Scientific endeavour isn’t always as glamorous as crashing atoms into each other deep underground in Switzerland, or sending rovers to roam the surface of Mars looking for clues to life. For Dirk Schmeller, a researcher at the CNRS research centre in Moulis, southern France, there are more fundamental problems to deal with – like how to get a stubborn ass to shift.

Donkeys and mules may not be the most hi-tech option for lugging large volumes of water down a Pyrenees mountain, but it’s the most effective… until you get an uncooperative animal, of course. “We can thank Gaston and Justin, but we aren’t thanking Emile,” says biologist Adeline Loyau, Schmeller’s co-worker and wife, while discussing who to include in the acknowledgements section when writing up their research. Gaston, Justin and Emile were the donkeys Loyau and Schmeller hired to carry lake-water samples down from the mountain last summer. Her displeasure with Emile stems from a stressful day she spent unsuccessfully trying to get the beast to budge, in turn condemning Loyau to a backbreaking hike to and from a lake with up to 30 kilos (66 pounds) of water.

The donkeys have a key role in trying and save some of the world’s most vulnerable amphibians from their plight. Schmeller, Loyau and their colleagues are part of a Europe-wide project, RACE (Risk Assessment of Chytridiomycosis to European Amphibian Biodiversity), investigating – and trying to halt the spread of – a fungal disease that threatens to devastate amphibian populations across the globe.

The disease is already responsible for the mass death of over 350 amphibian species, pushing many to the brink of extinction. “If a single pathogen were causing the death, decline and extinction of 30% of mammal species (including humans), the entire world would be paying attention,” wrote the evolutionary biologists Valerie McKenzie and Anna Peterson from the University of Colorado last month. “This is what has been happening to the world's amphibians.”

Jaime Bosch first encountered the amphibian threat in Europe just over a decade ago. He was studying communication between midwife toads (Alytes obstreticans) in a lake in the Peñalara national park outside Madrid, when his worst nightmare began to appear before his eyes. The toads were beginning to die off in huge numbers, a once thriving lake became a graveyard for countless floating bodies. By 1999, Bosch and his colleagues at the Museo Nacional de Ciencias Naturales in Madrid reported that no living animals were found in 86% of the toad’s usual mating ponds in Central Spain.

But this wasn’t just happening in Spain. Thousands of dead amphibians were found floating in waters in North and Central America, and Australia. What was causing this mass slaughter seemed to have little regard for borders or which species it infected. In 1999, scientists finally tracked down the culprit and gave the disease a name: Batrachochytrium dendrobatidis, or Bd.

“Lakes of death”

Since then, a handful of scientists have got together to track its spread throughout Europe. Fungal epidemiologist Matthew Fisher, now at Imperial College, London, recalls how he first got involved in Europe’s Bd fight in 2002. He had just returned to the UK from studying the evolution of killer fungi at the University of Berkeley, California, when he read Bosch’s work on this mysterious new disease. Fisher got in touch, little suspecting the gruesome nature of what he was about to encounter.

“Jaime started FedEx-ing me these boxes of rotting frogs,” Fisher explains. As well as stinking out a lab, rotten bodies aren’t very useful for studying the genetic roots of the disease. Fisher needed a live sample of the fungus, but even Bd has its limits, and is easily killed by the bacteria in rotting flesh. “You have to get it from a frog that is either close to death, or recently dead,” he says.

Once he started to receive very fresh animals, Fisher managed to successfully grow the fungus in culture. “From that point on we could look at it's virulence, and genetics, and to start to do proper epidemiology,” he says. Fisher was finally in a position to study the genome of the disease, to try and understand how it evolved.

In 2003, Fisher and Bosch began driving around Spain looking for amphibians to sample for the disease. At the end of a long days’ hiking in the Spanish Pyrenees they arrived at a mountain lake called Ibon Acherito. They were confronted by an amphibian horror scene, a “lake of death” Fisher says. “We saw dead animals everywhere,” says Fisher. They found more gruesome scenes in other lakes in the Aspe and Lescun valleys of the French Western Pyrenees. Ever since that grisly hiking trip Fisher and his team have been regularly revisiting the lakes to monitor the health of the animals and the spread of the disease.

