Tissue Distribution and Metabolization of Ciguatoxins in an Herbivorous Fish following Experimental Dietary Exposure to Gambierdiscus polynesiensis.

Ciguatoxins (CTXs), potent neurotoxins produced by dinoflagellates of the genera Gambierdiscus and Fukuyoa, accumulate in commonly consumed fish species, causing human ciguatera poisoning. Field collections of Pacific reef fish reveal that consumed CTXs undergo oxidative biotransformations, resulting in numerous, often toxified analogs. Following our study showing rapid CTX accumulation in flesh of an herbivorous fish, we used the same laboratory model to examine the tissue distribution and metabolization of Pacific CTXs following long-term dietary exposure. Naso brevirostris consumed cells of Gambierdiscus polynesiensis in a gel food matrix over 16 weeks at a constant dose rate of 0.36 ng CTX3C equiv g-1 fish d-1. CTX toxicity determination of fish tissues showed CTX activity in all tissues of exposed fish (eight tissues plus the carcass), with the highest concentrations in the spleen. Muscle tissue retained the largest proportion of CTXs, with 44% of the total tissue burden. Moreover, relative to our previous study, we found that larger fish with slower growth rates assimilated a higher proportion of ingested toxin in their flesh (13% vs. 2%). Analysis of muscle extracts revealed the presence of CTX3C and CTX3B as well as a biotransformed product showing the m/z transitions of 2,3-dihydroxyCTX3C. This is the first experimental evidence of oxidative transformation of an algal CTX in a model consumer and known vector of CTX into the fish food web. These findings that the flesh intended for human consumption carries the majority of the toxin load, and that growth rates can influence the relationship between exposure and accumulation, have significant implications in risk assessment and the development of regulatory measures aimed at ensuring seafood safety.

Comparative Study on the Performance of Three Detection Methods for the Quantification of Pacific Ciguatoxins in French Polynesian Strains of Gambierdiscus polynesiensis.

Gambierdiscus and Fukuyoa dinoflagellates produce a suite of secondary metabolites, including ciguatoxins (CTXs), which bioaccumulate and are further biotransformed in fish and marine invertebrates, causing ciguatera poisoning when consumed by humans. This study is the first to compare the performance of the fluorescent receptor binding assay (fRBA), neuroblastoma cell-based assay (CBA-N2a), and liquid chromatography tandem mass spectrometry (LC-MS/MS) for the quantitative estimation of CTX contents in 30 samples, obtained from four French Polynesian strains of Gambierdiscus polynesiensis. fRBA was applied to Gambierdiscus matrix for the first time, and several parameters of the fRBA protocol were refined. Following liquid/liquid partitioning to separate CTXs from other algal compounds, the variability of CTX contents was estimated using these three methods in three independent experiments. All three assays were significantly correlated with each other, with the highest correlation coefficient (r2 = 0.841) found between fRBA and LC-MS/MS. The CBA-N2a was more sensitive than LC-MS/MS and fRBA, with all assays showing good repeatability. The combined use of fRBA and/or CBA-N2a for screening purposes and LC-MS/MS for confirmation purposes allows for efficient CTX evaluation in Gambierdiscus. These findings, which support future collaborative studies for the inter-laboratory validation of CTX detection methods, will help improve ciguatera risk assessment and management.

Occurrence of three dominant epibenthic dinoflagellates (Ostreopsis spp., Coolia monotis and Prorocentrum lima) in relation to biotic substrates and environmental factors in a highly dynamic ecosystem, the Strait of Gibraltar (Southwestern Mediterranean).

