White-nose syndrome initiates a cascade of physiologic disturbances in the hibernating bat host


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White-nose syndrome initiates a cascade of physiologic disturbances in the hibernating bat host

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Title:
White-nose syndrome initiates a cascade of physiologic disturbances in the hibernating bat host
Series Title:
BMC Physiology
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Verant, Michelle L
Meteyer, Carol U
Speakman, John R
Cryan, Paul M
Lorch, Jeffery M.
Blehert, David S
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English

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White-Nose Syndrome ( local )
Bats ( local )
Doubly Labeled Water ( local )
Wns ( local )
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serial ( sobekcm )

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Abstract:
The physiological effects of white-nose syndrome (WNS) in hibernating bats and ultimate causes of mortality from infection with Pseudogymnoascus (formerly Geomyces) destructans are not fully understood. Increased frequency of arousal from torpor described among hibernating bats with late-stage WNS is thought to accelerate depletion of fat reserves, but the physiological mechanisms that lead to these alterations in hibernation behavior have not been elucidated. We used the doubly labeled water (DLW) method and clinical chemistry to evaluate energy use, body composition changes, and blood chemistry perturbations in hibernating little brown bats (Myotis lucifugus) experimentally infected with P. destructans to better understand the physiological processes that underlie mortality from WNS.
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BMC Physiology, Vol. 14, no. 10 (2014-12-09).

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K26-00046 ( USFLDC: LOCAL DOI )
k26.46 ( USFLDC: LOCAL Handle )

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White-nosesyndromeinitiatesacascadeof physiologicdisturbancesinthehibernating bathost Verant etal. Verant etal.BMCPhysiology 2014, 14 :10 http://www.biomedcentral.com/1472-6793/14/10

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RESEARCHARTICLEOpenAccess White-nosesyndromeinitiatesacascadeof physiologicdisturbancesinthehibernating bathost MichelleLVerant 1 ,CarolUMeteyer 2,5 ,JohnRSpeakman 3 ,PaulMCryan 4 ,JeffreyMLorch 1 andDavidSBlehert 2* Abstract Background: Thephysiologicaleffectsofwhite-nosesyndrome(WNS)inhibernatingbatsandultimatecausesof mortalityfrominfectionwith Pseudogymnoascus (formerly Geomyces ) destructans arenotfullyunderstood.Increased frequencyofarousalfromtorpordescribedamonghibernatingbatswithlate-stageWNSisthoughttoaccelerate depletionoffatreserves,butthephysiologicalmechanismsthatleadtothesealterationsinhibernationbehavior havenotbeenelucidated.Weusedthedoublylabeledwater(DLW)methodandclinicalchemistrytoevaluate energyuse,bodycompositionchanges,andbloodchemistryperturbationsinhibernatinglittlebrownbats ( Myotislucifugus )experimentallyinfectedwith P.destructans tobetterunderstandthephysiologicalprocessesthat underliemortalityfromWNS. Results: Thesedataindicatedthatfatenergyutilization,asdemonstratedbychangesinbodycomposition,was two-foldhigherforbatswithWNScomparedtonegativecontrols.Thesedifferenceswereapparentinearlystages ofinfectionwhentorpor-arousalpatternswereequivalentbetweeninfectedandnon-infectedanimals,suggesting that P.destructans hascomplexphysiologicalimpactsonitshostpriortoonsetofclinicalsignsindicativeof late-stageinfections.Additionally,ba tswithmildtomoderateskinlesionsassoci atedwithearly-stageWNSdemonstrated achronicrespiratoryacidosischaracterizedbysignifican tlyelevateddissolvedcarbondioxide,acidemia,andelevated bicarbonate.Potassiumconcentrationswerealsosignificantlyhigheramonginfectedbats,butsodium,chloride,andother hydrationparameterswereequivalenttocontrols. Conclusions: Integratingthesenovelfindingsonthephysiologicalchangesthatoccurinearly-stageWNSwiththose previouslydocumentedinlate-stageinfections,weproposeam ulti-stagediseaseprogressi onmodelthatmechanistically describesthepathologicandphysiologiceffectsunderlyin gmortalityofWNSinhibernatingbats.Thismodelidentifies testablehypothesesforbetterunderstandingthisdisease,knowledgethatwillbecriticalfordefiningeffectivedisease mitigationstrategiesaimedatreducingmor bidityandmortalitythatresultsfromWNS. Keywords: White-nosesyndrome,Bats,Doublylabeledwater Background Sinceemergenceofwhite-nosesyndrome(WNS)in 2007,batpopulationsofeasternNorthAmericahave declinedprecipitouslyduetodisease-relatedmortality [1-3].ThecausativeagentofWNSisthefungus Pseudogymnoascus (formerly Geomyces ) destructans [4-6], whicherodesunfurredskincomprisingwingmembranes,muzzles,andearsofhibernatingbats,inducing physiologicalperturbations,alteredbehavior,anddeath [7].Althoughunderlyingcausesformortalityfromthis invasivecutaneousmycosisremainunclear,proposed mechanismsincludedisruptionstovitalhomeostatic functionssuchasthermoregulationandwaterbalance [8].Forexample,waterandelectrolytelossesacrossthe ulceratedwingepitheliumhavebeenproposedtocause hypotonicdehydration[9]andacid ? basedisturbances [10].Consequently,alterationsinbehaviorhavebeen observedininfectedbats,includingincreasedfrequency ofarousalfromtorporduringhibernation[11,12]and unusualdayflightsduringwinter[3].Highmetabolic *Correspondence: dblehert@usgs.gov 2 USGeologicalSurvey ? NationalWildlifeHealthCenter,6006SchroederRd., Madison,Wisconsin,USA Fulllistofauthorinformationisavailableattheendofthearticle ?2014Verantetal.;licenseeBioMedCentral.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle, unlessotherwisestated. Verant etal.BMCPhysiology 2014, 14 :10 http://www.biomedcentral.com/1472-6793/14/10

