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

 

In unprecedented effort, more than 40 organizations from 13 countries come together to protect and restore harlequin toads, the jewels of South and Central America, hard hit by a deadly amphibian disease

With the formation of the Atelopus Survival Initiative (ASI)–a new alliance of more than 40 organizations from 13 countries–comes a new day for harlequin toads, the jewels of South and Central America’s forests and creeks and a group of amphibians hardest hit by the deadly chytrid fungus Batrachochytrium dendrobatidis (Bd). 

While amphibian researchers and conservationists have worked for many years to save harlequin toads (which make up the Atelopus genus) and groups of species in individual countries, the ASI is bringing them together for the first time to pool the resources, decades of experience and knowledge necessary to prevent the extinction of the entire genus of harlequin toads across the region where these species still survive. 

“As an incredibly diverse group of amphibians facing a number of threats, harlequin toads require innovative solutions coming from a diverse group of individuals and organizations with different expertise, knowledge and capacities,” said Lina Valencia, ASI founder, co-coordinator of the IUCN SSC Amphibian Specialist Group Atelopus Task Force and Andean countries coordinator for Re:wild, one of the primary ASI conveners. “More than ever before, we need a constellation of champions working together to bring harlequin toads back from the brink of extinction. The ASI underscores the vital need to implement on-the-ground conservation actions that will mitigate the main threats to this beautiful group of amphibians.”

Over the past few decades, many harlequin toad species have suffered severe population declines and extinctions throughout their range. Today, of the 94 harlequin toad species that have been assessed by the IUCN, 83 percent are threatened with extinction, while about 40% of Atelopus species have disappeared from their known homes and have not been seen since the early 2000s, despite great efforts to find them. Four harlequin toad species are already classified as extinct, according to the IUCN Red List of Threatened Species, but this number is likely higher. 

The fungus Batrachochytrium dendrobatidis (Bd) causes the lethal disease chytridiomycosis, which has resulted in amphibian declines all around the world, including in South and Central America, Australia and the western United States. Although Bd may likely be the primary driver of these declines, a number of other threats are exacerbating the precipitous drops in population numbers. This includes habit destruction and degradation (as the result of animal agriculture, logging, mining and infrastructure development), the introduction of invasive species such as the rainbow trout that prey on harlequin toad tadpoles, pollution, illegal collection for the pet trade, and the effects of climate change.

The ASI and its members, including governments, local communities and Indigenous peoples, will collaboratively address each of these threats–and new ones as they arise–across the genus’s full range, taking into account the social, political and cultural realities of each of the 11 countries where harlequin toads are found. 

“With their beautiful songs and unique lifestyles, amphibians are among the most extraordinary animals on Earth, and among them, harlequin toads stand out for their amazing colors,” said Luis Fernando Marin da Fonte, coordinator of the ASI and director of partnerships and communications for the Amphibian Survival Alliance. “But these colorful and delicate jewels are becoming increasingly rarer. Harlequin toads must be protected not only because of their beauty and uniqueness, but also because of their intrinsic value and biological, ecological and even cultural importance.”

The initiative’s newly developed Harlequin Toad (Atelopus) Conservation Action Plan (HarleCAP) provides the roadmap for conserving and restoring harlequin toads as a genus and their habitat. The action plan’s goals, which ASI aims to achieve by 2041 (the 200th anniversary of the description of the genus Atelopus), include:

  • developing and implementing innovative methods to mitigate chytrid’s impacts on harlequin toad populations and better understanding why some species are less susceptible to the effects of chytrid;
  • protecting and restoring harlequin toads’ forests and watersheds;
  • creating and maintaining conservation breeding programs;
  • searching for species that are lost to science and filling in other gaps in scientific knowledge about harlequin toads;
  • sharing stories that will transform harlequin toads into symbols of hope for the region and the world and a flagship for conservation success, and demonstrate a commitment to the conservation of harlequin toads; 
  • ensuring the Atelopus conservation network has the technical, logistical, and financial support to secure the long-term conservation of harlequin toads

“The establishment of collaborative initiatives at the international and regional level is essential to coordinate efforts and obtain tangible results that have an efficient and real impact on the conservation of an endangered species,” said Gina Della Togna of the Universidad Interamericana de Panamá, Panamá. “The Atelopus Survival Initiative is a concrete example, which not only aims to conserve one species, but an entire genus, perhaps the most threatened by the global amphibian extinction crisis.”

Harlequin toads are found from Costa Rica in the north to Bolivia in the south, and Ecuador in the west and French Guiana to the east. They are known as the jewels of South and Central America in part because of their beautiful and varied colors, which range from orange, green, yellow, brown, black, red, and sometimes even purple. They are celebrated in a number of Latin American cultures, including Indigenous cultures, and across entire countries, like in Panama, where the national animal is the Panamanian golden toad.

