Abstract
The dinoflagellates Karenia mikimotoi and Prorocentrum donghaiense are both important causative species of harmful algal blooms (HABs) in the East China Sea. The ichthyotoxic K. mikimotoi, which occasionally leads to large-scale HABs in the East China Sea, is difficult to be discriminated from other morphologically similar species in the family Kareniaceae by light microscope observation. To improve the accuracy and efficiency in detection of K. mikimotoi, a real-time quantitative PCR (qPCR) assay was developed in this study. The qPCR assay has high specificity and sensitivity, which allows the detection of K. mikimotoi at the lower detection limit of one cell. The qPCR assay target K. mikimotoi, together with another qPCR assay previously developed for P. donghaiense, was applied to study a bloom of dinoflagellates in the coastal waters of Fujian province from April 25 to June 11 in 2019. Cells of K. mikimotoi were detected in about half of the samples, and the maximum abundance was lower than 30 cells L−1. Abundance of P. donghaiense cells (maximum abundance above 106 cells L−1) were determined using both qPCR assay and light microscope cell counting, and the results of the two methods were consistent with each other. The qPCR assays of the blooming dinoflagellates offer reliable and accurate approaches for the detection of HABs species.
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The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.
References
Adolf JE, Bachvaroff TR, Deeds JR, Place AR (2015) Ichthyotoxic Karlodinium veneficum (Ballantine) J Larsen in the upper Swan River Estuary (Western Australia): Ecological conditions leading to a fish kill. Harmful Algae 48:83–93
Al-Kandari MA, Highfield AC, Hall MJ, Hayes P, Schroeder DC (2011) Molecular tools separate harmful algal bloom species, Karenia mikimotoi, from different geographical regions into distinct sub-groups. Harmful Algae 10:636–643
Anderson DM, Cembella AD, Hallegraeff GM (2012) Progress in understanding harmful algal blooms: paradigm shifts and new technologies for research, monitoring, and management. Annu Rev Mar Sci 4:143–176
Aoki K, Kameda T, Yamatogi T, Ishida N, Hirae S, Kawaguchi M, Syutou T (2017) Spatio-temporal variations in bloom of the red-tide dinoflagellate Karenia mikimotoi in Imari Bay, Japan, in 2014: factors controlling horizontal and vertical distribution. Mar Pollut Bull 124:130–138
Aoki K, Kuroda H, Setou T, Okazaki M, Yamatogi T, Hirae S, Ishida N, Yoshida K, Mitoya Y (2019) Exceptional red-tide of fish-killing dinoflagellate Karenia mikimotoi promoted by typhoon-induced upwelling. Estuar Coast Shelf Sci 219:14–23
Barkallah M, Elleuch J, Smith KF, Chaari S, Neila IB, Fendri I, Michaud P, Abdelkafi S (2020) Development and application of a real-time PCR assay for the sensitive detection of diarrheic toxin producer Prorocentrum lima. J Microbiol Meth 178:106081
Barnes MK, Tilstone GH, Smyth TJ, Widdicombe CE, Gloël J, Robinson C, Kaiser J, Suggett DJ (2015) Drivers and effects of Karenia mikimotoi blooms in the western English Channel. Prog Oceanogr 137:456–469
Bergholtz T, Daugbjerg N, Moestrup Ø, Fernández-Tejedor M (2005) On the identity of Karlodinium veneficum and description of Karlodinium armiger sp. nov. (Dinophyceae), based on light and electron microscopy, nuclear-encoded LSU rDNA, and pigment composition. J Phycol 42:170–193
Bowers HA, Tengs T, Glasgow HBJR, Burkholder JM, Rublee PA, Oldach DW (2000) Development of real-time PCR assays for rapid detection of Pfiesteria piscicida and related dinoflagellates. Appl Environ Microbiol 66:4641–4648
Brand LE, Campbell L, Bresnan E (2012) Karenia: the biology and ecology of a toxic genus. Harmful Algae 14:156–178
