Paralytic Shellfish Toxins Research Articles

The Caspase Homologues in Scallop Chlamys farreri and Their Expression Responses to Toxic Dinoflagellates Exposure.

The cysteine aspartic acid-specific protease (caspase) family is distributed across vertebrates and invertebrates, and its members are involved in apoptosis and response to cellular stress. The Zhikong scallop (Chlamys farreri) is a bivalve mollusc that is well adapted to complex marine environments, yet the diversity of caspase homologues and their expression patterns in the Zhikong scallop remain largely unknown. Here, we identified 30 caspase homologues in the genome of the Zhikong scallop and analysed their expression dynamics during all developmental stages and following exposure to paralytic shellfish toxins (PSTs). The 30 caspase homologues were classified as initiators (caspases-2/9 and caspases-8/10) or executioners (caspases-3/6/7 and caspases-3/6/7-like) and displayed increased copy numbers compared to those in vertebrates. Almost all of the caspase-2/9 genes were highly expressed throughout all developmental stages from zygote to juvenile, and their expression in the digestive gland and kidney was slightly influenced by PSTs. The caspase-8/10 genes were highly expressed in the digestive gland and kidney, while PSTs inhibited their expression in these two organs. After exposure to different Alexandrium PST-producing algae (AM-1 and ACDH), the number of significantly up-regulated caspase homologues in the digestive gland increased with the toxicity level of PST derivatives, which might be due to the higher toxicity of GTXs produced by AM-1 compared to the N-sulphocarbamoyl analogues produced by ACDH. However, the effect of these two PST-producing algae strains on caspase expression in the kidney seemed to be stronger, possibly because the PST derivatives were transformed into highly toxic compounds in scallop kidney, and suggested an organ-dependent response to PSTs. These results indicate the dedicated control of caspase gene expression and highlight their contribution to PSTs in C. farreri. This work provides a further understanding of the role of caspase homologues in the Zhikong scallop and can guide future studies focussing on the role of caspases and their interactions with PSTs.

Chlorophyll-a and Sea Surface Temperature Changes in Relation to Paralytic Shellfish Toxin Production off the East Coast of Tasmania, Australia

Toxic phytoplankton have been detrimental to the fishing and aquaculture industry on the east coast of Tasmania, causing millions of dollars in loss due to contaminated seafood. In 2012–2017, shellfish stocks were poisoned by Alexandrium catenella, a dinoflagellate species that produces paralytic shellfish toxins (PST). Remote sensing data may provide an environmental context for the drivers of PST events in Tasmania. We conducted spatial and temporal trend analyses of the Multi-Scale Ultra-High-Resolution Sea Surface Temperature (MUR SST) and Ocean Color Climate Change Initiative chlorophyll-a (OC-CCI chl-a) to determine if SST and chl-a correlated with the major toxin increases from 2012 to 2017. Along with the trends, we compare the remotely sensed oceanographic parameters of SST and chl-a to toxin events off the east coast of Tasmania to provide environmental context for the high-toxin period. Spatial and temporal changes for chl-a differ based on the north, central, and southeast coast of Tasmania. For sites in the north, chl-a was 5.3% higher from the pre-PST period relative to the PST period, 5.1% along the central part of the coast, and by 6.0% in the south based on deviations from the coastal study area time series. Overall, SST has slightly decreased from 2007 to 2020 (tau = −0.011, p = 0.827) and chl-a has significantly decreased for the east coast (tau = −0.164, p = 1.58 × 10−3). A negative relationship of SST and PST values occurred in the north (r = −0.530, p = 5.32 × 10−5) and central sites (r = −0.225, p = 0.157). The correlation between satellite chl-a (from OC-CCI, Visible Infrared Imaging Radiometer Suite (VIIRS), and Moderate-Resolution Imaging Spectrometer (MODIS) Aqua) and in situ data is weak, which makes it difficult to assess relationships present between chl-a and toxin concentrations. Moving forward, the development of a regional chl-a algorithm and increased in situ chl-a collection and plankton sampling at a species level will help to improve chl-a measurements and toxic phytoplankton production monitoring around Tasmania.

Temporal Prediction of Paralytic Shellfish Toxins in the Mussel Mytilus galloprovincialis Using a LSTM Neural Network Model from Environmental Data.

