Edging Extinction
UMaine mycologist's research essential in the global race to stem amphibian die-offs

About the Photo: Nearly a decade ago, University of Maine mycologist Joyce Longcore identified a particular species of chytrid, B. dendrobatidis, left, that has been implicated in massive amphibian die-offs around the world. The aquatic fungus was first thought to kill all amphibians it infected. However, Longcore's study of frogs and toads throughout the Northeast, and especially in Maine, found the "frog chytrid" statewide in healthy amphibians. Why the chytrid kills some and not others remains a mystery that researchers worldwide are racing to solve.

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From cool, swirling streams in California's High Sierra to foggy mountaintops in western Peru, frogs have begun to disappear, wiped out by a mysterious fungus that can cause entire populations — even entire species — to vanish.

The pandemic threat has helped to drive more than 100 species to extinction in the past 25 years, leaving researchers and conservationists to wonder how frogs can be saved.

Some of the answers may well be found in a refrigerator in a biological sciences laboratory at the University of Maine.

Dressed in faded jeans and a "Deep Hyphae" tee shirt, UMaine mycologist and associate research professor Joyce Longcore explained the fine points of fungal phylogeny with her good-natured and characteristically direct approach. A respected researcher in a highly specialized

field, Longcore's techniques have become required reading for anyone wishing to study one of the world's deadliest fungi, Batrachochytrium dendrobatidis.

Patient and unassuming, Longcore has the dedication and experience to guide the operation of a tiny, two-room lab in Deering Hall, which could easily be described as command central for the global battle to stop the fungus that threatens the world's frogs.

Longcore has become one of the world's leading authorities on an unusual group of fungi casually known as the chytrids. That fact alone might have left her to work in relative anonymity outside of mycology circles, had it not been for the discovery of a particular species of chytrid, B. dendrobatidis, that has been implicated in massive amphibian die-offs around the world.

The first to isolate a pure culture of B. dendrobatidis nearly 10 years ago, Longcore has made numerous trips to remote locales to teach other researchers her methods for culturing the problematic pathogen. Her laboratory refrigerator has become the repository of numerous strains of the so-called "frog chytrid" from around the world.

"The chytrids are different from most other types of fungi. You have to use very particular methods to be successful because they just don't grow well in pure culture," says Longcore, pointing out a tiny white smear growing inside a test tube. "B. dendrobatidis is peculiarly adapted to amphibian skin."
 

The affinity of the fungus for frog skin is key to its growing lethality. From California to Australia, entire populations of frogs, toads and other amphibians have been wiped out by the chytrid, leaving researchers scrambling to determine how it kills and, perhaps more importantly, how it spreads among populations.

Longcore maintains more than 100  different strains of the frog fungus and distributes them to scientists for research projects ranging from small field studies to full-blown genome projects. Despite considerable attention in recent years, the enigmatic organism has led to more questions than answers.

"We know that it grows in the skin, causing it to thicken, and we suspect that it interferes with the movement of air and water through the skin, but no one is exactly sure how it kills the host. We know that it does not need an amphibian host to survive, but we have never seen it in a resting stage," says Longcore. "There's still a lot that we just don't know."

One of the most perplexing questions surrounding B. dendrobatidis infections has to do with the degree to which the pathogen affects the host. Working with her husband Jerry Longcore, a field biologist with the U.S. Geological Survey, now retired, Longcore determined that the same organism that had caused near 100 percent mortality in amphibian populations elsewhere was present in frogs throughout Maine, causing only a mild, sublethal infection.

"We looked at road-killed animals and were surprised to see frogs that had been healthy with the frog chytrid. Until then, we had always associated an infection with death," says Longcore. "We now know that most species of anuran amphibians in Maine are infected, but as far as we know there have been no die-offs attributed to the disease."

Longcore first isolated the deadly chytrid in 1997 in response to a die-off of exotic frogs in captivity. At that time, the Smithsonian National Zoological Park in Washington, D.C., was raising blue poison dart frogs. But during metamorphosis, the change from tadpole to frog, the amphibians native to South American rainforests were dying of unknown causes. The only clues were the spherical bodies inside their skin cells.

The search for answers led to Longcore.

"As soon as I saw the photographs, I knew (the culprit) was a chytrid," says Longcore. "I had spent the last 10 years isolating chytrids and growing them in pure culture. The blue poison dart frog was the first (amphibian) from which I isolated this particular chytrid, then we showed that it is capable of causing disease and death."

It turns out that around the same time, scientists in Australia found the organism decimating populations of frogs in the wild. In addition, a researcher doing frog surveys in Central America returned to the rainforest for a consecutive year to find the wilds eerily quiet, with frogs dead along the streams.

The big questions have to do with what the deaths mean environmentally and ecologically. Are frogs that are dying of chytrids harbingers of a yet unseen shift in the ecosystem, much like a canary in a coal mine? Or is this an invasive disease that has spread from a different continent?

Longcore is quick to point out that she is not a frog biologist. Her focus is on the fungi. Her current projects focus on the relationships between chytrid species and their place among other fungi. She cooperatively published the first phylogeny of chytrid fungi in 2000, and she and colleagues are currently making use of recent advances in molecular classification methods to improve and expand on previous work. By determining how the strange and highly mobile chytrids are related, Longcore hopes to contribute to their reclassification and make it possible to study their role in the ecosystem.
 

Constantly looking for ways to improve chytrid culture and identification techniques, Longcore has become an expert on the behavior of the fungi. As a result, one of her primary roles in many research programs by other scientists is to locate and identify chytrid species in soil and water samples. To do that, she goes "fishing."

Using tiny bits of shrimp chitin, onion skin, pollen and other baits, she attracts the zoospores of carbohydrate-and-protein-loving chytrids, growing them so they can then be isolated and identified. It's fishing on micrometer scale, and a prize catch means a pure culture of a chytrid for someone's research project.

"A big part of what we do is hunt for different chytrids in samples that are sent here," says Longcore "Right now, we have samples sent from mountain tops on three different continents. With the baiting technique, instead of looking for a needle in a haystack, we can draw the chytrids in."

Once the individual chytrids grow on the baits, Longcore and graduate student Rabern Simmons can begin the daunting task of culturing the uncooperative fungi. When working with the frog chytrid, they must maintain strict safety protocols to ensure that the cultures don't escape into the environment.

Established cultures are kept in two refrigerators in Longcore's lab; back-up specimens are frozen in liquid nitrogen in case the active cultures fail. Establishing and rotating the ever-growing collection is time-consuming work, but there is certainly no shortage of researchers requiring samples and Longcore's expertise.

Recently, one of Longcore's cultures was used by researchers at Duke University to isolate high-quality DNA, which was sent to the Broad Institute, where scientists from MIT and Harvard University are sequencing the B. dendrobatidis genome.

Longcore's frog chytrid cultures also are being used by Cornell University researchers who are examining the role of repeats in the organism's genetic code, and by U.S. Fish and Wildlife Service technicians seeking to determine the risk of spreading the disease to new bodies of water through fish stocking programs.

"We have a lot of different strains and there are a lot of different projects. Some researchers will ask for one and some will ask for multiple strains from different areas," Longcore says as she checks some samples under the microscope.

"Right now we have isolates from Africa going out for a study in population genetics. Just yesterday we sent an isolate to Montana for some DNA research. It definitely keeps us busy."
 

By David Munson

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