Grazing impact of the calanoid copepods Acartia spp. on the toxic dinoflagellate Alexandrium pseudogonyaulax in the western coastal waters of Korea

Marine dinoflagellate species in the genus Alexandrium are well known to produce paralytic shellfish poison as well as common coastal species with cosmopolitan distribution. However, few studies on the feeding of copepods on Alexandrium species have been conducted. The toxic dinoflagellate Alexandrium pseudogonyaulax contains goniodomin A and causes red tides in many countries. To investigate the relationship between the toxic dinoflagellate A. pseudogonyaulax and the calanoid copepods Acartia spp., we quantified the ingestion rates of Acartia spp. feeding on A. pseudogonyaulax as a function of prey concentration. Additionally, we estimated grazing coefficients by integrating data from field observations of Acartia spp. and coexisting A. pseudogonyaulax with laboratory measurements of ingestion rates obtained during this investigation. Furthermore, we compared the ingestion rates of Acartia spp. and other predators feeding on Alexandrium species as previously reported. The ingestion rates of Acartia spp. on A. pseudogonyaulax increased continuously with increasing mean prey concentration. The highest values among the ingestion rate of Acartia spp. feeding on A. pseudogonyaulax was 3,407 cells predator−1 d−1 (4,872 ng C predator−1 d−1) at the given prey concentration. The calculated grazing coefficients for Acartia spp. on A. pseudogonyaulax in Shiwha Bay, Korea, were up to 0.073 d−1. The results of this study suggest that A. pseudogonyaulax may decrease or maintain the population of Acartia spp. in marine food webs.


Preparation of experimental organisms
For isolation and culture of Alexandrium pseudogonyaulax, plankton samples collected with Niskin sampler were taken from Shiwha Bay, Korea when the water temperature and salinity were 25.4°C and 23.9, respectively (Table 1).These samples were screened through a 202-Nitex mesh and placed in 6-well tissue culture plates (SPL lifesciences, Gyeonggido, Korea).A clonal culture of A. pseudogonyaulax was established by performing two serial single-cell isolations.As the concentration of A. pseudogonyaulax increased, this species was subsequently transferred to 50-mL and 500-mL polycarbonate (PC) bottles containing fresh f/2-Si medium (Guillard and Ryther, 1962).Freshly filtered seawater was used to fill bottles containing the f/2-Si medium and A. pseudogonyaulax.The capped bottles were then incubated at 20°C under illumination of 20 μmol photons m −2 s −1 of cool white fluorescent light on a 14:10 h light:dark cycle.Once dense cultures of A. pseudogonyaulax were obtained, the cells were transferred to new 2-L PC bottles containing fresh f/2-Si medium approximately 3 weeks before the feeding experiments were conducted at a temperature of 15°C.
Copepods were collected Shiwha Bay, Korea, using a 303 μm mesh net when water temperature and salinity were 7.3°C and 27.2, respectively (Table 1).The copepods were acclimatized in a 15°C room in the presence of Prorocentrum cordatum for 10 days.Adult female Acartia spp.(A.hongi and A. omorii) were used in the experiments.A. hongi and A. omorii which co-occur in the western coastal waters of Korea, are very similar and it is impossible to distinguish between these two species when they are alive (Soh and Suh, 2000).
The mean equivalent spherical diameter (ESD) of live A. pseudogonyaulax was measured using an electron-particle counter (Coulter Multisizer II; Coulter Corporation, Miami, Florida, United States).The carbon content of this species was estimated based on the cell volume according to Menden-Deuer and Lessard (2000).

Swimming speed
A dense culture (ca.1,500 cells mL −1 ) of A. pseudogonyaulax, which grew photosynthetically under a 14:10 h light:dark cycle at 20 μmol photons m −2 s −1 in f/2-Si medium was transferred to a 250-mL PC bottle.Subsequently, an aliquot from the bottle was transferred to a 50-mL cell culture flask and allowed to acclimate for 30 min.The observations were conducted at 20°C using a video analyzing system (SV-C660, Samsung) and a CCD camera (KP-D20BU, Hitachi).The video camera was focused on a field of view within the cell culture flask and observed as a single field under a dissecting microscope at 50× magnification.The mean and maximum swimming velocities of all A. pseudogonyaulax cells in motion within the first 10 min were recorded and analyzed.The linear displacement of the cells within a single-frame playback was measured to calculate the average swimming speed.The swimming velocities of 30 cells were assessed.

