RESUMEN
Tetrodotoxin (TTX) is a potent neurotoxin causing human intoxications from contaminated seafood worldwide and is of emerging concern in Europe. Shellfish have been shown to contain varying TTX concentrations globally, with concentrations typically higher in Pacific oysters Crassostrea gigas in Europe. Despite many decades of research, the source of TTX remains unknown, with bacterial or algal origins having been suggested. The aim of this study was to identify potential source organisms causing TTX contamination in Pacific oysters in French coastal waters, using three different techniques. Oysters were deployed in cages from April to September 2021 in an estuary where TTX was previously detected. Microscopic analyses of water samples were used to investigate potential microalgal blooms present prior or during the peak in TTX. Differences in the bacterial communities from oyster digestive glands (DG) and remaining flesh were explored using metabarcoding, and lastly, droplet digital PCR assays were developed to investigate the presence of Cephalothrix sp., one European TTX-bearing species in the DG of toxic C. gigas. Oysters analysed by liquid chromatography-tandem mass spectrometry contained quantifiable levels of TTX over a three-week period (24 June-15 July 2021), with concentrations decreasing in the DG from 424 µg/kg for the first detection to 101 µg/kg (equivalent to 74 to 17 µg/kg of total flesh), and trace levels being detected until August 13, 2021. These concentrations are the first report of the European TTX guidance levels being exceeded in French shellfish. Microscopy revealed that some microalgae bloomed during the TTX peak, (e.g., Chaetoceros spp., reaching 40,000 cells/L). Prokaryotic metabarcoding showed increases in abundance of Rubritaleaceae (genus Persicirhabdus) and Neolyngbya, before and during the TTX peak. Both phyla have previously been described as possible TTX-producers and should be investigated further. Droplet digital PCR analyses were negative for the targeted TTX-bearing genus Cephalothrix.
Asunto(s)
Reacción en Cadena de la Polimerasa , Tetrodotoxina , Tetrodotoxina/análisis , Animales , Francia , Microscopía , Crassostrea , Código de Barras del ADN Taxonómico , Monitoreo del Ambiente/métodos , Microalgas , Estaciones del AñoRESUMEN
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.
Asunto(s)
Dinoflagelados , Océano Atlántico , Dinoflagelados/química , Dinoflagelados/metabolismo , Espectrometría de Masas , Cromatografía Liquida , MetabolómicaRESUMEN
To understand environmental effects affecting paralytic shellfish toxin production of Centrodinium punctatum, this study examined the growth responses, and toxin contents and profiles of a C. punctatum culture exposed to drastic changes of temperature (5-30 °C) and salinity (15-40). C. punctatum grew over a temperature range of 15-25 °C, with an optimum of 20 °C., and over a salinity range of 25-40, with optimum salinities of 30-35. This suggests that C. punctatum prefers relatively warm waters and an oceanic habitat for its growth and can adapt to significant changes of salinity levels. When C. punctatum was cultivated at different temperature and salinity levels, the PST profile included four major analogs (STX, neoSTX, GTX1 and GTX4, constituted >80 % of the profile), while low amounts of doSTX and traces of dc-STX and dc-GTX2 were also observed. Interestingly, though overall toxin contents did not change significantly with temperature, increases in the proportion of STX, and decreases in proportions in GTX1 and GTX4 were observed with higher temperatures. Salinity did not affect either toxin contents or profile from 25 to 35. However, the total toxin content dropped to approximately half at salinity 40, suggesting this salinity may induce metabolic changes in C. punctatum.
Asunto(s)
Dinoflagelados , Toxinas Biológicas , Temperatura , Salinidad , Océanos y MaresRESUMEN
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.
