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: Azaspiracids (AZAs) are a group of lipophilic biotoxins responsible for the azaspiracid shellfish poisoning syndrome (AZP) in humans after consumption of contaminated shellfish. AZAs are produced by four representatives of the marine nanoplanktonic family Amphidomataceae (Dinophyceae), i.e. Azadinium spinosum, Az. poporum, Az. dexteroporum and Amphidoma languida. Among those species, Az. spinosum producing AZA-1, -2 and -33 (as known in 2017) and, to lesser extent, Az. poporum producing AZA-37, are known from the North Atlantic. These toxigenic species pose a major concern, especially for the coastal shellfish production in Ireland, and are thus frequently monitored along with AZA toxins by the regulatory authorities of the Irish government. A third North Atlantic AZA producer, Amphidoma languida, has been described based on an isolate obtained from Irish coastal waters, but the actual threat by this species and the respective AZA variants (AZA-38, -39) is unknown. In contrast to AZAs produced by Az. spinosum and Az. poporum, these AZA congeners are currently not regulated within the EU. The three AZA producers have been confirmed in the North Sea as well, but current knowledge on the biogeography of toxigenic Amphidomataceae relies on a limited number of observations and studies. The lack of data impedes an assessment of the actual risk of AZP in the North Sea and adjacent waters at present. However, shellfish farming in European coastal waters including the North Sea is of increasing importance for seafood supply, and enhanced production capacities are heavily advocated by the European Commission (EC). The goal of this thesis study was to increase knowledge about the current biogeography of toxigenic Amphidomataceae in the eastern North Atlantic, and to evaluate the risk potential of AZP in the area under the perspective of global change. Interpretations of the results should help to improve safety and sustainable use of coastal seafood production sites in the North Sea and adjacent areas. Major difficulties for reliable species detection and identification are the small cell size and inconspicuousness of nanoplanktonic Amphidomataceae, as well as the sympatric occurrence of toxigenic and non-toxigenic representatives. Multiple methods, i.e. light microscopy (LM) and scanning electron microscopy (SEM) for morphological inspection, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for AZA analysis, and quantitative polymerase chain reaction (qPCR) for DNA-based cell detection, were applied to respond to these challenges and to gain a broad spectrum of new insights into (toxigenic) Amphidomataceae. The isolation and characterization of (in total) 102 new Az. spinosum and Am. languida strains from the North Atlantic in 2016 and 2018 yielded increased knowledge on variation in AZA profiles and cell quotas of these toxigenic species. Samples from the North Sea provided 30 new Am. languida strains, all confirming previous morphological, phylogenetic and toxinological (i.e. AZA-38 and -39) records from the area. The 72 new Az. spinosum strains represented both Ribotype A in the North Sea and Irish Sea, but Ribotype B was only detected from the North Sea. For the first time, variability in the toxin profile of Ribotype A was confirmed, with different combinations of the three AZA variants (AZA-1 always present, combined with presence/absence of AZA-2 and/or -33), whereas the toxin profile of Ribotype B (AZA-11 and -51) was consistent in all strains. Multiple analyses over 18 months revealed that the AZA profile within all given strains remained stable. In contrast, AZA cell quotas were highly variable among and within Az. spinosum strains, and variability of single analogs was as high as 330-fold. These findings confirmed previous studies, but the reasons for the cell quota variability remain unclear. Five new amphidomatacean strains isolated from the 2018 field survey displayed the morphological characteristics of Az. spinosum, but exhibited significant DNA sequence differences (clustering closer to Az. obesum in phylogenetic trees) and no AZA production. The final taxonomic assignment remains undetermined, and the strains were thus designated as Az. cf. spinosum. The newly identified Az. cf. spinosum and the description of four new non-toxigenic Azadinium species (i.e. Az. galwayense, Az. perforatum, Az. perfusorium and Az. pseudozhuanum) highlighted in fact that amphidomatacean biodiversity is still underestimated and that AZA production is rather exceptional within this dinophyte family. Although qPCR assays for Az. spinosum and Az. poporum were already available prior to this study, the respective assay for quantification of toxigenic Amphidoma languida cells was developed and extensively evaluated in the course of this doctoral thesis project. A quick, cost-effective and high throughput application, coupled with high specificity and quantification limit down to 10 target gene copies per reaction, enables this sensitive assay to detect even single Am. languida cells per liter of seawater, and thus is a valuable tool for subsequent biogeographical studies. With respect to multiple newly discovered species and isolated amphidomatacean strains, specificity testing of the three alternative qPCR assays was of upmost importance to test for false-positive or falsenegative amplification and therefore to assure reliable detection and quantification in monitoring programs. None of the three assays showed false-positive signals, including for the new nontoxigenic Az. cf. spinosum, except for rDNA amplification from a new non-toxigenic Az. poporum isolate from the Danish coast. The most concerning result, however, was the significant amplification efficiency difference between Az. spinosum Ribotype A and B