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.

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.

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.

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.

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.

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.

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.

Characterization of toxin-producing strains of Dinophysis spp. (Dinophyceae) isolated from French coastal waters, with a particular focus on the D. acuminata-complex.

Dinoflagellates of the genus Dinophysis are the most prominent producers of Diarrhetic Shellfish Poisoning (DSP) toxins which have an impact on public health and on marine aquaculture worldwide. In particular, Dinophysis acuminata has been reported as the major DSP agent in Western Europe. Still, its contribution to DSP events in the regions of the English Channel and the Atlantic coast of France, and the role of the others species of the Dinophysis community in these areas are not as clear. In addition, species identification within the D. acuminata complex has proven difficult due to their highly similar morphological features. In the present study, 30 clonal strains of the dominant Dinophysis species have been isolated from French coasts including the English Channel (3 sites), the Atlantic Ocean (11 sites) and the Mediterranean Sea (6 sites). Morphologically, strains were identified as three species: D. acuta, D. caudata, D. tripos, as well as the D. acuminata-complex. Sequences of the ITS and LSU rDNA regions confirmed these identifications and revealed no genetic difference within the D. acuminata-complex. Using the mitochondrial gene cox1, two groups of strains differing by only one substitution were found in the D. acuminata-complex, but SEM analysis of various strains showed a large range of morphological variations. Based on geographical origin and morphology, strains of the subclade A were ascribed to 'D. acuminata' while those of the subclade B were ascribed to 'D. sacculus'. Nevertheless, the distinction into two separate species remains questionable and was not supported by our genetic data. The considerable variations observed in cultured strains suggest that physiological factors might influence cell contour and bias identification. Analyses of Dinophysis cultures from French coastal waters using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) revealed species-conserved toxin profiles for D. acuta (dinophysistoxin 2 (DTX2), okadaic acid (OA), pectenotoxin 2 (PTX2)), D. caudata (PTX2) and D. tripos (PTX2), irrespective of geographical origin (Atlantic Ocean or Mediterranean Sea). Within the D. acuminata-complex, two different toxin profiles were observed: the strains of 'D. acuminata' (subclade A) from the English Channel and the Atlantic Ocean contained only OA while strains of 'D. sacculus' (subclade B) from Mediterranean Sea/Atlantic Ocean contained PTX2 as the dominant toxin, with OA and C9-esters also being present, albeit in lower proportions. The same difference in toxin profiles between 'D. sacculus' and 'D. acuminata' was reported in several studies from Galicia (NW- Spain). This difference in toxin profiles has consequences in terms of public health, and consequently for monitoring programs. While toxin profile could appear as a reliable feature separating 'D. acuminata' from 'D. sacculus' on both French and Spanish coasts, this does not seem consistent with observations on a broader geographical scale for the D. acuminata complex, possibly due to the frequent lack of genetic characterization.

Effect of a short-term salinity stress on the growth, biovolume, toxins, osmolytes and metabolite profiles on three strains of the Dinophysis acuminata-complex (Dinophysis cf. sacculus).

Dinophysis is the main dinoflagellate genus responsible for diarrheic shellfish poisoning (DSP) in human consumers of filter feeding bivalves contaminated with lipophilic diarrheic toxins. Species of this genus have a worldwide distribution driven by environmental conditions (temperature, irradiance, salinity, nutrients etc.), and these factors are sensitive to climate change. The D. acuminata-complex may contain several species, including D. sacculus. The latter has been found in estuaries and semi-enclosed areas, water bodies subjected to quick salinity variations and its natural repartition suggests some tolerance to salinity changes. However, the response of strains of D. acuminata-complex (D. cf. sacculus) subjected to salinity stress and the underlying mechanisms have never been studied in the laboratory. Here, a 24 h hypoosmotic (25) and hyperosmotic (42) stress was performed in vitro in a metabolomic study carried out with three cultivated strains of D. cf. sacculus isolated from the French Atlantic and Mediterranean coasts. Growth rate, biovolume and osmolyte (proline, glycine betaine and dimethylsulfoniopropionate (DMSP)) and toxin contents were measured. Osmolyte contents were higher at the highest salinity, but only a significant increase in glycine betaine was observed between the control (35) and the hyperosmotic treatment. Metabolomics revealed significant and strain-dependent differences in metabolite profiles for different salinities. These results, as well as the absence of effects on growth rate, biovolume, okadaic acid (OA) and pectenotoxin (PTXs) cellular contents, suggest that the D. cf. sacculus strains studied are highly tolerant to salinity variations.

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.

Effects of pH and Nutrients (Nitrogen) on Growth and Toxin Profile of the Ciguatera-Causing Dinoflagellate Gambierdiscus polynesiensis (Dinophyceae).

