Happy 13th Golden Frog Day!

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Every year on August 14th, Panamanians officially celebrate golden frog day as a symbol of Panama’s incredible biodiversity and cultural diversity. This year we are thrilled to share the original song “La Rana Dorada” by National Geographic explorer Janni Benavides of Jacana Jacana. The song is one of four original scores developed as part of a National Geographic Meridian Grant supporting the Atelopus Survival Initiative.

On spotify https://open.spotify.com/album/3C3J2FJXRyxYGKLffGXyEe

On you tube https://youtu.be/LwxzgzKa40A 

Following reintroduced frogs

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The Panama Amphibian Rescue and Conservation Project completed the first reintroduction trial of the Limosa Harlequin frog (Atelopus limosus) in 2017, and our findings were published in the journal Frontiers in Amphibian and Reptile Science. Reintroducing a species comes with a lot of unknowns and questions, just a few are: Where will the frogs go? What is their life like outside of a terrarium? Will they become infected with amphibian chytrid fungus? The purpose of this first reintroduction trial was to begin to unravel some of these questions so the researchers could adapt their strategies and improve the odds of the frogs in the wild.

We were able to get a detailed life of the frogs post-reintroduction by radiotracking the frogs and checking in on them daily. We found that when frogs were provided a 30-day acclimation period in a predator-free rainforest mesocosm, their probability of survival significantly increased and they did not disperse as far as the hard-released animals. We know from other studies that more movement can increase the likelihood of predators finding animals, and that likely happened in this study too. Frogs that were released without radio transmitters were 44x less likely to be reencountered during stream surveys (finding a frog in the rainforest isn’t easy!). We were able to follow the lives of these frogs in the wild for up to 56 days after release and developed a method that can use both radio-tracked animals and non-radiotracked animal encounters to estimate survivorship by assigning different detection probabilities in the model.

We observed a couple predation events of reintroduced frogs and some became infected with amphibian chytrid fungus. However, we learned a lot and has no shortage of questions to continue researching to get this species (and others in Panama) back into the wild.

Read the open access paper here:
Klocke, B., Estrada, A., Mataya, M., Medina, D., Baitchman, E., Belden, L., Guerrel, J., Evans, M., Baughman, J. and Connette, G., Illueca, E., Ibáñez, R., Gratwicke, B. (2023) Movement and survival of captive-bred Limosa Harlequin frog (Atelopus limosus) released into the wild. Frontiers in Amphibian and Reptile Science, 1, p.1205938. https://doi.org/10.3389/famrs.2023.1205938 

by Blake Klocke

The Golden Frog Song

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Enjoy this performance of “La Rana Dorada” one of four original songs by National Geographic explorer Jani Benavides of Jacana Jacana as part of the Atelopus Survival Initiative and National Geographic Meridian Grant meeting hosted by the Fundacion Atelopus and Re:wild in Sierra Santa Marta.

Insectarium

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Construction of our new insect facility was completed and fire suppression systems installed. This new 1,600 square feet insectarium has two climate-controlled rooms that can be maintained at different temperatures, allowing a diversity of food items with varying sizes and nutritional properties to meet the needs of our diverse amphibian collection. A huge thanks to the STRI Office of Facilities and Engineering who oversaw this project and to the many individual donors, the Cheyenne Mountain Zoo, Zoo New England, the Houston Zoo, the Holtzman Foundation, the Shared Earth Foundation for enabling us to complete it.

Under the leadership of Nancy Fairchild and Jennifer Warren, our insect production capacity is now one of the incredible success stories of our project. There are no local sources of captive-reared insects, so we are 100% reliant on our own production and have redundant capacity to accommodate any unexpected changes in the populations of the various springtails, fruitflies, crickets, pantry moths, soldier flies, cockroaches and earthworms that we produce. This team has also conducted original research to develop gutloading propocols for springtails and demonstrated improved outcomes of juvenile frogs reared on this nutritionally supplemented prey, compared to their normal yeast-based diets.

