Pseudogymnoascus destructans transcriptome changes during white-nose syndrome infections


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Pseudogymnoascus destructans transcriptome changes during white-nose syndrome infections

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Title:
Pseudogymnoascus destructans transcriptome changes during white-nose syndrome infections
Series Title:
Virulence
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Reeder, Sophia M.
Palmer, Jonathan M.
Prokkola, Jenni M.
Lilley, Thomas M.
Reeder, DeeAnn M.
Field, Kenneth A.
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Dual RNA-Seq ( local )
Fungal Virulence ( local )
Host-Pathogen Interactions ( local )
Transcriptomics ( local )
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serial ( sobekcm )

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White nose syndrome (WNS) is caused by the psychrophilic fungus Pseudogymnoascus destructans that can grow in the environment saprotrophically or parasitically by infecting hibernating bats. Infections are pathological in many species of North American bats, disrupting hibernation and causing mortality. To determine what fungal pathways are involved in infection of living tissue, we examined fungal gene expression using RNA-Seq. We compared P. destructans gene expression when grown in culture to that during infection of a North American bat species, Myotis lucifugus, that shows high WNS mortality. Cultured P. destructans was grown at 10 to 14 C and P. destructans growing in vivo was presumably exposed to temperatures ranging from 4 to 8 C during torpor and up to 37 C during periodic arousals. We found that when P. destructans is causing WNS, the most significant differentially expressed genes were involved in heat shock responses, cell wall remodeling, and micronutrient acquisition. These results indicate that this fungal pathogen responds to host-pathogen interactions by regulating gene expression in ways that may contribute to evasion of host responses. Alterations in fungal cell wall structures could allow P. destructans to avoid detection by host pattern recognition receptors and antibody responses. This study has also identified several fungal pathways upregulated during WNS infection that may be candidates for mitigating infection pathology. By identifying host-specific pathogen responses, these observations have important implications for host-pathogen evolutionary relationships in WNS and other fungal diseases.

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RESEARCHPAPER Pseudogymnoascusdestructans transcriptomechangesduringwhite-nose syndromeinfectionsSophiaM.Reedera,JonathanM.Palmerb,JenniM.Prokkolaa,ThomasM.Lilleya,DeeAnnM.Reedera, andKennethA.Field aaDepartmentofBiology,BucknellUniversity,Lewisburg,PA,USA;bCenterforForestMycologyResearch,NorthernResearchStation,USForest Service,Madison,WI,USAARTICLEHISTORYReceived17November2016 Revised11May2017 Accepted10June2017ABSTRACTWhitenosesyndrome(WNS)iscausedbythepsychrophilicfungus Pseudogymnoascusdestructans that cangrowintheenvironmentsaprotrophicallyorparas iticallybyinfectinghibernatingbats.Infections arepathologicalinmanyspeciesofNorthAmericanbats,disruptinghibernationandcausing mortality.Todeterminewhatfungalpathwaysarein volvedininfectionoflivingtissue,weexamined fungalgeneexpressionusingRNA-Seq.Wecompared P.destructans geneexpressionwhengrownin culturetothatduringinfectionofaNorthAmericanbatspecies, Myotislucifugus ,thatshowshigh WNSmortality.Cultured P.destructans wasgrownat10to14Cand P.destructans growing invivo was presumablyexposedtotemperaturesranging from4to8Cduringtorporandupto37Cduring periodicarousals.Wefoundthatwhen P.destructans iscausingWNS,themostsigni cantdifferentially expressedgeneswereinvolvedinheatshockresponses,cellwallremodeling,andmicronutrient acquisition.Theseresultsindicatethatthisfunga lpathogenrespondstohost-pathogeninteractions byregulatinggeneexpressioninwaysthatmaycontributetoevasionofhostresponses.Alterations infungalcellwallstructurescouldallow P.destructans toavoiddetectionbyhostpatternrecognition receptorsandantibodyrespon ses.Thisstudyhasalsoidenti edseveralfungalpathwaysupregulated duringWNSinfectionthatmaybecandidatesformitigatinginfectionpathology.Byidentifyinghostspeci cpathogenresponses,theseobservationshaveimportantimplicationsforhost-pathogen evolutionaryrelationshipsi nWNSandotherfungaldiseases.KEYWORDSdualRNA-Seq;fungal virulence;host-pathogen interactions;transcriptomicsIntroductionFungalpathogenshaveemergedasmajorthreatstobiodiversity1andhumanhealth.2Thediversityoftheseinfectiouseukaryotesandtheirhostspresentnewchallengesin characterizingtheinteractionsbetweenhost,pathogen, andtheenvironmentthatleadtopathogenesis.Onesuccessfulapproachistousesystemsbiologytocompare whole-transcriptomechangesingeneexpressionbetween thepathogeninfectingthehost,thehostwithoutthe pathogen,andthepathogenwithoutthehost.3-5Thisdual RNA-Seqapproachcanbeusedtoidentifygeneticfactors fromthepathogenthatcontributetohostcolonization andmanipulationofhost-pathogeninteractions. Amongfungalemerginginfectiousdiseases,white-nose syndrome(WNS)inbatshasspreadfromEurasia,where itisendemic,toNorthAmerica,6-8whereitisdecimating severalspeciesofhibernatingbats.Susceptiblespecies, suchasthelittlebrownmyotis( Myotislucifugus )have shownpopulationdeclinesupto90%inaffectedhibernacula.9-11WNSiscausedby Pseudogymnoascusdestructans ,apsychrophilicfungusthatgrowsincold hibernaculaandcausescutaneousinfectionsinbatswhile