No studies have been carried out on the benthic harmful algal blooms (BHABs) along the Strait of Gibraltar in the Mediterranean, and little is known about the diversity of blooming species. Here, epibenthic dinoflagellates were monitored at least biweekly over 18 months (May 2019-November 2020) in Oued Lihoud, Cap Malabata and Dalia on the thalli of five dominant macrophytes and in the water column. This is the first report on the seasonal distribution of BHAB species hosted by natural biotic substrates in the Strait of Gibraltar, which is known for high hydrodynamics, major entry of Atlantic waters and important maritime traffic. Three BHAB dinoflagellates were observed in the surveyed areas: Ostreopsis spp., Coolia monotis and Prorocentrum lima. The analysis of all data at the three sites showed that Dictyota dichotoma was the most favourable macroalgae host for these benthic dinoflagellates. The highest cell densities were observed in Cap Malabata for Ostreopsis spp. (2.7 × 105 cells/g fresh weight in September 2020), P. lima (4.57 × 104 cells/g FW in September 2020) and C. monotis (4.07 × 104 cells/g FW in June 2019). Phosphate and temperature were positively correlated to the abundances of the studied thermophilic BHAB species. In contrast, negative correlations were recorded with salinity, ammonium, nitrite, nitrate, DIN, nitrogen/phosphate ratio and suspended material, attesting of the complex relationships between environmental factors and BHAB species dynamic in each marine ecosystem. Toxin analyses of the natural phytoplankton assemblage during BHABs showed the presence of only lipophilic toxins, namely okadaic acid and dinophysistoxins produced by P. lima. These BHABs species have to be isolated to establish monoclonal cultures for ribotyping and ecophysiological investigations.

Toxicity Screening of a Gambierdiscus australes Strain from the Western Mediterranean Sea and Identification of a Novel Maitotoxin Analogue.

Dinoflagellate species of the genera Gambierdiscus and Fukuyoa are known to produce ciguatera poisoning-associated toxic compounds, such as ciguatoxins, or other toxins, such as maitotoxins. However, many species and strains remain poorly characterized in areas where they were recently identified, such as the western Mediterranean Sea. In previous studies carried out by our research group, a G. australes strain from the Balearic Islands (Mediterranean Sea) presenting MTX-like activity was characterized by LC-MS/MS and LC-HRMS detecting 44-methyl gambierone and gambieric acids C and D. However, MTX1, which is typically found in some G. australes strains from the Pacific Ocean, was not detected. Therefore, this study focuses on the identification of the compound responsible for the MTX-like toxicity in this strain. The G. australes strain was characterized not only using LC-MS instruments but also N2a-guided HPLC fractionation. Following this approach, several toxic compounds were identified in three fractions by LC-MS/MS and HRMS. A novel MTX analogue, named MTX5, was identified in the most toxic fraction, and 44-methyl gambierone and gambieric acids C and D contributed to the toxicity observed in other fractions of this strain. Thus, G. australes from the Mediterranean Sea produces MTX5 instead of MTX1 in contrast to some strains of the same species from the Pacific Ocean. No CTX precursors were detected, reinforcing the complexity of the identification of CTXs precursors in these regions.

Sulfo-Gambierones, Two New Analogs of Gambierone Produced by Gambierdiscus excentricus.

Ciguatera poisoning is caused by the ingestion of fish or shellfish contaminated with ciguatoxins produced by dinoflagellate species belonging to the genera Gambierdiscus and Fukuyoa. Unlike in the Pacific region, the species producing ciguatoxins in the Atlantic Ocean have yet to be definitely identified, though some ciguatoxins responsible for ciguatera have been reported from fish. Previous studies investigating the ciguatoxin-like toxicity of Atlantic Gambierdiscus species using Neuro2a cell-based assay identified G. excentricus as a potential toxin producer. To more rigorously characterize the toxin profile produced by this species, a purified extract from 124 million cells was prepared and partial characterization by high-resolution mass spectrometry was performed. The analysis revealed two new analogs of the polyether gambierone: sulfo-gambierone and dihydro-sulfo-gambierone. Algal ciguatoxins were not identified. The very low ciguatoxin-like toxicity of the two new analogs obtained by the Neuro2a cell-based assay suggests they are not responsible for the relatively high toxicity previously observed when using fractionated G. excentricus extracts, and are unlikely the cause of ciguatera in the region. These compounds, however, can be useful as biomarkers of the presence of G. excentricus due to their sensitive detection by mass spectrometry.

Deep-Water Fish Are Potential Vectors of Ciguatera Poisoning in the Gambier Islands, French Polynesia.

