Pan-European Distribution of White-Nose Syndrome Fungus (Geomyces destructans) Not Associated with Mass Mortality


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Pan-European Distribution of White-Nose Syndrome Fungus (Geomyces destructans) Not Associated with Mass Mortality

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Title:
Pan-European Distribution of White-Nose Syndrome Fungus (Geomyces destructans) Not Associated with Mass Mortality
Series Title:
PLOS One
Creator:
Puechmaille, Sebastien J.
Wibbelt, Gudrun
Korn, Vanessa
Fuller, Hubert
Forget, Frédéric
Mühldorfer, Kristin
Kurth, Andreas
Bogdanowicz, Wieslaw
Borel, Christophe
Bosch, Thijs
Cherezy, Thomas
Drebet, Mikhail
Görföl, Tamás
Haarsma, Anne-Jifke
Herhaus, Frank
Hallart, Guénael
Hammer, Matthias
Jungmann, Christian
Le Bris, Yann
Lutsar, Lauri
Masing, Matti
Mulkens, Bart
Passior, Karsten
Starrach, Martin
Wojtaszewski, Andrzej
Zöphel, Ulrich
Teeling, Emma C.
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English

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Mass Mortalities ( local )
Wns ( local )
Hibernating Bats ( local )
Northeastern America ( local )
G. Destructans ( local )
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serial ( sobekcm )

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Abstract:
The dramatic mass mortalities amongst hibernating bats in Northeastern America caused by “white nose-syndrome” (WNS) continue to threaten populations of different bat species. The cold-loving fungus, Geomyces destructans, is the most likely causative agent leading to extensive destruction of the skin, particularly the wing membranes. Recent investigations in Europe confirmed the presence of the fungus G. destructans without associated mass mortality in hibernating bats in six countries but its distribution remains poorly known. We collected data on the presence of bats with white fungal growth in 12 countries in Europe between 2003 and 2010 and conducted morphological and genetic analysis to confirm the identity of the fungus as Geomyces destructans. Our results demonstrate the presence of the fungus in eight countries spanning over 2000 km from West to East and provide compelling photographic evidence for its presence in another four countries including Romania, and Turkey. Furthermore, matching prevalence data of a hibernaculum monitored over two consecutive years with data from across Europe show that the temporal occurrence of the fungus, which first becomes visible around February, peaks in March but can still be seen in some torpid bats in May or June, is strikingly similar throughout Europe. Finally, we isolated and cultured G. destructans from a cave wall adjacent to a bat with fungal growth. G. destructans is widely found over large areas of the European continent without associated mass mortalities in bats, suggesting that the fungus is native to Europe. The characterisation of the temporal variation in G. destructans growth on bats provides reference data for studying the spatio-temporal dynamic of the fungus. Finally, the presence of G. destructans spores on cave walls suggests that hibernacula could act as passive vectors and/or reservoirs for G. destructans and therefore, might play an important role in the transmission process.

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k26.4854 ( USFLDC: LOCAL Handle )

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Pan-EuropeanDistributionofWhite-NoseSyndrome Fungus(Geomycesdestructans)NotAssociatedwith MassMortalitySe ´ bastienJ.Puechmaille1,2*.,GudrunWibbelt3 .,VanessaKorn4,HubertFuller1,Fre ´ de ´ ricForget5,Kristin Mu ¨ hldorfer3,AndreasKurth6,WieslawBogdanowicz7,ChristopheBorel8,ThijsBosch9,Thomas Cherezy10,MikhailDrebet11,Tama ´ sGo ¨ rfo ¨ l12,Anne-JifkeHaarsma13,FrankHerhaus14,Gue ´ naelHallart15, MatthiasHammer16,ChristianJungmann17,YannLeBris18,LauriLutsar19,MattiMasing20,Bart Mulkens21,KarstenPassior22,MartinStarrach23,AndrzejWojtaszewski24,UlrichZo ¨ phel25,EmmaC. Teeling1,21 SchoolofBiologyandEnvironmentalScience,UniversityCollegeDublin,Dublin,Ireland, 2 ConwayInstituteofBiomolecularandBiomedicalResearch,Dublin,Ireland, 3 LeibnizInstituteforZooandWildlifeResearch,Berlin,Germany, 4 OfficeforFaunisticandLandscapeEcology,Scho ¨ neberg,Germany, 5PlecotusWorkingGroup, AssociationNatagora,Brussels,Belgium, 6 RobertKochInstitute,Berlin,Germany, 7 MuseumandInstituteofZoology,PolishAcademyofSciences,Warszawa,Poland, 8 CommissiondeProtectiondesEaux,duPatrimoine,del’Environnement,duSous-soletdesChiropte ` res–Lorraine,Velaine-en-Haye,France, 9 DutchBatWorkersGroup, Nijmegen,TheNetherlands, 10 CoordinationMammalogiqueduNorddelaFrance,Be ´ thune,France, 11 PodilskiTovtryNationalNaturePark,Kamenets-Podilsky,Ukraine, 12 NatureConservationFoundationofTolnaCounty,Szeksza ´ rd,Hungary, 13 CentreforEcosystemStudies,AlterraandWageningenUniversity,Wageningen,The Netherlands, 14 BiologyStationOberberg,Nu ¨ mbrecht,Germany, 15 Socie ´ te ´ d’EtudeetdeProtectiondelaNatureenThie ´ rache,LeChaudron,Origny-en-Thie ´ rache, France, 16 DepartmentofBiology,CenterforBatConservationinNorthernBavaria,ErlangenUniversity,Erlangen,Germany, 17 NatureandBiodiversityConservation UnionRhineland-Palatine,Birkenfeld,Germany, 18 BretagneVivanteSEPNB,Roussimel,Gle ´ nac,France, 19 EstonianFundforNature,Tartu,Estonia, 20 Sicista DevelopmentCentre,Tartu,Estonia, 21 BatWorkingGroup,NatuurpuntVZW,Belgium, 22 NatureandBiodiversityConservationUnionSouthernLower-Saxony, Nordstemmen,Germany, 23 BiotopeMappingCooperation,Herford,Germany, 24 InstituteofNaturalFibresandMedicinalPlants,Poznan,Poland, 25 