Should Symbionts Be Nice or Selfish? Antiviral Effects of Wolbachia Are Costly but Reproductive ...

20 Pages·2015·2 MB·English

RESEARCHARTICLE
Should Symbionts Be Nice or Selfish? Antiviral
Effects of Wolbachia Are Costly but
Reproductive Parasitism Is Not
JulienMartinez☯*,SuzanOk☯,SophieSmith,KianaSnoeck,JonP.Day,Francis
M.Jiggins
DepartmentofGenetics,UniversityofCambridge,Cambridge,UnitedKingdom
☯Theseauthorscontributedequallytothiswork.
* [email protected]
Abstract
Symbiontscanhavemutualisticeffectsthatincreasetheirhost’sfitnessand/orparasitic
effectsthatreduceit.Whichofthesestrategiesevolvesdependsinpartonthebalanceof
theircostsandbenefitstothesymbiont.WehaveexaminedthesequestionsinWolbachia,a
OPENACCESS
verticallytransmittedendosymbiontofinsectsthatcanprovideprotectionagainstviralinfec-
Citation:MartinezJ,OkS,SmithS,SnoeckK,Day tionand/orparasiticallymanipulateitshosts’reproduction.Acrossmultiplesymbiontstrains
JP,JigginsFM(2015)ShouldSymbiontsBeNiceor
wefindthattheparasiticphenotypeofcytoplasmicincompatibilityandantiviralprotectionare
Selfish?AntiviralEffectsofWolbachiaAreCostlybut
ReproductiveParasitismIsNot.PLoSPathog11(7): uncorrelated.Strongantiviralprotectionisassociatedwithsubstantialreductionsinotherfit-
e1005021.doi:10.1371/journal.ppat.1005021 ness-relatedtraits,whereasnosuchtrade-offwasdetectedforcytoplasmicincompatibility.
Editor:ElizabethAnnMcGraw,MonashUniversity, Thereasonforthisdifferenceislikelythatantiviralprotectionrequireshighsymbiontdensities
AUSTRALIA butcytoplasmicincompatibilitydoesnot.TheseresultsareimportantfortheuseofWolbachia
Received:February24,2015 toblockdenguevirustransmissionbymosquitoes,asnaturalselectiontoreducethesecosts
mayleadtoreducedsymbiontdensityandthelossofantiviralprotection.
Accepted:June11,2015
Published:July1,2015
Copyright:©2015Martinezetal.Thisisanopen
accessarticledistributedunderthetermsofthe
AuthorSummary
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany Arthropodsarecommonlyinfectedwithheritablebacteria,andsomeofthesesymbionts
medium,providedtheoriginalauthorandsourceare
canprotecttheirhostsagainstinfectionand/orbereproductiveparasites.Whichofthese
credited.
traitsevolveswilldependonwhetherthetraitiscostlytothesymbiontandthehost.Using
DataAvailabilityStatement:Allrelevantdataare apanelofstrainsofthesymbiontWolbachiainthefruitflyDrosophilasimulans,wefound
withinthepaperanditsSupportingInformationfiles.