Ciguatera poisoning (CP) cases linked to the consumption of deep-water fish occurred in 2003 in the Gambier Islands (French Polynesia). In 2004, on the request of two local fishermen, the presence of ciguatoxins (CTXs) was examined in part of their fish catches, i.e., 22 specimens representing five deep-water fish species. Using the radioactive receptor binding assay (rRBA) and mouse bioassay (MBA), significant CTX levels were detected in seven deep-water specimens in Lutjanidae, Serranidae, and Bramidae families. Following additional purification steps on the remaining liposoluble fractions for 13 of these samples (kept at -20 °C), these latter were reanalyzed in 2018 with improved protocols of the neuroblastoma cell-based assay (CBA-N2a) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Using the CBA-N2a, the highest CTX-like content found in a specimen of Eumegistus illustris (Bramidae) was 2.94 ± 0.27 µg CTX1B eq. kg-1. Its toxin profile consisted of 52-epi-54-deoxyCTX1B, CTX1B, and 54-deoxyCTX1B, as assessed by LC-MS/MS. This is the first study demonstrating that deep-water fish are potential ciguatera vectors and highlighting the importance of a systematic monitoring of CTXs in all exploited fish species, especially in ciguatera hotspots, including deep-water fish, which constitute a significant portion of the commercial deep-sea fisheries in many Asian-Pacific countries.

Characterization of maitotoxin-4 (MTX4) using electrospray positive mode ionization high-resolution mass spectrometry and UV spectroscopy.

RATIONALE: The dinoflagellate genera Gambierdiscus and Fukuyoa are producers of toxins responsible for Ciguatera Poisoning (CP). Although having very low oral potency, maitotoxins (MTXs) are very toxic following intraperitoneal injection and feeding studies have shown they may accumulate in fish muscle. To date, six MTX congeners have been described but two congeners (MTX2 and MTX4) have not yet been structurally elucidated. The aim of the present study was to further characterize MTX4. METHODS: Chemical analysis was performed using liquid chromatography coupled to a diode-array detector (DAD) and positive ion mode high-resolution mass spectrometry (LC/HRMS) on partially purified extracts of G. excentricus (strain VGO792). HRMS/MS studies were also carried out to tentatively explain the fragmentation pathways of MTX and MTX4. RESULTS: The comparison of UV and HRMS (ESI+ ) spectra between MTX and MTX4 led us to propose the elemental formula of MTX4 (C157 H241 NO68 S2 , as the unsalted molecule). The comparison of the theoretical and measured m/z values of the doubly charged ions of the isotopic profile in ESI+ were coherent with the proposed elemental formula of MTX4. The study of HRMS/MS spectra on the tri-ammoniated adduct ([M - H + 3NH4 ]2+ ) of both molecules gave additional information about structural features. The cleavage observed, probably located at C99 -C100 in both MTX and MTX4, highlighted the same A-side product ion shared by the two molecules. CONCLUSIONS: All these investigations on the characterization of MTX4 contribute to highlighting that MTX4 belongs to the same structural family of MTXs. However, to accomplish a complete structural elucidation of MTX4, an NMR-based study and LC/HRMSn investigation will have to be carried out.

Chemically mediated interactions between Microcystis and Planktothrix: impact on their growth, morphology and metabolic profiles.

Freshwater cyanobacteria are known for their ability to produce bioactive compounds, some of which have been described as allelochemicals. Using a combined approach of co-cultures and analyses of metabolic profiles, we investigated chemically mediated interactions between two cyanobacterial strains, Microcystis aeruginosa PCC 7806 and Planktothrix agardhii PCC 7805. More precisely, we evaluated changes in growth, morphology and metabolite production and release by both interacting species. Co-culture of Microcystis with Planktothrix resulted in a reduction of the growth of Planktothrix together with a decrease of its trichome size and alterations in the morphology of its cells. The production of intracellular compounds by Planktothrix showed a slight decrease between monoculture and co-culture conditions. Concerning Microcystis, the number of intracellular compounds was higher under co-culture condition than under monoculture. Overall, Microcystis produced a lower number of intracellular compounds under monoculture than Planktothrix, and a higher number of intracellular compounds than Planktothrix under co-culture condition. Our investigation did not allow us to identify specifically the compounds causing the observed physiological and morphological changes of Planktothrix cells. However, altogether, these results suggest that co-culture induces specific compounds as a response by Microcystis to the presence of Planktothrix. Further studies should be undertaken for identification of such potential allelochemicals.

Metabolomic Profiles of Dinophysis acuminata and Dinophysis acuta Using Non-Targeted High-Resolution Mass Spectrometry: Effect of Nutritional Status and Prey.

