The Cell From Hell
Scientific controversy surrounds elusive fish-killing microorganism

By Harold G. Marshall and Andrew S. Gordon

During a six-week period in 1991, more than 1 billion Atlantic menhaden died in North Carolina’s Pamlico River estuary. The subsequent hypothesis of this event was that the dinoflagellate Pfiesteria piscicida (microscopic single-celled algae) was the organism responsible for these deaths, as well as the cause of associated cases of human illness due to exposure to one or more Pfiesteria toxins.

Newspaper accounts of the massive Pfiesteria-related fish kill and the potential for human health problems stirred the imagination to the dangers of these organisms. The species’ myriad life cycle forms added mystique to this organism, which one reporter dubbed the “cell from hell.” The developing notoriety of Pfiesteria initiated further scientific interest in the toxins produced by this species, and raised the possibility of similar toxic species that might also be lurking in local waterways.

JoAnn Burkholder and her associates at North Carolina State University were the pioneer investigators in the Pfiesteria research, which our group of Old Dominion colleagues has since pursued. Burkholder was the first scientist to link the 1991 fish deaths to Pfiesteria, based on her team’s on-site investigations and controlled laboratory studies. In addition, she observed a complicated life cycle in the organism, including numerous morphological forms such as motile flagellated cells, amoebae and cysts that are able to survive in the sediment of estuaries until activated by the presence of fish to produce toxic motile cells.

Pfiesteria shumwayae, a second toxic member of this genus, was also identified by Burkholder and her associates, who have published their findings extensively over the past decade. Their research has shown that Pfiesteria species:
• Can produce an exotoxin that kills fish;
• Have some strains that produce and others that do not produce toxins;
• Have cells that act as predators, which can attack and kill fish; and
• Have a complex life cycle that includes amoeboid and cyst stages.

Further, their findings indicate that Pfiesteria toxins are harmful to humans, and that high concentrations of these cells, known as algal blooms, and fish kills associated with Pfiesteria have occurred in nutrient-rich waters.

The link between pollution and harmful algal blooms
Toxin-producing microscopic algae, including many dinoflagellates and diatoms, have long been associated with fish and shellfish poisoning, as well as human illness. This has been of great concern lately due to a worldwide increase in the occurrence of toxic and non-toxic algal blooms in our rivers and coastal regions. Many of these blooms have been associated with increased water pollution from agricultural, commercial and industrial sources. Among the pollutants, of primary concern are increased concentrations of nitrogen and phosphorus. Also, algae introduced through the ballast water discharge of ships from other regions have been known to produce harmful blooms. In the Chesapeake Bay, we have identified 19 algal species that can produce toxins and which represent potential threats to the regional fish and shellfish populations. To date, only Pfiesteria piscicida has been linked to the approximately 50,000 fish deaths that have occurred since 1997 in several Chesapeake Bay tributaries in Maryland. Further research is needed to understand this unique organism. Because it has been studied only in the past decade, its manner of toxicity to fish is still not clearly understood and its toxin has not been fully identified.

Pfiesteria research at Old Dominion University
At Old Dominion, we have conducted research on Pfiesteria since 1997. This effort initially was a component of our phytoplankton studies under the Chesapeake Bay Monitoring program, but later expanded to a multidimensional program involving faculty, technicians and graduate students from several departments in the College of Sciences. Under the sponsorship of the Centers for Disease Control and Prevention, the Virginia Department of Health and the Virginia Department of Environmental Quality, our Pfiesteria research has centered on the environmental relationships and life cycle studies of Pfiesteria species, the detection of Pfiesteria in Virginia estuaries, fish bioassays of Pfiesteria-associated toxins, investigations using various strains of Pfiesteria species, and the isolation, characterization and detection of Pfiesteria toxins. Recently, another component of this program has embarked on a study of animal behavioral response to Pfiesteria toxin exposure. Co-investigators in our Pfiesteria studies are Keith Carson, associate professor of biology, Slawomir Cerbin, postdoctoral research assistant in biological sciences, Perry Duncan, associate professor of psychology, William Dunstan, professor and eminent scholar of ocean, earth and atmospheric sciences, Wayne Hynes, associate professor of biology, and David Seaborn, visiting assistant professor in biological sciences.

