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.

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.

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.