CHEMICALECOLOGYPepperWeevilAttractiontoVolatilesfromHostandNonhostPlantsKARLAM.ADDESSO1ANDHEATHERJ.MCAUSLANEEntomologyandNematologyDepartment,UniversityofFlorida,POBox110620,Gainesville,FL32611-0620 Environ.Entomol.38(1):216Ã224(2009)ABSTRACT Thelocationofwildandcultivatedhostplantsbypepperweevil( Anthonomuseugenii Cano)maybeaidedbyvisualcues,themale-producedaggregationpheromone,herbivore-induced, orconstitutivehostplantvolatiles.Theattractivenessofconstitutiveplantvolatilestopioneerweevils isimportantinunderstanding,andperhapscontrolling,dispersalofthisinsectbetweenwildand cultivatedhosts.Ten-day-oldmaleand2-and10-day-oldfemaleweevilsweretestedinshort-range Y-tubeassays.Ten-day-oldmaleandfemaleweevilswereattractedtothevolatilesreleasedbywhole plantsofthreeknownovipositionhosts,Ã”Jalapeno'pepper,Americanblacknightshade,andeggplant, aswellastomato,acongener,whichsupportsfeedingbutnotoviposition.Two-day-oldfemaleswere attractedtoallplantstested,includinglimabean,anunrelated,nonhostplant.Fruitvolatilesfromall threehostsandÃŸowervolatilesfromnightshadeandeggplantwerealsoattractive.Inchoicetests, weevilsshoweddifferentpreferencesfortheovipositionhosts,dependingonageandsex.Upwind responseof10-day-oldmaleandfemaleweevilstohostplantvolatileswasalsotestedinlong-range windtunnelassays.Weevilsrespondedtopepper,nightshade,andeggplantvolatilesbymoving upwind.Therewasnodifferenceintheobservedupwindresponseoftheweevilstothethreehost plantsunderno-choiceconditions.Reproductivelymaturepepperweevilscandetect,orientto,and discriminatebetweenthevolatileplumesofhostplantsintheabsenceofvisualcues,conspeciÃžc feedingdamage,orthepresenceoftheiraggregationpheromone. KEYWORDS Anthonomuseugenii ,hostplantvolatiles, Capsicumannuum , Solanumamericanum , Solanummelongena Phytophagousinsectsuseawiderangeofgeneraland host-speciÃžccuestolocatetheirhostplantswithina heterogeneousenvironment(Schoonhovenetal. 1998).Aninsect,movingrandomlythroughspace,will uselong-rangevisualand/orolfactorycuestoleadit tothevicinityoftheplant.Oncetheinsectmakes contactwithapotentialhost,short-rangeolfactory, mechanicalandgustatorycuesverifytheplant'ssuitabilityasafeedingorovipositionsite.Otherfactors, suchasspecies-speciÃžcpheromonesignals,addtothe complexityofhostplantselectionpatternsasthese signalsmayalsoworktodrawaninsectintoahabitable patch,aswellasaidinmatelocation. Determiningthesequenceofeventsleadingtohost acceptanceisparticularlyimportantforphytophagous insectpests.Thepepperweevil, Anthonomuseugenii Cano,commonlyinfestscultivatedpepper( Capsicum spp.)ÃželdsinthesouthernUnitedStates,Central America,andtheCaribbean(GoffandWilson1937, O'BrienandWibmer1982,AbreuandCruz1985).In additiontofeedingon Capsicum spp.,theweevilisalso capableofreproducinginthesouthernUnitedStates onanumberofwildandcultivatedplantsinthegenus Solanum, includingAmericanblacknightshade, Solanumamericanum Mill.(PatrockandSchuster1987) andeggplant, Solanummelongena L.Ã‘aspeciesof Asianorigin(Diazetal.2004).Theabilityofthe pepperweeviltosurvivethefallowseasononwild nightshadesmakesthisinsectdifÃžculttoeradicate becausenightshade-residingpopulationsareableto reinfestpepperÃželdsthefollowingseason.Although thesegeneralizeddispersalpatternsoftheweevilhave beendescribedbypreviousauthors(Patrockand Schuster1987),noonehasyetaddressedhowpepper weevilsinitiallylocatetheircultivatedandwildhost plants. Althoughnoresearchdescribingthepepperweevil'sresponsetohostplantvolatilesexists,thereisa bodyofliteratureonthevolatileattractantsforthree ofitscongeners,thebollweevil( Anthonomusgrandis Boheman),theappleblossomweevil( Anthonomus pomorum L.),andthestrawberryblossomweevil( Anthonomusrubi Herbst).Previousbehavioralbioassays andelectroantennogramstudieshaveshownthat thesecongenerscandetectandorienttohostplant volatiles.Thebollweevilwasattractedtocottonplant volatileoilsandcottonsquareextractsinbehavioral bioassays(Hardeeetal.1971,McKibbenetal.1977). Later,Dickens(1984,1986,1989,1990)veriÃžedboll weevildetectionofsix-carbon Â greenleafvolatiles ÂŽ 1Correspondingauthor:EntomologyandNematologyDepartment,UniversityofFlorida,POBox110620,Gainesville,FL326110620,(e-mail:addesso@uÃŸ.edu). 0046-225X/09/0216Ã0224$04.00/0 2009EntomologicalSocietyofAmerica
andhost-speciÃžcvolatilesusingelectroantennography(EAG)andsinglecellrecording(SCR)techniques.Anexaminationofappleblossomweevilresponsetovolatileblendsofvariousapplecultivarsalso suggeststhatthisspeciesusesvolatilesascuestolocate itshostplants(Kalinovaetal.2000).Morerecently, studiesoftheolfactoryneuronsofthestrawberry blossomweevilidentiÃžed15receptortypesthatdetectedatotalof54hostandnonhostplantvolatiles (Bicha oetal.2005b),aswellasÃžveadditionalreceptorsdetectingvolatilesfromstrawberryplantsthatare inducedbyweevilfeeding(Bicha oetal.2005a). Ourgoalistobetterunderstandthecomplexitiesof pepperweevilhostplantselection.WedidthisbyÃžrst addressingtheresponseofweevilstoconstitutivevolatilecuesreleasedfromhostandnonhostplantsinthe absenceofvisualcuesorpheromonesignals.Removingthesealternatesourcesofinformationwillallowus tofocussolelyontheimportanceofplantvolatilesto theweevil'sabilitytolocateahostplant.Inaddition, anydifferencesbetweenmaleandfemaleresponseto plantvolatilescanbeidentiÃžedwithoutconfounding differencesinsensitivitytothemale-producedaggregationpheromone(Elleretal.1994).Wewerealso abletoidentifychangesinvolatilediscriminationby