Article | Published:

Microbiota and skin defense peptides may facilitate coexistence of two sympatric Andean frog species with a lethal pathogen

The ISME Journalvolume 13pages361373 (2019) | Download Citation


Management of hyper-virulent generalist pathogens is an emergent global challenge, yet for most disease systems we lack a basic understanding as to why some host species suffer mass mortalities, while others resist epizootics. We studied two sympatric species of frogs from the Colombian Andes, which coexist with the amphibian pathogen Batrachochytrium dendrobatidis (Bd), to understand why some species did not succumb to the infection. We found high Bd prevalence in juveniles for both species, yet infection intensities remained low. We also found that bacterial community composition and host defense peptides are specific to amphibian life stages. We detected abundant Bd-inhibitory skin bacteria across life stages and Bd-inhibitory defense peptides post-metamorphosis in both species. Bd-inhibitory bacteria were proportionally more abundant in adults of both species than in earlier developmental stages. We tested for activity of peptides against the skin microbiota and found that in general peptides did not negatively affect bacterial growth and in some instances facilitated growth. Our results suggest that symbiotic bacteria and antimicrobial peptides may be co-selected for, and that together they contribute to the ability of Andean amphibian species to coexist with the global pandemic lineage of Bd.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    Zilber-Rosenberg I, Rosenberg E. Role of microorganisms in the evolution of animals and plants: the hologenome theory of evolution. FEMS Microbiol Rev. 2008;32:723–35.

  2. 2.

    Li M, Wang B, Zhang M, Rantalainen M, Wang S, Zhou H, et al. Symbiotic gut microbes modulate human metabolic phenotypes. PNAS. 2008;105:2117–22.

  3. 3.

    Becker MH, Walke JB, Murrill L, Woodhams DC, Reinert LK, Rollins-Smith LA, et al. Phylogenetic distribution of symbiotic bacteria from Panamanian amphibians that inhibit growth of the lethal fungal pathogen Batrachochytrium dendrobatidis. Mol Ecol. 2015a;24:1628–41.

  4. 4.

    Breznak JA, Brune A. Role of microorganisms in the digestion of lignocellulose by termites. J Wild Dis. 2003;39:453–87.

  5. 5.

    Fujimura KE, Slusher NA, Cabana MD, Lynch SV. Role of the gut microbiota in defining human health. Expert Rev Anti Infect Ther. 2010;8:435–54.

  6. 6.

    Fujimura-Yamamoto M, Matsumoto S. Probiotics in prevention of lifestyle disorders. In: Ramakrishna BS, Balakrish Nair G, Takeda Y(eds). Intestinal microbiota and colon cancer. India: Elsevier; 2014. p. 75–83.

  7. 7.

    Gilbert SF, Sapp J, Tauber AI. A symbiotic view of life: we have never been individuals. Q Rev Biol. 2012;87:325–41.

  8. 8.

    McFall-Ngai M, Hadfield MG, Bosch TCG, Carey HV, Domazet-Lošo T, Douglas AE, et al. Animals in a bacterial world, a new imperative for the life sciences. PNAS. 2013;110:3229–36.

  9. 9.

    Dong Y, Manfredini F, Dimopoulos G. Implication of the mosquito midgut microbiota in the defense against malaria parasites. PLoS Pathog. 2009;5(5):e1000423.

  10. 10.

    Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL, et al. Emerging fungal threats to animal, plant and ecosystem health. Nature. 2012;484:186–94.

  11. 11.

    Longcore JE, Pessier AP, Nichols DK. Batrachochytrium dendrobatidis gen. et sp. nov., a chytrid pathogenic to amphibians. Mycologia. 1999;91:219–27.

  12. 12.

    Berger L, Speare R, Daszak P, Green DE, Cunningham AA, Goggin CL, et al. Chytridiomycosis causes amphibian mortality associated with population declines in the rain forest of Australia and Central America. PNAS. 1998;95:9031–6.

  13. 13.

    Fisher MC, Garner TWJ, Walker SF. Global emergence of Batrachochytrium dendrobatidis and amphibian chytridiomycosis in space, time, and host. Annu Rev Microbiol. 2009;63:291–10.

  14. 14.

    Skerratt LF, Berger L, Speare R, Cashins SD, McDonald KR, Philott AD, et al. Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs. EcoHealth. 2007;4:125–34.

  15. 15.

