The White-Nose Syndrome Transcriptome: Activation of Anti-fungal Host Responses in Wing Tissue of Hibernating Little Brown Myotis

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White-nose syndrome is the most devastating epizootic wildlife disease of mammals in history, having killed millions of hibernating bats in North America since 2007. We have used next-generation RNA sequencing to provide a survey of the gene expression changes that accompany this disease in the skin of bats infected with the causative fungus. We identified possible new mechanisms that may either provide protection or contribute to mortality, including inflammatory immune responses. Contrary to expectations that hibernation represents a period of dormancy, we found that gene expression pathways were responsive to the environment. We also examined which genes were expressed in the pathogen and identified several classes of genes that could contribute to the virulence of this disease. Gene expression changes in the host were associated with local inflammation despite the fact that the bats were hibernating. However, we found that hibernating bats with white-nose syndrome lack some of the responses known to defend other mammals from fungal infection. We propose that bats could be protected from white-nose syndrome if these responses could be established prior to hibernation or if treatments could block the virulence factors expressed by the pathogen.

Vyšlo v časopise: The White-Nose Syndrome Transcriptome: Activation of Anti-fungal Host Responses in Wing Tissue of Hibernating Little Brown Myotis. PLoS Pathog 11(10): e32767. doi:10.1371/journal.ppat.1005168
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1005168

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Zdroje

1. Coleman JT, Reichard JD. (2014) Bat White-Nose Syndrome in 2014: A Brief Assessment Seven Years After Discovery of a Virulent Fungal Pathogen in North America. Outlooks on Pest Management 25: 374–377.

2. Blehert DS. (2012) Fungal disease and the developing story of bat white-nose syndrome. PLoS Pathog 8: e1002779. doi: 10.1371/journal.ppat.1002779 22829763

3. Blehert DS, Hicks AC, Behr M, Meteyer CU, Berlowski-Zier BM, et al. (2009) Bat white-nose syndrome: an emerging fungal pathogen? Science 323: 227–227. doi: 10.1126/science.1163874 18974316

4. Lorch JM, Meteyer CU, Behr MJ, Boyles JG, Cryan PM, et al. (2011) Experimental infection of bats with Geomyces destructans causes white-nose syndrome. Nature 480: 376–378. doi: 10.1038/nature10590 22031324

5. Warnecke L, Turner JM, Bollinger TK, Lorch JM, Misra V, et al. (2012) Inoculation of bats with European Geomyces destructans supports the novel pathogen hypothesis for the origin of white-nose syndrome. Proc Natl Acad Sci U S A 109: 6999–7003. doi: 10.1073/pnas.1200374109 22493237

6. Verant ML, Boyles JG, Waldrep Jr W, Wibbelt G, Blehert DS. (2012) Temperature-dependent growth of Geomyces destructans, the fungus that causes bat white-nose syndrome.

7. Cryan PM, Meteyer CU, Boyles JG, Blehert DS. (2010) Wing pathology of white-nose syndrome in bats suggests life-threatening disruption of physiology. BMC Biol 8: 135-7007-8-135.

8. Meteyer CU, Buckles EL, Blehert DS, Hicks AC, Green DE, et al. (2009) Histopathologic criteria to confirm white-nose syndrome in bats. Journal of Veterinary Diagnostic Investigation 21: 411–414. 19564488

9. Turner GG, Reeder DM, Coleman JTH. (2011) A five year assessment of mortality and geographic spread of white-nose syndrome in North American bats and a look to the future. Bat Research News 52: 13–27.

10. Frick WF, Puechmaille SJ, Hoyt JR, Nickel BA, Langwig KE, et al. (2015) Disease alters macroecological patterns of North American bats. Global Ecol Biogeogr 24: 741–749.

11. Frank CL, Michalski A, McDonough AA, Rahimian M, Rudd RJ, et al. (2014) The Resistance of a North American Bat Species (Eptesicus fuscus) to White-Nose Syndrome (WNS). PloS One 9: e113958. doi: 10.1371/journal.pone.0113958 25437448

12. Wibbelt G, Puechmaille SJ, Ohlendorf B, Mühldorfer K, Bosch T, et al. (2013) Skin Lesions in European Hibernating Bats Associated with Geomyces destructans, the Etiologic Agent of White-Nose Syndrome. PloS One 8: e74105. doi: 10.1371/journal.pone.0074105 24023927