femalesatdifferentages.Fromalife-historyperspective,newlyemergedfemalesshouldspendthemajorityoftheirtime-seekingmatesandfeedinglocations, whereasolder,previouslymatedfemalesshouldbe moremotivatedtosearchforovipositionsites.These changesinfemalebehaviorovertimemayleadto changesinvolatilepreferencesifplanthostssufÃžcient formatingandadultfeedingarenotnecessarilyoptimalsitesforlarvaldevelopment. WeassayedÃžveplantspecies(pepper,eggplant, Americanblacknightshade,tomato,andlimabean) withvaryingdegreesofsuitabilityforadultfeeding andovipositionÃ‘threespeciesthatsupportadult feedingandlarvaldevelopment,onespeciesthatcan supportadultfeedingandonespeciesthatsupports neitherfeedingnoroviposition.Thethreehostplants differedintheirlevelofacceptabilityandlengthof associationwithpepperweevil.Bothpepperand AmericanblacknightshadeareNewWorldspecies thathavehistoricallyservedashostsplantsforthe weevil,whereaseggplantisanAsianspecieswith whichthepepperweevilhasonlyrecentlybeenin contact.AlthoughtheweevilhasbeenobservedfeedingandovipositingineggplantÃŸowers(Diazetal. 2004),theweevilhasnotbeenamajorsourceof economicinjurytocultivatedcropsandeggplantdoes notappeartobeapreferredhostinthepresenceof pepper(P.A.Stansly,personalcommunication). Tobeginunravelingthecomplexitiesofpepper weevilhostplantselectionweseektoanswerthe followingquestions:(1)willpepperweevilsorientto hostplantvolatiles?;(2)willpepperweevilsorientto generalplantvolatiles(i.e.,emanatingfromanonhost)?;(3)willpepperweevilsshowapreferencefor thevolatilesofparticularhostplants?;(4)willmales andfemalesresponddifferentlytoplantvolatiles?;(5) willnewlyemergedfemalesresponddifferentlythan reproductivelymaturefemalestohostplantvolatiles?; and(6)willpepperweevilsrespondtohostplant volatilesinbothshort-andlong-rangeorientation assays? MaterialsandMethods InsectsandPlants. Apepperweevilcolonywas establishedattheUniversityofFlorida,Gainesville, FL,inthespringof2004frominsectscollectedfrom pepperÃželdsinClewiston,FL.Additionalwildinsects fromImmokalee,Bradenton,andWimaumawereintroducedintothecolonyinthefallof2005and2006 tomaintaincolonyhealth.Thecolonywasheldunder a14:10L:Dlightregimenat27 Cand30%RH.Weevils wererearedongreenhouse-grownÃ”Jalapen o'peppers ( Capsicumannuum L.),withwaterandhoneysupplements.Gravidfemales( 10dold)wereplaced singlyinovipositioncupswithasinglepepperfruit, whichwasreplacedevery2d.Ovipositioncupswith screenedlidsweremadefromwaxedcardboardcans (250ml,8.5cmdiameter).Infestedfruitwereheldin emergencecontainerswithscreenlids(1.5-literTupperwarecontainers)untilallweevilsemerged.Newly emergedweevilswerecollectedandtransferredinto acolonycageforuseinassays. Jalapen opepper( Capsicumannuum L.),Ã”Ghostbuster'eggplant,Americanblacknightshade,Ã”Better Boy'tomato( Solanumlycopersicum L.),andÃ”Fordhook242 bushlimabean( Phaseoluslunatus L.)plants (IllinoisFoundationSeeds,Champagne,IL)were growninagreenhouse.Plantsweregrownin12-cmdiameterpotsin50:50mixtureofMetro-Mix200and 500andfertilizedusingOsmocote14Ã14-14slowreleasepellets(TheScottsCompany,Marysville,OH). Y-tubeOolfactometerExperimentalDesign. BioassayswereconductedinaglassY-tubeolfactometer (AnalyticalResearchSystems,Gainesville,FL)with TeÃŸontubingconnections.BreathingqualitycompressedairwaspushedthroughacharcoalÃžlterand humidiÃžedwithdeionizedwaterbeforesplittinginto twoholdingchambers.Threetypesofholdingchamberswereusedinourassays,dependingonthesizeof theplantpartused:(1)Plexiglaschambers(60cmtall, 15cminternaldiameter),(2)largeglasschambers(21 cmlength,3cminternaldiameter),and(3)smallglass chambers(6cmlength,2cminternaldiameter).AirÃŸowwasmaintainedat250ml/minbytwoinlineÃŸow meters(Manostat,NewYork,NY).TheglassY-tube (12-cmcommontube,10-cmarms,2.5-cminternal diameter)washeldata30 angleabovehorizontal insideathree-walledcardboardenclosure(46by28 by42cm).Holdingchamberswereplacedoutside theenclosuretoeliminatevisualcues.TheY-tube assemblywasilluminatedbyaÃŸuorescentlightÃžxture(four85-Wbulbs)suspended70cmabovethe tablesurface.Theassayroomwasmaintainedat 25Ã27 Cand40%RH. WeevilsweresexedaccordingtoEller(1995).Male andfemaleweevilswerestarvedovernight( 12h) beforeassaywithoutaccesstowater.Twoageclasses offemales(newlyemergedand 10daysold)andone February2009ADDESSOANDMCAUSLANE:PEPPERWEEVILATTRACTIONTOPLANTVOLATILES217
maleageclass( 10daysold)weretestedundereach setofexperimentalconditions.Olderfemaleshad beenconÃžnedwithmalesinthecolonycagesince theiremergenceandwerepresumedtohavemated. Fortyinsectsineachage/sexclasswereassayedindividuallyoringroupsof5and10perdayon4separate days.Tenweevilsineachofthethreeclasseswere assayedeachday.Insectsweregiven15mintomake achoiceofarmsintheolfactometer.Weevilsthat passedhalfwayorfurtherintoonearmoftheY-tube wererecordedasmakingachoice.Ifnochoicewas madein15min,theassaywasconcluded.AfterÃžve assays,theairÃŸowwasreversedtotheoppositesideto controlforright-orleft-handedbias.Afteronesex/ ageclassofweevilswasassayed,theY-tubewas cleanedwithsoapywater,rinsedwithethanol,and driedonthebenchbeforethenextclasswasassayed. Thestartingclasswasrandomlyselectedeachdayand assayswererunwithinthepreviouslyestablishedactivityperiodforovipositionof1000Ã1700hours (PatrockandSchuster1992). Y-tubeAttractiontoHostandNonhostPlantVolatiles. Threehostplants(pepper,eggplant,andnightshade)andtwononhostplants,onesolanaceousplant thatsupportedfeedingbutnotoviposition(tomato) andonenonsolanaceousspeciesthatwasnotacceptableasafeedingorovipositionhost(limabean)were