When Defenses Fail: Atelopus zeteki Skin Secretions Increase Growth of the Pathogen Batrachochytrium dendrobatidis

Abstract To combat the threat of emerging infectious diseases in wildlife, ecoimmunologists seek to understand the complex interactions among pathogens, their hosts, and their shared environments. The cutaneous fungal pathogen Batrachochytrium dendrobatidis (Bd), has led to the decline of innumerable amphibian species, including the Panamanian golden frog (Atelopus zeteki). Given that Bd can evade or dampen the acquired immune responses of some amphibians, nonspecific immune defenses are thought to be especially important for amphibian defenses against Bd. In particular, skin secretions constitute a vital component of amphibian innate immunity against skin infections, but their role in protecting A. zeteki from Bd is unknown. We investigated the importance of this innate immune component by reducing the skin secretions from A. zeteki and evaluating their effectiveness against Bd in vitro and in vivo. Following exposure to Bd in a controlled inoculation experiment, we compared key disease characteristics (e.g., changes in body condition, prevalence, pathogen loads, and survival) among groups of frogs that had their skin secretions reduced and control frogs that maintained their skin secretions. Surprisingly, we found that the skin secretions collected from A. zeteki increased Bd growth in vitro. This finding was further supported by infection and survival patterns in the in vivo experiment where frogs with reduced skin secretions tended to have lower pathogen loads and survive longer compared to frogs that maintained their secretions. These results suggest that the skin secretions of A. zeteki are not only ineffective at inhibiting Bd but may enhance Bd growth, possibly leading to greater severity of disease and higher mortality in this highly vulnerable species. These results differ from those of previous studies in other amphibian host species that suggest that skin secretions are a key defense in protecting amphibians from developing severe chytridiomycosis. Therefore, we suggest that the importance of immune components cannot be generalized across all amphibian species or over time. Moreover, the finding that skin secretions may be enhancing Bd growth emphasizes the importance of investigating these immune components in detail, especially for species that are a conservation priority.

[1]  Christopher A. Voigt,et al.  Genetically modifying skin microbe to produce violacein and augmenting microbiome did not defend Panamanian golden frogs from disease , 2021, ISME Communications.

[2]  N. Callewaert,et al.  Epidermal galactose spurs chytrid virulence and predicts amphibian colonization , 2021, Nature Communications.

[3]  A. P. Rothstein,et al.  Divergent regional evolutionary histories of a devastating global amphibian pathogen , 2021, Proceedings of the Royal Society B.

[4]  Gregory P. Brown,et al.  Host defense or parasite cue: Skin secretions mediate interactions between amphibians and their parasites. , 2021, Ecology letters.

[5]  E. Rosenblum,et al.  Whole exome sequencing identifies the potential for genetic rescue in iconic and critically endangered Panamanian harlequin frogs , 2020, Global change biology.

[6]  H. McCallum,et al.  Immunological Aspects of Chytridiomycosis , 2020, Journal of fungi.

[7]  E. Rebollar,et al.  The Amphibian Skin Microbiome and Its Protective Role Against Chytridiomycosis , 2020, Herpetologica.

[8]  L. Rollins‐Smith Global Amphibian Declines, Disease, and the Ongoing Battle between Batrachochytrium Fungi and the Immune System , 2020, Herpetologica.

[9]  P. Sharma,et al.  Mini Review on Antimicrobial Peptides, Sources, Mechanism and Recent Applications , 2019, Protein and peptide letters.

[10]  P. Houser,et al.  Conserving Panamanian harlequin frogs by integrating captive-breeding and research programs , 2019, Biological Conservation.

[11]  J. Voyles,et al.  Quantifying Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans Viability , 2019, EcoHealth.

[12]  P. Chaurand,et al.  Probiotics Modulate a Novel Amphibian Skin Defense Peptide That Is Antifungal and Facilitates Growth of Antifungal Bacteria , 2019, Microbial Ecology.

[13]  Mark Wilkinson,et al.  Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity , 2019, Science.

[14]  Maxwell P. Bui-Marinos,et al.  Frog Skin Innate Immune Defences: Sensing and Surviving Pathogens , 2019, Front. Immunol..

[15]  H. McCallum,et al.  Review of the Amphibian Immune Response to Chytridiomycosis, and Future Directions , 2018, Front. Immunol..

