Ectomycota Associated with Arthropods from Bat Hibernacula in Eastern Canada, with Particular Reference to Pseudogymnoascus destructans


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Ectomycota Associated with Arthropods from Bat Hibernacula in Eastern Canada, with Particular Reference to Pseudogymnoascus destructans

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Title:
Ectomycota Associated with Arthropods from Bat Hibernacula in Eastern Canada, with Particular Reference to Pseudogymnoascus destructans
Series Title:
Insects
Creator:
Vanderwolf, Karen J.
Malloch, David
McAlpine, Donald F.
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English

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Subjects / Keywords:
Pseudogymnoascus Destructans ( local )
Cave Fungi ( local )
Meta Ovalis ( local )
Nelima Elegans ( local )
Scoliopteryx Libatrix ( local )
Exechiopsis Sp. ( local )
Anatella Sp. ( local )
Entomopathogens ( local )
White-Nose Syndrome Decontamination ( local )
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serial ( sobekcm )

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Abstract:
The introduction of Pseudogymnoascus destructans (Pd) to North America, agent of white-nose syndrome in hibernating bats, has increased interest in fungi from underground habitats. While bats are assumed to be the main vector transmitting Pd cave-to-cave, the role of other fauna is unexplored. We documented the fungi associated with over-wintering arthropods in Pd-positive hibernacula, including sites where bats had been recently extirpated or near-extirpated, to determine if arthropods carried Pd, and to compare fungal assemblages on arthropods to bats. We isolated 87 fungal taxa in 64 genera from arthropods. Viable Pd was cultured from 15.3% of arthropods, most frequently from harvestmen (Nelima elegans). Fungal assemblages on arthropods were similar to those on bats. The different fungal assemblages documented among arthropods may be due to divergent patterns of movement, aggregation, feeding, or other factors. While it is unlikely that arthropods play a major role in the transmission dynamics of Pd, we demonstrate that arthropods may carry viable Pd spores and therefore have the potential to transport Pd, either naturally or anthropogenically, within or among hibernacula. This underlines the need for those entering hibernacula to observe decontamination procedures and for such procedures to evolve as our understanding of potential mechanisms of Pd dispersal improve.
Original Version:
Insects, Vol. 7, no. 2 (2016-04-22).

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University of South Florida Library
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University of South Florida
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K26-00043 ( USFLDC: LOCAL DOI )
k26.43 ( USFLDC: LOCAL Handle )

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Article EctomycotaAssociatedwithArthropodsfromBat HibernaculainEasternCanada,withParticular Referenceto Pseudogymnoascusdestructans KarenJ.Vanderwolf 1,2, *,DavidMalloch 1 andDonaldF.McAlpine 1 1 NewBrunswickMuseum,277DouglasAve,SaintJohnE2K1E5,NB,Canada; dmalloch@xplornet.comD.M.;Donald.McAlpine@nbm-mnb.caD.F.M. 2 CanadianWildlifeFederation,350PromenadeMichaelCowplandDrive,KanataK2M2G4,ON,Canada * Correspondence:kjvanderw@gmail.com;Tel.:+1-608-270-2394 AcademicEditor:DrionG.Boucias Received:12February2016;Accepted:14April2016;Published:22April2016 Abstract:TheintroductionofPseudogymnoascusdestructansPdtoNorthAmerica,agentofwhite-nosesyndromeinhibernatingbats,hasincreasedinterestinfungifromundergroundhabitats.WhilebatsareassumedtobethemainvectortransmittingPdcave-to-cave,theroleofotherfaunaisunexplored.Wedocumentedthefungiassociatedwithover-winteringarthropodsinPd-positivehibernacula,includingsiteswherebatshadbeenrecentlyextirpatedornear-extirpated,todetermineifarthropodscarriedPd,andtocomparefungalassemblagesonarthropodstobats.Weisolated87fungaltaxain64generafromarthropods.ViablePdwasculturedfrom15.3%ofarthropods,mostfrequentlyfromharvestmenNelimaelegans.Fungalassemblagesonarthropodsweresimilartothoseonbats.Thedifferentfungalassemblagesdocumentedamongarthropodsmaybeduetodivergentpatternsofmovement,aggregation,feeding,orotherfactors.WhileitisunlikelythatarthropodsplayamajorroleinthetransmissiondynamicsofPd,wedemonstratethatarthropodsmaycarryviablePdsporesandthereforehavethepotentialtotransportPd,eithernaturallyoranthropogenically,withinoramonghibernacula.Thisunderlinestheneedforthoseenteringhibernaculatoobservedecontaminationproceduresandforsuchprocedurestoevolveasourunderstandingofpotentialmechanismsof Pd dispersalimprove. Keywords: Pseudogymnoascusdestructans ;cavefungi; Metaovalis ; Nelimaelegans ; Scoliopteryxlibatrix ; Exechiopsis sp.; Anatella sp.;entomopathogens;white-nosesyndromedecontamination 1.IntroductionTheintroductionofPseudogymnoascusdestructansPdtoNorthAmerica,thecauseofthefataldiseasewhite-nosesyndromeWNSinhibernatingbats[1],haspromptedincreasedinterestinfungifromundergroundhabitatssuchascavesandmines.WNShasrapidlyspreadthroughtheeasternUnitedStatesandCanada,killing>6.7millionbatsanestimatemadein2012sinceitwasrstreportedin2006inAlbany,NewYork[2].ItisthoughtthatbatsarethemainvectortransmittingPdcave-to-cavewithinNorthAmerica[3],butthepossibleroleofotherfaunaasvectorsislargelyunexplored.Usingculture-independentmethods,Lucan,etal.[4]foundthattheexternalsurfaceofwingmitesSpinturnixmyoti;removedfromPd-positiveMyotismyotiswerePd-positiven=33mites,100%positive.SinceS.myotiareknowntoswitchhosts,Lucan,etal.[4]suggestthatbatectoparasitesmayplayaroleinthetransmissiondynamicsofPd.RaudabaughandMiller[5]foundthatPdgrewonautoclavedMigratoryLocustLocustamigratoriainthelab,butitisunclearifPdcancompetewiththenativemicroorapresentonarthropodsundereldconditions.Additionally,somearthropodsincavesareknowntoproduceanti-microbialsubstancesthatpreventfungalinfection[6].ThemicrooraInsects 2016 , 7 ,16;doi:10.3390/insects7020016www.mdpi.com/journal/insects

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Insects 2016 , 7 ,16 2of16onarthropodsincavesmaydifferfromthatfoundonbats,andthismayalsoimpacttheabilityofPdtopropagate.Thefungiassociatedwitharthropodsincavesarerelativelywellstudiedcomparedtoothercavefauna;arecentreviewoffungiincaveslisted201speciesoffungiin89generaisolatedfromarthropods,mostofwhichwereascomycetesandzygomycetes[7].Moststudiesoffungionarthropodsincavesfocusonentomopathogenicfungi[7–9],whichcanreducepopulationsofcavearthropods.ForexampleTolypocladiumsp.occasionallyreducesglowwormArachnocampaluminosapopulationsinaNewZealandshowcave[10],andthephenomenonof“cricketmarshmallows”BeauveriacaledonicacolonizingHadenoecusspp.iswellknownincavesintheUnitedStates[11].Conversely,arthropodsconsumefungiincaves[12],andmayplayaroleinregulatingnumbersofcavemicrofungi[13].Arthropodsmayintroducefungiintocavesbytransportingsporesbothexternallyandinternally.CaveCricketsCeuthophilusgracilipesgracilipesarebelievedtobevectorsfordictyostelidcellularslimemoldsintoandwithincaves[14].Entomopathogenicfungalsporescanbetransmittedamongandbetweeninsectspecies,orbeacquiredfromthecaveenvironment[15,16],similartothehypothesizedtransmissiondynamicsforPd[17].Incaveswithlimitedairowandnorainfall,arthropodsmaybedisproportionallyimportantassporedispersers.Dickson[18]notedthatpopulationsofinvertebrateswerepositivelycorrelatedwithpopulationsoffungiinVirginiacavesediments.Arthropodexoskeletonspossesshairsandcrevicesthatpromotesporeadherence,andsomespeciesoffungihaveadaptationsforarthropoddispersal,suchassporesinstickydropsatthetipoffungalfruitingstructures[19].Outsidecaves,arthropodsareknowntobeimportantvectorsoffungithatcauseplantdiseases,suchasDutchElmdisease,transportingviablesporesbothexternallyandinternally[20].FungiassociatedwithhibernatingbatsinNewBrunswick,CanadaMyotislucifugus,M.septentrionalis,andPerimyotissubavushavebeendocumentedoverseveralyears[21,22].WNSwasrstobservedinNewBrunswickinMarch2011,leadingtomassmortalityofthehibernatingbatpopulation[23].AdiverseassemblageofarthropodshavebeendocumentedfromcavesineasternCanada[24,25],wherePdisnowwidespread.Ourobjectivesinthestudyreportedherewereto:determineifviablePdcanbeculturedfromarthropodspresentinPd-positivemines/cavesduringtheperiodwhenbatsarehibernating;comparethefungalassemblagefromdifferentspeciesofarthropodsamongmines/caves;andcomparethefungalassemblageonarthropodstothatfoundonbatsatthesameundergroundsites. 2.MaterialsandMethods 2.1.FieldCollectionsWesampledarthropodsinfourPd-positivebathibernaculainNewBrunswick:GlebeMineabandonedmanganesemineandDorchesterMineabandonedcoppermineinMarch/April2012,andMarkhamvilleMineabandonedmanganesemineandDallingsCavelimestoneinApril2013.LocationsofsitescanbefoundinVanderwolf,etal.[26].Thenumberofbatspresentatindividualsiteswerecountedduringeachsamplingtrip.VisiblePd-growthhadbeenobservedonhibernatingMyotislucifugusandM.septentrionalisinGlebeMineandDorchesterMinepriortoarthropodsamplingin2012,andinMarkhamvilleMineandDallingsCavepriorto2013.Pd/WNSpresencewasalsoconrmedonbatsatallsitesfromsamplessubmittedtotheCanadianWildlifeHealthCo-operative.Hibernatingbatswerepresentduringallsamplingperiods,exceptinDorchesterMinein2013andGlebeMinein2014.Arthropodsweresampled~10mfromtheentranceofeachstudysite.ThemeanSDtemperatureduringwinterNovember13,2009toApril30,2010athibernaculaentranceswasasfollows:DallingsCave3.472.20C,GlebeMine1.651.90C,andMarkhamvilleMine1.591.47C[26].DorchesterMinewasnotstudiedin2010.TemperatureloggeriButtonsmodelDS1921G,MaximIntegratedProducts,Inc.,Sunnyvale,CA,USAwereplacedinsitesandsettorecordairtemperaturetwiceadayat0230and1430.TwoiButtonsweredeployed

