The impact of sexual reproduction-induced vertical migration on the complexity of harmful algal blooms in Heterosigma akashiwo.

Vertical migration behaviour, which is integral to marine energy circulation, is a prevalent trait among marine organisms. However, the behaviour of phytoplankton, particularly beyond diel vertical migration (DVM), remain underexplored compared to groups like zooplankton. Through the lens of the harmful alga Heterosigma akashiwo, which exhibits unique vertical migrations and fluctuating red tide patterns, this study aimed to explore the ecological intricacies and diverse benefits of phytoplankton vertical migration behaviours. During the bloom period of H. akashiwo, we unexpectedly observed a dense concentration of cells at bottom layer during daytime. This phase coincided with the emergence of cells related to this species' sexual reproduction. Laboratory experiments further showed an elevated frequency of sexual reproduction in the cell populations that migrated to deeper depths compared to those at the surface. This finding implies a connection between dense bottom accumulation (BA) and the life cycle transitions of the species. This BA phase persisted for two days, after which the populations returned to their standard DVM behaviour, providing insight into the unique fluctuating red tide patterns of H. akashiwo. Our study suggests that phytoplankton vertical migrations are not strictly dictated by DVM, revealing diverse vertical migration behaviours that may contribute to the complexity of harmful algal bloom patterns.

Can algicide (the thiazolidinedione derivative TD49) truly contribute to the restoration of microbial communities?

Harmful algal blooms (HABs) are becoming a more serious ecological threat to marine environments; they not only produce toxins, resulting in the death of marine organisms, but they also adversely affect biodiversity, which is an indicator of the health of an ecosystem. Thus, to mitigate HABs, numerous studies have been conducted to develop an effective algicide, but few studies have elucidated the effect of algicides on marine environmental health. In this study, thiazolidinedione derivative 49 (TD49), which has been developed as an algicide for the dinoflagellate Heterocapsa circularisquama, was used, and we investigated changes in phytoplankton biomass (abundance, chlorophyll a, and carbon biomass) and biodiversity (diversity, evenness, and richness) following the application of TD49. To gain deeper understanding, a large-scale mesocosm (1300 L) experiment containing control and treatment with four different concentrations (0.2, 0.4, 0.6 and 1 µM) was conducted for 10 days. Based on a previous study, TD49 shows algicidal activity against H. circularisquama depending on its concentration. The phytoplankton biomass in the TD49 treatments was generally lower than that in the control due to the algicidal effect of TD49 on H. circularisquama. The biodiversity indices (e.g., the Shannon-Weaver index) in the treatments were consistently higher than those in the control before depletion of nitrite + nitrate. Interestingly, the 0.6 µM TD49 treatment had higher biodiversity indices than the high-concentration treatment (1 µM), which appeared to show a better algicidal effect. These findings suggest that mitigation of H. circularisquama blooms with TD49 treatment may enhance phytoplankton biodiversity, but treatment with excessively high concentrations can lead to harmful effects. During the study period, regardless of the control and TD49 treatments, the total biomass of phytoplankton gradually decreased from the midpoint of the experiment to the end of the experiment. This was more likely caused by the depletion of nutrients than by the toxicity of TD49.

Distinct Bloom Dynamics of Toxic and Non-toxic Microcystis (Cyanobacteria) Subpopulations in Hoedong Reservoir (Korea).

