A. australis, and to assess the ability of A. australis to pollinate crops in greenhouse and field settings. This research is incomplete and ongoing, and therefore, is cited here as unpublished data. Although Australian stingless bees are not as diverse in size or morphology as Neotropical or Paleotropical species, our bees are proving to be diverse and resilient in their behavior. Their native range is mostly limited to the northern half of the continent; however, T. (Heterotrigona) carbonaria has a distribution that reaches the southernmost range of any stingless bee species (Dollin et al. 1997). Austroplebeia occur in some of the most arid areas of Australia, where the climate extremes are harsh and the food resources are often scarce. Australian stingless bees have evolved diverse behaviors to survive under such conditions. While few scientific studies have been conducted on the behavior of Australian stingless bees, amateur beekeepers often have a wealth of knowledge and their experience is extremely valuable. Communication with experienced beekeepers is of utmost importance when initiating research, and anecdotal accounts are appropriately cited here. While there is great potential for further research on stingless bees in Australia, this chapter aims to provide an overview of current knowledge and suggest areas for further study. 3.2 Indigenous Australians and Their Relationship with Stingless Bees Indigenous Australians have been collecting the strong, tangy honey from stingless bee nests 1sugarbag for centuries. Hockings (1883) first reports the Australian Trigona (s.l.) and Austroplebeia from his visit to northern regions of Queensland, where local Aboriginal people call these bees “karbi” and “kootchar,” respectively. It is unclear which tribal language Hockings refers to in his paper. There are many different Australian Aboriginal tribes. The Aurukun on Cape York, in far north Queensland, is the homeland for the Wik Mungkan people. In 2003, an industry based on stingless bees and traditional culture was the inspiration for a group of 50 Wik school children, aged between 12 and 16 years. Using the natural resources of their homeland, the sugarbag “may man-pathan” provided the prospect of making real money and building a culturally based business. The children within this indigenous community developed a business plan and become more motivated and engaged in learning (Yunkaporta 2009). Anecdotal accounts of indigenous bee hunting methods are described in bush tales, and these include: placing a fine hair or grass into the terminal abdominal segment of a forager, which is used as a flag to follow it back to the nest; sprinkling foragers with flour to make them easier to see and follow; and bee hunters relying on the loud humming sound of a predatory wasp (Bembix) which hovers outside the nest entrance, waiting for foragers to leave (A. Beil, personal communication). 3 Australian Stingless Bees 37 Fig. 3.1 Indigenous Australian axes. Photo: G. Walsh—http://www.hogartharts.com.au Traditionally, honey “may at” or “may kuyan” is used for medicinal and culinary purposes, while the cerumen “wom” is used as a waterproofing agent for baskets, as a wood preservative, as glue to secure axe heads “thayan” (Fig. 3.1), and for personal and artifact decoration (Rayment 1935; Yunkaporta 2009; Welch 2010). Cerumen has also been found in protective covers, fashioned around ancient rock paintings, to protect them from rain and erosion (Rayment 1935). Pellets of cerumen are used in some rock art, notably in the Kimberley Ranges in Western Australia, to create shapes of humans, dingoes, turtles, and spirit figures on the rock surface (Welch 1995). This collage technique (Brandl 1968) permitted incorporation of organic materials in a normally inert, inorganic rock face. Cerumen and plant resins are extremely amenable to carbon dating because storage of fresh products within hives, and consequent use by indigenous craftsmen, enables accurate estimates of when the collages were created, thus dating the artwork (Bednarik 2002). Interest in indigenous culture and art has increased over the past 20 years and is at a peak in popularity (Artlandish 2010). Cerumen is still used by Australian Aboriginal artists and craftsmen to manufacture hunting tools such as spears “kek” and woomeras “thul,” as well as firesticks “thum pup” and mouth pieces for didgeridoos, a traditional musical instrument (Yunkaporta 2009). Sugarbag honey can fetch very high prices in comparison to honey bee honey. In 2005, Russell and Janine Zabel commenced a training program in sugarbag harvest and colony transfer within the Aurukun, with the aim to develop a sustainable industry based on sale of sugarbag honey and cerumen (Zabel 2008). An Australian government grant was received to assist development of this new enterprise, which had potential to boost local employment and would be consistent with the rapidly developing ecotourism industry. In 2010, an industry based on sugarbag is seen as an option for inclusion in a preliminary proposal for the Department of Aboriginal Business Development, in Grafton, in northern New South Wales. This proposal is investigating indigenous 38 M. Halcroft et al. land development in the Northern Rivers region using traditional cultures and sustainable practices (Lain 2010). Another initiative is the Thamarrurr Development Corporation proposal to develop a wildlife industry in Wadeye, Northern Territory, including health products containing sugarbag honey (Adlam 2010). Potentially, stingless bees could provide sustainable income for both Australian indigenous and non-indigenous communities through production of honey, cerumen, bee colonies, and pollination service. 3.3 Australian Stingless Bees There is much needed change regarding classification of the genus/subgenus group name of Trigona (Heterotrigona), which includes a portion of the native Australian Meliponini. At present, according to Michener (1990), species of Trigona (s.l.) that occur in the Indoaustralian regions are of the subgenus Heterotrigona. Recent molecular studies, and also morphology, suggest this taxonomic classification is incorrect and that Australian species previously named Trigona (subgenus Heterotrigona) should be changed to the genus Tetragonula Moure, 1961 (Rasmussen and Cameron 2007, 2010). There are many species and subgenera to consider in Asia and Australia, with 15 species in Australian comprising two genera. We have chosen to preserve the group name Trigona (Heterotrigona) in this chapter, until further taxonomic and systematic research is decisive. The bees in Australia are small (<4.5 mm) and black. However, Austroplebeia can be distinguished from Trigona (s.l.) by colored body markings, thoracic shape, and nest architecture. The highest rainfall areas within Australia occur in the northern, eastern, and far south eastern coasts (BOM 2010a) (Fig. 3.2), resulting in tropical, subtropical, and temperate forest and woodland vegetation. The natural range for Australian stingless bees is in the tropical and subtropical regions of northern Australia, with the exception of T. (Heterotrigona) carbonaria, which has, by far, the southernmost distribution. The temperature threshold for flight activity in T. (Heterotrigona) carbonaria (Heard and Hendrikz 1993) is >18°C, and for A. australis >20°C (Halcroft, unpublished data), which means foraging periods are substantially reduced for colonies in the most southerly range of their distribution. 3.3.1 Castes and Genders of the Australian Stingless Bees As with all stingless bees species there are two castes—queen and worker. All Australian species are thought to be monogynous. However, the incidence of virgin queen imprisonment in queenright colonies of A. australis has been observed (MH, personal observation) (Fig. 3.3). Queens can be identified by their long, pale abdomen and short wings (Fig. 3.4). They are usually found on the brood, although extensive nest patrolling is not uncommon in A. australis (MH, personal observations). 3 Australian Stingless Bees 39 Fig. 3.2 Average annual rainfall charted for Australia, including the reported distribution of Australian stingless bees (Dollin et al. 1997; BOM, 2010a; Dollin, 2010, unpublished data) Fig. 3.3 Imprisoned A. australis virgin queen. Photo: M. Halcroft 40 M. Halcroft et al. Fig. 3.4 A. australis queen with workers. Photo: M. Halcroft Fig. 3.5 A. australis drone showing cream markings on legs and thorax. Photo: M. Halcroft Trigona (s.l.) drones are difficult to identify within the hive, without the aid of a magnifying glass, as they have no defining markings (Dollin 2010a). Their bodies are slightly more slender and the antennae are longer, having one additional segment, compared to females or workers. They frequently form drone swarms outside nests and sometimes aggregate on foliage at night. These aggregations and swarms can be seen for a number of days when conditions are favorable (Klumpp 2007). Austroplebeia drones are easier to identify within the nest because the cream-colored markings on their thorax are more pronounced, and they also have markings on the abdomen and legs (Dollin 2010a) (Fig. 3.5). Their apparently 3 Australian Stingless Bees 41 slimmer bodies and constant movement of the antennae, as they move, also distinguish them (MH, personal observation). A. australis drones also form mating swarms and aggregations, although these are not as large as those of Trigona (s.l.) (MH, personal observation). A. australis Au. australis colonies appear to produce drones in “batches” or “male-producing periods” (MPP) (Velthuis et al. 2005), with drones being present only periodically in a single colony. It is not clear whether drone production is curtailed during periods of resource scarcity. 3.3.2 Brood Production In the Meliponini, brood production is an elaborate procedure and involves a sequence of interactions between the queen and a group of workers (Sakagami et al. 1973; Sakagami 1982). This temporal sequence is termed the “provisioning and ovipositing process” or “POP” (Sakagami and Zucchi 1963; Michener 1974; Wittmann et al. 1991). Cells are mass-provisioned with a mixture of honey, pollen, and protein-rich secretions from the hypopharyngeal glands (Michener 1974; Silva de Moraes et al. 1996). Some species provision cells successively while others provision synchronously (Sommeijer and Bruijn 1984). Once a cell is provisioned, the queen oviposits and workers seal the cell (operculation) (Drumond et al. 1999). Trigona carbonaria constructs and provisions brood cells synchronously, and the queen oviposits in batches (Yamane et al. 1995). Austroplebeia australis and A. symei construct and provision brood cells in a successive pattern, while the queen does not oviposit in batches (Drumond et al. 1999). Meliponine queens normally mate only once (Kerr et al. 1962; Michener 1974), returning to the nest with the male genitalia still caught in the vagina (Michener 1974). The incidence of low frequency polyandry has been reported in Melipona beecheii and Scaptotrigona postica, (Paxton et al. 1999); however, it is thought that most stingless bees are monandrous, including the Australian species (Drumond et al. 2000; Green and Oldroyd 2002). Sperm is stored in her spermatheca. A diploid female is produced when a sperm cell is released to fertilize the egg as it passes through the oviduct. If sperm is not released, the egg is not fertilized and a haploid male is produced (Michener 2000). While drones are normally produced by the queen, laying workers have been reported in some Brazilian species of Melipona (Koedam et al. 2005, 2007). Although this is rare in Australian stingless bees (Michener 1974; Drumond et al. 1999; Tóth et al. 2004), A. australis and A. symei workers have been observed laying small numbers of trophic eggs in queenright colonies. On all recorded occasions the queen consumed those eggs (Drumond et al. 1999). Microsatellite analysis determined that workers were not responsible for drone production in queenright colonies of A. australis, A. symei, or T. (Heterotrigona) carbonaria (Drumond et al. 2000; Gloag et al. 2007). Drone production has been observed in some queenless colonies (Klumpp 2007; MH, personal observation); however, this has not been studied in sufficient detail. 42 3.4 M. Halcroft et al. Characteristics of Australian Stingless Bees 3.4.1 Austroplebeia Nine species of Austroplebeia are listed in the Zoological Catalogue of Australia (Cardale 1993), and the most commonly domesticated and studied species are A. australis and A. symei. Species descriptions for this genus are inadequate for effective identification and no working key exists at present. Ongoing research in the areas of molecular, morphological, and morphometric analysis suggests that there are only 3–6 species of Austroplebeia in Australia (Halcroft and Dollin, unpublished data). Only one of these, A. cincta, occurs outside Australia, in Papua New Guinea (PNG) (Moure 1961; Rasmussen 2008). Current classification is based mainly on variations in body markings. Mature adult bees are black, with varying levels of cream/yellow markings on the scutellum of their thorax and on their face (Michener 2000). Bees measure between 3.5 and 4.5 mm, and species characteristics are presented in Table 3.1 (Michener 1961; Dollin 2010a). 3.4.1.1 Natural Distribution Dollin (2010b) found that Austroplebeia occurs throughout northern Australia (Fig. 3.2). A. australis and A. symei have the widest distribution. Specimens currently considered to be A. symei have been collected along the east coast from Cape York (11°04¢ S) to Kilcoy in Queensland (26°57¢ S) as well as the northern areas of the Northern Territory. Austroplebeia australis is found coastally, as far south as Kempsey, New South Wales (31.08ºS, 152.82ºE, elevation 10 m) and inland near Inverell, New South Wales (29.46°S, 151.06°E, elevation 584 m) and also occurs in arid regions of inland Queensland. The remaining species are found mainly in northern Queensland, Northern Territory, and Western Australia, with A. percincta originally described from an arid region of central Australia (Cockerell 1929). While Trigona (s.l.) is commonly found in areas of high rainfall, many Austroplebeia thrive in areas that experience low annual rainfall (300–600 mm) and extreme temperature ranges (3–40.5°C) (A. Dollin, 2009, personal communication; BOM 2009). Until recently, it was thought that Austroplebeia were more sensitive to low temperatures, resulting in their northerly restricted distribution. Current research has revealed that colonies of A. australis are able to survive subzero temperatures, without actively thermoregulating the nest. These colonies were shown to contain developing brood throughout the year (Halcroft, unpublished data). 3.4.1.2 Nest Architecture, Colony Population, and Brood Structure Similar to Trigona (s.l.), Austroplebeia chooses tree hollows, but cavity diameter is usually smaller. A. australis is found in cavities 50–110 mm in diameter (Halcroft, 43 3 Australian Stingless Bees Table 3.1 Explanation of color markings used to classify species in the genus Austroplebeia (Cardale 1993; Dollin 2010a,b,c) Species name Native range Description Markings Austroplebeia symei (Rayment 1932) Qld and NT A. australis (Friese 1898) A. cassiae (Cockerell 1910) Qld and NSW 4 mm, four distinct cream markings on the scutellum. Minimal facial markings NT 3.5–4 mm. Facial markings more extensive but vary in degree. NT Broad cream markings on thorax, Cape York, mesothorax Qld narrow stripes each side Central NT A. cockerelli (Rayment 1930) A. essingtoni (Cockerell 1905) A. ornata (Rayment 1932) A. percincta (Cockerell 1929) A. websteri (Rayment 1932) A. cincta (Mocsary, in Friese 1898) 4.5 mm, darkest with little or no markings on the face and thorax WA PNG and possibly Qld 3.5 mm. Distinct facial and thoracic markings Dark markings represent cream/yellow markings on black bees unpublished data). A smaller species found near Normanton, Queensland, may occupy cavities in coolabah (Eucalyptus coolabah, Myrtaceae) trees with a diameter of only 35 mm (A. Beil, 2009, personal communication). Some colonies of A. australis have been found in narrow tree limb hollows up to 6 m in length (R. Zabel, 2008, personal communication). A recent nest survey conducted in southeast Queensland showed that dead trees comprised over 87% of nest cavities chosen by Austroplebeia in that area (M. Halcroft, unpublished data). Estimates of colony populations in Austroplebeia have not been studied in detail; however, recent studies have shown that, within natural nests, brood 44 M. Halcroft et al. Fig. 3.6 Australian stingless bee brood structures. (a) Austroplebeia australis (b) Trigona carbonaria, (c) Trigona hockingsi, (d) Trigona clypearis. Photos: (a–b) M. Halcroft, (c–d) R. Brito populations can range from 2,000 to 13,000, averaging of 5,000 (M. Halcroft, unpublished data). All Austroplebeia construct spherical brood cells and, with the exception of A. cincta (see Table 3.1), make simple cell clusters (Michener 1961; Dollin 2010a) (Fig. 3.6a). Open cells face outwards from the leading edge of the cluster, in irregular directions. Clustered brood cells can be constructed to fit into the narrow, irregular cavities of the smaller trees or large limbs favored by Austroplebeia. The New Guinea species, A. cincta, is the only Austroplebeia found outside Australia (Moure 1961). Recently, however, some colonies resembling A. cincta have been found in Queensland (Dollin 2010a). Nests of these newly discovered colonies have not been examined, and studies are in progress. Unfortunately, no photographs of A. cincta nests or brood structures are currently available. 3.4.2 Trigona (s.l.) Identification of Australian Trigona (s.l.) is very difficult in the field. Some species, especially T. carbonaria, can vary considerably in size according to geographic 3 Australian Stingless Bees 45 location (Dollin et al. 1997). The largest bee is T. hockingsi, measuring approx. 4.5 mm in length, while the smallest is T. clypearis, 3.5 mm in length (Klumpp 2007). Species within the carbonaria species group are difficult to separate on their body size or morphology. Thus, nest architecture is an invaluable tool in the accurate identification of species (see “Nest and brood architecture”). The currently described Australian Trigona (s.l.) are classified into three species groups (Dollin et al. 1997; J. Klumpp, 2010, personal communication; A. Dollin, 2010, personal communication), namely: • Iridipennis group Sakagami 1978 – T. (Heterotrigona) clypearis Friese 1908 • Laeviceps group Sakagami 1978 – T. (Heterotrigona) sapiens Cockerell 1911 • Carbonaria group Dollin et al. 1997 – – – – T. (Heterotrigona) carbonaria Smith 1854 T. (Heterotrigona) hockingsi Cockerell 1929 T. (Heterotrigona) mellipes Friese 1898 T. (Heterotrigona) davenporti Franck 2004 3.4.2.1 Natural Distribution of Trigona (s.l.) in Australia Dollin et al. (1997) report that T. clypearis and T. sapiens are restricted to the Cape York Peninsula in northern Queensland (18°0¢ S–10°56¢ S) compared to the carbonaria species group, distributed throughout northern and eastern Australia. The most recently described Trigona (s.l.), T. davenporti, was discovered by Peter Davenport, a local beekeeper who helped to pioneer stingless beekeeping in Australia (Klumpp 2007; Dollin 2010c). So far, this species has only been reported within a restricted area around the Gold Coast in south eastern Queensland (A. Dollin, 2008, personal communication). T. carbonaria is the most widely distributed species, occurring along much of the east coast of Australia. It is found as far north as the Atherton Tablelands in Queensland (17°15¢ S) and as far south as Bega, in New South Wales (36°40¢ S) (Fig. 3.2). Trigona carbonaria chooses large tree cavities that may provide superior insulation against the weather extremes experienced in its most southerly locale. Tse (unpublished data) found that both T. (Heterotrigona) carbonaria and T. (Heterotrigona) hockingsi maintain the brood chamber at significantly higher temperatures than the nest cavity or ambient temperature. These studies were not, however, conducted during periods of temperature extremes and further studies would be beneficial to better understand temperature regulation, especially by T. (Heterotrigona) carbonaria. 46 3.4.2.2 M. Halcroft et al. Nest Architecture, Colony Population, and Brood Structure Tree cavities are the most commonly chosen nest substrate for Trigona (s.l.) in Australia. They can also be found inside water meter boxes, stone walls, beneath concrete foot paths, and within door and wall cavities. Nest entrance modifications vary, depending on species; however, environmental factors such as weather and predators can also influence those structures (Dollin et al. 1997). Trigona (Heterotrigona) carbonaria often daub the area around the entrance with significant amounts of resin, whereas T. (Heterotrigona) hockingsi and T. (Heterotrigona) davenporti generally leave their entrances unadorned (Dollin 2010a). Trigona (Heterotrigona) mellipes, T. (Heterotrigona) sapiens, and T. (Heterotrigona) clypearis build entrance tubes of varying sizes (Table 3.2), although they do not always do so. It has been estimated that a strong colony of T. (Heterotrigona) carbonaria has a population of approximately 11,000 workers (Hoffmann, unpublished data). Brood volume can vary 940–3,535 ml in T. (Heterotrigona) carbonaria and 1,100–2,550 ml in T. hockingsi (Dollin et al. 1997); however, T. (Heterotrigona) hockingsi is able to build much larger nests if provided with the appropriate nest cavity (A. Dollin, 2010, personal communication). Both T. (Heterotrigona) davenporti and T. (Heterotrigona) hockingsi build brood areas with similar structure; however, T. davenporti has a smaller adult population. T. (Heterotrigona) mellipes, T. (Heterotrigona) sapiens, and T. (Heterotrigona) clypearis have much smaller nests and average brood volumes measure 595, 224, and 464 ml, respectively (Dollin et al. 1997). All Australian Trigona (s.l.) build elongated, vertically oriented brood cells in regular, or nearly regular, structures (Dollin et al. 1997). There are, however, distinguishing features within these structures that can aid in species identification. Trigona (Heterotrigona) carbonaria (Fig. 3.6b) builds single layers of comb, arranged in a horizontal spiral. Brood cells are constructed on the outer rim of up to three circular spirals, at a time. The spiral formation can be clockwise or counterclockwise. Brood construction can become erratic if the nest is disturbed, e.g., if the tree is felled (A. Dollin, 2010, personal communication). Trigona (Heterotrigona) hockingsi (Fig. 3.6c) builds a regular, horizontal brood structure with hexagonal comb, which is best described as terraced or stepped; it is not in a single layer. Both T. (Heterotrigona) davenporti and T. (Heterotrigona) mellipes build brood comb similar to that of T. (Heterotrigona) hockingsi; however, the brood comb area of T. (Heterotrigona) mellipes is considerably smaller (J. Klumpp, personal communication). Neither T. (Heterotrigona) sapiens nor T. (Heterotrigona) clypearis (Fig. 3.6d) have a hexagonal comb structure because individual cells are arranged irregularly, in horizontal or diagonal layers. Brood structure T. (Heterotrigona) hockingsi 145 Horizontal steps/terraces. Hexagonal comb 198 Flat spiral, single layer. Hexagonal comb T. (Heterotrigona) mellipes T. (Heterotrigona) sapiens None Seldom smear entrance with resin None Smear entrance with resin +++ 16 6 82 58 T. (Heterotrigona) clypearis 28 78 A. australis A. ornata or cockerelli A. cincta (PNG) None to ~20 mm None to ~20 mm 20–80 52–110 35 45 Similar to T. hockingsi but smaller Irregular, horizontal, or diagonal layers. No hexagonal comb Roughly arranged in diagonal rows No hexagonal comb Clustered Clustered Irregular concentric layers of one cell thickness, with bee space between layers T. (Heterotrigona) carbonaria 3 Australian Stingless Bees Table 3.2 Comparative description of nest entrance characteristics within Trigona and Austroplebeia species Species Average entrance tube length (mm) Average nest cavity diameter (mm) Trigona species (Dollin et al. 1997; Klumpp 2007), Austroplebeia species (Halcroft and Dollin, 2010, unpublished data) and A. cincta (Michener 1961) 47 48 3.5 3.5.1 M. Halcroft et al. Behavior of Australian Stingless Bees Guard and Forager Behavior Australian Trigona (s.l.) colonies usually employ at least 4–5 guards at the entrance (Yamane et al. 1995; MH, personal observation), with higher numbers occurring around the front of the nest on warm days (Klumpp 2007). Guards are not normally aggressive towards human onlookers; however, if the nest is opened workers can become moderately to strongly aggressive (Michener 1961). Austroplebeia guards occur in small numbers within the entrance of the nest but they withdraw into the entrance tube if observed too closely. When colonies are opened, workers are not aggressive (Michener 1961), they buzz around the heads of human “predators” and daub their hair with globules of resin until the nest is sealed (MH, personal observation). Australian Trigona (s.l.) have evolved mostly in high rainfall areas (Fig. 3.2), which provide consistent, reliable floral resources. Austroplebeia, on the other hand, have evolved mainly in arid regions, with evidently unreliable resources (Fig. 3.2). Based on detailed observations, T. (Heterotrigona) carbonaria and T. (Heterotrigona) hockingsi workers appear to be “curious and flighty,” whereas A. australis and A. symei are “shy and cryptic.” In 2009 (M. Halcroft, unpublished data) a parallel study was conducted to compare foraging behavior and energy efficiency of three Australian stingless bees: T. (Heterotrigona) carbonaria, A. australis, and A. symei. The following information is based on this study. When provided with the same floral resources, T. (Heterotrigona) carbonaria sent out nine times as many foragers as A. australis and four times as many as A. symei. Even when the floral resources were completely depleted, T. (Heterotrigona) carbonaria continued to send foragers from the nest, while Austroplebeia colonies ceased to do so. This study also showed that T. (Heterotrigona) carbonaria foragers spend over 30% of their foraging time hovering in close proximity to flowers, before finally alighting to collect pollen or nectar (Fig. 3.7). Conversely, A. australis and A. symei spend over 90% of their foraging time exploring flowers and collecting pollen and nectar, while only 10% of their time is spent in flight between flowers. 3.5.2 Austroplebeia: Adapted to the Harsh Australian Outback Floral resources in the Australian outback are often unreliable. Regions may experience periods of drought that can last 1–4 years (BOM 2010b) (Fig. 3.8). Alternatively, they can also experience occasional extensive flooding. Colonies of Austroplebeia have presumably evolved and adapted in order to survive such conditions. These behavioral adaptations ensure surviving nestmates exist within the colony after the drought has broken and a long-awaited floral bloom arrives. Austroplebeia australis is an extremely long-lived worker bee, with a mean maximum worker longevity of 161.4 ± 6.1 days and a maximum longevity of 240 days 3 Australian Stingless Bees 49 Fig. 3.7 T. carbonaria forager hovering near a citrus flower. Photo: M. Halcroft Fig. 3.8 Arid native range of A. australis, Tara Queensland. Photo: M. Halcroft (M. Halcroft, unpublished data). The colonies forgo a “high rate of living” when floral resources are unavailable. Only small numbers of foragers (4 returning/2 min) are recruited during times of limited floral resources, whereas recruitment greatly increases (250 returning/2 min) during floral abundance (A. Beil, personal communication; M. Halcroft, unpublished data). Colonies have also been observed closing their nest entrance with a resin curtain during periods of dearth (MH, personal observation; A. Beil, personal communication), presumably reducing the need to guard the nest entrance. Many nest sites chosen by Austroplebeia are within dead trees (see “Nest and brood architecture”), which provide no canopy protection against frosts in winter or 50 M. Halcroft et al. Fig. 3.9 Typical dead tree chosen by A. australis colonies. Colonies in Tara, Qld, being sampled for further studies. Photo: S. Ruttley searing heat in summer (Fig. 3.9). While the insulation of natural logs is superior to that of most artificial hives, exposed trunks and limbs still allow temperature extremes to penetrate (R. Luttrell, unpublished data). Under such circumstances, it might be expected that Austroplebeia has developed thermoregulatory mechanisms. This, however, is, not the case and studies have shown that A. australis brood temperatures parallel those of the empty nest cavity and the ambient conditions (M. Halcroft, unpublished data). Prior to the onset of the cold season, colonies begin constructing a layer of involucrum over the brood, on top of which honey pots are built and filled. Those structures provide some level of protection, as the brood beneath remains undamaged. Colonies that have not been prepared for cold exposure suffer chill damage and brood death (MH, personal observation). Brood can survive at temperatures as low as −1°C (although larval development is probably delayed) and as high as 38°C, indicating the possible development of physiological resistance to temperature extremes (Halcroft, unpublished data). Austroplebeia australis colonies do not become broodless during the cold winter months, although they build a smaller number of brood cells during this time. The bees may be stimulated 3 Australian Stingless Bees 51 to build brood during the winter months when the colony is artificially warmed and provided with supplemental food (Halcroft 2007). Austroplebeia australis, and possibly other Austroplebeia species, have evolved in the unforgiving environmental conditions of arid inland Australia. Their ability to conserve energy through improved foraging efficiency and thermoconformity, and by reducing workers’ exposure to high-risk activities and high rates of living, has resulted in a well-adapted and resilient bee species. It is not only capable of surviving conditions most other species could not; it thrives in them. 3.6 The Australian Stingless Bee Industry The Australian stingless beekeeping industry is still very much in its infancy, especially when compared to many South American countries. However, comparative surveys conducted in 1998 (Heard and Dollin 2000) and 2010 (Halcroft, unpublished data) show the industry is expanding and developing. Information provided below is based upon data compiled in 1998 and 2010. In recent years there has been growing interest in Australian native bees, especially stingless bees. The honey and other hive products support an industry that has grown from 257 beekeepers in 1998 to 637 in 2010. Half of them owned just one hive and, in 2010, a quarter had less than 3 years of experience. The number of hives owned by the 637 beekeepers totally almost 5,000. Over two-thirds of the beekeepers maintain their hives on suburban blocks, although many of them also live near some form of remnant natural vegetation or “bushland.” The most commonly kept bees are T. (Heterotrigona) carbonaria, T. (Heterotrigona) hockingsi, A. australis, and A. symei. In 2010, all but three survey respondents resided in New South Wales and Queensland. Enjoyment and conservation were, by far, the most popular reasons for keeping stingless bees. The pollination of nearby vegetable and flower gardens, as well as bushland, was reported to be of considerable benefit. Only eight respondents provided pollination services on a professional basis (see “Pollination”). 3.6.1 Colony Production Australian stingless bee-keepers use a variety of hive designs ranging from a simple, wooden box to a complex, insulated (or even heated), PVC-constructed, cylindrical hive. The most commonly used hive is based on the original Australian Trigona (s.l.) hive (OATH) design (Dollin 2002; Klumpp 2007) and has a capacity of 6–7 L. Most hives are constructed so that they can be divided into two equal sections. Colony propagation techniques and hive design are discussed briefly here, because these topics are detailed elsewhere (Klumpp 2007; Dollin and Heard 2010; Heard 2010). Colony propagation of Austroplebeia is easier than for Trigona (s.l.). Small sections of brood containing a queen cell can be removed from an Austroplebeia colony 52 M. Halcroft et al. Fig. 3.10 Splitting OATH box with T. carbonaria colony. Photo: T.A. Heard and placed in a small hive, together with food stores and workers (A. Beil, personal communication; MH, personal observation). Queenright colonies with as few as 200 workers can survive and build strong colonies, if provided with the right conditions, which may include supplemental warmth and feeding (MH, personal observation). Austroplebeia colonies can also be strengthened during winter if maintained in artificially warmed rooms and provided with supplemental food (Halcroft 2007). Trigona (s.l.) species, on the other hand, are more particular. Colonies need to be very strong before they are divided for propagation. Propagation involves dividing the brood mass or inducing colony “budding.” The quickest and, therefore, the most popular technique is “splitting,” and the success of a division is dependent upon the strength of the mother colony. Colonies (not including the hive or box) should weigh at least 2 kg or 3 kg for those kept in the cooler southern regions (Klumpp 2007). The hive, containing the brood and nest structures, is split horizontally into two sections, and the occupied sections are united with new, empty half-boxes (Heard 1988a) (Fig. 3.10). This results in two half-filled hives, one with a queen (mother colony) and one with several developing queen cells. Colony survival is dependent on adequate worker number for nest repair, foraging, and defense, and the ability of the daughter colony to successfully “re-queen” (Klumpp 2007). Colony budding is a noninvasive form of hive propagation. An empty hive is attached to the nest entrance of an existing colony via a black polyethylene pipe. 3 Australian Stingless Bees 53 This technique is often used when the nest is located in an inaccessible structure, such as a wall or living tree (Klumpp 2007). An observation lid on top of the hive box is required to monitor progress of the “budded” colony. The colony provisions the attached hive, and after several weeks or, more likely several months, a virgin queen leaves the colony and mates. If successful, she returns to the “front hive” and begins laying eggs within several days. At the same time, the beekeeper intervenes by creating an opening in the connecting tube to allow foragers from the mother colony direct access to the nest. Eventually, the tube is disconnected or the bees close the connection themselves (Klumpp 2007). This technique is ideal for those beekeepers who wish to increase their colony number but are not confident with the splitting technique. It does, however, require considerable patience. The number of beekeepers involved in hive propagation has doubled. Those practicing hive division have increased colony number eightfold since 1998. More than 8,000 colonies have been produced. The number of beekeepers who sell colonies has doubled, while the number of colonies sold each year has more than quadrupled. Although this development sounds impressive, the overall annual increase in colony number since 1998 is only 9% (Halcroft, unpublished data). The retail value of a strong stingless bee colony in Australia has increased from $AU200 to between $AU350 and $AU450 per hive. Demand is high and many producers report that they are unable to keep up with demand. 3.6.2 Australian Stingless Bee Honey and Other Hive Products Honey harvesting techniques vary. Often pots are removed from the hive and honey is squeezed through a cloth or sieve. Beekeepers in Queensland and northern New South Wales, where bees can forage all year round, are able to harvest approximately 1 kg/year per hive. Beekeepers who reside in the cooler, southern regions are only able to harvest every 2–3 years, and almost not at all if they are in the Sydney basin or farther south (A. Ashhurst, 2010, personal communication). It is recommended that honey not be harvested if hive propagation is being practiced, because colonies require good stores to rebuild their strength (Dollin 2002; Heard 2010). Beekeepers who produce honey on a large scale utilize a honey super on top of hives. The OATH has a honey super with a capacity of 1.5 L, and the following technique is used by Tim Heard and many other beekeepers. The honey hive is fitted with a “floorless” super that sits on top of a thin hive ceiling, which separates the main nest from stored honey (Fig. 3.11). The separator provides access for the bees to all nest structures but still allows honey to be stored away from the brood. For easy, non-destructive honey harvest, the super is removed from the hive, exposing the intact pots (Fig. 3.12). Excess pollen stored in the super is cut out before harvest, to reduce contamination and the possibility of fermentation. The super is inverted and pots are pierced with a hand-tool similar to a small bed-of-nails. The super is placed over a plastic tray, into which fresh honey drains. After the honey is completely drained, the super is replaced and the bees can clean 54 M. Halcroft et al. Fig. 3.11 Honey super placed on top of OATH box, with separator in place. Photo: T.A. Heard Fig. 3.12 Honey super filled with honey, ready for harvest. Photo: M. Halcroft 3 Australian Stingless Bees 55 and rebuild the pots (Dollin 2002; Heard 2010). The only processing that occurs with sugarbag honey is straining out debris such as cerumen or bees. Honey harvesting is carried out by a small number of Australian beekeepers and production is low. Although the number of beekeepers has more than doubled over the last decade, overall production of Australian sugarbag honey is <300 kg/year. Of the 63 beekeepers who stated they harvest honey, only five reported selling their product, and they accounted for approximately half of overall production (Halcroft, unpublished data). Sugarbag honey caters to a “niche market” in Australia and its price is indicative of its rarity. The wholesale price has increased from $AU40/kg in 1999 to $AU70/ kg in 2010; however, the retail price remains the same, approximately $AU160/kg (Heard 2010). In comparison, honey bee—Apis mellifera—honey sells for only $AU6.50/kg (Shaw 2010). Sugarbag honey is sold in local markets, restaurants, and via Internet, and two producers export to Japan (Halcroft, unpublished data). Limited research has been conducted on Australian stingless bee honey, but T. carbonaria has similar specifications to those of other Meliponine (Persano Oddo et al. 2008). Preliminary studies on antioxidant and antimicrobial activities have shown some promise for nutritional and pharmaceutical uses (Irish et al. 2008; Persano Oddo et al. 2008; Boorn et al. 2010). Trigona carbonaria honey has a moisture content of around 26% (Persano Oddo et al. 2008) and should be stored in the refrigerator to avoid fermentation (Heard 2010). The production of cerumen and resin supplies an extremely small market in Australia. Some beekeepers are able to sustainably harvest around 200 g of cerumen per hive each year. It is sold to “didgeridoo” manufacturers (see “Introduction”), artists, and hobbyists, for $AU5/25 g, which is sufficient to make up to four didgeridoo mouth pieces (Heard 2010). 3.6.3 Pollination Pollination of commercial crops by stingless bees is rare in Australia and growers of over 35 commercial crops rely heavily on managed honey bee colonies (RIRDC 2007). Free pollination services are also provided by colonies of feral honey bees in Australia, with 40–150 colonies/km2 present in some surveyed areas (Oldroyd et al. 1997). The Australian honey bee pollination industry is currently strong and reliable (RIRDC 2007); therefore, little funded research has been undertaken on native bees as alternative pollinators. However, the reliability of the honey bee industry is now in question because managed and feral bee populations are declining due to pests and disease, as well as possible pesticide problems. For example, between 2002 and 2006 more than 4,500 colonies died out due to African small hive beetle Aethina tumida Murray (Nitidulidae) infestation (Rhodes and McCorkell 2007). Australia is the only major country without varroa mite Varroa destructor, but the likelihood of an incursion has raised major concerns about the future reliability of the honey bee pollination industry in this country (RIRDC 2007). 56 M. Halcroft et al. A rapidly expanding almond Prunus dulcis (Rosaceae) industry in Australia has resulted in industrial migration of thousands of managed honey bee colonies, transported in from the northern regions for their pollination service. It is estimated that the almond industry requires one-half of all managed honey bees in the eastern states of Australia (RIRDC 2010). This continued development, together with the predicted overall reduction in colony number, is likely to stimulate increasing interest in alternative pollinators in the warmer regions of northern New South Wales and Queensland, from which many honey bee colonies will be taken. There is already a small group of stingless bee-keepers that provide pollination services in these regions. The Australian stingless bee pollination industry had its beginnings in the late 1980s when it was found that yields of macadamia nut Macadamia integrifolia (Proteaceae) grown near remnant native vegetation were noticeably higher than for crops situated in cleared land (Heard 1988a; Heard and Exley 1994). The main pollinators of macadamia are honey bees and stingless bees (Vithanage and Ironside 1986), and presence of these insects is extremely important for maximum seed set (Wallace et al. 1996). Although the temperature threshold for Trigona (s.l.) flight activity is 18°C (Heard and Hendrikz 1993), resulting in shorter foraging days compared to honey bees (7 vs. 10 h/day, Heard and Exley 1994), Trigona (s.l.) are superior pollinators of macadamia flowers. Their small bodies are able to make more intimate contact with stigmata while collecting pollen (Heard 1994), thus aiding pollen transfer. Trigona carbonaria are opportunistic foragers that use group strategies to independently search for resources and rapidly recruit nest mates once rewards are located. Foragers demonstrate floral constancy (White et al. 2001) and resources are harvested, often by groups of bees, until they are depleted (Bartareau 1996). Few studies have been carried out on Australian stingless bee communication. Bartareau (1996) reports that T. (Heterotrigona) carbonaria foragers leave a marker of glandular secretions near the food resource, but do not leave scent trails from the resource to the nest (Nieh et al. 2000). Heard (1987) also demonstrated that Trigona (s.l.) foragers returned to hives with 100% macadamia pollen, compared to honey bees, carrying only 24%. Interestingly, Trigona (s.l.) prefer warm flowers (Norgate et al. 2010) and this is demonstrated by their attraction to flowers on outer, sun drenched racemes (Heard and Exley 1994). Macadamia also benefit from varietal interplanting for cross-pollination (Rhodes 1986) as their flowers are mostly self-incompatible and protandrous (providing pollen before stigmata are receptive) (Sedgley et al. 1985). Heard (1988b) developed a technique whereby colonies could be transferred into artificial hives for use in managed crop pollination. The use of stingless bees for pollination of macadamia has grown since then, and several macadamia farmers have purchased their own hives to improve crop yield. Some growers were originally honey bee keepers, but found it easier to move small Trigona (s.l.) hives to their macadamia crops, which are often grown on steep slopes. Those farmers have since become reputable stingless bee-keepers in their own right (F. Adcock, personal communication). The demand for stingless bee pollination service by the macadamia nut industry, as well as other crops, is growing. At present, there are not enough hives available to meet this demand (M. Grosskopf, 2010, personal commu- 3 Australian Stingless Bees 57 nication). Further effort is required to improve colony propagation. This would ensure that enough colonies are available in the future for suitable stocking rates and satisfactory pollination service (T. Carter, personal communication). It is estimated that Australian stingless bees have an average flight range of only 500 m (Heard and Dollin 1998). This is advantageous for crop pollination, because bees are more likely to forage within the crop area than to venture farther afield in search of other floral resources, as is often the case with honey bees (Graham 1992). Hive placement is important, and the 15–20 hives per hectare (compared to seven honey bee hives per hectare) should be interspersed throughout the crop if possible, especially if cross-pollination is required (Heard and Dollin; F. Adcock, personal communication; T. Carter, personal communication). Crops other than macadamia can also benefit from stingless bee pollination. Anderson et al. (1982) showed stingless bees to be effective pollinators of mango (Mangifera indica; Anacardiaceae) and anecdotal accounts of increased crop quality and yield have been reported for other crops such as lychee Litchi chinensis (Sapindaceae), avocado Persea americana (Lauraceae), and watermelon Citrullus lanatus (Cucurbitaceae) (T. Carter, personal communication). Although no scientific studies have been conducted on the effectiveness of stingless bees as pollinators in Australian crops other than macadamia and mango, estimates of improved crop yield have been assessed by one beekeeper. Stingless bees have also been introduced into blueberry (Vaccinium corymbosum; Ericaceae) and bees are able to collect pollen and nectar more efficiently than honey bees (F. Adcock, S. Maginnity, M. Grosskopf, personal communication). Blueberry flowers are small, with a deep corolla and narrow terminal orifice (Rhodes 2006). Unfortunately, there is no experimental design or statistical analysis associated with these trials. Although the role of stingless bees in pollination of native flora is well documented, their efficacy in horticultural and agricultural crops of Australia needs further study (Heard 1987; Heard 1999; Slaa et al. 2006). Of the eight beekeepers who reported that they provided pollination services on a professional basis during 2010, only four charged a service fee. One beekeeper charged only $AU10 per hive, while the other three charged $AU35–40 per hive (Halcroft, unpublished data). 3.7 3.7.1 Management Issues Pests of Australian Stingless Bees Australian stingless bees seem to be relatively disease-free and no reports of brood disease have been seen. They do, however, suffer from predation, parasitism, and colony infestation. There are many general predators such as flies, ants, spiders, mites, wasps, birds, lizards, toads, and, of course, humans, which are common pests of social bees worldwide. Australia has its own unique species of stingless bee parasites and predators; however, little is known about most species. Usually, strong hives 58 M. Halcroft et al. Fig. 3.13 Syrphid fly adult. Photo: J. Klumpp are at minimal risk of hive invasion but weakened or newly propagated colonies are vulnerable to attack from pests. It is of critical importance, when managing colonies, that all means of access to the nest cavity are well sealed and that colonies are divided or transferred as quickly and efficiently as possible, to minimize pest infestation. One of the most serious pests of stingless bee colonies in Australia is the syrphid fly Ceriana ornata australis Macquar. Ceriana ornata is 12 mm long, with bright orange-yellow and black markings (Fig. 3.13) and is frequently observed hovering near nests during summer (MH, personal observation). This fly has been observed in all regions where stingless bee colonies are present and is seldom observed in areas where stingless bees do not occur (Klumpp 2007). Ceriana ornata is most destructive when colonies are divided or damaged. The female lays eggs directly on nest structures if the hive is left open and unattended, or eggs are laid in unsealed joints or cracks in the hive surface. The eggs hatch and the larvae (Fig. 3.14) make their way into the nest cavity and food stores. If fly larval numbers are high the colony will die as stores and immatures are consumed. The phorid fly Dohrniphora trigonae Disney can also cause problems in Australian stingless bees, especially Trigona (s.l.) species (Disney and Bartareau 1995), similar to phorid fly pests overseas (genus Pseudohypocera). D. trigonae lays its eggs within the colony stores and are most problematic following colony division (Klumpp 2007; Dollin and Heard 2010). These tiny flies (2.5 mm) (Fig. 3.15) enter nests more easily than the larger syrphid fly and can do so in high numbers (Klumpp 2007). Once inside, flies run along the surface of the structures, laying eggs in honey and pollen pots. This pest is less of a problem in Australia than overseas. Stingless bee predators that are unique to Australia include Bembix flavipes Smith and Bembix musca Handlirsch (Crabronidae) (Fig. 3.16). These Bembix hunt singly and hover outside the entrance, waiting for bees to exit. Once a bee leaves the 3 Australian Stingless Bees Fig. 3.14 Syrphid fly larvae in nest of dead colony. Photo: M. Halcroft Fig. 3.15 Trigona worker (left) beside a phorid fly (right). Photo: J. Klumpp Fig. 3.16 Bembix wasp. Photo: J. Klumpp 59 60 M. Halcroft et al. Fig. 3.17 (a) Braconid wasp (right) lying in wait near Trigona foragers. (b) Braconid wasp (left) preparing to oviposit into Trigona forager. Photos: J. Klumpp nest the wasp swoops from behind the unsuspecting worker and drags it to its own nest (A. Beil, 2009, personal communication). Evans et al. (1982) observed mass provisioning of B. flavipes nests with over 25 freshly collected Austroplebeia, and B. musca provision nests with T. (Heterotrigona) carbonaria. Drones are the main prey during the stingless bee mating season (Evans and O’Neill 2007). The only known parasitoid of Australian stingless bees is the braconid wasp (Syntretus trigonaphagus) (Gloag et al. 2009). Syntretus trigonaphagus has only been reported in the Brisbane area. The distribution of its host, T. carbonaria, suggests that it may be found more widely along the east coast of Australia. Wasps wait near the hive entrance or on flowers where bees are foraging (Fig. 3.17). When close enough to the posterior of an individual bee the wasp projects her abdomen under and in front of hers and oviposits onto the abdomen of the bee. The hatching larva grows inside the abdomen of the living bee, for an unknown period of time. Parasitized bees are easily identified because the abdomen is 2–2.5 times larger than that of normal bees. The fully developed larva emerges from the bee, which then usually flies away, presumably to die. Gloag et al. (2009) were unable to successfully rear pupae from emerged larvae and it is thought that pupation may take place in the soil (Klumpp 2007). Australian native beetles in the genus Brachypeplus (Coleoptera: Nitidulidae) have been observed in stingless bee hives (MH, personal observation) although it is thought they are not a major problem for strong colonies (A. Dollin, 2010, personal communication). Adult beetles are commonly observed on the outer surfaces of hives. These beetles are smaller and more slender than the worker bees (Fig. 3.18) and are, therefore, able to gain access through unsealed cracks and joints in hives. They lay eggs in inaccessible cracks and crevices within the hive. Beetle larvae have not been observed in high numbers within hives and the main signs of their presence are the accumulation of dry debris in the bottom of the hive and a reduction in worker bee number (MH, personal observation). Little is known about these native beetles and it is unclear what the larvae consume within the stingless bee nests. Pupating beetle larvae have been observed under the transparent lid of A. australis 3 Australian Stingless Bees 61 Fig. 3.18 Adults and larva of Brachypeplus sp. beside an A. australis worker. Photo: M. Halcroft Fig. 3.19 Adult small hive beetles beside an A. australis worker. Photo: M. Halcroft hives, indicating that these beetles can complete their life cycle within the nest (MH, personal observation). The African small hive beetle (Aethina tumida Murray; Nitidulidae) is a newly introduced honey bee pest in Australia (Fig. 3.19) and can devastate newly divided, or damaged, stingless bee colonies. Adult beetles are frequently found near hives (MH, personal observation) and enter nest openings whenever possible. If left unchallenged, the beetle lays eggs in food stores and brood. The larvae hatch and begin feeding, defecating throughout the nest, and cause hive contents to ferment. Eventually the entire colony collapses into a slimy mass. As with syrphid fly invasion, strong colonies are usually able to remove larvae from an infested nest, but prevention is always better than cure. A strong, undamaged colony can defend against small hive beetle invasion, and studies have shown that T. (Heterotrigona) carbonaria can incapacitate invading adult beetles within 10 min of being introduced to the nest entrance (Greco et al. 2010). Halcroft et al. (2011) showed that A. australis was effective in removing or destroying all life stages (eggs, larvae and adults) from hives and that efficiency in entrance defense and invader removal increased with frequency of exposure to beetle invasion. Both T. (Heterotrigona) carbonaria and A. australis utilize resin to entomb adult 62 M. Halcroft et al. Fig. 3.20 Adult small hive beetle entombed alive in cerumen while an A. australis worker guards the interloper. Photo: M. Halcroft beetles within the nest (Fig. 3.20). Austroplebeia australis later dismembers the remains and removes them from the nest. Another exotic predator of Australian stingless bees is the cane toad (Rhinella marina—formerly known as Bufo marinus Linneaus; Bufonidae). Introduced into Australia in 1935 as a biological control agent for the pest cane beetle this highly toxic pest has spread from coastal northern Queensland to the central coast of New South Wales and across northern Australia to Kakadu National Park in Northern Territory (Australian Museum 2010). Cane toads are quite ingenious and may stand on each other’s backs in order to reach bee hive entrances. The toad will stay at the hive entrance and consume incoming and outgoing workers until forager numbers dwindle, to the point that the colony may be in danger of perishing (R. Zabel, personal communication) (Fig. 3.21). 3.7.2 Seed Dispersal by Stingless Bees Corymbia torelliana F. Mueller (Myrtaceae), or cadaghi tree, as it is commonly known, is native to the rainforest margins of the Atherton Tablelands in northern Queensland. This species of Australian gum tree has spread extensively outside its native range as it has been used for plantation timber. Its abundant blooms (Fig. 3.22) and showy gum nuts (Fig. 3.23) have also resulted in it being used in street plantings and parks, especially in the Brisbane area (AWC 2010). Although it is a source of abundant pollen and nectar in spring, it has become a major management problem for some stingless bee-keepers. Corymbia torelliana seeds are mainly dispersed by gravity; 88% of seeds drop to the ground soon after the fruit opens. However, one or two seeds remain within the gum nut and all are dispersed by Trigona (s.l.) (Wallace et al. 2008). 3 Australian Stingless Bees 63 Fig. 3.21 Cane toad waiting at the entrance of a stingless bee hive. Photo: R. Zabel Fig. 3.22 C. torelliana flowers are an abundant pollen and nectar source. Photo: J. Klumpp Resin is produced in the gum nut, behind the valve (Fig. 3.24). When the bee enters the nut to collect resin, the seeds attach to the sticky corbicular load (Fig. 3.25). Seeds are dispersed by bee vectors, or “mellitochory,” and may be spread during the flight back to the nest or transported to the nest itself. This may be up to 1 km away from the tree (Klumpp 2007; Wallace et al. 2008; A. Beil, R. Luttrell, J. Klumpp, personal communication). Trigona (s.l.) are strongly attracted to the resin from C. torelliana and the colonies stop normal foraging 64 M. Halcroft et al. Fig. 3.23 The attractive gum nuts of C. torelliana make it an ideal amenities tree. Photo: J. Klumpp Fig. 3.24 Cross section of C. torelliana fruit, showing seeds and resin in close proximity. Photo: R. Luttrell activity to collect as much of this resource as possible (Klumpp 2007). Trigona (s.l.) are known to collect and store large amounts of resin, with up to 10% of foragers returning with resin loads (Wallace and Lee 2010). Analysis of the chemical profiles of body surfaces of five Trigona (s.l.) species showed that 51% 3 Australian Stingless Bees 65 Fig. 3.25 Trigona forager on a C. torelliana fruit, with a seed adhered to her corbicular load. Photo: R. Luttrell of these compounds were derived from plant resins. Conversely, Austroplebeia, which collect only small amounts of plant resin, had little or no resinous compounds on their bodies (Leonhardt et al. 2010). As foragers return to the nest some attempt to dislodge seeds on the nest exterior, while others transport seeds directly into the nest cavity. The colony removes some, but not all, of the introduced seeds and these are either disposed of, up to 10 m outside the nest (Wallace and Trueman 1995), or adhere to the sticky surface of the nest entrance (Wallace et al. 2008) (Fig. 3.26). Seeds collect around the entrance (Wallace and Trueman 1995), reducing airflow within the nest. Resin from C. torelliana may have a lower melting point than many other plant resins. Collection of the resin and its seed occurs during the hottest months of the year in Australia—December to February (Wallace and Lee 2010); and as temperatures rise, the resin begins to soften. Reports of structural collapse due to seed weight and resin softening are not uncommon, particularly if ambient temperatures exceed 39°C (J. Klumpp, M. Duncan, 2006, personal communication). As a result, some beekeepers remove their hives from C. torelliana areas during resin flow to prevent colonies from collecting the resin and seed mixture (Klumpp 2007; T. Carter, 2010, personal communication). While many Australian beekeepers consider C. torelliana to be a major management problem, others consider it to be a useful source of pollen, nectar, and resin (Klumpp 2007). 3.7.3 Fighting Swarms Nest defense is widely reported in stingless bees around the world. Incapacitation of intruders is achieved by biting, resin daubing, chemical repellents, and locking onto the wing or body with their mandibles, thus grounding invaders and rendering them harmless (Roubik et al. 1987; Wittman et al. 1990; Lehmberg et al. 2008; Halcroft 66 M. Halcroft et al. Fig. 3.26 C. torelliana seed collection around the entrance of a Trigona nest entrance. Photo: R. Luttrell et al. 2011). Nest defense against conspecifics is, however, more specialized and involves recognition of nestmates from non-nestmates, using recognition cue compounds (Buchwald and Breed 2005). Trigona carbonaria demonstrate a collective defense behavior known as a “fighting swarm,” during which time hundreds to thousands of workers, usually from two colonies, become entwined in an aerial battle, to the death. The fight takes place outside the defending nest and may result in the usurpation of the defending colony. In flight, two workers lock together by biting each other and immediately drop to the ground. Sometimes the opponents mistakenly attack their own nestmate and, once they recognize this, the pair will usually unlock mandibles and take to the air again, rejoining the fight. Typically, the combatants remain locked together until death, after which they are dragged away by opportunistic scavengers, such as ants. The battle, which may begin each morning and can last for days, results in a carpet of thousands of dead bees locked together by the mandibles (Fig. 3.27) (Wagner and Dollin 1982; Heard 1996; Klumpp 2007; Dollin 2008; Gloag et al. 2008). Gloag et al. (2008) found that most “fighting swarms” involved only two colonies. However, there were instances where up to seven colonies were identified 3 Australian Stingless Bees 67 Fig. 3.27 T. carbonaria hive entrance showing just a proportion of the coupled bees that will die in battle. Photo: R. Gloag in a single battle. Alarm pheromones are probably responsible for attracting neighboring colonies into the “fighting swarm.” This may potentially increase overall losses within a meliponary or orchard. Trigona (Heterotrigona) carbonaria is the most popular species kept by Australian stingless bee-keepers (Halcroft, unpublished data), and “fighting swarms” are a major management problem. While there are reports of other stingless bee species forming defensive groups, “fighting swarms” are regularly reported in T. (Heterotrigona) carbonaria (ANBees 2010). Colony strength is greatly reduced after a fight and colonies may not be divisible for another season. Gloag et al. (2008) also find that approximately one in five of the paired combatants were nestmates, contributing further to the cost of battle. Gloag et al. (2008) tested the theory that returning workers may become disorientated, especially when moved into a crop area for pollination. Workers were forced to enter a foreign nest, which quickly provoked a “fighting swarm.” In the field, disoriented workers mistakenly entered another nest, thus prompting a “fighting swarm.” Management practices that are used to reduce the incidence of “fighting swarm” due to disorientation include: separation of hives by 5 m, positioning hives at different heights and directions, and identification of hives with different colors or symbols (Gloag et al. 2008). Fortunately, colonies involved in “fighting swarms” usually recover (Heard 1996) and in the case of usurpation, the weaker colony may even increase in vigor (Dollin 2008). More information on fighting swarms can be found at http://www.aussiebee. com.au/video-fighting-swarms-1.html. 68 M. Halcroft et al. Acknowledgements We would like to sincerely thank the following people for their generous contributions to the information within this chapter: John Klumpp for his technical, field, and photographic support; Tim Heard for his technical and photographic support; Robert Luttrell and RosGloag for their photographic support; Mark Grosskopf, Thomas Carter, and Steve Maginnity for their expertise in the area of pollination and colony management; Peter Lain for information pertaining to indigenous employment opportunities, and Alan Beil for his incredible enthusiasm and expertise in the field. Thank you also to those involved in the long and arduous editorial process. We thank the reviewers for their time and constructive input and to Patricia Vit and David Roubik for their patience and guidance in this process. References Adlam N. 2010. Taking tucker to town. Northern Territory News. Available at: http://www.ntnews. com.au ANBees. 2010. Australian Native Bees. 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Wallace HM, Vithanage V, Exley EM. 1996. The effect of supplementary pollination on nut set of Macadamia (Proteaceae). Annals of Botany 78:765–773. Welch D. 1995. Beeswax rock art in the Kimberley, Western Australia. Rock Art Research 12:23–28. Welch D. 2010. Aboriginal culture. Available at: www.aboriginalculture.com.au White D, Cribb BW, Heard TA. 2001. Flower constancy of the stingless bee Trigona carbonaria Smith (Hymenoptera: Apidae: Meliponini). Australian Journal of Entomology 40:61–64. Wittmann D, Bego LR, Zucchi R, Sakagami SF. 1991. Oviposition behavior and related aspects of the stingless bees XIV. Plebeia (Mourella) caerulea, with comparative notes on the evolution of the oviposition patterns (Apidae, Meliponinae). Japanese Journal of Entomology 59:793–809. Wittman D, Radtke R, Zeil J, Lübke G, Francke W. 1990. Robber bees (Lestrimelitta limao) and their host: chemical and visual cues in nest defense by Trigona (Tetragonisca) angustula (Apidae: Meliponinae). Journal of Chemical Ecology 16:631–641. Yamane S, Heard T, Sakagami SF. 1995. Oviposition behavior of the stingless bees (Apidae, Meliponinae) XVI. Trigona (Tetragonula) carbonaria endemic to Australia, with a highly integrated oviposition process. Japanese Journal of Entomology 63:275–296. Yunkaporta T. 2009. Decolonising education: an Indigenous learning journey. PhD Thesis, Aboriginal pedagogies at the cultural interface, James Cook University; Cairns, Australia. 85 pp. Zabel R. 2008. Aurukun newsletter. Available at: http://uqconnect.net/~zzrzabel/aurukun-2005newsletter-2.html Chapter 4 Stingless Bees from Venezuela Silvia R.M. Pedro and João Maria Franco de Camargo* 4.1 Introduction The stingless bees of Venezuela, or “abejas criollas” as known by locals, have aroused the interest of native and foreign people since long ago. According to Rivero Oramas (1972) the first records about the biology of these bees dated 1578 when the Governor Juan Pimentel wrote about the province of Caracas mentioning the use of tree trunk hollows of the “jobo” (Spondias mombin L.—Anacardiaceae) as a place for nesting by bees (probably stingless bees). He also commented about the commerce of honey and cerumen, which is always black in that region. In the years 1612–1613 Father Pedro Simón gave more detailed accounts of the bees in the region of Los Llanos. He made observations about the honey stored in pots arranged in clusters, not in combs, the quality of honey and cerumen and their use by native people, and the docile behavior of the bees. Reports about traditional meliponiculture (beekeeping with stingless bees) are even older. Venezuelan Indians kept stingless bees in large calabashes in their houses according to reports of Rodrigo de Bastidas dated from the 1540s (Oviedo 1550 apud Crane 1999), who also mentioned the presence of many bees without stinging organs in the wild woods. It is interesting to note that the European bee Apis mellifera Linnaeus, 1758 was not present in Venezuela at least until 1866, according to oral information by Prof. Karsten (Gerstaker 1866 apud Nogueira-Neto 1962). Nowadays despite the great diversity of stingless bee species in Venezuela and the traditional meliponiculture widespread in that country, there are few studies dealing with the taxonomic diversity of the Venezuelan native stingless bees. *João M.F. Camargo—In memoriam. S.R.M. Pedro (*) • J.M.F. Camargo Departamento de Biologia, Faculdade de Filoso fi a, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, CEP 14040-901, Ribeirão Preto, SP, Brazil e-mail: silviarmp@ffclrp.usp.br P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_4, © Springer Science+Business Media New York 2013 73 S.R.M. Pedro and J.M.F. Camargo 74 The species more intensively reared are known only by their common names, most of them from indigenous origin, and sometimes the same name is applied for different species, or one species can receive different names depending on the region (Rivero Oramas 1972). On the other hand, there is a field of scientific research that has meaningfully contributed to the improvement of knowledge of the taxonomic biodiversity of the stingless bee fauna from Venezuela: honey and propolis analyses. For the last 25 years, Dr. Patricia Vit and collaborators have been sending Venezuelan specimens of stingless bees, associated with honey and propolis samples, for identification and deposit in the Camargo Collection—RPSP (see Vit 2008). They have been working to create the quality standards of stingless bee honey through determination of their botanical and geographical origins, melissopalynology, biochemical composition, and physicochemical, sensory, and bioactive properties—antibacterial activity, antioxidant capacity, acidity, electrical conductivity, diastase and invertase activities, and levels of ash, nitrogen, flavonoids, hydroxymethylfurfural, reducing sugars, sucrose, and water (e.g., Vit Olivier 1992; Vit and Ricciardelli d’Albore 1994a, b; Vit et al. 1994, 1997, 1998a, b, 2011; Bogdanov et al. 1996; Vit and Pulcini 1996; Vit and TomásBarberán 1998; Rodríguez-Malaver et al. 2009; Vit 2005, 2009). Propolis collected from nests of Venezuelan stingless bees has been also analyzed concerning phenolic compounds (e.g., Tomás-Barberán et al. 1993; Vit et al. 1993). The identification was provided mainly by one of the present authors, the late Prof. João MF Camargo, specialist in taxonomy, biology, and biogeography of Meliponini. The bees sampled by Prof. P. Vit have provided valuable information about the meliponine fauna from Venezuela, such as new records of species and geographical records, as well as taxonomic information for future revisions. The present chapter introduces a preliminary checklist of stingless bees from Venezuela, including common names, geographic records, and studies concerning honey. This is only a preliminary treatment and certainly there are many more species in Venezuela than the ones listed here, considering that we have not studied material deposited in other collections. Other constraints involve the lack of intensive and periodic surveys comprising the large diversity of habitats distributed throughout the Venezuelan territory as well as taxonomic limitations. Nonetheless, this can be useful in future faunistic surveys as well as in taxonomic revisions of Venezuelan Meliponini bees. 4.2 Data Sources The data were obtained from material studied by the authors, mostly collected by Prof. P Vit (Universidad de Los Andes, Mérida, Venezuela), during the last 25 years, and sent to RPSP (Camargo Collection, housed in the Department of Biology, FFCLRP-USP, in Ribeirão Preto, São Paulo) for identification. Other studied specimens in RPSP from Venezuela were collected by JMF Camargo—who traveled across the states of Merida, Barinas, and Zulia studying nests of Meliponini through 4 Stingless Bees from Venezuela 75 March 2008, accompanied by P Vit. Material has also been collected by RW Brooks and collaborators, D Wittmann, and others. Popular names listed here were obtained from traditional stingless bee-keepers and locals by P Vit when collecting the bees. Some popular names mentioned by Rivero Oramas (1972), such as “bayures” (probably the same as “guayures”), “araguatas,” and “mabas,” could not be associated with the scientific names of the species. Additional information was obtained from literature, mainly from Schwarz (1932, 1948) and Camargo and Pedro (2007, 2008). 4.3 Diversity and Distribution of Stingless Bees in Venezuela Species recorded in Venezuela are listed in Table 4.1. Geographical records are listed by states following two-letter abbreviations (AM Amazonas, AP Apure, ME Mérida, etc.). From the 83 species of stingless bees that occur in Venezuela, here listed, 18% have their honeys already analyzed (references in Table 4.1). Nests of some species are represented in the Fig. 4.1a–f. The total of 83 species included in 19 genera is certainly an underestimate, mainly considering that material deposited in collections, other than RPSP, was not examined. Also, some genera are currently under revision or need to be revised, and the identity of some species could not be determined for this work. These are mainly Frieseomelitta, Nannotrigona, Scaptotrigona, Tetragona, Tetragonisca (Fig. 4.1d–f), Plebeia, and Scaura. Despite the exhaustive revisions by Schwarz (1932, 1948), the taxonomy of Melipona and Trigona deserves a reevaluation as well. Melipona Illiger, 1806 is the most diversified Neotropical stingless bee genus, divided in four subgenera, all represented in Venezuela, and with about 70 known species (Camargo and Pedro 2007, 2008), some of them extensively reared by beekeepers. Trigona is also widely diversified with about 32 valid species, besides at least other 10–20 new to science and in some cases there are complexes of different species now frequently identified under the same epithet (e.g., Trigona fulviventris, T. guianae, T. fuscipennis, T. hypogea, T. pallens). Some species were only recorded in Venezuela in the literature and we could not confirm the identity of this material. Some of them are certainly misidentifications or junior synonyms of other species [e.g., Scaptotrigona polysticta Moure, 1950, Trigona hyalinata (Lepeletier, 1836) (probably T. branneri), Trigona spinipes (Fabricius, 1793) (probably T. amazonensis), Trigona alfkeni Friese, 1900, Trigona silvestriana (Vachal, 1908), Trigona trinidadensis (Provancher, 1888) (junior synonym of T. amalthea); see Camargo and Pedro (2007, 2008)] and were not included in the Table 4.1. Other names were listed in Table 4.1 with some uncertainty such as Geotrigona subnigra, Lestrimelitta glaberrima, Nannotrigona perilampoides, and Plebeia fraterna, recorded in Guárico by Rodríguez-Parilli et al. (2010). These authors also mentioned one unnamed Friesella, recorded in Portuguesa, but the material was probably misidentified. Friesella schrottkyi (Friese, 1900), the only species of the genus, is restricted to the southern part of Brazil (Camargo and Pedro 2007, 2008). There is no other record of Friesella between southern Brazil and Venezuela. 76 Table 4.1 Stingless bees from Venezuela: Common names, geographic records (by states in Venezuela), and honey analysis Scientific names Common names Geographic records Honey analysis Aparatrigona impunctata (Ducke, 1916) Cephalotrigona capitata (Smith, 1854) Duckeola pavani Moure, 1963 Frieseomelitta paupera (Provancher, 1888) “guanotica” 5, 6 Frieseomelitta spp. (at least two species) “angelita,” “erica” 7 8, 9 10 Geotrigona subnigra (Schwarz, 1940) Geotrigona spp. (at least two species) Lestrimelitta glaberrima Oliveira & Marchi 2005 Lestrimelitta maracaia Marchi & Melo, 2006 Melipona (Eomelipona) concinnula # Cockerell, 1919 Melipona (Eomelipona) illustris Schwarz, 1932 Melipona (Eomelipona) ogilviei Schwarz, 1932 Melipona (Melikerria) compressipes (Fabricius, 1804) 11 12 13 14 15 16 Melipona (Melikerria) interrupta (Latreille, 1811) “abejita,” “negrito” AM BO, DA AM AM, AR, BA, BO, FA, GU, MO, NE, PO, ZU AM, BA, BO, MO, TA 2, 4, 5, 6, 7, 9, 10, 14 [as Trigona (Frieseomelitta) nigra paupera] 2, 4, 5, 6, 9, 10, 11, 12, 13, 14, 15 [as Frieseomelitta sp. aff. varia, F. aff. varia or F. sp. group varia, Frieseomelitta officinalis varia, Trigona (Frieseomelitta) sp aff varia, Friesomelitta (sic) varia] GU* AM, LA GU* “limoncita” “guanota” AM BO AM AM AM, AP, BA, BO, GU, LA, PO, TA, ZU ZU 1, 2, 3, 4, 5, 6, 9, 10, 12, 13, 14 S.R.M. Pedro and J.M.F. Camargo 1 2 3 4 Geographic records Honey analysis 17 Melipona (Melipona) favosa (Fabricius, 1798) “abejita,” “abejita casera,” “angelita,” “arica,” “criollita,” “erica,” “mabita” AM, AP, BA, BO, FA, GU, LA, ME, MO, NE, PO, SU, TA 1, 2, 3, 4, 5, 6, 9, 10, 11, 12, 13, 14, 15 18 Melipona (Michmelia) apiformis# (Buysson, in Du Buysson & Marshall, 1892) Melipona (Michmelia) cramptoni Cockerell, 1920 Melipona fasciata cramptoni duidae# Schwarz, 1932 (junior synonym) Melipona (Michmelia) crinita Moure & Kerr, 1950 Melipona (Michmelia) fulva Lepeletier, 1836 AR AR, LA, TA, YA 23 Melipona (Michmelia) indecisa# Cockerell, 1919 Melipona (Michmelia) lateralis Erichson, 1848 “ñuriño” AM, BO, NE, SU 24 25 Melipona (Michmelia) paraensis Ducke, 1916 Melipona (Michmelia) trinitatis Cockerell, 1919 “isabitto” “guanota” 26 Melipona (Michmelia) sp. 1 “cigarroncito,” “sabite,” “tobillo morrocoy,” “isabitto” AM, BO AN, DA, MO, NE, SU, ZU AM 27 28 29 Melipona (Michmelia) sp. 2 Nannotrigona melanocera (Schwarz, 1938) Nannotrigona perilampoides (Cresson, 1878) 19 20 21 22 AM “moscochola” AM 2, 4, 5, 6, 14 BO 2, 4, 5, 6, 14 (as Melipona sp. group fulva, Melipona sp. aff. fulva) Stingless Bees from Venezuela Common names 4 Scientific names 2, 4, 5, 6, 14 [as Melipona lateralis kangarumensis Cockerell, 1920] 2, 4, 5, 6, 14 1, 9, 10, 11 2, 4, 5, 6, 8, 14 [as Melipona fuscopilosa Moure & Kerr, 1950] AR, BA AM, AP, AR, DC, PO GU* (continued) 77 78 Table 4.1 (continued) Scientific names Nannotrigona schultzei (Friese, 1901) Nannotrigona tristella# Cockerell, 1922 Nannotrigona sp. 33 Oxytrigona mellicolor (Packard, 1869) 34 Paratrigona anduzei (Schwarz, 1943) 35 36 Paratrigona pannosa Moure, 1989 Paratrigona permixta# Camargo & Moure, 1994 Partamona ailyae Camargo, 1980 Partamona auripennis Pedro & Camargo, 2003 Partamona epiphytophila Pedro & Camargo, 2003 Partamona ferreirai Pedro & Camargo, 2003 Partamona nigrior (Cockerell, 1925) Partamona pearsoni (Schwarz, 1938) Partamona peckolti (Friese, 1901) 37 38 39 40 41 42 43 44 45 46 47 Partamona vicina Camargo, 1980 Partamona vitae# Pedro & Camargo, 2003 Plebeia fraterna# Laroca & Rodriguez-Parilli, 2009 Plebeia goeldiana# (Friese, 1900) “zamurita” “abejita,” “cortacabello,” “españolita,” “pegón” Geographic records AM FA, YA, ZU AP, AR, BA, DA, ME, PO, YA, ZU AR, BA, GU, ME, PO, YA, ZU BO, ME, TA Honey analysis 2, 4, 5, 6, 14 [as Nannotrigona sp. aff. chapadana (Schwarz, 1938)] 12, 13 AM SU AM AM AM “pegona,” “pegón,” “churrusca” BO AR, BO, SU AM AM, AR, BO, DC, FA, GU, LA, ME, TA, TR, ZU AM AM, BO GU* BO, ME, TR S.R.M. Pedro and J.M.F. Camargo 30 31 32 Common names Geographic records Honey analysis 48–54 Plebeia spp. (at least more seven species) “mosquito” 7, 9, 10 55 56 57–62 Ptilotrigona lurida (Smith, 1854) Scaptotrigona ochrotricha# (Buysson, in Du Buysson & Marshall, 1892) Scaptotrigona spp. (at least more six species) AR, BO, GU, LA, NE, TR, ZU AM, BO AM, AR 63 “guaracho,” “isabitto,” “pico,” “sonquette” AM, AR, NE, ZU Scaura sp. “pegoncito” BO, TA 64 Tetragona clavipes (Fabricius, 1804) “ajabite,” “ajavitte,” “ajavitta” AM 65 66, 67 Tetragona ziegleri (Friese, 1900) Tetragonisca spp. (at least two species) 68 Trigona amalthea (Olivier, 1789) “abejita,” “eriquita,” “españolita,” “guayure,” “lambeojitos,” “pañuelita,” “princesita,” “rubita” “pegón” ME BA, GU, LA, ME, PO, YA 2, 4, 5, 6, 14 [as Scaptotrigona ochrotica (sic = ochrotricha)] 2, 4, 5, 6, 7, 9, 10, 12, 13, 14 [as S. polystica (sic = S. polysticta), Scaptotrigona officinalis (sic) depilis, Scaptotrigona sp aff depilis, Scaptotrigona depilis] 7, 9, 10 [as Scaura aff. latitarsis, Scaura latitarsis, Plebeia (Scaura) latitarsis] 8 Stingless Bees from Venezuela Common names 4 Scientific names 2, 4, 5, 6, 7, 8, 9, 10, 14 [as Trigona (Terragonisca) (sic = Tetragonisca) Angustula angustula, Trigona (Tetragonisca) angustula angustula, Tetragonisca angustula angustula, Trigona (Frieseomelitta) angustula angustula] AR, BO, GU, LA, ME, SU, TR, YA, ZU (continued) 79 69 70 71 72 73 74 75 Trigona amazonensis (Ducke, 1916) Trigona branneri Cockerell, 1912 Trigona cilipes (Fabricius, 1804) Trigona dallatorreana Friese, 1900 Trigona fulviventris Guérin, 1844 Trigona fuscipennis Friese, 1900 Trigona guianae Cockerell, 1910 76 77 78 79 80, 81 82, 83 Trigona pallens (Fabricius, 1798) Trigona truculenta Almeida, 1984 Trigona venezuelana# Schwarz, 1948 Trigona williana Friese, 1900 Trigona spp. (at least +2 species) Trigonisca spp. (at least + 2 species) 80 Table 4.1 (continued) Scientific names Common names “pegón” “pegón” “pegona,” “pegón” “pegón” Geographic records Honey analysis AM AM, BO, MO AM AM TA, YA AM, GU, TA, ZU AM, AR, BA, LA, ME, PO, TA, ZU AM AM AR, DA, DC, MI, YA AM, BO AM, LA, YA GU, TR, ZU S.R.M. Pedro and J.M.F. Camargo References for honey and propolis analyses: Bogdanov et al. (1996) (1); Vit and Pulcini (1996) (2); Vit et al. (1997) (3); Vit and Tomás-Barberán (1998)(4); Vit et al. 1998a, b) (5, 6); Vit (2009) (7); Vit et al. (2011) (8); Vit Olivier (1992) (9); Vit et al. (1994) (10); Vit and Ricciardelli d’Albore 1994b (11); TomásBarberán et al. (1993) (12); Vit et al. (1993) (13); Vit and Ricciardelli d’Albore 1994a (14); Vit (2005) (15); *recorded by Rodríguez-Parilli et al. (2010); # species described from Venezuela. Bees were recorded from AM Amazonas, AN Anzoátegui, AP Apure, AR Aragua, BA Barinas, BO Bolívar, DA Delta Amacuro, DC Distrito Capital (= Distrito Federal), FA Falcón, GU Guárico, LA Lara, ME Mérida, MI Miranda, MO Monagas, NE Nueva Esparta, PO Portuguesa, SU Sucre, TA Táchira, TR Trujillo, YA Yaracuy, ZU Zulia 4 Stingless Bees from Venezuela 81 Fig. 4.1 Nests of stingless bees from Venezuela. (a, b) Paratrigona anduzei (Schwarz, 1943) among roots of epiphyte, Garden of Medicinal Plants, Universidad de Los Andes, Mérida, Venezuela. (c) Nest entrances of Frieseomelitta paupera (Provancher, 1888) in wall of cement bricks, Trail Peña de La Yuca, Barinas, Venezuela. (d) Nest entrance of Tetragonisca sp., in the base of a trunk of mango tree, Garden of Medicinal Plants, Universidad de Los Andes, Mérida, Venezuela. (e, f) Tetragonisca sp., nest in artificial cavity of funnel, Food Science Department, Universidad de Los Andes, Mérida, Venezuela; the arrow indicates the nest entrance built with cerumen in the open funnel it is possible to see the layers of involucrum protecting the nest. Photos: J.M.F. Camargo 82 S.R.M. Pedro and J.M.F. Camargo Among the genera with species represented in Venezuela and recently revised, including not only taxonomy but also information about biology, are Paratrigona and Aparatrigona (Camargo and Moure, 1994), Geotrigona (Camargo and Moure, 1996), Partamona (Camargo and Pedro, 2003, Pedro and Camargo, 2003), and Ptilotrigona (Camargo and Pedro, 2004). Lestrimelitta and Oxytrigona were partially revised by Marchi and Melo (2006) and Gonzalez and Roubik (2008). Several species were described from Venezuela and some of them remain known only from the type locality. Melipona concinnula Cockerell, 1919 is known only from a single specimen (the holotype) from Rio Mato, Caura District, Bolívar, Venezuela. However, it is possible that M. ogilviei is a junior synonym of this species [see comments in Schwarz (1932) and Camargo and Pedro (2007, 2008)]. Melipona apiformis (Buysson, in Buysson & Marshall, 1892) was originally described in the genus Trigona and later included in Melipona based on its description (Camargo and Pedro 2007, 2008) and it was recorded only in the type locality (Colonia Tovar, AM, Venezuela). Its true identity, however, remains unknown because the whereabouts of type material is unknown. Plebeia fraterna was described by Laroca and Rodríguez-Parilli (2009) from San Juan de Los Morros, Guárico, Venezuela, and is known only from there now. Other species described from Venezuela are Melipona indecisa Cockerell, 1919 from Lagunita de Aroa, Yaracuy (Camargo and Pedro 2007, 2008); Paratrigona permixta Camargo & Moure, 1994 from San Rafael, Cumanacoa, Sucre; Nannotrigona tristella Cockerell, 1922 and Trigona venezuelana Schwarz, 1948 both from Lagunita de Aroa, Yaracuy; Plebeia goeldiana (Friese, 1900) from Mérida; Scaptotrigona ochrotricha (Buysson, in Du Buysson & Marshall, 1892); and Melipona apiformis, described from Colonia Tovar, Aragua. Melipona fasciata cramptoni duidae (Schwarz, 1932), junior synonym of Melipona cramptoni Cockerell, 1920, was described from the Mt. Duida region, between La Esmeralda and Cerro Duida, Amazonas, near the Orinoco River. According to Camargo and Pedro (2007, 2008), it is possible that M. cramptoni is only a dark form of M. fulva. Geographic records of these species in Venezuela are listed in Table 4.1. Although Friese (1900) has included specimens of Melipona fasciata Latreille, 1811 from Venezuela in the type series of M. fuscipes Friese, 1900 (junior synonym of M. fasciata) this species is not present in the Venezuelan stingless bee fauna. Indeed, the type series of M. fuscipes was composed of different species and its identity was interpreted by Moure (1971) on the basis of specimens from central Mexico (Morelos). Melipona fasciata was also described based on specimens from Mexico (Veracruz) (see notes in Camargo and Pedro 2007, 2008). Some species are widely distributed in Venezuela, such as Frieseomelitta paupera (Fig. 4.1c), Melipona favosa, Partamona peckolti, Trigona amalthea, and T. guianae. Most of the species recorded in Venezuela (Table 4.1), however, seem to have a more restricted distribution. 4 Stingless Bees from Venezuela 4.4 83 Biogeographic Patterns of Venezuelan Meliponini Despite the taxonomic problems and restriction of studied material allied to the lack of consistent surveys in Venezuela, we can make a first attempt to relate the known geographical records of Venezuelan Meliponini with biogeographic patterns already recognized, including other stingless bees (Camargo and Pedro 2003; Camargo 2008; Camargo Chap. 2, this book). The Venezuelan fauna of stingless bees is quite diversified in terms of biogeographic origins, including predominantly elements from NAm (Amazon region, on north of the Negro and Amazonas rivers) and SWAm (a component delimited, on the north, by the alignment of the Uaupés/Negro rivers; on the south, by the Madeira/ Mamoré rivers; and on the west, by the Andean mountain range) components. Species from Central America (Choco-CA component) that reach Venezuela are Tetragona ziegleri, Trigona fulviventris, Trigona fuscipennis, Partamona peckolti, and Frieseomelitta paupera (Fig. 4.1c). However, the taxonomic status of some of these must be reviewed. Oxytrigona mellicolor is also from Chocó-CA component [as interpreted by Schwarz (1948), followed by Camargo and Pedro (2007, 2008)], although the holotype is from somewhere between Quito and the Napo River (Schwarz 1948), on the eastern side of the Andes; it can be found from Honduras to Esmeraldas, Ecuador, and in Venezuela, in Lagunita de Aroa, Aragua, and other states listed in Table 4.1 (Schwarz 1948; Camargo and Pedro 2007, 2008; Gonzalez and Roubik 2008). Nannotrigona perilampoides was only recorded in Venezuela in Guárico by Rodríguez-Parilli et al. (2010), but it was possibly misidentified considering this species is only known from Mexico to Panama (Camargo and Pedro 2007, 2008). The other two components, Atl (Atlantic area, from Bahia to Paraná, Brazil) and SEAm (area to the south of the Madeira/Amazonas rivers to northwestern Argentina), are apparently not represented in Venezuela except, perhaps, by Cephalotrigona capitata, from the SEAm, implying that Amazon River represents an important faunal divisor (geographic barrier) for stingless bees. Species with more restricted distribution patterns are apparently associated with areas of endemism related with Venezuelan terranes (Venezuela–Trinidad, Imeri, and others not named yet) included in the NAm biogeographic component. They are Trigona venzuelana, Partamona vitae, Paratrigona anduzei (Fig. 4.1a, b), Paratrigona pemixta, Plebeia fraterna, P. goeldiana, Scaptotrigona ochrotricha, Melipona concinnula, M. apiformis, M. indecisa (probably junior synonym of M. apiformis), M. ogilviei, M. trinitatis, and Nannotrigona tristella. Other species from the NAm component, but more widely distributed nevertheless, are Duckeola pavani, Partamona nigrior, Geotrigona subnigra, Lestrimelitta glaberrima, Melipona compressipes, M. interrupta, M. cramptoni, M. fulva, M. lateralis, M. paraensis, Paratrigona pannosa, Partamona auripennis, P. ferreirai, P. nigrior, and P. pearsoni. Melipona favosa is also widespread throughout Venezuela, Guianas, Trinidad, and Tobago, reaching Colombia (Magdalena), but its wider distribution can be a result of extensive beekeeping and transportation of hives. 84 S.R.M. Pedro and J.M.F. Camargo Some examples of species from the SWAm component that are represented in Venezuela are Lestrimelitta maracaia, Melipona crinita, M. illustris, Nannotrigona melanocera, N. schultzei, and Partamona epiphytophila. Many species present in Venezuela have wider distribution in South America occupying two or three of the large components and it is difficult to determine their biogeographic origins. Some examples are: NAm + SWAm: Aparatrigona impunctata, Ptilotrigona lurida, Trigona amalthea, T. amazonensis, T. branneri, T. cilipes [the material listed from Costa Rica and Panamá by Schwarz (1948):346 is probably another species], T. dallatorreana, T. guianae, T. pallens, and T. venezuelana. NAm + SWAm + SEAm: Partamon ailyae, Partamona vicina, Scaura latitarsis (different species included), Tetragona clavipes (different species included), Trigona hypogea (different species included), and T. truculenta. In summary, information on distribution patterns must be improved with the increase of surveys in the country and adequate taxonomic treatment of the material sampled, which will allow more precise interpretation of biogeographic patterns. Acknowledgements We are indebted to all researchers that kindly donate the bees collected in Venezuela to Collection Camargo—RPSP, especially to Prof. Patricia Vit who also invited JMF Camargo to carry out the expedition to Venezuela in March, 2008, with financial support from Programa de Formación de Personal e Intercambio Científico, Universidad de los Andes, Mérida, Venezuela. We are very grateful to Charles D. Michener (University of Kansas), David W. Roubik (Smithsonian Institution), and Patricia Vit (Universidad de Los Andes) for suggestions and corrections. References Bogdanov S, Vit P, Kilchenmann V. 1996. Sugar profiles and conductivity of stingless bee honeys from Venezuela. Apidologie 27:445–450. Camargo JMF. 2008. Biogeografia histórica dos Meliponini (Hymenoptera, Apidae, Apinae) da região Neotropical. pp. 13–26. In Vit P, ed. Abejas sin aguijón y valorización sensorial de su miel. APIBA-DIGECEX, Universidad de los Andes; Mérida, Venezuela. 148 pp. Camargo JMF, Moure JS. 1994. Meliponinae neotropicais: os gêneros Paratrigona Schwarz, 1938 e Aparatrigona Moure, 1951 (Hymenoptera, Apidae). Arquivos de Zoologia 32:33–109. Camargo JMF, Moure JS. 1996. Meliponini neotropicais: o gênero Geotrigona Moure, 1943 (Apinae, Apidae, Hymenoptera), com especial referência à filogenia e biogeografia. Arquivos de Zoologia 33:95–161. Camargo JMF, Pedro SRM. 2003. Meliponini neotropicais: o gênero Partamona Schwarz, 1939 (Hymenoptera, Apidae, Apinae) – bionomia e biogeografia. Revista Brasileira de Entomologia 47:311–372. Camargo JMF, Pedro SRM. 2004. Meliponini neotropicais: o gênero Ptilotrigona Moure (Hymenoptera, Apidae, Apinae). Revista Brasileira de Entomologia 48:353–377. Camargo JMF, Pedro SRM. 2007. Meliponini Lepeletier, 1836. pp. 272–578. In Moure JS, Urban D, Melo GAR, Eds. Catalogue of Bees (Hymenoptera, Apoidea) in the Neotropical Region. Sociedade Brasileira de Entomologia; Curitiba, Brasil. 1958 pp. Camargo JMF, Pedro SRM. 2008. Meliponini Lepeletier, 1836. In Moure JS, Urban D, Melo GAR, Eds. Catalogue of Bees (Hymenoptera, Apoidea) in the Neotropical Region – online version. Available at: http://www.moure.cria.org.br/catalogue. 4 Stingless Bees from Venezuela 85 Crane E. 1999. The world history of beekeeping and honey hunting. Routledge; New York, United States. 682 pp. Friese H. 1900. Neue Arten der bienengattungen Melipona Ill, und Trigona Jur. Természetrajzi füzetek 23:381–394. Gonzalez VH, Roubik DW. 2008. Espécies nuevas y filogenia de las abejas de fuego Oxytrigona (Hymenoptera: Apidae, Meliponini). Acta Zoológica Mexicana (n.s.) 24:43–71. Laroca S, Rodríguez-Parilli S. 2009. Descipción de uma nueva espécie de Plebeia de los Llanos Centrales de Venezuela (Anthophila, Meliponini). Acta Biológica Paranaense 37:211–215. Marchi P, Melo GAR. 2006. Revisão taxonômica das espécies brasileiras de abelhas do gênero Lestrimelitta Friese (Hymenoptera, Apidae, Meliponina). Revista Brasileira de Entomologia 50:6–30. Moure JS. 1971. Nota sobre algumas espécies duvidosas de Melipona. Hymenoptera – Apidae. Arquivos do Museu Nacional 54:193–201. Nogueira-Neto P. 1962. O início da apicultura no Brasil. Boletim de Agricultura. Secretaria da Agricultura do Estado de São Paulo, Diretoria de Publicidade Agrícola; São Paulo, Brasil. Offprint. 14 pp. Pedro SRM, Camargo JMF. 2003. Meliponini neotropicais: o gênero Partamona Schwarz, 1939 (Hymenoptera, Apidae). Revista Brasileira de Entomologia 47:1–117. Rivero Oramas R. 1972. Abejas criollas sin aguijón. Monte Ávila Editores; Caracas, Venezuela. 112 pp. Rodríguez-Malaver AJ, Rasmussen C, Gutiérrez MG, Gil F, Nieves B, Vit P. 2009. Properties of honey from ten species of Peruvian stingless bees. Natural Product Communications 4:1221–1226. Rodríguez-Parilli S, Velázquez M, Laroca S. 2010. Análisis de la estructura biológica de la comunidad de abejas (Hymenoptera, Apoidea) em um bosque seco tropical del Estado de Guárico (Venezuela). Acta Biológica Paranaense 39:26–60. Schwarz HF. 1932. The genus Melipona. The type genus of the Meliponidae or stingless bees. Bulletin of the American Museum of Natural History 63:231–460, plates I-X. Schwarz HF. 1948. Stingless bees (Meliponidae) of the Western Hemisphere. Bulletin of the American Museum of Natural History 90: i-xvii + 1–546. Tomás-Barberán F, García-Viguera C, Vit-Olivier P, Ferreres F, Tomás-Lorente F. 1993. Phytochemical evidence for the botanical origin of tropical propolis from Venezuela. Phytochemistry 34:191–196. Vit P. 2005. Melissopalynology Venezuela. APIBA – CDCHT, Universidad de los Andes; Mérida, Venezuela. 205 pp. Vit P. 2008. Colaboración entre Venezuela y Brazil: La diversidad de abejas sin aguijón neotropicales. Investigación, Julio – Diciembre, 18:28–29. Vit P. 2009. Caracterización físicoquímica de mieles de abejas sin aguijón (Meliponini) de Venezuela. Revista Del Insituto Nacional de Higiene “Rafael Rangel” 40:7–12. Vit Olivier P. 1992. Caracterización de mieles de abejas sin aguijón producidas en Venezuela. Trabajo presentado como requisito para optar a la categoria de Profesor Asociado en la Universidad de Los Andes, Facultad de Farmacia, Universidad de Los Andes; Mérida, Venezuela. 125 pp. Vit P, Pulcini P. 1996. Diastase and invertase activities in Meliponini and Trigonini honeys from Venezuela. Journal of Apicultural Research 35:57–62. Vit P, Ricciardelli d’Albore G. 1994a. Palinología comparada en miel y polen de abejas sin aguijón (Hymenoptera: Apidae: Meliponinae) de Venezuela. pp.121–132. Trabajos de Palinología Básica y Aplicada, X Simposio de Palinología (A.P.L.E.), Universitat de Valencia; Valencia, Spain. 313 pp. Vit P, Ricciardelli d’Albore G. 1994b. Melissopalynology for stingless bees (Apidae: Meliponinae) from Venezuela. Journal of Apicultural Research 33:145–154. Vit P, Tomás-Barberán FA. 1998. Flavonoids in Meliponinae honeys from Venezuela related to their botanical, geographical and entomological origin to assess their putative anticataract activity. Zeitschrift für Lebensmittel-Untersuchung und -Forschung 206:288–293. 86 S.R.M. Pedro and J.M.F. Camargo Vit P, Tomás-Barberán FT, García-Viguera C, Ferreres F, Camargo J. 1993. Caracterización de propóleos venezolanos. Revista del Instituto Nacional de Higiene “Rafael Rangel” 24:38–46. Vit P, Bogdanov S, Kilchenmann V. 1994. Composition of Venezuelan honeys from stingless bees (Apidae: Meliponinae) and Apis mellifera L. Apidologie 25:278–288. Vit P, Soler C, Tomás-Barberán FA. 1997. Profiles of phenolic compounds of Apis mellifera and Melipona spp. honeys from Venezuela. Zeitschrift für Lebensmittel-Untersuchung und-Forschung 204:43–47. Vit P, Fernandez-Maeso MC, Ortiz-Valbuena A. 1998a. Potential use of the three frequently occurring sugars in honey to predict stingless bee entomological origin. Journal of Applied Entomology 122:5–8. Vit P, Persano Oddo L, Marano ML, Salas de Mejias E. 1998b. Venezuelan stingless bee honeys characterized by multivariate analysis of physicochemical properties. Apidologie 29: 377–389. Vit P, Deliza R, Pérez A. 2011. How a Huottuja (Piaroa) community perceives genuine and false honey from the Venezuelan Amazon, by free-choice profile sensory method. Revista Brasileira de Farmacognosia 21:786–792. Chapter 5 Stingless Bees (Hymenoptera: Apoidea: Meliponini) of French Guiana Alain Pauly, Silvia R.M. Pedro, Claus Rasmussen, and David W. Roubik 5.1 Introduction Stingless bees (Hymenoptera: Apoidea; Meliponini) are found worldwide in tropical and subtropical regions (Michener 2007), but are most diverse and numerous in tropical South and Central America, where they often are the most commonly encountered bees. The stingless bees have long played an important role for inhabitants of these areas as the suppliers of excellent honey (Schwarz 1948; Nogueira-Neto 1997; Stearman et al. 2008; Guerrini et al. 2009; Rodríguez-Malaver et al. 2009) and crop pollinators (Heard 1999; Slaa et al. 2006). They are also the focus for scientific research on sociality and colony evolution (e.g., Nieh 2004; Rasmussen and Camargo 2008; Lichtenberg et al. 2010). Further comparative studies are encouraged by a robust phylogeny of the entire group (Rasmussen and Cameron 2007, 2010; Ramírez et al. 2010). The stingless bees have also been of concern for conservation biologists, because most nest in living trees and therefore they may be more susceptible to habitat disturbance than other bees and insects (Brown and Albrecht 2001; Kerr et al. 2001; Samejima et al. 2004; Roubik 2006). A. Pauly (*) Department of Entomology, Royal Belgian Institute of Natural Sciences, Rue Vautier 29, B-1000, Brussels, Belgium e-mail: alain.pauly@brutele.be S.R.M. Pedro Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, CEP 14040-901, Ribeirão Preto, SP, Brazil C. Rasmussen Department of Bioscience, Aarhus University, Ny Munkegade 114-116, DK-8000, Aarhus C, Denmark D.W. Roubik Smithsonian Tropical Research Institute, Ancón, Balboa, Republic of Panamá MRC 0580-12, Unit 9100, Box 0948, DPO AA, 34002-9998, USA P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_5, © Springer Science+Business Media New York 2013 87 88 A. Pauly et al. The lowland forest of French Guiana contains extensive and undisturbed habitats, and the smallest human population density on earth. The land area is little more than 80,000 km2. No high mountains occur, and the rainfall varies between 4,000 mm annually in the Cayenne and Kaw mountain area, gradually diminishing to half this amount to the south and west. The forests are of white sand soils or of laterites, as well as the extensive mangrove of the coastal and estuarine areas. In order to conduct research in a given area, it is often essential to have some background on the species encountered in that area. An introduction to the local species is often provided in the form of checklists for families or orders of insects or for smaller units (e.g., genera) in taxonomic revisions or species descriptions. However, no complete ecological perspective is given in such lists, because relative abundances or phenologies of the different species are unknown. In the case of stingless bees, the bees are ecologically active every day and the colonies are perennial (Roubik 1989). For basic information, stingless bee researchers have access to three checklists. Camargo and Pedro (2007, 2008a) and Rasmussen (2008) in their catalogues of stingless bees listed all species from the Neotropical and Indo-Malayan/ Australasian region, respectively, including bibliographic references, synonymies, and distribution records based on their literature surveys. Eardley (2004) in a taxonomic revision of the Afrotropical stingless bees provided keys, synonymies, and distribution records for all stingless bees of that region, excluding Madagascar. Much of the information was also included in the later catalogue to the Afrotropical bees, including Madagascar (Eardley and Urban 2010). Distribution records in all three catalogues (Camargo and Pedro 2007, 2008a; Rasmussen 2008; Eardley and Urban 2010) were largely based on the literature, and given the sporadic collection effort of these regions, it is not surprising that several countries are poorly sampled, incompletely known, or that even well-known regions can provide new records of species. We surveyed the literature and a comprehensive collection of stingless bees from French Guiana to compile a list of all known taxa from that country. Whereas the stingless bee fauna of French Guiana was first studied extensively by Roubik (1979, 1980), century old scattered records (Dominique 1898; Rasmussen et al. 2007) were the first to document the fauna from the country, some even dating to the time of Linnaeus and his students, namely, JC Fabricius who named seven new stingless bee taxa likely collected in French Guiana (primarily Cayenne) and Suriname (Moure 1960; Papavero 1971). 5.1.1 Data Compiled from Preserved Material from Four Collections Data were compiled from the following sources (collecting sites on Fig. 5.1): 1. Material preserved at the Royal Belgian Institute of Natural Sciences (RBINS), collected by Société Entomologique Antilles Guyane (SEAG) (Brulé et al. 2011), and identified by Silvia RM Pedro: Saut Pararé (4°02 N 52°41¢ W) à Nouragues, Montagne des Chevaux (4°43¢ N 52°26¢ W) à Roura (RN2 PK22), Iracoubo (5°29¢ N 53°13¢ W). 5 Stingless Bees (Hymenoptera: Apoidea: Meliponini) of French Guiana 89 Fig. 5.1 Map of collecting sites in French Guiana (extracted from www.atlashymenoptera.net). (1) Saint Laurent du Maroni, (2) Yalimapo, (3) Iracoubo, (4) Sinnamary, (5) Soumourou, (6) Kourou, (7) Degrad Saramaca, (8) Cayenne, (9) Roura Montagne des Chevaux, (10) Relais Patawa, (11) Kaw, (12) Regina, (13) Saut Pararé, (14) Petit Saut, (15) Mt Galbao, (16) Saul, (17) Saint Georges de l’Oyapock 2. Material preserved at the Royal Belgian Institute of Natural Sciences [RBINS] and identified by Claus Rasmussen: Kaw, Relais Patawa (4°32¢ N 52°09 W) leg. Y Braet, leg. J Cerda; Kourou (5°09¢ N 52°39¢ W) leg. Y Braet; Maroni (5°30¢ N 54°02¢ W) (= Saint Laurent du Maroni) leg. Y Braet; Saül (3°37¢ N 53°12¢ W) leg. Y Braet; Piste Soumourou (5°09¢ N 52°44¢ W) leg. D Faure; Sinnamary, Pointe Combi (5°19¢ N 52°57¢ W) leg. P Cerdan; Sinnamary, barrage de Petit Saut (4°04¢ N 53°03¢ W) leg. P Cerdan; Yalimapo, Les Hattes (5°44¢ N 53°57¢ W), Ecloserie du WWF, leg. R Babin. 3. Material preserved at the [DWR] Collection, Smithsonian Tropical Research Institute, Panamá, collected and identified by David W. Roubik, JMF Camargo, and JS Moure: Kourou-Sinnamary area, in addition to Cayenne, St. Laurent and St. George areas (1976–2009). 4. Material preserved at Division of Entomology, University of Kansas Natural History Museum, Lawrence, Kansas, USA (SEMK). Various localities collected by RW Brooks and identified by JMF Camargo. 90 A. Pauly et al. 5. Material preserved at Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil (RPSP), identified by JMF Camargo and SRM Pedro. Duplicates of collections a, c, and d are preserved in this institution. In addition we included literature records (Camargo and Pedro 2005, 2008b, 2009; Moure 1989; Moure and Camargo 1982; Moure et al. 1988; Oliveira and Marchi 2005; Roubik 1980, 1990; Smith Pardo and Engel 2001) and a record of Celetrigona manauara collected by R. Snyder and preserved in American Museum of Natural History (AMNH). 5.1.2 A List of Stingless Bee Species Found in French Guiana Aparatrigona impunctata (Ducke 1916) [DWR, RBINS, RPSP, SEMK] Camargoia camargoi Moure 1989 [DWR, RBINS, RPSP, SEMK] Celetrigona manauara Camargo and Pedro 2009) [AMNH] Cephalotrigona capitata (Smith 1854) [DWR, RBINS, RPSP, SEMK] Dolichotrigona longitarsis (Ducke 1916) [SEMK] Duckeola ghilianii (Spinola 1853) [DWR, RPSP] Duckeola pavani (Moure 1963) [DWR, RPSP] Frieseomelitta flavicornis (Fabricius 1798) [DWR, RBINS, RPSP, SEMK] (=Tetragona savannensis (Roubik 1980)) Frieseomelitta portoi (Friese 1900) [DWR, RBINS, RPSP] Frieseomelitta sp. A aff. varia (Lepeletier 1836) [RBINS] Frieseomelitta sp. B [RBINS] Frieseomelitta sp. C [RBINS] Lestrimelitta glaberrima Oliveira and Marchi 2005 [DWR, RBINS, RPSP] Lestrimelitta guyanensis Roubik 1980 [DWR, RBINS, RPSP] Lestrimelitta monodonta Camargo and Moure 1989 [RBINS] Leurotrigona pusilla Moure and Camargo 1988 in Moure et al. 1988 [DWR, RPSP] Melipona (Eomelipona) bradleyi Schwarz 1932 [RBINS] Melipona (Eomelipona) ogilviei Schwarz 1932 [DWR, RBINS, RPSP] Melipona (Eomelipona) puncticollis Friese 1902 [DWR, RPSP] Melipona (Melikerria) compressipes Fabricius 1804 [DWR] Melipona (Melikerria) interrupta Latreille 1811 [DWR, RBINS, RPSP] Melipona (Melipona) favosa Fabricius 1798 [DWR, RBINS, RPSP] Melipona (Michmelia) captiosa Moure 1962 [DWR, RBINS] Melipona (Michmelia) fuliginosa Lepeletier 1836 [DWR] Melipona (Michmelia) fulva Lepeletier 1836 [DWR, RBINS, RPSP] Melipona (Michmelia) lateralis Erichson 1848 [DWR, RBINS, RPSP, SEMK] Melipona (Michmelia) melanoventer Schwarz 1932 [DWR] Melipona (Michmelia) paraensis Ducke 1916 [DWR, RBINS] Melipona (Michmelia) sp. [RBINS] Nannotrigona punctata (Smith 1854) [DWR, RBINS, RPSP, SEMK] Nannotrigona schultzei (Friese 1901) [DWR, RPSP, SEMK] 5 Stingless Bees (Hymenoptera: Apoidea: Meliponini) of French Guiana 91 Nogueirapis minor (Moure and Camargo 1982) [DWR, RBINS, RPSP, SEMK] Oxytrigona obscura Friese 1900 [DWR, RBINS, RPSP, SEMK] Paratrigona femoralis Camargo and Moure 1994 [DWR, RBINS, RPSP, SEMK] Paratrigona pannosa Moure 1989 [DWR, RBINS, RPSP, SEMK] Partamona auripennis Pedro and Camargo 2003 [DWR, RBINS, SEMK] Partamona ferreirai Pedro and Camargo 2003 [DWR, RBINS] Partamona mourei Camargo 1980 [DWR, RBINS] Partamona pearsoni Schwarz 1938 [DWR, RBINS, RPSP] Partamona testacea (Klug 1807) [DWR, RBINS, RPSP, SEMK] Partamona vicina Camargo 1980 [DWR, RBINS, RPSP, SEMK] Plebeia minima (Gribodo 1893) [DWR, RBINS] Plebeia mosquito (Smith 1863) [RBINS] Plebeia sp. 1 [DWR, RBINS] Plebeia sp. 2 [DWR, RBINS] Plebeia sp. 3 [DWR, RBINS] Plebeia sp. 4 [DWR, RBINS] Plebeia sp. 5 [DWR, RBINS] Ptilotrigona lurida (Smith 1854) (Fig. 5.2d) [DWR, RBINS, RPSP, SEMK] Scaptotrigona cf. depilis (Moure 1942) [RBINS] Scaptotrigona fulvicutis (Moure 1964) [DWR, RBINS, RPSP] Scaptotrigona sp. 1 (gr. tubiba Smith 1863) [RBINS] Scaptotrigona sp. 2 (gr. tubiba) [RBINS] Scaura latitarsis (Friese 1900) [DWR, RBINS, SEMK] Scaura longula (Lepeletier 1836) [DWR, RPSP] Scaura tenuis (Ducke 1916) [DWR, RBINS, RPSP] Tetragona beebei (Schwarz 1938) [DWR, RBINS, RPSP] Tetragona clavipes (Fabricius 1804) [DWR, RBINS, RPSP, SEMK] Tetragona dorsalis (Smith 1854) [DWR, RPSP, SEMK] Tetragona handlirschii (Friese 1900) [DWR, RBINS, RPSP, SEMK] Tetragona kaieteurensis (Schwarz 1938) [RBINS, SEMK] Tetragona sp. [RBINS] Tetragonisca angustula (Latreille 1811) [DWR, RPSP, SEMK] Trigona branneri Cockerell 1912 [DWR, RBINS, RPSP, SEMK] Trigona cilipes (Fabricius 1804) (Fig. 5.2c) [DWR, RBINS, RPSP, SEMK] [= T. mazucatoi Almeida 1992] Trigona crassipes (Fabricius 1793) [DWR, RBINS, RPSP, SEMK] Trigona sp. 1 (gr. crassipes (Fabricius 1793)) [DWR, RPSP] Trigona sp. 2 (gr. crassipes (Fabricius 1793)) [RPSP, SEMK] Trigona sp. 3 (gr. crassipes (Fabricius 1793)) [RBINS] Trigona sp. 1 (gr. fuscipennis Friese 1900) [DWR, RBINS] Trigona sp. 2 (gr. fuscipennis Friese 1900) [DWR, RBINS] Trigona sp. 3 (gr. fuscipennis Friese 1900) [RBINS] Trigona guianae Cockerell 1910 [DWR, RBINS, RPSP, SEMK] Trigona pallens (Fabricius 1798) (Fig. 5.2a, b) [DWR, RBINS, RPSP, SEMK] Trigona permodica Almeida 1995 [DWR, RBINS, SEMK] 92 A. Pauly et al. Trigona sp. (gr. recursa Smith 1863) [RPSP, SEMK] Trigona sesquipedalis Almeida 1984 [DWR, RPSP] Trigona williana Friese 1900 [DWR, RBINS, RPSP, SEMK] Trigonisca dobzhanskyi (Moure 1950) [DWR, RPSP] Trigonisca sp. [DWR, RBINS] Some common species of stingless bees from French Guiana are illustrated in Fig. 5.2. Fig. 5.2 Some Meliponini of French Guiana. (a) Ptilotrigona lurida, (b) Trigona cilipes, (c) Trigona pallens (at nest entrance), (d) Trigona pallens. Photos: Stéphane Brulé Table 5.1 presents a list of Neotropical genera of stingless bees, including total number of described species and distribution. Table 5.1 A list of Neotropical genera of stingless bees Number French Genus of species Guiana Notes on distribution Aparatrigona Moure 1951 Camargoia Moure 1989 Celetrigona Moure 1950 Cephalotrigona Schwarz 1940 Dolichotrigona Moure 1950 Duckeola Moure 1944 Friesella Moure 1946 Frieseomelitta Ihering 1912 Geotrigona Moure 1943 2 3 4 5 10 2 1 16 21 1 1 1 1 1 2 0 5 0 NW Brazil to Panamá E and Central Brazil to French Guiana Bolivia, Peru, Brazil to Guianas Argentina and S Brazil to Trinidad Peru to Mexico and W and N Brazil Bolivia and Peru to Guianas Only found in SE Brazil SE Brazil to Mexico A widespread genus, from Argentina to Mexico, but so far not reported from French Guiana (continued) 5 Stingless Bees (Hymenoptera: Apoidea: Meliponini) of French Guiana 93 Table 5.1 (continued) Number of species French Guiana Notes on distribution 21 4 71a 3 1 13 1 0 1 0 Nannotrigona Cockerell 1922 Nogueirapis Moure 1953 10 3 2 1 Oxytrigona Cockerell 1917 Parapartamona Schwarz 1948 11 7 1 0 Paratrigona Schwarz 1938 Paratrigonoides Camargo and Roubik 2005 Partamona Schwarz 1939 30 1 2 0 32 6 Plebeia Schwarz 1938 40 7 3 1 Scaptotrigona Moure 1942 22 4 Scaura Schwarz 1938 Schwarziana Moure 1943 5 2 3 0 Schwarzula Moure 1946 2 0 Tetragona Lepeletier and Serville 1828 Tetragonisca Moure 1946 13 6 4 1 Genus Lestrimelitta Friese 1903 Leurotrigona Moure 1950 Melipona Illiger 1806 Including subgenera Eomelipona Moure 1992, Melikerria Moure 1992, Melipona, and Michmelia Moure 1975 Meliwillea Roubik et al. 1997 Mourella Schwarz 1946 Ptilotrigona Moure 1951 Trichotrigona Camargo and Moure 1983 Trigona Jurine 1807 1 0 32 14 Trigonisca Moure 1950 25 2 S Brazil to Mexico S Brazil to Guianas Widely distributed, from S Brazil and Argentina to Mexico Only found in the higher parts of Costa Rica and Panama Only found in S Brazil and south to Argentina, Paraguay, and Uruguay S Brazil and Argentina to Mexico Amazon region and W Andes from Ecuador to Costa Rica S Brazil (SC) and Paraguay to Mexico Endemic to the Andean region (Peru to Colombia) at altitudes between app. 1,400 and 3,400 m Widely distributed, Argentina to Mexico Narrow endemic from Colombia (Antioquia, Bolívar) Widely distributed, S Brazil (SC) to Mexico Widely distributed, Uruguay and Argentina to Mexico Amazon region and W Andes from Ecuador to Costa Rica Widely distributed, S Brazil and Argentina to Mexico SE Brazil to Mexico Restricted to SE and S Brazil, Paraguai, and Argentina SE Brazil to Amazon region (Bolivia, Peru Ecuador, and Brazil) Widely distributed, Uruguay to Mexico Widely distributed, Argentina, Paraguai, S Brazil to Mexico Narrow endemic from Brazil (Amazonas) Widely distributed, Argentina, Paraguai, S Brazil to Mexico Widely distributed, S Brazil to Mexico Including total number of described species, presence (number of described/number of undescribed species in French Guiana), and notes on the distribution of the genera (based on Camargo and Pedro 2007; 2008a, b) a Or 77, if subspecies are involved 94 5.1.3 A. Pauly et al. A Unique and Intact Stingless Bee Fauna We report from our survey all of the genera of stingless bees otherwise expected to be found in French Guiana (Table 5.1), with the exception of Geotrigona, a widespread genus of exclusively ground nesting bees. Geotrigona can be difficult to locate as they have a shy nest entrance defense, but Geotrigona subnigra (Schwarz 1940) was described from Guyana and this and other species of Geotrigona could be found with additional collecting in the country. At least two elements are outstanding in this small tropical country—the large number of Frieseomelitta and Duckeola in the white sand forests and the large number of Melipona species, especially in the interior of the country. We surmise that Trichotrigona inhabits the southern portion of French Guiana, due to the high number of potential host Frieseomelitta—which is the host to parasitic Trichotrigona extranea (Camargo and Moure 1983) as neighboring inquilines and thief—apparently never foraging outside its nest (Camargo and Pedro 2007). Trigona amalthea (Olivier 1789) was described from Cayenne (Olivier 1789); however, it is not included in the present list because this species has not been collected anywhere near Cayenne or in other parts of French Guiana, despite the intensive surveys in the region during 30 years (DWR). The only record of this species is the lectotype in Kiel collection, presently in the Zoological Museum, University of Copenhagen, Copenhagen Denmark (Camargo and Pedro 2007, 2008a) collected before 1789. It is possible that the type locality is an error; local extinction is another possibility. The total number of species recorded here from French Guiana is 80 and is the highest number for any of the countries in the Guiana Shield probably in part due to incomplete sampling of the region, and certainly a much lower number than the fauna from better known areas, such as Brazil (178 spp., excluding those undescribed) and Peru (175 spp. including those undescribed, C. Rasmussen, unpublished). There were still no European bees (Apis mellifera (Linnaeus 1758)) in French Guiana and probably not in other South American countries (Bolivia, Ecuador, Venezuela, Guyana, Peru, and Suriname) until the late 1800s (Crane 1999). Fougères Marquis de (1902) reported that in French Guiana most honey was harvested either from natural nests or from hives of stingless bees, but there were apiaries of modern hives, and in Cayenne Mme Cablat’s 40 hives gave a colony average of about 40 kg of honey a year. We have no knowledge of stingless bee keeping for honey production. One of us (D.W. Roubik) observed that a beekeeper in Sinnamary, the largest in French Guiana (40 hives), occasionally harvests honey from Melipona favosa and Frieseomelitta flavicornis—two common savanna-forest edge bees—which he has in small hives at his home. The Africanized honey bee arrived in French Guiana during 1975, and this was the earliest date at which there were honey bees living in the wild (D.W. Roubik, personal observation). The great number of meliponine species recorded from a relatively small area like French Guiana gives an idea of the many sources of honey that must be available there, even though almost no use of them is recorded. 5 Stingless Bees (Hymenoptera: Apoidea: Meliponini) of French Guiana 95 Acknowledgments A. Pauly thanks Stéphane Brulé, Pierre-Henri Dalens, Eddy Poirier, Serge Fernandez, and Marc Tussac (all SEAG), Yves Braet, Jean-Aimé Cerda, and Philippe Cerdan for providing material of bees collected in French Guiana and preserved in RBINS. We thank also Yvan Barbier (University of Mons, Belgium) for the distribution map of collecting sites in French Guiana and Patricia Vit (Universidad de Los Andes, Venezuela) for constructive comments on the manuscript. References Brown JC, Albrecht C. 2001. The effect of tropical deforestation on stingless bees of the genus Melipona (Insecta: Hymenoptera: Apidae: Meliponini) in central Rondonia, Brazil. Journal of Biogeography 28:623–634. Brulé S, Touroult J, Dalens PH, eds. 2011. Résultats de l’inventaire entomologique du site de Saut Pararé, réserve des Nouragues (Guyane), 2009–2010. Rapport de la Société entomologique Antilles-Guyane, SEAG, ONF. [Cayenne, French Guiana]. 120 pp. + annexes. Camargo JMF, Pedro SRM. 2005. Meliponini Neotropicais: o gênero Dolichotrigona Moure (Hymenoptera, Apidae, Apinae). Revista Brasileira de Entomologia 49:69–92. Camargo JMF, Pedro SRM. 2007. Meliponini Lepeletier 1836. pp. 272–578. In Moure JS, Urban D, Melo GAR, eds. Catalogue of bees (Hymenoptera, Apoidea) in the Neotropical region. Sociedade Brasileira de Entomologia; Curitiba, Brazil. 1958 pp. Camargo JMF, Pedro SRM. 2008a. Meliponini Lepeletier, 1836. In Moure JS, Urban D, Melo, GAR, eds. Catalogue of Bees (Hymenoptera, Apoidea) in the Neotropical Region - online version. Available at: http://www.moure.cria.org.br/catalogue. Camargo JMF, Pedro SRM. 2008b. Revisão das espécies de Melipona do grupo fuliginosa (Hymenoptera, Apoidea, Apidae, Meliponini). Revista Brasileira de Entomologia 52:411–427. Camargo JMF, Pedro SRM. 2009. Neotropical Meliponini: the genus Celetrigona Moure (Hymenoptera: Apidae, Apinae). Zootaxa 2155:37–54. Crane E. 1999. The world history of beekeeping and honey h unting. Duckworth; London, UK. 682 pp. Dominique J. 1898. Coup d’oeil sur les mellifères sud-américains du muséum de Nantes. Bulletin de la Société des Sciences Naturelles de l’Ouest de la France [Nantes] 8:57–65. Eardley CD. 2004. 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Behavioral suites mediate group-level foraging dynamics in communities of tropical stingless bees. Insectes Sociaux 57:105–113. Michener CD. 2007. The bees of the world, second edition. Johns Hopkins University Press; Baltimore, USA. xvi+953 pp Moure JS. 1960. Notes on the types of the neotropical bees described by Fabricius (Hymenoptera: Apoidea). Studia Entomologica 3:97–160. 96 A. Pauly et al. Moure JS. 1989. Camargoia, un novo gênero neotropical de Meliponinae (Hymenoptera Apoidea). Boletim do Museu Paraense Emilio Goeldi, Serie. Zoologia 5:71–78. Moure JS, Camargo JMF. 1982. Partamona (Nogueirapis) minor, nova species de Meliponinae (Hymenoptera: Apidae) do Amazonas e notas sobre Plebeia variicolor (Ducke). Boletim do Museu Paraense Emilio Goeldi, Nova Serie. Zoologia, Belem 120:1–10. Moure JS, Camargo JMF, Garcia MVB. 1988. A new species of Leurotrigona (Hymenoptera, Apidae, Meliponinae). Boletim do Museu Paraense Emilio Goeldi, Nova Serie. 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Properties of honey from ten species of peruvian stingless bees. Natural Product Communications 4:1221–1226. Roubik DW. 1979. Nest and colony characteristics of stingless bees from French Guiana (Hymenoptera: Apidae). Journal of the Kansas entomological Society 52:443–470. Roubik DW. 1980. New species of Trigona and cleptobiotic Lestrimelitta from French Guyana (Hymenoptera: Apidae). Revista de Biologia Tropical 28:263–269. Roubik DW. 1989. Ecology and natural history of tropical bees. Cambridge University Press, New York, USA. 514 pp. Roubik DW. 1990. Niche Preemption in Tropical Bee Communities: A Comparison of Neotropical and Malesian Faunas. pp. 245–258. In Sakagami SF, Ohgushi R, Roubik DW, eds. Natural History of Social Wasps and Bees in Equatorial Sumatra: Hokkaido University Press, Sapporo, Japan. 274 pp. Roubik DW. 2006. Stingless bee nesting biology. Apidologie 37:124–143 Samejima H, Marzuki M, Nagamitsu T, Nakasizuka T. 2004. The effects of human disturbance on a stingless bee community in a tropical rainforest. Biological Conservation 120:577–587. Schwarz HF. 1948. Stingless bees (Meliponidae) of the western hemisphere. Lestrimelitta and the following subgenera of Trigona: Trigona, Paratrigona, Schwarziana, Parapartamona, Cephalotrigona, Oxytrigona, Scaura, and Mourella. Bulletin of the American Museum of Natural History 90:1–546. 5 Stingless Bees (Hymenoptera: Apoidea: Meliponini) of French Guiana 97 Slaa EJ, Chaves LAS, Malagodi-Braga KS, Hofstede FE. 2006. Stingless bees in applied pollination: practice and perspectives. Apidologie 37:293–315. Smith Pardo AH, Engel MS. 2001. Distribution records for Trigona subgenus Duckeola outside of Brazil. Journal of the Kansas Entomological Society 74:115–117. Stearman AM, Stierlin E, Sigman ME, Roubik DW, Dorrien D. 2008. Stradivarius in the jungle: traditional knowledge and the use of “black beeswax” among the Yuquí of the Bolivian Amazon. Human Ecology 36:149–159. Chapter 6 Stingless Bees of Guatemala Carmen Lucía Yurrita Obiols and Mabel Vásquez 6.1 Introduction In many areas of their tropical distribution, the meliponines are the most common bees and hence are considered to play an important role as pollinators of native and crop vegetation (Slaa et al. 2006). This fact has been taken advantage of by local human populations, who have learned to harvest the honey (Villanueva et al. 2005; Posey 1982). In Guatemala, as in other Central American countries, the inhabitants of some regions keep a few of the stingless bee species in a traditional way and use the honey and the pollen as a medicine and food source. However, despite their importance these and other bees are at risk due to a combination of factors, including deforestation and presumably competition with nonnative species (Villanueva et al. 2005). In the case of the stingless bees destruction of colonies to extract honey and pollen represents an additional threat. In this chapter we present an overview of the stingless bee species native to Guatemala, the species richness of the group, their distribution in the country, floral resources visited, stingless bee beekeeping activity, and uses of stingless bee-derived products, particularly honey. C.L. Yurrita Obiols (*) • M. Vásquez Unidad de Conocimiento, Uso y Valoración de la Biodiversidad. Centro de Estudios Conservacionistas, Universidad de San Carlos de Guatemala, Avenida Reforma 0-63 zona 10, Guatemala 01010, Guatemala e-mail: clyurrita@gmail.com P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_6, © Springer Science+Business Media New York 2013 99 100 6.2 C.L. Yurrita Obiols and M. Vásquez Taxonomy and Distribution of Stingless Bees in Guatemala The bee family Apidae includes the only two groups of highly eusocial corbiculate bees: the tribes Apini and Meliponini. These two tribes are characterized by the presence of a pollen carrying structure on the hind legs called “corbicula.” This feature is shared with other corbiculate bees of the same family: the primitively eusocial bumble bees (Bombini) and the mostly solitary orchid bees (Euglossini). From a behavioral point of view Meliponini, like Apini, differ from other eusocial bees in that they form permanent colonies in which queens and workers are morphologically very different, the queen never forages, and neither the queen nor the workers can establish colonies by themselves (Michener 2007). The Meliponini exhibit a worldwide tropical and subtropical distribution and are the most diverse group of the corbiculate bees, with several hundred species (Rasmussen and Cameron 2010). Different classifications of the group have been proposed. In one classification system, a few genera and many subgenera are recognized, in order to emphasize the relationships between the groups (Michener 2007). In the other classification system many subgenera are elevated to the generic level to stress the full taxonomic diversity of the tribe (Moure 1961; Rasmussen and Cameron 2007, 2010; Camargo and Pedro 2008). Here, we use the classification system proposed by Moure (1961) and Camargo and Pedro (2008). In this system the entire tribe consists of at least 59 genera (Moure 1961; Camargo and Pedro 2008; Rasmussen and Cameron 2007). The greatest diversity of stingless bees is found in the Tropical America where 33 genera have been recognized that include approximately 400 species (Camargo and Pedro 2008). Here we present an updated list of the stingless bees of Guatemala that has been prepared using the identification key for the Meliponini of Mexico (Ayala 1999) and the specimens included in the entomological Guatemalan Native Bee Collection “Colección de Abejas Nativas de Guatemala” (CANG), of the Biodiversity Research Unit at the Conservation Studies Center (CECON) of the University of San Carlos of Guatemala. In Guatemala the diversity of Apoidea is estimated as at least 500 species (Enríquez et al. 2012), belonging to the families Andrenidae, Apidae, Colletidae, Halictidae, and Megachilidae. Of these the family Apidae has the highest diversity with 227 reported species (Enríquez et al. 2012). Currently, 33 stingless bee species are included in the collection (Table 6.1). Even though this number may increase with additional taxonomic and collecting work, it is not expected to exceed either the Costa Rican richness (50 species, Ortiz 1998) nor the Mexican one (46 species, Ayala 1999). Indeed a bibliographic survey produced a list of approximately 40 species of meliponines already reported for the country (Enríquez et al. 2012), which covers records from the literature (Camargo and Pedro 2008) like Paratrigona opaca (Cockerell, 1917), Geotrigona lutzi Camargo & Moure, 1996, Geotrigona terricola Camargo & Moure, 1996 and Scaptotrigona wheeleri (Cockerell, 1913), as well as material from other collections that was not included here. 6 Stingless Bees of Guatemala 101 Table 6.1 List of the Guatemalan stingless bees in the native bee collection (CANG). Departments (geopolitical division), number of collecting sites, and altitudinal ranges where they have been collected (from CANG database) No. Stingless bee species Departments of occurrence No. of sites Altitudinal ranges of collecting 1 Cephalotrigona zexmeniae (Cockerell, 1912) AV, CHIQ, SR 4 0–500, 501–1,000, 1,001–1,500 2 Dolichotrigona schulthessi (Friese, 1900) AV, QUE, REU, SM 7 0–500, 501–1,000, 1,001–1,500 3 Frieseomelitta nigra (Cresson, 1878) PR 3 0–500 4 Geotrigona acapulconis (Strand, 1919) G, SR 2 1,001–1,500 5 Lestrimelitta niitkib Ayala, 1999 AV, G, PR 5 0–500, 1,001–1,500, 1,501–2,000 6 Melipona beecheii Bennett, 1831 AV, BV, CHIQ, QUI, PE, ESC, 36 0–500, 501–1,000, 1,001–1,500, 1,501–2,000 G, I, JUT, REU, SR, SOL 7 Melipona solani Cockerell, 1912 AV, I, QUI, PE, HUE, QUE, REU, SM 19 0–500, 501–1,000, 1,001–1,500 8 Melipona yucatanica Camargo, HUE, JUT, SAC, SR 5 501–1,000 Moure & Roubik, 1988 9 Nannotrigona perilampoides (Cresson, 1878) AV, BV, PE, G, I, JUT, SR, ZAC 16 0–500, 501–1,000, 1,001–1,500, 1,501–2,000 10 Oxytrigona mediorufa (Cockerell, 1913) CHIM, I, QUE, SUCH 4 501–1,000, 1,001–1,500 11 Paratrigona guatemalensis (Schwarz, 1938) AV, SR 4 0–500, 1,001–1,500 12 Partamona bilineata (Say, 1837) AV, BV, CHIQ, G, HUE, I, JUT, JAL, 35 0–500, 501–1,000, 1,001–1,500, 1,501–2,000, QUE, QUI, REU, SAC, SM, 2,001–2,500 SOL, SUCH 13 Partamona orizabaensis (Strand, 1919) AV, CHIQ, QUI, QUE, REU, 9 0–500, 501–1,000, 1,001–1,500, 2,001–2,500 SM, SOL, SUCH 14 Plebeia frontalis (Friese, 1911) CHIQ, PE, I, ZAC 7 0–500, 1,001–1,500 15 P. fulvopilosa Ayala, 1999 CHIQ 1 1,501–2,000 16 P. jatiformis (Cockerell, 1912) AV, G, SR 10 0–500, 1,001–1,500, 1,501–2,000 17 P. llorentei Ayala, 1999 AV 2 0–500 18 P. melanica Ayala, 1999 BV, CHIQ, QUI 5 1,501–2,000 19 P. moureana Ayala, 1999 AV, JUT, SM 5 0–500, 501–1,000 20 P. parkeri Ayala, 1999 AV, G, QUE, SM, SR 7 0–500, 501–1,000, 1,001–1,500 21 P. pulchra Ayala, 1999 AV, QUI, SR 6 0–500, 1,001–1,500 (continued) 27 Trigona corvina Cockerell, 1913 28 T. fulviventris Guérin-Méneville, 1844 29 30 T. fuscipennis Friese, 1900 T. nigerrima Cresson, 1878 Departments of occurrence AV, CHIM, QUI, QUE, REU, SM, SR QUI, REU, SM, SR AV, PE CHIQ, I CHIM, SUCH, QUE, REU, PR, G, JUT, I, CHIQ, AV AV, BV, CHIQ, PR, JUT, I, SUCH, SR, ZAC AV, CHIQ, PE, PR, QUI, G, I, JUT, QUE, SAC, SM, SR, SOL, SUCH, ZAC AV, CHIM, PR, I, JUT, SUCH AV, PE, QUE, SR, SOL, SUCH 31 32 33 T. silvestriana (Vachal, 1908) Trigonisca maya Ayala, 1999 T. pipioli Ayala, 1999 AV, I PR BV, CHIQ No. of sites 11 Altitudinal ranges of collecting 0–500, 501–1,000, 1,001–1,500, 1,501–2,000 5 11 3 19 0–500, 501–1,000, 1,001–1,500 0–500, 501–1,000 0–500, 501–1,000 0–500, 501–1,000, 1,001–1,500, 1,501–2,000 23 0–500, 501–1,000, 1,001–1,500, 1,501–2,000 31 0–500, 501–1,000, 1,001–1,500, 1,501–2,000 8 7 0–500, 501–1,000, 1,001–1,500 0–500, 1,001–1,500, 1,501–2,000, 2,001–2,500 0–500, 501–1,000 0–500 501–1,000, 2,001–2,500 10 1 2 C.L. Yurrita Obiols and M. Vásquez AV Alta Verapaz, BV Baja Verapaz, CHIQ Chiquimula, CHIM Chimaltenango, ESC Escuintla, G Guatemala; HUE Huehuetenango, I Izabal, JAL Jalapa, JUT Jutiapa, PR El Progreso, PE Petén, QUE Quetzaltenango, QUI Quiché, REU Retalhuleu, SAC Sacatepéquez, SM San Marcos, SR Santa Rosa, SUCH Suchitepéquez, SOL Sololá, ZAC Zacapa 102 Table 6.1 (continued) No. Stingless bee species 22 Scaptotrigona mexicana (Guérin-Méneville, 1844) 23 S. pectoralis (Dalla Torre, 1896) 24 Scaura argyrea (Cockerell, 1912) 25 Tetragona mayarum (Cockerell, 1912) 26 Tetragonisca angustula (Latreille, 1811) 6 Stingless Bees of Guatemala 103 The species included in the CANG belong to 17 different genera of those recognized by Camargo and Pedro (2008) for the neotropical region. Plebeia and Trigona are the most diverse genera with eight and five species, respectively. Melipona include three species and Partamona, Scaptotrigona, and Trigonisca two. The remaining 11 genera are represented by a single species each (Table 6.1). The species Tetragona dorsalis (Smith, 1854) and Scaura latitarsis (Friese, 1900) included in Ayala (1999) now correspond to Tetragona mayarum (or ziegleri) and Scaura argyrea, respectively (Camargo and Pedro 2008). The species cited here as Trigona silvestriana has also been interpreted as T. amalthea (Olivier, 1789) (Roubik and Moreno 2009; DW Roubik, personal communication). Of the species listed, Lestrimelitta niitkib is the only cleptobiotic one. Most of the species reported are mainly Mesoamerican, with distributions spanning from Mexico to Colombia (Camargo and Pedro 2008). The exceptions are Trigona nigerrima, Trigona silvestriana, and Tetragonisca angustula which are found farther south than Colombia. The presence of Trigonisca maya in Guatemala represents a new distributional record, since it was previously reported only in Mexico (Ayala 1999; Camargo and Pedro 2008). The distributional analysis presented in this work is based on a geopolitical division of the territory. However, in order to provide a more realistic geographic approach we made reference to the altitudinal range and the type of forest where species were collected. Unfortunately, the available information does not represent the actual distribution of species, given that the collecting effort has not been systematic across the country. Indeed, most of the collecting sites correspond to places where the research group has carried out other studies. Nevertheless, the available data show that Meliponini have a wide distribution in Guatemala, since the species have been collected in all but one of the 22 Departments (administrative division equivalent to Province) of the country. The distributional data correspond to 323 unique localities in which at least one of the 33 species recorded has been collected (Fig. 6.1 and Table 6.1). Some species can be considered more common since they have been collected in more sites (Table 6.1). For instance, Partamona bilineata and Trigona fulviventris have been collected in 15 Departments at 31 and 35 localities within them, respectively, while Trigonisca maya, Plebeia fulvopilosa, P. llorentei, and Frieseomelitta nigra have been collected in one Department and one or two localities. The departments of Alta Verapaz (North Central region), Santa Rosa (Central South), and Chiquimula (East) show the highest diversity, with more species recorded (22, 14, 12, respectively) (Table 6.1). Nonetheless, this result might be biased by the fact that they correspond to areas where a larger collecting effort has been made. Additionally, the collecting sites within them are very localized (Fig. 6.1). The stingless bee species in Guatemala are found in a wide variety of forests (pine-oak, dry, thornscrub, montane, and moist) at elevations that range from near sea level to as high as 2,353 m in the mountainous areas. The majority of species do not show a very clear distribution in relation to a certain forest type. Indeed, even species for which we have only a few records can be found in very different areas. For instance, Plebeia pulchra and Paratrigona guatemalensis have been collected 104 C.L. Yurrita Obiols and M. Vásquez Fig. 6.1 Occurrence localities of the stingless bees in Guatemala. (•) Collecting sites; altitudinal ranges (masl): 0–500, 501–1,000, 1,001–1,500, 1,501–2,000, 2,001–2,500, 2,501–4,000 in six and four different sites, respectively, located in moist (department of Alta Verapaz) and montane (Santa Rosa) forests. However, a few species like Trigona silvestriana that appear in ten different localities exhibit a distribution restricted to very moist forests (Alta Verapaz and Izabal). Most species (31) occur between sea level and 1,500 m. Four of these (Trigonisca pipioli, Oxytrigona mediorufa, Geotrigona acapulconis, and Melipona yucatanica) have not been collected at the lowest elevation range (0–500 m), while Frieseomelitta nigra, Trigonisca maya, and Plebeia llorentei were captured exclusively in this altitudinal range. However, according to Ayala (1999) F. nigra can be found in altitudes over 1,500 m. Fourteen of these species were also collected up to 2,000 m elevation, and in different kinds of forests. Plebeia fulvopilosa and P. melanica were restricted to elevations of 1,500–2,000 m, as Ayala (1999) reports. Only four species appeared in the highest altitudinal range, and all of them (Partamona bilineata, P. orizabaensis, Trigona nigerrima, and Trigonisca pipioli) exhibit a very wide altitudinal range of distribution and habitat preference, occurring from the lowest to the highest elevations and distributed in different habitat types. Figure 6.1 indicates that collection is few in the southern coast and in northern part of the country, as well as in the central east and central west regions. Nonetheless, given the wide altitudinal and habitat tolerance of some species we expect that most will appear in these areas in future studies. 6 Stingless Bees of Guatemala 6.3 105 Floral Resources of Stingless Bees Animal-mediated pollination is an important ecosystem service for sexually reproducing plants. Bees are considered the most important pollinators for major agricultural crops (Klein et al. 2007) and wild plants (Cane et al. 2006). In the tropics, the stingless bees constitute an important portion of the flower-visiting fauna (Lorenzon et al. 2003; Wilms et al. 1996), having been reported as the major pollinators of 18 crops (Slaa et al. 2006; Heard 1999) and contributing to different degrees to the pollination of many others (Heard 1999). The stingless bee species stored at CANG were collected on flowers from at least 117 different species (data not shown) that belong to 47 plant families, having information about the resource they provide to the bees (polen and/or nectar) only for a small portion (Table 6.2). Most of the visited families (70%) can be considered Table 6.2 Plant families visited by the stingless bee species in Guatemala. Number of bee species visiting and the number of plant species visited per family (from CANG database) No. of visiting No. of plant Pollen (P)/nectar Plant families visited bee species species visited (N) source Asteraceae Zingiberaceae Fabaceae (Papilionoideae, Mimosoideae, Caesalpinioideae) Bixaceae Malpighiaceae Lamiaceae Onagraceae Cucurbitaceae Poaceae Malvaceae Melastomataceae Rubiaceae Violaceae Solanaceae Convolvulaceae Commelinaceae Cyperaceae, Piperaceae Cactaceae, Euphorbiaceae Bignoniaceae, Passifloraceae Bromeliaceae, Salicaceae, Vitaceae, Zygophyllaceae Apocynaceae, Arecaceae, Boraginaceae, Costaceae, Lythraceae, Acanthaceae, Fagaceae, Musaceae, Nyctaginaceae, Orchidaceae Asparagaceae Anacardiaceae, Apiaceae, Brassicaceae, Caryophyllaceae, Myrtaceae, Phytolaccaceae, Ranunculaceae, Rosaceae, Sapindaceae, Verbenaceae 21 16 13 30 1 11 P, N P P, N 11 11 10 10 9 9 8 6 6 6 5 4 4 4 3 3 3 1 1 4 1 1 1 5 3 4 1 5 3 2 1 3 2 1 P P P, N P, N P 2 2 2 1 P, N 1 1 2 1 P, N P, N P, N P P, N P, N P P, N 106 C.L. Yurrita Obiols and M. Vásquez occasional floral resources, since only a few species (<5) forage on their flowers. In addition, for each of these families, only a few species were visited (<5 species per family) (Table 6.2). On the other hand, Asteraceae and Fabaceae can be suggested to be an important food source for the stingless bees as the diversity of the visited species within these families was higher (30 and 11 visited species, respectively). Moreover, approximately 60% (21) and 40% (13) of the identified bee species, respectively, have been collected while foraging on their flowers (Table 6.2). Other studies have already reported that the family Asteraceae is one of the main food resources for the stingless bees (Wilms et al. 1996). Other plant families visited by more than five bee species show that seven of them are represented by a single species (Table 6.2). This is the case of “achiote” Bixa orellana (Bixaceae), “nance” Byrsonima crassifolia (Malpighiaceae), “cardamomo” Elettaria cardamomum (Zingiberaceae), and watermelon Citrullus lanatus (Cucurbitaceae). These species, all important economic and/or food resources for human populations in Guatemala, were part of a more detailed survey. A palynological analysis was carried out to assess the potential of the stingless bees foraging on their flowers as pollen vectors. For Elettaria cardamomum eight of the 16 visiting species are suggested as potential pollinators. In the case of Bixa orellana six out of 11 can be considered possible pollinators, and for Citrullus lanatus and Byrsonima crassifolia six and one stingless bee species, respectively, were detected as potential pollinators (Enríquez 2007). Previous studies had already registered these plants as effectively or occasionally being pollinated by stingless bees in other regions (Slaa et al. 2006; Heard 1999). The flowers of maize Zea mays (Poaceae), the only recorded species from the Poaceae family, were visited by nine stingless bee species, but there is no evidence proving that these visiting species are acting as potential pollinators. Eleven meliponines were collected on less than five plant species, and five were not collected on any flower (Table 6.3). Among the latter Lestrimelitta niitkib is not expected to collect pollen (or visit flowers) since it has a cleptobiotic behavior. In Guatemala, this bee has been seen attacking colonies of at least two stingless bee species, Melipona beecheii and Tetragonisca angustula (CL Yurrita 2011, personal observation). Trigona fulviventris is the species that visited the widest array of plants (45) (Table 6.3); nonetheless, it has been documented that sometimes it may not act as a pollinator but rather as a nectar or pollen robber (Barrows 1976; CL Yurrita 2010, personal observation). Melipona spp. are capable of buzz pollination (Heard 1999), a feature that makes them potential pollinators of many plants. Finally there is a record of Partamona orizabaensis captured on feces. 6.4 Stingless Beekeeping in Guatemala There is a long tradition of stingless beekeeping, or meliponiculture, in the Mesoamerican region (Kent 1984; Crane 1992; Cortopassi-Laurino et al. 2006) and in the Amazon (Posey 1982; Posey and Camargo 1985), in comparison with other 6 Stingless Bees of Guatemala 107 Table 6.3 Number of plant species visited by the stingless bees (from CANG database) Stingless bee species Plant species visited Plebeia parkeri 5 Scaptotrigona mexicana 5 Scaptotrigona pectoralis 5 Trigonisca maya 5 Trigonisca pipioli 5 Trigona silvestriana 7 Melipona solani 8 Scaura argyrea 8 Tetragona mayarum 8 Partamona orizabaensis 8 Melipona beecheii 13 Trigona nigerrima 14 Cephalotrigona zexmeniae 15 Nannotrigona perilampoides 16 Plebeia jatiformis 17 Trigona fuscipennis 17 Tetragonisca angustula 29 Trigona corvina 29 Partamona bilineata 33 Trigona fulviventris 45 Dolichotrigona schultessi, Frieseomelitta nigra, Melipona aff. yucatanica, <5 Paratrigona guatemalensis, Plebeia frontalis, P. fulvopilosa, P. melanica, P. moureana, P. pulchra The following species were not collected on flowers and were not included in the table: Geotrigona acapulconis, Lestrimelitta niitkib, Oxytrigona mediorufa, Plebeia llorentei regions of the world (Cortopassi-Laurino et al. 2006). This is probably due to the great diversity of meliponines found in Tropical America. In Mesoamerica, stingless bee beekeeping has been culturally important since the precolonial era. Indeed, the Maya codices and some colonial writings record the importance of the stingless bees in the Mayan culture. This importance is revealed by the existence of bee gods (Maya codices) and the rituals of beekeeping and use of hive products documented in the writings of the Bishop Diego de Landa (apud Kent 1984). The Mayan region including the Yucatán Peninsula and northern Guatemala and Belize were suggested as a place of intense stingless bee rearing activity in pre-Columbian days, particularly Melipona beecheii. Furthermore, this region has been considered the possible place of origin of the practice (Kent 1984; Crane 1992). Thus, both the beekeeping technique and the hive design most commonly employed in the Yucatán Peninsula are considered the original ones (Crane 1992). Nowadays the traditional practice of meliponiculture in the Yucatán Peninsula and in other regions of México (GonzálezAcereto and De Araujo-Freitas 2005), as well as in areas throughout Mesoamerica (Enríquez et al. 2005; Kent 1984), has not changed much over time. In Guatemala, stingless bee beekeeping is practiced by different ethnic groups across the country. Kent (1984) has documented that the activity takes place in the 108 C.L. Yurrita Obiols and M. Vásquez Common names of stingless bees used in Guatemala [modified from Enríquez et al. Table 6.4 (2005)] No. 1 2 3 Scientific name Cephalotrigona zexmeniae Lestrimelitta niitkib Melipona beecheii 4 5 6 7 8 9 10 11 12 13 14 15 16 Melipona solani Melipona yucatanica Nannotrigona perilampoides Oxytrigona mediorufa Partamona sp. Plebeia sp. Scaptotrigona mexicana Scaptotrigona pectoralis Tetragonisca angustula Trigona fulviventris Trigona nigerrima Trigona silvestriana Geotrigona acapulconis Folk name “congo” “limoncillo” “colmena grande,” “criolla,” “abeja maya,” “xuna’n cab,” “bichi” “chac chow” “tinzuca” “serenita” “tamagás,” “pringador” “sacar,” “cushpun” “chelerita,” “serenita,” “boca de sapo,” “sarquita” “magua negro,” “congo,” “congo negro” “magua canche,” “alazán,” “congo canche,” “shuruya” “chumelo,” “doncellita” “mandinga,” “culo de chucho” “cushusho,” “homo,” “joloncán” “homo” “talnete” Q’eqchi (Alta Verapaz), Maya-chortí (Jocotán, Chiquimula), and Jacaltec (Jacaltenango, Huehuetenango) areas. Our research group has worked with beekeepers in different regions of the country. The most important group dedicated to rearing the stingless bees are the Ladinos or Mestizos even though the practice is also carried out by Q’eqchí, Chortí, Mam, and Ixil-Quiché populations. For most of these people keeping the stingless bees remains a family tradition inherited for generations, although for others it is a recent activity, initiated as a result of their attendance at workshops carried out by different organizations, including our research group. People identify at least 16 stingless bee species, some of which have different regional names (Table 6.4). Given the great variety of local names that meliponines receive, we can deduce that they constitute a well-known part of the insect fauna in Guatemala, even if the number of species used in meliponiculture is limited. Meliponiculture is still practiced in a traditional way in Guatemala. The beekeepers for whom the activity is an inherited family tradition still employ the original techniques (Crane 1992) which involve the use of hollow logs closed at both ends with discs made of wood. Usually the hives are hanging from the roof of houses and less frequently people construct shelters to keep them. The most important species reared with a honey-harvesting purpose are Melipona beecheii and Tetragonisca angustula. Another important bee species from which the honey is used is Geotrigona acapulconis. However, its nesting behavior (nest constructed deep underground) makes it difficult for people to keep them in hives, and the only way to extract the honey is by destroying the nest. A larger number of bee species are reared with ornamental purposes (because “they are nice”), but eventu- 6 Stingless Bees of Guatemala 109 ally their honey can be extracted. Occasionally, people harvest honey from nests kept in their original location without destroying them. This is the case for Trigona nigerrima which constructs its nest on tree branches and not in hollows, making it easier to harvest the honey in place. Scaptotrigona mexicana and S. pectoralis are two species with a special potential in meliponiculture due to the low management requirements and high yields they provide. Finally, the honey of some other species, like that of Trigona fulviventris, is avoided due to their anti-hygienic behavior (they collect feces) (M Vásquez 2010, personal observation). The main product harvested from the stingless bees in Guatemala is the honey, but the pollen and the cerumen are also used. The honey is used mainly for medicinal practices or as an energy supplement, but it is not an important food item, probably due to the small yield. The medicinal properties attributed to the stingless bee honey are very diverse and depend on the species producing it, even though some uses are common to all of them. The honey of Melipona beecheii is the most appreciated, probably due to the fact that this species produces larger amounts of honey in comparison with Tetragonisca angustula (Vit et al. 2004). Usually the honey is not for sale; if someone in the community needs some, a beekeeper will provide it without any cost. In Guatemala, our research group has undertaken studies aiming to investigate the pollen species content, the antibacterial activity, the physicochemical properties, and the sensory attributes of the honey of nine of the 32 stingless bee species used for honey production (almost 30% of the honey diversity) (Dardón and Enríquez 2008, and Dardón et al., Chap. 28 in this book). 6.5 Final Comments Given the diversity of stingless bees in Guatemala and the wide distributional range of the majority of the species, promoting the use of the honey as an alternative energetic or medicinal supplement or perhaps as a food complement could be a great opportunity. Nonetheless, as it has been suggested for other regions (Villanueva et al. 2005), bees like Melipona and other species that nest in tree hollows may be at risk in Guatemala. One important reason causing this situation is the loss of nesting sites as a consequence of the high deforestation rate, which reaches 1.53% each year in Guatemala (Tuy et al. 2009). Also, as was pointed out for Yucatán (Villanueva et al. 2005), the stingless bee beekeeping practice itself may be in decline in Guatemala. Therefore, the potential loss of the stingless bee diversity as well as that of the meliponiculture hampers the use of the great diversity of honeys for medical or food complement purposes. Moreover, the lack of quality standards for the honey prevents the marketing of the product. It is therefore necessary to promote programs aiming to preserve the species habitats as well as programs to enhance the practice of meliponiculture to transform it in a certifiably hygienic and productive activity. That initiative has to be complemented by continuing studies on honey composition, as well as by educating people on improving meliponiculture techniques. 110 C.L. 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Bee World 73: 29–42. Dardón MJ, Enríquez E. 2008. Caracterización fisicoquímica y antimicrobiana de la miel de nueve especies de abejas sin aguijón (Meliponini) de Guatemala. Inteciencia 33 (12):916–922. Enríquez E. 2007. Diversidad de potenciales polinizadores del grupo de los insectos en el Parque Nacional Laguna Lachuá y su zona de influencia a lo largo de un año. Informe final Proyecto Fodecyt 017-2006, Guatemala. 66 pp. Enríquez E, Yurrita CL, Aldana C, Ocheíta J, Jáuregui R, Chau P. 2005. Conocimiento tradicional acerca de la biología y manejo de abejas nativas sin aguijón en Chiquimula. Apicultura 69:27–30. González-Acereto J, De Araujo-Freitas Ch. 2005. Manual de Meliponicultura Mexicana. Impresos Gramma. Mérida Yucatán, México. 45 pp. Heard TA. 1999. The role of stingless bees in crop pollination. Annual Review of Entomology 44:183–206. Kent R. 1984. Mesoamerican stingless beekeeping. Journal of Cultural Geography 4:14–28. Klein AM, Vaissière BE, Cane JH, Steffan-Dewenter I, Cunningham SA, Kremen C, Tscharntke T. 2007. Importance of pollinators in changing landscapes for world crops. Proceedings of the Royal Society B 274:303–313 Lorenzon MAC, Matrangolo CAR, Schoereder JH. 2003. Flora visitada pelas abelhas eussociais (Hymenoptera, Apidae) na Serra da Capivara, em caatinga do sul do Piauí. Neotropical Entomology 32:027–036. Michener CD. 2007. The Bees of the World. 2nd edn. The John Hopkins University Press, Baltimore, USA. 972 pp. Moure JS. 1961. A preliminary supra-specific classification of the old world meliponine bees (Hymenoptera, Apoidea). Studia Entomologica 4:181–242. Ortiz R. 1998. Biodiversidad de las abejas sin aguijón (Apidae: Meliponini) de Costa Rica. CINATPRAM Universidad Nacional, Costa Rica. 23 pp. Posey DA. 1982. The importance of bees to Kayapó Indians of the Brazilian Amazon. The Florida Entomologist 65(4):452–458. Posey DA, Camargo JMF. 1985. Additional notes on the classification and knowledge of stingless bees (Meliponinae, Apidae, Hym.) by the Kayapo Indians of Gorotide (Para, Brazil). Annals of the Carnegie Museum 54:247–274. Rasmussen C, Cameron S. 2007. A molecular phylogeny of the Old World stingless bees (Hymenoptera: Apidae: Meliponini) and the non-monophyly of the large genus Trigona. Systematic Entomology 32:26–39 DOI: 10.1111/j.1365-3113.2006.00362.x Rasmussen C, Cameron S. 2010. Global stingless bee phylogeny supports ancient divergence, vicariance, and long distance dispersal. Biological Journal of the Linnean Society 99:206–232. 6 Stingless Bees of Guatemala 111 Roubik DW, Moreno-Patiño JE. 2009. Trigona corvina: An ecological study based on unusual nest structure and pollen analysis. Psyche. 1–7 DOI:10.1155/2009/268756 Slaa EJ, Sánchez Chaves LA, Malagodi-Braga K, Hofstede FE. 2006. Stingless bees in applied pollination: practice and perspectives. Apidologie 37:293–315. 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Studies on the Neotropical Fauna & Environment 31:137–151. Chapter 7 Stingless Bees of Costa Rica Ingrid Aguilar, Eduardo Herrera, and Gabriel Zamora 7.1 Introduction The keeping of stingless bees (Apidae, Meliponini), or meliponiculture, is carried out in a rustic and traditional way in tropical America by a variety of ethnic groups and rural populations. This practice has been maintained over time in regions of Mexico, Central America, and South America in countries such as Brazil, Venezuela, Colombia, Ecuador, Bolivia, Peru, and Argentina (Mahecha and Nates-Parra 2002; Elizalde et al. 2007; Flores and Sánchez 2010; Jiménez 2011). This is due in part to the cultural value, which this practice holds, but also reflects the interest that has been aroused in consumers of stingless bee honey as a medicinal alternative. The product of these bees that is mostly used is the honey (De Jong 1999; Aguilar 2010; Herrera and Aguilar 2011) and this has generated much interest in the scientific community due to the results achieved by microbiological tests, which have shown that stingless bee honey has antimicrobial properties (Gonçalves et al. 2005; Aguilera et al. 2009; Vit et al. 2009). Paradoxically, in Costa Rica the growing demand for these products coincides with a decrease in the populations of some species of this group, e.g., Melipona beecheii (Villanueva-Gutiérrez et al. 2005; Genaro 2006). This reduction is due to the environmental damage caused by the process of urbanization, the direct consequences of such include loss of forest areas, among other effects. We are thus making scientific progress in recognizing the usefulness of stingless bees in terms of their products and uses in natural folk medicine, as well as pollination services, but at the same time we are losing the natural resource that provides these products and services. This becomes a problem for fulfilling the demands of the market (environmental services of pollination, acquisition of colonies and products such as honey or propolis). Consequently, we must act I. Aguilar (*) • E. Herrera • G. Zamora Centro de Investigaciones Apícolas Tropicales (CINAT), Universidad Nacional, Apartado Postal, 475-3000 Heredia, Costa Rica e-mail: iaguilar@una.ac.cr P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_7, © Springer Science+Business Media New York 2013 113 114 I. Aguilar et al. to strengthen the conservation and sustainable use of these bees. This chapter refers to the stingless bees of Costa Rica, with attention to stingless beekeeping, and it has two goals: first, we document the information that exists about the past and current state of meliponiculture in our country, and second we record the bee species as well as the tree species that bees use for establishing nests. 7.1.1 Stingless Beekeeping in Costa Rica Stingless beekeeping has its origins in the culture of the Maya of the Yucatan Peninsula and this practice spread to other groups in Mesoamerica, extending to the northern part of Costa Rica (southern tip of Mesoamerica, Nicoya, Guanacaste Province) under the influence of the indigenous Nahuatl and Mestizos (Kent 1984). Another study of the pre-Hispanic cultures (Tous 2002) based on ethnographic descriptions of the region known as “La Gran Nicoya”—16th-17th centuries— that among the products obtained from the harvest were honey and cerumen. Honey was used for human consumption; the cerumen was very abundant and used for lighting and silver work , the “lost wax” technique. Tous (2002) also mentions that the practice of trade and exchange with local products, such as honey and cerumen, were of vital importance in the Nicoya Peninsula to resolve situations of scarcity; at the same time these products were used for exchange with other indigenous groups since its redistribution ensured a more diversified access. Kent (1984) mentions that in the Nicoya of the 1500s the indigenous people delivered 55 L of honey every 6 months as a tribute to the priests. The Boruca of the Central Valley (Province of San José) and the Térraba (native of the Atlantic coast, Limón Province) used cerumen on their spears and arrows (Stone and Gabb cited by Kent 1984). Despite the data mentioned above, there are very few records concerning the use of this natural resource by the indigenous people of Costa Rica. We believe this is because the ethnic groups in Costa Rica suffered from eighteenth century slavery and resettlement to areas far from their original territory (Montoya et al. 2008). Possibly, as in other Mesoamerican areas, stingless bee keeping in Costa Rica did not play an important role in the religion of indigenous cultures. Yet, an image of a bee found in Costa Rica (De Jong 1999) and other reports cited by Kent (1984) that allude to the use of words such as honeycomb, wax, and honey in the language of the Bribri and Cabécar suggest that the bees had a meaning for them. It was reported that an abundance of honey and cerumen was produced around the beginning of the twentieth century (Kent 1984; De Jong 1999). Kent (1984), at the beginning of the 1980s in the area of the Central Valley, described the existence of a more advanced meliponiculture. The author mentioned the use of Tetragonisca angustula and at least three species of Melipona. The greatest numbers recorded of colonies were in the Provinces of Guanacaste and San José with T. angustula and Melipona beecheii. There are no reports indicating the use of these bees by local indigenous groups during this period. According to our records, the beginning of the twenty-first century is marked by a widespread use of T. angustula (Aguilar 2009; Herrera and Aguilar 2011). 7 Stingless Bees of Costa Rica 115 Fig. 7.1 Map of Costa Rica with the location of stingless bee keepers (red dots), showing the boundaries of the provinces A large number of stingless bee keepers are situated in the rural areas of San José, mostly on the Pacific coast rather than the Atlantic region (see Fig. 7.1). Currently in San José Province, in the canton of San José (the capital of Costa Rica), it is very rare to find rational boxes with nests of stingless bees. We have occasionally found boxes with T. angustula and Nannotrigona in the cantons of Santa Ana, Montes de Oca, Moravia, and Escazú, more frequently in the cantons of Puriscal, Tarrazú, Aserrí, Acosta and Perez Zeledón. In the canton of San José the genus Melipona has not been reported in the last three decades, which is linked with the urban development of this area. In addition, we have observed in the remnants of riparian forests, coffee plantations, playgrounds of the urban areas of this and other provinces a variable but important number of nests of T. angustula, T. corvina, and Nannotrigona spp. Other areas belonging to Guanacaste and Puntarenas provinces, for example Santa Cruz, Hojancha, Philadelphia, and Miramar, are known for traditional meliponiculture (De Jong 1999). We have also observed a few stingless bee keepers in Heredia, Cartago, and Limón provinces (see Fig. 7.1). Some of them have received motivation during recent workshops. 7.1.2 Management of Native Stingless Bee Species A total of 20 different hived or semi-domesticated species have been reported (see Table 7.1) in the provinces of Guanacaste, Puntarenas, San José, Cartago and Heredia (Arce et al. 1994; Ramírez and Ortiz 1995; De Jong 1999; Herrera and Aguilar 2011). It is mainly T. angustula that is being kept, followed by M. 116 Table 7.1 Stingless bees of Costa Rica. Nomenclature according to Camargo and Pedro (2007, 2008). Information on domesticated species, common names and distribution is given Domesticated Common Species species name Distribution (province of Costa Rica) (c) “tamaga amarillo” Dolichotrigona schulthessi (Friese, 1900) Frieseomelitta nigra (Cresson, 1878) Frieseomelitta paupera (Provancher, 1888) Geotrigona chiriquiensis (Schwarz, 1951) Geotrigona lutzi Camargo & Moure, 1996 Lestrimelitta danuncia Oliveira & Marchi, 2005 Lestrimelitta mourei Oliveira & Marchi, 2005 Melipona beecheii Bennett, 1831 Melipona carrikeri Cockerell, 1919 Melipona costaricensis Cockerell, 1919 Melipona fuliginosa Lepeletier, 1836 Melipona panamica Cockerell, 1912 Melipona torrida Friese, 1916 Melipona yucatanica Camargo, Moure & Roubik, 1988 Meliwillea bivea Roubik, Lobo & Camargo, 1997 Nannotrigona mellaria (Smith, 1862) Nannotrigona perilampoides (Cresson, 1878) Nogueirapis mirandula (Cockerell, 1917) Oxytrigona daemoniaca Camargo, 1984 Oxytrigona mellicolor (Packard, 1869) Paratrigona lophocoryphe Moure, 1963 Paratrigona opaca (Cockerell, 1917) Paratrigona ornaticeps (Schwarz, 1938) ND (f) ND ND ND (c, f) ND (a, b) ND (a) (a, c) ND ND ND ND (d) (a, b) ND ND (c) ND (d) (d) ND “ala blanca” “chupaojos” ND ND “jicote limón” ND “jicote gato” ND “jicote barcino” “jicote” ND ND ND ND ND “chicopipe” ND ND “peladora” ND ND ND Alajuela, Cartago, Guanacaste, Heredia, Puntarenas, San José Cartago, Puntarenas Guanacaste Guanacaste Alajuela, Cartago, Puntarenas, San José Alajuela, Cartago, Guanacaste, Limón, Puntarenas, San José Cartago San José Puntarenas, San José Guanacaste, San José Guanacaste, Heredia, Limón, Puntarenas, San José Cartago, Guanacaste, Limón, Puntarenas, San José Costa Rica San José Puntarenas Cartago, Puntarenas, San José Heredia, Puntarenas Alajuela, Cartago, Guanacaste, Heredia, Limón, Puntarenas Guanacaste, Puntarenas, San José Costa Rica Alajuela, Guanacaste, Limón, Puntarenas, San José Cartago, Limón Limón, Puntarenas, San José Cartago, Limón, Puntarenas, San José I. Aguilar et al. Cephalotrigona zexmeniae (Cockerell, 1912) Common name Partamona grandipennis (Schwarz, 1951) ND ND Partamona musarum (Cockerell, 1917) Partamona orizabaensis (Strand, 1919) ND ND ND ND Plebeia franki (Friese, 1900) Plebeia frontalis (Friese, 1911) ND ND ND ND Plebeia jatiformis (Cockerell, 1912) Plebeia llorentei Ayala, 1999a Plebeia minima (Gribodo, 1893)a Plebeia pulchra Ayala, 1999a Plebeia tica (Wille, 1969) Ptilotrigona occidentalis (Schulz, 1904) Scaptotrigona luteipennis Friese, 1902 Scaptotrigona mexicana (Guérin, 1844) Scaptotrigona (ex. barrocoloradensis) Scaptotrigona pectoralis (Dalla Torre, 1896) (d) ND ND ND (e) ND (c,b) ND ND (a) “chupa ojos” ND ND ND “bocarena” ND “soncuano” ND ND “soncuano” Scaptotrigona subobscuripennis (Schwarz, 1951) ND ND Scaptotrigona wheeleri (Cockerell, 1913) Scaura argyrea (Cockerell, 1912) Tetragona perangulata (Cockerell, 1917) Tetragona ziegleri (Friese, 1900) Tetragonisca angustula (Latreille, 1811) Tetragonisca buchwaldi (Friese, 1925) Trigona cilipes (Fabricius, 1804) ND (d) ND (a,c) (a,b,e,d) ND ND ND ND ND “miel de leche” “mariola” ND ND Distribution (province of Costa Rica) Alajuela, Cartago, Guanacaste, Heredia, Puntarenas, San José Cartago, Heredia, Limón, San José Alajuela, Cartago, Guanacaste, Heredia, Limón, Puntarenas, San José Costa Rica Alajuela, Cartago, Guanacaste, Heredia, Limón, Puntarenas, San José Cartago, Puntarenas ND ND ND Guanacaste, Heredia Puntarenas Costa Rica Cartago Guanacaste, Puntarenas, San José Alajuela, Cartago, Guanacaste, Limón, Puntarenas, San José Alajuela, Cartago, Guanacaste, Heredia, Limón, Puntarenas, San José Costa Rica Cartago, Guanacaste, Puntarenas, San José Guanacaste, Puntarenas Alajuela, Guanacaste, Heredia, Limón, Puntarenas Alajuela, Cartago, Heredia, Limón, Puntarenas, San José Puntarenas Costa Rica Stingless Bees of Costa Rica Domesticated species 7 Species (continued) 117 Species Domesticated species Common name Trigona corvina Cockerell, 1913 ND ND Trigona ferricauda Cockerell, 1917 Trigona fulviventris Guérin, 1844 ND (c) ND “culo de buey” Trigona fuscipennis Friese, 1900 Trigona muzoensis Schwarz, 1948 Trigona necrophaga Camargo & Roubik, 1991 Trigona nigerrima Cresson, 1878 Trigona silvestriana (Vachal, 1908) ND ND ND ND (c) ND ND ND ND “congo” Trigonisca atomaria (Cockerell, 1917) Trigonisca discolor (Wille, 1965) Trigonisca pipioli Ayala, 1999a ND ND ND ND ND ND 118 Table 7.1 (continued) Distribution (province of Costa Rica) Alajuela, Cartago, Guanacaste, Heredia, Limón, Puntarenas, San José Cartago, Heredia, Limón Alajuela, Cartago, Guanacaste, Limón, Puntarenas, San José Alajuela, Guanacaste, Limón, Puntarenas, San José Limón CR Alajuela, Limón, Puntarenas, San José Alajuela, Cartago, Guanacaste, Heredia, Limón, Puntarenas, San José Alajuela, Guanacaste, Puntarenas San José ND Sources: (a) Arce et al. (1994); (b) Wagner (1958); (c) Ramírez and Ortiz (1995); (d) Herrera and Aguilar (2011); (e) Aguilar (2009); (f) Aguilar (personal observation); ND no data a Species not cited in the catalogue of Camargo and Pedro (2007) but found in the entomological collections (Ayala, personal communication) I. Aguilar et al. 7 Stingless Bees of Costa Rica 119 beecheii. The breeding of Frieseomelitta sp., T. fulviventris, Lestrimelitta sp., and Plebeia tica is less common. Lestrimellita sp., a robber bee that does not visit flowers, is not suitable for stingless bee keeping. In addition, Nannotrigona perilampoides and T. angustula can be considered as alternatives to honey bees for commercial crop pollination in Costa Rica (Slaa et al. 2000). According to van Veen et al. (1990) meliponiculture in Costa Rica is basically practiced in two ways: (1) maintaining the nests in tree trunks, from which the honey by a lateral opening is extracted, this is typically used for M. beecheii; and (2) keeping the colonies in small boxes, pieces of bamboo or hollow logs, common practice with T. angustula. As stated by Arce et al. (1994), stingless bee keeping in Costa Rica has been practiced at a low technical level, almost without equipment, and the type of hive mainly used has been the hollow log. They observed that stingless bee keepers kept their colonies in log hives, generally hanging under the roof of their houses. On the other hand, from 99 interviews carried out during the period 2006–2011, we recorded a total of 720 colonies. Stingless bee keepers with some sort of technical or higher education degree represented the main social group involved (29%, N = 25), followed by peasants and beekeepers (28%, N = 24; 27%, N = 23 respectively). The most commonly kept species was T. angustula (N = 523 colonies). Most beekeepers maintained meliponaries and bees for a long time, an average of 14 years, but ranging from 1 to 81 years. The average number of hives per bee keeper was 3.8 (SD = 5.08), with a maximum of 35 colonies. In regard to the design and dimensions of the hive boxes, van Veen et al. (1993) recommended for M. beecheii a hive with a volume of 10 L, with internal dimensions of 15 cm height, 15 cm width and 45 cm length. For T. angustula the recommended box dimensions were 15 cm × 15 cm × 20 cm long, which provided a volume of 4.5 L. In practice we have observed that the stingless beekeepers modify these dimensions according to the species and the size of the colony. The interest in stingless bees and their honey has increased over the past few years. Today, commerce of this honey in Costa Rica commands high prices; 1 L sells for US$ 20–50 and small containers of 10 ml cost US$ 2–4 due to an increasing interest mostly in its medicinal properties as treatment for cataracts (Aguilar 2007). Finally, the average production was 836 ml of honey/hive/year (SD = 839, N = 37). 