Ingestion rates of Acartia spp. on Alexandrium pseudogonyaulax
This experiment was designed to measure the ingestion and clearance rates of Acartia spp. on A. pseudogonyaulax as a function of prey concentration (Table 2).Adult female Acartia spp.(a combination of A. hongi and A. omorii) were used in the present study.
For the feeding experiment, the initial concentrations of A. pseudogonyaulax were dertermined using an autopipette to deliver predetermined volumes of known cell concentrations to the bottles and those of Acartia spp.were obtained by individually transferring Acartia spp.using a pasteur pipette.Triplicate 500-mL PC bottles (mixtures of predator and prey) and triplicate control bottles (A.pseudogonyaulax prey only) were set up for each predator-prey combination.In order to maintain consistent water conditions, the water from the predator culture was passed through a 0.7 μm GF/F filter before being added to the prey control bottles.The volume of the filtered predator culture added to the experimental bottles for each predator-prey combination was matched with an equal amount of filtered water added to the prey control bottles.All the bottles were filled to capacity with freshly filtered seawater and capped.To determine the initial predator and prey densities, a 10-mL sample was extracted from each bottle and fixed with 1% Lugol's solution for fixation.The fixed sample were examined using a light microscope to determine the abundance of predator and prey species.The cells in three 1-mL Sedgwick-Rafter chambers (SRCs) were counted to determine the actual densities of predator and prey species.The bottles were refilled to capacity with freshly filtered seawater, capped, and placed on rotating wheels at a temperature of 15°C, following the conditions outlined earlier.We considered any dilution of the cultures associated with refilling the bottles when determining the clearance rate.A 10-mL aliquot was taken from each bottle after 24 and 48-h incubation periods and fixed with 1% Lugol's solution.The abundance of prey species was determined by counting all or > 200 cells in three 1-mL SRCs.Following sub-sampling, the bottles were filled with freshly filtered seawater and placed back on rotating wheels.After incubation for 48 h, the Acartia spp.were counted.The mortality of Acartia spp.occurred until the end of the incubation period.The ingestion and clearance rates were calculated using the equations of Frost (1972).

Grazing impact
We calculated the grazing coefficients attributable to Acartia spp. on A. pseudogonyaulax by combining field data on the abundances of Acartia spp.and A. pseudogonyaulax with the ingestion rates of Acartia spp. on A. pseudogonyaulax obtained in the present study.Data on the abundance of Acartia spp.and co-occurring A. pseudogonyaulax used in this estimation were obtained from water samples from Shiwha Bay, Korea using real-time PCR for A. pseudogonyaulax and cell counting for Acartia spp.
The grazing coefficients (g, d −1 ) were calculated as follows: where CR is the clearance rate (mL predator −1 h −1 ) of a Acartia spp. on A. pseudogonyaulax at a given prey concentration and GC is the predator concentration (cells mL −1 ).The CR values were calculated as follows: where IR is the ingestion rate (cells predator −1 h −1 ) of the predator on the prey and X is the prey concentration (cells mL −1 ).

Species-specific primer and probe design and specificity analysis
We developed species-specific primer and probe set for A. pseudogonyaulax and obtained the sequences of the internal transcribed spacer region of ribosomal DNA (ITS rDNA) of A. pseudogonyaulax and other dinoflagellate species belonging to the Alexandrium genus and related dinoflagellate species from GenBank.These sequences were aligned using MEGA v.11. A. pseudogonyaulax specific primers and probe were developed by searching the arrangement for unique portions of the ITS rDNA sequences for A. pseudogonyaulax.Primer and probe sequences were analyzed using Primer 4 (Whitehead Institute for Biomedical Research, Cambridge, MA, United States) and Oligo Calc: Oligonucleotide Properties Calculator (Kibbe, 2007) to investigate the optimal melting temperature and secondary structure, respectively.Primers and probe were synthesized by Bioneer (Table 3).The probe was dual labeled with the fluorescent dyes FAM and BHQ1 at the 5′ and 3′ ends.
Specificity analysis of the primer and probe sets for A. pseudogonyaulax was performed using DNA extracts of A. pseudogonyaulax and related dinoflagellate species in the Family Pyrocystaceae.The qPCR reaction mixture contained 1 μL of DNA template, 0.2 μM of specific forward and reverse primers, 0.15 μM of the specific probe, 5 μL of qPCRBIO Probe Separate-ROX (Genepole, Gwangmyeong, Korea), and deionized sterilized water (DDW; Bioneer), with a final total volume of 10 μL.The qPCR assay was conducted using the Rotor-Gene Q (Qiagen, Hilden, Germany).The    cycling conditions were initialized with a denaturation step at 95°C for 3 min, followed by 40 cycles of 10 s at 95°C for 10 s, and 58°C for 40 s.