    Wake DB, Vredenburg VT. Are we in the midst of the sixth mass extinction? A view from the world of amphibians. PNAS. 2008;105:11466–73.

  16. 16.

    Daszak P, Strieby A, Cunningham AA, Longcore JE, Brown CC, Porter D. Experimental evidence that the bullfrog (Rana catesbeiana) is a potential carrier of chytridiomycosis, an emerging fungal disease of amphibians. Herpetol J. 2004;14:201–7.

  17. 17.

    Reeder NMM, Pessier AP, Vredenburg VT. A reservoir species for the emerging amphibian pathogen Batrachochytrium dendrobatidis thrives in a landscape decimated by disease. PLoS ONE. 2012;7:e33567.

  18. 18.

    Farrer RA, Weinert LA, Bielby J, Garner TWJ, Balloux F, Clare F, et al. Multiple emergences of genetically diverse amphibian-infecting chytrids include a globalized hypervirulent recombinant lineage. PNAS. 2011;108:18732–6.

  19. 19.

    Harris RN, Brucker RM, Walke JB, Becker MH, Schwantes CR, Flaherty DC, et al. Skin microbes on frogs prevent morbidity and mortality caused by a lethal skin fungus. ISME J. 2009a;3:818–24.

  20. 20.

    Rollins-Smith LA, Woodhams DC, Reinert LK, Vredenburg VT, Briggs CJ, Nielsen PF, et al. Antimicrobial peptide defenses of the mountain yellow-legged frog (Rana muscosa). Dev Comp Immunol. 2006;30:831–42.

  21. 21.

    Rowley JJL, Alford RA. Hot bodies protect amphibians against chytrid infection in nature. Sci Rep. 2013;3.

  22. 22.

    Rollins-Smith LA, Woodhams DC. Amphibian immunity: staying in tune with the environment. In: Demas GE, Nelson RJ(eds). Ecoimmunology. New York, NY: Oxford University Press; 2012. p. 92–143.

  23. 23.

    Woodhams DC, Rollins-Smith LA, Carey C, Reinert LK, Tyler MJ, Aford RA. Populational trend associated with skin peptides defenses against chytridiomycosis in Australian frogs. Oecologia. 2006;146:531–40.

  24. 24.

    Woodhams DC, Ardipradja K, Alford RA, Marantelli G, Reinert LK, Rollins-Smith LA. Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses. Anim Conserv. 2007a;10:409–17.

  25. 25.

    Harris RN, Lauer A, Simon MA, Banning JL, Alford RA. Addition of antifungal skin bacteria to salamanders ameliorates the effects of chytridiomycosis. Dis Aquat Organ. 2009b;83:11–16.

  26. 26.

    Kueneman JG, Woodhams DC, Harris R, Archer HM, Knight R, McKenzie VJ. Probiotic treatment restores protection against lethal fungal infection lost during amphibian captivity. Proc R Soc B. 2016;283:20161553.

  27. 27.

    Woodhams DC, Alford RA, Antwis RE, Archer H, Becker MH, Belden LK, et al. Antifungal isolates database of amphibian skin-associated bacteria and function against emerging fungal pathogens. Ecology. 2015;96:595.

  28. 28.

    Becker MH, Walke JB, Cikanek S, Savage AE, Mattheus N, Santiago CN, et al. Composition of symbiotic bacteria predicts survival in Panamanian golden frogs infected with a lethal fungus. Proc R Soc B. 2015b;282:20142881.

  29. 29.

    Bletz MC, Loudon AH, Becker MH, Bell SC, Woodhams DC, Minbiole KPC, et al. Mitigating amphibian chytridiomycosis with bioaugmentation: characteristics of effective probiotics and strategies for their selection and use. Ecol Lett. 2013;16:807–20.

  30. 30.

    Woodhams DC, Bosch J, Briggs CJ, Cashins SD, Davis LR, Lauer A, et al. Mitigating amphibian diseases: strategies to maintain wild populations and control chytridiomycosis. Front Zool. 2011;8:8.

  31. 31.

    Woodhams DC, Bletz M, Kueneman J, McKenzie VJ. Managing amphibian disease with skin microbiota. Trends Microbiol. 2016;24:161–4.

  32. 32.