theyhibernate.DuringWNS, P.destructans invadesthe skintissue,formingsubcutaneouslesionsidenti edas cuppingerosionsbyhistopathology.12Theinfectiondisruptsthehibernationbehaviorofsusceptiblebatsand leadstomorefrequentarousalsfromtorpor,premature energydepletion,electrolyteimbalance,anddeath.13-16WNSdoesnotaffectallspeciesofbatsequally.Many,but notall,NorthAmericanspeciesarebeingseverelyaffected,17,18whilemostEuropeanbatscanhost P.destructans infections, buthavelowmortalityfromWNS.19-22Coevolutionof P. destructans andEurasianbats,suchasDaubenton ’ smyotis ( M.daubentonii ),appearstohaveadaptedthesepopulations toacommensalorparasiticrelationshipwithlowerpathology.8NorthAmericanbats,ontheotherhand,haveyetto CONTACT KennethA.Field k eld@bucknell.edu BiologyBuilding,BucknellUniversity,Lewisburg,PA17837,USA. Supplementaldataforthisarticlecanbeaccessedon FigShare .©2017SophiaM.Reeder,JonathanM.Palmer,JenniM.Prokkola,ThomasM.Lilley,DeeAnnM.Reeder,andKennethA.Field.PublishedwithlicensebyTayl or&Francis. ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense( http://creativecommons.org/licenses/by/4.0/ ),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.VIRULENCE 2017,VOL.8,NO.8,1695 – 1707 https://doi.org/10.1080/21505594.2017.1342910

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bene tfromsuchselectionagainstextirpationofthehostspecies23andsomespeciesfacethepossi bilityofregionalextinctions.10,18,24Thevirulenceofthe P.destructans infectionis controlledbyacombinationoftheenvironment(i.e.,temperatureandhumidityofthehibernaculum),thehost(andthe host ’ sresponsetoinfection),andthepathogen(andthepathogen ’ sresponsetothehost).25Inthisstudy,wefocusonthethird componentofthisepidemiologicaltrianglebydissectingthe geneticcomponentsthatallow P.destructans toinfecthosts andbecomeavirulentpathogen. Whether P.destructans remainsacommensalparasite orbecomespathogenicisdeterminedbyhost-pathogen interactions.8,26Wehavepreviouslyexaminedthehost responseoftheWNS-susceptible M.lucifugus to P. destructans infectioninthewingmembraneandfound robustgeneexpressionchangesinthehostduringhibernation.27Wenowshiftourfocustocharacterizepreviouslyhypothesizedvirulenceattributesofthefungus thatincludeimmuneevasion,nutrientacquisition,stress responses,andtissueinvasion.28,29Wemeasured P. destructans geneexpressionatthewhole-transcriptome level,comparingexpressionpatternsbetweenthefungus whengrowingincultureandwheninfectingaNorth Americanbatspecies.ResultsTwodifferentgroupsofsampleswereusedtomeasure geneexpressionin P.destructans forthisstudy( Table1 ). Geneexpressionduringinfectionof M.lucifugus was measuredintheMyLusamplesofwingtissuefrom6 individual P.destructansinfected M.lucifugus collected 60 – 120minutesafterarousalfromhibernationincavesin Kentucky,USA.Geneexpressionin P.destructans during infectionwascomparedwith4samplesfromthe20631 – 21strainof P.destructans growingincultureat10 – 14C for23donSabourauddextroseagarplates( Table1 ). Comparisonofinfectedanduninfectedbats Priortocomparingtheexpressionof P.destructans genesduringhostinfectiontothoseinculture,we con rmedthatinfectionlevelsinhosttissuesweresufcienttomeasurepathogengeneexpressionbyquantifyingthenumberofRNA-Seqreadsthatmappedto the P.destructans transcriptome(TableS1).Compared toagroupofsamplesfrom M.lucifugus notinfected with P.destructans (FigureS1),thesamplesfromthe infectedbatsfromKentuckyshowedsigni cantly higherlevelsof P.destructans transcripts(t D 8.84,p < 0.00001).Inthewingsamplesfrominfectedbats, wefoundthat5990 § 324 P.destructans geneswere expressedataminimumcountof1,representing63% ofall P.destructans genes(TableS1).Thesesamples expressed13512 § 357 M.lucifugus genes,representing52%ofallbatgenes.Usingaminimumof1count inanysample,theculturedsamplesexpressed8825 genesandthewingsamplesexpressed7264genesof 9575known P.destructans genes(TableS1).These resultsindicatethatsuf cientreaddepthwasobtained inthisdatasettomeasure P.destructans geneexpression,atleastforthemajorityofgenes. Comparisonof P.destructans geneexpression duringWNSandculture Usingbothhierarchicalclustering( Fig.1A )andprincipalcomponentanalysis( Fig.1B ),wefoundthatthepatternsof P.destructans geneexpressionweresimilarin eachgroupofsamples(culturedorWNS).Weobserved asmallbatcheffectbetweentheculturedsamplesthat weregrownatdifferenttimesandsequenceddifferently ( Table1 ).WealsofoundthatsamplesfrombatsKY19 andKY23,whichcamefromadifferentcaveinthesame countyasbatsKY06,KY07,andKY11,27clusteredseparatelyfromthesesamplesandfromsampleKY39,which camefromadifferentcounty.Theseresultssuggestthat someofthedifferencesingeneexpressionthatwe observewithinthe2groupscouldbeduetovariationsin theenvironmentalconditionsorgeneticdifferences betweenthe P.destructans isolatedgrowingindifferent hibernacula.However,thelargestdifferencesappearto beduetothedifferentgrowthconditionsbetweencultureandgrowthonbats. Table1. RNA-SeqdatasetsusedforanalysisandRSEMexpectedcounts.GroupSampleSRAAccessionSequencingReadspost-trim P.destructans counts M.lucifugus countsPercent Pd MyLuKYMYLU06WSRR1916825PE101bp192898259905551901251.9% KYMYLU07WSRR1916826PE101bp1886252012183853793702.2% KYMYLU11WSRR1916827PE101bp193025169887850345001.9% KYMYLU19WSRR1916842PE101bp1764246012313945351562.6% KYMYLU23WSRR1916830PE101bp149979568524935997872.3% KYMYLU39WSRR1916832PE101bp176099945988842524021.4% CultureSRR1270148PE50bp2279242313820072 SRR1270408PE50bp2440030815080834 SRR1270412PE101bp10725095563836991 SRR1270417SE51bp27402575183019301696 S.M.REEDERETAL.