Asunto(s)
Ecosistema , Mariscos , Humanos , Estaciones del Año , Nueva Caledonia , Mariscos/análisis , Ácido OcadaicoRESUMEN
BACKGROUND: Given the recent detection of tetrodotoxin (TTX) in bivalve molluscs but the absence of a full collaborative validation study for TTX determination in a large number of shellfish samples, interlaboratory assessment of method performance was required to better understand current capabilities for accurate and reproducible TTX quantitation using chemical and immunoassay methods. OBJECTIVE: The aim was to conduct an interlaboratory study with multiple laboratories, using results to assess method performance and acceptability of different TTX testing methods. METHODS: Homogenous and stable mussel and oyster materials were assessed by participants using a range of published and in-house detection methods to determine mean TTX concentrations. Data were used to calculate recoveries, repeatability, and reproducibility, together with participant acceptability z-scores. RESULTS: Method performance characteristics were good, showing excellent sensitivity, recovery, and repeatability. Acceptable reproducibility was evidenced by HorRat values for all LC-MS/MS and ELISA methods being less than the 2.0 limit of acceptability. Method differences between the LC-MS/MS participants did not result in statistically different results. Method performance characteristics compared well with previously published single-laboratory validated methods and no statistical difference was found in results returned by ELISA in comparison with LC-MS/MS. CONCLUSION: The results from this study demonstrate that current LC-MS/MS methods and ELISA are on the whole capable of sensitive, accurate, and reproducible TTX quantitation in shellfish. Further work is recommended to expand the number of laboratories testing ELISA and to standardize an LC-MS/MS protocol to further improve interlaboratory precision. HIGHLIGHTS: Multiple mass spectrometric methods and a commercial ELISA have been successfully assessed through an interlaboratory study, demonstrating excellent performance.
Asunto(s)
Bivalvos , Ostreidae , Humanos , Animales , Tetrodotoxina/análisis , Cromatografía Liquida/métodos , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem , Bivalvos/química , Ostreidae/química , Ensayo de Inmunoadsorción Enzimática/métodosRESUMEN
The comprehension of microbial interactions is one of the key challenges in marine microbial ecology. This study focused on exploring chemical interactions between the toxic dinoflagellate Prorocentrum lima and a filamentous fungal species, Aspergillus pseudoglaucus, which has been isolated from the microalgal culture. Such interspecies interactions are expected to occur even though they were rarely studied. Here, a co-culture system was designed in a dedicated microscale marine-like condition. This system allowed to explore microalgal-fungal physical and metabolic interactions in presence and absence of the bacterial consortium. Microscopic observation showed an unusual physical contact between the fungal mycelium and dinoflagellate cells. To delineate specialized metabolome alterations during microalgal-fungal co-culture metabolomes were monitored by high-performance liquid chromatography coupled to high-resolution mass spectrometry. In-depth multivariate statistical analysis using dedicated approaches highlighted (1) the metabolic alterations associated with microalgal-fungal co-culture, and (2) the impact of associated bacteria in microalgal metabolome response to fungal interaction. Unfortunately, only a very low number of highlighted features were fully characterized. However, an up-regulation of the dinoflagellate toxins okadaic acid and dinophysistoxin 1 was observed during co-culture in supernatants. Such results highlight the importance to consider microalgal-fungal interactions in the study of parameters regulating toxin production.
Asunto(s)
Dinoflagelados , Microalgas , Toxinas Marinas , Dinoflagelados/metabolismo , Aspergillus , Cromatografía Líquida de Alta Presión/métodos , Microalgas/metabolismoRESUMEN
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.
