10.1111/eea.12362
SexualdifferencesinelectrophysiologicalandbehavioralresponsesofCydiamolestatopeachandpearvolatiles
Peng-FeiLu1,2#,RongWang1#,Chen-ZhuWang2,You-QingLuo1*&Hai-LiQiao3*
1BeijingKeyLaboratoryforForestPestControl,BeijingForestryUniversity,Beijing100083,China,2StateKeyLaboratoryofIntegratedManagementofPestInsectsandRodents,InstituteofZoology,ChineseAcademyofSciences,Beijing100101,China,and3InstituteofMedicinalPlantDevelopment,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing100193,China
epted:22July2015
Keywords:orientalfruitmoth,Lepidoptera,Tortricidae,male,female,hostplant,windtunnel,fieldtrial,Rosaceae
Theorientalfruitmoth(OFM),Cydia(=Grapholita)molesta(Busck)(Lepidoptera:Tortricidae),isaseriousinvasivepest.Thestonefruitpeach,Prunuspersica(
L.)Batsch,isitsprimaryhost,andthepomefruitpear,PyrusbretschneideriRehder(bothRosaceae),isitssecondaryhost.ElectrophysiologicalandbehavioralresponsesofC.molestafemalesandmalestopeachshootandpearfruitvolatilesparedinlaboratoryandfieldbioassays.Basedongaschromatography–electroantennographicdetection(GC–EAD)activity,poundsintheheadspacesofpeachandpearvolatileselicitedfemaleantennalresponses.Ofthese,poundsalsoelicitedmaleantennalresponse.FourluresweredevelopedbasedonmaleandfemaleEADresponsestopearandpeach-derivedpounds(VOCs).Moremalesthanfemaleswerecapturedforallfourluresduringfieldtrials,evenintrapswithluresthatemittedVOCsbasedonfemaleEADresponses.LuresbasedonfemaleEADresponsestopearfruitVOCsconsistentlycaughtmorefemalesthanluresbasedonmaleEADresponsestopearfruitVOCsineitherpeachorpearorchards.PeachshootVOCluresbasedonfemaleEADresponsesdidnotattractmorefemalesthanluresbasedonmaleEADresponsetopeachshoots.Thetwopear-derivedVOClureswerehighlyattractivetobothsexesinpeachorchards,whereasconversely,thetwopeach-derivedVOCluresshowedstrongerattractioninpearorchards.Seasonalpopulationmonitoringindicatedbothsexesmadeinter-orchardflightsduringthelatepeach-andpear-fruitingperiods.Apossiblehypothesisthatcouldexplaindifferentresponseprofilesinfemalesandmalesandseasonalmigrationforherbivoreswithmultiplegenerationsperyearisdiscussed. Introduction Theorientalfruitmoth(OFM),Cydia(=Grapholitha)molesta(Busck)(Lepidoptera:Tortricidae),presumablyoriginatingfromnorth-westChina,hasewidely *Correspondence:You-QingLuo,BeijingKeyLaboratoryforForestPestControl,BeijingForestryUniversity,35QinghuaDongRoad,HaidianDistrict,Beijing100083,China.E-mail:luoyouqing224@;Hai-LiQiao,InstituteofMedicinalPlantDevelopment,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,151MalianwaNorthRoad,HaidianDistrict,Beijing100193,China.E-mail:qhl193314@#Peng-FeiLuandRongWangcontributedequallytothiswork. distributedthroughouttheworld(Rothschild&Vickers,1991).Untilthelate1970sthespecieswasconsideredtobeoligophagousandonlytodamagestonefruitinWesternEurope(Bovey,1979).Inrecentyears,however,OFMdamagetopomefruitorchardshasbeenwidelyreportedinAsia,Europe,Australia,andAmerica(Rothschild&Vickers,1991;Dornetal.,2001;Myersetal.,2006;Il’ichevetal.,2009;Luetal.,2012).InNorthChina,peachshootsandpearfruitsaretheprimaryOFMhostsubstratesearlyandlateinthegrowingseason(Luetal.,2012),andthismayresultinmovementfromstonefruitorchardstopomefruitorchards(Zhaoetal.,1989;Yang&Liu,2010;Luetal.,2012). ©2015TheNetherlandsEntomologicalSocietyEntomologiaExperimentalisetApplicata157:279–290,2015 279 280Luetal. TheflightoffemaleC.molestasignificantlyexceedsthatofmales(Hughes&Dorn,2002).Femalesaredirectlyresponsibleforinfestationsbymakinginter-orchardflightsandlayingeggsonpearfruits(Yetter&Steiner,1932;Steiner&Yetter,1933),sothisstudyfocusesontheinteractionbetweenadultOFMresponsetohostplantderivedvolatileswithemphasisonparisonbetweenfemaleandmaleresponses. OlfactorycuesforOFMfemalesfrombothshootsandfruitsofpeachandapplehavebeenstudiedextensively(Nataleetal.,2003,2004;Pin~ero&Dorn,2007,2009;Najar-Rodriguez&Dorn,2013).Excisedpeachshootsemitting(Z)-3-hexenylacetate,(Z)-3-hexenol,andbenzaldehydeata4:1:1ratiowasattractivetomatedOFMfemales(Nataleetal.,2003).Volatilesemittedfrompeachandapplefruitswerealsoattractivetomatedfemalesinadualchoicearena(Nataleetal.,2004).Behavioralexperimentshaveidentified(Z)-3-hexenylacetate,(Z)-3-hexenol,(E)-2-hexenal,benzonitrile,andbenzaldehyde,emittedbypeachshoots,toactinasynergisticmanner,resultinginahighlyattractiveodormixturetofemaleOFM(Pin~ero&Dorn,2007).Recentstudiespayparticularattentiontotheseasonaldynamicsofvolatileemissionsfromdifferenthosts.Pin~ero&Dorn(2009)reportedthatC.molestafemalesselectedshoots,twigwithleaves,andfruitsofpeachandappletreesatdifferenttimesoftheseason.Najar-Rodriguez&Dorn(2013)reportedvolatileemissionsfrominsitupeachandpeartreesoveranentireseason,alongwiththeirolfactoryattractivenesstoC.molestafemales.SomestudieshavebeenconductedtoindicatehighattractivenessofparticularbinationstofemaleOFMinthelaboratory(Pin~eroetal.,2008),butfielddataarelacking.ForthefewstudiesthatreportfieldresponsesofOFMtolures,thereseemstobenocorrespondencebetweenthelaboratoryandfieldfindings.Forexample,Il’ichevetal.(2009)carriedoutheadspaceanalysisofvolatilesfromtheintactyoungshoottipsofpeach.Theyfoundthatamixtureof(Z)-3-hexenylacetate,(E)-bocimene,and(E)-b-farneseneataratioof1:2:2wasthebestattractant,butonlytoOFMmales,nofemaleswerecapturedinthefield.Morerecently,basedonfemaleOFMresponsesrecordedusinggaschromatography–electroantennographicdetection(GC–EAD),theprofileofbioactivevolatilesemittedfromimmatureandmaturepearfruitwerecharacterized(Luetal.,2012)andreportedtwopear-derivedponentattractantsthatcanbeusedformonitoringbothsexesoforientalfruitmothinthefield.Subsequently,theycarriedparativeanalysesofpeachandpearfruitvolatilesattractivetobothOFMsexes,andevaluatedtheattractivenessofvolatilemixturesmimickingpeachandpearfruitvolatilestoOFMmalesandfemales(Luetal.,2014).Despiteusingmixtures containingvolatilesattractivetofemalesinthelaboratory,malecaptureswerestillhigherunderfieldconditionsinthesestudies. Theseresultsraisedtwoimportantquestions.WhyaremoremaleOFMsthanfemalescapturedbytrapswithluresemittingplant-derivedpounds(VOCs)attractivetofemalesinthelaboratory?
Whatisthedifferenceintheelectrophysiologicalandbehavioralresponsesofbothsexestopeachandpearvolatilesbetweenthelaboratoryandfieldbioassays?
Theanswersshouldhelptoelucidatethebehavioralmechanismsunderlyinghighermalecatcheswhenusingvolatilesselectedonthebasisoffemaleelectroantennography(EAG)responses. Theobjectivesofthestudywere(1)toidentifyandanalyzetheVOCsfromyoungpeachshoots[Prunuspersica(
L.)Batsch]andpearfruits[PyrusbretschneideriRehder(bothRosaceae)]attractivetoOFMs,and(2)paretheresponsesofmaleandfemaleOFMtopeachshootandpearfruitvolatilesusinglaboratoryandfieldbioassays. Materialsandmethods Insects Larvaewerecollectedfrominfestedshootsofpeach,PrunuspersicaL.Batsch‘Wuyuexianbiangan’,inanexperimentalorchardattheInstituteofForestryandPomology(IFP),BeijingAcademyofAgricultureandForestry(BAAF),Beijing,China(39°580N,116°130E)inlateJune.Theselarvaeweremaintainedinaclimaticchamberat24Æ1°C,65–70%r.h.,andL16:D8eriod,withthehasestartingat05:00hours.Untilthethirdinstar,larvaeweremass-rearedonapple,MalusdomesticaL.Borkh.‘Hongfushi’(Rosaceae),inaglasscontainer(27cmdiameter,13cmhigh)andthentransferredtosmallerindividualglasscontainers(2.5cmdiameter,8cmhigh)untileclosion.Abell-shapedglasscontainer(6-and15-cm-diameteropenings,41cmhigh)wasusedtoraisetheadults,andcoveredwithfinenylonmeshatbothsides.A15%(wt/vol)honeysolutionwasprovidedonwater-soakedcottonviaaholepiercedthroughthemeshinthesmallsideofcontainer.Themothswererearedinthelaboratoryforthreegenerationsbeforetesting.Forelectrophysiologicalexperiments(GC–EAD),2-to3-dayoldfemalesandmaleswereused.Forthewind-tunnelbioassay,matedfemaleandmalemothswereselectedrandomly.Matedmothswereobtainedbyplacinggroupsofca.20newlyemergedfemalestogetherwith30malesinthesamecagefortwoeriodiccyclestoensuremating.Eachadultmothwasusedinonlyoneassayandmothswerenotexposedtosyntheticodorsourcesbeforethebioassay. SexualdifferencesofOFMinresponsestohosts281 ChemicalsTetradecane(99%purity),pentadecane(99%),hexadecane(99%),octadecane(99%),nonadecane(99%),(Z)-3hexen-1-ol(98%),butylbutanoate(98%),(Z)-3-hexenylacetate(98%),benzaldehyde(99%),methylsalicylate(99%),(E)-b-ocimene(60%),and(
E,E)-a-farnesene(49%)wereobtainedfromSigma-Aldrich(St.Louis,MO,USA).Theremainingfarnesenefractionconsistedofmainly(E)-b-farnesene(26%),(Z)-b-farnesene(18%),and(
