Quantification of the paralytic shellfish poisoning dinoflagellate Alexandrium species using a digital PCR

Harmful Algae 92 (2020) 101726

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Quantification of the paralytic shellfish poisoning dinoflagellate Alexandrium species using a digital PCR

T

Hyun-Gwan Leea,b, Hye Mi Kima,b, Juhee Mina,b, Chungoo Parkb,c, Hae Jin Jeongd, Kitack Leee, Kwang Young Kima,b,* a

Department of Oceanography, College of Natural Sciences, Chonnam National University, Gwangju 61186, Republic of Korea Marine Ecosystem Disturbing and Harmful Organisms (MEDHO) Research Center, Gwangju 61186, Republic of Korea c School of Biological Sciences and Technology, College of Natural Sciences, Chonnam National University, Gwangju 61186, Republic of Korea d School of Earth and Environmental Sciences, Seoul National University, Seoul 08826, Republic of Korea e School of Environmental Science and Engineering, Pohang University of Science and Technology, Pohang 37673, Republic of Korea b

ARTICLE INFO

ABSTRACT

Keywords: Abundance Alexandrium spp. ddPCR Identification ITS copy number Paralytic shellfish toxin (PST) Quantification

A ubiquitous dinoflagellate, Alexandrium, produces paralytic shellfish toxin (PST), and its outbreaks have negative impacts on aquaculture, fisheries, human health, and the marine ecosystem. To minimize such damages, a routine monitoring program of toxic species must be implemented with a suitable analytical technique for their identification and quantification. However, the taxonomic identification and cell quantification of Alexandrium species based on their external morphology under a light microscope, or by using conventional molecular approaches have limited sensitivity and reproducibility. To address these challenges, we have developed an advanced protocol using droplet-digital PCR (ddPCR) for the discrimination and enumeration of three co-occurring Alexandrium species (A. affine, A. catenella, and A. pacificum) in environmental samples. Copies of species-specific internal transcribed spacer (ITS) per cell, which were calculated from environmental samples spiked with various numbers of culture cells, were used to estimate the abundance of species in the field samples. There were no significant differences in ITS copies estimated by the digital PCR assay between environmental samples from different localities, spiked artificially with a consistent number of cells from Alexandrium cultures. This sensitive assay was applied to determine the abundance and vertical distribution of those populations in the southern coastal waters of Korea. In spring, A. catenella was the dominant species, followed by the non-toxic A. affine in summers. A novel digital PCR assay can also be used to monitor other harmful marine protists that require high sample throughput and low detection limit with high accuracy and precision.

1. Introduction Harmful algal blooms (HABs) cause significant economic losses in the aquaculture and fisheries industry, and have negative impacts on marine ecosystems and human health. In Korea, economic losses caused by HAB events have exceeded 600 million US$ in the past two decades (Korean Ministry of Ocean and Fisheries (KMOF, 2019). This devastating impact of HAB is generally linked to eutrophication, environmental degradation, and increasing temperature, and is increasing in severity, frequency, and intensity (Anderson et al., 2002; Grattan et al., 2016; Morabito et al., 2018). Algal-derived biotoxins accumulate in filter-feeding organisms and can lead to food poisoning, including various neurological and gastrointestinal illnesses in humans, such as

paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP), amnesic shellfish poisoning (ASP), ciguatera shellfish poisoning (CFP), and azaspiracid shellfish poisoning (AZP) (Grattan et al., 2016). Toxic dinoflagellate Alexandrium species, which produce paralytic shellfish toxins (PSTs) responsible for PSP, have caused mass mortality in invertebrates, fishes, mammals, and birds by infecting shellfish consumed in many countries (Shumway et al., 2018). These toxins can also lead to human diseases and sometimes to death (Band-Schmidt et al., 2019). For example, five people died after consuming shellfish contaminated with PSTs in Korea between the 1980s and 2000s (Park et al., 2013). There are more than 30 species in the genus Alexandrium (previously Gonyaulax and Protogonyaulax), about half of which produce PST (Anderson et al., 2012). Since saxitoxin (STX) is naturally

Abbreviations: HAB, harmful algal bloom; ITS, internal transcribed spacer; PCR, polymerase chain reaction; ddPCR, droplet-digital PCR; qPCR, quantitative realtime polymerase chain reaction ⁎ Corresponding author at: Department of Oceanography, Chonnam National University, Gwangju 61186, Republic of Korea. E-mail address: [email protected] (K.Y. Kim). https://doi.org/10.1016/j.hal.2019.101726 Received 2 July 2019; Received in revised form 1 December 2019; Accepted 3 December 2019 1568-9883/ © 2019 Elsevier B.V. All rights reserved.

