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: 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 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.

Description: We establish the cyst-theca relationship of the cyst species Trinovantedinium pallidifulvum Matsuoka 1987 based on germination experiments of specimens isolated from the Gulf of Mexico. We show that the motile stage is a new species, designated as Protoperidinium louisianensis. We also determine its phylogenetic position based on single-cell PCR of a single cell germinated from the Gulf of Mexico cysts. To further refine the phylogeny, we determined the LSU sequence through single-cell PCR of the cyst Selenopemphix undulata isolated from Brentwood Bay (Saanich Inlet, BC, Canada). The phylogeny shows that P. louisianensis is closest to P. shanghaiense, the motile stage of T. applanatum, and is consistent with the monophyly of the genus Trinovantedinium. Selenopemphix undulata belongs to a different clade than Selenopemphix quanta (alleged cyst of P. conicum), suggesting that the genus Selenopemphix is polyphyletic.

Description: The marine dinoflagellate Azadinium poporum produce azaspiracids (AZA) and has been recorded widely in the world. However, information on its biogeography is still limited, especially in view of the fact that A. poporum comprises several genetically differentiated groups. A total of 18 strains of A. poporum were obtained from the Eastern Mediterranean area by incubating surface sediment collected from Ionian Sea of Greece. The morphology of these strains was examined with light microscopy and scanning electron microscopy. Small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences were obtained from all cultured strains. Molecular phylogeny based on concatenated SSU, LSU and ITS sequences confirmed three ribotypes within A. poporum and revealed two subclades within ribotypes A and C. Greek strains of A. poporum ribotype A were nested within ribotype A2 together with strains from Western Mediterranean Sea and French Atlantic, and Greek strains of A. poporum ribotype C were nested within ribotype C2 together with a strain from the Gulf of Mexico. Growth experiments on four selected strains revealed that ribotypes A and C from Greece differed in their growth at higher temperatures, indicating that they are physiologically differentiated. Azaspiracid profiles were analyzed for 15 cultured A. poporum strains using LCMS/MS and demonstrate that the A. poporum ribotype A from Greece produce low level or no AZA and A. poporum ribotype C from Greece produces predominantly AZA-40 (9.6–30.2 fg cell−1) followed by AZA-2 (2.1–2.6 fg cell−1). The first record of AZA-40 producing A. poporum from the Mediterranean suggests that this species is a potential source for azaspiracid contaminations in shellfish from the Eastern Mediterranean Sea.

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: 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.