J Anim Ecol. 2019;88:591–600. wileyonlinelibrary.com/journal/jane  | 591© 2019 The Authors. Journal of Animal Ecology © 2019 British Ecological Society

Received:10September2018  |  Accepted:30November2018DOI:10.1111/1365-2656.12954

R E S E A R C H A R T I C L E

Higher fat stores contribute to persistence of little brown bat populations with white- nose syndrome

Tina L. Cheng1,2  | Alexander Gerson3  | Marianne S. Moore4 | Jonathan D. Reichard5 | Joely DeSimone3 | Craig K. R. Willis6 | Winifred F. Frick1,2 | Auston Marm Kilpatrick1

1Department of Ecology and Evolutionary Biology, University of California, UC Santa Cruz, California; 2BatConservationInternational,Austin,Texas;3DepartmentofBiology,UniversityofMassachusetts,Amherst,Amherst,Massachusetts;4CollegeofIntegrativeScienceandArts,ArizonaStateUniversity,Mesa,Arizona;5US Fish and Wildlife Service, Hadley, Massachusetts and 6Department of Biology and Centre for Forest Interdisciplinary Research, University of Winnipeg, Winnipeg, Manitoba, Canada

CorrespondenceTinaL.ChengEmail: tinalcheng@gmail.com

Funding informationU.S. Fish and Wildlife Service, Grant/AwardNumber:WNS2015-08;DivisionofEnvironmentalBiology,Grant/AwardNumber:1115895and1336290

HandlingEditor:AnnTate

Abstract1. Thepersistenceofpopulationsdecliningfromnovelstressorsdepends,inpart,ontheirabilitytorespondbytraitchangeviaevolutionorplasticity.White-nosesyn-drome(WNS)hascausedrapiddeclinesinseveralNorthAmericabatspeciesbydisrupting hibernation behaviour, leading to body fat depletion and starvation. However, some populations of Myotis lucifugusnowpersistwithWNSbyunknownmechanisms.

2. WeexaminedwhetherpersistenceofM. lucifiguswithWNScouldbeexplainedbyincreased body fat in early winter, which would allow bats to tolerate the increased energeticcostsassociatedwithWNS.Wealso investigatedwhetherbatswereescapinginfectionorresistanttoinfectionasanalternativemechanismexplainingpersistence.

3. Wemeasuredbodyfat inearlyand latewinterduring initialWNS invasionand8yearslateratsixsiteswherebatsarenowpersisting.Wealsomeasuredinfec-tion prevalence and intensity in persisting populations.

4. Infectionprevalencewasnotsignificantlylowerthanobservedindecliningpopu-lations. However, at two sites, infection loads were lower than observed in declin-ingpopulations.BodyfatinearlywinterwassignificantlyhigherinfourofthesixpersistingpopulationsthanduringWNSinvasion.

5. Physiological models of energy use indicated that these higher fat stores could reduceWNSmortalityby58%–70%.TheseresultssuggestthatdifferencesinfatstorageandinfectiondynamicshavereducedtheimpactsofWNSinmanypopu-lations. Increases in body fat provide a potential mechanism for management in-tervention to help conserve bat populations.

K E Y W O R D S

emerging infectious disease, evolution, plasticity, resistance, tolerance, trait change

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1  | INTRODUC TION

Anthropogenic processes have resulted in rapid environmentalchange, resulting in novel environments and multiple stressors (Vitousek, Mooney, Lubchenco, & Melillo, 1997). The persistenceof populations in the face of anthropogenic change depends, to some degree, on their ability to respond demographically or by trait change at the population level—whether through selection or phe-notypicplasticity(Cattau,Fletcher,Kimball,Miller,&Kitchens,2017;Darimont etal., 2009; Daszak, Cunningham, &Hyatt, 2001; Dirzoetal.,2014;Gomulkiewicz&Holt,1995;Hendry,Farrugia,&Kinnison,2008; Kilpatrick etal., 2006; Williams, Shoo, Isaac, Hoffmann, &Langham,2008).Novelpathogenscanexertstrongselectivepres-sures on populations and cause rapid evolutionary responses in hosts (Altizer,Harvell,&Friedle,2003)andhaveallowedsomespeciestopersist and recover (e.g., rabbits and Myxoma virus (Ratcliffe, Myers, Fennesy,&Calaby,1952),house finchesandMycoplasma gallisepti-cum (Bonneaudetal.,2011)),whileotherspecieshavebeendrivenextinct(e.g.,chytridiomycosis;Skerrattetal.,2007).Identifyinghosttraits that enable persistence can be critical to help facilitate manage-ment efforts to recover populations following perturbations (Hobbs, Hallett,Ehrlich,&Mooney,2011).

