A deadly fungus is attacking this fire salamander, producing tiny circular ulcers with black rims.

PHOTO: © FRANK PASMANS

A fungus is attacking Europe’s most beloved salamander. It could wreak havoc if it gets to North America

GHENT, BELGIUM—Until recently, the Bunderbos was the best place in the Netherlands to find fire salamanders. With tall broadleaf trees shading small streams, the small forest was home to thousands of the 20-centimeter-long creatures, glistening black with bright yellow spots. "It's a very charismatic animal," says Annemarieke Spitzen-van der Sluijs, a conservation biologist with Reptile, Amphibian & Fish Conservation Netherlands (RAVON), a nonprofit group based in Nijmegen. "It's like a dolphin among amphibians, always smiling, with pretty eyes."

But starting around 2008, the population in the Bunderbos began to plummet for no apparent reason. When Frank Pasmans and An Martel, veterinarians here at Ghent University, heard about the enigmatic deaths, they recalled extinctions caused by Batrachochytrium dendrobatidis (Bd), a highly lethal fungus that infects more than 700 species of amphibian. Yet tests for Bd at their lab were negative.

The declines became so alarming that RAVON removed 39 fire salamanders (Salamandra salamandra) from the park, safe-guarding them temporarily in an employee's basement. When these animals began to die as well, Spitzen-van der Sluijs rushed them here, about 2 hours away, where Martel and Pasmans cultured a fungus from a salamander clinging to life. It was a new pathogen, related to Bd. They named it B. salamandrivorans (Bsal) for the ulcers that voraciously eat away at the animals' skin.

So began a bittersweet odyssey for the couple, partners in life as well as work. Studies they have led since their initial discovery show that Bsal—probably introduced from Asia by the pet trade—has the potential to wipe out salamander populations across Europe. An even bigger fear is that the pathogen will reach North America, which holds the world's greatest diversity of salamanders. (Tennessee alone has 57 species.)

The work has brought Martel and Pasmans funding and scientific recognition. "They're doing amazing work, in the right place, at the right time," says Vance Vredenburg, an ecologist at San Francisco State University in California. But the couple worries they have front row seats to the extinction of rare species they love. "This would be a true loss," Pasmans says. Nobody knows how to slow Bsal's spread, although Martel, Pasmans, and many others are discussing measures such as trade restrictions, habitat protection, and even enlisting other organisms to fight the pathogen. "It will be a race," says Dirk Schmeller, a conservation biologist with the Helmholtz Centre for Environmental Research in Leipzig, Germany. "And there may not be enough time."

An Martel and Frank Pasmans are deciphering a fungal threat to fire salamanders and other species.

PHOTO: © MANON BRUININGA

As a child, Pasmans, 42, played with newts in the ditches of a park near his home in the suburbs of Antwerp, Belgium, and he has been fascinated by amphibians and reptiles ever since. "I just loved watching their behavior, seeing the larvae grow and go through metamorphosis," he says. At 17, he joined a group of herpetologists and enthusiasts, which focused on amphibian and reptile diseases. Martel, 41, loved mammals at first, growing up with cats, dogs, and guinea pigs, but she caught Pasmans's enthusiasm for amphibians after they started dating in graduate school. They live in a farmhouse with two dogs, 15 sheep, and about 50 fire salamanders in the cellar.

Veterinary science was a natural career choice for both of them. Pasmans joined the faculty at Ghent in 2005, followed by Martel 2 years later. Because funding for research on amphibians and reptiles was scarce, they spent several years researching infectious diseases of poultry and pigs, while working on turtles and salamanders after hours.

Pasmans had long been aware of threats to amphibians, whose habitat—moist woodlands, ponds, and streams—makes them vulnerable to development and water pollution. By now, nearly one-third of the more than 7600 known amphibian species are endangered, a higher proportion than in any other major group of vertebrates. Lately, diseases have emerged as a major concern, among them ranaviruses, which have led to a few documented cases of mass mortality and local extirpations worldwide since the 1990s.

The worst infectious disease has been Bd, which in 1999 was implicated in amphibian declines in rain forests in Panama and Australia but is thought to have started spreading and harming populations at least 2 decades earlier. Where the fungus came from is unknown. It infects the skin of susceptible species, causing problems with respiration and fluid regulation, and, eventually, triggering heart attacks. Bd drove to extinction many species of frogs in the Americas and the northern gastric brooding frog of Australia. It has been found across North America, where it threatens several species of frogs. Although Bd wiped out salamanders in Mexico, Guatemala, and Costa Rica, it does not seem to have caused significant problems for the highly diverse salamanders in the southeastern United States.

In 2008, Ghent University awarded Martel and Pasmans a grant to study Bd and skin pathology in amphibians. That same year, while on vacation in Costa Rica, they got their first look at the power of the fungus. Visiting the mountain cloud forests, which once echoed with the chirps of harlequin frogs, they were struck by the silence. "They're completely deserted," Pasmans says. "It was really dramatic."