Despite the importance of understanding the bloom mechanisms that influence cyanobacterial toxin production, the dynamics of toxic Microcystis subpopulations are largely unknown. Here, we quantified both toxic and entire (i.e., toxic and non-toxic) Microcystis populations based on the microcystin synthetase E (mcyE) and 16S ribosomal RNA genes. Samples were collected from pelagic water and sediments twice per week from October to December 2011, and we investigated the effects of physicochemical factors (pH, water temperature, dissolved oxygen, nutrients, etc.) and biological factors (ciliates and zooplankton) on the abundance of toxic and non-toxic Microcystis. During the study period, Microcystis blooms were composed of toxic and non-toxic subpopulations. Resting stage Microcystis in sediment may be closely linked to Microcystis populations in pelagic water and may contribute to the toxic subpopulation composition in surface Microcystis blooms. In pelagic water, the toxic and entire Microcystis population had a significant positive correlation with the pH and water temperature (p < 0.05). However, their responses to changes in environmental factors were thought to be distinct. The ratio of the toxic to non-toxic Microcystis subpopulations was significantly (p < 0.05) enhanced by a lower pH and water temperature and an increase in protozoan grazers, reflecting environmental stresses. These results suggest that the toxic and non-toxic subpopulations of Microcystis have distinct tolerance levels against these stressors. The intracellular microcystin (MC) concentration was positively associated with the abundance of the mcyE-positive Microcystis. By comparison, the MC concentration in pelagic water body (extracellular) increased when Microcystis was lysed due to environmental stresses.

Use of microcatheters for suction thrombectomy of acute distal occlusions.

We report our initial experience of using a microcatheter for contact aspiration of acute distal occlusions for recanalization. Endovascular technique and a case using Excelsior XT-27 microcatheter are presented. After manual suction within distal middle cerebral artery segments using a 50-ml syringe, instant and complete recanalization was obtained. Microcatheter suction thrombectomy using a 50-ml syringe can be considered a feasible treatment option for acute distal small artery occlusions.

Improvement of cyanobacterial-killing biologically derived substances (BDSs) using an ecologically safe and cost-effective naphthoquinone derivative.

In previous studies, naphthoquinone (NQ) compounds have been shown to be effective, selective, and ecologically safe algicides for controlling harmful algal blooming species (HABs) or winter bloom species, such as Stephanodiscus hantzschii. However, there are no reports on NQ-based algicides for use with cyanobacterial blooming species. In this study, we developed 31 NQ compounds to investigate algicides for mitigating cyanobacterial blooms. In addition, to better apply these compounds in the field, we reduced the number of production steps to develop a cost-effective algicide. In preliminary testing, we screened NQ compounds that showed the best algicidal activity on target cyanobacteria, including Aphanizomenon, Dolichospermum, Microcystis, Oscillatoria, and Nostoc species. The compound NQ 2-0 showed the highest algicidal activity (90%) at a low concentration (≥1µM) on target algae. These were very limiting algicidal effects of 1µM NQ 2-0 observed against non-target algae, such as diatoms (Stephanodiscus hantzschii, Cyclotella meneghiniana, Synedra acus, and Aulacoseira granulata) or green algae (Cosmarium bioculatum and Scenedesmus quadricauda), and the effect did not exceed 15-25% (except against S. quadricauda). NQ 2-0 (1µM) showed no eco-toxicity, as represented by the survival rates of Pseudokirchneriella subcapitata (100%), Daphnia magna (100%), and Danio rerio (100%). Additionally, a chronic eco-toxicity assessment showed no toxicity toward the survival, growth or reproduction of D. magna. Moreover, NQ 2-0 quickly dissipated from field water samples and had a half-life of approximately 3.2 days. These results suggest that NQ 2-0 could be a selective and ecologically safe algicide to mitigate harmful cyanobacterial blooms.

Intraspecific diversity and distribution of the cosmopolitan species Pseudo-nitzschia pungens (Bacillariophyceae): morphology, genetics, and ecophysiology of the three clades.

Three clades of Pseudo-nitzschia pungens, determined by the internal transcribed space (ITS) region, are distributed throughout the world. We studied 15 P. pungens clones from various geographical locations and confirmed the existence of the three clades within P. pungens, based on ITS sequencing and described the three subgroups (IIIaa, IIIab, and IIIb) of clade III. Clade III (clade IIIaa) populations were reported for the first time in Korean coastal waters and the East China Sea. In morphometric analysis, we found the ultrastructural differences in the number of fibulae, striae, and poroids that separate the three clades. We carried out physiological tests on nine clones belonging to the three clades growing under various culture conditions. In temperature tests, only clade III clones could not grow at lower temperatures (10°C and 15°C), although clade I and II clones grew well. The estimated optimal growth range of clade I clones was wider than that of clades II and III. Clade II clones were considered to be adapted to lower temperatures and clade III to higher temperatures. In salinity tests, clade II and III clones did not grow well at a salinity of 40. Clade I clones were regarded as euryhaline and clade II and III clones were stenohaline. This supports the hypothesis that P. pungens clades have different ecophysiological characteristics based on their habitats. Our data show that physiological and morphological features are correlated with genetic intraspecific differentiation in P. pungens.