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demandsofsuchactivities[13-15]likelyalsocontributeto mortalityofbatspriortospringemergencebyaccelerating depletionoffatreserves.However,physiologicaldatalinkingalteredbehaviortoincreasedenergydemandsinbats withWNSarelacking. Thedoublylabeledwater(DLW)methodiswidely applicabletothestudyofenergeticsinrelationtohomeostasis,behavioraladaptations,andresourceallocationin bothanimalsandhumans[16].Thismethodisbasedon dynamicfluxofhydrogenandoxygenthroughthebody andabilitytomeasurethesefluxratesoveraperiodof timeusinglabeledisotopes,2Hand18O[17].Following administrationoftheseexogenousisotopes,theyequilibratethroughoutthebodywaterpool.Thetotalbody watervolume(TBW)canthenbeestimatedfromthedilutionspacesoftheisotopeswhenintroducedatknown concentrationsandservesasavaluableindicatorofbody composition(ratioofleanbodymasstofat)[18].Notably theDLWmethodhasbeenusedintemperate-zoneinsectivorousbatsinthewild( e.g. , Myotislucifugus [19]and Eptesicusfuscus [20]),buttherearenopublishedreports ofthismethodbeingusedinbatshibernatingovera protractedtimeperiod( i.e. ,months). ToevaluateproposedcausesofmortalityfromWNS, weusedtheDLWmethodtoquantifyenergyexpenditure andchangesinbodycompositionofhibernatinglittle brownbats( M.lucifugus )experimentallyinfectedwith P. destructans totestthehypothesisthatWNSincreases metabolicdemandsduringhibernation.Wepredictedthat infectedbatswouldexhibitgreaterchangesinbodycomposition,specificallydecreasedproportionoffatmass, overthecourseoftheexperimentcomparedtonegative controlbatsasaresultofhigherdailyenergyexpenditures andfatutilization.Tofurthercharacterizepreviously reportedphysiologicoutcomesassociatedwithWNS,we analyzedbloodchemistriesofallbatsattheendofthe experimenttoassessacid ? basebalance,electrolytes,and hydrationstatus.ResultsInfectionstatusandtorporpatternsOfthe39batstreatedwithconidiafrom P.destructans , 32bats(14male,18female)developedepidermalwing lesionscharacteristicofWNSbytheendofthe98-d experiment.Themajorityofthesebats(n=30)hadmild tomoderateWNS(severityscores1or2withmedian scoreof1),whiletheremainingtwohadmoderateto severeWNS(severityscores3and4).Sexhadnoeffect ontheprobabilitythatabatdevelopedWNS(Fisher ? s ExactTest,p=0.4075).Allinfectedbats,including animalsthatdidnotdevelopdetectableWNSbyhistology (n=7),werePCR-positivefor P.destructans ;allbatsin thecontrol(non-infected)groupwerePCR-negativefor thefungalpathogen.Fourinfectedbatsandfivecontrol batsdiedpriortotheendoftheexperiment. Averagetorporboutdurationforinfectedbatsfollowing DLWinjectionwas9.13(2.31)dwithaveragearousaldurationof54(10)min.Averagetorporboutdurationfor controlbatsfollowingDLWinjectionwas8.52(2.34)d withaveragearousaldurationof55(11)min.Differencesintorpor-arousalpatternsofbatsbetweentreatmentgroupswerenotsignificant(torporboutduration, p=0.5337;arousaldurationp=0.6508).BloodchemistryBloodchemistryparameterswereanalyzedfor27infectedbats(10male,17female)and11controlbats (6male,5female)fromwhichsufficientbloodsample volumeswerecollected.Oneinfectedbatdidnothave skinlesionscharacteristi cofWNSonhistopathology, buttherewerenodiscernibledifferencesacrossparameterswhencomparedtoinfectedbatswithconfirmedWNS.Additionally,wewereunabletocollect sufficientbloodvolumeforanalysisfromthetwobats withmostadvancedpathology(WNSseverityscores3 and4).Thus,bloodchemistryvaluespresentedhereinfor infectedbatsrepresentbatswithmildWNSpathology (medianseverityscoreof1). InfectedbatshadsignificantlylowerbloodpHthan controls(Table1,Figure1a).ThisacidemiawasassociatedwithasignificantelevationofpCO2ininfectedbats comparedtocontrols(Table1,Figure1a),indicating thatbatshadrespiratoryacidosis.Bicarbonatelevelsof infectedbatswerealsosignificantlyhigherthanthoseof non-infectedcontrols(Table1,Figure1a),evidentofa compensatoryrenalresponsetoachronicacidosis.The accumulationofbicarbonateinbloodofinfectedbats wasalsoreflectedinanelevatedbaseexcesscompared tocontrols(Table1).Therewerenodifferencesinsodiumorchlorideconcentrationsbetweentreatment groups,butpotassiumconcentrationwassignificantly higherintheinfectedbatsthanitwasinthecontrols (Table1,Figure1b).Glucoseconcentrationswerelower ininfectedbatsbutnotsignificantly(Table1),andanion gapvalueswerenotdifferentfromcontrolsnorelevated aswouldbeexpectedifacidemiaresultedfrommetaboliclacticorketo-acidosis.Othermeasuresofhydrationstatus(hematocrit,bloodureanitrogen,andtotal protein)wereequivalentbetweentreatmentgroups (Table1,Figure1c).Overall,therewerenoeffectsofsex (two-wayMANOVA,Pillais ? Trace=0.48,F(9,14)=1.46, p=0.25)ortheinteractionofsexandtreatment(two-way MANOVA,Pillais ? Trace=0.53,F(9,14)=1.79,p=0.16) onmeasuredbloodparameters.SeeAdditionalfile1fora completetableofbloodchemistryparameterestimations comparedtoavailablereferencevalues.Verant etal.BMCPhysiology 2014, 14 :10 Page2of10 http://www.biomedcentral.com/1472-6793/14/10

