Science to the Rescue in the #FightForFrogs

Gina Della Togna with a Panamanian golden frog, a beloved species at the center of her research. (Photo by Pei-Chih Lee, Smithsonian Conservation Biology Institute)

Gina Della Togna with a Panamanian golden frog, a beloved species at the center of her research. (Photo by Pei-Chih Lee, Smithsonian Conservation Biology Institute)

When SCBI conservation biologist Brian Gratwicke started the Panama Amphibian Rescue and Conservation Project with partners in 2009, it was a mad dash to find and collect frogs representing the very last best hope for their species, rapidly vanishing at the hands of an amphibian chytrid fungus (Batrachochytrium dendrobatidis, or Bd) that causes a disease called chytridiomycosis.

If that was the opening chapter of the rescue project’s story, seven years later the story reads like a manuscript for an initiative set up to be among the most successful comprehensive conservation projects to date.

Today the rescue project has provided a stable safe haven for 12 of the most imperiled Panamanian frog species, requiring keepers to learn the complex husbandry, behavior and reproductive physiology unique to each individual species. In the meantime, rescue project scientists are making strides in developing and refining assisted reproduction protocols, while also conducting experiments in a resolute search for a cure for Bd.

“We are entering a new phase,” Gratwicke says. “We’ve brought together some of the world’s leading animal husbandry experts, veterinarians, reproductive biologists, disease ecologists and herpetologists. With all of the talented scientific minds working on this one, we have great hope that we may someday be able to return these species safely to their home in the wild.”

Searching for a Cure

Things in Matt Becker’s lab can sometimes get a bit…strange. Take, for instance, an experiment the SCBI postdoctoral researcher conducted a year ago with unexpected results. Becker’s research focuses on the use of probiotics—or beneficial bacteria—to help frogs fight off Bd. Last year Becker applied five different probiotics with anti-fungal properties to the skin of five groups of Panamanian golden frogs, hoping to discover which probiotic gives them an effective shield against the pathogen.

Matt Becker prepares-probiotic baths. (Photo by Brian Gratwicke)

Matt Becker prepares-probiotic baths. (Photo by Brian Gratwicke, Smithsonian Conservation Biology Institute)

What he found surprised him. In past experiments, the probiotics were ineffective and all of the frogs died after the researchers infected them with Bd. This time, though, about 25 percent of the individuals survived. And those surviving frogs didn’t come from just one group with one kind of probiotics, but from every group, even the one that had been infected with Bd without a probiotic protectant.

So Becker and Gratwicke needed to determine what it was that the frogs did have in common to help them fight the disease. They started by looking at the frogs’ microbial community, or the complex community of bacteria on the skin. All of the frogs that survived had a greater abundance of specific bacteria on their skin.

In June of this year, the team launched a new experiment, this time using frogs from the Species Survival Plan collection at the Maryland Zoo in Baltimore that have similar abundances and types of bacteria as those that survived last year. The researchers have given the frogs a cocktail of eight bacteria that seem to strongly ward off Bd.

Looking at which immune system genes turn on or off to fight off a chytrid infection can help scientists discover why some frogs aren't as susceptible. (Photo by Mehgan Murphy, Smithsonian's National Zoo)

Looking at which immune system genes turn on or off to fight off a chytrid infection can help scientists discover why some frogs aren’t as susceptible. (Photo by Mehgan Murphy, Smithsonian’s National Zoo)

“At the start of every experiment, you’re really optimistic,” says Becker, who has been working on golden frog probiotics since 2007. “It’s been a great journey and we’re really learning a lot about golden frogs and how chytrid affects these guys. Every little bit of information really goes a long way for the conservation of this species and similar species.”

For the first time during a probiotics study on frogs, the researchers will also be looking at the gene expression—or combination of genes in an individual frog that gets turned on or turned off—while the frog mounts an immune response to fight off Bd.

“We’re throwing everything we’ve got at this,” Becker says. “We want to be able to use these tools to determine which frogs in the overall captive population share those same strengths—either their microbial community or gene expression—that keep them alive. There are so many questions we need to answer, but through the scientific process, we’re getting there.”

Frogs for the Future

While Becker is focused on getting frogs safely back into the wild, this goal is only possible if there are actually future generations of frogs to release into the wild. That’s where Smithsonian researcher and Panamanian native Gina Della Togna comes in.

Gina Della Togna in the lab. (Photo by Brian Gratwicke)

Gina Della Togna in the lab. (Photo by Brian Gratwicke, Smithsonian Conservation Biology Institute)

Della Togna is working on a number of complex assisted reproduction techniques for Panamanian frog species. She is the first scientist to develop protocols for extracting and freezing sperm from the Panamanian golden frog, a species that is extinct in the wild and a cultural icon in her home country. Scientists could someday use the sperm to infuse populations with additional genetic diversity, key to a species’ overall health.

