ALBERT

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

Description: 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.