The seafood illness, ciguatera poisoning (CP) is a significant public health concern in tropical and subtropical regions worldwide. In Japan, CP is particularly prevalent in Okinawa Prefecture, where is the subtropical region of Japan, due to the consumption of fish contaminated with Pacific ciguatoxins (P-CTXs). The benthic dinoflagellate Gambierdiscus polynesiensis is known to produce P-CTXs in the South Pacific, but P-CTX-producing microalgae have not been identified in Japanese waters. A practical method applicable to field samples containing diverse benthic microalgae is therefore required. To identify a P-CTX producer in Japan, an indirect immunofluorescence (IIF) assay using P-CTX-specific monoclonal antibodies that can be applied to various benthic microalgae such as diatoms and dinoflagellates was developed. The optimal conditions for cell membrane permeabilization were determined using the IIF assay with an anti-actin antibody on benthic diatoms and dinoflagellates treated with various concentrations of Triton X-100. Treatment of the cells with 5.0% (v/v) Triton X-100 resulted in the highest fluorescence and the lowest cell loss. When an IIF assay with an anti-P-CTX antibody (8H4) was performed on cells of Gambierdiscus australes , G. jejuensis , G. polynesiensis , and G. silvae treated with 5.0% Triton X-100, only G. polynesiensis showed fluorescence. The simultaneous application of two anti-P-CTX antibodies (8H4 and 10C9) doubled the fluorescence compared to 8H4 alone. Extending the antibody reaction time from one hour to 16 hours increased the fluorescence by about sevenfold. These optimized conditions enabled visualization of P-CTXs-derived fluorescence in G. polynesiensis cells under both not only a confocal laser scanning microscope but also a conventional inverted fluorescence microscope having deeper depths of focus. Combining the IIF assay with observing the cells under an inverted microscope provides a practical tool for detecting P-CTX producers in samples of various sizes of benthic microalgae collected from coastal waters. This will contribute to identifying unknown P-CTX-producing microalgae in Japanese and other coastal waters in the future.
Ohara et al. (Wed,) studied this question.