“When we started, we didn’t know anything about anything,” Della Togna says. “We needed to learn which hormones at what concentrations to use, how to keep the sperm alive long enough to freeze it and the best techniques to freeze it so that the sperm is viable when we thaw it, even years later. It was a challenge, but I love a good challenge.”

Now Della Togna is working on developing similar protocol for other rescue project species, including the mountain harlequin frog, Pirre harlequin frog, variable harlequin frog, limosa harlequin frog and the rusty robber frog. In the future, she plans to get out into the field to capture genetic lineages from frogs in the wild. As she continues to perfect these protocols, Della Togna also aims to collect eggs from female Panamanian golden frogs to use for artificial fertilization with the frozen sperm. And most recently in Panama, she successfully applied a hormone treatment to help six pairs of the limosa harlequin frog and Pirre harlequin frogs breed that hadn’t laid eggs before.

“Breeding frogs is the fundamental step to sustaining captive populations and growing the numbers for release trials,” Gratwicke says. “Gina’s work is of huge applied value to us because we have some very challenging species to breed, and hormone dosing may help us to get them to cycle reproductively, even if we can’t figure out the external reproduction cues.”

For Della Togna, Gratwicke and Becker, the goal is the same: to give these unique species a fighting chance against Bd.

“If these frogs go extinct, nothing can replace them,” Della Togna says. “They are important to the ecosystem and essential to our planet’s equilibrium. There’s no doubt that we’re responsible for getting them back to where they belong.”

From now until the end of August, you can help us #FightForFrogs! Our generous sponsor Golden Frog—a global online services provider with a terrific name—will match donations to the rescue project up to $20,000, helping us raise money critical to our fight for frogs. Your donations during the Fight for Frogs campaign will buy us equipment to care for the frogs in the rescue pods, help us continue to conduct experiments to find a cure, ensure crucial breakthroughs, and ultimately one day see the return of these incredible species to their home in the wild.

The Panama Amphibian Rescue and Conservation Project is a project partnership between the Cheyenne Mountain Zoo, the Houston Zoo, Zoo New England and Smithsonian Institution. You can follow the Fight for Frogs campaign on Twitter using the #FightForFrogs hashtag or on the rescue project’s Facebook page.

Join us as together we Fight for Frogs

Brian swabs frogs in the field_Brian GratwickeFrogs matter. As a kid in nursery school, I remember observing tadpoles metamorphose into froglets right before our eyes in the classroom. It was like watching a magic trick over and over again. As I grew more interested in these cool little creatures, I learned that some frogs reproduce using pouches, others by swallowing their own eggs and regurgitating their young, others still by laying eggs that hatch directly into little froglets. It was like discovering not one magic trick, but an entire magical world—except this world was no illusion, it was real. My formative experiences both in the classroom and out rummaging around cold rainy ponds at night with my best friend and a headlamp spurred me into a career in the biological sciences. They also instilled in me a deep appreciation for the incredible diversity of life.

Panamanian golden frog_Brian GratwickeToday I am focused on conserving that incredible diversity specifically among amphibians in Panama, which is home to an astounding 214 amphibian species. Or at least it was. When a deadly amphibian chytrid fungus swept through, nine species disappeared entirely, including the country’s national animal, the beautiful Panamanian golden frog.

graphical abstractSince 2009, the Panama Amphibian Rescue and Conservation Project has spearheaded efforts to bring at-risk species into rescue pods to ride out the storm while we work on finding a cure. We’ve worked with partners to conduct several experiments in search of a cure and to better understand why some frogs resist infection and others do not. We have built new facilities that house highly endangered species of amphibians as part of a bigger global push to create an “Amphibian Ark.” These efforts and those of our colleagues around the world give me profound hope for our amphibian friends.

But we need your help.

Although frogs are the orchestral backdrop to every pond and forest, frogs have no voice to represent themselves, and they certainly can’t write checks. It’s up to professional conservationists, including the rescue project’s 12 talented conservationists in Panama, to save frogs so that others can enjoy them. This, however, requires money. From now until the end of August, our generous sponsor Golden Frog—a global online services provider with a terrific name—will match donations to the rescue project up to $20,000, helping us raise money critical to our fight for frogs. Your donations during the Fight for Frogs campaign will buy us equipment to care for the frogs in the rescue pods, help us continue to conduct experiments to find a cure, ensure crucial breakthroughs, and ultimately one day see the return of these incredible species to their home in the wild.