SaxonianState OfficeforEnvironmentAgricultureandGeology,Dresden-Pillnitz,GermanyAbstractBackground:ThedramaticmassmortalitiesamongsthibernatingbatsinNortheasternAmericacausedby‘‘whitenosesyndrome’’(WNS)continuetothreatenpopulationsofdifferentbatspecies.Thecold-lovingfungus, Geomycesdestructans , isthemostlikelycausativeagentleadingtoextensivedestructionoftheskin,particularlythewingmembranes.Recent investigationsinEuropeconfirmedthepresenceofthefungus G.destructans withoutassociatedmassmortalityin hibernatingbatsinsixcountriesbutitsdistributionremainspoorlyknown.Methodology/PrincipalFindings:Wecollecteddataonthepresenceofbatswithwhitefungalgrowthin12countriesin Europebetween2003and2010andconductedmorphologicalandgeneticanalysistoconfirmtheidentityofthefungusas Geomycesdestructans .Ourresultsdemonstratethepresenceofthefungusineightcountriesspanningover2000kmfrom WesttoEastandprovidecompellingphotographicevidenceforitspresenceinanotherfourcountriesincludingRomania, andTurkey.Furthermore,matchingprevalencedataofahibernaculummonitoredovertwoconsecutiveyearswithdata fromacrossEuropeshowthatthetemporaloccurrenceofthefungus,whichfirstbecomesvisiblearoundFebruary,peaksin MarchbutcanstillbeseeninsometorpidbatsinMayorJune,isstrikinglysimilarthroughoutEurope.Finally,weisolated andcultured G.destructans fromacavewalladjacenttoabatwithfungalgrowth.Conclusions/Significance:G.destructans iswidelyfoundoverlargeareasoftheEuropeancontinentwithoutassociated massmortalitiesinbats,suggestingthatthefungusisnativetoEurope.Thecharacterisationofthetemporalvariationin G. destructans growthonbatsprovidesreferencedataforstudyingthespatio-temporaldynamicofthefungus.Finally,the presenceof G.destructans sporesoncavewallssuggeststhathibernaculacouldactaspassivevectorsand/orreservoirsfor G.destructans andtherefore,mightplayanimportantroleinthetransmissionprocess.Citation: PuechmailleSJ,WibbeltG,KornV,FullerH,ForgetF,etal.(2011)Pan-EuropeanDistributionofWhite-NoseSyndromeFungus( Geomycesdestructans ) NotAssociatedwithMassMortality.PLoSONE6(4):e19167.doi:10.1371/journal.pone.0019167 Editor: Raphae ¨ lArlettaz,UniversityofBern,Switzerland Received November30,2010; Accepted March21,2011; Published April27,2011 Copyright: 2011Puechmailleetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: ThisprojectwassupportedbyagrantfromthePresidentofIrelandYoungResearcherAward,ScienceFoundationIrelandtoE.C.T.,anIRCSET-Marie CurieInternationalMobilityFellowshipsinScience,EngineeringandTechnologytoS.J.PandagrantfromtheClara-Samariter-Foundation,German y,toG.W.and K.M.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript. CompetingInterests: Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:s.puechmaille@gmail.com . Theseauthorscontributedequallytothiswork. PLoSONE|www.plosone.org1April2011|Volume6|Issue4|e19167

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IntroductionWhitenose-syndrome(WNS)isadevastatingdiseasecausing massmortalitiesinhibernatingbatsinNorth-America.InMay 2009,itwasestimatedthatoveronemillionbatshaddiedfromthe diseasewhichwasfirstdocumentedinFebruary2006atHowe’s Cave,WestofAlbany,NewYork[1].Avisuallyconspicuouswhite fungusgrowsontheface,ears,orwingsofstrickenbatswith hyphaepenetratingdeepintotheconnectivetissueofglabrousskin andsnout[2]andcausingseveredamage[3].Thefungus associatedwithWNSisanewlydescribed,psychrophilic(coldloving)species( Geomycesdestructans )[4],closelyrelatedtoother psychrophilicspeciesof Geomyces [5,6].Althoughitisnotyet conclusivelyprovenwhether G.destructans isthecausativeagentof thediseaseorifotherco-factorsarenecessaryfordiseasetooccur, thefungusisalwaysfoundonbatsatWNSsiteswherehibernating batsexperiencemassmortalities[7].Todate,bacteriological, virological,parasitologicalandpathologicalevaluationsaswellas studiesoftoxiccontaminantshavenotidentifiedtheconsistent presenceofanyotheragents/causeofdeath.Thelackofevidence fortheinvolvementofotheragentsorcompoundsreinforcesthe suspicionthat G.destructans isthecausativeagentofWNSmortality [2,7,8,9]. Geomycesdestructans hasbeenfoundinninespeciesofbatsin North-America,fromtheprovincesofOntarioandQuebecin CanadasouthandwesttothestatesofTennesseeandOklahoma intheUSA[10].Threerecentstudiesinvestigatingsamples collectedin2008–2010haveshownthat G.destructans wasalso presentinsixEuropeancountries(France,Germany,Switzerland, CzechRepublic,Slovakia&Hungary)[6,11,12].Nevertheless,the geographiccoverageofthesestudieswaslimitedandtheextentof thedistributionof G.destructans inEuroperemainspoorlyknown. Inthispaper,wecombinepreviouslypublisheddataonthe distributionof G.destructans inEurope[6,11,12]withnewdata fromtwelvecountriescovering2,400kmfromWesttoEast (FrancetoTurkey)and1,900kmfromNorthtoSouth(Estoniato Turkey)todemonstratethewidespreadpresenceof G.destructans onmultiplespeciesofhibernatingbatsinEuropewithout associatedmassmortality.Results Reviewofdataon Geomycesdestructans inEuropean bats,2008–2010Althoughphotographsofbatswithfungalgrowthsimilarto G. destructans werepublishedinGermanyinthe1980 9 s[13],andalso takeninthe1990 9 sintheCzechRepublic[11],therehavebeen noconfirmedrecordsof G.destructans inEuropepriorto2008 [6,12].In2010, G.destructans hasbeenconfirmedbymorphological andgeneticanalysesfromsamplescollectedduringthewinters 2007/2008,2008/2009and2009/2010insixEuropeancountries [6,11,12].InFrance,Hungary,SwitzerlandandSlovakia,the fungushasbeenconfirmedfrom1–2location(s)percountry, whereasithasbeenconfirmedat8sitesinGermanyand23sitesin theCzechRepublic[6,11,12].Allconfirmeddetectionsof G. destructans inEuropehavebeenmadebyisolatingand/or geneticallyidentifyingthefungusfromhairs,swabsortouch imprintsfrombats[6,11,12].InEurope,eightspeciesof Myotis havebeenobservedbeingcolonisedby G.destructans : M.myotis , M. blythii (referredtoas M.oxygnathus in[12]), M.mystacinus , M. daubentonii , M.dasycneme , M.nattereri , M.bechsteinii and M.brandtii . Speciesfromotherbatfamilieswerepresentinthecaveswith infectedindividuals(e.g.Miniopteridae: Miniopterusschreibersii ; Rhinolophidae: Rhinolophushipposideros and R.ferrumequinum ),but no G.destructans hasbeenconfirmedfromthesespecies.Previous extensivesurveysofcavefungiinEurope(i.e.