Temperature-dependent growth of Geomyces destructans, the fungus that causes Bat White-Nose Syndrome


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Temperature-dependent growth of Geomyces destructans, the fungus that causes Bat White-Nose Syndrome
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PLOS One
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Verant, Michelle L.
Boyles, Justin G.
Waldrep Jr., William
Wibbelt, Gudrun
Blehert, David S.
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White-nose syndrome ( lcsh )
Bats ( lcsh )
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White-nose syndrome (WNS) is an emergent disease estimated to have killed over five million North American bats. Caused by the psychrophilic fungus Geomyces destructans, WNS specifically affects bats during hibernation. We describe temperature-dependent growth performance and morphology for six independent isolates of G. destructans from North America and Europe. Thermal performance curves for all isolates displayed an intermediate peak with rapid decline in performance above the peak. Optimal temperatures for growth were between 12.5 and 15.8°C, and the upper critical temperature for growth was between 19.0 and 19.8°C. Growth rates varied across isolates, irrespective of geographic origin, and above 12°C all isolates displayed atypical morphology that may have implications for proliferation of the fungus. This study demonstrates that small variations in temperature, consistent with those inherent of bat hibernacula, affect growth performance and physiology of G. destructans, which may influence temperature-dependent progression and severity of WNS in wild bats.
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Temperature-DependentGrowthofGeomyces destructans,theFungusThatCausesBatWhite-Nose SyndromeMichelleL.Verant1 .,JustinG.Boyles2 . ¤,WilliamWaldrepJr3,GudrunWibbelt4,DavidS.Blehert5*1 DepartmentofPathobiologicalSciences,UniversityofWisconsin,Madison,Wisconsin,UnitedStatesofAmerica, 2 DepartmentofEcologyandEvolutionaryBiology, UniversityofTennessee,Knoxville,Tennessee,UnitedStatesofAmerica, 3 DepartmentofComputerScience,UniversityofChicago,Chicago,Illinois,UnitedStatesof America, 4 LeibnizInstituteforZooandWildlifeResearch,Berlin,Germany, 5 USGeologicalSurvey–NationalWildlifeHealthCenter,Madison,Wisconsin,UnitedStatesof AmericaAbstractWhite-nosesyndrome(WNS)isanemergentdiseaseestimatedtohavekilledoverfivemillionNorthAmericanbats.Caused bythepsychrophilicfungus Geomycesdestructans ,WNSspecificallyaffectsbatsduringhibernation.Wedescribe temperature-dependentgrowthperformanceandmorphologyforsixindependentisolatesof G.destructans fromNorth AmericaandEurope.Thermalperformancecurvesforallisolatesdisplayedanintermediatepeakwithrapiddeclinein performanceabovethepeak.Optimaltemperaturesforgrowthwerebetween12.5and15.8 u C,andtheuppercritical temperatureforgrowthwasbetween19.0and19.8 u C.Growthratesvariedacrossisolates,irrespectiveofgeographicorigin, andabove12 u Callisolatesdisplayedatypicalmorphologythatmayhaveimplicationsforproliferationofthefungus.This studydemonstratesthatsmallvariationsintemperature,consistentwiththoseinherentofbathibernacula,affectgrowth performanceandphysiologyof G.destructans ,whichmayinfluencetemperature-dependentprogressionandseverityof WNSinwildbats.Citation: VerantML,BoylesJG,WaldrepWJr,WibbeltG,BlehertDS(2012)Temperature-DependentGrowthof Geomycesdestructans ,theFungusThatCausesBat White-NoseSyndrome.PLoSONE7(9):e46280.doi:10.1371/journal.pone.0046280 Editor: MatthewCharlesFisher,ImperialCollegeFacultyofMedicine,UnitedKingdom Received June4,2012; Accepted August28,2012; Published September28,2012 Thisisanopen-accessarticle,freeofallcopyright,andmaybefreelyreproduced,distributed,transmitted,modified,builtupon,orotherwiseus edbyanyonefor anylawfulpurpose.TheworkismadeavailableundertheCreativeCommonsCC0publicdomaindedication. Funding: FinancialsupportforthisprojectwasprovidedbytheUSGeologicalSurvey,theUSFishandWildlifeService,andBatConservationInternational.Th e fundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript. CompetingInterests: Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:dblehert@usgs.gov . Theseauthorscontributedequallytothiswork. ¤Currentaddress:CooperativeWildlifeResearchLaboratory,DepartmentofZoology,SouthernIllinoisUniversity,Carbondale,Illinois,United StatesofAmericaIntroductionWhite-nosesyndrome(WNS)isanemergentdiseaseofhibernating, insectivorousbats[1].Thecausativeagent, Geomycesdestructans [2,3],isa psychrophilic(cold-loving)fung uswithactivegrowthlimitedtocool environmentssuchasthosechara cteristicofundergroundbat hibernacula[4,5].WNSischaracterizedbyinvasivegrowthof G. destructans onthemuzzleandwingsofhiber