Cary SC, Coyne KJ, Rueckert A, Wood SA, Kelly S, Gemmill CEC, Vieglais C, Hicks BJ (2014) Development and validation of a quantitative PCR assay for the early detection and monitoring of the invasive diatom Didymosphenia geminata. Harmful Algae 36:63–70
Cen JY, Wang JY, Huang LF, Ding GM, Qi YZ, Cao RB, Cui L, Lu SH (2019) Who is the “murderer” of the bloom in coastal waters of Fujian, China, in 2019? J Oceanol Limnol 38:722–732
Chen BH, Wang K, Guo HG, Lin H (2021) Karenia mikimotoi blooms in coastal waters of China from 1998 to 2017. Estuar Coast Shelf Sci 249:107034
Coyne KJ, Handy SM, Demir E, Whereat EB, Hutchins DA, Portune KJ, Doblin MA, Cary SC (2005) Improved quantitative real-time PCR assays for enumeration of harmful algal species in field samples using an exogenous DNA reference standard. Limnol Oceanogr Methods 3:381–391
Dai XF, Lu DD, Guan WB, Xia P, Wang HX, He PX, Zhang DS (2013) The correlation between Prorocentrum donghaiense blooms and the Taiwan Warm Current in the East China Sea - evidence for the “Pelagic Seed Bank” hypothesis. PLoS ONE 8:e64188
Davidson K, Miller P, Wilding TA, Shutler J, Bresnan E, Kennington K, Swan S (2009) A large and prolonged bloom of Karenia mikimotoi in Scottish waters in 2006. Harmful Algae 8:349–361
Deng H, Guan WB, Cao ZY, Bao M, Chen Q (2016) Analysis of hydrological and meteorological factors causing Karenia mikimotoi bloom in 2012 along Fujian coast. J Mar Sci 34:28–38 (in Chinese with English abstract)
Dhar SS, Chakraborty B, Chaudhuri P (2014) Comparison of multivariate distributions using quantile–quantile plots and related tests. Bernoulli 20:1484–1506
Ding GM, Zhang SF (2018) Ecological characteristics and the causes of Karenia mikimotoi bloom in the Sansha Bay in 2012. Haiyang Xuebao 40:104–112 (in Chinese with English abstract)
Dyhrman ST, Erdner D, Du JL, Galac M, Anderson DM (2006) Molecular quantification of toxic Alexandrium fundyense in the Gulf of Maine using real-time PCR. Harmful Algae 5:242–250
Eckford-Soper LK, Daugbjerg N (2015) Development of a multiplex real-time qPCR assay for simultaneous enumeration of up to four marine toxic bloom-forming microalgal species. Harmful Algae 48:37–43
Elleuch J, Barkallah M, Smith KF, Ben Neila I, Fendri I, Abdelkafi S (2020) Quantitative PCR assay for the simultaneous identification and enumeration of multiple Karenia species. Environ Sci Pollut Res Int 27:36889–36899
Enjoji N, Katano T, Yoshinaka Y, Furuoka F, Ando Y, Yamada M, Hamasaki T, Miyamura E, Otsubo M, Yokoyama K (2019) Development of primer sets for multiplex and qPCR assays targeting Skeletonema species and their application to field samples. J Oceanogr 75:319–334
Erdner DL, Percy L, Keafer B, Lewis J, Anderson DM (2010) A quantitative real-time PCR assay for the identification and enumeration of Alexandrium cysts in marine sediments. Deep Sea Res II 57:279–287
Galluzzi L, Penna A, Bertozzini E, Vila M, Garcés E, Magnani M (2004) Development of a real-time PCR assay for rapid detection and quantification of Alexandrium minutum (a Dinoflagellate). Appl Environ Microbiol 70:1199–1206
Gao Y, Yu RC, Chen JH, Zhang QC, Kong FZ, Zhou MJ (2015) Distribution of Alexandrium fundyense and A. pacificum (Dinophyceae) in the Yellow Sea and Bohai Sea. Mar Pollut Bull 96:210–219
Gillibrand PA, Siemering B, Miller PI, Davidson K (2016) Individual-based modelling of the development and transport of a Karenia mikimotoi bloom on the North-west European continental shelf. Harmful Algae 53:118–134
GlobalHAB (2017) Global harmful algal blooms, science and implementation plan. SCOR and IOC, Delaware
Godhe A, Otta SK, Rehnstam-Holm AS, Karunasagar I, Karunasagar I (2001) Polymerase chain reaction in detection of Gymnodinium mikimotoi and Alexandrium minutum in field samples from southwest India. Mar Biotechnol 3:152–162