Together, let’s make a stand. Together, let’s #FightForFrogs.

The Panama Amphibian Rescue and Conservation Project is a project partnership between the Cheyenne Mountain Zoo, the Houston Zoo, Zoo New England and Smithsonian Institution. You can follow the Fight for Frogs campaign on Twitter using the #FightForFrogs hashtag or on the rescue project’s Facebook page.

Mission Critical: Amphibian Rescue

This award-winning documentary featuring our race to find a cure for a deadly amphibian disease and to build an amphibian ark in Panama is now available for FREE.  Watch the trailer below and download the full feature if you would like to see more on the itunes store for a limited time only.

CLICK HERE to download the full episode of Smithsonian Networks Mission Critical: Amphibian Rescue film. FREE FOR A LIMITED TIME ONLY!

Rescue Project Successfully Breeds Endangered Frog Species

 

Limosa harlequin frog (Atelopus limosus) baby on a U.S. quarter.

Limosa harlequin frog (Atelopus limosus) baby on a U.S. quarter. (Photo by Brian Gratwicke, Smithsonian Conservation Biology Institute)

The limosa harlequin frog (Atelopus limosus), an endangered species native to Panama, now has a new lease on life. The Panama Amphibian Rescue and Conservation Project is successfully breeding the chevron-patterned form of the species in captivity for the first time. The rescue project is raising nine healthy frogs from one mating pair and hundreds of tadpoles from another pair.

“These frogs represent the last hope for their species,” said Brian Gratwicke, international coordinator for the project and a research biologist at the Smithsonian Conservation Biology Institute, one of six project partners. “This new generation is hugely inspiring to us as we work to conserve and care for this species and others.”

Nearly one-third of the world’s amphibian species are at risk of extinction. The rescue project aims to save priority species of frogs in Panama, one of the world’s last strongholds for amphibian biodiversity. While the global amphibian crisis is the result of habitat loss, climate change and pollution, a fungal disease, chytridiomycosis, is likely responsible for as many as 94 of 120 frog species disappearing since 1980.

Between its facilities at the Smithsonian Tropical Research Institute in Gamboa, Panama, and the El Valle Amphibian Conservation Center in El Valle, Panama, the rescue project currently cares for 55 adult limosa harlequin frogs of the chevron-patterned form and 10 of the plain-color form. The project has had limited success breeding the plain-color form of this species, and has successfully bred other challenging endangered species, including crowned treefrogs (Anotheca spinosa), horned marsupial frogs (Gastrotheca cornuta) and toad mountain harlequin frogs (A. certus).

Each species requires its own unique husbandry to thrive and breed. The project’s animal care team and scientists learn husbandry techniques as they work with a limited number of individuals. Jorge Guerrel, conservation biologist at the Smithsonian Tropical Research Institute, arranged rocks in the breeding tank to create the submerged caves that appear to be the preferred egg deposition sites for limosa harlequin frogs. Like other Atelopus species, tadpoles require highly oxygenated, gently flowing water between 22 and 24 degrees Celsius. The tadpoles’ natural food is algal film growing on submerged rocks, which Guerrel and his colleagues re-created by painting petri dishes with a solution of powdered spirulina algae, then allowing it to dry.

The mission of the Panama Amphibian Rescue and Conservation Project is to rescue amphibian species that are in extreme danger of extinction throughout Panama. The project’s efforts and expertise are focused on establishing assurance colonies and developing methodologies to reduce the impact of the amphibian chytrid fungus so that one day captive amphibians may be reintroduced to the wild. Current project partners include Cheyenne Mountain Zoo, Houston Zoo, Smithsonian’s National Zoological Park, Smithsonian Tropical Research Institute and Zoo New England.

Lindsay Renick Mayer, Smithsonian’s National Zoo

Frog Love on Valentine’s Day

This Valentine’s Day we asked some of the rescue project’s researchers why they love frogs. Here’s what we got back from a few biologists at the Smithsonian’s National Zoo. The common link? A life-long love of wildlife.

Matt Evans, biologist, Smithsoinan's National Zoo

Matt Evans, biologist, Smithsonian

“My favorite childhood memories revolve around my mother taking me down to the nearest stream and letting me get dirty playing with frogs and salamanders. I loved it so much, I get to do that for a living now! The diversity within all amphibians still amazes me as there is so much we still have  to learn. I consider being able to work on projects, which may help save frogs from extinction, to be the absolute coolest part of my job.”