Meanwhile, Schmeller arrived at the tiny French Pyrenean village of Moulis, tucked away in the lush, remote Ariege area of Southern France. Moulis hosts a French national research centre, and Schmeller started work there in 2007 to monitor the area's biodiversity. Around the time Fisher and Bosch were getting to grips with rotten toads and frogs, Schmeller was investigating the population genetics of waterfrogs at the University of Mainz, Germany, and met PhD student Trent Garner through his work. Garner moved to Fisher’s lab, and realised that Schmeller, newly landed in Moulis, was perfectly poised to investigate this mysterious disease in the Pyrenees. “I was working with amphibians and genetics before, but diseases was a whole new thing to me,” says Schmeller.

They gathered together other researchers, and initiated the Europe-wide RACE project, with €1.5 million funding from the EU’s Biodiversa network, as a concerted, organised effort to understand Bd in Europe. Schmeller’s team work with Fisher in the Aspe and Lescun valleys, and also monitor eastern Pyrenean lakes every year, to see whether Bd has reached them. There they hike up to the Bassies mountain refuge in the Auzat-Vicdessos valley, and spend hours waist-deep in cool mountain lakes, collecting frogs, toads and newts. They swab them, weigh and measure them and sometimes mark them with tiny electronic tags.

Cold comfort

So far, even though Bd has reached the western Pyrenees, the lakes Schmeller and his colleagues are monitoring in the east are disease-free, although Schmeller doesn’t expect them to remain so for much longer. “You can run but you can’t hide from Bd. It will get there in the end,” Fisher told a meeting of all RACE participants in May this year.

The end is pretty horrific for frogs, toads and other amphibians. Once Bd infection sets in, the skin thickens. This is deadly for amphibians because they absorb water and nutrients through their skin, not through the mouth like we do. The disruption in fluid balance leads to heart failure, and eventually death.

As suspects go, Bd is probably one that you’d have least pointed the finger at. The group of fungi it belongs to is an obscure species called chytrids, which normally feed on dead, decaying plants. Something clearly converted them from a vegetarian to a carnivore diet. What’s also interesting is that the disease doesn’t always wipe out amphibian populations, so something else is at play too.

Both the Penalara and the Pyrenean work show that altitude is critical to the devastating effect of Bd. Usually, midwife toad eggs laid in spring hatch into tadpoles after a few weeks. The next weeks and months see the tadpoles metamorphose into toadlets, at which point they hop off to enjoy life out of the pond. But tadpoles born in high-altitude mountain lakes do something unique: they stay as tadpoles over winter. Once summer warms the mountains briefly the following year, they continue to grow slowly. Sometimes this process can go on for several years.

There seems to be a link between overwintering and the disease's deadliness. Tadpoles in frozen-over lakes all huddle together in the deepest part of the lake where the water isn’t frozen – it usually stays around 4C. The disease tends to gather around the tadpoles’ mouths, and as these chilly tadpoles wriggle around they pass on the infection to other tadpoles they come into contact with. “At 4 degrees there’s not much the immune system can do about it,” says Schmeller. The Bd spores become concentrated over the years, leading to infections that are much more severe at high altitudes than at lower altitudes. When these high-altitude tadpoles finally metamorphose into toads, the disease overwhelms them and the effort is too much: they die.

To dig into the effects of the disease, Schmeller carries out experiments in the more comfortable conditions of his lab in Moulis. A concrete shed houses row upon row of small plastic water-filled tanks the size of office water cooler bottles, each home to a midwife toad at some stage in its development, monitored regularly for the disease and any changes in physiology or behaviour. Some of these experiments are done with Bd-infected water from the Aspe valley, hence Schmeller’s need for those donkeys.

Infected tadpoles apparently need to invest some energy in fighting the infection, and they are smaller when they reach the toadlet stage, says Schmeller. They’ve also seen that when they cool down infected toadlets to 4 degrees for just five or ten minutes, they die very quickly, while the ones that were not infected don’t. In other words, the animals can live with Bd until there’s an additional pressure, Schmeller explains. “This is something we need to understand better, especially under climate change scenarios,” he says.

