Description: Azadinium poporum is a small dinoflagellate from the family Amphidomataceae which is known for the production potential of azaspiracids (AZAs) causative of azaspiracid shellfish poisoning (AZP). A. poporum has been recorded from European and western Pacific waters. Here we report on the high variability of toxin profiles within this species in Chinese coastal waters. Out of 16 analyzed strains of A. poporum from different geographic locations along the Chinese coastline, three strains proved not to contain AZAs, whereas 13 strains contained different combinations of AZA-2, AZA-11, AZA-37, a yet unknown isomer of AZA-1 (named AZA-40) and new AZA with yet unreported molecular mass of 853 Da (named AZA-41). The new AZA-40, other than AZA-1 itself, belongs to the recently discovered “348-type” group, which in tandem mass spectrometry displays a group 4 fragment with m/z 348 instead of the group 4 fragment of the classic AZAs with m/z 362, indicating a shift of a methyl group from the C24-C40 part of the molecule (rings F-I) to the C2-C9 part (carboxylic side chain and ring A). AZA-41 apparently is a dehydro variant of AZA-2. In addition a previously reported AZA with a molecular mass 871 DA could be unambiguously assigned to AZA-11, which is known to be a shellfish metabolite of AZA-2. This is the first report of AZA-11 being also de novo synthetized by dinoflagellates.

Description: Background A major cause of phytoplankton mortality is predation by zooplankton. Strategies to avoid grazers have probably played a major role in the evolution of phytoplankton and impacted bloom dynamics and trophic energy transport. Certain species of the genus Pseudo-nitzschia produce the neurotoxin, domoic acid (DA), as a response to the presence of copepod grazers, suggesting that DA is a defense compound. The biosynthesis of DA comprises fusion of two precursors, a C10 isoprenoid geranyl pyrophosphate and l-glutamate. Geranyl pyrophosphate (GPP) may derive from the mevalonate isoprenoid (MEV) pathway in the cytosol or from the methyl-erythritol phosphate (MEP) pathway in the plastid. l-glutamate is suggested to derive from the citric acid cycle. Fragilariopsis, a phylogenetically related but nontoxic genus of diatoms, does not appear to possess a similar defense mechanism. We acquired information on genes involved in biosynthesis, precursor pathways and regulatory functions for DA production in the toxigenic Pseudo-nitzschia seriata, as well as genes involved in responses to grazers to resolve common responses for defense strategies in diatoms. Results Several genes are expressed in cells of Pseudo-nitzschia when these are exposed to predator cues. No genes are expressed in Fragilariopsis when treated similarly, indicating that the two taxa have evolved different strategies to avoid predation. Genes involved in signal transduction indicate that Pseudo-nitzschia cells receive signals from copepods that transduce cascading molecular precursors leading to the formation of DA. Five out of seven genes in the MEP pathway for synthesis of GPP are upregulated, but none in the conventional MEV pathway. Five genes with known or suggested functions in later steps of DA formation are upregulated. We conclude that no gene regulation supports that l-glutamate derives from the citric acid cycle, and we suggest the proline metabolism to be a downstream precursor. Conclusions Pseudo-nitzschia cells, but not Fragilariopsis, receive and respond to copepod cues. The cellular route for the C10 isoprenoid product for biosynthesis of DA arises from the MEP metabolic pathway and we suggest proline metabolism to be a downstream precursor for l-glutamate. We suggest 13 genes with unknown function to be involved in diatom responses to grazers.

Description: Thirteen isolates of Prorocentrum species were established from the coral reefs of Perhentian Islands Marine Park, Malaysia and underwent morphological observations and molecular characterization. Six species were found: P. caipirignum, P. concavum, P. cf. emarginatum, P. lima, P. mexicanum and a new morphotype, herein designated as P. malayense sp. nov. Prorocentrum malayense, a species closely related to P. leve, P. cf. foraminosum, P. sp. aff. foraminossum, and P. concavum (Clade A sensu Chomérat et al. 2018), is distinguished from its congeners as having larger thecal pore size and a more deeply excavated V-shaped periflagellar area. Platelet arrangement in the periflagellar area of P. malayense is unique, with the presence of platelet 1a and 1b, platelet 2 being the most anterior platelet, and a broad calabash-shaped platelet 3. The species exhibits consistent genetic sequence divergences for the nuclear-encoded large subunit ribosomal RNA gene (LSU rDNA) and the second internal transcribed spacer (ITS2). The phylogenetic inferences further confirmed that it represents an independent lineage, closely related to species in Clade A sensu Chomérat et al. Pairwise comparison of ITS2 transcripts with its closest relatives revealed the presence of compensatory base changes (CBCs). Toxicity analysis showed detectable levels of okadaic acid in P. lima (1.0–1.6 pg cell˗1) and P. caipirignum (3.1 pg cell˗1); this is the first report of toxigenic P. caipirignum in the Southeast Asian region. Other Prorocentrum species tested, including the new species, however, were below the detection limit.

