Identification of Gambierdiscus species from La Réunion and evaluation of toxicity and toxin profile

Five different species of Gambierdiscus have been identified in La Réunion (G. belizeanus, G. balechii, G. pacificus, G. silvae and G. ribotype 2) by morphological observations in Scanning Electron Microscopy (SEM) and molecular identification and phylogenetic analysis. Growth rates of cultures have also been evaluated showing values from 0.09 to 0.36 d^-1. The toxicity and toxin profile of thirteen strains have been analysed by a multidisciplinary approach with Neuro-2a cell-based assay (CBA), magnetic bead-based immunoassay, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and LC coupled to high-resolution mass spectrometry (LCHRMS). G. balechii showed the highest toxicity by CBA (∼627 fg equiv. CTX1B·cell ^-1) followed by G. ribotype 2 (76 to 13 fg equiv. CTX1B·cell ^-1), G. balechii (63 to 7 fg equiv. CTX1B·cell ^-1), G. belizeanus (30 to 20 fg equiv. CTX1B·cell ^-1) and G. pacificus with values close to LOQ but not conclusive. The toxin profile for the 13 strains was evaluated by LC-MS/MS using seven different methods and being gambierone and 44-methylgambierone the two only known compounds, found in high concentrations in all samples. Gambierone was detected from 2.05 pg ·cell^-1 in G. balechii (P-0414B) to 12.91 pg·cell^-1 in G. balechii (P-0414A) and 44-methylgambierone was detected from 1.93 pg·cell^-1 in G. belizeanus (P-0414B) to 14.95 pg·cell^-1 in G. pacificus (P-0304). These samples were analysed also by LCHRMS, confirming gambierone and 44-methylgambierone the main compounds detected. Additionally, a potential polyether sulphur-containing compound corresponding to the novel molecular formula C₆₂H₉₄O₂₃S ([M+NH₄]⁺, m/z 1256.6234) were tentatively identified. This study combining morphological and molecular data is the first to mention such diversity in the area. It is also the first time that toxicity and toxin profile of Gambierdiscus from La Réunion have been evaluated.