Abstract
Host-associated microbiomes are increasingly recognized to contribute to host disease resistance; the temporal dynamics of their community structure and function, however, are poorly understood. We investigated the cutaneous bacterial communities of three newt species, Ichthyosaura alpestris, Lissotriton vulgaris and Triturus cristatus, at approximately weekly intervals for 3 months using 16S ribosomal RNA amplicon sequencing. We hypothesized cutaneous microbiota would vary across time, and that such variation would be linked to changes in predicted fungal-inhibitory function. We observed significant temporal variation within the aquatic phase, and also between aquatic and terrestrial phase newts. By keeping T. cristatus in mesocosms, we demonstrated that structural changes occurred similarly across individuals, highlighting the non-stochastic nature of the bacterial community succession. Temporal changes were mainly associated with fluctuations in relative abundance rather than full turnover of bacterial operational taxonomic units (OTUs). Newt skin microbe fluctuations were not correlated with that of pond microbiota; however, a portion of community variation was explained by environmental temperature. Using a database of amphibian skin bacteria that inhibit the pathogen Batrachochytrium dendrobatidis (Bd), we found that the proportion of reads associated with ‘potentially’ Bd-inhibitory OTUs did not vary temporally for two of three newt species, suggesting that protective function may be maintained despite temporal variation in community structure.
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References
Allison SD, Martiny JBH . (2006) Resistance, resilience, and redundancy in microbial communities. In: Avise JC, Hubbel SP, Ayala FJ (eds). Vol. II. In the Light of Evolution. The National Academies Press: Washington DC, USA, pp 11512–11519.
Altizer S, Dobson A, Hosseini P, Hudson P, Pascual M, Rohani P . (2006). Seasonality and the dynamics of infectious diseases. Ecol Lett 9: 467–484.
Antwis RE, Preziosi RF, Harrison XA, Garner TW . (2015). Amphibian symbiotic bacteria do not show universal ability to inhibit growth of the global pandemic lineage of Batrachochytrium dendrobatidis. Appl Environ Microbiol 81: 3706–3711.
Bates D, Maechler M, Bolker M, Walker S . (2015). Fitting Linear Mixed-Effects Models Using lme4. Journal of Statistical Software 67: 1–48.
Becker MH, Brucker RM, Schwantes CR, Harris RN, Minbiole KPC . (2009). The bacterially produced metabolite violacein is associated with survival of amphibians infected with a lethal fungus. Appl Environ Microbiol 75: 6635–6638.
Becker MH, Harris RN . (2010). Cutaneous bacteria of the redback salamander prevent morbidity associated with a lethal disease. PLoS One 5: e10957.
Becker MH, Walke JB, Cikanek S, Savage AE, Mattheus N, Santiago CN et al. (2015). Composition of symbiotic bacteria predicts survival in Panamanian golden frogs infected with a lethal fungus. Proc R Soc B 282: 20142881.
Belden LK, Hughey MC, Rebollar EA, Umile TP, Loftus SC, Burzynski EA et al. (2015). Panamanian frog species host unique skin bacterial communities. Front Microbiol 6: 1171.
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.
Blooi M, Pasmans F, Longcore JE, Spitzen-Van Der Sluijs A, Vercammen F, Martel A . (2013). Duplex real-time PCR for rapid simultaneous detection of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans in amphibian samples. J Clin Microbiol 51: 4173–4177.
Bokulich NA, Subramanian S, Faith JJ, Gevers D, Gordon I, Knight R et al. (2013). Quality-filtering vastly improves diversity estimates from illumina amplicon sequencing. Nat Methods 10: 57–59.
Burke C, Steinberg P, Rusch DB, Kjelleberg S, Thomas T . (2011). Bacterial community assembly based on functional genes rather than species. Proc Natl Acad Sci USA 108: 14288–14293.
Bustamante HM, Livo LJ, Carey C . (2010). Effects of temperature and hydric environment on survival of the Panamanian golden frog infected with a pathogenic chytrid fungus. Integr Zool 5: 143–153.
Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK et al. (2010). QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7: 335–336.
Caporaso JG, Lauber CL, Costello EK, Berg-Lyons D, Gonzalez A, Stombaugh J et al. (2011). Moving pictures of the human microbiome. Genome Biol 12: R50.
