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

 

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

A vigorous immune response to the chytrid fungus is associated with susceptibility to the disease

For frogs dying of the invasive chytridiomycosis disease, the leading cause of amphibian deaths worldwide, the genes responsible for protecting them may actually be leading to their demise, according to a new study published today in the journal Molecular Ecology by Smithsonian Conservation Biology Institute (SCBI) and University of Central Florida researchers.

The lowland leopard frog, found in river drainages in Arizona, is one of a few amphibian species in which some individuals survive infection by Batrachochytrium dendrobatidis chytrid fungus (Bd) while other individuals do not—even when they live in the same local population.

In a study of lowland leopard frogs infected with Bd, the fungus that causes the disease chytridiomycosis or chytrid, researchers found that frogs that died from the disease had higher expression of major histocompatibility complex and other immune system genes than frogs that survived it. Those genes help organisms fight off infections and foreign substances.

“This result was totally counterintuitive and the opposite of the pattern we expected to recover,” said Anna Savage, the study’s lead author, an associate professor in UCF’s Department of Biology and former postdoctoral fellow at SCBI’s Center for Conservation Genomics (CCG).

Comparison of differential gene expression in control, early infected, surviving and susceptible frogs in both the spleen (left) and skin (right). 

“My previous research on these immune genes showed that some variants were associated with higher survival to Batrachochytrium dendrobatidis, so I hypothesized that those genes were enabling the frogs to have a stronger immune response that would kill the fungus,” she said. “Instead, it seems like those stronger responses are linked to susceptibility, and the genes associating with survival are linked to reduced immune function.”

Savage said acquired immune responses can be very potent, require a lot of energy from the body and can sometimes produce toxic byproducts that harm the host and the pathogen.

“Immune responses are much more complex than just an on-off switch,” she said. “A big part of the immune system is regulating the type, timing and dosage of a particular response, and if any of those components get dysregulated, it can have extremely negative consequences.”

She said, for instance, Batrachochytrium dendrobatidis suppresses the host immune system by killing B and T lymphocytes. “Because those are the same cells that proliferate during acquired immune responses, producing lots of those cells might just be wasting energy on something that chytrid can easily destroy,” she said.

Amphibian populations are in decline around the world, with two-thirds of the world’s 8,000 species considered to be threatened and nearly 200 species that have already gone extinct in the last two decades. In the U.S., amphibian populations overall are declining at a rate of nearly 4 percent a year, with some areas, such as the Rocky Mountains and the West Coast, facing a higher rate of decline, according to the U.S. Geological Survey.

Although the researchers studied immune gene expression in lowland leopard frogs with chytridiomycosis, the findings may be useful for studying the disease in other frog species due to genetic similarities they share, Savage said.

 

Lowland leopard frogs were chosen for the study because their responses to chytridiomycosis vary from one individual to the next, unlike many other frog species that are completely susceptible to the disease or are completely resistant or tolerant.

This allowed the researchers to rule out genetic variation between species and pinpoint specific differences in lowland leopard frogs’ immune genes that predicted different responses to infection.

The frogs were collected in Arizona and shipped overnight to the Smithsonian’s National Zoo in Washington, D.C., where the infection experiments were conducted. Subsequent analyses of gene expression occurred at the SCBI’s Center for Conservation Genomics. Statistical analyses of the data were performed at UCF.

Robert Fleischer, senior scientist and head of the SCBI’s CCG, co-authored the study and was Savage’s main advisor for the research when she was a postdoctoral fellow at the Smithsonian. Fleischer said the results help in understanding why some frogs survive the disease and others do not.

“If we can solve this mystery, and we have taken a big step in that direction with this study, our hope and plan is to use this information to develop resources and strategies to mitigate the disease in the more susceptible species, and to counter the worldwide tide of extinction and endangerment caused by chytrid,” he said.

The researcher said the findings also show that acquired immune responses, such as those generated by vaccination, may not always be useful in combating invasive diseases of conservation concern.

Brian Gratwicke, a conservation biologist with SCBI; Katherine Hope, an associate veterinarian with the Smithsonian’s National Zoo; and Ed Bronikowski, senior curator of the Smithsonian’s National Zoo, were study co-authors as well.

The research was funded by a Smithsonian Institution Competitive Grants Program for Science grant, the Smithsonian’s Center for Conservation Genomics and a Smithsonian Institution Molecular Evolution Postdoctoral Fellowship.

Anna E. Savage, Brian Gratwicke, Katharine Hope, Edward Bronikowski, Robert C. Fleischer. Sustained immune activation is associated with susceptibility to the amphibian chytrid fungusMolecular Ecology, 2020; DOI: 10.1111/mec.15533

written by Robert Wells (University of Central Florida)

Panamanian toads may harbor a cure for Chagas disease

Panamanian toads Rhinella centralis are distinguished by their dorsal skin covered with pointed warts. They are common along the Pacific coastal areas, often in urban areas around Panama City and small towns, and form large choruses on rainy nights. The small but strongly swollen poison glands on their heads secrete a white toxic goop. This effective defense mechanism makes predators spit them out, or froth at the mouth, vomit and it may even kill them if they try to eat the toad.