Application of a Quantitative PCR to Investigate the Distribution and Dynamics of Two Morphologically Similar Species, Karenia mikimotoi and K. papilionacea (Dinophyceae) in Korean Coastal Waters.

Species of the marine dinoflagellate genus Karenia are known to produce various potent biotoxins and can form noxious blooms that cause mass mortalities of fish and shellfish. To date, harmful blooms of the species K. mikimotoi have been reported in Korea, but K. papilionacea was recently recorded off the southern coast of Korea. Here, we developed a quantitative real-time PCR (qRT-PCR) assay with specific primer pairs for the accurate detection and quantification of these two similar-looking unarmored species, K. mikimotoi and K. papilionacea, and investigated their distribution and dynamics in Korean coastal waters. Overall, K. papilionacea had not only a wider distribution, but also higher cell abundances (15-2553 cells L-1) than K. mikimotoi (3-122 cells L-1) in surface waters. Of 18 sampling sites, the two Karenia species were found to coexist at two sites. During monitoring at a fixed station (S5), K. papilionacea was generally predominant over K. mikimotoi; however, the two species exhibited similar dynamics and occasionally co-occurred. Both Karenia species showed similar physiological responses to temperature and salinity, requiring similar conditions for optimum growth. These results suggest that blooms of the two species may co-occur and induce a synergistic adverse effect on marine environments.

Comprehensive understanding of the life history of harmful raphidophyte Heterosigma akashiwo: Integrating in situ and in vitro observations.

Heterosigma akashiwo (Raphidophyceae) is widely recognized as a species responsible for harmful algal blooms worldwide. The species has long been speculated to possess a more complex life history, attributed to the diverse morphological variations observed during cell cultivation. However, the understanding of its life history has remained insufficient due to limitations in observing transitions between life cycle stages in vitro and challenges associated with in situ investigations. In this study, a combination of in vitro (laboratory-based) and in situ (field-based) observations was employed to define the life cycle stages of H. akashiwo and elucidate the pathways of transition between these stages. Notably, novel homothallic sexual reproduction processes involving the fusion of hologametes and the subsequent formation of zygotes were observed for the first time in vitro. These zygotes were found to either divide into vegetative cells (Pathway I) or undergo enlargement, resulting in the formation of multiple cells with multiple nuclei (Pathway II). Furthermore, this study provides the first documentation of large cells and cell clusters in situ, including intermediate stages referred to as large cells with ongoing cytoplasmic division that serve as a bridge between these two cell types. The observed zygotes in vitro exhibited a large size (21.9-51.8 µm) and multinucleated characteristics, similar to the large cells (38.2-45.8 µm) and cell clusters observed in situ. This finding suggests that the large cells observed in situ were zygotes undergoing cell division to form cell clusters (Pathway III). Moreover, based on the striking similarities in cell morphology and nuclear size between the cells comprising the cell cluster (2.7-4.4 µm) and the cyst clusters of this species, along with the synchronized germination characteristics of cyst clusters, it is proposed that the cell cluster serves as a precursor to cysts. By integrating the in situ and in vitro observations, this study provides a comprehensive understanding of the previously poorly understood life history of H. akashiwo.

Bloom development of toxic dinoflagellate Alexandrium catenella (Group I) in Jinhae-Masan Bay, Korea: Germination strategy of resting cysts in relation to temperature and salinity.

