RESUMO
Pfiesteria piscicida and P. shumwayae reportedly secrete potent exotoxins thought to cause fish lesion events, acute fish kills and human disease in mid-Atlantic USA estuaries. However, Pfiesteria toxins have never been isolated or characterized. We investigated mechanisms by which P. shumwayae kills fish using three different approaches. Here we show that larval fish bioassays conducted in tissue culture plates fitted with polycarbonate membrane inserts exhibited mortality (100%) only in treatments where fish and dinospores were in physical contact. No mortalities occurred in treatments where the membrane prevented contact between dinospores and fish. Using differential centrifugation and filtration of water from a fish-killing culture, we produced 'dinoflagellate', 'bacteria' and 'cell-free' fractions. Larval fish bioassays of these fractions resulted in mortalities (60-100% in less than 24 h) only in fractions containing live dinospores ('whole water', 'dinoflagellate'), with no mortalities in 'cell-free' or 'bacteria'-enriched fractions. Videomicrography and electron microscopy show dinospores swarming toward and attaching to skin, actively feeding, and rapidly denuding fish of epidermis. We show here that our cultures of actively fish-killing P. shumwayae do not secrete potent exotoxins; rather, fish mortality results from micropredatory feeding.