Z,E)-a-farnesene(7%).WeusedthismixtureoffarneseneisomersforourEAGandfieldstudiesandrefertoitas‘farnesene’.Heptadecane(99%),1-hexanol(99%),2ethyl-1-hexanol(99%),nonanal(97%),hexylacetate(99%),andracemiclinalool(97%)wereobtainedfromFlukaProduction(Buchs,Switzerland).Ethylbutanoate(99%)andbutylacetate(99%)wereboughtfromAcrosOrganics(MorrisPlains,NJ,USA).Ethylhexanoate(98%)andhexylbutanoate(98%)werepurchasedfromTokyoChemicalIndustry(Tokyo,Japan).Compoundsthatdidnotelicitantennalresponses,andforwhichnostandardswereavailable,weretentativelyidentifiedusingtheNIST-database(Tasinetal.,2005). Plantmaterials Ten-year-oldplantsofpeachesandpearswerecultivatedinanexperimentalorchardattheIFP.Thepeachvarietyusedwasflatpeach(FP),‘Wuyuexianbiangan’.ThepearvarietyusedwasJimi(JM).BothvarietiesmonlygrownintheBeijingarea.NoinsecticidesoranyspecifictreatmentagainstOFMwereusedintheorchardduringthetests. CollectionofVOCsfromdetachedplanttissuesPeachshoots(ca.30cmlong)werecutfromselectedtreesandsealedwithliquidparaffin(FlukaProduction),beforebeingtransferredtothelaboratorynearby(Anforaetal.,2009).Plantmaterialwasusedforvolatilecollectionorwind-tunnelassayswithin20minaftercutting.PearandpeachfruitswerehealthyandwerepickedfromtheexperimentalorchardtreeswhenfruitsofthecorrespondingvarietieswereripeintheBeijingarea(Caoetal.,2000,2006;Jiangetal.,2002). Apush–pullsystemwasusedtocollectheadspaceVOCs.Peachshoots(ca.1500g)andpearfruits(ca.1500g)wereplacedintoa2lglassjarforextraction.Airaspiratedwithavacuumpump(QianxiAir,Beijing,China)andfilteredthroughanactivatedcharcoalfilterwaspassedthroughthejarat300mlpermin,andfinallythroughasorbentcartridge(PorapakQ,50mg,80/100mesh;Supelco,Bellefonte,PA,USA).Thesorbentcartridgeswereheldbetweenplugsofglasswoolinaglass tube(10cmlong,0.5cminnerdiameter).VOCsampleswerecollectedfor8hat24Æ1°Cand65–70%r.h. Volatilesweredesorbedbyelutingthesorbentcartridgewith500llhexane(HPLCgrade,Sigma-Aldrich)atroomtemperature.Fivesampleseachwerecollectedfrompeachshootsandripepearfruits.ThesampleswereanalyzedbyGC–EADandgaschromatography–massspectroscopy(GC–MS).Forquantitativeanalyses,0.5lgethylpentanoate(99%;FlukaProduction)wasaddedasaninternalstandardtoeachsample.PreliminaryanalysesshowedthatpoundwasnotdetectableintheheadspaceVOCsofthepeachorpearvarietiesstudiedhere.Samplevolumeswerereducedto50llusingaslowstreamofnitrogenandthenanalyzed.Ifnotusedimmediately,extractsweresealedinglassvialsandstoredatÀ18°Cuntilused. GC–MSHeadspaceVOCsofpeachshootsandpearfruitswereanalyzedwithanAgilent5973MScoupledtoanAgilent6890NGC(AgilentTechnologies,PaloAlto,CA,USA)equippedwithapolarDB-WAX-fusedsilicacolumnoranon-polarDB-5-fusedsilicacolumn(both30m90.25mmi.d.,0.25lmfilm;J&WScientific,Folsom,CA,USA).Thetemperatureprogramwasasfollows:50°Cfor1min,thenincreasing3°Cperminto120°C,thenincreasing10°Cperminto240°C,andfinallyheldat240°Cfor10min.WindowsNT/MASSSpectralSearchProgramsoftware(version1.7)wasusedfordataanalysis.Injectionsweremadeinthesplitlessmode.Heliumwasusedasthecarriergas(1.0mlpermin).Forelectronimpact(EI)massspectra,theionizationvoltagewas70eV,andthetemperaturesoftheionsourceandoftheinterfacewere230and280°C,respectively.Theemissioncurrentwas34.6lA.IdentificationofVOCswasverifiedparisonwithauthenticsamples. GC–EADAmicromanipulatorassembly(MP-15)wasconnectedtoastimuluscontroller(CS-55;bothSyntech,Hilversum,TheNetherlands).Allsignalsourceswereconnectedtoaserialdataacquisitioninterface(IDAC-4;Syntech).TheantennaeofOFMmalesandfemaleswereexcisedusingmicro-scissors.Afewsegmentsfromthetipsofantennaewereclippedoffandmountedontheantennaholderwithtwometalelectrodesusingconductivegel(Spectra360;ParkerLaboratories,Fairfield,NJ,USA),andthentheelectrodeholderwasinsertedintotheEADprobe.Testingbeganafterarelativestablebaselinehadbeenachieved.TheoutletoftheGCcolumnwassplitina2:1ratiobetweenacutantennaandtheflameionizationdetector(FID).Themountedantennawaspositionedinthecharcoal-filteredandhumidifiedairstreamthatcarriedthe 282Luetal. VOCsfromtheGCcolumn.TheantennalandFIDsignalswereamplifiedandrecordedsimultaneouslyusingSyntechsoftware(GC-EAD32,version4.4).Eachsamplewastestedwithsixantennae,eachderivedfromadifferentmoth.Identitiesofpoundswereverifiedparisonofmassspectraandretentiontimeswiththoseofsyntheticstandards. TheVOCswereanalyzedwithanAgilent6890NGCwithaflameionizationdetector,coupledwithaSyntechEAD.ColumntypeandoventemperatureprogramwerethesameasintheGC–MSanalysis.Nitrogenwasusedasthecarriergas(1.0mlpermin). FieldexperimentBasedontheresultsoftheGC–EADanalyses,VOCsfromthepeachshootsandpearfruitsthatelicitedantennalresponsesinfemaleandmaleOFMswereusedtoformulatefourseparateblendsforthefieldtests.Thefoursyntheticblendsderivedfromthetwohostplantswereevaluatedinadjacentorchardsofthetwohostcropsfromthetimeoflatepeachfruitingtolatepearfruiting. ThefourVOCblendswereasfollows:
(1)Sh-F(shootfemale):VOCsfrompeachshootstowhichfemaleshadpositiveEADresponses;
(2)Sh-M(shoot-male):VOCsfrompeachshootstowhichmaleshadpositiveEADresponses;
(3)Fr-F(fruit-female):VOCsfrompearfruitstowhichfemaleshadpositiveEADresponses(Luetal.,2012);and
(4)Fr-M(fruit-male):VOCsfrompearfruitstowhichmaleshadpositiveEADresponses.ThefourblendsofpoundswerepreparedinthesameratiosofGC–EAD-activeVOCsasemittedbythecorrespondingplanttissue(Table1).Thesewerepreparedusing100mgofthemostpoundandaddingtheothersinthesameproportionasinthenaturallyurringvolatileblend. Goblet-shapedrubbersepta(10mmdeep,6mmi.d.)withamaximumvolumeof400ll(ShunyiRubber,Beijing,China)wereusedtodispersetheVOCblends.ThisvolumewassufficientforalltheVOCblendsinourstudy.TheVOCblendswereprepared1–2hpriortotheiruseinfieldbioassays.ForeachVOCblend,rubberseptawerefilledandthenfixedupwardonthebottomofastickydeltatrap.Trapswereplacedinthefieldatdusk,whenOFMflightintensitypeaks,forallthetreatments.Wedeployedtherubberseptawithsolutionsdirectlyinthetrap,beforethemixturesolutionwasfullyimpregnatedintotherubbersepta.Allodorblendsweredeployedinthisway(Luetal.,2012).Unbaitedtraps(HPLC-gradehexane;Sigma-Aldrich)andOFMsex-pheromonelures(GeruibiyuanTechnology,Beijing,China)wereusedascontrols. Table1Components,theiramountsloadedonrubbersepta(mg),andtheirratiosfortheblendsusedinthefieldexperiment.Theblendsarebasedonfemale(F)ormale(M)EADresponsetodetachedpeach(Prunuspersicacv.Wuyuexianbiangan)shoots(Sh)andmaturepear(Pyrusbretschneidericv.Jimi)fruits(Fr) Compound1 Sh-FSh-MFr-F2Fr-
M Alcohols 1-Hexanol
1 1 (Z)-3-Hexen-1-ol
4 Aldehydes Nonanal
1 Esters Ethylbutanoate 100 100 Butylacetate 70 70 Ethylhexanoate
7 7 Hexylacetate
5 5 (Z)-3-Hexenylacetate100100 Hexylbutanoate
1 1 Benzenoids Benzaldehyde
4 Terpenoids (E)-b-Ocimene 85 Linalool
1 Farnesene3
4 4 1In
orderofelutionduringgaschromatographyonapolarDB- Wax-fusedsilicacolumn.2TheblendsofFr-Fwerebasedonheadspaceposition fromdetachedmaturepearfruits(Luetal.,2012).3Farnesene=mixtureof(
E,E)-a-farnesene(49%),(E)-b-farnesene(26%),(Z)-b-farnesene(18%),and(
Z,E)-a-farnesene(7%). Thefieldtestwasconductedina7-hapeachorchardanda6.5-hapearorchardattheIFPwithca.30–50mopenspacebetweentheorchards.BothorchardshadahistoryofOFMinfestation.Twotrialswereconducted,eachof2weeks:5–18August(Fieldexperiment1)and5–18September(Fieldexperiment2)2013.ThesecoincidedwithpeakOFMactivityintheBeijingarea.Stickydeltatraps(35920920cm)wereusedtotraptheinsects.Ineachorchard,thetrialswerecarriedoutinarandomizedblockdesigncontainingsixreplicateblocks.Ineachofthesixblocks,thedifferenttreatmentswererepeatedonlyonce.Blockswereatleast120mapart.Trapswereinstalledca.1.5mabovethegroundandwereseparatedbyatleast30mtominimizeinterferencebetweentraps. Thetrapsweremonitoredtwiceweekly,capturedOFMsweretransferredtothelaboratory,andtheirnumberandsexwererecorded.Thedataofcapturesovereach2-weekperiodwerepooled. Wind-tunnelassayThewind-tunneltestsweredesignedtotestforsexualdifferencesinflightabilitytowardthefourVOCblends. SexualdifferencesofOFMinresponsestohosts283 