Like other amphibians, harlequin toads support healthy ecosystems. Their tadpoles depend on clean water and, because of this, the presence of harlequin toads indicates better quality water in an ecosystem, while their decline or absence is often the first sign of an ecosystem in trouble. 

“Protecting and restoring harlequin toads and their habitats will also benefit the species that share the ecosystems in which they live and that provide water to tens of millions of people, and ultimately all life on Earth,” Valencia said. “And we’re hoping that the ASI will be a successful model that conservationists can emulate for other groups of threatened species.”

 The Atelopus Survival Initiative includes national and international conservation groups, zoos, captive breeding centers, academic institutions, governments and local communities. Its current members represent the following organizations: Amphibian Ark, Amphibian Survival Alliance, Asociación Pro Fauna Silvestre  – Ayacucho, Bioparque Municipal Vesty Pakos, Bolivian Amphibian Initiative, Centre National de la Recherche Scientifique, Centro de Conservación de Anfibios AMARU, Centro Jambatu de Investigación y Conservación de Anfibios/Fundación Jambatu, CORBIDI, DoTS, El Valle Amphibian Conservation Center Foundation, Facultad Latinoamericana de Ciencias Sociales, Florida International University, Fort Worth Zoo, Fundación Atelopus, Fundación Zoológica de Cali, Universidad del Tolima (GHEE), Grupo de Trabajo Atelopus Venezuela, Image Conservation, Instituto Nacional de Pesquisas da Amazônia, Instituto Venezolano de, Investigaciones Científicas, Ministerio del Ambiente de Perú, MUBI (Museo de Biodiversidad del Perú), Parque Explora, Parque Nacional Natural Puracé, Photo Wildlife Tours, Pontificia Universidad Católica del Ecuador, Pontificia Universidad Javeriana, Re:wild, San Diego State University, Smithsonian Tropical Research Institute, Trier University, Universidad de Antioquia, Universidad de Costa Rica, Universidad de los Andes, Universidad del Tolima, Universidad del Magdalena, Universidade Federal do Pará, Universidad Nacional, Universidad Interamericana de Panamá, Universidad Nacional de Colombia, Universidad San Francisco de Quito, Universidade Estadual de Campinas, Universidade Federal do Oeste do Pará, University of Nevada, Reno, University of Notre Dame, University of Pittsburgh, WCS (Wildlife Conservation Society), WCS Colombia, Zoológico Cuenca Bioparque Amaru

New publication! IUCN Guidelines for amphibian reintroductions and other conservation translocations

An exciting new publication has just been released by the International Union for the Conservation of Nature (IUCN) of best practice guidelines for a wide range of amphibian conservation translocations. The project was many years in development through the coordinated effort of numerous translocation specialists across the globe, but the project the led by the Panama Amphibian Rescue and Conservation Project’s post-doctoral research fellow Dr. Luke Linhoff. The guidelines cover the reasons for conducting amphibian translocations, pre-translocation planning and risk assessment, and also cover important topics such as disease, welfare, human social dimensions, post-release monitoring and reporting results.

A free digital download and more information on the guidelines can be found at: https://www.iucn-amphibians.org/iucn-guidelines-for-amphibian-reintroductions-and-other-conservation-translocations/

Meet the spectacular Borderlander frog Atelopus fronterizo, Panama’s newest frog species!

Milan Vesely and Abel Batista scientifically described Panama’s seventh harlequin frog species in the journal Zoological Research in April 2021. This beautiful harlequin frog species has been in biological collections for some time, the first museum specimen was first collected by Henri Pittier at Puerto Obaldlia in 1911, but has previously been assumed to be related to other sister species that it closely resembles. Historically collected formalin-preserved specimens were unsuitable for genetic analyses and so the frog remained undescribed.

Vesely and Batista conducted expeditions to the Darien to collect new specimens and this allowed them to conduct genetic analysis showing that this species is most closely related to Atelopus certus and Atelopus glyphus, but is genetically distinct enough to warrant recognition as a species. It also has a slightly different call and morphological characteristics that are also described in the paper.

Borderlander Harlequin Frog

Atelopus fronterizo Photo by Abel Batista, UNACHI-Fundación Los Naturalistas-SNI (SENACYT)

They named the frog Atelopus fronterizo to refer to borderland inhabitants and the Panamanian border security force who protect the Darien mountain range in NE Panama on the border where this frog is found. Like other harlequin frog species in Panama, even though they live in well-protected habitat, they are Likely Critically Endangered due to the threat of the amphibian chytrid fungus that has caused the declines of other Atelopus species in Panama.

Vesely, M. and Batista, A., 2021. A new species of Atelopus (Amphibia: Bufonidae) from eastern Panama. Zoological research, pp.272-279.

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