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: The gonyaulacean family Protoceratiaceae is characterised by five precingular plates. It currently encompasses the type genus Ceratocorys and the fossil genus Atopodinium. Fourteen strains of Ceratocorys, Pentaplacodinium, and Protoceratium were established from Malaysian and Hawaiian waters, and their morphologies were examined using light and scanning electron microscopy. Two new species, Ceratocorys malayensis sp. nov. and Pentaplacodinium usupianum sp. nov., were described from Malaysian waters. They share a Kofoidean plate formula of Po, Pt, 3?, 1a, 6??, 6C, 6S, 5???, 1p, 1????. Ceratocorys malayensis has a short first apical plate (1?) with no direct contact with the anterior sulcal plate (Sa) whereas Pentaplacodinium usupianum had a parallelogram-shaped 1? plate which often contacted the Sa plate. The genera Ceratocorys and Pentaplacodinium were emended accordingly to incorporate species bearing five or six precingular plates. The Protoceratium strain from Hawaii was morphologically similar to P. reticulatum, but differed in the lack of a ventral pore in plate 1? and slight or lack of contact between plates 1? and Sa, and is here designated as P. cf. reticulatum. The maximum-likelihood and Bayesian inference analyses based on SSU, LSU and ITS ribosomal DNA sequences revealed that these three genera are monophyletic and form a well-resolved group. Our results support Protoceratium and Pentaplacodinium as members of the family Protoceratiaceae, characterised by the presence of one anterior intercalary plate. Seven strains of Protoceratium cf. reticulatum, Ceratocorys malayensis and Pentaplacodinium usupianum were examined for yessotoxin production by LC-MS/MS but none produced a detectable amount of toxin.

Description: Azaspiracids (AZA) are a group of lipophilic toxins, which are produced by a few species of the marine nanoplanktonic dinoflagellates Azadinium and Amphidoma (Amphidomataceae). A survey was conducted in 2018 to increase knowledge on the diversity and distribution of amphidomatacean species and their toxins in Irish and North Sea waters (North Atlantic). We here present a detailed morphological, phylogenetic, and toxinological characterization of 82 new strains representing the potential AZA producers Azadinium spinosum and Amphidoma languida. A total of ten new strains of Am. languida were obtained from the North Sea, and all conformed in terms of morphology and toxin profile (AZA-38 and-39) with previous records from the area. Within 72 strains assigned to Az. spinosum there were strains of two distinct ribotypes (A and B) which consistently differed in their toxin profile (dominated by AZA-1 and -2 in ribotype A, and by AZA-11 and -51 in ribotype B strains). Five strains conformed in morphology with Az. spinosum, but no AZA could be detected in these strains. Moreover, they revealed significant nucleotide differences compared to known Az. spinosum sequences and clustered apart from all other Az. spinosum strains within the phylogenetic tree, and therefore were provisionally designated as Az. cf. spinosum. These Az. cf. spinosum strains without detectable AZA were shown not to cause amplification in the species-specific qPCR assay developed to detect and quantify Az. spinosum. As shown here for the first time, AZA profiles differed between strains of Az. spinosum ribotype A in the presence/absence of AZA-1, AZA-2, and/or AZA-33, with the majority of strains having all three AZA congeners, and others having only AZA-1, AZA-1 and AZA-2, or AZA-1 and AZA-33. In contrast, no AZA profile variability was observed in ribotype B strains. Multiple AZA analyses of a period of up to 18 months showed that toxin profiles (including absence of AZA for Az. cf. spinosum strains) were consistent and stable over time. Total AZA cell quotas were highly variable both among and within strains, with quotas ranging from 0.1 to 63 fg AZA cell-1. Cell quota variability of single AZA compounds for Az. spinosum strains could be as high as 330-fold, but the underlying causes for the extraordinary large variability of AZA cell quota is poorly understood.

Description: Gymnodinium catenatum is able to produce paralytic shellfish toxins (PSTs) and was responsible for a massive bloom in the Taiwan Strait, East China Sea, in June 2017, which resulted in serious human poisoning and economic losses. To understand the origin of the bloom and determine the potential for blooms in subsequent years, water and sediment samples collected in the Taiwan Strait from 2016 to 2019 were analyzed for cells and cysts using light microscopy (LM) and/or quantitative polymerase chain reaction (qPCR). The morphology of both cells and cysts from the field and cultures was examined with LM and scanning electron microscopy (SEM). Large subunit (LSU) and/or internal transcribed spacer (ITS)-5.8S rRNA gene sequences were obtained in 13 isolates from bloom samples and five strains from cysts. In addition, cells of strains TIO523 and GCLY02 (from the Taiwan Strait and Yellow Sea of China, respectively) were subjected to growth experiments, and cysts from the field were used for germination experiments under various temperatures. Our strains shared identical LSU and ITS-5.8S rRNA gene sequences with those from other parts of the world, and therefore belonged to a global population. A low abundance of G. catenatum cells were detected during most of the sampling period, but a small bloom was encountered in Quanzhou on June 8, 2018. Few cysts were observed in 2016 but a marked increase was observed after the bloom in 2017, with a highest density of 689 cysts cm−3. Cysts germinated at temperatures between 14 and 23 °C with a final germination rate over 93%. Strains TIO523 and GCLY02 displayed growth at temperatures between 17 and 26 °C and 14 and 26 °C, respectively, with both strains displaying the highest growth rate of ca. 0.5 divisions d–1 at 23 °C. The PSTs of the three strains and cysts from the sediments were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). All strains were able to produce PSTs, which were dominated by N-sulfocarbamoyl C toxins (C1/2, 53.0–143.5 pg cell−1) and decarbamoyl gonyautoxins (dcGTX2/3, 26.7–52.1 pg cell−1), although they were not detected in cysts. However, hydroxybenzoyl (GC) toxins were detected in both cells and cysts. Our results suggested that the population in the Taiwan Strait belonged to a warm water ecotype and has a unique toxin profile. Our results also suggested that the persistence of cells in the water column may have initiated the bloom.