In the past decade, the disease white-nose syndrome (WNS),caused by the fungal pathogen, Pseudogymnoascus destructans (Pd), has causedwidespread declines in hibernating bats inNorthAmericaandthreatensseveralspecieswithextinction (Fricketal.,2010,2015,2017;Langwigetal.,2012).Pd infects the epidermis of hibernatingbatsduringwintercausinggrosslesionsininfectedskintissue (Meteyeretal., 2009), a cascadeofphysiological responses(including hypotonic dehydration, hypovolaemia and metabolic ac-idosis), disruption of hibernation patterns and sickness behaviour(Bohn etal., 2016; Verant etal., 2014; Warnecke etal., 2013). Inhealthy bats, arousals during winter hibernation are infrequent (rep-resenting <1% of hibernation time expenditure) but necessary inordertocounteractcostsoftorpor,includingthebuild-upofmeta-bolic waste and dehydration, despite also being energetically costly (using 80%–90% of total stored body fat throughout the winter)(Kayser,1965;Thomas,Dorais,&Bergeron,1990).Pd-infectedbatsarouseapproximatelytwiceasfrequentlyasuninfectedbats(Lilleyetal.,2016;Lorchetal.,2011;Reederetal.,2012),whichresultsinpremature depletion of body fat and starvation.

WhilemanycoloniesofmultiplespecieshavebeenextirpatedorcontinuetodeclinefromWNS,somecoloniesofthelittlebrownbat Myotis lucifugus in the north-eastern United States appeartohave stabilized following initial declines (Langwig etal., 2012,2017;Maslo,Valent,Gumbs,&Frick,2015).Themechanismsal-lowing each colony to persist with Pdarenotfullyknownbutmayinclude:(a)reducedtransmissionfromdensity-dependentdiseasedynamics (Anderson, & May, 1992), (b) attenuated virulence ofthe pathogen (Alizon, Hurford, Mideo, & Van Baalen, 2009), (c)environmental refugia that reduce pathogen growth rate, such as lower temperatures or humidity (Langwig etal., 2012;Verantetal.,2014),(d)resistancetoinfectionbybats(e.g.,viatheimmune

system or cutaneous barriers, including antimicrobial peptides or the skinmicrobiome (Hoyt etal., 2015)), as suggested by recentwork showing lower pathogen loads in some colonies (Langwigetal.,2017);or(e)toleranceofinfection(Fricketal.,2017),eitherviaalteredhibernationbehaviour(Lilleyetal.,2016)orgreaterfatstores, which could allow animals to tolerate more frequent arous-alsorsicknessbehaviour.

Given the impact of WNS on hibernation energetics (Reederetal.,2012;Warneckeetal.,2012)andtheimportanceofbodyfatin overwinter survival (Kunz et al., 1998; Supporting Information Figure S5), greater body fat ofWNS-affectedbats should also beassociated with higher overwinter survival, but evidence for fatter batsfollowingWNSdeclineshasbeenmixed.Asingleanalysisthatgroupedalldataacross13sitesinVirginiasuggestedthatbodymassindex(BMI) infemalesofthreebatspecies,M. lucifugus, Perimyotis subflavus and M. sodalis, during early hibernation showed little temporal variation over a 5-year period followingWNS detection(Powers,Reynolds,Orndorff,Ford,&Hobson,2015).Incontrast,atasinglesite inKentucky,bodymassandBMIofM. lucifugus in fall swarmwere significantly higher in year followingWNS detectionthanthethreepreviousyears(Lackietal.,2015).

Here,weexaminewhetherM. lucifugus in colonies that are now persistingatleast8yearsafterWNSdetectionhavegreaterearlywinter body fat than bats from these same colonies nearly a decade earlierwhenWNSwasfirstdetected.Wehypothesizedthatbatsinpersisting colonies would have greater early winter fat stores than whenWNSemerged, andweused energeticmodels to examinewhether differences in fat could explain thepersistenceof littlebrown bats at these sites. We also tested the alternate hypotheses that bats in persisting sites were escaping infection, or resistant to infection, by measuring infection prevalence and intensity of Pd. Finally, we gathered data on temporal patterns of variation in body mass for M. lucifugus to put our results in a broader spatial and temporalcontext.

