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Title:
White-nose syndrome fungus, Pseudogymnoascus destructans, on bats captured emerging from caves during winter in the southeastern United States
Series Title:
BMC Zoology
Creator:
Bernard, Riley F.
Willcox, Emma V.
Parise, Katy L.
Foster, Jeffrey T.
McCracken, Gary F.
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BMC
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English
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Subjects / Keywords:
Bats -- Mortality ( lcsh )
White-nose syndrome ( lcsh )
Hibernation ( lcsh )
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serial ( sobekcm )
Location:
North America -- United States -- Tennessee

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Abstract:
Background Emerging infectious diseases in wildlife are an increasing threat to global biodiversity. White-nose syndrome (WNS) in bats is one of the most recently emerged infectious diseases in North America, causing massive declines in eastern bat populations. In the Northeast, winter behavior of bats during the hibernation period, such as flying during the day or in cold weather, has been attributed to WNS. However, winter emergence of bats in the southeastern United States, where winters are warmer, has received little attention. The goals of this study were to determine if winter emergence results from infection by Pseudogymnoascus destructans, the causative pathogen of WNS, and to investigate how pathogen load and prevalence vary by species, site, and over time. Results We collected epidermal swab samples from 871 active bats of 10 species captured outside of hibernacula in Tennessee during winters 2012–2013 and 2013–2014. Deoxyribonucleic acid (DNA) from P. destructans was not detected on 54% of these bats, suggesting that winter emergence occurs regardless of fungal infection. Among infected bats, Perimyotis subflavus (tri-colored bats) had the highest mean fungal load, whereas Myotis lucifugus (little brown bats) had the highest infection prevalence of all individuals captured. Less than 18% (n = 59 of 345 individuals sampled) of all M. grisescens (gray bats) captured had detectible P. destructans DNA on their forearms and muzzle. Hibernacula with large M. grisescens populations had lower fungal loads than sites used by other species; however, mean load per species did not significantly differ between M. grisescens and non-M. grisescens sites. Conclusions We found that pathogen load and prevalence were higher on bats captured during winter 2012–2013 than in the following winter, indicating that fungal loads on bats did not increase the longer a site was presumably contaminated. Repeated low-dose exposure, mild temperatures, and availability of prey during winter in the Southeast may provide a regional refuge for surviving bat populations.

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University of South Florida
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K26-05343 ( USFLDC DOI )
k26.5343 ( USFLDC Handle )

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RESEARCHARTICLEOpenAccess White-nosesyndromefungus,Pseudogymnoascusdestructans,onbatscapturedemergingfromcavesduringwinterinthesoutheasternUnitedStatesRileyF.Bernard1,2*,EmmaV.Willcox3†,KatyL.Parise4,5†,JeffreyT.Foster4,5†andGaryF.McCracken1†AbstractBackground:Emerginginfectiousdiseasesinwildlifeareanincreasingthreattoglobalbiodiversity.White-nosesyndrome(WNS)inbatsisoneofthemostrecentlyemergedinfectiousdiseasesinNorthAmerica,causingmassivedeclinesineasternbatpopulations.IntheNortheast,winterbehaviorofbatsduringthehibernationperiod,suchasflyingduringthedayorincoldweather,hasbeenattributedtoWNS.However,winteremergenceofbatsinthesoutheasternUnitedStates,wherewintersarewarmer,hasreceivedlittleattention.ThegoalsofthisstudyweretodetermineifwinteremergenceresultsfrominfectionbyPseudogymnoascusdestructans,thecausativepathogenofWNS,andtoinvestigatehowpathogenloadandprevalencevarybyspecies,site,andovertime.Results:Wecollectedepidermalswabsamplesfrom871activebatsof10speciescapturedoutsideofhibernaculainTennesseeduringwinters2012–2013and2013–2014.Deoxyribonucleicacid(DNA)fromP.destructanswasnotdetectedon54%ofthesebats,suggestingthatwinteremergenceoccursregardlessoffungalinfection.Amonginfectedbats,Perimyotissubflavus(tri-coloredbats)hadthehighestmeanfungalload,whereasMyotislucifugus(littlebrownbats)hadthehighestinfectionprevalenceofallindividualscaptured.Lessthan18%(n=59of345individualssampled)ofallM.grisescens(graybats)capturedhaddetectibleP.destructansDNAontheirforearmsandmuzzle.HibernaculawithlargeM.grisescenspopulationshadlowerfungalloadsthansitesusedbyotherspecies;however,meanloadperspeciesdidnotsignificantlydifferbetweenM.grisescensandnon-M.grisescenssites.Conclusions:Wefoundthatpathogenloadandprevalencewerehigheronbatscapturedduringwinter2012–2013thaninthefollowingwinter,indicatingthatfungalloadsonbatsdidnotincreasethelongerasitewaspresumablycontaminated.Repeatedlow-doseexposure,mildtemperatures,andavailabilityofpreyduringwinterintheSoutheastmayprovidearegionalrefugeforsurvivingbatpopulations.Keywords:Bats,White-nosesyndrome,Hibernation,Tennessee,Bodycondition *Correspondence:rbernar3@vols.utk.edu†Equalcontributors1DepartmentofEcologyandEvolutionaryBiology,UniversityofTennessee,Knoxville,TN,USA2DepartmentofEcosystemScienceandManagement,PennsylvaniaStateUniversity,UniversityPark,Pennsylvania,USAFulllistofauthorinformationisavailableattheendofthearticle BMC Zoolo gy TheAuthor(s).2017OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.Bernardetal.BMCZoology (2017) 2:12 DOI10.1186/s40850-017-0021-2

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BackgroundEmerginginfectiousdiseasesinwildlifeposeanincreas-ingthreattoglobalbiodiversityandconservation[1,2].Asignificantproportionofthesediseasesaretheresultof“pathogenpollution”:theintroductionbyhumansorlivestockofnovelpathogensintonavewildlifepopula-tions[2,3].ProminentexamplesofpathogenpollutioncausingmassmortalityareAfricanrinderpestandam-phibianchytridiomycosis.Inthe1880’srinderpestkilled90%ofKenya’sbuffalopopulation,resultingindown-streameffectsonpredatorpopulationsandecosystemhealth[2].Chytridiomycosishasinfectedover50%ofallamphibianspeciesandcankill80%ofapopulationwithin4–5monthsofitsintroduction[4].Suchemer-ginginfectiousdiseasesaredevastatingtonativespecies,withdeleteriouseffectsthatpervadeecosystems[2,5].White-nosesyndrome(WNS)isarecentlyemergedin-fectiousdiseasethathasrapidlyspreadthrougheasternpopulationsofcavehibernatingbatsinNorthAmerica.ItiscausedbythepsychrophilicfungusPseudogymnoascusdestructans,andwasfirstdocumentedinNorthAmericainFebruary2006atacaveinupstateNewYork[6,7].Thisinvasivepathogen,whichishypothesizedtohaveoriginatedinEurasia[8–10],hassincespreadtomorethanhalfoftheUnitedStates(U.S.)andfiveCanadianprovincesandhaskilledover5.7millionbats[11].Cur-rently,atleastsixbatspeciesareexperiencingdetectablepopulationlossesduetoWNS,whereinonceabundantspeciesarenowthreatenedwithregionalextinction[11–14].PopulationdeclinesandthelossofbatspeciesduetoWNSarelikelytohavemajorecologicalandeconomicconsequences,withexpectedincreasesincropandforestpestpopulations[15,16].Pseudogymnoascusdestructanscolonizesthecutane-ousmembranesofthemuzzle,ears,wingsandtailofbats,erodingtheepidermisandinvadingtheunderlyingskinandconnectivetissue[17].Onceinvasionoccurs,P.destructansdisruptscriticalphysiologicalfunctionssuchascutaneousrespiration,bloodcirculation,andwaterbalance[18–21].Thesephysiologicalchangesresultinmorefrequentarousalsfromtorporandincreasedde-pletionofenergyreservesneededforhibernation[21,22].Recentstudiessuggestinfectedindividualscanelicittheinitialstagesofanimmuneresponse(e.g.transcriptionofcytokines);however,aprotectiveresponsedoesnotoccurduetohibernation[23–25].BatswithWNSalsoexhibitaberrantbehaviorinwinter,includingmovementfromthermallystablecaveenvironmentstolocationsnearthecaveentrance,daytimeemergence,andflyingincoldwin-tertemperatures[7,12,26].Species-specificbehaviorsduringhibernation,suchasmicroclimatepreference,mayalsoplayaroleindiseasesusceptibilityandsurvival[14,27,28].InNorthAmerica,smallbodiedbatshavebeenknowntohibernateatmicroclimatetemperaturesrangingfrom0to10C[20]andrelativehumidityashighas90–100%,whichfallwithintheoptimalgrowingconditionsforP.destructans.Whereaslargerbodiedspecies,likeE.fus-cus(mean=12g)andM.grisescens(mean=10g),oftenroostincolder,driersitesinahibernacula[29].Europeanbats,suchasM.myotis,a30gspecies,havebeenfoundtohibernateatmicroclimatetemperaturesrangingfrom4to12C[30],suggestingthatthereisnooptimalmicroclimatetemperatureforhibernatingbatspecies,withindividual-specificmicroclimatepref-erenceswithinaspeciesrangingwidely[31,32].MyotismyotisisthemostfrequentbatinEuropedoc-umentedwithP.destructansandulcerationsleadingtothemanifestationofWNS[27,33–35].NaturallyoccurringP.destructansinfectionsonM.myotishavebeenfoundtobequiteextensive,yethavenotleadtowidespreadmortalityofthespecies[33].Overall,themostaffectedbatspeciesinEuropearelargerbodiedspecies,whereasinNorthAmerica,smallbodiedindi-vidualshaveexperiencedthelargestpopulationde-clinesduetoWNS[13,14,36].InnortheasternNorthAmerica,wherewintersaresevereandpreyislimited,batsflyingoutsideduringthehibernationperiodarelikelysufferingtheeffectsofWNS.However,batsinthesoutheasternU.S.areknowntoleavehibernaculatofeedonwarmwinternights[Bernardetal.unpublished],suggestingthatwinteractivityintheSouthmaynotbeaconsequenceofdisease[37].Asanexample,minimumnighttimetemperaturesthroughoutJanuaryinTennesseeoverthelastfouryearsrangedfrom17Cto6C,whereasexternalcavetemperaturesinVermontrangedfrom27Cto17C,consistently10Ccolder[38].AsWNShasnowspreadthroughoutmuchofthesoutheasternU.S.[39]thepossibleeffectsofwinteractiv-ityontheepizootiologyofWNSremainunknown.Winterforagingoninsectsmayprovidebatshibernatinginsouth-ernlatitudeswithenergynotavailabletobatsintheNorth.Further,arousingfromtorportoengageinepisodicfeedingduringwinterwillraisebodytemperature,whichshouldactivatetheimmunesystemandpossiblybolsterimmunologicaldefensesagainstP.destructans.Evidencefromrabiesinbats[40],aswellasotherhost-pathogensystems[41,42],demonstratesthathostimmunitycanresultfromrepeatedlow-levelexposuretopathogens.Behaviorallyandphysiologically,batsintheSouthmaybedifferentfromnorthernbatsinwaysthatenablethemtosurviveWNS.ToinvestigatepossibleeffectsofwinteractivityonP.destructansinfectionsonbatsinsouthernlatitudes,weexaminedfungalloadandprevalenceonbatscapturedoutsideofhibernaculaduringwinter.Inthisstudy,weassessedprevalenceandfungalloadofP.destructansandidentifiedlesionsandulcerationscausedbypenetrationofP.destructansintowingandBernardetal.BMCZoology (2017) 2:12 Page2of11

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tailmembranesfortenspeciesofbatscapturedwhileactiveoutsideofhibernaculaduringtwowintersinTennessee.OurgoalsweretodetermineifemergenceduringwinteriscausedbythepresenceofP.destructansandtoexamineiftherearerelationshipsbetweenwinteractivity,fungalloadandprevalence,andbatspecies.Toaddressthesegoals,wetestedthefollowinghypotheses:1)activebatsleavingcavesduringwinterintheTennesseewillshowsignsofWNSasdemonstratedbyfungalloadorultravioletfluorescence;2)fungalloadandprevalencewillbehigheronsmall-bodiedcavehibernatingspecies,suchasM.lucifugus(littlebrownbats),M.septentrionalis(northern-longearedbats)andPerimyotissubflavus(tri-coloredbats),thanlargerbodiedspecies,suchasEptesicusfuscus(bigbrownbats)andM.grisescens(graybats).MethodsWeconductedourstudyattheentrancesoffivehiber-naculainTennesseefromOctobertoApril2012–2013and2013–2014(Fig.1).PriortotheemergenceofWNS,BlountCavewasthelargestknownendangeredM.sodalis(Indianabat)hibernaculuminthestateofTennessee,withanestimatedpopulationof9500indi-vidualsinFebruary2013[43].SmallnumbersofM.lucifugusandP.subflavusalsooccurinthecave.HawkinsandWarrenCavesaretwoofthelargesthi-bernaculaforendangeredM.grisescensinthestate,withestimatedpopulationsof150,000and400,000M.grisescens,respectively.BothcavesalsocontainasmallpopulationofM.sodalisduringwinter[44].CampbellandWhiteCavescontainpopulationsofM.leibii(easternsmall-footedbats),M.lucifugus,M.septentrionalis,andM.sodalis,withfewerthan1000individualsineachcave[45].BatsinBlountandHawkinsCaveswereconfirmedpositiveforP.destructansinthewintersof2009–2010and2010–2011,respectively,withallothersitesconfirmedbywinter2012–2013[44,46].Wecapturedbatsateachsiteonceamonthusingmist-nets(AvinetInc.,Dryden,NY;meshdiameter:75/2,2.6mhigh,4shelves,6–12mwide).Site-specificsin-gle-,double-andtriple-highnetsweredeployed30minbeforecivilsunsetatcaveentrancesandalongcorri-dorswithin100mofthecave.Wekeptthenetsopenfor5horuntilwecaptured30batsandclosedthemwhentemperaturesdroppedbelow0C.Aftercapture,individualbatswereplacedinpaperbagsandheldfor30to60mininaninsulatedboxwithfourhand-warmers(HotHands,Dalton,GA).MyotisgrisescensandM.sodaliswereheldforamaximumof30min.Werecordedspecies,reproductivecondition,forearmlength(mm),weight(g),miteload[47]andwing-damageindex(WDI,[48]),andcollectedepidermalswabsamplesfromeachbatfollowingestablishedpro-tocols(seebelow).Duringthewinterof2013–2014,weexaminedbatsforthepresenceofWNS-relatedfluor-escencebytransilluminatingthewingswithultraviolet Fig.1CavelocationswithintheircountyboundariesinTennessee,UnitedStates.Batswerecapturedateachsiteoncepermonthduringwinter(OctobertoApril)2012–2013and2013–2014.AtleastonebatateachsitewasfoundpositiveforP.destructansbywinter2012–2013Bernardetal.BMCZoology (2017) 2:12 Page3of11