Standard curve construction
A standard curve for exploring the abundance of A. pseudogonyaulax was constructed using a qPCR.DNA was extracted from the culture of A. pseudogonyaulax (4,200 cells mL −1 ) in the growth phase using the AccuPrep Genomic DNA Extraction Kit (Bioneer), targeting 1, 10, 100, 1,000, 2,000, and 4,000 A. pseudogonyaulax cells.The qPCR assay was performed using the reaction mentioned above under the following thermal cycling conditions: 95°C for 3 min, followed by 45 cycles of 10 s at 95°C for 10 s, and 58°C for 40 s.

Quantification using qPCR
We developed species-specific primer and probe set for A. pseudogonyaulax, and obtained the sequences of the internal transcribed spacer region of ribosomal DNA (ITS rDNA) of A. pseudogonyaulax and related dinoflagellates.
The previously mentioned qPCR assay conditions were used to analyse the abundance of A. pseudogonyaulax in field samples.The DNA from each sample was amplified four times to ensure the accuracy of results.The sample using DDW as the template was used as a negative control, whereas the one used to construct a standard curve was used as positive and standard control.

Ingestion rates of Acartia spp. on Alexandrium pseudogonyaulax
The ingestion rate of Acartia spp. on A. pseudogonyaulax continuously increased with increasing prey concentration (Figure 1).The highest ingestion and clearance rates of Acartia spp. on A. pseudogonyaulax at the given prey concentration was 3,407 cells Acartia −1 d −1 (4,872 ng C Acartia −1 d −1 ) and 192 mL Acartia −1 h −1 , respectively.