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Insects 2016 , 7 ,16 3of16withineachcave:oneintheentrance-twilightzoneandthesecondinthedarkzone.Athirdibuttonwasplacedabovegroundatchestheight50mfromeachhibernaculumentrance.Generally,wesampledarthropodsinthesameareatemperaturewasrecorded,butsomesamplingalsooccurreddeeperinhibernaculawherethetemperaturewasseveraldegreeswarmerandmorestablethanatentrances.WefollowedtheprotocoloftheUnitedStatesFishandWildlifeService[27]forminimizingthespreadofWNSduringallvisitstocaves.Wesampled17CaveOrbWeaversMetaovalisGertsch1933,15harvestmenNelimaelegansWeed1889,17HeraldMothsScoliopteryxlibatrixLinnaeus1758,and>46fungusgnatsExechiopsissp.withafewAnatellasp..Figure1showsthethreelargerarthropodspeciessampled,withNelimaelegansindividualsina“looseaggregation”asdenedbyHolmberg,etal.[28].FungusgnatswereidentiedusingVockeroth[29].Onlyadultarthropodsweresampled.Otherarthropodsobservedatourstudysites,includingcricketsCeuthophilusspp.,woodliceOniscusasellus,largerdipterans,craneiesTipulidae,andTissueMothsTriphosahaesitata,weretoorareduringthestudyperiodtoprovidesufcientsamplesizes. Figure1.PrincipalarthropodspeciesinNewBrunswickbathibernaculafromwhichfungiwerecultured. A Metaovalis ; B Nelimaelegans inlooseaggregation; C Scoliopteryxlibatrix.WeinoculatedtwopetriplatesperindividualM.ovalis,N.elegans,andS.libatrix;oneplatewithdextrose-peptone-yeastextractagarDPYA[30]andtheotherwithSabouraud-dextroseagarSAB,bothofwhichwereinfusedwiththeantibioticschlortetracyclinemg/Landstreptomycinmg/L.ThesemediawerepreviouslyusedtoculturefungifromhibernatingbatsinNewBrunswick[21,22].Pre-pouredpetriplateswereusedtodirectlycaptureM.ovalis,N.elegans,andS.libatrixfromcavewallsontothehardenedagarsurface,sonoindividualwashandled.Eachindividualwasencouragedtomoveacrosstheagarsurfacefor~2min,duringwhichweensuredthattheabdomentouchedtheagaratleastonce,beforebeinggentlyshakenintothenextpetridish.Arthropodswerethenreleasedbackontothecavewall.Themediatypeinoculatedrstwasalternatedforeachindividualarthropod.WecapturedExechiopsis/Anatellasp.withtweezerssterilizedin95%ethanolandembedded2individualsdirectlyintotheagarsurfaceperplatewhileinthecave,usingequalnumbersofbothmediatypes.Unlikeotherarthropodssampled,Exechiopsis/Anatellasp.werenotremovedfromthepetriplates.Allplatesweresealedinsituwithparalm. 2.2.LaboratoryMethodsInthelaboratory,sampleswereincubatedinalow-temperatureincubatorModel2015,VWRInternational,Mississauga,ON,Canadainthemannerreportedpreviouslyinverted,inthedarkat7Cforstudiesoffungiassociatedwithbatshibernatingatthesamesites[21,22].Samplesweremonitoredover4monthsuntileithernonewcultureshadappearedfor3weeksonaplate,orplateshadbecomeovergrownwithhyphae.Oncefungibegangrowingonplates,eachdistinctcolonywassub-culturedtoanewplate.DPYAwithoutoxgallandsodiumpropionatewasusedformaintainingpurecultures.

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Insects 2016 , 7 ,16 4of16Identicationswereaccomplishedbycomparingthemicro-andmacro-morphologicalcharacteristicsofmicrofungitothosetraitsappearinginthetaxonomicliteratureandcompendia[31,32].IdenticationswerealsomadebycomparisontoanexistingreferencecollectionoffungalculturesfromMyotislucifugusandM.septentrionalisthatwerepreviouslyidentiedin2010usingamixofsequencingandmorphologicalfeatures[21].IdenticationsofPdwereconrmedbysequencingaspartofotherstudies[33]for2012samples;[34]for2012,2013,and2014samples.PermanentculturesarehousedintheUniversityofAlbertaMicrofungusCollectionandHerbariumUAMH11726-11728,anddesiccant-driedsamplesareintheNewBrunswickMuseumNBM#F-04966,05028,05144,05156,05162,05170,05175,05177,05184;05351,05360,05371,05401,05426,05435,05559,05627,05639.Wetotaledthenumberoffungaltaxaperindividualarthropodspecimen,anddeterminedthenumberofindividualsofeacharthropodspeciesthateachfungaltaxonwasidentiedfrom. 2.3.StatisticalAnalysisThenumberoffungaltaxa/individualarthropodweresquareroottransformedandcomparedamongstsites,years,andarthropodspecieswithagenerallinearmodelandTukeypost-hoctests.Usingpreviouslyassembleddataonfungifromover-winteringbatsMyotislucifugus,M.septentrionalis,andPerimyotissubavusinGlebeMine[21,22],thenumberoffungaltaxa/individualweresquareroottransformedandcomparedamongstarthropodandbatspeciesfromGlebeMineusinga1-wayANOVAwithaTukeypost-hoctest.ThenumberoftimeseachfungaltaxonwasisolatedwascomparedamongstarthropodspecieswithaKruskal-Wallistest.ThenumberoffungaltaxaobtainedfromtherstplateinoculatedwascomparedtothesecondplateforN.elegans,M.ovalis,andS.libatrixusingapairedt-testafterthedataweresquareroottransformed.AMann-WhitneytestwasusedtocomparethenumberoffungaltaxaobtainedperplateforDPYAversusSAB.AllstatisticalanalysiswereperformedusingMinitabMinitabInc.,StateCollege,PA,USA. 3.ResultsFungiwereculturedfrom71of72arthropodssampledcountingeachplateofExechiopsis/Anatellasp.asanindividualandfrom114of119.8%eld-inoculatedplates.Thisproducedatotalof559isolates.OneN.elegansescapedbeforethesecondplatecouldbecompleted.Ameanof6.63.8fungaltaxawereisolatedfromeacharthropodTable1.Themeannumberoffungaltaxa/individualarthropodwasnotsignicantlydifferentacrossyearsF2,71=2.24,p=0.115,butwassignicantlyhigheronNelimaelegansandScoliopteryxlibatrixwhencomparedtoMetaovalisandExechiopsis/Anatella sp.F 3,71 =17.98, p <0.001,Table1.Themeannumberoffungaltaxa/individualarthropodwassignicantlyhigherinGlebeMinewhencomparedtotheotherthreesitesF3,71=12.21,p<0.001.WithinGlebeMine,themeannumberoffungaltaxa/individualwassignicantlyhigheronN.eleganswhencomparedtoMyotislucifugus,Metaovalis,andExechiopsis/Anatellasp.,andsignicantlyloweron Exechiopsis/Anatella sp.comparedto Perimyotissubavus F 6,48 =5.10, p =0.001;Table1.Duringthisstudy,87fungaltaxain64generaplus20sterilefungalmorphswereisolatedfromfourarthropodspeciesTable2.Thirty-seven.5%ofthe87fungaltaxawereisolatedfromasingleindividualarthropodeach.Thirty-onefungaltaxawereisolatedfromExechiopsis/Anatellasp.n=~46individuals,23plates,33fromM.ovalisn=17individuals,34plates,46fromS.libatrixn=17individuals,34plates,and53fromN.elegansn=15individuals;29plates.ViablePdwasculturedfrom15.3%ofarthropodsn=72,with26.7%ofN.elegans,17.7%ofM.ovalis,17.4%ofExechiopsis/Anatellasp.,and0%ofS.libatrixPd-positiveTable2.Pdwasisolatedfrom16.0%ofarthropodsin2012n=25,27.3%in2013n=22,and4.2%in2014n=24;Table3.Theseguresdonotshowconsistenttrendswiththenumberofhibernatingbatspresentduringsamplingoryear-to-yeartemperaturevariationTable3.WedidcultureviablePdfromsomearthropodsinhibernaculafromwhichbatshadbeenextirpatedornear-extirpatedintheyearspriortosampling.ThemostcommonfungaltaxawereCladosporiumspp.isolatedfrom52.8%ofarthropods;n=72,

