Blake Klocke, a student at George Mason University is studying where frogs go once we release them, how long it takes them to contract the chytrid fungus, if there are any effects of releasing chytrid susceptible frogs on the existing frog community & what other non chytrid fungus sources of mortality might affect reintroduction work.
In addition to our primary project partners the Houston Zoo, Zoo New England and the Cheyenne Mountain zoo we thanks the National Geographic Society, Mohammed Bin Zayed Species Conservation Fund, and the Smithsonian Women’s Committee for their support of these release trials.
Many infectious diseases can fade away after initial outbreaks. Bubonic plague, cholera, and influenza are examples from recent human history. The same phenomenon occurs for wildlife diseases as well. How does this happen? One popular explanation is that the pathogen evolves to become less deadly, so that it doesn’t completely wipe out its hosts, ensuring pathogen survival. While this scenario does sometimes play out, we know that there are other reasons why the severity of diseases can change over time.
Cori Richards and Jamie Voyles
For amphibians, we’ve known about a highly lethal disease called “chytridiomycosis” since the 1990s. This disease was especially devastating in Central America, where it may have wiped out entire species. In this study, we made the exciting discovery that some amphibian species – frogs that were thought to be extinct – are persisting, and even recovering, after lethal disease outbreaks. We wanted to understand how it was happening. Was it a change in the pathogen, the frogs, or both?
To answer these questions, we did two things. To begin with, we surveyed frogs in Panama before and after the disease outbreak. In addition, we collected samples of the pathogen at multiple time points: during initial outbreaks and ~10 years later. We found that nearly a decade after the outbreak, the pathogen was just as deadly. However, the frogs are surviving and have better defenses against it. Panama’s frogs are fighting back! Understanding how amphibian communities are recovering after this disease outbreak is important multiple reasons. First, resolving how this works will help us develop more informed conservation strategies to protect amphibians from disease-induced extinctions. Second, clarifying how disease outbreaks subside will help us predict, and respond to, other emerging pathogens in plants, wildlife, and in humans. These goals are increasingly important in a time when rapid globalization has increased the introduction of pathogens to naïve host populations.
Atelopus varius is one species that appears to have evolved antifungal skin secretions
Scientists from the Smithsonian Tropical Research Institute (STRI) and officials from Panama’s Ministry of the Environment (MiAmbiente) visited the Mamoní Valley Reserve, where release trials are underway to release Limosa Harlequin (Atelopus limosus) frogs. These frogs were bred in captivity and are the descendants of frogs collected a few years ago in the same area.
MiAmbiente was represented by biologists Erick Núñez and Anthony Vega, technicians from the Department of Biodiversity of the Office of Protected Areas and Wildlife. They were accompanied by STRI staff scientist Roberto Ibáñez, director of the Panama Amphibian Rescue and Conservation Project (PARC), and Juan Maté, STRI’s manager for scientific affairs and operations and institutional liaison with MiAmbiente. PhD student at the Smithsonian-Mason School of Conservation at George Mason University, Blake Klocke, who is currently conducting this research, hosted and guided this visit, together with his field assistant, Mirjana Mataya.
Some of the frogs are from the initial release trial conducted in the El Valle del Mammoní Reserve by Panamanian PhD students from Virginia Tech, Daniel Medina and Angie Estrada. They kept the frogs inside enclosures in this reserve to protect them from predators and so that they could obtain samples to determine if they were infected by the chrytrid fungus (Batrachochytrium dendrobatidis). In a second release trial, Blake Klocke freed these and additional frogs. In this phase of the investigation, one of the objectives is to determine if it is necessary to keep in enclosures before releasing them completely. Some of the frogs were equipped with radio transmitters that allow researchers to follow their movements and estimate the size of their territories.
The visit with MiAmbiente officials was aimed at monitoring the release trials and the progress of research. Blake Klocke showed them how the radio-telemetry tracking system works using mini-transistors. This technological tool, applied to scientific research, allows us to follow the movements of these small frogs. Frogs without radio transmitters were certainly harder to observe. Likewise, measurements of the size and weight of frogs were obtained and samples of their skin were collected with swabs for detection of the chytrid fungus. At this point, the frogs have been kept close to the point of release. However, based on the data being collected, researchers will be able to estimate the future dispersion and survival of the frogs.
The initiative to advance the release trials is part of the Panama Amphibian Rescue and Conservation Project, known colloquially as PARC and administered by the Smithsonian Tropical Research Institute under the supervision of MiAmbiente. This project is a collaboration among several organizations including Cheyenne Mountain Zoo, Houston Zoo, Smithsonian National Zoological Park and New England Zoo. It has been supported by a long list of sponsors, among them Minera Panama SA.
