Frequent Arousal from Hibernation Linked to Severity of Infection and Mortality in Bats with White-Nose Syndrome


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Frequent Arousal from Hibernation Linked to Severity of Infection and Mortality in Bats with White-Nose Syndrome

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Title:
Frequent Arousal from Hibernation Linked to Severity of Infection and Mortality in Bats with White-Nose Syndrome
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PlosONE
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Reeder, DeeAnn M.
Frank, Craig L.
Turner, Gregory G.
Meteyer, Carol U.
Kurta, Allen et al
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Bats ( local )
Hibernation ( local )
Fungal Disease ( local )
Wings ( local )
Histology ( local )
White-Nose Syndrome ( local )
Seasons ( local )
Body Temperature ( local )
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serial ( sobekcm )

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White-nose syndrome (WNS), an emerging infectious disease that has killed over 5.5 million hibernating bats, is named for the causative agent, a white fungus (Geomyces destructans (Gd)) that invades the skin of torpid bats. During hibernation, arousals to warm (euthermic) body temperatures are normal but deplete fat stores. Temperature-sensitive dataloggers were attached to the backs of 504 free-ranging little brown bats (Myotis lucifugus) in hibernacula located throughout the northeastern USA. Dataloggers were retrieved at the end of the hibernation season and complete profiles of skin temperature data were available from 83 bats, which were categorized as: (1) unaffected, (2) WNS-affected but alive at time of datalogger removal, or (3) WNS-affected but found dead at time of datalogger removal. Histological confirmation of WNS severity (as indexed by degree of fungal infection) as well as confirmation of presence/absence of DNA from Gd by PCR was determined for 26 animals. We demonstrated that WNS-affected bats aroused to euthermic body temperatures more frequently than unaffected bats, likely contributing to subsequent mortality. Within the subset of WNS-affected bats that were found dead at the time of datalogger removal, the number of arousal bouts since datalogger attachment significantly predicted date of death. Additionally, the severity of cutaneous Gd infection correlated with the number of arousal episodes from torpor during hibernation. Thus, increased frequency of arousal from torpor likely contributes to WNS-associated mortality, but the question of how Gd infection induces increased arousals remains unanswered.
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PlosONE, Vol. 7, no. 6 (2012-06-20).

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FrequentArousalfromHibernationLinkedtoSeverityof InfectionandMortalityinBatswithWhite-Nose SyndromeDeeAnnM.Reeder1* ,CraigL.Frank2,GregoryG.Turner3,CarolU.Meteyer4,AllenKurta5,EricR.Britzke6, MeganE.Vodzak1,ScottR.Darling7,CraigW.Stihler8,AlanC.Hicks9,RoymonJacob1, LauraE.Grieneisen1,SarahA.Brownlee1,LauraK.Muller4,DavidS.Blehert41 DepartmentofBiology,BucknellUniversity,Lewisburg,Pennsylvania,UnitedStatesofAmerica, 2 DepartmentofBiologicalSciences,FordhamUniversity,Armonk,New York,UnitedStatesofAmerica, 3 PennsylvaniaGameCommission,Harrisburg,Pennsylvania,UnitedStatesofAmerica, 4 U.S.GeologicalSurvey–NationalWildlifeHealth Center,Madison,Wisconsin,UnitedStatesofAmerica, 5 DepartmentofBiology,EasternMichiganUniversity,Ypsilanti,Michigan,UnitedStatesofAmerica, 6 U.S.Army EngineerResearchandDevelopmentCenter,Vicksburg,Mississippi,UnitedStatesofAmerica, 7 VermontFishandWildlifeDepartment,Rutland,Vermont,UnitedStatesof America, 8 WestVirginiaDivisionofNaturalResources,Elkins,WestVirginia,UnitedStatesofAmerica, 9 NewYorkStateDepartmentofEnvironmentalConservation, Albany,NewYork,UnitedStatesofAmericaAbstractWhite-nosesyndrome(WNS),anemerginginfectiousdiseasethathaskilledover5.5millionhibernatingbats,isnamedfor thecausativeagent,awhitefungus( Geomycesdestructans (Gd))thatinvadestheskinoftorpidbats.Duringhibernation, arousalstowarm(euthermic)bodytemperaturesarenormalbutdepletefatstores.Temperature-sensitivedataloggerswere attachedtothebacksof504free-ranginglittlebrownbats( Myotislucifugus )inhibernaculalocatedthroughoutthe northeasternUSA.Dataloggerswereretrievedattheendofthehibernationseasonandcompleteprofilesofskin temperaturedatawereavailablefrom83bats,whichwerecategorizedas:(1)unaffected,(2)WNS-affectedbutaliveattime ofdataloggerremoval,or(3)WNS-affectedbutfounddeadattimeofdataloggerremoval.HistologicalconfirmationofWNS severity(asindexedbydegreeoffungalinfection)aswellasconfirmationofpresence/absenceofDNAfromGdbyPCRwas determinedfor26animals.WedemonstratedthatWNS-affectedbatsarousedtoeuthermicbodytemperaturesmore frequentlythanunaffectedbats,likelycontributingtosubsequentmortality.WithinthesubsetofWNS-affectedbatsthat werefounddeadatthetimeofdataloggerremoval,thenumberofarousalboutssincedataloggerattachmentsignificantly predicteddateofdeath.Additionally,theseverityofcutaneousGdinfectioncorrelatedwiththenumberofarousalepisodes fromtorporduringhibernation.Thus,increasedfrequencyofarousalfromtorporlikelycontributestoWNS-associated mortality,butthequestionofhowGdinfectioninducesincreasedarousalsremainsunanswered.Citation: ReederDM,FrankCL,TurnerGG,MeteyerCU,KurtaA,etal.