2  | MATERIAL S AND METHODS

2.1 | Sites

Westudiedbatsatsixsitesinthenorth-easternUnitedStatesduringinitialWNS invasion (2009) andagain roughly8years later (2016)incoloniespersistingwithWNS(SupportingInformationTableS1).SpecificdatesofWNSarrivalateachof thesesitesareunknown,but Pd is presumed to have invaded between 2008 and 2009, with themostseveredeclinesoccurringwhenmanysickbatswereob-servedleavingthesiteduringthefirst2–3yearsfollowingPd arrival (SupportingInformationFigureS1;Fricketal.,2010;Langwigetal.,2012).

2.2 | Data collection

We measured body fat in early winter (November to December)andagaininlatewinter(FebruarytoMarch)toassessfatlossover

     |  593Journal of Animal EcologyCHENG Et al.

hibernation (Supporting Information Table S1). We sampled anaverage of 22 ± SD =8(range:2009:4–39;2016:19–20)M. lucifugus persitepervisitwitharoughlyevensexratio(SupportingInformationTableS1).

In the winter of 2009, which encompasses the hibernation sea-sonfromOctober2008toApril2009(exceptforlatewintersam-plingatWilliamsconductedinFebruary2008),wecollectedbatsandmeasuredfatmassusingSoxhletanalyses.Wecollectedbatsduringearly (November through December) and late winter (FebruarythroughMarch)fromroostinglocationsinsidehibernacula,recordedbodymassandkilled themviaoverdoseof isoflurane followedbycervicaldislocation.Westoredspecimensat4°Cforupto2weeksand then transferred them to Boston University where we measured totalbodyfatmassbylipidextractionwithaSoxhletapparatususing3:1solutionof95%ethylalcoholandpetroleumether(Reynolds&Kunz,2001).Wecalculatedfatmass(g)asthedifferencebetweendrymass before lipid extraction and lean drymass after lipid ex-traction. We also calculated proportion body fat as the total body fat (g)dividedbybodymass(g)attimeofcollection.

Inthewinterof2016(October2015–April2016),wefirstsam-pled bats for Pd infection at their roosting location by swabbing batsusingapolyester-tippedswab(FisherScientific23-400-116)five times across the forearm and five times across the muzzle (Langwig etal., 2015). We stored swabs in RNAlater until pro-cessingtopreservegenomicDNA(Puechmaille,Fuller,&Teeling,2011).WemeasuredPd prevalence and fungal loads on bats from swabs using quantitative polymerase chain reaction (Muller et al., 2013)andquantifiedPdDNAfromCt scores as described previ-ously (Frick etal., 2017). After collecting swab samples andUVphotographs of wings in situ, we transported bats in cloth bags inside coolers (maintained at room temperature, 17–24°C) fromthe hibernacula to a sprinter van housing a quantitative magnetic resonance (QMR) scanner tomeasure body fat.We usedQMR,ratherthanSoxhletanalysesformeasuringfatmassinwinter2016sampling because it can be done on live bats and in a relatively short period of time, which minimizes disturbance to bats. QMR has been validated for a range of species, including little brown bats(Guglielmo,McGuire,Gerson,&Seewagen,2011),providingbodycompositionmeasurements(inourcase,bodyfat)thataredi-rectlycomparable(1:1)toSoxhletanalyses(McGuire&Guglielmo,2010). TheQMR scannerwasmaintained at 17–24°C inside thetemperature-controlledvan.Wecalibratedandvalidated theac-curacy of the QMR using 5.02 and 15 g canola oil standards be-fore and after sampling at each site. Prior to QMR measurement, we measured forearm length of each bat using digital callipers (±0.01mm) and weighed each individual using a digital scale(±0.01g).Weallowedeachbattofullyarouseandreacheuther-mic body temperature prior to QMR processing. Once aroused, eachbatwasgentlyinsertedintoandrestrainedbya3-cminter-naldiameterplastictubeoutfittedwithbreathingholes(EchoMRI-Bird-Mini-HLDR-ASM,productnumber600-000533B).ThetubewasthenplacedinsidetheQMRscanner.Eachscantook2–3min,and we scanned each bat twice. We averaged fat measurements

