An amphibious rescue mission

A few years back, a message from a frog-loving friend dropped unexpectedly into my mailbox. “Toughie,” he wrote, “is dead.” Toughie the frog had fans the world over, and I was one of them. Impossibly cute, he was the most famous frog on the planet, for the worst possible reason: The last of his kind, he was enjoying a brief but stellar career as the boggle-eyed face of the amphibian extinction crisis.

In 2005, a conservation team had plucked Toughie and four more Rabb’s fringe-limbed tree frogs from a remote Panamanian cloud forest and taken them to the US to start a breeding program in a last-ditch effort to fend off extinction. The species vanished from the wild soon after; the breeding program failed. By 2012, Toughie was alone, and in September 2016, he died. It was bound to happen sometime, but that didn’t soften the blow.

This year, I’ve been following the fortunes of Romeo, a Sehuencas water frog from Bolivia. For a decade, this handsome orange-bellied frog lived alone in an aquarium at the natural history museum in Cochabamba. Repeated attempts to find others had failed and by the end of 2017, Romeo gave up calling for females that never came. His species seemed doomed to join Toughie’s, yet thanks to Internet dating and a little extra help from his friends, the Sehuencas water frog has a chance.

In one final heroic stunt, as Valentine’s Day approached last year, Romeo’s keepers posted his profile on the dating site Match.com, bringing a flood of donations to fund one more expedition in search of wild water frogs. In January, the museum announced the discovery of five more frogs, including two females, and in light of their gloomy prospects in the wild, they were brought back to the aquarium. Romeo finally has his Juliet, and a shot at fatherhood.

Each spring, the common frogs in my urban backyard pump out prodigious amounts of spawn seemingly at the drop of a hat, so I naively supposed that the course of Romeo’s romance would now run smooth and looked forward to news of tadpoles. But no: A little reading revealed that captive frogs aren’t always so obliging. What floats one frog’s boat leaves another unmoved — and the rarer the species, the less chance of discovering what gets them in the mood. So, what to do if rarities like Romeo and Juliet don’t or won’t do what’s needed to save their species?

For some frogs and toads, reproductive technologies could be the answer. Advances in the rapidly evolving fields of amphibian hormone therapy, artificial fertilization and cryopreservation are beginning to improve the odds of tiny captive populations producing healthy tadpoles. So far, the number of threatened species benefiting from these technologies is small, but as Australian reproductive biologist Aimee Silla of the University of Wollongong told me, the lessons being learned are paving the way to help many more. “These technologies have a lot of potential,” says Silla, who reported on their mounting achievements with her colleague Phillip Byrne in the Annual Review of Animal Biosciences. “And there are exciting breakthroughs all the time, so we have reason to be optimistic.”

Amphibian Armageddon

Earth’s biodiversity is shrinking at unprecedented speed and amphibians are in the front line, losing a greater proportion of species than any other vertebrate group. In 2017, the International Union for Conservation of Nature listed some 2,100 amphibians as at imminent risk of extinction — almost 32 percent of all the species known — and it estimates the figure could be as high as 55 percent.

Behind the losses are the usual suspects — disappearing habitat, changing climate, pollution and overexploitation for food and the exotic animal trade — but with the addition of the worst wildlife disease ever documented, chytridiomycosis. A recent assessment of this catastrophic fungal disease concludes that it has led to the decline of at least 500 amphibian species and the extinction of at least 90.

Herpetologists began sounding the alarm in the late 1980s, and their fears prompted the first global assessment of frogs and toads and their kin. The results, published in 2004, were shocking. Populations everywhere were plummeting; some species had vanished. The following year, international experts held a summit to thrash out an action plan, and one key recommendation was that zoos, aquariums and other organizations with suitable facilities should “rescue” the species closest to extinction and breed them in captivity.

If all went well, those “assurance colonies” would maintain themselves and preserve what remained of their species’ genetic diversity. Where suitable habitat remained, surplus offspring could be returned to the wild to replenish dwindling populations or restore species to old haunts.

That was the plan. Conservationists scooped up rare frogs and toads, and the number of captive colonies proliferated. And some breeding programs have been a triumph. The Mallorcan midwife toad, for example, is now off the critical list, with 18 breeding populations reestablished in the wild. Tanzania’s Kihansi spray toad was declared extinct in the wild in 2009 after a hydro project destroyed its misty microhabitat alongside the waterfalls of the Kihansi Gorge. Painstaking efforts to replicate that misty atmosphere in breeding colonies paid off, and thousands of captive-bred tadpoles — along with an artificial misting system — have been returned to the gorge.

