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Amphibians on the edge: AAAS members on trail of chytrid fungus

About a third of the world's 6,260 known amphibian species are threatened by Bd and nearly 170 species are believed to have gone extinct in the last few decades. (Photo: Edward Kabay/ University of Maryland)

The once-frog-rich forest was eerily quiet when biologist Karen Lips, associate professor at The University of Maryland, returned to Las Tablas, Costa Rica in 1993, where she had been studying the stream ecology of frogs for three years. "I came back, and there were no frogs," recalls Lips, who puzzled over why she did find a handful of dead amphibians. "[It was] impossible to believe that they just went extinct from one year to the next."

So, due to the lack of abundant frog populations to study, she shifted her research from Costa Rica to Panama. And again, a few years later, she and students returned to the western Reserva Forestal of Fortuna, Panama to find another big die-off: 50 dead frogs within two weeks. "Every time you'd go out you'd see a dead frog on the ground," she recalls. "Things were just not right."

Years later, Lips and other scientists discovered exactly what the mystery killer was: a chytrid fungi called Batrachochytrium dendrobatidis, known simply as 'Bd,' that has threatened amphibians around the world — in some cases, to the point of extinction.

This fast-moving fungal disease has swept many of the world's amphibian populations, leaving silence and missing amphibians in its wake. About a third of the world's 6,260 known amphibian species are threatened by Bd and nearly 170 species are believed to have gone extinct in the last few decades.

View photos of Lips and her team searching for the chytrid fungus in Panama

Bd favors cool, moist conditions at higher elevations. Biologists would be hard pressed to find frogs to sample in the South American mountains, since there have been so many extinctions among endemic species, according to Lips.

"Bd is almost everywhere," Lips says. And it's likely our fault: Bullfrog farming, zoo missteps and the amphibian pet trade have transported the fungi around the world. Conservationists advocate increased biosecurity to halt Bd's entrance to the few places it hasn't yet touched, including Madagascar, Papua New Guinea, and parts of Asia.

AAAS followed up with three member biologists who are solving the mystery of chytrid's global invasion and searching for clues to help frog populations survive.

Deconstructing a mystery

To identify this strange infection on the frogs' skin, Lips says The Declining Amphibian Populations Task Force referred her to pathologist David Green, now at the USGS National Wildlife Health Center. A New York Times article reported on it, and other groups — including an Australian group and pathologists at the National Zoo — realized they were dealing with the same problem. In 1997, researchers and conservationists gathered to compare cases, and discovered that they were all looking at the same pathogen.

The following year, when frog chytrid showed up at the National Zoo, pathologists Allan Pessier and Donald Nichols, and mycologist Joyce Longcore, determined that it was Bd, a chytrid fungus.

Around the world, scientists puzzled over the sudden loss.

"The changes have been nothing short of dramatic, from very abundant populations to seeing tens of thousands of dead carcasses on lake shorelines," says biologist Vance Vredenburg, assistant professor at San Francisco State University and AAAS member, who's worked with yellow-legged frogs in the Sierra Nevada mountains in California for more than 15 years.

These frogs — which have lived in the area for some 5 million years — dwell in highly protected, 'pristine' habitats with roadless areas, Vredenburg explains. Soon after publishing studies that showed that frog populations would rebound if invasive species were removed, the chytrid appeared.

It was 2004 when chytrid hit Sixty Lake Basin in the Sierra Nevada mountains, where Vredenburg was studying populations. He returned to the area to find hundreds, if not thousands, of dead frogs washed up on the lakeshores.

The sight was devastating, Vredenburg recalls: "I had spent a decade trying to restore these populations, and they had come back, and now this microscopic killer was coming through and killing everything off."

During the next four years, Bd spread at a rate of about 700 meters per year. "So we went from tens of thousands of frogs in this one basin, to just a little over a hundred frogs in 2008," Vredenburg reports.

Also in 2004, Lips and her students witnessed chytrid hit Parque Nacional Omar Torrijos, near El Cope in central Panama: "It was like overnight, we went from hundreds of frogs to dozens of frogs, and it kept getting worse," she says.

A pattern began to emerge: a wave of frog fatalities moving from Costa Rica westward through Panama. Lips co-authored a paper that looked at the movement of Bd across Central and South America, showing chytrid's spread over time with a sequential loss of populations. "We were able to recreate our hypothesis of how we think Bd affected South America in the 1970s and '80s, before Bd was known," she says.

