Resolving the intra-specific succession within Cochlodinium polykrikoides populations in southern Korean coastal waters via use of quantitative PCR assays

Harmful Algae 37 (2014) 133–141

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Resolving the intra-specific succession within Cochlodinium polykrikoides populations in southern Korean coastal waters via use of quantitative PCR assays Bum Soo Park a, Pengbin Wang a, Jin Ho Kim a, Joo-Hwan Kim a, Christopher J. Gobler b, Myung-Soo Han a,c,* a

Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791, South Korea School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY 11794-5000, USA c Research Institute for Natural Sciences, Hanyang University, Seoul 133-791, South Korea b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 9 September 2013 Received in revised form 30 April 2014 Accepted 30 April 2014

While the toxic dinoflagellate Cochlodinium polykrikoides is known to form blooms that are maintained for extended periods, the genetic differentiation of these blooms are currently unknown. To assess this, we developed a real-time PCR assay to quantify C. polykrikoides at the intra-specific level, and applied this assay to field samples collected in Korean coastal waters from summer through fall. Assays were successfully developed to target the large-subunit ribosomal RNA region of the three major ribotypes of C. polykrikoides: Philippines, East Asian, and American/Malaysian. Significant linear relationships (r2  0.995) were established between Ct and the log of the copy number for each ribotype qPCR assay. Using these assays, C. polykrikoides blooms in Korean coastal waters were found to be comprised of Philippines and East Asian ribotypes but not the American/Malaysian ribotype. The Philippines ribotype was found to be highly abundant during summer bloom initiation and peak, whereas the East Asian ribotype became the dominant ribotype in the fall. As such, this newly developed qPCR assay can be used to quantify the cryptic ecological succession of sub-populations of C. polykrikoides during blooms that light microscopy and previously developed qPCR assays cannot resolve. ß 2014 Published by Elsevier B.V.

Keywords: Cochlodinium polykrikoides Ribotype Quantitative real-time PCR Field application

1. Introduction The ichthyotoxic unarmored dinoflagellate Cochlodinium polykrikoides a taxa of harmful algal blooms (HAB) responsible for substantial mortality to both wild and farmed fish (Kudela and Gobler, 2012). The presence of C. polykrikoides has been reported in tropical, subtropical and temperate waters, such as British Columbia, Canada (Whyte et al., 2001), the US east coast (Gobler et al., 2008), Mexico in the eastern Pacific, the coastal waters of Costa Rica (Vargas-Montero et al., 2004, 2006), Japan (Yuki and Yoshimatsu, 1989), China (Qi et al., 1993), and Korea (Kim, 1998a; Cho and Costas, 2004). In recent decades, harmful algal blooms caused by C. polykrikoides Margalef have exhibited an apparent increase in harmful impacts worldwide (Kudela and Gobler, 2012).

* Corresponding author at: Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791, South Korea. Tel.: +82 2 2220 0956; fax: +82 2 2296 1741. E-mail address: [email protected] (M.-S. Han). http://dx.doi.org/10.1016/j.hal.2014.04.019 1568-9883/ß 2014 Published by Elsevier B.V.

At the same time, the economic damage to fisheries and aquaculture due to massive mortality has also increased sharply, particularly in Korea (Kim et al., 2001, 2007). In 1995, a particularly severe and widespread C. polykrikoides bloom persisted for nearly eight weeks along the entire south coast of Korea, ultimately resulting in economic losses of up to 95 million US dollars (Kim, 1998b). Since then, harmful algal blooms of this species have been an annual feature along southern Korean coastal waters. Therefore, intensive investigation, including studies of growth characteristics and vertical migration, have been conducted to obtain a better understanding of the factors influencing the formation of C. polykrikoides blooms (Park et al., 2001; Kim et al., 2004; Jeong et al., 2004). In addition, various studies have been undertaken to clarify the mechanisms of C. polykrikoides bloom formation, such as overwintering strategies, hyaline cysts (temporary cyst), and resting cysts (Matsuoka and Fukuyo, 2000; Kim et al., 2002; Tang and Gobler, 2012), and the current-driven movement of C. polykrikoides blooms in the Andaman Sea, the East China Sea, and the East Sea/the Sea of Japan have been evaluated using oceancolor satellite imagery (Azanza and Baula, 2005; Miyahara et al.,