Grazing impact
The grazing coefficients attributable to Acartia spp. on co-occurring A. pseudogonyaulax in Shiwha Bay, Korea were affected by the abundance of Acartia predators (Figure 2).The abundance of Acartia spp.and A. pseudogonyaulax were 1.5-126.0cells mL −1 and
Among the MIRs of Acartia grazers on Alexandrium prey species, the MIR of A. hudosonica on the toxic of A. tamarense strain was higher than that on the non-toxic strain of A. tamarense (Colin and Dam, 2007;Teegarden et al., 2008).Additionally, the MIR of A. tonsa on the toxic of A. fundyense strain was lower than that on the non-toxic strain of A. tamarense (Teegarden, 1999).Thus, the toxicity of Alexandrium prey species probably did not affect the MIRs of Acartia grazers.
Among the MIRs of Acartia grazers on Alexandrium prey species, the MIR of Acartia spp. on A. pseudogonyaulax was higher than A. bifilosa on A. ostenfeldii, but lower than that of A. grani on A. minutum (da Costa and Fernández, 2002;da Costa et al., 2008).Many Alexandrium species contain PSP toxins; however, the subgenus Gessnerium does not produce PSP toxins (Balech, 1995).A. pseudogonyaulax belonging to the subgenus Gessnerium may not produce PSP toxins, but may produce goniodomin A (Balech, 1995;Matsuoka and Fukuyo, 2003;Bravo et al., 2006).Furthermore, A. pseudogonyaulax is a mixotrophic species when mucus traps are used to immobilize prey cells prior to ingestion (Blossom et al., 2012).Therefore, the mucus trap excreted by A. pseudogonyaulax may not only function to effectively accumulate toxins but also be used to avoid encounters and ingestion by potential predators.
The motility of dinoflagellates is not only relevant to potential predators but is also important for resource availability (Buskey, 1997;Tillmann and Reckermann, 2002;Jeong et al., 2015Jeong et al., , 2017)).The Maximum ingestion rates of Acartia spp.feeding on Alexandrium prey species as a function of prey size (ESD: equivalent spherical diameter, μm).The p-value was p > 0.1 (linear regression ANOVA).Af (T): toxic strain of A. fundyense; Am (T): toxic strain of A. minutum; Ao (T): toxic strain of A. ostenfeldii; Ap (T): toxic strain of A. pseudogonyaulax; At (T): toxic strain of A. tamarense; At (NT): nontoxic strain of A. tamarense.The data were obtained from Calbet et al. (2003), Colin and Dam (2002, 2003, 2007), da Costa andFernández (2002), da Costa et al. (2008), Sopanen et al. (2011), Teegarden (1999), and Teegarden et al. (2003, 2008).The average (±standard error) swimming speed of A. pseudogonyaulax was 372 (±12) μm s −1 .The maximum swimming speed of A. pseudogonyaulax was faster than that of A. minutum but slower than that of A. tamarense (Kang et al., 2018).Thus, the swimming speed of Alexandrium prey species probably did not affect the MIRs of Acartia grazers.Other properties, such as the biochemical factors of Alexandrium species, may affect the ingestion of Acartia grazers more than their physical and behavior properties.Grazing impacts calculated by using field observation data of Acartia spp.and coexisting A. pseudogonyaulax with laboratory measurements of ingestion rates suggest that up to 7% of A. pseudogonyaulax populations may be eliminated in a day by the copepods Acartia spp.Therefore, the copepod Acartia species could have a considerable potential grazing impact on Alexandrium populations in Shiwha Bay.
Few studies have been conducted on the grazing effects of copepods on Alexandrium species in the field.The grazing effect of A. hudsonica on Alexandrium spp. was 0.8 d −1 at Cundy's Harbor (Campbell et al., 2005).In Cape Cod embayment, the grazing pressure of A. hudsonica feeding on A. tamarense was less than 1% (Turner and Anderson, 1983).Additionally, the grazing coefficients of the copepods such as Acartia granii and Oithona davisae on A. minutum were 0.00003-0.00007d −1 (i.e., up to 0.007% of Alexandrium populations could be removed by the copepod populations in a day) in the Arenys de Mar harbor (Calbet et al., 2003).Thus, the copepod Acartia species sometimes have a considerable potential grazing impact on populations of Alexandrium.

Conclusion
The present study investigated the grazing by calanoid copepods Acartia spp. on the toxic dinoflagellate Alexandrium pseudogonyaulax.The grazing of Acartia spp.can affect the abundance of Alexandrium populations in many countries.A total of 34 Alexandrium species have been reported, but there have been few studies on grazing by metazooplankton on Alexandrium spp.(Calbet et al., 2003;Campbell et al., 2005;Guiry and Guiry, 2020).Therefore, constant investigation of feeding by dominant copepods on Alexandrium species would be worthwhile to enhance our understanding of the interactions and population dynamics between the copepods and dinoflagellates in natural marine ecosystems.

FIGURE 2
FIGURE 2 Calculated grazing coefficients (g, d −1 ) of the Acartia spp.(A.hongi and A. omorii) in relation to the population of cooccurring Alexandrium pseudogonyaulax in the western coastal waters of Korea.

TABLE 1
Isolation and maintenance conditions of the experimental organisms.

TABLE 2
Experimental design for feeding by the copepods Acartia spp. on Alexandrium pseudogonyaulax.
The numbers are the initial abundances (cells mL −1 for A. pseudogonyaulax and inds.L −1 for Acartia spp.) of A. pseudogonyaulax and Acartia spp.Values in the parentheses in A. pseudogonyaulax are the abundances in ng C mL −1 .

TABLE 3
Sequences of the primers and probe for Alexandrium pseudogonyaulax used in this study.
,570 ind.m −3 , respectively.The grazing coefficients attributable to Acartia spp. on co-occurring A. pseudogonyaulax were 0.001 to 0.073 d −1 (i.e., up to 7% of A. pseudogonyaulax population could be removed by the copepod Acartia spp. in a day).

TABLE 4
Comparison of maximum ingestion rates of Acartia species on Alexandrium species.