thatthebeneficialeffectofantiviralprotectionandtheparasiticphenotypeofcytoplasmic
Funding:ThisstudywasfundedbytheWellcome incompatibilityoccurindependentlyacrossthestrains.Wefoundthathighantiviralpro-
TrustgrantWT094664MA(http://www.wellcome.ac. tectionisassociatedwithhighsymbiontdensitiesandstrongreductionsinotherlife-his-
uk/).FMJreceivedthefunding.FMJisalsosupported
torytraitsaffectingthefitnessofboththesymbiontandthehost.Incontrastcytoplasmic
byaRoyalSocietyResearchFellowship.Thefunders
incompatibilitydidnotinducecostsonthesetraits.Thistrade-offbetweenantiviralpro-
hadnoroleinstudydesign,datacollectionand
analysis,decisiontopublish,orpreparationofthe tectionandotherfitnesscomponentsmayselectforreducedantiviralprotection,which
manuscript. wouldendangerthelong-termsuccessofprogramsusingWolbachiatoblockthetrans-
missionofmosquito-borneviruses.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
PLOSPathogens|DOI:10.1371/journal.ppat.1005021 July1,2015 1/20
CostofMutualismandParasitisminWolbachia
Introduction
Heritablesymbiontsarefrequentininsectsandtheirevolutionarysuccessreliesonvarious
strategies.Bysharingacommonrouteoftransmissionwiththeirhost’sgenes,theybenefit
fromincreasinghostfitness.Consequently,numerousendosymbioticbacteriaevolvedtowards
mutualism,forexamplebycomplementingtheirhostdiet[1,2],increasingtolerancetoenvi-
ronmentalstresses[3]orprotectingagainstnaturalenemies[4–9].However,becausemostof
theseheritablebacteriaarematernally-transmitted,theevolutionaryinterestsofhostandsym-
biontarenotperfectlyalignedsinceonlyfemalestransmitthesymbiont.Thishasledtomany
symbiontsevolvingselfishstrategiesthatconsistofparasiticmanipulationoftheirhost’srepro-
ductionbyinducingfemale-biasedsex-ratiosorcytoplasmicincompatibility(CI)[10].CIisa
spermmodificationthatresultsinembryonicmortalityincrossesbetweenuninfectedfemales
andmalesharboringthesymbiont,thusgivingacompetitiveadvantagetoinfectedfemales
thatcanrescuethespermmodification.Mutualismandreproductivemanipulationarenot
mutuallyexclusive,andsomesymbiontsdisplayboth[11].However,thebalancebetweenthe
benefitsandcostsoftheseextendedphenotypestothesymbiont’sfitness,aswellasthegenetic
correlationsbetweenthem,willdeterminewhichofthesestrategiesisfavouredbynatural
selection.
Wolbachia,whicharecommonmaternally-transmittedbacterialsymbiontsofarthropods,
canbebothparasitesandmutualists.WolbachiahasbeenshowntoprotectDrosophilaand
mosquitoesagainstseveralRNAviruses—includingDengueandChikungunyaviruses[7,9,12–
15].Somestrainsalsoprotectinsectsagainstfilarialnematodes[16],Plasmodiumparasites
[12,17,18]andpathogenicbacteria[19].Althoughitisunclearhowimportantantiviralprotec-
tionisinnatureandwhetheritisunderstrongselection,someprotectiveWolbachiastrains
areabletoinvadehostpopulationswhileinducingnootherknownphenotypes[20,21].In
addition,Wolbachiahastheabilitytospreadrapidlythroughinsectpopulationsbyparasit-
icallymanipulatingreproduction,inparticularbyCI[22].Thiscombinationoftraitsmakes
Wolbachiaanattractivetoolforblockingdiseasetransmissionbymosquitoes,asCIallowsitto
spreadthroughvectorpopulationswhileitsantiviraleffectscanpreventthemfromtransmit-
tingarboviruses[23,24].
LevelsofbothantiviralprotectionandCImayevolverapidly.Duringthe20thcenturyin
naturalpopulationsofD.melanogastertheWolbachiastrainwMelCS,whichprovidesstrong
antiviralprotection,waspartiallyreplacedbywMel[25,26],whichprovidesweakerprotection
[27].InNorthAmericanpopulationsofD.simulans,fieldandexperimentaldatasuggestthat
thestrainwRihasevolvedtoproduceweakerlevelsofCIwithinafewdecades[28].
EffortstouseWolbachiatoblockthetransmissionofviruseshavefocusedlargelyonthe
mosquitoAedesaegypti,whichistheprimaryvectorofdenguevirus.Wolbachiahasbeensuc-
cessfullyintroducedintotwoAustralianpopulationsofAedesaegypti[29],andthreeyears
post-releaseithadreachedastableandhighprevalenceinthefielddespitehavinganegative
effectonthefecundityofmosquitoes[30].BothantiviralprotectionandlevelsofCIwere
maintainedovertime[30,31].