But for many rescued species, it was a different story. Some never developed eggs or sperm, or they produced them out of synch; some flunked the necessary mating behavior; a few produced embryos that rarely made it to tadpoles or toadlets. To add to the extinction crisis there was now a captive breeding crisis, I was told by conservation biologist Andy Kouba of Mississippi State University, who has worked with some of the US’s rarest amphibians.

Why was it so hard to produce tadpoles? In large part, it comes down to how little is known about the finicky requirements of rare species. Frogs and toads are an extraordinarily diverse group of animals with many, often highly specialized reproductive strategies and mechanisms. Some breed in water, from placid lakes to fast-running streams and shallow temporary pools; others breed on land, from the rainforest floor to the canopy, beneath the moss of an alpine bog or balanced on reeds in a foamy nest of bubbles; most fertilize eggs externally though a few bear live young; some are monogamous, others promiscuous with multiple males piling in on spawning females.

What’s more, the changes in physiology and behavior that culminate in mating are prompted by a mix of environmental and social cues: changes in humidity, temperature or pressure; brighter, longer days; the calls of other frogs; a spell of hibernation or a sudden downpour that signals the start of the rainy season. Once a species is near extinction, there’s little chance of studying it in the wild to identify the triggers. “Sometimes we can figure it out and it works, but for other species, no matter what we try we still can’t get it right,” Kouba says.

That’s where the three arms of assisted reproductive technology (ART) come in: hormone treatments to persuade reluctant pairs to mate or to induce development and release of eggs and sperm; in vitro fertilization to increase the number and diversity of offspring; and cryopreservation of gametes so breeders can draw on them when they’re needed.

A handful of endangered species have already benefited from one or more of this trio of technologies. In Australia, for instance, hormone treatment has transformed unwilling pairs of northern corroboree frogs into ardent lovers, ensuring that they all contribute their genes to the next generation. The Wyoming toad from the Laramie Plains is officially extinct in the wild but has begun to make a comeback thanks to hormones and IVF. And the endangered boreal toad from the southern Rockies has gained some extra toadlets thanks to frozen sperm.

But it’s been hard going. Though science labs have used hormones and IVF for almost 70 years to supply amphibian eggs and embryos for basic research, when conservation biologists turned to the technology in the 1990s in an attempt to boost output, they found they couldn’t just adopt procedures worked out for a narrow set of lab models. It took years of tweaking and testing hormones at different doses to establish the first effective therapies; even the simpler task of mixing gametes in a dish needed work.

With the number of rescue colonies rapidly proliferating — and many failing to breed naturally — there was a mounting sense of urgency. “We realized we had to ramp up this research,” Silla told me. And that’s now paying off. “The past few years have seen things progressing faster.”

Most efforts to develop hormone treatments to stimulate gonad maturation and release of eggs and sperm have focused on injection of two hormones: gonadotropin-releasing hormone and human chorionic gonadotropin (whose ability to trigger spawning in toads was the basis of human pregnancy tests until the 1970s). Some species respond best to one hormone, some to the other, and the dose and regimen of shots is critical. “Every species is different and it can take several years to get the right suite of hormones and figure out the timing and frequency of treatment,” Kouba says. For a long time, finding the right treatment for a new species was mostly a matter of trial and error. Today, recipes laying out the what, how and when are in place for some two dozen species.

And as the number grows, patterns are emerging that should help speed the process. For example, it’s starting to look as if an amphibian’s ancestral line might dictate choice of hormone, with members of some frog and toad families responding best to one hormone, and those of other groups to the second.

Evolutionary considerations might also prove key to predicting the best time for sperm collection, suggest Silla and Byrne. That’s because the quantity of sperm males make, and the speed at which they release it, ties in to a species’ mating strategy.

In promiscuous species, competition between males for females drives the evolution of larger testes that make more sperm and release them faster. Take Western Australia’s quacking frog (yes, it quacks like a duck): It exhibits simultaneous polyandry, where many males cluster round a spawning female. Those males have huge testes packed with tens of thousands of sperm. Inject hormones and they start to release sperm within minutes, although the best time to collect is when sperm output peaks seven hours after treatment, Silla found.

