Protecting amphibians from a deadly fungus

"I loved being outside and found amphibians fascinating," Molly Bletz, PhD says of her childhood.

She enjoyed collecting frogs and observing them in buckets of water before returning them back to their pond or lake home.

Today, as an assistant professor of disease ecology at Penn State College of Agricultural Sciences, she is deeply concerned about their future. Amphibians are dying at an alarming rate. Since 1980, at least 37 species have gone extinct and another 40% are threatened by extinction (1).

And the loss of amphibians like frogs, for example, alters entire ecosystems. Without tadpoles that feast on algae, blooms might choke streams. Frogs eat insects. Without them, some disease-carrying species may become more common. And birds and other predators that eat frogs would also be forced to find alternatives or starve.

The usual culprits, such as habitat loss and climate change, contribute to amphibian decline, but there is also a unique pandemic. In 1999, researchers identified a deadly fungus, Batrachochytrium dendrobatidis (Bd), as a key cause of the dramatic declines in frog populations around the globe.

To boost immunity to pathogens, Bletz is researching the microbiomes of amphibians. Similar to how imbalanced microbes can weaken the human immune system and lead to disease, disruptions in the amphibian microbiome can increase their susceptibility to Bd infection. A microbial treatment could potentially prevent infection and limit the spread of the pathogen.

The great die-off

Around the late 1990s, researchers noticed unusual patterns of amphibian declines across several continents. Through extensive fieldwork and laboratory analyses, they identified Bd as one major culprit. Over time, the infection disrupts amphibian motor function and the ability to breathe, triggering a heart attack by depletion of electrolytes.

Bletz first learned about the fungal pathogens threatening amphibians as an undergraduate student studying ecology, and then further as a graduate student. She grew increasingly frustrated by the lack of action. “We were basically studying the dynamics of the pathogen and documenting where it was spreading,” she recalls. “But instead of just studying those patterns, I wanted to start thinking about how to actually fix it.”

Because Bd is found in such a diverse range of environments, eradicating the fungus seems unlikely, says Bletz, so she has been focusing her research on how to lessen its impact. Bletz was inspired by a study from 2011 (2) in which researchers collected a group of threatened yellow-legged frogs that live in the Californian Sierra Nevada mountains and bathed them in a probiotic solution.

Probiotics for frogs

One particular question Bletz is pursuing is the role the environment plays in shaping a frog’s immune function.  Tadpoles grow in an aquatic environment and have a completely different immune system than an adult frog, she says. Many external factors can affect immunity across development, such as environmental degradation. “Tadpoles may not be maturing in the most robust habitat, and that might be interfering with their immune function directly, or indirectly by changing how the microbiome contributes to immunity.”

Bletz studies both wild and captive amphibian colonies throughout their different developmental stages to learn how environmental conditions affect their immune function. This research might offer clues on how to modulate their microbiomes by changing their environment, she says.

Treating amphibian habitats with a probiotic would offer a practical way to protect them. In the 2011 study, researchers collected frogs in containers and treated them individually. “Even though the study was positive, we know it’s not going to work on a global scale,” says Bletz. So, she is studying ways to introduce a probiotic into the environment, such as adding it to pond water where frogs congregate.

“That would be much more feasible and finding probiotics that work for specific species or certain areas might be the best strategy.” Any environmental probiotic treatment would focus on increasing the abundance of locally occurring bacteria, Bletz emphasizes, not bringing in microbes from other places.

Where did this fungus even come from? And what now?

One project that Bletz is “most excited about,” is studying the microbiomes of frogs native to Madagascar – a tropical biodiversity hotspot that has not yet been devastated by Bd fungus.

In 2013, Bletz collaborated with researchers there to study frog microbiomes to learn more about the local microbial species that might confer protection against fungal infections. The research could potentially protect frogs on the island from Bd infection, saving them before it’s too late.

“The idea is to be proactive, to create a toolbox of potential probiotics to remedy and help populations if Bd does arrive there and starts causing problems,” she says.

No one knows for sure how Bd became so widespread. The fungus appears to be native to South Asia (3), where amphibians seem to better tolerate it. There is some evidence that birds can carry it on their feet, says Bletz, but it’s not clear that birds actually spread it. It has also been suggested that it traveled the world as part of the international amphibian trade.

In Madagascar, Bletz and her colleagues spend their time collecting frogs from different areas, swabbing their skin for microbial samples and then plating the samples in culture to observe what grows. She uses the DNeasy DNA extraction kits from QIAGEN and sequences microbial DNA to identify the species. She then looks for specific bacterial species that have antifungal properties.

“We have identified over 4,000 bacteria,” Bletz says, “about 30 to 40% of those show strong fungal inhibition.” She and her colleagues then experiment with different bacterial species as potential probiotic treatments. Ideally, any probiotic should exist in harmony with other microbes in an organism’s microbiome or environment, and it should persist for long enough to be of benefit.

"By understanding and harnessing beneficial microbes, we can offer a glimmer of hope for amphibian conservation,” says Bletz.