Inthelong-term,thepresenceoffitnesscostsisexpectedtoselectforbothhostgenesand
bacterialgenesthatreducethesecosts[32].Inaccordancewiththisprediction,theWolbachia
strainwRievolvedfromreducingthefecundityofthefliestoincreasingitwithintwodecades
inNorthAmericanpopulationsofD.simulans[33].Itispossiblethattheevolutionoflower
costscouldbeachievedbyadecreaseinbacterialdensities,ascostlyWolbachiatendtohave
highbacterialdensities[27,34,35].SinceahighWolbachiadensitymayberequiredforthe
expressionofbothantiviralprotection[14,27,34,36–38]andCI[35,39–42],theevolutionof
PLOSPathogens|DOI:10.1371/journal.ppat.1005021 July1,2015 2/20
CostofMutualismandParasitisminWolbachia
reducedWolbachiadensitymighttranslateintoacorrelateddecreaseintheabilitytoblock
arbovirustransmissionandinvadeinsectpopulations.
Toinvestigatethesequestions,weusedsixteenWolbachiastrainsinacommonhostgenetic
backgroundtomeasurethelevelofCIinducedandeffectsonotherfitness-relatedtraits,and
havetestedforcorrelationsbetweenthesetraitsandantiviralprotection.Ourresultsdemon-
stratethatantiviralprotectionisindependentofCIbutthatitisassociatedwithreductionon
otherfitnesscomponents.Furthermore,thistrade-offcanbeexplainedbythedensityofthe
bacteriainthesomatictissuesoftheinsect.Overall,ourstudysuggeststhatnewlyintroduced
Wolbachiainfectionsmayevolvetowardsweakerprotectioninthefield.
Results
Tocomparemultiplesymbiontstrainsindependentofhostgeneticeffects,weusedapanelof
WolbachiastrainsthathadbeentransferredfromdifferentDrosophilaspeciesintoasingle
inbredlineofD.simulans(Fig1F).Toavoideffectsofusinganinbredflyline,wecrossedthese
fliestoadifferentinbredflylineandusedtheF1progenyinourexperiments.Verticaltrans-
missionrateswerepreviouslyestimatedandwere100%forallWolbachiastrainsusedinthis
study[14].
Cytoplasmicincompatibilityandantiviralprotectionareindependent
traits
Cytoplasmicincompatibilitycausesanexcessofembryonicmortalityincrossesbetweensym-
biont-infectedmalesanduninfectedfemales.Therefore,inordertomeasurelevelsofCI
inducedbydifferentWolbachiastrains,wecrossedinfectedmalesofeachstrainwithunin-
fectedfemalesandcountedthenumberofeggsthathatched(9,432eggsfrom380females).
TherewasasignificanteffectofWolbachia(Deviance=681.81;df=16;P<0.0001)witha
cleardivisionbetween10strainsthatinduceCIandsixthatdonot(Fig1B).ThestrengthofCI
alsovariedamongthe10CIstrains,rangingfromjust0.5%oftheeggshatchinginincompati-
blecrossesinvolvingthewMelstrain,to38.7%oftheeggshatchingwithwStv.
Wehavepreviouslyshownthatthesestrainsprovidevaryinglevelsofprotectionagainstthe
virusesDCVandFHV[14],andusingthisdatawefoundthattherewasnocorrelationbetween
CIandtheantiviraleffectsofWolbachia.Thiswasthecaseregardlessofwhichvirustheflies
areinfectedwithorwhetherantiviralprotectionismeasuredintermsofincreasedsurvival
(blacklineinFig2Aand2B)orreducedviraltitre(blacklineinS1AandS1BFig).Thisconclu-
sionalsoholdsifweonlyanalysethe10strainsthatinducesignificantCI(redlineinFig2A
and2B;S1AandS1BFig).Sinceadecreaseinhatchrateinincompatiblecrossescanbedue
notonlytoCIbutalsotoaninducedcostonmalefertility,wealsoanalysedthecorrelation
betweenprotectionandlevelsofCIcorrectedfordifferencesinmalefertility(thehatchratesof
infectedfemalesmatedwithinfectedmalesrelativetohatchrateswhenmatedwithuninfected
males).Similartotheuncorrectedestimate,thesecorrectedlevelsofCIdidnotshowanysig-
nificantcorrelationwithantiviralprotection,whethermeasuredassurvivalafterinfection
(Pearson’scorrelationtest:Allstrains:DCV:P=0.28andFHV:P=0.86;CI-inducingstrains:
DCV:P=0.67andFHV:P=0.71)orasviraltitre(Pearson’scorrelationtest:Allstrains:DCV:
P=0.58andFHV:P=0.95;CI-inducingstrains:DCV:P=0.87andFHV:P=0.75).