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Wethencompared P.destructans geneexpression duringWNSinfectionof M.lucifugus tothe20631 – 21 strainof P.destructans grownincultureusingboth edgeR( Fig.2 , 3A , 3B )andDESeq2( Fig.3C ).Because ofthelowerdepthofsequencingfortheWNSsamples,wethen lteredtheresultstoexcludeany P. destructans genesthatwerenotexpressedinatleast2 ofthe6MyLusamples.Withacutoffof0.001for FDRanda2-foldminimumchange,similarresults wereobtainedusingthese2differentanalysismethods ( Fig.3D ),withthemajorityofthegenesidenti edas differentiallyexpressedbyedgeRalsobeingidenti ed byDESeq2. Usingthesubsetofgenesidenti edbybothedgeR andDESeq2,94 P.destructans geneswereidenti edas morehighlyexpressedduringWNSinfectionof M.lucifugus ,and117genesweremorehighlyexpressedin P. destructans growinginculture( Fig.3D ,TableS2).Using ourTrinotateannotation,weidenti ed39genesthat showedsigni cantchangesinexpressionduringWNS whoseputativefunctionscouldcontributetovirulence byaffectingtissueinvasion,theheatshockresponse, nutrientacquisition,immuneevasion,andotherpathways( Table2 ). Wespeci callyexaminedtheexpressionlevelsof secretedproteases,becausetheyhavebeenimplicatedin thepathogenesisofWNS.30,31Proteasegeneswereidenti edbyhomologyandbyPFAManalysis32andthe expressionofthesegeneswascomparedinthe5culture samplesand6 M.lucifugus WNSsamples(TableS2). Table3 listsselectedproteasegenesanddemonstrates thatthegenesforsubtilase-familyproteasesaremore highlyexpressedduringculturethanduringtissueinvasion.Otherproteasesarehighlyexpressedduringhost infection,suchas VC83_01361 ,the P.destructans homologofthe Aspergillusfumigatus majorallergenAspf2, showlowergeneexpressionwhen P.destructans isgrowinginculture. Tofurtherexplorethefunctionalpathwaysthatregulateinfection,geneontologyenrichmentanalysiswas performedusingthegenesidenti edbyedgeRatamaximumFDRof0.05andminimumfold-changeof2.We examinedtheannotatedfunctionsof P.destructans genes upregulatedineither M.lucifugus infectionsorinculture ( Table4 ).Thisanalysisdeterminedthatseveralpathways involvedinpeptideandnitrogenmetabolismaresigni cantlyenrichedin P.destructans duringinfection(FDR < 0.05).Whilegrowinginculture, P.destructans showed enrichmentofoxidation-reductionandtransportpathways(FDR < 0.001)anddepletionofothermetabolic pathways(FDR < 0.05).DiscussionWedeterminedhowparasitismaffectstheexpression patternsof P.destructans genesbycomparingexpression levelsbetweenthefungusincultureandduringhost infection.WeuseddualRNA-Seqdataandanapproach thatsimultaneouslymappedthereadstobothhostand pathogentranscriptomesfollowedbytheremovalof readsthatmappedtohosttranscripts.Thisapproach allowedfortheestimationofexpressionlevelsof P. destructans geneswithhighlevelsofcon dencebyusing RSEMtocontrolfortheuncertaintyofmulti-mapped Figure1. Geneexpressionof P.destructans incultureandwhen infecting M.lucifugus .(a)Hierarchicalclusteringofdifferentially expressed P.destructans trimmedmeanofM-values(TMM)-normalizedgeneexpressionlevelsusingPearsoncorrelationcomplete-linkageclusteringwithEuclideandistances.Scaleshows Pearsoncorrelationcoef cient.Verticalbreaksintheheatmap indicateclusteringsupportedbybootstrapanalysisatacon denceof99%andthehorizontalbreakindicatesseparateclusteringofthedifferentgroupsofsamples.(b)Principalcomponent analysisofglobal P.destructans geneexpressionusinglog2-transformedTMM-normalizedexpressionlevels.TheprincipalcomponentsPC1andPC2represent96%and2%ofthevarianceinthe data,respectively.TrianglesrepresenttheMyLusamplesand circlesrepresenttheculturesamples.VIRULENCE 1697

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reads.Wecomparedgeneexpressionchangesofthecultured20631 – 21NorthAmericanstrainof P.destructans toinfectionofana € õ veNorthAmericanspecies.Although thedatasethadlimitedreaddepthfor P.destructans genesinthe M.lucifugus samples,weobservedsigni cantdifferentialgeneexpressionin211genes,or2.2%of the9575known P.destructans genes.Thisinitialstudy hasvalidatedthisapproachtoidentifyingchangingpatternsofpathogengeneexpression.Futurestudieswillbe neededtoovercomesomeofthelimitationsofthecurrentlyavailabledatasetsbyusinggreaterreaddepthfor thedualRNA-Seqdata,bettermatchingenvironmental conditions invitro tothoseinhibernacula,andusingthe identicalisolateof P.destructans forbothdatasets. Figure2. Expressionlevelsofdifferentiallyexpressed P.destructans genes.Heatmapsshowtheexpressionlevelincountspermillion (CPM)of(a)the94 P.destructans genesupregulatedintheMyLusamplescomparedwiththeCulturesamplesor(b)the117genesupregulatedintheCulturesamplescomparedwiththeMyLusamples.Geneswereidenti edasdifferentiallyexpressed(FDR < 0.001)by bothedgeRandDESeq2andexpressed(CPM > 0)inatleast2oftheMyLusamples.Thescaleislog10CPMwithamaximumof4.5(a) or4.1(b).1698 S.M.REEDERETAL.

