Description: The data represent species counts (cells L-1) of the three AZA-producing dinoflagellate species Azadinium spinosum, Az. poporum and Amphidoma languida (all members of the taxonomic family Amphidomataceae) of water samples taken during in total six different field expeditions on several research vessels (RV Heincke, RV Uthörn, RV Polarstern) and on in total five stationary sampling stations (Scapa Flow/Scotland, Cuxhaven/Germany, Helgoland/Germany, Wilhelmshaven/Germany, Sylt/Germany) between 2015 and 2019. The water samples have been taken using Niskin bottles (on research vessels attached to a CTD). After DNA extraction, the species cell numbers have been calculated by quantitative PCR (qPCR) analysis using respective standard curves. These samples gained from different geographical areas in the eastern North Atlantic have been analyzed as part of the RIPAZA Project (funded by the German BMBF; in cooperation with the Third Institute of Oceanography, Xiamen/China) and the results are presented and discussed in the doctoral thesis of Stephan Wietkamp (Suppl.Tab.S6, Suppl.Tab.S7). Aim of the project and especially of this data set was to provide first reference data on the biogeography (geographical distribution and seasonality) of toxigenic Amphidomataceae in the eastern North Atlantic.

Description: The data represent species counts (cells L-1) of the three AZA-producing dinoflagellate species Azadinium spinosum, Az. poporum and Amphidoma languida (all members of the taxonomic family Amphidomataceae) of water samples taken during in total six different field expeditions on several research vessels (RV Heincke, RV Uthörn, RV Polarstern) and on in total five stationary sampling stations (Scapa Flow/Scotland, Cuxhaven/Germany, Helgoland/Germany, Wilhelmshaven/Germany, Sylt/Germany) between 2015 and 2019. The water samples have been taken using Niskin bottles (on research vessels attached to a CTD). After DNA extraction, the species cell numbers have been calculated by quantitative PCR (qPCR) analysis using respective standard curves. These samples gained from different geographical areas in the eastern North Atlantic have been analyzed as part of the RIPAZA Project (funded by the German BMBF; in cooperation with the Third Institute of Oceanography, Xiamen/China) and the results are presented and discussed in the doctoral thesis of Stephan Wietkamp (Suppl.Tab.S6, Suppl.Tab.S7). Aim of the project and especially of this data set was to provide first reference data on the biogeography (geographical distribution and seasonality) of toxigenic Amphidomataceae in the eastern North Atlantic.

Description: The temporal & spatial variability of various Azaspiracid-producing microalgae and their toxins in the North Sea is investigated by quantitative polymerase-chain-reaction (qPCR) and liquid gas chromatography/mass spectrometry (LC-MS/MS). Temperature effects on the individual life cycles of AZA-producing species and their toxin production are conducted to simulate global warming conditions. The aim of the research is to improve food safety for sea food & sustainable use of coastal Areas. Results of the studies will be used for a report for the national reference laboratory for marine biotoxins 

Description: The almost globally distributed, marine dinoflagellate genera Azadinium and Amphidoma (Amphidomataceae) produce a variety of lipophilic phycotoxins known as Azaspiracids (AZA). These toxins are accumulated mostly by filter-feeders like the blue mussel (Mytilus edulis) and may lead to the azaspiracid-shellfish-poisoning (AZP) syndrome in humans after consumption of contaminated seafood. With respect to the impacts on humans health, AZA-concentrations above the EU-regulatory limit (0.16 mg AZA Kg-1 mussel flesh) go along with closures of shellfish farms and are therefore a threat to the aquaculture industry, as well. Thus, there is a need for a rapid, sensitive and reliable detection and quantification of these microalgae and their toxigenic products. However, this is challenging, as the small-sized cells (12-16 µm) are hardly possible to be identified by traditional light microscopy. Even more challenging, only a few amphidomatacean species produce toxins, and toxigenic and non-toxigenic species can co-occur in the same area. In 2018, a seagoing expedition took place in the North Sea, the English Channel and Irish coastal waters, combining onboard light microscopy, quantitative real-time PCR (qPCR) and liquid-chromatography, coupled with tandem mass-spectrometry (LC-MS/MS), to search for the three azaspiracid-producing species known from the North Atlantic: Azadinium spinosum, Az. poporum and Amphidoma languida. Findings revealed that AZA-producers and respective toxins were widely distributed in the survey area, with high cell densities in the North Sea area and along the Irish coastline. Highlight was a bloom stage of Am. languida with 1.2 × 105 cells L-1, observed on a central North Sea station. Results of microscopy, molecular and chemical analyses matched well, which increased the confidence about species and toxin detection. This study supports again the recommendation to include toxigenic Amphidomataceans into regular monitoring programs and further demonstrated the advantage of real-time, multi-method approaches to investigate inconspicuous, harmful microalgae species in the field.

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: Protists are single-celled organisms, which are very sensitive to changes in environmental parameters. They show a high diversity and occur under a huge variety of environmental conditions – also in polar regions. They live in and on the ice flows, as well as in the water column beneath. The knowledge about the interchange of marine protists between sea ice and the water surface is still insufficient, whereas more and more studies pay attention to the cryopelagic coupling of these microorganisms. Recently in the context of global change, where sea ice minima are observed more frequently - especially in the Arctic Ocean. The central hypothesis of this thesis refers to the coupling of the protist communities in the sea ice and the water column. During the freezing process, the salt leaves the ice through a channel system (“brine channels”), which contains high salinities and offers many habitats for different organisms to coexist on small scales. Therefore, we assume a higher diversity in the sea ice than in the under-ice water. Although the distance between both habitats is relatively small, results of other studies in the Arctic Ocean showed already differences in the community composition. To address this hypothesis, a molecular approach has been chosen. The protist community in the ice and the water shows a similarity of ~ 60-70%. This result indicates, that the exchange between ice and water is relatively high, which confirms former studies about cryo-pelagic coupling. The second part of this thesis is about the comparison of the cryo-pelagic coupling between the Arctic and the Southern Ocean, to get insides into potentially different mechanisms in both polar regions. Data for the Southern Ocean are still scarce in this context. Therefore, we include Antarctic samples from the equivalent season. A taxonomic overlap of ~ 60-70% between the sea ice and the under-ice water is remarkable. Therefore, we conclude similar mechanisms like in the Arctic Ocean. In total, ~ 60% of the taxa are found in both, the Arctic and the Southern Ocean. Consequently, a global exchange of marine protists is imaginable, but true bipolarity has to be proven by sampling in latitudes between both poles. The focus of the last part is on freshwater taxa and especially the comparison between the land-surrounded Arctic Ocean and the ocean-surrounded Southern Ocean. The Arctic Ocean is influenced by a higher amount of freshwater input (e.g. rivers), and our results confirm more freshwater taxa in the Arctic samples than in the samples of the Southern Ocean. The results of this study bring inside into a variety of aspects of cryo-pelagic coupling in the Arctic and Southern Ocean. The high exchange of taxa between the sea ice and under-ice water, as well as the occurrence of one taxon at both poles, might be more common than assumed by previous studies and need to get more attention in the future, when a further impact of climate change on ice extension takes place.

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.