First release trial of Atelopus limosus shows that animals rapidly recover a wild-type skin microbiome.

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In 2017, Virginia Tech PhD students Angie Estrada and Daniel Medina conducted the first release trial of captive-bred Limosa Harlequin frogs at the Mamoni Valley Preserve. Their study aimed to closely observe how 1-year-old captive-bred animals would transition from captive conditions back into the wild. To soften the blow of the changing conditions, and to allow the researchers to capture the frogs again, they designed 30 square shaped mesocosms from plastic mesh. In reintroduction biology placing animals in a temporary field enclosure prior to release is known as a soft release. They filled the bottom layer with leaf litter collected from the forest floor that was rich in leaf-litter invertebrates, a diet quite different from the crickets and fruit-flies they are usually fed by staff at the Panama Amphibian Rescue and Conservation Center. Each mesocosm housed a single animal that was monitored daily  for a month and weighed and swabbed weekly.

It is known that captivity can alter skin microbiomes of amphibians and other captive animals, and that this is an important component of amphibian immune defenses. This study found that wild and captive Atelopus limosus had very different microbiomes, but that after a month living in mesocosms, the skin microbiome had rapidly changed to resemble the microbiome of wild individuals. One concern about bringing animals into captivity for prolonged periods is that animals might lose symbiotic microbes that help them to survive in the wild, which might reduce the fitness of captive-bred animals, but this study found that the skin microbiome was rapidly rewilded.

The mesocosms were a useful tool that protected the animals from larger predators, though one was killed by army ants. Female animals lost body condition more rapidly than males, but at the end of the trial their condition resembled that of wild-caught animals. About 15% of the animals became infected with the amphibian chytrid fungus in the first month compared to 13-27% Bd prevalence in wild amphibian community.

Estrada, A., Medina, D; Gratwicke, B, Ibáñez, R, Belden, L (2022) Body condition, skin bacterial communities and disease status: Insights from the first release trial of the Limosa harlequin frog, Atelopus limosus. Proceedings of the Royal Society B. https://doi.org/10.1098/rspb.2022.0586

It’s world frog day and we are celebrating Harlequin frogs!

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There are more than 100 brightly colored Harlequin toads in the genus Atelopus, and 90% of them are threatened with extinction, and are highly susceptible to the deadly amphibian chytrid fungus. The Panama Amphibian Rescue and Conservation Project is working to save 5 critically endangered Panamanian Atelopus species from extinction and is part of a a larger global conservation effort called the Atelopus Survival Initiative.

The Panamanian Golden frog Atelopus zeteki.

The Panamanian Golden Frog Atelopus zeteki is a national symbol of Panama, and one of the most recognizable harlequin frogs, but they are extinct in the wild. Fortunately this species breeds in 50 AZA zoos and aquaria and at the Panama Amphibian Rescue and Conservation Project. The National Zoo has conducted intense research efforts to develop a probiotic cure for the amphibian chytrid fungus, but these efforts have not been successful to date.

The Variable harlequin frog Atelopus varius

As its name suggests the variable harlequin frog  comes in lots of different colors, from solid gold like Panamanian golden frogs to brown, green or orange! This species is a very close genetic sister species to the Panamanian golden frog Atelopus zeteki, and both species are referred to as golden frogs. Atelopus varius that have survived the amphibian chytrid epidemic seem to have experienced recent migration or gene flow into their populations, indicating the potential for genetic rescue in this species. We are actively exploring this idea with a grant from Revive and Restore.

The Pirre harlequin frog Atelopus glyphus

Some people call Atelopus the stub-foot toads because the etymology of word ‘Atelopus’ translates to “incomplete end of toe”. However, a more commonly used name for this group of animal is harlequin toads because their bright patterns and colors are reminiscent of a court jester costume. Atelopus are true toads because they are in the toad family Bufonidae and while they don’t resemble the more familiar brown, warty toad species, they do lay the double-stranded strings of eggs characteristic of toads. All toads are also members of the tail-less amphibian order Anura (Frogs), so sometimes they are also called harlequin frogs.