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2005; Ahn et al., 2006; Kim et al., 2007). However, the bloom formation mechanism of this species is not yet fully understood. In particular, it is unclear how C. polykrikoides blooms are maintained for such long periods (8 weeks; Kim et al., 2001; Gobler et al., 2008). Many researchers have suggested that genetic differentiation within C. polykrikoides may be one explanation for long-term bloom maintenance (Kudela and Gobler, 2012), as genetic differentiation may lead to variations in physiological and ecological characteristics. Similarly to C. polykrikoides bloom, the toxic dinoflagellate Alexandrium fundyense bloom occurred during 8 weeks (May to July, 2005) and affected over 700 km of coastline on the U.S. Northeast Coast (Anderson et al., 2005). According to Erdner et al. (2011), A. fundyense bloom in the northeastern U.S. harbor composed by at least two genetically distinct sub-populations. Moreover, two sub-populations of A. fundyense are responsible for early- and late bloom and originated from the northern and southern areas of the bloom, respectively. Therefore, clarification of genetic differentiation of sub-populations could allow for improved understanding of the mechanisms of C. polykrikoides blooms. Recently, Iwataki et al. (2008) reported that Cochlodinium polykrikoides had significant genetic differentiation in the large ribosomal subunit, and could therefore be separated into three distinct sub-clades in the phylogenetic tree generated in that study. These clades or ‘‘ribotypes’’ can be phylogenetically diverse groups of C. polykrikoides differentiated based on their large-subunit (LSU) ribosomal RNA gene sequences. The three ribotypes are the East Asian (Hong Kong, Japanese, and Korean), Philippines, and American/Malaysian ribotypes. Monitoring the dynamics of these ribotypes in field during blooms would significantly advance our understanding of C. polykrikoides blooms. However, light microscopy is incapable of discriminating between these ribotypes due to nearly identical morphologies (Iwataki et al., 2008). Even current quantitative real-time PCR assays (Park and Park, 2010) cannot distinguish among the three ribotypes as the molecular markers used are optimized for species-, not clonal-level, distinction. Hence, the aim of this study was to develop a sensitive and accurate assay for the quantitative analysis of all three C. polykrikoides ribotypes

and examine the suitability of these qPCR assays for tracking the dynamics of these ribotypes during blooms in Korean waters. 2. Materials and methods 2.1. Algal cultures Algal strains were obtained from the CCMP (Provasoli-Guillard National Center for Marine Algae and Microbiota, ME, USA), the National Research Laboratory for Water Environmental Ecology and Restoration of Hanyang University (Seoul, South Korea), the KIOST (Korea Institute of Ocean Science and Technology, Ansan, South Korea), the NIES (National Institute for Environmental Studies, Tsukuba, Japan), and the Gobler laboratory (School of Marine and Atmospheric Sciences, Stony Brook University, NY, USA) (Table 1). All strains were cultured at 20 8C in f/2 growth medium (Guillard, 1975) or GSe medium (Doblin et al., 1999) with a salinity of 31–33 under cool-white fluorescent lamps (photon flux of 100 mE m2 s1) on a 12-h light:12-h dark photoperiod. Cryptoperidiniopsis brodyi (CCMP 2781, 2782), Luciella masanensis (CCMP 1835, 1873), Pfiesteria piscicida (CCMP1830, 1831), and Pseudopfiesteria shumwayae (CCMP 2089, 2807) were maintained in f/2 medium with a salinity of 15 at 20 8C and fed Rhodomonas sp. (CCMP 768). Table 1 shows the list of the strains used in this study. 2.2. Collection and processing of environmental samples Field sampling was performed from August to November 2009 on Geum-o Island (St. Y) near Yeosu in Jeollanam-do and Mi-jo Harbor (St. M) in Gyeongsangnam-do, which are both located in the southern part of Korea (Fig. 1). The water depths were as follows: (i) St. Y, 29–31 m, and (ii) St. M, 3–4 m. Water samples were collected at six water depths at St. Y (0, 3, 5, 10, 20, and 30 m) and two water depths at St. M (0 and 3 m), in this sequence, using a 4.2 l Van Dorn water sampler (Wildlife supply company, MI, USA). One liter of each water sample was fixed with 1% Lugol’s solution. After gentle mixing, the preserved field samples were counted

Table 1 Specificity of the primer sets used in qPCR with EvaGreen. Species

Strain

CPSF2

PhiCPSF

AMCPSF

CPSR3

PhiCPSR

AMCPSR

CPSF2

PhiCPSF

AMCPSF

CPSR3

PhiCPSR

AMCPSR

n.d

n.d

n.d

n.d

n.d

n.d

HY981028M

n.d

n.d

n.d

Fibrocapsa japonica

Akashiwo sanguinea

0806-HYPH-AS10

n.d

n.d

n.d

Gymnodinium catenatum

D-133 /CCMP 1661 GnCt-K01

Amphidinium sp.

CCMP 1684

n.d

n.d

n.d

Gymnodinium impudicum

NF-F-GIM-1

n.d

n.d

n.d

Chattonella antiqua

CCMP 2050 /YSIP0806 CCMP 2049 /CMGM

n.d

n.d

n.d

Heterocapsa triquetra

HtTq_K01

n.d

n.d

n.d

n.d

n.d

n.d

Heterosigma akashiwo

CCMP 452 /NIES 298

n.d

n.d

n.d

Chattonella ovata

JH0805

n.d

n.d

n.d

Prorocentrum micans

KMCC D-086

n.d

n.d

n.d

Chattonella subsalsa

CCMP 217

n.d

n.d

n.d

Cryptoperidiniopsis ribotype

CCMP 1828

n.d

n.d

n.d

Pfiesteria piscicida

CCMP 1830 /CCMP 1831 CCMP 1835 /CCMP 1873

n.d

n.d

n.d

n.d

n.d

n.d

CCMP 2089 /CCMP 2807 CCMP 2781 /CCMP 2782

n.d

n.d

n.d

n.d

n.d

n.d

Cochlodinium polykrikoides East Asian ribotype

Philippines ribotype American/ Malaysian ribotype n.d: Not detected. Pos: Positive.

y

Strain

Alexandrium sp.

