Azaspiracids Research Articles

Toxigenic strains of Azadinium poporum (Amphidomataceae, Dinophyceae) from Japan and Vietnam, with first reports of A. poporum (ribotype A) and A. trinitatum in Asian Pacific

SUMMARYMorphology and phylogeny of small armored dinoflagellate Azadinium species isolated from Japanese and Vietnamese coasts were examined by light microscopy, scanning electron microscopy and molecular phylogeny inferred from ITS and LSU rDNA (D1–D3) sequences. Dominant azaspiracids (AZAs) were analyzed by using liquid chromatography/triple quadrupole mass spectrometer (LC/MS/MS). Sixteen and four culture strains were established from Japan and Vietnam, respectively. A phylogenetic tree confirmed our species identifications of Japanese cultures as Azadinium poporum (ribotypes A, B and C), A. trinitatum and A. zhuanum, and Vietnamese cultures as A. poporum (ribotype B). Typical cells of A. poporum had the thecal tabulation of Po, cp, X, 4′, 3a, 6″, 6C, 5S, 6″′, 2″″, A. trinitatum had Po, cp, X, 4–5′, 3a, 6–7″, 6C, 5S, 5–6″′, 2″″ and A. zhuanum had Po, cp, X, 3–4′, 2a, 6″, 6C, 5S, 6″′, 2″″. Cellular morphology of the three species were congruent with those in the original reports, in terms of the presence/absence of antapical spine and the position of the ventral pore, nucleus and pyrenoids, although small differences were also found in the nucleus position of A. trinitatum and A. zhuanum. AZA‐2 was the dominant component in Japanese A. poporum (ribotypes A, B and C), and AZA‐40 in Vietnamese A. poporum (ribotype B). AZA‐2 and AZA‐40 were not detected in Vietnamese and Japanese A. poporum, respectively. AZA analogs were not detected from A. trinitatum and A. zhuanum. This is the first report of the toxigenic A. poporum from Japan and Vietnam, with identifications of the major AZAs. The ribotype A of A. poporum and A. trinitatum were the first records in Asia.

Diversity and regional distribution of harmful algal events along the Atlantic margin of Europe

The IOC-ICES-PICES Harmful Algal Event Database (HAEDAT) was used to describe the diversity and spatiotemporal distribution of harmful algal events along the Atlantic margin of Europe from 1987 - 2018. The majority of events recorded are caused by Diarrhetic Shellfish Toxins (DSTs). These events are recorded annually over a wide geographic area from southern Spain to northern Scotland and Iceland, and are responsible for annual closures of many shellfish harvesting areas. The dominant causative dinoflagellates, members of the morphospecies ‘Dinophysis acuminata complex’ and D. acuta, are common in the waters of the majority of countries affected. There are regional differences in the causative species associated with PST events; the coasts of Spain and Portugal with the dinoflagellates Alexandrium minutum and Gymnodinium catenatum, north west France/south west England/south Ireland with A. minutum, and Scotland/Faroe Islands/Iceland with A. catenella. This can influence the duration and spatial scale of PST events as well as the toxicity of shellfish. The diatom Pseudo-nitzschia australis is the most widespread Domoic Acid (DA) producer, with records coming from Spain, Portugal, France, Ireland and the UK. Amnesic Shellfish Toxins (ASTs) have caused prolonged closures for the scallop fishing industry due to the slow depuration rate of DA. Amendments to EU shellfish hygiene regulations introduced between 2002 and 2005 facilitated end-product testing and sale of adductor muscle. This reduced the impact of ASTs on the scallop fishing industry and thus the number of recorded HAEDAT events. Azaspiracids (AZAs) are the most recent toxin group responsible for events to be characterised in the ICES area. Events associated with AZAs have a discrete distribution with the majority recorded along the west coast of Ireland. Ciguatera Poisoning (CP) has been an emerging issue in the Canary Islands and Madeira since 2004. The majority of aquaculture and wild fish mortality events are associated with blooms of the dinoflagellate Karenia mikimotoi and raphidophyte Heterosigma akashiwo. Such fish killing events occur infrequently yet can cause significant mortalities. Interannual variability was observed in the annual number of HAEDAT areas with events associated with individual shellfish toxin groups. HABs represent a continued risk for the aquaculture industry along the Atlantic margin of Europe and should be accounted for when considering expansion of the industry or operational shifts to offshore areas.