By then, European herpetologists were alarmed as well. In 2001, Bd had been linked to a severe decline of the midwife toad in Spain. Over the decade, Bd was detected in northern Europe as well. Spitzen-van der Sluijs found it in amphibians all over the Netherlands and in Belgium. Yet it didn't seem to cause die-offs. "The dogma was: When Bd enters, everything dies," says Pasmans, who felt the impacts had been a bit hyped.

Then salamanders began dying in the Bunderbos. In most other forests, the outbreak might have gone unnoticed. But volunteers have systematically surveyed the 1.4-square-kilometer reserve and surrounding woods since 1997 to keep tabs on what was the biggest of three populations of fire salamanders in the Netherlands. When Pasmans and Martel first heard about the deaths in 2008, they were not particularly alarmed. Wild animals die all the time, after all, and northern Europe's amphibians seemed resistant to Bd. But year after year, the news got worse, with estimated population declines of nearly 20% a year.

In 2012, with die-offs reaching a crisis, Spitzen-van der Sluijs drove two dozen living salamanders to the lab at Ghent. From one visibly sick animal, Pasmans and Martel could tell that this was something new. Unlike frogs suffering from Bd, which thickens and hardens their skin, the salamander had ulcers all over its body. (Other symptoms, such as lethargy and loss of appetite, matched those of Bd.) Martel and Pasmans took skin samples and after 3 weeks managed to culture a fungus on substrates of agar and broth.

A genetic analysis revealed that, like Bd, it was a chytrid—a cousin to the frog scourge. "My face went white when I heard about it," Schmeller recalls. Found around the world, this varied group of fungi typically feeds on pollen or the degraded remains of plants and insects in ponds, streams, or moist soil. In a unique—and for some amphibians deadly—adaptation, they release so-called zoospores that can swim a few centimeters by whipping a flagellum.

When Bd zoospores land on a susceptible amphibian, they grow a skinny tube that penetrates the outer skin. The end of the tube swells into a round body that sends another tube even deeper. The burrowing disrupts the amphibian's ability to regulate its fluids. After a few days or weeks, structures called sporangia develop, which migrate to the skin surface, then burst to release massive new batches of zoospores. (The ulcers caused by Bsal suggest its behavior differs in some respects.)

To confirm that Bsal was a pathogen rather than a secondary infection, the duo took zoospores from a laboratory culture and dripped them onto the backs of healthy fire salamanders. The animals developed the symptoms seen in the first sick salamander, and all died a few days later, as they reported in 2013 in the Proceedings of the National Academy of Sciences. "This thing is as pathogenic as it gets," says Joe Mendelson, a herpetologist at Zoo Atlanta.

Individual fire salamanders can be identified by their spots, which has helped researchers chart their decline as the pathogenic fungus Bsal rampages through populations.

PHOTO: © MANON BRUININGA

But was Bsal as broadly destructive as Bd? To find out, Martel and Pasmans conducted experiments on 35 species of amphibians from around the world. All seven European salamander species they tested were highly susceptible. So was one from Turkey and two from North America, including the widespread eastern spotted newt. Frogs resisted or tolerated infection.

The study also identified Bsal's birthplace. Martel and Pasmans detected the fungus in samples of salamanders that other researchers had collected in Thailand, Vietnam, and Japan—including a museum specimen more than 150 years old—but not in salamanders from other parts of the world. During the infection experiments, they found that some Asian salamanders developed symptoms and then recovered, whereas others were completely resistant. The team concluded that Bsal and salamanders probably have coexisted in Asia for millions of years.

In the Netherlanders, however, Bsal was on the warpath. By the time Martel and Pasmans identified the fungus, it had annihilated the population in the Bunderbos. They had missed the chance to study the outbreak firsthand. Then in April 2014, they got a tip from a Dutch man vacationing in Belgium who had come across a dead fire salamander in a forest near Robertville. Aware of the Bunderbos catastrophe, he emailed the lab at Ghent.

Wasting no time, Martel and Pasmans began monitoring the population. "Every week we went and found more diseased animals," Martel recalls. "It was really heartbreaking." Within 6 months, Ph.D. student Gwij Stegen, who took over the fieldwork, had trouble spotting any fire salamanders at all. The study quantified the rate of decline and also showed that sexually mature fire salamanders are much more likely than juveniles to get infected (probably during fights with rivals or mating), which prevents them from reproducing and makes the population less likely to recover.

Back in the lab, there was more bad news. Martel and Pasmans tested other common European amphibians and found that two species can act as a reservoir for the fungus. Alpine newts (Ichthyosaura alpestris) and midwife toads (Alytes obstetricans) both recovered from mild infections during which they shed spores for weeks to months. By ensuring that the pathogen will continue to circulate, these wild reservoirs make it more likely that highly susceptible species such as the fire salamander will go extinct.