    Rollins-Smith LA, Carey C, Longcore J, Doersam JK, Boutte A, Bruzgal JE, et al. Activity of antimicrobial skin peptides from ranid frogs against Batrachochytrium dendrobatidis, the chytrid fungus associated with global amphibian declines. Dev Comp Immunol. 2002;26:471–9.

  33. 33.

    Rollins-Smith LA, Carey C, Conlon JM, Reinert LK, Doersam JK, Bergman T, et al. Activities of temporin family peptides against the chytrid Fungus (Batrachochytrium dendrobatidis) associated with global amphibian declines. Antimicrob Agents Chemother. 2003;47:1157–60.

  34. 34.

    Rollins-Smith LA, Conlon JM. Antimicrobial peptide defenses against chytridiomycosis, an emerging infectious disease of amphibian populations. Dev Comp Immunol. 2005;29:589–98.

  35. 35.

    Rollins-Smith LA, Reinert LK, OLeary CJ, Houston LE, Woodhams DC. Antimicrobial peptide defenses in amphibian skin. Inter Comp Biol. 2005;45:137–42.

  36. 36.

    Rollins-Smith LA. The role of amphibian antimicrobial peptides in protection of amphibians from pathogens linked to global amphibian declines. Biochim Biophys Acta. 2009;1788:1593–9.

  37. 37.

    Annis SL, Dastoor FP, Ziel H, Daszak P, Longcore JE. A DNA-Based assay identifies Batrachochytrium dendrobatidis in amphibians. J Wildl Dis. 2004;40:420–8.

  38. 38.

    Hyatt AD, Boyle DG, Olsen V, Boyle DB, Berger L, Obendorf D, et al. Diagnostic assays and sampling protocols for the detection of Batrachochytrium dendrobatidis. Dis Aquat Organ. 2007;73:175–92.

  39. 39.

    Boyle DG, Boyle DB, Olsen V, Morgan JAT, Hyatt AD. Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay. Dis Aquat Organ. 2004;60:141–8.

  40. 40.

    Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010;7:335–6.

  41. 41.

    Amir A, McDonald D, Navas-Molina JA, Kopylova E, Morton JT, Zech Xu Z, et al. Deblur rapidly resolves single-nucleotide community sequence patterns. mSystems. 2017;2:e00191–16.

  42. 42.

    R Core Team. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2013.

  43. 43.

    Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS, et al. Metagenomic biomarker discovery and explanation. Genome Biol. 2011;12(6):R60.

  44. 44.

    Flechas SV, Sarmiento C, Cárdenas ME, Medina EM, Restrepo S, Amézquita A. Surviving chytridiomycosis: differential anti-Batrachochytrium dendrobatidis activity in bacterial isolates from three lowland species of Atelopus. PLoS ONE. 2012;7:e44832.

  45. 45.

    Bell SC, Alford RA, Garland S, Padilla G, Thomas AD. Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay. Dis Aquat Organ. 2013;103:77–85.

  46. 46.

    Daum JM, Davis LR, Bigler L, Woodhams DC. Hybrid advantage in skin peptide immune defenses of water frogs (Pelophylax esculentus) at risk from emerging pathogens. Infect Genet Evol. 2012;12:1854–64.

  47. 47.

    Flechas SV, Medina EM, Crawford AJ, Sarmiento C, Cárdenas ME, Amézquita A, et al. Characterization of the first Batrachochytrium dendrobatidis isolate from the Colombian Andes, an amphibian biodiversity hotspot. EcoHealth. 2013;10:72–76.

  48. 48.

    Holden WM, Reinert LK, Hanlon SM, Parris MJ, Rollins-Smith LA. Development of antimicrobial peptide defenses of southern leopard frogs, Rana sphenocephala, against the pathogenic chytrid fungus, Batrachochytrium dendrobatidis. Dev Comp Immunol. 2015;48:65–75.

  49. 49.

    Oliveros, JC. VENNY. An interactive tool for comparing lists with Venn Diagrams. 2007.

  50. 50.

    Oksanen J, Blanchet FG, Kindt R, Legendre P, Minchin PR, O’Hara RB, et al. Community ecology package. R package version 2.0-2. 2015.

  51. 51.

    Vredenburg VT, Knapp RA, Tunstall TS, Briggs CJ. Dynamics of an emerging disease drive large-scale amphbian population extinctions. PNAS. 2010;107:9689–94.

  52. 52.