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produced by several dinoflagellates and cyanobacteria, the PSP toxins, including STX, can be identified during the routine monitoring and surveillance of coastal waters. Currently, PSTs in shellfish tissues are analyzed using a mouse bioassay and are monitored at least twice per month during the spring and summer when they occur most frequently in Korean waters (Korean Ministry of Ocean and Fisheries (KMOF, 2019). Once the PST level exceeds the permitted standard (80 μg per 100 g of shellfish tissue), further harvesting is not carried out in the shellfish beds in Korea. To date, there is no validated method for early PSP detection by monitoring the causative HAB species. Nonetheless, continued development of more reliable detection methods will be facilitated by new emerging technologies. To minimize economic, ecosystem, and human health risks, the methods of identifying, preventing, and controlling or mitigating HAB are major targets for many geographic areas and ecosystems (Sellner and Rensel, 2018). The precise quantification of low-density HAB species during pre- and initial bloom periods is critical to reduce or prevent the deleterious consequences and to develop an early warning system to manage detrimental effects. In addition, a large number of samples should be analyzed quickly to determine the identity and quantity of causal organisms for precise monitoring, control, and management of HABs. Conventional light microscopy is used routinely to provide early warning of bloom events or to track the development of a blooming population. However, a microscopic approach for cell identification and enumeration is labor-intensive, and subjected to human error. Alexandrium species are difficult to identify based on their morphological features and can form amorphous cysts that lack surface features, particularly under unfavorable environmental conditions (Anderson et al., 2012; Erdner et al., 2010; Lilly et al., 2005; Touzet et al., 2008). Because these difficulties may result in the failure to detect early bloom stages, identification via species-specific DNA sequences or one of the key genes to produce paralytic shellfish toxin sxtA4 has been applied (Cusick and Sayler, 2013; Galluzzi et al., 2004; Murray et al., 2011; Savela et al., 2016). Ribosomal DNA (rDNA) and mitochondrial markers, such as 18S, 28S, and the cytochrome c oxidase subunit 1 (COI), are widely used to identify and quantify Alexandrium through fluorescence in situ hybridization (FISH), sandwich hybridization assay (SHA), and PCR-based assay (Sako et al., 2004; Touzet et al., 2009). During the last decade, quantitative real-time PCR (qPCR) has been actively developed and used widely as a tool to identify and monitor HAB species (Galluzzi et al., 2004; Garneau et al., 2011; Hosoi-Tanabe and Sako, 2005). qPCR is an excellent tool that can be used to measure the relative amount of target species by analyzing fold changes based on known control, or by comparing threshold cycles (Ct). However, quantitative measurements using qPCR should be performed carefully: Ct values are easily affected by molecules that interact with PCR, which could be contaminated with salts, pigments, exopolysaccharides, humic acids, and other substances in seawater (Ellison et al., 2011; Flekna et al., 2007; Galluzzi et al., 2004; Scollo et al., 2016; Touzet et al., 2009). The digital-droplet PCR (ddPCR) assay has recently been used to enumerate the fish-killing dinoflagellate Margalefidinium (Cochlodinium) polykrikoides in environmental samples, with high levels of sensitivity and accuracy (Lee et al., 2017). Absolute quantification of target DNA in the ddPCR mixture can be calculated by assuming a Poisson distribution (Majumdar et al., 2015), which allows the estimation of the number of positive droplets containing more than one target molecule after endpoint PCR. The abundance of target sequences in environmental samples can be calculated based on the frequency of positive to negative droplets, the total number of droplets, and the droplet volumes in the reaction mixture. In the Bio-Rad ddPCR system, 20 μL of reaction mixture is partitioned into up to 20,000 droplets, prior to thermal cycling, and allows droplets to be classified as positive or negative based on their fluorescence amplitudes. Therefore, digital PCR is a promising a new tool with advantages over quantitative real-time PCR (RT-qPCR)

for the quantification of DNA fragments without the need of an external reference or calibration curve. Moreover, as it relies on an endpoint fluorescence measurement, ddPCR results are generally much less sensitive to the presence of PCR inhibitors within the sample (Hindson et al., 2011; Huggett et al., 2013; Sanders et al., 2011; Scollo et al., 2016). Following the successful enumeration of M. polykrikoides cells in complex environmental samples, quantification of Alexandrium species is another challenge; however, this will help us better manage the harmful algal species. The aim of this work was to develop and apply a digital PCR assay suitable for the accurate and rapid quantification of A. affine, A. catenella, and A. pacificum in seawater samples. Specific primers were designed based on the nucleotide sequences of internal transcribed spacer (ITS) regions. Using the digital PCR protocol and simplified DNA prepared from the field samples on cellulose acetate membrane filters, the abundance of three species of Alexandrium was assessed in the coastal waters of the South Sea of Korea during 2017–2018. 2. Materials and methods 2.1. Alexandrium cultures Alexandrium affine (Aa-MEDHO0710), A. catenella (AcMEDHO1504), and A. pacificum (Ap-MEDHO0210) were isolated from the southern coast waters of Korea, Mijo Harbor in September 2007, Masan Bay in April 2015, and Jangmok Bay in February 2010, respectively. The strains were grown separately in 32–34 psu f/2 medium lacking silica at 20–23 °C under a 12:12 h light-dark cycle. Light was simulated with ∼300 μmol photons m−2 s-1 provided by four 36 W daylight fluorescent lamps (Dulux L 36 W/865; Osram, Münich, Germany). The cultures were replenished every 5–7 days by adding half a volume of fresh f/2 medium and the density was kept under 5000 cells mL-1 to minimize the number of dead cells (Guillard and Ryther, 1962). 2.2. Sampling for laboratory cultures and environmental sample To acquire the number of species-specific ITS fragments from a single cell of each Alexandrium species using ddPCR (Bio-Rad Co. USA), the following procedure was used: Alexandrium-free seawaters spiked artificially with a known number of cells from A. affine, A. catenella, and A. pacificum cultures were filtered through a 47 mm cellulose acetate filter with a pore size of 0.45 μm (CA, Advantech Inc. Tokyo, Japan) under a vacuum (< 100 mmHg). The number of cells spiked in each sample was determined by counting cells in 1 mL using a SedgwickRafter counting (SRC) chamber and Muse Cell Analyzer (Merck, MA, USA). Reproducibility of the species-specific copies determined by digital PCR assay was validated in terms of precision using a field seawater sample, which was spiked with the same number of cells of each species. The field seawater samples (1 L) were collected from three localities on the southern coast of Korea: Mokpo (MP, 34°77′N, 126°38′E), Namhae (NH, 34°70′N, 128°05′E), and Busan (BS, 35°06′N, 129°08′E) in the winter of 2017 (Fig. 1). Alexandrium species were not observed in these field samples under light microscopy examination. Alexandrium cell abundance determined by the digital PCR assay and via light microscopy was compared using samples (1 L) collected from the near-shore waters of Namhae (NH, 34°70′N, 128°05′E) and Tongyeong (TY, 34°86′N, 128°41′E) in August 2017 and April 2018 (Fig. 1). Shellfish and HAB species have been monitored during spring and summer on these sites by the Korean National Institute of Fisheries Science (KNIFS) to prevent possible toxic effects to humans and aquaculture industries. Samples were concentrated by sedimentation in 5 % Lugol’s iodine solution and then Alexandrium were identified and counted at the genus level in the SRC chamber with an inverted 2