(2012)FrequentArousalfromHibernationLinkedtoSeverityofInfectionandMortalityin BatswithWhite-NoseSyndrome.PLoSONE7(6):e38920.doi:10.1371/journal.pone.0038920 Editor: Raphae ¨ lArlettaz,UniversityofBern,Switzerland Received September20,2011; Accepted May16,2012; Published June20,2012 Thisisanopen-accessarticle,freeofallcopyright,andmaybefreelyreproduced,distributed,transmitted,modified,builtupon,orotherwiseus edbyanyonefor anylawfulpurpose.TheworkismadeavailableundertheCreativeCommonsCC0publicdomaindedication. Funding: ThetemperaturetrackingportionofthisstudywassupportedbyStateWildlifeGrantfundsawardedthroughtheNortheastAssociationofFishand WildlifeAgencies(NEAFWA)RegionalConservationNeedsgrantprogramtoDMR.(PI),CLF,GGT,ACH,andERB,byfundsfromthePennsylvaniaDepartment of ConservationandNaturalResourcesandtheWoodtigerFundtoDMR,andbyGraduateFellowshipsfromBucknellUniversitytoLEG,SAB,andRJ.Thisgrant and itsassociatedconservationactivitiesaredonetosupportimplementationofapriorityactionoftheStateWildlifeActionPlansfrommembersofthe NEAFWA.The fundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript. CompetingInterests: Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:dreeder@bucknell.eduIntroductionWhite-nosesyndrome(WNS)isestimatedtoberesponsiblefor thedeathsofatleast5.7to6.7millionhibernatingbatsinthe easternUnitedStatesandCanada[1,2].ClinicalsignsofWNS werefirstobservedatasinglecaveinNewYorkStateduringthe winterof2006–2007andasofApril2012,WNShasspreadto over200hibernaculain19U.S.statesandfourCanadian provinces(Fig.1[2,3]).BatswithWNSdisplayanumberof aberrantbehaviors,andinmanyinstancestheyhavedepletedfat stores.Thusfar,WNSaffectsatleastsix(andpossiblynine)species ofhibernatinginsectivorousbats[2],includingsomeclassifiedas endangeredorthreatened.Thelittlebrownbat(or,littlebrown myotis, Myotislucifugus ),whichwasoncethemostcommon hibernatingbatintheAmericanNortheast(NE),hasincurred anaverageof91%mortalityinsitesthathavebeenaffectedforat leasttwoyears[2]andmathematicalmodelsindicatethatthis speciesmaygoextinctintheNEwithin16years[4].Awhite fungusidentifiedas Geomycesdestructans (Gd)growsonthemuzzle, wings,andearsofbatssufferingfromWNSstartinginlate January/earlyFebruary[3,5,6].Recentlaboratoryexperiments havedemonstratedthatcutaneousinfectionwiththisfungusisthe causeofWNS,butitisnotfullyunderstoodhowsuchaninfection producesmortalityduringhibernation[7].Itishypothesizedthat infectionbyGddisruptsnormalphysiologicalfunctions,suchas waterbalance[8]orotheraspectsofhibernationphysiology, includinguseoftorpor[9]. Forinsectivorousbatsthatliveinnortherntemperatezones, suchasthoseaffectedbyWNS,foodisprimarilyavailablefrom latespringtoearlyautumnandabsentduringwinter.Batssurvive PLoSONE|www.plosone.org1June2012|Volume7|Issue6|e38920

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thiswinterenergeticbottleneckbybuildingstoresofbodyfat (depotfat)inlatesummerandearlyautumnandbyconserving metabolicenergythroughhibernation.Inlittlebrownbats,body fatincreasesfromapproximately7%oftotalmass( , 6g)during summerto27%oftotalmass( , 9g)priortohibernation,an increaseof3gormoreinbodymass[10,11].Thisdepotfatisthe soleenergysourceduringthehibernatingperiod,whenbody temperature(Tb)andmetabolicratearebothgreatlyreduced. Becausetheirenergeticcostsinthesubsequentspringaregreater thanthoseofmales,femalelittlebrownbatsenterhibernationwith higherbodymassindexes(BMI)andmanagetheirenergystores duringhibernationmoreefficientlythanmales[12].Minimum metabolicratesduringmammaliantorporcanbe , 5%ofbasal metabolicratewithTbclosetoambienttemperature(2 u to8 u for bats)[13,14].However,hibernatorsdonotremaintorpid throughouthibernation;insteadboutsoftorporlastfromdaysto weeks,interruptedbybriefarousalepisodesinvolvingperiodsof highmetabolicrateandeuthermicTb[15].Earlierstudies demonstratedthathealthy,free-ranginglittlebrownbatshibernatingatambienttemperaturesof5–6 u Chavetorporboutslasting between12.4and19.7days[16,17],witharousalepisodeslasting 1–2hours. Althougheuthermicperiodsaccountforapproximately1%of thetotaltimebudgetduringwinter,about80–90%oftheenergy (depotfat)usedduringhibernationisconsumedduringthese periodicarousalsfromtorpor,becausemetabolicrategreatly increaseswithincreasedTb[13,18].Theamountofdepotfat expendedduringeacharousalepisode(notincludingflight)for hibernatinglittlebrownbatsisabout107.9mg[18].Whilethe functionofarousalepisodesinhibernatorsispoorlyunderstood andlikelymultifactorial[19],thefactthateverymammalian Figure1.DistributionandspreadofWNSthroughoutNorthAmerica. SpreadofWNSbyhibernationseasonthroughthewinterof2010– 2011isshownalongwithlocationsofstudysites,indicatedbystars(seealsoTable1).Confirmedsiteshavebeenofficiallyreportedbyeachstateor provincebaseduponhistologicalconfirmationofinfectionwiththefungalpathogen Geomycesdestructans (Gd);batsfromsuspectsiteshaveclinical