natingbats[6].Sincefungal infectionconsistentwithWNSwasfirstphoto-documentedamong hibernatingbatsinapopulartouristcaveineast-centralNewYork duringwinter2006–2007,overfivemillionbatsareestimatedtohave diedfromthisdisease(http://www.fws.gov/whitenosesyndrome/pdf/ WNS_Mortality_2012_NR_FINAL.pdf).Populationmodelspredict thatWNScouldcausethelittlebrownbat( Myotislucifugus ),onceoneof themostcommonbatsacrossNorthAmerica,tobeextirpatedfrom thenortheasternUnitedStatesby2026[7]. Followinginitialdiscoveryanddescriptionof G.destructans in NorthAmerica[1],thefunguswasalsoidentifiedonhibernating batsintwelveEuropeancountries[8,9,10,11].Whileinfectionof Europeanbatsby G.destructans hasbeendocumentedtocause lesionsdiagnosticforWNS[12],infectionshavenotbeenlinked tounusualmortalityeventsamongwildEuropeanbats[13].A recentlaboratorystudy,however,demonstratedthatisolatesof G. destructans fromGermanyandNewYorkwerebothlethaltoan experimentallyinfectedNorthAmericanbatspecies,thelittle brownbat[3].ReasonsforthedisparateeffectsofWNSonbat mortalitybetweenthetwocontinentsareunknown,andwhile intercontinentaldifferencesinbatphysiologyorbehaviormaybe responsible,growthpropertiesofindividualfungalisolates,and/ orenvironmentalconditionsmayalsoplayacontributingrole [3,14]. Thepsychrophilicnatureof G.destructans likelyrestrictsits fundamentalniche,butdetailedinvestigationsoftemperaturedependentgrowthofthefungusarelacking.Activegrowthof G. destructans hasbeenpreviouslyreportedbetween0and20 u C,but optimaltemperatureforgrowthremainsunclearbecauseprevious measurementswereconductedatlowthermalresolution[1,4]. Thus,theobjectivesofthisstudywereto:1)definetheupper criticaltemperatureforgrowthof G.destructans ;2)characterize temperature-dependentgrowthperformanceofthefungusfrom approximately0 u Ctotheuppercriticaltemperature;3)compare temperature-dependentgrowthperformanceforsixisolatesof G. destructans fromNorthAmericaandEurope;and4)describe temperature-dependentvariationinfungalmorphologyacrossa rangeoftemperatures. PLOSONE|www.plosone.org1September2012|Volume7|Issue9|e46280

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Results Thermaloptimaanduppercriticaltemperaturesfor growthThermalperformancecurvesweregeneratedforsixindependentisolatesof G.destructans culturedfromskinorhairofbats collectedinNewYork,Pennsylvania,Virginia,Germany, Hungary,andSwitzerland.Performancecurvesforallfungal isolateswerecharacteristicinshape,withanintermediatepeakat thethermaloptimumforgrowth(Topt)andrapiddeclinein performanceattemperaturesabovethepeak(Figs.1and2). Growthratesforeachisolateincreasedslowlytoapeakratebefore decreasingtozerogrowthoverarangeof3to5 u CaboveTopt. Therewasvariationinthebest-fitfunctionamongisolatesand weeklyreplicates,butskewedfunctions,particularlytheBrie `re2 function,bestdescribedthegrowthdatainmostcases(TableS1). Followingfiveweeksofincubation,Toptvariedacrossisolates, rangingfrom12.5to13.8 u C,withtheexceptionoftheSwiss isolate,whichgrewbestat15.8 u C(Table1). Geomycesdestructans did notgrowat21.4 u C,butgrowthwasobservedat18.9 u C,indicating theuppercriticaltemperatureiswithinthisrange.Estimatesfrom functionsthatprovidedthemeanstocalculateuppercritical temperatureforgrowthplacedthevaluebetween19.0and 19.8 u C.Eightypercentperformancebreadths(therangeacross whichgrowthis . 80%ofmaximalgrowth)forallisolates followingfiveweeksofincubationwerebetween6.1and8.0 u C (Table1).TheSwissisolatehadanarrower80%performance breadthof3.6 u C(Table1).Inourinitialexperiment,which evaluatedgrowthoftwoisolateseachweekforfiveweeks, performancebreadthincreasedovertime(Table1)withsymmetricalfunctionsbecomingreasonablefitstothedata(i.e., D AICc , 2)inthelastweekofsampling(TableS1).Variationsin estimatesofToptandperformancebreadthamongsubsetswithin eachanalysiswereminimal,suggestinglittleintra-platevariation incolonygrowth.Thissmallamountofvariationinadditiontothe largenumberofrandomizationsledtosmall84%CIs,andthere wasnooverlapinCIsforanycomparisonsbetweenreplicates.Independentisolatesexhibiteddifferentialgrowth performanceInaninitialexperimentcomparinggrowthpropertiesoftwo isolatesof G.destructans previouslyshowninthelaboratorytobe lethaltolittlebrownbats[3],anisolatefromNewYork consistentlygrewfasterthanaGermanisolateacrosstheentire rangeoftemperaturesexamined(Fig.1).Twoindependentbest-fit functionswereabetterfittothedata( D AICc=0)forthefinal weekofmeasurementsthanwasasinglefunctiondescribingthe combineddata( D AICc=32.1),indicatingsubstantialdifferences intemperature-dependentgrowthbetweentheseisolates. Theresultsofthisinitialexperimentledtothehypothesisthat isolatesof G.destructans fromNorthAmericagrowfasterthanthose fromEurope.However,continent-dependentdifferencesin growthperformancewerenotverifiedbyfollow-upanalysisin