    Kriger KM, Pereoglour F, Hero J-M. Latitudinal variation in the prevalence and intensity of chytrid (Batrachochytrium dendrobatidis) infection in eastern Australia. Conserv Biol. 2007;21:1280–90.

  53. 53.

    Langhammer PF, Burrowes PA, Lips KR, Bryant AB, Collins JP. Susceptibility to the amphibian chytrid fungus varies with ontogeny in the direct-developing frog Eleutherodactylus coqui. J Wildl Dis. 2015;50:438–46.

  54. 54.

    Rollins-Smith LA, Ramsey JP, Pask JD, Reinert LK, Woodhams DC. Amphibian immune defenses against chytridiomycosis: impacts of changing environments. Integr Comp Biol. 2011;51:552–62.

  55. 55.

    Bakar AA, Bower DS, Stockwell MP, Clulow S, Clulow J, Mahony MJ. Susceptibility to disease varies with ontogeny and immunocompetence in a threatened amphibian. Oecologia. 2016;181:997–10091.

  56. 56.

    Kueneman JG, Parfrey LW, Woodhams DC, Archer HM, Knight R, McKenzie VJ. The amphibian skin-associated microbiome across species, space and life history stages. Mol Ecol. 2014;23:1238–50.

  57. 57.

    Prest TL, Kimball AK, Kueneman JG, McKenzie VJ. Host-associated bacterial community succession during amphibian development. Mol Ecol. 2018;27:1992–2006.

  58. 58.

    Woodhams DC, Bell SC, Bigler L, Caprioli RM, Chaurand P, Lam BA, et al. Life history linked to immune investment in developing amphibians. Conserv Physiol. 2016;4(1):cow025

  59. 59.

    Burkart D, Flechas SV, Vredenburg VT, Catenazzi A. Cutaneous bacteria, but not peptides, are associated with chytridiomycosis resistance in Peruvian marsupial frogs. Anim Conserv. 2017;20:483–91.

  60. 60.

    Woodhams DC, LaBumbard BC, Barnhart KL, Becker MH, Bletz MC, Escobar LA, et al. Prodigiosin, violacein, and volatile organic compounds produced by widespread cutaneous bacteria of amphibians can inhibit two Batrachochytrium fungal pathogens. Microb Ecol. 2018;75:1049–62.

  61. 61.

    Antwis RE, Preziosi RF, Harrison XA, Garner TWJ. Amphibian symbiotic bacteria do not show universal ability to inhibit growth of the global pandemic lineage of Batrachochytrium dendrobatidis. Appl Environ Microbiol. 2015;81:3706–11.

  62. 62.

    Kearns P, Fischer S, Fernández-Beaskoetxea S, Gabor C, Bosch J, Bowen JL, et al. Fighting fungi with fungi: Antifungal properties of the amphibian mycobiome. Front Microbiol. 2017;8:1–12.

  63. 63.

    Vredenburg VT, Briggs CJ, Harris RN. Host pathogen dynamics of amphibian chytridiomycosis: the role of the skin microbiome in health and disease. In: Olsen L, Choffnes ER, Relman DA, Pray L(eds). Fungal diseases: an emerging threat to human, animal and plant health. Washington, D.C.: The National Academies Press IOM (Institute of Medicine); 2011. p. 342–55.

  64. 64.

    Becker MH, Harris RN, Minbiole KPC, Schwantes CR, Rollins-Smith LA, Reinert LK, et al. Towards a better understanding of the use of probiotics for preventing chytridiomycosis in panamanian golden frogs. EcoHealth. 2011;8:501–6.

  65. 65.

    Woodhams DC, Vredenburg VT, Simon MA, Billheimer D, Shakhtour B, Shyr Y, et al. Symbiotic bacteria contribute to innate immune defenses of the threatened mountain yellow-legged frog Rana muscosa. Biol Conserv. 2007b;138:390–8.

  66. 66.

    Funke G, Aravena-Roman M, Frodl R. First description of Curtobacterium spp. isolated from human clinical specimens. J Clin Microbiol. 2005;43:1032–6.

  67. 67.

    Briggs CJ, Knapp RA, Vredenburg VT. Enzootic and epizootic dynamics of the chytrid fungal pathogen of amphibians. PNAS. 2010;107:9695–700.

  68. 68.