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Measurementsofdailyenergyexpenditureandtotal bodywaterUponcollectionoffinalbloodsamples,67dafterinjection withDLW,labeledisotopeconcentrationshaddecreased tolevelsstatisticallyindistinguishablefrombackground levels.Consequently,isotopeturnoverratesanddaily energyexpenditure(DEE)couldnotbedeterminedfor batseuthanizedattheendoftheexperiment.However, onebatinthecontrolgroupthatdied35dafterinitial injectionofDLWhaddetectableisotopeconcentrations attimeofdeath.Forthisbat,kdandkowere0.004,calculatedrespiratoryCO2production( r CO2)was0.01ml/min, andresultantDEEwas0.44kJ/day. Measurementsoftotalbodywater(TBW)wereavailablefor26bats(19infected,7controls)followinginitialinjectionofDLWandfor24bats(17infected,7 controls)afterfinalinjection(Table2).Thereduction insamplesizeisattributedtomortalityofbatsduringthe experimentorinabilitytocollectasufficientamountof bloodforisotopeanalyses.AtthetimeofinitialDLW injection,meanTBWaspercentofbodymass(TBW% BM)wassignificantlylowerforinfectedbatsthan controls(Tables2and3),butmeanbodymasswasnot differentbetweengroups(t= 0.5925,df=30,p=0.558). After67dhibernation,changesinTBWandbodymass werecomparedforinfectedandcontrolbatsforwhich pairedmeasurementswereavailable.Bodymassdecreased significantlyinbothtreatmentgroups(infected:t=11.32, df=13,p<0.0001;control:t=14.24,df=3,p=0.0008), butthelossinbodymasswasequalbetweengroups. ChangesinTBW%BMweremarginallysignificant betweeninfectedandcontrolbats,butnotwhencorrected formultiplecomparisons(Tables2and3).Withingroups, infectedbats,whichalldevelopedmildWNS(n=14; medianWNSseverityscore1,range1 ? 2)exhibiteda significantincreaseinTBW%BM(Tables2and3);increaseinTBW%BMamongcontrolbats(n=4)was notsignificant(Tables2and3). AstrendsinTBW,namelyTBW%BM,reflectchanges inbodycomposition,netfatenergyutilizationwascalculatedfromthechangeinTBW(ing)andbodymassmeasurementsforallbatswithpaireddataassumingconstant 73%watercontentofleanmass(seeAdditionalfile2). Baseduponthesecalculations,meantotalfatenergy utilizationforthe67dafterwhichbatswereadministered DLWwassignificantlyhigherforbatswithWNScomparedtonegativecontrols(Tables2and3).DiscussionResultsofthisstudysupportthehypothesisthatinfection with P.destructans andsubsequentdevelopmentofWNS increasesenergy(fat)useinhibernatingbatsandprovide keyinformationforunderstandingtheprogressionof physiologicdisturbancesthatultimatelyleadtomortality fromthisdisease.Specifically,isotope-basedestimatesof changesinbodycompositionprovidedevidencethat hibernatinglittlebrownbatswithWNSutilizedtwiceas muchenergyasnon-infectedcontrolbatshousedunder equivalentexperimentalconditions.However,thegreater energyusebyinfectedbatswasnotassociatedwithan increasedrateordurationofarousalsfromtorpor.This impliesthatbats,evenwithmildWNSlesions,havean elevatedmetabolismpriortotheonsetofaltered arousalpatternscharacteris ticoflate-stageinfections [12].Additionally,batswithearly-stageWNSdeveloped severe,chronicrespiratoryacidosisandhyperkalemia Table1BloodchemistrycomparisonsParameter k dfUnadjustedp-value =0.05Holm-Bonferronicorrectedp-value( / k ) pCO21238<0.0001*.0042 K1135<0.0001*.0045 pH1038<0.0001*.0050 HCO3 936<0.0001*.0056 BE837<0.0001*.0063 Glucose726.0144.0071 AG626.0483.0083 Cl522.0646.01 BUN436.2093.0125 TP336.6085.0167 Na+236.7079.025 Hct123.8396.05Abbreviations : pCO2dissolvedcarbondioxide, K potassium, HCO3 bicarbonate, BE baseexcess, AG aniongap, Clchloride, BUN bloodureanitrogen, TP total protein, Na+sodium, Hct hematocrit. Resultsof t -testsusedtocomparemeanbloodchemistryparametersforlittlebrownbats( Myotislucifugus )eitherexperimentallyinfectedwith Pseudogymnoascus destructans ornegative(non-infected)controls.Significantdifferencesbetweentreatmentgroups(*)weredeterminedbyp-values
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(highpotassiumconcentrationsintheblood).Integrating theseresultswiththosereportedbyothers[7,9-12,21],we proposeamechanisticmulti-stagediseaseprogression modelforWNSthatencompassesourcurrentknowledge ofdiseasepathologyandphysiologicsequelae,including death,thatresultfollowinginfectionby P.destructans (Figure2). Asshowninthisstudy,earlystagesofWNSinvolving fungalcolonizationofthewingmembranewithprogressiontoerosionandulcerationoftheepidermis,are characterizedbyincreasedCO 2 levelsinblood,resultantacidemia,andhyperkalemia.Theaccumulationof CO 2 maystemfromeitherincreasedCO 2 production fromanelevatedmetabolicrateassociatedwithinfection,decreasedCO 2 expiration,possiblyduetoinhibitionofdiffusionacrossthedamagedwingepithelium [7],and/oracompensatoryresponsebythehostto attempttofurtherlowertorpormetabolicrates(andconservefatreserves)byinducingahypercapnic(highpCO 2 ) acidosis[15,22-24].Thisacidosismaythencontributeto theobservedhyperkalemiabyanacidosis-inducedextracellularshiftofpotassium.Intracellularpotassiumions mayalsoleakintothebloodthroughdamagedandnecroticcellmembranescausedbyhyphalinvasionofthe Figure1 Bloodchemistryparameters. Box-and-whiskerplotsof bloodchemistryvaluesforhibernatinglittlebrownbats( Myotis lucifugus )experimentallyinfectedwith Pseudogymnoascusdestructans andnegative(non-infected)controls.Parametersforacid-basebalance (a) ,electrolytes (b) andhydrationstatus (c) areshown.Themedian (boldline),upperandlowerquartiles(box),andmaximumand minimumvalues(whiskers)areshown.Potentialoutliers(points)shown werenotconfirmedbyBonferonnioutliertests.Significantdifferences (*)weredeterminedat =0.05correctedformultiplecomparisonsby theHolm-Bonferonnimethod. Table2Bodycompositionmeasurements Parameter Pd infectedbatsControlbats Alldatan=22n=10 InitialTBW%BM53.4(4.3)58.8(2.4) FinalTBW%BM60.5(3.9)61.0(4.6) InitialBM(g)7.64(0.75)7.48(0.58) FinalBM(g)6.60(0.68)6.41(0.57) Paireddatan=14n=4 ChangeinTBW%BM+9.1(3.5)+4.0(3.9) Fatenergyuse(kJ)43.9(13.6)20.5(14.7) Valuesofbodymass(BM)andtotalbodywateraspercentageofbodymass (TBW%BM)wereusedtoestimatenetfatenergyuseover67dforindividual littlebrownbats( Myotislucifugus )experimentallyinfectedwith Pseudogymnoascusdestructans andnegativecontrol(non-infected)bats.Values inthetablearemean(SD)andn=samplesize.InitialTBW%BMwas significantlydifferentbetweentreatmentgroups.Overthecourseofthe experiment,infectedbatsdemonstratedasignificantincreaseinTBW%BM andusedsignificantlymorefatenergythannon-infectedbats.Bodymass decreasedsignificantlyovertheexperimentininfectedandcontrolbatsbut therewasnodifferencebetweengroups. Verant etal.BMCPhysiology 2014, 14 :10 Page4of10 http://www.biomedcentral.com/1472-6793/14/10