Antiviralprotectioniscostly
AsWolbachiaisverticallytransmitted,reductionsinthesurvivalorfecundityofWolbachia-
infectedfemaleswillreducethefitnessofboththehostandthesymbiont.Toestimatethese
costs,wemeasuredegghatchrates(inparalleltotheCIcrosses,16,469eggsfrom555females),
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CostofMutualismandParasitisminWolbachia
Fig1.PhylogeneticdistributionofCIlevelsandWolbachiaeffectsonegghatchrates,fecundityandlifespan.(A)ThephylogenybasedontheMLST
genes16SrRNA,aspC,atpD,ftsZ,sucB,groEL,coxAandfbpAwasinferredusingClonalFramev1.2[43]asin[14].Strainsinboldconferredsignificant
antiviralprotection[14].Branchlabelsrepresentposteriorsupportvalues.Nodeswithlessthan50%supportwerecollapsed.Branchlengthsindicaterelative
time.(B)CImeasuredasegghatchratesincrossesbetweenuninfectedfemalesandWolbachia-infectedmales.(C)Egghatchratesincrossesbetween
Wolbachia-infectedfemalesandWolbachia-infectedmales(bluebars)oruninfectedmales(greybars).(D)FecundityofWolbachia-infectedfemales.(E)
LifespanofWolbachia-infectedfemales.Errorbarsarestandarderrors.*:significancerelativetotheWolbachia-freeline(Dunnett’stest;*:P<0.05;**:
P<0.01;***:P<0.001).ThedottedlineindicatesforeachtraitthemeanvalueintheWolbachia-freecontrols.(F)OriginalhostspeciesoftheWolbachia
strains.
doi:10.1371/journal.ppat.1005021.g001
early-lifefecundity(280,260eggsfrom1,548females)andfemalelifespan(913females)offlies
infectedwiththe16differentWolbachiastrains.
Wefoundsignificantvariationinegghatchratesbetweenflylinesinfectedwithdifferent
Wolbachiastrains(Fig1C;Deviance=340,97;df=16;P<0.0001).Whenthefatherwasunin-
fected,fourstrainscausedasignificantreductioninhatchrates,withthreeofthemresultingin
lessthan40%oftheeggshatching(Fig1C,greybars).Additionally,whenboththemotherand
fatherwereinfected,therewasatrendtowardsevenlowerhatchrates,withtwomorestrains
becomingsignificant(Fig1C,bluebars).Thissuggeststhatmalefertilityisalsobeingreduced
byWolbachiaorthatrescueofCIisnotperfectforsomeofthestrains(iethemodification
ofsperminmalesthatisrequiredforCIstillcausesembryonicmortalitywhentheeggis
infected).
FecundityandlifespanarealsoaffectedbyWolbachia.Forfecundity,twostrainsincreased
andtwostrainsreducedthenumberofeggslaid(Deviance=250.55;df=16;P<0.0001;Fig
1D).Wolbachiaalsoaffectedfemalesurvival(Deviance=52.37;df=16;P<0.0001),withfive
ofthesixteenstrainssignificantlyshorteninglifespan(Fig1E).
Thestrainsthatprovidethegreatestprotectionagainstviruses(measuredassurvival)
tendedtocausethegreatestreductionsintheotherlife-historytraitsoftheflies.Hatchratesof
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CostofMutualismandParasitisminWolbachia
Fig2.CorrelationbetweenCIandantiviralprotection.LevelsofCIestimatedasthepercentageofunhatchedeggsrelativetothemeanhatchratein
crossesbetweenuninfectedfemalesanduninfectedmales.Levelofprotectionmeasuredassurvivalin[14]uponinfectionwith(A)DCVand(B)FHV(0and
positivevaluesmeannodifferenceandincreaseinsurvivalcomparedtoWolbachia-freecontrolrespectively).Meansandstandarderrorsareshown.Solid
linesshowpredictedvaluesfromlinearregressionsusingallstrains(black)oronlyCI-inducingstrains(red).risthePearson’scorrelationcoefficientbetween
traits.