    Catenazzi A, Lehr E, Rodriguez LO, Vredenburg VT. Batrachochytrium dendrobatidis and the collapse of anuran species richness and abundance in the upper Manu National Park, Peru. Conser Biol. 2011;25:382–91.

  69. 69.

    Catenazzi A, Swei A, Finkle J, Foreyt E, Wyman L, Vredenburg VT. Epizootic to enzootic transition of a fungal disease in tropical Andean frogs: are surviving species still susceptible? PLoS ONE. 2017;12(10):e0186478.

  70. 70.

    Davis LR, Bigler L, Woodhams DC. Developmental trajectories of amphibian microbiota: response to bacterial therapy depends on initial community structure. Environ Microbiol. 2017;19:1502–17.

Download references


This study was partially completed with financial support from The Rufford Foundation (RSG15305-1 to S.V.F.), Sciences Faculty at Universidad de los Andes (Proyecto Semilla to S.V.F.), the National Science Foundation (1258133 & 1633948 to V.T.V.). J.G.K. was supported by the University of Massachusetts Boston and the Simons Foundation (429440, WTW). We thank COLCIENCIAS for supporting doctoral studies of S.V.F. A.A.G. was a recipient of “Es Tiempo de volver 2015–2016” postdoctoral fellowship from COLCIENCIAS and the Universidad de La Sabana (Bogotá, Colombia). Procedures for handling animals were approved by the Colombian National Authority under resolution N°0528. For assistance in the field, we thank L.M. Arenas, J. Méndez-Narvaez, P. Palacios, C. Rodríguez, C.M. Betancourth, A. Paz, E. Lasso, R. Márquez, M.E. Cárdenas, E.M. Medina, C. Esquivel, M. González, A.J. Crawford, J.A. Hernández, M. Guevara, C. Sarmiento, M. Anganoy, A. Zarling, J. Sunyer, and D. Galindo. We thank Mr. Mauricio Moreno for allowing us to work at his property. For help in the laboratory, we thank S. Ellison, T. Nguyen, and B. LaBumbard, and for analysis advice, we thank M. Bletz.

Author information

Author notes

    • Sandra V. Flechas

    Present address: Instituto de Investigación de Recursos Biológicos Alexander von Humboldt, Bogotá, Colombia


  1. Department of Biological Sciences, Universidad de los Andes, Bogotá, 111711, Colombia

    • Sandra V. Flechas
    •  & Adolfo Amézquita
  2. Faculty of Engineering, Universidad de la Sabana, Chía, AA, 53753, Colombia

    • Alejandro Acosta-González
  3. Department of Microbiology, Faculty of Sciences, Pontificia Universidad Javeriana, Bogotá, AA 56710, Colombia

    • Laura A. Escobar
    • , Zilpa Adriana Sánchez-Quitian
    •  & Claudia M. Parra-Giraldo
  4. Biology Department, University of Massachusetts Boston, Boston, MA, 02125, USA

    • Jordan G. Kueneman
    •  & Douglas C. Woodhams
  5. Smithsonian Tropical Research Institute, Panama, Apartado 0843-03092, Republic of Panama

    • Jordan G. Kueneman
    •  & Douglas C. Woodhams
  6. Environmental Management Group, Department of Biology and Microbiology, Universidad de Boyacá, Tunja, 150000003, Colombia

    • Zilpa Adriana Sánchez-Quitian
  7. Department of Pathology, Microbiology and Immunology, Vanderbilt University, School of Medicine, Nashville, TN, 37232, USA

    • Louise A. Rollins-Smith
    •  & Laura K. Reinert
  8. Department of Biology, San Francisco State University, San Francisco, CA, 94132-1722, USA

    • Vance T. Vredenburg


  1. Search for Sandra V. Flechas in:

  2. Search for Alejandro Acosta-González in:

  3. Search for Laura A. Escobar in:

  4. Search for Jordan G. Kueneman in:

  5. Search for Zilpa Adriana Sánchez-Quitian in:

  6. Search for Claudia M. Parra-Giraldo in:

  7. Search for Louise A. Rollins-Smith in:

  8. Search for Laura K. Reinert in:

  9. Search for Vance T. Vredenburg in:

  10. Search for Adolfo Amézquita in:

  11. Search for Douglas C. Woodhams in:

Conflict of interest

The authors declare that they have no conflict of interest.

Corresponding author

Correspondence to Sandra V. Flechas.

Electronic supplementary material

About this article

Publication history