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

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

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Title:
The White-Nose Syndrome Transcriptome: Activation of Anti-fungal Host Responses in Wing Tissue of Hibernating Little Brown Myotis
Series Title:
PLOS Pathogens
Creator:
Field, Kenneth A.
Johnson, Joseph S.
Lilley, Thomas M.
Reeder, Sophia M.
Rogers, Elizabeth, J.
Behr, Melissa J.
Reeder, DeeAnn M.
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PLOS One
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White-nose syndrome ( lcsh )
Gene expression
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serial ( sobekcm )

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Abstract:
White-nose syndrome (WNS) in North American bats is caused by an invasive cutaneous infection by the psychrophilic fungus Pseudogymnoascus destructans (Pd). We compared transcriptome-wide changes in gene expression using RNA-Seq on wing skin tissue from hibernating little brown myotis (Myotis lucifugus) with WNS to bats without Pd exposure. We found that WNS caused significant changes in gene expression in hibernating bats including pathways involved in inflammation, wound healing, and metabolism. Local acute inflammatory responses were initiated by fungal invasion. Gene expression was increased for inflammatory cytokines, including interleukins (IL) IL-1β, IL-6, IL-17C, IL-20, IL-23A, IL-24, and G-CSF and chemokines, such as Ccl2 and Ccl20. This pattern of gene expression changes demonstrates that WNS is accompanied by an innate anti-fungal host response similar to that caused by cutaneous Candida albicans infections. However, despite the apparent production of appropriate chemokines, immune cells such as neutrophils and T cells do not appear to be recruited. We observed upregulation of acute inflammatory genes, including prostaglandin G/H synthase 2 (cyclooxygenase-2), that generate eicosanoids and other nociception mediators. We also observed differences in Pd gene expression that suggest host-pathogen interactions that might determine WNS progression. We identified several classes of potential virulence factors that are expressed in Pd during WNS, including secreted proteases that may mediate tissue invasion. These results demonstrate that hibernation does not prevent a local inflammatory response to Pd infection but that recruitment of leukocytes to the site of infection does not occur. The putative virulence factors may provide novel targets for treatment or prevention of WNS. These observations support a dual role for inflammation during WNS; inflammatory responses provide protection but excessive inflammation may contribute to mortality, either by affecting torpor behavior or causing damage upon emergence in the spring.

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K26-05061 ( USFLDC DOI )
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RESEARCHARTICLETheWhite-NoseSyndromeTranscriptome: ActivationofAnti-fungalHostResponsesin WingTissueofHibernatingLittleBrown MyotisKennethA.Field1* ,JosephS.Johnson1,ThomasM.Lilley1,SophiaM.Reeder1,Elizabeth J.Rogers1,MelissaJ.Behr2,DeeAnnM.Reeder11 DepartmentofBiology,BucknellUniversity,Lewisburg,Pennsylvania,UnitedStatesofAmerica, 2 DepartmentofPathobiologicalSciences,SchoolofVeterinaryMedicine,UniversityofWisconsin-Madison, Madison,Wisconsin,UnitedStatesofAmerica kfield@bucknell.eduAbstractWhite-nosesyndrome(WNS)inNorthAmericanbatsiscausedbyaninvasivecutaneous infectionbythepsychrophilicfungus Pseudogymnoascusdestructans ( Pd ).Wecompared transcriptome-widechangesingeneexpressionusingRNA-Seqonwingskintissuefrom hibernatinglittlebrownmyotis( Myotislucifugus )withWNStobatswithout Pd exposure. WefoundthatWNScausedsignificantchangesingeneexpressioninhibernatingbats includingpathwaysinvolvedininflammation,woundhealing,andmetabolism.Localacute inflammatoryresponseswereinitiatedbyfungalinvasion.Geneexpressionwasincreased forinflammatorycytokines,includinginterleukins(IL)IL-1 ,IL-6,IL-17C,IL-20,IL-23A,IL24,andG-CSFandchemokines,suchasCcl2andCcl20.Thispatternofgeneexpression changesdemonstratesthatWNSisaccompaniedbyaninnateanti-fungalhostresponse similartothatcausedbycutaneous Candidaalbicans infections.However,despitethe apparentproductionofappropriatechemokines,immunecellssuchasneutrophilsandT cellsdonotappeartoberecruited.Weobservedupregulationofacuteinflammatorygenes, includingprostaglandinG/Hsynthase2(cyclooxygenase-2),thatgenerateeicosanoidsand othernociceptionmediators.Wealsoobserveddifferencesin Pd geneexpressionthatsuggesthost-pathogeninteractionsthatmightdetermineWNSprogression.Weidentifiedseveralclassesofpotentialvirulencefactorsthatareexpressedin Pd duringWNS,including secretedproteasesthatmaymediatetissueinvasion.Theseresultsdemonstratethathibernationdoesnotpreventalocalinflammatoryresponseto Pd infectionbutthatrecruitmentof leukocytestothesiteofinfectiondoesnotoccur.Theputativevirulencefactorsmayprovide noveltargetsfortreatmentorpreventionofWNS.Theseobservationssupportadualrole forinflammationduringWNS;inflammatoryresponsesprovideprotectionbutexcessive inflammationmaycontributetomortality,eitherbyaffectingtorporbehaviororcausingdamageuponemergenceinthespring. PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 1/29 a11111 OPENACCESS Citation: FieldKA,JohnsonJS,LilleyTM,Reeder SM,RogersEJ,BehrMJ,etal.(2015)TheWhiteNoseSyndromeTranscriptome:ActivationofAntifungalHostResponsesinWingTissueofHibernating LittleBrownMyotis.PLoSPathog11(10):e1005168. doi:10.1371/journal.ppat.1005168 Editor: BruceSKlein,UniversityofWisconsinMadison,UNITEDSTATES Received: July17,2015 Accepted: August25,2015 Published: October1,2015 Copyright: 2015Fieldetal.Thisisanopen accessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense ,whichpermits unrestricteduse,distribution,andreproductioninany medium,providedtheoriginalauthorandsourceare credited. DataAvailabilityStatement: AllRNA-Seqfilesare availablefromtheSRAdatabase(accessionnumber SRP055976).Allmetagenomefilesareavailablefrom theMG-RASTdatabase(project11709). Funding: ThisworkwassupportedbytheUnited StatesFishandWildlifeServicegrantsF12AP01210 toDMR,F14AP00739toDMRandKAF,andalsothe WoodtigerFundtoDMRandKAFandBucknell University.Thefundershadnoroleinstudydesign, datacollectionandanalysis,decisiontopublish,or preparationofthemanuscript.

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AuthorSummaryWhite-nosesyndromeisthemostdevastatingepizooticwildlifediseaseofmammalsin history,havingkilledmillionsofhibernatingbatsinNorthAmericasince2007.Wehave usednext-generationRNAsequencingtoprovideasurveyofthegeneexpressionchanges thataccompanythisdiseaseintheskinofbatsinfectedwiththecausativefungus.We identifiedpossiblenewmechanismsthatmayeitherprovideprotectionorcontributeto mortality,includinginflammatoryimmuneresponses.Contrarytoexpectationsthat hibernationrepresentsaperiodofdormancy,wefoundthatgeneexpressionpathways wereresponsivetotheenvironment.Wealsoexaminedwhichgeneswereexpressedinthe pathogenandidentifiedseveralclassesofgenesthatcouldcontributetothevirulenceof thisdisease.Geneexpressionchangesinthehostwereassociatedwithlocalinflammation despitethefactthatthebatswerehibernating.However,wefoundthathibernatingbats withwhite-nosesyndromelacksomeoftheresponsesknowntodefendothermammals fromfungalinfection.Weproposethatbatscouldbeprotectedfromwhite-nosesyndrome iftheseresponsescouldbeestablishedpriortohibernationoriftreatmentscouldblockthe virulencefactorsexpressedbythepathogen.IntroductionWhite-nosesyndrome(WNS)isanepizooticdiseasethathaskilledmillionsofbatsinNorth America[ 1 2 ].WNSiscausedbythepsychrophile Pseudogymnoascusdestructans ( Pd )(formerly Geomycesdestructans ),anascomycetefungalpathogen[ 3 – 5 ]thataffectsbatsduring hibernation. Pd growsattemperaturesbetween2and18Candcaninfectbatswhilethey hibernate[ 4 6 ]. Pd isinvasiveanddamagesthecutaneoustissuesofbats,includingthewing [ 7 ],formingcharacteristiccuppingerosionsthatarediagnosticof Pd infection[ 8 ].Mortality ratesduetoWNSvarybyspecies.Inthelittlebrownmyotis, Myotislucifugus ,themortality rateisupto91%inaffectedcaves[ 9 10 ]whereasWNSresistancehasbeenreportedinthebig brownbat, Eptesicusfuscus [ 11 ].BatsinEuropeareexposedtoendemic Pd ,butdonotexhibit WNSmortalityandappeartoberesistanttothedisease[ 12 ],despitecutaneousinvasionby Pd [ 13 ]. Cutaneousinfectionby Pd causessomespeciesofbatstoarousemorefrequentlyfromtorpor[ 5 14 15 ].Althoughhibernatingmammalsspendlessthan1%oftheirtimeeuthermic [ 16 ],theyuseupto90%oftheirstoredenergyduringtheseperiods[ 17 18 ].Becauseeach arousalinlittlebrownmyotisutilizesanestimated108mgofstoredfat[ 18 ],theincreasein arousalfrequencycausedbyWNSexplains58%ofthemorbidityrateassociatedwith Pd infection[ 14 ].OtherfactorsthatarealsoassociatedWNSpathologyincludeeffectsof Pd infection ontheintegrityofwingtissue[ 7 19 ],electrolytebalanceandhydration[ 7 20 21 ],chronic respiratoryacidosis[ 22 ],oxidativestress[ 23 ],andimmunefunction[ 24 ].Therelativeimpor-tanceofeachofthesemechanismsincausingdeathinWNSisnotclear,andthemostlikely modelthathasemergedisamulti-stageprogressionofWNSwithcontributionsofseveralof thesefactors[ 22 ].DifferencesinsusceptibilitytoWNSbetweenspeciesinNorthAmericamay beexplainedinpartbydifferentresponsesto Pd infectionsuchaschangesinthermoregulatory behavior.Understandinghostresponsesto Pd infectionmayprovideinsightthatcouldbeusefulforimprovingsurvivalofaffectedspecies. Cutaneousfungalinfectionsinmammalsarefirstrecognizedbycomponentsoftheinnate immunesystem,includingC-typelectinreceptorsandToll-likereceptors[ 25 ].Conserved componentsofthefungalcellwallactivatepatternrecognitionreceptorsonphagocytessuchas TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 2/29 CompetingInterests: Theauthorshavedeclared thatnocompetinginterestsexist.

