Complete transition from chromosomal to cytoplasmic sex determination during prolonged Wolbachia symbiosis

Domain: biology
Published: 2026-01-08
Editorial outcome: minor_revision

Abstract
Wolbachia infection causes male-specific death in Ostrinia furnacalis, but its removal from infected strains results in female-specific death instead of restoring 1:1 sex ratio, suggesting that cytoplasmic Wolbachia, not the host genome, primarily determines femaleness in infected strains. This phenomenon is a striking example of the evolutionary outcome of cytoplasmic sex determination, potentially arising from prolonged host-symbiont co-evolution. Although we recently identified Oscar, the Wolbachia-encoded male-killing effector targeting the host masculinizing factor OfMasc in Ostrinia moths, inactivation or loss of the host’s endogenous feminizer remains unknown. Here we identify a W-linked primary feminizer, OfFem piRNA, which targets an mRNA encoding an OfMasc-interacting protein Ofznf-2. We demonstrate that Ofznf-2 is essential for both masculinization and dosage compensation. We also show that OfFem piRNA is entirely absent in the Wolbachia-infected lineage, providing molecular evidence that a male-killing Wolbachia hijacks the host feminizing piRNA function by acquiring the Oscar protein during prolonged endosymbiosis.

Introduction
Some bacterial endosymbionts, such as Wolbachia species, manipulate the reproductive systems of arthropod hosts through male killing, feminization, cytoplasmic incompatibility, or parthenogenesis1. In various host-endosymbiont combinations, male killing causes infected mothers to produce only female progeny by selectively killing the male offspring2. In the interaction between Ostrinia species and male-killing Wolbachia strains (wSca or wFur, Fig. S1), an evolutionary phenomenon termed “cytoplasmic sex determination” has been observed, as in a few other arthropod species3–5. In wFur-infected Ostrinia furnacalis, the Wolbachia male-killing protein Oscar essentially determines femaleness, as the host’s endogenous feminizing factor is considered to have been inactivated or lost during prolonged symbiosis6–9. While several bacterial effectors responsible for reproductive manipulations have recently been identified9–12, no molecular evidence has yet demonstrated the inactivation or absence of the host’s endogenous feminizing factor in symbiont-dependent cytoplasmic sex determination systems.

Lepidopteran sex chromosomes are female-heterogametic, with female-specific W chromosomes that are enriched in repetitive elements and transposons13–16. These elements serve as sources of PIWI-interacting RNAs (piRNAs), a class of small RNAs that silence transposons17. In Bombyx mori and Plutella xylostella, female-specific piRNAs originating from the W chromosome, called Feminizer (Fem) piRNAs, determine femaleness18,19. Fem piRNAs target an mRNA of Masculinizer (Masc), a lepidopteran-specific CCCH-tandem zinc finger protein gene essential for both masculinization and dosage compensation18,20–30. The Fem piRNA-PIWI protein complex cleaves Masc mRNA18, inhibiting male-type splicing of doublesex (dsx) and dosage compensation in male embryos18,31, making Fem piRNAs the primary determinant of sex in Lepidoptera. In contrast, we discovered that in O. furnacalis, Masc (OfMasc) is not regulated by female-specific piRNAs during sex determination, indicating a unique sex determination system distinct from that of B. mori32.

In Japan, the infection rate of Wolbachia in O. furnacalis is relatively low33 (~15%), where infected individuals consistently harbor a single strain of wFur (wFur9)33,34. Because this infection causes complete male killing, where Wolbachia-infected females can only mate with uninfected males, the autosomal gene pool is shared between uninfected and Wolbachia-infected strains. Unlike male killing observed in other lepidopteran insects35–39, depleting wFur from infected moths using antibiotics induces female killing instead of restoring a 1:1 sex ratio6,8. This suggests that the primary feminizing factor, which may be located on the matrilineally inherited W chromosome, has been lost or inactivated in the Wolbachia-infected strain. Recently, we demonstrated that the Wolbachia male-killing factor Oscar degrades OfMasc, thereby inhibiting male-type splicing of O. furnacalis dsx (Ofdsx) and dosage compensation9,40. Ectopic overexpression of Masc in females has been shown to cause female-specific death during larval stages41,42, as observed in female killing in antibiotics-treated Wolbachia-infected O. furnacalis, suggesting that Oscar directs the female-determining pathway by inactivating OfMasc in infected strains where the primary feminizing factor may be missing or nonfunctional. To unravel the molecular mechanism underlying Wolbachia-dependent cytoplasmic sex determination, identifying the endogenous feminizing factor of O. furnacalis and validating its absence or inactivation in infected females is essential.

Here, we identify a single feminizing piRNA derived from the W chromosome of O. furnacalis. Unlike Fem piRNAs in B. mori or P. xylostella, this piRNA targets an mRNA encoding Ofznf-2, a protein that interacts with OfMasc. Knockdown experiments reveal that Ofznf-2 is crucial for male-type dsx splicing and dosage compensation. Additionally, we find that this piRNA is entirely absent in the Wolbachia-infected lineage of O. furnacalis. These findings provide molecular evidence that a male-killing Wolbachia hijacks the role of a W-linked feminizing piRNA by acquiring the Oscar protein during prolonged endosymbiosis.

