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Gymnodimines and spirolides are cyclic imine phycotoxins and known antagonists of nicotinic acetylcholine receptors (nAChRs). We investigated the effect of gymnodimine A (GYM A) and 13-desmethyl spirolide C (SPX 1) from Alexandrium ostenfeldii on rat pheochromocytoma (PC12) cells by monitoring intracellular calcium levels ([Ca] i ). Using whole cells, the presence of 0.5 µM of GYM A or SPX 1 induced an increase in [Ca] i mediated by acetylcholine receptors (AChRs) and inhibited further activation of AChRs by acetylcholine (ACh). To differentiate the effects of GYM A or SPX 1, the toxins were applied to cells with pharmacologically isolated nAChRs and muscarinic AChRs (mAChRs) as mediated by the addition of atropine and tubocurarine, respectively. GYM A and SPX 1 activated nAChRs and inhibited the further activation of nAChRs by ACh, indicating that both toxins mimicked the activity of ACh. Regarding mAChRs, a differential response was observed between the two toxins. Only GYM A activated mAChRs, resulting in elevated [Ca] i, but both toxins prevented a subsequent activation by ACh. The absence of the triketal ring system in GYM A may provide the basis for a selective activation of mAChRs. GYM A and SPX 1 induced no changes in [Ca] i when nAChRs and mAChRs were inhibited simultaneously, indicating that both toxins target AChRs.

Spirolides belong to a group of marine phycotoxins produced by the marine planktonic dinophyte Alexandrium ostenfeldii . Composed of an imine moiety and a spiroketal ring system within a macrocylcle, spirolides are highly diverse with toxin types that vary among different strains. This study aims to characterize the spirolides from clonal A. ostenfeldii strains collected from the Netherlands, Greenland and Norway by mass spectral techniques. The structural characterization of unknown spirolides as inferred from high-resolution mass spectrometry (HR-MS) and collision induced dissociation (CID) spectra revealed the presence of nine novel spirolides that have the pseudo-molecular ions m/z 670 ( 1 ), m/z 666 ( 2 ), m/z 696 ( 3 ), m/z 678 ( 4 ), m/z 694 ( 5 ), m/z 708 ( 6 ), m/z 720 ( 7 ), m/z 722 ( 8 ) and m/z 738 ( 9 ). Of the nine new spirolides proposed in this study, compound 1 was suggested to have a truncated side chain in lieu of the commonly observed butenolide ring in spirolides. Moreover, there is indication that compound 5 might belong to new spirolide subclasses with a trispiroketal ring configuration having a 6:5:6 trispiroketal ring system. On the other hand, the other compounds were proposed as C- and G-type SPX, respectively. Compound 7 is proposed as the first G-type SPX with a 10-hydroxylation as usually observed in C-type SPX. This mass spectrometry-based study thus demonstrates that structural variability of spirolides is larger than previously known and does not only include the presence or absence of certain functional groups but also involves the triketal ring system., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cyclic imine toxins are neurotoxic, macrocyclic compounds produced by marine dinoflagellates. Mass spectrometric screenings of extracts from natural plankton assemblages revealed a high chemical diversity among this toxin class, yet only few toxins are structurally known. Here we report the structural characterization of four novel cyclic-imine toxins (two gymnodimines (GYMs) and two spirolides (SPXs)) from cultures of Alexandrium ostenfeldii . A GYM with m / z 510 ( 1 ) was identified as 16-desmethylGYM D. A GYM with m / z 526 was identified as the hydroxylated degradation product of ( 1 ) with an exocyclic methylene at C-17 and an allylic hydroxyl group at C-18. This compound was named GYM E ( 2 ). We further identified a SPX with m / z 694 as 20-hydroxy-13,19-didesmethylSPX C ( 10 ) and a SPX with m / z 696 as 20-hydroxy-13,19-didesmethylSPX D ( 11 ). This is the first report of GYMs without a methyl group at ring D and SPXs with hydroxyl groups at position C-20. These compounds can be conceived as derivatives of the same nascent polyketide chain, supporting the hypothesis that GYMs and SPXs are produced through common biosynthetic genes. Both novel GYMs 1 and 2 were detected in significant amounts in extracts from natural plankton assemblages ( 1 : 447 pg; 2 : 1250 pg; 11 : 40 pg per mL filtered seawater respectively).