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Fig. 1. Maps of the study area and sampling sites in the South Sea of Korea. The southern area of the Korean map, on the right, was enlarged from the area indicated by the dashed red box on the left panel. Mokpo (MP), Namhae (NH), Tongyeong (TY), and Busan (BS) at the near-shore sampling sites are represented as red dots, and at the off-shore stations along two west-east transects (205-01, 400-16, 206-01, 207-01 and 205-02, 400-00, 206-00, 207-01) in blue dots (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

microscope system (Olympus, IX73, Japan). In the South Sea of Korea, the digital PCR assay was applied to enumerate each Alexandrium species from the off-shore transects at St. 205-01 (34°22′N, 127°48′E), St. 400-16 (34°31′N, 128°12′E), St. 206-01 (34°35′N, 128°34′E), and St. 207-01 (35°01′N, 129°07′E) in April, August, and December 2017, and at St. 205-02 (34°15′N, 127°52′E), St. 400-00 (34°36′N, 128°04′E), St. 206-00 (34°37′N, 128°25′E), and St. 207-01 in April 2018 (Fig. 1). The off-shore samples (1 L) were collected during cruises of the KNIFS research vessel Tamgu No.8. To determine species-specific ITS copies, the same protocols were used for sampling and DNA extraction in both cultures and field samples, as shown in the schematic workflow for Alexandrium species using digital PCR in Fig. 2. The filter was stored at −80 °C after freezing in liquid nitrogen until DNA was prepared. 2.3. DNA preparation DNA was extracted from both cultures and field samples on a CA filter by modifying previously reported methods, in two steps (Lee et al., 2017). To minimize sample-to-sample variance in DNA preparation, a whole CA filter membrane was transferred into a 5 mL microfuge tube. First, cells were lysed by bead-beating (FastPrep-24™, MP Biomedicals Inc, USA) with 2 mm Zirconia beads (Watson Co., Tokyo, Japan) in 2 mL of lysis buffer (25 mM NaOH and 2 mM EDTA), and incubated at 95 °C for 30 min. Second, an equal volume of 40 mM TrisHCl (pH 5.5) was added to neutralize the sample DNA. The DNA-containing supernatant (5 μL) was used as the template for each ddPCR. These steps involved bead-beating to mechanically disrupt the cells, resulting in a higher yield of extracted DNA shredded into smaller fragments, less than 10 kbp (see Suppl. fig. S2). Before analyzing the samples by digital PCR, aliquots of the supernatant were stored at −80 °C and used later to quantify other target species.

Fig. 2. Schematic workflow from sampling to assessing the abundance of Alexandrium species using digital PCR. The whole procedure was adopted and modified from the protocol used to quantify Margalefidinium (Cochlodinium) polykrikoides via droplet-digital PCR (ddPCR) (Lee et al., 2017). The workflow includes four-steps: sampling, DNA preparation, ddPCR, and analysis of ddPCR data to estimate the cell abundance. Cells collected on a 0.45 μm celluloseacetate (CA) filter under a low-pressure vacuum were lysed by bead-beating in the alkaline-solution with EDTA and neutralized by adding Tris-buffer (pH 5.5). After eliminating cell debris by brief centrifugation, DNA in the supernatant was used as a template in ddPCR. The internal transcribed spacer (ITS) copy number per cell in Alexandrium species was acquired with a known number of cells spiked into the Alexandrium-free environmental sample, and was applied to quantify the abundance of species in complex environmental samples.

2.4. Design and validation of species-specific primers rDNA genes possess highly conserved sequences and are used widely in molecular phylogenetic studies. The sequences are organized as a tandemly repeated multigene family separated by an ITS in the genome, which provides a target area that can be used to identify dinoflagellate species (LaJeunesse, 2001). Laboratory-cultured Alexandrium species were genotyped based on the rDNA sequences between the small 18S subunit and the large 28S subunit rDNA. The sequences were used to design each Alexandrium species-specific primer sets in the region (Table 1) (Adachi et al., 1996; John et al., 2014). Additionally, PCR was performed for the Saxitoxin A4 gene (sxtA4) to confirm that the

Alexandrium strains produced STX (Murray et al., 2011). Thermal cycling was performed for 30 cycles for genotyping, or 40 cycles to test primer specificity with denaturation at 95 °C for 30 s, 3

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Table 1 Oligodeoxynucleotides used for genotyping, the Saxitoxin A4 gene used to identify Alexandrium strains, and species-specific primers targeting ITS for the digital PCR assay. Name

Target region

Primer sequences (5′ to 3′)

Annealing temp. (°C)

Amplicon size (bp)

ITS-A ITS-B sxtA4-F sxtA4-R A. affine-F A. affine-R A. catenella-F A. catenella-F A. pacificum-F A. pacificum-R

18S ∼ 28S

CCA AGC TTC TAG ATC GTA ACA AGG HTC CGT AGGT CCT GCA GTC GAC AKA TGC TTA ART TCA GCR GG CTG AGC AAG GCG TTC AAT TC TAC AGA TMG GCC CTG TGA RC TGT GCT TGA CTT TTA CAT GTA A GAG CAG CAC AGA TAC ACT AAT GCA ATA CAC ATT GAC TCT CT GAG TGG TGC TGT GTT TGT G TTC AAT GCA AAA CAT TGA CCT TTA CTG TTT GCA TTT CTC TAG TTG C