Chattonella marina

*

Species

y

EA-CP 01 /KORDI-CP /Regular-CP HYID1108-CP

Pos

n.d

n.d

Luciella masanensis

n.d

Pos

n.d

Pfiesteria shumwayae

CPMHC-4/ CPMC-40C/ CP 01/CPOFP-11

n.d

n.d

Pos

Cryptoperidiniopsis brodyi

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cold 70% ethanol was added, and the samples were centrifuged at 14,000  g for 15 min. The pellets were dried in air before being dissolved in 100 ml of TE buffer (10 mM Tris–HCl, 1 mM EDTA; pH 8). 2.4. Specific primer design

Fig. 1. Map indicating the locations of sampling stations Y and M in southern Korean coastal waters.

using a Sedgwick-Rafter counting chamber with a light microscope at 200 magnification (Axioplan, Carl Zeiss, Jena, Germany). The phytoplankton in the field samples were also identified under a light microscope, though at a higher magnification (Axioplan, Carl Zeiss, Jena, Germany, 400 and 1000 magnification). Samples for subsequent DNA extraction were filtered onto a 3-mm, 47-mm diameter ISOPORE membrane filter (MILLIPORE, Cork, Ireland). Loaded filters were placed in a 2-ml microtube (Axygen Sciences, CA, USA) that contained 800 ml of extraction buffer (100 mM Tris– HCl, 100 mM Na2-EDTA, 100 mM sodium phosphate, 1.5 M NaCl, 1% CTAB) and were stored at 80 8C until use. 2.3. DNA extraction DNA extraction of samples was performed following the EX DNA extraction protocol (Harder et al., 2003). To summarize, the 2-ml microtubes containing membrane filters were immersed in liquid N2 until completely frozen, then thawed in a 65 8C water bath. After the addition of 8 ml of proteinase K (10 mg ml1 in TE buffer), the samples were incubated at 37 8C for 30 min. Following the addition of 80 ml of 20% sodium dodecyl sulfate (SDS) made using doubledistilled water, the samples were incubated at 65 8C for 2 h, shaken with an equal volume of chloroform-isoamyl alcohol (24:1), then centrifuged at 10,000  g for 5 min. The aqueous phase of the mixture was transferred to a new tube, into which 88.8 ml of 3 M sodium acetate, pH 5.1, made with double-distilled water, and 586.08 ml of isopropanol (99%) were added. Following centrifugation at 14,000  g for 20 min, the supernatant was decanted, 1 ml of

The unique regions of the target sequences for each ribotype of Cochlodinium polykrikoides were identified from data obtained from Genbank (www.ncbi.nih.gov) and from alignments made using Clustal W (Thompson et al., 1997). The specific primer sets for the 28S ribosomal RNA region produced products (Table 2) that were 219 bp for the East Asian ribotype (CPSF2-CPSR3), 177 bp for the Philippines ribotype (PhiCPSF-PhiCPSR), and 148 bp for the American/Malaysian ribotype (AMCPSF-AMCPSR). To compare rRNA copy numbers in C. polykrikoides, we designed a primer set for all ribotypes (UNICPF-UNICPR), which yielded a product size of 154 bp. The melting temperatures of the specific primer sets were estimated using Primer 3 (Whitehead Institute and Howard Hughes Medical Institute, USA), and the formation of primer dimers was analyzed using Beacon Designer (PREMIER Biosoft International Inc., CA, USA). The specificities of the forward and reverse primers were confirmed using BLAST (Altschul et al., 1997). 2.5. PCR amplification and DNA sequencing To assess the specificity of the designed primer set, competitive PCR was carried out in 1 PCR buffer, which contained less than 0.1 mg genomic DNA template of target and non-target algae (Table 1), 0.3 mM of each primer, dNTP mixture (0.25 mM of each dNTP), 0.2 units Takara Ex Taq polymerase (TaKaRa, Osaka, Japan), and PCR-grade water to a final volume of 20 ml. Using a thermoblock (iCycler, Bio-Rad, CA, USA), thermocycling was conducted as follows: 95 8C for 4 min, followed by 35 cycles of denaturation at 95 8C for 20 s, annealing at 61.5 8C (East Asian ribotype), 62 8C (Philippines ribotype), or 64 8C (American/Malaysian ribotype), and extension at 72 8C for 50 s. After denaturation, the final extension was completed at 72 8C for 5 min. PCR-amplified products were analyzed using 1.2% agarose gel electrophoresis by the standard methods (Sambrook and Russell, 2001). For direct DNA sequencing, PCR amplicons were purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany). DNA sequencing reactions were performed using the ABI PRISM1 Big DyeTM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, CA, USA), with the PCR product as the template. 2.6. Real-time PCR assay for standard curve construction Real-time PCR assays were performed in a total reaction volume of 15 ml, which contained 7.5 ml of 1 SsoFastTMEvaGreen1 Supermix (Bio-Rad, CA, USA), 0.3 mM of the primer set, 2 ml of genomic DNA (approximately 0.1 mg), and double-distilled water. qPCR reactions were run using a Chromo 4 Detection System (BioRad, CA, USA) at 98 8C for 2 min, followed by 40 cycles at 98 8C for 5 s, then 61.5 8C (East Asian ribotype), 62 8C (Philippines ribotype), 64 8C