To better understand the role of resting cysts in the outbreak of paralytic shellfish poisoning and bloom dynamics in Jinhae-Masan Bay, Korea, this study investigated the germination features of ellipsoidal Alexandrium cysts isolated from sediments collected in winter and summer under different combinations of temperature and salinity. Morphology and phylogeny of germling cells revealed that the ellipsoidal Alexandrium cysts belong to Alexandrium catenella (Group I). The cysts could germinate across a wide range of temperature (5-25 °C) with germination success within 5 days, indicating that continuous seeding for the maintenance of vegetative cells in the water column may occur through the year without an endogenous clock to regulate germination timing. In addition, the cyst germination of A. catenella (Group I) was not controlled by seasonal salinity changes. Based on the results, this study provides a schematic scenario of the bloom development of A. catenella (Group I) in Jinhae-Masan Bay, Korea.

Abiotic and biotic factors controlling sexual reproduction in populations of Pseudo-nitzschia pungens (Bacillariophyceae).

Pseudo-nitzschia pungens is a widely distributed marine pennate diatom. Hybrid zones, regions in which two different genotypes may interbreed, are important areas for speciation and ecology, and have been reported across the globe for this species. However, sexual reproduction between differing clades in the natural environment is yet to be observed and is difficult to predict. Here we carried out experiments using two mono-clonal cultures of P. pungens from different genotypes to measure the frequency and timing of sexual reproduction across varying biotic (growth phases and cell activity potential) and abiotic conditions (nutrients, light, turbulence). We found the mating rates and number of zygotes gradually decreased from exponential to late stationary growth phases. The maximum zygote abundance observed was 1,390 cells mL-1 and the maximum mating rate was 7.1%, both which occurred during the exponential growth phase. Conversely, only 9 cells mL-1 and a maximum mating rate of 0.1% was observed during the late stationary phase. We also found the higher the relative potential cell activity (rPCA) in parent cells, as determined by the concentration of chlorophyll a per cell and the ratio of colony formation during parent cultivations, revealed higher mating rates. Furthermore, sexual events were reduced under nutrient enrichment conditions, and mating pairs and zygotes were not formed under aphotic (dark) or shaking culture conditions (150 rpm). In order to understand the sexual reproduction of Pseudo-nitzschia in the natural environment, our results highlight that it is most likely the combination of both biotic (growth phase, Chl. a content) and abiotic factors (nutrients, light, turbulence) that will determine the successful union of intraspecific populations of P. pungens in any given region.

Detection and Quantification of the Harmful Dinoflagellate Margalefidinium polykrikoides (East Asian Ribotype) in the Coastal Waters of China.

As a marine ichthyotoxic dinoflagellate, Margalefidinium polykrikoides, previously named Cochlodinium polykrikoides, have caused mass mortalities of fish worldwide during blooms. Rapid detection of target species is a prerequisite for the timely monitoring and early warning of harmful algal blooms (HABs). However, it is difficult to achieve rapid identification with traditional methods. The technology of using quantitative real-time PCR (qPCR) to detect and quantify microalgae is relatively mature. Based on the accuracy, rapidity, and sensitivity of qPCR technology, it can be used in the monitoring and development of early warning systems for HABs. From 2017 to 2020, samples were collected from 15 locations off the Chinese coast or from local sea areas. Based on the qPCR detection and analysis, the target species, M. polykrikoides (East Asian ribotype, EAr), was found in samples from Tianjin, Yangtze River estuary, and offshore Fujian (East China Sea). This is the first time that M. polykrikoides (EAr) was detected in the coastal waters of Tianjin. The results reveal a distributive pattern of M. polykrikoides (EAr) along Chinese coastal waters. It is helpful to predict the future diffusion trend of M. polykrikoides (EAr) in the China Sea and provides a practical case for the future construction of monitoring and warning systems for M. polykrikoides and HABs.

Different Algicidal Modes of the Two Bacteria Aeromonas bestiarum HYD0802-MK36 and Pseudomonas syringae KACC10292T against Harmful Cyanobacteria Microcystis aeruginosa.