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(UV)light(wavelengths385–390nm,[49,50]).IfP.destructanshaspenetratedtheskin,lesionsfluoresceyellow-orangeunderUVillumination[49].Fungalsam-plesforeachindividualwerecollectedusingasterileepi-dermalswabdippedinsteriledeionizedwaterandrubbedonthebat’sforearmandmuzzlefivetimeseach[51].SwabswereplacedinRNAlaterTissueStabilizationSolu-tion(LifeTechnologies,GrandIsland,NY)andstoredat4C.Allcave-roostingspecieswerebandedwitheither2.4mmor2.9mmnumbered,lippedalloyforearmbands(Porzana,Ltd.,Icklesham,EastSussex,UK)andreleasedatthesiteofcapture.Duetothedistancebetweensitesandthelackofevidencetosuggestmovementoccursbe-tweencavesduringwinterintheregion,weassumedeachcavewasaclosedpopulation.WeextractedfungalDNAfromeachswabsampleusingDNeasy96Blood&Tissuekits(QiagenInc.,Valencia,CA;[52]).Allsamples,aswellasnegativecontrolwellsdistributedacrosseachpolymerasechainreaction(PCR)plate,weretestedforthepresenceofP.destructansDNAusingaReal-TimePCRassaytargetingtheintergenicspa-cer(IGS)regionoftheribosomalribonucleicacid(rRNA)genecomplex[53].AllplateswereruninduplicatewithaquantifiedstandardofisolateP.destructans20,631–21.Anyreactionthatcrossedthethresholdbaselineinfewerthan40cyclesoneitherplatewasconsideredpositiveforP.destructansDNAand,whenrelevant,theaverageP.destructansload,hereafterreferredtoasfungalload,innanograms(ng)wascalculatedineachsamplebasedonthecyclethreshold(Ct)valueandageneratedstandardcurvebasedonserialdilutions([34];nanogramsP.destructans=10–3.348xCt+22.049).FungalloadvaluesofP.destructanswereaveragedacrossbothruns.Wefollowedfielddecontaminationprotocolsinaccord-ancewiththeUnitedStatesFishandWildlifeServiceWNSDecontaminationGuidelinesandrecommendationsbythestateofTennessee[54].AllcaptureandhandlingtechniqueswereapprovedbytheUniversityofTennesseeInstituteofAnimalCareandUseCommittee(IACUC2026–0514)andwereconsistentwiththeguidelinesis-suedbytheAmericanSocietyofMammalogists[55].Weobtainedbothfederal(USFWSTE-71613A;GRSM-2013-SCI-1053;GRSM-2014-SCI-1053)andstate(TWRA3716;TDEC2011–031)permitstocaptureandhandlebatsatwinterhibernaculaforthisstudy.StatisticalanalysisFungalloaddatawerelogtransformedpriortoanalysestomeetassumptionsofnormalityandhomogeneityofvariance.Weusedseparategeneralizedlinearmodels(functionglminpackagelme4[56]inProgramRv3.1.2[57])tocomparechangesinloadandprevalenceofP.destructansforeachspeciesovertime.Modelswererunaseitherbinomial(prevalence)orGaussian(fungalloads)distributions,andweretestedforsignifi-canceusinglikelihoodratiotests.TodeterminethechangeinP.destructansloadandprevalenceovertimewithineachmodel,weusedamodifiedtimeaxissimilartoLangwigetal.2015[36]wheretime-0representedthestartofhibernation(October1).Infectionpreva-lencewascalculatedbydividingthetotalnumberofin-fectedindividualsbythetotalnumberofindividualscapturedduringthesametimeperiod.Allmeansarereportedstandarderror.Theresultspresentedhereinrepresentbatscapturedoutsideofeachsite,notofthehibernatingpopulationasawhole.ResultsWecapturedandswabbed871batsof10species(593males,276females,2unknownsduetoescape;Table1).Ofthese,408individualswerepositiveforthepresenceofP.destructansDNA(Pd+)byReal-TimePCRanalysis.AtleastoneindividualfromallspeciescapturedwasPd+,includingtwoCorynorhinusrafinesquii(Rafinesque’sbig-earedbat),twoLasiurusborealis(easternredbat)andoneLasionycterisnoctivagans(silver-hairedbat)([58];Table1).However,thesethreespecieswereexcludedfromthecomparativeanalysesduetosmallsamplesizes.CaptureratesofM.septentrionalis,M.sodalis,andP.subflavusdramaticallydeclinedduringwinter2013–2014,withM.septentrionalisrarelycapturedafterDecember2013.Fifty-onepercentofthebatscaptured(n=245/480)duringwinter2012–2013werePd+,whereasonly41.6%ofthebats(n=163/391)werePd+inwinter2013–2014.Whenpooledbyseason,meanfungalloadsweresignifi-cantlyhigherduringthefirstyearofsampling(likelihoodratiotest:X2=17.978,p<0.0001).Excludingspecieswithlowsamplesize,thereweresignificantdifferencesinload Table1TotalbatscapturedandswabbedatfivecavesinTennesseeduringwinters2012–2013and2013–2014Winter2012–2013Winter2013–2014SpeciesPd+Pd-TotalPd+Pd-TotalCorynorhinusrafinesquiia123145Eptesicusfuscus8111981119Lasiurusborealisa202033Lasionycterisnoctivagansa000123Myotisgrisescens2413916335147182Myotisleibii212344242751Myotislucifugus1111214519Myotisseptentrionalis843111546955Myotissodalis38114914822Perimyotissubflavus551873211132Talliesareprovidedforthetotalnumberofindividualsdeterminedpositive(Pd+)ornegative(Pd-)forP.destructansthroughreal-timePCRanalysisaDataforthesespeciesareexplainedfurtherinBernardetal.[58]Bernardetal.BMCZoology (2017) 2:12 Page4of11