from repeated scans to produce a single estimate of total body fat for each bat (the mean difference between the two measurements was0.03g).Wecalculated theper centbody fat as theamountof fat measured by QMR divided by the body mass of each bat. We measured body fat using QMR at all sampling visits in 2016 except for latehibernationsamplingatHiberniaMinewherewemeasured forearm length and body mass. We estimated body fat for this visit based on the relationship between measured QMR fatandbodymass(SupportingInformationFigureS2).AfterQMRsampling, we placed bats in bags inside coolers, and transported thembacktotheirroostinglocationandreleasedthem.

2.3 | Statistical analyses

AllanalyseswereruninprogramRversion3.3.0(Brack&Twente,1985;RCoreTeam,2016;Twente,Twente,&Brack,1985).

2.3.1 | Pd prevalence and fungal load on bats in 2016

WeexaminedPdprevalenceusingageneralizedlinearmixed-effectsmodel(withabinomialdistributionandalogitlink)andPd load using alinearmixed-effectsmodelusingpackagelme4.Wefirstexaminedwhether Pd prevalence and load increased over hibernation time (de-finedasdayssincethefallequinox—September22or23dependingon theyear)asacontinuous fixedeffectandsiteasa randomef-fect.WeexaminedwhetherPd prevalence or load differed between sexesorwascorrelatedwithbodyfatofabatwithineachhiberna-tionsamplingtimeperiod (earlyor latewinter),and includedbodyfat,sexandhibernationtimeasfixedeffectsandsiteasarandomeffect.WeusedtheAkaikeinformationcriteriawithacorrectionforsmallsamplesize(functionAICcinpackageMuMIn;Bartoń,2016)tocompare models.

We also compared trends in Pd prevalence and Pd loads over winter hibernation at our six sites with continent-wide patterns(Frick etal., 2017) to determine whether differences in Pd trans-mission (lower Pd prevalence in early winter) or host resistance(decreased change in Pd loads over hibernation and lower Pd loads at theendof hibernation; Langwig etal., 2017)were contributingtopersistenceofbatsatoursixsites.WecomparedPd prevalence andloadsatoursixsiteswith>5,600Pdsamplestakenacross130sites acrossNorthAmerica (Frick etal., 2017).Weused the best-supported models describing continental trends in Pd prevalence and loads (generalized linear mixed-effects model with binomialdistributionandlinearmixed-effectsmodelusingpackagelme4 and lmerTest),whichincludedsiteasarandomeffectanddaysinhiber-nation, years since first Pddetectionatasiteandspeciesasfixedef-fects(Fricketal.,2017).InordertocomparetrendsinPd prevalence andloadsatoursitesagainstcontinentaltrends(Fricketal.,2017),we included an additional grouping variable with one level for each ofoursixsitesplusanadditionalgroupingvariableforcontinentaldata (designated as “continental”).Weused this grouping variableas a fixedeffect in thismodelwith “continental” as the reference

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level. For Pd prevalence analysis,we subtracted32days from thedate (the mean hibernation day in early winter when our samples weretaken) inordertocomparetheearlywinterPd prevalence at oursitesagainstthecontinent-widedatasetonthisdate.ForPd load analysis, we adjusted days in hibernation to the end of hibernation (day192)inordertocomparelatewinterPd loads at our sites with thecontinent-widedatasetonthisdate.

2.3.2 | Site- specific differences in body condition between sampling periods

We compared body fat between bat colonies sampled in winter 2009and2016ateachsitetoaccountforsite-specificdifferencesinwinterbodyfatinbats.Foreachofthesixsampledsites,weex-amined predictors of body fat with linear beta regression models (function betareg inpackagebetareg;Cribari-Neto&Zeileis,2010).We included the two sampling timeperiods (2009and2016), sexof the sampled bat, hibernation time (also defined as days since thefallequinox)andinteractionsbetweenthesetermsasfixedef-fects(SupportingInformationTableS8).WeusedAICc, as described above, to compare models. We used beta regression because the re-sponsevariable(fractionbodyfat)wasconstrainedbetween0and1andbecauseanalyseswithlog-orarcsine-square-root-transformeddata resulted in residuals that deviated significantly from normality usingaShapiro–Wilknormalitytest(functionshapiro.test).

