small frog sits on a person's fingerA diverse group of WSU scientists share a common, critial goal: to prevent the occurrence of a second fungal pandemic—an explosive threat looming just over the horizon.

Their collective efforts have put WSU in the national spotlight as an emerging center for amphibian research.

Excerpted from “Where have all the frogs gone?” in Washington State Magazine

Along a muddy path around the little pond at Virgil Phillips Farm Park just outside Moscow, Idaho,  Caren Goldberg assembles her eDNA collection system. Dressed in jeans and tall rubber boots, she kneels in faded grass near the edge of the water where two male Columbia spotted frogs have staked out territories.

researcher near a pond
Caren Goldberg at Virgil Phillips Farm Park (Photo Robert Hubner/WSU)

She quietly lowers a plastic tube into the pond and uses a hand pump to draw water up through a filter and into a flask. With tweezers, she carefully removes the wafer-like filter and stuffs it into a test tube.

Goldberg, an assistant professor in the School of the Environment, is a pioneer in the development of environmental DNA (eDNA) techniques that simplify the ability to screen for pathogens like Batrachochytrium dendrobatidis (Bd) that can be spread through the international pet trade.

Back in her laboratory, she will extract DNA from the skin cells, feces, urine, and other bits of material left behind by aquatic inhabitants. The DNA is then run through assays to identify target species of fish, amphibians, snails, turtles, and other creatures. With that one sample, Goldberg can also detect rare and invasive species as well as disease-causing organisms like Bd and ranavirus.

As one of the world’s leading amphibian eDNA researchers, Goldberg analyzes more than a thousand samples each year from all over the world. She and her team have developed nearly 50 assays, each uniquely designed for a particular species.

Not only does eDNA improve and simplify the process of monitoring aquatic species, it’s also safer, more efficient, and minimally invasive. Now, instead of tromping through fragile wetlands—turning over rocks and kicking up mud, which can harm the animals living there—scientists can get answers with only a few water samples.

When Goldberg first learned of the concept as a graduate student in 2008, it transformed her world.

“As an ecologist, I spend a lot of time looking for rare species out in the field and not always finding them even though we know they are probably there,” she says. “When I heard about eDNA that detected amphibians in water, I was so excited. I knew it could have huge implications for managing and conserving rare species.”

By 2011, Goldberg had a contract with the Department of Defense to bring eDNA surveillance into the real world as a practical tool for wildlife conservation. Joined by fellow researchers Katherine Strickler and Alex Fremier in the School of the Environment, they set out to develop reliable techniques that would enable them to detect rare amphibians and fish on military bases across the United States.

“Our military bases are some of the last preserved parts of ecosystems that have otherwise been developed or plowed under,” Goldberg says. “They contain a lot of the nation’s endangered species. If you think about it, even a bombing range, for example, is much less disturbance to a salamander than is a shopping mall.”

She began the project by adapting protocols for working with poor-quality DNA that she’d learned as a doctoral student at the University of Idaho. In 2015, she joined WSU and designed her lab to use these new methods for processing eDNA samples.

Recently, Goldberg, Strickler, and WSU wildlife biologist Jeff Manning were awarded another $1.4 million DoD contract to continue improving eDNA detection especially for species that are very rare and present in low numbers. They want to increase test sensitivity to handle some of nature’s most challenging conditions such as highly acidic water or very large ponds.

The biggest challenge for eDNA surveillance, however, may lie in the frontline battle to prevent a deadly salamander fungus from entering the United States and other vulnerable parts of the world.

In 2013, scientists were alarmed to discover massive salamander die-offs occurring throughout Europe from a new strain of chytrid fungus similar to Bd. Known as Batrachochytrium salamandrivorans, Bsal, or salamander chytrid, the disease is especially threatening to the United States, a global hotspot for salamander biodiversity.

Thanks to lessons learned during the frog Bd pandemic, the new infection was quickly identified and international barriers were established to prevent spread of the pathogen. By 2016, the U.S. Fish and Wildlife Service had banned imports of 201 salamander species.

researcher holds a turtle near a pond
Jesse Brunner with painted turtle. (Courtesy Jesse Brunner)

Jesse Brunner, associate professor in the School of Biological Sciences and another member of the National Bsal Task Force says the fungus has not yet been detected in North America.

“That’s really a good thing,” he says. “Allan Pessier (a pathologist in the Washington Animal Disease Diagnostic Laboratory (WADDL) and clinical associate professor in the College of Veterinary Medicine), Caren Goldberg, and I are working on developing better approaches to screen animals and try to prevent it getting here. Millions of amphibians are imported into the U.S. every year, mostly through the pet trade.

“It’s very unregulated—we know Bd is found in some of these animals,” Brunner says. “We want to use eDNA testing to screen a whole shipment at a time rather than test each animal individually. The idea is that we can collect a handful of samples from the water and have a high probability of ensuring there isn’t infection in that group of animals.”

Worldwide surveys indicate these infectious fungi likely emerged from Asia where over millions of years, the local amphibian species developed a resistance to it.

“The exact origin may be uncertain but what is clear is that the movement of animals for the pet trade is moving pathogens like Bd and Bsal around the world,” says Brunner. “So, we can expect to see more emerging dangerous pathogens in the future rather than fewer.”

And while Asian frogs and salamanders seem to have a natural immunity, the fungus can wreak havoc when moved to a new location or into a novel species, he says. “That’s when you often see some of the worst outcomes.”