signsofWNSbutlacklaboratoryconfirmation.TheinsetshowsalittlebrownbatinfectedwithGdfromsite # 1inVermont.ThissitewasWNS confirmedin2008–2009,whenbatswerestudied.Batsfromsite # 2inPennsylvaniawerestudiedin2008–2009(for8weeksonlyinthespring), whennosignsofWNSwerepresent,in2009–2010,whenasinglebatfromthissiteshowedinfectionwithGdwithoutmassmortalityandin2010– 2011,whenbatsinthissitewereheavilyinfected.Batsfromsite # 3inPennsylvaniawerestudiedin2008–2009(noWNS),2009–2010(whenGdwas notedbutwithoutmassmortality)andin2010–2011,whenbatsinthissitewereheavilyinfected.Batsfromsite # 4inPennsylvaniawerestudiedin 2009–2010(for8weeksonlyinthespring),whenbatswereheavilyinfected.Batsfromsite # 5inWestVirginiawerestudiedin2008–2009,when therewasnoevidenceofGdpresence–whichwasalsothecaseforbatsfromsite # 6inMichigan,whichwerestudiedallthreeyears. doi:10.1371/journal.pone.0038920.g001 AlteredHibernationPatternsinWNS-AffectedBats PLoSONE|www.plosone.org2June2012|Volume7|Issue6|e38920

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hibernatorperiodicallyarousesfromtorporatgreatenergeticcost indicatesthebenefitsmustbesignificant. WetestedthehypothesisthatWNSreducesthelengthoftorpor boutsduringhibernationinfree-ranginglittlebrownbats.We predictedthataprimarycauseoftheincreasedmortality/disease stateassociatedwithWNSisabnormallyshortenedtorporbouts, duetomorefrequentarousalepisodes,aswasshownpreviouslyfor oneaffectedfree-rangingbatinlatehibernation[20]andrecently foragroupofexperimentallyinfectedbatsheldincaptivity[21]. Wealsopredictedthatgreaterbodyfatstoresatthebeginningof hibernation,asestimatedbyBMI,wouldmediatethenegative effectsoffrequentarousals.Thesepredictionsweretestedinfield studiesonfree-ranginglittlebrownbatsconductedatmultiplesites (Fig.1)overthreehibernationseasons.Skintemperature(Tsk), whichcorrelateswellwithTbinsmallinsectivorousbats,and whichhasbeenusedextensivelytostudymammalianhibernation [22],wasmeasuredwithtemperature-sensitivedataloggers attachedtothebacksofWNS-affectedandunaffectedbats. HibernationpatternsinrelationtothestageofinfectionbyGd werealsoanalyzedforasmallsampleofbatsforwhichdatawere availableonfungalpresence(PCR)anddegreeofinfection (histopathology).MaterialsandMethods PermitsandPermissionsThisstudywascarriedoutinstrictaccordancewiththe recommendationsintheGuidefortheCareandUseof LaboratoryAnimalsoftheNationalInstitutesofHealth.The protocolwasapprovedbytheInstitutionalAnimalCareandUse CommitteeatBucknellUniversity(protocolnumberDMR-02).In thestatesofVTandWV,researchwasconductedbystatewildlife officials(SRDwithVermontFishandWildlifeDepartmentand CWSwithWVDepartmentofNaturalResources)onnonendangeredbats;thusnumberedpermitswerenotrequiredor issued.InMichigan,researchwasconductedeachyearunderMI ScientificCollector’sPermitSC620fromtheMichiganDepartmentofNaturalResourcestoAK.InPA,researchwas conductedeachyearunderPAGameCommissionpermitsto DMR(84-2008;70-2009;183-2010),incollaborationwithGGT, awildlifebiologistforthestateofPA.Inaccordancewiththe permitsandwithstatewildlifepolicies,researchwaseither conductedonstatelandoronprivateproperty,withtheexplicit permissionofprivatelandowners.TemperatureTrackingTemperature-sensitivedataloggerswereprogrammedtoread skintemperature(Tsk)every30minandwereattachedto504bats overthecourseofthreewintersatsixdifferenthibernaculausing standardmethods[22].Temperaturereadingscouldnotbe collectedmorefrequentlyduetoconstraintsondatalogger memoryandtheneedtorecordcontinuousdataforuptofive months.Tomaximizerecapturerates,batswithloggerswere recapturedinMarchofeachyear,severalweekspriortothe ‘normal’timeofemergencefromhibernation.Loggersweighted about1.1gandwereeitherpurchasedcommercially(iBBator WeeTagLites,AlphaMach,Inc.,BritishColumbia,Canada)or wereconstructedbytheauthors(DMRandGGT).AppendixS1 describesandillustratesthemethodsformakingthesedataloggers fromThermochronDS1922LiButtons(MaximIntegratedProducts,Inc.,California,USA),modifiedfromthetechniquesof Lovegrove[23].Table1providesasummaryofloggersdeployed, retrieved,anddownloadedsuccessfully,bysite,year,andsex. Table1. Temperature-sensitivedataloggerdeploymentandretrieval(batrecapture)data,bystudyyear,hibernaculasitenumber(seeFig.1),andsexforth e504loggers deployed.Site # 2008–20092009–20102010–2011 deployed*retrieved* downloaded includedinfinal analyses**deployed*retrieved* downloaded includedinfinal analyses**deployed*retrieved* downloaded includedin finalanalyses** 1(VT)16/14[11/6/08]6/7[3/17/09]6/75/7 2(PA)20/19 + [1/27/09]13/13[3/24/09]7/73/341/41[11/13/09]25/26[3/25/10]13/813/822/18[11/18/10]4/4[3/10/11]3/13/1 3(PA)15/15[11/3/08]9/4[3/23/09]8/28/240/30[11/12/09]9/8[3/17/10]7/27/222/7[11/19/10]8/1[3/2/11]7/17/1 4(PA) 35/25 + [1/6/10]7/1[3/11/10]7/04/0 5(WV)21/21 + [1/29/09]7/7[3/23/09]2/42/4 6(MI)15/15[11/7/08]7/9[3/21/09]7/57/513/13[11/14/09]9/6[3/27/10]4/24/214/12[11/6/10]10/8[3/26/11]0/10/1 Whetherdataweresuccessfullydownloadedfromtheloggerandultimatelyusedintheanalysesofthispaper,arealsodescribed. * # ofmales/ # offemalesanddateofdeploymentorretrievalofloggers. **batswereoccasionallyexcludedfromanalysesduetoincompletedata(e.g.,BMInotrecorded)orproblemswithdownloadeddata.+loggersonlydeployedmid-winter(January-March)asopposedtothefull-hibernationseason(November-March). doi:10.1371/journal.pone.0038920.t001 AlteredHibernationPatternsinWNS-AffectedBats PLoSONE|www.plosone.org3June2012|Volume7|Issue6|e38920