2.4 | Predicted WNS survival from greater body fat

InordertoexaminehowincreasedbodyfatmightaffectsurvivalofbatswithWNS,weusedanenergeticmodel (Thomasetal.,1990)toestimateWNSmortalityprior to,duringWNS invasion in2009andafterWNSinvasionin2016(SupportingInformationAppendixS1).Wecalculatedoverwintermortalityforbatsstartingwithearlyhibernation body fat from our measurements in 2009 versus 2016, andexpendingenergyover thecourseofhibernationatpre-WNSand peak-WNS rates (Lilley etal., 2016; Supporting InformationAppendix S1). We compared overwinter mortality for three sce-narios:(a)batsstartingwithearlyhibernationbodyfatobservedin2009andexperiencingnoWNSimpacts,(b)batswithearlyhiberna-tionbodyfatobservedin2009andexperiencinghigherarousalfre-quenciesfromWNSand(c)batswithearlyhibernationfatobservedin2016andalsoexperiencinghigherarousalfrequenciesfromWNS.

3  | RESULTS

3.1 | Pd prevalence and fungal loads on bats in 2016

Inearlywinterof2016,prevalenceaveraged82.3%±7.8%,whichwasnot significantly lower than prevalence observed at 5,659 sampling eventsacrosscontinentalNorthAmerica(Fricketal.,2017)wherePd has become established (Figure1a; Table S3). Prevalence increasedto >90% at five of six sites by late winter (Figure1a), and changesin Pdprevalenceoverhibernationatour six sitesdidnotdiffer from

continentaltrends(Fricketal.,2017;SupportingInformationTableS3).Pd loads in early winter were similar to those from declining sites in the continentalstudy(Fricketal.,2017),butwerelowerthanexpectedattheendofhibernationintwo(AeolusandBartonHill)outofsixsites(Figure1bandSupportingInformationTableS4).Pd loads increased over hibernationatcomparableratestocontinentaltrendsexceptatBartonHill where Pd loads increased at a lower rate compared to continental trends (Figure1b and Supporting Information Table S4). AtWilliamsHotel, Pd loads decreased over hibernation (Figure 1b and Supporting InformationTableS4),butthesedatamaynotbecomparableasbatsinearly and late winter were sampled from different sections of the mine. Pdpresenceorloaddidnotvarysignificantlywithbodyfatorsexofthebat(SupportingInformationTableS5).

3.2 | Site- specific changes in body condition

Fat stores in early winter were significantly greater in bats sampled in 2016thanin2009atfouroutofsixsites(Figures2and3,Supporting

F IGURE  1 Pd prevalence and fungal loads in bats sampled in2016inearlyandlatewinterinsixpersistingMyotis lucifugus populations. Points show Pd prevalence ±1 SE based on a binomial distribution(a)andmeanlog10-transformedfungalloadsinattograms ±1 SE(b)atoursixsites(colours).Greypointsandgreydashedlinesrepresentsamplingfromapreviousstudyof130sitesacrossNorthAmerica(Fricketal.,2017)

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F IGURE  2 Mean per cent body fat (±1 SE)ofMyotis lucifugusoverhibernationtime(dayssincefallequinox)sampledin2009and2016atAeolusCave,HiberniaMine,GraphiteMine,WilliamsPreserveMine,BartonHillMine,WilliamsHotelMine.Ineachpanel,fittedlinesarebasedonbest-supportedbetaregressionmodels(SupportingInformationTableS6),andincludevariationbetweensexeswheresignificant

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Information Tables S6 and S8) and when examined collectivelyacrossallsites(SupportingInformationTableS8).Atsiteswherebatswere fatter, they were fatter by 0.8 ± 0.009 g (mean ± SE; Aeolus,Graphite, Hibernia, Williams Preserve) and had greater body massby 1.08±0.07g (mean±SE; Aeolus, Graphite, Hibernia), exceedingnatural inter-annualvariation inbodymass,whichwas0.19±0.05g(mean ± SE; BayCityMine,Wise1,Williams Preserve) (Figure4 andFigureS3).Atthreeofthesefoursites(Aeolus,Graphite,andWilliamsPreserve),therateoffatlossoverwinterdidnotdifferbetween2009and 2016 (Supporting Information Tables S6 and S7), whereas atHibernia Mine, bats lost fat more slowly over winter in 2016 than 2009 (Supporting InformationTablesS6andS7).At twosites (BartonHillandWilliamsHotel),fittedmodelssuggestedthatearlywinterbodyfatwaslowerin2016than2009(Figure2;SupportingInformationTablesS6andS7).Inaddition,atWilliamsHotel,thebest-fitmodelsuggestedthat 2016 bats started with less fat in early winter but lost fat more slowlythan2009bats(Figure2;SupportingInformationTablesS6andS7).Bodyfatwassignificantlyhigherinfemalebatsthanmalesatthreeofthesixsites(SupportingInformationFigureS4;TablesS6andS7).