PARC has two facilities, one at El Níspero Zoo in El Valle de Antón, Coclé, now known as PARC El Valle and the other in Gamboa in the Panama Canal Watershed, known as PARC Gamboa. The PARC El Valle facility received the first rescued frogs, which had been temporarily kept at the Hotel Campestre in 2007, an initiative of the El Nispero Zoo and the Houston Zoo, with funding from the latter as an emergency response to the reduction in numbers of amphibians due to the mortality caused when the fungus arrived in El Valle de Antón in 2006. In 2009, this facility in El Valle came under the umbrella of the PARC project, and has continued its operation within the El Níspero Zoo. In addition, in 2009, PARC Gamboa initially began with the building of facilities at Summit Municipal Park, before relocating to its current location in Gamboa in 2012 to improve and expand capacity for ex-situ conservation of amphibians and create an additional backup at a second site. The PARC project is characterized by the result of the joint effort of multiple organizations and large numbers of people who, over the years, have contributed to the conservation of Panama’s amphibians.
MiAmbiente and the STRI have an inter-agency collaborative agreement under which they work closely on the implementation of the Amphibian Conservation Action Plan in Panama approved in 2011. The Plan aims to ensure the conservation of amphibian populations through the implementation of actions that promote research and management, both in situ (in their habitat) and ex situ (outside their habitat) in addition to promoting the education of society in general. This plan integrates specific research, conservation and education activities in the short and medium term with the goal of safeguarding our natural heritage.
This visit served as a joint verification by STRI and MiAmbiente on the progress of the project. An important milestone has been reached in the implementation of this Action Plan, as this is the first time in Panama that an amphibian conservation project is executing the phase where the behavior and survival of frogs reared in captivity is being investigated by exposing the animals to their natural environment. The results of these release trials will be of great use in guiding future efforts to re-establish the populations of certain species of frogs at sites where they have decreased in abundance or disappeared.
The global conservation movement has reached a turning point. We have documented the fast pace of habitat loss, the growing number of endangered and extinct species, and the increasing speed of global climate change. Yet while the seriousness of these threats cannot be denied, there are a growing number of examples of improvements in the health of species and ecosystems, along with benefits to human well-being, thanks to our conservation actions. Earth Optimism is a global initiative that celebrates a change in focus from problem to solution, from a sense of loss to one of hope, in the dialogue about conservation and sustainability.
Gina Della Togna with a Panamanian golden frog, a beloved species at the center of her research. (Photo by Pei-Chih Lee, Smithsonian Conservation Biology Institute)
When SCBI conservation biologist Brian Gratwicke started the Panama Amphibian Rescue and Conservation Project with partners in 2009, it was a mad dash to find and collect frogs representing the very last best hope for their species, rapidly vanishing at the hands of an amphibian chytrid fungus (Batrachochytrium dendrobatidis, or Bd) that causes a disease called chytridiomycosis.
If that was the opening chapter of the rescue project’s story, seven years later the story reads like a manuscript for an initiative set up to be among the most successful comprehensive conservation projects to date.
Today the rescue project has provided a stable safe haven for 12 of the most imperiled Panamanian frog species, requiring keepers to learn the complex husbandry, behavior and reproductive physiology unique to each individual species. In the meantime, rescue project scientists are making strides in developing and refining assisted reproduction protocols, while also conducting experiments in a resolute search for a cure for Bd.
“We are entering a new phase,” Gratwicke says. “We’ve brought together some of the world’s leading animal husbandry experts, veterinarians, reproductive biologists, disease ecologists and herpetologists. With all of the talented scientific minds working on this one, we have great hope that we may someday be able to return these species safely to their home in the wild.”
Searching for a Cure
Things in Matt Becker’s lab can sometimes get a bit…strange. Take, for instance, an experiment the SCBI postdoctoral researcher conducted a year ago with unexpected results. Becker’s research focuses on the use of probiotics—or beneficial bacteria—to help frogs fight off Bd. Last year Becker applied five different probiotics with anti-fungal properties to the skin of five groups of Panamanian golden frogs, hoping to discover which probiotic gives them an effective shield against the pathogen.
Matt Becker prepares-probiotic baths. (Photo by Brian Gratwicke, Smithsonian Conservation Biology Institute)
What he found surprised him. In past experiments, the probiotics were ineffective and all of the frogs died after the researchers infected them with Bd. This time, though, about 25 percent of the individuals survived. And those surviving frogs didn’t come from just one group with one kind of probiotics, but from every group, even the one that had been infected with Bd without a probiotic protectant.
