Algal ciguatoxin identified as source of ciguatera poisoning in the Caribbean

Abstract

Ciguatera poisoning (CP) is a severe seafood-borne disease, caused by the consumption of reef fish contaminated with Caribbean ciguatoxins (C-CTXs) in the Caribbean and tropical Atlantic. However, C-CTXs have not been identified from their presumed algal source, so the relationship to the CTXs in fish causing illness remains unknown. This has hindered the development of detection methods, diagnostics, monitoring programs, and limited fundamental knowledge on the environmental factors that regulate C-CTX production. In this study, in vitro and chemical techniques were applied to unambiguously identify a novel C-CTX analogue, C-CTX5, from Gambierdiscus silvae and Gambierdiscus caribaeus strains from the Caribbean. Metabolism in vitro by fish liver microsomes converted algal C-CTX5 into C-CTX1/2, the dominant CTX in ciguatoxic fish from the Caribbean. Furthermore, C-CTX5 from G. silvae was confirmed to have voltage-gated sodium-channel-specific activity. This finding is crucial for risk assessment, understanding the fate of C-CTXs in food webs, and is a prerequisite for development of effective analytical methods and monitoring programs. The identification of an algal precursor produced by two Gambierdiscus species is a major breakthrough for ciguatera research that will foster major advances in this important seafood safety issue.

Keywords: Caribbean ciguatoxin; Ciguatera poisoning; Gambierdiscus caribaeus; Gambierdiscus silvae; In vitro hepatic biotransformation; LC–HRMS; Voltage-gated sodium channel.

Fig. 1.

Structures of the known Caribbean ciguatoxins C-CTX1 to C-CTX4 and the novel C-CTX5, along with their elemental formulae and the exact masses of their [M + H]⁺ ions. Selected polyether rings A–N and carbons 1, 3, and 56 are marked, and the ketone and hemiketal oxygen atoms are coloured blue and red, respectively.

Fig. 2.

Microscopy, N2a–MTT activity, and LC–HRMS profiling of G. silvae 1602 SH-6. (A) Light microscopy image of G. silvae 1602 SH-6 in culture. (B) Normalized N2a–MTT response (mean ± SD) in cells sensitized with (+OV; red) and without (−OV; black) ouabain and veratrine, revealing the CTX-like response of the G. silvae 1602 SH-6 extracts dosed across a dilution series. (C) Total-ion chromatogram of G. silvae 1602 SH-6 extract (m/z 1000–1250), with the C-CTX5 peak marked in red. (D) Extracted-ion chromatogram of m/z 1121.6043 ([M + H–H₂O]⁺ for C-CTX5) in G. silvae 1602 SH-6. (E) Full-scan mass spectrum of C-CTX5 (m/z 1100–1180). (F) Product-ion spectrum of C-CTX5 [M + H – H₂O]⁺ (m/z 1121.6043), with identification of product-ions based on the proposed structure (3-oxoC-CTX1/2).

Fig. 3.

Chemical and enzymatic conversions of C-CTX5 into known fish C-CTXs. (A) Overlaid extracted-ion chromatograms of C-CTX1/2 (m/z 1123.6200, [M + H–H₂O]⁺), C-CTX3/4 (m/z 1143.6462, [M + H]⁺) and C-CTX5 (m/z 1121.6043, [M + H–H₂O]⁺) in ciguatoxic fish (S. barracuda) from the Caribbean (black), semi-purified C-CTX5 isolated from G. silvae 1602 SH-6 (red), C-CTX5 chemically reduced with NaBH4 (blue) and NaBH₃CN (green), and C-CTX5 metabolized with liver microsomes prepared from sheepshead (A. probatocephalus; amber). (B) Product-ion spectra of C-CTX3 from fish (black) and from NaBH₄ reduction of C-CTX5 (blue). (C) Scheme showing chemical and enzymatic reductions of C-CTX5 to known C-CTXs.

Fig. 4.

CTX-like activity of semi-purified C-CTX1/2 (blue) and C-CTX5 (black) based on N2a–MTT activity with (+OV; solid lines), and without (−OV; dashed lines), ouabain and veratrine. Fractions (FRC) tested had approximately equivalent peak areas for each toxin based on LC–HRMS analysis. Data shown are the means of triplicate doses per treatment with standard error, across 9-point serial dilutions at approximate toxin equivalency across four independent assays.