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Insects 2016 , 7 ,16 5of16Penicilliumspp..8%,Mortierellaspp..9%,Verticilliumsp.cf.Gabarnaudia.1%,Acremoniumspp..3%,Mucorspp..9%,Cephalotrichumstemonitis.2%,andLeuconeurosporapolypaeciloides.2%.ThenumberoftimeseachfungaltaxonwasisolatedfromarthropodswassignicantlydifferentamongarthropodspeciesH3,347=12.55,P=0.006.However,whenfungaltaxathatoccurred3timeswereexcluded,thedifferencewasnotsignicantH3,115=5.02,P=0.17.Therefore,commonfungaltaxaoccurredatsimilarfrequenciesonallfourarthropodspecies,whileuncommonfungidifferedamongstarthropodspecies.Somefungiappearedtobeassociatedwithspecicarthropodspeciesmoreoftenthanothers,suchasBotrytissp.,Fusariumsp.,andPhomasp.onS.libatrixandAcrodontiumspp.onN.elegansTable2.ThelackofCladosporiumspp.onM.ovalisisalsonotable.Visiblefungalgrowthonarthropodswasnotobservedduringoursamplingperiod. Table1.Themeannumberstandarddeviationoffungaltaxaperindividualarthropodperplatefor Exechiopsis/Anatella sp.overthreeyearsinthreeminesandonecaveinNewBrunswick,Canada. Arthropod&Bat Species GlebeMineDorchesterMine Markhamville Mine DallingsCaveAllsitesRange Metaovalis 7.0 1.9 BC 2.8 2.2NDND4.76 2.95*0 Scoliopteryxlibatrix 9.7 4.3 ABC 8.4 2.56.0 1.46.5 4.2 8.12 3.59 & 2 Nelimaelegans 13.6 3.0 A 8.3 3.17ND10.003.89& 4 Exechiopsis/Anatella sp. 5.8 1.8 C 3.3 1.2NDND4.55 1.97*1 Perimyotissubavus 10.2 2.2 AB NDNDNDND7 Myotislucifugus 7.7 3.7 BC NDNDNDND2 Myotisseptentrionalis 9.3 4.0 ABC NDNDNDND6 Allarthropodspecies8.2 3.85.2 3.46.3 1.26.5 4.26.61 3.780 Thenumberofarthropodssampledareinparentheses.MeansfromGlebeMinethatdonotsharealetteraresignicantlydifferentp<0.001.Pooledmeansfromallsiteswithdifferentsymbols*,&aresignicantlydifferentfromeachotherp<0.001.TherangeisthenumberoffungaltaxaperindividualperplateforExechiopsis/Anatellasp.acrossallsites.DatafromMyotislucifugus,M.septentrionalis,andPerimyotissubavustakenfromVanderwolf, etal. [21,22]areincludedforcomparison.ND=nodata. Table2.Fungaltaxaisolatedfromtheexternalsurfaceofover-winteringarthropodsincavesandminesinNewBrunswick,Canadaoverthreeyears.ColumnguresindicatethenumberofindividualsplatesforExechiopsis/Anatellasp.culturingpositiveforeachfungaltaxon.Gl=GlebeMine,Do=DorchesterMine,Ma=MarkhamvilleMine,andDa=DallingsCave. FungalTaxon MetaovalisScoliopteryxlibatrixNelimaelegans Exechiopsis/ Anatellasp. GlDoGlDoMaDaGlDoMaGlDoall Ascomycota Acremonium sp.2034013604124 Acremonium sp.hyaline000000010001 Acrodontium spp.0010004900216 Alternaria sp.001000000001 Aureobasidium sp.000000010001 A.pullulans DeBaryG.ArnaudexCif., Ribaldi&Corte 001000000001 Beauveriabassiana Bals.-Criv.Vuill.000000000022 Beauveria sp.penicillate001000000001 Botrytis sp.003311010009 Cephalotrichumstemonitis Pers.Link8011002018021 Chaetomidium sp.000000010001 Cladosporium sp.0065245414738 C.cladosporioides complexFresen.G.A. deVries 000001000001 cf.Conioscypha sp.010000000001 cf.Cylindrocarpon sp.000100000001 Dactylella sp.100000000001 Dendryphiella sp.010000000001 Exophiala sp.001001010003 Fusarium sp.002102000005 Fusicladiumcf.carpophilum Thum.Oudem100000000001 Hormonema sp.002000100003

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Insects 2016 , 7 ,16 6of16 Table2. Cont. FungalTaxon MetaovalisScoliopteryxlibatrixNelimaelegans Exechiopsis/ Anatellasp. GlDoGlDoMaDaGlDoMaGlDoall Ascomycota Humiculacf. UAMH115952110202013113 Hyalodendriella sp.000000100001 Isariafarinosa Holmsk.Fr.001200000003 Lecanicillium sp.000000000022 L.muscarium PetchZare&W.Gams001000010002 Leuconeurosporacapsici J.F.H.Beyma Malloch,Sigler&Hambleton 000001000315L.cf.pulcherrimaG.WinterMalloch&Cain000000010001 L.polypaeciloides Malloch,Sigler& Hambleton 4110104117121 Malbranchea sp.100000000001 Mammaria sp.000000000101 Microascus sp.100000000001 M.caviariformis Malloch&Hubart000000000011 M.cf.giganteus Malloch101000100003 Monodictys sp.000000100001 Myceliophthora sp.100000100002 Oidiodendron sp.000000000101 O.truncatum G.L.Barron111000020005 Paecilomyces sp.000200100003 P.inatus BurnsideJ.W.Carmich.000000030003 cf.Penicillifer sp.000000100001 Penicillium sp.4234022505229 P.cf.brevicompactum Dierckx000010000001 P.cf.decumbens Thom000000100001 P.cf.soppii K.M.Zalessky000001000001 P.cf.thomii Maire002100300017 Phaeotrichumhystricinum Cain&M.E.Barr100000100114 Phialemonium sp.000000010001 Phoma sp.0043222000013 Preussia sp.200000100104 Pseudogymnoascusdestructans Blehert& GargasMinnis&D.L.Lindner 1200000404011 P.pannorum LinkMinnis&D.L. Lindner sensolato 5112024003220 Sarocladiumstrictum W.GamsSummerbell001000000001 Scopulariopsis sp.000001010002 Simplicillium sp.001200110005 Stachybotrys sp.000000000011 sterile1432012401220 Streptomyces sp.000000000011 Thelebolus sp.100000000001 Theleboluscrustaceus FuckelKimbr.000000101002 Thysanophora sp.001000000001 T.penicilliodes Roum.W.B.Kendr.0210002401111 Tolypocladiumcf.cylindrosporum W.Gams000000100001 T.inatum W.Gams100000010013 Trichoderma sp.000001010002 Trichosporiella sp.000000110103 Verticillium sp.000201110005 Verticilliumcf.alboatrum Reinke&Berthold001100000002 Verticillium sp. cf.Gabarnaudia 4611002704126 Wardomyces sp.000000000101 W.cf.columbinus DemeliusHennebert000000000101 W.humicola Hennebert&G.L.Barron100000000001 W.inatus MarchalHennebert100000200205 Zopellapleuropora Malloch&Cain100000100002 Zythia sp.000100000001 Basidiomycota Asterotremella sp.000000300104 Basidiomyceteunidentied012000100004 Cystolobasidium sp.100000000001 Trichosporon sp.101010210006 T.dulcitum BerkhoutWeijman100010000002