Results
AsingleW-linkedpiRNAtargetsmRNAsforCCCH-typezincfingerproteinsinO.furnacalis
WehypothesizedthatthefeminizingfactorinO.furnacalishadbeeninactivatedorlostintheWolbachia-infectedlineage.ToidentifytheprimaryfeminizingfactorinO.furnacalis,wesequencedthetranscriptomesofmolecularlysexedearlyembryosduringtheperiodofsexdetermination32;thesex-specificsplicingofOfdsxisfirstobservedafter12hourspost-oviposition(hpo)andisestablishedat48hpo.Fromthedenovoassembledtranscriptome,14contigswerefoundtobesignificantlymorehighlyexpressedinfemalesthaninmalesacrossallembryonicstages(Fig.S2a).Amongthese,twocontigsmetthefollowingcriteria:(i)higherexpressioninuninfectedembryosandovariescomparedtoinfectedones,and(ii)higherexpressioninuninfectedovariescomparedtouninfectedtestes(Fig.S2b).Intwoin-houseassembledwhole-genomeassemblies(version#1andversion#2,derivedfromO.furnacaliscollectedinJapan;TableS1)andthepublishedgenomeofO.furnacalis43(collectedinChina),thesetwocontigsweremappedtoasinglelocusontheWchromosomes.FragmentsfromthislocuswerePCR-amplifiedexclusivelyfromfemalegenomesamples(Fig.S3a,b).Combinedwiththeobservationthatitsexpressionwasrestrictedtofemales,notmales,duringthesexdeterminationperiod(Figs.1a,b,S3c,andS4a,b),thislocusemergedasastrongcandidateforencodingthefeminizingfactor.WedesignatedthislocusasO.furnacalisFeminizer(OfFem).TheOfFemlocusconsistedofnumerousrepeatelements(Fig.S5a),atypicalfeatureofpiRNAclusters44–46.SmallRNAsequencing(smallRNA-seq)revealedthatasinglefemale-specificpiRNA(OfFempiRNA)wasabundantlyproducedfromtheOfFemlocusinembryosandgonads(Figs.1a,candS4a,c).ThemostplausibletargetgeneofOfFempiRNA,namedOfznf-2,isaputativehomologoftheO.scapulaliszincfingergeneOsznf-247,48.WithintheOfFemlocus,sequencesof162–197basepairs(bp)homologoustoOfznf-2wererepeated17times(Fig.S5a,b).SixlocicontainingOfznf-2geneswereidentifiedacrosstheO.furnacalisgenome(version#2)(Fig.S6).TwocopiesofOfznf-2ontheWchromosomeappearedpseudogenizedduetoa6-kbp-longinsertion(Fig.S6),sotheywereexcludedfromfurtheranalysis.Theremainingfourcopieswerecategorizedintotwoexpressiontypes:latetype(Ofznf-2-L)andearlytype(Ofznf-2-E)(Fig.1d,e).Additionally,Ofznf-2-Econtainedaspecific75–131bp-longinsertionabsentinOfznf-2-L(Fig.S6).AcopyofOfznf-2-Lonthe28thchromosomewasexpressedthroughoutembryogenesisafter12hpo,whiletwocopiesonthe28thchromosomeandoneontheZchromosome,classifiedasOfznf-2-E,wereexpressedexclusivelyaround12hpo,whichistheonsetofsexdetermination32(Figs.1dandS7a).Boththeversion#1genome(derivedfromadifferentindividualfromthatusedforversion#2genome)andthepublishedgenome43alsoencodedatleastonecopyofOfznf-2-L and Ofznf-2-E, respectively (Fig. S8, Table S2), suggesting variability in the copy number of Ofznf-2-E among O. furnacalis populations.

Acrossthegenome,OfFempiRNAperfectlymatchedtheantisenseorientationofOfznf-2sequences(Figs.1eandS6).AllcopiesofOfznf-2-EandOfznf-2-Lencodedshortproteins(126–187aaand107aa,respectively)withtwoCCCH-typezincfingerdomains(Figs.1eandS9a).TheseproteinsshowedhighhomologytoBmznf-2,whichisrequiredforpropermaledifferentiationinB.mori49,50.InacelllinefromO.scapulalis,male-typeO.scapulalisdsx(Osdsx)wasexpressed,buttransinfectionwithmale-killingWolbachia(wSca)inducedtheexpressionoffemale-typeOsdsxandalteredOsznf-2expression47,48.TheseobservationssupporttheroleofOfznf-2proteinsinsexdetermination.ABLASTpsearchidentifiedhomologsofOfznf-2acrossLepidoptera,allcontainingtwoCCCH-typezincfingerdomainssimilartoMaschomologs(Fig.S10).ArapidamplificationofcDNAends(RACE)experimentconfirmedthatOfFempiRNA-mediatedcleavageofOfznf-2mRNAoccurredinearlyembryos(Figs.1fandS11).Atthisstage, Ofznf-2 mRNAexpression was higherinmalesthaninfemales(Fig.S7b,c).ThesefindingssuggestthatOfFemgeneratesafemale-specificpiRNAthatsuppressesOfznf-2mRNA,whichmayplayacriticalroleinmale-determiningfunctions.

Ofznf-2isrequiredformaledeterminationinO.furnacalis
TotestwhetherOfznf-2isessentialformaledetermination,weperformedembryonicknockdownexperimentsusingsmallinterferingRNA(siRNA)designedtotargettheconservedsequencesharedacrossallcopiesofOfznf-2.MicroinjectionofOfznf-2siRNAinhibitedthemale-typesplicingofOfdsx(Fig.2a,b)andledtotheupregulationofZ-linkedgenesineggmasses(Fig.2c).ThesefindingsindicatethatOfznf-2isnecessaryforbothmale-typesplicingofOfdsxanddosagecompensation.Furthermore,embryonicknockdownofOfznf-2producedadultmothswithafemale-biasedsexratio(Fig.2d),suggestingthatOfznf-2playsacriticalroleinmaleviability.AllobservedphenotypesmirroredthoseresultingfromtheknockdownordegradationofOfMasc8,9,21,32,40.Additionally,twoofthe26maleadultsthatemergedfromeggsinjectedwithOfznf-2siRNAdisplayedfemale-likewingmorphology(Fig.S12),furthersupportingtheroleofOfznf-2inmasculinization.