Table 2 Sequence information of each ribotype-specific primer set and the primer set (UNICPF-UNICPR) for the comparison of the rRNA gene copy number within C. polykrikoides strains. Target species

Region

Cochlodinium polykrikoides

Large subunit (28S)

Ribotype East Asian (Korean/Japanese) Philippines American/Malaysian East Asian, Philippines, American/Malaysian

Primer

Sequence (50 –30 )

CPSF2 CPSR3 PhiCPSF PhiCPSR AMCPSF AMCPSR UNICPF UNICPR

AAC GCA AGT GTG AGT GTG AGT T GGA CCC ACG ATC AAC CCA TGC AAG TTT CAA CCA TCT CTC GC GAA AGC AAG TTC AAT CGA CGG TTT CTC AAT CGC CTT TCG CCT GAT ACC GGA CAC CTC GGA TAT GAT ACG ACC AAA TGG TTC TTT CC CTC AAA CGT CTC GCA ATT GA

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(American/Malaysian ribotype), or 60 8C (all ribotypes) for 10 s. After denaturation, melting curves were monitored from 65 to 95 8C in 0.2 8C increments or 0.5 8C increments with a 10-s hold at each step. To construct a standard curve, KORDI-CP (East Asian ribotype), HYID1108-CP (Philippines ribotype) and CPOFP-11 (American/ Malaysian ribotype) were harvested between 9 and 11 a.m. to minimize rRNA gene copy variability due to diurnal cell cycle. Then, each ribotype of Cochlodinium polykrikoides was counted using a Sedgwick-Rafter counting chamber with a light microscope at 200 magnification (Axioplan, Carl Zeiss, Jena, Germany), placed on filters, and DNA was extracted as described above. Serial 10-fold dilutions of the DNA extracts were used to construct the standard curve. 2.7. Comparison of rRNA gene copies for Cochlodinium polykrikoides To examine the rRNA gene copy variability, we counted eight strains of Cochlodinium polykrikoides by light microscopy and qPCR assay which calculated cell numbers using standard curve of one strain (CPMHC-4), and compared two measurements following Park et al. (2012). All samples were harvested cells from cultures in log phase growth between 9 and 11 a.m. to minimize gene copy variability due to cell cycle. To examine the rRNA gene copy variability, eight strains of C. polykrikoides, including the East Asian (EA-CP01, KORDI-CP, and Regular-CP), Philippines (HYID1108-CP), and American/Malaysian (CPMHC-4, CPMC-40C, CP01, and CPOFP-11) ribotypes, were preserved and counted using a Sedwick-Rafter chamber under a light microscope at 200x magnification. Concurrently, samples for DNA extraction were filtered through a 3-mm, 47-mm diameter ISOPORE membrane filter (Millipore, Cork, Ireland). Genomic DNA was extracted through the EX method described above (Harder et al., 2003). Diluting samples 10- or 20-fold effectively minimized PCR inhibitors. Using the standard curve generated by plotting and CPMHC-4 (American/Malaysian ribotype), the cell counts of other strains were quantified. To assess rRNA gene copy variability among C. polykrikoides strains, we compared the cell number results from both the direct microscopic counting and qRT-PCR assay.

designed primer set were 177 bp (Philippines ribotype), 219 bp (East Asian ribotype), and 148 bp (American/Malaysian ribotype; Table 2). To verify the specificity, three primer sets were used for competitive PCR with genomic DNA from other algae containing sequences similar to that of Cochlodinium polykrikoides. The algae used for the specificity test consisted of 24 dinoflagellate and 10 raphidophyte strains (Table 1). Each of the primer sets aligned only to the target ribotype of C. polykrikoides, and did not produce nonspecific amplicons from the non-target algae (Table 1). Analysis of the melting curves of the EvaGreen-based PCR assay can be useful for the detection of false positives due to unexpected products or primer dimers. All PCR products resulting from the alignment with each ribotype-specific primer set had one informative melting curve peak (Fig. 2). The melting temperatures were 85 8C (Philippines ribotype), 88 8C (East Asian ribotype), and 86 8C (American/ Malaysian ribotype), and peak widths were 1.75 8C (Philippines ribotype), 1.62 8C (East Asian ribotype), and 1.83 8C (American/ Malaysian ribotype; Fig. 2A–C). On gel analysis, only one amplified band was detected, and neither primer dimers nor unexpected amplicons were present. These results suggested that the designed primers were highly specific to each Cochlodinium polykrikoides strain and was acceptable for our qPCR field studies. 3.2. Accuracy of quantitative real-time PCR assay To assess the suitability of the primer set for accurately quantifying the East Asian, Philippines, and American/Malaysian ribotypes of Cochlodinium polykrikoides, a standard curve was constructed with ten-fold serial dilutions of the genomic DNA extracted from each ribotype of C. polykrikoides. Quantitative real-time PCR assays were performed in triplicate, and the mean value of the Ct (threshold cycle) was obtained. There was a strong linear relationship between the Ct value and the log of the copy number (r2  0.995) in all standard curves plotted for each ribotype of C. polykrikoides (Fig. 3A–C). The reaction efficiencies (E), as calculated by the formula E = 10(1/m)  1, where m is the slope of the standard curve (Rebricov and Trofimov, 2006), were 95% (Philippines ribotype), 91% (East Asian ribotype), and 94% (American/Malaysian ribotype; Fig. 3A–C).