Ciguatera poisoning is a foodborne disease caused by the consumption of seafood contaminated with ciguatoxins (CTXs) produced by dinoflagellates in the genera Gambierdiscus and Fukuyoa. Ciguatera outbreaks are expected to increase worldwide with global change, in particular as a function of its main drivers, including changes in sea surface temperature, acidification, and coastal eutrophication. In French Polynesia, G. polynesiensis is regarded as the dominant source of CTXs entering the food web. The effects of pH (8.4, 8.2, and 7.9), Nitrogen:Phosphorus ratios (24N:1P vs. 48N:1P), and nitrogen source (nitrates vs. urea) on growth rate, biomass, CTX levels, and profiles were examined in four clones of G. polynesiensis at different culture age (D10, D21, and D30). Results highlight a decrease in growth rate and cellular biomass at low pH when urea is used as a N source. No significant effect of pH, N:P ratio, and N source on the overall CTX content was observed. Up to ten distinct analogs of Pacific ciguatoxins (P-CTXs) could be detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in clone NHA4 grown in urea, at D21. Amounts of more oxidized P-CTX analogs also increased under the lowest pH condition. These data provide interesting leads for the custom production of CTX standards.

Evidence for the Range Expansion of Ciguatera in French Polynesia: A Revisit of the 2009 Mass-Poisoning Outbreak in Rapa Island (Australes Archipelago).

Ciguatera poisoning (CP) results from the consumption of seafood contaminated with ciguatoxins (CTXs). This disease is highly prevalent in French Polynesia with several well-identified hotspots. Rapa Island, the southernmost inhabited island in the country, was reportedly free of CP until 2007. This study describes the integrated approach used to investigate the etiology of a fatal mass-poisoning outbreak that occurred in Rapa in 2009. Symptoms reported in patients were evocative of ciguatera. Several Gambierdiscus field samples collected from benthic assemblages tested positive by the receptor binding assay (RBA). Additionally, the toxicity screening of ≈250 fish by RBA indicated ≈78% of fish could contain CTXs. The presence of CTXs in fish was confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The potential link between climate change and this range expansion of ciguatera to a subtropical locale of French Polynesia was also examined based on the analysis of temperature time-series data. Results are indicative of a global warming trend in Rapa area. A five-fold reduction in incidence rates was observed between 2009 and 2012, which was due in part to self-regulating behavior among individuals (avoidance of particular fish species and areas). Such observations underscore the prominent role played by community outreach in ciguatera risk management.

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.

Cultures of Dinophysis sacculus, D. acuminata and pectenotoxin 2 affect gametes and fertilization success of the Pacific oyster, Crassostrea gigas.

Harmful algal blooms (HABs) of toxic species of the dinoflagellate genus Dinophysis are a threat to human health as they are mainly responsible for diarrheic shellfish poisoning (DSP) in the consumers of contaminated shellfish. Such contamination leads to shellfish farm closures causing major economic and social issues. The direct effects of numerous HAB species have been demonstrated on adult bivalves, whereas the effects on critical early life stages remain relatively unexplored. The present study aimed to determine the in vitro effects of either cultivated strains of D. sacculus and D. acuminata isolated from France or their associated toxins (i.e. okadaic acid (OA) and pectenotoxin 2 (PTX2)) on the quality of the gametes of the Pacific oyster Crassostrea gigas. This was performed by assessing the ROS production and viability of the gametes using flow cytometry, and fertilization success using microscopic counts. Oocytes were more affected than spermatozoa and their mortality and ROS production increased in the presence of D. sacculus and PTX2, respectively. A decrease in fertilization success was observed at concentrations as low as 0.5 cell mL-1 of Dinophysis spp. and 5 nM of PTX2, whereas no effect of OA could be observed. The effect on fertilization success was higher when both gamete types were concomitantly exposed compared to separate exposures, suggesting a synergistic effect. Our results also suggest that the effects could be due to cell-to-cell contact. These results highlight a potential effect of Dinophysis spp. and PTX2 on reproduction and recruitment of the Pacific oyster.

Diversity and Toxicity of the Genus Coolia Meunier in Brazil, and Detection of 44-Methyl Gambierone in Coolia tropicalis.

Coolia is a genus of marine benthic dinoflagellates which is widely distributed in tropical and temperate zones. Toxicity has been reported in selected Coolia species, although the identity of causative compounds is still controversial. In this study, we investigated the taxonomical and toxicological aspects of Coolia species from Brazil. Since light- and electron microscopy-based morphology was not enough to distinguish small-celled species, ITS and LSU D1-D3 phylogenetic analyses were used for species definition. Cultures of Coolia palmyrensis and Coolia santacroce were established from samples collected along the northeastern Brazilian coast, the first record of both species in South Atlantic waters. Cultures of Coolia malayensis and Coolia tropicalis were also established and exhibited acute in vivo toxicity to adults of Artemia salina, while C. palmyrensis and C. santacroce were non-toxic. The presence of 30 yessotoxin analogues, 7 metabolites of Coolia and 44 Gambierdiscus metabolites was screened in 14 strains of Coolia. 44-methyl gambierone (formerly referred to as MTX3) and a new isomer of this compound were detected only in C. tropicalis, using both low- and high-resolution LC-MS/MS. To our knowledge, this is the first report of gambierone analogues in dinoflagellates other than Gambierdiscus; the role of C. tropicalis in ciguatera poisoning thus deserves to be considered in further investigations.