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Futureworkcouldalsocomparechangesin P.destructans geneexpressionduringinfectionofNorthAmerican orEuropeanbatspeciesthatshowmoreresistanceto WNSmortalitythan M.lucifugus .8,19,20,33,34Asexpected,wefoundthatthetransitionfromabiotic toparasiticgrowthwasaccompaniedbymanychanges in P.destructans geneexpression.Differencesintemperatureandhumiditycouldalsocontributetothedifferencesingeneexpressionthatweobserved.Someofthe geneexpressionchangesarealsopresumablydueto alterationsinnutrientavailability,suchastheincreased expressionoflipase( VC83_00616)invivo duetothe highlipidcontentofmammalianskin.Althoughthecultured P.destructans wasnotgrownonthesamesubstrate thatitwould ndintheenvironment,manyofthegene expressionchangesthatweobservedappearconsistent withadaptationtothehostenvironment,ratherthan changesduetonutrientsources.Forexample,the increasedexpressionofheatshockgenesisconsistent withtheresponsetoarousalfromtorportoeuthermic Figure3. Differential P.destructans geneexpressionincultureandwheninfecting M.lucifugus . (a) Expressionof P.destructans genesis comparedbyedgeRbetweencultureand M.lucifugus infectionwithanMAplot.Themeanexpressionlevel(log2countspermillion (CPM))andthefoldchange(log2FC)areshownforeachgene.Genesmorehighlyexpressedincultureareontheupperhalfofthe graphandthosemorehighlyexpressedin M.lucifugus tissueinthelowerhalf.Bluepointsindicatedifferentialexpression(FDR 0.001 determinedbyedgeR)thatareexpressedinatleast2MyLusamples.Redpointsindicatesigni cantdifferentialexpressionforgenes thatwerenotexpressedinatleast2MyLusamples.AninteractiveversionofthisgraphisavailableasDataSetS2.AfterunzippingFile S2andopeningthehtml leinawebbrowser,hoverovereachpointtoviewtheannotationmetadataforthatgeneandtheexpression level(inlog2CPM)foreachsample.Individualgenescanbefoundbysearching,forexamplebyenteringVC83_01361inthesearchbox. (b) Expressionof P.destructans genesiscomparedbyedgeRbetweencultureand M.lucifugus infectionwithavolcanoplot.Thefold change(log2)andtheFDR(log10)areshownforeachgene.Genesmorehighlyexpressedincultureareontherighthalfofthegraph andthosemorehighlyexpressedin M.lucifugus tissueinthelefthalf.Bluepointsindicatedifferentialexpression(FDR 0.001determinedbyedgeR),withcolorsasfor(a).AninteractiveversionofthisgraphisavailableasDataSetS3andcanbemanipulatedas describedabove. (c) Expressionof P.destructans genesiscomparedbyDESeq2betweencultureand M.lucifugus infectionwithanMA plot.Themeanexpressionlevelandthefoldchange(log2)areshownforeachgene.Theredlineindicatesequalexpressionandthe bluelineindicatea2-foldchange.Genesmorehighlyexpressedincultureareontheupperhalfofthegraphandthosemorehighly expressedin M.lucifugus tissueinthelowerhalf.Redpointsindicatedifferentialexpression(FDR 0.001determinedbyDESeq2). (d) A Venndiagramcomparesthenumberof P.destructans genesidenti edasdifferentiallyexpressedbyedgeRandDESeq2.Thenumberof genessharedbyedgeRandDESeq2,oruniquetoeachmethod,areshownusingamaximumFDRof0.001andminimumfoldchange of2forgenesupregulatedin M.lucifugus infectionsorupregulatedincultureafterremovinggenesnotexpressedinatleast2ofthe MyLusamples.TableS2listsresultsforall P.destructans genes.VIRULENCE 1699

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bodytemperaturesthatoccurred60to120minutes beforecollectingthe M.lucifugus samples.27Correspondingly,asinglesamplefromabatthatwasallowedto becomeeuthermiconlybrie ydidnotshowupregulationof P.destructans heatshockgenes(unpublished results).Thermalstresscausedbyafebrileresponsein thehumanhosthasbeenshowntoactivateaheatshock responsein Candidaalbicans ,preventingdeleterious proteinunfoldingandaggregation.35Thisheatshock responsecouldbeimportantforfungalsurvivalinour system,asbatsarousetoeuthermictemperaturesseveral timesthroughouthibernation(thusseveraltimes throughout P.destructans infection),andsusceptible populationsarousefromtorpormorefrequentlyduring WNS.13,14,36Consistentwitharesponseofthepathogentoevade hostimmunerecognition,wealsofoundlargeincreases anddecreasesinexpressionofgenesinvolvedinfungal cellwallstructures( Table2 ).Thefungalcellwalliscomposedofaninnerlayerofchitin,amiddlelayerof Table2. Selected P.destructans genesdifferentiallyexpressedbetweencultureandWNS-affected M.lucifugus thathaveputativefunctionsimplicatedinfungalvirulence.edgeRDESeq2 GeneaFullNameBLASTXbFCcFDRdCulteWNSeFDRfSecretedEnzymes VC83_01361MajorallergenAspf2ALL2_ASPFU36.01.11E-0953.22044.87.80E-16 VC83_00616Lipase1LIP1_GEOCN9.11.84E-0531.3290.51.27E-07 HeatShockResponse VC83_0255330kDaheatshockproteinHSP30_NEUCR29.49.69E-07249.47737.63.63E-09 VC83_07843Hsp70nucleotideexchangefactorFES1FES1_NEUCR19.08.14E-0819.1378.49.52E-12 VC83_00970Heatshockprotein78,mitochondrialHSP78_SCHPO12.44.17E-06126.81643.31.15E-08 VC83_00522Proteinpsi1PSI1_SCHPO9.95.21E-0642.2431.61.90E-08 VC83_01964Heatshockproteinhsp88HSP88_NEUCR9.52.15E-05291.42874.62.03E-07 VC83_08137Heatshockproteinhsp98HSP98_NEUCR9.07.52E-05449.04178.23.60E-06 VC83_01046Heatshock70kDaprotein2HSP72_PARBA7.32.04E-042430.7185648.05E-06 VC83_02466UncharacterizedproteinC1711.08YNY8_SCHPO5.31.41E-04104.3571.72.61E-07 VC83_08187Heatshockprotein82HSP82_AJECA4.31.91E-03989.34490.97.48E-05 