ALBERT

Description: Species of the dinophyte genus Alexandrium are widely distributed and are notorious bloom formers and producers of various potent phycotoxins. The species Alexandrium taylorii is known to form recurrent and dense blooms in the Mediterranean, but its toxin production potential is poorly studied. Here we investigated toxin production potential of a Mediterranean A. taylorii clonal strain by combining state-of-the-art screening for various toxins known to be produced within Alexandrium with a sound morphological and molecular designation of the studied strain. As shown by a detailed thecal plate analysis, morphology of the A. taylorii strain AY7T from the Adriatic Sea conformed with the original species description. Moreover, newly obtained Large Subunit (LSU) and Internal Transcribed Spacers (ITS) rDNA sequences perfectly matched with the majority of other Mediterranean A. taylorii strains from the databases. Based on both ion pair chromatography coupled to post-column derivatization and fluorescence detection (LC-FLD) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis it is shown that A. taylorii AY7T does not produce paralytic shellfish toxins (PST) above a detection limit of ca. 1 fg cell−1, and also lacks any traces of spirolides and gymnodimines. The strain caused cell lysis of protistan species due to poorly characterized lytic compounds, with a density of 185 cells mL−1 causing 50% cell lysis of cryptophyte bioassay target cells (EC50). As shown here for the first time A. taylorii AY7T produced goniodomin A (GDA) at a cellular level of 11.7 pg cell−1. This first report of goniodomin (GD) production of A. taylorii supports the close evolutionary relationship of A. taylorii to other identified GD-producing Alexandrium species. As GD have been causatively linked to fish kills, future studies of Mediterranean A. taylorii blooms should include analysis of GD and should draw attention to potential links to fish kills or other environmental damage.

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: Alexandrium ostenfeldii is among the most intensely studied marine planktonic dinophytes and in the last few years blooms have become a recurrent phenomenon mainly in brackish coastal waters. Since 2012, A. ostenfeldii recurs annually in the Ouwerkerkse Kreek, a Dutch brackish water creek discharging into an estuary with large stocks of mussels, oysters and cockles. The creek is characterized by highly dynamic abiotic conditions, notably salinity. Here, we investigated the impacts of salinities ranging from 3 to 34 on growth and toxin content of an A. ostenfeldii isolate from the creek. Our results demonstrate a broad salinity tolerance of the Dutch A. ostenfeldii population, with growth rates from 0.13 to 0.2 d−1 over a salinity range from 6 to 34. Highest paralytic shellfish toxin and cyclic imine toxin cell quotas were observed for the lowest and highest salinities, and were associated with increases in cell size. Lytic activity was highest at the lowest salinity, and was 5-fold higher in the cell-free supernatants compared to cell extracts. Together our results demonstrate a tight coupling between salinity and A. ostenfeldii growth rate, cell size and toxin synthesis, which may have consequences for the seasonal dynamics of bloom toxicity.

Description: From the German Bight along Jutland to the western Skagerrak, we found representatives of almost all groups of phycotoxins known to occur in North Sea plankton. Identification was by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in plankton size fractions, with domoic acid and 20-me G the most abundant toxins. The dominance of 20-me G in the spirolide (SPX) composition of plankton from the Jutland current system matched very well with that of an isolate of the dinoflagellate Alexandrium ostenfeldii. The SPXs of the A. ostenfeldii strain S6_P12_E11, previously isolated from the western North Sea along the Scottish coast, comprised 100% 20-me G, suggesting toxin homogeneity among North Sea populations of this species. We detected highest amounts of azaspiracid-1 in the 3–20-mm size fraction at offshore stations, where the Jutland coastal current converges with the westward North Sea flow off Skagerrak. Azadinium spinosum was subsequently identified by clonal isolation from crude cultures established from these stations. Except for lipophilic toxins usually produced by the dinoflagellate Dinophysis spp., dinophysistoxin-1 (DTX-1) and DTX-2, we detected no other phycotoxins in plankton from the southern German Bight. The spatial distribution of the phycotoxins in the eastern North Sea was apparently related to the hydrographical conditions, identified from salinity and coloured dissolved organic matter profiles. The biogeographical distribution of phycotoxins indicates a strong association with the northward advection by the Jutland current and the mixing of German Bight and North water masses along the northwest Danish coast towards the Skagerrak.

Description: Molecular probes were developed for the dinoflagellate genus Azadinium to discriminate among three taxa difficult to differentiate by light microscopy. This genus contains azaspiracid toxin-producing Azadinium spinosum, but also non-toxigenic species. Quantitative polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) assays were applied to cultured isolates and Azadiniumspiked field plankton. Molecular methods were highly specific and sensitive in the unambiguous detection of Azadinium, and thus are valuable for routine plankton, biogeographic and phylogenetic investigations.

Description: Species of the planktonic dinoflagellates Azadinium and Amphidoma are small, inconspicuous and difficult, if not impossible to be identified and differentiated by light microscopy. Within this group, there are some species that produce the marine biotoxin azaspiracid (AZA) while others are non-toxigenic, therefore a requirement exists for precise species identification. A quantitative polymerase chain reaction (qPCR) assay for molecular detection and quantification of one of the toxigenic species, Amphidoma languida, was designed and extensively tested. The assay was highly specific and sensitive to detect and quantify down to 10 target gene copies (corresponding to ca. 0.05 cells) per reaction. DNA cell quota and copy number cell−1 were constant for four different Am. languida strains, and for one strain they were shown to be stable at various time points throughout the growth cycle. Recovery of known cell numbers of Am. languida spiked into natural samples was 95–103%, and the assay was successfully tested on field samples collected from Irish coastal waters. This new qPCR assay is a valuable tool for routine monitoring for the prevention of AZA-shellfish-poisoning caused by the consumption of contaminated shellfish and is a supportive tool for studies on the biogeography of this AZA-producing species.