7.1.3 Costa Rican Stingless Bees According to Roubik (1992) and Griswold et al. (1995) there are 12 stingless bee genera in Costa Rica, with a total of 40–60 described species. However, the latest classification by Camargo and Pedro (2007, 2008) and the revision by Ayala (personal communication) of the entomological collections of University of Costa Rica (UCR), Instituto Nacional de Biodiversidad (INBIO), and the Tropical Beekeeping Research Center (CINAT) of National University (UNA) show that there are 20 stingless bee genera and 58 species present in the country (see Table 7.1, nomenclature as given by (Camargo and Pedro 2007, 2008). If we consider these data and the recent classification 120 I. Aguilar et al. of Camargo and Pedro (2007, 2008), Costa Rica possesses approximately 60% of the 33 Neotropical genera of Meliponini. The number of stingless bee species recorded in Table 7.1 is nearly 8% of the known bee fauna of Costa Rica (Griswold et al. 1995). There are about 5000 Neotropical species, and about 800 from Costa Rica. With about 60 species of meliponines, the proportion of total bee species in Costa Rica that are Meliponini is about 8% (see Roubik 2000). In the tropical wet forests of Costa Rica, higher bee diversity is found at elevations below 500 m (Lobo 1996). Most species occur in the lowland rain forests of the Caribbean and the Osa Peninsula (Hanson 2000). In the Golfo Dulce region, southwestern (Pacific coast) Costa Rica 26 species were identified (Jarau and Barth 2008), which is nearly 54% of the stingless bee species reported for the country. In contrast, stingless bee species richness on the slopes of the Cordillera of Tilarán (Guanacaste province) declines dramatically above 1,000 m, and at altitudes of 700–1,000 m they are rare (Ortiz-Mora and van Veen 1995). The only known stingless bee genus endemic to Central America, Meliwillea bivea, is found in the Costa Rican highlands above 1,500 m of elevation (Roubik et al. 1997). 7.1.4 Tree Species Used for Nesting by Stingless Bees The architecture of stingless bees nests of Costa Rica has been well studied by Wille and Michener (1973), in their work at least nine categories of nesting cavities are described. They find that the cavities in trees can be very variable, but stingless bees nonetheless use them. Owing to the importance for the establishment of nests, it is necessary to identify the species of trees used for nesting. In addition, severe deforestation affects the density of nests and could lead to significant changes in the composition of species; some species may disappear, e.g., Scaptotrigona pectoralis, while others could become abundant, e.g., T. angustula (Slaa 2003). On this regard, the work carried out in Costa Rica (Berrocal 1998; Arce et al. 2001; Slaa 2003) shows that a total of 36 identified botanical species correspond to timber species of high commercial value, which in turn are sources of nectar and pollen for these bees (see Table 7.2, modified from Aguilar 2001). Furthermore, most of them have multiple uses in our society (forage, wood, shade, crops, pollination, medicinal, etc.) and nowadays are at risk of disappearing. On the other hand, they are suitable for being embedded in tropical agroforestry systems (Aguilar 2001). The latter is a valuable recommendation. If implemented, it would allow the preservation of bee communities (Gordon et al. 2004) and adequate resources for food, protection, and new niches, enabling stingless bees to increase their populations. 7.1.5 Future Trends of the Stingless Bee Keeping in Costa Rica Stingless bee keeping in Costa Rica is an activity that is present in several regions of the country, especially among the inhabitants of rural areas. The honey produced is used mainly as a medicine and ongoing investigations confirm the indigenous 7 121 Stingless Bees of Costa Rica Table 7.2 Species of trees used by the Costa Rican stingless bees (Apidae, Meliponini) as a substrate to establish nests and importance for the bees Species tree/substrate Common name Bee species Importance Acrocomia viniferae Anacardium excelsuma,c,e Andira inermise Astronium graveolense Citrus sp.e Bombacopsis quinatae Bravaisia integerrimaa Brosimum alicastrume ND “espavel, rabito” “almendro de montaña” ND “cítricos” “pochote” “mangle blanco” “ojoche” Bursera simarubaa Cedrela odoratae Clarisia bifloraa,e Coccoloba caracasanae Combretum fruticosume Copaifera aromaticae Cordia alliodoraa,e Diphysa americanaa,c Enterolobium cyclocarpume Ficus sp.a,e Ficus goldmaniie Ficus trachelosycea Gliricidia sepiuma,e Inga sapindoidese Lonchocarpus costaricensisc Luehea seemanniie Minquartia guianensisd Myrospermum frutescensa,e Ocotea veraguensise Pentaclethra macrolobad Persea americanae Pseudosamanea guachapelee Psidium guajavab “jiñocuabe” “cedro amargo” ND “papaturro blanco” ND ND “laurel” “guachipelín” “guanacaste” “higuerón” ND “higuerón” “madero negro” “guaba” “siete cueros” ND “manú” ND “aguacatillo” “gavilán” “aguacate” ND “guayaba, guayabo” Rehdera trinervise Spondias mombine Tabebuia ochraceaa,c Tabebuia roseae Terminalia oblongae ND “jobo” “corteza amarilla” “roble de sabana” ND Ts Sp, Ts, Tan, Mb, Tc Mb, Tan Tc Np Tan, Tc, Tf Sp, Tan, Np Tan, Sp, Tz, Cz, Om, Pf, Tfs, Tf Mb, Tz, Sp, Tan, Np Tz Sp, Om, Tan, Pf Tz Tfs Mb, Fn Tz, Tan, Np Tan, Tz, Sp Tz, Tan Sp, Mb, Tan Tan, Tz Tan Tan, Tz, Cz, Om, Pf Om Np Tan, Cz Tz Cz, Om, Tan, Fn Tz Tan Tc Tan, Tc, Sp, Cz Te.sp, Mb, Sp, Tc, Tan Tan, Np, Pf Tc Cz, Mb, Tan Tan, Tc Tan, Sp, Om ND N, 1, 3 N, 1, 2, 3 ND ND N, P, 1, 2, 3 N, P, 2 P, 1, 2, 3 P, 1, 2, 3 P, 1, 2, 3 ND N, P, 1, 2, 3 ND ND P, N, 1, 2, 3 P, N, 1, 2, 3 P, 1, 2, 3 1, 3 ND 1 N, 1, 2, 3 ND ND ND 1, 3 N, 1, 3 P, 1, 2, 3 1, 3 ND ND N, P, 1, 3 ND N, P, 1, 2, 3 N, P, 1, 2, 3 N, P, 1, 2, 3 ND Modified from Aguilar (2001) Sources: aBerrocal (1998) (for dry tropical forest), bAguilar personal observation, cArce et al. (2001), dRincón (1997) (for premontane humid tropical and humid forest), eSlaa (2003) (for tropical dry forest) N: nectar; P: pollen; ND: no data; 1: used by stingless bees; 2: used by Apis mellifera; 3: timber and other uses (according to Arce et al. 2001) Species code: Sp, Scaptotrigona pectoralis; Ts, Trigona silvestriana (amalthea); Tan, Tetragonisca angustula; Mb, Melipona beecheii; Tc, Trigona corvina; Np, Nannotrigona perilampoides; Tz, Tetragona ziegleri; Om, Oxytrigona mellicolor; Pf, Plebeia frontalis; Cz, Cephalotrigona zexmeniae; Fn, Frieseomelitta nigra; Tfs, Trigona fuscipennis; Tf, Trigona fulviventris; Te.sp., Tetragona sp. 122 I. Aguilar et al. view that honeys from the stingless bees have medicinal potential. According to our most recent data, the number of stingless bee keepers has progressively increased. However, compared to previous studies, the number of colonies per person has declined. This partially is due to the practice, in many cases, of keeping bees as a hobby more than as a source of improvement in family income. In Costa Rica, nearly 26% of the territory is preserved as national parks and reserves, but these areas are scattered and increasingly becoming isolated. We must continue efforts to preserve stingless bees, learn more about their ecology and populations, which are threatened by the loss of forest areas (Kevan 1999). Among other weaknesses confronting the successful development of Costa Rican meliponiculture are included: the lack of appropriate collections covering great part of the country, the absence of a good inventory of the existing stingless bee keepers and the fear that many keepers have of dividing nests. When carried out in a careless way, nest division results in parasite attack by phorid flies and eventually in the loss of the colony. There is an important lack in up-to-date information concerning the use of stingless bees by the natives of Costa Rica, which is noticed due to the few studies performed on meliponiculture after the nineteenth century. Therefore, further research is required in this field and more action should be taken to continue the work initiated by Wille (1961) on the biology, biodiversity conservation and management of stingless bees in Costa Rica. Acknowledgments We thank Dr. Ricardo Ayala B (Universidad Autónoma de México) and Dr. Paul Hanson (Universidad de Costa Rica) for advice and valuable additions to the manuscript. References Aguilar I. 2001. ¿Cómo manejar abejas nativas sin aguijón (Apidae: Meliponinae) en sistemas agroforestales?. Agroforestería en las Américas 85:0–55. Aguilar I. 2007. Estado actual de la Meliponicultura en Costa Rica. Notas Apícolas [Heredia, Costa Rica] 136:–8. Aguilar I. 2009. Meliponicultura en Costa Rica. pp. 332–336. In Memorias del VI Congreso Mesoamericano sobre Abejas Nativas. Antigua, Guatemala, Guatemala. 368 pp. Aguilar I. 2010. Meliponicultura en Costa Rica. Notas Apícolas [Heredia, Costa Rica] 144:5–47. Aguilera G, Gil Fl, González AC, Nieves B, Rojas Y, Rodríguez AM, Vit P. 2009. Evaluación antibacteriana de mieles de Apis mellifera, contra Escherichia coli y Staphylococcus aureus. Revista del Instituto Nacional de Higiene 402:1–25. Arce HG, van Veen JW, Sommeijer MJ, Ramírez JF. 1994. Aspectos técnicos y culturales de la crianza de abejas sin aguijón (Apidae: Meliponinae) en Costa Rica. pp. 7–17. In Memorias III Congreso Nacional de Apicultura. San José, Costa Rica. 75 pp. Arce HG, Sánchez LA, Slaa EJ, Sánchez-Vindas PE, Ortiz RA, van Veen JW, Sommeijer MJ. 2001. Árboles melíferos nativos de Mesoamérica, eds. PRAM / CINAT / UNA / UU; Heredia, Costa Rica. 207 pp. Berrocal SE. 1998. Determinación de las especies arbóreas utilizadas por las abejas sin aguijón (Apidae: Meliponinae) para anidar. Tesis de Licenciatura. Escuela de Ciencias Agrarias. Universidad Nacional, Heredia, Costa Rica. 100 pp. 7 Stingless Bees of Costa Rica 123 Camargo JMF, Pedro SRM. 2007. Meliponini Lepeletier, 1836. pp. 272–578. In Moure JS, Urban D, Melo GAR, eds. Catalogue of bees (Hymenoptera, Apoidea) in the Neotropical Region. Sociedade Brasilera de Entomologia; Curitiba, Brasil. 1958 pp. Camargo JMF, Pedro SRM. 2008. Meliponini Lepeletier, 1836. In Moure JS, Urban D, Melo GAR, Eds. Catalogue of Bees (Hymenoptera, Apoidea) in the Neotropical Region – online version. Available at: http://www.moure.cria.org.br/catalogue. De Jong H. 1999. The land of corn and honey. The keeping of stingless bees (meliponiculture) in the ethno-ecological environment of Yucatan (Mexico) and El Salvador. PhD thesis. 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In Frankie GW, Mata A, Vinson SB, eds. Biodiversity conservation in Costa Rica: learning the lessons in a seasonal dry forest. University of California Press; London, England. 341 pp. Griswold T, Parker FD and Hanson PE. 1995. The bees (Apidae). pp. 650–691. In Hanson PE and Gauld ID, eds. The Hymenoptera of Costa Rica. Oxford, UK. 893 pp. Hanson PH. 2000. Insects and Spiders. pp. 95–147. In Nalini MN, Wheelwright NT, eds. Monteverde: Ecology and Conservation of a Tropical Cloud Forest. Oxford University Press; New York, USA. 573 pp. Herrera E, Aguilar I. 2011. Situación actual de la meliponicultura en el Pacífico Sur de Costa Rica. pp. 55–58. In Memorias VII Seminario Mesoamericano sobre Abejas Nativas. Cuetzalan, México. 242 pp. Jarau S, Barth FG. 2008. Stingless bees of the Golfo Dulce region, Costa Rica (Hymenoptera, Apidae, Meliponini). Stapfia 88, zugleich Kataloge der oberösterreichischen Landesmuseen Neue Serie 802:67–276. Jiménez M. 2011. Guía para la crianza de abejas nativas. Fundación ALTROPICO. Quito, Ecuador. 20 pp. Kent RB. 1984. Mesoamerican stingless bees. Journal of Cultural Geography 41:4–28. Kevan PG. 1999. Pollinators as bioindicators of the state of the environment: species, activity and diversity. Agriculture, Ecosystems & Environment 743:73–393. Lobo JA. 1996. Notes of the biogeography of the stingless bees of Costa Rica. p.14. Abstracts of the Sixth IBRA Conference on Tropical Bees: Management and Diversity. San José, Costa Rica. 45 pp. Mahecha O, Nates-Parra G. 2002. Cría y Manejo de abejas sin aguijón (Hymenoptera: Meliponini) en Colombia. p. 59. In Memorias I Encuentro Colombiano sobre Abejas Silvestres; Bogotá, Colombia. 70 pp. Montoya FG, Carvajal K, Salas U. 2008. Descripción de la cultura del agua en Costa Rica: Pueblo Terraba. Available at http://www.unesco.org.uy/phi/aguaycultura/fileadmin/phi/foro2008/ Costa_Rica/Pueblo_Terraba.pdf Ortiz-Mora RA, van Veen JW. 1995. Influence of altitude on the distribution of stingless bees (Hymenoptera Apidae: Meliponinae). Apiacta 4:01–105. Ramírez JF, Ortiz RA. 1995. Crianza de las abejas sin aguijón. Centro de Investigaciones Apícolas Tropicales (CINAT). Universidad Nacional (UNA). Heredia, Costa Rica. 22 pp. Rincón RM. 1997. Riqueza y composición de especies de abejas (Hymenoptera:Apoidea) de sotobosque y de los recursos florales que utilizan, de un bosque neotropical manejado para producción de madera. Disertación de Maestría, CATIE. Turrialba, Costa Rica. 72 pp. 124 I. Aguilar et al. Roubik DW. 1992. Stingless bees: a guide to Panamanian and Mesoamerican species and their nests (Hymenoptera: Apidae: Meliponinae). pp. 495–524. In Quintero D, Aiello A, eds. Insects of Panamá and Mesoamerica. Oxford University Press; Oxford, UK. 692 pp. Roubik DW, Lobo AJ, Camargo JMF. 1997. New stingless bee genus endemic to Central American cloud forests: phylogenetic and biogeographic implications (Hymenoptera: Apidae: Meliponini). Systematic Entomology 226:7–80. Roubik DW. 2000. The de-flowering of Central America: a current perspective. In: Proceedings of the sixth international bee research conference on tropical bees IBRA, Cardiff, UK., pp 144–151 (p 289) Slaa EJ. 2003. Foraging ecology of stingless bees: from individual behavior to community ecology. Doctoral dissertation. Department of Behavioural Biology, Faculty of Biology, Utrecht University; Utrecht, The Netherlands. 181 pp. Slaa EJ, Sanchez LA, Sandí M, Salazar W. 2000. A scientific note on the use of stingless bees for commercial pollination in enclosures. Apidologie 311:41–142. Tous MM. 2002. De la gran Nicoya precolombina a la provincia de Nicaragua, s. XV y XVI. Tesis de Doctorado. Departamento de Historia, Universidad Autónoma de Barcelona. Barcelona, España. 525 pp. van Veen JW, Arce HG and Sommeijer MJ. 1993. Manejo racional de la abeja sin aguijón Melipona beecheii (Apidae: Meliponinae), I. Cómo transferir la colonia de un tronco hueco a una caja. pp. 41–45. In Memorias II Congreso Nacional de Apicultura: Perspectivas para una Apicultura Sostenible. San José, Costa Rica. 53 pp. van Veen JW, Bootsma MC, Arce H, Hallim MKI, Sommeijer MJ. 1990. Biological limiting factors for the beekeeping with stingless bees in the Caribbean and Central America. Social insects and Environment. pp. 772–773. In 11th International Congress IUSSI; Bangalore, India. 765 pp. Villanueva-Gutiérrez R, Roubik DW, Collin-Ucán W. 2005. Extinction of Melipona beecheii and traditional beekeeping in the Yucatan Peninsula. Bee World 863:5–41. Vit P, Gutiérrez MG, Rodríguez-Malaver AJ, Aguilera G, Fernández-Díaz C, Tricio AE. 2009. Comparación de mieles producidas por la abeja yateí (Tetragonisca fiebrigi) en Argentina y Paraguay. Acta Bioquímica Clínica Latinoamericana 432:19–26. Wagner PL. 1958. Nicoya, a cultural geography. Geography 121:95–250. Wille A. 1961. Las abejas jicótes del género Melipona (Apidae: Meliponini) de Costa Rica. Revista de Biología Tropical 241:23–147. Wille A, Michener CD. 1973. The nest architecture of the stingless bees with special reference to those of Costa Rica. Revista de Biología Tropical 211:–278. Chapter 8 Stingless Bees in Argentina Arturo Roig-Alsina, Favio Gerardo Vossler, and Gerardo Pablo Gennari 8.1 Introduction Stingless bees in Argentina are found throughout the northern portions of the territory, with the highest diversity in the humid forests of the northeast. Although the knowledge of these bees is deeply rooted in the cultural practices and the use that aboriginal peoples made of them, formal studies of stingless bees in Argentina are scattered over time and rather fragmentary. The first described species was Plebeia molesta (Puls, in Strobel 1868). Later, Holmberg (1887) recorded the habits and characteristics of several species in his article “Viaje a Misiones,” but referred to most of them by their vernacular names. The single most extensive account has been that of Silvestri (1902), who also traveled the province of Misiones. He surveyed the fauna recording and characterizing nine species, registered common names, and studied the nesting habits. His material, housed at the University of Portici, Naples, Italy, has been critically examined by Camargo and Moure (1988). Since that date there has been no other comprehensive treatment of Meliponini in Argentina. Other early work, but narrower in scope, consisted in the description of a few new species (Holmberg 1903; Vachal 1904; Schrottky 1911). By the time Schrottky (1913) published his “Distribución de los himenópteros argentinos” 17 species were known to occur. Schwarz, in his A. Roig-Alsina (*) Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Av. Angel Gallardo 470, 1405, Buenos Aires, Argentina e-mail: arturo@macn.gov.ar F.G. Vossler CONICET, Laboratorio de Sistemática y Biología Evolutiva (LASBE), Museo de La Plata, Paseo del Bosque s/n, 1900, La Plata, Argentina G.P. Gennari INTA Estación Experimental Agropecuaria Famaillá, Instituto Nacional de Tecnología Agropecuaria, Ruta Provincial 301 km 32, Famaillá, Tucumán, Argentina P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_8, © Springer Science+Business Media New York 2013 125 126 A. Roig-Alsina et al. Table 8.1 Distribution of stingless bees in Argentina. Nomenclature follows Camargo and Pedro (2007) Species Genus Northeast Cephalotrigona Frieseomelitta Geotrigona Lestrimelitta capitata (Smith)10,12 varia (Lepeletier)14 argentina Camargo & Moure4,12 sulina Marchi & Melo12, rufipes (Friese)14 muelleri (Friese)14 bicolor schencki Gribodo9,12, obscurior Moure4,12, quadrifasciata Lepeletier3,12, quinquefasciata Lepeletier3,12 caerulea (Friese)10,12 testaceicornis (Lepeletier)7,14 tataira (Smith)14 Leurotrigona Meliponaa Mourella Nannotrigona Oxytrigona Paratrigona Partamona Plebeia helleri (Friese)14 droryana (Friese)4,12, nigriceps (Friese)4,12 Scaptotrigona depilis (Moure)4,12, aff. postica (Latreille)14 quadripunctata (Lepeletier)3,12 clavipes (Fabricius)9,14 fiebrigi (Schwarz)4,12 Schwarziana Tetragona Tetragonisca Trigona Trigonisca spinipes (Fabricius)4,12 Chaco Northwest argentina chacoana Roig Alsina13 rufipes13 orbignyi (Guérin)12 baeri Vachal1,12, fuliginosa Lepeletier11 glabella Camargo & Moure glabella6,12 catamarcensis catamarcensis5,12, (Holmberg), wittmanni Moure molesta (Puls)2,12 & Camargo12 jujuyensis jujuyensis8,13 (Schrottky) fiebrigi aff. angustula (Latreille)14 spinipes sp.14 Superscript numbers refer to first citation of species for Argentina and nomenclatural updates 1 Burmeister (1861); 2Puls, in Strobel (1868); 3Holmberg (1887); 4Silvestri (1902) 5Holmberg (1903) 6Friese (1908); 7Bertoni (1911); 8Schrottky (1911); 9Schrottky (1913); 10Schwarz (1948); 11 Moure (1992); 12Camargo and Pedro (2007); 13Roig Alsina (2010); 14Museum specimens a Melipona titania, described by Gribodo from La Rioja in 1893, is most probably an erroneous record, since the xeric conditions of La Rioja are extremely different from the tropical conditions of the areas where the species actually occurs (see Camargo and Pedro 2008) revisionary works on Neotropical meliponines, added further records to the Argentinean fauna, mainly for the province of Misiones; he recorded this province as the southern limit of distribution of several of the species he studied (Schwarz 1932, 1948). More recently, Almeida and Laroca (1988) studied the single species of Trigona present in Argentina, and Camargo and Moure (1994, 1996) described two new species in the genera Paratrigona and Geotrigona. Camargo and Pedro (2007), in their comprehensive catalog of the Neotropical Meliponini, listed all the known records for every species, updating their systematics, and mentioning for Argentina 22 species in 12 genera. Later additions (Roig Alsina 2010), and a scrutiny of museum specimens, indicate the presence of 33 species in 18 genera (Table 8.1). 8 Stingless Bees in Argentina 127 When these figures are compared to those of the Neotropical region as a whole (391 species in 32 genera, Camargo and Pedro 2007), the low species richness (8.4%) is evident, but it is striking that over half of the genera (56.2%) are present in the Argentinean fauna. Thirteen of these genera are represented by a single species. This is in agreement with the observation made in Brazil by Biesmeijer and Slaa (2006) that local meliponine assemblages tend to consist of one or a few species of many different genera. In this chapter we compile current information on the systematics, distribution, traditional knowledge, use as a resource, and recent studies on the biology and ecology of meliponines in Argentina. 8.2 Distribution of Stingless Bees in Argentina Argentina represents in South America a marginal area for the rich tropical fauna of meliponines. This is particularly the case of the northeast, where the Paranaense forest enters the province of Misiones and the northern part of the province of Corrientes (Cabrera and Willink 1973). This area has the highest record of species (Table 8.1), all of which also occur in Brazil and most of them also in eastern Paraguay. A second tropical forest, the Yungas, occurs in the northwestern mountain region of Argentina. The Yungas extends southward, penetrating as a slender wedge in the provinces of Jujuy, Salta, and Tucumán and reaching northern Catamarca (Cabrera and Willink 1973). The fauna of this region is the least surveyed, and the one that may provide new additions to the number of species present in the country. Between these two regions is the Chaco, a biogeographic unit with xeromorphic forests and savannas (Cabrera and Willink 1973; Prado 1993). Here the precipitations diminish to the west, so the central and western areas have a long, unfavorable, dry season. This region harbors the most distinctive fauna of meliponines in Argentina, although the poorest in number of species (Table 8.1). The Chaco not only occupies north-central Argentina but also western Paraguay, southeastern Bolivia, and the extreme western edge of the state of Mato Grosso do Sul in Brazil (Prado 1993). The distinctiveness of its fauna has been noted by Camargo and Moure (1994, 1996). The boundaries of the three regions just mentioned are not absolutely distinct when the meliponine fauna is considered, and some species range into neighboring areas. This is the case of Tetragonisca fiebrigi, which occurs both in the Chaco and in the Paranaense forest. Scaptotrigona jujuyensis, a species characteristic of the Chaco, cohabits in Tucumán with Trigona spinipes, a species that does not occur in the xeromorphic central region. The southernmost records of meliponines in the western hemisphere are represented by four species of Plebeia that manage to survive in temperate climates. Two of them are elements of the Chacoan fauna. Plebeia molesta was described from San Luis (Strobel 1868), but without indication of whether the province or the city of San Luis was meant. Specimens with sound data come from the northern part of the province of San Luis at 32°30¢ S latitude. The second species, Plebeia catamarcensis, has been recorded as far south as 31°20¢ S latitude in the province of Santa Fe 128 A. Roig-Alsina et al. (Dalmazzo 2010). The two other species are elements of the Paranaense fauna, which extends its range southwards through the gallery forests growing along the Paraná and Uruguay Rivers, reaching the western margin of the Río de La Plata in the province of Buenos Aires. The two species, the identity of which is currently under study, are found as far south as 34°37¢ S latitude. 8.3 Traditional Knowledge on Stingless Bee Biodiversity Stingless bees were exploited and well known by different cultures in northern Argentina before the arrival of Europeans. This knowledge is reflected in the many and accurate names by which different species were known by local people. Holmberg (1887), Silvestri (1902), and Bertoni (1911) recorded Guaraní vernacular names for the bees that they surveyed in Misiones, and Bertoni also in Paraguay. The alimentary customs of the Guaraní people in Misiones have been documented by Martínez Crovetto (1968). In the early eighteenth century Jesuit missionaries described the abundance of bees and honey in the Chaco region, and the importance of these insects for the Guaycurú people, as well as the uses that they made of the honey and other products of stingless bees (Medrano and Rosso 2010a, b). Arenas (2003), in a comprehensive ethnographic study of the Wichi and Toba peoples of central Chaco, describes the prominent role that stingless bees have played, and still play presently, in their culture. Nearly all of the species present in the Chaco are individually recognized and have their own names in both ethnic groups. Honey has been important in the production of alcoholic beverages, particularly for festivities, besides being used as a nourishment and as a sweetener for other foods and diluted with water for children. There is an oral tradition regarding which honeys have curative effects for various ailments. Pollen masses and larvae were also consumed, and cerumen was used to mend water containers, as well as in the making of various utensils. The Quechuan lexicon compiled by Bravo (1975) in the province of Santiago del Estero includes the names of several species of meliponines, although some such names refer to the hives rather than to the bees themselves. Names such as “yana” (Scaptotrigona jujuyensis), “ashpamishki” (Geotrigona argentina), “tíu simi” (Melipona spp.), and “ckella” and “pusquellu” (Plebeia spp.) are broadly used nowadays in northern Argentina. The creole population has also developed their own vernacular names in Spanish, such as “negrito,” “peluquerito,” and “rubita.” Some of them are indicated in Table 8.2. 8.4 Meliponini as a Natural Resource We present here preliminary results of a survey aimed at knowing which of the many species of stingless bees are exploited or reared nowadays by the local population in northern Argentina. The survey is being carried out in the provinces Region Species Common name Harvested in the field Kept near dwellings in logs, or rustic hives Northeast Nannotrigona testaceicornis Tetragona clavipes Tetragonisca fiebrigi Scaptotrigona aff. postica Melipona obscurior Plebeia spp. Geotrigona argentina Scaptotrigona jujuyensis Tetragonisca fiebrigi Plebeia spp. Melipona orbignyi Tetragonisca aff. angustula Scaptotrigona jujuyensis Plebeia spp. * * *** *** ** * * *** *** * * *** ** * * “borá” “yateí” “tapezuá,” “tobuna” “mandurí” “mirim” “alpamiski” “negrito,” “tapezuá” “rubiecito” “apynguarei,” “shimilo” “moro-moro” “rubiecito,” “mestizo” “yana” “pusquello,” “quella” Chaco Northwest Kept in man-made hives ** ** * * *** ** * * * *** *** * * * ** *** * ** 8 Stingless Bees in Argentina Table 8.2 Species of stingless bees exploited in northern Argentina A higher degree of exploitation is indicated by an increased number of asterisks 129 130 A. Roig-Alsina et al. of Misiones, Chaco, Formosa, Salta, Jujuy, and Tucumán, under a project leaded by INTA (Gennari 2009). The nests of several species are known to be harvested in the field when they are spotted. This practice includes both species with subterraneous and arboreal habits (Table 8.2). The data in the table reflect the present survey, but other species are known to be collected in the field as they are encountered (e.g., Arenas 2003). Trees are frequently felled to obtain arboreal nests, an undesirable practice. In some cases, logs containing the nests are cut down and then kept near the dwellings, so they can be opened, harvested, and resealed, becoming rustic hives. The survey indicates that this type of extractive exploitation is frequent and widespread. Rustic hives of Tetragonisca, Scaptotrigona, and, to a lesser extent, Plebeia are common in Misiones, Formosa, and Chaco, and less frequent in the northwest. Although extractive exploitation is a traditional undertaking in rural communities, formal meliponiculture is a recent development in the area. The interest in the use of man-made hives and in the manipulation of the colonies is steadily growing in Argentina, but only a few species are being reared rationally. The most widely cultivated stingless bees are Tetragonisca fiebrigi, T. aff. angustula, and Scaptotrigona jujuyensis. The colonies of Tetragonisca are small, and the harvest modest, but these bees are highly esteemed because of the quality of their honey. Scaptotrigona are favored because of the relatively large colony size, strength of the colonies, and large honey harvest. Both governmental and nongovernmental organizations are engaged in projects to promote sustainable beekeeping of stingless bees. The government of the province of Misiones promotes the culture of meliponines (CEDIT 2005) and supports regular meetings of producers of honey of Tetragonisca fiebrigi. The national government also promotes projects through several agencies, such as the Consejo Federal de Ciencia y Tecnología, the Secretaría de Ambiente y Desarrollo Sustentable (Meriggi et al. 2008), and the Instituto Nacional de Tecnología Agropecuaria (Gennari 2009). There are several nongovernmental organizations engaged in community-level development of meliponiculture. Some such organizations are the Asociación para la Promoción de la Cultura y el Desarrollo (APCD), in the province of Formosa, which works with the Wichi people. The Fundación Proyungas works to qualify individuals in the management of stingless bees in the northwest (Stamatti 2006; Baquero and Stamatti 2007). 8.5 Recent Studies on Biology and Ecology of Argentine Meliponines There has been an upsurge of interest in the study of meliponines in Argentina in recent years. Most contributions have been oriented to the study of the pollen resources used by stingless bees. Palynological analyses of honeys and the contents of pollen pots have been carried out in the Northwest for Tetragonisca aff. angustula (Flores and Sánchez 2010); in the Chaco region for Geotrigona argentina (Vossler 2007a; Vossler and Tellería 2009b; Vossler et al. 2010), for Tetragonisca fiebrigi (Cabrera 2007; Vossler 2007a, b, 2011; Vossler and Tellería 2009a), and for 8 Stingless Bees in Argentina 131 Scaptotrigona jujuyensis (Basilio et al. 2006; Vossler 2007a, b; Vossler and Tellería 2009a; Basilio et al. 2011; Vossler 2011); and in the Northeast for Tetragonisca fiebrigi (Fabbio et al. 2007; Dallagnol et al. 2007; Paul et al. 2009, 2011; Flores et al. 2011). Flower visitation has been also used to study the resources harvested by stingless bees. Vossler (2009, 2012) investigated with this methodology six species of Meliponini in the Chaco (Tetragonisca fiebrigi, Scaptotrigona jujuyensis, Geotrigona argentina, Melipona orbignyi, Plebeia catamarcensis, and Plebeia molesta). Other studies deal with nesting ecology (Basilio et al. 2006; Colleselli et al. 2008; Vossler 2012), management (Achával et al. 2006), medicinal uses of honeys (Zamudio et al. 2011; Kujawska et al. 2012), physicochemical characterization of honeys (Vit et al. 2009, Sgariglia et al. 2010, Salomón et al. 2011, Basilio et al. 2011), antimicrobial properties of honeys (Dallagnol et al. 2007; Sgariglia et al. 2010), and ethnobiology (Zamudio and Hilgert 2012). 8.6 Future Research Knowledge on the biodiversity of stingless bees in Argentina is not satisfactory. The systematics of some genera, such as Scaptotrigona and Plebeia, which have several species in the region, is poorly resolved. Some areas have not been adequately surveyed. A more intensive exploration of the Yungas may uncover additional species for the Argentinean fauna. Studies on several aspects of biology, such as nesting behavior, reproduction, caste development, feeding habits, as well as practical matters such as multiplication and management of colonies, and handling of their products, are almost nonexistent for many species. Even for those species that occur in Brazil and have received much attention, their behavior in southern marginal areas may reveal particular issues that merit further study. Undoubtedly, a better knowledge of the fauna will help decide which species can be selected for meliponiculture in specific areas. Acknowledgements This contribution is part of the project Abejas nativas con importancia social, económica y ambiental, INTA, Argentina, PNAPI-123032. ARA acknowledges support of grants ANPCyT Argentina, 2007/1238 Préstamo BID, and CONICET Argentina, PIP 2011-288. We appreciate the invitation extended by Dr. Silvia RM Pedro to contribute this chapter, and the comments of referees and editors. 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Vossler FG. 2012. Flower visits, nesting and nest defence behaviour of Stingless bees (Apidae: Meliponini): suitability of the bee species for Meliponiculture in the Argentinean Chaco region. Apidologie 43:139–161. Vossler FG, Tellería MC. 2009a. Origen botánico de la miel y el polen almacenado por abejas “meliponinas” nativas del Chaco Seco de Argentina. p. 133. In Libro de resúmenes 1º Jornada Patagónica de Biología y 3º Jornadas estudiantiles de Ciencias Biológicas, Trelew (Chubut). Vossler FG, Tellería MC. 2009b. Estudio palinológico de las reservas alimentarias de la abeja Geotrigona argentina en el bosque de “palosantal” chaqueño (Apidae, Meliponini). Boletín Sociedad Argentina Botánica 42:138. Vossler FG, Tellería MC, Cunningham M. 2010. Floral resources foraged by Geotrigona argentina (Apidae, Meliponini) in the Argentine Dry Chaco forest. Grana 49:142–153. Zamudio F, Hilgert NI. 2012. Descriptive attributes used in the characterization of stingless bees (Apidae: Meliponini) in rural populations of the Atlantic forest (Misiones-Argentina). Journal of Ethnobiology and Ethnomedicine, 8:9, as DOI:10.1186/1746-4269-8-9. Zamudio F, Kujawska M, Hilgert NI. 2011. Honey as Medicinal and Food Resource. Comparison between Polish and Multiethnic Settlements of the Atlantic Forest, Misiones, Argentina. The Open Complementary Medicine Journal 2:1–16. Chapter 9 Mexican Stingless Bees (Hymenoptera: Apidae): Diversity, Distribution, and Indigenous Knowledge Ricardo Ayala, Victor H. Gonzalez, and Michael S. Engel 9.1 Introduction Stingless bees (Meliponini) are highly eusocial apine bees restricted to the tropical and subtropical areas of the world but are most diverse in the Western Hemisphere, where about 80% of the nearly 500 known species worldwide are found (Michener 2007; Camargo and Pedro 2007). In the Western Hemisphere, stingless bees occur from Mexico to Brazil and northern Argentina, and also on Caribbean and Pacific Islands, inhabiting a diverse variety of ecosystems, including both humid and xeric lowlands to cloud forests and Páramos in the Andes reaching up to 4,000 m in elevation (Gonzalez and Engel 2004; Nates-Parra 2005; Michener 2007; Camargo and Pedro 2007). Stingless bees are ecologically, economically, and culturally important. They are considered among the major pollinators of many native and cultivated tropical plants (e.g., Slaa et al. 2006), while pollen, honey, and cerumen of some species have also been used traditionally by indigenous and non-indigenous people in rural areas across the Americas, thus representing an important source of income for these communities (e.g., Nates-Parra 2005; Michener 2007; and references therein). In addition, the shared cultural heritage of these people is integrally tied, in some respects, to the stingless bees which they exploit, representing an inestimable value well beyond modern fiscal concerns. Despite the importance of stingless bees and R. Ayala (*) Estación de Biología Chamela, Instituto de Biología, Universidad Nacional Autónoma de México (UNAM), Apartado Postal 21, San Patricio, Jalisco, 48980, Mexico e-mail: rayala@ibiologia.unam.mx V.H. Gonzalez Southwestern Oklahoma State University, Biological Sciences, USA M.S. Engel Division of Entomology, Natural History Museum, University of Kansas, 1501 Crestline Drive – Suite 140, Lawrence, KS 66045, USA P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_9, © Springer Science+Business Media New York 2013 135 136 R. Ayala et al. several decades of research, most species hypotheses remain to be tested and many species await discovery. Each species, as recognized by an individual systematist, is a hypothesis subject to rigorous testing when other species, specimens, or characters (morphological or molecular) are discovered (e.g., Wheeler 2004, 2009). A vast majority of stingless bee species have never been tested since they were proposed at the beginning of the nineteenth century by scientists who either had an obsolete species concept or a limited knowledge of their biology, distribution, and morphology, given the specimens and technology available at the time. Stingless bees are characterized by an abundance of cryptic species (i.e., morphologically very similar species), only distinguished by fine morphological characters as well as by differences in nesting biology and distribution (Michener 2007). Moreover, large areas in tropical America have never been or are poorly explored and their stingless bee fauna remains practically unknown. Likewise, there remains no consensus on the generic and subgeneric classification of stingless bees. Some authors prefer to emphasize the differences, recognizing species or groups of species with unusual characters at the generic or subgeneric rank, resulting in some 60 supraspecific taxa. As in Gonzalez and Griswold (2011), herein we follow Michener’s classification for Neotropical Meliponini, except that we recognize at the generic level those taxa he placed as subgenera of Trigona Jurine and Plebeia Schwarz (Table 9.1). Exploring, discovering, testing species hypotheses, and building sound phylogenies that allow us to understand the evolutionary process and develop stable classifications with predictive values for diverse biological and ecological traits is a dynamic, complex process that requires years of professional training, substantial knowledge of the bees and their environment, time, and an investment in both financial and human capital. Such processes are vital not only to understand the true diversity and evolution of the group but also to promote their conservation and sustainable use. In Mexico, stingless bees represent a relatively small portion (2.6%) of the highly diverse bee fauna of the country, but the economic, social, and cultural impacts they have are like no other in the world. A large percentage (41.3%) of the comparatively small Mexican stingless bee fauna has been used since pre-Colombian times when compared to other countries with more diverse meliponine faunas (Table 9.2). Mexican stingless bees played a significant role in the religion of the Mayans, one of the most important ancient civilizations of the world; and stingless bees and their products are used for diverse purposes, including managed pollination, folk medicine, art, and honey, cerumen and pollen extraction (Fig. 9.1a–h). Herein, we provide a synopsis of the diversity, biogeography, origin, and traditional uses of the stingless bees in Mexico. 9.2 Diversity Studies on the Mexican stingless bee fauna started as early as the beginning of the nineteenth century (Latreille 1811; Guérin-Méneville 1844; Bennett 1831; Say 1837; Cresson 1878; Dalla Torre 1896; Friese 1900; Cockerell 1913; Strand 1917; Schwarz 9 Mexican Stingless Bees (Hymenoptera: Apidae)... 137 Table 9.1 Stingless bee genera present in Mexico with the total number of species in the neotropics and in Mexico Genus Total Mexico Cephalotrigona Schwarz Frieseomelitta Ihering Geotrigona Moure Lestrimelitta Friese Melipona Illiger Nannotrigona Cockerell Nogueirapis Moure Oxytrigona Cockerell Paratrigona Schwarz Partamona Schwarz Plebeia Schwarz Proplebeia Michenerc Scaptotrigona Moure Scaura Schwarz Tetragona Lepeletier de Saint Fargeau & Audinet-Serville Tetragonisca Moure Trigona Jurine Trigonisca Moure 5 16 21 21 70 10 4 11 34 39 42 4 22 7 19 4 32 43 3 (2) 1 1 2 (1) 6 (3) 1 1a (1) 1 2b 2 11 (4) 1d 3 (1) 1 1 1 5 5 (2) A number of endemic species to the country are given in parentheses. Generic classification follows Michener (2007) except by those taxa he placed as subgenera of Trigona and Plebeia which are herein recognized at the generic level. The approximate number of species in the neotropics is based on Michener (2007), Camargo and Pedro (2007), Ascher and Pickering (2011), and SRM Pedro (personal communication) a One extinct species is known in Miocene Chiapas amber b Camargo and Moure (1994) listed P. opaca for Chiapas but we have not seen yet specimens of this species c Extinct, early Miocene Dominican and Chiapas amber d Engel (2004a) recorded an unidentified species in Mexican amber Table 9.2 Total number of bee species, stingless bees, and species of stingless bees used in some Latin American countries. Bee diversity per country is based on Ascher and Pickering (2011), while estimations for the exploited number of stingless bee species are based on the corresponding citation Total bee Stingless Stingless bees Country species bees (%) used (%) Reference Mexico Costa Rica 1,795 785 46 (2.6) 58 (7.3) 19 (41.3) 2 (4.2) Colombia French Guiana 541 210 101 (20.0) 80 (38) 17 (16.8) 2 (2.5) Peru 688 100 (14.5) 12 (12) Brazil 1,814 236 (13.0) 21 (8.9) Herein Roubik (2000), Aguilar et al., this volume Nates-Parra (2005) Roubik (1979), and Pauly et al., this volume C. Rasmussen (personal communication) Crane (1992) 138 R. Ayala et al. Fig. 9.1 Economic and cultural importance of stingless bees in Mexico. (a) Workers of Melipona beecheii on a brood comb; (b, c) nest entrance and managed hives of Scaptotrigona mexicana; (d) worker of Nogueirapis silacea preserved in Early Miocene amber from Chiapas; (e) temple of the “descending god” in Tulum, Quintana Roo. Ah-muzen-cab, one of the Mayan gods of bees and honey, is enlarged in the box of the lower left corner; (f) Huichol artisan using Scaptotrigona hellwegeri cerumen on a piece of wood for his work with chaquira beads; (g) Huichol art depicting stingless bees; (h) feather art. Photos: (a-c) C. Balboa, J. Mérida, M. Guzmán; (d) V. Gonzalez; (e-h) R. Ayala 9 Mexican Stingless Bees (Hymenoptera: Apidae)... 139 1948, 1949; Camargo et al. 1988; Ayala 1988, 1997, 1999; Ayala et al. 1993, 1996), although earlier biological accounts before Linnaean nomenclature or standardized concepts of species were given (e.g., Hernandez 1648; Purchas 1657). The most recent synthesis on the diversity of the Mexican stingless bee fauna is that of Ayala (1999). In that work, the status of species was clarified, and several species, accounting for 36% of the total number of Mexican meliponines known to date, were discovered to Science. Today, 46 species belonging to nearly all extant Neotropical stingless bee genera are known in Mexico (Table 9.3), except Meliwillea Roubik et al., endemic to the mountains of Costa Rica and Panama, and the genera Duckeola Moure, Paratrigonoides Camargo and Roubik, Schwarziana Moure, and Trichotrigona Camargo and Moure, which occur in South America. Plebeia, Trigona, Melipona Illiger, and Trigonisca are the most diverse genera in Mexico with 12, nine, six, and five species, respectively. Because Mexico is located in the northernmost range of the stingless bees in the Americas, it possesses a relatively low number by comparison to that of much smaller countries that are closer to the equator (Tables 9.2 and 9.3). The Pacific Coast, from Guerrero to Chiapas, and southern Veracruz are areas that contain the greatest number of stingless bee species (Fig. 9.2a, b). An undescribed species of the extinct genus Proplebeia Michener, one of the two known extinct stingless bee lineages in the Americas, and a single extinct species of the presently living South American genus Nogueirapis Moure, N. silacea (Wille 1959) (Fig. 9.1d), are known from the Early Miocene (17–19 myo) Chiapas amber, near Simojovel (Wille 1959; Engel 2004a). The attribution of N. silacea to Proplebeia by Camargo et al. (2000) and Camargo and Pedro (2007) is based upon a misinterpretation between the former and a true Proplebeia species in the Mexican amber fauna. The holotype of N. silacea was not examined and their remarks were based upon Wille’s account (1959:850, 851). Examination of the holotype of N. silacea by MSE reveals it to be a true Nogueirapis. 9.3 Distribution Based on the distribution and type of vegetation in their habitat, Mexican stingless bees can be divided into three large ecological groups: (I) species widely distributed and associated with both tropical deciduous and evergreen forests; (II) species associated with tropical evergreen forest; and (III) endemic species associated with various forest types. Group I. These species follow three distinct distribution patterns: 1. Wide montane and tropical distribution. Partamona bilineata is the only representative of this pattern. This species is present in the Sierra Madre del Sur, from Michoacán to Oaxaca, in the southern slope of the transverse volcanic axis (Eje Volcánico Transversal), Balsas River Basin; it reaches Sinaloa and San Luis Potosí through the Pacific and Gulf slopes. The species occurs in lowlands with tropical deciduous and evergreen forests, and in montane pine-oak forests. Uses Distribution Cephalotrigona eburneiventer (Schwarz, 1948)a C. oaxacana Ayala, 1999a C. zexmeniae (Cockerell, 1912) H Colima, Guerrero, Michoacán, Morelos, Puebla Oaxaca Campeche, Chiapas, Quintana Roo, San Luis Potosí, Tabasco, Tamaulipas, Veracruz, Yucatán Campeche, Chiapas, Colima, Guerrero, Jalisco, Michoacán, Nayarit, Oaxaca, Puebla, Quintana Roo, Sinaloa, Yucatán Chiapas, Estado de México, Guerrero, Michoacán, Morelos, Oaxaca Colima, Jalisco, Guerrero, Nayarit Chiapas, San Luis Potosí, Quintana Roo, Veracruz, Yucatán Campechec, Chiapasc, Jalisco, Nayarit, Oaxaca, Quintana Rooc, San Luís Potosí, Sinaloa, Tabasco, Tamaulipas, Veracruzc, Yucatánc Colima, Jalisco Estado de México, Michoacán, Guerrero, Morelos, Oaxaca, Veracruz Michoacán Chiapas Oaxaca, Yucatán Campeche, Chiapas, Jaliscoc, Michoacán, Morelos, Nayaritc, Oaxaca, Puebla, Quintana Roo, San Luis Potosí, Sinaloac, Veracruz, Yucatán Chiapas Chiapasc, Veracruz Chiapas Campeche, Chiapas, Colima, Distrito Federal, Durango, Guerrero, Jalisco, Michoacán, Morelos, Nayarit, Oaxaca, Puebla, Quintana Roo, San Luis Potosí, Sinaloa, Tabasco, Veracruz, Yucatán Veracruz Zacatecas, Sinaloa Campeche, Chiapas, Colima, Jalisco, Michoacán, Nayarit, Nuevo León, Oaxaca, Puebla, Quintana Roo, San Luis Potosí, Veracruz, Yucatán H Frieseomelitta nigra Cresson, 1878 Geotrigona acapulconis (Strand, 1919) Lestrimelitta chamelensis Ayala, 1999a L. niitkib Ayala, 1999 Melipona beecheii Bennett, 1831 Hb M. colimana Ayala, 1999a M. fasciata Latreille, 1811a M. lupitae Ayala, 1999a M. solani Cockerell, 1912 M. yucatanica Camargo et al., 1988 Nannotrigona perilampoides (Cresson, 1878) H H H Oxytrigona mediorufa (Cockerell, 1913) Paratrigona guatemalensis (Schwarz, 1938) P. opaca (Cockerell, 1917) Partamona bilineata (Say, 1837) P. orizabaensis (Strand, 1919) Plebeia cora Ayala, 1999a P. frontalis (Friese, 1911) M, H, C, P H, P H Hb R. Ayala et al. Stingless bee species 140 Table 9.3 Stingless bees of Mexico with information on state distribution and uses. Uses: Cerumen used for feather and bead arts (A), honey (H), Meliponiculture (M), pollination (P), cerumen (C). S. mexicana (Guérin-Méneville, 1844) M, H, C, P S. pectoralis (Dalla Torre, 1896) Scaura argyrea (Cockerell, 1912) Tetragona mayarum (Cockerell, 1912) Tetragonisca angustula (Latreille, 1811) Trigona corvina Cockerell, 1913 T. fulviventris Guérin-Méneville, 1844 Hb T. fuscipennis Friese, 1900 T. nigerrima Cresson, 1878 T. silvestriana (Vachal, 1908) Trigonisca azteca Ayala, 1999a T. maya Ayala, 1999 T. mixteca Ayala, 1999a T. pipioli Ayala, 1999 T. schulthessi (Friese, 1900) M, H, C Hb, Cb Estado de México, Guerrero, Morelos, Oaxaca, Puebla Chiapas, Oaxaca, Puebla, Quintana Roo, San Luis Potosí, Veracruz Chiapas: Hidalgo; Puebla, Quintana Roo, San Luis Potosí, Veracruz Colima, Durango, Estado de México, Guerreroc, Jaliscoc, Michoacánc, Morelos, Nayaritc, Oaxaca, Puebla, Chiapas Chiapas, Guerrero, Hidalgo, Morelos, Oaxaca, Pueblac, San Luis Potosí, Veracruz, Guerrero Campeche, Chiapas, Oaxaca, Quintana Roo, Veracruz, Yucatán Chiapas, Veracruz Chiapas, Tabasco Chiapas, Veracruz Campeche, Chiapas, Oaxaca, Quintana, Veracruz Campeche, Chiapas, Colima, Jalisco, Michoacán, Nayarit, Oaxaca, Quintana Roo, Veracruz, Yucatán Campeche, Chiapas, Oaxaca, Puebla, Quintana Roo, Veracruz, Yucatán Chiapas, Oaxaca, Tabasco, Veracruz Chiapas, Campeche y Quintana Roo Guerrero, Estado de México, Morelos, Puebla Quintana Roo, Yucatán Chiapas, Oaxaca Chiapas, Jalisco, Quintana Roo, Oaxaca Veracruz, Yucatán Chiapas, Oaxaca Endemic species Species that are rarely used for a given purpose are indicated after the abbreviation for that use c Localities where the species is used Mexican Stingless Bees (Hymenoptera: Apidae)... A, H, C, Mb, P H Guerrero Chiapas, Quintana Roo, Veracruz Chiapas, Veracruz Colimac, Jaliscoc Chiapas, Quintana Roo, San Luis Potosí, Veracruz 9 P. fulvopilosa Ayala, 1999a P. jatiformis (Cockerell, 1912) P. llorentei Ayala, 1999 P. manantlensis Ayala, 1999a P. melanica Ayala, 1999 P. mexica Ayala, 1999a P. moureana Ayala, 1999 P. parkeri Ayala, 1999 P. pulchra Ayala, 1999 Scaptotrigona hellwegeri (Friese, 1900)a a 141 b 142 R. Ayala et al. Fig. 9.2 Distribution of stingless bees in Mexico. (a) Relative density of the number of species. The darker the area, the more species coexist in the same place as indicated by the color legend. (b) Areas of endemism indicating some of the 16 endemic species of the country (see text for explanation) 9 Mexican Stingless Bees (Hymenoptera: Apidae)... 143 2. Wide coastal distribution. Consists of Melipona beecheii (Fig. 9.1a), Plebeia frontalis, Trigona fulviventris, and Trigonisca pipioli, which reach Sinaloa and San Luis Potosí through the Pacific and Gulf slopes; they also occur in Chiapas (probably not in the mountains) with a few isolated records from the Balsas River Basin (M. beecheii and T. pipioli). The species appear to be stenothermic (living only within a narrow temperature range), sometimes reaching areas of transition between tropical and mountain vegetation, including cloud forests. Plebeia frontalis is the most widely distributed of the four species, reaching the state of Nuevo León through the Gulf Coastal Plain. 3. Special cases of species with wide distribution. This pattern is exhibited by Nannotrigona perilampoides and Frieseomelitta nigra. The former species reaches more northern areas (up to 29°N) than the latter, through the Pacific slope (Bennett 1964). It is also present in the southern slope of the transverse volcanic axis, between 1,000 and 1,500 m, primarily in areas with cloud forests. Nannotrigona perilampoides is absent from areas with tropical dry or xerophytic vegetation such as those in Chamela, Jalisco (Ayala 1988), and east of the Balsas River Basin. Frieseomelitta nigra is found in the Pacific coast, Balsas River Basin, and Yucatán Peninsula but does not reach them through the Gulf coast. Both species seem to have a broad ecological valence that allows them to survive in areas with food resources and nesting sites available year round. Group II. This group comprises 50% of the Mexican stingless bee species. The following four distribution patterns can be recognized: 1. Species restricted to Chiapas: M. solani, T. mayarum, and T. silvestriana. 2. Species that follow the distribution of the tropical evergreen forest but are absent from Yucatán, reaching central Veracruz or southeastern San Luis Potosí. Species of this group are found in the mountains above 1,000 m, occurring in conifer and cloud forests, such as P. llorentei, P. melanica, P. pulchra, S. argyrea, and T. nigerrima. 3. Species distributed as above but present throughout the Yucatán Peninsula, occupying drier areas with tropical deciduous forests, such as L. niitkib and S. pectoralis. 4. Species presumably restricted to the Pacific coast of Chiapas, near Tapachula. Only Oxytrigona mediorufa and Trigonisca schulthessi are known to exhibit this distribution. Group III. Thirteen species are endemic to Mexico (Tables 9.1 and 9.3). The distribution of such endemism defines areas that are often disjunct, suggesting possible vicariance events that have resulted in sister or closely related species. The following are the recognized areas with endemisms: 1. Southern half of the Tehuantepec Isthmus. Three species (C. oaxacana, M. yucatanica, and T. mixteca), adapted to tropical deciduous and semi-deciduous forests, are found in that area. Melipona yucatanica is also found in southern Yucatán, as well as Belize and Quintana Roo (DW Roubik, personal communication). It is possible that this species is associated with tropical savannah vegetation (Fig. 9.2b). 144 R. Ayala et al. 2. Southern mountains (transverse volcanic axis and Sierra Madre del Sur) with four species (M. colimana, M. fasciata, P. fulvopilosa, and P. manantlensis) of insular distribution and present in the mountains between 1,000 and 3,000 m. They appear to be phylogenetically related to those species associated with the tropical evergreen forest from southeastern Mexico and Central America. Plebeia fulvopilosa is restricted to the Sierra Madre del Sur in Guerrero; P. manantlensis and M. colimana are restricted to the mountains of southeastern Jalisco (North of Colima, Volcán Colima, Sierra de Manantlán, and Sierra del Tigre), which represent an isolated group of mountains from the remaining transverse volcanic axis; M. fasciata is a montane species widely distributed in Mexico, occurring from the southern slope of the transverse volcanic axis to west of Michoacán, and in the Sierra Madre del Sur, from Guerrero to Oaxaca. 3. Balsas River Basin. Cephalotrigona eburneiventer, P. mexica, and T. azteca occur in the lower basin (Guerrero, Morelos, Puebla, and the central region, east of Michoacán), while M. lupitae occurs in the upper basin (Michoacán). The dominant vegetation types of the area are tropical deciduous or semi-deciduous forests and xeric vegetation. The species of this river basin seem to be closely related with those of tropical evergreen forests or deciduous forests from the Tehuantepec Isthmus. 4. Northern Nayarit, southern Sinaloa, and southeastern Zacatecas. Plebeia cora occurs in this area, a species presumably closely related to P. mexica from the lower basin of the Balsas River (Fig. 9.2b). 5. Pacific Coast between southern Oaxaca and Sinaloa. Scaptotrigona hellwegeri, L. chamelensis, and Geotrigona acapulconis are endemic to this area; the first and last species are also found in the Balsas River Basin and in the mountains up to 2,000 m. Several species are often found at mid- and high elevations in the mountains. Melipona fasciata, P. bilineata, and G. acapulconis are often found at elevations above 2,000 m. Other species, such as N. perilampoides, T. corvina, T. fulviventris, and T. fuscipennis, occur from sea level up to 1,500 m. Melipona fasciata, M. colimana, P. fulvopilosa, and P. manantlensis are only found above 1,500 m and only in some areas, such as in the Sierra de Atoyac (southeastern slope of Sierra Madre del Sur in Guerrero); M. fasciata is frequently found at elevations around 2,400 m in northern Morelos (southern slope of the transverse volcanic axis) and has been collected at 3,000 m in the Sierra Madre del Sur, Guerrero, the highest elevation record for stingless bees in Mexico. 9.4 Origin of the Mexican Stingless Bees The extant stingless bee fauna of Mexico seems to be the result of recent migrations of Central or South American taxa during the Pliocene and Pleistocence when the Mexican plateau and its surrounding mountains were already present, such as that described for vegetation and other organisms (e.g., Halffter 1976; Simpson and Neff 9 Mexican Stingless Bees (Hymenoptera: Apidae)... 145 1985). If that is the case, then the current areas of endemism likely resulted from vicariance events that occurred during the climatic changes of the Pleistocene (e.g., Toledo 1982), as evidenced by the presence of endemic species or species with disjunct or insular distributions. The presence of N. silacea in Chiapas amber not only indicates that Nogueirapis, now known only from Bolivia to Costa Rica, occurred as far north as southern Mexico but also that it must have reached it well before the Central American land bridge was formed during the Pliocene (e.g., Moure and Camargo 1982). Halffter (1978, 1987) suggests that migrations between South and North America during the Oligocene– Miocene transition were possible, yet difficult. However, given that Cretotrigona prisca (Michener and Grimaldi) is known from the latest Cretaceous New Jersey amber in North America (Michener and Grimaldi 1988a,b; Engel 2000) and Proplebeia from both Dominican and Chiapas amber (e.g., Wille and Chandler 1964; Wille 1977; Greco et al. 2011), alternatively it is possible that Nogueirapis is a remnant of a more northern meliponine lineage that inhabited southern Mexico or present-day Guatemala and Honduras (Donnelly 1988), during the latest Cretaceous or Early Tertiary (Michener and Grimaldi 1988b). In other words, it is possible that some Mesoamerican stingless bees may have evolved from otherwise North American lineages, not from extant South American taxa (Michener and Grimaldi 1988a,b; Camargo et al. 2000); also, it is likely that some of those taxa diversified in South America as a consequence of climatic events during the Pliocene and Pleistocene. Certainly, the North American fauna of meliponines, as evidenced by C. prisca, suffered considerably from the Chixulub impact (65 Ma) and resulting northern projection of ejecta (Schulte et al. 2010), but remnants may have persisted and move southward during the Early Tertiary. Certainly extensive paleomelittological work needs to be done in additional North American amber deposits (e.g., Eocene Arkansas amber, additional Mexican amber, etc.). In the absence of a phylogenetic hypothesis that includes all stingless bee fossils worldwide, it is difficult to know which taxa evolved from ancient North American lineages, but, given their distribution and diversity, Cephalotrigona, Trigona, Nannotrigona, and Melipona seem to be good candidates. Evidence suggestive of this pattern is found in Melipona, such as the presence of M. yucatanica and M. lupitae in Mexico, the diversification of the fasciata species group in Mexico and in northern Central America, and the presence of M. beecheii Bennett in Mexico, as well as M. variegatipes Gribodo in Mexico as well as in some islands of the Caribbean (Camargo et al. 1988). In addition to the Mexican fauna likely being composed of some relics of that tropical North American fauna, there is no doubt that a large part of the Mexican taxa are South American in origin, some lineages of which evolved well before the separation of that continent from Africa. 9.5 Traditional Uses and Indigenous Knowledge Indigenous knowledge demonstrates how traditional cultures have organized cultural beliefs, linguistic practices, and historical interpretations that have given meaning to their lives. This form of knowledge construction comes directly from 146 R. Ayala et al. experience with the environment, is transmitted through oral tradition, and is based on holistic perspectives of the interconnectedness of all areas of life, as seen by indigenous perceptions of the world (Cajete 2000; Semali and Kincheloe 1999; Ortiz 2009). Such indigenous knowledge may also inform conservation practices (e.g., Posey 1993). The use of stingless bees by the Mayan people since pre-Colombian times is a good example of ethnobiological knowledge that has been transformed, innovated, and revitalizated. A number of researchers have emphasized the close relationship between the stingless bees and the Mayan culture and how such a practice was almost lost when the Spaniards introduced the Western hive honey bee, Apis mellifera Linneaus (e.g., Bennett 1964; Dixon 1987; Labougle and Zozaya 1986; Schwarz 1949). Mayans used honey as a sweetener, antibiotic, and an ingredient of “balché”, a culturally important fermented drink still used today. Aztecs also regularly used honey from stingless bees to sweeten and flavor the drink of the gods and one of the most appreciated beverages in the world today: chocolate (Coe and Coe 1996). It is no wonder stingless bees were important, regarded as gifts from the gods, handled with care, or even considered as gods outright, such as “ah-muzencab” (Fig. 9.1e), one of the Mayan gods of bees and honey usually appearing landing or taking off in ceremonial temples in the Yucatán Peninsula. Melipona beecheii, locally known as xunan kab or kolil kab in Mayan, meaning “royal lady”, is one of the most culturally and socially important stingless bees in Mexico, and perhaps in the world, given its traditional value for the Mayans, one the most important ancient civilizations of humanity (e.g., Villanueva-G et al. 2005; and references therein). Some works that document the traditional knowledge and use of stingless bees in Mexico, including names in local languages are those of Murillo (1981), Dixon (1987), and González (1983, 1989). Stingless bees are currently used for crop pollination at local scales in Mexico. For example, S. mexicana is used in the pollination of avocado [Persea americana (Lauraceae)], rambutan [Nephelium lappaceum (Sapindaceae)], and coffee [Coffea arabica (Rubiaceae)] in Hidalgo, Puebla and Tapachula, Chiapas; N. perilampoides is used for pollination of habanero chile [Capsicum chinense (Solanaceae)], one of the most piquant (spicy hot) species of peppers; and Melipona are used in other more traditional crops, such as tomatoes [Solanum lycopersicum (Solanaceae)] (May-Itzá et al. 2008). Also, many towns with ethnic Nahuatl populations around Cuetzalan in northen Puebla have developed and depend almost entirely on stingless beekeeping, particularly S. mexicana or “pisilnekmej” (Fig. 9.1b, c); the honey of this species is highly appreciated locally and internationally, and it is estimated that up to two tons of honey are exported each year to Europe, principally Germany (Guzmán et al. 2011). A total of 19 of 46 known species in Mexico are currently used for crop pollination, crafts, folk art, medicine, honey, pollen, and cerumen some are used more regularly than others, depending on local abundance (Table 9.3). Of the species used, six are endemic and restricted to particular regions. The cerumen of endemic S. hellwegeri for feather, strands of yarn, and glass beads (locally known as “chaquiras” or “kuka”) arts, developed by the Huichol people from western central Mexico, is a remarkable 9 Mexican Stingless Bees (Hymenoptera: Apidae)... 147 traditional use of stingless bees. The cerumen, sometimes mixed with pine resin, is spread over a piece of wood onto which feathers, beads, or yarn are pressed (Fig. 9.1f–h). (R Ayala, personal observation). Mexico has a relatively small number of stingless bee species but they appear more heavily used, when compared to other countries in the Americas. For example, Colombia has at least twice the number of species of Mexico but available information suggests that only a small fraction is regularly exploited (Table 9.2). It is possible that this is a mere coincidence of the technological and cultural advancement of the Mayan and Nahuatl civilizations with the need and availability of the bees in the region. The comparable pre-Colombian civilization in South America was the Incas, but did not have immediate access to stingless bees, because only a few species reach high altitudes in the Andes. Another explanation is that the reduced number of stingless bees may have been the cause of the more exhaustive exploitation, progressively becoming more culturally important with iterative generations. Numerous records indicate that native people in South America (e.g., Colombia: Nates-Parra 2005; Bolivia: Stearman et al. 2008; Brazil: Posey and Camargo 1985; Camargo and Posey 1990) also used stingless bees, but none of them developed such a strong cultural relationship or relied as heavily on stingless bees such as those of the Mayas and Nahuatl, possibly because resources appeared to be limitless; they could sample many more species and as regularly as they pleased. However, archeological records are better preserved and documented in Central America than in the humid, tropical lowlands of South America, where meliponines are especially more diverse and abundant. Also, stingless bees are still poorly studied in most countries of the Americas and their uses poorly documented. Whatever the reason, it is clear that meliponines were, and are, a vital resource for ancient Mexicans and their descendants; for many indigenous groups now pursuing an urban life, stingless bees and their products still play an important role in the material and symbolic artwork that has facilitated their engagement to the regional and national market economies. 9.6 Future Directions Despite the relatively small number of stingless bee species and several decades of research in Mexico, a significant amount of work remains to be done. For example, the common M. beecheii is highly variable morphologically and it is still not clear whether it is composed of several cryptic species (which seems likely to be the case). Conversely, M. solani, M. fasciata, and M. belizeae (Schwarz, 1932) may be the same species. All three species appear to be geographically separated; the first is primarily found in lowlands whereas the second in highlands; the last species is only known from a few old specimens collected in Belize. Also, as discussed by Ayala (1999), similar cases to those described for Melipona are likely to be found in Trigonisca and Plebeia given our limited knowledge on the distribution and variation 148 R. Ayala et al. of these groups. Further studies using molecular characters, such as DNA barcodes may help to test those hypotheses. Some areas of Mexico need to be explored in more detail to obtain a better understanding of individual species distributions. Records are scarce from the mountains north of Oaxaca, Campeche, the mountains north of Chiapas, areas near to the Guatemalan border, and the mountains north of Puebla. Alongside this, Mexican amber remains to be explored more fully (Engel 2004a). The study of the extinct Mexican stingless bees will shed light on the evolutionary history and diversification of modern meliponines in the Americas. Indeed, paleomelittological investigation often greatly overturns our preconceived dogmas as they relate to bee diversity, biogeography, or the evolution of particular biological phenomena (e.g., Engel 2004b). Examples include the decreasing disparity and diversity of highly eusocial bees (e.g., Engel 2001a,b; Kotthoff et al. 2011) or the discovery of true honey bees (Apis spp.) natively occurring in western North America (Engel et al. 2009). It is exciting to imagine what kind of revelations await in the paleontological record of Mexico and surrounding countries. Multidisciplinary studies are needed to estimate the economic value of the bee products used in crafts, particularly those employed for the feather and bead arts. We do not know the ecological impact of stingless bee exploitation for crafts and other activities on local bee populations, and whether indigenous people are using colonies in a sustainable fashion for their and the bees’ maximal benefit. Special attention to these and other traditional activities related with meliponines, including beekeeping, are critical because such techniques and experiences accumulated by generations can be useful when replicating or promoting them in other countries that do not possess similar indigenous knowledge or tradition. Indigenous knowledge defines indigenous identity and how indigenous people perceive and transmit their understanding of the world (e.g., Ortiz 2009). The ancestral ethnobiological knowledge on stingless bees is an invaluable component of the cultural capital of Mexico and humanity; its preservation ultimately depends on assuring the survival of the bees. Acknowledgments We are indebted to Amy Comfort de Gonzalez, Claus Rasmussen, David W Roubik, Miguel Ortega, and Silvia RM Pedro for constructive comments and suggestions that improved this contribution, to Patricia Vit for inviting us to contribute to the present chapter, and to Carlos Balboa, Jorge Mérida, and Manuel Guzman for the images of Melipona and Scaptotrigona. Partial support was provided by US National Science Foundation grant DBI-1057366 (to MSE). This is a contribution of the Division of Entomology, University of Kansas Natural History Museum. References Ascher JS, Pickering J. 2011. Bee Species Guide (Hymenoptera: Apoidea: Anthophila). Available at: http://www.discoverlife.org/mp/20q?guide=Apoidea_species. Ayala R. 1988. 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Revista de Biología Tropical 25:43–46. Chapter 10 The Role of Useful Microorganisms to Stingless Bees and Stingless Beekeeping Cristiano Menezes, Ayrton Vollet-Neto, Felipe Andrés Felipe León Contrera, Giorgio Cristino Venturieri, and Vera Lucia Imperatriz-Fonseca 10.1 Introduction The close relationship between bees and microorganisms is unquestionable (Cano et al. 1994; Gilliam 1997). As in many insects, bacteria, molds, and yeasts seem to play an important role for bee nutrition and protection against harmful microorganisms (Roubik 1989; Gilliam et al. 1990; Gilliam 1997; Mueller et al. 2005; Anderson et al. 2011). The microorganisms are transferred from one bee generation to the next—while associated with their hosts, they find suitable microenvironments in which to live and reproduce (Sachs et al. 2011). The subject of this chapter has been extensively explored in Apis mellifera, from which more than 6,000 microbial strains were isolated and identified (Gilliam 1997). Most studies focus on identification, while a few studies consider biochemical contributions of the microbes (Gilliam 1997; Teixeira et al. 2003; Promnuan et al. 2009; Kroiss et al. 2010). However, the biology and roles of microorganisms associated with bees are still unclear and sometimes controversial (Herbert and Shimanuki 1978; Loper et al. 1980; Standifer et al. 1980; Fernandes-da-Silva and Serrao 2000; Anderson et al. 2011). C. Menezes (*) • G.C. Venturieri Embrapa Amazônia Oriental, Belém, PA, Brazil e-mail: menezes.cristiano@gmail.com A. Vollet-Neto Universidade de São Paulo Ribeirão Preto, SP, Brazil F.A.F. León Contrera Universidade Federal do Pará, Belém, PA, Brazil V.L. Imperatriz-Fonseca Universidade de São Paulo, Ribeirão Preto, SP, Brazil Universidade Federal Rural do Semi-árido, Mossoró, RN, Brazil 153 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_10, © Springer Science+Business Media New York 2013 154 C. Menezes et al. Although stingless bees (Apidae: Meliponini) share many similarities with Apis mellifera, this diverse group (Roubik 1989; Michener 2000) still conceals many particularities that have not been explored. Here we discuss the role of non-pathogenic microorganisms in stingless bee colonies and focus on their importance to stingless bee keeping. Our aim is to stimulate further studies on functional aspects of microorganisms associated with stingless bees and their nests or managed hives. 10.2 Known Microorganisms Living in Stingless Bee Colonies The main microorganisms living in stingless bee colonies are yeasts, molds, and bacteria. However, the knowledge about this biodiversity is very limited, since most of papers only mention their occurrence, not their function. Furthermore, information is available only for a few stingless bee species. Our aim in this section is to present the most common microorganisms, where they live and what they may provide for the host colonies. 10.2.1 Bacteria Two genera of bacteria have been identified in stingless bee colonies. The most common and always present are from the Bacillus genus. Some DNA of this group was even found in fossils of the extinct Proplebeia dominicana which is about 20 million years in age (Cano et al. 1994; Camargo et al. 2000). This suggests a very old relationship between bees and Bacillus. These microorganisms seem to play an important role by secreting enzymes that cause fermentation and conversion of pollen constituents (Gilliam et al. 1985, 1989, 1990). Apparently, the enzymes have two main functions—pre-digestion of the pollen (softening of the exine wall) before it is ingested and altering the stored pollen so that it is less susceptible to harmful microorganism proliferation. The acetic and lactic fermentations, which occur in pollen and honey, are also realized by these bacteria (Gilliam 1979b). Besides the apparent function in food digestion, Yoshiyama and Kimura (2009) found strong evidence that Bacillus species also secrete antibiotics. By using in vitro inhibition assays, those authors demonstrated that strains of Bacillus from the digestive tract of Apis cerana japonica inhibit Paenibacillus larvae, which cause American foulbrood disease. Similar effects may also be found in stingless bees’ Bacillus. A classic study in stingless bee biology indicated that Melipona quadrifasciata could not survive without a Bacillus species found in the nest (Machado 1971). Bacillus was found in stored pollen, brood provisions, digestive tracts of larvae and adult bees, and less abundantly in honey. During 1 month, the study colony was fed with sugar syrup mixed with streptomycin, an antibiotic that killed Bacillus species in vitro. After that treatment, the new brood cells were continuously destroyed, and the colony died after 30 days. 10 The Role of Useful Microorganisms to Stingless Bees… 155 The other genus of bacteria recently found in brood cells and nest materials from stingless bee colonies is the actinomycete Streptomyces (Promnuan et al. 2009). This genus is well known for secreting antibiotics (Kroiss et al. 2010) and those found in the stingless bees Trigona (Tetragonula) laeviceps and T. fuscobalteata showed high inhibitory activity against Paenibacillus larvae and Melisococcus plutonius, pathogens of A. mellifera, responsible for American foulbrood and European foulbrood, respectively. Recent contributions have clearly demonstrated the potential of the relationships between bees and Streptomyces and suggest this kind of relationship may also be found in stingless bees. Kaltenpoth et al. (2006) and Goettler et al. (2007) found a symbiotic relationship between a wasp (Philanthus triangulum) and bacteria from the genus Streptomyces which live inside antennal glands of female wasps. The bacteria are spread inside brood cells before larval provisioning and secrete nine different antibiotic substances that protect larvae from fungi and other pathogens (Kroiss et al. 2010). 10.2.2 Yeasts Ten yeast genera are known in stingless bee colonies so far. The most representative are Candida and Starmerella, which occur very frequently in pollen and honey (Camargo et al. 1992; Rosa et al. 2003; Teixeira et al. 2003). Other genera were found in adult bees, propolis, the colony trash deposit area and, rarely, in the honey (Rosa et al. 2003). Because they are less frequently found in parts of the nest associated with external materials, such as propolis, it can be assumed that they are occasional contaminants from external environment and from plants visited by bees (Lachance et al. 2001a,b; Rosa et al. 2003). The significance of yeasts and their potential roles to meliponine colonies are similar to the bacterial roles; i.e., they secrete enzymes, which convert substances from stored food and help to conserve it. Alcoholic fermentation is also a process initiated with yeast. It is still unclear how yeasts influence bee nutrition, but the changes seen within stored pollen are striking. An interesting role of yeasts was described by Camargo et al. (1992). Yeasts of Candida genus seem to dehydrate the pollen stored by the stingless bee Ptilotrigona lurida. This dehydration process is efficient to avoid spoilage and prevent Phoridae (mainly Pseudohypocera) from consuming pollen and causing serious damage to the colony. 10.2.3 Other Fungi A recent paper has described foragers of Tetragonula collina harvesting spores of Rhizopus sp. in lieu of pollen (Eltz et al. 2002). The same behavior was also observed 156 C. Menezes et al. Fig. 10.1 Filamentous fungus growing on the surface of larval food and at the borders of brood cells of Scaptotrigona depilis. The larvae eat the fungus as it grows and seem to depend upon this fungus to survive (Menezes 2010). Photo: C. Menezes in Partamona bees (G. Azevedo, cited as personal communication in Oliveira and Morato 2000). Similar observations were also noted by Roubik (1989), Burr et al. (1996), and Oliveira and Morato (2000). They found that workers of stingless bees lick or harvest a mucilaginous mass of spores of stinkhorn species (Fungi, Phaleles). It is still not known what motivates this behavior. The nutritive value of spores is low compared to pollen, but could complement their diet if availability is high and harvest is relatively easy (Oliveira and Morato 2000; Eltz et al. 2002). Indeed, plant trichomes (sometimes called pseudopollen) are harvested from orchid flowers by Neotropical Partamona, Plebeia, Melipona, and Trigona, and may have a similar role (Davies 2009). Another recent paper reports the occurrence of several filamentous fungi isolated from individual workers of Melipona subnitida (Ferraz et al. 2008). The bees were already dead from natural causes when collected, and most of those microorganisms must be opportunistic in exploiting the carcasses. An interesting relationship between a fungus and bees has recently been discovered. A filamentous fungus grows inside brood cells of Scaptotrigona depilis at the surface of larval food and is eaten by developing larvae (Fig. 10.1) (Menezes 2010). Apparently, the presence of this fungus was known (Flechtmann and Camargo 1974). It was then considered a disease because the brood of the studied colony presented a high mortality rate. However, recent observations have demonstrated that this fungus is very abundant in healthy colonies of S. depilis and also occurs with other stingless bee species, such as Tetragona clavipes and Melipona flavolineata (Menezes, unpublished data). The fungus proliferates before the egg hatches and 10 The Role of Useful Microorganisms to Stingless Bees… 157 grows intensively until the larva reaches 3 days of age. Larvae eat the fungus as it grows and preliminary tests show that the larvae depend on this fungus to survive, because all of them died when fungal growth was inhibited experimentally. We are investigating whether the fungus is providing nutritional benefits or protection against undesirable microorganisms. 10.3 Fermentation and Biochemical Processes Fermentation is a biochemical process that transforms carbohydrates into other organic substances, providing energy to microorganisms. There are three main categories of fermentation: (1) alcoholic, in which carbohydrates are transformed into alcohol; (2) acetic, when alcohol is transformed into acetic acid; and (3) lactic, in which carbohydrates are transformed into lactic acid and other organic byproducts. Mixed fermentations also occur in nature. 10.3.1 Fermentation of Honey Stingless bee honey is stored in pots made of cerumen (a mixture of wax and resins). To become honey, nectar undergoes three kinds of change: (1) physical, by the evaporation of a large part of its water, (2) biological, by the fermentation of yeast and bacteria, (3) chemical, when enzymes secreted by cephalic glands are added by the workers, transforming the sucrose of nectar into glucose and fructose (Beutler 1954 apud Zucoloto 1975; Nogueira-Neto 1997; Venturieri et al. 2007). Stingless bee honey is different in many ways from the honey of A. mellifera. Although its organoleptic and physicochemical characteristics vary according to the bee species and floral resources, we can assume that the main difference is the water content, generally higher than A. mellifera honey (Gonnet et al. 1964; Cortopassi-Laurino and Gelli 1991; Vit et al. 2004; Bijlsma et al. 2006; Venturieri et al. 2007; reviewed by Souza et al. 2006). This relatively abundant water in stingless bee honey allows microorganisms to survive and to be active (Sanz et al. 1995). Additionally, some species of microbes isolated from stingless bee provisions survive under acidic conditions and at high osmotic pressure (Gilliam et al. 1985, 1989, 1990; Rosa et al. 2003; Teixeira et al. 2003). There is some evidence that stingless bee honey may ferment naturally inside sealed honey pots. It is very common to see foam on the surface of the honey inside honey pots (Souza et al. 2007; Menezes, personal observations), indicating that gas bubbles are escaping from the honey, probably from alcoholic fermentation (Fig. 10.2). In the figure there are evident particles floating on the surface of the honey, which may be yeasts, bacteria, and residue of pollen. If the honey is kept at room temperature after being harvested, this layer of particles increases considerably and the honey becomes more acidic. In addition, fresh honey that was stored 158 C. Menezes et al. Fig. 10.2 Pot-honey of Melipona fasciculata. Foam and floating particles (probably bacteria, yeasts, and pollen grains) are frequently found on the surface of pot-honey which indicates that fermentation naturally occurs. Photo: C. Menezes recently by the bees is generally not sour, but very sweet (Alves et al. 2007). On the contrary, when honey is harvested from natural colonies living inside tree trunks, or from colonies not managed for long periods, it seems to be more sour (Menezes, personal observation). The alcoholic fermentation is generally performed by yeasts (Rosa et al. 2003; Teixeira et al. 2003). Sugar molecules are transformed into alcohol and CO2. Bubbles and foam at the honey indicate alcoholic fermentation. Afterward, under aerobic conditions, certain strains of bacteria can convert alcohol molecules and O2 into acetic acid and water. This kind of fermentation is generally performed by Bacillus, which is common in stingless bee honey (Machado 1971; Gilliam et al. 1985, 1990). In addition, lactic fermentation can also occur, whereupon sugars are converted into lactic acid and water, or other organic molecules. Bacteria are the main agent responsible for this kind of fermentation, although yeasts and other fungi can perform the same function. In honey from A. mellifera, the main biochemical reaction is catalyzed by the enzyme glucose-oxidase, which converts glucose + O2 + H2O into gluconic acid and hydrogen peroxide (White et al. 1963; Nogueira-Neto 1997). The gluconic acid is the main acid in honey bee honey and hydrogen peroxide is an important (Burgett 1978), but not the only, anti-microbial substance (Kwakman et al. 2010). The glucose-oxidase enzyme is produced by bee glands, but it is possible that some microorganisms can also produce it (Gilliam 1997). Many other biochemical reactions occur during honey storage. Workers can add many enzymes to the honey, which are produced by their glands (Costa and CruzLandim 2005), but the microorganisms living in honey can also secrete many proteolytic, glycolytic, and lipolytic enzymes, which will convert, ferment, enhance, and/ or preserve the honey (Gilliam et al. 1990). 10 The Role of Useful Microorganisms to Stingless Bees… 159 To our knowledge there are only two studies about changes in physicochemical characteristics in stingless bee honey over time. One of them does not allow strong generalizations to other stingless bees, since the studied species was Trigona hypogea, a obligately necrophagous stingless bee that does not harvest nectar from flowers, only from fruits and extra-floral nectaries (Noll et al. 1996), and also from homopteran bugs (DW Roubik, personal communication). They observed that there are no changes in amounts of sugar and protein traces in the course of time, but other parameters, such as pH, were not studied. The other study showed that, after harvest, fermentation increases the antioxidant activity of T. angustula honey, increases the amount of alcohol, and diminishes the amount of sugar (Pérez-Pérez et al. 2007). Although this is a preliminary study with small sample size and does not represent a natural situation, these results show that fermentation may add important substances to honey. Due to the high diversity of stingless bees and limited studies on their microorganisms, the honey maturation process is still not understood. Physicochemical analysis of honey in the course of time would be of great value to understand the biological and biochemical processes involved in honey storage by stingless bees. 10.3.2 Fermentation of Pollen When harvesting pollen, foragers transfer and accumulate pollen grains on their corbicula using nectar and salivary secretions (Herbert and Shimanuki 1978; Leonhardt et al. 2007). Workers return to their colonies with the pollen on their corbicula and leave the pellets inside pollen pots (made of cerumen), which are closed when they are full (Nogueira-Neto 1997). The pollen is stored for about 2 weeks before being consumed (Loper et al. 1980). In honey bees the pollen is stored in the same cells used for brood rearing and then sealed with a drop of honey. Under this condition of storage the pollen is subjected to the action of microorganisms: pollen stored in combs by honey bees is named bee bread; whereas pollen stored in pots by stingless bees is called “saburá” by indigenous people in Brazil (Fig. 10.3). The characteristics of the pollen such as flavor, odor, color, and texture, change considerably after being stored and vary among bee species (Camargo et al. 1992; Souza et al. 2004). A few bee species, such as Tetragonisca angustula and Frieseomelitta varia, produce dry and relatively sweet fermented pollen. However, other meliponines, such as Melipona and Scaptotrigona, produce and store moist and sour pot-pollen. Few studies have investigated the transformation process of stored pollen in stingless bees, thus we will base most of our discussion on A. mellifera, although even in this bee there is no clear consensus. The most consistent change during pollen storage in A. mellifera is the decrease of pollen pH (from 4.8 to 4.1—Herbert and Shimanuki 1978) caused by lactic acid fermentation (Haydak 1958). Apparently, bacteria of Streptococcus, Bifidobacterium, and Lactobacillus are the main microorganisms responsible for lactic fermentation (Pain and Maugenet 1966; Gilliam 1979b; Vásquez and Olofsson 2009). Yeasts may 160 C. Menezes et al. Fig. 10.3 Young workers of Scaptotrigona depilis feeding on natural fermented pollen “saburá” stored in cerumen pots. Photo: C. Menezes also ferment pollen, and their population increases after pollen fermentation, supposedly increasing nutritional quality (Pain and Maugenet 1966). Machado (1971) isolated Bacillus from pots of pollen and larval food of M. quadrifasciata and verified that stored pollen had more proteins cleaved into free amino acids than did pollen removed directly from the bees’ corbiculae. He found those bacteria in the larval food of 13 more stingless bee species. Gilliam et al. (1990) also studied four species of Bacillus in Melipona (currently known as the species M. panamica) and found that these microorganisms were able to secrete enzymes related to cleavage of lipids, carbohydrates, and proteins. Bacillus spp. are known for secreting several extracellular enzymes, antibiotics, and fatty acids, which could act directly on the chemical conversion of pollen and on the control of competing microorganisms that could spoil the pollen. This may explain why Bacillus are predominant in pollen and other microorganisms are less abundant (Gilliam et al. 1990). Moreover, some Bacillus species are known to ferment glucose when isolated, so pollen fermentation may also be attributed to these microbes. For a long time it was hypothesized that fermentation increased the nutritional quality and accelerated the digestion of pollen grains. However, this may not be the main function of microbial activity in pollen. Some studies show that the nutritional quality increases (Beutler and Opfinger 1949 apud Herbert and Shimanuki 1978; Cremonez et al. 1998) and others demonstrate that the quality can remain the same (Herbert and Shimanuki 1978; Fernandes-Da-Silva and Serrão 2000) or even decrease after pollen storage (Human and Nicolson 2006). Likewise, studies about chemical differences between newly collected pollen and bee bread show that protein content and free amino acids remain the same (Herbert and Shimanuki 1978) or decrease after some time (Standifer et al. 1980; Human and Nicolson 2006). Some kinds of nutrients increase in concentration, like vitamin K (Haydak and Vivino 10 The Role of Useful Microorganisms to Stingless Bees… 161 1950 apud Loper et al. 1980), vitamin E (Haydak and Palmer 1938 apud Loper et al. 1980), and some fatty acids (Loper et al. 1980). Other vitamins, however, can decrease in concentration, like vitamins C and B6 (Loper et al. 1980). Only the increase of lactic acid and the decrease of starch on bee bread appear to be consistent among the studies (Herbert and Shimanuki 1978). Moreover, when nutritional quality was tested, results were controversial. Some studies show that longevity increases when workers feed on bee bread, compared to newly collected pollen (Beutler and Opfinger 1949 apud Herbert and Shimanuki 1978), in addition to studies that show that bee bread increases the amount of protein in haemolymph (Cremonez et al. 1998) and increases digestibility (Gilliam 1979a), when compared to fresh pollen. Nevertheless, many studies show no significant differences in hypopharyngeal gland development and pollen digestion (Herbert and Shimanuki 1978) when compared to the consumption of bee bread and newly collected pollen in A. mellifera. Fernandes-da-Silva and Serrão (2000) also showed that in S. depilis, a Brazilian stingless bee, the storage of pollen does not increase nutritional quality for workers. They verified the effect of fermented pot pollen and newly collected corbicular pollen on the development of hypopharyngeal glands and the degree of digestion of pollen grains, and found no significant difference between treatments. Fermentation may therefore have greater importance in the conservation of stored pot-pollen than in altering its nutritional condition (Herbert and Shimanuki 1978; Fernandes-da-Silva and Serrão 2000). The presence of lactic acid, combined with other microorganism metabolites, could stabilize the stored pollen, preventing the development of other microorganisms that could spoil the pollen (Herbert and Shimanuki 1978; Gilliam 1997), in the same way that this process is used in industrial conservation and stabilization of fermented food, such as cheese, pickles, and wine (Gilliam 1997). There are still no detailed investigations in this area. Vollet-Neto et al. (unpublished data) verified that newly emerged workers of S. depilis are more attracted to fermented pollen stored in pots than newly collected pollen from the corbicula. This behavior could indicate, at first, an instinctive behavior caused by the nutritional advantage in feeding on fermented pollen. However, according to studies of Fernandes-da-Silva and Serrão (2000), who found no nutritional advantage in the processed pot-pollen, the attraction and higher consumption of the fermented pollen could be explained by its strong and distinctive odor, which could attract worker bees. Other parameters to be analyzed include the amount of protein in the haemolymph, nutritional quality of the larval food, and development of immatures, besides studies on chemical composition. An interesting example described by Camargo et al. (1992) suggests that pollen fermentation can provide additional advantages for stingless bees. They observed intense proliferation of Candida on stored pollen of Ptilotrigona lurida, an Amazonian stingless bee. These yeasts seem to dehydrate the stored pollen to 13.9% water content (while they found 52.2% pollen water content for Melipona seminigra and 24.1% for Trigona dallatorreana). These physicochemical changes could prevent the development of undesirable microorganisms that could spoil\ the food. Moreover, they verified that phorid flies (Diptera, Phoridae), parasites that lay their 162 C. Menezes et al. eggs on the larval food and stored pollen of stingless bees, do not lay their eggs on pollen of P. lurida, but lay on stored pollen of Melipona seminigra. Several other stingless bee species also possess relatively dry stored pollen in nests (e.g., Frieseomelitta varia; Tetragonisca angustula; Menezes, Cristiano), but the function of dehydration for these species is not known. In summary, we may assume that the storage of pollen in cerumen pots is associated with inoculation of microorganisms, which promote biochemical changes that alter nutritional quality and enhance digestion and absorption of nutrients, but probably the main function is to prevent spoilage and diseases (Anderson et al. 2011). We still need much more information to draw valid conclusions about the advantages brought about by microorganisms living in pollen. 10.4 Practical Applications for Stingless Bee-Keepers (Problems, Solutions, and Peculiar Products Generated from Fermentation) Given the above considerations, it is impossible to harvest stingless bee products without including their natural microorganisms (Souza et al. 2009). Therefore, it is very difficult to avoid the consequences, such as fermentation. The use of hygienic procedures while managing, harvesting, and processing stingless bee products considerably reduces the risk of contamination by unnatural microorganisms, from other parts of the nest or from the external environment (Fonseca et al. 2006; Venturieri et al. 2007; Souza et al. 2009). 10.4.1 Proliferation of Microorganisms after Harvesting the Honey The high water content of most stingless bee honey is a big challenge to stingless beekeeping (Vit et al. 2004; reviewed by Souza et al. 2006). If it is kept at room temperature, honey will ferment after being harvested, even if extremely hygienic procedures are adopted (Nogueira-Neto 1997). Thus, four different solutions, refrigeration, dehydration, pasteurization, and maturation, have been developed by researchers and stingless bee-keepers to increase the post-harvest stability and extend the shelf life of pot-honey (Nogueira-Neto 1997; Fonseca et al. 2006; Alves et al. 2007; Venturieri et al. 2007; Drummond 2010; Contrera et al. 2011). Refrigeration is the easiest process and preserves the natural characteristics and substances of honey. There are two disadvantages of this method. First is the high cost of storage until sale. Second, if honey was harvested with poor hygienic procedures, pathogens will remain alive in the honey. The honey must be kept refrigerated (approximately 4–8°C) just after harvesting and until consumption (Venturieri et al. 2007). 10 The Role of Useful Microorganisms to Stingless Bees… 163 Honey can be kept refrigerated for long periods, even for years. However, pot-honey produced by different species may behave differently; sometimes off-flavors develop after refrigeration of Melipona quadrifasciata honey (P Vit, personal communication). The dehydration process consists of removal of water from the honey, which can be accomplished by means of ventilation in a dry room (Nogueira-Neto 1997; Alves et al. 2007). Fonseca et al. (2006) describe a method whereby honey is spread upon flat containers in a relatively dry room with a dehumidifier, then bottled when the honey moisture content diminishes to 20% or less, which normally takes up to 3 days. Some advantages are that the honey can be stored at room temperature until consumption, without fermentation, and the natural substances and flavor of honey are not lost, because it has not been heated. A disadvantage is that the honey becomes more viscous than normal for stingless bees, thus becomes very similar to commercial honey bee honey. Crystallization is enhanced, and produces sharp crystals, as observed in some Melipona species (P Vit, personal communication). Pasteurization is a viable option in order to keep honey at room temperature without fermentation and to eliminate pathogenic microbes. The honey should be heated for 15 s at 72°C or 30 min at 63°C (Nogueira-Neto 1997), and bottled just after cooling to room temperature. If the process cannot be done just after harvesting, the honey should be cooled until pasteurization. This process does not kill all microorganisms and spores in the honey, but eliminates pathogens. The disadvantage of this process is that some natural enzymes are lost, like glucose-peroxidase (Nogueira-Neto 1997). Pasteurization offers three great advantages compared to other post-harvest methods. First, it is possible to store the honey at room temperature, without any fermentation. Second, it controls pathogens. Third, the natural flavor and texture of stingless bee honey are maintained (Nogueira-Neto 1997; Venturieri et al. 2007). After opening a bottle, it should be stored under 8°C and should be consumed before 1 year. In the maturation process, fermentation after harvest will naturally occur at room temperature (Drummond 2010). The honey is kept inside closed bottles, which are opened once a week to release the gases generated by fermentation, and closed again. Honey can also be kept in bottles with lids that allow gas exchange. This process takes up to 3 months or until gas is no longer released. After this period fermentation stops, and the stabilized honey can be bottled. The main advantage of this method is that matured honey does not ferment at room temperature after the process and the costs are very low. The honey becomes more acidic after maturation, and acquires some peculiar odors and aromas (Drummond 2010). Sensory characteristics of matured honey, compared to fresh honey, may be perceived as an advantage or a disadvantage, according to personal tastes and use by the consumer. The above mentioned possibility has been widely used in Maranhão, Brazil. Although it seems to be an interesting post-harvest alternative to preserve honey, especially for rural communities, it is still very controversial since we remain ignorant regarding its consequences at biochemical and microbial levels. They may provide healthy sub-products for human consumption (Pérez-Pérez et al. 2007), but also conceivably generate toxic substances. 164 10.4.2 C. Menezes et al. Harvesting Fermented Pollen and Unfermented Pollen Stingless bee pollen is very nutritious and is an ‘alternative’ healthful food source (Souza et al. 2004). Pollen extracts inhibit oxidizing agents and free radicals, and this property seems to be important in the prevention of various human diseases (Lins et al. 2003; Silva et al. 2006, 2009). However, the only way to harvest stingless bee pollen is by removing it directly from the pollen pots, because pollen traps used for A. mellifera do not work for stingless bees (Menezes et al. 2012). Pot-pollen alone is sour in most stingless bee species. A Brazilian stingless beekeeper, Wilson Melo, who manages more than 600 colonies of Scaptotrigona spp. for pollen production, suggests consuming it as a honey-pollen jelly or as a creamy pollen milk shake. Both recipes neutralize the acid from the pollen and produce a pleasant flavor. Although fermented pollen is relatively easy to harvest, we have developed a method to harvest pollen before fermentation (Menezes et al. 2012). We noticed that if we harvest the pollen a week after it has been stored, it is still sweet and not yet fermented. Because it would be impossible to distinguish fresh from fermented pollen in a bee nest or hive, a solution is moving a strong colony to a different place and replacing it with an empty hive, where the foragers will return from the field and store the pollen in new pots. After a week, the pollen can be harvested and will not be fermented. We tested this method with ten colonies of S. depilis and they produced an average of 60 g unfermented pollen in a week. This pollen can be used as it is, stored frozen or dehydrated. Another solution is harvesting the pollen from the honey super every week, so it has yet to ferment. It is important to emphasize that some stingless bees, such as the Scaptotrigona species, harvest much more pollen than honey and produce a substantial amount of pollen. 10.4.3 Stingless Bee Mead A popular beverage since antiquity, consumed by several civilizations like the Chinese, Greeks, Romans, and Vikings (McGovern et al. 2004; Bishop 2005), mead (also known as honey wine) is basically a drink produced with fermented honey and water, which is also produced with pot-honey from stingless bees, known as “balché” by the Mayans (Villanueva et al. 2004). The elaboration of a mead beverage based upon melipona honey is a recent research line from Embrapa Amazônia Oriental, in Belém, Brazil. Preliminary results show that the high acidity of pot-honey needs pH control with calcium carbonate. This procedure allows yeast Saccharomyces cerevisiae to better perform its function. Otherwise, the resulting mead will have an unstable and acidic taste, because of its lesser quantity of alcohol. In order to stop the action of undesirable microorganisms on the fermentation process by S. cerevisiae, pasteurization (65°C during 5 min) is performed before the yeast is added. The fermentation process can last from 2 and up to 4 weeks, depending 10 The Role of Useful Microorganisms to Stingless Bees… 165 on the proportion of water, honey, acids, and yeast, and should be done under anaerobic conditions allowing CO2 release. After the fermentation cycle, the mead must be filtered and decanted. A further clarification with bentonite facilitates the precipitation of suspended particles. After bottled and sealed, the mead must be pasteurized (65°C during 5 min), in order to increase its stability and for safety reasons. 10.4.4 Pollen Substitutes for Artificial Feeding The nutritional base of natural feeding by stingless bees, like in the majority of Apoidea, is nectar and pollen, with few exceptions. Nectar is the source of sugars while pollen, besides carbohydrates, also supplies them with protein, lipids, vitamins, and minerals (Michener 1974). Pollen is stored in pots and undergoes an intense fermentation caused by bacteria and yeasts. These microorganisms seem to be essential to pre-digest and conserve the stored pollen (see the above sections for more details, and also Morais et al. this book). Honey and pollen substitutes are extremely important to stingless beekeeping, especially during dearth periods and after colony division or artificial multiplication. The nectar is easily substituted by sugar syrup and its acceptance and consumption are very good (Nogueira-Neto 1997), but pollen has been more difficult to substitute and frequently the workers throw it away (reviewed by Vollet-Neto et al. 2010). The first study on a semi-artificial diet for the substitution of pollen was made by Camargo (1976). She mixed pollen of Typha dominguensis with honey and natural pollen from the bee that received the supplementary diet. The artificial food was stored in a glass covered by gauze at temperatures from 28 to 32°C during 10–15 days, leading to fermentation. She concluded that if the pollen substitute is not fermented, the workers reject it. Vollet-Neto et al. (unpublished data) also verified that young workers of S. depilis prefer fermented pollen instead of fresh pollen from foragers, and prefer a fermented artificial diet instead of a an unfermented one (Fig. 10.4). Several pollen substitute formulations were later developed using different ingredients, such as commercial yeasts (S. cerevisiae) and soybean extracts (Penedo et al. 1976; Fernandes-Da-Silva and Zucoloto 1990; Pires et al. 2009). For Scaptotrigona postica, mixture of 25% commercial yeast and 75% pollen was found to be a good substitute, based on the development of hypopharingeal glands and oocytes (Penedo et al. 1976). Costa and Venturieri (2009) and Pires et al. (2009) also developed and tested the consumption and nutritional value of pollen substitutes for M. fasciculata. They found that soybean extract mixed with sugar, water and about 20% pollen of the same bee species was a good pollen substitute, consumed by workers in a normal colony (Pires et al. 2009). The nutritional value was confirmed by development of worker hypopharyngeal glands and queen oocytes in a laboratory assay with the same bee species (Costa and Venturieri 2009). 166 C. Menezes et al. Fig. 10.4 Young workers of Scaptotrigona depilis are more attracted to fermented food (left plate) than unfermented food (right plate) (Vollet-Neto et al., unpublished). Photo: C. Menezes Most stingless bee species are not very tolerant of pollen substitutes and, if it is inadequate, workers discard the artificial food in the colony trash pile. However, some species, such as F. varia, show the opposite behavior. Foragers of this species are very attracted to artificial food even if offered outside the nest (Vollet-Neto, personal observation). They harvest a large amount and store it inside the nest (Fig. 10.5). Surprisingly, the worker bees were also attracted by food fermented by microorganisms from other stingless bee species. Although in such a diverse group as Meliponini, generalizations are always difficult, we can conclude that a good substitute for pollen must have characteristics similar to the natural pot-pollen stored in the nest. The main factor to be considered is that a pollen substitute must be fermented, and we conclude that stingless bees prefer a pollen substitute fermented by microorganisms found in pot-pollen of their own species. 10.5 Conclusions 1. The main microorganisms living in stingless bee colonies are yeasts, molds, and bacteria. However, knowledge about this biodiversity is very limited, because most papers only mention their occurrence, not their function. 2. Due to the high diversity of stingless bees and limited studies on their microorganisms, the honey maturation process is still poorly understood. Physicochemical analysis of honey in the course of time would be of great value to understand the biological and biochemical processes involved in honey storage of stingless bees. 3. We may assume that the storage of pollen in cerumen pots is associated with inoculation of microorganisms, which promote biochemical changes that alter 10 The Role of Useful Microorganisms to Stingless Bees… 167 Fig. 10.5 Frieseomellita varia storing artificial food. (a) Outside the nest, (b) inside the pollen pots made of cerumen. Green dye was used in the artificial food to distinguish them from natural pollen inside the nests. Photos: C. Menezes 168 C. Menezes et al. nutritional quality and enhance digestion and absorption of nutrients, but probably the main function is to prevent spoilage and disease. We still need much more information to draw valid conclusions about the advantages brought about by microorganisms living in pollen. 4. The high water content of most pot-honey is a necessary challenge to stingless bee keeping. If honey is kept at room temperature, it will ferment after being harvested, even if extremely hygienic procedures are applied. Thus, four different solutions, refrigeration, dehydration, pasteurization, and maturation, have been developed by researchers and stingless bee-keepers to increase the postharvest stability and extend the shelf life of pot-honey. 5. Microorganisms from stingless bees can be very useful for stingless beekeepers because peculiar products may be produced by them, such as mead, honey-pollen jelly, or a creamy pollen milk-shake. References Alves, RMO, Sodré GS, Souza BA, Carvalho CAL, Fonseca AAO. 2007. Desumidificação: uma alternativa para a conservação do mel de abelhas sem ferrão. Mensagem Doce 91:1–5. 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Ciência e Cultura 28:430–431. Camargo JMF, Garcia MVB, Junior ERQ, Castrillon A. 1992. Notas previas sobre a bionomia de Ptilotrigona lurida (Hymenoptera, Apidae, Meliponinae): associação de leveduras em pólen estocado. Boletim do Museu Paraense Emílio Goeldi 8:391–395. Camargo JMF, Grimaldi D, Pedro SRM. 2000. The extinct fauna of stingless bees (Hymenoptera: Apidae: Meliponini) in Dominican amber: two new species and redescription of the male of Proplebeia dominicana (Wille and Chandler). American Museum Novitates 3293:1–24. Cano RJ, Borucki MK, Higby-Schweitzer M, Poinar HN, Poinar GO, Pollard KJ. 1994. Bacillus DNA in fossil bees: an ancient symbiosis? Applied and Environmental Microbiology 60:2164–2167. Contrera FAL, Menezes C, Venturieri GC. 2011. New horizons on stingless beekeeping (Apidae, Meliponini). Revista Brasileira de Zootecnia 40:48–51. Cortopassi-Laurino M, Gelli DS. 1991. Analyse pollinique, propriétés physico-chimiques et action antibactérienne des miels d’abeilles africanisées Apis mellifera et de Méliponinés du Brésil. Apidologie 22:61–73. Costa L, Venturieri GC. 2009. Diet impacts on Melipona flavolineata workers (Apidae, Meliponini). Journal of Apicultural Research 48:38–45. 10 The Role of Useful Microorganisms to Stingless Bees… 169 Costa RAC, Cruz-Landim C. 2005. Hydrolases in the hypopharyngeal glands of workers of Scaptotrigona postica and Apis mellifera (Hymenoptera, Apinae). Genetics and Molecular Research 4:616–623. Cremonez TM, De Jong D, Bitondi MMG. 1998. Quantification of hemolymph proteins as a fast method for testing protein diets for honey bees (Hymenoptera: Apidae). Journal of Economic Entomology 91:1284–1289. Davies KL. 2009. Food-Hair Form and Diversification in Orchids. pp. 159–184. In Kull T, Arditti J and Wong SM, eds. Orchid Biology-Reviews and Perspectives. Springer Netherlands; New York, USA. 452 pp. Drummond MS. 2010. Maturação do mel de abelhas nativas sem ferrão: novo panorama de consumo no mercado gastronômico. pp. 1–3. In Anais do XVIII Congresso Brasileiro de Apicultura, Cuiabá, Mato Grosso, Brasil. Eltz T, Brühl CA, Görke C. 2002. Collection of mold (Rhizopus sp.) spores in lieu of pollen by the stingless bee Trigona collina. Insectes Sociaux 49:28–30. Fernandes-da-Silva PG, Zucoloto FS. 1990. A semi-artificial diet for Scaptotrigona depilis Moure (Hymenoptera, Apidae). Journal of Apicultural Research 29:233–235. Fernandes-da-Silva PG, Serrao JE. 2000. Nutritive value and apparent digestibility of bee-collected and bee-stored pollen in the stingless bee, Scaptotrigona postica Latr. (Hymenoptera, Apidae, Meliponini). Apidologie 31:39–45. Ferraz RE, Lima PM, Pereira DS, Freitas CCO, Feijó FMC. 2008. Microbiota Fúngica de Melipona subnitida Ducke (Hymenoptera: Apidae). Neotropical Entomology 37:345–346. Flechtmann CHW, Camargo CA. 1974. Acari associated with stingless bees (Meliponidae, Hymenoptera) from Brazil. pp. 315–319. In Proceedings of the 4th International Congress of Acarology. Saalfelden, Austria. Fonseca AAO, Sodré GS, Carvalho CAL, Alves RMO, Souza BA, Silva SMPC, Oliveira GA, Machado CS, Clarton L. 2006. Qualidade do mel de abelhas sem ferrão: uma proposta para boas práticas de fabricação. Série Meliponicultura, n. 5. UFRB/SECTI-FAPESB; Cruz das Almas, Brasil. 2006. 70 pp. Gilliam M. 1979a. Microbiology of pollen and bee bread: the yeasts. Apidologie 10:43–53. Gilliam M. 1979b. Microbiology of pollen and bee bread: genus Bacillus. Apidologie 10:269–274. Gilliam M. 1997. Identification and roles of non-pathogenic microflora associated with honey bees. FEMS Microbiology Letters 155:1–10. Gilliam M, Buchmann SL, Lorenz BJ, Roubik DW. 1985. Microbiology of the larval provisions of the stingless bee, Trigona hypogea, an obligate necrophage. Biotropica 71:28–31. Gilliam M, Prest DB, Lorenz BJ. 1989. 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Nutritional content of fresh, bee-collected and stored pollen of Aloe greatheadii var. davyana (Asphodelaceae). Phytochemistry 67:1486–1492. Kaltenpoth M, Goettler W, Dale C, Stubblefield JW, Herzner G, Roeser-Mueller K, Strohm E. 2006. ‘Candidatus Streptomyces philanthi’, an endosymbiotic streptomycete in the antennae of Philanthus digger wasps. International Journal of Systematic and Evolutionary Microbiology 56:1403–1411. 170 C. Menezes et al. Kroiss J, Kaltenpoth M, Schneider B, Schwinger MG, Hertweck C, Maddula RK, Strohm E, Svatos A. 2010. Symbiotic streptomycetes provide antibiotic combination prophylaxis for wasp offspring. Nature Chemical Biology 6:261–263. Kwakman PHS, te Velde AA, de Boer L, Speijer D, Vandenbroucke-Grauls CMJE, Zaat SAJ. 2010. How honey kills bacteria. FASEB Journal 24:2576–2582. Lachance MA, Bowles JM, Chavarria-Diaz MM, Janzen DH. 2001a. Candida cleridarum, Candida tilneyi, and Candida powellii, three new yeast species isolated from insects associated with flowers. International Journal of Systematic and Evolutionary Microbiology 51:1201–1207. Lachance MA, Starmer WT, Rosa CA, Bowles JM, Barker JSF, Janzen DH. 2001b. Biogeography of the yeasts of ephemeral flowers and their insects. FEMS Yeast Research 1:1–8. Leonhardt SD, Dworschak K, Eltz T, Blüthgen N. 2007. Foraging loads of stingless bees and utilization of stored nectar for pollen harvesting. Apidologie 38:125–135. Lins ACS, Silva TMS, Camara CA, Silva EMS, Freitas BM. 2003. Flavonoides isolados do pólen coletado pela abelha Scaptotrigna bipunctata (canudo). Revista Brasileira de Farmacognosia 13:40–41. Loper GM, Standifer LN, Thompson MJ, Gilliam M. 1980. Biochemistry and microbiology of bee-collected almond (Prunus dulcis) pollen and bee bread. I. Fatty acids, sterols, vitamins and minerals. Apidologie 11:63–73. Machado JO. 1971. Simbiose entre as abelhas sociais brasileiras (Meliponinae, Apidae) e uma espécie de bactéria. Ciência e Cultura 23:625–633. McGovern PE, Zhang J, Tang J, Zhang Z, Hall GR, Moreau RA, Nuñez A, Butrym ED, Richards MP, Wang CS, Cheng G, Zhao Z, Wang C. 2004. Fermented beverages of pre- and proto-historic China. Proceedings of the National Academy of Sciences 101:17593–17598. Menezes C. 2010. A produção de rainhas e a multiplicação de colônias em Scaptotrigona aff. depilis (Hymenoptera, Apidae, Meliponini). Tese de doutorado, Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras, Universidade de São Paulo, Ribeirão Preto, Brasil. 97 pp. Michener CD. 1974. The Social Behavior of the Bees. Harvard University Press; Cambridge, USA. 404 pp. Michener CD. 2000. The Bees of the World. 2nd ed. Johns Hopkins University Press; Baltimore, USA. 913 pp. Mueller UG, Gerardo NM, Aanen DK, Six DL, Schultz TR. 2005. The evolution of agriculture in insects. Annual Review of Ecology, Evolution, and Systematics 36:563–595. Nogueira-Neto P. 1997. Vida e criação de abelhas indígenas sem ferrão. Nogueirapis; São Paulo, Brasil. 445 pp. Noll FB, Zucchi R, Jorge JA, Mateus S. 1996. Food Collection and Maturation in the Necrophagous Stingless Bee, Trigona hypogea (Hymenoptera: Meliponinae). Journal of the Kansas Entomological Society 69:287–293. Oliveira ML, Morato EF. 2000. Stingless bees (Hymenoptera, Meliponini) feeding on stinkhorn spores (Fungi, Phallales): robbery or dispersal? Revista Brasileira de Zoologia 17:881–884. Pain J, Maugenet J. 1966. Recherches biochimiques et physiologiques sur le pollen emmagasiné par les abeilles. Annales Abeille 9:209–236. Penedo MCT, Testa PR, Zucoloto FS. 1976. Valor nutritivo do gevral e do levedo de cerveja em diferentes misturas com pólen para Scaptotrigona (Scaptotrigona) postica (Hymenoptera, Apidae). Ciência e Cultura 28:536–538. Pérez-Pérez E, Rodríguez-Malaver A, Vit P. 2007. Efecto de la fermentación postcosecha en la capacidad antioxidante de miel de Tetragonisca angustula Latreille, 1811. BioTecnología 10:14–20. Pires NVCR, Venturieri GC, Contrera FAL. 2009. Elaboração de uma dieta artificial protéica para Melipona fasciculata. Embrapa Amazônia Oriental, Belém, PA, Brasil. Documentos / Embrapa Amazônia Oriental; 363. Promnuan Y, Kudo T, Chantawannakul P. 2009. Actinomycetes isolated from beehives in Thailand. World Journal of Microbiology and Biotechnology 25:1685–1689. 10 The Role of Useful Microorganisms to Stingless Bees… 171 Rosa CA, Lachance MA, Silva JO, Teixeira AC, Marini MM, Antonini Y, Martins RP. 2003. Yeast communities associated with stingless bees. FEMS Yeast Research 4:271–275. Roubik D. 1989. Ecology and Natural History of Tropical Bees. Cambridge University Press New York; USA. 514 pp. Sachs JL, Essenberg CJ, Turcotte MM. 2011. New paradigms for the evolution of beneficial infections. Trends in Ecology and Evolution 26:202–209. Sanz S, Gradillas G, Jimeno F, Perez C, Juan T. 1995. Fermentation problem in Spanish NorthCoast honey. Journal of Food Protection 58:515–518. Silva TMS, Camara CA, Lins ACS, Barbosa-Filho JM, Silva EM, Reis, IT, Freitas BM, Santos FAR. 2006. Chemical composition and free radical scavenging activity of pollen loads from stingless bee Melipona subnitida Ducke. Journal of Food Composition and Analysis 19:507–511. Silva TMS, Camara CA, Lins ACS, Agra, MF, Silva EM, Reis, IT, Freitas BM. 2009. Chemical composition, botanical evaluation and screening of radical scavenging activity of collected pollen by the stingless bees Melipona rufiventris (uruçu-amarela). Anais da Academia Brasileira de Ciências 81:173–178. Souza B, Roubik D, Barth O, Heard T, Enríquez E, Carvalho C, Villas-Bôas J, Marchini L, Locatelli J, Persano-Oddo L, Almeida-Muradian L, Bogdanov S, Vit P. 2006. Composition of stingless bee honey: Setting quality standards. Interciencia 31:867–875. Souza BA, Alves RMO, Carvalho CAL. 2007. Diagnóstico da arquitetura de ninho de Oxytrigona tataira (Smith, 1863) (Hymenoptera: Meliponinae). Biota Neotropica 7:83–86. Souza BA, Marchini LC, Dias CTS, Oda-Souza M, Carvalho CAL, Alves RMO. 2009. Avaliação microbiológica de amostras de mel de trigoníneos (Apidae: Trigonini) do Estado da Bahia. Ciência e Tecnologia de Alimentos 29:798–802. Souza RCS, Yuyama LKO, Aguiar JPL, Oliveira FPM. 2004. Valor nutricional do mel e pólen de abelhas sem ferrão da região amazônica. Acta Amazonica 34:333–336. Standifer LN, McCaughey WF, Dixon SE, Gilliam M, Loper GM. 1980. Biochemistry and microbiology of pollen collected by honeybees (Apis mellifera) from almond, Prunus dulcis. II Protein, amino-acids and enzymes. Apidologie 11:163–171. Teixeira ACP, Marini MM, Nicoli JR, Antonini Y, Martins RP, Lachance MA, Rosa CA. 2003. Starmerella meliponinorum sp. nov., a novel ascomycetous yeast species associated with stingless bees. International Journal of Systematic and Evolutionary Microbiology 53:339–343. Vásquez A, Olofsson TC. 2009. The lactic acid bacteria involved in the production of bee pollen and bee bread. Journal of Apicultural Research 48:189–195. Venturieri GC, Oliveira PS, Vasconcelos MAM, Mattietto RA. 2007. Caracterização, colheita, conservação e embalagem de méis de abelhas indígenas sem ferrão. Embrapa Amazônia Oriental; Belém, Brasil. 51 pp. Villanueva-G R, Roubik DW, Colli-Ucán W. 2004. Extinction of Melipona beecheii and traditional beekeeping in the Yucatán peninsula. Bee World 86:35–41. Vit P, Medina M, Enríquez ME. 2004. Quality standards for medicinal uses of Meliponinae honey in Guatemala, Mexico and Venezuela. Bee World 85:2–5. Vollet-Neto A, Maia-Silva C, Menezes C, Venturieri GC, De Jong D, Imperatriz-Fonseca VL. 2010. Dietas protéicas para abelhas sem ferrão. pp. 121–129. In Anais do IX Encontro Sobre Abelhas de Ribeirão Preto, Ribeirão Preto, São Paulo, Brasil. White JW, Subers MH, Schepartz AI. 1963. The identification of inhibine, the antibacterial factor in honey, as hydrogen peroxide and its origin in a honey glucose-oxydase system. Biochimica et Biophysica Acta 73:57–70. Yoshiyama M, Kimura K. 2009. Bacteria in the gut of Japanese honeybee, Apis cerana japonica, and their antagonistic effect against Paenibacillus larvae, the causal agent of American foulbrood. Journal of Invertebrate Pathology 102:91–96. Zucoloto FS. 1975. Valor nutritivo de pólens usados por diferentes espécies de abelhas para Nannotrigona (Scaptotrigona) postica (Hymenoptera, Apoidea). Revista Brasileira de Biologia 35:77–82. Chapter 11 Microorganisms Associated with Stingless Bees Paula B. Morais, Paula S. São Thiago Calaça, and Carlos Augusto Rosa 11.1 Introduction Evidence for the great biodiversity associated with stingless bees is obtained from the variety of materials and structures used to build their nests. Inside the nest, there are different shapes and arrangements of brood cells and food storage containers. Wax secreted by stingless bees is mixed with plant resins to produce cerumen (Wille and Michener 1973; Michener 1974; Roubik 1983). Honey and pollen are stored in separate cerumen pots (Fig. 11.1). The size and shape of these pots vary among bee species. Stored nectar or ripened honey is found in the extremes of the nest cavity (for storage during heavy flowering periods), while pollen and some honey surround the brood area (Roubik 2006). Diverse ethnomedicinal properties attributed to stingless bee honeys are known in Brazil, Ecuador, Guatemala, Mexico, and Venezuela (Vit et al. 2004; Mendes and Antonini 2008; Guerrini et al. 2009), where pot-honey is worth up to 20 times more than Apis mellifera honey (Nogueira-Neto 1997; Vit et al. 1998). Most of the studies of the microorganisms associated with stingless bees were carried out with the objective of describing the bacterial and fungal communities associated with these bees. However, data on the functional relationship between P.B. Morais Laboratório de Microbiologia Ambiental e Biotecnologia, Campus Universitário de Palmas, Universidade Federal do Tocantins Palmas, TO 77020220, Brazil P.S. São Thiago Calaça Fundação Ezequiel Dias (FUNED), Rua Conde Pereira Carneiro 80, Gameleira, Belo Horizonte, MG 30510-010, Brazil C.A. Rosa (*) Departamento de Microbiologia, ICB, CP 486, Universidade Federal de Minas Gerais, Belo Horizonte, MG 31270-901, Brazil e-mail: carlrosa@icb.ufmg.br 173 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_11, © Springer Science+Business Media New York 2013 174 P.B. Morais et al. Fig. 11.1 Honey and pollen of Melipona quinquefasciata stored in separate cerumen pots. Photo: P.S. São Thiago Calaça microorganisms and stingless bees are scarce. Although honey has some distinct properties that inhibit the growth of microorganisms, such as high sugar concentrations and high acidity (Snowdon and Cliver 1996), microbial fermentation has been suspected to contribute to the transformation of pollen into bee bread and in the formation of the honey itself. Microorganisms may also have a role in honey maturation and in the biochemical modification of stored pot pollen. After its collection by bees from flowers, the pollen stored inside meliponine nests becomes biochemically distinct due to fermentation processes, but it is not clear if yeasts or bacteria (or both) are responsible for these processes (Ganter 2006). The association of microorganisms with honey, pollen, immature, and adult bees is indicative of a functional relationship with these insects. In this chapter, we will discuss the presence of different species of bacteria, molds, and yeasts associated with stingless bees and the possibility of the existence of a symbiotic relationship between these organisms. 11.2 Bees and Microbes Insects engage in a vast array of symbiotic relationships with a wide diversity of microorganisms, in which some of them benefit the host nutritionally and provide protection from natural enemies (Klepzig et al. 2009). Yeasts, for example, are a food source for insects and are known to be the main source of sterols, vitamins, and protein for adult and larval stages of Drosophila (Morais et al. 1995b). The number of symbionts of the ground-dwelling ants and termites is large compared to that of social wasps and bees (Wilson 1971; Kistner 1982). According to Peruquetti (2000), the highly social stingless bees (Apidae, Meliponini) seem to be an exception to this rule. Their nests have many guests, including mites, moths, cockroaches, flies, beetles, 11 Microorganisms Associated with Stingless Bees 175 fungi, and bacteria, some of which are obligate symbionts (Wasmann 1904; Salt 1929; Nogueira-Neto 1970; Machado 1971; Flechtmann and Camargo 1974; Aponte 1996; Kerr et al. 1996). Insect species are important vectors of microorganisms, including bacteria, fungi, and protozoans (Starmer and Lachance 2011; Redak et al. 2004; Purcell 1982). For example, the distribution and habitat specificity of yeasts depend primarily on the insect vectors but are also dependent on the substrate composition and the presence of inhibitory compounds (Morais and Rosa 2000; Morais et al. 1995a; Starmer et al. 1976). Various studies have aimed to characterize the microbial community associated with bees (Gilliam et al. 1984; Gilliam 1997; Inglis et al. 1993; Rosa et al. 1999, 2003; Teixeira et al. 2003). The microbiota of the European honey bee (Apis mellifera) has been isolated and identified (Gilliam 1997; Gilliam and Morton 1978; Piccini et al. 2004; Rada et al. 1997). These microbes are believed to help chemical conversion in the intestinal tract, preservation of pollen stored in comb cells, and production of antimycotic substances against the chalkbrood pathogen (Gilliam 1997). Most of the bacteria isolated from brood combs and hive floors of the honey bee belong to the genera Bacillus and Corynebacterium (Piccini et al. 2004). Studies on the microbiota of the alfalfa leafcutting bee showed a dominance of fungi (e.g., Aspergillus niger, Penicillium sp., and Saccharomyces sp.) and bacteria (e.g., Bacillus circulans, B. mycoides, Enterobacter agglomerans, and Pseudomonas sp.) (Goerzen 1991). Other spore-forming bacteria belonging to the genus Bacillus were found to be prevalent in larval populations of two solitary bees (Centris pallida and Anthophora sp.) (Gilliam et al. 1984, 1990a). Bacteria of the genus Lactobacillus were identified in A. mellifera and A. mellifera scutellata (Mohr and Tabbe 2006; Jeyaprakash et al. 2003). Recently, a novel bacterial flora composed of lactic acid bacteria of the genera Lactobacillus and Bifidobacterium was found in the stomach of A. mellifera (Olofsson and Vásquez 2008). In contrast, Evans and Armstrong (2006) failed to find Lactobacillus species in A. mellifera, suggesting that the gut microbial population is not constant even within the same species. Yoshiyama and Kimura (2009) did not find Lactobacillus species in the gut of A. cerana japonica, but they detected the following gut bacterial groups that had not been found in other Apis species: Staphylococcus saprophyticus (Firmicutes), Kocuria sp., Tsukamurella tyrosinosolvens, Microbacterium sp. (Actinobacteria), Sphingomonas melonis, Mesorhizobium sp. (Alphaproteobacteria), Janthinobacterium sp. (Betaproteobacteria), Escherichia coli, Pseudomonas sp., Providencia alcalifaciens, Erwinia tasmaniensis, and Moraxella sp. (Gammaproteobacteria). Honey bees visit flowers of many types, which vary geographically and seasonally. Furthermore, honey bees of different species tend to visit flowers of a particular species. Thus, Yoshiyama and Kimura (2009) suggest that variation of a characteristic gut bacterial flora in Apis species is likely to be related to variation in the food source, and this may be also true for other plant pollinators such as meliponines. The bacteria Streptomycetes sp. have also frequently been found in pollen, provisions, and alimentary canals of alfalfa leafcutter bees (Megachile rotundata), and these bacteria are considered to be part of the resident microbiota of the bee 176 P.B. Morais et al. (Inglis et al. 1993). Streptomyces fradiae was isolated from the hive materials of A. florea, and S. drozdrwiczii, S. albidoflavus, and S. badius were isolated from A. cerana in Thailand. According to Promnuan et al. (2009), Streptomyces species show a symbiotic relationship with some insects. A unique association between a new Streptomyces species and the European beewolf (Philanthus triangulum), a solitary hunting wasp, was reported. The beewolf females harbor the Streptomyces bacteria in specialized antennal glands and apply them to the brood cell prior to oviposition. The bacteria are taken up by the larva and are also found on the walls of the cocoon. Bioassays indicated that the streptomycetes protect the cocoon from fungal infestation and significantly enhance the survival probability of the larva, possibly by producing antibiotics (Kaltenpoth et al. 2005). Rosa et al. (1999) found that a killer toxin-producing Mucor species was a dominant fungus, together with the yeast C. batistae, in nearly 100 nests of the solitary bee, Diadasina distincta. This fungus may play a role in pollen maturation because it presents proteolytic and pectinolytic ability that could be combined with the yeast fermentative and lipolytic function for pollen transformation (Rosa et al. 1999). Inglis et al. (1993) showed that Candida bombicola (Starmerella bombicola) is frequently found in nectar, pollen, and provisions of the solitary bee Megachile rotundata. Rosa et al. (1999) isolated Candida batistae from the solitary bees D. distincta and Ptilothrix plumata in Brazil, and the authors suggested a possible mutualistic interaction between this yeast species and the bees. Pimentel et al. (2005) described two new species of yeasts, Candida riodocensis and Candida cellae, associated with two solitary bees, Megachile sp. and Centris tarsata, in the Atlantic rain forest of Brazil. At this time, the Starmerella clade contains more than 40 yeast species, most of which were isolated from bees (Table 11.1). This clade is defined as a single branch in the Ascomycetes that present the common ecological traits of the association with insects and ephemeral flowers. Species belonging to this clade, such as C. magnoliae, C. batistae, S. bombicola, and S. meliponinorum, are thought to be involved in a mutualistic relationship with bees (Gilliam 1979a; Inglis et al. 1993; Rosa et al. 1999). In addition to the two Starmerella species, Candida bombi is common in European bumble bees (Brysch-Heberg 2004). Candida davenportii, C. apicola, C. bombi, C. powellii, C. floricola, C. tilneyi, C. vaccinii, C. sorbosivorans, C. magnoliae, and C. apis have been isolated from bees, wasps, substrates that these insects visit and from other insects that visit the same substrates (Lachance et al. 2001a,b; Trindade et al. 2002). 11.3 Bacteria Associated with Stingless Bees and Their Ecological Roles Bacteria maintain a symbiotic relationship with various groups of bees (Roubik 1989). Although the interior of the nests of stingless bees has a high relative humidity and contains mud and large quantities of feces and other detritus, relatively few 11 177 Microorganisms Associated with Stingless Bees Table 11.1 Some yeast species in the Starmerella clade and their association with beesa Yeast species Bee species or bee substrate of isolation Locality Candida apicola C. apis C. batistae C. bombi C. cellae C. davenportii C. etchellsii C. floricola C. floris C. geochares C. magnolia C. powellii C. riodocensis C. tilneyi Starmerella bombicola S. meliponinorum a Bee gut Melipona quadrifasciata, M. rufiventris, Trigona spp., and their hives and pollen Trachea of a bee Ground nesting solitary bee Bombus terrestris, B. hortorum, B. cryptarum, Bombus sp. Centris tarsata (solitary bee) Dead wasp Trigona Unknown bee in Opuntia flowers Ipomoea flowers visited by bees Trigona spp. Honey of T. angustula and M. quinquefasciata Bee gut and pollen (Apis mellifera) Unknown bee on Ipomoea Pollen and nectar provision of Megachile sp. Halictid bee in Ipomoea carnea Honey and pollen of T. angustula, M. quinquefasciata, M. quadrifasciata, and F. varia Bombus sp. Trigona fulviventris Honey and pollen of T. angustula, M. quadrifasciata, M. rufiventris, and F. varia Trigona sp. Croatia Brazil, Costa Rica, Malaysia UK Brazil France, Germany Brazil UK Costa Rica USA Brazil Costa Rica Brazil, South Africa Croatia, USA Costa Rica Brazil Costa Rica Brazil Canada Costa Rica Brazil Costa Rica Data from Lachance (2011) bacteria are found in the nest, probably due to antibiotic substances in the nest materials and inhibitors produced by the bees themselves to suppress competitors (Roubik 1983). Bacteria present in the bee nests seem to have an important role in pot-honey maybe by inhibiting spoilage bacteria. In the intestinal tract of M. quadrifasciata, five different types of Bacillus spp. are found, although only one species may maintain a close relationship with the bee because it is found in bee’s intestines and also in pot-honey (Cruz-Landim 1996). It is possible that Bacillus meliponotrophicus is responsible for a type of pre-digestion of honey and pollen produced by M. quadrifasciata (Nogueira-Neto 1997). Machado (1971) has shown that B. meliponotrophicus is associated with Trigona and Melipona but not with Apis and Bombus, which are phylogenetically related to the stingless bees. In the M. quadrifasciata colonies, bacteria are present in high concentrations in larval food and honey pots, where they take part in the fermentation process. The relationship between the bacterial species and the bee is obligatory because the use of antibiotics/streptomycin in the food led to the disappearance of the colony. 178 P.B. Morais et al. Spore-forming bacteria belonging to the genus Bacillus were found in some nests of stingless bees Melipona panamica (B. alvei, B. circulans, and B. megaterium) and Trigona necrophaga (B. circulans, B. licheniformis, P. megaterium, B. pumilis, and B. subtilis) in Panama (Gilliam et al. 1985, 1990b). Lactic acid bacteria (probably Lactobacillus species) were isolated in high numbers from honey and pollen samples of T. angustula and M. quadrifasciata (C.A. Rosa, unpublished results). These bacteria likely have a role in the honey maturation of these bees by suppressing spoilage bacteria, as we speculate above. Two stingless bees, Tetragourla laeviceps and Tetragourla fuscobalteata, commonly found in the northern region of Thailand, are known to construct nests inside forest trees. Bacterial communities of T. laeviceps included Streptomyces pseudogriseolus, S. rochei, S. drozdowiczii, S. mutabilis, S. minutiscleroticus, S. albus, S. tosaensis, and S. malaysiensis. In contrast, in the T. fuscobalteata hives, S. ambofaciens, S. mutabilis, S. coalescens, and S. violaceoruber were isolated from brood cells (Promnuan et al. 2009). The ecological role of the bacterial community still needs to be determined. Although beneficial endosymbiosis has been described in many solitary and colonial insects that vary from obligate and intracellular to facultative and extracellular within the gut lumen (Kikuchi 2009). Anderson et al. (2011) point that virtually nothing is known about beneficial symbionts of bees. Mohr and Tabbe (2006) suggest the existence of cosmopolitan gut bacteria in bees, although Koch and Schmid-Hempel (2011) affirm that bumble bee gut presents a highly specific microflora largely different from bacteria associated with guts of honey bees. Killer et al (2009) described a new species Bifidobacterium bombi among gram-positivestaining, anaerobic, non-spore-forming, lactate- and acetate-producing bacteria isolated from the digestive tracts of different bumble bee species (Bombus lucorum, Bombus pascuorum, and Bombus lapidarius). Recent studies on the microbial flora of the honey bee gut have revealed an apparently highly specific community of resident bacteria that might play a role in immune defense and food preservation for their hosts. As pointed by Anderson et al. (2011), honey bees used in agriculture are stressed by a plethora of agricultural chemicals and their associated by products, and this may be a general situation for most bees including wild meliponing, and those antibacterial agents may kill bacterial symbionts resulting in the decline of bee populations as seen for honey bees in part of the world. 11.4 Molds Associated with Stingless Bees There are few reports on molds associated with stingless bees. Roubik and Wheeler (1982) report the presence of Stemphylium (similar to those that decompose wood) in nests of M. panamica. Fungal identification was performed by observation of spores and hyphae found in the stomach of a beetle of the genus Scotocryptus that inhabits the nests of stingless bees. Gilliam et al. (1990b) reported the presence of a 11 Microorganisms Associated with Stingless Bees 179 green fungus in the honey of M. Panamica. Melo (1996) also reported a dark purple fungus in the cerumen of M. capixaba. However, the ecological roles of these fungi have not been determined. Early mycological studies recognized that certain molds are common saprophytes both on and inside dead honey bees and brood combs and are probably unable to become established within the bee or the hive (Betts 1920). Fungusassociated spoilage of provisions and mortality of honey bees are rare (Batra et al. 1973). Gilliam et al. (1988) showed that only Ascosphaera apis, which causes chalkbrood disease, is of economic importance. Egorova (1971) isolated Aspergillus flavus, A. versicolor, Mucor alboalter, Penicillium granulatum, P. solitum, and Sporotrichum olivecum from bee bread. Two studies, Chevtchik (1950) and Pain and Maugnet (1966), did not mention molds in pollen or bee bread (the actual food consumed by bee larvae). However, Gilliam et al. (1989) isolated Aureobasidium pullulans, P. corylophilum, P. crustosum, and Rhizopus nigricans (R. stonolifer) in pollen and bee bread but not from floral pollen. These authors determined that these isolates may have been introduced by the bees. They noticed that the number of isolates decreased after storage by the bees, and Mucor sp., the dominant mold in floral pollen, was not found in corbicular pollen or bee bread. They concluded that, as with yeasts (Gilliam 1979a) and Bacillus spp. (Gilliam 1979b), the mold biota of corbicular pollen and bee bread may be the result of microbial inoculation by the bees and chemical changes in pollen that allow some species but not others to survive, as noted by Klungness and Peng (1983). In the course of a study on pollen diets of three sympatric species of stingless bees Heterotrigona collina, Tetragonnla melina, and T. melanocephala in Sabah, Malaysia, Eltz et al. (2002) observed that large fractions of the foragers of three colonies of H. collina collected corbicular loads of fungal spores in lieu of pollen. Collection of spores continued for at least three consecutive days. The spores were brought to germination in the laboratory, and the culture was identified as mold of the genus Rhizopus. Their observations represent the first reported case of the collection of Rhizopus mold spores in lieu of pollen by bees and a rare case of the collection of fungal spores by bees other than honey bees (Apis) (Eltz et al. 2002). Yeasts and molds are found naturally in honey, according to Gilliam (1997), who argues that microorganisms associated with bees are non-pathogenic and that most of these microorganisms are not yet known. Eltz et al. (2002) affirm that the fungi collection sometimes replaces pollen harvesting in Apis, Trigona, and Partamona. Ferraz et al. (2006) detected Aspergillus sp., A. niger, Penicillium sp., A. terreus, Curvularia sp., Monilia sp., Nigrospora sp., Cladosporium sp., and Trichoderma sp. in “jandaíra” Melipona subnitida, which inhabit the semiarid rocky areas of Brazilian Northeast. A species of Curvularia was reported as an inhabitant of Trigona sp. inhabiting the dry Caatinga ecosystem of Northeastern Brazil (Ferraz et al. 2006). However, the ecological role of these filamentous fungal species in the bee nests has not been determined. Indeed, Gibson and Hunter (2005) noted that the distinction between commensal and mutualistic interactions is often difficult to discern. 180 P.B. Morais et al. 11.5 Yeasts Associated with Stingless Bees Bee nests harbor a diversified yeast microbiota, and their role in biochemistry, nutrition, and physiology of bees has been investigated (Teixeira et al. 2003). According to Gilliam (1997), in social species, yeasts may have an important role in the conversion of pollen into available nutrients. Early studies showed that microbiota of pollen taken directly from flowers, corbicular pollen, and pollen stored in comb cells in the hive (bee bread) are similar. Foraging bees add microbes to pollen during collection and the same species of bacteria and yeasts are found in guts of worker bees and in corbicular pollen (Gilliam 1979a; Gilliam et al. 1984; Gilliam and Prest 1987). These microorganisms may be involved in the metabolic conversion, fermentation, and preservation of the stored food. The conversion of pollen to bee bread has often been postulated to be the result of microbial action, principally a lactic acid fermentation caused by bacteria and yeasts (Haydak 1958). Yeasts have been isolated from honey bees, stingless bees, and solitary bees (Gilliam 1997; Rosa et al. 2003; Brysch-Heberg 2004). The Amazonian species Ptilotrigona lurida maintains mutualistic interactions with an unidentified yeast species that is believed to be responsible for dehydrating and retarding the deterioration of the pollen in the bee nest (Camargo et al. 1992). Starmerella meliponinorum was described in association with nests of the eusocial stingless bee, T. angustula, and could also be associated with food, both honey and pollen, propolis, detritus, and adult individuals of M. quadrifasciata, M. rufiventris, T. angustula, and T. fulviventris (Rosa et al. 2003; Teixeira et al. 2003). Starmerella meliponinorum and C. apicola, also part of the Starmerella clade, have been consistently isolated from T. angustula adults, honey, pollen provisions and refuse, M. quadrifasciata and M. rufiventris in Brazil, and Heterotrigona Tetragonula sp. in Malaysia. Therefore, they may have a mutualistic relationship with stingless bees. Most of the described species in the Starmerella clade are associated with bees or related habitats (Rosa et al. 2003). Some species in the clade are also found in other environments. In addition to the two Starmerella species, S. bombicola and S. meliponinorum, C. apicola and closely related types are found in tropical meliponine bees worldwide (Lachance 2011). Rosa et al. (2003) showed that the yeast community associated with T. angustula, M. quadrifasciata, and Frieseomelitta varia is specific to these bee species, although the ecological roles of the yeasts have not yet been defined. A large number of other yeast species were isolated from various adults of these three bee species, including Aureobasidium pullulans, Pseudozyma antarctica, and various species of Cryptococcus and Rhodotorula that may represent a transient mycota vectored by bees. Debaryomyces hansenii was isolated from adults and garbage pellets of M. quadrifasciata and from a propolis sample of T. angustula. This halotolerant and osmotolerant generalist is a frequent contaminant of human food and usually rare on the phylloplane (Fonseca and Inácio 2006; Kurtzman 2011a, 2011b). It was reported to cause spoilage of A. mellifera honey (Snowdon and Cliver 1996). Highly osmotolerant species of Zygosaccharomyces were isolated from the honey of T. angustula, from an adult M. quadrifasciata and from a garbage pellet of F. varia. Zygosaccharomyces machadoi was isolated from a garbage pellet of T. angustula 11 Microorganisms Associated with Stingless Bees 181 Fig. 11.2 Ripe honey of Melipona quinquefasciata (Rosa and Lachance 2005). The new species Zygosaccharomyces siamensis was isolated from raw honey of A. mellifera, A. dorsata, and Tetragonula pagdeni in Thailand (Saksinchai et al. 2012). These yeasts might act as an agent of pot-honey spoilage for these bees, as argued by Rosa et al. (2003). Other yeasts already isolated from stingless bees are Hyphopichia burtonii (Kurtzman 2011a) and Priceomyces mellissophilus (Kurtzman 2011b), whereas M. kunwiensis and M. reukaufii are consistently isolated from Bombus bee species (Lachance 2011). Calaça (2011) reported that the number of yeast cells was higher in unripe pothoney than in ripe honey of M. quinquefasciata (Fig. 11.2) collected in Brazil, which indicates that abundance and diversity of yeasts decreases during honey ripeness. Candida sp. MUCL 4571, a new undescribed species sister of C. apicola, was the prevalent species in the samples and could have a mutualistic association with this bee. 11.6 A Possible Mutualistic Interaction Between Yeasts and Bees? High yeast counts in larval provisions suggest that these microorganisms are metabolically active, and that the enzymes they produce may be important for the improvement of the nutritional characteristics of pollen. Both social and solitary bees introduce yeasts into their nests (Gilliam 1997), which possibly bring nutritional benefits to larvae. Bees require nutrients, such as proteins, lipids, and vitamins, from pollen and carbohydrates from nectar (Standifer et al. 1980). Corbicular pollen is transformed into bee bread (comb pollen) through a fermentative process that is carried out primarily by yeasts (Pain and Maugnet 1966) and brings a higher nutritional value and availability of amino acids in the bee bread compared to corbicular pollen (Loper et al. 1980; Standifer et al. 1980). 182 P.B. Morais et al. Gibson and Hunter (2005) defined five stages in the pathway to obligate mutualism: (1) consistent and extended contact; (2) avoidance of lethal harm during contact; (3) coadaptation, leading to increased tolerance; (4) further coadaptation, leading to dependence and/or interdependence; and (5) permanent association. In studies of the association of yeasts and Chrysoperla lacewings, Gibson and Hunter (2005) argue that the ease with which the yeasts can be cultured suggests that these two organisms are not interdependent obligate mutualists, as in case of bacterial symbionts (Douglas 1998). Although they could not find evidence that resident yeasts bring nutritional benefits to the lacewings, they were not able to cultivate yeast-free lacewings and, therefore, could not reach a conclusion on the role of yeasts in the interaction. Our own studies on the yeasts associated with the bees M. quinquefasciata in Minas Gerais (Southeastern Brazil) and M. compressipes, M. scutellaris, Plebeia sp., Scaptotrigona polysticta, and S. tubiba in Cerrado ecosystems of Central North Brazil indicate that those yeast strains are very difficult to maintain in culture collections, and various strains die before a complete identification is reached, raising the possibility that association with the bees is important for survival of those yeasts. Further investigation is needed to reach any conclusions on the mutualistic interactions between stingless bees and yeasts. Records of yeast-insect associations in which the role of the yeasts is not well understood include: green June beetles (Vishniac and Johnson 1990), nitidulid beetles (Lachance et al. 2003), clerid beetles (Lachance et al. 2001a), encyrtid parasitoids (Lebeck 1989), ichneumonid parasitoids (Middeldorf and Ruthmann 1984), fire ants (Ba and Phillips 1996), leafcutting bees (Teixeira et al. 2003), solitary digger bees (Rosa et al. 1999), vespid wasps and bumble bees (Stratford et al. 2002), honey bees (Spencer and Spencer 1997), and the green lacewings in the genus Chrysoperla (Hagen et al. 1970; Gibson and Hunter 2005). Although we cannot rule out the possibility that stingless bees are simply vectors for yeasts, Lachance et al. (2011) affirm that the insect vectors appear to be the primary agents responsible for the organization of the yeast communities, a role of great importance for the understanding of yeast ecology in all ecosystems. 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Silveira FA, Melo GAR, Almeida EAB. 2002. Abelhas brasileiras. Sistemática e identificação. Fundação Araucária; Belo Horizonte, Brasil. 253 pp. Snowdon JA, Cliver D. 1996. Microorganisms in honey. International Journal of Food Microbiology 31:1–26. Spencer JFT, Spencer DM. 1997. Ecology: where yeasts live. pp. 33–67. In Spencer JFT, Spencer DM, eds. Yeasts in Natural and Artificial Habitats. Springer-Verlag; Berlin, Germany. 381 pp. Standifer LN, McCaughey WF, Dixon SE, Gilliam M, Loper GM. 1980. Biochemistry and microbiology of pollen collected by honey bees (Apis mellifera) from almond (Prunus dulcis). II. Protein, aminoacids and enzymes. Apidologie 11:163–171. Starmer WT, Heed WB, Miranda M, Miller MW, Phaff HJ. 1976. The ecology of Yeast flora associated with Cactiphilic. Drosophila and their host plants in the Sonoran Desert. Microbial Ecology 3:11–30. Starmer WT, Lachance MA. 2011. Yeast ecology. pp. 33–67. In Kurtzman CP, Fell JW, Boeukhout T, eds. The Yeasts, a Taxonomic Study. Elsevier Science; Amsterdam, The Netherlands. 2354 pp. Stratford M, Bond CJ, James SA, Roberts IN, Steels H. 2002. Candida davenportii sp. nov., a potential soft-drinks spoilage yeast isolated from a wasp. International Journal of Systematic and Evolutionary Microbiology 52:1369–1375. Saksinchai S, Suzuki M, Chantawannakul P, Ohkuma M, Lumyong S. 2011. A novel ascosporogenous yeast species, Zygosaccharomyces siamensis, and the sugar tolerant yeasts associated with raw honey collected in Thailand. 2012. Fungal Diversity 52:123–139 Teixeira ACP, Marini MM, Nicoli JR, Antonini Y, Martins RP, Lachance MA, Rosa CA. 2003. Starmerella meliponinorum sp. nov., a novel ascomycetous yeast species associated with stingless bees. International Journal of Systematic and Evolutionary Microbiology 53:339–343. Trindade RC, Resende MA, Silva CM, Rosa CA. 2002. Yeasts associated with fresh and frozen pulps of Brazilian tropical fruits. Systematic and Applied Microbiology 25:294–300. Vishniac HS, Johnson DT. 1990. Development of a yeast flora in the adult green June beetle (Cotinis nitida, Scarabaeidae). Mycologia 82:471–479. Vit P, Medina M, Enríquez ME. 2004. Quality standards for medicinal uses of Meliponinae honey in Guatemala, Mexico and Venezuela. Bee World 85:2–5. Vit P, Persano Oddo L, Marano ML, Mejias, ES. 1998. Venezuelan stingless bee honeys characterized by multivariate analysis of physicochemical properties. Apidologie 29:377–389. Wasmann E. 1904. Contribuição para o estudo dos hóspedes de abelhas brasileiras. Revista do Museu Paulista 6:482–487. Wille A, Michener CD. 1973. The nest architecture of stingless bees with special reference to those of Costa Rica. Revista de Biologia Tropical 21:1–278. Wilson EO. 1971. The Insect Societies. Harvard University Press; Massachusetts. 548 pp. Yoshiyama M, Kimura K. 2009. Bacteria in the gut of Japanese honeybee Apis cerana japonica, and their antagonistic effect against Paenibacillus larvae, the causal agent of American foulbrood. Journal of Invertebrate Pathology 102:91–96. Chapter 12 Stingless Bee Food Location Communication: From the Flowers to the Honey Pots Daniel Sánchez and Rémy Vandame 12.1 Introduction Colonies of social insects lack a central control yet they function as a coherent whole, adjusting their activities in response to a changing environment (Seeley 1995; Visscher 1998; Wilson 2000). How such biological systems are organized has been one of the biggest questions raised by researchers in this field. Honey bees have been studied since ancient times. Aristotle noted that honey bees may recruit nestmates to rich food sources (Nieh 1999). It was the Austrian scientist, Karl von Frisch, at the end of World War I, who described a series of behavioral patterns in the honeybee Apis mellifera (Hymenoptera: Apidae, Apini) that seemed related to the organization of the colonies of this species (von Frisch 1967). To observe their behaviors inside the colony, he designed a glass-walled hive, which allowed him to notice that some bees were performing particular behaviors which he called dances. These dances apparently had information about where the dancing forager had found pollen or nectar. Von Frisch discovered what it is now known as the honeybee dance language. Later, with his book “The dance language and orientation of bees” published in 1967, von Frisch described in detail the communication behaviors observed in A. mellifera and briefly discussed similar behaviors in other insects. Subsequently, other researchers raised the possibility that recruits may orient only to the smells of the food brought back by the explorer. They hypothesized that the dance behavior was actually an experimental artifact, or a behavior that did not convey location information to nestmates (Wenner et al. 1969; Gould and Gould 1988; Wenner 2002). However, subsequent studies provided detailed, convincing evidence that bees can use the spatial information encoded in the dance language D. Sánchez (*) • R. Vandame El Colegio de la Frontera Sur, Carretera Antiguo Aeropuerto Km 2.5, Tapachula, Chiapas CP 30700, Mexico e-mail: dsanchez@ecosur.mx 187 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_12, © Springer Science+Business Media New York 2013 188 D. Sánchez and R. Vandame and that a correct interpretation of this information is beneficial for colony fitness (Robinson 1986; Dyer 2002b; Dornhaus et al. 2006). Parallel to the research on the honeybee language, a rising interest in unveiling the ultimate and the proximal mechanisms involved in its evolution led researchers to investigate other species, like the stingless bees (Hymenoptera, Apidae, Meliponini). Stingless bees have proven to have mechanisms of communication as remarkable as the honeybee’s, although behaviors identical to the honeybee waggle dance have not been observed in studied species. However, stingless bees consist of hundreds of species that display a diversity of behaviors and ecological adaptations. Thus, they deserve to be studied in their own right, given their importance in their respective ecological niches. In the following pages, the reader is acquainted with elementary knowledge about stingless bee food location communication. First, we give a general view of the topic. Then, several communication mechanisms are described. External and internal factors that affect the communication system in stingless bees are detailed. Finally, as a result of integration of these elements, the food communication systems and their influence on the foods collected become evident. The characteristics of the pot-honey and pot-pollen are of course affected by the food matter thus collected. 12.2 Food Location Communication Systems in Highly Social Bees (Apidae) After the initial discovery of the honeybee dance, von Frisch turned his attention to the evolutionary origins of this behavior. Because the meliponines (stingless bees) are similar to honeybees, Martin Lindauer, one of von Frisch’s students, began to study stingless bee recruitment communication (Lindauer and Kerr 1960; Lindauer 1967). Together with the Brazilian scientist, Warwick Kerr, Lindauer found a wide range of potential recruitment and communication behaviors in the several meliponine species that they studied, including behaviors that were not observed in honeybees: random searching (no location communication) and odor trails, to name two. They hoped to help elucidate the evolution of the A. mellifera waggle dance. Whether stingless bee and honey bee recruitment communication derived from a common ancestor or evolved independently is unclear, although molecular evidence suggests that the two groups are not as closely related as once thought (Cameron and Mardulyn 2001). Nonetheless, even if their recruitment communication systems have evolved convergently, they exhibit certain similarities that suggest common pathways, perhaps deriving from traits shared by both groups of bees and similarities in the ecological niches that they occupy. More recently, it has been demonstrated that the meliponine bees have communication systems as complex, in their own ways, as those described by von Frisch for A. mellifera (Dyer 2002a; Nieh 2004). In general, social insects use communication for various purposes, such as to ensure the cohesion of the colony, 12 Stingless Bee Food Location Communication... 189 to warn the presence of danger, to find mates, and to communicate the spatial location of resources, to name a few (Wille 1983; Gould and Gould 1988; Collins et al. 1989; Wilson 2000). With respect to foraging communication systems, the focus of von Frisch’s work, it has been observed that highly social bees such as A. mellifera and stingless bees have developed sophisticated mechanisms to recruit nestmates to resources such as pollen, nectar, water, resins, and places to establish new colonies (von Frisch 1967; Nieh 2004; Seeley 2010). With these mechanisms, scouts can send recruits to specific sites that offer profitable resources, a process often referred to as “food recruitment”. In fact, the arrival of recruits to an advertised food source is the conclusion of a series of processes that occur at various levels of the colony and the individual (Biesmeijer and Slaa 2004). Meliponines are a good model to study the evolution of recruitment because they are a highly diverse taxon and display correspondingly diverse strategies to reach the same goal: recruit nestmates to rich food sources. 