Quantitative Real-Time Polymerase Chain Reaction for Cochlodinium Fulvescens (Dinophyceae), a Harmful Dinoflagellate from California Coastal Waters1
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J. Phycol. 48, 384–393 (2012) Ó 2012 Phycological Society of America DOI: 10.1111/j.1529-8817.2012.01120.x QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION FOR COCHLODINIUM FULVESCENS (DINOPHYCEAE), A HARMFUL DINOFLAGELLATE FROM CALIFORNIA COASTAL WATERS1 Meredith D. A. Howard 2 Southern California Coastal Water Research Project, Costa Mesa, California 92626, USA Department of Biological Sciences, University of Southern California, Los Angeles, California 90089, USA Adriane C. Jones, Astrid Schnetzer Department of Biological Sciences, University of Southern California, Los Angeles, California 90089, USA Peter D. Countway Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, Maine 04575, USA Carmelo R. Tomas Center for Marine Science, University of North Carolina Wilmington, Wilmington, North Carolina 28409, USA Raphael M. Kudela, Kendra Hayashi Department of Ocean Sciences and Institute for Marine Sciences, University of California Santa Cruz, Santa Cruz, California 95064, USA Pamela Chia and David A. Caron Department of Biological Sciences, University of Southern California, Los Angeles, California 90089, USA Harmful blooms formed by species of the dinofla- dynamic abundance range of seven orders of magni- gellate Cochlodinium have caused massive fish kills tude. Initial application of the method to archived and substantial economic losses in the Pacific field samples collected during blooms in Monterey Ocean. Recently, prominent blooms of Cochlodinium Bay revealed no statistically significant correlations have occurred in central and southern California between gene copy number and environmental (2004–2008), and Cochlodinium cells are now rou- parameters. However, the onset of Cochlodinium tinely observed in microscopical analysis of algal blooms in central California was consistent with pre- assemblages from Californian coastal waters. The viously reported findings of correlations to first documented economic loss due to a Cochlodini- decreased surface temperature and increased inputs um bloom in California occurred in Monterey Bay of nitrogenous nutrients. and resulted in the mortality of commercially Key index words: 18S rDNA; Cochlodinium;harmful farmed abalone. Increasing occurrences of Cochlodi- algal blooms; protist; qPCR nium blooms, the fact that these cells preserve poorly using standard techniques, and the difficulty Abbreviation: BLAST, basic local alignment search of identifying preserved specimens using morpho- tool logical criteria make Cochlodinium species prime candidates for the development of a quantitative real-time polymerase chain reaction (qPCR) Harmful blooms of dinoflagellates within the approach. The 18S rDNA gene sequenced from genus Cochlodinium have been increasing in Cochlodinium cells obtained from California coastal frequency worldwide and have caused substantial waters, as well as GenBank sequences of Cochlodini- economic losses and fish kills in the Pacific Ocean um, were used to design and test a Molecular off the coasts of Japan (Onoue and Nozawa 1989, Ò Beacon approach. The qPCR method developed in Yuki and Yoshimatsu 1989), China (Onoue and this study is species specific, sensitive for the detec- Nozawa 1989, Yuki and Yoshimatsu 1989), Korea tion of C. fulvescens that has given rise to the recent (Kim 1998), Malaysia (Anton et al. 2008), the Phil- blooms in the eastern Pacific Ocean, and spans a ippines (Azanza et al. 2008), and Canada (Whyte et al. 2001). Blooms of this genus have also been reported in the eastern Pacific including Costa Rica 1Received 19 January 2011. Accepted 5 August 2011. (Vargas-Montero et al. 2006), Mexico (Morales- 2Author for correspondence: e-mail [email protected]. Blake et al. 2001, Ga´rate-Liza´rraga et al. 2004), and 384 qPCR FOR COCHLODINIUM FULVESCENS 385 the Caribbean Sea (Margalef 1961). A persistent studies and indicated that C. fulvescens was the spe- bloom in 2008–2009 in the Arabian Gulf and Gulf cies present in those waters (Iwataki et al. 2008). of Oman had severe consequences including fish However, a robust culture of C. fulvescens isolated mortality, damage to coral reefs, impacts to coastal from North America has not yet been established tourism, and most significantly, the forced closure despite numerous attempts by several groups of desalination plants in the region (Richlen et al. (R. Kudela, personal observation and C. Tomas, per- 2010). sonal observation) limiting material for comparative The presence of Cochlodinium off the California studies of morphology. coast has been unremarkable until recently. How- In addition to difficulties associated with morpho- ever, prominent blooms of Cochlodinium have been logical identification