whichgrowthperformanceoffouradditionalisolates(twofrom NorthAmericaandtwofromEurope)wasmeasured(Fig.2). Baseduponthisfollow-upanalysis,anisolatefromVirginia exhibitedslowestgrowthwhilethefastestgrowingisolatewasfrom Switzerland.Fourindividualfunctions( D AICc=0)collectively describedthedatabetterthanonefunctionforeachcontinent ( D AICc=35.7),orthanonefunctionforallfourisolatescombined ( D AICc=60.8),furthervalidatingvariationbetweenisolates. However,withtheexceptionoftheSwissisolate,smallvariations ingrowthparametersoftheremainingfiveisolates(Topt=12.5to 13.6 u C;uppercriticaltemperatureforgrowth=19.0to19.8 u C; 80%performancebreadths=6.6to8.0 u C)indicatethatoverall, growthperformancewassimilar(Table1).MorphologyofGeomycesdestructansvarieswith temperatureWecharacterizedmorphologyofhyphaeandconidiaproduced bytheNorthAmericantypeisolateof G.destructans following Figure1.WeeklygrowthcurvesfortwoisolatesofGeomycesdestructans. Inaninitialexperiment,twoisolatesof G.destructans (onefrom NewYorkandonefromGermany)exhibiteddifferencesingrowthperformancebuthadsimilarthermaloptimaanduppercriticaltemperaturesfor growth.Toptanduppercriticaltemperatures(CLu)forgrowthatweek5aremarkedonthegraphswitharrows.Forthisfigure,eachcurveis representedusingaBrie ` re2function,althoughinsomecases,otherfunctionswereequallyparsimonious(TableS1).ToptandCLuinthisfigure representthevaluesspecifictotheBrie ` re2functionshowninthegraph;thereforeToptdoesnotmatchtheweightedaveragespresentedinTable1. TheisolatesweregrownonSabourauddextroseagar.Twenty-onereplicatecoloniesofeachwereincubatedacrossarangeofninetemperatures from0.8to21.4 u C.Theareaofeachexpandingcolonywasmeasuredweekly,forfiveweeks,andagrowthcurvewasfittoeachweeklydataset. doi:10.1371/journal.pone.0046280.g001 ThermalPerformanceof Geomycesdestructans PLOSONE|www.plosone.org2September2012|Volume7|Issue9|e46280

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incubationatarangeoftemperatures(Fig.3).Qualitative evaluationsoftheotherisolatescompletedoverthesame temperaturerangerevealedmorphologiessimilartothoseofthe typeisolate.Consistentwithpreviousreports[4],whenthetype isolatewasgrownbetweenapproximately0and7 u C,itdisplayed colonymorphologycharacteristicfor G.destructans —whiteand smoothdomedcolonieswithmostdevelopinglightgreycentersover time.Microscopically,hyphaewereslim(averagediameter=1.5mm)andyieldedabundant,characteristicallycurved conidiafromthetipsofbranchedconidiophores(Fig.3a,3b). Atypicalmorphologiesofhyphaeandconidiawereseenat approximately12 u Candabove(Fig.3c,3d,3e).Atthesehigh temperatures,hyphaewerethicker(averagediameter=2.2mmat < 12 u C;3mmat < 18 u C)anddiffuselyseptatewithdifferential sequestrationofcellularmaterialamongsegments.Conidiawere mostlypyriformtogloboidinshape,primarilyproducedinshort chains(Fig.3d;arrows),andreleasedbyrhexolyticdehiscence. Detachmentofhyphalsegmentsproducedshortarthrospore-like fragments(Fig.3c,arrows),anddensesingle-cellstructures resemblingchlamydospores(Fig.3c,3e;arrowheads)wereseen. Attemperaturesaboveapproximately15 u C,hyphaeweremarkedly deformed,andhyphaltipsexhibitedbranchedantler-likemorphology(Fig.3e;arrows).Thesechangesbecamemorepronouncedat highertemperatureswithcompletelossofcharacteristichyphal structureandcurvedconidiaaboveapproximately18 u C(Fig.3d). Grossly,coloniesgrownaboveapproximately15 u Cwerevariably tantodarkbrown,andcolonysurfacesweremarkedlycreased. Grossandmicroscopicmorphologyofcoloniesgrownatmid-range temperatures(approximately7to12 u C)includedamixtureofthese Figure2.Five-weekgrowthcurvesforfourisolatesofGeomycesdestructans. Inafollow-upexperiment,differencesingrowthperformance wereconfirmedamongfouradditionalisolatesof G.destructans ,twofromNorthAmericaandtwofromEurope.Aconsistentintercontinentaltrendin growthperformancewasnotobservedamongtheisolates.TheisolatesweregrownonSabourauddextroseagar.Twenty-onereplicatecoloniesof eachisolate(Pennsylvania,Virginia,Hungary,andSwitzerland)wereincubatedacrossarangeoffivetemperaturesfrom1.9to17.7 u C.Theareaof eachexpandingcolonywasmeasuredafterfiveweeks,andagrowthcurvewasfittoeachdataset.Eachcurveisrepresentedusingthebest-fit function.Forcomparisontotheweeklygrowthcurveanalysis(Fig.1),21replicatecolonieseachoftheisolatesfromNewYorkandGermanywere incubatedwiththeotherisolatesat6.7,12.2,and17.7 u C.Theareaofeachexpandingcolonywasmeasuredafterfiveweeks,anddescriptivedata (mean 6 SD)arerepresented.IsolatesfromNewYorkandGermanygrewproportionallyfasterateachofthetemperaturesusedforthisanalysisthan intheinitialweeklyanalysis.Althoughthecurveshapesforbothanalyseswereconsistent,thetwoanalysescannotbedirectlycompared. doi:10.1371/journal.pone.0046280.g002 Table1. Weightedaveragesofthethermaloptimafor growth(Topt)and80%performancebreadthforeachisolate