strains, revealing a degree of uncertainty for quantification from natural field samples by application of the current Az. spinosum assay because both ribotypes have been shown to co-occur in the Norwegian Sea and the North Sea. Although the current Az. spinosum and Az. poporum assays have not completely lost their validity for field applications, they should be redesigned for improved reliability. Multiple DNA sample sets, comprising more than 200 field samples from various expeditions between 2015 and 2019 to the eastern North Atlantic, were analyzed by qPCR for the presence and cell abundance of the three toxigenic amphidomatacean species. All three AZA-producers were found to be widely distributed in the area. In terms of positive geographical hits and cell densities (up to 8.3 x 104 cells L-1) Az. spinosum was the dominant toxigenic species in Irish coastal waters in summer 2018, underlining the threat for Irish shellfish production. Multiple hits and relatively high cell abundances of Az. spinosum were frequently found in the North Sea, as well. Amphidoma languida was also widely present and relatively abundant (2.3 x 104 cells L-1) around Ireland at that time, but highest cell density was found in the central North Sea, with an extraordinary abundance of ~ 1.2 x 105 cells L-1. This represents the highest ever recorded field abundance for this species and for North Atlantic Amphidomataceae in general. This finding, together with multiple further geographical records, indicated that Am. languida may be the dominant AZA producer in the North Sea. On this basis, incorporation of this species is recommended for both the national Irish- and official EU monitoring programs. Several amphidomatacean species have been found in Arctic and Subarctic waters before, and this finding was confirmed in the course of this study. Amphidoma languida was the only AZA producing species detected in the Arctic (〉 75 °N) close to Spitzbergen in 2015, indicating that this species is able to cope with colder (around 5 °C) water temperatures. In contrast to Az. spinosum and Am. languida, Az. poporum was found in only a few locations and at low cell densities usually 〈 100 cells L-1, but with one extraordinary signal at Scapa Flow, Orkney Islands in June 2016, corresponding to ~ 3 x 103 cells L-1. This indicates an overall much lower potential contribution of this species to AZA contamination in recent years. Due to continuous sampling at several fixed North Sea stations, this thesis contains detailed qPCR data (in total 245 samples) on the seasonality of all three toxigenic species. The subsequent analysis revealed recurrent occurrence from July to October, consistent with observations at the Irish coastline (Marine Institute, Galway, Ireland), and indicating higher AZP risk in summer and fall. In addition, weekly sampling at the North Sea islands Helgoland and Sylt suggested relatively rapid population increases, demonstrating that sudden bloom events of toxigenic Amphidomataceae leading to rapid shellfish toxicity should be considered for respective monitoring frequency. First data on the vertical distribution of toxigenic Amphidomataceae presented here revealed no distinct distributional pattern in the water column, and hence pooling of water samples from various depths is an appropriate sampling method. Simultaneous on-board application of alternative technologies during an expedition in 2018 revealed a highly significant correlation between the results of light microscopy of plankton cells and qPCR assays for the detection and enumeration of toxigenic Amphidomataceae, and chemical analysis of AZA composition in the field. Detailed method-specific advantages and disadvantages are presented herein, but in particular the qPCR approach has proven to give solid results by combining high specificity with convenient detection limits. Laboratory experiments with North Atlantic strains representing all three toxigenic Amphidomataceae (including the first study on Am. languida) targeted temperature dependent growth and AZA production. Growth rates and AZA cell quota were inversely related: whereas higher temperatures led to higher growth rates, AZA content per cell decreased with increasing temperatures. Nevertheless, faster growth was shown to overcompensate for lower toxin cell quotas, leading to similar or even higher total AZA content per seawater volume (μg AZA L-1) at higher temperatures. This suggests a potentially increasing AZP risk under expected rising ocean temperatures. Highest AZA production was found in Az. spinosum Ribotype A (with a characteristic toxin profile of AZA-1, -2 and -33), highlighting a major role of this taxon determining AZP risk in the eastern North Atlantic. Except for Az. spinosum Ribotype B strain (containing AZA-11 and -51), all investigated strains showed lower extracellular than intracellular AZA levels. This suggests that AZA is predominantly retained intracellularly, and that screening for cells and intracellular AZAs is an appropriate monitoring method for AZP risk assessment. In conclusion, extensive research in this doctoral study, including development of a reliable qPCR assay for toxigenic Am. languida, with the description of new amphidomatacean species, strains, AZA variants, toxin profiles, adds considerably to the knowledge base on biogeography and variability within the Amphidomataceae. Combining data on AZA cell quota variability with the comprehensive data set on biogeography, seasonality and vertical distribution of the three toxigenic representatives in the North Sea has redefined our view of the role and importance of (toxigenic) Amphidomataceae and AZAs in the North Sea and adjacent areas. Thus, this doctoral thesis study provides a highly valuable baseline for official monitoring and future studies on toxigenic Amphidomataceae.

