Image credit: Aimee Silla
Today, amphibians, including frogs, are becoming extinct faster than any other vertebrate in the world. Scientists and environmentalists have built multiple artificial breeding bases for them, but they have not been able to alleviate this trend. How to make the few remaining breeding offspring of endangered species and alleviate the species crisis has become a new problem. But with the development of breeding technology and the diversification of artificial habitats, we may still hear some endangered frogs in the future.
A large number of frogs are endangered
A few years ago, I unexpectedly received an email from a friend. The friend, a frog lover, wrote in an email: "Toughie is dead. "Toughie is a Rabb's fringe-limbed tree frog that has a huge following around the world, and I'm one of them. Toughie is cute, but it's famous for a harsh truth: it's the last Panamanian tree frog in the world. As the only survivor of the extinction crisis population, Toughie lived a very short life of stars and moons.
Toughie (Image: wikipedia)
In 2005, an animal protection team captured Toughie and four other Panamanian frogs from a remote cloud forest in Panama and brought them to the United States for artificial breeding in hopes of stopping the extinction of Panamanian frogs. Due to the infection of Chytrid frog fungus, the species quickly disappeared from the wild, and subsequent artificial breeding also failed. By 2012, Toughie was the last survivor, and it died in September 2016. Although we already knew that Toughie was leaving, that wouldn't have eased the blow.
This year, I've been following the fate of a Sehuencas water frog from Bolivia. The handsome water frog, named Remeo, lived alone for ten years in the aquarium of the Natural History Museum of Cochabamba, Bolivia. It has repeatedly tried to find a partner, but without success. At the end of 2017, Remeo gave up the call to the opposite sex, and it seemed destined to face the same fate as Toughie. Fortunately, thanks to the help of others on the internet, this West Vinks water frog has a chance to avoid ending up alone.
On Valentine's Day last year, Remeo's keepers posted Remeo's profile on the website. This move led to a large donation that funded and supported another expedition to find work for wild water frogs. In January, the museum announced that the expedition had found five water frogs, including 2 females. Given the bleak prospects for survival in the wild, the expedition brought them back to the aquarium. Remeo finally found his other half, and it was named Juliet.
Remeo (Credit: American Physical Society)
Every spring, the frogs in my backyard seem to take every opportunity to lay their eggs in large numbers, so I naively guessed that Remeo's "post-marriage" life would also be very smooth, and eagerly awaited news of the little tadpoles. However, the results are not optimistic, and the captive water frogs do not seem to have much enthusiasm for breeding. If endangered animals like the West Vinks water frog don't mate and lay their eggs, how can they be saved?
For frogs and toads, we can solve this problem with reproductive technology. With the rapid development of amphibian hormone therapy, artificial insemination and cryopreservation techniques, the chances of captive frogs giving birth to healthy offspring have also begun to increase. Few endangered species are currently benefiting from these technologies, but as Aimee Silla, a reproductive biologist at the University of Wollongong in Australia, says, the technologies are expected to help more endangered animals. Silla and colleague Phillip Byrne report on achievements in the field in the Annual Review of Animal Biosciences. "These technologies have a lot of potential for application, and exciting breakthroughs are emerging, so we have reason to be optimistic," she said. ”
Biodiversity on Earth is being decimated at an unprecedented rate, with amphibians bearing the brunt of the decline in numbers faster than any vertebrate population. In 2017, the International Union for Conservation of Nature (IUCN) listed about 2,100 endangered amphibians, accounting for almost 32 percent of known amphibian species. According to IUCN estimates, this number is likely to continue to rise to around 55 percent.
In fact, we are familiar with the culprits behind the sharp decline in amphibian populations – habitat loss, climate change, environmental pollution, over-prep as food and the growing wildlife trade. But now it's added to the worst amphibian epidemic ever recorded, chytridiomycosis. Recently, scientists have evaluated the impact of the disease and have shown that this terrible fungal infection disease has led to a decline in the number of more than 500 amphibians, and the extinction of 90 more amphibians.
In the late 1980s, reptilians first sounded alarm bells of extinction and eventually led to a global assessment of frogs, toads and their close relatives. The results, published in 2004, were shocking: amphibian populations were plummeting everywhere, and some species had even disappeared. In 2005, international experts held a summit to develop appropriate protective measures. One of the key proposals is that zoos, aquariums and other capable organizations should seek to "save" endangered species and breed in captivity.
If all goes well, these "man-made conservation habitats" will preserve the genetic diversity of endangered species to some extent. If suitable habitat is available, the remaining offspring can be released into the wild to help increase or restore dwindling populations.