[14,15,16])orfungi associatedwithinsectshibernatinginundergroundsites[17]never reported G.destructans intheirinventory,althoughsomeother speciesof Geomyces wererecovered[14,15,16].Newdataon G.destructans inEurope2003–2010Duringwinterhibernationcounts,atotalof107batsfrom56 sitesintwelveEuropeancountrieswerereportedtohavevisible whitefungalgrowth(Tables1,2,3andFigure1).Thisrepresents thefirstrecordsfromninecountries(Austria,Belgium,Denmark, Estonia,TheNetherlands,Poland,Romania,Turkeyand Ukraine).Onehundredandfivebatswerealiveandtwoofthem werefounddeadinhibernacula.These107batsbelongedtoeight differentspeciesof Myotis : M.myotis (59), M.dasycneme (26), M. mystacinus (9), M.daubentonii (4), M.myotis/blythii (3), M.blythii (3), M. nattereri (1), M.escalerai /sp. A (1)and M.brandtii (1).Ofthese, molecularandmorphologicalidentificationsofthecolonising funguswerecarriedoutin23cases(Table1),whileonly photographicevidencewasobtainedforafurther50cases (Table2andFigure2).Theremaining34caseswerebasedon reportsofvisualobservationsofawhitefungalgrowthonbat snoutsand/orears(Table3),whichwasverysimilartopictures presentedinFigure2.All84batsreportedinTables2and3are consideredasGd-suspects(batsshowingfungalgrowththatis thoughttobe G.destructans ).Geomycesdestructans identificationOutofatotalof107batswithfungalgrowth,22weresampled, 16withtouchimprintsand6withcottonswabs.The22bats sampled(20aliveand2dead)belongedtothespeciesof Myotis from which G.destructans hadbeenpreviouslyisolated(seelistabove).In somecases,wewerenotabletodiscriminatebetween M.myotis and M.blythii (referredtoas M.myotis/blythii )aswellasbetweenthe newlyrecognised M.escalerai [18,19]and Myotis sp. A [20],ayet undescribedcrypticspeciesfromthe M.nattereri speciescomplex [18,21].Additionally,swabsampleswerecollectedfromthetunnel wallofanEstonianhibernaculum.Onthe23rdMay2010,a M. brandtii wasobservedinthishibernaculumwithwhitefungalgrowth onitssnout(Figure2A)butnosamplewascollectedatthetime. Whenthesitewasrevisitedforsamplecollectiononthe1stofJune 2010,thebathadleftthesitesosampleswerecollectedbyswabbing thewallofthetunnelwherethebatwasseenninedaysbefore.Four cottonswabswereusedtosampledifferentareasafewcentimetres aroundthelocationwherethebatwasobserved.Thefourswabs werethenstreakedontofourSabouraud’sagarplateseachand monitoredregularlytophysicallyremoveanyfungalgrowththat wasnotsimilarto G.destructans .Althoughtheamountoffungivaried perswabsample, G.destructans wasrecoveredfromallfourswabs, henceforthconsideredasasinglesample,bringingthetotalof samplesanalysedto23.Nomassmortalitywasreportedatanyof thesitesinvestigated. Outof23samplesinvestigatedinthelaboratory,14ofthe16 touchimprintsamplespresentedcharacteristicconidiawhen observedunderamicroscopeandtwoofthemweredoubtful; noneofthecottonswabswereinspectedunderamicroscopeprior toculture.Culturesfrom22ofthesesamplesweresuccessful.The twodeadbatsinvestigateddidnotrevealthepresenceof G. destructans butotherfungalspeciessuchas Mucor sp.and Cladosporium sp.wereidentified(datanotshown). DNAwasisolatedfromthe22culturesofwhich20showed morphologicalsimilarityto G.destructans (e.g.curvedconidia)and fromonetouchimprintwithunsuccessfulcultureattempts. AmplificationandsequencingoftheinternaltranscribedspacerGeomycesDestructansWidespreadinEurope PLoSONE|www.plosone.org2April2011|Volume6|Issue4|e19167

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(ITS)region(ITS1,5.8S,andITS2)waspreferredoverthesmall subunit(SSU)rDNAasitwasshowntobemoreinformativeand wascomparabletobothEuropean[6,12]andNorthAmerican G. destructans [7,22].Allsequencesobtainedwereidenticaland showed100%similaritywithpreviouslypublishedITSsequences of G.destructans availableonGenBank(retrievedonOctober13th) [6,7,12,22].Seasonaldistributionof G.destructansThemonitoringofonesiteovertwoconsecutivewintersshowed anabsenceofGd-suspectbatsfromSeptemberuntiltheendof January(FigureS1).ThefirstGd-suspectbatswerereportedin Februaryeachyear(16/02/2007and07/02/2008)andtheir numberspeakedinMarch(Figure3).InApril,thetotalnumberof batsandthenumberofGd-suspectbatsdecreasedasbatsleftthe hibernacula.However,asthenumberofGd-suspectbats decreasedmoreslowlythanthetotalnumbersofbats,thehighest prevalencewasobservedinApril(FigureS1).Prevalencevaried betweenyearsforthesameperiodoftheyearandreachedvalues intherangeof18–25%in2007(14th–28thMarch)or28–55%in 2008(13th–28thMarch)whenthenumbersofGd-suspectbatsare atthehighest.Thedistributionsofreportedcasesweresimilar betweenthetwoyears,althoughmorecaseswerereportedinApril inthewinter2007/2008(Figure3).InApril2008,themonitoring ofthreemarkedbatswithwhitefungalgrowthclearlyshowedthat afterabathadchangeditspositionwithinthehibernaculumor whenitwasleavingthehibernaculum,thevisiblewhitefungal growthdisappeared(Figure4),mostlikelyasaresultofselfgrooming. ThetemporaldistributionofreportedcasesofliveGd-suspect batsfromthroughoutEurope(thisstudy,n=105)wascombined withinformationavailablefrompreviouslyreportedcasesof G. destructans [6,12](n=22)toinvestigatetheseasonalvariation acrossmultiplesitesinEurope.Thetemporalrangeofreported