Description: The AZAHAB HE516 survey aboard the R/V Heincke (Helgoland) was conducted during summer 2018 to study the coastal oceanographic processes and mechanisms underlying the dynamics of Amphidomatacean species and the biogeographical distribution of their toxins in the water column. The survey transects were from Bremerhaven, Germany across the southern North Sea and the British Channel with detailed sampling initiated in the Celtic Sea and West Irish coastal waters. From Irish waters the transects continued along the Outer Hebrides and the northern Scottish coast to the North Sea, which again was sampled in more detail. In addition to the primary transect, five transects perpendicular to the coast were performed in along the Irish coast. Standard physical oceanographic parameters (temperature: ˚C, salinity: psu, σt ) plus current velocity were supplemented with bio-optical measurements with multiple profiling fluorometers and various passive optical profilers (for turbidity and diffuse attenuation), including hyperspectral radiometers and microscopic plankton analyses, on-board phycotoxin measurements, and real time polymerase chain reaction (PCR).

Description: Representatives of the marine dinophyte family Amphidomataceae produce lipophilic phycotoxins called azaspiracids (AZA) which may cause azaspiracid shellfish poisoning (AZP) in humans after consumption of contaminated seafood. Three of the four known toxigenic species are observed frequently in the eastern North Atlantic. In 2018, a research survey was performed to strengthen knowledge on the distribution and abundance of toxigenic Amphidomataceae and their respective toxins in Irish coastal waters and in the North Sea. Species-specific quantification of the three toxigenic species (Azadinium spinosum, Azadinium poporum and Amphidoma languida) was based on recently developed qPCR assays, whose performance was successfully validated and tested with specificity tests and spike experiments. The multi-method approach of on-board live microscopy, qPCR assays and chemical AZA-analysis revealed the presence of Amphidomataceae in the North Atlantic including the three targeted toxigenic species and their respective AZA analogues (AZA-1, -2, -33, -38, -39). Azadinium spinosum was detected at the majority of Irish stations with a peak density of 8.3 x 104 cells L-1 and AZA (AZA-1, -2, -33) abundances up to 1,274 pg L-1. Amphidoma languida was also present at most Irish stations but appeared in highest abundance in a bloom at a central North Sea station with a density of 1.2 x 105 cells L-1 and an AZA (AZA-38, -39) abundances of 618 pg L-1. Azadinium poporum was detected sporadically at the Irish south coast and North Sea and was rather low in abundance during this study. The results confirmed the wide distribution and frequent occurrence of the target species in the North Atlantic area and revealed, for the first time, bloom abundances of toxigenic Amphidomataceae in this area. This emphasizes the importance of future studies and monitoring of amphidomatacean species and their respective AZA analogues in the North Atlantic.

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: Shellfish contamination with azaspiracids (AZA), which are lipophilic marine biotoxins produced by marine dinoflagellates, is a major and recurrent problem for the Irish shellfish industry. AZA are produced by certain species of Amphidomataceae, but the species diversity of this group of microalgae in Irish waters is poorly known. Here we present a morphological and molecular characterization of multiple new strains of non-toxigenic Azadinium isolated on an oceanographic survey in 2018. A lack of AZA production for all strains presented here was demonstrated by LC-MS/ MS analysis. One strain of Azadinium caudatum var. margalefii (first strain for the area) confirmed nontoxigenicity of Atlantic populations of this species. One strain designated as Azadinium cf. zhuanum was similar to Az. zhuanum described from China but differed from the type strain in nucleus position, by the dominant number of apical plates, and by significant differences in rRNA gene sequences. Finally, two new non-toxigenic Azadinium species are described from the North East Atlantic: Azadinium galwayense sp. nov. and Azadinium perfusorium sp. nov. Azadinium galwayense differed from other Azadinium by a characteristic combination regarding presence and location of the ventral pore (vp; on the right side of the pore plate), of a pyrenoid (located in the episome), and by a pentagonal shape of the median anterior intercalary plate 2a, and lack of contact between plates 1´´ and 1a. Azadinium perfusorium shared the same vp position as Az. galwayense and differed by a characteristic combination of a pyrenoid located in the hyposome, a tetragonal shape of plate 2a, and a relatively large size of the two lateral anterior intercalary plates. Molecular phylogeny confirmed the distinctiveness of these two new species and their placement in Azadinium. The present findings significantly increased knowledge on the diversity of Azadinium species in the North East Atlantic.