of live or preserved Cochlodini- documented in the coastal waters of central and um species, cells of this genus often burst or become southern California in 2004 and from 2006 to 2008 deformed during preservation (Curtiss et al. 2008). (Curtiss et al. 2008, Kudela et al. 2008). Blooms in Therefore, determinations of cell abundances based Monterey Bay during 2004 and 2006 and off San on microscopy of preserved samples would be Diego during 2004 attained cell abundances of 104 expected to underestimate actual cell abundances. ) cellsÆL 1 (Curtiss et al. 2008, Kudela et al. 2008). LM is currently the most commonly employed Abundances in Santa Monica Bay during 2006 method for identifying and enumerating coastal phy- ) reached an even greater cell abundance of 106 cellsÆL 1 toplankton including HAB-forming species in moni- ) and a chl a concentration of 67 mgÆm 3 (Reifel toring and research programs, but molecular 2009). Cells belonging to the genus Cochlodinium techniques are being adopted rapidly as alternative are now routinely identified as members of algal or complementary approaches for determining the assemblages at many locations along the central and abundances of important protistan species including southern California coastline. many HAB taxa. The latter methods require less tax- Concurrent with these recent outbreaks of Coch- onomic expertise for identification than microscopi- lodinium, harmful events presumably linked to these cal analyses and are less time consuming for large dinoflagellates have been documented. The first numbers of samples, although they require consider- reported ecological damage and economic loss due able front-end work to design and verify approaches to a Cochlodinium bloom in Californian coastal (Miller and Scholin 1996, Countway and Caron waters occurred in Monterey Bay (2007) and 2006). Quantitative real-time polymerase chain reac- resulted in a loss of commercially farmed abalone tion (qPCR) based on rDNA can provide species- (http://www.cencoos.org/documents/about/Abalone_ specific identification and has now been successfully success.pdf). Additional reports of mussel mortali- developed and employed for a number of HAB ties were observed in Monterey Bay during blooms species (Coyne et al. 2001, Moorthi et al. 2006). (Curtiss et al. 2008). These increases in cell abun- The increasing frequency of C. fulvescens blooms dances of Cochlodinium, and their resulting harmful on the United States, West Coast, the lack of an effects, raise concern for more effective monitoring established culture of this species, and the difficulty of these species and the need for tools to study the of identifying Cochlodinium species based on mor- ecology of this harmful organism. phological criteria made this species a prime candi- Species of Cochlodinium are morphologically simi- date for the development of a qPCR approach. We lar and difficult to differentiate by light microscopy exploited the unique sequence signature of the 18S except by an expert taxonomist. Recently, the mor- rDNA gene from local Cochlodinium sp. as well as phologies of C. polykrikoides Margalef, C. catenatum two GenBank sequences (AB288381 and AB288380, Okamura, C. convolutum Kof. et Swezy, and C. fulves- identified as C. fulvescens in GenBank) to design cens Iwataki, Kawami et Matsuoka were compared and test a Molecular BeaconÒ approach to identify (Matsuoka et al. 2008), and phylogenetic relation- and quantify abundance of C. fulvescens in natural ships were also proposed for some Cochlodinium spe- water samples. The approach was then applied to cies based on DNA sequence information (Iwataki samples from local coastal waters to begin to eluci- et al. 2007, 2008). Results of these studies con- date the environmental conditions conducive to firmed that differences in morphological character- algal blooms, such as temperature, nutrient concen- istics among C. polykrikoides, C. convolutum, and trations, rainfall, and river runoff (Hallegraeff et al. C. fulvescens species were subtle, whereas C. catena- 1995, Thompson et al. 2008), and to determine if tum Okamura (reported from La Jolla CA by Kofoid any of these conditions correlate to blooms of C. ful- and Swezy 1921) is a different species than C. catena- vescens in California. tum sensu Okamura (Matsuoka et al. 2008). Phyloge- netic results based on LSU rDNA gene sequences MATERIALS AND METHODS revealed that C. polykrikoides can be distinguished Collection and processing of environmental samples. Cochlodinium from C. fulvescens, but that they are closely related cells were obtained from a natural seawater sample collected sister species. Samples from a 2006 bloom of Coch- during a Cochlodinium bloom on 4 December 2006, in Santa lodinium in Monterey Bay as well as samples from Monica Bay in southern California (33º54.2¢ N, 118º28.22¢ W). British Columbia, Canada were included in those