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

Variable Harlequin Frog release trials begin in Panama

Once common along highland streams from western Costa Rica to western Panama, the variable harlequin frog is endangered throughout its range, decimated by a disease caused by the amphibian chytrid fungus. On Jan. 17, Smithsonian researchers released approximately 500 frogs at Cobre Panama concession site in Panama’s Colon province as a first step toward a potential full-scale reintroduction of this species. This release trial is included in Cobre Panama’s biodiversity conservation plan as an important part of their environmental commitments.

Composite image showing variation in coloration within this population of frogs

The variable harlequin frog, Atelopus varius, takes its name from the variety of neon colors—green, yellow, orange or pink—juxtaposed with black on its skin. In order to monitor the released frogs over time, 30 are wearing miniature radio transmitters. The scientific team also gave each frog an elastomer toe marking that glows under UV light to mark individuals as part of a population monitoring study.


“Before we reintroduce frogs into remote areas, we need to learn how they fare in the wild and what we need to do to increase their chances of survival in places where we can monitor them closely,” said Brian Gratwicke, international coordinator of the Panama Amphibian Rescue and Conservation project (PARC) at the Smithsonian Conservation Biology Institute. “Release trials may or may not succeed but the lessons we learn will help us to understand the challenges faced by a frog as it transitions from captivity into the wild.”

Heidi Ross and her team at our facilities in the Nispero Zoo successfully bred and reared these animals for the release trial

Variable harlequin frogs are especially sensitive to the amphibian chytrid fungus, which has pushed frog species to the brink of extinction in Central America. PARC brought a number of individuals into the breeding center between 2013 and 2016 as chytrid continued to impact wild populations.

 

The field team all assembled with frogs ready for the release trial

“The variable harlequin frog is one of the closest relatives of Atelopus zeteki, Panama’s iconic golden frog, another target species in our captive breeding program,” said Roberto Ibañez, PARC project director at the Smithsonian Tropical Research Institute in Panama. “We’ll be monitoring the surrounding amphibian community and the climate at this site, and comparing this to the amphibian community at another, control site. This kind of intensive monitoring will help us to understand disease dynamics in relation to the release trials”

One of our Atelopus varius wearing a mini radio-transmitter

PARC hopes to secure the future for this and other endangered amphibians by reintroducing animals bred in captivity according to an action plan developed with Panama’s Ministry of the Environment and the International Union for the Conservation of Nature (IUCN) and other stakeholders. “It took us several years to learn how to successfully breed these frogs in captivity,” said Ibañez. “As the number of individuals we have continues to increase, it provides new research opportunities to understand factors influencing survival that will ultimately inform long-term reintroduction strategies.”

The PARC project thanks Cobre Panama, National Geographic Society, Mohammed bin Zayed Species Conservation Fund and The WoodTiger Fund for their generous support.

PARC is a partnership between the Cheyenne Mountain Zoo, the Houston Zoo, the Smithsonian Tropical Research Institute, the Smithsonian Conservation Biology Institute and Zoo New England. It has two facilities in Panama: the Gamboa Amphibian Rescue and Conservation Center at STRI and the El Valle Amphibian Conservation Center at El Nispero. Combined, these facilities have a full-time staff caring for a collection of 12 endangered species.

SCBI plays a leading role in the Smithsonian’s global efforts to save wildlife species from extinction and train future generations of conservationists. SCBI spearheads research programs at its headquarters in Front Royal, Va., the Smithsonian’s National Zoo in Washington, D.C., and at field research stations and training sites worldwide. SCBI scientists tackle some of today’s most complex conservation challenges by applying and sharing what they learn about animal behavior and reproduction, ecology, genetics, migration and conservation sustainability.

The Smithsonian Tropical Research Institute, headquartered in Panama City, Panama, is a unit of the Smithsonian Institution. The Institute furthers the understanding of tropical biodiversity and its importance to human welfare, trains students to conduct research in the tropics and promotes conservation by increasing public awareness of the beauty and importance of tropical ecosystems.

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. http://dx.doi.org/10.1016/j.theriogenology.2016.12.033