57

629

Adachi et al. (1996)

55

125

Murray et al. (2011)

56

206

This study　

62

252

This study　

58

257

This study　

sxtA ITS ITS ITS

ITS, internal transcribed spacer. sxtA4, Saxitoxin A4 gene.

annealing at 58 °C for 20 s, and extending at 72 °C for 20 s using the T100 Thermal Cycler (Bio-Rad Lab Inc., Hercules, CA, USA). The following reagents were added to each PCR tube, for a final volume of 20 μL containing 50–100 ng DNA templates: 10 μL of 2× prime Taq premix (Genet Bio, Daejeon, Korea) and 1 μL each of 2 pmol forward and reverse primer. The purified PCR amplicons were sequenced (Macrogen Co., Seoul, South Korea), and BLAST searches were performed to identify species with highly similar sequences (megablast) blast option in nr database for all organisms (https://blast.ncbi.nlm.nih. gov/Blast.cgi?LINK_LOC=blasthome&PAGE_TYPE=BlastSearch& PROGRAM=blastn). To evaluate the specificity of PCR primers for Alexandrium species, whole genomic DNA (gDNA) from 11 laboratory-cultured dinoflagellates, which are common and widely distributed in Korean waters, was extracted using DNeasy Plant Mini kit (Qiagen, Germany). Following the extraction, the DNA quantity and quality of all samples were checked by NanoDrop2000 (Thermo Fisher Scientific, Waltham, USA).

2.6. Data and statistical analysis

2.5. Performing digital PCR

To determine the abundance of three Alexandrium species, copies of target DNA fragments in a sample were analyzed via the following steps: collection of organisms on a CA filter by vacuum, DNA preparation by alkaline lysis and mechanical bead-beating to disrupt cells, digital PCR using the species-specific primer set, and estimating the abundance of target species. A schematic diagram of such a workflow is shown in Fig. 2.

Estimates of Alexandrium species abundance obtained by the ddPCR were compared with the total PST content of PSP monitoring reports by the KNIFS (http://www.nifs.go.kr/). The number of ITS copies was represented as mean ± standard error of the mean, except for cell abundance data from the field samples. One-way analysis of variance (ANOVA) was performed to compare the number of ITS copies per cell with the various numbers of cells and different sample locations. The means were compared using Duncan’s multiple range test or StudentNewman-Keuls test at a 5 % probability level. A normality test was performed for all variables prior to ANOVA using Minitab statistical software Ver. 18.1 (Minitab LLC, Pennsylvania State University, PA, USA). During 2017–2018, the spatial distribution of abundance in the South Sea of Korea was determined using Surfer® (Ver. 16, Golden software, LLC, CO, USA). 3. Results

A QX200 ddPCR system including a droplet generator, droplet reader, and T100 Thermal Cycler were used to determine the average single-cell copy numbers of each Alexandrium species by following the manufacturer’s protocols (Bio-Rad Laboratories Inc., CA, USA). Each ddPCR was performed in a total volume of 20 μL containing: 10 μL of EvaGreen Supermix (Bio-Rad Laboratories Inc., Munich, Germany); 1 μL each of 2 pmol of forward and reverse primer; 5 μL of sample DNA; and sterile water make up to the final volume. The reaction mixture was fractionated into approximately 20,000 droplets with a volume of one nanoliter using the Bio-Rad QX200™ Droplet Generator (Bio-Rad Lab Inc., Hercules, CA, USA) following the manufacturer’s instructions, and transferred to a 96-well PCR plate for PCR amplification using the T100 Thermal Cycler (Bio-Rad Lab Inc., Hercules, CA, USA). After PCR amplification, the plate was transferred to the Droplet Reader (Bio-Rad Lab Inc., Hercules, CA, USA), which reads each droplet to determine its signal (positive or negative) amplitude according to a threshold of fluorescence signal intensity. The QuantaSoft™ software version 1.7.4 (Bio-Rad Laboratories Inc., CA, USA) calculated the absolute quantity of target DNA fragments from the fraction of positive end-point reaction droplets using Poisson statistics. More than 10,000 droplets were required to accept a signal reading from a sample. The threshold for positive droplets was set by manually adding arbitrary amplitudes (ca. 5000) to the average amplitudes of negative droplets on a two-dimensional ddPCR amplitude scatter plot (Huggett et al., 2013). All solvents and chemicals for the ddPCR assay were used as received from SigmaAldrich Co. (St. Louis, MO, USA) or Bio-Rad (Bio-Rad Laboratories Inc., USA) unless otherwise specified.

3.1. Identification and toxigenicity of laboratory Alexandrium strains The cellular morphology of three laboratory-cultured strains was similar, and identifying Alexandrium species by the overall morphology of cells was not possible under a light microscope (Fig. 3A–C). Therefore, three species were distinguished by analyzing differences in the nucleotide sequences between the small subunit 18S and the large subunit 28S rDNA (Fig. 3D). The toxigenicity of three Alexandrium species was confirmed by detecting one of the STX biosynthesis enzyme genes, sxtA4, through conventional PCR (Fig. 3E). A. catenella (A. tamarense complex group I) and A. pacificum (A. tamarense complex group IV) are regarded as the causative agents of PSP, but A. affine is not closely associated with toxic events. 3.2. Design and validation of the species-specific primers targeting the ITS of Alexandrium The DNA sequences of species of the Alexandrium genus covering the ITS were collected from the NCBI nucleotide database (https:// www.ncbi.nlm.nih.gov/nucleotide/) to design species-specific PCR primers targeting ITS of each Alexandrium species. The Alexandrium 4

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Fig. 3. The morphology, identification, and paralytic shellfish toxins (PSTs) production of three Alexandrium species. A–C. Representative images of three Alexandrium obtained under a light microscope are indistinguishable based on the external morphological characteristics of live cells as well as cells fixed with Lugol’s solution. The scale bar in C for AeC is 20 μm. D. The internal transcribed spacer (ITS) sequences of three Alexandrium strains cultured in the laboratory, were used for genotyping and were identified as A. affine, A catenella (A. tamrense Group I), and A. pacificum (A. tamrense Group IV). E. A catenella and A. pacificum are PST-producing species, as confirmed by the presence of the sxtA4 gene in conventional PCR.