Malesandfemaleswereusedinthewind-tunnelassay.Thelaboratorywindtunnelwas1.6mlong,0.5mwide,and0.5mhigh.Afanattheupwindendgeneratedasteadyairflowintothetunnel,setat0.3msÀ1atthepointofreleaseofmoths.Thelightintensityinthetunnelwasca.250lx.Theroomwaskeptat23Æ2°Cand50–70%r.h. FourVOCblendswerepreparedinproportionsofGC–EAD-activeVOCsfoundinthenaturalblendsemittedbythecorrespondingvarieties(Table1).ThepredominantVOCinthemixturewasdosedat0.5mg.TheVOCsweredilutedwithhexane(HPLCgrade;SigmaAldrich).Preliminarytestsinthewindtunnelshowedthattheseconcentrationswereadequatetoelicitmothresponses.Theseptumloadedwithoneoftheblendswasplacedonaholderattheupwindendinthecenterofthetunnel,10cmfromtheupwindend.Individualmothswerescoredforthefollowingbehaviors:(1)departurefromthereleasecageandflightupwind;(2)arrivalwithin10cmoftheVOCsource;and(3)landingonthesource.Wecategorizedeachmothasthebehaviorassociatedwiththefurthestdistancetraveledwithin20min. Testsbegan2hbeforethebeginningofthehaseandlasted3h.Mothsweresexedandwerethentransferredintothetestroom2hbeforetheexperiments.Batchesof10mothswereplacedinasmall,screenedmetalreleasecage(7cmdiameter,9cmhigh)withasidedoorthroughwhichtheairfromthewindtunnelcouldflow.Thereleasecagewasplacedonaholderatthedownwindendofthetunnel,30cmaboveitsfloorandca.140cmfromtheVOCsource.Thedoor,facingtheupwindendofthetunnel,wasopenedtoallowthemothstoleavethecage.Eachbatchof10matedmothswastestedfor20min,and6batchesofmothswereusedperday.EachVOCblendwastestedwithninebatchesofmoths,eachonadifferentday.Individualmothsweretestedonlyonce. Dataanalysis MeannumbersofOFMmalesandfemalescapturedintrapsbaitedwitheachVOCblendinthefieldandtheperformancescoresofOFMfemalesandmalestotheVOCsourceinthewindtunnelwereanalyzedbyone-wayANOVA.Tukey’smultiplerangetestwasusedtotestforsignificantdifferencesamongVOCblendsinthemeannumbersofOFMscaptured(a=0.05).Unpairedsamplet-testswereusedtotestforsignificantdifferencesbetweensexesinthreeflightbehaviorsinthewind-tunnelassay.AlldatawereanalyzedwiththestatisticalprogramSPSSv.16.0(SPSS,Chicago,IL,USA). Results Characterizationoftheheadspacevolatilesfrompeachshootsand pearfruits Compoundsbelongingtovariouschemicalclasseswereidentifiedfrompeachshoots:hydrocarbons,alcohols,aldehydes,esters,benzenederivatives,andterpenoids(Table2).ponents—octadecane,(Z)-3-hexen1-ol,(Z)-3-hexenylacetate,benzaldehyde,methylsalicylate,(E)-b-ocimene,andlinalool—werecharacteristicofpeachshootsandabsentfromripepearfruits.ponents—tetradecane,6-methyl-octadecane,1-hexanol,2-ethyl-1-hexanol,2-methyl-1-hexadecanol,(
E,E)a-farnesene,andsevenesters—werecharacteristicofripepearfruitsandabsentfrompeachshoots.Inparticular,(Z)-3-hexenylacetateandethylbutanoatewerethemostabundantVOCsidentifiedinpeachshootsandpearfruits,respectively. FemaleandmaleantennalresponsestoVOCsfrompeachshootsand pearfruits Fromdetachedpeachshoots,(Z)-3-hexenylacetateelicitedantennalresponsesinbothsexes(Figure1AandB),whereas(Z)-3-hexen-1-ol,benzaldehyde,(E)-b-ocimene,andlinaloolonlyelicitedresponsesinfemales.Frommaturepearfruits,nonanalelicitedonlyresponsesinfemales,and1-hexanol,ethylbutanoate,butylacetate,ethylhexanoate,hexylacetate,hexylbutanoate,and(
E,E)a-farneseneelicitedantennalresponsesinbothsexes(Figure1CandD). poundsfromtheheadspaceVOCsofdetachedpeachshootsandmaturepearfruitselicitedantennalresponsesinOFMfemales:1-hexanol,(Z)-3hexen-1-ol,ethylbutanoate,butylacetate,ethylhexanoate,hexylacetate,hexylbutanoate,(Z)-3-hexenylacetate,nonanal,benzaldehyde,(E)-b-ocimene,linalool,and(
E,E)-afarnesene(Figure1AandC).Ofthese,poundselicitedantennalresponsesinmales:1-hexanol,ethylbutanoate,butylacetate,ethylhexanoate,hexylacetate,hexylbutanoate,(Z)-3-hexenylacetate,and(
E,E)-a-farnesene(Figure1BandD). BasedonmaleandfemaleEADresponsestopear-andpeach-derivedVOCsthefollowingfourluresweredeveloped:
(1)Sh-F:(Z)-3-hexen-1-ol,(Z)-3-hexenylacetate,benzaldehyde,(E)-beta-ocimene,andlinalool(ratio4:100:4:85:1);
(2)Sh-M:(Z)-3-hexenylacetate;
(3)Fr-F:1hexanol,nonanal,ethylbutanoate,butylacetate,ethylhexanoate,hexylacetate,hexylbutanoate,andfarnesene(1:1:100:70:7:5:1:4);and
(4)Fr-M:1-hexanol,ethylbutanoate,butylacetate,ethylhexanoate,hexylacetate,hexylbutanoate,andfarnesene(1:100:70:7:5:1:4). 284Luetal. Peach Pear Compound Amount Relativeamount Amount Relativeamount Hydrocarbons Tetradecane* Pentadecane* 0.81Æ0.10
1 Hexadecane* 0.72Æ0.07
1 Heptadecane* 0.62Æ0.13
1 Octadecane* 0.29Æ0.03<
1 6-Methyl-octadecane Nonadecane* 0.30Æ0.10<
1 Alcohols 1-Hexanol* (Z)-3-Hexen-1-ol* 3.15Æ0.63
4 2-Ethyl-1-hexanol* 2-Methyl-1-hexadecanol Aldehydes Nonanal* 0.34Æ0.15<
1 Esters Ethylbutanoate* Butylacetate* Butylbutanoate* Ethylhexanoate* Hexylacetate* (Z)-3-Hexenylacetate*71.45Æ3.50100 Hexylbutanoate* Hexylhexanoate Benzenoids Benzaldehyde* 3.15Æ0.14
4 Methylsalicylate* 0.52Æ0.07<
1 Terpenoids (E)-b-Ocimene* 61.02Æ2.6685 Linalool* 0.33Æ0.06<
1 (
E,E)-a-Farnesene* 1.50Æ0.11 <
1 2.05Æ0.25
1 3.07Æ0.28
1 1.70Æ0.20
1 1.41Æ0.12 <
1 1.32Æ0.10 <
1 2.95Æ0.25
1 0.73Æ0.23 <
1 0.52Æ0.28 <
1 0.48Æ0.22 <
1 302.11Æ13.00100 212.32Æ11.2970 0.93Æ0.18 <
1 19.75Æ4.25
7 13.75Æ2.25
5 3.25Æ0.25
1 0.53Æ0.23 <
1 12.54Æ2.23
4 Compoundsmarkedwithan*hadbeenconclusivelyidentifiedparisonofspectraandretentiontimeswiththoseofanauthenticstandard.CompoundsinboldfacetypeelicitedantennalresponsesinGC–EADexperiments.CompoundswithineachclasswerelistedordingtoretentiontimesonapolarDB-Wax-fusedsilicacolumn. Table2Quantitiesofpoundscollectedintheheadspaceofpeach(Prunuspersicacv.Wuyuexianbiangan)shootsandmaturepear(Pyrusbretschneidericv.Jimi)fruits:mean(ÆSD)amount(ng100gÀ1plantmaterialhÀ1)andrelativeamount(expressedrelativetothemostpound,settoavalueof100) Fieldexperiment1 Thefirstfieldtestswerecarriedoutduringthelatepeachfruitingandearlypear-fruitingstages,respectively.Basedonthenumberofmalemothstrappedwiththecontrolsexpheromone,itseemsalargerOFMpopulationwaspresentinthepeachorchardatthistimethaninthepearorchard(Figure2AandB). Inpeachorchards,luresthatcontainedpear-derivedVOCs(Fr-
F,Fr-M)caughtsignificantlymoreOFMmalesandfemalesthanthecontrolortheluresthatcontainedpeach-derivedVOCs(Sh-
F,Sh-M)(Figure2A;one-wayANOVA:males:F4,25=105.80;females:F4,25=43.59,bothP<0.01).Fr-MdidnotcatchmoremalesorfemalesthanFr-F,andSh-MdidnotcatchmoremalesthanSh-
F (Figure2A).EventhoughtheSh-Fblendwasbasedonfemaleresponse,nomorefemaleswerecaughtthanwithSh-M(Figure2A). Inthepearorchard,Sh-McaughtmoreOFMmalesthanthecontrolandtheotherlures,buttherewerenosignificantdifferencesamongSh-
F,Fr-M,andFr-Fblends(Figure2B;F4,25=33.67,P<0.01).Allfourblendswereequallyattractivetofemales(Figure2B). Fieldexperiment2Duringthepeachharvestandthelatefruitingstageforpear,asignificantlylargerOFMpopulationwaspresentinthepearorchardthaninthepeachorchard,basedonthenumberofmalemothstrappedwiththecontrolsex SexualdifferencesofOFMinresponsestohosts285 Figure1SimultaneouslyrecordedCydiamolesta(
A,C)femaleand(
B,D)maleGC–EADresponsestoVOCscollectedfrom(
A,B)detachedshootsofpeachvarietyWuyuexianbianganand(
C,D)maturefruitsofthepearvarietyJimiusingapolarDB-WAXcapillarycolumn.Ineachpanel,theuppertraceindicatestheflameionizationdetectorresponses(FID),thelowertracetheantennalresponses(EAD).poundsfoundconsistentlyinsixtestswere(A)(E)-b-ocimene
(1),(Z)-3-hexenylacetate
(2),(Z)-3-hexen-1-ol
(3),benzaldehyde
(4),linalool
(5);(B)(Z)-3-hexenylacetate
(1),(C)ethylbutanoate
(1),butylacetate
(2),ethylhexanoate
(3),hexylacetate
(4),1-hexanol
(5),nonanal
(6),hexylbutanoate
(7),(
E,E)-a-farnesene
(8);(D)ethylbutanoate
(1),butylacetate
(2),ethylhexanoate
(3),hexylacetate
(4),1-hexanol
(5),hexylbutanoate
(6),(
E,E)-a-farnesene
(7). pheromone(Figure2CandD).Inthepeachorchard,muchfewerOFMwerepresentbasedonthemonitoringbysexpheromone-baitedtraps,asalmostnomothswerecaughtinthetraps.Inthepearorchard,luresthatcontainedpeach-derivedVOCs(Sh-
F,Sh-M)caughtsignificantlymoreOFMmalesthanthecontrolandthelures thatcontainedpear-derivedVOCs(Fr-
F,Fr-M)(Figure2D;one-wayANOVA:F4,25=65.67,P<0.01).AlthoughtheSh-Fblendwasbasedonfemaleresponses,Sh-
M,Fr-F,andSh-FblendscaughtsignificantlymorefemalesthanSh-Finpearorchards.(Figure2D;F4,25=47.38,P<0.01). 286Luetal. No.moths/trap 220400A 160120 80 50 Peach 40 AA 30 20 a
B a 10 BC b b
0 240 Sh-FSh-MFr-FFr-
M 200 160 120 80 50 40 30 20 100 1-Hexanol AaSh-
F AaSh-
M AaFr-
F Aa Fr-
M Cb SP CK
C Aa SP CK 240200
B 160 120 80 50 Pear 40 30 20A 10
B a Ba
B ab ab
0 240 Sh-FSh-MFr-FFr-MSP 200 160 120 80 50 40 30AA 20 aBabB bc 10 c 0Sh-FSh-MFr-FFr-MSP MaleFemale Fieldexperiment1 CbCK
D Fieldexperiment2 CdCK (Z)-3-Hexen-1-ol Nonanal Ethylbutanoate Butylacetate Ethylhexanoate Hexylacetate (Z)-3-HexenylacetateHexylbutanoateBenzaldehyde (E)-£-OcimeneLinalool FarneseneSexpheromone Figure2Mean(ÆSD;n=6)totalnumberofCydiamolestamalesandfemalescapturedintrapsin(
A,C)peachand(
B,D)pearorchards,during(
A,B)5-18August(Fieldexperiment1)and(
C,D)5-18September(Fieldexperiment2).EachtrapwasbaitedwitharubberseptumcontainingsyntheticVOCblendscorrespondingtothoseemittedbypeachshoots(Sh-
F,Sh-M),pearfruits(Fr-
F,Fr-M),ahexanecontrol(CK),andsexpheromone(SP).TheratiosoftheponentsaregiveninTable1.Eachlurecontained100mgofthemostponent.Meanswithinapanelcappedwithdifferentletters(capitalformales,lowercaseforfemales)aresignificantlydifferent(one-wayANOVAfollowedbyTukey’sparisontest:P<0.05). 100A 8060 a*b UpwindCloseContact %respondingfemales A* A* 40 c* 20 *d