Carrino-Kyker SR, Swanson AK . (2008). Temporal and spatial patterns of eukaryotic and bacterial communities found in vernal pools. Appl Environ Microbiol 74: 2554–2557.
Cheatsazan H, de Almedia APLG, Russell AF, Bonneaud C . (2013). Experimental evidence for a cost of resistance to the fungal pathogen, Batrachochytrium dendrobatidis, for the palmate newt, Lissotriton helveticus. BMC Ecol 13: 27.
Chung H, Pamp J, Hill JA, Surana NK, Edelman SM, Troy EB et al. (2012). Gut immune maturation depends on colonization with a host-specific microbiota. Cell 149: 1578–1593.
Clarke KR, Gorley RN . (2015). PRIMER v7: User Manual/Tutorial. PRIMER-E, Plymouth, p296.
Colombo BM, Scalvenzi T, Benlamara S, Pollet N . (2015). Microbiota and mucosal immunity in amphibians. Front Immunol 6: 1–15.
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.
Costello EK, Stagaman K, Dethlefsen L, Bohannan BJM, Relman DA . (2012). The application of ecological theory toward an understanding of the human microbiome. Science 336: 1255–1262.
Cramp RL, Mcphee RK, Meyer EA, Ohmer ME, Franklin CE . (2014). First line of defence: the role of sloughing in the regulation of cutaneous microbes in frogs. Conserv Biol 2: 1–12.
Crump BC, Hobbie JE . (2005). Synchrony and seasonality in bacterioplankton communities of two temperate rivers. Limnol Oceanogr 50: 1718–1729.
Daskin JH, Bell SC, Schwarzkopf L, Alford RA . (2014). Cool temperatures reduce antifungal activity of symbiotic bacteria of threatened amphibians – Implications for disease management and patterns of decline. PLoS One 9: e100378.
Dethlefsen L, McFall-Ngai M, Relman DA . (2007). An ecological and evolutionary perspective on human–microbe mutualism and disease. Nature 449: 811–818.
Douglas B, Martin M, Ben B, Steve W . (2015). Fitting Linear Mixed-Effects Models Using lme4. J Stat Softw 67: 1–48.
Drechsler A, Bock D, Ortmann D, Steinfartz S . (2010). Ortmann’s funnel trap – a highly efficient tool for monitoring amphibian species. Herpetol Notes 3: 13–21.
Eberl G . (2010). A new vision of immunity: homeostasis of the superorganism. Mucosal Immunol 3: 450–460.
Engel P, Moran NA . (2013). The gut microbiota of insects-diversity in structure and function. FEMS Microbiol Rev 37: 699–735.
Fierer N, Ferrenberg S, Flores GE, González A, Kueneman J, Legg T et al. (2012). From animalicules to an ecosystem: application of ecological concepts to the human microbiome. Annu Rev Ecol Evol Syst 43: 137–155.
Fitzpatrick BM, Allison AL . (2014). Similarity and differentiation between bacteria associated with skin of salamanders (Plethodon jordani and free-living assemblages. FEMS Microbiol Ecol 88: 482–494.
Flechas SV, Sarmiento C, Cardenas ME, Medina EM, Restrepo S, Amezquita A . (2012). Surviving chytridiomycosis: differential anti-Batrachochytrium dendrobatidis activity in bacterial isolates from three lowland species of Atelopus. PLoS One 7: e44832.
Franzenburg S, Walter J, Künzel S, Wang J, Baines JF, Bosch TCG et al. (2013). Distinct antimicrobial peptide expression determines host species-specific bacterial associations. Proc Natl Acad Sci USA 110: E3730–E3738.
Fraune S, Anton-Erxleben F, Augustin R, Franzenburg S, Knop M, Schröder K et al. (2014). Bacteria-bacteria interactions within the microbiota of the ancestral metazoan Hydra contribute to fungal resistance. ISME J 9: 1543–1556.
Frossard A, Gerull L, Mutz M, Gessner MO . (2011). Disconnect of microbial structure and function: enzyme activities and bacterial communities in nascent stream corridors. ISME J 6: 680–691.