Description: We report on the investigation of a mass stranding of 68 short-beaked common dolphins (Delphinus delphis) that occurred in Golfo Nuevo, Península Valdés, Argentina in March 2018. Twenty-one of the stranded dolphins were returned alive to the sea, while 47 animals died. Dead dolphins included all ages, with more males than females (29 males and 18 females). The cause of death investigation reported here is restricted to 15 adult individuals and one fetus on which a full set of diagnostics was prioritized due to limited funding. Our results demonstrate that the death of 16 dolphins assessed in this study was not due to obvious human effects (e.g. bycatch) or underlying pathologies, as all animals were in good body condition and had no external evidence of injuries. Infections by Morbillivirus, Influenza A virus, Sarcocystis spp., Toxoplasma gondii, or Neospora caninum, as well domoic acid (DA) toxicity were ruled out as ethiologies in this event. Notably, results on exposure to paralytic shelfish toxins (PSP) were the only investigated cause of death found positive. This is the first documentation of exposure to PSP toxins in short-beaked common dolphins from the Argentine Sea. At present our results are insufficient to assess whether PSP toxin exposure played a role in the death of the stranded dolphins. Notwithstanding, the full documentation and investigation of the most commonly reported pathogens and toxins involved in cetacean mass strandings allowed us to clear the most relevant health differentials and suggests areas for future study. Additional potential hypothesis related to factors known or speculated to cause cetacean mass strandings are currently being explored within the ecological context at the time of the event.

Description: In the Argentine Sea, marine phycotoxins of microalgal origin associated with five shellfish poisoning syndromes have been reported. The most problematic in terms of toxicity and geographic distribution is paralytic shellfish poisoning (PSP), followed by diarrhetic shellfish poisoning (DSP). In contrast, amnesic shellfish poisoning (ASP), spiroimine shellfish poisoning (SSP), and azaspiracid shellfish poisoning (AZP) have not been reported to cause human illness or closures of shellfish harvest sites in Argentina to date but pose a potential risk, as associated toxins and producing organisms are present in Southwest Atlantic waters and were detected at subregulatory levels in mollusks. Alexandrium catenella and Gymnodinium catenatum have been identified as producers of the PSP toxins C1/2, gonyautoxins (GTX1-4), saxitoxin (STX), and neosaxitoxin (NEO) in the Argentine Sea. Nine potentially toxigenic species of the diatom genus Pseudo-nitzschia have been reported for Argentinean coastal waters: P. australis, P. brasiliana, P. delicatissima, P. fraudulenta, P. multiseries, P. pseudodelicatissima, P. pungens, P. seriata, and P. turgidula, all of which are known to produce the neurotoxin domoic acid that causes ASP. Two genera have been identified as producers of DSP toxins in Argentina: the benthic dinoflagellate Prorocentrum lima and several species of the pelagic dinoflagellate genus Dinophysis: D. acuminata, D. caudata, D. fortii, D. norvegica, and D. tripos. The occurrence of these species in Argentine waters is associated with okadaic acid (OA), dinophysistoxin-1 (DTX-1), pectenotoxin-2 (PTX-2), and pectenotoxin-2 seco acid (PTX-2sa). Historically, yessotoxins (YTXs) were also included in DSP syndrome and all three known YTX-producers have been confirmed in Argentinean waters: Gonyaulax spinifera, Lingulodinium polyedra, and Protoceratium reticulatum, but of these only P. reticulatum could be associated with YTX production to date. Several species of the family Amphidomataceae, which cause AZP, have been reported for Argentina: Amphidoma languida, Azadinium dexteroporum, Az. luciferelloides, Az. poporum, and Az. spinosum. In Argentinean coastal waters, out of these species only Az. poporum has been identified as toxigenic to date, as it produces azaspiracid-2 (AZA-2) and its phosphorylated form. Currently in Argentina, seafood is monitored for the risk of ASP, AZP, DSP, and PSP.

Description: Copepods are important grazers on toxic phytoplankton and serve as vectors for algal toxins up the marine food web. Success of phytoplankton depends among other factors on protection against grazers like copepods, and same way copepod survival and population resilience relies on their ability to escape predators. Little is, however, known about the effect of toxins on the escape response of copepods. In this study we experimentally tested the hypothesis that the neurotoxin domoic acid (DA) produced by the diatom Pseudo-nitzschia affects escape responses of planktonic copepods. We found that the arctic copepods Calanus hyperboreus and C. glacialis reduced their escape response after feeding on a DA-producing diatom. The two species were not affected the same way; C. hyperboreus was affected after shorter exposure and less intake of DA. The negative effect on escape response was not related to the amount of DA accumulated in the copepods. Our results suggest that further research on the effects of DA on copepod behavior and DA toxicity mechanisms is required to evaluate the anti-grazing function of DA.