So Becker and Gratwicke needed to determine what it was that the frogs did have in common to help them fight the disease. They started by looking at the frogs’ microbial community, or the complex community of bacteria on the skin. All of the frogs that survived had a greater abundance of specific bacteria on their skin.
In June of this year, the team launched a new experiment, this time using frogs from the Species Survival Plan collection at the Maryland Zoo in Baltimore that have similar abundances and types of bacteria as those that survived last year. The researchers have given the frogs a cocktail of eight bacteria that seem to strongly ward off Bd.
Looking at which immune system genes turn on or off to fight off a chytrid infection can help scientists discover why some frogs aren’t as susceptible. (Photo by Mehgan Murphy, Smithsonian’s National Zoo)
“At the start of every experiment, you’re really optimistic,” says Becker, who has been working on golden frog probiotics since 2007. “It’s been a great journey and we’re really learning a lot about golden frogs and how chytrid affects these guys. Every little bit of information really goes a long way for the conservation of this species and similar species.”
For the first time during a probiotics study on frogs, the researchers will also be looking at the gene expression—or combination of genes in an individual frog that gets turned on or turned off—while the frog mounts an immune response to fight off Bd.
“We’re throwing everything we’ve got at this,” Becker says. “We want to be able to use these tools to determine which frogs in the overall captive population share those same strengths—either their microbial community or gene expression—that keep them alive. There are so many questions we need to answer, but through the scientific process, we’re getting there.”
Frogs for the Future
While Becker is focused on getting frogs safely back into the wild, this goal is only possible if there are actually future generations of frogs to release into the wild. That’s where Smithsonian researcher and Panamanian native Gina Della Togna comes in.
Gina Della Togna in the lab. (Photo by Brian Gratwicke, Smithsonian Conservation Biology Institute)
Della Togna is working on a number of complex assisted reproduction techniques for Panamanian frog species. She is the first scientist to develop protocols for extracting and freezing sperm from the Panamanian golden frog, a species that is extinct in the wild and a cultural icon in her home country. Scientists could someday use the sperm to infuse populations with additional genetic diversity, key to a species’ overall health.
“When we started, we didn’t know anything about anything,” Della Togna says. “We needed to learn which hormones at what concentrations to use, how to keep the sperm alive long enough to freeze it and the best techniques to freeze it so that the sperm is viable when we thaw it, even years later. It was a challenge, but I love a good challenge.”
Now Della Togna is working on developing similar protocol for other rescue project species, including the mountain harlequin frog, Pirre harlequin frog, variable harlequin frog, limosa harlequin frog and the rusty robber frog. In the future, she plans to get out into the field to capture genetic lineages from frogs in the wild. As she continues to perfect these protocols, Della Togna also aims to collect eggs from female Panamanian golden frogs to use for artificial fertilization with the frozen sperm. And most recently in Panama, she successfully applied a hormone treatment to help six pairs of the limosa harlequin frog and Pirre harlequin frogs breed that hadn’t laid eggs before.
“Breeding frogs is the fundamental step to sustaining captive populations and growing the numbers for release trials,” Gratwicke says. “Gina’s work is of huge applied value to us because we have some very challenging species to breed, and hormone dosing may help us to get them to cycle reproductively, even if we can’t figure out the external reproduction cues.”
For Della Togna, Gratwicke and Becker, the goal is the same: to give these unique species a fighting chance against Bd.
“If these frogs go extinct, nothing can replace them,” Della Togna says. “They are important to the ecosystem and essential to our planet’s equilibrium. There’s no doubt that we’re responsible for getting them back to where they belong.”
From now until the end of August, you can help us #FightForFrogs! Our generous sponsor Golden Frog—a global online services provider with a terrific name—will match donations to the rescue project up to $20,000, helping us raise money critical to our fight for frogs. Your donations during the Fight for Frogs campaign will buy us equipment to care for the frogs in the rescue pods, help us continue to conduct experiments to find a cure, ensure crucial breakthroughs, and ultimately one day see the return of these incredible species to their home in the wild.
The Panama Amphibian Rescue and Conservation Project is a project partnership between the Cheyenne Mountain Zoo, the Houston Zoo, Zoo New England and Smithsonian Institution. You can follow the Fight for Frogs campaign onTwitterusing the #FightForFrogs hashtag or on the rescue project’sFacebook page.