Fukami T, Ian A, Wilkie JP, Paulus C, Park D, Roberts A et al. (2010). Assembly history dictates ecosystem functioning: evidence from wood decomposer communities. Ecology 13: 675–684.
Gallo RL, Hooper LV . (2012). Epithelial antimicrobial defence of the skin and intestine. Nat Rev Immunol 12: 503–516.
Grassly NC, Fraser C . (2006). Seasonal infectious disease epidemiology. Proc Biol Sci 273: 2541–2550.
Grice EA, Segre JA . (2011). The skin microbiome. Nat Rev Microbiol 9: 244–253.
Harris RN, Brucker RM, Walke JB, Becker MH, Schwantes CR, Flaherty DC et al. (2009). Skin microbes on frogs prevent morbidity and mortality caused by a lethal skin fungus. ISME J 3: 818–824.
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, 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.
Holden WM, Hanlon SM, Woodhams DC, Chappell TM, Wells HL, Glisson SM et al. (2015). Skin bacteria provide early protection for newly metamorphosed southern leopard frogs (Rana sphenocephala against the frog-killing fungus, Batrachochytrium dendrobatidis. Biol Conserv 187: 91–102.
Jones SE, Cadkin TA, Newton RJ, Mcmahon KD, Lynch RC . (2012). Spatial and temporal scales of aquatic bacterial beta diversity. Front Microbiol 3: 1–10.
Kaiser C, Koranda M, Kitzler B, Fuchslueger L, Schnecker J, Schweiger P et al. (2010). Below ground carbon allocation by trees drives seasonal patterns of extracellular enzyme activities by altering microbial community composition in a beech forest soil. New Phytol 187: 843–858.
Kent AD, Yannarell AC, Rusak JA, Triplett EW, Mcmahon KD . (2007). Synchrony in aquatic microbial community dynamics. ISME J 1: 38–47.
Khosravi A, Mazmanian SK . (2013). Disruption of the gut microbiome as a risk factor for microbial infections. Curr Opin Microbiol 16: 221–227.
Kinney VC, Heemeyer JL, Pessier AP, Lannoo MJ . (2011). Seasonal pattern of Batrachochytrium dendrobatidis infection and mortality in Lithobates areolatus: affirmation of Vredenburg’s ‘10,000 zoospore rule’. PLoS One 6: e16708.
Kohl KD, Yahn J . (2016). Effects of environmental temperature on the gut microbial communities of tadpoles. Environ Microbiol 18: 1–18.
Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD . (2013). Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the Miseq Illumina sequencing platform. Appl Environ Microbiol 79: 5112–5120.
Krediet CJ, Ritchie KB, Alagely A, Teplitski M . (2013). Members of native coral microbiota inhibit glycosidases and thwart colonization of coral mucus by an opportunistic pathogen. ISME J 7: 980–990.
Kueneman JG, Parfrey LW, Woodhams DC, Archer HM, Knight R, McKenzie VJ . (2014). The amphibian skin-associated microbiome across species, space and life history stages. Mol Ecol 23: 1238–1250.
Kueneman JG, Woodhams DC, Harris R, Archer HM, Knight R, Mckenzie VJ . (2016). Probiotic treatment restores protection against lethal fungal infection lost during amphibian captivity. Proc Biol Sci 283: 20161553.
Kueneman JG, Woodhams DC, Van Treuren W, Archer HM, Knight R, McKenzie VJ . (2015). Inhibitory bacteria reduce fungi on early life stages of endangered Colorado boreal toads (Anaxyrus boreas. ISME J 10: 934–944.
Küng D, Bigler L, Davis LR, Gratwicke B, Griffith E, Woodhams DC . (2014). Stability of microbiota facilitated by host immune regulation: informing probiotic strategies to manage amphibian disease. PLoS One 9: e87101.
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.
Langwig KE, Frick WF, Reynolds R, Parise KL, Drees KP, Hoyt JR et al. (2015). Host and pathogen ecology drive the seasonal dynamics of a fungal diseas, white-nose syndrome. Proc R Soc B 282: 20142335.
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.
Longo AV, Burrowes Pa, Joglar RL . (2010). Seasonality of Batrachochytrium dendrobatidis infection in direct-developing frogs suggests a mechanism for persistence. Dis Aquat Organ 92: 253–260.