VC83_09034Unchar.Jdomain-containingproteinC63.13YCJD_SCHPO4.33.20E-0361.6272.26.18E-04 VC83_06435Heatshockproteinsti1homologSTI1_SCHPO4.12.03E-03192.3830.46.31E-05 IonHomeostasis VC83_01360Zinc-regulatedtransporter1ZRT1_YEAST18.65.62E-0891.11801.31.61E-12 VC83_07026Calcium-transportingATPase3ATC3_SCHPO11.72.53E-04125.61481.67.19E-05 VC83_00191PutativeCoppertransporterprotein(PFAM)10.32.37E-05112.51225.76.78E-07 VC83_06862Calcium-transportingATPase3ATC3_SCHPO6.24.74E-0536.3231.69.40E-07 VC83_01014Calcium-transportingATPase2ATC2_SCHPO3.42.64E-03193.9698.95.31E-06 VC83_04094MetalhomeostasisfactorATX1ATX1_YEAST ¡ 3.64.37E-0387.625.11.06E-03 VC83_00736Na( C )/H( C )antiporter1NAH1_ZYGRO ¡ 6.24.25E-07522.888.41.23E-09 CellWallRemodeling VC83_03500Spherulin-1ASR1A_PHYPO22.29.41E-0531.4758.36.99E-06 VC83_07867UncharacterizedproteinAFUA_6G02800YA280_ASPFU21.28.97E-08169.03783.05.93E-11 VC83_00788Endochitinase1CHI1_APHAL11.62.07E-04106.91327.31.93E-05 VC83_07327Probableglucanendo-1,3b -glucosidaseeglCEGLC_NEOFI6.51.93E-03221.51489.44.95E-04 VC83_04729Endochitinase1CHI1_COCIM6.38.24E-0583.2553.75.69E-07 VC83_07145Mannanendo-1,6a -mannosidaseDCW1DCW1_YEAST5.92.51E-0574.0450.77.40E-09 VC83_05104Chitinsynthase4CHS4_NEUCR ¡ 3.48.74E-04397.1121.71.56E-05 VC83_09076Glucan1,3b -glucosidaseEXG1_COCCA ¡ 3.51.99E-041722.2512.66.20E-10 VC83_00261Mannanendo-1,6a -mannosidaseDFG5DFG5_CANAL ¡ 3.83.97E-03145.439.13.12E-03 VC83_08448ProteinSUR7SUR7_CANAL ¡ 10.11.43E-03726.271.15.55E-03 VC83_05292CellwallmannoproteinCIS3CIS3_YEAS7 ¡ 14.83.30E-0597.96.81.76E-04 VC83_01650Mannanendo-1,6a -mannosidaseDCW1DCW1_YEAST ¡ 15.41.18E-06106.07.08.53E-07 Other VC83_06039Putativeheme-bindingperoxidaseCCPR2_ASPFU7.54.90E-0342.7340.32.49E-03 VC83_00225PutativecryptochromeDASH,mitochondrialCRYD_NEUCR7.52.15E-0518.2140.45.05E-07 VC83_06307SqualenemonooxygenaseERG1_CANAL4.52.51E-0440.5188.41.24E-07 VC83_03222ProbableGTPcyclohydrolase-2RIB1_SCHPO4.17.67E-0444.1189.42.58E-06 VC83_01624LeptomycinBresistanceproteinpmd1PMD1_SCHPO3.83.55E-03164.9662.74.06E-04 VC83_06509ThioredoxinreductaseTRXB_NEUCR3.12.03E-0349.8163.18.71E-07 VC83_08771ProbabletransporterMCH5MCH5_YEAST ¡ 4.22.18E-04177.545.38.53E-07 Notes.aP.destructans gene(Dreesetal.2016).bBLASThitwiththelowestE-valueintheSwissprotdatabase.OnlyhomologswithE < 1E-04wereconsidered.cFoldchangeingeneexpressionoftheWNSsamplescomparedwiththeculturesamplesdeterminedbyEdgeR.Negativevaluesindicatehigherexpressioni nthe culturesamples.DashedlinesseparategeneswithhigherexpressioninWNSfromgeneswithhigherexpressioninculture.dAdjustedp-valueofdifferentialexpressiondeterminedbyedgeRafterBenjamini-HochbergFDRcorrection.eMeannormalizedexpressionlevel(TPM)incultureorWNSsamplesdeterminedbyDESeq2.fAdjustedp-valueofdifferentialexpressiondeterminedbyDESeq2afterBenjamini-HochbergFDRcorrection.1700 S.M.REEDERETAL.

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b -glucans,andanouterlayerofmannose.Thecellwall providesrigidityandstructure,howeverisalsohighly dynamic.ThepatternrecognitionreceptorDectin-1has beenshowntobeareceptorforfungal1,3b glucansand 1,6b glucans,37,38thuscellwallcomponentsserveto alertthemammalianimmunesystemofafungalpathogen.WehaveobservedthatDectin-1andseveralother C-typelectindomainfamilymembersaresigni cantly upregulatedinbattissuesinfectedwith P.destructans .27Consistentwiththishostobservation,wedetectedsignificantalterationsin P.destructans enzymespredictedto beinvolvedinfungalcellwallremodeling( Table2 ). VC83_00788 and VC83_04729 ,homologsofEndochitinase1,anenzymewhichrandomlycleavesandbreaks downchitin,areupregulated11.6and6.3-fold,respectively,while VC83_05104 ,ahomologofChitinsynthase Table3. Expressionofselected P.destructans proteasegenes.edgeRDESeq2 GeneaFullNameBLASTXbFCcFDRdCulteWNSeFDRfSubtilase-familyProteases VC83_09074Subtilisin-likeprotease3(Destructin-3)SUB3_PSED21.80.6629.351.60.67 VC83_06062Subtilisin-likeprotease2(Destructin-1)SUB2_PSED2 ¡ 1.30.78502.4375.40.80 VC83_07090Subtilisin-likeproteaseSpm1SPM1_MAGO7 ¡ 1.50.302056.91421.40.12 VC83_06607ProteaseKexin2KEX2_CANAW ¡ 2.30.05147.866.90.04 VC83_04892Subtilisin-likeprotease1(Destructin-2)SUB1_PSED2 ¡ 3.00.335800.61962.11.00 VC83_02181Tripeptidyl-peptidasesed2SED2_ASPFU ¡ 5.50.0011791.6152.60.0045 OtherPutativeSecretedProteases VC83_01361MajorallergenAspf2ALL2_ASPFU36.01.11E-0953.22044.87.80E-16 VC83_03800Disintegrinandmetalloproteasedomain-containingproteinBADMB_ASPFU2.80.004494.6277.41.50E-10 VC83_02385ZincmetalloproteaseZmpBZMPB_STRPN2.50.1523.658.70.01 VC83_08633Threonineaspartase1TASP1_HUMAN2.20.2710.424.20.14 VC83_05359Calpain-likeproteasepalBPALB_EMENI1.80.2441.774.80.05 VC83_03810CarboxypeptidaseYhomologARB_06361SCPE_ARTBC1.90.5311.020.50.37aP.destructans gene(Dreesetal.2016).bBLASThitwiththelowestE-valueintheSwissprotdatabase.OnlyproteinswithE < 1E-04wereconsidered.cFoldchangeingeneexpressionoftheWNSsamplescomparedwiththeculturesamplesdeterminedbyEdgeR.Negativevaluesindicatehigherexpressioni nthe