1999 Progress Report: The Toxic Dinoflagellate, Pfiesteria piscicida, as a Potential Biosensor of Estuarine StressEPA Grant Number: R825551
Title: The Toxic Dinoflagellate, Pfiesteria piscicida, as a Potential Biosensor of Estuarine Stress
Investigators: Burkholder, Joann M. , Rublee, Parke , Shumway, Sandra E.
Institution: North Carolina State University , Long Island University - Southampton College , University of North Carolina at Greensboro
Current Institution: North Carolina State University , Long Island University - Southampton College , University of North Carolina at Greensboro
EPA Project Officer: Hiscock, Michael
Project Period: May 19, 1997 through May 18, 2000 (Extended to May 18, 2001)
Project Period Covered by this Report: May 19, 1999 through May 18, 2000
Project Amount: $500,000
RFA: Harmful Algal Blooms (1997) RFA Text | Recipients Lists
Research Category: Aquatic Ecosystems , Water Quality , Water , Ecosystems
Objective:The objectives of this project are to: (1) examine controlling influences of both inorganic and organic nitrogen (N) and phosphorus (P) on growth of Pfiesteria piscicida zoospores (toxic and nontoxic), when given food resources as dissolved or particulate (algal) carbon; (2) improve detection of Pfiesteria piscicida through development of a molecular probe for this species; and (3) assess Pfiesteria's acute and sublethal/chronic impacts on life stages of commercially/ecologically valuable shellfish species.
Progress Summary:We obtained research-quality clones of toxic Pfiesteria piscicida as well as the second known toxic Pfiesteria species, P. shumwayae sp. nov., from a fish kill site on the Neuse Estuary, NC (repeated twice in this project year; toxicity of the clones, and all other steps cross-confirmed by independent laboratories having demonstrated expertise in toxic Pfiesteria research).
In nutrient ecology research, we experimentally demonstrated that the functional type of Pfiesteria (actively toxic - TOX-A; temporarily nontoxic [no access to live fish] but capable of toxin production - TOX-B; and non-inducible or incapable of toxin production-NON-IND) used in experiments is extremely important in data interpretations about the behavior and ecological responses of Pfiesteria. The three functional types were found to differ significantly in their responses to N and P enrichments, algal prey, and fish prey. Response to N or P enrichments by NON-IND Pfiesteria spp. was highest, with intermediate response by TOX-B (no recent access to fish), and lowest response by TOX-A Pfiesteria (recently in fish-killing mode). Responses to algal prey were highest for TOX-B Pfiesteria, and intermediate for NON-IND Pfiesteria (which was "plantlike," with extended retention of kleptochloro-plasts and strongest apparent reliance on photosynthesis in carbon acquisition). The TOX-A functional type (both Pfiesteria spp.) rapidly consumed algal prey, but did not translate that consumption into cell production so that cell densities of TOX-A Pfiesteria remained low. In marked contrast, TOX-A Pfiesteria showed strong, rapid attraction to fresh substances (mucus, secreta) in a microcapillary tube assay, whereas TOX-B Pfiesteria showed low response, and NON-IND Pfiesteria did not respond at all to the fish materials. In addition to the insights gained about the comparative ecology of the three functional types of Pfiesteria, the data raise a "red flag" in warning against use of the available NON-IND strains in research to gain insights about environmental controls on toxic Pfiesteria. In molecular probe development, PCR and fluorescent in situ hybridization (FISH) probes were used to assay for the presence of Pfiesteria piscicida in 170 estuarine water samples collected from New York to northern Florida. Approximately 20 percent of the sites tested positive for the presence of P. piscicida, including sites where fish kills due to Pfiesteria have occurred and sites where there was no historical evidence of such events. This study extended the known range of P. piscicida northward to Long Island, NY. The results also suggested that P. piscicida is a common, and normally benign, inhabitant of estuarine waters of the eastern United States. We also worked to employ a sequencing strategy directed by heteroduplex mobility assay to detect P. piscicida 18S rDNA "signature" sequences in complex pools of DNA. We used those data as the basis for determination of the complete P. piscicida 18S rDNA sequence. Specific PCR assays for P. piscicida and other estuarine heterotrophic dinoflagellates were developed, permitting their detection in algal cultures and estuarine water samples collected during fish kill and fish lesion epizootic events.
In shellfish research, we assessed the response of representative adult and pediveliger eastern oysters to zoospores of Pfiesteria piscicida. We also examined behavioral responses of Pfiesteria to larval oysters and bay scallops (n = 5), and survival of Pfiesteria zoospores consumed by the adults. Both TOX-A and TOX-B zoospores sometimes attacked and consumed oyster and scallop pediveliger larvae that had discarded their vela (only adductor muscle tissue remained in minutes). Apart from this predation effect, there also was a toxicity impact. When P. piscicida was maintained within dialysis membrane to prevent direct contact with pediveligers, there was high pediveliger mortality when exposed (1 hour) to TOX-A Pfiesteria, but negligible mortality with NON-IND Pfiesteria. In another experiment, oyster pediveligers grazing activity was highest on NON-IND zoospores and lowest on TOX-A zoospores. In contrast, adult oysters were able to graze high quantities of toxic Pfiesteria, although grazing was much higher on NON-IND zoospores. However, 90 percent of the TOX-A zoospores survived passage through the adult oyster digestive tract by forming temporary cysts, with moderate survival by TOX-B and NON-IND zoospores as well. The data indicate that toxic Pfiesteria zoospores can potentially affect recruitment and survival of commercially important shellfish species. The demonstrated ability of adult oysters to remove toxic zoospores from the water column indicates a potential, as well, for trophic mitigation/control of toxic Pfiesteria outbreaks.