Toxicology of Gambierdiscus spp. (Dinophyceae) from Tropical and Temperate Australian Waters.

Ciguatera Fish Poisoning (CFP) is a human illness caused by the consumption of marine fish contaminated with ciguatoxins (CTX) and possibly maitotoxins (MTX), produced by species from the benthic dinoflagellate genus Gambierdiscus. Here, we describe the identity and toxicology of Gambierdiscus spp. isolated from the tropical and temperate waters of eastern Australia. Based on newly cultured strains, we found that four Gambierdiscus species were present at the tropical location, including G. carpenteri, G. lapillus and two others which were not genetically identical to other currently described species within the genus, and may represent new species. Only G. carpenteri was identified from the temperate location. Using LC-MS/MS analysis we did not find any characterized microalgal CTXs (P-CTX-3B, P-CTX-3C, P-CTX-4A and P-CTX-4B) or MTX-1; however, putative maitotoxin-3 (MTX-3) was detected in all species except for the temperate population of G. carpenteri. Using the Ca2+ influx SH-SY5Y cell Fluorescent Imaging Plate Reader (FLIPR) bioassay we found CTX-like activity in extracts of the unidentified Gambierdiscus strains and trace level activity in strains of G. lapillus. While no detectable CTX-like activity was observed in tropical or temperate strains of G. carpenteri, all species showed strong maitotoxin-like activity. This study, which represents the most comprehensive analyses of the toxicology of Gambierdiscus strains isolated from Australia to date, suggests that CFP in this region may be caused by currently undescribed ciguatoxins and maitotoxins.

Cyclodextrin polymer clean-up method for the detection of ciguatoxins in fish with cell-based assays.

Ciguatoxins (CTXs) are marine toxins produced by microalgae of the genera Gambierdiscus and Fukuyoa, which are transferred through the food webs, reaching humans and causing a poisoning known as ciguatera. The cell-based assay (CBA) is commonly used for their detection because of its high sensitivity and the provided toxicological information. However, matrix effects may interfere in the CBA. In this work, γ-cyclodextrin-hexamethylene diisocyanate (γ-CD-HDI), γ-cyclodextrin-epichlorohydrin (γ-CD-EPI) and γ-CD-EPI conjugated to magnetic beads (γ-CD-EPI-MB) have been evaluated as clean-up materials for fish flesh extracts containing CTXs. The best results were achieved with γ-CD-HDI in column format, which showed a CTX1B recovery of 42% and 32% for Variola louti and Seriola dumerili, respectively, and allowed exposing cells to at least 400 mg/mL of fish flesh. This clean-up strategy provides at least 4.6 and 3.0-fold higher sensitivities to the assay for V.louti and S.dumerili, respectively, improving the reliability of CTX quantification.

Predictive score and probability of CTX-like toxicity in fish samples from the official control of ciguatera in the Canary Islands.

This research identifies factors associated with the contamination by ciguatoxins (CTXs) in a population of fish and proposes a predictive score of the presence of CTX-like toxicity in amberjack samples from the official control program of ciguatera in the Canary Islands of the Directorate-General (DG) Fisheries (Canary Government). Out of the 970 samples of fish studied, 177 (18.2%) samples showed CTX-like toxicity. The fish were classified according to the species, amberjack (Seriola dumerili and S. rivoliana) (n = 793), dusky grouper (Epinephelus marginatus) (n = 145) and wahoo (Acanthocybium solandri) (n = 32). The data were separated by species category and statistically examined, resulting in 137 (17.3%) amberjack and 39 (26.9%) grouper samples showing CTX-like toxicity; regarding wahoo species, only 1 toxic sample (3.1%) was found. According to fishing location the contamination rates suggested grouping the islands in four clusters; namely: {El Hierro: HI; La Gomera: LG; La Palma: LP}, {Gran Canaria: GC; Tenerife: TF}, {Fuerteventura: FU} and {Lanzarote: LZ}. For the amberjack species, the multivariate logistic regression showed the factors that maintained independent association with the outcome, which were the warm season (OR = 3.617; 95% CI = 1.249-10.474), the weight (per kg, 1.102; 95% CI = 1.069-1.136) and the island of fish catching. A prediction score was obtained for the probability of contamination by CTX in amberjack fish samples. The area under de curve (AUC) obtained using the validation data was 0.747 (95% CI = 0.662-0.833). Regarding grouper species, the island of fishing was the only factor that showed significant differences associated with the presence of CTX-like toxicity. We provide herein data for a better management and prediction of ciguatera in the Canary Islands, suggesting a review of the minimum limits of fish weight established by the Canary Government for the control program.

Design of experiments for the optimization of electrospray ionization in the LC-MS/MS analysis of ciguatoxins.

Liquid chromatography (LC) coupled to mass spectrometry (MS) is being widely applied as an analytical tool in the field of marine biotoxins both for regulated and for new and emerging compounds. LC-MS/MS recently became the reference method for the control of lipophilic toxins in the European Union, and new methods are being developed and optimized to extend the applicability of this technique to other toxin groups. In this work, conditions for the analysis of ciguatoxins (CTXs) by LC-MS/MS were investigated using standard solutions of CTX1B and CTX3C, which are structurally representative compounds for the rest of the main congeners of Pacific group toxins (P-CTXs). Preliminary studies were carried out for the selection of precursor and product ions used for multiple reaction monitoring. Two transitions based on the chemical structures of CTXs were set up, and mass spectrometer parameters were adjusted for selected reactions monitored. The electrospray ionization source has been carefully optimized through a design of experiments that consisted of a two-level fractional factorial design of resolution IV for the screening of adequate source conditions and of response surface designs for optimization of the main interactions between factors. The statistical approach allowed maximizing the sensitivity on the MS analyzer that provides a good specificity in P-CTX detection, which can be also used for confirmation purposes.