culturesamples.AdashedlineseparategeneswithhigherexpressioninWNSfromgeneswithhigherexpressioninculture.dAdjustedp-valueofdifferentialexpressiondeterminedbyedgeRafterBenjamini-HochbergFDRcorrection.eMeannormalizedexpressionlevel(TPM)incultureorWNSsamplesdeterminedbyDESeq2.fAdjustedp-valueofdifferentialexpressiondeterminedbyDESeq2afterBenjamini-HochbergFDRcorrection. Table4. Geneontologyanalysisof P.destructans pathwaysalteredduringWNS.GOCategoryBiologicalProcessE/P1Ratioinstudy2pFDR3UpregulatedduringWNSinfection GO:0006518peptidemetabolicprocesse23/4102.49E-08 < 0.001 GO:0006412translatione21/4109.07E-08 < 0.001 GO:0043043peptidebiosyntheticprocesse21/4101.90E-07 < 0.001 GO:0043603cellularamidemetabolicprocesse23/4105.57E-070.002 GO:0042254ribosomebiogenesise8/4101.04E-060.002 GO:0043604amidebiosyntheticprocesse21/4101.58E-060.002 GO:0022613ribonucleoproteincomplexbiogenesise8/4102.59E-060.002 GO:0044085cellularcomponentbiogenesise8/4101.16E-050.008 GO:0034645cellularmacromoleculebiosyntheticprocesse28/4102.62E-050.012 GO:1901566organonitrogencompoundbiosyntheticprocesse31/4102.82E-050.012 GO:0044271cellularnitrogencompoundbiosyntheticprocesse34/4109.20E-050.028 Upregulatedinculture GO:0055114oxidation-reductionprocesse75/8462.42E-07 < 0.001 GO:0055085transmembranetransporte56/8461.09E-06 < 0.001 GO:0044710single-organismmetabolicprocesse119/8468.21E-060.002 GO:0090304nucleicacidmetabolicprocessp18/8463.19E-050.004 GO:0006396RNAprocessingp1/8465.71E-050.01 GO:0046483heterocyclemetabolicprocessp31/8466.80E-050.01 GO:0072350tricarboxylicacidmetabolicprocesse7/8467.88E-050.012 GO:1901360organiccycliccompoundmetabolicprocessp33/8460.000110.024 GO:0006139nucleobase-containingcompoundmetabolicprocessp27/8460.000130.032 GO:0034641cellularnitrogencompoundmetabolicprocessp42/8460.000130.032 GO:0016070RNAmetabolicprocessp11/8460.000170.0461Enrichment(e)orpuri cation(p)detected.EnrichmentindicatesthattheGOcategoryismorehighlyrepresentedthanexpectedbychanceandpuri cationindicatesthatthecategoryisunderrepresented.2Numberofdifferentiallyexpressedgenesinthiscategorycomparedwithtotaldifferentiallyexpressedgenes.3Adjustedp-valueofenrichmentorpuri cationafterBenjamini-HochbergFDRcorrection.VIRULENCE 1701

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4isdownregulated3.4-foldin P.destructans during infectioncomparedwithculture.TwohomologstoGlucanendo-1,3b glucosidasesweredifferentiallyregulated; VC83_07327 wasupregulatedin P.destructans during infectionwhile VC83_09076 wasupregulatedduringculture.Theseenzymespresumablyregulatecellwall b -glycanturnoverandcatabolismof b -glycans39byremoval ofnon-reducingterminalglucosylresiduesfromsaccharidesandglycosides. Additionally,3Mannanendo-1,6a mannosidases thatweredifferentiallyexpressedbetween P.destructans activelyinfectingahostandgrowinginculture( Table2 ). Twowereupregulatedinculture( VC83_00261 and VC83_01650 ),andonewasupregulatedduringWNS ( VC83_07145 ).Mannanendo-1,6a mannosidasesare requiredfornormalsynthesisofthecellwallandalkaline pH-inducedhyphaformation,aswellasbeingresponsibleforrandomhydrolysisof a -mannosidiclinkagesin unbranchedmannans.40Itislikelythatthechangesin Glucanendo-1,3b glucosidaseandMannanendo-1,6a mannosidasegeneexpressionthatweobserveduponthe switchfromabioticgrowthtohostcolonizationleadsto substantialalterationsinthecellwallstructures.The resultingdifferencesinsaccharideandglycosidebranchingpatternsinthecellwallcouldmakethepathogenless recognizabletothemammalianimmunesystem. Alternatively,thesechangesincellwallenzymegene expressioncouldbeduetochangesinmetabolicpathwaysthataccompanytheshiftfromabiotictoinfectious niches.Differentcarbonsourcescanmodulatecellwall structureandvirulencein C.albicans .41,42Itispossible thatchangesincellwallstructuresarecausedbydifferencesinmetabolismwheninfectingbats,ratherthan directadaptationtothehost. Alterationsincellwallstructuresalsoaccompany shiftsinthemorphologicalgrowthtypeoffungi,suchas ashiftfromyeasttohyphalphasein C.albicans .37However, P.destructans growsvegetativelyashyphaeonboth Sabouraud ’ sdextroseagarmediuminculture,43,44and whenformingcuppingerosionsinthewingtissueofthe host.12,19Thusthereisnodifferenceinmorphotype betweenourculturedandWNS P.destructans samples thatmightexplainthedramaticalterationsinexpression ofcellwallremodelingenzymesthatweobserved.Consequently,weproposethatchangesinthe b ¡ glucan landscapeonthefungalsurfaceviacellwallremodeling areamechanismofimmuneevasionfor P.destructans , similartootherfungalpathogens.45Alterationsofthecellwallduringinfectioncould explaintheineffectivenessofantibodiesthatrecognize thecellwallofcultured P.destructans inprovidingprotectionfromWNS.46,47Theseresultsmayalsoexplain whyimmunizationwitheithercultured P.destructans or a b -glucanvaccine48didnotaffectthesusceptibilityof M.lucifugus toWNS(J.Johnson,J.McMichael,D. Reeder,andK.Field,unpublished).Theantigensprovidedbytheseimmunizationsmaynotbepresentonthe surfaceof P.destructans duringinfectionbecauseof changesinthecellwallstructurethataccompanythe transitionfromabiotictoparasiticgrowth. Becausetissueinvasionisahallmarkcharacteristicof P.destructans infectionsduringWNS,weexpectedthat expressionofgenesinvolvedindegradationoftheextracellularmatrixwouldbeupregulated.Unexpectedly,we foundthatthepreviouslycharacterizedsubtilase-familyof secretedproteases30,31showedlowerexpressionin