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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 Chanaral, 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.

An external standard of goniodomin A (GDA) was prepared from a strain of Alexandrium pseudogonyaulax originating from New Zealand and its chemical structure was confirmed by nuclear magnetic resonance (NMR) spectroscopy. Using the GDA standard, an ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method in selected reaction monitoring (SRM) mode was developed for separation and quantification of GDA. This method was successfully applied to planktonic field samples collected during an oceanographic expedition conducted with R/V Uthorn along the Danish west coast, Limfjord and Kattegat in June 2016. In addition, this method was used to characterize goniodomin (GD) profiles of 17 A. pseudogonyaulax strains from the coastal North Sea and from Limfjord. Highest GDA levels were found in Limfjord (up to 590 ng NT−1 m−1), but GDA was also detected in the North Sea appearing at the latitude of Sylt Island northwards and in Kattegat from the eastern mouth of Limfjord down to the Kiel Bight, but at lower abundances than within Limfjord. This is the first reported detection of GDA in planktonic field samples. Chemical analysis of 17 strains of A. pseudogonyaulax revealed that all strains were producers of GDA (5–35 pg cell−1) as well as in most cases minor amounts (0.01–0.07 pg cell−1, expressed as GDA equivalents) of goniodomin B (GDB).

Amphidoma is an old though poorly studied thecate dinophyte that has attracted attention recently as a potential producer of azaspiracids (AZA), a group of lipophilic phycotoxins. A new species, Amphidoma parvula, sp. nov. is described from the South Atlantic shelf of Argentina. With a Kofoidean thecal plate pattern Po, cp, X, 6′, 6′′, 6C, 5S, 6′′′, 2′′′′, the cultivated strain H-1E9 (from which the type material of Am. parvula, sp. nov. was prepared) shared the characteristic plate arrangement of Amphidoma each with six apical, precingular and postcingular plates. Amphidoma parvula, sp. nov. differs from other species of Amphidoma by a characteristic combination of small size (10.7–13.6 µm in length), ovoid shape, high length ratio between epitheca and hypotheca, and small length ratio between apical and precingular plates. Other morphological details, such as the number and arrangement of sulcal plates and the fine structure of the apical pore complex support the close relationship between Amphidoma and the other known genus of Amphidomataceae, Azadinium. However, Am. parvula, sp. nov. lacks a ventral pore, a characteristically structured pore found in all contemporary electron microscopy studies of Amphidoma and Azadinium. As inferred from liquid chromatography coupled with tandem mass spectrometry, Am. parvula, sp. nov. did not produce AZA in measurable amounts. Molecular phylogenetics confirmed the systematic placement of Am. parvula, sp. nov. in Amphidoma (as sister species of Amphidoma languida) and the Amphidomataceae. The results of this study have improved the knowledge of Amphidomataceae biodiversity. Fil: Tillmann, Urban. Alfred Wegener Institut; Alemania Fil: Gottschling, Marc. Universität München; Alemania Fil: Guinder, Valeria Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto Argentino de Oceanografía. Universidad Nacional del Sur. Instituto Argentino de Oceanografía; Argentina Fil: Krock, Bernd. Alfred Wegener Institut; Alemania

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.

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.

Shellfish aquaculture has become an increasingly important factor in Bulgarian economy in the recent years. Marine biotoxins, produced by some phytoplankton species, may accumulate in mussels and present an important challenge in commercialization of shellfish. The aim of this study was to determine the occurrence and variability of hydrophilic toxins – paralytic shellfish poisoning (PSP) and amnesic shellfish poisoning (ASP) as well as lipophilic toxins – including diarrheic shellfish poisoning (DSP), pectenotoxins (PTXs) and yessotoxins (YTXs) in plankton, wild and farmed mussel samples from the Southern Black Sea coast, an important shellfish farming area in spring 2017. PSP toxins were determined by HPLC with postchromatographic oxidation and fluorescence detection and domoic acid and lipophilic toxins by liquid chromatograph coupled to tandem mass spectrometry. DA and pectenotoxin-2 were detected in plankton, wild and farmed mussel samples. Yessotoxins were detected only in cultivated mussels and no PSP toxins were detected. The occurrence of phycotoxins differed over both space and time. Toxin profile includes prevalent domoic acid, scarce presence of pectenotoxin-2 and yessotoxins in the samples.

Alexandrium ostenfeldii is an emerging harmful algal bloom species forming a global threat to coastal marine ecosystems, with consequences for fisheries and shellfish production. The Oosterschelde estuary is a shallow, macrotidal and mesotrophic estuary in the southwest of The Netherlands with large stocks of mussels, oysters, and cockles. These shellfish stocks were threatened by a recent A. ostenfeldii bloom in the Ouwerkerkse Kreek, which is a brackish water creek discharging water into the Oosterschelde. Little is yet known about the characteristics of the A. ostenfeldii population in this creek. We therefore isolated 20 clones during an A. ostenfeldii bloom in 2013, and characterized these clones on their growth and toxin profile in their exponential growth phase. The cyclic imines were identified by comparison of A. ostenfeldii extracts with the retention time and CID spectra of standard solutions, or with published CID spectra. We furthermore assessed the allelochemical potency and phylogeny of a selection of 10–12 clones. Morphology and molecular phylogeny showed that all clones belong to Group 1 of A. ostenfeldii. All clones showed comparable growth rates of on average 0.22 ± 0.03 d−1. During exponential growth, they all produced a unique combination of paralytic shellfish poisoning toxins, spirolides and gymnodimines, of which particularly the latter showed a high intra-specific variability, with a 25-fold difference between clones with the lowest and highest cell quota. Furthermore, the selected 12 clones showed high allelopathic potencies with EC50 values based on lysis assays against the cryptophyte Rhodomonas salina between 212 and 525 A. ostenfeldii cells mL−1. Lytic activities were lower for cell extracts, indicating an important extracellular role of these compounds. A high intra-specific variability may add to the success of genotypically diverse A. ostenfeldii blooms, and make populations resilient to changes in environmental and climatic conditions.

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.

The paralytic shellfish toxin (PST) profiles of Gymnodinium catenatum Graham have been reported for several strains from the Pacific coast of Mexico cultured under different laboratory conditions, as well as from natural populations. Up to 15 saxitoxin analogues occurred and the quantity of each toxin depended on the growth phase and culture conditions. Previous analysis of toxin profiles of G. catenatum isolated from Mexico have been based on post-column oxidation liquid chromatography with fluorescence detection (LC-FLD), a method prone to artefacts and non-specificity, leading to misinterpretation of toxin composition. We describe, for the first time, the complete toxin profile for several G. catenatum strains from diverse locations of the Pacific coast of Mexico. The new results confirmed previous reports on the dominance of the less potent sulfocarbamoyl toxins (C1/2); significant differences, however, in the composition (e.g., absence of saxitoxin, gonyautoxin 2/3 and neosaxitoxin) were revealed in our confirmatory analysis. The LC-MS/MS analyses also indicated at least seven putative benzoyl toxin analogues and provided support for their existence. This new toxin profile shows a high similarity (> 80%) to the profiles reported from several regions around the world, suggesting low genetic variability among global populations.