Asunto(s)
Ciguatoxinas , Dinoflagelados , Animales , Humanos , Ciguatoxinas/toxicidad , Distribución Tisular , Exposición Dietética , PecesRESUMEN
Gymnodimines (GYMs) are a group of fast-acting phycotoxins and their toxicological effects on human beings are still unclear due to the lack of sufficiently well-characterized large quantities of purified toxins for toxicology studies. In this study, a certified reference material (CRM) of GYM-A was prepared from the dinoflagellate Karenia selliformis, followed by multi-step chromatography separation and purification. Subsequently, the stability of GYM-A in methanolic media was evaluated at different temperature (-20, 4, and 20 °C) and pH (3, 5, and 7) conditions for 8 months, and the conversion products of GYM-A were explored by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The results show that the stability of GYM-A decreased with increasing temperature and pH values. The GYM-A was stable during storage at -20 °C regardless of pH, but it decreased rapidly (81.8% ± 9.3%) at 20 °C in pH 7 solution after 8 months. Moreover, the concentrations of GYM-A did not significantly change at all temperatures in solutions with pH 3 (p > 0.05). It is recommended that GYM-A should be stored at low temperature (≤-20 °C) and pH (≤3) conditions for long-term storage in aqueous methanolic media. In addition, two conversion products of GYM-A, tentatively named as GYM-K (m/z 540) and GYM-L (m/z 524), were identified in the samples stored at high levels of pH and temperature. Based on the LC-HRMS data, the hypothetical chemical structures of both converting derivatives were proposed. A useful strategy for long-term storage of GYM-A CRM in aqueous methanolic media was suggested and two hypothesized conversion products of GYM-A were discovered in this study.
Asunto(s)
Dinoflagelados , Hidrocarburos Cíclicos , Humanos , Temperatura , Hidrocarburos Cíclicos/química , Iminas/química , Dinoflagelados/química , Concentración de Iones de HidrógenoRESUMEN
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.
Asunto(s)
Intoxicación por Ciguatera , Ciguatoxinas , Dinoflagelados , Animales , Cromatografía Liquida , Intoxicación por Ciguatera/etiología , Ciguatoxinas/análisis , Dinoflagelados/química , Polinesia , Espectrometría de Masas en TándemRESUMEN
The taxonomy of the extant dinoflagellate genus Gonyaulax is challenging since its thecate morphology is rather conservative. In contrast, cysts of Gonyaulax are varied in morphology and have been related with the fossil-based genera Spiniferites and Impagidinium. To better understand the systematics of Gonyaulax species, we performed germination experiments on cysts that can be identified as S. ristingensis, an unidentified Spiniferites with petaloid processes here described as Spiniferites pseudodelicatus sp. nov. and Impagidinium variaseptum from Chinese and Portuguese waters. Despite marked differences in cyst morphology, motile cells of S. pseudodelicatus and I. variaseptum are indistinguishable from Gonyaulax baltica. Motile cells hatched from S. ristingensis are morphologically similar to G. baltica as well but differ in the presence of one pronounced antapical spine. Three new species, Gonyaulax amoyensis (cyst equivalent S. pseudodelicatus), Gonyaulax bohaiensis (cyst equivalent I. variaseptum), and Gonyaulax portimonensis (cyst equivalent S. ristingensis), were erected. In addition, a new ribotype (B) of G. baltica was reported from South Korea and a bloom of G. baltica ribotype B is reported from New Zealand. Molecular phylogeny based on LSU and SSU rRNA gene sequences revealed that Gonyaulax species with minute or short antapical spines formed a well-resolved clade, whereas species with two pronounced antapical spines or lack of antapical spines formed the sister clade. Six strains of four above species were examined for yessotoxin production by liquid chromatography coupled with tandem mass spectrometry, and very low concentrations of yessotoxin were detected for one G. bohaiensis strain.
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Dinoflagelados , Cromatografía Liquida , Dinoflagelados/genética , Filogenia , República de Corea , Espectrometría de Masas en TándemRESUMEN
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.
Asunto(s)
Dinoflagelados , Éteres/farmacología , Toxinas Marinas/farmacología , Animales , Organismos Acuáticos , Océano Atlántico , Línea Celular Tumoral/efectos de los fármacos , Intoxicación por Ciguatera , Éteres/química , Humanos , Toxinas Marinas/químicaRESUMEN
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.
