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: 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: 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: 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.

Description: Two novel azaspiracids (AZA) with a molecular mass of 869 Da were found in Pacific strains of Azadinium poporum and characterized by tandem mass spectrometry and high resolution mass spectrometry (HRMS). One compound, AZA-42, was found in Az. poporum strains AZFC25 and AZFC26, both isolated from the South China Sea. AZA-42 belongs to the 360-type AZA that in comparison to AZA-1 has an additional double bond in the F–I ring system of AZA comprising C28-C40. The other compound, AZA-62, was detected in Az. poporum strain 1D5 isolated off Chañaral, Northern Chile. Mass spectral data indicate that AZA-62 is a variant of AZA-11 with an additional double bond in the C1-C9 region of AZA. In addition to the description of the two novel AZA, a comprehensive list of all AZA known to be produced by species of the genera Azadinium and Amphidoma comprising 26 AZA variants is presented.

Description: The widespread diatom Pseudo-nitzschia can produce domoic acid (DA). DA is a compound with well described neurotoxic effects on vertebrates including humans known as amnesic shellfish poisoning (ASP) syndrome. It has also been suggested to serve as an organic ligand that binds to iron and copper. By binding these trace elements, DA may increase their solubility and bioavailability. In order to serve this function, DA has to be excreted and reabsorbed by the cells. Only few records of dissolved domoic acid (dDA) concentrations in the ocean exist. To accomplish quantification by ultra performance liquid chromatography (UPLC), samples have to be pre-concentrated and desalted using solid-phase extraction, a procedure commonly applied for dissolved organic matter. Our major goals were to quantify dDA in a basin-wide assessment in the East Atlantic Ocean, to determine extraction efficiencies for complexed and uncomplexed dDA, and to assess whether domoic acid is represented by its molecular formula in direct-infusion high resolution mass spectrometry. Our results showed that dDA was extracted almost quantitatively and occurred ubiquitously in the ocean surface but also in deeper (and older) water, indicating surprisingly high stability in seawater. The maximum concentration measured was 173 pmol L−1 and the average molar dDA carbon yield was 7.7 ppm. Both carbon yield and dDA concentration decreased with increasing water depth. Providing quantification of dDA in the water column, we seek to improve our understanding of toxic bloom dynamics and the mechanistic understanding of DA production.

Description: Blooms of Alexandrium spp. are a well-known phenomenon in Northern European waters. While A. tamarense/catenella, and A. pseudogonyaulax have been reported from marine waters, high densities of A. ostenfeldii are mainly observed at lower salinities in North Sea estuaries and the Baltic Sea, suggesting salinity as a driver of Alexandrium species composition and toxin distribution. To investigate this relationship, an oceanographic expedition through a natural salinity gradient was conducted in June 2016 along the coasts of Denmark. Besides hydrographic data, phytoplankton and sediment samples were collected for analyses of Alexandrium spp. cell and cyst abundances, for toxin measurement and cell isolation. Plankton data revealed the predominance of A. pseudogonyaulax at all transect stations while A. ostenfeldii and A. catenella generally contributed a minor fraction to the Alexandrium community. High abundances of A. pseudogonyaulax in the shallow enclosed Limfjord were accompanied by high amounts of goniodomin A (GDA). This toxin was also detected at low abundances along with A. pseudogonyaulax in the North Sea and the Kattegat. Genetic and morphological characterization of established strains showed high similarity of the Northern European population to distant geographic populations. Despite low cell abundances of A. ostenfeldii, different profiles of cycloimines were measured in the North Sea and in the Limfjord. This field survey revealed that salinity alone does not determine Alexandrium species and toxin distribution, but emphasizes the importance of habitat conditions such as proximity to seed banks, shelter, and high nutrient concentrations. The results show that A. pseudogonyaulax has become a prominent member of the Alexandrium spp. community over the past decade in the study area. Analyses of long term monitoring data from the Limfjord confirmed a recent shift to A. pseudogonyaulax dominance. Cyst and toxin records of the species in Kiel Bight suggest a spreading potential into the brackish Baltic Sea, which might lead to an expansion of blooms under future climate conditions.