Description: The dinoflagellate genus Azadinium includes species with a plate formula of po, cp, X, 4´, 3a, 6´´, 6C, 5S, 6´´´, 2´´´´ and is part of the family Amphidomataceae with an uncertain order affiliation. Among six species, at least two produce azaspiracids (AZAs), a group of lipophilic toxins that accumulate in shellfish and can cause human health problems. Diversity within the genus might be underestimated at present due to its small size. In the present study, we searched for Azadinium by incubating freshly collected sediments from the Yellow Sea off China and succeeded in detecting a new species, here described as Azadinium dalianense sp. nov. It shared identical hypothecal, cingular and sulcal plates with the other Azadinium species, but it was unique in having only three apical and two anterior intercalary plates. Up to two stalked pyrenoids were present but their location in the cell varied. Phylogenetic analyses based on concatenated smallsubunit, partial large-subunit, and internal transcribed spacer sequences revealed that A. dalianense was nested within Azadinium and formed a strongly supported clade with A. poporum. Liquid chromatography–mass spectrometry analyses did not detect any known AZAs.

Description: The toxigenic genus Alexandrium includes ∼30 species, but information about its biogeography at a regional scale is limited. In this study, we explored the diversity of Alexandrium along the coast of China by incubating resting cysts collected from 7 sites. A total of 231 strains of Alexandrium belonging to 7 morphospecies were found. Among them, Alexandrium andersonii, Alexandrium fraterculum, Alexandrium leei, Alexandrium pseudogonyaulax, and Alexandrium tamutum were recorded from the China Sea for the first time. Partial large subunit (LSU) and/or internal transcribed spacer region (ITS1, ITS2, and 5.8S rDNA) sequences revealed two ribotypes of Alexandrium andersonii, Alexandrium leei, and Alexandrium tamarense: Atama complex Group I and IV. Atama complex Group I was exclusively distributed in the Yellow Sea and the Bohai Sea, whereas Group IV was restricted to the East China Sea and South China Sea. Atama complex Group I produced mainly N-sulfocarbamoyl toxins (C1/C2, 61–79% of total toxins) and gonyautoxins (GTX1/4, 17–37%). Alexandrium ostenfeldii strain ASBH01 produced NEO and STX exclusively (65% and 35%, respectively). Our results support the premise that Atama complex Group I is endemic to the Asian Pacific and includes cold water species, whereas Atama complex Group IV tends to inhabit warmer waters.

Description: Azaspiracids (AZA) are lipophilic marine biotoxins associated with shellfish poisoning which are produced by some species of Amphidomataceae. Diversity and global biogeography of this family are still poorly known. In summer 2017 plankton samples were collected from the central Labrador Sea and western Greenland coast from 64° N (Gothaab Fjord) to 75° N for the presence of Amphidomataceae and AZA. In the central Labrador Sea, light microscopy revealed small Azadinium-like cells (9200 cells l−1). Clonal strains established from plankton samples and scanning electron microscopy of fixed plankton samples revealed at least eight species of Amphidomataceae: Azadinium obesum, Az. trinitatum, Az. dexteroporum, Az. spinosum, Az. polongum, Amphidoma languida, Azadinium spec., and a new species described here as Azadinium perforatum sp. nov. The new species differed from other Azadinium species by the presence of thecal pores on the pore plate. All samples, including cultured strains, filtered seawater samples, and solid phase adsorption toxin tracking (SPATT) samplers deployed during the expedition in a continuous water-sampling system (FerryBox), were negative for AZA. DNA samples and PCR assays were positive for Amphidomataceae from most stations, whereas species-specific assays for three toxigenic species were rarely positive (two stations for Az. poporum, one station for Am. languida). The results highlight the presence of Amphidomataceae in the area but the lack of toxins and low abundance of toxigenic species currently indicate a low risk of toxic Amphidomataceae blooms in Arctic coastal waters.

Description: Azadinium poporum is a small dinoflagellate from the family Amphidomataceae which is known for the production potential of azaspiracid toxins. A. poporum has been recorded from European and Korean waters. Here we present the first report of its occurrence along the coast of China. Morphology of Chinese A. poporum is similar to those from Europe and Korea. Several stalked pyrenoids surrounded by a starch sheath were revealed with light microscopy and confirmed by transmission electron microscopy. Among 25 strains from the China Sea we identified two distinct ribotypes (referred to as ribotypes B and C). ITS sequences of strains within the same ribotype are identical, whereas ribotype B and C differ from each other at 11 positions (98.3% similarity). A. poporum ribotypes B and C type differ from European strains (referred to as ribotype A) at 16 and 15 positions (97.5% and 97.7% similarity). The ITS region pairwise distance within A. poporum ranged from 0.017 to 0.022. Among all three ribotypes, no hemicompensatory based changes were found within helix III of ITS indicating that they are conspecific. Azaspiracid profiles were analyzed for six strains and turned out to be unexpectedly diverse. Whereas no AZAs could be detected for one strain, another strain was found to contain a m/z 348 fragment type AZA previously found in a Korean Isolate and traces of two other unknown AZAs of higher masses. A third strain produced a novel AZA with a molecular mass of 871 Da. Three strains were found to contain considerable amounts of toxic AZA-2 as the sole AZA, a finding that might elegantly explain the detection of AZA-2 in sponges in the Sea of Japan and which underline the risk potential of A. poporum blooms with subsequent shellfish intoxication episodes for the Asian Pacific.

Description: 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.