Paralytic shellfish toxins (PSTs) are produced mainly by Alexandrium catenella (formerly A. tamarense). Since 2000, the National Institute of Fisheries Science (NIFS) has been providing information on PST outbreaks in Korean coastal waters at one- or two-week intervals. However, a daily forecast is essential for immediate responses to PST outbreaks. This study aimed to predict the outbreak timing of PSTs in the mussel Mytilus galloprovincialis in Jinhae Bay and along the Geoje coast in the southern coast of the Korea Peninsula. We used a long-short-term memory (LSTM) neural network model for temporal prediction of PST outbreaks from environmental data, such as water temperature (WT), tidal height, and salinity, measured at the Geojedo, Gadeokdo, and Masan tidal stations from 2006 to 2020. We found that PST outbreaks is gradually accelerated during the three years from 2018 to 2020. Because the in-situ environmental measurements had many missing data throughout the time span, we applied LSTM for gap-filling of the environmental measurements. We trained and tested the LSTM models with different combinations of environmental factors and the ground truth timing data of PST outbreaks for 5479 days as input and output. The LSTM model trained from only WT had the highest accuracy (0.9) and lowest false-alarm rate. The LSTM-based temporal prediction model may be useful as a monitoring system of PSP outbreaks in the coastal waters of southern Korean.

Massive bloom of the paralytic shellfish toxin producing dinoflagellate Alexandrium spp. and its mechanisms in Onagawa Bay during spring 2018

Massive bloom of the paralytic shellfish toxin producing dinoflagellate <i>Alexandrium</i> spp. and its mechanisms in Onagawa Bay during spring 2018

Marine paralytic shellfish toxins: chemical properties, mode of action, newer analogues, and structure-toxicity relationship.

Up to the end of 2020Every year, the appearance of marine biotoxins causes enormous socio-economic damage worldwide. Among the major groups of biotoxins, paralytic shellfish toxins, comprising saxitoxin and its analogues (STXs), are the ones that cause the most severe effects on humans, including death. However, the knowledge that currently exists on their chemistry, properties and mode of toxicological action is disperse and partially outdated. This review intends to systematically compile the dispersed information, updating and complementing it. With this purpose, it addresses several aspects related to the molecular structure of these toxins. Special focus is given to the bioconversion reactions that may occur in the different organisms (dinoflagellates, bivalves, and humans) and the possible mediators involved. A critical review of the most recently discovered analogues, the M-series toxins, is presented. Finally, a deep discussion about the relationship between the molecular structure (e.g., effect of the substituting groups and the net charge of the molecules) and the toxic activity of these molecules is performed, proposing the concept of "toxicological traffic light" based on the toxicity equivalency factors (TEFs).

Paralytic shellfish poisoning toxins in butter clams (Saxidomus gigantea) from the Kodiak Archipelago, Alaska.

Consumption of toxic butter clams (Saxidomus gigantea) is the most frequent cause of paralytic shellfish poisoning (PSP) in Alaskan coastal communities. This study examines seasonal variation in total paralytic shellfish toxin concentrations and congener distribution in tissues of butter clams collected in three communities in the Kodiak Islands, Alaska: the City of Kodiak, Ouzinkie and Old Harbor. In response to questions from local harvesters, the efficacy of removing particular clam tissues on total toxin levels was also assessed. Butter clam samples were collected ∼monthly during 2015–2020 in each community to monitor shellfish toxin levels. Results were combined with clam monitoring data collected previously (2013–2015) to document the seasonal distribution of saxitoxin (STX) and its congeners (neosaxitoxin, gonyautoxin) in clam tissues. Seasonally, paralytic shellfish toxin levels in butter clams were highest in summer, declined in winter, but often remained above regulatory limits throughout the year in the three Kodiak communities. Butter clams collected from Ouzinkie (2013–2020) averaged 165 ± 87 µg STX equivalents (Eq.) 100 g − 1, compared to Kodiak 73 ± 54 µg STX Eq. 100 g − 1 and Old Harbor 143 ± 103 µg STX Eq. 100 g − 1. STX accounted for 59–71% of the total toxin concentration in clams at Ouzinkie, Kodiak, and Old Harbor, while neosaxitoxin (neoSTX) accounted for 12–18%. Gonyautoxins (GTXs) represented 31–60% of the total toxin concentration during the seasonal Alexandrium catenella bloom in June-July, with lower percentages in other months. The fraction of total toxin varied among clam tissues: the siphon tip (2–29%), the neck (3–56%), the gut (3–65%) and the body (6–85%). Removal of the siphon tip reduced total toxin content substantially in some samples but had little effect in others. Saxitoxin congeners varied greatly and somewhat unpredictably among clam tissues, and the results indicate removal of specific tissues was not an effective strategy for reducing paralytic shellfish toxin levels in butter clams for safe consumption.

