Wolbachia Infection in a Natural Parasitoid Wasp Population

16 Pages·2015·1.2 MB·English

RESEARCHARTICLE
Wolbachia Infection in a Natural Parasitoid
Wasp Population
AnneDuplouy1*,ChristelleCouchoux2,IlkkaHanski1,SaskyavanNouhuys1,3
1 UniversityofHelsinki,MetapopulationResearchCentre,DepartmentofBiosciences,P.O.Box65,FI-
00014,Helsinki,Finland,2 UniversityofSussex,SchoolofLifeSciences,BrightonBN19QG,United
Kingdom,3 CornellUniversity,DepartmentofEcologyandEvolutionaryBiology,Ithaca,NewYork,14853,
UnitedStatesofAmerica
* [email protected]
Abstract
ThematernallytransmittedbacteriumWolbachiapipientisiswellknownforspreadingand
persistingininsectpopulationsthroughmanipulationofthefitnessofitshost.Here,weiden-
tifythreenewWolbachiapipientisstrains,wHho,wHho2andwHho3,infectingHyposoter
horticola,aspecialistwaspparasitoidoftheGlanvillefritillarybutterfly.ThewHhostrain
(ST435)infectsabout50%oftheindividualsintheÅlandislandsinFinland,withadifferent
infectionrateinthetwomitochondrial(COI)haplotypesofthewasp.Theverticaltransmis-
sionrateofWolbachiaisimperfect,andlowerinthehaplotypewithlowerinfectionrate,sug-
OPENACCESS
gestingafitnesstrade-off.WefoundnoassociationofthewHhoinfectionwithfecundity,
Citation:DuplouyA,CouchouxC,HanskiI,van longevityordispersalabilityoftheparasitoidhost.However,preliminaryresultsconveyspa-
NouhuysS(2015)WolbachiaInfectioninaNatural
tialassociationsbetweenWolbachiainfection,hostmitochondrialhaplotypeandparasitism
ParasitoidWaspPopulation.PLoSONE10(8):
ofH.horticolabyitshyperparasitoid,Mesochoruscf.stigmaticus.Wediscussthepossibility
e0134843.doi:10.1371/journal.pone.0134843
thatWolbachiainfectionprotectsH.horticolaagainsthyperparasitism.
Editor:KostasBourtzis,InternationalAtomicEnergy
Agency,AUSTRIA
Received:March3,2015
Accepted:July15,2015
Published:August5,2015
Copyright:©2015Duplouyetal.Thisisanopen Introduction
accessarticledistributedunderthetermsofthe
Wolbachiapipientisisestimatedtoinfectupto60%ofallinsectspecies[1–4].Thematernally
CreativeCommonsAttributionLicense,whichpermits
inheritedbacterialendosymbiontoftenmanipulateshostreproductiontoincreasethefre-
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare quencyofinfectedhostindividualsinthepopulation[5].Moreover,Wolbachiacanmanipulate
credited. otheraspectsofhostbiologyviainteractionsrangingfromparasitismtomutualism.Inpara-
siticinteractions,Wolbachiainfectiondecreaseshostfecundity[6–10],longevity[11],and
DataAvailabilityStatement:Datafilesareavailable
fromDryadunderthedoi:10.5061/dryad.34sv3. othercomparablephysiologicaltraits[12,13],whileinmutualisticinteractionsWolbachia
increasesfitnesscomponentsofthehost,suchasresistancetopathogens(reviewedin[14]).In
Funding:TheprojectwasfundedbytheAcademyof
parasitoidwasps,Wolbachiahasbeenshowntoimposephysiologicalcosttothehost(asin
Finland(grant#255553toSvN,213547and250444
toSvNandIH,and266021toAD)andtheOttoMalm Leptopilinaheterotoma[15])andtomanipulatehostreproduction,suchasthelytokousparthe-
foundation(granttoAD). nogenesisinTrichogramma[16,17]andAsobarajaponica[12]orcytoplasmicincompatibility
inthewaspsLeptopilina[18]andNasonia[19].Incontrast,someAsobarawaspshaveastrong
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. mutualisticrelationshipwiththeirWolbachia,whichplaysanessentialroleinthedevelopment
PLOSONE|DOI:10.1371/journal.pone.0134843 August5,2015 1/16
WolbachiainHyposoterhorticola
ofthehostovaries[20].Inmanycases,exactlyhowWolbachiaaffectshostbiologyandpersists
inthehostpopulationisunknown.
