Multiple New Strains of Amphidomataceae (Dinophyceae) from the North Atlantic Revealed a High Toxin Profile Variability of Azadinium spinosum and a New Non-Toxigenic Az. cf. spinosum.

Urban Tillmann,Stephan Wietkamp,Rafael Salas,Haifeng Gu,Bernd Krock,Dave Clarke

doi:10.3390/microorganisms9010134

Abstract

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.

Highlights

Azaspiracids (AZA) are polyether lipophilic marine biotoxins that accumulate in filter-feeding bivalves
Within strains designated as Az. spinosum we identified strains of toxigenic ribotypes A and B, as well as five new strains without detectable AZA
Azadinium spinosum Ribotype A and B. These results presented here confirm the presence of two different Az. spinosum ribotypes (A and B) in the North Atlantic and their different AZA profiles

Summary

Introduction

Azaspiracids (AZA) are polyether lipophilic marine biotoxins that accumulate in filter-feeding bivalves. AZA have been associated with human incidents of shellfish poisoning since the first intoxication case in 1995 attributed to Irish mussels [1,2]. Seven human azaspiracid shellfish poisoning (AZP) events have been confirmed in The. Microorganisms 2021, 9, 134. Netherlands, Ireland, Italy, France, the UK, and the US [3,4], and each of these AZP events have been traced to contaminated Irish shellfish. AZA concentrations above the EU threshold level of 0.16 mg kg−1 shellfish meat have exceptionally been recorded in Norway in 2002/2003 [16] and along the Atlantic coast of southern Spain in 2009 [17]. Elevated AZA levels in Ireland have led to recurrent and extended production site closures with severe economic consequences for the shellfish industry since 2002 [18,19,20]. There is a need to increase knowledge on the diversity and distribution of AZA-producing species in order to allow better identification and quantification of these species in national monitoring programs

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