Solution Story: Freezing DNA to Bring Amphibians Back to Life

Huge strides have been made in the effort to biobank endangered amphibians.
Left: Young gopher frog metamorphs produced by in-vitro fertilization. Right: Depositing frozen amphibian samples into the biobank at the Memphis Zoo. (Photos: Courtesy of the Memphis Zoo)
Jul 8, 2013
Richard Conniff is the author of The Species Seekers: Heroes, Fools, and the Mad Pursuit of Life on Earth and other books.

For one of the most colorful animal groups on Earth, the 21st century is looking a lot like the end of the world. Scientists suspect that 165 amphibian species—frogs, toads, newts, salamanders, and caecilians—have gone extinct over the past few decades. Another 2,000 species—roughly a third of the 6,260 known amphibians—are either threatened or endangered. That’s not counting the 1,500 species for which scientists lack adequate data. Habitat loss is the usual culprit, together with the worldwide amphibian pandemic commonly known as chytrid fungus.  

But a team of researchers from eight countries, writing in the journal Biological Conservation, now proposes a slender thread of hope in the form of “biobanking,” the preservation of genetic material for amphibian species. In the face of “what seems to be an inevitable march of destruction and loss,” the authors write, their proposal amounts to “an eleventh hour, last-ditch effort” to save what could amount to the entire taxonomic class Amphibia.

It may sound a bit like science fiction: A liquid nitrogen tank taking up half the space of a typical desk can hold sperm samples from hundreds of individuals. Some may be used next month, or ten years from now, to help save a species. Others may be stored away for 200 or 300 years, to be resurrected in some future world that, with luck, will be more hospitable than the one amphibians now face on Earth. Like candidates for flight to a distant planet, donor amphibians must have the right stuff: Lab technicians screen them to ensure that they do not carry the chytrid fungus and also to see if they have specific resistance genes associated with survival should chytrid fungus recur.

Biobanking itself is not a particularly new idea. Up to now, genome resource banks (GRBs) have been reserved mainly for charismatic mammals, including giant pandas and large cats. (Biobanking of tissue samples and genetic material is also becoming an increasingly important factor in human medicine.) But until now, cryopreserving amphibian reproductive material has simply been too difficult. 

The first successful attempt to freeze amphibian sperm took place more than 40 years ago, according to Andy Kouba, a reproductive physiologist specializing in conservation research at the Memphis Zoo. But researchers have struggled in the decades since then to get the technology right. So far, they’ve managed to apply it to just 10 or 12 model amphibian species, none of them at risk of extinction. But according to Kouba, lead author of the Biological Conservation article, the picture has changed dramatically over the past two years.

In the past, the only way to get sperm from amphibians was to kill the donor animal, not a good option with threatened and endangered species.In the past, the only way to get sperm from amphibians was to kill the donor animal, not a good option with threatened and endangered species. But researchers have recently learned to administer hormones that cause males to produce sperm-laden urine. With some species, says Kouba, “you pick up the animal and it just pees into a Petri dish.” With others, “it’s not so simple, so we insert a tiny catheter into the cloaca.” 

This solution addresses only half the puzzle. Female genetic materials, in the form of eggs that are 80 to 90 percent yolk and therefore difficult to freeze, have so far eluded efforts at cryopreservation. Getting the female half of the equation right could take 10 or 20 years, Kouba admits, “or we could have a breakthrough next year. I think it will happen, but it’s not going to be easy.” Even so, he says, “There are amazing things we can do in science,” including “the possibility to resurrect a species without the female genome.” One technique involves taking eggs of a related species, inactivating the genome of that species, and replacing it with the genome from male donors of the desired species.      

Biobanking also makes it possible to ship frozen sperm samples around the world in place of live animals, and a single sample can now be broken up and distributed to fertilize eggs from multiple females for greater genetic diversity. Because it can be tricky to induce a female to lay her eggs, or predict when she will do so, having a bank of frozen sperm available at all times also makes in vitro fertilization easier. In recent experiments using fresh sperm samples, researchers have produced and released 5,000 Colorado boreal toad and 2,000 Wyoming toad tadpoles as a way of rebuilding wild populations of these imperiled species. Despite all the efforts being made in mammal sperm cryopreservation, says Kouba, “you’d be lucky to find 10 examples” of similar success using the technology to reintroduce any mammal species, and any such examples would typically involve no more than one or two individuals.

According to the Biological Conservation article, the cost to maintain a single cryopreservation tank with 9,000 specimens is about $3,000 a year. The article does not calculate how many specimens it would take to adequately preserve the genetic diversity of a single species, or how many tanks would be needed to preserve all the amphibian species that are now threatened or endangered. Skeptics sometimes suggest that making the required effort is pointless since there may not be any habitat for these species to repopulate, says Kouba. But we have no way of knowing what the world will look like in the future, and hope is a deeply ingrained human trait. Species that represent millions of years of evolutionary development deserve the kind of heroic effort we would expect for our own species. 

The challenge now, says Kouba, is to get zoos that are biobanking mammals to expand their efforts to include amphibians. Natural history museums that already maintain frozen reproductive material for making taxonomic distinctions among species could also expand their facilities to save those species from extinction. Because relatively few threatened or endangered species are being protected in zoos, it’s also important for field biologists to begin collecting reproductive material. (The technology for getting specimens back to the lab can be as simple, says Kouba, as a Thermos with an ice pack at the bottom.)

Even if Kouba and his colleagues can make this last-ditch biobanking effort actually happen, many amphibians will still vanish. The hope is that, in some better world 200 or 300 years from now, they may come back to life and once again populate the Earth.

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