Scientists at the Smithsonian Institution and partners have published a paper that optimizes sperm collection protocols from the critically endangered Panamanian Golden Frog Atelopus zeteki. It also improves our understanding of reproduction in endangered harlequin frogs. The research, to be published published 15 March 2017, in Theriogenology, was conducted by Dr. Gina DellaTogna, a Panamanian biologist who studied this charismatic animal at the National Zoological Park in Washington DC. The study characterizes the dose-response patterns for several artificial hormone treatments and describes the sperm morphology for the first time in this species.
Atelopus zeteki spermatazoan
“This study is important, because it contributes towards the basic understanding of reproduction of a highly endangered group of frogs in Latin America,” said DellaTogna, who performed the experiments for her PhD at the University of Maryland. “This study has already helped us to solve critical reproduction problems in captive Atelopus collections in Panama and allowed us to repeatedly collect high-quality sperm samples for genome resource banking at any time of the year, without harming the frogs.”
“Basic reproductive research is something that has yielded huge conservation dividends for the successful care and management of other endangered species like Pandas and Black Footed Ferrets,” said Pierre Comizzoli, a co-author of the paper and reproduction specialist at the National Zoo. “Gina’s research opens the door to develop methods like sperm freezing and storage to preserve the long term genetic integrity and diversity in small populations.”
The research is particularly relevant to current amphibian conservation efforts in Panama where the Panama Amphibian Rescue and Conservation Project has captive-breeding colonies of five species of Atelopus that are threatened with extinction from the deadly fungal disease chytridiomycosis.
Roberto Ibáñez, and Gina DellaTogna working on hormonal stimulation of frogs at the Panama Amphibian Rescue and Conservation Project
“Successful reproduction is key to any captive assurance program,” said Roberto Ibáñez, the director of the Panama Amphibian Rescue and Conservation project at the Smithsonian Tropical Research Institute in Panama. “Gina has already begun applying what she has learned to successfully help us to produce offspring from four other endangered harlequin frog species. I hope that she will eventually extend it to species with different modes of reproduction that are also difficult to breed”.
The research was made possible with assistance from the Maryland Zoo in Baltimore who manage the Golden Frog Species Survival Plan. Funding was provided from the Panamanian Government’s Secretaría Nacional de Ciencia y Tecnología (SENACYT), The WoodTiger Fund, the Smithsonian Endowment for Science and the University of Ottawa Research Chairs Program.
Della Togna G, Trudeau VL, Gratwicke B, Evans M, Augustine L, Chia H, Bronikowski EJ, Murphy JB, Comizzoli P. 2017 Effects of hormonal stimulation on the concentration and quality of excreted spermatozoa in the critically endangered Panamanian golden frog (Atelopus zeteki). Theriogenology. http://dx.doi.org/10.1016/j.theriogenology.2016.12.033
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.
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.
The first captive-bred Andinobates geminisae at the Gamboa Amphibian Research and Conservation Center. Photo by Jorge Guerrel, Smithsonian Tropical Research Institute.
Smithsonian Conservation Biology Institute (SCBI) and Smithsonian Tropical Research Institute (STRI) scientists working as part of the Panama Amphibian Rescue and Conservation Project hatched the first Andinobates geminisae froglet born in captivity. The tiny dart frog species only grows to 14 millimeters and was first collected and described last year from a small area in central Panama. Scientists collected two adults to evaluate the potential for maintaining the species in captivity as an insurance population.
“There is a real art to learning about the natural history of an animal and finding the right set of environmental cues to stimulate successful captive breeding,” said Brian Gratwicke, amphibian conservation biologist at SCBI and director of the Panama Amphibian Rescue and Conservation Project. “Not all amphibians are easy to breed in captivity, so when we do breed a species for the first time in captivity it is a real milestone for our project and a cause for celebration.”