The Bd fungus is a devastating example. Brunner says frogs rely on their skin for breathing as well as electrolyte balance. When Bd invades skin cells, it disturbs the frog’s ability to regulate water and electrolytes, which leads to changes in the blood that essentially cause a heart attack.

“It’s sort of like whole-body athlete’s foot that ends up killing them,” he says.

Though most salamanders breathe using both lungs and skin, it’s a similar story when they’re infected with Bsal—within days the fungus causes ulcers and sloughing tissue that lead to apathy, loss of appetite, and death. As one researcher put it, “It’s death by a thousand holes.”

Besides the fungus, Brunner is also concerned about one more “cold-blooded killer” called ranavirus that can cross-infect fish, reptiles, and amphibians.

“Ranavirus has a global distribution now,” he says. “It can be a really nasty infection—the virus gets into every bit of tissue they have, every cell, where it causes massive damage and organ failure. Thankfully, it doesn’t replicate at warm-blooded temperatures.”

The curious question is how some animals manage to control these viral and fungal infections so they don’t cause severe illness or death. Part of Brunner’s research is aimed at determining the factors that lead to this resistance and why catastrophic losses occur in some places and not others. He and his fellow scientists are following several clues.

Erica Crespi studying wood frogs in New York. (Courtesy Erica Crespi)

Erica Crespi, a physiologist and associate professor in the School of Biological Sciences, studies the way stress affects an amphibian’s early development. Frogs and salamanders are very sensitive to environmental changes which can trigger spikes in their stress hormone corticosterone.

“Just as in pregnant mammals where elevated stress hormones can cause premature birth, high corticosterone can shorten an amphibian’s development time and affect how the brain and lungs develop and cause other lifelong impacts,” she says.

Brunner says the idea that long-term chronic stress can suppress the immune system and make it harder for an animal to fight off infections has been studied by biologists for decades.

“In its simple form, the hypothesis says that anywhere we see human activities or other stressors, we should see big outbreaks of disease, but it’s not that simple. Stress doesn’t always translate into outbreaks.”

He and Crespi are trying to determine how individual animals respond to environmental stressors such as increased salinity or water temperature, and how that scales up to negative population outcomes like a pond full of floating frogs.

“The underlying stress mechanisms we’re studying apply to all sorts of animals like elk, fish, or any other species—and disease outbreaks in general,” says Brunner.

Jonah Piovia-Scott (left) and his student research assistants study amphibians in a WSU Vancouver wetland.

The investigation continues at WSU Vancouver, where Jonah Piovia-Scott, assistant professor in the School of Biological Sciences and a member of the National Bsal Task Force, is exploring the effects of climate change on chytrid fungal diseases.

“Neither Bd nor Bsal tend to do well when it’s hot,” he says. “So, some aspects of climate change may actually help amphibians with these pathogens. But other aspects may make them more susceptible. For example, if ponds dry up earlier in the season, it will decrease the amount of time amphibians have to develop. The stress will force them to develop faster, which may make them more susceptible to disease later in life.”

Piovia-Scott is often asked why we should care about amphibians and his answer is unequivocal.

“These amazing, beautiful, and wonderful organisms have intrinsic value, and are a part of our world we’re losing quite rapidly,” he says. “They are also integral components of the ecosystem—an important food source for some animals and they themselves eat insects, worms, and snails. Like salmon who are eaten by bears and fertilize the forest, amphibians are also an important link between aquatic and terrestrial systems.”

Indeed, isolated and far away, every frog and salamander die-off creates a domino effect that ultimately impacts the planet. Streams that were once crystal clear turn green without tadpoles to eat the algae. Human infections like malaria and dysentery spread more rapidly without amphibians to eat mosquitos and flies.

“It’s a very good example of how small the world has gotten,” says Pessier, who also specializes in biosecurity and reintroduction programs for endangered species.

“Diseases like Bd and Bsal are moved around by people. Domestic cows don’t move from Asia to the U.S. without a huge number of diagnostic tests. But for frogs, you just need the right permits and you can move them all over the world without concerns about disease.

“Once Bd has moved through an area, the amphibian biodiversity drops to virtually nothing and there is no way to mitigate the fungus in the wild,” he says. “So, the last resort strategy is to develop survival assurance populations (SAP). We capture threatened species to preserve their genetic diversity and then try to breed and maintain a colony in captivity until they can be reintroduced to the wild, once we have a way to deal with the fungus.”

Pessier works with SAP in Madagascar, Ecuador, Panama, and many other areas around the world to diagnose disease issues such as vitamin A deficiency in captive Panamanian golden frogs.

Closer to home, he is joining Crespi and Goldberg to protect Washington’s last surviving remnant of northern leopard frogs in the Columbia Basin. Working with the Washington Department of Fish and Wildlife, they hope to reintroduce and expand populations within the state.

Their intentions clearly reach beyond academia to a deeper love of the Pacific Northwest and our amphibian wildlife. Drafted in the spirit of Teddy Roosevelt, their proposal reads, “Do what you can, with what you have, where you are.”

It’s a philosophy all five faculty members stand behind. Their shared interests and mutual support have multiplied efforts to protect frogs and salamanders throughout the world.

“Conservation is an interdisciplinary science,” Crespi says. “Having Jesse, Caren, Allan, and Jonah here allows me to do projects I could never do in isolation.”

Read the full story in the Fall 2019 issue of Washington State Magazine

Top image: Allan Pessier holds a Pacific chorus frog (Photo Henry Moore).