Unknown Extracellular and Bioactive Metabolites of the Genus Alexandrium: A Review of Overlooked Toxins.

Various species of Alexandrium can produce a number of bioactive compounds, e.g., paralytic shellfish toxins (PSTs), spirolides, gymnodimines, goniodomins, and also uncharacterised bioactive extracellular compounds (BECs). The latter metabolites are released into the environment and affect a large range of organisms (from protists to fishes and mammalian cell lines). These compounds mediate allelochemical interactions, have anti-grazing and anti-parasitic activities, and have a potentially strong structuring role for the dynamic of Alexandrium blooms. In many studies evaluating the effects of Alexandrium on marine organisms, only the classical toxins were reported and the involvement of BECs was not considered. A lack of information on the presence/absence of BECs in experimental strains is likely the cause of contrasting results in the literature that render impossible a distinction between PSTs and BECs effects. We review the knowledge on Alexandrium BEC, (i.e., producing species, target cells, physiological effects, detection methods and molecular candidates). Overall, we highlight the need to identify the nature of Alexandrium BECs and urge further research on the chemical interactions according to their ecological importance in the planktonic chemical warfare and due to their potential collateral damage to a wide range of organisms.

Magnetic Amino-Modified Multiwalled Carbon Nanotube (MWCNT) Based Magnetic Dispersive Solid-Phase Extraction (m-dSPE) for the Determination of Paralytic Shellfish Toxins in Bivalve Mollusks with Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry (HILIC-MS/MS)

A sensitive and efficient magnetic amino modified multi-walled carbon nanotubes (m-MWCNT-NH2)-based magnetic dispersive solid-phase extraction (m-dSPE) coupled with hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) strategy is reported for the simultaneous determination of 13 paralytic shellfish toxins (PSTs) in bivalve mollusks. The samples were extracted twice with 1% acetic acid and purified using optimized m-dSPE procedures with Fe3O4 coated MWCNT-NH2 composites as magnetic adsorbents. The adsorbed PSTs on the m-MWCNT-NH2 adsorbents were separated using a Ni-coated neodymium magnet and subsequently eluted with 2 mL water–acetonitrile–acetic acid (80:20:1, v/v/v) and analyzed by HILIC-MS/MS. The correlation coefficients (r) of the targeted toxins obtained in matrix-matched external standard curves were from 0.997 to 0.999. The PSTs were acquired and quantified in the multiple reaction-monitoring mode and the limits of detection and quantitation in bivalve mollusks were 1.10 to 4.51 μg/kg and 3.67 to 13.5 μg/kg, respectively. Spiked recoveries at three concentrations in negative samples were from 73.4% to 92.5% with precision less than 10.6%. The method validation was in accordance with 2002/657/EC guidelines and method application in commercial bivalve mollusk samples. The developed method was rapid, convenient and economical for the determination of the targeted toxins.

The value of toxin profiles in the chemotaxonomic analysis of paralytic shellfish toxins in determining the relationship between British Alexandrium spp. and experimentally contaminated Mytilus sp.

Although phytoplankton is ubiquitous in the world's oceans some species can produce compounds that cause damaging effects in other organisms. These include the toxins responsible for paralytic shellfish poisoning, which, in UK waters, are produced by dinoflagellates from the Alexandrium genus. Within Great Britain (GB) a monitoring programme exists to detect this harmful genus as well as the Paralytic Shellfish Poisoning (PSP) toxins in the flesh of shellfish from classified production areas. The techniques used for toxin analysis allow for detailed analysis of the toxin profiles present in contaminated shellfish. It is possible to compare the toxin profiles of contaminated shellfish with the profiles from toxin producing algae and use this information to infer the causative microalgal species responsible for the contamination. This study sought to evaluate the potential for this process within the GB monitoring framework. Two species of toxic Alexandrium, A. catenella from Scotland and A. minutum from Southern England, were fed to mussels (Mytilus sp.) under controlled conditions. The toxin profile in mussels derived from feeding on each species independently, when mixed and when introduced sequentially was analysed and compared to the source algal cultures using K means cluster analysis. Toxin profiles in contaminated shellfish clustered with those of the causative algae and separately from one another during toxin accumulation and, where A. catenella was the sole toxin source, during depuration. During depuration after feeding with A. minutum and where mixed or sequential feeding was undertaken deviant toxin profiles were observed. Finally, data generated within this experimental study were compared to monitoring data from the GB official control programme. These data indicated that the causative algal species in sole source contaminations could be inferred from toxin profile analysis. This technique will be of benefit within monitoring programmes to enhance the value of data with minimal additional expense, where the toxin profiles of causative microalgae have been well described.

