A frog’s story of surviving a fungal pandemic offers hope for other species microbiologystudy

Roland Knapp, a biologist at the University of California, Santa Barbara, was there to witness the carnage. “I saw these massive frog die-offs in which over the course of two weeks or so entire frog populations were wiped out before my eyes.”

The killer: the chytrid fungus Batrachochytrium dendrobatidis, or Bd for short. It can destroy frog skin, choke off the frog’s supply of electrolytes and induce a fatal heart attack within a couple weeks.

Probably originating in East Asia, the fungus is thought to have gone global through the pet trade (SN: 5/10/18; 3/13/14). Conservationists have searched in vain for a silver bullet solution. Antifungal ointment can save individuals, and Bd can be scrubbed from isolated bodies of water. But these Band-Aids have done little to stop the hemorrhaging losses of frog biodiversity around the world.

All told, Bd has been implicated in the population declines of at least 500 amphibian species, including 90 possible extinctions — making it perhaps the most devastating pathogen on record to ever afflict vertebrates, researchers reported in 2019 (SN: 3/28/19). And that’s on top of habitat loss, pollution and climate change, which also beset amphibians. At last count, about 200 species of frogs have gone extinct since the 1970s.

Like a load-bearing Jenga piece, if frogs go, entire ecosystems may collapse. A world devoid of frogs will leave a gap in the food chain no other class of organisms can fill. Without these insect eaters, swarms of bugs could overrun wild places like Yosemite. In the tropics, more people could get diseases like malaria, spread by mosquitoes (SN: 10/5/22). The algae normally eaten by tadpoles could grow out of control. And deprived of frogs as meals, snakes, carnivorous birds and furry predators of all kinds, including the occasional down-on-its-luck bear, could starve.

But “amphibians are incredibly tough in many ways,” says Vance Vredenburg, an ecologist at San Francisco State University who along with Knapp tracked the decline of Sierra Nevada yellow-legged frogs. “If you look at the big, big picture, they’ve made it through the last four major mass extinctions on Earth as a lineage.”

Indeed, some yellow-legged frogs survive Bd, and since 2006, Knapp has been using them to found new colonies. On foot or by helicopter, he ferries frogs to remote mountain lakes and drops off his mottled brown cargo in waters swimming with fungal spores. Ultimately, he hopes the transplants go forth, multiply and return the High Sierra to its natural state —a landscape jumping with frogs.

Knapp and colleagues say these survivors evolved immunity to Bd. Other species in Australia and Central America may be doing the same, though none seem to be rebounding at the rate of the Sierra Nevada frogs. If researchers can identify individuals carrying the genetic mutations driving this adaptation, they may be able to help other struggling frog populations grow.

“We’re at a critical point where if we can start linking these candidate resistance genes in frogs with their immune system functions, we could go for bolder conservation actions such as breeding for specific genetic variation we are confident will increase resilience,” says Anna Savage of the University of Central Florida in Orlando, who studies how genes influence frogs’ immune response to Bd.

How the Sierra Nevada yellow-legged frog survived Bd

The High Sierra was once a Shangri-la for Sierra Nevada yellow-legged frogs (Rana sierrae) — perhaps the most abundant vertebrate above 1,800 meters. The frogs arrived at this haven sometime within the last 10,000 years, when they climbed the waterfalls that kept out fish and reached crystal pools surrounded by glacier-carved granite peaks. The newcomers adapted to live nine months of the year under thick ice at near-freezing temperatures. In summer, the frogs emerge from the depths to bask on rocky shorelines or hang out in clear shallows to soak up the sun.

The water became more dangerous for the frogs in the late 1800s. Back then, sportsmen were angling to fish in the High Sierra. But there was one problem — no fish. So fish stocking commenced, first by hauling up trout in milk canisters slung over mules and then eventually by airplanes bombing lakes with trout hatchlings.

Knapp set out in the mid-1990s to investigate why the frogs were disappearing. He and Vredenburg collected data revealing that trout have a ravenous appetite for tadpoles and bite-size froglets. The pair convinced the then-named California Department of Fish and Game plus the National Park Service to remove every trout from numerous lakes and ponds in protected areas of the High Sierra. By the late 1990s, frog populations showed signs of recovery. But then Bd fungus crept into the water in the early 2000s, erasing the gains.