Scientists working at INDICASAT, the University of Panama and STRI began screening wild frogs for substances with pharmacological potential to treat various tropical diseases. When they analyzed secretions from these toads they discovered a chemical in the poison glands called 19-hydroxy-bufalin. They found that this chemical was very potent at killing the parasites that cause Chagas disease, and that it was not very toxic to cells. Chagas disease is a neglected tropical disease that kills 10,000 people per year, and current medications used to treat the disease are not very effective, particularly in acute cases. The fact that this chemical is quite selective with low cell toxicity means it is a promising compound that make it a candidate compound to further explore as a potential way to treat Chagas disease.

Read the research here:

Rodriguez, C., Ibáñez, R., Ng, M., Spadafora, C., Durant-Archibold, A.A. and Gutiérrez, M. 2020. 19-Hydroxy-bufalin, a major bufadienolide isolated from the parotoid gland secretions of the Panamanian endemic toad Rhinella centralis (Bufonidae), inhibits the growth of Trypanosoma cruzi. Toxicon 177:89-92.

Strawberry poison dart frogs prefer mates that look like their parents

A new research paper published on strawberry poison dart frogs in Bocas del Torro found that one of the reasons we have polymorphism or so many different color forms within one species of frog.  Female tadpoles prefer to mate with males that have the same color as their parents (sexual imprinting), and males defend their territories more vigorously from other males that are the same color as their parents (rival imprinting). The researchers demonstrated this experimentally by using foster parents of different color forms to raise offspring, and then tested mate or rival preference of the adult offspring.

This process of sexual selection can lead to sexual isolation even in populations that live in the same places. From an evolutionary perspective this would be a rare example of sympatric speciation, or the evolution through natural selection without geographical isolation.

Read the paper here  Yang, Y., Servedio, M.R., Richards-Zawacki, C.L., 2019. Imprinting sets the stage for speciation. Nature 574, 99–102.

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)

Update from Release trial Research at Centro Mamoni

Blake Klocke, a student at George Mason University is studying where frogs go once we release them, how long it takes them to contract the chytrid fungus, if there are any effects of releasing chytrid susceptible frogs on the existing frog community & what other non chytrid fungus sources of mortality might affect reintroduction work.

In addition to our primary project partners the Houston Zoo, Zoo New England and the Cheyenne Mountain zoo we thanks the National Geographic Society, Mohammed Bin Zayed Species Conservation Fund, and the Smithsonian Women’s Committee for their support of these release trials.

Defying disease: Panama’s frogs are fighting back

Many infectious diseases can fade away after initial outbreaks. Bubonic plague, cholera, and influenza are examples from recent human history. The same phenomenon occurs for wildlife diseases as well. How does this happen? One popular explanation is that the pathogen evolves to become less deadly, so that it doesn’t completely wipe out its hosts, ensuring pathogen survival. While this scenario does sometimes play out, we know that there are other reasons why the severity of diseases can change over time.

Cori Richards and Jamie Voyles

For amphibians, we’ve known about a highly lethal disease called “chytridiomycosis” since the 1990s. This disease was especially devastating in Central America, where it may have wiped out entire species. In this study, we made the exciting discovery that some amphibian species – frogs that were thought to be extinct – are persisting, and even recovering, after lethal disease outbreaks. We wanted to understand how it was happening. Was it a change in the pathogen, the frogs, or both?

To answer these questions, we did two things. To begin with, we surveyed frogs in Panama before and after the disease outbreak. In addition, we collected samples of the pathogen at multiple time points: during initial outbreaks and ~10 years later. We found that nearly a decade after the outbreak, the pathogen was just as deadly. However, the frogs are surviving and have better defenses against it. Panama’s frogs are fighting back! Understanding how amphibian communities are recovering after this disease outbreak is important multiple reasons. First, resolving how this works will help us develop more informed conservation strategies to protect amphibians from disease-induced extinctions. Second, clarifying how disease outbreaks subside will help us predict, and respond to, other emerging pathogens in plants, wildlife, and in humans. These goals are increasingly important in a time when rapid globalization has increased the introduction of pathogens to naïve host populations.

Atelopus varius is one species that appears to have evolved antifungal skin secretions

by Jamie Voyles and Cori Richards-Zawacki

Read the paper: Shifts in disease dynamics in a tropical amphibian assemblage are not due to pathogen attenuation BY JAMIE VOYLES, DOUGLAS C. WOODHAMS, VERONICA SAENZ, ALLISON Q. BYRNE, RACHEL PEREZ, GABRIELA RIOS-SOTELO, MASON J. RYAN, MOLLY C. BLETZ, FLORENCE ANN SOBELL, SHAWNA MCLETCHIE, LAURA REINERT, ERICA BREE ROSENBLUM, LOUISE A. ROLLINS-SMITH, ROBERTO IBÁÑEZ, JULIE M. RAY, EDGARDO J. GRIFFITH, HEIDI ROSS, CORINNE L. RICHARDS-ZAWACKI SCIENCE 30 MAR 2018 : 1517-1519