Symbiotic bacteria contribute to innate immune defenses of the threatened mountain yellow-legged frog, Rana muscosa

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Abstract

Symbiotic microorganisms influence health and disease and may contribute to the innate immune defenses of amphibians. The mountain yellow-legged frog, Rana muscosa, is currently undergoing unprecedented population declines. One cause of recent declines is the pathogenic chytrid fungus, Batrachochytrium dendrobatidis (Bd). Skin swabs for detection of Bd, skin peptide secretions, and symbiotic skin bacteria were collected from 70 adult R. muscosa from two populations designated “Sixty Lake” and “Conness” in 2004–2005. The Conness population has persisted with the presence of Bd for at least 6 years whereas the Sixty Lake population is newly infected and declining. Of the frogs sampled at Conness, 67.5% were infected; whereas 96.7% of the Sixty Lake frogs were infected. Sixty Lake frogs were also more intensely infected than frogs at Conness. We isolated symbiotic bacteria that may contribute to immune defense. A significantly greater proportion of individuals with at least one anti-Bd bacterial species present were found at Conness (85%) than at Sixty Lake (62%). We observed no apparent differences in total skin peptides recovered; however, peptide mixtures from frogs at Sixty Lake showed better growth inhibitory activity against Bd than peptides from frogs at Conness. By MALDI-TOF MS analysis, there were no differences between the two populations in the previously described antimicrobial peptides (ranatuerin-2Ma, ranatuerin-2Mb, and temporin-1M). Antimicrobial skin peptides are only one factor in the resistance of R. muscosa to Bd infection. We suggest that symbiotic bacteria with the ability to persist in the presence of mucosal peptides may inhibit infection and colonization of the skin by Bd and increase the effectiveness of innate defense mechanisms in the skin.

Introduction

The mountain yellow-legged frog, Rana muscosa, is native to cold and remote lakes in the California Sierra Nevada mountains. This once very abundant species is currently undergoing unprecedented population declines (Bradford, 1991, Drost and Fellers, 1996, Sparling et al., 2001, Vredenburg et al., 2005). Although previous declines were attributed to the introduction of non-native trout (Bradford, 1989, Bradford et al., 1993, Knapp and Matthews, 2000, Vredenburg, 2004) or to pesticides (Davidson, 2004, Fellers et al., 2004), recent declines have continued even in apparently unpolluted fishless lakes. One cause of recent declines is the pathogenic chytrid fungus, Batrachochytrium dendrobatidis (Bd) (Briggs et al., 2005, Rachowicz et al., 2006). This fungus is associated with amphibian population declines on several continents (Berger et al., 1998, Waldman et al., 2001, Weldon et al., 2004, Garner et al., 2005, Lips et al., 2006). The factors that lead to extinction or to persistence of amphibian populations with Bd are currently under examination (Briggs et al., 2005, Bosch and Martinez-Solano, 2006, Woodhams et al., 2006a, Woodhams et al., 2006b).

One factor that may contribute to disease resistance is innate immune defense. Innate skin defenses including antimicrobial peptides and symbiotic microbial barriers may be crucial for defending amphibians against the skin-invasive fungus, Bd (Harris et al., 2006, Woodhams et al., 2006a, Woodhams et al., 2006b, Lauer et al., in press). Granular glands in the skin of R. muscosa secrete a mixture of peptides including antimicrobial peptides (ranatuerin-2Ma, 2Mb, and temporin-1M). These peptides inhibit the growth of Bd in vitro (Rollins-Smith et al., 2006). Symbiotic bacteria of amphibians are not well-known and have not been previously described in R. muscosa. However, the salamanders Plethodon cinereus and Hemidactylium scutatum host beneficial bacteria that inhibit growth of Bd in vitro and may be important for resisting Bd colonization of skin (Harris et al., 2006, Lauer et al., in press). Here we describe the microbiota of R. muscosa that contribute to resistance against Bd, and suggest that populations which host beneficial bacteria may be more likely to persist with Bd.

Section snippets

Study species and sites

Adult R. muscosa were sampled from two populations in August, 2005. These populations were Lake 11 (elevation 3390 m, depth 3.5 m) in Sixty Lake Basin, Kings Canyon National Park, California (36.82°N, 118.43°W) and Conness (elevation 3175 m, depth 3.9 m) in Yosemite National Park, California (37.97°N, 119.34°W). Populations were in similar high alpine habitats and presumably experienced similar environmental conditions. Using a hand net, we sampled 30 adults from the Sixty Lake site on August

Assessment of Bd infection status

The prevalence and intensity of infection with Bd differed between the two populations. Of the Conness frogs, 67.5% were infected, whereas 96.7% of the Sixty Lake frogs were infected (Fisher’s exact test, p = 0.0024). Infections of frogs at Sixty Lake (median = 6723.9 zoospore equivalents) were significantly more intense than infections at Conness (median = 9.5 zoospore equivalents) (Mann–Whitney U-test, Z = 6.583, p < 0.0001). Of the infected frogs only, the infection intensity of the Sixty Lake

Batrachochytrium dendrobatidis and population declines of R. muscosa

The emerging infectious disease, chytridiomycosis, is linked to population declines and extinctions of R. muscosa (Briggs et al., 2005). The prevalence of Bd infection varies among sites; frogs can persist with the fungus at some sites but not others. For example, the disease history of Sixty Lake and Conness populations with respect to chytridiomycosis is quite different. The Conness population has persisted in the presence of Bd for about 6 years whereas individuals from the Sixty Lake

Acknowledgements

The authors thank J. Morgan, M. Stice, and T. Tunstall for field assistance and B.A. Lam for assistance in the laboratory. L.A. Rollins-Smith was funded by an Integrated Research Challenges in Environmental Biology (IRCEB) grant IBN-9977063 (J.P. Collins, P.I.) and grant IOB-0520847 from the U.S. National Science Foundation. R.N. Harris was funded by NSF grant 0413981, and C.J. Briggs was funded by the NIH/NSF Ecology of Infectious Disease Program grant R01 ES12067 from the National Institute

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