Gray M, Wawrik B, Paul J, Casper E (2003) Molecular detection and quantitation of the red tide dinoflagellate Karenia brevis in the marine environment. Appl Environ Microbiol 69:5726–5730
Guillard RRL, Ryther JH (1962) Studies of marine planktonic diatoms I. Cyclotella nana Hustedt, and Detonula confervacea(Cleve) Gran. Can J Microbiol 8:229–240
Guillou L, Nezan E, Cueff V, Denn EE, Cambon-Bonavita MA, Gentien P, Barbier G (2002) Genetic diversity and molecular detection of three toxic dinoflagellate genera (Alexandrium, Dinophysis, and Karenia) from French coasts. Protist 153:223–238
Guiry MD, Guiry GM (2010) AlgaeBase. https://www.algaebase.org. Searched on 30 December 2021
Handy SM, Hutchins DA, Cary SC, Coyne KJ (2006) Simultaneous enumeration of multiple raphidophyte species by quantitative real-time PCR: capabilities and limitations. Limnol Oceanogr Methods 4:193–204
Hansen G, Daugbjerg N, Henriksen P (2003) Comparative study of Gymnodinium mikimotoi and Gymnodinium aureolum, comb. nov. (= Gyrodinium aureolum) based on morphology, pigment composition, and molecular data. J Phycol 36:394–410
Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6:986–994
Heil CA, Steidinger KA (2009) Monitoring, management, and mitigation of Karenia blooms in the eastern Gulf of Mexico. Harmful Algae 8:611–617
Huang B, Shao JB, Wei N, Wang YM (2014) Ecological studies during occurrence of dinoflagellate blooms in East China Sea spring 2014. Ecology and Environmental Sciences 23:1457–1462 (in Chinese with English abstract)
Huang HL, Gao WF, Zhu P, Zhou CX, Qiao LL, Dang CY, Pang JH, Yan XJ (2020) Molecular method for rapid detection of the red tide dinoflagellate Karenia mikimotoi in the coastal region of Xiangshan Bay. China J Microbiol Methods 168:105801
Huang HY, Kang LC, Yang Y, Yang L, Liu SM, Wang QL, Li X (2016) Species composition and distribution of red tide causative organisms in the coastal waters of China in 2013. Marine Sciences 40:17–27 (in Chinese with English abstract)
Joanes DN, Gill CA (1998) Comparing measures of sample skewness and kurtosis. J R Stat Soc Ser D 47:183–189
Kavanagh S, Brennan C, O’Connor L, Moran S, Salas R, Lyons J, Silke J, Maher M (2010) Real-time PCR detection of Dinophysis species in Irish coastal waters. Mar Biotechnol 12:534–542
Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120
Li HM, Tang HJ, Shi XY, Zhang CS, Wang XL (2014) Increased nutrient loads from the Changjiang (Yangtze) River have led to increased harmful algal blooms. Harmful Algae 39:92–101
Li XD, Yan T, Lin JN, Yu RC, Zhou MJ (2017) Detrimental impacts of the dinoflagellate Karenia mikimotoi in Fujian coastal waters on typical marine organisms. Harmful Algae 61:1–12
Liang YB (2012) Investigation and evaluation of red tide disasters in China (1933–2009). China Ocean Press, Beijing
Lin JN, Song JJ, Yan T, Zhang QC, Zhou MJ (2015) Large-scale dinoflagellate bloom species Prorocentrum donghaiense and Karenia mikimotoi reduce the survival and reproduction of copepod Calanus sinicus. J Mar Biol Assoc U K 95:1071–1079
Lin JN, Yan T, Zhang QC, Wang YF, Liu Q, Zhou MJ (2014) In situ detrimental impacts of Prorocentrum donghaiense blooms on zooplankton in the East China Sea. Mar Pollut Bull 88:302–310
Lin YR, Cen JY, Wang JY, Liang QY, Lu SH (2020) Preliminary study on interspecific relationship and hemolytic activity of four Karenia species from South China Sea. Oceanol Limnol Sinica 51:1402–1411
Liu RY (2008) Checklist of marine biota of China seas. Science Press, Beijing
Liu ZG, Wang JH, Cai P, Wang Y, Qin YT, Liu SH (2014) Distribution of Karenia mikimotoi and rules of its causing red tide. Territory & Natural Resources Study 1:38–41 (in Chinese with English abstract)