In the program, many conservationists brought back rare frogs and toads, and the number of amphibians in captivity spiked. Some of these breeding programs have been a great success. Mallorcan midwife toad, for example, has re-established 18 breeding populations in the wild, off the endangered species list. Tanzania's Kihansi spray toad was declared extinct in the wild in 2009 after a local water project destroyed its mist-filled habitat next to the Chihansi Gorge waterfall. So efforts were paid off by managing to create a niche water mist environment for captive-bred spray toads, and thousands of captive-reared spray toads were released into artificial water mist habitats in Chihansea Canyon.
Mallorcan midwife toad. (Image source: Wikipedia)
Assisted reproductive technologies
But for many other species that have been saved, the situation is less optimistic. Some species are unable to produce eggs or sperm, or the time for male and female individuals to produce gametes is out of sync; some species are unable to perform the necessary mating behaviors; and a few species produce embryos, but embryos are difficult to develop further. Andy Kouba, a conservation biologist at Mississippi State University who has studied some of America's rarest amphibians, said there is now a captive breeding crisis in addition to the extinction crisis.
Why is it so hard for amphibians to reproduce offspring? In large part, this is mainly because we do not understand the demanding requirements for the reproduction of rare species. Frogs and toads are extremely diverse animals, often with many highly differentiated reproductive strategies and habits. Some breed in water, including calm lakes, rushing streams, and shallow pools; others breed on land, from the ground and canopy of tropical rainforests, down the moss of alpine swamps to the foam pools of reeds. Most of them reproduce through in vitro fertilization, but a small percentage are in vivo fertilization. Some are monogamous, while others employ a very confusing many-to-many mating approach.
What's more, physiological and behavioral changes associated with mating can be linked to a range of environmental and social factors, including changes in humidity, temperature, or pressure, brighter, longer days, calls from companions, the end of hibernation, or a sudden downpour that marks the beginning of a rainy season. Once a species is on the verge of extinction, we have little opportunity to study it in the wild, so it's hard to find triggers for mating. "There are times when we can figure out the triggers for mating and succeed in getting them to mate, but for a lot of other species, no matter how much we try, we can't find the right answer." Kouba explains.
Thus three assisted reproductive technologies (ART) emerged: (1) hormone therapy, which can prompt reluctant partners to mate, or induce the development and release of eggs and sperm; (2) in vitro fertilization, which increases the number and diversity of offspring; and (3) sperm or egg freezing techniques in case of emergency.
A small number of endangered species have already benefited from these three technologies. In Australia, for example, hormone therapy turns reluctant northern corroboree frogs into passionate lovers, ensuring that they all contribute their genes to the next generation. Wyoming toads from the Laramie Plains of the United States are extinct in the wild, but thanks to hormone and artificial fertilization techniques, their populations are beginning to recover again. In addition, frozen sperm contributed some offspring to the endangered western toad (Anaxyrus boreas) in the southern Rocky Mountains.
Western toad. (Image source: Wikipedia)
But it's not easy to get these jobs done. Although scientists have been conducting basic research for nearly 70 years to grow amphibian embryos using hormones and ivory fertilization techniques, by the 1990s, when biologists tried to increase the number of amphibians with these techniques, they found that the lab's process was only suitable for specific objects and could not be used directly in the wild. After several years of adjusting and testing the dose of the hormone, the first effective treatment was finally established. In addition, even the simple task of fusing sperm and egg takes a lot of energy.
With the rapid increase in the number of man-made conservation habitats (many of which are still unable to reproduce naturally), there is a growing sense of urgency. "We realized that this research had to be done in a hurry," Silla said. And now, the hard work paid off. She added: "Assisted reproductive technologies have evolved rapidly in recent years. ”
Hormone therapy stimulates gonadal maturation and egg and sperm release by injecting two hormones: gonadotropin-releasing hormone and human chorionic gonadotropin (until the 1970s, these hormone experiments were mainly used to study human pregnancy behavior). Some species respond best to the first hormone, while others are more appropriate for the second, and injection doses and regimens are also crucial. "Every species is different, so it can take several years to find the right hormone and determine the timing and frequency of treatment," Kouba said. "Finding the right treatment for a new species has long been a trial-and-error process. Today, for about 20 species, scientists have identified corresponding hormone therapies.
As the number of hormone regimens increases, some patterns gradually surface, and this helps scientists find more suitable hormone therapies. For example, the ancestral lineage of amphibians seems to determine the choice of hormone, with some members of the frog and toad families responding better to the first hormone, while members of other families are more suited to the second hormone.
