Golden frogs with unique communities of skin bacteria survive exposure to frog-killing fungus  

Chytridiomycosis is an amphibian disease that has wiped out populations of many frog species around the world, including the charismatic Panamanian golden frog, which now exists only in captivity in the United States and Panama.

Research published this week in the Proceedings of the Royal Society found unique communities of skin bacteria on golden frogs that survived chytridiomycosis. The original experiment was designed to test the idea that antifungal probiotic bacteria may be used to prevent chytridiomycosis in captive golden frogs. Approximately 25 percent of the golden frogs eventually cleared infection, but their survival was not associated with the probiotic treatment, rather it was associated with bacteria that were present on their skin prior to the start of the experiment. In fact, the probiotic antifungal bacteria did not appear to establish on the golden frog skin at all.

Study authors Matt Becker and Shawna Cikanek work to inoculate frogs with beneficial bacteriaMatthew Becker, a fellow at the Smithsonian Conservation Biology Institute who conducted the experiment as part of his PhD research at Virginia Tech University, says it is unclear why the microbes did not linger on the skin, but he thinks that the way he treated the frogs – with a high dose of bacteria for a short duration – may be part of the reason.

“I think identifying alternative probiotic treatment methods that optimize dosages and exposure times will be key for moving forward with the use of probiotics to mitigate chytridiomycosis,” Becker said.

Brian Gratwicke, amphibian conservation biologist at the Smithsonian Conservation Biology Institute where the experiment was conducted, says that he was disappointed that they did not find a ‘silver bullet’ to cure chytridiomycosis in this species, but noted that the results do advance our understanding of this disease.

“Previous experiments found that golden frogs are highly susceptible to chytridiomycosis, so any survival is cause for hope,” said Reid Harris, director of disease mitigation at the Amphibian Survival Alliance. “The tricky piece is figuring out the survival mechanism, and this exciting research gives some new insights in that direction.”

This research also provides additional support for the importance of symbiotic microbes, or the ‘microbiome,’ for the health of their hosts, ranging from sponges and corals to humans.

“In all multi-cellular organisms, we have suites of microbes performing critical functions for their hosts, and the same appears to be true for golden frogs,” said Lisa Belden, who supervised the study at Virginia Tech University.

The team, led by Becker, now plans to determine if this study is repeatable by investigating whether the golden frog’s skin microbiota can predict the susceptibility to chytridiomycosis. They will also investigate whether the bacteria associated with the surviving frogs from this study can be used as a probiotic treatment to prevent infections of golden frogs without a ‘protective’ microbiota.

“The ultimate goal of this research is to identify a method to establish healthy populations of golden frogs in their native habitat, despite the presence of chytridiomycosis in the environment,” Becker said.

Citation: Matthew H. Becker, Jenifer B. Walke, Shawna Cikanek, Anna E. Savage, Nichole Mattheus, Celina N. Santiago, Kevin P. C. Minbiole, Reid N. Harris, Lisa K. Belden, Brian Gratwicke (2015) Composition of symbiotic bacteria predicts survival in Panamanian golden frogs infected with a lethal fungus. Proc. R. Soc. B: 2015 282 20142881; DOI: 10.1098/rspb.2014.2881. Published 18 March 2015

National Zoo Successfully Collects Sperm Samples to Save Endangered Frog

Gina Della Togna, an SCBI PhD student and native Panamanian, is one of the researchers in charge of the sperm collection procedure.

Gina Della Togna, an SCBI PhD student and native Panamanian, is one of the researchers in charge of the sperm collection procedure. (Photo courtesy of the Smithsonian Conservation Biology Institute)

With nearly one-third of all amphibian species at risk of extinction as the result of the deadly chytrid fungus, the Smithsonian’s National Zoo has taken a bold step toward preserving amphibian genes and the world’s incredible amphibian biodiversity. Researchers at the Smithsonian Conservation Biology Institute in Washington, DC, have begun to collect sperm samples from the Zoo’s collection of Panamanian golden frogs (Atelopus zeteki), which are extinct in the wild.

Although researchers have collected sperm samples from other amphibian species such as Mississippi gopher frogs and leopard frogs, there are no publications detailing sperm collection methods from Panamanian golden frogs. SCBI’s colleagues at the Maryland Zoo have aided in the process, providing advice to the SCBI researchers about the method to collect the frogs’ spermatozoa using hormonal stimulations.

“We currently have three other species of Atelopus in captive assurance colonies in Panama,” said Brian Gratwicke, an SCBI conservation biologist who leads the Zoo’s amphibian conservation program to curb global amphibian declines. “If we can freeze some of their sperm, golden frogs will be a model to secure the long-term genetic integrity of other toad species in similar situations.”

Gina Della Togna, an SCBI PhD student and native Panamanian, is one of the researchers in charge of the sperm collection procedure. Even though this is still a fairly new endeavor, Della Togna said she felt that it was easy compared to collecting sperm from mammals. After hormonal stimulation, spermatozoa are excreted in the urine from the frog’s cloaca, a multipurpose opening from which feces, urine and gases are expelled. This is in contrast to mammals, which possess specialized structures for the expulsion of waste and reproduction.

Atelopus zeteki sperm

A Panamanian golden frog sperm

Although sperm collection from this species has been successful, finding the most efficient and repeatable stimulation protocol is critical. Then, identifying the right cryoprotectant and freezing method will be another challenge. Researchers suspect that the cell component most likely responsible for the movement of the sperm, called a mitochondrial vesicle, has a unique structure compared to that of other animals.

“The mitochondrial vesicle is a very fragile structure,” Della Togna said. “Protecting this structure will definitely be one of our greatest challenges.”

Even in the face of numerous challenges, the research team overseeing the sperm collection and storage of the samples remains optimistic.