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neutrophils,macrophages,anddendriticcells,andonepithelialcells[ 26 ].Activationofthese cellscanleadtoinductionoftheinflammasome,theproductionofinflammatorycytokines, andgenerationofreactiveoxygenspeciesthatcanmediatefungalcellkilling[ 25 ].Theimportanceoftheinnateimmuneresponsetotheinitialrecognitionoffungalinfectionsisdemonstratedbytheobservationthatdeficienciesinthesesignalingpathwayscanleadtochronic fungalinfectionsinhumans[ 27 28 ].Intheabsenceofinvasion,colonizationbycommensal fungicanbemaintainedthroughtolerancemechanismsmediatedbyinteractionswithdendriticcellsandepithelialcellsintheskin[ 29 ].Localactivationofinnateimmunepathwayscan slowthegrowthofinvasivepathogenicfungiandpromotetolerance,possiblyleadingtoacommensalrelationshipwiththefungus[ 30 ],butisnotusuallysufficienttoclearinfections.ClearanceofinfectionstypicallyrequiresThelper(Th)cells,asdemonstratedbythesusceptibilityof patientswithacquiredimmunedeficiencysyndrome,immunosuppressanttherapy,orchemotherapytofungalinfections[ 31 ].TheseTcellresponsescanbemediatedbyTh17cells[ 32 33 ] or,insomecases,Th1cells[ 34 ],withTh2responsestypicallyassociatedwithgreatersusceptibility[ 35 ].Th17responsescancontributetoclearanceofinvasivefungalinfectionsthrough theactionsofIL-17AandIL-22[ 36 ]andthefurtherrecruitmentandactivationofneutrophils [ 37 ].TheseTcellsubsetshavenotbeenwellcharacterizedinbats,butthoseT-cellmediated immunemechanismsthathavebeenstudiedappeartobeconservedbetweenbatsandother mammals[ 38 – 41 ]. Fungalinfectionsinanimalsaretypicallylife-threateningonlyuponsuppressionofadaptive immuneresponsesinthehost,suchaswhenchytridfungus( Batrachochytriumdendrobatidis ) blockslymphocyte-mediatedinflammatoryresponses[ 42 ].Hibernationproducesanatural suppressionofsomeimmuneresponsesinmammalspecieswhereithasbeenstudied.During hibernation,whentheconservationofenergyiscritical,certainimmunologicalmechanisms aredownregulatedwhileothersremainunaffected[ 43 – 51 ].Changesduringhibernationcan includedepressedantibodyresponses[ 44 52 ],decreasedabilityofTandBlymphocytesto proliferateinresponsetochallenge[ 53 54 ],andreducedcomplementactivity[ 47 ].Hibernationdoesnotaffectallimmuneresponsesequally,asshowninthirteen-linedgroundsquirrels ( Ictidomystridecemlineatus )thathaveasuppressedT-independentantibodyresponsebutare capableofmountingaTcell-dependentresponseduringhibernation[ 44 ].Studiesoftranscriptome-widechangesduringhibernationinsquirrels[ 55 – 59 ]haveshownexpressionchangesin genesinvolvedinmetabolism,oxidativestress,proteinfolding,ischemia/hypoxia,andother processes,butthesestudieswerenotexamininganactiveimmuneresponse.Hibernationis alsoknowntoaffectthedistributionofleukocytes[ 45 60 ]andplatelets[ 61 ].However,we haveanincompleteunderstandingofhowhibernationaffectsthesuppression,orsubsequent recovery,ofimmuneresponses[ 43 ],orhowimmunephysiologyinbatsduringhibernation maydifferfromthatofrodents. Thecostofimmunesuppressionduringtorporispresumablyoutweighedbythebenefitsof energyconservationbecausemostpathogensarenotcapableofproliferatingatthelowbody temperaturesofhibernatinganimals.However,thepsychrophilicnatureof Pd allowsittoinfectbatswithinhibernacula[ 2 4 ].Thebriefeuthermicboutsofhibernatingbatsareshorter thanmostotherhibernatingmammalianspecies[ 14 62 ]anditmaynotbepossibleforabat navetoapathogentomountaprimaryimmuneresponseinthefewhoursthatitiseuthermic throughoutthehibernationseason.Wehaveobservedantibodyresponsesto Pd inbats,but theseresponsesarestrongestinactivebatsexposedto Pd afteremergencefromhibernation [ 63 ].Therefore,hibernatingbatsmaykeeppathogensincheckbyrelyingonhypothermia, innateimmuneresponses,and/ormemoryimmuneresponses.Thepsychrophilicnatureof Pd overcomesthefirstofthesebarrierstoinfectionandthedifficultyinfightingfungalpathogens TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 3/29

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withinnatemechanismsalonemayallow Pd toproliferateandinvadethecutaneoustissuesof bats. TheWNSpanzootichascreatedanurgentneedtounderstandifNorthAmericanbatpopulationscanpersistinthepresenceofthefungalpathogen[ 1 10 ].Understandingthecomplete arrayofhostresponsesmountedbybatsafflictedwithWNSmayhelpilluminatesourcesof variationinsurvivalwithinandamongbatspecies.Todeterminewhichhostresponsesare activatedby Pd infection,wemeasuredtranscriptome-widegeneexpressionlevelsinbatwing tissuefromhibernatingbatsaffectedbyWNS.Geneexpressionwascomparedtobatsthat werehibernatedincaptivityintheabsenceof Pd exposure.Wehypothesizedthat Pd infection wouldcausechangesingeneexpressionthatwouldrevealphysiologicalresponsesduring WNSthatmightbeeitherprotectiveorpathological.Byusingnext-generationRNAsequencingtoexaminetranscriptome-widegeneexpressionchangesweexpectedtodiscoverconsistent patternsofhostresponsesthatoccurin Pd -infectedtissues.Combinedwithchangesingene expressionwithinthe Pd pathogen,theseresultshaveprovidedasurveyofthehostandpathogeninteractionsoccurringduringWNS.Results GeneExpressionChangesRevealedbyNextGenerationRNA SequencingTodeterminethehostresponsemountedbylittlebrownmyotisto Pd duringhibernation,we measuredchangesingeneexpressionatthewholetranscriptomelevel.Wingtissuesamples wereobtainedfromhibernatinglittlebrownmyotiswithnoknownexposureto Pd andbats exhibitingphysicalsignsofWNS,asshownin Table1 .Histopathology[ 8 ]andquantitative PCR(qPCR)for Pd [ 64 ]wereusedtoconfirmtheWNSstatusofeachbat( Table1 ).Cupping erosionsdiagnosticofWNSwerefoundonall6batscapturedinKentucky,butonnoneofthe 5batsfromstatesnegativeforWNSatthetimeofcapture.Lowlevelsofneutrophilicinflammationwerefoundinall11wingsamples( Table1 ;Infl),althoughthisinflammationwasnot associatedwithsitesof Pd infection.All6WNS-affectedbatstestedpositivefor Pd byqPCR, althoughthefungalloadmeasuredonwingswabs( Table1 ;qPCR)didnotcorrelatewiththe numberofcuppingerosionsfoundbyhistology( Table1 ;WNS).Aspreviouslyshown[ 5 14 15 ],WNS-affectedbatshadsignificantlylowerbodycondition( Table1 ;SMI;p=0.017, t=2.9255,df=9). NextgenerationRNAsequencing(RNA-Seq)wasperformedusingpoly-AselectedRNA isolatedfromeachRNAlater-preservedwingtissuesample( S1Table ).Usingexpressionlevels of Pd -derivedtranscripts,weconfirmedthatall6WNS-affectedbatshadabundantexpression of Pd genes.The Pd -derivedtranscriptswerenotpresentatsignificantlevelsinanyofthe5 samplesfromunaffectedbats( S2Table ;p=2.2x10-6,t=21.5,df=5.33),includingtheMN090 samplethathadtestedpositivefor Pd byqPCRinoneofthetworeplicates( Table1 ).Because highlevelsofdifferentialexpressionof Pd transcriptswouldmakeitmoredifficulttodetectsignificantchangesinhostgeneexpression,theassemblywasfiltered[ 65 ]toremove Pd -derived sequences.Comparisonofthefilteredassemblywiththeoriginalrevealedthatremovingthe Pd sequencesdidnotsignificantlydecreasethecompletenessoftheassembly( S3Table )asdeterminedbyBUSCO[ 66 ].Thisfilteredassembly( S1Dataset )wasusedtocalculatedifferential expressioninhostgenesbetweentheunaffectedandWNS-affectedsamples. Wecomparedhostgeneexpressionacrossallsamples( S2Dataset )usingDESeq2[ 67 ]to identifytranscriptclustersthatwereexpressedataminimumof2-folddifferenceandsignificantatafalsediscoveryrate(FDR)of0.05( S1Fig ).Wefound1804transcriptclustersthat wereexpressedathigherlevels,and1925transcriptclustersexpressedatlowerlevels,inWNSTranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 4/29