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Acuicultura , Intoxicación por Ciguatera/prevención & control , Peces , Animales , Organismos Acuáticos , Humanos , Ratones , PolinesiaRESUMEN
Tetrodotoxins (TTXs) are potentially lethal paralytic toxins that have been identified in European shellfish over recent years. Risk assessment has suggested comparatively low levels (44 µg TTX-equivalent/kg) but stresses the lack of data on occurrence. Both bacteria and dinoflagellates were suggested as possible biogenic sources, either from an endogenous or exogenous origin. We thus investigated TTXs in (i) 98 shellfish samples and (ii) 122 bacterial strains, isolated from French environments. We optimized a method based on mass spectrometry, using a single extraction step followed by ultrafiltration without Solid Phase Extraction and matrix-matched calibration for both shellfish and bacterial matrix. Limits of detection and quantification were 6.3 and 12.5 µg/kg for shellfish and 5.0 and 10 µg/kg for bacterial matrix, respectively. Even though bacterial matrix resulted in signal enhancement, no TTX analog was detected in any strain. Bivalves (either Crassostrea gigas or Ruditapes philippinarum) were surveyed in six French production areas over 2.5-3 month periods (2018-2019). Concentrations of TTX ranged from 'not detected' to a maximum of 32 µg/kg (Bay of Brest, 17 June 2019), with events lasting 2 weeks at maximum. While these results are in line with previous studies, they provide new data of TTX occurrence and confirm that the link between bacteria, bivalves and TTX is complex.
Asunto(s)
Bivalvos/química , Microbiología de Alimentos , Tetrodotoxina/análisis , Animales , Cromatografía Liquida/métodos , Crassostrea/química , Francia , Extracción en Fase Sólida/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
Gymnodimine-A (GYM-A) is a fast-acting microalgal toxin and its production of certified materials requires an efficient harvesting technology from the large-scale cultures of toxigenic microalgae. In this study the recoveries of GYM-A were compared between several liquid-liquid extraction (LLE) treatments including solvents, ratios and stirring times to optimize the LLE technique for harvesting GYM-A from Karenia selliformis cultures, of which the dichloromethane was selected as the extractant and added to microalgal cultures at the ratio 55 mL L-1 (5.5%, v/v). The recovery of GYM-A obtained by the LLE technique was also compared with filtration and centrifugation methods. The stability of GYM-A in culture media were also tested under different pH conditions. Results showed that both the conventional filter filtration and centrifugation methods led to fragmentation of microalgal cells and loss of GYM-A in the harvesting processes. A total of 5.1 µg of GYM-A were obtained from 2 L of K. selliformis cultures with a satisfactory recovery of 88%. Interestingly, GYM-A obviously degraded in the culture media with the initial pH 8.2 and the adjusted pH of 7.0 after 7 days, but there was no obvious degradation in the acidic medium at pH 5.0. Therefore, the LLE method developed here permits the collection of large-volume cultures of K. selliformis and the high-efficiency extraction of GYM-A. This work provides a simple and valuable technique for harvesting toxins from large-scale cultures of GYM-producing microalgae.