Longo AV, Savage AE, Hewson I, Zamudio KR . (2015). Seasonal and ontogenetic variation of skin microbial communities and relationships to natural disease dynamics in declining amphibians. R Soc Open Sci 2: 140377.
Louca S, Jacques SMS, Pires APF, Leal JS, Srivastava DS, Parfrey LW et al. (2016). High taxonomic variability despite stable functional structure across microbial communities. Nat Ecol Evol 1: 15.
Loudon AH, Woodhams DC, Parfrey LW, Archer HM, Knight R, McKenzie V et al. (2014). Microbial community dynamics and effect of environmental microbial reservoirs on red-backed salamanders (Plethodon cinereus. ISME J 8: 830–840.
Marsh PD . (2000). Role of the oral microflora in health. Microb Ecol Health Dis 12: 130–137.
Martel A, Blooi M, Adriaensen C, Rooij P, Van, Beukema W, Fisher MC et al. (2014). Recent introduction of a chytrid fungus endangers western Palearctic salamanders. Science 6209: 630–631.
Martel A, Spitzen-van der Sluijs A, Blooi M, Bert W, Ducatelle R, Fisher MC et al. (2013). Batrachochytrium salamandrivorans sp. nov. causes lethal chytridiomycosis in amphibians. Proc Natl Acad Sci USA 110: 15325–15329.
McKenzie VJ, Bowers RM, Fierer N, Knight R, Lauber CL . (2012). 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.
Nemergut DR, Schmidt SK, Fukami T, O’Neill SP, Bilinski TM, Stanish LF et al. (2013). Patterns and processes of microbial community assembly. Microbiol Mol Biol Rev 77: 342–356.
Perrotta I, Sperone E, Bernabo I, Tripepi S, Brunelli E . (2012). The shift from aquatic to terrestrial phenotype in Lissotriton italicus: larval and adult remodelling of the skin. Zoology 115: 170–178.
Phillott AD, Grogan LF, Cashins SD, McDonald KR, Berger L, Skerratt LF . (2013). Chytridiomycosis and seasonal mortality of tropical stream-associated frogs 15 years after introduction of Batrachochytrium dendrobatidis. Conserv Biol 27: 1058–1068.
Portillo MC, Anderson SP, Fierer N . (2012). Temporal variability in the diversity and composition of stream bacterioplankton communities. Environ Microbiol 14: 2417–2428.
Purahong W, Schloter M, Pecyna MJ, Kapturska D, Daumlich V, Mital S et al. (2014). Uncoupling of microbial community structure and function in decomposing litter across beech forest ecosystems in Central Europe. Sci Rep 4: 7014.
R Core Team. (2016). R: A language and environment for statistical computing. R Foundation for Statistical Computing. Vienna, Austria. URL. https://www.R-project.org/.
Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R . (2004). Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 118: 229–241.
Rebollar EA, Hughey MC, Medina D, Harris RN, Ibáñez R, Belden LK . (2016). Skin bacterial diversity of Panamanian frogs is associated with host susceptibility and presence of Batrachochytrium dendrobatidis. ISME J 10: 1682–1695.
Robinson CJ, Bohannan BJM, Young VB . (2010). From structure to function: the ecology of host-associated microbial communities. Microbiol Mol Biol Rev 74: 453–476.
Rohr JR, Raffel TR, Romansic JM, McCallum H, Hudson PJ . (2008). Evaluating the links between climate, disease spread, and amphibian declines. Proc Natl Acad Sci USA 105: 17436–17441.
Rollins-Smith LA . (2009). The role of amphibian antimicrobial peptides in protection of amphibians from pathogens linked to global amphibian declines. Biochim Biophys Acta 1788: 1593–1599.
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.
Rosenthal M, Goldberg D, Aiello A, Larson E, Foxman B . (2011). Skin microbiota: microbial community structure and its potential association with health and disease. Infect Genet Evol 11: 839–848.
Sanchez E, Bletz MC, Dunsch L, Bhuju S, Geffers R, Jarek M et al. (2016). Cutaneous bacterial communities of a poisonous salamander: a perspective from life stages, body parts and environmental conditions. Microb Ecol 73: 455–465.