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epidermis.Overall,thesephysiologiceffectsresultina chronicrespiratoryacidosis,hyperkalemia,andreduction offatreservesamongbatsduringearlystagesofWNS. Consistentwithresultsobservedinbatsatlaterand moreseverestagesofWNS[9,10],weproposethatonce pCO 2 elevatesbeyondatolerancethreshold,chemoreceptorsstimulatehyperventilation,andresultingincreased arousalsfromtorporservetoremoveexcessCO 2 , returningbloodpHtonormal[22,25-28].Thehighenergy demandofthesearousalsthenlikelyfurthercontributes toaccelerateddepletionoffatreserves.Additionally,increasedventilatoryratesandgreatervaporpressuredifferencewithincreasedbodytemperaturesduringarousals wouldcontributetogreaterevaporativewaterloss[29] anddehydration.Together,theseoutcomesareconsistent withdatapreviouslypublishedforlittlebrownbatswith Table3Doublylabeledwatercomparisons Measurement k dfUnadjustedp-value =0.05Holm-Bonferronicorrectedp-value( / k ) Fatenergyuse(I)813<0.0001*0.0063 ChangeinTBW%BM(I)713<0.0001*0.0071 InitialTBW%BM(IvsC)6250.0041*0.0083 Fatenergyuse(IvsC)5160.0088*0.01 ChangeinTBW%BM(IvsC)4160.02430.0125 Fatenergyuse(C)330.06870.0167 ChangeinTBW%BM(C)230.13530.025 FinalTBW%BM(IvsC)1220.78410.05 Resultsof t -testsusedtocomparedoublylabeledwatermeasurementsforlittlebrownbats( Myotislucifugus )eitherexperimentallyinfectedwith Pseudogymnoascusdestructans (I)ornon-infectedcontrols(C).Totalbodywaterisrepresentedaspercentageofbodymass(TBW%BM).Significantdifferences betweentreatmentgroups(*)weredeterminedbyp-values
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moresevereWNSpathologythatexhibitedincreasedfrequencyofarousalfromtorpor,decreasedpCO2,normal bloodpH,anddehydration[10]. AsWNSprogressestowardsmoreextensiveandsevere winglesions,dehydrationmaybefurtherexacerbatedby waterandelectrolytelossacrossthedamagedepidermis ofthewing[9,30],furtherstimulatingarousalfromhibernationtodrink[29,31,32].Positivefeedbackloopsare thenestablishedthatlinkworseningdisease-associated wingpathologytofurtherincreasesinarousalfrequency,waterloss,andenergyuseresultinginadditionalobservedacutephysiologicchanges,including hypocapnia,hypoglycemia,hyponatremia,hypochloremia, andemaciation[9-11].Oncecompensatorymechanisms suchascellularbuffering,respiratoryandmetabolicregulation,and/orbehavioraladaptationsareexhausted,this suiteofdisturbancesultimatelyleadstomortalityunless thebathassufficientenergyreservestopersistuntilspring emergenceandcleartheinfectionfollowingareturntoa metabolicallyactivestate[33]. Althoughnormalreferencerangesforbloodchemistry valuesinmicrochiropteranspeciesaregenerallylacking [34],deviationsofmeasuredparametersininfectedbats fromthenegativecontrolsinthisstudy,togetherwith publishedinformationforapparentlyhealthyhibernating littlebrownbats[10,34-39],suggestpathologicandpotentiallylife-threateningphysiologicaldisturbancesassociated withearly-stageWNSinfections[seeAdditionalfile1]. BatswithWNSinthisstudyhadalmost40%highermean pCO2thannegativecontrolbats(99.4and37.1mmHg respectively),andpCO2valuesabove90mmHgaregenerallyconsideredtobelethalinothernon-hibernatinganimalsandhumans.ElevatedpCO2levelsandassociated acidemiaareknowntointerferewithenzymaticfunctions, reducemetabolicactivity,andcausecentraldepressionof respiration;inseverecases,suchelevatedlevelscanleadto comaanddeath. Directcalculationsofenergyexpenditurecouldnotbe determinedformostbatsinthisstudybecausefinalisotopeconcentrationswereindistinguishablefrombackgroundlevels.However,theincreaseinTBWasapercent ofbodymassobservedininfectedbatsindicatedthatbats withWNShadhigherproportionsofleantissuemassto fattissuemassattheendofthestudy.Thisfindingimplies thatbatswithWNSusedsignificantlymorefatenergyreservescomparedtonegativecontrolsdespitehibernating underequivalentconditions.Fromtheestimatedchanges infatcontentoverthe67-dmeasurementperiod,infectedbatsutilized0.65kJ/d,whilecontrolbatsutilized 0.31kJ/d.TheseresultsindicatethatbatswithWNS expendedapproximatelyt wiceasmuchenergyduring hibernationasnon-infectedcontrolbats.Somecaution mustbetakenininterpretingtheseresultsduetothe smallnumberofcontrolbatsforwhichpairedTBW measurementswereavailabletocalculateestimatesoffat use.However,therangeofexpecteddailyenergyuseof 0.27to0.51kJ/dpredictedforhealthyhibernating M.lucifugus atthemeantemperatureofourstudy[40]isconsistentwiththedailyratesofenergyuseobservedincontrol batsinthisexperiment,andlowerthanratesobservedin infectedbats.Therewerenodifferencesintorpor-arousal patternsbetweentreatmentgroupsinthisstudysuggesting thatWNScausesanincreaseinmetabolismthatisnot directlyassociatedwitharousalfromtorporandoccursat earlystagesindiseaseprogression.Additionally,therewere nodifferencesbetweeninfectedandcontrolbatsinTskinmaintainedduringtorpororarousalbouts.Alternatively, metaboliccostsassociatedwithinfectionanddevelopment ofwingpathologymaybelinkedtoincreasedcostsof thermoregulationcausedbyinhibitionofperipheralvasoconstrictionduringtorporandarousal[25],catabolismof fattogeneratemetabolicwaterinresponsetoincreased waterloss,oradditionalenergeticcostsassociatedwiththe host-pathogeninteraction. RisingconcentrationsofpCO2inamammalwouldnormallystimulateincreasedrespirationtoreleaseexcess CO2.However,theuniquephysiologyofmammalianhibernatorsallowsforactivesuppressionofrespirationduring torpor,andundertheseconditions,bloodpCO2increases tolevelshigherthanthoseobservedinmetabolicallyactive mammals[22,28,41,42].ThiselevationofpCO2isthought tobeanintegralpartofhibernationphysiologyasinductionofanacidoticstateservestoreducemetabolicrate andthermogenesis[25,43,44].Additionally,theresultant highpCO2gradientimprovesventilationefficiency,thereby minimizingenergycostsofrespirationduringtorpor[23]. Despitethistoleranceforarespiratoryacidosis[45],a hibernatingmammalmuststi llbeabletoregulatepCO2forproperphysiologicfunction.IfCO2eliminationroutes, suchaspassivediffusionofCO2acrossthewingepithelium[46,47]arecompromisedbydisease,ashypothesized forbatswithWNS[7],persistentlyrisingbloodpCO2levelswouldcausetheseverechronicrespiratoryacidosis wehaveobserved.Thus,underlyingcausesforthehigh pCO2levelsinbatswithWNSarelikelyacombinationof theuniquelyadaptedphysiologyofahibernatingmammal compoundedbypathologicallyinducedinsult(s)tothese physiologicalmechanisms. BatWNSpresentsanewparadigmforthestudyof infectiousdisease.Neverbeforehasafungalskinpathogen beenknowntospecificallyinfectahibernatingmammal, causingseverephysiologicdisturbancesandmortality. Althoughsubstantialeffortshavebeendevotedtounderstandingchytridiomycosis,alethalfungalskindisease ofamphibians,priortoWNStherehadbeennoindepthstudyofdiseaseproces sesinametabolicallyrepressedanimalcausedbyapsych rophilicandmetabolically activepathogen.ThepoorlycharacterizedcapacityofbatsVerant etal.BMCPhysiology 2014, 14 :10 Page6of10 http://www.biomedcentral.com/1472-6793/14/10