Description: Azaspiracids (AZA), a group of lipophilic phycotoxins, are produced by some species of the marine dinophycean genus Azadinium. AZA have recently been detected in shellfish from the Southeast Pacific, however, AZA-producing species have not been recorded yet from the area. This study is the first record of the genus Azadinium and of the species Azadinium poporum from the Pacific side of South America. Three strains of A. poporum from Chañaral (Northern Chile) comply to the type description of A. poporum by the presence of multiple pyrenoids, in thecal plate details, and in the position of the ventral pore located on the left side of the pore plate. Molecular phylogeny, based on internal transcribed spacer and large subunit ribosomal DNA sequences, revealed that Chilean strains fall in the same ribotype clade as European and strains from New Zealand. Analyses of AZA profiles using LC–MS/MS showed an identical profile for all three strains with the presence of AZA-11 and two phosphorylated AZA. This is the first confirmation of the presence of AZA producing Azadinium in the Chilean coastal area and underlines the risk of AZA shellfish and concomitant human contamination episodes in the Southeast Pacific region.

Description: Alexandrium ostenfeldii is a toxic dinoflagellate that has recently bloomed in Ouwerkerkse Kreek, The Netherlands, and which is able to cause a serious threat to shellfish consumers and aquacultures. We used a large set of 68 strains to the aim of fully characterizing the toxin profiles of the Dutch A. ostenfeldii in consideration of recent reports of novel toxins. Alexandrium ostenfeldii is known as a causative species of paralytic shellfish poisoning, and consistently in the Dutch population we determined the presence of several paralytic shellfish toxins (PST) including saxitoxin (STX), GTX2/3 (gonyautoxins), B1 and C1/C2. We also examined the production of spiroimine toxins by the Dutch A. ostenfeldii strains. An extensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed a high intraspecific variability of spirolides (SPX) and gymnodimines (GYM). Spirolides included 13-desMethyl-spirolide C generally as the major compound and several other mostly unknown SPX-like compounds that were detected and characterized. Besides spirolides, the presence of gymnodimine A and 12-Methyl-gymnodimine A was confirmed, together with two new gymnodimines. One of these was tentatively identified as an analogue of gymnodimine D and was the most abundant gymnodimine (calculated cell quota up to 274 pg cell−1, expressed as GYM A equivalents). Our multi-clonal approach adds new analogues to the increasing number of compounds in these toxin classes and revealed a high strain variability in cell quota and in toxin profile of toxic compounds within a single population.

Description: Harmful effects caused by the exposure to paralytic shellfish toxins (PSTs) and bioactive extracellular compounds (BECs) on bivalves are frequently difficult to attribute to one or the other compound group. We evaluate and compare the distinct effects of PSTs extracted from Alexandrium catenella (Alex5) cells and extracellular lytic compounds (LCs) produced by A. tamarense (NX-57-08) on Mytilus edulis hemocytes. We used a 4 h dose–response in vitro approach and analyzed how these effects correlate with those observed in a previous in vivo feeding assay. Both bioactive compounds caused moderated cell death (10–15%), being dose-dependent for PST-exposed hemocytes. PSTs stimulated phagocytic activity at low doses, with a moderate incidence in lysosomal damage (30–50%) at all tested doses. LCs caused a dose-dependent impairment of phagocytic activity (up to 80%) and damage to lysosomal membranes (up to 90%). PSTs and LCs suppressed cellular ROS production and scavenged H2O2 in in vitro assays. Neither PSTs nor LCs affected the mitochondrial membrane potential in hemocytes. In vitro effects of PST extracts on M. edulis hemocytes were consistent with our previous study on in vivo exposure to PST-producing algae, while for LCs, in vivo and in vitro results were not as consistent.

Description: Various species of Alexandrium can produce a number of bioactive compounds, e.g., paralytic shellfish toxins (PSTs), spirolides, gymnodimines, goniodomins, and also uncharacterised bioactive extracellular compounds (BECs). The latter metabolites are released into the environment and affect a large range of organisms (from protists to fishes and mammalian cell lines). These compounds mediate allelochemical interactions, have anti-grazing and anti-parasitic activities, and have a potentially strong structuring role for the dynamic of Alexandrium blooms. In many studies evaluating the effects of Alexandrium on marine organisms, only the classical toxins were reported and the involvement of BECs was not considered. A lack of information on the presence/absence of BECs in experimental strains is likely the cause of contrasting results in the literature that render impossible a distinction between PSTs and BECs effects. We review the knowledge on Alexandrium BEC, (i.e., producing species, target cells, physiological effects, detection methods and molecular candidates). Overall, we highlight the need to identify the nature of Alexandrium BECs and urge further research on the chemical interactions according to their ecological importance in the planktonic chemical warfare and due to their potential collateral damage to a wide range of organisms.