PrevalenceofWolbachiainfectionvariesfrom100%toextremelylowbetweenhostspecies
andamongpopulationswithinahostspecies[21,22].Thisrangeofprevalencecomesabout
becausehost-symbiontassociationseitheroccurunderdifferentenvironmentalconditions
[23–25];areatdifferentstagesofinfectionhistory[26];orareaffectedbyvariablegeneticfac-
torsofeitherthesymbiontorthehost[27,28].Furthermore,throughmanipulationofitshost
reproductivesystemWolbachiahavethepotentialtopersistintheirhostpopulationdespite
obviouscoststohostfecundity.CIinducing-Wolbachiadothisbycausingtotalorpartial
reproductivefailurebetweenmalesandfemaleswithadifferentinfectionstatus,providinga
reproductiveadvantagetofemalehosts,whichcanproduceviableoffspringregardlessof
whethertheymatewithinfectedoruninfectedmales.Wolbachiathatdistortpopulationsex-
ratio(male-killing,parthenogenesisandfeminization)promotetheproductionandfitnessof
infectedfemales,ensuringthetransmissionofinfectionthroughgenerations[5].Finally,the
coexistenceofinfectedanduninfectedindividualsinapopulationispossibleifthereisperfect
verticaltransmissionofthesymbiont[29–32].
IntheÅlandislandsinSouthwestFinland,thepopulationdynamicsoftheGlanvillefritil-
larybutterfly,Melitaeacinxia,anditsspecializedparasitoidwasp,Hyposoterhorticola,are
closelylinked.Thebutterflylivesasametapopulationinalandscapeconsistingof~4000small
habitatpatches(drymeadows),ofwhich500to800areoccupiedyearly.Thehabitatpatches
areclusteredinto~100semi-independentnetworks(SINs)[33]withlittlegeneflowofthebut-
terflybetweenthenetworks.Incontrast,H.horticolaishighlydispersiveamonglocalbutterfly
populationsresultinginhighgeneflowandlowisolationbydistance[34].Hyposoterhorticola
isitselfparasitizedbyasimilarlydispersivespecialisthyperparasitoid,Mesochorusc.f.stigmati-
cus[35].Ofthethreespecies,H.horticolaistheonlyoneyetfoundtobeinfectedwithWolba-
chia(Duplouypers.obs.).Insuchaspatiallyandtrophicallystructuredsystem,Wolbachia
epidemiologydependsbothontheeffectsofthebacteriumonthewasphost,aswellasonthe
demographyandpopulationdynamicsofitshost.
Here,wereportthreenewWolbachiastrains,anddescribeone,wHho,whichispresentin
theÅlandislands,atintermediateprevalence.Inanattempttoexplaintheintermediatelevelof
infection,wemeasuredtherateofverticaltransmissionandtheassociationofinfectionwith
severallife-historytraits.WereportspatialassociationssuggestingthatWolbachiainfection
mayinfluencetheresistanceofH.horticolatoitscommonlarvalspecialisthyperparasitoidM.
c.f.stigmaticus.
MaterialsandMethods
Insectmaterial
Hyposoterhorticola(Gravenhorst)(Hymenoptera:Ichneumonidae:Campoplaginae)isasoli-
taryegg-larvalparasitoidoftheGlanvillefritillarybutterfly,Melitaeacinxia(L.)(Lepidoptera:
Nymphalidae)[36].ThewaspparasitizesaboutathirdofthehostcaterpillarsintheÅland
islands[37].WeusedH.horticolarearedfromnaturallyparasitizedhostcaterpillarsthatwere
systematicallysampledintheÅlandislands(60°07’N,19°54’E)between2008and2013asa
partofthelong-termecologicalstudyoftheGlanvillefritillary[33].TodocumentWolbachia
infectionoveralongerperiod,wealsoscreenedindividualssampledlesssystematically
between1993and2005andpreservedfrozen(-20°C)inethanol.Moreover,toplacethesam-
plesinageographicalcontext,weanalysedtheinfectionstatusofH.horticolacollectedfrom
Estonia(58°41’N,22°50’E&59°35’N,24°05’E),France(43°61’N,3°87’E),Spain(37°38’N,5°
98’E)andSweden(60°00’N,18°00’E&56°73’N,16°67’E).Altogether751waspswerescreened
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WolbachiainHyposoterhorticola
Table1. WolbachiainfectionrateofthetwomitotypesofHyposoterhorticolainsamplescollected
fromFinland,SwedenandEstoniabetween1993and2013(fordetailsseeS1andS2Tables).