“To have the dark cloud of Bd arrive on the scene and make the situation almost infinitely more complicated…it was nightmarish,” Knapp says.

But in the midst of his despair, he noticed something “wildly different.” Hardy descendants of frogs that had survived the onslaught of both trout and Bd, he says, were “recovering to a point where the populations had hundreds or even thousands of adult frogs and thousands of tadpoles.”

And they were doing it in water suffused with the fungus. “They were clearly able to suppress the infection,” he says, “and as a result they were surviving.”

Knapp wanted to see if these survivors could live in places where the species had disappeared. From 2006 through 2020, taking 30 or so Bd-immune frogs at a time, he moved them to 12 lakes spread across Yosemite National Park for a total of 24 reintroductions, trekking nearly 15 kilometers in some instances across difficult terrain. A few frogs died in the wet cloth bags he initially used for transport. Switching to plastic containers and using a helicopter to shorten the longest journeys saved lives.

In 2016, he stood on the shore of one of those lakes — names and locations haven’t been disclosed to protect the transplants — and watched a new cohort of hundreds of Sierra Nevada yellow-legged frogs jump into the water. They weren’t the big, old frogs he had brought 10 years earlier. They were smaller, younger adults — the progeny of that initial generation.

Knapp knew the experiment had been a success. “That was the first indication that a population had in fact become established. It was head-exploding,” he says. Since then, he has had similar successes at other lakes. To be exact, nine new colonies out of the 12 reintroduced populations flourished.

A statistical model based on the ebb and flow of frog density predicts that more than half of transplanted colonies will last for 50 years or more, Knapp and colleagues reported in a paper posted in 2023 to bioRxiv.org.

But even after inspecting over 2,000 potential new habitats, selecting the right one for translocation is still difficult, Knapp says. And the failures stick with him. “It’s a super frustrating outcome. I’m basically throwing frog lives away because I lack some bit of understanding that would’ve told me I was missing something that’s constraining the ability of frogs to persist,” he says. For instance, finding lakes that don’t freeze to the bottom is vital to ensure the frogs have a place to spend the winter. “It’s pretty clear to me,” he says, “that we need to learn from these naturally recovering populations.”

Finding the genes that help fend off Bd

Of course, the most pressing thing to learn is, how does the Sierra Nevada yellow-legged frog fight off Bd? One possible explanation: Communities of beneficial bacteria that live on the frog’s skin outcompete the fungus. Another: Glands in the skin secrete antifungal chemicals, though Bd may be able to suppress this defense.

Erica Bree Rosenblum, a molecular geneticist at the University of California, Berkeley, argues the answer hides in the frog’s genetic code. In studying the DNA of Bd survivors, she’s found mutations that don’t appear in the DNA of Sierra Nevada yellow-legged frogs from areas untouched by the epidemic. These differences in DNA sequence — the order of the chemical letters, or bases, that make up the genetic code — show up in statistical patterns called signatures of selection. It’s a sign that a region of the genome has evolved due to some selective pressure, say, a deadly disease.

“The frogs that survive better have certain variations in their genomes,” Rosenblum says. “Since they’re the ones surviving, they’re passing their genes down, and over time the whole population is changing towards having these more favorable genetic mutations.” It’s a classic case of evolution by natural selection.

One gene stands out as the blueprint for tiny sentries on cell surfaces called glycoproteins, which bind to pathogens and present them to white blood cells for destruction. Another gene instructs the building of interferons. These proteins, active in frog skin, sound alarm bells when they detect an intruder like Bd, mobilizing other immune defenses.

In the Sierra Nevada yellow-legged frogs, Rosenblum identified eight genes in a region of the genome called the major histocompatibility complex, or MHC, that show up as a signature of selection. MHC genes play important roles in the immune system, and any of the eight genes could be giving frogs an edge against Bd.

Rosenblum’s findings explain why Sierra Nevada yellow-legged frogs are recovering, but not how. At this stage she can’t pinpoint the biological mechanism that’s saving frogs’ lives — that would be a leap.