Description: The identification of a new suite of toxins, called azaspiracids (AZA), as the cause of human illnesses after the consumption of shellfish from the Irish west coast in 1995, resulted in interest in understanding the global distribution of these toxins and of species of the small dinoflagellate genus Azadinium, known to produce them. Clonal isolates of four species of Azadinium, A. poporum, A. cuneatum, A. obesum and A. dalianense were obtained from incubated sediment samples collected from Puget Sound, Washington State in 2016. These Azadinium species were identified using morphological characteristics confirmed by molecular phylogeny. Whereas AZA could not be detected in any strains of A. obesum, A. cuneatum and A. dalianense, all four strains of A. poporum produced a new azaspiracid toxin, based on LC–MS analysis, named AZA-59. The presence of AZA-59 was confirmed at low levels in situ using a solid phase resin deployed at several stations along the coastlines of Puget Sound. Using a combination of molecular methods for species detection and solid phase resin deployment to target shellfish monitoring of toxin at high-risk sites, the risk of azaspiracid shellfish poisoning can be minimized.

Description: In the Argentine Sea, blooms of toxigenic dinoflagellates of the Alexandrium tamarense species complex have led to fish and bird mortalities and human deaths as a consequence of paralytic shellfish poisoning (PSP). Yet little is known about the occurrence of other toxigenic species of the genus Alexandrium, or of their toxin composition beyond coastal waters. The distribution of Alexandrium species and related toxins in the Argentine Sea was determined by sampling surface waters on an oceanographic expedition during austral spring from ~39°S to 48°S. Light microscope and SEM analysis for species identification and enumeration was supplemented by confirmatory PCR analysis from field samples. The most frequent Alexandrium taxon identified by microscopy corresponded to the classical description of A. tamarense. Only weak signals of Group I from the A. tamarense species complex were detected by PCR of bulk field samples, but phylogenetic reconstruction of rDNA sequences from single cells from one station assigned them to ribotype Group I (Alexandrium catenella). PCR probes for Alexandrium minutum and Alexandrium ostenfeldii yielded a positive signal, although A. minutum morphology did not completely match the classical description. Analysis of PSP toxin composition of plankton samples revealed toxin profiles dominated by gonyautoxins (GTX1/4). The main toxic cyclic imine detected was 13-desMe-spirolide C and this supported the association with A. ostenfeldii in the field. This study represents the first integrated molecular, morphological and toxinological analysis of field populations of the genus Alexandrium in the Argentine Sea.

Description: The marine dinoflagellate Lingulodinium polyedra is a toxigenic species capable of forming high magnitude and occasionally harmful algal blooms (HABs), particularly in temperate coastal waters throughout the world. Three cultured isolates of L. polyedra from a fjord system on the Skagerrak coast of Sweden were analyzed for their growth characteristics and to determine the effects of a strong salinity gradient on toxin cell quotas and composition. The cell quota of yessotoxin (YTX) analogs, as determined by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS), ranged widely among strains. For two strains, the total toxin content remained constant over time in culture, but for the third strain, the YTX cell quota significantly decreased (by 32%) during stationary growth phase. The toxin profiles of the three strains differed markedly and none produced YTX. The analog 41a-homo-YTX (m/z 1155), its putative methylated derivative 9-Me-41a-homo-YTX (m/z 1169) and an unspecified keto-YTX (m/z 1047) were detected in strain LP29-10H, whereas strain LP30-7B contained nor-YTX (m/z 1101), and two unspecified YTX analogs at m/z 1159 and m/z 1061. The toxin profile of strain LP30-8D comprised two unspecified YTX analogs at m/z 1061 and m/z 991 and carboxy-YTX (m/z 1173). Strain LP30-7B cultured at multiple salinities (10, 16, 22, 28 and 34) did not tolerate the lowest salinity (10), but there was a statistically significant decrease (by 21%) in toxin cell quota between growth at the highest versus lower permissible salinities. The toxin profile for strain LP30-7B remained constant over time for a given salinity. At lower salinities, however, the proportion of the unspecified YTX analog (m/z 1061) was significantly higher, especially with respect to nor-YTX (m/z 1101). This study shows high intra-specific variability in yessotoxin composition among strains from the same geographical region and inconsistency in toxin cell quota under different environmental regimes and growth stages in culture. This variation has important implications for the kinetics of YTX production and food web transfer in natural bloom populations from diverse geographical regions.