Population wHho-infected Uninfected
C T C T
Estonia - - 43 -
Finland(Åland) 75 128 185 27
Sweden 9 15 11 -
doi:10.1371/journal.pone.0134843.t001
(Table1,S1–S3Tables).Thebutterflyanditsparasitoidarenotclassifiedasthreatenedspecies
andhencenopermitsarerequiredfortheircollectioninEurope.
WolbachiastrainsandHyposoterhorticolamitotypediversity
DNAwasextractedfromthewaspabdomenusingaQiagenDNeasybloodandtissuesextrac-
tionkit,followingthemanufacturer’sprotocol(Qiagen,USA).TheDNAqualitywastestedby
PCRamplifyingthemitochondrialCOIgene(primerpairLCO/HCO[38]).Anysamplewith
noamplificationforCOIwasremoved.TheCOIlocuswassequencedfrom514samples(422,
45,6,6and35fromFinland,Estonia,France,SpainandSweden,respectively,S2Table).Wol-
bachiainfectionwasassessedthroughtheamplificationoftwotosevenWolbachialoci,includ-
ingthewspgene(primerpair81F/691R[39]),the16S-RNAgene(553F_W/1334R_W[40])
andthefiveMLSTloci(MultiLocusSequenceTypinggenes:CoxA,FbpA,HCpA,GatBand
FtSZusingrespectiveprimersfrom[41]).WealsosequencedtwotosevenWolbachialoci
(MLST,wspand16sloci)from249(218,5and26fromFinland,FranceandSweden,respec-
tively)ofthe340waspsinfectedwithWolbachia.TheFstvalues,basedontheCOIsequences,
werecalculatedusingArlequin3[42](S5Table).Eachconsensussequencewassubmittedto
GenBank(KF722987–94,KJ150624,KM598264–68&KR185318–21).
Phylogenies
Phylogenies(Fig1,S1FigandS1File)werebuiltusingtheonlinetree-buildingprogramPhy-
logeny.fr[43,44]intheOne-clickmodewithdefaultsettings.ForWolbachia,weusedthe
concatenatedwsp,16SandMLSTconsensussequencesfromourthreestrainsandeightaddi-
tionalWolbachiastrains(GenBankAM999887,CP001391,CP003883,AE017321,AE017196,
EF025179to-183,EF078895,AB474245to-249,AB094382,AB052745).Werootedthetree
usingwBmasanoutgroup.ForH.horticolamtDNA,weusedthesevenmitotypesobtained
fromoursamplestogetherwithsequencesfromfiveotherspeciesofHyposoter(GenBank
JF963449,JQ576453,JQ576979&DQ538855to-58)andtwoCampoletissonorensis(Ichneu-
monidae,GenBankDQ538849&-50).WerootedthetreeusingtwoCotesiamelitaearum(Bra-
conidae,GenBankKM598269&-70)asoutgroups.
MappingoftheinfectionintheÅlandislandpopulation
Eachwaspwascollectedfromaknowngeographiclocation,allowingustoanalyseWolbachia
prevalenceatthelevelsoflocalbutterflypopulations,semi-independentpatchnetwork(SIN),
andinthelandscapeasawhole.Weusedlog-linearmodelstoinvestigatetemporalvariationin
prevalencefrom2008until2012inthepooledmaterialfortheÅlandislands(variables:year&
infectedversusuninfected)andvariationinprevalenceamongthefiveSINswiththelargest
samplesizes(variables:SIN&infectedversusuninfected).Finally,wecomparedinfectionlev-
elsbetweenSwedenandtheÅlandislandsusingFisher’sexacttestusingpooleddatafrom
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WolbachiainHyposoterhorticola
Fig1.Rootedphylogrambasedontheconcatenatedsequencesofthewsp,MLST(CoxA,FbpA,FtsZ,GatBandHcpA)and16Sgenesfromnine
Wolbachiastrains,includingwHho,andontheconcatenatedsequencesofwspand16SofwHo2andwHho3,withbranchsupportvalues.The
wBmstrainfromtheparasiticnematodeBrugiamalayiwasusedasanoutgroup.A,BandDarethreeWolbachiasuper-groups.
doi:10.1371/journal.pone.0134843.g001
2008to2012(categoriescomparedwereinfectedanduninfectedindividualsfromSwedenand
Finland).