The toxic diatom Pseudo-nitzschia seriata was exposed directly and indirectly (separated by a membrane) to copepods, Calanus hyperboreus and C. finmarchicus, to evaluate the effects of the copepods on domoic acid production and chain formation in P. seriata. The toxicity of P. seriata increased in the presence of the copepods. This response was chemically mediated without physical contact between the organisms suggesting that it was induced by potential waterborne cues from the copepods or changes in water chemistry. Domoic acid production may be related to defense against grazing in P. seriata although it was not shown in the present study. To evaluate if the induction of domoic acid production was mediated by the chemical cues from damaged P. seriata cells, live P. seriata cells were exposed to a P. seriata cell homogenate, but no effect was observed. Chain formation in P. seriata was affected only when in direct contact with the copepods. This study suggests that the presence of zooplankton may be one of the factors affecting the toxicity of Pseudo-nitzschia blooms in the field.

In fjord systems, Harmful Algal Blooms (HABs) not only constitute a serious problem when affecting the wildlife and ecosystems, but also human health and economic activities related to the marine environment. This is mostly due to a broad spectrum of toxic compounds produced by several members of the phytoplankton. Nevertheless, a deep coverage of the taxonomic diversity and composition of phytoplankton species and phycotoxin profiles in HAB prone areas are still lacking and little is known about the relationship between these fundamental elements for fjord ecosystems. In this study, a detailed molecular and microscopic characterization of plankton communities was performed, together with an analysis of the occurrence and spatial patterns of lipophilic toxins in a HAB prone area, located in the Southeastern Pacific fjord region. Microscopy and molecular analyses based on the 18S rRNA gene fragment indicated high diversity and taxonomic homogeneity among stations. Four toxigenic genera were identified: Pseudo-nitzschia, Dinophysis, Prorocentrum, and Alexandrium. In agreement with the detected species, liquid chromatography coupled with mass spectrometry revealed the presence of domoic acid (DA), pectenotoxin-2 (PTX-2), dinophysistoxin-2 (DTX-2), and 13-desmethyl spirolide C (SPX-1). Furthermore, a patchy distribution among DA in different net haul size fractions was found. Our results displayed a complex phytoplankton-phycotoxin pattern and for the first time contribute to the characterization of high-resolution phytoplankton community composition and phycotoxin distribution in fjords of the Southeastern Pacific region.

espanolLas especies dinoficeas del genero Azadinium son conocidas por la produccion potencial de azaspiracidos (AZAs), un grupo de toxinas microalgales que pueden causar intoxicacion a traves de mariscos. El incremento de registros globales de Azadinium indican una distribucion bastante amplia de este genero, sin embargo los datos cuantitativos de abundancia de las especies de familia Amfidomataceae son dificiles de encontrar. Como parte del programa de monitoreo bio-oceanografico en aguas costeras peruanas frente a Chancay, se detecto y muestreo en febrero de 2014 una floracion densa de Azadinium, con una temperatura de agua de mar alrededor de 20,5 °C y una salinidad de 35. Este es el primer reporte de una floracion de Azadinium en el oceano Pacifico con densidades celulares de hasta un millon de celulas por litro causante de una decoloracion del agua de mar. Usando microscopia electronica de barrido, se determino como taxon responsable a Azadinium polongum. Esta especie previamente habia sido detectada solo en las Islas Shetland (Atlantico norte), localidad tipo y descrita como no productora de AZA. La poblacion observada en campo se ajusto al cultivo tipo Shetland en la mayoria de los aspectos morfologicos. Sin embargo, a diferencia de este tipo de cultivo de Az. polongum, la primera placa intercalar de la poblacion observada en el Pacifico, en ningun caso, estuvo en contacto con la primera placa precingular. Mas aun, el tamano y la forma de la segunda placa intercalar fue ligeramente diferente y muy variable en la poblacion del Pacifico, ocurriendo en casi igual abundancia en configuracion cuadra (es decir, en contacto con cuatro placas) o configuracion penta (en contacto con cinco placas), un rasgo no reportado para el cultivo tipo de Az. polongum. La cromatografia liquida acoplada con espectrometria de masa no detecto AZA por encima del nivel de deteccion en la muestra de la floracion, indicando que la especie peruana de Az. polongum puede considerarse como no productora de AZA. EnglishSpecies of the dinophycean genus Azadinium are known for their potential production of azaspiracids (AZAs), a group of microalgal toxins that can cause shellfish poisoning. Increase of global Azadinium records indicates a rather wide distribution, but quantitative abundance data of species of Amphidomataceae are hardly available. Within the Peruvian bio-oceanographic monitoring program we detected and sampled a dense bloom of Azadinium in Peruvian coastal waters off Chancay in February 2014 at a water temperature of about 20.5 °C and a salinity of 35. With water discoloration and cell densities of Azadinium up to 1 million cells per liter this is the first Azadinium bloom record for the Pacific. The causative taxon was determined using scanning electron microscopy as Azadinium polongum, a species previously recorded only from the type locality, the Shetland Islands (north Atlantic) and described as non-AZA producer. The field population conformed to the Shetland type culture in most morphological aspects. However, the first intercalary plate of the Pacific field population without exception was not in contact with the first precingular plate, which is different to the type culture of Az. polongum. Moreover, the size and shape of the median intercalary plate was slightly different and very variable in the Pacific population, and occurred in about equal abundance in quadra- (i.e., in contact with four other plates) or in penta-configuration (i.e., in contact with five other plates), a feature not reported for the type culture of Az. polongum. Liquid chromatography coupled with mass spectrometry revealed no AZA above detection level in the bloom sample, indicating that the Peruvian Az. polongum can be regarded as a non-AZA producer.

The present work reports the first data set on particulate organic carbon (POC) and nitrogen (PON), and the high-resolution modelling of their stable isotope variability in the Patagonian Cold Estuarine System (PCES), with focus on particulate organic matter (POM) origin and distribution in dependence on physical, chemical and biological parameters. POC, PON, stable carbon (δ¹³C) and nitrogen isotopes (δ¹⁵N), dissolved organic nitrogen, phaeopigments, diatom, dinoflagellate and heterotrophic bacteria (HB) abundance are reported for 17 stations in different waters masses in the southern end of the Argentine shelf in late summer 2012. Most parameters denote clear differences between Beagle - Magellan Water (BMW), Subantarctic Shelf Water (SSW) and Subantarctic Water (SAW). POC and PON decreased from maxima in BMW to intermediate values in SSW and minima in SAW. There was a highly significant correlation among POC, PON and fluorescence indicators of diagenetic maturity of dissolved humic matter. This, together with the inverse correlations of salinity with POC and PON, and the wide range of C:N ratios indicate that POM in the study area is partly derived from terrestrial runoff, superimposed by autochthonous components from plankton of different life stages. HB abundance was significantly correlated with POC and dissolved organic matter (DOM), likely reflecting a resource control of HB and a significant contribution of bacterial biomass to POM in the nanoparticle fraction. The direct relationship between HB and dissolved humics suggests bacterial uptake of DOM fractions otherwise considered refractory. POM complexity was reflected in a wide variation of δ¹³C, despite the narrow temperature range of this region. The variability of stable isotopes of POC could be accounted for by a model with a degree of detail hitherto not reported in the literature. A multiple regression including C:N ratio, ammonium and the quotient between log abundance of diatoms, dinoflagellates and HB explained 92% of δ¹³C variance, mostly produced by ammonium. Despite the strong effect of ammonium on δ¹³C, δ¹⁵N variability was largely explained by a strong inverse relationship with the fraction of unutilized nitrate, suggesting dominance of nitrate uptake. However, the proportion of presumably isotopically heavier ammonium derived from continental runoff in the marine δ¹⁵N-POM pool is unknown and requires investigation of the isotopic composition of dissolved inorganic nitrogen in the PCES. The presented new information and its comparison with data from other sectors of the Argentine shelf constitute a contribution to an approach for the understanding of the organic matter dynamics that can be potentially expanded to the entire Southwest Atlantic., Facultad de Ciencias Naturales y Museo