Assessment of the Presence of Lipophilic Phycotoxins in Scallops (Argopecten purpuratus) Farmed along Peruvian Coastal Waters

Some harmful algal blooms produce lipophilic marine biotoxins (LMTs) such as okadaic acid (OA; and its analogs dinophysistoxins [DTXs]), yessotoxins (YTXs), pectenotoxins (PTXs), and azaspiracids (AZAs), all of which may accumulate in filter-feeding bivalve mollusks. European health regulations stipulate a limit of 160 μg/kg for OA or DTXs, PTXs, and AZAs and 3.75 mg/kg for YTXs. Argopecten purpuratus is a valuable commercial marine bivalve exploited in Peru. Despite its importance and the periodic reports of the presence of harmful algal blooms in Peruvian coastal waters, information regarding potential contamination of these scallops by LMTs is lacking. We evaluated LMTs in 115 samples of A. purpuratus collected between November 2013 and March 2015 from 18 production areas distributed along the Peruvian coast. The hepatopancreas, which accumulates most of the toxins in the scallop, was analyzed with liquid chromatography-tandem mass spectrometry to quantify OA in its free form, YTX, AZA-1, and PTX-2. Baseline separation was achieved in 19 min. Linearity (R2 > 0.997), precision (coefficient of variation < 15%), and limits of quantification (0.155 to 0.479 ng/mL) were satisfactory. YTX was found in 72 samples, and PTX-2 was found in 17 samples, but concentrations of both biotoxins were below the regulatory limits. Free OA and AZA-1 were not detected in the scallop samples. This atypical profile (i.e., presence of PTX-2 and absence of OA) may be linked to the presence of the dinoflagellate Dinophysis acuminata. The production of YTX could be associated with the phytoplankton Gonyaulax spinifera and Protoceratium reticulatum. This is the first systematic assessment of the four types of LMTs in shellfish from Peruvian coastal waters. The results suggest low prevalence of LMTs in Peruvian bay scallops but support continued surveillance and analysis of LMTs in Peru.

Targeting Chloride Ion Channels: New Insights into the Mechanism of Action of the Marine Toxin Azaspiracid.

Azaspiracids (AZAs) are marine toxins produced by dinoflagellates belonging to the genera Azadinium and Amphidoma that caused human intoxications after consumption of contaminated fishery products, such as mussels. However, the exact mechanism for the AZA induced cytotoxic and neurotoxic effects is still unknown. In this study several pharmacological approaches were employed to evaluate the role of anion channels on the AZA effects that demonstrated that cellular anion dysregulation was involved in the toxic effects of these compounds. The results presented here demonstrated that volume regulated anion channels (VRACs) are affected by this group of toxins, and, because there is not any specific activator of VRACs besides the intracellular application of GTPγ-S molecule, this group of natural compounds could represent a powerful tool to analyze the role of these channels in cellular homeostasis. In addition to this, in this work, a detailed pharmacological approach was performed in order to elucidate the anion channels present in human HEK293 cells as well as their regulation by the marine toxins azaspiracids. Altogether, the data presented here demonstrated that the effect of azaspiracids in human cells was completely dependent on ATP-regulated anion channels, whose upregulation by these toxins could lead to regulatory volume decrease and underlie the reported toxicity of these compounds.

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.

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.

