"DINO-TOXIN" OF FISH FOUND IN CHESAPEAKE BAY

TORONTO, Can. (BIO2002)--A species of marine algae, once misidentified as Pfiesteria by researchers investigating the cause of mid-Atlantic fish kills, has been found to be the more likely culprit in 1996 massive kills at HyRock Fish Farm on Maryland's Eastern Shore.

Scientists of the University of Maryland Biotechnology Institute (UMBI) have identified a potent toxin of Karlodinium micrum from algal cultures isolated from HyRock, open waters of the Chesapeake Bay, and from water samples collected during a fish kill in South Carolina.

The Chesapeake finding is the first confirmed toxin found in Bay waters from a dinoflagellate algae, the type often associated with algae blooms. Pfiesteria piscicida, another and better known dinoflagellate, was identified initially by several laboratories as the apparent cause of the HyRock fish kills. In extensive tests at UMBI, however, no toxin has ever been found from P. piscicida.

In recent years, scientists at UMBI's Center of Marine Biotechnology (COMB) in Baltimore have gathered evidence that K. micrum could have a primary role in fish kills. Since it was originally described in 1950, K. micrum has seldom been associated with adverse aquatic events such as fish kills, although it has commonly been misidentified as other similarly sized organisms. But COMB researchers found the toxin, after abandoning their investigation of a suspect fatty acid of the microbe--a false lead.

COMB marine biologists Jonathan R. Deeds and Allen R. Place found toxin in a sample of water sent to COMB by fellow researchers from the South Carolina Department of Natural Resources and the Belle W. Baruch Institute for Marine Biology. Fish had been dying as water turned progressively greener in a brackish water retention pond of a housing development near Charleston. The 1.6 L sample contained a remarkably high 68,000 K. micrum cells per ml. and yielded 1.3 milligrams of toxin. Deeds says, "This was much higher than I had gotten in cultures of the organism. We thought, 'What was going on there?'" In COMB laboratory tests, such concentrations of the toxin killed fish in 5 minutes.

In previous laboratory tests while investigating the HyRock algae bloom, they had established that K. micrum has a toxin associated with it. But now, COMB tests of the South Carolina samples showed K. micrum toxin in the water while fish were dying. The water conditions of the housing development pond were similar to those of the aquaculture ponds in 1996. In retrospect, says COMB's Allen Place, water samples from HyRock had yielded 65,000 cells per ml of K. micrum algae, known at the time as Gyrodinium galatheanum, but only 300 counts of P. piscicida. In initial studies of fish kills in the late 1990's, which were prior to molecular probe diagnostics by UMBI and others that clearly distinguish their identities, the two algae and a third species had all been lumped together and called "Pfiesteria-like" dinoflagellates-and blamed widely for the fish kills in the aquaculture ponds and similar fish kills in rivers in Maryland, Virginia and North Carolina.

COMB cooperating researcher Daniel E. Terlizzi, University of Maryland Sea Grant Extension, recalls raising some concerns about the high numbers of K. micrum from the ponds, but no toxin had yet been identified.

Further analysis at COMB yielded the toxin in HyRock algal cultures and the researchers learned that the organism only releases the toxin when agitated. In the laboratory, filtering or centrifuge spinning of a sample will release the toxin. In nature, it could possibly happen when cells are broken, sucked through fish gills, or chemically treated, offered the researchers. And that is just what had happened at HyRock in 1996. The majority of the fish died soon after managers treated the pond with copper sulfate to arrest a dense dinoflagellate bloom. Evidence now points to the treatment chemically breaking open the K. micrum cells and releasing the toxin.

[NOTE: in a related story, experiments both at HyRock and at COMB with ozone treatments of the ponds have been found to control the algae blooms and further to destroy the released toxins, say Deeds and Place.]

With the K. micrum toxin in hand, the COMB researchers asked officials at Maryland's Department of Natural Resources to be on alert for any high counts of K. micrum. In February, in a routine check for Pfiesteria and other toxic algae-a DNR monitoring operation intensified in recent years-they found and sent to COMB a sample of only 7,000 K. micrum cells per ml. It was from, ironically, Bloody Point, Md., a regular monitoring site for DNR in the southern area of the Chesapeake.

Deeds and colleagues at COMB used a molecular probe to confirm the presence of K. micrum and detected trace amounts of the toxin in the sample.

The researchers add that the two closest microbial relatives of K. micrum are the well-known toxic dinoflagellates: Karenia brevis (formerly Gymnodinium breve) which causes red tides and fish kills off the coast of Florida and Karenia mikimotoi (formerly Gyrodinium mikimotoi), a toxic fish killing algae in Japan.

Scientists now conclude that K. micrum has been an important component of the phytoplankton community in both the Maryland and Virginia portions of the Chesapeake Bay and perhaps in other mid-Atlantic states.

Meanwhile, it has been P. piscicida that has been implicated as the causative agent in numerous fish kills in both the mid-Atlantic and southeastern U.S. estuaries. P. piscicida's involvement in the incidents at HyRock still can't be ruled out, say the researchers. But considering now the repeated associations between high K. micrum cell numbers and fish mortality, combined with laboratory confirmation of toxic materials with effects similar to those observed during numerous kills, K. micrum does appear to have been a contributing factor in the HyRock fish kills.

Currently, it only seems to be a problem when in blooms in very high numbers in shallow, poorly flushed systems. The findings open a new concern for the estuarine aquaculture industry. "We don't yet know the true mode of toxicity. It does appear to be a membrane disrupter," explains Deeds. "We do know it's an easily released endotoxin and that it breaks up the tissue and sloughs off cells from small fish."

The research was conducted in cooperation with Jason W. Kempton of the South Carolina Department of Natural Resources, Marine Resources Research Institute, Charleston SC, and Alan J. Lewitus of the Belle W. Baruch Institute for Marine Biology and Coastal Research, Georgetown, SC.

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The University of Maryland Biotechnology Institute was mandated by the state of Maryland legislature in 1985 as "a new paradigm of state economic development in biotech-related sciences." With five major research and education centers across Maryland, UMBI is dedicated to advancing the frontiers of biotechnology. The centers are the Center for Advanced Research in Biotechnology in Rockville; Center for Agricultural Biotechnology in College Park; and Center of Marine Biotechnology, Medical Biotechnology Center, and the Institute of Human Virology, all in Baltimore.