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

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


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)

Love potion for frogs

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.