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: Azaspiracids (AZA) are a group of lipophilic toxins, which are produced by a few species of the marine nanoplanktonic dinoflagellate genera Azadinium and Amphidoma (Amphidomataceae). Amphidomataceae were found to be globally distributed in coastal waters and new areas of occurrence are regularly discovered. The AZA toxins accumulate mainly in shellfish and - when consumed by humans - can lead to the so-called azaspiracid shellfish poisoning syndrome (AZP). Given this serious threat to seafood production and to deepen knowledge about the distribution and risk potential of AZA-producing algae, an appropriate detection method enabling a fast identification and quantification for these toxigenic species is needed. Traditional light microscopy is time-consuming, requires expertise and is getting rather difficult when it comes to the detection, identification and quantification of small-sized plankton. To overcome this challenges, quantitative real-time PCR (qPCR) assays are increasingly used as a molecular additive. Basically, when amplifying the extracted DNA and using DNA standards, the amplification threshold (CT) gives information about the number of target species in the sample. For two AZA-producing species, Azadinium spinosum and Azadinium poporum, quantitative PCR assays have already been developed and successfully applied in the field. Another AZA-producing species, Amphidoma languida, was discovered in 2012 in Irish coastal waters and discovered as a new species within the group of Amphidomataceae - in close relationship with Azadinium spp. All available strains from Ireland, Iceland, Norway, Denmark and Spain produce azaspiracids. Moreover, Am. languida from the Atlantic coast of southern Spain was responsible for AZA amounts in shellfish above the EU regulatory limit, emphasizing the need for further investigations. We thus developed a quantitative TaqMan PCR assay, amplifying 60bp of the D2 region (located on the LSU/28S) of the ribosomal DNA (rDNA) to detect toxic Am. languida. To confirm assay specificity in vitro, cross-reactivity tests with DNA of a variety of related organisms were performed. This included 12 different Am. languida strains as positive controls, Amphidoma parvula, 10 Azadinium species (each including different strains), as well as 10 further related dinoflagellate species (Alexandrium spp., Gymnodinium spp., Heterocapsa spp., Karlodinium sp., Prorocentrum spp. & Scripsiella sp.). The developed probe and primer set successfully detected only A. languida strains. Currently, we perform tests of the newly-designed assay on spiked field samples to test and optimize the quantification ability of the assay. With this assay, we provide a tool for the rapid and distinctive quantification of the toxic dinoflagellate Amphidoma languida to be used in monitoring programs and bio-geographic studies.

Description: Amphidomataceae in the Labrador Sea and western Greenland waters Azaspiracids (AZA) are the most recently discovered group of lipophilic marine biotoxins of microalgal origin associated with human incidents of shellfish poisoning. Over the last couple of years, four out of 27 described species of Amphidomataceae have been identified as primary source of AZA. Diversity and global biogeography of species of Amphidomataceae, however, still are poorly known. In summer 2017 we sampled the central Labrador Sea and the western Greenland coast from Gothaab Fjord (64° N) to 75° N for the presence of Amphidomataceae and AZA. In the central Labrador Sea, light microscopy revealed the presence of small Azadinium-like cells at fairly high densities of 9,200 cells L-1. Single cell isolation from that station yielded 14 clonal strains representing four different species, Azadinium obesum, Az. trinitatum, Az. dexteroporum, and a new species which is currently described taxonomically. For all cultured strains, no known AZA at measureable amounts were detected. From all stations along the cruise, filtered seawater samples were negative for AZA. Specific qPCR assays for DNA samples, targeting ribosomal genes of Azadinium and Amphidoma, revealed positive signals from the majority of stations along the Greenland west coast indicating a not yet recorded widespread occurrence, albeit at low densities, of Amphidomataceen species in that area. Solid phase adsorption toxin tracking (SPATT) samplers were long-term deployed during the expedition in a continuous water sampling system (FerryBox) and were negative for known AZA. The results highlight the presence of Amphidomataceae in the area but the lack of toxins in the field samples indicate a currently low risk of toxic Amphidomataceae blooms in arctic coastal waters.

Description: Planktonic dinophyte species of the familiy Amphidomataceae attract attention as producers of azaspiracids, lipophilic phycotoxins that accumulate in shellfish and cause human health problem for shellfish consumers. About 30 species are describe, which are small and difficult to differentiate with routine light microscopy. These group of species is thus an obvious case where molecular methods for detection and quantification are needed. An overview is given on the state of the art and current use of qPCR assays for Amphidomataceae (Dinophyceae). New challanges related to quantification and assay specificity due to the continuously increasing diversity of species and strains are highlighted and discussed.

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: Species of the planktonic dinoflagellates Azadinium and Amphidoma are small, inconspicuous and difficult, if not impossible to be identified and differentiated by light microscopy. Within this group, there are some species that produce the marine biotoxin azaspiracid (AZA) while others are non-toxigenic, therefore a requirement exists for precise species identification. A quantitative polymerase chain reaction (qPCR) assay for molecular detection and quantification of one of the toxigenic species, Amphidoma languida, was designed and extensively tested. The assay was highly specific and sensitive to detect and quantify down to 10 target gene copies (corresponding to ca. 0.05 cells) per reaction. DNA cell quota and copy number cell−1 were constant for four different Am. languida strains, and for one strain they were shown to be stable at various time points throughout the growth cycle. Recovery of known cell numbers of Am. languida spiked into natural samples was 95–103%, and the assay was successfully tested on field samples collected from Irish coastal waters. This new qPCR assay is a valuable tool for routine monitoring for the prevention of AZA-shellfish-poisoning caused by the consumption of contaminated shellfish and is a supportive tool for studies on the biogeography of this AZA-producing species.

Description: Azaspiracids (AZA) are lipophilic marine biotoxins associated with shellfish poisoning which are produced by some species of Amphidomataceae. Diversity and global biogeography of this family are still poorly known. In summer 2017 plankton samples were collected from the central Labrador Sea and western Greenland coast from 64° N (Gothaab Fjord) to 75° N for the presence of Amphidomataceae and AZA. In the central Labrador Sea, light microscopy revealed small Azadinium-like cells (9200 cells l−1). Clonal strains established from plankton samples and scanning electron microscopy of fixed plankton samples revealed at least eight species of Amphidomataceae: Azadinium obesum, Az. trinitatum, Az. dexteroporum, Az. spinosum, Az. polongum, Amphidoma languida, Azadinium spec., and a new species described here as Azadinium perforatum sp. nov. The new species differed from other Azadinium species by the presence of thecal pores on the pore plate. All samples, including cultured strains, filtered seawater samples, and solid phase adsorption toxin tracking (SPATT) samplers deployed during the expedition in a continuous water-sampling system (FerryBox), were negative for AZA. DNA samples and PCR assays were positive for Amphidomataceae from most stations, whereas species-specific assays for three toxigenic species were rarely positive (two stations for Az. poporum, one station for Am. languida). The results highlight the presence of Amphidomataceae in the area but the lack of toxins and low abundance of toxigenic species currently indicate a low risk of toxic Amphidomataceae blooms in Arctic coastal waters.