Connections and clustering of Paralytic Shellfish Toxin events among coastal embayments in an archipelago partly mediated by advection

The potential for advection to influence harmful algal bloom (HAB) spread in adjacent embayments and islands has not been investigated in the Philippines as previous studies have focused on HAB dynamics within specific embayments. Advection of HABs may be relevant in the Samar-Leyte region where adjacent sites are subject to recurrent blooms of Pyrodinium bahamense. We used different analyses to extract the potential role of advection in blooms in the region. First, we used Bayesian and information theoretic approaches applied to historical data on shellfish bans to quantify spatial dependencies in HAB occurrences between sites. Then, to determine whether such dependencies are related to circulation patterns in the region, we analyzed connectivity using a hydrodynamic model coupled with a conservative tracer-based HAB model. The Bayesian network showed that in 7 out of 11 sites, the probability of a shellfish ban depended on the state of an adjacent site. Site pairs with direct dependence relations also shared relatively high similarity in HAB occurrences over time. In the modelled network, bans tend to occur sequentially, spreading from a few sites with relatively high probabilities for ban events. A subset of sites (sources) were found to be informative of future HAB event probabilities in other sites (destinations) over time lags that are generally longer the farther the destination. Modelled surface advection showed high connectivity strengths between sources and destinations associated with circulation features, e.g., an anticyclonic current in Leyte, wind-driven coastal current in western Samar, and tidally-driven flow in the shallow embayments in southwest Samar. High connectivities were correlated with direct dependence relations in the Bayesian network. Connectivity explained up to about 1/3 of the variance in statistical dependencies between ban signals. Our results show that Paralytic Shellfish Toxin events within this region can be due to advection of blooms originating from nearby areas rather than localized cyst dynamics within an embayment. Particular sites were also identified as potential indicator sites within spatial clusters for monitoring since they are typically where blooms originate and spread from.

The effects of two sized polystyrene nanoplastics on the growth, physiological functions, and toxin production of Alexandrium tamarense

Micro- and nano-plastics (MNPs) are increasingly prevalent pollutants in marine ecosystems and result in various deleterious effects on marine organisms. There have been studies evaluated the toxic effects of MNPs on marine microalgae, but few of them focused on the effects of MNPs on dinoflagellate species and their toxins production, which could have significant implications on human health and ecological safety in coastal areas. In this study, the common harmful algal blooms-causing dinoflagellate Alexandrium tamarense was exposed to 0.1 and 1 μm sized polystyrene nanoplastics (NPs) to investigate the responding patterns of population growth, multiple physiological functions, as well as the intracellular paralytic shellfish toxins (PSTs) productions. The results indicated the population growth, photosynthetic parameters, nutrients (nitrate and phosphate) uptake rates and extracellular carbonic anhydrase activities (CAext) were all inhibited by the two sized NPs, accompanied by the prolonged and more aggregated microalgal cells under the observation of scanning electron microscope (SEM), and the inhibition effects were more severe under 1 μm sized NPs than 0.1 μm sized NPs. Finally, we found the intracellular PSTs contents increased 73.59% exposed to 0.1 μm sized NPs while decreased 85.50% exposed to 1 μm sized NPs comparing the controls at 96 h, without significant changes of relative compositions. These results provided evidence that MNPs were toxic to A. tamarense and affected their intracellular PSTs productions within 96 h, which is critical to consider when evaluating the potential risks of MNPs in marine ecosystems.