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StudysiteswerewidelydistributedandlocatedinVermont, WestVirginia,Pennsylvania,andtheUpperPeninsulaof Michigan(Fig.1).Amongloggersretrieved,successratesvaried. WeeTagLitesfailedatarateofupto90%whereasloggers constructedbytheauthorsfailedabout20%ofthetime.Overall 111of190loggersretrievedyieldedusabledata,anaverageof 58.4%.Weexpectedtorecoverlessthanhalftheloggersplacedin thefieldandexpecteddataloggerfailureaswell,whichiswhyso manyloggersweredeployed.Ofthe190batsfromwhichloggers wereretrieved,17werefounddead(fourofwhichwereinsuitable post-mortemconditiontoperformhistologyanalysis).Forthe173 livebatsrecapturedinthespring,loggerswereremoved,andthe animalwaseitherreleased(N=126)oreuthanizedformeasurementofimmunefunctionandotherphysiologicalparametersfor aseparatestudy(N=25)orforhistologyanalysis(N=22),as describedbelow.PCRandHistologyWingskinsamples(approximately3mmX3mmeach)were collectedfromasubsetoffreshlyeuthanizedanimals(N=26). NucleicacidwasextractedfromeachskinsampleusingtheGentra PuregenegenomicDNApurificationkit(QiagenInc.,Valencia, CA)perthemanufacturer’sinstructions(solidtissuesprotocol), withthefollowingmodifications:proteinaseKwasaddedtoafinal concentrationof0.5mg/mlduringthecelllysisprocedureandno RNasetreatmentwasperformed.Todeterminepresence/absence ofDNAfromGdoneachsampleofwingskin(withinthedefined sensitivitylimitationsofthetechniqueused),extractednucleicacid wasanalyzedbyPCRaspreviouslydescribedbyLorchetal.[24]. Wingmembranefromthesesameanimalswasalsoanalyzedby histology[5]todetermineWNSinfectionstatus.Theentirewing membranewasstrippedfromtherightforearmanddigits,rolled onto2dowels2.5cminlength,trimmedintothreeapproximately 0.8cm-widesections,placedontrimmededge,sectionedat 0.4 m m-thickness,andstainedwithPeriodicAcidSchiff[5].This preparationtechniqueyieldssixwhorlsofwingmembraneon eachslide.White-nose-syndromewasdiagnosedbasedonpreviouslypublishedmicroscopiccriteria[5].Ahistologicscoring systemwasdevelopedtoclassifyseverityofWNSonascaleof0to 4asdescribedandillustratedinAppendixS2.Briefly,ascoreof 0indicatesthesampleisnegativeforWNS,andthereareno diagnosticcuppingerosionsinthetissues.Ascoreof1indicatesthe tissuesarepositiveforWNSwithcuppingerosionsdiagnosticfor WNSbuterosionsaremild,occasional,andarelimitedinboth depthandextentofwingmembraneinvolved.Thepresenceof evenonecharacteristicWNSerosionissufficientforadiagnosisof WNS.Aseverityscoreof2indicatesmoderateWNSwithmore frequentanddeeperfungalcuppingerosionsdiagnosticofWNS, butdistributionoverwingmembraneisstilllimited.AWNS severityscoreof3indicatesmoderatelyseverefungalinfection withdeeperandcoalescingcuppingerosionsthataredeepenough tobeconsideredulcers,andtheextentofthewingmembranewith fungalinvasionisgreater.Aseverityscoreof4indicatesasevere fungalinfectionwithdeeptissueinvasionandcoalescingof cuppingerosions;asmanyas100ormoreerosions/ulcerscanbe presentinonerollofwingmembrane.Scoresrangingfrom1to4 wereidentifiedasWNS.AnalysesCalculationsandinitialstatistics.Usabledataforour analyseswererecoveredfrom99ofthe504loggersdeployed(see Table1).Althoughdatadownloadedfrom111loggers,datafrom 12ofthesebatswereremovedfromfinalanalysesforavarietyof reasons,includinghavingtemperaturedatarecordedfortooshort ofatimeperiodtobecomparabletoothergroupsandmissing bodymassdata.Priortodataloggerattachment,eachbatwas weighedusingaportablebattery-operatedscale(accuracyto 0.1g),andthelengthoftheirrightforearmwasmeasured(in triplicate)tothenearestmmusingcalipers;fromthesedataBMI (weighting/lengthofrightforearminmm)[10]wascalculated. AsmostanalysesincludedBMIasacovariate,onlybatsforwhich wewereabletocalculateBMIatthebeginningofhibernation (November)wereincludedinthefinalanalysis(N=83).Datafrom anadditional16batsforwhichwehadrecordingsfromonly JanuarythroughMarch(seeTable1)arealsodescribedinthe results. Torporwasdefinedaswhenabat’sTskwas10 u Cormore belowitshighesttemperature(Tmax).Durationofanarousal episode(whenTskwaswithin10 u CofTmax)wascalculatedtothe nearest30min.AlthoughrecordingTskevery30minwas sufficienttodetectarousalepisodes,itdidnotprovidesufficient resolutiontodescribepreciselythetruelengthofanarousalbout, asarousalepisodesaveragedlessthan90mininlength(see results).Thus,wedidnotattempttodetermineiftherewere significantdifferencesinarousalepisodelengthbyWNSstatus. Torporboutlength(TBL,indays)wasdefinedastheperiod betweentwoarousalepisodes.Forbotharousalboutlengthand TBL,valueswerefirstaveragedforeachbatandthenaveraged acrossallbats.DataonTBLwerelog(10)transformedtoachieve normalityandhomogeneityofvariance,asdeterminedby Shapiro-Wilk’stestfornormalityandexaminationofskewand kurtosisandbyLevene’stestforequalityofvariances.BMIdata werenormallydistributed.TBLdatafrommultipleyearsare combinedinouranalysis,whichissupportedbythelackofayearto-yeardifferenceinTBLinbatsfromagivenhibernaculumwhen