B a0 Sh-
F 100B B*a* Sh-
M a*Fr-F b a* Fr-Ma
A dBa CK 80 Bb 60C c40 aa %respondingmales 20
D b bc
0 Sh-
F Sh-
M Fr-
F 1-Hexanol Fr-
M d Dc CK (Z)-3-Hexen-1-olNonanal EthylbutanoateButylacetate EthylhexanoateHexylacetate (Z)-3-HexenylacetateHexylbutanoateBenzaldehyde(E)-£-OcimeneLinaloolFarnesene Figure3Mean(ÆSD)attraction(%)ofmatedCydiamolesta(A)femalesand(B)malesinawindtunneltosyntheticVOCblendsmimickingtheheadspaceofdetachedshootsofpeachvarietyWuyuexianbiangan,maturefruitsofpearvarietyJimi,andahexanecontrol(CK).TheratiosoftheponentsaregiveninTable1.Farnesene=mixtureof(
E,E)a-farnesene(49%),(E)-b-farnesene(26%),(Z)-b-farnesene(18%),and(
Z,E)-a-farnesene(7%).Meanswithinapanelwiththesamecolorcappedwithdifferentlettersaresignificantlydifferent(one-wayANOVAfollowedbyTukey’sparisontest:P<0.05).Mothswerescoredfororientation(upwind),flighttowithin10cmoftheVOCsource(close),andlandingontheVOCsource(contact).Differencesbetweensexesinthethreeflightbehaviorswereanalyzedbyunpaired-samplettests(*P<0.05). SexualdifferencesofOFMinresponsestohosts287 Wind-tunnelbioassaysAllfourblendsstimulatedupwindflightinbothfemalesandmales(Figure3AandB),butonlymalesreachedandcontactedthesource(Figure3B).MalesweremorestronglyattractedthanfemalestothesyntheticVOCblends.Inthelaboratory,pear-derivedluresweremoreattractivetobothsexesthanpeach-derivedlures.TheFr-Fblendprovedtobethemostattractivetofemalesinthewindtunnel:67%ofthefemalesflewupwind(one-wayANOVA:F4,10=183.43)and37%arrivedwithin10cmofthesource(F4,10=83.11,bothP<0.01),butnonelandedonthesource(Figure3A).Similarly,Fr-Mwasmostattractivetomalesinthewindtunnel:99%ofthemalesflewupwind(F4,10=130.22),87%arrivedwithin10cmofthesource(F4,10=510.10),and36%landedonthesource(F4,10=64.57,allP<0.01)(Figure3B).AlthoughtheSh-Fblendwasbasedonfemaleresponses,nomorefemalesthanmalesflewupwindandarrivedwithin10cmofthesource(Figure3A). Discussion Femaleorientalfruitmothsrepresentamoreseriousthreattoadjacentorchardsthanmales,becausetheirflightexceedsthatofmales(Hughes&Dorn,2002),andbecausefemaleslaytheeggsthatdevelopintothedamaginglarvae.PreviousstudiesmainlyfocusedontheinteractionbetweenadultOFMfemalesandhostplant-derivedvolatiles(Nataleetal.,2003,2004;Pin~ero&Dorn,2007,2009;Najar-Rodriguez&Dorn,2013).Mostrecently,thediscoverythatmixturescontainingvolatilesattractivetofemalesinthelaboratorycouldcapturemoremalesunderfieldconditionsstimulatedparisonofresponsesofmaleandfemaleOFMtohostplant-derivedvolatiles. Wefoundthatpoundsfromtheheadspacesofpearfruitsweresimilarforfemalesandmales—eightandpounds,respectively—indicatingasimilarchemosensorysystemofOFMmaleandfemaleantennae.Inthefield,malecapturessignificantlyoutnumberedfemalecatches,evenintrapswithpoundluresbasedonfemaleresponsestopearfruitVOCs(Fr-F). Responsestohostplant-derivedvolatileswerealsofoundtodifferbetweenthesexes.Theblendwithoutnonanal(Fr-M)wasmoreattractivetomalesthantheblendcontainingnonanal(Fr-F),suggestingthatnonanalactsasarepellentorattraction-inhibitorforOFMmalesunderfieldconditions.Nonanalhasbeencharacterizedasanositiondeterrentforfemalecodlingmoth,Cydiapomonella(
L.)(Yokoyama&Miller,1991).Otherstudiesalsosuggestedasexdifferencein 288Luetal. responsetosemiochemicals.Forturnipmoths,Agrotissegetum(Denis&Schiffermu€ller),Hanssonetal.(1989)characterizedthepheromoneandplantvolatileperceptioninmalesandfemalesbyEADandsinglesensillumtechniquesandfoundfemalereceptorsarespecializedforplantvolatilereceptionandareinsensitivetopheromones.Incontrast,thespecializedpheromonereceptorsonmaleantennaearesensitivenotonlytopheromonesbutalsotoplantvolatiles.Inaddition,maleshavespecializedplantvolatilereceptors(Hanssonetal.,1989).Codlingmothmalesandfemaleswerefoundtodifferinthenumberofolfactoryreceptorneuronsintheantennae(B€ackmanetal.,2000). Overall,thebehavioralresponseofmalestowardhostplant-derivedvolatilesbothinthelaboratoryandinthefieldwasstrongerthanthatoffemales,demonstratinggoal-directedmaleorientationtowardsexpheromonesources.SomeauthorshavesuggestedthatmalesuseplantVOCstodistinguishenvironmentswheretheycanfindfemalesmoreeasily(Anseboetal.,2004;Il’ichevetal.,2009).Femalesontheotherhandneedtoconsidermorefactors,suchasflightmode,responseprofile,orflightstrategytowardhostplantsunderfieldconditions.Theflightmodeofmalesmaybedifferentfromthatoffemales.Inourwind-tunnelstudy,femaleswereattractedbysyntheticlures,buttheperformanceofmalesexceededthatoffemalesespeciallywhenapproachingthesource.Theorientationmechanismsofmalesflyinginsearchoffemalesareadaptedtolocateapoint-sourceofsexpheromone.Bycontrast,femalessearchingforsuitableositionsitesmightnotbeasstronglyattractedtopointsourcesofplantVOCs,andthereforetrapdesigncouldhavealargeinfluenceontheirabilitytoattractfemales.Similarresultswerefoundforthecodlingmoth,animportantpestofapple(Coracinietal.,2004).Femalecodlingmothshavefrequentlybeenobservedtoflyupwindoverseveralmeterstowardbrancheswithgreenapples,butcontactedtheapplelessfrequently,whichsuggeststhatthefemalesemployadifferentsearchstrategythanmales,especiallyatcloserange,lookingforasuitableositionsite(Witzgalletal.,1999).Also,femalesmaylocateositionsitesbasedonbothchemicalandvisualcues.ThegreatestflightactivityofmatedandunmatedmaleandfemaleOFMsurredduringthefirsthourofdusk,whenlightintensitydecreasedfrom3750to57lx,suggestingthatthefemalesuseopticalcuestolocateositionsites(Hughes&Dorn,2002).Similarresultshavebeenfoundingrapeberrymoth,Paralobesiaviteana(Clemens),acrepuscularspecies,whichositslessintheabsenceoflight(Clark&Dennehy,2002).Thephysicalstructureandlowvolatilityof VOCspresentonfruitsurfacescouldalsoaffectfemaleperformance.Inourstudy,OFMsalwayslaideggsonthesmoothsurfaceofwaxedpaperinthelaboratory.Thestickybaseofthetrapsmightrepelfemalelanding. Aspredicted,theVOCblendbasedonmaleresponsestopeachshootVOCs(Sh-M)caughtmoremalesthantheblendbasedonfemaleresponsestothesamesource(Sh-F);however,theblendbasedonfemaleresponses(Sh-F)didnotattractmorefemalesthanthemaleresponse-derivedblend(Sh-M).OftheVOCsfrompeachshoots,(Z)-3-hexenylacetateelicitedantennalresponsesfrombothsexes,whereastheotherponents,(Z)-3-hexen-1-ol,benzaldehyde,(E)b-ocimene,andlinalool,onlyelicitedresponsesfromfemales.Fieldassaysindicatedtheotherponentswereredundantinattractingnotonlymalesbutalsofemales.Wehypothesizethatvolatilescollectedfromcutyoungpeachshootsmaydifferfromtheheadspaceofintactpeachshoots.ThiscouldgreatlyaffecttheolfactoryorientationofOFMs.Inourstudy,besideahydrocarbon,ponents—(Z)-3-hexen-1-ol,(Z)3-hexenylacetate,benzaldehyde,methylsalicylate,(E)b-ocimene,andlinalool—werecharacteristicofdetachedpeachshoots.Nataleetal.(2003)foundpoundsintheheadspaceofexcisedpeachshoots.(Z)-3-Hexen-1-ol,(Z)-3-hexenylacetate,benzaldehyde,methylsalicylate,and(Z)-b-ocimenewerealsodetectedintheirstudy.Il’ichevetal.(2009)analyzedintactyoungshoottipsofpottedpeachtreesbyGC-MS.Theyfound(Z)-3-hexenylacetateand(Z)-b-ocimeneintheheadspaceoflivingpeachshoots,but(Z)-3-hexen-1-ol,benzaldehyde,andmethylsalicylatewerenotdetected.Wededucethat(Z)-3-hexen-1-ol,benzaldehyde,andmethylsalicylatearecharacteristicofdetachedpeachshootsandmaybeinducedbytheartificialcuttingofshoots.Theseponentsareassociatedwithplantstresscausedbymanualdamageandcouldindicatealow-qualityhostoranon-host.Activeavoidanceofnon-hostodorsandevennutritionallyunsuitablehostsappearstobeanimportantpartoftheinsecthostlocationprocess. Inconclusion,ourstudysuggestedsimilaritiesinthechemosensorysystemofOFMmaleandfemaleantennae,butalsodifferencesinresponseswerefoundbetweenthesexes.FurtherstudyisneededtoidentifythevolatilesfromintactpeachshootsthatattractOFMmalesorfemales.Both,malesandfemalescoulddisperseintosecondaryhostorchardsbetweenthelatepeach-fruitingandlatepear-fruitingstage.Thetwopear-derivedVOCblends(FrFandFr-M)caughtsignificantlymoreOFMmalesandfemalesthantheotherblendsinpeachorchards;conversely,thetwopeach-derivedblendsweremoreattractive SexualdifferencesofOFMinresponsestohosts289 thantheotherblendsinpearorchards.Together,theblendsderivedfromoneplantspeciesshouldbeusedinorchardsoftheotherspecies.ThismaybevaluablefordesigningfurthercandidateattractantblendsforC.molesta.Inaddition,weshouldseektobetterunderstandthedevelopmentaldifferencesbetweenthesexesofC.molestaandamongitshostcropsandexplorebetterpestmanagementapproachesbasedonoptimizingthepeach–pearplantingsysteminthefield. Acknowledgements WearegratefultoEditage(CactusCommunications,Trevose,PA,USA)forlanguageimprovement.ThisworkwassupportedbyBeijingNaturalScienceFoundation(grantno.6142015),BeijingYoungEliteProject(grantno.2013D009046000002),FundamentalResearchFundsfortheCentralUniversities(grantno.YX2015-09),NationalNaturalScienceFoundationofChina(grantnos.31570643,31270693,81102747),andPublicWelfareProjectfromtheMinistryofAgricultureofChina(grantno.201103024). References AnforaG,TasinM,CristofaroAD,IoriattiC&hiA(2009)SyntheticgrapevolatilesattractmatedLobesiabotranafemalesinlaboratoryandfieldbioassays.JournalofChemicalEcology35:1054–1062. AnseboL,CoraciniMDA,BengtssonM,LiblikasI,RamirezMetal.