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tocompensateforandrespondtoinfectionduringhibernationdemonstratesthedifficultyofunderstandinghow host-pathogeninteractionsinfluencediseasemanifestation andmortality.ConclusionsThisstudydemonstratedthatinfectionwith P.destructans andsubsequentdevelopmentofWNSincreasedenergy (fat)useinhibernatingbatspriortotheonsetofalteredarousalpatternsassociatedwithlaterstagesof WNS.Severe,chronicrespiratoryacidosisandhyperkalemiawerealsoapparentinbatsthatdevelopedmildWNS. Withtheseresults,wepresentamulti-stagediseaseprogressionmodelforWNSasaframeworkforunderstandingthepathogenesisandunderlyingcausesofmortality duetoWNS(Figure2).Thismodelintegratesarangeof publishedwork[7,9-12,21]withdatafromthisstudyinto thefirstattempttomechanisticallydefineacomprehensiveconceptualmodelofwhatmayoccurduring developmentofWNSinahibernatingbat ? frominitial colonizationofwingskinby P.destructans untilthe deathoftheanimal.Thismodelidentifieskeytestable hypothesesnecessarytodevelopacomprehensiveunderstandingofthephysiologiceffectsofWNSonhibernating bats.Ultimately,thisknowledgewillbecriticalforguiding effectiveandproperlytimedmanagementactionsto moderatephysiologiceffectsofWNSandminimize morbidityandmortalityfromthisdevastatingdisease.MethodsBatsThisstudywasconductedinaccordancewithexperimentalprotocol#110921approvedbytheInstitutionalAnimal CareandUseCommitteeoftheUSGS ? NationalWildlife HealthCenter(NWHC).Sixty(30maleand30female) littlebrownbats( Myotislucifugus )werecollectedfroma hibernaculuminWisconsinonDecember21,2012and transportedtoNWHC.Batswereheldindividuallywithin tubesocksmaintainedincoolersatapproximately7?C duringtransport.Bodymassandrightforearmlength wererecordedforeachbat,andbothwingsofeachbat wereevaluatedforpre-existinginjuries.Allanimalswere confirmednegativefor P.destructans bypolymerasechain reaction(PCR)[48]analysisofwing-skinswabsamples (PurFlocknylon-flockedswabs,Puritan,Guilford,ME) collectedfromeachbat.Anarchivaltemperaturelogger (iBBat,AlphaMach)wasaffixedbetweenthescapulaeof eachbatusingalatex-basedadhesive(Ostobond,M.O.C., Vaudreuil,Quebec,Canada)aftertrimminga1cm?1cm patchoffurtowithin1mmoftheskinsurface.Loggers wereprogrammedtorecordskintemperature(Tskin) every15minutesstartingatthetimeofDLWinjection untiltheendoftheexperiment.Torpor-arousalpatternswereassessedusingTskindatadownloadedfrom iBBattemperatureloggersfollowingterminationofthe experiment.Arousalthresholdsweredefinedas10%of maximumTskinforeachindividual[11]. Batsweresortedbysexandrandomlyassignedtoinfected(n=39;19males,20females)andcontrol(n=21; 11males,10females)groups.Conidiaof P.destructans (5?105in20 lofPBSwith0.5%Tween-20)wereapplied totheskinofthedorsalsurfaceofbothwingsofeachbat intheinfectedgroupaspreviouslydescribed[49].PBS Tween-20lackingconidiawassimilarlyappliedtothe wingsofcontrol(non-infected)bats.Eachgroupwas placedintoameshenclosure(22 ? H?14 ? W?14 ? D; ApogeeReptaria,Reptiledirect.com)withinseparateenvironmentalincubators(PercivalScientific,Perry,IA)and maintainedat7.5?Cand90%RHfor98days.Batswere monitoredeveryotherdayforthedurationoftheexperimentandanydeadanimalsremoved. Attheendoftheexperiment,allremainingbatswere euthanized.Polymerasechainreaction(PCR)analysesof wingskinwereperformedaspreviouslydescribed[48]to confirmthepresenceof P.destructans .Additionally,the entiremembraneofonewingwasexaminedbyhistology toidentifylesionsdiagnosticforWNS[21]andtoassign severityscores[from0(nolesions)to4(severe,extensive lesions)]basedonextentoffungalinfection[11].DoublylabeledwaterTheDLWmethodusedatwo-sampleapproachbymeasuringisotopicconcentrationsatequilibrium(1hfollowing DLWinjection)andoncemoreattheendoftheexperiment[17].Thedurationoftheeliminationperiodwas determinedfromestimatesofisotopewashoutratesmodeledusingparametersofenergyexpenditureforhibernatinglittlebrownbats[40],assumingtorporboutsof8to 16dinterspersedby0.5to1.5harousals,asrecordedfor littlebrownbatswithandwithoutWNSinpreviousexperiments[12].Oneadditionalarousalanda3-heuthermic periodwereincludedinthemodeltoaccountforinitial DLWinjectionandbloodcollectionattheendofthe experiment.Baseduponanenergyexpenditurebudgetof approximately60kJ,isotopewashoutwaspredictedto occur89to171dpost-administrationofDLW.To increasethelikelihoodthatlabeledisotopeconcentrations wouldremainmeasurableoveradurationoftimesufficient fordevelopmentofexperimentallyinducedWNS(90to 120d;[12,49]),weadministeredthefirstdoseofDLW28 daftertreatmentofbatswithfungalconidia. ForadministrationofDLW,batswereremovedfrom incubatorsandarousedatroomtemperaturefor20to 30min.Oncefullyaroused,bothinfected(n=34)and negativecontrol(n=16)batswereinjectedintraperitoneallywithapproximately70 lofamixtureofenriched18O (approximately19atom%)and2H(approximately11 atom%).DoseenrichmentswerequantifiedusingaVerant etal.BMCPhysiology 2014, 14 :10 Page7of10 http://www.biomedcentral.com/1472-6793/14/10