Wolbachiatransmissionrate
TheverticaltransmissionofWolbachiaisnotalwaysperfect[32].Wemeasuredtheproportion
ofWolbachia-infectedprogenyfrom15femalewaspsfromtheÅlandislands,eightinfected
andsevenuninfected.Eachfemaleparasitizedabutterflyhostegg-clutchinthelaboratory(see
[45]formethods).Thecaterpillarswerereareduntilthe4thinstar(15caterpillarsperfemale)
oruntilwaspsemergedasadults.Wedetectedparasitisminthe4thinstarcaterpillarsbyampli-
fyingboththeparasitoidwaspDNA(microsatelliteregionHho10[46])andasectionofthe
repeatelementprotein-b11fromthewasp-integratedichnovirus(length:429bp,PCRanneal-
ingtemperature:50°C,primers:34F5’-GGCGTATGCARTAGTGRTGAAand463R5’-GAT
TATCGGAACCTGRTYGAA,thisarticle).Additionally,becausemostH.horticolaemerging
fromthesamefamilygroupofGlanvillefritillarylarvaearefullsiblings[47],weestimated
transmissionrateinalargersampleusingtheinfectionstatusofwaspsemergingfromsamples
oftwoorthreehostindividualtakenfromeachof51hostfamilygroupsduringthefieldsurvey
(S4Table).Therateoftransmissionassociatedwiththetwowaspmitotypeswascompared
usingaFisher’sexacttest.
Hyposoterhorticolasexratio
Insomesystems,Wolbachiaskewsthesexratioofthehostpopulationtowardfemales
(throughkillingorfeminizationofmales,orviaparthenogenesis)[5].Aseachparasitized
M.cinxialarvalgroupcontainsprimarilyoffspringofasingleH.horticolafemale[47],we
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WolbachiainHyposoterhorticola
determinedtheper-motherallocationtomaleandfemaleprogeny,andthepopulation-level
sexratio,byrecordingthesexofeachindividualrearedfrom53larvalgroupsparasitizednatu-
rallyintheÅlandislandsbetween2007and2010.Theirinfection-statusisnotknown.How-
everyearlyrandomcollectionsshowthathalfofallcollectedwaspscarrywHhointheÅland
islands(S1Table),soweassumethataroundhalfofthese53larvalgroupsoriginatefrom
infectedwasps.Themeanbroodsexratio(fractionmale)wascomparedwithanequal(0.5)sex
ratiousingt-test.Thedistributionofbroodsexratioswascomparedwithanormaldistribution
usingtheShapiro-Wilkgoodnessoffittest.
Hyposoterhorticolaegg-load
Wolbachiaisknowntoaffecthostfecundity[7,9].Wetestedforanassociationbetweenthe
infectionstatusandegg-load(asproxyforfecundity)of213females(including111infected
and102uninfected)rangingfromage0(dayofemergence)to51days(maximumlifespanin
thelaboratory).Thewaspswererearedfromnaturallyparasitizedcaterpillarsandrandomly
killedatdifferentages,yieldingasimilarsampleofinfectedanduninfectedindividualsateach
agepoint.Theovariesweredissectedinsterileconditionsandmatureeggsintheoviductand
calyxorganswerecountedaspartofanotherstudy[45].Theinfectionstatusofeachwaspwas
determinedupondeath.Theeggloadwasthenanalysedusingalinearmodel,withtheinfec-
tionstatusandageofthewaspasexplanatoryvariables.
Hyposoterhorticolalongevity
PresenceofWolbachiacandecreasehostlongevity[11].Fifteenfemalesand33males,reared
underlaboratoryconditionsfromcaterpillarsnaturallyparasitizedintheÅlandislandsin2010
and2011,werekeptinindividualvials(V=100ml)inanincubatorat12:12L/Dand18/10°C
day/nighttemperature.Waspswerefed30%honeysolutioneveryotherdayuntilnaturaldeath
(asdescribedin[45]),andlaterassessedforWolbachiainfection.Wetestedtheassociationof
longevitywithinfectionstatususingalinearmodel,withsexincludedasanextraexplanatory
variable.
AssociationofWolbachiainfectionwithhabitatconnectivity
IfWolbachiainfectiondecreasesdispersalabilityofwasps,wewouldexpectlowprevalenceof
thebacteriumwherelocalbutterflypopulationsaresparseinthelandscape(lowconnectivity)
[48],asthechancesforWolbachia-infectedwaspstoreachthesurroundinghabitatpatches
wouldbereduced.Alternatively,ifthebacteriumincreaseddispersalability[49],wemight
expectprevalencetobelowerinhighlyconnectedhabitatpatches.Foreachyear(2008–2013),
wecalculatedanindexofconnectivityofeachlocalbutterflypopulationfromwhichwasps
werereared,accountingforthedistancefromeachlocalbutterflypopulationtoallotherpopu-
lations,thesizesofthepopulationsandthedispersalabilityofthebutterfly[50,51].Usinga
linearmodelwetestedpossibleassociationoftheinfectionrateofH.horticolainalocalbutter-
flypopulationinyeartwiththeconnectivityofthepopulationinyeart-1,aswaspscollectedin
thecurrentyearweretheprogenyofadultsfromtheprevioussummer.