Long H, Du Q (2005) Prmiary Research on Karenia mikimotoi bloom in Fujian Coast. J Fujian Fisheries 4:22–26 (in Chinese with English abstract)
Loret P, Tengs T, Villareal TA, Singler H, Richardson B, Mcguire P, Morton S, Busman M, Campbell L (2002) No difference found in ribosomal DNA sequences from physiologically diverse clones of Karenia brevis (Dinophyceae) from the Gulf of Mexico. J Plankton Res 24:735–739
Lu DD, Qi YZ, Gu HF, Dai XF, Wang HX, Gao YH, Shen PP, Zhang QC, Yu RC, Lu SH (2014) Causative species of harmful algal blooms in Chinese coastal waters. Algol Stud 145–146:145–168
Lu SH, Hodgkiss IJ (2004) Harmful algal bloom causative collected from Hong Kong waters. Hydrobiologia 512:231–238
Luo ZH, Wang L, Chan L, Lu SH, Gu HF (2018) Karlodinium zhouanum, a new dinoflagellate species from China, and molecular phylogeny of Karenia digitata and Karenia longicanalis (Gymnodiniales, Dinophyceae). Phycologia 57:401–412
Ministry of Natural Resources of the People’s Republic of China (2009–2020) Bulletin of China marine disaster. http://www.mnr.gov.cn/sj/sjfw/hy/gbgg/zghyzhgb/. Accessed 18 Jul 2021
Ministry of Natural Resources of the People’s Republic of China (2000–2017) Bulletin of China marine ecological and environment status. http://www.mnr.gov.cn/sj/sjfw/hy/gbgg/zghyhjzlgb/. Accessed 18 Jul 2021
Mitchell SO, Rodger H (2007) Pathology of wild and cultured fish affected by a Karenia mikimotoi bloom in Ireland. Bull Eur Ass Fish Pathol 27:39–42
Moorthi SD, Countway PD, Stauffer BA, Caron DA (2006) Use of quantitative real-time PCR to investigate the dynamics of the red tide dinoflagellate Lingulodinium polyedrum. Microb Ecol 52:136–150
Nézan E, Siano R, Boulben S, Six C, Bilien G, Chèze K, Duval A, Panse SL, Quéré J, Chomérat N (2014) Genetic diversity of the harmful family Kareniaceae (Gymnodiniales, Dinophyceae) in France, with the description of Karlodinium gentienii sp. nov.: a new potentially toxic dinoflagellate. Harmful Algae 40:75–91
Oda M (1935) Gymnodinium mikimotoi Miyake et Kominami n. sp. (MS.) no akashiwo to ryusando no koka. (The red tide of Gymnodinium mikimotoi Miyake et Kominami and the influence of copper sulfate on the red tide of November 1972). Doubutsugaku Zasshi 47:35–48
Pearson LA, D’Agostino PM, Neilan BA (2021) Recent developments in quantitative PCR for monitoring harmful marine microalgae. Harmful Algae 108:102096
Perini F, Casabianca A, Battocchi C, Accoroni S, Totti C, Penna A (2011) New approach using the real-time PCR method for estimation of the toxic marine dinoflagellate Ostreopsis cf ovata in marine environment. PLoS One 6:e17699
Pitcher GC, Calder D (2000) Harmful algal blooms of the southern Benguela Current: a review and appraisal of monitoring from 1989 to 1997. Afr J Mar Sci 22:255–271
Raine R, O’Boyle S, O’Higgins T, White M, Patching J, Cahill B, McMahon T (2001) A satellite and field portrait of a Karenia mikimotoi bloom off the south coast of Ireland, August 1998. Hydrobiologia 465:187–193
Shimada H, Kanamori M, Yoshida H, Imai I (2016) First record of red tide due to the harmful dinoflagellate Karenia mikimotoi in Hakodate Bay, southern Hokkaido, in autumn 2015. Nippon Suisan Gakkaishi 82:934–938
Shin HH, Li Z, Mertens KN, Seo MH, Gu HF, Lim WA, Yoon YH, Soh HY, Matsuoka K (2019) Prorocentrum shikokuense Hada and P. donghaiense Lu are junior synonyms of P. obtusidens Schiller, but not of P. dentatum Stein (Prorocentrales, Dinophyceae). Harmful Algae 89:101686
Silke J, O’Beirn F, Cronin M (2005) Karenia mikimotoi: an exceptional dinoflagellate bloom in Western Irish Waters, Summer 2005. Marine Institute, Galway, Marine Environment and Health Series 21.