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ofP.destructansperspeciesovertime(likelihoodratiotest:X2=278.06,p<0.0001,Fig.2).FungalloadswerelowestwhenbatsenteredhibernationinOctober(4.990.328log10ng)andpeakedformostspeciesdur-ingmid-hibernation(December–February;2.800.095log10ng;Fig.2andAdditionalfile1).Thereafter,meanfungalloadsonsixofthesevenspeciesremainedstablethroughtheendofthehibernationperiodinApril.However,meanfungalloadonP.subflavus,theseventhspecies,continuedtoincreasethroughtheendofhiber-nation,reachinglevelstwiceashighasthoserecordedinDecember(Fig.2).PerimyotissubflavushadthehighestmeanP.destructansload(2.340.091log10ng),whereasM.grisescenshadthelowestmeanP.destructansloadofallspeciessampled(4.890.075log10ng).Infectionprevalencevariedamongspecies,withlarge-bodiedspecies,suchasE.fuscusandM.grisescens,experiencingthelowestprevalenceonaverage(Fig.3).FungalloadsonactivebatsintheSoutheastwerealsolowerthanontorpidbatssampledinaseparatestudyinthenortheasternU.S.(Table2).Duringthesecondseasonofsampling,weexamined481batsforWNS-relatedfluorescence.Ultravioletfluorescencerevealedvaryingdegreesofdamageduetothefungus,fromsmallpin-sizedlesionstolargecoalescingregionsoffluorescenceandinfiltrationcorrespondingwithincreasedpathogenloads.Only15batsthatfluorescedshowedsomesignsofwingdamage,varyingfromslightde-pigmentationtopinholes(WDI=1).Allbatscapturedduringearlyhibernation(OctoberandNovember)werenegativeforUVfluorescence.ThehighestpercentofUV-infectedbatswerecapturedduringmid-hibernation(December35.9%,January29.5%).Atotalof66batswerepositivebybothPCRandUV,withonlytwoUVpositivesnotdetectedasPd+byPCR.Atotalof181individualswerePd+fromPCRbutUVnegative,whereas232batswerenegativeforbothP.destructansandUV.BatsthatwerePd+bybothPCRandUVhadhigherfungalloadsthanindividualsthatweredeterminedP.destructansposi-tiveonlybyPCR(t200.6=8.83,p<0.0001).AsnotedinZukaletal.2016[8],wedidnotobserveathresholdwithwhichthepresenceofUVfluorescencecorrespondedtoaminimumfungalload(UVpositive:range4.74–0.22log10ng;UVnegative:range5.73–0.36log10ng).DiscussionOurstudydemonstratesthattheemergenceofbatsduringwinterinTennesseeisnotindicativeofWNS,aslessthanhalfofallbatscapturedoutsideofthecavessampledduringthehibernationperiodwerepositiveforP.destructans.Althoughwecanonlymakeinferencedir-ectlytobatscapturedoutsideofhibernaculainTennessee,wefindawiderangeofP.destructansloadsoncapturedbats.Aswearenotonlysamplinghigh-loadindividualsleavingthehibernaculawe,therefore,canassumethatourresultsarerepresentativeoftheentirepopulation.BycapturingbatsactiveduringwinterandcouplingwinteractivitywithmeasuresofprevalenceandloadofP.destructansonbats,wehighlighttheregionaldifferencesintheresponsesofWNSaffectedspecieswithintheirgreatergeographicrange.ThisstudydemonstratesthatasWNScontinuestospreadthroughoutNorthAmerica,it Fig.2MeanmonthlyloadofPseudogymnoascusdestructansperspecies.Meanload(SE)ofP.destructans(Pd)permonthforsevenspeciescapturedatfivecavesitesinTennesseeduringOctober–April;2012–2013(closedcircles)and2013–2014(opencircles).CirclesindicatemonthswherePdpositiveindividualswerecapturedBernardetal.BMCZoology (2017) 2:12 Page5of11

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shouldnotbeassumedthatallindividualswithinaspecieswillreactsimilarlytothedisease.IntheNortheast,asecondarysymptomofthediseaseisactivityduringwin-ter,specificallyaberrantbehavior,orbatsflyingduringcoldweatherorduringdaylighthours[12,13,26,59].Although,casesofunusualwinterbehaviorhavebeenreportedintheSoutheast[37,46],ourdatahighlightthatnighttimeemergencesfromhibernaculainTennesseearenotalwaysassociatedwithP.destructansinfection.There-fore,theeffectsofWNSonbatsinthesoutheasternU.S.arenotdirectlycomparabletothoseintheNorth,asregionalandspecies-specificdifferences,likedegreeofwinteractivity,bodycondition[60]andsusceptibilitytodisease[14,36],likelyvarysignificantlyfromthoseintheNortheast.Bothinfectionprevalenceandloadsvariedconsider-ablyamongspecies,withallsmall-bodiedbats,exceptM.leibii,havinghigherfungalloadsandprevalencethanE.fuscusandM.grisescens.Species,suchasM.septentrionalisandP.subflavus,withhighratesoffun-galloadsandprevalence,wereconsistentlyfoundwiththelargestregionsoffluorescenceandwingdamage,indicatinghighratesoftissueinvasionbyP.destructans[48].Whereas,M.grisescens,whichhavelowfungalloadsandprevalence,wereoftenfoundwithsubstantialdiscoloration,wingdamage,andtissuelossunrelatedtoWNSbasedonnegativeUVandPCRresults,aswellasWNSWDIscoring[48,61].Accordingtosurveyre-cordsinTennessee,M.grisescenswasoftenobservedwithdiscoloredwingmembranesandsignificantscar-ringandtissuelosspriortoP.destructansNorthAmericanintroduction(JohnLambandTroyBest,per-sonalcommunication).AlthoughtransmissionofP.destructansamongindi-vidualscouldbeassociatedwiththeaccumulationofthefunguswithinahibernaculumbasedonthetimesinceinitialintroduction,numberofbats,andinternalcaveconditions,itisimportanttoalsoconsiderspeciesspecificbiologyandbehaviors.WhentheNortheastwastheleadingedgeofWNSinfection,small,solitarybats,suchasM.septentrionalisandP.subflavus,hadsignificantlyhigherfungalloadsthansimilarsizedcolo-nialspecies[11,28,41],suggestingP.destructanswasspreadingviadensity-dependenttransmissioninthesetwospeciesasclustersizeincreased[14].Incontrast,ourfindingssuggestthatM.sodalisandM.lucifugus,speciesknowntoclusterintightaggregationsduringhibernation,hadfungalloadsandprevalencesimilartothoseofsolitaryspecies.Therefore,transmissionofP.destructansamongmorecolonialspeciesinsouthernhibernaculamaybeafunctionofthefrequency,orrate,ofinfectionamongindividualswithinthecluster,ratherthantheclustersize[14,34,62].Interestingly,M.grises-cens,thelargestbatthathibernatesexclusivelyincavesintheMidwestandsoutheasternU.S.,hadthelowestfungalloadsandprevalenceofP.destructansamongallspeciessampled(Figs.2and3).Thisfindingcontrastswithpat-ternsobservedinEurope,whereM.myotisandotherlargebodiedhibernatingbatshavethehighestincidenceofP.destructans[8,35,63].IntheNortheast,diseaseimpactsonM.lucifugus,M.septentrionalisandP.subflavusin-creasedwithhigherhumidityandtemperaturewithin Fig.3InfectionprevalenceofPseudogymnoascusdestructansonactivebatscapturedinTennessee.Infectionprevalence(#infectedindividuals/total#individualssampled)ofPseudogymnoascusdestructans(SE)forsevenbatspeciescapturedatfivehibernaculainTennesseeduringwinters2012–2013(closedcircles)and2013–2014(opencircles)Bernardetal.BMCZoology (2017) 2:12 Page6of11

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Table2P.destructansloads(log10ng)onbatscapturedintheSoutheastcomparedtovaluescollectedfromtorpidbatsintheNortheastSpeciesEPFUMYLEMYLUMYSEMYSOPESUMonthSoutheastNortheastSoutheastNortheastSoutheastNortheastSoutheastNortheastSoutheastNortheastSoutheastNortheastOct.4.940.163.960.845.000.235.184.834.830.215.200.074.200.95Nov.1.995.734.120.592.750.353.810.992.030.984.844.430.363.120.53Dec.4.720.382.234.860.122.342.240.231.80Jan.3.304.400.234.172.780.343.112.030.122.380.292.280.13Feb.2.980.694.280.382.520.172.520.442.400.12Mar.4.120.292.740.254.390.292.480.492.700.481.190.182.680.241.350.502.700.112.430.212.400.120.820.12Apr.4.190.453.803.962.570.141.640.223.500.222.860.262.890.601.860.171.460.45Valuesforfungalloadsonnortheasternbatsarebasedonthepublishedreportsofsamplescollectedfrom30hibernaculacoloniesinNewYork,Vermont,Massachusetts,Virginia,NewHampshireandIllinoiswhereP.destructanshasbeenpresentforatleastoneyear[36,82].Allvaluesarepresentedwithstandarderror,unlessonlyoneindividualwassampled.EmptycellsindicatenoP.destructanswasdetectedonthespeciesduringthesamplemonth.Boldvaluessignifywhichloadislowestinregionalcomparisonperspeciespermonth,withsimilarloadsinbothregionsitalicized.Onlyspeciesthatwerecapturedinbothregionsareincludedinthetable.Samplesfrombothstudieswereanalyzedatthesamelabbytwooftheauthors(KLPandJTF)Bernardetal.BMCZoology (2017) 2:12 Page7of11