DNA sequences were also aligned with analyzed ITS sequences of laboratory-cultured A. affine, A. catenella, and A. pacificum (https://www. ebi.ac.uk/Tools/msa/clustalo/). Based on the nucleotide sequence of the ITS region, species-specific primers were designed with an average length of 20 bases and a melting temperature of 56–62 °C (Table 1). Before the newly designed primers were applied to ddPCR, their specificity was confirmed using the NCBI Primer-Blast tool (Suppl. table S1, https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and conventional PCR, which showed high specificity to target species and no cross-reactivity with other dinoflagellates (Fig. 4). The presence of DNA in each PCR was confirmed by performing a PCR for cytochrome oxidase subunit I (COI), a gene commonly used to identify eukaryotic algae groups. The PCR confirmed that the specificity was not due to the absence of DNA fragments in other reactions (data not shown).

3.3. Acquisition and confirmation of species-specific copies of ITS of Alexandrium Prior to the simplified preparation of DNA, an environmental sample containing a known number of cells (1 × 104–1 × 105 cells L−1) was filtered, and then the unique number of the target ITS copies for each Alexandrium species was determined by the ddPCR assay with the species-specific primer set. The ddPCR procedure resulted in digital PCR data with over 15,000 droplets in a total read, and a difference in amplitude of about 1.0 × 104 between negative and positive signals. The positive droplets are represented in blue, whereas the negative droplets are represented in grey (see Suppl. fig. S1). The average fluorescence amplitudes of positive or negative droplets clearly differed for each species-specific primer set. The average fluorescence value of negative droplets was 6378, 6205, and 4975 for A. affine, A. catenella, 5

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Fig. 4. Specificity of the newly designed species-specific primers targeting internal transcribed spacer (ITS) DNA fragments from each Alexandrium species. Conventional PCRs were performed using the template DNA from the target and non-target species to confirm the specificity of the new primers targeting the ITS of each species for a digital PCR. Only the specific PCR amplicons were detected in the targeted species.

and A. pacificum, respectively. By defining the fluorescence threshold line (a red line in Suppl. fig. S1C, G, K), the specific number of target DNA copies was calculated by QuantaSoft™ (Bio-Rad Lab Inc., USA). The specific copy number of ITS in Alexandrium species was validated by the ddPCR assay based on a known number of cells spiked into the Alexandrium-free environmental sample. The ITS copy number per cell of A. affine, A. catenella, and A. pacificum was 1492 ± 91 (n = 25), 515 ± 36 (n = 43), and 3894 ± 152 (n = 42), respectively (Fig. 5A–C). The total copy numbers were positively correlated to the number of cells in A. affine ( r2 = 0.859 with p < 0.01), A. catenella ( r2 = 0.920 with p < 0.01), and A. pacificum ( r2 = 0.868 with p < 0.01), showing the reliability of the ddPCR assay for quantification over a substantial range of cell numbers. There were no significant differences in the specific ITS copies from three Alexandrium species between environmental samples, spiked artificially with the same number of cells, from three localities in the coastal waters of Mokpo (MP), Namhae (NH), and Busan (BS) (all with p > 0.05, specified p-values in Fig. 5D–F), while relatively low p-values were observed for A. catenella between MP and NH, and between NH and BS, 0.353 and 0.445 respectively, in Fig. 5E.

3.4. Use of digital PCR to estimate the abundances of Alexandrium species The abundance of Alexandrium species determined using ddPCR was compared with the cell counts generated using light microscopy from the same samples, and with the total PST content of PSP monitoring reports by the KNIFS (Table 2). In Namhae (NH) and Tongyeong (TY) coastal waters, there have been frequent warnings of toxic dinoflagellate blooms over the past decade. In August 2017, A. affine and A. catenella occurred at very low abundances (less 12 cells L−1), precluding any meaningful comparison. In April 2018, only A. catenella occurred at both sites, and its abundance was relatively high, with 427 and 242 cells L−1 at NH and TY, respectively, according to the digital PCR outcomes (Table 2). These values were generally similar to those obtained by direct enumeration under light microscopy. Although it was possible to identify Alexandrium at a genus level by microscopic observation, microscopy-based enumeration was found to compared with the results of ddPCR quantification in terms of estimated cell abundance. According to PSP monitoring reports by the KNIFS, the total PST content in April 2018 was 134 and 2424 μg 100g−1 in shellfish tissue at NH and TY, respectively. Fig. 5. Validation of the specific copy number of internal transcribed spacer (ITS) DNA fragments of Alexandrium species by digital PCR with various numbers of cells (A–C) and samples from different localities (D–F), which were generated based on a known and consistent number of cells spiked into the Alexandrium-free environmental sample. Linear, positive correlations were observed between the total copies and number of cells in A. affine (A), A. catenella (B), and A. pacificum (C). There was no significant difference in the cell copies among samples from MP, NH, and BS in A. affine (D), A. catenella (E), and A. pacificum (F). Error bars indicate ± standard error (SE). NS, not statistically significant (p > 0.05). Location abbreviations are given in Fig. 1.

6

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otherwise insignificant A. affine throughout the entire water column. A. catenella was the most abundant species in near-shore as well as offshore waters in April 2018, as shown in Table 2. The abundance of A. pacificum was very low or negligible in August and December 2017, while it was relatively high, albeit not as high, in A. catenella in April of consecutive years. All three Alexandrium species presented a patchy distribution along a transect and no consistent distributional pattern with depth or time of year.

