Description: Amphidomataceae in the Labrador Sea and western Greenland waters Azaspiracids (AZA) are the most recently discovered group of lipophilic marine biotoxins of microalgal origin associated with human incidents of shellfish poisoning. Over the last couple of years, four out of 27 described species of Amphidomataceae have been identified as primary source of AZA. Diversity and global biogeography of species of Amphidomataceae, however, still are poorly known. In summer 2017 we sampled the central Labrador Sea and the western Greenland coast from Gothaab Fjord (64° N) to 75° N for the presence of Amphidomataceae and AZA. In the central Labrador Sea, light microscopy revealed the presence of small Azadinium-like cells at fairly high densities of 9,200 cells L-1. Single cell isolation from that station yielded 14 clonal strains representing four different species, Azadinium obesum, Az. trinitatum, Az. dexteroporum, and a new species which is currently described taxonomically. For all cultured strains, no known AZA at measureable amounts were detected. From all stations along the cruise, filtered seawater samples were negative for AZA. Specific qPCR assays for DNA samples, targeting ribosomal genes of Azadinium and Amphidoma, revealed positive signals from the majority of stations along the Greenland west coast indicating a not yet recorded widespread occurrence, albeit at low densities, of Amphidomataceen species in that area. Solid phase adsorption toxin tracking (SPATT) samplers were long-term deployed during the expedition in a continuous water sampling system (FerryBox) and were negative for known AZA. The results highlight the presence of Amphidomataceae in the area but the lack of toxins in the field samples indicate a currently low risk of toxic Amphidomataceae blooms in arctic coastal waters.

Description: Planktonic dinophyte species of the familiy Amphidomataceae attract attention as producers of azaspiracids, lipophilic phycotoxins that accumulate in shellfish and cause human health problem for shellfish consumers. About 30 species are describe, which are small and difficult to differentiate with routine light microscopy. These group of species is thus an obvious case where molecular methods for detection and quantification are needed. An overview is given on the state of the art and current use of qPCR assays for Amphidomataceae (Dinophyceae). New challanges related to quantification and assay specificity due to the continuously increasing diversity of species and strains are highlighted and discussed.

Description: Azadinium poporum produces a variety of azaspiracids and consists of several ribotypes, but information on its biogeography is limited. A strain of A. poporum (GM29) was incubated from a Gulf of Mexico sediment sample. Strain GM29 was characterized by a plate pattern of po, cp, x, 4′, 3a, 6″, 6C, 5S, 6‴, 2⁗, a distinct ventral pore at the junction of po and the first two apical plates, and a lack of an antapical spine, thus fitting the original description of A. poporum. The genus Azadinium has not been reported in waters of the United States of America before this study. Molecular phylogeny, based on large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences, reveals that strain GM29 is nested within the well-resolved A. poporum complex, but forms a sister clade either to ribotype B (ITS) or ribotype C (LSU). It is, therefore, designated as a new ribotype, termed as ribotype D. LSU and ITS sequences similarity among different ribotypes of A. poporum ranges from 95.4% to 98.2%, and from 97.1% to 99.2% respectively, suggesting that the LSU fragment is a better candidate for molecular discrimination. Azaspiracid profiles were analyzed using LC–MS/MS and demonstrate that strain GM29 produces predominantly AZA-2 with an amount of 45 fg/cell. The results suggest that A. poporum has a wide distribution and highlights the risk potential of azaspiracid intoxication in the United States.

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: Around 30 epibenthic Prorocentrum species have been described, but information about their biogeography is limited. Some species are able to produce okadaic acid (OA) and its derivatives, which are responsible for diarrheic shellfish poisoning (DSP). In the present study, we examined the diversity of epibenthic Prorocentrum in the northern South China Sea by isolating single cells from sand, coral, and macroalgal samples collected from 2012 to 2015. Their morphology was examined using light microscopy and scanning electron microscopy. Among 47 Prorocentrum strains, seven morphospecies were identified as P. lima, P. rhathymum, P. concavum, P. cf. emarginatum, P. fukuyoi, P. cf. maculosum and P. panamense. The latter five species have not been previously reported in Chinese waters, and this is the first record of P. panamense outside its type locality. Partial large subunit (LSU) ribosomal DNA and internal transcribed spacer region sequences were obtained and molecular phylogenetic analysis was carried out using maximum likelihood and Bayesian inference. Chinese P. cf. maculosum strains share 99.5% similarity of LSU sequences with the strain from Cuba (close to the type locality), but Chinese P. lima strains share only 96.7% similarity of LSU sequences with the strain from the type locality. P. cf. emarginatum differs from P. fukuyoi mainly in the presence/absence of marginal pores and they form a well-resolved clade together with P. sculptile. OA was detected in all Chinese strains of P. lima and P. cf. maculosum based on liquid chromatography-mass spectrometry analysis, but dinophysistoxin was produced only by two P. lima strains. Chinese strains of P. concavum, P. rhathymum, and P. panamense do not produce detectable level of OA. Our results support the wide distribution of epibenthic Prorocentrum species and highlight the potential risk of DSP in the northern South China Sea.

Description: The marine planktonic dinophyceaen genus Azadinium is a primary source of azaspiracids, but due to their small size its diversity may be underestimated and information on its biogeography is still limited. A new Azadinium species, A. zhuanum was obtained from the East China Sea and Yellow Sea of China by incubating surface sediments. Five strains were established by isolating single germinated cells and their morphology was examined with light microscopy and scanning electron microscopy. Azadinium zhuanum was characterized by a plate pattern of Po, cp, X, 4′, 2a, 6′′, 6C, 5S, 6′′′, 2′′′′, by a distinct ventral pore at the junction of Po, the first and fourth apical plates, and a conspicuous antapical spine. Moreover, Azadinium poporum was obtained for the first time from the Mediterranean by incubating surface sediment collected from Diana Lagoon (Corsica) and a new strain of Azadinium dalianense was isolated from the French Atlantic. The morphology of both strains was examined. Small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences were obtained from cultured strains. In addition, LSU sequences were obtained by single cell sequencing of two presumable A. poporum cells collected from the French Atlantic. Molecular phylogeny based on concatenated SSU, LSU and ITS sequences revealed that A. zhuanum was closest to A. polongum. French A. poporum from Corsica (Mediterranean) and from the Atlantic showed some genetic differences but were nested within one of the A. poporum ribotypes together with other European strains. Azadinium dalianense from France together with the type strain of the species from China comprised a well resolved clade now consisting of two ribotypes. Azaspiracid profiles were analyzed for the cultured Azadinium strains using LC–MS/MS and demonstrate that the Mediterranean A. poporum strain produced AZA-2 and AZA-2 phosphate with an amount of 0.44 fg cell−1. Azadinium zhuanum and A. dalianense did not produce detectable AZA. Results of the present study support the view of a high diversity and wide distribution of species belonging to Azadinium. The first record of AZA-2 producing A. poporum from the Mediterranean suggests that this species may be responsible for azaspiracid contaminations in shellfish from the Mediterranean Sea.

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.