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

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

Spindly leg syndrome is reduced by increasing calcium hardness of water used to rear tadpoles

Atelopus varius metamorph with spindly leg (left) and without spindly leg (right)

Rearing frogs in captivity has its own unique challenges, one problem that has been a persistent issue in the Panama Amphibian Rescue and Conservation Project is spindly leg syndrome (SLS). This common musculoskeletal disease is mostly associated with captive amphibian breeding. SLS is a condition where legs of newly metamorphed amphibians, with otherwise healthy and typical development, are poorly developed and cannot support the weight or newly metamorphed froglets. Ultimately, SLS leads to death as the animal is unable to move or feed themselves. A brief review online will reveal a host of theories and potential remedies for the condition ranging from parental nutrition to water quality and dietary supplements, but there are very few replicated peer-reviewed experiments identifying the cause of this disease.

Elliott Lassiter and Orlando Garcés with the experimental rearing setup

As an intern with the Panama Amphibian Rescue and Conservation Project I teamed up with Orlando Garcés a graduate of the University of Panama and employee of the project to conduct an experiment primarily funded by the Morris Animal Foundation. We had observed that SLS was most prevalent in water that did not have any supplementary calcium and we knew that incoming water to our facility was very soft (lacking in calcium hardness). Bone growth is the symptom of SLS, therefore, we decided to look at the principle minerals affecting bone growth: calcium and phosphate. Tadpoles can gain calcium through their diet but they absorb about 70% of their calcium from the water through their gills and skin. The collected calcium is then stored in endolymphatic sacs in their heads and used during metamorphosis when tadpoles’ skeleton turns from cartilage into bone and limbs begin to grow.

We took 600 Atelopus varius tadpoles and divided them into three calcium treatments (low, medium, high) and then divided those into two groups one with added phosphate and one without added phosphate.  We monitored our tadpoles until they metamorphosed, at which point we looked at their legs and body posture to determine whether or not they had SLS. We found that calcium supplementation drastically increased survivorship overall and that the medium and high calcium groups had less SLS than the low calcium groups. Addition of phosphate also decreased the prevalence of SLS in low calcium treatment.

Based on the results of this study we were able to determine that SLS in harlequin frogs, is linked to an imbalance in calcium and phosphate homeostasis. Therefore, our current husbandry recommendation to reduce SLS in frogs and toads is to consider checking water hardness to determine if it is too soft. We also advise against over feeding tadpoles which has been shown to cause an increase in SLS prevalence in another experiment. We hope that our findings can guide future SLS research and help to lower the prevalence of SLS in captive amphibians, improving animal welfare. This research will help to improve the long-term sustainability of captive populations while researching solutions for the amphibian chytrid fungus and eventual reintroduction of these frogs back into the wild.

Lassiter, E., Garcés, O., Higgins, K., Baitchman, E., Evans, M., Guerrel, J., Klaphake, E., Snellgrove, D., Ibáñez, R. and Gratwicke, B., 2020. Spindly leg syndrome in Atelopus varius is linked to environmental calcium and phosphate availabilityPloS one15(6), p.e0235285.

By Elliot Lassiter and Orlando Garcés

Update on the conservation status of Harlequin Frogs in Panama

On May 30, 2019 a special issue of the Journal Biological Conservation entitled ‘Amphibian conservation in the Anthropocene: Progress and challenges‘ Edited by Vincent Devictor, Evan Grant, Erin Muths, Benedikt Schmidt, Silviu Petrovan was published. The focus of this issue is on examples of potential solutions to the amphibian crisis that are directly relevant to, and integrated with conservation management actions.

The issue features a case study on Atelopus in Panama, updating the known historical distribution records and modeling potentially suitable habitat (below).

The paper also updates the IUCN conservation status for each Panamanian Atelopus species and the status of each of these species in captive populations, with commentary on the potential use of the captive population in research to find solutions that may be useful in restoring wild populations.

Atelopus conservation status in Panama 2019

Lewis CHR., Richards-Zawacki CL., Ibáñez R., Luedtke J., Voyles J., Houser P., Gratwicke B. 2019 Conserving Panamanian harlequin frogs by integrating captive-breeding and research programs. Biol. Conserv. 236, 180–187. (doi:10.1016/J.BIOCON.2019.05.029)