Ofznf-2cooperateswithOfMascinthemasculinizingpathwayinO.furnacalis
GiventhatOfMascandOfznf-2arebothrequiredformaledeterminationinO.furnacalis,wehypothesizedthattheseproteinsphysicallyinteract.Totestthis,weusedacelllinederivedfromO.furnacalisembryos9.ExpressionofGFP-taggedOfMascorOfMascNLS(aderivativeofOfMascLackingtheactivenuclearlocalizationsignalbutretainingmasculinizingactivity51)intheO.furnacaliscellline,followedbyimmunoprecipitation(IP)andLC-MS/MSanalysis,identifiedOfznf-2asaninteractingpartnerforbothOfMascandOfMascNLS(Fig.3a).WhenmCherry-taggedOfznf-2-EorOfznf-2-Lwasco-expressedwithOfMasc-GFPorOfMascNLS-GFPinB.moriBmN-4cells,onlyOfznf-2-E-mCherryco-localizedwithOfMasc-GFPorOfMascNLS-GFPinthenucleusorcytoplasm(Figs.3b,S9bandS13).Aco-IPexperimentfurtherconfirmedtheproteininteractionbetweenOfMascandOfznf-2-E,butnotbetweenOfMascandOfznf-2-L(Fig.3c).TheseresultssuggestthatOfznf-2-E,ratherthanOfznf-2-L,physicallyassociateswithOfMasctofacilitatemale-specificsplicingofOfdsxanddosagecompensationinmales.Infemales,theseprocessesareinhibitedthroughOfFempiRNA-mediatedcleavageofOfznf-2mRNA(Fig.3d).

AWolbachiahijacksthehostfeminizingpiRNAfunctioninO.furnacalis
PreviousstudiessuggestedthattheendogenousfeminizingfactoriseitherabsentorinactivatedintheWolbachia-infectedlineageofOstriniaspecies6–8.Inthislineage,theWolbachiamale-killingproteinOscarinactivatesOfMascprotein,effectivelysubstitutingforthehostfeminizingfactor9,40.Whole-genomesequencingwithshortreadsfromaWolbachia-infectedfemalerevealedthatalmostnoreadsmappedtotheOfFemlocus(Fig.4a).InthedraftassemblyofWolbachia-infectedO.furnacalis,thecontigtig00004611containedasingle222bp-longregionhomologoustotheOfFempiRNA-producingregion(80.2%identity),butitlackedtherepeatstructurehomologoustoOfznf-2(Fig.S14).Additionally,fragmentsoftheOfFemlocuscouldnotbePCR-amplifiedfromfield-collectedWolbachia-infectedOstriniaindividuals(Fig.4b).Consistently,theexpressionlevelsofOfFemandOfFem-derivedpiRNAwerenearlyundetectableintheWolbachia-infectedlineage(Figs.4a,c,dandS15).ThesefindingsindicatethattheOfFemlocusisabsentfromtheWchromosomeintheWolbachia-infectedlineage.Asaresult,thisWchromosomelacksfeminizingabilitythroughthedownregulationofOfznf-2mRNA.Thisabsenceexplainsthesex-reversalphenomenoninO.furnacalisinfectedwithmale-killingWolbachia,wherethedepletionofWolbachiafrominfectedmothersleadstofemalekilling(Fig.S16).

Methods
Insectrearingandtissuecollection
ThefounderO.furnacalismothsofthelaboratorystrainwerecollectedduringtheearlysummerseasonsof2014–2021inJapan.Thelaboratorystrainsweremaintainedaccordingtopreviouslydescribedmethods32.Eggmasseswereharvestedwithin2-htimewindowatdesignatedtimepointsforfurtherexperiments.GenomicDNAandtotalRNAwereextractedusingtheDNeasyBlood&TissueKit(QIAGEN)andTRIreagent(MolecularResearchCenter),respectively.

GenomicPCRandreversetranscription-PCR(RT-PCR)
TheOfFemlocuswasamplifiedusingKODOneDNApolymerase(TOYOBO,Japan)withtheprimersdesignedtomatchthehomologousrepeatstoOfznf-2intheOfFemlocus.Thesex-specificsplicingpatternofOfdsxwasexaminedbyRT-PCRusingKODFX–neoDNApolymerase(TOYOBO,Japan)aspreviouslydescribed32.QuantitativeRT-PCR(RT-qPCR)wasperformedwiththeKAPASYBRFASTqPCRkit(KapaBiosystems).ExpressionlevelsofOfdsxMwerenormalizedtoOfdsxC,thecommonregionofthemale-andfemale-typeOfdsx.RT-qPCRprimersspecificallytargetingOfznf-2-EaredetailedinFig.S6,whileallprimersusedinthisstudyarelistedinTableS3.

ModifiedrapidamplificationofcDNAends(RACE)
ModifiedRACEexperimentsfollowedapreviouslyestablishedprotocolwithminoradjustments18,57.TheGeneRacerKit(Invitrogen)wasemployedaccordingtothemanufacturer’sinstructions,excludingcalfintestinephosphatase(CIP)treatment.Omittingthisstepenabledadapteroligonucleotideligationtoboththecappedand5′-OHmRNAmoleculesforcomprehensive5′-endanalysis.