casesofGd-suspectlivebatsandbatsconfirmedwith G.destructans (n=127)wasnotevenlydistributedthroughoutthewinter/ spring,withabout2/3rdofthecasesreportedinMarch(81/127; Figure3).Thenumberofreportedcasesmorethandoubled betweenFebruary(30cases)andMarch(81cases).Theearliest casewasreportedonJanuary17thfromBelgiumandthethree latestcaseswereobservedonMay23rdinEstonia,inJune24thin theNetherlandsandJune25thinFrance(Tables1,2,3, Figures2Aand2O).Discussion Presenceof G.destructans inEuropeG.destructans wasfirstidentifiedinEuropein2008–2009[6,12] butincreasingphotographicevidencesuggestthatthefunguswas presentinEuropewellbeforethisdate(thisstudy,[11,13]).Most previousstudiesinvestigatingfungiinEuropeancaves,including batguano[14,15,23,24]reported Geomyces species,butnonehad Table1. Confirmedrecordsof Geomycesdestructans onhibernatingbatsinEuropeanddetailsofthecultureandgeneticanalyses.CountryLatLonDateHostspeciesCulturePCRGenBankNo. France*49.94.104/03/2010 Myotismystacinus GuH-04032010{ n/a France*50.62.522/02/2010 Myotisnattereri ThC-22022010{ n/a France{47.7-2.104/03/2010 Myotismyotis Mmyo-FR-1 + JF502415 Belgium49.85.303/04/2010 Myotismyotis Mmyo-BE-1 + JF502414 Belgium{50.85.618/03/2010 Myotismystacinus Mmys-BE-1 + JF502407 Belgium{50.85.618/03/2010 Myotismystacinus n/a + n/a Netherlands{52.05.809/03/2010 Myotisdaubentonii Mdau-NL-1 + JF502411 Netherlands52.14.327/02/2010 Myotisdasycneme Mdas-NL-1 + JF502410 Germany{49.77.410/03/2010 Myotismyotis Mmyo-DE-12 + JF502401 Germany49.89.622/03/2010 Myotismyotis Mmyo-DE-14 + JF502403 Germany50.713.720/03/2010 Myotismyotis Mmyo-DE-13 + JF502402 Germany50.97.518/04/2009 Myotismyotis Mmyo-DE-10 + JF502399 Germany51.28.121/03/2010 Myotismystacinus Mmys-DE-2 + JF502409 Germany51.28.121/03/2010 Myotismystacinus Mmys-DE-3 + n/a Germany51.28.107/03/2010 Myotismyotis Mmyo-DE-11 + JF502400 Germany51.28.107/03/2010 Myotismyotis Mmyo-DE-16 + n/a Germany{52.39.523/03/2010 Myotismyotis Mmyo-DE-15 + JF502404 Germany{52.39.423/03/2010 Myotismystacinus Mmys-DE-1 + JF502408 Hungary47.117.624/03/2010 Myotismyotis Mmyo-HU-2 + JF502405 Hungary47.117.624/03/2010 Myotismyotis Mmyo-HU-3 + n/a Poland50.816.707/03/2010 Myotismyotis Mmyo-PL-1 + JF502413 Estonia# , {59.324.601/06/2010 Myotisbrandtii EsT-01062010 + JF502412 Ukraine48.726.617/03/2010 Myotismyotis Mmyo-UA-1 + JF502406 *Deadbat.#Environmentalsample(individualobserved23/05/2010;seetextforfurtherexplanations).{PhotographofthebatshowninFigure2. { Sampleswerenegativefor G.destructans butamplifiedanotherfungus. doi:10.1371/journal.pone.0019167.t001 GeomycesDestructansWidespreadinEurope PLoSONE|www.plosone.org3April2011|Volume6|Issue4|e19167

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curvedconidiasofartypicalof G.destructans .IntheCzech Republic,Kuba ´tova ´&Dvor a ´k[17]investigatedfungiassociated withinsectshibernatinginundergroundsitesbutdidnotfind Geomyces species.Toourknowledge,onlyonestudyinEuropehas investigatedfungipresentinbats’skinandhairsampleswhere, basedonourcurrentknowledge, G.destructans ismostlikelytobe found.Duringthewinter1999/2001,Larcheretal.[25]collected 25samplesofhairandskinswabsfromsixspecies,includingthree Myotismyotis ,butdidnotfindany Geomyces species.Itisimportant tonotethatmostfungalcultureshavebeencarriedoutat temperaturesabove24–25 u C,temperaturesatwhich G.destructans doesnotgrow[4,22],whichcouldexplainwhyalthoughpresent, thisfungalspecieshadneverbeenreportedinEuropebeforethe studyofPuechmailleetal.[6]. CombiningpreviouslypublisheddatafromFrance,Germany, Switzerland,Hungary,TheCzechRepublicandSlovakia [6,11,12],additionaldatacollectedfromFrance,Germanyand Hungary(thisstudy),andnewdatafromBelgium,TheNetherlands,Poland,EstoniaandUkraine(thisstudy),wedemonstrate herethat G.destructans iswidespreadinEurope.Weconsiderthe photographicevidenceofbatswithwhitefungusmatchingthe characteristicgrowthpattern(e.g.Figure2;picturesfrom RomaniaandTurkey)tomostlikelyrepresent G.destructans , becausesofaralltestedliveEuropeanbatswithsuchwhitefungal growthontheirnose,similartoFigure2,havebeenconfirmedto carrythatspeciesoffungus.Thesefindingsfurthersupportthefact that G.destructans iswidespreadacrossEurope.However,to confirmthepresenceof G.destructans inEuropepriorto2008, historicalcollectionsofbatspecimens(orcavesoilsamples), especiallyspecimenscollectedduringthehibernationperiod, shouldbescreenedforthefungus. AsdepictedinFigure1,mostcasesofbatswith G.destructans (confirmedandsuspected)havebeenfoundfromNorth-eastern FrancethroughBelgium,TheNetherlands,Germanyandthe CzechRepublic.However,itisnotclearwhetherthispattern reflectsanactualhigheroccurrenceand/orprevalenceofthefungus intheseregionsorifitisatleastpartlyduetosamplingbias, wherebythefungusismorelikelytobedetectedinregionswitha highernumberofundergroundsitesvisitedeverywinterorin Table2. Suspectedphotographicrecordsof Geomyces destructans onhibernatingbatsinEurope.CountryLat.Lon.DateHostspeciesNo.Individual France44.81.625/04/2008 Myotismyotis 1 France{42.62.226/06/2010 Myotisescalerai/sp.A 1 France47.7 2 2.104/03/2010 Myotismyotis 1 France{45.02.013/02/2010 Myotismyotis 2 France47.36.204/03/2010 Myotismyotis 3 France50.43.501/03/2008 Myotismystacinus 1 France47.21.424/02/2010 Myotismyotis 2 Belgium50.85.609/02/2008 Myotisdasycneme 1 Belgium50.85.620/03/2008 Myotisdaubentonii 1 Belgium50.85.617/01/2010 Myotisdasycneme 1 Belgium{50.35.907/03/2010 Myotismyotis 1 Belgium50.85.713/03/2010 Myotisdasycneme 1 Netherlands52.14.326/03/2008 Myotisdasycneme 1 Netherlands52.14.318/02/2008 Myotisdasycneme 1 Netherlands52.05.704/03/2010 Myotismystacinus 1 Denmark{56.49.114/03/2010 