Morphological and molecular characterization of multiple new Azadinium strains revealed a high diversity of non‐toxigenic species of Amphidomataceae (Dinophyceae) including two new Azadinium species in Irish waters, North East Atlantic

SUMMARYShellfish 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 non‐toxigenicity 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.

Fatty acid esters of azaspiracids identified in mussels (Mytilus edulis) using liquid chromatography-high resolution mass spectrometry.

Azaspiracids (AZAs) are lipophilic polyether toxins produced by Azadinium and Amphidoma species of marine microalgae. The main dinoflagellate precursors AZA1 and AZA2 are metabolized by shellfish to produce an array of AZA analogues. Many marine toxins undergo fatty acid esterification in shellfish, therefore mussel tissues contaminated with AZAs were screened for intact fatty acid esters of AZAs using liquid chromatography-high resolution mass spectrometry. Acyl esters were primarily observed for AZAs containing hydroxy groups at C-3 with 3-O-palmitoylAZA4 identified as the most abundant acyl ester, while other fatty acid esters including 18:1, 16:1, 17:0, 20:2 and 18:0 acyl esters were detected. The structures of these acyl derivatives were determined through LC-MS/MS experiments, and supported by periodate cleavage reactions and semi-synthesis of palmitate esters of the AZAs. Esters of the hydroxy groups at C-20 or C-21 were not observed in mussel tissue. The relative proportion of the most abundant AZA ester was less than 3% of the sum of the major free AZA analogues. These findings reveal an additional metabolic pathway for AZAs in shellfish.

Distribution and abundance of azaspiracid-producing dinophyte species and their toxins in North Atlantic and North Sea waters in summer 2018.

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.

A Rapid Method for the Detection of Diarrhetic Shellfish Toxins and Azaspiracid Shellfish Toxins in Washington State Shellfish by Liquid Chromatography Tandem Mass Spectrometry.

Diarrhetic shellfish toxins (DSTs) in domestic shellfish and azaspiracids (AZAs) in imported products are emerging seafood safety issues in the United States. In addition to causing gastrointestinal illnesses, some of these toxins are also carcinogenic and genotoxic. Efficient analytical strategies are needed for their monitoring in U.S. domestic and imported shellfish. In the US, DSTs and AZAs are the only lipophilic shellfish toxins addressed in regulations. Streamlining of existing methods for several classes of lipophilic toxins, based on liquid chromatography coupled with triple quadrupole mass spectrometry, was pursued. The resulting simplified LC-MS/MS method is focused on the separation and detection of just the AZAs and total DSTs using a C18 Hypersil gold column. Filter vials are used to expedite and simplify sample handling. The method has a run time of 7.25 min. LOQs for the AZAs and DSTs in shellfish were 0.3-0.4 µg/kg. Recoveries (AZAs and total DSTs) for three spiking levels in three matrixes ranged from 68 to 129%. Trueness was established using certified reference materials. Method equivalence was established using shellfish provided blind by the Washington State Department of Health Public Health Laboratory (WA DOH PHL). Data obtained from these samples agreed well with data from another LC-MS/MS method used in harvest control by WA DOH PHL (R = 0.999; P < 0.0001). The LC-MS/MS method described offers more rapid sample handling and has excellent sensitivity, linearity, and repeatability.

Field observations of the dinoflagellate genus Azadinium and azaspiracid toxins in the south-west Atlantic Ocean