Wolbachiainfectionandhostmetabolicrate
Wolbachiacaninfluencebasicphysiologicalandmetabolicfunctionsofitshost.Thiswouldbe
especiallyrelevantforthedispersalabilityofthewasp,asflightismetabolicallyexpensive[52,
53].Wecomparedtheactiveandrestingmetabolicrates(AMRandRMR,respectively)of33
Wolbachia-infectedand29uninfectedwaspsrearedfromcaterpillarscollectedfromthewild
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in2013.Themetabolicratesweremeasuredusingflow-throughrespirometryattheconstant
temperatureof30°C[53].TheAMRwasmeasuredasthemaximumrateofCO emission
2
underabrightlightduring7minofforcedactivityinducedbyflickingthe100mlmetabolic
chambereachtimethewasp’swingswerestill(H.horticolamayflyformorethan30minwith-
outexhaustion,Duplouy,pers.obs.).WeusedtheaverageCO emissionrateduring60secpre-
2
cedingtheactiveperiod,underacovertoblocklight,asameasureoftheofthebaselineRMR.
MeasuresofAMRandRMRwerecorrectedforvariationinbodymassbyusingresidualsfrom
linearregressionbetweenthemetabolicrateandtheweight.Linearmodelswereusedtoana-
lyseassociationsofCO emissionratewiththeinfectionstatusandsexofindividuals.
2
PrevalenceofWolbachiaandtherateofhyperparasitism
IfWolbachiaconfersresistanceagainstparasitoids,wemightexpectthebacterialinfectionto
behighwheretheriskofparasitismishigh[54].Weusedlinearregressiontocomparethe
prevalenceofWolbachiainfectioninH.horticolaandtherateofhyperparasitismbyMeso-
chorusc.f.stigmaticususingdatafromtensemi-independentpatchnetworks(SINs)inthe
Ålandislands.ForeachSIN,wecalculatedtherelativeabundanceofthehyperparasitoidas
thenumberofhyperparasitoidsinthesampledividedbythesumoftheparasitoidsand
hyperparasitoids.
Dataaccessandanalysessoftware
DatafilesareavailableinDryadunderthedoi:10.5061/dryad.34sv3.Statisticalanalyseswere
performedusingR64[55].
Results
WolbachiainfectioninHyposoterhorticola
TheFinnishandSwedishpopulationsofH.horticolacarryacommonWolbachiastrainthat
belongstotheWolbachiaB-supergroup(Fig1),andwhichwenamewHho(ST435).Asingle
specimenfromtheÅlandislands(sampledin1998)wasinfectedbyanA-supergroupstrain,
wHho3.SamplesfromEstonia(n=43)andSpain(n=6)wereuninfected,whilesamplesfrom
France(n=6)carriedathirdstrain,calledwHho2,alsointheA-supergroup.
IntheÅlandislandsandSweden,Wolbachiainfectionrateisrelatedtothemitochondrial
haplotype(mitotype)ofthehost.BasedontheCOIsequence,therearetwocommonmito-
types,whichdifferbyasinglebasepairsubstitution(synonymoussubstitutionbetweenCand
Tatthenucleotideposition364,thecodonencodingforLeucineatposition121).Individuals
withmitotypeTaremorelikelytobeinfectedbyWolbachiathanindividualswithmitotypeC
(Åland:87%vs.30%;Sweden:100%vs.56%,Fisher’sexacttestsP=0.0001andP=0.0068,
respectively,Fig2).TheoverallinfectionrateissignificantlyhigherinsamplesfromSweden
thanfromtheÅlandislands(P=0.012).WithintheÅlandislands,fromwherewehavesam-
plesfrom2008until2013,theoverallinfectionratehasremainedstable(non-significantinter-
actionbetweenyearandinfectionrate,df=4,P=0.21).However,therearehighlysignificant
differencesintheinfectionratebetweenSINs(df=16,P<0.001,Fig2),andtheoverallinfec-
tionrateiscorrelatedwiththefrequenciesofthetwomitotypes(df=1,P=0.039,Fig3).