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Insects 2016 , 7 ,16 7of16 Table2. Cont. FungalTaxon MetaovalisScoliopteryxlibatrixNelimaelegans Exechiopsis/ Anatellasp. GlDoGlDoMaDaGlDoMaGlDoall Zygomycota Mortierella sp.3131013814328 Mucor sp.4013232214123 Thamnidiumelegans Link001000100002 Umbelopsisisabellina Oudem.W.Gams001000100002 ThemostcommonfungaltaxaisolatedfrombatsinGlebeMinewereCephalotrichumstemonitisisolatedfrom84.21%ofbats,n=19;datatakenfrom[21,22],Leuconeurosporapolypaeciloides.42%,Baeosporasp..16%,Humicolacf.UAMH11595.16%,Wardomycesspp.63.16%,Preussiasp..37%,Mortierellaspp..11%,andPenicilliumspp..84%.ThemostcommonfungaltaxaisolatedfromarthropodsinGlebeMinewerePenicilliumspp.isolatedfrom70.00%ofarthropods,n=30, Cephalotrichumstemonitis .33%, Cladosporium spp..33%, Leuconeurosporapolypaeciloides.33%,Mortierellaspp..33%,Acremoniumspp..00%,Mucorspp..67%,andVerticilliumsp.cf.Gabarnaudia.67%.Somefungaltaxaoccurredmoreoftenorexclusivelyonbats,suchasBaeosporasp..2%onbats,n=19;0%onarthropods,n=30,unidentiedBasidiomycetes.8%,10%,Humicolacf.UAMH11595.2%,26.7%,andPreussiasp..4%,13.3%.Otherfungaltaxaoccurredmoreoftenorexclusivelyonarthropods,suchasVerticilliumsp.cf.Gabarnaudia.7%onarthropods,0%onbats,Botrytissp.%,0%,Acrodontiumsp..7%,0%,Phomasp.%,5.3%,Acremoniumspp..0%,10.5%,Penicilliumspp..0%,36.8%,Cladosporiumspp..3%,5.3%,Hormonema sp.%,0%,and Mucor spp..7%,5.3%.Atotalof65fungaltaxawereisolatedfromarthropodsinGlebeMinewhereas50fungaltaxawereculturedfromarthropodsinDorchesterMine.Thecompositionofthemostcommonfungaltaxaweresimilarbetweenthetwosites,butGlebeMinehadagreaterdiversityoffungithatwereisolatedonasingleoccasioncomparedtoDorchesterMine.Inparticular,highernumbersofCephalotrichumstemonitis,Leuconeurosporapolypaeciloides,Wardomycesspp.,andHumicolacf.UAMH11595wereisolatedfromGlebeMine.33%,53.33%,26.67%,and26.67%ofarthropods,respectively,n=30comparedtoDorchesterMine.94%,8.82%,0%,and5.88%,n=34.DPYAproduced301isolatesn=58plateswhileSABproduced258isolatesn=61plates,butthenumberofisolatesperplatewasnotsignicantlydifferentbetweenthetwomediatypesW1,118=3767,p=0.125.TherstplateinoculatedforN.elegans,M.ovalis,andS.libatrixdidnothaveasignicantlyhigherfungaldiversitythanthesecondplateT1,71=0.89,p=0.382.Thesecondplatecontributedameanof2.081.83fungaltaxafromM.ovaliswhichwerenotdetectedwiththerstplate.Likewise,4.082.31fungaltaxaonN.elegans,and2.422.19fungaltaxaculturedfromS.libatrixweredetectedwiththesecondplatebutnottherst.PdwasisolatedfromsixSABplatesandveDPYAplates,buttendedtoappearontherstplateused,regardlessofagartypeonrstplate,2onsecondplate.

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Insects 2016 , 7 ,16 8of16 Table3.ThepercentageofarthropodsinbathibernaculainNewBrunswick,CanadafromwhichPseudogymnoascusdestructansPdwasculturedoverthreeyears. BatHibernacula 201220132014 Pd yield#ofbatsTemperature Pd yield#ofbatsTemperature Pd yield#ofbatsTemperature GlebeMine 30% 1 3 M, 3 6 A, 0 3 S 1745.83 1.48 a D 0% 0 2 M, 0 2 N 225.98 1.94D 7.1% 0 2 M, 0 2 N, 1 4 A, 0 2 S 0 5.33 2.09D 0.06 1.33 b E 2.67 7.66O DorchesterMine 0% 0 3 M, 0 1 N, 0 7 A, 0 2 S 1 6.63 0.00 c D 1.08 2.67 c E 0.97 7.12 c O 54.5% 2 4 M, 4 6 N, 0 1 S 0 6.59 0.12D 2.19 6.91 c O 0% 0 3 M, 0 3 N, 0 5 A, 0 3 S 2 6.61 0.07D 3.93 7.29 c OMarkhamvilleMineND5.33 0.52 a D 0% 0 1 N, 0 2 S 165.28 0.37DND 4.89 0.69D 0.64 2.68E 3.71 7.69O DallingsCaveND 3.55 1.29 d D 0% 0 4 S 2 4.26 2.07D 0.12 6.03O ND 3.59 2.26D 0.84 3.20E 2.96 7.79O SpeciesofarthropodsareinparentheseswiththenumberofPd-positiveindividualsplatesfordipteransovertotalindividualssampled.M=Metaovalis;N=Nelimaelegans;A=Exechiopsis/Anatellasp.;S=Scoliopteryxlibatrix.Thenumber#ofbatsMyotislucifugus,M.septentrionalis,andPerimyotissubavuspresentduringsamplingisindicated,althoughbatsroosteddeeperwithinsitesthanarthropods.ThemeantemperatureSDinCattheentranceE,deepD,andjustoutsideOofsitesislisted.TemperaturesweretakenNov1toApr30exceptwhereindicated.Missingtemperaturevaluesindicatefailedibuttons.ND=nodata. a Nov30toApr30; b Dec5toApr30; c Dec8toApr30; d Dec16toApr30.

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Insects 2016 , 7 ,16 9of16 4.DiscussionViablePdwasisolatedfromNelimaelegans,Metaovalis,andExechiopsis/Anatellasp.,butnotScoliopteryxlibatrix.Nelimaelegansappeartoacquireagreaterdiversityoffungalsporeswhencomparedtotheotherarthropodspeciessampled.ThismaybeonereasonwhyharvestmenproducedthehighestPdyield.Nelimaspp.areknowntooverwinterinthetwilightzoneofcavesineasternandsouthwesternCanadafromOctobertoMay.Althoughthisspeciesmayformaggregationsofuptotensofthousands[25,28,35],wemostcommonlyobservedgroupsof2.Harvestmenarethoughttoaggregateincavestooptimizetheirimmediatemicroclimate,particularlytomaintainexposuretohighhumidity[28].However,aggregationmayalsopromotetheacquisitionandtransmissionoffungi,includingPd.AsamobilespeciesN.elegansmayencounterorganicmatterincavesasourceoffungalspores;[36]morefrequentlythanotherarthropods.Someharvestmenspecieshavebeenobservedconsumingfungigrowingonwoodincaves[37].Acrodontiumspp.wereparticularlyabundantonN.eleganscomparedtootherarthropodssampledduringthisstudy,andpreliminaryITSsequencingindicatethatatleastthreeAcrodontiumspecieswereisolatedKeithSeifert,AgricultureCanada,per.comm..Acrodontiumspp.havebeenfoundinsoil,air,andonmitesandspiders[38,39],andsomespeciesareconsideredplantpathogens[40].Acrodontiumsp.havebeenisolatedfromsoilinAntarctica[41],indicatingthatsomespeciesinthegenusarecoldtolerant.Previousstudiesoffungionharvestmenincavesarefew:Meyer-RochowandLiddle[42]observedMetarrhiziumanisopliaegrowingondeadMegalopsalistumidainacaveinNewZealandandHolmberg,etal.[28]notedunidentiedfungigrowingondeadLeiobunumpaessleriincavesinBritishColumbia,Canada.Machado,etal.[43]reportedthatGoniosomalongipeswerefrequentlyinfectedwithunidentiedfungiincavesinBrazil.Outsidecavesadiversityofentomopathogenicfungihavebeenisolatedfromavarietyofharvestmenspecies,includingAspleniumtrichomanes,Cordycepsgonylepticida,Engyodonthiumaranearum,Entomophthoraphalangicida,Hymenostilbeverrucosa,Metarrhiziumanisopliae,Nomuraeaatypicola,Pandoraphalangicida,andPhyllactiniaguttata[42,44–49].EntomophagabatkoiwasdescribedfromharvestmenandisknowntocauseconsiderablemortalityinEuropeanharvestmen,withepizooticsobservedduringlatesummer[50,51].Harvestmenproduceexocrinesecretionsthatmayactasfungicides[48],whichmayenablethemtocopewithhighdiversitiesoffungi.AlthoughS.libatrixisbelievedtobelargelysedentaryduringthewinter,whenthisspeciesoccupieshibernacula,S.libatrixnonethelesscarriedarelativelydiversefungalassemblage.WhyPdwasnotculturedfromS.libatrixisunclear.UnlikeN.elegans,over-winteringS.libatrixdonotaggregateinlargenumbers,althoughgroupsof2and2havebeenobservedincavesinManitoba[52]andPoland[53].Duringthepresentstudy,mostS.libatrixroostedindividuallynearorontheceilingwithgroupsof2rarelyobserved.Thisisolationmaydecreasethetransmissionoffungi,includingPd,whencomparedtoaggregatingspeciessuchasN.elegans.Additionally,over-winteringS.libatrixrarelymoveanddonotfeedinhibernacula.Combined,thesebehaviorsmaylimitthenumberofsporesmothsencounter.IncavesinManitobaandOntario,S.libatrixwerenotobservedmovinginoroutofcaves,butnumbersincreasedintheautumnanddeclinedinspring[52,54].Movementofindividualswithincaveswaslimitedtoafewcmoverthewinter,withthemajorityofindividualsfoundwithin10mofthecaveentranceneartheceiling[52].InPoland,Kowalski[53]notedgreaterwithin-cavemovementsupto10mofover-winteringS.libatrixincavessubjecttorapidtemperatureshifts,andoccasionallyobservedS.libatrixjustoutsidethehibernationsite.Ofthearthropodsexaminedduringthisstudy,S.libatrixhasthegreatestpotentialofmovingsporescave-to-cave.Individualmothscanhibernatefortwosuccessivewintersinthesamecave[52].ScoliopteryxlibatrixproducestwogenerationsayearandindividualsoftherstgenerationappearinJuneandytogetherwiththosewhichhavehibernated[53].PreviousstudiesoffungionS.libatrixincaveshavefocusedonmothcadavers,withunidentiedfungiobservedgrowingoncadaversincavesinPoland[53]andManitoba,Canada[52].IncavesintheCzechRepublic,Engyodontiumrectidentatum,Lecanicilliummuscarium,Paecilomycesfarinosusmostcommon,P.fumosoroseus,Simplicilliumcf.lamellicola,and2sterilemorphswereculturedfrom30deadS.libatrix[55].AsidefromEngyodontium,thesegenerawere