theWNSstatusdidnotchangebetweenyears(e.g.,fromsite6 (Table1;Figure1):10.52 6 1.62days(2008–2009)vs. 12.47 6 3.09days(2009–2010);F(1,16)=3.091,p=0.098;partial etasquared=0.162,power=0.380).Forallanalyses,powerand effectsizearereportedfornon-significantresults.Alldataare presentedasthemean 6 standarddeviation(SD).WNSstatusandTBL.Fortheinitialanalysis,batsforwhich wehaddataonTBL,BMI,andsexweregroupedintothree ‘WNSstatus’categories:(1)unaffected[N=57],(2)WNS-affected (asdeterminedbyhistologyand/orvisiblefungus)andaliveat timeofdataloggerremoval[N=14],and(3)WNS-affectedand founddeadattimeofdataloggerremoval[N=12].Batswere assignedtothe‘unaffected’categoryeitherwhenthepresenceof fungalinfectionwithGdwasnotdetectedwithPCRorhistology [N=10]orwhentheywerefromahibernaculumpresumedtobe unaffectedandnotlocatedintheWNSzoneatthetimeofstudy [N=47](Fig.1).Combiningthetwogroupsof‘unaffected’bats forfurtheranalysesissupportedbythelackofadifferenceinTBL betweenthem(17.55 6 4.56days(PCR/histology)vs. 16.06 6 7.03days(presumedunaffected); F(1,55)=1.111,p=0.297;partialetasquared=0.020,power =0.179).EffectsofWNSstatusonTBLweretestedwith ANCOVA,withBMI(random),siteidentity(fixed),andsex (fixed)ascovariates.Post-hocexaminationofsexdifferencesin BMIwasconductedusingaStudent’s t -test(withdfandpvalues adjustedforunequalvariance).TBLanddateofdeath.WithintheWNS-affectedbatsthat werefounddeadatthetimeofdataloggerremoval,the relationshipsbetweenTBLandBMIanddateofdeathwere analyzedusingPearsonProductMomentCorrelations(PPMC) (afterconfirmingnormalityandhomoscedasticityforeachvariable).DateofdeathwasmeasuredasthedateonwhichTsk, 0 u C forthefirsttime,sincetheTskoflittlebrownbatsalwaysremainsAlteredHibernationPatternsinWNS-AffectedBats PLoSONE|www.plosone.org4June2012|Volume7|Issue6|e38920

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above0 u Cduringtorpor[17,18].Pvalueswereadjustedfor multiplecomparisonsusingsequentialBonferronicorrection[25], andthecoefficientofdetermination(r2)wascalculatedbysquaring significantcorrelations.TBLandWNSseverityscore.Usingasubsetofanimalsfor whicha‘WNSseverityscore’couldbecalculatedandforwhich BMIatthestartofhibernationwasavailable(N=26),theeffects ofseverityscore,BMI,andsiteonTBLwereexaminedwith ANCOVA.Asignificantrelationshipbetweenseverityscoreand TBLwasexaminedusingtheGammaCorrelationStatistic,which allowsformultiple‘tiedrankings’[26].Ofthese26bats,10were classifiedinthefirstanalysisas‘‘unaffected’’13wereclassifiedin thefirstanalysisas‘‘WNS-affectedandaliveattimeofdatalogger removal’’(ofthesethreebatsreceivedaseverityscoreof1,four batsaseverityscoreof2,twobatsaseverityscoreof3,andfour batsaseverityscoreof4),andthreewereclassifiedinthefirst analysisas‘‘WNS-affectedandfounddeadattimeofdatalogger removal’’(ofthesetwobatsreceivedaseverityscoreof2andone bataseverityscoreof3).Results ArousingtoEuthermicTemperaturesDuringthecourseofthisstudy,whenbatsarousedfromtorpor, theyremainedateuthermictemperaturesforashortperiod, averaging78.3 6 27.3min.Therangeofaveragearousalbout lengthperbatwasfrom38.18to180min(N=83bats),whilethe shortestrecordedarousalboutlasted30min(theshortestperiod thatcouldbediscernedbyourmethods)andthelongest330min. Wewereunabletotestfordifferencesinarousalboutlengthin relationtoWNSstatus(orseverityscore)duetothelimiteddata storagecapacityofourdataloggers(andthusinsufficientresolution forpreciselyquantifyingarousalboutlength).WNSStatusandTBLAlthoughfemalebatswereinsignificantlygreaterbody conditionthanmalesatthestartofhibernation(BMI: 0.2284 6 0.0283g/mm(N=32)vs.0.2073 6 0.0210g/mm (N=51);t= 2 3.633,adjusteddf=52.2,p=0.001),therewere nodetectableinfluencesofsexonTBL(F(1,76)=0.031,p=0.861; partialetasquared=0.000,power=0.053).Likewise,wedidnot detectarelationshipbetweenBMIatthestartofhibernationand TBL(F(1,76)=0.140,p=0.710;partialetasquared=0.000, power=0.066).OurBMIanalyseswerenotbiasedbyrecapture dynamicsastherewasnosignificantdifferenceinBMIatthetime ofdataloggerattachmentbetweenbatsforwhichloggerswere retrievedandbatsthatwerenotrecovered(Mann-Whitney U=3.339,Z=1.259,p=0.208).However,bothWNS-status andsiteidentitysignificantlyinfluencedTBL.Siteidentityheavily influencedthemodel(F(1,78)=25.027,p , 0.001)astwoofthesites containedonlyonecategoryofbat(site1hadonly‘WNSdeadat timeofdataloggerremoval’bat,andsite6hadonly‘unaffected’ bats).Despitethestronginfluenceofsiteidentity,asignificant WNSstatusmaineffectwasstillapparent(F(1,78)=7.569, p=0.007). UnaffectedbatshadameanTBLof16.32 6 6.65days(Fig.2). Limiteddatacollectedfromanadditional12unaffectedbatsfrom fieldsiteswheredataloggersweredeployedforonlyeightweeks lateinthehibernationseasonin2009aresimilarwithamean TBLof15.62 6 8.07days(sites2and5,Fig.1).Aspredicted, havingWNSwasassociatedwithdecreasedTBL(Fig.2).Batsthat wereaffectedbyWNSbutstillaliveatthecollectionofdataloggers (March)hadshorterTBLsthanunaffectedbats,althoughthe differencewassmallandnotstatisticallysignificant (13.96 6 4.30daysvs.16.32 6 6.65days;F(1,69)=1.491,p=0.226, partialetasquared=0.021,power=0.226).However,these affectedbutalivebatshadsignificantlylongerTBLsthanWNSaffectedbatsthatwerefounddeadatthetimeofdatalogger collection(7.93 6 