RNA-seqandsmallRNA-seq
TotalRNAsampleswereisolatedfromindividualeggsat18,24,and48hpoandweremolecularlysexedfollowingestablishedprotocols32.Sexedeggsampleswereeitherpooledbysexoranalyzedindividually.RNA-seqlibrarieswereconstructedfromthesesamplesusingtheStrandedmRNAPrep,LigationKit(Illumina),andindexedwithIDTforIlluminaRNAUDIndexesSetA.ThelibrariesweresequencedontheNovaSeq6000SequencingSystem(Illumina,SanDiego,CA,USA)with100-bppaired-endreads.SmallRNA-seqwasperformedonsamplesdependingontheexperimentalsetup.Forsex-pooledeggsamples,RNAfractionsof18–30nucleotidesinlengthwereisolatedandusedtoconstructsmallRNA-seqlibraries.SequencingoftheselibrarieswasconductedontheDNBSEQplatform,withlibrarypreparationandsequencingservicesprovidedbyBGI(Shenzhen,China).Forgonadandeggmasssamples,totalRNAwasisolatedfromthetestesofeightmales,theovariesoftwotosevenfemales,andeggmassescollectedfromfivefemalesateachtimepoint.SmallRNAfractions(20–30nucleotidesinlength)wereisolatedforthesesamplesasdescribedpreviously32.SmallRNA-seqlibrarieswereconstructedandsequencedusingtheIlluminaHiSeq2500platform.ForRNA-seqanalysisofeggmasssamples,RNA-seqlibrarieswereconstructedusingtheRibo-ZororRNARemovalEpidemiologyKitandIlluminaTruSeqRNAPrepKit,whicharenon-strand-specificandomitpoly(A)selection.TheselibrariesweresequencedontheIlluminaHiSeq2500platform.

IdentificationandexpressionanalysisofOfFemlocusandOfFempiRNA
ThedenovotranscriptomeassemblywasperformedusingtheTrinitysoftwarepackage58.ContigexpressionlevelswerequantifiedusingSalmon59,andtheoutputfileswereprocessedwiththeRpackageWasabi(https://github.com/COMBINE-lab/wasabi).Sex-biasedcontigswithstatisticallysignificantexpressiondifferencesbetweenmaleandfemalesamples(qvalue<0.05)wereidentifiedusingtheSleuthsoftwaretool60.Twocontigsthatmetseveralcriteria(seeResults)weremappedonthesingleOfFemlocusidentifiedfromanin-houseassembledwhole-genomeassembly(version#2),whichisusedforfurtheranalysis.RNA-seqdataweremappedtotheOfFemlocususingHISAT261.CoveragedepthatthelocuswascalculatedwithSAMtools62andBEDTools63.SmallRNA-seqdataweremappedtotheOfFemlocususingBowtie64,allowinguptotwonucleotidemismatchesandpermittingmulti-maps.OfFempiRNAreadswerespecificallyquantifiedbymappingsmallRNA-seqdataontoasinglerepresentativerepeatoftheOfznf-2sequencewithintheOfFemlocususingBWA65withnomismatchesallowed.ToinferthestructureoftherepeatelementsintheOfFemlocus,acustomrepeatlibrarywasconstructeddenovofromtheO.furnacaliswhole-genomesequence(version#2)usingRepeatModeler266.ThegenomewassubsequentlyannotatedwithRepeatMasker67,employingthiscustomrepeatlibrary.HomologousrepeatstotheOfznf-2sequencewithintheOfFemlocuswereidentifiedbyperformingaBLASTnsearchwiththenucleotidesequenceofOfznf-2-Lasthequery.

Phylogeneticanalysis
HomologsofOfznf-2,limitedtothetophitfromeachspecies,wereidentifiedthroughaBLASTpsearchagainsttheNCBInon-redundantproteindatabase(TableS4).BLASTpsearchwasperformedviatheNCBIBLASThomepage(https://blast.ncbi.nlm.nih.gov/)withdefaultparameters,usingasthequerythepartialproteinsequenceofOfznf-2(“MCKDWVRGTCARGAACIYAHELDKDQLKGVYRFCRDFENDRCERQVCYFVHATTFEKEHFFRTAFLPPHALHHLKT”).PhylogeneticanalysisofOfznf-2homologswasconductedalongsidepreviouslypublishedMascproteinsequences(TableS4).AminoacidsequenceswerealignedusingMAFFT68withdefaultsettings,andthealignmentsweretrimmedwithtrimAl69in“automated1”mode.AmaximumlikelihoodtreewasconstructedviatheIQ-TREEwebserver70,withnodesupportcalculatedfrom1000ultrafastbootstrapreplicates71.

Analysisofdosagecompensation
RNA-seqlibrarieswerepreparedfromtotalRNAusingtheIlluminaplatformprotocolsandsequencedasstrandedlibrariesontheNovaSeq6000SequencingSystem(Illumina,SanDiego,CA,USA)with150-bppaired-endreads.LibrarypreparationandsequencingwereperformedbyNovogene(Beijing,China).RNA-seqdataweremappedtotheannotatedO.furnacalisgenome(version#2)usingHISAT261.GeneexpressionlevelswerequantifiedwithfeatureCounts72.Countspermillionvaluesandfold-changemetricswerecalculatedusingtheedgeRpackage,employingnormalizationbythetrimmedmeanofM-valuesmethod73.

Cellcultureandtransfection
TheDNAfragmentsencodingOfMasc-GFP,Ofznf-2,andtheirderivativeswereclonedintothepIZ/V5-His-g3vector51.B.moriBmN-4cellswereculturedat26°CinIPL-41medium(Applichem,Germany)supplementedwith10%fetalbovineserum(FBS;Gibco,USA).BmN-4cells(4.0×105cellsper35-mmdiameterdish)weretransfectedwithtwoplasmidDNAs(1µg each) using FuGENE HD transfection reagent (Promega, USA). The localization of GFP-fused and mCherry-fused proteinswasexaminedat72hpost-transfectionusingeitheraFLoidTMcellimagingstationoranEVOSM5000cellimagingstation(ThermoFisherScientific).O.furnacalisOfT1C/tetcells,derivedfromWolbachia-infectedO.furnacalisembryos9,wereculturedat26°CinExpressFiveTMSFMmedium(Gibco,USA)supplementedwith18mML-glutamine,10%FBS,and3μg/mLtetracycline.WeisolatedthreeRT-PCRclonesofOfznf-2-EfromO.furnacalisembryoniccDNA.Theseclonesencodedproteinswithslightdifferencesinaminoacidsequences,butalldisplayedsimilarlocalizationpatternsinBmN-4cells(Fig.S9).Ofthese,weusedOfznf-2-E_clone#1astherepresentativecloneforallsubsequentcell-basedexperiments.