evaluatedinone-waychoicetestsagainstapuriÃžedair control.Inasecondstudy,threeknownpepperweevil hostplants(pepper,eggplant,nightshade)wereevaluatedinpairwisechoiceteststodeterminepreference amongtheplantodors.Floweringplantsusedinboth assayswerebetween2and3monthsoldandwere presentedinPlexiglascylinders. Y-tubeAttractiontoHostFlowerorFruitVolatiles. Pepper,eggplant,andnightshadeÃŸowersandfruit wereassayedinone-waychoicetestsagainstapuriÃžed aircontrol.Flowersandfruitwerepickedimmediately beforeuse.Flowerstemswerewrappedwithmoistenedcottonballstopreventwilting.IntheÃŸower assays,5pepperÃŸowers,2eggplantÃŸowers,or40 nightshadeÃŸowerswereplacedinsmallglass(pepper)orlargeglass(eggplantandnightshade)chambers.Inthefruitassays,2pepperfruit( 5cmlong), 2eggplantfruit( 2cmdiameter),or25nightshade fruit( 0.5cmdiameter)wereplacedinsmallglass (nightshade)orlargeglass(pepperandeggplant) chambers.DifferentnumbersofÃŸowersorfruitswere presentedtoapproximateequivalentmass. WindTunnelDesign. Bioassayswereconductedin twosizesofPlexiglaswindtunnelsattheUSDAÃARS CMAVEfacilityinGainesville,FL.Thelargewind tunnel(160by45by45cm)wasusedforassaysthat requiredwholeplantstobeplacedinthetunneland weevilstobeintroducedinthecenterofthetunnel. Thelargewindtunnelwashousedinasmallgreenhouse(approximatetemperature30 C,75%RH).AirÃŸowof0.2m/swascreatedbypullingairthroughthe windtunnelusingavacuum.Volatileswerereleased fromaplantplaceddirectlyinsidethetunnel,thus exposinginsectstovisualandvolatileplantcues.Once weevilsmadecontactwiththeplantsource,theydid notleave,andnotrapswereusedintheseassays. Foursmallwindtunnels(120by30by30cm)were housedinaseparategreenhouse(approximatetemperature30 C,66Ã75%RH).Allremainingplantvolatileassayswereconductedinthesmalltunnels.In theseassays,puriÃžedairpassedthroughtwoPlexiglas cylinders(60cmtall,15cminternaldiameter)before enteringthewindtunnels.Cylinderscontainedplants orwereempty,dependingontheexperiment.Air fromeachcylinderwassplitwithTeÃŸontubinginto fourstreams,whichwereattachedtoodorportsin eachofthefourwindtunnels.Compressedairentered thewindtunnelsat0.6liters/min,maintainedbyinline ÃŸowmeters,andairinthetunnelswaspulledbya vacuumat0.2m/s.Allassayswerecarriedoutbetween0900and1500hours. Trapswereplacedattheupwindendofthesmall windtunnelstocaptureanyweevilsattemptingto contacttheodorport.Twovialtrapswereconstructed from25-dramplasticvials(5.0cmdiameter,8.5cm height;Bioquip,Gardena,CA)placedhorizontally andÃžttedwitha1-mlplasticcentrifugetubetoallow weevilstoenterthetrapbutnotescape.Trapswere attachedtotheodorports25cmabovetheÃŸoor.In preliminaryassays,itwasdiscoveredthatweevilspreferredtowalkratherthanÃŸyinthewindtunneland arampleadinguptothevialtrapswasconstructed fromwhiteTOMCATglueboards(10.0by24.0cm; Motomco,Clearwater,FL)totraptheweevilsasthey attemptedtowalkuptothevialtraps.Thus,weevils couldbetrappedattheodorsourceeitherinthevial traps(iftheyÃŸewtothesource)oronthestickyboard (iftheywalkeduptothesource). WindTunnelBioassays. Maleandmatedfemale weevils(10Ã20dold)wereusedinthefollowing assays.Weevilsweresexedandheldseparatelyin groupsof10in7-dramplasticvialswithairholes. Weevilswereheldovernight( 15h)withnofoodor waterbeforeassay.Thesamevialswereplacedinto thedownwindendofthewindtunnel,unlessstated otherwise(120or60cmfromtheodorsourceinthe largeorsmallwindtunnel,respectively).Weevils werereleasedingroupsof10byremovingtheviallid atthestartofeachassay. Weevillocationwascategorizedandtheirupwind orientationtoplantvolatileswasrecorded15,30,60, and300minaftertheirrelease,unlessstatedotherwise.Weevillocationsrecordedwere(1)weevilsthat movedmorethanhalfwayupthetunnel(60cmupwindinthelargetunnel,45cminthesmalltunnel; 50%)and(2)thosethatmadecontactwiththeodor source(SC).SourcecontactwasdeÃžnedascontacting thesourceplant,ifpresentinthewindtunnel,or enteringthevialtraporbeingcaughtonthesticky boardrampleadingtothetrap,ifthehostplantwasnot present.Totalupwindresponsewascalculatedby summingthevaluesinthe 50%andSCcategories.All experimentswererepeatedonfourseparatedaysfor atotalof40weevilspersexpertreatment. 218ENVIRONMENTALENTOMOLOGYVol.38,no.1
WindTunnelOrientationofWeevilsintheAbsenceofOdors. Thisassaywasconductedinthelarge windtunneltodetermineweevilorientationinawind tunnelintheabsenceofplantodors.Weevilswere placedinthecenterofthewindtunnelwithnoolfactorystimulibutwithwindspeedsetat0.2m/s. Weevilorientationwasobserved15and30minafter theirintroductiontodeterminewhethertheinsects preferredtomoveupwindordownwindintheabsenceofodors.Weevilswererecordedashavingorientedinonedirectionortheotheriftheymoved60 cmormorefromthecenterofthetunnel.Thisdistancewaschosenbecauseitrepresentedthehalfway distanceofthelargetunnel(thedistanceusedto deÃžneorientationinthevolatileassays).Thisassay permittedustojustifythe Â orientationdistance ÂŽ for ourplantvolatileassaysbydeterminingwhetherweevilswillwalk60cmupwindintheabsenceofvolatiles. Differencesinupwindanddownwindorientation werecomparedwithinandbetweensexes. WindTunnelOrientationofWeevilstoConstitutivePepperVolatiles. Twoexperimentswereconductedtoquantifyweevilorientationtowardconstitutivehostplantvolatiles.TheÃžrstexperimentwas carriedoutinthelargewindtunnel.Inthisassay,a 3-mo-oldpepperplantwasplacedupwindinsidethe largewindtunneltoseeifmaleandfemaleweevils wouldmoveupwindinthepresenceofbothvisualand olfactorystimuli.Maleorfemaleweevilswerereleasedingroupsof10atthedownwindendofthe