[16]  J. Schipper,et al.  From hope to alert: demography of a remnant population of the Critically Endangered Atelopus varius from Costa Rica , 2018 .

[17]  E. Rosenblum,et al.  Shifts in disease dynamics in a tropical amphibian assemblage are not due to pathogen attenuation , 2018, Science.

[18]  Lynn B. Martin,et al.  An Introduction to Ecoimmunology , 2018, Advances in Comparative Immunology.

[19]  Peter Daszak,et al.  One Health, emerging infectious diseases and wildlife: two decades of progress? , 2017, Philosophical Transactions of the Royal Society B: Biological Sciences.

[20]  A. Blaustein,et al.  Virulence variation among strains of the emerging infectious fungus Batrachochytrium dendrobatidis (Bd) in multiple amphibian host species. , 2017, Diseases of aquatic organisms.

[21]  P. Chaurand,et al.  Life history linked to immune investment in developing amphibians , 2016, Conservation physiology.

[22]  F. Haesebrouck,et al.  Amphibian chytridiomycosis: a review with focus on fungus-host interactions , 2015, Veterinary Research.

[23]  Anna E. Savage,et al.  Composition of symbiotic bacteria predicts survival in Panamanian golden frogs infected with a lethal fungus , 2015, Proceedings of the Royal Society B: Biological Sciences.

[24]  Graziella V. DiRenzo,et al.  More than Skin Deep: Functional Genomic Basis for Resistance to Amphibian Chytridiomycosis , 2014, Genome biology and evolution.

[25]  T. Raffel,et al.  Amphibians acquire resistance to live and dead fungus overcoming fungal immunosuppression , 2014, Nature.

[26]  D. Bates,et al.  Fitting Linear Mixed-Effects Models Using lme4 , 2014, 1406.5823.

[27]  Anna E. Savage,et al.  Fighting a Losing Battle: Vigorous Immune Response Countered by Pathogen Suppression of Host Defenses in the Chytridiomycosis-Susceptible Frog Atelopus zeteki , 2014, G3: Genes, Genomes, Genetics.

[28]  Pieter T. J. Johnson,et al.  Experimental infection dynamics: using immunosuppression and in vivo parasite tracking to understand host resistance in an amphibian–trematode system , 2013, Journal of Experimental Biology.

[29]  Tawnya L Cary,et al.  Skin peptides protect juvenile leopard frogs (Rana pipiens) against chytridiomycosis , 2013, Journal of Experimental Biology.

[30]  R. Harris,et al.  Towards a Better Understanding of the Use of Probiotics for Preventing Chytridiomycosis in Panamanian Golden Frogs , 2011, EcoHealth.

[31]  L. Reinert,et al.  Amphibian immune defenses against chytridiomycosis: impacts of changing environments. , 2011, Integrative and comparative biology.

[32]  K. Zamudio,et al.  MHC genotypes associate with resistance to a frog-killing fungus , 2011, Proceedings of the National Academy of Sciences.

[33]  J. Voyles Phenotypic profiling of Batrachochytrium dendrobatidis, a lethal fungal pathogen of amphibians , 2011 .

[34]  J. Ragle,et al.  IUCN Red List of Threatened Species , 2010 .

[35]  A. J. Crawford,et al.  Epidemic disease decimates amphibian abundance, species diversity, and evolutionary history in the highlands of central Panama , 2010, Proceedings of the National Academy of Sciences.

[36]  R. Alford,et al.  Adaptations of skin peptide defences and possible response to the amphibian chytrid fungus in populations of Australian green‐eyed treefrogs, Litoria genimaculata , 2010 .

[37]  L. Reinert,et al.  Immune Defenses against Batrachochytrium dendrobatidis, a Fungus Linked to Global Amphibian Declines, in the South African Clawed Frog, Xenopus laevis , 2010, Infection and Immunity.

[38]  David Cook,et al.  Pathogenesis of Chytridiomycosis, a Cause of Catastrophic Amphibian Declines , 2009, Science.

[39]  C. Kenneth Dodd,et al.  Amphibian Ecology and Conservation: A Handbook of Techniques , 2009 .

[40]  M. Eisen,et al.  Genome-Wide Transcriptional Response of Silurana (Xenopus) tropicalis to Infection with the Deadly Chytrid Fungus , 2009, PloS one.