2.8. Removal of PCR inhibitors analysis of field samples via qPCR assay PCR inhibitors affect the accuracy of the values measured in real-time PCR assays. The absence of PCR inhibitors was confirmed using a qPCR assay with primers specific for Chattonella subsalsa. Because C. subsalsa has not been reported in Korean coastal waters. C. subsalsa DNA spiked into the serial dilutions of DNA extracted from the field samples (dilution factors: 0, 10, 50, 100) and C. subsalsa without field sample DNA as a positive control were amplified using real-time PCR (Park et al., 2012). The optimal DNA dilution factor for the field samples was calculated by comparing their Ct values and PCR efficiency with positive control. Genomic DNA was extracted from field samples, as described above. Each ribotype of Cochlodinium polykrikoides in field samples was quantified via qPCR assay with a specific primer set. qPCR was performed under the same reaction conditions and PCR program as described above. While highly unlikely due to the specificity of our primers, false positives were assessed by performing sequence analyses of PCR products and melting curve analysis were undertaken using a 4200 Dual Dye Automated Sequencer (Li-Cor, NE, USA), following the manufacturer’s instructions, and a qPCR assay. 3. Results 3.1. Specificity of primer set for each ribotype The sequence lengths of the 28S ribosomal RNA-LSU (large subunit) region amplified by our primers yielded from the

3.3. Seasonal fluctuation of Cochlodinium polykrikoides via light microscopy and real-time PCR assay While PCR efficiency and accuracy can be low in field samples due to the presence of PCR inhibitors, dilution can be utilized to overcome this challenge (Park et al., 2012). In our results, there was no PCR inhibitor effect resulting in the underestimation of spiked Chattonella subsalsa in field samples when dilution factor was higher than ten and thus DNA extracts of field samples were diluted by 10-fold. During our sampling around Geum-O Island (St. Y) and Mi-jo Harbor (St. M), qPCR assay detected the Philippines ribotype in all samples at St. Y (42 samples in total) and 13 samples at St. M, while the East Asian ribotype was detected in 31 samples at St. Y and 8 samples at St. M (Figs. 4 and 5). However, the American/Malaysian ribotype was not detected in any of the samples. Sequencing of PCR products confirmed that the amplified PCR products matched each Cochlodinium polykrikoides ribotype targeted by the specific primer sets. Positive samples were quantified using an EvaGreen-based real-time PCR assay. The Philippines ribotype had a high abundance in September, with cell densities of 80 cells per liter at St. Y and 355 cells per liter at St. M (Figs. 4 and 5). The East Asian ribotype, conversely, had the highest abundance in November, with cell densities of 243 cell per liter at St. Y and 17,844 cells per liter at St. M. Hence, these results showed that the Philippines and East Asian ribotypes had different seasonal bloom patterns, despite belonging to the same species, C. polykrikoides (Figs. 4 and 5).

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Fig. 2. Melting curves obtained using the EvaGreen-based assay with DNA extracts from each C. polykrikoides ribotype. A: East Asian ribotype. B: Philippines ribotype. C: American/Malaysian ribotype. D: CPMHC-4, belonging to the American/Malaysian ribotype. Melting temperatures were 88 8C (East Asian ribotype), 85.0 8C (Philippines ribotype), 86 8C (American/Malaysian ribotype), and 85.0 8C (CPMHC-4).