Transmissionrate
Ofthefifteenfemalewaspsusedinthetransmissionexperiment,eightwereinfectedby
Wolbachia.FiveoftheeightshowedhighWolbachiatransmission(>85%),whiletheremain-
ingthreeshowedlowertransmission(between33and66%).Noneoftheoffspringoftheseven
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WolbachiainHyposoterhorticola
Fig2.GeographicalpatternofWolbachiainfectionrate(ingrey)insamplesatthe(A)regionalscaleinFinland,SwedenandEstonia,and(B)
amongnetworksofhabitatpatchesintheÅlandislandsinFinland.Thedataincludessamplescollectedbetween2008and2013.Largecirclesshow
theinfectionrateintheTmitotypeandsmallcirclesintheCmitotype.Whenonemitotypeisabsent,therespectivecircleisalsoabsent(e.g.onlytheC
mitotypeoccursinEstonia).
doi:10.1371/journal.pone.0134843.g002
uninfectedmotherswereinfected(S4Table).Asonlyoneoftheeightinfectedfemaleshad
mitotypeC,thismaterialcannotbeusedonitsowntocomparethetwomitotypes.Wethere-
foreaugmentedthelab-rearedmaterialwiththefield-collectedsamples.Amongthepresumed
Fig3.RelationshipsbetweenHyposoterhorticolamitotype,WolbachiainfectionandhyperparasitismbyMesochorusc.f.stigmaticusin10sub-
regionsintheÅlandislands.Thedatawerecollectedfrom2008to2013.Panel(A)showstheassociationbetweenWolbachiainfectionrateandthe
relativeabundanceoftheTmitotypeinH.horticola(P=0.01).Thenexttwopanelsshowtherelationshipsbetweentherelativeabundanceofthe
hyperparasitoidand(B)therelativeabundanceoftheTmitotype(P=0.039)and(C)Wolbachiainfectionrate(P=0.11).
doi:10.1371/journal.pone.0134843.g003
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WolbachiainHyposoterhorticola
siblinggroupsproducedbyaninfectedindividualoftheCmitotype,nineoutof17(53%)
includedoneuninfectedoffspring(twotosevenoffspringpermother).Incontrast,amongthe
presumedsiblingsgroupsfrominfectedfemaleswithmitotypeT,onlyfiveoutof30(17%)sib-
linggroupshadatleastoneuninfectedindividual.Theseresultssuggestthattransmissionis
lessefficientintheCmitotype(Fisher’sexacttest,P=0.018).Theabsolutevaluesofuninfected
individualsarelikelytobebiased,assomeinfectedfemalesofeithermitotypemayhavehad
onlyuninfectedoffspring,andhencethefemalewaswronglyscoredasuninfected.However,
comparisonofthetwomitotypessuggestsmoreefficienttransmissionintheTmitotype.
Life-historyandfitnessconsequencesofWolbachiainfection
IntheÅlandislands,H.horticolahasanequalpopulation-levelsexratio(0.54,SD=0.24),
whichdoesnotdifferfrom0.5(t=1.01,P>0.05).Moreover,thebroodlevelsexratiowasnor-
mallydistributedaroundthemeaninwaspsfrom53hostlarvalfamilygroups(Shapiro-Wilk
Goodness-of-fittest,W=0.9827,P=0.78).Thereisthusnoevidenceforextremesexratiodis-
tortion.However,theseresultsdonotruleoutthepossibilityofotherreproductivemanipula-
tionsapartfromsexratiodistortion,suchasCI.
WefoundnoassociationofWolbachiainfectionwithfitness-relatedtraitsinH.horticola.
Infectedanduninfectedfemaleshadsimilaregg-load(520and522,df=1,P>0.05,Fig4).
Femaleslivedlongerthanmales(37versus31days,df=1,P=0.036),andWolbachiainfection
wasunrelatedtolongevityineithersex(df=1,P>0.05,S2Fig).
WefoundnoassociationofWolbachiainfectionwithhabitatconnectivityintheprevious
year(FigAinS2File),suggestingthattheinfectiondoesnotinfluencewaspmobility.Consis-
tentwiththisnegativeresult,therewasnoassociationofWolbachiainfectionwithactiveor
restingmetabolicrate(FigsBandCinS2File).