of Geomycesdestructans growninculture.IsolateTopt( ± 84%CI)PerformanceBreadth( ± 84%CI) NewYork Week116.98(0.008)4.37(0.007) Week216.27(0.006)5.06(0.006) Week314.92(0.007)6.39(0.004) Week414.20(0.005)6.67(0.004) Week513.05(0.004)7.62(0.005) Germany Week116.87(0.012)4.61(0.011) Week216.63(0.008)5.20(0.007) Week316.20(0.003)5.59(0.002) Week415.38(0.002)6.03(0.001) Week513.79(0.003)6.42(0.002) Pennsylvania 12.45(0.002)7.03(0.003) Virginia 13.22(0.004)7.99(0.006) Switzerland 15.79(0.001)3.64(0.003) Hungary 13.96(0.005)6.07(0.004) Overlapin 6 84%CIvaluesindicatesnon-significantdifferencesatthe a =0.05 level.GrowthofisolatesfromPennsylvania,Virginia,Switzerland,andHungary wereonlymeasuredat5weeks.AllvaluesareindegreesCelsius. doi:10.1371/journal.pone.0046280.t001 ThermalPerformanceof Geomycesdestructans PLOSONE|www.plosone.org3September2012|Volume7|Issue9|e46280

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characteristics.Culturesof G.destructans maintainedatapproximately7 u Cforuptooneyeardidnotexhibitmorphological characteristicsofculturesincubatedatelevatedtemperatures.DiscussionWeprovideadetailedcharacterizationoftemperature-dependentgrowthofsixisolatesof G.destructans fromNorthAmericaand Europeanddemonstrateinter-isolatevariationingrowthperformance.Thermalperformancecurvesgeneratedforeachisolate indicatedthermaloptimaforgrowthbetween12.5and15.8 u C andanuppercriticaltemperatureforgrowthbetween19.0and 19.8 u C.Althoughweobserveddifferencesinestimatedgrowth curvesamongisolates,ouranalysesdidnotindicatethatfungal isolatesfromNorthAmericaandEuropehaveinherentlydifferent growthcharacteristics.Furthermore,apreviouslypublished experimentalstudydemonstratedthatisolatesof G.destructans fromNorthAmericaandEuropewerebothlethaltoaNorth Americanbatspecies[3].Thus,observedinter-continental differencesinWNSdiseasemanifestation,progression,and mortalityamongwildbatsdonotlikelyresultfromcontinentspecificvariationsingrowthperformanceofthepathogen. Theresultsofthisstudydo,however,demonstratethat environmentalconditions,specificallytemperature,exertastrong influenceongrowthperformanceof G.destructans (Figs.1and2). Consequently,differencesintemperatureatlocationswithin undergroundsitesoccupiedbyhibernatingbatsmayinfluence bothprogressionandseverityofWNSamonginfectedbatsand environmentalpersistenceandtransmissionofthefungus. Hibernationtemperaturespreferredbybatsvaryconsiderably dependingonspecies,hibernaculumcharacteristics,geographic location,durationofthehibernationseason,andenergetic conditionoftheanimal[15,16,17].Forexample,inastudyof temperaturepreferencesofhibernatingbatsinalimestoneminein Ohio[16],themajorityoflittlebrownbatshibernatedat 7.2 6 2.6 u C(mean 6 SD),althoughsomeindividualshibernated acrossawiderrangeoftemperatures(1.1to16.4 u C)[15].During hibernation,corebodytemperaturesofbatsarenearorslightly aboveambienttemperature.Thus,acommonbodytemperature ofhibernatinglittlebrownbatsofNorthAmerica,approximately 7.2 u C,isbelowthethermaloptimumforgrowthof G.destructans (12.5to15.8 u C). Figure3.MorphologyofGeomycesdestructansvarieswithincubationtemperature. (a)Acharacteristicallybranchedconidiophore followinggrowthatapproximately7 u C.(b)Curvedconidiatypicalofthoseproducedfollowingincubationatapproximately7 u C.(c)Hyphaewere thickened,fragmentedintoarthrospores(arrows),andproducedchlamydospore-likestructures(arrowhead)followingincubationatapproximate ly 12 u C.(d)Conidiawereprimarilypyriformtogloboidinshapeandfrequentlyformedshortchains(arrow)followingincubationatapproximately12 u C. (e)Atelevatedtemperatures(above15 u C),thickened,deformedhyphaeshowedevidenceofdegeneration,andhyphaltipsexhibitedbranched antler-likemorphology(arrow).Chlamydospore-likestructureswerealsocommon(arrowhead).(f)Thickirregularhyphalfragmentswereproduced by coloniesgrownatapproximately18 u C;conidiawerenotobserved.Scalebars,10mm. doi:10.1371/journal.pone.0046280.g003 ThermalPerformanceof Geomycesdestructans PLOSONE|www.plosone.org4September2012|Volume7|Issue9|e46280

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Withintherangeoftemperaturesoccupiedbyhibernatingbats, variationislikelytobebiologicallyimportantforindividual animalsinfectedby G.destructans .Forexample,inlaboratory culture,theNewYorkisolateof G.destructans exhibiteda93% reductioningrowthperformancefromtheupper(approximately 16 u C)tolower(approximately1 u C)temperaturesreportedsuitable forhibernationoflittlebrownbats.Fromthemeanhibernation temperatureusedbythesebats(7.2 u C),toonestandarddeviation belowthemean(4.6 u C)[16],ourlaboratory-basedmeasurements indicatethatgrowthperformanceof G.destructans isreducedby 40%.Ifthesereductionsingrowthperformanceofthefungusare ultimatelyshowntoyieldsimilarreductionstotheseverityof