Scientists simulated breeding conditions for the adult frogs in a small tank. The frogs laid an egg on a bromeliad leaf, which scientists transferred to a moist petri dish. After 14 days, the tadpole hatched. Scientists believe adult A. geminisae frogs may provide their eggs and tadpoles with parental care, which is not uncommon for dart frogs, but they have not been able to determine if that is the case. In the wild, one of the parents likely transports the tadpole on his or her back to a little pool of water, usually inside a tree or on a bromeliad leaf.
After the tadpole hatched, scientists moved it from the petri dish to a small cup of water, mimicking the small pools available in nature. On a diet of fish food, the tadpole successfully metamorphosed into a froglet after 75 days and is now the size of a mature adult.
Panama Amphibian Rescue and Conservation Project scientists are unsure if A. geminisae is susceptible to the amphibian-killing chytrid fungus. However, since it is only found in a small area of Panama and is dependent on primary rain forests, which are under pressure from agricultural conversion, they have identified it as a conservation-priority species.
The Panama Amphibian Rescue and Conservation Project breeds endangered species of frogs in Gamboa, Panama and El Valle, Panama. The Panama Amphibian Rescue and Conservation Project is a partnership between the Houston Zoo, Cheyenne Mountain Zoo, Zoo New England, SCBI and STRI. This study was supported by Minera Panama.
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.”
Before beginning my research at the Smithsonian, I knew surprisingly little about the status of the world’s amphibians. I was fortunate to be awarded an internship through Kansas State University College of Veterinary Medicine based at the Smithsonian Conservation Biology Institute in Front Royal, VA. Once I arrived in Front Royal, I learned quickly about the plight of amphibians around the globe and I remember listening in awe when I was first told about the Amphibian Arks in Gamboa and El Valle in Panama and how they hold the last remaining individuals of some of Panama’s most precious amphibian species. They had been brought into these assurance colonies because of the threat of a devastating fungal pathogen Batrachochytrium dendrobatidis (Bd) that is responsible for the disappearance of many species around the world. Initially, the harlequin frogs in the Amphibian Arks in Panama were housed individually, because when placed together keepers observed that males would fight and they were worried the fighting would unduly stress these precious animals. However, this quickly led to a space shortage as the amphibian ark filled up. The Maryland Zoo in Baltimore, which helps coordinate the Species Survival Plan for Panamanian Golden frogs in the USA, suggested that we group the animals together in same-sex groups. They noted that this was working out just fine for frogs reared in captivity, but we wanted to evaluate whether wild-collected harlequin frogs would adapt to this group-housing situation.
Ethogram showing different types of aggressive interactions
My collaborators in Panama conducted a behavioral study of frogs placed in groups and monitored the number of aggressive interactions between the frogs, making sure that there were no injuries. Back in Front Royal, I worked with my colleagues in the endocrine labs to measure the amount of cortisol, a steroid hormone that the frog produces and can be detected in its poop. We did this by adapting existing stress hormone monitoring methods that the SCBI uses for many other wildlife species, like pandas and elephants. We found that, initially, the frogs housed together would interact aggressively towards one another. This aggression was mirrored by an elevation in stress hormones in the feces during the first week of the study. However, after the first 2 weeks, the frequency of aggressive behaviors declined dramatically, and the concentration of cortisol dropped back down to normal. Based on these results, we determined that wild caught harlequin frogs could be safely housed together in same-sex groups over the longer term, and this study helped us to greatly reduce space constraints in our ex-situ collection of amphibians. We hope that our new method may be useful to others wanting to evaluate husbandry issues in captive amphibian collections. Read the full paper in PLoS One here By Shawna Cikanek, Kansas State University
Our frog conservation team in pod #6 Jorge Guerrel, Dr. Roberto Ibáñez, Dr. Della Garelle, Angie Estrada and Dr. Brian Gratwicke happy to see the product of years of hard work.
Matt Evans, Angie Estrada and Rigoberto Díaz moving frogs out of the quarantine room at Summit Municipal Park
Amphibian rescue pods at the Gamboa ARC
Our awesome volunteers Dr. Myra Hughey and Dr. Justin Touchon literally doing some heavy lifting for conservation.