The chain-forming marine dinoflagellate Gymnodinium catenatum Graham has a remarkable capacity to produce a wide array of neurotoxic alkaloids associated with Paralytic Shellfish Poisoning (PSP). More than a decade ago, a completely new group of benzoyl saxitoxin analogs produced exclusively by this species was discovered, but the exact structural assignments and diversity among global population has remained elusive and nconfirmed in most cases. In the current study, fifteen among eighteen hypothetical benzoyl analogs were partially purified and identified from cultured isolates of G. catenatum from the Pacific coast of Mexico. Combined serial application of flash chromatography, preparative liquid chromatography and tandem mass spectrometry (LC-MS/MS) in multiple steps yielded a richness of benzoyl analogs that has not been reported nor confirmed before. Two sub-fractions were analyzed by 1H-NMR; results from one fraction showed a probable AMX pattern for three protons, consistent with the presence of a 3,4-dihydroxylated benzoyl ring. These findings could be interpreted to correct the 2,4-dihydroxylated structure previously proposed for the GCa benzoyl analog series. The revised and enhanced structural information on proposed benzoyl derivatives is necessary to provide further insights into biogeographical diversity of these potentially potent toxins produced by marine dinoflagellates and their role in seafood safety.

Alexandrium ostenfeldii is a toxic dinoflagellate that has recently bloomed in Ouwerkerkse Kreek, The Netherlands, and which is able to cause a serious threat to shellfish consumers and aquacultures. We used a large set of 68 strains to the aim of fully characterizing the toxin profiles of the Dutch A. ostenfeldii in consideration of recent reports of novel toxins. Alexandrium ostenfeldii is known as a causative species of paralytic shellfish poisoning, and consistently in the Dutch population we determined the presence of several paralytic shellfish toxins (PST) including saxitoxin (STX), GTX2/3 (gonyautoxins), B1 and C1/C2. We also examined the production of spiroimine toxins by the Dutch A. ostenfeldii strains. An extensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed a high intraspecific variability of spirolides (SPX) and gymnodimines (GYM). Spirolides included 13-desMethyl-spirolide C generally as the major compound and several other mostly unknown SPX-like compounds that were detected and characterized. Besides spirolides, the presence of gymnodimine A and 12-Methyl-gymnodimine A was confirmed, together with two new gymnodimines. One of these was tentatively identified as an analogue of gymnodimine D and was the most abundant gymnodimine (calculated cell quota up to 274 pg cell(-1), expressed as GYM A equivalents). Our multi-clonal approach adds new analogues to the increasing number of compounds in these toxin classes and revealed a high strain variability in cell quota and in toxin profile of toxic compounds within a single population.

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 0.44 fg cell−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.

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.

The marine diatom genus Pseudo-nitzschia, the major known producer of the neurotoxin domoic acid (DA) responsible for the amnesic shellfish poisoning (ASP) syndrome in humans and marine mammals, is globally distributed. The genus presents high species richness in the Argentine Sea and DA has been frequently detected in the last few years in plankton and shellfish samples, but the species identity of the producers remains unclear. In the present work, the distribution and abundance of Pseudo-nitzschia species and DA were determined from samples collected on two oceanographic cruises carried out through the Argentine Sea (⁓39–47ºS) during summer and spring 2013. Phytoplankton composition was analysed by light and electron microscopy while DA was determined by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The genus Pseudo-nitzschia was recorded in 71 and 86% of samples collected in summer and spring, respectively, whereas DA was detected in only 42 and 21% of samples, respectively. Microscopic analyses revealed at least five potentially toxic species (P. australis, P. brasiliana, P. fraudulenta, P. pungens, P. turgidula), plus putatively non-toxigenic P. dolorosa, P. lineola, P. turgiduloides and unidentified specimens of the P. pseudodelicatissima complex. The species P. australis showed the highest correlation with DA occurrence (r = 0.55; p < 0.05), suggesting its importance as a major DA producer in the Argentine Sea. In the northern area and during summer, DA was associated with the presence of P. brasiliana, a species recorded for the first time in the Argentine Sea. By contrast, high concentrations of P. fraudulenta, P. pungens and P. turgidula did not correspond with DA occurrence. This study represents the first successful attempt to link toxigenicity with Pseudo-nitzschia diversity and cell abundance in field plankton populations in the south-western Atlantic., Facultad de Ciencias Naturales y Museo

Azadinium dexteroporum is the first species of the genus described from the Mediterranean Sea and it produces different azaspiracids (AZA). The aims of this work were to characterize the toxin profile of the species and gain structural information on azaspiracids produced by the A. dexteroporum strain SZN-B848 isolated from the Gulf of Naples. Liquid chromatography-mass spectrometry (LC-MS) analyses were carried out on three MS systems having different ion source geometries (ESI, TurboIonSpray®, ESI ION MAX) and different MS analyzers operating either at unit resolution or at high resolution, namely a hybrid triple quadrupole-linear ion trap (Q-Trap MS), a time of flight (TOF MS), and a hybrid linear ion trap Orbitrap XL Fourier transform mass spectrometer (LTQ Orbitrap XL FTMS). As a combined result of these different analyses, A. dexteroporum showed to produce AZA-35, previously reported from Azadinium spinosum, and six compounds that represent new additions to the AZA-group of toxins, including AZA-54 to AZA-58 and 3-epiAZA-7, a stereoisomer of the shellfish metabolite AZA-7. Based on the interpretation of fragmentation patterns, we propose that all these molecules, except AZA-55, have the same A to I ring system as AZA-1, with structural modifications all located in the carboxylic side chain. Considering that none of the azaspiracids produced by the Mediterranean strain of A. dexteroporum is currently regulated by European food safety authorities, monitoring programs of marine biotoxins in the Mediterranean area should take into account the occurrence of the new analogues to avoid an underestimation of the AZA-related risk for seafood consumers. Graphical Abstract A multi-platform MS approach reveals known and new azaspiracids in a Mediterranean strain of Azadinium dexteroporum.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the detection and quantitation of karlotoxins in the selected reaction monitoring (SRM) mode. This novel method was based upon the analysis of purified karlotoxins (KcTx-1, KmTx-2, 44-oxo-KmTx-2, KmTx-5), one amphidinol (AM-18), and unpurified extracts of bulk cultures of the marine dinoflagellate Karlodinium veneficum strain CCMP2936 from Delaware (Eastern USA), which produces KmTx-1 and KmTx-3. The limit of detection of the SRM method for KmTx-2 was determined as 2.5 ng on-column. Collision induced dissociation (CID) spectra of all putative karlotoxins were recorded to present fragmentation patterns of each compound for their unambiguous identification. Bulk cultures of K. veneficum strain K10 isolated from an embayment of the Ebro Delta, NW Mediterranean, yielded five previously unreported putative karlotoxins with molecular masses 1280, 1298, 1332, 1356, and 1400 Da, and similar fragments to KmTx-5. Analysis of several isolates of K. veneficum from the Ebro Delta revealed small-scale diversity in the karlotoxin spectrum in that one isolate from Fangar Bay produced KmTx-5, whereas the five putative novel karlotoxins were found among several isolates from nearby, but hydrographically distinct Alfacs Bay. Application of this LC-MS/MS method represents an incremental advance in the determination of putative karlotoxins, particularly in the absence of a complete spectrum of purified analytical standards of known specific potency. info:eu-repo/semantics/publishedVersion

Azaspiracids (AZA), a group of lipophilic phycotoxins, are produced by some species of the marine dinophycean genus Azadinium. AZA have recently been detected in shellfish from the Southeast Pacific, however, AZA-producing species have not been recorded yet from the area. This study is the first record of the genus Azadinium and of the species Azadinium poporum from the Pacific side of South America. Three strains of A. poporum from Chanaral (Northern Chile) comply to the type description of A. poporum by the presence of multiple pyrenoids, in thecal plate details, and in the position of the ventral pore located on the left side of the pore plate. Molecular phylogeny, based on internal transcribed spacer and large subunit ribosomal DNA sequences, revealed that Chilean strains fall in the same ribotype clade as European and strains from New Zealand. Analyses of AZA profiles using LC–MS/MS showed an identical profile for all three strains with the presence of AZA-11 and two phosphorylated AZA. This is the first confirmation of the presence of AZA producing Azadinium in the Chilean coastal area and underlines the risk of AZA shellfish and concomitant human contamination episodes in the Southeast Pacific region.