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Table1.SamplesusedfornextgenerationRNAsequencing. SampleLocationDateCapturedDateSampledSexMassSMI1Pd LoadbyqPCR2Histology WNS3In 4MI011MineinDickinsonCo,MI5-Nov-201122-Mar-2012M6.706.69Negative09 MN064MineinSaintLouisCo,MN16-Nov-201122-Mar-2012F7.417.15Negative02 MN075MineinSaintLouisCo,MN16-Nov-201122-Mar-2012F7.477.68Negative050 MN090MineinSaintLouisCo,MN16-Nov-201122-Mar-2012M7.667.51Neg/Pos?013 IL114MineinLaSalleCo,IL17-Nov-201122-Mar-2012F7.137.40Negative02 KY06Cave1inBreckinridgeCo,KY12-Mar-201412-Mar-2014F6.046.1712000035225 KY07Cave1inBreckinridgeCo,KY12-Mar-201412-Mar-2014F6.906.891300043857 KY11Cave1inBreckinridgeCo,KY12-Mar-201412-Mar-2014M5.476.121690002883 KY19Cave2inBreckinridgeCo,KY12-Mar-201412-Mar-2014M6.286.93640001179 KY23Cave2inBreckinridgeCo,KY12-Mar-201412-Mar-2014F6.586.892100023410 KY39CaveinJacksonCo,KY13-Mar-201413-Mar-2014M6.286.69120000197101Scaledmassindex:(mass(ing)) (38.01/(forearmlength(inmm))^1.4062WingswabsfromMI,MN,andILweremeasuredinduplicateanddeterminedtobepositivefor Pd ifthecycle-thresholdwaslessthan40.Samplesfrom KYwerequanti edin Pd genomicequivalentsrelativetoswabsspikedwith10000 Pd conidia.3CuppingerosionscharacteristicofWNSperrollofwingtissue.4Fociofneutrophilicin ammationperrollofwingtissue. doi:10.1371/journal.ppat.1005168.t001 Fig1.HierarchicalclusteringofgeneexpressioninWNS-affectedandunaffectedbats. Hierarchical clusteringofdifferentiallyexpressedgenesusingPearsoncorrelationcomplete-linkageclustering.Scale showsPearsoncorrelationcoefficient. doi:10.1371/journal.ppat.1005168.g001 TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 5/29

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affectedbattissues( S4Table ).Hierarchicalclustering( Fig1 )revealedthatexpressionofthese transcriptsfromall5batswithoutWNSwassimilartoeachother.GeneexpressioninwingtissuefromWNS-affectedbatswasdifferentfromunaffectedbatsandmoresimilartoeachother, aspredicted.Thenormalizedexpressionlevelsofthe3729identifiedtranscriptclustersdifferentiallyexpressedarelistedin S4Table DifferentialexpressionofindividualgeneisoformswasfurtheranalyzedusingEBSeq[ 68 ], anempiricalBayesianapproachtomodelinggeneexpression.ForeachtranscriptclusteridentifiedasdifferentiallyexpressedbyDESeq2,weusedEBSeqtodetermineifanyoftheindividualtranscriptsweredifferentiallyexpressedataposteriorprobabilitygreaterthan0.99( S4 Table ).Ofthe3729differentiallyexpressedtranscriptclustersidentifiedbyDESeq2,EBSeq identifiedatleastonedifferentiallyexpressedtranscriptfor1427(38%oftotal,43%ofupregulatedgenesand33%ofdownregulatedgenes).Theseresultsindicatethatdifferencesingene expressionarelikelyduetoalternativesplicingorotherisoformdifferencesformanyofthedifferentiallyexpressedgenes. Toannotatethefunctionsofthesegenesandidentifythoselikelytobeinvolvedwithhost responsesto Pd infection,weusedtheTrinotatepipeline.BLASTwasusedtoidentify1365 upregulatedtranscriptsand325downregulatedtranscriptsinWNS-affectedtissueswithsignificanthomologytoknowngenesfromvertebratesintheSwissprotdatabase.Ofthe2295 remainingtranscripts,13weremappedtogenesfromnon-vertebratesintheSwissprotdatabase,presumablyduetoenvironmentalcontaminationorincompleteremovalof Pd transcript sequences.Ofthe2842trinitytranscriptclusterswithoutaBLASTxmatchinSwissprot,2731 (96%)werefoundtoaligntosequences(e-value < 0.0001)inthelittlebrownmyotisgenome. Ofthealignedtranscripts,204(7.4%)werefoundtocorrespondtopreviouslyidentifiednoncodingRNAsequences.Ofthe111transcriptclusterswithoutatranscriptthatalignedtothe littlebrownmyotisgenomeorSwissprot,BLASTwasusedtoaligntheirtranscriptstotheUniRef90database.Wefoundthat7genesalignedtovertebratehomologs,9alignedtofungal homologs,and15alignedtoothermetagenomicsequences.Wewereunabletoidentifyhomologoussequencesforanytranscriptsfrom80(2.1%)ofthetranscriptclustersthatweredifferentiallyexpressed. ExpressionlevelsfortheSwissprot-identifiedtranscriptclusterswiththe100lowest adjustedpvaluesareshownin Fig2 (see S4Table forallresults).Someofthedifferentially expressedgeneswithputativefunctionsthatwerepredictedtoassociatewithhostresponsesto afungalpathogenarelistedin Table2 .WNScauseddramaticchangesinexpressionofgenes involvedininflammation,immuneresponses,woundhealing,metabolism,andoxidative stress,eventhoughthebatswerehibernatingduringthe Pd infection.Mostofthesegeneswere upregulatedinWNS-affectedtissues,whileamuchsmallernumberofidentifiedgeneswith putativefunctionsinthesecategoriesweredownregulated(Tables 2 and S4 ). Todetermineifall6littlebrownmyotiswithWNSexhibitsimilarchangesingeneexpression,weperformedclusteringanalysisofthedifferentiallyexpressedtranscripts( Fig1 ).To confirmthesignificanceofthesepatternsofgeneexpression,bootstrapanalysisofclustering wasperformed[ 69 ].Theclusteringoftheunaffectedsamplestogetherandtheclusteringofthe WNS-affectedsamplestogetherwasverifiedwithaconfidenceof99%( Fig3A ).Principalcomponentanalysiswasperformedtobetterunderstandtherelationshipsbetweenthetranscripts expressedinthe11samples( Fig3B ).All5samplesfromunaffectedbatswereverysimilar basedonthefirstthreeprincipalcomponentsidentified,whichaccountfor71%ofthevariance inthesetranscripts.TheWNS-affectedbatsampleshavemorediversegeneexpression( S5 Table )andPC1(accountingfor44%ofthevariance)differentiatesall6fromtheunaffected batsamples.ThegenesrepresentedbyPC1includethosethataremorehighlyexpressedin unaffectedthanWNS-affectedwingtissue( Fig2 ).PC2(17%ofthevariance)andPC3(10%of TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 6/29

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Fig2.GlobaltranscriptionalanalysisofWNS-affectedandunaffectedbatsbyRNA-Seq. Centeredlog2foldchangesareshownforthe100most significantdifferentiallyexpressedidentifiedgenes.Adjustedpvaluesrangedfrom3.3x10-5to2.8x10-18.TheheatmapofTMM-normalizedFPKMexpression TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 7/29

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estimatesiscenteredandlog2scaledfromaminimumof-4.8toamaximumof4.8.TranscriptswereidentifiedbyBLASTalignmenttotheSwissProt database. doi:10.1371/journal.ppat.1005168.g002 Table2.SelectedgenesdifferentiallyexpressedinWNS-affectedtissues. Gene1FullNameFC2pvalue3padj4EBFC5PPEE6In ammation IL23AInterleukin-23subunitalpha32.64.8E-092.0E-0633.61.9E-08 PGH2ProstaglandinG/Hsynthase2(Cyclooxygenase-2)30.51.2E-142.1E-1142.33.2E-12 IL6Interleukin-630.31.1E-109.9E-0850.95.2E-09 MMP25Matrixmetalloproteinase-2525.75.1E-092.0E-0625.55.7E-06 CSF3RGranulocytecolony-stimulatingfactorreceptor23.71.7E-099.1E-0731.36.9E-08 CCL20C-Cmotifchemokine2022.01.4E-073.0E-0521.13.3E-06 IL20Interleukin-2020.22.9E-075.3E-0521.51.5E-06 CSF3Granulocytecolony-stimulatingfactor19.16.8E-081.8E-0544.42.3E-07 IL1BInterleukin-1beta17.75.6E-081.5E-0513.66.6E-06 IL1AInterleukin-1alpha15.44.5E-052.1E-0311.43.1E-04 PA21PhospholipaseA213.83.7E-049.1E-03NANA CCL2C-Cmotifchemokine212.36.5E-065.3E-0416.72.7E-05 IL17CInterleukin-17C10.36.5E-079.6E-0512.73.0E-05 IL19Interleukin-199.01.4E-044.7E-03NANA IL24Interleukin-247.63.4E-081.0E-0518.91.3E-05 NCF2Neutrophilcytosolfactor25.79.9E-053.8E-035.05.6E-04 PG12AGroupXIIAsecretoryphospholipaseA22.79.4E-071.3E-043.15.7E-07 S10ACProteinS100-A122.39.0E-041.7E-02NANA OtherImmuneGenes ABC3GDNAdC> dU-editingenzymeAPOBEC-3G32.43.9E-124.7E-0954.24.1E-10 LIRA6Leukocyteimmunoglobulin-likereceptorsubfamilyAmember623.11.5E-059.5E-04NANA HPTHaptoglobin18.93.3E-051.7E-0372.23.9E-08 CD3GT-cellsurfaceglycoproteinCD3gammachain14.86.7E-065.5E-04NANA CLC4DC-typelectindomainfamily4memberD12.57.0E-052.9E-03NANA PTPRCReceptor-typetyrosine-proteinphosphataseC12.31.4E-073.1E-0516.01.1E-04 CLC4EC-typelectindomainfamily4memberE12.33.6E-076.0E-05NANA CLC7AC-typelectindomainfamily7memberA10.95.4E-078.3E-05NANA CO3ComplementC310.12.7E-033.4E-0264.98.0E-08 TLR9Toll-likereceptor98.91.4E-061.7E-046.22.5E-05 S10A3ProteinS100-A38.44.8E-041.1E-02NANA CLC6AC-typelectindomainfamily6memberA7.01.5E-045.0E-037.03.9E-03 CLC1AC-typelectindomainfamily1memberA6.52.4E-062.6E-04NANA D103ABeta-defensin103A6.15.4E-064.7E-046.33.2E-03 CLC5AC-typelectindomainfamily5memberA5.13.3E-048.3E-03NANA BIRC3BaculoviralIAPrepeat-containingprotein33.47.9E-053.2E-034.63.3E-04 UNGUracil-DNAglycosylase-3.81.0E-031.8E-02NANA LEG3Galectin-3-3.45.0E-081.4E-050.281.0E-10 WoundHealing SPRR1Corni n184.64.1E-171.0E-1366.4 < 1E-16 LCE3CLatecorni edenvelopeprotein3C17.52.1E-091.1E-0615.06.2E-05 FIBBFibrinogenbetachain15.91.8E-045.6E-0367.25.2E-08 ( Continued ) TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 8/29