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Insects 2016 , 7 ,16 10of16alsoisolatedfromliveS.libatrixinthecurrentstudy.Phomasp.,Botrytissp.,andFusariumsp.,whicharegenerallyconsideredplantpathogens[31],wereisolatedmorefrequentlyfromS.libatrixinNewBrunswickcomparedtotheotherthreearthropodspeciessampledandmayhavebeentransportedintohibernaculaonthemoths.Metaovalisdonotaggregateandindividualsaregenerallyfound12cmfromeachother,butcanoccasionallybefoundincloseproximitycm[56].Rector[56]foundlittlemovementof M.ovaliswithincaves,andnomovementbetweencaves.Whilethismaylimitthenumberoffungalsporesspidersencounter,clearlyitdoesnotpreventacquisitionofPdsporesbyM.ovalis.Yoder,etal.[9]notedthatM.ovalisgenerallyappearinactive,butcanoccasionallybefoundoutsidecaves.SpiderlingsofacloselyrelatedEuropeanspecies,M.menardi,dispersefromnatalundergroundsitesinspringandreturntothesamesiteinlatesummerorndnewsitesviaballooning[57].Therefore,spiderlingsmaybemoreimportantthanadultsinspreadingfungicave-to-cave.Asobservedduringthisstudy,M.ovalisoccursmostcommonlyintheentranceandtwilightzonesnearorontheceiling,butmaysometimesbeencounteredindeeperpartsofcaves[9,56].Metaovalismayacquiresporesfromotherarthropodscaughtinspiderwebs.Spiderwebbingoutsidecavesisknowntotrapfungalspores,includingspeciesofAlternaria,Cladosporium,andFusarium,amongotherAscomycotaandBasidiomycota[58],withthispotentiallycontributingtothefungaldiversityfoundontheexternalsurfaceofspiders.Yoder,etal.[9]sampled40freshlykilledM.ovalisinaKentuckycaveandcommonlyculturedAspergillussp.,Mucorsp.,Penicilliumsp.,andRhizopussp.,withfeweroccurrencesofAbsidiasp.,Beauveriasp.,Cladosporiumsp.,Paecilomycessp.,Trichodermasp.,andsterilemorphs.ThelowfungaldiversityYoder,etal.[9]reportedfromM.ovaliscomparedtothepresentstudyislikelyduetotheshorterincubationtimesfollowedbytheseauthorsdays,whichwouldfavorthefast-growingfungitheydocumented.TheoveralllowfungaldiversitywefoundassociatedwithExechiopsis/Anatellasp.maybeduetothesmallbodysizeandshortlifespanofthesedipteranscomparedtotheotherarthropodsandbatswesampled.Fungusgnatadultslivedaystoweeks,whileadultmoths,harvestmen,andspiderssurvivemonthstoyears[53,59,60].AlthoughfewerplateswereinoculatedwithExechiopsis/Anatellasp.,agreaternumberofindividualsweresampled.Fungiassociatedwithfungusgnatsincaveshavenotbeenpreviouslystudied,butKeates,etal.[61]sampledfungusgnatsBradysiasp.inconifernurseriesinBritishColumbiaandculturedspeciesofPenicillium,Cladosporium,Mucor,Rhizopus,Cephalosporium,Alternaria,Ulocladium,Fusarium,Botrytiscinerea,andPhoma.Fungiassociatedwithaninsectofsimilarsizetothefungusgnat,themosquitoCulexpipiens,havebeenstudiedincaves.Teernstra-EekenandEngel[62]sampledC.pipiensandHeleomyzidaeiesincavesintheNetherlandsandculturedLecanicilliumlecanii,Beauveriabassiana,Isariafarinosa,Aspergillussp.,Polycephalomycesformosus,Hirsutellaentomophila,Hirsutellasaussurei,Paecilomycessp.,andEntomophthorasp.mostcommon.Thesefungi,largelyEntomophthorasp.,caused56%%mortalityofoverwinteringC.pipiens,withthehighestmortalityinFebruary,justpriortotheemergenceofC.pipiensfromcaves[62].IncavesinCzechoslovakia,over-winteringC.pipienshad85%mortalityduetoinfectionwithEntomophthoradestruens[63].AlthoughwedidculturePdfromExechiopsis/Anatellasp.withafrequencyverysimilartothatofM.ovalis,theshortlife-span,smallbodysize,andperhapslimiteddispersalabilityoffungusgnats,probablylimitsthepossibleroleofthesedipteransintransportingPdsporeswithinorbetweencaves.Eighty-ninefungalgenerahavepreviouslybeendocumentedfromarthropodsincaves,mostcommonlyBeauveria,Aspergillus,Laboulbenia,Penicillium,Rhachomyces,Mucor,Mortierella,Cladosporium,Paecilomyces,Lecanicillium,Isaria,andHirsutella[7].Manyofthesegeneraareknownentomopathogensandaretargetedbyinvestigators,particularlyLaboulbeniaandRhachomyces,suggestingtheirrelativeabundanceonarthropodsinthecaveenvironmentmaybeoverestimated.Entomopathogens,althoughpresent,werenotcommonlyisolatedduringthisstudy,whichwascarriedoutduringthewintermonthswhentemperaturesarelow.Entomopathogens,suchasspeciesofTolypocladium,Paecilomyces,Beauveria,andIsaria,haveoptimalgrowthtemperaturesof20C[64].However,some,suchasBeauveriabassiana,areabletogrowattemperaturesaslowas5C,albeitataslowrate[65].

