Biogeography, diversity and risk potential of toxigenic Amphidomataceae (Dinophyceae) in the North Sea and adjacent areas

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

A Mediterranean Alexandrium taylorii (Dinophyceae) Strain Produces Goniodomin A and Lytic Compounds but Not Paralytic Shellfish Toxins

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

Prorocentrum pervagatum sp. nov. (Prorocentrales, Dinophyceae): A new, small, planktonic species with a global distribution

Prorocentrum comprises a unique group of dinophytes with several apomorphic traits, such as an apical insertion of flagella and the presence of two major, large thecal plates. Species delimitation is challenging, especially for morphologically very similar, small planktonic species. Contemporary analyses, including SEM studies and molecular phylogenetics of type material, are not available for many described species. Based on six strains isolated from Antarctic, subarctic and North Atlantic waters, Prorocentrum pervagatum sp. nov. is described. Prorocentrum pervagatum was small (12–16 μm long and deep), oval to round in outline, and moderately compressed. One small, pyrenoid-like structure was faintly visible in some cells. Rod-like, long trichocysts were present. Cells had one distinct apical spine (1.1–1.7 μm in length) visible in light microscopy. The plate surface appeared smooth in light microscopy with few pores located close to the plate margin visible in empty thecae. Electron microscopy revealed plates to be densely covered by small projections and two size classes of thecal pores. Cells had a row of mostly four large pores in apical-ventral position on the right thecal plate. The periflagellar area consisted of eight small platelets. The apical spine was formed by platelet six. In molecular phylogenetics, P. pervagatum was part of a species group generally exhibiting small size and spiny thecal ornamentation, together with Prorocentrum cordatum and Prorocentrum obtusidens. The new species is distinct in DNA trees and differs from the protologues of other small species of Prorocentrum by the unique combination of size, shape (i.e. only moderately compressed or round), presence of a distinct apical spine, and position of thecal pores (i.e. located at the plate margins only). Its clear description and illustration may stimulate similar work of other small species of Prorocentrum, particularly including the re-investigation of taxa with historical names collected at the corresponding type localities

Occurrence and distribution of Amphidomataceae (Dinophyceae) in Danish coastal waters of the North Sea, the Limfjord and the Kattegat/Belt area

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

Molecular detection and quantification of the azaspiracid-producing dinoflagellate Amphidoma languida (Amphidomataceae, Dinophyceae)

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

New real-time PCR assay for toxigenic Amphidoma languida

Azaspiracids (AZA) are a group of lipophilic toxins, which are produced by a few species of the marine nanoplanktonic dinoflagellate genera Azadinium and Amphidoma (Amphidomataceae). Amphidomataceae were found to be globally distributed in coastal waters and new areas of occurrence are regularly discovered. The AZA toxins accumulate mainly in shellfish and - when consumed by humans - can lead to the so-called azaspiracid shellfish poisoning syndrome (AZP). Given this serious threat to seafood production and to deepen knowledge about the distribution and risk potential of AZA-producing algae, an appropriate detection method enabling a fast identification and quantification for these toxigenic species is needed. Traditional light microscopy is time-consuming, requires expertise and is getting rather difficult when it comes to the detection, identification and quantification of small-sized plankton. To overcome this challenges, quantitative real-time PCR (qPCR) assays are increasingly used as a molecular additive. Basically, when amplifying the extracted DNA and using DNA standards, the amplification threshold (CT) gives information about the number of target species in the sample. For two AZA-producing species, Azadinium spinosum and Azadinium poporum, quantitative PCR assays have already been developed and successfully applied in the field. Another AZA-producing species, Amphidoma languida, was discovered in 2012 in Irish coastal waters and discovered as a new species within the group of Amphidomataceae - in close relationship with Azadinium spp. All available strains from Ireland, Iceland, Norway, Denmark and Spain produce azaspiracids. Moreover, Am. languida from the Atlantic coast of southern Spain was responsible for AZA amounts in shellfish above the EU regulatory limit, emphasizing the need for further investigations. We thus developed a quantitative TaqMan PCR assay, amplifying 60bp of the D2 region (located on the LSU/28S) of the ribosomal DNA (rDNA) to detect toxic Am. languida. To confirm assay specificity in vitro, cross-reactivity tests with DNA of a variety of related organisms were performed. This included 12 different Am. languida strains as positive controls, Amphidoma parvula, 10 Azadinium species (each including different strains), as well as 10 further related dinoflagellate species (Alexandrium spp., Gymnodinium spp., Heterocapsa spp., Karlodinium sp., Prorocentrum spp. & Scripsiella sp.). The developed probe and primer set successfully detected only A. languida strains. Currently, we perform tests of the newly-designed assay on spiked field samples to test and optimize the quantification ability of the assay. With this assay, we provide a tool for the rapid and distinctive quantification of the toxic dinoflagellate Amphidoma languida to be used in monitoring programs and bio-geographic studies

New Knowledge on Distribution and Abundance of Toxic Microalgal Species and Related Toxins in the Northwestern Black Sea

Numerous potentially toxic plankton species commonly occur in the Black Sea, and phycotoxins have been reported. However, the taxonomy, phycotoxin profiles, and distribution of harmful microalgae in the basin are still understudied. An integrated microscopic (light microscopy) and molecular (18S rRNA gene metabarcoding and qPCR) approach complemented with toxin analysis was applied at 41 stations in the northwestern part of the Black Sea for better taxonomic coverage and toxin profiling in natural populations. The combined dataset included 20 potentially toxic species, some of which (Dinophysis acuminata, Dinophysis acuta, Gonyaulax spinifera, and Karlodinium veneficum) were detected in over 95% of the stations. In parallel, pectenotoxins (PTX-2 as a major toxin) were registered in all samples, and yessotoxins were present at most of the sampling points. PTX-1 and PTX-13, as well as some YTX variants, were recorded for the first time in the basin. A positive correlation was found between the cell abundance of Dinophysis acuta and pectenotoxins, and between Lingulodinium polyedra and Protoceratium reticulatum and yessotoxins. Toxic microalgae and toxin variant abundance and spatial distribution was associated with environmental parameters. Despite the low levels of the identified phycotoxins and their low oral toxicity, chronic toxic exposure could represent an ecosystem and human health hazard

Response of toxin-producing Amphidomataceans on changing environmental conditions and risk potential of azaspiracid shellfish poisoning (AZP) in the North Sea and adjacent areas.

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 biotoxin

A comparative study of cryo-pelagic coupling of protist communities in the Arctic and Southern Ocean

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