2.49days;F(1,24)=17.191,p , 0.0001).Limited datacollectedfromanadditionalfourWNS-affectedbatsfound deadfromafieldsitewheredataloggersweredeployedforonly eightweekslateinthehibernationseasonin2010aresimilarwith ameanTBLof6.17 6 1.79days(site4,Fig.1).TBLandDateofDeathWithinthe12WNS-affectedbatsfounddeadatthetimeof dataloggercollection,therewasaverystrongpositiverelationship betweenTBLandthenumberofdaysthatabatlived(Fig.3; PPMC,r=0.763,correctedp=0.012).Baseduponthecalculated coefficientofdetermination(r2=0.582),TBLsignificantlypredictedthedateofdeath,explaining58%ofthevariance.Similar tothefindingsofourfullANCOVA,wedidnotdetect arelationshipbetweenBMIatthestartofhibernationandTBL (PPMC,r=0.178,p=0.580)orbetweenBMIatthestartof hibernationanddateofdeath(PPMC,r= 2 0.026,p=0.936). Whilethepowertodetectsignificantdifferencesattheseloweffect sizes(correlationcoefficientsof0.178and0.026)isextremelylow ( , 0.05),eveniftheywerestatisticallysignificant,theyarenot biologicallysignificant.Ineachbat,mortalitywasobserved immediatelyafterthelastarousaltoeuthermictemperatures. Whileseveralbats(Fig.2C)displayedfrequentarousalsjustbefore death,mostdidnot,andarousalswerespreadthroughouttheir hibernationperiod.TBLandWNSSeverityScoreInthesubsetofanimalsforwhichtheWNSseverityscorecould becalculated(N=26),TBLwasnotrelatedtoBMI (F(1,21)=0.111,p=0.743,partialetasquared=0.005,power =0.062)orsiteidentity(F(2,22)=2.515,p=0.104,partialeta squared=0.186,power=0.045),butwasrelatedtoseverityscore (F(1,24)=6.509,p=0.018).Batswithmoreseverefungalinfections hadsignificantlyshortertorporbouts(gammacorrelationstatistic = 2 0.383,p=0.022;Fig.4).DiscussionOurresultssupportthehypothesisthatWNScausesalterations inbattorporpatternsthatlikelycontributetodeath.Our predictionthatincreasedmortality/diseasestateisassociatedwith abnormallyshorttorporboutsduetofrequentarousalepisodes wassupportedbyourlargerdataset,inwhichbatswereplaced intotheWNSstatuscategoriesof‘unaffected,’‘WNS-affectedand aliveattimeofdataloggercollectionattheendofhibernation,’ and‘WNS-affectedanddeadatthetimeofdataloggercollection.’ Whileour‘unaffected’batshadanaverageTBLthatfallswithin thepreviouslydocumentedrangeforthisspecies(16.32days) [16,17],TBLwasshortened(atthelowendofpreviously describedTBLs)inWNS-affectedbats(13.96days),andsignificantlyreducedinWNS-affectedbatsthatdiedbetweenmidDecemberandlate-February(7.93days).Anaveragetorporbout lengthof7.93daysispresumablynotsustainable.Infact,within thoseWNS-affectedbatsfounddeadatthetimeofdatalogger removal,TBLwasaverystrongpredictorofthedateofdeath, explaining58%ofthevarianceintimingofmortality.The distributionofdeathdatesforthesebats(Fig.3)isearlierthanthat reportedintheUSA[7]andearlierthanseasonalchangesinGd prevalencereportedforEurope[27,28].However,thiswasatleast thesecondyearofinfectionatthissite,whichmightshifttheAlteredHibernationPatternsinWNS-AffectedBats PLoSONE|www.plosone.org5June2012|Volume7|Issue6|e38920

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Figure2.Torporboutlength(TBL)indaysbyWNSstatus. WNSwasassociatedwithdecreasedTBL:batsthatwereaffectedbyWNSbutstill aliveatthecollectionofdataloggers(March)hadshorterTBLsthanunaffectedbats(butthisdifferencewasnotsignificant).Significantlyshort erTBLs wereseeninWNS-affectedbatsthatwerefounddeadatthetimeofdataloggercollectioncomparedtoaffectedbutalivebats(2A).Batswere categorizedas:unaffected,WNS-affectedandaliveattimeofdataloggerremoval(‘WNS-alive’),andWNS-affectedanddeadwhenloggerswere removedinthespring(‘WNS-dead’).Numbersinbracketsindicatesamplesizeandboxessharingthesameletterarenotsignificantlydifferentfrom eachother.Boxesdepictthe25thand75thpercentiles,lineswithinboxesmarkthemedian,andwhiskersrepresent95thandthe5thpercentiles. Outliersareindicatedwithopencircles.Additionalpanelsillustratesampletemperatureprofileofanunaffected(B)andanaffected(C)bat,duri ng thewinterof2009.ThebatillustratedinCdisplayeddailyarousalsattheendofitslife,whichwasseeninseveraloftheseanimals.Eachofthe‘WNSdead’batsdiedattheendoftheirlastarousal. doi:10.1371/journal.pone.0038920.g002 AlteredHibernationPatternsinWNS-AffectedBats PLoSONE|www.plosone.org6June2012|Volume7|Issue6|e38920

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Figure3.Torporboutlength(TBL)asafunctionofdateofdeathandBMI. Forthe12batsthatdiedfromWNS,BMIatthebeginningof hibernationwasnotrelatedtoTBL(3A),norwasBMIpredictiveofthedateofdeath(3B).However,TBLsignificantlypredicteddateofdeathinWNSaffectedbatsthatwerefounddeadatthetimeofdataloggerretrieval(3C)(r2=0.58).Batsthatdiedsoonerwerearousingtoeuthermictemperatures muchmorefrequentlythanthosethatlivedlonger. doi:10.1371/journal.pone.0038920.g003 AlteredHibernationPatternsinWNS-AffectedBats PLoSONE|www.plosone.org7June2012|Volume7|Issue6|e38920