Contrast that with the southern corroboree frog, a critically endangered species from the Snowy Mountains of New South Wales. This frog is monogamous which, according to theory, means it needn’t produce many sperm or be in such a rush about it. Its testes are tiny. By Silla and Byrne’s theory, after hormone injection they’d have a long wait for few sperm. And that’s what they saw. “It was 36 hours to peak sperm release, and we only collected a few hundred,” Silla says.

Tricks of the fertility toolbox

Hormone treatment and getting gametes is just the start. Tool number two is IVF, which provides a way to optimize genetic diversity in tiny colonies by giving breeders control over the parentage of offspring. Simple in theory, less so in practice. The traditional technique honed on aquatic frogs and toads is to put eggs and sperm from minced testes into a petri dish, leave for a few minutes then flood with water: The sudden dilution shocks the sperm into activity.

But in conservation, there’s no question of sacrificing animals for their testes. Instead, breeders must collect sperm after it’s traveled to the cloaca and mixes with urine. Some toads respond to mere handling by dumping the sperm-urine cocktail – just pick them up and hold them over a dish, and if that doesn’t work, a little gentle pressure on the abdomen should do the trick. Frogs are less obliging and may require the use of fine catheters.

That’s the easy bit. Success depends on mixing the right proportion of sperm and eggs — in fluid with just the right properties to activate sperm, and as with so much else, every species seems to need something different.

The third tool, cryopreservation, is about boosting output and ensuring the genetic health of threatened species. Hormone-treated amphibians frequently produce sperm and eggs out of synch, so the solution is to store one sex’s gametes until the other’s are available.

Long-term storage of sperm also opens up new possibilities for optimizing genetic diversity. A single clutch of eggs can be fertilized with sperm from many males; zoos and aquariums can exchange sperm; it should even be possible to crossbreed wild and captive animals. And particularly valuable individuals, such as those with resistance to disease, can keep contributing their genes long after they die.

Although — you guessed — it’s all a lot trickier than it sounds. Storing sperm is proving the easier option: Trials with assorted cryoprotective agents, storage media and rates of freezing and thawing have seen success for a growing number of species. “We’ve gone from 5 percent recovery after thawing to between 50 and 70 percent,” says Kouba, who with colleagues is working to create a National Amphibian Genome Bank in the US. “We’ve also shown that we can preserve sperm from live animals and produce offspring with it.”

Eggs are another matter: Their large size, yolk, high water content and thick coat of jelly have so far defeated attempts to freeze them. Solving that problem is a must, Kouba told me. “Freezing sperm will make a big difference but we’ll only be preserving male genomes — and that’s only half the picture.”

So far, the number of endangered species benefiting from ART is small even as the need is huge. In some programs, many or all of the resulting offspring remain in captivity, helping to sustain the assurance colonies. But others have provided a multitude of tadpoles and toadlets for reintroduction to the wild: More than 300,000 Puerto Rican crested toadlets have been released on their native island, for instance, and hundreds of northern corroborees are settling back into their subalpine bogs. Silla tells me how proud she is that all the northern corroborees produced during her research have been released. “I’m excited that this isn’t just a lab-based study but conservation in action.”

There are more sophisticated reproductive technologies on the horizon, but little prospect of using them to generate amphibians any time soon. In the meantime, hundreds of amphibians are edging ever closer to extinction. “Right now,” Kouba tells me, “one of the biggest needs is to set up more breeding programs to save more species.”

With luck, patience, and plenty of TLC, some will be able to help themselves; for others, there’s the prospect of a helping human hand. “In the overall scheme of things, we are making a small dent compared to the number of amphibians that are threatened,” says Kouba. “But for the frogs and toads we are actively working on, we are making a difference.”

That’s music to my ears. From my desk I can hear a common frog calling. I feel lucky that three of the UK’s seven species of amphibians wander into my backyard, and even luckier when some decide to stay. There are thousands more of these fascinating animals I’d like to see — from beauties like the tiny jewel-colored dart frogs and ethereal glass frogs, to big beasts like the wrinkled scrotum frog of Lake Titicaca — and although I know I probably never will, I’d be bereft if I never could. And Romeo? He’s started calling again — and I’m still checking his Twitter feed for news.