doi:10.1371/journal.ppat.1005021.g002
Wolbachia-infectedfemalesweresignificantlyreducedinfliescarryingthesymbiontsprovid-
ingthehighestlevelsofprotectionagainstbothDCVandFHV,whatevertheWolbachia-infec-
tionstatusofmales(Fig3Aand3B;S2AandS2BFig).Becausethetestedtraitsarenot
phylogeneticallyindependent,wereanalyzedthesecorrelationsusingphylogeneticindepen-
dentcontrasts(seemethods).Thecorrelationsbetweenhatchratesandlevelofprotectionwere
robusttothephylogeneticnon-independenceofthedata(S1Table).Higherlevelsofantiviral
protectionwerealsoassociatedwithreducedmalefertility(Fig3Cand3D)andlowerfecundity
(Fig3Eand3F),butthesecorrelationswereonlysignificantincaseofDCV.Phylogeneticinde-
pendentcontrastsanalysesalsoshowedthatcorrelationswithmalefertilityandfecunditywere
significantbutitstronglydependedonthebranchlengthusedinthelinearmodels(S1Table).
Nocorrelationwiththelevelofprotectionandfemalelifespanwasdetected(S2CandS2DFig;
notethesmallersamplesizesforthistrait).Interestingly,wAu,whichisanativestrainofD.
simulans,provideshighantiviralprotectionyetinducedlittlereductioninhatchratesor
fecundity.
IftheantiviraleffectsofWolbachiaweremeasuredaschangesinviraltitresratherthansur-
vival,mostofthecorrelationsbecamenon-significantormarginally-significant,butthedirec-
tionoftherelationshipsremainedthesame,withlowviraltitresassociatedwithstrongercosts
(S3A–S3JFig).Again,costsinducedbywAuonhatchratesweregenerallylowerthanexpected
bythecorrelationswithviraltitres.
Cytoplasmicincompatibilityisnotcostly
Similartoantiviralprotection,wetestedforcorrelationsbetweenlevelsofCIandother
componentsofhostfitness.TherewasnosignificantcorrelationbetweenthelevelofCI
andmalefertility,femalefecundity,lifespanorthehatchrateofeggsfromcrossesbetween
PLOSPathogens|DOI:10.1371/journal.ppat.1005021 July1,2015 5/20
CostofMutualismandParasitisminWolbachia
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CostofMutualismandParasitisminWolbachia
Fig3.Correlationsbetweenantiviralprotectionandotherhostlife-historytraits.AandB:correlationbetweensurvivalafterviralinfectionandegg
hatchratesincrossesbetweenWolbachia-freemalesandWolbachia-infectedfemales.Virusinfectionsused(A)DCVand(B)FHV[14](0andpositive
valuesmeannodifferenceandincreaseinsurvivalcomparedtoWolbachia-freecontrolrespectively).CandD:correlationbetweendecreaseinmalefertility
incrossesbetweenWolbachia-infectedparentsandsurvivalafterinfectionwith(C)DCVand(D)FHV.EandF:correlationbetweeneggnumberandsurvival
afterinfectionwith(E)DCVand(F)FHV.Meansandstandarderrorsareshown.Solidlinesshowpredictedvaluesfromlinearregressions.risthePearson’s
correlationcoefficientbetweentraits.
doi:10.1371/journal.ppat.1005021.g003
Wolbachia-infectedfemalesanduninfectedmales(Fig4A–4C;S4BFig).Incrosseswhereboth
parentswereWolbachia-infected,thelevelofCIwasnegativelycorrelatedwithhatchrates
(S4AFig).ThiswasonlythecasewhenbothCIinducingandnon-CIinducingstrainswere
analyzed,anditmayreflectincompleterescueofcytoplasmicincompatibility.
Wolbachiadensitymediatesthetrade-offbetweenprotectionandcost
WehypothesizedthatWolbachiamustinfectthegermlinetoinduceCIandsomatictissuesto
provideantiviralprotection,sodifferencesintissuetropismbetweensymbiontstrainsmay
partlyexplainwhytheyhavedifferentphenotypiceffectsontheirhosts.Toexaminethis,we
measuredWolbachiadensityinsomatictissues(headandthoraxoffemales),testesandfreshly
laideggs(asaproxyforthefemalegermline).
