Abstract
Host-microbe symbioses rely on the successful transmission or acquisition of symbionts in each new generation. Amphibians host a diverse cutaneous microbiota, and many of these symbionts appear to be mutualistic and may limit infection by the chytrid fungus, Batrachochytrium dendrobatidis, which has caused global amphibian population declines and extinctions in recent decades. Using bar-coded 454 pyrosequencing of the 16S rRNA gene, we addressed the question of symbiont transmission by examining variation in amphibian skin microbiota across species and sites and in direct relation to environmental microbes. Although acquisition of environmental microbes occurs in some host-symbiont systems, this has not been extensively examined in free-living vertebrate-microbe symbioses. Juvenile bullfrogs (Rana catesbeiana), adult red-spotted newts (Notophthalmus viridescens), pond water and pond substrate were sampled at a single pond to examine host-specificity and potential environmental transmission of microbiota. To assess population level variation in skin microbiota, adult newts from two additional sites were also sampled. Cohabiting bullfrogs and newts had distinct microbial communities, as did newts across the three sites. The microbial communities of amphibians and the environment were distinct; there was very little overlap in the amphibians’ core microbes and the most abundant environmental microbes, and the relative abundances of OTUs that were shared by amphibians and the environment were inversely related. These results suggest that, in a host species-specific manner, amphibian skin may select for microbes that are generally in low abundance in the environment.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Amato KR, Yeoman CJ, Kent A, Righini N, Carbonero F, Estrada A et al. (2013). Habitat degradation impacts black howler monkey (Alouatta pigra) gastrointestinal microbiomes. ISME J 7: 1344–1353.
Anderson MJ . (2001). A new method for non-parametric multivariate analysis of variance. Austral Ecol 26: 32–46.
Banning JL, Weddle AL, Wahl GW, Simon MA, Lauer A, Walters RL et al. (2008). Antifungal skin bacteria, embryonic survival, and communal nesting in four-toed salamanders, Hemidactylium scutatum. Oecologia 156: 423–429.
Becker MH, Harris RN . (2010). Cutaneous bacteria of the redback Salamander prevent morbidity associated with a lethal disease. PLoS One 5: e10957.
Belden LK, Harris RN . (2007). Infectious diseases in wildlife: the community ecology context. Front Ecol Environ 10: 533–539.
Berger L, Speare R, Daszak P, Green DE, Cunningham AA, Goggin CL et al. (1998). Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proc Natl Acad Sci USA 95: 9031–9036.
Bletz MC, Loudon AH, Becker MH, Bell SC, Woodhams DC, Minbiole KPC et al. (2013). Mitigating amphibian chytridiomycosis with bioaugmentation: characteristics of effective probiotics and strategies for their selection and use. Ecol Lett 16: 807–820.
Bokulich NA, Subramanian S, Faith JJ, Gevers D, Gordon JI, Knight R et al. (2013). Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat Methods 10: 57–59.
Bray JR, Curtis JT . (1957). An ordination of upland forest communities of southern Wisconsin. Ecol Monogr 27: 325–349.
Briggs CJ, Vredenburg VT, Knapp RA, Rachowicz LJ . (2005). Investigating the population-level effects of chytriomycosis: an emerging infectious disease of amphibians. Ecology 86: 3149–3159.
Bright M, Bulgheresi S . (2010). A complex journey: transmission of microbial symbionts. Nat Rev Microbiol 8: 218–230.
Bulgheresi S . (2011). Microbial symbiont transmission: basic principles and dark sides. In: Rosenberg E and Gophna U (eds) Beneficial Microorganisms in Multicellular Life Forms. Springer: Berlin, Heidelberg, Germany, pp 299-311.
Caporaso JG, Bittinger K, Bushman FD, DeSantis TZ, Andersen GL, Knight R . (2010a). PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics 26: 266–267.
Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK et al. (2010b). QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7: 335–336.
Conlon JM . (2011). The contribution of skin antimicrobial peptides to the system of innate immunity in anurans. Cell Tissue Res 343: 201–212.
Costello EK, Lauber CL, Hamady M, Fierer N, Gordon JI, Knight R . (2009). Bacterial community variation in human body habitats across space and time. Science 326: 1694–1697.
Culp CE, Falkinham JO, Belden LK . (2007). Identification of the natural bacterial microflora on the skin of eastern newts, bullfrog tadpoles and redback salamanders. Herpetologica 63: 66–71.
Damiani C, Ricci I, Crotti E, Rossi P, Rizzi A, Scuppa P et al. (2008). Paternal transmission of symbiotic bacteria in malaria vectors. Curr Biol 18: R1087–R1088.