Nair Cabezón, helps get cages cleaned for the move.
Veterinarian Dr. Della Garelle from Cheyenne Mountain Zoo moves frogs into their new digs!
Lanki Cheucarama inspecting the fruit-fly cultures.
Captive-bred Atelopus limosus getting settled into their new tanks at the Gamboa ARC
Atelopus certus checking out her new digs in pod#6
November was the culmination of a year of incredibly hard work for us in Panama. We finally moved into our beautiful our beautiful new frog conservation facility in Gamboa. Maersk Line generously donated 7 shipping containers that that once ferried ice cream and frozen vegetables around the world, but they now house a most precious collection of endangered Panamanian frogs. The new Gamboa ARC (Amphibian Rescue Center) is an incredible leap forward enabling us to more effectively tackle the amphibian conservation crisis in Panama.
We are incredibly grateful to the Summit Municipal Park, who have been our generous hosts for the first 4 years of our project, and to our project partners Cheyenne Mountain Zoo, Houston Zoo, Zoo New England, the Smithsonian Conservation Biology Institute and the Smithsonian Tropical Research Institute. We have relied intensively on each other for help over the last 4 years and it has truly been a team effort! Generous grants from USAID and Minera Panama were the primary source of funds used for the construction of phase I and thanks to them, we now have a world class amphibian conservation facility. We have essential back-up systems such as an emergency generator power, and backup air-conditioning so that frogs can be kept in simulated tropical montane forest environments, even in the event of a power failure. We are now getting ready to break ground on phase II, a new NSF-funded amphibian research lab, quarantine and office building that will be the hub of our new research facility for the conservation of endangered Panamanian amphibians.
Juvenile Panamanian golden frog, Atelopus zeteki, reared at the El Valle Amphibian Conservation Center
Now in its fourth year, Panama’s Golden Frog Day, August 14, is a salute to Panama’s cultural and ecological heritage with the golden frog, one of the most iconic symbols of Panama. The national legislation promotes species preservation and maintains an objective to promote conservation and protection of this amphibian species. This year the country can celebrate the successful breeding of the Panamanian golden frog at the El Valle Amphibian Conservation Center (EVACC), located inside the Níspero Zoo in El Valle de Antón. The egg clutch laid on November 24, 2012 successfully developed into tadpoles and were raised to form a group of 42 healthy young golden frogs.
“Bringing wild animals into captivity is only the beginning of the work that we do in our facility. Fusing applied technology, available resources, and human innovation to create Mother Nature, inside, is the challenge, “ said Heidi Ross, Director of EVACC. “Learning from our past experiences we focused a lot of energy on diet, and as the saying “you are what you eat” applies to humans, it also is essential for amphibians”.
EVACC director, Heidi Ross with a box of juvenile captive-reared golden frogs (Atelopus varius).
“We are extremely proud of our conservation team in Panama,” said Peter Riger, director of conservation programs at the Houston Zoo, and one of the principal sponsors of this project. “EVACC has successfully bred both golden frog species in captivity and they have aggressive population management goals to grow the captive population to at least five hundred individuals for each species that I’m sure they will meet.”
The EVACC facility forms part of the Panama Amphibian Rescue and Conservation Project. The Project collects frogs in areas threatened by the devastating chytrid fungal disease that has decimated amphibians worldwide. The hope is to learn to raise these animals in captivity until enough is known about the disease to allow researchers to release amphibians into the wild once again. Project partners include the Smithsonian Tropical Research Institute, the Smithsonian’s National Zoological Park, the Cheyenne Mountain Zoo, the Houston Zoo, and Zoo New England. To learn more about the project please visit the project’s website.
This award-winning documentary featuring our race to find a cure for a deadly amphibian disease and to build an amphibian ark in Panama is now available for FREE. Watch the trailer below and download the full feature if you would like to see more on the itunes store for a limited time only.