Unfortunately, there is limited reliable, long-term population data on amphibian populations. But anecdotal evidence abounds: Every time Lips gives a talk, listeners approach her and report that they used to have many frogs in their backyard, and these days they don't.

Now, Lips says, "from Mexico to the tip of South America, there really are no places that have not been affected by Bd."

Research hope

Biologists have found that Bd affects different amphibians in different ways: While some frog populations go extinct, others, such as the American bull frog (a known carrier of chytrid), seem to be able to cope with the pathogen.

Some biologists and conservationists believe that the answer to fighting chytrid is in the frogs' own biology. Reid Harris, professor of biology at James Madison University and a AAAS member, and his team are examining whether some amphibians have something that others don't.

Each amphibian species has its own set of symbiotic bacteria that dwell on the skin, explains Harris. And since Bd is a skin fungus, Harris set to work studying amphibians' skin bacteria.

"In a nutshell, what we're trying to do is find a skin probiotic that works for frogs, in order to inhibit this chytrid fungus," says Harris.

Get a look at Harris' work in the lab on anti-Bd metabolites

To find such a probiotic, Harris and his team are looking at the mutualistic bacteria's production of anti-Bd metabolites — such as violacein — that seem to kill off Bd.  They found the bacterially-produced metabolites on the skin of local Virginia salamanders in high enough concentrations to inhibit Bd.

Harris and his team are looking at what makes a good probiotic — one that sticks to amphibians and works with the amphibians' own immune system, including antimicrobial peptides produced by poison glands that can kill Bd. Such peptides, combined with the metabolites, would pack an extra-powerful punch to Bd. And, one probiotic may work on multiple species, Harris says, although research is still under way.

To put the probiotic Janthinobacterium lividum to the test, Harris collaborated with Vredenburg, who found that in Sierra Nevada field sites, populations that were surviving chytrid had a higher proportion of individuals that had the same bacteria species — Janthinobacterium lividum — that Harris had found on the East Coast.  Vrendenburg swabbed the frogs and sent samples back to Harris, who analyzed the probiotic. Then, in a clinical trial, individually marked yellow-legged frogs that were bathed in the probiotic in the field seemed to survive a year later.

"I think it has potential to work in other areas," says Vrendenburg, who had planned a larger experiment, but chytrid spread so fast that all but one population had been killed off in the basin.

"We still have a long way to go to understand more about the microbiome of these species," says Vrendenburg, "and to understand how important that is for the survival of these host species in the face of this new pathogen."

Harris and colleagues are looking at the possibility of putting the probiotic in soil or water to protect amphibians. This concept is already used in agriculture to fight pathogenic fungus in soils. "We're not talking about introducing anything new to the environment, you would be taking a bacteria that's already there and augmenting its abundance," says Harris. "We're taking it slowly, to see if works in a laboratory situation and perhaps in a localized stream bank."

Looking forward

While Lips and Vredenburg had the opportunity to witness the wave of Bd move through an area, they're also working to retrospectively pick up the pieces from chytrid's trail.

Since waves of Bd swept places such as South America before scientists could study its movement, Vredenburg and grad student Tina Cheng figured out a way to test museum specimens for Bd with a simple skin swab. "This is a way to look back through time," Vredenburg says. Both Vredenburg and Lips and their students are looking back through museum specimen collections from both Americas from the 1950s through the '80s, in hopes of finding out more about chytrid's history.

One way researchers and conservationists are trying to save struggling populations is by putting amphibians into ark survival assurance colonies. "The idea is to rescue these [species] before they go extinct," says Harris, who hopes a probiotic treatment would allow such species to be safely released back into nature.

View photos of Vredenburg's frog research in the Sierra Nevada mountains

Vredenburg's research has pointed towards evolution, as he tries to figure out why some host species in some populations are so susceptible to this pathogen, while others are not.  Lips agrees, seeing how much has been lost already, that the best course of action is to assist evolution as much as possible — whether it's a genetic protection or a naturally occurring microbe that resists infection.

"The more genetic combinations we can throw back out into the field, the better the chance of coming across something," says Lips, who advocates breeding frogs to build up numbers for wild populations. "Frogs should be able to evolve."

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About a third of the world's 6,260 known amphibian species are threatened by Bd and nearly 170 species are believed to have gone extinct in the last few decades. (Photo: Edward Kabay/ University of Maryland)
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