Differentiating toxic and nontoxic congeneric harmful algae using the non-polar metabolome

Recognition and rejection of chemically defended prey is critical to maximizing fitness for predators. Paralytic shellfish toxins (PSTs) which strongly inhibit voltage-gated sodium channels in diverse animal taxa are produced by several species of the bloom-forming algal genus Alexandrium where they appear to function as chemical defenses against grazing copepods. Despite PSTs being produced and localized within phytoplankton cells, some copepods distinguish toxic from non-toxic prey, selectively ingesting less toxic cells, in ways that suggest cell surface recognition perhaps associated with non-polar metabolites. In this study LC/MS and NMR-based metabolomics revealed that the non-polar metabolomes of two toxic species (Alexandrium catenella and Alexandrium pacificum) vary considerably from their non-toxic congener Alexandrium tamarense despite all three being very closely related. Toxic and non-toxic Alexandrium spp. were distinguished from each other by metabolites belonging to seven lipid classes. Of these, 17 specific metabolites were significantly more abundant in both toxic A. catenella and A. pacificum compared to non-toxic A. tamarense suggesting that just a small portion of the observed metabolic variability is associated with toxicity. Future experiments aimed at deciphering chemoreception mechanisms of copepod perception of Alexandrium toxicity should consider these metabolites, and the broader lipid classes phosphatidylcholines and sterols, as potential candidate cues.

Performance of different extraction methods for paralytic shellfish toxins and toxin stability in shellfish during storage.

Accurate analysis of paralytic shellfish toxins (PSTs) in shellfish is important to protect seafood safety and human health. In this study, the performance of different extraction protocols for PSTs from scallop tissues is compared and discussed, including regular extraction solvents hydrochloric acid (HCl) and acetic acid (AcOH) followed by heating and solid-phase extraction (SPE) purification, and a novel technique of matrix solid-phase dispersion (MSPD) without heating. The possible conversion of C1/2 and GTX2/3 standards after heating, and the stability of PSTs in wet scallop tissues stored at -20°C for a 6-month period are also explored. Results showed that the MSPD technique could effectively mitigate matrix interference, but its recoveries of PSTs were significantly lower than those of the HCl and AcOH extraction methods followed by carbon SPE purification. The molar concentrations of M-toxins obtained by the MSPD method were generally lower than those analyzed by the HCl and AcOH extraction methods, which demonstrated a weak chemical conversion of C1/2 and GTX2/3 due to the heating process. Most of the PSTs were relatively stable in scallop tissues during 1-month storage at -20°C, while the concentrations of PSTs in scallop tissues obviously changed after 6 months due to the degradation and transformation of PSTs during long-term storage at -20°C. This work helps improve our understanding of the performance of different extraction methods and the stability of PSTs in scallop tissues stored at -20°C.

Acute toxicity of decarbamoyl gonyautoxin 1&4 to mice by various routes of administration

Saxitoxin and its derivatives, the paralytic shellfish toxins (PSTs), are well known to be toxic to humans, and maximum permitted levels in seafood have been established by regulatory authorities in many countries. Monitoring of PSTs is typically performed using chemical methods which quantify the concentration of the individual PST analogues, of which there are many. However, since the toxicities of analogues are different, they do not equally contribute to the overall toxicity of the sample. To account for these differences, toxicity equivalency factors (TEFs) need to be determined for each analogue and applied. Currently there are no established TEFs for decarbamoyl gonyautoxin 1&4 (dcGTX1&4), which occurs in some clam species such as Mactra chinensis contaminated with PSTs due to metabolism within the shellfish. In this study the median lethal dose of purified, equilibrated epimeric mixture of dcGTX1&4 has been determined by intraperitoneal injection (i.p.) (4.75 μmol/kg) and by feeding (34.9 μmol/kg). The most relevant route of exposure is orally with feeding being more representative of human consumption and more reliable than gavage. Based on the median lethal dose by feeding, a TEF of 0.1 is recommended for dcGTX1&4. Receptor binding activity and i.p. toxicity results showed dcGTX1&4 to be much less toxic than STX (140–170-fold). However, by feeding a much smaller difference in toxicity was observed with dcGTX1&4 being only 11-fold less toxic than STX. Analysis of the gut contents of mice dosed with dcGTX1&4 showed the presence of decarbamoyl gonyautoxin 2&3, decarbamoyl saxitoxin and decarbamoyl neosaxitoxin, all of which are of greater toxicity. This conversion of dcGTX1&4 within the digestive track to more toxic congeners may explain the high relative toxicity of dcGTX1&4 by feeding compared to that determined by i.p. and by sodium channel activity.

