The curious quest to make this frog poisonous again

Deep in the Panamanian jungle, conservation biologist Brian Gratwicke and his team hunted a radio transmitter signal that led them to a ferocious-looking whip scorpion in the middle of a meal.

Some days earlier the team had attached a tiny tracker to the back of a small Atelopus limosus, or limosa frog, and released the animal near a stream in Central Panama’s Mamoní Valley. They weren’t sure if they’d find the frog again. But they certainly weren’t expecting to find it in the spiny clutches of a whip scorpion.

That’s because frogs belonging to the Atelopus genus are extremely toxic, with a single individual able to kill so many thousands of mice that “at some point they upped it to horses,” says Gratwicke, who works at the Smithsonian’s National Zoo and Conservation Biology Institute in Virginia and is also a National Geographic Explorer. As such, the frogs’ predator list is very short. It doesn’t typically include whip scorpions.

When Gratwicke and his colleagues at the Panama Amphibian Rescue and Conservation Project released 83 captive-bred limosa frogs into the wild back in 2017—including the ill-fated individual with the radio tracker—they were concerned with a totally different threat: Batrachochytrium dendrobatidis, or Bd. The microscopic fungus has been infecting and killing frogs since it first emerged in Central America in the 1970s. As it swept across the world, Bd became the deadliest wildlife pathogen in recorded history, causing at least 90 extinctions—including another Atelopus species and Panama’s national mascot, the Panamanian golden frog (Atelopus zeteki). Most other species in the Atelopus genus are now critically endangered.

Fortunately, researchers realized early on that the Atelopus frogs, also called harlequin frogs or harlequin toads, were particularly susceptible to the fungus. Anticipating the worst, they collected wild individuals from five Atelopus species, including the golden frog, to start building insurance populations in zoos and aquariums across the U.S. and in Panama that could one day be released back into the wild.

Decades later, as the fungus still runs rampant, their reintroduction remains a distant dream. But small release trials like the one in 2017 allow scientists to study how the frogs interact with the pathogen in the wild and what tools they might need to survive—essentially, “how to stick a thumb on the scale in favor of these frogs,” Gratwicke says.

Yet the amphibians didn’t even have time to get Bd before becoming dinner, not just for a whip scorpion, but also a fishing spider and a very large Savage's thin-toed frog. Subsequent chemical testing revealed the reason why: Born in captivity, Atelopus frogs had lost their toxicity. That’s extremely bad news for an animal that uses toxins to avoid getting eaten.

The revelation that the previously-deadly Atelopus frogs are now susceptible to a host of predators—on top of the whole fungus situation—has led to a full-blown side quest involving scientists from around the world, all working to answer a simple question that Gratwicke puts like so: “How can we make these frogs spicy again?”

Where do poisonous frogs get their poison?

In the pet trade, the phenomenon of frogs losing their toxicity is well-documented. The Dendrobatidae family, more commonly known as poison dart frogs, acquire their toxins from the ants and mites they eat in the wild. Feed them different insects, and their toxicity will dissipate over time. Captive-bred poison dart frogs never eat the insects that make them poisonous, so they are totally toxin-free—which is, of course, desirable for pet owners.

(How one man is working to save the world’s most poisonous animals.)

But it’s not ideal for a frog in the wild that depends on its toxins for self-defense. Mix that with a genus of frogs whose numbers are dwindling in the wild: living predators may have never come across an Atelopus frog, let alone had a negative interaction with them (many toxins are not only dangerous, but very bitter). Whereas bright colors might have previously served as a warning, Gratwicke says, they’re now an invitation to a delicious meal. “They’re like blinking little McDonald’s signs.”

As it happens, there isn’t much precedent for restoring toxicity to wild frogs. Even if there were, the five Atelopus species in question were decimated by Bd before scientists had time to deeply study their toxin makeups. To make Atelopus adequately spicy again, chemists would have to start from the basics: Which toxins do the wild individuals have, and in what amount? And then, how does that compare to the captive-bred ones—did they retain any of their original toxins, or were they totally spice-free?

Chemist Phillip Jervis wanted to answer those questions, so he set out to find some wild Atelopus frogs. It’s tough work, given their scarcity. During a stint at the Smithsonian’s lab in Panama in June 2019, it took Jervis, visiting from Imperial College London, weeks of surveying stream banks to find just a couple dozen limosa frogs—many more than he expected to find, though still a relatively small number. He placed each one in a plastic bag with 20 milliliters of water and a few drops of norepinephrine, a stress hormone that causes them to release some of their toxins. Now he’s analyzing those samples in hopes of finally describing the recipe of toxins that wild limosa frogs carry, and how it stacks up to those in captivity.

Though the full scope of of that recipe remains a mystery, scientists have previously documented a few types of toxin in Atelopus species, including: tetrodotoxin (TTX), a neurotoxin found in a few frogs, as well as some newts, the blue-ringed octopus, and pufferfish in Japan—which poses a risk for sushi enthusiasts—among other animals. Some of Gratwicke’s earlier work revealed that TTX is entirely missing in frogs born in captivity. The kicker is that “no one really knows how this toxin is made,” says Jervis. “It just sort of appears on random animals across the tree of life, and no one really knows how it’s acquired.”