We have been fortunate to be engaged in numerous inter-laboratory studies involving investigators from several universities and state agencies within and outside of Virginia (e.g., Delaware, Maryland, North Carolina and South Carolina). We have been able to exchange toxic cultures, share research findings and conduct external verification of investigative results. These cooperative studies are particularly helpful in addressing two major concerns associated with Pfiesteria research initiatives. The first is that since there are strains of this species that produce and do not produce toxins, a distinction between the two is imperative and requires the appropriate culturing protocols to maintain the toxin-producing strains. The second concern is that because Pfiesteria toxins are suspected of causing human illness, including respiratory ailments, temporary memory loss and skin lesions, they must be maintained under strict health control measures. Experiments must be conducted in a Biohazard Level III facility, where researchers are required to wear appropriate protective respiratory gear. In 1998, we were able to create a Biohazard Level III facility on campus, where we have maintained toxic cultures of both Pfiesteria piscicida and Pfiesteria shumwayae. These cultures are the source of the cells used in our investigations and have been exchanged with our fellow scientists who are conducting similar research.

Controversy over the life cycle and toxicity of Pfiesteria species
This past year, a controversy arose in the scientific community regarding the toxicity and life cycle of Pfiesteria shumwayae. The authors of one publication questioned this species’ ability to produce a toxin and stated that it causes fish deaths by micro-predatory feeding. Their conclusion that Pfiesteria predation can kill fish larvae under certain conditions was previously documented by Burkholder. Another publication questioned whether Pfiesteria piscicida has an amoeboid form in its life cycle, where the investigators did not find evidence of amoeboid stages in their cultures or through genetic analysis. Prior to these publications, Burkholder and her associates reported that Pfiesteria species possess a complex life cycle with numerous optional paths of development, several including the formation of a temporary amoeboid stage. From the hundreds of Pfiesteria strains they have studied since 1991, they also documented that there are numerous strains of Pfiesteria species that vary in their ability to produce toxins, and that there are strains which produce no toxins.

We approach the observation of different life cycle forms by beginning with clonal cell cultures of the species at the motile stage. By subjecting these cells to different environmental stimuli, different life forms can be established. In our study of Pfiesteria clonal cultures, various life forms have been identified, including amoeboid forms which were verified as Pfiesteria using molecular protocols from our laboratory. This was also confirmed by an independent, external laboratory. Studies in our lab have also identified other life forms of Pfiesteria from our cell cultures, supporting Burkholder’s earlier findings. Utilizing clonal cultures she supplied, we have confirmed the presence of fish-killing toxins in cell-free filtrates (filtered liquids) from fish-killing cultures of Pfiesteria piscicida and Pfiesteria shumwayae. In contrast, other strains from our culture collection have proven to be non-toxic to fish. However, toxin-containing filtrates are not as lethal to fish as intact cultures containing active Pfiesteria cells. Thus, a combination of predation and toxin excretion may contribute to fish death in these cultures. Furthermore, studies conducted by ODU psychologist Perry Duncan have confirmed that the Pfiesteria toxin affects learning performance in rats. With support from the National Oceanic and Atmospheric Administration, we are in the process of developing a quantitative analysis for Pfiesteria toxins that can be used at fish-kill sites and which will allow us to rigorously test the connection between the toxins and fish kills in the field.

Inaccurate media coverage and efforts to promote the dissenting viewpoint in reference to Pfiesteria toxicity and its life cycle have produced some negative repercussions. Aspersions have been cast on the integrity of reputable scientists, and certain reported statements have reduced the public concern regarding a potential threat from this toxic organism. Our evidence, however, suggests that when the environmental conditions are suitable, the Pfiesteria organism is, indeed, potentially capable of producing extensive fish kills in our estuaries.

Fortunately, when controversies of this type develop and different opinions are expressed, the natural recourse in science is to resolve these questions, or doubt, through further investigative efforts. At Old Dominion, as well as other schools and agencies, the research will continue.

Harold G. Marshall is Morgan Professor Emeritus and eminent scholar of biological sciences at Old Dominion University. His particular interests include marine ecology and marine and freshwater phytoplankton.

Andrew S. Gordon is a professor in the Department of Biological Sciences at Old Dominion University. His teaching and research interests include marine microbial ecology, general microbiology and bacteria.