(2004)AntennalandbehaviouralresponseofcodlingmothCydiapomonellatoplantvolatiles.JournalofAppliedEntomology128:488–493. 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L.)Batsch,isitsprimaryhost,andthepomefruitpear,PyrusbretschneideriRehder(bothRosaceae),isitssecondaryhost.ElectrophysiologicalandbehavioralresponsesofC.molestafemalesandmalestopeachshootandpearfruitvolatilesparedinlaboratoryandfieldbioassays.Basedongaschromatography–electroantennographicdetection(GC–EAD)activity,poundsintheheadspacesofpeachandpearvolatileselicitedfemaleantennalresponses.Ofthese,poundsalsoelicitedmaleantennalresponse.FourluresweredevelopedbasedonmaleandfemaleEADresponsestopearandpeach-derivedpounds(VOCs).Moremalesthanfemaleswerecapturedforallfourluresduringfieldtrials,evenintrapswithluresthatemittedVOCsbasedonfemaleEADresponses.LuresbasedonfemaleEADresponsestopearfruitVOCsconsistentlycaughtmorefemalesthanluresbasedonmaleEADresponsestopearfruitVOCsineitherpeachorpearorchards.PeachshootVOCluresbasedonfemaleEADresponsesdidnotattractmorefemalesthanluresbasedonmaleEADresponsetopeachshoots.Thetwopear-derivedVOClureswerehighlyattractivetobothsexesinpeachorchards,whereasconversely,thetwopeach-derivedVOCluresshowedstrongerattractioninpearorchards.Seasonalpopulationmonitoringindicatedbothsexesmadeinter-orchardflightsduringthelatepeach-andpear-fruitingperiods.Apossiblehypothesisthatcouldexplaindifferentresponseprofilesinfemalesandmalesandseasonalmigrationforherbivoreswithmultiplegenerationsperyearisdiscussed. Introduction Theorientalfruitmoth(OFM),Cydia(=Grapholitha)molesta(Busck)(Lepidoptera:Tortricidae),presumablyoriginatingfromnorth-westChina,hasewidely *Correspondence:You-QingLuo,BeijingKeyLaboratoryforForestPestControl,BeijingForestryUniversity,35QinghuaDongRoad,HaidianDistrict,Beijing100083,China.E-mail:luoyouqing224@;Hai-LiQiao,InstituteofMedicinalPlantDevelopment,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,151MalianwaNorthRoad,HaidianDistrict,Beijing100193,China.E-mail:qhl193314@#Peng-FeiLuandRongWangcontributedequallytothiswork. distributedthroughouttheworld(Rothschild&Vickers,1991).Untilthelate1970sthespecieswasconsideredtobeoligophagousandonlytodamagestonefruitinWesternEurope(Bovey,1979).Inrecentyears,however,OFMdamagetopomefruitorchardshasbeenwidelyreportedinAsia,Europe,Australia,andAmerica(Rothschild&Vickers,1991;Dornetal.,2001;Myersetal.,2006;Il’ichevetal.,2009;Luetal.,2012).InNorthChina,peachshootsandpearfruitsaretheprimaryOFMhostsubstratesearlyandlateinthegrowingseason(Luetal.,2012),andthismayresultinmovementfromstonefruitorchardstopomefruitorchards(Zhaoetal.,1989;Yang&Liu,2010;Luetal.,2012). ©2015TheNetherlandsEntomologicalSocietyEntomologiaExperimentalisetApplicata157:279–290,2015 279 280Luetal. TheflightoffemaleC.molestasignificantlyexceedsthatofmales(Hughes&Dorn,2002).Femalesaredirectlyresponsibleforinfestationsbymakinginter-orchardflightsandlayingeggsonpearfruits(Yetter&Steiner,1932;Steiner&Yetter,1933),sothisstudyfocusesontheinteractionbetweenadultOFMresponsetohostplantderivedvolatileswithemphasisonparisonbetweenfemaleandmaleresponses. OlfactorycuesforOFMfemalesfrombothshootsandfruitsofpeachandapplehavebeenstudiedextensively(Nataleetal.,2003,2004;Pin~ero&Dorn,2007,2009;Najar-Rodriguez&Dorn,2013).Excisedpeachshootsemitting(Z)-3-hexenylacetate,(Z)-3-hexenol,andbenzaldehydeata4:1:1ratiowasattractivetomatedOFMfemales(Nataleetal.,2003).Volatilesemittedfrompeachandapplefruitswerealsoattractivetomatedfemalesinadualchoicearena(Nataleetal.,2004).Behavioralexperimentshaveidentified(Z)-3-hexenylacetate,(Z)-3-hexenol,(E)-2-hexenal,benzonitrile,andbenzaldehyde,emittedbypeachshoots,toactinasynergisticmanner,resultinginahighlyattractiveodormixturetofemaleOFM(Pin~ero&Dorn,2007).Recentstudiespayparticularattentiontotheseasonaldynamicsofvolatileemissionsfromdifferenthosts.Pin~ero&Dorn(2009)reportedthatC.molestafemalesselectedshoots,twigwithleaves,andfruitsofpeachandappletreesatdifferenttimesoftheseason.Najar-Rodriguez&Dorn(2013)reportedvolatileemissionsfrominsitupeachandpeartreesoveranentireseason,alongwiththeirolfactoryattractivenesstoC.molestafemales.SomestudieshavebeenconductedtoindicatehighattractivenessofparticularbinationstofemaleOFMinthelaboratory(Pin~eroetal.,2008),butfielddataarelacking.ForthefewstudiesthatreportfieldresponsesofOFMtolures,thereseemstobenocorrespondencebetweenthelaboratoryandfieldfindings.Forexample,Il’ichevetal.(2009)carriedoutheadspaceanalysisofvolatilesfromtheintactyoungshoottipsofpeach.Theyfoundthatamixtureof(Z)-3-hexenylacetate,(E)-bocimene,and(E)-b-farneseneataratioof1:2:2wasthebestattractant,butonlytoOFMmales,nofemaleswerecapturedinthefield.Morerecently,basedonfemaleOFMresponsesrecordedusinggaschromatography–electroantennographicdetection(GC–EAD),theprofileofbioactivevolatilesemittedfromimmatureandmaturepearfruitwerecharacterized(Luetal.,2012)andreportedtwopear-derivedponentattractantsthatcanbeusedformonitoringbothsexesoforientalfruitmothinthefield.Subsequently,theycarriedparativeanalysesofpeachandpearfruitvolatilesattractivetobothOFMsexes,andevaluatedtheattractivenessofvolatilemixturesmimickingpeachandpearfruitvolatilestoOFMmalesandfemales(Luetal.,2014).Despiteusingmixtures containingvolatilesattractivetofemalesinthelaboratory,malecaptureswerestillhigherunderfieldconditionsinthesestudies. Theseresultsraisedtwoimportantquestions.WhyaremoremaleOFMsthanfemalescapturedbytrapswithluresemittingplant-derivedpounds(VOCs)attractivetofemalesinthelaboratory?
Whatisthedifferenceintheelectrophysiologicalandbehavioralresponsesofbothsexestopeachandpearvolatilesbetweenthelaboratoryandfieldbioassays?
Theanswersshouldhelptoelucidatethebehavioralmechanismsunderlyinghighermalecatcheswhenusingvolatilesselectedonthebasisoffemaleelectroantennography(EAG)responses. Theobjectivesofthestudywere(1)toidentifyandanalyzetheVOCsfromyoungpeachshoots[Prunuspersica(
L.)Batsch]andpearfruits[PyrusbretschneideriRehder(bothRosaceae)]attractivetoOFMs,and(2)paretheresponsesofmaleandfemaleOFMtopeachshootandpearfruitvolatilesusinglaboratoryandfieldbioassays. Materialsandmethods Insects Larvaewerecollectedfrominfestedshootsofpeach,PrunuspersicaL.Batsch‘Wuyuexianbiangan’,inanexperimentalorchardattheInstituteofForestryandPomology(IFP),BeijingAcademyofAgricultureandForestry(BAAF),Beijing,China(39°580N,116°130E)inlateJune.Theselarvaeweremaintainedinaclimaticchamberat24Æ1°C,65–70%r.h.,andL16:D8eriod,withthehasestartingat05:00hours.Untilthethirdinstar,larvaeweremass-rearedonapple,MalusdomesticaL.Borkh.‘Hongfushi’(Rosaceae),inaglasscontainer(27cmdiameter,13cmhigh)andthentransferredtosmallerindividualglasscontainers(2.5cmdiameter,8cmhigh)untileclosion.Abell-shapedglasscontainer(6-and15-cm-diameteropenings,41cmhigh)wasusedtoraisetheadults,andcoveredwithfinenylonmeshatbothsides.A15%(wt/vol)honeysolutionwasprovidedonwater-soakedcottonviaaholepiercedthroughthemeshinthesmallsideofcontainer.Themothswererearedinthelaboratoryforthreegenerationsbeforetesting.Forelectrophysiologicalexperiments(GC–EAD),2-to3-dayoldfemalesandmaleswereused.Forthewind-tunnelbioassay,matedfemaleandmalemothswereselectedrandomly.Matedmothswereobtainedbyplacinggroupsofca.20newlyemergedfemalestogetherwith30malesinthesamecagefortwoeriodiccyclestoensuremating.Eachadultmothwasusedinonlyoneassayandmothswerenotexposedtosyntheticodorsourcesbeforethebioassay. SexualdifferencesofOFMinresponsestohosts281 ChemicalsTetradecane(99%purity),pentadecane(99%),hexadecane(99%),octadecane(99%),nonadecane(99%),(Z)-3hexen-1-ol(98%),butylbutanoate(98%),(Z)-3-hexenylacetate(98%),benzaldehyde(99%),methylsalicylate(99%),(E)-b-ocimene(60%),and(
E,E)-a-farnesene(49%)wereobtainedfromSigma-Aldrich(St.Louis,MO,USA).Theremainingfarnesenefractionconsistedofmainly(E)-b-farnesene(26%),(Z)-b-farnesene(18%),and(