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thevariance)distinguishtheKY19,KY23,andKY39samplesfromtheothertwoWNSaffectedsamplesandfromtheunaffectedsamples.Therotationvaluesofprincipalcomponent analysis( S5Table )revealthatinflammatorygenesmadethegreatestcontributiontoPC2. Clusteringanalysisrevealeddiversehostresponsesamongthebatsinfectedwith Pd .MetabolicandInflammatoryImmunePathwaysAssociatedwithWNSWenextexaminedthefunctionalpathwaysthatweremostaffectedinlittlebrownmyotis infectedwith Pd .Forthisgeneontologyanalysis,DESeq2resultsontranscriptisoformswere usedwithahigherFDRthresholdof0.1,asistypicalforthistypeofanalysis.FromWNSaffectedbattissue,3104upregulatedtranscriptswerealignedwithBLASTtothehumanUniprotdatabase.Homologsforthesetranscriptswereidentifiedandalistof1937uniqueEnsembl IDsassociatedwithupregulatedgeneswasgenerated( S6Table ).GOrilla[ 70 ]wasusedto Table2. ( Continued ) Gene1FullNameFC2pvalue3padj4EBFC5PPEE6FIBAFibrinogenalphachain12.41.1E-031.9E-0288.11.6E-08 ARGI1Arginase-111.94.8E-041.1E-02NANA FIBGFibrinogengammachain11.81.4E-032.2E-0248.91.6E-07 EPGNEpigen10.32.0E-091.1E-0613.17.3E-06 EREGProepiregulin8.86.9E-071.0E-0410.74.8E-04 KLK6Kallikrein-68.13.4E-075.9E-059.14.2E-04 K1C17Keratin,typeIcytoskeletal175.91.3E-058.7E-046.63.7E-03 P63Tumorprotein633.12.0E-051.2E-0316.99.8E-05 Metabolism PLAC8Placenta-speci cgene8protein37.01.5E-098.4E-0725.91.3E-05 LIPPPancreatictriacylglycerollipase22.31.0E-061.3E-0419.42.7E-06 ANGL3Angiopoietin-relatedprotein317.21.5E-044.9E-03NANA APOC4ApolipoproteinC-IV16.17.9E-053.2E-03NANA APOC3ApolipoproteinC-III15.81.8E-045.5E-0342.58.7E-07 APOC2ApolipoproteinC-II14.53.7E-049.2E-0345.06.8E-07 FFAR2Freefattyacidreceptor27.71.8E-051.1E-037.39.1E-04 HCAR2Hydroxycarboxylicacidreceptor24.06.9E-041.4E-02NANA IP6K2Inositolhexakisphosphatekinase2-5.03.8E-063.6E-040.192.2E-03 ACACAAcetyl-CoAcarboxylase1-4.21.1E-031.9E-020.166.7E-03 OtherOxidativeStress MMP3Stromelysin-134.03.4E-102.5E-0757.76.6E-08 PERTThyroidperoxidase3.72.7E-033.4E-02NANA PRDX2Peroxiredoxin-23.61.1E-057.8E-04NANA HMOX1Hemeoxygenase13.05.4E-041.2E-028.21.7E-031BLASThitwiththelowestE-valueintheSwissprotdatabase.OnlygeneswithE < 1E-05wereconsidered.2FoldchangeingeneexpressionoftheWNS-affectedsamplescomparedtotheunaffectedsamplesasdeterminedbyDESeq2.Negativevaluesindicate higherexpressionintheunaffectedsamples.3ProbabilityofdifferentialexpressiondeterminedbyDESeq2.4AdjustedprobabilityofdifferentialexpressionafterBenjamini-HochbergFDRcorrection.5PosteriorprobabilityfoldchangeinEBSeq-estimatedexpressionofeachtranscriptinWNS-affectedtissuesoverunaffectedtissues.NAindicates thatno isoformforthatgenewasdifferentiallyexpressedatanFDR < 0.0016PosteriorprobabilityestimatebyEBSeqthattheisoformisdifferentiallyexpressed. doi:10.1371/journal.ppat.1005168.t002 TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 9/29

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determinesignificantlyupregulatedgeneontologycategoriesfromtheUniprotGOIDdatabase( Table3 and S7Table )andREVIGO[ 71 ]wasusedtovisualizebiologicalprocessesthat weresignificantlyoverrepresentedintheWNS-affectedtranscriptome( Fig4 ).Thefunctional analysisrevealedthat Pd infectionincreasesexpressionofgenesinvolvedinmetabolism, defenseresponses,andotherpathways( Table3 ). Fig3.Clusteringofgeneexpressionpatterns. (A)Bootstrapanalysisofregularized-logtransformed countstoassignconfidencelevelstosampleclustering.Redvaluesareapproximatelyunbiasedpvalues andgreenvaluesarebootstrapprobabilities.Redboxesindicateclusterssupportedataconfidencelevelof 99%.(B)Principalcomponentanalysisofregularized-logtransformedcountsofdifferentiallyexpressed genes.BlackspheresrepresentunaffectedbatsandwhitespheresrepresentWNS-affectedbats. doi:10.1371/journal.ppat.1005168.g003 TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 10/29

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ForthetranscriptsthatshowedlowerexpressioninWNS-affectedtissue( Fig2 ),thesame analysiswasperformed.Ofthe1152identifiedtranscriptsthatweredownregulatedatanFDR of0.1,694homologoushumangeneswereidentifiedbyBLASTandmappedtoEnsemblgene IDs( S6Table ).GOrilladidnotidentifyanyBiologicalProcesscategoriesthatweresignificantly downregulatedintheWNS-affectedbattissue.Host-PathogenInteractionsduringWNSToexaminethegeneexpressionofthe Pd pathogenusingadualRNA-Seqapproach[ 72 ],we separatelygeneratedagenome-guidedTrinityassembly( S3Dataset )withtheBroadInstitute G destructans genome.ThereadsfromeachoftheWNS-affectedtissuesweremappedonto thisassemblywithBowtieandgeneexpressionestimatedusingRSEM( S4Dataset ).Expression levelsforthe Pd geneswiththegreatestvarianceareshownin Fig5 .Hierarchicalclustering ( Fig6 )andprincipalcomponentanalysis( S2Fig )ofthedifferentiallyexpressedtranscripts indicatedthat Pd geneexpressionwasmostsimilarinthewingtissuesfrombatsobtainedfrom thesamehibernaculum( Table1 ).Theexpressionpatternsof Pd genesweremoresimilarfor KY06,KY07,andKY11,whichcorrespondstobatscapturedinCave1inKentucky,andfor KY19andKY23,whichwerecapturedfromCave2. Table3.Selectedover-representedgeneontologybiologicalprocesscategories. CategoryGO:BiologicalProcessTermp1FDR2Enrich3#DE4#Cat50051246regulationofproteinmetabolicprocess4.2E-155.3E-111.463441810 0006952defenseresponse2.3E-102.2E-071.56179881 0051248negativeregulationofproteinmetabolicprocess4.3E-102.9E-071.61156746 0080134regulationofresponsetostress9.9E-094.6E-061.461941019 0044403symbiosis,encompassingmutualismthroughparasitism3.4E-081.2E-051.64113528 0006953acute-phaseresponse3.5E-078.9E-054.111528 0034097responsetocytokine1.0E-062.1E-041.8858237 0045089positiveregulationofinnateimmuneresponse1.1E-062.1E-041.8957232 0006954in ammatoryresponse1.1E-062.1E-041.8164271 0030216keratinocytedifferentiation3.8E-065.4E-043.141844 0070555responsetointerleukin-17.5E-069.5E-042.921950 0002526acutein ammatoryresponse8.4E-061.0E-033.111742 0002755MyD88-dependenttoll-likereceptorsignalingpathway1.7E-051.8E-032.562266 0034162toll-likereceptor9signalingpathway4.1E-053.5E-032.562060 0050860negativeregulationofTcellreceptorsignalingpathway5.7E-054.5E-034.72813 2000378negativeregulationofreactiveoxygenspeciesmetabolicprocess8.7E-056.3E-033.521124 0009913epidermalcelldifferentiation9.0E-056.5E-032.442063 0030593neutrophilchemotaxis1.3E-049.1E-033.021333 0002223stimulatoryC-typelectinreceptorsignalingpathway2.3E-041.4E-022.0127103 0050878regulationofbody uidlevels2.7E-041.6E-021.4388472 0051005negativeregulationoflipoproteinlipaseactivity2.9E-041.7E-027.6844 0032480negativeregulationoftypeIinterferonproduction3.7E-042.1E-022.7713361Over-representedpvalue.2FalsediscoveryrateafterBenjamini-Hochbergadjustmentformultiplecomparisons.3Enrichmentofdifferentiallyexpressedgenesinthiscategory.4Numberofdifferentiallyexpressedgenesinthiscategoryidenti edbyGOrillaatapvaluecutoffof0.001.5NumberofgenesinthisGOcategoryrepresentedinthebackgroundset. doi:10.1371/journal.ppat.1005168.t003 TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 11/29

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Fig4.REVIGOtreemapsummarizinggeneontologybiologicalprocesscategoriesover-representedinWNS-affectedtissues. GOrillawasusedto identifyGeneOntologyBiologicalProcessesthatwereover-representedamongtranscriptsmorehighlyexpressedinWNS-affectedtissuesatanFDRc utoff of0.1( S6Table ).Over-representedcategorieswithpvaluesoflessthan0.001(290terms)wereusedtogenerateatreemapcoloredbyfunctionalcategory. Thesizeofeachrectangleisproportionaltothepvalueforthatcategory. doi:10.1371/journal.ppat.1005168.g004 TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 12/29

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Fig5.Transcriptionalanalysisof Pd geneexpressiononbatswithWNS. Centeredlog2foldchangesare shownfor100 Pd geneswiththegreatestvarianceandaminimumTMM-normalizedFPKMexpressionof1 inall6samples.Theheatmapisscaledfromaminimumof-4.4toamaximumof4.4. doi:10.1371/journal.ppat.1005168.g005 TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 13/29