tunnel.Theirlocationinthewindtunnelwasrecorded 15,30,and60minaftertheirrelease.Maleandfemale responsetothevisualandolfactorystimuluswascompared. Inthesecondexperiment,thesmallwindtunnels wereused.Totestweevilresponsetovolatilesinthe absenceofvisualstimuli,pepperplantvolatileswere pipedinthroughtheodorportofeachchamber.Ten malesorfemaleswerereleasedatthedownwindend ofthewindtunnel.Weevillocationwasrecordedat 15,30,60and300minandmaleandfemaleresponse toplantvolatilesintheabsenceofvisualstimuliwas compared. WindTunnelOrientationofWeevilstoAmerican BlackNightshadeandEggplant. Inseparateassays, nightshadeoreggplantvolatileswerepipedintosmall windtunnelsinno-choicetests.Tenmaleorfemale weevilswerereleasedatthedownwindendofeach windtunnel.Weevillocationwasrecordedat300min onlyandmaleandfemaleresponsetotheplantvolatileswithoutvisualstimuliwascompared.Upwind movementtoeggplantandnightshadevolatileswas comparedwithorientationtopepperplantvolatilesin no-choicetestsrunsimultaneously. DataAnalysis. Y-tubeandwindtunneldatawere analyzedastotalpercentresponsetoeachsource using2analysis(SASInstitute2006).FortheYtubeandwindtunneldata,thepercentageofnonresponders(i.e.,thoseweevilsnotmakingachoice [Ytube]ormovingupwind[windtunnel]inthe allottedtimeperiod)ineachsex/classassayedwas alsocomparedusing2analyseswithinandamong theexperiments. Results Y-tubeAttractiontoHostandNonhostPlantVolatiles. Inone-waychoicetests,10-d-oldmalesandfemalespreferredpepper,eggplant,nightshade,and tomatovolatilesoverpuriÃžedairbutshowednopreferenceforlimabean(Fig.1).Two-day-oldfemales wereattractedtovolatilesfromallplants,including thenonhostlimabean. Inpairwisechoicetests,10-d-oldfemalespreferred peppervolatilesovernightshade,whereas10-d-old malesshowednopreference(Fig.2).Ten-day-old malesandfemalespreferrednightshadevolatilesto eggplantvolatiles.Ten-day-oldmalespreferredpepperovereggplantbut10-d-oldfemalesshowedno preferences.Two-day-oldfemalesshowednopreferencebetweenanyofthethreehostplants. Y-tubeAttractiontoHostFlowerorFruitVolatiles. Ten-day-oldmalesand2-and10-d-oldfemales showednopreferenceforpepperÃŸoralodorover puriÃžedair(Fig.3).However,allthreeage/sexcategorieswereattractedtonightshadeÃŸowers.Tenday-oldmalesandfemaleswerehighlyattractedto volatilesfromeggplantÃŸowers,whereas2-d-oldfemaleswereonlymarginallyattracted(2 1 3.24, P 0.0719). Ten-day-oldmalesand2-dand10-d-oldfemales wereattractedtopepperfruitvolatiles(Fig.4).Only 10-d-oldmalesandfemaleswereattractedtonightshadeandeggplantfruitvolatiles. WindTunnelOrientationofWeevilsintheAbsenceofOdors. Pepperweevilsoccasionallymade shortdistanceÃŸightsfromthereleasepointandsometimesfromÃŸoortoceiling(orviceversa);however, themajorityofmovementwasthroughwalking.A signiÃžcantlygreaterpercentageoffemalesmoved downwind(27%)ratherthanupwind(5%)at15 (2 1 15.12, P 0.0001)and30min(47.5%versus5%;2 1 34.40, P 0.0001)afterrelease.Therewasno differenceinmalelocation15minafterrelease(12.5% downwindversus7.5%upwind;2 1 1.25, P 0.2636),butafter30minalargerpercentageofmales hadmoveddownwind(25%)thanhadmovedupwind (12.5%;2 1 4.17, P 0.0412). WindTunnelOrientationofWeevilstoConstitutivePepperVolatiles. Whenapepperplantwasplaced inthelargewindtunnel,femaletotalupwindresponse (movement 50% SC)increasedovertimewith signiÃžcantdifferencesobservedamongthe15-,30-, and60-mintimeintervals(2 2 9.13, P 0.0104).The totalupwindresponseofmalesalsoincreasedwith time(2 2 10.77, P 0.0046).Therewasnodiffer encebetweenmaleandfemaletotalupwindresponses topeppervolatilesatanyobservationtime(15min:2 1 0.11, P 0.7416;30min:2 1 3.46, P 0.0628; 60min:2 1 1.45, P 0.2280),with47.5%offemales and60.5%ofmalesshowinganupwindresponseby60 min.Slightlymoremalesthanfemaleswereincontact February2009ADDESSOANDMCAUSLANE:PEPPERWEEVILATTRACTIONTOPLANTVOLATILES219
withthevolatilesourceat60min(SC;female 25.0%, male 40.5%;2 1 3.67, P 0.0555). Insmallwindtunnels,upwindresponseoffemale (2 3 53.06, P 0.0001)andmaleweevils(2 3 58.11, P 0.0001)topeppervolatilesincreasedwith time.Therewasnodifferenceintotalresponseof malesandfemalesafter300min(female 77.5%, male 77.4%;2 1 0.03, P 0.8651).However,more femalesthanmaleswereincontactwiththeodor sourceat300min(female 57.5%,male 37.4%;2 1 4.26, P 0.0391). WindTunnelOrientationofWeevilstoAmerican BlackNightshadeandEggplant. Pepperweeviltotal upwindresponsedidnotdifferbysexwheninsects werepresentedthecontrol(pepper),nightshadeor eggplantvolatiles(Table1).Inno-choicecomparisons,totalupwindresponsetopepperandnightshade volatilesdidnotdifferformalesorfemales.Therewas alsonodifferenceinsourcecontactinthetwohost planttreatmentsforeithersex.Inthesecondnochoiceassay,therewasnodifferenceintotalupwind responseorsourcecontacttopepperandeggplant volatilesformalesorfemales. NonresponderData. Someinsectsremainedatthe baseoftheY-tubeordidnotmakeachoiceinthe15 minallotted.Ten-day-oldmalesand2-d-and10-d-old femalesallshowedvariationinnonrespondersacross assays.Malenonrespondersrangedfromalowof5% inthepepperÃŸowerassaytoahighof33%inthe nightshadewholeplantassay(2 13 33.36, P 0.0001).Ten-day-oldfemalesnonrespondersranged fromalowof10%inthepepperÃŸowerandfruitassays toahighof42%intheeggplantfruitassay(2 13 52.23, P 0.0001).Two-day-oldfemalenonrespondersrangedfrom2.5%intheeggplantandpepper ÃŸowerassaysto22%inthebeanwholeplantvolatile assay(2 13 42.32, P 0.0001).AsigniÃžcantdiffer encewasalsoobservedamongsex/classes,with2-doldfemaleshavingthelowestpercentageofweevils