Daszak P, Strieby A, Cunningham AA, Longcore JE, Brown CC, Porter D . (2004). Experimental evidence that the bullfrog (Rana catesbeiana) is a potential carrier of chytridiomycosis, an emerging fungal disease of amphibians. Herpetol J 14: 201–207.
Daum JM, Davis LR, Bigler L, Woodhams DC . (2012). Hybrid advantage in skin peptide immune defenses of water frogs (Pelophylax esculentus) at risk from emerging pathogens. Infect Genet Evol 12: 1854–1864.
DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K et al. (2006). Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 72: 5069–5072.
Dethlefsen L, McFall-Ngai M, Relman DA . (2007). An ecological and evolutionary perspective on human–microbe mutualism and disease. Nature 449: 811–818.
Edgar RC . (2010). Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26: 2460–2461.
Ewald PW . (1987). Transmission modes and evolution of the parasitism-mutualism continuum. Ann N Y Acad Sci 503: 295–306.
Fierer N, Hamady M, Lauber CL, Knight R . (2008). The influence of sex, handedness, and washing on the diversity of hand surface bacteria. Proc Natl Acad Sci USA 105: 17994–17999.
Grice EA, Kong HH, Conlan S, Deming CB, Davis J, Young AC et al. (2009). Topographical and temporal diversity of the human skin microbiome. Science 324: 1190–1192.
Harris RN, James TY, Lauer A, Simon MA, Patel A . (2006). Amphibian pathogen Batrachochytrium dendrobatidis is inhibited by the cutaneous bacteria of amphibian species. Ecohealth 3: 53–56.
Harris RN, Brucker RM, Walke JB, Becker MH, Schwantes CR, Flaherty DC et al. (2009a). Skin microbes on frogs prevent morbidity and mortality caused by a lethal skin fungus. ISME J 3: 818–824.
Harris RN, Lauer A, Simon MA, Banning JL, Alford RA . (2009b). Addition of antifungal skin bacteria to salamanders ameliorates the effects of chytridiomycosis. Dis Aquat Organ 83: 11–16.
Heath-Heckman EAC, Peyer SM, Whistler CA, Apicella MA, Goldman WE, McFall-Ngai MJ . (2013). Bacterial bioluminescence regulates expression of a host cryptochrome gene in the squid-Vibrio symbiosis. mBio 4 2: e00167–13.
Hong P-Y, Wheeler E, Cann IKO, Mackie RI . (2011). Phylogenetic analysis of the fecal microbial community in herbivorous land and marine iguanas of the Galápagos Islands using 16S rRNA-based pyrosequencing. ISME J 5: 1461–1470.
Hosokawa T, Kikuchi Y, Fukatsu T . (2007). How many symbionts are provided by mothers, acquired by offspring, and needed for successful vertical transmission in an obligate insect-bacterium mutualism? Mol Ecol 16: 5316–5325.
Jones KM, Kobayashi H, Davies BW, Taga ME, Walker GC . (2007). How rhizobial symbionts invade plants: the Sinorhizobium–Medicago model. Nat Rev Microbiol 5: 619–633.
Kaltenpoth M, Engl T . (2013). Defensive microbial symbionts in Hymenoptera. Funct Ecol 28: 315–327.
Kikuchi Y, Hosokawa T, Fukatsu T . (2007). Insect-microbe mutualism without vertical transmission: a stinkbug acquires a beneficial gut symbiont from the environment every generation. Appl Environ Microbiol 73: 4308–4316.
Kruskal JB . (1964). Multidimensional scaling by optimizing goodness of fit to a nonmetric hypothesis. Psychometrika 29: 1–27.
Lam BA, Walke JB, Vredenburg VT, Harris RN . (2010). Proportion of individuals with anti-Batrachochytrium dendrobatidis skin bacteria is associated with population persistence in the frog Rana muscosa. Biol Conserv 143: 529–531.
Lauer A, Simon MA, Banning JL, André E, Duncan K, Harris RN . (2007). Common cutaneous bacteria from the eastern red-backed salamander can inhibit pathogenic fungi. Copeia 2007: 630–640.
Lauer A, Simon MA, Banning JL, Lam BA, Harris RN . (2008). Diversity of cutaneous bacteria with antifungal activity isolated from female four-toed salamanders. ISME J 2: 145–157.
Lauzon CR, McCombs SD, Potter SE, Peabody NC . (2009). Establishment and vertical passage of Enterobacter (Pantoea) agglomerans and Klebsiella pneumoniae through all life stages of the Mediterranean Fruit Fly (Diptera: Tephritidae). Ann Entomol Soc Am 102: 85–95.
Lee OO, Qian P-Y . (2004). Potential control of bacteria epibiosis on the surface of the sponge Mycale adhaerens. Aquat Microb Ecol 34: 11–21.