13. Bandouchova H, Bartonicka T, Berkova H, Brichta J, Cerny J, et al. (2015) Pseudogymnoascus destructans: evidence of virulent skin invasion for bats under natural conditions, Europe. Transboundary and Emerging Diseases 62: 1–5. doi: 10.1111/tbed.12282 25268034

14. Reeder DM, Frank CL, Turner GG, Meteyer CU, Kurta A, et al. (2012) Frequent arousal from hibernation linked to severity of infection and mortality in bats with white-nose syndrome. PLoS One 7: e38920. doi: 10.1371/journal.pone.0038920 22745688

15. Johnson JS, Reeder DM, McMichael JW III, Meierhofer MB, Stern DW, et al. (2014) Host, pathogen, and environmental characteristics predict white-nose syndrome mortality in captive little brown myotis (Myotis lucifugus). PLoS ONE 9: e112502. doi: 10.1371/journal.pone.0112502 25409028

16. Geiser F. (2004) Metabolic rate and body temperature reduction during hibernation and daily torpor. Annu Rev Physiol 66: 239–274. 14977403

17. Thomas DW, Cloutier D. (1992) Evaporative water loss by hibernating little brown bats, Myotis lucifugus. Physiol Zool: 443–456.

18. Thomas DW, Dorais M, Bergeron J. (1990) Winter energy budgets and cost of arousals for hibernating little brown bats, Myotis lucifugus. J Mammal 71: 475–479.

19. Fuller NW, Reichard JD, Nabhan ML, Fellows SR, Pepin LC, et al. (2011) Free-ranging little brown myotis (Myotis lucifugus) heal from wing damage associated with white-nose syndrome. Ecohealth 8: 154–162. doi: 10.1007/s10393-011-0705-y 21922344

20. Cryan PM, Meteyer CU, Blehert DS, Lorch JM, Reeder DM, et al. (2013) Electrolyte depletion in white-nose syndrome bats. J Wildl Dis 49: 398–402. doi: 10.7589/2012-04-121 23568916

21. Willis CK, Menzies AK, Boyles JG, Wojciechowski MS. (2011) Evaporative water loss is a plausible explanation for mortality of bats from white-nose syndrome. Integr Comp Biol 51: 364–373. doi: 10.1093/icb/icr076 21742778

22. Verant ML, Meteyer CU, Speakman JR, Cryan PM, Lorch JM, et al. (2014) White-nose syndrome initiates a cascade of physiologic disturbances in the hibernating bat host. BMC Physiol 14: 10. doi: 10.1186/s12899-014-0010-4 25487871

23. Moore MS, Reichard JD, Murtha TD, Nabhan ML, Pian RE, et al. (2013) Hibernating little brown myotis (Myotis lucifugus) show variable immunological responses to white-nose syndrome. PloS One 8: e58976. doi: 10.1371/journal.pone.0058976 23527062

24. Moore MS, Reichard JD, Murtha TD, Zahedi B, Fallier RM, et al. (2011) Specific alterations in complement protein activity of little brown myotis (Myotis lucifugus) hibernating in white-nose syndrome affected sites. PLoS One 6: e27430. doi: 10.1371/journal.pone.0027430 22140440

25. LeibundGut-Landmann S, Wüthrich M, Hohl TM. (2012) Immunity to fungi. Curr Opin Immunol 24: 449–458. doi: 10.1016/j.coi.2012.04.007 22613091

26. Brown GD. (2011) Innate antifungal immunity: the key role of phagocytes. Annu Rev Immunol 29: 1. doi: 10.1146/annurev-immunol-030409-101229 20936972

27. Holland SM, DeLeo FR, Elloumi HZ, Hsu AP, Uzel G, et al. (2007) STAT3 mutations in the hyper-IgE syndrome. N Engl J Med 357: 1608–1619. 17881745

28. Liu L, Okada S, Kong X, Kreins AY, Cypowyj S, et al. (2011) Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis. J Exp Med 208: 1635–1648. doi: 10.1084/jem.20110958 21727188

29. Gow NA, van de Veerdonk, Frank L, Brown AJ, Netea MG. (2012) Candida albicans morphogenesis and host defence: discriminating invasion from colonization. Nature Reviews Microbiology 10: 112–122.