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Thepossiblefunctionsofthe Pd genesexpressedamongtheWNS-affectedsampleswere analyzedbysequencehomology.Wefirstexaminedtheexpressionlevelsofafamilyofsecreted proteasesthathavebeenproposedtobeinvolvedinPdvirulence[ 73 74 ]andfoundthatthese alkalineproteaseswereexpressedby Pd inall6wingsamples( Table4 ).Destructin-2wasthe mosthighlyexpressedisoforminallWNS-affectedbat Pd samples. Wenextexaminedthe Pd transcriptclustersforadditionalfactorsthatcouldaffectvirulence.AlignmentbyBLASTtotheSwissprotandUniprot90databasesidentified12056transcriptswithsignificanthomologytoknownfungalgenes( S8Table ).Fortheremaining67 Pd transcriptclusters,Trinotatewasnotabletoidentifyknownfunctionaldomainsorsignalpeptidespresentinthesepreviouslyuncharacterized Pd transcripts.TheresultsfromtheBLAST alignmentwereexaminedforgenesknowntobeinvolvedinprocessesthatcouldaffect Pd virulence,suchassecretedproteases[ 73 – 75 ],metalbindingproteins[ 76 ],fungalcellwallremodeling[ 76 77 ],andothervirulencefactors[ 75 77 78 ].Thisanalysisidentified46 Pd genesthat couldbeinvolvedinpathogenesis( Table5 ),includingadditionalsecretedproteasesthatcould beinvolvedintissueinvasion. Fig6.Hierarchicalclusteringof Pd geneexpressiononbatswithWNS. Hierarchicalclusteringof differentiallyexpressedPdgenesusingPearsoncorrelationcomplete-linkageclustering.Scaleshows Pearsoncorrelationcoefficient. doi:10.1371/journal.ppat.1005168.g006 Table4.Expressionofdestructintranscriptsin Pd growingonWNS-affectedbats. IsoformUniProt Match ProteinNamePutativeFunction(s)KY061KY07KY11KY19KY23KY39 GG4320| c0_g1_i1 ALP_ACRCHDestructin-2(Alkaline protease-2) Subtilisin-familyalkaline protease 1004711.71038369644371157 GG6167| c0_g1_i1 ORYZ_ASPCLDestructin-1(Alkaline protease-1) Subtilisin-familyalkaline protease 516.8107.4720.0779.4192.4128.5 GG5612| c0_g1_i1 ORYZ_NEOFIDestructin-3Subtilisin-familyalkaline protease 8.319.223.6231.126.212.91TrimmedmeanofM-values-(TMM-)normalizedexpressiondeterminedbyRSEMinfragmentsperkilobaseoftranscriptpermillionmappedreads (FPKM). doi:10.1371/journal.ppat.1005168.t004 TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 14/29

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Table5.Possiblevirulencegenesexpressedin Pd growingonWNS-affectedbats. Pd GeneUniProtMatchProteinNamePutativeFunctionTMM-normalizedexpressionbyRSEM (FPKM) KY06KY07KY11KY19KY23KY39 GG1839| c0_g1 MSBP2_ARATHMembranesteroid-bindingprotein2Antimicrobialpeptideresponse63.6146.430.124.865.348.2 GG5000| c0_g1 DUR3_YEASTUreaactivetransporterAntimicrobialpeptide susceptibility 65.779.5107.051.619.814.6 GG5000| c1_g1 DUR31_SCHPOProbableureaactivetransporter1Antimicrobialpeptide susceptibility 079.5081.1102.10 GG680| c0_g1 GMDG_00178UncharacterizedproteinDrugresistancetransporter029.724.4141.0175.239.8 GG2580| c0_g1 SOL5_ALTSOBifunctionalsolanapyronesynthasePhytotoxinsynthesis, Pathogenesis 101.400157.399.20 GG6092| c0_g1 SOL5_ALTSOBifunctionalsolanapyronesynthasePhytotoxinsynthesis, Pathogenesis 66.435.8113.6714.71116352.5 GG3668| c0_g1 TOXA_COCCAPutativeHC-toxinef uxcarrier TOXA Toxintransporter20.4077.5143.885.062.7 GG3668| c1_g1 TOXA_COCCAPutativeHC-toxinef uxcarrier TOXA Toxintransporter78.6074.4122.540.10 GG432| c0_g1 TOXA_COCCAPutativeHC-toxinef uxcarrier TOXA Toxintransporter114.035.154.289.5131.953.1 GG432| c1_g1 TOXA_COCCAPutativeHC-toxinef uxcarrier TOXA Toxintransporter42.549.020.266.5132.50 GG680| c1_g1 TOXA_COCCAPutativeHC-toxinef uxcarrier TOXA Toxintransporter27.120.912.9148.7198.90 GG1568| c0_g1 CHI1_COCP7Endochitinase1Fungalcellwall305.226.9348.1581.946.027.2 GG2156| c0_g1 YCZ2_SCHPOPutativemannanendo-1,6-alphamannosidase Fungalcellwall32.56.37.757.4214.3113.9 GG1843| c0_g1 ENG1_SCHPOEndo-1,3(4)-beta-glucanase1Fungalcellwall77.7062.1196.48.626.0 GG2744| c0_g1 BGLE_NEOFIProbablebeta-glucosidaseEFungalcellwall115.2355.184.665.877.68.2 GG2744| c1_g1 BGLE_NEOFIProbablebeta-glucosidaseEFungalcellwall0238.000124.60 GG4455| c0_g1 AVR4_PASFURace-speci celicitorA4Fungalcellwall,Pathogenesis106.35.1158.0866.6309.2166.0 GG2938| c0_g1 BGBP_PENMOBeta-1,3-glucan-bindingproteinInnateimmuneactivation121.285.2150.434.568.4104.5 GG2938| c0_g2 BGBP_PENMOBeta-1,3-glucan-bindingproteinInnateimmuneactivation840.1332.2768.8783.6476.6339.3 GG6498| c0_g1 SR1A_PHYPOSpherulin-1AManganesebinding1067629.21202217.1373.46328 GG3236| c0_g1SBNA_RALMEPduncharacterizedproteinL8FZ85Siderophorebiosynthesis68.3205.460.548.3192.00 GG4315| c0_g2 FRE3_YEASTFerricreductasetransmembrane component3 Siderophoretransport,Iron binding 157.986.4135.776.186.495.6 GG5300| c0_g1 FRE3_YEASTFerricreductasetransmembrane component3 Siderophoretransport,Iron binding 242214411623510.9400.3197.0 GG6235| c0_g1 SIT1_YEASTSiderophoreirontransporter1Siderophoretransporter00023.1135.30 GG6235| c0_g2 SIT1_YEASTSiderophoreirontransporter2Siderophoretransporter115.7133.8170.1191.274.4107.0 ( Continued ) TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 15/29

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Becausethetissuesampleswerecollectedfrombatsfrom6differenthibernaculaforthis study,itispossiblethatdifferencesinhostorpathogengeneexpressionreflectdifferencesin theenvironmentalconditionspresentineachlocation,includingthemicrobiome.Inaddition, thehousingoftheunaffectedbatsincaptivityfor13weekspriortoanalysiscouldalsohave affectedthemicrobiome.Toexaminethedifferencesintheskinmicrobiomebetweenthebats, weusedMG-RASTtoidentifythelowestcommonancestorofmetagenomicsequencespresent Table5. ( Continued ) Pd GeneUniProtMatchProteinNamePutativeFunctionTMM-normalizedexpressionbyRSEM (FPKM) KY06KY07KY11KY19KY23KY39 GG2118| c0_g2 ZRT1_YEASTZinc-regulatedtransporter1Zinctransporter187316371447924.51211501.3 GG5016| c0_g2 ZRT1_YEASTZinc-regulatedtransporter2Zinctransporter457.4310.7449.366.9137.0564.1 GG3064| c0_g1 MIRB_EMENISiderophoreirontransportermirBResponsetoironion starvation 547.5169.3220.7136.1425.8259.6 GG861| c0_g1 MIRB_EMENISiderophoreirontransportermirBResponsetoironion starvation 656.0410.1712.3913.41324541.4 GG4694| c1_g1 LAC2_PODASLaccase-2Melaninbiosynthesis64.219.8329.4553.869.1179.0 GG6611| c0_g1 UREA_ASPFUUreaseNitrogenmetabolism49.80038.8151.1222.7 GG6611| c1_g1 UREA_ASPFUUreaseNitrogenmetabolism028.234.828.6112.60 GG6611| c3_g1 UREA_ASPFUUreaseNitrogenmetabolism054.8055.7142.60 GG6612| c0_g1 UREA_ASPFUUreaseNitrogenmetabolism000499.600 GG3518| c0_g1 ALL2_ASPFUMajorallergenAspf2Metallopeptidase,Fungal allergen 27894105102135105373449.6 GG2311| c0_g1 PRTA_ASPNGAspergillopepsin-2Asparticendopeptidase59.379.984.5709.2114968.8 GG2082| c0_g1 PEPA_ASPORAsparticproteasepep1Secretedaspartic endopeptidase 64.531.045.995.5370.10 GG2082| c0_g2 PEPA_ASPORAsparticproteasepep1Secretedaspartic endopeptidase 0.10.10.1189.75.10 GG4492| c0_g1 CARP_CRYPAEndothiapepsinSecretedaspartic endopeptidase 648.7460.5311.8406.5423.6216.0 GG448| c0_g1 LAP1_SCLS1Leucineaminopeptidase1Secretedleucylendopeptidase100.784.095.7283.3265.6183.0 GG788| c0_g1 SPM1_MAGO7Subtilisin-likeproteinaseSpm1Secretedserine endopeptidase 956.8952.7525.512601176919.4 GG2765| c0_g1 PEPS_ASPPHCarboxypeptidasecpdSSerinecarboxypeptidase83.2114.5146.9775.2981.3408.6 GG3562| c1_g1 SED4_ARTOCTripeptidyl-peptidaseSED4Serineendopeptidase, Pathogenesis 715.7366.5690.317032009191.9 GG2259| c0_g1 SOD6_CANALCellsurfacesuperoxidedismutase [Cu-Zn]6 Superoxidemetabolism, Pathogenesis 43.544.613.8556.7880.6179.3 GG4408| c0_g1 CCPR2_ASPFUPutativeheme-bindingperoxidaseOxidativestressresponse,Iron binding 1044842.8477.163.042.3421.4 GG6788| c0_g1 HOG1_CRYPAMitogen-activatedproteinkinase HOG1 Virulenceandconidia formation 735.1816.2959.1325.7373.5772.8 doi:10.1371/journal.ppat.1005168.t005 TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 16/29