LC-MS/MS-basedidentificationofOfMasc-interactingproteins
OfMasc-GFP, OfMascNLS-GFP, or GFP were transiently expressed in OfT1C/tetcells(3.2×106cells)seededin10cm-diameterculturedishesbytransfectionusingFuGeneHD(Promega).OfMasc-interactingproteinswereco-immunoprecipitatedwithOfMasc-GFPusingGFP-Trapmagneticagarosebeads(ChromoTek,Germany).ProteinsboundtothebeadsweredigestedbyTrypsin/Lys-Cmix(Promega).LC-MS/MSanalysisoftheresultantpeptideswasperformedusinganEASY-nLC1200UHPLCconnectedtoanOrbitrapFusionmassspectrometerequippedwithananoelectrosprayionsource(ThermoFisherScientific).Thepeptideswereseparatedona75μminnerdiameter×150mmC18reversed-phasecolumn(NikkyoTechnos,Japan)withalinear4–32%acetonitrilegradientfor0–100minfollowedbyanincreaseto80%acetonitrilefor10min.Themassspectrometerwasoperatedindata-dependentacquisitionmodewithamaximumdutycycleof3s.MS1spectraweremeasuredwitharesolutionof120,000,anautomaticgaincontrol(AGC)targetof4e5,andamassrangefrom375to1500m/z.Higher-energycollisionaldissociationMS/MSspectrawereacquiredinthelineariontrapwithanAGCtargetof1e4,anisolationwindowof1.6m/z,amaximuminjectiontimeof35ms,andanormalizedcollisionenergyof30.Dynamicexclusionwassetto20s.Themassspectrometryexperimentswereperformedonceforeachsample.TherawLC-MS/MSdataweredirectlyanalyzedagainstO.furnacalisproteindata(GCF_004193835.1_ASM419383v1_protein.faa)downloadedfromNCBI,supplementedwithsequencesforOfMasc-GFPandGFP-Trap.AnalyseswereperformedusingProteomeDiscovererversion2.5software(ThermoFisherScientific)withSequestHTsearchengine.Thesearchparameterswereasfollows:(a)trypsinasanenzymewithuptotwomissedcleavages;(b)precursormasstoleranceof10ppm;(c)fragmentmasstoleranceof0.6Da;(d)carbamidomethylationofcysteineasafixedmodification;and(e)acetylationofproteinN-terminusandoxidationofmethionineasvariablemodifications.Peptidesandproteinswerefilteredatafalsediscoveryrate(FDR)of1%usingthepercolatornodeandtheproteinFDRvalidatornode,respectively.Label-freeprecursorionquantificationwasperformedusingtheprecursorionsquantifiernode,andnormalizationwasperformedsuchthatthetotalsumofabundancevaluesforeachsampleoverallpeptideswasthesame.

Westernblottingandimmunoprecipitation
Westernblottingandimmunoprecipitationexperimentswereperformedaspreviouslydescribed9.LysatesfromBmN-4cellsexpressingGFP-taggedproteinswereincubatedwithGFP-Trapmagneticagarosebeads.Afterbinding,thebeadswerewashedthreetimesandboiledtoelutetheboundproteins.TheelutedproteinswereseparatedbySDS-PAGEandtransferredontoPVDFmembranes.Forimmunodetection,ananti-GFPantibody(1:5000dilution,MBL,598,Japan)orananti-RFPantibody(1:5000dilution,MBL,PM005,Japan)wasusedastheprimaryantibody.

MicroinjectionofsiRNA
MicroinjectionofsiRNAwascarriedoutaspreviouslydescribed32.Specifically,100μMsiRNAsolutionswereinjectedintoO.furnacalisembryoswithin2hpo.ForFigs.2dandS12,however,50µMsiRNAwasused.EggmassesinjectedwithsiRNAwerecollectedat48hpoforanalysisorallowedtohatch.Hatchedlarvaewereraisedtothepupalstageandsexedbasedonpupalterminalabdominalmorphology.siRNAswerepurchasedfromFASMACCorp.(Atsugi,Japan),preparedinannealingbuffer(100mMpotassiumacetate,2mMmagnesiumacetate,30mMHEPES-KOH,pH7.4)atconcentrationsof100–500μM,andstoredat−80°C.NucleotidesequencesfortheOfznf-2siRNAusedinthisstudyarelistedinTableS3.