Toad mountain harlequin frog Atelopus certus

Male harlequin toads can be very clingy, hanging on to females for up to 100 days before they lay their eggs, it is a kind of mate-guarding behavior.  Egg laying can take 8-9 hours, and once the eggs have been laid the males swiftly lose interest and abandon the females fairly quickly.

The Limosa harlequin frog Atelopus limosus

Limosa harlequin frogs are found in central Panama and has declined rapidly due to the amphibian chytrid fungus. Researchers at the Pamama Amphibian Rescue and Conservation Project and Virginia Tech conducted the first release trials for this species. We reared captive frogs and released them to understand how animals transition from captive conditions to the wild and attached tiny radiotransmitters to the frogs to understand where they disperse.

New study genetically engineers skin bacteria in attempt to control the amphibian chytrid fungus

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In our latest research project the Smithsonian Conservation Biology Institute and the Synthetic Biology Center Dept of Biological Engineering at MIT collaborated to test two technically challenging ideas using probiotic approaches to protect highly susceptible amphibians from the amphibian chytrid fungus (Bd). The paper was recently published in the Journal ISME Communications. We isolated a core frog skin bacteria that is found in high numbers on most golden frogs and genetically engineered it to produce an antifungal metabolite that kills the pathogen called violacein. By using a bacterium well-adapted to thrive on the frogs’ skin, that also produces antifungal metabolites we hoped to protect the frogs from disease. We were able to genetically modify one core skin microbe to produce violacein, but it did not persist well on the frog skin and was displaced after 4 weeks by the unmodified native bacteria strain. Treating the frogs with this genetically modified core skin microbe did not prevent the frogs from Bd or reduce infections.

In this new experiment, we tested two new probiotic strategies to protect frogs from Bd. 1) Using a consortium of antifungal bacteria isolated from the frogs 2) Using a core skin microbe found on all golden frog skin that was genetically modified to produce antifungal metabolites.

In a second experimental group we mixed a consortium of seven antifungal bacteria that had been isolated from golden frog skin and supplemented the skin microbiome with these potentially beneficial microbes. Three of the seven bacteria persisted on the skin after 4 weeks, but this probiotic treatment also failed to protect the frogs from disease. While these results are disappointing, we were able to successfully test two technically-challenging ideas that have been discussed in the amphibian conservation community for many years. Furthermore, this research illustrates some of the challenges we still face in understanding and manipulating microbiomes and in using synthetic biology to solve real environmental problems.

The research was led by Dr. Matthew Becker, Rob Fleischer and Brian Gratwicke (Smithsonian’s National Zoo and Conservation Biology Institute) and Dr. Jennifer Brophy and Christopher Voigt (Synthetic Biology Center Dept of Biological Engineering at MIT). Other collaborators include Ed Bronikowski, Matthew Evans, Blake Klocke, Elliot Lassiter, Alyssa W. Kaganer, Carly R. Muletz-Wolz (Smithsonian’s National Zoo and Conservation Biology Institute). Kevin Barrett (Maryland Zoo in Baltimore), Emerson Glassey & Adam J. Meyer (MIT). The work was funded by individual donors, the Smithsonian Institution Competitive Grants Program for Science, the Smithsonian Postdoctoral Fellowship, the U.S. Fish and Wildlife Service Division of International Conservation Amphibians in Decline Fund, the U.S. Defense Advanced Research Projects Agency’s Biological Robustness in Complex Settings program. The Maryland Zoo in Baltimore and the AZA Golden Frog Species Survival Program provided surplus-bred animals for research.

By Brian Gratwicke

Becker, M.H., Brophy, J.A.N., Barrett, K, Bronikowski, E., Evans, M., Glassey, E., Klocke, B. Lassiter, E., Meyer, A.J., Kaganer, A.W., Muletz-Wolz, C.R.,  Fleischer, R.C., Voigt, C.A., and Gratwicke, B. Genetically modifying skin microbe to produce violacein and augmenting microbiome did not defend Panamanian golden frogs from disease. ISME Communications