Martel and Pasmans discovered another reason why Bsal will probably persist. Zoospores usually survive a few days at most before they're consumed by microscopic predators, but Bsal creates a second, much hardier type of spore that has a sturdy cell wall and can survive in pond water for more than 2 months. These spores float, which helps them avoid being eaten.

All of that means Bsal could rapidly devastate susceptible species of salamander, the team concluded in a Nature paper in April. Species with small populations are especially vulnerable. Ten European species, including five on Sardinia in Italy, each live in an area smaller than 5000 square kilometers, and a species named Calotriton arnoldi makes its home in less than 10 square kilometers in a Spanish nature park.

A looming threat

Accidentally introduced from Asia, the fungus Batrachochytrium salamandrivorans (Bsal) sickens or kills several amphibian species with large ranges in Europe, such as the fire salamander (left), so it will likely spread across the continent. Scientists are especially worried about rare species, as they are more likely to go extinct. Sardinia (right) alone has five such species, most with ranges smaller than 500 square kilometers.

CREDITS: (GRAPHIC) J. YOU/SCIENCE; (DATA, TOP TO BOTTOM) THE IUCN RED LIST OF THREATENED SPECIES; RAVON

The buildup of alarming results has been depressing, Martel and Pasmans acknowledge. To boost morale, Martel and other lab members bring cake and other treats to the lab. (On a recent visit, there was a large box of chocolate-covered Belgian waffles.) Losing herself in the lab research is a consolation as well. "You always find out new little things," she says. "You forget about the animals, and work." For Pasmans, it's walks in nature. "Seeing salamanders active on the forest floor is still magic and gives me a thrill."

The threat from Bsal is clear, the battle plan less so. One top priority, researchers say, is to prevent further introductions from Asia. Last year the United States banned imports of 201 species of salamander to guard against Bsal, and Canada did so for all species in May. In Europe, some are calling for a complete ban on the trade in live amphibians, but Martel and Pasmans believe that would just drive it underground. They favor banning imports of wild-caught amphibians but allowing the sale of animals bred in captivity, even if they come from Asia, as long as they are quarantined and tested. (Commercial breeders in Europe and elsewhere already supply many salamander species.) Encouragingly, a consortium of European pet trade organizations said in April that it supported quarantines and testing of imported salamanders for Bsal.

As for slowing Bsal's spread, experts met in January in Zurich, Switzerland, to evaluate the options. "None of them looked very hopeful," says Matthew Fisher, a geneticist at Imperial College London. Small, isolated populations could be protected by amphibian-proof fences. Testing their efficacy would be "quite complicated," Martel says. And culling healthy fire salamanders around outbreaks, which might delay Bsal's spread, could face public objections.

If Bsal continues to march through Europe, one hopeful scenario is that it will reach an equilibrium with at least some species, as hosts evolve better immunity while the pathogen becomes less virulent. In Australia and California, some frog species are recovering despite continued infections with Bd. So far, however, there are no signs of this happening with fire salamanders, which apparently can't mount any immune response to Bsal. It's possible that other species could put up more of a fight.

One way to boost the odds of survival might be to treat salamanders with probiotic bacteria that release antifungal compounds. Researchers have isolated bacteria from frogs that resisted Bd; when applied to the skin of vulnerable species, the microbes protected many animals from infection. A small field trial with probiotics in 2010, led by Vredenburg, helped protect a small population of mountain yellow-legged frogs in California's Sierra Nevada against Bd. Pasmans and Martel are collaborating with researchers in Germany to identify Bsal-fighting bacteria.

Another idea is to boost the numbers of zooplankton in ponds where amphibians breed. Although many microorganisms consume Bd zoospores, it's not clear how effective they will be with the hardier variety of Bsal spores. And Martel and Pasmans hope to learn at a molecular level what makes adult fire salamanders so susceptible and how larvae resist infection. Identifying genetic markers might help accelerate selective breeding for resistance.

Meanwhile, reducing other threats can make a difference. Mildly infected populations of mountain yellow-legged frogs in California have been slowly rebounding, Vredenburg and his colleagues reported last year, because stocked fish that prey on the larvae were removed from their habitat. The frogs also appear to be acquiring some resistance. "I'm more hopeful about what will happen with Bsal than I would have been without this example," Vredenburg says. Improving the fire salamander's habitat by reducing water pollution might make the animals more resilient, Pasmans says, and removing invasive crustaceans or fish that prey on larvae could boost populations.

Any strategy to make salamanders more resistant to Bsal will likely take years to develop. For now, Martel and Pasmans are monitoring its inevitable spread. With colleagues in seven countries, they have set up an EU-funded program that enlists volunteers to survey for the disease. They hope it will not be a death watch for the smiling salamander.