deadly or not?
by Angela Spivey

Pfiesteria stands charged with killing fish and endangering people, as well. But could this be a case of mistaken identity?

emember Pfiesteria? In the late 1990s, North Carolina newspaper headlines warned that this single-celled water creature was killing fish. And maybe, they said, it could even harm you. Fishermen and tourists exposed to suspect waters, as well as researchers studying Pfiesteria in laboratories, had reported memory loss and other cognitive impairment.

The tiny creature seemed not only dangerous, but bizarre. The first scientific studies reported a 24-stage life cycle never before seen in this type of organism (a marine dinoflagellate). Pfiesteria's shapeshifting included unusual amoeba and cyst stages. According to the reports, Pfiesteria showed its amoeba forms only in the presence of fish — when it was in killing mode. Many of these amoeba stages were reported to be toxic.

.: TOP LEFT: a Pfiesteria dinospore fluorescing after binding with a Pfiesteria-specific genetic probe. CENTER and LOWER RIGHT: Amoebas from the same sample do not fluoresce when tested with the Pfiesteria probe — they don't contain unique Pfiesteria DNA and are not Pfiesteria amoebas. Used by permission of the Journal of Phycology.Click to enlarge. :.

Now a team of researchers, having reexamined Pfiesteria using some new tools of medicine, are questioning the idea that the animal produces a toxin. In June 2002, research biologist Wayne Litaker and colleagues released results of their meticulous effort to precisely characterize Pfiesteria using a tool that has been around for only three years — peptide nucleic acid (PNA) probes. The team's findings suggest that Pfiesteria isn't so complex after all. The creature, the researchers say, shows a normal life cycle much like those of other dinoflagellates. The team published their findings in the June 20 issue of the Journal of Phycology, known as the leading journal on algae.

he project began with Pat Tester, plankton research team leader at the National Oceanic and Atmospheric Administration's (NOAA) Center for Coastal Fisheries and Habitat Research in Beaufort, N.C. She had originally studied Pfiesteria in collaboration with N.C. State University scientist Joann Burkholder, who first identified Pfiesteria and observed the unusual life cycles. Tester began working on the organism on her own after discovering that there were many look-alike species living in estuary waters with Pfiesteria, which made it impossible to measure. She brought Litaker on board to develop molecular tests to definitively detect the creature.

For years, Litaker has worked in two different disciplines and in two different towns. Until recently he spent part of his week working for the Program on Molecular Biology and Biotechnology at Carolina, organizing and teaching two-week, hands-on training sessions in molecular biology techniques. He spent the rest of his time as a research biologist at NOAA's Beaufort research center. That's a lot of traveling and juggling, but Litaker calls it fun. "I'm one of those people who gets paid to do what they love to do," he says. "You're welcome to come and play in my lab anytime."

Constantly fidgeting, Litaker explains his favorite toys — the molecular biology techniques he has spent years teaching — and how he and his colleagues applied them to marine biology.

The team started by carefully isolating the creature — they wanted to make sure they were studying Pfiesteria and nothing else. They started pure cultures of Pfiesteria from different sources, including water from North Carolina's Pamlico River, where Pfiesteria was reported to be killing fish. Using a capillary tube that they melted into a fine, hollow thread, the researchers drew up the cultures one cell at a time. They cleaned the cell by transferring it into a new drop of sterile media and then drew the cell up into another pipette. They repeated this process until they were sure they had isolated a single cell. Now their Pfiesteria culture contained only two things — Pfiesteria and a species of algae known as Rhodomonas, which the researchers added as Pfiesteria's food.

Next the team observed their cultures — in tanks all alone, in tanks with fish, in tanks with algae. They waited and waited to see the 24 life stages that other researchers had attributed to Pfiesteria. "Two years — nothing," Litaker says. The researchers used video microscopy to record Pfiesteria's normal dinoflagellate activities — the creature swam around using flagella, and when it had eaten enough algae, it dropped to the bottom and temporarily formed a round cyst. Some forms also went through mitosis — cell division or asexual reproduction — while others reproduced sexually.

f they hadn't before, the researchers began to doubt the 24-stage life cycle. But how could they get to know this creature? Pfiesteria is notoriously hard to distinguish from other species with light microscopy and other techniques. Litaker's team decided to bring out their medical tools — molecular probes. "These probes work because no matter what life stage your critter is in, the DNA stays the same," Litaker says.

To make such a probe, scientists sequence the DNA of a creature until they find a piece of DNA that is unique to that organism. Litaker's group sequenced a large segment of DNA from Pfiesteria and from a number of related species until they found a unique piece of Pfiesteria DNA. Instead of DNA probes, the team made PNA probes — synthetic cousins of DNA probes that work the same way but bind tighter and longer and aren't easily degraded.