Fig4.Theegg-loadoffemalewaspsfromtheÅlandislandsinrelationtotheirageandwhethertheyareinfectedbyWolbachia(triangles,n=111)
ornot(circles,n=102).
doi:10.1371/journal.pone.0134843.g004
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Parasitoid-hyperparasitoidinteractionandWolbachiainfection
WeanalysedtherelativeabundancesofH.horticolaanditshyperparasitoidMesochorusc.f.
stigmaticus,aswellasWolbachiainfectionrate,intenregionsintheÅlandislands(Fig2B).
ThehyperparasitoidhadgreaterrelativeabundancewheretheprevalenceofthehostTmito-
typewaslower(P=0.039).RecallingthatWolbachiainfectionrateishigheramongT-than
C-mitotypewasps(above),thisresultsuggeststhehypothesisthatwHhoinfectionprotects
H.horticolaagainstthehyperparasitoid.Indeed,inthesedataWolbachiainfectionrateislow
wherehyperparasitismishigh,thoughtheassociationisnotstatisticallysignificant(P=0.11,
Fig3C).InEstonia,whereWolbachiaisentirelyabsent,allthewaspsareofmitotypeCandthe
hyperparasitoidisabsent.UnderthehypothesisthatWolbachiaprotectsagainsthyperparasit-
ism,wewouldexpectWolbachiatobeabsentwherethereisnohyperparasitoidbecause,asit
wouldbenoadvantagetoitshost,itwouldnotspread.
Discussion
WedescribethreeWolbachiastrainsintheichneumonidwaspHyposoterhorticola,aparasit-
oidoftheGlanvillefritillarybutterfly:twoA-groupstrains,wHho2andwHho3,detected
fromFrenchsamplesandauniqueFinnishsample,respectively,andaB-groupstrain,wHho
(ST435),whichiswidespreadintheÅlandislandsinFinlandandinSweden.NoWolbachia
wasidentifiedinsamplesfromEstonia(n=47).ThehostH.horticolahassevenCOImitotypes
inourmaterial,ofwhichthetwocommonones(mitotypesCandT)occurinFinlandandSwe-
den,oneraremitotypeoccursinEstonia,andfourotherdivergentmitotypescamefromFrance
andSpain.Thepost-glacialrangeexpansionofH.horticolaisunclear,butitmayhavespread
tonorthernEurope,possiblywithdifferentWolbachiastrains,frombothAsianandsouthern
Europeanrefugia,ashasitshostbutterfly[56]andanotherspecialistparasitoidCotesiameli-
taearum[57].
RateoftransmissionandassociationofWolbachiainfectionwithhost
mitotype
WefoundthatintheÅlandislandstherateofverticaltransmissionofWolbachiaisimperfect,
suggestingthattheinfectionpersistsinanintermediatestate,inbalancebetweenfitnessbene-
fitsandcosts[58,59].Moreover,thetwoH.horticolamitotypesarenotequallyinfected.Inthe
FinnishandSwedishpopulations,WolbachiaiscommoninthehostmitotypeT(92%)but
muchlesscommonintheCmitotype(43%).InEstonia,allwaspshadtheCmitotypeand
wereuninfected.
BothmitochondriaandWolbachiaaretransmittedmaternally.HencepatternsofmtDNA-
Wolbachiaassociationreflectinfectionbiogeography,strengthoftransmission,andhistoryof
Wolbachiaspreadinhostpopulations[60,61].Whenverticaltransmissionisefficient,Wolba-
chiainfectionmayresultinaselectivesweepleadingtodistinctinfectedanduninfectedmatri-
lines([26,62]e.g.inbutterflies[63]andfigwasps[64]).Whentransmissionisimperfector
whenthereishorizontaltransfer,infectedanduninfectedindividualsmaysharethesame
mitotypes[65].InfectionrateofthewHhostraininFinlandandSwedenmaybehigherinthe
TthantheCmitotypebecausewHhoinfectedthecommonancestralmatrilineofbothmito-
types,buttransmissionrateishigherintheTmitotype.Alternatively,thewHhostrainmay
havefirstarisenintheTmitotypeandlatercolonizedtheCmitotypebylateraltransfer.Such
lateraltransmissioncouldoccuriftwoindividualsparasitizethesamehost(superparasitism),
whichcanoccasionallyhappeninH.horticola[37].AlternativelythehyperparasitoidM.c.f.
stigmaticuscouldtransmitthebacteriumbetweenhosts(e.g.inaphids[66]).Finally,itisalso
PLOSONE|DOI:10.1371/journal.pone.0134843 August5,2015 9/16
WolbachiainHyposoterhorticola
possiblethatthemitotypediversityishigherthanwhattheCOIgenealoneindicates,which
wouldincreasethecomplexityofthesystem.