Savage AE, Sredl MJ, Zamudio KR . (2011). Disease dynamics vary spatially and temporally in a North American amphibian. Biol Conserv 144: 1910–1915.
Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS et al. (2011). Metagenomic biomarker discovery and explanation. Genome Biol 12: R60.
Shade A, Caporaso JG, Handelsman J, Knight R, Fierer N . (2013). A meta-analysis of changes in bacterial and archaeal communities with time. ISME J 7: 1493–1506.
Shade A, Peter H, Allison SD, Baho DL, Berga M, Burgmann H et al. (2012). Fundamentals of microbial community resistance and resilience. Front Microbiol 3: 1–19.
Singmann H, Bolker B, Westfall J . (2015). afex: Analysis of Factorial Experiments. R package version 0.13-145.
Spitzen-van der Sluijs A, Martel A, Asselberghs J, Bales EK, Beukema W, Bletz MC et al. (2016). Expanding distribution of lethal amphibian fungus Batrachochytrium salamandrivorans in Europe. Emerg Infect Dis 22: 1286–1288.
Stappenbeck TS, Hooper LV, Gordon JI . (2002). Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells. Proc Natl Acad Sci USA 99: 15451–15455.
Stecher B, Hardt WD . (2008). The role of microbiota in infectious disease. Trends Microbiol 16: 107–114.
Stecher B, Robbiani R, Walker AW, Westendorf AM, Barthel M, Kremer M et al. (2007). Salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota. PLoS Biol 5: 2177–2189.
Strickland MS, Lauber CL, Fierer N, Bradford MA . (2009). Testing the functional significance of microbial community composition. Ecology 90: 441–451.
Vredenburg VT, Briggs CJ, Harris RN . (2011) Host-pathogen dynamics of amphibian chytridiomycosis: the role of the skin microbiome in health and disease. In: Olson L, Choffnes E, Relman D, Pray L . (eds). Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health. National Academy Press: Washington, DC, USA, pp 342–355.
Waldrop MP, Firestone MK . (2006). Response of microbial community composition and function to soil climate change. Microb Ecol 52: 716–724.
Walke JB, Becker MH, Loftus SC, House LL, Cormier G, Jensen RV et al. (2014). Amphibian skin may select for rare environmental microbes. ISME J 8: 2207–2217.
Walter J, Ley R . (2011). The human gut microbiome: ecology and recent evolutionary changes. Annu Rev Microbiol 65: 411–429.
Woodhams DC, Alford RA, Antwis RE, Archer HM, Becker MH, Belden LK et al. (2015). Antifungual isolates database of amphibian skin-associated bacteria and function against emerging fungal pathogens. Ecology 96: 595.
Woodhams DC, Bigler L, Marschang R . (2012). Tolerance of fungal infection in European water frogs exposed to Batrachochytrium dendrobatidis after experimental reduction of innate immune defenses. BMC Vet Res 8: 197.
Woodhams DC, Brandt H, Baumgartner S, Kielgast J, Küpfer E, Tobler U et al. (2014). Interacting symbionts and immunity in the amphibian skin mucosome predict disease risk and probiotic effectiveness. PLoS One 9: e96375.
Woodhams DC, Vredenburg VT, Simon M-A, Billheimer D, Shakhtour B, Shyr Y et al. (2007). Symbiotic bacteria contribute to innate immune defenses of the threatened mountain yellow-legged frog Rana muscosa. Biol Conserv 138: 390–398.
Acknowledgements
We are grateful to the conservation authorities of Helmstedt and Braunschweig for research permits. Special thanks are due to Uwe Kirchberger for continued assistance. This study was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG) to MV (VE247/9-1), and by a fellowship of the Deutscher Akademischer Austauschdienst (DAAD) to MCB. We are indebted to Meike Kondermann for her assistance with laboratory work and Joana Sabino Pinto for performing qPCR reactions.
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Bletz, M., Perl, R., Bobowski, B. et al. Amphibian skin microbiota exhibits temporal variation in community structure but stability of predicted Bd-inhibitory function. ISME J 11, 1521–1534 (2017). https://doi.org/10.1038/ismej.2017.41
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DOI: https://doi.org/10.1038/ismej.2017.41
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