12.3 Food Recruitment in Stingless Bees Stingless bees are a monophyletic group found in tropical and subtropical areas of the world, in America, Asia, Africa, and Australia (Roubik 1989). Unlike honeybees, which consist of approximately 11 species in one genus (Apis), stingless bees consist of hundreds of species distributed in 36 genera (Michener 2000). In addition, stingless bees have multiple lifestyles, including necrophagy, and can recruit to resources such as dead animals, nectar sources, and even the food reserves of other bee species (Roubik 1989). Also, stingless bees exhibit a great diversity of behaviors for transferring information about the location of a resource. These range from pheromone trails to the referential coding through sounds (Nieh 2004). Unfortunately, no studies on stingless bees have been conducted as intensively as in A. mellifera, so the understanding of their biology is in an early stage compared to what is known in the Apini. Fortunately, the meliponines have recently drawn the attention of researchers in animal communication, since their study could have implications for understanding the evolution of communication within the Apidae. It is useful at this point to define some key terms for understanding the processes that arise during food recruitment in social bees. An individual is considered a forager if it is collecting resources for the colony. A scout is a forager that leaves the colony to find resources on its own. A forager is considered to be a recruit if it receives information from the scout about the location of a rich food source (von Frisch 1967). Food recruitment is a communication system that refers to a set of behaviors involved in the transfer of information between scouts and recruits; these behaviors are known as mechanisms for information transfer or simply communication mechanisms. The latter explanation is more specific because communication generally occurs through signals whereas information transfer involves both signals and cues. In general, we can classify communication according to where it occurs: inside the colony (recruitment movements, trophallaxis, and sounds) and outside the 190 D. Sánchez and R. Vandame colony (social facilitation, pheromones). This, however, is not sufficient to understand the complexity that occurs in the communication systems. Biesmeijer and de Vries (2001) proposed the following classification of the individuals involved in food recruitment in order to better understand the phenomenon of communication: 1. Naïve forager: forager without any previous experience in collecting resources. 2. Explorer (also known as a scout): forager using only internal information to search for resources not previously known to it. 3. Recruit: forager using external information, generally from scouts, to find resources not previously known by her. 4. Engaged recruit (also called employed recruit): forager collecting resources in a known location; it does not usually follow external information while collecting resources. 5. Unemployed experienced foragers: individuals that are temporarily idle because the resource they were visiting was depleted. 6. Inspector: individual temporarily idle that periodically revisits depleted food sources expecting to find them profitable again. 7. Reactivated forager: individual that resumes its foraging activities after having received external information on the availability of resources it previously collected. The information delivered by communication about resources outside the nest along with other information such as weather and the external experiences of foraging outside the nest are jointly referred to as external information (Biesmeijer and Slaa 2004). Thus there are two types of external information according to its source: information from other bees and information from the environment. The other source of information used by foragers, which has not received sufficient attention yet, is internal information, which can be more precisely defined as the physiological and genetic status of the individual. A bee’s experience, genetic variation, age, and hormone levels are examples of internal information (Biesmeijer and Slaa 2004). Although it is not a communication mechanism, internal information has a significant influence on the decision of recruits and experienced bees (Biesmeijer et al. 1998). The overall strategy that colonies use to gather resources is thus the result of the interaction between the communication system, the conditions inside and outside the colony, and forager internal information. In the end, this results in either the recruitment or non-recruitment of foragers to a specific location. 12.4 External Sources of Information: Mechanisms of Communication and Recruitment Insects search for and gather food in unpredictable environments (Goulson 1999). This makes it difficult to exploit efficiently those resources. To keep foragers from wasting time and energy in the tasks of resource gathering, highly social bee species 12 Stingless Bee Food Location Communication... 191 have developed organization systems that allow them to make continuous adjustments in the number of individuals performing certain tasks inside or outside the colony. This is achieved through behaviors that enable bees to communicate with each other, establishing the conditions for the colony to survive in cohesion, in addition to providing a competitive advantage, in some cases, over other species that do not communicate or coordinate to the same degree (Dornhaus et al. 2006). Thus, by understanding the mechanisms of foraging communication we will be able to understand more in general about communication systems in social insects. 12.4.1 Mechanisms of Communication Inside the Nest Successful foragers of most stingless bee species produce sounds and execute particular behaviors inside the nest or hive after returning from a good food source. In some species, these sounds may indicate the distance from the colony to the food source. Lindauer and Kerr (1960), Esch et al. (1965), and Esch (1967) were the first researchers to describe in detail the patterns of dances, the sound pulses, and the trophallactic interactions in colonies of stingless bees, with special attention paid to explorers returning from profitable resources. The general method is based on training bees to a feeder placed at a known distance and direction from the colony and recording the behavior (trophallaxis, dances, and sounds) of the foragers returning to the colony. In fact, this is the same method currently used to investigate possible correlations between a particular behavior and spatial parameters such as distance, direction, and height of stingless bees (Nieh 2004). 12.4.1.1 Behavioral Rituals (Dances) in Meliponini In several species of recruiting bees, including Apis spp. and meliponines, successful foragers display specific behaviors inside the colony to draw the attention of their fellow foragers in order to transfer information related to the site where they discovered resources (Lindauer and Kerr 1960; von Frisch 1967). The dances in Melipona scutellaris and M. quadrifasciata consist of agitated running and jostling, without any discernible pattern that can be associated with the location of resources found by the scouts (Hrncir et al. 2000). In other species, like M. panamica (Nieh 1998a) and M. beecheii (Sánchez and Vandame, unpublished data) the returning foragers display both clockwise and counterclockwise turns while emitting sounds. But so far, no dance similar to the honeybee waggle dance has been described in stingless bees. It has been shown that variations in the intensity of the dance of Apis and some meliponine species are related to the quality of the resource (Aguilar and Briceño 2002; Dyer 2002a; Nieh et al. 2003b). However, the recruitment “dance” movements of meliponines apparently do not communicate the polar coordinates of resources (distance and direction) as the dance of Apis does (Nieh 2004). In studies with M. panamica, Nieh (1998a) found no effects of food distance, direction, or 192 D. Sánchez and R. Vandame height on forager movement patterns inside the nest. In the species M. scutellaris and M. quadrifasciata, Hrncir et al. (2000) also found no clear correlation between the dances observed in these species and any parameter of the resource’s location. This suggests that meliponines are unable to encode direction, distance, or height in recruitment dance movements. Similarly, bumble bee foragers evidently do not communicate resource location and instead forage individually after being activated by the return of a successful forager (Dornhaus and Chittka 2004). Thus, the recruitment dance of meliponines appears to work as a mechanism to alert potential recruits about the presence of a highly profitable resource. 12.4.1.2 Sounds The pioneering work of Esch et al. (1965) and Esch (1967) suggested that the stingless bee species M. quadrifasciata and M. seminigra were able to communicate the distance at which the resource was located through sound pulses inside the colony, produced by the flight muscles of successful scouts. Other work has shown similar results, describing in M. panamica the production of sound pulses by successful explorers; for instance, the duration of individual pulses correlated well with the distance at which the resource is found (Nieh and Roubik 1998). Moreover, they distinguished sound pulses produced during unloading food (trophallaxis) and pulses produced after unloading food (during the dance) in M. panamica. While the duration of the first type of pulses correlated negatively with food quality, the duration of the second type of sound correlated positively with the distance of the resource from the hive. That is, M. panamica may be able to communicate through sound pulses both the quality of the resource and its distance. However, the pulse durations were highly variable and thus it is unclear if they could provide the level of precise information observed in how recruits find the indicated food sources. Thus, this area requires further investigation. In a different species, M. quadrifasciata, no clear correlation has been found between the recruitment sound pulses and any parameter of the resource’s location (Hrncir et al. 2000), although they were correlated with the quality of the food source (Hrncir et al. 2004). Thus, there are many aspects of recruitment communication in the genus Melipona that require further study, including the possibility of significant interspecific variation in communication mechanisms. In addition, it is necessary to conduct experiments where the sounds recorded in the colony are played back with high fidelity in order to see whether there is any effect of recruitment to a specific distance. 12.4.1.3 Trophallaxis When a successful honey bee forager enters the colony, it can produce recruitment dances to attract potential recruits, some of which extend their proboscis to make contact with the mandible of the explorer. When the forager stops dancing it begins to share the collected nectar with her nestmates, resulting in a trophallactic 12 Stingless Bee Food Location Communication... 193 interaction. Trophallaxis thus refers to the exchange of liquid food between individuals of the same colony (Wilson 1971). Trophallactic contact is a primary form of information transfer. It can give information about the quality and odor of food resources. Trophallaxis is believed to have evolved with the need to communicate. However, not all the bees that receive nectar follow the dancer, and vice versa. The bees that both follow the dance and get nectar, on the other hand, receive more information about the resource the explorer just visited. Many of these bees follow to receive the forager’s dance information and may decide to visit the resource (Farina and Nunez 1995; Stabentheiner 1996; Wainselboim and Farina 2000; De Marco and Farina 2003). 12.5 Mechanisms of Communication Outside the Nest Foragers have to make decisions about where and when to explore new places in search of resources. They can make decisions based on innate behavior, their experience, or their interactions with other bees through communication mechanisms. These interactions can occur, as previously stated, inside the nest or outside the nest. Social facilitation and pheromone deposition are mechanisms of communication outside the nest that have been observed in several species of meliponines. 12.5.1 Social Facilitation In stingless bees, the phenomenon of social facilitation occurs when the behavior of executers influences the behavior of observers (Slaa and Hughes 2009). Social facilitation has also been studied in vertebrates, in which it seems to be one of the most important mechanisms to learn how to gather food, how to build nests, etc. (Wilson 2000). In social vertebrates, social facilitation provides further advantages: it makes it easier to find and handle resources and improves both the recruitment of nestmates and the collection of food, which may additionally reduce the individual probability of being preyed upon (Galef 1976; Burger and Gochfeld 1992; Galef and Giraldeau 2001). Social insects other than stingless bees also exhibit social facilitation, which has been shown to influence decisions about where to gather resources. In social bees, there are two types of social facilitation: local inhibition (foragers avoid places already occupied by other individuals) and local promotion (foragers are attracted to and learn about rewarding resources based upon the presence of other individuals already performing a task). Both have been described in meliponine species (Slaa 2003). Experience and learning also play an important role in the development of these two types of social facilitation. For example, the selection of patches of resources, or even the selection of individual flowers within a patch, can be guided by the physical presence of other bees on the basis of prior learning, modulating the final decision. 194 12.5.2 D. Sánchez and R. Vandame Pheromonal Signaling Several sources of olfactory information can influence bees’ orientation: the smell of the resource itself, pheromones and potentially locale odors (Aguilar and Sommeijer 2001; Nieh 2004; Arenas et al. 2007; Barth et al. 2008). Even though resource odors, such as floral scents, have proven to be very important in guiding foragers little has been studied regarding the importance of locale odors (the odors of the environment immediately surrounding the rewarding food source). Pheromones are mixtures of chemical compounds secreted externally by bees. They convey critical information about many aspects of the status of the individual or of the colony. Pheromones used in recruitment are mainly secreted in glands located in the abdomen, head, and in the legs. In addition to the diversity in the chemical composition of pheromones in stingless bees, there is also a great variation among species in the way they are deposited. These behavioral differences in the ways of depositing pheromones may, in part, be adaptations to the different ecological needs of each species. 12.5.2.1 Complete Pheromone Routes Some meliponine species can deposit an odor trail extending from the nest to the food source. Successful foragers lay a pheromone trail upon their return from the food source to the nest by depositing pheromone droplets on vegetation (Lindauer and Kerr 1960; Kerr et al. 1981). In some species, the distance between the marks ranges 1–8 m (Nieh 2004). In this way direction and distance to the food source are communicated. 12.5.2.2 Incomplete Pheromonal Routes Some species leave incomplete pheromone trails that extend from the food source to part of the distance towards the nest. In this case, successful foragers deposit pheromone droplets nearby the advertised resource, but not all the way back to the nest, up to 8 m from the target in M. rufiventris and M. compressipes and up to 27 m in Trigona spinipes (Nieh 2004). By doing this, foragers signal the direction where the resource is located, but not the distance. Such partial odor trails appear to provide partial guidance for a swarm of foragers that is recruited at the nest and guided towards the food source. 12.5.2.3 Polarization of Pheromone Trails This is an interesting behavior observed in T. spinipes and T. hyalinata and that may occur in other species (Nieh et al. 2003a, 2004). Basically, foragers deposit larger 12 Stingless Bee Food Location Communication... 195 amounts of pheromones as they reach the resource, thus decreasing towards the nest. In this way recruits can determine with high precision where the food is located, because this is indicated with the highest concentration of pheromones. 12.5.2.4 Odor-Marking the Resource This strategy refers to the deposition of pheromones on the resource itself. This behavior is frequently found together with pheromone trails, either complete or incomplete. Melipona panamica and M. favosa, however, only odor-mark the resource, without laying any pheromone trail (Nieh 1998b; Aguilar and Sommeijer 2001). 12.5.2.5 Aerial Pheromones This is a hypothesis not tested rigorously to date (Kerr 1994). It refers to the releasing of pheromones during the flight back to the resource from the nest, creating a sort of tunnel filled with pheromones that recruits follow as they fly to the food. 12.6 Effect of Internal Information on Communication Systems The decision to continue or to stop visiting a resource depends on a balance between external and internal information. However, the food recruitment process, as studied until recently, only considered the information from the scout bees and the nutritional needs of the colony to describe the phenomenon, without considering the internal status of recruits. In fact, the influence of internal factors, such as age and experience, has been little studied in meliponines. However, we do know that there are several behavioral stages that scouts and recruits go through, depending upon their experience with resources previously visited. These experiences in turn largely determine the effect that recruitment information will exert upon foragers (Biesmeijer and de Vries 2001). More detailed investigations revealed that naïve bees follow most of the information conveyed by scouts, contrary to experienced bees, which only need an indication that the resource is available once again (Biesmeijer et al. 1998). Other internal sources of information, such as individual’s hormone levels, genetic load and experience, affect decisions about what foragers do and where and when to collect resources (Biesmeijer et al. 1998; Robinson 1998; Johnson et al. 2002). The genetic variability among individuals within a colony may give rise to different preferences: some honey bees have a tendency to collect pollen while others prefer nectar (Robinson and Page 1989; Page et al. 1995). Thus food recruitment information may have different influences on the recipients. 196 12.7 D. Sánchez and R. Vandame Efficiency and Accuracy of Communication Systems The purpose of the recruitment systems is to concentrate foragers into a profitable resource trying to bring the majority of recruits to the exact site, preventing their spread in areas where there may be no resources to exploit (Sánchez et al. 2004). To achieve this goal, communication between individuals must be efficient. Efficiency in the context of communication may be defined as the amount of time and energy that explorers use to be “understood” by recruits. The cost of communication should therefore be much less than the energy gained by retrieving the resource, i.e., it must be profitable to communicate. The accuracy of the communication systems is part of their efficiency, and can be defined as the ability of recruits to choose the resource over other non-communicated alternatives (Sánchez et al. 2004). Choosing only one option is therefore the end result of the transfer of information made through the communication systems. Evaluating the accuracy is thus a practical way to measure the adaptation of communication systems in evolutionary time (Towne and Gould 1988). 12.8 Concluding Remarks Previous studies on the accuracy of the communication system of A. mellifera focused on the waggle dance, in an attempt to find an adaptive explanation of this behavior in relation to the size of resource patches that A. mellifera foragers visit and their distribution in time and space (Towne and Gould 1988; Weidenmuller and Seeley 1999). However, we now know that additional factors, such as social facilitation, are an essential part of bee foraging communication systems. In fact, more recent studies with stingless bees have revealed high accuracy, even greater than that observed in A. mellifera, where bees are allowed to use all means and modalities of communication (Schmidt et al. 2003; Sánchez et al. 2004). However, communication mechanisms are not the only factors that affect accuracy. There is evidence that experience changes the decision making in bees (Sánchez et al. 2007) inexperienced bees being more accurate than experienced ones. Thus, it seems to be more appropriate to study recruitment systems from a multimodal perspective that incorporates information about individual forager experiences to understand the evolution of communication in highly social bee species. The characteristics of the pot-honey, the pot pollen, and the cerumen the colonies generate are the result of decisions made by the foragers and the resources within the flight range of foragers. For some species that are highly efficient at recruiting nestmates, like S. mexicana (Sánchez et al. 2004), it is expected that the pot-honey they produce is less nectar-diverse than that produced by a less efficient bee, like Tetragonisca angustula (Aguilar et al. 2005), provided they occur in the same spot. Pot-honey characteristics may thus be inherently different between stingless bee species depending upon the specific recruitment mechanisms used by each bee species. 12 Stingless Bee Food Location Communication... 197 In this chapter, we briefly explained some of the processes involved in the organization of the foragers, which are the responsible for bringing resources to the colony. Those resources become the goods that beekeepers obtain from their colonies and that make stingless bees so appreciated by rural farmers, their families and until recently considered a delicacy in many international cuisines. Acknowledgments We would like to thank the opportune suggestions made by Dr. James Nieh which greatly improved this manuscript, and to the financial support of CONACYT agreement no. 128702 “Evolución de la Cleptobiosis en Lestrimelitta (Apidae, Meliponini)”. References Aguilar I, Briceño D. 2002. Sounds in Melipona costaricensis (Apidae : Meliponini): effect of sugar concentration and nectar source distance. Apidologie 33:375–388. Aguilar I, Fonseca A, Biesmeijer JC. 2005. Recruitment and communication of food source location in three species of stingless bees (Hymenoptera, Apidae, Meliponini). Apidologie 36:313–324. Aguilar I, Sommeijer M. 2001. The deposition of anal excretions by Melipona favosa foragers (Apidae: Meliponinae): behavioural observations concerning the location of food sources. Apidologie 32:37–48. Arenas A, Fernandez VM, Farina WM. 2007. Floral odor learning within the hive affects honeybees’ foraging decisions. Naturwissenschaften 94:218–222. 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Pulsed mass-recruitment by a stingless bee, Trigona hyalinata. Proceedings of the Royal Society of London B: Biological Sciences. 270:2191–2196. Nieh JC, Contrera FAL, Rangel J, Imperatriz-Fonseca VL. 2003b. Effect of food location and quality on recruitment sounds and success in two stingless bees, Melipona mandacaia and Melipona bicolor. Behavioral Ecology and Sociobiology 55:87–94. Nieh JC, Contrera FAL, Yoon RR, Barreto LS, Imperatriz-Fonseca VL. 2004. Polarized short odor-trail recruitment communication by a stingless bee, Trigona spinipes. Behavioral Ecology and Sociobiology 56:435–448. Nieh JC, Roubik DW. 1998. Potential mechanisms for the communication of height and distance by a stingless bee, Melipona panamica. Behavioral Ecology and Sociobiology 43:387–399. Page RE, Jr., Robinson GE, Fondrk MK, Nasr ME. 1995. Effects of worker genotypic diversity on honeybee colony development and behavior (Apis mellifera L.). Behavioral Ecology and Sociobiology 36:387–396. 12 Stingless Bee Food Location Communication... 199 Robinson GE. 1986. The dance language of the honeybee: the controversy and its resolution. American Bee Journal 126:184–189. Robinson GE. 1998. Colony integration in honeybees: genetic, endocrine and social control of division of labor. Apidologie 29:159–170. Robinson GE, Page RE, Jr. 1989. Genetic determination of nectar foraging, pollen foraging, and nest-site scouting in honeybee colonies. Behavioral Ecology and Sociobiology 24:317–323. Roubik DW. 1989. Ecology and natural history of tropical bees. Cambridge University Press; New York, USA. 514 pp. Sánchez D, Kraus F, Hernández M, Vandame R. 2007. Experience but not distance influences the recruitment precision in the stingless bee Scaptotrigona mexicana. Naturwissenschaften 94:567–573. Sánchez D, Nieh JC, Hénaut Y, Cruz L, Vandame R. 2004. High precision during food recruitment of experienced (reactivated) foragers in the stingless bee Scaptotrigona mexicana (Apidae, Meliponini). Naturwissenschaften 91:346–349. Schmidt VM, Zucchi R, Barth FG. 2003. A stingless bee marks the feeding site in addition to the scent path (Scaptotrigona aff. depilis). Apidologie 34:237–248. Seeley TD. 1995. The wisdom of the hive: the social physiology of honey bee colonies. Harvard University Press; Cambridge, USA. 295 pp. Seeley T. 2010. Honeybee Democracy. Princeton University Press; Princeton NJ, USA. 280 pp. Slaa EJ. 2003. Foraging ecology of stingless bees: from individual behaviour to community ecology. In. PhD Thesis. Utrecht University; Utrecht, Netherlands, 181 pp. Slaa EJ, Hughes WO. 2009. Local enhancement, local inhibition, eavesdropping, and the parasitism of social insect communication. pp. 147–164. In: Jarau S, Hrncir M, (eds). Food Exploitation by Social Insects: Ecological, Behavioral, and Theoretical Approaches. CRC Press, Taylor and Francis Group; Boca Raton, FL, USA. 360 pp. Stabentheiner A. 1996. Effect of foraging distance on the thermal behaviour of honeybees during dancing, walking and trophallaxis. Ethology 102:360–370. Towne WF, Gould JL. 1988. The spatial precision of the honey bees’ dance communication. Journal of Insect Behavior 1:129–155. Visscher PK. 1998. Colony integration and reproductive conflict in honey bees. Apidologie 29:23–45. von Frisch K. 1967. The Dance Language and Orientation Of Bees. Belknap Press of Harvard University Press; Cambridge, MA, USA. 566 pp. Wainselboim AJ, Farina WM. 2000. Trophallaxis in the honeybee Apis mellifera (L.): the interaction between flow of solution and sucrose concentration of the exploited food sources. Animal Behaviour 59:1177–1185. Weidenmuller A, Seeley TD. 1999. Imprecision in waggle dances of the honeybee (Apis mellifera) for nearby food sources: error or adaptation? Behavioral Ecology and Sociobiology 46:190–199. Wenner AM. 2002. The elusive honey bee dance “language” hypothesis. Journal of Insect Behavior 15:859–878. Wenner AM, Wells PH, Johnson DL. 1969. Honey bees: do they use the direction and distance information provided by their dancers? Science 158:1076–1077. Wille A. 1983. Biology of the stingless bees. Annual Review of Entomology 28:41–64. Wilson EO. 1971. The Insect Societies. Belknap Press of Harvard University Press; Cambridge, USA. 548 pp. Wilson EO. 2000. Sociobiology: The New Synthesis, 25th anniversary ed. Belknap Press of Harvard University Press; Cambridge, USA. 697 pp. Chapter 13 On the Diversity of Foraging-Related Traits in Stingless Bees Michael Hrncir and Camila Maia-Silva 13.1 Introduction When thinking about bees and flowers, frequently an image of a balmy spring-meadow where honey bees, and sometimes maybe a bumble bee, peacefully buzz from flower to flower almost automatically pops up in our minds. Yet, as so often, nature is much more realistic than our soft-focus-lens imagination, for there is tough competition for available food in the insects’ world. Thus, our romantic summer-meadow is far from being an amicable place, but rather resembles a free cold buffet at which all invited and uninvited guests, each one equipped with his/her particular little vicious tricks and strategies, struggle to get the major portion. Due to the rich diversity of both flowering plants and flower-visiting insects, the tropics have been an ideal evolutionary playground to develop a spectacular diversity of plant–insect, plant–plant, and insect–insect interactions, governed by the continuous struggle for survival and successful reproduction. Plants, on the one hand, have evolved a fascinating variety of floral shapes, flowering traits, and phenological strategies in order to prevail in the inter- and intraspecific competition for pollinators (Bawa 1983; Frankie et al. 1983; Waser 1983; Caruso 2000). Flowervisiting insects, on the other hand, have developed a no less impressive diversity of strategies and mechanisms aiming at maximising the exploitation of floral foraging bonanzas (Johnson 1983; Roubik 1989; Goulson 1999). M. Hrncir (*) Laboratório de Ecologia Comportamental, Departamento de Ciências Animais, Universidade Federal do Semi-Árido, Avenida Francisco Mota 572, Mossoró-RN 59625-900, Brazil e-mail: michael@ufersa.edu.br C. Maia-Silva Faculdade de Filosofia, Ciências e Letras, Universidade de São Paulo, Avenida Bandeirantes 3900, Ribeirão Preto-SP 14040-901, Brazil 201 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_13, © Springer Science+Business Media New York 2013 202 M. Hrncir and C. Maia-Silva In virtually all tropical habitats, the most abundant flower visitors are bees, in particular the eusocial corbiculate bees: the stingless bees (Apidae, Meliponini), bumble bees (Apidae, Bombini), and honey bees (Apidae, Apini) (Roubik 1989; Bawa 1990; Biesmeijer and Slaa 2006). In contrast to solitary insects, which collect food for their individual and direct fitness, foragers of social insect colonies gather food to guarantee the successful rearing of the brood and to satisfy the energetic demands of all non-foraging colony members (Wilson 1971; Michener 1974; Jarau and Hrncir 2009). The survival of a bee colony, therefore, largely depends upon the success of the foragers in collecting carbohydrates (usually nectar) and proteins (usually pollen). Both these food items are stored within the nest to insure a constant food supply, thus preventing potentially fatal colony-weakening during periods of resource scarcity. Most stingless bees are generalist foragers, and even those species with a relatively low niche breath usually collect at a wide array of food plants (Wilms et al. 1996; Ramalho 2004; Biesmeijer and Slaa 2006). Thus, and due to the fact that tropical habitats are frequently shared by several dozen meliponine species, diet overlap in terms of food sources used is considerable. The generalised utilisation of common resources among stingless bees results in both interference and scramble competition between species, which reduces not only the foraging efficiency at food patches but also diminishes the pollen and nectar harvest of colonies (Johnson 1983; Johnson and Hubbell 1974; Roubik 1980; Roubik et al. 1986; Wilms and Wiechers 1997; Biesmeijer et al. 1999a; Nagamitsu and Inoue 2005; Maia-Silva et al. 2010a). Thus, selective pressure to maximise food collection led to the evolution of a rich variety of foraging strategies among meliponine bees that differ according to variation in different foraging-related traits, among them morphology, foraging strategy, aggressiveness, and recruitment efficiency (Lindauer and Kerr 1958; Johnson 1983; Roubik 1989; Biesmeijer et al. 1999a; Biesmeijer and Slaa 2004; Nieh 2004; Willmer and Stone 2004; Nagamitsu and Inoue 2005; Barth et al. 2008; Hrncir 2009; Jarau 2009). With the present chapter, we want to give a brief overview of some of this diversity found among stingless bees shaped by the competition for food. 13.2 Food Niches If we want to understand the diet breath of stingless bees, why they collect at particular plant species while ignoring others, we need to differentiate between a species’ fundamental food niche and its realised food niche (Biesmeijer and Slaa 2006). The fundamental niche, on the one hand, is the ecological niche occupied by a species in the absence of competitors. Its breath is determined by both the morphological and physiological characteristics of the respective organism. A species’ realised niche, on the other hand, is determined through the interactions with other organisms and, thus, depends on the competitor-community of the respective habitat. In the following, we discuss some morphological traits, tongue length, body colour, and size, which putatively play a major role for the separation of fundamental food niches among stingless bees. Further, concerning the realised food niche, 13 On the Diversity of Foraging-Related Traits... 203 we discuss how differences in foraging strategy with regard to aggression, recruitment ability, and recruitment precision may influence dominance relationships at a feeding site and, thus, the partitioning of resources. 13.3 The Fundamental Food Niche: Tongue Length as Predictor of Flower Choice A major trait for the segregation of stingless bee species in order to reduce competition for food is their morphology. At least since Charles Darwin (1859) it has become clear that the body shape of a bee species is adapted to the plants at which it collects floral resources. In “The Origin of Species” (1859), Darwin wrote: “The tubes of the corollas of the common red and incarnate clovers (Trifolium pratense and incarnatum) do not on a hasty glance appear to differ in length; yet the hive-bee [honey bee; authors’ note] can easily suck the nectar out of the incarnate clover, but not out of the common red clover, which is visited by humble-bees [bumble bees; authors’ note] alone; so that whole fields of the red clover offer in vain an abundant supply of precious nectar to the hive-bee”. More recent, detailed studies investigating possible correlations between bee morphology and flower choice corroborate Darwin’s observations indicating in both stingless bees and bumble bees a morphological matching between tongue length and corolla depth of the preferentially visited flowers (Heinrich 1976; Pleasants 1983; Harder 1985; Johnson 1986; Nagamitsu and Inoue 1998) (Fig. 13.1). Yet, as demonstrated for bumble bees, the relationship between glossa length and corolla depth is not a straight one: long-tongued bees are able to collect nectar at flowers with both long and short corollas, whereas short-tongued species are restricted to shallow flowers. Consequently, species with a long glossa, hypothetically, have access to nectar in a greater diversity of food plants (larger fundamental food niche breath) than those with a short glossa (Heinrich 1976; Harder 1985; Johnson 1986). Increasing corolla depth raises the energetic costs of foraging due to an increase in probing time. Probing time comprises, in essence, two components: access time, which increases linearly with corolla depth, and ingestion time, which increases with corolla depth only in those flowers that are deeper than the bee’s glossa due to a reduced nectar uptake rate (Harder 1983, 1985). Thus, given that bee species with long tongues have the choice to collect nectar from flowers with both shallow and long corollas, why should they bother feeding at deep flowers, thereby unnecessarily increasing their foraging costs? In an investigation of 13 bumble bee-visited plant species, Harder (1985) demonstrated that the average 12-h sugar production was positively correlated with corolla depth. This elevated offer of sugar, and, consequently, energetic gain, putatively is the crucial incentive for bees to visit deepflower plants as long as the net energetic profit of nectar collection remains positive. Thus, when available, bees should preferentially feed from flowers with corollas approximately as deep as their glossae (Harder 1985). The high sugar reward of several deep flowers certainly is interesting for most nectar-feeding animals, and several species evolved strategies to circumvent the elevated energetic costs associated with probing time. Several bee species, for 204 M. Hrncir and C. Maia-Silva Fig. 13.1 Bee morphology, nectar feeding, and illegitimate flower-visits. Since floral morphology determines the accessibility to floral resources, stingless bees with different tongue length should specialise on different plant species. (a) Example of bee tongue-flower-matching: Trigona spinipes collecting nectar at Waltheria rotundifolia (Malvaceae). (b) Example of an illegitimate flowervisit: Melipona subnitida collecting nectar at Tarenaya spinosa (Capparaceae), which is pollinated by bats. (c) Flowers of Tarenaya spinosa: note the protruding stamina. (d) Example of nectar robbing: Trigona spinipes collecting nectar through a hole at the flower-base of Hibiscus sp. (Malvaceae). Photos: M. Hrncir instance, easily enter the flowers designed for larger animals, such as bats or humming birds, without even getting anywhere close to the plant’s reproductive units (Heard 1999) (Fig. 13.1). The extremists among these illegitimate flower-visitors are bees who steal nectar and pollen by entering the flowers through piercing or biting (Wille 1963; Inouye 1980; Roubik 1982) (Fig. 13.1). Among the Meliponini, species of the genus Trigona have brought this larceny-technique to perfection. Through goal-directed mass-recruitment, these bees are able to activate a large number of nestmates to profitable food patches and, subsequently, defend them against other flower-visitors. Thus, after perforating a flower, and recruiting additional foragers to the food source, the bees aggressively dominate the flower patch, repelling other bees or even hummingbirds through joint attacks. The detrimental effect of these robbers for the plants, therefore, is not so much the damaging of the floral structures, but the fact that they prevent potentially effective pollinators from visiting the flower (Roubik 1982; Heard 1999). 13.4 The Fundamental Food Niche: Body Colour, Body Size, and Thermal Tolerance In addition to the, since Darwin well-established, relation between flower morphology and bee tongues, two morphological traits, related to thermal tolerance, are considered responsible for the spatio-temporal separation of niches among bee 13 On the Diversity of Foraging-Related Traits... 205 species: body size and colouration (Biesmeijer et al. 1999a, b; Pereboom and Biesmeijer 2003). Tropical and subtropical bees, such as the Meliponini, are constrained by high ambient temperatures and heat production when foraging (Heinrich 1993; Biesmeijer et al. 1999a; Pereboom and Biesmeijer 2003). Due to the production of excess temperature when flying, many bees are exposed to the danger of overheating, some even operating close to their lethal limit. In full sunlight, generally, small bees heat up and cool down more rapidly than large bees (Fig. 13.2), but, in contrast to large bees, they will not attain excessively high body temperatures due to an elevated convective heat loss (Digby 1955; Pereboom and Biesmeijer 2003) (Fig. 13.2). Large species, therefore, run a higher risk of overheating than small species when foraging in sunshine. Here, body coloration comes into play. Physically, temperature excess and overheating are proportional to absorptivity (radiation absorbed by an object). Consequently, since absorptivity is lower for light than for dark colours (pale-coloured insects: 63–86%; dark-coloured insects: 71–117%1; Digby 1955), pale-coloured bees heat up more slowly in full sunlight than dark-coloured bees (Digby 1955; Pereboom and Biesmeijer 2003) (Fig. 13.2). Stingless bees show both a spatial and temporal segregation concerning sunlit flower-patches in compliance with the thermal characteristics assigned to body size and colouration (Fig. 13.3). Meliponine species of similar size, but differing in body colour, partition patches of the same floral resource according to sunlight incidence.2 In consequence of differential evaporation, inter-patch differences in illumination result in more concentrated nectar in sunlit flower patches as compared to shaded patches (Willmer and Corbet 1981; Biesmeijer et al. 1999a, b). Consequently, lightcoloured Meliponini, which favour sunlit patches, collect more concentrated nectar from the same plant species and at the same time of day as do dark-coloured species that prefer the shaded patches (Biesmeijer et al. 1999b) (Fig. 13.4). Concerning the temporal partitioning of floral resources among bee species, it has been repeatedly demonstrated that large Meliponini start foraging earlier during the day than smaller species (Fig. 13.5). The first stingless bees to initiate foraging early in the morning are species of the genus Melipona, some of which start their activity even before sunrise and at low ambient temperatures (de Bruijn and Sommeijer 1997; Pereboom and Biesmeijer 2003; Teixiera and Campos 2005; Maia-Silva et al. 2010a, b). Their capacity to fly at low temperatures is putatively related to their larger body size as compared to other stingless bee species. Due to their elevated mass of thoracic flight muscles (responsible for heat production), The explanation for this apparent absorptivity in excess of 100% probably lies in the site of absorption. Heat produced is carried away by conduction and convection to the air, and by conduction to the underlying body of the insect and to the other cooling surfaces (radiation being very slight). Where the surface is highly absorbing, the heat is produced at the surface where it will readily be carried away; but where the surface absorbs little of the heat, more radiation will be available for absorption throughout the thickness of the thorax. In this case, as cooling is only at the outer surface, the inside will be hotter than the outside” (Digby 1955, pp 287–288). 2 In an experimental study on the foraging choice of the sympatrically occurring dark-coloured Melipona costaricensis (former: M. fasciata) and light-coloured M. beecheii, the dark species clearly preferred shaded food patches and avoided sunlit ones (Biesmeijer et al. 1999a) (Fig. 13.3). 1 206 M. Hrncir and C. Maia-Silva b 14 Dark coloured 12 10 8 6 4 Light coloured 2 0 0 1 2 3 4 Thorax width (mm) 5 Passive cooling/heating (°C/s) Temperature excess (°C) a 0.55 0.45 Dark coloured 0.35 0.25 Light coloured 0.15 0 1 2 3 4 Thorax width (mm) 5 Fig. 13.2 The importance of body size and colouration for heat gain and heat loss of stingless bee foragers. Scatter plots showing the correlation between body temperature (thorax width) and temperature excess (maximum difference between thoracic and ambient temperature) (a) as well as passive cooling/heating (cooling constant K) (b) of 24 species of stingless bees. Light-coloured bees (grey-filled circles) have a lower temperature excess and cool down (and warm up) less rapidly than dark bees (black-filled circles) of similar size. Dashed lines indicate linear regressions for light-coloured and dark-coloured bees (data from Pereboom and Biesmeijer 2003) Shade Sun 0 Sun 20 40 20 0 Shade 40 60 Sun 60 80 Shade 80 100 Sun b Melipona beecheii Melipona costaricensis Foragers choosing patch (%) 100 Shade Foragers choosing patch (%) a Fig. 13.3 Spatial niche differentiation among stingless bee species differing in body colouration. (a) Under clear sky-conditions, foragers of the light-coloured Melipona beecheii (grey bars) preferentially collect at sunlit patches whereas the dark-coloured M. costaricensis (black bars) prefer food patches in the shade. (b) Under changing weather conditions, foragers of M. costaricensis react immediately with respect to their patch preference in response to switches from sunny to cloudy weather or vice versa (data from Biesmeijer et al. 1999a) large species are capable of attaining ideal flight temperatures even at low ambient temperatures (Heinrich 1993), and can initiate foraging long before the small species warmed up sufficiently. Concerning the onset of flight activity, body colouration might play a decisive role for smaller species, since dark-coloured bees absorb thermal radiation more efficiently (Digby 1955) and, consequently, heat up quicker than light-coloured species (Fig. 13.5). a b Percentage of loads 20 15 10 5 66-70 56-60 46-50 36-40 26-30 16-20 <10 0 60 50 40 30 20 10 0 7 11 9 13 Time of day (hours) 15 Melipona beecheii Melipona costaricensis Sugar concentration (weight/weight) c Sugar concentration (Brix) 207 On the Diversity of Foraging-Related Traits... Sugar concentration (weight/weight) 13 80 60 40 20 0 [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] Plant species Fig. 13.4 Sugar concentration of nectars collected by stingless bee species differing in body colouration. Light-coloured Melipona beecheii (grey-filled bars and squares) collect nectars of significantly higher sugar concentration than dark-coloured M. costaricensis (black-filled bars and squares). (a) Percentage of foragers returning with loads of the respective sugar concentration. (b) Variation of sugar concentration (mean ± 1 SD) of nectar collected in the course of a day. (c) Sugar concentration (mean + 1 SD) of nectar of different botanic origin obtained from foragers at the nest entrance. [1] Oyedaea verbesinoides (Asteraceae); [2] Vernonia patens (Asteraceae); [3] Bidens squarrosa (Asteraceae); [4] Type 11; [5] cf. Heliocarpus (Malvaceae); [6] Hyptis capitata (Lamiaceae); [7] Serjania sp. (Sapindaceae); [8] Mikania micrantha (Asteraceae); [9] Bravaisia integerrima (Acanthaceae); [10] Schlegelia parviflora (Schlegeliaceae); [11] cf. Celtis (Cannabaceae); [12] Type 9; [13] Type 16; [14] Type 42; [15] Type 50 (data from Biesmeijer et al. 1999b). Photos: M. Hrncir 13.5 The Realised Food Niche: Aggression and Dominance at a Feeding Site Stingless bee colonies are, in essence, sessile. Consequently, both the food sources available in space and time and the presence of potential competitors are determined by the nest’s location. In bee assemblages, competition for food putatively is strongest among coexisting colonies of the same species and among species of the same genus, which tend to be similar in body size, colony size, and foraging strategy, and, therefore, tend to have similar fundamental food niches (Biesmeijer and Slaa 2006). 208 M. Hrncir and C. Maia-Silva a b c e Returning foragers (%) d Melipona scutellaris (B) 100 Scaptotrigona aff. depilis (C) 80 60 40 20 0 06:00 Onset of foraging (hour) Melipona quadrifasciata (A) 12:00 PL 11:00 10:00 FS PD 9:00 NT SX 8:00 7:00 MB PH 6:00 MQ 1.0 08:00 10:00 Time of day (hours) FV 1.5 2.0 2.5 3.0 Intertegular width (mm) Fig. 13.5 Temporal niche differentiation among stingless bee species differing in body size and colouration. (a–d) Foraging of big, dark-coloured Melipona quadrifasicata (a), big, pale-coloured M. scutellaris (b), and small, dark-coloured Scaptotrigona aff. depilis (c) at mass-flowering Eugenia uniflora. (d) Onset, maximum, and end of the foraging processes are influenced by body size and colouration of the respective bee species. Data collected in August 2009 at the campus of the University of São Paulo in Ribeirão Preto, Brazil (given are the proportions of bees returning to colonies with pollen loads relative to the maximum number of foragers; data are presented as mean ± 1 SD of 12 observations per species; CMS and MH, unpublished data). (e) Onset of foraging in nine stingless bee species differing in body size (given as intertegular width); MQ, Melipona quadrifasciata; MB, Melipona bicolor, PH, Partamona helleri; SX, Scaptotrigona xanthotricha; NT, Nannotrigona testaceicornis; PD, Plebeia droryana; FV, Frieseomelitta varia; FS, Friesella schrottkyi; PL, Plebeia lucii. Note earlier foraging start of dark-coloured PH compared to the similar-sized, light-coloured SX (data from Teixiera and Campos 2005). Photos: M. Hrncir In these cases, common resources might be shared either through spatio-temporal differences in foraging activity among congeneric species (see above) or through scramble competition. Consistent with the idea of limiting similarity (MacArthur and Levins 1967), eusocial bee assemblages in the tropics tend to consist largely of species from different genera. Even so, food niches overlap, and there is strong association among several coexisting taxa with respect to food sources used (Biesmeijer and Slaa 2006). Here, differences in foraging strategies and underlying recruitment mechanisms between different genera might be important factors concerning the partitioning of common resources. In stingless bees, foraging strategies can be described in terms of three basic foraging traits: recruitment ability (solitary or group foraging), individual aggressiveness (present or absent), and local enhancement in heterospecific encounters (attraction or 13 On the Diversity of Foraging-Related Traits... 209 avoidance) (Biesmeijer and Slaa 2004). Among the possible combinations of these traits, a highly successful strategy is aggressive group foraging, as found in several Trigona and Oxytrigona species (Nagamitsu and Inoue 1997; Johnson 1983; Slaa 2003). These mass-recruiting aggressive species form dense forager groups through local enhancement, and attack everything at or near the exploited food patch. Consequently, these bees “extirpate” less aggressive group foragers or solitary foraging species at the food patch, and, thus, monopolise clumped and rich resources (Johnson and Hubbell 1974, 1975; Johnson 1983; Biesmeijer and Slaa 2004; Lichtenberg et al. 2010). However, due to a low independent scouting activity, aggressive mass-recruiters have a limited capacity of discovering new food sources or even neighbouring food patches independently (Hubbell and Johnson 1978; Biesmeijer and Slaa 2004). Although aggressiveness can lead to dominance at a food patch, it should not be used as a direct measure for dominance. Rather, dominance should be interpreted as the suppression or exclusion of one species or individual by another (Johnson and Hubbell 1974; Lichtenberg et al. 2010). In solitarily foraging animals, like many vertebrates, larger and stronger species, or individuals within a species, tend to dominate at a feeding site. In social insects, however, the strength often lies in numbers. When a large group of foragers of a single colony arrives at a feeding site, other species are often at a loss due to the sheer fact that they cannot find a free spot to land and feed (Johnson 1983; Biesmeijer et al. 1999a; Hrncir 2009; Lichtenberg et al. 2010). Consequently, non-aggressive mass-recruiters, such as Scaptotrigona, Partamona, and some Trigona species, are able to numerically dominate rich clumped patches, excluding other species even without aggressive interactions3 (Johnson 1983; Biesmeijer and Slaa 2004; Lichtenberg et al. 2010). Scrambler species that forage individually or in small groups, therefore, would need to move to less disputed, often poorer feeding sites or, alternatively, arrive at rich patches ahead of the mass-recruiting species. 13.6 The Realised Food Niche: First Come First Served Many medium-sized, unaggressive Meliponini share similar floral resources (Biesmeijer and Slaa 2006) and, therefore, experience scramble competition when foraging. Scramble competition among colonies is highest at rich clumped food sources, such as mass flowering plants (Biesmeijer and Slaa 2006), which produce 3 Johnson (1983) described a situation in which two non-aggressive mass-recruiters, Partamona orizabaensis (as Trigona testacea) and Scaptotrigona mexicana (as Trigona mexicana), numerically dominated the inflorescences of a Bactris palm tree. Although both these scrambler species did not exclude each other from the food patch, insinuators (small, unaggressive, and mostly solitarily foraging bees, such as many Plebeia species) did not find space to land at the inflorescences. More surprisingly, even an aggressive group-foraging species, Trigona silvestriana, was competitively outnumbered by the scrambling mass of bees and, consequently, left the patch (Johnson 1983). 210 M. Hrncir and C. Maia-Silva a large amount of new flowers each day over a short period of time (“big-bang” or “mass-flowering” strategy) (Augspurger 1980; Bawa 1983). Within plant populations, in general, mass-flowering individuals of a species bloom synchronously. Slight differences in the onset of flowering among individuals result in an extended blooming period on the population level (Bawa 1983). Mass-flowering plants, therefore, offer a great opportunity for colonies to hoard large amounts of food within a short period of time, and represent the predominant source of both nectar and pollen for stingless bees, contributing up to 90% of the annual nutritional input into the colonies (Wilms et al. 1996; Wilms and Wiechers 1997; Ramalho 2004). Fully grown mass-flowering trees are usually too big to be monopolised by a single colony of mass-recruiting bees (aggressive or unaggressive). Individual or group-foraging scramblers, consequently, can exploit such kind of resource virtually undisturbed (Biesmeijer and Slaa 2006). The situation, however, might be different with small mass-flowering trees or shrubs, which can be easily defended by aggressive colonies (Johnson and Hubbell 1975) or numerically dominated by nonaggressive mass-recruiters (Johnson 1983). Here, in order to be able to profit from such foraging bonanzas, non-aggressive scramblers that forage individually or in small groups should get to the food patch prior to others, or as long as the population density of potential competitors is low. An important trait that allows bees to arrive at a food patch ahead of competitors is their capability to learn both the position of a potential collecting site and the time of resource availability (Johnson 1983; Biesmeijer and Slaa 2004; Schorkopf et al. 2004; Murphy and Breed 2008). Food-patch-experienced foragers, consequently, arrive at familiar feeding sites far quicker than inexperienced bees, which still have to search for it. So far, however, few studies investigated the time–place–memory of stingless bees (Biesmeijer and Slaa 2004). An important topic for future research, therefore, is to investigate whether the capacity to memorise the spatio-temporal characteristics of food sources differs among species with fundamentally different foraging strategies (aggressive mass-recruiters, unaggressive mass-recruiters, group-foraging scramblers, solitary scramblers, insinuators). For group-foraging bees, a second parameter important for the efficient exploitation of resources is recruitment velocity (Jarau et al. 2003). Here, we have to distinguish, in essence, between mass-recruiting species (aggressive and unaggressive) and species that forage in small groups. The strategy of mass-recruiting species relies on the rapid mobilisation of a huge number of foragers to one particular feeding site. In aggressive mass-recruiters, the overwhelming multitude of recruits extirpates other species at a feeding site and, subsequently, defends this patch against other aggressive colonies (Hubbell and Johnson 1978; Johnson 1983). Through similar fast and goal-oriented recruitment, unaggressive mass-recruiters are able to dominate food patches numerically, thereby diminishing exploitative competition by other scramblers or even keeping off aggressive species (see footnote 3). In contrast to mass-recruiters, the strategy of unaggressive scrambler colonies that forage in small groups, such as Melipona or Nannotrigona species, relies on a quick mobilisation of all available recruits, yet without indicating the position of a particular food patch. Due to this lack of vector information, the foraging force spreads out over the surroundings to find any patch that carries the odour that has been brought 13 211 On the Diversity of Foraging-Related Traits... NEST FOOD SOURCE A DC ID PD Sugar concentration Sugar flow Nectar volume Energetic gains Energetic gains Flight costs Handling costs Search costs DC PD Energetic costs ID FOOD SOURCE VALUE Energetic costs Fig. 13.6 Activation signals of stingless bees. The nest-internal recruitment signals of stingless bees, the thoracic vibrations, are directed at the fast activation of additional foragers. The temporal pattern of the foragers’ pulsed vibrations is influenced by the value of the visited food source. Increasing energetic gains at the food patch result in longer pulses (PD), shorter intervals (ID), and, consequently, an increasing duty cycle (DC = PD/[PD + ID]). Increasing energetic costs, by contrast, result in shorter pulses, longer intervals, and a decreasing duty cycle (figure adapted from Hrncir 2009) back to the colony by successful scouts (Hubbell and Johnson 1978; Jarau et al. 2000; Slaa 2003; Biesmeijer and Slaa 2006; Hrncir 2009). Thus, when excluded from one feeding site by a mass-recruiting species (aggressive or unaggressive), the colonies are still able to profit from a rich food source by switching their foraging focus to less disputed patches (Hubbell and Johnson 1978; Johnson 1983; Biesmeijer and Slaa 2006). Based on the differences in necessity to guide the foraging force to a specific food patch, recruitment strategies should differ between mass-recruiters and scramblers that forage in small groups with respect to the information about the exact position of a feeding site (important for mass-recruiters, useless for unaggressive scramblers) but not necessarily concerning the velocity of mobilising the foraging force. So far, few meliponine species have been analysed in detail concerning their recruitment strategies. In both mass-recruiters (Scaptotrigona aff. depilis) and unaggressive scramblers that forage in small groups (Melipona spp., Nannotrigona testaceicornis), the temporal pattern of thoracic vibrations generated by recruiting scouts within the nest is related to the profitability of a food source (Fig. 13.6). These vibrations, putatively, are an alerting signal, activating the foraging force (Hrncir 2009). Although these nest-internal recruitment signals are similar for massrecruiters and small-group-scramblers, only the mass-recruiting species have been shown to be able to guide recruits to a specific food patch (aggressive massrecruiters: Trigona corvina, T. hyalinata, T. spinipes; unaggressive mass-recruiters: Geotrigona mombuca, Scaptotrigona aff. depilis, S. postica, S. mexicana, Trigona recursa). In contrast to honey bees, which indicate the position of a feeding site 212 M. Hrncir and C. Maia-Silva through their waggle dance (Grüter and Farina 2009), mass-recruiting stingless bees achieve this goal-directed recruitment through species- or even colony-specific pheromone trails or pheromone marks at and near the feeding site (Jarau 2009; Stangler et al. 2009; Jarau et al. 2010; Schorkopf et al. 2011). 13.7 Concluding Remarks Stingless bee pot-honey is a valuable product with a long tradition of harvest and consumption (Camargo and Posey 1990; Crane 1999). A large diversity of stingless bee species is kept by meliponiculturists all over Latin America to provide this precious gold. The differences found among meliponine honeys with respect to their physiochemical composition, sugar content, and floral origin depend not only on the geographic region where it has been harvested but also on the stingless bee species being used for honey production (Barth 1989; Souza et al. 2006; see related chapters in this book). Tropical habitats are frequently shared by several dozen meliponine species. Consequently, diet overlap in terms of food sources used is considerable. The selective pressure to maximise food collection led to the evolution of a rich variety of foraging-related traits among the stingless bees. In our chapter, we wanted to give a brief overview of this diversity, discussing the importance of morphological characteristics (tongue length, body colour, and body size) for the separation of fundamental food niches among the Meliponini. In contrast to a species’ fundamental niche, which is delimited by the morphological and physiological characteristics of an organism, the food niche realised by a species is determined through the interactions with other organisms that share the same fundamental food niche. Here, differences in foraging strategy among the stingless bees with regard to aggression, recruitment ability, and recruitment precision influence dominance relationships at a feeding site and, thus, are important factors concerning the partitioning of resources. To be sure, our overview is far from being complete, since our description of the foraging strategies used by stingless bees almost entirely omitted the unaggressive solitary foragers, often very small species that remain competitive through an “insinuation strategy” (Johnson 1983). These insinuators fly off a food patch when threatened by dominant species, yet they quickly return to the same site or nearby flowers and continue feeding as if indifferent to the aggressors (Biesmeijer and Slaa 2006). Several of these insinuator species, like Tetragonisca angustula or Frieseomelitta varia, are bees important for meliponiculture (Souza et al. 2006). Yet, knowledge about the foraging strategies of the small Meliponini is rather poor, probably because the large bees, like Melipona spp., and the aggressive ones, like Trigona spp., are more attractive to scientists. Acknowledgements We would like to thank Rubens Teixeira de Queiroz for identifying the flowers in Fig. 13.1, and four anonymous reviewers for valuable comments on the manuscript. 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Wilms W, Imperatriz-Fonseca, VL, Engels W. 1996. Resource partitioning between highly eusocial bees and possible impact of the introduced Africanized honey bee on native stingless bees in the Brazilian Atlantic rainforest. Studies on Neotropical Fauna and Environment 31:137–151. Wilson, EO. 1971. The Insect Societies. Belknap Press of Harvard University Press; Cambridge, MA, USA. 548 pp. Part II Stingless Bees in Culture, Traditions and Environment Chapter 14 Stingless Bees: A Historical Perspective Richard Jones This chapter is dedicated to the memory of Dr. Eva Crane who, in over 50 years of worldwide research, produced the seminal texts on the history of beekeeping and honey hunting. Everyone advancing these studies today owes her a tremendous debt. 14.1 Introduction Stingless bees are native to all tropical regions although they closely resemble another familiar honey-making bee, Apis, which ranges naturally through most tropical and temperate regions of the Old World. The honey bee, Apis mellifera, was introduced into many areas, especially in the New World and on islands, by European explorers and settlers in the sixteenth century in the Americas, and as late as the nineteenth century in Indoaustralia. The main distribution of stingless bees in historical times has been described by Kerr and Maule (1964) and summarised by Michener (2007, and in the present book). It is safe to assume that the connection between bees and man began then when the first honey hunters ripped open nests to release the sweet golden treasure of honey and also perhaps to benefit from the protein provided by the bee brood. Between 15,000 and 10,000 years ago, when people first inhabited the New World, they exploited its tropical honey-making bees. Far before this, in Africa, Asia, and Australia, there were humans taking honey from wild bees and this can be seen in some of the earliest records of human culture (Crane 1999). Until the introduction to the Americas of the honey bee, Apis mellifera, stingless bees were the source of cerumen and honey and therefore played a significant role in native civilisations. Honey bees later provided a much bigger return for the effort of management, but pot-honey is undoubtedly a legacy of stingless bees. R. Jones (*) International Bee Research Association (IBRA), 16 North Road, Cardiff CF10 3DY, UK e-mail: joneshr@ibra.org.uk 219 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_14, © Springer Science+Business Media New York 2013 220 14.2 R. Jones Bee Hunting to Beekeeping Honey hunters were able to harvest the honey stores of bees by tolerating their defensive biting or stinging, using tools to access the native bee nests in tree trunks or in the ground, or even using plants that diminish their aggressiveness, e.g. the Andaman islanders’ use of tranquilising plants to harvest nests of giant honey bees (Crane 1990), while the Kayapó Indians of Brazil employ crushed toxic leaves to extract honey from some of the fiercely biting stingless bees (Posey and Camargo 1985). It is but a short evolutionary step from honey hunting to beekeeping. This involves providing a suitable nest site in a location that is easily accessible for exploitation. So a hollow tree becomes a hollow log; the log is cut in such a way that it can be opened and resealed by the owner and thus beekeeping is born. This first step certainly occurred in the area dominated by the Maya Civilisation, between 10° and 23°N in Mesoamerica, but remained comparatively rare in the rest of tropical America. In 1492 Columbus recorded that there was honey and cerumen in Cuba and Santo Domingo (Schwarz 1949). These must have been the products of stingless bees but it is not known if they were derived from kept or wild bees, although it was probably the latter. Bishop Diego de Landa writing at the time of the Spanish Conquest of Mesoamerica said: “honey was often consumed together with bee maggots” and that the honey was contained in “wax pots as large as doves’ eggs” (Kent 1984). 14.3 Commercial and Cultural Importance of Honey and Cerumen The cerumen was as important as the honey to many early Pre-Columbian societies. No stingless bee builds its nest of pure wax, but uses cerumen mixed with resin, called “cerumen” (as noted in several book chapters herein). These civilisations are famed for their treasures of gold. Indeed the legend of El Dorado—the Golden Man—impelled the Spanish as they exploited the newly discovered lands and people. The cerumen was used to cast exquisite jewellery, usually made from pure gold. The process known as “lost wax casting” allows quite intricate objects to be sculpted in cerumen or wax. The resultant object is then surrounded by clay hardened by drying in the sun. The ball of clay was then heated so that the cerumen could drain away through vents and molten gold was poured in to take its place and thereby assume the shape of the desired object. This method was mostly used for small objects such as jewellery but artisans doing the work would require a constant and reliable supply of cerumen which would make heavy demands on honey (cerumen) hunters. Such a need might have encouraged more organised beekeeping: a simple example of the economic principle of supply and demand. The people of South and Central America were expected to pay tribute to their European Conquerors—preferably in gold that was then taken back to the Old 14 Stingless Bees: A Historical Perspective 221 World to reward those who had financed the exploratory expeditions. However, there are several records showing that for the poorer communities some of these tributes were paid in honey and cerumen (Georghiou 1955; Landa 2008). 14.4 Historical Production and Management One of the first European travellers to report stingless bees in detail was the German Ulrich Schmidel (Crane 1999). Between 1536 and 1545 he traveled extensively in what is now called Northern Argentina. Many years later he wrote: “An Indian goes into a wood with an axe and the first tree he comes to that has an entrance hole to a bees’ nest. By boring other holes he gets five or six jugs of pure honey. These bees are small and have no sting …” Similarly, Jesuit priest Bernabé Cobo (1892) traveled in Central America and as far as present-day Peru. In “Historia del Nuevo Mundo” (Cobo 1653) he wrote about stingless bees including: “The smallest bee is the size of a fly that breeds in wine; another is somewhat larger … neither can sting, but they burrow in the hair and in the beard”. The first reference in the literature to stingless bees in Australia was made by Dutch explorer, Abel Tasman (1603–1659), in 1648 when he noted that the indigenous people on the island now named after him (Tasmania) cut notches in some trees and used these to help them climb and gain access to individual bees’ nests (Wills 1970). A reference to the importance of cerumen is to be found in Reyne (1962) quoting a 1769 report of two and a half tonnes being exported from what is now Surinam in the year 1745. It seems likely that most of this would have been supplied from stingless bees as it is unlikely that imported Apis mellifera would have been established in sufficient numbers to generate this quantity of wax, but we have no certain data on that point. The records of the amount of honey and cerumen yielded by a single nest vary considerably: one rather dubious 1657 report (Purchas 1657) tells of a nest providing “enough honey to fill a firkin”—an old barrel measurement equating to about 40 L! Goudot (1846) describing the situation in Columbia explains the seasonal cycle and that a nest harvested in April/May or October might yield 3 L of honey and 1 kg of cerumen. Many more reports are available now [see Barceló (Chap. 17) and Ocampo Rosales (Chap. 15) in this book]. Interestingly he also mentions that the honey was often sold in markets using bamboo internodes as containers. For some tribes brood was an important food source. So the honey and brood were eaten while the wax and propolis (cerumen) that constitute the walls of the storage cells—the honey pots—were chewed and stored in soft balls. The mixture could be heated and used for a multitude of purposes, from fixing feathered flights to arrows (Stearman et al. 2008), to making toys and ceremonial objects. Mesoamerica was the area directly affected by Mayan culture and this advanced culture certainly embraced beekeeping. The stingless bee of the Maya—Melipona 222 R. Jones Fig. 14.1 Symbols of Melipona beecheii in the Mayan Tro-Cortesian Codex. (a) Effigy censer from Cozumel, in the shape of the descending Mayan bee god of honey Ah Mucen Cab, with brood of M. beecheii, in the Archaeological Museum of Yucatán, Mérida, Mexico (Darchen and Darchen 1978). (b) Bees icons and god-like figure on the right, holding stingless bee brood with the hands, like the Ah Mucen Cab censer. (c) Upper portion of page 104, the lower row shows two bee gods facing left (Villacorta and Villacorta 1977), each with a hive of M. beecheii. Itzamná grandfather god is working in summer close to the bee queen and Chaac (god of rain) fixes a honey supper (Cappas e Sousa 1995) (permission granted by the International Bee Research Association) beecheii—was known as “colecab” or “xunan cab” (lady bee). Melipona beecheii is painted in the Tro-Cortesianus Codex, Museum América, in Madrid. The sacred world of this goddess bee was represented by knowledgeable priests and painters (Fig. 14.1). Mayan codices are folding books written in Maya hieroglyphic script on papersheets obtained from the inner bark of wild-growing fig tree. Tro-Cortesianus is derived from the two fragments Troano, owned by the Spanish palaeographer Don Juan Tro y Ortolano (pp 22–56, 78–112), and Cortesianus (pp 1–21, 57–77), united in the Madrid Codex since 1888, after León de Rosny identified that Troano and Cortesianus were two parts of the same book (FAMSI 2012). The united manuscript is 6.7 m long with 56 leaves, and page dimensions are 12 cm × 24 cm (The University of Arizona Library. Mayan Codex facsimiles. http://www.library.arizona.edu/exhibits/ mexcodex/maya.htm). The cerumen from stingless bees is of lower quality than honey bee wax for candle making, because the resin burns and sputters, emitting some smoke. However, in 1549, 3 tonnes of honey and an amazing 277 tonnes of cerumen, known as “cera de Campeche”, were paid in tribute to the conquerors and exported from Yucatan to Spain (Calkins 1974). Such production was only possible because M. beecheii were kept on a commercial, almost industrial, scale. This stingless bee is amenable to 14 Stingless Bees: A Historical Perspective 223 hive management and gives worthwhile honey yields, but the reason could be more cultural than biological (D. Roubik, personal communication). The Nicoya peninsula in Costa Rica marks the southern limit of M. beecheii and, as it happens, that of Mayan influence. In the 1500s the Spanish referred to traditional hive beekeeping here, so it is likely that the design of the equipment and the necessary accompanying skills had been in existence for centuries. To this day traditional log hives can be seen hanging in the eves of houses (Imperatriz-Fonseca 1989) or, if there are ten or more hives together, sheltered in a specially constructed “A” frame structure near the house. In Australia cerumen was also used to paint animal and human figures on rock faces. Some of these pictures of the life of the indigenous people have been dated back to 2000 BC. There are no records of any such applications in the Americas although often, similar discoveries, abilities, and cultural mores developed simultaneously, thousands of kilometres apart and without any contact whatsoever between those people concerned. In Central America there is a musical percussion instrument, the marimba, which in its traditional form uses stingless bee cerumen to adjust the pitch and so control the sound produced from the gourd resonators that are to be found below the wooden keys. While in Australia the mouth piece of the didgeridoo was made of cerumen so as to make an airtight seal with the mouth of the player. 14.5 Recent History and Transitions Today log hives are used, along with boards fashioned into “rational hives”, in the Yucatan peninsula. They have a central flight entrance and closures at each end made from disks of wood or soft stone that can be easily cut to shape. Archaeological digs have revealed many similar stone disks, which shows that this type of hive and its associated beekeeping management techniques existed over a thousand years ago. Many of these finds have been in close proximity indicating that then, as now, some beekeeping was on a grand scale with hundreds of hives in some meliponaries (Calkins 1974). The reader is invited to see the short film “Honey for the Maya” by Buchmann (2011), to appreciate Melipona beecheii honey making and meliponiculture. The Maya valued cerumen as they did not use the cerumen for candles but used, instead, reed torches for lighting. In the latter part of the twentieth century stingless beekeeping has been under threat and suffered some setbacks. Spreading urbanisation and in some regions heavy deforestation have reduced forage and potential nest sites from which the stock for beekeeping activities could be obtained. Indiscriminate application of pesticides and general pollution have killed many colonies. However, one of the biggest problems is competition for forage. This began with the introduction of A. mellifera with the European settlers in the sixteenth century but was greatly exacerbated by the Africanised honey bee from 1956 onwards. Despite early demonstration of competition at flower patches between 224 R. Jones meliponines and honey bees (Roubik 1978), there is little certainty about what influence Africanised honey bees will ultimately have on native bees; what is certain is that they provide a pollination service which may benefit the native bees (Roubik and Villanueva 2009; Roubik 2000). Arrival of the Africanised honey bee also heavily affected hive bees of European varieties, with reduced yields from 15 to 2–3 L in one Brazilian apiary (Imperatriz-Fonseca 1989). Traditional hives by definition mean that the designs, and indeed often the actual hives, have been handed down from generation to generation. On the other hand the word rational is used for a hive based on reasoning and thought after a study of the stingless bees’ needs (Mariano-Filho 1910). Mariano-Filho (1910) devised a hive consisting of three-tiered boxes. However, Paulo Nogueiro-Neto in São Paulo has undertaken some of the most intuitive and constructive developments in stingless beekeeping over the last 60 years. In 1948 he designed hives for Trigona and Melipona species, and over the years he has refined the design and, from his own tireless observations, added copious information and instructions for harvesting honey, transferring nests, and dividing colonies. Much of this work has been published on various occasions but it all comes together in one seminal text book “Vida e Criação de Abelhas Indigenas Sem Ferrão” (Nogueira-Neto 1998). Kempff Mercado (1966) in Bolivia and Nates-Parra (1978) in Colombia, for example, have also promoted rational hives. An interesting modern hive has been developed in Tobago by the University of Utrecht (Sommeijer 1999) which allows harvesting of honey without disturbance of the brood chamber. Sadly the rapid and almost universal growth of honey bee beekeeping throughout Hispanic America, at both commercial and hobbyist levels, especially over the last 100 years, has been to the detriment of stingless bees. Traditions and the special management skills that are required are being lost almost daily. These bees evolved with the natural ecology and crops of the area and so have a valuable role to play in the pollination of those crops with all the resultant benefits in improved yields and food security. They are valuable bio-indicators of the state of the environment and provide not just honey and cerumen but also, as the nature of these products is being more deeply understood, medicaments that could provide pharmaceutical benefits where so far synthetic substitutes have failed. Only eusocial bees store honey and pollen as a provision for the brood and for times of dearth. Properly managed and by using rational hives, the honey can be harvested from the stingless bees without damage to the colony. The quantities produced are much smaller than those produced by honey bees. The honey has a higher water content than honey bee honey and is a little more acidic but still very sweet and pleasant. Many stingless bees do not confine their foraging to nectar, pollen, and honeydew—the basis of honey bee honey. However, throughout history to the present time it has been used in its natural state as a pleasurable eating experience or as a sweetener with other food. There is evidence that the Mayan civilisation used considerable stingless bee honey for production of a fermented drink—“balché”—roughly the equivalent of mead (Crane 1975; Ocampo Rosales Chap. 15 in this book). 14 Stingless Bees: A Historical Perspective 14.6 225 Value of Pot-Honey Some of the stingless bees from Brazil were included in the song of Caetano Veloso “Mel” (http://letras.terra.com.br/caetano-veloso/44746/), honey in English (Souza 2008). In his song about honey (available in the Internet), there are no Africanised bees but the worth of three stingless bees “lambe-olhos” Leurotrigona muelleri, “torce cabelos” Scaptotrigona depilis, and “vamo-nos embora” Lestrimelita limao, is appreciated by the public. In Venezuela, “arica” Melipona favosa is present in the poem “Miel de arica” by Guillermo Jiménez Leal (T. Castro, personal communication) and in the novel Doña Bárbara (Gallegos 1976). These are bees of high value since ancient times and expanding legacy of cultural expressions. Although the quantities produced are small (see Alves Chap. 40 in this book), pot-honey is believed to have healing qualities and plays an important role in folk medicine, particularly in South and Central America. The use in different treatments for coughs and throat infections is well known but it can also be used in fertility treatment and in combination with jungle herbs to treat fever. Preliminary research shows that the honey has many potential benefits in the treatment of ocular cataracts (Vit and Jacob 2008), besides the putative treatment of pterygium with eye drops. The value of stingless bees is highly prized, but has been somewhat dismissed in pot-honey standards and overshadowed by the commercial honey bees for many years. Now there is a resurgence of interest in these bees and their honey (Main 2012). Efforts are being made to establish controls and standards for the honey produced (Vit et al. 2004; Souza et al. 2006) so that it can take place as a marketable product. This would give a great boost to many areas that would benefit from economic input but above all would be a clear statement of the value of stingless bees and so an important step in ensuring their conservation. Acknowledgements I wish to thank the editor, Professor Patricia Vit for her patience, tolerance, constant guidance, and above all friendship without which I would have given up the task, and most appreciated editorial comments from Dr. David W Roubik, and also those who refereed the material for their knowledge and intellectual rigour which is vital to a publication of this kind. Finally, I repeat the dedication at the beginning of this chapter. So much research work into the history of beekeeping owes everything to Dr. Eva Crane and the foundations she laid in her works. References Buchmann S. 2011. Honey for the Maya. The Drylands Institute & Wildtime Media Inc., Tucson. Available at: http://www.youtube.com/watch?v=d_pjoDxwYS8 Calkins CF. 1974. Beekeeping in Yucatan: a study in historical-cultural zoogeography. PhD thesis, University of Nebraska, Lincoln, Nebraska, USA Cappas e Sousa JP. 1995. Os Maias e a Meliponicultura O Apicultor [Cascais, Portugal] 9:15–17. Cobo B. 1653. Historia del Nuevo Mundo. (published 1890, Seville: Sociedad de Bibliófilos Andaluces) vol. 2 226 R. Jones Cobo B. 1892. Historia del Nuevo Mundo. Sociedad de Bibliófilos Andaluces, E Rasco, Bustos Tavera, Seville, Spain. 350 pp. Crane E. 1975. Honey, A Comprehensive Survey, Hienemann; London, UK. 466 pp. Crane E. 1990. Bees and Beekeeping - Science, Practice and World Resources. Heinemann Newnes; Oxford, England. 614 pp. Crane E. 1999. The World History of Beekeeping and Honey Hunting. Duckworth; London, UK. 682 pp. Darchen B, Darchen R. 1978. Le comportement constructeur des abeilles sociales. Centre National de la Recherche Scientifique [Montpellier, France] 30:38–45 FAMSI. Foundation for the Advancement of Mesoamerican Studies, Inc. 2012. The Madrid Codex. Available at: http://www.famsi.org/mayawriting/codices/madrid.html Gallegos R. 1976. Doña Bárbara.Colección Austral. Espasa-Calpe; Argentina. 284 pp. Georghiou G. 1955. The history of beekeeping. Gleanings in Bee Culture 38:87–89. Goudot J. 1846. Observations relatives a l’histoire des meliponites. In comptes rendu hebdomadaire des seances de l’Academie des Sciences 22:710–713. Imperatriz -Fonseca VL. 1989. The developemnt of Meliponinae culture in Brazil. Proceedings 32nd International Apicultural Congress pp. 66–67. Kempff Mercado N. 1966. Abejas indígenas: su explotación racional. Revista Universidad Autónoma G.R. Moreno 23/24:47–53. Kent RB. 1984. Mesoamerican stingless beekeeping. Journal of Cultural Geography 10:14–28. Kerr W, Maule V. 1964. Geographic distribution of stingless bees and its implications. Journal of the New York Entomological Society 72:2–18. Landa D. 2008. Relación de las cosas de Yucatán (1566) republished as: Yucatan before and after the conquest. Forgotten Books; Charlston, South Carolina, USA. 270 pp. Main D. 2012. A different kind of beekeeping takes flight. The New York Times. Available at: http://green.blogs.nytimes.com/2012/02/17/a-different-kind-of-beekeeping-takes-flight/ Mariano-Filho J. 1910. Keeping indigenous bees in a type of hive for commercial harvesting. Entomologica Brazilia 3:14–18. Michener CD. 2007. The Bees of the World. Second edition. Johns Hopkins University Press; Baltimore, USA. 953 pp. Nates-Parra G. 1978. La meliponicultura en Colombia. Revista Nacional de Apicultura 71:23 pp. Nogueira-Neto P. 1997. Vida e criação de abelhas indigenas sem ferrão. Editora Nogueirapis: São Paulo, Brazil. 446 pp. Posey DA, Camargo JMF. 1985. Additional notes on the classification and knowledge of stingless bees (Meliponinae, Apidae, Hymenoptera) by the Kayapó Indians of Gorotire, Pará, Brazil. Annals of Carnegie Museum 54:247–274. Purchas S. 1657. A Theatre of Political Flying Insects. Thomas Parkhurst; London. 202–207 pp. Reyne A. 1962. Stingless bees occuring in Surinam. Entomologischer Berichten, Amsterdam 22:30–37. Roubik DW. 1978. Competitive interactions between neotropical pollinators and Africanized honeybees. Science 201:1030–1032. Roubik DW. 2000. Pollination system stability in tropical America. Conservation Biology 14:1235–1236. Roubik DW, Villanueva GR. 2009. Invasive Africanized honey bee impact on native solitary bees: a pollen resource and trap nest analysis. Biological Journal of the Linnean Society 98:152–160. Schwarz HA. 1949. The stingless bees (Meliponidae) of Mexico. American Institute of Biology 20:357–370. Sommeijer MJ. 1999. Beekeeping with stingless bees: a new type of hive. IBRA, Cardiff. Bee World 80:70–79. Souza BA. 2008. Meliponicultura nas Américas: Aspectos culturais. pp. 27–30. In Vit P, ed. Abejas sin aguijón y valorización sensorial de su miel. APIBA, Facultad de Farmacia y Bioanálisis, Dirección General de Cultura y Extensión, Universidad de Los Andes; Mérida, Venezuela. 146 pp. 14 Stingless Bees: A Historical Perspective 227 Souza B, Roubik D, Barth O, Heard T, Enriquez E, Carvahlo C, Villas-Bôas, Locateli J, PersanoOddo L, Almeida-Muradian L, Bogdanov S, Vit P. 2006. Composition of stingless bee honey: setting quality standards. Interciencia 31:867–875. Stearman AM, Stierlin E, Sigman ME, Roubik DW, Dorrien D. 2008. Stradivarius in the jungle: Traditional knowledge and the use of “black beeswax” among the Yuquí of the Bolivian Amazon. Human Ecology 36:149–159. Villacorta JA, Villacorta CA. 1977. Codicies Mayas reproducidos y desarrollados, Guatemala. 2nd ed. Tipografía Nacional; Ciudad de Guatemala, Guatemala. pp. Vit P, Jacob TJ. 2008. Putative anticataract properties of honey studied by the action of flavonoids on a lens culture. Journal of Health Science 54:196–202. Vit P, Medina M, Enriquez ME. 2004. Quality standards for medicinal uses of Meliponinae honey in Guatemala, Mexico and Venezuela. Bee World 85:2–5. Wills AP. 1970. Dinna…giraga…warrul (Honey or honey comb in three aboriginal languages) Niddrine, Victoria, Australia. Australian Bee Journal 51:12. Chapter 15 Medicinal Uses of Melipona beecheii Honey, by the Ancient Maya Genoveva R. Ocampo Rosales La mayor gloria que al secreto oficio de la abeja se da, a la qual los discretos deven imitar, es que todas las cosas por ella tocadas convierte en mejor de lo que son. (La Celestina, Fernando de Rojas) The greatest glory that is given to the secret craft of the bee, which those that are prudent must imitate, is that all things touched by it are converted into something better than they are. (La Celestina, Fernando de Rojas) 15.1 Introduction In the Yucatan peninsula, the bee Melipona beecheii was named “cab” or “kab” in the Mayan language. It was considered of such importance by the Mayan people that, after a long process of appropriation, the bees were deified and named “xunan cab,” or “xunan kab.” The word “xunan” means principal lady (Barrera Vázquez 1980). With this word, we perceive that the bees were docile, gentle, well born, belonging to the lineage, and, because of this last quality, direct descendants of the Mayan gods. Thus, the deity, “Ah mucen kab,” was granted to the native stingless bees, so that he would take care of their nests and hives, due to the delicacy required in all the breeding and collecting activities. “Hobones” is the Mayan name for the traditional nests of bees, built within the hollow trunks of certain tropical trees that the meliponas found in the forest during their reproductive phase, and then colonized. G.R.O. Rosales (*) Facultad de Filosofía y Letras, Universidad Nacional Autónoma de México, Moctezuma 28, Col. Toriello Guerra, Del., Tlalpan México 14050, Mexico DF, Mexico e-mail: paredeso@prodigy.net.mx 229 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_15, © Springer Science+Business Media New York 2013 230 G.R. Ocampo Rosales Within the family lands, the bees received protection from many natural enemies that did not dare to come close to the hobones to feed on the sweet honey that the bees produced, destroying the hives and killing the larvae and adults. Also benefiting from the closeness of the hives, the Mayan families that had lodged the nests had easy access to the products that the stingless bees manufactured, honey, cerumen, and pollen, and to the benefits they provided. The melipona honey was an especially coveted product, valued for its medicinal properties and for its ritual importance in the elaboration of beverages used in religious ceremonies. This is documented in the manuscripts carefully preserved throughout the difficult centuries of Colonial domination. The anonymous texts that deal with medical practices, the “Ritual de los Bacabes” (Arzápalo Marín 1987), the “Recetario de Indios en Lengua Maya” (Roys 1976), and the “Libros del Judío” (Barrera and Barrera Vásquez 1983), include a great number of healing incantations and prescriptions for the preparation of remedies based on the honey of the native bee M. beecheii, which could be used either alone, as the main ingredient, or as a vehicle for other healing products. Many years before the Spanish conquest, honey and cerumen were important products exported to other regions of Mesoamerica, Central America, and the Caribbean. We find mention of this trade in the manuscripts known as “Relaciones Histórico-Geográficas de la Gobernación de Yucatán.” This translation of quotes was made respecting the style used in the manuscripts: In these provinces there are not mines of any type. The profit they give are some cotton sheets and wax and honey, that is the land’s trade, and in order to be valuable, they are taken to Mexico, Honduras and other parts. (Garza et al. 1983) From a thriving industry and trade that survived three centuries of colonial exploitation, meliponiculture is now on the verge of extinction. The activity has suffered from the current economical and social pressures experienced by the Maya people and from the introduction of Apis mellifera. Apiculture with that bee has become a very important agroindustry in the Yucatan peninsula. It is probable that due to the medicinal properties and ritual use of the honey and other products of the native bees, meliponiculture continued in practice in a reduced scale in the backyards of Mayan homes and has barely survived. Studies carried out by bacteriologists have proven that the honey of Melipona beecheii has high levels of Bacillus that inhibit pathogenic bacterial growth (Quezada Euán 2005; Catzin Ventura et al. 2009). This fact may contribute to its medicinal action, as well as a higher acidity compared with the honey of Apis mellifera (Vit et al. 2004). The hydrogen peroxide, an antibiotic found in all honeys, acts as an hypotonic medium that, dehydrates microbes or inhibits their growth (Menezes et al., Chap. 10 in present book). We will make a brief review of some ideas that the Mayans had about the diseases, the literature where we find notes on how this honey was used as medicine, and will indicate the ways in which the Pre-Hispanic Indians used honey as well as the bee nests and brood. Finally, a description of the maladies cured with honey and its application by the “ah dzaco’ob,” general medics of ancient times, will be given. 15 Medicinal Uses of Melipona beecheii Honey, by the Ancient Maya 15.2 231 Mayan Ideas of Disease For the Mayan people, a disease was a serious state of physical, mental, and spiritual alteration. The sick person suffered physically in an intense way, and presented mental alterations and emotional or spiritual unsteadiness. A sick man or woman was defenseless and incapable to carry out his or her everyday labors and personal, familiar, and social duties. In the emblematic book of Mayan medicine, the “Ritual de los Bacabes” (Arzápalo Marín 1987), we find that for these people, the diseases were supernatural beings, with origins in a remote mythical time, born to a mother and father in a “temazcal,” the traditional steam bath, located in a selected spot of the sacred geography. They also possessed clothes, pieces of gold jewelry, and symbols, which provided them with character. Human beings fell sick for a number of reasons; most of these had to do with the supernatural world and beings. A man that was negligent, cruel, or naughty with his family or neighbors, or with defenseless people such as youngsters or elders, was prone to anger the gods and to receive their punishment in the form of a sickness. Dangerous places such as caves, rivers, water springs, lakes, and the forest were abodes of great energy that could affect man in a negative way. The men that dared go into these places were either owners of enough power to arrest the energy that prevailed, or carried out rituals to appease the supernatural beings and forces that prowled there. If a person was at fault during the rituals that were due to the deities, he or she could also be chastised with a disease. The gods of Mesoamerican religions were capricious creatures that would equally bestow great luck or the worst of fortunes, pain, and maladies on a human being, despite his or her good conduct and respect to his or her obligations to them. These were the main causes of disease and, as we proceed through the texts to see how the honey of Melipona beecheii helped to cure many of them, we will recognize a few of these ideas that persisted in spite of years of cultural repression during the Colonial period. More information regarding these subjects can be found in López Austin (1980) and Ocampo Rosales (2005). Why was the honey endowed with such power to cure? For the Mayans, the energy was a force called “kinam,” whose various meanings are (1) strength, robustness, rigor, and fortitude; (2) virtue, as in the stones, or herbs, etc.; and (3) venom or poison from animals, or pain caused by the poison or the ulcer, and that which is very painful (Ciudad Real 2001). It is probable that the Maya word “kinam” derives from the word “kin,” sun, which might indicate that for these people, a certain kind of power was like that of the sun, or provided from it, thus being especially strong. The Mayans considered that the sun’s power concentrated in the plants’ reproductive organ, the flower, in the form of a sweet liquid, the nectar. That strength or energy was transmitted to the bee and from the insect to the honey. That is why “kab,” honey, was so powerful that it was even considered as a sacred food, used in rituals. 232 G.R. Ocampo Rosales 15.3 Traditional Literature on the Use of Melipona beecheii Honey for Medicinal Purposes In the Mayan literature, written in Latin characters, that has been preserved, there are a few texts that comprise medical aspects of great importance. In some, the health of Mayan populations and their unfortunate contact with the epidemics brought by the Spanish conquerors were recollected, as well as the years when the conditions were most severe. In others, the illnesses were described with their name in Mayan and, occasionally, the translation for this name was provided in Spanish. We also find very complete lists of plants, their medicinal properties, and their use against different maladies. The main purpose that the Mayan specialists had in writing these texts was the preservation of the part of their culture that dealt with the recovery of health and the prescriptions by means of which the patients were treated. Obviously, in most of these books, the ritual parts, fundamental in the treatment carried out by the “ah dzac,” and which had a deep religious background, were scarcely mentioned. To demonstrate this omission, the important collection of prayers and invocations gathered in the “Ritual de los Bacabes” (Arzápalo Marín 1987) is a complete manual of the rituals by means of which the Mayan doctors healed their sick. Characteristic of this manuscript is the use of a language that was only known to the initiated, and the description of complicated ceremonies. Here, the use of honey to heal certain diseases is recorded, but the examples are few. It is in the collection of manuscripts known as “Libro del Judío” (Barrera and Barrera Vásquez 1983) where the Mayan informants wrote widely about the use of honey as a powerful healing agent. The “Libro del Judío” is a complex, detailed, and long compilation of several manuscripts that was accomplished by an Italian physician, Ricardo Ossado, who lived in the Yucatan Peninsula during the eighteenth century. Probably due to an acute professional curiosity, a considerable knowledge of medicine and the vegetation of the region, the Jew, as Ricardo Ossado was nicknamed, used the “ah dzaco’ob,” general doctors, as informants and translators to compile many prescriptions to cure several diseases that were common among the Mayan population. His knowledge of the maladies is clear in this minute register of medical practices of the time. The manuscripts were named after the village where they were recovered and because of their characteristics, we consider that they are copies of prescriptions that were handed down from teacher to disciple since remote times; many exhibit a clear Pre-Hispanic tradition. From this extensive corpus, we extracted those prescriptions in which honey is one of the main ingredients incorporated to act effectively against an illness, particularly, virgin honey, taken directly from the honey pots inside the hives or “hobones,” named “hobnil cab,” honey of “hobon.” 15.4 Preparation of Prescriptions The honey of the meliponas was used for its effectiveness as a curative product, and for its religious and mythical powers. Due to its properties, honey was used to treat all kinds of diseases, prepared and dosed adequately, but always as a principal ingredient, 15 Medicinal Uses of Melipona beecheii Honey, by the Ancient Maya 233 capable of restoring a patient’s lost balance. Honey appears in the prescriptions as the fundamental curative element, added to plants that were macerated, cooked, roasted, or burnt to ashes. Honey was rubbed or anointed alone, or with plants to form a paste or a liquid that was applied over the sick member, skin bruises, ulcers, wounds, on the eyes, inside the ear, or covering the region of the organ to be treated. The “ah dzac” is also advised to use the nests of certain bees or wasps to cure certain illnesses. The way it was done was to burn down the nest of the insect, extract the larvae from it, grind them, and administer all with the ashes in the form of a beverage (Roys 1976). In the case of burnt skin, honey was applied alone. It is also used in many of the prescriptions as basis of anti-inflammatory liquids or ointments. For “chuchup calil,” swollen neck: You take the Malachra palmata (Malvaceae), mallows and honey. Let them be mashed and let him drink it. Or else let him drink milk and cinnamon mixed to honey, and let a little of it be applied wherever the swelling is. (Roys 1976) 15.5 Diseases Treated with Honey of Melipona beecheii In order to make the copious information of the medicinal properties of the melipona honey more comprehensible, we will use a classification of diseases according to the organs that were affected. In these prescriptions, honey, “kab,” was used to cure diseases of respiratory, digestive, circulatory, and immunological systems. It was also used as a remedy for maladies of the sensory organs, such as the skin, eyes, ears, mouth, tongue, gums, and teeth. An important part of the literature is dedicated to a group of diseases that were named fevers which due to their high incidence, importance, and negative effects, were considered as a unit in their particular classification by the Mayans. Another part refers to those illnesses typical of the Mayan worldview, with defined traits and supernatural etiology that are called syndromes of cultural filiation. In these regions characterized by a high biodiversity, another important application of honey was as a remedy against the stings and bites of scorpions, spiders, tarantulas, bugs, ants, and venomous serpents (Barrera and Barrera Vásquez 1983). 15.5.1 “Cold” Diseases In the Mayan classification of diseases, an important part is dedicated to those considered cold diseases, sent by gods or entities that inhabited the cold, dark, damp portion of the Mayan universe—the underworld. The gods and forces that inhabited this place exhibited traits that reflected their surroundings. They were cold, damp, and dark. Many of the respiratory maladies were considered cold diseases. To cure the white phlegm, whose symptoms make us suspect tuberculosis, the elements of the prescription included expectorants like pepper (Piperaceae, a recent import from the 234 G.R. Ocampo Rosales Old World), chilli Capsicum annuum (Solanaceae), and tobacco Nicotiana tabacum, N. rustica (Solanaceae). In this particular case, as well as in other prescriptions, it is clear that the ingredients were prepared searching for a balance between the intrinsic qualities of the disease and the properties of the remedies. The phlegm disease was cold and the constituents of the medicine were hot. Honey was a hot product due to its origin and properties, and this made it especially valuable to treat the problems that women experienced before, during, and after giving birth. In this situation, the parturient was in an extremely cold and dangerous state, because she had come close to death and to the underworld; consequently, she was invaded by the negative forces and spirits that dwelled in this place. Honey was used to expel the placenta, “kal ybin”: The remedy is honey heated with a little sugar, not much, roasted, powdered and stirred thoroughly into the hot honey. Let it be given to drink to the patient. It will be good to put immediately the blood of a chicken in it, the blood from the leg of the chicken. When for two days the after-birth may be retarded in part, administer the other remedy for the afterbirth, grated “chaya” Cnidoscolus chayamansa, with horse-dung and honey and chilli Capsicum annuum. Let it be drunk warm. (Roys 1976) In this prescription, we observe the addition of an element that is hot in its very nature, the blood, to counteract the placenta’s coldness and promote its detachment. Its second part seems elaborated under the dictations of the “medicine of filth,” typical of the knowledge of medieval physicians, medical procedures that had probably been brought to New Spain by the doctors that emigrated from the Spanish territories in Europe and had become popular in America or the American continent. It also involves the use of two plants that originated in this continent, “chaya” Cnidoscolus chayamansa (Euphorbiaceae) and chilli Capsicum annuum (Solanaceae). In another prescription, honey was rubbed on the woman’s abdomen before birth and was also taken as a beverage. To this day, in the Mexican states of Campeche and Yucatán, women who are attended during labor by traditional midwives also receive this treatment before giving birth. The midwife anoints honey over the woman’s stomach to help increase the contractions, to correct the position of the child, and to protect both from the coldness of the labor. This is accompanied by other rituals in which help is summoned from supernatural beings to make the labor short and the delivery successful (González-Acereto et al. 2011). In the manuscript called “Manuscrito de Chan Cah,” recovered from the so-called Maya village, the compiler refers to a problem of the placenta in a few lines, unfortunately incomplete: When the unhealthy afterbirth is retained by the woman _____ the afterbirth that is tangled his _____ put honey on them. (Grupo Dzibil 1982) In the group of diseases that came from the cold places of the universe, a dangerous case of heart failure, “chibal puczik,” heart pain, is treated with the integration of three different constituents in the prescription, which are all hot remedies: honey, anise Pimpinella anisum (Apiaceae), and wine. They were mixed and placed on a piece of cloth and while still hot applied over the region of the heart. The mixture was probably used as an effort to reanimate this organ in case of heart failure. 15 Medicinal Uses of Melipona beecheii Honey, by the Ancient Maya 235 We believe that due to the seriousness of this disease, this prescription is one of the longest and most complex. Three or four different remedies are provided to apply in case of “chibal puczik.” We have to consider, in addition the inclusion of European elements such as anise and wine and their use in Mayan medicine. We only quote the part in which “kab,” honey, is used: Or else you burn honey with roasted anise, (mix) with wine and put it on a cloth like a thick cake baked in hot ashes. Then you bind it on the heart, hot… (Roys 1976). Among the indications given in “El Libro del Judío” to treat heart diseases, we find the following: “Chiople” Eupatorium hemipteropodum (Asteraceae), “xhóch” Ricinus communis (Euphorbiaceae), green tobacco Nicotiana tabacum (Solanaceae). An infusion of these three herbs is sweetened [with honey] and you imbibe two spoonfuls, every three hours, and it is very effective to cure heart disease and palpitations of this organ; it is taken for three, six or nine days, continually, and you will be cured. (Barrera and Barrera Vásquez 1983) 15.5.2 Fevers and “Hot” Diseases In all ancient texts on the subject of medicine, fevers are amply cited. We now know that a fever is an abnormally high body temperature, symptom of infection, autoimmune disease, intoxication, and parasitosis, but even now they are considered as a group, and, in the Mesoamerican world they were known as “hot” diseases. According to the Mayan worldview, these illnesses were sent by gods, beings, or forces that belonged to the hot, dry, luminous part of the universe, the supranatural world, above the terrestrial stratum. These beings possessed a very powerful constitution that could damage humans in a severe way. In some of the prescriptions to treat these maladies, we do not fully understand the nature of the products that are required. For nocturnal fever, “akab chacuil,” the “ah dzac” recommends administration of “hobnil haa,” “hobon water,” with “kanlecay,” dodder, Cuscuta americana (Convolvulaceae) in a tepid bath so that the fever disappears (Roys 1976). At present, it is difficult for us to know exactly what the doctor means by “hobnil haa.” In the hives of Melipona beecheii, there are small water reservoirs collected by the bees that are probably utilized, amongst other uses, to regulate the hive temperature (Quezada Euán 2005). It could be that the “hobnil haa” required was, alternatively, waste liquid from the hive, but its quantity is minimal. Perhaps the empty “hobones” or logs were used to collect “virgin” water, that is, the rain gathered in the forest and that had never been touched by human hand. This water was profusely used in rituals and treatments by the Mayan priests. It is also possible that the “ah dzac” referred to the “kab,” honey, in a metaphorical way whose meaning still remains obscure to us. In these books, certain children’s diseases are mentioned repeatedly: for example, nocturnal fevers, convulsions, and shivers, which bring to our attention the fact that 236 G.R. Ocampo Rosales children were more likely to catch maladies and were defenseless against a great many of them. Honey was used for several diseases that had fevers as symptoms. For example, in the case of a skin eruption accompanied by fever, “u chacuil hobonte kak,” three herbs, lemon juice, and fresh honey were integrated to prepare a beverage for the patient (Roys 1976). 15.5.3 Syndromes of Cultural Origin The name of syndromes of cultural origin has been given to particular diseases that still exist in indigenous communities, related to their ancient medical traditions by Carlos Zolla and his investigative team (Mellado Campos et al. 1994). The Mesoamerican cultures believed in the existence of a complex collection of diseases that were due to the direct action of the deities or other forces, such as an evil wind. The sick person lost one or several faculties like the ability of speech. They had a sad, anguished heart, “okom puczikal.” They suffered from dizziness or vertigo and consequently were exposed to the danger of falling during a journey; they had pain in the legs, or walker’s tiredness and many others. Some of these patients were treated with a variety of plants integrated with the honey. Found in sixteenth-century dictionaries, this group of diseases, “tamcaz,” translated as frenzy, madness, could probably be epileptic seizures. Antonio de Ciudad Real, the Franciscan friar who collected thousands of terms to compile the first “calepino” Maya-Spanish dictionary, registers for “tamcaz”: stiffness or numbness, epilepsy or frenzy, that strikes dumb and deaf those who suffer tamcaz (Ciudad Real 2001). The Chan Cah manuscript records a remedy for this illness consisting of a mixture of the root of “kulche” Cedrela mexicana (Meliaceae) and the root of “cat” Parmentiera edulis (Solanaceae), water, and honey (Grupo Dzibil 1982). With respect to a malady where the patient fell, we might speculate over its multiple causes. It could be a simple faint or swoon, or a complication of a cardiovascular disease, epilepsy, or a diabetic coma. There are several entries that refer to this disorder, in which the “ah dzac” specified multiple symptoms. In one of the prescriptions, the doctor referred to a blood movement in the bowels, the sick person fell, and spitted or vomited blood. These symptoms remind us of a gastric ulcer. The prescription was integrated with a handful of “xucul”: leaves, stem, and root of purslane (imported from the Old World with the Spanish conquest), Portulaca oleracea (Portulacaceae), that were boiled with one-third drachma of honey. It was left to cool, sugar was added, and it was administered to the patient at sunrise, under abstinence, for 3 or 4 days (Roys 1976). 15.5.4 Maladies of the Digestive Tract Several diseases of the digestive tract were treated with honey. In the first place, diarrhea with severe colic, named “u lom tokil hubnak” with “othcehil,” was treated with the tender tips of the cualote tree Guazuma polybotrya (Malvaceae) and green 15 Medicinal Uses of Melipona beecheii Honey, by the Ancient Maya 237 leaves of “taamaay” Zuelania roussoviae (Salicaceae), “ixim-che” Casearia nitida (Salicaceae), “muloch” Triumfetta semitriloba (Malvaceae, Tilioideae), and “buhumkak” Cordia geraschanthoides (Boraginaceae) ground and dissolved in a “tumin,” Mayan measure, of hot honey. The liquid was left to cool and was given as a drink although it could provoke vomiting or colic (Roys 1976). A prescription to treat dysentery, “kik choch,” bloody diarrhea, appears in the “Ritual de los Bacabes.” Honey extracted from the logs was added to the plants required for this medicinal beverage (Arzápalo Marín 1987). Not only honey had the strength called “kinam” that the Mayans imagined came from the energy that the sun bestowed upon the earth’s creatures and plants. For other digestive diseases, an indication was given to the specialist to use bees’ or wasps’ nests. For yellow stools and spasms, or colic, the nest of a wasp, “kanal,” was boiled with some plants. This prescription is an example of sympathetic medicine in which color is fundamental. The malady’s signs were yellow, and so were the wasps and their nests. The plants required for the remedy were also yellow, thus having an additional healing power, which could depend on the color division of the Mayan universe (Roys 1976). Another prescription to treat dysentery required burning a nest of “kan-kub,” a bee, taking the larvae, grinding them, and mixing all to administer as a beverage, with honey (Roys 1976). To cure diarrhea, the “ah dzac” could use: “Lucal”. Residue that is collected in the honey pots or in the hive and dissolved in water cures diarrhea, even chronic ones. (Barrera and Barrera Vásquez 1983) To eliminate intestinal worms, honey was also applied in an effective enema: Take milk and honey and vinegar and apply to the rectum (or lower abdomen). It will draw them out immediately. (Roys 1976) One of the most important ritual drinks that the Maya manufactured was “balché.” This beverage was prepared with water, honey, and the bark of the tree called “balché” Lonchocarpus longistylus (Fabaceae, Faboideae), which were mixed and left to ferment for 2 days. The beverage was used by all the members of the Mayan society, according to the ritual that was being enacted. Only children were sometimes exempted from its drinking. Balché was used like a very good purgative, to promote health, strength, and longevity (Garza 1987). The Catholic priests tried to ban the production and use of this beverage mainly because of its close connection with the idolatrous rituals that the Mayans still had fresh in their memories. To this day, balché is commonly drunk in all the Yucatán peninsula. 15.5.5 Diseases of the Sensory Organs In the past, honey was used against ear and eye infections and it is still used by the Mayan traditional specialists called “ah men” or “h men” to heal these ailments. In the literature, we find a prescription to use plants like Hibiscus tubiflorus (Malvaceae) “tupkin,” hibiscus, sorrel, or black mustard Brassica nigra, another European import 238 G.R. Ocampo Rosales (Brassicaceae), whose leaves were roasted and introduced in the ear. If it did not get better, the specialist suggested the use of a ripe, red chilli, without seeds. The doctor took a small quantity of honey directly from the “hobon” and added water. Both substances were put into the chilli that was roasted over hot ashes. When the liquid was tepid, it had to be squeezed into the ear (Roys 1976). When there was pain in the eyes, the medicinal treatment was: It is good also to take fresh honey from the hive and the tender tips of the Carica papaya (Caricaceae), covered with banana leaves and cooked, add a little salt, then wrap it in cotton–wool and squeeze it into the eye. (Roys 1976) Some of the most notorious symptoms of a great number of hot diseases are rashes, spots, pustules, and abscesses on the skin. In the documents that support this investigation, a very serious disease called “ek pedz kak,” smallpox, is mentioned. The prescription indicates: There is also black confluent smallpox “ek pedz kak”. This is the remedy, the blossom and the leaf and the outside of the red Plumeria rubra (Apocynaceae), frangipani. Let these all be roasted, then you mash them and you add a little honey from the hive, raw honey. Then you heat it to just the right temperature and you give it to drink to anyone who has this eruption, in order that it may put an end to the burning and the throbbing. (Roys 1976) In another case of infectious rash, “canal kak,” the informant registered the months and years when the disease appeared and the symptoms as well as the remedies. The word “kak” means fire and “kak cimil,” fire, disease, smallpox in general (Ciudad Real 2001). Contagious skin eruptions were treated with an emetic drink made up with crushed fresh leaves of Bravaisia tubiflora (Acanthaceae) “ek-huleb”; the Croton niveus (Euphorbiaceae) croton “chuy-che”; the Zuelania roussoviae (Salicaceae), “taamaay”; the Castilla elastica (Moraceae) rubber tree; the Alvaradoa amorphoides (Picramniaceae) “besinikche”, Sapindales stub [sic]; and the Leucaena glauca (Fabaceae, Mimosoideae) “uaxim”, white leadtree and mixed with honey (Roys 1976). To cure skin burns, “chuhul,” the injuries had to be covered with honey: “… let it be anointed with honey fresh from the hive, immediately” (Roys 1976). The prescription is long and complex, but honey was the first product that was used to treat these accidents. 15.6 Conclusions In Yucatan, the Mayan traditional doctors, “ah dzaco’ob,” used honey produced by the stingless bee, Melipona beecheii, as a medicinal product of great importance. This fact was rigorously registered in the Colonial chronicles that deal with traditional Mayan medicine. But the complete information on which the practice of these specialists was based gradually disappeared under the pressure of the Spanish culture that was imposed on the native people. The prescriptions lost Pre-Hispanic tradition, and the use of honey was modified from being a curative element of great 15 Medicinal Uses of Melipona beecheii Honey, by the Ancient Maya 239 power or kinam, elaborated by deities, to being used only to sweeten the remedies. Reading the prescriptions that were compiled by Ricardo Ossado and comparing them with the invocations of “El Ritual de los Bacabes” (Arzápalo Marín 1987), we realize the loss of medical, ritual, and religious information that the former underwent. It is also clear that many “traditional remedies” in fact included plants introduced by the Europeans to the Mayans. There are a number of reasons to support the extensive use of this bee’s honey to treat a great number of diseases: first, its unequivocal properties, considering its antimicrobial capacity; second, the “kinam” of its origin that makes it a hot product, gift of long forgotten gods; and third, the fact that it is a natural product, with almost null toxicity on the human organism. Much research remains to be done on the medicinal properties of “kab,” based on the ancient texts. The prescriptions reviewed above were quoted as they were written to provide insight into the logical structure of Mayan thought regarding the power of honey as medicine—ideas that led to its extensive use for the many diseases against which human applications of honey were effective. They may also instruct us regarding the Mayan worldview, an issue of great complexity and interest. Efforts seeking to inform present-day tropical people on the existence and importance of the native stingless bees are very valuable. Let this work be an open invitation to learn more about the native stingless bees of America, their honey stored in pots, the people that have protected them for centuries, and the countries which they inhabit. Acknowledgements I wish to sincerely thank all the anonymous referees who dedicated their time and effort to read and comment this chapter, suggesting changes that really improved it, and the editors that helped me in every possible way. References Arzápalo Marín R, ed. 1987. El Ritual de los Bacabes. Universidad Nacional Autónoma de México, Instituto de Investigaciones Antropológicas; México, DF, México. 1109 pp. Barrera Vázquez A, ed. 1980. Diccionario Maya-Español, Español-Maya. Ediciones Cordemex; Mérida, México. 360 pp. Barrera A, Barrera Vásquez A, eds. 1983. El Libro del Judío. Su ubicación en la tradición botánica y en la medicina tradicional yucatanense. Instituto Nacional de Investigaciones sobre Recursos Bióticos; Xalapa, Veracruz, México. 53 pp. Ciudad Real A. 2001. Calepino Maya de Motul. Plaza y Valdés Editores; México, DF, México. 602 pp. Catzin Ventura GA, Bates A, Medina L, Delgado M. 2009. Actividad antimicrobiana y origen botánico de mieles de Melipona beecheii, Scaptotrigona pectoralis y Apis mellifera del estado de Yucatán”. pp. 84–90, Memorias VI Congreso Mesoamericano sobre Abejas Nativas. Antigua Guatemala, Guatemala, 367 pp. Garza M de la, Izquierdo A, León M, Figueroa T, eds. 1983. Relaciones histórico-geográficas de la Gobernación de Yucatán, (Mérida, Valladolid y Tabasco) vol I. Universidad Nacional Autónoma de México, Instituto de Investigaciones Filológicas; México, DF, México. 448 pp. González- Acereto JA, De Araujo-Freitas, Ch, González-Freyre, J. 2011. Los productos de las abejas nativas, la salud, la vida y la magia: Elementos asociados en la realidad comunitaria 240 G.R. Ocampo Rosales entre los campesinos mayas de la península de Yucatán. pp. 18–22. Memorias del VII Seminario Mesoamericano sobre Abejas Nativas. Cuetzalan, Puebla, México. 242 pp. Grupo Dzibil, ed. 1982. Manuscrito de Chan Cah. Compañía Editorial Impresora y Distribuidora CEID; Mérida, Yucatán, México. 128 pp. López Austin A. 1980. Cuerpo humano e ideología. Universidad Nacional Autónoma de México; México, DF, México. 490 pp. Mellado Campos V, Sánchez A, Femia P, Navarro A, Erosa E, Bonilla D, Domínguez M. 1994. La medicina tradicional de los pueblos indígenas de México, vol II. Instituto Nacional Indigenista; México, DF, México. 623 pp. Ocampo Rosales G. 2005. La salud y la enfermedad en las Relaciones Geográficas del siglo XVI (1579–1585). Tesis de Maestría en Estudios Mesoamericanos, Facultad de Filosofía y Letras. Universidad Nacional Autónoma de México; México, DF, México. 392 pp. Quezada Euán JJ. 2005. Biología y uso de las abejas sin aguijón de la Península de Yucatán, México (Hymenoptera: Meliponini). Ediciones de la Universidad Autónoma de Yucatán; Yucatán, Mérida. 112 pp. Roys RL. 1976. The Ethno-Botany of the Maya. Institute for the Study of Human Issues; Philadelphia, USA. 380 pp. Vit P, Medina M, Enriquez ME. 2004. Quality standards for medicinal uses of Meliponinae honey in Guatemala, Mexico and Venezuela. Bee World 85:2–5. Chapter 16 Staden’s First Report in 1557 on the Collection of Stingless Bee Honey by Indians in Brazil Wolf Engels Dedicated to my colleague and friend Paulo Nogueira-Neto on the occasion of his 90th birthday, April 18, 2012. 16.1 Introduction Honey has presumably been much in demand by people since prehistoric times. To procure this unique, delicious food, many modes of honey hunting were developed, of which several are still in use today. To facilitate access to this delicacy, several ancient cultures invented modes of beekeeping, in particular with two species of honey bees, Apis mellifera in Europe and Africa, and Apis cerana in Asia (Crane 1999). In the Americas, management of stingless bees in artificial hives has only been reported for the culturally advanced Mayans and Aztecs, a tradition of meliponiculture now continued by the indigenous population of the Mexican peninsula, Yucatán (Inoue 1990). As far as we know, the early Brazilians never developed similar techniques, although their methods of honey hunting include sustainable removal without destroying the nest (Posey 2002). Nevertheless, that they knew very well where to find stingless bee colonies was already reported by Hans Staden in the sixteenth century (see also Cobo 1653, in Roubik 2000). W. Engels (*) Zoological Institute, University of Tübingen, Tübingen, Germany Departamento de Genética, Universidade de São Paulo, Ribeirão Preto, Brazil e-mail: wolf.engels@uni-tuebingen.de 241 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_16, © Springer Science+Business Media New York 2013 242 16.2 W. Engels The Oldest Written Report on Brazilian Honey Collection The first book on Brazil, the “Warhaftig Historia” by Hans Staden (Fig. 16.1), was published in Marburg in 1557. The author was a German adventurer who served as a mercenary on Spanish and French ships exploring the Atlantic coast from the La Plata region north to Cabo Frio near Rio de Janeiro. During two journeys he spent Fig. 16.1 Frontispiece of Hans Staden’s book, original edition 1557 16 Staden’s First Report in 1557 on the Collection of Stingless... 243 about 10 years in the New World, including 10 months as a prisoner of the Tupinambá tribe in the São Paulo region. In his 178-page book he described in New High German language the coastal geography in great detail, based on his experience cruising the coast. In addition, he reported on the life of the indigenous people. Especially because it included description of an anthropophagic cult, the book immediately became a bestseller. 16.3 Hans Staden’s Contribution to the Knowledge of Stingless Bees in Brazil The original publication of Staden’s book as well as early illegal editions, and also recent literature on Hans Staden and on stingless bees in Brazil, were consulted. The figures shown here are copies from online facsimiles prepared by the University of São Paulo. At the very end of his book, in only six pages, some peculiarities of Brazilian nature (Engels and Heinle 2011) were recorded (Fig. 16.2). In the second part of the book, the last chapters discuss nature in Brazil, beginning with Chap. 30, titled “Bericht etlicher Thier im lande” (record on several animals in Figs. 16.1 and 16.2). Chapter 35 is entitled “Von Binen oder Imen des lands” (from bees or “ims” of the land), including remarks on stingless bees and the collection of their honey (Fig. 16.3). With a mere 140 words Hans Staden described stingless bees, mentioned their typical behavior, and noted that nests with honey stores are found in hollow trees. He had observed how the Indians collected the honey and participated in the process, and was attacked vigorously by the non-stinging but biting bees. He wrote [in translation]: There are three species of bees in the land. According to their nature, the first are almost like those in our land. The others are black and as large as flies. The third are small like midges. All these bees have honey in hollow trees. Together with the wild men, I frequently collected the honey. Among the three species, we usually found better honey from the smallest bees than from the others. They do not sting so hard as the bees in our country. As I have Fig. 16.2 Title of Chapter 30 on Brazilian animals 244 W. Engels Fig. 16.3 Chapter 35 on Brazilian stingless bees, their behavior, and how the Indians in Brazil collect their honey often seen, when the wild people take honey, the bees fly upon them, so that they had much to do in striking them off from their naked bodies. I myself also took honey naked. The first time I had to run with great pain to water and wash them off, merely to get rid of the bees from my body. 16.4 Forward-Thinking Based on the Precise Bee Descriptions of Staden The original text of this short chapter in German is very precise (Fig. 16.3). I will comment on the above-mentioned sentences. First of all, it was possible for me to deduce the genera and the probable species mentioned by Hans Staden. These are most likely Melipona quadrifasciata, Scaptotrigona postica, and Tetragonisca angustula (Engels 2009), all today still occur in the São Paulo region (NogueiraNeto 1997; Marcolin 2009). According to Staden, these stingless bees use hollow trees as nesting sites, a correct observation (Nogueira-Neto 1997). The Indians collected the honey by removing it 16 Staden’s First Report in 1557 on the Collection of Stingless... 245 from the colony after cutting the trunk open. Presumably they only took the honey pots, because it is known from recent studies on apicultural traditions of the North Brazilian Kayapó Indians (Posey and Camargo 1985; Posey 2002) that honey hunting is done by repeated removal of sealed pots from the storage area of stingless bee nests without destroying the colony. In comparing the honey of the three species, Staden favored that from T. angustula, and in fact this “jataí” honey also yields the highest price on today’s Brazilian market. It is delicious and also is used for medicinal purposes. The term “stingless bee” was unknown in the sixteenth century; however, Staden mentioned correctly that the Brazilian bees did not sting. In particular, S. postica colonies very actively defend their nest. Any enemy is immediately attacked, the workers hang onto hairs and eyelashes, bite into the skin, enter the ears, nostrils, and mouth, and chase the intruder. I experienced this behavior during field work in Brazil, as documented in our film on their nest biology (Engels and Engels 1980). Staden reported that it is not easy to get rid of these defenders, which also recruit many nestmates by releasing an alarm pheromone (Smith and Roubik 1983). 16.5 Conclusions In summary, Hans Staden’s book provided the first published information on stingless bees, unknown then in Europe. He described their nesting habit, non-stinging defense strategy, and in particular, stingless bee honeys of different qualities. This precise record was until recently (Engels 2009; Marcolin 2009) not quoted in the scientific literature on stingless bees (Nogueira Neto 1997; Michener 2007; Moure et al. 2007). The cultural traditions of South American Indians evidently allowed them to harvest honey as a valuable product of the native meliponine bees, similar to various forms of honey hunting developed in Europe, Africa, Asia, and both Americas (Crane 1999). We can assume that detailed knowledge on stingless bee biology was present in the indigenous Brazilian tribes and practiced in the sustainable use of the resources available in the tropical forests. Honey hunting from stingless bees presumably was common long before the Europeans arrived in South America. Acknowledgements I thank David De Jong, Klaus Hartfelder, and David Roubik for critical reading of the manuscript, and Sabine Heinle for cooperation in our search for Staden literature in the rare books collection of the University of Tübingen library, and for preparation of the figures. References Crane E. 1999. The World History of Beekeeping and Honey Hunting. Routledge; New York, USA. 682 pp. Engels W. 2009. The first record on Brazilian stingless bees published 450 years ago by Hans Staden. Genetics and Molecular Research 8:738–743. 246 W. Engels Engels W, Engels E. 1980. Nest biology of the Stingless Bee Scaptotrigona postica. Farbtonfilm 16 mm, 18 min. IWF C 1351; Göttingen, Germany. Engels W, Heinle S. 2011. Hans Staden als Tropen-Biologe: Erste Beschreibungen andersartiger Tiere und Pflanzen in seiner Warhaftig Historia - Stadens 22 Beispiele der Biodiversität Brasiliens - Martius-Staden-Jahrbuch 58: im Druck. Inoue T. 1990. A trip in Yucatan, Mexico - meliponiculture of the Maya. Journal of Honeybee Science 11:49–58. Marcolin N. 2009. Hans Staden naturalista. Pesquisa Fapesp (São Paulo) 164:10–11. Michener CD. 2007. The Bees of the World, 2nd edn. The Johns Hopkins University Press; Baltimore, Maryland, USA. 953 pp. Moure JS, Urban D, Melo GAR, eds. 2007. Catalogue of Bees (Hymenoptera, Apoidea) in the Neotropical Region. Sociedade Brasileira de Entomologia; Curitiba, Paraná, Brasil. 1058 pp. Nogueira-Neto P. 1997. Vida e criação das abelhas indígenas sem ferrão. Editora Nogueirapis; São Paulo, Brasil. 442 pp. Posey DA. 2002. Kayapó Ethnoecology and Culture. Routledge; New York, USA. 304 pp. Posey DA, Camargo JMF. 1985. Additional notes on the classification and knowledge of stingless bees (Meliponinae, Apidae, Hymenoptera) by Kayapó Indians of Gorotire, Pará, Brazil. Annals of Carnegie Museum, Pittsburgh, 54 (8):247–274. Roubik DW. 2000. Pollination system stability in Tropical America. Conservation Biology 14:1235–1236. Smith B H, Roubik D W. 1983. Mandibular glands of stingless bees (Hymenoptera: Apidae): chemical analysis of their contents and biological function in two species of Melipona. Journal of Chemical Ecology 9:1465–1472. Staden H. 1557. Warhafftig Historia. Andreas Kolbe; Marburg, Germany. 178 pp. Chapter 17 Melipona Bees in the Scientific World: Western Cultural Views Raquel Barceló Quintal and David W. Roubik 17.1 Introduction To study the tiny world of insects, a microscope is a necessary tool. Insects were made large by their deed, in the case of stingless bees, by manufacturing honey from the nectar of flowers—or other sugary resources—in their environment. To follow up on such a novel discovery, many entomologists and natural historians had to use a microscope. Further exploration and taxonomic expertise were required, as illustrated here in examples from the Western World and literature. 17.1.1 Early Studies on the Stingless Bees For centuries, humans have used honey from bees known as meliponas or the stingless bees (Schwarz 1932, 1948; Lutz 1933; Friese 1903; Ducke 1924), tribe Meliponini, as a natural source of food, as a healing element, and as a product for commercial exchange. It was not, however, until the nineteenth century when European scientific studies on the aforementioned bees began in earnest (e.g., Spinola 1853). This was not the case for Apis mellifera because its study was closely linked to the development of optical devices, such as the microscope. According to the Italians, this instrument was invented, in 1610, by Galileo Galilei, but the Dutch attribute it to Zacharias Jansen, in the year 1602. Later, at the workshop of Cornelius Drebbel R.B. Quintal (*) History and Anthropology Area, Social Sciences and Human Studies Institute, Universidad Autónoma del Estado de Hidalgo, Pachuca, Mexico e-mail: rbarceloquintal@gmail.com D.W. Roubik Smithsonian Tropical Research Institute, Ancón, Balboa, Republic of Panamá MRC 0580-12, Unit 9100, Box 0948, DPO AA, 34002-9998, USA 247 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_17, © Springer Science+Business Media New York 2013 248 R.B. Quintal and D.W. Roubik Fig. 17.1 Stelluti’s book with compound eye of the honey bee. From Stelluti (1625) (1572–1633), a similar device was created that was called the microscopium. With this instrument, a new age of biology arose. It was in Francesco Stellutti’s workshop (1577–1651) that the honey bee Apis mellifera and its compound eyes were first observed under the microscope (see Fig. 17.1, from Stelluti 1625). Such observations revealed various new characteristics. Nevertheless, Francesco Redi (1621–1679) may be considered the “father of insect biology,” thanks to interesting observations gathered in his work written in 1668 “Esperienze intorno alla generazione degl’ insetti,” translated in the book “Experiments on the generation of insects” (Redi 1909). Despite several conceptual mistakes in the seventeenth century, there were a number of direct observations that influenced early treatises on bees and fostered emergence of modern science as a system of approaching reality, whose historical achievements included publication, around 1637, of the work “Discourse on Methods,” by René Descartes, who distinguished Physics from Biology. Descartes included, among natural facts, behavioral responses from living beings as events obeying general laws, similar to those that govern inanimate objects. With Descartes’ text, science moved ahead, since the old controversy on spontaneous generation of small animals was challenged by the sound experiments of Redi, when for the first time insects were demonstrated not to come from flesh through spontaneous generation. 17.1.2 Enlightenment and the Study of Insects Even though scientific studies in the seventeenth century were devoted to Apis mellifera, in the eighteenth century the Western World became interested in stingless 17 Melipona Bees in the Scientific World: Western Cultural Views 249 bees. In that century, best known as the enlightenment century, there was an optimistic attitude in minority European circles about the possibilities and benefits of reason, education, and science as means of solving mankind’s problems. There was important progress from a peculiar constitutive and operational principle, which, in its turn, was conceived as a vital force—ontologically and operationally superior to other cosmic natural forces (mechanics, thermodynamics, electricity, chemistry, and magnetism). In the eighteenth century, observers of the natural world were concerned about ordering living diversity by means of taxonomy, that is to say a hierarchical system. In 1731, Carl Linnaeus (1758) (1707–1778) invented a biological classification system, presented in its 10th edition in 1758, and considered the origin of modern taxonomy. He developed the modern scheme of binomial nomenclature, first, indicating genus, and second, species. After that, diverse taxonomists added other categories: family, order, class, phylum or division, and kingdom. According to the sociologists Émile Durkheim (1858–1917) and Marcel Mauss (1872–1950), primitive classifications emerged not only from the ability to recognize groups but also as a projection of social organization; they said “man classified things because he was divided into clans” […]. The first categories were the social ones; the first classes of things were human classes. This was because men were grouped, and they thought about themselves in the form of groups, and in their minds appeared the idea of grouping things […]. Man was the first genus; clans were the first species (Durkheim and Mauss 1963). On 22 March 1803, Aimé Bonpland (1793–1858), aboard a Spanish frigate, sailed from Guayaquil (Ecuador) to Acapulco, the most important Pacific Mexican port. He visited and described the places in his diary before leaving for Chilpancingo and Taxco, on 29 March, and then to Mexico City, where he arrived in on 12 April. There, Alexander von Humboldt (1984) (1769–1859) traveled to nearby places. When he published his work “Political Essay on the Kingdom of New Spain,” and related Campeche’s honey and cerumen production, he wondered if this bee was the same one that Bonpland found on the Eastern slopes of the Venezuelan Cordillera, mentioned in their book “Recueil d’observations de zoologie et anatomie comparée,” published in 1811 (Freites 2000). Baron Alexander von Humboldt knew about melipona bees through the entomologists Johann Karl Wilhelm Illiger (1811) (1775–1813), Pierre André Latreille (1762–1833), and Louis Jurine (1775–1819). In 1806 Illiger was the person who described the characteristics of the genus Melipona (Wille 1983), as he mentions in his work “Prodomus systematis mammalium et avium” (1811), which is a treatise on systematics or Linnaean Taxonomy. Another entomologist, Latreille, arranged the entomological collection of the National Museum of Natural History in Paris; in 1814, as a member of the French Academy of Sciences, he studied Melipona scutellaris. In 1819, he published his work “Mémoires sur divers sujets de l’histoire naturelle des insectes, de géographie ancienne et de chronologie.” He went further, by subdividing the tropical American stingless bees into two genera; Melipona, in which the mandibles are not toothed; and Trigona, in which mandibles are dentate. The basis of these subdivisions seemed to be supported by the general appearance of the insects (see Schwarz 1932, 1948; Michener 2007). R.B. Quintal and D.W. Roubik 250 Fig. 17.2 Portrait of Captain Frederik William Beechey. From Christian Young (n/d) 17.1.3 The Nineteenth Century and Melittology During the nineteenth century, there were many formal studies on insects in Mexico. Indeed, nearly a century after Illiger and Latreille established the ground plan of studies on Neotropical stingless bees, a number of publications appeared on regional fauna (Cockerell 1900; von Ihering 1902; Friese 1903; Marianno 1911; von Ihering 1912; Ducke 1924). This century saw the origin of an accredited entomological profession; centers of teaching and research were founded, and museums and collections initiated, together with societies and periodic publications devoted to insects. Meanwhile in Europe, studies on Apis mellifera proliferated, among them works on pollen contained in honey, which gave a new impulse to apiculture. In 1827, Frederik William Beechey (1796–1856; see Fig. 17.2) British naval officer, artist and geographer, went across the Bering Strait with the purpose of meeting John Franklin and William Edward Parry. Although his voyage was unsuccessful, on his return he explored the Pacific Coast, where he discovered several islands and visited the ports, such as San Francisco and Mazatlán, where he arrived in on 3 February 1828, and drew one of the first known maps of the city. He had the good fortune to bring together a variety of rare species from distant localities, some of which had been seldom, if ever, visited by any collector. In 1831, as a result of this travel, Beechey published his work “Narrative of a voyage to the Pacific and Bering Strait to co-operate with the Polar Expeditions, 1825–1828.” Later, in 1831, Edward Turner Bennett (1797–1836), British zoologist, reviewed the notes of captain Beechey1 on the domestication of the bee that he knew in British zoologists studied the notes of captain Beechey and published the book “Zoology of Beechey’s Voyage.” In 1891 the stingless bee, whose culture in hollow logs was developed by the Mayans, acquired the name Melipona beecheii Bennett, named in his honor. 1 17 Melipona Bees in the Scientific World: Western Cultural Views 251 Fig. 17.3 Herbert F. Schwarz. Image reproduced courtesy of J. Ascher and E. Wyman Mexico. The interconnection between human and stingless bees was typified in the following paragraph: In the domestications of the bees of Mexico but little violence is done to their natural habits. In habitants, in their wild state of cavities in trees, a hollow tree is selected to form their hive. A portion of it, of between two and three feet in length; is cut off, and a hole is bored trough the side into the hollow, at about its middle. The ends of the hollow are then stopped to with clay, and the future hive is suspended on a tree, in a horizontal position, with the hole opening the cavity directed also horizontally. Of the hive, this prepared, a swarm of bees speedily take possession, and commence their operations by forming cells for receptions of their larvae, and sacs that contain the superabundant honey collected by them in their excursions (Bennett 1831). The final decades of the nineteenth century saw several entomologists who describe species of Meliponini from Mexico, among them Ezra Townsend Cresson (1838–1926), Theodore OA Cockerell (1866–1948), and Karl Wilhem von Dalla Torre (1858–1928). Studies in Brazil also produced meliponines new to science (Spinola 1853). 17.1.4 The Meliponas in Twentieth Century Science Behavior and ecology of stingless bees was beginning to be explored, particularly in regard to the foraging flights and recruitment of individual bees to food sources by others from their colony (Salt 1929; Lutz 1933; see also Lindauer 1961; Wille 1983; Roubik 1989). The foundations of meliponine taxonomy were further extended to other portions of the world, and intensive country-wide surveys continued (Schwarz 1932, 1934, 1937, 1948; Moure and Kerr 1950; Michener 1954; Moure 1961). See Fig. 17.3 with the portrait of HF Schwarz, ca. 1935, from the American Museum of Natural History, New York. 252 R.B. Quintal and D.W. Roubik Paleontologists soon joined in stingless bee studies from their external morphology captured in amber, focusing on bees from both Dominican Republic and Mexico (Wille 1983, and see also present book Chap. 9 by Ayala et al.). Regarding this last subject certain specimens that have been found allow observing or inferring relationships. Such is the case of specimens of Proplebeia dominicana which became trapped while collecting resin for their nests. The most ancient amber fossil meliponine Cretotrigona prisca dating as early as the Upper Cretaceous Period2 was found in New Jersey, United States, and it is roughly 67 million years old. The first fossils of Apis were discovered in Western Germany, and they date back to the Early Miocene Period, from 22 to 25 million years ago (Engel et al. 2009). A bee that looks like Apis dorsata, but is smaller, similar to the current size of Apis mellifera, was present in the Upper Miocene period, ca. 12 million years ago, in Western North America (Engel et al. 2009). It is thought that Apis florea and Apis dorsata might have existed as separate species or lineages since the Oligocene period. With regard to paleontological studies, João María Franco de Camargo (1941– 2009), Brazilian entomologist, proposed biogeographical barriers or geological compartments in hierarchies defined by sequences of vicariance and cladogenesis among the fossil and extant stingless bees (Camargo 2008; Vit 2010; Camargo, Chap. 2 in this book). In the twentieth century, after some paleontological discoveries, several researchers, such as Joachim C. Evenius (1896–1933), Guido Grandi3 (1886–1970), and Edward Butler (1881–1963) devoted themselves to the study of pollen carried by bees (Apis and Melipona). Methods of melissopalynology (pollen identification of pollen in honey) were published by Louveaux et al. (1978). As a result of the discontinuity produced by the Revolution, entomological research in Mexico was disturbed, and it was not until the twentieth century, after 1921, when it regained vitality. During the decades of 1940 and 1950 the proper means for the development of this discipline were established. More recent years were characterized by some important achievements: well-equipped laboratories and proper salaries have allowed entomologists to work on research full-time (Pacheco 1989). Regarding taxonomy, two major genera were long used for stingless bees. In 1951, Jesús Santiago Moure (1912–2010) and Warwick Estevam Kerr (1922–) proposed 12 genera and 19 subgenera for the Neotropical region (Moure and Kerr 1950). In 1967, Kerr et al. proposed the subgenus Micheneria; and Moure, in 1975, changed it to Michmelia. Nevertheless, Charles Duncan Michener4 (1918–) does not consider In that time, continents were already separate and had a form similar to now, but they presented distinctive attributes, for example, the inner part of North America contained a sea which divided the continent, known as Cretaceous Seaway. 3 Italian entomologist, who founded, in 1928, the Institute of Entomology in the University of Bologna. 4 In 1944, he published a classification system for bees that would be soon adopted by melittologists, and was used until 1995, when he was the co-author of new classifications; again modernized for the world in 2000 and in a revised work, “The bees of the World,” in 2007. 2 17 Melipona Bees in the Scientific World: Western Cultural Views 253 Fig. 17.4 C. Rasmussen, J.M.F. Camargo, and Father J.S. Moure. Three of the twenty-first century entomologists most devoted to stingless bee taxonomic and systematic studies, in the library of the Claretian Home in Batatais, São Paulo, Brazil, 2008. Photo P. Vit that Melipona is heterogeneous enough to be divided into subgenera (Michener 1990). It is important to note that Moure, known as the “Father of bees,” was a priest who created a catalogue of Neotropical bees, together with Danuncia Urban, Gabriel AR Melo, and individual authors of large sections, e.g., Camargo and Pedro (2007) Chapter Meliponini Lepeletier, 1836. This catalogue was a product initiated with compilation of Moure’s notes about bees, dating back to 1938. In 1975, the catalogue contained over 11,200 typed cards.5 During a short stopover in Ribeirão Preto, Brazil, while Dr. Rasmussen was invited for a talk, Professor Camargo suggested a visit to his very appreciated mentor Padre Moure in the Claretian Retirement Home in Batatais, during the local holiday known as “tiradentes” in 2008. Three generations of stingless bee scholars are shown including Padre Moure in Fig. 17.4. Studies were directed toward discovering Brazilian stingless bee communication by meliponologists Martin Lindauer6 (1918–2008) and Warwick Estevam Kerr Padre Moure’s catalogue consisted of handwritten cards; carbon copies can be found at the University of Kansas, where they were deposited by Padre Moure; 11,200 typed cards, which in large part relate to the family Halictidae (around 2,000 cards), were published as a catalogue in 1987 by Moure and Paul David Hurd (1921–1982), for the Smithsonian Institution. Recently, the part containing information about Colletidae (around 750 cards) was published in five articles in the Magazine of Zoology, reaching a total of 161 pages. Therefore, most of Padre Moure’s catalogue was unpublished until 2007 when the whole catalogue of bees in the Neotropical region was edited by Moure, Urban, and Melo. 6 German neurobiologist, who was a Zoology professor at Frankfurt University. As a scientist, he discovered communication among bees; their sense of orientation to find their way and live in a society. 5 254 R.B. Quintal and D.W. Roubik (1922–), in Piracicaba, and elsewhere in Brazil. The communication procedure is partly chemical, when the foragers find an important source of nectar, pollen, and presumably resin, they make from six to ten journeys to the hive carrying it as a demonstration of a harvestable resource. Then, bees suddenly change their behavior, they leave the nest, and fly towards the resource, but this time they do not pick it up when returning to the nest; instead they start “marking” the foraging site, leaving signals from place to place. These substrates differ according to bee species; for example, Trigona spinipes “irapuã” marks stones, leaves, flowers, or any other objects before entering the nest. The mark that these bees leave consists of tiny drops of the pheromone produced by certain glands in the head. Recent research reveals different combinations of zigzag dances in the nest, or use of marking pheromones, in Melipona, Scaptotrigona, Cephalotrigona, and Partamona (QuezadaEuán 2005 and various chapters in the present book). In the 1970s, in addition to cataloguing native bees, biological studies were extended to the nesting biology, beekeeping, and behavior of stingless bees, for example, by Paulo Nogueira-Neto (1922–)7 who studied nesting colonies, the fertilization of the queen, and the foraging of worker bees, and published a comprehensive manual on stingless beekeeping (Nogueira-Neto 1970). In addition, the nest architecture and varied biological details of nesting colonies were rendered with detailed drawings and field observations (Camargo 1970). The 1980s witnessed not only the first detailed ethnography of stingless bee specialists within indigenous American tribes (Posey 1980; Posey and Camargo 1985) but also an integration of literature on tropical bees, highlighting many of the biological features of Meliponini (Roubik 1989). In the 1990s, study of the Meliponini has been concerned with risk of extinction, crops and their pollination, the impact of pesticides, the devastation of forests, the introduction of non-native species, and reduction of stingless bee abundance. In the same decade there was consideration of stingless bee “re-population” in forests, in such a way that the trees will receive pollination and the latter obtain food and protection (Svensson 1991; Méndez 1999). Other research showed that the stingless bees produce more honey under conditions of ecological balance (Hill and Webster 1995). Currently, a growing number of studies that consider physicochemical composition of honey from stingless bees are being carried out. Moreover, standards are being devised for their honey quality in different ways, as shown in the present book. 17.1.5 Cultural Studies on the Stingless Bees Claude Lévi-Strauss (1908–2009) exposed to the eyes of Western scientists the existence of indigenous knowledge about nature and its societies, what he called “sciences of the concrete,” that is to say, traditional knowledge, with the aim of 7 First Secretary of the Special Secretariat for the Environment, Brazil. 17 Melipona Bees in the Scientific World: Western Cultural Views 255 Fig. 17.5 Levi-Strauss in the Brazilian Amazon. From Wilcken (2011) validating its principles and establishing its cultural rights (Lévi-Strauss 1964). He was one of the social scientists who recovered popular knowledge about stingless bees (Fig. 17.5). Lévi-Strauss, as an anthropologist, focused on popular knowledge and/or peoples’ primitive thought. From 1930 to 1935, he lived in Brazil, where he performed his first ethnographical work, in Mato Grosso, and the Amazon. In 1955, he published his work “Tristes tropiques” (Sad tropics), which is an ethnographical work; and in 1972 “De la miel a la cenizas” (From honey to ashes), second part of his series “Les mythologiques,” where he undertakes a structural analysis about diverse myths created over honey by the tribes he visited in Brazil. In brief, through the study of Brazilian indigenous cultures, he understood that despite the differences existing among diverse parts of mankind, human mind is one and the same thing everywhere, with the same abilities (Lévi-Strauss 1972). The current trend seems to be more holistic, regarding all or a great part of intellectual elements: cognitive, symbolic, economic, cultural, and ecological. In Latin America, in general, and Mexico and certainly several Tropical American countries there is a large cultural diversity that contains a wide knowledge which may be highly correlated to scientific knowledge. Within the fauna of Tropical America, bees have been of great cultural value, since before America’s discovery by European explorers, and further conquest. They have been part of religious, festive, and trade customs of several indigenous peoples, the Mayans were the main ethnic group who developed, through Melipona beecheii beekeeping and husbandry, the science and art of meliponiculture (see the Ocampo Rosales Chap. 15 in this book). The other meliponas are ever present in the culture of Latin American people. With regard to research on Melipona beecheii, in the culture of the ancient Mayans, Ernst Förstemann (1822–1906), librarian of Dresden, was one of the pioneers in trying to decipher the “calendar of the meliponary” of the Codice Madrid. Later, Alfred Marston Tozzer (1877–1954) spent several seasons in Yucatán to 256 R.B. Quintal and D.W. Roubik Fig. 17.6 Bees from the Mayan Codex. From Tozzer and Allen (1910) investigate the Mayan culture. Among the folk stories, he drew attention to those in which the bees were of high value between cultures. In 1910, together with Glover Morrill Allen (1879–1942) he published “Animal Figures in the Mayan Codices” (Tozzer and Allen 1910), see Fig. 17.6. Another pioneering work was that of Édouard Bunge, member of the Société des Américanistes of Paris, published in the Journal of this Society, in 1936, as “Les pages des abeilles du Codex Tro” (Bunge 1936). At the end of the 1950s, Wolfang Cordan (1908–1966) traveled to Mexico 17 Melipona Bees in the Scientific World: Western Cultural Views 257 where he studied the language and the Mayan writing. In 1966, he studied the rite of harvest of honey in the codice Madrid (Cordan 1966). And finally, among recent works, is that of Mary A. Ciaramella, who interprets the beekeepers in the same codex (Ciaramella 2002). Studies of the native stingless bees and their relations to humans, because of the complexity of their biological history and cultures, require interdisciplinary research, combining biology, anthropology, cultural ecology, ethnomedicine, ethnozoology, biochemistry, genetics, and combinations thereof. The stingless bees have produced many things, products such as honey, pollen, cerumen, and propolis. They have tangible intellectual and economic value, as well as providing a unique source of food and medicines. Acknowledgments We thank Professor Charles D. Michener for early comments on the manuscript, Professor Patricia Vit and Dr. Silvia R.M. Pedro for their attention and help with this chapter. References Beechey FW. (ed) 1831. 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Annual Review of Entomology 28:41–64. Chapter 18 Taxonomy as a Tool for Conservation of African Stingless Bees and Their Honey Connal Eardley and Peter Kwapong We dedicate this chapter to Professor Charles Duncan Michener who has been a huge inspiration throughout our career. As ecology embodies taxonomy, the background created by Mich will endure and always be treasured. 18.1 Introduction In Africa stingless bees are most diverse in the equatorial regions. To the north the Sahara Desert abruptly delimits their distribution. Southwards they become progressively less diverse reaching more or less the Tropic of Capricorn in the interior of the Subcontinent. Their distribution extends farther south along the East coast, and to a lesser extent along the west coast (Eardley 2004). Several species appear confined to the tropical wet forests. Most species, however, occur in both savannah and tropical forests, including the east African coastal forest (Eardley 2004). Two species have been recorded from desert areas, one occurs in the south-western Sahara (Hypotrigona penna Eardley) and there is an unpublished record of Liotrigona from the Richtersveld, South Africa. The African stingless bees are smaller than indigenous African honey bees Apis mellifera L. and their approximately 30 recognised subspecies (Ruttner 1988; C. Eardley (*) Agricultural Research Council, Private Bag X134, Queenswood, 0121 Pretoria, South Africa School of Biological and Conservation Sciences, University of KwaZulu-Natal, Private Bag X01, Scottsville, Pietermaritzburg 3209, South Africa e-mail: eardleyc@arc.agric.za P. Kwapong Department of Entomology & Wildlife—International Stingless Bee Centre, School of Biological Sciences, University of Cape Coast, Cape Coast, Ghana 261 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_18, © Springer Science+Business Media New York 2013 262 C. Eardley and P. Kwapong Michener 2007). They also do not produce as much honey as the honey bee does, which has a larger flight range than meliponines. The robbing of honey bee nests by indigenous people has been practiced for millennia in Africa, as documented in rock paintings (Crane 1999; Johannsmeier 2001), and they are still being robbed in Africa (Eardley C, personal observations). Currently meliponiculture is practiced in tropical Africa, but for the most part its history has not been documented and its age is unknown. In Ghana, current stingless beekeeping only recently began as an activity complementary to beekeeping with Apis (Kwapong et al. 2010). It does not appear to have been practiced in southern Africa. However, stingless bee honey, although less in quantity, is highly sought in all of tropical Africa—primarily for its medicinal uses. It fetches higher prices than honey bee honey, and is culturally important. The current value of stingless bees, as pollinators, to biodiversity conservation and agriculture is unknown, but they do visit flowers of many different plants and crops, as seen in the field and often indicated on museum specimen labels. Being social they can possibly be more easily managed than solitary bees and the expansion of meliponiculture to agriculture should be further investigated (Roubik 1995). Vernacular names for stingless bees in South Africa are “mopani” bees or “mocca” bees. In Ghana several of the species are known by their common names: “anihammoa”, “duro kokoo”, “duro tuntum”, “mimina” and “tifuie”. 18.2 Taxonomy of Stingless Bees Prior to Eardley (2004) research articles on stingless bees of the Afrotropical Region were relatively few, and by a handful of authors (Ambougo-Atisso 1990; Darchen 1966, 1969a, b, 1970, 1971a, b, 1972a, b, 1973, 1977a, b, 1981, 1985; Darchen and Louis 1961; Darchen and Pain 1966, Fletcher and Crewe 1981a, b; Kajobe 2006, 2007a, b; Kajobe and Echazarreta 2005; Kajobe and Roubik 2006; Lobreau-Callen et al. 1986, 1990, 1994; Michener 1959; Moure 1961; Moritz and Crewe 1988; Portugal-Araújo 1955a, 1955b, 1956, 1958, 1963; Portugal-Araújo and Kerr 1959; Sakagami et al. 1977), excluding those that described new species. Moure (1961) provides keys for the identification of many African stingless bees, but understanding the small differences between species together with intraspecific variation still prevented confident identification of many species. Consequently a taxonomic revision, based on worker bee morphology, was undertaken (Eardley 2004). Since then a lot of interest has been shown in developing meliponiculture in West Africa (Kwapong et al. 2010). Meliponiculture has been practiced for a long time in East Africa but more recently research into foraging and nesting has been undertaken (Kajobe 2006, 2007a, b; Kajobe and Echazarreta 2005), while little interest has been generated in southern Africa. There has also been interest in documenting meliponines as pollinators and the medicinal use of their honey, but to date there are no substantial data for Africa. Eardley (2004) found that the material available in museums and comparative biological information in the literature were scant, in contrast with the great abundance 18 Taxonomy as a Tool for Conservation of African Stingless Bees and Their Honey 263 of these bees in the wild. As now recognised, differentiating many stingless bee species and some genera require microscopic or molecular studies (see Rasmussen and Cameron 2010, Chap. 1, in the present book), and cryptic species have been noted (Camargo and Pedro 2007). It is now widely believed that Eardley (2004) underrepresented the true diversity of the taxon (Macharia J, personal communication). Portugal-Araújo and Kerr (1959) discovered Hypotrigona araujoi (Michener) to be a distinct species through observation in a meliponary, and Michener (1959) subsequently discovered subtle differences between it and Hypotrigona gribodoi (Magretti). Darchen (1970, 1981) studied stingless bee biology in West Africa that led to the description of three new species; Meliponula (Axestotrigona) sawadogoi (Darchen), Meliponula (Axestotrigona) richardsi (Darchen) and Meliponula (Axestotrigona) eburnensis (Darchen) whose types have not yet been located. Joseph Macharia found differences in the nest architecture in the species that Eardley documented as Meliponula bocandei (Spinola) (Macharia J, personal communication) suggesting that this taxon is composite. Katherine Krause found size differences in the species that Eardley documented as H. gribodoi (Magretti) which indicate that H. gribodoi comprises more than one species. Further, the fact that the majority of species (10 out of a total of 18 species) occur in distinct habitats, such as tropical forest and dry savannah, suggests a potential greater species diversity than recorded by Eardley (2004) (Table 18.1). The difficulty in separating stingless bees based on morphology necessitates the need for new diagnostic tools. Nest architecture and host plant preferences pose logistical problems in gathering material for taxonomic revisions of genera and would be better suited to studies on differences between identified species rather than being used to recognise different species. The most promising tool for identifying morphologically similar species is evidently DNA barcodes, a method using a short genetic sequence to identify an organism, as suggested by Packer et al. (2009). 