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Insects 2016 , 7 ,16 11of16Eilenberg,etal.[66]foundthattheentomophthoraleanfungusEntomophthoraschizophoraesurvivesthewinterinitshost,theadultdipteranPolleniasp.,throughslowdiseasedevelopmentandtransmissionamonghostshibernatinginclustersinunheatedattics.Overwinteringnearentrancesandinthetwilightzoneofcavesandmines,wheretemperaturesarerelativelylow,canreducemetabolismandhenceextendfoodreservesinharvestmenandotherarthropods[28].Inaddition,arthropodsmayavoidfungalinfectionswheretheyoverwinterclosetotheentranceofcold,northernhibernacula.Overwinteringarthropodsareknowntoventuredeeperintocaves[56]wheretemperaturesarehigherthanatentrances,butmaybemoresusceptibletofungalinfectionsinthesewarmertemperatures.Higherrelativehumidity,asfoundinthedarkzoneofcavescomparedtothelightzone,increasesfungalsurvival,germination,andsporulation,aswellasfacilitatingpenetrationofinsectcuticlesbyentomopathogenicfungi[67,68].Wehavepreviouslyisolatedentomopathogensfrombatsinthesehibernacula[21,22],conrmingtheirpresenceinthedarkzoneatourstudysites.Thecompositionandnumberoffungaltaxaperindividualwassimilarwhencomparingarthropodstobatsatthesamehibernaculum.Otherstudieshavefoundahigherdiversityoffungiclosertocaveentrancescomparedtothedarkzone[69,70].However,thispatternwasnotapparentatoursiteswhenarthropodsfromthecaveentrancewerecomparedtobatshibernatinginthedarkzone.Currentlythereislittleinformationonhowindividualarthropodorbatspeciesinteractwithsourcesoffungalsporesintheenvironment,however,manyoftheseinuencesareprobablysite-specic.Forexample,wefoundthatbatshibernatinginGlebeMineharboredgreaternumbersofbasidiomycetes,particularlyBaeosporasp.,thanarthropodsatthesamesite.Thismaybebecausebatsroosteddeeperwithinthemine,whereBaeosporasp.andothermushroomswereobservedgrowingonwood,whilearthropodsoverwinteredintheentrance-twilightzonewheremushroomswereabsent.Severalofthefungalgenerathatweremorecommononarthropodsthanonbats,suchasVerticilliumsp.,Botrytissp.,Hormonemasp.,andPhomasp.,areoftenassociatedwithgreenplantsandmayhavebeenblownintothehibernaculumentrance,wherearthropodsareconcentrated,ortransportedundergroundfromthesurfaceonthearthropodsthemselves.Althougharthropodsandbatsover-winterindifferentareasofundergroundhibernacula,arthropodsareintheightpathsofbatsarrivingandleavingcavesandmines.Furthermore,duringautumnswarming,batsmayroostoralightatcaveentrances.ArthropodsmaythereforeacquirePdsporesfromcavewallswherefungalsporeshavebeenshedfrompassingbats.However,thegrowthofPdatcaveentrances,whetherintheenvironmentoronarthropodsthemselves,cannotberuledoutatthistimeandcouldcontributetothelong-termpersistenceofPdincavesintheabsenceofbats[71].Pdcangrowattemperaturesaslowas0.8C,albeitmoreslowlythanatitsoptimumgrowthtemperatureof12C[72].ThevariationintheproportionsofPd-positivearthropodsoverthecourseofthisstudydidnotseemtoberelatedtoyear-to-yeartemperaturevariation.ThehighestyieldsofPddidnotoccurduringthewarmestwinters.TheproportionofPd-positivearthropodsdecreasedfrom2012to2014inGlebeMineastheover-winteringbatpopulationdeclinedduetoWNS-relatedmortality.ThissuggeststhatbatsareasourceofPdsporesathibernaculaentrances.However,wedidnotobservesuchadecreaseinDorchesterMine,wherethenumberofPd-positivearthropodspeakedin2013,despitetheextirpationofover-winteringbatsfromahighof140M.lucifugusandM.septentrionalisin2011duetoWNSmortality.OursamplingofN.elegans,thearthropodspecieswiththehighestyieldofPd,inDorchesterMinein2012and2014waslimitedcomparedto2013in2012,6in2013,and2in2014,andthismaypartlyexplainthepattern.ResultssuggestareservoirofviablePdsporesremainedintheenvironment,availablefortransmissiontoarthropods,foratleastayearfollowingextirpation,ornear-extirpation,oftheoverwinteringbatpopulation.Likewise,werecordedPd-positivearthropodsatGlebeMinein2014,whenhibernatingbatswerenolongerpresent.WhilethedatapresentedheresuggestsitisunlikelythatarthropodsplayamajorroleinthetransmissiondynamicsofPd,wedemonstratethatarthropodsmaycarryPdsporesandthereforehavethepotentialtotransportPdwithinoramonghibernaculashouldtheydispersenaturallyorbemovedanthropogenicallyfromPd-positivesites.Thelatterunderlinestheneedforthoseenteringhibernaculatoobserveaccepted

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Insects 2016 , 7 ,16 12of16decontaminationproceduresandforsuchprocedurestoevolveasourunderstandingofthepotentialmechanismsofPddispersalimprove.Althoughnaturaldispersalofarthropodsfromhibernaculaisunlikelytooccurovergreatdistances,itdoesappearthatthemovementofPdsporesviaarthropodsfromspeciccavesorminescanpotentiallycontinuetooccurforatleastayearaftertheextirpationofoverwinteringbatpopulations. 5.ConclusionsWeisolated87fungaltaxain64generafromfourspeciesofarthropodsoverwinteringineasternCanadianbathibernacula.Thesesiteswereoccupied,orformerlyoccupied,bybatsinfectedwiththefungusPseudogymnoascusdestructans,thecauseofwhite-nosesyndrome.ViablePdwasculturedfrom15.3%ofarthropodsuptoayearafterextirpationornear-extirpationofresidentbatpopulationsandmostfrequentlyfromharvestmenNelimaelegans.Fungalassemblagesonarthropodsweresimilartothoseonbatsinthesamesites.WhileitisunlikelythatarthropodsplayamajorroleinthetransmissiondynamicsofPd,wedemonstratethatarthropodsmaycarryviablePdsporesandthereforehavethepotentialtotransportPd,eithernaturallyoranthropogenically,withinoramonghibernacula.ThisunderlinestheimportanceforthoseenteringhibernaculatoobservedecontaminationproceduresandforsuchprocedurestoevolveasourunderstandingofpotentialmechanismsofPddispersalimprove.Acknowledgments:WeappreciatetheeffortsofJohnKlymkoinidentifyingthefungusgnatsandforeldassistance.ThankstoScottMcBurney,LiamMcBurney,andMirelleGeervlietforeldassistance.D.Robertskindlyallowedaccesstooneofthestudysites.AaronFairweatherandthreeanonymousreviewersprovidedhelpfulcommentsduringthewritingprocess.ResearchfundingwasprovidedbytheNewBrunswickWildlifeTrustFund,CrabtreeFoundation,NewBrunswickEnvironmentalTrust,NewBrunswickDepartmentofNaturalResources,andParksCanada. AuthorContributions:KarenJ.Vanderwolf,DavidMalloch,andDonaldF.McAlpineconceivedanddesignedtheexperiments;KarenJ.VanderwolfandDonaldF.McAlpineperformedtheexperiments;KarenJ.VanderwolfandDavidMallochanalyzedthedata;KarenJ.VanderwolfandDonaldF.McAlpinewrotethepaper. ConictsofInterest:Theauthorsdeclarenoconictofinterest.Thefundingsponsorshadnoroleinthedesignofthestudy;inthecollection,analyses,orinterpretationofdata;inthewritingofthemanuscript,andinthedecisiontopublishtheresults. Abbreviations Thefollowingabbreviationsareusedinthismanuscript:PdPseudogymnoascusdestructansWNSwhite-nosesyndromeDPYAdextrose-peptone-yeastextractagarSABSabouraud-dextroseagar References 1.Lorch,J.M.;Meteyer,C.U.;Behr,M.J.;Boyles,J.G.;Cryan,P.M.;Hicks,A.C.;Ballmann,A.E.;Coleman,J.T.;Redell,D.N.;Reeder,D.M.;etal.ExperimentalinfectionofbatswithGeomycesdestructanscauseswhite-nosesyndrome. Nature 2011 , 480 ,376.[CrossRef][PubMed] 2.NorthAmericanbatdeathtollexceeds5.5millionfromwhite-nosesyndrome.Availableonline:http://www.batcon.org/pdfs/USFWS_WNS_Mortality_2012_NR_FINAL.pdfaccessedon1February2012. 3.Turner,G.G.;Reeder,D.M.;Coleman,J.T.Ave-yearassessmentofmortalityandgeographicspreadofwhite-nosesyndromeinNorthAmericanbatsandalooktothefuture. BatRes.News 2011 , 52 ,13. 4.Lucan,R.K.;Bandouchova,H.;Bartonicka,T.;Pikula,J.;ZahradnikovaJr,A.;Zukal,J.;Martinkova,N.Ectoparasitesmayserveasvectorsforthewhite-nosesyndromefungus. Parasit.Vectors 2016 .[CrossRef] 5.Raudabaugh,D.B.;Miller,A.N.NutritionalcapabilityofandsubstratesuitabilityforPseudogymnoascusdestructans ,thecausalagentofbatwhite-nosesyndrome. PLoSONE 2013 , 8 ,e78300.[CrossRef][PubMed]