Myotisdasycneme 2 Germany51.810.802/02/2008 Myotismyotis 1 Germany51.610.507/02/2010 Myotismyotis 1 Germany51.710.320/03/2010 Myotismyotis 1 Germany{52.39.523/03/2010 Myotismystacinus 1 Germany52.39.523/03/2010 Myotisdasycneme 1 Germany52.18.221/03/2007 Myotisdaubenonii 1 Germany52.18.214/03/2007 Myotisdasycneme 1 Germany52.28.004/02/2008 Myotismyotis 1 Austria{46.816.007/02/2007 Myotismyotis 1 Hungary47.117.624/02/2007 Myotismyotis 1 Hungary47.117.623/02/2009 Myotismyotis 1 Hungary46.218.103/03/2009 Myotismyotis/blythii 1 Hungary48.520.518/02/2010 Myotisblythii 1 Hungary47.117.619/02/2010 Myotisblythii 1 Hungary{47.117.619/02/2010 Myotismyotis 2 Poland{50.816.707/03/2010 Myotismyotis 1 Ukraine { 48.826.613/02/2010 Myotismyotis 1 Ukraine48.826.617/03/2010 Myotismyotis 8 Romania{46.822.629/03/2008 Myotisblythii 1 Romania45.425.214/03/2009 Myotismyotis/blythii 1 Turkey{41.927.922/03/2009 Myotismyotis/blythii 1 { PhotographofthebatshowninFigure2. doi:10.1371/journal.pone.0019167.t002 Table3. Suspectedvisualrecordsof Geomycesdestructans on hibernatingbatsinEurope.CountryLat.Lon.DateHostspeciesNo.Individual France49.16.606/04/2009 Myotismyotis 1 France48.56.928/02/2009 Myotismyotis 1 France48.37.129/03/2009 Myotismyotis 1 France48.35.716/03/2008 Myotismyotis 1 France47.96.803/03/2010 Myotismyotis 2 France49.55.204/03/2010 Myotismyotis 1 France48.90.306/02/2010 Myotismyotis 1 France47.25.720/02/2010 Myotismyotis 3 Netherlands52.14.310/03/2005 Myotisdasycneme 2 Netherlands52.14.324/06/2006 Myotisdasycneme 1 Netherlands52.14.307/03/2007 Myotisdasycneme 1 Netherlands52.14.315/03/2008 Myotisdasycneme 3 Netherlands52.14.330/03/2008 Myotisdasycneme 2 Netherlands52.14.305/04/2008 Myotisdasycneme 1 Netherlands52.14.312/04/2008 Myotisdasycneme 1 Netherlands52.14.313/02/2004 Myotisdasycneme 2 Netherlands52.14.305/04/2003 Myotisdasycneme 1 Netherlands52.14.326/03/2008 Myotisdasycneme 1 Netherlands52.14.310/03/2005 Myotisdasycneme 1 Germany50.913.323/03/2010 Myotisdaubentonii 1 Germany49.97.414/03/2010 Myotismyotis 1 Ukraine48.826.617/03/2010 Myotismyotis 4 Romania47.022.408/04/2008 Myotismyotis 1 doi:10.1371/journal.pone.0019167.t003 GeomycesDestructansWidespreadinEurope PLoSONE|www.plosone.org4April2011|Volume6|Issue4|e19167

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regionswerethefungusisspecificallysought.Inouropinion,itis mostlikelythatthislarge-scalepatternisduetoasamplingbias.For example,thelargestnumberofsiteswith G.destructans inany EuropeancountrywasreportedfromtheCzechRepublic(76 localitieswithsuspectedorconfirmed G.destructans )weremostsites havebeensearchedforsignsofthefungus( . 800hibernacula)[11].G.destructans growthonbatsTheclearseasonalpeakinthenumberofobservationsofbats withwhitefungalgrowthindicatesanincreasingprevalenceor detectabilityof G.destructans aswinterpasses.Thissuggeststhat batsmightacquire G.destructans lateduringthehibernationperiod orthatthefungusiscarriedbybatsattheonsetofhibernationbut needstimetodevelopthevisiblewhitefungalgrowthduetothe phenologyofthefungus.Therefore,theabsenceofvisiblewhite fungalgrowthonbatswhenobservedwiththenakedeyemaynot directlyreflecttheabsenceof G.destructans ,butratherjustthe absenceofvisiblefungalcolonies.Furthercomplicatingmatters, ourabilitytodetect G.destructans growthonbatscansubstantially differwithproximitytothebats(i.e.lowceiling versus highceiling) orthelocationofthebat(ceiling versus crevices). OurresultsconfirmthesuggestionofMart ´ nkova ´etal.[11]by showingthatduringthehibernationperiod,batscanremovethe fungusfromtheirsnout,earsandwingstoapointwherethe fungusisnolongervisibletothenakedeye,althoughsomespores mightstillbepresentontheirskin.Duringhibernation,bats arouseeverytwoweeksonaverage[26,27]andifbatsconsistently groomoffthefungusontheseoccasions,ourabilitytovisually detectthefungus,ifpresent,willbeconsiderablyreduced.Wealso showedthattowardstheendofthehibernationperiod,batswere emergingfromthehibernaculumwithoutvisiblesignsofthe fungusdespiteshowingvisiblewhitefungalgrowthfromtwoweeks tofivedaysbeforeleavingthehibernaculum.Itwouldbe importanttoinvestigatewhetherbatscarrysporesoutof hibernaculaandasaresultcouldpossiblycontaminatematernity roostsandmaternitymatesassuggestedbyHallamand McCracken[28].Factorsaffecting G.destructans prevalenceAlthoughitisnotpossibletoclearlyidentifythemechanism responsibleforthesuddenincreaseintheprevalenceof G. destructans inlateFebruaryandMarch,thesedatasuggestthat shorterwinterperiodsshouldbeassociatedwithlowerprevalence. ThispredictionseemstoholdasintheMediterraneanregion, wherehibernationperiodsareshorter[29],nobatswithvisually conspicuousfungalgrowthhaveyetbeenreportedduringwinter cavesurveys.ThecasereportedfromSouthernFrance(June25th2010,Figure2O)wasfoundinthePyreneesmountainsat ca. 1700ma.s.l.andhence,isnotconsideredtypicalofthe Mediterraneanclimate.Itisneverthelesstooearlytoconclude onthisassociationbetween G.destructans prevalenceandthe hibernationduration,asotherfactorswouldneedtobeconsidered suchasforexample,thehighertemperatureobservedin hibernaculaintheMediterraneanregioncomparedtoother regionsinEurope[29].Highertemperaturesinhibernaculahave beenassociatedwithmorefrequentarousalsin Rhinolophus ferrumequinum [30,31,32].Consideringthatthisassociationholds forotherspecies,asaconsequenceofmorefrequentarousals,bats areexpectedtogroommoreoftenandtherefore,reducethe probabilityofavisiblefungalgrowthtodevelop.Moresurveysand strategicsamplingeffortsareneededtouncoverwhetherthe lengthofthehibernationperiodand/orclimaticconditionshavea directorindirecteffectonthegrowthrates,prevalence,and detectabilityof G.destructans