Blooms of harmful cyanobacteria Microcystis aeruginosa lead to an adverse effect on freshwater ecosystems, and thus extensive studies on the control of this cyanobacteria's blooms have been conducted. Throughout this study, we have found that the two bacteria Aeromonas bestiarum HYD0802-MK36 and Pseudomonas syringae KACC10292T are capable of killing M. aeruginosa. Interestingly, these two bacteria showed different algicidal modes. Based on an algicidal range test using 15 algal species (target and non-target species), HYD0802-MK36 specifically attacked only target cyanobacteria M. aeruginosa, whereas the algicidal activity of KACC10292T appeared in a relatively broad algicidal range. HYD0802-MK36, as a direct attacker, killed M. aeruginosa cells when direct cell (bacterium)-to-cell (cyanobacteria) contact happens. KACC10292T, as an indirect attacker, released algicidal substance which is located in cytoplasm. Interestingly, algicidal activity of KACC10292T was enhanced according to co-cultivation with the host cyanobacteria, suggesting that quantity of algicidal substance released from this bacterium might be increased via interaction with the host cyanobacteria.

Effect of marine heatwaves on bloom formation of the harmful dinoflagellate Cochlodinium polykrikoides: Two sides of the same coin?

In 2018, the bloom of harmful dinoflagellate Cochlodinium polykrikoides occurred under abnormally high water temperature (WT) conditions caused by heatwaves in Korean coastal water (KCW). To better understand C. polykrikoides bloom at high WTs in 2018, we conducted field survey and laboratory experiments (the physiological and genetic differences between the two strains, CP2013 and CP2018). The heatwave increased the WT from 24.1°C to 29.2°C for two weeks, leading to strong stratification even in mid July (p < 0.01, Chi square = 94.656, Kruskal-Wallis test). Under early stratification conditions, patch blooms formed more earlier than the average outbreak in the last 17 years in KCW, despite high WT reaching 30°C. In laboratory experiments, although there were no genetic differences in the LSU rDNA, both strains showed a significant different growth response to high WTs; above 28°C, CP2013 did not survive, but CP2018 was able to grow, suggesting that CP2018 had potential growth capacity at high WTs. However, the growth rate and yield of the culture (CP2018) were lowered at 30°C. Also, the blooms of C. polykrikoides in 2018 lasted only 3 weeks, which is unusual short compared to the average duration since 2002. The negative correlation between the average WT and duration of C. polykrikoides bloom in previous 17 years (R2 = 0.518, p < 0.01) supports that high WT approaching 30°C is not favorable for C. polykrikoides in KCW. Thus, our findings indicated that in relation to heatwaves, early stratification condition plays a critical role in developing C. polykrikoides blooms, but maintaining bloom are negatively affected under high WT conditions.

Alexandrium catenella (Group I) and A. pacificum (Group IV) cyst germination, distribution, and toxicity in Jinhae-Masan Bay, Korea.

To better understand the outbreaks of paralytic shellfish poisoning and bloom dynamics caused by Alexandrium species in Jinhae-Masan Bay, Korea, the germination and distributions of ellipsoidal Alexandrium cysts were investigated, and paralytic shellfish toxins (PSTs) profiles and contents were determined using strains established from germling cells. The phylogeny and morphological observations revealed that the germinated vegetative cells from ellipsoidal cysts collected from the surface sediments in Jinhae-Masan Bay belong to Alexandrium catenella (Group I) and A. pacificum (Group IV) nested within A. tamarense species complex. Cyst germinations of A. catenella (Group I) were observed at only 10 °C, whereas cysts of A. pacificum (Group IV) could germinate at temperature ranges of 10 to 25 °C. Maximum germination success (85%) for isolated cysts occurred at 15 °C, and the germling cells were A. pacificum (Group IV). The results indicate that the variation in water temperature in Jinhae-Masan Bay can control the seasonal variations in germination of cysts of A. catenella (Group I) and A. pacificum (Group IV). The germination rates of ellipsoidal Alexandrium cysts were different among sampling sites in Jinhae-Masan Bay, probably because of differences in distribution and abundance of A. catenella (Group I) and A. pacificum (Group IV) in the sediments. The ellipsoidal Alexandrium cyst concentrations were much higher in February than in August, however the distributions were similar. Gonyautoxins 3 and 4 (GTX-3 and GTX-4) contributed a large proportion (>90%) of the toxins produced by strains A. catenella (Group I) and A. pacificum (Group IV) established from germling cells, and the total cellular contents were higher in A. catenella (Group I) than in A. pacificum (Group IV).

