Using new molecular detection methods, a study headed by a University of North Carolina at Chapel Hill scientist found no indication that the Pfiesteria life cycle as originally described is unusually complex with many amoeba-like toxic stages.
Many of the stages were reported to be associated with fish kills in the Chesapeake Bay area and North Carolina estuary waters during the 1990s. Pfiesteria stages and their putative neurotoxin also were blamed for health problems, particularly cognitive impairment, among people exposed to the organism, local crabbers and several research laboratory technicians.
Instead, the new study showed a simple life cycle for Pfiesteria piscicida similar to many other marine organisms of its kind, the dinoflagellates.
The new findings are detailed in the June issue of the Journal of Phycology, a leading publication in marine algae sciences. Authors of the report are from UNC; the National Oceanic and Atmospheric Administration, which funded the research; and North Carolina State University College of Veterinary Medicine.
Dr. Wayne Litaker, a member of the Program in Molecular Biology and Biotechnology at the UNC School of Medicine, is principal author of the study.
In the 1990s, much of the regional news coverage portrayed Pfiesteria against a backdrop of fear and near-hysteria among coastal residents of Maryland and North Carolina. Later, Rodney Barker's book on the subject, "And the Waters Turned to Blood," raised a horrific specter of potential human disaster.
As many as 24 life cycle stages were originally described for Pfiesteria piscicida in a report published in the journal Limnology and Oceanography in 1997.
"These stages included amoebae and cyst forms not previously known in free-living marine dinoflagellates. Our study found no evidence to support the presence of these amoebae stages, many of which were originally reported to be toxic," said Litaker.
Litaker, also a National Oceanic and Atmospheric Administration fisheries research biologist at the Center for Coastal Fisheries and Habitat Research in Beaufort, N.C., said subsequent funding from the Beaufort Laboratory helped his study team develop the molecular tools to detect and monitor Pfiesteria.
Study co-author and colleague at Beaufort, Dr. Patricia A. Tester, said these tools arose "from an exciting transfer of technology from medical science to environmental science" and were based on finding a genetic sequence unique to the dinoflagellate. "Once a unique DNA sequence was identified, it was used to develop molecular detection methods that distinguished Pfiesteria piscicida from the many similar organisms found in the same environment," Tester said.
In their report, Litaker and his co-authors said that they had previously been unable to confirm many of the reported life cycle stages by using clonal cultures of Pfiesteria piscicida. One of these clones originated from a tank inoculated with water from North Carolina's Pamlico River in 1991 that had been killing fish since that time.
"Following two years of culture, isolates of P. piscicida were never observed to form amoeba. Only life cycle stages typical of free-living marine dinoflagellates were observed," said the authors.
They stress that the fish tanks sampled during their studies were maintained under conditions similar to those described by previous researchers to produce "Pfiesteria amoebae."
The researchers said amoebae were found in all tanks once fish were introduced, even during the acclimation period prior to introducing Pfiesteria. Fish almost universally harbor a number of microorganisms, they added, which means the tanks invariably contain many different contaminant species including amoebae.
"Thus, it is not possible to determine life cycle stages of P. piscicida from fish tanks without the use of species-specific molecular probes," they said. "Consequently, any ecological role or potential toxicity ascribed to specious P. piscicida stages based on the original fish tank studies should be reexamined."
To confirm the simplified Pfiesteria life cycle, the researchers "undertook a rigorous examination of the Pfiesteria piscicida life cycle using nuclear stains combined with traditional light microscopy, high-resolution video microscopy, electron microscopy and in situ hybridization with a suite of fluorescent labeled peptide nucleic acid (PNA) probes." This was one of the first applications of PNA technology in phytoplankton research.
Both the microscopy and molecular studies "did not support the life cycle of Pfiesteria piscicida" reported in 1992, 1993 and 1997 the authors said. "We describe instead, a very typical dinoflagellate life cycle," Litaker said.
Litaker and his collaborators acknowledged that their life cycle findings from fish tank microcosms would not have been possible without the carefully controlled use of the species-specific molecular probes. The PNA probes used in this study had not yet been developed when the original Pfiesteria life cycle study was undertaken.
What killed the fish? Some research has associated fish kills with hypoxic events, a lack of oxygen from bacterial overload and stratified water conditions in estuary waters at certain times of the year, Litaker said. "More recent studies in Virginia find Pfiesteria is common in many estuaries where there's absolutely no evidence of fish kill activity," he said. "In short, toxic Pfiesteria life cycle stages that don't exist can't be toxic."
Other co-authors with Litaker and Tester are Dr. Victoria J. Madden of the UNC School of Medicine, Dr. Edward J. Noga of N.C. State's College of Veterinary Medicine and Mark W. Vandersea and Steven R. Kibler from National Oceanic and Atmospheric Administration.
Note: Contact Litaker at 252-728-8774, 919-672-8881 or wayne_litaker@med.unc.edu. Contact Tester at 252-728-8792 after 19 June. School of Medicine contact: Les Lang at 919-843-9687 or llang@med.unc.edu
By LESLIE H. LANG
UNC School of Medicine
Journal
Journal of Phycology