A simple sample of the protective mucus layer that coats a frog's skin can now be analyzed to determine how susceptible the frog is to disease, thanks to a technique developed by a researcher at the University of Colorado Boulder.
The same method can be used to determine what kind of probiotic skin wash might be most effective at bolstering the frog's defenses without actually exposing the frog to disease, according to a journal article published today in the journal PLOS ONE.
The findings could help scientists successfully reintroduce endangered amphibians into the wild by reducing the chance that the amphibians will be killed by diseases that may have played a role in the species' original declines.
"We have all these amphibians in captivity now, like the golden frog in Panama, a really beautiful species that is now extinct in the wild," said Douglas Woodhams, a postdoctoral researcher at CU-Boulder and lead author of the paper.
"We want to be able to reintroduce them, but the pathogen that attacked them is still out there," he said. "Now we can determine what probiotic treatment might work best to protect the frogs without infecting them with the pathogen and seeing how many die."
The mucus that coats amphibians is home to a teeming community of microbes and also contains biochemical defenses secreted by the animal itself. The interplay between these microbes—which include beneficial and pathogenic species of bacteria and fungi—and the biochemical defenses determine how susceptible the amphibian is to a particular disease.
"What we find is that the amphibian skin is really a micro-ecosystem," Woodhams said. "All these things are interacting together."
The research team, led by Woodhams, studied midwife toads, which live in Europe and are highly susceptible to the chytrid fungus, a skin disease that is devastating amphibian populations across the globe.
In the new study, the scientists collected samples from the frogs' skins and then incubated spores from the chytrid fungus in the mucus. The ability of the mucus samples to kill the fungal disease was related to how prevalent the infections were among the frog population in the field as well as the survival rate of frogs raised in the laboratory that were exposed to the disease.
The correlations mean that scientists no longer have to expose frogs to the disease to determine whether the frog is susceptible to it.
"We can assess the risk of disease in different species and populations by this method, and focus resources were they are most needed," Woodhams said. "Now we can just test the mucus to see how susceptible the amphibian is."
The research team also used mucus tests to determine how effective probiotic treatments would be at increasing amphibian defenses against disease in a range of environmental conditions and at a range of life stages.
Because the microbial community in the mucus of each amphibian species differs, adding a particular type of bacteria to the mucus through a probiotic wash will have different impacts on different types of frogs.
To determine which probiotic would work best for which frog, the researchers added the probiotic to the mucus before incubating the fungal spores in the sample. The researchers could then determine the effect of the probiotic by observing whether the spores were able to survive in the mucus or whether they were killed off.
The researchers also found that the survival rate of the fungal spores differed depending on the environmental conditions. For example, the bacteria Serratia plymuthica, which can be found on the skin of midwife toads, is known to fight off fungal infections. But when the researchers increased the temperature at which the fungal spores were incubated in a mucus sample containing the bacteria, the results flipped. An increase of about 5 degrees Celsius caused the bacteria to actually encourage the growth of the fungus instead of inhibiting it.
This type of environmental information can help scientists determine where the best location to reintroduce an endangered amphibian species might be by understanding what environmental conditions might bolster the frog's own defenses, Woodhams said.
The study, available at http://dx.plos.org/10.1371/journal.pone.0096375, was funded by the Swiss National Science Foundation. Other CU-Boulder co-authors are Professor Rob Knight and Assistant Professor Valerie McKenzie. Co-authors from the University of Zurich are Hannelore Brandt, Simone Baumgartner, Eliane Kupfer, Ursina Tobler, Leyla Davis, Benedikt Schmidt, Christian Bel and Sandro Hodel. Jos Kielgast from the University of Copenhagen also is a co-author.
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