Assessment of Ciguatera and Other Phycotoxin-Related Risks in Anaho Bay (Nuku Hiva Island, French Polynesia): Molecular, Toxicological, and Chemical Analyses of Passive Samplers.

Ciguatera poisoning is a foodborne illness caused by the consumption of seafood contaminated with ciguatoxins (CTXs) produced by dinoflagellates from the genera Gambierdiscus and Fukuyoa. The suitability of Solid Phase Adsorption Toxin Tracking (SPATT) technology for the monitoring of dissolved CTXs in the marine environment has recently been demonstrated. To refine the use of this passive monitoring tool in ciguateric areas, the effects of deployment time and sampler format on the adsorption of CTXs by HP20 resin were assessed in Anaho Bay (Nuku Hiva Island, French Polynesia), a well-known ciguatera hotspot. Toxicity data assessed by means of the mouse neuroblastoma cell-based assay (CBA-N2a) showed that a 24 h deployment of 2.5 g of resin allowed concentrating quantifiable amounts of CTXs on SPATT samplers. The CTX levels varied with increasing deployment time, resin load, and surface area. In addition to CTXs, okadaic acid (OA) and dinophysistoxin-1 (DTX1) were also detected in SPATT extracts using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), consistent with the presence of Gambierdiscus and Prorocentrum species in the environment, as assessed by quantitative polymerase chain reaction (qPCR) and high-throughput sequencing (HTS) metabarcoding analyses conducted on passive window screen (WS) artificial substrate samples. Although these preliminary findings await further confirmation in follow-up studies, they highlight the usefulness of SPATT samplers in the routine surveillance of CP risk on a temporal scale, and the monitoring of other phycotoxin-related risks in ciguatera-prone areas.

Use of Mass Spectrometry to Determine the Diversity of Toxins Produced by Gambierdiscus and Fukuyoa Species from Balearic Islands and Crete (Mediterranean Sea) and the Canary Islands (Northeast Atlantic).

Over the last decade, knowledge has significantly increased on the taxonomic identity and distribution of dinoflagellates of the genera Gambierdiscus and Fukuyoa. Additionally, a number of hitherto unknown bioactive metabolites have been described, while the role of these compounds in ciguatera poisoning (CP) remains to be clarified. Ciguatoxins and maitotoxins are very toxic compounds produced by these dinoflagellates and have been described since the 1980s. Ciguatoxins are generally described as the main contributors to this food intoxication. Recent reports of CP in temperate waters of the Canary Islands (Spain) and the Madeira archipelago (Portugal) triggered the need for isolation and cultivation of dinoflagellates from these areas, and their taxonomic and toxicological characterization. Maitotoxins, and specifically maitotoxin-4, has been described as one of the most toxic compounds produced by these dinoflagellates (e.g., G. excentricus) in the Canary Islands. Thus, characterization of toxin profiles of Gambierdiscus species from adjacent regions appears critical. The combination of liquid chromatography coupled to either low- or high-resolution mass spectrometry allowed for characterization of several strains of Gambierdiscus and Fukuyoa from the Mediterranean Sea and the Canary Islands. Maitotoxin-3, two analogues tentatively identified as gambieric acid C and D, a putative gambierone analogue and a putative gambieroxide were detected in all G. australes strains from Menorca and Mallorca (Balearic Islands, Spain) while only maitotoxin-3 was present in an F. paulensis strain of the same region. An unidentified Gambierdiscus species (Gambierdiscus sp.2) from Crete (Greece) showed a different toxin profile, detecting both maitotoxin-3 and gambierone, while the availability of a G. excentricus strain from the Canary Islands (Spain) confirmed the presence of maitotoxin-4 in this species. Overall, this study shows that toxin profiles not only appear to be species-specific but probably also specific to larger geographic regions.

Liquid Chromatography Coupled to High-Resolution Mass Spectrometry for the Confirmation of Caribbean Ciguatoxin-1 as the Main Toxin Responsible for Ciguatera Poisoning Caused by Fish from European Atlantic Coasts.

Ciguatera poisoning (CP) is a common seafood intoxication mainly caused by the consumption of fish contaminated by ciguatoxins. Recent studies showed that Caribbean ciguatoxin-1 (C-CTX1) is the main toxin causing CP through fish caught in the Northeast Atlantic, e.g., Canary Islands (Spain) and Madeira (Portugal). The use of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) combined with neuroblastoma cell assay (N2a) allowed the initial confirmation of the presence of C-CTX1 in contaminated fish samples from the abovementioned areas, nevertheless the lack of commercially available reference materials for these particular ciguatoxin (CTX) analogues has been a major limitation to progress research. The EuroCigua project allowed the preparation of C-CTX1 laboratory reference material (LRM) from fish species (Seriola fasciata) from the Madeira archipelago (Portugal). This reference material was used to implement a liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) for the detection of C-CTX1, acquisition of full-scan as well as collision-induced mass spectra of this particular analogue. Fragmentation pathways were proposed based on fragments obtained. The optimized LC-HRMS method was then applied to confirm C-CTX1 in fish (Bodianus scrofa) caught in the Selvagens Islands (Portugal).