Therewerelargebetween-straindifferencesindensity(Fig5A–5C).Forexample,insomatic
tissuestheWolbachiacopynumbervariesovera19-foldrange.Furthermore,thestrainshave
differenttissuetropisms,withasignificantstrain-by-tissueinteraction(Fig5A–5C).Theden-
sityinthetestesandhead+thoraxtendedtobetightlycorrelated(Pearson’scorrelationtest:
r=0.89;P<0.0001),andfrequentlydifferedfromthedensityineggs(Pearson’scorrelation
test:head+thorax–eggs:r=0.63;P=0.01;testes–eggs:r=0.61;P=0.013).
VariationinWolbachiadensitycanexplainbetween-straindifferencesinantiviralprotec-
tionbutnotdifferencesinCI.ProtectionagainstDCVandFHVwaspositivelycorrelatedwith
Wolbachiadensityinheadandthorax,whethermeasuredassurvival(Fig6Aand6B)orviral
titres(S5AandS5BFig),evenafterremovingpotentialphylogeneticeffects(S1Table).This
holdswhenboththedensityinthesomaandeggsareincludedaspredictivevariables:protec-
tionshowsasignificantpartialcorrelationwithdensityinthesomabutnotwithdensityinthe
eggs(onlymarginallysignificantforFHVtitre;S2Table).Onthecontrary,thereisnocorrela-
tionbetweenlevelsofCIanddensityinthesomatictissues(Fig6C),inthetestesorintheeggs
(S6A–S6BFig).TheonlyexceptiontothiswaswhenonlyanalyzingCI-inducingstrains,levels
ofCIwerepositivelycorrelatedtotheWolbachiadensityineggs(redlineinS6BFig;noteeggs
areuninfectedintheCIcross).
ThenegativeeffectsofWolbachiaonhostlife-historytraitsarerelatedtothesymbiontden-
sity,withhatchrates,malefertilityandfecundityallnegativelycorrelatedtotheWolbachia
densityinthesomatictissues(Fig6D–6F)butnotwiththedensityintheeggs(Pearson’scorre-
lationtest:Hatchratewithuninfectedfather:P=0.08;hatchratewithinfectedfather:P=0.06;
malefertility:P=0.58;fecundity:P=0.27).Thesameconclusionholdswhencontrollingfor
theWolbachiaphylogeny(S1Table),althoughformalefertilityandfecunditysignificance
dependsonthebranchlengthusedforthelinearmodel.Whenthesetraitsareanalyzedwitha
multipleregression,theyshowsignificantpartialcorrelationswithdensityinthesomabutnot
withdensityintheeggs(S2Table).TherewasnocorrelationbetweenfemalelifespanandWol-
bachiadensityinanyofthetissues(Pearson’scorrelationtest:head+thorax:P=0.73;testes:
P=0.32;eggs:P=0.13).
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CostofMutualismandParasitisminWolbachia
Fig4.CorrelationsbetweenCIandotherhostlife-historytraits.ThelevelofCIiscorrelatedwith(A)theegghatchratesincrosseswithWolbachia-free
males,(B)thedecreaseinmalefertilityand(C)theeggnumber.Meansandstandarderrorsareshown.Solidlinesshowpredictedvaluesfromlinear
regressionsusingallstrains(black)oronlyCI-inducingstrains(red).risthePearson’sorSpearman’s(*)correlationcoefficientbetweentraits.
doi:10.1371/journal.ppat.1005021.g004
Discussion
Heritablebacterialsymbiontshavesuccessfullycolonizedawiderangeofarthropodsbyusing
adiversityofstrategiesrangingfrommutualismtoparasitism.Typicallytheevolutionofthese
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CostofMutualismandParasitisminWolbachia
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CostofMutualismandParasitisminWolbachia
Fig5.Wolbachiatissuetropism.MeanWolbachiadensityin(A)headandthoraxoffemales,(B)testesand(C)freshlylaideggs.Errorbarsarestandard
errors.LettersindicatesignificantdifferencesbasedonaTukey’shonestsignificancetestonln-transformeddata.Alltissueswereanalyzedinasinglelinear
modeltotestfordifferenceintissuetropism:straineffect:F =131.1;P<0.0001;tissueeffect:F =4448.8;P<0.0001;strain×tissueeffect:F
15,427 2,427 30,427
=11.5;P<0.0001.