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( S8Table ).Althoughthereweresomedifferencesobservedinthebacterialmicrobiomespresentonthewingsofthe11bats,therewerenosignificantchangesbetweentheWNS-affected andunaffectedsampleswhenbacteriawereidentifiedattheclasslevel.Severalstrainsof Pseudomonasfluorescens isolatedfrombattissueshavebeenidentifiedwith Pd growthinhibiting properties[ 79 ].MG-RASTanalysisshowedthat Pseudomonas speciesarepresentinall11 samples( S8Table ). P fluorescens transcriptsrepresented2.80.6%oftranscriptsidentified fromgammaproteobacteriaand0.400.05%ofallbacteriaonthewingsofunaffectedbatsand 0.370.07%ofallbacteriaonWNS-affectedbats. P fluorescens waspresentonalllittlebrown myotissampled,butwasrareandrelativeabundancewasnotstatisticallydifferentbetween WNS-affectedandunaffectedbats(p=0.49,t=-0.71,df=9).DiscussionThecomparisonofhostgeneexpressionbetweenWNS-affectedandunaffectedlittlebrown myotisclearlydemonstratesthat Pd infectioncausesphysiologicalresponsesinwingtissue, wheresubstantialfungalinvasionoftheskinoccursinWNS-affectedbats[ 8 ].Thechangesin transcriptlevelsthatwehaveobservedindicatethathostresponsestofungalinfectionremain intactduringhibernationandaresimilartothoseobservedduringtheinitialstagesoffungal infectionineuthermicmammals[ 32 ].Thesehostresponsesincludeacuteinflammation, woundhealing,andmetabolicchanges.Pathogengeneexpressionvariesamongbatswith WNS,suggestinghost-pathogeninteractionsthatmediatepathogenesis.Together,theseresults layafoundationtodeterminewhichhostandpathogenresponsescontributetoWNSresistanceandsusceptibilityandidentifytargetstoincreasesurvival.HostResponseto Pd InfectionThegeneexpressionchangesweobservedinthewingtissueofWNS-affectedbatsaresimilar tothoseobservedinothercutaneousfungalinfections[ 80 ].Cutaneous Candidaalbicans infectionsinhumansandmicetypicallyinitiateanimmuneresponsebyactivatingpatternrecognitionreceptorsoftheC-typelectinfamily[ 81 – 83 ]andthetoll-likereceptorfamily,bothof whichwefoundupregulatedinWNS-affectedbatwingtissue( S4Table ).TheseincludedCtypelectindomain(CLEC)familymembersCLEC4D(MCL),CLEC4E(MINCLE),CLEC7A (Dectin-1),CLEC6A(Dectin-2),andToll-likereceptor9.Inmiceandhumans,protectivehost responsesto C albicans areusuallycharacterizedbymanyofthesamecytokinesandchemokines[ 29 ]thatwehavefoundupgregulatedinWNS-affectedwingtissue,includingthecytokinesIL-1 ,IL-6,G-CSF,IL-23A,andIL-17C.Littlebrownmyotisinfectedwith Pd are increasingtranscriptionofthekeygenesnecessaryforinitiatingahostresponsethatprovides protectionfromfungalinfection.Thisclearlydemonstratesthathibernationdoesnotprevent innateimmuneresponsesinbatsinfectedwith Pd andthat,althoughtheyarenotclosely relatedtorodentsandprimates[ 41 ],batsrespondtofungalinfectionssimilarlytotheseother mammals. Theresponsesto Pd infectionwithinbatwingtissuemaybemediatedbykeratinocytesin theepithelialtissue.Activationofpatternrecognitionreceptorsbyfungalligandsisexpectedto inducekeratinocytestoproducemanyofthecytokinesthatwehavefoundupregulatedatthe transcriptlevelinWNS-affectedbatwingtissue[ 84 ].Inadditiontothecytokinestypically involvedin C albicans responsesdescribedabove,keratinocytesarealsoknowntoexpressthe chemokineCcl2andthecytokinesIL-20andIL-24inresponsetopatternrecognitionreceptor activation[ 85 ].KeratinocytesandfibroblastsarealsoknowntoexhibitaparacrineloopofIL-1 andIL-6activation[ 86 ]thatenhanceswoundhealingandhostdefensetomicrobialinfection andwefoundevidenceofIL-1andIL-6receptoractivationintheincreasedRNAlevelsfor TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 17/29

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transcriptionfactorp65,NF B,andP-selectinglycoproteinligand1( S4Table ).Another importantcytokineproducedbyepithelialcellsinresponsetoinfectionisIL-17C[ 87 ].Thisis anatypicalIL-17familymemberthatisexpressedbyepithelialcellsandcausesautocrine responsesintheepithelialcellsthatalsoexpresstheIL-17RAandIL-17REheterodimericIL-17 receptor[ 87 ].ThewingtissuetranscriptomesfromWNS-affectedandunaffectedbatsshow similarexpressionlevelsofbothIL-17RAandIL-17RE( S2Dataset )andwould,therefore,be expectedtoberesponsivetoIL-17C.Thegeneontologyanalysisalsofoundevidenceforfunctionalenrichmentofgenesinvolvedinkeratinocytedifferentiation,presumablyduetowound healingresponses.Keratinocytesorotherepithelialcellsinbatwingtissueappeartohave respondedtotheinvasionoftheepidermisbyfungalhyphae. Genesforpro-inflammatorymediatorscharacterizedtheinnateimmuneresponsethatwe observedinthewingtissueof Pd infectedbats.Undereuthermicconditionsthiswouldbe expectedtoprovideprotectionbytherecruitmentofmonocytesandneutrophils,mediatedby G-CSF,IL-23A,Ccl2,IL-17CandIL-6[ 88 ],andtheinitiationofanadaptiveTh17orTh1 response.However,undertheconstraintsofhibernation,responsesthatrequireleukocyte migrationdonotappeartooccurin Pd -infectedbats.Wedonotfindstrongevidenceof increasedexpressionforgenescharacteristicofeitherinnateoradaptiveleukocytes,exceptfor L-selectin,whichisexpressedonTcells,andCD177,whichisexpressedonneutrophils.Lower thanexpectedlevelsofmonocyte,neutrophil,Th1,andTh17cellrecruitmentmayberelatedto thesequestrationofleukocytesduringhibernation[ 45 ].However,wehaveobservedneutrophil recruitmentinhibernatinglittlebrownmyotisinresponsetoanotherfungalinfection ( Table1 ).Inthehistologicalexaminationofthecurrentsamples,wefoundneutrophilic inflammationinbothWNS-affectedandunaffectedwingtissue( Table1 ).However,thisinflammationdidnotoccuratthesitesof Pd infection.Curiously,wefoundasignificantincreasein WNS-affectedtissuefortranscriptsforCD3 andCD45thatcouldbeexpressedbygammadeltaTcellsorotherinnatelymphocytesthatresideintheskin[ 89 ].Itispossiblethat Pd isspecificallysuppressingneutrophiland/orTcellrecruitmentbyinterferingwithchemotacticsignals,similartothesuppressionofinflammatoryimmuneresponsesduringchytridiomycosisin amphibians[ 42 ].However,analysisoftissuelevelsofthecytokinesandchemokinesisnecessary toconfirmthesecretionoftheseproteins.BecauseneutrophilsandTcellsdonotappeartobe recruitedtositesof Pd infectionduringhibernation,onlylocalinflammatorymediatorsmaybe availableandtheyappeartobeunabletocontroltheinfectioninlittlebrownmyotis. Inadditiontoimmuneresponses,hibernatingbatsalsorespondto Pd infectioninother ways.Wefoundtranscriptsforproteinsfrommanypathwaysinvolvedinmetabolism,signaling,geneexpression,transport,migration,anddifferentiationthatwerealteredinWNSaffectedbats( Fig4 ).Wecannotexcludethepossibilitythatsomeofthesedifferencesweredue tothedifferenthibernationconditionsofthetwogroupsofbats.However,thedifferential expressionofthegenesinthesepathwaysdemonstratesthattheyaresubjecttoregulationduringhibernationandcanrespondtoinfection,tissuedamage,and/orenvironmentalchanges. Hostresponsestofungalinfectioncanbeinfluencedbychangesinthepathogen,including geneexpressionchangesinthecolonizingfungus,suchas C albicans [ 29 ].Wefoundsignificantvariabilityinthegeneexpressionby Pd ,whichisparticularlyinterestingbecauseall Pd in NorthAmericaispresumedtobeacloneofthesamematingtype[ 90 ].Thepathogenhas adopteddifferentgeneexpressionprofilesinthe6battissues( Fig5 ),perhapsinresponseto differencesinthehostenvironments.Correspondingly,hostgeneexpressionpatternsalso showdifferencesbetweentheWNS-affectedtissuesamples.Ofparticularinterestistheobservationthatthecytokineandchemokinegenesfoundinprincipalcomponent2ofourPCA analysis( Fig3B and S5Table )areexpressedatverydifferentlevelsinthe6 Pd -infectedsamples.Fromthisstudywecannotdeterminewhetherthedifferencesinpathogengeneexpression TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 18/29