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roosts,suchthatindividualssampledinthecoldestanddriestroostshadsignificantlylowerfungalloads[14,27].Myotisgrisescens,however,hibernateinaggregationsof100,000to1,500,000individuals[64]incoldairtrapsvary-ingfrom1to9C[65],whicharethelowesttemperaturesatwhichP.destructansgrowthoccurs[66].Asofspring2017,M.grisescenshaveyettoexperienceanyWNS-relateddeclinesandtheirpopulationsappeartohaveremainedstablewithinTennessee.AlthoughsomeM.gri-sescensthatwecapturedhavebeenidentifiedwithsecond-aryfungalinfections,skindiscoloration,and/orsubstantialtissueloss[Bernardunpublisheddata,JohnLambandTroyBestpersonalcommunication],wehaveyettoiden-tifyhowthespeciesissurvivingWNS.Severalbehavioraltraits,suchaspreferredmicroclimateswithinhibernacula,sustainedactivityandforagingthroughoutwinter[37]andyear-roundcaveuse[67,68],mayenablethisspeciestopreventorminimizethecolonizationofP.destructansduringtorpor.Whenallsevenspecieswithsamplessizes12individ-ualswerecombined,meanfungalloadwashighestduringmid-hibernation,DecemberthroughFebruary,thecoldestperiodoftheyearintheSoutheast.Perimyotissubflavus,however,continuedtoexperienceanincreaseinfungalloadthroughtheendofhibernation,whichcouldbeattributedtothemicroclimate(11to23C;80%Rela-tiveHumidity)usedbythespeciesduringhibernation[69–71].Alternatively,invitrogrowthcurvessuggestthatP.destructansmayreproducemorequicklyincaveenvironmentsthatmaintainmoremoderatetemperaturesof10to15Cinwinter[66],whichcouldresultinin-creasedgrowthratesofthefungusinsouthernhibernac-ula,andthereforeleadtoapeakinfungalloadwithinspecieshibernatingwithinthattemperaturerange.Contrarytoourpredictionandthefindingsofstudiesfromnorthernhibernacula[69],bothpathogenloadandprevalencewerelowerinthesecondyearofthestudyforsevenofthetenbatspeciescaptured[58].Bythesecondsurveyyear,allcaveshadbeencontami-natedbyP.destructansforatleasttwoyears.Althoughthiscouldbeduetothedecreaseinthecaptureofhighlysusceptiblespecies(Table1)causedbyWNSre-lateddeclineswithineachcave[72],climaticvariationbetweenyearscouldalsoimpactdiseasespread.SimilartrendshavebeendocumentedafterthearrivalofBatra-chochytriumdendrobatidis,thepathogenicfungalagentofchytridiomycosisinfrogs.Pathogenloadsinnavefrogpopulationsincreaseddramaticallyinthefirstyear,causingarapidriseininfectionintensityandpreva-lenceindenselypopulatedhabitats[73].Asthepatho-genloadoninfectedfrogsincreased,manypopulationssufferedfromhighratesofmortality.However,thesur-vivalofinfectedindividualsledtopathogenendemismandpopulationpersistenceonthefrogs.AsimilardynamicmaybeoccurringinhibernaculacontaminatedbyP.destructans[74,75].Individualswithhighpathogenloadsperishinthefirstyear,perhapsallowingforindivid-ualswithminorP.destructansinfectionstoreturntothehibernaculathefollowingwinter.Further,somescientistshypothesizethatincreasedincidencesofchytridiomycosisarelinkedtoincreasesinglobaltemperaturescreatingop-timalsitesforthepathogen[76,77].SimilarresponsesmayoccurwithWNSifregionsoftheSoutheastexperi-encemoreextremewinters,creatingmorefavorablecon-ditionsforP.destructansgrowthorlimitedopportunitiesforbatstoreplenishfatstores.Repeatedlow-levelexpos-uretoP.destructansorendemismofthepathogen,mildwinters,andepisodicfeedingmayallowforpersistenceofbatpopulationshibernatingintheSoutheast.RecentevidencefromtheNortheastsuggestssomepop-ulationsofbatshavetheabilitytopersistandreproducedespitecontinuedexposuretoWNS[42,75,78,79].Com-paringP.destructansloadsonthesamespeciessampledwhiletorpidintheNortheasternandactiveintheSouth-easternU.S.,wefindtheaveragefungalloadsoverthesea-sonwereconsistentlyloweronactivebats(Table2).EptesicusfuscusandM.lucifuguscapturedinTennesseehadlowerloadsthanthosesampledinnortheasternhiber-nacula,whereasfungalloadsonM.septentrionalisandM.sodalisweresimilartowardsthebeginningandendofhibernation.PerimyotissubflavusintheNortheast,how-ever,hadhigherloadsthanindividualssampledintheSoutheast.MeaningfuldirectcomparisonsarelackingduetoinsufficientnumberssampledintheNortheast.Ultim-ately,weareseeingthatPd+batscapturedinTennesseehavesimilarloadstotorpidindividualssampledinmorenorthernareasoftheirrange,indicatingthatactivityandsurvivalintheSoutheastmaybemorecloselylinkedwithshort,mildwintersandmoderatepreylevelsduringwinter.ConclusionsThedepopulationofnavebathostsbyWNSwilllikelyleadtochronicpopulationdepression[2]duetothelong-termpersistenceofP.destructanswithincaveenvironments.Whereas,mortalityintheNortheastcanreach90%withintwoyearsofWNSconfirmation[12,13],populationdeclineslikelyattributedtoWNSintheSoutheastoccurfourtofiveyearsafterconfirmationandtendtobelesssevereinsomespecies[26,27,40,80].Ourfindingssupportthehypothesisthatemergencefromcavesduringwintermayinfluencethevariationseeninpathogenloadandinfectionintensityamongspecies.Byunderstandingthespecies-specificdynamicsofP.destruc-tanswithinactivewinterpopulations,managementstrat-egies,suchasregionalareaclosuresandbio-controltreatmentscanbeimplementedmoreeffectively.IntheSoutheast,mitigationmeasures,suchascaveareaclosuresBernardetal.BMCZoology 