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: Representatives of the marine dinophyte family Amphidomataceae produce lipophilic phycotoxins called azaspiracids (AZA) which may cause azaspiracid shellfish poisoning (AZP) in humans after consumption of contaminated seafood. Three of the four known toxigenic species are observed frequently in the eastern North Atlantic. In 2018, a research survey was performed to strengthen knowledge on the distribution and abundance of toxigenic Amphidomataceae and their respective toxins in Irish coastal waters and in the North Sea. Species-specific quantification of the three toxigenic species (Azadinium spinosum, Azadinium poporum and Amphidoma languida) was based on recently developed qPCR assays, whose performance was successfully validated and tested with specificity tests and spike experiments. The multi-method approach of on-board live microscopy, qPCR assays and chemical AZA-analysis revealed the presence of Amphidomataceae in the North Atlantic including the three targeted toxigenic species and their respective AZA analogues (AZA-1, -2, -33, -38, -39). Azadinium spinosum was detected at the majority of Irish stations with a peak density of 8.3 x 104 cells L-1 and AZA (AZA-1, -2, -33) abundances up to 1,274 pg L-1. Amphidoma languida was also present at most Irish stations but appeared in highest abundance in a bloom at a central North Sea station with a density of 1.2 x 105 cells L-1 and an AZA (AZA-38, -39) abundances of 618 pg L-1. Azadinium poporum was detected sporadically at the Irish south coast and North Sea and was rather low in abundance during this study. The results confirmed the wide distribution and frequent occurrence of the target species in the North Atlantic area and revealed, for the first time, bloom abundances of toxigenic Amphidomataceae in this area. This emphasizes the importance of future studies and monitoring of amphidomatacean species and their respective AZA analogues in the North Atlantic.

Description: Shellfish contamination with azaspiracids (AZA), which are lipophilic marine biotoxins produced by marine dinoflagellates, is a major and recurrent problem for the Irish shellfish industry. AZA are produced by certain species of Amphidomataceae, but the species diversity of this group of microalgae in Irish waters is poorly known. Here we present a morphological and molecular characterization of multiple new strains of non-toxigenic Azadinium isolated on an oceanographic survey in 2018. A lack of AZA production for all strains presented here was demonstrated by LC-MS/ MS analysis. One strain of Azadinium caudatum var. margalefii (first strain for the area) confirmed nontoxigenicity of Atlantic populations of this species. One strain designated as Azadinium cf. zhuanum was similar to Az. zhuanum described from China but differed from the type strain in nucleus position, by the dominant number of apical plates, and by significant differences in rRNA gene sequences. Finally, two new non-toxigenic Azadinium species are described from the North East Atlantic: Azadinium galwayense sp. nov. and Azadinium perfusorium sp. nov. Azadinium galwayense differed from other Azadinium by a characteristic combination regarding presence and location of the ventral pore (vp; on the right side of the pore plate), of a pyrenoid (located in the episome), and by a pentagonal shape of the median anterior intercalary plate 2a, and lack of contact between plates 1´´ and 1a. Azadinium perfusorium shared the same vp position as Az. galwayense and differed by a characteristic combination of a pyrenoid located in the hyposome, a tetragonal shape of plate 2a, and a relatively large size of the two lateral anterior intercalary plates. Molecular phylogeny confirmed the distinctiveness of these two new species and their placement in Azadinium. The present findings significantly increased knowledge on the diversity of Azadinium species in the North East Atlantic.