International audience; Toxin-producing microalgae are thriving worldwide due to coral reef destruction and global warming with major consequences on ecosystems, international trade and human health. Microalgae belonging to the family of flagellate protists, in particular dinoflagellates, secrete a variety of high-molecular-weight polyether toxins that accumulate through the marine food chain to cause disease in humans by acting as sodium channel activator toxins; ciguatera is the most frequent seafood-borne illness worldwide with 50,000 to 500,000 global incidences per annum and is usually limited to endemic areas located between 35 degrees northern and 35 degrees southern latitude. The rising global incidence frequency renders it a major human health problem, because no curative treatment is available yet and reliable detection assays are lacking. During the last decade ciguatera has increasingly become endemic in previously unaffected areas for two reasons: first global warming has contributed to the emergence of dinoflagellate species in subtropical and even temperate regions that previously had been constrained to tropical areas and second: in Europe globalization of fishing industry and tourism has led to a progressive increase in the number of ciguatera cases and a lack of awareness among medical personnel contributes to under-reporting. We review, through a recent ciguatera outbreak in Germany, the risk for ciguatera poisoning in Europe and highlight characteristic symptoms, current knowledge about disease pathomechanisms and treatment options.

A unialgal culture of a Pseudo-nitzschia species dominant in the plankton of Algoa Bay in the spring of 2012 was established by isolation of clonal chains of cells. Identification of the species as Pseudo-nitzschia multiseries was based on frustule morphometrics provided by light and scanning electron microscopy, and confirmed by phylogenetic analysis of the LSU rDNA gene. Cultures were shown to produce domoic acid (DA) as measured by ELISA and LC/MS-MS methods, and levels of cellular DA were ∼0.1 pg cell–1. Although it is recognised as a cosmopolitan species, these observations provide the first account of this toxic diatom in the coastal waters of South Africa.

Azadinium poporum is a small dinoflagellate from the family Amphidomataceae which is known for the potential production of azaspiracids (AZAs) causative of azaspiracid shellfish poisoning (AZP). A. poporum has been recorded from European and western Pacific waters. Here we report on the high variability of toxin profiles within this species in Chinese coastal waters. Out of 16 analyzed strains of A. poporum from different geographic locations along the Chinese coastline, three strains proved not to contain AZAs, whereas 13 strains contained different combinations of AZA-2, AZA-11, AZA-36, a yet unknown isomer of AZA-1 (named AZA-40) and new AZA with yet unreported molecular mass of 853 Da (named AZA-41). The new AZA-40, other than AZA-1 itself, belongs to the recently discovered “348-type” group, which in tandem mass spectrometry displays a group 4 fragment with m/z 348 instead of the group 4 fragment of the classic AZAs with m/z 362, indicating a shift of a methyl group from the C 24 –C 40 part of the molecule (rings F–I) to the C 2 –C 9 part (carboxylic side chain and ring A). AZA-41 apparently is a dehydro variant of AZA-2. In addition, a previously reported AZA with a molecular mass 871 DA could be unambiguously assigned to AZA-11, which is known to be a shellfish metabolite of AZA-2. This is the first report of AZA-11 being also de novo synthetized by dinoflagellates.

Alexandrium ostenfeldii (Paulsen) Balech and Tangen and A. peruvianum (Balech and B.R. Mendiola) Balech and Tangen are morphologically closely related dinoflagellates known to produce potent neurotoxins. Together with Gonyaulax dimorpha Biecheler, they constitute the A. ostenfeldii species complex. Due to the subtle differences in the morphological characters used to differentiate these species, unambiguous species identification has proven problematic. To better understand the species boundaries within the A. ostenfeldii complex we compared rDNA data, morphometric characters and toxin profiles of multiple cultured isolates from different geographic regions. Phylogenetic analysis of rDNA sequences from cultures characterized as A. ostenfeldii or A. peruvianum formed a monophyletic clade consisting of six distinct groups. Each group examined contained strains morphologically identified as either A. ostenfeldii or A. peruvianum. Though key morphological characters were generally found to be highly variable and not consistently distributed, selected plate features and toxin profiles differed significantly among phylogenetic clusters. Additional sequence analyses revealed a lack of compensatory base changes in ITS2 rRNA structure, low to intermediate ITS/5.8S uncorrected genetic distances, and evidence of reticulation. Together these data (criteria currently used for species delineation in dinoflagellates) imply that the A. ostenfeldii complex should be regarded a single genetically structured species until more material and alternative criteria for species delimitation are available. Consequently, we propose that A. peruvianum is a heterotypic synonym of A. ostenfeldii and this taxon name should be discontinued.

Marine biotoxins can be accumulated in shellfish and in turn can lead to severe illness or chronical consequences in human shellfish consumers. The aim of this study was to assess the levels of hydrophilic marine biotoxins in both farmed and wild mussels from the Bulgarian coast sampled in 2017 and to estimate the exposure (acute and chronic) of detected toxins to Bulgarian consumers if investigated mussels were consumed. The hydrophilic toxin - domoic acid was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). Mean domoic acid in whole mussel meat was estimated 0.139 mg/kg which is below the regulatory limit of 20 mg/kg. Among all paralytic shellfish (PSP) toxins evaluated only gonyautoxin-2 (GTX2) was detected via high performance liquid chromatography with fluorescence detection (HPLC-FD). Mean GTX2 level in whole mussel meat was calculated to be 0.151 mg STX.2HCl eq/kg which is far beneath the legislative limit of 800 mg STX.2HCl eq/kg. Estimation of acute exposure for both detected toxins - domoic acid and GTX2 and of chronic exposure to domoic acid showed similar results among male and female, as well as among wild and cultivated mussel consumers. This study showed an overall low contamination level of wild and farmed mussels with hydrophilic marine biotoxins compared to the regulatory limits. This is concluding in low acute and chronic exposure via consumption of contaminated mussels.

The widespread diatom Pseudo-nitzschia can produce domoic acid (DA). DA is a compound with well described neurotoxic effects on vertebrates including humans known as amnesic shellfish poisoning (ASP) syndrome. It has also been suggested to serve as an organic ligand that binds to iron and copper. By binding these trace elements, DA may increase their solubility and bioavailability. In order to serve this function, DA has to be excreted and reabsorbed by the cells. Only few records of dissolved domoic acid (dDA) concentrations in the ocean exist. To accomplish quantification by ultra performance liquid chromatography (UPLC), samples have to be pre-concentrated and desalted using solid-phase extraction, a procedure commonly applied for dissolved organic matter. Our major goals were to quantify dDA in a basin-wide assessment in the East Atlantic Ocean, to determine extraction efficiencies for complexed and uncomplexed dDA, and to assess whether domoic acid is represented by its molecular formula in direct-infusion high resolution mass spectrometry. Our results showed that dDA was extracted almost quantitatively and occurred ubiquitously in the ocean surface but also in deeper (and older) water, indicating surprisingly high stability in seawater. The maximum concentration measured was 173 pmol L−1 and the average molar dDA carbon yield was 7.7 ppm. Both carbon yield and dDA concentration decreased with increasing water depth. Providing quantification of dDA in the water column, we seek to improve our understanding of toxic bloom dynamics and the mechanistic understanding of DA production.