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distributionofdeathdatesearlierrelativetocompileddatafrom multiplesites[7,27,28].Recaptureofbatsfordataloggerremoval inMarchofeachyear(Table1),thetimewhenpeakmortalityhas beennotedinthefield[7],mayhavepreventedusfromdetecting othermortalityeventswithinourstudyanimals. OuranalysisofWNSseveritybaseduponhistologicalconfirmationofthedegreeoffungalinvasionandinfectionfurther supportedandstrengthenedourconclusion–astheseverityof infectionincreased,sodidthefrequencyofarousalsfromtorpor. Ourdatamirrortheindependentlyderivedmathematicalmodel ofBoylesandWillis[9],forwhichanestimatedshiftinTBLto every8.33daysresultedinapredictionof81.9%mortality. Relativetothismodel,ourfindingofaTBLof7.93daysfor WNS-affectedbatsfounddead,andfieldobservationsof91% mortalitysupportthelinkagebetweenTBLanddeath,as significantbodyfatislostwitheacharousal[13,18].Boylesand Willis[9]alsoproposedthatsignificantchangesinarousalbout durationinWNS-affectedbatscouldleadtomortality.Batsare unlikeotherhibernators[13,18]inthattheirarousalboutsare typicallymeasuredinminutesratherthanhours(orevendays). Thus,anincreaseinthedurationofeuthermywouldincur significantenergeticcosts.Althoughwewereunabletostatistically validatedifferencesinarousalboutlengthinbatsofvariableWNS status,ourfindingofanaveragearousalboutof78.3 6 27.3minutesforallbatstestedindicatethatbiologicallyimportantshiftsin arousalboutlengthdonotoccurinWNS-affectedanimals. WealsopredictedthatrelationshipsbetweenWNSandtorpor patternswouldbeinfluencedbytheamountofenergystores availabletothebat.Inapreviousstudyoflittlebrownbats,BMI significantlyinfluencedhibernationenergeticssuchthatbatswith lowerbodymassesatthebeginningofhibernationselectedcolder roostingsites,whichallowsfordecreasedmetabolicratesandthus lowerenergyexpenditure[29].Otherstudieshavedemonstrated thatbatsroostingatcoldertemperaturesarousefromtorporless often,allowingthemtoconserveevenmoreenergy[19,30,31]. Thus,itisreasonabletoexpectthatbatswithlowerBMIswould displaygreaterTBLandexpendlessenergy. TheseenergeticargumentsunderlaythemodelofBoylesand Willis[9]thatourdatasocloselymatch.However,contrarytoour predictions,wedidnotfindarelationshipbetweenBMIandTBL orBMIand‘WNSstatus’,deathdate,or‘severityscore’.Asthe powerforBMIeffectsinourmodelswaslow(drivenbythestrong siteeffects),BMImaystillplayaroleinhibernationpatternsand inabat’sabilitytowithstandGdinfection.However,evenwithin asite(WNS-affectedbatsthatwerefounddeadatthetimeof dataloggerattachmentfromsite1inVermont),wefailedtofind arelationshipbetweenBMIandWNS.IfahigherBMIcould ‘buffer’abatfromtheeffectsofWNSbyallowingittowithstand morearousalstoeuthermy,thenweshouldhavedetected arelationshipbetweenBMIandthenumberofarousalspriorto death–butwedidnot. Althoughstatisticalanalysesconfirmedthesignificanceofour findings,studiesofbehaviorandphysiologyinfree-ranging animalsareoftenfraughtwithunknownsandpotentialbiases, whichlikelyunderliethesignificantsiteeffectsinourstatistical models.OnepotentialsourceofbiasinourdatasetisBMIatthe startofthehibernationseason.Whileonecouldpredictthatbats inpoorerbodyconditionwouldfinddataloggerattachmentmore physiologicallystressfulthanbatsingreaterbodycondition(and thusbelesslikelytoberecaptured),therewasnodifferencein startingBMIbetweenbatsthatwererecapturedandthosethat werenot.AnothersourceofbiasinourWNS-affectedbatscould havebeenambienttemperatureofhibernacula,becauseTBL generallydecreaseswithincreasedambienttemperature[30]. Althoughtheexactambienttemperatureattheexactroostingsite ofeachindividualstudiedduringhibernationwasunknown,our WNS-affectedfieldsitesweregenerallycolderthanourunaffected sites(e.g.,7.29 u Cvs.9.77 u C).Thiswouldpresumablybiasbats withWNStowardlongerTBLs,butweobservedtheopposite pattern.Withinourunaffectedbats,TBLsvariedgreatly(Fig.2A), likelyduetoanumberofsite-,individual-,andpopulation-specific factors.However,thesefactorsappeartobeoverriddeninthe WNSaffectedbats,especiallythosefounddeadatthetimeof dataloggerremoval–asvariabilitydecreasedandallbats exhibitedshortenedTBLs. Collectively,ourdataindicatethatoneproximatemechanismof themortalityassociatedwithWNSisdecreasedTBL.Warnecke etal.[21],inastudyofcaptivebatsexperimentallyinfectedwith Gdduringthethirdyearofourfieldstudy,foundasimilarTBL shift.Thechallengethatliesbeforeusistodeterminehow infectionbyGdinducesalteredtorporpatternsandwhy significantvariationinTBLbetweenaffectedbatsoccurs.While too-frequentarousalisclearlyassociatedwithWNS,notallbats thatdieddisplayedtheseverelyshortenedTBLcharacteristicof somethatdied,andsomebatsthatdisplayedveryshortTBLdid notdie. Inothermammalianhibernators,mechanismsassociatedwith immunityarereducedduringhibernation,whentheconservation ofenergyiscritical[32,33],andtheperiodicarousalsfrom hibernationmayactivatethedormantimmunesystem.Thus, immunologicalresponsestofungalinfectionmaybetriggering arousalsmorefrequentlythannormal[34].Additionally,physical damagetowingskincausedbyfungalinfectionmaydisruptother physiologicalfunctions,suchwaterbalance,resultingindehydration,anothertriggerforarousalfromtorporinhibernating animals[8].EquallyimportanttounderstandinghowGdinfection leadstoalteredtorporpatternsistheneedtounderstandhow Figure4.Torporboutlength(TBL)asafunctionofWNS severityscore. Wingtissuewasassignedadiseaseseverityscore(SS0 toSS4)baseduponhistology,asfollows:SS0=nofungisuggestiveof WNS;SS1=occasionalbutlimitedsuperficialfungalinfection;SS2= moreextensivesuperficialfungalinfectionwithlimitedinvasion;SS3= moreextensivefungalinfectionwithfrequentcuppingerosions;and SS4=severefungalinfectionwithdeeptissueinvasion.Detailsofthe scoringsystemcanbefoundinAppendixS2andscores1through4 wereidentifiedasWNS.Individualdatapointsareshownasopen circles,themedianisindicatedbyaline.Asseverityofinfection increased,torporboutlengthsignificantlydecreased(batsaroused morefrequentlyfromtorpor. doi:10.1371/journal.pone.0038920.g004 AlteredHibernationPatternsinWNS-AffectedBats PLoSONE|www.plosone.org8June2012|Volume7|Issue6|e38920