30. Netea MG, Brown GD, Kullberg BJ, Gow NA. (2008) An integrated model of the recognition of Candida albicans by the innate immune system. Nature Reviews Microbiology 6: 67–78. 18079743

31. Romani L. (2011) Immunity to fungal infections. Nat Rev Immunol 11: 275–288. doi: 10.1038/nri2939 21394104

32. Hernández-Santos N, Gaffen SL. (2012) Th17 cells in immunity to Candida albicans. Cell Host & Microbe 11: 425–435.

33. Kagami S, Rizzo HL, Kurtz SE, Miller LS, Blauvelt A. (2010) IL-23 and IL-17A, but not IL-12 and IL-22, are required for optimal skin host defense against Candida albicans. J Immunol 185: 5453–5462. doi: 10.4049/jimmunol.1001153 20921529

34. Smeekens SP, Ng A, Kumar V, Johnson MD, Plantinga TS, et al. (2013) Functional genomics identifies type I interferon pathway as central for host defense against Candida albicans. Nature Communications 4: 1342. doi: 10.1038/ncomms2343 23299892

35. Blanco JL, Garcia ME. (2008) Immune response to fungal infections. Vet Immunol Immunopathol 125: 47–70. doi: 10.1016/j.vetimm.2008.04.020 18565595

36. De Luca A, Zelante T, D'Angelo C, Zagarella S, Fallarino F, et al. (2010) IL-22 defines a novel immune pathway of antifungal resistance. Mucosal Immunology.

37. Conti HR, Shen F, Nayyar N, Stocum E, Sun JN, et al. (2009) Th17 cells and IL-17 receptor signaling are essential for mucosal host defense against oral candidiasis. J Exp Med 206: 299–311. doi: 10.1084/jem.20081463 19204111

38. Brook CE, Dobson AP. (2015) Bats as ‘special’reservoirs for emerging zoonotic pathogens. Trends Microbiol 23: 172–180. doi: 10.1016/j.tim.2014.12.004 25572882

39. Cogswell-Hawkinson AC, McGlaughlin ME, Calisher CH, Adams R, Schountz T. (2011) Molecular and phylogenetic characterization of cytokine genes from Seba’s short-tailed bat (Carollia perspicillata). Open Immunology Journal 4: 31–39.

40. Iha K, Omatsu T, Watanabe S, Ueda N, Taniguchi S, et al. (2009) Molecular cloning and sequencing of the cDNAs encoding the bat interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12p40, and tumor necrosis factor-alpha. Journal of Veterinary Medical Science 71: 1691–1695. 20046044

41. Zhang G, Cowled C, Shi Z, Huang Z, Bishop-Lilly KA, et al. (2013) Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science 339: 456–460. doi: 10.1126/science.1230835 23258410

42. Fites JS, Ramsey JP, Holden WM, Collier SP, Sutherland DM, et al. (2013) The invasive chytrid fungus of amphibians paralyzes lymphocyte responses. Science 342: 366–369. doi: 10.1126/science.1243316 24136969

43. Bouma HR, Carey HV, Kroese FG. (2010a) Hibernation: the immune system at rest? J Leukoc Biol 88: 619–624.

44. Bouma HR, Henning RH, Kroese FG, Carey HV. (2012) Hibernation is associated with depression of T-cell independent humoral immune responses in the 13-lined ground squirrel. Dev Comp Immunol.

45. Bouma HR, Strijkstra AM, Boerema AS, Deelman LE, Epema AH, et al. (2010b) Blood cell dynamics during hibernation in the European Ground Squirrel. Vet Immunol Immunopathol 136: 319–323.

46. Kurtz CC, Carey HV. (2007) Seasonal changes in the intestinal immune system of hibernating ground squirrels. Dev Comp Immunol 31: 415–428. 16930701

47. Maniero GD. (2002) Classical pathway serum complement activity throughout various stages of the annual cycle of a mammalian hibernator, the golden-mantled ground squirrel, Spermophilus lateralis. Dev Comp Immunol 26: 563–574. 12031416

48. Maniero GD. (2005) Ground squirrel splenic macrophages bind lipopolysaccharide over a wide range of temperatures at all phases of their annual hibernation cycle. Comp Immunol Microbiol Infect Dis 28: 297–309. 16182368

49. Jaroslow BN, Serrell BA. (1972) Differential sensitivity to hibernation of early and late events in development of the immune response. J Exp Zool 181: 111–116. 4556623

50. Larsen B. (1971) Antibody formation in hedgehogs. II. Hibernating animals. Comp Biochem Physiol A Comp Physiol 38: 571–580. 4396827

51. Manasek FJ, Adelstein SJ, Lyman CP. (1965) The effects of hibernation on the in vitro synthesis of DNA by hamster lymphoid tissue. J Cell Physiol 65: 319–324. 5891143