Table 2 The abundance of Alexandrium species as determined by digital PCR assay and light microscopic cell counts, and the concentration of paralytic shellfish toxins (PSTs) determined via mouse assay in the southern coastal waters of Korea in August 2017 and April 2018. Methods

Time Sites

Digital PCR assay (cells L−1) Light microscopy (cells L−1) PST contentb (μg 100 g−1 tissue)

a

A. affine A. catenella A. pacificum Alexandrium spp. Mouse assay

August 2017 a

April 2018 a

NH

TY

NHa

TYa

8 9 – –

– 12 – 67

– 427 1 245

– 242 – 177

134

2424

4. Discussion 4.1. Identification and toxigenicity of Alexandrium Digital PCR was successfully applied to quantify M. polykrikoides in complex environmental samples, using species-specific ITS primer (Lee et al., 2017). Quantification of Alexandrium species using this technique is another challenge, which needs to be critically analyzed at the initial stages of its development and be implemented into decision-support activities for the management of PST-producing species. This study explored the advantages of a digital PCR approach, which is much less sensitive to potential contaminants and allows the absolute quantification of Alexandrium species without the need for a calibrant in marine environmental samples (Hindson et al., 2011; Sanders et al., 2011; Scollo et al., 2016). In routine monitoring of HABs, the early detection of causative species in real or near-real time is critical for timely decision-making and for forecasting its development and trajectories (Doucette et al., 2018). The results obtained in this study delineate the potential suitability of an early warning method for PSP, as an alternative molecular method to monitor PSTs in shellfish only coarsely or indirectly by quantifying toxic Alexandrium species. Quantification of PST-producing species in

- not detected. a Location abbreviations are given in Fig. 1. b The values represent the maximum in the month reported by Korean National Institute of Fisheries Science (KNIFS).

The abundance and vertical distribution of three Alexandrium species along the transects were investigated at four stations at 10 m interval from the surface to a depth of 30 m (Fig. 6). The abundance of A. affine was the highest (50 cells L−1) at 20 m at St. 205-01 in August, but very low (5 cells L−1) or negligible in the entire water column in December 2017 and continued into April 2018, except for the surface at St. 205-01 in April 2017 and 10 m at St. 207-01 in April 2018. The abundance of A. catenella ranged from 1 to 30,074 cells L−1, and the highest value was recorded at 0 m at St. 205-02 in April 2018. In that month, A. catenella and A. pacificum cells were observed along the transects and were highly concentrated in the surface (0–20 m) layer,

Fig. 6. The abundance and vertical distribution of three Alexandrium species obtained via digital PCR in the South Sea of Korea in April, August, December 2017, and April 2018. The color-coded contour maps of the cells are shown on a logarithmic scale. Locations of the transects are given in Fig. 1. 7

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environmental samples is challenging, because the total PST content of farmed shellfish is frequently higher than the regulation sanitary threshold (80 μg 100g−1 tissue) even when present in very low or undetectable levels in the southern coastal waters of Korea (Jeong et al., 2017). Overall this novel assay can detect the initial symptoms of PSP outbreaks early, and seems to have a great advantage for PSP monitoring programs as an earlier predictor of PSP compared with the mouse bioassay. Indeed, the abundance of Alexandrium cells determined by digital PCR was enhanced by the microscopy-based enumeration, which cannot resolve species-level identification, and there was a clear correlation between digital PCR outcomes and total PST content reported by KNIFS (Table 2). Moreover, the results generated by digital PCR suggest that the species-specific assay is more sensitive and has better specificity for Alexandrium species than other methods reported to date. When PSP outbreaks occurred in the southern coast of Korea, which includes the coastal waters of NH and TY (see Fig. 1) in April 2018, the PSTs content was about 30-times higher than the regulation threshold at TY (Table 2). We can only suggest that this devastating episode was attributed to A. catenella, which was confirmed by the digital PCR assay. This approach, with high validity and reliability, can therefore be a suitable alternative for use in regulatory PSP monitoring programs. The primer targeting sxtA4 in a STX biosynthesis pathway detected only the PST toxin-producing species, A. catenella and A. pacificum (Fig. 3E). The primer was used to quantify the STX-producing species (John et al., 2014), and was used in the digital PCR assay to determine the copies of sxtA4 in A. catenella and A. pacificum. Although this primer amplified the sxtA4 gene of Alexandrium species producing STX, the number of copies of sxtA4 (18.7 [n = 5] for A. catenella and 2.4 [n = 5] for A. pacificum), varied four-fold between the two species. Therefore, additional studies are required to identify the sources of the gene in the samples and to prove the relationship between gene copies and the STX concentration per cell. However, it is possible that our digital PCR assay with the primer targeting sxtA4 enumerates the single STX species in the sample (John et al., 2014; Savela et al., 2016). Moreover, it is unclear whether sxtA4 originated from PST-producing Alexandrium species only, or also from other species, such as Gymnodinium and Pyrodinium in complex marine communities (Shumway, 1990).