It is generally known that qPCR is more sensitive than the direct counting method. The difference in the sensitivities between the two assays was demonstrated well in our study. The direct counting method via light microscopy was not able to detect Cochlodinium polykrikoides in most samples, whereas the qPCR assay detected C. polykrikoides in all samples, even though the cell densities were 46 cells per liter on November 19th in a surface sample obtained at St. Y (Fig. 4). However, in the case of the samples from November 11th, which were obtained from a depth of 3 m (St. M), the value from direct counting (2496 cells per liter) was higher than that from the qPCR assay (about 712 cells per liter). In addition, qPCR yielded results exceeding those of microscopic counts on September 10th (3 m depth) and 23rd (20 m depth) at St. Y, and September 3rd (on the surface) at St. M (Figs. 4 and 5). 3.4. Variability of rRNA gene copies in Cochlodinium polykrikoides The rRNA gene copy number of Cochlodinium polykrikoides strains within the Philippines ribotype (HYID1108-CP), East Asian ribotype (EA-CP01, KORDI-CP, and Regular-CP), and American/ Malaysian ribotype (CP01, CPMS-40C, and CPOFP-11) were compared to C. polykrikoides (CPMHC-4; American/Malaysian ribotype). The qPCR standard curve of CPMHC-4 showed a significant linear relationship (r2  0.999) between Ct and the log of the cell count (Fig. 3D). On the basis of melting curve analysis, the species-specific primer set (UNICPF-UNICPR) aligned successfully with all ribotypes of C. polykrikoides (Fig. 2D). Therefore, qPCR assay was reliable for quantifying and comparing rRNA copy number among C. polykrikoides strains.

The cell number of the East Asian ribotype measured slightly higher (1.22–2.2-fold) by direct counting than the value calculated using the standard curve of CPMHC-4 (Table 3). The cell number of the American/Malaysian ribotype was calculated to be slightly lower (1.26–2.2-fold) than the value determined from the standard curve of CPMHC-4 (Table 3). For the Philippines ribotype (HYID1108-CP), the cell number calculated using a standard curve was almost 10 times lower than the value obtained by direct counting (Table 3). Consequently, on average, the rRNA gene copy number of the American/Malaysian ribotype was about 2-fold higher than that of the East Asian ribotype and 10-fold higher than that of the Philippines ribotype (Table 3). 4. Discussion Cochlodinium polykrikoides has been shown to adapt to diverse environments, such as those found offshore, and possibly even to tropical and subtropical waters with warm temperatures (>20 8C) and moderate (a salinity of 30–33) salinities (Kim et al., 2004; Nagai et al., 2009; Kudela and Gobler, 2012). Some C. polykrikoides blooms have occurred in low water temperatures (<17 8C) and brackish water, even though these environmental conditions are unfavorable for growth (Lee and Lee, 2006; Lee et al., 2009; Gobler et al., 2008). These studies showed that C. polykrikoides had wide eco-physiological traits. However, none of these studies can fully explain how C. polykrikoides is able to sustain extended blooms in various environments. According to previous studies, high level of genetic differentiation can allow a population to rapidly adapt to environmental changes, since genetic differentiations typically lead to physiological variability in traits such as irradiance, salinity

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Fig. 3. Standard curves of the EvaGreen-based assay using ten-fold serial dilutions of C. polykrikoides DNA extracts. A: East Asian ribotype. B: Philippines ribotype. C: American/Malaysian ribotype. D: CPMHC-4, belonging to the American/Malaysian ribotype. Error bars indicate standard deviation.

and temperature tolerance (Moore et al., 1998; Lebret et al., 2012). Such genetic differentiation could account for C. polykrikoides blooms under diverse environmental conditions for extended periods of time. Prior to this study, there have been no methods to investigate how genetic differentiation of C. polykrikoides may change during blooms. Quantitative real-time PCR is the most powerful and sensitive assay for the detection and quantification of microorganisms (Walker, 2002; Coyne et al., 2005). Since its development, qPCR

assays has been developed to detect and quantify harmful algae in the field (Bowers et al., 2006; Gray et al., 2003; Popels et al., 2003; Galluzzi et al., 2004; Handy et al., 2005; Park et al., 2007). However, in previous HAB studies, qPCR assays have not discriminated subpopulation as the molecular markers were typically designed for detection at the species level. In this study, we developed quantitative real-time PCR assays with molecular markers capable of distinguishing and quantifying sub-population of Cochlodinium polykrikoides.

Fig. 4. The seasonal fluctuation of each C. polykrikoides ribotype at St. Y from September to November in 2009 based on light microscopy (top) and quantitative real-time PCR (middle: East Asian ribotype; bottom: Philippines ribotype). The American/Malaysian ribotype was not detected.

Fig. 5. The seasonal fluctuation of each C. polykrikoides ribotype at St. M from August to November in 2009 based on light microscopy (left) and quantitative real-time PCR (middle: East Asian ribotype. Right: Philippines ribotype). The American/ Malaysian ribotype was not detected.