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standarddilutionexperiment[50].Batswerethenheldat roomtemperatureforapproximately1htoallowforisotopicequilibration[20].Fol lowing1hateuthermictemperatures,50to75 lofbloodwascollectedfromthe ventralaspectoftheuropatagialveinofeachbat[51]into twoheparinized100 lcapillarytubesfordeterminationof initialisotopeconcentrationsandTBW.Endsofthecapillarytubeswereimmediatelyflame-sealed,dippedinsealing wax,andstoredat4?Cuntilanalysis.Batswerethen returnedtoincubatorsandleftundisturbedforthedurationoftheexperiment.Additionally,fivebatsfromeach treatmentgroupwereusedtomeasurebackgroundisotope levels[52];thesebatsweretreatedidenticallyasthosedescribedabove,buttheydidnotreceiveinjectionsofDLW. At95dayspost-treatmentwithconidia(67dafter injectionofDLW)batswereremovedfromhibernation chambers,arousedtoeuthermicbodytemperatures,and bloodwascollected(asabove)todeterminefinalisotope concentrations.BloodfrombatsthatdidnotreceiveinitialDLWinjectionswasalsosampledatthistimetoobtainaconcurrentmeasureofbackgroundisotopelevels. SinceTBWcontentofeachbatwasassumedtohave changedsincesamplingatthebeginningoftheexperiment,adoseoftheDLWsolutionusedpreviouslywas administeredtoeachbat,forwhichanon-terminal bloodsamplewascollected,todetermineTBWatthe endoftheexperimentforinfected(n=22)andcontrol (n=12)bats.FollowingtheinjectionwithDLW,bats wereheldatroomtempfor1hforisotopicequilibration,afterwhichtimetheywereanesthetizedwith5% isofluraneanddecapitated.Wholebloodwascollected intoheparinizedcapillarytubes,and50to75 lofblood wasimmediatelysealedincapillarytubes(asdescribed above)forisotopeanalysis.Rectaltemperaturewasrecordedatthetimeofeuthanasiausingaminiatureprobe anddigitalthermometer(ModelsRET-4&BAT7001H, PhysitempInstruments,Inc.,Clifton,NJ).BloodchemistryFollowingeuthanasia,bloodch emistryparameterswereanalyzedforeachbataspreviouslydescribed[9].Briefly,95 l ofwholebloodwascollectedfollowingdecapitationandanalyzedwithinoneminuteusingani-STATportableclinical analyzer(EC8+diagnosticcartridge,Abaxis,UnionCity, California,USA)toassesssodium(Na+,mmoll 1),potassium(K+,mmoll 1),chloride(Cl,mmoll 1),pH,dissolved carbondioxide(pCO2,mmHg),bicarbonate(HCO3 ,mmol l 1),baseexcess(BE,mmoll 1),aniongap(AG,mmoll 1), bloodureanitrogen(BUN,mgdL 1),hematocrit(Hct,% PCV),andglucose(mgdL 1).Remainingbloodwascentrifugedinmicrotubes(Stat-Spin,IrisSampleProcessing, Westwood,MA)for90s,andplasmaprotein(gdL 1)ofthe serumwasmeasuredusingahand-heldrefractometer(Pulse Instruments,VanNuys,CA).Temperature-correctedvalues forpHandpCO2werecalculatedusingrectaltemperature ofthebatatthetimeofbloodcollection,andHCO3 ,AG, andBEwerethencalculatedusingthesetemperaturecorrectedvaluesaccordingtothei-STATmanual[53]and specificationsoftheClinicalLa boratoryStandardsInstitute forBloodGasandpHAnalysisandRelatedMeasurements [54](seealsoAdditionalfile3forequations).IsotopeanalysisCapillarytubescontainingthebloodsampleswerevacuum distilled[55],andwaterfromtheresultingdistillatewas usedtoproduceCO2andH2(methodsin[56]forCO2and [57]forH2).Theisotoperatios18O:16Oand2H:1Hwere analyzedusinggassourceisotoperatiomassspectrometry (Optima,MicromassIRMSandIsochrom G,Manchester, UK).Sampleswererunalongsidethreelabstandardsfor eachisotope(calibratedtoInternationalstandards)tocorrectdeltavaluestoppm.Isotopeenrichmentswereconvertedtovaluesofdailyenergyexpenditureusingasingle poolmodelasrecommendedforthissizeofanimal[58]. Dilutionspacesforoxygen(N0)andhydrogen(Nd)were calculatedbytheplateaumethod(SpeakmanandKrol, [59]).CO2productionwascalculatedusingequation7.17 ofSpeakman[50]andusedtoestimateDEEaccordingto theWeirequation[59](seeAdditionalfile2fordetails).StatisticalanalysesNormalityofmeasuredparameterswasassessedvisually usinghistogramsandQ-Qplotsforeachparameter.Assumptionsofnormalityandhomogeneityofvariancewere satisfiedsonotransformationswereperformed.Mean valuesofmeasuredparametersforinfectedbatswere comparedtocontrolbatsusingindependenttwo-sample t -tests.PaireddatasetsfromTBWestimateswerecomparedusingpaired t -tests.All t -testsweretwo-tailedwith =0.05,andcriticalvaluesforsignificancewereadjusted tocontrolthefamily-wiseerrorrateusingtheHolmBonferonnimethod[60]appliedseparatelytoeachdata set:bloodchemistryparameters(12comparisons), DLWmeasurements(8comparisons),andtorporprofiles (2comparisons).Allstatisticalanalyseswereconductedin R [61].Datainthetextarepresentedasmeans(SD).AdditionalfilesAdditionalfile1: TableofBloodChemistryParameters. Comparisons ofbloodchemistryparametervaluesforhibernatinglittlebrownbats( Myotis lucifugus )experimentallyinfectedwith Pseudogymnoascusdestructans ( Pd ) andnegative(non-infected)controlstoavailablereferencevalues. Additionalfile2: DoublyLabeledWater. Meanisotopeenrichmentsof administeredDLWformulationsandbackgroundisotopeconcentrations inbatsatthestartandendoftheexperiment.Equationsusedfor calculationofdailyenergyrequirementsandfatenergyuse. Additionalfile3: BloodChemistryCalculations. Equationsusedto calculatetemperature-correctedacid ? basebloodparameters.Verant etal.BMCPhysiology 2014, 14 :10 Page8of10 http://www.biomedcentral.com/1472-6793/14/10