Killer trade

What also needs to be understood is how to treat the disease. There have been various unsuccessful attempts; for instance, Bosch and Fisher’s teams have tried treating animals in Spain, the Western Pyrenees, and the Balearic island of Majorca with the anti-fungal agent itraconazole. While this can get rid of the disease, it often ends up killing infected toads as well. And in other cases, in Mallorca for example, early experiments to treat infected larvae cleared the disease, but once they were returned to their lakes or rivers, they became re-infected.

Over in the small town of Rascafria in the Penalara park, Bosch has built a breeding centre to try another another tack – repopulate the area with animals that have survived Bd outbreaks. Work has been slow going, and again results have been mixed, but he is beginning to see some success. “We are using the last surviving animals after 10 years of the outbreak to breed with them, and their offspring look more resistant,” he says. The big test will come when these animals are released into the wild.

That’s because we still don’t know how Bd arrived into these mountainous areas in the first place. In the western Pyrenees, there have been at least two separate infections, with several theories about their origins. The disease could have been unwittingly spread on the muddy boot of a hiker, or stuck to the feet of ducks or other water fowl. Or perhaps stocking lakes with fish, or hydroelectric pumping brought Bd in.

Another possibility is that the fungus was introduced into countries through trade: two of the main suspects being the African clawed frog (Xenopus laevis), which is used in biological research; and the North American bullfrog (Rana catesbeina), which is prized for its meat. Trade laws are in urgent need of updating, if Bd’s global spread is to be halted, says RACE participant Mark Auliya, a herpetologist and trade-policy expert at the Helmholtz Centre for Environmental Research in Leipzig, Germany. Pet fairs are big business, and exotic amphibians go for handsome sums. Yet these animals aren’t tightly regulated and could easily be carrying Bd or other infectious diseases.

The importance of regulating trade really hit home recently after Fisher and his team published a detailed look at Bd’s genome. When Fisher and his team deciphered the DNA from 20 fungus samples, they found three distinct strains of Bd. One is isolated in South Africa, another in Switzerland, both of which aren’t as deadly as the third.

The bad news for the European team is that the third strain, the one found in the Pyrenees, is hypervirulent – extra deadly. This strain, BdGPL, is also responsible for infections in Central and North America, and Australia. It seems likely the only way the fungus travelled such distances is through human movements – mainly through trade. That trade could also be responsible for BdGPL’s deadly characteristics – its genome reveals that it was formed from the recombination of different, possibly harmless, strains of Bd to become the lethal frog-killing fungus. “We’re dealing with a much more complex evolutionary history than previously expected,” says Fisher.

Stay vigilant

So far the story seems gloomy at best. Amid all the monitoring experiments, mitigation efforts and genomic analyses, it’s hard to see a rosy prognosis for amphibians, particularly those destined to live at high altitudes. “The reintroduction doesn’t work, disinfection is very difficult, cleaning of ponds in Mallorca didn’t work,” says Schmeller. “It’s very difficult.”

Time will tell. Optimism is essential for researchers in this field, and Fisher thinks there are reasons to be cheerful. “I’d be very sceptical of a shock horror story in Europe,” he says, despite having witnessed “lakes of death” for himself. Bd will continue to cause population declines in high altitude places, including the Pyrenees, he predicts, but things could change.

One glimmer of light is that Fisher’s genome work revealed something else about Bd – “the fungus has an extraordinarily dynamic genome,” he says, “This means there could be very rapid selection for less virulent lineages,” Fisher explains. It makes sense: the disease can’t propagate if it kills off all its hosts. What would be a much better scenario for the fungus’s survival is a milder version that can live together with the amphibians it infects.

Bosch, who first witnessed the devastating effects of Bd, agrees. “In the end, most amphibian species here will co-exist with Bd,” he says. That’s not to say that amphibians are safe. “Just for a few of them, in high elevation areas, most populations will disappear,” Bosch adds. “In my opinion, the key is just to try to keep these populations alive for a few generations to allow natural selection to act.” This is why he believes his breeding centre is so important.

Schmeller is about to take up a new post at the Helmholtz institute in Leipzig, but he’s determined to keep a close eye on amphibians in the Pyrenees, even if natural selection might end up being the only answer. “We need to stay vigilant,” he says. So until Bd is better understood, or trade rules tightened to control its spread, Schmeller and his team will continue wading through chilly mountain lakes swabbing toads and newts. With or without the help of those stubborn donkeys.

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