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

stephan.wietkamp [ at ]


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.

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Thesis (PhD)
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DOI 10.26092/elib/350

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Wietkamp, S. (2020): Biogeography, diversity and risk potential of toxigenic Amphidomataceae (Dinophyceae) in the North Sea and adjacent areas , PhD thesis, doi: 10.26092/elib/350

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