18.3 Host Plants and Nests of Stingless Bees Knowing bee host plant usage is important for understanding pollination as well as the medicinal use of bee honey, as explained among the chapters in the present book, which include studies in Africa, Asia, Australia and the Neotropics. Stingless bees focus their foraging activities on a wide range of food plants. As a group they have been recorded visiting 135 plant genera (Eardley and Urban 2010). A preliminary survey of the data suggests that food plant overlap is greater within bee genera than between the genera. However, the data do not indicate if the bees are collecting pollen or nectar. Until the taxonomy is properly resolved, the degree of host plant specificity will not be understood. In Ghana, stingless bees have been collected from tropical rain forest canopies (Nuttman et al. 2011), crops growing on agricultural landscapes as well as on flowers of vegetables and medicinal plants. The most important native and introduced fruit crops on which stingless bees forage include mango, cashew, avocado, citrus, coconut, oil palm, shea butter tree, passion fruit, pepper and many others. 264 Tropical forest including east coast forest Savannah including dry savannah and desert x x x x x x x x x x x x x x x x x x x x x x x x x x x x C. Eardley and P. Kwapong Table 18.1 African stingless bees and vegetation type in which they occur Ghanaian common Taxon names of bees Cleptotrigona cubiceps (Friese, 1912) Dactylurina schmidti (Stadelmann, 1895) Dactylurina staudingeri (Gribodo, 1893) “tifuie” Hypotrigona araujoi (Michener, 1959) “mimina” “anihammoa” Hypotrigona gribodoi (Magretti, 1884) “mimina” “anihammoa” Hypotrigona penna Eardley, 2004 “mimina” “anihammoa” Hypotrigona ruspolii (Magretti, 1898) “mimina” “anihammoa” Liotrigona bottegoi (Magretti, 1895) Meliponula (Axestotrigona) cameroonensis (Friese, 1990) Meliponula (Axestotrigona) ferruginea (Lepeletier, 1841) “duro tuntum” Meliponula (Meliplebeia) beccarii (Gribodo, 1879) Meliponula (Meliplebeia) griswoldorum Eardley, 2004 Meliponula (Meliplebeia) lendliana (Friese, 1900) Meliponula (Meliplebeia) nebulata (Smith, 1854) Meliponula (Meliplebeia) ogouensis (Vachal, 1903) Meliponula (Meliplebeia) roubiki Eardley, 2004 Meliponula (Meliponula) bocandei (Spinola, 1853) “duro kokoo” Plebeina hildebrandti (Friese, 1900) 18 Taxonomy as a Tool for Conservation of African Stingless Bees and Their Honey 18.4 265 Challenges to Stingless Bee Survival Kwapong et al. (2010) discuss some of the challenges stingless bees face in Ghana in their booklet on their management and utilisation. Conservation of stingless bees is threatened by loss of habitat from logging, bush fires and wild honey hunting, pests and predators. As most stingless bees are arboreal, when trees are cut the colonies are lost. Bush fires which constantly sweep through tropical forest during dry season burn up trees or meliponary rustic hives harbouring stingless bee colonies. Quite a number of rural communities are aware of stingless bee nests. When harvesting honey they often burn the bees and thereby destroy the colonies. The most important obstacles facing domesticated colonies of stingless bees are predators and pests, notably the small hive beetle Aethina tumida Murray (Coleoptera: Nitidulidae) whose larvae destroy entire colonies. Hive beetle adults live in close association with both honey bees and stingless bees. If hive beetles get an opportunity to oviposit in a colony the eggs hatch and the larvae quickly destroy the colony or cause the bees to abandon the nest. Other predators such as lizards, ants and spiders also threaten stingless bee colonies. 18.5 Justification for Further Taxonomic Research The species name is the main tool to access the existing information on biology. If the taxonomy is inadequate, accurate biological information cannot be disseminated. The increasing demands of the human population result in the need for more food. Many foods result from pollination, and therefore pollinators need to be properly studied (Roubik 1995). As agriculture intensifies, pollination management will become more important. Increased agriculture and urban sprawl will most likely also place more pressure on the natural environment, resulting in a greater need to conserve biodiversity and the habitat of these organisms. Social bees have an advantage in pollination management in that many individuals live in a colony and they can be moved more easily than solitary bees, but similarly the loss of a colony results in the loss of many pollinators. The ability to move pollinators also introduces the risk of moving them to areas where they do not naturally occur. Moving honey bees in South Africa has had some disastrous consequences, such as the production of the pseudoclone (Neumann and Hepburn 2002) which is a social parasite of Apis scutellata Lepeletier. 18.6 Conclusions Through personal observations the authors’ impressions are that in East Africa the importance of stingless bees in traditional medicine is well appreciated and widely used by traditional healers. Here meliponiculture is practiced, but the detailed uses for the honey appear to be trade secrets. In other parts of Africa stingless bee honey 266 C. Eardley and P. Kwapong appears to be less widely used for medical purposes, and if meliponiculture exists it is uncommon. Its wide use for food is mainly through nest robbing. Meliponiculture for agriculture is limited and very recent. Consequently, little is published on stingless bees in Africa. Nevertheless it appears from the limited studies that have recently taken place that stingless bees are an invaluable resource in Africa for biodiversity conservation, agriculture and medicine. A number of scientists throughout the continent are showing an interest in studying these bees and in the future their biology and honey should become better documented. There is clearly a need for an updated taxonomic revision of the African stingless bees, following the recent advance made by Eardley (2004). This need is justified by their apparent importance as pollinators for agriculture and biodiversity conservation. DNA barcoding could be introduced as a complementary tool for separating stingless bee taxa and facilitate the recognition of those morphological characters that are useful in separating species. A study should be undertaken that systematically surveys the stingless bees of Africa to maximise the likelihood of discovering the entire fauna and to document their biogeography. Where possible, host plants and nest architecture should also be documented, which provides a tool for identifying bees in the field. The data should be stored, using relational database technology, in such a way that they will be useful for research including biogeographic analyses, phylogeny and pollination ecology. Finally, before the honey, its composition and uses can be studied, the taxonomy of all living species needs further consideration. Acknowledgements Dr. Janine Kelly and Dr. Claus Rasmussen are thanked for critical reading of the manuscript. Editorial support was kindly provided by P. Vit, S.R.M. 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Journal of the Kansas Entomological Society 84:260–270. Packer L, Sheffield CS, Gibbs J, de Silva N, Best LR, Ascher J, Ayala R, Martins D, Roberts SPM, Tadauchi O, Kuhlmann M, Williams PH, Eardley C, Droege S, Levchenko TV. 2009. The campaign to barcode the bees of the world: Progress, problems, prognosis. pp.178–180 In Yurrita CL, ed. Memorias: VI Congreso Mesoamericano Sobre Abejas Nativas. Universidad de San Carlos de Guatemala – Centro de Estudios Conservacionistas; Guatemala. 368 pp. Portugal-Araújo V. 1955a. Notas sôbre colônias de meliponineos de Angola - África. Dusenia 6:97–114. Portugal-Araújo V. 1955b. Colméias para “abelhas sem ferrão” “Meliponini”. Boletin do Instituto de Angola 7:9–34. Portugal-Araújo V. 1956. La Culture des Mélipones et son Introduction en Europe. Meliponiculture. Rapport presente au XVIeme Congrés International d’Apiculture Vienne Aoǔt 1956. Instituto de Angola; Luanda, Angola. 11 pp. Portugal-Araújo V. 1958. A contribution to the bionomics of Lestrimelitta cubiceps (Hymenoptera, Apidae). Journal of the Kansas Entomological Society 31:203–211. Portugal-Araújo V. 1963. Subterranean nests of two African stingless bees (Hymenoptera: Apidae). Journal of the New York Entomological Society 71:130–141. Portugal-Araújo V, Kerr WE. 1959. A case of sibling species among social bees. Revista Brasileira de Biologia 19:223–228. Rasmussen C, Cameron SA. 2010. Global stingless bee phylogeny supports ancient divergence, vicariance, and long distance dispersal. Biological Journal of the Linnean Society 99: 206–232. Roubik D. 1995. Pollination of Cultivated Plants in the tropics. Vol. 118. FAO Agricultural Service Bulletin; Rome, Italy. 1–196 pp. Ruttner F. 1988. Biogeography and Taxonomy of Honeybees. Springer; Berlin, Germany. 284 pp. Sakagami SF, Zucchi R, Portugal-Araújo V. 1977. Oviposition behavior of an aberrant African stingless bee Meliponula bocandei, with notes on the mechanism and evolution of oviposition behavior in stingless bees [X]. Journal of the Faculty of Science, Hokkaido University, series VI Zoology [Hokkaido, Japan] 20:647–690. Chapter 19 Effects of Human Disturbance and Habitat Fragmentation on Stingless Bees Virginia Meléndez Ramírez, Laura Meneses Calvillo, and Peter G. Kevan 19.1 Introduction Nowadays, deforestation and the consequent loss of natural and semi-natural habitats is one of the most important causes for the decline of biodiversity and key species, such as pollinators, in terrestrial ecosystems around the world (Kevan 1999, 2001; Kevan and Imperatriz-Fonseca 2002; Aizen and Feinsinger 2003; Fahrig 2003; Foley et al. 2005; Brown and Paxton 2009). The rate of world deforestation is decreasing, but still continues at an alarmingly high rate in many countries (FAO 2011). Thus, various human activities, like agriculture, cattle ranching, selective logging, timber harvesting, urbanization, and other human activities that cause deforestation, ultimately contribute to habitat fragmentation. With those processes different habitats are reduced or divided into fragments. The degree of disturbance, coupled with the composition and structure of the original and remaining habitat and their physical characteristics are expected to influence the populations and faunal composition of the bee biota in different ways. One would expect that species restricted to fragmented sites disappear in the short, medium, or long term, depending on the type and extent of disturbance and characteristics of the species. The rate of reduction of population would be affected by dispersal ability and potential for colonization, gene flow (e.g., Allee effect), and changes in the inter-specific interactions (Araújo et al. 2004). At present, insect conservation is based generally on species and specific habitats but ecological data V. Meléndez Ramírez (*) • L. Meneses Calvillo Departamento de Zoología, Campus de Ciencias Biológicas y Agropecuarias, Universidad Autónoma de Yucatán, Mérida, Yucatán, México e-mail: virmelen@uady.mx P.G. Kevan Canadian Pollination Initiative, School of Environmental Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada 269 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_19, © Springer Science+Business Media New York 2013 270 V. Meléndez Ramírez et al. are essential to integrating strategies into the larger landscape scale (dynamic and within interconnected habitats) through which bee conservation can be facilitated in the perspective of global environmental change (Murray et al. 2009). The pollination of plants in tropical regions is mainly carried out by wild bees. Many different species of the social bees called stingless bees comprise ecologically important communities because of their diversity, colony size, and social habits. In fact, social bees are the dominant species in tropical bee communities (Roubik 1992; Nates-Parra et al. 2008) as well as being major pollinators of wild and cultivated plants (Heard 1999; Meléndez et al. 2002; Brosi et al. 2008). The first research on the impact of different perturbations on bees in tropical ecosystems and their fragmentation suggests that stingless bees are affected both in abundance and diversity (Brosi et al. 2007), with some species possibly endangered. For example, in the Yucatán Peninsula, Mexico, stingless bees are evidently suffering decline, as in Melipona beecheii, an economically and culturally important species (Cairns et al. 2005). In this chapter, we first explain the effects of human disturbance and fragmentation on the bee communities and their interactions, as now known in particular for stingless bees and then we suggest strategies for conserving these species for their ecology and economic importance. 19.2 Disturbance, Habitat Fragmentation, and Bee Communities The different human activities like agriculture, livestock management, selective or other timber harvesting, urbanization, and generally all human disturbances that cause deforestation have the ultimate effect of fragmenting habitats. The result is a reduction of continuous habitat into spatially isolated remnants separated from each other by vegetation different from the original. Thus, plant and animal populations are diminished and become spatially isolated. Fragmentation has different effects on various habitat components through time. The total area of fragments may decrease further, the number of fragments may increase as larger tracts become further fragmented, isolation becomes more severe, and fragment shapes become increasingly dominated by straight borders (Bennett and Saunders 2010). Each of those components affects processes within and between resident populations and biotic communities (Fahrig 2003). The effects of human disturbance and fragmentation on bee communities are little studied (Cane 2001; Aizen and Feinsinger 2003; Taki et al. 2007), although it is understood that ecological interactions, such as the mutualisms in pollination, are adversely affected—the occurrence and/or abundance of the mutualistic partners notwithstanding (Bennett and Saunders 2010). Despite current concerns and controversy over the “global pollination crisis” (Kearns et al. 1998; Kevan and Imperatriz-Fonseca 2002; Ghazoul 2005; NASU 2007) there is little information on the responses of bees to land-use change and effects of tropical fragmentation on entire bee communities (Brosi et al. 2008). 19 Effects of Human Disturbance and Habitat Fragmentation on Stingless Bees 271 Tropical bee communities comprise many species, including stingless bees (Meliponini) and Apis spp., which dominate and determine the structure of the communities because of their perennial and large colonies (Roubik 1992; Appanah and Kevan 1995). Bee communities in the Mexican tropics show similar patterns with the composition of species changing between vegetation types and even between cultivated areas (Meléndez et al. 2002; Novelo Rincón et al. 2003). In the on-going current study in a fragmented landscape in this area it was found that bee communities were structurally similar across fragments regardless of size, but species richness and diversity increased with fragment size. It was also found that the greater difference in species composition could be explained by greater degrees of isolation (Meneses et al. 2010). It is important to understand ecologically that species are embedded in complex webs with mutualistic and antagonistic interactions and nowhere are these webs more complex and diverse than in tropical forest ecosystems. Differences in species interactions between ecosystems and regions reflect the particular sets of species present and the nature of the physical environment (Bennett and Saunders 2010). Extinction cascades are particularly likely to occur in degraded landscapes with reduced native vegetation, low connectivity, and intensive land use, especially if keystone species or entire functional groups of species are lost (Fischer and Lindenmayer 2007). In addition, disrupted inter-specific interactions may have exacerbating effects through other trophic levels in ecology, dispersion is a static feature, and dispersal is a process or action (Bennett and Saunders 2010). We now know that mutualistic networks, such as pollination and seed dispersal provide well-defined and predictable patterns of interdependence between species and they are highly heterogeneous and nested (Bascompte and Jordano 2007). In such networks, a greater number of links provides greater resilience of the web through buffering between individual species against disruption of any particular interaction (Okuyama and Holland 2008). Because mutualistic webs are highly asymmetric and nested, adding to the robustness of the networks, when invasive species are inserted, web structure can be altered, with consequences for species persistence. Analysis in temperate forests of the southern Andes and oceanic islands revealed that invasive species became integrated into the networks and did not alter the overall connectivity. However, some links were replaced from generalist native species to super-generalist alien species during invasion so that connectivity among native species declined. These alterations in the structure of pollination networks, due to the dominance of alien species, can leave many native species in a new ecological and evolutionary context (Aizen et al. 2008). Until now, the effect of alien mutualists on the architecture of plant–pollinator webs and fragmentation has not been investigated in the tropics. The stingless bees in the mutualistic networks are mostly super-generalist species and could be displaced by alien species, like Apis mellifera, at the levels of habitat and floral interactions (Pinkus-Rendon et al. 2005; Meléndez 2006; Roubik and Villanueva-Gutiérrez 2009). In the context of island biogeography, it is suggested that the number of links of species present in pollination webs increases twice as fast, in comparison to species richness when area increases, as a consequence of decreasing dominance 272 V. Meléndez Ramírez et al. (i.e., increasing evenness) of any particular interaction. This could indicate a faster loss of interaction links than of species as isolated habitats become reduced, and also has implications for conservation (Sabatino et al. 2010). In addition, theoretically one would expect negative effects of fragmentation on wild bee species to arise and cause profound structural and functional changes in plant communities (e.g., Aizen and Feinsinger 1994a; Steffan-Dewenter et al. 2006; Taki et al. 2007). Indeed, recent work has shown that pollination limitation results from the interruption of some plant–pollinator interactions in fragmented areas with high plant diversity, such as in the tropics (Brosi et al. 2008). 19.3 How Can Habitat Fragmentation Affect Stingless Bee Biology? Stingless bees are the most diverse group (over 500 species worldwide) of all eusocial bees. They found in tropical and southern subtropical areas throughout the world (Roubik 2006; Michener 2007). They have a particular distinctiveness that must be considered to understand how human disturbance and fragmentation could impact them. Stingless bees occur in colonies from a few dozen to one hundred thousand or more workers. They live in permanent colonies, being the only highly eusocial bees together with Apis spp. (Michener 2007). Different species have different densities of nests in given landscapes and also differ in their capacities of flight and strategies of foraging, as shown in several book chapters herein. 19.3.1 Stingless Bee Nesting Most species of stingless bees nest in cavities in live trees, others nest in the ground and some establish within nests of termites or ants (Salmah et al. 1990; Roubik 2006; Michener 2007). Some trees are used by several species, and sometimes several can coexist. Stingless bee nesting in natural forest has been studied in various tropical countries (Kajobe and Roubik 2006). They occur in high numbers in Borneo, Thailand, and Brazil (840, 115, and 1,500, respectively) in small areas of the moist forests there (2.8, 4, and 11.3 ha, respectively). Deforestation and fragmentation cannot but have a negative effect on species richness, abundance, and dispersal. Given that nesting resources are limited the negative effects of deforestation cannot be denied even though there is little numerical evidence to prove the scale, frequency, or severity (Roulston and Goodell 2011). In Sabah, Malaysia, the nest density of stingless bees in undisturbed and loggedover dipterocarp forests was evaluated (Eltz et al. 2002). It was generally high in the 19 Effects of Human Disturbance and Habitat Fragmentation on Stingless Bees 273 fragments of primary forest (mean 8.4 nests/ha) but extremely low nest densities (0.5–0.7 nests/ha) in newly logged areas reflected direct impact of availability of food. According to Roulston and Goodell (2011), there is strong evidence that food availability regulates bee populations. Moreover, it has been suggested that some species appear not to be affected by disturbances. Batista et al. (2003) find 16 species of stingless bees but Tetragonisca angustula is the most abundant, occurring in all habitats (disturbed and undisturbed), with 31% of all nests. The ecological plasticity of this species is associated with aggressive patrolling of potential nests cavities, as documented in numerous studies (Roulston and Goodell 2011) that serve to explain its capacity to withstand perturbations. However, some species of stingless bees are restricted to forests, at least for nesting. There, nests and/or individual bees in deforested habitats may be prone to greater incidences of diseases, parasites, or predation (Brosi et al. 2007). New research could identify the main factors driving interactions that determine the nesting sites of each species and those could include human activities in the mosaic of tropical environments. 19.3.2 Stingless Bees and Potential Flight Ranges Another important issue is the potential flight ranges of bees in fragmented areas. When a habitat is fragmented dispersal and potential for colonization is often reduced, especially as fragments become more and more isolated by degraded and highly modified areas between them. The maximum flight ranges in bees, including stingless bees, are a function of body size especially with wing dimensions (Table 19.1). Because they are central place foragers they occupy a maximum effective space proportional to this, thus presenting strong constraint on local populations restricted to forest fragments (Araújo et al. 2004). From the foregoing, it can be predicted that the risk of extinction is greater for smaller stingless bees than for larger ones. For example, colonies of Plebeia droryana (1.35 mm, maximum length of the forewing) could be effectively isolated if inter-fragment distances were greater than 600 m. In contrast, larger species, such as Melipona compressipes (3.25 mm) and Melipona quadrifasciata (2.90 mm), could be effectively isolated if forest fragments were greater than 2 km apart (Table 19.1). In theory even though larger species have a greater capacity to migrate between forest fragments their doing so but would also depend on other factors (e.g., resources requirements). Additionally, swarming in stingless bees could also act as a limiting factor in nest dispersion because new colonies of stingless bees depend strongly on the parental nest which generally provides the new nest with food and material. Thus, long-distance dispersal by individual reproductive or by swarms is impossible (Michener 2007), unlike the situation for Apis spp. However, the effects of fragmentation in this context have not been investigated. 274 V. Meléndez Ramírez et al. Table 19.1 Bee species and flight distances (according to Araújo et al. 2004) Bee size Flight distances Small bees Nannotrigona testaceicornis (Lepeletier, 1836) Plebeia droryana (Friese, 1900)a Plebeia poecilochroa Moure and Camargo, 1993 Scaura latitarsis (Friese, 1900) Tetragonisca angustula (Latreille, 1811) Trigona sipinipes (Frabricius, 1793)a Medium-sized species bees Cephalotrigona capitata (Smith, 1854)b Frieseomelitta varia (Lepeletier, 1836) Geotrigona inusitata Moure and Camargo, 1992 Partamona cupira (Smith, 1863) Scaptotrigona postica (Latreille, 1807) Trigona hypogea Silvestri, 1902 Trigona recursa Smith, 1863 Larger bees Melipona bicolor Lepeletier, 1836 Melipona compressipes (Fabricius, 1804)a Melipona marginata Lepeletier, 1836c Melipona quadrifasciata Lepeletier, 1836a Melipona scutellaris Latreille, 1811 Maximal flight distances ranged From 621 to 951 m 540 m From 621 to 951 m 840 m 1,650 m From 1,159 to 1,710 m Greater than 2 km 2,470 m 800 m 2,000 m Greater than 2 km With the fitted linear regression, maximum flight distance = 1,383.333 ± 645.185 (generalized wing size) ± error, they estimate the maximum flight distance for 12 species of stingless bees from their generalized wing size. Each estimated value represents a mean expectation of the maximum flight distance for each species with an associated error a Using mark-recapture method: Kerr (1987), bRoubik and Aluja (1983), cWille (1983) 19.3.3 Stingless Bee Foraging Habitat fragmentation could affect foraging by stingless bees, the colonies of which are largely self-organized. Some species (solitary foragers) trust individual forager decision making in the field. Other species belong to the obligate foragers group that relies largely on collective decision making, with foragers following each other and even communicating in the nest. The species-specific balance between individual and collective decision-making determines the foraging niche of each species. The coexistence of multiple species with different foraging strategies indicates that the various strategies are complementary in as to how food is extracted from their ever-changing habitat (Beismeijer and Slaa 2004). Within a community of stingless bees the species overlap extensively in foraging range (e.g., Eltz et al. 2002; Slaa 2003), and inter-specific encounters are common. Reactions vary widely from avoidance to attack, depending on the species combination. Aggressive species are sometimes attracted to a heterospecific, generally leading to 19 Effects of Human Disturbance and Habitat Fragmentation on Stingless Bees 275 the departure of the latter. However, avoidance seems more common, and is accurately predicted by relative body size of the two species. Thus, unaggressive species avoid aggressive species and smaller species generally avoid larger species (Slaa et al. 2003). The complex interactions between small and large, aggressive and unaggressive species and between species with similar sizes and behaviors suggest that deforestation and fragmentation change the insect–plant and insect–insect interactions with negative results for both species diversity and functional diversity, although some species could be more favored than others. 19.3.4 Stingless Bees, Disturbance and Habitat Fragmentation In the tropics few studies have investigated factors like deforestation, logging and shifting cultivations, fragmentation, and their relationship with diversity and abundance of stingless bees. Early studies in Sumatra indicate that species diversity and abundance of stingless bees decreased along anthropogenic disturbance gradients in secondary forests and at higher altitudes (Salmah et al. 1990). In Rondônia, species richness in Melipona increased with increasing forest cover and proximity to forests and adverse effects of deforestation were detectable, despite the fact that significant areas of tropical forest cover remained (Brown and Albrecht 2001). Samejima et al. (2004) in Sarawak, Malaysia, reported that for stingless bees nest density is positively related to the density of large trees (>50 cm DBH) and that some species were abundant in the primary forests, whereas others in disturbed forests. Nevertheless, in this study, species richness was not affected by human disturbance, but the relative abundance of the species may have been affected both by nest site availability and food resource limitations. Thus, it is suggested that changes in the composition of pollinator community may also affect tree community composition in the long term. In Costa Rica, Brosi et al. (2007) studied the effects of distance to forest, tree management, and floral resources on bee communities. They found no clear differences in bee diversity or abundance regarding pasture management or floral resources. However, the bee community composition was evidently different at forest edges than in deforested countryside only a few hundred meters away. The sites at the edge of a relatively large forest contained a much higher proportion of social stingless bees and a relatively low proportion of Apis, whereas non-edge sites showed the opposite pattern. The eusocial bee fauna of the study area comprised principally stingless bees and honey bees together; they are distinctive in quickly recruiting foragers to high-quality resources. Thus, it is necessary to emphasize the importance of the diverse assemblage of native stingless bees that cover a wide range of body sizes and flower foraging behavior not found in honey bees. Bee community responses to forest fragment size, shape, isolation, and landscape contexts including pastures adjacent are examined by Brosi et al. (2008) in southern Costa Rica. This study suggests no effects of forest variables on bee diversity and abundance, although strong changes in bee community composition are 276 V. Meléndez Ramírez et al. noteworthy. In particular, tree-nesting stingless bees are associated with larger fragments, smaller edge: area ratios and greater proportions of forest surrounding sample points. Community composition is also markedly different between forests and pastures, despite their spatial proximity. In forests, even in the smallest patches, stingless bees comprise a large proportion of bee communities. On the other hand, in the Yucatán Peninsula, particularly in Quintana Roo, Mexico, changes in the communities of stingless bees illustrate the effects of humaninduced ecosystem disturbance. The community with the greatest anthropogenic disturbance had lower overall species richness of stingless bees and the highest degree of dominance of the Africanized honey bee (A. mellifera), while the area with the most ecosystem conserved had the highest diversity of stingless bees, though A. mellifera was still the dominant species where in general bee numbers are lowest, richness of stingless bee species and evenness were higher in ecosystem conserved than in the more disturbed sites (Cairns et al. 2005). Similarly, Roubik (2009) found the greater abundance of honey bees in disturbed sites and lower abundance in the forest in neotropical areas. Although some changes in habitat are directly perceptible after fragmentation (e.g., shifts in habitat pattern, forest structure and composition at edges, changes in population sizes) other changes may emerge only after a long time. For example, genetically related changes on populations and lost or extinction of species often take years to become evident. In stingless bees, as in many organisms, genetic drift is a process frequently exacerbated by the isolation of small, local populations. For example, populations Melipona spp. are highly susceptible to the effects of genetic drift. In M. scutellaris within a population based on extended breeding from a small number of founder colonies there was a great reduction in the number of alleles even though with low genetic variability the population could be maintained for nearly 10 years (Alves et al. 2011). Thus, in some species of stingless bees, breeding from a small reserve of colonies may have less drastic consequences than previously assumed. Additional studies of genetic variability in other species are urgently needed to support strategies for the conservation of stingless bees. Recently, an overview of studies in tropical ecosystems on how bees are affected by human disturbances (Winfree et al. 2009) indicated that stingless bees and solitary bees are the most affected (Table 19.2). 19.4 Conservation and Importance of Stingless Bees Despite the fact that there are few studies in the tropics, all indicate that the local bee communities are negatively affected by human disturbance and fragment size. For stingless bees conservation is essential to identify that the sizes of fragment from medium to large are those in which that maintenance of the greatest number of susceptible species and in this way are adequate to design conservation strategies (Meneses et al. 2010). Also, it is important to consider the establishment of corridors to improve the connectivity between fragments in any conservation strategy for reducing the impacts of fragmentation on wild bee community (Bennett and Saunders 19 Effects of Human Disturbance and Habitat Fragmentation on Stingless Bees 277 Table 19.2 Effect of anthropogenic disturbance on tropical bees (from data base of Winfree et al. 2009) BTx A/R Ea DT BT BS Country Reference Apis A + F Tsdbf Social Argentina Aguilar (2005) A − F Tsmbf Social Argentina Chacoff and Aizen (2006) A − F Tsmbf Social Costa Rica Ricketts (2004) A + F Tsdbf Social Argentina Aizen and Feinsinger (1994b) A − F Tsgssh Social Australia Blanche et al. (2006) Bombus A − F Tsdbf Social Argentina Aguilar (2005) Beeb A − F Tsdbf Solitary Argentina Aguilar (2005) A − F Tsgssh Solitary Australia Blanche et al. (2006) A − F Tsmbf Solitary Brazil Becker et al. (1991) A − F Tsmbf Solitary Brazil Powell and Powell (1987) R + F Tsmbf Solitary Brazil Becker et al. (1991) R − F Tsmbf Solitary Indonesia Klein et al. (2006) A − F Tsmbf Social Argentina Chacoff and Aizen (2006) A + F Tsmbf Social Malaysia Eltz et al. (2002) A − F Tsgssh Social Argentina Blanche et al. (2006) A − Lg Tsmbf Social Malaysia Eltz et al. (2002) R + F Tsmbf Social Malaysia Eltz et al. (2002) R − Lg Tsmbf Social Malaysia Eltz et al. (2002) Beec A − F Tsmbf All Costa Rica Ricketts (2004) A + F Tsmbf All Indonesia Klein et al. (2003a) A − F Tsdbf All Argentina Aizen and Feinsinger (1994b) R − F Tsmbf All Argentina Aguilar (2005) R − F Tsmbf All Costa Rica Ricketts (2004) R − F Tsmbf All Indonesia Klein et al. (2003a) R − F Tsdbf All Argentina Aizen and Feinsinger (1994b) R + F Tsgssh All Australia Blanche et al. (2006) R + Ag Tsmbf Solitary Indonesia Klein et al. (2002) R − F Tsmbf Social Indonesia Klein et al. (2002) R − F Tsmbf Social Brazil Brown and Albrecht (2001) Beed A − F Tsmbf All Indonesia Klein et al. (2003b) R − F Tsmbf All Indonesia Klein et al. (2003b) BTx = bee taxon, A = abundance/R = species richness, Ea = effect, DT = disturbance type, BT = biome type, BS = bee sociality, Ag = agriculture, F = fragmentation, Lg = logging, Tsdbf = tropical and subtropical dry broadleaf forests, Tsmbf = tropical and subtropical moist broadleaf forests, Tsgssh = tropical and subtropical grasslands, savannahs, and scrublands a The effect was measured by Hedge’s unbiased standardized mean difference (Hedge’s d ). Positive values of the effect size (d) imply positive effects of anthropogenic disturbance on bee abundance or richness, whereas negative (d) values imply negative effects b Non-Apis and non-Bombus, cNon-Apis, dNon-Bombus 2010; Meneses et al. 2010). Although bee populations are known to fluctuate temporally (e.g., Roubik 2001; Roubik and Wolda 2001), they need resources throughout the year. The strong correlation between body size and flight range in stingless bees could be useful for developing strategies to conserve tropical bee diversity (Araújo et al. 2004). 278 V. Meléndez Ramírez et al. In addition, the conservation of these bees requires a continual assessing of their genetic diversity where effects of genetic drift (Allee effect) could be operating, as in fragmented areas. A simulation model has been developed to determine the number of colonies needed to maintain a certain number of sex alleles in a population, thereby providing useful guidelines for stingless bee breeding and conservation (Alves et al. 2011). The decline of stingless bees in the central area Quintana Roo, Mexico, particularly for the once-economically important M. beecheii suggests that both habitat change and increased competition with an invasive species (A. mellifera) have contributed to this decline. Moreover, selective logging affects several important nesting tree species for stingless bees in general, and other changes in the vegetation mosaic may also have contributed to the decline of M. beecheii (Cairns et al. 2005). Thus, habitat management strategies are needed. Conserving stingless bees require maintenance of natural forest because distance between fragments and overall isolation probably explains the presence of a large proportion of rare species restricted to only one fragment (Meneses et al. 2010), but the habitat requirements of wild bees are largely unknown and need investigation. The potential of native habitat to provide bee pollination services to agriculture is particularly most important in the neotropics, where also stingless bees are the principal pollinators (Kevan and Imperatriz-Fonseca 2002). Although beekeeping is promoted as an agroeconomic activity, the honey bees (A. mellifera) used cause the displacement of stingless bees from resources floral (Pinkus-Rendon et al. 2005; Roubik and Villanueva-Gutiérrez 2009). For local agriculture, the synergistic effects of combinations of species of pollinating bees are becoming recognized (Meléndez et al. 2002). Even in a crop as important and well known as coffee wild bee diversity and abundance in association with forest patches have been correlated with larger crops (Ricketts et al. 2004, Klein et al. 2003a). In addition, stingless bees have proven efficient pollinators in crops in greenhouses (e.g., Cauich et al. 2006; Palma et al. 2008a, b) and their domestication has great potential (Meléndez et al. 2004). Finally, key species, such as stingless bees, in the tropics are required for ecosystem function and ecosystem health, floral resources and nesting sites (i.e., as trees of sufficient thickness) will sustain the bees and their pollination interactions with the wild biodiversity of flowering plants and crops. References Aguilar R. 2005. Effects of forest fragmentation on the reproductive success of native plant species from the Chaco Serrano forest of Central Argentina. PhD thesis. National University of Cordoba, Cordoba, Argentina. 169 pp. Aizen MA, Feinsinger P. 1994a. Forest fragmentation, pollination and plant reproduction in Chaco dry forest, Argentina. Ecology 75:330–351. Aizen MA, Feinsinger P. 1994b. Habitat fragmentation, native insect pollinators, and feral honey bees in Argentine “chaco serrano”. Ecological Applications 4:378–392. 19 Effects of Human Disturbance and Habitat Fragmentation on Stingless Bees 279 Aizen MA, Feinsinger P. 2003. Bees not to be? Responses of insect pollinator faunas and flower pollination to habitat fragmentation. pp. 111–129. In Bradshaw GA, Marquet PA, eds. How landscapes change. Springer-Verlag, Berlin, Germany. 384 pp. Aizen MA, Morales CL, Morales JM. 2008. Invasive mutualists erode native pollination webs. PLoS Biology 6:1–8. Appanah S, Kevan PG. 1995. Bees and the natural ecosystem. pp. 19–26. In: Kevan PG. ed. The Asiatic Hive Bee: Apiculture, Biology and Role in Sustainable Development in Tropical and Subtropical Asia. Enviroquest Ltd., Cambridge. 315 pp. Araújo ED, Costa M, Chaud-Netto J, Fowler HG. 2004. Body size and flight distance in stingless bees (Hymenoptera: meliponini): inference of flight range and possible ecological implications. 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Hokkaido University Press; Sapporo, Japan. 274 pp. Samejima H, Marzuki M, Nagamitsu T, Nakasizuka T. 2004. The effects of human disturbance on a stingless bee community in a tropical rainforest. Biological Conservation 120:577–587. Slaa EJ. 2003. Foraging ecology of stingless bees: from individual behaviour to community ecology. PhD thesis, Department of Behavioural Biology, Utrecht University; Utrecht, Netherlands. 181 pp. Slaa EJ, Wassenberg J, Biesmeijer JC. 2003. The use of field-based social information in eusocial foragers: local enhancement among nestmates and heterospecifics in stingless bees. Ecology Entomology 28:369–379. 282 V. Meléndez Ramírez et al. Steffan-Dewenter I, Klein AM, Gaebele V, Alfert T, Tschrantke T. 2006. Bee diversity and plantpollinator interation in fragmented landscape. pp. 387–407. In Waser N, Ollerton J. Plant pollinator interactions. From Specialization to Generalization. University of Chicago Press; Chicago, USA. 488 pp. Taki H, Kevan PG, Ascher JS. 2007. Landscape effects of forest loss in a pollination system. Landscape Ecology 22:1574–1587. Wille A. 1983. Biology of the stingless bees. Annual Review of Entomology 28:41–64. Winfree R, Aguilar R, Vázquez DP, LeBuhn G, Aizen MA. 2009. A meta-analysis of bees responses to anthropogenic disturbance. Ecology 90:2068–2076. Part III What Plants Are Used by the Stingless Bees? Chapter 20 Palynology Serving the Stingless Bees Ortrud Monika Barth 20.1 Introduction Like the honey bees, stingless bees collect nectar, pollen grains, and resins from a large group of plant species. Palynological analysis of several bee products, such as honey, bee pollen, bee bread (brood provisions), geopropolis (resin collected by stingless bees), and royal jelly, allows one to identify the associated pollen species, and to understand composition of vegetation used by the bees. 20.2 20.2.1 Bees, Vegetation, and Pollen Grains The Bees The stingless bees (Meliponini) and honey bees (Apini) both are pollinators of native and exotic plant species and harvest honey and pollen appreciated by humans—and also by predatory animals. It is of interest to know more about bee food preference and floral choice, and also of economic interest. Pollen grains obtained directly from bees or taken from nests and colonies indicate foraging activities during a day, a week, a month, or even a year. These data deserve detailed investigation and evaluation in order to assess quality or quantity of bee products and to exploit the bee preferences for flowering plant and crop pollination. Pollen analysis is a refined scientific approach for investigating these subjects. O.M. Barth (*) Fiocruz and Department of Botany, Instituto Oswaldo Cruz, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil e-mail: monikabarth@gmail.com 285 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_20, © Springer Science+Business Media New York 2013 286 O.M. Barth Two methods of pollen analysis are normally pursued. Physicochemical methods provide information about honey and pollen grain composition, including the moisture, pH values, acidity, ash, sugars, proteins, and more. On the other hand, palynological methods detect where bees obtain nectar and pollen, and some other materials such as resin. Stingless bee honey properties depend upon the bee species. The bees consume nitrogen compounds of the plant phloem, while sugars and minerals are maintained in the processed nectar stored as concentrated sugar, in honey. Their honey presents a higher water content and more saccharose and mineral elements than honey of honey bees. On the other hand, the quality of Apis mellifera honeys depends upon the plant resources, and the honeys often have lower concentration of water, sucrose, and minerals (Bazlen 2000). 20.2.2 Vegetation to Benefit Bees Stingless bees occur in several continents, mainly in tropical and subtropical regions, and are adapted to different types of vegetation including forests, savannas, fields, marshes, and mountains. Honey and harvested pollen change in composition depending upon fluctuating plant species flowering. In South America, Brazil is of continental size and its types of vegetation (Veloso et al. 1991) change across the landscape. The majority of tropical plants depend upon pollination activities of insects, birds, and bats, and the stingless bees play a major role (Roubik 1978, 1980). Absy and Kerr (1977), using pollen analysis of honey, began the study of stingless bee floral visitation in the Amazon region, which comprises different forest types, savannas and riversides, as well as human-disturbed landscapes and urban areas. Absy and collaborators pursue observations on stingless bees over several years (Oliveira et al. 2009), demonstrating that a great variability of pollen resources, not commonly dominated by a unique plant species, are used by the bee species studied. Furthermore, the trophic niches of stingless bee species frequently overlap (Silva et al. 2004). Similar investigations in the Brazilian Northeast region concentrate in the state of Bahia, including semiarid localities. A sequence of investigations for 10 years by Bazlen (2000) and Carvalho et al. (2001) analyzed honey samples of Meliponini. Although representing a diversity of flowers, the honey revealed a significant monofloral element. The source of pollen loads (Ramalho et al. 2007) and residual nest pollen (Dórea et al. 2010) is known from different localities during different years and months in this region. Palynological investigation of pollen loads and honey of stingless bees in the Brazilian Southeast region has a long tradition (Barth 2004), starting with honey analysis by Iwama and Melhem (1979), and more recently with the analysis of forage pollen (Hilario and Imperatriz-Fonseca 2009) and pollen contained in storage pots 20 Palynology Serving the Stingless Bees 287 (Malagodi-Braga and Kleinert 2009). The last investigation compares the results obtained from corbicular loads on returning foragers to the pollen in storage pots. Larval food pollen analysis provides additional information about southern Brazil in Santa Catarina (Cortopassi-Laurino et al. 2009). Human activity degrades natural vegetation and is readily revealed by pollen analysis of honey and bee corbicular loads. Pot-honey and pot-pollen of Meliponini outside Brazil have also been investigated. Recently, Flores and Sánchez (2010) obtained the first results for Tetragonisca angustula from Salta, Argentina, showing some monofloral honey. Freitas et al. (2010) compared the food resources of Meliponini in different regions of Brazil and Venezuela using pollen analysis of honey samples. 20.2.3 Pollen Grains What is the difference between pollen analysis and palynological analysis? When considering pollen grains in honey, pollen loads, bee bread, and propolis/geopropolis, the study involves pollen analysis. On the other hand, when work considers additional structures found among honey, pollen, and nest products, such as bacteria, spores, and fungal hyphae, yeast, oil, wood, plant hairs (trichomes), and other materials, it is a palynological analysis. This enables us to make a better interpretation of the phytogeographic origin, cleanliness, bee storage, and manipulation of these products (Barth 1989). Research efforts consider in general the pollen grains alone. Distinction between pollen grains of nectariferous, polleniferous, and anemophilous plant species must be made to obtain a valid result and diagnosis. Super- and sub-representation of pollen grains of some plant species, abortive pollen grains (e.g., Citrus Rutaceae), and amyloplasts (e.g., Zea mays Poaceae), protoplasts (e.g., Persea Lauraceae), and gemma (e.g., Bauhinia Fabaceae, Caesalpinioideae) inside the palynological preparations must be recognized, and considered for diagnosis. Pollen morphology overlaps frequently between plant species and genera, so an exact identification cannot be made. For this reason, the usual technical terminology may relate to a pollen type at family, genus, or species level. A detailed knowledge of local plants visited by bees, however, may allow recognition of the pollen and nectar sources. The knowledge of pollen morphology is most often an accurate instrument with which to analyze bee products. Several publications illustrating pollen and spore morphology and terminology are available. Based upon pollen structure definition in Erdtman (1952) and Faegri and Iversen (1950), ordinary terms of pollen morphology are translated and illustrated in Portuguese by Barth (1965, 1975) and Barth and Melhem (1988). The standard English version today follows Punt et al. (2007). 288 20.3 O.M. Barth Palynological Analysis of Honey Honey of Meliponini stored in pots, when compared with honey of A. mellifera stored in combs, is more liquid, presenting a higher degree of water, sucrose, hydroxymethylfurfural (HMF), and ash, as noted in the literature, considering several phytogeographical regions of honey production. These properties depend mainly on the bee species. Melissopalynological studies of stingless bee honey are rare, limited to Melipona seminigra merrillae and Melipona rufiventris paraensis (Absy and Kerr 1977), Melipona compressipes, Melipona favosa, Melipona trinitatis, Frieseomelitta nigra, Frieseomelitta sp. aff. varia, Plebeia sp., Scaptotrigona sp. aff. depilis, Scaura latitarsis and T. angustula (Vit and Ricciardelli D’Albore 1994), Melipona scutellaris (Carvalho et al. 2001), Melipona mandacaia (Alves et al. 2006), T. angustula (Flores and Sánchez 2010), and M. favosa (Vit et al. 2012). Specific information about the use of Eucalyptus species by the bees was detailed in the thesis of Bazlen (2000). Stingless bee honey can be divided into two groups. One shows dominance of a unique pollen type (more than 45% of all counted nectariferous pollen grains). Such monofloral (or unifloral) honey maintains similar physicochemical and sensory properties, while heterofloral honey varies in its characteristics (Ferreira et al. 2007, 2009). Visiting flowers to collect nectar, Meliponini were considered sometimes to be specialists, producing monofloral honeys, and sometimes generalists, producing heterofloral honeys. The observed results depend upon several factors, including the blooming plant species and the available number of flowers, the content of nectar sugars and water, and the weather, as well as the bee species. Bazlen (2000) studied 92 meliponine honey samples, from the Brazilian states of Bahia, São Paulo, and Rio Grande do Sul, and considered physicochemical and palynological characteristics. Seventy-five samples (81.5%) comprised monofloral honeys. The main dominant pollen type was of Myrtaceae (without species or genus identification) in 27 samples (36%), followed by Fabaceae, Mimosoideae in 11 samples (14.5%) which mostly lacks nectar. Altogether 12 plant families were responsible for these monofloral honeys. Pollen analysis of bimonthly collected honeys of M. scutellaris in 15 colonies at Bahia State is presented by Carvalho et al. (2001). Eucalyptus was the dominant pollen type in all samples, except one of Psidium. In parallel, pollen analysis of 11 honey samples of M. mandacaia (Alves et al. 2006), obtained in a semiarid region at the state of Bahia, reveals that Piptadenia rigida (Fabaceae, Mimosoideae) is the dominant nectariferous pollen in six samples, and Ricinus communis, an anemophilous plant species, in one sample. Piptadenia moniliformis pollen grains were dominant inside one sample (97.6%) from Paraguassu, Bahia (Junior and Santos 2003), and a species of Euphorbiaceae (51%) in Trigona spinipes honey at São Cristóvão, Sergipe State (Oliveira et al. 2008). T. angustula was considered to be a generalist bee in foraging choice, although four honey samples from a total of eight presented dominant pollen types, two of Mitracarpus (Rubiaceae), one of Ziziphus joazeiro (Rhamnaceae), and one of Zornia (Fabaceae, Faboideae), in an arid region of Bahia State (Novais et al. 2006). 20 Palynology Serving the Stingless Bees 289 Table 20.1 Original and corrected pollen percentages >3%, in Amazonian Melipona honey Samples Families Pollen types Common names Original % Corrected % Amazonas 1 Brassicaceae Brassica mostarda – 3.3 Fabaceae – – 3.3 10.0 Gesneriaceae – – 4.9 15.1 Melastomataceae – – 66.8 – Solanaceae Solanum lobeira 22.7 69.8 Amazonas 2 Lythraceae Cuphea sete-sangrias 5.8 18.9 Melastomataceae – – 52.1 – Fabaceae, Mimosa bracatinga 17.3 – Mimosoideae scabrella Solanaceae Solanum lobeira 24.8 81.1 Amazonas 3 Anacardiaceae – – 4.1 26.4 Burseraceae Protium almecegueira 9.8 62.6 Fabaceae, Crudia jutairana – 3.3 Caesalpinioideae Gesneriaceae – – – 3.3 Melastomataceae – – 81.2 – Fabaceae, Mimosa bracatinga 3.1 – Mimosoideae scabrella – Non-identified pollen types, unknown common names, and frequency below 3%, bold = dominant pollen type, frequency >45% Three honey samples of Melipona obtained in the Brazilian Amazonas region (Table 20.1) were analyzed by Freitas et al. (2010). All of them contained dominant pollen grains. Two samples of M. compressipes manaosensis and M. seminigra, obtained at the Manacapuru region, were from a nectariferous Solanaceae; this result was based upon a correction of the percentages of counted pollen grains, when those of polleniferous plants (Melastomataceae and Mimosa scabrella pollen type) were excluded. The third sample of M. seminigra, obtained in Porangaba, was from Protium (Burseraceae). A similar result was obtained by Absy et al. (1980) in the Amazon region of Manaus. Two of the four honey samples of Melipona obtained at Paraíba State (Freitas et al. 2010) showed a dominant pollen type of Crotalaria (Fabaceae, Faboideae) that comprises several species and genera of the Fabaceae presenting the same pollen morphology. One honey sample of T. angustula, obtained at the region of Içara, Santa Catarina State, presents a dominant pollen type of Hovenia dulcis (Rhamnaceae), analyzed by Freitas et al. (2010). 20.4 Palynological Analysis of Bee Pollen Pollen harvested by Meliponini is known from recent work in the Brazilian states of Amazonas and São Paulo. Different methodologies of pollen load collection and of palynological analyses were utilized, and are not readily compared. A standard 290 O.M. Barth methodology of pollen load analysis was proposed recently by Barth et al. (2010). In summary, two grams of bee pollen are washed with ethanol, homogenized, and five hundred or more pollen grains of one drop of this well-mixed suspension are considered. It was demonstrated that pollen grain color is not plant species/genus/ family specific (Barth et al. 2009). Papers by Marques-Souza et al. (2002, 2007) analyzed bee pollen in Amazonas State, finding Stryphnodendron guianense, Schefflera morototoni, Miconia myriantha, and Myrcia amazonica to be the most frequent. Oliveira et al. (2009) reported no dominance of any pollen type in the same area. Pollen loads taken off from M. scutellaris at several localities of Bahia State (Salvador, Cruz das Almas, Alagoinhas) are investigated and compared with those obtained from Apis (Ramalho et al. 2007). Pollen types were recognized at family level only, and no dominance signalized, but an overlapping of resource plants is noted. Chemical composition of pollen loads study was based upon pollen identification, resulting in a predominance of yellow-colored loads of Mimosa gemmulata (98.95%) and bright colored loads of a Fabaceae (Silva et al. 2006), also in Bahia. Dórea et al. (2010) investigate pollen residues in nests of Centris tarsata at the Canudos Biological Station, a semiarid locality in Bahia State. The caesalpinoid Fabaceae Chamaecrista ramosa (46.5%) was the unique dominant taxon among the 31 pollen types identified. Pollen of pot samples investigated by Oliveira et al. (2008) at the locality of São Cristóvão, Sergipe State, shows a dominance of Celastraceae (46.9%) pollen grains. Pollen resources of Melipona are largely known from studies in the Brazilian state of São Paulo, mainly inside the campus of the State University, São Paulo city. Most recently, Malagodi-Braga and Kleinert (2009) present studies in the same locality, and emphasize the importance of Eucalyptus pollen nearly throughout the year, and of isolated plant pollen for alternative resources. Floral origin of pollen harvested by Plebeia saiqui inside pots was investigated by Pick and Blochtein (2002) during 1 year in São Francisco de Paula, Rio Grande do Sul state. No dominant plant species could be detected, but species of the Asteraceae were prevalent. 20.5 Palynological Analysis of Geopropolis (Meliponine Propolis) Plant exudates, resins, waxes, plant tissues, and trichomes, mixed with more or less 5% pollen grains, were the main ingredients of honey bee propolis (Barth 1998; Barth et al. 1999). On the other hand, meliponine geopropolis does not contain trichomes, but in addition these bees mix resins and waxes with earth, and frequently collect mud or clay, small pebbles, seeds, and sometimes sand. Spores and fungal hyphae, soot, and amorphous organic material are commonly present (Fig. 20.1 and Table 20.2). Further, the pollen grain spectrum reflects the vegetation of the phytogeographical regions or localities (Barth 2006; Barth and Luz 2003). 291 Fig. 20.1 Structured elements of geopropolis sediments. (a) Eucalyptus (Myrtaceae) pollen grain inside a complex structured sediment of geopropolis (Meliponinae) before acetolysis treatment. (b) Glandular trichomes of propolis (Apis) before acetolysis treatment (for comparison with a geopropolis sediment). (c) Geopropolis sediment after acetolysis treatment, polarized illumination. (d–v) Pollen grains; (d and e) tetrads of M. scabrella pollen type; (f) Piperaceae; (g and h) Schinus (Anacardiaceae); (i and j) Eucalyptus; (k–n) Melastomataceae; (o) Protium (Burseraceae); (p and q) Solanum (Solanaceae) pollen type; (r and s) Myrcia (Myrtaceae) pollen type; (t) Cecropia (Cecropiaceae); (u and v) Cyperaceae. All figures of 1,000× magnification, except figures 1–3 of nearly half of this magnification. Photos: O.M. Barth 292 O.M. Barth Table 20.2 Evaluation of nest entrance geopropolis of six sediment constituents, except pollen grains, after acetolysis Organic Sandy Bee species material fragments Lestrimellita cf. limao + ++ (crystals) Trigona recursa ++ + (crystals) Tetragonisca angustula + +++ (sandy powder) Melipona quadrifasciata +++ + (crystals) Nannotrigona testaceicornis + – Frieseomelitta varia + – bee species considering frequency of Spores and hyphae of fungi + + ++ Soot (burned organic material) + + +++ + ++ + + + + (+++) Very frequent, (++) frequent, (+) few, (–) not detected (Barth 2006) Dominant pollen types were Eucalyptus (Myrtaceae) in samples from São Paulo state (Barth 2006), Schinus (Anacardiaceae) in one sample of Minas Gerais, and Myrcia (Myrtaceae) in samples of several states (Barth and Luz 2003). Anemophilous and polleniferous pollen, as of Cecropia (Urticaceae), M. scabrella (Fabaceae, Mimosoideae) pollen type, and Piper (Piperaceae), were sometimes well represented in geopropolis samples. 20.6 Conclusions Summarizing the actual knowledge about pollen analysis of honey, pollen loads, and harvested pollen of the stingless bees, Meliponini, in Brazil, and considering the great size of this undertaking in such a remarkably large tropical country, scant data are available on the plant species offering the bees nectar and pollen. Most investigations recognize only the plant family. Detailed field study, followed by standard laboratory processing of samples and phytogeographic characterization of study sites and regions, will be the most promoting way to provide better resolution of meliponine behavior within the vast Brazilian regions. Acknowledgments I thank Professor Patricia Vit (Universidad de Los Andes, Mérida, Venezuela) for encouragement to write this chapter and Dr. David Roubik (Smithsonian Tropical Research Institute, Ancon, Panama) for careful editing, and also the Conselho Nacional de Desenvolvimento Científico e Tecnológico/CNPq for financial support. References Absy ML, Kerr WE. 1977. Algumas plantas visitadas para obtenção de pólen por operárias de Melipona seminigra merrillae em Manaus. Acta Amazônica 7:309–315. Absy ML, Bezerra EB, Kerr WE. 1980. Plantas nectaríferas utilizadas por duas espécies de Melipona da Amazônia. Acta Amazônica 10:271–281. 20 Palynology Serving the Stingless Bees 293 Alves RMO, Carvalho CAL, Souza BA. 2006. Espectro polínico de amostras de mel de Melipona mandacaia Smith, 1863 (Hymenoptera: Apidae). Acta Scientiarum Biological Sciences 28:65–70. Barth OM. 1965. Glossário palinológico. Parte complementar ao Catálogo sistemático dos pólens. Memórias do Instituto Oswaldo Cruz 63:133–161. Barth OM. 1975. Glossário palinológico (reedição atualizada). Leandra (UFRJ) 6:141–164. Barth OM. 1989. O Pólen no Mel Brasileiro. Editora Luxor, Rio de Janeiro, Brasil. 151 pp. Barth OM. 1998. Pollen analysis of Brazilian propolis. Grana 37:97–101. Barth OM. 2004. Melissopalynology in Brazil: a review of pollen analysis of honeys, propolis and pollen loads of bees. Scientia Agricola 61:342–350. Barth OM. 2006. Palynological analysis of geopropolis samples obtained from six species of Meliponinae in the Campus of the Universidade de Ribeirão Preto, USP, Brazil. Apiacta 41:71–85. Barth OM, Luz CFP. 2003. Palynological analysis of Brazilian geopropolis sediments. Grana 42:121–127. Barth OM, Melhem TS. 1988. Glossário Ilustrado de Palinologia. Editora da UNICAMP, Campinas, Brazil. 75 pp. Barth OM, Dutra VML, Justo RL. 1999. Análise polínica de algumas amostras de própolis do Brasil Meridional. Ciência Rural, Santa Maria 29:663–667. Barth OM, Munhoz MC, Luz CFP. 2009. Botanical origin of Apis pollen loads using color, weight and pollen morphology data. Acta Alimentaria 38:133–139. Barth OM, Freitas AS, Oliveira, ES, Silva RA, Maester FM, Andrella RRS, Cardozo GMBQ. 2010. Evaluation of the botanical origin of commercial dry bee pollen load batches using pollen analysis: A proposal for technical standardization. Anais da Academia Brasileira de Ciências 82:893–902. Bazlen K. 2000. Charakterisierung von Honigen stachelloser Bienen aus Brasilien. Thesis. Faculty of Biology, Eberhard-Karl University of Tübingen. 141 p. Carvalho CAL, Moreti ACCC, Marchini LC, Alves RMO, Oliveira PCF. 2001. Pollen spectrum of honey of “uruçu” bee (Melipona scutellaris Latreille, 1811). Revista Brasileira de Biologia 61:63–67. Cortopassi-Laurino M, Zillikens A, Stein J. 2009. Pollen sources of the orchid bee Euglossa annectans Dressler 1982 (Hymenoptera: Apidae, Euglossini) analyzed from larval provisions. Genetics and Molecular Research 8:546–556. Dórea MC, Aguiar CML, Figueiroa LER, Lima LCLE, Santos FAR. 2010. Pollen residues in nests of Centris tarsata Smith (Hymenoptera, Apidae, Centridini) in a tropical semiarid area in NE Brazil. Apidologie 41:557–567. Erdtman, G. 1952. Pollen Morphology and Plant Taxonomy - Angiosperms. Almqvist and Wicksell; Stockholm, Sweden. 539 pp. Faegri K, Iversen J. 1950. Textbook of Modern Pollen Analysis. Munksgaard; Copenhagen, Denmark. 168 pp. (reedited in 1996, 237 pp.). Ferreira EL, Leoncini C, Benassi MT, Barth OM, Bastos DHM. 2007. Avaliação sensorial de mel de abelhas indígenas de diferentes localidades do Brasil. Mensagem Doce 93:16–24. Ferreira EL, Lencioni C, Benassi MT, Barth OM, Bastos DHM. 2009. Descriptive sensory analysis and acceptance of stingless bee honey. Food Science and Technology International 15:251–258. Flores FF, Sánchez AC. 2010. Primeros resultados de la caracterización botánica de mieles producidas por Tetragonisca angustula (Apidae, Meliponinae) en Los Naranjos, Salta, Argentina. Boletín de la Sociedad Argentina de Botánica 45:81–91. Freitas AS, Barth OM, Luz CFP. 2010. Análise polínica comparativa e origem botânica de amostras de mel de Meliponinae (Hymenoptera, Apidae) do Brasil e da Venezuela. Mensagem Doce 106:2–9. Hilario SD, Imperatriz-Fonseca VL. 2009. Pollen foraging incolonies of Melipona bicolor (Apidae, Meliponini): effects of season, colony size and queen number. Genetics and Molecular Research 8:664–671. 294 O.M. Barth Iwama S, Melhem TS. 1979. The pollen spectrum of the honey of Tetragonisca angustula angustula Latreille (Apidae, Meliponinae). Apidologie 10:275–295. Junior MCS, Santos FAR. 2003. Espectro polínico de amostras de méis coletadas na microrregião do Paraguassu, Bahia. Magistra 15:70–85. Malagodi-Braga KS, Kleinert AMP. 2009. Comparative analysis of two sampling technique of pollen gathered by Nannotrigona testaceicornis Lepeletier (Apidae, Meliponini). Genetics and Molecular Research 8:596–606. Marques-Souza AC, Miranda IPA, Moura CO, Rabelo A, Barbosa EM. 2002. Características morfológicas e bioquímicas do pólen coletado por cinco espécies de Meliponíneos da Amazônia Central. Acta Amazônica 32:217–229. Marques-Souza AC, Absy ML, Kerr WE. 2007. Pollen harvest features of the Central Amazonian bee Scaptotrigona fulvicutis Moure 1964 (Apidae: Meliponinae), in Brazil. Acta Botanica Brasilica 21:11–20. Novais TS, Lima LCL, Santos FAR. 2006. Espectro polínico de méis de Tetragonisca angustula Latreille, 1811 coletados na caatinga de Canudos, Bahia, Brazil. Magistra:18:257–264. Oliveira MEC, Poderoso JCM, Ferreira AF, Lessa ACV, Dantas PC, Ribeiro GT, Araújo ED. 2008. Análise melissopalinológica e estrutura de ninhos de abelhas Trigona spinipes (Fabricius, 1793) (Hymenoptera: Apidae) encontradas no campus da Universidade Federal de Sergipe, São Cristóvão, Sergipe. EntomoBrasilis 1:17–22. Oliveira FPM, Absy ML, Miranda IS. 2009. Recurso polínico coletado por abelhas sem ferrão (Apidae, Meliponinae) em um fragmento de floresta na região de Manaus—Amazonas. Acta Amazônica 39:505–518. Pick RA, Blochtein B. 2002. Atividade de coleta e origem floral do pólen armazenado em colônias de Plebéia saiqui (Holmberg) (Hymenoptera, Apidae, Meliponinae) no sul do Brasil. Revista Brasileira de Zoologia 19:289–300. Punt W, Hoen PP, Blackmore S, Nilsson S, Le Thomas A. 2007. Glossary of pollen and spore terminology. Review of Palaeobotany and Palynology 143:1–81. (second and revised edition of 1994). Ramalho M, Silva MD, Carvalho CAL. 2007. Dinâmica de uso de fontes de pólen por Melipona scutellaris Latreille (Hymenoptera:Apidae): uma análise comparativa com Apis mellifera L. (Apidae), no domínio tropical atlântico. Neotropical Entomology 36:38–45. Roubik DW. 1978. Competitive interactions between neotropical pollinators and Africanized honeybees. Science 201:1030–1032. Roubik DW. 1980. Foraging behavior of competing Africanized honeybees and stingless bees. Ecology 61:835–845. Silva AC, Kinupp VF, Absy ML, Kerr WE. 2004. Pollen morphology and study of the visitors (Hymenoptera, Apidae) of Solanum stramoniifolium Jacq. (Solanaceae) in Central Amazon. Acta Botanica Brasilica 18:653–657. Silva TMS, Câmara CA, Silva AC. 2006. Chemical composition and free radical scavenging activity of pollen loads drom stingless bee Melipona subnitida Ducke. Journal of Food Composition and Analysis 19:507–511. Veloso HP, Rangel Filho LR, Lima JCA. 1991. Classificação da vegetação brasileira adaptada a um sistema universal. Instituto Brasileiro de Geografia; Rio de Janeiro, Brazil. 124 pp. Vit P, Ricciardelli D’Albore G. 1994. Melissopalynology for stingless bees (Hymenoptera: Apidae: Meliponinae) in Venezuela. Journal of Apicultural Research 33:145–154. Vit P, Mejías A, Rial L, Ruíz J, Peña S, González AC, Rodríguez-Malaver A, Arráez M, Gutiérrez C, Zambrano A, Barth OM. 2012. Conociendo la miel de Melipona favosa en la Península de Paraguaná, Estado Falcón, Venezuela. Revista del Instituto Nacional de Higiene Rafael Rangel 43:15–19. Chapter 21 How to Be a Bee-Botanist Using Pollen Spectra David W. Roubik and Jorge Enrique Moreno Patiño 21.1 Introduction To better understand tropical biology, we would like to see the world as a bee does (Fig. 21.1). Two central tasks exist for a foraging bee. The first is to find rewarding resources, and the second to remember the route between the nest or home base and the resource. Once a bee is successful finding food, it can move back and forth until the food is depleted, both within a single day and within the flowering period of that plant. In tropical wild lands, that course of action includes primarily the forest canopy (Roubik et al. 1984). And because so many flowers are not observable, despite the labors of field biologists, we are still woefully ignorant of which flowering plants are most important to the honey-making social bees, especially stingless bees and honey bees (Roubik 1989, 1993; Roubik and Hanson 2004; Roubik et al. 2003; Corlett 2011). Such bees are termed “generalists” because they use many floral species, but this term is qualitative, not quantitative. Substantial research has attempted to give quantitative pollen data and its potentially important role in understanding which plant species are most important to bees (classic studies by Louveaux 1968; Barth 1970a, b; Maurizio 1975; Iwama and Melhem 1979; see also Roubik et al. 1984; Roubik 1989; Villanueva-Gutiérrez and Roubik 2004; Roubik and Moreno 2009). Palynology and its specialized subdisciplines of melittopalynology and melissopalynology (see present book chapters and Roubik 2009)—more simply termed bee-botany and bee-palynology—provide the best approach to connect bees with their food sources, whereby pollen taxonomy is applied to plants—used opportunistically and steadfastly pollinated by bees. Pollen taxonomy, we believe, D.W. Roubik (*) • J.E.M. Patiño Smithsonian Tropical Research Institute, Ancón, Balboa Republic of Panamá MRC 0580-12, Unit 9100, Box 0948, DPO AA, 34002-9998, USA e-mail: roubikd@si.edu 295 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_21, © Springer Science+Business Media New York 2013 296 D.W. Roubik and J.E. Moreno Patiño Fig. 21.1 A tropical lowland forest, Barro Colorado Island (9° North latitude) viewed from above the canopy. Photo: Archives at the Smithsonian Tropical Research Institute is the first step in a process of training persons who study bees to study the plants that bees depend upon and also to extract the most information possible from bee biology field and laboratory studies, using the identification of pollen as a foundation. If we fail to interpret our own data, then we have not been able to graduate as “bee-botanists,” which is that to which we must aspire. We studied the honey of two social bees in tropical American forests and semiforested areas, and also the actual pollen loads brought in the nest by returning foragers, to demonstrate how melittopalynology (bee–pollen studies) and melissopalynology (honey–pollen studies) can be organized to answer biologically meaningful questions, such as the translation of pollen identification and counts to resource importance for bees, and their ecology. 21.1.1 Pollen and Bee-Botany Bee resources dominant in pollen counts, both in honey and in pollen provisions in bee nest cells, are often small woody plants, plants thought to be anemophilous, or herbs growing on the forest edge. Their grain number, instead of concentration, weight, or volume in a bee “pollen spectrum” (see Barth 1970a, chapter in present book, and below) indicates they are present, but little else. This presence was determined 21 How to Be a Bee-Botanist Using Pollen Spectra 297 to be either “isolated” (very rare) or “accessory” (intermediate), when not dominant. We employ a different analytical technique and methods, in an attempt to simplify the categorization of pollen choice, abundance, and importance, especially in honey (nectar) sources. Furthermore, unless careful field studies are made of whether bees or other nectar drinkers, such as flies, butterflies, wasps, or birds, are foraging nectar at a flower, the nature of the floral resource is unknown from its pollen detection in a bee product. Dioecious plants—many palms, for example—and up to 25% of tropical forest tree species in a given natural, mature forest (Henderson 1986), provide only pollen (and scent) at one sex of flower and often only scent at the other. They are deceptive mimics. Variation in the floral resource-pollinator theme (e.g., Latham and Mbuta 2011), without adequate field study, also deceives researchers on bee-flower ecology. Indeed, Iwama and Melhem (1979) summarize the findings of researchers and indicate that, even within a single genus (Alchornea, Euphorbiaceae), some species have nectar, but many do not, and this also occurs in Miconia (Melastomataceae) and Acacia (Fabaceae) (Sornsathapornkul and Owens 1998; Stone et al. 2003; Dos Santos, et al. 2010). How can we accurately demonstrate the specialization we perceive (Roubik 1992) in the resources a generalist bee uses? Like the foraging bee, the biologist must try to find a way to establish important links. Pollen quantification can be misleading. As already mentioned, pollen does not always indicate a nectar source (and the female flower of dioecious species may often have nectar, but never pollen). Furthermore, because different bee–pollen species have grains ranging 7–300 mm in diameter (Roubik 1989; Roubik and Moreno 1991) there is a difference between individual grains of almost a quarter million (216,000) volumes (see Roubik 1989). In other words, the largest and smallest grains are potentially of identical importance to a bee as harvested food if there is one of one species and 216,000 of the other. When pollen slides are prepared and pollen grains counted along a transect, omission of one of the large grains constitutes a serious loss of biological data. Consider, for example, grains of Cucurbitaceae (e.g., Cayaponia spp. 200 mm diameter) versus those of Miconia or Piper (7 mm), see Roubik and Moreno (1991). Because generalist bees use large numbers of plant species, but not all species are used evenly—either in raw volume as protein or in quality—(Roubik 1988, 1989; Biesmeijer et al. 1992; Roulston et al. 2000) there is certainly a potential and sometimes large degree of specialization for “generalist” foraging bee species. 21.1.2 Quantitative Methods for Bee-Palynology In response to the challenges mentioned above, our first step was to determine pollen volume, either as it comes mixed in honey or as pollen gathered by individual bees as pollen loads. Pollen volumes are quantified, in the case of honey bees, either by counting the pellets trapped from the hind legs and then identified (Roubik et al. 1984), by dry weight, or by computing individual grain volume of the plant species (Villanueva-Gutiérrez and Roubik 2004). Another method, which we use here, is an 298 D.W. Roubik and J.E. Moreno Patiño internal standard from spore tablets of Lycopodium, with a known number of spores per tablet batch (Stockmarr 1971). This is a straightforward method, much like weighing individual pollen pellet loads of a single species, to quantify the portion per weight or volume of sample pollen species (Roubik and Moreno 2009). Three analytical methods are introduced here. First is that of determining pollen concentration, which we call “importance” in a pollen spectrum. The second is to document high importance across sites or apiaries, which we call “consistency.” Both methods require corrections from raw pollen counts, based either on number of grains, relative to spore counts, per gram or per cubic centimeter. For corbicular pollen loads, those of Apis mellifera at one apiary (from which honey also was collected) were sorted by color. The pollen species, and concentrations of different pollen types per gram, were determined for each designated color variety. In addition to providing a comparison of corbicular pollen to pollen found in honey, this method tests whether color is a valid index of pollen species and whether the density of different pollens in a pellet (pollen load from one bee’s leg) is similar across species. We obtained honey samples from apiaries and meliponaries with collaboration of local beekeepers and also used colonies maintained by the first author in Panama. The two bee species studied were Tetragonisca angustula (Latreille 1811) (which may include other cryptic species, Camargo and Pedro 2007) and the Africanized honey bee, Neotropical Apis mellifera—close to African A. mellifera scutellata, but no longer identified as that subspecies (Francoy et al. 2008). Honey was collected in clean 50 ml plastic vials from a sample of the entire nest honey, or in the case of Apis from multiple colonies, at the normal honey harvest time and usually stored under refrigeration until analysis. Pollen pellets of the corbicular load of Apis were taken at the hive entrance with an exterior screen commercial pollen trap and collecting pan. To obtain an estimate of pollen volumes for different species, Lycopodium spores were added before carrying out the acetolysis process, with a known weight and volume, to provide an internal standard that allows calculation of relative proportions of the same pollen species in different samples or in multiple slides prepared from a single processed sample (Roubik and Moreno 2009). Eighteen samples of honey removed from bee hives of Apis mellifera at 17 lowland (<500 m elevation) localities in Bolivia, Brazil, Venezuela, Mexico, French Guiana, and Panama and from 11 lowland localities for Tetragonisca angustula in Panama, Bolivia, and Peru (Tables 21.1 and 21.2) were used for pollen analysis. For Apis mellifera, Step 1 was only applied to nine samples of Venezuelan honeys from seven sites and for two apiary samples from two sites, near Sinnamary, French Guiana. Venezuelan sites varied considerably and were located in agro-ecosystems with some natural vegetation patches available. Both the French Guiana sites were in forest–savanna or along a mangrove areas with coastal forest, with very little human disturbance of vegetation, aside from the roads and seasonal savanna burnings. Peru Bolivia Plant taxa San Martín Isozog A Kopere Isozog B Karapari Pampas A Beni Pampas B Beni Yungas Ixiamas Panama Sacramento Chaco STRI Curundu Families Genera Species 24 28 34 13 13 15 36 43 52 21 27 31 36 53 62 20 22 23 29 33 41 22 23 27 18 20 22 25 25 27 34 47 57 21 How to Be a Bee-Botanist Using Pollen Spectra Table 21.1 Numbers of plant taxa in pollen spectra, by locality, from honey of Tetragonisca angustula in tropical lowlands Locality Colony samples from: Peru: San Martín; Bolivia: Isozog A—Kopere, Isozog B—Karapari, Beni—Pampas A, Beni—Pampas B, Coroico, Yungas, Ixiamas, Sacramento, Chaco; Panama: Ancon area, Curundu Flats (see Supplemental Data) 299 Table 21.2 Numbers of plant taxa in pollen spectra, by locality, from honey of Africanized Apis mellifera in tropical lowlands Locality Brazil Plant taxa Bolivia French Guiana México Panama Venezuela Bord Forêt et Curundu Barinas Barinas Barinas Curitiba Ixiamas Isozog Pampas Coroico du Mer Savanne Chetumal Flats A B C Cojedes Anzoátegui Bolivar Lara Miranda Trujillo Families 14 Genera 19 Species 19 34 43 52 16 20 24 28 32 37 27 34 37 23 24 27 17 19 24 23 29 36 25 33 39 22 24 31 20 21 23 22 27 34 27 37 44 16 18 26 36 58 71 34 46 60 20 24 30 30 36 46 Colony samples from: Brazil, Curitiba; Bolivia, Ixiamas, Isozog, Pampas, Coroico; French Guiana, Sinnamary—bord du mer, Forêt et Savanne; Mexico, Chetumal; Panama, Curundu Flats; Venezuela, Barinas—Altamira de Cáceres A, Barinas—Altamira de Cáceres B, Barinas—Guanare-Barrancas, Cojedes, Anzoátegui, Bolívar, Lara, Trujillo, and Miranda (see Supplemental Data) 21 How to Be a Bee-Botanist Using Pollen Spectra 301 The following general methods describe our acetolysis procedure for honey and pollen pellet samples: 1. One Lycopodium tablet was added to each sample (a “batch” of the tablets is accompanied by information on its mean spore count per tablet; batch 938934 had a mean = 10.700 spores (T. angustula and A. mellifera), batch 124961 a mean = 12.500 spores (A. mellifera only)). 2. Samples were dissolved in water and sieved with mesh (250 mm). 3. Samples were concentrated at 2,700 rpm/5 min and supernatant discarded. 4. Residues were dried with glacial acetic acid. 5. Samples were concentrated at 2,700 rpm/5 min and supernatant discarded. 6. Solution of Acetolysis was added (nine parts of anhydride acetic acid and one part of sulfuric acid concentrated)/heated 5 min, to destroy all cellulose content and to clean pollen grains. 7. Samples were concentrated at 2,700 rpm/5 min and the solution of Acetolysis discarded. 8. Samples were then washed with distilled water and their residues concentrated. 9. Ethanol was used as dehydratant and samples were concentrated at 2,700 rpm/5 min. 10. The ethanol was discarded and some drops of glycerol were added. 11. Finally, permanent microscope preparations were made using glycerin jelly as mounting media and paraffin as sealant. To identify all pollen grain types, transects of all slides were made at ×40 magnification using an Olympus BH-2 binocular scope. Electronic microphotographs of material were obtained at ×100 magnification using a Pixera Camera System attached to the Olympus scope. The botanical names of families, genera, and species were established by comparisons with pollen atlases (see References). Species names preceded with “cf.” Or “prob.” indicate the identification based on neotropical pollen collections kept at the Center for Tropical Paleoecology and Archaeology (CTPA) of the Smithsonian Tropical Research Institute (STRI) in Panama require further confirmation, using collections of the local flora at particular sampling sites. The procedure for analyzing botanical species importance to a bee species was as follows: pollen concentrations or counts were determined for each pollen type. Given that the volume and weight of a subsample from the entire collected pollen were determined and the number of Lycopodium spores added to the sample known, each subsample has an exact pollen and spore concentration, revealed by the number of Lycopodium counted and its ratio to other pollen types. For example, if 10,000 spores of Lycopodium had been added to 1 g or 1 cc of acetolyzed pollen sample, and a transect count had produced six spores with 12, 60, and 300 grains of pollen types A, B, and C, then the total of those grains would be estimated as 20,000, 100,000, and 500,000. The concentration method, described by Stockmarr (1971)—see also O’Rourke and Buchmann (1991)—was used in one group of honey bee samples from Venezuela, the largest for one region in this study (N = 9), pollen of corbicular loads, 302 D.W. Roubik and J.E. Moreno Patiño and in the honey samples of Tetragonisca, to calculate the ratio of Lycopodium spores to the pollen in question. We provide details here on the methods used to compare the importance of a given botanical resource among sites. The assumption is made that the spores are distributed evenly among all the pollen types on the microscope slide preparation. To compare between sites or different colony samples, the total number of spores counted is also inversely proportional to the total amount of sample grains present. That is, if one sample returned 20 grains of a pollen type and recorded ten spores, and a second sample returned ten grains of the same pollen type with five spores, the proportional representation or concentration of the pollen in the two subsamples was identical. Our procedure for identification and quantification of pollen indicates three possible categories. The first is the number of grains counted in transects, the second consists of cases in which only one grain was found in transects, and the third category, signifying “presence,” occurs when one or more grains were found on the microscope slide preparation, but none within transects used to count the spores and total pollen (see Supplemental Data). Pollen resource importance is examined first as a histogram, in which species are ranked by their total concentrations or counts, from highest to lowest. This may be done for a single colony or honey sample, or combined samples from several colonies, different areas, or regions. The idea is to see whether certain species, genera, or families stand out as important resources. Pollen resource consistency is next examined, for those resources that registered high importance. This step requires precise taxonomy, so that the same taxa can be registered as present or absent. We also evaluate relative concentration (proportion of the total sample) across sites. Here we chose to make the comparisons using the plant genera scored as important, then expanded that category to include species, as discussed below. Resource counts and concentration are graphed after ranking in descending order. An overall list of important resources is made on the basis of plants that are both important and consistently used by the bees (see Supplemental Data for individual colonies and honey samples from apiaries of A. mellifera). In this way, we try to establish whether in a majority of sites, or samples, the pollen spectrum and relative importance were similar. In addition, also based on the concept of consistency, we examine the plant resources (family or genus) which were used in most sites, but were not among the high-volume resources. As will be made clear in the following sections, the analysis of importance using a histogram is an intuitive approach. A simple curve-fitting procedure was applied to the ranked pollen resource histograms testing logarithmic, exponential, and power functions for goodness of fit, using Microsoft Excel. 21.1.3 Application to Stingless Bees and Honey Bees After genus and mostly species identifications were made of pollen, resource richness was categorized and analyzed. For the stingless bee Tetragonisca angustula, 21 How to Be a Bee-Botanist Using Pollen Spectra 303 Fig. 21.2 Pollen consistency—representation across sites—for Tetragonisca angustula and Apis mellifera at 11 and 18 sites, in three and six countries, respectively and including all pollen identified, the 11 samples produced 175 species and 134 genera in 69 plant families (Table 21.1 and Fig. 21.2). For Apis mellifera, the 18 honey samples contained 206 species, 156 genera, and 76 families (Table 21.2). Plant species enumerated from pollen in honey samples from Africanized Apis mellifera in largely forested areas of natural vegetation. Honey bee colonies had a mean of 46 species in their honey (range 19–71), while those of the stingless bee averaged 35 species (range 15–62). An index of pollen diversity, the number of botanical species divided by the number of samples, yielded approximately 16 for T. angustula and 12 for A. mellifera. However, little difference was found between the means of the averages for each region, 33.3 for T. angustula and 34.6 for A. mellifera. There was an “outlier,” with considerably lower pollen richness, in each bee study―that of Curitiba, Brazil for A. mellifera and that of San Martín, Peru for T. angustula. Pollen corbicular pellets from Africanized honey bees in French Guiana, sampled during April from one apiary near the coast and one in the interior forest and savanna, included 22 color categories. A total of 1,048 pellets was analyzed, an average of 24 of each color (SD = 35). The average pellet weight was 4.39 mg (SD = 2.12 mg). The color subsamples of each apiary, to which one Lycopodium tablet was added, averaged 134.59 mg (SD = 237 mg). Total concentrations of pollen 304 D.W. Roubik and J.E. Moreno Patiño Fig. 21.3 Pollen species as indicated by counted pollen grains of different species in honey of Tetragonisca angustula at 11 sites, 3 Neotropical countries grains per mg in those pellet samples were calculated to range from 4.54 × 106 to 1.573 × 1010. Thus, individual pollen species grains differed in weight by over 3,000-fold. There were 37 pollen types in the corbicular pollen samples, and each pellet color contained an average of four (range 1–7) species. A single pollen of the shrub, Mimosa pudica, was 89% of total pollen pellets, represented by 1.59 × 109 grains in 5 g. Corbicular pellets strongly dominated by M. pudica ranged in color from almost white to light brown to light yellow (see Supplemental Data, pollen loads of A. mellifera spreadsheet). Those color differences may be due to the degree to which the pellets had been dried. Botanical resources of Tetragonisca angustula included plants with one or more grains counted in a sample and are ranked in total volume in Fig. 21.3. There is a clear break in the curve after the 11th species, with those below that rank counted as less than 200 grains. The “top 10” species are considered in Table 21.3, further modified for actual pollen volume(Fig. 21.4). The total number of sites and the total volume are given with the plant taxonomy (see also Supplemental Data). From pollen counts alone, summed among the diverse lowland sites, a papilionaceous legume (Machaerium) and a genus of Rubiaceae (Macrocnemum) were far more common than the next most common families—but Anacardiaceae clearly predominated in consistency and would appear equally important (but not as a source of nectar). The summed pollen concentrations better quantify taxonomic preferences. An Anacardiaceae (Spondias) was the most important pollen source (it has no nectar, see Carneiro and Martins 2012), with Gouania (Rhamnaceae), Machaerium, and Macrocnemum following, then the palm Scheelea (a pollen-only source), Anacardium, Eugenia, Alternanthera, Miconia, and Calopogonium. These were the top ten plants, after which the remainder drop well below the predictive distributional 305 21 How to Be a Bee-Botanist Using Pollen Spectra Table 21.3 Pollen counts, consistency, and concentration (volume) of major resources indicated by honey pollen analysis for Tetragonisca angustula Pollen counts and sites present Pollen concentration Plant genus Plant family Sum pollen count Sites Plant genus Sum % conc. 136 6 Spondiasa Anacardiaceae 2 4 3 8 3 1 3 6 4 Gouania Machaerium Macrocnemum Scheeleaa Anacardium Eugenia Alternanthera Miconiaa Calopogoniumb 200 2 Rubiaceae Rhamnaceae 120 Fabaceae 97 Rubiaceae 78 Arecaceae 66 Anacardiaceae 59 Myrtaceae 58 Amaranthaceae 47 Melastomataceae 40 Fabaceae, 35 Papilionoideae Rubiaceae 24 168 Hyptisb Acaciaa Fabaceae, 2,767 Papilionoideae Macrocnemum Rubiaceae 1,210 Gouania Rhamnaceae 446 Eugenia Myrtaceae 401 Anacardium Anacardiaceae 381 Alternanthera Amaranthaceae 321 Rubiaceaeb Rubiaceae 252 Miconiaa Melastomataceae 250 Spondiasa Anacardiaceae 248 Scheeleaa Arecaceae 203 Machaerium Guazumab Poaceaea Fabaceae, Mimosoideae Sterculiaceaec Poaceae – 2 7 Cecropiaa Asteraceae Euphorbia Celtis Urticaceae Asteraceae Euphorbiaceae Cannabaceae – – – – 6 7 6 11 Acaciaa Plant family Pipera Serjania 2 Syzygium Triumfetta Guazuma Asteraceae Arrabidaea Lamiaceae Fabaceae, Mimosoideae Piperaceae Sapindaceae Myrtaceae Tiliaceaec Sterculiaceaec Asteraceae Bignoniaceae 19 18 17 15 14 11 11 11 11 The cutoff range was determined at 200-grain counts (see Fig. 21.4) and at two portions of the pollen spectrum curve—concentration a Nectarless flowers b Concentration rank changed presence and ranking from raw count data c Sterculiaceae and Bombacaceae are now included in Malvaceae sensu APG III curve (Fig. 21.5). The potentially nectarless Acacia, and the solely nectarless and Piper were very low in the overall ranking. Botanical resources of Apis mellifera quantified by our ranking methods followed a logarithmic curve, and two relatively minor cutoff points were found for the Venezuelan honey samples (Fig. 21.6). Remarkably, one floral species was the single most important resource for Apis in this research. Apis mellifera in both Venezuela and French Guiana lowlands used Mimosa pudica heavily, a plant with no floral nectar. Identification of corbicular pollen from French Guiana and honey samples of both countries (Table 21.3, Fig. 21.7) ranked this plant species highest, excessively so as a seasonal pollen source. In Fig. 21.6 this species is depicted far 306 D.W. Roubik and J.E. Moreno Patiño Fig. 21.4 Pollen concentrations provided by internal calibration marker spores of Lycopodium in Neotropical honey of Tetragonisca angustula (see Fig. 21.3). The “cutoffs” are points where the importance declines greatly between sequential ranked species Fig. 21.5 Pollen concentrations and cutoff points for floral resource importance in Neotropical honey of Africanized Apis mellifera above the predictive curve ranking pollen found in honey. The simple pollen counts without concentration marker Lycopodium also registered relatively abundant Mimosa in four of the additional six regions—southern Brazil, Mexico, Panama, and Bolivia. In Suriname, in contrast (Biesmeijer et al. 1992; Kerkvliet and Beerlink 1991) there was a predominance of Cecropia among pollen species, which indicates recently disturbed habitats, like forest edges that have been cleared or burned 21 How to Be a Bee-Botanist Using Pollen Spectra 307 Fig. 21.6 French Guiana corbicular pollen data and honey data for Apis mellifera data at two natural sites, with the combined sites shown for honey pollen species concentrations (Roubik 2009), and little in common, aside from various palms in the sample, with the forest, coastal, and savanna samples taken in French Guiana. The consistency of pollen genera across sites averaged 0.25, SD 0.02, so that we would expect each recorded genus to occur in one of four lowland sites. The most consistently scored genera and the summed pollen concentrations (the concentrations summed across sites) indicated that eight floral resources were outstanding in their importance to Apis mellifera; half of them do not produce nectar (Table 21.4). The potentially most important nectar sources, those not obviously pollen-only flowers, were Psidium (Myrtaceae), Alchornea (Euphorbiaceae), Hyptis (Lamiaceae), and Roystonea (Arecaceae). Many palms have no nectar in their flowers (Henderson 1986). The Alchornea are dioecious, and nectar of female flowers therefore leaves no trace of pollen (but flowers of both sexes, at least of some species, have nectar; Latham and Mbuta 2011). 308 D.W. Roubik and J.E. Moreno Patiño Fig. 21.7 Corrected (summed) pollen concentrations combining all Neotropical sites sampled for Tetragonisca angustula and Apis mellifera, shown for honey pollen species concentrations Pollen pellets had similar pollen types of major importance, in proximal habitats of French Guiana—each with some natural savanna and extensive forest, and were combined (Fig. 21.6). These showed a power function ranking, with only one type far more abundant than the more uniformly abundant remainder. In their honey pollen types, the forest–savanna area had ten abundant pollen types, while that of the coastal forest contained only five. The following significant forage species, indicated in Fig. 21.7 as decreasing gradually in importance, contained scattered pollen-only flowers in the ranking— Cecropia, Doliocarpus, Poaceae, Mimosa, and Anthurium. This leaves, as likely important nectar sources, two Myrtaceae, one Sapindaceae, one Bignoniaceae, two Asteraceae, one Euphorbiaceae, one Rutaceae, one Ulmaceae, two papilionaceous legumes, one Anacardiaceae, one Melastomataceae, and one Elaeocarpaceae. Corrections for consistency and volume of pollen types across sites provide distributions plotted in Fig. 21.7, which returned a power function for Apis and a logarithmic one for Tetragonisca, each highly significant (R2 = 0.96–0.98). There is 309 21 How to Be a Bee-Botanist Using Pollen Spectra Table 21.4 Pollen concentration indicating rank in importance of floral nectar and pollen sources, and consistency (no. sites present) for honey of Apis mellifera from seven Venezuelan sites (see Table 21.2) Plant Family Sum pollen concentration No. sites Mimosa pudicaa Psidium Pipera Alchorneaa Hyptis Roystonea Cecropiaa Doliocarpusa Poaceae 1a Eugenia Serjania Poaceae 2a Asteraceae Bignoniaceae Asteraceae Mimosa castaa Croton Zanthoxylum Celtis Syzygium Fabaceae, Papilionoideae Desmodium Mangifera Spondiasa Anthuriuma Miconiaa Sloanea a Fabaceae, Mimosoideae Myrtaceae Piperaceae Euphorbiaceae Lamiaceae Arecaceae Urticaceae Dilleniaceae Poaceae Myrtaceae Sapindaceae Poaceae Asteraceae Bignoniaceae Asteraceae Fabaceae, Mimosoideae Euphorbiaceae Rutaceae Cannabaceae Myrtaceae Fabacaeae, Papilionoideae Fabaceae, Papilionoideae Anacardiaceae Anacardiaceae Araceae Melastomataceae Elaeocarpaceae 667 500 269 251 217 170 166 162 131 130 105 96 88 70 69 62 60 57 57 54 52 48 42 40 40 37 36 6 3 4 2 5 2 2 4 1 1 1 2 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 Nectarless flowers a sudden drop in importance for Apis after the first and sixth-ranked species. Tetragonisca displays a smooth curve with no sudden decline in rank, except after the tenth species in the ranking. The rankings and consistency overviews, including raw counts, are given for T. angustula in Table 21.3, and the consistently major pollen, based on volume (concentration), for Apis mellifera is given in Table 21.4. Some of our photomicrographs of the most important pollen types are given for comparison in Fig. 21.8. 21.1.4 Pitfalls of Pollen Analysis and Need for Field Observation We frequently observe plants flowering over many weeks, or even all year, and this may explain their abundance in seasonal or yearly samples. Moreover, those pollen 310 D.W. Roubik and J.E. Moreno Patiño Fig. 21.8 Most important pollen resources. Dicotyledoneae: Anacardiaceae: (a) Anacardium sp., (b) Spondias sp. Asteraceae: (c) Undetermined. Boraginacae: (d) Cordia sp. Euphorbiaceae: (e) Alchornea sp. Lamiaceae: (f) Hyptis sp. Fabaceae-Faboideae: (g) Machaerium sp. FabaceaeMimosoideae: (h) Acacia sp., (i) Mimosa sp. Myrtaceae: (j) Eugenia sp. Melastomataceae: (k) Miconia sp. Rhamnaceae: (l) Gouania sp. Rutaceae: (m) Zanthoxyllum sp. Sapindaceae: (n) Serjania sp. Urticaceae: (o) Cecropia sp. Monocotyledoneae: Arecaceae: (p) Scheelea sp. Poaceae: (q) Undetermined (×100) (photos not in same scale). Photos: J.E. Moreno Patiño grains are often relatively small, smaller than 10 mm in diameter, and may occur in high density in a honey sample. The same kind of small and numerous pollen is sometimes incorrectly associated with a nectar source. Although the pollen is found in honey in a hive comb or food pot, the plant does not have nectar and cannot be a honey source (e.g., Piper, in Kerkvliet and Beerlink 1991, or Cecropia, in VillanuevaGutiérrez and Roubik (2004), or Spondias (Carneiro and Martins 2012), or Acacia (apparently, from a detailed study of a hybrid, Sornsathapornkul and Owens 1998)); numerous small grains do not signify importance (Biesmeijer et al. 1992). Further, 21 How to Be a Bee-Botanist Using Pollen Spectra 311 we believe there is nectar in Alchornea, Trema, Theobroma, and many palms, but more study is needed. We found the Africanized honey bees and Tetragonisca angustula tend to use diverse but distinctive groups of floral resources (typically three or four dozen families, genera, and species) in lowland Neotropical areas, and they specialize heavily among them—shown by pollen ranking using power, logarithmic, and exponential functions. Dominant pollen is often no indication of a nectar source, if flowers are nectarless, thus the less abundant pollen types in honey may serve to indicate some important nectar plants. The consistencies with which resources were utilized across a range of sites were predictable for both bees by a logarithmic curve, but for pollen pellets (pollen analysis alone) a power function was superior, and A. mellifera showed high consistency. Simple pollen counts for T. angustula were difficult to fit with a regression model as to rank (R2 = 0.44). Pollen counts corrected for density in the sample—concentration and total volume—produced different species ranks and even introduced or removed species from consideration in the top 20 floral species. We do not yet have a comprehensive picture for annual pollen use and floral visitation for an entire year at any site. However, the combination of sites, during wet season, dry season, and primarily, the time in which most honey is harvested, or peak “honeyflow” (see Villanueva-Gutiérrez et al. 2009) give us some confidence that the data are representative of floral importance. The pollen types of honey from the nest, and from pollen loads, were dominated by a nectarless “roadside weed,” Mimosa pudica, which provides pollen to diverse bees, primarily Melipona in vast forest regions, in early morning, but is often monopolized by Africanized A. mellifera near de-forested areas (Roubik 1996). Nine of the most widespread honey bee sources were nectarless or dioecious. This was not true for the stingless bee, although it had nectarless flowers of Spondias as one of its major, consistent resources. In addition, the honey bee used many grasses and, although T. angustula also uses nectarless grasses, sedges, and palms (see Chap. 23 by Obregón et al. in this book) these were not among its main resources. The vast majority of both bee resource spectra were trees (in the semi-forested and forested habitats, see Supplemental Data). Pollen content, presented as a list of species (see Supplemental Data, pollen pellets of A. mellifera), provides basic information on flowering plants used by bees, but often, particularly in botanically rich environments, does not lead to any particular insight or prediction. Honey bees and solitary bees both are generalists (Roubik and Villanueva 2009) but this is not a guide toward understanding either ecology or management. Counts of grains as indices of resource importance, with no further quantification, are likely to be inadequate or misleading. The pollen concentration in honey and nectar varies greatly (Bryant and Jones 2001). As mentioned in the pollen and bee-botany section, grain volumes vary widely, and pollen importance (concentration) in one sample may not be comparable to that in another. We believe the Lycopodium density marker can be used to make adequate corrections for the different grain sizes found in melittopalynological samples. As for simply examining pollen taken from the field or the bees―with no chemical treatment to remove the interior protoplasm and expose exine characteristics—in the tropics, where there 312 D.W. Roubik and J.E. Moreno Patiño are roughly 1,000 species in a given habitat, and 20% have the same gross pollen characteristics (Roubik and Moreno 1991), correct identification, even at the family level, is very often impossible. On the other hand, some playnologists, if they have an adequate reference collection, discern tropical genera or families with only pollen grains taken from bees and slide-mounted in water (M. Burgett, personal communication). Our organization of the pollen obtained from honey has made clear, for both a stingless bee and the Africanized Apis of the Neotropics, the difference between an abundant pollen source and the source of nectar. This kind of information is seldom regarded in melissopalynology, but now there is easy access to literature with Internet search engines, but there is still, above all, the need for direct observation, in the field, of bees visiting flowers. If they are using the tongue to extract nectar, it should be noted. Apis uses large amounts of pollen to support its brood production and swarming, while the stingless bees do not often swarm and should use less pollen (Roubik 2006). Therefore, the honey bee leaves much more pollen from nectarless sources within its nest, and that pollen finds its way into the honey. For honey, honey bee samples contained large quantities of pollen that may be called “contaminants” in nectar (Iwama and Melhem 1979) of other species which provide the liquid source of honey. The flowers of Mimosa, Acacia, Piper, Senna, Cassia, Cecropia, Spondias, Doliocarpus, all grasses (Poaceae), most Solanaceae, most Melastomaceae, and many palms have no nectar foraged by bees, and some dioecious flowers may be exploited solely for pollen. Yet pollen of this botanical origin, at species or higher level, is common in honey among Apis (Kiew 1997; Roubik 1989, 2005; Adekanmbi and Ogundipe 2009; and chapters of the present book). In lowlands from sea level to several hundred meters altitude, tropical flowers first open near 6 a.m. or sunrise, and flowers that were open during the night also present their surplus nectar and pollen (Roubik 1989, see Corlett 2011). Our lowland samples reflected these trends, but it remains to be seen whether similar findings would apply to tropical highlands. Pollen usually is depleted at flowers in the morning and before nectar, because it is not continuously secreted. With most foraging in early morning for pollen, loose pollen is distributed throughout the bee nest. That pollen can easily be carried all day, on the bodies of active nest bees and foragers, into areas of nectar storage. Why is so much pollen from non-nectar species contained in honey of certain tropical bees? The timing and intensity of foraging are likely the key. Pollen is present in large quantities in the early morning, for example, from Mimosa pudica, Piper, or grasses (Roubik 1996). For a seasonal pollen in Venezuela, Apis had 89% of its pollen volume or mass one nectarless type—also a major food for Melipona—among 37 species identified. It was Mimosa pudica (Roubik 1996). Barth (1970a) also found much Mimosa pollen in the honey of Apis mellifera in Brazil, as did Iwama and Melhem (1979) in the honey of Tetragonisca angustula there. The value of the present comparative study is this: the stingless bee used fewer major pollens but used them more evenly than did Apis. We suggest a generally more even distribution of resource types for stingless bees, and predominance of non-nectariferous pollen in honey of Africanized honey bees contrasted to stingless bees, is due to their extensive swarming and greater demand for pollen. 21 How to Be a Bee-Botanist Using Pollen Spectra 313 The stingless bees take a long time to reproduce, whereas Apis does so freely, and apparently at least once a year (Roubik 1989, 2006). However, both groups take advantage of pollen and also nectar, that is ostensibly to feed large nocturnal animals that visit large flowers, dioecious species or those with no nectar, and dense inflorescences. This still appears to be the general situation for tropical honey-making bees, when importance and not only species-lists are considered (Roubik 1989). Acknowledgments We thank Dr. R. Villanueva for comments and for providing Mexican honey samples. Dr. R. Harrison helped collect Bolivian samples, for which we also thank E. Stierlin. B. Gaucher and G. El Alaoui sampled French Guianan bees for both pollen and honey. References Adekanmbi O, Ogundipe O. 2009. Nectar sources for the honey bee (Apis mellifera adansonii) revealed by pollen content. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 37:211–217. Barth M. 1970a. Análise microscópica de algunas amostras de miel; 1, pólen dominante. Anais Academia Brasileira de Ciencia 42:351–366. Barth M. 1970b. Análise microscópica de algunas amostras de miel; 2, pólen acessório. Anais Academia Brasileira de Ciencia 42:571–590. Biesmeijer JC, van Marwijk B, van Deursen K, Punt W, Sommeijer MJ. 1992. Pollen sources for Apis mellifera L (Hym, Apidae) in Surinam, based on pollen grain volume estimates. Apidologie 23:245–256. Bryant VM Jr, Jones GD. 2001. The R-values of honey: pollen coefficients. Palynology 25:11–28. Camargo JMF, Pedro SM. 2007. Meliponini Lepeletier 1836. pp. 272–578. In: Moure JS, Urban D, Melo GAR, eds. Catalogue of bees (Hymenoptera, Apoidea) in the neotropical region. Sociedade Brasileira de Entomologia; Curitiba, Brazil. 1958 pp. Carneiro LT, Martins CF. 2012. Africanized honey bees pollinate and preempt the pollen of Spondias mombin (Anacardiaceae) flowers. Apidologie 43:474–486. Corlett RT. 2011. Honeybees in natural ecosystems. pp. 215–225. In: Hepburn HR, Radloff SE, eds. Honeybees of Asia. Springer Verlag; Berlin, Germany. 612 pp. Dos Santos APM, Romero R, de Oliveira PEAM. 2010. Biologia reprodutiva de Miconia angelana (Melastomataceae), endémica da Serra da Canastra, Minas Gerais. Revista Brasileira de Botanica 33:333–341. Francoy T, Wittmann D, Drauschke M, Müller S, Steinhage V, Becerra-Laure MAF, De Jong D, Gonçalves LS. 2008. Identification of Africanized honey bees through wing morpholometrics: two fast and efficient procedures. Apidologie 39:488–494. Henderson A. 1986. A review of pollination studies in the Palmae. Botanical Review 52:221–259. Iwama S, Melhem TS. 1979. The pollen spectrum of the honey of Tetragonisca angustula angustula Latreille (Apidae; Meliponinae) Apidologie 10:275–295. Kiew R. 1997. Analysis of pollen from combs of Apis dorsata in peninsular Malaysia. pp. 174– 186. In: Mardan M, Sipat A, Yousoff KM, Kew HMSR, Abdullah MM, eds. Proceedings of the international conference on tropical bees and the environment. Beenet Asia; Penang, Malaysia. Kerkvliet JD, Beerlink JG. 1991. Pollen analysis of honey from the coastal plain of Surinam. Journal of Apicultural Research 20:249–253. Latham P, Mbuta K. 2011. Some honeybee plants of Bas-Congo Province, Democratic Republic of Congo. 2nd edition. 248 pp. (published by authors). Louveaux J. 1968. L’analyse pollinque des miels. pp. 325–362. In: Chauvin R, ed. Traité de biologie de l’abeille, vol. 3. Masson; Paris, France. 152 pp. 314 D.W. Roubik and J.E. Moreno Patiño Maurizio A. 1975. Microscopy of honey. pp. 240–257. In: Crane E, ed. Honey: A comprehensive survey. Heinemann; London, UK. 608 pp. O’Rourke MK, Buchmann SL. 1991. Standardized analytical techniques for bee-collected pollen. Environmental Entomology 20:507–513. Roubik DW. 1988. An overview of Africanized honey bee populations: reproduction, diet and competition. pp. 45–54. In: Needham G, Page R, Delfinado-Baker M, eds. Proceedings of the international conference on Africanized honey bees and bee mites. E. Horwood Ltd.; Chichester, UK. 572 pp. Roubik DW. 1989. Ecology and natural history of tropical bees. Cambridge University Press; New York, USA. 514 pp. Roubik DW. 1992. Loose niches in tropical communities: why are there so many trees and so few bees? pp. 327–354. In: Hunter MD, Ohgushi T, Price PW, eds. Resource distribution and animal-plant interactions. Academic Press, Ltd.; London, UK. 505 pp. Roubik DW. 1993. Direct costs of forest reproduction, bee-cycling and the efficiency of pollination modes. Journal of Bioscience 18:537–552. Roubik DW. 1996. African honey bees as exotic pollinators in French Guiana. pp. 173–182. In: Matheson A, Buchmann SL, O’Toole C, Westrich P, Williams IH, eds. The conservation of bees. Academic Press, Ltd.; London, UK. 254 pp. Roubik DW. 2005. Honeybees in Borneo. pp. 89–103. In Roubik DW, Sakai S, Hamid Karim A, eds. Pollination ecology and the rain forest: Sarawak studies. Springer-Verlag; New York, USA. 307 pp. Roubik DW. 2006. Stingless bee nesting biology. Apidologie 37:124–143. Roubik DW. 2009. Ecological impact on native bees by the invasive Africanized honey bee. Acta Biologica Colombiana 14:115–124. Roubik DW, Villanueva GR. 2009. Invasive Africanized honey bee impact on native solitary bees: a pollen resource and trap nest analysis. Biological Journal of the Linnean Society 98:152–160. Roubik DW, Schmalzel RJ, Moreno E. 1984. Estudio apibotanico de Panama: Cosecha y fuentes de polen y nectar usados por Apis mellifera y sus patrones estacionales y anuales. Technical Bulletin 24. Organización Internacional Regional de Sanidad Agropecuaria (OIRSA); San Salvador, El Salvador. 74 pp. Roubik DW, Moreno JE. 1991. Pollen and Spores of Barro Colorado Island. Missouri Botanical Garden Monographs in Systematic Botany, No. 36. 269 pp. Roubik DW, Moreno JE. 2009. Trigona corvina: an ecological study based on unusual nest structure and pollen analysis. Psyche, vol. 2009. Available at: http://www.hindawi.com/journals/ psyche/Article ID 268756, DOI:10.1155/2009/268756. Roubik DW, Sakai S, Gattesco F. 2003. Canopy flowers and certainty: loose niches revisited. pp. 360–368. In: Basset Y, Kitching R, Mijller S, Novotny V, eds. Arthropods of tropical forests: spatio-temporal dynamics and resource use in the canopy. Cambridge University Press; Cambridge, UK. 474 pp. Roubik DW, Hanson PE. 2004. Orchid bees of tropical America: biology and field guide. Spanish/ English edition. InBIO Press (Editorial INBio); Heredia, Costa Rica. 370 pp. Roulston T, Cane JH, Buchmann SL. 2000. What governs protein content of pollen: pollinator preferences, pollen–pistil interactions, or phylogeny? Ecological Monographs 70:617–643. Sornsathapornkul P, Owens JN. 1998. Pollination biology in a tropical Acacia Hybrid (A. mangium Willd. x A. auriculiformis A. Cunn. ex Benth.) Annals of Botany 81:631–645. Stockmarr J. 1971. Tablets with spores used in absolute pollen analysis. Pollen et Spores 13:615–621. Stone G, Raine NE, Prescott M, Willmer PG. 2003. Pollination ecology of acacias (Fabaceae, Mimosoideae). Australian Systematic Botany 16:103–118. Villanueva-Gutiérrez R, Roubik DW. 2004. Why are African honey bees and not European bees invasive? Pollen diet determination in community experiments. Apidologie 35:550–560. Villanueva-Gutiérrez R, Moguel-Ordóñez YB, Echazarreta-González C, Arana LG. 2009. Monofloral honeys in the Yucatán península, Mexico. Grana 48:214–223. Chapter 22 Important Bee Plants for African and Other Stingless Bees Robert Kajobe 22.1 Introduction Stingless bees are distributed throughout the tropical parts of the world (Michener 1979, 2000; Camargo and Pedro 1992; Roubik 1992). Their highest abundance may be in the neotropical region. There are hundreds of stingless bee species existing in the world and these vary in colony size, body size, body color, and biology (Roubik 1989, 1992; Michener 2000; Eardley 2004). The fauna of Afrotropical meliponines is smaller than that in neotropical or Indo-Malayan/Australasian faunas (Rasmussen and Cameron 2006). The Afrotropical meliponines have relatively fewer species (Eardley 2004) and genera (Moure 1961). The African meliponine has also a relatively low abundance in most parts of Africa (Darchen 1972; Kajobe and Roubik 2006). Stingless bee colonies have a single queen (Sakagami 1982; Velthuis et al. 2001). The founding of a new colony occurs by colony fission and swarming. Stingless bees, like solitary bees, produce brood, with an egg placed on top of a food mass in a sealed cell (Sakagami 1982). Unlike Apis, meliponines have no sting, mate only once, and do not use pure wax to build their nests or use water to cool the nests. Meliponines cannot freely swarm to reproduce and the males feed at flowers, while the gravid queens cannot fly (Roubik 2006). Stingless bees are dependent on flowering plants because plants offer bees food in the form of nectar and pollen. The colonies of stingless bees make less honey, as compared to honey bees. In the tropical regions, there are a variety of families and species of trees, shrubs, and agricultural crops that provide pollen and nectar to the bees. Most of the plants in this chapter were obtained from what the various authors considered to be important nectar and pollen source for the bees, and offering shelter or nesting tree cavities. R. Kajobe (*) National Agricultural Research Organisation (NARO), Rwebitaba Zonal Agricultural Research and Development Institute (ZARDI), P.O. Box 96, Fort Portal, Uganda e-mail: robertkajobe@gmail.com 315 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_22, © Springer Science+Business Media New York 2013 316 22.2 R. Kajobe Plants Used by Stingless Bees for Food Pollen and nectar are a primary reward to insect pollinators in general and to the bees in particular (Roubik 1989). As honey bees require large quantities of pollen and nectar at specific times, they utilize particular plant species for a limited period of time. During the flowering period, there is a significant movement by bees between plants of the same species. This in turn favors the successful cross-pollination of plants (Faegri and der Pijl 1979; Free 1970). The utilization of plant resources as food (pollen and nectar) by tropical bees has been extensively studied. Studies have been made in areas with different types of vegetation such as natural forests, grasslands, agricultural land, pasture land, or urban areas. Most of the methods for obtaining information about bee plants in an area are based on direct field observation of foraging bees on flowers. The analysis of bee plant pollen loads and palynological analysis of honey samples can provide the accurate depiction of the bee flora of an area (Johnson and Hubbell 1974; Hubbell and Johnson 1977; Sommeijer et al. 1983; Kleinert-Giovannini and Imperatriz-Fonseca 1987; Roubik 1989, 1992; Ramalho et al. 1990; Ramalho et al. 1994; Eltz et al. 2001; Kajobe 2008; Hilario and Imperatriz-Fonseca 2009). Many of such stingless bee foraging behavior studies are based on analysis of pollen and nectar diets, and bee foraging behavior. Our review provided the list of selected important bee plants (Table 22.1). The genera of stingless bees (Meliponula, Melipona and Trigona) and Apis melifera were used for this analysis. The important pollen plant species include Mimosaceae, Caesalpiniaceae, Myrtaceae, Asteraceae (sometimes called Compositae), and Moraceae. The other important families include Anacardiaceae, Euphorbiaceae, and Solanaceae. The most important plant species used were trees followed by shrubs, herbs, climbers, and runners in order of importance. 22.2.1 Pollen and Nectar Plant Sources Pollen is extensively used by many species of insects, and by bees for brood rearing. Many studies consider chemical composition and nutritive value of pollen, effect on brood rearing growth, and longevity of bees or colonies. Pollen ordinarily provides bees with their only natural source of protein, which is needed for larval development and also satisfies other dietary needs for lipids, sterols, vitamins, and minerals (Roubik 1989; Herbert 1992). The protein content of the pollen is a direct measure of pollen quality in the diet of the bee (Pernal and Currie 2001). Foraged pollen loads are good indicators of the surrounding flowering plant species that provide pollen for the bees. They also indicate availability of dominant food resources for the different pollinators in an ecosystem. Stingless bees collect nectar from flowering plants and transport it to the nest, to be used in feeding larvae and for preparing honey. Kajobe (2006b) found that nectar concentration of plant species differs considerably in the amount and concentration of sugar they produce. He found that certain plant species produce large quantities of nectar to attract more pollinators, or 22 Trigona A. mellifera N P x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x Important Bee Plants for African and Other Stingless Bees Table 22.1 Some of the important bee plants, their life form, nectar (N) and pollen (P) sources Genera of stingles bees Bee plants Plant form Reference Meliponula Melipona Acanthaceae Acanthaceae Type Vit and Ricciardelli D’Albore (1994b) x Avicennia Vit and Ricciardelli D’Albore (1994b) x Amaranthaceae Alternanthera Dórea et al. (2010) Amaryllidaceae Allium cepa Crop Adjare (1990) Anacardiaceae Anacardium occidentale Tree Katende et al. (1995) Astronium fraxinifolium D’Apolito et al. (2010) Lannea barteri Tree Katende et al. (1995) Mangifera indica Tree Mouga (1984) Spondias mombim Tree Sommeijer et al. (1983) and Dórea et al. (2010) x Spondias radlkoferri Tree Dórea et al. (2010) Tapirira guianensis Tree Absy et al. (1984) and Dórea et al. (2010) x Apocynaceae Adenium obesum Shrub Crane et al. (1984) Couma utilis Rech and Absy (2011) Rauvolfia caffra Tree Crane et al. (1984) Arecaceae Attalea maripa Rech and Absy (2011) Bactris gasipaes Rech and Absy (2011) Cocos nucifera Tree Adjare (1990) Elaeis guineensis Tree Dórea et al. (2010) (continued) 317 Bee plants Reference Tree Rech and Absy (2011) Rech and Absy (2011) Crane et al. (1984) Genera of stingles bees Meliponula Melipona Crop Shrub Shrub Shrub x Orth (1983) Orth (1983) Orth (1983) Kajobe and Roubik (2006) Dórea et al. (2010) Dórea et al. (2010) Horn (2004) Dórea et al. (2010) Kajobe and Roubik (2006) Kajobe and Roubik (2006) Vit and Ricciardelli D’Albore (1994a) Leonhardt et al. (2007) Tree Tree Tree Ramalho et al. (1985) Ramalho et al. (1985) Ramalho et al. (1985) Tree Tree Katende et al. (1995) Kajobe (2006a) N P x x x x x x x x A. mellifera x x Herb Herb Trigona x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x R. Kajobe Euterpe precatoria Leopoldinia pulchra Phoenix reclinata Asparagaceae Agave sisalana Asteraceae Baccharis sp. Baccharis erioclada Baccharis semiserrata Bidens pilosa Elephantopus Eupatorium Helianthus annuus Mikania Vernonia amygdalina Vernonia auriculifera Vernonia pauciflora Wedelia trilobata Balsaminaceae Impatients balsamina Impatients sultanii Impatiens walleriana Bignoniaceae Jacaranda mimosifolia Markhamia lutea Plant form 318 Table 22.1 (continued) Bee plants Reference Tree Tree Tree Tree Crane et al. (1984) Crane et al. (1984) and Katende et al. (1995) Crane et al. (1984) and Katende et al. (1995) Crane et al. (1984) and Katende et al. (1995) Genera of stingles bees Meliponula Melipona Trigona A. mellifera N P x x x x x x x x x x x x x x x x 22 Vit and Ricciardelli D’Albore (1994b) x x Herb Orth (1983) x Tree Orth (1983) x Tree Tree Shrub Tree Vit and Ricciardelli D’Albore (1994b) Horn (2004) Horn (2004) Kajobe (2006b) Vit and Ricciardelli D’Albore (1994a) Climber Katende et al. (1995) x x x Vine-like Vine Vine Katende et al. (1995) Katende et al. (1995) Katende et al. (1995) x x x x x x x x x Vit and Ricciardelli D’Albore (1994b) Tree Leonhardt et al. (2007) Katende et al. (1995) x x x x x x x x x x x x x x x x x x Important Bee Plants for African and Other Stingless Bees Boraginaceae Cordia africana Cordia millenii Cordia monoica Cordia sinensis Burseraceae Protium Type Cannaceae Canna indica Caricaceae Carica papaya Combretaceae Combretaceae Type Combretum collinum Combretum molle Combretum Combretum Commelinaceae Commelina africana Cucurbitaceae Citrullus lanatus Cucumis sativus Cucurbita pepo Cunoniaceae Weinmannia Ebenaceae Diospyros Diospyros mespiliformis Plant form x x x 319 x x x (continued) Bee plants Plant form Vit and Ricciardelli D’Albore (1994b) Absy et al. (1984) and Rech and Absy (2011) Kleinert-Giovannini (1989) Rech and Absy (2011) Kajobe (2006b) Genera of stingles bees Meliponula Melipona x x x Leonhardt et al. (2007) Vit and Ricciardelli D’Albore (1994b) Adjare (1990) Vit and Ricciardelli D’Albore (1994b) Vit and Ricciardelli D’Albore (1994b) Adjare (1990) Vit and Ricciardelli D’Albore (1994b) A. mellifera x x x N x x x x Kajobe (2006a) Kajobe (2006a) Kajobe (2006a) Vit and Ricciardelli D’Albore (1994b) Leonhardt et al. (2007) Rech and Absy (2011) Kajobe (2006a) Vit and Ricciardelli D’Albore (1994b) Crane et al. (1984) Leonhardt et al. (2007) Kajobe (2006b) Trigona x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x P x x x x x x x x x x R. Kajobe Euphorbiaceae Acalypha Alchornea discolor Alchornea sidifolia Aparisthmium cordatum Croton macrostachyus Tree Euphorbia splendens Mallotus Ricinus Fabaceae, Caesalpinioideae Acrocarpus fraxinifolius Tree Afzelia africana Tree Caesalpinia decapetala Shrub Cassia Cassia fistula Cassia undulata Cynometra alexandri Tree Julbernardia Parkinsonia aculeata Tree Peltophorum pterocarpum Tamarindus indica Tree Fabaceae, Faboideae Cajanus cajan Shrub Crotalaria Crotalaria Type Gliricidia sepium Tree Machaerium Type Reference 320 Table 22.1 (continued) Machaerium Vicia Fabaceae, Mimosoideae Acacia sp. Albizia coriaria Albizia gummifera Archidendron jiringa Calliandra calothyrsus Faidherbia albida Leucaena leucocephala Mimosa bimucronata Mimosa caesalpiniaefolia Mimosa pudica Mimosa scabrella Mimosa scabrella Schrankia Lamiaceae Gmelina arborea Vitex doniana Lythraceae Malvaceae Grewia Grewia bicolor Triumfetta Meliaceae Azadirachta indica Carapa guianensis Ekebergia capensis Melia azedarach Shrub Vit and Ricciardelli D’Albore (1994a) Vit and Ricciardelli D’Albore (1994b) Tree Tree Tree Katende et al. (1995) and Dórea et al. (2010) Katende et al. (1995) Katende et al. (1995) Leonhardt et al. (2007) Kajobe (2006b) Adjare (1990) Adjare (1990) Vit and Ricciardelli D’Albore (1994a) Vit and Ricciardelli D’Albore (1994b) Vit and Ricciardelli D’Albore (1994a, b) Vit and Ricciardelli D’Albore (1994a) Adjare (1990) Vit and Ricciardelli D’Albore (1994b) Shrub Tree Tree Tree Herb Tree Tree Tree Tree Tree Tree Tree Tree Adjare (1990) Adjare (1990) Vit and Ricciardelli D’Albore (1994b) Vit and Ricciardelli D’Albore (1994b) Adjare (1990) Vit and Ricciardelli D’Albore (1994b) Adjare (1990) Rech and Absy (2011) Adjare (1990) Adjare (1990) Genera of stingles bees Meliponula Melipona Trigona A. mellifera x x x x x x x x x x N P x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x (continued) 321 Reference Important Bee Plants for African and Other Stingless Bees Plant form 22 Bee plants Bee plants Moraceae Artocarpus heterophyllus Moraceae Type Morus alba Moringaceae Moringa oleifera Musaceae Musa sp. Myrtaceae Eucalyptus Reference Tree D’Apolito et al. (2010) Vit and Ricciardelli D’Albore (1994b) Adjare (1990) Tree Adjare (1990) Herb Kajobe and Roubik (2006) Vit and Ricciardelli D’Albore (1994b) Kajobe and Roubik (2006) and D’Apolito et al. (2010) Kajobe (2006b) Tree Tree Adjare (1990) Vine Kajobe and Roubik (2006) Tree Vit and Ricciardelli D’Albore (1994a) Vit and Ricciardelli D’Albore (1994b) Climber Adjare (1990) Crop Rech and Absy (2011) Kajobe and Roubik (2006) Vit and Ricciardelli D’Albore (1994b) Vit and Ricciardelli D’Albore (1994b) Genera of stingles bees Meliponula Melipona Trigona N P x x x x x x x x A. mellifera x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x R. Kajobe Syzygium Oleaceae Olea capensis Passifloraceae Passiflora Piperaceae Piper Piperaceae Type Phytolaccaceae Phytolacca dodecandra Poaceae Pariana Zea mays Polygonaceae Antigonon Plant form 322 Table 22.1 (continued) Bee plants Reference Tree Adjare (1990) Genera of stingles bees Meliponula Melipona Trigona A. mellifera N P x x x x x x x x x x x x 22 Shrub Tree Tree Vit and Ricciardelli D’Albore (1994b) Adjare (1990) Vit and Ricciardelli D’Albore (1994b) Adjare (1990) Adjare (1990) Kajobe (2006a) Leonhardt et al. (2007) D’Apolito et al. (2010) Tree Tree D’Apolito et al. (2010) Vit and Ricciardelli D’Albore (1994b) Vit and Ricciardelli D’Albore (1994b) x x x x x Tree Tree Shrub Leonhardt et al. (2007) Adjare (1990) Adjare (1990) Vit and Ricciardelli D’Albore (1994b) Adjare (1990) x x x x x x x x x x x x x x x x x x x x x Shrub Adjare (1990) Shrub or tree Adjare (1990) x x x x x x x x x x x (continued) 323 x x Important Bee Plants for African and Other Stingless Bees Proteaceae Faurea saligna Rhamnaceae Rhamnaceae Type Ziziphus abyssinica Rosaceae Eriobotrya japonica Prunus africana Rubiaceae Coffea Ixora javanica Richardia brasiliensis Rutaceae Calodendrum capense Citrus Citrus Zanthoxylum Salicaceae Dovyalis abyssinica Flacourtia indica Sapindaceae Allophylus rubifolius Dodonaea angustifolia Sapotaceae Butyrospermum paradoxum Scrophulariaceae Solanaceae Datura suaveolens Plant form Bee plants Solanaceae Type Solanum Stilbaceae Nuxia congesta Urticaceae Cecropia Verbenaceae Aloysia triphylla Plant form Reference Genera of stingles bees Meliponula Melipona Vit and Ricciardelli D’Albore (1994b) Vit and Ricciardelli D’Albore (1994b) Tree Kajobe (2006a) Vit and Ricciardelli D’Albore (1994b) D’Apolito et al. (2010) 324 Table 22.1 (continued) Trigona A. mellifera N x x P x x x x x x x x x R. Kajobe 22 Important Bee Plants for African and Other Stingless Bees 325 large pollinators such as birds or mammals, while others produce little. Nectar secretion among plant species differs, depending on the time of day and environmental factors. The frequencies of visitors for nectar gathering have been strongly correlated with the quantity of sugar and chemical constituents of the nectar (Biesmeijer 1997). The characterization and quantification of pollen is an important indicator of the geographical and botanical origin of bee-collected forage. The pollen spectrum of honey depends on the plants (agricultural and forest) where it is collected. In tropical regions there are some important palynological studies (Eltz et al. 2001; Villanueva and Roubik 2004; Hilario and Imperatriz-Fonseca 2009). For the African countries, some palynological studies have been undertaken. There are also lists of bee plants made from field observations at flowers (Kajobe 2008; Gikungu 2006; Munyuli 2009). Results from the palynological studies show that stingless bees and honey bees collect pollen from a wide range of plant species. This may not be surprising because these eusocial bees have perennial colonies which make them forage for food throughout the year. The bees cannot therefore specialize on one or a few food plant resources. Rather, they are generalists, using a wide range of food plant resources (Michener 1974; Roubik 1989; Biesmeijer 1997; Hilario and Imperatriz-Fonseca 2009). In a comparative palynological study among stingless bees and honey bees in Uganda, Kajobe (2006a) finds a considerable overlap in pollen resources for three bee species (Apis mellifera, Meliponula bocandei, and Meliponula nebulata), and concludes that the overlap represented the bees’ attraction to few sources or lack of attractive alternatives for other pollen sources. Such overlap may also be a partial adjustment according to local conditions, including kinds and abundance of competitors and distance to pollen sources (Sommeijer et al. 1983; Roubik et al. 1986; Kleinert-Giovannini and Imperatriz-Fonseca 1989; Roubik 1989; Biesmeijer 1997; Slaa 2003; Roubik and Villanueva-Gutiérrez 2009). 22.3 22.3.1 Plants Used for Stingless Bee Nests Nesting Sites in Trees Nests are notable points of bee activity which exhibit spectacular examples of animal architecture. Nesting biology is therefore a highly visible aspect of stingless bee behavior (Michener 1974; Roubik 2006). Stingless bees nest in tree cavities (Figs. 22.1 and 22.2), house wall crevices, and underground, with trees containing more nests as compared to the other nesting substrate (Michener 1974; Hubbell and Johnson 1977; Roubik 1989, 2006; Eltz et al. 2003; Slaa 2003; Martins et al. 2004; Kajobe 2007). Many Melipona (such as M. quadrifasciata and M. marginata), Scaptotrigona (such as S. postica and S. xanthotricha), and Plebeia build their nests inside living trees or branch hollows. Other meliponine nests, such as those built by Frieseomelitta, Friesella schrottkyi, and Tetragonisca angustula, can be found in available hollows in dead trees, fences, and walls. Geotrigona, such as Geotrigona mombucae, Schwarziana 326 R. Kajobe Fig. 22.1 Two nest entrances of Meliponula ferruginea in Bwindi Impenetrable National Park (BINP), Uganda. Photo: R. Kajobe Fig. 22.2 Nest of Meliponula bocandei in a live tree predated upon by Batwa Pygmies in Bwindi Impenetrable National Park (BINP), Uganda. Photo: R. Kajobe quadripunctata, Melipona quinquefasciata, and some Paratrigona, nest in underground existing cavities, often abandoned ant and termite nests. Melipona bicolor chooses to nest in damp places with fresh air, including tree trunks near soil. Some Partamona and Scaura latitarsis build their nests inside living termite nests. Further, nests of Trigona spinipes, Trigona truculenta, and other Trigona species are built in exposed areas, supported by tree branches, walls, or similar places (Kerr et al. 1967; Michener 1974; Sakagami 1982). 22 Important Bee Plants for African and Other Stingless Bees 327 A broad range of trees (194 trees of 57 species) may be used as nesting sites. The predominant plant families include Anacardiaceae, Euphorbiaceae, Fabaceae, Dipterocarpaceae, and Lauraceae (Eltz et al. 2003; Slaa 2003; Martins et al. 2004; Kajobe 2007). Generally, nest tree diversity is high and many tree species are represented by a single individual (Table 22.2). Stingless bees are known to be generalists in relation to nest site selection (Hubbell and Johnson 1977; Roubik 1989). Relatively few bee species have a clear preference to nest in a particular tree species. The non-specificity normally results in high overlap in the use of nest tree species (Hubbell and Johnson 1977). In few cases where there appears to be some nest site specificity, the preference is for the location or the structure of that particular tree and not for the species of the tree (Kajobe 2007). Most stingless bee nests are located in or under large to very large canopy trees. For example, Eltz et al. (2003) found that a total of 86.1% of nest trees were larger than 60 cm dbh and 73.0% were between 60 and 120 cm dbh. Kajobe (2007) found that over 79% of nests of all the tree cavity nesting species were situated in large trees of dbh above 60 cm. These authors concluded that tree species probably differed in their tendency to form suitable cavities due to differences in wood and growth characteristics. These stingless bees are of greatly different worker and colony sizes but build nests in tree cavities of roughly the same dimensions. Roubik (1989) explained that this may probably be because cavity-nesting meliponines can significantly reduce the size of a tree cavity by blocking part of it. Nests in large trunks are in most cases well insulated. In most cases stingless bees nest on either living trees or dead wood randomly. However, Eltz et al. (2003) found that at least 8.5% of the nest trees were dead while 91.5% were living trees. Stingless bees nest in any type of tree species suggesting that they are opportunistic in selection of a nest site and use whatever tree species that presents a cavity of the correct dimensions and purpose. In the forest, unoccupied tree cavities are fairly common (Johnson and Hubbell 1986). However, the size of the tree hole leading to the nest cavity markedly influences acceptability to bees (Roubik 1983) and whether resident colonies saturate their environment with bees, regardless of nest abundance, is an open question. In some nest trees, there are cases of multiple nests in one tree. In most cases the mature nest tree of such species is characteristically big, and has cavities left after its core of living tree is rotten from rainwater entering through the scars left by its fallen branches (Roubik 1989). The availability of such cavities can account for the clumping of the stingless bee nests. Eltz et al. (2003) reported that over 40% of nest trees contained more than one (maximum: 8) stingless bee nest in an undisturbed forest in Malaysia. 22.3.2 Tree Nest Height Partitioning Kajobe and Roubik (2006) found some degree of height partitioning with regard to the larger stingless bee species. In general, the mean height given for a species building nests in tree cavities was biased towards lower heights, since nests are mostly 328 R. Kajobe Table 22.2 Trees used for nesting by stingless bee species in the Afrotropical, Indo-Malayan, and Neotropical regions (reference: Eltz et al. 2001 = Indo-Malayan; Martins et al. 2004 = Neotropical; Kajobe 2007 = Afro-tropical) Nest tree species Stingless bee species Afrotropical Indo-Malayan Neotropical Achariaceae Hydnocarpus sp. Not named x Alangiaceae Alangium chinense Mb Mn Mf x Anacardiaceae Gluta oba Not named x Gluta sabahana Not named x Gluta Not named x Myracrodruon urundeuva Fv x Schinopsis brasiliensis Fv x Spondias tuberosa Ms Fd Fv x Apocynaceae Aspidosperma pyrifolium Ms x Araliaceae Polyscias fulva Mn x Schefflera barteri Mf x Bignoniaceae Tabebuia caraiba Fd x Burseraceae Commiphora leptophloeos Ms Fv x Calophyllaceae Calophyllum sp. Not named x Celastraceae Lophopetalum sp. Not named x Maytenus acuminata Mn Mf x Chrysobalanaceae Licania rigida Ms x Mb Mn Mf Afrotropical x Mb Mn Mf x Not named Not named Not named Mn Mf Indo-Malayan Neotropical Important Bee Plants for African and Other Stingless Bees Stingless bee species Parinari excelsa Cupressaceae Cupressus lusitanica Dipterocarpaceae Dipterocarpus grandiflorus Dipterocarpus Shorea sp. Ericaceae Agauria salicifolia Euphorbiaceae Chaetocarpus castanocarpus Cnidoscolus phyllacanthus Trigonopleura malayana Fabaceae, Caesalpiniodeae Caesalpinia pyramidalis Intsia palembanica Sympetalandra borneensis Fabaceae, Mimosoideae Albizia gummifera Anadenanthera colubrina Dialium Mimosa acutistipula Piptadenia communis Lamiaceae Premna angolensis Lauraceae Dehaasia Eusideroxylon zwageri Litsea caulocarpa 22 Nest tree species x x x x Not named x Not named x Not named Not named x x Ms Ms Fd Mb Ms Fv x Mf x x Not named x Fd x x Ms Mn x x x x (continued) 329 Not named Not named Not named Stingless bee species Afrotropical Not named Not named Mb Mb x x Mb Mn Mn Mf x Mf Mf x x x x Mb Mn x Mn x Mn x x Mf Not named Mb Neotropical x Not named Mb Indo-Malayan x x x Mn Mf x Mn Mf Mf x x Not named x R. Kajobe Litsea Phoebe macrophylla Malvaceae Glyphaea brevis Scaphium affine Triumfetta macrophylla Melastomataceae Dichaetanthera corymbosa Meliaceae Carapa grandiflora Ekebergia capensis Entandrophragma cylindricum Entandrophragma excelsum Melianthaceae Bersama abyssinica Monimiaceae Xymalos monospora Moraceae Ficus natalensis Ficus Ficus Myricaceae Myrica salicifolia Myrtaceae Eucalyptus Syzygium guineense Syzigium Olacaceae 330 Table 22.2 (continued) Nest tree species Nest tree species Stingless bee species Afrotropical Indo-Malayan Neotropical 22 Important Bee Plants for African and Other Stingless Bees Scorodocarpus borneensis Not named x Strombosia scheffleri Mb Mn Mf x Penaeaceae Olinia usamberensis Mn Mf x Podocarpaceae Podocarpus milanjianus Mn x Primulaceae Maesa lanceolata Mb Mf x Proteaceae Faurea saligna Mn Mf x Putranjivaceae Drypetes gerrardii Mb Mf x Rosaceae Hagenia abyssinica Mn x Prunus africana Mb Mn Mf x Rutaceae Zanthoxylum gilletii Mn Mf x Zanthoxylum macrophyllum Mf x Sapotaceae Chrysophyllum albidum Mb Mn x Chrysophyllum gorungosanum Mn Mf x Theaceae Ficalhoa laurifolia Mb Mn Mf x Thymelaeaceae Wikstroemia Not named x Key: MB = Meliponula bocandei, Mn = Meliponula nebulata, Mf = Meliponula ferruginea, Ms = Melipona subnitida, Ma = Melipona asilvai, Fd = Frieseomelitta doederleini, Fv = Frieseomelitta varia 331 332 R. Kajobe found near ground level. Roubik (1979, 1983) found that eusocial bee species do not often make their nest entrances level with the ground or in the tallest branches of forest trees but a height of 30 m seems to be their normal limit. The differences in height are explained by the fact that different species are most often active at different strata above the ground. 22.4 Conclusions The importance of plants to stingless bees was discussed based on available literature in tropical areas of the world. In this chapter emphasis was made on resources needed by stingless bees to survive, mainly food (pollen and nectar) and shelter (tree nest) availability. The data showed that stingless bees collect pollen and nectar from a wide range of plant species. Also, a broad range of tree species were used as nesting sites for stingless bees. Acknowledgements I acknowledge the important contribution of Prof. Carlos Rosa who helped by providing me with relevant literature for this chapter. 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(Hymenoptera: Apidae) in the Dourados region, Mato Grosso do Sul state (Brazil). Acta Botanica Brasilica 24(4):898–904. Darchen R. 1972. Ecologie de quelques trigones (Trigona sp.) de la savane de Lamto (Cote D’Ivoire). Apidologie 3:341–367. Dórea MC, Novais JS, Santos FSR. 2010. Botanical profile of bee pollen from the southern coastal region of Bahia, Brazil. Acta Botanica Brasilica 24:862–867. Eardley CD. 2004. Taxonomic revision of the African stingless bees (Apoidea: Apidae: Apinae: Meliponini). African Plant Protection 10:63–96. Eltz T, Bruhl CA, van der Kaars S, Chey VK, Linsenmair KE. 2001. Pollen foraging and resource partitioning of stingless bees in relation to flowering dynamics in a Southeast Asian tropical rainforest. Inspects Sociaux 48:273–279. Eltz T, Bruhl CA, Imiyabir Z, Linsenmair KE. 2003. Nesting and nest trees of stingless bees (Apidae: Meliponini) in lowland dipterocarp forests of Sabah, Malaysia, with implications for forest management. 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The influence of Pollen quality on foraging behavior in honeybees (Apis mellifera L.). Behavioral Ecology and Sociobiology 51:53–68. Ramalho M, Imperatriz-Fonseca VL, Kleinert-Giovannini A, Cortopassi-Laurino M. 1985. Exploitation of floral resources by Plebeia remota Holmberg (Apidae, Meliponinae). Apidologie 16:307–330. Ramalho M, Kleinert-Giovannini A, Imperatriz-Fonseca VL. 1990. Important bee plants for stingless bees (Melipona and Trigonini) and Africanized honeybees (Apis mellifera) in Neotropical habitats: a review. Apidologie 21:469–488. Ramalho M, Giannini TC, Malagodi-Braga KS, Imperatriz-Fonseca VL. 1994. Pollen harvest by stingless bee foragers (Hymenoptera, Apidae, Meliponinae). Grana 33:239–244. Rasmussen C, Cameron SA. 2006. A molecular phylogeny of the Old World stingless bees (Hymenoptera: Apidae: Meliponini) and the nonmonophyly of the large genus Trigona. Systematic Entomology 32:26–39. Rech AR, Absy ML. 2011. 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Invasive africanized honey bee impact on native solitary bees: a pollen resource and trap nest analysis. Biological Journal of the Linnean Society. 98:152–160. Roubik DW, Moreno JE, Vergara C, Wittmann D. 1986. Sporadic food competition with the African honey bee: projected impact on neotropical social bees. Journal of Tropical Ecology 2:97–111. Sakagami SF. 1982. Stingless bees. pp. 361–423. In: Hermann HR, ed. Social insects, vol. 3. Academic Press; New York, USA. 459 pp. Slaa J. 2003 Foraging ecology of stingless bees: from individual behaviour to community ecology. PhD Dissertation, Department of Biology, Faculty of Science, Utrecht University, Elinkwijk, Utrecht, The Netherlands. 181 pp. Sommeijer MJ, de Rooy GA, Punt W, de Bruijn LLM. 1983. A comparative study of foraging behaviour and pollen resources of various stingless bees (Hym., Meliponinae) and honeybees (Hym., Apidae) in Trinidad, West-Indies. Apidologie 14:205–224. Velthuis HHW, Roeling A, Imperatriz-Fonseca VL. 2001. Repartition of reproduction among queens in the polygynous stingless bee Melipona bicolor. Proceedings of the Section Experimental and Applied Entomology of the Netherlands Entomological Society 12:45–49. Villanueva RG, Roubik DW. 2004. Why are African honey bees and not European bees invasive? Pollen diet diversity in community experiments. Apidologie 35:481–491. 22 Important Bee Plants for African and Other Stingless Bees 335 Vit P, Ricciardelli D’Albore G. 1994a. Melissopalynology for stingless bees (Apidae: Meliponinae) from Venezuela. Journal of Apicultural Research 33:145–154. Vit P, Ricciardelli D’Albore G. 1994b. Palinología comparada en miel y polen de abejas sin aguijón (Hymenoptera: Apidae: Meliponinae) de Venezuela. pp. 121–132. In: Mateu Andrés I, Dupré Ollivier M, Güemes Heras J, Burgaz Moreno ME, eds. Trabajos de palinología básica y aplicada, X Simposio de Palinología (APLE). Universitat de Valencia, Valencia, Spain. 313 pp. Chapter 23 Botanical Origin of Pot-Honey from Tetragonisca angustula Latreille in Colombia Diana Obregón, Ángela Rodríguez-C, Fermín J. Chamorro, and Guiomar Nates-Parra 23.1 Introduction Tetragonisca angustula, known in Colombia as “angelita”, is the stingless bee most widely distributed in the country, found in all natural regions below 1,800 m elevation (Nates-Parra 2001). Tetragonisca angustula is widely kept and recognized for medicinal value attributed to its honey, commercialized in various local markets (Cepeda et al. 2009). However, so far there has been no complete characterization of the honey’s botanical origin. In studies conducted in other countries, T. angustula shows a broad pollen spectrum, classifying it as a bee with a generalist foraging habit (Cortopassi-Laurino 1982), but with some plant families represented by many species, such as Euphorbiaceae, Asteraceae, and Myrtaceae (Carvalho and Marchini 1999; Braga et al. 2009; Flores and Sánchez 2010). In this context, our investigation aims to provide knowledge about the plants that are nectar sources for this bee and help to identify the honey in different regions. 23.2 Honey Collection and Pollen Frequency Classes The study was conducted between 2008 and 2010, in different regions and agroecosystems. Seventy-six honey samples were collected in the Andean region, in the following states: Tolima (1), Antioquia (6), Cauca (1), Cundinamarca (12), and Santander (29), and in the Caribbean region: Cesar (1), Magdalena (24), and Sucre (2). D. Obregón • Á. Rodríguez-C • F.J. Chamorro • G. Nates-Parra (*) Laboratorio de Investigaciones en Abejas LABUN 128, Departamento de Biología, Universidad Nacional de Colombia, Edificio 421, Carrera 30 No. 45-03, Ciudad Universitaria, Bogotá, DC, Colombia e-mail: mgnatesp@unal.edu.co 337 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_23, © Springer Science+Business Media New York 2013 338 D. Obregón et al. Pollen in honey was prepared using the acetolysis method (Erdtman 1952) and mounted on a slide. To calculate the frequency of pollen types, 250 and 400 grains were counted per sample (depending upon the pollen content and sample volume). Identification of pollen species was carried out by comparison to pollen collections in the Bee Research Laboratory LABUN, and the Palynology and Paleoecology Laboratory of Instituto de Ciencias Naturales, both located at Universidad Nacional de Colombia, and also by using pollen atlases: Moreno and Devia (1982); Roubik and Moreno (1991); Colinvaux et al. (1999); Bush and Weng (2007). Frequency classes (predominant pollen “D” (> 45%); secondary pollen, “S” (16–45%); important minor pollen, “M” (3–15%); minor pollen, “m” (between >1 and <3%)) (Louveaux et al. 1970) were estimated for the pollen types to determine the types of honeys. Honey was characterized as “monofloral” when a species was predominant, “bifloral” when two pollen types had secondary percentages, and “multifloral” for other honeys. Pollen belonging to plants without nectar production was excluded from the counts of the honey because it is considered irrelevant to the botanical origin of nectar sources (von der Ohe et al. 2004; Barth 2005). The botanical names of pollen types were based on “The plant list.” The samples were classified in groups according to similarity by a cluster analysis based on Euclidean distances and correspondence analysis using the statistical package Past (Hammer et al. 2001). 23.3 Botanical and Geographic Origin of Pot-Honey A total of 306 pollen types, belonging to 49 families, were identified. Twenty-five pollen types were not included in the analysis because they belong to plants that do not produce nectar, such as Piper aduncum, Cecropia, Acalypha, Trema micrantha, Myrsine, Theobroma cacao, Alchornea, Fraxinus uhdei, Ricinus communis, Amaranthaceae, Moraceae, Poaceae, Arecaceae, and Cyperaceae types. Frequency classes in 76 honey samples of Tetragonisca angustula are presented in Table 23.1. The families with a higher representation in the number of pollen types were Asteraceae (47), Fabaceae (39), Malvaceae (11), Rubiaceae (11), Melastomataceae (11), and Euphorbiaceae (11). The samples had an average of 13 ± 5.30 pollen types. The most frequent pollen types were Heliocarpus americanus occurring in 46% of the samples, followed by Coffea arabica 35.50%, Citrus 32.89%, and Myrcia Type 30.26%. The most important pollen types are illustrated in Fig. 23.1. The general palynological spectrum used by T. angustula, according to the analyzed samples, is large. At the plant family level, pollen types are similar to those found in other studies (Iwama and Melhem 1979; Cortopassi-Laurino 1982; ImperatrizFonseca et al. 1984; Knoll 1990; Carvalho and Marchini 1999; Vossler 2007; Flores and Sánchez 2010), but generic and specific levels differ greatly, probably due to the plant composition of the ecosystems in which samples were taken. This has also been observed in other studies when comparing different locations and seasons, where 23 Botanical Origin of Pot-Honey from Tetragonisca angustula Latreille in Colombia 339 Table 23.1 List of pollen types with frequency of occurrence percentage >10% and with the respective frequency classes in 76 honey samples of Tetragonisca angustula Family Pollen type FO D S IM m Acanthaceae Justicia 10.5 1.3 9.2 Apiaceae Apiaceae Type 18.4 9.2 9.2 Boraginaceae Cordia alliodora 14.5 2.6 2.6 9.2 Caryophyllaceae Stellaria Type 30.3 1.3 13.2 15.8 Cleomaceae Cleome Type 19.7 7.9 9.2 2.6 Asteraceae Asteraceae Type 10.5 3.9 6.6 Austroeupatorium 17.1 2.6 5.3 9.2 inulifolium Critonia aff. morifolia 10.5 1.3 9.2 Hypochaeris radicata 10.5 1.3 9.2 Euphorbiaceae Croton sp. 26.3 9.2 17.1 Euphorbia cotinifolia 11.8 1.3 1.3 1.3 7.9 Euphorbia hirta 15.8 2.6 5.3 5.3 2.6 Euphorbiaceae Type 15.8 1.3 1.3 3.9 9.2 Lamiaceae Hyptis brachiata 10.5 1.3 2.6 6.6 Hyptis 14.5 1.3 13.2 Fabaceae, Fabaceae Type 1 15.8 1.3 3.9 10.5 Caesalpinioideae Fabaceae Type 2 13.2 1.3 1.3 2.6 7.9 Fabaceae, Mimosa 30.3 1.3 6.6 22.4 Mimosoideae Fabaceae, Faboideae Fabaceae Type 3 17.1 3.9 6.6 6.6 Loranthaceae Oryctanthus sp. 17.1 1.3 1.3 5.3 9.2 Lythraceae Adenaria floribunda 15.8 2.6 7.9 5.3 Malpighiaceae Tetrapteris 11.8 1.3 3.9 6.6 Malvaceae/ Heliocarpus 46.1 5.3 5.3 21.1 14.5 Grewioideae americanus Muntingiaceae Muntingia calabura 25.0 1.3 3.9 5.3 14.5 Myrtaceae Myrcia Type 30.3 1.3 3.9 15.8 9.2 Myrtaceae Type 25.0 1.3 9.2 14.5 Syzygium jambos 15.8 2.6 5.3 7.9 Rhamnaceae Gouania polygama 21.1 11.8 2.6 6.6 Rubiaceae Coffea arabica 35.5 11.8 1.3 9.2 13.2 Rutaceae Citrus 32.9 1.3 2.6 11.8 17.1 Verbenaceae Lantana aff. fucata 11.8 3.9 6.6 1.3 Vitaceae Vitis tiliifolia 15.8 1.3 3.9 10.5 FO frequency of occurrence percentage. Frequency classes: Value indicating the number of samples in which different pollen types appear in the following percentages: D dominant pollen (>45%), S Secondary pollen (16–45%), IM Important minor pollen (3–15%), m minor pollen (<3%) T. angustula has to take advantage of all food sources it has within its reach (Landaverde et al. 2004). Analyzing the individual samples, each of which corresponds to a certain locality and specific sampling date, we can observe that they each contain only a few pollen types. This may indicate that the worker bees of the same colony have a tendency to be constant in their visits to flowers of the same species, and that they have some favorite sources for nectar, especially those with massive blooms. 340 D. Obregón et al. Fig. 23.1 Some important pollen types found in honey samples of Tetragonisca angustula: (a, b) Austroeupatorium inulifolium, (c, d) Calycolpus moritzianus, (e, f) Citrus Type, (g, h) Coffea arabica, (i, j) Euphorbia hirta, (k, l) Gouania polygamya, (m, n) Heliocarpus americanus, (o, p) Muntingia calabura, (q, r) Spananthe paniculata, (s, t) Toxicodendron striatum. Scale: 10 mm. Photos: LABUN (Laboratorio de Investigaciones en Abejas) Archives at Universidad Nacional de Colombia 23 Botanical Origin of Pot-Honey from Tetragonisca angustula Latreille in Colombia 341 According to the frequency classes, 40 monofloral, 13 bifloral, and 23 multifloral honeys (Table 23.2) were detected from different regions of the country (Fig. 23.2). Based on results of the multivariate analyses of similarity and correspondence, some groupings of samples were found that allow us to classify groups by botanical and geographical origin, as follows: – Honey from coffee agroecosystems: Forty-four samples from coffee agroecosystems: nine monofloral Coffea arabica honeys, nine monofloral Gouania polygama honeys, four monofloral Heliocarpus americanus honeys, 16 honeys from Sierra Nevada de Santa Marta group (Magdalena state), and six mixed honeys similar in composition but with different geographical origin. – Honey from dry ecosystems: Twelve samples from a specific locality in Santander called Giron group. – Undifferentiated honey: Nineteen samples from different places, which were not clearly differentiated by the analysis. Among the honey samples, palynological composition categorized honey from coffee regions as a typical Colombian agroecosystem, located between 1,000 and 2,000 m elevation (García and Vallejo 2002). Some samples show a clear botanical origin of typical regional plants and can be classified as monofloral honey of Coffea arabica, Gouania polygama, or Heliocarpus amercianus. There is also one group with a clear geographic origin from the region called Sierra Nevada de Santa Marta, which also belongs to the coffee area. The remaining samples were not clearly grouped (mixed honeys). There were nine monofloral samples of Coffea arabica, with an average relative abundance of 76.26 ± 17.70%, coming from different locations in the states of Antioquia and Magdalena (Sierra Nevada de Santa Marta). Within these honeys, 27 complementary pollen types occur in low proportion, the most frequent being Heliocarpus americanus, Stellaria type, and Cleome Type. In addition, there were nine monofloral samples of Gouania polygama, with an average relative abundance of 72.34 ± 14.35% coming from different locations in the states of Santander (Charalá, Floridablanca and Socorro) and Magdalena (Sierra Nevada de Santa Marta). In these honeys there were 20 complementary pollen types in low proportion, the most frequent being Heliocarpus americanus, Muntingia calabura, Coffea arabica, and Myrcia Type. Finally, there were four monofloral samples of Heliocarpus americanus, with an average relative abundance of 69.4 ± 21% in the states of Santander (Charalá) and Antioquia (Medellín). In these honeys were 25 complementary pollen types in low proportion, the most frequent being Myrcia Type, Eucalyptus, and Oryctanthus. The floral preference of T. angustula for Heliocarpus has been reported elsewhere (Landaverde et al. 2004; Martínez-Hernández et al. 1994). H. americanus is a pioneer species, common in secondary forests, and blooms during several months of the year (Cole et al. 2010; Riaño 2005). Sierra Nevada de Santa Marta is a mountainous region located in the state of Magdalena, where all samples in this group originate. This includes 16 samples, some of monofloral origin: Astronium (1), Asteraceae Type 1 (1), Fabaceae/ Caesalpinoideae Type (1), and Euphorbiaceae Type 1 (1). The rest include a wide spectrum with 113 pollen types, within which the most frequent were Cleome (11), 342 D. Obregón et al. Table 23.2 Honey types according to botanical origin and geographical distribution Honey type Pollen types Monofloral (40 samples) Asteraceae Type 1 Asteraceae Type 2 Astronium graveolens Austroeupatorium inulifolium Calycolpus moritzianus Citrus Coffea arabica Euphorbia cotinifolia Euphorbia hirta Euphorbia Euphorbia thymifolia Euphorbiaceae Type Gouania polygama Heliocarpus americanus Bifloral (13 samples) Multifloral (23 samples) Undetermined Type 1 Fabaceae, Caesalpinioideae Type Muntingia calabura Oryctanthus Rosaceae Type Spananthe paniculata Cleome Type—Fabaceae, Caesalpinioideae Type Coffea arabica—Asteraceae Type Euphorbia hirta—Citrus Euphorbiaceae type—Vitis tilifolia Heliocarpus americanus—Lantana aff. fucata Hyptis brachiata—Cordia spinescens Lantana aff. fucata—Adenaria floribunda Melastomataceae type—Undeter mined Type 2 Myrcia—Cuphea racemosa Rosaceae Type—Asteraceae Type Fabaceae, Caesalpinioideae Type 1—Solanaceae Type 12 pollen types 11 pollen types 13 pollen types 16 ± 1.4 pollen types 15.7 ± 5.7 pollen types 15.3 ± 1.4 pollen types 14 pollen types Number of samples States in Colombia 1 1 1 2 1 1 3 6 1 1 1 1 1 2 7 1 3 1 1 1 1 1 1 1 Santander Magdalena Magdalena Santander Santander Santander Antioquia Magdalena Cundinamarca Santander Sucre Santander Magdalena Magdalena Santander Antioquia Santander Santander Magdalena Santander Santander Cundinamarca Cundinamarca Magdalena 1 2 1 2 Antioquia Santander Magdalena Cundinamarca 1 1 Magdalena Cundinamarca 1 Sucre 1 1 1 Cundinamarca Magdalena Santander 1 1 1 5 8 6 1 Antioquia Cauca Cesar Cundinamarca Magdalena Santander Tolima 23 Botanical Origin of Pot-Honey from Tetragonisca angustula Latreille in Colombia 343 Fig. 23.2 Honey types of Tetragonisca angustula found in the study area. The number inside each symbol indicates the number of samples in each state Coffea arabica (8), Euphorbiaceae Type 1 (8), and Toxicodendron striatum (6). As reflected in the samples and the characterization of vegetation (Carbonó and Lozano-Contreras, 1997; Rangel and Garzón, 1995), this is a region with great diversity and many endemic flora species, allowing the production of unique and varied honey. 344 D. Obregón et al. A group of six samples from different localities (4 Santander, 2 Antioquia) were similar in composition and characterized by typical pollen types present in coffeegrowing areas, including Coffea arabica, Myrcia Type, Heliocarpus americanus, and Gouania polygama, but occurring in low proportion. However, this group also contains two monofloral samples from Myrcia Type and from Oryctanthus. The Santander-Girón region is between 150 and 1,200 m elevation, and the climate is dry with a tendency toward desertification (UIS 2009). The vegetation is of low stature and much of the area is degraded, reflected in the palynological spectrum. This group includes 12 samples. The pollen types with the highest average relative abundance were Euphorbia hirta 19 ± 20% and Muntingia calabura 12 ± 20% (typical plants of disturbed ecosystems). The most frequent pollen types were Stellaria Type (11), Euphorbia hirta (8), Muntingia calabura (8), Euphorbiaceae type (8), and Citrus (6). Within the group there were monofloral honeys of Citrus (1), Euphorbiaceae Type (1), Undetermined (1), Euphorbia hirta (1), and Muntingia calabura (1). We named undifferentiated honey, 19 samples from different localities that were not clearly separated by multivariate analysis or geographic or botanical origin. Cundinamarca (Fusagasugá) had 11 samples and they were taken in this location. The most frequent pollen types were Myrcia Type 90%, Heliocarpus americanus 90%, Eucalyptus 90%, Citrus 90%, Fabaceae, Faboideae 1 80%, Lantana fucata 80%, and Adenaria floribunda 80%. Santander (Oiba) had three samples and they were taken in this location. The most frequent pollen types were Asteraceae Type 15.33%, Mimosa Type 1 33%, Stellaria Type 33%, and Spermacoce 33%. Two samples were taken from Sucre (Colosó, Sincelejo) where pollen types with the highest average relative abundance were Euphorbia Type 1 28.8 ± 40% and Austroeupatorium inulifolium 12.3 ± 17%. Tolima (Dolores), with a single location and sample, contained pollen of Adenaria floribunda 16%, Croton 13.50%, Syzygium jambos 10%, and Dalechampia 10%. Similarly, with a single sample Cauca (Popayán) contained Asteraceae Type 1, 36.7%; Myrcia Type, 20%; and Bignoniaceae Type 2, 16%. With one sample, Cesar (Pueblo Bello) had Syzygium jambos, 41.5%; Asteraceae Type 13, 21.1%; and undetermined, 17%. 23.4 Conclusions Multivariate analysis of palynological composition helped to identify the geographical origin T. angustula honey. We distinguished honey from coffee agroecosystems and from other localities. The honey from coffee areas contains Coffea arabica, Gouania polygama, Heliocarpus americanus, Muntingia calabura, and Myrcia Type, which are useful as pollen indicators because they have a high frequency in the samples and they are characteristic components of those areas. Honey from Santander, specifically from a dry region called Girón, can be also recognized by pollen analysis due to the occurrence of Euphorbia hirta and Muntingia calabura. The detection of monofloral and bifloral honeys from specific pollen types such as Coffea arabica or Heliocarpus americanus allowed characterizing the 23 Botanical Origin of Pot-Honey from Tetragonisca angustula Latreille in Colombia 345 botanical origin. This information is useful for stingless bee-keepers because it helps to characterize the products of this species and to recognize the plants that provide nectar and contribute to the maintenance of colonies. This information contributes to recognition of floral preferences of T. angustula in areas where it is mostly kept in Colombia. It is desirable to expand sampling from different regions of the country to continue the characterization of honey from T. angustula by botanical and geographical origin. Acknowledgments We thank all the stingless bee-keepers for allowing study in their meliponaries. We thank the team of the Bee Research Laboratory (LABUN) for collaboration. We also thank Scott Bridges for editorial help and Jorge Velez for assistance in identifying plants. We thank Patricia Vit, David Roubik for editorial observations, Monika Barth, and Jorge Enrique Moreno Patiño for constructive comments. We thank Ministerio de Agricultura y Desarrollo Rural and the Universidad Nacional de Colombia (Departamento de Biología) for funding the study. We also thank the laboratory of Palynology and Paleoecology of the Instituto de Ciencias Naturales for allowing us to consult the pollen collection. References Barth M. 2005. Análise polínica de mel: avaliação de dados e seu significado. Mensagem Doce n° 81. 