Some dinoflagellate species of the genera Azadinium and Amphidoma (Amphidomataceae) produce azaspiracids (AZA), a group of toxins responsible for gastrointestinal disorders in humans following the consumption of contaminated shellfish. In this study, we investigated the diversity, distribution and abundance of Azadinium and AZA from field plankton samples collected during four oceanographic expeditions that covered an extended area of the Argentine Sea during different seasons. Scanning electron microscopy analyses indicated the presence of five Azadinium species: Az. dexteroporum, Az. luciferelloides, Az. obesum, Az. asperum and Az. cf. poporum. Azadinium-like cells were frequently found and were even an abundant component of plankton assemblages, showing a wide latitudinal distribution, from ~38 to ~55.5°S, and occurring in a wide temperature and salinity range. High cell densities (up to 154000cellsL–1) occurred in northern slope and external shelf waters during spring. AZA-2 was detected in net samples from the 20- to 200-µm fractions by tandem mass spectrometry–liquid chromatography analysis, suggesting a transfer of AZA through the food web. Our results contribute to the knowledge of the worldwide occurrence of Azadinium species and AZA, and highlight the importance of amphidomatacean species as a potential source of AZA shellfish poisoning in the south-west Atlantic Ocean.

Biological Effects of the Azaspiracid-Producing Dinoflagellate Azadinium dexteroporum in Mytilus galloprovincialis from the Mediterranean Sea.

Azaspiracids (AZAs) are marine biotoxins including a variety of analogues. Recently, novel AZAs produced by the Mediterranean dinoflagellate Azadinium dexteroporum were discovered (AZA-54, AZA-55, 3-epi-AZA-7, AZA-56, AZA-57 and AZA-58) and their biological effects have not been investigated yet. This study aimed to identify the biological responses (biomarkers) induced in mussels Mytilus galloprovincialis after the bioaccumulation of AZAs from A. dexteroporum. Organisms were fed with A. dexteroporum for 21 days and subsequently subjected to a recovery period (normal diet) of 21 days. Exposed organisms accumulated AZA-54, 3-epi-AZA-7 and AZA-55, predominantly in the digestive gland. Mussels’ haemocytes showed inhibition of phagocytosis activity, modulation of the composition of haemocytic subpopulation and damage to lysosomal membranes; the digestive tissue displayed thinned tubule walls, consumption of storage lipids and accumulation of lipofuscin. Slight genotoxic damage was also observed. No clear occurrence of oxidative stress and alteration of nervous activity was detected in AZA-accumulating mussels. Most of the altered parameters returned to control levels after the recovery phase. The toxic effects detected in M. galloprovincialis demonstrate a clear biological impact of the AZAs produced by A. dexteroporum, and could be used as early indicators of contamination associated with the ingestion of seafood.

The effect of temperature and salinity on growth rate and azaspiracid cell quotas in two strains of Azadinium poporum (Dinophyceae) from Puget Sound, Washington State

Azaspiracids (AZA) are novel lipophilic polyether marine biotoxins associated with azaspiracid shellfish poisoning (AZP). Azaspiracid-59 (AZA-59) is a new AZA that was recently detected in strains of Azadinium poporum from Puget Sound, Washington State. In order to understand how environmental factors affect AZA abundances in Puget Sound, a laboratory experiment was conducted with two local strains of A. poporum to estimate the growth rate and AZA-59 (both intra- and extracellular) cell quotas along temperature and salinity gradients. Both strains of A. poporum grew across a wide range of temperatures (6.7 °C to 25.0 °C), and salinities (15 to 35). Growth rates increased with increasing temperature up to 20.0 °C, with a range from 0.10 d−1 to 0.42 d−1. Both strains of A. poporum showed variable growth rates from 0.26 d−1 to 0.38 d−1 at salinities from 15 to 35. The percentage of intracellular AZA-59 in both strains was generally higher in exponential than in stationary phase along temperature and salinity gradients, indicating higher retention of toxin in actively growing cells. Cellular toxin quotas varied by strain in both the temperature and salinity treatments but were highest at the lowest growth rates, especially for the faster growing strain, NWFSC1011.Consistent with laboratory experiments, field investigations in Sequim Bay, WA, during 2016–2018 showed that A. poporum was detected when salinity and temperature became favorable to higher growth rates in June and July. Although current field data of A. poporum in Puget Sound indicate a generally low abundance, the potential of local A. poporum to adapt to and grow in a wide range of temperature and salinity may open future windows for blooms. Although increased temperatures, anticipated for the Puget Sound region over the next decades, will enhance the growth of A. poporum, these higher temperatures will not necessarily support higher toxin cell quotas. Additional sampling and assessment of the total toxicity of AZA-59 will provide the basis for a more accurate estimation of risk for azaspiracid poisoning in Puget Sound shellfish.