52. Cahill JE, Lewert RM, Jaroslow BN. (1967) Effect of hibernation on course of infection and immune response in Citellus tridecemlineatus infected with Nippostrongylus brasiliensis. J Parasitol 53: 110–115. 6017221

53. Sieckmann DG, Jaffe H, Golech S, Cai D, Hallenbeck JM, et al. (2014) Anti-lymphoproliferative activity of alpha-2-macroglobulin in the plasma of hibernating 13-lined ground squirrels and woodchucks. Vet Immunol Immunopathol 161: 1–11. doi: 10.1016/j.vetimm.2014.05.006 25113962

54. Maniero GD. (2000) The influence of temperature and season on mitogen-induced proliferation of ground squirrel lymphocytes. In: Heldmaier G, Klingenspor M, editors. Life in the Cold. New York: Springer. pp. 493–503.

55. Hampton M, Melvin RG, Kendall AH, Kirkpatrick BR, Peterson N, et al. (2011) Deep sequencing the transcriptome reveals seasonal adaptive mechanisms in a hibernating mammal. PloS One 6: e27021. doi: 10.1371/journal.pone.0027021 22046435

56. Hampton M, Melvin RG, Andrews MT. (2013) Transcriptomic analysis of brown adipose tissue across the physiological extremes of natural hibernation. PloS One 8: e85157. doi: 10.1371/journal.pone.0085157 24386461

57. Yan J, Burman A, Nichols C, Alila L, Showe LC, et al. (2006) Detection of differential gene expression in brown adipose tissue of hibernating arctic ground squirrels with mouse microarrays. Physiol Genomics 25: 346–353. 16464973

58. Schwartz C, Hampton M, Andrews MT. (2013) Seasonal and regional differences in gene expression in the brain of a hibernating mammal. PloS One 8: e58427. doi: 10.1371/journal.pone.0058427 23526982

59. Williams DR, Epperson LE, Li W, Hughes MA, Taylor R, et al. (2005) Seasonally hibernating phenotype assessed through transcript screening. Physiol Genomics 24: 13–22. 16249311

60. Bouma HR, Kroese FG, Kok JW, Talaei F, Boerema AS, et al. (2011) Low body temperature governs the decline of circulating lymphocytes during hibernation through sphingosine-1-phosphate. Proc Natl Acad Sci U S A 108: 2052–2057. doi: 10.1073/pnas.1008823108 21245336

61. de Vrij EL, Vogelaar PC, Goris M, Houwertjes MC, Herwig A, et al. (2014) Platelet dynamics during natural and pharmacologically induced torpor and forced hypothermia. PloS One 9: e93218. doi: 10.1371/journal.pone.0093218 24722364

62. Melvin RG, Andrews MT. (2009) Torpor induction in mammals: recent discoveries fueling new ideas. Trends in Endocrinology & Metabolism 20: 490–498.

63. Johnson JS, Reeder DM, Lilley TM, Czirják GÁ, Voigt CC, et al. (2015) Antibodies to Pseudogymnoascus destructans are not sufficient for protection against white-nose syndrome. Ecology and Evolution 5: 2203–2214. doi: 10.1002/ece3.1502 26078857

64. Muller LK, Lorch JM, Lindner DL, O’Connor M, Gargas A, et al. (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. doi: 10.3852/12-242 22962349

65. Schmieder R, Edwards R. (2011) Fast identification and removal of sequence contamination from genomic and metagenomic datasets. PloS One 6: e17288. doi: 10.1371/journal.pone.0017288 21408061

66. Simao FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM. (2015) BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics.

67. Love MI, Huber W, Anders S. (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15: 550. 25516281

68. Leng N, Dawson JA, Thomson JA, Ruotti V, Rissman AI, et al. (2013) EBSeq: an empirical Bayes hierarchical model for inference in RNA-seq experiments. Bioinformatics 29: 1035–1043. doi: 10.1093/bioinformatics/btt087 23428641

69. Suzuki R, Shimodaira H. (2006) Pvclust: an R package for assessing the uncertainty in hierarchical clustering. Bioinformatics 22: 1540–1542. 16595560

70. Eden E, Navon R, Steinfeld I, Lipson D, Yakhini Z. (2009) GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists. BMC Bioinformatics 10: 48. doi: 10.1186/1471-2105-10-48 19192299

71. Supek F, Bošnjak M, Škunca N, Šmuc T. (2011) REVIGO summarizes and visualizes long lists of gene ontology terms. PloS One 6: e21800. doi: 10.1371/journal.pone.0021800 21789182