reads can be detected by ddPCR due to unknown species or possible contaminations in marine environmental samples (Mora et al., 2011). Multiple primers targeting regions other than the ITS, along with marine genome data accumulated by advanced next-generation sequencing technologies, are required to improve the specificity of the digital PCR assay (Porter and Hajibabaei, 2018). Unique ITS copies of each species were verified by a known number of cells in the experimental samples and in various environmental samples from different locations (Fig. 5). Despite some inevitable variation in the ITS copies between seawater samples as those in Fig. 5E, overall this technique allows to Alexandrium species to be identified and quantified with excellent reliability owing to the insensitivity of cell copies to different species composition, abundance, and diversity, as well as environmental conditions. Like in other measuring devices, errors in the quantity of the ddPCR read (copy number) may stem from the presence of too few or too many positive droplets. The errors suggest the limitation of the cell count assay using the specific copies of target DNA fragments. The theoretical maximum cell counts of A. affine, A. catenella, and A. pacificum in 20 μL of a single ddPCR are about 67, 194, and 25 cells, respectively. These values were calculated using the acquired specific ITS copies of A. affine (1492 ± 91), A. catenella (515 ± 36), and A. pacificum (3894 ± 152) under the Poisson distribution assumption (Majumdar et al., 2015). To broaden the range to the abundance of naturally occurring species, the following options are suggested: dilute sample DNA to the specified limits of maximum cell counts in a single ddPCR, or use a primer set targeting another DNA sequence with fewer copies than the ITS repeats. The digital PCR assay targeting specific DNA fragments might overestimate the abundance of interesting species because of contamination with dead cells or resting cysts in field samples. This hampers the efforts to prevent and manage the further development and spread of harmful species. Thus, the accuracy and sensitivity of Alexandrium monitoring could be improved by targeting transcripts specifically present in live cells that are actively proliferating, even a whole-genome sequence and transcriptome analyses are prerequisites. Rapid and precise identification and quantification of HABs are crucial to predict species distribution, and to prevent or reduce the potential damages from their blooms.

4.2. Alexandrium species-specific ITS primers and cell copies

4.3. Use of digital PCR to estimate the abundances of Alexandrium species

Species-specific nucleotide sequences can be used to identify species. For example, DNA regions of cytochrome oxidase subunit I (COI) and ribulose bisphosphate carboxylase large chain (rbcL) have been used widely to design primers for taxonomic markers. The number of subcellular organelles containing their own genomic DNA can vary depending on the biological state of a given cell. Therefore, mitochondria and chloroplast genes are not suitable targets for assessing the quantity of eukaryotic species (Bereiter-Hahn, 1990; Sato et al., 2003). While whole-genomic data are not available for most marine species, nuclear rDNA, including the ITS region, has been sequenced for phylogenetic analysis in many species (LaJeunesse, 2001). Nuclear ribosomal ITS sequences can be used to design species-specific primers that can be successfully used to identify species and determine their abundance via PCR-based environmental DNA (eDNA) amplification methods. The ITS consists of multiple repeated copies in a genome, which can be used to distinguish between species with a higher detection sensitivity (Gardes and Bruns, 1993). The ITS regions of rDNA, which diverge rapidly during speciation, and are most widely sequenced, along with whole genome sequences. Sequenced data from Alexandrium strains cultured in our laboratory were integrated into the ITS sequences of dinoflagellates in NCBI to design species-specific PCR primers. The newly designed species-specific ITS primers presented no cross-reactivity to other species, which confirms the specificity of the ddPCR to target species of Alexandrium (Fig. 4). However, false positive

For the safe harvest of shellfish and the mitigation of risks associated with bioaccumulation of PSTs, programs including a mouse bioassay of shellfish tissue and HAB species monitoring have been routinely conducted in the coastal waters of Korea, along with other major environmental parameters since the 1990s. Local government officers and shellfish harvesters, however, would expect to obtain more reliable and early predictive indicators than PST content to plan their harvesting operations to minimize any potential risk. In Korea, as well as in many other countries, shellfish growing areas are closed for harvesting when total PST content in tissue, which is determined by mouse bioassays, exceeds the regulatory threshold or the permitted standard. The mouse bioassay is still the most common method used to test for PSP, however, at the present, its use is being reconsidered due to ethical issues associated with animals, as well as for practical considerations. Routine methods of monitoring HAB species are time consuming and have high limits of detection with low specificity (Hatfield et al. 2019). Particularly, it is not possible to accurately identify and reliably quantify Alexandrium cells to species level using light microscopy in a SRC chamber. Therefore, we applied the digital PCR assay to quantify three Alexandrium species in coastal waters, and their abundances were compared with those reported by the KNIFS. The abundance of Alexandrium species obtained by the dPCR assay was broadly reflected in the data of HAB species or PSP monitoring. The higher abundance of A. catenella in the near-shore and off-shore waters of Korea coincided with the time when KNIFS had issued PSP warnings against the collection of 8