DNAextractionandgenomesequencing
Whole-genomeshotgunsequencingwasperformedusingPacBioandIlluminasequencingplatforms.GenomicDNAwasextractedfromfemalelarvaeoflaboratorystrains(T1-AandT1-C1)ofO.furnacalisusingtheBlood&CellCultureDNAMidiKitandaGenomic-tipKit,respectively(QIAGEN,Hilden,Germany).DNAqualityandconcentrationwereassessedusingaQubit4Fluorometer(ThermoFisherScientific,MA,USA)andaPippinPulseelectrophoresissystem(AgilentTechnologies,CA,USA).
ForPacBiosequencing,acontinuouslongread(CLR)librarywasconstructedfromtheT1-AsampleusingtheSMRTbellExpressTemplatePrepKit2.0(PacificBiosciences,CA,USA),followedbysizeselectionwiththeBluePippinsystem(SagaScience,MA,USA)usinga30kbcutoff.ThelibrarywassequencedonthePacBioSequelIIsystemwithaBindingKit2.0andSequencingKit2.0(PacificBiosciences,CA,USA)using30-hmovies.Rawsequencingdatafromasingle8MSMRTcellwereprocessedusingthePacBioSMRTLinkv11.0.0.144466software.ForIlluminasequencing,apaired-endlibrarywasgeneratedusingtheIlluminaDNAPCR-FreeLibraryPrepFragmentationKit(Illumina,CA,USA)andsequencedontheNovaSeq6000platform(Illumina,CA,USA)witha2×150bpreadlength.
ForHighFidelity(HiFi)sequencing,genomicDNAfromtheT1-C1samplewasfragmentedinto15–20kb sizes using a g-tube device (Covaris Inc., MA, USA). A HiFi library was prepared with the SMRTbell Prep Kit 3.0 (Pacific Biosciences, CA, USA) and subsequently size-selected with 35% AMPure PB beads (Pacific Biosciences, CA, USA). Sequencing was conducted on the PacBio Sequel II system using the Binding Kit 3.2 and Sequencing Kit 2.0 (Pacific Biosciences, CA, USA) with 30-h movies. HiFi reads were generated and processed from one 8M SMRT cell using the DeepConsensus v1.2 program under the default parameters74. Genomic DNA was also fragmented to an average size of 500 bp with the focused-ultrasonicator M220 (Covaris Inc., MA, USA). A paired-end library was prepared with the TruSeq DNA PCR-Free Library Prep Kit (Illumina, CA, USA) and size-selected using an agarose gel and the Zymoclean Large Fragment DNA Recovery Kit (Zymo Research, CA, USA). Sequencing library was then conducted on the NovaSeq 6000 system (Illumina, CA, USA) with a read length of 2×150 bp.

Hi-Clibrarypreparationandsequencing
TheHi-Clibrary,derivedfromtheT1-C8larvalbodysample,waspreparedfollowingtheOmni-CKitprotocol(Dovetail,CA,USA).Briefly,chromatinwasfixedinthenucleususingdisuccinimidylglutarateandformaldehyde,thendigestedwithDNaseI.ExtractedchromatinDNAfragmentswereend-repairedandligatedtoabiotinylatedbridgeadapter.Afterproximityligation,crosslinkswerereversed,andDNAwaspurified.SequencinglibraryconstructioninvolvedIllumina-compatibleadapters,withbiotin-labeledfragmentsenrichedviastreptavidinmagneticbeadsandPCRamplification.ThefinallibrarywassequencedontheIlluminaNovaSeq6000systemwith2×150bppaired-endreads,yieldingatotalof98.3Gbofsequencedata.

Chromosome-levelgenomeassembly
TwoversionsoftheO.furnacalisgenomeassembly(version#1andversion#2)wereconstructedusingPacBioCLRreadsandHiFireads,respectively.Forversion#1,PacBioCLRreadswereassembledwithCanuv2.1.175,withtheparameters“genome_size=400m corOutCoverage=200batOptions=\"-dg3-db3-dr1-ca500-cp50\"-pacbio-raw.”TheresultingcontigswerepolishedwithPilonv1.4.076usingIlluminareads.Toaddressredundancycausedbytheindependentassemblyofhaplotypes,haplotigswereremovedwithPurge_Dupsv1.2.577.Hi-CscaffoldingwasthenperformedusinganOmni-Cdatasettoproduceachromosome-levelassembly.
BeforeHi-Cscaffolding,11Wchromosome-derivedcontigs,initiallyremovedbythePurge_Dupsprocess,wererescuedusingHi-Ccontactinformation.Hi-CscaffoldingwasconductedwithYaHSv1.2a78,utilizingOmni-CreadmappinginformationgeneratedbyHiLinev0.2.4(https://github.com/wtsi-hpag/HiLine).TheHi-CcontactmapwasvisualizedwithJuiceboxv2.16.0079,underwentdetailedmanualcurationtoresolveassemblyerrorsandmis-scaffoldings.
Forversion#2,thegenomeassemblywasgeneratedusingHifiasmv0.19.680withtheparameter“--primary-s0.30,”basedonPacBioHiFireads.The“-s”parameterwasoptimizedbycomparingassemblystatisticsfromseveraltrialruns,withtheprimarygoalofreducingredundancycausedbyunresolvedhaplotypesintheprimaryassembly.ObtainedcontigswerepolishedwithNextPolish2v0.2.081usingPacBioHiFireadsandIlluminareads.SubsequentHi-Cscaffoldingandmanualcurationwereperformedontheprimarycontigs(p_ctg)usingthesameproceduresasforversion#1.
Bothassembliesdemonstratedrobustresults,withN50valuesgreaterthan16Mbpanddemonstrating>99%completenessofconservedgenes,asevaluatedbyBUSCO82.DetailedassemblystatisticsforbothversionsaresummarizedinTableS1.ThechromosomenumberswereassignedbasedonsyntenywiththechromosomesofB.mori(TableS5).