Atelopus frogs might get their TTX from bacteria, algae, something in the food chain; perhaps they manufacture it in their bodies. The most popular hypothesis at the moment is that it comes from the diet. While developing methodologies to detect small amounts of TTX, chemist Candelario Rodriguez, currently at Coiba Scientific Station in Panama, says he found the toxin not just in the frogs’ skin but also the gastrointestinal tract, kidney, and liver, which supports that idea.

(This extinct toad rediscovery offers hope amid the amphibian apocalypse.)

From frog tents to online shopping for poison

Whether Atelopus’ mix of toxins came from their diet or some other mechanism that only transpires in the wild, scientists wondered if the frogs could regain their toxicity by simply being back in their natural environment. So in a subsequent release trial in 2018, the Panama Amphibian Rescue and Conversation Project team set up a dozen Purrfect Play cat tents (unfortunately no longer in stock, Gratwicke notes) in the rainforest, filled them with three inches of leaf litter, and then placed an Atelopus varius, or variable harlequin frog, in each one. The tents would make the frogs easier to recover for testing and also keep them safe from predators, while still integrating them back into their natural environment.

After 79 days amongst the leaf litter, testing revealed that the frogs had quickly reestablished their wild microbiome—including with bacteria that could potentially produce TTX, Gratwicke says. But TTX was nowhere to be seen. 

The researchers decided to try another direction: Buy synthetic tetrodotoxin online (sold by suppliers for analytical chemistry work) and feed it directly to the frogs. After six months of paperwork to legally transport the toxin to the lab in Panama, the team injected it into pantry moth larvae that they fed to some frogs. The moth contained enough toxin to kill thousands of mice—and was about half the amount that used to be found on an Atelopus, says Gratwicke. The frogs were totally fine. Jervis is still reviewing the findings to see whether they reached their wild toxicity levels.

That has been one of the hardest parts of this puzzle: How is one supposed to restore a frog’s toxicity back to the “before”—when the “before” is not well understood? It’s a question particularly pertinent to the Panamanian golden frog, or Atelopus zeitiki, because of the completely unique toxin it’s named for. Zetekitoxin was described by chemists back in 2003 before the frog went extinct in the wild in 2009. But there are no living frogs that carry the toxin today, so it simply does not exist in nature.  No other origin of the toxin has been found.

In search of a long-lost molecule

Up at the University of Michigan, chemist Tim Cernak looks for an answer in the archives. He’s currently studying frogs in jars that were caught back in April 1965, some of them by entomologist and naturalist James Zetek for whom the toxin was named. The frogs have been stored in ethanol for 60 years, which has made trying to sift out the toxin “a ridiculously challenging chemistry problem,” says Cernak. It’s one shot at finding a long-lost molecule, and the odds are long.

“We’re chasing a ghost,” Cernak says.

Luckily, he might not need to find zetekitoxin to restore it. Based on similarities in the chemical structure, Cernak thinks it’s possible to create zetekitoxin from a different type of toxin called saxitoxin, an ancient compound that’s relatively common in nature. Cernak’s hunch is that the frogs eat something in their diet containing saxitoxins—likely some kind of insect—that then transforms into zetekitoxin. How, he can’t yet say. But he recently sifted through their gut contents and found hints of saxitoxin; ongoing analysis will hopefully reveal what insect species it may have come from. In the meantime, he’s running artificial intelligence simulations to unravel the steps for turning one toxin into another.

The holy grail would be to find a golden frog in the wild and test its toxins. But while limosa frogs and variable harlequin frogs still exist in the wild—albeit in small numbers—golden frogs haven’t been seen since 2009. Even releasing captive golden frogs into the wild for the first time last August was a big deal.

In an undisclosed spot of jungle in Panama, Gratwicke’s team put 100 golden frogs in the enclosed cat tents. As with the other Atelopus species, the goal with these trials is to study the disease dynamics in the wild to increase the frogs’ future odds of survival. Researchers are considering a swatch of potential mitigation strategies, including selective breeding of individuals that are more resistant to Bd, and targeted releases in areas called “climatic refuges” that have conditions that are suboptimal for the fungus.

(Read more about how “frog saunas” could protect amphibians from Bd.)

After 12 weeks in the tents, about 70 percent of the frogs had died of Bd—a sobering reminder of the fungus’s persistent presence.  But the trial allowed the scientists to take skin samples from the closest thing to a golden frog in the wild. Now they’re working to get the swabs from Panama to Michigan so Cernak can test his saxitoxin hypothesis. He plans to incubate synthetic saxitoxin with the frogs’ special soup of skin microbes to see if that might produce zetekitoxin.

For now, there’s still more questions than answers, but the important thing is that the search is on. “Many researchers know that this problem exists with the frogs, but so far only this project has tried to understand the system,” says Rodriguez, the Panamanian chemist. “You cannot preserve if you don't understand the system.” Captivity may have saved these species. But as the researchers learn more, the frogs are one step closer to returning home.