The marine dinoflagellate Azadinium poporum produce azaspiracids (AZA) and has been recorded widely in the world. However, information on its biogeography is still limited, especially in view of the fact that A. poporum comprises several genetically differentiated groups. A total of 18 strains of A. poporum were obtained from the Eastern Mediterranean area by incubating surface sediment collected from Ionian Sea of Greece. The morphology of these strains was examined with light microscopy and scanning electron microscopy. Small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences were obtained from all cultured strains. Molecular phylogeny based on concatenated SSU, LSU and ITS sequences confirmed three ribotypes within A. poporum and revealed two subclades within ribotypes A and C. Greek strains of A. poporum ribotype A were nested within ribotype A2 together with strains from Western Mediterranean Sea and French Atlantic, and Greek strains of A. poporum ribotype C were nested within ribotype C2 together with a strain from the Gulf of Mexico. Growth experiments on four selected strains revealed that ribotypes A and C from Greece differed in their growth at higher temperatures, indicating that they are physiologically differentiated. Azaspiracid profiles were analyzed for 15 cultured A. poporum strains using LCMS/MS and demonstrate that the A. poporum ribotype A from Greece produce low level or no AZA and A. poporum ribotype C from Greece produces predominantly AZA-40 (9.6-30.2 fg cell -1 ) followed by AZA-2 (2.1-2.6 fg cell -1 ). The first record of AZA-40 producing A. poporum from the Mediterranean suggests that this species is a potential source for azaspiracid contaminations in shellfish from the Eastern Mediterranean Sea., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The identification of a new suite of toxins, called azaspiracids (AZA), as the cause of human illnesses after the consumption of shellfish from the Irish west coast in 1995, resulted in interest in understanding the global distribution of these toxins and of species of the small dinoflagellate genus Azadinium, known to produce them. Clonal isolates of four species of Azadinium, A. poporum, A. cuneatum, A. obesum and A. dalianense were obtained from incubated sediment samples collected from Puget Sound, Washington State in 2016. These Azadinium species were identified using morphological characteristics confirmed by molecular phylogeny. Whereas AZA could not be detected in any strains of A. obesum, A. cuneatum and A. dalianense, all four strains of A. poporum produced a new azaspiracid toxin, based on LC-MS analysis, named AZA-59. The presence of AZA-59 was confirmed at low levels in situ using a solid phase resin deployed at several stations along the coastlines of Puget Sound. Using a combination of molecular methods for species detection and solid phase resin deployment to target shellfish monitoring of toxin at high-risk sites, the risk of azaspiracid shellfish poisoning can be minimized., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The marine planktonic dinophyceaen genus Azadinium is a primary source of azaspiracids, but due to their small size its diversity may be underestimated and information on its biogeography is still limited. A new Azadinium species, A. zhuanum was obtained from the East China Sea and Yellow Sea of China by incubating surface sediments. Five strains were established by isolating single germinated cells and their morphology was examined with light microscopy and scanning electron microscopy. Azadinium zhuanum was characterized by a plate pattern of Po, cp, X, 4', 2a, 6'', 6C, 5S, 6''', 2'''', by a distinct ventral pore at the junction of Po, the first and fourth apical plates, and a conspicuous antapical spine. Moreover, Azadinium poporum was obtained for the first time from the Mediterranean by incubating surface sediment collected from Diana Lagoon (Corsica) and a new strain of Azadinium dalianense was isolated from the French Atlantic. The morphology of both strains was examined. Small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences were obtained from cultured strains. In addition, LSU sequences were obtained by single cell sequencing of two presumable A. poporum cells collected from the French Atlantic. Molecular phylogeny based on concatenated SSU, LSU and ITS sequences revealed that A. zhuanum was closest to A. polongum. French A. poporum from Corsica (Mediterranean) and from the Atlantic showed some genetic differences but were nested within one of the A. poporum ribotypes together with other European strains. Azadinium dalianense from France together with the type strain of the species from China comprised a well resolved clade now consisting of two ribotypes. Azaspiracid profiles were analyzed for the cultured Azadinium strains using LC-MS/MS and demonstrate that the Mediterranean A. poporum strain produced AZA-2 and AZA-2 phosphate with an amount of 0.44fgcell -1 . Azadinium zhuanum and A. dalianense did not produce detectable AZA. Results of the present study support the view of a high diversity and wide distribution of species belonging to Azadinium. The first record of AZA-2 producing A. poporum from the Mediterranean suggests that this species may be responsible for azaspiracid contaminations in shellfish from the Mediterranean Sea., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Azaspiracids (AZA) are a group of food poisoning phycotoxins that are known to accumulate in shellfish. They are produced by some species of the planktonic dinophycean taxon Amphidomataceae. Azaspiracids have been first discovered in Ireland but are now reported in shellfish from numerous global sites thus showing a wide distribution. In shellfish samples collected in 2009 near Huelva (Spain), AZA was also found along the Andalusian Atlantic coast for the first time. Analysis using LC-MS/MS revealed the presence of two different AZA analogues in different bivalve shellfish species (Chamelea gallina, Cerastoderma edule, Donax trunculus, and Solen vagina). In a number of samples, AZA levels exceeded the EU regulatory level of 160μg AZA-1 eq. kg -1 (reaching maximum levels of >500μg AZA-1 eq. kg -1 in Chamelea gallina and >250μg AZA-1 eq. kg -1 in Donax trunculus) causing closures of some local shellfish production areas. One dinophyte strain established from the local plankton during the AZA contamination period and determined as Amphidoma languida was in fact toxigenic, and its AZA profile disclosed it as the causative species: it contained AZA-2 as the main compound and the new compound AZA-43 initially detected in the shellfish. AZA-43 had the same mass as AZA-3, but produced different collision induced dissociation (CID) spectra. High resolution mass spectrometric measurements indicated that there is an unsaturation in the H, I ring system of AZA-43 distinguishing it from the classical AZA such as AZA-1, -2, and -3. Furthermore, the Spanish strain was different from the previously reported AZA profile of the species that consist of AZA-38 and AZ-39. In molecular phylogenetics, the Andalusian strain formed a monophyletic group together with other strains of Am. languida, but ITS sequences data revealed surprisingly high intragenomic variability. The first Andalusian case of AZA contamination of shellfish above the EU regulatory limit reported here clearly revealed the risk of azaspiracid poisoning (AZP) for this area and also for the Atlantic coast of Iberia and North Africa. The present study underlines the need for continuous monitoring of AZA and the organisms producing such toxins., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Azadinium poporum produces a variety of azaspiracids and consists of several ribotypes, but information on its biogeography is limited. A strain of A. poporum (GM29) was incubated from a Gulf of Mexico sediment sample. Strain GM29 was characterized by a plate pattern of po, cp, x, 4', 3a, 6″, 6C, 5S, 6‴, 2⁗, a distinct ventral pore at the junction of po and the first two apical plates, and a lack of an antapical spine, thus fitting the original description of A. poporum. The genus Azadinium has not been reported in waters of the United States of America before this study. Molecular phylogeny, based on large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences, reveals that strain GM29 is nested within the well-resolved A. poporum complex, but forms a sister clade either to ribotype B (ITS) or ribotype C (LSU). It is, therefore, designated as a new ribotype, termed as ribotype D. LSU and ITS sequences similarity among different ribotypes of A. poporum ranges from 95.4% to 98.2%, and from 97.1% to 99.2% respectively, suggesting that the LSU fragment is a better candidate for molecular discrimination. Azaspiracid profiles were analyzed using LC-MS/MS and demonstrate that strain GM29 produces predominantly AZA-2 with an amount of 45fg/cell. The results suggest that A. poporum has a wide distribution and highlights the risk potential of azaspiracid intoxication in the United States., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Two strains of Azadinium poporum, one from the Korean West coast and the other from the North Sea, were mass cultured for isolation of new azaspiracids. Approximately 0.9 mg of pure AZA-36 (1) and 1.3 mg of pure AZA-37 (2) were isolated from the Korean (870 L) and North Sea (120 L) strains, respectively. The structures were determined to be 3-hydroxy-8-methyl-39-demethyl-azaspiracid-1 (1) and 3-hydroxy-7,8-dihydro-39-demethyl-azaspiracid-1 (2) by ¹H- and (13)C-NMR. Using the Jurkat T lymphocyte cell toxicity assay, (1) and (2) were found to be 6- and 3-fold less toxic than AZA-1, respectively.

Azaspiracids (AZAs) are a group of lipophilic polyether toxins implicated in incidents of shellfish poisoning in humans, particularly in northern Europe, which are produced by the small marine dinoflagellate Azadinium spinosum. Other related species/strains of the Amphidomataceae have not been proven to date to contain any of the known azaspiracids. Closer analyses of these species/strains by triple quadrupole mass spectrometry in the precursor and product ion mode now revealed four new compounds with high similarity to azaspiracids, all of them with a characteristic m/z 348 fragment but with absence of the m/z 362 fragment. These compounds were detected in three species/strains, i.e. in North Sea isolates of Azadinium poporum (molecular mass: 845.5 Da), in a Korean isolate which has been designated as A. cf. poporum (molecular mass: 857.5 Da) and in Amphidoma languida isolated from Bantry Bay, Ireland (molecular masses: 815.5 and 829.5 Da). Cell quotas of roughly 2-20 fg per cell were in the same range as found for AZA-1/-2 in A. spinosum. Structures for all compounds were proposed by interpretation of fragmentation patterns and high resolution mass measurements using Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS)., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Azaspiracids (AZAs) are secondary metabolites of Azadinium spinosum that can accumulate in shellfish and cause food poisoning when consumed. We describe here an analytical procedure for the determination of AZAs in cultures of A. spinosum with a focus on the formation of AZA methyl esters as artefacts during extraction and sample pre-treatment. A. spinosum cells were collected from bioreactor cultures using centrifugation or filtration. Different extraction procedures were evaluated for formation of methyl ester artefacts, yield, and matrix effects. Filtration of cultures using glass-fibre filters led to increased formation of methyl esters, and centrifugation is recommended for recovery of cells. The extraction solvent (methanol (MeOH), acetone, and acetonitrile (MeCN)) did not significantly affect the yield of AZAs as long as the organic content was 80% or higher. However, the use of MeOH as extraction solvent led to increased formation of methyl esters. AZA1 recovery over two successive extractions was 100% at the 95% confidence level for acetone and MeOH. In standard-addition experiments, no significant matrix effects were observed in extracts of A. spinosum or Azadinium obesum up to a sample size of 4.5 × 10(9) μm(3). Moreover, experiments carried out to clarify the formation and structure of methylated AZA analogues led to the description of two AZA methyl esters and to the correction of the chemical structures of AZAs29-32.