Photosynthetic species of the genus Dinophysis are obligate mixotrophs with temporary plastids (kleptoplastids) that are acquired from the ciliate Mesodinium rubrum, which feeds on cryptophytes of the Teleaulax-Plagioselmis-Geminigera clade. A metabolomic study of the three-species food chain Dinophysis-Mesodinium-Teleaulax was carried out using mass spectrometric analysis of extracts of batch-cultured cells of each level of that food chain. The main goal was to compare the metabolomic expression of Galician strains of Dinophysis acuminata and D. acuta that were subjected to different feeding regimes (well-fed and prey-limited) and feeding on two Mesodinium (Spanish and Danish) strains. Both Dinophysis species were able to grow while feeding on both Mesodinium strains, although differences in growth rates were observed. Toxin and metabolomic profiles of the two Dinophysis species were significantly different, and also varied between different feeding regimes and different prey organisms. Furthermore, significantly different metabolomes were expressed by a strain of D. acuminata that was feeding on different strains of the ciliate Mesodinium rubrum. Both species-specific metabolites and those common to D. acuminata and D. acuta were tentatively identified by screening of METLIN and Marine Natural Products Dictionary databases. This first metabolomic study applied to Dinophysis acuminata and D.acuta in culture establishes a basis for the chemical inventory of these species.

Toxicological Investigations on the Sea Urchin Tripneustes gratilla (Toxopneustidae, Echinoid) from Anaho Bay (Nuku Hiva, French Polynesia): Evidence for the Presence of Pacific Ciguatoxins.

The sea urchin Tripneustes gratilla (Toxopneustidae, Echinoids) is a source of protein for many islanders in the Indo-West Pacific. It was previously reported to occasionally cause ciguatera-like poisoning; however, the exact nature of the causative agent was not confirmed. In April and July 2015, ciguatera poisonings were reported following the consumption of T.gratilla in Anaho Bay (Nuku Hiva Island, Marquesas archipelago, French Polynesia). Patient symptomatology was recorded and sea urchin samples were collected from Anaho Bay in July 2015 and November 2016. Toxicity analysis using the neuroblastoma cell-based assay (CBA-N2a) detected the presence of ciguatoxins (CTXs) in T.gratilla samples. Gambierdiscus species were predominant in the benthic assemblages of Anaho Bay, and G.polynesiensis was highly prevalent in in vitro cultures according to qPCR results. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses revealed that P-CTX-3B was the major ciguatoxin congener in toxic sea urchin samples, followed by 51-OH-P-CTX-3C, P-CTX-3C, P-CTX-4A, and P-CTX-4B. Between July 2015 and November 2016, the toxin content in T.gratilla decreased, but was consistently above the safety limit allowed for human consumption. This study provides evidence of CTX bioaccumulation in T.gratilla as a cause of ciguatera-like poisoning associated with a documented symptomatology.

Relative molar response of lipophilic marine algal toxins in liquid chromatography/electrospray ionization mass spectrometry.

RATIONALE: Accurate quantitative analysis of lipophilic toxins by liquid chromatography/mass spectrometry (LC/MS) requires calibration solution reference materials (RMs) for individual toxin analogs. Untargeted analysis is aimed at identifying a vast number of compounds and thus validation of fully quantitative untargeted methods is not feasible. However, a semi-quantitative approach allowing for profiling is still required and will be strengthened by knowledge of the relative molar response (RMR) of analogs in LC/MS with electrospray ionization (ESI). METHODS: RMR factors were evaluated for toxins from the okadaic acid (OA/DTXs), yessotoxin (YTX), pectenotoxin (PTX), azaspiracid (AZA) and cyclic imine (CI) toxin groups, in both solvent standards and environmental sample extracts. Since compound ionization and fragmentation influences the MS response of toxins, RMRs were assessed under different chromatographic conditions (gradient, isocratic) and MS acquisition modes (SIM, SRM, All-ion, target MS/MS) on low- and high-resolution mass spectrometers. RESULTS: In general, RMRs were not significantly impacted by chromatographic conditions (isocratic vs gradient), with the exception of DTX1. MS acquisition modes had a more significant impact, with PnTX-G and SPX differing notably. For a given toxin group, response factors were generally in the range of 0.5 to 2. The cyclic imines were an exception. CONCLUSIONS: Differences in RMRs between toxins of a same chemical base structure were not significant enough to indicate major issues for non-targeted semi-quantitative analysis, where there is limited or no availability of standards for many compounds, and where high degrees of accuracy are not required. Differences in RMRs should be considered when developing methods that use a standard of a single analogue to quantitate other toxins from the same group.