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Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ? contributions MLV,PMC,JML,andDSBconceivedanddesignedthestudy.MLVcoordinated andconductedtheexperiment,performedmolecularanalyses,andanalyzed thedata.JRSformulatedthedoublylabeledwater(DLW),supervisedanalyses ofDLWsamples,andprovidedexpertconsultonstudydesignand interpretationofresults.MLV,PMC,JML,CUM,andDSBparticipatedindata collection.CUMperformedthehistopathologyexaminationsfordisease confirmation.MLVwrotethemanuscript,allco-authorsprovidedinput,and DSBeditedthemanuscript.Allauthorsreadandapprovedthemanuscript. Acknowledgments ThisprojectwasfinanciallysupportedbytheUSGeologicalSurveythrougha cooperativeagreementwiththeUniversityofWisconsin ? Madison.Weare indebtedtoDaveandJenniferRedellandPaulWhitefromtheWisconsin DepartmentofNaturalResourcesforcollectingtheanimalsusedto completethisstudyandforassistingwithdatacollection.WethankMelissa BehrforassistancewithnecropsiesandNWHCAnimalCareStafffortheir helpwithset-upandmaintenanceofanimals.WethankLobkeVaanholtand CatherineHambly(UniversityofAberdeen,Scotland)fortheirexpertiseand coordinationintheanalysesoftheDLWbloodsamples.Fundswereusedfor directprojectcostsonly.Useoftrade,product,orfirmnamesisfordescriptive purposesonlyanddoesnotimplyendorsementbytheUSGovernment. Authordetails 1 DepartmentofPathobiologicalSciences,SchoolofVeterinaryMedicine, UniversityofWisconsin-Madison,2015LindenDr.,Madison,Wisconsin,USA. 2 USGeologicalSurvey ? NationalWildlifeHealthCenter,6006SchroederRd., Madison,Wisconsin,USA. 3 InstituteofBiologicalandEnvironmentalSciences, UniversityofAberdeen,Aberdeen,Scotland,UK. 4 USGeologicalSurvey ? Fort CollinsScienceCenter,2150CentreAve.BuildingC,FortCollins,Colorado, USA. 5 USGeologicalSurvey ? NationalCenter,EnvironmentalHealth,12201 SunriseValleyDr.,Reston,Virginia,USA. Received:4June2014Accepted:18November2014 References 1.BlehertDS,HicksAC,BehrMJ,MeteyerCU ,Berlowski-ZierBM,BucklesEL,Coleman JTH,DarlingSR,GargasA,NiverR,O koniewskiJC,RuddRJ,StoneWB: Bat white-nosesyndrome:anemergingfungalpathogen? Science 2009, 323: 227. 2.FrickWF,PollockJF,HicksAC,LangwigK,ReynoldsDS,TurnerGG,Butchkoski CM,KunzTH: Anemergingdiseasecausesregionalpopulationcollapseofa commonNorthAmericanbatspecies. Science 2010, 329: 679 ? 682. 3.TurnerGG,ReederDM,ColemanJC: Afive-yearassessmentofmortality andgeographicspreadofwhite-nosesyndromeinNorthAmericanbats andalooktothefuture. BatResNews 2011, 52: 13 ? 27. 4.GargasA,TrestMT,ChristensenM,VolkTJ,BlehertDS: Geomyces destructanssp.nov.asssociatedwithbatwhite-nosesyndrome. Mycotaxon 2009, 108: 147 ? 154. 5.MinnisAM,LindnerDL: Phylogeneticevaluationof Geomyces andallies revealsnocloserelativesof Pseudogymnoascusdestructans ,comb.nov.,in bathibernaculaofeasternNorthAmerica. FungalBiol 2013, 117: 638 ? 649. 6.LorchJM,MullerLK,RussellRE,O ? ConnorM,LindnerDL,BlehertDS: Distributionandenvironmentalpersistenceofthecausativeagentof white-nosesyndrome, Geomycesdestructans ,inbathibernaculaofthe easternUnitedStates. ApplEnvironMicrobiol 2012, 79: 1293 ? 2839. 7.CryanPM,MeteyerCU,BoylesJG,BlehertDS: Wingpathologyof white-nosesyndromeinbatssuggestslife-threateningdisruptionof physiology. BMCBiol 2010, 8: 135. 8.WillisCKR,MenziesAK,BoylesJG,WojciechowskiMS: Evaporativewater lossisaplausibleexplanationformortalityofbatsfromwhite-nose syndrome. IntegrCompBiol 2011, 51: 364 ? 373. 9.CryanPM,MeteyerCU,BlehertDS,LorchJM,ReederDM,TurnerGG,Webb J,BehrM,VerantML,RussellRE,CastleKT: Electrolytedepletionin white-nosesyndromebats. JWildlifeDis 2013, 49: 398 ? 402. 10.WarneckeL,TurnerJM,BollingerTK,Mi sraV,CryanPM,BlehertDS,WibbeltG, WillisCKR: Pathophysiologyofwhite-nosesyndromeinbats:amechanistic modellinkingwingdamagetomortality. BiolLett 2013, 9: 20130177. 11.ReederDM,FrankCL,TurnerGG,MeteyerCU,KurtaA,BritzkeER,Vodzak ME,DarlingSR,StihlerCW,HicksAC,JacobR,GrieneisenLE,BrownleeSA, MullerLK,BlehertDS: Frequentarousalfromhibernationlinkedto severityofinfectionandmortalityinbatswithwhite-nosesyndrome. PloSOne 2012, 7: e38920. 12.WarneckeL,TurnerJM,BollingerTK,LorchJM,MisraV,CryanPM,Wibbelt G,BlehertDS,WillisCKR: InoculationofbatswithEuropean Geomyces destructans supportsthenovelpathogenhypothesisfortheoriginof white-nosesyndrome. ProcNatlAcadSciUSA 2012, 109: 6999 ? 7003. 13.KayserC: ThePhysiologyofNaturalHibernation. Oxford:PergamonPress;1961. 14.ThomasDW,DoraisM,BergeronJM: Winterenergybudgetsandcostof arousalsforhibernatinglittlebrownbats, Myotislucifugus . JMammal 1990, 71: 475 ? 479. 15.GeiserF: Metabolicrateandbodytemperaturereductionduring hibernationanddailytorpor. AnnuRevPhysiol 2004, 66: 239 ? 274. 16.ButlerPJ,GreenJA,BoydIL,SpeakmanJR: Measuringmetabolicrateinthe field:theprosandconsofthedoublylabelledwaterandheartrate methods. FunctEcol 2004, 18: 168 ? 183. 17.LifsonN,GordonGB,McClintockR: Measurementoftotalcarbondioxide productionbymeansofD 2 O 18 . JApplPhysiol 1955, 7: 704 ? 710. 18.SpeakmanJR,VisserGH,WardSE,Kr?lE: TheIsotopeDilutionMethodforthe EvaluationofBodyComposition. In BodyCompositionAnalysisofAnimals. EditedbySpeakmanJR.Cambridge:CambridgeUniversityPress;2001:56 ? 98. 19.KurtaA,BellGP,NagyKA,KunzTH: Energeticsofpregnancyandlactation infree-ranginglittlebrownbats( Myotislucifugus ). PhysiolZool 1989, 62: 804 ? 818. 20.KurtaA,KunzTH,NagyKA: Energeticsandwaterfluxoffree-rangingbig brownbats( Eptesicusfuscus )duringpregnancyandlactation. JMammal 1990, 71: 59 ? 65. 21.MeteyerCU,BucklesEL,BlehertDS,HicksAC,GreenDE,Shearn-BochslerV, ThomasNJ,GargasA,BehrMJ: Histopathologiccriteriatoconfirm white-nosesyndromeinbats. JVetDiagnInvest 2009, 21: 411 ? 414. 22.MalanA,ArensH,WaechterA: Pulmonaryrespirationandacid ? basestate inhibernatingmarmotsandhamsters. RespirPhysiol 1973, 17: 45 ? 61. 23.SzewczakJM: Matchinggasexchangeinthebatfromflighttotorpor. AmZool 1997, 37: 92 ? 100. 24.MalanA,MioskowskiE,CalgariC: Time-courseofbloodacid ? basestate duringarousalfromhibernationintheEuropeanhamster. JCompPhysiolB 1988, 158: 495 ? 500. 25.SnappBD,HellerHC: Suppressionofmetabolismduringhibernationin groundsquirrels( Citelluslateralis ). PhysiolZool 1981, 54: 297 ? 307. 26.BicklerPE: CO 2 balanceofaheterothermicrodent:comparisonofsleep, torpor,andawakestates. AmJPhysiol 1984, 246: 49 ? 55. 27.SzewczakJM,JacksonDC: Acid ? basestateandintermittentbreathingin thetorpidbat, Eptesicusfuscus . RespirPhysiol 1992, 88: 205 ? 215. 28.ThomasDW,CloutierD,GagneD: Arrhythmicbreathing,apneaand non-steadystateoxygenuptakeinhibernatinglittlebrownbats ( Myotislucifugus ). JExpBiol 1990, 149: 395 ? 406. 29.ThomasDW,CloutierD: Evaporativewater-lossbyhibernatinglittle brownbats, Myotislucifugus . PhysiolZool 1992, 65: 443 ? 456. 30.Munoz-GarciaA,Ben-HamoM,PinshowB,WilliamsJB,KorineC: The relationshipbetweencutaneouswaterlossandthermoregulatorystate inKuhl ? spipistrelle Pipistrelluskuhlii ,aVespertillionidbat. PhysiolBiochemZool 2012, 85: 516 ? 525. 31.SpeakmanJR,RaceyPA: HibernalecologyofthePipistrellebat-energyexpenditure,waterrequirementsandmass-loss,implicationsforsurvival andthefunctionofwinteremergenceflights. JAnimEcol 1989, 58: 797 ? 813. 32.Ben-HamoM,Munoz-GarciaA,WilliamsJB,KorineC,PinshowB: Wakingto drink:ratesofevaporativewaterlossdeterminearousalfrequencyin hibernatingbats. JExpBiol 2013, 216: 573 ? 577. 33.MeteyerCU,ValentM,KashmerJ,BucklesEL,LorchJM,BlehertDS,LollarA, BerndtD,WheelerE,WhiteCL,BallmannAE: Recoveryoflittlebrownbats ( Myotislucifugus )fromnaturalinfectionwith Geomycesdestructans , white-nosesyndrome. JWildlifeDis 2011, 47: 618 ? 626. 34.RiedeselML: BloodPhysiology. In BiologyofBats.Volume3. Editedby WimsattW.NewYork:AcademicPress;1977:651. 35.CryanPM,WolfBO: Sexdifferencesinthethermoregulationand evaporativewaterlossofaheterothermicbat,Lasiuruscinereus,during itsspringmigration. JExpBiol 2003, 206: 3381 ? 3390. 36.RiedeselML,FolkGEJr: Serumelectrolytelevelsinhibernatingmammals. AmNat 1958, 92: 307 ? 312. Verant etal.BMCPhysiology 2014, 14 :10 Page9of10 http://www.biomedcentral.com/1472-6793/14/10