Effects of Temperature, Growth Media, and Photoperiod on Growth and Toxin Production of Azadinium spinosum.

Azaspiracids (AZAs) are microalgal toxins that can accumulate in shellfish and lead to human intoxications. To facilitate their study and subsequent biomonitoring, purification from microalgae rather than shellfish is preferable; however, challenges remain with respect to maximizing toxin yields. The impacts of temperature, growth media, and photoperiod on cell densities and toxin production in Azadinium spinosum were investigated. Final cell densities were similar at 10 and 18 °C, while toxin cell quotas were higher (~3.5-fold) at 10 °C. A comparison of culture media showed higher cell densities and AZA cell quotas (2.5–5-fold) in f10k compared to f/2 and L1 media. Photoperiod also showed differences, with lower cell densities in the 8:16 L:D treatment, while toxin cell quotas were similar for 12:12 and 8:16 L:D treatments but slightly lower for the 16:8 L:D treatment. AZA1, -2, and -33 were detected during the exponential phase, while some known and new AZAs were only detected once the stationary phase was reached. These compounds were additionally detected in field water samples during an AZA event.

Occurrence and distribution of Amphidomataceae (Dinophyceae) in Danish coastal waters of the North Sea, the Limfjord and the Kattegat/Belt area

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.

Variability and profiles of lipophilic toxins in bivalves from Great Britain during five and a half years of monitoring: azaspiracids and yessotoxins

Cefas has been responsible for the delivery of official control biotoxin testing of bivalve molluscs from Great Britain for just over a decade. Liquid chromatography tandem mass spectrometric (LC–MS/MS) methodology has been used for the quantitation of lipophilic toxins (LTs) since 2011. The temporal and spatial distribution of okadaic acid group toxins and profiles in bivalves between 2011 and 2016 have been recently reported. Here we present data on the two other groups of regulated lipophilic toxins, azaspiracids (AZAs) and yessotoxins (YTXs), over the same period. The latter group has also been investigated for a potential link with Protoceratium reticulatum and Lingulodinium polyedra, both previously recognised as YTXs producing phytoplankton.On average, AZAs were quantified in 3.2% of all tested samples but notable inter-annual variation in abundance was observed. The majority of all AZA contaminated samples were found between July 2011 and August 2013 in Scotland, while only two, three-month long, AZA events were observed in 2015 and 2016 in the south-west of England. Maximum concentrations were generally reached in late summer or early autumn. Reasons for AZAs persistence during the 2011/2012 and 2012/2013 winters are discussed. Only one toxin profile was identified, represented by both AZA1 and AZA2 toxins at an approximate ratio of 2 : 1, suggesting a single microalgal species was the source of AZAs in British bivalves. Although AZA1 was always the most dominant toxin, its proportion varied between mussels, Pacific oysters and surf clams.The YTXs were the least represented group among regulated LTs. YTXs were found almost exclusively on the south-west coast of Scotland, with the exception of 2013, when the majority of contaminated samples originated from the Shetland Islands. The highest levels were recorded in the summer months and followed a spike in Protoceratium reticulatum cell densities. YTX was the most dominant toxin in shellfish, further strengthening the link to P. reticulatum as the YTX source. Neither homo-YTX, nor 45−OH homo-YTX were detected throughout the monitored period. 45−OH YTX, thought to be a shellfish metabolite associated with YTX elimination, contributed on average 26% in mussels. Although the correlation between 45−OH YTX abundance and the speed of YTX depuration could not be confirmed, we noted the half-life of YTX was more than two-times longer in queen scallops, which contained 100% YTX, than in mussels. No other bivalve species were affected by YTXs.This is the first detailed evaluation of AZAs and YTXs occurrences and their profiles in shellfish from Great Britain over a period of multiple years.