Potential effects of bacterial communities on the formation of blooms of the harmful dinoflagellate Prorocentrum after the 2014 Texas City "Y" oil spill (USA).

The association between phytoplankton blooms and oil spills is still controversial despite numerous studies. Surprisingly, to date, there have been no studies on the effect of bacterial communities (BCs) exposed to crude oil on phytoplankton growth, even though crude oil changes BCs, which can then affect phytoplankton growth and species composition. Co-culture with crude oil-exposed BCs significantly stimulated the growth of Prorocentrum texanum in the laboratory. To gain more direct evidence, oil-degrading bacteria from oil-contaminated sediment collected after the Texas City "Y" oil spill were isolated, and changes in dinoflagellate growth when co-cultured with single bacterial isolates was investigated. The oil-degrading bacterial isolates significantly stimulated the growth of dinoflagellates (axenic and xenic cultures) through releasing growth-promoting substances. This study provides new evidence for the potential role of oil-degrading bacteria in the formation of phytoplankton blooms after an oil spill.

Potential Cause of Decrease in Bloom Events of the Harmful Dinoflagellate Cochlodinium polykrikoides in Southern Korean Coastal Waters in 2016.

Blooms of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides are responsible for massive fish mortality events in Korean coastal waters (KCW). They have been consistently present in southern KCW over the last two decades, but they were not observed in 2016, unlike in the previous years. Despite extensive studies, the cause of this absence of this dinoflagellate bloom remains largely unknown. Thus, we compared physico-chemical and biological data from along the Tongyeong coast between 2016 and the previous four years (2012-2015). The averages of water temperature and salinity in August, 2016 were significantly (p < 0.001) different from those in the previous years. The amount of Changjiang River discharge, which can affect the environmental conditions in the southern Korean coastal area via ocean currents, was larger than in the previous years, resulting in a reduction in the salinity level in August when blooms of C. polykrikoides usually occurred. Moreover, compared to previous years, in 2016, there was a weak expansion of C. polykrikoides blooms in the Goheung-Oenarodo area where C. polykrikoides blooms were annually initiated in KCW. Lastly, the strong winds from the typhoon Lionrock may also have contributed to the early termination of this dinoflagellate bloom. Together with these findings, the combination of these environmental conditions in 2016, unlike in previous years, may have inhibited the formation of C. polykrikoides blooms along the Tongyeong coast.

Sexual reproduction and genetic polymorphism within the cosmopolitan marine diatom Pseudo-nitzschia pungens.

Different clades belonging to the cosmopolitan marine diatom Pseudo-nitzschia pungens appear to be present in different oceanic environments, however, a 'hybrid zone', where populations of different clades interbreed, has also been reported. Many studies have investigated the sexual reproduction of P. pungens, focused on morphology and life cycle, rather than the role of sexual reproduction in mixing the genomes of their parents. We carried out crossing experiments to determine the sexual compatibility/incompatibility between different clades of P. pungens, and examined the genetic polymorphism in the ITS2 region. Sexual reproduction did not occur only between clades II and III under any of experimental temperature conditions. Four offspring strains were established between clade I and III successfully. Strains established from offspring were found interbreed with other offspring strains as well as viable with their parental strains. We confirmed the hybrid sequence patterns between clades I and III and found novel sequence types including polymorphic single nucleotide polymorphisms (SNPs) in the offspring strains. Our results implicate that gene exchange and mixing between different clades are still possible, and that sexual reproduction is a significant ecological strategy to maintain the genetic diversity within this diatom species.

Change in Paralytic Shellfish Toxins in the Mussel Mytilus galloprovincialis Depending on Dynamics of Harmful Alexandrium catenella (Group I) in the Geoje Coast (South Korea) during Bloom Season.