fungalinfectionofhibernatingbats,smallreductionstohibernaculumtemperatures( e.g. ,2to3 u C)couldservetomoderatedisease progressionininfectedanimals. Inadditiontotemperature-dependentvariationingrowth performance,wealsoobservedchangesinmorphologicalcharacteristicsof G.destructans whengrownincultureattemperatures aboveapproximately12 u C.Atypicalcharacteristicsweremost apparentfollowingincubationaboveapproximately15 u C.These characteristicswerenotseeninculturesincubatedatapproximately7 u Cforuptooneyear,indicatingthatobserved morphologicalchangesweretemperature-,notage-dependent. Consistentwithmorphologicalchangesobservedinotherfungal speciesgrownatelevatedtemperatures[18,19,20], G.destructans exhibitedincreasedseptationandthickeningofhyphae,altered conidialshapes,productionofarthrospores,andformationof chlamydospore-likestructures.Hyphaltipsalsoexhibited branchedantler-likemorphologyconsistentwith‘‘favicchandeliers’’characteristicofsomepathogenic Trichophyton species[18]. Although G.destructans exhibitedincreasedgrowthperformance followingincubationatelevatedtemperatures(12to15 u C),the associatedmorphologicalchangesweobservedhavebeenlinkedto stressresponseinotherfilamentousfungi[19,20]. Morphologicalchangesobservedinculturesof G.destructans grownathighertemperaturesmaybebiologicallyimportantfor propagationandpersistenceofthefungusfollowingtransferto warmenvironments.Forexample,underadverseconditions, arthrosporescanbecomeaprimarymeansoffungalpropagation [21,22],andchlamydosporesserveasdesiccation-resistant,resting structures[19].Consequently,ifproductionofthesefungal reproductivestructuresispromoteduponemergenceofinfected batsfromhibernation,theymayfacilitateenhancedpersistenceof G.destructans withinenvironmentsotherthanunderground hibernacula.However,thesealteredreproductivestructuresdo notcontributetofungaldispersalinasrobustamannerasthe abundantmicroconidiaproducedby G.destructans atcooler temperatures.Thus,althoughproductionofchlamydosporesand arthrosporesmayfacilitatesurvivalof G.destructans outsideofbat hibernacula,warmertemperatureseitherinsideoroutsideof hibernaculamaydecreasetheoverallreproductivecapacityof G. destructans .Abetterunderstandingofthebasicbiologyandlife cycleof G.destructans willbeimportantfordefiningthe epidemiologyofWNSacrossthelandscape. Ourlaboratory-basedinvestigationshaverevealedvariationin physiologicalcharacteristicsof G.destructans andhighlightaneedto determinehowthesedifferencesmayinfluenceperformanceand disseminationofthisfungusinnaturalenvironments.Similar phenotypicvariationhasbeendemonstratedamongisolatesof otherfungi,includingtheamphibianpathogen, Batrachochytriumdendrobatidis ,andtheplantpathogen, Phytophthoraramorum ,resulting indifferentialpathogenesisandriskfordiseaseamongsusceptible hostspecies[23,24,25].Currently,littleisknownabouthow growthcharacteristicsandperformanceof G.destructans on artificialmediumcomparetothoseof G.destructans onbatsoras itpersistsorgrowsinassociationwithenvironmentalsubstrates. Likewise,itisunknownhownaturalbodytemperaturepatternsof batsduringhibernation,whichconsistoflongboutsofcold (torpor)interspersedbyshortperiodsofwarmth(inter-torpor arousals),mayaffectgrowthperformanceandothercharacteristics of G.destructans asitcolonizestheirskin.Finally,littleisknown aboutmicroclimateselectionofbatsthroughoutthewinterwithin ahibernaculumandhowmicroclimateselectionvarieswithina speciesacrossitsgeographicrange.Consideringthedemonstrated variationsintemperature-dependentgrowthof G.destructans , microclimateselectionmaybeacriticalfactorintheepidemiology andmanifestationofWNS.Thus,amoredetailedunderstanding ofhowenvironmentalvariationwithinhibernaculainfluences physiologyandgrowthperformanceof G.destructans acrossits geographicrangemaybekeytounderstandingandmanagingthe progressionofWNSinhibernatingbats.MaterialsandMethods FungalisolatesIsolatesof G.destructans usedinthisstudywereculturedduring diagnosticinvestigationsfromskinorhairofbatscollectedinNew York[4](February,2008;AmericanTypeCultureCollection numberATCCMYA-4855),Pennsylvania(March,2009),Virginia(March,2009),Germany[9](March,2009),Hungary[9] (March,2009),andSwitzerland[9](April,2009).GrowthcurvesStarterculturesforgrowthcurveswereinitiatedfromfrozen ( 2 80 u C)glycerolculturestocksbytransfertoSabourauddextrose agarplatessupplementedwithgentamycinandchloramphenicol (SDA)andincubatedat7 u Cforapproximatelyeightweeks.For eachincubationtemperatureevaluated,starterculturematerial wastransferredusingaflame-sterilizedinoculatingneedletothree evenlyspacedlocationsonthesurfaceofeachofsevenSDAplates, yielding21replicatesofeachisolatepertemperaturecondition evaluated.Thishighlevelofcolonyreplicationwasusedto accountforpotentialvariabilityinherentinourmethodof