onbats. Itiscrucialthatthechangeinprevalenceordetectabilityover thehibernationperiodisconsideredwhencomparingprevalence Figure1.DistributionofconfirmedandsuspectedrecordsofG.destructansonhibernatingbatsinEurope. Dataarepresentedfor geneticallyconfirmedrecordsof G.destructans inred(circles,thisstudy;triangles,publishedrecords),photographicevidenceinyellow,visualreports ingreen.DeadbatsfromNorthernFrancewhichcultureandgeneticanalysisdidnotrevealthepresenceof G.destructans aredepictedasblackdots. Countriesabbreviatednamesareasfollows:AUT:Austria,BEL:Belgium,CHE:Switzerland,CZE:CzechRepublic,DEU:Germany,DNK:Denmark,EST: Estonia,FRA:France,HUN:Hungary,NLD:Netherlands,POL:Poland,ROM:Romania,SVK:Slovakia,TUR:Turkey,UKR:Ukraine. doi:10.1371/journal.pone.0019167.g001 GeomycesDestructansWidespreadinEurope PLoSONE|www.plosone.org5April2011|Volume6|Issue4|e19167

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acrosssitesand/oryears.Ourresultsfrommonitoringonesite throughoutthehibernationperiodovertwoconsecutiveyearsas wellasreportedcasesfrommultiplesitesinmultipleyearsshow thatbatswithfungalgrowtharefirstseeninJanuary,thenumber ofcasesslowlyincreasesintoFebruaryandpeaksinMarch,then inAprilwhenbatsemergefromhibernationitdropsagain.Our resultsareinagreementwithrecentresultsfromtheCzech Republicwhereinthewinter2009/2010,thenumberofsiteswith batswithwhitefungalgrowthincreasedfrom4.1%inJanuary/ February(33/800sites;regularbatmonitoring)to77.5%inlate February/March(76/98sites;additionalinspections)[11].The Czechstudyreportedthatthisincreasein G.destructans prevalence was‘‘ suggestiveofanepizooticspreadofthefungus ’’[11];weproposean alternativeexplanationwherebytheincreaseinprevalenceof G. destructans inlatewinter(March)mightregularly(yearly)occurin Europebuthasgoneunnoticed.Nearlyallhibernationcountsin previousyearswerecarriedoutbetweenDecemberandmidFebruarywhenprevalence/detectabilityof G.destructans islow,but notinMarch[33]whentheprevalence/detectabilityof G. destructans isatitshighest(Figure3).Althoughthetotalnumbers ofbatsinthehibernaculadecreasedthroughAprilasbatsleftfor thematernitycolonies,ourresultsshowthatthereisahigh probabilityoffungalgrowthdevelopingontheremaining individuals.Thisfurthersupportsourhypothesisproposedabove andlinksthedurationofthehibernationperiodwiththe prevalenceof G.destructans .Byincreasingthesamplesize,some casesmightbereportedearlierinthehibernationseasonorlater throughthesummer,butweexpectthatthegeneralpattern observedwillnotchange.Despitethesedifficultiesinassessingthe occurrenceofthefungusonbats,ourdataareconsistentwith otherstudies[6,11,12],andalsodemonstratethatthemost commonlyencounteredbatspecieswith G.destructans inEuropeis thelargestspeciesof Myotis onthecontinent, Myotismyotis .In countries/regions(i.e.theNetherlands,NorthwestGermany) where M.dasycneme ismorecommonlyencounteredinhibernacula, G.destructans prevalencecanreachhighlevelsinthatspecies.Itis interestingtonotethatneither Pipistrelluspipistrellus nor Miniopterus schreibersii havebeenobservedwith G.destructans [6,11,12], althoughthesetwospeciesareknowntohibernateinaggregations oftensofthousandsofindividuals,especiallythelatter [34,35,36,37].Althoughrare,hibernaculaofafewthousands anduptoabout34,000individualsarealsoknownforspeciesof Myotis inEurope[34,35,38,39,40,41].G.destructans outsideofthehibernationperiodWeobservedthreeindividualbatswithwhitefungalgrowth aroundtheirnose(oneconfirmedas G.destructans )fromMayand June,whentheywerestilltorpidincoldundergroundsites.This representsthefirstmentionofindividualswith G.destructans colonisationoutsideofthehibernationperiodandraisesquestions abouttheroleoftheseindividualsinthepersistenceofthefungus inbatpopulations.Duringthesummerperiod,whilefemales aggregateincoloniestoraisetheiryoung,itremainslargely unknownwheremalesareroosting(e.g.[42]).Furthermore, duringtheswarmingseasoninlatesummer/autumn,large numbersofindividualsaggregateincaves,minesortunnelsand comeinclosecontactwitheachother(chasing,mating) [42,43,44,45,46,47],whichcouldrepresentanopportunityfor G. destructans tobetransmittedbetweenindividuals. Weisolated G.destructans fromtheenvironmentsurrounding hibernatingbats.Thepresenceofviablesporesof G.destructans on thesurfacesofhibernationsiteshashugeimplicationsforthe understandingofdiseasetransmissionmechanismsanddisease modelling[28].Itseemslikelythatcavewallscouldserveasa passivevectorand/orreservoirfor G.destructans spores.Itisnotyet knownhowlongthesesporescanremainviablebutfungalspores generallyremainviableforextendedperiods.Batsenteringthese sitesinautumn(forswarmingand/orhibernation)couldbecome contaminatedwithsporesof G.destructans leftfrombatsinfected duringthepreviouswinter.InNorth-America,Lindneretal.[48] successfullyamplifiedITSsequencesidenticalto G.destructans DNAfromsoilsamplescollectedduringthewinter2008–2009at threebathibernaculaandstressedtheimportanceofconsidering theenvironmentasareservoirfor G.destructans andinthe dynamicsofWNStransmission.Ourresultsconfirmthisand furthersuggestthatmoreworkisneededtounderstandthe persistenceof G.destructans onhibernaculawalls(reservoiror passivevector)wheretheyareinphysicalcontactwithbats.Figure2.PhotographicevidenceshowingbatswithconfirmedorsuspectedgrowthofG.destructans. Photographsofcasesconfirmed bygeneticanalysis,from(A)Estonia( M.brandtii ,May23rd2010, L.Lutsar),(B)Poland( M.myotis ,March7th2010, A.Wojtaszewski),(C)Germany ( M.myotis ,March10th2010, C.Jungmann),(D)France( M.myotis ,March4th2010, Y.LeBris),(E)Netherlands( M.daubentonii ,March9th2010, T.Bosch),(F)Germany( M.myotis ,March23rd2010, K.Passior)(G)Belgium( M.mystacinus ,March18th2010, B.Mulkens),(H)Germany( M. mystacinus ,March23rd2010, K.Passior)orbatswithwhite-fungalgrowthsuspectedas G.destructans from(I)Denmark( M.dasycneme ,March14th2010, B.Ohlendorf),(J)Austria( M.myotis ,February2nd2007, O.Gebhardt),(K)Hungary( M.myotis ,February19th2010, T.Go ¨ rfo ¨ l),(L)Belgium ( M.myotis ,March7th2010, F.Forget),(M)France( M.myotis ,February13th2010, J.Vittier),(N)Ukraine( M.myotis ,February13th2010, A.