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