inoculation.Eachgroupofsevenplateswassealedintoaplastic containercontainingmoistpapertowelstomaintainhumidity.A temperaturelogger(iButtonmodel1923,MaximSemiconductor, Dallas,TexasUSA)programmedtorecordtemperatureeveryfive minuteswasmaintainedinonecontainerperincubation temperatureforthedurationofeachgrowthexperiment.WeeklygrowthcurveanalysisIsolatesof G.destructans fromNewYorkandGermanywere incubatedforfiveweeksatninedifferenttemperatureseach.Mean incubationtemperatures(calculatedfromrecordedtemperature logs)overthecourseofthefiveweekgrowthperiodfortheNew Yorkisolatewere0.8,2.0,4.6,7.2,11.9,16.0,17.6,19.0,and 21.4 u C;meanincubationtemperaturesfortheGermanisolate were0.8,2.0,4.6,7.2,11.9,14.7,17.6,19.0,and21.4 u C.The environmentalchambersmaintainedrelativelyconstanttemperatureswithstandarddeviationsrangingfrom0.11to1.3 u C.Aswe lackedsufficientincubatorstotestalltemperaturessimultaneously, astandardizedgrowthcurveanalysis(21coloniesincubatedat approximately7 u C)wasgeneratedintandemwitheachgroupof temperaturesevaluatedtoprovideameanstocomparenonsimultaneousevaluationsofgrowth.Therewerenosignificant differencesingrowthperformanceamongthreetemporally independentgrowthcurvesgeneratedfromculturesincubatedatThermalPerformanceof Geomycesdestructans PLOSONE|www.plosone.org5September2012|Volume7|Issue9|e46280

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approximately7 u CforeithertheNewYorkisolate( P =0.20)or theGermanisolate( P =0.17).Five-weekgrowthcurveanalysisAsecondgrowthperformanceanalysiswascompletedwith isolatesof G.destructans fromPennsylvania,Virginia,Hungary,and Switzerland.Meanincubationtemperaturestestedfortheseisolates were1.9,2.6(Hungarianisolateonly;thisisolatewasnotgrownat 1.9 u C),6.7,12.2,14.6,and17.7 u C.Theenvironmentalchambers maintainedrelativelyconstanttemperatureswithstandarddeviationsrangingfrom0.2to0.4 u C.Forcomparativepurposes,isolates of G.destructans fromNewYorkandGermanyweregrowninparallel andincubatedat6.7,12.2,and17.7 u C.Usingbothfrequentistand Bayesiananalyticaltechniques,theNewYorkandGermanisolates grewfasterduringthesecondexperimentthanthefirst(datanot shown).Thissuggestedthattherewasanundefinedfactorthat affectedoverallgrowthrates,sowecouldnotdirectlycompare growthperformanceoftheseisolatesfromthetwoindependent analyses.Thus,inthesecondanalysis,weonlydirectlycompared theisolatesforwhichwehadgrowthmeasurementsacrossthefull rangeoftemperatures(Pennsylvania,Virginia,Hungary,and Switzerland).However,thisundefinedfactoraffectedonlythe absolutegrowthratesofeachisolateandnottherelativegrowth rateswithinanisolate;thusitshouldnothaveaffectedestimationof Toptandperformancebreadth.MeasurementofgrowthFortheweeklygrowthcurveanalysis,wedeterminedtemperature-dependentgrowthratesoftheNewYorkandtheGerman isolatesbymeasuringtotalsurfaceareaofvisiblegrowth(colony expansion)ofeachcolonyatweeklyintervalsforfiveweeks (referredtothroughoutasweeklyreplicates).Measurementsof eachcolonywerecollectedaftervisiblegrowthwasfirstapparent attheinoculationpoint.Forthefour-isolateanalysis,colonyarea wasanalyzedatweekfiveonly.Ateachmeasurementinterval,we captureddigitalgray-scaleimagesofeachcultureplateusinga FOTO/Analyst H InvestigatorCCDcamerasystem(FOTODYNE Inc.,Hartland,Wisconsin,USA)andImageJVersion1.34S (NationalInstitutesofHealth,Bethesda,Maryland,USA).A metricscalewasincludedineachphotographforcalibration.We calculatedthesurfaceareaofeachcolonyusingimageanalysis software(AnalyzingDigitalImagesVersion11,Museumof Science,Boston,Massachusetts,USA).Adigitalcolormaskwas appliedtoallfungalcoloniesonaplate,andtheareaofmasked pixelscorrespondingtoeachcolonywasmeasuredincm2.Gross colonymorphologywasalsorecorded.AnalysisofgrowthcurvesWeusedacurve-fittingprocedurebasedoninformationcriteria [26]todeterminethefunctionthatbestdescribedtemperaturedependentgrowthratesof G.destructans ateachofthemeasurementsforisolatesintheweeklyanalysisandforthesingle measurementforeachisolateinthefive-weekanalysis.Weused Akaike’sInformationCriterionadjustedforsmallsamplesize (AICc)[27]tocomparerelativefitofsevenfunctions(TableS2): quadratic,Gaussian,modifiedGaussian,Logan1,beta,performance,andBrie `re2[26,28,29,30,31]. Toaccountforpossibleplateeffectsintheinitialexperiment( i.e. , theweeklygrowthcurveanalysis;threecoloniesweregrownon eachplate),weanalyzedrandomlydrawnsubsetsofdata.Tocreate eachsubset,werandomlyselectedthevalueofonecolonyfrom eachplateateachtemperature.Weexcludedanytemperaturesat whichnocoloniesgrew( e.g. ,nocoloniesgrewat21.4 u C,andno coloniesgrewatthecoldesttemperaturesforthefirsttwoweeks) [32];however,weusedthisinformationtoconstrainthevaluesfor criticalminimumormaximumtemperaturesinthefunctionsthat specifiedthoseparameters(Logan1,beta,performance,and Brie `re2).Ifacolonywaschosenthatdidnotgrow,itwasnot includedandthesamplesizeforthatparticularsubsetwasreduced. Ifacolonynevergrew,weexcludedthatcolonyfromsampling,and thesamplesizesforallsubsetswerereduced.Wemade10,000such subsetsofdataforeachisolateduringeachweek. Wefitthesevencurvestoeachsubsetusingacustom-written scriptbasedontheLSQCURVEFITfunctioninMATLAB Version7.12(TheMathWorks,Inc.,Natick,Massachusetts,USA). Forsomerandomsubsets,especiallyduringthefirsttwoweeksof incubationwhengrowthwaspoor,someoftheresultingcurvesdid notshowthecharacteristicintermediatepeakandwerethus excluded.Todoso,weremovedanycurveforwhichthesecond derivativewaspositive,orforwhichthefirstderivatewasnegative belowthepeakorpositiveabovethepeak.Thisensuredthatonly curveswiththecorrectshapewereincluded.Wethenaveraged parameterandAICcvaluesacrosstheremainingsubsetsineach analysis.Wechosethebest-fitcurveforeachanalysisbasedon averageAICcvalues.Anycurveswith D AICc , 2wereconsidered equallyparsimonious.Iflessthan50%ofsubsetswerefitproperly byagivenfunction,thatfunctionwasremovedfromconsideration asthebestfit.Wecalculatedtwocommondescriptivecharacteristicsforeachsubset,thermaloptimum(Topt)and80% performancebreadth,andaveragedthecharacteristicstogeta valueforeachcurve.Wethenaveragedparameterestimates acrossthefunctionsforwhich50%ofthesubsetsweregoodfitsto calculateasinglevalueforeachcharacteristicineachisolateor weeklymeasurement.AsperstandardAICtechnique[26],we weightedtheparameterestimatesbyAICcweightssothatbetter fittingmodelsmorestronglyaffectedtheaverageparameter estimatethandidweakermodels.The‘‘optimaltemperature’’we estimateddescribesthe‘‘optimaltemperatureformaximum growth,’’butweuseToptforconsistencywithliteratureinthe field.Incontextofourmeasurements,optimaltemperatureisonly optimalforgrowthonSDAandnotnecessarilyforreproduction orfitness.Theuppercriticaltemperaturesforgrowthdescribedfor G.destructans arerepresentedonlyforthosefunctionsthatprovide explicitestimatesofthisparameterandarethereforeexpressedas arangeratherthananaverage. Tofurtherevaluateinitialdifferencesingrowthcharacteristics observedbetweenisolatesineachexperiment,weconducted severaladditionalanalyses.Fortheweeklygrowthcurveanalysis, wedeterminedifthetwoisolatesfollowing35daysofincubation werebestdescribedusingtwoseparatecurvesorasinglecurve. Forthefive-weekgrowthcurveanalysis,wedeterminedifisolates werebestdescribedusingseparatecurves,onecurveforeach continent,oronecurveforallisolatescombined.Asobservedplate effectsintheinitialanalysiswereminimal,weassumedequalplate effectsacrossallreplicatesforthisanalysis,andwefitcurves directlytothedatawithoutperformingtherandomization procedureonsubsetsofdataasdescribedabove.Theorderof bestfitmodelsdifferedslightlyusingthistechniquefromtheorder obtainedbytherandomizationprocedure,butparametervalues foreachcurveweresimilar.WealsocomparedtheToptand performancebreadthsineachexperiment[33].Asabove,weused growthmeasurementscollectedafter35daysofincubation.We evaluatedoverlapofthesecharacteristicsinthe84%confidence intervals,whichresultsinaTypeIerrorrateof0.05[33,34].ObservationofmorphologyMicroscopiccharacteristicsofeachfungalisolatewereexaminedfollowing8weeksincubationatapproximately4,12,15,andThermalPerformanceof Geomycesdestructans PLOSONE|www.plosone.org6September2012|Volume7|Issue9|e46280

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18 u C.Priortoexamination,fungalelementswerestainedusing 1%Phloxineorlactophenol-cottonblue.Digitalimageswere capturedusingaLeicaDM2500microscope(LeicaMicrosystems, GmbH,Wetzlar,Germany)withRetiga2000RFast1394camera (QImaging,Surrey,BritishColumbia,Canada).Averagedimensionsofhyphaeandconidiaateachincubationconditionwere calculatedfrommeasurementsoftenrandomlyselectedstructures asdeterminedusingImage-ProAnalysisVersion6.0(Media Cybernetics,Inc.,Bethesda,Maryland,USA).SupportingInformationTableS1Descriptivestatisticsandparametervaluesforbest-fit functionsofeachisolate. (DOCX)TableS2Mathematicalformulasofthesevenfunctionsusedfor analyses. (DOCX)AcknowledgmentsWethankD.Lindner(USForestService)forprovidingtechnical assistance.Useoftrade,product,orfirmnamesisfordescriptivepurposes onlyanddoesnotimplyendorsementbytheUSGovernment.AuthorContributionsConceivedanddesignedtheexperiments:MLVJGBDSB.Performedthe experiments:MLVDSB.Analyzedthedata:JGBWWMLV.Contributed reagents/materials/analysistools:GW.Wrotethepaper:MLVJGBDSB.References1.BlehertDS,HicksAC,BehrMJ,MeteyerCU,Berlowski-ZierBM,etal.(2009) Batwhite-nosesyndrome:anemergingfungalpathogen?Science323:227. 2.LorchJM,MeteyerCU,BehrMJ,BoylesJG,CryanPM,etal.(2011) Experimentalinfectionofbatswith Geomycesdestructans causeswhite-nose syndrome.Nature480:376–378. 3.WarneckeL,TurnerJM,BollingerTK,LorchJM,MisraV,etal.