Z,E)-a-farnesene(7%).WeusedthismixtureoffarneseneisomersforourEAGandfieldstudiesandrefertoitas‘farnesene’.Heptadecane(99%),1-hexanol(99%),2ethyl-1-hexanol(99%),nonanal(97%),hexylacetate(99%),andracemiclinalool(97%)wereobtainedfromFlukaProduction(Buchs,Switzerland).Ethylbutanoate(99%)andbutylacetate(99%)wereboughtfromAcrosOrganics(MorrisPlains,NJ,USA).Ethylhexanoate(98%)andhexylbutanoate(98%)werepurchasedfromTokyoChemicalIndustry(Tokyo,Japan).Compoundsthatdidnotelicitantennalresponses,andforwhichnostandardswereavailable,weretentativelyidentifiedusingtheNIST-database(Tasinetal.,2005). Plantmaterials Ten-year-oldplantsofpeachesandpearswerecultivatedinanexperimentalorchardattheIFP.Thepeachvarietyusedwasflatpeach(FP),‘Wuyuexianbiangan’.ThepearvarietyusedwasJimi(JM).BothvarietiesmonlygrownintheBeijingarea.NoinsecticidesoranyspecifictreatmentagainstOFMwereusedintheorchardduringthetests. CollectionofVOCsfromdetachedplanttissuesPeachshoots(ca.30cmlong)werecutfromselectedtreesandsealedwithliquidparaffin(FlukaProduction),beforebeingtransferredtothelaboratorynearby(Anforaetal.,2009).Plantmaterialwasusedforvolatilecollectionorwind-tunnelassayswithin20minaftercutting.PearandpeachfruitswerehealthyandwerepickedfromtheexperimentalorchardtreeswhenfruitsofthecorrespondingvarietieswereripeintheBeijingarea(Caoetal.,2000,2006;Jiangetal.,2002). Apush–pullsystemwasusedtocollectheadspaceVOCs.Peachshoots(ca.1500g)andpearfruits(ca.1500g)wereplacedintoa2lglassjarforextraction.Airaspiratedwithavacuumpump(QianxiAir,Beijing,China)andfilteredthroughanactivatedcharcoalfilterwaspassedthroughthejarat300mlpermin,andfinallythroughasorbentcartridge(PorapakQ,50mg,80/100mesh;Supelco,Bellefonte,PA,USA).Thesorbentcartridgeswereheldbetweenplugsofglasswoolinaglass tube(10cmlong,0.5cminnerdiameter).VOCsampleswerecollectedfor8hat24Æ1°Cand65–70%r.h. Volatilesweredesorbedbyelutingthesorbentcartridgewith500llhexane(HPLCgrade,Sigma-Aldrich)atroomtemperature.Fivesampleseachwerecollectedfrompeachshootsandripepearfruits.ThesampleswereanalyzedbyGC–EADandgaschromatography–massspectroscopy(GC–MS).Forquantitativeanalyses,0.5lgethylpentanoate(99%;FlukaProduction)wasaddedasaninternalstandardtoeachsample.PreliminaryanalysesshowedthatpoundwasnotdetectableintheheadspaceVOCsofthepeachorpearvarietiesstudiedhere.Samplevolumeswerereducedto50llusingaslowstreamofnitrogenandthenanalyzed.Ifnotusedimmediately,extractsweresealedinglassvialsandstoredatÀ18°Cuntilused. GC–MSHeadspaceVOCsofpeachshootsandpearfruitswereanalyzedwithanAgilent5973MScoupledtoanAgilent6890NGC(AgilentTechnologies,PaloAlto,CA,USA)equippedwithapolarDB-WAX-fusedsilicacolumnoranon-polarDB-5-fusedsilicacolumn(both30m90.25mmi.d.,0.25lmfilm;J&WScientific,Folsom,CA,USA).Thetemperatureprogramwasasfollows:50°Cfor1min,thenincreasing3°Cperminto120°C,thenincreasing10°Cperminto240°C,andfinallyheldat240°Cfor10min.WindowsNT/MASSSpectralSearchProgramsoftware(version1.7)wasusedfordataanalysis.Injectionsweremadeinthesplitlessmode.Heliumwasusedasthecarriergas(1.0mlpermin).Forelectronimpact(EI)massspectra,theionizationvoltagewas70eV,andthetemperaturesoftheionsourceandoftheinterfacewere230and280°C,respectively.Theemissioncurrentwas34.6lA.IdentificationofVOCswasverifiedparisonwithauthenticsamples. GC–EADAmicromanipulatorassembly(MP-15)wasconnectedtoastimuluscontroller(CS-55;bothSyntech,Hilversum,TheNetherlands).Allsignalsourceswereconnectedtoaserialdataacquisitioninterface(IDAC-4;Syntech).TheantennaeofOFMmalesandfemaleswereexcisedusingmicro-scissors.Afewsegmentsfromthetipsofantennaewereclippedoffandmountedontheantennaholderwithtwometalelectrodesusingconductivegel(Spectra360;ParkerLaboratories,Fairfield,NJ,USA),andthentheelectrodeholderwasinsertedintotheEADprobe.Testingbeganafterarelativestablebaselinehadbeenachieved.TheoutletoftheGCcolumnwassplitina2:1ratiobetweenacutantennaandtheflameionizationdetector(FID).Themountedantennawaspositionedinthecharcoal-filteredandhumidifiedairstreamthatcarriedthe 282Luetal. VOCsfromtheGCcolumn.TheantennalandFIDsignalswereamplifiedandrecordedsimultaneouslyusingSyntechsoftware(GC-EAD32,version4.4).Eachsamplewastestedwithsixantennae,eachderivedfromadifferentmoth.Identitiesofpoundswereverifiedparisonofmassspectraandretentiontimeswiththoseofsyntheticstandards. TheVOCswereanalyzedwithanAgilent6890NGCwithaflameionizationdetector,coupledwithaSyntechEAD.ColumntypeandoventemperatureprogramwerethesameasintheGC–MSanalysis.Nitrogenwasusedasthecarriergas(1.0mlpermin). FieldexperimentBasedontheresultsoftheGC–EADanalyses,VOCsfromthepeachshootsandpearfruitsthatelicitedantennalresponsesinfemaleandmaleOFMswereusedtoformulatefourseparateblendsforthefieldtests.Thefoursyntheticblendsderivedfromthetwohostplantswereevaluatedinadjacentorchardsofthetwohostcropsfromthetimeoflatepeachfruitingtolatepearfruiting. ThefourVOCblendswereasfollows:
(1)Sh-F(shootfemale):VOCsfrompeachshootstowhichfemaleshadpositiveEADresponses;
(2)Sh-M(shoot-male):VOCsfrompeachshootstowhichmaleshadpositiveEADresponses;
(3)Fr-F(fruit-female):VOCsfrompearfruitstowhichfemaleshadpositiveEADresponses(Luetal.,2012);and
(4)Fr-M(fruit-male):VOCsfrompearfruitstowhichmaleshadpositiveEADresponses.ThefourblendsofpoundswerepreparedinthesameratiosofGC–EAD-activeVOCsasemittedbythecorrespondingplanttissue(Table1).Thesewerepreparedusing100mgofthemostpoundandaddingtheothersinthesameproportionasinthenaturallyurringvolatileblend. Goblet-shapedrubbersepta(10mmdeep,6mmi.d.)withamaximumvolumeof400ll(ShunyiRubber,Beijing,China)wereusedtodispersetheVOCblends.ThisvolumewassufficientforalltheVOCblendsinourstudy.TheVOCblendswereprepared1–2hpriortotheiruseinfieldbioassays.ForeachVOCblend,rubberseptawerefilledandthenfixedupwardonthebottomofastickydeltatrap.Trapswereplacedinthefieldatdusk,whenOFMflightintensitypeaks,forallthetreatments.Wedeployedtherubberseptawithsolutionsdirectlyinthetrap,beforethemixturesolutionwasfullyimpregnatedintotherubbersepta.Allodorblendsweredeployedinthisway(Luetal.,2012).Unbaitedtraps(HPLC-gradehexane;Sigma-Aldrich)andOFMsex-pheromonelures(GeruibiyuanTechnology,Beijing,China)wereusedascontrols. Table1Components,theiramountsloadedonrubbersepta(mg),andtheirratiosfortheblendsusedinthefieldexperiment.Theblendsarebasedonfemale(F)ormale(M)EADresponsetodetachedpeach(Prunuspersicacv.Wuyuexianbiangan)shoots(Sh)andmaturepear(Pyrusbretschneidericv.Jimi)fruits(Fr) Compound1 Sh-FSh-MFr-F2Fr-
M Alcohols 1-Hexanol
1 1 (Z)-3-Hexen-1-ol
4 Aldehydes Nonanal
1 Esters Ethylbutanoate 100 100 Butylacetate 70 70 Ethylhexanoate
7 7 Hexylacetate
5 5 (Z)-3-Hexenylacetate100100 Hexylbutanoate
1 1 Benzenoids Benzaldehyde
4 Terpenoids (E)-b-Ocimene 85 Linalool
1 Farnesene3
4 4 1In
orderofelutionduringgaschromatographyonapolarDB- Wax-fusedsilicacolumn.2TheblendsofFr-Fwerebasedonheadspaceposition fromdetachedmaturepearfruits(Luetal.,2012).3Farnesene=mixtureof(
E,E)-a-farnesene(49%),(E)-b-farnesene(26%),(Z)-b-farnesene(18%),and(
Z,E)-a-farnesene(7%). Thefieldtestwasconductedina7-hapeachorchardanda6.5-hapearorchardattheIFPwithca.30–50mopenspacebetweentheorchards.BothorchardshadahistoryofOFMinfestation.Twotrialswereconducted,eachof2weeks:5–18August(Fieldexperiment1)and5–18September(Fieldexperiment2)2013.ThesecoincidedwithpeakOFMactivityintheBeijingarea.Stickydeltatraps(35920920cm)wereusedtotraptheinsects.Ineachorchard,thetrialswerecarriedoutinarandomizedblockdesigncontainingsixreplicateblocks.Ineachofthesixblocks,thedifferenttreatmentswererepeatedonlyonce.Blockswereatleast120mapart.Trapswereinstalledca.1.5mabovethegroundandwereseparatedbyatleast30mtominimizeinterferencebetweentraps. Thetrapsweremonitoredtwiceweekly,capturedOFMsweretransferredtothelaboratory,andtheirnumberandsexwererecorded.Thedataofcapturesovereach2-weekperiodwerepooled. Wind-tunnelassayThewind-tunneltestsweredesignedtotestforsexualdifferencesinflightabilitytowardthefourVOCblends. SexualdifferencesofOFMinresponsestohosts283 