-T. Bashta),(O)France( M.escalerai /sp. A ,June25th2010, F.Blanc),(P)Turkey( M.myotis / blythii ,March22nd2009, M.Doker),and(Q)Romania( M. blythii ,March29th2008, B.Szila ´ rd). doi:10.1371/journal.pone.0019167.g002 Figure3.Seasonalchangesofthenumberoflivebatsreported withwhitefungalgrowthinEurope. Thenumberofbatswith visiblewhitefungalgrowthatanhibernaculuminGermanywas monitoredduringthewinter2006/2007(blueline)andthewinter2007/ 2008(greenline).TheverticalredlinesrepresentthenumberofGdsuspectbats(orconfirmed)observedacrosstwelveEuropeancountries (n=127)from2003until2010.IntheX-axis,thethickmarksrepresent thestartofeachmonth. doi:10.1371/journal.pone.0019167.g003 GeomycesDestructansWidespreadinEurope PLoSONE|www.plosone.org7April2011|Volume6|Issue4|e19167

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Insightsintotheoriginof G.destructans andWNSThewidedistributionof G.destructans inEuropeandtheabsence ofassociatedmortalitysupportsthehypothesisthat G.destructans hasco-evolvedwithEuropeanbatsandonlyrecentlyarrivedin NorthAmericawhereitiscausingunprecedentedmassmortalities [6,7,11,12].Alternatively, G.destructans couldhavebeenpresenton bothcontinentsandavirulentstraincouldhaveevolvedinNorthAmerica.Untiltherelationshipsbetween G.destructans populations acrosscontinentsareclarified,precautionsshouldbetakento minimisethechancesoftranscontinentalmovementofviable G. destructans [49]. DuringthetwoyearsmonitoringatonesiteinGermanywhere G.destructans prevalencereachedhighlevelsinMarch-April,nota singledeadbatwasfound.Thisisinagreementwithprevious studies[6,11,12]reportingthatthepresenceof G.destructans inbats fromEuropeisnotassociatedwithmassmortality.Thissharply contrastswithmassmortalitiesreportedinNorthAmericawhere hundredsorthousandsofdeadbatsarefoundinhibernacula towardstheendofthehibernationperiod.Recentpathological investigationsofbatsdyingfromWNSinNorthAmericaled Cryanetal.[50]toproposethatmortalitywascausedby importantdisruptionsofwing-dependantphysiologicalfunctions duetoinfectionby G.destructans .InNorthAmerica,thefungus deeplyinvadeswingstissues[2]andcausesdamagesthatare thoughttoalterhomeostasisandwaterbalance,resultinginmore frequentarousalsthanbatscanaffordwiththeirfatreserves, leadingtodeathbystarvation[50].Thepathologyassociatedwith G.destructans colonisationinEuropeisnotyetknown.Webelieve thatthefirststepinunderstandingmortalitydifferencesbetween batsfromEuropeandNorthAmericarelyonunderstanding Figure4.IndirectevidenceofbatsgroomingoffG.destructansduringhibernation. Photographicevidenceshowingthreedifferent M. dasycneme individuals(A–B,C–DandE–F)observedattwodifferentdates,firstwithvisiblefungalgrowth(A,C,E)andlaterwithoutvisiblefungal growth(B,D,F).ThebatinA–Bchangeditspositionwithinthehibernaculumwhereastheothertwo(C–DandE–F)werecapturedwhenleavingthe hibernaculum( V.Korn). doi:10.1371/journal.pone.0019167.g004 GeomycesDestructansWidespreadinEurope PLoSONE|www.plosone.org8April2011|Volume6|Issue4|e19167

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pathologicaldifferencesincurredbythefungusonthebats’wings. Asaresult,weurgethenecessitytocarryoutpathological investigationoflivebatsfromEuropecolonisedby G.destructans . Despitetheabsenceofmortalityassociatedwiththepresenceof G. destructans inEurope,itwouldbenecessarytoinvestigatewhether chronicinfectionswiththefungusarecompromisingthehealthof individuals,especiallyin M.myotis and M.dasycneme ,whichshow highprevalenceofthefungustowardstheendofthehibernation period. PhylogeographicstudiesofEuropeanbatspecieshaveshown thatinthelast100,000years,somespeciescolonisedEuropefrom WesternAsia[51],including Myotisblythii [52,53]whichhasbeen foundwith G.destructans [12].Assumingthat G.destructans canbe transportedoverlongdistancesbybats,wespeculatethatthe distributionof G.destructans isprobablynotlimitedtoEuropeand possiblyextendseastwardsintoRussia,WesternandCentralAsia. Furthersurveysarenecessarytoclarifytheglobaldistributionof G. destructans .ConclusionsWehaveshownherethat G.destructans ,themostlikelycausative agentofWNSinNorthAmerica,iswidespreadinEurope,butis notassociatedwithmassmortality.Theprevalenceofvisible fungalgrowthonbatsincreasesinFebruary/Marchbeforesharply decreasingwhenbatsemergefromhibernation.Wealsoisolated viable G.destructans fromthewallsofanundergroundsite suggestingthatthehibernaculacouldactaspassivevectorsand/ orreservoirsfor G.destructans andtherefore,mightplayan importantroleinthetransmissionprocess.Furtherresearchis neededtoclarifytheglobalprevalenceof G.destructans andidentify variables(e.g.temperature,humidityandhibernationlength) explainingregionaldifferences.Finally,furtherresearchisneeded indifferentpartsoftheglobe,especiallytemperateregionofthe NorthernandSouthernhemispheres,topreciselydeterminethe globaldistributionof G.destructans .MaterialsandMethods SamplecollectionDuringongoingpopulationcensusescarriedoutathibernacula indifferentcountriesacrossEuropeandduringadditional hibernaculasurveyscarriedoutforthepurposeofthisstudy, informationonbatswithvisiblewhitefungalgrowthonsnouts and/orearswasrecorded.Wheneverpossible,steriledrycotton swabs[6]oradhesivetapetouchimprints[12]wereusedtocollect