Ed Smith, biologist, Smithsonian's National Zoo

Ed Smith, biologist, Smithsonian

“Since childhood it was snakes, not frogs, that were the focus of hours spent searching woodlands, streams and ponds. Unlike other more noticeable  creatures, snakes are decidedly unapparent; unless they happen to be eating a frog! Although, the thrill of finding a snake by following the plaintive screams of a leopard frog never diminished, what increased was my interest in the gradually disappearing prey. So, thanks in part to the appetite of a few alluring garter snakes the equally marvelous world of frogs, toads, salamanders, sirens, and hellbenders opened to me and now inform and enhance days at work and in the field.”

Brian Gratwicke, rescue project international coordinator

Brian Gratwicke, rescue project international coordinator

“I fell in love with frogs growing up as a child in Zimbabwe. There was something exhilarating about discovering the translucent, musical jewels that are responsible for the familiar nocturnal soundtrack of my childhood. Unlike any computer games or TV shows, I will carry those first memories of catching frogs around a pond with me for the rest of my life. For me, seeing frogs in the wild stirs emotions of wonder and discovery, and they are accessible to everyone who is willing to take a little extra effort to open their eyes and look.”

–Brian Gratwicke, rescue project international coordinator

Genetic Matchmaking Saves Endangered Frogs

Marsupial frog

The casque headed tree frog (Hemiphractidae: Hemiphractus fasciatus), is one of 11 species of highest conservation concern now being bred in captivity in Panama. Females carry eggs on their backs where the young complete development hatching out as miniature frogs. DNA barcoding data suggest that populations of H. fasciatus may comprise more than one taxonomic group.

What if Noah got it wrong? What if he paired a male and a female animal thinking they were the same species, and then discovered they were not the same and could not produce offspring? As researchers from the Panama Amphibian Rescue and Conservation Project race to save frogs from a devastating disease by breeding them in captivity, a genetic test averts mating mix-ups.

At the El Valle Amphibian Conservation Center, project scientists breed 11 different species of highland frogs threatened by the chytrid fungus Batrachochytrium dendrobatidis, which has already decimated amphibian populations worldwide. They hope that someday they will be able to re-release frogs into Panama’s highland streams.

Different frog species may look very similar.

“If we accidentally choose frogs to breed that are not the same species, we may be unsuccessful or unknowingly create hybrid animals that are maladapted to their parents’ native environment,” said Andrew J. Crawford, research associate at the Smithsonian Tropical Research Institute in Panama and professor at Colombia’s Universidad de los Andes. Crawford and his colleagues make use of a genetic technique called DNA barcoding to tell amphibian species apart. By comparing gene sequences in a frog’s skin cells sampled with a cotton swab, they discover how closely the frogs are related.

The Smithsonian Tropical Research Institute, headquartered in Panama City, Panama, is a unit of the Smithsonian Institution. The Institute furthers the understanding of tropical nature 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.

Beth King, Smithsonian Tropical Research Institute

Photo by Edgardo Griffith.

Frog Poetry and the Washington Post

Washington Post

On Dec. 30, the Washington Post ran a front-page story about the rescue project.

The year ended on a high note for the Panama Amphibian Rescue and Conservation Project. William Booth, a science writer for the Washington Post, joined rescue project researchers on a field expedition and his story about the rescue project came out on the front page of the Post on Dec. 30. The story inspired one reader, Tim Torkildson, to share a lovely poem about frogs and the disease that is wiping them out.

Booth also did this NPR interview about his recent trip to Panama.

If you saw the story and are interested in making a donation to the rescue project, please follow this link to the National Zoo’s website.

THE FROG
by Tim Torkildson

The frog is an amphibian
Who thrives most ev’rywhere,
From the dry Namibian
To just off ol’ Times Square.
The ones who have a bumpy skin,
With warts and pits and nodes,
Are the closest Phylum kin;
We simply call them toads.
The bullfrogs in the early spring
give ponds reverberation
With their raucous verbal fling,
Attempting procreation.
The have a courtship ritual
that’s called, I think, amplexus,
Which gives them fits conniptual
Between the two odd sexes.
A little boy will manage to
Corral a tadpole, yes,
And give it quite a slimy view
Right down his sister’s dress!
And did you know the urine from
a pregnant lady will
cause some frogs to lay a scum
of eggs, with no male thrill?
And so they’re useful critters,
As the French will tell you so;
Their legs taste good in fritters,
Are mistaken for turbot.
And what of cane toads, mind you,
Where, if you lick the skin,
The psychedelics blind you
To sorrow, grief and sin?
But frogs, those little gargoyles,
Which are funny in cartoons,
Are engaged in lethal broils
That leaves their lives in ruins.
A fungus known as “Bd” kills
The frogs down in Belize,
Then jumps the valleys and the hills
So others it may seize.
The Costa Rica Golden Toad
Is now extinct, alack.
More are headed down that road,
Since habitat is slack.
Toxins give some frogs three legs,
Which doesn’t help them jump.
Instead they are like clumsy kegs
Who in the water flump.
Scientists preserve some frogs
In habitats in labs.
Dressed in their starched, stiff white togs
They keep meticulous tabs.
To save the frogs, oh please donate
A dollar or a yuan,
So the polliwog birth rate
Will someday be a shoo-in!