Statisticsandreproducibility
Allstatisticaltestsweretwo-sided.Thesignificancethresholdandtestusedareindicatedineachfigurelegend.Experimentswereindependentlyreplicatedasfollows:Fig.2a,threeindependentbiologicalreplicates;Fig.3b,c,twoindependentbiologicalreplicates;Fig.1f,twotechnicalreplicates;Figs.1d(n=3)and4b(n=2),onetechnicalreplicate.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In a previous study, Susumu Katsuma's team identified a protein called Oscar, through which the endosymbiotic Wolbachia inhibits the Masculinizer (OfMasc) protein in Ostrinia furnacalis, thus explaining the molecular mechanism of Wolbachia-induced male killing, which probably also applies to other lepidopteran species (Katsuma et al. 2022). In this study, S. Katsuma’s team answered another fundamental question by showing that Wolbachia completely takes over the feminizing function in the Wolbachia-infected lineage of O. furnacalis, which explains why the tetracycline-cured Wolbachia male-killing strains of O. furnacalis produce only male offspring while the females die. Furthermore, the authors discovered that in a Wolbachia-free O. furnacalis population, feminization is controlled by the W chromosome-derived OfFem piRNA, which surprisingly does not target the OfMasc mRNA but the mRNA of the Ofznf-2 gene and inhibits the OfMasc/Ofznf-2 masculinizing protein complex. Thus, there are two sex-determining pathways in O. furnacalis, one with the dominant role of the W chromosome, controlled by the feminizing piRNA produced by the W-linked gene, and the other in the Wolbachia-infected lineage, where feminization is taken over by Wolbachia and the W chromosome has lost the feminizing gene and plays no role in sex determination. Overall, this is a fascinating story that deserves to be published in this highly respected journal.\nThe essential role of the Ofznf-2 gene in sex determination is an important part of this study and the name of this gene should therefore appear in the abstract. Below are my comments and minor suggestions for you to consider when making some revisions to the text.\nSpecific comments\n(1) Results, lines 88-89: The timing of sex determination in embryos of Ostrinia furnacalis, which is given here and in several other places in the results as “during the period of sex determination”, is referred to Fukui et al. (2023). As this is important for a better understanding of your results, could you define this period? Do you mean the period from the first appearance of the male-specific splice variant of Ofdsx (12 h) to the completion of the feminization and masculinization processes (48 h)?\n(2) Fig. S3a: Strains D, E, F and α, β, γ are not mentioned in the Methods and should therefore be explained in the figure legend. What is the difference between the strains (D, E, F) and (α, β, γ)? Yes, fragments of the OfFem locus were amplified in all female samples, but especially in the (α, β, γ) strains, several bands can also be seen in the male DNA samples. Make this clear.\n(3) Table S2: The Ofznf-2 copies identified in chromosome 24 in the genome sequenced by Dai et al. (2024) have the same length as those in genome sequence version 2, where the copies, however, are located on chromosome 28. It is obvious that they are the same copies, but that there is chaos in the chromosome numbering. For O. furnacalis, did you use the numbering according to the silkworm where chr 28 matches chr 26 and chr 24 matches chr 28+31 in the O. furnacalis assembly of Dai et al. (2024)? See Fig. 2B in Dai et al. (2024). Is it possible that chr 28 in the version 2 genome sequence is identical to chr 24 in the Dai et al. (2024) assembly? The same applies to Fig. S8. Make this clear.\n(4) References in Supplementary Tables 2, 3", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_2": "The manuscript titled “Complete transition from chromosomal to cytoplasmic sex determination during prolonged Wolbachia symbiosis” by Fukui et al is reviewed. This study comprehensively investigates the molecular mechanism of sex determination of Ostrinia furnacalis with and without infection of Wolbachia and provides strong evidence that male-killing Wolbachia hijacks the feminizing piRNA function in Ostrinia furnacalis. I appreciate the amount of effort in this work and its achievement and only have some minor suggestions to the manuscript.\nLine 63: This is optional since it has been mentioned in the discussion but I was wondering if such sex determination in the wild could cause any intraspecific diversification. So I think having information about the infection rate here in the introduction may provide clear picture about how important such sex determination mechanisms are to the evolution of the species.\nLine 182: I find this figure (S16) very helpful in summarizing the whole picture, I think it would be better to have this figure in the main paper but not supplementary.\nLine214-216: Here comes inference about genetic drift, I guess it is related to the next section about the prevalence but I think it would be better to have more context about how the fixation of the mutation to the evolutionary history of the population in Japan.\nLine 311-318: I was a bit confused here as I thought OfFem was identified based on the expression difference, but how the “expression analysis of the OfFem locus” section comes after the identification of OfFem? I wonder if merging these two sections could make it easier for readers to understand the flow of the work.\nLine 333: was there blast criterion applied in blastp? If so, please specify.\nLine 400: were these samples known for their sex? Are they from the lab breeding lines?\nLine458: I wonder if it is required to lower “-s” here, I assume the samples are quite inbred and should have low heterozygosity? If now, maybe it will be better to provide the evaluated heterozygosity and to make sense the “-s” parameter change.", "reviewer_3": "This study reports fascinating research that will be of wide interest with respect to the evolution of insect sex determination systems and of symbiont reproductive manipulations. The authors achieve two objectives; firstly, they identify a new primary Lepidoteran feminising factor in O. furnacalis ‘OfFem’, a W-linked piRNA that silences a constitutively expressed masculinising factor, and secondly, they demonstrate that the OfFem locus has been lost in a O. furnacalis lineage carrying the male-killing Wolbachia strain wFur. These are important, novel contributions, explaining convincingly how ‘cytoplasmic sex determination’ has arisen in this species. The research is of a very high standard, robustly conducted and the results convincing. The data are clearly presented, the manuscript is well written and is for the most part easy to follow, with an excellent discussion.\nIt would be useful to orient the reader to provide in the introduction a short overview of how widespread is the wFur strain in O. furnacalis in Japan, and where it occurs what is the typical prevalence. Are there any wFur lineages that have produced 1:1 sex ratios on tet treatment to remove Wolbachia?