P.destructans duringinfectionthaninculture.Instead, thehomologofthe A.fumigatus vacuolarprotease,major allergenAspf2,showedhighlevelsofexpressionduring infectionof M.lucifugus andwassigni cantlyupregulated comparedwithculturecondi tions.Thissuggeststhat otherproteasesmaybebettertargetsforpreventingcolonizationthanthesubtilase-familyproteases,althoughthe possibleroleofAspf2intissueinvasionremains unknown.Itisalsoplausiblethatsubtilase-familyproteasesareregulatedatapost-transcriptionallevelorare usedbythefungusprimarilyduringinitialcolonization. Therefore,furtherproteomic andexpressiontime-course experimentsmayproveusefultofurtherdissecttheinfection.Nevertheless,theabundantexpressionof Aspf2 , knowntobean A.fumigatus allergeninhumans,49suggeststhatfurtherinvestigationofIgE-mediatedallergic reactionsduringWNSmaybewarranted. Infectionofhostswasalsoassociatedwithchangesin expressionforseveralgenesinvolvedinthetransportor homeostasisofmetalions,includingzinc,iron,andcopper.Thisfungalresponsemaybeduetolimitedavailabilityofsomeofthesemicronutrientsinthehost,whichis likelysequesteringmetalionsasaformofnutritional immunity.27,50Changesinmicronutrientacquisitiongene expressionappeartobeassoci atedwithhostcolonization, includingincreasedexpressionofthezinctransporter Zrt1 ,thecopperhomeostasisfactor ATX1 ,andaputative coppertransporter,aswellastheunexpectedlossofsiderophoreimportusing MirB .51Homeostasisofthese micronutrientsisessentialfornormalfungalmetabolism andfortheabilityofthepathogentorespondtotheoxidativestressactivatedbythehostimmuneresponse.50However,ourgeneontologyanalysis( Table4 )indicates thatgenesinvolvedinoxidation-reductionpathwaysare morehighlyexpressedduringgrowthinculturethanhost colonization.Enrichmentofp athwaysinvolvingpeptide metabolismandtranslationin P.destructans infecting bats( Table4 )indicatesthathostcolonizationdemands higherlevelsofproteinexpressionthanabioticgrowth. Competitionbetweenthehostandpathogenfor1702 S.M.REEDERETAL.

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micronutrientsandthegenerationofoxidativestress likelyvariesoverthecourseofinfection52andfurther studyisneededtodissectthistimecourse. Together,theseresultsprovideamodelofgene expressionchangesin P.destructans thataccompanythe transitionsfromabiotictoparasiticgrowth( Figure4 ). Thismodelprovidesaframeworktounderstandhowthe pathogenrespondswithphenotypicplasticitytothe environmentanditshosttoadoptavirulentphenotype. Ourresultsalsosuggestapproachestominimizevirulenceand/orcolonizationbytargetingimmuneevasion, micronutrientacquisition,tissueinvasion,ortheheat shockresponse.Effortstounderstandwhysomespecies aremoresusceptibletoWNSthanotherswillrequire furtherexaminationofhost-pathogeninteractionsto determineifthepathogenrespondsdifferentlyinhosts thatexhibitlowerWNSsusceptibility.MaterialsandmethodsSamplecollection Twodifferentdatasetswereusedforthisstudy( Table1 ). Thesamplesforthe rstdataset(MyLu)consistedof wingtissuefrom6individual P.destructansinfected M. lucifugus (littlebrownmyotis)collected60 – 120minutes afterarousaltoeuthermyfr omhibernationfromcavesin Kentucky,USA,asdescribedpreviously.27Hibernacula temperatureswere4 – 6Catthetimeofcollectionand, basedonourpreviousexperience,weestimatethatskin temperaturevariedbetween4and8Cduringtorporand upto37Cduringperiodicarousals.Theseconddataset wasobtainedfromtheNorthAmerican20631 – 21strain of P.destructans growinginculturebyD.Akiyoshiand A.Robbins(DepartmentofInfectiousDiseaseandGlobal Health,CummingsSchoolofVeterinaryMedicine,Tufts University).The20631 – 21strainof P.destructans was obtainedfromD.Blehert(NationalWildlifeHealthCenter,US.GeologicalSurvey,Madison,WI,USA).Thefunguswasgrownincultureat10 – 14Cfor23don Sabourauddextroseagarplates(BDDiagnostics, #221180)( Table1 ).Sabourauddextroseagarcontains nutrientsourcesofdextrose,pancreaticdigestofcasein, andpepticdigestofanimaltissue.RNAwasisolatedusing aQiagenRNeasyLipidTissueKitafterdisruptionofthe cellsusingZymosBashingBeadLysisTubesandabead beateronmaximumspeedfor30secfor3timesand then20seconce,withcoolingonicebetweeneach. RNAsequencing RNAsequencingwasperformedusingIlluminasequencingassummarizedin Table1 .Priortoanalysisalldata setswerequalitytrimmedusingTrimmomaticv.0.3553withtheparametersSLIDINGWINDOW:4:5LEADING:5TRAILING:5MINLEN:25.Forsampleswith paired-endsequencing,onlyreadswithbothpairs remainingaftertrimmingwereusedforfurtheranalysis. AnalysisofthereadsusingFastQCv0.11.554andthe resultsofSTARmappingindicatethattherearenosigni cantdifferencesinthequalityoftheRNAinanyof theculturedsamplesfromtheMyLusamples. Differentialexpressionanalysis ThequalitytrimmedreadswerealignedusingSTAR v.2.5.1b55totheconcatenatedgenomesof M.lucifugus and P.destructans .For M.lucifugus ,weusedgenome assemblyMyoluc2.0andgenemodelsfromEnsembl release84.56For P.destructans ,weusedthegenome assemblyandgenemodelsfromDreesetal..57RSEM v1.2.2958wasthenusedtoapplyanexpectationmaximizationalgorithmtopredictgeneexpressioncountsfor eachtranscript.TheexpectedcountmatrixforallsamplesisavailableinDataSetS1.Todetermineifthenumberofreadsmappedto P.destructans transcripts providedsuf cientstatisticalpowertodetectdifferential expressionofthesegenes,weusedScotty59toanalyzethe expectedcountsgeneratedbyRSEM.Wedetermined that65%of P.destructans genesexpressedataminimum of4-foldchangecouldbedetectedwitha p -valuecutoff Figure4. Modelofthe P.destructans geneexpressionchanges thataccompanyWNS.Geneexpressionchangesby P.destructans arecomparedforabioticandparasiticgrowth.Thechangesin geneexpressionthatwefoundareassociatedwiththesephases areindicated.VIRULENCE 1703