Paralytic shellfish toxins (PSTs) produced by Alexandriumcatenella (formerly A. tamarense) in Korean coastal waters caused the deaths of four people (in 1986 and 1996) who consumed contaminated mussels (Mytilus edulis). This led to more detailed consideration of the risks of PST outbreaks and incidents in Korea, including the introduction of shellfish collection bans. In this study, we investigated the relationships between A. catenella population dynamics and PST accumulation in the mussel M. galloprovincialis. Discharges from the Nakdong River affect the environmental conditions along the Geoje coast, resulting in low salinity and high nutrient levels that trigger blooms of A. catenella. At the toxin peak on 24 April 2017, the toxins detected in A. catenella cells were C1, gonyautoxin (GTX)1 and GTX2, whereas the concentrations of PSTs in M. galloprovincialis were high and in the order of GTX4 > GTX1 > GTX3 > saxitoxin (STX) > GTX2 > neoSTX > decarbamoylgonyautoxin (dcGTX)2 > dc GTX3. The PST level in mussels was also high. At 15 °C, the PSTs are constantly found to be higher (10-fold higher in 2017 and 30-fold higher in 2018) than safe levels for human consumption (80 µg STX diHCl equivalents 100 g-1).

Centrodinium punctatum (Dinophyceae) produces significant levels of saxitoxin and related analogs.

Centrodinium punctatum is a fusiform dinoflagellate with a global marine distribution. Due to a close phylogenetic relationship of one C. punctatum strain to Alexandrium species, toxin production of this C. punctatum strain was assessed using liquid chromatography coupled to tandem mass spectrometry. The paralytic shellfish toxin (PST) profile of C. punctatum was dominated by six analogs, i.e. STX (30%), GTX-1 (20%) and neoSTX (24%), followed by GTX-2 (9%), GTX-4 (9%) and GTX-3 (8%); deoxy-STX was also putatively identified while no gymnodimines, spirolides or goniodomins were detected. This is the first record of C. punctatum producing saxitoxins. The estimated cellular toxicity was rather elevated, between 91 and 212 pg cell-1 (or 259 and 601 fmol cell-1). When considering the toxicity equivalent factors, results suggest that this species can produce high cellular toxicity compared to other STX-producing dinoflagellates. Morphological details of the sulcal area and the hypotheca of Centrodinium punctatum were re-examined by scanning electron microscopy (SEM); this revealed that in the sulcal area, the left posterior sulcal plate (Ssp) is larger and longer than the left posterior sulcal plate and extended into the hypotheca. Based on the morphological observation, a revised interpretation of the sulcus and hypotheca is proposed.

Dynamics of Amoebophrya parasites during recurrent blooms of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides in Korean coastal waters.

During the bloom events of the harmful dinoflagellate Cochlodinium polykrikoides in August and October, 2012, infections by two different Amoebophrya species were observed in Korean coastal waters. To investigate the dynamics of the two parasites and their relative impact on the host populations, a quantitative real-time PCR (qPCR) method was applied to detect and quantify the parasites in the free-living and parasitic stages. Each specific primer set of the target species, Amoebophrya sp. 1 and sp. 2 was designed on the large subunit (LSU) and the first internal transcribed spacer (ITS1) of ribosomal RNA (rRNA) gene, respectively. Dynamics of the two Amoebophrya species via qPCR assay showed distinct patterns during the C. polykrikoides bloom events. Amoebophrya sp. 1 showed peaks during both bloom events in August and October with relatively low copies (106 to 107 copies L-1), while Amoebophrya sp. 2 appeared only during the bloom event in October with very high copies (109 to 1010 copies L-1). Overall, the qPCR measurements for the dynamics of two Amoebophrya species in the parasitic stage (> 5 µm fractions) were consistent with parasite prevalence through microscopic observations. Amoebophrya sp. 1 infections were observed during both bloom events in August and October with relatively low parasite prevalence (0.1-1.5%), while Amoebophrya sp. 2 infections were detected only during the bloom event in October with high prevalence (up to 45%). Taken together, Amoebophrya sp. 1 may be a generalist and C. polykrikoides may not be its primary host, while Amoebophrya sp. 2 may be a specialist which can substantially impact host population dynamics.