Paralytic shellfish toxins (PSTs) and tetrodotoxin (TTX) of Korean pufferfish

Paralytic shellfish toxins (PSTs) and tetrodotoxin (TTX) are neurotoxins that display pharmacological activity that is similar to that of specific sodium channel blockers; they are the principle toxins involved in shellfish and puffer fish poisoning. In Korea, puffer fish is a very popular seafood, and several cases of accidental poisoning by TTX have been reported. Therefore, it is necessary to determine whether puffer fish poisoning incidents are caused by PSTs or by TTX. In this study, we used mouse bioassay (MBA) and liquid chromatograph-tandem mass spectrometry (LC-MS/MS) to determine the presence of PSTs and TTX in puffer fish from an area near Mireuk-do, Tong-Yeong on the southern coast of Korea from January through March, 2014. The toxicity of PSTs and TTX extracts prepared from three organs of each specimen was analyzed by MBA. Most of the extracts killed mice with typical signs of TTX and PSTs. The LC-MS/MS analysis of seven specimens of Takifugu pardalis and Takifugu niphobles, each divided into muscles, intestines, and liver, were examined for TTX. In T. pardalis, the TTX levels were within the range of 1.3–1.6 μg/g in the muscles, 18.8–49.8 μg/g in the intestines, and 23.3–96.8 μg/g in the liver. In T. niphobles, the TTX levels were within the range of 2.0–4.5 μg/g in the muscles, 23.9–71.5 μg/g in the intestines, and 28.1–114.8 μg/g in the liver. Additionally, the toxicity profile of the detected PSTs revealed that dcGTX3 was the major component in T. pardalis and T. niphobles. When PSTs were calculated as saxitoxin equivalents the levels were all less than 0.5 μg/g, which is below the permitted maximum standard of 0.8 μg/g. These findings indicate that the toxicity of T. pardalis and T. niphobles from the southern coast of Korea is due mainly to TTX and that PSTs do not exert an effect.

The effect of iron on Chilean Alexandrium catenella growth and paralytic shellfish toxin production as related to algal blooms

The dinoflagellate Alexandrium catenella is a well-known paralytic shellfish toxin producer that forms harmful algal blooms (HABs) worldwide. Blooms of this species have repeatedly brought severe ecological and economic impacts to Chile, especially in the southern region, where the shellfish and salmon industries are world-famous. The mechanisms of such HABs have been intensively studied but are still unclear. Nutrient overloading is one of the often-discussed drivers for HABs. The present study used the A. catenella strain isolated from southern Chile to investigate how iron conditions could affect their growth and toxin production as related to HAB. Our results showed that an optimum concentration of iron was pivotal for proper A. catenella growth. Thus, while excess iron exerted a toxic effect, low iron media led to iron insufficiency and growth inhibition. In addition, the study shows that the degree of paralytic shellfish toxin production by A. catenella varied depending on the iron concentration in the culture media. The A. catenella strain from southern Chile produced GTX1-4 exclusively in the fmol cell−1 scale. Based on these findings, we suggest that including iron and paralytic shellfish toxin measurements in the fields can improve the current HAB monitoring and contribute to an understanding of A. catenella bloom dynamics in Chile.

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).

Changing Trends in Paralytic Shellfish Poisonings Reflect Increasing Sea Surface Temperatures and Practices of Indigenous and Recreational Harvesters in British Columbia, Canada.

Paralytic shellfish poisoning (PSP) occurs when shellfish contaminated with saxitoxin or equivalent paralytic shellfish toxins (PSTs) are ingested. In British Columbia, Canada, documented poisonings are increasing in frequency based on 62 investigations identified from 1941–2020. Two PSP investigations were reported between 1941 and 1960 compared to 31 since 2001 (p < 0.0001) coincident with rising global temperatures (r2 = 0.76, p < 0.006). The majority of PSP investigations (71%) and cases (69%) were linked to self-harvested shellfish. Far more investigations involved harvests by indigenous communities (24%) than by commercial and recreational groups. Single-case-exposure investigations increased by more than 3.5 times in the decade 2011–2020 compared to previous periods. Clams (47%); mussels (26%); oysters (14%); scallops (6%); and, in more recent years, crabs (4%) were linked to illnesses. To guide understanding of self-harvesting consumption risks, we recommend collecting data to determine when PST-producing algae are present in high concentrations, improving the quality of data in online shellfish harvest maps to include dates of last testing; biotoxin testing results; and a description of bivalve species tested. Over reliance on toxin results in biomonitored species may not address actual consumption risks for unmonitored species harvested from the same area. We further recommend introducing phytoplankton monitoring in remote indigenous communities where self-harvesting is common and toxin testing is unavailable, as well as continuing participatory education about biotoxin risks in seafoods.