Frogs matter. As a kid in nursery school, I remember observing tadpoles metamorphose into froglets right before our eyes in the classroom. It was like watching a magic trick over and over again. As I grew more interested in these cool little creatures, I learned that some frogs reproduce using pouches, others by swallowing their own eggs and regurgitating their young, others still by laying eggs that hatch directly into little froglets. It was like discovering not one magic trick, but an entire magical world—except this world was no illusion, it was real. My formative experiences both in the classroom and out rummaging around cold rainy ponds at night with my best friend and a headlamp spurred me into a career in the biological sciences. They also instilled in me a deep appreciation for the incredible diversity of life.
Today I am focused on conserving that incredible diversity specifically among amphibians in Panama, which is home to an astounding 214 amphibian species. Or at least it was. When a deadly amphibian chytrid fungus swept through, nine species disappeared entirely, including the country’s national animal, the beautiful Panamanian golden frog.
Since 2009, the Panama Amphibian Rescue and Conservation Project has spearheaded efforts to bring at-risk species into rescue pods to ride out the storm while we work on finding a cure. We’ve worked with partners to conduct several experiments in search of a cure and to better understand why some frogs resist infection and others do not. We have built new facilities that house highly endangered species of amphibians as part of a bigger global push to create an “Amphibian Ark.” These efforts and those of our colleagues around the world give me profound hope for our amphibian friends.
But we need your help.
Although frogs are the orchestral backdrop to every pond and forest, frogs have no voice to represent themselves, and they certainly can’t write checks. It’s up to professional conservationists, including the rescue project’s 12 talented conservationists in Panama, to save frogs so that others can enjoy them. This, however, requires money. From now until the end of August, our generous sponsor Golden Frog—a global online services provider with a terrific name—will match donations to the rescue project up to $20,000, helping us raise money critical to our fight for frogs. Your donations during the Fight for Frogs campaign will buy us equipment to care for the frogs in the rescue pods, help us continue to conduct experiments to find a cure, ensure crucial breakthroughs, and ultimately one day see the return of these incredible species to their home in the wild.
The Panama Amphibian Rescue and Conservation Project is a project partnership between the Cheyenne Mountain Zoo, the Houston Zoo, Zoo New England and Smithsonian Institution. You can follow the Fight for Frogs campaign on Twitter using the #FightForFrogs hashtag or on the rescue project’s Facebook page.
Scientists at the Smithsonian Institution and partners have published a paper that will help them save Panamanian frog species from extinction due to a deadly fungal disease called Chytridiomycosis (chytrid). The study, which was published Jan. 4 in Animal Conservation, draws on the expertise of amphibian biologists and scientists the Panama Amphibian Rescue and Conservation Project to mathematically determine which frog species have the best probability of escaping extinction with the rescue project’s help.
“We don’t want to arbitrarily decide which species lives and which species don’t, nor do we want to waste our time on species that don’t need our help,” said Brian Gratwicke, co-author on the paper and international coordinator of the rescue project out of the Smithsonian Conservation Biology Institute. “This study took into account the differences in opinions among amphibian experts in Panama and found consensus in a systematic away. This has allowed us to focus on the species where we have the best chance of making a difference.”
The study also found that eight Panamanian species are likely now extinct in the wild due to disease-related declines. About 80 of Panama’s frog species were too rare for conservationists to prioritize their need for help or the likelihood of successful rescue. The new prioritization scheme, however, will allow the scientists to adapt to new information as it becomes available.
“Over the years, several frog populations—and even species—have vanished or nearly vanished from Panama,” said Roberto Ibáñez, the in-country director of the rescue project at the Smithsonian Tropical Research Institute, “Unfortunately, it is impossible to save them all through conservation programs. With this study, we can focus our limited resources on those species that we are more likely to find in the wild and breed in captivity, while we simultaneously look for a way to manage chytrid.”
Since 2009, the rescue project has been building and maintaining insurance populations of frog species susceptible to chytrid, bringing small groups into captivity to breed as the species crashes in the wild. For each of Panama’s 214 known frog species, the paper’s authors asked amphibian experts to determine the probability that: 1) the rescue project could locate an adequate founding population (20 males and 20 females), 2) the rescue project could successfully breed the species and 3) without the rescue project’s help, the species would go extinct.
While most of the rescue project’s original priority species ranked high based on the new prioritization scheme, the conservationists have already started making some changes. They have determined that the likelihood of successfully breeding La Loma tree frogs (Hyloscirtus colymba) is low and they are instead shifting resources to the recently discovered Craugastor evanesco and the Rusty robber frog (Strabomantis bufoniformis), both of which came up as high priorities.
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.
Matthew 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.