4 pp. Available at: http://www.apacame.org.br/mensagemdoce/81/artigo.htm. Braga J, Nunes R, Neto J, Conde M, Sales E, Barth O, Lorenzon M. 2009. Floral sources and pollen morphology of Tetragonisca angustula (Apidae: Meliponina) in fragments of Atlantic rain forest vegetation, in southeastern Brazil. In: Proceedings of Apimondia Congress. Montpellier, France. Bush M, Weng C. 2007. Introducing a new (freeware) tool for palynology. Journal of Biogeography 34:377–380. Carbonó E, Lozano-Contreras G. 1997. Endemismos y otras singularidades de la Sierra Nevada de Santa Marta; posibles causas de origen y necesidad de conservarlos. Revista de la Academia Colombiana de Ciencias XXI:409–419. Carvalho CAL, Marchini LC. 1999. Tipos polínicos coletados por Nannotrigona testaceicornis e Tetragonisca angustula (Hymenoptera, Apidae, Meliponinae). Scientia Agricola 56:717–722. Cepeda-G M, Nates-Parra G, Téllez-I G. 2009. Comercialización de los productos de la meliponicultura en Colombia. Memorias IV Encuentro Colombiano de Abejas Silvestres Bogotá, DC, Colombia. Acta Biológica Colombiana 14:1–181. Cole RJ, Holl KD, Zahawi RA. 2010. Seed rain under tree islands planted to restore degraded lands in a tropical agricultural landscape. Ecological Applications 20:1255–1269. Colinvaux P, De Oliveira P, Moreno J. 1999. Amazon pollen manual and atlas. Harwood Academic Publishers; Amsterdam, Netherlands. 332 pp. Cortopassi-Laurino M. 1982. Divisão de recursos tróficos entre abelhas sociais principalmente em Apis mellifera Linné e Trigona (Trigona) spinipes Fabricius (Apidae, Hymenoptera). Tese (Doutorado), Instituto de Biociências, Universidade de São Paulo; São Paulo, Brasil. 180 pp. Erdtman G. 1952. Pollen morphology and plant taxonomy-Angiosperms. Almqvist & Wiksell; Stockholm. 539 pp. Flores F, Sánchez AC. 2010. Primeros resultados de la caracterización botánica de mieles producidas por Tetragonisca angustula (Apidae, Meliponinae) en Los Naranjos, Salta, Argentina. Boletín Sociedad Argentina de Botánica 45:81–91. García CJ, Vallejo RJ. 2002. Sostenibilidad económica de las pequeñas explotaciones cafeteras. Ensayos sobre Economía Cafetera 15:73–89. 346 D. Obregón et al. Hammer O, Harper D, Ryan P. 2001. PAST: paleontological statistics software for education and data analysis. Paleontología Electrónica 4:1–9. Imperatriz-Fonseca VL, Kleinert-Giovannini A, Cortopassi-Laurino M, Ramalho M. 1984. Hábitos de coleta de Tetragonisca angustula angustula Latreille (Hymenoptera, Apidae, Meliponinae). Boletim de Zoologia da Universidade de São Paulo 8:115–131. Iwama S, Melhem TS. 1979. The pollen spectrum of the honey of Tetragonisca angustula angustula Latreille (Apidae, Meliponinae). Apidologie 10:275–295. Knoll FRN. 1990. Abundância relativa, sazonalidade e preferências florais de Apidae (Hymenoptera) em uma área urbana. Tese (Doutorado), Instituto de Biociências, Universidade de São Paulo; São Paulo, Brasil. 127 pp. Landaverde V, Sánchez LA, Ruano C, Smeets M. 2004. Temporary dominance of pollen of nectiferous and polliniferous plants collected by Melipona beecheii in El Salvador and pollen of polliniferous plants collected by Tetragonisca angustula and M. beecheii in Costa Rica. In: Proceedings beekeeping in tropical countries, research and development for pollination and conservation. San José, Costa Rica. CD-ROM. 44–52. Louveaux J, Maurizio A, Vorwohl G. 1970. Methods of Melissopalynology. Bee World 51:125–138. Martínez-Hernández E, Cuadriello-Aguilar JI, Ramírez-Arriaga E, Medina-Camacho M, SosaNájera MS, Melchor-Sánchez JE. 1994. Foraging of Nannotrigona testaceicornis, Trigona (Tetragonisca) angustula, Scaptotrigona mexicana and Plebeia sp. in the Tacaná region, Chiapas, Mexico. Grana 33:205–217. Moreno J, Devia W. 1982. Procedencia del polen y la miel almacenados por las abejas Apis mellifera Linneo, Melipona eburnea Friese y Trigona (Tetragonisca) angustula Latreille (Hymenoptera: Apidae) en el municipio de Arbeláez (Colombia: Cundinamarca). Tesis de Pregrado, Departamento de Biología, Universidad Nacional de Colombia; Bogotá, DC, Colombia. 272 pp. Nates-Parra G. 2001. Guía para la cría y manejo de la abeja angelita o virginita Tetragonisca angustula Illiger. Convenio Andrés Bello, Serie Ciencia y Tecnología No. 84; Bogotá. 43 pp. Rangel O, Garzón A. 1995. Sierra Nevada de Santa Marta (Colombia). pp. 155–170. In: Rangel O, ed. Colombia diversidad biótica I. Instituto de Ciencias Naturales, Universidad Nacional de Colombia; Bogotá. 442 pp. Riaño K. 2005. Aspectos ecológicos de diez especies pioneras arbóreas en corredores de conexión Barbas-Bremen, Quindío Colombia. Trabajo de grado, Licenciatura en Biología y Educación Ambiental, Facultad de Educación, Universidad del Quindío; Armenia, Quindío, Colombia. 72 pp. Roubik DW, Moreno JE. 1991. Pollen and spores of Barro Colorado Island. Monographs in systematic botany No. 36. Missouri Botanical Garden; St. Louis, Missouri. 268 pp. The Plant List. 2010. Version 1. Available at: http://www.theplantlist.org/. Universidad Industrial de Santander (UIS). 2009. Plan de Ordenamiento Territorial de San Juan de Girón 2000–2009. Documento de Diagnóstico. Centro de Estudios Regionales-UIS. Available at: http://giron-santander.gov.co. Von der Ohe W, Persano Oddo L, Piana ML, Morlot M, Martin P. 2004. Harmonized methods of melissopalynology. Apidologie 35:S18-S25. Vossler FG. 2007. Las preferencias alimentarias de Tetragonisca angustula y Scaptotrigona aff. depilis durante la floración temprana del bosque xerófilo chaqueño. Boletín de la Sociedad Argentina de Botánica 42:236 pp. Part IV Sensory Attributes and Composition of Pot-Honey Chapter 24 Sensory Evaluation of Stingless Bee Pot-Honey Rosires Deliza and Patricia Vit To Michel Gonnet, for the first imprinting with the sensory message of a lavanda honey served in a crystal gobblet in Monfavet, France 24.1 Introduction The sensory characteristics of honey play an important role in producing quality standards, as they determine consumer acceptance. The sensory attributes in terms of appearance, aroma, flavor, and texture vary from product to product, revealing the need for investigating every honey in order to better understand their characteristics. When one evaluates honey sensory quality, several perspectives are taken into account, and among them is the consumer perception that leads to different honey evaluations. Consumers are more and more concerned about health and wellness and, consequently, they are more interested in the benefits from food and beverage (Sloan 2011). Honey is a health product (Amtmann 2010), and therefore, a thorough investigation of honey sensory properties is desirable. Sensory analysis as a discipline uses the five human senses (sight, smell, taste, touch, and hearing) to analyze food, beverages, and other products. By using human panels to sample the products, with an adequate experimental design and statistical R. Deliza Embrapa Agroindústria de Alimentos, Av. das Américas, 29501, CEP 23.020—470 Rio de Janeiro—RJ, Brazil P. Vit (*) Apitherapy and Bioactivity, Food Science Department, Faculty of Pharmacy and Bioanalysis, Universidad de Los Andes, Mérida 5101, Venezuela Cancer Research Group, Discipline of Biomedical Science, The University of Sydney, Cumberland Campus C42, 75 East Street, Lidcombe, NSW 1825, Australia e-mail: vitolivier@gmail.com 349 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_24, © Springer Science+Business Media New York 2013 350 R. Deliza and P. Vit Table 24.1 Main characteristics of qualitative and quantitative sensory methods for investigating honey Sensory issues in qualitative studies How do you perceive this honey? What did you specifically like and dislike about it? Please explain what do think about this honey color compared to the other one Please describe the consistency of this honey Please tell me more about why the color of this honey is unacceptable to you Why in your opinion honey 371 is nicer than product 775? Why your family preferred this honey instead of the others? Characteristics of sensory quantitative studies The honey consumer is invited to participate: A relatively large group of consumers participates (depending on the statistical power required) Careful honey preparation for a large number of participants Written questionnaire with attributes and scales to score consumer response. Sensory questions may include overall liking, liking and perceived intensity of attributes, and preference The selection of attributes in the questionnaire is critical Data are statistically analyzed Adapted from Deliza and Glória (2009) analysis, it is possible to evaluate products in terms of appearance, aroma, flavor, texture, and aftertaste (Meilgaard et al. 1999). Assessment can derive from a panel selected according to specific criteria and trained to evaluate product sensory attributes, or from a consumer panel, i.e., any person who consumes the product under investigation or matches predefined recruitment criteria, normally based on demographics (e.g., gender, age, education, product consumption). Several quantitative sensory methods are available and well defined in terms of application procedures (Stone and Sidel 2004). The choice regarding panel selection (trained people or consumers) will depend on the study objective. Three distinct methods are applied, as follows: the descriptive method (used when the aim is to have a sensory characterization of samples), discriminative tools (useful to investigate whether there are sensory differences between products), and affective tests, which investigate how much a product is liked/accepted by consumers. Consumer studies can be carried out through qualitative and quantitative studies. Qualitative research often has an exceptional value, since the consumer can be queried to obtain information not easily obtainable in quantitative studies. Qualitative information can provide the most important data and cannot be easily measured through a written questionnaire. The qualitative studies do not replace quantitative ones, but complement them (Muñoz 1998). Quantitative studies, on the other hand, are geared to collect data that can be summarized and analyzed statistically. The main characteristics quantitative and qualitative sensory studies are indicated in Table 24.1. Consumers may have subjective impressions regarding product quality, and several aspects contribute to their product evaluation. Among them are psychological processes. Such processes are influenced by many factors, including the level of previous knowledge and cognitive competencies of each consumer (Deliza and MacFie 1996; McBride and MacFie 1990). Thus, from a consumer perspective, 24 Sensory Evaluation of Stingless Bee Pot-Honey 351 quality refers to the perceived quality and not to quality in an objective sense (Deliza and Glória 2009; Steenkamp 1990). We illustrate a number of distinctive sensory characteristics of pot-honey. Comparisons between honey produced by Apis (in combs) or meliponines (in pots) are presented and discussed, as well as the sensory evaluation of fermented honey. The latter is, objectively, fairly common for honey in different stingless bee species. Sensory implications based on the extractive techniques are also included considering the new odor–aroma families needed to describe such a product. Preliminary data on acceptance of pot-honey produced by different species are given. A Freechoice profile described is a useful method to group honeys according to their entomological origin, by untrained panels. 24.2 Sensory Characteristics of Pot-Honey Honey consumers in the cities can find honey from A. mellifera on supermarket shelves. In tropical villages where many of the stingless bees are appreciated, as well as the several species of tropical Apis produced in combs, there is also a great variety of honey produced in pots. Familiarity with local species of meliponines is also reflected in the cultural uses of honey by stingless bee honey hunters and stingless bee keepers. Their honeys were widely relished in tropical America before Columbus (Schwarz 1948). Honey is as varied as the different species that produce it and the different seasons and habitats in which it is harvested. Therefore, when we taste honey it is like a communication between man and the habits of bees through the human senses. Honey produced in pots by Meliponini shares compositional properties with A. mellifera honey produced in combs, but differs in others such as higher water content and free acidity (Vit et al.; Souza et al. 2006). Therefore, their sensory attributes vary accordingly. For example, a higher acidity increases the sour taste perceived in pot-honey, as observed since Gonnet et al. (1964). The higher water content causes a lower visual viscosity, and has different implications in the perception of odors and aromas, caused by a flavor dilution factor. A wide range of applications derives from the perception of a paradoxical honey, so far the most ancient honey in the planet (Camargo, personal communication) but a new product in the honey market, with few recent sensory studies (Ferreira et al. 2009; Vit et al. 2011a, d). Classical work on sensory characteristics and defects of honey from A. mellifera (Gonnet and Vache 1984) were expanded towards perception evaluation by human consumers. Persano Oddo et al. (1995) characterized honey by visual, olfactory, and flavor attributes, later organized in complete sheets of 20 European honey types (Persano Oddo and Piro 2004). Anupama et al. (2003) developed a specific lexicon for Indian honey by quantitative descriptive analysis (QDA). They applied Principal Component Analysis (PCA) to appearance, aroma, mouthfeel, and flavor descriptors and physicochemical variables. Galán-Soldevilla et al. (2005) developed a sensory lexicon for floral honey with 15 descriptors, in categories of odor, flavor, 352 R. Deliza and P. Vit texture, and trigeminal sensations, i.e., more associated with the sense of touch, perceived through the action of specific compounds on the trigeminal receptors (e.g., the tingling effect of citric acid, cooling sensation from menthol, fizzy feeling of carbonated beverages, astringency caused by unripe persimmons and bananas, or the hotness perceived after eating chilli). Additionally, postharvest conservation methods (see Menezes et al. chapter in this book) cause variable sensations according to the stingless bee species, which leads to the human reaction and distinctive sensory perception, that needs to be considered. A number of distinctive sensory characteristics of honey derive from extractive techniques. As we will discuss, some new odor–aroma families are needed to describe this product. The sensory interpretation of fermented honey, preliminary data on acceptance of pot-honey produced by different species, and the free-choice profile as a useful method to group honey according to their entomological origin are explored by untrained panels. 24.3 New Odor–Aroma Families for Pot-Honey The system used to describe the honey of A. mellifera has identified and arranged seven families of sensory attributes in the odor–aroma wheel (Piana et al. 2004). This was adapted to eight odor–aroma families for pot-honey produced by stingless bees (Table 24.2), as follows: (1) Floral-fruity, (2) Vegetable, (3) Fermented, (4) Wood, (5) Bee hive, (6) Mellow, (7) Primitive, and (8) Industrial chemicals (Vit et al. 2007a, b). For the public the family bee hive makes sense, but for scholars bee nest would be a better expression. 24.4 Pot-Honey Extraction by Pressure or By Suction? Compression of mature honey pots is the traditional method of extraction. Compared to modern honey extraction by suction after piercing sealed pots, more pollen is added to the honey by squeezing the storage pots, which may include adjacent pollen pots. The extractive technique has implications related to the fermented pollen (see Menezes et al., chapter this book) added to the honey. Using descriptors of Table 24.2, eight assessors tasted pressed pot-honeys of Melipona aff. fuscopilosa [= Melipona (Michmelia) sp. 1, see Table in Pedro and Camargo chapter, this book, until the revision of Melipona is done] and Tetragona clavipes from the Venezuelan Amazon (Vit et al. 2007a, b). The intense fermented odor and aroma reduced the relative frequencies of descriptors from the other seven sensory families. Fermented odor was perceived more frequently than fermented aroma, somehow associated to volatile components of fermentation. For honey of A. mellifera, fermentation is considered an off-odor, something that is not normal (Gonnet and Vache 1984). It represents not only a sensory defect, 24 Sensory Evaluation of Stingless Bee Pot-Honey 353 Table 24.2 Organized odor–aroma descriptors for pot-honey Family Subfamily Sensory descriptors 1. Floral-fruity Floral Orange blossom, jasmine, rose, violet Citrus fruit Citrus zesty, lemon, mandarine, orange, grapefruit Fresh fruit plum, coconut, apricot, berries, apple, melon, passion fruit, watermelon, pear, pineapple, rose apple, fig, peach, grape Processed fruit Candied fruit, dehydrated fruit, syrup fruit, fruit jam 2. Vegetable Fresh Sugar cane, raw beans, fresh leaves, sweet corn, sweet parsnip, bitter plants, vegetation Dry Dry hay, malted, chamomile, straw, tea Aromatic Lemongrass, eucalyptus, bay leaves, peppermint, oregano, rue, lime, liquorice 3. Fermented Acetic Vinegar, meliponine pollen pots Alcoholic Aguardiente, fermented fruit, yeast, liqueur, must, sake, vinasse, white wine, red wine Lactic Miso, cheese, yogurt 4. Wood Woody Sawdust, cork, wood flakes Resinous Cedar, incense, pine resin Spicy Anise, cocoa, coffee, cinnamon, clove, nutmeg, tobacco, vanilla Seeds Sesame, almond, marzipan, chestnut, hazelnut 5. Bee hive Stingless bee Bee, batumen, cerumen, pot-honey Apis mellifera Beeswax, bee excrement, honey, bee pollen, propolis, moth 6. Mellow Sugary White sugar, brown sugar, syrup, tablets, chocolate Caramelized Arequipe, burned sugar, candy, caramel, maple, molasses, jaggery, toffee, malt Pastry Pudding, butter 7. Primitive Animal Formic acid, pet food, leather, stable, manure, fat, eggs, cat urine, sweat Smoke Smoked food, burned straw Wet Floor mop, after the rain, humus, moldy Sulfate Artichoke, cabbage Mineral Water, clay, ice, water Marine Nori seaweed, fish Oily Oil, rancid 8. Industrial Petrochemical Engine oil, book glue, rubber, paint, plastic, Chemical photographic film, solvent Medicinal Ascorbic acid, soap, quinine, soap, vitamin B1 Vit et al. (2007) but is considered to result from harvesting unripe honey which has a higher water content which causes fermentation. Meliponini process honey differently. Fermentation is accomplished by associated microorganisms inside the storage pots and also after harvest. Therefore, fermentation of pot-honey is not a defect but an aspect of honey maturation by meliponines and a human sensory attribute that needs 354 R. Deliza and P. Vit further consideration. The consumer’s preferences are related to cultural backgrounds, and tropical cultures value sour tastes, possibly because tropical fruits are soursweet. A group of 20 Venezuelan assessors tasted compressed pot-honey and honey extracted by suction. Despite the very small number of participants in this preliminary study, the results demonstrated that the acceptance was higher for the compressed honey than for the honey extracted by suction. Honey compressed with surrounding sour pollen pots contains fermented pollen, and was perceived with a more intense “lemon-like” flavor (unpublished data) i.e., the honey was perceived as having a citrusy note similar to lemon. This result suggests that such characteristic (“lemon-like” flavor) might have contributed to increase the compressed honey acceptance by consumers, compared to the honey extracted by suction. The sensory evaluation and interpretation of fermented pot-honey is a challenge for those who work in the field. A transition from defect to value could be based on a direct preference for a more fruity-sour characteristics, a complex perception of fermentation patterns, and also an indicator of medicinal properties derived from the fermentive process. Stingless bees have associations with microorganisms that transform and help to preserve honey and pollen (see Menezes et al. and Rosa et al. chapters in this book). Different microorganisms have a characteristic fermentation pathway. The presence of lactic acid was confirmed in honey of Meliponini (Vit et al. 2011c). Honey of Tetragonisca angustula was studied during a 30-day-postharvest experiment. The gradual increase of ethanol enhanced the antioxidant activity in fermented honey stored at 30ºC (Pérez-Pérez et al. 2007). 24.5 Acceptance of Pot-Honeys from Different Species of Meliponini Considering that food acceptance depends on several consumers’ and individual cultural background, the stingless bee honey’s acceptance has been evaluated in different populations. In separate studies, participants from Spain, Venezuela, Mexico, and Australia rated how much they liked the honeys on 10-cm unstructured line scales anchored with the expressions “dislike it a lot” and “like it a lot”, in the left (1 cm) and right ends (9 cm), respectively. The acceptance scores were measured and the data were analyzed, with ANOVA, followed by a Tukey test to check differences between means. The results are presented in Tables 24.3, 24.4, 24.5, 24.6 and 24.7. Spanish consumers tasted pot-honey from Australia, Bolivia, Brazil, Mexico, and Venezuela (Vit et al 2010b). The results in Table 24.3 reveal that on average Spanish consumers did not like the pot-honeys, as the higher acceptance mean was 6.2, which is situated slightly above of the neutral score 5 (neither like nor dislike). Stratified sampling is suggested to see if any type of consumer emerges and we can identify people who most like the products. Little is known about the perception of pot-honey from the forest by native communities of stingless bee-hunters and stingless bee-keepers. For this reason, the acceptance of honey was evaluated in a Huottuja group in Paria Grande, Amazonas 24 Sensory Evaluation of Stingless Bee Pot-Honey 355 Table 24.3 Average honey acceptance evaluated by Spanish consumers Acceptance1 Common name of the bee Country of origin Bee species (Mean ± SD) “negrita” Mexico Scaptotrigona mexicana 4.3 ± 2.5a “suro negro” Bolivia Scaptotrigona polysticta 4.9 ± 2.1a “carby” Australia Tetragonula carbonaria 5.1 ± 2.3a “uruçú” Brazil Melipona scutellaris 5.6 ± 2.4a “erica” Venezuela Melipona favosa 6.2 ± 2.2a 1 Evaluated in 10-cm unstructured line scales varying from “dislike it a lot” (1) and “like it a lot” (9). Significant differences between honeys (P < 0.05, ANOVA) are indicated by different superscripts Table 24.4 Average acceptance of honey by Huottuja consumers in Amazonas State, Venezuela Acceptance1 Common name of the bee Bee species (Mean ± SD) honey bee Apis mellifera 5.4 ± 3.3a “angelita” artificial – 6.5 ± 3.1a “isabitto” Melipona aff. fuscopilosa2 6.9 ± 3.6a “ajavitte” Tetragona clavipes 7.9 ± 2.2a “angelita” artificial – 8.4 ± 1.5a 1 Evaluated in 10-cm unstructured line scales varying from “dislike it a lot” (1) and “like it a lot” (9). Significant differences between honeys (P<0.05, ANOVA) are indicated by different superscripts 2 Melipona aff. fuscopilosa [= Melipona (Michmelia) sp. 1, see Table in Pedro chapter, this book] Table 24.5 Average acceptance of “tiúba” M. fasciculata honey from different locations Acceptance1 Location (Mean ± SD) Todos os Santos 3.5 ± 2.9a Limoeiro 4.4 ± 0.8a,b Tabocas 4.8 ± 1.4a,b Moura 5.1 ± 1.1b Preazinho 6.5 ± 2.6c 1 Evaluated in 10-cm unstructured line scales varying from “dislike it a lot” (1) and “like it a lot” (9). Significant differences between honeys (P < 0.05, ANOVA) are indicated by different superscripts State, Venezuela (Vit et al. 2010a). Two artificial honeys sold as “angelita” Tetragonisca angustula in the indigenous market from Puerto Ayacucho, one honey bee and two genuine stingless bee honeys of “isabitto” Melipona aff. fuscopilosa and “ajavitte” from Tetragona clavipes, were evaluated. The acceptance results are given in Table 24.4. Another study was carried out with commercial pot-honey produced by “tiúba” Melipona fasciculata in five different places: Limoeiro, Moura, Preazinho, Tabocas, and Todos os Santos, all located in Maranhão state, Brazil. In that honey, natural fermentation was completed, as the postharvest processing aiming at stabilizing the 356 R. Deliza and P. Vit Table 24.6 Average Mexican acceptance scores for pot-honey from different stingless bees Acceptance1 Common name of the bee Bee species Year of harvest (Mean ± SD) “ala blanca” Frieseomelitta nigra 2011 4.7 ± 2.4a “uruçú” Melipona scutellaris 2011 4.8 ± 2.5a “criolla” Melipona solani 2011 5.2 ± 3.3a,b “colmena real” Melipona fasciata 2010 5.3 ± 2.2a,b “abeja bermeja” Scaptotrigona 2010 5.5 ± 1.9a,b hellwegeri “mijui” Scaptotrigona polysticta 2011 5.7 ± 2.3a,b “pisilnekmej” Scaptotrigona mexicana 2009 6.5 ± 2.1a,b “abeja bermeja” Scaptotrigona 2009 6.6 ± 2.0a,b hellwegeri “abeja real” Melipona beecheii 2011 6.8 ± 2.3a,b “pisilnekmej” Scaptotrigona mexicana 2010 6.8 ± 1.9a,b “pisilnekmej” Scaptotrigona mexicana 2011 7.3 ± 2.2b 1 Evaluated in 10-cm unstructured line scales varying from “dislike it a lot” (1) and “like it a lot” (9). Significant differences between honeys (P < 0.05, ANOVA) are indicated by different superscripts Table 24.7 Average Australian acceptance scores of pot-honey from different stingless bee species and unifloral A. mellifera honeys Acceptance1 (Mean ± SD) Stingless bee species Melipona fasciata 3.7 ± 2.6a Scaptotrigona mexicana 4.0 ± 3.0a Tetragonula carbonaria 4.1 ± 2.6a Frieseomelitta nigra 4.1 ± 2.8a Melipona beecheii 4.7 ± 3.2a Unifloral honey Passion fruit 4.1 ± 2.7a Lychee 5.1 ± 2.5a Leatherwood 5.5 ± 2.6a,b Manuka 6.0 ± 2.5a,b Avocado 7.3 ± 0.2b 1 Evaluated in 10-cm unstructured line scales varying from “dislike it a lot” (1) and “like it a lot” (9). Significant differences between honeys (P < 0.05, ANOVA) are indicated by different superscripts honey prior to packaging. The word “natmel” was created for naming this type of honey. Honey was collected during the X IberoLatinamerican Congress of Apiculture held in Natal, Brazil 2010. The honey was taken to Venezuela to be tasted by Venezuelan honey consumers. Table 24.5 presents the acceptance results (Vit et al. 2011b). During the VII Mesoamerican Seminar on Native Bees held in Cuetzalán, Puebla, Mexico, May 2011, the Municipality of Cuetzalán was declared Sanctuary of S. mexicana “pisilnekmej” (from the Nahuatl “pisil” small, “nektsin” bee). 24 Sensory Evaluation of Stingless Bee Pot-Honey 357 Pot-honeys from eight species of stingless bees were tasted by a panel of Mexican creole, Mayan, and Nahuatls. Two species had honeys harvested in different years. Higher acceptance mean scores were observed for recently harvested S. mexicana (2011) (Table 24.6). Another study investigated the acceptance of pot-honeys produced by five species of stingless bees (M. beecheii, M. fasciata guerreroensis, S. mexicana, T. carbonaria, and T. nigra) and five unifloral honeys: avocado Persea americana (Lauraceae), lychee Litchi chinensis (Sapindaceae), passion fruit Passiflora edulis (Passifloraceae), leatherwood Eucriphia lucida (Cunoniaceae), and manuka Leptospermum scoparium (Myrtaceae) of A. mellifera from Kuranda forest, Queensland, Australia. Table 24.7 shows the average acceptance results achieved in the study. 24.6 Descriptive Sensory Studies of Pot-Honey Descriptive studies were also carried out with pot-honey, to investigate the relationship between sensory attributes and the bee origin of the honey produced in pots by Vit et al. (2011a and 2011d). Samples were analyzed by free-choice profiling (FCP) (Deliza et al. 2005), a quick and inexpensive method in which participants are asked to both identify attributes in the sample, and score their intensities on appropriate scales. They should be provided with adequate instruction on how to perform this test, and possibly given product categories to describe them in terms of appearance, aroma, flavor, texture, etc. Each participant will generate his/her own set of attributes, and consumers should be recruited as product users, age/gender/education level. It is important to note that consumers may use terms in different ways. Researchers may be able to separate consumers into groups, aiming at better identifying which characteristics are most important for that consumer segment. Generalized Procrustes Analysis (GPA) is a common statistical tool for analysis of FCP data. Figures 24.1 and 24.2 present the results of the studies conducted with the Huottuja (Piaroa) community and Spanish consumers, respectively. 24.7 Final Considerations Perception is a multifactorial process that needs to be considered to explain any sensory response, in our case the pot-honey results. Orthonasal (breathing, nasal mucosal tissues, nasal metabolism) and retronasal (physicochemical release, salivation, oral metabolism, oral and pharyngeal) peripheral factors, besides chewing and swallowing patterns, and tongue movements affect the tasting process (Buettner and Beauchamp 2010). Odor, aroma, and taste are released from the honey matrix according to chemical and physical features. Although we are interested in comparing honeys—not assessors, we cannot forget the individual 358 R. Deliza and P. Vit Fig. 24.1 Honey descriptive sensory evaluation by Huottuja community (from Vit et al. 2011a). Used by permission of Sociedade Brasilera de Farmacognosia differences of participants regarding honey perception with a strong cultural imprinting since their childhood (Barthomeuf et al. 2009). In addition, due to today’s market competitiveness, it is necessary to understand the factors influencing consumers at the emotional level. Identifying the emotional elements that consumers experience and expect in a product can help providing a complete perspective on consumer affective behaviors, and contributing to the identification of the products most liked by consumers. In this context, scales for measuring different emotions associated to food product have been developed to test food by consumers (King and Meiselman 2010), and may be a useful tool to help better understand consumer’s honey perception. 24 359 Sensory Evaluation of Stingless Bee Pot-Honey Biplot (axes F1 and F2: 60.00%) 20 Honey 1 Var10 Var13 Var7 Var24 Var16 Var8 Honey 5 10 F2 (27.25 %) 0 Var12 Var14 Var9 Honey 2 Var17 Honey 3 Var22 Var23 Var18 Var5 Var20 Var3 Var15 -10 Var11 -20 Var6 Melipona -30 America Var21 Var2 Var1 Var4 Var19 Scaptotrigona Honey 4 Australia -40 -50 -40 Tetragonula -30 -20 -10 0 10 20 30 40 F1 (32.75 %) Fig. 24.2 Pot-honey descriptive sensory evaluation by Spanish consumer (from Vit et al. 2011d). Permission granted by the International Bee Research Association Acknowledgments The authors thank the great generosity of stingless bee keepers who provided the pot-honey used in the sensory tests. To Dr. Tim Heard from CSIRO Ecosystem Sciences, Brisbane, Queensland, Australia for providing honey from Tetragonula carbonaria, and for its identification. To Dr. Urbelinda Ferrufino, from Asociación Ecológica de Oriente, Santa Cruz, Bolivia, for providing Scaptotrigona polysticta honey. To Mr. Fini Opa Carrasquel from Asociación Cooperativa de Meliponicultores Warime, Paria Grande, Estado Amazonas, Venezuela, for the honey of Melipona aff. fuscopilosa and the Tetragona clavipes honeys. To MSc. Jerônimo Khan Villas-Boâs collaborator of the Universidade Federal da Paraíba, Brazil, for providing the honey of Melipona scutellaris, and Scaptotrigona polysticta from João Pessoa and Xingú, Brazil. The honey of Melipona fasciculata was received from Prof. Murilo Sergio Drummond, Universidade Federal do Maranhão, Brazil. We also thank Mr. José Reyes from the Tosepan Titaniske Cooperative, Cuetzalan, Puebla, Mexico, for providing honey of Scaptotrigona mexicana; Mrs. Liliana Castro from Mujeres Juntas Enfrentando Retos, Guerrero, Mexico, for the three honey samples of Melipona fasciata guerreroensis, Scaptotrigona hellwegeri, and Frieseomelitta nigra; Mr. Emmanuel Pérez de León and Mr. Ramiro García Farfán from the Soconusco group, Chiapas, México, for providing honey of Melipona solani and Melipona beecheii, respectively. Honey of Melipona favosa was collected by Prof. Patricia Vit, and the bee was identified by Prof. João MF Camargo. Scaptotrigona polysticta was kindly identified by Dr. Silvia RM Pedro from the Biology Department, Universidade de São Paulo, Ribeirão Preto, Brasil. Both Camargo and Pedro identified the M. fuscopilosa and Tetragona clavipes from Venezuela. The Mexican bees were identified by Prof. Ricardo Ayala from Chamela, Jalisco, Mexico. Finally, we would like to thank the Intercambio Científico, Universidad de Los Andes for a stage at Universidad de Burgos, Spain (with Prof. María Teresa Sancho). We thank Endeavour Awards from Australia for a Research Fellowship at The University of Sydney (with Prof. Fazlul Huq) to P Vit, anonymous reviewers who kindly improved the manuscript, and Dr. DW Roubik for his careful English style editing. 360 R. Deliza and P. Vit References Amtmann M. 2010. The chemical relationship between the scent features of goldenrod (Solidago canadensis L.) flower and its unifloral honey. Journal of Food Composition and Analysis 23:122–129. Anupama D, Bhat KK, Sapna VK. 2003. Sensory and physico-chemical properties of commercial samples of honey. Food Research International 36:183–191. Barthomeuf L, Rousset S, Droit-Volet S. 2009. Emotion and food. Do the emotions expressed on other people faces affect the desire to eat liked and disliked food products? Appetite 52:27–33. Buettner A, Beauchamp J. 2010. Chemical input – sensory output: diverse modes of phisiology – flavour interactions. Food Quality and Preference 21:915–924. Deliza R, MacFie HJH. 1996. The generation of sensory expectation by external cues and its effect on sensory perception and hedonic ratings: a review. Journal of Sensory Properties 11:103–128. Deliza R, MacFie H, Hedderley D. 2005. The consumer sensory perception of passion-fruit juice using free-choice profiling. Journal of Sensory Studies 20:17–27. Deliza R, Glória MBA. 2009. Sensory perception. pp. 525–548. In: Nollet LML, Toldrá F, eds. Handbook of muscle food analysis. CRC Press Taylor & Francis Group; Boca Ratón, USA. 967 pp. Ferreira EL, Lencioni C, Benassi MT, Barth MO, Bastos DHM. 2009. Descriptive sensory analysis and acceptance of stingless bee honey. Food Science and Technology International 15:251–258. Galán-Soldevilla H, Ruíz-Pérez-Cacho MP, Serrano Jiménez S, Jodral Villarejo M, Bentabol Manzanarez A. 2005. Development of a preliminary sensory lexicon for floral honeys. Food Quality and Preference 16:71–77. Gonnet M, Lavie P, Nogueira-neto P. 1964. Étude de quelques characteristiques des miels récoltés para certains Méliponines brésiliens. Comptes Rendu Academic Sciences Paris 258:3107–3109. Gonnet M, Vache G. 1984. L’Analisi Sensoriale dei Mieli. Come degustare e individuare le qualitá organolettiche di un miele. Federazione Apicoltori Italiani; Roma, Italia. 62 pp. King SC, Meiselman HL. 2010. Development of scales to measure consumer emotions associated with food products. Food Quality and Preference 21:168–177. McBride RL, MacFie HJH. 1990. Psychological basis of sensory evaluation. Elsevier Science Publishing; New York, USA. 212 pp. Meilgaard M, Civille GV, Carr BT. 1999. Sensory evaluation techniques. 3rd ed. CRC Press; Boca Raton, USA. 354 pp. Muñoz AM. 1998. Consumer perceptions of meat. Understanding these results through descriptive analysis. Meat Science 1:S287. Pérez-Pérez E, Rodríguez-Malaver J, Vit P. 2007. Efecto de la fermentación en la capacidad antioxidante de miel de Tetragonisca angustula Latreille, 1811. BioTecnología 10:14–22. Persano Oddo L, Piana L, Sabatini AG. 1995. Conoscere il Miele. Guida all’Analisi Sensoriale. Istituto Nazionale di Apicoltura; Bologna, Italia. 398 pp. Persano Oddo L, Piro R. 2004. Main European unifloral honeys: descriptive sheets. Apidologie 35:S38-S81. Piana ML, Persano Oddo L, Bentabol A, Bruneau E, Bogdanov S, Guyot DC. 2004. Sensory analysis applied to honey. Apidologie 35:S26-S37. Schwarz HF. 1948. Stingless Bees (Meliponidae) of the Western Hemisphere. Bulletin of the American Museum of Natural History 90:1–546. Sloan AE. 2011. Top ten food trends. Food Technology 65:24–41. Souza B, Roubik D, Barth O, Heard T, Enríquez E, Carvalho C, Marchini L, Villas-Bôas J, Locatelli J, Persano Oddo L, Almeida-Muradian L, Bogdanov S, Vit P. 2006. Composition of stingless bee honey: setting quality standards. Interciencia 31:867–875. Steenkamp JBEM. 1990. Conceptual model of the quality perception process. Journal of Business Research 21:309–333. 24 Sensory Evaluation of Stingless Bee Pot-Honey 361 Stone H, Sidel JL. 2004. Sensory evaluation practices. Elsevier Academic Press; San Diego, USA. 377 pp. Vit P, Carvalho CAL, Enríquez E, González I, Moreno E, Roubik DW, Souza BA, Villas-Bôas JK. 2007. Descripción sensorial de mieles de abejas sin aguijón de Argentina, Australia, Brasil, Guatemala y Venezuela. pp. 102–117. In: Vit P, ed. Cría de Abejas sin Aguijón y Valorización sensorial de sus Mieles. APIBA-FFB-DIGECEX-ULA; Mérida, Venezuela. 146 pp. Vit P, Deliza R, Pérez A. 2010a. Una experiencia de perfil de libre elección para valorar las mieles de Meliponini en Paria Grande, Estado Amazonas. LX Convención Anual de AsoVAC; Ciudad Bolívar, Venezuela. Vit P, Deliza R, Pascual A, Heard TA, Villas-Boas JK, Ferrufino U, Fernández-Muiño MA, SanchoOrtiz MT. 2010b. Spanish perception of pot honey from Australia, Bolivia, Brazil, Mexico, Venezuela. In: Internacional conference on beekeeping, development and honey marketing. Hanoi, Vietnam. Vit P, Deliza R, Pérez A. 2011a. How a Huottuja (Piaroa) community perceives genuine and false honey from the Venezuelan Amazon, by free-choice profile sensory method. Brazilian Journal of Pharmacognosy 21:786–792. Vit P, Drummond MS, Barth OM. 2011b. Venezuelan perception of tiúba Melipona fasciculata pot honeys from different locations in the state of Maranhão, Brazil. In: 9th Pangborn Sensory Science Symposium. Toronto, Canada. Vit P, González I, Carvalho CAL, Enríquez E, Moreno E, Roubik DW, Souza BA, Villas-Bôas JK. 2007. Tabla olor-aroma. Taller evaluación sensorial de mieles de abejas sin aguijón; Mérida, Venezuela. Available at: www.saber.ula.ve/stinglessbeehoney/odour-aroma.php. Vit P, Rojas LB, Usubillaga A, Aparicio R, Meccia G, Sancho MT. 2011c. Presencia de ácido láctico y otros compuestos semivolátiles en mieles de Meliponini. Revista del Instituto Nacional de Higiene Rafael Rangel 42:58–63. Vit P, Sancho T, Pascual A, Deliza R. 2011d. Sensory perception of tropical pot honeys by Spanish consumers, using free choice profile. Journal of ApiProduct and ApiMedical Science 3:174–180. Chapter 25 Melipona favosa Pot-Honey from Venezuela Patricia Vit To the memory of Father Santiago López Palacios of Universidad de Los Andes, Venezuela, for his inspiration to investigate bee botany and honey. To the retired Dr. Livia Persano Oddo and Dr. Stefan Bogdanov for their truthful interest and timely scientific collaboration to study this unknown honey in Europe. To the memory of Professor João MF Camargo for identifying stingless bee species to name pot-honey beyond expectations. 25.1 Introduction During his visit to Venezuela in 2008, Prof. JMF Camargo could not observe the Melipona favosa (Fabricius 1798) that he kindly identified, in their cactus wild nests (see Fig. 25.1). However, he informed us that this was the first species of Meliponini accurately described, probably with a specimen from French Guiana. Prof. Camargo also authored and anchored the idea of pot-honey as the first honey on planet Earth, dating back to the late Cretaceous, before comb honey was produced by Apis mellifera. He had studied the oldest bee fossil, Cretotrigona prisca, preserved in amber from New Jersey (Michener and Grimaldi 1988a, b), and knew that dinosaurs and stingless bees shared landscapes 97–74 million years before present. This bee from the Paraguaná Peninsula (Falcón state, Venezuela) was undisturbed by Apis mellifera, until honey bee swarms were seen after the floods caused by el Niño at the end of 1999. But the Africanized honey bee colonized Venezuela since 1975 in southern Amazon state, and 1976 in Santa Elena de Uairén, Bolívar state (Gómez Rodríguez 1986). P. Vit (*) Apitherapy and Bioactivity, Food Science Department, Faculty of Pharmacy and Bioanalysis, Universidad de Los Andes, Mérida 5101, Venezuela Cancer Research Group, Discipline of Biomedical Science, The University of Sydney, Cumberland Campus C42, 75 East Street, Lidcombe, NSW 1825, Australia e-mail: vitolivier@gmail.com 363 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_25, © Springer Science+Business Media New York 2013 364 P. Vit Fig. 25.1 Entrance of Melipona favosa nest in a columnar cactus “cardón” Stenocereus griseus, Paraguaná Peninsula, Falcón state, Venezuela Photo: P. Vit M. favosa is mostly known as “erica” but is also named “maba” in a few places. It is a smaller bee than other Venezuelan Melipona such as M. compressipes and M. trinitatis, named “guanota.” However, it is bigger than stingless bees from other genera different from Melipona, like Tetragonisca and Scaptotrigona. The honey pots also have an intermediate size. This honey is reported in the classic novel “Doña Bárbara” (Gallegos 1973), the Venezuelan book on creole bees (Rivero Oramas 1972), and the chapter on Meliponini in the Catalogue of Bees in the Neotropical Region (Camargo and Pedro 2007), but is not considered in Venezuelan honey standards (Vit 2008a). It was available during field work in the plains and coastal regions of Venezuela. The honey harvest is traditionally made by removing sealed pots from the storage area in the hive, on a dish. The honey pots are compressed with forks or hands, and honey is decanted, and bottled, as learned from Venezuelan stingless bee-keepers, also known as “meliponicultors” (Vit 1994a, b). Our analytical pot-honey harvests were done by extraction with rubber tube adapted to a syringe, after piercing sealed honey pots, to avoid contamination from pollen pots. However, in a preliminary sensory trial in the Food Science Department at Universidad de Los Andes held in 2007, the additional sour taste, derived from fermented pollen in honey extracted by compression (by hand, with honey and pollen pot contents admixed), was highly appreciated (Vit et al. 2010b). Currently, suction pumps are used for meliponine honey extraction in Brazil (see Alves chapter in this book), while piercing and decantation are used in Australia (TA Heard and M Halcroft, personal communication). Comb honey from A. mellifera is different from pot-honey of Melipona. However, both honey types have practical applications as sweeteners, and prototypical medicinal uses conferred by the high osmotic pressure, and the action of minor components of botanical (see Tomás-Barberán chapter in this book) and bee origin. The enormous biodiversity of Meliponini, and their associated microorganisms (see chapters 25 Melipona favosa Pot-Honey from Venezuela 365 by Menezes et al., and Morais et al., this book), may add further functional properties to pot-honey, unknown for comb honey. Here I analyze the M. favosa honey composition in a collection of five samples from the Paraguaná Peninsula and review a database of 40 M. favosa pot-honeys from Venezuela, including their bioactive and sensory properties. 25.2 A Peculiar Honey, with Similarities to and Differences from Apis mellifera Since 1985, the collection of M. favosa honey has steadily increased. Only recently, a false M. favosa honey invaded the Venezuelan market (Vit et al. 2011). This fact should be of interest for Venezuelan sanitary authorities, and not ignored, as is often the case for fraudulent or adulterated honey of A. mellifera. It remains the responsibility of the consumer to determine the authenticity of honey, when needed for medicinal use. Venezuelan norms for honey created in 1984 (Comisión Venezolana de Normas Industriales 1984a, b) have not been revised, in contrast to the recent assessment of Colombian norms, in which honey produced by native bees was included for the first time in a honey regulation (ICONTEC 2007). This is a promising example for other countries to join the quest of setting standards for the honey produced by Meliponini, instead of searching for a new word such as “divine elixir” (Vit et al. 1998b). The word honey is not a trademark for that made by bees in combs and can be used for both the honey produced in pots and in combs (Vit 2010a). A number of collaborators were attracted by this honey processed in pots, and from that of other stingless bee species (their ability of transporting and storing the energy of the sun—as watery sugars—in flexible pots built up with cerumen, able to expand and reduce volumes during fermentive process) (see Fig. 25.2). Fig. 25.2 Melipona favosa (a) on a bottle of fermenting honey harvested from sealed honey pots and (b) her storage pots in the nest Photo: P. Vit P. Vit 366 This sour–sweet honey, with its own sugar spectra (Bogdanov et al. 1996; Vit et al. 1998a), has very low diastase activity, higher moisture and higher free acidity than that of A. mellifera, but similar ash, sucrose, and nitrogen content (Vit et al. 1994, 1998b). Possibly, it is a honey finished and stored with lower processing of nectar, causing nose perceptions resembling those of the foraged flowers, from a more diluted sugar matrix less transformed than A. mellifera comb honey, which is permitted by resin, which kills the bacteria, and by mutualistic microbes in the gut— just like Apis—which kill pathogenic microbes that would otherwise ruin both the honey and pollen (DW Roubik, personal communication). 25.3 Composition of Melipona favosa Honey Collected in Rational Hives Honey pots of M. favosa from Paraguaná Peninsula, Falcón state, Venezuela, were pierced to collect the honey by syringe extraction. Honey samples were harvested from five M. favosa hives, the same day. Physicochemical parameters were analyzed in duplicate according to the methods recommended by the Venezuelan regulations (COVENIN 1984a). Parameters measured included ash (gravimetric method), water content (refractometric method), reducing sugars and sucrose (titrimetric method), pH, free acidity (titrimetric method). Color was measured by optical comparison (instrumental method). Nitrogen was determined by a standard micro Kjeldahl method (AOAC 1984). The analytical results on chemical composition of the five samples of M. favosa honey are shown in Table 25.1. The honey produced by M. favosa is light in color. In the five samples analyzed here, the color varied between 20 and 27 mm Pfund. The moisture content varied between 29.7 and 30.2 g water/100 g honey, which is higher than the honey standard for A. mellifera, and typical for the values in meliponine honeys reported since Gonnet et al. (1964). The ash content varied between 0.07 and 0.14 g ash/100 g honey, falling below the maximum 0.5 g/100 g A. mellifera honey standard. The pH Table 25.1 Composition of Melipona favosa pot-honey from the Paraguaná Peninsula of Venezuela, n = 5 Physicochemical parameters Mean ± SD Min Max Color (mm Pfund) 23.2 ± 2.7 20 27 Moisture (g/100 g honey) 30.0 ± 0.2 29.7 30.2 Ash (g/100 g honey) 0.10 ± 0.02 0.07 0.14 pH 3.7 ± 0.2 3.5 3.9 Free acidity (milliequivalents/kg honey) 50.6 ± 18.3 34.2 85.2 Nitrogen (mg/100 g honey) 41.7 ± 8.1 30.0 53.4 Sugars (g/100 g honey) Reducing sugars 64.6 ± 2.3 61.4 69.0 Apparent sucrose 1.3 ± 0.5 0.7 2.0 25 Melipona favosa Pot-Honey from Venezuela 367 values are in the same range of A. mellifera honey, whereas the average free acidity (50.6 meq/kg honey) is higher than the maximum 40 meq/kg A. mellifera honey standard (COVENIN 1984b). This indicates the presence of higher amounts of weak acids, such as organic acids with low ionization. The nitrogen content varied between 30.0 and 53.4 mg N/100 g honey with an average of 41.7, similar to 40.66 mg N/100 g reported for M. favosa honey in a previous work, and slightly lower than 57.1 mgN/100 g found in A. mellifera honey from Venezuela (Vit et al. 1994). The average concentration of reducing sugars is into the limit of the minimum 65 g/100 g and of the maximum 5 g/100 g prescribed by the A. mellifera honey standards (COVENIN, 1984b). This means that some M. favosa honey samples do not fulfill this parameter due to a slightly lower concentration of reducing sugars, which is consistent with previous results (Vit et al. 1998b). 25.4 Sensory Attributes of Melipona favosa Honey A honey tasting sensory assay was initiated with the system used for A. mellifera. Sensations in the nose are called “odor,” whereas the multimodal sensations in the mouth—differing from taste and trigeminal sensations, are called “aroma.” Seven families of sensory attributes in the odor-aroma wheel (Piana et al., 2004) were adapted to eight sensory odor-aroma families in a table for stingless bees: (1) Floralfruity. (2) Vegetable. (3) Fermented. (4) Wood. (5) Bee hive. (6) Mellow. (7) Primitive. (8) Industrial chemicals (Vit et al. 2007). This is a cognitive construct to facilitate the perception of pot-honey in this system. A histogram of odor-aroma families perceived in one sample of M. favosa honey by eight assessors is shown in Fig. 25.3. The highest count for odor was halved with a dotted line, and for aromas with a straight line. Bars above the lines are considered primary odors and aromas, respectively, and below the lines are considered secondary odors and aromas. The family floral-fruity described both primary odor and aroma. The peculiar smell of the M. favosa nest is a primary attribute more frequent than woody, mellow, and primitive odors. Fermented, vegetable, and primitive secondary aromas are more frequent than woody, nest, and mellow. Overall, this M. favosa is a floral-fruity and fermented honey with the bouquet of the hive (given by the bees, collected substances and products). Other secondary odors and aromas were less frequent. The fermenting honey, noted as a sensory attribute, is interpreted as an indication that Meliponini process their food with microorganisms, possibly as evolutionary ability. The sensory concept, that fermented meliponine honey is not spoiled, was recently assessed during the 8th Pangborn Sensory Science Symposium (Vit et al. 2009b). In fact, meliponine honey is not to be considered a spoiled honey, even if it may ferment in the storage pots inside the hive and after harvest, due to the high water content and associated microorganisms. On the contrary, fermentation contributes to the typical sensory profile of this honey and also increased the antioxidant activity of T. angustula honey (Pérez-Pérez et al. 2007). P. Vit 368 O-A 40 odor aroma 30 c o u n 20 t s primary 10 secondary 0 1 2 3 4 5 6 7 odor-aroma families 1FF 2V 3F 4W 5N 6M 7P 8IC (no counts) Fig. 25.3 Sensory profile of Melipona favosa honey with a trained panel. FF floral-fruity, V vegetable, F fermented, W woody, N bee hive, M mellow, P primitive. The highest count for odor was halved with a dotted line, and for aromas with a straight line. Bars above the lines are considered primary odors and aromas, respectively, and below the lines are considered secondary odors and aromas. No counts for the industrial chemical family 8IC Modified from: Vit (2008b). Permission granted by Revista de la Facultad de Farmacia 25.5 Database of Melipona favosa Honey from Venezuela Settings of honey standards were suggested for the most studied stingless bees, four species of Melipona (M. asilvai, M. compressipes, M. favosa, M. mandacaia) and Tetragonisca angustula. The averages values found for 20 samples of M. favosa honey in a previous review were free acidity 49.9 meq/kg, 0.22 g ash/100 g, 55.8 mg nitrogen/100 g, 71.2 g reducing sugars/100 g, 1.7 g apparent sucrose/100 g, and 24.8 g water/100 g (Souza et al. 2006). The seven physicochemical standards in the Venezuelan norm COVENIN 2191– 84 are set for A. mellifera but not for Meliponini pot-honey: (1) Moisture (Max 20%), (2) Reducing sugars (min 65%), (3) Sucrose (max. 5%), (4) Free acidity (max 40 meq/100 g), (5) Ash (max 0.5%), (6) Hydroxymethylfurfural HMF (negative), (7) Diastase activity (positive). These last two parameters are qualitative and refer 25 Melipona favosa Pot-Honey from Venezuela 369 Table 25.2 Composition of Melipona favosa pot-honey from Venezuela highlighted values are different from Apis mellifera honey standards Physicochemical parameter N Mean ± SD Min Max Moisture (g/100 g honey) 40 28.0 ± 2.7 22.1 32.0 Ash (g/100 g honey) 40 0.14 ± 0.13 0.01 0.61 Diastase (DN)a,b 6 2.86 ± 0.36 2.64 3.50 Free acidity (milliequivalents/kg honey) 40 51.7 ± 25.2 12.7 97.1 Invertase (IU)c 6 90.08 ± 48.03 31.80 150.70 Nitrogen (mg/100 g honey) 39 45.7 ± 18.3 10.5 102.0 HMF (mg/kg honey) 21 17.7 ± 8.5 5.04 24.69 Sugars (g/100 g honey) Reducing sugars 40 67.3 ± 4.1 60.9 78.6 Apparent sucrose 40 2.1 ± 1.3 0.5 5.1 a The Diastase Number (DN) indicates g starch hydrolyzed/100 g honey/h, at pH 5.2 and 40°C b Semiquantitative data not included c An Invertase Unit (IU) indicates mmoles p-nitrophenyl glucopyranoside hydrolyzed/kg honey/ min, at pH 6.0 and 40°C to the heating and aging of the honey. Findings in previous works indicated the low diastase activity of M. favosa honey, as well as an HMF content similar to that of A. mellifera honey (Vit et al. 1994, 1998b). The natural low diastase activity values found in previous qualitative (Vit 1992) and quantitative (Vit et al. 1994, 1998b) measurements suggest this is not a quality indicator for M. favosa honey. For this reason, diastase activity was measured in half of the samples. The average composition and variations of 40 samples of M. favosa honey studied from samples taken over 20 years are indicated in Table 25.2. Free acidity, ash, reducing sugars, sucrose, and water content of honey are useful quality indicators for M. favosa, as they are for A. mellifera, although standards may differ. Flavonoid and polyphenol contents, antioxidant and antibacterial activities, and sensory analysis are biochemical, biological, and consumer analyses which also contributed to M. favosa honey characterization. 25.6 Suggested Standards for Melipona favosa Honey Compared to Apis mellifera Compared to Venezuelan honey standards for A. mellifera (COVENIN 1984b), the following changes in reference values may be adopted for M. favosa honey (see Table 25.3): (1) No variation for HMF values, (2) Increased maximum values for water content (up to a maximum of 35%), apparent sucrose (up to a maximum of 6%), free acidity (up to a maximum of 100 meq/100 g), and ash (up to a maximum of 1.0%), (3) Decreased minimum for reducing sugars (down to a minimum of P. Vit 370 Table 25.3 Suggested standards for Melipona favosa honey, compared to A. mellifera Melipona favosa Apis mellifera Quality factor suggested standard Relation standard Moisture (g/100 g) Max 35.0 > Max 20.0 Ash (g/100 g) Max 0.5 = Max 0.5 Free acidity (meq/100 g) Max 100.0 > Max 40.0 Nitrogen (mg/100 g) 10.0–105.0 New – Reducing sugars (g/100 g) Min 60.0 < Min 65.0 Apparent Sucrose (g/100 g) Max 6.0 > Max 5.0 HMF (mg/kg) Max 40.0 = Max 40.0 60%), (4) The nitrogen content is not included in the standards for A. mellifera honey, but a range 10–100 mg N/100 g honey would be useful for protection against adulteration and falsification, (5) Diastase activity is not included because the activity of this enzyme is very low in M. favosa honey; therefore, it is not a practical quality factor to measure freshness or heating. 25.7 The Inclusion of Biological Activity Descriptors In addition to compositional quality factors, the biological activity of honey could also become a useful descriptor for medicinal use. However, there are no simple descriptors for that purpose. For instance, the variable contents of flavonoids and polyphenols in A. mellifera unifloral honeys (Frankel et al. 1998) did not correlate with antioxidant capacity. The flavonoid content is lower than the polyphenols, as generally observed in the honey produced by other species of stingless bees, such as T. carbonaria from Australia (Persano Oddo et al. 2008), M. beecheii and M. solani from Guatemala (Gutiérrez et al. 2008), M. crinita, M. eburnea, M. grandis, M. illota, Nannotrigona melanocera, Partamona epiphytophyla, Ptilotrigona lurida, Scaptotrigona polysticta, Scaura latitarsis, and Tetragonisca angustula from Peru (Rodríguez-Malaver et al. 2009), Tetragonisca fiebrigi from Argentina and Paraguay (Vit et al. 2009a), and also in M. favosa from Venezuela (Vit et al. 2012). This means that other polyphenol types in pot-honey may explain their antioxidant activities. Seminal findings on greater contents of flavonoid glycosides than aglycones in M. favosa honey strongly differentiate them from A. mellifera honey. Pothoney of M. favosa has more aglycones, from hydrolyzed O-glycosides in the nectar and propolis (Truchado et al. 2011). Values of 45.9–227.92 mmole Trolox equivalents/100 g honey, positioned M. favosa honeys in the categories low (0–100) and high (200–300) reported for unifloral A. mellifera Czech honeys (Vit et al. 2008a). Considering antibacterial activity, a successful marker of antibacterial activity is the unique manuka factor (UMF) described by Prof. Peter Molan from Waikato University in New Zealand (Molan 2005). However, this is a useful marker for 25 Melipona favosa Pot-Honey from Venezuela 371 honey of a botanical origin including only Myrtaceae, genus Leptospermum. More conservative are the tests to measure inhibition of bacterial growth under controlled condition. The Gram positive S. aureus is more resistant to these honeys than the Gram negative E. coli, because lower MICs of honey were needed to kill E. coli than S. aureus. This was also observed in Venezuelan honeys of A. mellifera (Vit et al. 2008b) and M. favosa (Vit et al. 2012), other stingless bee species from Argentina (Vit et al. 2009a), and Geotrigona acapulconis from Guatemala (Dardón and Enríquez 2008). Although E. coli and S. aureus MICs were similar to those found in other Melipona species, E. coli was more resistant than S. aureus to Tetragonisca angustula honey from Guatemala. The anticancer activity of two M. favosa honeys (IC50 3.39–16.50 mg/mL) was measured in vitro using a model based on ovarian cancer (see Vit et al. chapter 35, this book). Considering that both samples were collected in the same meliponary but in different months, the effect of the botanical origin (see Obregón et al. chapter 23 in this book) becomes relevant to the bioactive properties of pot-honey. Melissoplaynology will be useful in the future, for understanding the contribution of botanical origin to the composition, sensory and biological properties of M. favosa honey. Denomination of unifloral honeys of each stingless bee species is not envisaged, but some exceptions may be valid, as well as for the geographical origin. 25.8 Contemporary Interactions to Value Melipona favosa Honey Expert scientists, technicians, and keepers of traditional meliponiculture can benefit consumers in search of information. Emotion, cognition, and communication are relevant components to spread the tradition and to foster technological progress. Observing a living stingless bee hive is the ultimate learning experience concerning pot-honey and the meliponines. M. favosa is a gentle bee that could be easily kept in schools, where young people can observe them. However, this bee thrives in the plains and coastal regions, and other species will be needed in different locations of Venezuela. The M. favosa bee can be kept by women, children, and the elderly. The entomological origin of honey should be on the label (common and scientific name of the bee). Consumers and stingless bee-keepers should be protected from producers of false meliponine honeys without stingless bee apiaries (meliponaries) to back up their honey production. Labels of organic certified honey may help to safeguard the reputation of pot-honey and be useful to promote this industry, but they demand great organization to be reliable. Acknowledgments To a 10-year-old child—my youngest brother Leonardo Vit, who found a hole with the face of a bee living inside a brick, in a wall of my parents’ garden. To the memory of Mr. Ramón Álvarez, who carefully kept the “erica” meliponary in the Paraguaná Peninsula. To the late Prof. João MF Camargo, Biology Department, Universidade de São Paulo, Ribeirão Preto, 372 P. Vit Brazil, for the identification of the bee. To “erica” keepers throughout Venezuela for their essential role in transmitting the tradition and the valuable pot-honeys that made this research possible: Mr. Rafael Obregón (Guasdualito, Apure state), Mr. Simón Cananeo (Vía Elorza, Apure state), Mr. Esteban Locsi (Barrancas, Barinas state), Mr. Francisco Oronoz (Guasipati, Bolívar state), Mr. Jacinto Cabrera (Las Manoas de Cariaco, Sucre state), Mr. Santana Obando (Vericallar, Sucre state), Mr. Amadeo Zavala (San Francisco de Macanao, Nueva Esparta state), Mr. Ramón Campos (Salamanca, Nueva Esparta state), Mr. Luis Martínez (Araguaimujo, Delta Amacuro state), Mrs. Natacha Ceccarelli (Acarigua, Portuguesa state). To the careful advice of Dr. Livia Persano Oddo after reading this manuscript, Dr. Tim Heard from CSIRO Ecosystem Science, Brisbane, Queensland, Australia, and Dr. D.W. Roubik for reviewing the English expressions. References Association of Official Analytical Chemists (AOAC). 1984. Official methods of analysis. 14th ed. AOAC; Arlington (TX), USA. 1375 pp. Bogdanov S, Vit P, Kilchenmann V. 1996. Sugar profiles and conductivity of stingless bee honey from Venezuela. Apidologie 27:445–450. Camargo JMF, Pedro SRM. 2007. Meliponini Lepeletier 1836. pp. 272–578. In: Moure JS, Urban D, Melo GAR, eds. Catalogue of bees (Hymenoptera, Apoidea) in the neotropical region. Sociedade Brasilera de Entomologia; Curitiba, Brasil. 1958 pp. COVENIN. 1984a. Comisión Venezolana de Normas Industriales. Miel de Abejas. Métodos de Ensayo. COVENIN 2136–84. Fondonorma; Caracas, Venezuela. 39 pp. COVENIN. 1984b. Comisión Venezolana de Normas Industriales. Miel de Abejas. COVENIN 2194–84. Fondonorma; Caracas, Venezuela. 5 pp. Dardón MJ, Enríquez E. 2008. Caracterización físicoquímica y antimicrobiana de la miel de nueve especies de abejas sin aguijón (Meliponini) de Guatemala. Interciencia 33:916–922. Frankel S, Robinson G, Berembaum M. 1998. Antioxidant capacity and correlated characteristics of 14 unifloral honeys. Journal of Apicultural Research 37:27–31. Gallegos R. 1973. Doña Bárbara. Colección Austral. Espasa Calpe; Buenos Aires, Argentina. 255 pp. Gómez-Rodríguez R. 1986. Apicultura venezolana. Manejo de la abeja africanizada. Edicanpa; Caracas, Venezuela. 280 pp. Gonnet M, Lavie P, Nogueira-Neto P. 1964. Étude de quelques charactéristiques des miels récoltés par certains Méliponines brésiliens. Comptes Rendus de l’ Academie des Sciences, Paris 258:3107–3109. Gutiérrez MG, Enríquez E, Lusco L, Rodríguez-Malaver A, Persano Oddo L, Vit P. 2008. Caracterización de mieles de Melipona beecheii y Melipona solani de Guatemala. Revista de la Facultad de Farmacia 50:2–6. ICONTEC. 2007. Instituto Colombiano de Normas Técnicas y Certificación. Norma Técnica Colombiana. Miel de Abejas. NTC 1273; Bogotá, Colombia. Available at: http://www.sinab. unal.edu.co/ntc/NTC1273.pdf. Michener CD, Grimaldi DA. 1988a. A Trigona from late Cretaceous amber of New Jersey (Hymenoptera: Apidae: Meliponinae). American Museum Novitates 2917:10 pp. Michener CD, Grimaldi DA. 1988b. The oldest fossil bee: Apoid history, evolutionary stasis, and antiquity of social behavior. Proceedings of the National Academy of Sciences of the United States of America 85:6424–6426. Molan P. 2005. Manuka honey as a medicine. Public Service Review 5:52–54. Available at: www. bio.waikato.ac.nz/pdfs/honeyresearch/bioactives.pdf. Pérez-Pérez E, Rodríguez-Malaver J, Vit P. 2007. Efecto de la fermentación en la capacidad antioxidante de miel de Tetragonisca angustula Latreille, 1811. BioTecnología 10:14–22. Persano Oddo L, Heard TA, Rodríguez-Malaver A, Pérez RA, Fernández-Muiño M, Sancho MT, Sesta G, Lusco L, Vit P. 2008. Composition and antioxidant activity of Trigona carbonaria honey from Australia. Journal of Medicinal Food 11:789–794. 25 Melipona favosa Pot-Honey from Venezuela 373 Piana ML, Persano Oddo L, Bentabol A, Bruneau E, Bogdanov S, Guyot Declerck C. 2004. Sensory analysis applied to honey. Apidologie 35:S26-S37. Rivero Oramas R. 1972. Abejas Criollas Sin Aguijón. Monte Ávila Editores, Colección Científica; Caracas, Venezuela. 110 pp. Rodríguez-Malaver AJ, Rasmussen C, Gutiérrez MG, Gil F, Nieves B, Vit P. 2009. Properties of honey from ten species of Peruvian stingless bees. Natural Product Communications 4:1221–1226. Souza B, Roubik DW, Barth O, Heard T, Enríquez E, Carvalho C, Marchini L, Villas-Bôas J, Locatelli J, Persano Oddo L, Almeida-Muradian L, Bogdanov S, Vit P. 2006. Composition of stingless bee honey: Setting quality standards. Interciencia 31:867–875. Truchado P, Vit P, Ferreres F, Tomás-Barberán F. 2011. Liquid chromatography-tandem mass spectrometry analysis allows the simultaneous characterization of C-glycosyl and O-glycosyl flavonoids in stingless bee honeys. Journal of Chromatography A 1218:7601–7607. Vit P. 1992. Caracterización de mieles de abejas sin agijón producidas en Venezuela. Trabajo de Ascenso a la Categoría de Profesor Asociado. Facultad de Farmacia, Universidad de Los Andes; Mérida, Venezuela. 125 pp. Vit P. 1994a. Las abejas criollas sin aguijón. Vida Apícola 63:34–41. Vit P. 1994b. Los meliponicultores venezolanos. Vida Apícola 64:26–33. Vit P. 2008a. La miel precolombina de abejas sin aguijón (Meliponini) aún no tiene normas de calidad. Revista Boletín Centro de Investigaciones Biológicas 42:415–423. Vit P. 2008b. Valorización de la miel de abejas sin aguijón (Meliponini). Revista de la Facultad de Farmacia 50:20–28. Vit P. 2010a. The word “honey” is not a trade mark for combs. Available at: http://www.saber.ula. ve/bitstream/123456789/31449/1/honey_is_not_a_trademark.pdf. Vit P, Bogdanov S, Kilchenman V. 1994. Composition of Venezuelan honeys from stingless bees and Apis mellifera L. Apidologie 25:278–288. Vit P, Deliza R, Pérez A. 2011. How a Huottuja (Piaroa) community perceives genuine and false honey from the Venezuelan Amazon, by free-choice profile sensory method. Brazilian Journal of Pharmacognosy 21:786–792. Vit P, Fernández-Maeso MC, Ortiz-Valbuena A. 1998a. Potential use of the three frequently occurring sugars in honey to predict stingless bee entomological origin. Journal of Applied Entomology 122:5–8. Vit P, González I, Carvalho CAL, Enríquez E, Moreno E, Roubik DW, Souza BA, Villas-Bôas JK. 2007. Tabla olor-aroma. Taller evaluación sensorial de mieles de abejas sin aguijón; Mérida, Venezuela. Available at: www.saber.ula.ve/stinglessbeehoney/odour-aroma.php. Vit P, González I, Deliza R. 2010b. Contributions of two sensory methods to differentiate Meliponini pot honey. XIX Italo-Latinoamerican Congress of Etnomedicine “Fernando Cabieses Molina”. Tanka Village Resort, Villasimíus, Cagliari, Sardegna, Italia. Vit P, Gutiérrez MG, Rodríguez-Malaver AJ, Aguilera G, Fernández-Díaz C, Tricio AE. 2009a. Comparación de mieles producidas por la abeja yateí (Tetragonisca fiebrigi) en Argentina y Paraguay. Acta Bioquímica Clínica Latinoamericana 43:219–226. Vit P, Gutiérrez M, Titera D, Bednár M, Rodríguez-Malaver A. 2008a. Mieles checas categorizadas según su actividad antioxidante. Acta Bioquímica Clínica Latinoamericana 42:237–244. Vit P, Mejías A, Rial L, Ruíz J, Peña S, González AC, Rodríguez-Malaver A, Arráez M, Gutiérrez C, Zambrano A, Barth OM. 2012. Conociendo la miel de Melipona favosa en la Península de Paraguaná, Estado Falcón, Venezuela. Revista del Instituto Nacional de Higiene Rafael Rangel 43:15–19. Vit P, Persano Oddo L, Marano ML, Salas de Mejías E. 1998b. Venezuelan stingless bee honeys characterised by multivariate analysis of compositional factors. Apidologie 29:377–389. Vit P, Rodríguez-Malaver AJ, Pérez-Pérez E, Enríquez E, Pérez A. 2009b. Fermented Meliponini honey is not spoiled honey. In: 8th Pangborn sensory science symposium, Firenze, Italia, 26–30 July. Vit P, Rodríguez-Malaver A, Roubik DW, Moreno E, Souza BA, Sancho MT, Fernández-Muiño M, Almeida-Anacleto D, Marchini LC, Gil F, González C, Nieves B. 2008b. Expanded parameters to assess the quality of honey from Venezuelan bees (Apis mellifera). Journal of ApiProduct and ApiMedical Science 1:72–81. Chapter 26 Tetragonisca angustula Pot-Honey Compared to Apis mellifera Honey from Brazil Ligia Bicudo de Almeida-Muradian 26.1 Introduction According to Brazilian legislation (Brasil 2000), honey is considered a food product produced by bees from floral nectar, plant secretions and sap-feeding insects, collected from living plants that bees transform, combine with specific substances, and store. Commercial honey is usually produced from floral nectar. Honeydew is produced from exudates of some insects and is called in Portuguese “mel de melado” (Campos et al. 2003). In Brazil there are two types of beekeeping: (1) the commercial kind with Apis mellifera, and (2) meliponiculture, which uses stingless bees. Honey from stingless bees is more expensive than commercial honey. However, it is sold without proper regulation. There are no identity and quality parameters, or regulation, for this type of honey which is popularly known by its beneficial properties to human health (Vit et al. 2004; Sousa 2008). The characteristics of beekeeping products have specific laws for quality control of honey (Brasil 2000), pollen, propolis and royal jelly (Brasil 2001). Meliponiculture is the art of dealing with indigenous stingless bees, obtaining honey as the primary product (Nogueira-Neto 1997). As cited by Kerr et al. (2005), stingless bees were the only species producing commercial honey in Brazil, until 1838. Because they are traditionally kept by indigenous people, they can be also referred to as indigenous bees. Tetragonisca angustula (Latreille 1811), a small bee known as “jataí”, is the most abundant stingless bee in the southeast and southern regions of Brazil L.B. Almeida-Muradian (*) Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, Brazil e-mail: ligiabi@usp.br 375 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_26, © Springer Science+Business Media New York 2013 376 L.B. Almeida-Muradian (Villas Bôas and Malaspina 2005) and produces an excellent honey with distinct characteristics from A. mellifera. It is difficult to establish a single standard for all of Brazil, a country rich in stingless bee species and characterized by great environmental diversity (AlmeidaMuradian 2009). Honey samples produced by A. mellifera and T. angustula in the same region of Brazil are compared here. 26.2 Beekeeping and Meliponiculture in Brazil Beekeeping with honey bees is practiced in Brazil since the immigration of Europeans, mostly Italians and Germans, who brought the bees in the middle of the nineteenth century, introducing them in Rio Grande do Sul, Santa Catarina and Paraná states (Kerr et al. 2005); but stingless beekeeping potentially originated 15,000 years ago (David W Roubik, personal communication). Meliponiculture can be a sustainable activity and economically viable, since honey produced by native bees has a guaranteed market (Oliveira 2002). There are about 400 species of meliponines in Brazil, and many others are found in the tropical world, with more than 70% occurring in the Americas (Carvalho et al. 2003) and Camargo, Michener, in this book. Although the amount of honey obtained from a colony of stingless bees is not much, compared with the Africanized honey bee, stingless bees have advantages such as: (1) they are more suitable for the pollination of trees from Brazilian forest and cultures and (2) their honey has the best price in the market, as a special organic product, with particularities of taste and aroma, which depend on flora and bee species (Venturieri 2003). According to Nogueira-Neto (1997), the different food collection habits of native bees, compared to the Africanized bees, varies the composition of their honey. 26.3 Honey Composition of Brazilian Tetragonisca angustula The stingless bee jataí has a characteristic nest and a cerumen entrance tube. This is one of the meliponine species most adaptable in nesting. They live in cities and towns, virgin forests, and secondary vegetation, under the ground, in trees, and in the hollows between rocks (Nogueira-Neto 1970). Jataí honey is collected by piercing the honey pots. Honey is removed with a large syringe, or a suction pump, and later filtered. As a sanitary precaution, the honey is removed from closed pots, considered “mature honey”, to prevent absorption of moisture and consequently deterioration. Although they produce honey in lesser amount, the meliponines supply a varied product compared to common honey from A. mellifera, because of their special flavours (Carvalho et al. 2005). T. angustula 26 Tetragonisca angustula Pot-Honey Compared... 377 produces a honey well appreciated by the consumers. Jataí honey is used also for therapeutic treatments including ophthalmic and pulmonary uses (Iwama 1977). 26.4 Legislation for Apis mellifera Honey and Quality Parameters Honey is considered food that provides energy, being elaborated from the dehydration and transformation of floral nectar. For human consumption, honey needs to comply with the minimum requirements of identity and quality demanded by proper regulation (Sousa 2008). For A. mellifera, Brazilian honey standardization employs Normative Instruction 11 October 2000 (Brasil 2000). This regulation was based on European legislation and honey from A. mellifera, not the honey from native bees, which presents differences in some physicochemical parameters (Azeredo et al. 2000; Sousa 2008). However, some works had suggested quality standards for the regulation of the stingless bee honeys. Some researchers suggested maximum and minimum values for each quality parameter for stingless bee honey. Vit et al. (2004) proposed quality standards for stingless bee honey from Venezuela, divided in three groups: Melipona, Scaptotrigona and Trigona. Villas-Bôas and Malaspina (2005) suggested parameters for Brazilian stingless bee honey. The values defined for Brazilian legislation of quality control concerning A. mellifera and values suggested for stingless bee honey, by VillasBôas and Malaspina (2005), are shown in Table 26.1. The Brazilian Legislation standardizes the quality of the honeys evaluating the parameters indicating physicochemical characteristics of maturity (reducing sugars, apparent sucrose and moisture) of authenticity (insoluble solids in water and minerals) and of deterioration (acidity, diastase activity and hydroxymethylfurfural). The recommended method for sugar analyses in honey, using Lane and Eynon method which consists of the reduction of copper ions in alkaline Fehling solution (Brasil 2000). Beyond the physicochemical traditional methods, other methods more selective have been studied that can be applied like high performance liquid chromatography Table 26.1 Legislation standards for quality control of Apis mellifera, suggested values for honey of Meliponini and Tetragonisca angustula in Brazil Apis mellifera Meliponini honey (VillasChemical parameters honey (Brasil 2000) Bôas and Malaspina 2005) Reducing Sugars (%) Min. 65.0 Min. 50.0 Moisture (%) Max. 20.0 Max. 35.0 Apparent Sucrose (%) Max. 6.0 Max. 6.0 Insoluble Solids (%) Max. 0,1 Max. 0.4 Minerals (%) Max. 0.6 Max. 0.6 Acidity (meq/Kg) Max. 50.0 Max. 85.0 Diastase activity (DN) Min. 8.0 Min. 3.0 Hydroxydometylfurfural (mg/kg) Max. 60.0 Max. 40.0 378 L.B. Almeida-Muradian (HPLC), which is capable of identifying a high variety of carbohydrates in the sample. This method is more sensible, reducing the time of the analysis (Cano et al. 2006). Another important parameter for the determination of honey quality is the amount of moisture (water content), responsible for conservation of the honey. Water content of honey presents a great variation (14–25%), with the ideal values between 17 and 18%, when it is not prone to fermentation (Louveaux 1968). Moreover, sugars and water correspond to a major part of honey composition, but there are also small amounts of enzymes, whose presence vary compared with the substances producing sweetness. The main enzymes presented in honey are invertase, diastase and glucose oxidase (White 1975). Invertase originates from the hypopharyngeal glands of the bees. It is the main factor responsible for the chemical transformation of the nectar in honey (Maurizio 1959). It is added to the nectar and its activity can continue in the product after extraction. Invertase hydrolyzes sucrose into glucose and fructose; other more complex sugars are also transformed under the action of this enzyme (Iwama 1977). The reduction of this enzyme can be caused by the processing, heat and shelf life (Huidobro et al. 1995). The activity of the enzyme diastase is used as a quality parameter for authenticity of honey from A. mellifera. This quantification indicates the intensity of heating and natural degradation of the product. Another indicative characteristic from adulteration of the honey is the quantity of hydroxymethylfurfural (HMF). It is a cyclic aldehyde (C6H6O3) formed by decomposition of fructose in the presence of acid (pH 3.8–3.9). The process of dehydration from fructose indicate ageing and heating of the honey (González 2002; White 1975; Gonnet 1963). The identification of this compound is used to verify honey adulteration with commercial sugar (beetroot or maize), inadequate storage and overheating (Vilhena and Almeida-Muradian 1999). Another quality parameter is the free acidity of honey. The acid found in honey is responsible for its stability against microorganisms (White 1975). The pH of honey varies from 3.2 to 4.2 (average of 3.9) being influenced by the mineral percentage. Generally the honeys rich in ash present high values of pH (White 1975). The quantification of insoluble solids is another quality parameter demanded by legislation used to verify the pureness from honey and the efficiency in the extraction process (Leite and Santos 2001). The maximum allowed by Brazilian legislation for insoluble solids in honey is of 0.1%, except the pressed honey that tolerates 0.5% (Brasil 2000). Brazilian honey possesses a large variety of colours, which can influence the preference of the consumers. Honey colour can be correlated with its floral origin, processing storage, climatic factors and the temperature which the honey ripens in the beehive (Seemann 1988). Dark honeys have largest amounts of minerals compared with light ones. The percentage of mineral (total ash) varies from 0.02 to 0.6%. Ashes constitute mainly of salts from calcium, sodium, potassium, magnesium, iron, chlorine, phosphorus, sulphur and iodine (Sepúlveda Gil 1980). 26 Tetragonisca angustula Pot-Honey Compared... 26.5 379 Physicochemical Properties of T. angustula and A. mellifera Honey in Brazil Samples from T. angustula (n = 6) and A. mellifera (n = 6) honey were collected in six cities from São Paulo state, Brazil (Amparo, Itaberaba, Lins, Marília, Pedreira, Santo Antonio de Posse). The honey was kept frozen until analysis. Moisture was measured with an Abbe refractometer, and refraction index was converted into humidity using the Chataway table at 20°C (Brasil 2000; AOAC 1990; Almeida-Muradian and Bera 2008). Reducing sugar content and apparent sucrose were determined by titration using Fehling reagent (CAC 1989; Bogdanov et al. 1997). Insoluble solids were analyzed by gravimetry according to Brazilian regulation (Brasil 2000) and the Codex Alimentarius Commission (CAC 1989). Minerals (ash) were determined by gravimetric methods (oven at 550°C) (CAC 1989; Brasil 2000). Free acidity was measured by potentiometric titration (AOAC 1990; Brasil 2000; Bogdanov et al. 1997). Diastase employed the spectrophotometric method—wavelength 660 nm (Brasil 2000; CAC 1989). Hydroxymethylfurfural (HMF) content was measured by spectrophotometry at 284 nm, subtracting the back absorbance at 336 nm according to AOAC (1990) and Brazilian regulation (Brasil 2000). All measurements were made in triplicate. The composition of the two types of honeys (A. mellifera and T. angustula) from Brazil obtained by Sousa (2008) as well as the values used for honey quality control required by the Brazilian regulation for A. mellifera Brasil 2000) can be seen in Table 26.2. Lower moisture offers some security against fermentation, because below 18%, this process does not occur (Crane 1975; Rodrigues et al. 2005). Campos et al. (2003), analyzing floral and honeydew samples from A. mellifera, obtained variation between 15 and 20.8% moisture content. Azeredo and Azeredo (1999), working with honeys from São Fidelis (RJ) found levels between 18.96 and 19.6%. In Bahia State (Brazil), Sodré (2000) obtained moisture values between 18 and 21.9% for coastal region honey. Brazilian regulation for A. mellifera (Brasil, 2000) establishes a maximum of 20% moisture. Sousa (2008) gave honey of T. angustula values varying from 23.40 to 25.60% for São Paulo state (in Lins, Amparo, Pedreira, Itaberaba, Marília and Santo Antônio de Posse) which are adequate if we use the suggested values for stingless bee honey of Villas-Bôas and Malaspina (2005) (<35% moisture). Similar values were found by Souza et al. (2006), between 26.10 and 26.62, and by Denadai et al. (2002), 23.70%. However, Iwama (1977) found wide variation (22.70–35.4%). In Table 1, Sousa (2008) jataí honey presents values not meeting standards for honey of A. mellifera: moisture (23.40–25.60%), acidity (21.65–63.85 meq/kg) and reducing sugars (44.78–67.54%). However, they are in accordance with the values suggested for stingless bee honey (Villas-Bôas and Malaspina 2005). Regarding the honey free acidity values obtained for T. angustula (Table 26.2), they were similar to Cortopassi-Laurino and Gelli (1991) (acidity between 30.0 and 90.0 meq/ kg) for different species of stingless bees. Reducing sugars were similar to data from Almeida—Anacleto (2007) (48.66–57.94%) and Rodrigues et al. (1998) 58.19% average 380 L.B. Almeida-Muradian Table 26.2 Composition of T. angustula and Apis mellifera honey, compared to the Brazilian legislation parameters Brazilian regulation for Tetragonisca A. mellifera angustula honeya Apis mellifera honeyb n=6 honeya n = 6 Parameters Reducing sugars (g/100 g honey) Moisture (g/100 g honey) Apparent sucrose (g/100 g honey) Insoluble solids (g/100 g honey) Minerals/Ash (g/100 g honey) Free acidity (meq/kg) Diastase activity (DN) hydroxymethylfurfural (mg/kg) (HMF) a Sousa (2008) b Brasil (2000) Mean ± SD (Min–Max) 57.09 ± 7.83 (44.78–67.54) 24.37 ± 0.77 (23.4–25.6) 2.14 ± 1.80 (0.43–4.46) 0.06 ± 0.03 (0.02–0.10) 0.28 ± 0.11 (0.17–0.42) 37.34 ± 16.74 (21.65–63.85) 16.93 ± 3.94 (11.01–22.45) 0.65 ± 0.25 (0.30–0.93) Mean ± SD (Min–Max) 71.50 ±10.45 (52.98–84.24) 17.29 ± 1.23 (15.40–19.00) 2.99 ± 2.60 (0.56–7.64) 0.04 ± 0.03 (0.01–0.08) 0.20 ± 0.06 (0.11–0.26) 25.48 ± 5.66 (16.82–32.47) 7.32 ± 3.50 (2.20–11.49) 11.37 ± 7.78 (2.0–21.0) Standard Min. 65.0 Max. 20.0 Max. 6.0 Max. 0.1 Max. 0.6 Max. 50.0 Min. 8.0 Max. 60.0 for T. angustula; apparent sucrose values were similar to those obtained by Souza et al. (2006) who analyzed 152 samples of different stingless bee honey (1.1–4.8%). HMF values for honey of T. angustula were similar to “uruçú” M. scutellaris (mean = 0.38 mg/kg) (Marchini et al., 1998). Diastase values (Table 26.2) were in accordance with the values obtained by Vit et al. (1998) for stingless bees (excluding Melipona) from Venezuela (2.60–36.60). Insoluble solids obtained were low, indicating there are no impurities in samples, similar to values obtained from M. fasciculata known as “uruçú cinzenta” (Silva 2006), with a mean value of 0.02%. Ash content was in accordance with that presented by Carvalho et al. (2005), varying between 0.04 and 0.50% for different stingless bees. Acknowledgements The author is grateful to FAPESP, CNPq, anonymous referees, and careful editorial support constantly received, and also greatly thanks G.L. Sousa. References Almeida-Muradian LB. 2009. Qualidade dos produtos apícolas e otimizaçãoquimiométrica dos métodos de análise do mel por espectroscopia no infravermelho (FT-IR ATR). Tese de LivreDocência, Faculdade de Farmacêuticas da Universidade de São Paulo, Brasil, 119 pp. Association of Official Analytical Chemists [AOAC] (1990). Official methods of analysis of the association of official analytical chemists. 15th edition. Arlington: AOAC. 26 Tetragonisca angustula Pot-Honey Compared... 381 Azeredo LC, Azeredo MA, Beser LBO. 2000. Características físico-químicas de amostras de méis de melíponas coletados no Estado de Tocantins. In 13 Congresso Brasileiro de Apicultura, Florianópolis, Santa Catarina, Brazil. Azeredo MAA, Azeredo LC. 1999. Características físico-químicas dos méis do município de São Fidélis-RJ. Ciência e Tecnologia de Alimentos 19:3–7. Bogdanov S, Martin P, Lullmann C. 1997. Harmonized methods of the European Honey Commission. Apidologie (extra issue):1–59. Brasil. 2000. Ministério da Agricultura, Pecuária e Abastecimento. Legislação. SisLegis—Sistema de Consulta à Legislação. Instrução Normativa n.11, de 20 de outubro de 2000. Aprova o regulamento técnico de identidade e qualidade do mel. Available at: http://extranet.agricultura.gov. br/sislegisconsulta. Brasil. 2001. Ministério da Agricultura, Pecuária e Abastecimento. Legislação. SisLegis – Sistema de Consulta à Legislação. Instrução Normativa n.3, de 19 de janeiro de 2001. Aprovar os regulamentos técnicos de identidade e qualidade de apitoxina, cera de abelha, geléia real, geléia real liofilizada, pólen apícola, própolis e extrato de própolis. Available at: http://extranet.agricultura.gov.br/sislegis-consulta. Campos G, Campos G, Della-Modesta RC, Silva, TJP, Baptista KE, Gomides MF, Godoy RL. 2003. Classificação de mel em floral ou mel de melato. Ciência e Tecnologia de Alimentos 123:1–5. Cano CB, Felsner ML, Bruns RE, Matos JR, Almeida-Muradian LB. 2006. Optimization of mobile phase for separation of carbohydrates in honey by high performance liquid chromatography using a mixture design. Journal of the Brazilian Chemical Society 17:588–593. Carvalho CAL, Alvez RMO, Souza BA. 2003. Criação de abelhas sem ferrão: aspectos práticos. SEAGRI; Cruz das Almas, Bahia, Brasil. 42 pp. Carvalho CAL, Souza BA, Sodre GS, Marchini LC, Alves RMO. 2005. Mel de abelha sem ferrão: contribuição para a caracterização físico-química. SEAGRI; Cruz das Almas, Bahia, Brasil. 32 pp. Codex Alimentarius Commission. 1989. Codex standards for sugars (honey). Rome: FAO. Cortopassi-Laurino M, Gelli DS. 1991. Analysepollinique, propriétesphysico-chimiques et action antibactérienne des miels d’ abeilles africanisées Apis mellifera et de Méliponinés du Brésil. Apidologie 22:61–73. Crane E, ed. 1975. Honey: a comprehensive survey. Heinemann; London, UK. 605 pp. Crane E. 1990. Bees and beekeeping: science, practice, and world resources. Cornell University Press; London, UK. 720 pp. Denadai JM, Ramos Filho MM, Costa DC. 2002. Caracterização físico-química de mel de abelha Jataí (Tetragonisca angustula) do município de Campo Grande - MS: obtenção de parâmetros para análises de rotina. p. 80. 14 Congresso Brasileiro de Apicultura, Campo Grande, MS, Brasil. Gonnet M. 1963. L’hydroxymethylfurfural dans les miels: mise au point d’une méthode de dosage. Annales Abeille 61:53–67. González MM. 2002. El origem, la calidad y la frescura de una miel: la interpretación hidroximetilfurfural de amostras de méis de flores silvestres produzidos por Apis melliferano estado de São Paulo. 14º Congresso Brasileiro de Apicultura, Campo Grande, MS, Brasil. Heard TA. 1999. The role of stingless bees in crop pollination. Annual Review of Entomology 44:183–206. Huidobro JF, Santana FJ, Sanches MP, Sancho MT, Muniategui S, Simal-Lozano J. 1995. Diastase, invertase and ß-glucosidase activities in fresh honey from north-west Spain. Journal of Apicultural Research 34:39–44. Iwama S. 1977. Coleta de alimentos e qualidade do mel de Tetragoniscaangustula Latreille (Apidae, Meliponinae). Dissertação de Mestrado, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brasil. 134 pp. Kerr WE, Carvalho GA, Silva AC, Assis MGP. 2005. Aspectos pouco mencionados da biodiversidade Amazônica. Mensagem Doce, n. 80, mar. Available at: http://www.apacame.org.br/ index1.htm. 382 L.B. Almeida-Muradian Leite FF, Santos AAR. 2001. Estudo exploratório da cadeia produtiva do mel de abelhas no estado do Pará, Monografia do Curso de Especialização em Agricultura Integrada da Amazônia, Faculdade de Ciências Agrárias do Pará, Pará, Brasil. 67 pp. Louveaux J. 1968. Composition, propriétés et technologie du miel. pp. 277–324. In Chauvin R, ed. Traité de biologie de l’abeille: les produits de la ruche. Masson; Paris, France. v.3, 152 pp. Marchini LC, Carvalho CAL, Alves RMO, Teixeira GM, Oliveira PCF, Rubia VR. 1998. Características físico-químicas de amostras de méis da abelha uruçú (Melipona scutellaris). p. 201. In 12º Congresso Brasileiro de Apicultura, Salvador, Bahia, Brasil. Maurizio A. 1959. Breakdown of sugar by inverting enzymes in the pharyngeal glands and midgut of the honeybee. 2. Winter bees (Carniolan and Nigra). Bee World 40:275–283. Nogueira-Neto P. 1970. Criação de abelhas indígenas sem ferrão (Meliponinae). Tecnapis; São Paulo, Brasil. 365 pp. Nogueira-Neto P. 1997. Vida e criação de abelhas indígenas sem ferrão. Editora Nogueirapis; São Paulo, Brasil. 446 pp. Oliveira F. Meliponicultura: Projeto Iraquara: Promovendo a “Arte de manejar abelhas indígenas sem ferrão na região Amazônica”. 2002. Mensagem Doce n.69. Available at: http://www.apacame.org.br/mensagemdoce/69/meliponicultura.htm. Rodrigues ACL, Marchini LC, Carvalho CAL. 1998. Análises de mel de Apis mellifera L. 1758 e Tetragoniscaangustula (Laitreille, 1811) coletado em Piracicaba - SP. Revista de Agricultura 73:255–262. Rodrigues AE, Silva SEM,Beserra EMF; Rodrigues ML. 2005. Análise físico-químico dos méis das abelhas Apis mellifera e Melípona scutellarisproduzidos em duas regiões no Estado da Paraíba. Ciência Rural 35:1166–1171. Seemann P, Neira M. 1988. Tecnología de la producción apícola. Valdivia: Universidad Austral de Chile/Facultad de Ciencias Agrarias Empaste, p. 202. Sepulveda Gil JM. 1980. Apicultura. AEDOS, Barcelona, Spain. p 418. Silva EVC. 2006. Caracterização e pasteurização de méis de abelhas Melipona fasciculata (uruçu cinzenta) e Apis mellifera (Africanizadas). Dissertação de Mestrado, Faculdade de Engenharia Química em Alimentos - Universidade Federal do Pará, Belém, Pará, Brasil. 49 pp. Sodré GS. 2000. Características físico-químicas e análises polínicas de amostras de méis de Apis mellifera L. 1758 (Hymenoptera: Apidae) da região litoral norte do Estado da Bahia. Dissertação de Mestrado, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba, Brasil. 83 pp. Sousa GL. 2008. Composição e qualidade de méis de abelhas Apis mellifera e méis de abelha jataí (Tetragonisca angustula). Dissertação de Mestrado, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, Brasil. 86 pp. Souza B, Roubik D, Barth O, Heard T, Enríquez E, Carvalho C, Villas-Bôas J, Locateli J, PersanoOddo L, Almeida-Muradian L, Bogdanov S, Vit P. 2006. Composition of stingless bee honey: setting quality standards. Interciencia 31:867–875. Venturieri GC. 2003. Meliponicultura: criação racional de abelhas indígenas sem ferrão. Embrapa; Belém, Pará, Brasil. 21 pp. Villas Bôas JK,Malaspina O. 2005. Parâmetros físico-químicos propostos para o controle de qualidade do mel de abelhas indígenas sem ferrão no Brasil. Mensagem Doce, n. 82. Available at: http://www.apacame.org.br/index1.htm. Vilhena F, Almeida-Muradian LB. 1999. Manual de análise físico-químicas do mel. Apacame; São Paulo, Brasil. 16 pp. Vit P, Medina M, Enriquez ME. 2004. Quality standards for medicinal uses of Meliponinae honey in Guatemala, Mexico and Venezuela. Bee World 85:2–5. Vit P, Persano Oddo LP, Marano ML, Mejías ES. 1998. Venezuelan stingless bee honeys characterized by multivariate analysis of physiochemical properties. Apidologie 29:377–389. White Jr. JW. 1975. Composition of honey. pp. 157–206. In Crane E, ed. Honey: a comprehensive survey. Heinemenn; London, England. 608 pp. Chapter 27 Honey of Colombian Stingless Bees: Nutritional Characteristics and Physicochemical Quality Indicators Carlos Alberto Fuenmayor, Amanda Consuelo Díaz-Moreno, Carlos Mario Zuluaga-Domínguez, and Martha Cecilia Quicazán 27.1 Introduction The geographic location of Colombia and its mega-biodiversity have been identified as advantages for beekeeping and for meliponiculture. Beekeeping is a potentially sustainable activity and presents an interesting opportunity to identify new products—mostly yet-to-be-discovered—with unique features related to their natural origin and functional characteristics. There are certainly more than an estimated 900 native bee species in Colombia (Freitas et al. 2009). As among other Latin American countries, pre-Hispanic cultures that lived in different territories now located in Colombia practiced meliponiculture (especially of the genera Melipona and several others), for the extraction and processing of honey and the use of cerumen in metalwork. The European colonization of Central and South America minimized the practice of meliponiculture, introduced beekeeping with hives of Apis mellifera, and largely ended meliponiculture in Colombia. More recently, the trends of increased consumption of natural foods and health products have played an important role in the renewed interest in bee products, particularly honey from stingless bee species, and the recovery of traditional knowledge. Because of this, meliponiculture in Colombia has recently developed. Products such as honey produced by T. angustula, called “angelita” (“little angel” in English), is available in traditional markets and commands a significantly higher price relative to A. mellifera honey (e.g., because of its scarcity and because it is commonly thought to have medicinal features, the price of T. angustula honey can reach over ten times the price of honey from A. mellifera) (Rosso and Nates-Parra 2005). Although data on the marketing of pot-honeys in Colombia is not available, this product, known in Spanish as “miel de pote,” is mainly sold in natural foods stores. C.A. Fuenmayor • A.C. Díaz-Moreno (*) • C.M. Zuluaga-Domínguez • M.C. Quicazán Instituto de Ciencia y Tecnología de Alimentos—ICTA, Universidad Nacional de Colombia, Carrera 30 # 45-03 Ed. 500-C, Ciudad Universitaria, Bogotá, Colombia e-mail: amcdiazmo@unal.edu.co 383 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_27, © Springer Science+Business Media New York 2013 384 C.A. Fuenmayor et al. Beekeepers generally maintain relatively few nests, without financial gain, and often express a desire to make them a source of income, but they often lack knowledge about breeding techniques and maintenance. Technological and environmental issues, such as complex ecosystem interactions, the susceptibility of some species to human practices and relatively low honey production yields of individual nests, must be studied and overcome in order to make meliponiculture feasible in Colombia. Sustainable meliponiculture must be based on the generation of knowledge about native bee biology, their environment and characteristics of their products; therefore, the assessment of physical and chemical features of various honeys of Colombian stingless bees is of great interest. This chapter summarizes existing information regarding the physicochemical properties, nutritional information and quality of Colombian pot-honey. 27.2 Physicochemical Characteristics of Colombian Pot-Honey Only very recently have data been obtained on composition and physicochemical properties of Colombian pot-honeys. In fact, the Colombian technical standard for A. mellifera honey was extended from the stingless bee data published by Souza et al. (2006) and lacks information regarding Colombian stingless bee honeys (ICONTEC 2007). This general lack of knowledge has had several consequences. For example, there are difficulties in regulating the adulteration and falsification of stingless bee honey. The objective differentiation between authentic pot-honey and adulterated honey is especially interesting. Adulteration is often achieved by mixing pot-honey with common A. mellifera honey, and even by using adulterated honey of that species, containing added molasses and fructose syrup. Therefore, physicochemical characteristics are useful for regulating adulteration, and that knowledge will allow the development of regulatory standards. Previous physicochemical characterization of Colombian pot-honey (Zuluaga et al. 2009) has focused on T. angustula or non-compositional analysis (Torres et al. 2004, 2007), or remained unpublished. Information provided in this chapter derives from studies performed in the Institute of Food Science and Technology (ICTA), Universidad Nacional de Colombia, since 2008. The data are compared to those of Zuluaga (2010). Among the hundreds of Colombian stingless bee species (belonging to more than 13 genera; Nates-Parra 2001; Nates-Parra et al. 2006), the chemical composition of honey from seven genera has been explored. The species for which honey has been analyzed, as well as the number of samples and location for each are shown in Table 27.1. Several samples identified only to taxonomic group, e.g. genus. Often, the small amount of pot-honey that can be harvested at one time limits parameters assessed for a sample; therefore, some physicochemical characteristics are evaluated for few species or samples. In most cases, the analytical methods for 27 Honey of Colombian Stingless Bees... 385 Table 27.1 Physicochemical composition of pot-honey from Colombia (general information about the samples) Number of Taxon Geopolitical regions pot-honey samples a Frieseomelitta sp. Magdalena, Santander, Caldas 6 Melipona compressipes Santander, Caldas 12 Melipona favosa Sucre, Magdalena, Cundinamarca 7 Melipona eburnea Cundinamarca 7 Melipona sp. Santander 14 Nannotrigona testaceicornis Santander, Cundinamarca 3 Nannotrigona sp. Cundinamarca, Boyacá, Sucre, Santander 4 Paratrigona opaca Santander 4 Partamona peckolti Santander 1 Partamona sp. Santander 1 Plebeia spp. Santander 1 Scaptotrigona limae Sucre, Santander 2 Scaptotrigona sp. Cundinamarca, Caldas, Magdalena, 4 Santander Tetragona sp.a Santander 21 Tetragonisca angustulaa Magdalena, Santander, Cundinamarca, 45 Sucre, Caldas, Tolima, Huila a Previously denominated as a subgenus of Trigona (Rasmussen and Cameron 2010) pot-honey are the same as for A. mellifera honey (AOAC 1998). The number of analyzed honey samples varies according to the genus and the species; the largest number of samples corresponds to the genera Melipona and Tetragonisca because there is ample breeding of those species (see Souza et al. 2006). 27.2.1 Main Composition (Water and Sugars) The honey from stingless bees, like A. mellifera honey, is composed primarily of simple reducing sugars (mainly fructose and glucose), and non-reducing sugars (mainly sucrose and maltose), water and ash. These quality parameters depend on many factors, even for the same species, such as the maturity achieved in the bee nest or hive during the harvesting season, climatic and geographic factors, and other elements affecting floral abundance. The concentration of sugars and water for Colombian varieties of pot-honey are given in Table 27.2. Mean moisture content values ranged from 24.3 g/100 g for T. angustula to 42.7 g/100 g for Partamona pecktolti. The high water concentration in the former species is consistent with the relatively low total sugar content (°Brix) reported by Souza et al. (2006); such large moisture values had only been reported for Melipona quadrifasciata (Gonnet et al. 1964; Pamplona 1989) and Plebeia (Bijlsma et al. 2006; Carvalho et al. 2005). Most honey moisture content values 386 Table 27.2 Water and sugar contents of stingless bee honey from Colombia b b Fructose + Glucose (g/100 g) 29.7 ± 7.5 (5) 71.1 ± 8.1 (11) 72.2 ± 7.4 (3) 72.2 ± 7.4 (7) 67.6 ± 7.5 (14) 65.8 ± 35.1 (2) 50.8 ± 7.4 (4) 58.1 ± 12.4 (4) 40.6 (1) 38.3 (1) 36.7 (1) 67.7 ± 4.1 (2) 55.7 ± 5.0 (4) 60.8 ± 10.7 (19) 53.6 ± 11.8 (41) Disaccharidesb, c (g/100 g) 3.1 ± 2.7 (5) 3.4 ± 2.2 (11) 3.1 ± 1.8 (3) 3.6 ± 1.5 (7) 6.0 ± 2.3 (14) 7.9 ± 4.3 (2) 9.7 ± 4.3 (4) 3.9 ± 2.8 (4) 6.1 (1) 13.1 (1) 0.9 (1) 6.6 ± 4.6 (2) 12.1 ± 7.4 (4) 4.4 ± 5.6 (19) 4.2 ± 2.4 (41) C.A. Fuenmayor et al. Taxon Moisture (g/100 g) Fructose (g/100 g) Glucose (g/100 g) 33.1 ± 3.3 (6) 17.1 ± 6.6 (5) 12.6 ± 7.5 (5) Frieseomelitta sp.d Melipona compressipes 25.8 ± 2.0 (12) 36.9 ± 3.7 (11) 34.2 ± 4.4 (11) Melipona favosa 24.8 ± 1.8 (3) 39.3 ± 7.0 (7) 33.5 ± 3.1 (3) Melipona eburnea 27.6 ± 2.1 (7) 39.3 ± 7.0 (7) 38.5 ± 7.5 (7) Melipona sp. 26.2 ± 1.8 (14) 36.7 ± 3.5 (14) 30.9 ± 4.0 (14) Nannotrigona testaceicornis 27.5 ± 4.2 (3) 40.1 ± 18.1 (2) 25.7 ± 17.0 (2) Nannotrigona sp. 25.7 ± 1.8 (4) 33.1 ± 4.1 (4) 17.7 ± 3.7 (4) Paratrigona opaca 26.6 ± 1.2 (4) 30.9 ± 2.4 (4) 27.2 ± 10.7 (4) Partamona peckolti 42.7 (1) 26.6 (1) 14.0 (1) Partamona sp. 28.9 (1) 29.0 (1) 9.3 (1) Plebeia spp. 28.6 (1) 17.4 (1) 19.3 (1) Scaptotrigona limae 25.8 ± 2.2 (2) 39.0 ± 0.7 (2) 28.7 ± 3.4 (2) Scaptotrigona sp. 26.9 ± 2.9 (4) 31.8 ± 2.9 (4) 23.9 ± 3.1 (4) Tetragona sp.d 25.8 ± 3.6 (21) 31.8 ± 3.9 (19) 29.0 ± 6.8 (19) Tetragonisca angustulad 24.3 ± 2.3 (44) 30.1 ± 5.4 (41) 23.5 ± 6.4 (41) Mean values, ± standard deviation and (number of samples) are presented a Measured by refractometry according to the AOAC 969.38B standard methodology (AOAC 1998) b Assessed using an HPLC method based on the AOAC 979.23 and 983.22 standard methodologies (AOAC 1998) c Sucrose plus maltose d Previously denominated as a subgenus of Trigona (Rasmussen and Cameron 2010) a 27 Honey of Colombian Stingless Bees... 387 ranged between 24 and 27 g/100 g; this parameter maybe a promising distinctive criterion for this kind of honey. It is important to mention that this assessment is performed via the indirect refractometric methodology (AOAC 1998), and thus, equations originally developed for A. mellifera honey are used as an approximation; the accuracy of this methodology should be scrutinized for each honey. To obtain more reliable data on this important feature, methods such as vacuum drying (an official and a low cost procedure), the Karl-Fischer method, and similar techniques are recommended. Because of their floral origin, the main sugars present in stingless bee honey are glucose, fructose, maltose and sucrose; other disaccharides and oligosaccharides occur in lower proportion and in trace quantities. The sugar composition shown in Table 27.2 includes the most important sugars, all of which were evaluated using an HPLC (high pressure liquid chromatography) method, which does not differentiate sucrose and maltose. Therefore, the sum of these sugars is presented as disaccharides. Mean glucose content varied between 9.3 g/100 g (Partamona sp.) and 38.5 g/100 g (Melipona eburnea), mean fructose content between 17.1 g/100 g Frieseomelitta, and 40.1 g/100 g (Nannotrigona testaceicornis). The disaccharides varied between 0.9 g/100 g (Plebeia) and 13.1 g/100 g (Partamona). Honey from all Melipona had mean glucose content >30 g/100 g and mean fructose content >36 g/100 g. The mean fructose–glucose ratio for all species is >1 with an exception of one sample of Plebeia. An exceptionally high fructose/glucose value was found for Partamona, accompanied by the lowest total reducing sugars value and a relatively low value of total sugars. The fructose–glucose ratio for this species had not been previously reported as an unusually high value, although the low total sugar content has an antecedent in the study by Roubik (1983) (cited by Souza et al. 2006) in which honey of P. pecktolti had the lowest values of total sugars (°Brix) from among more than 25 types of stingless bee honey from Panama. Torres et al. (2004) reported values of fructose (36.1–37.6 g/100 g) and glucose (29.8– 31.8 g/100 g) for honey of T. angustula from Colombia that are at the higher end of the range shown in Table 27.2. Unusually low glucose content occurred in honey of Frieseomelitta (12.6 ± 7.5 g/100 g) and Nannotrigona (17.7 ± 3.7 g/100 g), whereas M. eburnea had the highest mean glucose content (38.5 ± 7.5 g/100 g). Moreover, high disaccharide content was found for Scaptotrigona (12.1 g/100 g) and Partamona (13.1 g/100 g). These values differ from those reported by Santiesteban-Hernández et al. (2003) in Mexico for the former genus (1.1 g/100 g). Such divergent values have high variability and probably too few samples analyzed, and thus, further characterization must be performed to better establish sugar concentration value as an origin denomination criterion, and to set regulatory quality standards. 27.2.2 Ash and Minerals The ash and mineral contents depend strongly not only on botanical and geographical origin, but also on the species (Vit et al. 1994, 2004, 2005; Vit 2005; Souza et al. 388 C.A. Fuenmayor et al. 2006). The concentration of ash and some minerals (Na, K, Ca, Mg, Fe, Cu, and Zn) for Colombian pot-honey from four genera is shown in Table 27.3. For Colombian honey known thus far, obvious differences exist between species or genera. According to mean ash content value, most analyzed honey meets the standard for Codex Alimentarius proposed by Vit et al. (2004), which is a maximum of 0.5 g/100 g (for honey from A. mellifera, Melipona, Scaptotrigona and Tetragonisca (formerly labeled a subgenus of Trigona), with the exception of honey from Tetragona) which had a mean content of 0.495 g/100 g and a standard deviation of ±0.077 g/100 g. This difference implies that some samples would not meet the suggested standard, in spite of authenticity, unless only one decimal place was used. In this case, the value could be approximated as 0.5 g/100 g. Some 40% of the Tetragona samples that were characterized exceeded 0.5 g of ash/100 g. Therefore, this suggestion needs to be clarified, at least for pot-honey from this species. For all types of honey, the most concentrated mineral element yet quantified is potassium (188.3–1,669.4 ppm), and the least concentrated element is copper (0.8–1.2 ppm). Other minerals, in increasing order of concentration, are iron (3.3–49.6 ppm), zinc (6.7–19.6 ppm), magnesium (4.7–85.5 ppm), sodium (63.6–178.3 ppm), and calcium (51.0–267.8 ppm). This order of concentration is the same found for Colombian A. mellifera honey (Zuluaga 2010). In general, the honey that exhibits higher ash concentration has higher concentration of each mineral element, as may be expected. High variability indicates that this parameter can be used as a differentiation criterion, since it has been widely used for A. mellifera honey, and other apicultural products. 27.2.3 Other Physicochemical Quality Parameters Physicochemical analyses are important for establishing the identity of each variety of pot-honey, according to bee species and geographical origin, and to provide regulatory organizations with objective tools for preventing honey falsification in commerce. The quality parameters of honey produced by A. mellifera are not directly related to nutritive value (i.e., water, sugar and mineral content), but to authenticity, denomination of origin, and safety (pH, acidity, content of hydroxymethylfurfural, diastase activity, specific rotation, conductivity and color). They have been widely assessed for several types of this product, throughout the world. Such characterization, together with the need to avoid adulteration and falsification, have led food regulation agencies to set standards, which are generally very accurate for A. mellifera honey but regularly exclude the honey of other species from the legal definition of honey. This situation occurs in several countries, including Colombia. To set accurate quality standards for Colombian stingless bee honey, an extensive knowledge base regarding the behavior of these variables for each species must be gathered in the same manner currently used by other countries such as Venezuela, 27 Honey of Colombian Stingless Bees... Table 27.3 Ash and mineral contents of Colombian stingless pot-honey Asha Sodiumb Potassiumb Calciumb Magnesiumb Ironb Copperb Zincb Taxon (g/100 g) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) Melipona 0.09 (1) 63.6 (1) 299.8 (1) 55.0 (1) 20.0 (1) 4.8 (1) 1.2 (1) 10.8 (1) compressipes Melipona favosa 0.01 ± 0.01 (2) – – – – – – – Melipona sp. 0.20 ± 0.00 (2) 67.7 ± 33.6 (2) 545.7 ± 138.2 (2) 150.3 ± 0.9 (2) 32.5 ± 1.8 (2) 3.3 ± 0.3 (2) 0.8 ± 0.4 (2) 6.7 ± 2.5 (1) Nannotrigona sp. 0.33 (1) 154.5 (1) 961.2 (1) 117.0 (1) 4.7 (1) 49.6 (1) 1.9 (1) 14.9 (1) Scaptotrigona 0.04 (1) – – – – – – – limae Scaptotrigona sp. 0.06 (1) – 188.3 (1) 51.5 (1) 37.4 (1) 15.1 (1) 0.6 (1) 19 (1) 0.50 ± 0.08 (5) 178.3 ± 29.5 (5) 1669.4 ± 388.8 (5) 267.8 ± 113.3 (5) 85.5 ± 7.1 (5) 6.2 ± 0.8 (5) 1.2 ± 0.7 (5) 18.1 ± 3.1 (5) Tetragona sp.c Tetragonisca 0.21 ± 0.70 (12) 155.0 ± 65.1 (9) 576.6 ± 177.6 (9) 199.6 ± 63.4 (9) 56.0 ± 27.5 (9) 5.8 ± 2.3 (9) 0.9 ± 0.3 (9) 19.6 ± 8.3 (9) angustulac Mean values, ± standard deviation and (number of honey samples) are presented a Ash content was determined according to the AOAC 920.181 standard methodology (AOAC 1998) b Mineral elements (Na, K, Ca, Mg, Fe, Cu, and Zn) were quantified by atomic absorption spectrometry according to the AOAC 979.23 standard methodology (AOAC 1998) c Previously denominated as a subgenus of Trigona (Rasmussen and Cameron 2010) 389 390 C.A. Fuenmayor et al. Mexico, Guatemala, and Brazil (Vit et al. 2004; Souza et al. 2006). In Colombia, little knowledge on these quality parameters is published (Torres et al. 2004; Quicazán et al. 2009). However, such studies signal differences between honey from a stingless bee species in different countries (see Chap. 21). In addition, although our results agree with other reports in most cases, some values fell outside the suggested ranges. Table 27.4 presents the existing information regarding color, pH, acidity, diastase activity, HMF, conductivity and specific rotation of honeys of Colombian stingless bees. Color was assessed using the Pfund scale, which is the most common color scale for A. mellifera honey, using a colorimeter (HI C221 Hanna Instruments). For Melipona honey, color is highly variable and may correspond to the particular species. Among the Melipona, some lacking current taxonomic certainty have the darkest honey, which can be considered light amber to amber according to the USDA color standard designation, whereas most honey of other Melipona ranges from very white to very light amber. Nannotrigona honey is considered to be light amber, and Paratrigona and Scaptotrigona honeys vary from white to light amber (high variability is found for these genera). For the former genus Trigona (here considered among the three genera Tetragona, Tetragonisca and Frieseomelitta) the lighter honeys appear to be those of T. angustula, even though they range from very white to light amber, and the darker honey is that of Frieseomelitta. The free acidity in honey of Meliponini is usually significantly higher than that of A. mellifera, reflected in pH, and in the flavor (Vit et al. 1994, 2004, 2005, 2006; Souza et al. 2004, 2006; Sosa López et al. 2004; Zuluaga 2010). This is likely associated with a higher tendency to spontaneously ferment due to a higher water content; fermentation is not necessarily an undesirable process, even though is typically not controlled (Vit et al. 1994, 2004). All of the analyzed Colombian honey meets the standards proposed by Vit et al. (2004) for pot-honey varieties from Venezuela, Guatemala, and Mexico. An unusual value of acidity was found for M. compressipes. Such low acidity has only been reported in honey from Melipona beecheii and Melipona scutellaris (Souza et al. 2006); therefore, because of the low number of samples, further assessment needs to establish whether this is normal in Colombia or only among analyzed samples. Currently, the diastase activity of Colombian meliponine honey is known for only a few species. Melipona and Scaptotrigona pot-honey presented lower values than Frieseomelitta, Tetragona, and Tetragonisca for diastase activity, which is consistent with previously reported information (Vit et al. 1994, 2004). Unlike the activities of A. mellifera and Tetragonisca, these results indicate a lack of high enzyme activity, not due to possible heating of the product. It is important to note that the diastase activity for Melipona and Scaptotrigona honey was less than 3.0 DN, which is the lower detection limit of the Schade method (Bogdanov et al. 1997) used in this assessment; therefore, the diastase activity is not a standard to be considered for the quality of pot-honey. The hydroxymethylfurfural (HMF) contents for Colombian pot-honey were much lower than the maximum accepted content for A. mellifera (40 mg/kg) 27 Honey of Colombian Stingless Bees... Table 27.4 Physicochemical quality parameters of Colombian stingless bee honey Free acidityb Diastase HMFd Conductivitye Specific Taxon Colora (mm Pfund) pHb (meq/kg) activityc (DN) (mg/kg) (mS/cm) rotatione f 82 ± 7 (3) – – – – – – Frieseomelitta sp. Melipona 42 ± 19 (10) – 7.0 (1) n.d. (2) 3.0 (1) 1049 ± 56 (2) −12.6 ± 2.6 (2) compressipes Melipona favosa 36 ± 4 (3) – – n.d. (1) – – – Melipona eburnea 34.4 ± 8 (7) – – – – – – Melipona sp. 45.2 ± 27.8 (13) – – – – – – Nannotrigona sp. 65 ± 4 (2) – – – – – – Paratrigona opaca 36 ± 15 (4) 4.1 (1) 31.7 (1) – – – – Plebeia spp. 62 (1) – – – – – – Scaptotrigona sp. 54 ± 27 (4) 4.5 (1) 57.83 (1) 2.4 (1) 6.0 (1) 392 (1) – Tetragona sp.f 70 ± 15 (18) 4.2 ± 0.3 (4) 44.3 ± 21.8 (4) 17.8 ± 5.5 (2) 1.0 ± 1.1 (2) 1183 ± 122 (3) −1.1 (1) Tetragonisca 49 ± 19 (23) 4.2 ± 0.3 (12) 39.2 ± 22.9 (12) 16.7 ± 9.2 (8) 1.3 ± 2.1 (6) 658 ± 57 (2) 2.6 ± 1.3 (3) angustulaf Mean values, ± standard deviation and (number of honey samples) are presented a Estimated photometrically on the Pfund scale using a C-221 colorimeter (Hanna Instruments, USA) b pH was measured at 20 °C (10 g of honey/75 ml water); free acidity was assessed by neutralization according to the AOAC 962.19 standard methodology (AOAC 1998) c Diastase activity assessed by the method of Schade (Bogdanov et al. 1997); DN: diastase number d Hydroxymethilfurfural (HMF) evaluated spectrophotometrically according to the White method as described by Bogdanov et al. (1997) e Electrical conductivity and specific rotation evaluated according to methods described by Bogdanov et al. (1997) f Previously denominated as a subgenus of Trigona (Rasmussen and Cameron 2010) 391 392 C.A. Fuenmayor et al. (Table 27.4). It is interesting to note changes of this variable during long-term storage, considering that meliponine honey should be kept refrigerated, and the high moisture content could eventually enhance product appearance. Electrical conductivity has not been commonly assessed for stingless bee honey. In the case of T. angustula, conductivity (0.66 ± 0.06 mS/cm) was different from values reported by Vit et al. (1994) for Venezuelan honey (7.32 mS/cm), but similar to the value reported by Santiesteban-Hernández et al. (2003) for Mexican honey of this species (0.78 mS/cm), although there may be several species involved (see Chap. 21). The singular honey of Scaptotrigona. for which conductivity has been assessed had a particularly low value (0.39 mS/cm), which to the best of our knowledge is the lowest reported value for any stingless bee honey; a conductivity of 0.49 mS/cm for Scpatotrigona mexicana (reported erroneously as S. luteipennis) in Mexico was apparently the previous minimum reported value (Santiesteban-Hernández et al. 2003). The specific rotation is also a property that is not widely explored for stingless bee honey. The data presented in Table 27.4 indicate that specific rotation is a potential criterion for differentiating honeys because values for each species seem to stay within a consistent range. This property is related to the concentration of levorotary (as fructose) and dextrorotary (as glucose) compounds. However, the correlation is not known for pot-honey that has been evaluated and may be due to the presence of other sugars that have not been quantified, and other compounds with rotation capacity. 27.3 Conclusions Even though most of the Colombian pot-honey display physicochemical properties within the range of values previously reported for diverse stingless bee species, the values show that physicochemical data can potentially be used as criteria to differentiate the honey from adulterated products, A. mellifera honey, other stingless bees honey, and even honey of the same species from different regions. Nevertheless, it is necessary to continue the characterization process that leads to a better knowledge of this valuable product, and the establishment of laws that regulate falsification and adulteration. The result will enable or stimulate sustainable meliponiculture across Colombia. In the Zuluaga-Domínguez et al. chapter of the present book, we tackle a further classification and differentiation of stingless bee honey with multivariate statistical analysis of physicochemical properties and the novel analytical methodology known as an “electronic nose.” Acknowledgements Authors wish to thank specially the Colombian Ministry of Agriculture, Professor Guiomar Nates-Parra and her research group for their contribution regarding the taxonomical classification, ASOAPIS, ASOAPIBOY, ASOAPICOM and APISIERRA for their guidance in sample collection and Dr. Juliana Barrios for her important contribution during validation of the analytical methodologies. 27 Honey of Colombian Stingless Bees... 393 References AOAC. 1998. Official methods of analysis. AOAC International; Washington, USA. 1298 pp. Bijlsma L, de Brujin L, Martens E, Sommeijer M. 2006. Water content of stingless bee honeys (Apidae, Meliponini): interspecific variation and comparison with honey of Apis mellifera. Apidologie 37:480–486. Bogdanov S, Martin P, Lüllmann C. 1997. Harmonized methods of the European Honey Commission. Apidologie (extra issue):1–59. Carvalho C, Souza B, Sodré GS, Marchini LC, Alves R. 2005. Mel de abelhas sem ferrão: contribução para caracterização físico-química. Série Meliponicultura Nº4. Universidade Federal da Bahia. Cruz das Almas; Brazil. 32 pp. Euán JJG. 2009. Diversity, threats and conservation of native bees in the Neotropics. Apidologie 40:332–346. Freitas BM, Imperatriz-Fonseca V, Medina LM, Kleinert AMP, Galetto L, Nates-Parra G, QuezadaEuán, JJG. 2009. Diversity, threats and conservation of native bees in the Neotropics. Apidologie 40:332–346. Gonnet M, Lavie P, Nogueira-Neto P. 1964. Étude de quelques characteristiques des miels récoltés para certains Méliponines brésiliens. Reports of the Paris Academy of Sciences 258:3107–3109. Instituto Colombiano de Normas Técnicas y Certificación (ICONTEC). 2007. NTC 1273: Norma Técnica Colombiana de Miel de Abejas. ICONTEC; Bogotá, Colombia. 21 pp. Nates-Parra G. 2001. Las abejas sin aguijón (Hymenoptera: Apidae: Meliponini) de Colombia. Biota Colombiana 2:233–249. Nates-Parra G, Rodríguez A, Vélez ED. 2006. Abejas sin aguijón (Hymnoptera: Apidae: Meliponini) en cementerios de la cordillera oriental de Colombia. Acta Biológica Colombiana 11:25–35. Pamplona BC (1989) Exame dos elementos inorgânicos encontrados em méis de Apis mellifera e suas relações físico-biológicas. Master Thesis (Entomology), Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil, 133 pp. Quicazán MC, Zuluaga CM, Fuenmayor CA. 2009. Perspectivas para la caracterización físicoquímica de productos apícolas de variedades de abejas nativas en Colombia. Acta Biológica Colombiana 14:185–186. Rasmussen C, Cameron SA. 2010. Global stingless bee phylogeny supports ancient divergence, vicariance, and long distance dispersal. Biological Journal of the Linnean Society 99:206–232. Rosso JM, Nates-Parra G. 2005. Meliponicultura: una actividad generadora de ingresos y servicios ambientales. LEISA Revista de Agroecología 21:14–17. Roubik DW (1983) Nest and colony characteristics of stingless bees from Panama (Hymenoptera: Apidae). Journal of the Kansas Entomological Society 56:327–355. Santiesteban-Hernández A, Cuadriello JA, Loper G. 2003. Comparación de parámetros físicoquímicos de mieles de abejas sin aguijón y Apis mellifera de la región del Sonocusco, Chiapas, México. pp. 60–61. In: III Seminario Mesoamericano sobre Abejas sin Aguijón. Tapachula, México. Sosa López AA, Subovsky MJ, Castillo AE, Aumer AO, Burdyn L, Mambrin VM, Rojas JM. 2004. Caracterización de mieles de meliponas. In: F. d. C. A. XV Reunión de Comunicaciones Científicas y Técnicas, Universidad Nacional del Nordeste. Available at: http://agr.unne.edu.ar/ Extension/Res2004/Quimica/qca-004.pdf. Souza BA, Carvalho CAL, Sodré GS, Marchini LC. 2004. Características físico-químicas de amostras de mel Melipona asilvai (Hymenoptera: Apidae). Ciência Rural 34:1623–1624. Souza B, Roubik D, Barth O, Heard T, Enríquez E, Carvalho C, Villas-Bôas J, Locateli J, PersanoOddo L, Almeida-Muradian L, Bogdanov S, Vit P. 2006. Composition of stingless bee honey: setting quality standards. Interciencia 31:867–875. Torres A, Garedew A, Schmolz E, Lamprecht I. 2004. Calorimetric investigation of the antimicrobial action and insight into the chemical properties of “angelita” honey—a product of the stingless bee Tetragonisca angustula from Colombia. Thermochimica Acta 415:107–114. 394 C.A. Fuenmayor et al. Torres A, Hoffmann W, Lamprecht I. 2007. Thermal investigations of a nest of the stingless bee Tetragonisca angustula Illiger in Colombia. Thermochimica Acta 458:118–124. Vit P. 2005. Denominaciones de Origen de la Miel de Abejas en Venezuela. APIBA-CDCHT Universidad de Los Andes. Mérida, Venezuela. 28 pp. Vit P, Almeida-Muradian LB, Hitomi Matsuda A, Enríquez E, Barth OM. 2005. Iniciando una base de datos para proponer estándares de calidad de mieles de abejas sin aguijón. pp. 1–7. In: IV Seminario y Taller Mesoamericano sobre Abejas sin Aguijón. San Ignacio, Chalatenango, El Salvador. Vit P, Bogdanov S, Kilchenmann V. 1994. Composition of Venezuelan honeys from stingless bees (Apidae: Meliponinae) and Apis mellifera L. Apidologie 25:278–288. Vit P, Enríquez E, Barth OM, Matsuda AH, Almeida-Muradian LB. 2006. Necesidad del Control de Calidad de la Miel de Abejas sin Aguijón. MedULA 15:789–796. Vit P, Medina M, Enríquez E. 2004. Quality standards for medicinal uses of Meliponinae honey in Guatemala, Mexico and Venezuela. Bee World 85:2–6. Zuluaga CM. 2010. Análisis quimiométrico para diferenciar la huella digital de los productos de las abejas en Colombia. Master Thesis (Food Science and Technology). Instituto de Ciencia y Tecnología de Alimentos, Universidad Nacional de Colombia, Bogotá, Colombia, 246 pp. Zuluaga CM, Quicazán MC, Díaz-Moreno C, Fuenmayor CA, Cadena A. 2009. Classification and differentiation of Colombian stingless bee honey by using an electronic nose and multivariate analysis. In: 41st World Congress of Apiculture APIMONDIA. Montpellier, France. Chapter 28 The Pot-Honey of Guatemalan Bees María José Dardón, Carlos Maldonado-Aguilera, and Eunice Enríquez 28.1 Introduction In Guatemala there are at least 32 species of native stingless bees that produce honey. Guatemalan beekeepers have developed, since Pre-Columbian times, skills for bee breeding and nowadays refer to about 15 species by their common name. However, the species with superior realized breeding potential and honey production are Melipona beecheii Bennett, 1831, Tetragonisca angustula (Latreille, 1811), Scaptotrigona pectoralis (Dalla Torre, 1896), and Scaptotrigona mexicana (GuérinMéneville, 1844). Geotrigona acapulconis (Strand, 1919) is also greatly appreciated for its honey, which is believed to have medicinal properties. However, the bee nests underground and is not kept in hives easily thus no traditional breeding apparently exists (Yurrita et al. 2004; Enríquez et al. 2001, 2004, 2005). In some regions stingless bee breeding and artificial feeding, in the rainy season, are practiced. This is an economic alternative currently promoted by nongovernmental organizations, to benefit families in the rural area. However, there are still regions of Guatemala where stingless bee colonies are kept in traditional log hives, and beekeeping practical knowledge is transmitted orally, from generation to generation (Yurrita et al. 2004; Enríquez et al. 2001, 2004, 2005). Honey is the hive’s most coveted product; there are few reports on the use of wax (i.e., cerumen—a mixture of wax with resin), pollen and no reports on the use of propolis (i.e., pure resin). Most of the beekeepers use the honey only for their own consumption, either as medicine and food, because of the scarcity of the product. Only those who have many hives sell the honey, but M.J. Dardón (*) • C. Maldonado-Aguilera • E. Enríquez Unidad para el Conocimiento, Uso y Valoración de la Biodiversidad, Centro de Estudios Conservacionistas, Universidad de San Carlos de Guatemala, Guatemala City, Guatemala e-mail: dardon.mariajose@usac.edu.gt 395 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_28, © Springer Science+Business Media New York 2013 396 M.J. Dardón et al. always locally. The honey of stingless bees is priced three times higher than that of Apis mellifera L., as in other countries of the region (Yurrita et al. 2004; Enríquez et al. 2001, 2004, 2005). Popularly, the honey of stingless bees is claimed to have a great number of medicinal properties that together with cultural, historic, and biologic components, gives an added value to it (Enríquez et al. 2001, 2004, 2005 and chapters in this book). However, the exact composition of the honey is unknown, which represents a challenge that has to be overcome to encourage the conservation of these species and their honey. There are many characteristics to study in honey, for example physicochemical, pollen composition, nutrition and taste or sensorial evaluation. Also the sanitary quality of the product and popular beliefs regarding properties and uses require validation, before marketing can be pursued. Part of this work has already begun, and the results are discussed below. 28.2 Physicochemical Characteristics of Guatemalan Pot-Honeys Honey presents a great variety of physicochemical characteristics that have been used to determine its quality. In A. mellifera some useful parameters are acidity, ash, sucrose, reducing sugars, moisture content, diastase and hydroxymethylfurfural. These parameters may also be used to establish quality control and to avoid adulteration of stingless bee honey. However, the composition of honey should be known, throughout the regions from which it comes, to define normal values for such parameters and lead to its commercialization. Composition has been studied, preliminarily, in 18 samples of honey from Melipona beecheii, M. solani Cockerell, 1912, M. yucatanica Camargo, Moure and Roubik 1988, Tetragonisca angustula, Plebeia sp., Nannotrigona perilampoides Cresson 1878, Scaptotrigona mexicana and Geotrigona acapulconis (Dardón and Enríquez 2008). 28.2.1 Reducing Sugars The principal reducing sugars found in honey, generally in almost equal proportions, are glucose and fructose (Alves et al. 2005). The reducing sugars in the honey of Guatemalan stingless bees (Table 28.1) are of higher content than the minimum proposed by Vit et al. (2004) (50 g/100 g) and Souza et al. (2006) (58.0–75.7 g/100 g), as honeys show values between 57.22 and 75.97 g/100 g. Average values of reducing sugars are not very different among honeys of different stingless bee genera. The honey of the genera Melipona and Trigona present a higher quantity of reducing sugars, while honey of Scaptotrigona has about 20% less reducing sugars, compared to honey of Melipona, so their honey is usually less sweet. 28 The Pot-Honey of Guatemalan Bees Table 28.1 Sugars content of stingless bees honey from Guatemala Honey Reducing sugars Apparent sucrose Bee species samples n (g/100 g) (g/100 g) Melipona beecheii 7 68.77 ± 3.82 3.50 ± 4.14 Melipona solani 1 75.97 1.7 Scaptotrigona mexicana 1 57.22 0.06 Tetragonisca angustula 1 65.78 4.83 After Dardón and Enríquez (2008) 28.2.2 397 Total sugars (g/100 g) 72.45 ± 6.10 76.19 57.28 70.86 Sucrose Sucrose represents about 2–3% of the carbohydrates in honey of A. mellifera (Swallow and Low 1990); high values of this disaccharide are related with premature honey harvest, where sucrose has not been converted into glucose and fructose by the action of invertase (Alves et al. 2005). The sucrose in honey of Guatemalan stingless bees (Table 28.1) is in the allowed parameters for the Codex alimentarius (maximum of 5 g/100 g) and the values coincide with those reported by Souza et al. (2006) for stingless bees (1.1–4.8 g/100 g). Values for sucrose in the honey of Scaptotrigona are lower than those of Melipona and Geotrigona, as suggested by Vit et al. (2004) and Dardón and Enríquez (2008). 28.2.3 pH The pH values in honey refer to the hydrogen ions present in solution that participate in formation of other components (e.g., hydroxymethylfurfural) (Carvalho et al. 2005). According to Alves et al. (2005), pH is determined by nectar, the cephalic secretions of the bees while they carry the nectar to the hive, by the origin of the honey and the concentration of different ions like calcium, potassium, and sodium. Most (Table 28.2) are found in the ranges reported by Souza et al. (2006), with values between 3.71 and 5.18, with the highest pH in the honey of Geotrigona (Dardón and Enríquez 2008). 28.2.4 Free Acidity Honey contains acids that contribute to its stability and retard development of microorganisms; gluconic acid is the most common (Mato et al. 1997). This acid is formed by the action of glucose-oxidase on glucose, this enzyme is produced in the hypopharyngeal glands of bees, acting even after the honey is stored (Alves et al. 2005). Acids found in smaller quantities include acetic, benzoic, butyric, citric, phenylacetic, formic, isovaleric, lactic, maleic, oxalic, propionic, pyroglutamic, succinic, and valeric acids (Carvalho et al. 2005). The values were less than 20 meq/100 g 398 M.J. Dardón et al. Table 28.2 Physicochemical parameters of stingless bees honey from Guatemala Physicochemical parameters Moisture Bee Honey Ash content Diastase HMF Free acidity content species samples n pH activity (DN) (mg/kg) (meq/100 kg) (g/100 g ) (g/100 g) Mb 7 3.67 ± 0.12 23.2 ± 30.0 17.3 ± 2.6 0.07 ± 0.05 21.3 ± 32.8 n.d. Ta 4 5.18 ± 1.35 17.4 ± 10.4 17.5 ± 2.8 0.35 ± 0.26 12.3 ± 10.3 n.d. Sm 2 4.04 ± 0.4 12.7 ± 3.0 18.7 ± 0.2 0.10 ± 0.04 18.6 ± 12.7 n.d. Ms 1 3.81 4.95 19.66 0.06 8.3 n.d. Ga 1 3.06 85.53 32.09 0.09 2.6 n.d. Pl 1 3.8 15.31 30.26 1.25 7.6 n.d. My 1 3.79 10.59 20.37 0.06 10.0 n.d. Np 1 3.8 9.93 16.54 0.33 6.8 n.d. Mb = Melipona beecheii, Ms = Melipona solani, My = Melipona aff. yucatanica, Ta = Tetragonisca angustula, Pl = Plebeia sp., Np = Nannotrigona perilampoides, Sm = Scaptotrigona mexicana, Ga = Geotrigona acapulconis After Dardón and Enríquez (2008) in our study, although in G. acapulconis the value is four times higher and tends to reach values above 80 meq/100 g (Table 28.2) (Dardón and Enríquez 2008). Vit et al. (2004) proposed maximum values between 70 and 85 meq/100 g for the genera Melipona, Scaptotrigona and Trigona. The free acidity range was (5.9–109.0), with averages between 36.6 and 49.7 in the most studied species (Souza et al. 2006). 28.2.5 Moisture Content The moisture content, besides water, has a relation with the viscosity, specific weight, maturity, crystallization and taste of honey. The honey of Guatemalan stingless bees (Table 28.2) is, on average, below 20 g/100 g, which is an acceptable value for commercial A. mellifera honey. There is also an exception for Geotrigona acapulconis and Plebeia sp., which acquire moisture values above 30 g/100 g and give honey the lowest viscosity. Souza et al. (2006) point out that, in honey of these species, the most common range is 19.9–41.9 g/100 g. However, Vit et al. (2004) proposed a maximum of 30 g/100 g for Melipona, Scaptotrigona and Trigona. According to observations on honey of Plebeia and Geotrigona with higher moisture values, an extension of the parameter should be considered. 28.2.6 Ash Content The amount of ash found in honey is a quality criterion influenced by botanical origin. This parameter is correlated with the color of the honey; darker honeys have more ash and, consequently, more minerals (González-Miret et al. 2005). Our honey 28 The Pot-Honey of Guatemalan Bees 399 (Table 28.2) contains, an average of 0.23 g/100 g of ash content. However, the high quantity of ash in the honey of Plebeia sp. stands out, acquiring values above 1.25 g/100 g. Vit et al. (2004) propose a maximum of 0.5 g/100 g for ash of stingless bee honey, while Souza et al. (2006) list the common values of stingless bee honey at 0.01–1.18 g/100 g. 28.2.7 Diastase (a-Amylase) Enzymes present in honey are formed by bee hypopharyngeal glands in the head and are found in small proportions in collected pollen (Moritz and Crailsheim 1987). Diastase is a heat-sensitive enzyme, so it is recommended for testing honey quality. The diastase activity is calculated as diastase number (DN = units of diastase activity). One unit is defined as the amount of enzyme that will convert 0.01 g of starch to the prescribed end point in that 40°C under the condition of the test (Vorlová and P idal, 2002). The stingless bee honey in Guatemala is highly variable in diastase number. This is reflected in the values of standard deviations presented in Table 28.2, particularly in M. beecheii honey. Vit et al. (1998) reported diastase values 2.9–23.0 DN for Melipona favosa honey, somewhat similar to values found in some Guatemalan stingless bees, 2.6–21.3 DN (Table 28.2), in agreement with the minimum of 3 DN for Melipona honey, initially proposed by Vit et al. (2004). 28.2.8 Hydroxymethylfurfural (HMF) HMF is a degradation compound formed by the reaction of certain sugars with acids, principally by the decomposition of fructose (Spano et al. 2006). Its presence is an indicator of honey quality because it is found in small quantities in recently collected honey, and also because the quantity increases with time and overheating. HMF was not detected in honey of Guatemalan stingless bees (Table 28.2). Vit et al. (2004) proposed a maximum of 40 mg/kg. For Souza et al. (2006) the averages for the stingless bee honey most studied varied between 2.4 and 16.0 mg/kg, although the highest HMF value known so far is 78.5 mg/kg from an abstract meeting (Grajales et al. 2001). 28.3 Nutritional Characteristics The honey of A. mellifera is recognized as a high-energy and nutritive food, and for being a sugar substitute of wide use in the food industry. The honey is principally composed by carbohydrates, which are about the 95–99% of the solids, and of those, 85–95% corresponds to reducing sugars that give honey its sweet taste and energy. 400 M.J. Dardón et al. Table 28.3 Nutritional characteristics of stingless bees honey from Guatemala Honey Carbohydrates Bee species samples n (g/100 g) Proteins (g/100 g) Calories kcal/100 g Scaptotrigona pectoralis 2 70.22 0.41 283 Melipona beecheii 3 75.08 0.07 300 Tetragonisca angustula 3 70.22 1.19 286 Scaptotrigona mexicana 1 71.73 0.47 289 After Rodas et al. (2008) The protein content of honey, in A. mellifera, presents a maximum of 0.1% and 7 proteins have been identified, five from the bees and two from plants. Of these proteins, enzymes are the most important for their role in the conservation of honey. Proline is the most abundant protein amino acid in honey (Carvalho et al. 2005). Honey also contains most of the essential chemical elements for the organism, such as K, Na, Ca, Mg, Mn, Ti, Co, Mo, Fe, Cu, Li, Ni, Pb, Sn, Zn, Os, Ba, Ga, Bi, Ag, Au, Ge, Sr, Be, and Ba (Freitas et al. 2006). Other compounds are found in smaller quantities, like organic acids, vitamins and aromatic substances, which play an important role in nutrition. Preliminary studies of the honey of four Guatemalan stingless bees (Table 28.3) demonstrate an energy value of 280–300 kcal/100 g, 70–75% carbohydrate, each lower values than honey of A. mellifera. The percentage of protein in the honey of stingless bees varies between 0.073 and 1.19, for M. beecheii and T. angustula with the lowest and highest protein contents, respectively. 28.4 Antibacterial Properties of Guatemalan Pot-Honey Honey has been used since ancient times in efforts to cure many diseases. It has been utilized by Chinese, Egyptian, Hebrew, Greek, Hindu, Persian, Roman, and Mayan cultures (see the Ocampo Rosales chapter in this book). The scientific mechanism known for the antibacterial activity in honey is hydrogen peroxide (H2O2), slowly released by the action of glucosidase and ingredients including antioxidant activity, vitamins, osmotic pressure, and polyphenol content, etc., which are of botanical origin (Aguilera et al. 2006). The study of antibacterial activity of honey validates its therapeutic use and has shown activity against some pathogenic bacteria. There should be valid reasons for medicinal use of this hive product, and its derivatives, in the treatment of infectious disease (Aguilera et al. 2006). After evaluating the antibacterial activity (Table 28.4) it was found that honey of eight among nine species shows antibacterial activity, against eight pathogen microorganisms, at concentrations of 2.5–10%. The honey of M. solani, however, had no such activity. The least susceptible microorganisms to the honey were Candida albicans and Salmonella tiphy. However, in dilutions of 2.5%, the honey of S. pectoralis was effective (Table 28.4). The stingless bee honey inhibited growth of Staphylococcus aureus, in 28 401 The Pot-Honey of Guatemalan Bees Table 28.4 Antimicrobial activity of stingless bees honey from Guatemala Stingless bee speciesa Mb Ms My Ta Pl Np Sample size 12 3 1 5 1 6 Bacterias and yeasts Dilutions with microbial growth Staphylococcus aureus 5 – 5 Salmonella typhi 5 – 10 Mycobacterium smegmatis 5 – 5 Bacillus subtilis 5 – 5 Pseudomonas aeuroginosa 5 – 5 Escherichia coli 5 – 5 Candida albicansb 10 – 5 Criptococcus neoformansb 5 – 5 a Stingless bee species are indicated in the Table 28.2 b Yeast After Dardón and Enríquez (2008) 10 10 5 5 10 5 10 5 5 5 5 5 5 5 10 5 5 5 2.5 2.5 5 5 5 2.5 Sm 1 Sp 1 Ga 1 5 5 5 5 5 5 5 5 2.5 2.5 2.5 2.5 2.5 5 5 2.5 10 10 5 5 5 5 – 5 dilutions ranging from 2.5 to 10%, with exception of S. pectoralis, which inhibited at 2.5%, and M. solani, which had no activity. Mycobaterium smegmatis was inhibited by honey of eight species, at an average dilution of 5% (Table 28.4). 28.5 Sensory Characteristics of Guatemalan Pot-Honey Sensory characteristics are those perceived through the sense organs (eyes, nose, tongue, skin, or ears) to evaluate the color, size, shape, smell, aroma, taste, texture, malleability, and sound of consumables. Honey has a wide range of qualities that are very useful for detecting or describing its attributes (Vit 2007; Vit et al. 2008). The honey of five Guatemalan stingless bees was analyzed in color, smell, taste, and viscosity. Color allowed recognition of four descriptors ranging from transparent white (honey of M. solani) to orange (honey of T. angustula) (Table 28.5). Generally, the honey of Melipona is characterized for color ranging from pale yellow to white, or “white honey”. In addition, refrigerated honey, stored for 10 years, changes color, giving rise to many colors of the same origin but different age. For the taste of honey, of Guatemalan stingless bees, 10 descriptors were identified: strong acetic acid, sugar, sugarcane, sweet, slightly sweet, floral, formaldehyde, fruity, slightly acetic acid, and “nance” (the sour, edible fruit from a tree, Byrsonima crassifolia, Malpighiaceae). For the smell, 11 descriptors were recognized: accentuated acetic acid, sugar, “panela” (jaggery), fermented, floral, slightly formaldehyde, slightly fat, slightly acetic acid, slightly alcoholic, slightly fruity, and hive (Table 28.5). Both the smell and taste varied between the samples analyzed, influenced possibly by their location of origin. According to these results we can say that the pot-honey of Guatemalan stingless bees present sweet smell and taste, but the smell is also slightly acetic acid because of the relatively high water content, which triggers the fermentation processes. 402 Table 28.5 Sensory characteristics of stingless bees honey from Guatemala Honey Bee species samples n Color Melipona beecheii 5 Pale yellow Scaptotrigona mexicana Melipona solani 3 3 Trigona angustula 2 Pale yellow Transparent white and pale yellow Yellow and orange Geotrigona acapulconis After Rodas et al. (2008) 1 Yellow Odor/aroma Slightly fat, floral, hive, slightly acetic acid, slightly frutal Slightly ethanolic, floral Slightly acetic acid, slightly formaldehyde Fermented, jaggery, strong acetic acid Strong acetic acid Taste Slightly sweet Viscosity 78.8 Slightly sweet Sweet 72 76 Sweet, slightly acid Sweet, strong acid 81 64 M.J. Dardón et al. 28 403 The Pot-Honey of Guatemalan Bees 28.6 Pollen Composition of Guatemalan Pot-Honey Melissopalynology considers pollen types found in honey and information on botanical origin, sometimes used for honey classification as unifloral or multifloral (Louveaux et al. 1970). A unifloral honey is the one that presents at least 45% of a single species, while a multifloral honey presents a high number of pollen resources or, at least, three different species in similar proportion. Honey characteristics are strongly influenced by botanical origin due to bee-plant interaction (i.e., bee foraging preferences), and it is useful to apply palynology for understanding bee flora. Our 53 honey samples of 9 different species revealed 20 botanical families (Table 28.6). The families Asteraceae, Fagaceae, Melastomataceae, and Tiliaceae were found in the honey of at least five different species and were the most commonly visited families. The honey of T. angustula presented a higher richness of families (18), while the honey of S. mexicana and G. acapulconis were the poorest (3). Melipona honey in Guatemala did not exceed eight plant families in pollen content. Table 28.6 Floral resources of stingless bee honey from Guatemala Bee speciesa Mb Ms M Ta P Np Sample size 13 6 1 21 1 4 Botanical Family Sm 4 Sp 2 Ga 1 Pollen types Acanthaceae Amaranthaceae Asclepiadaceae Asteraceae X X Begoniaceae X Bignoniaceae Cochlospermaceaeb Convolvulaceae Fabaceae X X Fagaceaeb X X Lamiaceae Malvaceae Melastomataceae X X Myrsinaceae Myrtaceae X X Onagraceae Piperaceaeb X Rutaceae Solanaceaeb X X Malvaceae (Tilioideae) X X Total 8 8 a Bee species are indicated in Table 28.2 b Pollen is not indicator of nectar origin X X X X X X X X X X X X X X X X X X X X X X X X X 2 X X X X X X 18 X X X X X 5 X X 5 4 X 3 X 3 404 28.7 M.J. Dardón et al. Sanitary Quality of the Honey of Guatemalan Stingless Bees The sanitary quality control of a product insures a safe product by detecting the presence of components that may negatively affect human health. Honey of stingless bees has been studied to detect presence or absence of insecticides. During flight and foraging, as well as in search of water, nectar, and/or honey, a bee may have contact with agricultural pesticides and other artificial chemical sources. This is why they are considered excellent bioindicators of the distribution of pesticides (Kevan 1999). One type of the most common pesticides is the organophosphates, which have been detected in low levels in the honeys of A. mellifera. The presence of pesticides represents a major risk to public health and maximum values allowed in honey have not been established, although some acaricide residues are regulated (Blasco et al. 2004). In Guatemala, organochlorides, organophosphates, pyrethroids, bipiridils, glyphosate, and atrazines are used around apiaries and meliponaries (Rodas et al. 2008). Therefore, there may be pesticide contamination of honey from agricultural areas. Four Guatemalan stingless bees studied by gas chromatography/mass spectrophotometry revealed no contaminants (Rodas et al. 2008). Detectable levels of pesticides were not found in six samples of honey from M. beecheii, 3 T. angustula, 2 G. acapulconis, and 1 of S. pectoralis. There is no detectable risk, at present, of pesticide in the honey, despite the fact that these compounds are used in the immediate environment. 28.8 28.8.1 Honey Attributes of the Four Most Appreciated Stingless Bee Species in Guatemala Melipona beecheii This species is popularly known in Guatemala as the creole bee “abeja criolla,” large beehive “colmena grande,” “bichi,” and, in Mayan language, “sak’q qaw.” This species has been used extensively since PreColumbian times. Its pot-honey, denominated “white honey,” is very prized in Guatemala and is used against various maladies, such as stomach, respiratory and ocular disease or sickness, bumps, sores, and skin wounds. Due to its physicochemical components, the honey of M. beecheii presents a high degree of acidity, 23.2 meq/kg honey, in comparison with the other species studied (excluding G. acapulconis). The ash content is relatively low, possibly the reason for the pale yellowish color, also reflected in low protein content (in comparison with T. angustula). The floral-fruity, fermented and woody odors and aromas make this honey very pleasant to the consumer. The price of M. beecheii honey 28 The Pot-Honey of Guatemalan Bees 405 ranges from Q75.00 to Q300.00 (US$ 10–40), per L, which is a price two to eight times higher than the local honey of A. mellifera. When evaluated against various microbial pathogens, M. beecheii honey inhibited their growth at dilutions of 5–10% and was least effective against the yeast C. albicans (Table 28.6). 28.8.2 Geotrigona acapulconis This species is commonly called “talnete”. It produces a considerable amount of honey that is popularly used to treat broken bones, internal injuries, eye diseases, cleaning the kidneys, and as a purgative. Due to the biology of this bee and its strict nesting habits, captive breeding is not practiced. Honey is obtained by digging up underground nests. The free acidity, 85.5 meq/kg honey of one sample, was at least four times higher than other Guatemalan stingless bees. Accordingly, the smell of the honey has relatively high acetic acid content and its flavor is described as sweet and strong acetic acid. The moisture content is high, making it a very liquid honey, and ash values are similar than those found in the genus Melipona. It has low diversity in pollen content, with only three plant families recorded. These families are often visited by stingless bees kept in our country. The honey, of yellow color when extracted, is not very well known and its sale is by a prior agreement. It is strictly a product of “honey hunting,” not rational beekeeping. It is also a highly prized honey, and it is conducive to fraud and adulteration. It has been observed that some people offer a honey prepared with panela and lemon, as “talnete” honey. 28.8.3 Scaptotrigona mexicana The breeding of this bee, commonly named “magua negro” or “congo negro,” has advanced because it produces a considerable amount of honey. The honey has a pale yellow color and its smell is alcoholic and slightly floral. All the sensory families described by Vit (2007) for the aroma and smell of stingless bee honeys were found here floral-fruity, fermented, woody, mellow, primitive, industrial chemicals, hive, and vegetable. The honey of S. mexicana, as in S. pectoralis, presents a higher percentage of protein, more than honey of M. beecheii, although it shows lower values for carbohydrates and this is reflected in its kilocalorie content. The study of four honey samples of this species allowed identification of four plant families in its pollen composition. With respect to the biotic activity of honey, S. mexicana was effective against all the evaluated microorganisms, in a dilution of 5%, being therefore one of the most active pot-honey (Table 28.6). Curiously, beekeepers report little medicinal use, even though the honey shows a potential for therapy. 406 28.8.4 M.J. Dardón et al. Tetragonisca angustula T. angustula is a very small and normally docile bee, commonly known as “chumelo,” “doncella,” “doncellita,” in Mayan language it is known as “an us” and “qán us.” It can form big colonies, but due to the small size of the honey pots, the quantities of honey produced are considerably less than those obtained in species like M. beecheii, with larger honey pots. This honey is very popular for the treatment of eye diseases (cataract and pterygium) but is also used for stomach illness, wounds and ulcers, and sometimes as an energy food or drink. The honey of T. angustula has yellow to orange color, with the aroma and smell families: floral-fruity, fermented, woody, mellow, primitive, industrial chemicals, hive, and vegetable. Its honey contains 19 families identified in Guatemala, reflected in color variation and high values of ash and protein. Its physicochemical composition stands out from the other stingless bees, having the highest pH (>5) and the highest sucrose content (4.8 g/100 g). Antibacterial activity occurs at 5–10% honey dilution and was least effective of all evaluated honey. The microorganisms Staphylococcus aureus, Salmonella typhi, Pseudomonas aeruginosa, and Candida albicans were the most resistant (Table 28.6). Popularly, this honey is considered useful for the treatment of eye diseases, so it has to be evaluated to confirm this putative medicinal property. 28.9 Conclusions The honey of stingless bees is a patrimony for tropical regions, especially for Latin America, where most of these bees exist. The honey of each varies among species and also within the same species, depending on the region where they are found and the plant resources they utilize. Determining the composition of this greatly varied honey, and knowing its attributes, is a difficult task. However, the challenge has been taken by research from Argentina, Bolivia, Brazil, Colombia, Costa Rica, Perú, Venezuela, and us, in Guatemala. We have 33 species of stingless bees, 32 produce honey and of these, only 9 species have been studied: all of them in manners considering antibacterial activity and pollen composition, 8 in physicochemical properties, 5 in sensory attributes and 4 for its sanitary quality. There are still 23 species that have not been studied, this corresponding to 60% of the entomological diversity of honey in the country. Efforts for understanding more about the pot-honey of stingless bees have begun, and for the moment, boosted stingless bee keeping. However, it is necessary to continue, to get to know all the diversity of honey, and promote its commercialization, and to validate potential therapeutic use. Acknowledgments The authors express their gratitude for the editorial assistance received from P Vit and DW Roubik and for referee comments. 28 The Pot-Honey of Guatemalan Bees 407 References Aguilera G, Gil F, González AC, Nieves B, Rojas Y, Vit P. 2006. In Vit P, ed. Iniciación a la Apiterapia. Universidad de los Andes. APIBA-CDCHT; Mérida, Venezuela. 32 pp. Alves R, Carvalho C, Souza B, Sodre G, Marchini L. 2005. Características Físico-Químicas de amostras de mel de Melipona mandacaia Smith (Hymenoptera: Apidae). Ciéncia e Tecnologia de Alimentos, Campinas 25:644–650. Blasco C, Lino C, Picó Y, Pena A, Font G, Silveira M. 2004. Determination of organochlorine pesticide residues in honey from the central zone of Portugal and the Valencian community of Spain. Journal of Chromatography 1049:155–160. Carvalho CA, Souza B, Sodré G, Marchini L, Alves R. 2005. Mel de abelhas sem ferrao: contribuicao para a caracterizacao fisico-química. Serie meliponicultura No. 04. Insecta-Nucleo de Estudo dos Insectos, Centro de Ciencias Agrarias, Ambientales e Biológicas. Cruz das Almas, Bahia, Brasil. 32 pp. Dardón MJ, Enríquez E. 2008. Caracterización fisicoquímica y antimicrobiana de la miel de nueve especies de abejas sin aguijón (Meliponini) de Guatemala. Interciencia 33:916–922. Enríquez E, Monroy C, Solis A. 2001. Situación actual de la meliponicultura en Pueblo Nuevo Viñas, Santa Rosa, Guatemala. pp. 36–39. II Seminario mexicano sobre abejas sin aguijón. Mérida, Yucatán, México. 120 pp. Enríquez E, Yurrita C, Aldana C, Ocheita J, Jáuregui R, Chau P. 2004. Desarrollo de la crianza de abejas nativas sin aguijón (meliponicultura). Revista Agricultura 68:27–30. Enríquez E, Yurrita C, Aldana C, Ocheita J, Jáuregui R, Chau, P. 2005. Conocimiento tradicional acerca de la biología y manejo de abejas nativas sin aguijón en Chiquimula. Revista Agricultura 69:27–30. Freitas M, Pacheco A, Ferreira E. 2006. Nutrients and other elements in honey from Azores and mainland Portugal. Journal of Radioanalytical and Nuclear Chemistry 270:123–130. González-Miret M, Terrab A, Hernanz D, Fernández-Recamales M, Heredia F. 2005. Multivariate correlation between color and mineral composition of honeys and by their botanical origin. Journal of Agricultural and Food Chemistry 53:2574–2580. Grajales-C J, Rincón-R M, Vandame R, Santiesteban-N A, Guzmán-D M. 2001. Características físicas, químicas y efecto microbiológico de mieles de Meliponinos y Apis mellifera de la región Soconusco, Chiapas. pp. 61–66. II Seminario Mexicano sobre Abejas sin Aguijón. Mérida, Mexico. Kevan P. 1999. Pollinators as bioindicators of the state of the environment: species, activity and diversity. Agriculture, Ecosystems & Environment 74:373–393. Louveaux J, Maurizio A, Vorwohl G. 1970. Methods of melissopalynology. Bee World 51:125–138. Mato I, Huidobro J, Sánchez P, Muniategui S, Fernández-Muiño M, Sancho T. 1997. Enzymatic determination of total d-gluconic acid in honey. Journal of Agricultural and Food Chemistry 45:3550–3553. Moritz B, Crailsheim K. 1987. Physiology of protein digestion in the midgut of the honeybee (Apis mellifera L.). Journal of Insect Physiology 33:923–931. Rodas A, Enríquez E, Maldonado C. 2008. Determinación de insecticidas y estudio nutricional de las mieles de las abejas nativas sin aguijón, Melipona beecheii y Tetragonisca angustula (Hymenoptera: Apidae: Meliponinae). Informe final. Dirección General de Investigación (DIGI). Universidad de San Carlos de Guatemala. 30 pp. Souza B, Roubik D, Barth O, Heard T, Enríquez E, Carvalho C, Villas-Bôas J, Marchini L, Locatelli J, Persano-Oddo L, Almeida-Muradian L, Bogdanov S, Vit P. 2006. Composition of stingless bee honey: Setting quality standards. Interciencia 31:867–875. Spano N, Casula L, Panzanelli A, Pilo M, Piu P, Scanu R, Tapparo A, Sanna G. 2006. An RP-HPLC determination of 5-hydroxymethylfurfural in honey. The case of strawberry tree honey. Talanta 68(4):1390–1395. 408 M.J. Dardón et al. Swallow K, Low N. 1990. Analysis and quantitation of the carbohydrates in honey using highperformance liquid chromatography. Journal of Agricultural and Food Chemistry 38: 1828–1832. Vit P, Carvalho CAL, Enríquez E, González I, Moreno E, Roubik DW, Souza BA, Villas-Bôas JK. 2008. Descripción sensorial de mieles de abejas sin aguijón de Argentina, Australia, Brasil, Guatemala y Venezuela. pp. 102–117. In Vit P, ed. Cría de abejas sin aguijón y valorización sensorial de sus mieles. APIBA-FFB-DIGECEX-ULA; Mérida, Venezuela. 146 pp. Vit P, Persano Oddo L, Marano M, Salas E. 1998. Venezuelan stingless bee honeys characterized by multivariate analysis of physicochemical properties. Apidologie 29:377–389. Vit P, Medina M, Enríquez E. 2004. Quality standards for medicinal uses of Meliponinae honey in Guatemala, Mexico and Venezuela. Bee World 85:2–5. Vit P. 2007. Práctica análisis sensorial. Ciencia de los Alimentos. Facultad de Farmacia y Bioanálisis. Universidad de los Andes; Mérida, Venezuela. 26 pp. Vorlová L, P idal A. 2002. Invertase and diastase activity in honeys of Czech provenience. Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis 50:57–65. Yurrita CL, Enríquez E, Monroy C, Marroquín A. 2004. Study of stingless bee diversity in Guatemala. pp. 402–408. Proceedings of the 8th IBRA International Conference on Tropical Bees and VI Encontro sobre Abelhas. 710 pp. Chapter 29 Pot-Honey of Six Meliponines from Amboró National Park, Bolivia Urbelinda Ferrufino and Patricia Vit Our forests wait to be discovered, shaped by David W. Roubik’s interpretations of foraging bees and seminal work of Francisco Tomás-Barberán and Federico Ferreres on flavonoids of tropical honey 29.1 Introduction A project on sustainable meliponiculture promoted by the Ecological Association of the East (ASEO, “Asociación Ecológica de Oriente”) initiated the Association of Native Honey Producers (APROMIN, “Asociación de Productores de Miel Nativa”) in Amboró National Park, S17°43¢–17°53¢ W60°30¢–0°04¢, 637,600 ha, located in the eastern lowlands of Bolivia, near San Carlos. Forty families became stingless bee-keepers to improve their economy with a new product from the forest. Each associate started with one hive and added up to 40. The web site “Amazonia Boliviana” advertises stingless bee honey on the web at prices ranging from 30 to 300 USD/l. The highest value in the Amboró community corresponds to “señorita” honey, produced by the widespread Tetragonisca fiebrigi, used to treat ocular diseases. Stingless bee honey yield is about 1–15 kg/year, and the fact that the honey is highly appreciated for potential medicinal use increases the price up to 10–25 times that of Apis mellifera honey. Packaging of pot-honey for commercial distribution U. Ferrufino Asociación Ecológica de Oriente, Santa Cruz, Bolivia P. Vit (*) Apitherapy and Bioactivity, Food Science Department, Faculty of Pharmacy and Bioanalysis, Universidad de Los Andes, Mérida 5101, Venezuela Cancer Research Group, Discipline of Biomedical Science, The University of Sydney, Cumberland Campus C42, 75 East Street, Lidcombe, NSW 1825, Australia e-mail: vitolivier@gmail.com 409 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_29, © Springer Science+Business Media New York 2013 410 U. Ferrufino and P. Vit includes a creative approach, based on a traditional spheroidal ceramic jar called “puño,” which simulates darkness inside the hive. In the Carmen Surutú community, Amboró National Park, six species of stingless bees have been selected by stingless bee-keepers (meliponicultors) to be kept in hives: Melipona brachychaeta, M. grandis, Scaptotrigona depilis, Scaptotrigona polysticta, S. near xanthotricha, and T. fiebrigi. A general comparison of honey, pollen and propolis production is given for each species. The chemical composition (moisture, ash, pH, free acidity, reducing sugars, sucrose), minerals (Ca, Cu, Fe, K, Mg, P, Zn), and microbiological counts (mesophilic bacteria, molds, yeasts) are compared here. 29.2 Species of Stingless Bees Producing Pot-Honey in Amboró National Park Bolivian stingless bees were collected and sent to Dr. Silvia RM Pedro at the Biology Department, Universidade de São Paulo, Ribeirão Preto, Brazil, for identification. Additional data including location, behavior and images were also submitted to the Camargo Collection RPSP (São Paulo, Ribeirão Preto), as stingless bee-keeper information. From ten species of stingless bees identified in a brief and incomplete survey of Amboró National Park (see Table 29.1), only six are kept by stingless bee-keeper. Pot-honey was extracted by syringe and collected in PET recycled bottles, from M. brachychaeta, M. grandis, S. depilis, S. polysticta, S. sp. aff. xanthotricha, T. fiebrigi of stingless bees kept in Amboró National Park. Nest entrances are shown in Fig. 29.1. Each species of stingless bee produces honey, pollen, and propolis in different ratios. In Table 29.2 an annual yield for stingless bee products in Amboró National Park is characterized, and relative stingless bee species abundance. S. polysticta “suro negro” is the most abundant, and T. fiebrigi “señorita” also is abundant, but is the lowest producer because this is as small bee with small storage pots. The Melipona “erereú barcina” and “erereú choca” are less abundant. S. depilis “obobosí” Table 29.1 Scientific and common names of Bolivian stingless bees Scientific names of Bolivian stingless bees Honey Common names Melipona brachychaeta Moure, 1950 1 “erereú choca”a Melipona grandis Guérin, 1834 2 “erereú barcina”a Melipona aff. crinita Moure and Kerr, 1950 “unknown” Plebeia droryana (Friese, 1920) “lambeojitos” Plebeia kerri Moure, 1950 “boca de vieja” Scaptotrigona depilis (Moure, 1942) 3 “obobosí”a Scaptotrigona polysticta Moure, 1950 4 “suro negro”a Scaptotrigona aff. xanthotricha Moure, 1950 5 “suro choco”a Tetragonisca fiebrigi (Schwarz, 1938) 6 “señorita”a Trigona chanchamayoensis Schwarz, 1948 “sicae amarilla” a Pot-honey studied here 29 Pot-Honey of Six Meliponines from Amboró National Park, Bolivia 411 Fig. 29.1 Nest entrances of Bolivian stingless bees in hives in the Carmen Surutú community, Amboró National Park, Bolivia. (a) Melipona brachychaeta, (b) Melipona grandis, (c) Scaptotrigona depilis, (d) Scaptotrigona polysticta, (e) Scaptotrigona aff. xanthotricha, (f) Tetragonisca fiebrigi, not shown to scale Photos: P. Vit Table 29.2 Relative annual yield of stingless bee products Common name Average Average pollen of the bees honey (l/year) (kg/year) Erereú barcina 1 0.5 Erereú choca 1 0.5 Obobosí 3 2 Suro negro 2 1 Suro choco 3 1.5 Señorita 0.5 0.5 Average propolis (kg/year) 0.5 0.5 1 3 4 0.25 Abundance in the park Very low Very low Abundant Abundant Medium Very high produces 2 kg pollen/year and 3 kg honey/year, like “suro negro,” but this is a rare species. S. near xanthotricha “suro choco” is a remarkable propolis producer with 4 kg/year and also yields an average of near 3 kg honey/year. Local common names of the bees, familiar to consumers, are used for marketing purposes. 