Effect of biotic and abiotic factors on in vitro proliferation, encystment, and excystment of Pfiesteria piscicida.

Pfiesteria spp. are mixotrophic armored dinoflagellates populating the Atlantic coastal waters of the United States. They have been a focus of intense research due to their reported association with several fish mortality events. We have now used a clonal culture of Pfiesteria piscicida and several new environmental isolates to describe growth characteristics, feeding, and factors contributing to the encystment and germination of the organism in both laboratory and environmental samples. We also discuss applied methods of detection of the different morphological forms of Pfiesteria in environmental samples. In summary, Pfiesteria, when grown with its algal prey, Rhodomonas sp., presents a typical growth curve with lag, exponential, and stationary phases, followed by encystment. The doubling time in exponential phase is about 12 h. The profiles of proliferation under a standard light cycle and in the dark were similar, although the peak cell densities were markedly lower when cells were grown in the dark. The addition of urea, chicken manure, and soil extracts did not enhance Pfiesteria proliferation, but crude unfiltered spent aquarium water did. Under conditions of food deprivation or cold (4 degrees C), Pfiesteria readily formed harvestable cysts that were further analyzed by PCR and scanning electron microscopy. The germination of Pfiesteria cysts in environmental sediment was enhanced by the presence of live fish: dinospores could be detected 13 to 15 days earlier and reached 5- to 10-times-higher peak cell densities with live fish than with artificial seawater or f/2 medium alone. The addition of ammonia, urea, nitrate, phosphate, or surprisingly, spent fish aquarium water had no effect.

Characterization of ichthyocidal activity of Pfiesteria piscicida: dependence on the dinospore cell density.

The ichthyocidal activity of Pfiesteria piscicida dinospores was examined in an aquarium bioassay format by exposing fish to either Pfiesteria-containing environmental sediments or clonal P. piscicida. The presence of Pfiesteria spp. and the complexity of the microbial assemblage in the bioassay were assessed by molecular approaches. Cell-free water from bioassays that yielded significant fish mortality failed to show ichthyocidal activity. Histopathological examination of moribund and dead fish failed to reveal the skin lesions reported elsewhere. Fish larvae within "cages" of variable mesh sizes were killed in those where the pore size exceeded that of Pfiesteria dinospores. In vitro exposure of fish larvae to clonal P. piscicida indicated that fish mortality was directly proportional to the dinospore cell density. Dinospores clustered around the mouth, eyes, and operculi, suggesting that fish health may be affected by their direct interaction with skin, gill epithelia, or mucous surfaces. Molecular fingerprinting revealed the presence of a very diverse microbial community of bacteria, protists, and fungi within bioassay aquaria containing environmental sediments. Some components of the microbial community were identified as potential fish pathogens, preventing the rigorous identification of Pfiesteria spp. as the only cause of fish death. In summary, our results strongly suggest (i) that this aquarium bioassay format, which has been extensively reported in the literature, is unsuitable to accurately assess the ichthyocidal activity of Pfiesteria spp. and (ii) that the ichthyocidal activity of Pfiesteria spp. is mostly due to direct interactions of the zoospores with fish skin and gill epithelia rather than to soluble factors.

Characterization of the rRNA locus of Pfiesteria piscicida and development of standard and quantitative PCR-based detection assays targeted to the nontranscribed spacer.

Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 micro g of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.