Fish gill damage by the dinoflagellate Alexandrium catenella from Chilean fjords: synergistic action of ROS and PUFA

Outbreaks of the dinoflagellate <i>Alexandrium catenella</i> have caused significant economic losses to the salmon industry in the south of Chile. However, the precise ichthyotoxic mechanism by this paralytic shellfish toxin (PST) – producing dinoflagellate remains poorly understood. The rainbow trout cell line RTgill-W1 assay was used to investigate fish gill damage by multiple <i>A. catenella</i> strains under different environmental conditions, and potency of whole cells, lysed cells and culture medium were compared. Cytotoxic potency was highly variable among strains and strongly correlated to dinoflagellate cell-abundance. Lysed cells produced more gill damage than intact cells and supernatant, reducing gill cell viability down to 20% of controls at 4000 cells mL⁻¹. However, gill cells exposed to pure PST fractions (C1&C2, STX, GTX 1&4) exhibited only very limited loss of viability (<30%), even at cell concentrations equivalent to exceeding those detected in southern Chilean fjords. <i>A. catenella</i> lytic compounds rapidly (1–4 days) degraded in the light, but were pH (7–9) and temperature stable (17–85 °C), thus arguing against the involvement of proteinaceous compounds. Superoxide production by Chilean dinoflagellate strains was as high as by the raphidophyte <i>Chattonella marina</i> (max. 8.67 ± 0.14 pmol O₂⁻ cell⁻¹ h⁻¹), variable among strains and enhanced by cell disruption and environmental stress (low nutrients, low salinity, low or high pH). Fatty acid profiles included high concentrations of the long-chain (≥C₂₀) polyunsaturated fatty acids (PUFA) – docosahexaenoic acid (DHA, 22:6ω3; 16–20% of total fatty acids) in addition to several other PUFA, which were highly cytotoxic against gill cells as a purified fraction (LC₅₀ 1.30 μg mL⁻¹). In addition, potency was enhanced 9-fold in combination with superoxide anion (LC₅₀ 0.15 μg mL⁻¹). In conclusion, the present study demonstrates that fish gill damage during <i>A. catenella</i> fish-kill events in Chilean fjords cannot be explained by PST toxins, but can to a large extent be accounted for by the synergistic interaction between ROS (superoxide anion) and DHA, and potentially other PUFA, notably under conditions that promote cell lysis.