Enigmatic Fire salamander (Salamandra salamandra) declines in the Netherlands have been attributed to the recently described fungal pathogen Batrachochytrium salamandrivorans (Bs). Since 2010, the S. salamandra population at Bunderbos, Netherlands has decreased by 96%. An Martel et al’s recent Science paper showed that some US salamander species are highly susceptible to Bs, confirmed its occurrence in the pet trade, and noted that it has not yet been detected in the US. Large numbers of live salamanders are legally imported into the US each year for the pet trade. In the first 6 months of 2014, for example, 3,445 fire salamanders imported into the US, mostly from Slovenia.
The genus Batrachochytrium, which before the discovery of Bs solely included Batrachochytrium dendrobatidis (Bd), has gained an infamous reputation for global amphibian declines. Biologists believe that we are witnessing the sixth mass extinction in part because of the virulence and global spread of Bd among the world’s amphibians. The discovery of this new pathogen and our improved understanding of the ravaging effects of emerging wildlife disease raise concerns that US salamanders could share the same fate.
The US is a biodiversity hotspot for salamanders
Appalachia is a global salamander biodiversity hotspot (Source: http://www.biodiversitymapping.org/amphibians.htm)
The Appalachian Mountains are a renowned biodiversity hotspot for salamanders. The potential threat of this emerging pathogen in the US is therefore magnified, and it is imperative that we keep this disease out of the US. Salamander genetic diversity in the Appalachians is the highest in the world with 72 salamander species that are mostly endemic. The United States is home to nine out of ten salamander families and four of the ten extant salamander families are endemic to the United States including amphiumas, Pacific giant salamanders, torrent salamanders and sirens. Mole salamanders are also found in Canada and Mexico, but nearly all of their biodiversity is contained with U.S. borders. Giant salamanders are a primitive lineage of giant salamanders with three extant species, located in the U.S., Japan, and China. The hellbender is one of the giants and has found refuge in the Appalachian Mountains since amphibians originated, some 360 million years ago.
The ecological role of salamanders, the smaller majority, can often go unnoticed, but consider this biomass assessment of salamanders in Appalachia. One classic mark-recapture study in the eastern US noted “The biomass of salamanders is about twice that of birds during the bird’s peak breeding season and is about equal to the biomass of small mammals” (Burton and Likens 1975). With densities this high, a novel salamander-specific pathogen to which these animals have never been exposed have the potential be able to spread like wildfire, much like Bd spread through naïve Neotropical amphibian populations.
Immediate action is needed
We should immediately halt the importation of salamanders from any overseas sources, unless they can be certified free from Bs and Bd. In May 2008 the OIE, which is the organization created to mitigate zoonotic diseases (i.e., anthrax, mad cow disease, etc.), recognized Bd as a notifiable disease. Stricter trade regulations recommended by OIE would substantially reduce the spread of both Bs and Bd, however the OIE changes have not been adopted by the US Department of Agriculture and Interior and until doing so there are no legal means to reject infected shipments. A joint statement from the Amphibian Specialist Group and Amphibian Survival Alliance calls for immediate policy actions to stop the further spread of devastating wildlife diseases, and this time it is not too late to do something about it.
This new method could help us to test out new probiotic therapies. It can predict a captive-bred frog’s survival from exposure to chytrid fungus, without ever having to expose them to it experimentally.
Researchers at the University of Boulder Colorado, University of Zurich and Copenhagen University have developed a new method to predict how susceptible an amphibian is to a frog-killing fungus wiping out amphibians all over the world. The test looks at the antifungal properties of skin mucus that contains skin bacteria and chemicals secreted by the frog itself. Together the interactions between the skin bacteria and chemical secreted from glands on the frog skin are the frog’s first line of defense against skin disease.
Their paper, just published in PLOS One, sampled 8,500 frogs across Europe. They found that antifungal properties of the mucus were related to the prevalence of amphibian chytrid infection in natural populations. They found that when they experimentally exposed frogs to the chytrid fungus in a lab that they could predict survival of frogs based on an independent mucus sample. The researchers also found that when they added beneficial skin bacteria to the frogs that the anti-fungal properties of the skin were improved.
This study may help us to develop tools that we could use to reintroduce frogs back into areas affected by the frog-killing fungus, including Panama. “We have all these amphibians in captivity now, like the golden frog in Panama, a really beautiful species that is now extinct in the wild,” said Douglas Woodhams, a postdoctoral researcher at CU-Boulder and lead author of the paper. “We want to be able to reintroduce them, but the pathogen that attacked them is still out there,” he said. “Now we can determine what probiotic treatment might work best to protect the frogs without infecting them with the pathogen and seeing how many die.”