Insights into the in vitro ichthyotoxicity on fish gill cells and toxin production of Karlodinium veneficum (Dinophyceae) strains from French waters

The dinoflagellate Karlodinium veneficum is regularly detected in French coastal waters; however, the ichthyotoxic potential of French strains and their ability to produce karlotoxins (KmTxs) remain unclear. This study aims to compare the bioactivity and KmTx profiles of four French strains and one previously characterized American strain of K. veneficum (used here as a reference), using the RTgill-W1 rainbow trout gill cell line. Both intracellular and releasable toxins (INT, EXT) in methanolic extracts were assessed for their effects using a resazurin-based assay, lactate dehydrogenase (LDH) release, mitochondrial membrane potential (TMRE dye), while liquid chromatography coupled to mass spectrometry was used to detect KmTxs. Cytotoxic activity varied depending on the strain and fraction, with IFR-CC-20–44 EXT (French) and CCMP 2936 INT (American) being the most toxic, while CBC7 (French) showed no toxicity. The French strains generally exhibited different cytotoxic mechanisms compared to the American strain. The KmTx profiles of the toxic strains showed an unexpectedly high chemodiversity with 93 KmTx-like molecules detected. Three French strains had similar KmTx profiles, dominated by KmTx-5 and a KmTx-5 analog. In contrast, the KmTx profile of the American CCMP 2936 was completely different to the French strains, with no KmTx in common. Interestingly, strain CBC7, which was non-toxic, did not produce any KmTxs. However, among the other K. veneficum strains, no clear link was established between the cytotoxicity of their extracts and total KmTx content. This may reflect differences in potency among KmTx analogs, although the involvement of other uncharacterized compounds cannot be excluded.