Ley RE, Hamady M, Lozupone C, Turnbaugh PJ, Ramey RR, Bircher JS et al. (2008a). Evolution of mammals and their gut microbes. Science 320: 1647–1651.
Ley RE, Lozupone CA, Hamady M, Knight R, Gordon JI . (2008b). Worlds within worlds: evolution of the vertebrate gut microbiota. Nat Rev 6: 776–788.
Lillywhite HB, Licht P . (1975). A comparative study of integumentary mucous secretions in amphibians. Comp Biochem Physiol A Physiol 51: 937–941.
Lips KR, Brem F, Brenes R, Reeve JD, Alford RA, Voyles J et al. (2006). Emerging infectious disease and the loss of biodiversity in a Neotropical amphibian community. Proc Natl Acad Sci USA 103: 3165–3170.
Longcore JE, Pessier AP, Nichols DK . (1999). Batrachochytrium dendrobatidis gen et sp nov, a chytrid pathogenic to amphibians. Mycologia 91: 219–227.
Lozupone C, Lladser ME, Knights D, Stombaugh J, Knight R . (2011). UniFrac: an effective distance metric for microbial community comparison. ISME J 5: 169–172.
McKenzie VJ, Bowers RM, Fierer N, Knight R, Lauber CL . (2011). Co-habiting amphibian species harbor unique skin bacterial communities in wild populations. ISME J 6: 588–596.
Meyer EA, Cramp RL, Bernal MH, Franklin CE . (2012). Changes in cutaneous microbial abundance with sloughing: possible implications for infection and disease in amphibians. Dis Aquat Organ 101: 235–242.
Moran NA . (2003). Tracing the evolution of gene loss in obligate bacterial symbionts. Curr Opin Microbiol 6: 512–518.
Muegge BD, Kuczynski J, Knights D, Clemente JC, González A, Fontana L et al. (2011). Diet drives convergence in gut microbiome functions across mammalian phylogeny and within humans. Science 332: 970–974.
Muletz CR, Myers JM, Domangue RJ, Herrick JB, Harris RN . (2012). Soil bioaugmentation with amphibian cutaneous bacteria protects amphibian hosts from infection by Batrachochytrium dendrobatidis. Biol Conserv 152: 119–126.
Myers JM, Ramsey JP, Blackman AL, Nichols AE, Minbiole KPC, Harris RN . (2012). Synergistic inhibition of the lethal fungal pathogen Batrachochytrium dendrobatidis: The combined effects of symbiotic bacterial metabolites and antimicrobial peptides of the frog Rana muscosa. J Chem Ecol 38: 958–965.
Nayak SK . (2010). Role of gastrointestinal microbiota in fish. Aquac Res 41: 1553–1573.
Nyholm SV, Stabb EV, Ruby EG, McFall-Ngai MJ . (2000). Establishment of an animal-bacterial association: Recruiting symbiotic vibrios from the environment. Proc Natl Acad Sci USA 97: 10231–10235.
Nyholm SV, McFall-Ngai M . (2004). The winnowing: establishing the squid-vibrio symbiosis. Nat Rev Microbiol 2: 632–642.
Oksanen J, Blanchet FG, Kindt R, Legendre P, Minchin PR, O'Hara RB et al. (2013). vegan: Community Ecology Package. R package version 2.0-7 http://CRAN.R-project.org/package=vegan.
Oliveros JC . (2007). VENNY. An interactive tool for comparing lists with Venn Diagrams http://bioinfogp.cnb.csic.es/tools/venny/index.html.
Reeder J, Knight R . (2010). Rapidly denoising pyrosequencing amplicon reads by exploiting rank-abundance distributions. Nat Methods 7: 668–669.
Retallick RWR, McCallum H, Speare R . (2004). Endemic infection of the amphibian chytrid fungus in a frog community post-decline. PLoS Biol 2: e351.
Roeselers G, Mittge EK, Stephens WZ, Parichy DM, Cavanaugh CM, Guillemin K et al. (2011). Evidence for a core gut microbiota in the zebrafish. ISME J 5: 1595–1608.
Rosenberg E, Koren O, Reshef L, Efrony R, Zilber-Rosenberg I . (2007). The role of microorganisms in coral health, disease and evolution. Nat Rev Microbiol 5: 355–362.
Sachs JL, Essenberg CJ, Turcotte MM . (2011a). New paradigms for the evolution of bacterial infections. Trends Ecol Evol 26: 202–209.
Sachs JL, Skophammer RG, Regus JU . (2011b). Evolutionary transitions in bacterial symbiosis. Proc Natl Acad Sci USA 108: 10800–10807.