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37.RiedeselML: Serummagnesiumlevelsinmammalianhibernators. TransKansAcadSci 1957, 60: 99 ? 141. 38.KallenFC: Plasmaandbloodvolumesinthelittlebrownbat. AmJPhysiol 1960, 198: 999 ? 1005. 39.BloodFR,DodgenCL: Energysourcesinthebat. AmJPhysiol 1956, 187: 151 ? 154. 40.HumphriesMM,ThomasDW,SpeakmanJR: Climate-mediatedenergetic constraintsonthedistributionofhibernatingmammals. Nature 2002, 418: 313 ? 316. 41.MalanA: RespirationandAcid ? BaseStateinHibernation. In Hibernation andTorporinMammalsandBirds. EditedbyLymanCP,WillisJS,MalanA, WangLCH.NewYork:AcademicPress;1982:237 ? 282. 42.HochachkaPW,GuppyM: Metabolicarrestandthecontrolofbiologicaltime. Cambridge,MA:HarvardUniversityPress;1987. 43.MalanA: pHandhypometabolisminmammalianhibernation. CanJZool 1988, 66: 95 ? 98. 44.SchaeferKE,WunnenbergW: Thresholdtemperaturesforshiveringin acuteandchronichypercapnia. JApplPhysiol 1976, 41: 67 ? 70. 45.StudierEM,FresquezAA: Carbondioxideretention:amechanismof ammoniatoleranceinmammals. Ecology 1969, 50: 492 ? 494. 46.HerreidCF2nd,BretzWL,Schmidt-NielsenK: Cutaneousgasexchangein bats. AmJPhysiol 1968, 215: 506 ? 508. 47.MakanyaAN,MortolaJP: Thestructuraldesignofthebatwingweband itspossibleroleingasexchange. JAnat 2007, 211: 687 ? 697. 48.MullerLK,LorchJM,LindnerDL,O ? ConnorM,GargasA,BlehertDS: Bat white-nosesyndrome:areal-timeTaqManpolymerasechainreaction testtargetingtheintergenicspacerregionof Geomycesdestructans . Mycologia 2012, 105: 253 ? 259. 49.LorchJM,MeteyerCU,BehrMJ,BoylesJG,CryanPM,HicksAC,BallmannAE, ColemanJTH,RedellD,ReederDM,BlehertDS: Experimentalinfectionof batswith Geomycesdestructans causeswhite-nosesyndrome. Nature 2011, 480: 376 ? 378. 50.KunzTH,NagyKA: MethodsofEnergyBudgetAnalysis. In Ecologicaland BehaviorMethodsfortheStudyofBats. EditedbyKunzTH.Washington,D.C: SmithsonianInstitutionPress;1988:277 ? 302. 51.SpeakmanJR: DoublyLabelledWater-TheoryandPractice. London:Chapman andHall;1997. 52.SpeakmanJR,RaceyPA: TheequilibriumconcentrationofO-18in body-water:implicationsfortheaccuracyofthedoubly-labeledwater techniqueandapotentialnewmethodofmeasuringRQinfree-living animals. JTheorBiol 1987, 127: 79 ? 95. 53.AbbottPointofCareInc: iStatInstructionManual. AbbottPark,IL:Abbott PointofCareInc;2008. 54.ClinicalLaboratoryStandardsInstitute: BloodgasandpHAnalysisandRelated Measurements:ApprovedGuideline-SecondEdition. Wayne,PA:CLSI;2009. 55.NagyKA: TheDoublyLabeledWater 3 HH 18 OMethod:AGuidetoitsuse. Los Angeles:UniversityofCalifornia;1983. 56.SpeakmanJR,NagyKA,MasmanD,MookWG,PoppittSD,StrathearnGE, RaceyPA: Interlaboratorycomparisonofd ifferentanalyticaltechniques forthedeterminationofoxygen-18abundance. AnalChem 1990, 62: 703 ? 708. 57.SpeakmanJR,KrolE: Comparisonofdifferentapproachesforthe calculationofenergyexpenditureusingdoublylabeledwaterinasmall mammal. PhysiolBiochemZool 2005, 78: 650 ? 667. 58.SpeakmanJR: HowshouldwecalculateCO 2 productionindoubly labelledwaterstudiesofanimals? FunctEcol 1993, 7: 746 ? 750. 59.WeirJB: Newmethodsforcalculatingmetabolicratewithspecial referencetoproteinmetabolism. JPhysiol 1949, 109: 1 ? 9. 60.HolmS: Asimplesequentiallyrejectivemultipletestprocedure. ScandJStat 1979, 6: 65 ? 70. 61. R:ALanguageandEnvironmentforStatisticalComputing. [http://www. R-project.org] doi:10.1186/s12899-014-0010-4 Citethisarticleas: Verant etal. : White-nosesyndromeinitiatesa cascadeofphysiologicdisturbancesinthehibernatingbathost. BMC Physiology 2014 14 :10. Submit your next manuscript to BioMed Central and take full advantage of: ? Convenient online submission ? Thorough peer review ? No space constraints or color ?gure charges ? Immediate publication on acceptance ? Inclusion in PubMed, CAS, Scopus and Google Scholar ? Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Verant etal.BMCPhysiology 2014, 14 :10 Page10of10 http://www.biomedcentral.com/1472-6793/14/10


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