Intraspecific Variability in the Toxin Production and Toxin Profiles of In Vitro Cultures of Gambierdiscus polynesiensis (Dinophyceae) from French Polynesia

Abstract

Ciguatera poisoning (CP) is a foodborne disease caused by the consumption of seafood contaminated with ciguatoxins (CTXs) produced by dinoflagellates in the genera Gambierdiscus and Fukuyoa. The toxin production and toxin profiles were explored in four clones of G. polynesiensis originating from different islands in French Polynesia with contrasted CP risk: RIK7 (Mangareva, Gambier), NHA4 (Nuku Hiva, Marquesas), RAI-1 (Raivavae, Australes), and RG92 (Rangiroa, Tuamotu). Productions of CTXs, maitotoxins (MTXs), and gambierone group analogs were examined at exponential and stationary growth phases using the neuroblastoma cell-based assay and liquid chromatography-tandem mass spectrometry. While none of the strains was found to produce known MTX compounds, all strains showed high overall P-CTX production ranging from 1.1 ± 0.1 to 4.6 ± 0.7 pg cell^-1. In total, nine P-CTX analogs were detected, depending on strain and growth phase. The production of gambierone, as well as 44-methylgamberione, was also confirmed in G. polynesiensis. This study highlighted: (i) intraspecific variations in toxin production and profiles between clones from distinct geographic origins and (ii) the noticeable increase in toxin production of both CTXs, in particular CTX4A/B, and gambierone group analogs from the exponential to the stationary phase.

Keywords: 44-methylgambierone; CBA-N2a; Gambierdiscus polynesiensis; LC-MS/MS; ciguatera; ciguatoxins; gambierone; toxin profiles.

Figure 1

Growth curve of Gambierdiscus polynesiensis RAI-1 as measured by mean natural in vivo fluorescence (rfu). Data represent the mean ± SD of three experiments (with each fluorescence reading run five times).

Figure 2

Dose-response curve of Neuro-2a cells in OV⁺ conditions when exposed to increasing concentrations of dichloromethane-soluble fractions of G. polynesiensis strains. Data represent the mean ± SD of three replicate cultures per clonal isolate, with each concentration run in triplicate.

Figure 3

Chromatogram of scheduled Multiple Reaction Monitoring (MRM) liquid chromatography–tandem mass spectrometry (LC-MS/MS) showing the different P-CTX analogs detected in crude extracts (methanol) of strain NHA4 tested at the stationary growth phase. The retention time (RT) of each analog is indicated close to the corresponding peak and the different P-CTX3B/C group isomers 1, 2, and 3 are indicated in brackets. NB: P-CTX3B peak is cut off to show all analogs.

Figure 4

Comparison of the toxin profiles in batch cultures of G. polynesiensis RIK7, NHA4, RAI-1, and RG92 as determined by LC-MS/MS at the exponential vs. the stationary phase. The relative abundance of each analog is expressed in percentage relative to the total toxin content. Only quantifiable compounds are included in this representation.

Figure 5

Chromatogram of non-scheduled MRM LC-MS/MS (negative mode) showing the presence of gambierone and 44-methylgambierone in crude extracts (methanol) of strain NHA4 at the stationary growth phase.

Figure 6

Linear relationship between G. polynesiensis RAI-1 cell concentration (cells.mL⁻¹) and in vivo fluorescence (rfu, relative fluorescence units). Filled circles represent individual measurements to which a linear regression line has been fitted.