Maitotoxin-4, a Novel MTX Analog Produced by Gambierdiscus excentricus.

Maitotoxins (MTXs) are among the most potent toxins known. These toxins are produced by epi-benthic dinoflagellates of the genera Gambierdiscus and Fukuyoa and may play a role in causing the symptoms associated with Ciguatera Fish Poisoning. A recent survey revealed that, of the species tested, the newly described species from the Canary Islands, G. excentricus, is one of the most maitotoxic. The goal of the present study was to characterize MTX-related compounds produced by this species. Initially, lysates of cells from two Canary Island G. excentricus strains VGO791 and VGO792 were partially purified by (i) liquid-liquid partitioning between dichloromethane and aqueous methanol followed by (ii) size-exclusion chromatography. Fractions from chromatographic separation were screened for MTX toxicity using both the neuroblastoma neuro-2a (N2a) cytotoxicity and Ca2+ flux functional assays. Fractions containing MTX activity were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to pinpoint potential MTX analogs. Subsequent non-targeted HRMS analysis permitted the identification of a novel MTX analog, maitotoxin-4 (MTX4, accurate mono-isotopic mass of 3292.4860 Da, as free acid form) in the most toxic fractions. HRMS/MS spectra of MTX4 as well as of MTX are presented. In addition, crude methanolic extracts of five other strains of G. excentricus and 37 other strains representing one Fukuyoa species and ten species, one ribotype and one undetermined strain/species of Gambierdiscus were screened for the presence of MTXs using low resolution tandem mass spectrometry (LRMS/MS). This targeted analysis indicated the original maitotoxin (MTX) was only present in one strain (G. australes S080911_1). Putative maitotoxin-2 (p-MTX2) and maitotoxin-3 (p-MTX3) were identified in several other species, but confirmation was not possible because of the lack of reference material. Maitotoxin-4 was detected in all seven strains of G. excentricus examined, independently of their origin (Brazil, Canary Islands and Caribbean), and not detected in any other species. MTX4 may therefore serve as a biomarker for the highly toxic G. excentricus in the Atlantic area.

Targeted and non-targeted mass spectrometry to explore the chemical diversity of the genus Gambierdiscus in the Atlantic Ocean.

Dinoflagellates of the genus Gambierdiscus have been associated with ciguatera, the most common non-bacterial fish-related intoxication in the world. Many studies report the presence of potentially toxic Gambierdiscus species along the Atlantic coasts including G. australes, G. silvae and G. excentricus. Estimates of their toxicity, as determined by bio-assays, vary substantially, both between species and strains of the same species. Therefore, there is a need for additional knowledge on the metabolite production of Gambierdiscus species and their variation to better understand species differences. Using liquid chromatography coupled to mass spectrometry, toxin and metabolomic profiles of five species of Gambierdiscus found in the Atlantic Ocean were reported. In addition, a molecular network was constructed aiming at annotating the metabolomes. Results demonstrated that G. excentricus could be discriminated from the other species based solely on the presence of MTX4 and sulfo-gambierones and that the variation in toxin content for a single strain could be up to a factor of two due to different culture conditions between laboratories. While untargeted analyses highlighted a higher variability at the metabolome level, signal correction was applied and supervised multivariate statistics performed on the untargeted data set permitted the selection of 567 features potentially useful as biomarkers for the distinction of G. excentricus, G. caribaeus, G. carolinianus, G. silvae and G. belizeanus. Further studies will be required to validate the use of these biomarkers in discriminating Gambierdiscus species. The study also provided an overview about 17 compound classes present in Gambierdiscus, however, significant improvements in annotation are still required to reach a more comprehensive knowledge of Gambierdiscus' metabolome.

Influence of environmental factors on the paralytic shellfish toxin content and profile of Alexandrium catenella (Dinophyceae) isolated from the Mediterranean Sea.