3.3 | Predicted WNS survival from greater body fat

Across all sites, we estimated that averagewintermortality priortoWNSarrivalwas6%±3%,increasedto52%±12%duringinitialmortality fromWNS in2009anddecreased to36%±9% in2016(Figure5).Atsiteswherebatswerefatterin2016(Aeolus,Graphite,Hibernia,WilliamsPreserve),mortalitypriortoWNSarrivalwases-timatedat4%±3%,butroseto69%±16%duringWNSinvasionin2009.In2016,estimatedmortalitydecreasedto25%±4%forbatsexperiencingsustainedimpactsfromWNSbutwithgreaterbodyfat(Figure5). Thus, these energeticmodels suggest that higher bodyfatobservedinpersistingbatswouldreduceWNSmortalitybyanaverageof64%(Figure5).

4  | DISCUSSION

White-nose syndromehas causedwidespreaddeclines andextirpa-tionsinmanycoloniesofhibernatingbatsasithasspreadacrossNorth

F IGURE  4 Body mass and colony counts of Myotis lucifugusovertime.BodymassandWhite-nosesyndrome(WNS)mortality(toppanel):meanbodymasswithstandarderror(solidshapes,leftaxes)andcolonycount(log-transformed;bluesquares,rightaxes)ofM. lucifugus areplottedbywinteryear(e.g.,winteryear2009includesthehibernationseasonfromSeptember2008toApril2009).ThethinredbarindicatesthefirstdetectionofWNS,andthethickredbarindicatesthefirstyearofmassmortalityateachsite.Lettersabovesolidshapesdenote comparisons in body mass among years, with significant differences (α =0.05)indicatedbydifferentletters.Historicalcounts(priortowinteryear2009)aredenotedbyasterisks.Inter-annualvariationinbodymass(bottompanel):meanbodymasswithstandarderror(closedcircles,leftaxes)isshownfordifferentyearseitherbeforeorafterWNSdetection(thinredline)orWNSmortality(thickredline).Lettersabovesolidshapesdenotecomparisonsinbodymassamongyears,withsignificantdifferences(α =0.05)indicatedbydifferentletters

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America,butsomecoloniesofM. lucifugus are now persisting with the fungusinpartsofthenorth-easternUnitedStates(Fricketal.,2015,2017;Langwigetal.,2012,2017).Wefoundthat infectionwiththefungusinsixpersistingcolonieswasnotlowerthanexpected,butthatinfection intensity was lower than in declining populations at two per-sistingcoloniesattheendofwinter,andatfourofsixsitesbatsweresignificantly fatter in early hibernation in 2016 than bats at these sites nearly a decade earlier during initial invasion of the fungus. Energetic models suggested that the increased fat stores observed at these sites couldreduceWNSmortalityby58%–70%.

AlthoughincreasedbodyfatlikelyreducedWNSmortality,othermechanismsarenecessarytofullyexplainpersistenceofbatswithWNSatoursites.Pd prevalence in bats sampled during early hiber-nationwas10%–35% lower thansiteswherePd has been present foratleast5years(Fricketal.,2017).TheseresultssuggestthatPd transmission was lower in early winter at these sites, either due to density-dependentmechanisms(Anderson&May,1979),reductionof the environmental reservoir (and therefore environment-to-battransmission) over time, or increased resistance in bats (Langwigetal., 2017). Lower transmissionwould increase survival by (a) al-lowingsomebatstoescapeinfectionentirely(5%–15%ofbatswerePd-negativein latewinteratoursites)and(b)delayinginfectioninbats, some of which could survive until spring since death often oc-curs70–100daysafterinfectioninthelaboratory(Warneckeetal.,2012).WedidnotfindevidencethatPd transmission was lower at persisting sites than in declining populations. However, we found that late winter Pdloadswerelowerthanexpectedattwositesandincreased at a slower rate over hibernation at one of these sites com-pared to declining populations (Frick etal., 2017), suggesting thatsomebatsmaybe exhibiting resistance, as has beendocumented