Using genetic tools to understand and manage chytridiomycosis

Lowland leopard frog (Lithobates yavapaiensis)

The lowland leopard frog is the focus of SCBI postdoc Anna Savage's work looking at the relationship between genes and chytrid resistance.

Chytridiomycosis, the disease wiping out frog species around the world, was described in 1999 by a team of researchers at the Smithsonian and the University of Maine. Today, in addition to creating an insurance population for various Panamanian species, the Smithsonian Conservation Biology Institute aims to find a way to manage this disease. One of the most promising solutions to stopping the killer may rest in genetics. SCBI scientists are looking to identify genes within frogs that provide resistance to the pathogen or that make them more susceptible to it.

Researchers pursuing this approach are optimistic that genetics could provide different answers than those offered by probiotics, which SCBI is also pursuing. The genetic approach is one based on natural selection.

“If you look across species, including humans, diseases rarely wipe out an entire species without any evidence of resistance, regardless of how virulent they are,” said Anna Savage, an postdoctoral fellow in SCBI’s Center for Conservation and Evolutionary Genetics studying the genetics of chytrid. “Immune systems are so complex that there’s a strong possibility for the development of resistance. The probability that a species develops no resistance to a disease is rather small.”

Genetics research within the last decade has identified frogs with varying degrees of resistance to chytrid. Savage is focusing her research on the lowland leopard frog (Lithobates yavapaiensis), which exhibits an intermediate level of resistance to the pathogen. Some individuals demonstrate resistance while others of the same species die if infected with chytrid. The identification of genetic variations between the individuals holds the potential of being the answer between life and death. One of the new frontiers for genetic research is the examination of a frog species’ immune system genes.

“If we can identify the genes responsible for resistance, we can breed the animals in captivity to ‘spread’ this genetic resistance and give adaptation a headstart,” Savage said. “This area of research holds the potential for creating populations of self-sustaining frogs that confer resistance to the rest of the population through reproduction.”

Anna Savage with bullfrog

Savage's research could hold the key to putting a stop to a deadly pathogen killing frogs around the world.

In order to understand a frog’s response to chytrid, the focus must be broadened to understanding how immune system genes interact with one another and which ones are being expressed during resistance. Savage is using this approach to understand chytrid susceptibility in lowland leopard frogs that are being raised at the Smithsonian’s National Zoo. This approach could potentially help scientists identify genes responsible for chytrid resistance. While this type of research shows considerable potential, researchers are only just beginning to scratch the surface.

“Disease outcomes from chytridiomycosis can depend on several factors,” said Brian Gratwicke, SCBI wildlife biologist and Panama Amphibian Rescue and Conservation Project international coordinator.  “Some frogs appear to have innate resistance to the disease and produce antimicrobial peptides from glands in their skin. It is possible that some might acquire resistance through exposure, while other frogs have beneficial anti-fungal skin bacteria or a behavioral preference for temperatures that are bad for the fungus. As the fight against chytrid continues, we remain hopeful that the answers are out there and that Dr. Savage’s work will give us some insight into how this system really functions.”        

–Will Lazaro, Smithsonian’s National Zoo

(Frog photo by Jared Grummer; photo of Anna Savage by Dennis Caldwell)

Things we are thankful for: Amphibian baby boom at EVACC and Summit Zoo

Reflecting on our achievements this last year, we would like to thank you, our generous supporters. You have provided moral support; social media likes and retweets generously; more than 40 of you have given us your time to help us save frogs; and even more have sacrificed your hard-earned money to help us achieve this important mission. Thank you! In particular I would like to recognize the extraordinary dedication and above-and-beyond service from our devoted conservation staff seen above proudly displaying the the captive-bred products of their hard work, including a second generation of captive-bred frogs at EVACC, and our first grown-up generation of captive-born frogs at the Summit Zoo. We salute you all.

Brian Gratwicke – International project coordinator for the Panama Amphibian Rescue and Conservation Project, Smithsonian Conservation Biology Institute