Laboratory experiments were designed to study the toxin content and profile of the Alexandrium catenella strain ACT03 (isolated from Thau Lagoon, French Mediterranean) in response to abiotic environmental factors under nutrient-replete conditions. This dinoflagellate can produce various paralytic shellfish toxins with concentrations ranging from 2.9 to 50.3 fmol/cell. The toxin profile was characterized by carbamate toxins (GTX3, GTX4 and GTX5) and N-sulfocarbamoyl toxins (C1, C2, C3 and C4). C2 dominated at 12-18 °C, but only for salinities ranging from 10 to 25 psu, whereas GTX5 became dominant at temperatures ranging from 21 to 30 °C at almost all salinities. There was no significant variation in the cellular toxin amount from 18 °C to 27 °C for salinities ranging between 30 and 40 psu. At salinities of 10 to 25 psu, the toxin concentrations always remained below 20 fmol/cell. Toxin content was stable for irradiance ranging from 10 to 70 µmol photons/m2/s then slightly increased. Overall, the toxin profile was more stable than the toxin content (fmol/cell), except for temperature and/or salinity values different from those recorded during Alexandrium blooms in Thau Lagoon.

Seasonal Single-Site Sampling Reveals Large Diversity of Marine Algal Toxins in Coastal Waters and Shellfish of New Caledonia (Southwestern Pacific).

Algal toxins pose a serious threat to human and coastal ecosystem health, even if their potential impacts are poorly documented in New Caledonia (NC). In this survey, bivalves and seawater (concentrated through passive samplers) from bays surrounding Noumea, NC, collected during the warm and cold seasons were analyzed for algal toxins using a multi-toxin screening approach. Several groups of marine microalgal toxins were detected for the first time in NC. Okadaic acid (OA), azaspiracid-2 (AZA2), pectenotoxin-2 (PTX2), pinnatoxin-G (PnTX-G), and homo-yessotoxin (homo-YTX) were detected in seawater at higher levels during the summer. A more diversified toxin profile was found in shellfish with brevetoxin-3 (BTX3), gymnodimine-A (GYM-A), and 13-desmethyl spirolide-C (SPX1), being confirmed in addition to the five toxin groups also found in seawater. Diarrhetic and neurotoxic toxins did not exceed regulatory limits, but PnTX-G was present at up to the limit of the threshold recommended by the French Food Safety Authority (ANSES, 23 µg kg-1). In the present study, internationally regulated toxins of the AZA-, BTX-, and OA-groups by the Codex Alimentarius were detected in addition to five emerging toxin groups, indicating that algal toxins pose a potential risk for the consumers in NC or shellfish export.

Quantitative analysis of azaspiracids in Azadinium spinosum cultures.

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.

Ovatoxin-a and palytoxin accumulation in seafood in relation to Ostreopsis cf. ovata blooms on the French Mediterranean coast.

Dinoflagellates of the genus Ostreopsis are known to cause (often fatal) food poisoning in tropical coastal areas following the accumulation of palytoxin (PLTX) and/or its analogues (PLTX group) in crabs, sea urchins or fish. Ostreopsis spp. occurrence is presently increasing in the northern to north western Mediterranean Sea (Italy, Spain, Greece and France), probably in response to climate change. In France, Ostreopsis. cf. ovata has been associated with toxic events during summer 2006, at Morgiret, off the coast of Marseille, and a specific monitoring has been designed and implemented since 2007. Results from 2008 and 2009 showed that there is a real danger of human poisoning, as these demonstrated bioaccumulation of the PLTX group (PLTX and ovatoxin-a) in both filter-feeding bivalve molluscs (mussels) and herbivorous echinoderms (sea urchins). The total content accumulated in urchins reached 450 µg PLTX eq/kg total flesh (summer 2008). In mussels, the maximum was 230 µg eq PLTX/kg (summer 2009) compared with a maximum of 360 µg found in sea urchins during the same period at the same site. This publication brings together scientific knowledge obtained about the summer development of Ostreopsis spp. in France during 2007, 2008 and 2009.

First evidence of palytoxin and 42-hydroxy-palytoxin in the marine cyanobacterium Trichodesmium.