72. Westermann AJ, Gorski SA, Vogel J. (2012) Dual RNA-seq of pathogen and host. Nature Reviews Microbiology 10: 618–630. doi: 10.1038/nrmicro2852 22890146

73. Pannkuk EL, Risch TS, Savary BJ. (2015) Isolation and identification of an extracellular subtilisin-like serine protease secreted by the bat pathogen Pseudogymnoascus destructans. PLoS One 10: e0120508. doi: 10.1371/journal.pone.0120508 25785714

74. O'Donoghue AJ, Knudsen GM, Beekman C, Perry JA, Johnson AD, et al. (2015) Destructin-1 is a collagen-degrading endopeptidase secreted by Pseudogymnoascus destructans, the causative agent of white-nose syndrome. Proc Natl Acad Sci U S A 112: 7478–7483. doi: 10.1073/pnas.1507082112 25944934

75. Smyth C, Schlesinger S, Overton B, Butchkoski C. (2013) The alternative host hypothesis and potential virulence genes in Geomyces destructans. Bat Res News 54: 17–24.

76. Whittington A, Gow NA, Hube B. (2014) From commensal to pathogen: Candida albicans. In: Anonymous Human Fungal Pathogens.: Springer. pp. 3–18.

77. Casadevall A, Steenbergen JN, Nosanchuk JD. (2003) ‘Ready made’virulence and ‘dual use’virulence factors in pathogenic environmental fungi—the Cryptococcus neoformans paradigm. Curr Opin Microbiol 6: 332–337. 12941400

78. Swidergall M, Ernst JF. (2014) Interplay between Candida albicans and the antimicrobial peptide armory. Eukaryot Cell 13: 950–957. doi: 10.1128/EC.00093-14 24951441

79. Hoyt JR, Cheng TL, Langwig KE, Hee MM, Frick WF, et al. (2015) Bacteria isolated from bats inhibit the growth of Pseudogymnoascus destructans, the causative agent of white-nose syndrome. PLoS ONE 10: e0121329. doi: 10.1371/journal.pone.0121329 25853558

80. Rizzetto L, Kuka M, De Filippo C, Cambi A, Netea MG, et al. (2010) Differential IL-17 production and mannan recognition contribute to fungal pathogenicity and commensalism. J Immunol 184: 4258–4268. doi: 10.4049/jimmunol.0902972 20228201

81. Reid DM, Gow NA, Brown GD. (2009) Pattern recognition: recent insights from Dectin-1. Curr Opin Immunol 21: 30–37. doi: 10.1016/j.coi.2009.01.003 19223162

82. Yamasaki S, Matsumoto M, Takeuchi O, Matsuzawa T, Ishikawa E, et al. (2009) C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia. Proc Natl Acad Sci U S A 106: 1897–1902. doi: 10.1073/pnas.0805177106 19171887

83. Zhu L, Zhao X, Jiang C, You Y, Chen X, et al. (2013) C-type lectin receptors Dectin-3 and Dectin-2 form a heterodimeric pattern-recognition receptor for host defense against fungal infection. Immunity 39: 324–334. doi: 10.1016/j.immuni.2013.05.017 23911656

84. Bernard F, Morel F, Camus M, Pedretti N, Barrault C, et al. (2012) Keratinocytes under fire of proinflammatory cytokines: bona fide innate immune cells involved in the physiopathology of chronic atopic dermatitis and psoriasis. J Allergy 2012.

85. Kunz S, Wolk K, Witte E, Witte K, Doecke W, et al. (2006) Interleukin (IL)-19, IL-20 and IL-24 are produced by and act on keratinocytes and are distinct from classical ILs. Exp Dermatol 15: 991–1004. 17083366

86. Werner S, Krieg T, Smola H. (2007) Keratinocyte–fibroblast interactions in wound healing. J Invest Dermatol 127: 998–1008. 17435785

87. Ramirez-Carrozzi V, Sambandam A, Luis E, Lin Z, Jeet S, et al. (2011) IL-17C regulates the innate immune function of epithelial cells in an autocrine manner. Nat Immunol 12: 1159–1166. doi: 10.1038/ni.2156 21993848

88. Taylor PR, Roy S, Leal SM Jr, Sun Y, Howell SJ, et al. (2014) Activation of neutrophils by autocrine IL-17A-IL-17RC interactions during fungal infection is regulated by IL-6, IL-23, ROR [gamma] t and dectin-2. Nat Immunol 15: 143–151. doi: 10.1038/ni.2797 24362892

89. Heath WR, Carbone FR. (2013) The skin-resident and migratory immune system in steady state and memory: innate lymphocytes, dendritic cells and T cells. Nat Immunol 14: 978–985. doi: 10.1038/ni.2680 24048119

90. Palmer JM, Kubatova A, Novakova A, Minnis AM, Kolarik M, et al. (2014) Molecular characterization of a heterothallic mating system in Pseudogymnoascus destructans, the fungus causing white-nose syndrome of bats. G3 (Bethesda) 4: 1755–1763.