failingtomakeachoice(2-d-oldfemales 8.1 1.7 [SE]SE;10-d-oldfemales 26 2.7%;10-d-old males 22.9 2.1%;2 2 135.1, P 0.0001).Sexually maturemalesandfemalesdidnotdifferinpercentage ofnonresponders;however,2-d-and10-d-oldfemales diddifferwithyoungerfemalesrespondingmoreoften(2 1 131.68, P 0.0001). Forthethreewindtunnelassays,weanalyzednonrespondingmalesandfemalesacrosstreatmentsat300 min.Overall,slightlymorefemalesthanmalesfailed torespond(females 30.4 2.6%,males 22.2 1.5%;2 2 3.86, P 0.0493).Neithermalesnorfe malesdifferedinthepercentageofnonrespondersto pepper,eggplant,ornightshadewholeplantvolatiles. Fig.1. PepperweevilattractiontohostandnonhostplantvolatilesinaY-tubeolfactometer.Dataarerepresentedas percentageresponding. * P 0.05, ** P 0.01, *** P 0.001in2analysis.220ENVIRONMENTALENTOMOLOGYVol.38,no.1
Discussion Ourbioassaysshowedthatpepperweevilscanorienttoconstitutivehostplantvolatilesintheabsence ofvisualandpheromonestimuli.Intheshort-range Y-tubeassays,malesandfemaleswereattractedtothe volatilesofknownhostplantsbutalsorespondedto tomato,anonhostplantwithinthegenus Solanum. In aseriesoffeedingandovipositionassays,Patrockand Schuster(1992)observedpepperweevilfeedingon tomatoplantsandotherspecieswithinthefamily Solanaceae.However,theweevilsovipositedononly asmallsubsetofthe Solanum spp.(whichdidnot includetomato)butovipositedonall Capsicum spp. tested.ThissuggeststhattheremaybevolatilesspeciÃžctoplantsinthefamilythatcandrawweevilsin fromadistance,whereasfurthercontactorshortrangecuesdeterminetheacceptabilityoftheplant speciesforoviposition. Wealsofounddifferencesbetweentheresponseof newlyemergedfemales 2doldandresponsesofthe reproductivelymaturefemalesandmales.Inthesingle-choiceplantY-tubeassays,10-d-oldmalesand femaleswerenotattractedtothegeneralplantvolatilesoflimabeanbut2-d-oldfemalesdidrespondto thisnonhostplant.Thereasonsforthedifferencein responsemaybecausedbyexperienceordevelopmentaldifferencesinneurologicalresponse.Inadditiontoshowingapositiveresponsetoanonhostplant, 2-d-oldfemalesalsofailedtoshowthesamediscriminatorypatternsasolderfemalesinpairwisecomparisons.Allweevilsusedintheseassayswererearedon Ã”Jalapen o'pepperfruitandnonehadpreviousexperiencewiththealternativehostandnonhostplantsor pepperplantandÃŸowervolatiles.Therefore,naivety shouldnotbeafactoraffectingthedifferencesobservedinthebehaviorof2-d-oldfemalesbecause 10-d-oldmalesandfemaleswerealsounfamiliarwith thealternativeplantvolatiles.Itisthereforemost likelythatdifferencesincentralprocessingordevelopmentofolfactoryneuronswasresponsibleforthe observeddifferencesinresponse.DickensandMoorman(1990)cametothesameconclusionwhenaddressingthechangingresponseofmaleandfemale bollweevilstohostplantvolatileswithage.Between Fig.2. Pepperweevilattractioninpairwisechoiceof hostplantvolatilesinaY-tubeolfactometer.Dataarerepresentedaspercentresponding * P 0.05, ** P 0.01, *** P 0.001in2analysis. Fig.3. PepperweevilattractiontohostplantÃŸowervolatilesinaY-tubeolfactometer.Dataarerepresentedaspercent. * P 0.05, ** P 0.01 *** P 0.001in2analysis.February2009ADDESSOANDMCAUSLANE:PEPPERWEEVILATTRACTIONTOPLANTVOLATILES221
0and4dafteremergence,bollweevilsshoweda signiÃžcantincreaseinsensitivitytothegeneralhost plantvolatile1-hexanolandthecotton-speciÃžcvolatile-bisabolol.Itmaybethat,whereasnewly emergedpepperweevilscandetectthepresenceof plantvolatiles,theirreceptorneuronsarenotyet sensitiveenoughtodifferentiatebetweentwocomplexodorplumes. Ten-day-oldmalesandfemalesshowedpreferences fordifferenthostplantsintheY-tubeolfactometer, andthesepreferenceschangeddependingonsexand whichtwospecieswereoffered.Itisimportanttonote that,whereas10-d-oldfemaleshadastrongafÃžnityfor Americanblacknightshadevolatilesinthesinglechoiceassays(Fig.1b),whenputincompetitionwith peppervolatiles,thefemalesoverwhelminglychose pepper(Fig.2a).Thisoccurredeventhoughasmall percentageoffemaleswalkedtotheair-ladenarmin thesingle-choiceassaywithpepperinthealternative arm,whereas100%offemaleswalkedtonightshadein thesameseriesofassay.Itislikelythedifferencein preferenceiscausedbythelargersizeandpresumably betterqualityofpepperfruitasalarvalhostsource makingitselectivelyadvantageousforfemalestoorienttowardpeppervolatiles.Femalesarerarelyobserveddepositingmorethanoneegginnightshade fruit,whereastheweevilsdepositmanymoreinthe largerÃ”Jalapen o'fruit(unpublisheddata).Femalesare thereforelikelytohavemoreegglayingopportunities inpepperthaninnightshade.Also,ourcolonyinsects wererearedonpepperfruit,andalthoughtheyhadno priorexperiencewithwholepepperplantvolatiles, thepotentialsimilaritiesinthevolatileplumesoffruit andfoliagemayhavebiasedtheweevilstowardpepperplants. Becausepepperweevilsfeedprimarilyandoviposit solelyonÃŸowerbudsandsmallfruits,wemighthave expectedtheweevilstobeattractedtovolatilesfrom hostplantÃŸowersandfruits.Thisdoesnotseemtobe trueinallcases.Although10-d-oldmaleandfemale weevilsorientedtowardtheimmaturefruitofpepper, nightshade,andeggplant,theyonlyrespondedto ÃŸowersofnightshadeandeggplant.Noattractionto pepperÃŸowervolatileswasobserved.Itispossiblethat theattractivecompoundspresentintheÃŸowersofthe two Solanum spp.areabsentfrom C.annuum, alternatively,theamountofvolatilesbeingreleasedfrom thepepperÃŸowersmayhavebeenbelowthebehavioralthresholdforresponse.FurtheranalysisofÃŸower volatileproÃžles,GC-EAD,andbehavioralbioassays arebeingplannedtotestthishypothesis. Fig.4. PepperweevilattractiontohostplantfruitvolatilesinaY-tubeolfactometer.Dataarerepresentedaspercentresponding. * P 0.05, ** P 0.01, *** P 0.001in2analysis.Table1.Responseofpepperweevilmalesandfemalestohostplantvolatilesinno-choicetestsconductedinawindtunnel Orientationclass(percentresponsea) Control(pepper)bNightshadeEggplant 50%cSCdTotal response 50%SC Total response 50%SC Total response Female16.447.363.722.343.665.9Ã‘Ã‘Ã‘ 15.951.267.1Ã‘Ã‘Ã‘25.348.974.2 Male18.155.673.629.746.075.7Ã‘Ã‘Ã‘ 20.960.981.8Ã‘Ã‘Ã‘37.043.980.9a300-minassays.bTrialstoassessattractiontonightshadeoreggplantwererunsimultaneouslywithpeppercontrols.c50% weevilswalked 45cmupwindinsmallwindtunnel.dSC sourcecontact,weevilscontactedthestickyboardorenteredthevialtrap.222ENVIRONMENTALENTOMOLOGYVol.38,no.1