in four colonies of M. lucifugus inNewYork (Langwigetal.,2017),including two that were studied here (Williams Preserve where fat storeswere higher, andWilliamsHotelwhere theywere not).Increased torpor bout duration could also contribute to continued persistence ofWNS-affected bats, and has been suggested as anexplanationforpersistenceatonesite(Lilleyetal.,2016).Althoughtemperatures were also cold at this site (and torpor bout duration decreases loglinearlywith roosting temperature (Brack& Twente,1985;Twenteetal.,1985)),thetorporboutdurationwas29%lon-ger than expected based on temperature (Supporting InformationFigureS6).However,batsat thissitehadmuch longerarousaldu-rations;thus,itisnotclearwhethertheirenergyexpenditurewouldbe lower than bats exhibiting disturbed hibernation behaviourcharacteristicofWNS(Reederetal.,2012;Warneckeetal.,2012).Finally, M. lucifugus in hibernacula with lower temperatures had less severedeclinesduetoWNS(Langwigetal.,2012),possiblyduetoreduced Pd growth (Verant etal., 2012) or lower energy expendi-turebybatsduringhibernation(Brack&Twente,1985;Twenteetal.,1985), suggesting that coolerhibernacula temperaturescouldalsofacilitate bats persisting with Pd.Additional research isneeded todetermine the relative contribution of these different mechanisms to persistence of M. lucifuguswithWNS.

Our results are consistent with previous studies showing increased body condition in early winter in M. lucifugusfollowingWNSdeclinesat themajorityof sites, but not all.We re-examineddifferences inbody mass from a study in Virginia of M. lucifugus (Powers et al., 2015), which had data from three sites where bats were sampled in earlywinterbothbeforeandafterWNSdetectionandhad>4batspersample (Supporting InformationAppendixS2).Wefound thatbodymasswas15%–23%higher intwo(Highland2,Bath2)outofthree

F IGURE  5 Estimated winter mortality from energetic models based on fat and hibernation energetics for bat populations prior to Pdinvasion(pinkbars),duringPdinvasionin2009(greenbars),andfollowing Pdinvasionin2016(bluebars).Arrowsandtextindicatewheregreaterearly winter body fat in 2016 resulted in reducedWhite-nosesyndrome(WNS)-relatedmortalitycomparedtopeak-WNSmortality in 2009

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colonies following declines of 88%–99% (Figure4 and SupportingInformationFigureS3),and7%higher inanother (Bland1;Figure4andSupportingInformationFigureS3).AseparatestudyinKentucky(ColossalCave)foundthatbodymassofM. lucifugus measured during fallswarmwashigherby15%–20%1yearfollowingWNSdetectionandan80%decline(Figure4andFigureS3;Lackietal.,2015).Thesedata suggest that body fat increases following mass mortality due to WNSinmany,butnotall,coloniesofM. lucifugus, and are contributing topersistenceofbat colonieswithWNS.Whether thevariation inincreased fat stores is due to differences in site quality near hibernac-ula(e.g.,higherinsectabundance),orthestrengthofselectionduetoWNSmortality,orotherfactorsremainstobedetermined.

Several mechanisms could result in bats having higher fat compo-sitioninearlywinterfollowingWNSdeclines.First,inter-annualvari-ation in prey availability in fall, due to natural variability or decreased resourcecompetition,couldleadtohigherfatcompositionifbatsex-hibit phenotypic plasticity in body fat and forage to higher fat stores when prey abundance is higher. However, temporal data from col-onycountsinNewYork,Virginia,WisconsinandKentuckysuggestthat inter-annualvariation inearlywinterbodymass is lower thanwe observed (Figure4 and Supporting Information Figure S3) andtheweather(precipitationandtemperature)wassimilarin2009and2016(SupportingInformationFigureS7).Second,iflevelsoffatstor-age in fall is a heritable trait, our results suggest that natural selection wouldincreasefatstoresthroughincreasedsurvivalwithWNS,andpossibly increased reproduction due to higher fat stores at the end of winter.Althoughbothphenotypicplasticityandevolutionarychangecouldincreasefatstores,ifhigherfatstorespost-WNSdeclinesareduetoreducedcompetition,thenbatpopulationsare less likelytoreachtheirpreviouspre-WNSabundance.Regardless,managementactions that allow bats to obtain higher fat stores in fall before begin-ning hibernation will aid in conservation and recovery.