Marine pelagic diazotrophic cyanobacteria of the genus Trichodesmium (Oscillatoriales) are widespread throughout the tropics and subtropics, and are particularly common in the waters of New Caledonia. Blooms of Trichodesmium are suspected to be a potential source of toxins in the ciguatera food chain and were previously reported to contain several types of paralyzing toxins. The toxicity of water-soluble extracts of Trichodesmium spp. were analyzed by mouse bioassay and Neuroblastoma assay and their toxic compounds characterized using liquid chromatography coupled with tandem mass spectrometry techniques. Here, we report the first identification of palytoxin and one of its derivatives, 42-hydroxy-palytoxin, in field samples of Trichodesmium collected in the New Caledonian lagoon. The possible role played by Trichodesmium blooms in the development of clupeotoxism, this human intoxication following the ingestion of plankton-eating fish and classically associated with Ostreopsis blooms, is also discussed.

Deeper insight into Gambierdiscus polynesiensis toxin production relies on specific optimization of high-performance liquid chromatography-high resolution mass spectrometry.

Ciguatera food poisoning affects consumer health and fisheries' economies worldwide in tropical zones, and specifically in the Pacific area. The wide variety of ciguatoxins bio-accumulated in fish or shellfish responsible for this neurological illness are produced by marine dinoflagellates of the genus Gambierdiscus and bio-transformed through the food web. The evaluation of the contents of ciguatoxins in strains of Gambierdiscus relies on the availability of standards and on the development of sensitive and specific tools to detect them. There is a need for sensitive methods for the analysis of pacific ciguatoxins with high resolution mass spectrometry to ensure unequivocal identification of all congeners. We have applied a fractional factorial design of experiment 2^8-3 for the screening of the significance of eight parameters potentially influencing ionization and ion transmission and their interactions to evaluate the behavior of sodium adducts, protonated molecules and first water losses of CTX4A/B, CTX3B/C, 2-OH-CTX3C and 44-methylgambierone on a Q-TOF equipment. The four parameters that allowed to significantly increase the peak areas of ciguatoxins and gambierones (up to a factor ten) were the capillary voltage, the sheath gas temperature, the ion funnel low pressure voltage and the ion funnel exit voltage. The optimized method was applied to revisit the toxin profile of G. polynesiensis (strain TB92) with a confirmation of the presence of M-seco-CTX4A only putatively reported so far and the detection of an isomer of CTX4A. The improvement in toxin detection also allowed to obtain informative high resolution targeted MS/MS spectra revealing high similarity in fragmentation patterns between putative isomer (4) of CTX3C, 2-OH-CTX3C and CTX3B on one side and between CTX4A, M-seco-CTX4A and the putative isomer on the other side, suggesting a relation of constitutional isomerism between them for both isomers.

Molecular networking as a novel approach to unravel toxin diversity of four strains of the dominant Dinophysis species from French coastal waters.

Some species of the genus Dinophysis contain Diarrhetic shellfish Poisoning (DSP) toxins and are the main threat to shellfish farming in Europe including France. Dinophysis species are known to produce two families of bioactive lipophilic toxins: (i) okadaic acid (OA) and their analogues dinophysistoxins (DTXs) and (ii) pectenotoxins (PTXs). Only six toxins (OA, DTX1, DTX2, DTX3, PTX1 and PTX2) regulated by the European Union Legislation (EC No. 15/2011; 3) are routinely monitored using targeted chemical analysis by liquid chromatography coupled to mass spectrometry (LC-MS/MS) while toxic species of Dinophysis produce many other analogues. To tentatively identify unknown toxin analogues, a recent approach (Molecular Networking, MN) was used based on fragmentation data obtained by untargeted high resolution mass spectrometry (HRMS). An optimization of the data-dependent LC-HRMS/MS acquisition conditions was conducted to obtain more informative networks. The MN was applied to provide an overview of the chemical diversity of four strains belonging to three major Dinophysis species isolated from French coastal waters (D. acuta, D. caudata and the "D. acuminata complex" species D. acuminata and D. sacculus). This approach highlighted species-specific chemical patterns and also that Dinophysis chemical diversity is largely unexplored. Using MN allowed to identify directly known toxins and their relationship between species of Dinophysis, leading to the discovery of five new putative PTX analogues.