Description: The cosmopolitan, potentially toxic dinoflagellate Protoceratium reticulatum possesses a fossilizable cyst stage which is an important paleoenvironmental indicator. Slight differences in the internal transcribed spacer ribosomal DNA (ITS rDNA) sequences of P. reticulatum have been reported, and both the motile stage and cyst morphology of P. reticulatum display phenotypic plasticity, but how these morpho-molecular variations are related with ecophysiological preferences is unknown. Here, 55 single cysts or cells were isolated from localities in the Northern (Arctic to subtropics) and Southern Hemispheres (Chile and New Zealand), and in total 34 strains were established. Cysts and/or cells were examined with light microscopy and/or scanning electron microscopy. Large subunit ribosomal DNA (LSU rDNA) and/or ITS rDNA sequences were obtained for all strains/isolates. All strains/isolates of P. reticulatum shared identical LSU sequences except for one strain from the Mediterranean Sea that differs in one position, however ITS rDNA sequences displayed differences at eight positions. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference based on ITS rDNA sequences. The results showed that P. reticulatum comprises at least three ribotypes (designated as A, B, and C). Ribotype A included strains from the Arctic and temperate areas, ribotype B included strains from temperate regions only, and ribotype C included strains from the subtropical and temperate areas. The average ratios of process length to cyst diameter of P. reticulatum ranged from 15% in ribotype A, 22% in ribotype B and 17% in ribotype C but cyst size could overlap. Theca morphology was indistinguishable among ribotypes. The ITS-2 secondary structures of ribotype A displayed one CBC (compensatory change on two sides of a helix pairing) compared to ribotypes B and C. Growth response of one strain from each ribotype to various temperatures was examined. The strains of ribotypes A, B and C exhibited optimum growth at 15 °C, 20 °C and 20–25 °C, respectively, thus corresponding to cold, moderate and warm ecotypes. The profiles of yessotoxins (YTXs) were examined for 25 strains using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The parent compound yessotoxin (YTX) was produced by strains of ribotypes A and B, but not by ribotype C strains, which only produced the structural variant homoyessotoxin (homoYTX). Our results support the notion that there is significant intra-specific variability in Protoceratium reticulatum and the biogeography of the different ribotypes is consistent with specific ecological preferences.

Description: Some species of the dinophytes Azadinium and Amphidoma (Amphidomataceae) produce azaspiracids (AZA), lipophilic polyether compounds responsible for Azaspiracid Shellfish Poisoning (AZP) in humans after consumption of contaminated seafood. Toxigenic Amphidomataceae are known to occur in the North Atlantic and the North Sea area, but little is known about their importance in Danish coastal waters. In 2016, 44 Stations were sampled on a survey along the Danish coastline, covering the German Bight, Limfjord, the Kattegat area, Great Belt and Kiel Bight. Samples were analysed by live microscopy, liquid chromatography-tandem mass spectrometry (LC–MS/MS) and quantitative polymerase-chain-reaction (qPCR) on the presence of Amphidomataceae and AZA. Amphidomataceae were widely distributed in the area, but were below detection limit on most of the inner Limfjord stations. Cell abundances of the three toxigenic species, determined with species-specific qPCR assays on Azadinium spinosum, Az. poporum and Amphidoma languida, were generally low and restricted to the North Sea and the northern Kattegat, which was in agreement with the distribution of the generally low AZA abundances in plankton samples. Among the toxigenic species, Amphidoma languida was dominant with highest cell densities up to 3×103 cells L−1 on North Sea stations and at the western entrance of the Limfjord. Azaspiracids detected in plankton samples include low levels of AZA-1 at one station of the North Sea, and higher levels of AZA-38 and -39 (up to 1.5 ng L−1) in the North Sea and the Limfjord entrance. Furthermore, one new AZA (named AZA-63) was discovered in plankton of two North Sea stations. Morphological, molecular, and toxinological characterisation of 26 newly established strains from the area confirmed the presence of four amphidomatacean species (Az. obesum, Az. dalianense, Az. poporum and Am. languida). The single new strain of Az. poporum turned out as a member of Ribotype A2, which was previously only known from the Mediterranean. Consistent with some of these Mediterranean A2 strains, but different to the previously established AZA-37 producing Az. poporum Ribotype A1 strains from Denmark, the new strain did not contain any AZA. Azaspiracids were also absent in all Az. obesum and Az. dalianense strains, but AZA-38 and -39 were found in all Am. languida strains with total AZA cell quotas ranging from 0.08 up to 94 fg cell−1. In conclusion, AZA-producing microalgae and their respective toxins were low in abundance but widely present in the area, and thus might be considered in local monitoring programs to preserve seafood safety in Danish coastal waters.