(2012) InoculationofbatswithEuropean Geomycesdestructans supportsthenovel pathogenhypothesisfortheoriginofwhite-nosesyndrome.ProcNatlAcadSci USA109:6999–7003. 4.GargasA,TrestMT,ChristensenM,VolkTJ,BlehertDS(2009) Geomyces destructans sp.nov.asssociatedwithbatwhite-nosesyndrome.Mycotaxon108: 147–154. 5.ChaturvediV,SpringerDJ,BehrMJ,RamaniR,LiX,etal.(2010) Morphologicalandmolecularcharacterizationsofphychrophilicfungus Geomyces desctructans fromNewYorkbatswithwhitenosesyndrome(WNS).PLoSOne5: e10783. 6.MeteyerCU,BucklesEL,BlehertDS,HicksAC,GreenDE,etal.(2009) Histopathologiccriteriatoconfirmwhite-nosesyndromeinbats.JVetDiagn Invest21:411–414. 7.FrickWF,PollockJF,HicksAC,LangwigK,ReynoldsDS,etal.(2010)An emergingdiseasecausesregionalpopulationcollapseofacommonNorth Americanbatspecies.Science329:679–682. 8.PuechmailleSJ,WibbeltG,KornV,FullerH,ForgetF,etal.(2011)PanEuropeandistributionofwhite-nosesyndromefungus( Geomycesdestructans )not associatedwithmassmortality.PLoSOne6:e19167. 9.WibbeltG,KurthA,HellmannD,WeishaarM,BarlowA,etal.(2010)Whitenosesyndromefungus( Geomycesdestructans )inbats,Europe.EmergingInfectDis 16:1237–1243. 10.Mart ´ nkova ´N,Bac korP,Bartonic kaT,Blaz kova ´P,C erveny ´J,etal.(2010) Increasingincidenceof Geomycesdestructans fungusinbatsfromtheCzech RepublicandSlovakia.PLoSOne5:e13853. 11.PuechmailleSJ,VerdeyrouxP,FullerH,GouilhMA,BekaertM,etal.(2010) White-nosesyndromefungus( Geomycesdestructans )inbat,France.Emerging InfectDis16:290–293. 12.PikulaJ,BandouchovaH,NovotnyL,MeteyerCU,ZukalJ,etal.(2012) Histopathologyconfirmswhite-nosesyndromeinbatsinEurope.JWildlDis48: 207–211. 13.PuechmailleSJ,FrickWF,KunzTH,RaceyPA,VoigtCC,etal.(2011)Whitenosesyndrome:isthisemergingdiseaseathreattoEuropeanbats?TrendsEcol Evol26:570–576. 14.FloryAR,KumarS,StohlgrenTJ,CryanPM(2012)Environmentalconditions associatedwithbatwhite-nosesyndromemortalityinthenorth-easternUnited States.JApplEcol. 15.BoylesJG,DunbarMB,StormJJ,BrackVJr(2007)Energyavailability influencesmicroclimateselectionofhibernatingbats.JExpBiol210:4345– 4350. 16.BrackVJr(2007)Temperaturesandlocationsusedbyhibernatingbats, including Myotissodalis (Indianabat),inalimestonemine:implicationsfor conservationandmanagement.EnvironManage40:739–746. 17.TwenteJWJr(1955)Someaspectsofhabitatselectionandotherbehaviorof cavern-dwellingbats.Ecology36:706–732. 18.St-GermainG,SummerbellR(2011)IdentifyingFungi.Belmont,CA:Star Publishing. 19.LinX,HeitmanJ(2005)Chlamydosporeformationduringhyphalgrowthin Crypococcusneoformans .EukaryoticCell4:1746–1754. 20.RobsonGD,PuciJ,ThrondsetW,Dunn-ColemanN(2008)Chapter9 Oxidativestress,fragmentationandcelldeathduringbioreactorgrowthof filamentousfungi.In:SimonV.AveryMS,PieterVanW,editors.British MycologicalSocietySymposiaSeries:AcademicPress.pp.129–142. 21.RashidA(2001)Arthroconidiaasvectorsofdermatophytosis.Cutis67:23. 22.YazdanparastSA,BartonRC(2006)Arthroconidiaproductionin Trichophyton rubrum andanewexvivomodelofonychomycosis.JMedMicrobiol55:1577– 1581. 23.KasugaT,KozanitasM,BuiM,Hu ¨berliD,RizzoDM,etal.(2012)Phenotypic diversificationisassociatedwithhost-inducedtransposonderepressioninthe SuddenOakDeathpathogen Phytophthoraramorum .PlosOne7:e34728. 24.PiotrowskiJS,AnnisSL,LongcoreJE(2004)Physiologyof Batrachochytrium dendrobatidis,achytridpathogenofamphibians.Mycologia96:9–15. 25.BrasierC(2003)SuddenOakDeath: Phytophthoraramorum exhibitstransatlantic differences.MycolRes107:257–259. 26.AngillettaMJ(2006)Estimatingandcomparingthermalperformancecurves. JThermBiol31:541–545. 27.BurnhamKP,AndersonDR(2002)Modelselectionandmultimodalinference:a practicalinformation-theoreticapproach.NewYork:Springer. 28.ShiP,GeF(2010)Acomparisonofdifferentthermalperformancefunctions describingtemperature-dependentdevelopmentrates.JThermBiol35:225– 231. 29.LoganJA,WolkindDJ,HoytSC,TanigoshiLK(1976)Ananalyticmodelfor descriptionoftemperaturedependentratephenomenainarthropods.Environ Entomol5:1133–1140. 30.BriereJF,PracrosP,LeRouxAY,PierreJS(1999)Anovelratemodelof temperature-dependentdevelopmentforarthropods.EnvironEntomol28:22– 29. 31.HueyRB,StevensonRD(1979)Integratingthermalphysiologyandecologyof ectotherms:adiscussionofapproaches.AmZool19:357–366. 32.OksanenJ(1997)Whythebeta-functioncannotbeusedtoestimateskewnessof speciesrichness.JVegSci8:147–152. 33.SchulerMS,CooperBS,StormJJ,SearsMW,AngillettaJMJ(2011)Isopods failedtoacclimatetheirthermalsensitivityoflocomotorperformanceduring predictableorstochasticcooling.PLoSOne6:e20905. 34.PaytonME,GreenstoneMH,SchenkerN(2003)Overlappingconfidence intervalsorstandarderrorintervals:whatdotheymeanintermsofstatistical significance?JInsectSci3:34.ThermalPerformanceof Geomycesdestructans PLOSONE|www.plosone.org7September2012|Volume7|Issue9|e46280


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