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aredrivenbydifferencesinthehostenvironmentorviceversa.Althoughall6WNS-affected batshadvisiblesignsofWNS,hadsimilar Pd burdens,andsimilarhistopathology,itispossible thatthedifferencesinhostorpathogengeneexpressionthatweobservedmayhaveaffected progressionofWNSandsurvival.ResponsesthatMayContributetoWNSMortalityBecausetheincreasedfrequencyofarousalsfromtorporappearstobeaprimarycauseofWNS mortality[ 5 14 22 ],weconsideredpossiblemechanismsthatcouldaffecttorporboutlength. TheincreasedgeneexpressionofIL-1,IL-6,andotherpro-inflammatorycytokinesmediatesa localacuteinflammatoryresponseto Pd .Thesecytokinesalsohavesystemiceffectsthatmodify behaviorandthermoregulation[ 91 ].Inadditiontocytokineandchemokinetranscript increases,wealsofoundincreasedtranscriptsfortheenzymecyclooxygenase-2(prostaglandin G/Hsynthase2)andbothsecretedandcytosolicphospholipaseA2thatformcriticalinflammatorylipidmediatorssuchasprostaglandinH2.Theeicosanoidsgeneratedbytheseenzymes, alongwiththeactionsoftheupregulatedgeneskallikrein-6andcathepsinS,areexpectedto generatepainanditchingbylocallyactivatingneuronalnociceptors[ 92 93 ].This,inturn, couldaffecttorporboutlengthand/orbehaviorduringperiodicarousals.Indeed,wehavedocumentedsignificantlymoregroominginWNS-affectedbatsinfectedinthewild[ 94 ],although adifferentstudyonlaboratory-infectedbatsdidnotfindsimilarbehaviorchanges[ 95 ]. Together,theupregulatedgeneswilllikelygenerateaninflammatorymicroenvironmentwithin thewingthatmaycontributetotherobustwoundhealingresponsethatweobserveinWNSaffectedbats.However,inflammationcanalsoplayadetrimentalroleinsomediseases[ 96 ]. Furthertissuedamageandsubsequentwoundhealingoccursinsurvivingbatsuponemergence fromhibernation[ 19 ].Theselocalaffectsofinflammation(painanditching)aswellassystemiceffectsarelikelytoplayakeyroleinWNSpathology. Inadditiontothegeneexpressionchangesthatmaycontributetoacuteinflammation locallywithintheepithelialtissuesinvadedby Pd ,thesystemicreleaseoffebrilecytokinessuch asIL-6couldaffectthesignalsthatcontrolhibernationarousal.However,anexogenouspyrogen,lipopolysaccharide,isnotabletoprovokearousalsinhibernatinggolden-mantledground squirrels[ 97 ],soitmaybeunlikelythatinflammationorfebrilecytokinescandirectlytrigger arousalinWNS-affectedbats.IntracerebroventricularinjectionofprostaglandinE2ingoldenmantledgroundsquirrelsinducesarousalfromtorporandafebrileresponseduringan extendedperiodicarousal[ 97 ].Ourobservationofincreasedexpressionoftheenzymethat generatesprostaglandinH2mayprovideamechanismthatexplainstheshortenedtorporbouts inWNS-affectedbats,ifitcanbeshownthatthisenzymeisactiveinthetissueandproduces enoughprostaglandinH2toactsystemically. Inadditiontothechangesinexpressionofgenesinvolvedinimmuneresponsesandwound healing,wealsofoundsignificantchangesinmetabolicgenes.Wefoundevidenceofgene expressionchangesconsistentwithincreasedfatmetabolism,includingchangesintranscripts forapolipoproteins,lipidtransportproteins,proteinmetabolism,andcarbohydratemetabolism.Ofparticularinterest,wefoundincreasesintheexpressionofhydroxycarboxylicacid receptors2and3thatareknowntomediateadiponectinsecretion[ 98 ].Thissuggeststhat infectionwith Pd maydirectlytriggerchangesinlipidandcarbohydratemetabolismthatcontributetoWNSpathology.Thesechangesstandincontrasttothechangesthathavebeenseen inthebraintranscriptomeofhibernatinghorseshoebats( Rhinolophusferrumequinum )[ 99 ] andthebrainproteomeofhibernatingRickett Â’ sbig-footedbats( Myotispilosus )[ 100 ],which showdecreasedfatmetabolismduringtorpor.Byleadingtoprematuredepletionoffatstores, thesegeneexpressionchangescouldcontributetoWNSmortality. TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 19/29

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TheotherchangesinhostgeneexpressionthatweobservedareconsistentwithamultistageprogressionmodelofWNS[ 22 ].Wealsofoundsupportforchangesingenesinvolvedin oxidativestress[ 23 ]andbodyfluidlevels,whichmaycontributetoWNSprogression. Together,thepatternofgeneexpressionchangesthatwefindinlittlebrownmyotiswithWNS suggeststhatacombinationofmaladaptiveresponsesmaycontributetomortality.However, thenumberofupregulatedgenesinvolvedintheacuteinflammatoryresponsesuggeststhat excessiveinflammationmayalsobeafactorcontributingtopathologyevenpriortoemergence fromhibernationwhenitissuspectedtocontributetowingdamage[ 101 ].ImplicationsforFutureStudiesThechangesinhosttranscriptlevelsthatwehavefoundarepresumablycausedbyphysiologicalresponsesofthehosttoinfection.However,cautionmustbeusedwhenextendingthese transcriptionalresponsestofunctionalmechanismsbecausethecurrentstudydoesnotmeasureproteinormetabolitelevelsdirectly.Futurestudieswillbenecessarytodeterminewhich ofthegeneexpressionchangesobservedaffectwhichhostresponsemechanisms. ThelittlebrownmyotischosenfortheWNS-affectedsampleswereexhibitingWNSpathologyandappearedunlikelytosurviveatthetimeofsamplecollection.Forthisreason,itispresentlyuncertainwhichofthegeneexpressionchangesthatwehaveobservedarecontributing toprotectionandwhicharepathological.Anotherfactorthatlikelycontributestothevariation ingeneexpressionthatweobservedamongthesamplescollectedfromfree-rangingbatsisthe timesincethemostrecentarousalfromtorpor.Priortocollectionofeachwingtissuesample, batswereartificiallyarousedfor30to120minutes.Thisperiodofarousalissimilarinduration tothenaturalarousalsduringhibernationforlittlebrownmyotis[ 14 ],andpresumablyofsufficientdurationforsomeinnateimmuneresponsestooccurandfortranscriptlevelstobe altered.Onereasonforthisprocedurewastoavoiddisparitiesbetweentheelapsedtimefrom themostrecentarousalboutuntiltissuecollection.FortheWNS-affectedbatswecouldnot determinewhenthemostrecentnaturalarousalwouldhaveoccurred,butitwouldhavelikely beenmorerecentlythaninunaffectedanimals,asaffectedanimalsarousefromtorpormore frequently[ 14 ].Inthecurrentstudywecannotresolvewhetherthechangesingeneexpression thatweobservedoccurredduringthemostrecentarousal,duringpreviousperiodicarousals, orduringtorpor.Futurestudieswillbeneededtodeterminewhichofthechangesingene expressionthatweobservedduringWNSinbatsinthewildalsovaryincontrolledcaptive hibernationconditionswhenpriorarousalpatternsareknown.Furtherstudiesarealsoneeded tocomparethephysiologicalresponsesinbatsexhibitingWNSmorbiditytoresponsesinless susceptiblebats,suchasEuropeanspecies,NorthAmericanspeciesthatarelesssusceptible likethebigbrownbat[ 11 ],andtheremnantpopulationsoflittlebrownmyotisthatappearto havedevelopedtoleranceorresistanceto Pd [ 1 ].Suchstudiesshouldpointtoapathforward forbatsinNorthAmericatopersistinalandscapewhere Pd isendemic.ConclusionsLittlebrownmyotismountahostresponseto Pd infectionduringhibernation.WhichcomponentsofthisresponseareprotectiveorcontributetoWNSpathologyremainstoberesolved. TheinnateimmuneresponsewehaveobservedwouldbeexpectedtopromoteaTh17-directed adaptiveimmuneresponsethatcouldcleartheinfection.However,theenergeticconstraintsof hibernationmaypreventlittlebrownmyotisfromexecutionoftheTh17-andneutrophilmediatedphasesoftheimmuneresponse.Thismayleadtoexcessiveinflammatoryresponses, eitherduringhibernationoruponemergence.Thechangesinhostgeneexpressionthatwe observeddemonstratethatduring Pd infection,littlebrownmyotisalsoalterotherdefense TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 20/29

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responses,metabolicpathways,andtranscription.Numerous Pd genesthatmaycontributeto virulencewereidentifiedandtheserepresentpotentialpathogenresponsestohostdefense. Hibernationdoesnotpreventahostresponsetoinfectionandabetterunderstandingofthe differencesbetweenhostandpathogenresponsesinbatssusceptibletoWNSandthoseresistantmayleadtowaysforincreasingsurvival.MaterialsandMethods EthicsStatementThisstudywascarriedoutonbatsfromnon-endangeredspeciesinstrictaccordancewiththe recommendationsintheGuidefortheCareandUseofLaboratoryAnimalsoftheNational InstitutesofHealth.AllmethodswereapprovedbytheInstitutionalAnimalCareandUse CommitteeatBucknellUniversity(protocolDMR-016).Animalswerehumanelyeuthanized byisofluraneanesthesiaoverdosefollowedbydecapitation.InIllinois,animalcollectionwas conductedbystatewildlifeofficialsandanumberedpermitwasnotrequired.Scientificcollector ’ spermitswereobtainedinMichigan(SC1448),Minnesota(201174),andKentucky (SC1411147).SamplesWecollectedhibernatinglittlebrownmyotisfromcaveorminewallsatthelocationslistedin Table1 .Batscollectedfromalllocationsareexpectedtobefromthesamegeneticpopulation ofeasternlittlebrownmyotis[ 102 ].ForbatsunaffectedbyWNS,littlebrownmyotiswerefirst swabbedontheleftforearmforquantitativePCRanalysis.Aftermeasurementsweretaken, batswereindividuallyplacedinclothbagsandhunginconstanttemperaturethermoelectric coolers(KoolatronPC-3)maintainedat~7C.Water-saturatedspongeswereplacedinthebottomofeachcoolertomaintainhumidityduringtransportationtoBucknellUniversity.Bats werehousedfor13weeksinaPercival(modelI36VLC8)environmentalchamberwithconditionssetto4Cand95%relativehumidity.Batswereprovidedwaterthroughouthibernation. Batswerearousedfromhibernationfor30 – 120minutespriortoeuthanasia.ForWNS-affected bats,littlebrownmyotiswerecollectedinthefield,measured,swabbedforquantitativePCR, andhumanelyeuthanizedafterbeingarousedfromhibernationfor60 – 120minutes.Scaled massindex(SMI)wascalculatedusingtheformula(mass(ing))(38.01/(forearmlength(in mm))^1.406[ 103 ].WingtissuewasplacedinformalinforhistologyandplacedinRNAlater (Sigma-Aldrich)forgeneexpressionanalysis.RNAlatersampleswerestoredatambienttemperatureforupto24hoursbeforelong-termstorageat-80C.RNAwaspurifiedfrom50mg ofwingtissueusingaQIAGENRNeasyMiniKit.AllsamplesusedforRNAsequencinghad RNAintegrityvaluesgreaterthan7.0usinganAgilentBioanalyzer.VerificationofWNSStatusWingskintissuewasremovedfromthebonesofthearmanddigitsandrolledonto2cmparaffinwaxlogs.Thelogswerethenfixedin10%neutralbufferedformalinforatleast24hours. Eachlogwascutinto3piecesthatwereprocessedintoparaffinblocksovernightinaTissueTekVIPprocessor(SakuraFinetek).Thepieceswereembeddedinparaffinblocks,sectionedat 3microns,andstainedwithperiodicacidSchiffwithahematoxylincounterstain[ 8 ].WNS lesions( Table1 ;WNS)wereidentifiedascuppingerosionswithfungalhyphaeandconida present.Inflammatoryfoci( Table1 ;Infl)wereidentifiedasclustersofinfiltratingneutrophils andwerenotassociatedwiththeasymmetricalcurvedconidiaof Pd TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 21/29