Azaspiracids Increase Mitochondrial Dehydrogenases Activity in Hepatocytes: Involvement of Potassium and Chloride Ions

Background: Azaspiracids (AZAs) are marine toxins that are produced by Azadinium and Amphidoma dinoflagellates that can contaminate edible shellfish inducing a foodborne poisoning in humans, which is characterized by gastrointestinal symptoms. Among these, AZA1, -2, and -3 are regulated in the European Union, being the most important in terms of occurrence and toxicity. In vivo studies in mice showed that, in addition to gastrointestinal effects, AZA1 induces liver alterations that are visible as a swollen organ, with the presence of hepatocellular fat droplets and vacuoles. Hence, an in vitro study was carried out to investigate the effects of AZA1, -2, and -3 on liver cells, using human non-tumor IHH hepatocytes. Results: The exposure of IHH cells to AZA1, -2, or -3 (5 × 10−12–1 × 10−7 M) for 24 h did not affect the cell viability and proliferation (Sulforhodamine B assay and 3H-Thymidine incorporation assay), but they induced a significant concentration-dependent increase of mitochondrial dehydrogenases activity (MTT reduction assay). This effect depends on the activity of mitochondrial electron transport chain complex I and II, being counteracted by rotenone and tenoyl trifluoroacetone, respectively. Furthermore, AZAs-increased mitochondrial dehydrogenase activity was almost totally suppressed in the K+-, Cl−-, and Na+-free media and sensitive to the specific inhibitors of KATP and hERG potassium channels, Na+/K+, ATPase, and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. Conclusions: These results suggest that AZA mitochondrial effects in hepatocytes derive from an imbalance of intracellular levels of K+ and, in particular, Cl− ions, as demonstrated by the selective reduction of toxin effects by CFTR chloride channel inhibition.

High abundance of Amphidomataceae (Dinophyceae) during the 2015 spring bloom of the Argentinean Shelf and a new, non-toxigenic ribotype of Azadinium spinosum

Azaspiracids (AZA) are the most recently discovered group of lipophilic marine biotoxins of microalgal origin, and associated with human incidents of shellfish poisoning. They are produced by a few species of Amphidomataceae, but diversity and occurrence of the small-sized dinophytes remain poorly explored for many regions of the world. In order to analyze the presence and importance of Amphidomataceae in a highly productive area of Argentinean coastal waters (El Rincón area, SW Atlantic), a scientific cruise was performed in 2015 to sample the early spring bloom. In a multi-method approach, light microscopy was combined with real-time PCR molecular detection of Amphidomataceae, with chemical analysis of AZA, and with the establishment and characterization of amphidomatacean strains. Both light microscopy and PCR revealed that Amphidomataceae were widely present in spring plankton communities along the El Rincón area. They were particularly abundant offshore at the shelf front, reaching peak densities of 2.8 × 105 cells L−1, but no AZA were detected in field samples. In total, 31 new strains were determined as Az. dalianense and Az. spinosum, respectively. All Az. dalianense were non-toxigenic and shared the same rRNA sequences. The large majority of the new Az. spinosum strains revealed for the first time the presence of a non-toxigenic ribotype of this species, which is otherwise the most important AZA producer in European waters. One of the new Az. spinosum strains, with a particular slender shape and some other morphological peculiarities, clustered with toxigenic strains of Az. spinosum from Norway and, exceptionally for the species, produced only AZA-2 but not AZA-1. Results indicate a wide diversity within Az. spinosum, both in terms of sequence data and toxin profiles, which also will affect the qualitative and quantitative performance of the specific qPCR assay for this species. Overall, the new data provide a more differentiated perspective of diversity, toxin productivity and occurrence of Amphidomataceae in a poorly explored region of the global ocean.