Theshort-rangevolatileattractionexhibitedinthe Y-tubeassayshowedthatthepepperweevilcandiscriminatebetweenthevolatilesofdifferenthost plants.Althoughshort-rangestudiesofferussome informationaboutpepperweevilpreferences,awind tunnelgivesustheopportunitytoobserveweevil behavioronalargerscale.Oneconcernaboutinsect behaviorinawindtunneliswhethertheinsectsare cueinginonattractivevolatilesoriftheyaremerely positivelyanemotactic.Weshowedherethat,inthe absenceofplantvolatiles,bothmaleandfemaleweevilsaremoreinclinedtomovedownwindorremain stationarythantomoveupwind.Therefore,theupwindresponseobservedinthewindtunnelassayswith plantscanbeattributedtoattractiontoplantvolatiles. IntheÃžrstthreepeppervolatileassays,bothmalesand femalesorientedupwind,regardlessofwhetherthe plantsourcewasvisibleornot.Therewasnodifferenceinthetotalnumbersofmalesandfemalesorientingtotheplantvolatiles.Inaddition,bothmaleand femaleweevilsshowedsimilarresponsestopepper, eggplantandnightshadeplantvolatilesinthewind tunnelunderno-choiceconditions.Ideally,wewould liketorepeatourchoicetests(conductedintheYtubeolfactometer)inthelarger-scalewindtunnelas well;howeverweranintoproblemswhenattempting torunchoicetests.AlthoughpepperweevildoesÃŸy, itpreferstowalkinthewindtunnelasitdoesinthe Y-tube.Thismakestrapcapturesunreliablebecause theweevilsdonotÃŸyintothemouthofthevialtrap; theyprefertowalkalongthewallsofthetunneland climbontothetrap. TheexperimentsdescribedinthisstudywerespeciÃžcallydesignedtoexcludealternativecuesoften usedinhostplantlocation.Othervolatileattractants excludedincludethemale-producedpepperweevil aggregationpheromone(CoudrietandKishaba1988, Elleratal.1994)aswellasplantvolatilesinducedby weevilfeedingand/oroviposition.Whilearguably, theveryÃžrstweeviltoarriveatanuninfestedplant mustrelysolelyonconstitutiveplantcuestolocatethe host,subsequentarrivalsmaybeorientingtoacombinationofplantandinsect-speciÃžcvolatiles.Ifthe Ãžrstarrivalisfemale,herfeedingorovipositionwill inducechangesinthevolatileplumeoftheplant, whichmaypotentiallyincreasetheplant'sattractivenesstootherweevils.IftheÃžrstarrivalismale,a combinationofplantvolatilesandmale-produced pheromonewillbeavailabletoaidotherweevilsin locatingtheplant. Withregardtothesixquestionsposedatthebeginningofthisstudy,wefoundthat(1)pepperweevils willorienttohostplantvolatiles,(2)thatsexually maturepepperweevilswillnotorienttogeneralplant volatilesemanatingfromanonhostoutsidetheSolanaceaebutmaybeattractedtoothernonhostSolanaceae,(3)thatpepperweevilsshowpreferences forthevolatilesofdifferenthostplants,but(4)that malesandfemalesresponddifferentlytodifferent plants,(5)thatnewlyemergedfemales 2dold responddifferentlythanreproductivelymaturefemales,showingnoneofthepreferencesobservedin olderfemales,and(6)thatpepperweevilswillrespondtohostplantvolatilesinbothshort-andlongrangeorientationassays. Moreworkisneededtounderstandtheintricacies ofpepperweevilhostplantlocationand,morespeciÃžcally,theroleplantvolatilesplayinthiscomplex chainofevents.Moreinterestingistherelationship betweenalternativehostplantsandthevolatilecues thatcausemaleandfemaleweevilstoselectonespeciesoveranotherandallowpopulationstosurvive eradicationbymigratingbackandforthbetweencultivatedandcloselyrelatedwildspecies.Workiscurrentlybeingconductedontheeffectoffeedingdamagebymaleandfemaleweevilsonattractionof conspeciÃžcsinY-tubeandwindtunnelassays.Wewill alsouseGC-EADandbehavioralbioassaystoidentify themostattractivehost-speciÃžcvolatilescommonto thepepperweevil'shostplants.Ourtwo-foldpurpose istounderstandhowaninsectdiscriminatesbetween thevolatileplumesofmultiplehostspeciesinclose proximityandtousethatknowledgetodevelopecologicallyfriendlytrappingsystemsformonitoringand managementofpepperweevil. AcknowledgmentsWethankR.TanayandJ.Meyerforassistancewithcolony maintenanceandF.EllerandP.Stanslyforcommentsonan earlierdraftofthemanuscript.WealsothanktheUSDAÃ ARS,CenterforMedical,AgriculturalandVeterinaryEntomology(Gainesville,FL)fortheuseoftheirwindtunnels. ThisresearchwassupportedbytheFloridaAgricultural ResearchStation.ReferencesCitedAbreu,E.,andC.Cruz.1985. Theoccurrenceofthepepper weevil,AnthonomuseugeniiCano(Coleoptera:Curculionidae)inPuertoRico.J.Agric.Univ.P.R.69:223Ã224. Bicha o,H.,A.K.Borg-Karlson,J.Araujo,andH.Mustaparta. 2005a. Fivetypesofolfactoryreceptorneuronsinthe strawberryblossomweevil Anthonomusrubi: Selective responsestoinduciblehost-plantvolatiles.Chem.Senses. 30:153Ã170. Bicha o,H.,A.K.Borg-Karlson,A.Wibe,J.Araujo,andH. Mustaparta.2005b. Molecularreceptiverangesofolfactoryreceptorneuronesrespondingselectivelytoterpenoids,aliphaticgreenleafvolatilesandaromaticcompounds,inthestrawberryblossomweevil Anthonomus rubi. Chemoecology15:211Ã226. Coudriet,D.L.,andA.N.Kishaba.1988. Bioassayprocedureforanattractantofthepepperweevil(Coleoptera: Curculionidae).J.Econ.Entomol.81:1499Ã1502. Diaz,F.G.,P.J.McLeod,andD.T.Johnson.2004. Seasonal occurrenceofeggplantÃŸeabeetle, Epitrixfuscula Crochet(Coleoptera:Chrysomelidae)oneggplantinArkansas.J.Kans.Entomol.Soc.77:80Ã88. Dickens,J.C.1984. Olfactioninthebollweevil Anthonomus grandis Boheman.(Coleoptera:Curculionidae):electroantennogramstudies.J.Chem.Ecol.10:1759Ã1785. Dickens,J.C.1986. Orientationofbollweevil, Anthonomus grandis Boheman(Coleoptera,Curculionidae),topheromoneandvolatilehostcompoundinthelaboratory. J.Chem.Ecol.12:91Ã98.February2009ADDESSOANDMCAUSLANE:PEPPERWEEVILATTRACTIONTOPLANTVOLATILES223
