By Peter Moon | Agência FAPESP – Researchers at São Paulo State University (UNESP) in Brazil are investigating how deforestation can affect the action of pathogens that cause diseases such as chytridiomycosis, which has devastated frog populations worldwide in recent decades.
In an article published in the Proceedings of the Royal Society of London B – Biological Sciences, the researchers analyze how the interaction between deforestation and skin microbiome can affect amphibians infected by fungi such as Batrachochytrium dendrobatidis, which causes chytridiomycosis.
“There’s a suspicion that it may be harder for this fungus to establish itself and proliferate in an animal whose skin biota is intact,” said Célio Haddad, Full Professor of Vertebrates at UNESP’s Bioscience Institute. The research is part of the Thematic Project “Diversity and conservation of Brazilian amphibians”, with Haddad as principal investigator and funding from FAPESP under its Research Program on Biodiversity Characterization, Conservation, Restoration & Sustainable Use (BIOTA-FAPESP).
The skin microbiome is a kind of ecosystem that hinders the action of invading pathogens. To analyze the skin microbiome of Atlantic Rainforest amphibians that inhabit areas of continuous or degraded forest, the researchers needed to choose a species that was not exclusive to either but lived in both.
It also had to be a species that tolerated the fungus B. dendrobatidis (Bd) to some extent, so that a higher or lower level of tolerance could be associated with the diversity of each individual’s skin microbiome and evaluated according to the type of habitat.
The species they chose was the Golden lesser treefrog (Dendropsophus minutus), with moderate tolerance of the fungus and wide distribution in both continuous areas and fragmented remnants of Atlantic Rainforest.
In 2010, the researchers studied ten populations of D. minutus inhabiting Atlantic Rainforest areas in São Luiz do Paraitinga (São Paulo State) and ten populations inhabiting continuous and degraded Araucaria Rainforest areas in Serra Gaúcha (Rio Grande do Sul).
They sampled approximately 600 individuals and selected 187 for molecular analysis. “We used disposable gloves to handle these individuals and cleaned them with distilled water. We then collected skin swabs, which were stored in sterile flasks,” said another author of the study, Guilherme Becker, then a postdoctoral scholar at UNESP and now a visiting professor at the University of Campinas (UNICAMP) under its Graduate Studies Program in Ecology.
The skin swabs were genetically sequenced. “The process generated a list of the bacteria present in each individual and their abundance. The result was a huge database, as each individual had hundreds of bacteria,” Becker said.
The researchers used statistical techniques to establish correlations and infer patterns from the data. “Our molecular approach enabled us to determine a link between infection load and skin bacterial diversity for each individual. We also generated other diversity indicators from the database, such as the number of bacterial species, their relative abundance, and their phylogenetic diversity,” he explained.
According to Haddad, skin microbiome composition was found to be less diversified in terms of bacterial species and less homogeneous across individuals inhabiting open and degraded areas.
“In continuous closed-canopy areas, on the other hand, microbiome composition was more homogeneous across individuals and more diversified in terms of microorganisms,” he said.
The authors of the study found higher microbiome diversity in frogs from natural forest areas. “Deforestation reduces the diversity of these animals’ skin microbiota, but it’s hard to say categorically whether this impoverishment of the microbiota increases the risk of infection by the fungus,” Becker said.
He added that once an amphibian has been infected by Bd, the number of bacteria increases sharply at first, possibly because attacks by opportunistic bacteria damage the immune system.
“The frogs begin to fall sick. Their skin thickens and is covered by the fungus,” Becker said. “Once they become very sick, bacterial load drops sharply. This is a bad sign. It means the microbiome is in dysbiosis – microbially unbalanced. When the number of bacteria drops dramatically, the amphibian usually dies.”
The ecology of chytridiomycosis is even more complex. The fungus spreads through the environment by means of spores suspended in the water of lakes and rivers.
“This is one of the worst epidemics today. No other vertebrate disease acts like the fungus Bd. It’s a generalist pathogen that proliferates best in natural environments, which is bad news for amphibians. That’s why chytridiomycosis is so devastating,” Becker said.
Endemic to Atlantic Rainforest
Not only known amphibian species but also hundreds of species unknown to science are being decimated by chytridiomycosis. The fungus establishes itself in the skin of sick animals, affecting their respiration and physiology. Bd is endemic to the Brazilian Atlantic Rainforest biome, where it infects countless species with greater or lesser susceptibility.
The susceptibility of amphibians to Bd varies considerably. Some species, like the American bullfrog (Lithobates catesbeianus) are highly tolerant, whereas others are only moderately so. Mortality can reach 100% in many species. The disease is found throughout the Americas, as well as in Europe, Australia, New Zealand, and parts of Africa.
Amphibians have different respiratory systems at different stages of their lives. Tadpoles and larvae use gills to breathe, like fish, and do not leave the water. Adults depend mainly on cutaneous respiration, which may or may not be associated with pulmonary and oral cavity breathing, depending on the species.
Bd attacks keratin, the main structural protein in cutaneous tissue, making the amphibian’s skin impermeable and hindering gas exchange with the environment.
Chytridiomycosis is widespread in the Brazilian Atlantic Rainforest biome, although it is not yet as devastating there as in the forests of Costa Rica, for example, where several species of amphibians have been wiped out by it. Researchers have reported areas free of the disease and full of amphibians in a given year but with none at all a year later.
It is not known why chytridiomycosis is more severe in Costa Rica and is apparently milder among amphibians in the Atlantic Rainforest. It may not always have been thus. Amphibian populations in Atlantic Rainforest areas are known to have declined sharply in the late 1970s.
“In the late 1970s, we observed a peak in the prevalence of the Bd fungus in the skin of amphibians deposited in museums, compared with animals deposited earlier or later. It may have been this fungus that caused the decline in populations of Atlantic Rainforest amphibians at that time. All this coincides with the occurrence of mass declines at the same time in other places, such as the United States, the Andes, and Australia,” Becker said.
The next step in the research project, he added, will entail finding out whether any bacteria in the amphibian microbiome confer greater resistance to the proliferation of Bd in the Atlantic Rainforest. If an agent that combats the fungus can be discovered, it may be possible to formulate probiotics in an attempt to protect endemic amphibian populations that are not yet affected by helping the skin microbiome combat Bd.
The article “Land cover and forest connectivity alter the interactions among host, pathogen and skin microbiome” (doi: 10.1098/rspb.2017.0582) by C. G. Becker, A. V. Longo, C. F. B. Haddad and K. R. Zamudio can be retrieved from: rspb.royalsocietypublishing.org/content/284/1861/20170582.