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

doi:10.1073/pnas.0500168102

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

^*Center for Applied Aquatic Ecology and ^¶College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606; ^‡Department of Biology, Old Dominion University, Norfolk, VA 23529; ^§Marine Biotoxins Program, National Oceanic and Atmospheric Administration–National Ocean Service, Charleston, SC 29412; ^∥College of Marine Studies, University of Delaware, Lewes, DE 19958; ^**Baruch Institute, University of South Carolina, Georgetown, SC 29442; ^††South Carolina Department of Natural Resources, Charleston, SC 29412; and ^‡‡Department of Biology, University of North Carolina, Greensboro, NC 27402

Communicated by Marye Anne Fox, University of California at San Diego, La Jolla, CA, January 7, 2005 (received for review May 20, 2004)

Abstract

Toxicity and its detection in the dinoflagellate fish predators Pfiesteria piscicida and Pfiesteria shumwayae depend on the strain and the use of reliable assays. Two assays, standardized fish bioassays (SFBs) with juvenile fish and fish microassays (FMAs) with larval fish, were compared for their utility to detect toxic Pfiesteria. The comparison included strains with confirmed toxicity, negative controls (noninducible Pfiesteria strains and a related nontoxic cryptoperidiniopsoid dinoflagellate), and P. shumwayae strain CCMP2089, which previously had been reported as nontoxic. SFBs, standardized by using toxic Pfiesteria (coupled with tests confirming Pfiesteria toxin) and conditions conducive to toxicity expression, reliably detected actively toxic Pfiesteria, but FMAs did not. Pfiesteria toxin was found in fish- and algae-fed clonal Pfiesteria cultures, including CCMP2089, but not in controls. In contrast, noninducible Pfiesteria and cryptoperidiniopsoids caused no juvenile fish mortality in SFBs even at high densities, and low larval fish mortality by physical attack in FMAs. Filtrate from toxic strains of Pfiesteria spp. in bacteria-free media was cytotoxic. Toxicity was enhanced by bacteria and other prey, especially live fish. Purified Pfiesteria toxin extract adversely affected mammalian cells as well as fish, and it caused fish death at environmentally relevant cell densities. These data show the importance of testing multiple strains when assessing the potential for toxicity at the genus or species level, using appropriate culturing techniques and assays.

Footnotes

↵ † To whom correspondence should be addressed. E-mail: joann_burkholder{at}ncsu.edu.
Author contributions: J.M.B., A.S.G., P.D.M., J.M.L., K.J.C., A.J.L., H.G.M., N.J.D., S.C.C., J.W.K., and P.A.R. designed research; A.S.G., P.D.M., J.M.L., K.J.C., N.J.D., J.W.K., and S.L.M. performed research; J.W.K. contributed new reagents/analytic tools; J.M.B., A.S.G., P.D.M., J.M.L., K.J.C., A.J.L., J.S.R., H.G.M., N.J.D., S.C.C., J.W.K., and P.A.R. analyzed data; and J.M.B., A.S.G., P.D.M., J.M.L., K.J.C., A.J.L., and H.G.M. wrote the paper.
Abbreviations: CAAE, Center for Applied Aquatic Ecology; CCMP, Culture Center for Marine Phytoplankton; DGGE, denaturing gradient gel electrophoresis; FMA, fish microassay; LC₅₀, 50% lethal concentration; NON-IND, noninducible strain, unable to kill fish with toxin; PfTx, hydrophilic Pfiesteria toxin; SFB, standardized fish bioassay; TOX-A, actively toxic.
↵ §§ P. piscicida CCMP1832 rDNA sequence is GenBank accession no. AF077055; P. shumwayae CCMP2089 rDNA sequence is GenBank accession no. AF218805.