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Diving deeper into the Pfiesteria mystery. (Marine Science).

Although the dinoflagellate Pfiesteria has not triggered any major U.S. fish kills or human illness since 1998, controversy continues to swirl around the perplexing organism. In the latest challenge to the developing knowledge of the organism, two teams of researchers, with four overlapping members, say that one species of Pfiesteria does not kill by emitting a toxin, but by maiming and killing through other methods, possibly in conjunction with other organisms. Their studies were published 5 August 2002 online in Nature and in the 20 August 2002 Proceedings of the National Academy of Sciences (PNAS). Hoverer, an established Pfiesteria researcher is highly critical of their methods and conclusions.

The teams investigated the potential toxin-emitting properties of one strain of P. shumwayae through several avenues. The PNAS team began by culturing a clonal isolate on both algal prey and tilapia. They then exposed test fish to solutions from the-cultures and found that the fish died in about 12-72 hours. They centrifuged those tank waters to separate liquids and other materials--which presumably would have contained any toxin--from: the water, and exposed additional test fish to those redissolved substances. None died. They also exposed test fish to solutions that had been freeze-dried, extracted sequentially with dichloromethane and methanol, and redissolved in water, which again would presumably contain any toxin. Again, no fish died.

As additional tests, they used genetic analysis techniques to look for evidence of polyketides, which are synthesized by a family of enzymes known as PKSs and are the only group of ichthyotoxins known to be produced by dinoflagellates. They found evidence of some PKS-encoding genes, but concluded that these genes are involved in production of polyunsaturated fatty acids, not toxins. Based on these angles of investigation, the team concluded that P. shumwayae does not produce a toxin, although principal investigator Robert Gawley, a professor of chemistry at the University of Miami, says other species of Pfiesteria could still be producing a toxin.

The Nature team used similar methods, as well as other techniques, to expose test fish to waters containing P. shumwayae, but did not perform any genetic analysis. They observed relatively low mortality of test fish, up to about 25%, within 24 hours, and 92-100% mortality within 48 hours. Through use of scanning electron microscopy, they concluded that fish died after dinospores fed extensively on their skin. They also concluded that no toxins existed in the test waters, because none passed through membranes that were known to be permeable to several known toxins.

However, JoAnn Burkholder, a North Carolina State University professor of aquatic botany who has studied Pfiesteria for more than a decade, isn't surprised the teams found no toxins. "These people have no idea how to culture Pfiesteria," she says, "They have never worked with toxic Pfiesteria. You can't just throw a fish in a beaker and call it a day."

She points to differences between the peer-reviewed protocols published elsewhere and the methodology used in these studies to culture Pfiesteria and to allow expression of toxicity. She says the teams had higher ammonia concentrations and low pH in cultured solutions, and allowed excessively lengthy time periods for observing mortality. They studied only a single strain of the more than 400 known to exist. She also notes that the toxicity data for Pfiesteria have been cross-confirmed by multiple laboratories and published in more than 50 peer-reviewed articles. Several groups have found Pfiesteria toxin in pure cultures grown under conditions that allow them to express toxicity, she adds.

When asked if his team's methods were sound, Gawley says, "Yes, in a word." However, he acknowledges that something still unknown is occurring in fish-killing waters. "There's all kinds of stuff in [the water]. It may well be there's a toxin in the cultures the Burkholder group has. My question would be, who made it?"

Many facets of Pfiesteria, from its life cycle to its lethality, likely will continue to be controversial until ongoing research at numerous laboratories provides additional information. Tests for the organism continue to turn up positive in waters along the eastern U.S. seaboard and elsewhere in the world.
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Author:Weinhold, Bob
Publication:Environmental Health Perspectives
Date:Nov 1, 2002
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