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thesetoo-frequentarousalstoeuthermymaybecontributingto death–inwaysthatarenotclearlyrelatedtoenergybalance,but arepotentiallyrelatedtothedisruptionofotherhomeostatic mechanisms[8]. Adetailedunderstandingofthemechanism(s)bywhich infectionwithGdcausesmortalityinhibernatingbatsmay provideinsightstodevelopinterventionalstrategiestomitigatethis unprecedentedwildlifedisease.Insectivorousbatsperformsignificantecosystemservicesbecausetheyareprimarypredatorsof nocturnalinsects[35–37].Assuch,webelievethatthelossofcavedwellinghibernatingbatsinNorthAmericawillbeecologically significant.SupportingInformationAppendixS1Instructionsforproducingtemperaturesensitive dataloggersforattachmenttobats,includingfigures. (PDF)AppendixS2DescriptionofWNShistopathologyandassignmentofwingdamageseverityscores(SS),includingfigures. (PDF)AcknowledgmentsForfieldassistance,wethankR.Arndt,L.DeWolski,B.Douglas,R. Doyle,J.Fregonara,C.Hauser,H.Kaarakka,J.Hajenga,J.Kobilis,M. Kurta,K.Langwig,K.O’Malley,C.Patterson,D.Redell,B.Roelle,C. Rockey,A.Rolfe,B.Scullon,P.Sewell,B.Smith,A.Stauffer,J.Wallace, andP.White.Wealsothanktheindividualsiteownersforaccessto hibernacula.C.Musante,N.White,K.Weaver,C.Meade,M.Furze,P. Reamey,S.Wade,andS.Alfanoassistedintheconstructionof dataloggers.W.M.Fordprovidedareviewofanearlyversionofthe manuscript;M.J.BehrreviewedandprovidedcommentsforAppendixS2 (DescriptionofWNShistopathologyandassignmentofwingdamage severityscores(SS)).D.R.Dougherty,P.T.Reamey,J.Glathar,andC. ButchkoskiassistedwithFigure1.Useoftrade,product,orfirmnamesis fordescriptivepurposesonlyanddoesnotimplyendorsementbytheUS Government.AuthorContributionsConceivedanddesignedtheexperiments:DMRCLFGGTAKACH ERBSRDCWSDSB.Performedtheexperiments:DMRCLFGGTAK ACHERBSRDCWSDSBCUMRJLEGSABMEVLKMDSB. Analyzedthedata:DMRCLFDSBCUMRJSABMEVLEG. Contributedreagents/materials/analysistools:DMRCLFGGTAK ACHSRDCWSDSBCUMDSB.Wrotethepaper:DMRCLFGGT DSBCUMMEV.References1.USFishandWildlifeService(2012)NorthAmericanbatdeathtollexceeds 5.5millionfromwhite-nosesyndrome.PressRelease(January17,2012). Available:http://www.fws.gov/white nosesyndrome/pdf/WNS_Mortality_ 2012_NR_FINAL.pdf.Accessed2012Apr02. 2.TurnerGG,ReederDM , ColemanJTH(2011)Afive-yearassessmentof mortalityandgeographicspreadofwhite-nosesyndromeinNorthAmerican bats,withalookatthefuture.BatResearchNews52:13–27. 3.BlehertDS,HicksAC,BehrMJ,MeteyerC,Berlowski-ZierBM,etal.(2009) Batwhite-nosesyndrome:anemergingfungalpathogen?Science323:227. 4.FrickWF,PollockJF,HicksAC,LangwigKE,ReynoldsDS,etal.(2010)An emergingdiseasecausesregionalpopulationcollapseofacommonNorth Americanbatspecies.Science329:679–682. 5.MeteyerCU,BucklesEL,BlehertDS,HicksAC,GreenDE,etal.(2009). 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BMCBiology8:135.Available:http://www.biomedcentral.com/1741-7007/ 8/135.Accessed2011Jul28. 9.BoylesJG,WillisCKR(2010)Couldlocalizedwarmareasinsidecoldcaves reducemortalityofhibernatingbatsaffectedbywhite-nosesyndrome?Frontiers inEcologyandtheEnvironment8:92–98. 10.KunzTH,WrazenJA,BurnettCD(1998)Changesinbodymassandbody compositioninpre-hibernatinglittlebrownbats( Myotislucifugus ).Ecoscience5: 8–17. 11.ReynoldsDS,KunzTH(2000)Changesinbodycompositionduring reproductionandpostnatalgrowthinthelittlebrownbat, Myotislucifugus (Chiroptera:Vespertilionidae).Ecoscience7,10–17. 12.JonassonKA,WillisCKR(2011)ChangesinBodyConditionofHibernating BatsSupporttheThriftyFemaleHypothesisandPredictConsequencesfor PopulationswithWhite-NoseSyndrome.PLoSONE6(6):e21061.Available: http://www.plosone.org/a rticle/info%3Adoi%2F10.1371%2Fjournal.pone. 0021061.Accessed2011Aug25. 13.KayserC(1965)Hibernation.In:MayerW,VanGelderR,editors. 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33.BoumaHR,CareyHV,KroeseFG(2010)Hibernation:Theimmunesystemat rest?JournalofLeukocyteBiology88:619–624. 34.PuechmailleSJ,FrickWF,KunzTH,RaceyPA,VoigtCC,etal.(2011)Whitenosesyndrome:IsthisemergingdiseaseathreattoEuropeanbats?Trendsin Ecology&Evolution26(11):570–576. 35.ClevelandCJ,BetkeM,FedericoP,FrankJD,HallamTG,etal.(2006) EconomicvalueofthepestcontrolserviceprovidedbyBrazilianfree-tailedbats insouth-centralTexas.FrontiersinEcologyandtheEnvironment4:238–243. 36.BoylesJG,CryanPM,McCrackenGF,KunzTH(2011)Economicimportance ofbatsinagriculture.Science332:41–42. 37.KunzTH,BraundeTorrezE,BauerD,LobovaT,FlemingTH(2011) Ecosystemservicesprovidedbybats.AnnalsoftheNewYorkAcademyof Sciences1223:1–38.AlteredHibernationPatternsinWNS-AffectedBats PLoSONE|www.plosone.org10June2012|Volume7|Issue6|e38920


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