fungalmaterialfromthebats.InEstonia,sampleswerecollected fromthewallofthetunnelwhereabatwithcharacteristicwhite funguswasobservedninedayspriortothesampling.Whereno samplecollectionwaspossible,aphotographwastakenofthebat (photographicrecord).Incaseswhereneithersamplecollection norphotographicevidencewasobtained,therecordwasclassified asvisualobservation.Livehibernatingbatswithpowdery,white fungalgrowthontheirnoseswereconsideredsuspectsofinfection by G.destructans (Gd-suspects)butnotsuspectedofhavingWNS. ThereispresentlynodatasupportingtheoccurrenceofWNSin Europeandtheco-occurrenceofthefunguswithlesions characteristicofWNS[2]hasnot(yet)beenreportedinEurope [12,54].Although,prevalenceof G.destructans canreachhighlevels insomeEuropeanspecies(i.e. Myotismyotis , M.dasycneme )inlate winter(especiallyinMarch),itcanbeexpectedthatbychance alonesomebatsdyingfromcausesunrelatedtothepresenceof G. destructans willalsobecarryingthefungus.Unlessthecriteriafor thediagnosisofWNSaremet(confirmationbyhisto-pathology andPCR)[2]WNSshouldnotbeassumedasacauseofmortality indeadbatsfoundinhibernaculaofEurope.Variousspeciesof fungihavebeenidentifiedondeadbats[12,55],mostofthem likelybeingsaprophytesthatcolonisebatcarcasses post-mortem .FungalculturesInthelaboratory,samplesweretreatedasin[6]forswabsand following[12]fortouchimprints.Briefly,swabswerestreak-plated ontoplatesofSabouraud’sagar,supplementedwith0.1% mycologicalpeptone.Fortouchimprints,smallareaswithfungal conidiacharacteristicof G.destructans wereidentifiedbylight microscopyandthetapewasdisinfectedandexcisedbeforebeing transferredforculturetoSabouraud’sagar.Theplatesweresealed withparafilmandincubatedinvertedinthedarkat10 u C.Afungal growthdevelopedwithin14days,fromwhichsinglesporecultures wereestablished.MolecularidentificationEachculturewassequencedforonemolecularmarker,the rRNAgeneinternaltranscribedspacer(ITS, ca. 930bp.)region (ITS1,5.8S,andITS2)tofurtherconfirmspeciesidentity.The DNAextraction,PCRamplificationandDNAsequencing followedprotocolsdescribedinPuechmailleetal.[6].Briefly, DNAwasextractedusingtheQiagenBloodandTissuekit followingthemanufacturer’sinstructionswithslightmodifications (afterstep3,weaddedanincubationtimeof10minutesat70 u C). PCRreactionswerecarriedoutin25mLcontaining1mLofDNA extract(at10–75ng/mL),1.5mmol/LMgCl2,0.4mmol/Leach primer(Forward:ITS4,5 9 -TCCTCCGCTTATTGATATGC3 9 ;Reverse:ITS5,5 9 -GGAAGTAAAAGTCGTAACAAGG-3 9 ; [56]),0.2mmol/LdNTP,1xPCRbufferand1UPlatinumTaq DNAPolymeraseHighFidelity(Invitrogen).PCRcycling conditionswere:initialstep15 9 at95 u C,then10cyclesof30 0 at 95 u C,1 9 45 0 at60 u C(reduceof2 u Cevery2cycles),1 9 at72 u C, followingby30cyclesof30 0 at95 u C,1 9 45 0 at50 u Cand1 9 at 72 u C.PCRproductswerepurifiedandsequencedbyMacrogen Inc.(Seoul,Korea)inbothdirectionsusingthePCRprimers. Complementarysequenceswereassembledandeditedfor accuracyusingCodonCodeAligner3.0.3(www.codoncode.com/ aligner/download.htm).MonitoringofvisiblefungalgrowthonbatsOnesitesituatedinNorthwestGermany(Latitude:52.1; Longitude:8.2)nearthecityofOsnabru ¨ckwasmonitoredover twoconsecutivewinters,2006/2007(5thSeptemberuntil19thMay)and2007/2008(28thAugustuntil23rdApril).The monitoringconsistedofcountingthetotalnumberofbatsatthe siteaswellasthenumberofbatswithvisiblewhitefungalgrowth similartothepicturespresentedinFigures2and4.Thecounts weredonebythesameperson(V.Korn)every4daysonaverage duringthefirstyearandevery2.5daysonaverageduringthe secondyear.Theprocedurescompliedwithguidelinesofthe AmericanSocietyofMammalogistsandwerecarriedoutunder permitnumberFBD7.260fromtheAdministrationoftheCounty ofOsnabru ¨ck,DepartmentofEnvironment.SupportingInformationFigureS1MonitoringofbatsatanhibernaculuminGermany during(A)thewinter2006/2007(September5th2006untilApril 19th2007)and(B),thewinter2007/2008(August28th2007until April23rd2008).Thebluelinerepresentsthetotalnumberofbats countedwhereasthegreenlinerepresentsthenumberofbatswith visiblewhitefungalgrowth(Gd-suspects).Dottedverticallines separatecountsfromeachmonth.NotethatthenumberofcountsGeomycesDestructansWidespreadinEurope PLoSONE|www.plosone.org9April2011|Volume6|Issue4|e19167

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permonthwasnotequalbetweenmonths.In(B),theblackline representsthetotalnumberofbatscountedwhereastheblueline representsthetotalnumberofbatsbaroneportionofthe hibernaculumwherebatsgroupeddensely( ca .20individuals)and didnotallowareliableidentificationofthenumberofbatswith whitefungalgrowth.Thegreenlinerepresentsthenumberofbats withvisiblewhitefungalgrowth(Gd-suspects)countedatthe hibernaculumwithoutconsideringindividualsdenselygroupingat oneplaceinthehibernaculum.Thegroupofabout20individuals formedwhilethehibernaculumwaspartiallyflooded,likelyasa resultofbatschangingpositiontoavoiddrowning.Notethatthe rightY-axisscaleisdifferentbetween(A)and(B). (PDF)AcknowledgmentsWewouldliketothankDo ´czyAnnama ´ria,Andriy-TarasBashta,Fre ´de ´ric Blanc,Sa ´ndorBoldogh,GabyBollen,ThomasChatton,EmrahCoraman, Je ´reCsaba,SimonDutilleul,MehmetDoker,OliverGebhardt,Lena Godlevska,Rene ´Janssen,DanielLefe `vre,BartiLevente,Vadim Martyniuk,GerhardMa ¨scher,MykolaMatveev,BerndOhlendorf,Rian Pulles,TonyRock,WolfgangRackow,Se ´bastienRoue ´,Bu ¨csSzila ´rd, AbigelSzodoray-Para ´di,FarkasSzodoray-Para ´diandJulienVittierfor providinguswiththeirfieldobservations.ThecommentsofPaulCryan, PaulRacey,NataliaMart ´ nkova ´andananonymousreviewerhelpedto improveapreviousversionofthemanuscript.AuthorContributionsConceivedanddesignedtheexperiments:SJPGWVKECT.Performed theexperiments:SJPGWHFVKKMAK.Analyzedthedata:SJPGW. 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