29.3 Chemical and Microbial Composition of Bolivian Pot-Honey The chemical composition (quality factors and mineral contents) and the microbiological analysis were performed with a sample of 300 g pot-honey, for each meliponine species, by Quality Control Laboratory, Food and Natural Products Centre, Faculty of Science and Technology, Universidad Mayor de San Simón, in Cochabamba, Bolivia (report number CAPN M197/08-3/6). Physicochemical parameters were analyzed in duplicate: ash (gravimetric method), water content (refractometric method), reducing sugars and sucrose (titrimetric method), pH, and free acidity (titrimetric method) (AOAC 1984). The minerals were measured by spectrophotometry (Cu, Mg, Zn) (Perkin Elmer 1996), flame (Ca, K) (Vogel 1978), and colorimetry (Fe, P) (AOAC 1984) methods. 412 U. Ferrufino and P. Vit Microbiological spectra of mesophilic bacteria, molds, and yeasts were measured in colony-forming units (cfu)/g, using plate count agar. The analytical results of pothoney produced by six species are shown in Table 29.3. In Table 29.3, three sets of data are given for quality factors, mineral contents, and microbe content. Compared to A. mellifera honey standards (Codex Standard for Honey 1981), those of the meliponines were often different, including: (1) moisture (24.1–26.5 g water/100 g) for M. grandis and S. polysticta, all values higher than the honey standard <20%, (2) ash content (0.01–0.33 g ash/100 g) for M. brachychaeta and T. fiebrigi, complies with the honey standard of not more than 0.5%, (3) pH (3.4–4.5) for S. depilis and T. fiebrigi, as reference values not included in the honey standards, (4) free acidity (10.4–49.4 meq./kg) for M. brachychaeta and S. depilis, included in the maximum of 50 meq./kg for honey standards, (5) reducing sugar content (58.6–73.4 g reducing sugars/100 g) for T. fiebrigi and M. brachychaeta; standards are >60% glucose and fructose, and (6) sucrose content (0.0–1.5 g sucrose/100 g) for S. aff. xanthotricha, and M. brachychaeta, like the standards, <5%. Honey mineral content was measured, for the first time, for Bolivian meliponines. The Ca, Cu, Fe, K, Mg, P, and Zn content was lower in honey produced by M. grandis and higher in the honey of T. fiebrigi, in agreement with ash content (0.01–0.33 g ash/100 g). Microbe composition information is useful for sanitary quality control and is a routine analysis in the Brazilian Norm for honey (Brasil 1997). The counts for total aerobic mesophilic bacteria varied between 9.6 × 102 and 3.2 × 105 cfu/g for T. fiebrigi and M. brachychaeta, respectively. These values cover a wider range than the 1.0 × 103 and 5.0 × 103 cfu/g for Nigerian A. mellifera, where mold and yeasts were not detected in the honey (Omafuvbe and Akanbi 2009). Yeasts are usually present in honey, while other fungi were found only in the honey produced by three species: S. depilis, S. near xanthotricha and T. fiebrigi, in concentrations of 1.0 × 10 to 1.6 × 102 cfu/g. Souza (2008) also reported molds and yeasts in S. xanthotricha (2.5 × 10 to 4.6 × 102) and T. angustula (3.5 × 10 to 4.4 × 103) Brazilian pot-honeys. Molds were absent in the Melipona and S. polysticta honey. Yeast concentration varied between 3.0 × 10 and 4.1 × 103 cfu/g for S. depilis and M. grandis. A similar range, 1.3 × 10 to 1.6 × 103 cfu/g, was found in two samples of M. mandacaia from Brazil. Melipona such as M. asilvai, M. quadrifasciata anthidioides, and M. scutellaris were also within that range (Souza 2008). This author also observes increasing mold and yeast counts in pot-honey of M. asilvai, M. quadrifasciata anthidioides, T. angustula, and M. scutellaris, respectively. Therefore, molds and yeasts are fairly common in pot-honey. Association of microorganisms with Meliponini is discussed elsewhere in this book (see Chaps. 10 and 11). The identification of yeasts, molds, and bacteria associated with the six bees is needed, in order to explain their function for the bees and for human health. The fact that meliponines cannot migrate (Roubik 2006) may lead to eventual fermentation and regulation of this factor within stingless bee nests. Flexible cerumen pots are ideal containers to do that, in contrast with the more rigid besswax combs, with 29 Chemical parameters Quality factors Moisture (g/100 g honey) Ash (g/100 g honey) pH Free acidity (milliequivalents/kg honey) Sugars (g/100 g honey) Reducing sugars Sucrose Minerals (mg/100 g honey) Calcium Cupper Iron Magnesium Phosphorus Potassium Zinc Microbe composition (cfu/g) Mesophilic bacteria Molds Yeasts a See Table 29.1 for species 1 erereú choca 2 erereú barcina 3 obobosí 4 suro negro 5 suro choco 6 señorita 24.9 0.01 3.8 10.4 24.1 0.02 3.6 16.0 26.0 0.03 3.4 49.4 26.5 0.06 3.5 49.1 24.9 0.09 3.8 34.5 25.1 0.33 4.5 43.8 73.4 1.5 72.5 0.9 67.7 1.0 67.8 1.0 67.0 0.0 58.6 1.8 2.10 n.d. 0.02 0.36 0.97 9.63 0.02 2.47 0.04 0.06 0.71 1.32 12.52 0.02 2.97 0.01 0.09 1.58 3.00 14.75 0.01 2.97 0.01 0.13 1.48 5.38 29.1 0.68 2.98 0.08 0.27 2.77 7.01 43.58 0.15 10.99 0.11 0.40 4.97 16.85 144.92 0.63 3.2 × 105 Absent 2.8 × 102 2.3 × 104 Absent 4.1 × 103 4,6 × 103 2.0 × 10 3.0 × 10 1.4 × 104 Absent 3.1x102 1.5 × 103 1.0 × 10 6.4 × 102 9.6 × 102 1.6 × 102 4.7 × 102 Pot-Honey of Six Meliponines from Amboró National Park, Bolivia Table 29.3 Average values in chemical composition and antibacterial activity for six species of Bolivian meliponine pot-honeys from Amboró National Park Stingless beesa 413 414 U. Ferrufino and P. Vit potentially thicker walls and aggregated arrangements to store higher quantities of honey. Honey microbes may be used to set sanitary standards for meliponines. For organic honey (Sereia et al. 2010), it has been suggested that microbe counts are of primary importance, but we believe this is still undetermined for meliponine honey. One example of the possible relationship between a yeast and health is the protective role of S. cerevisiae, acting as a probiotic able to colonize and survive in the mice enteron, and the immune modulation exerted against Salmonella infection (Martins et al. 2007). MICs of T. fiebrigi honey from Argentina and Paraguay are lower for the Gram negative E. coli than the Gram positive S. aureus (Vit et al. 2009). For Bolivian pothoney, only mesophilic bacteria, molds, and yeast concentrations were measured here. The measurement of antibacterial activity and probiotic action will be useful analyses to include with prospective medicinal value in these honeys. 29.4 Sensory Approaches to Evaluate Pot-Honey from Bolivia The sensory evaluation for consumer acceptance included a Spanish panel of students and staff at the University of Burgos, Spain, who had never tasted meliponine honey before (Vit et al. 2010). The panel consisted of honey users with adequate physiological conditions. The six honey samples were evaluated at the same time, in an individual booth of the sensory room, under natural daylight. Water and toast were provided to clean the palate between samples. Instructions suggested trying all honeys first from left to right, and then to rank each one in a free order, and describe a short reason for this choice. Participants rated how much they liked each honey, manually, on a 10-cm line anchored with the words “dislike it a lot” and “like it a lot,” in the left and right ends. This procedure provided a baseline rating the following averages of acceptance ± SD: “suro negro” S. polysticta 5.6 ± 2.2, “obobosí” 5.5 ± 2.5, “ereureú choca” M. brachychaeta 5.0 ± 2.5, “suro choco” 4.9 ± 2.2, “señorita” T. fiebrigi 4.8 ± 2.4, and “erereú barcina” M. grandis 3.7 ± 2.1. Although M. grandis honey was very light amber color, similar to acacia honey, it was the honey with the lowest score, due to a bitter taste, and animal notes. This average acceptance could be improved by a better knowledge of the honey and would be very interesting to compare with acceptance by consumers from urban and rural Bolivia. Another sensory approach compared one pot-honey of S. polysticta from Bolivia with that from four species in Australia, Brazil, Mexico, and Venezuela, using the free-choice profile method. In this method there is no need of a trained panel, because sensory descriptors of honey are elicited from the assessors, and then quantified. The S. polysticta honey in this international set was characterized by fresh fruit aroma, sour taste and an astringent trigeminal sensation, and was grouped with another species of Scaptotrigona, S. mexicana (Vit et al. 2011). 29 Pot-Honey of Six Meliponines from Amboró National Park, Bolivia 29.5 415 Need for Networking to Market Bolivian Pot-Honey The main honey importers in the world are Germany, the USA, UK, Japan, and France, and commercial interest is growing for organic and special honey (Hernández 2010). However, producing pot-honey and achieving a market niche are two different aspects of the business. Most projects, assisted or not, attain successful production and community interest. The chain of marketing needs to fulfill a system and a philosophy, valid in all the steps of the process, from raw materials in the environment to packaged honey as a commodity for the consumer and the “cradle to grave” perspective to attract consumers of organic honey (Hilmi, n.d.). Small to medium-sized enterprises (SMEs) like meliponiculture are not focused on conventional marketing. The additional lack of marketing resources makes alternative marketing approaches necessary, which benefit from a variety of networking processes (Gilmore 2001). The Bolivian effort of 11 years with this meliponiculture project in Santa Cruz de la Sierra Department, Ichilo Province and three counties (Buena Vista, San Carlos and Yapacaní) was successfully coordinated by ASEO (Aguilera Peralta and Ferrufino Arnéz 2004; Ferrufino Arnéz and Aguilera Peralta 2006). Seven communities with 40 associates evidence the cooperative organization of APROMIN. Acknowledgments To Dr. Silvia R.M. Pedro from the Biology Department, at Universidade de São Paulo, Ribeirão Preto, Brazil, for the entomological identification of the Bolivian stingless bees, and to Dr. David W. Roubik for appreciated editorial care. To stingless bee-keepers from Amboró National Park in Bolivia. References Aguilera Peralta FJ, Ferrufino Arnéz U. 2004. Cómo criar abejas melíferas sin aguijón. Asociación Ecológica del Oriente (ASEO), Unión Mundial para la Naturaleza (UICN); Santa Cruz de la Sierra, Bolivia. 140 pp. Amazonia Boliviana. Abejas, las obreras de la conservación ecológica. Available at: http://www. amazonia.bo/amazonia_bo.php?id_contenido=135&opcion=detalle_des AOAC. Association of Official Analytical Chemists. 1984. Official methods of analysis. 14th. Edition. AOAC; Arlington, TX, USA. 1375 pp. Brasil. 1997. Leis, decretos, etc. Decreto no 30.691, de 08 de setembro 1997. Diário Oficial, Brasilia. Seção 1, pp. 19696–19697. Aprova o Regulamento Técnico para Fixação de Identidade e Qualidade do Mel. Codex Standard for Honey. 1981. Codex Stan 12–1981. Revisions 1987 and 2001. pp. 1–8. Ferrufino Arnéz U, Aguilera Peralta FJ. 2006. Producción rural sostenible con abejas melíferas sin aguijón. Asociación Ecológica del Oriente (ASEO); Santa Cruz de la Sierra, Bolivia. 101 pp. Gilmore A. 2001. SME marketing in practice. Marketing Intelligence & Planning 19:6–11. Available at: http://www.sie.ed.ac.uk/resources/SIE%20Gilmore%20et%20al.pdf Hernández MA. 2010. Perfil de mercado: Miel de abejas nativas. Instituto Boliviano de Comercio Exterior. 24 pp. Available at: http://www.ibce.org.bo//documentos/perfil_mercado_miel_abejas_CB16.pdf Hilmi M. n.d. The marketing of organic honey. Available at: http://www.beekeeping.com/new/ books/martin.htm 244 pp. 416 U. Ferrufino and P. Vit Martins FS, Rodrigues ACP, Tiago FCP, Penna FJ, Rosa CA, Arantes RME, Nardi RMD, Neves MJ, Nicoli JR. 2007. Saccharomyces cerevisiae strain 905 reduces the translocation of Salmonella enterica serotype Typhimurium and stimulates the immune system in gnotobiotic and conventional mice. Journal of Medical Microbiology 56:352–359. Omafuvbe BO, Akanbi OO. 2009. Microbiological and physico-chemical properties of some commercial Nigerian honeys. African Journal of Microbiology Research 3:891–896. Perkin Elmer. 1996. Standard conditions for the determination of individual elements. Model Analysis 200. The Perkin Elmer Corporation; Wellesley, USA. Roubik DW. 2006. Stingless bee nesting biology. Apidologie 37:124–143. Sereia MJ, Arnaut de Toledo VA, Marchini LC, Machado Alves E, Faquinello P, Arnaut de Toledo TCSO. 2010. Microorganisms in organic and non organic honey samples of Africanized honeybess. Journal of Apicultural Science 54:49–54. Souza BA. 2008. Physico-chemical characterization and microbiological quality of stingless bees (Apidae, Meliponinae) honey samples from the State of Bahia, Brazil, with emphasis on Melipona Illiger, 1806. Tese de Doutorado, Escola Superior de Agricultura “Luiz de Queiroz”; Piracicaba, Brasil. 107 pp. Vit P, Gutiérrez MG, Rodríguez-Malaver AJ, Aguilera G, Fernández-Díaz C, Tricio AE. 2009. Comparación de mieles producidas por la abeja yateí (Tetragonisca fiebrigi) en Argentina y Paraguay. Acta Bioquímica Clínica Latinoamericana 43:219–226. Vit P, Ferrufino U, Pascual A, Fernández-Muiño MA, Sancho Ortiz MT. 2010. How Spanish perceive Bolivian pot honeys from six Meliponini species. Fourth European Conference of Apidology, Eur Bee, Metu, Ankara, Turkey. Vit P, Sancho T, Pascual A, Deliza R. 2011. Sensory perception of tropical pot honeys by Spanish consumers, using free choice profile. Journal of ApiProduct and ApiMedical Science 3:174–180. Vogel AI, 1978. Vogel’s textbook of quantitative inorganic analysis. 4th. Edition. Bassett J, Denney RC, Jeffery GH, Mendham J, eds. Longman; London, UK. 962 pp. Chapter 30 An Electronic Nose and Physicochemical Analysis to Differentiate Colombian Stingless Bee Pot-Honey Carlos Mario Zuluaga-Domínguez, Amanda Consuelo Díaz-Moreno, Carlos Alberto Fuenmayor, and Martha Cecilia Quicazán 30.1 Introduction Honey derived from Apis mellifera (Linnaeus, 1758) are well known by consumers worldwide. Honey has been valued since ancient times and has been used as a nutritional and therapeutic supplement in many cultures (Vit et al. 1994). Previous studies have focused research on defining distinctive characteristics of honey from A. mellifera to obtain quality and authenticity labels (Acquarone et al. 2007; Kaškoniené et al. 2008; Baroni et al. 2009; Cajka et al. 2009; Truchado et al. 2009; Castro-Vázquez et al. 2010; Kaškoniené et al. 2010; Kropf et al. 2010; Stanimirova et al. 2010; Wang and Li 2011). Geographical differentiation and the establishment of quality standards give added value to bee products such as honey and facilitate their commercial exploitation. Protected Geographical Status (PGS) is a legal framework defined in the European Union law to protect the names of regional foods, which ensures that only products genuinely originating in that region are allowed to be identified as such in commerce (EC 2008). The purpose of this law is to protect the reputation of regional foods, to promote rural and agricultural activity, to help producers obtain a premium price (or fair price) for their authentic products, and to eliminate unfair competition and the deception of consumers by false or adulterated products, which may be of inferior quality. According to these laws, the quality of bee products—especially honey—can be defined by providing additional information about floral and geographic origin. As of 2011, more than 24 different kinds of European honey have been registered with PGS (EC 2011). In addition to that of A. mellifera, honey from stingless bees (Meliponini, or meliponines) is found in Latin America. Meliponini live in tropical and subtropical areas, C.M. Zuluaga-Domínguez • A.C. Díaz-Moreno (*) • C.A. Fuenmayor • M.C. Quicazán Instituto de Ciencia y Tecnología de Alimentos—ICTA, Universidad Nacional de Colombia, Carrera 30 #45-03 Ed. 500-C, Ciudad Universitaria, Bogotá, Colombia e-mail: amcdiazmo@unal.edu.co 417 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_30, © Springer Science+Business Media New York 2013 418 C.M. Zuluaga-Domínguez et al. often acting as pollinators. Stingless bees have been on the continent far longer than A. mellifera; the latter was introduced during the Hipsanic period of conquest, mostly in the 1500s. Historical reports show that honey from the stingless bees was considered to be a treasure of great value for the indigenous population and that it was used as a trading instrument (see Chap. 14). In Latin American culture, honey has traditionally been conferred with different therapeutic effects in addition to its nutritional properties. Stingless bee honey from Mexico, Guatemala, Venezuela, Brazil, Ecuador, Colombia, and Bolivia (Vit et al. 2004; Souza et al. 2006; Almeida-Muradian et al. 2007; Guerrini et al. 2009; see Chaps. 7, 28, 29) have physical and chemical properties of interest. On the other hand, due to reduction in forests where stingless bees thrive, there is a severe decrease in stingless bee populations, even to the point where they may be in danger of extinction. Different government entities have made efforts to increase the population of the bees by encouraging beekeepers to breed them and to commercialize their honey (ImperatrizFonseca and Peixoto 2006). The distribution of stingless bee honey in the market is limited, compared with the honey from A. mellifera, as a consequence of limited production, shorter shelf life and lack of an institutional quality standard, due to the scant knowledge about the products. The main objective of this research was to establish quality attributes of stingless bee honey based on its physicochemical properties and the application of an “electronic nose” to monitor the volatile components of honey. This preliminary research determines if an ‘electronic nose’ is a valuable device for determining the quality and authenticity of stingless bee pot-honey. An electronic nose analysis has been conducted for A. mellifera honey (Benedetti et al. 2004; Lammertyn et al. 2004; Zuluaga et al. 2011). In this chapter we report for the first time, an electronic nose multivariate approach to pot-honey from Colombia. 30.2 Physicochemical and Electronic Nose Analysis of Honey Fifty-five honey samples were collected from Melipona sp. (10 samples), Tetragona sp. (21 samples), Melipona compressipes (10 samples), Melipona favosa (7 samples), and Melipona eburnea (7 samples). The samples were immediately stored at 4°C in airtight containers in the dark to prevent degradation prior to analysis. To make a comparison of analyzed properties, 15 honey samples were collected from A. mellifera and processed in the same manner. 30.2.1 Physicochemical Analysis The water content was determined by measuring the refraction index according to AOAC 969.38B (AOAC 2005) using a table refractometer ABBE (Euromex, The Netherlands) at 20°C. The water content (g/100 g) was obtained by correlation with a Chataway table (Chataway 1932). 30 An Electronic Nose and Physicochemical Analysis to Differentiate Colombian... 419 Sugars analysis included the quantification of disaccharide (maltose–sucrose) and monosaccharide (glucose and fructose) content. This procedure was performed according to AOAC 979.23 and 983.22 (AOAC 2005) by high performance liquid chromatography (JASCO CO-2065, Japan) with a refraction index detector (JASCO RI-2031, Japan) and a calcium cationic exchange resin column Metacarb Ca Plus (VARIAN A5205, USA). In the mobile phase, distilled, degassed, and deionized water was used, with a flow of 0.5 mL/min; column temperature was kept at 80°C, and the detector at 45°C. Sugars results are expressed as g/100 g. 30.2.2 The Electronic Nose Analysis The electronic nose consists of an array of weakly specific or broad-spectrum chemical sensors that mimic human olfaction and convert sensor signals into data that can be analyzed with appropriate statistical software. Such characteristics greatly facilitate monitoring volatile components of food, providing real-time information about the various characteristics of food under study (Schaller et al. 1999). A number of potential applications of an electronic nose in the food industry have been reported, such as quality parameters for A. mellifera honey (Benedetti et al. 2004; Lammertyn et al. 2004) and quality assessment of meat (García et al. 2005; García et al. 2006), fruit and vegetables (Lebrun et al. 2008; Pani et al. 2008), wines (Aleixandre et al. 2008; Berna et al. 2008; Lozano et al. 2008), and dairy products (Pillonel et al. 2003; Brudzewski et al. 2004; Benedetti et al. 2005; Labreche et al. 2005). Analyses were performed with an Airsense PEN 3 electronic nose (Germany) that consisted of three parts: a sampling apparatus, a detector unit containing the sensor array, and software for pattern recognition. Samples were introduced to the sampling apparatus randomly and after an adequate sensor flush time to avoid undesirable effects caused by sensor drift on readings. The sensor array was composed of ten Taguchi type sensors (metal oxide semiconductors—MOS). Sensors were kept at 400–500°C during all of the process phases. The MOS sensors are the most suitable for food headspace analysis as they work at high temperatures and thus are not sensitive to humidity (Benedetti et al. 2004). The sensors used in this work are: W1C (aromatic compounds), W5S (wide range of compounds, especially nitrogen), W3C (aromatic compounds), W6S (mainly hydrogen), W5C (aromatic and aliphatic compounds), W1S (short chain hydrocarbons), W1W (sulphur compounds), W2S (alcohols), W2W (sulphur–chlorine compounds), and W3S (short chain aliphatic compounds). The operative procedure was standardized and optimized as reported by Zuluaga et al. (2011). Three grams of each sample were placed in 40 mL Pyrex® vials with silicone caps and then introduced to the sampling unit of the electronic nose. Preliminary trials indicated that using larger sample volumes did not significantly increase signal intensities and reproducibility. After an equilibration time of 20 min at 40°C, the measurement sequence began (Zuluaga et al. 2011). 420 C.M. Zuluaga-Domínguez et al. The measurement procedure consisted of pumping reference air over the sensors (the air in the room filtered through active carbon) at a constant flow rate (1 cm3/s) for 10 s to set a stable baseline. Then the honey gas headspace sampled with a syringe was pumped over the sensor surfaces for 150 s. The sensors were then exposed to the reference air to recover the baseline. The total cycle time for each measurement was 7.5 min. Sensor drift was not experienced during the measurement period. 30.2.3 Data Analysis The data obtained from the sensor array and physicochemical analyses for all of the honey samples were analyzed by partial least squares-discriminant analysis (PLS-DA) performed with MATLAB (v. 7.0 The Mathworks, Natick, MA, USA). PLS-DA is a combination of the PLS technique, and regression to correlate an experimental response with a calculated response from a model, and DA analysis, which discriminates the experimental response among classes. The dimensions (components) extracted are composed such that they exhibit the maximum correlation with Y (class membership, e.g., origin and species) (van Ruth et al. 2010). This technique is a “supervised method,” thus validated to obtain a reliable classification model. Some indicators were used to evaluate the robustness and prediction capacity of this model: non-error rate, specificity, sensibility, and precision. For a better understanding of PLS-DA techniques, see Beebe et al. (1998), Wold et al. (2001), Gemperline (2006), Bereton (2007), and Aguilera et al. (2010). 30.3 Aromatic Profile and Physicochemical Results for the Genus Melipona The physicochemical results for stingless bee and A. mellifera honey are presented in Table 30.1. To create the classification models, data were organized in two matrices, analyzed separately with PLS-DA. The first data matrix grouped stingless bee honey from M. compressipes, M. favosa, and M. eburnea. The second data matrix grouped stingless bee honey from Melipona and Tetragona and A. mellifera. The PLS-DA results are shown in Fig. 30.1 for the sample plot and the loading plot, respectively. Melipona are well classified in three defined classes. Samples from M. compressipes have high sugar values and an appreciable response from the sensors identified as W1W, W2W, and W3S. The same analysis for M. eburnea shows higher glucose content and a specific response for the sensors identified as W1C, W3C, and W5C. M. favosa has the highest moisture content. The validation model shows adequate results for non-error rate and error rate for both the fitting and the cross-validation stages (Table 30.2), which indicates 30 An Electronic Nose and Physicochemical Analysis to Differentiate Colombian... 421 Table 30.1 Physicochemical results for analyzed honeys from Colombia Moisture (g/100 g) Genus/species Melipona Melipona compressipes 25.8 ± 2.0 Melipona eburnea 27.6 ± 2.1 Melipona favosa 24.8 ± 1.8 Melipona sp. 26.8 ± 5.3 Tetragona Tetragona sp. 25.8 ± 3.6 Apis Apis mellifera 18.6 ± 1.5 Mean values ± standard deviation a Sucrose plus maltose Glucose (G) Fructose (F) Disaccharides (g/100 g) (g/100 g) (D)a (g/100 g) Sugars (G + F + D) (g/100 g) 34.2 ± 4.4 38.5 ± 7.5 33.5 ± 3.1 30.5 ± 5.6 36.9 ± 3.7 39.3 ± 7.0 38.7 ± 4.3 36.9 ± 5.7 3.4 ± 2.2 3.6 ± 1.5 3.1 ± 1.8 6.5 ± 3.2 75.2 ± 8.0 73.0 ± 3.4 75.3 ± 6.2 73.5 ± 8.0 29.0 ± 6.8 31.8 ± 3.9 4.4 ± 5.6 69.1 ± 4.3 32.6 ± 4.4 40.1 ± 3.9 6.8 ± 2.1 82.6 ± 9.3 Fig. 30.1 PLS-DA result for Melipona pot-honey that the model has a good capacity for recognizing classes and should be tested for prediction in future. The other parameters, such as specificity, sensibility, and precision, established that the capacity of prediction is very accurate for M. compressipes and M. eburnea. However, for M. favosa the model has a fair capacity to differentiate samples from this class, but a low capacity to predict new, unknown samples. 422 Table 30.2 PLS-DA model fitting and validation results for species Melipona Fitting Error rate: 0.08 Non-error rate: 0.83 Cross-validation Error rate: 0.17 Non-error rate: 0.75 Class Specificity Sensibility Precision Class Specificity Sensibility Precision M. compressipes M. favosa M. eburnea 1.00 0.94 0.94 0.80 0.71 1.00 1.00 0.83 0.88 M. compressipes M. favosa M. eburnea 0.93 0.88 0.94 0.80 0.43 1.00 0.89 0.60 0.88 C.M. Zuluaga-Domínguez et al. 30 An Electronic Nose and Physicochemical Analysis to Differentiate Colombian... 423 Fig. 30.2 PLS-DA results for Melipona, Tetragona, and Apis mellifera honey 30.4 Aromatic Profile and Physicochemical Results for the Species Melipona, Tetragona and A. mellifera A. mellifera is included to establish differences from Melipona and Tetragona. The results from PLS-DA (Fig. 30.2) show differentiation from A. mellifera and separation between Melipona and Tetragona. A. mellifera is distinguished by high levels of fructose and low moisture content, also by responses of the sensor identified by the manufacturer as W3S. These results corroborate with those reported in the chapter of Deliza and Vit in this book, using assessors to evaluate pot-honey. Tetragona is characterized by the response of sensors W1C, W3C, and W5C; the same analysis concluded that the Melipona was characterized by W1S, W2S, W5S, W6S, W1W, and W2W. The model evaluation (Table 30.3) shows a well-adjusted classification and a robust prediction capacity, especially for the Tetragona and the A. mellifera species. In the case of the Melipona, the model is adequate in differentiating samples of this species, but according to the results from cross-validation, the model has a low prediction of new unknown samples for this class. 30.5 Classification Model Honey classification was made possible with sensor responses and data from simple chemical analysis. Both results showed that it is possible to create a model that facilitates the differentiation and classification of honey according to 424 Table 30.3 PLS-DA model fitting and validation results for species Melipona, Tetragona, and Apis mellifera Fitting Cross-validation Error rate: 0.09 Error rate: 0.20 Non-error rate: 0.80 Non-error rate: 0.76 Class Specificity Sensibility Precision Class Specificity Sensibility Precision Melipona Tetragona Apis mellifera 0.92 0.96 1.00 0.70 0.81 0.87 0.70 0.94 1.00 Melipona Tetragona Apis mellifera 0.86 0.84 1.00 0.60 0.76 0.87 0.55 0.80 1.00 C.M. Zuluaga-Domínguez et al. 30 An Electronic Nose and Physicochemical Analysis to Differentiate Colombian... 425 bee species—in this case, from Colombian stingless bees. The PLS-DA model can be implemented as a useful tool for classification to guarantee the quality and the authenticity of honey. Data from the electronic nose analysis confirmed that volatile and semi-volatile organic compounds present in the headspace contributed significantly to the honey aroma and to the aroma variation in relation to the bee species. Aroma is a very important parameter for defining the quality of apicultural products (Ampuero et al. 2004; Benedetti et al. 2004). Pot-honey has different flavors depending on various factors, one of which is the bee species (Vit et al. 2011a, b). However, in Colombia, there have been no studies aimed at characterizing and differentiating honeys from an objective point of view. It is clear that other types of analyses exist that facilitate the discrimination of honey according to species (e.g., gas chromatography), but using an electronic nose has shown that the proposed methodology is simple, rapid and does not require isolation of the volatile components. This makes the technique particularly useful for online quality control because any alteration that causes changes in the volatile fraction can be detected, which is of great importance to control adulteration and counterfeiting (very common activities in stingless bee honey sales). Despite the fact that PLS-DA model classification parameters for M. favosa and Melipona could not achieve 100 % prediction, the results confirm the influence of the variables analyzed here for creating new models. It is advisable to increase the number of samples to enhance the fitting and predictive capacity of the statistical method to ensure reliability of results. Acknowledgement The authors would like to express their thanks to the Universidad Nacional de Colombia’s Institute of Food Science and Technology (ICTA), the Minisitry of Agriculture and Rural Development, the Sumapaz Region Beekeepers’ Association, the Boyaca Beekeepers’ Association, Comunera Beekeeping Association, the Conservationist Beekeepers Association from the Sierra Nevada de Santa Marta, the Colombian Science, Technology and Innovation Department (COLCIENCIAS), the Italo-Latin American Institute and the Unversity of Milan’s Food and Microbiological Science and Technology Department (DISTAM). References Acquarone C, Buera P, Elizalde B. 2007. Pattern of pH and electrical conductivity upon honey dilution as a complementary tool for discriminating geographical origin of honeys. Food Chemistry 101:695–703. Aguilera A, Escabias M, Preda C, Saporta G. 2010. Using basis expansions for estimating functional PLS regression: applications with chemometric data. 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Journal of Chromatography A 1216:1458–1462. Castro-Vázquez L, Díaz-Maroto M, de Torres C, Pérez-Coello M. 2010. Effect of geographical origin on the chemical and sensory characteristics of chestnut honeys. Food Research International 43:2335–2340. Chataway H. 1932.Determination of moisture in honey. Canadian Journal Research 6:532–547. EC. 2008. Reglamento (CE) No 628/2008. Diario Oficial de la Unión Europea; Luxemburgo. EC. 2011.Geographical indications and traditional specialities. Available at: http://ec.europa.eu/ agriculture/quality/schemes/index_en.htm García M, Aleixandre M,Gutiérrez J, Horrillo M. 2006. Electronic nose for ham discrimination. Sensors and Actuators B 114:418–422. García M, Aleixandre M, Horrillo M. 2005. Electronic nose for the identification of spoiled Iberian hams. Spanish on Conference Electron Devices. pp. 537–540. Gemperline P. 2006. Practical Guide To Chemometrics. Taylor & Francis Group; Boca Raton, USA. 521 pp. Guerrini A, Bruni R, Maietti S, Poli F, Rossi D, Paganetto G, Muzzoli M, Scalvenzi L, Sacchetti G. 2009. Ecuadorian stingless bee (Meliponinae) honey: a chemical and functional profile of an ancient health product. Food Chemistry 114:1413–1420. Imperatriz-Fonseca V, Peixoto A. 2006. As abelhas e as iniciativas internacionais de polinizadores. Memorias II Encuentro Colombiano Sobre Abejas Silvestres. Bogotá, Colombia, pp. 28–35. Kaškoniené V, Venskutonis P, eksteryt V. 2008. Composition of volatile compounds of honey of various floral origin and beebread collected in Lithuania. Food Chemistry 111:988–997. Kaškoniené V, Venskutonis P, eksteryt V. 2010. Carbohydrate composition and electrical conductivity of different origin honeys from Lithuania. LWT - Food Science and Technology 43:801–807. Kropf U, Korošec M, Bertoncelj J, Ogrinc N, Ne emer M, Kump P, Golob T. 2010. Determination of the geographical origin of Slovenian black locust, lime and chestnut honey. Food Chemistry 121:839–846. Labreche S, Bazzo S, Cade S, Chanie E. 2005. Shelf life determination by electronic nose: application to milk. Sensors and Actuators B 106:199–206. Lammertyn J, Veraverbeke E, Irudayaraj J. 2004. zNose technology for the classification of honey based on rapid aroma profiling. Sensors and Actuators B 1:54–62. Lebrun M, Plotto A, Goodner K, Ducampa M, Baldwin E. 2008. Discrimination of mango fruit maturity by volatiles using the electronic nose and gas chromatography. Postharvest Biology and Technology 48:122–131. 30 An Electronic Nose and Physicochemical Analysis to Differentiate Colombian... 427 Lozano J, Arroyo T, Santos J, Cabellos J, Horrillo M. 2008. Electronic nose for wine ageing detection. Sensors and Actuators B 133:180–186. Pani P, Leva A, Riva M, Maestrelli A, Torreggiani D. 2008. 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Sensory perception of tropical pot honeys by Spanish consumers, using free choice profile. Journal of ApiProduct and ApiMedical Science 3(4): 174–180. Wang J, Li Q. 2011. Chapter 3 - Chemical Composition, Characterization, and Differentiation of Honey Botanical and Geographical Origins. pp. 89–137. In Steve L, eds. Advances in Food and Nutrition Research, Academic Press; Nebraska, USA. 270 pp. Wold S, Sjöström M, Eriksson L. 2001. PLS-regression: a basic tool of chemometrics. Chemometrics and Intelligent Laboratory Systems 58:109–130. Zuluaga C, Díaz C, Quicazán M. 2011. Standardising and validating aromatic profile analysis by an electronic nose. Ingenieria e Investigacion 31:65–73. Chapter 31 Nuclear Magnetic Resonance as a Method to Predict the Geographical and Entomological Origin of Pot-Honey Elisabetta Schievano, Stefano Mammi, and Ileana Menegazzo 31.1 Introduction Nuclear Magnetic Resonance (NMR) is a powerful spectroscopic method, traditionally used as a very important tool in chemistry for structure verification, elucidation and purity analysis. However, driven by the needs of multidisciplinary topics such as biochemistry, medicine, pharmaceutical sciences, food chemistry, and others, NMR has rapidly expanded its applications to many other fields, and recent examples are the analysis of complex mixtures and screening applications (Lindon et al. 2000; Spraul et al. 2009). NMR is an especially suited detector in the analysis of fluids of biological origin, food materials or drinks. It combines truly quantitative and structural information with high throughput (a 1D spectrum can be measured in a few minutes) and excellent reproducibility, which depends mostly on the minimal sample preparation required and the absence of any derivatization step. For these reasons, it can be used to detect small molecules to generate global metabolite profiles in metabolomic studies, which aim to categorize or classify samples and to understand the basic underlying principles that contribute to the differences among them (Kang et al. 2008). Pattern recognition is followed by related multivariate statistical approaches to analyze the latent structures in the multivariate data. Principal Component Analysis (PCA) and Partial Least-Squares Discriminant Analysis (PLS-DA) have often been used to identify sample groups and to relate specific biochemical compounds to the group separation. 1 H NMR-based metabolomic studies have been applied also to food science (Cevallos-Cevallos et al. 2009), including for example assessments of green tea E. Schievano (*) • S. Mammi • I. Menegazzo Department of Chemical Sciences, University of Padova, Via Marzolo 1, 35100 Padova, Italy e-mail: elisabetta.schievano@unipd.it 429 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_31, © Springer Science+Business Media New York 2013 430 E. Schievano et al. (Tarachiwin et al. 2007), rosemary (Xiao et al. 2008), honey (Schievano et al. 2012), and grape wine (Son et al. 2008). In this chapter, a study of the honey matrix is illustrated, performed using an NMR-based metabolomic approach combined with multivariate analysis. 31.2 Nuclear Magnetic Resonance NMR is a branch of spectroscopy which uses radio waves, with a frequency between 20 MHz and 1 GHz on sensitive nuclei. The most common NMR experiments are performed on 1H nuclei, but spectra on many other nuclei (13C, 31P, 19F, and 15N are the most common ones) are frequently acquired. The principles of NMR spectroscopy are well known nowadays; they are available in many textbooks (Günther 1995; Claridge 1999) and they are not discussed in detail here. Some basic principles will be quickly illustrated, to enhance the comprehension of this work. Nuclei with an intrinsic magnetic moment may be oriented by a strong magnetic field; two orientations are possible for 1H nuclei. A consequence is the tendency to absorb and emit energy at a specific resonance frequency. Based on this phenomenon, a very large number of different NMR experiments have been developed, which explore different properties of the material under study. Samples can be analyzed in the solid (CP-MAS NMR), semisolid (HR-MAS NMR), and solution state (HR-NMR): the last one has been utilized in this work. The 1H 1D spectrum is the simplest NMR experiment: a radio frequency pulse inverts the orientation of some of the 1H nuclei in the magnet; then, relaxation toward the original situation results is an electric signal (free induction decay: FID), which can be processed with a Fourier Transform to give a resonance peak. Samples must be completely dissolved in a solvent. In the solvents for NMR analyses, protons are normally replaced with deuterium atoms to avoid saturation of the NMR receiver with the solvent protons, which would otherwise hide the signals of the protons of the solute. Each peak in the spectrum is the signal of a particular kind of proton in the mixture and its resonance position, the chemical shift, is measured in ppm units on the x-axis of the spectrum. The y-axis is an intensity scale, relative to the amount of protons. Integration of a peak area is directly proportional to the number of protons resonating at that same frequency. 31.3 Metabolomic Analysis Metabolomics is the study of the global metabolic profile in a system (cell, tissue, or organism) under a given set of conditions. Metabolic profiling first appeared in the literature in the 1950s, and developed throughout the following decades (Rochfort 2005). The metabolome is formally defined as a collection of small 31 Nuclear Magnetic Resonance as a Method to Predict the Geographical... 431 molecules, including a range of endogenous and exogenous chemical entities such as peptides, amino acids, nucleic acids, carbohydrates, organic acids, vitamins, polyphenols, alkaloids, minerals, and just about any other chemical that can be used, synthesized, or ingested by a given cell or organism. Over the past few years, two schools of thought have emerged for processing and interpreting metabolomic data: the chemometric and the quantitative metabolomics (or targeted profiling) approaches (Wishart 2008). The chemometric approach (untargeted metabolomics) includes the analysis of multiple samples (for example by NMR) and statistical comparison of the results, without identifying the chemical compounds, but only using the recorded spectral pattern to recognize the relevant spectral features that distinguish sample classes. This method involves unsupervised clustering (PCA) or supervised classification (e.g., PLS-DA). After discovering significant differences, the most informative peaks in the spectra are identified and these molecules can then be used as markers. In the quantitative metabolomics approach, most compounds in the sample are first identified and quantified, and this information is then used to perform multivariate statistical analyses and to find the most important markers and informative metabolic pathways. From the perspective of a metabolomics researcher, most foods can essentially be viewed as complex chemical mixtures consisting of various metabolites and chemical additives in a solid, semisolid, or liquid mixture. In food science, metabolomics has become a tool to assess the quality, the processing history, and the safety of raw materials and final products. Recent applications involve geographical or botanical origin, or authenticity, of several foods. In this work, a chemometric approach to differentiate the geographical and entomological origin of stingless bee honey has been used: 1H NMR spectra provided signals, which were integrated and used as inputs for PCA and PLS-DA studies. Formally, PCA is a clustering technique that reduces the dimensions of a complex data matrix to orthogonal linear combinations (Principal Components visualized as principal axes) which describe variation in the data. These components can be displayed graphically as a score plot, where the separation of the observations is visualized in the space between the two axes. Unsupervised PCA was initially used to explore variation in the NMR spectra dataset while PLS-DA was subsequently applied to maximize the separation among the samples. The score plot can be visualized also in 3D corresponding to three principal components. In the loading plot, the most influential variables are highlighted: the farther they are from the center of the graph, the more they influence cluster separation. The Hotelling’s T2 region, shown as an ellipse in score plots of the models, defines the 95% confidence interval of the modeled variation. The quality of the models is described by R2x and Q2 values. R2x is defined as the proportion of variance in the data explained by the models and indicates goodness of the fit. Q2 is defined as the proportion of variance in the data predictable by the model and indicates predictability (Eriksson et al. 2006). Thus, PCA is most commonly used to identify how one sample 432 E. Schievano et al. is different from another, and which variables contribute most to this difference. PLS-DA is based on the same basic principles as PCA, but it uses the labeled set of class identities, enhancing the separation between groups of observations. 31.4 NMR-Based Metabolomics Applied to Pot-Honey Because of the complexity and diversity of the metabolites present in a complex food matrix, it is unlikely that one single analytical method would generate information about all the metabolites present and it would probably be necessary to perform a wide range of chemical analyses, which should be both rapid and reproducible. 1H NMR has the potential to detect and identify a large number of compounds; as such, it is emerging as a leading technique in the area of metabolomic studies. An important advantage of the use of NMR spectroscopy in metabolomic studies is that the sample requires hardly any physical or chemical treatment prior to analysis. MS studies usually require separation of the metabolites, and for GC-MS it may be necessary to modify the metabolites to render them volatile. On the other hand, separation via HPLC requires conveniently detectable chromophores or functional groups. NMR methodologies overcome these problems, and the range of compounds that can be analyzed is not limited by their volatility, presence of chromophores, or polarity, or other properties. Although the detection limit of NMR is still higher than that of other techniques, new pulse sequences have been introduced that lower the detection limit to about 10 mM in the sample solution (Rastrelli et al. 2009). Moreover, NMR spectroscopy simultaneously gives definitive structural information on many different compounds in the sample, maximizing the chance to identify important but unexpected or previously unknown metabolites (Teresa and Fan 1996). 1 H NMR has been successfully used, for example, in the area of toxicology, clinical diagnostics, and in the field of plant metabolites; it is frequently applied to food samples that can be directly examined as liquids (Belton et al. 1996), but very simple extraction or sample preparation procedures may also be used (Schievano et al. 2008). In the last decades, specific chemical and physical properties of honey have been used to determine its botanical origin (Anklam 1998; Bogodanov et al. 2004; Arvanitoyannis et al. 2005), and new analytical techniques have been proposed to this aim. An improvement in determination of botanical origin can certainly be achieved by a multivariate analytical approach. Recently, NMR techniques have been proposed also to identify and classify honey of different floral sources (Beretta et al. 2008; Lolli et al. 2008; Schievano et al. 2010) or geographical origin (Donarski et al. 2008; Consonni and Cagliani 2008). The composition and properties of a particular honey sample depend strongly on the type of bee, on the type of flowers visited by the bees, as well as on the climatic conditions in which the plants grow and on contributions of the beekeeper (Al et al. 2009; Azeredo et al. 2003). In fact, Schievano et al. (2012) have shown that 1H NMR spectra of organic extracts of honey can be used as a fingerprint to differentiate the botanical origin, when coupled with chemometric analysis. 31 Nuclear Magnetic Resonance as a Method to Predict the Geographical... 433 Fig. 31.1 The work flow of the NMR-based metabolomic approach applied to honey The extraction method is simple and reproducible: a water/chloroform mixture was used as extracting solvent, with the advantage to eliminate the compounds most present in the honey mixture, i.e., the carbohydrates, with the water layer. The aroma compounds and those hydrophobic substances that differ the most in honeys of various sources are retained in the organic solution. Also, the extraction procedure yields a concentrated solution amenable to rapid NMR analysis. In more detail, portions of honey samples (6 g) were weighted in a centrifuge tube and dissolved with 15 ml of deionized water. 15 ml of CHCl3 were added and the mixture was mechanically stirred for 10 min. The biphasic mixture was then centrifuged at 10,000 rpm for 15 min at 4 °C. The lower chloroform phase was collected and the solvent was evaporated under a gentle stream of nitrogen. The solid residue was dissolved in 600 ml of CDCl3 and put in an NMR tube. The scheme of this NMR-based metabolomic approach is shown in Fig. 31.1. The 1H spectrum provides a fingerprint for each honey type showing many characteristic peaks in all spectral regions. Figure 31.2 shows a representative NMR spectrum from a Melipona fuscopilosa honey sample from the Amazon. Generally, the strongest signals in a honey spectrum are in the aliphatic region (0.0–2.5 ppm) while signals of comparable intensities rise in the other regions. All the regions appear very crowded. Specifically, many peaks are present in the 3.0–3.5 ppm region (–CH2OH resonances), in the 4.0–4.5 ppm (–CH2O–CO– signals), in the olefinic proton region (4.5–5.5 ppm), and in the aromatic region (6.5–8.5 ppm); also aldehydic and acidic proton signals are present (9.0–13.0 ppm). The 1D spectra were acquired at 298 K, with a 600 MHz NMR instrument, using a modified double pulsed field gradient spin echoes (DPFGSE) sequence (Rastrelli et al. 2009). The introduction of a p pulse in the DPFGSE sequence allows the removal of the strongest signals present in the 0–2 ppm region, and this results in improved digitization of the weaker peaks, lower integration errors, and eventually, 434 E. Schievano et al. 11 10 9 8 7 6 5 4 3 2 1 Chemical Shift (ppm) Fig. 31.2 Representative 1H NMR spectrum of a M. fuscopilosa honey sample from the Amazon. The extract was dissolved in deuterochloroform and acquired with a 600 MHz NMR instrument better quantification of the number of resonant spins. The spectra collection, processing, and analysis require 30 min. The choice of chloroform as a solvent offers great advantages compared to other solvents previously used in NMR studies of honey. The residual chloroform signal is very sharp, and obscures a very small region at 7.26 ppm, which does not affect the analysis. On the other hand, solvents such as DMSO and MeOH are less suitable since they exhibit large signals in very important areas (around 3.4 ppm for MeOH and around 2.5 ppm for DMSO). Data were processed using the ACD software (ACD/Specmanager 7.00 software, Advanced Chemistry Development Inc., 90 Adelaide Street West, Toronto, Ont., Canada M5H 3V9). Principal component analysis (PCA) and PLS-DA were conducted using the software SIMCA-P11 (Umetrics, Umea Sweden). 31.5 Geographical and Entomological Differentiation of Pot-Honey by NMR The present study was performed on a total of 67 honey samples: 63 were obtained from stingless bees (see Table 31.1), one pot-honey was bought at the Indigenous market of Puerto Ayacucho, Amazonas state, Venezuela, as “erica” honey, one additional pot-honey sample was obtained after sugar feeding M. quadrifasciata bees, São Paulo state, Brazil, and two commercial honeys from Apis mellifera (one from Venezuela and one from Italy). 31 Nuclear Magnetic Resonance as a Method to Predict the Geographical... 435 Table 31.1 Table of stingless bee pot-honey samples tested in this study Common name Stingless bee species Geographical origin Honey samples “carby” Tetragonula carbonaria Australia, Brisbane 1–10 “uruçú” Melipona scutellaris Brazil, João Pessoa 11 “mijui” Scaptotrigona polysticta Brazil, Xingú 12 Melipona sp. Brazil 13 “tiúba” Melipona fasciculata Brazil, Maranhão 14–18 “jandaíra” Melipona subnitida Brazil, Rio Grande 19–22 du Norte “erica” Melipona favosa Venezuela, Falcón 23–30 “isabitto” Melipona aff. Venezuela, Amazon 31–33 fuscopilosaa “ajavitte” Tetragona clavipes Venezuela, Amazon 34–37 “pisilnekmej” Scaptotrigona mexicana Mexico 38–40 “colmena real” Melipona fasciata Mexico 41 guerreroensis “abeja real roja” Melipona fasciata Mexico 42 guerreroensis “criolla” Melipona solani Mexico 43 “abeja bermeja” Scaptotrigona hellwegeri Mexico 44–46 “ala blanca” Frieseomelitta nigra Mexico 47 “abeja real” Melipona beecheii Mexico 48–50 “erereú barcina” Melipona grandis Bolivia, Amborό 51 National Park “erereú choca” Melipona brachychaeta Bolivia, Amborό 52 National Park “obobosí” Scaptotrigona depilis Bolivia, Amborό 53–54 National Park Melipona sp. Bolivia, Amborό 55 National Park “suro choco” Scaptotrigona sp. aff. Bolivia, Amborό 56, 57 xanthotricha National Park “suro negro” Scaptotrigona polysticta Bolivia, Amborό 58, 59 National Park “señorita” Tetragonisca fiebrigi Bolivia, Amborό 60, 61 National Park “obobosí” Scaptotrigona depilis Bolivia, Amborό 62 National Park “abejita” Plebeia sp. Bolivia, Amborό 63 National Park a Melipona aff. fuscopilosa (= Melipona (Michmelia) sp. 1, see table in Pedro chapter 4, this book) The pot-honey samples in Table 31.1 are from different entomological and geographical origins: 10 came from Australia, 12 from Brazil, 15 from Venezuela, 13 from Mexico, and 13 from Bolivia. The principal bees are Melipona, Scaptotrigona, Tetragonisca, Tetragonula, and Frieseomelitta nigra. In the sample data set, the differences due to geographical and entomological origin are not 436 E. Schievano et al. easily separable. In fact, the different species of bees generally live in different ecosystems. This consideration suggests that it might be difficult to discriminate the effects of different geographical origin from bee identify. From the geographical point of view, our data set is composed of five main classes of honeys from five different regions. Furthermore, samples from Venezuela and Brazil can be divided in two other classes of samples collected in different regions of the same nation. Usually, projection methods for classification, such as PLS-DA, are able to produce efficient classification models for not more than four classes of samples. For this reason, we did not consider the entomological origin of our honey samples at first, and PLS-DA models were obtained from groups of honey samples of different geographical origin, compared three at a time. Figure 31.3 shows the PLS-DA score plots (in 3D, corresponding to PC1/PC2/PC3) derived from the NMR spectra of the honey extracts, and they visualize good separations among these extracts (R2x and Q2 value of 0.70 and 0.80 for the “a” plot, 0.63 and 0.56 for the “b” plot, 0.91 and 0.80 for the “c” plot). As a prediction test, we randomly selected two test samples from each region and built the PLS-DA prediction models without them. The approach yielded similar statistical characteristics to those previously obtained using the entire data set and correctly predicted the origins of the ten test samples. These results show that our method could be applicable to discriminate other unknown honey samples on the basis of their geographical origin. If we apply the same PLS-DA calculations to the classification of the different entomological origins, there can be some ambiguity because some bees are found only in a specific geographical zone (e.g., the Tetragonula carbonaria and the Melipona favosa honeys sampled here are found only in Australia and in the Amazon, respectively). When we considered restricted regions, we were able to achieve good discrimination based on the entomological origin. The best results were obtained with the honeys collected in Venezuela (n° 23–37 of Table 31.1). Within these samples, we have honey of the same geographical origin, but of different entomological origin. A PLS-DA model (Fig. 31.4) is able to discriminate T. clavipes (four samples), M. aff. fuscopilosa (three samples), and M. favosa (eight samples). Specifically, samples from the same ecosystem (the Amazon) are very clearly separated in two groups (R2x of 0.88, Q2 of 0.97) corresponding to honey produced by two different bees (M. aff. fuscopilosa and T. clavipes). The honey sample bought at the local indigenous market in Puerto Ayacucho (State Amazonas) as “erica” M. favosa honey was used to test the predictive capability of our model. In Fig. 31.4, PLS-DA assigns it to the Tetragona group, not to M. favosa as claimed. PCA of Mexican honeys (Fig. 31.5a) readily separated the groups of the most numerous samples from Melipona and Scaptotrigona. The remaining samples, produced by different bees, are in different regions of the plot. Pot-honey N° 48 is known to be produced by M. beecheii; however, it is found in a different area, and the most probable reason for that is the presence of a high content of hydroxymeth- 31 Nuclear Magnetic Resonance as a Method to Predict the Geographical... 437 Fig. 31.3 PLS-DA score plots derived from 600 MHz 1H NMR spectra of chloroform honey extracts. (a) PLS-DA on samples from Australia, Brazil, and Venezuela. (b) PLS-DA on samples from Bolivia, Brazil, and Venezuela. (c) PLS-DA on samples from Australia, Brazil, and Mexico. (Filled triangle) Australia, (filled circle) Brazil, (asterisk) Venezuela, (filled diamonds) Bolivia, (open diamonds) Mexico 438 E. Schievano et al. Fig 31.3 (continued) Fig. 31.4 PLS-DA on Venezuelan pot-honey samples. M. fuscopilosa (= Melipona aff. fuscopilosa = Melipona (Michmelia) sp. 1, see table in Pedro chapter 4, this book) 31 Nuclear Magnetic Resonance as a Method to Predict the Geographical... 30 a Mexico 30 Frieseomelitta 20 n°48 0 -10 Melipona -20 t[2] t[2] 10 Brazil 20 n°13 10 n°11 n°12 0 Scaptotrigona -10 M. solani -20 -30 b 439 M. subnitida M. fasciculata -30 -40 -30 -20 -10 0 t[1] R2X[1] = 0.390064 10 20 30 40 R2X[2] = 0.235903 Ellipse: Hotelling T2 (0.95) -60 -50 -40 -30 -20 -10 0 10 20 30 40 50 60 t[1] R2X[1] = 0.52662 R2X[2] = 0.168504 Ellipse: Hotelling T2 (0.95) Fig. 31.5 PC1 and PC2 scores on pot-honey from (a) Mexico (b) Brazil. See honey numbers in Table 31.1 ylfurfural (HMF), which indicates lack of freshness or bad storage conditions and substances from fermentation. The sample produced by M. solani is different from the other Melipona honeys. When a PCA on Brazil samples (n° 11–22 of Table 31.1) was performed (Fig. 31.5b), a clear differentiation, by the first PCA component, was seen between M. fasciculata and M. subnitida. The three samples outside the ellipses originated from different species. Again, samples produced by different bees are in different regions of the plot. Our NMR-based metabolomic approach, even if applied to a limited number of samples, confirmed the validity of the multivariate statistical analysis in discrimination. We developed an efficient tool to differentiate the honeys by their geographical origin; additionally, to highlight the entomological origin, we understood that the field of investigation must be restricted to a smaller geographical region. The following step was the identification of chemical shift resonances indicating specific marker molecules, responsible for the separation of origins. This was achieved by analyzing the loading plots of PCA, which explain the influence of the selected variables on the PCA model. As an example, we show here the assignment of a chemical compound in the Brazilian honeys. The loading plot is shown in Fig. 31.6a (the corresponding score plot is reported in Fig. 31.5b). M. subnitida honeys from Maranhão are characterized by the following NMR resonances: 5.79, 5.96, 5.89, 6.16, 6.29, 6.44, and 2.28 ppm (see continuous line spectra in Fig. 31.6c, in comparison with the dotted line from Rio Grande du Norte honey). Characteristic peaks for M. fasciculata honey resonate at 5.32, 5.36, 4.24, and 4.12 ppm (see dotted line spectra in Fig. 31.6c). In the case of M. subnitida honeys, the resonances were assigned and attributed to the cis and trans isomers of abscisic acid, which is present in large amounts (in comparison with the other compounds) in these samples. Unequivocal structural identification of this marker compound was obtained using homo- and hetero-nuclear 440 E. Schievano et al. Fig. 31.6 Assignment of abscisic acid. (a) Loading plot of PCA on Brazilian honey samples (the corresponding score plot is shown in Fig. 31.5b on the right). (b) Chemical structures of trans and cis abscisic acid. Asterisks and ellipsoids indicate protons and the corresponding resonances. (c) Expanded region of 1H spectra of three samples of M. subnitida (dotted line) and of three samples of M. fasciculata (continuous line) where the same resonances of abscisic acid are found 31 Nuclear Magnetic Resonance as a Method to Predict the Geographical... 441 Fig. 31.7 Comparison of 1H NMR spectra of chloroform extract of bees fed either sucrose syrup or floral resources (a) “sucrose honey” from M. quadrifasciata. (b) Expanded aromatic region of the “sucrose honey” extract. (c) Expanded aromatic region of a floral Scaptotrigona mexicana honey extract correlation 2D-NMR experiments, and MS analysis. In Fig. 31.6b, the molecular structure of abscisic acid is reported, with its resonance assignment. Concerning the Amazon honey samples, PCA led to a clear discrimination of the different bees present in the same ecosystem (M. aff. fuscopilosa and T. clavipes, as evident also in the PLS-DA of Fig. 31.4). According to the loading plot analysis, the discriminating region of the 1H NMR spectrum is between 2.3 and 5.4 ppm. Assuming that in the same territory bees visit the same kind of plants and flowers, these data suggest that signals in this region of the spectrum come from organic compounds secreted by the specific bees. To find the contribution of the bees and cerumen pots on the honey composition, a blank-trial probe was prepared, in which M. quadrifasciata were fed a sucrose syrup, to obtain a “sucrose honey”. The 1H NMR spectrum was acquired (see Fig. 31.7a,b) and compared with a typical pot-honey spectrum (as Scaptotrigona mexicana in Fig. 31.7c). The expanded aromatic region (6–8 ppm) of the sucrose honey (Fig. 31.7b) is poor of signals, in contrast to floral honey (Fig. 31.7c). However, most of the peaks in the aliphatic region (0–5.5 ppm), at frequencies typical of the fatty acid protons, are present in both sugar and floral honeys. It is evident that these aliphatic compounds must be part of the endogenous metabolism of the bee, rather than of the floral, exogenous resources. Therefore, discriminating signals that differentiate bee species are expected in the aliphatic region, whereas the foraging variation was observed in the aromatic region of the spectrum. 442 E. Schievano et al. Fig. 31.8 1H NMR spectra (region 2–5.5 ppm) of the chloroform extract of honey produced by A. mellifera, and stingless bees. M. fuscopilosa (= Melipona aff. fuscopilosa= Melipona (Michmelia) sp. 1, see table in Pedro chapter 4, this book) To substantiate this point, a comparison between the 2 – 5.5 ppm regions of the H NMR spectra of different species of meliponine honeys was conducted and is shown in Fig. 31.8. This region of the spectra shows the typical resonances of the free or bound fatty acids and many other signals of the glycerol esters. In particular, the very high similarity between A. mellifera from Venezuela and from Italy confirms that these resonances are not geographical but entomological markers, clearly characterizing honeys produced by A. mellifera. 1 31 Nuclear Magnetic Resonance as a Method to Predict the Geographical... 31.6 443 Conclusions In this work, 65 honey samples from Meliponini (63 in Table 31.1, one from the local market, one obtained from a sugar fed colony) and two from A. mellifera, were analyzed by our NMR-based metabolomic approach. The 1H NMR spectrum of the chloroform honey extract represents a mixture profile containing both endogenous bee metabolites and exogenous compounds coming from plants and flowers visited by the bees. For this reason, the data set is particularly suitable for a multivariate statistical analysis to distinguish both geographical and entomological origin. Moreover, as a preliminary work, the number of the analyzed samples was sufficient a higher number would allow us to have a test set to perform a prediction analysis to confirm these first results. Using an NMR-based metabolomic approach, we showed that: 1. Considering the entire data set samples, the stingless bee pot-honeys were well differentiated by their geographical origin. 2. The structural identification of abscisic acid, as an example of geographical marker compound for the Brazilian honeys, was achieved using 1D and 2D NMR spectroscopy. 3. If the analysis is restricted to a smaller region, it is possible to group honeys according to their entomological origin, because the entomological discriminant character becomes stronger than the geographical differences. 4. The application of NMR to authenticate the entomological origin of pot-honey (i.e., the market honey sold as “erica” was not a honey produced by M. favosa, but by T. clavipes), is demonstrated for the first time. 5. Sugar-fed M. quadrifasciata produced a honey with an unusual NMR profile, very poor in signals in the aromatic region (6–8 ppm), compared to natural floral honey. Therefore, the specific region in the NMR spectrum responsible for entomological separation seems to be the 0–5 ppm aliphatic region, where the protons from endogenous fatty chains resonate. 6. Discriminating signals to differentiate stingless bee species are expected in the aliphatic region of the NMR spectrum of honey. Acknowledgments See the chapter on anticancer activity by Vit et al. in this book for the pothoney samples received for this study. Prof. Paulo Nogueira-Neto provided the sucrose pot-honey of M. quadrifasciata, from São Paulo, Brazil. The Apis mellifera honeys were provided by Rigoni S.p.A. within the Veneto Region, Italy, UNIMIELE project 2008, and Miel La Encantada, Venezuela. We acknowledge Prof. Vit’s proposal to initiate this research in our lab. References Al ML, Daniel D, Moise A, Bobis O, Laslo L, Bogdanov S. 2009. Physico-chemical and bioactive properties of different floral origin honeys from Romania. Food Chemistry 112(4):863–867. Anklam E. 1998. A review of the analytical methods to determine the geographical and botanical origin of honey. Food Chemistry 63:549–562. 444 E. Schievano et al. Arvanitoyannis I S, Chalhoub C, Gotsiou P, Lydakis-Simantiris N, Kefalas P. 2005. 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WILEY; Guildford, UK. 581 pp. Kang J, Choi MY, Kang S, Kwon HN, Wen H, Lee CH, Park M, Wiklund S, Kim HJ, Kwon SW, Park S. 2008. Application of a 1H nuclear magnetic resonance (NMR) metabolomics approach combined with orthogonal projections to latent structure-discriminant analysis as an efficient tool for discriminating between Korean and Chinese herbal medicines. Journal of Agricultural and Food Chemistry 56:11589–11595. Lindon JC, Nicholson JK, Holmes E, Everett JR. 2000. Metabonomics: metabolic processes studied by NMR spectroscopy of biofluids. Concepts in Magnetic Resonance 12(5):289–320. Lolli M, Bertelli D, Plessi M, Sabatini AG., Restani C. 2008. Classification of Italian honeys by 2D HR-NMR. Journal of Agricultural and Food Chemistry 56:1298–1304. Rastrelli F, Schievano E, Bagno A, Mammi S. 2009. NMR Quantification of trace components in complex matrices by band-selective excitation with adiabatic pulses. Magnetic Resonance in Chemistry 47:868–872. Rochfort S. 2005. Metabolomics reviewed: a new “omics” platform technology for systems biology and implications for natural products research. Journal of Natural Products 68:1813–1820. Schievano E, Pasini G, Cozzi G, Mammi S. 2008. Identification of the production chain of asiago d’allevo cheese by nuclear magnetic resonance spectroscopy and principal component analysis. Journal of Agricultural and Food Chemistry 56:7208–7214. Schievano E, Peggion E, Mammi S. 2010. 1H nuclear magnetic resonance spectra of chloroform extracts of honey for chemometric determination of its botanical origin. Journal of Agricultural and Food Chemistry. 58:57–65. Schievano E, Stocchero M, Morelato E, Facchin C, Mammi S. 2012. An NMR-based metabolomic approach to identify the botanical origin of honey. Metabolomics 8:679–690. Son HS, Kim KM, Van De berg F, Hwang GS, Park WM, Lee CH, Hong YS. 2008. H-1 nuclear magnetic resonance-based metabolomic characterization of wines by grape vaieties and production areas. 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Journal of Agricultural and Food Chemistry 56:10142–10153. Chapter 32 Nonaromatic Organic Acids of Honeys María Teresa Sancho, Inés Mato, José F. Huidobro, Miguel Angel Fernández-Muiño, and Ana Pascual-Maté 32.1 Introduction The composition of stingless bee (Meliponini) honey, also called pot-honey, has been researched since the 1960s (Gonnet et al., 1964 apud Souza et al. 2006). Despite having particular organoleptic properties and being highly appreciated in tropical areas, stingless bee honeys are not commonly available for purchase by consumers in most parts of the world. Stingless bees have been widely studied by several researchers (Wille 1979; Kerr 1987; Camargo and Menezes Pedro 1992, 2007; Roubik 1995; Heard 1999; Michener 2000). As food commodities, some pot-honeys have been described as delicate and with delicious flavors (Kent 1984; van Veen et al. 1990), as well as honeys with sweet and sour flavors (Vit et al. 2010). Many researchers have studied the physical and chemical properties of stingless bee honeys, as reviewed by Souza et al. 2006. With regard to acidity, scientists have reported that in general, pH of these honeys ranges from 2.0 to 4.7, whereas the values of free acid may be close to 200 meq/kg (Souza et al. 2006; Persano Oddo et al. 2008; Sgariglia et al. 2010). Although high values of free acid have been sometimes related to honey fermentation, the high acidity shown by stingless bee honeys has not been characteristically associated with spoilage of this food, and therefore, a high free acid could be a normal parameter of pot-honeys. In fact, several researchers have pointed out that an organic acids profile could be a better parameter than free acidity to determine Apis mellifera honey spoilage (Mato et al. 2006a). M.T. Sancho (*) • M.A. Fernández-Muiño • A. Pascual-Maté Department of Biotechnology and Food Science, Faculty of Sciences, University of Burgos, Burgos, Spain e-mail: mtsancho@ubu.es I. Mato • J.F. Huidobro Department of Analytical Chemistry, Nutrition and Food Science, Faculty of Pharmacy, University of Santiago de Compostela,Santiago de Compostela, Spain 447 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_32, © Springer Science+Business Media New York 2013 448 M.T. Sancho et al. Stingless bee honeys are included neither in the revised codex standard for honey (CODEX 2001) nor in the European council directive 2001/110/EC relating to honey (OJEC 2002). Current studies on this food are needed because these standards would provide the consumers with a guarantee of food safety and food control by responsible laboratories. This chapter reviews the importance and methods of analysis of nonaromatic organic acids of honey, based mainly on data obtained for Apis mellifera, compared to Tetragonula carbonaria and Melipona favosa, as well as its relationship to other parameters of this food. 32.2 Importance of Nonaromatic Organic Acids in Honey More than 30 different nonaromatic organic acids have been identified in honey (Mato et al. 2003), most of them added by bees (Echigo and Takenaka 1974). Along with the concentration of sugars and hydrogen peroxide, nonaromatic organic acids are responsible for the excellent resistance of honey against microbial spoilage (White 1979a). Gluconic acid is the predominant nonaromatic organic acid in honey (Stinson et al. 1960), instead of malic or citric acids as previously thought (Nelson and Mottern 1931). Gluconic acid in equilibrium with gluconolactone is present in all honeys, in concentrations much higher than others (White 1978). Besides gluconic acid, other nonaromatic organic acids commonly present in honey are malic, citric, lactic, succinic, fumaric, maleic, formic, acetic, oxalic, and pyruvic, among others (Mato et al. 2003). Malic acid was one of the first acids identified in honey (Hilger 1904) and has been usually considered the second in importance after gluconic acid (Cherchi et al. 1994). Citric acid is a tricarboxylic acid, and the relationship between the acid forms and salt depends on honey pH, total citric acid content, and citric acid dissociation constants (Mato et al. 2000). The content of citric acid has been considered potentially useful to differentiate between nectar and honeydew honeys (Talpay 1988). Honey gluconic acid comes mainly from the action of bee glucose-oxidase on nectar or honeydew glucose. Part of this acid is also produced by Gluconobacter spp., bacteria that are common in a bee’s gut and stay throughout the ripening of honey. In aerobic environments with high glucose concentrations, Gluconobacter spp. microorganisms produce large amounts of gluconic acid (Ruiz-Argüeso and Rodríguez-Navarro 1973). The variation in the amounts of gluconic acid depends on the time required to completely transform the nectar or honeydew into honey; the longer it is, the greater the addition of glucose oxidase by the bee, and the greater therefore the amount of gluconic acid. Other factors that also influence the process are the strength of the colony and the quality and quantity of nectar coming into the hive (White 1979b). The origin of the other nonaromatic organic acids in honey is not fully known. They may come directly from nectar or honeydew, and some of them are produced from nectar and honeydew sugars by the action of enzymes secreted by worker bees and added to honey at ripening (Echigo and Takenaka 1974). Many honey nonaromatic organic acids are intermediates of such enzymatic 32 Nonaromatic Organic Acids of Honeys 449 pathways as Krebs cycle and others, being oxidized throughout the mentioned pathways (Echigo and Takenaka 1974; White 1979b; FAO 1990). Honey organic acids have been proposed as potentially useful to characterize the botanical and geographical origin of honeys (Steeg and Montag 1988; Talpay 1989; Cherchi et al. 1994; Anklam 1998; Del Nozal et al. 1998; Mato 2004; Kaskoniene and Venskutonis 2010). 2-Methoxybutanedioic and 4-hydroxy-3-methyl-trans-2pentenedioic acids were described as possible markers of Knightia excelsa (Proteaceae) honeys (Wilkins et al. 1995). In Erica sp. (Ericaceae) honeys, cis,trans-abscisic acid and trans,trans-abscisic acid (Ferreres et al. 1996), as well as high concentrations of quinic acid (Del Nozal et al. 1998), were found as possible markers, being the concentrations of cis,trans-abscisic acid about ten times higher than those found in honeys of other botanical origins (Gheldof et al. 2002). Low concentrations of pyruvic acid and high quantities of both malic and succinic acid were typical of Quercus sp. (Fagaceae) honeys, whereas high citric acid concentrations were described as a possible marker of Thymus sp. (Lamiaceae) honeys (Del Nozal et al. 1998). In Castanea sativa (Fagaceae) honey, high levels of formic acid were found, contrary to the low levels of formic acid described in Eucalyptus spp. (Myrtaceae) honey (Suárez-Luque et al. 2006). Acetic acid has been proposed as possible indicator of honey fermentation, when its levels are excessively high (Mato et al. 2003). Such osmophilic yeasts as Saccharomyces spp., Zygosaccharomyces spp., Torula spp. and others, produce alcohols and eventually organic acids from honey sugars (Gonnet 1982). These yeasts come from flowers, soil, air, or the equipment used for honey extraction and processing, and are very sensitive to heat, so many companies pasteurize their honeys in order to prevent fermentation (Piana et al. 1989). For unpasteurized honeys, the possible usefulness of nonaromatic organic acid profile as a fermentation indicator should be researched (Mato et al. 2003). Among other parameters such as phenolics, peptides, aminoacids, Maillard reaction products and enzymes, and nonaromatic organic acids, also contribute to antioxidant capacity observed in honeys (Gheldof et al. 2002). Such honey organic acids as citric, malic, and others act as metal ion chelators, and are considered as synergists of primary antioxidants enhancing antioxidant activity (Gheldof et al. 2002; Wanasundara and Shahidi 2005). There is evidence that some acidic components of honey show antibacterial activity (Russel et al. 1988; Wahdan 1998). Acidic substances identified to date as antibacterial in honeys are mainly aromatic organic acids; such as ferulic and caffeic acids (Wahdan 1998), benzoic acid derivatives (Russel et al. 1988; Weston et al. 1999), and acids of royal jelly (Isidorov et al. 2011). Possible relationships between honey acidity and antibacterial activity have been studied, as well as between honey pH and antibacterial activity (Yatsunami and Echigo 1984; Bogdanov 1997). Honey antibacterial activity was significantly correlated with free acid and total acidity, showing the acidic fraction of several honeys with the greatest non-peroxide antibacterial activity (Bogdanov 1997; Kirnpaul-Kaur et al. 2011). In an acidic medium, honeys show better antibacterial activity (Bogdanov 2011). Stingless bee honeys have been used in traditional and Mesoamerican aboriginal medicine (Vit and Tomás-Barberán 2004; Vit et al. 2004; Sgariglia et al. 2010). 450 M.T. Sancho et al. Pot-honeys show high free acid values, and antibacterial activity is found in them by many scientists (DeMera and Angert 2004; Dardon and Enríquez 2008; Irish et al. 2008; de Almeida et al. 2009; Rodríguez-Malaver et al. 2009; Vit et al. 2009a; Boorn et al. 2010; Sgariglia et al. 2010). Therefore, it would be very interesting to study antibacterial activity of stingless bee honeys in relation with their levels of organic acids. 32.3 Honey Components and Parameters Related to Nonaromatic Organic Acids Honey contains less than 0.5% of organic acids. Nevertheless, they are a group of constituents that contribute to several properties of this food, such as its color, aroma, taste, pH, acidity, and, to a lesser extent, electrical conductivity. Color is an optical property of honey, described as the result of different degrees of absorption of light at different wavelengths by honey compounds (FAO 1990). The color of honey varies widely, from nearly colorless to almost black. This variability depends heavily on its origin and thus on its composition. Dark honeys tend to have higher acidity and higher organic acids contents (White 1979b; Crane 1990) than light honeys. Aroma and flavor of honey are mainly due to a complex mixture of substances that are highly dependent on the botanical origin, but also influence the processing and storage conditions of this food (Anklam 1998). Among these substances organic acids are important, in particular for the taste of honey (Louveaux 1985; Crane 1990; Bogdanov 2009). Honey acidity depends mainly on the presence of organic acids (White 1979b). Lactones are internal esters of organic acids and do not contribute to honeys’ active acidity (Bogdanov 2009). Lactones hydrolyze over time, therefore increasing honey free acid. Total acidity is the sum of free acid and lactones. Honey pH depends on the amount of ionized acids, as well as the content in such minerals as potassium, sodium and calcium (White 1979b). Small oscillations in the range of pH in relation to the large swings in the free acid values were attributed to the buffer properties of honey, due to such mineral salts as phosphates, carbonates and others (Bogdanov 2009). Electrical conductivity is a physical property of honey mainly related to the content of mineral salts, and to a lesser extent to the content of organic acids, proteins, sugars, and polyols (Crane 1990). It was found that the electrical conductivity was directly proportional to ash content and acidity of honey (Vorwohl 1964). 32.4 Methods of Analysis of Nonaromatic Organic Acids in Honey The most important and frequently employed methods to determine honeys’ nonaromatic organic acids are enzymatic assays, chromatographic techniques, and electrophoretic procedures (Mato et al. 2006b). Enzymatic assays are based on spectrophotometric 32 Nonaromatic Organic Acids of Honeys 451 measurements, usually at 340 nm, of the increase or decrease in absorbance of the reduced form’s coenzymes nicotinamide adenine dicucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH), after the reaction of organic acids with specific enzymes. Enzymatic methods are precise and accurate. In addition, their specificity is excellent, allowing quantification of the d/l isomers of several organic acids. Furthermore, enzymatic procedures require very simple equipment, normally available in every quality control laboratory. Unfortunately, the stability of the enzymatic kits is not very long, and enzymatic procedures are tedious and timeconsuming, allowing the determination of only one organic acid each time. Enzymatic analyses were commonly used to determine nonaromatic organic acids in Apis mellifera honeys (Tourn et al. 1980; Stoya et al. 1986, 1987; Hansen and Guldborg 1988; Talpay 1988, 1989; Sabatini et al. 1994; Mato et al. 1997, 1998a, b; Mutinelli et al. 1997; Cossu and Alamanni 1999; Alamanni et al. 2000; Bogdanov et al. 2002; Gheldof et al. 2002; Pulcini et al. 2004; and Vit et al. 2009a, b, among others). In respect of honeys produced by stingless bees, total d-gluconic, citric, and l-malic acids were quantified enzymatically in honeys from Australian Tetragonula carbonaria (Persano Oddo et al. 2008) and Venezuelan Melipona favosa. Organic acids of honeys have been widely determined by chromatographic techniques. At first, these compounds were analyzed by paper and on-column ion exchange chromatography (Stinson et al. 1960). Gas chromatography–mass spectrometry (GCMS) and gas chromatography–flame ionization detector (GC-FID) were applied to analyze honey nonaromatic organic acids with a previous derivatization process, due to the fact that most of these acids are not volatile (Echigo and Takenaka 1974; Wilkins et al. 1995; Horváth and Molnár-Perl 1998; Pilz-Güther and Speer 2004; Sanz et al. 2005), albeit recently, 29 organic acids were analyzed by GC-MS in honeys and other food commodities, using a procedure based on continuous solid-phase extraction without prior derivatization (Jurado-Sánchez et al. 2011). Many researchers analyzed honey nonaromatic organic acids by high-performance liquid chromatography with ultraviolet detection (Cherchi et al. 1994, 1995; del Nozal et al. 1998, 2003a, b; Alamanni et al. 2000; Suárez-Luque et al. 2002a, b; SerraBonvehí et al. 2004; Hrobonová et al. 2007), although ionic chromatography with conductivity detection was also used to determine some nonaromatic organic acids in honeys (Pérez-Cerrada et al. 1989; Defilippi et al. 1995; del Nozal et al. 2000), as well as anionic exchange chromatography with UV detection (del Nozal et al. 1998) or constant voltage amperometric detection (Casella and Gatta 2001). Liquid chromatographic methods allow the simultaneous determination of several organic acids, showing a good versatility, reproducibility, and sensitivity. However, there are many interferences that must be removed by pretreatment of honey samples, or by using several columns in series, thus liquid chromatographic methods to determine honey nonaromatic organic acids are tedious and time-consuming. Capillary electrophoresis with ultraviolet detection is another method that was successfully employed to quantify nonaromatic organic acids in honeys (Boden et al. 2000; Navarrete et al. 2005; Mato et al. 2006a; Suárez-Luque et al. 2006). Capillary electrophoresis is a rapid and low cost procedure that allows the simultaneous determination of several nonaromatic organic acids with a very simple preparation of the honey sample. The drawbacks of this method, if compared with 452 M.T. Sancho et al. other procedures, are its lower reproducibility and sensitivity. Nevertheless, capillary electrophoresis is a very promising technique that should be intensively studied for future analysis of honey compounds. Its application to analyze nonaromatic organic acids of pot-honeys could contribute to their characterization, which would be very interesting to promote and improve the commercialization of stingless bee honeys. 32.5 Nonaromatic Organic Acids in Pot-Honey The content of d-gluconic, l-malic, and total citric acids was analyzed in eight samples of pot-honey produced by Tetragonula carbonaria, (Persano Oddo et al. 2008, as Trigona carbonaria, but see Rasmussen and Cameron 2007), and seven samples of Melipona favosa from Venezuela (Fig. 32.1). In all these pot-honeys, the quantities of l-malic and total citric acids were in general similar to those of Apis mellifera honeys described in the literature. As usual, d-gluconic acid values were one thousand times higher than l-malic and total citric acid concentrations. The quantities of d-gluconic acid in Trigona carbonaria honeys were in the same range of levels of d-gluconic acid of Castanea sp., Thymus sp., Arbutus sp. and honeydew honeys from Apis mellifera (Pulcini et al. 2004). The values of d-gluconic acid were about ten times higher in Melipona favosa samples (Fig. 32.1a), which might be indicative of a very high glucose oxidase activity at ripening (Persano Oddo et al. 2008), and could contribute to characterize Melipona favosa pot-honeys. Conversely, the concentrations of both l-malic and total citric acid were about ten times lower in honeys from Melipona favosa than in samples from Trigona carbonaria (Fig. 32.1b, c). It is interesting to highlight the fact that the Melipona favosa honey (sample 2) with the highest quantities of both l-malic and citric acid was the sample with the lowest concentration of d-gluconic acid. In contrast, the Melipona favosa sample with the lowest value of citric acid was the sample with the highest quantity of d-gluconic acid. In pot-honey from Trigona carbonaria it was observed that, in general, samples with the highest contents of d-gluconic acid contained the lowest quantities of total citric acid and vice versa. Most studies of pot-honey characterized the honey produced by different bee species of stingless bees (Vit et al. 1994; Souza et al. 2006; Persano Oddo et al. 2008; Sgariglia et al. 2010). It should be very interesting to research the nonaromatic organic acid profiles of these honeys, of particular interest the possible identification of the acid(s) responsible for the high free acid of pot-honey. Acknowledgments To Dr. Tim Heard from CSIRO, Brisbane Australia for providing the Tetragonula carbonaria honey, and for English proof reading of the manuscript. To Prof. Patricia Vit from the Food Science Department, Faculty of Pharmacy and Bioanalysis, Universidad de Los Andes, Mérida, Venezuela, for providing the samples of Melipona favosa honey, and editorial care. To Prof. João MF Camargo from the Biology Department, Universidade de São Paulo, Ribeirão Preto, Brazil, for the identification of the Melipona favosa bee. 32 453 Nonaromatic Organic Acids of Honeys Stingless bee species Trigona carbonaria Melipona favosa Non-aromatic organic acids (g/kg) average ± SD (min – max) a D-gluconic acid D-gluconic acid (g/kg) 80,00 70,00 60,00 40,00 63.6 ± 22.8 (14.6 – 79.9) 30,00 20,00 10,00 0,00 b 9.9 ± 1.3 (7.7 – 11.8) 50,00 0 1 2 3 4 5 Pot-honeys 6 7 8 Citric acid 0,40 Citric acid (g/kg) 0,35 0,30 0.23 ± 0.09 (0.11 – 0.36) 0,25 0,20 0.05 ± 0.05 (0.01 – 0.15) 0,15 0,10 0,05 0,00 0 1 2 3 4 5 Pot-honeys 6 7 8 c L-malic acid L-malic acid (g/kg) 0,25 0,20 0.12 ± 0.05 (0.04 – 0.20) 0,15 0.03 ± 0.01 (0.01 – 0.04) 0,10 0,05 0,00 0 1 2 3 4 5 Pot-honeys 6 7 8 Fig. 32.1 Content of nonaromatic organic acids in pot-honey. 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The Neotropical diversity of stingless bees, some 400 species reported by Camargo and Pedro (2007), is a challenge for any phytochemical investigation considering bee–plant interaction. The sugar and water acidic matrix of honey has a set of minor components used as quality indicators, such as hydroxymethylfurfural and diastase activity (Bogdanov 1999). All the natural products and minerals of nectar and plant exudates used for honey-making are concentrated in honey as such or transformed by the bees and associated microflora. Flavonoids are plant secondary metabolites that are associated with different physiological and ecological functions, such as protection of plant epithelial cells from ultraviolet rays, defense against biotic and abiotic stress, plant pigmentation, and signaling for interaction with animals, including bees, microbes, and other plants (Harborne 1982). Flavonoids from floral nectar, pollen (Tomás-Barberán et al. 1989), and different plant exudates (Tomás-Barberán et al. 1993a) are incorporated into honey by the bees, and the metabolites present in plants can be modified during the honey elaboration process, mainly by the action of bee enzymes, bee microbiota metabolism, and chemical transformations during honey maturation. Honey flavonoid profiles help to determine botanical (Ferreres et al. 1992, 1993, 1994, 1996b; Soler et al. 1995; Martos et al. 2000) and geographical F.A. Tomás-Barberán (*) • P. Truchado • F. Ferreres Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), Campus Espinardo, PO Box 164, 30100 Murcia, Spain e-mail: fatomas@cebas.csic.es 461 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_33, © Springer Science+Business Media New York 2013 462 F.A. Tomás-Barberán et al. (Tomás-Barberán et al. 1993b) origins of honey. It seems clear that honey contains complex phenolic and flavonoid profiles that could be associated with floral and geographical origin, although it is rather difficult to establish valid floral origin biomarkers, specific for a given plant. The study of potential phenolic fingerprints would be a very appropriate methodology for this purpose (Tomás-Barberán et al. 2001). Changes occur in the flavonoid profile with honey maturation in the bee nest and provide a method for evaluation of the degree of honey ripening (Truchado et al. 2010). The flavonoid content in Apis mellifera honey has been extensively studied (Frankel et al. 1998; Ferreres et al. 1996a; Martos et al. 1997). The content in stingless-bee honey, however, has only been recently reported for samples from Australia (Persano Oddo et al. 2008) and Venezuela (Truchado et al. 2011), although previous qualitative studies exist (Vit et al. 1997; Vit and Tomás-Barberán 1998). Recent research correlates flavonoid content (measured by a spectrophotometric method) to the antioxidant activity of honey produced by several species of stingless bees (Rodríguez-Malaver et al. 2007, 2009; Persano Oddo et al. 2008; Duarte et al. 2012). In this chapter the flavonoids of stingless-bee honey are reviewed, including new data presented here, from several countries. 33.2 Methods of Extraction and Analysis of Flavonoids in Honey In the analysis of flavonoids from honey, a major problem is the extraction of these minor compounds from a matrix very rich in polar compounds (sugars). This problem is successfully solved by filtration of the diluted honey in acidified water, through nonionic polymeric resins such as Amberlite XAD (Ferreres et al. 1991). This methodology is combined with a final liquid–liquid extraction in which the flavonoids are extracted from water with dyethyl ether. The extraction renders flavonoid extracts that contain most flavonoid aglycones present in Apis mellifera honey—the main flavonoids present. Recent studies reveal that some unifloral honey, e.g., Robinia pseudoacacia (Fabaceae, Papilionoideae), contains mainly flavonoid glycosides, considered an uncommon honey trait (Truchado et al. 2008). For its analysis, extraction using solid phase extraction cartridges, in combination with HPLC-MS analyses, is considered very useful. In fact, in a more recent paper, the widespread occurrence of flavonoid glycosides in A. mellifera honey from different floral origins is demonstrated (Truchado et al. 2009b) although in most cases, flavonoid aglycones are the main metabolites. For stingless-bee honey, since this type of honey contains glycosides in a higher proportion than aglycones (Vit et al. 1997), the same extraction methodology was applied to a number of samples collected in South America and Australia. 33 Flavonoids in Stingless-Bee and Honey-Bee Honeys 463 The methodology used was the following. Flavonoid compounds from honey samples (5 g) were isolated with a Sep-Pak solid phase extraction cartridge (reversed phase C18 cartridge). The samples were diluted with ultrapure water and centrifuged at 9,000 × g for 10 min. The supernatants were filtered through a cartridge previously activated with methanol (10 mL) followed by water (10 mL). Following this, the phytochemicals that remained adsorbed in the cartridge were eluted with 1 mL methanol. The methanol fractions were filtered through a 0.45 mm membrane filter and stored at −20°C until further analyzed by HPLC-DAD-MSn/ESI (Truchado et al. 2011). 33.3 Analysis of Honey Flavonoids Using Advanced HPLC-MS Methods Analysis of honey flavonoid glycosides and aglycones was carried out in an Agilent HPLC 1100 series equipped with a diode array detector and mass detector in series (Agilent Technologies, Waldbronn, Germany). The HPLC consisted of a binary pump (model G1312A), an autosampler (model G1313A), a degasser (model G1322A), and a photodiode array detector (model G1315B). The HPLC system was controlled by ChemStation software (Agilent, v. 08.03). The mass detector was an ion trap spectrometer (model G2445A) equipped with an electrospray ionization interface, controlled by LCMSD software (Agilent, v. 4.1). The ionization conditions were adjusted to 350°C and 4 kV for capillary temperature and voltage, respectively. The nebulizer pressure and flow rate of nitrogen were 65.0 psi and 11 L/min, respectively. The full scan mass covered the range from m/z 100 up to m/z 2,000. Collision-induced fragmentation experiments were performed in the ion trap using helium as the collision gas, with voltage camping cycles from 0.3 to 2.0 V. Mass spectrometry data were acquired in the negative ionization mode. MSn was carried out in the automatic mode on the more abundant fragment ion in MS(n−1). Chromatographic analyses were carried out on a LiChroCART column (250mm × 4 mm, RP-18, 5 mm particle size, LiChrospher®100 stationary phase, Merck, Darmstadt, Germany) protected with a LiChroCART guard column (4 mm × 4 mm, RP-18, 5 mm particle size, Merck, Darmstadt, Germany). The mobile phase consisted of two solvents: water–formic acid (1%) (A) and methanol (B) (99.9%, HPLC grade; Merck, Darmstadt, Germany), starting with 10% B and using a linear gradient to obtain 30% at 20 min, 60% at 40 min, 70% at 45 min, and 90% at 60 min. The flow rate was 1 mL/min, and the injection volume 20 mL. Spectral data from all peaks were accumulated in the range of 240–600 nm, and chromatograms were recorded at 280, 320, 330, 360, or 520 nm. The phenolic compounds were identified according to their UV spectra, molecular weights, retention times, and their MS–MS fragments, and whenever possible, with commercially available standards. 464 F.A. Tomás-Barberán et al. 33.4 Flavonoids Observed in Honey from Combs and Pots 33.4.1 Apis mellifera Comb Honey This type of honey contains flavonoid aglycones and other lipophylic compounds as the main plant secondary metabolites. Some honey samples of specific floral origin contain metabolites that may be considered biomarkers of the particular plant, as is the case of the flavanone hesperetin for citrus honey (Ferreres et al. 1993) and the alkaloid kinurenic acid for chestnut honey (Truchado et al. 2009a). Other honey samples contain specific compounds that are common to a number of different plant species, as in the case of the flavone tricetin and the flavonol myricetin in eucalyptus honey (Martos et al. 2000) and ellagic acid and abscisic acid in heather honey (Ferreres et al. 1996a). Some A. mellifera honey contains relatively high amounts of flavonoid aglycones from propolis (poplar bud exudates collected by bees) (Fig. 33.1) including the flavones chrysin, galangin and techtochrysin, the flavanones pinocembrin and pinobaknsin and the caffeic acid derivatives dimethyl-allyl-caffeate and phenylethyl-caffeate. Some of these compounds have also been reported in beeswax and in freshly secreted wax scales. It is suggested that bees may ingest propolis to incorporate these flavonoid metabolites in the secreted wax (Tomás-Barberán et al. 1993c). HO O OH HO OH O OH pinocembrin HO O O O pinobanksin HO O H3CO O OH OH O chrysin phenyl-ethyl-caffeate OH galangin O OH O 7-methyl-chrysin dimethyl-allyl-caffeate Fig. 33.1 Propolis-derived flavonoids and other phenolic compounds from Apis mellifera honey 33 Flavonoids in Stingless-Bee and Honey-Bee Honeys 465 Fig. 33.2 Nectar and pollen derived flavonoid aglycones in honey and pot-honey In addition, A. mellifera honey contains a large number of flavonoid aglycones derived from the naturally occurring flavonol-glycosides present in nectar, and probably pollen, from hydrolysis caused by bee saliva enzymes. These flavonoid aglycones include mainly polyhydroxylated flavones, but also their mono methyl ethers (i.e., isorhamnetin and 8-methoxykaempferol) and flavanones like hesperetin (Fig. 33.2). A good example to illustrate hydrolytic activity of bee saliva is found in eucalyptus nectar and honey which clearly shows the presence of flavonol glucosides and diglucosides in nectar, and the transformation of these polar metabolites into the corresponding aglycones in mature honey (Fig. 33.3) (Truchado et al. 2009b). When flavonoid rhamnosides or rhamnosyl-glucosides are present in nectar, those glycosides are not hydrolyzed by bee enzymes, as the bee does not have rhamnosidases in its saliva, and therefore the natural plant nectar glycosides are found in mature honey (Fig. 33.4). This occurs with Robinia pseudacacia honey, reported to contain mainly nectar flavonoid glycosides that bees cannot hydrolyze (Truchado et al. 2008). When the transformation of nectar flavonoid glycosides is followed during the maturation of nectar in the comb to produce mature honey, the original flavonoid glycosides that are present in freshly deposited nectar are hydrolyzed sequentially, This process releases the aglycones found in mature honey, as demonstrated in Diplotaxis tenuifolia (Brassicaceae) honey (Truchado et al. 2010) (Fig. 33.5). 466 F.A. Tomás-Barberán et al. a 25 36 20 15 Tc 10 17 37 5 40 38 27 39 My 39 Lt 0 0 10 20 30 40 50 Retention time (min) b 300 mAU Tc Q 200 Lt 100 Pc Ch Pb My G Tch Kf 0 Is 10 20 30 40 Retention time (min) 50 Fig. 33.3 Nectar (a) and honey (b) flavonoid profiles of Apis mellifera Eucalyptus honey. For flavonoid identification see Table 33.1 It can be concluded that, as a general rule, mature A. mellifera honey contains a larger amount of flavonoid aglycones than glycosides, although some specific honeys maintain large fractions of the original flavonoid glycosides, particularly when rhamnosides are present. 467 33 Flavonoids in Stingless-Bee and Honey-Bee Honeys OH OH HO OH O HO O Glucose O Rhamnose O Glucose OH O quercetin 3-O-rutinoside kaempferol 3-O-rutinoside OCH3 OH OH Rhamnose Rhamnose OH O O HO O O O Glucose OH Rhamnose OH O OH OH isorhamnetin 3-O-rutinoside kaempferol 7-O-rhamnoside Fig. 33.4 Apis mellifera honey representative flavonoid glycosides Intens. [mAU] 100 MDIPLO 12.D: UV Chromatogram, 330 nm Diplotaxis tenuifolia nectar 11 80 60 2+32 40 33 Ch Kf Is 29+30+31 20 0 Intens. [mAU] 125 34 35 Pb+Qc Pc+Pt DAC P_G00014.D: UV Chromatogram, 330 nm Diplotaxis tenuifolia honey Kf 33 100 Is 75 Ch 50 Pb 25 Pc+Pt Qc DAC 0 0 5 10 15 20 25 30 35 40 Time [min] Fig. 33.5 HPLC/DAD (330 nm) phenolic profile of Diplotaxis tenuifolia honey from Argentina. The chromatogram from nectar is immature honey. For compound identification see Table 33.1 33.4.2 Stingless-Bee Pot-Honey Pot-honey is generally characterized by a higher content of flavonoid glycosides than A. mellifera honey. This characteristic difference might be explained by the very low diastase activity of stingless bees compared to Apis (Persano Oddo et al. 2008). Recent studies report the occurrence of flavone di-C-glycosides and flavonoid O-glycosides in stingless-bee honey (Truchado et al. 2011) (Fig. 33.6). 468 F.A. Tomás-Barberán et al. Fig. 33.6 Stingless-bee honey representative flavonoid glycosides A collection of eight Tetragonula carbonaria honey samples collected from nests in various locations around Brisbane (Queensland, Australia), in suburban areas where the flora was composed mainly of ornamental shrubs and flowering trees (Persano Oddo et al. 2008) was studied to evaluate the content of flavonoid compounds. This screening showed a similar chromatographic profile for all samples (Fig. 33.7a), in which flavonoid aglycones [tricetin (Tc), pinobanksin (Pb), luteolin (Lt), kaempferol (Kf), apigenin (Ap), isorhamnetin (Is), and pinocembrin (Pc)], were identified together with large number of flavonoid glycosides derived from quercetin, kaempferol, and isorhamnetin and a possible tetrahydroxydihydroflavone (H). Six flavonoid triglycosides, namely, one flavonoid trihexoside (1), two compounds with a −3-O-(2hexosyl, 6-rhamnosyl)hexoside substitution (3, 9), another two with a −3-O-(2,6-dirhamnosyl)hexoside substitution (5, 14), and another compound isomeric of 3 and 9 with a tentative −3-O-(2-hexosyl, 3-rhamnosyl)hexoside substitution (7), were detected. In the same way several flavonoid diglycosides derived from the triglycosides mentioned above and with −3-O-(2-hexosyl)hexoside (2, 4, 11), −3-O-(2rhamnosyl)hexoside (6, 15, 16) (Fig. 33.7a), and −3-O-(6-rhamnosyl)hexoside (17) substitutions were, as well as two −3-O-(2-pentosyl)hexosides (10, 13) and one tentative −3-O-(3-pentosyl)hexoside (18), detected (Table 33.1). 469 33 Flavonoids in Stingless-Bee and Honey-Bee Honeys Intens. [mAU] a 31 AU.D: UV Chromatogram, 320 nm 60 8+9+10 6+7 40 2 4 3 5 1 20 12 + 13 Pb Tc Lt 17+18 Kf Ap EA Pc Is 10 Intens. [mAU] 15+16 11 14 15 20 25 30 35 40 b 45 Time [min] V16.D: UV Chromatogram, 320 nm 23 50 26 24 40 14+15+16 30 21+22 20 19 25 20 17 5 10 0 27 + EA 28 Tc Qc Pb 10 15 20 25 30 35 Kf 40 45 Time [min] Fig. 33.7 HPLC/DAD (320 nm) phenolic profile of stingless-bee honeys (a) Tetragonula carbonaria honey from Australia, (b) Melipona favosa honey from Venezuela. For compound identification see Table 33.1 In the same way, 12 stingless-bee (Melipona favosa) honey samples from Venezuela collected in the arid climate area of Moruy were analyzed. The vegetation of this area was rich in Cactaceae and Mimosaceae species (Truchado et al. 2011) and all of them showed a similar chromatographic profile (Fig. 33.7b). The samples were characterized by the occurrence of five flavonoid di-C-glycosides: three apigenin 6,8-di-C-hexoside isomers (19, 20, 21), apigenin 6-C-pentoside-8-Chexoside (23), and apigenin 6-C-hexoside-8-C-pentoside. Compounds with this C-glycosylation type had not been reported in honey (Truchado et al. 2011). In addition, these honey samples contained flavonol 3-O-glycosides, similar or identical to those reported from Australian stingless-bee honey described above. Compounds 5 and 14 and kaempferol 3-O-(2,6-di-rhamnosyl)hexoside (26) with a similar glycosylation to that of compound 5, the diglycosides 15 and 16, and the 3-O-(6rhamnosyl)hexoside derivatives 17, 27, and 28, in which only the aglycone was different, were detected and quantified. In addition, some propolis-derived aglycones, ellagic acid (EA), a flavonoid tetraglycoside [kaempferol 3-O-(2-hexosyl) rhamnosyl, 6-rhamnosyl)hexoside] (25), and a pentahydroxy-dihydroflavone, most likely dihydroquercetin (22), were detected (Fig. 33.7b). Several stingless-bee honeys from Bolivia were also studied [“erereú choca” Melipona brachychaeta Moure, 1950; “erereú barcina” Melipona grandis Guérin, 1834; “obobosí” Scaptotrigona depilis (Moure, 1942); “suro negro” Scaptotrigona polysticta Moure, 1950; “suro choco” Scaptotrigona sp., aff. xanthotricha Moure, 1950; “señorita” Tetragonisca fiebrigi (Schwarz, 1938)] from Parque Nacional Amboró at different geographical areas with different vegetation. Only one honey 470 F.A. Tomás-Barberán et al. Table 33.1 Flavonoids from nectar and honey samples from Tetragonula carbonaria (T), Melipona favosa (M), Apis mellifera (Diplotaxis tenuifolium) (D) and Apis mellifera (Eucalyptus globulus) (E) No. Compound T 1 Quercetin-O-trihexoside 2 Quercetin-3-O-sophorosidea 3 Isorhamnetin-3-O-(2-hexosyl, 6-rhamnosyl)hexosidea 4 Isorhamnetin-3-O-sophorosidea 5 Quercetin-3-O-(2,6-di-rhamnosyl)hexosidea 6 Quercetin-3-O-(2-rhamnosyl)hexosidea 7 Isorhamnetin-3-O-(2-hexosyl, 3-rhamnosyl)hexosidea 8 Tetrahydroxydihydroflavoneb 9 Kaempferol-3-O-(2-hexosyl, 6-rhamnosyl)hexosidea 10 Quercetin-3-O-(2-pentosyl)hexosidea 11 Kaempferol-3-O-sophorosidea 12 Isorhamnetin-3-O-(hexosyl)hexosideisomera 13 Kaempferol-3-O-(2-pentosyl)hexosidea 14 Isorhamnetin-3-O-(2,6-di-rhamnosyl)hexosidea 15 Kaempferol-3-O-(2-rhamnosyl)hexosidea 16 Isorhamnetin-3-O-(2-rhamnosyl)hexosidea 17 Quercetin-3-O-(6-rhamnosyl)hexosidea 18 Quercetin-3-O-hexosidea 19 Apigenin-6,8-di-C-hexosidec 20 Apigenin-6,8-di-C-hexoside isomerc 21 Apigenin-6,8-di-C-hexoside isomerc 22 Dihydroquercetinb 23 Apigenin-6-C-pentoside-8-C-hexosidec 24 Apigenin-6-C-hexoside-8-C-pentosidec 25 Kaempferol-3-O-(2-hexosyl)rhamnosyl, 6-rhamnosyl)hexosidea 26 Kaempferol 3-O-(2,6-di-rhamnosyl)hexosidea 27 Kaempferol-3-O-(6-rhamnosyl)hexosidea 28 Isorhamnetin-3-O-(6-rhamnosyl)hexosidea 29 Quercetin-3,3¢,4¢-O-triglucosidea 30 Isorhamnetin-3-O-glucoside-4¢-O-gentiobiosidea 31 Quercetin-3,4¢-O-diglucosidea 32 Kaempferol-3-O-diglucoside isomera 33 Isorhamnetin 4¢-O-gentiobiosidea 34 Isorhamnetin 4¢-O-glucosidea 35 Kaempferol-4¢-O-glucosidea 36 Tricetin 7-O-sophoroside (diglucoside)a 37 Tricetin 7,4¢-di-O-glucosidea 38 Quercetin 3-O-glucuronidea 39 Myricetin 3,7-di-O-glucosidea 40 Myricetin 3-O-sophoroside (diglucoside)a EA Ellagic acidd DAC Dimethylallylcaffeated My Myricetinb Qc Quercetinb a × × × × × × × × × × × × × × × × × × M D E × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × (continued) 471 33 Flavonoids in Stingless-Bee and Honey-Bee Honeys Table 33.1 (continued) No. Compound Lt Kf Ap Is G Ch Tch Tc Pb Pc Pt Luteolin Kaempferolb Apigeninb Isorhamnetinb Galanginb Chrysinb Techtochrysinb Tricetinb Pinobanksinb Pinocembrinb Pinostrobinb b T × × × × M D E × × × × × × × × × × × × × × × × × × × × O-glycoside Aglycone c C-glycoside d Phenolic acid derivative a b sample from each stingless-bee species was available for analysis and suggests strong limitations of this study. The flavonoid profile observed was not as consistent as observed in the pot-honey from Venezuela and Australia. Nevertheless, the flavonoid glycosides detected which were also derivatives of quercetin, kaempferol, and isorhamnetin showed a glycosidic combination similar to those reported above for other stingless-bee honeys: normally hexosyl-hexosides although the second sugar could also be rhamnose or a pentose. Flavonoid triglycosides were also detected and in this case the additional sugar was often rhamnose. Several of these glycosides are common to all the analyzed samples, and in some cases flavonoid aglycones were also observed. Several stingless-bee samples from Brazil were also analyzed: seven from “tiúba” Melipona fasciculata, four from “uruçú” M. scutellaris, and three from “jandaíra” M. subnitida, two from “mandaçaia” M. quadrifasciata and one from “uruçú amarela” M. rufiventris. All of them are characterized by having a very limited number of flavonoids, and in a very low quantity. These samples do not show a similar or common flavonoid profile, even for the same bee species, although this could be explained by different localities and therefore different floral origin. Some of them, and particularly the three samples from M. subnitida, have an abundant content of tt and ct-abscisic acid. In other samples they contained very small amounts of di-Cglycosyl flavonoids. Among the flavonoid O-glycosides, isorhamnetin and kaempferol derivatives, with a similar structure to those reported above, were detected, as well as other derivatives with glycosylations in the 3 and 7 positions. The aglycones pinobanksin and kaempferol were also detected. A recent study reports the flavonoid glycoside content of stingless-bee honey (2.7 mg/100 g honey) is considerably higher than the content of aglycones (0.3 mg/100 g) (Truchado et al. 2011), and this differs from previous studies on A. mellifera, with much higher aglycone content and smaller flavonoid-glycoside content. 472 33.5 F.A. Tomás-Barberán et al. Conclusions and Further Research Although the flavonoid content of A. mellifera honey has been extensively studied for potential use in determining botanical and geographical origin and also considering potential health benefit, the composition of stingless-bee honey is still largely unknown. An appealing topic of research is thus available due to the large number of bee species and the many and diverse plant sources used for honey production. The transformation of nectar flavonoids by bee enzymes is less relevant for the Meliponini, and therefore honey may better preserve the natural plant compounds. This observation deserves exploration in more detail. The fact that pot-honey is processed in storage pots containing resins may cause a transfer from the food container to the stored food which has never been measured, but certainly would add to its phytochemical spectra and bioactivity. Acknowledgments The authors are grateful to the European Commission FP7 for supporting the research on plant bioactive compounds collected by plants and their role in bee health (project BEEDOC, under grant agreement 244956). The stingless-bee honey samples were kindly provided by Patricia Vit (APIBA honey collection, Universidad de los Andes, Mérida, Venezuela); M. favosa from Venezuela, M. quadrifasciata and M. scutellaris from Paulo Nogueira-Neto Fazenda (São Simão, Brazil) were collected by herself. M. subnitida (Natal, Brazil), M. rufiventris (Pará, Brazil), other M. quadrifasciata and M. scutellaris honeys were received during the X Iberolatinamerican Congress in Natal, M. fasciculata maturated pot-honey from Sergio Murilo Drummond (Universidade Federal do Maranhão, Brazil), T. carbonaria honey was collected by Tim Heard (CSIRO, Brisbane, Australia), and honey from Bolivian species was collected by Urbelinda Ferrufino (ASEO, Santa Cruz, Bolivia). Useful editorial annotations by P. Vit and D.W. Roubik are appreciated. References Bogdanov S. 1999. Honey quality and international regulatory standards; a review by the International Honey Commission. Bee World 80:61–69. Camargo JMF, Pedro SRM. 2007. Meliponini Lepeletier 1836. pp. 272–578. In Moure JS, Urban D, Melo GAR, eds. Catalogue of bees (Hymenoptera, Apoidea) in the neotropical region. Sociedade Brasilera de Entomologia; Curitiba, Brasil. 1958 pp. Duarte AWF, dos Santos Vasconcelos MF, de Menezes APD, da Silva SC, Oda-Souza M, Quiejeiro López AM. 2012. Composition and antioxidant activity of honey from Africanized and stingless bees in Alagoas (Brazil): a multivariate analysis. Journal of Apicultural Research 51:23–35. Ferreres F, Tomás-Barberán FA, Gil MI, Tomás-Lorente F. 1991. An HPLC technique for flavonoid analysis in honey. Journal of the Science of Food and Agriculture 56:49–56. Ferreres F, Ortiz A, Silva C, García-Viguera C, Tomás-Barberán FA, Tomás-Lorente F. 1992. Flavonoids of “La Alcarria” honey. A study of their botanical origin. Zeitschrift für LebensmittelUntersuchung und –Forschung 194:139–143. Ferreres F, García-Viguera C, Tomás-Lorente F, Tomás-Barberán FA. 1993. Hesperetin, a marker of the floral origin of citrus honey. Journal of the Science of Food and Agriculture 61:121–123. 33 Flavonoids in Stingless-Bee and Honey-Bee Honeys 473 Ferreres F, Andrade P, Tomás-Barberán FA. 1994. Flavonoids from Portuguese heather honey. Zeitschrift für Lebensmittel-Untersuchung und –Forschung 199:32–37. Ferreres F, Andrade P, Tomás-Barberán FA. 1996a Natural ocurrence of abscisic acid in heather honey and floral nectar. Journal of Agricultural and Food Chemistry 44:2053–2056. Ferreres F, Andrade P, Gil MI, Tomás-Barberán FA. 1996b. Floral nectar phenolcis as biochemical markers for the botanical origin of heather honey. Zeitschrift für Lebensmittel –Untersuchung und –Forschung 202:40–44. Frankel S, Robinson GE, Berenbaun MR. 1998. Antioxidant capacity and correlated characteristics of 14 uniforal honeys. Journal of Apicultural Research 37:27–31. Harborne JB. 1982. Introduction to ecological biochemistry. Academic Press; London, UK. pp. 1–261. Martos I, Cossentini M, Ferreres F, Tomás-Barberán FA. 1997. Flavonoid composition of Tunisian honeys and propolis. Journal of Agricultural and Food Chemistry 45:2824–2829. Martos I, Ferreres F, Tomás-Barberán FA. 2000. Identification of flavonoid markers for the botanical origin of Eucalyptus honey. Journal of Agricultural and Food Chemistry 48:1498–1502. Persano Oddo L, Heard TA, Rodríguez-Malaver A, Pérez RA, Fernández-Muiño M, Sancho MT, Sesta G, Lusco L, Vit P. 2008 Composition and antioxidant activity of Trigona carbonaria honey from Australia. Journal of Medicinal Food 11:789–794. Rodríguez-Malaver AJ, Pérez-Pérez EM, Vit P. 2007. Capacidad antioxidante de mieles venezolanas de los géneros Apis, Melipona y Tetragonisca, evaluada por tres métodos. Revista del Instituto Nacional de Higiene Rafael Rangel 38:13–18. Rodríguez-Malaver AJ, Rasmussen C, Gutiérrez MG, Gil F, Nieves B, Vit P. 2009. Properties of honey from ten species of Peruvian stingless bees. Natural Product Communications 4:1221–1226. Schwarz HF. 1948. Stingless bees (Meliponidae) of the Western Hemisphere. Bulletin of the American Museum of Natural History 90:1–546. Soler C, Gil MI, García-Viguera C, Tomás-Barberán FA. 1995. Flavonoid patterns of French honeys with different floral origin. Apidologie, 26:53–60. Tomás-Barberán FA, García-Viguera C, Vit-Olivier P, Ferreres F, Tomás-Lorente F. 1993a. Phytochemical evidence for the botanical origin of tropical propolis from Venezuela. Phytochemistry 34:191–196. Tomás-Barberán FA, Ferreres F, García-Viguera C, Tomás-Lorente F. 1993b. Flavonoids in honey of different geographical origin. Zeitschrift für Lebensmittel-Untersuchung und -Forschung 196:38–44. Tomás-Barberán FA, Ferreres F, Tomás-Lorente F, Ortiz A. 1993c. Flavonoids from Apis mellifera beeswax. Zeitschrift für Naturforschung 48c:68–72. Tomás-Barberán FA, Tomás-Lorente F, Ferreres F, García-Viguera C. 1989. Flavonoids as biochemical markers of the plant origin of bee-pollen. Journal of the Science of Food and Agriculture 47:337–340. Tomás-Barberán FA, Martos I, Ferreres F, Radovic B, Anklam E. 2001. Flavonoids and floral origin of European monofloral honeys. Journal of the Science of Food and Agriculture 81:485–496. Truchado P, Ferreres F, Bortolotti L, Sabbatini AG, Tomás-Barberán FA. 2008. Nectar flavonol rhamnosides are floral markers of acacia (Robinia pseudacacia) Honey. Journal of Agricultural and Food Chemistry 56:8815–8824. Truchado P, Martos I, Bortolotti L, Sabatini AG, Ferreres F, Tomás-Barberán FA. 2009a. Use of quinoline alkaloids as markers of the floral origin of chestnut honey. Journal of Agricultural and Food Chemistry 57:5680–5686. Truchado P, Ferreres F, Tomás-Barberán FA. 2009b. Liquid chromatography-tandem mass spectrometry reveals the widespread occurrence of flavonoid glycosides in honey, and their potential as floral origin markers. Journal of Chromatography A 1216:7241–7248. Truchado P, Tourn E, Gallez L, Moreno DA, Ferreres F, Tomás-Barberán FA. 2010. Identification of botanical biomarkers in Argentinean Diplotaxis honeys: Flavonoids and glucosinolates. Journal of Agricultural and Food Chemistry 58:12678–12685. 474 F.A. Tomás-Barberán et al. Truchado P, Vit P, Ferreres F, Tomás-Barberán F. 2011. Liquid chromatography-tandem mass spectrometry analysis allows the simultaneous characterization of C-glycosyl and O-glycosyl flavonoids in stingless bee honeys. Journal of Chromatography A 1218:7601–7607. Vit P, Soler C, Tomás-Barberán FA. 1997. Profile of phenolic compounds of Apis mellifera and Melipona spp. honeys from Venezuela. Zeitschrift für Lebensmittel-Untersuchung und -Forschung 204:43–47. Vit P, Tomás-Barberán FA. 1998. Flavonoids in Meliponinae honey from Venezuela, related to their botanical, geographical and entomological origin to assess their putative anticataract properties. Zeitschrift für Lebensmittel-Untersuchung und -Forschung 206:288–293. Chapter 34 Antioxidant Activity of Pot-Honey Antonio Jesús Rodríguez-Malaver 34.1 Introduction Stingless bee honey has been used in traditional medicine for centuries. In countries including Peru, Guatemala, Mexico, and Venezuela, this honey is used widely and sold at local markets, often as a sweetener, but more often as an ingredient of folk medicine (Vit et al. 2004). This honey is a complex mixture that contains different botanical and entomological compounds. Such compounds contribute to honey’s bioactive properties and are important in apitherapy. Although there is a vast Neotropical biodiversity of 391 stingless bee species (Camargo and Pedro 2007), only the honey produced by a few species has been studied. In general, the main differences between stingless bee honey and Apis mellifera (honey bee) honey are a higher water content and acidity, lower diastase, and a different sugar content in the stingless bee honey compared to Apis mellifera honey (Vit et al. 2004; Souza et al. 2006). It has been demonstrated that fermentation increased the antioxidant bioactivity of Tetragonisca angustula honey. This observation, signaling the importance of antioxidants, could partly explain the reputed medicinal properties of stingless bee honey (Pérez-Pérez et al. 2007). Rodríguez-Malaver et al. (2007) measured the antioxidant capacity of Apis, Melipona, and Trigona honey from Venezuela with three oxidative systems, to test the effectiveness of honey at scavenging (i.e., removing) superoxide anions, hydroxyl radicals, and benzoate degradation. All the honey samples showed higher antioxidant capacity indicators than those of artificial honey and lipoic acid. The authors suggested that the antioxidant capacity could serve as a test to detect and then control adulterated honey on the commercial market. A.J. Rodríguez-Malaver (*) Department of Biochemistry, Faculty of Medicine, Universidad de Los Andes, Mérida 5101, Venezuela e-mail: anrod@ula.ve 475 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_34, © Springer Science+Business Media New York 2013 476 A.J. Rodríguez-Malaver In this chapter, the antioxidant capacity of pot-honey is reviewed, and further scrutinized using information available for stingless bee pollen and propolis. 34.2 Bioactivity of Stingless Bee Products (Honey, Propolis, Pollen) Among natural products, honey bee-derived apicultural products such as pollen and propolis have been applied for centuries in traditional medicine, as well as in food diets and supplementary nutrition (Kroyer and Hegedus 2002). Propolis has been used as a folk medicine and has been reported to possess therapeutic or preventive effects against inflammation, heart disease, diabetes mellitus, microbes hepatotoxity, and cancer (Burdock 1998). Kujumgiev et al. (1999) report no differences in the antibacterial, antifungal, and antiviral activities of propolis from different geographic origins, including four samples from Brazilian A. mellifera and two stingless bees. The flavonoids in propolis (mainly pinocembrin) are considered responsible for its inhibitory effect on bacteria and fungi, but only traces of these compounds have been found in propolis of South American origin (Tomás-Barberán et al. 1993); thus, propolis from that region may possess other active compounds. Farnesi et al. (2009) demonstrated that the antibacterial activity of green propolis from honey bee nests against Micrococcus luteus and Staphylococcus aureus was superior to that taken from nests of stingless bee, Melipona quadrifasciata and Scaptotrigona, propolis. Two samples of propolis (green propolis and Scaptotrigona propolis) were effective against Escherichia coli. Melipona quadrifasciata propolis was more active than green propolis and Scaptotrigona propolis against Pseudomonas aeruginosa, suggesting a potential importance for human and veterinary medicine. It was found that Fenton reagent causes a decrease in salivary total antioxidant activity (TAA) and Apis mellifera propolis protects and even increases salivary TAA. On the other hand, Melipona favosa propolis only protects salivary TAA against oxidative stress (Sánchez et al. 2010). Silva et al. (2009) show that the extracts of pollen from Melipona rufiventris are good scavengers of active oxygen species. Those authors suggest this property of pollen is important in prevention of diseases such as cancer, cardiovascular disease, and diabetes, among others. 34.3 Comparison of Pot-Honey and Apis mellifera Honey Pot-honey shows differences in antioxidant activity, in comparison to Apis mellifera honey. In a study on Peruvian stingless bee honey from ten species, the Trolox equivalent antioxidant capacity (TEAC) ranged from 93.84 to 569.65 mmol Trolox 34 Antioxidant Activity of Pot-Honey 477 equivalents (TE)/100 g (Rodríguez-Malaver et al. 2009). Some species (Nannotrigona melanocera) showed higher TEAC than both Czech A. mellifera honey (from 43.55 to 290.35 mmol TE/100 g) (Vit et al. 2008) and Venezuelan A. mellifera (from 34.90 to 203.21 mmol TE/100 g) (Vit et al. 2009a). In this work, flavonoid and polyphenol contents of stingless bee honey were measured; they ranged from 2.6 to 31.0 mg quercetine equivalents (QE)/100 g, and 99.7–464.9 mg gallic acid equivalents (GAE)/100 g, respectively. Those values were higher than Czech A. mellifera honey (from 1.90 to 15.74 mg QE/100 g and from 47.39 to 265.49 mg GAE/100 g) and Venezuelan A. mellifera honey (from 2.32 to 14.41 mg QE/100 g and 38.15 and 182.10 mg GAE/100 g). The antioxidant activity, flavonoid and polyphenol contents are compared in pothoney produced by several stingless bee genera. The highest values are found in Nannotrigona honey, followed by Scaura and Ptilotrigona. The lowest values are found in Melipona and Partamona, followed by Tetragonisca and Scaptotrigona. However, such comparisons are only preliminary, because more honey samples are needed. Only one honey was available for most of the genera, whereas 28 Melipona honeys and 18 Tetragonisca honeys were analyzed (Gutiérrez 2008). 34.4 Factors that Explain the Antioxidant Capacity and Possible Role for Authentication Persano Oddo et al. (2008) report that the TEAC of Tetragonula carbonaria (formerly named Trigona carbonaria) honey from Australia is higher (233.96 ± 50.95 mmol/100 g) than that reported for Czech floral honey of Apis mellifera, while the radical scavenging activity (RSA) (48.03 ± 12.58% ascorbic acid equivalents) is similar to that of floral and honeydew blends of Spanish honey (Pérez et al. 2007). The flavonoid content of T. carbonaria honey (10.02 ± 1.59 mg QE/100 g) is higher than those of Czech floral and honeydew honey (6.59 and 7.25 mg QE/100 g, respectively). In contrast, the polyphenol content is higher in the floral (115.03 mg GAE/100 g) and honeydew (129.03 mg GAE/100 g) Czech honeys than in T. carbonaria honey (55.74 ± 6.11 mg GAE/100 g) (Vit et al. 2008). The authors suggest that organic acids might explain its high antioxidant activity. The antioxidant capacity of T. carbonaria and other stingless bee honey represents an important added value, to encourage further research on medicinal attributes with both nutritional and pharmaceutical application. In a recent study, a high level of antibiotic activity was found in honey from T. carbonaria (Irish et al. 2008). In another study with pot-honey from Guatemala, M. beecheii “abeja criolla” and M. solani “chac chow” were compared. The antioxidant activity, flavonoid and polyphenol contents are given in Table 34.1. The TEAC values, flavonoid and polyphenol contents were significantly higher in M. beecheii than in M. solani honey (Gutiérrez et al. 2008). Such a difference could be explained by the floral species visited. Asteraceae and Melastomataceae were the most abundant plant families in the Melipona honey pollen spectrum in Guatemala (Dardón and Enríquez 2008). 478 A.J. Rodríguez-Malaver Table 34.1 Bioactivity of Melipona honey from Guatemala (permission granted by Revista de la Facultad de Farmacia) Stingless bee species Bioactive parameter M. beecheii, N = 4 M. solani, N = 2 Flavonoids* (mg QE/100 ghoney) 3.60 ± 0.61 1.88 ± 1.64 Polyphenols* (mg GAE/100 g honey) 107.35 ± 17.79 68.66 ± 15.11 TEAC* (mmol TE/100 ghoney) 87.38 ± 12.92 39.07 ± 10.52 Averages ± SD values * Significant differences between M. beecheii and M. solani (P < 0.05), t-test Tetragonisca fiebrigi Schwarz, 1938 is a stingless bee named “yateí” in Argentina and Paraguay. Vit et al. (2009b) compared a honey sample from both countries and found that TEAC was higher in honey from Argentina (160.15 ± 60.50 mmol TE/100 g) compared to Paraguay (120.91 ± 38.67 mmol TE/100 g). However, they did not find a difference in flavonoid (14.37 ± 11.11 and 12.66 ± 4.82 mg QE/100 g) and polyphenol (240.74 ± 94.05 and 148.29 ± 17.75 GAE/100 g) content. High nitrite content was found in Peruvian pot-honey (Rodríguez-Malaver et al. 2009). It was hypothesized that nitric oxide and/or nitrite might be responsible, in part, for the biological and therapeutic effects of honey (Al-Waili 2003). In addition, this metabolite could be used for authentication of honey. Also in this research, there were positive Pearson correlations (P < 0.01) between flavonoids-TEAC (0.879), polyphenols-TEAC (0.942), proteins-TEAC (0.911), color-TEAC (0.771), and nitrites-TEAC (0.422). Those correlations indicated compounds that could be involved in the antioxidant action of stingless bee honey. Similar results have been reported for polyphenols, flavonoids, and color in A. mellifera honey (Bertonceij et al. 2007; Frankel et al. 1998; Taormina et al. 2001; Vela et al. 2007, 2008). It has also been reported that the antioxidant activity of stingless bee honey increases with free acidity (r2 = 0.97, P < 0.01) (Vit et al. 2006). Due to a controversy about which compounds signify honey antioxidant activity, Gheldof et al. (2002) suggested that total antioxidant content of honey may be better explained by interactions of a wide range of compounds, including phenolics, peptides, organic acids, enzymes, and Maillard reaction products. 34.5 Conclusions Diversity of stingless bees in America is very high. Thus, bioactivities of stingless bee products are diverse because they depend on bee species, their habits, and also on external factors such as geography, climate, season, harvesting method, etc. Comparisons of bioactivities from bee products of native stingless bee species has been widely studied and reported. It was found that both internal and external factors affect classes, types, and contents of active compounds and their derivatives, which mainly belong to phenolic compounds and flavonoids. 34 Antioxidant Activity of Pot-Honey 479 The correlation between chemical compounds such as water, sugars and free acidity and the bioactivities has been widely studied. Standard control of stingless bee products in traditional medicine would require identifying new bioactive agents of interest in order to demonstrate their bee origin, and to avoid or reduce the side-effects of using present modern medicine. References Al-Waili NS. 2003. Identification of nitric oxide metabolites in various honeys: effects of intravenous honey on plasma and urinary nitric metabolites concentrations. Journal of Medicinal Food 6:359–364. Bertonceij J, Dobersek U, Jamnik M, Golob T. 2007. Evaluation of the phenolic content, antioxidant activity and colour of Slovenian honey. Food Chemistry 105:822–828. Burdock, GA. 1998. Review of the biological properties and toxicity of bee propolis (propolis). Food Chemistry and Toxicology 36:347–363. Camargo JMF, Pedro SRM. 2007. Meliponini Lepeletier 1836. pp. 272–578. In Moure JS, Urban D, Melo GAR. eds. Catalogue of Bees (Hymenoptera, Apoidea) in the Neotropical Region. Sociedade Brasilera de Entomologia; Curitiba, Brasil. 1958 pp. Dardón MJ, Enríquez E. 2008. Caracterización fisicoquímica y antimicrobiana de la miel de nueve especies de abejas sin aguijón (Meliponini) de Guatemala. Interciencia 33:916–922. Farnesi AP, Aquino-Ferreira R, De Jong D, Bastos JK, Soares AEE. 2009. Effects of stingless bee and honey bee propolis on four species of bacteria. Genetics and Molecular Research 8:635–640. Frankel S, Robinson GE, Berenbaun MR. 1998. Antioxidant capacity and correlated characteristics of 14 uniforal honeys. Journal of Apicultural Research 37:27–31. Gheldof N, Wang XH, Engeseth NJ. 2002. Identification and quantification of antioxidant components of honey from various floral sources. Journal of Agriculture and Food Chemistry 50:5870–5877. Gutiérrez MG. 2008. Actividad antioxidante, contenido de flavonoides y de polifenoles de mieles de abejas sin aguijón de Argentina, Brasil, Guatemala, Paraguay, Perú y Venezuela. Tesina de Grado. Escuela de Farmacia, Facultad de Farmacia y Bioanálisis, Universidad de Los Andes. Mérida, Venezuela. 57 pp. Gutiérrez MG, Enríquez E, Lusco L, Rodríguez-Malaver A, Persano Oddo L, Vit P. 2008. Caracterización de mieles de Melipona beecheii y Melipona solani de Guatemala. Revista de la Facultad de Farmacia 50:2–6. Irish J, Heard TA, Carter D, Blair S. 2008. Antibacterial activity of honey from the Australian stingless bee Trigona carbonaria. International Journal of Antimicrobial Agents 32:89–90. Kroyer G, Hegedus N. 2002. Evaluation of bioactive properties of pollen extracts as functional dietary food supplement. Innovative Food Science Emerging Technologies 2:171–174. Kujumgiev A, Tsvetkova I, Serkedjieva Y, Bankova V, Christov R, Popov S. 1999. Antibacterial, antifungal and antiviral activity of propolis of different geographic origin. Journal of Ethnopharmacology 64:235–240. Persano Oddo L, Heard TA, Rodríguez-Malaver A, Pérez RA, Fernández-Muiño M, Sancho MT, Sesta G, Lusco L, Vit P. 2008 Composition and antioxidant activity of Trigona carbonaria honey from Australia. Journal of Medicinal Food 11:789–794. Pérez RA, Iglesias MT, Pueyo E, González M, de Lorenzo C. 2007 Amino acid composition and antioxidant capacity of Spanish honeys. Journal of Agricultural Food Chemistry 55:360–365. Pérez-Pérez E, Rodríguez-Malaver A, Vit P. 2007. Efecto de la fermentación postcosecha en la capacidad antioxidante de miel de Tetragonisca angustula Latreille, 1811. Revista de la Sociedad Mexicana de BioTecnologia y Bioingeniería 10:14–20. 480 A.J. Rodríguez-Malaver Rodríguez-Malaver AJ, Pérez-Pérez E, Vit P. 2007. Capacidad antioxidante de mieles venezolanas de los géneros Apis, Melipona y Tetragonisca, evaluada por tres métodos. Revista del Instituto Nacional de Higiene Rafael Rangel 28:13–17. Rodríguez-Malaver AJ, Rasmussen C, Gutiérrez MG, Gil F, Nieves B. 2009. Properties of ten species of stingless bee honey from Peru. Natural Product Communications 4:1221–1226. Sánchez N, Miranda S, Vit P, Rodríguez-Malaver AJ. 2010. Propolis protects against oxidative stress in human saliva. Journal of ApiProduct and ApiMedical Science 2:72–76. Silva TMS, Camara CA, Lins ACS, Agra M, Silva EMS, Reis IT, Freitas BM. 2009. Chemical composition, botanical evaluation and screening of radical scavenging activity of collected pollen by the stingless bees Melipona rufiventris (Urucu-amarela). Anais da Academia Brasileira de Ciencias 81:173–178. Souza B, Roubik D, Barth O, Heard T, Enríquez E, Carvalho C, Villas-Bôas J, Persano-Oddo L, Almeida-Muradian L, Bogdanov S, Vit, P. 2006. Composition of stingless bee honey: setting quality standards. Interciencia 31:867–875. Taormina, P, Niemira, V, Beuchat, L. 2001. Inhibitory activity of honey against food borne pathogens as influenced by the presence of hydrogen peroxide and level of antioxidant power. International Journal of Food Microbiology 69:217–225. Tomás-Barberán FA, García-Viguera C, Vit-Olivier P, Ferreres F, Tomás-Lorente F. 1993. Phytochemical evidence for the botanical origin of tropical propolis from Venezuela. Phytochemistry 34:191–196. Vela L, de Lorenzo C, Pérez RA. 2007. Antioxidant capacity of Spanish honeys and its correlation with polyphenol content and other physicochemical properties. Journal of the Science of Food and Agriculture 87:1069–1075. Vit P, Medina M, Enriquez E. 2004. Quality standards for medicinal uses of Meliponinae honey in Guatemala, Mexico and Venezuela. Bee World 85:2–5. Vit P, Rodríguez-Malaver A, Almeida D, Souza BA, Marchini LC, Fernández Díaz C, Tricio AE, Villas-Bôas JK, Heard TA. 2006. A scientific event to promote knowledge regarding honey from stingless bees: 1. Physicalchemical composition. Magistra 18:270–276. Vit P, Gutiérrez MG, Tit ra D, Bedná M, Rodríguez-Malaver AJ. 2008. Mieles checas categorizadas según su actividad antioxidante. Acta Bioquímica Clínica Latinoamericana 42:237–244. Vit P, Gutiérrez MG, Rodríguez-Malaver A, Aguilera G, Fernández-Díaz C, Tricio AE. 2009a. Comparison of honey produced by the bee yateí (Tetragonisca fiebrigi) in Argentina and Paraguay. Acta Bioquímica Clínica Latinoamericana 43:219–226. Vit P, Rodríguez-Malaver A, Roubik DW, Moreno E, Souza BA, Sancho MT, Fernández-Muiño M, Almeida-Anacleto D, Marchini LC, Gil F, González C, Aguilera G, Nieves B. 2009b. Expanded parameters to assess the quality of honey from Venezuelan Apis mellifera. Journal of ApiProduct and ApiMedical Science 1:72–81. Chapter 35 Use of Honey in Cancer Prevention and Therapy Patricia Vit, Jun Qing Yu, and Fazlul Huq This chapter is dedicated to cancer sufferers and survivors, and researchers engaged in its prevention and therapy 35.1 Introduction The typical composition of honey (Codex Alimentarius Commission 2001) provides a generalization that misses variability in composition of an apparently homogeneous sugary product. Therefore, it was referred to as enigmatic honey in a book on melissopalynology (Vit 2005) meaning honey not being a standard syrup. Commonality and variability in properties of honey is considered to be useful in making informed healthcare choices (Gethin 2008). Honey composition and other factors may readily explain this variability, as shown in several chapters in this book. Variability in either composition of honey and characteristics of cancer raise a question: what type of honey for what cancer, at what stage of the disease, and in what dosage and timing? Further questions arise on the usefulness of honey intake alone or as an ingredient of natural remedies, or used in combination with conventional chemotherapy. Honey alone showed moderate murine antitumor activity and pronounced antimetastatic effects, but combined with anticancer drugs, 5-fluorouracil and cyclophosphamide, resulted in antitumor activity (Gribel and Pashinkii 1990). The use of honey with Aloe arborescens has been associated with tumor regression and survival time in patients P. Vit (*) Apitherapy and Bioactivity, Food Science Department, Faculty of Pharmacy and Bioanalysis, Universidad de Los Andes, Mérida 5101, Venezuela Cancer Research Group, Discipline of Biomedical Science, The University of Sydney, Cumberland Campus C42, 75 East Street, Lidcombe, NSW 1825, Australia e-mail: vitolivier@gmail.com J.Q. Yu • F. Huq Cancer Research Group, Discipline of Biomedical Science, The University of Sydney, Cumberland Campus C42, 75 East Street, Lidcombe, NSW 1825, Australia e-mail: vitolivier@gmail.com 481 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_35, © Springer Science+Business Media New York 2013 482 P. Vit et al. treated simultaneously with oncologic chemotherapy (Zago 2004; Lissoni et al. 2009). In a review of 131 studies, Aloe vera and honey prevented or reduced mucositis, varying with the type of cancer and treatment (Worthington et al. 2010). Aloe vera and honey were hepatoprotective, reduced cell proliferation, and increased apoptosis in murine tumors (Tomasin and Gomes-Marcondes 2011). Two recent reviews covered the ethnopharmacological uses of honey in northeastern Brazil, with a number of stingless bee species (Melipona scutellaris, Melipona subnitida, Partamona seridoensis, Scaptotrigona sp., and Tetragonisca angustula) (Oliveira et al. 2010; Souto et al. 2011). However, the term cancer was not included as a disease descriptor. Possibly cancer as such cannot be diagnosed in traditional medicine, but can only be related to inflammations and swellings. Cancer, the most dreaded disease of our time, is curable if detected in its early stages (Cantor 2008). The use of honey in cancer prevention and therapy has been tested both in vitro and in vivo, but the data do not cover the range of honey types or cancer symptoms known to exist. A number of cellular pathways in diverse cancer cell lines that are being investigated may eventually lead to a unified concept applying to the plethora of diseases termed cancer. The apoptotic ability (anti-proliferative potential, arresting cell growth at the subpopulation sub-G1, activation of the caspase cascade) of honey varies according to the cell type, e.g., in colon cancer cells (Jaganathan and Mandal 2009b), and involves nonprotein thiols, mitochondrial dysfunction, reactive oxygen species, and protein p53 (Jaganathan and Mandal 2010). The group of Nada Oršolić at the University of Zagreb in Croatia demonstrated growth inhibition of certain tumor types, reduction of metastases and prolonged survival in mice, after treatment with honey alone (Oršolić 2009), or propolis combined with chemotherapeutic agents (Benkovic et al. 2007). The ability of health scientists to measure the activity of honey in cancer is related to factors within a matrix of diverse botanical, entomological and geographical origin (major sugar components, water, polyphenols and other secondary plant metabolites, acids, enzymes, minerals, etc.), cancer type (adenoma, carcinoma, myeloma), organ site, cancer stage (initiation, metastasis, double tumor), cancer care (mucositis, radiation burns), patient age, and presence of other diseases. Cascades of molecular markers as indicators of cancer onset and anticancer action are actively investigated. Whether honey is useful to treat cancer is a question to be answered in relief of oncologic suffering and death. This study aims to provide an overview in the usefulness of honey in cancer prevention and therapy. Our data on the antiproliferative action of pot-honey from Frieseomelitta, Melipona, Scaptotrigona, and Tetragonula in three human ovarian cancer cell lines are described and evaluated here. 35.2 Cancer The name “cancer” originated with Hippocrates and the Greek word ‘carcinos’ “karkίnoV” to indicate tumors with the shape of a crab. All cancer cells in a patient originate from a unique cell starter among the 1014 cells in the human body (Pecorino 35 Use of Honey in Cancer Prevention and Therapy 483 2008) as the primordium of this progressive disease. One initial mutation accumulates in a single cell, causes unregulated cell growth, invasion of surrounding tissues, and eventually spreads. The disease is therefore clonal, and may evolve more than 10 years before clinical detection. The multistep process leading to the development of cancer is known as carcinogenesis. Proto-oncogenes are activated, while tumor suppressor and genomic stability genes are inactivated. A colon cancer model gave seminal evidence for cancer genetic and histological multistage progression (Volgestein et al. 1988). Age is the biggest risk factor for cancer (Tovey et al. 2007). The following six cell-markers differentiate cancer cell behavior from normal cells: (1) Evasion of apoptosis. (2) Growth signal autonomy, (3) Evasion of growth inhibitory signals, (4) Angiogenesis, (5) Unlimited replicative potential, and (6) Invasion and metastasis (Hanahan and Weinberg 2000). Molecular pathways and signaling used in cell function are considered to understand how a normal cell transforms into a cancer cell, and also how cancer cells alter tissue, organ and body functions. Any group of cells out of place is considered cancer in medical imaging. A new growth of cells is called a “neoplasm”. Oncology is the medical discipline specialized in cancer, and is also originated from a Greek word “onkos” “ogkoV”, which means bulky mass. Carcinomas are the most common tumors and occur in epithelial cells (e.g., brain, colon, kidney, lung, skin, stomach); sarcomas develop in mesoderm cells (e.g., bone, muscle), and adenocarcinomas develop in glandular tissue (e.g., breast, prostate, pancreas). The situation becomes more complex when examining molecular mechanisms, target tissues and cell types, patterns of metastasis, and causes. Besides the ability of cancer to invade other organs during final stages, secondary effects of cancer treatment also cause pain. Cancer patients tend to have wounds that fail to heal (Mc Nees and Dow Meneses 2007), causing suffering and death. Radiation-induced oral mucositis, stomatitis, malignant ulcers, infected lesions, and an infected oral cavity in head and neck cancer are common (Bardy et al. 2008). The feeling of helplessness is often the main cause of increasing pain in cancer (Toon 2008). Official labeling of a cancer drug contains approved information for the product. It covers a number of categories for precise use in terms of type and subtype of cancer, dose, association, schedule and route of administration, and duration of treatment according to the course of the disease. In medical practice, use outside this frame is considered “off-label” prescription (Levêque 2008) but does not apply to traditional use of phytochemicals, including honey. 35.3 Multidrug Resistance Caused by Chemotherapy Cells repeatedly exposed to anticancer drugs may develop drug resistance due to intrinsic or extrinsic factors of diverse nature. Tumor cells exposed to toxic agents increase their tolerance to drugs by adaptive response. Several molecular mechanisms that cause multidrug resistance have been described. First, there may be a reduced drug uptake and increased drug efflux at the membrane level. Second, enhanced drug 484 P. Vit et al. detoxification in cytoplasmic thiol systems, through glutathione S-transferases may occur. Third, there may be increased DNA repair by enzymes. Additionally, decreased apoptosis has three metabolic pathways; (1) overexpression of anti-apoptotic proteins, (2) underexpression of pro-apoptotic proteins, and (3) altered subcellular distribution of wild type p53 protein, called the “guardian of the genome”. Studies on sequenced combination of cisplatin and other platinum compounds with phytochemicals are being carried out in the cancer research laboratory at the Discipline of the Biomedical Science at The University of Sydney (F. Huq 2011, personal communication) with the aim of surmounting cisplatin resistance in ovarian cancer. 35.4 Honey and Cancer Because honey may be viewed as a medicinal dietary substance, scientific evidence on the benefits of honey have been growing since the ancient claims about health and longevity, e.g., by Hippocrates (Skiadas and Lascaratos 2001). Markers of human health suggest that honey consumption reduces the risk of diseases causing death (Cooper et al. 2010). The immunological activity mediated by cytokines is an important functional property modulated by honey (Tonks et al. 2001, 2003, 2007). Healing properties of bee products are related to the antioxidant, anti-inflammatory, antimicrobial, and anticancer activities of flavonoids. However, other substances such as amino acids, vitamins and organic acids can also contribute to the healing power of honey (Frankel et al. 1998) and its useful inclusion in the diet to complement other polyphenols (Blasa et al. 2006). One study indicated the presence of a tumor-promoting factor in honey (Upadhyay et al. 1980), but in current research honey is found to be healing. The antitumor activity of honey may occur through the activation of macrophages, T- and B-cells (Attia et al. 2008). The antiproliferative effect of honey in colon cancer cells is found to vary depending on honey’s botanical and geographical origin (Jaganathan and Mandal 2009b). Although Indian honey has been applied in culture media (Jaganathan et al. 2010), most studies use phenolic extracts of honey. Methanol extracts of Malaysian honey showed a higher phenolic content, whereas an ethyl acetate extract was more active to reverse the toxicity caused by tumor necrosis factor (Kassim et al. 2010). In research with human cancer cell lines, antiproliferative action of honey was observed by apoptosis with IC50 values (the concentration at which cell proliferation is inhibited by 50%) of 4, 10, and 14% after 24, 48, and 72 h, respectively, in a prostate PC-3 cell line (Samarghandian et al. 2010), and with an IC50 of 1.7 and 2.1 mg/mL after 48 and 72 h in renal cell carcinoma (Samarghandian et al. 2011). Therefore, the apoptotic nature of honey has potential for the treatment of prostate and kidney cancer. Honey of the giant honey bee Apis dorsata, reportedly from nesting in the large forest tree “Tualang” (Koompassia excelsa, Fabaceae) in Malaysia was found to induce apoptosis in human oral squamous cell carcinomas, osteosarcoma (Ghashm et al. 2010), and breast and cervical cancer cell lines by depolarization of the mitochondrial membrane (Fauzi et al.). 35 Use of Honey in Cancer Prevention and Therapy 485 Evidence of medicinal uses of honey in oncological care is found in reviews in the Journal of Clinical Nursing (Bardy et al. 2008; Gethin 2008). Nurses are directly involved in healthcare intervention, and have extensive contact with patients. They have often encountered secondary effects caused by conventional treatments of neoplasias. Honey is used to prevent neutropenia (Zidan et al. 2006), in pediatric hematology–oncology wound care (Wiszniewsky et al. 2006), for radiation induced skin toxicity (Moolenaar et al. 2006), mucositis (Motallebnejad et al. 2008), and as a potent antibacterial agent in cancer patients (Majtan et al. 2011). 35.4.1 The Botanical Diversity of Honey Plants visited by bees have been of great interest to diverse disciplines, and melissopalynology provides a tool to study the pollen residues of honey as a “fingerprint” potentially indicating botanical origin of nectar (but see Chap. 21, Roubik and Moreno in this book). Honey with more than 45% pollen counts of one taxon is considered unifloral (Louveaux et al. 1978). The honey of chestnut (Castanea sativa) has been studied for aroma composition (Castro-Vázquez et al. 2010), and manuka (Leptospermum) honey for its medicinal properties (Molan 2001; Tonks et al. 2007). Different plants may well confer different properties to honey. Sensory and physicochemical patterns described for 13 unifloral European honeys produced by Apis mellifera (Persano Oddo and Piro 2004) were further investigated for their aroma composition and medicinal properties. As an example, the antimutagenic activity of honey from seven different floral sources: acacia (Robinia pseudoacacia), buckwheat (Fagopyrum esculentum), clover (Melilotus), fireweed (Epilobium angustifolium), soybean (Glycine max), tupelo (Nyssa), and Christmas berry (Schinus terebinthifolius), and the sugars glucose, fructose, maltose, and sucrose, was measured against nonpolar heterocyclic amine Trp-p-1 (3-amino-1,4-dimethyl5H-pyrido[4,3-b]indole) and tested via Ames assay (Wang et al. 2002). Sucrose was not active, but fructose and glucose were more antimutagenic than honey and the weak maltose, against Trp-p-1. Buckwheat honey, which is extremely high in phenolics caused the greatest inhibition (52.1%) at 1 mg/mL, indicating its potential for use in anticancer therapy. 35.4.2 How Many Kinds of Bees Produce Honey? There are approximately 750 bee species that make honey, about 250 of which are in the genus Bombus, and not considered here (Michener 2007). Hymenoptera are one of the largest and most biologically diverse orders of phytophagous insects with various social grades, and a range of parasitic species (La Salle and Gauld 1993). Phylogenetic relationships of the hymenopteran superfamily, to which all types of bees belong, were initially resolved by sequenced mytochondrial genomes as a single 486 P. Vit et al. analytical approach (Dowton et al. 2009). However, mtDNA is not conservative enough to have any resolution power earlier than the Pliocene, needed to study bee phylogenies, as reviewed by Roubik 2012. In nature, honey is derived from water–sugar resources available in the environment, processed and accumulated for energy needs of the bee colony. Honey bees (Apis spp., Apini) store their honey in beeswax combs, while stingless bees (Meliponini) use cerumen pots of different sizes, shapes, and colors. Apini has 11 or 12 species in the single genus Apis, but Meliponini has more than 500 species in approximateley 61 genera (Rasmussen and Cameron 2010; Roubik 2012). The great biodiversity of Meliponini is treated in the contributions by Camargo and by Michener (Chaps. 1 and 2), in this book. Honey produced by Meliponini clustered naturally according to entomological origin, using compositional data (Vit et al. 1998). Therefore, the entomological origin of honey adds an important descriptor to any medicinal application of honey. 35.4.3 Flavonoids as Anticancer Components of Honey Cancer chemoprevention is an important issue concerning dietary components such as polyphenols, and their epigenetic role as modulating agents of gene expression (Jaganathan and Mandal 2009a; Link et al. 2010; Szic et al. 2010). Thus, flavonoids in honey have been studied for their chemopreventive action. Chemopreventive properties of dietary polyphenols (catechin, chrysin, epicatechin, epigallocatechin3-gallate, quercetin, rutin, myricetin, resveratrol, and xanthohumol) are associated with multiple molecular mechanisms of action against colorectal cancer cell lines (Araújo et al. 2011). Phytochemicals are also studied as agents that may help to counter multidrug resistance in combined treatments (Yunos et al. 2010). An hypothesis on the genotoxic role of honey flavonoids targeting cancer cells has been proposed (Jaganathan 2011). Flavonoids are a group of small molecules (C6-C3-C6, MW ~ 300) widely known to contribute to the colors of flowers and fruits. Five subclasses of dietary flavonoids were considered in selected food: flavones, flavonols, flavanones, flavan-3-ols, and anthocyanidins (USDA 2007). In this database there is an entry for a content of reference flavonoids in 100 g honey: 0.05 mg apigenin, 0.63 mg luteolin (flavones) and 0.17 mg isorhamnetin, 0.11 mg kaempferol, 1.03 mg myricetin, 0.51 g quercetin (flavonols). Over the past few years, a number of studies have used flavonoid profiles of honey to find botanical and other markers, such as bee species (Vit and Tomás-Barberán 1998), and locations of origin (Tomás-Barberán et al. 2001). The removal of free radicals—named scavenging, is one of the outstanding medicinal attributes of flavonoids (Havsteen 2002). Phosphorylation and dephosphorylation reactions that regulate the Na+/K+ ion pump are sensitive to flavonoids. Quercetin removes the phosphate ester from the phenol group tyrosine and restores the pH value in cancer cells (Spector et al. 1980). Apigenin and luteolin are potent inhibitors in human thyroid carcinoma cell lines (Yin et al. 1999). Polyphenols 35 Use of Honey in Cancer Prevention and Therapy 487 studied to characterize and differentiate bee products are a valuable background for predictions on what honey types may have anticancer value. The antiproliferative effects of honey are mainly explained by the presence of the flavonoid chrysin (5,7-dihydroxyflavone). Flow cytometry analysis indicated that cytotoxicity induced by honey or chrysin was mediated by G(0)/G(1) cell cycle arrest. Chrysin was therefore considered a potential candidate for both cancer prevention and treatment (Pichichero et al. 2010). Chrysin has been widely studied by several authors for its effect in suppressing inflammation caused by NF-kB and JNK activations (Ha et al. 2010), to trigger the unfolded endoplasmic reticulum resident protein GRP78 response (Sun et al. 2010), to enhance the apoptosis induced by a ligand (Li et al. 2011), p38 and Bax activation (Pichichero et al. 2011). However, in another study, chrysin inhibited the apoptosis induced by the antitumor-drug topotecan by inhibiting ATP-binding cassette (ABC) transporters (Schumacher et al. 2010). 35.5 Is Pot-Honey Cytotoxic to Human Ovarian Cancer Cells? Substances such as antioxidants that can be chemopreventive to normal cells can also be cytotoxic to cancer cells. Often, these opposing properties are manifested in different cell receptors. It is possible that honey can play both chemopreventive and cytotoxic roles, perhaps due to a variety of antioxidants. To answer this question, the survival of human ovarian cancer cells was measured in the presence of 200 mg honey/mL and three lower serial dilutions up to 1.6 mg honey/mL. The MTT reduction assay (Mosmann 1983) was carried out to determine cell kill due to 16 pothoney samples produced by 13 species of stingless bees (eight Melipona species, three Scaptotrigona species, Tetragonula carbonaria, and Frieseomelitta nigra obtained from Australia, Brazil, Mexico, or Venezuela). The IC50 values of honey samples against three human ovarian cancer cell lines (i.e., concentrations of honey required for 50% cell kill) are given in Table 35.1. The results show that honey samples vary widely in their ability to cause cell kill. The most active honey sample against parent A2780 cell line is Melipona solani (2.74 mg/ mL) and the least active one is Melipona scutellaris (24.37 mg/mL). The next two more active honey samples are Melipona favosa (3.39 mg/mL) and Scaptotrigona polysticta (3.60 mg/mL), followed by Scaptotrigona hellwegeri (4.19 mg/mL), Melipona beecheii (4.24 mg/mL), and Frieseomelitta nigra (4.58 mg/mL). The activity of cisplatin is found to be much lower in the resistant A2780cisR (3.88 mM) and A2780ZD0473R (3.44 mM) cell lines, as compared to that in the parent A2780 cell line (0.88 mM). Unlike that of cisplatin, generally the activity of the honey samples in the resistant cell lines is found to be comparable to that in the parent cell line or greater except in the case of Melipona subnitida (as applied to A2780ZD0473R) where the activity is some 50% lower in the resistant cell lines. Greater activities of some honey samples, especially Melipona solani (1.66 and 0.79 mg/mL) and Scaptotrigona polysticta (1.54 and 1.36 mg/mL) in the resistant A2780cisR and A2780ZD0473R cell 488 P. Vit et al. Table 35.1 IC50 values of pot-honeys in the human ovarian cancer cell lines Ovarian cancer cell lines A2780 Cisplatin (control) Geographical origin, city, country Pot-honey bee species Chetumal, Mexico Melipona beecheii El Reventón, Mexico Melipona fasciata Moura, Brazil Melipona fasciculata Tabocas, Brazil Melipona fasciculata Preazinho, Brazil Melipona fasciculata Moruy, Venezuela Melipona favosa Moruy, Venezuela Melipona favosa Belém, Brazil Melipona rufiventris João Pessoa, Brazil Melipona scutellaris Chiapas, Mexico Melipona solani Natal, Brazil Melipona subnitida El Reventón, Mexico Scaptotrigona hellwegeri Cuetzalan, Mexico Scaptotrigona mexicana Xingú, Brazil Scaptotrigona polysticta Brisbane, Australia Tetragonula carbonaria Guerrero, Mexico Frieseomelitta nigra IC50 honey (mg/mL), cisplatin (mM), RF resistance parent cell line A2780CisR A2780ZD0473R IC50 IC50 RF IC50 RF 0.88 3.88 4.42 3.44 3.91 4.24 6.17 6.18 8.00 13.56 16.50 3.39 5.10 24.37 2.74 17.54 4.19 7.71 3.60 8.96 4.58 factor as 3.35 4.72 5.83 3.97 6.69 4.21 3.68 4.68 25.72 1.66 27.60 4.59 4.43 1.54 4.76 4.72 the ratio 0.79 4.14 0.77 4.28 0.94 5.89 0.50 5.15 0.49 7.69 0.26 12.81 1.08 3.65 0.92 3.80 1.06 27.64 0.61 0.79 1.57 34.36 1.10 4.10 0.57 5.62 0.43 1.36 0.53 4.54 1.03 4.19 IC50 resistant cell 0.98 0.69 0.95 0.64 0.57 0.78 1.08 0.74 1.31 0.29 1.96 0.98 0.73 0.38 0.51 0.92 line/IC50 lines, respectively, than in the parent A2780 cell line, indicate that the pot-honey samples have been able to overcome (at least partially) cisplatin resistance operating in the cell lines. The lowest resistance factor in this set of experiments was achieved by honeys of Melipona favosa against A2780cisR (0.26) and Melipona solani against A2780ZD0473R (0.29). Further studies would be required to obtain information about the mechanisms of cell killing effect by the pot-honeys, and what active components confer their antiproliferative activity. The second honey of Melipona favosa (V12 in APIBA honey collection), was 4.5× richer in flavone C-glycosides than V9, and half in flavonol O-glycosides (Truchado et al. 2011). More precisely, enzymatic hydrolysis of flavone C-glycosides could produce cytotoxic metabolites, or a C-glycoside fit in a signaling molecular pocket to explain the observed higher cell kill. Much needed experiments should compare honey of the same species of bee fed from different kinds of flowers, and of different species of bees fed on the same species of flower. With bee colonies in greenhouses, so that the flowers available to them would be clearly known, such experiments would be possible. With such experiments, the sources of anticancer compounds, whether from flowers or bees or both, could be determined. The very different numbers sometimes shown in Table 35.1 for the same species of bees may suggest the great influence of the floral resources. 35 Use of Honey in Cancer Prevention and Therapy 489 35.6 Adaptive Response of Cancer and Normal Cells to Honey This review to approach the anticancer action of honey involved studies of a variety of mechanisms. We have highlighted three main issues. First, the complexity of the problem from both sides of honey and cancer biodiversity is discussed. Second, the role of honey in chemoprevention is shown. The action of some active components such as flavonoids and the well-known nature of high sugar concentration are discussed. Third, the therapy after cancer onset, with combined treatments using conventional chemotherapy and alternative medicine, is considered. Finally, the effect of pot-honey in a model based on human ovarian cancer cell lines was compared between the stingless bee genera Frieseomelitta, Melipona, Scaptotrigona, and Tetragonula. The adaptive response of cancer and normal cells to honey is a mosaic under construction, and we hope that it will lead to a model for a better understanding of flavonoid interactions with cells, as a chemopreventive and genotoxic tool. Generations of anticancer agents with reduced toxicity in cancer patients may have honey as an ingredient of preparations with other natural products such as Aloes, or combined with targeted therapy. Acknowledgments Persons and institutions that facilitated our work are as follows: Endeavour Awards from Australia for the 2011 Research Fellowship at The University of Sydney to Prof. P. Vit, during her sabbatical leave from Universidad de Los Andes. Prof. F. Huq scientific projects at The University of Sydney, BRIG and Cancer Research Donation Account. The supportive environment at the USYD Discipline of Biomedical Science. To the Ph.D. student Zaynab Al-Eisawi for her assistance. To Dr. Tim Heard from CSIRO Ecosystem Sciences, Brisbane, Queensland, Australia for honey of Tetragonula carbonaria. To M.Sc. Jerônimo Khan Villas-Boâs collaborator of Universidade Federal da Paraíba, Brazil, for honey of Melipona scutellaris and the Scaptotrigona polysticta from João Pessoa and Xingú, Brazil respectively. To Mr. José Reyes from the Tosepan Titaniske Cooperative, Cuetzalan, Puebla, Mexico, for honey of Scaptotrigona mexicana. To Mrs. Liliana Castro from Mujeres Juntas Enfrentando Retos, Guerrero, Mexico, for the three honey samples of Melipona fasciata, Scaptotrigona hellwegeri and Frieseomelitta nigra. To Mr. Emmanuel Pérez de León and to Mr. Ramiro García Farfán from the Soconusco group, Chiapas, México, for honey of Melipona solani and Melipona beecheii, respectively. To Dr. Giorgio Venturieri from Embrapa Amazônia Oriental, Belém, Pará, Brasil, for Melipona rufiventris honey. The Melipona fasciculata honey samples were received from Prof. Murilo Sergio Drummond, Universidade Federal do Maranhão, from Moura, Preazinho, and Tabocas, Brazil. The Melipona favosa honey samples were collected by Prof. Patricia Vit, and the bee was identified by Prof. João MF Camargo. Scaptotrigona polysticta was kindly identified by Dr. Silvia R.M. Pedro from the. The Mexican bees were identified by Prof. Ricardo Ayala from Chamela, Jalisco, Mexico. We are grateful to careful revision received from anonymous referees, Dr. David Roubik (Smithsonian Tropical Research Institute, Balboa, Panamá) and Dr. Silvia R.M. Pedro (Biology Department, Universidade de São Paulo, Ribeirão Preto, Brazil). 