[41]  L. Rollins‐Smith The role of amphibian antimicrobial peptides in protection of amphibians from pathogens linked to global amphibian declines. , 2009, Biochimica et biophysica acta.

[42]  L. Skerratt,et al.  BSA reduces inhibition in a TaqMan assay for the detection of Batrachochytrium dendrobatidis. , 2009, Diseases of aquatic organisms.

[43]  A. Hyatt,et al.  Chytridiomycosis and Amphibian Population Declines Continue to Spread Eastward in Panama , 2008, EcoHealth.

[44]  D. Wake,et al.  Are we in the midst of the sixth mass extinction? A view from the world of amphibians , 2008, Proceedings of the National Academy of Sciences.

[45]  J. Mendelson,et al.  The principles of rapid response for amphibian conservation, using the programmes in Panama as an example , 2008 .

[46]  M. S. San Francisco,et al.  Chemotaxis of the amphibian pathogen Batrachochytrium dendrobatidis and its response to a variety of attractants , 2008, Mycologia.

[47]  Jianxu Li,et al.  Anti-infection Peptidomics of Amphibian Skin *S , 2007, Molecular & Cellular Proteomics.

[48]  Richard Speare,et al.  Spread of Chytridiomycosis Has Caused the Rapid Global Decline and Extinction of Frogs , 2007, EcoHealth.

[49]  F. Gleason,et al.  Diagnostic assays and sampling protocols for the detection of Batrachochytrium dendrobatidis. , 2007, Diseases of aquatic organisms.

[50]  C. Carey,et al.  PREDICTED DISEASE SUSCEPTIBILITY IN A PANAMANIAN AMPHIBIAN ASSEMBLAGE BASED ON SKIN PEPTIDE DEFENSES , 2006, Journal of wildlife diseases.

[51]  R. Alford,et al.  Emerging infectious disease and the loss of biodiversity in a Neotropical amphibian community. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[52]  R. Harris,et al.  Amphibian Pathogen Batrachochytrium dendrobatidis Is Inhibited by the Cutaneous Bacteria of Amphibian Species , 2006, EcoHealth.

[53]  Fernando Castro,et al.  Catastrophic Population Declines and Extinctions in Neotropical Harlequin Frogs (Bufonidae: Atelopus) 1 , 2005 .

[54]  A. Hyatt,et al.  Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay. , 2004, Diseases of aquatic organisms.

[55]  L. Reinert,et al.  Antimicrobial peptide defenses of the Tarahumara frog, Rana tarahumarae. , 2002, Biochemical and biophysical research communications.

[56]  J. Longcore,et al.  BATRACHOCHYTRIUM DENDROBATIDIS GEN. ET SP. NOV., A CHYTRID PATHOGENIC TO AMPHIBIANS , 1999 .

[57]  D E Green,et al.  Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[58]  H. Vaudry,et al.  Dopamine inhibits corticosteroid secretion from frog adrenal gland, in vitro. , 1990, Endocrinology.

[59]  P. Rhodes Administration. , 1933, Teachers College Record: The Voice of Scholarship in Education.

[60]  Alexa,et al.  dendroBAtidis after develOPment under drying cOnditiOns , 2019 .

[61]  A. Dreher Modeling Survival Data Extending The Cox Model , 2016 .

[62]  Yvonne Neudorf Amphibian Medicine And Captive Husbandry , 2016 .

[63]  R. Huey,et al.  The Invasive Chytrid Fungus of Amphibians Paralyzes Lymphocyte Responses , 2015 .

[64]  R. Ibáñez,et al.  Field surveys in Western Panama indicate populations of Atelopus varius frogs are persisting in regions where Batrachochytrium dendrobatidis is now enzootic , 2014 .

[65]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[66]  J. Belant,et al.  Renewing hope: the rediscovery of Atelopus varius in Costa Rica , 2013 .

[67]  C. Mecklin,et al.  Larval growth in polyphenic salamanders: making the best of a bad lot , 2011, Oecologia.

[68]  J. Conlon The contribution of skin antimicrobial peptides to the system of innate immunity in anurans , 2010, Cell and Tissue Research.

[69]  L. Reinert,et al.  Antimicrobial Peptide Defenses in Amphibian Skin1 , 2005, Integrative and comparative biology.

[70]  A. Mor,et al.  Peptides as weapons against microorganisms in the chemical defense system of vertebrates. , 1995, Annual review of microbiology.