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Insects 2016 , 7 ,16 13of16 6.Ribeiro,J.M.;Labruna,M.B.;Mans,B.J.;Maruyama,S.R.;Francischetti,I.M.;Barizon,G.C.;deMirandaSantos,I.K.ThesialotranscriptomeofAntricoladelacruzifemaleticksiscompatiblewithnon-hematophagousbehaviorandanalternativesourceoffood.InsectBiochem.Mol.Biol.2012,42,332.[CrossRef][PubMed]7.Vanderwolf,K.J.;Malloch,D.;McAlpine,D.F.;Forbes,G.J.Aworldreviewoffungi,yeastsandslimemoldsincaves. Int.J.Spel. 2013 , 42 ,77.[CrossRef] 8.Benoit,J.B.;Yoder,J.A.;Zettler,L.W.;Hobbs,H.H.MycooraofatrogloxenicCaveCricket,HadenoecuscumberlandicusOrthoptera:Rhaphidophoridae,fromtwosmallcavesinnortheasternKentucky.Ann.Entomol.Soc.Am. 2004 , 97 ,989.[CrossRef] 9.Yoder,J.A.;Benoit,J.B.;Christensen,B.S.;Croxall,T.J.;HobbsIII,H.H.Entomopathogenicfungicarriedbythecaveorbweaverspider,MetaovalisAraneae,Tetragnathidae,withimplicationsformycooratransfertocavecrickets. J.CaveKarstStud. 2009 , 71 ,116. 10.Pugsley,C.EcologyoftheNewZealandglowworm, Arachnocampaluminosa Diptera:Keroplatidae,inthe GlowwormCave,Waitomo. J.R.Soc.NZ 1984 , 14 ,387.[CrossRef] 11.Yoder,J.A.;Benoit,J.B.;Hobbs,H.H.,III;Nelson,B.W.;Main,L.R.;Gibas,C.F.TheentomopathogenicfungusBeauveriacaledonica,anewlyidentiedpathogenofcavecrickets,Hadenoecusspp.Orthoptera:Rhaphidophoridae. SpeleobiologyNotes 2015 , 7 ,1. 12.Smr,J.;Kovc,L.;Mike,J.;ustr,V.;Lukeov,A.;Tajovsk,K.;Novkov,A.;Renkov,P.Foodsourcesofselectedterrestrialcavearthropods. Subterr.Biol. 2015 , 16 ,37.[CrossRef] 13.Estrada-Brcenas,D.A.;Palacios-Vargas,J.G.;Estrada-Venegas,E.;Klimov,P.B.;Martnez-Mena,A.;Taylor,M.L.BiologicalactivityofthemiteSancassaniasp.Acari:Acaridaefrombatguanoassociatedwiththepathogenicfungus Histoplasmacapsulatum . Mem.Inst.Oswaldo.Cruz. 2010 , 105 ,127. 14.Stephenson,S.;Slay,M.;Slay,C.;Tuggle,A.CavecricketsOrthoptera:RhaphidophoridaeasvectorsofDictyostelidsProtista:Dictyosteliida. Entomol.News 2007 , 118 ,292.[CrossRef] 15.Boyer-Lefevre,N.H.LesLaboulbnialesdesTrechinaecavernicolespyrnens.Ann.Splo.1966,21,775.16.Enghoff,H.;Santamaria,S.Infectiousintimacyandcontaminatedcaves—ThreenewspeciesofectoparasiticfungiAscomycota:LaboulbenialesfromblaniulidmillipedesDiplopoda:Julidaandinferencesabouttheirtransmittalmechanisms. Org.Divers.Evol. 2015 , 15 ,249.[CrossRef] 17.Langwig,K.E.;Frick,W.F.;Reynolds,R.;Parise,K.L.;Drees,K.P.;Hoyt,J.R.;Cheng,T.L.;Kunz,T.H.;Foster,J.T.;Kilpatrick,A.M.Hostandpathogenecologydrivetheseasonaldynamicsofafungaldisease,white-nosesyndrome. Proc.R.Soc.B. 2014 .[CrossRef][PubMed] 18.Dickson,G.W.Apreliminarystudyofheterotrophicmicroorganismsasfactorsinsubstrateoftroglobiticinvertebrates. NSS.Bull. 1975 , 37 ,89. 19.Malloch,D.;Blackwell,M.Dispersalbiologyoftheophiostomatoidfungi.InCertatocystisandOphiostoma.Taxonomy,EcologyandPathogenicity;Wingeld,M.J.,Seifert,K.A.,Webber,J.F.,Eds.;APS:SaintPaul,MN,USA,1993;pp.195. 20.Agrios,G.M. Plantpathology ,3rded.;AcademicPressInc.:SanDiego,CA,USA,1988;pp.416. 21.Vanderwolf,K.J.;McAlpine,D.F.;Malloch,D.;Forbes,G.J.EctomycotaassociatedwithhibernatingcavebatsineasternCanadapriortotheemergenceofwhite-nosesyndrome.Northeast.Nat.2013,20,115.[CrossRef] 22.Vanderwolf,K.J.;Malloch,D.;McAlpine,D.F.Fungiassociatedwithover-winteringTricoloredbats,Perimyotissubavus,inawhite-nosesyndromeregionofEasternCanada.J.CaveKarstStud.2015,77,145. 23.McAlpine,D.F.;Vanderwolf,K.J.;Forbes,G.J.;Malloch,D.ConsumptionofbatsMyotisspp.byraccoonsProcyonlotorduringanoutbreakofwhite-nosesyndromeinNewBrunswick:Implicationsforbatmortalityestimates. Can.FieldNat. 2011 , 125 ,257. 24.Peck,S.B.AreviewofthecavefaunaofCanada,andthecompositionandecologyoftheinvertebratefaunaofcavesandminesinOntario. Can.J.Zool. 1988 , 66 ,1197.[CrossRef] 25.Moseley,M.Acadianbiospeleology:compositionandecologyofcavefaunaofNovaScotiaandsouthernNewBrunswick,Canada. Int.J.Spel. 2007 , 36 ,1.[CrossRef] 26.Vanderwolf,K.J.;McAlpine,D.F.;Forbes,G.J.;Malloch,D.Winterbatpopulationsandcavemicroclimatepriortoandattheonsetofwhite-nosesyndromeinNewBrunswick. Can.FieldNat. 2012 , 126 ,125.