“My expectation with this situation is that it’s a pretty complex trait. There’s not going to be a single smoking gun. There’s lots of changes in the genomes that are happening,” Rosenblum says.

Is there hope for other frogs?

Other threatened frogs may be evolving immunity to Bd as well. And the hunt to find resistant individuals continues, especially in R. sierrae’s close cousin, the mountain yellow-legged frog (Rana muscosa). That species is in a much more precarious situation in Southern California, says Talisin Hammond, a conservation biologist at the San Diego Zoo Wildlife Alliance.

Habitat loss culled their ranks, and Bd’s arrival compounded their predicament. Now a few holdout populations totaling a couple hundred frogs eke out an existence amid invasive bullfrogs (which carry and transmit Bd), wildfires and perennial droughts (SN: 3/20/24). Occasionally, someone finds a doomed school of tadpoles in a dried-out stream bed and rushes it to the San Diego Zoo or other facilities that host R. muscosa breeding programs.

At the San Diego Zoo, large tanks beyond public view hold tadpoles that hatch and grow. Breeding occurs there too, with careful attention to degrees of kinship so the species’s genetic diversity is maintained. With so few R. muscosa frogs left, there’s a high risk of inbreeding.

Frogs reared in captivity are trained in simulated habitats to increase their chances of survival in the wild. Confront one with a rubber snake — and cues from a live one — and it becomes wary of predators. Add rushing water to the tank from time to time, and mobility improves.

The frogs’ immune systems can learn as well, so recently scientists purposely infected frogs with Bd and then bathed them in lifesaving antifungal ointment before release. The exposure in this safe environment is like a vaccine, priming the immune system for a future showdown with Bd. But unlike inborn immunity, the treatment may not last over a frog’s lifetime and can’t be passed on to offspring.

If researchers can identify the genes driving R. sierrae’s comeback, that might allow the San Diego Zoo and others to breed R. muscosa frogs with greater immunity to Bd, says Cynthia Steiner, associate director of conservation genetics at the San Diego Zoo Wildlife Alliance. She plans to compare the two species’ genomes and hopes “some of these genes — the genetic variants providing populations with more levels of resistance — are also present in our populations,” she says.

Meanwhile, frogs at the epicenter of Bd destruction in the misty, moss-covered cloud forests of Panama and Costa Rica are on the verge of mounting their own comeback. There, at least nine of the 40 or more frog species that mostly disappeared more than 20 years ago have begun to reemerge, including the dazzling harlequins (SN: 11/9/22), which come in an assortment of vibrant colors.

Jamie Voyles, a biologist at the University of Nevada, Reno, investigates their recovery, though she doesn’t have the resources to attempt relocations like Knapp’s. And besides, the frogs in these forests can be wildly difficult to catch, possibly complicating efforts to transplant survivors. Take the Panama rocket frog. “You have to sit in one spot, be as still as possible, and then lightning quick to catch them by hand or plastic bag once you learn their jumping pattern,” Voyles says. Other types are so elusive that catching one is mostly dumb luck.

Like Steiner, Voyles would like to replicate Knapp’s success once researchers like Rosenblum uncover the survivors’ secrets. “What is it that they had or were doing right to make it through that huge evolutionary selective sweep that wiped out everybody else?” she asks.

In 1998, biologist and veterinarian Lee Berger of the University of Melbourne was among the first scientists to discover the killer fungus (SN: 7/4/98). Since then, she has worked tirelessly to protect Bd-ravaged populations in Australia, such as the boldly yellow-and-black southern corroboree frogs. She celebrates Knapp’s work as an example of how humans can help frogs along: “We’ve only just begun figuring out ways to return ecologically important species to the landscape.”

As Knapp continues to transplant frogs, he wants their growing numbers to inspire other frog biologists. “In this world of Bd-caused declines,” he says, “it seemed to me really important to put out this positive example of how we can in fact, at least in this one system, and hopefully in many other systems in the future, use these naturally recovering populations to effect broader-scale, more sustainable long-term recovery.”