Sournia A (1978) Phytoplankton manual. UNESCO, Paris
Stern RF, Andersen RA, Jameson I, Kupper FC, Coffroth MA, Vaulot D, Le Gall F, Veron B, Brand JJ, Skelton H, Kasai F, Lilly EL, Keeling PJ (2012) Evaluating the ribosomal internal transcribed spacer (ITS) as a candidate dinoflagellate barcode marker. PLoS ONE 7:e42780
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol 30:2725–2729
Thyng KM, Hetland RD, Ogle MT, Zhang XQ, Chen F, Campbell L (2013) Origins of Karenia brevis harmful algal blooms along the Texas coast. Limnol Oceanogr Fluids Environ 3:269–278
Toldrà A, Andree KB, Fernández-Tejedor M, Diogène J, Campàs M (2018) Dual quantitative PCR assay for identification and enumeration of Karlodinium veneficum and Karlodinium armiger combined with a simple and rapid DNA extraction method. J Appl Phycol 30:2435–2445
Ulrich RM, Casper ET, Campbell L, Richardson B, Heil CA, Paul JH (2010) Detection and quantification of Karenia mikimotoi using real-time nucleic acid sequence-based amplification with internal control RNA (IC-NASBA). Harmful Algae 9:116–122
Utermöhl H (1958) Zur Vervollkommung der quantitativen Phytoplankton-Methodik. Mitt Int Ver Theor Angew Limnol 9:1–38
Vandersea M, Tester P, Holderied K, Hondolero D, Kibler S, Powell K, Baird S, Doroff A, Dugan D, Meredith A, Tomlinson M, Litaker RW (2020) An extraordinary Karenia mikimotoi “beer tide” in Kachemak Bay Alaska. Harmful Algae 92:101706
Vandersea MW, Kibler SR, Holland WC, Tester PA, Schultz TF, Faust MA, Holmes MJ, Chinain M, Litaker RW (2012) Development of semi-quantitative PCR assays for the detection and enumeration of Gambierdiscus species (Gonyaulacales, Dinophyceae). J Phycol 48:902–915
Wang JY, Cen JY, Li S, Lu SH, Moestrup Ø, Chan KK, Jiang T, Lei XD (2018) A re-investigation of the bloom-forming unarmored dinoflagellate Karenia longicanalis (syn. Karenia umbella ) from Chinese coastal waters. J Oceanol Limnol 36:2202–2215
Wang JY, Ho KC, Qi YZ, Lu SH, Yang J (2017) Progress in taxonomy study on Kareniaceae (Dinophyta). Oceanol Limnol Sinica 48:786–797 (in Chinese with English abstract)
Waters LG, Wolcott TG, Kamykowski D, Sinclair G (2015) Deep-water seed populations for red tide blooms in the Gulf of Mexico. Mar Ecol Prog Ser 529:1–16
Winnepenninckx B, Backeljau T, Dewachter R (1993) Extraction of high-molecular-weight DNA from mollusks. Trends Genet 9:407–407
Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP (1997) Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22:130–138
Yamaguchi H, Hirano T, Yoshimatsu T, Tanimoto Y, Matsumoto T, Suzuki S, Hayashi Y, Urabe A, Miyamura K, Sakamoto S, Yamaguchi M, Tomaru Y (2016) Occurrence of Karenia papilionacea (Dinophyceae) and its novel sister phylotype in Japanese coastal waters. Harmful Algae 57:59–68
Yang ZB, Takayama H, Matsuoka K, Hodgkiss IJ (2000) Karenia digitata sp. nov. (Gymnodiniales, Dinophyceae), a new harmful algal bloom species from the coastal waters of west Japan and Hong Kong. Phycologia 39:463–470