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Medical Oncology 23:549–552. Chapter 36 Bioactivity of Honey and Propolis of Tetragonula laeviceps in Thailand Chanpen Chanchao 36.1 Introduction Stingless bee products are used in traditional medicine in Thailand. The “chan-narong” Tetragonula laeviceps is of primary interest because of its wide distribution and management. Honey, propolis, bee pollen, royal jelly, and cerumen are among the many natural bee products that are applied for medicinal purposes (Riches 2000). For example, patients with hay fever and pollen-induced asthma purportedly alleviate their symptoms if they eat local honey. Litwin et al. (1997) suggest symptoms of ragwort hay fever are controlled by eating ragwort pollen present in honey. Natural medicines are a primary focus of one hospital in Thailand, Chao Phya Abhaibhubejhr Hospital, whose efforts are directed toward discovering information about the safety and efficacy of chemical raw materials which then can be applied and developed into traditional Thai medicines. Clinical uses of bee products have continued to increase in recent years. For example, Aburahma et al. (2010) surveyed 176 children who were patients at the pediatric neurology clinic of King Abdullah University Hospital in North Jordan during March to July of 2008. It was found that 29% of the children who used complementary and alternative medicine consumed honey products. It has been reported that honey can treat coughs better than the commercial drugs dextromethorphan and diphenhydramine (Shadkam et al. 2010; Paul et al. 2007). In Thailand, at Bangkok’s Ramathibodi Hospital, honey is successfully used to treat a wound after a radical operation for vulvar carcinoma, and efficiently works in a povidone-iodine solution to heal an abdominal wound disruption (Phuapradit and Saropala 1992; Phuapradit 2002). Stingless bee products are as economically important as honey bee products in Thailand. Interestingly, stingless bees can produce a large amount of propolis, which C. Chanchao (*) Department of Biology, Chulalongkorn University, Bangkok 10330, Thailand e-mail: chanpen@sc.chula.ac.th 495 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_36, © Springer Science+Business Media New York 2013 496 C. Chanchao is used as a sealant in the nest. It appears that propolis is the bee product most widely used for medical purposes (Butnariu and Giuchici 2011; Guney et al. 2011; Saxena et al. 2011). Here I address chemical properties and application of T. laeviceps honey and propolis extracts for medicinal purposes. Honey and propolis yields of this bee are approximately 300 g hive/year. The antiproliferative activity of propolis extracts was tested against cancer cell lines, and compared to normal cells. Besides the beneficial aspects of bee products, hygienic concern is also discussed, medical-grade honey is therefore suggested. 36.2 Composition of Honey and Propolis of Tetragonula laeviceps Honey pots, dark resin collected in the entrance, and propolis in the T. laeviceps hive are shown in Fig. 36.1. Although honey contains many monosaccharides and disaccharides that account for its sweet taste, it is very acidic. For example, the pH of honey from Apis dorsata is 3.81, that of Apis cerana is 3.87, Apis florea, 3.76, and Apis mellifera ranges 3.41–3.95, depending on foraged food sources (Chanchao et al. 2006), Fig. 36.1 Honey pots and propolis of T. laeviceps. (a) Honey pots. (b) Resins collected in the entrance. (c) Propolis outside the hive. (d) Propolis inside the hive. Photos: C. Chanchao 36 Bioactivity of Honey and Propolis of Tetragonula laeviceps in Thailand Table 36.1 Chemical components of WEPa and EEPb from T. laeviceps Propolis Total sugar Reducing Total polyphenols Flavonoids extracts (mg/ml) sugar (mg/ml) (mg/ml) (mg/ml) WEP 1.41 42.35 0.57 0.04 EEP 0.23 0.00 16.88 0.26 After Boonsai (2009) and Kaewwongwattana (2009) a WEP water extract of propolis b EEP ethanol extract of propolis 497 Total protein (mg/ml) 11.3 25.0 while T. laeviceps is 3.37 (Chanchao 2009). The honey of stingless bees tastes sour and bitter, and also smells different from honey of Apis; it is not as often consumed as honey bee honey. It has become widely used in traditional medicine instead. More bioactivities are obtained from the honey of stingless bees than from the honey of honey bees, since the honey pots of stingless bees are made from resin combined with beeswax, known as cerumen. In contrast, the honey cells of honey bees are made entirely of wax. Thus, the active chemical compounds in honey pots, many derived from terpenoids in natural resin (Langenheim 2003), may work together with those from honey. Honey of T. laeviceps has 1.72 mg proline/kg, 0.28 protein g/100 g (44.8 mg N/100 g), and 15.2 g invert sugars/100 g (Chanchao 2009). The chemical components of water extracts of propolis (WEP) and ethanol extracts of propolis (EEP) from T. laeviceps are shown in Table 36.1 (Boonsai 2009; Kaewwongwattana 2009). The data support the idea that propolis could provide bioactivity, based on plant-derived polyphenol and flavonoid contents, and other factors such as the extraction methods, collecting seasons, collecting sites, and other external factors (Gülçin et al. 2010; Li et al. 2010; Miguel et al. 2011). 36.3 36.3.1 Bioactivity of Tetragonula laeviceps Products Antimicrobial Activity Antimicrobial activity of T. laeviceps pot-honey was assayed against Staphylococcus aureus (a Gram-positive bacteria), Escherichia coli (a gram-negative bacterium), Candida albicans (yeast), and Aspergillus niger (fungus) (Wongchum 2007). In Fig. 36.2 the antibacterial, antiyeast, and antifungal activities of serial dilutions of T. laeviceps honey (0, 25, 50, 75, 100%) is determined by the diameter of a clear zone (no-growth area) in the agar-well diffusion method. It is obvious that the inhibition zone increased with higher doses of honey. Neat honey possesses the most effective antimicrobial activity. Using honey at a concentration of 50% (v/v) or higher, S. aureus was the most sensitive microorganism, followed by E. coli, C. albicans, and A. niger, respectively (Fig. 36.3). The yeast 498 C. Chanchao Fig. 36.2 Antimicrobial activity of Trigona laeviceps honey, by agar-well diffusion method, against (a) Staphylococcus aureus and (b) Escherichia coli. Photos: C. Chanchao Fig. 36.3 Antimicrobial activity of 0–100% honey from T. laeviceps (Modified from Wongchum 2007) C. albicans was more sensitive to the diluted honey at 25% (v/v) than the bacteria and the fungus. Honey extracts were tested for antimicrobial activity against Micrococcus luteus and Pseudomonas aeruginosa. Raw honey was partitioned with organic solvents of different polarities (nonpolar hexane, slightly polar dichloromethane, polar methanol). Considering the minimum inhibitory concentration (MIC in mg/ml) and the minimum bactericidal concentration (MBC in mg/ml), results showed active compounds of low polarity, since efficient antimicrobial activity was found in dichloromethane extract of honey (DEH) and hexane extract of honey (HEH), but not in the methanol extract of honey (MEH) (Fig. 36.4). The most efficient antimicrobial activity against M. luteus and P. aeruginosa was demonstrated by DEH at MIC of 10 mg/ml (Chartthai 2010). 36 Bioactivity of Honey and Propolis of Tetragonula laeviceps in Thailand 499 Fig. 36.4 Antimicrobial activity of partitioned extracts of honey. MIC and MBC of methanol (MeOH), dichloromethane (DCM), and hexane (HEX) honey extracts determined by Micrococcus luteus (ML) and Pseudomonas aeruginosa (PA) (Modified from Chartthai 2010) Table 36.2 Diameter of inhibition zones (cm) from ethanol extract of honey (EEH), showing antimicrobial activity against S. aureus and four isolates of MRSA Concentration (mg/ml) Isolates S. aureus MRSA 20645 MRSA 20646 MRSA 20651 MRSA 20652 0 0.00 0.00 0.00 0.00 0.00 64.5 1.60 ± 0.20 1.60 ± 0.05 1.48 ± 0.10 1.58 ± 0.13 1.70 ± 0.26 129 2.17 ± 0.21 2.20 ± 0.00 2.13 ± 0.32 2.17 ± 0.15 2.43 ± 0.13 193.5 2.33 ± 0.25 2.60 ± 0.10 2.53 ± 0.25 2.33 ± 0.15 2.63 ± 0.15 258 2.62 ± 0.24 2.83 ± 0.06 2.70 ± 0.20 2.60 ± 0.00 2.80 ± 0.20 After Jirakanwisal (2010) Not only pathogenic bacteria are susceptible to honey extract, methicillinresistant S. aureus (MRSA) is also susceptible (Jirakanwisal 2010). This indicates that honey may contain a promising new antibiotic. As shown in Table 36.2, the efficiency of an ethanol extract of honey (EEH) against S. aureus and MRSA increases with higher concentration. In addition to honey, the crude extract of propolis has presented antimicrobial activity. In 2009, Umthong et al. reported that both a water extract of propolis (WEP) and a methanol extract of propolis (MEP) from T. laeviceps inhibited the growth of A. niger, B. cereus, C. albicans, E. coli, and S. aureus. The T. laeviceps water extract of propolis was more active than the methanol extract, showing a remarkable anti-B. cereus, anti-Herpes simplex virus type 1, and anti-Mycobacterium tuberculosis activities inhibiting 25–33% of growth with a MIC of 50 mg/ml. WEP was no cytotoxic to Vero cells. Unlike WEP, EEP demonstrated antimalaria (Plasmodium falciparum, K1 strain) activity at an IC50 of 4.48 mg/ml (Kaewmuangmoon et al. 2012). 500 36.3.2 C. Chanchao Antiproliferative Activity Nowadays, cancer is one of the leading causes of death in the Thai population. From statistical records of the Thai Ministry of Public Health during 2005–2009, 13.57% of overall deaths were from cancer. A propensity for cancer is not only inherited, but it can also be triggered by environmental factors such as ultraviolet rays, carcinogens, etc. Research and development of treatments for this disease has been ongoing not only in Thailand but worldwide. Other than surgery, radiation, and chemotherapy—which are the most effective therapies at present—the search for a novel anticancer agent from natural products offers a promising alternative. In 2010, Tasaniyananda reported that honey of T. laeviceps could provide antiproliferative activity against breast tissue (BT474) cancers (Fig. 36.5). It was also found that this activity depended mainly on the type of organic solvent; a water extract of honey (WEH) provided better antiproliferation than an ethanol extract (EEH). Unlike EEH, EEP (IC50 of 25.54 mg/ml) demonstrated better anticancer activity against small-cell lung cancer (NCI-H187) than WEP, for which the percentage of inhibition was <50%. Moreover, EEP showed cytotoxicity against a human leukemia cell line (HL-60) at an IC50 of 29.29 mg/ml (Kaewmuangmoon et al. 2012). The antiproliferative action of T. laeviceps WEP and MEP on a colon cancer cell line (SW620) showed IC50 values of 60 and 80 mg/ml, respectively (Umthong et al. 2009). Not only could this be assayed by the percentage of cell viability, but DNA fragmentation and a change in morphology in SW620 cells were also observed. Later, purification was performed by partition and chromatography. The hexane extract of EEP, which showed the best antiproliferative activity against cancer cell lines from breast (BT474), lung (Chago), colon (SW620), hepatic (Hep-G2), and stomach (Kato-III), was further purified by column chromatography and size-exclusion Fig. 36.5 Percentage of cell viability of breast cancer cell lines (BT474) after being treated with water or ethanol extracts of T. laeviceps honey (From Tasaniyananda 2010) 36 Bioactivity of Honey and Propolis of Tetragonula laeviceps in Thailand 501 Fig. 36.6 Antiproliferative activity of crude extract and purified fractions of T. laeviceps propolis. The activity was tested against breast (BT474), lung (Chago), colon (SW620), hepatic (Hep-G2), and stomach (Kato-III) tissue cancers; liver (CH-liver) cells were used as a control (Modified from Umthong et al. 2011) chromatography. As shown in Fig. 36.6, IC50 values were lower for purified T. laeviceps propolis than the ethanol extract in all cancer cells except CH-liver. In addition, much lower cytotoxicity to normal cells (CH-liver) was found when using purified propolis at the IC50 value of 80.15 mg/ml, compared to EEP (IC50 value of 29.14 mg/ml) (Umthong et al. 2011). 36.4 Antimicrobial Peptides of Honey Antimicrobial peptides are ubiquitous gene-encoded peptide antibiotics (20–40 amino acids) with a folded size similar to the thickness of cellular membrane (Huang 2000). Honey also contains an antimicrobial peptide (AMP) (Kwakman and Zaat 2011; Kwakman et al. 2011a, b). Thus, its direct target is the microbial membrane, because the cationic domain of AMP specifically interacts with the negatively charged outer membrane. Later, a hydrophobic domain will act to disrupt the membrane and translocate into the cells (Epand and Vogel 1999). Several antimicrobial peptides have been reported for Apis. For example, Casteels-Josson et al. (1993) found the apidaecin in A. mellifera body. Later, in 2009, Viljakainen et al. (2009) reported the amino acid sequences of hymenoptaecin in A. mellifera body. Moreover, Yoshiyama and Kimura (2010) reported the amino acid sequences of defensin (GenBank: AB540997.1) and abaecin (GenBank: AB90717.1) from Apis cerana japonica. In Thailand, Wannakul (2007) reported epinecidin-1, which was another AMP, in honey of the giant honey bee (A. dorsata). These antimicrobial peptides could explain the antimicrobial action of honey and propolis. In 2011a, Kwakman et al. developed medical-grade honey containing 75 mM of the synthetic peptide known as bactericidal peptide 2 (BP2). It was able to rapidly 502 C. Chanchao inhibit the growth of many antibiotic-resistant strains of bacteria, including MRSA and extended-spectrum beta-lactamase-producing E. coli. Given the choices of medical-grade honey, BP2 alone, or honey alone, medical-grade honey clearly provided the best antimicrobial activity. Antibacterial peptides in stingless bees have not yet been reported, but considering the important function they have, it seems worthwhile to study them, in addition to known active compounds such as flavonoids (Tomás Barberán et al. 1993). 36.5 Awareness of Using Stingless Bee Products The main problem of using hive products concerns dosage and safety. This is especially true for honey, because it is usually consumed raw, and thus can easily be contaminated with plant pollen or spores of pathogens (Boukraa and Sulaiman 2009; Antúnez et al. 2004; Piccini et al. 2002). Interestingly, although honey is supersaturated, it does contain abundant water in which microorganisms can grow—including lactic acid bacteria of the genera Lactobacillus and Bifidobacterium (Olofsson and Vásquez 2008). Toxic or “mad honey” is also a possible concern. Grayanotoxin is a toxin known to be found in Rhododendron species and other Ericaceae, and can contaminate honey local to that area (Koca and Koca 2007). It can cause symptoms of bradycardia, atrioventricular block, and hypotension (Cagli et al. 2009; Dubey et al. 2009; Okuyan et al. 2010). Besides certain honeys, bee pollen should also be used with caution. For example, Akiyasu et al. (2010) reported that ingestion of bee pollen in nutritional supplements could cause renal failure. Moreover, it has been reported that propolis ointment could cause a dermatological problem: an enlarged, fluid-filled pruritic lesion on a minor trauma (Ting and Silver 2004). Thus, it is necessary to process honey. It can then safely be used to heal a wound or for other medical purposes. Good examples of processed honey are medicalgrade honey or “manuka” honey, which are produced under standardized conditions in a greenhouse. Also, the honey is sterilized by gamma irradiation which can kill bacterial spores efficiently without affecting the honey’s bioactivity (Postmes et al. 1995). More antimicrobial peptides can also be added to medical-grade honey for even more rapid bactericidal activity (Kwakman et al. 2011a, b). In addition, since the chemical compositions and bioactivities of bee hive products depend on seasonal variation and other external factors, it is very important to establish standards for types and amounts of active chemical compounds before selling them commercially (Salomão et al. 2008; Teixeira et al. 2010). Acknowledgments I wish to thank the following: the Thailand Research Fund (grant # RMU5180042); the National Research Council of Thailand; the Asahi Glass Foundation; the Thai Government Stimulus Package 2 (TKK2555), under the Project for the Establishment of a Comprehensive Center for Innovative Food, Health Products and Agriculture; the Ratchadapisek Somphot Endowment Fund (AG001B); and the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, 36 Bioactivity of Honey and Propolis of Tetragonula laeviceps in Thailand 503 for financial support. I also thank Dr. Orawan Duangphakdee, King Mongkut’s University of Technology Thonburi, Ratchaburi Campus, Bangkok, Thailand for honey collection. Finally, I express my gratitude to Professor Patricia Vit for the invitation to write this chapter, and all the editorial support received from her and Dr. David W Roubik. 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Medical-grade honey enriched with antimicrobial peptides has enhanced activity against antibiotic-resistant pathogens. European Journal of Clinical Microbiology and Infectious Diseases 30:251–257. Kwakman PH, te Velde AA, de Boer L, Vandenbroucke-Grauls CM, Zaat SA. 2011b. Two major medicinal honeys have different mechanisms of bactericidal activity. PLoS ONE as doi: 10.1371/journal.pone.0017709. Langenheim JH. 2003. Plant resins. Chemistry, evolution, ecology, ethnobotany. Timber Press; Portland, Oregon, USA. 586 pp. Li F, Awale S, Tezuka Y, Esumi H, Kadota S. 2010. Study on the constituents of Mexican propolis and their cytotoxic activity against PANC-1 human pancreatic cancer cells. Journal of Natural Products 73:623–627. Litwin A, Flanagan M, Entis G, Gottschlich G, Esch R, Gartside P, Michael JG. 1997. Oral immunotherapy with short ragweed extract in a novel encapsulated preparation: a double-blind study. Journal of Allergy and Clinical Immunology 100:30–38. 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Journal of Drugs in Dermatology 3:685–686. Tomás-Barberán FA, Ferreres F, Amparo Blazquez M, García-Viquera C, Tomas-Lorente F. 1993. High-performance liquid chromatography of honey flavonoids. Journal of Chromatography 634:41–46. Umthong S, Puthong S, Chanchao C. 2009. Trigona laeviceps propolis from Thailand: antimicrobial, antiproliferative and cytotoxic activities. The American Journal of Chinese Medicine 37:855–865. Umthong S, Phuwapraisirisan P, Puthong S, Chanchao C. 2011. In vitro antiproliferative activity of partially purified Trigona laeviceps propolis from Thailand on human cancer cell lines. BMC Complementary and Alternative Medicine. doi: 10.1186/1472-6882-11-37. Viljakainen L, Evans JD, Hasselmann M, Rueppell O, Tingek S, Pamilo P (2009) Rapid evolution of immune proteins in social insects. Molecular Biology and Evolution 26:1791–1801. Wannakul P (2007) Growth inhibition of microorganisms by Apis dorsata honey. 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Japanese Journal of Applied Entomology and Zoology 45:609–614. Chapter 37 Costa Rican Pot-Honey: Its Medicinal Use and Antibacterial Effect Gabriel Zamora, María Laura Arias, Ingrid Aguilar, and Eduardo Umaña 37.1 Introduction Honey is the natural sweet substance produced by honey bees from the nectar of flowers or extrafloral nectaries, or from excretions of plant sucking insects, which the bees collect and transform by adding specific substances of their own, dehydrate, and store in the honey comb to ripen and mature (Codex Alimentarius Commission 2001). Many studies have shown the honey of Apis mellifera possesses antimicrobial properties and also favors the healing of wounds and burns (Molan 1992; Bowler et al. 2001; Fournier et al. 2006; Aguilera et al. 2009). Nevertheless, stingless bee honey is locally considered to have stronger healing effects than the honey from A. mellifera of the same regions (de Jong 1999; Sommeijer 1999; Gonçalves et al. 2005; Boorn et al. 2009). The Mesoamerican region is the natural habitat for native stingless bees (Meliponini), acknowledged as indispensable pollinators with a key role in tropical forest conservation (Roubik et al. 1982; Roubik and Aluja 1983; Paxton 1995; Michener 2000; Slaa et al. 2006). Among them, the most commonly domesticated species are Melipona beecheii and Tetragonisca angustula. The Mayan and Aztec cultures started the keeping of these bees and used their honey for medicinal purposes (de Jong 1999; Vit et al. 2004). At present, treatment of infected wounds, digestive disorders, respiratory tract infection and eye problems like cataracts and G. Zamora • I. Aguilar (*) • E. Umaña Centro de Investigaciones Apícolas Tropicales (CINAT), Universidad Nacional, Apartado Postal, 475-3000 Heredia, Costa Rica e-mail: iaguilar@una.ac.cr M.L. Arias Centro de Investigaciones en Enfermedades Tropicales (CIET), Universidad de Costa Rica, 2060 Ciudad Universitaria Rodrigo Facio, San Jose, Costa Rica 507 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_37, © Springer Science+Business Media New York 2013 508 G. Zamora et al. conjunctivitis with the honey of stingless bees is widespread (Grajales et al. 2004; Vit et al. 2004, 2009). However, there are no studies that evaluate the medicinal properties of honey from stingless bees in Costa Rica. Due to the growing problem of antimicrobial resistance, it is of vital importance to discover innovative topical treatments for infected burns and wounds. This chapter provides updates on antibacterial activity of the pot-honey produced by several of our stingless bee species, and new data on M. beecheii and T. angustula, compared to A. mellifera. 37.2 Traditional Medicinal Use of Pot-Honey in Costa Rica The traditional use of honey collected by stingless bees as a medicine is deeply embedded in Costa Rican ethnopharmacology. This natural product remains a traditional medicine, since pre-Columbian times. At present, is still highly regarded as a burn and wound dressing and a topical treatment for cataracts and conjunctivitis (Kent 1984; de Jong 1999; Sommeijer 1999). Pot-honey collected by the stingless bee species T. angustula and M. beecheii have received the most commercial interest in Costa Rica. It is common to find stingless bee honey bottled in small dropper containers in natural medicine stores, sold at a substantially higher price than A. mellifera honey (Sommeijer 1996; Cortopassi-Laurino et al. 2006). Stingless bee honey in Costa Rica have the folk medicine reputation of having better medicinal properties as a burn and wound dressing than A. mellifera honey (DeMera and Angert 2004; Bijlsma et al. 2006). The ideal antimicrobial topical agent contains active constituents of a burn and wound dressing―inhibitory activity against common agents of infection, among other qualities (Bryskier 2005). In order to determine if the traditional value given to stingless bee honey over A. mellifera honey is valid, an evaluation over the antimicrobial activity of honey samples of T. angustula, M. beecheii, and A. mellifera was performed. 37.3 37.3.1 Comparative Study of Apis mellifera, Tetragonisca angustula, and Melipona beecheii Honey Honey Collection A total of 56 honey samples (500 g to 1 kg) collected from A. mellifera (n = 34), T. angustula (n = 14), and M. beecheii (n = 8) were obtained from producers. The honey under study belonged to several geographical locations were meliponiculture is practiced (see Table 37.1). All samples were kept in storage at 23°C, in a cool and dry place, away from light. 37 509 Costa Rican Pot-Honey: Its Medicinal Use and Antibacterial Effect Table 37.1 Geographical origin of 56 Costa Rican honey samples Bee species Region A. mellifera T. angustula Central Valley 8 7 Mountain South 12 – Central Pacific 2 – North Pacific 12 3 South Pacific – 4 Total honey samples 34 14 37.3.2 M. beecheii 1 – – 7 – 8 Evaluation of Antibacterial Activity Pot-honey solutions with final concentrations of 75, 50, 25, and 12.5% (w/v) were prepared in sterile peptone water 0.1%, pH 7.2. These solutions and pure honey were subjected to an antibacterial activity test following a Mueller-Hinton agar-well diffusion assay as described by Mitscher et al. (1972). A test solution was qualitatively considered antimicrobial if a clear zone without microbial growth was present surrounding the well after incubation. The analysis was conducted three times for all honey samples against the following American Type Culture Collection (ATCC) strains: Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Salmonella enteritidis (ATCC 13076), Listeria monocytogenes (ATCC 19166), and Pseudomonas aeruginosa (ATCC 9027). In addition, a clinical isolate of Staphylococcus epidermidis (UCR 2902) was included in the present trial. The results of antimicrobial activity evaluation are presented in Table 37.2. All descriptive and inferential statistics used InfoStat Software (InfoStat Group, Universidad Nacional de Córdoba, Argentina). A previous study performed by DeMera and Angert (2004) compared antimicrobial activity of honey produced by T. angustula and A. mellifera from Costa Rica. In their evaluation, S. aureus showed no susceptibility to any of the samples analyzed. In contrast, Estrada et al. (2005) reported 80% of A. mellifera honeys were active against S. aureus. By means of the same method, in our trial, all T. angustula, M. beecheii and 82% of A. mellifera honey exerted antibacterial activity against S. aureus. The present study shows no statistical difference (p > 0.05) from results presented by Estrada et al. (2005) for the inhibitory activity against S. aureus by A. mellifera honey. At a honey concentration of 25%, the differences observed in inhibition of S. aureus are statistically significant between A. mellifera and T. angustula (p < 0.05) and highly significant comparing A. mellifera to M. beecheii (p < 0.001). Hence, at lower concentration, stingless bee honey was more active against S. aureus. Moreover, at the lowest concentration tested, M. beecheii honey were the most active (p < 0.001). The results obtained for A. mellifera, T. angustula and M. beecheii honey, inhibitory against S. epidermidis and L. monocytogenes at a concentration of 50%, show 510 Table 37.2 Antibacterial activity of honey and pot-honey from Costa Rica Honey concentrations grouped by bee speciesa 100% 75% Bacterial strains Am Ta Mb Am Ta Mb Staphylococcus aureus 82 100 100 79 100 100 Staphylococcus epidermidis 85 100 100 76 100 100 Escherichia coli 97 100 100 85 86 89 Salmonella enteritidis 94 100 100 88 100 100 Listeria monocytogenes 79 100 100 47 100 89 Pseudomonas aeruginosa 9 93 100 0 86 100 Results are expressed as percentages of honey successful to inhibit bacterial growth a Am Apis mellifera, Ta Tetragonisca angustula, Mb Melipona beecheii 50% Am 71 38 74 85 9 0 Ta 100 93 7 7 50 21 Mb 100 100 67 56 67 78 25% Am 21 6 3 18 3 0 Ta 64 21 0 0 0 0 Mb 100 78 0 0 22 33 12.5% Am 0 0 0 0 0 0 Ta 7 0 0 0 0 0 Mb 78 0 0 0 0 0 G. Zamora et al. 37 Costa Rican Pot-Honey: Its Medicinal Use and Antibacterial Effect 511 significant differences (p < 0.05, p < 0.001 respectively). With 50% honey solutions, E. coli and S. enteritidis were the only cases in which A. mellifera was more active than T. angustula (p < 0.001). Nevertheless, there was no statistical difference between A. mellifera and M. beecheii (p > 0.05). Finally, the inhibitory effect on P. aeruginosa revealed a statistically significant difference in the results. The samples collected from both stingless bee species were more active than those of A. mellifera (p < 0.001, for 100 and 75% solutions). 37.4 Pot-Honey as Alternative Antibiotic The antibacterial effects presented herein invite further study of the nature of medicinal activity exerted by Costa Rican pot-honey. In general, these results exemplify the broad-spectrum antimicrobial activity of pot-honey from Costa Rica. Antibacterial activity towards S. aureus and P. aeruginosa was higher in T. angustula and M. beecheii pot-honey than in A. mellifera comb honey. The actual medical panorama reflects an increasing number of antibiotic resistant microorganisms that cause resilient disease (Bowler et al. 2001; Howell-Jones et al. 2005; Salyers and Whitt 2005). Under this turn of events, innovative therapies towards wound healing are urgent (Bryskier 2005) and pot-honey is an alternative treatment. References Aguilera G, Gil F, González AC, Nieves B, Rojas Y, Rodríguez AM, Vit P. 2009. Evaluación antibacteriana de mieles de Apis mellifera, contra Escherichia coli y Staphylococcus aureus. Revista del Instituto Nacional de Higiene 40:21–25. Bijlsma L, de Bruin LLM, Martens EP, Sommeijer MJ. 2006. Water content of stingless bee honeys (Apidae: Meliponini): interspecific variation and comparison with honey of Apis mellifera. Apidologie 37:480–486. Boorn KL, Khor YY, Sweetman E, Tan F, Heard TA, Hammer KA. 2009. Antimicrobial activity of honey from the stingless bee Trigona carbonaria determined by agar diffusion, agar dilution, broth microdilution and time-kill methodology. Journal of Applied Microbiology 108:1534–1543. Bowler PG, Duerden BI, Armstrong DG. 2001. Wound microbiology and associated approaches to wound management. Clinical Microbiology Reviews 14:244–269. Bryskier A. 2005. In pursuit of new antibiotics. pp.1242–1259. In: Bryskier A, ed. Antimicrobial agents: antibacterials and antifungals. American Society for Microbiology (ASM) Press. Washington, DC. 1456 pp. Codex Alimentarius Commission. 2001. Revised Codex Standard for honey. Codex STAN 12–1981, Rev.1 (1987), Rev.2 (2001). 24th Session of the Codex Alimentarius Commission. Cortopassi-Laurino M, Imperatriz-Fonseca VL, Roubik DW, Dollin A, Heard T, Aguilar I, Venturieri GC, Eardley C, Nogueira-Neto P. 2006. Global meliponiculture: challenges and opportunities. Apidologie 37:275–292. de Jong H. 1999. The land of corn and honey. The keeping of stingless bees (meliponiculture) in the ethno-ecological environment of Yucatan (Mexico) and El Salvador. Ph.D. thesis. Utrecht University. Utrecht, The Netherlands. 423 pp. 512 G. Zamora et al. DeMera JH, Angert ER. 2004. Comparison of the antimicrobial activity of honey produced by Tetragonisca angustula (Meliponinae) and Apis mellifera from different phytogeographic regions of Costa Rica. Apidologie 35:411–417. Estrada H, Gamboa M, Chaves C, Arias ML. 2005. Evaluación de la actividad antimicrobiana de la miel de abeja contra Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Salmonella enteritidis, Listeria monocytogenes y Aspergillus niger. Evaluación de la carga microbiológica. Archivos Latinoamericanos de Nutrición 55:167–171. Fournier AT, Gamboa M, Arias ML. 2006. Genes that encode botulism neurotoxins A,B,E and F in neotropical bee honey identified by the Polymerase Chain Reaction. Revista de Biología Tropical 54:29–34. Gonçalves AL, Alves Filho A, Menezes H. 2005. Actividade antimicrobiana do mel da abelha nativa sem ferrão Nannotrigona testacerconis (Hymenoptera: Apidae, Meliponini). Arquivos do Instituto Biológico. São Paulo. 72:445–459. Grajales CJ, Rincón M, Guzmán M, Vandame R. 2004. Propiedades físicas, químicas y antibacterianas de mieles de Scaptotrigona mexicana de la región Soconusco, Chiapas, México. Apitec. 42:22–24. Howell-Jones RS, Wilson MJ, Hill KE, Howard AJ, Price PE, Thomas DW. 2005. A review of the microbiology, antibiotic usage and resistance in chronic skin wounds. Journal of Antimicrobial Chemotherapy 55:143–149. Kent RB. 1984. Mesoamerican stingless bees. Journal of Cultural Geography 4:14–28. Michener CD. 2000. The bees of the world. John Hopkins University Press. Baltimore, MD. 913 pp. Mitscher LA, Leu RP, Bathala MS, Wu WN, Beal JL. 1972. Antimicrobial agents from higher plants. I. Introduction, rationale, and methodology. Lloydia 35:157–166. Molan P. 1992. The antibacterial activity of honey. International Beekeeping Research Association (IBRA). Cardiff, UK. 76 pp. Paxton R. 1995. Conserving wild bees. Bee World 76:53–55. Roubik DW, Aluja M. 1983. Flight ranges of Melipona and Trigona in tropical forest. Journal of the Kansas Entomological Society 56:217–222. Roubik DW, Ackerman JD, Copenhaver C, and Smith BH. 1982. Stratum, tree and flower selection by tropical bees: implications for the reproductive biology of outcrossing Coclospermum vitifolium in Panama. Ecology 63:712–720. Salyers AA, Whitt DD. 2005. Revenge of the microbes: how bacterial resistance is undermining the antibiotic miracle. American Society for Microbiology (ASM) Press. Washington, DC. 186 pp. Slaa J, Sánchez LA, Malagodi-Braga KS, Hofstede FE. 2006. Stingless bees in applied pollination: practice and perspectives. Apidologie 37:293–315. Sommeijer MJ. 1996. A regional programme for training and research on tropical beekeeping and tropical bees in Costa Rica. Bee World 77:3–7. Sommeijer MJ. 1999. Beekeeping with stingless bees: a new type of hive. Bee World 80:70–79. Vit P, Medina M, Enríquez E. 2004. Quality standards for medicinal uses of meliponinae honey in Guatemala, México and Venezuela. Bee World 85:2–5. Vit P, Gutiérrez MG, Rodríguez-Malaver AJ, Aguilera G, Fernández-Díaz C, Tricio AE. 2009. Comparación de mieles producidas por la abeja yateí (Tetragonisca fiebrigi) en Argentina y Paraguay. Acta Bioquímica Clínica Latinoamericana 43:219–226. Chapter 38 Immunological Properties of Bee Products José Angel Cova 38.1 Introduction Since ancient times, bee products have been used in medicine. Several reports have attributed anti-inflammatory, antitumoral and antioxidant properties to honey bee products (Majtán 2009; Attia et al. 2008; Bariliak et al. 1996; Rekka et al. 1990). Their mechanism of action often involves participation of the immune system, and it is important to know the impact of such substances in immune system defense, suppression and immunoregulation functions. In this chapter, we focus on the principal characteristics of the immune system and the impact of bee products on animal and human immune response. 38.2 Honey Bee Products and Innate Immune Response The immune system has been designed to protect animals from invader pathogenic microorganisms. Immunity—its main and unique function—has evolved until differentiating into two complementary forms: innate and adaptive. Innate immunity is considered the first line of defense against pathogenic microorganism such as bacteria, viruses, parasites and the cellular and humoral components of immunity are mainly located and distributed in the external surface of the body. Most of these components are present long before pathogen invasion or the infection’s settlement. Their molecular mechanisms are nonspecific and of short duration. They also cannot discriminate among different antigens, either nonself or J.A. Cova (*) Faculty of Medicine, Clinical Immunology Institute, Universidad de Los Andes, Mérida 5101, Venezuela e-mail: jacova@ula.ve 513 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_38, © Springer Science+Business Media New York 2013 514 J.A. Cova self in origin. They have no memory and their response remains unalterable even with the occurrence of repeated organism substance encounters (Abbas and Litchman 2005b; Goldsby et al. 2007). Innate immunity includes physical and anatomic barriers such as skin, the epithelial layer that covers intestinal, respiratory and urogenital tracts and some humoral and cellular components of the immune system. The humoral elements include complement system (CS) components, acute-phase proteins (APP) and Interferon, especially a and b interferon. The cells that participate in innate immunity are neutrophils, eosinophils, macrophages, natural killer cells (NK), dendritic cells (DC), mast cells, and endothelial cells, among others (Abbas and Lichtman 2005a). The complement system (CS) is a group of proteins, most of them synthesized in the liver, that circulate in an inactive state in sera and other body fluids. Several complement proteins are proteases that are self-activated by proteolytic cleavage (Janeway 2005; Trevani and Geffner 2005). The CS is activated through three different pathways: (1) the classical pathway (CP) which is activated through interaction between one of two isotypes of G or M immunoglobulin (IgG or IgM), bound to C1 complement component onto the microbial surface. As a cascade, activated C1 cleaves and activates C4, which activates C2 and subsequently activates C3. (2) The alternative pathway (AP) does not require antibody presence and is initiated in most cases by foreign cell-surface constituents: AP by means of factor D, factor B and properdin activates C3 to C5. (3) The mannan-binding-lectin (MBL) pathway is another route for complement activation. The MBL binds to mannose residues on glycoproteins or carbohydrates of the microbe surface and initiates complement activation in a similar manner than to C1, which resembles its structure. All these three pathways activate from C5 to C9 components of the system sequentially and form the membrane-attack-complex (MAC) which damages the membrane of pathogenic organisms. Thus, complement activation facilitates the clearance of bacteria through phagocytosis by macrophages and neutrophils. One of the most important complement activation components is C3 because of its role as a connector between the different pathways. The pharmacological intervention on C3 could switch all the system from an anti-inflammatory state to a pro-inflammatory state or vice versa (Trevani and Geffner 2005; Janeway 2005; Volanakis 1998). Acute-phase proteins (APP) are a family of proteins that include C-reactive protein, serum amyloid A protein, a-antiquimiotripsin, fibrinogen, and MBL and are produced by hepatocytes and macrophages in an inflammatory response. APP increase phagocytosis of opsonized bacteria, induce complement’s activation and inhibit bacterial proteases which help to eliminate dangerous microorganisms from the body (Goldsby et al. 2000). Interferon comprises a family of proteins produced by virus-infected cells. Interferon has many functions, especially one that enables its binding to nearby cells to induce a generalized antiviral state that prevents the spread of infection to other cells and organs. Other proteins involved in the humoral response belong to the cytokine and chemokins families such as tumor necrosis factor (TNF), interleukin 1b (IL-1), transforming growth factor-b (TGF-b), interleukin-12 (IL-12), interleukin-8 (IL-8), and others. These cytokines participate in the innate immune response and 38 Immunological Properties of Bee Products 515 their inhibition or increase by action of the honey bee products might modify the immune response. There is a growing interest to find molecules that induce the production of TGF-b by T regulatory cells in order to control several hypersensitivity reactions as arthritis and inflammatory bowel disease, among others. The function of cells that participate in the innate immunity is to recognize the pathogens when they invade the body. Cells have many different mechanisms to identify foreign invaders and most of these are based in the interaction between pathogens-associated-molecules-patterns (PAMP) present in the surface of microorganisms (viruses, bacteria, mycobacteria and parasites) and PAMP-recognizereceptors (PRR) also expressed in DC and macrophages surfaces. PAMP includes lypopolisaccharide (LPS), teicoic acid, non-methylated DNA, dsRNA, a class of molecules unique to microbes and are never found in multicellular organisms. Besides, PRR is placed in a different class of receptors that can activate a phagocyte cell after binding to PAMP as do the toll-like receptors (TLR) (Trevani and Geffner 2005, Akira et al. 2006). TLR4, as an example, recognizes the LPS of Gram-negative bacteria and initiates the activation of macrophages via MyD88-NFkB, which induces phagocytosis and secretion of proinflammatory cytokines (IL-1, IL-6, etc). Promising results of propolis usage to enhance TLR expression in cells have appeared as a new and exciting research area for natural medicine (Orsatti et al. 2010). Another mechanism to eliminate pathogens involves recognition of virusinfected cells and intracellular bacteria by the activation receptor (AR) expressed in natural killer (NK) cells. The AR includes NKp receptor group: NKR-P1, CD2, NKp30, and NKp44. Their binding to a specific ligand on target cells initiates a cytotoxic lysis. Whether the honey bee products can modify the expression of these receptors and enhance the lysis of cancer cells or virus by NK cells is a subject that requires further study. The results of studies about the effect of bee products from the honey bee in the immune system have been obtained under different conditions. These include varied botanical origin of compounds, extraction solvent (ethanol extraction vs. aqueous extraction), variable concentration of compounds, different times of incubation and different drug administration routes (peritoneal, subcutaneous, etc.). For this reason, interpretation of the cited evidence deserves to be analyzed very carefully. 38.2.1 Honey In humans, honey inhibits the basophil degranulation at high and low concentration levels of anti-IgE antibody used to stimulate them (Poitevin et al. 1988). With regard to this result, honey might be used as a homeopathic medicine in human allergic disease after controlled in vitro and in vivo assays. As a complex process, inflammation is studied using indicators of antiinflammatory activity, such as the lipoxigenase (LOX) essay. Salomón et al. (2011) studied the LOX inhibition by pot-honey of Tetragonisca fiebrigi, Scaptotrigona jujuyensis and Plebeia molesta from Northern Argentina (Chaco, Formosa, Misiones 516 J.A. Cova and Tucumán). The S. jujuyensis honey showed the most anti-inflammatory action, and positive correlations between radical scavenging activity and LOX inhibition (MI Isla, personal communication). 38.2.2 Propolis Honey bee products have been demonstrated to induce alteration in intracellular space and the cellular membrane. At the intracellular level, propolis decreases DNA synthesis in peripheral blood mononuclear cells (PBMC) including macrophages. In this report, propolis and its studied constituents were capable of suppressing DNA synthesis in dose-dependent phytohemagglutinin (PHA)-induced cells as well as in T cells. The production of cytokines (IL-1b, IL-2, IL-4, and IL-12) was also suppressed in these cells (Ansorge et al. 2003). However, when the macrophages are in the peritoneal compartment, propolis stimulates pro-inflammatory cytokine production, such as IL-1b and TNF-a in mice, after stimulation at a dose of 0.2–1 mg/ml (Moriyasu et al. 1994). These results show differences that could be explained based on the compartment in which cells are located. The immunosupressor effect of cyclophosphamide can be reversed at a dose of 50 mg/kg of propolis and could be possible via nonspecific immunity modulation through activation of macrophages (Dimov et al. 1991). In the complement system, propolis modulates the production of C1 complement component in macrophages after incubation at a dose of 0.150 mg/g (Dimov et al. 1992). It inhibits the classical and alternative pathways of the complement at higher doses (Ivanoska et al. 1995). Possibly, propolis causes inactivation or suppression of the one or more components of the complement and in this way diminishes the activity of these pathways. Georgieva et al. (1997) found compounds like flavonoids and phenolic substances with anticomplementary activities through inactivation of C3. Reactive-oxygen intermediate (ROI) and nitric oxide (NO) produce macrophages and activate neutrophils that help eliminate bacteria. Propolis increases generation of H2O2 in macrophages after incubation at doses of 5, 10 and 20 mg/ml. Otherwise, neutrophils obtained from rabbit decrease the superoxide anion (O2•−) production at different dose of propolis (range 2–25 mg/ml). In general, the production of NO is inhibited in macrophages treated with propolis (Krol et al. 1996). Also, in human neutrophils, propolis enhances the secretion of cytokines, both spontaneous and induced cytokine release, but plasma levels do not change (Orsi et al. 2000; Simoes et al. 2004). Commercial laying hens fed a diet supplemented with propolis show lower counts of heterophil cells (macrophage-like cells) than a control group. Likewise, this experiment demonstrates that supplementation with propolis improves performance and egg mass for commercial production (Galal et al. 2008). 517 38 Immunological Properties of Bee Products Table 38.1 Biological activity of pot-honey Effect Action mechanism Antiinflammatory Propolis and honey inhibits the production of nitric oxide by peritoneal macrophages Honey inhibits the oxidative burst in phagocytes cells Propolis and its component suppress prostaglandins and leukotriene production in murine peritoneal macrophages Antibacterial Propolis improves the bactericidal activity against Salmonella typhimurium on macrophages Propolis increases the bactericidal activity against Paracoccidioidis brasiliensis on macrophages Honey has antibacterial activity against Staphylococcus aureus Honey inhibits the H. pylori grow Antitumoral Propolis increases the NK cytotoxic activity against tumor Honey bee reduces tumor cells proliferation Adjuvant Propolis increases the specific antibodies production after vaccination with inactivated SuHV-1 vaccine preparation Reference Orsi et al. (2000); Kassim et al. (2010) Mesaik et al. (2008) Mirzoeva and Calder (1996) Orsi et al. (2005) Murad et al. (2002) Miorin et al. (2003) Ali et al. (1991) Sforcin et al. (2002) Attia et al. (2008) Fischer et al. (2007) Bullfrogs fed with propolis at 0.2, 0.5 and 1.0% of concentration in their diet significantly increase monocytes density in peripheral blood. However, other cellbasophils, neutrophils and eosinophils do not produce a statistical difference between groups (Romero et al. 2006). 38.2.3 Royal Jelly The antiinflammatory effects and immunomodulatory properties of glandular products secreted by worker honey bees (royal jelly) could ameliorate immunological disorders (ID) and act as an immunomodulatory agent. In fact, royal jelly treatment in lymphocytes from patients with Graves’ disease shifted the T helper cell Th1/Th2 cytokine ratio to the side of Th1 cytokine (Erem et al. 2006). Therefore, royal jelly may control tissue damage in the thyroid gland and induce remission in this disease. In conclusion, the major pharmacological activities of the products from bees have been focused on anti-inflammatory properties that induce immunosuppression. This effect modifies the innate immune response making it useful for treatment of hyper immune responses. Other biological activities attributed to bee-products are summarized in Table 38.1. 518 J.A. Cova 38.3 Action Mechanisms of Bee Products in the Adaptive Immune Response The adaptive immunity is a branch of the immune system developed to recognize and selectively eliminate foreign microorganisms (e.g., bacteria or viruses) and molecules. Unlike the innate immune response, adaptive immune responses are not the same in all members of a given species, but are reactions to specific antigenic challenges. Adaptive immunity displays four characteristic attributes: antigenic specificity, diversity, immunologic memory and self/nonself recognition (Goldsby et al. 2007). B-lymphocytes and T-lymphocytes are the cellular elements of the adaptive immune response, and they express specific receptors for antigens on the membrane: B-cell receptor (BCR) and T-cell receptor (TCR) in B-lymphocytes and T-lymphocytes, respectively. Each B cell or T cell clone will recognize only one antigenic structure (antigenic specificity). After the interaction between antigen and a specific cell receptor the cell is activated. After activation, the B or T lymphocyte will undergo a clonal expansion and produce millions daughter cells with identical antigenic specificity. In the case of B cells, the progeny undergo differentiation into memory B cells and effector B cells called plasma cells (Liu and Banchereau 1997). Memory B cells have a longer life span and circulate in the body until a reencounter with the antigen, followed by clonal expansion. Plasma cells live for a short time and produce enormous amounts of antibodies or immunoglobulins, secreted for binding to the antigen prior to their clearance by phagocytosis, and activation of complement and/or antibody-dependent cell-mediated cytotoxicity (ADCC). T-lymphocytes are divided into two well-defined populations known as T-helper (Th) or T/CD4+ and T-cytotoxic (Tc) or T/CD8+ cells. Following the stimulation, the Th cell can differentiate into Th1 cells (producing IL-2 and IFN-g), Th2 cells (producing IL-4 and IL-5), T regulatory cells (producing IL-10 and TGF-b) and Th17 cells (producing IL-17 and IL-6) (Mosmann and Coffman 1989; Murphy and Reiner 2002; Sakaguchi 2000; Harrington et al. 2005). Each subtype of Th cells and their pattern of secreted cytokines results in different types of immune response. For example, IFN-g—produced by Th1 cells—activates macrophages and helping to activation and expansion of naive CD8+ T cell transforms it into an effector cell called cytotoxic T lymphocyte (CTL). The CTL and macrophages play an important role in the defense against intracellular bacterial infection, virus-infected cells, tumor cells and cells of a foreign tissue graft (Abbas and Litchman 2005c). The Th2 cell and IL-4 secreted by them induces activation and differentiation of the B-lymphocyte into a plasma cell that secretes antibody into the extracellular space. This type of immune response is important in the control of helminth parasites, along with extracellular bacterial and some viral infections (Liu and Banchereau 1997). The immune response must terminate when the pathogen or parasite is eliminated or controlled if the antigen persists. The T regulatory cells (Treg) participate in regulating the immune response by at least two types of interaction. First, Treg produces immunosuppressive cytokines like IL-10 and TGF-b. Second, Treg interacts 38 Immunological Properties of Bee Products 519 with T effector cells through cell-cell contact and delivery inhibitory signal into activated Th cells (Thornton and Shevac 1998; Sakaguchi 2000). From this knowledge, new pharmacological applications for honey, propolis and royal jelly could be investigated through in vitro and in vivo studies. The influence of these social bee products after incubation of different duration and concentration with the immune cells can be measured using diverse techniques: expression of new molecules in the membrane of cells by flow cytometry, detection of RNA by molecular biology methods, and studies of protein by proteomics, among others. The compounds found in the honey may be useful to treat maladies in which immune system dysfunction is responsible for the disease. 38.3.1 Royal Jelly and Propolis Modify the Adaptive Immune Response In lymphocytes using proliferation assay, Del Valle-Pérez et al. (2001) do not observe changes in lymphocyte proliferation after incubation of cells with royal jelly. Instead, propolis diminishes DNA synthesis and is able to suppress IL-2 (pattern-Th1 cytokines) and IL-4 (pattern-Th2 cytokines) in T-lymphocytes, revealing an antinflammatory action. Moreover, TGF-b, an immunosuppressor cytokine, is enhanced after propolis incubation, indicating T regulatory cell activation (Ansorge et al. 2003). This could be the explanation for antinflammatory properties, inhibition of NO production and respiratory cell burst observed after incubation with propolis. The effect of stingless bee products in other Th cells (Th17, Th9, and Th22) has not been studied yet. On the other hand, Ivanoska et al (1995) observed a proliferative tendency in splenocytes incubated with propolis. Further propolis inhibited proliferation in Con A-stimulated cells compared to a control group in experiments with mice. Treatment with honey or propolis administered to Newcastle disease virus NDVinfected chicken produces an increase in the amount of antibodies as well as higher percentage of macrophages, both in sera. Likewise, the mortality rate is reduced in groups infected with virulent NDV and subsequently treated either with propolis or honey, if compared with the infected group only (Hegazi et al. 1995, 1996). With B lymphocytes, the evidence suggests an increase in antibody (Ab) production by cells after incubation with bee-products. Propolis increases Ab production in mice immunized with sheep red blood cell in different amounts (Scheller et al. 1998). Similarly, the administration of propolis at 10% concentration to rats significantly increases antibody titres, even after 15 days of immunization (Sforcin et al. 2005; Hegazi et al. 1997). Propolis and other bee products seem to have adjuvant activity in the adaptive immune response. The production of antibodies might be induced after the action of honey on macrophages which activate, directly or by soluble signal, B-cells that transform into antibody producing-cells. Further research will give insight to understanding the participation of honey bee-products in immune response. 520 38.4 J.A. Cova Future Perspectives to Use Honey Bee Products in Treatment of Immune Diseases The main immunological diseases comprise allergy, autoimmune disease and immunodeficiency. Both allergic and autoimmune diseases are mediated through a hypersensitivity mechanism and inflammation plays a critical role in pathogenesis. On the other hand, immunodeficiency occurs by defects in the elements or organs of the immune system. The inhibition on cyclooxygenase-2 (COX) induced by honey and propolis may improve the inflammatory process in autoimmune diseases (Viuda-Martos et al. 2008). Other targets to treat autoimmune diseases have been discovered, such as cytokines, cellular receptors, intracellular signals and pro-apoptotic molecules. One of them is IL-17secreted by Th17 cells that participate in chronic inflammation observed in the autoimmune diseases. IL-17 induces the production of inflammatory cytokines by synovial cells, recruitment of leukocytes into inflamed joints, upregulation of matrix metalloproteinase, and nitric oxide causing destruction of tissue and bone in rheumatoid arthritis (RA). The effect of propolis, royal jelly and honey in IL-17 secretion and Th17 cell proliferation need to be studied. For example, the expression of cellular markers and IL-17 secretion in T-lymphocyte culture from patients with RA using flow cytometry techniques and ELISA assays may provide insight for the treatment of RA. The production of antibodies against self-proteins is involved in pathogenesis of autoimmune diseases due the breakdown of tolerance mechanisms. Briefly, an autoreactive B-cell is activated following interaction of self-protein-MHC II complex and costimulatory signal (B7 and CD40) instead the inhibitory signal (BTLA). It induces transformation of B-cells into plasma cells that produce immunoglobulin which reacts against self-protein. Given the action of bee products over B-cells, new therapeutic approaches using these products seem highly promising. Allergic disease is a worldwide health problem and is increasing in many countries. The hypersensitivity reaction is initiated by antigen-presenting cells that internalize, process, and present allergic protein (allergen) to specific T-lymphocytes, inducing activation of those cells. By action of IL-4, cells proliferate and differentiate into Th2 cells secreting cytokines for the stimulation of B-cells. Following this, B-cells undergo immunoglobulin gene class switching, leading to their terminal differentiation into plasma cells that produce antigen-specific IgE antibodies. Once released by plasma cells, antigen specific IgE binds to the high-affinity IgE receptor in mast cells and basophils, leading to sensitization of these cell types. When mast cells and basophils with such IgE on their surface come in contact with native protein antigen, they are induced to degranulate, releasing histamine, tryptase, proteoglycans, serotonin, and other compounds. All of these are responsible for allergic symptoms. In allergic diseases, the influence of honey in inhibition of basophils and mast cells, lower expression of CD63, and lower levels of IgE, among others, will clearly be essential to demonstrate and understand, in order to design effective treatment and sound experiments. 38 Immunological Properties of Bee Products 38.5 521 Conclusions Bee products, known primarily from the honey bee but now starting to be investigated with stingless bees (as revealed in many chapters of the present book) contain various active compounds responsible for many positive effects in both normal and altered immune systems. A new avenue of experimental studies should be designed in order to evaluate the immunological effects of pot-honey in the different forms of the immune response, recently initiated in Argentina by evaluating the anti-inflamatory action on one key enzyme. Likewise, testing the effects of bee products on immunological disorders through clinical studies might provide us a new class of drugs to be employed in allergy and autoimmune disease treatment. References Abbas A, Lichtman A. 2005a. General properties of immune response, pp. 4–6. In Abul K. Abbas and Andrew H. Lichtman, eds. Cellular and molecular immunology 5th edition. Elsevier Saunders, Philadelphia, USA. 563 pp. Abbas A, Litchman A. 2005b. Innate immunity, pp. 275–292. In: Abbas AK, Andrew Lichtman AH, eds. Cellular and molecular immunology 5th edition. Elsevier Saunders; Philadelphia, USA. 563 pp. Abbas A, Litchman A. 2005c. Effector mechanism of cell-mediated immunity, pp. 298–317. In: Abbas AK, Andrew Lichtman AH, eds. Cellular and molecular immunology 5th edition. Elsevier Saunders; Philadelphia, USA. 563 pp. Akira S, Uematsu S, Takeuchi O. 2006. Pathogen recognition and innate immune. Cell 124:783–801. Ali A, Chowdhury M, Al-Humayyd M. 1991. 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Medical Immunology 20:9–32. Chapter 39 Chemical Properties of Propolis Collected by Stingless Bees Ömür Gençay Çelemli 39.1 Introduction Propolis is known in folk medicine since ancient times. Egyptians benefited from anti-putrefactive properties of propolis in order to embalm their dead. It was used as an antiseptic and healing agent by Greek and Roman physicians. Incas employed propolis as an anti-pyretic agent, and the London Pharmacopeia of the seventeenth century listed propolis as an official drug (Ghisalberti 1979). Studies on composition and biological properties of propolis reveal the interest of researchers on this bee product and its potential for the development of new drugs as well (Sforcin and Bankova 2011). Natural products are a promising source for the discovery of new pharmaceuticals. In the last decades, propolis has received regard for its potential in medicine and cosmetics, even if it is known primarily only in folk medicine and ancient times. The antimicrobial properties of propolis have been widely investigated, confirming its antibacterial, antiviral, and antifungal activities (Sforcin et al. 2000). Stingless bee propolis is used in folk medicine for the healing properties on digestive and respiratory systems, female fertility, skin and visual disorders. Pollen of stingless bees has also therapeutical uses, and the larvae of Melipona and other stingless bee genera are consumed in local diets (Freitas et al. 2008). Stingless bee honeys attract attention of researchers for their importance as foodstuffs and traditional remedies in folk medicines (Vit et al. 2004). For example, honey from Tetragonisca angustula bee is highly appreciated for its pleasant flavor and is used for the treatment of respiratory, eye infections, and anti-cataract properties (Torres et al. 2004). The aim of this study is to determine the chemical composition of ethanol extracts of propolis collected by the stingless bees Melipona favosa from Venezuela, O. Gençay Çelemli (*) Department of Biology, Hacettepe University, 06800 Beytepe, Ankara, Turkey e-mail: gencay@hacettepe.edu.tr 525 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_39, © Springer Science+Business Media New York 2013 526 O. Gençay Çelemli Melipona grandis, Scaptotrigona depilis, and Scaptotrigona polysticta from Bolivia, and Tetragonula biroi from Philippines. The chemical composition of the propolis of stingless bees is still not clear. Therefore our results will be a step toward the identification of the chemical profile of stingless bee propolis, needed for further applications. Also with this study we can compare the chemical profile of propolis collected by five different stingless bee species using gas chromatography–mass spectrometry (GC–MS). Using GC–MS analysis we can easily observe the volatile profile of terpenes to consider whether there is a variation in chemical composition of propolis samples among bee species. 39.2 Why Are Resins Collected by Honey Bees and Stingless Bees? Resin, a sticky plant substance, is produced by various plant families and is secreted in response to an injury or infection of plant parts. However, resin secretion can also occur spontaneously, as has been shown for the tropical legume Hymenaea (Fabaceae, Caesalpinioideae) (Langenheim et al. 1978). Resins of different botanical origin serve as a deterrent against herbivorous insects, such as lepidopteran larvae (Hymenaea resin), as well as against ants (Pinus Pinaceae resin), termites (“guayule” Parthenium argentatum Asteraceae, gray resin), bacteria ( floral Clusia Clusiaceae resin), and fungi (Dipterocarpus Dipterocarpaceae; guayule pine resin). This deterrent function is most likely due to the presence of terpenes, especially mono- and sesquiterpenes (Leonhardt and Blüthgen 2009). Some plant species use resin as an attractant for pollinators and seed dispersers (birds, mammals, reptiles, ants, bees) (Wallace and Trueman 1995). They secrete resin both to defend themselves against herbivores and to attract bees. In Borneo (Malaysia), bees use resin and resin-derived compounds not only to build and defend their nests but also to enrich their cuticular/chemical profiles (Leonhardt et al. 2011). Cuticular lipids are thought to preserve insects from desiccation, cuticle abrasion, and infection. In several insect taxa, cuticular lipids have become further involved in the communication system by enabling them to reliably differentiate between friend and foe or find a mate based on differences in the chemical composition of cuticular profiles (Leonhardt et al. 2009). The prominence of resin-derived compounds on the bees’ body is unique to stingless bees and has not been described in any other social insect (Leonhardt et al. 2011). Propolis is any resinous mixture or pure resin substance collected by bees and stored within the nest for construction and defense purposes (D. Roubik, personal communication). These natural resinous products collected by foraging bees, from various plant sources, are used to build, strengthen, isolate, and disinfect their nests to fill holes and to embalm dead predators inside the hives (Simone and Spivak 2010). Cerumen is a mixture of wax, and plant resins, potentially enriched with 39 Chemical Properties of Propolis Collected by Stingless Bees 527 stingless bee secretions. There is suggestive evidence that stingless bees add cephalic gland secretions during cerumen production (Massaro et al. 2011). Cerumen storage pots can expand and contract without breaking during fermentive processes (P. Vit personal communication). In some literature meliponine propolis is called geopropolis (Barth 2004). While propolis is prepared by Meliponini using resins of plants mixed with waxes and sometimes mud, honey bees (Apis mellifera) do not use soil material when preparing propolis (Barth and Luz 2003), and most stingless bees do not add wax or clay and earth. The presence of silica and clay and absence of trichomes was used, besides pollen grains, to differentiate propolis of Meliponini from propolis of A. mellifera (Barth 2004). However, Trigona workers (Meliponini) collect Maxillaria rufescens trichomes (Singer et al. 2004). On the contrary Barth and Luz (2003) investigated ten geopropolis samples collected from three meliponine species in Brazil. They observed pollen grains, hyphae and spores of fungi, organic material and burned plant fragments in most of the samples. Sandy or earth materials were present in all geopropolis sediments. Only one sample contained plant trichomes and was considered a propolis sample of A. mellifera. Propolis is mainly used by honey bees and stingless bees to protect the nest against infection and also as a multipurpose cement and varnish. Social immunity, which describes how individual behaviors of group members effectively reduce disease and parasite transmission at the colony level, is an emerging field in social insect biology (Finstrom and Spivak 2010). “Hygienic behavior” first described for honey bees is now a classical example of a social defense, whereby workers identify and remove infected larvae from among the healthy brood. Other defenses enabled by sociality include the construction of nests from antimicrobial materials, the raising of offspring in sterile nurseries, social “fever” in response to disease, transference of immune traits, and heightened risk-taking by infected individuals. Like most eukaryotes, colony members also possess individual defenses, including immune responses toward disease agents (Evans et al. 2006). 39.3 How Do Bees Collect Resins to Produce Propolis and Cerumen? The sticky resin known as propolis or bee glue is a material collected from plants by foraging workers. Bees break off pieces of the resinous exudates from the plants, using their mouth parts. The pieces are moistened with the tongue and formed into pellets by the mandibles with the help of the legs. The bees bring the pellets from the mouth along the hair of the tibia and into the corbiculae. While packing one piece of propolis into the corbicula, the bee is simultaneously collecting more. The collection of propolis could take a long time and might be interrupted by visits to the nest for feeding. When the corbicula has been filled, the bees deliver the propolis to the nest (Ghisalberti 1979). Resin foragers have shown a preference for O. Gençay Çelemli 528 young leaves and vegetative buds over older leaves. The process of obtaining a full corbicular load of resin has been noted to take about 7 min, but can extend up to 1 h depending on the weather. Unloading the resin from corbiculae in the nest is a process that typically takes approximately 15 min, but can extend to 7 h or even overnight (Finstrom and Spivak 2010). Stingless bees are the major visitors of many flowering plants in the tropics (Heard 1999). Some stingless bees may also incorporate mud, fecal material, chewed plant matter, and artificial products such as tar into their nest (Wallace and Trueman 1995; Roubik 2006). Plant resins are an essential resource for nest building and defense. In contrast with pollen and nectar flows, resin resources are generally unpredictable and short-lived and are aggressively defended by some species (Wallace and Lee 2010). To reveal factors that influence bee decisions about where and when to collect resin, resin collection was studied in ten stingless bee species by Leonhardt and Blüthgen (2009) in Southeast Asia. Bees prefer resins of particular trees and neglect resins of others. Most trees offering resins to be collected by bees belonged to the Dipterocarp family. Dipterocarps are highly resinous, and their resin is known to inhibit the growth of pollen-associated fungi (Leonhardt and Blüthgen 2009). Stingless bees appear to use the same mechanism and compounds to locate and recognize resin sources as honey bees do (Leonhardt et al. 2010). Stingless bees also important for seed dispersal of three plant species as of a rain forest eucalypt Corymbia torelliana (see Chap. 3). 39.4 Botanical Origin of Propolis Single or compound hairs from plants (trichomes), especially leaves, are commonly an additional component of propolis. Some of these indicate the plant species from which they were collected by their morphological characteristics (Ricciardelli D’Albore 1979). When bees prepare propolis from plant exudates pollen grains already are present, and with contact from bees and their nest, more are introduced. These pollen grains come from the flowers visited by bees for nectar and pollen and also from wind pollinated plants. Because of this, identification of the plant species, whose pollen occurs in propolis samples, allows a characterization of the surrounding vegetation, and frequently the geographical region from which the resin was collected (Warakomska and Maciejewicz 1992). Pollen analysis, besides chemical analysis, is a method used to characterize regionally different propolis samples (presenting different characteristics of hardness, elasticity, smell and colour). It is a good tool for defining the phytogeographical origin of resins and quality of the propolis (Barth and Luz 2003). Meliponini collect not only resin but also clay and, in separate loads, the latex of fruits of Coussopoa Moraceae (formerly thought to be Vismia Clusiaceae), for propolis confection (Barth 2004). It is possible to characterize the environmental conditions and the vegetation around the apiary using pollen from propolis, as well as the trophic preferences for 39 Chemical Properties of Propolis Collected by Stingless Bees 529 some of the bees (Barth 2006). Barth and Luz (2003) investigated ten samples of Brazilian propolis from three species Melipona quadrifasciata, Melipona orbygnii (sic, = orbignyi), and T. angustula, and two of the samples did not contain pollen grains. With one exception, propolis samples had no trichomes. Different sized grains of sand and/or small particles of soil were detected in all samples examined; these are of earth material utilized by the Meliponini in preparing propolis. With the exception of two samples, they observed pollen grains in all samples. About 64 pollen types could be identified, 22 occuring at a frequency of more than 3%. Pollen grains of Eucalyptus (Myrtaceae) and Schinus (Anacardiaceae) were dominant in several propolis samples. Barth (2006) analyzed six samples of propolis that showed different physicochemical properties. Only 21 pollen types occured with a frequency higher than 3% and only four with more than 25% of the pollen sum. According to their results, forest taxa were represented by Anacardiaceae, Anadenanthera (Fabaceae, Mimosoideae) and Aceraceae and open-land vegetation by species of Asteraceae, Poaceae, Alternanthera (Amaranthaceae), Scrophulariaceae, and Typha (Typhaceae). The chemical composition of propolis depends on the phytogeographic characteristics of the site of collection, because the bees choose different plants as sources of resins in different habitats. Thus, the complex standardization of propolis should relate biological properties to a detailed investigation of chemical composition and botanical sources (Bankova 2005; Sforcin and Bankova 2011). 39.5 Chemical Composition and Biological Properties of Propolis Most components of bud exudate are incorporated into propolis without alteration, although it is possible that some glucosides are subjected to enzymic hydrolysis by the bees either during collection of the bud exudate or during its addition to beeswax to make propolis (Greenaway et al. 1987). The compound groups identifed from propolis are: amino acids (researchers suggest that the traces of amino acid present in propolis come from the bees), aliphatic acids and their esters, alcohols (of these the a- and b-glycerophosphate probably derive from bee metabolism, the glycerol from wax and other components from bud exudate), aldehydes, chalcones (the chalcones are related to the flavanones and may be formed from them during propolis manufacture and during preparation and analysis of samples), dihydrochalcones, flavanones (these compounds, together with flavones, are often mentioned as having antimicrobial properties), flavones, hydrocarbons (the C 25 and C 27 hydrocarbons are common in poplar bud exudates), but it is likely that in propolis these compounds are derived both from bee metabolism and from bud exudates. It is believed that other hydrocarbons arise primarily from bee metabolism, ketones, terpenoids (the volatile C 10 terpenoids have strong odours and this group of compounds may be responsible for much of the odour of propolis), and sugars (such as glucose, fructose, and sucrose) are frequently present in propolis. 530 O. Gençay Çelemli It is suspected that these are due to contamination by honey (Greenaway et al. 1990). Therefore, some compounds of propolis originate from bee metabolism (e.g., alcohols, hydrocarbons) and others from plant exudates (e.g., terpenes, flavonoids). Researchers find it puzzling that European and Ecuadorian propolis are very different. Ecuadorian propolis contains neither the aromatic acids and esters nor the flavones and flavanones, indicated as the active antimicrobial principles of European propolis. Greenaway et al. (1990) compared propolis from colonies of native stingless bees (Melipona, Nannotrigona tristella, Scaptotrigona and Tetragonisca) and from A. mellifera. The unique phenolic compound in propolis of N. tristella and Melipona is 3,5-dihydroxybenzoic acid. They speculate exudates incorporated by Ecuadorian stingless bees probably come from flowers of Dalechampia and Clusia— although seldom visited by Nannotrigona and Melipona, which have evolved within their flowers special structures secreting a resin which is attractive to bees. There is another medical aspect of propolis: it may cause allergic reactions in susceptible persons. Prenyl caffeate (1,1-dimethylallyl caffeic acid ester) has been particularly identified as a contact allergen. This compound occurs in poplar bud exudates in varying amounts (Burdock 1998). Propolis from the honey bee A. mellifera is used in folk medicine in the countries of Eastern Europe as an antiseptic and anti-inflammatory agent for healing wounds and burns. There are limited indications that propolis from Meliponini can be used in the same way (Velikova et al. 2000). As a natural product of the bee colony, propolis possesses several biological activities such as anti-inflammatory, immunostimulatory, and antibacterial activity especially against Gram-positive bacteria. This activity is reported to be due to flavonoids, aromatic acids, and esters present in the resin (Marcucci et al. 2001). Ethanol extracts of propolis (EEP) are rich in various flavonoid aglycones, phenolic compounds, sesquiterpenes, steroids, amino acids, and inorganics—including trace—elements (Krol et al. 1993). Pereira et al. (2003) compared the propolis collected by A. mellifera and T. angustula, in southeastern Brazil. They found a total of 64 compounds. Both propolis samples were almost entirely comprised of pentacyclic triterpenes, mainly lupeol and lupeol acetate. On the other hand, polar compounds differed in propolis collected by A. mellifera and T. angustula. They identified seven amino acids (alanine, glicine, valine, isoleucine, leucine, proline, and threonine) from only propolis of A. mellifera. The main differences between the two propolis samples were the concentrations of an aldotetrol, characterized as erythritol (1.8% A. mellifera, T. angustula 4.0%). Analysis of propolis from Friesomellita varia, M. favosa, Melipona compressipes, Scaptotrigona depilis, and Paratrigona anduzei in tropical Venezuela revealed a phenolic profile characterized by polyprenylated benzophenones. In the chemical investigation of propolis of M. compressipes, M. quadrifasciata anthidioides, and Tetragona clavipes by GC–MS analysis, diterpenic acids were found in all samples, and their amounts were significant in M. quadrifasciata anthidioides and T. clavipes. On the other hand, the pentacyclic triterpene b-amyrin was identified as the main 39 Chemical Properties of Propolis Collected by Stingless Bees 531 component in T. clavipes, the flavonoid pinobanksin in M. compressipes and aromatic aldehydes in Melipona quadrifasciata anthidioides, respectively. Suprisingly, the prenylated benzophenones characteristic of propolis from Venezuela were absent in propolis from Brazil, including the one from M. compressipes that was analyzed in both tropical areas (Freitas et al. 2008). Farnesi et al. (2009) examined the antibacterial activities of several types of propolis, including Africanized honey bee green propolis and propolis produced by meliponine bees. They concluded that these resins have the potential for human and veterinary medicine. Massaro et al. (2011) contrasted the extensive research on therapeutic properties of honey bee propolis with the largely unknown biological and medicinal properties of stingless bee propolis. These authors investigated the chemical and biological properties of polar extracts of cerumen from Tetragonula carbonaria in South East Queensland, Australia using GC–MS analyses. Distinct GC–MS fingerprints of a mixed diterpenic profile typical of native bee cerumen were obtained with pimaric acid (6.31 ± 0.97%, w/w), isopimaric acid (12.23 ± 3.03%, w/w), and gallic acid (5.79 ± 0.81%, w/w) tentatively identified as useful chemical markers. Characteristic flavonoids and prenylated phenolics found in honey bee propolis were absent in cerumen of T. carbonaria. 39.6 39.6.1 Chemical Composition of Stingless Bee Propolis from Bolivia, Philippines, and Venezuela Propolis Samples Geographical origin and time of propolis collection are listed in Table 39.1. Eight propolis samples (Fig. 39.1) were investigated to determine their chemical composition by GC–MS. Three propolis samples were from Venezuela (M. favosa), one from Philippines (T. biroi), four from Bolivia (M. grandis, S. depilis and S. polysticta). Table 39.1 Stingless bee species and geographical origin of the propolis samples Sample no. Common name Stingless bee species Propolis type 1 “erica” Melipona favosa Hive 2 “erica” Melipona favosa Hive 3 “erica” Melipona favosa Hive 4 “kiwot” Tetragonula biroi Hive 5 “erereú barcino” Melipona grandis Hive 6 “obobosí” Scaptotrigona depilis Hive 7 “obobosí” Scaptotrigona depilis Hive 8 “suro negro” Scaptotrigona polysticta Entrance tube Country Venezuela Venezuela Venezuela Philippines Bolivia Bolivia Bolivia Bolivia O. Gençay Çelemli 532 Fig. 39.1 Propolis samples of stingless bees from Venezuela, Philippines, and Bolivia. See Table 39.1 for propolis sample numbers (Photos Omur Gençay Çelemli) 39.6.2 Propolis Extraction and Preparation Frozen propolis was pulverized and dissolved in 96% ethanol. This mixture was kept in the incubator at 30°C for 2 weeks, in a bottle closed tightly. After incubation, supernatant was filtered twice through Whatman No. 4 and No. 1 filter paper. The final filtered concentrated solution (1:10, w/v), ethanol extracts of propolis (EEP), was evaporated until dry. About 5 mg of residue was mixed with 75 ml of dry pyridine and 50 ml bis (trimethylsilyl) trifluoroacetamide (BSTFA), heated at 80°C for 20 min, then the final supernatant was analyzed by GC–MS. 39.6.3 GC–MS Analysis A GC 6890N from Agilent (Palo Alto, CA, USA) coupled with mass detector (MS5973, Agilent) was used for the analysis of EEP samples. Experimental conditions 39 Chemical Properties of Propolis Collected by Stingless Bees 533 of the GC–MS system were as follows: DB 5MS column (30 mm × 0.25 mm and 0.25 mm of film thickness), flow rate of mobile phase (He) set at 0.7 ml/min. For gas chromatography, temperature was kept for 1 min at 50°C and then increased to 150°C with a 10°C/min heating ramp. After this period, temperature was kept at 150°C for 2 min. Finally, temperature was increased to 280°C, with a 20°C/min heating ramp, then kept at 280°C for 30 min. Organic compounds in samples were identified using standard Wiley and Nist Libraries, available in the data acquisition system of GC–MS, if the comparison scores were higher than 95%, or our own library. For ethanol extracts, instead of internal or external standards, percentage sample compounds were used. This standard was primarily used to identify organic compounds in propolis samples; the error could not be higher than 5% (Gençay and Salih 2009). 39.6.4 Chemical Components of Stingless Bee Propolis Compounds of aliphatic acids and their esters, alcohols, aromatic acids and their esters, hydrocarbons, and terpenes were identified. In Venezuela, where Populus are not native plants, stingless bees and honey bees visit Clusia species in order to collect a resin excreted in a ring at the bases of their flower stamens. As a consequence, the chemical composition of both tropical propolis and stingless bees’ propolis is particularly characterized by the presence of polyprenylated benzophenones, in accordance with the chemical constituents identified from Clusia flowers (TomásBarberán et al. 1993; Freitas et al. 2008). But in our Venezuelan samples from M. favosa too few compounds were observed. Particularly in two samples we could not find any compound. In the third sample, 6,6,10-trimethyl-1-phenylthiospiro (3.6) dec-1-ene, a hydrocarbon, was the only compound identified. Due to these results we can say that these samples can be only clay, earth or soil, and did not include resin. The Philippine propolis ethanolic extract of T. biroi (sample 4, Fig. 39.1) contained aliphatic acids and their esters, alcohols, carboxylic acids and their esters. Terpenes also were observed. From aliphatic acids and their esters group; ethyl oleate, octadecanoic acid, ethyl ester, hexadecadien-1-ol acetate, linoleic acid ethyl ester, and ethyl tridecanoate compounds were identified. From these compounds, ethyl oleate showed the highest ratio of 4.51%. The T. biroi propolis (sample 4, Fig. 39.1) had lower terpene content than the propolis from Bolivia (samples 5–8, Fig. 39.1) but higher than the propolis of M. favosa (samples 1–3, Fig. 39.1) without terpenes. According to Table 39.2, we can say higher aliphatic acids and their esters group ratio could be a marker for Philippine propolis. However, there is a preliminary observation for Philippine propolis because we could investigate only one sample. Half of the propolis samples (samples 5–8) were collected by different bee species (Table 39.1) from Bolivia (Fig. 39.1). In these propolis we observed aliphatic acids, alcohols, carboxylic acids and their esters, hydrocarbons, and terpenes. A common trait of the Bolivian propolis was the presence of terpenes in high ratios 534 O. Gençay Çelemli Table 39.2 Chemical compound groups determined in the Meliponini propolis types Propolis typesa Compound groups 1 2 3 4 5 6 7 Aliphatic acids and – – – 5.48 2.35 3.62 0.34 their esters Alcohols – – – 0.45 5.64 0.06 4.89 Carboxylic acids and – – – 1.69 – 2.09 0.03 their esters Hydrocarbons – – 10.17 – – 3.47 0.34 Terpenes – – – 3.72 20.91 45.37 39.99 a See Table 39.1 for the stingless bee species that collected the propolis 8 0.06 22.22 0.08 0.35 6.92 (6.92–45.31%). The highest terpene ratio was observed in sample 6, from S. depilis (45.37%) and sample 7, from S. depilis, collected inside the hive (39.99%). Aliphatic acids and their esters, as we found in our study (see Table 39.2), are known for stingless bees (Velikova et al. 2000). From this group we found octadecanoic acid, as Pereira et al. (2003) found in propolis of T. angustula. Similiar to our results, in previous studies flavonoids were not observed in propolis of stingless bees (Massaro et al. 2011). These authors compared cerumen of T. carbonaria and propolis of A. mellifera and suggest that gallic acid and diterpenic acids of the pimaric and abietic type are chemical markers of T. carbonaria. Cinnamic acid, monosaccharide, gluconic acid, fructose, b-glucose, p-coumaric acid, and monosaccharide were present in both types of propolis. However, other propolis constituents such as p-hydroxybenzoic acid, hydroxybenzoic ester, monosaccharide, ferulic acid, caffeic acid, pentenyl ester iso-ferulic acid, pentenyl ester caffeic acid, pentenyl ester caffeic acid isomer, pinostrobin, pinocembrin, sterol, cinnamic acid ester, dihydroxy-2-methyl-anthroquinone, and galangin were not found in T. carbonaria cerumen. The researchers found mainly pimaric acid, isopimaric acid, and gallic acid in the cerumen of T. carbonaria. We did not find any of these compounds. We found the terpene delta-cadinene in two Bolivian samples from Scaptotrigona (samples 6 and 7). Patricio et al. (2002) observed this compound in the tibia of F. silvestrii and F. varia (Patricio et al. 2002). Another terpene observed in our results was germacrene D. This compound was found in the tibia of F. varia in previous studies (Patricio et al. 2002). We also observed mostly terpenic compounds. From this chemical group, similiar to our results, gamma-Terpinene, a-Amyrin, a-Caryophyllene, b-Amyrin, a-Gurjunene, a-Copaene were identified in previous studies (Freitas et al. 2008; Patricio et al. 2002). From terpenes, b-Amyrin was mostly observed in propolis of stingless bees in previous studies. Patricio et al. (2002) isolated b-Amyrin from the tibia of F. varia. Furthermore, Freitas et al. (2008) identified b-Amyrin as the main component of geopropolis of Tetragona clavipes, and Massaro et al. (2011) found this compound in cerumen of T. carbonaria. We observed b-Amyrin only in S. depilis (sample 7) but with a higher ratio (11.75%). With regard to our GC–MS results, we can say that the Bolivian ethanol extracts of Scaptotrigona have a richer and more concentrated chemical spectra 39 Chemical Properties of Propolis Collected by Stingless Bees 535 than the M. favosa from Venezuela and the T. biroi from the Philippines. Therefore, the thesis that different bee species use different plant sources to collect propolis is potentially correct, but needs much further study and corroboration. We also analyzed a commercial tincture of Bolivian stingless bee propolis. It presented major chemical differences when compared to the four Bolivian propolis samples as mentioned in Table 39.2 (samples 5–8). It presented fewer chemical compounds and the presence of sugars. We identified compounds from aliphatic acids and their esters with a minor amount (0.89%), hydrocarbons (1.42%), terpenes (6.39%), and sugars with quite higher amount (11.38%). From sugars only the compound Ethyl.alpha.-d-glucopyranoside was observed. Also we did not identify any alcohol compound in the commercial ticture like the other samples (samples 4–8). Most probably it is an alcohol-free product. We can argue that the dissimilarity of the propolis tincture from the Bolivian propolis analyzed here can be traced from its being mixed with other ingredients by its producers. They possibly added a sugar compound to make it sweeter. Also, the sugar content of the tincture can be attributed to contamination of propolis by honey. Besides these some producers put some plant extracts in these kind of products. According to previous work, propolis of A. mellifera shows a wide spectrum of chemical compounds. Generally, alcohols, acids, aldehydes, fatty acids, hydrocarbons, flavonoids, and terpenes form the chemical composition of propolis from A. mellifera. Flavonoids are found in high ratios in propolis. The high amount of flavonoids provides antioxidant activity (Gençay and Salih 2009). However, in propolis samples that we investigated, we could not find flavonoid components. 39.7 Conclusions The Kaur-16-ene (8.beta.13.beta.), Olean-12-ene (b-Amyrene), 3-KETO-URS-12ENE found in our samples are new constituents for stingless bee propolis. Kaur-16ene (8.beta.13.beta.) compound is found in S. depilis (0.24%), Olean-12-ene in M. grandis (4.07%), and 3-KETO-URS-12-ENE in S. depilis (21.66%). All new constituents belong to the Bolivian propolis. Acknowledgements The propolis samples were received from the collection of Apiterapia y Bioactividad (APIBA), Universidad de Los Andes, Mérida, Venezuela, seeking for collaborative research. The M. favosa propolis were collected by Professor Patricia Vit, Universidad de Los Andes, Mérida, Venezuela, and the bee was kindly identified by Prof. João M.F. Camargo from the Biology Department, Universidade de São Paulo, Ribeirão Preto, Brazil. The propolis of T. biroi was collected by Professor Cleofás Cervancia, Universidad Los Baños, Philippine, and she also identified the bee. The M. grandis, S. depilis and S. polysticta propolis from the National Park Amboró, Bolivia were collected by P. Vit and Dr. Urbelinda Ferrufino, Asociación Ecológica de Oriente (ASEO), Santa Cruz, Bolivia. The Bolivian stingless bees were kindly identified by Dr. Silvia R.M. Pedro from the Biology Department, Universidade de São Paulo, Ribeirão Preto, Brazil. I thank the anonymous reviewers to improve my chapter, as well as valued editorial interaction with P. Vit and Dr. David W. Roubik. 536 O. Gençay Çelemli References Bankova V. 2005. Chemical diversity of propolis and the proplem of standardization. Journal of Ethnopharmacology 100:114–117. Barth OM, Luz CF. 2003. Palynological analysis of Brazilian geopropolis sediments. Grana 42:121–127. Barth OM. 2004. 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Apidologie 23:277–283. Part VI Marketing and Standards of Pot-Honey Chapter 40 Production and Marketing of Pot-Honey Rogério Marcos de Oliveira Alves 40.1 Introduction A light yellow liquid, translucent, sour and slightly sweet. That description characterizes the honey of indigenous stingless bees or native honey-making bees in tropical America (Nunes 2009). The honey has a flavor different from that of Africanized bees used for beekeeping today, and was noticed by the early settlers of America when savoring honey that the native people used as a natural sweetener, medicine, and in religious rituals. Honey in the Neotropics came from stingless bees, before introduction of the Western honey bee, Apis mellifera (Kerr et al. 2005). This delicacy, found in different parts of Brazil and elsewhere, is still not well known to urban connoisseurs. Stingless bee honey carries a universe of components that go well beyond the traditional product of beekeeping. Another wealth is revealed when the honey is put in the mouth: an impressive array of flavors. Acidity, floral aromas and earthy notes are provided in honey from bees such as “jataí” and “tiúba,” among other stingless bees (Marques 2010). Although hundreds of bee species are known to make honey in the Americas, the entire consumption of honey in America is focused on exotic A. mellifera, considered the most productive per colony. However, this perception is changing, the market is becoming more selective, and now wants information on products it consumes. This means flavor, aroma, bouquet, and composition from bees such as “jataís,” “uruçús,” “tiúbas,” “canudos,” and “mandaçaia.” Stingless bee honey occupies a niche market with diverse value, added from natural sources of honey production. A major concern of the world market is the total elimination of waste and toxic antibiotics in honey, and organic farming is the most promising strategy to market R.M.O. Alves (*) Instituto Federal de Educação, Ciência e Tecnologia Baiano – IFBaiano Rua do, Rouxinol 115, Bairro do Imbuí, Salvador, Bahia, CEP 41.720-052, Brazil e-mail: eiratama@gmail.com 541 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7_40, © Springer Science+Business Media New York 2013 542 R.M.O. Alves such honey. The marketing of honey labeled “socially fair” is also known as “fairtrade,” with prices paid above the average market value to help poor communities that have a role in conservation (Paula Neto and Almeida Neto 2005). Honey of native species has the right profile for this segment because its production is developed in regions free of pesticide residues and is also free of antibiotics added by the beekeeper. The labor employed is from poor communities with low environmental impact. The product is stored in pots made with propolis and wax (meliponine cerumen). There are species of high productivity well-suited to fill a niche that includes only 3% of world trade (IBCE 2010). 40.2 Stingless Bee Species and Production of Pot-Honey Among hundreds of native bee species, some produce honey to satisfy the nutritional needs of the colony, others produce an excess available for humans. Only a few of them are excellent honey producers, like Melipona, with species of great potential and widely kept in Tropical America. The best known pot-honeys are produced by “mandaçaia” (Melipona quadrifasciata), “jataí” (Tetragonisca angustula), “jandaíra” (Melipona subnitida), “papaterra” (Melipona asilvai), “canudo” (Scaptotrigona sp.), “tiúba” (Melipona compressipes), “uruçú verdadeira” (Melipona scutellaris), “uruçú amarela” (Melipona rufiventris), “xunan cab” (M. beecheii). Some, like “jataí,” are widely distributed. Others— “papaterra” and “jandaíra,” are more restricted to some habitats, and live in savannas known as “caatinga” (Lopes et al. 2005). Few stingless bee species have been explored in all their technical potential, needed to increase pot-honey production. Stingless bee keeping should be optimized by bee management, genetic control and promotion of bee plant cultivars. The evaluation of meliponine honey production (Table 40.1) is difficult due to traditional practice differences. The colonies are mostly kept in logs or boxes (literally “tenements”), without management and inspection, insufficient forage, and rudimentary techniques of honey harvest. Therefore, it is easy to envisage greater honey production with adequate management. Estimated pot-honey annual yields were collected personally visiting meliponaries, and by searching the literature. Villar (2002) estimates that 4,000 tons of stingless bee honey is produced in Brazil every year, a volume ten times lower than the national production of 42,000 tons of total honey. About 1 ton of pot-honey is “Paulista” (produced in São Paulo), with the remarkable contribution of “jataí.” However, during visits to producers, the author estimates that harvests of native bee honey reach 100 tons per year. Research conducted by Londono (2011) using the internet, with 35 meliponiculturists, found that 40% of the producers were devoted to honey for sale, but only one was a full-time stingless bee keeper. This activity can be promoted by professional efforts. Well-supported stingless bee keeping may lead to high pot-honey yields, reduction of costs and greater benefits for the producer. The practice of migratory meliponiculture apparently increases honey production some 300%. 40 Production and Marketing of Pot-Honey 543 Table 40.1 Country of origin and estimated honey production by native stingless bees Country Species Pot-honey annual yield 1 kg (Wikipedia 2011) Australia Trigona carbonaria (s.l.) = Tetragonula carbonaria Austroplebeia australis Brazil Melipona asilvai 1 l (Carvalho et al. 2003) Brazil Melipona fasciculata 3–4 l (Magalhães and Venturieri 2010) Brazil Melipona flavolineata 2–3 l (Magalhães and Venturieri 2010) Brazil Melipona mandacaia 2.0 l (Carvalho et al. 2003) Brazil Melipona quadrifasciata anthidioides 2.0 l (Carvalho et al. 2003) Melipona quadrifasciata quadrifasciata 2.0 l (Carvalho et al. 2003) Brazil Melipona rufiventris 3.0 l (Carvalho et al. 2003) Brasil Melipona scutellaris 2–15 kg (Alves, personal observation) Brazil Melipona subnitida 2.5 kg (Bezerra 2002) Brazil Scaptotrigona 3.0 l (Carvalho et al. 2003) Brazil Tetragonisca angustula 1.0 l (Carvalho et al. 2003) Costa Rica Melipona “fasciata” = M. costaricensis 2.5 kg (Cortopassi-Laurino et al. 2006) Costa Rica Melipona beecheii 2.5 kg (Cortopassi-Laurino et al. 2006) Indonesia “Trigona” (s.l.) 1 kg (Soekartiko 2011) Mexico Melipona beecheii 2.5 kg (Cortopassi-Laurino et al. 2006) Paraguay Scaptotrigona 3.0 l (Carvalho et al. 2003) 40.3 Marketing of Meliponine Honey The world production of honey has increased 4.6%, honey export increased 35.6%, and honey import 38.8%, during 2002–2003. This fact means that lucrative external markets caused a decrease of internal honey consumption in several honey producing countries. Brazil doubled the value of honey exports in 2008, with a record of US$ 43.57 million, increasing by 42% the revenue of 2007, US$ 21.2 million. In 2010 Brazil increased its export by 54%. The exported honey volume also increased, from 12,900 to 18,270 tons in 2010 (IEA 2005). The world market of meliponine honey is still in its infancy and restricted to particular initiatives in Brazil, Mexico, Costa Rica, and Australia, with regional impact. According to the IBCE (2010), current tendencies in developed countries consider consumer attitude and preference toward organic honey and special honey. This interest to consume organic products is caused by consumer awareness of environmental protection, causing an increase in organic honey demand. Therefore meliponine honey is becoming better known while production increases. Pot-honey is becoming more familiar and consumed for its singular features and is widely appreciated, considered as an artisan bee product with organic origin, produced in natural environments of tropical nature. In Fig. 40.1 some pothoney packaging in sachets, bottles, and ceramic. R.M.O. Alves 544 Fig. 40.1 Commercial presentation of pot-honey. (a) Sachet presentation of Brazilian honey produced by M. compressipes, known as “tiúba” in Maranhão, (b) bottled Scaptotrigona honey also from Brazil, (c) bottled Australian honey produced by “sugarbag” Tetragonula carbonaria, (d) jar of “urucú,” bottle with artisanal cover of “tiúba” from Brazil, jar of “pisilnekmej” Scaptotrigona mexicana, from Mexico, and ceramic “puño” to bottle Bolivian honey produced by “suro negro” Scaptotrigona polysticta. Photos: (a-b) R.M.O. Alves, (c) T.A. Heard, (d) P. Vit 40.3.1 America Consumption of pot-honey in Mexico is as old as food, but mainly as medicine (see Chap. 15) and for use in religious rituals. However, due to low productivity of the colonies, the production is consumed mainly by the stingless bee keeper and the local community (Maganã 1998). The growth of beekeeping has caused a disincentive among farmers to raise native bees, but there is a movement now to preserve traditional stingless bee keeping. The bees considered best for honey are M. beecheii and Scaptotrigona (Quezada-Euán 2005). In Central America there are M. beecheii and M. fasciata (currently known as M. costaricensis, M. panamica, M. melanopleura, and others, Roubik D, personal communication) primarily in Costa Rica (Wikipedia 2011). 40 Production and Marketing of Pot-Honey 545 In South America, economic growth led to increased purchasing power and providing better education, which also increased the consumption of honey, no longer an unusual product in daily diet. Currently the market for bee products experiences tremendous growth, fueled mainly by exports and improvement in the internal market (Koshiyama et al. 2011). In Brazil, the consumption of honey from stingless bees is still small mainly due to availability constraints rather than ignorance about the product. In the north and northeast, despite low availability the honey is very popular and consumed often. However, the increase in consumption is subject to quality improvement and increased honey production. Meliponiculture is less developed in the south and southeast of the country; initiatives there are aimed at conserving colonies, except for the State of Paraná with the keeping of “jataí” (T. angustula) and “mandaçaia” (M. quadrifasciata) whose objective is the production of pot-honey (Laginsky 2011). 40.3.2 Africa, Asia and Australia No quantitative information is available for most of Asia. However, like Asia, Australia has no large bee like Melipona. In Australia, T. carbonaria and Austroplebeia australis are the main native honey sources (see chapter in present book). According to Klumpp (2007) a hive of Australian stingless bees produces <1 kg per year. The product is sold in jars of 50 ml at a price of AU$ 30.00 for consumers in urban centers, where it is appreciated for its taste and strong acidity. There is certainly a growing interest in meliponiculture in Africa (Kwapong et al. 2010). As Kajobe indicates (see chapter in present book) there is information being gathered on the biology of stingless bees, and the management techniques, many of them gleaned from work in the Neotorpics; there will soon be much more data on practices and commercial preference in pot-honey consumption. 40.3.3 Production and Consumption of Pot-Honey The market for native honey experienced an increase in recent years, accompanying the increase in consumption and insufficient production of honey of A. mellifera, especially in the greatest traditional Brazilian beekeeping areas (North–Northeastern Brazil). Traditionally known as the greatest producer of native honey, the Northeast serves as the development center, with the largest pot-honey producers located in the states of Maranhão, Bahia, Rio Grande do Norte, Pernambuco, and Piauí. This large region has highlighted the technical aspects of meliponiculture specialists who obtain the highest productivity per colony. Meanwhile the Northern Brazil states of Amazonas and Pará have outstanding potential, both in number of species and R.M.O. Alves 546 Relative price, demand or supply Supply, demand and native honey price for Brazil 1 2 3 4 5 6 7 8 Month of Year Price Supply 9 10 11 12 Demand Fig. 40.2 Market behavior, variation in supply, demand, and price of pot-honey production. The investments made by government and the private sector foster studies and projects that expand the number of species and honey production. Honey is marketed regionally and considered a seasonal, handcrafted or “artesinal” product. The statistics on colony productivity are estimates, mainly due to lack of product regulation and mode of regional marketing, without official records. Data collected on trips to interview beekeepers allows one to sketch a graph of market behavior of honey from stingless bees in the main producing regions of Brazil (Fig. 40.2). The consumption of honey from native bees is expanding, driven by the appeal health, social “fairness” and product appearance. With the range of color from white to light amber, honey from native species has a very strong appeal in consumer preference. Other factors that enhance value in production are a pollution-free environment and income, observing principles of sustainable environment. Oliveira et al. (2005), based on data analysis and research conducted in the State of Pernambuco (Brazil) observed that 86% of respondents said they had consumed honey. Of those, about 70% claim to eat honey from bees of the genus Apis while 30% consumed honey from native bees. In Bogotá (Colombia), a recent survey revealed that almost 70% of consumers would not buy pot-honey produced by Tetragonisca angustula because it is unknown. Indeed, consumers of “angelita” honey in this capital city are some 80% middle class of socioeconomic strata 3 and 4 (Rodríguez Reyes 2007). Frequency of native bee honey consumption showed around 8% on a monthly basis and 92% annually. When questioned as to use, the predominant response was that this was primarily used as a sweetener and folk remedy (Oliveira et al. 2005). Seasonality and unfamiliarity of the consumer with product characteristics constitute obstacles to increasing the consumption of pot-honey, as visualized in Table 40.2. 40 Production and Marketing of Pot-Honey 547 Table 40.2 Periods and causes guiding consumption according to pot-honey supply in north and northeast Brazil Month Supply Causes January to March Production and Hottest time of year high supply Habit of low traditional use Economic factors, school and other costs, fairs, festivals Lack of information on benefits of honey April to June Little supply Cool and rainy weather High honey consumption (folk remedy use) Honey crystalization, difficult to consume July to September Lack of supply Very cool and rainy weather High folk remedy consumption October to December Little supply Beginning of the hot season Reduced consumption related to heat From: Alves, personal observations during 30 years It is important that price remain stable throughout the year. The large difference between supply and demand causes disincentive to the farmer, who needs guidance on how to reduce the effects of factors that create this relationship. 40.3.4 Cultural Aspects of Pot-Honey Consumption Vilckas et al. (2001) reports that the frequency of honey consumption decreases in lower social strata. In the case of low-frequency, they argue that it is lack of custom, it is fattening, or they do not care for it, while some believe it increases their blood cholesterol, is too expensive a product, or is superfluous. Individuals in higher strata can be more knowledgeable and look for special honey types, such as unifloral and organic honey. However, pot-honey is better understood, from direct experience, in rural and forest villages. Native honey is well known to consumers in rural areas, usually people with lower incomes and little education, but for whom cultural factors are of great importance. Families generally maintain colonies in the yard for use in remedies as needed. In Bolivia, the honey of stingless bees, in addition to food characteristics, is widely used in traditional medicine to treat eye diseases, and respiratory and digestive maladies (IBCE 2010). Honey is characterized as a remedy, is consumed only in times of onset of colds and respiratory infections, yet in these times the production is lower. Attempts at honey processing using dehumidification caused an unusual situation. Accustomed to native bee honey as very fluid (less viscous) and sour, processed honey was initially refused because of similarities with the honey of A. mellifera (dense and sweet). Whereas honey of Apis might be eaten with a spoon, that of stingless bees is often drunk as though a liquor. R.M.O. Alves 548 Another important feature that restricts consumption is the crystallization of some honey that leads consumers to not accept the product, claiming it has been adulterated by addition of sugar. However, in southern Brazil the crystallized honey of T. angustula and M. rufiventris is usually consumed. 40.3.5 Low Production and Seasonality of Pot-Honey The low productivity of colonies is a significant consideration in the marketing of honey from stingless bees. Due to lack of technology to get the most of the colonies the producer realizes an average of 1 l per hive per year in species that have potential for 10 l per hive per year, as in M. scutellaris. In Manaus, M. fasciculata productivity reaches 3 kg per hive per year. In the state of Rio Grande do Norte meliponines produce about 2.5 l (Vollet Neto et al. 2011). A group of 30 native bee hives produces on average (depending on the flower) 5 pounds of honey per hive, totaling 150 pounds of honey from native bees in a year (CESMAG/COIMP 2007). The lack of product is a factor discouraging both the grower and the consumer and causes disruption in the consumption process. The major producing regions and greatest number of stingless bee species are in the humid forest biome with a dry season during the months at the end and beginning of the year. In the savanna biome (arid) and in the cerrado, production is greater in mid year, when the rains are reduced. Production of pot-honey is fundamentally different in its timing in the two regions. However, when human discomfort and virus-incuded colds are most frequent—leading to a consistently high demand for pot-honey as a remedy for sore throats and colds, tends to be seasonal and often associated with the beginning or end of the rainy season. Thus, the cerrado and the moist forest somewhat complement each other in the timing of honey production or demand. 40.3.6 Quality of Pot-Honey Methods of destroying the pots when turning the hive or box supers over to allow drainage into a sieve produce honey of lower quality, constituting a barrier to consumption in larger population centers. The honey harvest is done by piercing or squeezing the pots of honey and pollen, without the need for personal hygiene preparation, now required for the SIF (Federal Inspection Seal), i.e., requirement that harvest is done in a sterile place that is clean and has well ventilated facilities, and includes a sink (Melo 2010). Use of modern technology for honey extraction—such as a suction device—is frequent in Brazil. This allows improvement of product quality, as regulatory governmental bodies have been aiming to increase honey consumption. A major obstacle to honey of native bees in world trade is that the Codex Alimentarius Commission 40 Production and Marketing of Pot-Honey 549 Table 40.3 Price of honey according to the bee species and location of production Bee species Price (USD/kg) Price (BR$/L) Locality Melipona asilvai 7.00–10.00 20–30 Bahia Melipona compressipes 10.00–18.00 30.00–50.00 Maranhão Melipona fasciculata 7.00–9.00 20.00–25.00 Pará 7.00 20.00 Manaus Melipona flavolineata 10.00–13.00 30.00–40.00 Piauí 9.00–12.00 25.00–35.00 Amazonas and Pará Melipona mandacaia 7.00–12.00 20.00–35.00 São Paulo Melipona mondury 10.00–18.00 30.00–50.00 Bahia Melipona quadrifasciata 9.00–21.00 25.00–60.00 Bahia Melipona scutellaris 10.00–18.00 30.00–50.00 Bahia 25.00 70.00 Alagoas 35.00 100.00 Pernambuco Melipona subnitida 7.00–10.00 20.00–30.00 Bahia 12.00–18.00 35.00–50.00 Rio Grande do Norte Tetragonisca angustula 21.00–32.00 60–90 Bahia/Paraná Scaptotrigona sp. 7.00–10.00 20–30 Bahia/Pará From: Alves et al. 2005 (updated 2011) only recognizes honey produced by Apis (Vit et al. 2004; Quezada-Euán 2005; Souza et al. 2006), and pot-honey chapters in the present book (Vit, AlmeidaMuradian, Fuenmayor et al., Dardón et al., Ferrufino and Vit). 40.3.7 Cost-Value-Price The value of honey in the market is a function of quality, presentation, and more recently, certification as organic produce, which adds value and may raise the price by 50%. The price of honey varies according to the site and producing species (Table 40.3). The price of honey produced by stingless bees can reach a value up to 1,100% higher than the common honey, ranging between BR$40 and BR$100 a pound, against BR$3 a pound of honey from the traditional A. mellifera (Villar 2002). When asked about the value of a liter of honey, consumers shopping at “Garanhuns” in the State of Pernambuco indicated an average of BR$ 15.00 per kg as the ideal price for genuine honey of A. mellifera (Oliveira et al. 2005). The short supply of native honey places the product on the market with values that may exceed BR$ 30.00 per kg. In Manaus, 1 kg of honey costs BR$ 20.00 and production is 3 kg per hive per year (Portal Extraction 2011). In Maranhão prices range from BR$ 6.00 to BR$ 18.00 for packages of 200 g (INVESTENE 2011). On the west coast of the Yucatán Peninsula the price of honey from M. beecheii ranges from USD 2.00 to USD 15.00 per liter (Maganã 1998). R.M.O. Alves 550 Alves et al. (2005) reports that the difficulty in calculating the price of honey stems from the lack of standardization of protocols, which prevents establishment of basic pricing. Although stingless bee keepers sustain no losses by not marketing their product, the expansion of production could force down the price. As costs for maintaining the bees are low, the activity allows the production of a relatively inexpensive food with a strong commercial appeal (CESMAG/COIMP 2007). 40.3.8 Vending Locations of Pot-Honey The low yield associated with lack of regulation affects consumption of the product. Currently the production is sold “directly from the hive” or as on-site production, usually in the home, place or establishment of the producer, leading to a special relationship where trust in the product is more important than the amount paid. Another factor is the lack of registration by the government, which otherwise would allow honey to be sold at all pharmacies and supermarkets, considered by Magalhães et al. (2007) to be places of greater access to the product by buyers. 40.3.9 Packaging In producing regions honey is sold in glass or plastic containers with a capacity of 700–1,000 ml. Glass jars are the best and most suitable, but plastic predominates because it costs less. Modern beekeepers use narrow or wide mouth jars with a capacity of 50, 150, 200 or 500 ml, allowing higher consumption, better product presentation and higher added value. In Maranhão, Vilckas et al. (2001) found honey of “tiúba” (M. compressipes) sold in glass containers with a capacity of 205, 315 and 460 g. 40.3.10 Legislation The lack of regulation of native bee honey makes it impossible to trade efficiently, hindering consumer access to the product and discouraging activity. In Brazil there are initiatives for the characterization of honey in order to provide benefits for its regulation. In the state of Bahia, Brazil the legislation for pot-honey marketing is already in the planning design stage. To my knowledge, as informed by Vit P (see her chapter on M. favosa honey in this book), the information provided by NatesParra G during the regulation process for honey in Colombia included pot-honey produced by native bees in the annex (ICONTEC 2007) after the publication of their suggested standards (Souza et al. 2006). 40 Production and Marketing of Pot-Honey 40.4 40.4.1 551 Strategies to Increase the Production and the Consumption of Pot-Honey Production The production process in animal husbandry obeys the equality of the variables feeding-management-genetics. This implies that nourishment is provided in times of shortage of flowers, using deployment and improvement of “bee pastures,” performing management operations periodically, and performing selection of the best queens so that the producer is able to get the maximum possible production, and with lower costs. The deployment of meliponicultural “grazing” contributes to increased production and also to improving honey quality. The supply of trophic resources (nectar, pollen and resin) comes from existing plants, and maintenance of native species should encourage the beekeeper to introduce flowering plants recognized for increasing nectar production potential of the pasture. Unlike the honey bees, stingless bees have not such an extensive foraging area (Roubik 1989) thus improvement of available floral resources is a possibility that can be pursued profitably by an individual beekeeper. Otherwise, spreading plants that are profitable has a minimal impact on bee forage within foraging range. The utilization of efficient, timely and low-cost, hive inspection, calendars of beekeeping activities, control of enemies, colony division, equipment for harvesting and processing of honey allow for increased production through organization of time and less waste of product. The choice of the best queens allows better development of the colonies for more efficient storage of honey. The supplementation of nectar and pollen through the use of artificial food allows maintenance of colonies to be standardized, reducing losses and allowing more efficient management of colonies. 40.4.2 Consumption The current trend in developed countries, especially in the European Union includes consumers seeking organic and other special honey. For such consumers honey is a natural product, pure and healthy. It is a natural product that has several properties which improve health and has always been valued for its therapeutic qualities (IBCE 2010). There are several strategies to expand markets for honey of stingless bees, such as apitherapy, to stimulate consumption in various areas, greater media coverage, and reducing the price to the consumer (Paula Neto and Almeida Neto 2005). In addition, the use of standardized packaging—with labels and information—participation in fairs, exhibitions, and publicity in schools all are excellent tools for effective marketing and merchandising. R.M.O. Alves 552 40.4.3 Cooperative Marketing of Pot-Honey Established in regions distant from the consumer centers, perishable and seasonal native bee honey production forces the producer to seek other markets through trade shows and exhibitions. Marketing honey in smaller packages can increase income and encourages consumption with greater frequency. In the state of Rio Grande do Norte, a stingless bee keeper sells about 300 gallons of honey within the state only in packages of 200 ml (Lopes et al. 2005). In a survey conducted by the author, the honey sold in packs of 1 l is consumed within a year by family of three. The pack split into small sachets of honey is the best method for stimulating consumption and reaching mainly children—future consumers of honey. A strategy to increase consumption is to form associations or cooperatives of producers, making it easier and less costly to disseminate information and increase consumer confidence in the product. For an individual to produce and market a product is extremely difficult, even if they are a great producer. It may be that small producers, associating with each other, will accomplish the task. It may be an association or a group that shares the same interest (Melo 2010). 40.4.4 Processing and Storage of Pot-Honey The use of effective preservation methods provides honey quality insurance and allows longer shelf life. Currently used methods are refrigeration, maturation, pasteurization, and dehumidification (see Chap. 10), which conserve physicochemical and organoleptic properties (Alves et al. 2007). This activity could facilitate regulation by government agencies. 40.5 Major Initiatives of Pot-Honey Production in Brazilian States Maranhão—Commonly found in the State of Maranhão, the culture of tiúba proved viable commercial and socially. Each year, a colony can produce up to 300 kg of honey. In each community there are about ten families of “meliponicultors” as stingless bee keepers are called (INVESTENE 2011). Generating income, promoting social inclusion and preserving native species, meliponiculture with the tiúba bee (Melipona compressipes) has changed the lives of 18 communities in the semiarid region of Maranhão. The project was called “Native Bees,” developed by Maranhão for natural conservation and the Federal University of Maranhão since 2001 (INVESTENE 2011). The commercial manager of the Cooperative Agroecological Meliponary “Baixada Maranhaense,” Luis Pedro, reports that since 2005 a project was implemented aimed at increasing production and quality of honey from M. compressipes. 40 Production and Marketing of Pot-Honey 553 In 2011 there were 12,000 colonies in honey production. They produce 15 tons annually, sold in the regional market and in part in fairs, exhibitions and events across the country. Amazon—Honey production is quite impressive, though very large distances and logistic difficulties hinder the flow of the product and marketing. The number of colonies is about 80 colonies per individual stingless bee keeper, with a production of 2 kg annually per colony. Projects involving government agencies and associations have the objective of significantly increasing production and selling abroad as well as helping residents to find sustainable income. A liter of pot-honey produced by the community has a value of BR$ 40, but can reach BR$ 80. In each village 150 hives are maintained and each produces an average of 3 l of honey, which is little. This is because, apart from being marketed, it is also consumed by the indigenous people (INOVABRASIL 2011). Bahia—In the State of Bahia honey production is sourced from producers possessing few colonies, focused on productivity and honey quality. The largest producers are in the central, north, and northeast part of the state, with a mean of 30 colonies per producer and 2–5 l per hive per year. The bee species used are M. mandacaia, M. quadrifasciata, M. scutellaris, and M. mondury. Paraná—In 2007 the Breeders Association of Native Bee Conservation Area Guaraqueçaba (Acriapa) was created. We are already in the third honey harvest. The first two occurred in the summer of 2007–2008 and in late 2008 they were very modest, 30 and 40 pounds, respectively. The last harvest, in February, was 130 pounds, considered very good. The product is currently being sold in bottles of 65 g, priced at BR$ 7.00. According to our calculations, it is estimated that the stingless bee-keeper with the highest production should earn BR$ 1,200 annually (Laginsky 2011). Rio Grande do Norte—Paulo Menezes is one of the largest suppliers of pothoney to supermarkets and retail chains in the region. The stingless bee product is sold for up to BR$ 60.00 a gallon, compared to an average of BR$ 5.00 for A. mellifera honey. In 2004, Menezes produced 300 l of honey, an average of 1 l per hive. The entire product was sold to supermarkets in Rio Grande do Norte and Fortaleza, and to buyers from Brasilia and Rio de Janeiro. The sales yielded no less than BR$ 18,000 a year. “If you divide by 12 months, it was an income of BR$ 1,500 per month,” says the producer, satisfied with the result (SEBRAE 2005). Rio Grande do Sul—Stingless bee keeping for pot-honey production is already a reality for family farmers in the Sun Valley Center for the Support of Small Farmers. The bee leading the way is Tetragonisca angustula, which is popularly called “jataí.” Beekeeping there allows diversification and can be integrated into forest plantations, fruit and food crops, also contributing to the increase of agricultural production. Recently 1.5 pounds of honey has been harvested per colony (Mezziga 2011). The stingless bee keeper João Batista Ferreira, in the municipality of Belterra, Pará, is testimony to traditional knowledge and the conservation of Meliponini. Currently, “Mr. John” manages 23 species of stingless bees with an average production, among them, ranging from 0.5 to 5 kg per hive per year. The main producing species are M. fasciculata and Scaptotrigona. Beekeeping contributes a significant part of family income (Ferreira and Rebello 2005; Lopes et al. 2005). R.M.O. Alves 554 Sergipe—Bee keeping is encouraged in communities throughout the state. Courses and meliponary community building are the means used to organize and improve food quality, and generate income for residents in rural areas. The honey produced is totally consumed by the regional community and marketed in the same establishment. 40.6 Concluding Remarks Meliponiculture is a fundamental activity that maintains communities by revenues and improvement in quality of local products. Joint public and private strategies are needed for channel marketing to get pot-honey into the customer’s hands. Increased pot-honey demand will benefit meliponiculture. Integrating modern technology with traditional methods, and merchandising of native bee honey, promotes native species use and socially fair policies with a consumer product. Acknowledgments I thank Dr. David W. Roubik for careful translation and editing of my chapter, timely comments received by referees, and Professor Patricia Vit for earnest invitation and editorial commitment facilitating new references. References Alves RMO, Souza BA, Carvalho CAL, Justina G. 2005. Custo de produção de mel: uma proposta para abelhas africanizadas e meliponineos. Série Meliponicultura, No. 02. UFBA/SEAGRI-BA, Cruz das Almas, Bahia, Brazil. 14 pp. Alves, RMO, Sodré, GS, Souza, BA, Carvalho, CAL, Fonseca, AAO. 2007. Desumidificação: uma alternativa para a conservação do mel de abelhas sem ferrão. Mensagem Doce 91:2–8. Bezerra JA. 2002. A rainha do sertão. Revista Globo Rural [Rio de Janeiro, Brazil] 17:62–69. Carvalho CAL, Alves RMO, Souza BA. 2003. Criação de abelhas sem ferrão: aspectos práticos. Série meliponicultura, No. 1. UFBA/SEAGRI-BA, Cruz das Almas, Bahia, Brazil. 50 pp. CESMAG/COIMP. 2007. Projeto meliponicultura criação de abelhas indígenas sem ferrão. CESMAG/COIMP, Pará, Brazil. 9 pp. Cortopassi-Laurino M, Imperatriz-Fonseca VL, Roubik DW, Dollin A, Heard T, Aguilar I, Venturieri GC, Nogueira-Neto P. 2006. Global meliponiculture: challenges and opportunities. Apidologie 37:275–292. Ferreira JB, Rebello JFS. 2005. Belterra: o paraíso das abelhas indígenas sem-ferrão. Mensagem Doce. 83:23–24. IEA. 2005. Mel: exportações fazem produção aumentar de norte a sul. São Paulo. 3 pp. IBCE. 2010. Perfil de mercado de miel de abejas nativas. Boletin Técnico. Instituto Boliviano de Comércio Exterior. 27 p. ICONTEC. Instituto Colombiano de Normas Técnicas y Certificación. 2007. Norma Técnica Colombiana. Miel de Abejas. NTC 1273; Bogotá, Colombia. 6 pp. INOVABRASIL. 2011. Exportação de mel de abelhas sem ferrão. Available at http://inovabrasil. blogspot.com/2007/09/exportao-de-mel-de-abelhas-sem-ferro.html INVESTENE. 2011. Produção de mel ganha destaque no maranhão. Available at http://www. investne.com.br/noticias-maranhao/producao-de-mel-ganha-destaque-no-maranhao 40 Production and Marketing of Pot-Honey 555 Kerr WE, Carvalho GA, Silva AC, Assis MGP. 2005. Aspectos pouco mencionados da biodiversidade amazônica. Mensagem Doce 80:46–60 Klumpp J. 2007. Australian stingless bees. A guide to sugar bag beekeping. Earthilling Enterprise; Victoria, Australia. 110 pp. Koshiyama AS, Tassinari WS, Lorezon MCA. 2011. Perspectivas da produção melífera brasileira. Mensagem Doce 113:9–12. Kwapong P, Aidoo K, Combey R, Karikari A. 2010. Stingless bees: importance, management and utilization. A training manual for stingless beekeeping. Unimax Macmillan Ltd. Ghana: Accra North. 72 pp. Laginsky F. 2011. Projeto incentiva a produçao do mel no Paraná. 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Available at: http://en.wikipedia.org/wiki/Stingless_bee Appendix A Taxonomic Index of Bees* *The names of species (or subspecies) were organized by the specific (or subspecific) epithet (e.g., Apis mellifera must be searched as mellifera, Apis and Apis mellifera scutellata as scutellata, Apis mellifera). Species referred as sp. or spp. are indicated only by the generic name (e.g., Anthophora sp. appears only as Anthophora). Also, species named approximately to another one, mentioned as affinis, near, sp. gr., etc., can be found in the name of the affined species (e.g., Scaptotrigona aff. depilis appears as depilis, Scaptotrigona). Names in bold are junior synonyms (senior synonyms are indicated in brackets). acapulconis (Strand, 1919), Geotrigona, 101, 140, 395 acapulconis Strand, 1919, Trigona, 557 ailyae Camargo, 1980, Partamona, 78 alfkeni Friese, 1900, Trigona, 75 Alphaneura Gray, 1832 (= Trigona), 7 amalthea (Olivier, 1789), Trigona, 79, 94, 103 Amalthea Rafinesque, 1815 (= Trigona), 7 amazonensis (Ducke, 1916), Trigona, 80 Andrena Fabricius, 1775, 3 anduzei (Schwarz, 1943), Paratrigona, 78, 81 angustula (Latreille, 1811), Tetragonisca, 91, 102, 117, 141, 298, 375, 395 angustula (Latreille, 1811), Tetragonisca angustula, 91, 102, 117, 141, 274, 298, 375, 395 angustula Latreille, 1811, Trigona (Frieseomelitta) angustula, 557 angustula Latreille, 1811, Trigona (Tetragonisca), 79 angustula Latreille, 1811, Trigona (Tetragonisca) angustula, xiv, 11, 79, anthidioides Lepeletier, 1836, Melipona quadrifasciata, 412, 530, 531, 543 Anthophora Latreille, 1803, 175 Aparatrigona Moure, 1951, 7, 20, 92 Aphaneura Gray, 1832 (= Trigona), 7 apiformis (Buysson, in Du Buysson & Marshall, 1892), Melipona, 77, 82 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7, © Springer Science+Business Media New York 2013 557 558 Appendix A apiformis (Buysson, in Du Buysson & Marshall, 1892), Melipona (Michmelia) Apis Linnaeus, 1758, 77, 82 Apotrigona Moure, 1961[= Meliponula (Meliplebeia)], 8 araujoi (Michener, 1959), Hypotrigona, 263, 264 argentina, Camargo & Moure, 1996, Geotrigona, 100, 126 argyrea (Cockerell, 1912), Scaura, 102, 117, 141 asilvai Moure, 1971, Melipona, 368, 412, 542, 543, 549 atomaria (Cockerell, 1917), Trigonisca, 118 auripennis Pedro & Camargo, 2003, Partamona, 78, 91 australis (Friese, 1898), Austroplebeia, 43 australis Friese, 1898, Trigona, 5 Austroplebeia Moure, 1961, 8 Axestotrigona Moure, 1961, Meliponula (Axestotrigona), 8, 263 azteca Ayala, 1999, Trigonisca, 141, 144 baeri Vachal, 1904, Melipona, 126 batesi Pedro & Camargo, 2003, Partamona, 26, 27 beccarii (Gribodo, 1879), Meliponula (Meliplebeia), 264 beebei (Schwarz, 1938), Tetragona, 91 beecheii Bennett, 1831, Melipona, 101, 116, 140, 395 belizeae Schwarz, 1932, Melipona, 147 bicolor Lepeletier, 1836, Melipona, 274 bilineata (Say, 1837), Partamona, 101, 140 bipunctata (Lepeletier, 1836), Scaptotrigona, 558 biroi (Friese, 1898), Tetragonula, 526, 531, 533, 535 bivea Roubik, Lobo & Camargo, 1997, Meliwillea, 116 bocandei (Spinola, 1853), Meliponula, 263 bocandei (Spinola, 1853), Meliponula (Meliponula), 264 bottegoi (Magretti, 1895), Liotrigona, 264 Bombus Latreille, 1802, 181, 485 brachychaeta Moure, 1950, Melipona, 410, 469 bradleyi Schwarz, 1932, Melipona (Eomelipona), 90 branneri Cockerell, 1912, Trigona, 80, 91 buchwaldi (Friese, 1925), Tetragonisca, 117 caerulea (Friese, 1900), Mourella, 126 camargoi Moure, 1989, Camargoia, 90 Camargoia Moure, 1989, 20, 93 cameroonensis (Friese,1900), Meliponula (Axestotrigona), 264 capitata (Smith, 1854), Cephalotrigona, 76, 90, 274 capixaba Moure & Camargo, 1994, Melipona, 179 captiosa Moure, 1962, Melipona (Michmelia), 90 carbonaria (Smith, 1854), Tetragonula, 45 carbonaria Smith, 1854, Trigona, 41, 44, 45, 49, 52, 55, 56, 66, 67, 370, 451–453, 477, 545 carbonaria Smith, 1854, Trigona (Heterotrigona), 45 carrikeri Cockerell, 1919, Melipona, 116 Appendix A 559 cassiae (Cockerell, 1910), Austroplebeia, 43 catamarcensis (Holmberg, 1903), Plebeia, 126, 127, 131 Celetrigona Moure, 1950, 20, 92 Cephalotrigona Schwarz, 1940, 7, 20, 92 cerana Fabricius, 1793, Apis, 154, 175, 176, 241, 496, 501 chacoana Roig Alsina, 2010, Lestrimelitta, 126 chamelensis Ayala, 1999, Lestrimelitta, 140 chanchamayoensis Schwarz, 1948, Trigona, 410 chapadana (Schwarz, 1938), Nannotrigona, 78 chapadicola Pedro & Camargo, 2003, Partamona, 27 chiriquiensis (Schwarz, 1951), Geotrigona, 116 cilipes (Fabricius, 1804), Trigona, 80, 91, 117 cincta (Mocsáry in Friese, 1898), Austroplebeia, 43 clavipes (Fabricius, 1804), Tetragona, 79, 91 Cleptotrigona Moure, 1961, 8 clypearis Friese, 1909, Trigona, 44, 45 clypearis Friese, 1909, Trigona (Heterotrigona), 46, 47 coccidophila Camargo & Pedro, 2002, Schwarzula, 23, 24 cockerelli (Rayment, 1930), Austroplebeia, 43 colimana Ayala, 1999, Melipona, 140 collina Smith, 1857, Trigona, 179 collina (Smith, 1857), Tetragonula, 155 compressipes (Fabricius, 1804), Melipona, 274 compressipes (Fabricius, 1804), Melipona (Melikerria), 76, 90 concinnula Cockerell, 1919, Melipona, 82 concinnula Cockerell, 1919, Melipona (Eomelipona), 76, 82 cora Ayala, 1999, Plebeia, 140 corvina Cockerell, 1913, Trigona, 102, 118, 141 corvina Cockerell, 1913, Trigona (Trigona), 11 costaricensis Cockerell, 1919, Melipona, 116 cramptoni Cockerell, 1920, Melipona, 77, 82 cramptoni Cockerell, 1920, Melipona (Michmelia), 77 crassipes (Fabricius, 1793), Trigona, 91 Cretotrigona Engel, 2000, 14, 19, 145 crinita Moure & Kerr, 1950, Melipona, 410 crinita Moure & Kerr, 1950, Melipona (Michmelia), 77 cryptarum (Fabricius, 1775), Bombus, 177 cubiceps (Friese, 1912), Cleptotrigona, 264 cupira (Smith, 1863), Partamona, 274 cupira Smith, 1863, Trigona cupira [misidentification, = Partamona orizabaensis], 9 Dactylurina Cockerell, 1934, 8 daemoniaca Camargo, 1984, Oxytrigona, 116 dallatorreana Friese, 1900, Trigona, 80 danuncia Oliveira & Marchi, 2005, Lestrimelitta, 116 davenporti Franck, in Franck et al. 2004, Trigona, 559 560 Appendix A davenporti Franck, in Franck et al. 2004, Trigona (Heterotrigona), 45 depilis (Moure, 1942), Scaptotrigona, 91, 410, 469 Dioxys Lepeletier & Serville, 1825, 3 discolor (Wille, 1965), Trigonisca, 118 distincta (Holmberg, 1903), Diadasina, 176 dobzanhskyi (Moure, 1950), Trigonisca, 92 Dolichotrigona Moure, 1950, 20, 92 dominicana (Wille & Chandler, 1964), Proplebeia, 154, 252 dorsalis (Smith, 1854), Tetragona, 91, 103 dorsata Fabricius, 1793, Apis, 181, 252, 484, 501 droryana (Friese, 1900), Plebeia, 274 Duckeola Moure, 1944, 20, 92 Duckeola Moure, 1944, Trigona (Duckeola), 7 duidae Schwarz, 1932, Melipona fasciata cramptoni (= Melipona cramptoni), 77, 82 eburnea Friese, 1900, Melipona, 370, 385–387, 391, 418, 420–422 eburneiventer (Schwarz, 1948), Cephalotrigona, 140 eburnensis (Darchen, 1970), Meliponula (Axestotrigona), 263 eocenica (Kelner-Pillaut, 1970), Kelneriapis, 14 Eomelipona Moure, 1992, Melipona (Eomelipona), 7 epiphytophila Pedro & Camargo, 2003, Partamona, 78 essingtoni (Cockerell, 1905), Austroplebeia, 43 extranea Camargo & Moure, 1983, Trichotrigona, 24, 25, 94 fasciata Latreille, 1811, Melipona, 82, 140 fasciculata Smith, 1854, Melipona, 158, 165, 355, 380, 435, 439, 440, 471, 488, 543, 548, 549, 553 favosa (Fabricius, 1798), Melipona, 363 favosa (Fabricius, 1798), Melipona (Melipona), 77 femoralis Camargo and Moure, 1994, Paratrigona, 91 ferreirai Pedro & Camargo, 2003, Partamona, 78, 91 ferricauda Cockerell, 1917, Trigona, 118 ferruginea (Lepeletier, 1836), Meliponula (Axestotrigona), 264 ferruginea (Lepeletier, 1836), Meliponula, 326 fiebrigi (Schwarz, 1938), Tetragonisca, 410, 469, 478 flavicornis (Fabricius, 1798), Frieseomelitta, 90 flavolineata Friese, 1900, Melipona, 156, 543, 549 florea Fabricius, 1787, Apis, 176, 252, 496 franki (Friese, 1900), Plebeia, 117 fraterna Laroca & Rodriguez-Parilli, 2009, Plebeia, 78 Friesella Moure, 1946, 20, 92 Frieseomelitta Ihering, 1912, 20, 92 Frieseomelitta Ihering, 1912, Trigona (Frieseomelitta), 7 frontalis (Friese, 1911), Plebeia, 101, 117, 140 fuliginosa Lepeletier, 1836, Melipona, 116 fuliginosa Lepeletier, 1836, Melipona (Michmelia), 90 fulva Lepeletier, 1836, Melipona, 77, 90 Appendix A 561 fulva Lepeletier, 1836, Melipona (Michmelia), 77, 90 fulvicutis (Moure, 1964), Scaptotrigona, 91 fulviventris Guérin, 1844, Trigona, 80, 118 fulvopilosa Ayala, 1999, Plebeia, 101, 141 fuscipennis Friese, 1900, Trigona, 80, 91, 102, 118, 141 fuscipes Friese, 1900, Melipona (= Melipona fasciata), 82 fuscobalteata (Cameron,1908), Tetragonula, 11 fuscobalteata Cameron, 1908, Trigona, 155, 178 fuscopilosa Moure & Kerr, 1950, Melipona, 77 Geniotrigona Moure, 1961, Heterotrigona (Geniotrigona), 8 Geotrigona Moure, 1943, 20, 92 Trigona (Geotrigona), 7 ghilianii (Spinola, 1853), Duckeola, 90 glabella Camargo & Moure, 1994, Paratrigona, 126 glaberrima Oliveira & Marchi, 2005, Lestrimelitta, 76, 90 goeldiana (Friese, 1900), Plebeia, 78, 82 grandipennis (Schwarz, 1951), Partamona, 117 grandis Guérin, 1844, Melipona, 410, 469 gregaria Pedro & Camargo, 2003, Partamona, 27 gribodoi (Magretti, 1884), Hypotrigona, 264 griswoldorum Eardley, 2004, Meliponula (Meliplebeia), 264 guatemalensis (Schwarz, 1938), Paratrigona, 101, 140 guerreroensis Schwarz, 1936, Melipona fasciata [= Melipona (Michmelia) fasciata], 357, 435 guianae Cockerell, 1910, Trigona, 80, 91 guyanensis Roubik, 1980, Lestrimelitta, 90 handlirschii (Friese, 1900), Tetragona, 91 helleri (Friese, 1900), Partamona, 126, 208 hellwegeri (Friese, 1900), Scaptotrigona, 141 Heterotrigona Schwarz, 1939, 8 Heterotrigona Schwarz, 1939, Heterotrigona (Heterotrigona), 8 Heterotrigona Schwarz, 1939, Trigona (Heterotrigona), 38 hildebrandti (Friese, 1900), Plebeina, 264 hockingsi Cockerell, 1929, Trigona, 45 hockingsi Cockerell, 1929, Trigona (Heterotrigona), 35, 45–47, 51 Homotrigona Moure, 1961, 8 hortorum (Linnaeus, 1761), Bombus, 177 hyalinata (Lepeletier, 1836), Trigona, 75 hypogea Silvestri, 1902, Trigona, 274 hypogea Silvestri, 1902, Trigona (Trigona), 10 Hypotrigona Cockerell, 1934, 8 illota Cockerell, 1919, Melipona, 370 illustris Schwarz, 1932, Meliponam, 76 illustris Schwarz, 1932, Melipona (Eomelipona), 76 impunctata (Ducke, 1916), Aparatrigona, 76, 90 562 Appendix A indecisa Cockerell, 1919, Melipona, 77, 82 indecisa Cockerell, 1919, Melipona (Michmelia), 77 interrupta Latreille, 1811, Melipona (Melikerria), 76, 90 inusitata Moure & Camargo, 1992, Geotrigona [= Geotrigona mombuca (Smith, 1863)], 274 japonica Radoszkowski, 1877, Apis cerana, 154, 175, 501 jatiformis (Cockerell, 1912), Plebeia, 101, 117, 141 jujuyensis (Schrottky, 1911), Scaptotrigona, 126–131, 515, 516 kaieteurensis (Schwarz, 1938), Tetragona, 91 kangarumensis Cockerell, 1920, Melipona lateralis [=Melipona (Michmelia) lateralis], 77 kerri Moure, 1950, Plebeia, 410 laeviceps (Smith, 1857), Tetragonula, 495–503 laeviceps Smith, 1857, Trigona, 178 laeviceps Smith, 1857, Trigona (Tetragonula), 155 lapidarius (Linnaeus, 1758), Bombus, 178 lateralis Erichson, 1848, Melipona, 77, 90 lateralis Erichson, 1848, Melipona (Michmelia), 77, 90 latitarsis (Friese, 1900), Plebeia (Scaura), 11, 79 latitarsis (Friese, 1900), Scaura, 91, 274 lendliana (Friese, 1900), Meliponula (Meliplebeia), 264 Lepidotrigona Schwarz, 1939, 8 Lestrimelitta Friese, 1903, 7, 20, 93 Leurotrigona Moure, 1950, 20, 93 limae (Brèthes, 1920), Scaptotrigona, 385, 386, 389 limao (Smith, 1863), Lestrimelitta, 225, 292 Liotrigona Moure, 1961, 8 Lisotrigona Moure, 1961, 8 llorentei Ayala, 1999, Plebeia, 101, 117, 141 longitarsis (Ducke, 1916), Dolichotrigona, 90 longula (Lepeletier, 1836), Scaura, 91 lophocoryphe Moure, 1963, Paratrigona, 116 Lophotrigona Moure, 1961, 8 lucii Moure, 2004, Plebeia, 208 lucorum (Linnaeus, 1761), Bombus, 178 lupitae Ayala, 1999, Melipona, 140 lurida (Smith, 1854), Ptilotrigona, 79, 91 luteipennis (Friese, 1902), Scaptotrigona, 117 lutzi Camargo & Moure, 1996, Geotrigona, 116 manantlensis Ayala, 1999, Plebeia, 141 manaosensis Schwarz, 1932, Melipona compressipes (= Melipona interrupta), 289 manauara Camargo and Pedro, 2009, Celetrigona, 90 mandacaia Smith, 1863, Melipona, 288, 368, 412, 543, 549 maracaia Marchi & Melo, 2006, Lestrimelitta, 76 marginata Lepeletier, 1836, Melipona, 274 Appendix A 563 maya Ayala, 1999, Trigonisca, 102, 141 mayarum (Cockerell, 1912), Tetragona, 102, 141 mazucatoi (Almeida, 1992), Trigona (= Trigona cilipes), 91 mediorufa (Cockerell, 1913), Oxytrigona, 101, 140 Megachile Latreille, 1802, 176, 177 melanica Ayala, 1999, Plebeia, 101, 141 melanocephala Gribodo, 1893, Trigona, 179 melanocera (Schwarz, 1938), Nannotrigona, 77 melanopleura Cockerell, 1919, Melipona [= Melipona (Michmelia) costaricensis], 544 melanoventer Schwarz, 1932, Melipona (Michmelia), 90 Melikerria Moure, 1992 (= Melipona), 7 Melikerria Moure, 1992, Melipona (Melikerria), 93 melina Gribodo, 1893, Trigona, 179 Meliplebeia Moure, 1961, Meliponula (Meliplebeia), 8 Melipona Illiger, 1806, 7, 20, 75, 93 Melipona Illiger, 1806, Melipona (Melipona), 7, 20, 75, 93, 137, 139, 249 Meliponula Cockerell, 1934, 8 Meliponula Cockerell, 1934, Meliponula (Meliponula), 8 Meliwillea Roubik, Lobo & Camargo, 1997, 7, 20, 93 mellaria (Smith, 1862), Nannotrigona, 116 mellicolor (Packard, 1869), Oxytrigona, 78, 116 mellifera Linnaeus, 1758, Apis, 73, 94, 417 mellipes Friese (1898), Trigona, 45 mellipes Friese (1898), Trigona (Heterotrigona), 45–47 merrillae Cockerell, 1919, Melipona seminigra, 288 mexica Ayala, 1999, Plebeia, 141 mexicana (Guérin, 1844), Scaptotrigona, 117 Micheneria Kerr, Pisani & Aily, 1967, Melipona (Micheneria) [=Melipona (Michmelia)], 7 Michmelia Moure, 1975, Melipona (Michmelia), 93 minima (Gribodo, 1893), Plebeia, 91, 117 minor (Moure and Camargo, 1982), Nogueirapis, 91 mirandula Cockerell, 1917, Nogueirapis, 116 mixteca Ayala, 1999, Trigonisca, 141 molesta (Puls, in Strobel, 1868), Plebeia, 125 mombuca (Smith, 1863), Geotrigona, 211, 325 mondury Smith, 1863, Melipona, 549, 553 monodonta Camargo & Moure, 1989, Lestrimelitta, 90 moorei (Schwarz, 1937), Heterotrigona (Sundatrigona), 11 mosquito (Smith, 1863), Plebeia, 91 moureana Ayala, 1999, Plebeia, 101, 141 mourei Camargo, 1980, Partamona, 91 mourei Oliveira & Marchi, 2005, Lestrimelitta, 116 Mourella Schwarz, 1946, 20, 93 Mourella Schwarz, 1946 [= Plebeia (Plebeia)] 564 Appendix A muelleri (Friese, 1900), Leurotrigona, 126, 225 musarum (Cockerell, 1917), Partamona, 117 muzoensis Schwarz, 1948, Trigona, 118 Nannotrigona Cockerell, 1922, 7, 20, 93 nebulata (Smith, 1854), Meliponula (Meliplebeia), 264 nebulata (Smith, 1854), Meliponula, 325 necrophaga Camargo & Roubik, 1991, Trigona, 118 nigerrima Cresson, 1878, Trigona, 102, 118, 141 nigra (Cresson, 1878), Frieseomelitta, 101, 116, 140 nigra Cresson, 1878, Trigona, 357 nigriceps (Friese, 1901), Plebeia, 126 nigrior (Cockerell, 1925), Partamona, 78 niitkib Ayala, 1999, Lestrimelitta, 101, 140 Nogueirapis Moure, 1953, 7, 20, 93 oaxacana Ayala, 1999, Cephalotrigona, 140 obscura (Friese, 1900), Oxytrigona, 91 obscurior Moure, 1971, Melipona, 126, 129 occidentalis (Schulz, 1904), Ptilotrigona, 117 ochrotricha (Buysson, in Du Buysson & Marshall, 1892), Scaptotrigona, 79, 82 Odontotrigona Moure, 1961, 8 Odontotrigona Moure, 1961, Odontotrigona (Odontotrigona), 8 ogilviei Schwarz, 1932, Melipona, 76, 82, 83, 90 ogilviei Schwarz, 1932, Melipona (Eomelipona), 76, 90 ogouensis (Vachal, 1903), Meliponula (Meliplebeia), 264 opaca (Cockerell, 1917), Paratrigona, 100, 116, 140 orbignyi (Guérin,1844), Melipona, 126, 129, 131 orbygnii (Guérin, 1844), Melipona [sic = Melipona orbignyi] orizabaensis (Strand, 1919), Partamona, 101, 117, 140 ornata (Rayment, 1932), Austroplebeia, 43, 47 ornaticeps (Schwarz, 1938), Paratrigona, 116 Oxytrigona Cockerell, 1917, 7, 20, 93, 137 pagdeni (Schwarz, 1939), Tetragonula, 181 pallens (Fabricius, 1798), Trigona, 80, 92 pallida Fox, 1899, Centris, 175 panamensis (Cockerell, 1913), Scaptotrigona, 564 panamica Cockerell, 1912, Melipona, 116 pannosa Moure, 1989, Paratrigona, 78, 91 Papuatrigona Michener & Sakagami, 1990, 8 paraensis Ducke, 1916, Melipona, 90 paraensis Ducke, 1916, Melipona (Michmelia), 77, 90 paraensis Ducke, 1916, Melipona rufiventris, 288 Parapartamona Schwarz, 1948, 20, 93 Parapartamona Schwarz, 1948, Partamona (Parapartamona), 7 Paratetrapedia Moure, 1941, 4 Paratrigona Schwarz, 1938, 7, 20, 93 Appendix A Paratrigonoides Camargo & Roubik, 2005, 7, 20, 93 Pariotrigona Moure, 1961, 8 parkeri Ayala, 1999, Plebeia, 101, 141 Partamona Schwarz, 1939, 20, 93 Partamona Schwarz, 1939, Partamona (Partamona), 7 pascuorum (Scopoli, 1763), Bombus, 178 Patera Schwarz, 1938 (= Partamona), 7 paupera (Provancher, 1888), Frieseomelitta, 76, 81, 116 paupera (Provancher, 1888), Trigona (Frieseomelitta) nigra, 76 pavani (Moure, 1963), Duckeola, 76, 90 pearsoni (Schwarz, 1938), Partamona, 78, 91 peckolti (Friese, 1901), Partamona, 78 pectoralis (Dalla Torre, 1896), Scaptotrigona, 102, 117, 141, 395 peltata (Spinola, 1853), Paratrigona, 11 penna Eardley, 2004, Hypotrigona, 264 perangulata (Cockerell, 1917), Tetragona, 117 percincta (Cockerell, 1929), Austroplebeia, 42, 43 pereneae (Schwarz, 1943), Ptilotrigona, 22 perilampoides (Cresson, 1878), Nannotrigona, 77, 101, 116, 140, 396 permixta Camargo & Moure, 1994, Paratrigona, 78, 82 permodica Almeida, 1995, Trigona, 91 pipioli Ayala, 1999, Trigonisca, 102, 104, 118, 141 Platytrigona Moure, 1961, 8 Plebeia Schwarz, 1938, 20, 93 Plebeia Schwarz, 1938, Plebeia (Plebeia), 7 Plebeiella Moure, 1961 [= Meliponula (Meliplebeia)], 565 Plebeina Moure, 1961, 8 plumata Smith, 1853, Ptilothrix, 176 poecilochroa Moure & Camargo, 1993, Plebeia, 274 polysticta Moure, 1950, Scaptotrigona, 75, 410, 469 portoi (Friese, 1900), Frieseomelitta, 90 postica (Latreille,1807), Scaptotrigona, 274 prisca (Michener & Grimaldi,1988), Cretotrigona, 145 prisca Michener & Grimaldi,1988, Trigona, 14 Proplebeia Michener, 1982, 20 Ptilotrigona Moure, 1951, 20, 93 Ptilotrigona Moure, 1951 [= Trigona (Tetragona)] pulchra Ayala, 1999, Plebeia, 101, 117, 141 punctata (Smith, 1854), Nannotrigona, 90 puncticollis Friese, 1902, Melipona (Eomelipona), 90 pusilla Moure and Camargo 1988 in Moure et al., 1988, Leurotrigona, 90 quadrifasciata Lepeletier, 1836, Melipona, 274 quadrifasciata Lepeletier, 1836, Melipona quadrifasciata, 126, 177 quadripunctata (Lepeletier, 1836), Schwarziana, 126, 325–326 quinquefasciata Lepeletier, 1836, Melipona, 126, 174, 177, 181, 182, 326 565 566 Appendix A recursa Smith, 1863, Trigona, 92, 274, 292 remota (Holmberg, 1903), Plebeia, 565 richardsi (Darchen, 1981), Meliponula (Axestotrigona), 263 rotundata (Fabricius, 1787), Megachile, 175, 176 roubiki Eardley, 2004, Meliponula (Meliplebeia), 264 rozeni Engel, 2001, Liotrigonopsis, 14 rufipes (Friese, 1903), Lestrimelitta, 126 rufiventris Lepeletier, 1836, Melipona, 177, 180, 194, 288, 471, 476, 488, 542, 543, 548 ruspolii (Magretti, 1898), Hypotrigona, 264 saiqui (Friese, 1900), Plebeia, 290 Sakagamilla Moure, 1989 (= Scaptotrigona), 7 sapiens Cockerell, 1911, Trigona (Heterotrigona), 45 sapiens Cockerell, 1911, Trigona, 45 savannensis Roubik, 1980, Tetragona (= Friesomelitta flavicornis), 90 sawadogoi (Darchen, 1970), Meliponula (Axestotrigona), 263 Scaptotrigona Moure, 1942, 7, 20, 93 Scaura Schwarz, 1938, 20, 93 Scaura Schwarz, 1938, Plebeia (Scaura), 7 schencki Gribodo, 1893, Melipona bicolor, 208, 326 schmidti (Stadelmann, 1895), Dactylurina, 264 schrottkyi (Friese, 1900), Friesella, 75 schulthessi (Friese, 1900), Dolichotrigona, 101, 116 schulthessi (Friese, 1900), Trigonisca , 141 schultzei (Friese, 1901), Nannotrigona, 78, 90 Schwarziana Moure, 1943, 20, 93 Schwarziana Moure, 1943, Plebeia (Schwarziana), 7 Schwarzula Moure, 1946, 20, 93 scutellaris Latreille, 1811, Melipona, 274 scutellata Lepeletier, 1836, Apis mellifera, 175, 298 scutellata Lepeletier, 1836, Apis, 265 seminigra Friese, 1903, Melipona, 161, 162, 192, 288, 289 seridoensis Pedro & Camargo, 2003, Partamona, 482 sesquipedalis Almeida, 1984, Trigona, 92 silacea (Wille, 1959), Nogueirapis, 139 silvestriana (Vachal, 1908), Trigona, 75, 102, 118, 141 silvestrii (Friese, 1902), Frieseomelitta, 534 solani Cockerell, 1912, Melipona, 101, 140, 396 spinipes (Fabricius, 1793), Trigona, 75 staudingeri (Gribodo, 1893), Dactylurina, 264 subgrisea (Schwarz, 1940), Geotrigona, 566 subnitida Ducke, 1910, Melipona, 179, 204, 435, 439, 440, 471, 482, 487, 488, 542, 543, 549 subnuda Moure, 1947, Paratrigona, 566 subobscuripennis (Schwarz, 1951), Scaptotrigona, 117 sulina Marchi & Melo, 2006, Lestrimelitta, 126 Appendix A Sundatrigona Inoue & Sakagami, 1995, Heterotrigona (Sundatrigona), 8 symei (Rayment, 1932), Austroplebeia, 43 tarsata Smith, 1874, Centris, 176, 177, 290 tataira (Smith, 1863), Oxytrigona, 126 tenuis (Ducke, 1916), Scaura, 91 terrestris (Linnaeus, 1758), Bombus, 177 terricola Camargo & Moure, 1996, Geotrigona, 100 testacea (Klug, 1807), Partamona, 91 testaceicornis (Lepeletier, 1836), Nannotrigona, 274 Tetragona Lepeletier & Serville, 1828, 20, 93 Tetragona Lepeletier & Serville, 1828, Trigona (Tetragona), 7 Tetragonilla Moure, 1961, Tetragonula (Tetragonilla), 8 Tetragonisca Moure, 1946, 20, 93 Tetragonisca Moure, 1946, Trigona (Tetragonisca), 7 Tetragonula Moure, 1961, 38 Tetragonula Moure, 1961, Tetragonula (Tetragonula), 8 Tetrigona Moure, 1961, Odontotrigona (Tetrigona), 8 tica (Wille, 1969), Plebeia, 117 timida (Silvestri, 1902), Plebeia (Scaura), 9 timida (Silvestri, 1902), Scaura, 22 titania Gribodo, 1893, Melipona, 126 torrida Friese, 1916, Melipona, 116 Trichotrigona Camargo & Moure, 1983, 7, 20, 94 Trigona Jurine, 1807, 20, 93 Trigona Jurine, 1807, Trigona (Trigona), 7 Trigonella Sakagami & Moure, 1975 [= Heterotrigona (Sundatrigona)], 8 Trigonisca Moure, 1950, 20, 93 trinidadensis (Provancher, 1888), Trigona (= Trigona amalthea), 75 trinitatis Cockerell, 1919, Melipona, 83, 364 trinitatis Cockerell, 1919, Melipona (Michmelia), 77 tristella Cockerell, 1922, Nannotrigona, 78, 82 truculenta Almeida, 1984, Trigona, 80 tubiba (Smith, 1863), Scaptotrigona, 91 varia (Lepeletier, 1836), Frieseomelitta, 90, 274 variegatipes Gribodo, 1893, Melipona, 145 venezuelana Schwarz, 1948, Trigona, 80, 82 vicina Camargo, 1980, Partamona, 78, 91 vitae Pedro & Camargo, 2003, Partamona, 78 websteri (Rayment, 1932), Austroplebeia, 43 wheeleri (Cockerell, 1913), Scaptotrigona, 100, 117 williana Friese, 1900, Trigona, 80, 92 wittmanni Moure & Camargo, 1989, Plebeia, 126 xanthotricha Moure, 1950, Scaptotrigona, 410, 469 yucatanica Camargo, Moure & Roubik, 1988, Melipona, 101, 116, 140 zexmeniae (Cockerell, 1912), Cephalotrigona, 101, 116, 140 ziegleri (Friese, 1900), Tetragona , 79, 117 567 Appendix B List of Bee Taxa In this entry of bee taxa by genus, after the taxonomic index of bees, countries mentioned in this book are given. Broad distribitutions of the taxa are not included in this list, e.g., Table 5.1 in the French Guiana chapter. Names in bold are junior synonyms (senior synonyms are indicated in brackets or square brackets). Alphaneura Gray, 1832 [= Trigona], 7 Amalthea Rafinesque, 1815 [= Trigona], 7 Andrena Fabricius, 1775, 3 Anthophora Latreille, 1803, 175 Aparatrigona impunctata (Ducke, 1916) French Guiana, Venezuela, 76, 90 Aparatrigona Moure, 1951, 7, 20, 92 Aphaneura Gray, 1832 [= Trigona], 7 Apis Linnaeus, 1758 Argentina, Australia, Brazil, Costa Rica, Germany, 73, 94, 249, 417 Apis cerana Fabricius, 1793, 569 Apis cerana japonica Radoszkowski, 1877, 154, 175, 241, 496, 501 Apis dorsata Fabricius, 1793 Thailand, 252, 484 Apis florea Fabricius, 1787, 252, 496 Apis mellifera Linnaeus, 1758 Bolivia, Brazil, Czech Republic, French Guiana, Mexico, Panama, Uganda, Venezuela, 73, 94, 305, 417 Apis mellifera scutellata Lepeletier, 1836, 175, 298 Apis scutellata Lepeletier, 265 Apotrigona Moure, 1961[= Meliponula (Meliplebeia)], 8 Austroplebeia Moure, 1961 Australia, 8, 42 Austroplebeia australis (Friese, 1898) Australia, 43 Austroplebeia cassiae (Cockerell, 1910) Australia, 43 Austroplebeia cincta (Mocsáry in Friese, 1898) Australia, 43 Austroplebeia cockerelli (Rayment, 1930) Australia, 43 Austroplebeia essingtoni (Cockerell, 1905) Australia, 43 Austroplebeia ornata (Rayment, 1932) Australia, 43 Austroplebeia percincta (Cockerell, 1929) Australia, 42, 43 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7, © Springer Science+Business Media New York 2013 569 570 Appendix B Austroplebeia symei (Rayment, 1932) Australia, 43 Austroplebeia websteri (Rayment, 1932) Australia, 43 Bombus Latreille, 1802 Argentina, 177, 178, 181, 277, 485 Bombus cryptarum (Fabricius, 1775), 177 Bombus hortorum (Linnaeus, 1761), 177 Bombus lapidarius (Linnaeus, 1758), 178 Bombus lucorum (Linnaeus, 1761), 178 Bombus pascuorum (Scopoli, 1763), 178 Bombus terrestris (Linnaeus, 1758), 177 Camargoia Moure, 1989, 20 Camargoia Moure, 1989 [= Trigona (Tetragona)], 7 Camargoia camargoi Moure, 1989 French Guiana, 90, 92 Celetrigona Moure, 1950, 20, 92 Celetrigona Moure, 1950 [= Trigonisca], 7 Celetrigona manauara Camargo & Pedro, 2009 French Guiana, 90 Centris pallida Fox, 1899, 175 Centris tarsata Smith, 1874, 177, 290 Cephalotrigona Schwarz, 1940, 7, 20, 92, 137 Cephalotrigona capitata (Smith, 1854) Argentina, French Guiana, Venezuela, 76, 90, 274 Cephalotrigona eburneiventer (Schwarz, 1948) Mexico, 140 Cephalotrigona oaxacana Ayala, 1999 Mexico, 140 Cephalotrigona zexmeniae (Cockerell, 1912) Costa Rica, Guatemala, Mexico, 101, 116, 140 Cleptotrigona Moure, 1961, 8 Cleptotrigona cubiceps (Friese, 1912) Africa, 264 Cretotrigona Engel, 2000 {extinct}, 14 Cretotrigona prisca (Michener & Grimaldi,1988) {extinct}USA, 14, 19, 145, 252, 363 Dactylurina Cockerell, 1934, 8 Dactylurina schmidti (Stadelmann, 1895) Africa, 264 Dactylurina staudingeri (Gribodo, 1893) Africa, 264 Diadasina distincta (Holmberg, 1903), 176 Dioxys Lepeletier & Serville, 1825, 3 Dolichotrigona Moure, 1950, 20, 92 Dolichotrigona Moure, 1950 (= Trigonisca), 7 Dolichotrigona longitarsis (Ducke, 1916) French Guiana, 90 Dolichotrigona schulthessi (Friese, 1900) Costa Rica, Guatemala, 101, 116 Duckeola Moure, 1944, 20, 92, 139 Duckeola Moure, 1944, Trigona (Duckeola), 7 Duckeola ghilianii (Spinola, 1853) French Guiana, 90 Duckeola pavani (Moure, 1963) French Guiana, Venezuela, 90 Eomelipona Moure, 1992 (= Melipona), 7, 93 Friesella Moure, 1946, 20, 92 Friesella Moure, 1946 [= Plebeia (Plebeia)], 7 Friesella schrottkyi (Friese, 1900) Brazil, 75 Appendix B 571 Frieseomelitta Ihering, 1912 Colombia, Venezuela, 20, 92 Frieseomelitta Ihering, 1912, Trigona (Frieseomelitta), 7 Frieseomelitta flavicornis (Fabricius, 1798) French Guiana, 90 Frieseomelitta nigra (Cresson, 1878) Costa Rica, Guatemala, Mexico, 101, 116, 140 Frieseomelitta paupera (Provancher, 1888) Costa Rica, Venezuela, 76, 116 Frieseomelitta portoi (Friese, 1900) French Guiana, 90 Frieseomelitta silvestrii (Friese, 1902), 534 Frieseomelitta varia (Lepeletier, 1836) Argentina, Brazil, Venezuela, 274 Geniotrigona Moure, 1961, Heterotrigona (Geniotrigona), 8 Geotrigona Moure, 1943 Venezuela, 7, 20, 93 Geotrigona acapulconis (Strand, 1919) Guatemala, Mexico, 101, 140, 395 Geotrigona argentina, Camargo & Moure, 1996 Argentina, 126 Geotrigona chiriquiensis (Schwarz, 1951) Costa Rica, 116 Geotrigona inusitata Moure & Camargo, 1992 [= Geotrigona mombuca (Smith, 1863)], 274 Geotrigona leucogastra (Cockerell, 1914), 571 Geotrigona lutzi Camargo & Moure, 1996 Costa Rica, 100, 116 Geotrigona mombuca (Smith, 1863) Brazil, 211, 325–326 Geotrigona subgrisea (Schwarz, 1940), 571 Geotrigona subnigra (Schwarz, 1940) Venezuela, 76, 94 Geotrigona subterranea (Friese, 1901), 571 Geotrigona terricola Camargo & Moure, 1996, 100 Heterotrigona Schwarz, 1939, 8 Heterotrigona Schwarz, 1939, Heterotrigona (Heterotrigona), 8 Heterotrigona Schwarz, 1939, Trigona (Heterotrigona), 35, 36, 38, 41, 45–48, 51, 56, 60, 61, 67 Heterotrigona (Sundatrigona) moorei (Schwarz, 1937) Indonesia, Thailand, 8, 11 Homotrigona Moure, 1961, 8 Hypotrigona Cockerell, 1934, 8 Hypotrigona araujoi (Michener, 1959) Africa, 264 Hypotrigona gribodoi (Magretti, 1884) Africa, 264 Hypotrigona penna Eardley, 2004 Africa, 264 Hypotrigona ruspolii (Magretti, 1898) Africa, 264 Kelneriapis eocenica (Kelner-Pillaut, 1970), 14 Lepidotrigona Schwarz, 1939, 8 Lestrimelitta Friese, 1903, 7, 20, 93 Lestrimelitta chacoana Roig Alsina, 2010 Argentina, 126 Lestrimelitta chamelensis Ayala, 1999 Mexico, 140 Lestrimelitta danuncia Oliveira & Marchi, 2005 Costa Rica, 116 Lestrimelitta glaberrima Oliveira & Marchi, 2005 French Guiana, Venezuela, 76, 90 Lestrimelitta guyanensis Roubik, 1980 French Guiana, 90 Lestrimelitta limao (Smith, 1863) Brazil, 292 Lestrimelitta maracaia Marchi & Melo, 2006 Venezuela, 76 Lestrimelitta monodonta Camargo & Moure, 1989 French Guiana, 90 Lestrimelitta mourei Oliveira & Marchi, 2005 Costa Rica, 116 572 Appendix B Lestrimelitta niitkib Ayala, 1999 Guatemala, Mexico, 101, 140 Lestrimelitta rufipes (Friese, 1903) Argentina, 126 Lestrimelitta sulina Marchi & Melo, 2006 Argentina, 126 Leurotrigona Moure, 1950, 20, 93 Leurotrigona Moure, 1950 (= Trigonisca), 7 Leurotrigona muelleri (Friese, 1900) Argentina, Brazil, 126 Leurotrigona pusilla Moure and Camargo 1988 in Moure et al., 1988 French Guiana, 90 Liotrigona bottegoi (Magretti, 1895) Africa, 264 Liotrigona Moure, 1961, 8 Liotrigonopsis rozeni Engel, 2001, 14 Lisotrigona Moure, 1961, 8 Lophotrigona Moure, 1961, 8 Megachile Latreille, 1802, 176, 177 Megachile rotundata (Fabricius, 1787), 175, 176 Melikerria Moure, 1992 (= Melipona), 7, 93 Melipona Illiger, 1806 Brazil, Colombia, 7, 20, 93 Melipona apiformis (Buysson, in Du Buysson & Marshall, 1892), 82 Melipona asilvai Moure, 1971 Brazil, 368, 542, 543, 549 Melipona baeri Vachal, 1904 Argentina, 126 Melipona beecheii Bennett, 1831 Costa Rica, Guatemala, Mexico, 101, 106 Melipona belizeae Schwarz, 1932, 147 Melipona bicolor Lepeletier, 1836 Brazil, 274 Melipona bicolor schencki Gribodo, 1893 Argentina, 126 Melipona brachychaeta Moure, 1950 Bolivia, 469 Melipona capixaba Moure & Camargo, 1994, 179 Melipona carrikeri Cockerell, 1919 Costa Rica, 116 Melipona colimana Ayala, 1999 Mexico, 140 Melipona compressipes (Fabricius, 1804) Brazil, Colombia, Venezuela, 76, 90 Melipona compressipes manaosensis Schwarz, 1932 (= Melipona interrupta) Brazil, 289 Melipona concinnula Cockerell, 1919, 76, 82 Melipona costaricensis Cockerell, 1919 Costa Rica, 116 Melipona cramptoni Cockerell, 1920, 77, 82 Melipona crinita Moure & Kerr, 1950 Bolivia, 410 Melipona eburnea Friese, 1900 Colombia, 370, 385–387, 391, 420, 421, 422 Melipona fasciata Latreille, 1811 Mexico, Panama, 140 Melipona fasciata cramptoni duidae Schwarz, 1932 [= Melipona (Michmelia) cramptoni], 77, 82 Melipona fasciata guerreroensis Schwarz, 1[= Melipona (Michmelia) fasciata], 357, 435 Melipona fasciculata Smith, 1854 Brazil, 158, 165, 355, 380, 435, 439, 440, 471, 488, 543, 548, 549, 553 Melipona favosa (Fabricius, 1798) Colombia, Venezuela, 77, 90, 363 Melipona flavolineata Friese, 1900 Brazil, 56, 543, 549 Appendix B 573 Melipona fuliginosa Lepeletier, 1836 Argentina, Costa Rica, 90, 116 Melipona fulva Lepeletier, 1836, 77, 90 Melipona fuscipes Friese, 1900 (= Melipona fasciata), 82 Melipona fuscopilosa Moure & Kerr, 1950 Venezuela, 77 Melipona grandis Guérin, 1844 Bolivia, 370, 410–412, 414, 435, 469, 526, 531, 535 Melipona illota Cockerell, 1919, 370 Melipona illustris Schwarz, 1932, 76 Melipona indecisa Cockerell, 1919, 77, 82 Melipona lateralis Erichson, 1848, 77, 90 Melipona lateralis kangarumensis Cockerell, 1920 [= Melipona (Michmelia) lateralis], 77 Melipona lupitae Ayala, 1999 Mexico, 140 Melipona mandacaia Smith, 1863 Brazil, 288, 368, 412, 543, 549 Melipona marginata Lepeletier, 1836, 274 Melipona melanopleura Cockerell, 1919 [= Melipona (Michmelia) costaricensis], 544 Melipona mondury Smith, 1863 Brazil, 549, 553 Melipona obscurior Moure, 1971 Argentina, 126, 129 Melipona ogilviei Schwarz, 1932, 76, 90 Melipona orbignyi (Guérin,1844), Melipona [sic = Melipona orbignyi] Argentina, 126, 129, 131 Melipona panamica Cockerell, 1912 Costa Rica, 116 Melipona paraensis Ducke, 1916 , 77, 90 Melipona quadrifasciata Lepeletier, 1836 Argentina, Brazil, 274 Melipona quadrifasciata anthidioides Lepeletier, 1836 Brazil, 412, 530, 531, 543 Melipona quadrifasciata quadrifasciata Lepeletier, 1836 Brazil, 543 Melipona quinquefasciata Lepeletier, 1836 Argentina, Brazil, 126, 174, 177, 181, 182, 326 Melipona rufiventris Lepeletier, 1836 Brazil, 177, 180, 471, 476, 488, 542, 543, 548 Melipona rufiventris paraensis Ducke, 1916, 288 Melipona scutellaris Latreille, 1811 Brazil, 274 Melipona seminigra Friese, 1903 Brazil, 161, 162, 192, 288, 289 Melipona seminigra merrillae Cockerell, 1919, 288 Melipona solani Cockerell, 1912 Guatemala, Mexico, 101, 140, 396 Melipona subnitida Ducke, 1910 Brazil, 156, 179, 204, 331, 435, 439, 440, 471, 482, 487 Melipona torrida Friese, 1916 Costa Rica, 116 Melipona titania Gribodo, 1893, 126 Melipona trinitatis Cockerell, 1919, 77 Melipona variegatipes Gribodo, 1893, 145 Melipona yucatanica Camargo, Moure & Roubik, 1988 Costa Rica, Guatemala, Mexico, 101, 116, 140 Melipona (Melipona) Melipona Illiger, 1806, 7, 20, 75, 93 Melipona (Eomelipona) bradleyi (Schwarz, 1932) French Guiana, 90 Melipona (Eomelipona) concinnula Cockerell, 1919 Venezuela, 76 Melipona (Eomelipona) Eomelipona Moure, 1992, 7, 93 574 Appendix B Melipona (Eomelipona) illustris Schwarz, 1932 Venezuela, 76 Melipona (Eomelipona) ogilviei Schwarz, 1932 French Guiana, Venezuela, 76, 90 Melipona (Eomelipona) puncticollis Friese, 1902 French Guiana, 90 Melipona (Melikerria) compressipes (Fabricius, 1804) French Guiana, Venezuela, 76, 90, 274 Melipona (Melikerria) grandis Guérin, 1844, 573 Melipona (Melikerria) interrupta Latreille, 1811 French Guiana, Venezuela, 76, 90 Melipona (Melikerria) Melikerria Moure, 1992, 7, 93 Melipona (Melipona) favosa (Fabricius, 1798) French Guiana, Venezuela, 77, 90, 363 Melipona (Michmelia) apiformis (Buysson, in Du Buysson & Marshall, 1892) Venezuela, 77, 82 Melipona (Michmelia) captiosa Moure, 1962 French Guiana, 90 Melipona (Michmelia) cramptoni Cockerell, 1920 Venezuela, 77, 82 Melipona (Michmelia) crinita Moure & Kerr, 1950 Venezuela, 77, 410 Melipona (Michmelia) eburnea Friese, 1900, 370, 385–387, 391, 418, 420–422 Melipona (Michmelia) fasciata Latreille, 1811, 82, 140 Melipona (Michmelia) fuliginosa Lepeletier, 1836 French Guiana, 90, 116 Melipona (Michmelia) fulva Lepeletier, 1836 French Guiana, Venezuela, 77, 90 Melipona (Michmelia) indecisa Cockerell, 1919 Venezuela, 77, 82 Melipona (Michmelia) lateralis Erichson, 1848 French Guiana, Venezuela, 77, 90 Melipona (Michmelia) melanoventer Schwarz, 1932 French Guiana, 90 Melipona (Michmelia) Michmelia Moure, 1975 Venezuela, 7, 93 Melipona (Michmelia) paraensis Ducke, 1916 French Guiana, Venezuela, 77, 90 Melipona (Michmelia) trinitatis Cockerell, 1919 Venezuela, 77 Meliponula Cockerell, 1934, 8 Meliponula bocandei (Spinola, 1853) Uganda, 264 Meliponula ferruginea (Lepeletier, 1841), 264 Meliponula nebulata (Smith, 1854) Uganda, 264 Meliponula (Axestotrigona) Axestotrigona Moure, 1961, 8 Meliponula (Axestotrigona) cameroonensis (Friese,1900) Africa, 264 Meliponula (Axestotrigona) eburnensis (Darchen, 1970), 263 Meliponula (Axestotrigona) ferruginea (Lepeletier, 1841) Africa, 264 Meliponula (Axestotrigona) richardsi (Darchen, 1981), 263 Meliponula (Axestotrigona) sawadogoi (Darchen, 1970), 263 