The dinoflagellate genus Azadinium includes species with a plate formula of po, cp, X, 4′, 3a, 6′′, 6C, 5S, 6′′′, 2′′′′ and is part of the family Amphidomataceae with an uncertain order affiliation. Among six species, at least two produce azaspiracids (AZAs), a group of lipophilic toxins that accumulate in shellfish and can cause human health problems. Diversity within the genus might be underestimated at present due to its small size. In the present study, we searched for Azadinium by incubating freshly collected sediments from the Yellow Sea off China and succeeded in detecting a new species, here described as Azadinium dalianense sp. nov. It shared identical hypothecal, cingular and sulcal plates with the other Azadinium species, but it was unique in having only three apical and two anterior intercalary plates. Up to two stalked pyrenoids were present but their location in the cell varied. Phylogenetic analyses based on concatenated small-subunit, partial large-subunit, and internal transcribed spacer ...

The toxigenic genus Alexandrium includes ∼30 species, but information about its biogeography at a regional scale is limited. In this study, we explored the diversity of Alexandrium along the coast of China by incubating resting cysts collected from 7 sites. A total of 231 strains of Alexandrium belonging to 7 morphospecies were found. Among them, Alexandrium andersonii, Alexandrium fraterculum, Alexandrium leei, Alexandrium pseudogonyaulax, and Alexandrium tamutum were recorded from the China Sea for the first time. Partial large subunit (LSU) and/or internal transcribed spacer region (ITS1, ITS2, and 5.8S rDNA) sequences revealed two ribotypes of Alexandrium andersonii, Alexandrium leei, and Alexandrium tamarense: Atama complex Group I and IV. Atama complex Group I was exclusively distributed in the Yellow Sea and the Bohai Sea, whereas Group IV was restricted to the East China Sea and South China Sea. Atama complex Group I produced mainly N-sulfocarbamoyl toxins (C1/C2, 61–79% of total toxins) and gonyautoxins (GTX1/4, 17–37%). Alexandrium ostenfeldii strain ASBH01 produced NEO and STX exclusively (65% and 35%, respectively). Our results support the premise that Atama complex Group I is endemic to the Asian Pacific and includes cold water species, whereas Atama complex Group IV tends to inhabit warmer waters.

From the German Bight along Jutland to the western Skagerrak, we found representatives of almost all groups of phycotoxins known to occur in North Sea plankton. Identification was by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in plankton size fractions, with domoic acid and 20-me G the most abundant toxins. The dominance of 20-me G in the spirolide (SPX) composition of plankton from the Jutland current system matched very well with that of an isolate of the dinoflagellate Alexandrium ostenfeldii. The SPXs of the A. ostenfeldii strain S6_P12_E11, previously isolated from the western North Sea along the Scottish coast, comprised 100% 20-me G, suggesting toxin homogeneity among North Sea populations of this species. We detected highest amounts of azaspiracid-1 in the 3–20-mm size fraction at offshore stations, where the Jutland coastal current converges with the westward North Sea flow off Skagerrak. Azadinium spinosum was subsequently identified by clonal isolation from crude cultures established from these stations. Except for lipophilic toxins usually produced by the dinoflagellate Dinophysis spp., dinophysistoxin-1 (DTX-1) and DTX-2, we detected no other phycotoxins in plankton from the southern German Bight. The spatial distribution of the phycotoxins in the eastern North Sea was apparently related to the hydrographical conditions, identified from salinity and coloured dissolved organic matter profiles. The biogeographical distribution of phycotoxins indicates a strong association with the northward advection by the Jutland current and the mixing of German Bight and North water masses along the northwest Danish coast towards the Skagerrak.

Dinoflagellates of the Alexandrium ostenfeldii complex (A. ostenfeldii, A. peruvianum) are capable of producing different types of neurotoxins: paralytic shellfish toxins (PSTs), spirolides and gymnodimines, depending on the strain and its geographic origin. While Atlantic and Mediterranean strains have been reported to produce spirolides, strains originating from the brackish Baltic Sea produce PSTs. Some North Sea, USA and New Zealand strains contain both toxins. Causes for such intraspecific variability in toxin production are unknown. We investigated whether salinity affects toxin production and growth rate of 5 A. ostenfeldii/peruvianum strains with brackish water (Baltic Sea) or oceanic (NE Atlantic) origin. The strains were grown until stationary phase at 7 salinities (6–35), and their growth and toxin production was monitored. Presence of saxitoxin (STX) genes (sxtA1 and sxtA4 motifs) in each strain was also analyzed. Salinity significantly affected both growth rate and toxicity of the individual strains but did not change their major toxin profile. The two Baltic Sea strains exhibited growth at salinities 6–25 and consistently produced gonyautoxin (GTX) 2, GTX3 and STX. The two North Sea strains grew at salinities 20–35 and produced mainly 20-methyl spirolide G (20mG), whereas the strain originating from the northern coast of Ireland was able to grow at salinities 15–35, only producing 13-desmethyl spirolide C (13dmC). The effects of salinity on total cellular toxin concentration and distribution of toxin analogs were strain-specific. Both saxitoxin gene motifs were present in the Baltic Sea strains, whereas the 2 North Sea strains lacked sxtA4, and the Irish strain lacked both motifs. Thus sxtA4 only seems to be specific for PST producing strains. The results show that toxin profiles of A. ostenfeldii/peruvianum strains are predetermined and the production of either spirolides or PSTs cannot be induced by salinity changes. However, changes in salinity may lead to changed growth rates, total cellular toxin concentrations as well as relative distribution of the different PST and spirolide analogs, thus affecting the actual toxicity of A. ostenfeldii/peruvianum populations.

Diarrhetic shellfish poisoning (DSP) toxins constitute a severe economic threat to shellfish industries and a major food safety issue for shellfish consumers. The prime producers of the DSP toxins that end up in filter feeding shellfish are species of the marine mixotrophic dinoflagellate genus Dinophysis. Intraspecific toxin contents of Dinophysis spp. vary a lot, but the regulating factors of toxin content are still poorly understood. Dinophysis spp. have been shown to sequester and use chloroplasts from their ciliate prey, and with this rare mode of nutrition, irradiance and food availability could play a key role in the regulation of toxins contents and production. We investigated toxin contents, production and excretion of a Dinophysis acuta culture under different irradiances, food availabilities and growth phases. The newly isolated strain of D. acuta contained okadaic acid (OA), pectenotoxins-2 (PTX-2) and a novel dinophysistoxin (DTX) that we tentatively describe as DTX-1b isomer. We found that all three toxins were excreted to the surrounding seawater, and for OA and DTX-1b as much as 90% could be found in extracellular toxin pools. For PTX-2 somewhat less was excreted, but often >50% was found extracellularly. This was the case both in steady-state exponential growth and in food limited, stationary growth, and we emphasize the need to include extracellular toxins in future studies of DSP toxins. Cellular toxin contents were largely unaffected by irradiance, but toxins accumulated both intra- and extracellularly when starvation reduced growth rates of D. acuta. Toxin production rates were highest during exponential growth, but continued at decreased rates when cell division ceased, indicating that toxin production is not directly associated with ingestion of prey. Finally, we explore the potential of these new discoveries to shed light on the ecological role of DSP toxins.