doi:10.1371/journal.ppat.1005021.g005
symbiontstrategieshasbeenconsideredinisolation,butthiscanbemisleadingifthereare
trade-offsbetweenthesetraitsandothercomponentsofhostorsymbiontfitness.Identifying
thesetrade-offsisnotonlyaprerequisitetounderstandtheevolutionofsymbiosis,butwill
alsoinformtheuseofsymbiontsinappliedprograms.UsingasetofWolbachiastrainsthat
providevaryinglevelsofprotectionagainstviralpathogens,wefoundthatthismutualistic
effectwasindependentoftheabilitytoparasiticallymanipulatehostreproduction.Antiviral
protectionreliesonthebacteriareachinghighdensitiesinsomatictissuesandisassociated
withstrongreductionsinseveralhostlife-historytraits,whilereproductiveparasitismisnot
linkedtosymbiontdensityinsomatictissuesandnotcostlytoinfectedfemales.
Whilesomesymbiontsaremutualiststhatspreadthroughpopulationsbyincreasinghost
fitnessandothersareparasitesthatmanipulatehostreproduction,otherssimultaneouslyhave
botheffects[11].ItisalreadywellknownthatinWolbachiaantiviralprotectionandCIare
highlygeneticallyvariabletraits[14,27,37,44].However,toourknowledge,ourstudyisthe
firsttoassessbothtraitsinawidearrayofstrainsinacommonhostgeneticbackground.We
foundnocorrelationbetweentheexpressionsofthesephenotypes,withfourstrainsonlypro-
vidingprotection,twostrainsonlyinducingCI,eightstrainsinducingbothprotectionandCI,
andtwostrainsshowingneitherphenotype.Therefore,thesetraitshaveindependentevolu-
tionarytrajectories.Somestrainsmayalsorelyonalternativestrategiestobemaintainedin
populations,suchasenhancingthehostfecundityorotherfitnesscomponents[45].For
instance,twoofthetestedstrainsinourstudywereassociatedwithincreasedfecundity.
Besidesantiviralprotectionandreproductivemanipulation,Wolbachiainfectionscan
inducefitnesscosts,withimportantlife-historytraitsbeingaffectedsuchaslifespan,fecundity,
eggviabilityorlarvaldevelopmentandcompetitiveness[30,46–53].Inaccordancewithprevi-
ousstudies,wefoundWolbachia-inducedcostsonseveraltraitsthatshouldreduceboththefit-
nessofthehostandofWolbachia.Insomecasesthesecostscouldbeverylarge–forexample
somestrainsresultinthemajorityofinfectedeggsneverhatching,suggestingthatthosestrains
mightnotbeabletoinvadenaturalhostpopulations.
Wefoundthatantiviralprotectiontrade-offswithegghatchrates,femalefecundityand
malefertility.Inmanycaseshighlyprotectivestrainsinducedsubstantialreductionsinthese
fitnesscomponents.BecauseWolbachiareliesonhostreproductionforitstransmission,these
trade-offswillaffectboththehostandsymbiont,asbothpartnersbenefitfromantiviralprotec-
tionandbothwillsufferfromreducedfemalereproduction.Furtherevidencethatantiviral
protectioniscostlycomesfromacomparisonofthetwomainWolbachiagenotypesinD.mel-
anogasterpopulations,whichshowedthatthegenotypethatprovidedthegreatestantiviral
protectionalsoshortenedthelifespanofinfectedflies(Chrosteketal.2013).Similarly,when
wAuistransferredintoD.melanogasteritreacheshighdensities,providesstrongprotection
againstvirusesandshortensthelifespanofflies(Chrosteketal.2014).Interestingly,usinga
similarexperimentaldesigntoours,anotherstudyshowedthathighlevelsofprotectioncon-
ferredbythesymbiontHamiltonelladefensaagainstparasitoidsinaphidsareassociatedwith
lesscostlysymbiontstrainscontrarytowhatwefound[54].Whilethemechanismsofprotec-
tioninWolbachiaremaintobeelucidated,inH.defensaitisknownthatprotectionrelieson
thepresenceofabacteriophageencodingatoxin[55,56].Itislikelythatdifferentmechanisms
ofprotectionleadtodifferenttrade-offswithhostlife-historytraits.
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