Malesandfemaleswereusedinthewind-tunnelassay.Thelaboratorywindtunnelwas1.6mlong,0.5mwide,and0.5mhigh.Afanattheupwindendgeneratedasteadyairflowintothetunnel,setat0.3msÀ1atthepointofreleaseofmoths.Thelightintensityinthetunnelwasca.250lx.Theroomwaskeptat23Æ2°Cand50–70%r.h. FourVOCblendswerepreparedinproportionsofGC–EAD-activeVOCsfoundinthenaturalblendsemittedbythecorrespondingvarieties(Table1).ThepredominantVOCinthemixturewasdosedat0.5mg.TheVOCsweredilutedwithhexane(HPLCgrade;SigmaAldrich).Preliminarytestsinthewindtunnelshowedthattheseconcentrationswereadequatetoelicitmothresponses.Theseptumloadedwithoneoftheblendswasplacedonaholderattheupwindendinthecenterofthetunnel,10cmfromtheupwindend.Individualmothswerescoredforthefollowingbehaviors:(1)departurefromthereleasecageandflightupwind;(2)arrivalwithin10cmoftheVOCsource;and(3)landingonthesource.Wecategorizedeachmothasthebehaviorassociatedwiththefurthestdistancetraveledwithin20min. Testsbegan2hbeforethebeginningofthehaseandlasted3h.Mothsweresexedandwerethentransferredintothetestroom2hbeforetheexperiments.Batchesof10mothswereplacedinasmall,screenedmetalreleasecage(7cmdiameter,9cmhigh)withasidedoorthroughwhichtheairfromthewindtunnelcouldflow.Thereleasecagewasplacedonaholderatthedownwindendofthetunnel,30cmaboveitsfloorandca.140cmfromtheVOCsource.Thedoor,facingtheupwindendofthetunnel,wasopenedtoallowthemothstoleavethecage.Eachbatchof10matedmothswastestedfor20min,and6batchesofmothswereusedperday.EachVOCblendwastestedwithninebatchesofmoths,eachonadifferentday.Individualmothsweretestedonlyonce. Dataanalysis MeannumbersofOFMmalesandfemalescapturedintrapsbaitedwitheachVOCblendinthefieldandtheperformancescoresofOFMfemalesandmalestotheVOCsourceinthewindtunnelwereanalyzedbyone-wayANOVA.Tukey’smultiplerangetestwasusedtotestforsignificantdifferencesamongVOCblendsinthemeannumbersofOFMscaptured(a=0.05).Unpairedsamplet-testswereusedtotestforsignificantdifferencesbetweensexesinthreeflightbehaviorsinthewind-tunnelassay.AlldatawereanalyzedwiththestatisticalprogramSPSSv.16.0(SPSS,Chicago,IL,USA). Results Characterizationoftheheadspacevolatilesfrompeachshootsand pearfruits Compoundsbelongingtovariouschemicalclasseswereidentifiedfrompeachshoots:hydrocarbons,alcohols,aldehydes,esters,benzenederivatives,andterpenoids(Table2).ponents—octadecane,(Z)-3-hexen1-ol,(Z)-3-hexenylacetate,benzaldehyde,methylsalicylate,(E)-b-ocimene,andlinalool—werecharacteristicofpeachshootsandabsentfromripepearfruits.ponents—tetradecane,6-methyl-octadecane,1-hexanol,2-ethyl-1-hexanol,2-methyl-1-hexadecanol,(
E,E)a-farnesene,andsevenesters—werecharacteristicofripepearfruitsandabsentfrompeachshoots.Inparticular,(Z)-3-hexenylacetateandethylbutanoatewerethemostabundantVOCsidentifiedinpeachshootsandpearfruits,respectively. FemaleandmaleantennalresponsestoVOCsfrompeachshootsand pearfruits Fromdetachedpeachshoots,(Z)-3-hexenylacetateelicitedantennalresponsesinbothsexes(Figure1AandB),whereas(Z)-3-hexen-1-ol,benzaldehyde,(E)-b-ocimene,andlinaloolonlyelicitedresponsesinfemales.Frommaturepearfruits,nonanalelicitedonlyresponsesinfemales,and1-hexanol,ethylbutanoate,butylacetate,ethylhexanoate,hexylacetate,hexylbutanoate,and(
E,E)a-farneseneelicitedantennalresponsesinbothsexes(Figure1CandD). poundsfromtheheadspaceVOCsofdetachedpeachshootsandmaturepearfruitselicitedantennalresponsesinOFMfemales:1-hexanol,(Z)-3hexen-1-ol,ethylbutanoate,butylacetate,ethylhexanoate,hexylacetate,hexylbutanoate,(Z)-3-hexenylacetate,nonanal,benzaldehyde,(E)-b-ocimene,linalool,and(
E,E)-afarnesene(Figure1AandC).Ofthese,poundselicitedantennalresponsesinmales:1-hexanol,ethylbutanoate,butylacetate,ethylhexanoate,hexylacetate,hexylbutanoate,(Z)-3-hexenylacetate,and(
E,E)-a-farnesene(Figure1BandD). BasedonmaleandfemaleEADresponsestopear-andpeach-derivedVOCsthefollowingfourluresweredeveloped:
(1)Sh-F:(Z)-3-hexen-1-ol,(Z)-3-hexenylacetate,benzaldehyde,(E)-beta-ocimene,andlinalool(ratio4:100:4:85:1);
(2)Sh-M:(Z)-3-hexenylacetate;
(3)Fr-F:1hexanol,nonanal,ethylbutanoate,butylacetate,ethylhexanoate,hexylacetate,hexylbutanoate,andfarnesene(1:1:100:70:7:5:1:4);and
(4)Fr-M:1-hexanol,ethylbutanoate,butylacetate,ethylhexanoate,hexylacetate,hexylbutanoate,andfarnesene(1:100:70:7:5:1:4). 284Luetal. Peach Pear Compound Amount Relativeamount Amount Relativeamount Hydrocarbons Tetradecane* Pentadecane* 0.81Æ0.10
1 Hexadecane* 0.72Æ0.07
1 Heptadecane* 0.62Æ0.13
1 Octadecane* 0.29Æ0.03<
1 6-Methyl-octadecane Nonadecane* 0.30Æ0.10<
1 Alcohols 1-Hexanol* (Z)-3-Hexen-1-ol* 3.15Æ0.63
4 2-Ethyl-1-hexanol* 2-Methyl-1-hexadecanol Aldehydes Nonanal* 0.34Æ0.15<
1 Esters Ethylbutanoate* Butylacetate* Butylbutanoate* Ethylhexanoate* Hexylacetate* (Z)-3-Hexenylacetate*71.45Æ3.50100 Hexylbutanoate* Hexylhexanoate Benzenoids Benzaldehyde* 3.15Æ0.14
4 Methylsalicylate* 0.52Æ0.07<
1 Terpenoids (E)-b-Ocimene* 61.02Æ2.6685 Linalool* 0.33Æ0.06<
1 (
E,E)-a-Farnesene* 1.50Æ0.11 <
1 2.05Æ0.25
1 3.07Æ0.28
1 1.70Æ0.20
1 1.41Æ0.12 <
1 1.32Æ0.10 <
1 2.95Æ0.25
1 0.73Æ0.23 <
1 0.52Æ0.28 <
1 0.48Æ0.22 <
1 302.11Æ13.00100 212.32Æ11.2970 0.93Æ0.18 <
1 19.75Æ4.25
7 13.75Æ2.25
5 3.25Æ0.25
1 0.53Æ0.23 <
1 12.54Æ2.23
4 Compoundsmarkedwithan*hadbeenconclusivelyidentifiedparisonofspectraandretentiontimeswiththoseofanauthenticstandard.CompoundsinboldfacetypeelicitedantennalresponsesinGC–EADexperiments.CompoundswithineachclasswerelistedordingtoretentiontimesonapolarDB-Wax-fusedsilicacolumn. Table2Quantitiesofpoundscollectedintheheadspaceofpeach(Prunuspersicacv.Wuyuexianbiangan)shootsandmaturepear(Pyrusbretschneidericv.Jimi)fruits:mean(ÆSD)amount(ng100gÀ1plantmaterialhÀ1)andrelativeamount(expressedrelativetothemostpound,settoavalueof100) Fieldexperiment1 Thefirstfieldtestswerecarriedoutduringthelatepeachfruitingandearlypear-fruitingstages,respectively.Basedonthenumberofmalemothstrappedwiththecontrolsexpheromone,itseemsalargerOFMpopulationwaspresentinthepeachorchardatthistimethaninthepearorchard(Figure2AandB). Inpeachorchards,luresthatcontainedpear-derivedVOCs(Fr-
F,Fr-M)caughtsignificantlymoreOFMmalesandfemalesthanthecontrolortheluresthatcontainedpeach-derivedVOCs(Sh-
F,Sh-M)(Figure2A;one-wayANOVA:males:F4,25=105.80;females:F4,25=43.59,bothP<0.01).Fr-MdidnotcatchmoremalesorfemalesthanFr-F,andSh-MdidnotcatchmoremalesthanSh-
F (Figure2A).EventhoughtheSh-Fblendwasbasedonfemaleresponse,nomorefemaleswerecaughtthanwithSh-M(Figure2A). Inthepearorchard,Sh-McaughtmoreOFMmalesthanthecontrolandtheotherlures,buttherewerenosignificantdifferencesamongSh-
F,Fr-M,andFr-Fblends(Figure2B;F4,25=33.67,P<0.01).Allfourblendswereequallyattractivetofemales(Figure2B). Fieldexperiment2Duringthepeachharvestandthelatefruitingstageforpear,asignificantlylargerOFMpopulationwaspresentinthepearorchardthaninthepeachorchard,basedonthenumberofmalemothstrappedwiththecontrolsex SexualdifferencesofOFMinresponsestohosts285 Figure1SimultaneouslyrecordedCydiamolesta(
A,C)femaleand(
B,D)maleGC–EADresponsestoVOCscollectedfrom(
A,B)detachedshootsofpeachvarietyWuyuexianbianganand(
C,D)maturefruitsofthepearvarietyJimiusingapolarDB-WAXcapillarycolumn.Ineachpanel,theuppertraceindicatestheflameionizationdetectorresponses(FID),thelowertracetheantennalresponses(EAD).poundsfoundconsistentlyinsixtestswere(A)(E)-b-ocimene
(1),(Z)-3-hexenylacetate
(2),(Z)-3-hexen-1-ol
(3),benzaldehyde
(4),linalool
(5);(B)(Z)-3-hexenylacetate
(1),(C)ethylbutanoate
(1),butylacetate
(2),ethylhexanoate
(3),hexylacetate
(4),1-hexanol
(5),nonanal
(6),hexylbutanoate
(7),(
E,E)-a-farnesene
(8);(D)ethylbutanoate
(1),butylacetate
(2),ethylhexanoate
(3),hexylacetate
(4),1-hexanol
(5),hexylbutanoate
(6),(
E,E)-a-farnesene
(7). pheromone(Figure2CandD).Inthepeachorchard,muchfewerOFMwerepresentbasedonthemonitoringbysexpheromone-baitedtraps,asalmostnomothswerecaughtinthetraps.Inthepearorchard,luresthatcontainedpeach-derivedVOCs(Sh-
F,Sh-M)caughtsignificantlymoreOFMmalesthanthecontrolandthelures thatcontainedpear-derivedVOCs(Fr-
F,Fr-M)(Figure2D;one-wayANOVA:F4,25=65.67,P<0.01).AlthoughtheSh-Fblendwasbasedonfemaleresponses,Sh-
M,Fr-F,andSh-FblendscaughtsignificantlymorefemalesthanSh-Finpearorchards.(Figure2D;F4,25=47.38,P<0.01). 286Luetal. No.moths/trap 220400A 160120 80 50 Peach 40 AA 30 20 a
B a 10 BC b b
0 240 Sh-FSh-MFr-FFr-
M 200 160 120 80 50 40 30 20 100 1-Hexanol AaSh-
F AaSh-
M AaFr-
F Aa Fr-
M Cb SP CK
C Aa SP CK 240200
B 160 120 80 50 Pear 40 30 20A 10
B a Ba
B ab ab
0 240 Sh-FSh-MFr-FFr-MSP 200 160 120 80 50 40 30AA 20 aBabB bc 10 c 0Sh-FSh-MFr-FFr-MSP MaleFemale Fieldexperiment1 CbCK
D Fieldexperiment2 CdCK (Z)-3-Hexen-1-ol Nonanal Ethylbutanoate Butylacetate Ethylhexanoate Hexylacetate (Z)-3-HexenylacetateHexylbutanoateBenzaldehyde (E)-£-OcimeneLinalool FarneseneSexpheromone Figure2Mean(ÆSD;n=6)totalnumberofCydiamolestamalesandfemalescapturedintrapsin(
A,C)peachand(
B,D)pearorchards,during(
A,B)5-18August(Fieldexperiment1)and(
C,D)5-18September(Fieldexperiment2).EachtrapwasbaitedwitharubberseptumcontainingsyntheticVOCblendscorrespondingtothoseemittedbypeachshoots(Sh-
F,Sh-M),pearfruits(Fr-
F,Fr-M),ahexanecontrol(CK),andsexpheromone(SP).TheratiosoftheponentsaregiveninTable1.Eachlurecontained100mgofthemostponent.Meanswithinapanelcappedwithdifferentletters(capitalformales,lowercaseforfemales)aresignificantlydifferent(one-wayANOVAfollowedbyTukey’sparisontest:P<0.05). 100A 8060 a*b UpwindCloseContact %respondingfemales A* A* 40 c* 20 *d
