The other white-nose syndrome transcriptome: Tolerant and susceptible hosts respond differently to the pathogen Pseudogymnoascus destructans

Abstract

Mitigation of emerging infectious diseases that threaten global biodiversity requires an understanding of critical host and pathogen responses to infection. For multihost pathogens where pathogen virulence or host susceptibility is variable, host–pathogen interactions in tolerant species may identify potential avenues for adaptive evolution in recently exposed, susceptible hosts. For example, the fungus Pseudogymnoascus destructans causes white-nose syndrome (WNS) in hibernating bats and is responsible for catastrophic declines in some species in North America, where it was recently introduced. Bats in Europe and Asia, where the pathogen is endemic, are only mildly affected. Different environmental conditions among Nearctic and Palearctic hibernacula have been proposed as an explanation for variable disease outcomes, but this hypothesis has not been experimentally tested. We report the first controlled, experimental investigation of response to P. destructans in a tolerant, European species of bat (the greater mouse-eared bat, Myotis myotis). We compared body condition, disease outcomes and gene expression in control (sham-exposed) and exposed M. myotis that hibernated under controlled environmental conditions following treatment. Tolerant M. myotis experienced extremely limited fungal growth and did not exhibit symptoms of WNS. However, we detected no differential expression of genes associated with immune response in exposed bats, indicating that immune response does not drive tolerance of P. destructans in late hibernation. Variable responses to P. destructans among bat species cannot be attributed solely to environmental or ecological factors. Instead, our results implicate coevolution with the pathogen, and highlight the dynamic nature of the “white-nose syndrome transcriptome.” Interspecific variation in response to exposure by the host (and possibly pathogen) emphasizes the importance of context in studies of the bat-WNS system, and robust characterization of genetic responses to exposure in various hosts and the pathogen should precede any attempts to use particular bat species as generalizable “model hosts.”

REFERENCES Andrews, S. (2010). FastQC: A quality control tool for high throughput sequence data. Available online at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc. Benjamini, Y., & Hochberg, Y. (1995). Controlling the false discovery rate: A practical and powerful approach to multiple testing. Journal of the Royal Statistical Society. Series B (Methodological), 57, 289–300. Bolger, A. M., Lohse, M., & Usadel, B. (2014). Trimmomatic: A flexible trimmer for Illumina Sequence Data. Bioinformatics, 30(15): 2114–2120. Broad Institute of Harvard and MIT (2013). Geomyces destructans sequencing project. Retrieved from http://www.broadinstitute.org. Brown, M. J. F., Schmid-Hempel, R., & Schmid-Hempel, P. (2003). Strong context-dependent virulence in a host–parasite system: Reconciling genetic evidence with theory. Journal of Animal Ecology, 72(6), 994–1002. https://doi.org/10.1046/j.1365-2656.2003.00770.x Chen, Y. Y., Chao, C.-C., Liu, F.-C., Hsu, P.-C., Chen, H.-F., Peng, S.-C., … Wong, D. S. H.. (2013). Dynamic transcript profiling of Candida albicans infection in zebrafish: A pathogen-host interaction study. PLoS ONE, 8(9), e72483. https://doi.org/10.1371%2fjournal.pone.0072483 Cheng, T. L., Mayberry, H., McGuire, L. P., Hoyt, J. R., Langwig, K. E., Nguyen, H., … Frick, W. F. (2016). Efficacy of a probiotic bacterium to treat bats affected by the disease white-nose syndrome. Journal of Applied Ecology, 54(3): 701–708. Conesa, A., Götz, S., García-Gómez, J. M., Terol, J., Talón, M., & Robles, M. (2005). Blast2GO: A universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics, 21(18), 3674–3676. Retrieved from http://bioinformatics.oxfordjournals.org/content/21/18/3674.abstract. Cryan, P. M., Meteyer, C. U., Boyles, J. G., & Blehert, D. S. (2013). White-nose syndrome in bats: Illuminating the darkness. BMC Biology, 11(1), 47. Retrieved from http://www.biomedcentral.com/1741-7007/11/47. Cunningham, F., et al. (2015). Ensembl 2015. Nucleic Acids Research, 43(Database issue), D662–D669. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383879/. Daskin, J. H., & Alford, R. A. (2012). Context-dependent symbioses and their potential roles in wildlife diseases. Proceedings of the Royal Society B: Biological Sciences, 279(1733), 1457–1465. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282356/. Davy, C. M., Mastromonaco, G. F., Mayberry, H., Riley, J., Baxter-Gilbert, J. H., & Willis, C. K. R. (2016). Conservation implications of physiological carry-over effects in bats recovering from white-nose syndrome. Conservation Biology, 31(3), 615–624. Ellison, A. R., Tunstall, T., DiRenzo, G. V., Hughey, M. C., Rebollar, E. A., Belden, L. K., … Zamudio, K. R. (2015). More than skin deep: Functional genomic basis for resistance to amphibian Chytridiomycosis. Genome Biology and Evolution, 7(1), 286–298. https://doi.org/gbe.oxfordjournals.org/cgi/doi/10.1093/gbe/evu285 Enguita, F., Costa, M., Fusco-Almeida, A., Mendes-Giannini, M., & Leitão, A. (2016). Transcriptomic crosstalk between fungal invasive pathogens and their host cells: Opportunities and challenges for next-generation sequencing methods. Journal of Fungi, 2(1), 7. Retrieved from http://www.mdpi.com/2309-608X/2/1/7. Field, K. A., Johnson, J. S., Lilley, T. M., Reeder, S. M., Rogers, E. J., Behr, M. J., & Reeder, D. M. (2015). The white-nose syndrome transcriptome: Activation of anti-fungal host responses in wing tissue of hibernating little brown myotis. PLoS Pathogens, 11(10), e1005168. https://doi.org/10.1371%2fjournal.ppat.1005168 Fisher, M. C., Henk, D. A., Briggs, C. J., Brownstein, J. S., Madoff, L. C., McCraw, S. L., & Gurr, S. J. (2012). Emerging fungal threats to animal, plant and ecosystem health. Nature, 484(7393), 186–194. https://doi.org/10.1038/nature10947 Francl, K., Ford, W. M., Sparks, D. W., & Brack, J. V. (2012). Capture and reproductive trends in summer bat communities in West Virginia: Assessing the impact of white-nose syndrome. Journal of Fish and Wildlife Management, 2, 33–42. Frank, C. L., Michalski, A., McDonough, A. A., Rahimian, M., Rudd, R. J., & Herzog, C. (2014). The resistance of a North American bat species (Eptesicus fuscus) to white-nose syndrome (WNS). PLoS ONE, 9(12), 1–14. Frick, W. F., Puechmaille, S. J., Hoyt, J. R., Nickel, B. A., Langwig, K. E., Foster, J. T., … Kilpatrick, A. M. (2015). Disease alters macroecological patterns of North American bats. Global Ecology and Biogeography, 24(7), 741–749. https://doi.org/10.1111/geb.12290 Grabherr, M. G., Haas, B. J., Yassour, M., Levin, J. Z., Thompson, D. A., Amit, I., … Regev, A. (2011). Trinity: Reconstructing a full-length transcriptome without a genome from RNA-Seq data. Nature Biotechnology, 29(7), 644–652. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3571712/. Hayman, D. T. S., Pulliam, J. R. C., Marshall, J. C., Cryan, P. M., & Webb, C. T. (2016). Environment, host, and fungal traits predict continental-scale white-nose syndrome in bats. Science advances, 2(1),e1500831. Hoyt, J. R., Cheng, T. L., Langwig, K. E., Hee, M. M., Frick, W. F., & Kilpatrick, A. M. (2015). Bacteria isolated from bats inhibit the growth of Pseudogymnoascus destructans, the causative agent of white-nose syndrome. PLoS ONE, 10(4), e0121329. https://doi.org/10.1371%2fjournal.pone.0121329 Hoyt, J. R., Langwig, K. E., Sun, K., Lu, G., Parise, L., Jiang, T., … Hoyt, J. R. (2016a). Widespread bat white-nose syndrome fungus, Northeastern China. Emerging Infectious Diseases, 22(1), 140–142. Hoyt, J. R., Sun, K., Parise, K. L., Lu, G., Langwig, K. E., Jiang, T., … Feng, J. (2016b). Host persistence or extinction from emerging infectious disease: Insights from white-nose syndrome in endemic and invading regions. Proceedings of the Royal Society B, 283, 20152861. Huang, Z., Gallot, A., Lao, N. T., Puechmaille, S. J., Foley, N. M., Jebb, D., … Teeling, E. C. (2015). A non-lethal sampling method to obtain, generate and assemble whole-blood transcriptomes from small, wild mammals. Molecular Ecology Resources, 16(1), 150–162. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/26186236. James, T. Y., Toledo, L. F., Rödder, D., da Silva Leite, D., Belasen, A. M., Betancourt-Román, C. M., … Longcore, J. E. (2015). Disentangling host, pathogen, and environmental determinants of a recently emerged wildlife disease: Lessons from the first 15 years of amphibian chytridiomycosis research. Ecology and Evolution, 5(18), 4079–4097. https://doi.org/10.1002/ece3.1672 Johnson, J. S., Reeder, D. M., McMichael, J. W.III, Meierhofer, M. B., Stern, D. W. F., Lumadue, S. S., … Field, K. A. (2014). Host, pathogen, and environmental characteristics predict white-nose syndrome mortality in captive little brown myotis (Myotis lucifugus). PLoS ONE, 9(11), e112502. https://doi.org/10.1371%2fjournal.pone.0112502 Jones, K. E., Patel, N. G., Levy, M. A., Storeygard, A., Balk, D., Gittleman, J. L., & Daszak, P. (2008). Global trends in emerging infectious diseases. Nature, 451(7181), 990–993. Kim, D., Pertea, G., Trapnell, C., Pimentel, H., Kelley, R., & Salzberg, S. L. (2013). TopHat2: Accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biology, 14(4), R36–R36. Langmead, B., Trapnell, C., Pop, M., & Salzberg, S. L. (2009). Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biology, 10(3): R25. Langwig, K. E., Frick, W. F., Bried, J. T., Hicks, A. C., Kunz, T. H., & Kilpatrick, A. M. (2012). Sociality, density-dependence and microclimates determine the persistence of populations suffering from a novel fungal disease, white-nose syndrome. Ecology Letters, 15(9), 1050–1057. https://doi.org/10.1111/j.1461-0248.2012.01829.x Langwig, K. E., Frick, W. F., Reynolds, R., Parise, K. L., Drees, K. P., Hoyt, J. R., … Kilpatrick, A. M. (2015). Host and pathogen ecology drive the seasonal dynamics of a fungal disease, white-nose syndrome. Proceedings of the Royal Society of London B: Biological Sciences, 282(1799), 2014–2335. Retrieved from http://rspb.royalsocietypublishing.org/content/282/1799/20142335.abstract. Leopardi, S., Blake, D., & Puechmaille, S. J. (2015). White-nose syndrome fungus introduced from Europe to North America. Current Biology, 25(6), R217–R219. https://doi.org/10.1016/j.cub.2015.01.047 Li, B., & Dewey, C. N. (2011). RSEM: Accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics, 12, 323. Love, M. I., Huber, W., & Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-Seq data with DESeq2. Genome Biology, 15(12): 550. McGinnis, S., & Madden, T. L. (2004). BLAST: At the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids Research, 32(Web Server issue), W20–W25. Meteyer, C. U., Barber, D., & Mandl, J. N. (2012). Pathology in euthermic bats with white nose syndrome suggests a natural manifestation of immune reconstitution inflammatory syndrome. Virulence, 3(7), 583–588. https://doi.org/10.4161/viru.22330 Moore, M. S., Reichard, J. D., Murtha, T. D., Nabhan, M. L., Pian, R. E., Ferreira, J. S., & Kunz, T. H. (2013). Hibernating little brown myotis (Myotis lucifugus) show variable immunological responses to white-nose syndrome. PLoS ONE, 8(3), e58976. Muller, L., Lorch, J., Lindner, D., O’Connor, M., Gargas, A., & Blehert, D. (2013). Bat white-nose syndrome: A real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructans. Mycologia, 105, 253–259. Perez-Nadales, E., Almeida Nogueira, M. F., Baldin, C., Castanheira, S., El Ghalid, M., Grund, E., … Wendland, J. (2014). Fungal model systems and the elucidation of pathogenicity determinants. Fungal Genetics and Biology, 70(100), 42–67. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161391/. Poorten, T. J., & Rosenblum, E. B. (2016). Comparative study of host response to chytridiomycosis in a susceptible and a resistant toad species. Molecular Ecology, 25(22): 5663–5679. https://doi.org/10.1111/mec.13871 Puechmaille, S. J., Wibbelt, G., Korn, V., Fuller, H., Forget, F., Mühldorfer, K., … Teeling, E. C. (2011). Pan-European distribution of white-nose syndrome fungus (Geomyces destructans) not associated with mass mortality. PLoS ONE, 6(4), e19167. https://doi.org/10.1371%2fjournal.pone.0019167 Quinlan, A. R., & Hall, I. M. (2010). BEDTools: A flexible suite of utilities for comparing genomic features. Bioinformatics, 26(6), 841–842. Rapin, N., Johns, K., Martin, L., Warnecke, L., Turner, J. M., Bollinger, T. K., … Misra, V. (2014). Activation of innate immune-response genes in little brown bats (Myotis lucifugus) infected with the fungus Pseudogymnoascus destructans J. Sun, ed. PLoS ONE, 9(11), e112285. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4229191/. Reeder, D. M., Frank, C. L., Turner, G. G., Meteyer, C. U., Kurta, A., Britzke, E. R., … Blehert, D. S. (2012). Frequent arousal from hibernation linked to severity of infection and mortality in bats with white-nose syndrome. PLoS ONE, 7(6), e38920. https://doi.org/10.1371%2fjournal.pone.0038920 Reimand, J., Arak, T., Adler, P., Kolberg, L., Reisberg, S., Peterson, H., & Vilo, J. (2016). g:Profiler—a web server for functional interpretation of gene lists (2016 update). Nucleic Acids Research, 44(W1): W83–W89. Robinson, M. D., McCarthy, D. J., & Smyth, G. K. (2010). edgeR: A bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics, 26(1), 139–140. Rosenblum, E. B., Poorten, T. J., Settles, M., & Murdoch, G. K. (2012). Only skin deep: Shared genetic response to the deadly chytrid fungus in susceptible frog species. Molecular Ecology, 21(13), 3110–3120. https://doi.org/10.1111/j.1365-294x.2012.05481.x Sambrook, J., & Russell, D. W. (2001). Molecular cloning: A laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Savage, A., & Zamudio, K. R. (2011). MHC genotypes associate with resistance to a frog-killing fungus. Proceedings of the National Academy of Sciences of the United States of America, 108(40), 16705–16710. Smith, K. F., Sax, D. F., & Lafferty, K. D. (2006). Evidence for the role of infectious disease in species extinction and endangerment. Conservation Biology, 20(5), 1349–1357. Varet, H., Coppée, J.-Y., & Dillies, M.-A. (2016). SARTools: A DESeq2- and edgeR-based R pipeline for comprehensive differential analysis of RNA-Seq data. PLoS ONE, 11(6): e0157022. Verant, M. L., Boyles, J. G., Waldrep., W.Jr, Wibbelt, G., & Blehert, D. S. (2012). Temperature-dependent growth of Geomyces destructans, the fungus that causes bat white-nose syndrome. PLoS ONE, 7(9), e46280. https://doi.org/10.1371%2fjournal.pone.0046280 Warnecke, L., Turner, J. M., Bollinger, T. K., Lorch, J. M., Misra, V., Cryan, P. M., … Willis, C. K. R. (2012). Inoculation of bats with European Geomyces destructans supports the novel pathogen hypothesis for the origin of white-nose syndrome. Proceedings of the National Academy of Sciences of the United States of America, 109(18), 6999–7003. Retrieved from http://www.pnas.org/content/109/18/6999.abstract. Warnecke, L., Turner, J. M., Bollinger, T. K., Misra, V., Cryan, P. M., Blehert, D. S., … Willis, C. K. R. (2013). Pathophysiology of white-nose syndrome in bats: A mechanistic model linking wing damage to mortality. Biology Letters, 9(4), 20130177. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730627/. Wibbelt, G., Kurth, A., Hellmann, D., Weishaar, M., Barlow, A., Veith, M., … Blehert, D. S. (2010). White-nose syndrome fungus (Geomyces destructans) in bats, Europe. Emerging Infectious Diseases, 16(8), 1237–1243. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3298319/. Willis, C. K. R. (2015). Conservation physiology and conservation pathogens: White-nose syndrome and integrative biology for host–pathogen systems. Integrative and Comparative Biology, 55(4): 631–641. Retrieved from http://icb.oxfordjournals.org/content/early/2015/08/24/icb.icv099.abstract. Zukal, J., Bandouchova, H., Brichta, J., Cmokova, A., Jaron, K. S., Kolarik, M., … Martínková, N. (2016). White-nose syndrome without borders: Pseudogymnoascus destructans infection tolerated in Europe and Palearctic Asia but not in North America. Scientific Reports, 6(January), 19829.