Azadinium poporum is a small dinoflagellate from the family Amphidomataceae which is known for the production potential of azaspiracid toxins. A. poporum has been recorded from European and Korean waters. Here we present the first report of its occurrence along the coast of China. Morphology of Chinese A. poporum is similar to those from Europe and Korea. Several stalked pyrenoids surrounded by a starch sheath were revealed with light microscopy and confirmed by transmission electron microscopy. Among 25 strains from the China Sea we identified two distinct ribotypes (referred to as ribotypes B and C). ITS sequences of strains within the same ribotype are identical, whereas ribotype B and C differ from each other at 11 positions (98.3% similarity). A. poporum ribotypes B and C type differ from European strains (referred to as ribotype A) at 16 and 15 positions (97.5% and 97.7% similarity). The ITS region pairwise distance within A. poporum ranged from 0.017 to 0.022. Among all three ribotypes, no hemi-compensatory based changes were found within helix III of ITS indicating that they are conspecific. Azaspiracid profiles were analyzed for six strains and turned out to be unexpectedly diverse. Whereas no AZAs could be detected for one strain, another strain was found to contain a m/z 348 fragment type AZA previously found in a Korean Isolate and traces of two other unknown AZAs of higher masses. A third strain produced a novel AZA with a molecular mass of 871 Da. Three strains were found to contain considerable amounts of toxic AZA-2 as the sole AZA, a finding that might elegantly explain the detection of AZA-2 in sponges in the Sea of Japan and which underline the risk potential of A. poporum blooms with subsequent shellfish intoxication episodes for the Asian Pacific.

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 160 mg AZA-1 eq. kg�1 (reaching maximum levels of >500 mg AZA- 1 eq. kg�1 in Chamelea gallina and >250 mg 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 easurements 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.

Harmful Algal Bloom (HAB) surveillance is complicated by high diversity of species and associated phycotoxins. Such species-level information on taxonomic affiliations and on cell abundance and toxin content is, however, crucial for effective monitoring, especially of aquaculture and fisheries areas. The aim addressed in this study was to determine putative HAB taxa and related phycotoxins in plankton from aquaculture sites in the Ebro Delta, NW Mediterranean. The comparative geographical distribution of potentially harmful plankton taxa was established by weekly field sampling throughout the water column during late spring–early summer over two years at key stations in Alfacs and Fangar embayments within the Ebro Delta. Core results included not only confirmed identification of HAB taxa that are common for the time period and geographical area, but also provided evidence of potentially new taxa. At least 25 HAB taxa were identified to species level, and an additional six genera were confirmed, by morphological criteria under light microscopy and/or by molecular genetics approaches involving qPCR and next generation DNA pyrosequencing. In particular, new insights were gained by the inclusion of molecular techniques, which focused attention on the HAB genera Alexandrium, Karlodinium, and Pseudo-nitzschia. Noteworthy is the discovery of Azadinium sp., a potentially new HAB species for this area, and Gymnodinium catenatum or Gymnodinium impudicum by means of light microscopy. In addition, significant amounts of the neurotoxin domoic acid (DA) were found for the first time in phytoplankton samples in the Ebro Delta. While the presence of the known DA-producing diatom genus Pseudo-nitzschia was confirmed in corresponding samples, the maximal toxin concentration did not coincide with highest cell abundances of the genus and the responsible species could not be identified. Combined findings of microscopic and molecular detection approaches underline the need for a synoptic strategy for HAB monitoring, which integrates the respective advantages and compensates for limitations of individual methods.

Okadaic acid (OA), dinophysistoxins (DTX) and pectenotoxins (PTX) produced by the dinoflagellates Dinophysis spp. can accumulate in shellfish and cause diarrhetic shellfish poisoning upon human consumption. Shellfish toxicity is a result of algal abundance and toxicity as well as accumulation and depuration kinetics in mussels. We mass-cultured Dinophysis acuta containing OA, DTX-1b and PTX-2 and fed it to the blue mussel, Mytilus edulis under controlled laboratory conditions for a week to study toxin accumulation and transformation. Contents of OA and DTX-1b in mussels increased linearly with incubation time, and the net toxin accumulation was 66% and 71% for OA and DTX-1b, respectively. Large proportions (≈50%) of both these toxins were transformed to fatty acid esters. Most PTX-2 was transformed to PTX-2 seco-acid and net accumulation was initially high, but decreased progressively throughout the experiment, likely due to esterification and loss of detectability. We also quantified depuration during the subsequent four days and found half-life times of 5-6 days for OA and DTX-1b. Measurements of dissolved toxins revealed that depuration was achieved through excreting rather than metabolizing toxins. This is the first study to construct a full mass balance of DSP toxins during both accumulation and depuration, and we demonstrate rapid toxin accumulation in mussels at realistic in situ levels of Dinophysis. Applying the observed accumulation and depuration kinetics, we model mussel toxicity, and demonstrate that a concentration of only 75 Dinophysis cells l(-1) is enough to make 60 mm long mussels exceed the regulatory threshold for OA equivalents.

Alexandrium ostenfeldii is among the most intensely studied marine planktonic dinophytes and in the last few years blooms have become a recurrent phenomenon mainly in brackish coastal waters. Since 2012, A. ostenfeldii recurs annually in the Ouwerkerkse Kreek, a Dutch brackish water creek discharging into an estuary with large stocks of mussels, oysters and cockles. The creek is characterized by highly dynamic abiotic conditions, notably salinity. Here, we investigated the impacts of salinities ranging from 3 to 34 on growth and toxin content of an A. ostenfeldii isolate from the creek. Our results demonstrate a broad salinity tolerance of the Dutch A. ostenfeldii population, with growth rates from 0.13 to 0.2 d−1 over a salinity range from 6 to 34. Highest paralytic shellfish toxin and cyclic imine toxin cell quotas were observed for the lowest and highest salinities, and were associated with increases in cell size. Lytic activity was highest at the lowest salinity, and was 5-fold higher in the cell-free supernatants compared to cell extracts. Together our results demonstrate a tight coupling between salinity and A. ostenfeldii growth rate, cell size and toxin synthesis, which may have consequences for the seasonal dynamics of bloom toxicity.

