Pfiesteria shumwayae kills fish by micropredation not exotoxin secretion

Abstract

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.

Demonstration of toxicity to fish and to mammalian cells by Pfiesteria species: Comparison of assay methods and strains

“…strains (1,11,14,16), was toxic to fish and to mammalian cells, and at environmentally relevant cell densities. We suggest that previous conclusions that this strain did not produce exotoxin (21)(22)(23) 25) is not a sufficiently sensitive indicator of toxin presence͞absence. Here, when filtrates from CCMP2089 were analyzed for PfTx by using cytotoxicity and ichthyotoxin assays, PfTx was confirmed.…”

“…P. shumwayae strains CAAE1024C and CCMP2089 (algae-or fish-fed) were tested in FMAs (one fish per 10-ml well, n ϭ 6, in triplicate; supporting information) by using Cyprinodon variegatus (age Ͻ25 days). Two sets of FMAs were used, with (7,12,21) versus without (1, 12) a filter partition (0.4-m porosity, Costar 3450) to prevent physical contact of dinoflagellates and fish. A cell density gradient was imposed by adding filtered media (0.22-m porosity, 15 practical salinity units) to effect initial Pfiesteria densities of 0 (controls), 0.25 ϫ 10 2 , 0.5 ϫ 10 2 , 0.8 ϫ 10 2 , 1 ϫ 10 3 , 2 ϫ 10 3 , 3 ϫ 10 3 , and 5 ϫ 10 3 cells per ml.…”

“…In the interim, various fish assays have been used in attempts to detect toxic Pfiesteria strains (5,6,(10)(11)(12)(21)(22)(23)(24)(25). Although some techniques have provided evidence of toxicity to fish (5,10,14,24), researchers using other methods have concluded that Pfiesteria is not toxigenic at the species [P. shumwayae (21,22)] or genus (23,25) levels. A comparative technique analysis was warranted to resolve these divergent conclusions, which have important policy implications in coastal resource management (3,26).…”

“…Here we compare the two techniques most frequently used to detect toxic Pfiesteria, standardized fish bioassays (SFBs) with juvenile fish (10) and fish microassays (FMAs) with larval finfish (1,21) or shellfish (7), for utility in detecting actively toxic Pfiesteria strains. SFBs, often maintained for weeks, originally were calibrated by using Pfiesteria spp.…”

“…CCMP2089 was less toxic, indicated by no fish mortality or epidermal lesions (as in ref. 21) in culture filtrate. However, additional tests yielded low fish mortality in filtrate from SFBs with CCMP2089 (6%, n ϭ 13 trials, 8 fish per trial; significantly different from controls with 0% fish death, P Ͻ 0.05) (24).…”