Schmitt S, Angermeier H, Schiller R, Lindquist N, Hentschel U . (2008). Molecular microbial diversity survey of sponge reproductive stages and mechanistic insights into vertical transmission of microbial symbionts. Appl Environ Microbiol 74: 7694–7708.
Searle CL, Gervasi SS, Hua J, Hammond JI, Relyea RA, Olson DH et al. (2011). Differential Host Susceptibility to Batrachochytrium dendrobatidis, an Emerging Amphibian Pathogen. Conserv Biol 25: 965–974.
Skerratt LF, Berger L, Speare R, Cashins S, McDonald KR, Phillott AD et al. (2007). Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs. EcoHealth 4: 125–134.
Sullam KE, Essinger SD, Lozupone CA, O’Connor MP, Rosen GL, Knight R et al. (2012). Environmental and ecological factors that shape the gut bacterial communities of fish: a meta-analysis. Mol Ecol 21: 3363–3378.
Tennessen JA, Woodhams DC, Chaurand P, Reinert LK, Billheimer D, Shyr Y et al. (2009). Variations in the expressed antimicrobial peptide repertoire of northern leopard frog (Rana pipiens) populations suggest intraspecies differences in resistance to pathogens. Dev Comp Immunol 33: 1247–1257.
Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI . (2006). An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444: 1027–1031.
Walke JB, Harris RN, Reinert LK, Rollins-Smith LA, Woodhams DC . (2011). Social immunity in amphibians: evidence for vertical transmission of innate defenses. Biotropica 43: 396–400.
Wang Q, Garrity GM, Tiedje JM, Cole JR . (2007). Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73: 5261–5267.
Webster NS, Taylor MW, Behnam F, Lücker S, Rattel T, Whalan S et al. (2010). Deep sequencing reveals exceptional diversity and modes of transmission for bacterial sponge symbionts. Environ Microbiol 12: 2070–2082.
Wenzel M, Schönig I, Berchtold M, Kämpfer P, König H . (2002). Aerobic and facultatively anaerobic cellulolytic bacteria from the gut of the termite Zootermopsis angusticollis. J Appl Microbiol 92: 32–40.
Woodhams DC, Rollins-Smith LA, Carey C, Reinert L, Tyler MJ, Alford RA . (2006a). Population trends associated with skin peptide defenses against chytridiomycosis in Australian frogs. Oecologia 146: 531–540.
Woodhams DC, Voyles J, Lips KR, Carey C, Rollins-Smith LA . (2006b). Predicted disease susceptibility in a panamanian amphibian assemblage based on skin peptide defenses. J Wildlife Dis 42: 207–218.
Woodhams DC, Ardipradja K, Alford RA, Marantelli G, Reinert LK, Rollins-Smith LA . (2007a). Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses. Animal Conserv 10: 409–417.
Woodhams DC, Vredenburg VT, Simon MA, Billheimer D, Shakhtour B, Shyr Y et al. (2007b). Symbiotic bacteria contribute to innate immune defenses of the threatened mountain yellow-legged frog Rana muscosa. Biol Conserv 138: 390–398.
Woodhams DC, Kenyon N, Bell SC, Alford RA, Chen S, Billheimer D et al. (2010). Adaptations of skin peptide defences and possible response to the amphibian chytrid fungus in populations of Australian green-eyed treefrogs Litoria genimaculata. Divers Distrib 16: 703–712.
Acknowledgements
We thank the University of Virginia’s Mountain Lake Biological Station, Virginia Tech’s Kentland Farm, and the Jefferson National Forest for allowing us to conduct research on their lands. We also thank K. Walke for field assistance, and V. McKenzie for providing us with access to the sequence files from her 2011 study. This research was funded by the Morris Animal Foundation, the Fralin Life Science Institute at Virginia Tech and the National Science Foundation (DEB-1136640).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Rights and permissions
About this article
Cite this article
Walke, J., Becker, M., Loftus, S. et al. Amphibian skin may select for rare environmental microbes. ISME J 8, 2207–2217 (2014). https://doi.org/10.1038/ismej.2014.77
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/ismej.2014.77
Keywords
This article is cited by
-
First Report of Culturable Skin Bacteria in Melanophryniscus admirabilis (Admirable Redbelly Toad)
Microbial Ecology (2023)
-
A Variety of Fungal Species on the Green Frogs’ Skin (Pelophylax esculentus complex) in South Banat
Microbial Ecology (2023)
-
Signatures of functional bacteriome structure in a tropical direct-developing amphibian species
Animal Microbiome (2022)
-
Environment is associated with chytrid infection and skin microbiome richness on an amphibian rich island (Taiwan)
Scientific Reports (2022)
-
Microbial isolates with Anti-Pseudogymnoascus destructans activities from Western Canadian bat wings
Scientific Reports (2022)
