JMU biology research lending insight into amphibian extinction crisis

Frogs and salamanders are in trouble, and so are we
By Dr. Reid Harris

A deadly fungusMany amphibian species, such as frog and salamander species, are in trouble. Recent estimates place the percentage of threatened species worldwide at between 33 and 50 percent. These figures are staggering and indeed much has been written on the amphibian extinction crisis. There are some selfish reasons why losing so many species matter to us humans. For example, amphibian skin secretions of some species contain potent anti-HIV chemicals. Amphibians can control insect species in some areas and are generally important members of the food web. When frogs die out, tadpoles are not present to control algae in streams and ponds, altering the ecosystem. Yet, even if we were content to live with 50 percent fewer amphibian species, we might want to consider what such a massive loss is telling us about the condition of the environment in which we all live.

My laboratory in the Department of Biology is focusing on a perplexing problem in wildlife ecology: why are species of frogs and salamanders disappearing in seemingly pristine areas around the world? Amphibian species, like many other species, are imperiled by habitat destruction and fragmentation. However, in the western United States, Central and South America, and Australia, amphibians are dying in areas that seem untouched by habitat destruction, such as national parks and forests. One agent of mortality is a lethal skin fungus, Batrachochytrium dendrobatidis, which only attacks amphibians, and attacks many amphibian species. For example, this pathogen has caused losses of up to 70 percent of amphibian species in Panama. Perhaps no other pathogen in history has caused so much mortality to so many species. The fungus has a swimming spore stage that spreads the pathogen from one individual to the next. In Panama, the western United States, and perhaps other areas, the pathogen spreads in a wave-like fashion from one population to the next. Why this disease is emerging now is not fully understood, but recent evidence from Costa Rica links climate change to disease outbreaks. Importantly, not all species, populations or individuals are susceptible to the pathogen. This observation allows us to study the causes of variation in susceptibility.

Amphibians have chemical defenses, such as antimicrobial peptides, that can kill the fungal pathogen. These are the same chemicals that can kill the virus that causes AIDS and that show promise as pain-relief drugs for humans. Research by Dr. Doug Woodhams, formerly of Vanderbilt University, and now at JMU in my laboratory, has shown that amphibian species that co-exist with the fungal pathogen have different chemical secretions from those species that decline or go extinct once the pathogen arrives in their location. The chemical secretions of the persisting species show strong antifungal activity in laboratory assays. In addition to helping to explain why species do not go extinct when faced with this lethal fungal pathogen, many new antimicrobial chemicals have been discovered, some of which may be useful in human medicine. The amphibians' defensive secretions are important, but they may not be the whole story.

Can bacteria help?

Plants and animals, including humans, harbor a vast diversity and quantity of bacteria. Within and on our bodies, we are outnumbered by bacterial cells 10 to 1. It is increasingly recognized that these microbes play an often beneficial role in maintaining health. For example, bacteria in our digestive tracks are helpful in digestion and in preventing pathogenic bacteria from successfully colonizing or multiplying to high numbers. It turns out that amphibian skin hosts a diverse group of bacteria, many of which cannot even be cultured by current techniques. When a fungal pathogen arrives on an amphibian, it does not find a "blank slate" of skin. Rather, the pathogen's dispersal stage, which is a single cell, finds the amphibian's skin bacteria already in residence. We are exploring whether aspects of amphibians' skin bacteria can help prevent infection and death by the fungal pathogen.

In one study, we have collaborated with ecologists at the University of California at Berkeley on the threatened mountain yellow-legged frog, Rana muscosa. Populations in the northern part of its range in the high Sierra Nevada Mountains persist with the fungal pathogen, while those farther south along the ridge top succumb to the pathogen. In this case, the defensive chemical secretions did not differ between persisting populations and those predicted to go extinct once the pathogen arrives. Members of my laboratory, Mary Alice Simon and Brianna Lam, cultured skin bacteria from these frogs from both northern and more southern populations. We then grew the bacteria with the pathogen in laboratory challenge assays. Our results indicated that quite a few species of skin bacteria produced antifungal chemicals that killed the fungal pathogen. It turned out that members of the northern population that persists with the pathogen had a higher proportion of individuals with at least one antifungal species of skin bacteria. We were very excited about these results because they indicated that amphibians' skin bacteria might provide protection against fungal disease and opened up the possibility that perhaps antifungal bacteria could be added to amphibians as a means of protection.

Experiments and encouraging results

We decided to test the idea that adding an antifungal bacteria species to the skin would protect amphibians from B. dendrobatidis. Members of my laboratory, Antje Lauer, Jenifer Banning, Emily Andre and Karen Duncan, used a very common salamander species for this laboratory experiment because we wanted to work out the methods and see if our concept had any validity before moving on to a threatened species. Control groups were employed in the experimental design, such as having salamanders exposed to beneficial antifungal bacteria and not to the pathogen. However, our main interest was in comparing individuals exposed to the pathogen with individuals inoculated with beneficial bacteria before being exposed to the pathogen. We found that we were successful in getting the beneficial bacteria to colonize the skin, at least in the short term. We also found that this common salamander species did not die when exposed to the pathogen, indicating that it is one of the species that does not succumb to the fungus. However, we found that individuals exposed to the pathogen did lose about 30 percent of their body mass over a month-and-a-half, which is a symptom of the fungal disease. Those individuals inoculated first with beneficial bacteria before exposure to the fungus lost much less weight, which meant that the bacteria helped to prevent a symptom of the disease. This result is the first indication that we might be able to manage the disease, although certainly much more testing is necessary before field trials are conducted.

Given the encouraging results from this experiment, we are soon going to repeat the experiment with the mountain yellow-legged frog from the high Sierra Nevada Mountains in California. This species can be strongly affected by the disease and does die from the disease in laboratory trials. If we obtain results that suggest that the addition of beneficial bacteria prevents death from the fungal pathogen, then field trials would be a logical next step. The fungal pathogen in California moves in predictable directions, so we might be able to inoculate the frogs in the path of the disease and halt it. The beneficial bacteria already occur on the skins of some individuals in the populations, so we would not be adding new species to the ecosystem. Government officials would have the final say on how and when to proceed. We are cautiously optimistic that our ecological study of the interaction between skin bacteria of amphibians and their lethal skin fungal pathogen will give us a workable tool to manage and halt this horrible epidemic.

About the author
JMU biology professor Reid Harris' article appeared in the Fall 2007 Department of Biology newsletter Living Connection.

Read more about the research in Madison Scholar's "A Silver Bullet?"