Microcystis are known for their potential ability to synthesize toxins, mainly microcystins (MCs). In order to evaluate the effects of temperature on chlorophyll a (Chl a), growth, physiological responses and toxin production of a nativeMicrocystis aeruginosa, we exposed the cells to low(23 °C) and high (29 °C) temperature in addition to a 26 °C control treatment. Exponential growth ratewas significantly higher at 29 °C compared to 23 °C and control, reaching 0.43, 0.32 and 0.33 day−1 respectively. In addition, there was a delay of the start of exponential growth at 23 °C. However, the intracellular concentration of Chl a decreased significantly due to temperature change. A significant increase in intracellular ROS was observed in coincidencewith the activation of enzymatic antioxidant catalase (CAT) during the first two days of exposure to 23° and 29 °C in comparison to the control experiment, decreasing thereafter to nearly initial values. Five MCs were determined by LC-MS/MS analysis. In the experiments, the highest MC concentration, 205 fg [Leu1] MC-LR.cell−1 expressed as MC-LR equivalent was measured in the beginning of the experiment and subsequently declined to 160 fg.cell−1 on day 2 and 70 fg.cell−1 on day 4 in cells exposed to 29 °C. The same trend was observed for all other MCs except for the least abundant MC-LR which showed a continuous increase during exposure time. Our results suggest a high ability of M. aeruginosa to perceive ROS and to rapidly initiate antioxidant defenses with a differential response on MC production., Facultad de Ciencias Exactas

This study investigates whether feeding on the domoic acid (DA)-producing diatom Pseudo-nitzschia seriata affects the faecal pellet (FP) production (proxy for grazing) and fecundity of Calanus finmarchicus and Calanus glacialis. Female copepods were fed a saturating concentration of food (400 µg C L−1) in two combinations (i) natural phytoplankton spiked with 50% P. seriata and (ii) only the non-toxic phytoplankton community. The copepods ingested DA, as illustrated by DA accumulation in their FPs, and transferred a share of DA to their eggs and body tissue. DA was mainly excreted through FPs in both species. Compared with C. finmarchicus, C. glacialis accumulated substantially more DA in its body tissue. For both species, egg production and hatching success were unaffected by grazing on the toxic diatom. This suggests that feeding on DA-containing P. seriata does not affect copepod fecundity, despite increasing DA concentrations of P. seriata during the experiment.

Dinophysis is a cosmopolitan genus of marine dinoflagellates, considered as the major proximal source of diarrheic shellfish toxins and the only producer of pectenotoxins (PTX). From three oceanographic expeditions carried out during autumn, spring and late summer along the Argentine Sea (∼38–56°S), lipophilic phycotoxins were determined by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) in size-fractionated plankton samples. Lipophilic toxin profiles were associated with species composition by microscopic analyses of toxigenic phytoplankton. Pectenotoxin-2 and PTX-11 were frequently found together with the presence of Dinophysis acuminata and Dinophysis tripos. By contrast, okadaic acid was rarely detected and only in trace concentrations, and dinophysistoxins were not found. The clear predominance of PTX over other lipophilic toxins in Dinophysis species from the Argentine Sea is in accordance with previous results obtained from north Patagonian Gulfs of the Argentine Sea, and from coastal waters of New Zealand, Chile, Denmark and United States. Dinophysis caudata was rarely found and it was confined to the north of the sampling area. Because of low cell densities, neither D. caudata nor Dinophysis norvegica could be biogeographically related to lipophilic toxins in this study. Nevertheless, the current identification of D. norvegica in the southern Argentine Sea is the first record for the southwestern Atlantic Ocean. Given the typical toxigenicity of this species on a global scale, this represents an important finding for future surveillance of plankton-toxin associations., Facultad de Ciencias Naturales y Museo

Diarrhetic shellfish poisoning (DSP) toxins constitute a severe health risk to shellfish consumers and a serious economic risk to the shellfish industry. The most prominent producers of DSP toxins are the mixotrophic dinoflagellates Dinophysis spp., which rely on chloroplasts from their prey, the ciliate Mesodinium rubrum; i.e. they are kleptoplastidic. Studies based on field col- lected material have indicated that cellular toxin contents in Dinophysis spp. vary intraspecifically by several orders of magnitude, but the regulating factors of cellular toxin contents are still poorly understood. We isolated 7 strains of D. acuminata, all of which produced pectenotoxin-2 (PTX-2) in the range of 12.7 to 35.6 pg cell �1 when supplied with abundant nutrients, light and food. One strain was selected for detailed studies on the effects of irradiance and food availability on pho- toacclimation, photosynthesis, growth and toxin production. Photoacclimation was detected for the first time in this genus, indicating some genetic control of sequestered chloroplasts. Growth and photosynthesis increased 4-fold with irradiances from 7 to 130 µmol photons m �2 s �1 after a minimum of 2 wk under ample food conditions. Despite this, cellular PTX-2 content increased only slightly from (mean ±SE) 4.6 ± 0.3 to 7.8 ± 1.1 pg PTX-2 cell �1 . Toxin production continued for up to 30 d after food was depleted, leading to a build-up of toxin in D. acuminata cells to 24.7 ± 1.7 pg PTX-2 cell �1 . Thus, we demonstrate that PTX-2 content is not directly coupled to irradiance and that PTX-2 production is not directly associated with ingestion of ciliate prey. Intraspecific varia- tions in toxicity are instead most likely governed primarily by growth phase and genetic differ- ences between different populations of Dinophysis spp.

Azadinium is a dinophycean genus capable of producing azaspiracids (AZAs), a recently discovered group of lipophilic phycotoxins causing human intoxication via mussel consumption. Although initially described from the North Sea, the genus currently consisting of four described species is probably distributed worldwide. Here we report on Azadinium from the Shetland Islands, which are located in the northernmost part of the North Sea and are largely influenced by the Atlantic Ocean. Two strains of Azadinium were isolated from a single water sample. One strain was identified as Azadinium spinosum based on morphology and sequence data and had an AZA cell quota of about 20 fg per cell, similar to all other described strains of the species. The toxin profile consisted of AZA-1 and AZA-2 in a 2.3:1 ratio and a yet undescribed AZA of 715 Da. The other strain represents a new species and is here described as Azadinium polongum sp. nov. Like A. spinosum, but different to Azadinium obesum and Azadinium poporum, A. polongum has an antapical spine. A. polongum differs from A. spinosum by an elongated shape of the pore plate (Po), and X-plate, the location of the ventral pore, and the absence of a distinct pyrenoid with starch sheath. Molecular analysis based on SSU, LSU, and ITS sequencing supported separation of A. polongum at the species level. Detailed LC-MS analysis showed that A. polongum does not produce any known AZAs in measureable amounts.

The variability of toxigenic phytoplankton and the consequent uptake and loss of toxins by the mussel Choromytilus meridionalis was investigated in the southern Benguela at the event scale (3–10 days) in response to the upwelling–downwelling cycle. Phytoplankton and mussel samples were collected daily (20 March–11 April 2007) from a mooring station (32.04°S; 18.26°E) located 3.5 km offshore of Lambert's Bay, within the St Helena Bay region. Rapid changes in phytoplankton assemblages incorporated three groups of toxigenic phytoplankton: (1) the dinoflagellate Alexandrium catenella ; (2) several species of Dinophysis , including Dinophysis acuminata , Dinophysis fortii , Dinophysis hastata and Dinophysis rotundata ; and (3) members of the diatom genus Pseudo-nitzschia . Analysis of phytoplankton concentrates by LC–MS/MS or LC-FD provided information on the toxin composition and calculated toxicity of each group. Several additional in vitro assays were used for the analysis of toxins in mussels (ELISA, RBA, MBA for PSP toxins; and ELISA for DSP toxins). Good correspondence was observed between methods except for the MBA, which provided significantly lower (approximately 2-fold) estimates of PSP toxins. PSP and DSP toxins both exceeded the regulatory limits in Choromytilis meridionalis , but ASP toxins were undetected. Differences were observed in the composition of both PSP and DSP toxins in C. meridionalis from that of the ingested dinoflagellates (PSP toxins showed an increase in STX, C1,2, and traces of dcSTX and GTX1,4 and a decrease in NEO; DSP toxins showed an increased in DTX1, and traces of PTX2sa, and a decrease in OA). The rate of loss of PSP toxins following dispersal of the A. catenella boom was 0.12 d −1 . Variation in the loss rates of different PSP toxins contributed to the change in toxin profile in C. meridionalis . Prediction of net toxicity in shellfish of the nearshore environment in the southern Benguela is limited due to rapid phytoplankton community changes, high variability in cellular toxicity, and the selective uptake and loss of toxins, and/or transformation of toxins.