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of0.05.Transcriptspermillion(TPM)wascalculatedby normalizingreadcountsforthelengthofeachtranscript andadjustingforthelibrarysizeofmappedreadsfor eachsample.58The M.lucifugus transcriptswerethen removedfromtheanalysisanddifferentialexpression wasdeterminedusingonly P.destructans transcripts. DifferentialexpressionbetweenconditionswasdeterminedusingeitherDESeq2v1.10.160oredgeRv.3.12.161afternormalizingacrosssamplesusingthetrimmed meanofM-values(TMM)method62andaminimum expressionlevelof2TPMcombinedacrossallsamples. Falsediscoveryrate(FDR)wasusedtocontrolformultiplecomparisonsusingtheBenjamini-Hochbergprocedure.63Hierarchicalclusteringwasperformedusing Rstatspackagev3.3.1withPearsoncorrelation complete-linkageclusteringofEuclideandistances.Clusteringwascon rmedbybootstrapanalysisusingpvclust v2.0 – 064atan a levelof99%and100000iterations. Geneswithoutexpression(expectedcount < 1)inat least2MyLusampleswereexcludedfromthe nalanalysis.Annotationsforeachgeneweredeterminedby usingTrinotatev3.0,NCBIBLASTv2.2.29 C65withthe UniProtKB/SwissProtdatabase(E-valuecutoffof1 £ 10¡ 4),andInterProScanv.5.20 – 59.0.66Geneontology annotationswereextractedfromtheInterProScanresults andgeneontologyenrichmentanalysiswasperformed usingGOATOOLSv0.6.967withenrichmentorpuri cationmeasuredbyFisher ’ sexacttestafterFDRcorrection.Disclosureofpotentialcon ictsofinterestNopotentialcon ictsofinterestweredisclosed.AcknowledgmentsWearegratefultoHilaryMorrison(MarineBiologicalLaboratory,WoodsHole,Massachusetts),DonnaAkiyoshi(Tufts University,NorthGrafton,Massachusetts),andAlisonRobbins(TuftsUniversity)fordepositingRNA-Seqdatasetsinto theSequenceReadArchiveandforhelpfulcomments.We thankNat aliaMart õ nkov aandJi r õ CMoravec,InstituteofVertebrateBiology,AcademyofSciencesoftheCzechRepublic, Brno,CzechRepublicforvaluablediscussionsofthismanuscript.Wealsothankthefollowingforassistanceincollecting samplesthatwereusedinthisstudy:JosephJohnsonatOhio University,Athens,Ohio;ShayneLumadueatBucknellUniversity;andDonnaAkiyoshiandAlisonRobbinsatTufts University.FundingThisworkwassupportedbytheUnitedStatesFishandWildlifeServicegrantF14AP00739toDMRandKAF,andthe SvenskaKulturfondentoTML.JMPalmerwasfundedbythe USForestService(NorthernResearchStation).ORCIDKennethA.Field http://orcid.org/0000-0001-7417-4386References[1]FisherMC,HenkDA,BriggsCJ,BrownsteinJS,Madoff LC,McCrawSL,GurrSJ.Emergingfungalthreatstoanimal,plantandecosystemhealth.Nature2012;484 (7393):186-94;PMID:22498624;https://doi.org/10.1038/ nature10947 [2]NucciM,MarrKA.Emergingfungaldiseases.ClinInfect Dis2005;41(4):521-6;PMID:16028162;https://doi.org/ 10.1086/432060 [3]EnguitaF,CostaM,Fusco-AlmeidaA,Mendes-Giannini M,Leit ~ aoA.TranscriptomicCrosstalkbetweenFungal InvasivePathogensandTheirHostCells:Opportunities andChallengesforNext-GenerationSequencingMethods.JFungi2016;2(1):7;https://doi.org/10.3390/ jof2010007 [4]SchulzeS,SchleicherJ,GuthkeR,LindeJ.HowtoPredict MolecularInteractionsbetweenSpecies?FrontMicrobiol 2016;7(March):1-13. [5]WestermannAJ,GorskiSA,VogelJ.DualRNA-seqof pathogenandhost.NatureRevMicrobiol2012;10 (9):618-30;PMID:22890146;https://doi.org/10.1038/ nrmicro2852 [6]BlehertDS,HicksAC,BehrM,MeteyerCU,BerlowskiZierBM,BucklesEL,ColemanJT,DarlingSR,GargasA, NiverR,etal.Batwhite-nosesyndrome:anemerging fungalpathogen?Science2009;323(5911):227. [7]LeopardiS,BlakeD,PuechmailleSJ.White-nosesyndromefungusintroducedfromEuropetoNorth America.CurrBiol2015;25(6):R217-19;PMID:25784035; https://doi.org/10.1016/j.cub.2015.01.047 [8]ZukalJ,BandouchovaH,BrichtaJ,CmokovaA,Jaron KS,KolarikM,KovacovaV,Kub atov aA,Nov akov aA, OrlovO,etal.White-nosesyndromewithoutborders: Pseudogymnoascusdestructans infectiontoleratedin EuropeandPalearcticAsiabutnotinNorthAmerica.Sci Rep2016;6(January):19829;PMID:26821755;https:// doi.org/10.1038/srep19829 [9]FrickWF,PuechmailleSJ,HoytJR,NickelBA,Langwig KE,FosterJT,BarlowKE,Bartoni ckaT,FellerD, HaarsmaA-J,etal.DiseasealtersmacroecologicalpatternsofNorthAmericanbats.GlobalEcologyandBiogeography2015;24(7):741-9;https://doi.org/10.1111/ geb.12290 [10]LangwigKE,FrickWF,BriedJT,HicksAC,KunzTH, MarmKilpatrickA.Sociality,density-dependenceand microclimatesdeterminethepersistenceofpopulations sufferingfromanovelfungaldisease,white-nosesyndrome.EcolLett2012;15(9):1050-57;PMID:22747672; https://doi.org/10.1111/j.1461-0248.2012.01829.x [11]TurnerGG,ReederD,ColemanJTH.AFive-yearAssessmentofMortalityandGeographicSpreadofWhite-Nose SyndromeinNorthAmericanBats,withalooktothe future.UpdateofWhite-NoseSyndromeinbats.BatRes News2011;52(2):13-27. [12]MeteyerCU,BucklesEL,BlehertDS,HicksAC, GreenDE,Shearn-BochslerV,ThomasNJ,GargasA,1704 S.M.REEDERETAL.

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