\nSpecific comments:\nLine 108: states that there are 15 repeats of the Ofznf-2 target, but Fig. S5 seems to suggest that there are 17 repeats.\nPlease clarify.\nL214 ‘it must overcome random genetic drift’ - could there be a selective advantage associated with not expressing the W-linked feminizing factor in the presence of Wolbachia?\nFig. 3b\nIt is difficult to gauge the level of interaction between OfMasc and the Ofznf-2 variants from these images. Firstly, since the interpretation depends on co-localisation, a ‘merge’ panel for the fluorescent images could be provided. In addition, the images of cytoplasmic/nuclear co-localisation of Ofznf-2-E with OfMasc and OfMascNLS in Fig S13 are more convincing of interaction than the images in Fig 3b. and thus Fig 3b could be replaced with Fig S13 in the main text – especially if images can be added showing that Ofzbf-2-L does not show similar nuclear/cytoplasmic co-localisation.\nFig. S3\nwhat the different strains in panel A refer to – there does not seem to be an indication of what they represent anywhere in the ms.\nFig. S4C\nSince the presence of OfFem piRNAs is dependent on the host lineage rather than the Wolbachia infection status per se, rather than using Wol-, Wol+(Tet) designations it would be clearer to replace with ‘Wol- lineage’, ‘Wol+ lineage (tet-treated)’.\nSimilarly for the Figure legend of C, emphasise host lineage: e.g. ‘in the ovary of tetracycline-treated moths from the Wolbachia-infected lineage’.\nMinor / Typos\nFig. S7 legend: ‘expression’ missing e.\nLine 255: misspelling of O. furnacalis\n227 ‘both protein homologs’ would be clearer\n232 exhibited a little positive effect – remove little or replace with small"}, "author_response": "Point-by-point responses to the reviewer’s comments\nReviewer #1:\nIn a previous study, Susumu Katsuma's team identified a protein called Oscar, through\nwhich the endosymbiotic Wolbachia inhibits the Masculinizer (OfMasc) protein in\nOstrinia furnacalis, thus explaining the molecular mechanism of Wolbachia-induced\nmale killing, which probably also applies to other lepidopteran species (Katsuma et al.\n2022). In this study, S. Katsuma’s team answered another fundamental question by\nshowing that Wolbachia completely takes over the feminizing function in the Wolbachia-\ninfected lineage of O. furnacalis, which explains why the tetracycline-cured Wolbachia\nmale-killing strains of O. furnacalis produce only male offspring while the females die.\nFurthermore, the authors discovered that in a Wolbachia-free O. furnacalis population,\nfeminization is controlled by the W chromosome-derived OfFem piRNA, which\nsurprisingly does not target the OfMasc mRNA but the mRNA of the Ofznf-2 gene and\ninhibits the OfMasc/Ofznf-2 masculinizing protein complex. Thus, there are two sex-\ndetermining pathways in O. furnacalis, one with the dominant role of the W chromosome,\ncontrolled by the feminizing piRNA produced by the W-linked gene, and the other in the\nWolbachia-infected lineage, where feminization is taken over by Wolbachia and the W\nchromosome has lost the feminizing gene and plays no role in sex determination. Overall,\nthis is a fascinating story that deserves to be published in this highly respected journal.\n(Authors’ reply)\nWe thank Prof. František Marec for reviewing this manuscript and giving encouraging\ncomments.\nThe essential role of the Ofznf-2 gene in sex determination is an important part of this\nstudy and the name of this gene should therefore appear in the abstract.\n(Authors’ reply)\nAs suggested by this Reviewer, we added the gene name Ofznf-2 in the abstract.\nBelow are my comments and minor suggestions for you to consider when making some\nrevisions to the text.\nSpecific comments\n(1) Results, lines 88-89: The timing of sex determination in embryos of Ostrinia\nfurnacalis, which is given here and in several other places in the results as “during the\nperiod of sex determination”, is referred to Fukui et al. (2023). As this is important for a\nbetter understanding of your results, could you define this period? Do you mean the period\nfrom the first appearance of the male-specific splice variant of Ofdsx (12 h) to the\ncompletion of the feminization and masculinization processes (48 h)?\n(Authors’ reply)\nAs this Reviewer pointed out, we defined the sex determination period as the period when\nthe sex-specific splicing of Ofdsx is first observed after 12 hours post-oviposition (hpo)\nand is established at 48 hpo. This was added in the revised manuscript (line 97–98).\n(2) Fig. S3a: Strains D, E, F and α, β, γ are not mentioned in the Methods and should\ntherefore be explained in the figure legend. What is the difference between the strains (D,\nE, F) and (α, β, γ)? Yes, fragments of the OfFem locus were amplified in all female\nsamples, but especially in the (α, β, γ) strains, several bands can also be seen in the male\nDNA samples. Make this clear.\n(Authors’ reply)\nStrains D, E, F and strains α, β, γ were established from different founder moths and\nseparately maintained. And multiple bands amplified from the male sample of strains α,\nβ, and γ were non-specific. These were included in the figure legend of Fig. S3.\n(3) Table S2: The Ofznf-2 copies identified in chromosome 24 in the genome sequenced\nby Dai et al. (2024) have the same length as those in genome sequence version 2, where\nthe copies, however, are located on chromosome 28. It is obvious that they are the same\ncopies, but that there is chaos in the chromosome numbering. For O. furnacalis, did you\nuse the numbering according to the silkworm where chr 28 matches chr 26 and chr 24\nmatches chr 28+31 in the O. furnacalis assembly of Dai et al. (2024)? See Fig. 2B in Dai\net al. (2024). Is it possible that chr 28 in the version 2 genome sequence is identical to chr\n24 in the Dai et al. (2024) assembly? The same applies to Fig. S8. Make this clear.\n(Authors’ reply)\nWe used the chromosome numbering according to that of Bombyx mori, thus chr. 24 in\nDai et al. (2024) corresponds to chr. 28 in our newly assembled version #1 and version\n#2 genomes. This was added in Table S2, S5, and Fig. S8 in the revised manuscript.\n(4) References in Supplementary Tables 2, 3"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Well done! In the revised manuscript, the authors have satisfactorily addressed all my comments and suggestions. I look forward to the publication of this excellent study.\nNote: I have found two minor problems that can be fixed in the further editing of this article. These are:\n(1) Fig. 4a legend (line 796): The explanations for Wol-, Wol+ and Wol+(Tet) were added as suggested in my comment #5, but the explanations for Wol- and Wol+ were incorrectly switched [see “Wolbachia-infected (Wol−), uninfected (Wol+)"}, "author_response": ""}]

Decision letter