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: Some species of the dinophytes Azadinium and Amphidoma (Amphidomataceae) produce azaspiracids (AZA), lipophilic polyether compounds responsible for Azaspiracid Shellfish Poisoning (AZP) in humans after consumption of contaminated seafood. Toxigenic Amphidomataceae are known to occur in the North Atlantic and the North Sea area, but little is known about their importance in Danish coastal waters. In 2016, 44 Stations were sampled on a survey along the Danish coastline, covering the German Bight, Limfjord, the Kattegat area, Great Belt and Kiel Bight. Samples were analysed by live microscopy, liquid chromatography-tandem mass spectrometry (LC–MS/MS) and quantitative polymerase-chain-reaction (qPCR) on the presence of Amphidomataceae and AZA. Amphidomataceae were widely distributed in the area, but were below detection limit on most of the inner Limfjord stations. Cell abundances of the three toxigenic species, determined with species-specific qPCR assays on Azadinium spinosum, Az. poporum and Amphidoma languida, were generally low and restricted to the North Sea and the northern Kattegat, which was in agreement with the distribution of the generally low AZA abundances in plankton samples. Among the toxigenic species, Amphidoma languida was dominant with highest cell densities up to 3×103 cells L−1 on North Sea stations and at the western entrance of the Limfjord. Azaspiracids detected in plankton samples include low levels of AZA-1 at one station of the North Sea, and higher levels of AZA-38 and -39 (up to 1.5 ng L−1) in the North Sea and the Limfjord entrance. Furthermore, one new AZA (named AZA-63) was discovered in plankton of two North Sea stations. Morphological, molecular, and toxinological characterisation of 26 newly established strains from the area confirmed the presence of four amphidomatacean species (Az. obesum, Az. dalianense, Az. poporum and Am. languida). The single new strain of Az. poporum turned out as a member of Ribotype A2, which was previously only known from the Mediterranean. Consistent with some of these Mediterranean A2 strains, but different to the previously established AZA-37 producing Az. poporum Ribotype A1 strains from Denmark, the new strain did not contain any AZA. Azaspiracids were also absent in all Az. obesum and Az. dalianense strains, but AZA-38 and -39 were found in all Am. languida strains with total AZA cell quotas ranging from 0.08 up to 94 fg cell−1. In conclusion, AZA-producing microalgae and their respective toxins were low in abundance but widely present in the area, and thus might be considered in local monitoring programs to preserve seafood safety in Danish coastal waters.

Description: 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: 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.