Yu RC, Lu SH, Liang YB (2018) Harmful algal blooms in the coastal waters of China. In: Glibert PM, Berdalet E, Burford MA, Pitcher GC, Zhou MJ (eds) Global ecology and oceanography of harmful algal blooms. Springer, Cham, pp 309–316
Yu RC, Lu SH, Qi YZ, Zhou MJ (2020) Progress and perspectives of harmful algal bloom studies in China. Oceanol Limnol Sinica 51:769–788 (in Chinese with English abstract)
Yu RC, Zhang QC, Kong FZ, Zhou ZX, Chen ZF, Zhao Y, Geng HX, Dai L, Yan T, Zhou MJ (2017) Status, impacts and long-term changes of harmful algal blooms in the sea area adjacent to the Changjiang River estuary. Oceanol Limnol Sinica 48:1179–1186 (in Chinese with English abstract)
Yuan J, Mi TZ, Zhen Y, Yu ZG (2012a) Development of a rapid detection and quantification method of Karenia mikimotoi by real-time quantitative PCR. Harmful Algae 17:83–91
Yuan J, Mi TZ, Zhen Y, Yu ZG (2012b) Development of a real-time PCR method (Taqman) for rapid identification and quantification of Prorocentrum donghaiense. J Ocean Univ China 11:366–374
Zhang FY, Ma LB, Xu ZL, Zheng JB, Shi YH, Lu YN, Miao YP (2009) Sensitive and rapid detection of Karenia mikimotoi (Dinophyceae) by loop-mediated isothermal amplification. Harmful Algae 8:839–842
Zhang QC, Chen ZF, Zhao JY, Yu RC, Qiu LM, Kong FZ, Wang YF, Yan T, Zhou MJ (2019) Development of a sensitive qPCR method for the detection of pelagophyte Aureococcus anophagefferens. Limnol Oceanogr Methods 18:41–51
Zhou CX, Fu YJ, Yan XJ (2007) Hemolytic activity studies of several harmful alga strains. Asian Journal of Ecotoxicology 2:78–82
Zhou MJ, Shen ZL, Yu RC (2008) Responses of a coastal phytoplankton community to increased nutrient input from the Changjiang (Yangtze) River. Cont Shelf Res 28:1483–1489
Zhou MJ, Zhu MY (2006) Progress of the project “Ecology and Oceanography of Harmful Algal Blooms in China.” Advances in Earth Science 21:673–679 (in Chinese with English abstract)
Acknowledgements
We thank Professor Da-Zhi Wang in Xiamen University for providing the strain of Karenia mikimotoi isolated from the coastal water of Fujian province which was used in this study.
Funding
This study was supported by the project (No. 2017YFC1404304) of the National Key R&D Program of China, the project (grant XDA19060203) of the Strategic Priority Research Program of Chinese Academy of Sciences (CAS), the CAS-CSIRO BAU project (grant 133137KYSB20180141) of CAS, and the project (LY19D060007) of the Zhejiang Provincial Natural Science Foundation of China.
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Hu, XK., Lin, ZR., Zhang, QC. et al. Detection of bloom-forming dinoflagellates Karenia mikimotoi and Prorocentrum donghaiense using qPCR assays. J Appl Phycol 34, 1483–1496 (2022). https://doi.org/10.1007/s10811-022-02698-y
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DOI: https://doi.org/10.1007/s10811-022-02698-y