B a0 Sh-
F 100B B*a* Sh-
M a*Fr-F b a* Fr-Ma
A dBa CK 80 Bb 60C c40 aa %respondingmales 20
D b bc
0 Sh-
F Sh-
M Fr-
F 1-Hexanol Fr-
M d Dc CK (Z)-3-Hexen-1-olNonanal EthylbutanoateButylacetate EthylhexanoateHexylacetate (Z)-3-HexenylacetateHexylbutanoateBenzaldehyde(E)-£-OcimeneLinaloolFarnesene Figure3Mean(ÆSD)attraction(%)ofmatedCydiamolesta(A)femalesand(B)malesinawindtunneltosyntheticVOCblendsmimickingtheheadspaceofdetachedshootsofpeachvarietyWuyuexianbiangan,maturefruitsofpearvarietyJimi,andahexanecontrol(CK).TheratiosoftheponentsaregiveninTable1.Farnesene=mixtureof(
E,E)a-farnesene(49%),(E)-b-farnesene(26%),(Z)-b-farnesene(18%),and(
Z,E)-a-farnesene(7%).Meanswithinapanelwiththesamecolorcappedwithdifferentlettersaresignificantlydifferent(one-wayANOVAfollowedbyTukey’sparisontest:P<0.05).Mothswerescoredfororientation(upwind),flighttowithin10cmoftheVOCsource(close),andlandingontheVOCsource(contact).Differencesbetweensexesinthethreeflightbehaviorswereanalyzedbyunpaired-samplettests(*P<0.05). SexualdifferencesofOFMinresponsestohosts287 Wind-tunnelbioassaysAllfourblendsstimulatedupwindflightinbothfemalesandmales(Figure3AandB),butonlymalesreachedandcontactedthesource(Figure3B).MalesweremorestronglyattractedthanfemalestothesyntheticVOCblends.Inthelaboratory,pear-derivedluresweremoreattractivetobothsexesthanpeach-derivedlures.TheFr-Fblendprovedtobethemostattractivetofemalesinthewindtunnel:67%ofthefemalesflewupwind(one-wayANOVA:F4,10=183.43)and37%arrivedwithin10cmofthesource(F4,10=83.11,bothP<0.01),butnonelandedonthesource(Figure3A).Similarly,Fr-Mwasmostattractivetomalesinthewindtunnel:99%ofthemalesflewupwind(F4,10=130.22),87%arrivedwithin10cmofthesource(F4,10=510.10),and36%landedonthesource(F4,10=64.57,allP<0.01)(Figure3B).AlthoughtheSh-Fblendwasbasedonfemaleresponses,nomorefemalesthanmalesflewupwindandarrivedwithin10cmofthesource(Figure3A). Discussion Femaleorientalfruitmothsrepresentamoreseriousthreattoadjacentorchardsthanmales,becausetheirflightexceedsthatofmales(Hughes&Dorn,2002),andbecausefemaleslaytheeggsthatdevelopintothedamaginglarvae.PreviousstudiesmainlyfocusedontheinteractionbetweenadultOFMfemalesandhostplant-derivedvolatiles(Nataleetal.,2003,2004;Pin~ero&Dorn,2007,2009;Najar-Rodriguez&Dorn,2013).Mostrecently,thediscoverythatmixturescontainingvolatilesattractivetofemalesinthelaboratorycouldcapturemoremalesunderfieldconditionsstimulatedparisonofresponsesofmaleandfemaleOFMtohostplant-derivedvolatiles. Wefoundthatpoundsfromtheheadspacesofpearfruitsweresimilarforfemalesandmales—eightandpounds,respectively—indicatingasimilarchemosensorysystemofOFMmaleandfemaleantennae.Inthefield,malecapturessignificantlyoutnumberedfemalecatches,evenintrapswithpoundluresbasedonfemaleresponsestopearfruitVOCs(Fr-F). Responsestohostplant-derivedvolatileswerealsofoundtodifferbetweenthesexes.Theblendwithoutnonanal(Fr-M)wasmoreattractivetomalesthantheblendcontainingnonanal(Fr-F),suggestingthatnonanalactsasarepellentorattraction-inhibitorforOFMmalesunderfieldconditions.Nonanalhasbeencharacterizedasanositiondeterrentforfemalecodlingmoth,Cydiapomonella(
L.)(Yokoyama&Miller,1991).Otherstudiesalsosuggestedasexdifferencein 288Luetal. responsetosemiochemicals.Forturnipmoths,Agrotissegetum(Denis&Schiffermu€ller),Hanssonetal.(1989)characterizedthepheromoneandplantvolatileperceptioninmalesandfemalesbyEADandsinglesensillumtechniquesandfoundfemalereceptorsarespecializedforplantvolatilereceptionandareinsensitivetopheromones.Incontrast,thespecializedpheromonereceptorsonmaleantennaearesensitivenotonlytopheromonesbutalsotoplantvolatiles.Inaddition,maleshavespecializedplantvolatilereceptors(Hanssonetal.,1989).Codlingmothmalesandfemaleswerefoundtodifferinthenumberofolfactoryreceptorneuronsintheantennae(B€ackmanetal.,2000). Overall,thebehavioralresponseofmalestowardhostplant-derivedvolatilesbothinthelaboratoryandinthefieldwasstrongerthanthatoffemales,demonstratinggoal-directedmaleorientationtowardsexpheromonesources.SomeauthorshavesuggestedthatmalesuseplantVOCstodistinguishenvironmentswheretheycanfindfemalesmoreeasily(Anseboetal.,2004;Il’ichevetal.,2009).Femalesontheotherhandneedtoconsidermorefactors,suchasflightmode,responseprofile,orflightstrategytowardhostplantsunderfieldconditions.Theflightmodeofmalesmaybedifferentfromthatoffemales.Inourwind-tunnelstudy,femaleswereattractedbysyntheticlures,buttheperformanceofmalesexceededthatoffemalesespeciallywhenapproachingthesource.Theorientationmechanismsofmalesflyinginsearchoffemalesareadaptedtolocateapoint-sourceofsexpheromone.Bycontrast,femalessearchingforsuitableositionsitesmightnotbeasstronglyattractedtopointsourcesofplantVOCs,andthereforetrapdesigncouldhavealargeinfluenceontheirabilitytoattractfemales.Similarresultswerefoundforthecodlingmoth,animportantpestofapple(Coracinietal.,2004).Femalecodlingmothshavefrequentlybeenobservedtoflyupwindoverseveralmeterstowardbrancheswithgreenapples,butcontactedtheapplelessfrequently,whichsuggeststhatthefemalesemployadifferentsearchstrategythanmales,especiallyatcloserange,lookingforasuitableositionsite(Witzgalletal.,1999).Also,femalesmaylocateositionsitesbasedonbothchemicalandvisualcues.ThegreatestflightactivityofmatedandunmatedmaleandfemaleOFMsurredduringthefirsthourofdusk,whenlightintensitydecreasedfrom3750to57lx,suggestingthatthefemalesuseopticalcuestolocateositionsites(Hughes&Dorn,2002).Similarresultshavebeenfoundingrapeberrymoth,Paralobesiaviteana(Clemens),acrepuscularspecies,whichositslessintheabsenceoflight(Clark&Dennehy,2002).Thephysicalstructureandlowvolatilityof VOCspresentonfruitsurfacescouldalsoaffectfemaleperformance.Inourstudy,OFMsalwayslaideggsonthesmoothsurfaceofwaxedpaperinthelaboratory.Thestickybaseofthetrapsmightrepelfemalelanding. Aspredicted,theVOCblendbasedonmaleresponsestopeachshootVOCs(Sh-M)caughtmoremalesthantheblendbasedonfemaleresponsestothesamesource(Sh-F);however,theblendbasedonfemaleresponses(Sh-F)didnotattractmorefemalesthanthemaleresponse-derivedblend(Sh-M).OftheVOCsfrompeachshoots,(Z)-3-hexenylacetateelicitedantennalresponsesfrombothsexes,whereastheotherponents,(Z)-3-hexen-1-ol,benzaldehyde,(E)b-ocimene,andlinalool,onlyelicitedresponsesfromfemales.Fieldassaysindicatedtheotherponentswereredundantinattractingnotonlymalesbutalsofemales.Wehypothesizethatvolatilescollectedfromcutyoungpeachshootsmaydifferfromtheheadspaceofintactpeachshoots.ThiscouldgreatlyaffecttheolfactoryorientationofOFMs.Inourstudy,besideahydrocarbon,ponents—(Z)-3-hexen-1-ol,(Z)3-hexenylacetate,benzaldehyde,methylsalicylate,(E)b-ocimene,andlinalool—werecharacteristicofdetachedpeachshoots.Nataleetal.(2003)foundpoundsintheheadspaceofexcisedpeachshoots.(Z)-3-Hexen-1-ol,(Z)-3-hexenylacetate,benzaldehyde,methylsalicylate,and(Z)-b-ocimenewerealsodetectedintheirstudy.Il’ichevetal.(2009)analyzedintactyoungshoottipsofpottedpeachtreesbyGC-MS.Theyfound(Z)-3-hexenylacetateand(Z)-b-ocimeneintheheadspaceoflivingpeachshoots,but(Z)-3-hexen-1-ol,benzaldehyde,andmethylsalicylatewerenotdetected.Wededucethat(Z)-3-hexen-1-ol,benzaldehyde,andmethylsalicylatearecharacteristicofdetachedpeachshootsandmaybeinducedbytheartificialcuttingofshoots.Theseponentsareassociatedwithplantstresscausedbymanualdamageandcouldindicatealow-qualityhostoranon-host.Activeavoidanceofnon-hostodorsandevennutritionallyunsuitablehostsappearstobeanimportantpartoftheinsecthostlocationprocess. Inconclusion,ourstudysuggestedsimilaritiesinthechemosensorysystemofOFMmaleandfemaleantennae,butalsodifferencesinresponseswerefoundbetweenthesexes.FurtherstudyisneededtoidentifythevolatilesfromintactpeachshootsthatattractOFMmalesorfemales.Both,malesandfemalescoulddisperseintosecondaryhostorchardsbetweenthelatepeach-fruitingandlatepear-fruitingstage.Thetwopear-derivedVOCblends(FrFandFr-M)caughtsignificantlymoreOFMmalesandfemalesthantheotherblendsinpeachorchards;conversely,thetwopeach-derivedblendsweremoreattractive SexualdifferencesofOFMinresponsestohosts289 thantheotherblendsinpearorchards.Together,theblendsderivedfromoneplantspeciesshouldbeusedinorchardsoftheotherspecies.ThismaybevaluablefordesigningfurthercandidateattractantblendsforC.molesta.Inaddition,weshouldseektobetterunderstandthedevelopmentaldifferencesbetweenthesexesofC.molestaandamongitshostcropsandexplorebetterpestmanagementapproachesbasedonoptimizingthepeach–pearplantingsysteminthefield. 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