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Table 3 Calculated cell number per milliliter by direct counting and quantitative real-time PCR assay for the comparison of rRNA gene copy numbers among strains and ribotypes. qPCR values represent means  SD. Species C. polykrikoides

Ribotype East Asian

American/Malaysian

Philippines

Strain

Cells number via light microscope

Cells number via standard curve

EA-CP 01 KORDI-CP Regular-CP CP 01 CPMS-40C CPOFP-11 HYID1108-CP

1232 238 210 373 861 3056 98

571  13 117  2 167  4 567  9 1895  32 4456  579 9.82  0.46

Ribosomal RNA gene regions are commonly used to identify microorganisms due to the high degree of conservation within these regions (Ki et al., 2005, 2009; Ki and Han, 2006, 2007a). The D1–D3 region, located in the large subunit (LSU) ribosomal DNA, is best suited for identifying strain-level differences as this region is hyper-variable, even within the same species (Ki and Han, 2008). According to Iwataki et al. (2008), Cochlodinium polykrikoides is classified into three phylogenetic sub-clades on the basis of their distinct geographic locations (East Asian, Philippines, and American/Malaysian clades). These clades within C. polykrikoides, which are based on LSU rDNA sequences, are considered ‘‘ribotypes,’’ and are phylogenetically diverse groups. Therefore, specific primer sets for the detection and quantification of individual ribotypes were developed based on LSU rDNA (Table 2). While cross-reactivity tests for specificity were performed with species that had sequences most similar to those of C. polykrikoides, specific primer sets designed for this study did not amplify non-target species or different ribotypes (Table 1). On the basis of these results, each of the specific primer sets was deemed to be appropriate for the specific detection and quantification of each ribotype of C. polykrikoides. The rRNA gene has been PCR amplified more successfully than single-copy genes since the eukaryotic rRNA gene is organized in tandem repeats, with copy numbers up to the order of 10,000 (Schlo¨tterer, 1998). Therefore, based on this feature, a primer set for the rRNA gene can be amplified even if the extracted DNA sample contains less than one cell due to dilution. For example, Park et al. (2012) demonstrated that the sensitivity of PCR to amplify the rRNA gene of Heterosigma akashiwo was 0.25 cells. In this study, the rRNA genes of the Philippines (0.1 cells), East Asian (0.05 cells), and American/Malaysian (0.3 cells) ribotypes were successfully amplified, even at levels of less than one cell (Fig. 3). This result suggests that the LSU rRNA gene is a suitable region for qPCR assays of these ribotypes. Accurate enumeration of target algae via qPCR requires highly significant standard curves with strong correlations across serial dilutions of target DNA. In this study, all standard curves were highly significant (r2 > 0.995) (Fig. 3). All reaction efficiencies exceeded 91%, indicating successful polymerase chain reaction. Melting curve analysis is generally used to assess for the presence of unexpected amplicons or primer dimers. In our study, all melting curves had one informative and narrow peak (Fig. 2) suggesting that only the target region was successfully amplified. The comparison of microscopic and qPCR data, was useful to assess whether the qPCR assay is reliable for the enumeration of target algae in field samples. We compared the results of light microscopy and qPCR at the species level, as light microscope observations cannot discriminate ribotypes within Cochlodinium polykrikoides. Cell densities, determined by the qPCR assay were generally greater than those determined by direct counting via light microscopy for most samples (Figs. 4 and 5). Using direct counting, it can be difficult to detect and quantify microalgae at low densities, whereas qPCR can easily detect and quantify target algae, even at a low density (Park et al., 2012). Conversely, for

Ribotype (strain) for plotting standard curve American/Malaysian (CPMHC-4)

samples collected on November 11th at St. M at a depth of 3 m, the value from direct counting (2496 cells per liter) was higher than that detected by the qPCR assay (about 712 cells per liter). In addition, reverse sensitivity was observed in the samples collected on September 10th (3 m depth) and 23rd (20 m depth) from St. Y and on September 3rd (surface) sample from St. M (Figs. 4 and 5). Values quantified via direct counting may be higher than those quantified via qPCR assay if filtration pressure results in cell loss. Unarmored dinoflagellates such as C. polykrikoides can be fragile and may be more likely to lyse under pressure, than other cells (Ki and Han, 2007b). Alternatively, rRNA gene copy number variability within C. polykrikoides may also account for methodological differences in quantification. According to Galluzzi et al. (2010), variability in the rRNA gene copy number was observed in two Mediterranean Alexandrium species, A. catenella, and A. taylori, resulting in the low accuracy of quantitative real-time PCR assays. To investigate the variability in rRNA gene copies, we confirmed gene copies variability within C. polykrikoides strains by comparing rRNA gene copies (Park et al., 2012). As was the case with Alexandrium spp., there was a ten-fold difference in gene copy number between the HYID1108-CP and CPMHC-4 strains (Table 3). In addition, EA-CP01 and CP01 had similar calculated values via the qPCR assay, whereas cell numbers through direct counting differed by more than four-fold (Table 3). Interestingly, however, the difference in the rRNA gene copies was less apparent when C. polykrikoides strains did not belong to the same ribotype (Table 3). Although, strains belonging to the American/Malaysian or East Asian ribotype showed slightly rRNA gene copies variability respectively, these differences of rRNA gene copy number did not exceed 2-fold. The cause of slight rRNA gene copy variability among the same ribotype may be related to the cell cycle. During Sphase of the cell cycle, the DNA within the nucleus starts to replicate, and cells contain twice the amount of DNA contents prior to mitosis (Cooper, 2000). Dinoflagellates are generally haploid and phased division is synchronized by light-dark cycle. To prevent variability originating from cell cycle, all samples were harvested from culture in log phase between 9 and 11 a.m., respectively. Ideally, all cells in these cultures should have been in the same phase of the cell cycle at the same time, but in reality they cannot be completely synchronized. Hence, the slight rRNA gene copy variability within the same ribotype might be affected by DNA content differences associated with the cell cycle rather than the difference of gene copies number. Thus, rRNA gene copy number variability did not seem to significantly affect the accuracy of qPCR for quantifying each C. polykrikoides ribotype. Moreover, in our results, the cell numbers determined using the two assays were significantly correlated (p < 0.0001, n = 59) and had generally similar dynamic patterns (Figs. 4 and 5). All of these finding suggest these qPCR assays can reliably detect and quantify each ribotype of C. polykrikoides in field samples. The PCR efficiency was remarkably decreased due to PCR inhibitors in several samples extracted from our field samples (Park and Han, unpublished data). It is widely known that the main