Dynamics of the Toxic Dinoflagellate Alexandrium pacificum in the Taiwan Strait and Its Linkages to Surrounding Populations

The dinoflagellate Alexandrium pacificum can produce paralytic shellfish toxins and is mainly distributed in the Pacific. Blooms of A. pacificum have been frequently reported in offshore areas of the East China Sea, but not along the coast. To investigate the bloom dynamics of A. pacificum and their potential origins in the Taiwan Strait, we performed intensive sampling of both water and sediments from 2017 to 2020. Ellipsoidal cysts were identified as A. pacificum and enumerated based on microscopic observation. Their abundances were quite low but there was a maximum of 9.6 cysts cm−3 in the sediment near the Minjiang River estuary in May 2020, consistent with the high cell abundance in the water column in this area. Cells of A. pacificum were examined using a quantitative polymerase chain reaction, and they appeared to be persistent in the water column across the seasons. High densities of A. pacificum (103 cells L−1) were observed near the Jiulongjiang and Minjiang River estuary in early May 2020, where high nutrients (dissolved inorganic nitrogen and phosphate), and relatively low temperatures (20–21 °C) were also recorded. Strains isolated from the East and South China Sea exhibited the highest division rate (0.63 and 0.93 divisions d−1) at 20 and 23 °C, respectively, but the strain from the Yellow Sea showed the highest division (0.40 divisions d−1) at 17–23 °C. Strains from the East and South China Sea shared similar toxin profiles dominated by the N-sulfocarbamoyl toxins C1/2, but the strain from the Yellow Sea predominantly produced the carbamoyl toxins GTX1/4 and no C1/2. Our results suggest that both cyst germination and persistent cells in the water column might contribute to the bloom formation in the Taiwan Strait. Our results also indicate that the East and South China Sea populations are connected genetically through similar toxin formation but separated from the Yellow Sea population geographically.

Novel Non-paralytic Shellfish Toxin and Non-spirolide Toxicity to Finfish, Brine Shrimp, and Rotifer Observed in a Culture of the Dinoflagellate Alexandrium insuetum Isolated From the Coastal Water of China

The genus Alexandrium is one of the major harmful algal blooms (HABs)-forming dinoflagellate group and at least half of ~40 described species have been reported to produce paralytic shellfish toxins (PSTs). The potentially harmful species Alexandrium insuetum has been reported from many countries of Asia and Europe, and to have paralytic shellfish poisoning toxicity, but no mortality of marine animals was observed during its bloom. Therefore, it is ecologically important to characterize the possible toxicity and toxins of this organism. In this study, based on the establishment of two clonal cultures through cyst germination collected from the Yellow Sea, we identified A. insuetum from China as the first record via light microscopy (LM) and scanning electron microscopy (SEM) observations and phylogenetic analyses. The cultures of A. insuetum were further observed to be toxic to finfish and zooplankton and deleterious to rotifer eggs via laboratory bioassays. The exposure bioassays using rotifer (Brachionus plicatilis), brine shrimp (Artemia salina), and larval finfish (Oryzias melastigma) demonstrated that A. insuetum caused significant lethal effects on finfish and zooplankton species. Rotifer bioassays using cell-free culture medium, heat-treated cultures, and water, methanol, and trichloromethane extracts of algal cells revealed that A. insuetum produced heat-labile, water-soluble toxin(s) that could be excreted from A. insuetum cells and steadily accumulated in the medium during the growth phases. Hatching success of rotifer eggs was also found to be seriously affected by the exposure to A. insuetum. Importantly, ultra-high performance liquid chromatography-tandem mass spectrometry [UPLC (or LC)-MS-MS] analyses suggest the above-described toxicity of A. insuetum was caused by neither PSTs nor spiroimines (13-desmethyl spirolide C and gymnodimine). Collectively, our findings demonstrated the novel toxicity to finfish and zooplankton in A. insuetum, which is ecologically important in not only possibly contributing to population dynamics and even the formation of HABs of the species, but also affecting the on-the-spot survival and the reproduction potency of marine animals. The present work is believed to set a cornerstone for the monitoring and risk assessment of the species along the coastal waters of China and for understanding the general ecology of A. insuetum.