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usedtominimizeexternalcavedisturbances(e.g.GreatSmokyMountainsNationalPark)orbio-controlagents(e.g.Rhodococcusrhodochrous[81]andchitosan),mayworkbestwhentargetinghibernaculawithsmall-bodiedbatssuchasM.lucifugus,M.septentrionalis,andP.sub-flavus;speciesthatarebeinghitthehardestbyWNSintheSoutheast[80,61].Finally,ourstudysuggeststhatpopulationsofsomebatsarepersistingregardlessofre-peatedexposuretoP.destructans.AlthoughtheregioniscurrentlyexperiencingWNS-relatedmortalitywithinhighlyaffectedspecies,mildtemperaturesandthepersist-entavailabilityofpreyduringwintermayallowtheSouth-easttoserveasarefugeforsurvivingbatpopulations.AdditionalfileAdditionalfile1:FigureS1.PeakloadofP.destructansonbatscapturedleavinghibernacula.MaximummonthlyloadofP.destructansforsevenspeciescapturedatfivecavesitesinTennesseeduringthe2012–2013and2013–2014hibernationperiod(October–April).CirclesindicatemonthswherePdpositiveindividualswerecaptured.Speciesacronymcodes:EPFU–Eptesicusfuscus,MYGR–Myotisgrisescens,MYLE–Myotisleibii,MYLU–Myotislucifugus,MYSE–Myotisseptentrionalis,MYSO–Myotissodalis,PESU–Perimyotissubflavus.(PNG264kb)AbbreviationsCt:Cyclethreshold;DNA:Deoxyribonucleicacid;IGS:Intergenicspacer;ng:Nanograms;PCR:Polymerasechainreaction;Pd-:Pseudogymnoascusdestructansnegative;Pd+:Pseudogymnoascusdestructanspositive;rRNA:Ribosomalribonucleicacid;U.S.:UnitedStates;UV:Ultravioletlight;WDI:Wingdamageindex;WNS:White-nosesyndromeAcknowledgementsWewouldliketothankAnnaChow,MaxCox,NeilGiffen,ReillyJackson,DevinJones,KittyMcCracken,MariahPatton,andAnaReboredo-Segoviaforhelpinthefield;permittingandfieldhelpfromGreatSmokyMountainsNationalPark,TennesseeWildlifeResourcesAgency,TennesseechapterofTheNatureConservancy,TennesseeDepartmentofEnvironmentalConservation,andtheTennesseeregionalofficeoftheUSFishandWildlifeService.WewouldalsoliketothankColinSobekforassistingwithlabworkatNorthernArizonaUniversity,andtheWhite-nosesyndromeandNorthAmericanSocietyforBatResearch(NASBR)communitiesforcountlessdiscussions,advice,andcontinuedsupportthroughoutthisstudy.WewouldliketothankMelquisedecGamba-RiosforgraphicsupportandassistanceinR,aswellaskeepingRFBsanewhilefinishingherdegree.Finally,wewouldliketothankallreviewersfortheircommentsonearlierversionsofthemanuscript.FundingTheresearchpresentedinthismanuscriptwasfundedbytheWhite-nosesyndromeresearchgrantthroughBasicallyBatsWildlifeConservation,Inc.,UniversityofTennesseeInstituteofAgricultureCenterforWildlifeHealth,UniversityofTennesseeDepartmentofEcologyandEvolutionaryBiology,andtheUSGeologicalSurvey.AvailabilityofdataandmaterialsThedatasetsgeneratedand/oranalyzedduringthecurrentstudyareavailableinthefigsharerepository,https://figshare.com/s/e84c3d6178fbe7294d6b.Authors’contributionsRFBdesignedtheproject,collectedallfieldsamples,analyzedalldata,collaboratedonobtainingresearchfunding,andwrotethemanuscript.EVWassistedwithcollectionoffieldsamplesandcollaboratedonobtainingresearchfunding.JTFandKLPperformedthegeneticanalyses.GFMassistedwiththeprojectdesign,collaboratedonobtainingresearchfunding,andwasamajorcontributorinwritingthemanuscript.Allauthorsreadandapprovedthefinalmanuscript.EthicsapprovalandconsenttoparticipateWefollowedfielddecontaminationprotocolsinaccordancewiththeUnitedStatesFishandWildlifeServiceWNSDecontaminationGuidelinesandrecommendationsbythestateofTennessee[54].AllcaptureandhandlingtechniqueswereapprovedbytheUniversityofTennesseeInstituteofAnimalCareandUseCommittee(IACUC2026–0514)andwereconsistentwiththeguidelinesissuedbytheAmericanSocietyofMammalogists[55].Weobtainedbothfederal(USFWSTE-71613A;GRSM-2013-SCI-1053;GRSM-2014-SCI-1053)andstate(TWRA3716;TDEC2011–031)permitstocaptureandhandlebatsatwinterhibernaculaforthisstudy.ConsentforpublicationNotapplicable.CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.Publisher’sNoteSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.Authordetails1DepartmentofEcologyandEvolutionaryBiology,UniversityofTennessee,Knoxville,TN,USA.2DepartmentofEcosystemScienceandManagement,PennsylvaniaStateUniversity,UniversityPark,Pennsylvania,USA.3DepartmentofForestry,Wildlife,andFisheries,UniversityofTennessee,Knoxville,TN,USA.4CenterforMicrobialGeneticsandGenomics,NorthernArizonaUniversity,Flagstaff,AZ,USA.5DepartmentofMolecular,Cellular,andBiomedicalSciences,UniversityofNewHampshire,Durham,NH,USA.Received:21November2016Accepted:7August2017 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