Silla and Byrne believe that evolutionary factors may also be key to predicting optimal sperm collection times, as the number of sperm produced by males and the rate at which they are released are closely related to the species' mating strategy.
In non-monogamous species, competition between males pushes the testicles to evolve in greater directions, producing more sperm and releasing them more quickly. For example, the Georgian Toad in Western Australia (its nickname is quacking frog because it crunches like a duck) is polygamous, with many males flocking around an egg-laying female. Those males have huge testicles filled with tens of thousands of sperm. After injecting hormones, they begin to release sperm within minutes. However, Silla found that the optimal time to collect is 7 hours after the injection, when the release of sperm peaks.
In stark contrast is the southern corroboree frog in southern Australia, a critically endangered species that lives in the snowy mountains of New South Wales. This frog adopts monogamy, which also means that it does not need to produce many sperm and does not have to be anxious about mating, so its testicles are small. According to Silla and Byrne, after injecting the hormone, they wait a long time to get sperm. "In this frog, it takes 36 hours for injections to reach the peak of sperm release, and we ended up collecting only a few hundred sperm," Silla said. ”
Southern Australia Night Feast Frog. (Image: the conversation)
After gamete collection, the second technique needed is in vitro fertilization, which allows the breeder to control the kinship of the offspring, allowing genetic diversity to be optimized in small populations. While it sounds simple in theory, it is not easy in practice. The traditional method is to mix the eggs and sperm obtained by chopping the testicles into a Petri dish and let them stand for a few minutes, then flood them with water. Sudden dilution of the solution will cause the sperm to become active.
But when it comes to protecting animals, we can't go straight to the testicles. Instead, breeders must wait for sperm to move to the cloaca and mix with urine before they can collect them. For some toads, as long as they are picked up and placed on a plate, they will excrete a mixture of semen and urine. If this does not work, it can be done by applying a slight pressure to the abdomen. Frogs have a harder time obtaining sperm, and breeders may need to use thin catheters to complete the collection.
In addition, the success of breeding depends on whether the mixed ratio of sperm and egg is correct, whether a specific fluid that activates sperm is added, and many other reasons.
A third technique, cryopreservation, can also increase populations and ensure the genetic health of endangered species. Because amphibians undergoing hormone therapy often produce sperm and eggs at different times, cryopreservation techniques can store gametes of one sex until we get gametes of the other sex.
Long-term storage of sperm also opens up new possibilities for the optimization of genetic diversity. Some of the collected eggs can be fertilized with sperm from many different males, zoo and aquarium populations can exchange sperm, and we can even have wild and captive individuals hybridize. In particular, individuals with special value in fighting diseases can continue to contribute their genes long after death.
But, as you might think, the actual operation is much more complicated than it sounds. Sperm storage is easier than eggs, and experiments with sperm cryopreservation have been successful in a growing number of species. Kouba, who co-founded the U.S. National Amphibian Genome Library, said: "Initially the probability of sperm returning to activity after thawing was only 5 percent, and now it can reach 50 to 70 percent." We can also conserve sperm from living animals and use them to breed offspring. ”
Freezing eggs is another matter: Because of its large size, yolk content, high moisture content, and thick jelly-like outer layer, these characteristics have led scientists to fail to freeze them so far. Kouba said the problem must be solved. "Freezing sperm is indeed much simpler, but if only the male genome is preserved, that's only half the entire genome map."
Establish more breeding programs
So far, despite the huge demand, the number of endangered species benefiting from assisted reproductive technologies remains small. In some breeding programs, many or all of the resulting offspring are still kept in captivity to help keep artificial habitats running. Others have returned to the wild: more than 300,000 Puerto Rican crested toadlets have been released to their islands, and hundreds of northern Australian night-feast frogs have returned to their subalpine marshlands. She is proud that all of the northern Australian night feast frogs that Silla bred during the course of her research have been released: "I am thrilled that this is not only based on laboratory research, but also a real conservation action. ”
Puerto Rican crested toad. (Image source: Wikipedia)
In the near future, more advanced reproductive technologies will emerge, but we cannot apply them to amphibian breeding projects anytime soon. At the same time, hundreds of amphibian species are on the verge of extinction. "One of the most urgent needs right now is to set up more breeding programs to save more species," Kouba said. ”
With luck, patience, and other elements, some species may be able to recover on their own. But for other species, it may still be hoped that humans will help. "Overall, we've saved a fraction of what we've saved compared to the number of amphibians threatened." Kouba said, "But for some of the frogs and toads that we are actively studying, it will have a significant impact." ”