Pierre Comizzoli, an SCBI gamete biologist supervising the PhD project  is enthusiastic about the prospect of this endeavor and is charged with studying the complex golden frog sperm structure with Della Togna.

“It is always exciting to discover new biological mechanisms,” Comizzoli said. “Spermatozoa from each species have unique traits that needs to be well understood before developing preservation protocols.”

Other than its genetic and natural significance, the Panamanian golden frog is a meaningful symbol of culture for Panamanians. Pre-Columbian peoples used to make golden “huacas,” or sacred objects, in the image of these frogs, along with creating legends about these renowned frogs, which endure in the Panamanian countryside today, Della Togna said.

“This species does not exist anywhere else in the world,” Della Togna said. “You will find pictures and sculptures of it in local markets, in indigenous handcraft sales, and on lottery tickets, among places. Hopefully this project will help to ensure that one day you will be able to see them once again on the banks of Panamanian streams where they belong.”

Phil Jaseph, Smithsonian’s National Zoo

Press release: Smithsonian Scientists Find Deadly Amphibian Disease in the Last Disease-free Region of Central America

Toad Mountain harlequin frog (Atelopus certus)

The Panama Amphibian Rescue and Conservation Project has established an assurance colony for two species endemic to the Darien, including the Toad Mountain harlequin frog (Atelopus certus), shown here. (Brian Gratwicke, Smithsonian Conservation Biology Institute)

Smithsonian scientists have confirmed that chytridiomycosis, a rapidly spreading amphibian disease, has reached a site near Panama’s Darien region. This was the last area in the entire mountainous neotropics to be free of the disease. This is troubling news for the Panama Amphibian Rescue and Conservation Project, a consortium of nine U.S. and Panamanian institutions that aims to rescue 20 species of frogs in imminent danger of extinction.

Chytridiomycosis has been linked to dramatic population declines or even extinctions of amphibian species worldwide. Within five months of arriving at El Cope in western Panama, chytridiomychosis extirpated 50 percent of the frog species and 80 percent of individuals.

“We would like to save all of the species in the Darien, but there isn’t time to do that now,” said Brian Gratwicke, biologist at the Smithsonian Conservation Biology Institute and international coordinator for the Panama Amphibian Rescue and Conservation Project. “Our project is one of a few to take an active stance against the probable extinction of these species. We have already succeeded in breeding three species in captivity. Time may be running out, but we are looking for more resources to take advantage of the time that remains.”

The Darien National Park is a World Heritage site and represents one of Central America’s largest remaining wilderness areas. In 2007, Doug Woodhams, a research associate at the Smithsonian Tropical Research Institute, tested 49 frogs at a site bordering the Darien. At that time, none tested positive for the disease. In January 2010, however, Woodhams found that 2 percent of the 93 frogs he tested were infected.

“Finding chytridiomycosis on frogs at a site bordering the Darien happened much sooner than anyone predicted,” Woodhams said. “The unrelenting and extremely fast-paced spread of this fungus is alarming.”

The Panama Amphibian Rescue and Conservation Project has already established captive assurance colonies in Panama of two priority species endemic to the Darien—the Pirre harlequin frog (Atelopus glyphus) and the Toad Mountain harlequin frog (A. certus). In addition, the Smithsonian’s National Zoo maintains an active breeding program for the Panamanian golden frog, which is Panama’s national animal. The Panamanian golden frog is critically endangered, according to the International Union for Conservation of Nature, and researchers have not seen them in the wild since 2008.

Bd infection

Chytridiomycosis is a rapidly spreading amphibian disease that attacks the skin cells of amphibians (shown here) and is wiping out frog species worldwide. (Doug Woodhams, Smithsonian Tropical Research Institute)

“We would like to be moving faster to build capacity,” Gratwicke said. “One of our major hurdles is fundraising to build a facility to house these frogs. Until we jump that hurdle, we’re limited in our capacity to take in additional species.”

Nearly one-third of the world’s amphibian species are at risk of extinction. While the global amphibian crisis is the result of habitat loss, climate change and pollution, chytridiomycosis is at least partly responsible for the disappearances of 94 of the 120 frog species thought to have gone extinct since 1980.

“These animals that we are breeding in captivity will buy us some time as we find a way to control this disease in the wild and mitigate the threat directly,” said Woodhams, who was the lead author of a whitepaper Mitigating Amphibian Disease: strategies to maintain wild populations and control chytridiomycosis. This paper, published in Frontiers in Zoology, systematically reviews disease-control tools from other fields and examines how they might be deployed to fight chytrid in the wild. One particularly exciting lead in the effort to find a cure is that anti-chytrid bacteria living on frog skin may have probiotics properties that protect their amphibian host from chytrid by secreting anti-fungal chemicals. Woodhams recently discovered that some Panamanian species with anti-chytrid skin bacteria transmit beneficial skin chemicals and bacteria to their offspring. The paper, Social Immunity in Amphibians: Evidence for Vertical Transmission of Innate Defenses, was published in Biotropica in May.

“We are all working around the clock to find a cure,” Gratwicke said. “Woodhams’ discovery that defenses can indeed be transferred from parent to offspring gives us hope that if we are successful at developing a cure in the lab, we may find a way to use it to save wild amphibians.”

The Smithsonian Conservation Biology Institute serves as an umbrella for the Smithsonian Institution’s global effort to understand and conserve species and train future generations of conservationists. Headquartered in Front Royal, Va., SCBI facilitates and promotes research programs based at Front Royal, the National Zoo in Washington, D.C., and at field research stations and training sites worldwide.

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Media only: contact Lindsay Renick Mayer, Smithsonian’s National Zoo, 202-633-3081