Molecular detection and quantification of the azaspiracid-producing dinoflagellateAmphidoma languida(Amphidomataceae, Dinophyceae)

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.

Two novel azaspiracids from Azadinium poporum, and a comprehensive compilation of azaspiracids produced by Amphidomataceae, (Dinophyceae)

Two novel azaspiracids (AZA) with a molecular mass of 869 Da were found in Pacific strains of Azadinium poporum and characterized by tandem mass spectrometry and high resolution mass spectrometry (HRMS). One compound, AZA-42, was found in Az. poporum strains AZFC25 and AZFC26, both isolated from the South China Sea. AZA-42 belongs to the 360-type AZA that in comparison to AZA-1 has an additional double bond in the F–I ring system of AZA comprising C28-C40. The other compound, AZA-62, was detected in Az. poporum strain 1D5 isolated off Chañaral, Northern Chile. Mass spectral data indicate that AZA-62 is a variant of AZA-11 with an additional double bond in the C1-C9 region of AZA. In addition to the description of the two novel AZA, a comprehensive list of all AZA known to be produced by species of the genera Azadinium and Amphidoma comprising 26 AZA variants is presented.

Diversity, distribution, and azaspiracids of Amphidomataceae (Dinophyceae) along the Norwegian coast

Azaspiracids (AZA) are a group of lipophilic polyether compounds which have been implicated in shellfish poisoning incidents around Europe. They are produced by a few species of the dinophycean genera Azadinium and Amphidoma (Amphidomataceae). The presence of AZA toxins in Norway is well documented, but knowledge of the distribution and diversity of Azadinium and other Amphidomataceae along the Norwegian coast is rather limited and poorly documented. On a research survey along the Norwegian coast in 2015 from the Skagerrak in the South to Trondheimsfjorden in the North, plankton samples from 67 stations were analysed for the presence of Azadinium and Amphidoma and their respective AZA by on-board live microscopy, real-time PCR assays specific for Amphidomataceae, and liquid chromatography-tandem mass spectrometry (LC–MS/MS).Microscopy using live samples and positive real-time PCR assays using a general family probe and two species specific probes revealed the presence of Amphidomataceae distributed throughout the sampling area. Overall abundance was low, however, and was in agreement with a lack of detectable AZA in plankton samples. Single cell isolation and morphological and molecular characterisation of established strains revealed the presence of 7 amphidomatacean species (Azadiniun spinosum, Az. poporum, Az. obesum, Az. dalianense, Az. trinitatum, Az. polongum, Amphidoma languida) in the area. Azaspiracids were produced by the known AZA producing species Az. spinosum, Az. poporum and Am. languida only. LC–MS/MS analysis further revealed that Norwegian strains produce previously unreported AZA for Norway (AZA-11 by Az. spinosum, AZA-37 by Az. poporum, AZA-38 and AZA-39 by Am. languida), and also four novel compounds (AZA-50, -51 by Az. spinosum, AZA-52, -53 by Am. languida), whose structural properties are described and which now can be included in existing analytical protocols. A maximum likelihood analysis of concatenated rDNA regions (SSU, ITS1-ITS2, partial LSU) showed that the strains of Az. spinosum fell in two well supported clades, where most but not all new Norwegian strains formed the new Ribotype B. Ribotype differentiation was supported by a minor morphological difference with respect to the presence/absence of a rim around the pore plate, and was consistently reflected by different AZA profiles. Strains of Az. spinosum from ribotype A produce AZA-1, -2 and -33, whereas the new strains of ribotype B produce mainly AZA-11 and AZA-51. Significant sequence differences between both Az. spinosum ribotypes underline the need to redesign the currently used qPCR probes in order to detect all AZA producing Az. spinosum.The results generally underline the conclusion that for the Norwegian coast area it is important that amphidomatacean species are taken into account in future studies and monitoring programs.