A New Larval Fish Bioassay for Testing the Pathogenicity of Pfiesteria Spp. (dinophyceae)

Water quality, microbial contamination, prior fish health, and variable results have been major impediments to identifying the cause and mechanism of fish mortality in standard aquarium-format Pfiesteria bioassays. Therefore, we developed a sensitive 96-h larval fish bioassay for assessing Pfiesteria spp. pathogenicity using six-well tissue culture plates and 7-dayold larval cyprinodontid fish. We used the assay to test pathogenicity of several clonal lines of Pfiesteria piscicida Steidinger and Burkholder and P. shumwayae Glasgow and Burkholder that had been cultured with algal prey for 2 to 36 months. The P. shumwayae cultures exhibited 80%–100% cumulative mortality in less than 96 h at initial zoospore densities of approximately 1000 cells·mL 1 . No fish mortalities occurred with P. piscicida at identical densities or in controls. In a dose-response assay, we demonstrated a strong positive correlation between dinospore density and fish mortality in a highly pathogenic culture of P. shumwayae , generating a 96-h LD 50 of 108 zoospores·mL 1 . Additionally, we applied the assay to evaluate a 38-L P. shumwayae bioassay that was actively killing fish and compared results with those from exposures of juvenile tilapia ( Oreochromis niloticus ) in a 500-mL assay system. Water from the fish-killing 38-L assay was filtered and centrifuged to produce fractions dominated by dinoflagellates, bacteria, or presumed ichthyotoxin (cell-free fraction). After 96 h, the larval fish assay exhibited 50%–100% cumulative mortality only in fractions containing dinoflagellates, with no mortalities occurring in the other fractions. The 500-mL bioassay with tilapia produced inconsistent results and demonstrated no clear correlation between mortality and treatment. The new larval fish bioassay was demonstrated as a highly effective method to verify and evaluate dinoflagellate pathogenicity.