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Todeterminepresenceorabsenceof Pd onbatsunaffectedbyWNS,eachswabwastested twicebyquantitativePCR[ 64 ]byJeffreyT.FosteratUniversityofNewHampshire.Acyclethresholdlessthan40wasusedasapositiveresult.Oneofthe5unaffectedbatshadonepositiveandonenegativetest( Table1 ),buthistology( Table1 )andsubsequentRNAsequencing determinedthistomostlikelybeafalsepositive( S2Table ;p=2.2x10-6).Forbatsaffectedby WNS,weperformedquantitativePCRtomeasurethe Pd load,ingenomicequivalentsnormalizedtoswabsspikedwith10000 Pd conidia,thatweredetectedoneachbat[ 15 ].NextGenerationRNASequencingTheGenomeSequencingandAnalysisFacilityattheUniversityofTexasatAustinperformed alllibrarypreparationandqualitycontrolprocedures.DirectionalRNAlibrarieswereprepared withpoly-AmRNAenrichment,dUTP/UDGstrand-specificlabeling,fragmentation,and200 basepairsizeselection.RNA-SeqwasperformedintwolanesofanIlluminaHiSeq2500with 101basepairlengthreadsobtained.TranscriptomeAssembliesThepairedreadsfromallsampleswerepreprocessedbyremovingadaptersandusingtrimmomaticPE[ 104 ]withsettingsofIlluminaclip:2:30:10,seedmismatches:2,palindromethreshold:30,clipthreshold:10,leading:5,trailing:5,minlength:36.Theremainingpairedreadswere thencombinedandTrinity(v2.0.4)wasusedinstrand-specificmode(RF)toconstructade novoassembly[ 105 ].K-merinsilicoreadnormalizationwithmaximumcoverageof50 resultedin22482456readpairsthatwereusedforassemblyoutof177755004total.The assemblywasthenfilteredtoremove Pd sequencesusingtheprogramDeconseq[ 65 ]withthe BroadInstitute Geomycesdestructans genome20631 – 21usedtoidentifypathogensequences andwiththelittlebrownmyotisgenome(Myoluc2.0)usedtoretainhostsequences.Bowtie 1.0.1[ 106 ]wasusedtodeterminethenumberofreadsthatmappedtoeachtranscriptinthe assembly.DifferentialExpressionThescriptalign_and_estimate_abundance.plincludedintheTrinityv2.0.6distribution[ 105 ] wasusedtoestimateexpressionlevelsforeachtranscript.Bowtie1.0.1[ 106 ]wasusedtomap reads(includingunpairedreadsafterqualitytrimming)fromeachsampleontotheassembly. RSEMv1.2.20[ 107 ]wasusedtoapplyanexpectationmaximizationalgorithmtopredictgene expressioncountsforeachtranscript.Expressionlevelsarepresentedaftertrimmedmeanof M-values(TMM)normalizationinfragmentsperkilobaseoftranscriptpermillionmapped reads(FPKM).DESeq2v1.8.1[ 67 ]wasusedtodeterminetheprobabilityofdifferentialexpressionforeachTrinitytranscriptclusterthathadaminimumRSEM-estimatedcount,before normalization,of5acrossallsamples.ForDESeq2analysis,thedefaultvaluesforremoving outliersandfilteringlowlyexpressedtranscriptswereused.Analphavalueof0.05wasused insteadofthedefaultof0.1todecreasethenumberofdifferentiallyexpressedgenesidentified. PosteriorprobabilitiesofdifferentialexpressionforindividualtranscriptisoformswereestimatedusingaBayesianapproachwithEBSeqv1.8.0[ 68 ].Falsediscoveryrate[ 108 ]wasused tocontrolformultiplecomparisons.NCBIBLASTv2.2.29+[ 109 ]wasusedtoidentifythe highest-rankingmatchforeachisoformintheUniProtSwissprotdatabase(downloadedon Sep17,2014)withane-valuecutoffof1x10-5. HierarchicalclusteringofsamplesandgeneswasperformedwithinR3.1.2usingthehclust functionwiththecompletelinkagemethod.Bootstrapanalysisofclusteringwasperformed TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 22/29

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usingthepvclust1.3 – 2packageand1000replications[ 69 ].Principalcomponentanalysiswas performedusingtheprcompfunctionandvisualizedwiththergl0.93.1098package.GeneOntologyNCBIBLASTv2.2.29+[ 109 ]wasusedwithane-valuecutoffof1x10-5toidentifyhomologsin theUniprotSwissprothumanproteindatabase(downloadedonNov25,2014)fortranscripts significantlyupregulatedinWNS-affectedbatwingtissuewithanFDRoflessthan0.1(in ordertoincreasethenumberofgenespriortosubsequentanalysiswithhigherstringency FDR).UniqueEnsemblgeneIDswereidentifiedfor1144ofthe1922upregulatedtranscripts and481ofthe1356downregulatedtranscripts.GOrilla[ 70 ]wasusedwithapvaluecutoffof 0.001toidentifyupregulatedordownregulatedbiologicalprocessesbycomparisontothebackgroundlistof12828humangenesidentifiedbyBLASTintheTrinityassembly.Multipletestingcorrection[ 108 ]wasusedwithanFDRcutoffof0.01.Resultswerevisualizedasatreemap withREVIGO[ 71 ].Pd GeneAnalysisTrinityv2.0.4wasusedtogeneratea Pd assemblyingenome-guidedmodewithjaccardclippingandusingtheBroadInstitute G destructans genome20631 – 21.Thisassemblywasused toassesspathogengeneexpressioninthesamplesfromWNS-affectedbatsusingRSEM v1.2.20[ 107 ].Trinotatev2wasusedtoannotatethe Pd transcriptsbyusingNCBIBLAST v2.2.29+[ 109 ]andboththeSwissprotandUniref90databases(downloadedonSep17,2014).MetagenomeAnalysisReadsforeachsamplewereanalyzedusingMG-RASTv.3.5[ 110 ]toidentifymetagenomic sequencesafterfilteringagainstthe B taurus genome(thetaxonomicallyclosestgenomeavailableforfiltering).Forassignmentoforganismabundance,thebesthitclassificationwasused withtheM5NRdatabase,maximume-valuecutoffof1x10-5,minimumidentitycutoffof60%, andminimumalignmentlengthcutoffof15.SupportingInformationS1Fig.MAplotofgeneexpressionusingthetrinitytranscriptomeassembly. Expression levelsforeverygeneareshownbycomparingRSEM-estimatedcountstothefold-changein expressionbetweenunaffectedandWNS-affectedbattissues.Bluepointsindicatesignificant differentialexpressiondeterminedbyDESeq2usinganFDRcutoffof0.05.Genesthatare morehighlyexpressedinWNS-affectedtissuesarefoundinthelowersideofthegraph. (TIF) S2Fig.Principalcomponentanalysisof Pd genes. TheTrinityutilityPtRwasusedtoconduct principalcomponentanalysisonthe Pd geneswithaminimumexpressionof10FPKM. (PDF) S1Table.ReadstatisticsofRNA-Seqsamples. (DOCX) S2Table.FPKManalysisof Pd -derivedtranscriptspriortoremoval. (DOCX) S3Table.Transcriptomeassemblycomparison. (DOCX) TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 23/29

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S4Table.DifferentiallyexpressedgenesdeterminedbyRSEMandDESeq2combinedwith EBSeqandtrinotateresults. (XLSX) S5Table.Principalcomponentanalysisrotationvalues. (XLSX) S6Table.DifferentiallyexpressedgenesusedforGOrillaanalysis. (XLSX) S7Table.Geneontologybiologicalprocesscategoriesover-representedinWNS-affected tissues. (XLSX) S8Table. Pd geneexpressionestimatedbyRSEMcombinedwithtrinotateresults. (XLSX) S9Table.MG-RASTanalysisofbesthitclassificationforbacterialgenes. (XLSX) S1Dataset.FASTAfileofdenovoassemblyoflittlebrownmyotistranscriptome. (ZIP) S2Dataset.RSEMgeneexpressionmatricesusedfordifferentialhostgeneexpressioncalculations. (ZIP) S3Dataset.FASTAfileofgenome-guidedtrinityassemblyof Pd transcriptome. (ZIP) S4Dataset.RSEMgeneexpressionmatricesfor Pd transcripts. (ZIP)AcknowledgmentsWethankMarianneMoore,SarahBouboulis,MeganVodzak,AllenKurta,BrookeHines,Larisa Bishop-Boros,andShayneLumadueforassistanceincollectingsamples.JamesW.McMichael IIIprovidedtechnicalassistanceandperformedthe Pd qPCRoftheWNS-affectedsamples.JeffreyFosterperformed Pd qPCRoftheunaffectedsamples.CindyRhone,GretchenLong,andthe restoftheanimalcarestaffatBucknellUniversityassistedinprovidingexcellentcareforthecaptiveanimalsforthisstudy.WethankJeremyDreeseandMichaelHarveyfortechnicalsupport withperformingbioinformaticsanalysisontheBucknellLinuxcluster.WeacknowledgeBrian Haas,TiagoHori,andtherestofthetrinityrnaseq-usersmailinglistforhelpfulassistancewith dataanalysis.WethankScottHunicke-SmithandtheGenomeSequencingandAnalysisFacility attheUniversityofTexasatAustinforperforminglibrarypreparationandRNAsequencing.AuthorContributionsConceivedanddesignedtheexperiments:KAFJSJDMR.Performedtheexperiments:KAF SMREJRMJB.Analyzedthedata:KAF.Wrotethepaper:KAFJSJTMLDMR.References1. ColemanJT,ReichardJD.(2014)BatWhite-NoseSyndromein2014:ABriefAssessmentSeven YearsAfterDiscoveryofaVirulentFungalPathogeninNorthAmerica.OutlooksonPestManagement 25:374 – 377. TranscriptomeofBatswithWhite-NoseSyndrome PLOSPathogens|DOI:10.1371/journal.ppat.1005168October1,2015 24/29

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