Meliponula (Meliplebeia) beccarii (Gribodo, 1879) Africa, 264 Meliponula (Meliplebeia) griswoldorum Eardley, 2004 Africa, 264 Meliponula (Meliplebeia) lendliana (Friese, 1900) Africa, 264 Meliponula (Meliplebeia) Meliplebeia Moure, 1961, 8 Meliponula (Meliplebeia) nebulata (Smith, 1854) Africa, 264 Meliponula (Meliplebeia) ogouensis (Vachal, 1903) Africa, 264 Meliponula (Meliplebeia) roubiki Eardley, 2004 Africa, 264 Meliponula (Meliponula) bocandei (Spinola, 1853) Africa, 264 Meliponula (Meliponula) Meliponula Cockerell, 1934, 8 Meliwillea Roubik, Lobo & Camargo, 1997, 7, 20, 93, 116 Meliwillea bivea Roubik, Lobo & Camargo, 1997 Costa Rica, 116 Appendix B 575 Micheneria Kerr, Pisani & Aily, 1967 [= Melipona (Michmelia)], 7, 252 Michmelia Moure, 1975 (= Melipona), 7, 93 Mourella Schwarz, 1946, 20, 93 Mourella Schwarz, 1946 [= Plebeia (Plebeia)], 7 Mourella caerulea (Friese, 1900) Argentina, 126 Nannotrigona Cockerell, 1922 Colombia, Venezuela, 7, 20, 78, 93 Nannotrigona chapadana (Schwarz, 1938), 78 Nannotrigona melanocera (Schwarz, 1938) Venezuela, 77 Nannotrigona mellaria (Smith, 1862) Costa Rica, 116 Nannotrigona perilampoides (Cresson, 1878) Costa Rica, Guatemala, Mexico, 77, 101, 116, 140, 396 Nannotrigona punctata (Smith, 1854) French Guiana, 90 Nannotrigona schultzei (Friese, 1901) French Guiana, Venezuela, 78, 90 Nannotrigona testaceicornis (Lepeletier, 1836) Argentina, Brazil, Colombia, 274 Nannotrigona tristella Cockerell, 1922 Venezuela, 78, 82 Nogueirapis minor (Moure and Camargo, 1982) French Guiana, 91 Nogueirapis mirandula (Cockerell, 1917) Costa Rica, 116 Nogueirapis Moure, 1953, 7, 20, 93 Nogueirapis silacea (Wille, 1959), 139 Odontotrigona Moure, 1961, 8 Odontotrigona Moure, 1961, Odontotrigona (Odontotrigona), 8 Oxytrigona Cockerell, 1917, 7, 20, 93 Oxytrigona daemoniaca Camargo, 1984 Costa Rica, 116 Oxytrigona mediorufa (Cockerell, 1913) Guatemala, Mexico, 101, 140 Oxytrigona mellicolor (Packard, 1869) Costa Rica, Venezuela, 78, 116 Oxytrigona obscura (Friese, 1900) French Guiana, 91 Oxytrigona tataira (Smith, 1863) Argentina, 126 Papuatrigona Michener & Sakagami, 1990, 8 Parapartamona Schwarz, 1948, 20, 93 Parapartamona Schwarz, 1948, Partamona (Parapartamona), 7 Paratetrapedia Moure, 1941, 4 Paratrigona Schwarz, 1938 Colombia, 7, 20, 78 Paratrigona anduzei (Schwarz, 1943) Venezuela, 78, 81 Paratrigona femoralis Camargo & Moure, 1994 French Guiana, 91 Paratrigona glabella Camargo & Moure, 1994 Argentina, 126 Paratrigona guatemalensis (Schwarz, 1938) Guatemala, Mexico, 101, 140 Paratrigona lineata (Lepeletier, 1836), 575 Paratrigona lophocoryphe Moure, 1963 Costa Rica, 116 Paratrigona opaca (Cockerell, 1917) Costa Rica, Mexico, 100, 140 Paratrigona ornaticeps (Schwarz, 1938) Costa Rica, 116 Paratrigona pannosa Moure, 1989 French Guiana, Venezuela, 78, 91 Paratrigona peltata (Spinola, 1853) Costa Rica, 11 Paratrigona permixta Camargo & Moure, 1994 Venezuela, 78, 82 Paratrigona subnuda Moure, 1947, 334 Paratrigonoides Camargo & Roubik, 2005, 7, 20, 93 576 Appendix B Pariotrigona Moure, 1961, 8 Partamona Schwarz, 1939 Brazil, Colombia, 7, 20 Partamona Schwarz, 1939, Partamona (Partamona), 7 Partamona ailyae Camargo, 1980 Venezuela, 78 Partamona auripennis Pedro & Camargo, 2003 French Guiana, Venezuela, 78, 91 Partamona batesi Pedro & Camargo, 2003, 26, 27 Partamona bilineata (Say, 1837) Guatemala, Mexico, 101, 140 Partamona chapadicola Pedro & Camargo, 2003, 27 Partamona cupira (Smith, 1863), 274 Partamona epiphytophila Pedro & Camargo, 2003 Venezuela, 78 Partamona ferreirai Pedro & Camargo, 2003 French Guiana, Venezuela, 78, 91 Partamona grandipennis (Schwarz, 1951) Costa Rica, 117 Partamona gregaria Pedro & Camargo, 2003, 27 Partamona helleri (Friese, 1900) Argentina, Brazil, 126 Partamona mourei Camargo, 1980 French Guiana, 91 Partamona musarum (Cockerell, 1917) Costa Rica, 117 Partamona nigrior (Cockerell, 1925) Venezuela, 78 Partamona orizabaensis (Strand, 1919) Costa Rica, Guatemala, Mexico, 101, 117, 140 Partamona pearsoni (Schwarz, 1938) French Guiana, Venezuela, 78, 91 Partamona peckolti (Friese, 1901) Colombia, Venezuela, 78 Partamona seridoensis Pedro & Camargo, 2003, 482 Partamona testacea (Klug, 1807) French Guiana, 91 Partamona vicina Camargo, 1980 French Guiana, Venezuela, 78, 91 Partamona vitae Pedro & Camargo, 2003 Venezuela, 78 Patera Schwarz, 1938 (= Partamona), 7 Platytrigona Moure, 1961, 8 Plebeia Schwarz, 1938 Argentina, Brazil, Colombia, Guatemala, Venezuela, 7, 20 Plebeia Schwarz, 1938, Plebeia (Plebeia), 7 Plebeia (Scaura) latitarsis (Friese, 1900), 91, 103, 274 Plebeia (Scaura) timida (Silvestri, 1902), 9, 22 Plebeia catamarcensis (Holmberg, 1903) Argentina, 126 Plebeia cora Ayala, 1999 Mexico, 140 Plebeia droryana (Friese, 1900) Argentina, Bolivia, Brazil, 274 Plebeia emerina (Friese, 1900), 576 Plebeia franki (Friese, 1900) Costa Rica, 117 Plebeia fraterna Laroca & Rodriguez-Parilli, 2009 Venezuela, 78 Plebeia frontalis (Friese, 1911) Costa Rica, Guatemala, Mexico, 101, 117, 140 Plebeia fulvopilosa Ayala, 1999 Guatemala, Mexico, 101, 141 Plebeia goeldiana (Friese, 1900) Venezuela, 78, 82 Plebeia jatiformis (Cockerell, 1912) Costa Rica, Guatemala, Mexico, 101, 117, 141 Plebeia kerri Moure, 1950 Bolivia, 410 Plebeia lucii Moure, 2004 Brazil, 208 Plebeia llorentei Ayala, 1999 Costa Rica, Guatemala, Mexico, 101, 117, 141 Plebeia manantlensis Ayala, 1999 Mexico, 141 Plebeia melanica Ayala, 1999 Guatemala, Mexico, 101, 141 Appendix B 577 Plebeia mexica Ayala, 1999 Mexico, 141 Plebeia minima (Gribodo, 1893) Costa Rica, French Guiana, 91, 117 Plebeia molesta (Puls, in Strobel, 1868) Argentina, 125 Plebeia mosquito (Smith, 1863) French Guiana, 91 Plebeia moureana Ayala, 1999 Guatemala, Mexico, 101, 141 Plebeia nigriceps (Friese, 1901) Argentina, 126 Plebeia parkeri Ayala, 1999 Guatemala, Mexico, 101, 141 Plebeia poecilochroa Moure & Camargo, 1993, 274 Plebeia pulchra Ayala, 1999 Costa Rica, Guatemala, Mexico, 101, 117, 141 Plebeia remota (Holmberg, 1903), 334 Plebeia saiqui (Friese, 1900), 290 Plebeia tica (Wille, 1969) Costa Rica, 117 Plebeia wittmanni Moure & Camargo, 1989 Argentina, 126 Plebeiella Moure, 1961 [= Meliponula (Meliplebeia)], 577 Plebeina Moure, 1961, 8 Plebeina hildebrandti (Friese, 1900) Africa, 264 Proplebeia Michener, 1982 {extinct}Dominican Republic, Mexico, 20 Proplebeia dominicana (Wille & Chandler, 1964) {extinct} Dominican Republic, 154, 252 Ptilothrix plumata Smith, 1853, 176 Ptilotrigona lurida (Smith, 1854) Brazil, French Guiana, Venezuela, 79, 91 Ptilotrigona Moure, 1951, 20, 93 Ptilotrigona Moure, 1951 [= Trigona (Tetragona)], 7 Ptilotrigona occidentalis (Schulz, 1904) Costa Rica, 117 Ptilotrigona pereneae (Schwarz, 1943), 22 Sakagamilla Moure, 1989 (= Scaptotrigona), 7 Scaptotrigona Moure, 1942 Argentina, Brazil, Colombia, Paraguay, Venezuela, 7, 20 Scaptotrigona bipunctata (Lepeletier, 1836), 577 Scaptotrigona depilis (Moure, 1942) Argentina, Bolivia, Brazil, Venezuela, 91, 410 Scaptotrigona fulvicutis (Moure, 1964) French Guiana, 91 Scaptotrigona hellwegeri (Friese, 1900) Mexico, 141 Scaptotrigona jujuyensis (Schrottky, 1911) Argentina, 126–131, 515, 516 Scaptotrigona limae (Brèthes, 1920) Colombia, 385, 386, 389 Scaptotrigona luteipennis (Friese, 1902) Costa Rica, 117 Scaptotrigona mexicana (Guérin, 1844) Costa Rica, Guatemala, Mexico, 102, 117, 141, 395 Scaptotrigona ochrotricha (Buysson, in Du Buysson & Marshall, 1892) Venezuela, 79, 82 Scaptotrigona panamensis (Cockerell, 1913) Costa Rica, 117 Scaptotrigona pectoralis (Dalla Torre, 1896) Costa Rica, Guatemala, Mexico, 102, 117, 141, 395 Scaptotrigona polysticta Moure, 1950 Bolivia, Brazil, 75, 469 Scaptotrigona postica (Latreille,1807) Brazil, 274 Scaptotrigona subobscuripennis (Schwarz, 1951) Costa Rica, 117 Scaptotrigona tubiba (Smith, 1863) Brazil, 91 578 Appendix B Scaptotrigona wheeleri (Cockerell, 1913) Costa Rica, 100, 117 Scaptotrigona xanthotricha Moure, 1950 Brazil, 410, 469 Scaura argyrea (Cockerell, 1912) Costa Rica, Guatemala, Mexico, 102, 117, 141 Scaura latitarsis (Friese, 1900) French Guiana, 91, 103, 274 Scaura longula (Lepeletier, 1836) French Guiana, 91 Scaura Schwarz, 1938 Venezuela, 7, 20, 93 Scaura Schwarz, 1938, Plebeia (Scaura), 9, 11, 79 Scaura tenuis (Ducke, 1916) French Guiana, 91 Scaura timida (Silvestri, 1902), 9, 22 Schwarziana Moure, 1943, 7, 20, 93 Schwarziana Moure, 1943, Plebeia (Schwarziana), 7 Schwarziana quadripunctata (Lepeletier, 1836) Argentina, 126, 326 Schwarzula coccidophila Camargo & Pedro, 2002, 23, 24 Schwarzula Moure, 1946, 7, 20, 93 Schwarzula Moure, 1946 [= Plebeia (Scaura)], 7, 20, 93 Sundatrigona Inoue & Sakagami, 1995, Heterotrigona (Sundatrigona), 8 Tetragona Lepeletier & Serville, 1828 Colombia, 7, 20, 93, 137 Tetragona Lepeletier & Serville, 1828, Trigona (Tetragona), 7, 20, 93, 137 Tetragona beebei (Schwarz, 1938) French Guiana, 91 Tetragona clavipes (Fabricius, 1804) Argentina, Brazil, French Guiana, Venezuela, 79, 91 Tetragona dorsalis (Smith, 1854) French Guiana, 91, 103 Tetragona handlirschii (Friese, 1900) French Guiana, 91 Tetragona kaieteurensis (Schwarz, 1938) French Guiana, 91 Tetragona mayarum (Cockerell, 1912) [= Tetragona ziegleri (Friese, 1900)] Guatemala, Mexico, 102, 141 Tetragona perangulata (Cockerell, 1917) Costa Rica, 117 Tetragona savannensis Roubik, 1980 [= Frieseomelitta flavicornis], 90 Tetragona ziegleri (Friese, 1900) Costa Rica, Venezuela, 79, 117 Tetragonilla Moure, 1961, Tetragonula (Tetragonilla), 8 Tetragonisca Moure, 1946 Argentina, Venezuela, 7, 20, 93 Tetragonisca Moure, 1946, Trigona (Tetragonisca), 7, 20, 93 Tetragonisca angustula (Latreille, 1811) Argentina, Bolivia, Brazil, Colombia, Costa Rica, Guatemala, French Guiana, Mexico, Panama, Peru, 91, 102, 117, 141, 298, 375, 395 Tetragonisca angustula angustula (Latreille, 1811), 79 Tetragonisca buchwaldi (Friese, 1925) Costa Rica, 117 Tetragonisca fiebrigi (Schwarz, 1938) Argentina, Bolivia, 410, 469, 478 Tetragonula Moure, 1961, 8 Tetragonula Moure, 1961, Tetragonula (Tetragonula), 8 Tetragonula biroi (Friese, 1898) Philippines, 526, 531, 533, 535 Tetragonula carbonaria (Smith, 1854) Australia, 45 Tetragonula collina (Smith, 1857), 155, 179 Tetragonula fuscobalteata (Cameron,1908), 11 Tetragonula laeviceps (Smith, 1857), 155 Appendix B 579 Tetragonula pagdeni (Schwarz, 1939), 181 Tetrigona Moure, 1961, 8 Trichotrigona Camargo & Moure, 1983, 7, 20, 93, 139 Trichotrigona extranea Camargo & Moure, 1983 Australia, 93 Trigona Jurine, 1807 Brazil, Malaysia, Venezuela, 7, 20, 93, 136, 137 Trigona Jurine, 1807, Trigona (Trigona), 7, 20, 93, 136, 137 Trigona acapulconis Strand, 1919 (= Geotrigona acapulconis), 101, 140, 395 Trigona alfkeni Friese, 1900, 75 Trigona amalthea (Olivier, 1789) Venezuela, 79, 94, 103 Trigona amazonensis (Ducke, 1916) Venezuela, 80 Trigona australis Friese, 1898, 43 Trigona branneri Cockerell, 1912 French Guiana, Venezuela, 80, 91 Trigona carbonaria Smith, 1854 Australia, 45 Trigona chanchamayoensis Schwarz, 1948 Bolivia, 410 Trigona cilipes (Fabricius, 1804) Costa Rica, French Guiana, Venezuela, 80, 91, 117 Trigona clypearis Friese, 1909 Australia, 45 Trigona collina Smith, 1857 Malaysia, 155, 179 Trigona corvina Cockerell, 1913 Costa Rica, Guatemala, Mexico, 102, 118, 141 Trigona crassipes (Fabricius, 1793) French Guiana, 91 Trigona cupira cupira Smith, 1863 [misidentification, = Partamona orizabaensis], 274 Trigona dallatorreana Friese, 1900 Brazil, Venezuela, 80 Trigona davenporti Franck, 2004 Australia, 45 Trigona ferricauda Cockerell, 1917 Costa Rica, 118 Trigona fulviventris Guérin, 1844 Costa Rica, Guatemala, Mexico, Venezuela, 80, 118 Trigona fuscipennis Friese, 1900 Costa Rica, Guatemala, Mexico, Venezuela, 80, 91, 102, 118, 141 Trigona fuscobalteata Cameron, 1908 Thailand, 11, 155, 178 Trigona guianae Cockerell, 1910 French Guiana, Venezuela, 80, 91 Trigona hockingsi Cockerell, 1929 Australia, 45 Trigona hyalinata (Lepeletier, 1836) Brazil, 75 Trigona hypogea Silvestri, 1902 Brazil, Panama, 274 Trigona laeviceps Smith, 1857 Thailand, 178, 498 Trigona mazucatoi (Almeida, 1992) (= Trigona cilipes), 91 Trigona melanocephala Gribodo, 1893 Malaysia, 179 Trigona melina Gribodo, 1893 Malaysia, 179 Trigona mellipes Friese (1898) Australia, 45 Trigona muzoensis Schwarz, 1948, 118 Trigona necrophaga Camargo & Roubik, 1991 Costa Rica, 118 Trigona nigerrima Cresson, 1878 Costa Rica, Guatemala, Mexico, 102, 141 Trigona nigra Cresson, 1878, 101, 116, 140 Trigona pallens (Fabricius, 1798) French Guiana, Venezuela, 80, 91 Trigona permodica Almeida, 1995 French Guiana, 91 Trigona prisca Michener & Grimaldi, 1988, 14, 145 Trigona recursa Smith, 1863 Brasil, 92, 274 Trigona sapiens Cockerell, 1911 Australia, 45 580 Appendix B Trigona sesquipedalis Almeida, 1984 French Guiana, 92 Trigona silvestriana (Vachal, 1908) Costa Rica, Guatemala, Mexico, 75, 102, 118, 141 Trigona spinipes (Fabricius, 1793) Argentina, Brazil, 75 Trigona trinidadensis (Provancher, 1888) (= Trigona amalthea), 75 Trigona truculenta Almeida, 1984 Venezuela, 80 Trigona venezuelana Schwarz, 1948 Venezuela, 80, 82 Trigona williana Friese, 1900 French Guiana, Venezuela, 80, 92 Trigona (Frieseomelitta) angustula angustula Latreille, 1811, 79 Trigona (Frieseomelitta) nigra paupera (Provancher, 1888), 76 Trigona (Geotrigona) Geotrigona Moure, 1934, 7, 20, 92 Trigona (Heterotrigona) carbonaria Smith, 1854 Australia, 45 Trigona (Heterotrigona) clypearis Friese, 1909 Australia, 45 Trigona (Heterotrigona) davenporti Franck, 2004 Australia, 45 Trigona (Heterotrigona) hockingsi Cockerell, 1929 Australia, 45 Trigona (Heterotrigona) mellipes Friese, 1898 Australia, 45 Trigona (Heterotrigona) sapiens Cockerell, 1911 Australia, 45 Trigona (Tetragonisca) angustula (Latreille, 1811), 91, 102, 117, 141, 375, 395 Trigona (Tetragonisca) angustula angustula Latreille, 1811, 91, 117, 141, 298 Trigona (Tetragonula) laeviceps Smith, 1857, 155 Trigona (Trigona) corvina Cockerell, 1913, 102, 118, 141 Trigona (Trigona) hypogea Silvestri, 1902, 274 Trigonella Sakagami & Moure, 1975 [= Heterotrigona (Sundatrigona)], 8 Trigonisca Moure, 1950 Argentina, Venezuela, 7, 93 Trigonisca atomaria (Cockerell, 1917) Costa Rica, 118 Trigonisca azteca Ayala, 1999 Mexico, 141 Trigonisca discolor (Wille, 1965) Costa Rica, 118 Trigonisca dobzhanskyi (Moure, 1950) French Guiana, 92 Trigonisca maya Ayala, 1999 Guatemala, Mexico, 102 Trigonisca mixteca Ayala, 1999 Mexico, 141 Trigonisca pipioli Ayala, 1999 Costa Rica, Guatemala, Mexico, 102, 118, 141 Trigonisca schulthessi (Friese, 1900) Mexico, 101, 116, 141 Appendix C Common Names of Stingless Bees “abeja bermeja” Scaptotrigona hellwegeri Mexico, 356, 435 “abeja criolla” Melipona beecheii Guatemala, 404, 477 “abeja maya” Melipona beecheii Guatemala, 108 “abeja real” Melipona beecheii Mexico, 356, 435 “abeja real roja” Melipona fasciata guerreroensis Mexico, 435 “abejita” Cephalotrigona capitata Venezuela, Melipona (Melipona) favosa Venezuela, 76, 77 Paratrigona anduzei Venezuela, Plebeia sp. Bolivia, Tetragonisca spp. Venezuela, 78, “abejita casera” Melipona (Melipona) favosa Venezuela, 77 “ah-muzen-cab” Melipona beecheii Mexico,138 “ajabite” Tetragona clavipes Venezuela, 79 “ajavitta” Tetragona clavipes Venezuela, 79 “ajavitte” Tetragona clavipes Venezuela, 79, 355, 435 “ala blanca” Frieseomelitta nigra Costa Rica, Mexico, 116, 356, 435 “alazán” Scaptotrigona pectoralis Guatemala, 108 “alpamiski” Geotrigona argentina Argentina, 129 “an us” Tetragonisca angustula Guatemala, 406 “angelita” Frieseomelitta spp. Venezuela, Tetragonisca angustula Colombia, 76 “anihammoa” Hypotrigona araujoi, Hypotrigona penna, Hypotrigona ruspolii, Hypotrigona gribodoi Ghana, 264 “apynguarei” Plebeia spp. Argentina, 129 “arica” Melipona (Melipona) favosa Venezuela, 77, 225 “erica” Melipona favosa Venezuela, 77, 355, 363–364, 435, 531 “bichi” Melipona beecheii Guatemala, 108, 404 “boca de sapo” Plebeia Guatemala, 108 “boca de vieja” Plebeia kerri Bolivia, 410 “borá” Tetragona clavipes Argentina, 129 “bocarena” Plebeia tica Costa Rica, 117 “canudo” Scaptotrigona sp Brazil, 542 “carby” Tetragonula carbonaria Australia, 355, 435 “chac chow” Melipona solani Guatemala, 108, 477 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7, © Springer Science+Business Media New York 2013 581 582 Appendix C “chan-na-rong” Tetragonula laeviceps Thailand, 495 “chelerita” Plebeia Guatemala, 108 “chicopipe” Nannotrigona perilampoides Costa Rica, 116 “chumelo” Tetragonisca angustula Guatemala, 108, 406 “chupa ojos” Frieseomelitta paupera, Plebeia jatiformis Costa Rica, 116, 117 “churrusca” Partamona peckolti Venezuela, 78 “cigarroncito” Melipona (Michmelia) eburnea Venezuela, 77 “colecab” Melipona beecheii, 221–222 “colmena grande” Melipona beecheii Guatemala, 108, 404 “colmena real” Melipona fasciata Mexico, 356, 435 “congo” Cephalotrigona zexmeniae, Scaptotrigona mexicana Guatemala, Trigona silvestriana Costa Rica, 108, 118 “congo canche” Scaptotrigona pectoralis Guatemala, 108 “congo negro” Scaptotrigona mexicana Guatemala, 108, 405 “cortacabello” Paratrigona anduzei Venezuela, 78 “criolla” Melipona solani Mexico, Melipona beecheii Guatemala, 356, 435 “criollita” Melipona (Melipona) favosa Venezuela, 77 “culo de buey” Trigona fulviventris Costa Rica, 118 “culo de chucho” Trigona fulviventris Guatemala, 108 “cushusho” Trigona nigerrima Guatemala, 108 “doncella” Tetragonisca angustula Guatemala, 406 “doncellita” Tetragonisca angustula Guatemala, 108, 406 “duro kokoo” Meliponula (Meliponula) bocandei Ghana, 264 “duro tuntum” Meliponula (Axestotrigona) ferruginea Ghana, 264 “erereú barcina” Melipona grandis Bolivia, 410, 411, 435, 469 “erereú choca” Melipona brachychaeta Bolivia, 410, 411, 413, 435, 469 “erica” Melipona favosa, Melipona (Melipona) favosa Venezuela, 77, 355, 364, 435, 531 “eriquita” Tetragonisca spp. Venezuela, 79 “españolita” Paratrigona anduzei, Tetragonisca spp. Venezuela, 79 “guanota” Melipona (Melikerria) compressipes, Melipona (Michmelia) trinitatis Venezuela, 76, 77, 364 “guanotica” Frieseomelitta paupera Venezuela, 76 “guaracho” Scaptotrigona spp. Venezuela, 79 “guayure” Tetragonisca spp. Venezuela, 79 “homo” Trigona nigerrima, Trigona silvestriana Guatemala, 108 “isabitto” Melipona aff. fuscopilosa, Melipona (Michmelia) Venezuela, 77, 355, 435 “jandaíra” Melipona subnitida Brazil, 179, 435, 471, 542 “jataí” Tetragonisca angustula Brazil, 245, 375–376, 542 “jicote” Melipona fuliginosa Costa Rica, 116 “jicote barcino” Melipona costarricensis Costa Rica, 116 “jicote gato” Melipona beecheii Costa Rica, 116 “jicote limón” Lestrimelitta danuncia Costa Rica, 116 “joloncán” Trigona nigerrima Guatemala, 108 “kalulot” Tetragonula biroi Philippines, 531 Appendix C 583 “karbi” Tetragonula carbonaria Australia, 36 “kolil kab” Melipona beecheii Mexico, 146 “kootchar” Austroplebeia australis Australia, 36 “lambeojitos” Plebeia droryana Bolivia, Tetragonisca spp. Venezuela, 410 “lambe-olhos” Leurotrigona muelleri Brazil, 225 “limoncillo” Lestrimelitta niitkib Guatemala, 108 “limoncita” Lestrimelitta maracaia Venezuela, 76 “mabita” Melipona (Melipona) favosa Venezuela, 77 “magua canche” Scaptotrigona pectoralis Guatemala, 108 “magua negro” Scaptotrigona mexicana Guatemala, 108 “mandaçaia” Melipona quadrifasciata Brazil, 471, 542, 545 “mandinga” Trigona fulviventris Guatemala, 108 “mandurí” Melipona obscurior Argentina, 129 “mariola” Tetragonisca angustula Costa Rica, 117 “may man-pathan” Australian stingless bees, 36 “mestizo” Tetragonisca near angustula Argentina, 129 “miel de leche” Tetragona ziegleri Costa Rica, 117 “mijui” Scaptotrigona polysticta Brazil, 356, 435 “mimina” Hypotrigona araujoi, Hypotrigona penna, Hypotrigona ruspolii, Hypotrigona gribodoi Ghana, 264 “mirim” Plebeia spp. Argentina, 129 “mocca” South African stingless bees, 262 “mopani” South African stingless bees, 262 “moro-moro” Melipona orbignyi Argentina, 129 “moscochola” Nannotrigona melanocera Venezuela, 77 “mosquito” Plebeia, Venezuela, 79, 91 “negrita” Scaptotrigona mexicana Mexico, 355 “negrito” Cephalotrigona capitata Venezuela, 76 “negrito” Scaptotrigona jujuyensis Argentina, 129 “ñuriño” Melipona (Michmelia) lateralis Venezuela, 77 “obobosí” Scaptotrigona depilis Bolivia, 410–411, 435, 469, 531 “pañuelita” Tetragonisca spp. Venezuela, 79 “papaterra” Melipona asilvae Brazil, 542 “pegón” Paratrigona anduzei, Partamona peckolti, Trigona amalthea, Trigona branneri, Trigona fuscipennis, Trigona guianae, Trigona spp.Venezuela, 78–80 “pegona” Partamona peckolti, Trigona guianae Venezuela, 78, 80 “pegoncito” Scaura sp. Venezuela, 79 “peladora” Oxytrigona mellicolor Costa Rica, 116 “pico” Scaptotrigona spp. Venezuela, 79 “pisilnekmej” Scaptotrigona mexicana Mexico, 146, 356, 435, 544 “princesita” Tetragonisca spp. Venezuela, 79 “pringador” Oxytrigona mediorufa Guatemala, 108 “pusquello” Plebeia spp. Argentina, 129 “qán us” Tetragonisca angustula Guatemala, 406 “quella” Plebeia spp. Argentina, 129 584 Appendix C “rubiecito” Tetragonisca fiebrigi, Tetragonisca near angustula Argentina, 129 “rubita” Tetragonisca spp. Venezuela, 79 “sabite” Melipona (Michmelia) eburnea Venezuela, 77 “sacar” Partamona Guatemala, 108 “sak’q qaw” Melipona beecheii Guatemala, 404 “sarquita” Plebeia, Guatemala, Tetragonisca fiebrigi Bolivia, 108 “serenita” Nannotrigona perilampoides, Plebeia Guatemala, 108 “shimilo” Plebeia spp. Argentina, 129 “shuruya” Scaptotrigona pectoralis Guatemala, 108 “sicae amarilla” Trigona chanchamayoensis Bolivia, 410 “soncuano” Scaptotrigona luteipennis, Scaptotrigona pectoralis Costa Rica, 117 “sonquette” Scaptotrigona spp. Venezuela, 79 “sugarbag” Australian stingless bees, 36–38, 55, 544 “suro choco” Scaptotrigona near xanthotricha Bolivia, 410, 411, 435, 469 “suro negro” Scaptotrigona polysticta Bolivia, 355, 410, 414, 435, 469, 531, 544 “talnete” Geotrigona acapulconis Guatemala, 108, 405 “tamaga amarillo” Cephalotrigona zexmeniae Costa Rica, 116 “tamagás” Oxytrigona mediorufa Guatemala, 108 “tapezuá” Scaptotrigona jujuyensis, Scaptotrigona near postica Argentina, 129 “tifuie” Dactylurina staudingeri Ghana, 264 “tinzuca” Melipona yucatanica Guatemala, 108 “tiúba” Melipona fasciculata, Melipona compressipes Brazil, 355, 435, 471, 542, 544, 550, 552 “tobillo morrocoy” Melipona (Michmelia) eburnea Venezuela, 77 “tobuna” Scaptotrigona near postica Argentina, 129 “torce cabelos” Scaptotrigona depilis Brazil, 225 “uruçú amarela” Melipona rufiventris Brazil, 471, 542 “uruçú cinzenta” Melipona fasciculata Brazil, 380 “uruçú” Melipona scutellaris Brazil, 355, 356, 380, 435, 471, 542 “uruçú verdadeira” Melipona scutellaris Brazil, 542 “vamo-nos embora” Lestrimellita limao Brazil, 225 “xunan cab” Melipona beecheii Guatemala, Mexico, 221–222, 229, 542 “yana” Scaptotrigona jujuyensis Argentina, 128, 129 “yateí” Tetragonisca fiebrigi Argentina, 129, 478 “zamurita” Nannotrigona sp. Venezuela, 78 Appendix D Taxonomic Index of Plant Families Plant uses or mutualisms, thought to include but not restricted to: Mayan medicinal use (M), nectar (N) excluding extrafloral nectar, pollen (P), pollen only (PO) certain species or genera nectarless, trichomes (T) or resin source (R), used to make honey (H), build nests (B), or visited for nectar and/or pollen by stingless bees (S), and Apis mellifera (A). In parenthesis total number of genera and species per family. (691 morphospecies distributed into: Families = 125, Genera = 437, Species = 611, Varieties = 1) Angiospermae Dicotyledoneae Acanthaceae M, N, P, S, A (5–7), 105, 207, 238, 317, 339, 403 Avicennia P, S, 317 Bravaisia integerrima N, B, S, 121, 207 Bravaisia tubiflora M, 238 Justicia N, S, A, 339 Justicia adathoda A, 585 Mendoncia A, 585 Trichanthera gigantea N, S, 585 Achariaceae (1–1), 328 Hydnocarpus B, S, 328 Aceraceae R, S (1–1), 529 Adoxaceae (2–2), 585 Sambucus nigra P, S, 585 Viburnum N, S, 585 Amaranthaceae N, P, R, S, A (5–5), 305, 317, 338, 403, 529 Alternanthera P, R, S, A, 304, 305, 317, 529 Amaranthus A, 585 Chamissoa S, A, 585 Chenopodium S, A, 585 Gomphrena S, 585 Anacardiaceae N, P, PO, S, A (12–20), 57, 73, 105, 289, 291, 292, 304, 305, 308–310, 316, 317, 327, 328, 529 Anacardium excelsum B, S, A, 121 Anacardium occidentale N, P, S, A, 317 Astronium fraxinifolium P, A, 317 Astronium graveolens N, B, S, 121, 342 Gluta B, S, 328 Gluta oba B, S, 328 Gluta sabahana B, S, 328 Lannea barteri N, P, A, 317 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7, © Springer Science+Business Media New York 2013 585 586 Mangifera A, 309 Mangifera indica N, P, S, A, 57, 317 Metopium A, 585 Myracrodruon urundeuva B, S, 328 Schinopsis brasiliensis B, S, 328 Schinus N, P, R, S, 291, 292, 529 Spondias S, A, 304, 305, 309–312 Spondias mombin PO, B, S, A , 73, 121, 317 Spondias radlkoferi PO, S, 317 Spondias tuberosa B, S, 328 Tapirira guianensis P, S, A, 317 Toxicodendron striatum N, S, 340, 343 Annonaceae (2–2), 586 Annona S, 586 Unonopsis S, 586 Apiaceae M, N, P, S (3–3), 105, 234, 339 Coriandrum sativum N, S, 586 Pimpinella anisum M, 234 Spananthe paniculata N, S, 340, 342 Apocynaceae N, P, H, S, A (7–8), 105, 238, 317, 328 Adenium obesum N, P, A, 317 Aspidosperma S, 586 Aspidosperma pyrifolium B, S, 328 Couma utilis P, S, 317 Forsteronia S, 586 Plumeria rubra M, 238 Prestonia S, 586 Rauvolfia caffra N, P, A, 317 Aquifoliaceae (1–1), 586 Ilex A, 586 Araliaceae N, S (6–9), 328 Dendropanax A, 586 Didymopanax A, 586 Didymopanax morototoni S, 586 Hydrocotyle N, S, 586 Oreopanax N, S, 586 Polyscias fulva B, S, 328 Schefflera N, S, 586 Schefflera barteri B, S, 328 Schefflera morototoni N, P, S, 290 Appendix D Asteraceae M, N, P, R, S, A (23–35), 105, 106, 207, 235, 290, 305, 308–310, 316, 318, 337–339, 341, 342, 344, 403, 477, 526, 529 Austroeupatorium inulifolium N, S, 339, 340, 342, 344 Baccharis N, P, S, A, 318 Baccharis erioclada P, S, 318 Baccharis macrantha N, S, 586 Baccharis pedunculata N, S, 586 Baccharis semiserrata P, S, 318 Bidens N, S, 586 Bidens pilosa P, S, 318 Bidens squarrosa N, S, 207 Chaptalia S, 586 Critonia morifolia N, S, 339 Dalia N, S, 586 Eirmocephala brachiata N, S, 586 Elephantopus N, P, S, A, 318 Emilia sonchifolia N, S, 586 Eupatorium P, A, 318 Eupatorium hemipteropodum M, 235 Helianthus annuus N, P, A, 318 Hypochaeris radicata N, S,339 Mikania P, N, S, A, 318 Mikania micrantha N, S, 207 Oyedaea verbesinoides N, S, 207 Parthenium argentatum R, 526 Pentacalia N, S, 586 Piptocoma discolor N, S, 586 Steiractinia aspera N, S, 586 Taraxacum officinale N, S, 586 Tithonia diversifolia N, S, 586 Vernonanthura N, S, 586 Vernonia N, S, A, 586 Vernonia amygdalina P, S, 318 Vernonia auriculifera P, S, 318 Vernonia patens N, S, 207 Vernonia pauciflora N, P, S, 318 Wedelia trilobata N, S, 318 Balsaminaceae N, P, S (1–4), 318 Impatiens S, A, 586 Impatiens balsamina N, P, S, 318 Impatiens sultanii N, P, S, 318 Impatiens walleriana N, P, S, 318 Appendix D 587 Begoniaceae H, S (1–1), 403 Begonia S, A, 586 Buxaceae (1–1), 587 Buxus A, 587 Betulaceae (1–1), 586 Alnus acuminata S, 586 Cactaceae P, S (2–2), 105, 469 Epiphyllum A, 587 Selenicereus A, 587 Bignoniaceae M, N, P, S, A (6–9), 105, 305, 308, 309, 318, 328, 344, 403 Arrabidaea S, A, 305 Jacaranda mimosifolia N, P, A, 318 Markhamia lutea N, P, S, 318 Martinella obovata A, 586 Pithecoctenium crucigerum A, 586 Tabebuia S, A, 586 Tabebuia caraiba B, S , 328 Tabebuia ochracea B, S, 121 Tabebuia rosea N, B, S, 121 Bixaceae PO, S (1–1), 105, 106 Bixa orellana PO, S, 106 Boraginaceae M, N, P, S, A (2–12), 105, 237, 319, 339 Cordia A, 310 Cordia africana N, P, S, A, 319 Cordia alliodora B, S, A, 121, 339 Cordia bicolor S, 587 Cordia dentata N, S, 587 Cordia geraschanthoides M, 237 Cordia millenii N, P, S, A, 319 Cordia monoica N, P, S, A, 319 Cordia panamensis A, 587 Cordia sinensis N, P, S, A, 319 Cordia spinescens N, S, A, 342 Tournefortia A, 587 Brassicaceae M, N, P, S (3–3), 105, 238, 289, 465 Brassica N, P, S, 587 Diplotaxis tenuifolia N, H, A, 465, 467, 470 Sinapis nigra M, 237 Burseraceae P, S (3–4), 289, 291, 319, 328 Bursera A, 587 Bursera simaruba B, S, A, 121 Commiphora leptophloeos B, S, 328 Protium N, P, R, S, A, 289, 291, 319 Calophyllaceae (1–1), 328 Calophyllum B, S, 328 Cannabaceae (1–2), 207, 305, 309 Celtis B, S, A, 207, 305, 309 Celtis iguanaeus S, 587 Capparaceae (1–1), 204 Tarenaya spinosa N, S, A, 204 Caprifoliaceae (1–1), 587 Lonicera A, 587 Caricaceae M, N, P, S, A (1–1), 238, 319 Carica papaya M, N, P, S, A, 238, 319 Caryophyllaceae N, P, S, A (2–2), 105, 339 Drymaria cordata S, 587 Stellaria N, S, 339, 341, 344 Celastraceae P, S (4–5), 290, 328 Hippocratea volubilis N, S, 587 Hylenaea praecelsa S, 587 Lophopetalum B, S, 328 Maytenus S, A, 328 Maytenus acuminata B, S, 328 Chloranthaceae (1–1), 587 Hedyosmum A, 587 Chrysobalanaceae (3–4), 328 Hirtella S, 587 Licania A, 587 Licania rigida B, S, 328 Parinari excelsa B, S, 329 Cleomaceae (1–2), 339 Cleome N, S, A, 339, 341, 342 Cleome parviflora S, 587 Clusiaceae (1–1), 526 Clusia PO, R, S, A, E22, 526, 530, 533 Cochlospermaceae PO, S (1–1), 403 Appendix D 588 Combretaceae N, P, S, A (2–5), 319 Combretum N, P, S, A, 319 Combretum collinum N, P, A, 319 Combretum fruticosum B, S, 121 Combretum molle N, P, A, 319 Terminalia oblonga B, S, 121 Connaraceae (1–1), 587 Connarus S, 587 Convolvulaceae M, N, P, S, A (7–7), 105, 235, 403 Cuscuta americana M, 235 Evolvulus A, 587 Ipomoea A, 177 Iseia S, 587 Jacquemontia A, 587 Maripa S, A, 587 Merremia N, S, 587 Cornaceae (1–1), 588 Alangium chinense B, S, 328 Cucurbitaceae N, P, S, A (6–6), 57, 105, 106, 297, 319 Cayaponia A, 297 Citrullus lanatus N, P, L, S, A, 57, 106, 319 Cucumis sativus N, P, A, 319 Cucurbita pepo N, P, A, 319 Momordica S, 588 Sicyos A, 588 Cunoniaceae P, S (1–1), 319, 357 Weinmannia P, S, 319 Dilleniaceae (2–2), 309 Davilla nitida S, 588 Doliocarpus S, A, 308, 309, 312 Dipterocarpaceae (2–3), 327, 329, 526 Dipterocarpus R, B, S, 329, 526 Dipterocarpus grandiflorus B, S, 329 Shorea B, S, 329 Ebenaceae N, P, S, A (1–2), 319 Diospyros N, S, 319 Diospyros mespiliformis N, P, A, 319 Ericaceae (3–3), 57, 329, 449, 502 Agauria salicifolia B, S, 329 Erica H, A, 449 Vaccinium corymbosum L, S, 57 Escalloniaceae (1–1), 588 Escallonia pendula N, S, 588 Euphorbiaceae M, N, P, S, A (17–34), 105, 234, 235, 238, 288, 297, 305, 307– 310, 316, 320, 327, 329, 337–339, 341–344 Acalypha P, S, A, 320, 338 Acalypha discolor N, P, S, A, 588 Acalypha diversifolia P, S, 588 Acalypha macrostachya P, S, 588 Acalypha sidifolia N, P, S, A, 588 Alchornea PO, S, A, 297, 307, 309, 310, 311, 338 Alchonea discolor N, PO, S, 320 Alchonea sidifolia N, PO, S, A, 320 Aparisthmium cordatum P, S, 320 Chaetocarpus castanocarpus B, S, 329 Chamaesyce N, S, A, 588 Cnidoscolus chayamansa M, 234 Cnidoscolus phyllacanthus B, S, 329 Codiaeum A, 588 Croton S, A, 309, 339, 344 Croton macrostachyus N, P, 320 Croton leptostachyus N, S, 588 Croton niveus M, 238 Dalechampia R, S, E, 344, 530 Euphorbia S, A, 342, 344 Euphorbia cotinifolia N, S, 339, 342 Euphorbia cyatophora N, S, 588 Euphorbia hirta N, S, 339, 340, 342, 344 Euphorbia splendens N, P, S, 320 Euphorbia thymifolia N, S, 342 Hura crepitans S, 588 Hyeronima S, A, 588 Mabea A, 588 Mallotus N, S, 320 Ricinus P, S, 320 Ricinus communis M, N, P, S, 235, 288, 338 Sapium N, S, A, 588 Sapium caudatum S, A, 588 Appendix D Trigonopleura malayana B, S, 329 Fabaceae, Caesalpinioideae N, P, S, A (22–33), 287, 289, 320, 339, 342, 526 Acrocarpus fraxinifolius N, P, S, 320 Afzelia africana P, S, A, 320 Bauhinia N, P, S, A, 588 Bauhinia divaricata A, 588 Bauhinia guianensis S, A, 588 Bauhinia ungulata S, A, 588 Caesalpinia N, B, S, A, 588 Caesalpinia decapetala P, S, 320 Caesalpinia pyramidalis B, S, 329 Cassia PO, S, 312, 320 Cassia fistula PO, S, 320 Cassia fruticosa S, 588 Cassia obtusifolia S, 588 Cassia undulata PO, S, 320 Chamaecrista ramosa N, P, S, 290 Copaifera aromatica B, S, 121 Crudia N, P, S, 289 Cynometra alexandri N, P, S, A, 320 Daniellia oliveri P, S, 588 Delonix regia N, P, S, 588 Elizabetha A , 588 Elizabetha paraensis A, 589 Haematoxylon campechianum A, 589 Hymenaea R, A, 526 Intsia palembanica B, S, 329 Julbernardia P, S, 320 Parkinsonia aculeata P, S, 320 Peltogyne purpurea S, 589 Peltophorum inerme S, 589 Peltophorum pterocarpum N, S, 320 Senna P, S, 312 Sympetalandra borneensis B, S, 329 Tamarindus indica N, P, S, A, 320 Fabaceae, Faboideae M, N, P, S, A (20–28), 237, 288, 289, 320, 339, 344 Aeschynomene A, 589 Aeschynomene americana N, S, A, 589 Andira inermis B, S, 121 Arachis A, 589 Cajanus A, 589 Cajanus bicolor S, 589 589 Cajanus cajan N, P, A, 320 Calopogonium S, 304, 305 Crotalaria N, P, S, 289, 320 Dalbergia S, 589 Desmodium N, S, A, 309 Dioclea S, A, 589 Diphysa americana B, S, 121 Erythrina N, P, S, A, 589 Erythrina costaricensis A, 589 Gliricidia sepium N, P, B, S, 121, 320 Lonchocarpus A, 589 Lonchocarpus costaricensis B, S, 121 Lonchocarpus longistylus M, 237 Machaerium N, P, S, A, 304, 305, 310, 320, 321 Myrospermum frutescens B, S, 121 Pterocarpus A, 589 Robinia pseudoacacia H, A, 462, 485 Trifolium pratense N, S, 203 Trifolium repens N, S, 589 Vicia P, S, 321 Vicia faba N, S, 589 Zornia N, P, S, 288 Fabaceae, Mimosoideae M, PO, B, S (18–37), 238, 288, 289, 292, 305, 309, 321, 329, 339, 529 Acacia N, P, PO, S, A, 297, 305, 310, 312, 321, 414, 485 Acacia decurrens N, B, S, 589 Albizia coriaria N, P, A, 321 Albizia gummifera N, P, B, S, 321, 329 Anadenanthera R, S, 329, 529 Anadenanthera colubrina B, S, 329 Archidendron jiringa N, S, 321 Calliandra calothyrsus N, S, A, 321 Dialium B, S, 329 Entada monostachia S, 589 Enterolobium cyclocarpum B, S, 121 Faidherbia albida N, P, A, 321 Inga N, S, A, 589 Inga sapindoides B, S, 121 Leucaena A, 589 Leucaena glauca M, 238 Leucaena leucocephala N, P, A, 321 Appendix D 590 Mimosa PO, S, A, 306, 308, 310, 312, 339, 344 Mimosa acutistipula PO, B, S, 329 Mimosa bimucronata PO, S, 321 Mimosa caesalpineifolia PO, S, 589 Mimosa casta PO, A, 309 Mimosa gemmulata PO, S,290 Mimosa invisa PO, S, 589 Mimosa pigra PO, S, A, 589 Mimosa pudica PO, S, 304, 305, 309, 311, 312, 321 Mimosa pulcherrima PO, S, 589 Mimosa scabrella PO, S, 289, 291, 292, 321 Pentaclethra macroloba B, S, 121 Piptadenia communis B, S, 329 Piptadenia moniliformis N, P, S, 288 Piptadenia rigida N, P, S, 288 Pithecellobium S, A, 589 Pithecellobium dinizii A, 589 Pseudosamanea guachapele B, S, 121 Schrankia PO, S, 321 Stryphnodendron guianense N, P, S, 290 Fagaceae PO, H, S (2–2), 105, 403, 449 Castanea sativa H, A, 449, 485 Quercus H, A, 449 Humiriaceae (1–1), 590 Humiriastrum S, A, 590 Hydrangeaceae (1–1), 590 Hydrangea A, 590 Hypericaceae (1–1), 590 Vismia R, S, 528 Juglandaceae (1–1), 590 Juglans australis S, A, 590 Lamiaceae N, P, H, S (5–9), 105, 207, 305, 307, 309, 310, 321, 329, 339, 403, 449 Gmelina arborea N, P, A, 321 Hyptis N, S, A, 207, 305, 307, 309, 310, 339 Hyptis brachiata N, S, 339, 342 Hyptis capitata N, S, 207 Hyptis mutabilis N, S, 590 Premna angolensis B, S, 329 Thymus H, A, 449, 452 Vitex doniana N, P, A, 321 Vitex orinocensis N, S, 590 Lauraceae (6–8), 57, 146, 287, 327, 329, 357 Dehaasia B, S, 329 Eusideroxylon zwageri B, S, 329 Litsea B, S, 329, 330 Litsea caulocarpa B, S, 329 Ocotea veraguensis B, S, 121 Persea N, P, S, 287 Persea americana B, L, S, 57, 121, 146, 357 Phoebe macrophylla B, S, 330 Loranthaceae N, S, A (5–6), 339 Aetanthus S, 590 Gaiadendron S, A, 590 Oryctanthus N, S, 339, 341, 342, 344 Struthanthus S, 590 Struthanthus subtilis N, S, 590 Tristerix S, 590 Lythraceae P, S (3–4), 105, 289, 321, 339 Adenaria floribunda N, S, 339, 342, 344 Cuphea N, S, 289, 342 Cuphea racemosa N, S, 342 Lagerstroemia S, 590 Malpighiaceae P, S, A (6–7), 105, 106, 339, 401 Bunchosia S, 590 Byrsonima crassifolia P, S, 106, 401 Hiraea S, A, 590 Mascagnia hippocrateoides S, 590 Stigmaphyllon A, 590 Stigmaphyllon hypargyreum S, 590 Tetrapteris N, S, 339 Malvaceae M, N, P, H, S, A (27–39), 105, 204, 207, 233, 236, 237, 305, 321, 330, 338, 339, 403 (Bombacoideae), 590 Bombacopsis A, 121 Appendix D Bombacopsis quinata B, S, 121 Cavanillesia platanifolia A, 590 Ceiba aesculifolia S, 590 Ceiba pentandra N, B, S, 590 Ochroma A, 590 Ochroma pyramidale P, B, S, 590 Pachira aquatica A, 590 Pseudobombax septenatum N, S, A, 590 Scleronema A, 590 (Byttnerioideae), 590 Guazuma polybotra M, 236 Guazuma ulmifolia N, S, 590 Theobroma cacao N, P, S, 338 Waltheria glomerata A, 590 Waltheria rotundifolia N, S, 204 (Grewioideae), 339 Apeiba S, A, 590 Corchorus S, 590 Corchorus orinocensis N, S, 590 Glyphaea brevis B, S, 330 Grewia P, S, 321 Grewia bicolor N, P, A, 321 Heliocarpus N, S, A, 207, 341 Heliocarpus americanus N, S, 338, 339–342, 344 Luehea A, 590 Luehea seemannii B, S, 121 Trichospermum A, 590 Triumfetta P, S, 305, 321 Triumfetta macrophylla B, S, 330 Triumfetta semitriloba M, 237 (Malvoideae), 590 Abutilon S, A, 590 Hampea A, 590 Hampea trilobata A, 590 Hibiscus tubiflorus M, 237 Malachra palmata M, 233 Malvastrum A , 590 Pavonia N, S, 591 Sida N, S, A, 591 (Sterculioideae), 591 591 Scaphium affine B, S, 330 Sterculia apetala N, S, 591 Marcgraviaceae (1–1), 591 Souroubea S, 591 Melastomataceae N, P, H, S (3–4), 105, 289, 291, 297, 305, 308–310, 330, 338, 342, 403, 477 Dichaetanthera corymbosa B, S, 330 Miconia N, PO, S, A, 297, 304, 305, 309, 310 Miconia myriantha P, S, 591 Tibouchina N, S, 591 Meliaceae M, N, P, S, A (8–12), 236, 321, 330 Azadirachta indica N, P, A, 321 Carapa grandiflora B, S, 330 Carapa guianensis P, S, 321 Cedrela S, 591 Cedrela mexicana M, 236 Cedrela odorata B, S, 121 Ekebergia capensis N, P, B, A, 321, 330 Entandrophragma cylindricum B, S, 330 Entandrophragma excelsum B, S, 330 Guarea S, 591 Melia azedarach N, P, A, 321 Trichilia S, A, 591 Melianthaceae (1–1), 330 Bersama abyssinica B, S, 330 Menispermaceae (2–2), 591 Abuta A, 591 Cissampelos S, 591 Monimiaceae (1–1), 330 Xymalos monospora B, S, 330 Moraceae M, N, P, S, A (6–10), 238, 316, 322, 330, 338 Artocarpus heterophyllus P, A, 322 Brosimum S, 591 Brosimum alicastrum B, S, 121 Castilla elastica MM, 238 Clarisia biflora B, S, 121 Appendix D 592 Ficus B, S, 121, 330 Ficus goldmanii B, S, 121 Ficus natalensis B, S, 330 Ficus trachelosyce B, S, 121 Morus alba N, P, A, 322 Moringaceae N, P, A (1–1), 322 Moringa oleífera N, P, A, 322 Muntingiaceae (1–1), 339 Muntingia calabura N, S, 339–342, 344 Myricaceae (1–1), 330 Myrica salicifolia B, S, 330 Myrtaceae N, P, H, S (8–14), 43, 62, 105, 288, 291, 292, 305, 307–310, 316, 322, 330, 337, 339, 357, 371, 403, 449, 529 Callistemon N, S, 591 Calycolpus moritzianus N, S, 340, 342 Corymbia torelliana (Australian native) L, S, 62, 528 Eucalyptus N, P, E, R, H, B, S, A, 288, 290–292, 322, 330, 341, 344, 353, 449, 464, 466, 529 Eucalyptus coolabah (Australian native) L, S, 43 Eugenia S, A, 304, 305, 309, 310 Eugenia uniflora N, S, 208 Myrcia N, P, S, 291, 292, 338, 339, 341, 342, 344 Myrcia amazonica N, P, S, 290 Psidium N, P, S, A, 288, 307, 309 Psidium guajava N, B, S, 121 Syzygium N, B, S, A, 305, 309, 322 Syzygium guineense B, S, 330 Syzygium jambos N, B, S, 339, 344 Nyctaginaceae N, P, S (2–2), 105 Boerhavia coccinea S, 591 Guapira A, 591 Olacaceae (3–3), 330 Minquartia guianensis B, S, 121 Scorodocarpus borneensis B, S, 331 Strombosia scheffleri B, S, 331 Oleaceae N, P, A (2–2), 322 Fraxinus uhdei P, S, 338 Olea capensis N, P, A, 322 Onagraceae N, P, H, S (1–1), 105, 403 Ludvwigia S, A, 591 Passifloraceae N, P, S (2–2), 105, 322, 357 Passiflora N, P, S, A, 322, 357 Turnera panamensis S, 591 Penaeaceae (1–1), 331 Olinia usamberensis B, S, 331 Pentaphylacaceae (1–1), 592 Ternstroemia meridionalis N, S, 592 Phyllanthaceae (1–1), 592 Phyllanthus N, S, 592 Phytolaccaceae N, P, S, A (1–1), 105, 322 Phytolacca dodecandra N, P, A, 322 Picramniaceae (2–2), 238 Alvaradoa amorphoides M, 238 Picramnia latifolia S, 592 Piperaceae PO, H, S (2–2), 105, 291, 292, 305, 309, 322, 403 Peperomia PO, S, 592 Piper PO, S, A, 292, 297, 305, 309, 310, 312, 322 Polygonaceae P, S (5–6), xiii, 322 Antigonon P, N, S, A, 322 Coccoloba S, 592 Coccoloba caracasana B, S, 121 Polygonum acuminatum A, 592 Rumex Polygonaceae P, S, 592 Triplaris Polygonaceae N, S, 592 Portulacaceae M (1–2), 236 Portulaca N, S, 592 Portulaca oleracea M, 236 Primulaceae H, S (2–2), 331 Maesa lanceolata B, S, 331 Myrsine P, S, 338 Appendix D Proteaceae N, P, A (4–4), 56, 323, 331, 449 Euplassa A, 592 Faurea saligna N, P, B, S, A, 323, 331 Knightia excelsa H, A, 449 Macadamia integrifolia (Australian native) L, S, 35, 56 Putranjivaceae (1–1), 331 Drypetes gerrardii B, S, 331 Ranunculaceae N, P, S (1–1), 105 Rhamnaceae N, P, S, A (4–6), 288, 289, 304, 305, 310, 323, 339 Colubrina A, 592 Gouania S, A, 304, 305, 310 Gouania polygama N, S, 339–341, 342, 344 Hovenia dulcis N, P, S, 289 Ziziphus abyssinica N, P, A, 323 Ziziphus joazeiro N, P, S, 288 Rosaceae N, P, S (3–3), 56, 105, 323, 331, 342 Eriobotrya japonica N, P, A, 323 Hagenia abyssinica B, S, 331 Prunus africana N, P, B, S, A, 323, 331 Rubiaceae S, A (14–17), 105, 146, 288, 304, 305, 323, 338, 339 Alseis N, S, 592 Bertiera guianensis N, S, A, 592 Borreria S, A, 592 Coffea N, A, 323 Coffea arabica N, P, S, 146, 338, 339, 340–344 Genipa A, 592 Genipa americana S, 592 Ixora javanica N, S, 323 Macrocnemum S, A, 304, 305 Mitracarpus N, P, S, 288 Posoqueria A, 592 Psychotria A, 592 Randia N, S, 592 Richardia brasiliensis P, A, 323 Spermacoce verticillata N, S, 592 593 Warszewiczia S, A, 592 Warszewiczia coccinea N, S, 592 Rutaceae H, S, A (4–9), 287, 308–310, 323, 331, 339, 403 Adiscanthus A, 592 Calodendrum capense N, P, A, 323 Citrus N, P, B, S, A10, 49, 121, 263, 287, 323, 338–342, 344, 353, 464 Citrus aurantifolia A, 592 Citrus grandis S, 592 Citrus reticulate S, 592 Zanthoxyllum P, S, A, 310 Zanthoxylum gilletii B, S, 331 Zanthoxylum macrophyllum B, S, 331 Salicaceae M, S (6–8), 105, 237, 238, 323 Banara A, 592 Casearia S, A, 592 Casearia nitida M, 237 Dovyalis abyssinica N, P, A, 323 Flacourtia indica N, P, A, 323 Laetia A, 592 Zuelania guidonia A, 592 Zuelania roussoviae M, 237, 238 Sapindaceae N, P, S (9–11), 57, 105, 146, 207, 305, 308–310, 323, 357 Allophylus rubifolius N, S, 323 Cardiospermum S, A, 593 Cupania americana N, S, 593 Cupania cinerea N, S, 593 Dodonaea angustifolia N, P, A, 323 Litchi chinensis L, S, 57, 357 Nephelium lappaceum P, S, 146 Paullinia N, S, A, 593 Serjania N, S, A, 207, 305, 309, 310 Serjania racemosa A, 593 Talisia S, 593 Sapotaceae P, A (4–5), 323, 331 Butyrospermum paradoxum P, A, 323 Chrysophyllum albidum B, S, 331 Chrysophyllum gorungosanum B, S, 331 Elaeoluma A, 593 Pouteria S, A, 593 Appendix D 594 Schlegeliaceae (1–1), 207 Schlegelia parviflora N, S, 207 Scrophulariaceae P, R, S (1–1), 323, 529 Simaurobaceae M (1–2), 593 Simarouba N, S, 593 Simarouba amara S, 593 Sladeniaceae (1–1), 593 Ficalhoa laurifolia N, B, S, 331 Solanaceae M, N, PO, P, H, S (6–10–1), 105, 146, 234–236, 289, 291, 312, 316, 323, 324, 342, 403 Capsicum annuum M, 234 Capsicum annuum var. aviculare PO, S, 593 Capsicum chinense P, S, 146 Cestrum latifolia S, 593 Datura A, 593 Datura suaveolens N, P, A, 323 Nicotiana rustica M, 593 Nicotiana tabacum M, 234, 235 Parmentiera edulis M, 236 Solanum N, P, S, A, 289, 291, 324 Solanum lycopersicum P, S, 146 Stilbaceae N, P, S (1–1), 324 Nuxia congesta N, P, S, 324 Theaceae N, S (1–1), 331 Gordonia A, 593 Thymelaeaceae N, S (1–1), 331 Wikstroemia B, S, 331 Urticaceae P, S (1–1), 292, 305, 309, 310, 324 Cecropia PO, S, A, 291, 292, 305, 306, 308–310, 312, 324, 338 Verbenaceae N, P, S (5–5), 105, 324, 339 Aegiphila A, 593 Aloysia triphylla P, A, 324 Citharexylum N, S, 593 Lantana fucata N, S, 344 Rehdera trinervis B, S, 121 Violaceae S (1–1), 105, 178 Vitaceae S (1–1), 105, 339 Vitis tiliifolia N, S, 339 Zygophyllaceae S (1–1), 105 Angiospermae Monocotyledoneae Alismataceae (2–2), 593 Echinodorus S, A, 593 Sagittaria S, A, 593 Amaryllidaceae (1–1), 317 Allium cepa N, P, A, 317 Araceae (1–2), 309 Anthurium A, 308, 309 Anthurium bakeri S, 593 Arecaceae N, P, PO, S, A (16–23), 305, 309, 310, 317, 338 Acrocomia vinifera B, S, 121 Astrocaryum S, A, 593 Astrocaryum standleyanum A, 593 Attalea PO, S, 317 Attalea maripa P, S, 317 Bactris S, 209 Bactris gasipaes P, S, 317 Chamaedorea PO, S, A, 593 Cocos nucifera N, P, A, 317 Cryosophila A, 593 Elaeis A, 593 Elaeis guineensis N, P, A, 317 Elaeis oleifera S, A, 593 Euterpe precatoria P, S, 318 Geonoma A, 594 Iriartea gigantea S, A, 594 Leopoldinia pulchra P, S, 318 Mauritia A, 594 Mauritia flexuosa A, 594 Phoenix reclinata N, P, A, 318 Scheelea A, 304, 305, 310 Scheelea zonensis S, 594 Socratea durissima A, 594 Asparagaceae N, P, S (1–1), 105, 318 Agave sisalana N, P, S, A, 318 Appendix D Bromeliaceae S (1–1), 105 Tillandsia A, 594 Cannaceae P, S (1–1), 319 Canna indica P, S, 319 Commelinaceae S (1–2), 105, 319 Commelina N, P, A, 594 Commelina africana N, P, 319 Costaceae S (1–1), 105 Cyperaceae PO, S (3–3), 105, 291, 338 Cyperus S, A, 594 Rynchospora nervosa P, S, 594 Scleria A, 594 Orchidaceae N, S (1–1), 105 Maxillaria rufescens S, T, 527 595 Panicum PO, A, 594 Pariana PO, S, 322 Zea mays PO, S, A, 106, 287, 322 Pontederiaceae (1–1), 594 Eichhornia S, 594 Typhaceae (1–2), 529 Typha PO, R, S, 165, 529 Typha dominguensis PO, 165 Zingiberaceae P, S (1–1), 105, 106 Elettaria cardamomum P, S, 106 Gymnospermae Cupressaceae (1–1), 329 Cupressus lusitanica B, S, 329 Musaceae N, P, S (1–1), 105, 322 Musa N, S, 322 Pinaceae (1–2), 526 Pinus PO, R, S, A, 526 Pinus caribaea S, 594 Poaceae PO, R, S, A (3–3), 105, 106, 287, 305, 308–310, 312, 322, 338, 529 Podocarpaceae (1–1), 331 Podocarpus milanjianus B, S , 331 Appendix E List of Plant Taxa Used by Bees In this book, 691 plant taxa are referred to and are listed below, at species, genus, and/or family level. Nomenclature was checked and updated following the Missouri Botanical Garden database: Tropicos.org. Missouri Botanical Garden http://www. tropicos.org Major changes of family names (the currently preferred names appear in upper case letters) include: Agavaceae = ASPARAGACEAE, Asclepiadaceae = APOCYNACEAE, Bombacaceae = MALVACEAE, Cecropiaceae = URTICACEAE, Chenopodiaceae = AMARANTHACEAE, Compositae = ASTERACEAE, Flacourtiaceae = SALICACEAE, Gramineae = POACEAE, LEGUMINOSAE (Caesalpinioideae, Mimosoideae, Papilionoideae/Faboideae) = FABACEAE, Maesaceae = PRIMULACEAE, Myrsinaceae = PRIMULACEAE, Oliniaceae = PENDEACEAE, Papilionoideae = FABOIDEAE, Sterculiaceae = MALVACEAE, Tiliaceae = MALVACEAE, Umbelliferae = APIACEAE. In addition, transfer changes of some genera into different families include: Agave (Agavaceae) =ASPARAGACEAE, Alangium (Alangiaceae) = CORNACEAE, Alvaradoa (Simaroubaceae) = PICRAMNIACEAE, Avicennia (Avicenniaceae/ Verbenaceae) = ACANTHACEAE, Banara (Flacourtiaceae) = SALICACEAE, Calophyllum Guttiferae/Clusiaceae) = CALOPHYLLACEAE, Casearia (Flacourtiaceae) = SALICACEAE, Cecropia (Cecropiaceae) = URTICACEAE, Celtis (Ulmaceae) = CANNABACEAE, Chenopodium (Chenopodiaceae) = AMARANTHACEAE, Cleome (Capparaceae) = CLEOMACEAE, Cochlospermum (Cochlospermaceae) = BIXACEAE, Drypetes (Euphorbiaceae) = PUTRANJIVACEAE, Ficalhoa (Malvaceae) = SLADENIACEAE Glyphaea (Tiliaceae) = MALVACEAE, Gmelina (Verbenaceae) = LAMIACEAE, Heliocarpus (Tiliaceae) = MALVACEAE, Hydnocarpus (Flacourtiaceae) = ACHARIACEAE, Hydrangea (Saxifragaceae) = HYDRANGEACEAE, Hydrocotyle (Apiaceae) = ARALIACEAE P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7, © Springer Science+Business Media New York 2013 597 598 Appendix E Hyeronima (Euphorbiaceae) = PHYLLANTHACEAE, Maesa (Maesaceae) = PRIMULACEAE Myrsine (Myrsinaceae) = PRIMULACEAE, Nuxia (Loganiaceae) = STILBACEAE, Phyllanthus (Euphorbiaceae) = PHYLLANTHACEAE, Picramnia (Simaroubaceae) = PICRAMNIACEAE Sambucus (Caprifoliaceae) = ADOXACEAE, Strombosia (Oleaceae) = OLACACEAE, Ternstroemia (Theaceae) = PENTAPHYLACEAE, Trema (Ulmaceae) = CANNABACEAE Triumfetta (Tiliaceae) = MALVACEAE, Viburnum (Caprifoliaceae) = ADOXACEAE, Vismia (Guttiferae/Clusiaceae) = HYPERICACEAE, Vitex (Verbenaceae) = LAMIACEAE Plants with Mayan medicinal use (M), nectar (N), pollen (P), trichomes (T), or resin source (R) used to make honey (H), build nests (B), or pollinated (L) by stingless bees (S), and Apis mellifera (A) Abuta (Menispermaceae) A, 596 Abutilon (Malvaceae, Malvoideae) S, A, 596 Acacia (Fabaceae, Mimosoideae) N, P, S, A, 305, 321 Acacia decurrens (Fabaceae, Mimosoideae) N, B, S, 596 Acalypha (Euphorbiaceae) P, S, A, 320, 338 Acalypha discolor (Euphorbiaceae) N, P, S, A, 596 Acalypha diversifolia (Euphorbiaceae) P, S, 596 Acalypha macrostachya (Euphorbiaceae) P, S, 596 Acalypha sidifolia (Euphorbiaceae) N, P, S, A, 596 Acanthaceae M, N, P, S, A, 105, 317, 339, 403 Aceraceae R, S, 529 Acrocarpus fraxinifolius (Fabaceae, Caesalpinioideae) N, P, S, 320 Acrocomia vinifera (Arecaceae) B, S, 121 Adenaria floribunda (Lythraceae) N, S, 339, 344 Adenium obesum (Apocynaceae) N, P, A, 317 Adiscanthus (Rutaceae) A, 596 Aegiphila (Verbenaceae) A, 596 Aeschynomene (Fabaceae, Faboideae) A, 596 Aeschynomene americana (Fabaceae, Faboideae) N, S, A, 596 Aetanthus (Loranthaceae) S, 596 Agauria salicifolia (Ericaceae) B, S, 596 Agave sisalana (Asparagaceae) N, P, S, A, 318 Afzelia africana (Fabaceae, Caesalpinioideae) P, S, A, 320 Alangium chinense (Cornaceae) B, S, 328 Albizia coriaria (Fabaceae, Mimosoideae) N, P, A, 321 Albizia gummifera (Fabaceae, Mimosoideae) N, P, B, S, 321, 329 Alchornea (Euphorbiaceae) P, S, A, 297, 307, 309, 310 Alchonea discolor (Euphorbiaceae) N, P, S, 320 Alchonea sidifolia (Euphorbiaceae) N, P, S, 320 Appendix E 599 Allium cepa (Amaryllidaceae) N, P, A, 317 Allophylus rubifolius (Sapindaceae) N, S, 323 Alnus acuminata (Betulaceae) S, 597 Aloysia triphylla (Verbenaceae) P, A, 324 Alseis (Rubiaceae) N, S, 597 Alternanthera (Amaranthaceae) P, R, S, A, 304, 305, 317, 529 Alvaradoa amorphoides (Picramniaceae) M, 238 Amaranthaceae N, P, R, S, A, 305, 317, 338, 403 Amaranthus (Amaranthaceae) A, 597 Anacardiaceae N, P, S, A, 57, 105, 289, 304, 308, 316, 317, 327, 328, 529 Anacardium excelsum (Anacardiaceae) B, S, A, 121 Anacardium occidentale (Anacardiaceae) N, P, S, A, 317 Anadenanthera (Fabaceae, Mimosoideae) R, S, 529 Anadenanthera colubrina (Fabaceae, Mimosoideae) B, S, 329, 529 Andira inermis (Fabaceae, Faboideae) B, S, 121 Annona (Annonaceae) S, 597 Anthurium (Araceae) A, 308, 309 Anthurium bakeri (Araceae) S, 597 Antigonon (Polygonaceae) P, S, 322 Aparisthmium cordatum (Euphorbiaceae) P, S, 320 Apeiba (Malvaceae, Grewioideae) S, A, 597 Apiaceae M, N, P, S, 105, 234, 339 Apocynaceae N, P, H, S, A, 105, 317, 328 Arachis (Fabaceae, Faboideae) A, 597 Araliaceae N, S, 328 Archidendron jiringa (Fabaceae, Mimosoideae) N, S, 321 Arecaceae N, P, S, A, 105, 317, 338 Arrabidaea (Bignoniaceae) S, A, 305 Artocarpus heterophyllus (Moraceae) P, A, 322 Asparagaceae N, P, S, 105, 318 Aspidosperma (Apocynaceae) S, 328 Aspidosperma pyrifolium (Apocynaceae) B, S 328 Asteraceae M, N, P, R, S, A, 105, 106, 290, 305, 308–310, 316, 337–342, 344, 403, 477, 529 Astrocaryum (Arecaceae) S, A, 597 Astrocaryum standleyanum (Arecaceae) A, 597 Astronium fraxinifolium (Anacardiaceae) P, A, 317 Astronium graveolens (Anacardiaceae) N, B, S, 121, 342 Attalea (Arecaceae) S, 317 Attalea maripa (Arecaceae) P, S, 317 Austroeupatorium inulifolium (Asteraceae) N, S, 339, 340, 342, 344 Avicennia (Acanthaceae) P, S, 317 Azadirachta indica (Meliaceae) N, P, A, 321 Baccharis (Asteraceae) N, P, S, A, 318 Baccharis erioclada (Asteraceae) P, S, 318 600 Baccharis macrantha (Asteraceae) N, S, 597 Baccharis pedunculata (Asteraceae) N, S, 597 Baccharis semiserrata (Asteraceae) P, S, 318 Bactris (Arecaceae) S, 209 Bactris gasipaes (Arecaceae) P, S, 317 Balsaminaceae N, P, S, 318 Banara (Salicaceae) A, 598 Bauhinia (Fabaceae, Caesalpinioideae) N, P, S, A, 287 Bauhinia divaricata (Fabaceae, Caesalpinioideae) A, 598 Bauhinia guianensis (Fabaceae, Caesalpinioideae) S, A, 598 Bauhinia ungulata (Fabaceae, Caesalpinioideae) S, A, 598 Begonia (Begoniaceae) S, A, 598 Begoniaceae H, S, 403 Bersama abyssinica (Melianthaceae) B, S, 330 Bertiera guianensis (Rubiaceae) N, S, A, 598 Bidens (Asteraceae) N, S, 598 Bidens pilosa (Asteraceae) P, S, 318 Bidens squarrosa (Asteraceae) N, S, 207 Bignoniaceae M, N, P, S, A, 105, 308, 309, 318, 328, 344, 403 Bixa orellana (Bixaceae) P, S, 106 Bixaceae P, S, 105 Boerhavia coccinea (Nyctaginaceae) S, 598 Bombacopsis (Malvaceae, Bombacoideae) A, 598 Bombacopsis quinata (Malvaceae, Bombacoideae) B, S, 121 Boraginaceae M, N, P, S, A, 105, 319, 339 Borreria (Rubiaceae) S, A, 598 Brassica (Brassicaceae) N, P, S, 289 Brassicaceae M, N, P, S, 105, 238, 289, 465 Bravaisia integerrima (Acanthaceae) N, B, S, 121, 207 Bravaisia tubiflora (Acanthaceae) M, 598 Bromeliaceae S, 105 Brosimum (Moraceae) S, 598 Brosimum alicastrum (Moraceae) B, S, 121 Bunchosia (Malpighiaceae) S, 598 Bursera (Burseraceae) A, 598 Bursera simaruba (Burseraceae) B, S, A, 121 Burseraceae P, S, 289, 319, 328 Butyrospermum paradoxum (Sapotaceae) P, A, 323 Buxus (Buxaceae) A, 598 Byrsonima crassifolia (Malpighiaceae) P, S, 106, 401 Cactaceae P, S, 105 Caesalpinia (Fabaceae, Caesalpinioideae) N, B, S, A, 598 Caesalpinia decapetala (Fabaceae, Caesalpinioideae) P, S, 320 Caesalpinia pyramidalis (Fabaceae, Caesalpinioideae) B, S, 329 Cajanus (Fabaceae, Faboideae) A, 598 Appendix E Appendix E Cajanus bicolor (Fabaceae, Faboideae) S, 598 Cajanus cajan (Fabaceae, Faboideae) N, P, A, 320 Calliandra calothyrsus (Fabaceae, Mimosoideae) N, S, A 321 Callistemon (Myrtaceae) N, S, 599 Calodendrum capense (Rutaceae) N, P, A, 323 Calophyllum (Calophyllaceae) B, S, 328 Calopogonium (Fabaceae, Faboideae) S, 304, 305 Calycolpus moritzianus (Myrtaceae) N, S, 340, 342 Canna indica (Cannaceae) P, S, 319 Cannaceae P, S, 319 Capsicum annuum (Solanaceae) M, 234 Capsicum annuum var. aviculare (Solanaceae) S, 599 Capsicum chinense (Solanaceae), P, S, 146 Carapa grandiflora (Meliaceae) B, S, 330 Carapa guianensis (Meliaceae) P, S, 321 Cardiospermum (Sapindaceae) S, A, 599 Carica papaya (Caricaceae) M, N, P, S, A, 238, 319 Caricaceae M, N, P, S, A, 319 Caryophyllaceae N, P, S, A, 339 Casearia (Salicaceae) S, A, 599 Casearia nitida (Salicaceae) M, 237 Cassia (Fabaceae, Caesalpinioideae) P, S, 320 Cassia fistula (Fabaceae, Caesalpinioideae) S, 320 Cassia fruticosa (Fabaceae, Caesalpinioideae) S, 599 Cassia obtusifolia (Fabaceae, Caesalpinioideae) S, 599 Cassia undulata (Fabaceae, Caesalpinioideae) P, S, 320 Castanea sativa (Fagaceae) H, A, 449, 485 Castilla elastica (Moraceae) M, 238 Cavanillesia platanifolia (Malvaceae, Bombacoideae) A, 599 Cayaponia (Cucurbitaceae) A, 297 Cecropia (Urticaceae) P, S, A, 292, 305, 306, 309, 310, 324, 338 Cedrela (Meliaceae) S, 599 Cedrela mexicana (Meliaceae) M, 236 Cedrela odorata (Meliaceae) B, S, 121 Ceiba aesculifolia (Malvaceae, Bombacoideae) S, 599 Ceiba pentandra (Malvaceae, Bombacoideae) N, B, S, 599 Celastraceae P, S, 290, 328 Celtis (Cannabaceae) B, S, A, 207, 305, 309 Celtis iguanaeus (Cannabaceae) S, 599 Cestrum latifolia (Solanaceae) S, 599 Chaetocarpus castanocarpus (Euphorbiaceae) B, S, 329 Chamaecrista ramosa (Fabaceae, Caesalpiniaceae) N, P, S, 290 Chamaedorea (Arecaceae) S, A, 599 Chamaesyce (Euphorbiaceae) N, S, A, 599 Chamissoa (Amaranthaceae) S, A, 599 601 602 Chaptalia (Asteraceae) S, 599 Chenopodium (Amaranthaceae) S, A , 599 Chrysophyllum albidum (Sapotaceae) B, S, 331 Chrysophyllum gorungosanum (Sapotaceae) B, S, 331 Citharexylum (Verbenaceae) N, S, 600 Citrullus lanatus (Cucurbitaceae) N, P, L, S, A, 57, 106, 319 Citrus (Rutaceae) N, P, B, S, A, 287 Citrus aurantifolia (Rutaceae) A, 600 Citrus grandis (Rutaceae) S, 600 Citrus reticulate (Rutaceae) S, 600 Cissampelos (Menispermaceae) S, 600 Clarisia biflora (Moraceae) B, S, 121 Cleome (Cleomaceae) N, S, A, 339, 341, 342 Cleome parviflora (Cleomaceae) S, 600 Clusia (Clusiaceae) R, S, A, E, 22, 530, 533 Cnidoscolus chayamansa (Euphorbiaceae) M, 234 Cnidoscolus phyllacanthus (Euphorbiaceae) B, S, 329 Coccoloba (Polygonaceae) S, 600 Coccoloba caracasana (Polygonaceae) B, S, 121 Cochlospermaceae S, 403 Cocos nucifera (Arecaceae) N, P, A, 317 Codiaeum (Euphorbiaceae) A, 600 Coffea (Rubiaceae) N, P, A, 600 Coffea arabica (Rubiaceae) N, P, S, 342 Colubrina (Rhamnaceae) A, 600 Combretaceae N, P, S, A, 319 Combretum (Combretaceae) N, P, S, A, 319 Combretum collinum (Combretaceae) N, P, A, 319 Combretum fruticosum (Combretaceae) B, S, 121 Combretum molle (Combretaceae) N, P, A, 319 Commelina (Commelinaceae) N, P, A, 600 Commelina africana (Commelinaceae) N, P, 319 Commelinaceae S, 105, 319 Commiphora leptophloeos (Burseraceae) B, S, 328 Connarus (Connaraceae) S, 600 Convolvulaceae M, N, P, S, A, 105, 403 Copaifera aromatica (Fabaceae, Caesalpiniodeae) B, S, 121 Corchorus (Malvaceae, Grewioideae) S, 600 Corchorus orinocensis (Malvaceae, Grewioideae) N, S, 600 Cordia (Boraginaceae) A, 310 Cordia africana (Boraginaceae) N, P, S, A, 319 Cordia alliodora (Boraginaceae) B, S, A, 121, 339 Cordia bicolor (Boraginaceae) S, 600 Cordia dentata (Boraginaceae) N, S, 600 Cordia geraschanthoides (Boraginaceae) M, 237 Appendix E Appendix E Cordia millenii (Boraginaceae) N, P, S, A, 319 Cordia monoica (Boraginaceae) N, P, S, A, 319 Cordia panamensis (Boraginaceae) A, 601 Cordia sinensis (Boraginaceae) N, P, S, A, 319 Cordia spinescens (Boraginaceae) N, S, A, 342 Coriandrum sativum (Apiaceae) N, S, 601 Corymbia torelliana (Myrtaceae) (Australian native) L, S, 62, 528 Costaceae S, 105 Couma utilis (Apocynaceae) P, S, 317 Critonia morifolia (Asteraceae) N, S, 339 Crotalaria (Fabaceae, Faboideae) N, P, S, 289, 320 Croton (Euphorbiaceae) S, A, 309 Croton macrostachyus (Euphorbiaceae) N, P, 320 Croton leptostachyus (Euphorbiaceae) N, S, 601 Croton niveus (Euphorbiaceae) M, 238 Crudia (Fabaceae, Caesalpinioideae) N, P, S, 289 Cryosophila (Arecaceae) A, 601 Cucumis sativus (Cucurbitaceae) N, P, A, 319 Cucurbita pepo (Cucurbitaceae) N, P, A, 319 Cucurbitaceae N, P, S, A, 105, 297, 319 Cunoniaceae P, S, 319 Cupania americana (Sapindaceae) N, S, 601 Cupania cinerea (Sapindaceae) N, S, 601 Cuphea (Lythraceae) N, S, 289 Cuphea racemosa (Lythraceae) N, S, 342 Cupressus lusitanica (Cupressaceae) B, S, 329 Cuscuta americana (Convolvulaceae) M, 235 Cynometra alexandri (Fabaceae, Caesalpinioideae) N, P, S, A, 320 Cyperaceae P, S, 105, 291, 338 Cyperus (Cyperaceae) S, A, 601 Dalbergia (Fabaceae, Faboideae) S, 601 Dalechampia (Euphorbiaceae) R, S, E, 344, 530 Dalia (Asteraceae) N, S, 601 Daniellia oliveri (Fabaceae, Caesalpinioideae) P, S, 601 Datura (Solanaceae) A, 601 Datura suaveolens (Solanaceae) N, P, A, 323 Davilla nitida (Dilleniaceae) S, 601 Dehaasia (Lauraceae) B, S, 329 Delonix regia (Fabaceae, Caesalpinioideae) N, P, S, 601 Dendropanax (Araliaceae) A, 601 Desmodium (Fabaceae, Faboideae) N, S, A, 309 Dialium (Fabaceae, Mimosoideae) B, S, 329 Dichaetanthera corymbosa (Melastomataceae) B, S, 330 Didymopanax (Araliaceae) A, 601 Didymopanax morototoni (Araliaceae) S, 601 603 604 Dioclea (Fabaceae, Faboideae) S, A, 601 Diospyros (Ebenaceae) N, S, 319 Diospyros mespiliformis (Ebenaceae) N, P, A, 319 Diphysa americana (Fabaceae, Faboideae) B, S, 121 Diplotaxis tenuifolia (Brassicaceae) N, H, A, 465 Dipterocarpus (Dipterocarpaceae) R, B, S, 329, 526 Dipterocarpus grandiflorus (Dipterocarpaceae) B, S, 329 Dodonaea angustifolia (Sapindaceae) N, P, A, 323 Doliocarpus (Dilleniaceae) S, A, 308, 309, 312 Dovyalis abyssinica (Salicaceae) N, P, A, 323 Drymaria cordata (Caryophyllaceae) S, 602 Drypetes gerrardii (Putranjivaceae) B, S, 331 Ebenaceae N, P, S, A, 319 Echinodorus (Alismataceae) S, A, 602 Eichhornia (Pontederiaceae) S, 602 Eirmocephala brachiata (Asteraceae) N, S, 602 Ekebergia capensis (Meliaceae) N, P, B, A, 321, 330 Elaeis (Arecaceae) A, 602 Elaeis guineensis (Arecaceae) N, P, A, 317 Elaeis oleifera (Arecaceae) S, A, 602 Elaeoluma (Sapotaceae) A, 602 Elephantopus (Asteraceae) N, P, S, A, 318 Elettaria cardamomum (Zingiberaceae) P, S, 106 Elizabetha (Fabaceae, Caesalpinioideae) A, 602 Elizabetha paraensis (Fabaceae, Caesalpinioideae) A, 602 Emilia sonchifolia (Asteraceae) N, S, 602 Entada monostachia (Fabaceae, Mimosoideae) S, 602 Entandrophragma cylindricum (Meliaceae) B, S, 330 Entandrophragma excelsum (Meliaceae) B, S, 330 Enterolobium cyclocarpum (Fabaceae, Mimosoideae) B, S, 121 Epiphyllum (Cactaceae) A, 602 Erica (Ericaceae) H, A, 449 Eriobotrya japonica (Rosaceae) N, P, A, 323 Erythrina (Fabaceae, Faboideae) N, S, A, 602 Erythrina costaricensis (Fabaceae, Faboideae) A, 602 Escallonia pendula (Escalloniaceae) N, S, 602 Eucalyptus (Myrtaceae) N, P, E, R, H, B, S, A, 291, 292, 529 Eucalyptus coolabah (Myrtaceae) (Australian native) L, S, 43 Eugenia (Myrtaceae) S, A, 305, 309 Eugenia uniflora (Myrtaceae) N, S, 208 Eupatorium (Asteraceae) P, A, 318 Eupatorium hemipteropodum (Asteraceae) M, 235 Euphorbia (Euphorbiaceae) S, A, 305 Euphorbia cotinifolia (Euphorbiaceae) N, S, 339, 342 Euphorbia cyatophora (Euphorbiaceae) N, S, 602 Appendix E Appendix E Euphorbia hirta (Euphorbiaceae) N, S, 339, 340, 342, 344 Euphorbia splendens (Euphorbiaceae) N, P, S, 320 Euphorbia thymifolia (Euphorbiaceae) N, S, 342 Euphorbiaceae M, N, P, S, A, 105, 288, 305, 320, 327, 329, 337–339, 341–344 Euplassa (Proteaceae) A, 603 Eusideroxylon zwageri (Lauraceae) B, S, 329 Euterpe precatoria (Arecaceae) P, S, 318 Evolvulus (Convolvulaceae) A, 603 Fabaceae, Caesalpinioideae N, P, S, A, 289, 320, 329, 339, 342, 526 Fabaceae, Faboideae M, N, P, S, A, 237, 288, 289, 320, 339, 344 Fabaceae, Mimosoideae M, N, P, B, S, 238, 288, 289, 292, 321, 329, 339, 529 Fagaceae P, H, S, 105, 403, 449 Faidherbia albida (Fabaceae, Mimosoideae) N, P, A, 321 Faurea saligna (Proteaceae) N, P, B, S, A, 323, 331 Ficalhoa laurifolia (Sladeniaceae) N, B, S, 331 Ficus (Moraceae) B, S, 603 Ficus goldmanii (Moraceae) B, S, 121 Ficus natalensis (Moraceae) B, S, 330 Ficus trachelosyce (Moraceae) B, S, 121 Flacourtia indica (Salicaceae) N, P, A, 323 Forsteronia (Apocynaceae) S, 603 Fraxinus uhdei (Oleaceae) P, S, 338 Gaiadendron (Loranthaceae) S, A, 603 Genipa (Rubiaceae) A, 603 Genipa americana (Rubiaceae) S, 603 Geonoma (Arecaceae) A, 603 Gliricidia sepium (Fabaceae, Faboideae) N, P, B, S, 121, 320 Gluta (Anacardiaceae) B, S, 328 Gluta oba (Anacardiaceae) B, S, 328 Gluta sabahana (Anacardiaceae) B, S, 328 Glyphaea brevis (Malvaceae, Grewioideae) B, S, 330 Gmelina arborea (Lamiaceae) N, P, A, 321 Gomphrena (Amaranthaceae) S, 603 Gordonia (Theaceae) A, 603 Gouania (Rhamnaceae) S, A, 304, 305, 310 Gouania polygama (Rhamnaceae) N, S, 339–342, 344 Grewia (Malvaceae, Grewioideae) P, S, 321 Grewia bicolor (Malvaceae, Grewioideae) N, P, A, 321 Guapira (Nyctaginaceae) A, 603 Guarea (Meliaceae) S, 603 Guazuma polybotra (Malvaceae, Byttnerioideae) M, 236 Guazuma ulmifolia (Malvaceae, Byttnerioideae) N, S, 603 Haematoxylon campechianum (Fabaceae, Caesalpinioideae) A, 603 Hagenia abyssinica (Rosaceae) B, S, 331 Hampea (Malvaceae, Malvoideae) A, 603 605 606 Hampea trilobata (Malvaceae, Malvoideae) A, 604 Hedyosmum (Chloranthaceae) A, 604 Helianthus annuus (Asteraceae) N, P, A, 318 Heliocarpus (Malvaceae, Grewioideae) N, S, A, 207 Heliocarpus americanus (Malvaceae, Grewioideae) N, S, 338–342, 344 Hibiscus tubiflorus (Malvaceae, Malvoideae) M, 237 Hippocratea volubilis (Celastraceae) N, S, 604 Hiraea (Malpighiaceae) S, A, 604 Hirtella (Chrysobalanaceae) S, 604 Hovenia dulcis (Rhamnaceae) N, P, S, 289 Humiriastrum (Humiriaceae) S, A, 604 Hura crepitans (Euphorbiaceae) S, 604 Hydnocarpus (Achariaceae) B, S, 328 Hydrangea (Hydrangeaceae) A, 604 Hydrocotyle (Araliaceae) N, S, 604 Hyeronima (Euphorbiaceae) S, A, 604 Hylenaea praecelsa (Celastraceae) S, 604 Hymenaea (Fabaceae, Caesalpinioideae) R, A, 526 Hypochaeris radicata (Asteraceae) N, S, 339 Hyptis (Lamiaceae) N, S, A, 305, 307, 309, 310, 339 Hyptis brachiata (Lamiaceae) N, S, 339, 342 Hyptis capitata (Lamiaceae) N, S, 207 Hyptis mutabilis (Lamiaceae) N, S, 604 Ilex (Aquifoliaceae) A, 604 Impatiens (Balsaminaceae) S, A, 604 Impatiens balsamina (Balsaminaceae) N, P, S, 318 Impatiens sultanii (Balsaminaceae) N, P, S, 318 Impatiens walleriana (Balsaminaceae) N, P, S, 318 Inga (Fabaceae, Mimosoideae) N, S, A, 604 Inga sapindoides (Fabaceae, Mimosoideae) B, S, 121 Intsia palembanica (Fabaceae, Caesalpinioideae) B, S, 329 Ipomoea (Convolvulaceae) A, 177 Iriartea gigantea (Arecaceae) S, A, 604 Iseia (Convolvulaceae) S, 604 Ixora javanica (Rubiaceae) N, S, 323 Jacaranda mimosifolia (Bignoniaceae) N, P, A, 318 Jacquemontia (Convolvulaceae) A, 604 Juglans australis (Juglandaceae) S, A, 604 Julbernardia (Fabaceae, Caesalpinioideae) P, S, 320 Justicia (Acanthaceae) N, S, A, 339 Justicia adathoda (Acanthaceae) A, 604 Knightia excelsa (Proteaceae) H, A, 449 Laetia (Salicaceae) A, 604 Lagerstroemia (Lythraceae) S, 604 Lamiaceae N, P, H, S, 105, 321, 329, 403 Appendix E Appendix E 607 Lannea barteri (Anacardiaceae) N, P, A, 317 Lantana fucata (Verbenaceae) N, S, 344 Leopoldinia pulchra (Arecaceae) P, S, 318 Leucaena (Fabaceae, Mimosoideae) A, 605 Leucaena glauca (Fabaceae, Mimosoideae) M, 238 Leucaena leucocephala (Fabaceae, Mimosoideae) N, P, A, 321 Licania (Chrysobalanaceae) A, 328 Licania rigida (Chrysobalanaceae) B, S, 328 Litchi chinensis (Sapindaceae) L, S, 57, 357 Litsea (Lauraceae) B, S, 329, 330 Litsea caulocarpa (Lauraceae) B, S, 329 Lonchocarpus (Fabaceae, Faboideae) A, 605 Lonchocarpus costaricensis (Fabaceae, Faboideae) B, S, 121 Lonchocarpus longistylus (Fabaceae, Faboideae) M, 237 Lonicera (Caprifoliaceae) A, 605 Lophopetalum (Celastraceae) B, S, 328 Loranthaceae N, S, A, 339 Ludvwigia (Onagraceae) S, A, 605 Luehea (Malvaceae, Grewioideae) A, 605 Luehea seemannii (Malvaceae, Grewioideae) B, S, 121 Lythraceae P, S, 105, 289, 321, 339 Mabea (Euphorbiaceae) A, 605 Macadamia integrifolia (Proteaceae) (Australian native) L, S, 35, 56 Machaerium (Fabaceae, Faboideae) N, P, S, A, 304, 305, 310, 320, 321 Macrocnemum (Rubiaceae) S, A, 304, 305 Maesa lanceolata (Primulaceae) B, S, 331 Malachra palmata (Malvaceae, Malvoideae) M, 233 Mallotus (Euphorbiaceae) N, S, 320 Malpighiaceae P, S, A, 105, 106, 339, 401 Malvaceae M, N, P, H, S, A, 105, 204, 207, 305, 321, 330, 338, 339, 403 Malvastrum (Malvaceae, Malvoideae) A, 605 Mangifera (Anacardiaceae) A, 309 Mangifera indica (Anacardiaceae) N, P, L, S, A, 57, 317 Maripa (Convolvulaceae) S, A, 605 Markhamia lutea (Bignoniaceae) N, P, S, 318 Martinella obovata (Bignoniaceae) A, 605 Mascagnia hippocrateoides (Malpighiaceae) S, 605 Mauritia (Arecaceae) A, 605 Mauritia flexuosa (Arecaceae) A, 605 Maxillaria rufescens (Orchidaceae) S, T, 527 Maytenus (Celastraceae) S, A, 328 Maytenus acuminata (Celastraceae) B, S, 328 Melastomataceae N, P, H, S, 105, 289, 291, 297, 305, 308–310, 330, 338, 342, 403, 477 Melia azedarach (Meliaceae) N, P, A, 321 608 Appendix E Meliaceae M, N, P, S, A, 236, 321, 330 Mendoncia (Acanthaceae) A, 606 Merremia (Convolvulaceae) N, S, 606 Metopium (Anacardiaceae) A, 606 Miconia (Melastomataceae) N, S, A, 297, 305, 309 Miconia myriantha (Melastomataceae) P, S, 290 Mikania (Asteraceae) P, N, S, A, 318 Mikania micrantha (Asteraceae) N, S, 207 Mimosa (Fabaceae, Mimosoideae) N, S, A, 306, 308 Mimosa acutistipula (Fabaceae, Mimosoideae) B, S, 329 Mimosa bimucronata (Fabaceae, Mimosoideae) P, S, 321 Mimosa caesalpineifolia (Fabaceae, Mimosoideae) P, S, 321 Mimosa casta (Fabaceae, Mimosoideae) A, 309 Mimosa gemmulata (Fabaceae, Mimosoideae) P, S, 606 Mimosa invisa (Fabaceae, Mimosoideae) S, 606 Mimosa pigra (Fabaceae, Mimosoideae) S, A, 606 Mimosa pudica (Fabaceae, Mimosoideae) P, S, 304, 305, 309, 311, 312, 321 Mimosa pulcherrima (Fabaceae, Mimosoideae) S, 606 Mimosa scabrella (Fabaceae, Mimosoideae) P, S, 289, 321 Minquartia guianensis (Olacaceae) B, S, 121 Mitracarpus (Rubiaceae) N, P, S, 288 Momordica (Cucurbitaceae) S, 606 Moraceae M, N, P, S, A, 238, 316, 322, 330, 338 Moringa oleífera (Moringaceae) N, P, A, 322 Moringaceae N, P, A, 322 Morus alba (Moraceae) N, P, A, 322 Muntingia calabura (Muntingiaceae) N, S, 339–342, 344 Musa (Musaceae) N, P, S, 322 Musaceae N, P, S, 105, 322 Myracrodruon urundeuva (Anacardiaceae) B, S, 328 Myrcia (Myrtaceae) N, P, S, 291, 292 Myrcia amazonica (Myrtaceae) N, P, S, 290 Myrica salicifolia (Myricaceae) B, S, 330 Myrospermum frutescens (Fabaceae, Faboideae) B, S, 121 Myrsine (Primulaceae) P, S, 338 Myrtaceae N, P, H, S, 43, 62, 105, 316, 322, 330, 339, 403 Nephelium lappaceum (Sapindaceae) P, S, 146 Nicotiana rustica (Solanaceae) M, 234 Nicotiana tabacum (Solanaceae) M, 234, 235 Nuxia congesta (Stilbaceae) N, P, S, 324 Nyctaginaceae N, P, S, 105 Ochroma (Malvaceae, Bombacoideae) A, 606 Ochroma pyramidale (Malvaceae, Bombacoideae) N, P, B, S, 606 Ocotea veraguensis (Lauraceae) B, S, 121 Olea capensis (Oleaceae) N, P, A, 322 Appendix E Oleaceae N, P, A, 322 Olinia usambarensis (Penaeaceae) B, S, 331 Onagraceae N, P, H, S, 105, 403 Orchidaceae N, P, S, 105 Oreopanax (Araliaceae) N, S, 607 Oryctanthus (Loranthaceae) N, S, 339, 341, 342, 344 Oyedaea verbesinoides (Asteraceae) N, S, 207 Pachira aquatica (Malvaceae, Bombacoideae) A, 607 Panicum (Poaceae) A, 607 Pariana (Poaceae) P, S, 322 Parinari excelsa (Chrysobalanaceae) B, S, 329 Parkinsonia aculeata (Fabaceae, Caesalpinioideae) P, S, 320 Parmentiera edulis (Solanaceae) M, 236 Parthenium argentatum (Asteraceae) R, 526 Passiflora (Passifloraceae) N, P, S, A, 322 Passifloraceae N, P, S, 105, 322 Paullinia (Sapindaceae) N, S, A, 607 Pavonia (Malvaceae, Malvoideae) N, S, 607 Peltogyne purpurea (Fabaceae, Caesalpinioideae) S, 607 Peltophorum inerme (Fabaceae, Caesalpinioideae) S, 607 Peltophorum pterocarpum (Fabaceae, Caesalpinioideae) N, S, 320 Pentacalia (Asteraceae) N, S, 607 Pentaclethra macroloba (Fabaceae, Mimosoideae) B, S, 121 Peperomia (Piperaceae) S, 607 Persea (Lauraceae) N, P, S, 287 Persea americana (Lauraceae) B, L, S, 57, 121, 146, 357 Phoebe macrophylla (Lauraceae) B, S, 330 Phoenix reclinata (Arecaceae) N, P, A, 318 Phyllanthus (Phyllanthaceae) N, S, 607 Phytolacca dodecandra (Phytolaccaceae) N, P, A, 322 Phytolaccaceae N, P, S, A, 105, 322 Picramnia latifolia (Picramniaceae) S, 607 Pimpinella anisum (Apiaceae) M, 234 Pinus (Pinaceae) P, R, S, A, 526 Pinus caribaea (Pinaceae) S, 607 Piper (Piperaceae) P, S, A, 297, 310, 312 Piperaceae P, H, S, 105, 291, 292, 305, 309, 322, 403 Piptadenia communis (Fabaceae, Mimosoideae) B, S, 329 Piptadenia moniliformis (Fabaceae, Mimosoideae) N, P, S, 288 Piptadenia rigida (Fabaceae, Mimosoideae) N, P, S, 288 Piptocoma discolor (Asteraceae) N, S, 607 Pithecellobium (Fabaceae, Mimosoideae) S, A, 607 Pithecellobium dinizii (Fabaceae, Mimosoideae) A, 607 Pithecoctenium crucigerum (Bignoniaceae) A, 607 Plumeria rubra (Apocynaceae) M, 238 609 610 Poaceae P, R, S, A, 105, 106, 287, 305, 308–310, 312, 322, 338, 529 Podocarpus milanjianus (Podocarpaceae) B, S, 331 Polygonaceae P, S, 322 Polygonum acuminatum (Polygonaceae) A, 608 Polyscias fulva (Araliaceae) B, S, 328 Portulaca (Portulaceaceae) N, S, 236 Portulaca oleracea (Portulacaceae) M, 236 Portulacaceae M, 236 Posoqueria (Rubiaceae) A, 608 Pouteria (Sapotaceae) S, A, 608 Premna angolensis (Lamiaceae) B, S, 329 Prestonia (Apocynaceae) S, 608 Primulaceae H, S, 331 Proteaceae N, P, A, 56, 323, 331, 449 Protium (Burseraceae) N, P, R, S, A, 289, 291 Prunus africana (Rosaceae) N, P, B, S, A, 323, 331 Pseudobombax septenatum (Malvaceae, Bombacoideae) N, P, S, A, 608 Pseudosamanea guachapele (Fabaceae, Mimosoideae) B, S, 121 Psidium (Myrtaceae) N, P, S, A, 307, 309 Psidium guajava (Myrtaceae) N, B, S, 121 Psychotria (Rubiaceae) A, 608 Pterocarpus (Fabaceae, Faboideae) A, 608 Quercus (Fagaceae) H, A, 449 Randia (Rubiaceae) N, S, 608 Ranunculaceae N, P, S, 105 Rauvolfia caffra (Apocynaceae) N, P, A, 317 Rehdera trinervis (Verbenaceae) B, S, 121 Rhamnaceae N, P, S, A, 323, 339 Richardia brasiliensis (Rubiaceae) P, A, 323 Ricinus (Euphorbiaceae) P, S, 235 Ricinus communis (Euphorbiaceae) M, N, P, S, 235, 288, 338 Robinia pseudoacacia (Fabaceae, Faboideae) H, A, 462 Rosaceae N, P, S, 323, 331 Rubiaceae S, A, 105, 304, 305, 323, 339 Rubus (Rosaceae) N, S, 608 Rumex (Polygonaceae) P, S, 608 Rutaceae H, S, A, 308, 323, 331, 339, 403 Rynchospora nervosa (Cyperaceae) P, S, 608 Sagittaria (Alismataceae) S, A, 608 Salicaceae M, S, 323 Sambucus nigra (Adoxaceae) P, S, 608 Sapindaceae N, P, S, 308–310, 323 Sapium (Euphorbiaceae) N, S, A, 608 Sapium caudatum (Euphorbiaceae) S, A, 608 Sapotaceae P, A, 323, 331 Appendix E Appendix E 611 Scaphium affine (Malvaceae, Sterculioideae) B, S, 330 Scleria (Cyperaceae) A, 609 Scheelea (Arecaceae) A, 305 Scheelea zonensis (Arecaceae) S, 609 Schefflera (Araliaceae) N, S, 609 Schefflera barteri (Araliaceae) B, S, 328 Schefflera morototoni (Araliaceae) N, P, S, 290 Schinopsis brasiliensis (Anacardiaceae) B, S, 328 Schinus (Anacardiaceae) N, P, R, S, 291, 292, 529 Schlegelia parviflora (Schlegeliaceae) N, S, 207 Schrankia (Fabaceae, Mimosoideae) P, S, 321 Scleronema (Malvaceae, Bombacoideae) A, 609 Scorodocarpus borneensis (Olacaceae) B, S, 331 Scrophulariaceae P, R, S, 323 Selenicereus (Cactaceae) A, 609 Senna (Fabaceae, Caesalpinioideae) P, S, 312 Serjania (Sapindaceae) N, S, A, 207, 305, 309 Serjania racemosa (Sapindaceae) A, 609 Shorea (Dipterocarpaceae) B, S, 329 Sicyos (Cucurbitaceae) A, 609 Sida (Malvaceae, Malvoideae) N, S, A, 609 Simarouba (Simaroubaceae) N, S, 609 Simarouba amara (Simaroubaceae) S, 609 Simaurobaceae M, 609 Sinapis nigra (Brassicaceae) M, 237 Socratea durissima (Arecaceae) A, 609 Solanaceae M, N, P, H, S, 105, 146, 234–236, 289, 312, 316, 323, 324, 342, 403 Solanum (Solanaceae) P, S, A, 291, 324 Solanum lycopersicum (Solanaceae) P, S, 146 Souroubea (Marcgraviaceae) S, 609 Spananthe paniculata (Apiaceae) N, S, 340, 342 Spermacoce verticillata (Rubiaceae) N, S, 344 Spondias (Anacardiaceae) S, A, 305, 309, 317, 328 Spondias mombin (Anacardiaceae) P, B, S, A, 73, 121, 317 Spondias radlkoferi (Anacardiaceae) P, S, 317 Spondias tuberosa (Anacardiaceae) B, S, 328 Steiractinia aspera (Asteraceae) N, S, 609 Stellaria (Caryophyllaceae) N, S, 339 Sterculia apetala (Malvaceae, Sterculioideae) N, S, 609 Stigmaphyllon (Malpighiaceae) A, 609 Stigmaphyllon hypargyreum (Malpighiaceae) S, 609 Stilbaceae N, P, S, 324 Strombosia scheffleri (Olacaceae) B, S, 331 Struthanthus (Loranthaceae) S, 609 Struthanthus subtilis (Loranthaceae) N, S, 609 612 Stryphnodendron guianense (Fabaceae, Mimosoideae) N, P, S, 290 Sympetalandra borneensis (Fabaceae, Caesalpinioideae) B, S, 329 Syzygium (Myrtaceae) N, B, S, A, 305, 309 Syzygium guineense (Myrtaceae) B, S, 330 Syzygium jambos (Myrtaceae) N, B, S, 339, 344 Tabebuia (Bignoniaceae) S, A, 610 Tabebuia caraiba (Bignoniaceae) P, B, S, 328 Tabebuia ochracea (Bignoniaceae) P, B, S, 121 Tabebuia rosea (Bignoniaceae) N, P, B, S, 121 Talisia (Sapindaceae) S, 610 Tamarindus indica (Fabaceae, Caesalpinioideae) N, P, S, A, 320 Tapirira guianensis (Anacardiaceae) P, S, A, 317 Taraxacum officinale (Asteraceae) N, S, 610 Tarenaya spinosa (Capparaceae) N, S, A, 204 Terminalia oblonga (Combretaceae) B, S, 121 Ternstroemia meridionalis (Pentaphylacaceae) N, S, 610 Tetrapteris (Malpighiaceae) N, S, 339 Theaceae N, S, 331 Theobroma cacao (Malvaceae, Byttnerioideae) P, S, 338 Thymelaeaceae N, S, 331 Thymus (Lamiaceae) H, A, 449 Tibouchina (Melastomataceae) S, 610 Tillandsia (Bromeliaceae) A, 610 Tithonia diversifolia (Asteraceae) N, S, 610 Tournefortia (Boraginaceae) A, 610 Toxicodendron striatum (Anacardiaceae) N, S, 340, 343 Trichanthera gigantea (Acanthaceae) N, S, 610 Trichilia (Meliaceae) S, A, 610 Trichospermum (Malvaceae, Grewioideae) A, 610 Trifolium pratense (Fabaceae, Faboideae) N, S, 203 Trifolium repens (Fabaceae, Faboideae) N, S, 610 Trigonopleura malayana (Euphorbiaceae) B, S, 329 Triplaris (Polygonaceae) N, S, 610 Tristerix (Loranthaceae) S, 610 Triumfetta (Malvaceae Grewioideae) P, S, 237 Triumfetta macrophylla (Malvaceae, Grewioideae) B, S, 330 Triumfetta semitriloba (Malvaceae, Grewioideae) M, 237 Turnera panamensis (Passifloraceae) S, 610 Typha (Typhaceae) R, S, 529 Typha dominguensis (Typhaceae) P, 165 Unonopsis (Annonaceae) S, 610 Urticaceae P, S, 292, 305, 309, 310, 324 Vaccinium corymbosum (Ericaceae) L, S, 57 Verbenaceae N, P, S, 324 Vernonanthura (Asteraceae) N, S, 610 Appendix E Appendix E Vernonia (Asteraceae) N, S, A, 207 Vernonia amygdalina (Asteraceae) P, S, 318 Vernonia auriculifera (Asteraceae) P, S, 318 Vernonia patens (Asteraceae) N, S, 207 Vernonia pauciflora (Asteraceae) N, P, S, 318 Viburnum (Adoxaceae) N, S, 611 Vicia (Fabaceae, Faboideae) P, S, 321 Vicia faba (Fabaceae, Faboideae) N, S, 321 Violaceae S, 105 Vismia (Hypericaceae) R, S, 528 Vitaceae S, 105, 339 Vitex doniana (Lamiaceae) N, P, A, 321 Vitex orinocensis (Lamiaceae) N, S, 611 Vitis tiliifolia (Vitaceae) N, S, 339 Waltheria glomerata (Malvaceae, Byttnerioideae) A, 611 Waltheria rotundifolia (Malvaceae, Byttnerioideae) N, S, 204 Warszewiczia (Rubiaceae) S, A, 611 Warszewiczia coccinea (Rubiaceae) N, S, 611 Wedelia trilobata (Asteraceae) N, S, 318 Weinmannia (Cunoniaceae) P, S, 319 Wikstroemia (Thymelaeaceae) B, S, 331 Xymalos monospora (Monimiaceae) B, S, 330 Zanthoxyllum (Rutaceae) N, P, S, A, 310 Zanthoxylum gilletii (Rutaceae) B, S, 331 Zanthoxylum macrophyllum (Rutaceae) B, S, 331 Zea mays (Poaceae) P, S, A, 106, 287, 322 Zingiberaceae P, S, 105, 106 Ziziphus abyssinica (Rhamnaceae) N, P, A, 323 Ziziphus joazeiro (Rhamnaceae) N, P, S, 288 Zornia (Fabaceae, Faboideae) N, P, S, 288 Zuelania guidonia (Salicaceae) A, 611 Zuelania roussoviae (Salicaceae) M, 237, 238 Zygophyllaceae S, 105 613 Appendix F Common Names of Plants Used for Nesting by Stingless Bees “espavel, rabito” Anacardium excelsum (Anacardiaceae) Costa Rica, 121 “mangle blanco” Bravaisia integerrima (Acanthaceae) Costa Rica, 121 “jiñocuabe” Bursera simaruba (Burseraceae) Costa Rica, 121 “laurel” Cordia alliodora (Boraginaceae) Costa Rica, 121 “guachipelín” Diphysa americana (Fabaceae, Papilionoideae) Costa Rica, 121 “higuerón” Ficus (Moraceae) Costa Rica, 121 “higuerón” Ficus trachelosyce (Moraceae) Costa Rica, 121 “madero negro” Gliricidia sepium (Fabaceae, Papilionoideae) Costa Rica, 121 “siete cueros” Lonchocarpus lasiotropis (costaricensis) (Fabaceae, Faboideae), Costa Rica, 121 “guayaba, guayabo” Psidium guajava (Myrtaceae) Costa Rica, 121 “cortez amarillo” Tabebuia ochracea (Bignoniaceae) Costa Rica, 121 “gavilán” Pentaclethra macroloba (Fabaceae, Mimosoideae) Costa Rica, 121 “manú” Minquartia guianensis (Olacaceae) Costa Rica, 121 “ojoche” Brosimum alicastrum (Moraceae) Costa Rica, 121 “pochote” Bombacopsis quinata (Malvaceae, Bombacoideae) Costa Rica, 121 “cítricos” Citrus (Rutaceae) Costa Rica, 121 “aguacate” Persea americana (Lauraceae) Costa Rica, 121 “guanacaste” Enterolobium cyclocarpum (Fabaceae, Mimosoideae) Costa Rica, 121 “almendro de montaña” Andira inermis (Fabaceae, Faboideae) Costa Rica, 121 “cedro amargo” Cedrela odorata (Meliaceae) Costa Rica, 121 “papaturro blanco” Coccoloba caracasana (Polygonaceae) Costa Rica, 121 “guaba” Inga sapindoides (Fabaceae, Mimosoideae) Costa Rica, 121 “aguacatillo” Ocotea veraguensis (Lauraceae) Costa Rica, 121 “jobo” Spondias mombin (Sapindaceae) Costa Rica, 73, 121 “roble de sabana” Tabebuia rosea (Bignoniaceae) Costa Rica, 121 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7, © Springer Science+Business Media New York 2013 615 Appendix G Common Names of Medicinal Plants Used with Honey by Mayas “anis” Pimpinella anisum (Apiaceae) Mexico, 234 “balché” Lonchocarpus longistylus (Fabaceae, Faboideae) Mexico, 237 “besinikche” Mexican alvaradoa Alvaradoa amorphoides (Picramniaceae) Mexico, 238 “buhumkak” Cordia geraschanthoides (Boraginaceae) Mexico, 236–237 “cat” Parmentiera edulis (Solanacaeae) Mexico, 236 “chaya” Cnidoscolus chayamansa (Euphorbiaceae) Mexico, 234 “chilli” Capsicum annuum (Solanaceae) Mexico, 233–234 “chiople” Eupatorium hemipteropodum (Asteraceae) Mexico, 235 “chuy-che” Zuelania roussoviae (Salicaceae) Mexico, 238 “croton” Croton niveus (Euphorbiaceae) Mexico, 238, 309, 339 “cualote” Guazuma polybotrya (Malvaceae, Byttnerioideae) Mexico, 236–237 “ek-huleb” Bravaisia tubiflora (Acanthaceae) Mexico, 238 “ixim-che” Casearia nitida (Salicaceae) Mexico, 236–237 “k’uts” tobacco Nicotiana tabacum, N. rustica (Solanaceae) Mexico, 233–235 “kanlecay” dodder Cuscuta americana (Convolvulaceae) Mexico, 235 “kulche” Cedrela mexicana (Meliaceae) Mexico, 236 “malva” Malachra palmata (Malvaceae, Malvoideae) Mexico, 233 “mostaza” mustard Sinapis nigra (Brassicaceae) Mexico, 237–238 “muloch” Triumfetta semitriloba (Malvaceae, Tilioideae) Mexico, 236–237 “papaya” pawpaw Carica papaya (Caricaceae) Mexico, 238, 319 “plumeria” frangipani Plumeria rubra (Apocynaceae) Mexico, 238 “taamaay” rubber tree Castilla elastica (Moraceae) Mexico, 238 “tupkin” hibiscus Hibiscus tubiflorus (Malvaceae, Malvoideae) Mexico, 237–238 “uaxim” white leadtree Leucaena glauca (Fabaceae, Mimosoideae) Mexico, 238 “xhóch” Ricinus communis (Euphorbiaceae) Mexico, 235 “xucul” purslane Portulaca oleracea (Portulacaceae) Mexico, 236 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7, © Springer Science+Business Media New York 2013 617 Appendix H Microorganisms Associated to Stingless Bees or Used to Test Antimicrobial Activity Legend: H – Honey origin N – Nectar origin P – Pollen origin PI – Propolis origin L – Larval origin E – Adult bee origin G – Gut origin T – Nest/hive origin C – Brood comb origin R – Hive floor origin D – Bee bread origin GP – Garbage pellet origin SP – Spores stored in lieu of pollen B – Bacteria F – Mold O – Other Fungi LA – Cause lactic fermentation Y – Yeast S – Stingless bee A – Apis mellifera I – Solitary bees PA – Propolis antibacterial activity HA – Honey antibacterial activity HF – Honey antifungal activity HY- Honey antiyeast activity P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7, © Springer Science+Business Media New York 2013 619 620 List of microorganisms: Actinobacteria B, A, G, 175 Alphaproteobacteria B, A, G, 175 Ascosphaera apis F, A, 179 Aspergillus flavus F, A, D, 179 Aspergillus niger F, I, HF , 175, 179, 497, 499 Aspergillus niger F, S, 175, 179, 497, 499 Aspergillus sp. F, S, 179 Aspergillus terreus F, S, 179 Aspergillus versicolor F, A, D, 179 Aureobasidium pullulans Y, A, P, D, E, 179, 180 Bacillus B, H, P, G, L, C, S, 21, 154, 158, 160, 175, 177–179, 230 Bacillus alvei B, S, T, 178 Bacillus circulans B, I, S, T, 175, 178 Bacillus licheniformis B, S, 178 Bacillus megaterium B, S, T, 178 Bacillus meliponotrophicus B, S, G, H, P, T, 177 Bacillus mycoides B, I, 175 Bacillus pumilis nad B, S, 178 Bacillus spp. B, A, C, R, L, I, S, 160, 177 Bacillus cereus PA, 499 Bacillus subtilis B, S, HA, 178, 401 Betaproteobacteria B, A, G, 175 Bifidobacterium B, A, G, LA, 159, 175, 178, 502 Candida Y, P, H, 21, 155, 161, 181 Candida albicans Y, PA, HY, 400, 401, 405, 406, 497–499 Candida apicola Y, S, G, T, P, 176, 177, 180, 181 Candida apis Y, 176, 177 Candida batistae, Y, I, T, 176, 177 Candida bombi Y, I, 176, 177 Candida bombicola (Starmerella bombicola) Y, I, N, P, 176, 177, 180 Candida cellae Y, I, 176, 177 Candida davenportii Y, 176, 177 Candida etchellsii Y, S, 177 Candida floricola Y, 176, 177 Candida floris Y, S, 177 Candida geochares Y, H, S, 177 Candida magnoliae Y, G, A, P, 176 Candida powellii Y, 176, 177 Candida riodocensis Y, I, P, N, 176, 177 Candida sorbosivorans Y, 176 Candida tilneyi Y, I, 176, 177 Candida vaccinii Y, 176 Cladosporium sp. F, S, 179 Corynebacterium B, A, C, R, 175 Appendix H Appendix H 621 Cryptococcus Y, S, E, 180 Cryptococcus neoformans Y, HY, 401 Curvularia sp. F, S, 179 Debaryomyces hansenii Y, S, GP, A, 180 Enterobacter agglomerans B, I, 175 Erwinia tasmaniensis B, A, G, 175 Escherichia coli B, HA, PA, 175, 371, 401, 414, 476, 497–499, 501–502, 509–511 Firmicutes B, A, G, 175 Gammaproteobacteria B, A, G, 175 Hyphopichia burtonii Y, S, 181 Janthinobacterium sp. B, A, G, 175 Kocuria sp. B, A, G, 175 Lactobacillus B, S, T, G, LA, A, 159, 175, 178, 502 Listeria monocytogenes B, HA, 509–510 Melisococcus plutonius B, 155 Mesorhizobium sp. B, A, G, 175 Metschnikowia kunwiensis Y, I, 181 Metschnikowia lunata Y, S, 619 Metschnikowia reukaufii Y, I, 181 Mycobacterium smegmatis HA, 401 Mycobacterium tuberculosis PA, 233 Microbacterium sp. B, A, G, 175 Micrococcus luteus HA, 476, 498, 499 Monilia sp. F, S, 179 Moraxella sp. B, A, G, 175 Mucor alboalter F, A, D, 179 Mucor F, I, 176, 179 Mycobacterium smegmatis B, 401 Nigrospora sp. F, S, 179 Paenibacillus larvae B, 154, 155 Penicillium corylophilum F, A, P, D, 179 Penicillium crustosum F, A, P, D, 179 Penicillium granulatum F, A, D, 179 Penicillium solitum F, A, D, 179 Penicillium sp. F, I, S, D, 175, 179 Priceomyces mellissophilus Y, S, 181 Providencia alcalifaciens B, A, G, 175 Pseudomonas aeruginosa B, HA, 406, 476, 498, 499, 509–511 Pseudomonas sp. B, A, G, 175 Pseudomonas sp. B, I, 175 Pseudozyma antarctica Y, S, E, 180 Rhizopus F, SP, S, 179 Rhizopus nigricans F, A, P, D, 179 Rhodotorula Y, S, E, 180 Saccharomyces sp. Y, I, 175 Saccharomyces cerevisiae Y, 164, 165, 414 622 Appendix H Salmonella enteritidis B, HA, 509–511 Salmonella typhi B, HA, 401, 406 Sphingomonas melonis B, A, G, 175 Sporotrichum olivecum F, A, D, 179 Staphylococcus aureus B, HA, PA, 371, 400–401, 406, 414, 476, 497–499, 509– 511, 517 Staphylococcus epidermidis B, HA, 509–511 Staphylococcus saprophyticus B, A, G, 175 Starmerella P, H, Y, S, E, R, PI, GP, 155, 176, 177, 180 Starmerella bombicola Y, S, P, H, I, 176, 177, 180 Starmerella meliponinorum Y, S, P, H, I, E, GP, 176, 177, 180 Stemphylium F, S, 178 Stinkhorn species (Fungi, Phaleles) O, 156 Streptococcus B, LA, A, 159 Streptomyces albidoflavus B, A, 176 Streptomyces albus B, S, 178 Streptomyces ambofaciens B, S, C, 178 Streptomyces B, C, S, T, G, 155, 176 Streptomyces badius B, A, 176 Streptomyces coalescens B, S, C, 178 Streptomyces drozdowiczii B, S, A, 178 Streptomyces fradiae B, H, A, 176 Streptomyces malaysiensis B, S, 178 Streptomyces minutiscleroticus B, S, 178 Streptomyces mutabilis B, S, C, 178 Streptomyces pseudogriseolus B, S, 178 Streptomyces rochei B, S, 178 Streptomyces tosaensis B, S, 178 Streptomyces violaceoruber B, S, C, 178 Streptomycetes sp. B, P, I, C, T, 175–176 Trichoderma sp. F, S, 179 Tsukamurella tyrosinosolvens B, A, G, 175 Zygosaccharomyces machadoi Y, S, GP, 180–181 Zygosaccharomyces Y, S, H, E, GP, 180, 181, 449 Appendix I Summary of Meliponine and Apis Honey Composition [Number of Honey Samples Analyzed] P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7, © Springer Science+Business Media New York 2013 623 Physicochemical parametersa Bee species Chapter Country Number of honey samples analyzed (N) pH Frieseomelitta sp. 27 Colombia 5–6 Geotrigona acapulconis 28 Guatemala 1 3.06 85.53 0.09 2.6 – n.d. – – – – 32.09 Melipona beecheii 28 Guatemala 7 3.7 ± 0.1 23.2 ± 30.0 0.07±0.05 21.3± 32.8 – n.d. – – 68.8 ± 3.8 3.5 ± 4.1 17.3±2.6 Melipona brachychaeta 29 Bolivia 1 3.8 10.4 0.01 – – – – – 73.4 1.5 24.9 Melipona compressipes 27, 30 Colombia 1–12 – 7.0 [1] 0.09 [1] n.d. [2] – 3.0 [1] – – 71.1 ± 8.1 [11] 3.4 ± 2.2 [11] 25.8 ± 2.0 [12] Melipona eburnea 27, 30 Colombia 7 – – – – – – – – 77.8 ± 14.5 3.6 ± 1.5 27.6 ± 2.1 Melipona favosa 25 Venezuela 6–40 – 51.7 ± 25.2 (12.7–97.1) [40] 0.14 ± 0.13 2.86 ± 0.36 – (0.01–0.61) (2.65–3.50) [40] [6] 17.7 ± 8.5 90.08 ± 48.03 45.7 ± 18.3 67.3 ± 4.1 2.1 ±1.3 (5.04-24.69) (31.80-150.70) (10.5–102.00) (60.9–78.6) (0.5–5.1) [21] [6] [39] [40] [40] 28.0 ± 2.7 (22.1–32.0) [40] Melipona favosa 27, 30 Colombia 1–7 – – 0.01 ± 0.01 – [2] – n.d. [1] – – 72.2 ± 7.4 [7] 3.1 ± 1.8 [7] 24.8 ± 1.8 [7] Melipona grandis 29 Bolivia 1 3.6 16.0 0.02 – – – – – 72.5 0.9 24.1 Melipona solani 28 Guatemala 1 3.8 4.95 0.06 8.3 – n.d. – – 76.0 1.7 19.66 Melipona aff. yucatanica Guatemala 1 3.8 10.59 0.06 10.0 – n.d. – – – – 20.37 28 Reducing sugarsd (g/100 g honey) Apparent sucrosee Water (g/100 g (g/100 g honey) honey) – 29.7 ± 14.1 [5] 3.1 ± 2.7 [5] 33.1 ± 3.3 [6] Free Acidity (meq/Kg honey) Ash (g/100 g honey) Diastase activity (DN)b Insoluble solids (g/100 g honey) HMF (mg/kg honey) Invertase activity (IU)c Nitrogen (mg/100 g honey) – – – – – – – Melipona sp. 27 Colombia 2–18 – – 0.20 ± 0.00 – [2] – – – – 67.6 ± 7.5 [18] 6.0 ± 2.3 [18] 26.15 ± 1.8 [18] Melipona sp. 30 Colombia 10 – – – – – – – – 67.4 6.5 ± 3.2 26.8 ± 5.3 Nannotrigona perilampoides 28 Guatemala 1 3.8 9.93 0.33 6.8 – n.d. – – – – 16.5 Nannotrigona testaceicornis 27 Colombia 2–3 – – – – – – – – 65.8 ± 35.1 [2] 7.9 ± 4.3 [2] 27.5 ± 4.2 [3] Nannotrigona sp. 27 Colombia 1–4 – – 0.33 [1] – – – – – 50.8 ± 7.4 [4] 9.7 ± 4.3 [4] 25.7 ± 1.8 [4] Paratrigona sp. 27 Colombia 1–4 4.1 [1] 31.7 [1] – – – – – – 58.1 ± 12.4 4] 3.9 ± 2.8 [4] 26.6 ± 1.2 [4] Partamona peckolti 27 Colombia 1 – – – – – – – – 40.6 6.1 42.7 Partamona sp. 27 Colombia 1 – – – – – – – – 38.3 13.1 28.9 Plebeia sp. 27 Colombia 1 – – – – – – – – 36.7 0.9 28.6 Plebeia sp. 28 Guatemala 1 3.8 15.31 1.25 7.6 – n.d. – – – – 30.3 Scaptotrigona depilis 29 Bolivia 1 3.4 49.4 0.03 – – – – – 67.7 1.0 26.0 Scaptotrigona limae 27 Colombia 2 – – – – – – – – 67.7 ± 4.1 6.6 ± 4.6 25.8 ± 2.2 Scaptotrigona mexicana 28 Guatemala 1–2 4.0 ± 0.4 [2] 12.7 ± 3.0 [2] 0.10 ± 0.04 18.6 ± 12.7 [2] [2] – n.d. – – 57.2 [1] 0.1 [1] 18.7 ± 0.2 [2] Scaptotrigona polysticta 29 Bolivia 1 3.5 49.1 0.06 – – – – – 67.8 1.0 26.5 Scaptotrigona 29 near xanthotricha Bolivia 1 3.8 34.5 0.09 – – – – – 67.0 0.0 24.9 (continued) Physicochemical parametersa Bee species Number of honey samples analyzed (N) pH Free Acidity (meq/Kg honey) Ash (g/100 g honey) Diastase activity (DN)b Insoluble solids (g/100 g honey) HMF (mg/kg honey) Colombia 1–4 4.5 [1] 57.83 [1] 0.06 [1] 2.4 [1] – Chapter Country Scaptotrigona sp. 27 Invertase activity (IU)c Nitrogen (mg/100 g honey) Reducing sugarsd (g/100 g honey) Apparent sucrosee Water (g/100 g (g/100 g honey) honey) 6.0 [1] – – 55.7 ± 5 [4] 12.1 ± 7.4 26.9 ± 2.9 [4] [4] 1.0 ± 1.1 [2] – – 60.8 ± 10.7 [19] 4.4 ± 5.6 [19] 0.28 ± 0.11 16.9 ± 3.9 0.06 ± 0.03 0.65 ± 0.25 – (0.17– 0.42) (11.0– 22.5) (0.02– 0.10) (0.30– 0.93) – 57.1 ± 7.8 2.1 ± 1.8 (44.8–67.5) (0.4–4.5) 24.4 ± 0.8 (23.4–25.6) 0.21 ± 0.70 16.7 ± 9.2 [8] 1.3 ± 2.1 [6] [12] Tetragona sp. 27, 30 Colombia 2–21 4.2 ± 0.3 [4] 44.3 ± 21.8 [4] 0.50 ± 0.08 17.8 ± 5.5 [2] – [5] Tetragonisca angustula 26 Brazil 6 – Tetragonisca angustula 27 Colombia 6–44 4.2 ± 0.3 [12] 39.2 ± 22.9 [12] Tetragonisca angustula Tetragonisca fiebrigi 28 Guatemala 1–4 5.9±1.6 [4] 29 Bolivia 1 Tetragonula laeviceps 36 Thailand – 37.3 ± 16.7 (21.7–63.9) c – – – 53.6 ± 11.8 [44] 4.2 ± 2.4 [44] 24.3 ± 2.3 [44] 12.3±10.3 [4] – n.d. – – 65.78 [1] 4.83 [1] 4.5 17.4±10.4 [4] 0.35±0.26 [4] 43.8 0.33 – – – – – 58.6 1.8 17.5 ± 2.8 [4] 25.1 3.37 – – – – – 44.8 – 15.2 – – Mean values ± SD (minimum and maximum), and [number of honey samples tested, only where N varies] are presented The Diastase Number (DN) indicate g /100 g honey/h, at pH 5.2 and 40 °C An Invertase Unit (IU) indicates mmoles p-nitrophenyl glucopyranoside hydrolyzed/kg honey/min, at pH 6.0 and 40 °C d Chapters 27 and 30 measures of glucose + fructose are considered reducing sugars in this table e Chapters 27 and 30 measures of dissacharides (sucrose + maltose) are considered reducing sugars in this table a b 25.8 ± 3.6 [21] Appendix J Information of Collected Stingless Bees Studies of pot-honey need a backup of identified stingless bees by competent entomologists. Regulations to exchange insects for academic purposes between different countries should be met. If it helps, besides the entomological sample, it is useful to send available information such as that organized in the table below, including few images: No. 013-2008 Country VENEZUELA Nest Location La Vega del Corozo, Aricagua, Estado Mérida N 08° 14.581’ W 071° 08.336’ 3259 Adobe wall – – Geographic Coordinates (if possible with GPS) Height (feet) Type of hive (feral o meliponary) Type of meliponario (modern o crafted) Origin of the nest (location of nest collection transported to the hive, if possible with GPS) Substrate description (tree cavity, underground, termite Dark mass nest, ant nest, bee nest, exposed on tree branches, exposed on walls, inside walls, etc.) Entrance (tubular shape, etc.) and material Flat trumpet, resin, aprox. 1 m height (resin, cerumen, vegetal particles, clay, seeds, etc.) (continued) P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7, © Springer Science+Business Media New York 2013 627 Appendix J 628 Stingless bee Common name pegona negra Identification Partamona peckolti (Friese, 1901) jmafcama@ffclrp. usp.br Collector Name Date Address e-mail Patricia Vit, Marilin Pérez, Anacely Rivas, Llenis Toro 18.05.08 Departamento Ciencia de los Alimentos, Facultad de Farmacia y Bioanálisis, Universidad de Los Andes, Mérida 5101, Venezuela vit@ula.ve Meliponicultor Name Address e-mail Phone Mobile – – – – 0426-7772466 (Marilin), 0274-5116918 (Berta) Observations Flora Flight Behavior Chases, is disturbed by flash, and bites the head Index A Acidity and antibacterial activity, 449 Apis mellifera honey spoilage, 447 dark honeys, 450 electrical conductivity, 450 honey antibacterial activity, 449 honey fermentation, 447 lactones, 450 Acute-phase proteins (APP), 514 Adaptive immune response BCR and TCR, 518 CTL and macrophages, 518 foreign microorganisms and molecules, 518 royal jelly and propolis, 519 T/CD4+ and T/CD8+ cells, 518 Treg cells, 518–519 Advanced HPLC-MS methods, 463 Africanized honey bee. See also Pot-honey corbicular pollen pellets, 303 non-nectariferous pollen, 312 and Tetragonisca angustula, 311 African stingless bees Apis mellifera, 261 description, 261 host plants and nests, 263 survival, 265 taxonomic research, 265 taxonomy, 262–264 tropical wet forests, 261 vernacular names, 262 Afrotropical, 262 Altitudinal distribution geographic approach, 103 Guatemalan stingless bees, 100–102 and habitat tolerance, 104 American Type Culture Collection (ATCC) strains, 509 Anthophila, 4 Antibacterial activity antibiotic, pot-honey, 511 ATCC strains, 509 inhibitory vs. S. aureus, 509 Mueller-Hinton agar-well diffusion assay, 509 pot-honey, Costa Rica, 509, 510 Antibacterial properties, Guatemalan pot-honey. See also Pot-honey activity vs. pathogenic bacteria, 400 cure diseases, 400 stingless bee honey, 400, 401 Antibodies B lymphocytes, 519 high and low concentration levels, anti-IgE, 515 and immunoglobulins, 518 vs. self-proteins, autoimmune diseases, 520 Th2 cell and IL-4 secretion, 518 Anticancer. See also Flavonoids, 461–471, 476–478, 486–487, 497, 502 healing properties, bee products, 484 intrinsic/extrinsic factors, 483 molecular markers, 482 murine antitumor activity, 481 Antioxidant activity bioactivity, stingless bee products, 476 biological and therapeutic effects, 478 commercial market, 475 description, 475 Melipona honey from Guatemala, bioactivity, 477, 478 pot-honey vs. Apis mellifera honey, 476–477 RSA, 477 P. Vit et al. (eds.), Pot-Honey: A legacy of stingless bees, DOI 10.1007/978-1-4614-4960-7, © Springer Science+Business Media New York 2013 629 630 Apis mellifera Bolivia and Panama, 298 Brazil and Ecuador, 173 Brazilian honeys, 377 brood production and swarming, 312 comb honey honey representative flavonoid glycosides, 465, 467 HPLC/DAD phenolic profile, Diplotaxis tenuifolia, 465, 467 nectar and honey flavonoid aglycones, 465, 466 propolis-derived flavonoids and phenolic compounds, 464 secondary metabolites, 464 corrected/summed pollen concentrations, 308 description, 365 “divine elixir”, 365 Europe and Africa, 241 floral nectar and pollen sources and consistency, 309 Guatemala, Mexico and Venezuela, 173, 298 honey harvesting and storage pots, 365 logarithmic curve, 305 multiple colonies, 298 nose perceptions, 366 pollen concentrations and cutoff points, 305, 306 pollen consistency, 300 pollen trap and collecting pan, 298 tropical lowlands, 298, 300 Venezuela and French Guiana, 305 Venezuelan honeys, 298 wax and honey, 219 Apis mellifera scutellata, 298 Apoidea, 4 APP, 514 Argentinian stingless bees biology and ecology, 130–131 description, 125 distribution, 127–128 Geotrigona argentina, 126, 128 Meliponini, 128, 130–131 neotropical region, 126–127 Paratrigona, 126 Plebeia catamarcensis, 127–128 Plebeia molesta, 125 Scaptotrigona jujuyensis, 127, 128 stingless bee biodiversity, 128 Tetragonisca fiebrigi, 127, 130 Trigona spinipes, 127 Aromatic profile, Genus Melipona fitting and validation results, PLS-DA model, 420, 422 parameters, 421 physicochemical results, Colombia, 420, 421 Index PLS-DA results, 420, 421 Tetragona and A. mellifera honey, 423, 424 Ash and minerals, Colombian pot-honey. See also Pot-honey A. mellifera and apicultural products, 388 ash and mineral contents, 388, 389 botanical and geographical origin, 387 Australian stingless bees A. australis, 35–36 A. symei, 35–36 Austroplebeia, 42–44, 48–51 average annual rainfall charted, Australia, 38, 39 brood production, 41 castes and genders A. australis queen with workers, 38, 40 Austroplebeia drones, 40–41 imprisoned A. australis virgin queen, 38, 39 Trigona (s.l.) drones, 40 classification, genus/subgenus group, 38 description, 35 didgeridoo, 223 fighting swarms, 65–67 guard and forager behavior, 48, 49 indigenous people, 221 industry, 51–57 pests, 57–62 relationship aboriginal tribes, 36 anecdotal accounts, 36 axes, 37 beeswax and plant resins, 37 development, 37–38 hunting tools, 37 karbi and kootchar, 36 pellets, cerumen, 37 sugarbag honey, 37 seed dispersal, 62–65 Trigona (s.l.), 44–47 wax, 223 Austroplebeia behavior, Australian outback arid native range, A. australis, 48–49 brood, 50–51 colonies, 49 dead trees, 49–50 efficiency and thermoconformity, 51 thermoregulatory mechanisms, 50 brood structure, Australian stingless bee, 44 classification, 42, 43 colony population, 43–44 descriptions, 42 natural distribution, 42 nest architecture, 42–43 Index B Bacteria agricultural chemicals, 178 Bacillus meliponotrophicus, 177 description, 176–177 endosymbiosis, 178 lactic acid bacteria, 178 M. quadrifasciata, 177 spore-formation, 178 Trigona laeviceps and Trigona fuscobalteata, 178 types, Bacillus spp., 177 Barcodes complementary tool, 266 morphological identification, 262 B-cell receptor (BCR), 518 Bee bread amino acids, 160 haemolymph, 161 nutritional quality, 161 pollen stored, combs, 159 Bee honey and pollen resources, 297–302 Beekeeping and meliponiculture, 376 Beekeepings fermented and non-fermented pollen, 164 mead, 164–165 pollen substitutes, artificial feeding, 165–167 proliferation of microorganisms, 162–163 Bee-palynology acetolysis procedure, honey and pollen pellet, 301 Africanized Apis mellifera, tropical lowlands, 298, 300 analytical methods, 298 apiaries and meliponaries, 298 bee hives honey removal, 298 botanical species, 301 corbicular pollen loads, 298 description, 297–298 histogram, 302 identification and quantification, pollen, 302 Lycopodium spores, 298 pollen grain types identification, 301 pollen volumes, 297 ratio, Lycopodium spores, 301–302 resource counts and concentration, 302 Tetragonisca angustula, tropical lowlands, 298, 299 Bee pollen spectrum, 295–313 studies, 296 Bee products, 513–521 631 Bees anthropogenic disturbance, tropical, 275 honey and pollen, 285 hunting, 220 and man connection, 219 parental nest, 273 pollen analysis, 286 quality/quantity, products, 285 stingless bees, 272–278 tropical fragmentation, 270 vegetation, 286–287 wild and social, 270 Bees and microbes A. mellifera and A. mellifera scutellata, 175 Bacillus and Corynebacterium, 175 Candida bombicola/Starmerella bombicola, 176 ground-dwelling ants and termites, 174 insect species, 175 microbiota, 175 Mucor species, 176 Starmerella clade, 176, 177 Streptomycetes sp., 175–176 symbionts, 174–175 Beeswax bio-indicators, 224 “lost wax casting”, 220 Bioactivity, Tetragonula laeviceps antimicrobial activity agar-well diffusion method, 497, 498 diameter, inhibition zones, 499 0-100% honey, 497, 498 MEP and WEP, 499 vs. Micrococcus luteus and Pseudomonas aeruginosa, 498 partitioned extracts, honey, 498, 499 antiproliferative activity cell viability, breast cancer cell lines, 500 crude extract and purified fractions, 501 environmental factors, 500 Biogeography, Venezuelan Meliponini Atl and SEAm, 83 components, 83 NAm + SWAm, 84 NAm + SWAm + SEAm, 84 species, Central America, 83 Biological activity descriptors denomination, unifloral honeys, 371 gram positive S. aureus, 371 medicinal use, 370 melissopalinology, 371 pot-honey, 370 species, stingless bees, 370 UMF, 370 632 Biology, Meliponini colonies, 8 defense, 13 foraging, 9–10 nests, 10–13 reproduction, 9 Bolivia Apis mellifera, tropical lowlands, 298, 300 bee hives, Apis mellifera, 298 Mimosa, 306 Tetragonisca angustula, tropical lowlands, 298, 299 Botanical origin, propolis definition, 528 phytogeographic characteristics, 529 pollen analysis, 528 samples, Brazilian propolis, 528–529 Brazil Apis mellifera, tropical lowlands, 298, 300 bee hives, Apis mellifera, 298 Lycopodium, 306 Brazilian honey collection coastal geography, 242–243 “Warhaftig Historia”, 242 Byrsonima crassifolia “nance”, 401 C Cancer prevention and therapy adaptive response, 489 Aloe vera and honey, 482 antiproliferative action, 482 apoptotic ability, 482 carcinomas, 483 cell-markers differentiation, 483 commonality and variability, 481 definition, 482 ethnopharmacological use, 482 factors, health scientists, 482 and honey, 484–487 multidrug resistance, chemotherapy, 483–484 murine antitumor activity and antimeta-static effects, 481 official labeling, 483 pot-honey cytotoxic to human ovarian cancer cells, 487–488 proto-oncogenes, 483 Cell culture, 484 Central American stingless bees Cephalotrigona eburneiventer, 144 land bridge, 145 Mexico, 144 M. fasciata species, 145 M. colimana, 144 Index N. perilampoides, 144 Plebeia cora, 144 P. manantlensis, 144 Chaco Geotrigona argentina, 130 north-central Argentina, 127 Paranaense forest, 127 Checklist families/orders, insects, 88 stingless bees, 74 Chemical analysis, propolis botanical origin, 528–529 chemical composition and biological properties, 529–531 description, 525 ethanol extraction, 525–526 legislation standards, quality control of Apis mellifera, 377 natural products, 525 resins collection, 526–528 stingless bees, 531–535 Tetragonisca angustula bees, 525 Chemical and microbial composition, Bolivian pot-honey. See also Pot-honey A. mellifera honey standards, 412 antibacterial activity and probiotic action, 414 average values, antibacterial activity, 412, 413 description, 411 flexible cerumen pots, 412, 414 physicochemical parameters, 411 sanitary quality control, 412 sanitary standards, meliponines, 414 Chemical composition and biological properties, propolis antibacterial activities, 531 characteristic flavonoids and prenylated phenolics, 531 compound groups identification, 529 Ecuadoria, 530 EEP, 530 enzymic hydrolysis, bees, 529 honey bee A. mellifera, 530 prenyl caffeate, 530 species analysis, 530–531 Chemical composition, Melipona favosa physicochemical parameters, 366 pot-honey, Paraguaná Peninsula, 366–367 reducing sugars, 367 Citric acid description, 448 gluconic acid, 448 L-malic acid, 452 marker, Thymus sp., 449 Index Coffee agroecosystem, 341 Colombia, 337–345 Colombian stingless bee pot-honey. See also Pot-honey aromatic profile and physicochemical results, 420–424 classification model, 423–425 data analysis, 420 description, 418 electronic nose, 419–420 historical reports, 418 nutritional and therapeutic supplements, 417 PGS, 417 physical and chemical properties, 418 physicochemical analysis, 418–419 quality, bee products, 417 Colombian stingless bees beekeeping and meliponi culture, 383 defined, “angelita”, 383 “miel de pote”, 383 natural foods and health products, 383 physicochemical, 384–392 pre-Hispanic cultures, 383 technological and environmental issues, 384 Combs and pots, honey flavonoids. See also Pot-honey Apis mellifera comb honey, 464–467 stingless-bee pot honey, 467–471 Commercialization Maranhao, 552 pot-honey consumption, 545 presentation, pot-honey, 543, 544 Communication, 187–197 Competition coexisting colonies, 207 morphology, 203 pollinators, 201 soft-focus-lens imagination, 201 stingless bees shape, 202 Complement system (CS) humoral elements, 514 macrophages and neutrophils, 514 pathways, 514 Conservation, 100 Consumer acceptance and perception, 349 emotional level, 358 Huottuja consumers, 354, 355 Mexican and Australian, 354, 356 Spanish consumers, 354, 355 subjective impressions, 350 Cooperative marketing, 551–552 633 Corbicula bees, 100 Corbicular pellets Africanized honeybees, French Guiana, 303 M. pudica, 304 Corymbia torelliana abundant blooms, 62, 63 cadaghi tree, 62 cross section, 63, 64 seed collection, 65, 66 showy gum nuts, 62, 64 Trigona forager, 63, 65 Costa Rican pot-honey. See also Pot-honey A. mellifera, T. angustula and M. beecheii honey evaluation, antibacterial activity, 509–511 honey collection, 508–509 antibiotics, 511 description, 507–508 traditional medicinal use, 508 Costa Rican stingless bees classification, 119–120 description, 113 management, native, 115–119 Melipona beecheii, 113 microbiological tests, 113 stingless bee keeping, 114–115 tree species, 114, 120, 121 CS, 514 CTL, 518 Culture ancient Mayas, 255 apiculture, 250 Latin American people, 255 Mayan, 255–256 studies, stingless bees codice Madrid, 257 Levi-Strauss, Brazilian Amazon, 255 Mayan Codex, 256 Melipona beecheii, 255 “sciences of the concrete”, 254–255 tangible intellectual and economic value, 257 “Tristes tropiques”, 255 Cytokines autoimmune diseases, 520 and chemokins families, 514 production, propolis, 516 proinflammatory secretion, 515 royal jelly, 517 Treg cells, 518–519 Cytotoxic T lymphocyte (CTL), 518 634 D Decision external and internal information, 195 innate behavior, 193 social facilitation, 193 Defense, 13 De-forested habitat, 311 Diastase (a-amylase), 398, 399 Dioecious plants, 297 Distribution, 127–128 Diversity Apoidea, 100 meliponines, Tropical America, 100 stingless bees, Guatemala, 99–110 stingless bees, Tropical America, 100 Venezuelan stingless bees geographical records, species, 75–80, 82 nests, 75, 81 Double pulsed field gradient spin echoes (DPFGSE) sequence, 433 E EEP, 530, 532 Electronic nose analysis Airsense PEN 3 electronic nose, 419 broad-spectrum chemical sensors, 419 measurement procedure, 420 MOS sensors, 419 preliminary trials, 419 quality parameters, 419 Endemism, 83 Entomological origin, pot-honey. See also Pot-honey chemical structures, trans and cis abscisic acid, 439, 440 expanded aromatic region, “sucrose honey” extraction, 441 expanded region, 1H spectra, 439, 440 and geographical origins, 435–436 1 H NMR spectra, A. mellifera and M. fuscopilosa, 442 NMR-based metabolomic approach, 439 PCA loading plot, Brazilian honey samples, 439, 440 PC1 and PC2 scores, 436, 439 PLS-DA models, 436, 441 PLS-DA score plots, 436–438 stingless bee pot-honey samples, 434, 435 “sucrose honey”, M. quadrifasciata, 441 unequivocal structural identification, 439, 441 Venezuelan pot-honey samples, 436, 438 Index Ethanol extracts of propolis (EEP), 530, 532 Experience innate behavior, 193 and learning, social facilitation, 193 recruitment information, 195 unemployed foragers, 190 Exploitation of food sources, 201 External information description, 190–191 inside colony behavioral rituals/dances, Meliponine, 191–192 sounds, 192 trophallaxis, 192–193 outside colony pheromonal signaling, 194–195 social facilitation, 193 F Faunal list Argentinean, 126 Tetragonisca fiebrigi, 127 FCP, 351, 352, 357 Fermentation honey, 157–159 pollen, 159–162 Fighting swarms, Australian stingless bees colony strength, 67 management practices, 67 nest defense, 65–66 Trigona carbonaria, 66–67 Flavonoids advanced HPLC-MS methods, 463 antibacterial peptides, stingless bees, 502 antiproliferative effects, 487 bee–plant interaction, 461 bioactivity, propolis, 497 botanical and geographical origins, 462 cancer chemoprevention, 486 combs and pots, 464–471 extraction and analysis, 462–463 HMF and diastase activity, 461 honey maturation, 461 Pearson correlations, 478 physiological and ecological functions, 461 phytochemicals, 486 polyphenols, 486–487 polyphenol content, stingless bee honey, 477 in propolis, 476 scavenging, 486 subclasses, dietary, 486 Floral preferences, 105–106 Index Floral resources animal-mediated pollination, 105 palynological analysis, 106 plant families, 105–106 plant species visited, stingless bees, 106, 107 Folk medicine, 497 Food location communication Apis mellifera, 187 description, 187 efficiency and accuracy, 196 external and internal factors, 188 external sources, information, 190–195 honeybee language, 188 internal information, 195 nestmates, 187 recruitment, 189–190 social bees, 188–189 Food niches aggression and dominance, feeding site description, 207 eusocial bee, 208 foraging strategies, 208–209 solitarily foraging animals, 209 body colour, size and thermal tolerance flower morphology and bee tongues, 204 heat gain and heat loss, stingless bee, 205, 206 spatial niche differentiation, 205, 206 sugar concentration, 205, 207 temporal niche differentiation, 207, 208 tropical and subtropical bees, 204 competitor-community, 202 dominance relationships, 203 first come first serve activation signals, stingless bees, 211, 212 description, 209 food-patch-experienced foragers, 210 mass-flowering plants, 210 mass-recruiting species, 210–211 Melipona/Nannotrigona species, 210 morphological and physiological characteristics, 202 tongue length, predictor, 203–204 Food source partitioning mass flowering plants, 209–210 meliponine species, 202 Foraging strategies description, 201 eusocial corbiculate bees, 202 food niches, 202–212 inter-and intraspecific competition, pollinators, 201 pollen and nectar harvest, 202 tropical habitats, 202 635 Foraging vegetation, 286–287 Forest and semi-forested habitat, 296 Fragmentation, 269–278 Free-choice profile (FCP) attributes and score, 357 description, 351 entomological origin, 352 GPA, 357 French Guiana, stingless bees in Africanized honeybee, 94 beekeeper, Sinnamary, 94 collecting sites, map, 88–90 corbicular pollen data and honey data, 307 distribution records, 88 forest-savanna, 298 lowland forest, 88 Meliponini, 92 Mimosa pudica, 305 neotropical genera, 92–93 pollen corbicular pellets, 303 species, 90–92 Trigona amalthea, 94 Fungi Melipona flavolineata, 156 Melipona subnitida, 156 nutritional benefits/protection, 157 Partamona bees, 155–156 Scaptotrigona depilis, 156 Tetragona clavipes, 156 Tetragonula collina, 155 G GC-MS analysis experimental conditions, 532–533 GC 6890N from Agilent, 532 organic compounds, 533 Generalist bee, 297 Generalized procrustes analysis (GPA), 357 Geographical origin, 434–442 Geopropolis anemophilous and polleniferous pollen, 292 evaluation, nest entrance, 290, 292 structured elements, geopropolis sediments, 290, 291 Geotrigona acapulconis, 405 Gluconic acid bee glucose-oxidase, 448 nonaromatic organic acid, 448 GPA, 357 Guatemalan stingless bees antimicrobial activity, 401 apiaries and meliponaries, 404 deforestation rate, 109 636 Guatemalan stingless bees (cont.) description, 99 floral resources, 105–106 honey attributes, 404–406 meliponiculture, 106–109 meliponines, 99 nutritional characteristics, 400 physicochemical parameters, 398 promote programs, 109–110 sensory characteristics, 402 stingless bee colonies, 395 taxonomy and distribution, stingless bees, 100–104 H Habitat fragmentation. See also Human disturbance, 269–278 description, 270 mutualisms, pollination, 270 stingless bee, 272–278 tropical bee communities, 271 wild bee species, 272 Hans Staden’s report Brazilian honey collection, 242–243 description, 242 forward-thinking, bee description, 244 stingless bees, Brazil, 243 Hispanic America, 224 History early studies, stingless bees, 247–248 enlightenment, study of insects, 248–249 meliponas, twentieth century science behavior and ecology, stingless bees, 251 biogeographical barriers/geological compartments, 252 Brazilian stingless bee communication, 253–254 Cephalotrigona, 254 entomologists, stingless bee taxonomic and systematic studies, 252 “Father of bees”, 253 Melipona, 254 melissopalynology, 253 nesting colonies, 254 paleontologists, 253 Partamona, 254 “re-population”, forests, 254 Scaptotrigona, 254 taxonomy, 253 nineteenth century and melittology, 250–251 and transitions, 223–224 HMF, 288, 398, 399 Index Honey. See also pot-honey antimicrobial peptides antibiotic-resistant strains, bacteria, 501–502 Apis, 501 flavonoids, 502 gene-encoded antibiotics, 501 Apis cerana, 496, 501 Apis dorsata, 181, 484, 496, 501 Apis florea, 176, 496 Apis mellifera, 4, 55, 73, 153–155, 157–159, 173, 180, 286, 288, 298, 300–309, 311, 312, 317–325, 351–353, 355–357, 364–371, 375–380, 383–385, 387, 388, 390, 392, 396–400, 404, 405, 409, 412, 417–421, 423, 424, 442, 443, 447, 448, 451, 452, 462, 464–467, 470–472, 475–478, 485, 496, 501, 507–511, 541, 545, 547, 549 bee maggots, 220 beeswax, 220, 224 fermentation acidic and osmotic pressure, 157 alcoholic fermentation, yeasts, 158 glucose-oxidase, 158 nectar changes, 157 physicochemical characteristics, 159 storage, 158 HMF, 288 hunters, 220 innate immune response, 515–516 medicine and food source, 99 Melissopalynological studies, 288 monofloral honeys, 288 pollen analysis, 288 pollen percentages, 289 and propolis monosaccharides and disaccharides contents, 496 propolis analyses, 74–80 and Wax, 220–221 wild bees, 219 Honey and cancer antitumor activity, 484 botanical diversity, 485 flavonoids, anticancer components, 486–487 giant honey bee Apis dorsata, 484 kinds, bees, 485–486 markers, human health, 484 medicinal use, 485 methanol extract, 484 Index Honey and quality parameters, Apis mellifera Brazilian possess, 378 dehydration and transformation, floral nectar, 377 HMF, 378 invertase hydrolyzation, 378 legislation standards, 377 moisture and water content, 378 percentage of minerals, 378 physicochemical characteristics, 377–378 Honey attributes, stingless bee species Geotrigona acapulconis, 405 Melipona beecheii, 404–405 Scaptotrigona mexicana, 403, 405 Tetragonisca angustula, 403, 406 Honey bee products and innate immune response, 513–519 treatment, immune diseases, 520 Honey classification chemical analysis, 423 parameters, M. favosa and Melipona, 425 PLS-DA model, 425 pot-honey, 425 Honey collection, Indians, 242–244 Honey components and parameters, nonaromatic organic acids, 450 Honey composition, Brazilian Tetragonisca angustula, 376–377 Honey descriptive sensory evaluation, 357, 358 Honey removal from trunks, 244–245 Human disturbance characteristics, species, 269 conservation and importance, stingless bees, 276–278 description, 269 global environmental change, 270 habitat fragmentation and bee communities, 270–272 habitat fragmentation, stingless bee, 272–276 tropical bee communities, 270 Hydroxymethylfurfural (HMF), 288, 398, 399 I Immune disease, honey bee products allergic disease, 520 BTLA, 520 COX inhibition, 520 IL-17 secretion and TH17 cells, 520 pathogenesis, 520 Immune response and adaptive, 518–519 and innate, 513–517 637 Immunological diseases, 520 Immunological properties, bee products adaptive immune response, 518–519 description, 513 and innate immune response, 513–517 treatment, immune diseases, 520 Important bee plants Afrotropical meliponines, 315 colony fission and swarming, 315 description, 315 food, 316–325 stingless bee nests, 325–332 Indigenous people, 243 Industry, Australian stingless beekeeping beekeepers, 51 colony production brood mass, 52 budding, 52–53 hive, 51 OATH design, 51 queenright, 52 splitting OATH box, 52 honey hive beekeepers, 53 harvesting, 55 honey super, 53–55 “niche market”, 55 wax and resin supplies, 55 pollination, 55–57 Inflammation chronic, 520 and hypersensitivity mechanism, 520 indicators, anti-inflammatory activity, 515 Innate immune response APP, 514 CS, 514 honey, 515–516 interferons, 514–515 NKp receptor groups, 515 PAMP, 515 pathogenic microorganism, 513 physical and anatomic barriers, 514 propolis, 516–517 repeated organism substance encounters, 514 royal jelly, 517, 519 TLR4, 515 Internal information, 195 J Jaggery “panela” descriptors, Guatemalan stingless bees, 401 sensory characteristics, 401, 402 638 K kab “Ah mucen kab”, 229 ancient texts, 239 Melipona beecheii, Maya language, 229 sacred food, 231 “xunan kab”, 229 Kaur-16-ene (8.beta.13.beta), 535 3-KETO-URS-12-ENE, 535 L Lycopodium internal standard, 297–298 Lycopodium standard, 297–298 Lymphocytes BCR and TCR, 518 proliferation assay, 519 royal jelly treatment, 517 M Malic acid, 448, 451, 453 Marketing, Meliponine honey. See also Pot-honey Africa, Asia and Australia, 545 America, 544–545 commercial presentation, 543, 544 cost-value-price, 549–550 cultural aspects, 547–548 initiatives, 543 legislation, 550 low production and seasonality, 548 lucrative external markets, 543 packaging, 550 production and consumption, 545–547 quality, 548–549 vending locations, 550 Maya civilization, 220 Maya medicine colonial chronicles, 238 Pre-Hispanic Indians, 230 “Ritual de los Bacabes”, 231 and vegetation, 232 Mayan language M. beecheii, 404 T. angustula, 406 Mead honey-pollen jelly, 168 stingless bee, 164–165 Medicinal bee host plant usage, 263 pollinators and, 262 vegetables and, 263 Index Melipona beecheii honey. See also Pot-honey Apis mellifera, 230 Bacillus, 230 “cold” diseases, 233–235 description, 229, 404 fevers and “hot” diseases, 235–236 floral resources, 403, 405 honey and beeswax, 230 maladies, digestive tract, 236–237 Mayan ideas, disease, 231 medicinal purposes, 232 natural enemies, 230 physicochemical components, 404 prescriptions preparation, 232–233 “Ritual de los Bacabes”, 230 sensory organs, 237–238 syndromes, cultural origin, 236 Melipona bees. See also Melipona beecheii honey, 229–238, 403–405 cultural studies, stingless bees, 254–257 description, 247 early studies, stingless bees, 247–248 enlightenment and study, insects, 248–249 nineteenth century and melittology, 250–251 twentieth century science, 251–254 Melipona favosa pot-honey. See also Pot-honey A. mellifera, 365–366 applications, 364–365 biological activity descriptors, 370–371 composition, 366–367 consumers and stingless beekeepers, 371 database, Venezuela, 368–369 defined, 363–364 entrance nest, columnar cactus “cardón”, 363, 364 “meliponicultors”, 364 plains and coastal regions, 371 sensory attributes, 367–368 suggested standards, 369–370 Meliponiculture community-level development, 130 different regional names and beekeepers, 108 ethnic groups and rural population, 113 honey and wax, 114 honey-harvesting, 108–109 honey production, 542 Ladinos/Mestizos, 108 less development, 545 Mayan region and Mesoamerica, 107 medicinal properties, 109 Index Melipona compressipes bee, 552 quality, local products, 554 rural communities, 130 S. mexicana and S. pectoralis, 109 Meliponines biology and ecology, Argentine, 130–131 Indoaustralian region, 6 native and crop vegetation, 99 Tropical America, 107 Meliponini acceptance scores, 354 Alphaneura [= Trigona], 7 Amalthea [= Trigona], 7 Andrena, 3 “angelita” and Tetragonisca angustula, 355 Anthophora, 175 Aparatrigona impunctata French Guiana, Venezuela, 76, 90 Aparatrigona, 7, 20, 92 Aphaneura [= Trigona], 7 Apotrigona [= Meliponula (Meliplebeia)], 8 Austroplebeia Australia, 8, 42 Austroplebeia australis Australia, 43 Austroplebeia cassiae Australia, 43 Austroplebeia cincta Australia, 43 Austroplebeia cockerelli Australia, 43 Austroplebeia essingtoni Australia, 43 Austroplebeia ornata Australia, 43 Austroplebeia percincta Australia, 42, 43 Austroplebeia symei Australia, 43 Austroplebeia websteri Australia, 43 average Australian acceptance, pot-honey, 354, 356 average Mexican acceptance, pot-honey, 354, 356 biology, 8–13 Camargoia, 20 Camargoia [= Trigona (Tetragona)], 7 Camargoia camargoi French Guiana, 90, 92 Celetrigona, 20, 92 Celetrigona [= Trigonisca], 7 Celetrigona manauara French Guiana, 90 Cephalotrigona, 7, 20, 92, 137 Cephalotrigona capitata Argentina, French Guiana, Venezuela, 76, 90, 274 Cephalotrigona eburneiventer Mexico, 140 Cephalotrigona oaxacana Mexico, 140 Cephalotrigona zexmeniae Costa Rica, Guatemala, Mexico, 101, 116, 140 639 classification genus-group taxa, 7–8 Indoaustralian/Australasian, 8 Sub-Saharan/Afrotropical, 7 Trigona, 4–5 tropical zone, 5–6 Cleptotrigona, 8 Cleptotrigona cubiceps Africa, 264 corbicula, 4, 5 Cretotrigona {extinct}, 14 Cretotrigona prisca {extinct}USA, 14, 19, 145, 252, 363 Dactylurina, 8 Dactylurina schmidti Africa, 264 Dactylurina staudingeri Africa, 264 description, 128 Diadasina distincta, 176 Dioxys, 3 Dolichotrigona, 20, 92 Dolichotrigona (= Trigonisca), 7 Dolichotrigona longitarsis French Guiana, 90 Dolichotrigona schulthessi Costa Rica, Guatemala, 101, 116 Duckeola, 20, 92, 139 Duckeola, Trigona (Duckeola), 7 Duckeola ghilianii French Guiana, 90 Duckeola pavani French Guiana, Venezuela, 90 Eomelipona (= Melipona), 7, 93 eusocial apine bees, 135 extractive exploitation, 130 French Guiana, 87–94 Friesella, 20, 92 Friesella [= Plebeia (Plebeia)], 75 Friesella schrottkyi Brazil, 75 Frieseomelitta Colombia, Venezuela, 20, 92 Frieseomelitta, Trigona (Frieseomelitta), 7 Frieseomelitta flavicornis French Guiana, 90 Frieseomelitta nigra Costa Rica, Guatemala, Mexico, 101, 116, 140 Frieseomelitta paupera Costa Rica, Venezuela, 76, 116 Frieseomelitta portoi French Guiana, 90 Frieseomelitta silvestrii, 534 Frieseomelitta varia Argentina, Brazil, Venezuela, 274 Geniotrigona, Heterotrigona (Geniotrigona), 8 Geotrigona Moure, 1943 Venezuela, 7, 20, 92 640 Meliponini (cont.) Geotrigona acapulconis Guatemala, Mexico, 101, 140, 395 Geotrigona argentina, Argentina, 126 Geotrigona chiriquiensis Costa Rica, 116 Geotrigona inusitata [= Geotrigona mombuca (Smith, 1863)], 274 Geotrigona leucogastra, 571 Geotrigona lutzi Costa Rica, 100, 116 Geotrigona mombuca Brazil, 211, 325 Geotrigona subgrisea, 571 Geotrigona subnigra Venezuela, 76, 94 Geotrigona subterranea, 571 Geotrigona terricola, 100 governmental and nongovernmental organizations, 130 Heterotrigona, 8 Heterotrigona, Heterotrigona (Heterotrigona), 8 Heterotrigona, Trigona (Heterotrigona), 35, 36, 38, 41, 45–48, 51, 56, 60, 61, 67 Heterotrigona (Sundatrigona) moorei Indonesia, Thailand, 8, 11 Homotrigona, 8 and honeybees, 223–224, 285, 486 hunters and stingless bee keepers, 354 Huottuja consumers, 354, 355 hymenoptera, 3–4 Hypotrigona, 8 Hypotrigona araujoi Africa, 264 Hypotrigona gribodoi Africa, 264 Hypotrigona penna Africa, 264 Hypotrigona ruspolii Africa, 264 Kelneriapis eocenica, 14 Lepidotrigona, 8 Lestrimelitta, 7, 20, 93 Lestrimelitta chacoana Argentina, 126 Lestrimelitta, 1999 Mexico, 140 Lestrimelitta danuncia Costa Rica, 116 Lestrimelitta glaberrima French Guiana, Venezuela, 76, 90 Lestrimelitta guyanensis French Guiana, 90 Lestrimelitta limao Brazil, 292 Lestrimelitta maracaia Venezuela, 76 Lestrimelitta monodonta French Guiana, 90 Lestrimelitta mourei Costa Rica, 116 Lestrimelitta niitkib Guatemala, Mexico, 101, 140 Lestrimelitta rufipes Argentina, 126 Lestrimelitta sulina Argentina, 126 Leurotrigona, 20, 93 Leurotrigona (= Trigonisca), 7 Index Leurotrigona muelleri Argentina, Brazil, 126 Leurotrigona pusilla French Guiana, 90 Liotrigona bottegoi Africa, 264 Liotrigona, 8 Liotrigonopsis rozeni, 14 Lisotrigona, 8 Lophotrigona, 8 Megachile Latreille, 177 Megachile rotundata, 175, 176 Melikerria (= Melipona), 7, 93 Melipona Brazil, Colombia, 7, 20, 93 Melipona apiformis, 82 Melipona asilvai Brazil, 368, 542, 543, 549 Melipona baeri Argentina, 126 Melipona beecheii Costa Rica, Guatemala, Mexico, 101, 116 Melipona beecheii honey, 223 Melipona belizeae, 147 Melipona bicolor Brazil, 274 Melipona bicolor schencki Argentina, 126 Melipona brachychaeta Bolivia, 469 Melipona capixaba, 179 Melipona carrikeri Costa Rica, 116 Melipona colimana Mexico, 140 Melipona compressipes Brazil, Colombia, Venezuela, 76, 90 Melipona compressipes manaosensis (= Melipona interrupta) Brazil, 289 Melipona concinnula, 76, 82 Melipona costaricensis Costa Rica, 116 Melipona cramptoni, 77, 82 Melipona crinita Bolivia, 410 Melipona eburnea Colombia, 370, 385–387, 391, 420–422 Melipona fasciata Mexico, Panama, 140 Melipona fasciata cramptoni duidae [= Melipona (Michmelia) cramptoni], 77, 82 Melipona fasciata guerreroensis [= Melipona (Michmelia) fasciata], 357, 435 Melipona fasciculata, 355 Melipona fasciculata Brazil, 158, 165, 355, 380, 435, 439, 440, 471, 488, 543, 548, 549, 553 Melipona favosa Colombia, Venezuela, 77, 90, 363 Melipona flavolineata Brazil, 56, 543, 549 Melipona fuliginosa Argentina, Costa Rica, 90, 116 Melipona fulva, 1836, 77, 90 Melipona fuscipes (= Melipona fasciata), 82 Index Melipona fuscopilosa Venezuela, 77 Melipona grandis Guérin, 1844 Bolivia, 370, 410–412, 414, 435, 469, 526, 531, 535 Melipona illota, 370 Melipona illustris, 76 Melipona indecisa, 77, 82 Melipona lateralis, 77, 90 Melipona lateralis kangarumensis [= Melipona (Michmelia) lateralis], 77 Melipona lupitae Mexico, 140 Melipona mandacaia Brazil, 288, 368, 412, 543, 549 Melipona marginata, 274 Melipona melanopleura [= Melipona (Michmelia) costaricensis], 544 Melipona mondury Brazil, 549, 553 Melipona obscurior Argentina, 126, 129 Melipona ogilviei, 76, 90 Melipona orbignyi, Melipona [sic = Melipona orbignyi] Argentina, 126, 129, 131 Melipona panamica Costa Rica, 116 Melipona paraensis, 77, 90 Melipona quadrifasciata Argentina, Brazil, 274 Melipona quadrifasciata anthidioides Brazil, 412, 530, 531, 543 Melipona quadrifasciata quadrifasciata Brazil, 543 Melipona quinquefasciata Argentina, Brazil, 126, 174, 177, 181, 182, 326 Melipona rufiventris Brazil, 177, 180, 471, 476, 488, 542, 543, 548 Melipona rufiventris paraensis, 288 Melipona scutellaris Brazil, 274 Melipona seminigra Brazil, 161, 162, 192, 288, 289 Melipona seminigra merrillae, 288 Melipona solani Guatemala, Mexico, 101, 140, 396 Melipona subnitida Brazil, 156, 179, 204, 331, 435, 439, 440, 471, 482, 487 Melipona torrida Costa Rica, 116 Melipona titania, 126 Melipona trinitatis, 77 Melipona variegatipes, 145 Melipona yucatanica Costa Rica, Guatemala, Mexico, 101, 116, 140 Melipona (Melipona) Melipona, 7, 20, 75, 93 Melipona (Eomelipona) bradleyi French Guiana, 90 Melipona (Eomelipona) concinnula Venezuela, 76 641 Melipona (Eomelipona) Eomelipona, 7, 93 Melipona (Eomelipona) illustris Venezuela, 76 Melipona (Eomelipona) ogilviei French Guiana, Venezuela, 76, 90 Melipona (Eomelipona) puncticollis French Guiana, 90 Melipona (Melikerria) compressipes French Guiana, Venezuela, 76, 90, 274 Melipona (Melikerria) grandis, 573 Melipona (Melikerria) interrupta French Guiana, Venezuela, 76, 90 Melipona (Melikerria) Melikerria , 7, 93 Melipona (Melipona) favosa French Guiana, Venezuela, 77, 90, 363 Melipona (Michmelia) apiformis Venezuela, 77, 82 Melipona (Michmelia) captiosa French Guiana, 90 Melipona (Michmelia) cramptoni Venezuela, 77, 82 Melipona (Michmelia) crinita Venezuela, 77, 410 Melipona (Michmelia) eburnea, 370, 385–387, 391, 418, 420–422 Melipona (Michmelia) fasciata, 82, 140 Melipona (Michmelia) fuliginosa French Guiana, 90, 116 Melipona (Michmelia) fulva French Guiana, Venezuela, 77, 90 Melipona (Michmelia) indecisa Venezuela, 77, 82 Melipona (Michmelia) lateralis French Guiana, Venezuela, 77, 90 Melipona (Michmelia) melanoventer French Guiana, 90 Melipona (Michmelia) Michmelia Venezuela, 7, 93 Melipona (Michmelia) paraensis French Guiana, Venezuela, 77, 90 Melipona (Michmelia) trinitatis Venezuela, 77 Meliponula, 8 Meliponula bocandei Uganda, 264 Meliponula ferruginea, 264 Meliponula nebulata Uganda, 264 Meliponula (Axestotrigona) Axestotrigona, 8 Meliponula (Axestotrigona) cameroonensis Africa, 264 Meliponula (Axestotrigona) eburnensis, 263 Meliponula (Axestotrigona) ferruginea Africa, 264 642 Meliponini (cont.) Meliponula (Axestotrigona) richardsi, 263 Meliponula (Axestotrigona) sawadogoi, 263 Meliponula (Meliplebeia) beccarii Africa, 264 Meliponula (Meliplebeia) griswoldorum Africa, 264 Meliponula (Meliplebeia) lendliana Africa, 264 Meliponula (Meliplebeia) Meliplebeia, 8 Meliponula (Meliplebeia) nebulata (Smith, 1854) Africa, 264 Meliponula (Meliplebeia) ogouensis Africa, 264 Meliponula (Meliplebeia) roubiki Africa, 264 Meliponula (Meliponula) bocandei Africa, 264 Meliponula (Meliponula) Meliponula , 8 Meliwillea Roubik, 7, 20, 93, 116 Meliwillea bivea Costa Rica, 116 Micheneria Kerr, Pisani & Aily, 1967 [= Melipona (Michmelia)], 7, 252 Michmelia (= Melipona), 7, 93 monofloral honeys, 288 Mourella, 20, 93 Mourella [= Plebeia (Plebeia)], 7 Mourella caerulea Argentina, 126 Nannotrigona Colombia, Venezuela, 7, 20, 78, 93 Nannotrigona chapadana, 78 Nannotrigona melanocera Venezuela, 77 Nannotrigona mellaria Costa Rica, 116 Nannotrigona perilampoides Costa Rica, Guatemala, Mexico, 77, 101, 116, 140, 396 Nannotrigona punctata French Guiana, 90 Nannotrigona schultzei French Guiana, Venezuela, 78, 90 Nannotrigona testaceicornis Argentina, Brazil, Colombia, 274 Nannotrigona tristella Venezuela, 78, 82 natural pot pollen, 166 Neotropical, 136 Nogueirapis minor French Guiana, 91 Nogueirapis mirandula Costa Rica, 116 Nogueirapis Moure, 1953, 7, 20, 93 Nogueirapis silacea, 139 Odontotrigona, 8 Odontotrigona Odontotrigona (Odontotrigona), 8 Oxytrigona, 7, 20, 93 Oxytrigona daemoniaca Costa Rica, 116 Index Oxytrigona mediorufa Guatemala, Mexico, 101, 140 Oxytrigona mellicolor Costa Rica, Venezuela, 78, 116 Oxytrigona obscura French Guiana, 91 Oxytrigona tataira Argentina, 126 Papuatrigona Parapartamona, 8 Parapartamona Partamona (Parapartamona), 7 Paratetrapedia, 4 Paratrigona Colombia, 7, 20, 78 Paratrigona anduzei Venezuela, 78, 81 Paratrigona femoralis French Guiana, 91 Paratrigona glabella Argentina, 126 Paratrigona guatemalensis Guatemala, Mexico, 101, 140 Paratrigona lineata, 575 Paratrigona lophocoryphe Costa Rica, 116 Paratrigona opaca Costa Rica, Mexico, 100, 140 Paratrigona ornaticeps Costa Rica, 116 Paratrigona pannosa French Guiana, Venezuela, 78, 91 Paratrigona peltata Costa Rica, 11 Paratrigona permixta Venezuela, 78, 82 Paratrigona subnuda, 334 Paratrigonoides, 7, 20, 93 Pariotrigona, 8 Partamona Brazil, Colombia, 7, 20 Partamona, Partamona (Partamona), 7 Partamona ailyae Venezuela, 78 Partamona auripennis French Guiana, Venezuela, 78, 91 Partamona batesi, 26, 27 Partamona bilineata Guatemala, Mexico, 101, 140 Partamona chapadicola, 27 Partamona cupira, 274 Partamona epiphytophila Venezuela, 78 Partamona ferreirai French Guiana, Venezuela, 78, 91 Partamona grandipennis Costa Rica, 117 Partamona gregaria, 27 Partamona helleri Argentina, Brazil, 126 Partamona mourei French Guiana, 91 Partamona musarum Costa Rica, 117 Partamona nigrior Venezuela, 78 Partamona orizabaensis Costa Rica, Guatemala, Mexico, 101, 117, 140 Partamona pearsoni French Guiana, Venezuela, 78, 91 Partamona peckolti Colombia, Venezuela, 78 Index Partamona seridoensis, 482 Partamona testacea French Guiana, 91 Partamona vicina French Guiana, Venezuela, 78, 91 Partamona vitae Venezuela, 78 Patera (= Partamona), 7 phylogeny, 14–15 Platytrigona, 8 Plebeia Argentina, Brazil, Colombia, Guatemala, Venezuela, 7, 20 Plebeia, Plebeia (Plebeia), 7 Plebeia (Scaura) latitarsis, 91, 102, 274 Plebeia (Scaura) timida, 9, 22 Plebeia catamarcensis Argentina, 126 Plebeia cora Mexico, 140 Plebeia droryana Argentina, Bolivia, Brazil, 274 Plebeia emerina, 576 Plebeia franki Costa Rica, 117 Plebeia fraterna Venezuela, 78 Plebeia frontalis Costa Rica, Guatemala, Mexico, 101, 117, 140 Plebeia fulvopilosa Guatemala, Mexico, 101, 141 Plebeia goeldiana Venezuela, 78, 82 Plebeia jatiformis Costa Rica, Guatemala, Mexico, 101, 117, 141 Plebeia kerri Bolivia, 410 Plebeia lucii Brazil, 208 Plebeia llorentei Costa Rica, Guatemala, Mexico, 101, 117, 141 Plebeia manantlensis Mexico, 141 Plebeia melanica Guatemala, Mexico, 101, 141 Plebeia mexica Mexico, 141 Plebeia minima Costa Rica, French Guiana, 91, 117 Plebeia molesta Argentina, 125 Plebeia mosquito French Guiana, 91 Plebeia moureana Guatemala, Mexico, 101, 141 Plebeia nigriceps Argentina, 126 Plebeia parkeri Guatemala, Mexico, 101, 141 Plebeia poecilochroa, 274 Plebeia pulchra Costa Rica, Guatemala, Mexico, 101, 107, 141 Plebeia remota, 576 Plebeia saiqui, 290 Plebeia tica Costa Rica, 117 Plebeia wittmanni Argentina, 126 Plebeiella [= Meliponula (Meliplebeia)], 577 Plebeina, 8 643 Plebeina hildebrandti Africa, 264 pot-honey and pot-pollen, 287 Proplebeia {extinct}Dominican Republic, Mexico, 20 Proplebeia dominicana {extinct} Dominican Republic, 154, 252 Ptilothrix plumata, 176 Ptilotrigona lurida Brazil, French Guiana, Venezuela, 79, 91 Ptilotrigona, 20, 93 Ptilotrigona [= Trigona (Tetragona)], 7 Ptilotrigona occidentalis Costa Rica, 117 Ptilotrigona pereneae, 22 Sakagamilla Moure, 1989 (= Scaptotrigona), 7 Scaptotrigona Argentina, Brazil, Colombia, Paraguay, Venezuela, 7, 20 Scaptotrigona bipunctata, 577 Scaptotrigona depilis Argentina, Bolivia, Brazil, Venezuela, 91, 410 Scaptotrigona fulvicutis French Guiana, 91 Scaptotrigona hellwegeri Mexico, 141 Scaptotrigona jujuyensis Argentina, 126–131, 515, 516 Scaptotrigona limae Colombia, 385, 386, 389 Scaptotrigona luteipennis Costa Rica, 117 Scaptotrigona mexicana Costa Rica, Guatemala, Mexico, 102, 117, 141, 395 Scaptotrigona ochrotricha Venezuela, 79, 82 Scaptotrigona panamensis Costa Rica, 117 Scaptotrigona pectoralis Costa Rica, Guatemala, Mexico, 102, 117, 141, 395 Scaptotrigona polysticta Moure, 1950 Bolivia, Brazil, 75, 269 Scaptotrigona postica Brazil, 274 Scaptotrigona subobscuripennis Costa Rica, 117 Scaptotrigona tubiba Brazil, 91 Scaptotrigona wheeleri Costa Rica, 100, 117 Scaptotrigona xanthotricha Brazil, 410, 469 Scaura argyrea Costa Rica, Guatemala, Mexico, 102, 117, 141 Scaura latitarsis French Guiana, 91, 103, 274 Scaura longula French Guiana, 91 Scaura Venezuela, 7, 20, 93 Scaura, Plebeia (Scaura), 9, 11, 79 Scaura tenuis French Guiana, 91 Scaura timida, 9, 22 Schwarziana, 7, 20, 93 644 Meliponini (cont.) Schwarziana, Plebeia (Schwarziana), 7 Schwarziana quadripunctata Argentina, 126, 326 Schwarzula coccidophila, 2002, 23, 24 Schwarzula, 7, 20, 93 Schwarzula [= Plebeia (Scaura)], 7, 20, 93 S. mexicana, 356 Spanish consumers, 354 species, northern Argentina, 128–130 species, stingless bees, 357 sphecoidea, 4 subterraneous and arboreal habits, 130 Sundatrigona Heterotrigona (Sundatrigona), 8 Tetragona Colombia, 7, 20, 93, 137 Tetragona Trigona (Tetragona), 7, 20, 93, 137 Tetragona beebei French Guiana, 91 Tetragona clavipes Argentina, Brazil, French Guiana, Venezuela, 79, 91 Tetragona dorsalis French Guiana, 91, 103 Tetragona handlirschii French Guiana, 91 Tetragona kaieteurensis French Guiana, 91 Tetragona mayarum [= Tetragona ziegleri] Guatemala, Mexico, 102, 141 Tetragona perangulata Costa Rica, 117 Tetragona savannensis [= Frieseomelitta flavicornis], 90 Tetragona ziegleri Costa Rica, Venezuela, 79, 117 Tetragonilla, Tetragonula (Tetragonilla), 8 Tetragonisca Argentina, Venezuela, 7, 20, 93 Tetragonisca, Trigona (Tetragonisca), 7, 20, 93 Tetragonisca angustula Argentina, Bolivia, Brazil, Colombia, Costa Rica, Guatemala, French Guiana, Mexico, Panama, Peru, 91, 102, 117, 141, 298, 375, 395 Tetragonisca angustula angustula, 79 Tetragonisca buchwaldi Costa Rica, 117 Tetragonisca fiebrigi Argentina, Bolivia, 410, 469, 478 Tetragonula, 8 Tetragonula, Tetragonula (Tetragonula), 8 Tetragonula biroi Philippines, 526, 531, 533, 535 Tetragonula carbonaria Australia, 45 Tetragonula collina, 155, 179 Tetragonula fuscobalteata, 11 Tetragonula laeviceps, 155 Tetragonula pagdeni, 181 Tetrigona, 8 Index Trichotrigona, 7, 20, 93, 139 Trichotrigona extranea Australia, 94 Trigona Jurine, 1807 Brazil, Malaysia, Venezuela, 7, 20, 93, 136, 137 Trigona Jurine, 1807, Trigona (Trigona), 7, 20, 93, 136, 137 Trigona acapulconis (= Geotrigona acapulconis), 101, 140, 395 Trigona alfkeni, 75 Trigona amalthea Venezuela, 79, 94, 103 Trigona amazonensis Venezuela, 80 Trigona australis, 43 Trigona branneri French Guiana, Venezuela, 80, 91 Trigona carbonaria Australia, 45 Trigona chanchamayoensis Bolivia, 410 Trigona cilipes Costa Rica, French Guiana, Venezuela, 80, 91, 117 Trigona clypearis Australia, 45 Trigona collina Malaysia, 155, 179 Trigona corvina Costa Rica, Guatemala, Mexico, 102, 118, 141 Trigona crassipes French Guiana, 91 Trigona cupira cupira [misidentification, = Partamona orizabaensis], 274 Trigona dallatorreana Brazil, Venezuela, 80 Trigona davenporti Australia, 45 Trigona ferricauda Costa Rica, 118 Trigona fulviventris Costa Rica, Guatemala, Mexico, Venezuela, 80, 118 Trigona fuscipennis Costa Rica, Guatemala, Mexico, Venezuela, 80, 91, 102, 118, 141 Trigona fuscobalteata Thailand, 11, 155, 178 Trigona guianae French Guiana, Venezuela, 80, 91 Trigona hockingsi Australia, 45 Trigona hyalinata Brazil, 75 Trigona hypogea Brazil, Panama, 274 Trigona laeviceps Thailand, 178, 498 Trigona mazucatoi (= Trigona cilipes), 91 Trigona melanocephala Malaysia, 179 Trigona melina Malaysia, 179 Trigona mellipes Australia, 45 Trigona muzoensis, 118 Trigona necrophaga Costa Rica, 118 Trigona nigerrima Costa Rica, Guatemala, Mexico, 102, 141 Trigona nigra, 579 Trigona pallens French Guiana, Venezuela, 80, 92 Trigona permodica French Guiana, 92 Trigona prisca, 14, 579 Index Trigona recursa Brasil, 92, 274 Trigona sapiens Australia, 45 Trigona sesquipedalis French Guiana, 92 Trigona silvestriana Costa Rica, Guatemala, Mexico, 75, 102, 118, 141 Trigona spinipes Argentina, Brazil, 75 Trigona trinidadensis (= Trigona amalthea), 75 Trigona truculenta Venezuela, 80 Trigona venezuelana Venezuela, 80, 82 Trigona williana French Guiana, Venezuela, 80, 92 Trigona (Frieseomelitta) angustula angustula, 79 Trigona (Frieseomelitta) nigra paupera, 76 Trigona (Geotrigona) Geotrigona, 7, 20, 92 Trigona (Heterotrigona) carbonaria Australia, 45 Trigona (Heterotrigona) clypearis Australia, 45 Trigona (Heterotrigona) davenporti Australia, 45 Trigona (Heterotrigona) hockingsi Australia, 45 Trigona (Heterotrigona) mellipes Australia, 45 Trigona (Heterotrigona) sapiens Australia, 45 Trigona (Tetragonisca) angustula, 91, 102, 117, 141, 375, 395 Trigona (Tetragonisca) angustula angustula, 91, 117, 141, 298 Trigona (Tetragonula) laeviceps, 155 Trigona (Trigona) corvina, 102, 118, 141 Trigona (Trigona) hypogea Silvestri, 1902, 274 Trigonella [= Heterotrigona (Sundatrigona)], 8 Trigonisca Argentina, Venezuela, 7, 93 Trigonisca atomaria Costa Rica, 118 Trigonisca azteca Mexico, 141 Trigonisca discolor Costa Rica, 118 Trigonisca dobzhanskyi French Guiana, 92 Trigonisca maya Guatemala, Mexico, 102 Trigonisca mixteca Mexico, 141 Trigonisca pipioli Costa Rica, Guatemala, Mexico, 102, 118, 141 Trigonisca schulthessi Mexico, 101, 116, 141 tropical bee communities, 271 wings, Melipona fasciata and Euglossa cordata, 4, 6 Melissopalynology melittopalynology and, 295 645 pollen and nectar source, 312 pollen types, 403 Melittopalynology bee-pollen studies, 296 and melissopalynology, 295 MEP, 499, 500 Metabolomics chemometric approach, 431 definition, 430 endogenous and exogenous chemical entities, 430–431 1 H NMR spectra, 431 NMR-based, pot-honey (see also Pot-honey) chloroform solvents, 434 description, 432 DPFGSE sequence, 433 fingerprint, chemometric analysis, 432 1 H NMR, 432 representative 1H NMR spectrum, M. fuscopilosa, 433, 434 softwares, 434 structural information, 432 work flow, 433 PCA and PLS-DA, 431–432 quantitative metabolomics approach, 431 score plots, 431 Metal oxide semiconductors (MOS), 419 Methanol extract of propolis (MEP), 499, 500 Mexican stingless bees Africanized Apis mellifera, tropical lowlands, 298, 300 bee diversity per country, 136, 137 cryptic species, 136 distribution division, 139 Group I, 139, 143 Group II, 143 Group III, 143–144 diversity description, 136, 139 distribution and uses, 139–142 economic and cultural importance, 136, 138 eusocial apine bees, 135 generic and subgeneric classification, 136 honey removal, 298 Mimosa, 306 native and cultivated tropical plants, 135 origin, 144–145 stingless bee genera, 136, 137 traditional uses and indigenous knowledge, 145–147 Microbes, honey, 414 646 Microorganisms Apis mellifera, 153–154 applications, 162–167 bacteria, 154–155, 176–178 bees and microbes, 174–176 description, 153 ethnomedicinal properties, stingless bee, 173 fermentation and biochemical processes, 157–162 fungi, 155–157 honey and pollen, Melipona quinquefasciata, 173, 174 molds, 178–179 mutualistic interaction, yeasts and bee, 181–182 stored pot pollen, 174 yeasts, 155, 180–181 Minerals, 387–389 Molds corbicular pollen, 179 Curvularia, 179 mycological studies, 179 Stemphylium, 178 sympatric species, 179 Morphological traits competitor-community, 202 flower morphology and bee tongues, 204 MOS, 419 Mutualistic interaction, 181–182 N Native stingless bees “Ah mucen kab”, 229 existence and importance, 239 Nectar plants Apis mellifera honey, flavonoid glycosides, 465, 467 flavonoids, 461 honey-making, 461 hydrolytic activity, bee saliva, 465, 466 insect pollinators, 316 life form and, 316–324 and pollen derived flavonoid aglycones, 465 pollen loads and palynological analysis, 316 and pollen sources, 316, 325 rhamnosyl-glucosides, 465 transformation, nectar flavonoids, 472 Neotropical region, Meliponini Amazonian forest, 31, 33 area and biological cladograms, Geotrigona, 30 Index areas of endemism and biogeographical components, Partamona, 30, 31 collecting meat at dead lizard, Trigona hypogea, 21 Cretotrigona prisca, 19 diversification, 28 evolution, 20–21 genera and number, 19–20 hierarchy, 30 Kayapó Indians, 27–29 mutualism, 22–23 nest, 21–29 pantropical distribution, 19 phylogenetic systematics and vicariance biogeography, 29–30 pollen covered with yeast, Ptilotrigona lurida, 22, 24 principal biogeographic elements, 30, 32 resins, 22 Schwarzula coccidophila, 23–24 sequence of events, separation and vicariance, 30–32 storage pots, 21 Nest aggregation, Partamona batesi, 26 architecture, 25–26 entrances, Partamona, 26, 27 Leurotrigona pusilla, 27, 29 Meliponini Arboreal termites, 11 brood cells, 12, 13 cells, 12 cerumen, 10 Dactylurina and Melipona, 12 hollows and cavities, 11 hollow tree trunk, 11–12 involucrum and batumen, 13 mixtures, materials, 10–11 sites, 11 size and shape, 13 structure, 10 Partamona vicina, 26–28 Ptilotrigona lurida, 22, 23 species, 75, 81 Trichotrigona extranea, 24–25 Trigona hypogea, necrophagous bee, 21, 22 Venezuelan stingless bees, 74 Nest cavity meliponines, 327 size, tree hole, 327 Nest sites height partitioning, trees, 327, 332 trees Afrotropical, Indo-Malaya and Neotropical regions, 327–331 Index canopy trees, 327 living trees/dead wood, 327 Meliponula bocandei, 325, 326 Meliponula ferruginea, 325, 326 nest cavity and hole, 327 Partamona and Scaura latitarsis, 326 plant families, 327 Networking, Bolivian pot-honey, 415 Nonaromatic organic acids, honeys acetic, 449 Apis mellifera honey spoilage, 447 botanical and geographical origin, 449 capillary electrophoresis, 451–452 chromatographic techniques, 451 composition, stingless bee, 447 consumers, 448 enzymatic assays, 450–451 enzymatic pathways, 448–449 food commodities, 447 gluconic, 448 Gluconobacter spp., 448 honey acidity and antibacterial activity, 449 honey components and parameters, 450 low concentrations, pyruvic, 449 maillard reaction products and enzymes, 449 malic and citric, 448 in pot honey, 452, 453 traditional and Mesoamerican aboriginal medicine, 449–450 Nuclear magnetic resonance (NMR) method definition, 429 geographical and entomological, 434–442 1 H NMR-based studies, 429–430 metabolomic analysis, 430–434 pattern recognition, 429 PLS-DA and PCA, 429 quantitative and structural information, 429 radio waves, 430 samples, 430 Nutrition nutritional quality, 160–161 and protection, 153 O Olean-12-ene (b-Amyrene), 535 Ovarian cancer cells antioxidants, 487 cell killing effect, 488 enzymatic hydrolysis, flavone C-glycosides, 488 IC50 values, pot-honeys, 487, 488 647 MTT reduction assay, 487 sources, anticancer compounds, 488 P Palynology analysis, honey, 288–289 bee pollen, 289–290 bees, 285–286 description, 285 geopropolis, 290–292 pollen grains, 287 vegetation, 286–287 PAMP, 515 Panama apiaries and meliponaries, 298 Lycopodium and Mimosa, 306 Tetragonisca angustula, 298, 299 Paranaense forest, 127 Partial least squares-discriminant analysis (PLS-DA) classification, 1H NMR spectra, 431 data processing, 434 entomological origin, honey samples, 436 fitting and validation results, 422, 424 Melipona pot-honey, 421 physicochemical results, Colombia, 420, 421 score plots, 436, 437 sensor array and physicochemical, 420 Venezuelan pot-honey samples, 436, 438 Pathogens-associated-molecules-patterns (PAMP), 515 PCA, 431, 436, 439, 440 Peru, 298, 299 Pests, Australian stingless bees adult beetles, 60–62 Bembix wasp, 58–60 braconid wasp, 60 cane toad, 62, 63 dead nest, syrphid fly larvae, 58, 59 disease-free, 57 phorid fly Dohrniphora trigonae, 58, 59 predators, 57–58 syrphid fly adult, 58 pH activity and antibacterial activity, 449 citric acid, 448 ionized acids, 450 Pheromones aerial, 195 complete routes, 194 incomplete routes, 194 odor-marking, 195 polarization routes, 194–195 648 Phylogeny ancestral characters, 14–15 Eocene (44 Mya) Baltic amber, 14 fossil record, 14 phylogenetic relationships, 14 Physicochemical analysis, Colombian stingless bee pot-honey. See also Pot-honey sugars analysis, 419 water content, 418 Physicochemical characteristics Colombian pot-honey (see also Pot-honey) adulteration and falsification, 384 ash and minerals, 387–388 Colombian stingless bee honey, 390–392 colorimeter, 390 composition, 384, 385 description, 384 genera Melipona and Tetragonisca, 385 HMF contents, 390 levorotary and dextrorotary compounds, 392 M. beecheii and M. scutellaris, 390 quality standards, 388 regulatory organizations, 388 Schade method, 390 T. angustula/non-compositional analysis, 384 water and sugars, 385–387 Guatemalan pot-honey (see also Pot-honey) ash content, 398–399 diastase (a-amylase), 398, 399 free acidity, 397–398 HMF, 398, 399 moisture content, 398 pH values, 397, 398 quality determination, 396 reducing sugars, 396–397 samples, 396 sucrose, 397 PLS-DA, 420–422, 424, 431, 434, 436–438 Pollen analysis counts, consistency and concentration, 304, 305 and field observation Africanized honeybees and Tetragonisca angustula, 311 brood production and swarming, 312 description, 309 list of species, 311 Lycopodium density marker, 311–312 nectar source, 310–311 nocturnal animals, 313 non-nectar species, 312 Index pollen and nectar source, 312 types, honey, 311 honey, 286 larval food, 287 M. mandacaia, 288 scientific approach, 285 Tetragonisca angustula, 298, 299 Pollen composition, Guatemalan pot-honey, 403 Pollen fermentation description, 159 haemolymph, 161 hypopharyngeal glands, 161 Melipona seminigra, 162 M. quadrifasciata, 160 nutritional quality and pollen grains, 160 prevent spoilage and diseases, 162 Ptilotrigona lurida, 161 stored pot pollen, 161 Streptococcus, Bifidobacterium and Lactobacillus, 159 Tetragonisca angustula and Frieseomelitta varia, 159 young workers, Scaptotrigona depilis, 159, 160 Pollen loads M. scutellaris, 290 palynological investigation, 286–287 and residual nest pollen, 286 Pollen/nectar flowers, 528 Pollen plants, 316 Pollen species concentration corrected/summed, 308 French Guiana corbicular, 305, 307 Pollen spectra analysis and field observation, 309–313 “bee-botanists”, 296 and bee-botany, 296–297 bee-palynology, 297–302 description, 295 “generalists” bees, 295 melittopalynology and melissopalynology, 295 stingless bees and honeybees applications corbicular pellets, Africanized honeybees, 303 corrected/summed pollen concentration, 308 counted pollen grains, 304 cutoff points, floral resource, 305, 306 floral nectar and pollen sources, 309 French Guiana corbicular pollen, 305, 307 Lycopodium and Mimosa, 306 Mimosa pudica, 304 Index pollen consistency, 303 pollen counts, consistency and concentration, 304, 305 Tetragonisca angustula, 302–303 taxonomy, 295–296 tropical lowland forest, 295, 296 Pollen substitute artificial feeding, 165–166 fermentation, 159–162 M. fasciculata, 166 Pollination, Australian stingless bee industry advantageous, 57 blueberry, 57 commercial crops, 55 description, 55 macadamia, 56 Prunus dulcis, 56 Trigona carbonaria, 56 Varroa destructor, 55 Polyphenols bioactivity, Melipona honey, 477, 478 and flavonoids, antioxidant activity, 477 Pot-honey aroma families, 352 Austroplebeia australis, 543, 545 botanical and geographic origin Astronium, 341 Coffea arabica, 338, 343 coffee-growing areas, 344 Euphorbia hirta, 344 Heliocarpus americanus, 338 honey types, 341, 342 monofloral samples, 341 multivariate analysis, 344 Muntingia calabura, 344 palynological composition, 341 palynological spectrum, 338 pollen types, 338, 339 Tetragonisca angustula, 338–341, 343 Toxicodendron striatum, 343 description, 337 descriptive sensory evaluation, 357, 358 descriptive sensory studies, 357 extraction by pressure/suction, 352–354 Frieseomelitta sp., 76, 90, 119, 288, 385, 386, 391, 482, 489 Frieseomelitta sp. aff. varia, 288 Frieseomelitta nigra, 288, 435, 487, 488 Geotrigona acapulconis, 101, 104, 107, 108, 140, 144, 371, 395–398, 402–405 Guatemalan bees antibacterial properties, 400–401 Apis mellifera, 385–386 description, 385 649 honey attributes, 403–406 nutritional characteristics, 399–400 physicochemical, 396–399 pollen composition, 403 sanitary quality, 404 sensory characteristics, 401–402 traditional log hives, 385 honey collection and pollen frequency classes, 337–338 Melipona, 289, 435, 439, 482, 487, 489 Melipona asilvai, 543, 549 Melipona beecheii, 41, 101, 106–109, 113, 114, 116, 119, 121, 138, 140, 143, 145–147, 191, 205–207, 221–223, 229–239, 255, 270, 278, 356, 370, 390, 395–400, 402, 404–406, 435, 436, 477, 478, 487, 488, 507–511, 543, 544, 549 Melipona brachychaeta, 410–412, 414, 435, 469 Melipona compressipes, 76, 83, 90, 194, 273, 274, 288, 289, 363, 368, 385, 386, 389–391, 418, 420–422, 530–531, 542, 544, 549, 550, 552 Melipona costaricensis, 543, 544 Melipona eburnea, 370, 385–387, 391, 418, 420–422 Melipona fasciata, 544 Melipona fasciata guerreroensis, 435 Melipona fasciculata, 435, 439, 440, 471, 488, 543, 549, 553 Melipona favosa, 77, 82, 83, 90, 94, 195, 225, 288, 355, 363–371, 385, 386, 389, 391, 399, 418, 420–422, 425, 435, 436, 443, 448, 451–453, 469, 470, 476, 487, 488, 525–526, 530, 531, 533–535, 550 Melipona aff. fuscopilosa, 436, 441, 442 Melipona flavolineata, 543, 549 Melipona fuscopilosa, 433, 435 Melipona grandis, 370, 410–412, 414, 435, 469, 526, 531, 535 Melipona mandacaia, 288, 543, 549, 553 Melipona melanopleura, 544 Melipona mondury, 553 Melipona panamica, 544 Melipona quadrifasciata, 434, 441, 443, 471, 545, 549, 553 Melipona quadrifasciata anthidioides, 543 Melipona quadrifasciata quadrifasciata, 543 Melipona rufiventris, 471,488, 543 Melipona rufiventris paraensis, 288 650 Pot-honey (cont.) Melipona scutellaris, 288, 435, 471, 482, 487, 488, 543, 549, 553 Melipona seminigra merrillae, 288 Melipona solani, 101, 107, 108, 140, 143, 147, 356, 370, 396–398, 400–402, 435, 439, 477, 478, 487, 488 Melipona aff. yucatanica, 101, 104, 107, 108, 116, 140, 143, 145, 398 Melipona sp., 77, 106, 128, 163, 211, 212, 224, 276, 371, 385, 386, 389, 391, 418, 421, 435, 487 Melipona subnitida, 435, 439, 440, 471, 482, 487, 488, 543, 549 Melipona trinitatis, 288 Meliponines chemical and microbial composition, 411–414 networking to market, 415 packaging, commercial distribution, 409–410 sensory approaches, 414 species of stingless bees, 410–411 stingless beekeepers, 410 Meliponini, 9–13, 19–33 Nannotrigona perilampoides, 75, 77, 83, 101, 107, 108, 116, 119, 121, 140, 143, 144, 146, 396, 398 Nannotrigona testaceicornis, 126, 129, 208, 274, 292, 385–387 Nannotrigona sp., 78, 115, 210, 385, 386, 389, 391 nonaromatic organic acids, 452, 453 Paratrigona sp., 385, 386, 391 Partamona peckolti, 78, 82, 83, 385, 386 Partamona seridoensis, 482 Partamona sp., 108, 385–387 Plebeia sp., 79, 91, 108, 128, 129, 139, 182, 209, 288, 385, 386, 391, 396, 398, 399, 435 and pot pollen, 188 price, 549 production, 543 Scaptotrigona, 436, 482, 487, 489, 435, 543, 544, 549, 553 Scaptotrigona aff. depilis, 288 Scaptotrigona depilis, 43, 79, 91, 156, 160, 161, 164–166, 208, 211, 225, 288, 410–412, 435, 469, 526, 530, 531, 534, 535 Scaptotrigona hellwegeri, 435, 487, 488 Scaptotrigona jujuyensis, 515, 516 Scaptotrigona limae, 385, 386, 389 Scaptotrigona mexicana, 102, 107–109, Index 117, 138, 141, 146, 196, 209, 211, 355–357, 392, 395–398, 400, 402, 403, 405, 414, 435, 441, 488, 544 Scaptotrigona polysticta, 75, 79, 182, 355, 356, 370, 410–412, 414, 435, 469, 487, 488, 526, 531, 544 Scaptotrigona near xanthotricha, 410–412 Scaptotrigona sp., 79, 91, 164, 288, 385, 386, 389, 391, 435, 469, 482, 487, 542, 549 Scaptotrigona sp. aff. xanthotricha, 435, 469 Scaura latitarsis, 288 sensory characteristics, 351–352 stingless-bees isorhamnetin and kaempferol derivatives, 471 Melipona favosa honey, Venezuela, 468, 469 nectar and honey samples, 468, 470–471 representative flavonoid glycosides, 467, 468 Tetragonula carbonaria honey, Australia, 468, 469 triglycosides, 471 Sugarbag honey, 37, 55 Tetragona clavipes, 435, 436, 441, 443 Tetragona sp., 91, 121, 385, 386, 389, 391, 418, 421, 436 Tetragonisca, 435 Tetragonisca angustula, 11, 79, 91, 102, 103, 106–109, 114, 115, 117, 119–121, 129, 130, 141, 159, 162, 177, 178, 180, 196, 212, 244, 273, 274, 287–289, 292, 298, 299, 301–306, 308, 309, 311, 312, 325, 337–345, 354, 355, 368, 370, 371, 375–380, 383–387, 389–391, 395–398, 400, 401, 403–406, 412, 435, 468, 470, 475, 482, 507–511, 525, 529, 530, 534, 543, 545, 546, 548, 549, 553 Tetragonisca fiebrigi, 127, 129–131, 370, 409–412, 414, 435, 469, 478, 515 Tetragonula, 435,482,489 Tetragonula carbonaria, 435, 436, 448, 487, 488, 543, 544, 545 Tetragonula laeviceps, 155, 495–502 ‘Trigona’ (s.l.) = Tetragonula, 439 Trigona carbonaria, 452, 453 Trigona crassipes, 21 Trigona hypogea, 21, 159 Trigona necrophaga, 21 Index Pot pollen Candida, 161 characteristics, pot honey, 188 conservation, 161 decisions, 196 Melipona quadrifasciata, 180 Melipona rufiventris, 180 Melipona seminigra, 162, 288 Melipona scutellaris, 290 meliponines, 159 palynological analysis, honeys, 130 Plebeia saiqui, 290 and pot-honey, Meliponini, 287 Ptilotrigona lurida, 22, 24, 161, 180 Scaptotrigona depilis, 164 Tetragonisca angustula, 180 Tetragonisca rufiventris, 180 Trigona dallatorreana, 161 Ptilotrigona lurida, pollen covered with yeast, 22, 24 Precision, 195 Principal component analysis (PCA) Brazilian honey samples, 440 chemometric approach, 431 clustering technique, 431 1 H NMR spectra, 431 PC1 and PC2 scores, 436, 439 Production and management, stingless bees Australia wax, 223 “cera de Campeche”, 222 honey and brood, 221 individual bees’nests, 221 Mayan codices, 222 Melipona beecheii, 221–222 Production and marketing, pot-honey. See also Pot-honey acidity, floral aromas and earthy notes, 541 Africanized bees, 541 animal husbandry, 551 in Brazilian States, 552–554 consumption, 551 cooperative marketing, 551–552 meliponicultural “grazing”, 551 Meliponine honey, 543–550 processing and storage, 552 “socially fair”, 542 and stingless bee species, 542–543 supplementation, nectar and pollen, 551 waste and toxic antibiotics, 541–542 Propolis. See also Chemical analysis, propolis, 377, 525–535 adaptive immune response, 519 Apis mellifera, 527,535 Frieseomelitta varia, 292,530,534 651 Frieseomelitta silvestri, 534 innate immune response bullfrogs, 517 commercial laying hens, 516 cytokines, 516 flavonoids and phenolic substances, 516 honey bee products, 516 ROI and NO, 516 Lestrimellita cf. limao, 292 Melipona sp., 530 Melipona compressipes, 530, 531 Melipona favosa, 525, 530–533, 535 Melipona grandis, 526, 531, 532 Melipona obrbygnii, 529 Melipona quadrifasciata, 292, 529 Melipona quadrifasciata anthidioides, 530, 531 Nannotrigona, 530 Nannotrigona testaceicornis, 292 Nannotrigona tristella, 530 Paratrigona anduzei, 530 Scaptotrigona, 530, 534 Scaptotrigona depilis, 526, 530–532, 534 Scaptotrigona polysticta, 526, 531, 532 Tetragona clavipes, 530 Tetragonisca, 530 Tetragonisca angustula, 292, 529, 530 Tetragonula biroi, 526, 531–535 Tetragonula carbonaria, 531, 534 Trigona recursa, 292 Provisioning and ovipositing process (POP), 41 R Reactive-oxygen intermediate (ROI), 516 Recruitment mechanisms ability and precision, 203 and communication, 190–195 efficiency, 202 food, stingless bees, 189–190 internal information, 195 mechanisms, 208 velocity, 210 Resins collection, honey and stingless bees. See also Pot-honey cerumen, 527 definition, 526 deterrent vs. herbivorous insects, 526 dipterocarps family, 528 flowering plants, 528 foraging workers, 527–528 geopropolis, 527 nest construction and defense purposes, 526 social immunity, 527 Resource constancy, 302 652 ROI, 516 Royal jelly adaptive immune response, 519 biological activity, pot-honey, 517 description, 517 Rustic hives, 128–130 S “Saburá”, 159, 160 Scaptotrigona aff. xanthotricha, 410, 411 S. depilis, 410, 411 S. mexicana, 414 S. polysticta, 410, 411 Scaptotrigona mexicana bees description, 405 floral resources, 403, 406 pollen composition, 405 Seed dispersal, Australian stingless bees bee vectors/mellitochory, 63 C. torelliana, 62–65 resin, 65 Trigona (s.l.), 64–65 Sensory attributes, Melipona favosa honey, 367–368 Sensory characteristics, Guatemalan pot-honey. See also Pot-honey descriptors, 401 fermentation process, 401 stingless bees honey, 401, 402 Sensory evaluation characteristics, pot-honey, 351–352 consumer acceptance, 349 description, 349 free-choice profile (FCP), 351, 352, 357 human senses, 349 panel selection, 350 psychological processes, 350 qualitative and quantitative methods, honey, 350 qualitative studies, 350 Signals, 194–195 Social facilitation description, 193 experience and learning, 193 and pheromone deposition, 193 social insects, 193 South and Central America European Conquerors, 220–221 pot-honey, 225 Species occurrence, 90–93 Stingless bees advantages, 376 bee-keepers, 345 Index bee keeping, Costa Rica description, 114 honey and wax, 114 indigenous people, 114 map location, 114, 115 medicine and ongoing investigations, 120, 122 meliponiculture, 122 T. angustula, 114, 115 T. nannotrigona, 115 bee pollination, agriculture, 278 biology, 9 body size and flight range, 277 Bolivian species marketing purposes, 411 nest entrances, 410, 411 relative annual yield, products, 410, 411 scientific and common names, 410 colonies, 8 commercial honey, 375 composition, T. angustula and A. mellifera honey, 380 Costa Rican ethnopharmacology, 508 description, 3 disturbance and habitat fragmentation, 275–277 food, 461, 465, 467 food location communication, 187–197 foraging, 274–275 fossils, 14 history and transitions, 223–224 hollow tree trunk, 11–12 honey and wax, 220–221 hunting, 220 local bee communities, 276 medicinal properties, honey, 508 meliponiculture, 375 Mesoamerican region, 507 microorganisms, 153–168, 173–182 nesting, 272–273 nests, 325–332 and potential flight ranges, 273, 274 pot-honey, 225 and pot-honey production country of origin and native, 542, 543 description, 542 production and management, 221–223 propolis alcohol-free product, 535 Bolivian ethanol extract, Scaptotrigona, 534–535 compounds, aliphatic acids, 533 extraction and preparation, 532 flavonoids, 535 GC-MS analysis, 532–533 653 Index and geographical origin, 531 Meliponini types, 533, 534 octadecanoic acid, 534 terpenic compounds, 534 Venezuela, Philippines and Bolivia, 531, 532 simulation model, 278 taxonomy and distribution analysis, 103 classification system, 100 collection sites, 104 Mesoamerican, 103 native bee collection, 100–103 occurrence localities, 103, 104 Paratrigona guatemalensis, 103–104 Plebeia pulchra, 103–104 tribes Apini and Meliponini, 100 Tetragonisca angustula, 11, 79, 91, 102, 103, 106–109, 114, 115, 117, 119–121, 129, 130, 141, 159, 162, 177, 178, 180, 196, 212, 244, 273, 274, 287–289, 292, 298, 299, 301–306, 308, 309, 311, 312, 325, 337–345, 354, 355, 368, 370, 371, 375–380, 383–387, 389–391, 395–398, 400, 401, 403–406, 412, 475, 507–511, 525, 529, 530, 534, 543, 545, 546, 548, 549, 553 T Taxonomy, 261–266 T-cell receptor (TCR), 518 Tetragonisca, 298–299, 303, 305, 345, 375–380, 403, 406 Tetragonisca angustula angustula, 298 and Apis mellifera, 303 beekeeping and meliponiculture, 376 commercial honey, 375 environmental diversity, 376 honey and quality parameters, 377–378 honey composition, 376–377 meliponiculture, 375 physicochemical properties, 379–380 types, beekeeping, 375 description, 406 floral resources, 403, 406 pollen counts, consistency and concentration, 304, 305 tropical lowlands, 298, 299 Tetragonisca fiebrigi Bolivian stingless bees, 410 nest entrances, 410, 411 treatment, ocular diseases, 409 Tetragonula laeviceps antimicrobial peptides, honey, 501–502 antiproliferative activity, 496 bioactivity, 497–501 composition, honey and propolis, 496–497 description, 495 natural medicines, 495 stingless bee products, 502 Trigona (s.l.) classification, 45 identification, 44–45 natural distribution, 45 nest architecture, colony population, and brood structure, 46–47 V Venezuelan pot-honey, 436, 438. See also Pot-honey Venezuelan stingless bees agro-ecosystems, 298 biogeography, 83–84 Camargo collection—RPSP, 74 cutoff points, 305 data source, 74–75 description, 73 diversity and distribution, 75–82 honey and propolis analyses, 74 honey-bee samples, 301 propolis collection, 74 seasonal pollen, 312 Vernacular names alimentary customs, 128 creole population, 128 habits and characteristics, species, 125 Virgin honey, 232 W Water and sugars, Colombian pot-honey. See also Pot-honey contents, stingless bee honey, 385, 386 fructose-glucose ratios, 387 indirect refractometric methodology, 387 mean glucose contents, 387 quality parameters, 385 Water extract of propolis (WEP), 497, 499, 500 Wild honey, 265 654 Y Yeasts and bees, 181–182 Melipona quinquefasciata, 180, 181 microbiota, pollen, 180 pot honey spoilage, 181 Ptilotrigona lurida, 180 Starmerella meliponinorum, 180 T. angustula, M. quadrifasciata and Frieseomelitta varia, 180 Zygosaccharomyces machadoi, 180–181 Index Yeasts mutualistic interaction, 181–182 Yucatan “cera de Campeche”, 222 “rational hives”, 223 Yungas northwestern mountain, 127 slender wedge, 127