Wolbachiainfectionandhostfitness
Wolbachiaisknowntoinfluencehostfitness,reproductivesystemandbehaviourtoimprove
itstransmission[67].InthisstudytheprevalenceofwHhodifferedgeographicallybetweenthe
studyregions,from0to74%,atthescaleofseveralhundredkm(Fig2A),aswellasatasmaller
spatialscalewithintheÅlandislands,whereinfectionratevariesfrom8to100%regionally
(Fig2B).Withefficienttransmission,asappearstohappenintheTmitotype,thebacterium
wouldneedtoenhancehostfitnessonlylittle,ortoinduceastrongCI,topersist.IntheC
mitotypewithlessefficientverticaltransmission,thefitnessbenefitwouldneedtobegreaterto
compensateforthelowertransmissionrate.
Modelspredictthathighvariationinprevalenceamongpopulationsmayoccurwhenthere
ishighspatialpopulationdynamics[59,68].EnvironmentalheterogeneityintheÅlandislands
leadingtolargevariationandfluctuationoflocalhostpopulationsizes[69]andprevalenceof
thehyperparasitoidM.c.f.stigmaticus[35],mayinteractwiththeeffectsofWolbachiaonits
host,leadingtospatialvariationinprevalence.
Longevityandfecundityarecorrelatedwithfitnessinparasitoids[70],andareoftenaffected
byWolbachia[15,71].Wolbachiainfectioncanalsoinfluencebehaviouraltraits,suchasdis-
persalinmosquitos[49].However,wefoundnoevidenceforwHhoinfluencinghostwasplon-
gevity,fecundityordispersalinthefield.Thelackofanassociationofinfectionwithlife
historyorbehavioursuggeststhatthepatternofWolbachiainfectionintheÅlandislandsmay
simplybeinatransitionalstate,onitswaytowardfixationorextinction(e.g.inbutterflies
[26]).However,thespreadofaCI-inducingorabeneficialstraincouldalsoshowsuchhetero-
geneitybetweenlocalpopulations[72,73].
Alternatively,Wolbachiamayhavealesscommonlystudiedassociationwithfitness.One
interestinghypothesis,suggestedbyourresults,isthatWolbachiaconfersresistancetohyper-
parasitism.Severalsymbionts,includingWolbachia([74]butsee[75,76])havebeenfoundto
protecttheirhostsagainstparasitoids(e.g.inaphids[77,78]andfruitflies[79]).Thespecialist
hyperparasitoidM.c.f.stigmaticusisubiquitousintheÅlandislandsandSweden[35],where
Wolbachiaprevalenceisgreaterthan50%,whileM.c.f.stigmaticusisabsentfromEstonia[37],
whereWolbachiaisalsoabsent.Furthermore,withintheÅlandislands,therelativeabundance
ofthehyperparasitoiddecreasedwithincreasingprevalenceofthehostTmitotype.AstheT
mitotypeisalsomoreoftenassociatedwithWolbachiathantheCmitotype,wesuggestthat
thepresenceofWolbachiamayimproveresistancetohyperparasitism.Thus,wHhocouldbe
maintainedbecausetheinfectionreducesthelikelihoodofsuccessfulhyperparasitism.
Toconclude,inthiscomprehensivelandscape-levelstudyofWolbachiaprevalenceina
largespatiallystructuredwasppopulation,thereisastableintermediateWolbachiainfection
rate,whichissignificantlydifferentintwohostmitochondrialhaplotypes(mitotypes).This
patternhasseveralpossibleexplanations,includingimperfectbutdissimilarverticaltransmis-
sionrateinthetwomitotypes.Second,wefoundnodirecteffectofinfection,positiveornega-
tive,onfitness-relatedtraitsofthehost,includingegg-load(fecundity),adultlongevity,
metabolicrateandameasurereflectingdispersalrateinthefield.Third,wehavenoevidence
ofsex-ratiodistortioninthehost,thoughwecannotruleouttheexpressionofCIanditsconse-
quencesforthespreadofthebacteriuminthepopulation.Thelackofapositiveassociation
withhostfitnessmayindicatethatWolbachiaisinatransitionalstateinthelandscape,orthat
itismaintainedviasomeothermechanism.Onesuchmechanismisprotectionagainsthyper-
parasitism.Wedonothavedirectexperimentalevidenceforthishypothesis,butitissupported
PLOSONE|DOI:10.1371/journal.pone.0134843 August5,2015 10/16