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bivalve shellfish in April of 2017 and 2018 (Table 2, Fig. 6). However, the causative species of PST could not be confirmed, neither via HAB species monitoring by light microscopic observations, nor in PST monitoring by mouse bioassay. There are two possible interpretations for the high PST along with the low abundance of A. catenella in April: first, it could be a result of other PST-producing species, such as Alexandrium and Gymnodinium genera; second, PST-contaminated shellfish and crustaceans, particularly filter-feeding bivalves, could directly obtain PSTs by feeding on pelagic vectors, which vary significantly in their capacity to accumulate ingested PST in their tissues (Jester et al., 2009). In August 2017, a bloom of Alexandrium was reported in the southern coastal waters by the KNIFS without any information at the species level. According to the KNIFS reports of HAB species monitoring, cellular abundance of the Alexandrium genus reached up to 12 × 106 cell L−1 at NH and 15 × 106 cell L−1 at TY in August. Application of digital PCR revealed that the abundance of three Alexandrium species was only 12–17 cells L−1 at NH and TY sites in near-shore waters, and was maximal at 250 cells L−1 at four off-shore stations along the transects (Fig. 6). Unlike data from HAB species monitoring, it is characterized with considerably lower abundances of Alexandrium species, making it possible to confirm that A. affine is the only causative species of the bloom in the southern coastal waters in August 2017. There was a discrepancy in the abundance of the Alexandrium genus between data obtained through HAB species monitoring based on microscopic cell counts and our data of three Alexandrium species based on dPCR assay. It is possible that differences in the times and locations of sampling between the field surveys could have led to substantial differences in the abundance of Alexandrium species. Such variation seems to be inevitable, but also indicates limitations and failures of current methods used to monitor HAB species using light microscopy. Additionally, as more than 10 Alexandrium species have been found in the South Sea of Korea by metagenomic analysis using the SoEM (small-organelles enriched metagenomics) method (Jo et al., 2019), unidentified Alexandrium species may have contributed to the abundance of this genus examined by the KNIFS. Many HAB species are motile, and their swimming behavior or buoyancy may result in a non-random distribution in the patches (Glibert et al., 2005). These distributional features suggest that samples collected only at the surface of the near-shore beaches, or at the blooming sites, are insufficient to precisely monitor and estimate the accurate abundance. The digital PCR assay, allowing routine processing of 96 samples in parallel, is able to analyze a large amount (about 100) of complex seawater samples, from collecting cells on the filter to quantifying the abundance of target species, in a few hours. Therefore, a fast and highly efficient digital PCR assay might be a useful tool to analyze many samples from numerous sites and times simultaneously, and could reduce the spatial and temporal variation in abundance, as shown in Fig. 6. A new method for quantifying PSP-inducing Alexandrium species using ddPCR is presented in this paper. The technique enables DNA to be prepared from cells lysed by mechanical disruption with a beadbeater and includes species-specific primers for three Alexandrium species. The specific ITS copies per cell were consistent with the abundance gradients and reproducible when using different environmental DNA samples from various locations. Based on the abundance and distribution data of three Alexandrium species in the South Sea of Korea in 2017–2018, using the digital PCR assay, we found that A. catenella was the major species associated with PSP during the spring. Although various life-stages of Alexandrium cells as a function of the amount of the genomic DNA copies might be contained in the environmental samples, the quantification by our novel dPCR tool does not count more than two folds of that obtained from the sample with all diploid cells. Without separating each life-stage in the field, we could not be confident their real numbers, and also same issues took consideration through targeting DNA as a biomarker for quantification.

Therefore, simply comparing the copies would be a better way to present their abundances across the broad areas. It is also challenging in conventional count approaches with mixed diploid cells. Consequently, this assay is highly sensitive, can precisely quantify Alexandrium species in complex marine environmental samples, and might be appropriated for the early detection and monitoring of various invasive and harmful protists at very low levels of abundance. The approach may represent the first step for obtaining accurate abundance data of the target PST-producing species, which is valuable for developing a bloom prediction model to control possible damages, and to improve our HAB alarm system. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments We would like to thank Professor Park, Myung Gil for kindly offering Alexandrium lines, and Keunyong Kim for supporting the field sampling. This research was financially supported by NRF2016R1A6A1A03012647 to KYK, and by Basic Science Research Program by the Ministry of Science and ICT (NRF2015R1C1A1A02037499) to HGL.[CG] Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.hal.2019.101726. References Adachi, M., Sako, Y., Ishida, Y., 1996. Analyses of Alexandrium (Dinophyceae) species using sequences of the 5.8S ribosomal DNA and internal transcribed spacer regions. J. Phycol. 32, 424–432. Anderson, D.M., Alpermann, T.J., Cembella, A.D., Collos, Y., Masseret, E., Montresor, M., 2012. The globally distributed genus Alexandrium: multifaceted roles in marine ecosystems and impacts on human health. Harmful Algae 14, 10–35. Anderson, D.M., Glibert, P.M., Burkholder, J.M., 2002. Harmful algal blooms and eutrophication: nutrient sources, composition, and consequences. Estuaries 25, 704–726. Band-Schmidt, C.J., Durán-Riveroll, L.M., Bustillos-Guzmán, J.J., Leyva-Valencia, I., López-Cortés, D.J., Núñez-Vázquez, E.J., Hernández-Sandoval, F.E., RamírezRodríguez, D.V., 2019. Paralytic toxin producing dinoflagellates in Latin America: ecology and physiology. Front. Mar. Sci. 6, 42. https://doi.org/10.3389/fmars.2019. 00042. Bereiter-Hahn, J., 1990. Behavior of mitochondria in the living cell. Int. Rev. Cytol. 122, 1–63. Cusick, K.D., Sayler, G.S., 2013. An overview on the marine neurotoxin, saxitoxin: genetics, molecular targets, methods of detection and ecological functions. Mar. Drugs 11, 991–1018. Doucette, J., Medlin, K., McCarron, P., Hess, P., 2018. Detection and surveillance of harmful algal bloom species and toxins. In: Shumway, S.E., Burkholder, J.M., Morton, S.L. (Eds.), Harmful Algal Blooms: A Compendium Desk Reference. John Wiley & Sons, pp. 435–492. Ellison, S.L., Emslie, K.R., Kassir, Z., 2011. A standard additions method reduces inhibitor-induced bias in quantitative real-time PCR. Anal. Bioanal. Chem. 401, 3221–3227. Erdner, D.L., Percy, L., Keafer, B., Lewis, J., Anderson, D.M., 2010. A quantitative realtime PCR assay for the identification and enumeration of Alexandrium cysts in marine sediments. Deep Sea Res. Part 2 Top. Stud. Oceanogr. 57, 279–287. Flekna, G., Schneeweiss, W., Smulders, F.J., Wagner, M., Hein, I., 2007. Real-time PCR method with statistical analysis to compare the potential of DNA isolation methods to remove PCR inhibitors from samples for diagnostic PCR. Mol. Cell. Probes 21, 282–287. 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. Gardes, M., Bruns, T.D., 1993. ITS primers with enhanced specificity for basidiomycetes application to the identification of mycorrhizae and rusts. Mol. Ecol. 2, 113–118. Garneau, M.E., Schnetzer, A., Countway, P.D., Jones, A.C., Seubert, E.L., Caron, D.A., 2011. Examination of the seasonal dynamics of the toxic dinoflagellate Alexandrium catenella at Redondo Beach, California, by quantitative PCR. Appl. Environ.

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