Cell immunolocalization of ciguatoxins in the benthic dinoflagellate Gambierdiscus australes

Cell immunolocalization of ciguatoxins in the benthic dinoflagellate Gambierdiscus australes Giorgio Honsell1, Greta Gaiani2, Masahiro Hirama3, Marco Pelin4, Aurelia Tubaro4, Takeshi Tsumuraya3, Mònica Campàs2 1 Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine - via delle Scienze 91-93 Udine, 33100, Italy. 2 Institute of Agrifood Research and Technology (IRTA). Carretera Poble Nou km 5.5 Sant Carles de La Ràpita, Tarragona, 43540, Spain. 3 Department of Biological Sciences, Graduate School of Science, Osaka Prefecture University, 1-2, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Sakai, Japan.4 Department of Life Sciences, University of Trieste, Via E. Weiss, 2 34128 Trieste, Friuli-Venezia Giulia, Italy. Gambierdiscus species have been recognized as the producers of ciguatoxins (CTXs) which, after biotransformation in fish, are responsible of ciguatera fish poisoning (CFP).Cells from a culture of Gambierdiscus australes (clone IRTA-SMM-16-286) were fixed in paraformaldehyde and sectioned in a cryostat. Whole cells and sections were TritonX-100 permeabilized, BSA- blocked and incubated with 8H4 anti -CTX antibody, which binds to the right wing of CTX1B, CTX3C, 54-deoxyCTX1B and 51-hydroxyCTX3C, and then with a secondary Alexa Fluor 488-conjugated anti-mouse IgG. Cells and sections were mounted on slides and observed with a confocal microscope. Whole cells showed a strong positive reaction at cell surface, in correspondence with thecal pores (or poroids), while no reaction was detected inside the cells, as the antibody did not seem to be able to pass through the amphiesma. The observation of sections confirmed the positive reaction at cell surface and revealed positive reactions inside the cytoplasm. Small fluorescent dots grouped together in a reticulate pattern were visible between chloroplasts. Single small dots were observed also on nuclear surface. However, the strongest reaction was observed in two or three larger cytoplasmic globular structures (8 - 10 μm wide), which appeared to be formed by tiny filaments. The results confirm the reactivity of 8H4 anti - CTX antibody with G. australes toxins and suggest an involvement of cell endomembrane system in toxin production. Further research will be necessary to verify this hypothesis and to understand if the positive reaction observed at cell surface represents an effective release of toxins outside the cell.