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).

Toxic algae such as Alexandrium and Azadinium have an important ecological impact and have originated several times independently within the dinophytes. Their closest relatives, however, are mostly unknown at present. A new dinophyte species, Amphidoma languida sp. nov., was isolated from Bantry Bay (Ireland) during a period of elevated azaspiracid toxicity in mussels. The new species was described in detail, and its phylogenetic position was analysed, by using a combination of light and electron microscopy, chemical detection methods, and sequence comparison of concatenated ribosomal RNA sequence data. Morphological similarities, such as cingular and hypothecal plates, the number and arrangement of sulcal plates, and the characteristic apical pore complex with a small X-plate centrally invading the first apical plate, indicated a close relationship between Amphidoma and Azadinium. However, no known azaspiracid analogues were detected in A. languida by liquid chromatography coupled with tandem mass-spectrometry. In a molecular phylogeny, the Amphidomataceae including Amphidoma and Azadinium were an independent lineage among other monophyletic major groups of the dinophytes such as the Suessiales, Prorocentrales, Gonyaulacales, and Peridiniales. Thus, the taxonomic affiliation of Azadinium is clarified, and our data may prove helpful in the development of specific and reliable molecular detection methods of toxic Azadinium.

Detection of paralytic shellfish poisoning (PSP) toxins in scallops from the west coast of Greenland exceeding the 800 μg toxin/kg shellfish limit led to an investigation with the aim of finding the responsible organism(s). Three strains of Alexandrium Halim were established from single cell isolations. Morphological identification of the strains and determination of their position within the genus by LSU rDNA sequences was carried out. Light microscopy revealed that the three strains was of the Alexandrium tamarense morphotype, and bayesian and neighbor-joining analyses of the LSU rDNA sequences placed them within Group I of the A. tamarense species complex. The toxicity and toxin profiles of the strains were measured by liquid chromatography fluorescence detection (LC-FD) and their identity was confirmed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The three strains all turned out to be toxic and all produced large proportions (>60% total mol) of gonyautoxins 1 and 4 (GTX1/GTX4). This is the first record of saxitoxin producers from western Greenland. The toxin profiles were atypical for A. tamarense in their absence of N-sulfocarbanoyl C1/C2 or B1/B2 toxins. Rather the high molar percentage of GTX1/GTX4, the lesser amounts of only carbamoyl toxins and the absence of decarbamoyl derivatives are more characteristic features of A. minutum strains. This may indicate that the genetically determined toxin profiles in Alexandrium species are more complex than previously appreciated.

Certain strains of the toxigenic dinoflagellate Alexandrium tamarense produce potent allelochemicals with lytic activity against a wide variety of marine microorganisms. Our efforts to characterize these allelochemicals from a lytic strain focused on the less polar components because of their higher lytic activity. Fractionation and partial purification after solid phase extraction (SPE) were achieved via alternative chromatographic methods, namely HPLC separation on C8 and HILIC phases. Through MALDI-TOF mass spectrometry we compared the mass differences in SPE, C8 HPLC, and HILIC fractions between a lytic and non-lytic strain of A. tamarense. Several large species with masses between 7 kDa and 15 kDa were found in the HILIC lytic fraction by MALDI-TOF MS. Tryptic digestion and tryptic digestioncoupled size-exclusion chromatography (SEC) suggested that the lytic compounds are large nonproteinaceous molecules (

Phycotoxin distribution and abundance was determined during an oceanographic expedition along a latitudinal transect of 27° extent in the southeastern Pacific Ocean, from the fjords of Tierra del Fuego Island to offshore Copiapó in the Atacama region along the Chilean coast. Plankton samples were harvested at regular intervals during the entire cruise and later analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for domoic acid (DA) and lipophilic toxins. Although no evident toxic algal bloom was encountered during this transect, several phycotoxin analogues from distinct toxin groups were detected. These phycotoxins included DA, the pectenotoxins PTX-2, PTX-2sa and PTX-11, dinophystoxin-1 (DTX-1) and gymnodimine (GYM), which is the first report of this latter toxin in the southeast Pacific. A region-specific and rather disjunct distribution of GYM, DA and DTX-1 was observed, whereas PTX-2, PTX-2sa and PTX-11 were more widely distributed over almost the entire transect. This work represents the first assessment of lipophilic toxins through a wide latitudinal transect of the southeastern Pacific, revealing a patchy distribution of several phycotoxins and pointing out the specific geographical distribution of the putative toxigenic organisms.

Certain allelochemicals of the marine dinoflagellate Alexandrium tamarense cause lysis of a broad spectrum of target protist cells but the lytic mechanism is poorly defined. We first hypothesized that membrane sterols serve as molecular targets of these lytic compounds, and that differences in sterol composition among donor and target cells may cause insensitivity of Alexandrium and sensitivity of targets to lytic compounds. We investigated Ca 2+ influx after application of lytic fractions to a model cell line PC12 derived from a pheochromocytoma of the rat adrenal medulla to establish how the lytic compounds affect ion flux associated with lysis of target membranes. The lytic compounds increased permeability of the cell membrane for Ca 2+ ions even during blockade of Ca 2+ channels with cadmium. Results of a liposome assay suggested that the lytic compounds did not lyse such target membranes non-specifically by means of detergent-like activity. Analysis of sterol composition of isolates of A. tamarense and of five target protistan species showed that both lytic and non-lytic A. tamarense strains contain cholesterol and dinosterol as major sterols, whereas none of the other tested species contain dinosterol. Adding sterols and phosphatidylcholine to a lysis bioassay with the cryptophyte Rhodomonas salina for evaluation of competitive binding indicated that the lytic compounds possessed apparent high affinity for free sterols and phosphatidylcholine. Lysis of protistan target cells was dose-dependently reduced by adding various sterols or phosphatidylcholine. For three tested sterols, the lytic compounds showed highest affinity towards cholesterol followed by ergosterol and brassicasterol. Cholesterol comprised a higher percentage of total sterols in plasma membrane fractions of A. tamarense than in corresponding whole cell fractions. We conclude therefore that although the molecular targets of the lytic compounds are likely to involve sterol components of membranes, A. tamarense must have a complex self-protective mechanism that still needs to be addressed.

Diarrhetic shellfish poisoning (DSP) poses a significant threat to the safe consumption of shellfish in the southern Benguela ecosystem. The accumulation of DSP toxins was investigated in two cultivated bivalve species, the Pacific oyster Crassostrea gigas and the mussel Choromytilus meridionalis, suspended from a mooring located off Lambert’s Bay on the west coast of South Africa. The dinoflagellate Dinophysis acuminata, a known source of polyether toxins associated with DSP, was common through most of the study period. The toxin composition of the dinoflagellate was dominated by okadaic acid (OA) (91%), with lesser quantities of the dinophysistoxin DTX-1 (6.5%) and pectenotoxin PTX-2 (2.4%), and traces of PTX-2sa and PTX-11. The mean cell toxin quota ofD. acuminata was 7.8 pg OA cell–1. The toxin profile in shellfish was characterised by a notably higher relative content of DTX-1. The study showed the average concentration of DSP toxins in the mussels to exceed that in the oysters by approximately 20-fold. The results indicate a need to establish species-specific sampling frequencies in shellfish safety monitoring programmes. Keywords: Benguela Current, Dinophysis acuminata, DSP toxins, LC-MS/MS, shellfish toxicityAfrican Journal of Marine Science 2011, 33(2): 273–281