Thusfar,mostconservationeffortsforWNShavebeencentredon treating bats or reducing Pd in the environment during winter hibernation(Chengetal.,2016;Cornelisonetal.,2014;Hoytetal.,2015; Zhang, Chaturvedi, & Chaturvedi, 2015) when WNS mostheavilyimpactsbats(Langwigetal.,2015).Ourstudysuggeststhatmanagement efforts that increase bats’ ability to increase fat stores in autumn may help facilitate population persistence. M. lucifugus are dietary generalists, primarily consuming emerging aquatic insects (Anthony&Kunz,1977;Belwood&Fenton,1976;Clareetal.,2013).Management actions that protect and restore aquatic habitats, es-pecially near hibernacula, may facilitate greater fat accumulation duringthefallswarmperiod(Parkeretal.,2018).Forexample,statescould provide incentives for landowners not to remove beavers and beaver dams which create wetlands, and they could incentivize or implement wetland or riparian restoration by reducing river channel-izing, and by reducing invasive plants and restoring native vegetation which has recently been shown to be critical for some insectivorous birdpopulations(Narango,Tallamy,&Marra,2018).Managinghabi-tats to increase the ability of bats to increase fat stores before winter has several advantages as a management strategy. First, there is no associatedriskwithcausingadditionalmortalityduetodisturbance

during hibernation. Second, it can be done in combination with other conservation efforts occurring during winter. Improving body con-dition may facilitate both increased survival and reproduction, and thiscanfacilitatetheevolutionofresistanceortolerance(Kilpatrick,2006;Masloetal.,2015).Employingtheseconservationstrategiesmayprovide critical relief and facilitate recovery asWNS impactscontinuetothreatenNorthAmericanbatpopulations.

ACKNOWLEDG EMENTS

WegivespecialthankstoMariamarGutierrezRamirezandMichaelGriego for their monumental assistance in the field, without which thisworkwouldnothavebeenpossible.Wealso thank thehard-working biologists of the New York Department of Conservation(CarlHerzog,KathleenO'Connor,KatelynRitzko,MelanieBerger),AlyssaBennettoftheVermontFishandWildlifeDepartment,andMackenzie Hall of the New Jersey Division of Fish andWildlife.Wealso thankKatherineMcClure,TonyKovachandJoeHoyt forhelpfulcommentsonthepaper.WealsoacknowledgetheVirginiaDepartment of Game and Inland Fisheries for contribution of BMI datafromVirginia.Finally,wethankThomasKunzandtheBostonUniversityBatLab for supporting theworkconductedbyauthorsJ.ReichardandM.Mooreinwinter2009.ThisworkwasfundedbygrantWNS2015-08fromtheUSFishandWildlifeService,andNSFgrantsDEB-1115895andDEB-1336290.

AUTHORS’ CONTRIBUTIONS

T.L.C. conceived of the study idea following a presentation byG.Turner at a white-nose symposium; T.L.C., A.M.K. andW.F.F. de-signed and coordinated the study, conducted data analyses and drafted themanuscript; T.L.C., J.D.S. andA.G. collected the data;A.G.andC.K.R.W.advisedthestudydesignandparticipatedindataanalysis;J.D.R.andM.S.M.helpedwithstudydesignanddatacol-lection.Allauthorscontributedtothefinalversionofthemanuscriptand gave final approval for publication.

DATA ACCE SSIBILIT Y

Data used in this study are from the Dryad Digital Repository: https://doi.org/10.5061/dryad.sh487nh(Chengetal.,2019).

ORCID

Tina L. Cheng https://orcid.org/0000-0002-7930-8635

Alexander Gerson https://orcid.org/0000-0002-9082-1907

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SUPPORTING INFORMATION

Additional supporting information may be found online in theSupporting Information section at the end of the article.

How to cite this article:ChengTL,GersonA,MooreMS,etal.Higher fat stores contribute to persistence of little brown bat populationswithwhite-nosesyndrome.J Anim Ecol. 2019;88:591–600. https://doi.org/10.1111/1365-2656.12954