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Insects 2016 , 7 ,16 14of16 27.UnitedStatesFishandWildlifeService.ReviseddecontaminationprotocolJune25,2012.Availableonline:http://www.whitenosesyndrome.org/resource/revised-decontamination-protocol-june-25-2012accessedon15December2015. 28.Holmberg,R.G.;Angerilli,N.P.;LaCasse,L.J.OverwinteringaggregationsofLeiobunumpaessleriincavesandminesArachnida,Opiliones. J.Arachnol. 1984 , 12 ,195. 29.Vockeroth,J.R.Mycetophilidae.InManualofNearcticDipteraVolume1;McAlpine,J.F.,Peterson,B.V.,Shewell,G.E.,Teskey,H.J.,Vockeroth,J.R.,Wood,D.M.,Eds.;BiosystematicsResearchInstituteAgricultureCanada:Ottawa,Canada,1981;pp.223. 30.Papavizas,G.C.;Davey,C.B.Evaluationofvariousmediaandantimicrobialagentsforisolationofsoilfungi.Soil.Sci. 1958 , 88 ,112.[CrossRef] 31.Domsch,K.H.;Gams,W.;Anderson,T.H.CompendiumofSoilFungi,2nded.;IHW-Verlag:Regensburg,Germany,2007;p.672. 32.Seifert,K.;Morgan-Jones,G.;Gams,W.;Kendrick,B.TheGeneraofHyphomycetes;CBS-KNAWFungalBiodiversityCentre:Utrecht,theNetherlands,2011;p.997. 33.Khankhet,J.;Vanderwolf,K.J.;McAlpine,D.F.;McBurney,S.;Overy,D.P.;Slavic,D.;Xu,J.ClonalexpansionofthePseudogymnoascusdestructansgenotypeinNorthAmericaisaccompaniedbysignicantvariationinphenotypicexpression. PLoSONE 2014 , 9 ,e104684.[CrossRef][PubMed] 34.Foster,J.;Drees,K.UniversityofNewHampshire.PersonalCommunitcation,2014. 35.Moseley,M.;Hebda,A.OverwinteringLeiobunumelegansOpiliones:PhalangiidaeinCavesandMinesinNovaScotia. Proc.N.S.Inst.Sci. 2001 , 41 ,216. 36.Dickson,G.W.;Kirk,P.W.DistributionofheterotrophicmicroorganismsinrelationtodetritivoresinVirginiacaveswithsupplementalbibliographyoncavemycologyandmicrobiology.IntheDistributionalHistoryoftheBiotaoftheSouthernAppalachians.IV.AlgaeandFungi;Parker,B.C.,Roane,M.K.,Eds.;UniversityofVirginiaPress:Charlottesville,VA,USA,1976;pp.205. 37.Goodnight,C.J.;Goodnight,M.L.SpeciationamongcaveopilionidsoftheUnitedStates.Am.Midland.Nat.1960 , 64 ,34.[CrossRef] 38.Balazy,S.;Wisniewski,J.;Kaczmarek,S.Somenoteworthyfungioccurringonmites.B.Pol.Acad.Sci.Biol.1987 , 35 ,199. 39.Kubatova,A.;Cerny,M.;Novakova,A.NewrecordsofmicromycetesfromtheCzechRepublic.IV.Acrodontiumsalmoneum,Chaunopycnisalba,andCylindrocarpostylusgregarious,andnotesonDactylarialanosaand Trichodermasaturnisporum . Czech.Mycol. 2001 , 53 ,237. 40.Hwang,S.C.;Chen,C.L.Anewleaf-specklediseaseofbananacausedbyAcrodontiumsimplexinTaiwan.Plant.Protect.Bull. 1986 , 28 ,413. 41.Cabello,M.N.DeuteromycotinafromAntarctica—NewspeciesofhyphomycetesfromDancocoast,Antarcticpeninsula. Mycotaxon 1989 , 36 ,91. 42.Meyer-Rochow,V.B.;Liddle,A.R.StructureandfunctionoftheeyesoftwospeciesofopilionidfromNewZealandglow-wormcavesMegalopsalistumida:Palpatores,andHendeamyersicavernicola:Laniatores.Proc.R.Soc.Lond.B. 1988 , 233 ,293.[CrossRef] 43.Machado,G.;Raimundo,R.L.;Oliveira,P.S.Dailyactivityschedule,gregariousness,anddefensivebehaviourintheNeotropicalharvestmanGoniosomalongipesOpiliones:Gonyleptidae.J.Nat.Hist.2000,34,587.[CrossRef] 44.Mains,E.B.EntomogenousspeciesofAkanthomyces,HymenostilbeandInsecticolainnorthAmerica.Mycologia1950 , 42 ,566.[CrossRef] 45.Leatherdale,D.ThearthropodhostsofentomogenousfungiinBritain.Entomophaga1970,15,419.[CrossRef] 46.Greenstone,M.H.;Ignoffo,C.M.;Samson,R.A.SusceptibilityofspiderspeciestofungusNomuraeaatypicola.J.Arachnol. 1987 , 15 ,266. 47.Mitov,P.G.HarvestmenOpiliones,Arachnida—Carriersofplantandfungusspores.Acta.Zool.Bulg.1992,43 ,75. 48.Cokendolpher,J.C.PathogensandparasitesofOpilionesArthropoda:Arachnida.J.Arachnol.1993,21,120. 49.Cokendolpher,J.C.;Mitov,P.G.Naturalenemies.InHarvestmen:TheBiologyofOpiliones;Pinto-da-Rocha,R.,Machado,G.,Giribet,G.,Eds.;HarvardUniversityPress:Cambridge,MA,USA,2007;pp.339.

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Insects 2016 , 7 ,16 15of16 50.Balazy,S.AnewspeciesofentomophthoraceaeMycophyta:EntomophthoralesfromPoland.J.Invert.Path.1978,31 ,275.[CrossRef] 51.Keller,S.Arthropod-pathogenicEntomophthoralesofSwitzerland.I.Conidiobolus,Entomophaga,andEntomophthora . Sydowia 1987 , 40 ,122. 52.McKillop,W.B.ScoliopteryxlibatrixNoctuidaeandTriphosahaesitataGeometridaeincavesinManitoba,Canada. J.Lepid.Soc. 1993 , 47 ,106. 53.Kowalski,W.EthologicalandecologicalobservationsonLepidopteraintheirsubterraneanhibernatingplacesinthevicinityofCracow.ZeszytyNaukoweUniwersytetuJagiellonskiego.PraceZoologiczne,Zeszyt1965 , 103 ,97. 54.Roederk,K.D.;Fenton,M.B.AcousticresponsivenessofScoliopteryxlibatrixL.Lepidoptera:Noctuidae,amoththatshareshibernaculawithsomeinsectivorousbats. Can.J.Zool. 2011 , 51 ,681.[CrossRef] 55.Kubatova,A.;Dvorak,L.Entomopathogenicfungiassociatedwithinsectshibernatinginundergroundshelters.Czech.Mycol. 2005 , 57 ,221. 56.Rector,M.A.ForagingintheCaveEnvironment:TheEcologyoftheCaveSpiderMetaovalisAraneae:tetragnathidae.Master'sThesis,OhioStateUniversity,Columbus,OH,USA,2009. 57.Smithers,P.TheearlylifehistoryanddispersalofthecavespiderMetamenardiLatreille,1804Araneae:Tetragnathidae. Bull.Br.Arachnol.Soc. 2005 , 13 ,213. 58.DelFiol,F.;Solveig,T.;Riccardo,G.Fungalsporesandpollenaspotentialnutritionaladditivesforthecrossspider Araneusdiadematus ClerckAraneae,Araneidae. BoletinMicologico 2007 , 22 ,47. 59.Reed,C.F.;Witt,P.N.Growthrateandlongevityintwospeciesoforb-weavingspidersAraneae:Argiopidae.Bull.Brit.Arach.Soc. 1972 , 2 ,111. 60.Gnaspini,P.Development.InHarvestmen:TheBiologyofOpiliones;Pinto-da-Rocha,R.,Machado,G.,Giribet,G.,Eds.;HarvardUniversityPress:Cambridge,MA,USA,2007;pp.455. 61.Keates,S.E.;Sturrock,R.N.;Sutherland,J.R.PopulationsofadultfungusgnatsandshoreiesinBritishColumbiacontainernurseriesasrelatedtonurseryenvironment,andincidenceoffungiontheinsects.NewForest. 1989 , 3 ,1.[CrossRef] 62.Teernstra-Eeken,M.H.;Engel,A.NotesonentomophthorousfungionHeleomyzidaeandCulicidaeDiptera.J.Invert.Path. 1967 , 9 ,431.[CrossRef] 63.Weiser,J.;Batko,A.AnewparasiteofCulexpipiensEntomophthoradestruensnewspeciesPhycomycetesEntomophthoraceae. Folia.Parasitol.Praha. 1966 , 13 ,144. 64.Roberts,D.W.;Campbell,A.S.Stabilityofentomopathogenicfungi.Misc.Publ.Entomol.Soc.Am.1977,10,19. 65.Fernandes,E.K.;Rangel,D.E.;Moraes,A.M.;Bittencourt,V.R.;Roberts,D.W.ColdactivityofBeauveriaandMetarhizium ,andthermotoleranceof Beauveria . J.Invert.Path. 2008 , 98 ,69.[CrossRef][PubMed] 66.Eilenberg,J.;Thomsen,L.;Jensen,A.B.Athirdwayforentomophthoraleanfungitosurvivethewinter:Slowdiseasetransmissionbetweenindividualsofthehibernatinghost. Insects 2013 , 4 ,392.[CrossRef] [PubMed] 67.Rawlins,J.E.MycophagyinLepidoptera.InFungus-InsectRelationships:PerspectivesinEcologyandEvolution;Wheeler,Q.,Blackwell,M.,Eds.;ColumbiaUniversityPress:NewYork,NY,USA,1984;pp.382. 68.Steinkraus,D.C.Factorsaffectingtransmissionoffungalpathogensofaphids.J.Invert.Path.2006,92,125.[CrossRef][PubMed] 69.Mulec,J.;Vaupotic,J.;Walochnik,J.ProkaryoticandeukaryoticairbornemicoorganismsastracersofmicroclimaticchangesintheundergroundPostojnaCave,Slovenia. Environ.Microbiol. 2012 , 64 ,654. 70.Vaughan,M.J.;Nelson,W.;Soderlund,C.;Maier,R.M.;Pryor,B.M.AssessingfungalcommunitystructurefrommineralsurfacesinKartchnerCavernsusingmultiplexed454pyrosequencing.Environ.Microbiol.2015,70 ,175.[CrossRef][PubMed] 71.Reynolds,H.T.;Ingersoll,T.;Barton,H.A.ModelingtheenvironmentalgrowthofPseudogymnoascusdestructansanditsimpactonthewhite-nosesyndromeepidemic.J.Wildl.Dis.2015,51,318.[CrossRef][PubMed]

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Insects 2016 , 7 ,16 16of16 72.Verant,M.L.;Boyles,J.G.;Waldrep,W.,Jr.;Wibbelt,G.;Blehert,D.S.Temperature-dependentgrowthofGeomycesdestructans,thefungusthatcausesbatwhite-nosesyndrome.PLoSONE2012,7,e46280.[CrossRef][PubMed] 2016bytheauthors;licenseeMDPI,Basel,Switzerland.ThisarticleisanopenaccessarticledistributedunderthetermsandconditionsoftheCreativeCommonsAttributionCC-BYlicensehttp://creativecommons.org/licenses/by/4.0/.


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