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obstacle to obtaining accurate and reproducible results from qPCR of field samples is low PCR efficiencies or false-negative results due to the presence of various PCR inhibitors, such as mucopolysaccharides, phenolic compounds, humic acids, and heavy metals in field samples (Wilson, 1997; Audemard et al., 2004, 2006; Vadopalas et al., 2006; Faveri et al., 2009). Chemical treatments to eliminate PCR inhibitors include polyvinylpolypyrrolidone and polyvinylpyrrolidone, and purification of DNA extracts (Wilson, 1997; Guy et al., 2003; Zhang and Lin, 2005; Lin et al., 2006). During this study, ten-fold sample dilution proved to be a simple method to effectively remove PCR inhibitors from field samples that maintained the efficiency of PCR reactions. Previously, the East Asian ribotype was thought to be the only sub-population of Cochlodinium polykrikoides distributed across Korean coastal waters (Iwataki et al., 2008). However, our qPCR assays demonstrated that the Philippines and East Asian were also present in southern Korean coastal waters while the American/Malaysian ribotype was not. This represents the first report of appearance of Philippines ribotype in southern Korean coastal waters as well as the first report of two ribotypes of C. polykrikoides co-existing within the same water body. Matsuoka et al. (2010) suggested that C. polykrikoides was possibly transported into the northern part of East China Sea by Tsushima Warm Current each year. To better understand such transport mechanisms of C. polykrikoides, it will be necessary to investigate the diversity of ribotypes across the Kuroshio Warm Current. Rynearson and Armbrust (2005) reported that Ditylum brightwellii had a physiological variability among genotypes classified by microsatellite markers. In our study, Philippines and East Asian ribotypes displayed different seasonal patterns at our sampling sites (Figs. 4 and 5). The Philippines ribotype had a high abundance in September when water temperature was relatively high (>24 8C), while the East Asian ribotype showed a peak in cell density in November, when the water temperature was below 17 8C (see Fig. S1 in supplementary data). This pattern suggests that each ribotype has a specific ecological niche facilitated by different eco-physiological traits, even though they all were same species Cochlodinium polykrikoides. Kim et al. (2001) reported that outbreaks of Cochlodinium polykrikoides in Korean coastal waters were characterized by a wide bloom area, extremely high density (up to 48,000 cells per milliliter in 2003), and a long blooming period. The factors that permit this species to maintain ecological competitiveness across a diversity of environmental conditions are currently unknown. Recently, Erdner et al. (2011) reported that two sub-populations of Alexandrium fundyense bloom in U.S. coastal waters, persisted for eight weeks, and were responsible for early- and late blooms, respectively. Similarly, in our results, Philippines and East Asian ribotypes were predominant in September and November, respectively (Figs. 4 and 5). Interestingly, prior studies (Erdner et al., 2011) could not explain how the ecological succession between two sub-populations occurs, partly because quantitative data for two sub-populations were not available. In contrast, our qPCR assays revealed the quantitative, ecological succession that occurred between the two ribotypes of C. polykrikoides. Such ecological succession between these two ribotypes may play an important role in maintaining C. polykrikoides blooms over long periods. Future studies over a wider range of areas and over longer periods using these newly developed qPCR assays will further clarify the differential bloom dynamics of these ribotypes. 5. Conclusion To date, the genetic diversity of HAB events has been poorly studied and primarily investigated via restriction fragment length

polymorphism (RFLP) and amplified fragment-length polymorphism (AFLP). However, the qualitative data provided by such studies have not resolved how such diversity contributes to bloom formation and maintenance. The newly developed qPCR assays described here have provided novel insight regarding the dynamics of Cochlodinium polykrikoides ribotypes in Korean coastal waters. Future studies using these assays could further resolve the differential bloom ecology of these ribotypes as well as assess the origin of blooms in different geographic regions. Acknowledgements We thank Dr. Ying Zhong Tang for providing the Cochlodinium polykrikoides strain CP01, Myo-Kyung Kim, Zhun Li for assistance with field work, and Dr. Jang-Seu Ki and Dr. Sunju Kim for providing kind comments. This research was supported by a grant from Marine Biotechnology Program Funded by Ministry of Oceans and Fisheries, Korea.[SS]

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