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Dinoflagelados/metabolismo , Compuestos Heterocíclicos con 3 Anillos/metabolismo , Hidrocarburos Cíclicos/metabolismo , Iminas/metabolismo , Extracción Líquido-Líquido/métodos , Toxinas Marinas/metabolismoRESUMEN
Palytoxin (PLTX) and its congeners are emerging toxins held responsible for a number of human poisonings following the inhalation of toxic aerosols, skin contact, or the ingestion of contaminated seafood. Despite the strong structural analogies, the relative toxic potencies of PLTX congeners are quite different, making it necessary to isolate them individually in sufficient amounts for toxicological and analytical purposes. Previous studies showed poor PLTX recoveries with a dramatic decrease in PLTX yield throughout each purification step. In view of a large-scale preparative work aimed at the preparation of PLTX reference material, we have investigated evaporation as a critical-although unavoidable-step that heavily affects overall recoveries. The experiments were carried out in two laboratories using different liquid chromatography-mass spectrometry (LC-MS) instruments, with either unit or high resolution. Palytoxin behaved differently when concentrated to a minimum volume rather than when evaporated to complete dryness. The recoveries strongly depended on the solubility as well as on the material of the used container. The LC-MS analyses of PLTX dissolved in aqueous organic blends proved to give a peak intensity higher then when dissolved in pure water. After drying, the PLTX adsorption appeared stronger on glass surfaces than on plastic materials. However, both the solvents used to dilute PLTX and that used for re-dissolution had an important role. A quantitative recovery (97%) was achieved when completely drying 80% aqueous EtOH solutions of PLTX under N2-stream in Teflon. The stability of PLTX in acids was also investigated. Although PLTX was quite stable in 0.2% acetic acid solutions, upon exposure to stronger acids (pH < 2.66), degradation products were observed, among which a PLTX methyl-ester was identified.
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Acrilamidas/aislamiento & purificación , Cromatografía Liquida , Venenos de Cnidarios/aislamiento & purificación , Espectrometría de Masas , Solventes , Manejo de Especímenes , Solventes/química , Manejo de Especímenes/métodosRESUMEN
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.
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Dinoflagelados , Intoxicación por Mariscos , Cromatografía Liquida , Dinoflagelados/genética , Toxinas Marinas/análisis , Espectrometría de Masas en TándemRESUMEN
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.
Asunto(s)
Dinoflagelados , Intoxicación por Mariscos , Toxinas Marinas , Ácido Ocadaico , Estrés SalinoRESUMEN
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.
Asunto(s)
Intoxicación por Ciguatera , Dinoflagelados/química , Toxinas Marinas/química , Oxocinas/química , Animales , Organismos Acuáticos , Mar Mediterráneo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Intoxicación por Ciguatera , Ciguatoxinas , Dinoflagelados , Animales , Cromatografía Líquida de Alta Presión , Ciguatoxinas/análisis , Espectrometría de Masas en TándemRESUMEN
The marine dinoflagellate Vulcanodinium rugosum produces powerful paralyzing and cytotoxic compounds named pinnatoxins (PnTX) and portimines. Even though, no related human intoxication episodes following direct exposure in seawater or the ingestion of contaminated seafood have been documented so far. This study aimed at investigating a dinoflagellate bloom linked to acute dermatitis cases in two recreational beaches in Cienfuegos Bay, Cuba. We used epidemiological and clinical data from 60 dermatitis cases consisting of individuals in close contact with the bloom. Seawater physical-chemical properties were described, and the microorganism causing the bloom was identified by means of light and scanning electron microscopy. Morphological identification was confirmed genetically by sequencing the internal transcribed spacers ITS1 and ITS2, and the 5.8S rDNA region. Toxic compounds were identified from a bloom extract using liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS), and their concentrations were estimated based on low-resolution tandem mass spectrometry (LC-MS/MS). Sixty people who had prolonged contact with the dinoflagellate bloom suffered acute dermal irritation. Most patients (79.2%) were children and had to be treated with antibiotics; some required >5-day hospitalization. Combined morphological and genetic characters indicated V. rugosum as the causative agent of the bloom. rDNA sequences of the V. rugosum genotype found in the bloom aligned with others from Asia, including material found in the ballast tank of a ship in Florida. The predominant toxins in the bloom were portimine, PnTX-F and PnTX-E, similar to strains originating from the Pacific Ocean. This bloom was associated with unusual weather conditions such as frequent and prolonged droughts. Our findings indicate a close link between the V. rugosum bloom and a dermatitis outbreak among swimmers in Cienfuegos Bay. Phylogenetic evidence suggests a recent introduction of V. rugosum from the Pacific Ocean into Caribbean waters, possibly via ballast water.