91. Harden LM, du Plessis I, Poole S, Laburn HP. (2008) Interleukin (IL)-6 and IL-1β act synergistically within the brain to induce sickness behavior and fever in rats. Brain Behav Immun 22: 838–849. doi: 10.1016/j.bbi.2007.12.006 18255258

92. Moalem G, Tracey DJ. (2006) Immune and inflammatory mechanisms in neuropathic pain. Brain Res Rev 51: 240–264. 16388853

93. Couture R, Harrisson M, Vianna RM, Cloutier F. (2001) Kinin receptors in pain and inflammation. Eur J Pharmacol 429: 161–176. 11698039

94. Brownlee-Bouboulis SA, Reeder DM. (2013) White-nose syndrome-affected little brown myotis (Myotis lucifugus) increase grooming and other active behaviors during arousals from hibernation. J Wildl Dis 49: 850–859. doi: 10.7589/2012-10-242 24502712

95. Wilcox A, Warnecke L, Turner JM, McGuire LP, Jameson JW, et al. (2014) Behaviour of hibernating little brown bats experimentally inoculated with the pathogen that causes white-nose syndrome. Anim Behav 88: 157–164.

96. Tobin DM, Ramakrishnan L. (2013) TB: the Yin and Yang of lipid mediators. Current Opinion in Pharmacology 13: 641–645. doi: 10.1016/j.coph.2013.06.007 23849093

97. Prendergast BJ, Freeman DA, Zucker I, Nelson RJ. (2002) Periodic arousal from hibernation is necessary for initiation of immune responses in ground squirrels. Am J Physiol Regul Integr Comp Physiol 282: R1054–62. 11893609

98. Plaisance EP, Lukasova M, Offermanns S, Zhang Y, Cao G, et al. (2009) Niacin stimulates adiponectin secretion through the GPR109A receptor. Am J Physiol Endocrinol Metab 296: E549–58. doi: 10.1152/ajpendo.91004.2008 19141678

99. Lei M, Dong D, Mu S, Pan Y, Zhang S. (2014) Comparison of brain transcriptome of the greater horseshoe bats (Rhinolophus ferrumequinum) in active and torpid episodes. PloS One 9: e107746. doi: 10.1371/journal.pone.0107746 25251558

100. Zhang Y, Pan Y, Yin Q, Yang T, Dong D, et al. (2014) Critical roles of mitochondria in brain activities of torpid Myotis ricketti bats revealed by a proteomic approach. Journal of Proteomics 105: 266–284. doi: 10.1016/j.jprot.2014.01.006 24434588

101. Meteyer CU, Barber D, Mandl JN. (2012) Pathology in euthermic bats with white nose syndrome suggests a natural manifestation of immune reconstitution inflammatory syndrome. Virulence 3.

102. Vonhof MJ, Russell AL, Miller-Butterworth CM. (2015) Range-Wide Genetic Analysis of Little Brown Bat (Myotis lucifugus) Populations: Estimating the Risk of Spread of White-Nose Syndrome. PloS One 10: e0128713. doi: 10.1371/journal.pone.0128713 26154307

103. Peig J, Green AJ. (2009) New perspectives for estimating body condition from mass/length data: the scaled mass index as an alternative method. Oikos 118: 1883–1891.

104. Bolger AM, Lohse M, Usadel B. (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30: 2114–2120. doi: 10.1093/bioinformatics/btu170 24695404

105. Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, et al. (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nature Protocols 8: 1494–1512. doi: 10.1038/nprot.2013.084 23845962

106. Langmead B, Trapnell C, Pop M, Salzberg SL. (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10: R25. doi: 10.1186/gb-2009-10-3-r25 19261174

107. Li B, Dewey CN. (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12: 323-2105-12-323.

108. 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): 289–300.

109. McGinnis S, Madden TL. (2004) BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids Res 32: W20–5. 15215342

110. Meyer F, Paarmann D, D'Souza M, Olson R, Glass EM, et al. (2008) The metagenomics RAST server—a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics 9: 386-2105-9-386.