Dickens,J.C.1989. Greenleafvolatilesenhanceaggregationpheromoneofbollweevil, Anthonomusgrandis. Entomol.Exp.Appl.52:191Ã203. Dickens,J.C.1990. Specializedreceptorneuronsforpheromonesandhostplantodorsinthebollweevil, Anthonomusgrandis Boheman(Coleoptera:Curculionidae). Chem.Senses15:311Ã331. Dickens,J.C.,andE.E.Moorman.1990. Maturationand maintenanceofelectroantennogramresponsestopheromoneandhostodorsinboll-weevils,AnthonomusgrandisBoheman(Coleoptera:Curculionidae)fedtheirhost plantoranartiÃžcialdiet.J.Appl.Entomol.109:470Ã480. Eller,F.J.1995. Apreviouslyunknownsexualcharacterfor thepepperweevil(Coleoptera:Curculionidae).Fla.Entomol.78:180Ã183. Eller,F.J.,R.J.Bartelt,B.S.Shasha,D.J.Schuster,D.G. Riley,P.A.Stansly,T.F.Mueller,K.D.Schuler,B. Johnson,J.H.Davis,andC.A.Sutherland.1994. Aggregationpheromoneforthepepperweevil Anthonomiseugenii Cano(Coleoptera:Curculionidae):identiÃžcationof Ãželdactivity.J.Chem.Ecol.20:1537Ã1555. Goff,C.C.,andJ.W.Wilson.1937. Thepepperweevil.Fla. Agric.Exp.Sta.Bull.310. Hardee,D.D.,N.M.Wilson,E.B.Mitchell,andP.M. Huddleston.1971. Factorsaffectingactivityofgrandlure,thepheromoneofthebollweevil,inlaboratory bioassays.J.Econ.Entomol.64:1454Ã1456. Kalinova,B.,K.Stransky,J.Harmatha,R.Ctvrtecka,andJ. ZdÂarek.2000. Canchemicalcuesfromblossombuds inÃŸuencecultivarpreferenceintheappleblossomweevil ( Anthonomuspomorum )?Entomol.Exp.Appl.95:47Ã52. McKibben,G.H.,E.B.Mitchell,W.P.Scott,andP.A.Hedin. 1977. Bollweevilsareattractedtovolatileoilsfromcottonplants.Environ.Entomol.6:804Ã806. OÂBrien,C.W.,andG.J.Wibmer.1982. AnnotatedchecklistoftheweevilsofNorthAmerica,CentralAmericaand theWestIndies(Coleoptera:Curculionidae),number34. MemoirsoftheAmericanEntomologicalInstitute,Ann Arbor,MI. Patrock,R.J.,andD.J.Schuster.1987. Fieldsurveyforthe pepperweevil,Anthonomuseugenii,onnightshade. Proc.Fla.Hortic.Soc.100:217Ã220. Patrock,R.J.,andD.J.Schuster.1992. Feeding,oviposition, anddevelopmentofthepepperweevil( Anthonomiseugenii Cano),onselectedspeciesofSolanaceae.Trop.Pest Manage.38:65Ã69. SASInstitute.2006. SAS,version9.1.SASInstitute,Cary, NC. Schoonhoven,L.M.,T.Jermy,andJ.J.A.vanLoon.1998. Insect-plantbiology:fromphysiologytoevolution.Chapman&Hall,London,UnitedKingdom. Received10June2008;accepted16October2008. 224ENVIRONMENTALENTOMOLOGYVol.38,no.1
1 HOST PLANT VOLATILES FOR ATTRACTING AND TRAPPING PEPPER WEEVIL By CASEY REED A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SC IENCE UNIVERSITY OF FLORIDA 2014
2 Â© 2014 Casey Reed
3 To my family
4 ACKNOWLEDGMENTS I would like to thank Dr. Heather J. McAuslane, Dr. Hans T. Alborn, Dr. Peter Teal, and Lucy Skelley for their knowledge, su pport, and guidance. Thank you to my committee members Dr. Philip Stansly and Dr. Hugh Smith for all of their assistance and field trial guidance. I would also like to thank my family and friends for their constant patience and understanding.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 LITERATURE REVIEW ................................ ................................ .......................... 12 Economics of Pepper Production ................................ ................................ ............ 12 Pepper Production ................................ ................................ ............................ 12 Economic Importance of Pepper Weevil ................................ .......................... 13 Taxonomy of the Pepper Weevil ................................ ................................ ............. 14 Life History of the Pepper Weevil ................................ ................................ ............ 15 Adult Stage ................................ ................................ ................................ ....... 15 Life Cycle and Development ................................ ................................ ............. 15 Ecology of the Pepper Weevil ................................ ................................ ................. 18 Distribution ................................ ................................ ................................ ....... 18 Hosts ................................ ................................ ................................ ................ 18 Host Pla nt Location ................................ ................................ .......................... 20 Aggregation Pheromone ................................ ................................ ................... 23 Management Techniques ................................ ................................ ....................... 25 Cu ltural ................................ ................................ ................................ ............. 25 Chemical ................................ ................................ ................................ .......... 26 Biological ................................ ................................ ................................ .......... 26 Behavioral Manipulation with Se miochemicals ................................ ....................... 28 Research Objectives ................................ ................................ ............................... 30 2 INVESTIGATION OF EAG ACTIVE PEPPER PLANT VOLATILES AS ATTRACTANTS FOR PEPPER WEEVIL ................................ ............................... 31 Introduction ................................ ................................ ................................ ............. 31 Materials and Methods ................................ ................................ ............................ 34 Insects ................................ ................................ ................................ .............. 34 Plants ................................ ................................ ................................ ............... 34 Y Tube Olfactometer Assays ................................ ................................ ............ 35 Wind Tunnel Dual Choice Assays ................................ ................................ .... 37 Field Trials ................................ ................................ ................................ ........ 38 Results ................................ ................................ ................................ .................... 40 Y Tube Olfactometer Assays ................................ ................................ ............ 40 Wind Tunnel Dual Choice Assays ................................ ................................ .... 41
6 Field Trials ................................ ................................ ................................ ........ 41 Discussion ................................ ................................ ................................ .............. 43 3 INVESTIGATION OF COMMONALITY IN VOLATILES AMONG WEEVIL HOST PLANTS ................................ ................................ ................................ .................. 64 Introduction ................................ ................................ ................................ ............. 64 Ma terials and Methods ................................ ................................ ............................ 65 Plants ................................ ................................ ................................ ............... 65 Insects ................................ ................................ ................................ .............. 66 Headspace Collection an d Volatile Analysis ................................ ..................... 67 Behavioral Analysis ................................ ................................ .......................... 68 Results ................................ ................................ ................................ .................... 70 Discussion ................................ ................................ ................................ .............. 71 4 CONCLUSIONS ................................ ................................ ................................ ..... 93 LIST OF REFERENCES ................................ ................................ ............................... 96 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 103
7 LIST OF TABLES Table page 2 1 Response of adult pepper weevils to single host plant volatiles in a Y tube dual choice olfactometer when presented w ith a choice between the compound and purified air. (P< 0.05). ................................ ................................ 47 2 2 Response of adult pepper weevils to 3 component blends in a Y tube olfactometer when presented with a choice between the bl end and purified air. GLV indicates Z 3 hexen 1 ol and Z 3 hexenyl acetate. (P<0.05). .............. 48 2 3 Response of adult pepper weevils to 4 component blends in a Y tube olfactometer when presented with a choice between the blend and purified air. GLV indicates Z 3 hexen 1 ol and Z 3 hexenyl acetate. (P<0.05). ............. 49 2 4 ANOVA results for pepper weevils captured on traps in Immokalee field trial for Summer 2012. ................................ ................................ ............................... 50 2 5 ANOVA results for pepper weevils captured on traps in Wimauma field trial for Summer 2012. ................................ ................................ ............................... 51 2 6 ANOVA re sults for pepper weevils captured on traps in Immokalee field trial for Fall 2012. ................................ ................................ ................................ ....... 52 2 7 ANOVA results for pepper weevils captured on traps in Wimauma field trial for Fall 2012. ................................ ................................ ................................ ....... 53 3 1 Response of adult mated female pepper weevils to host plant volatile blends in a Y tube dual choice olfactometer when presented with a choice between the blend and purified air. ................................ ................................ ................... 74
8 LIST OF FIGURES Figure page 2 1 Y tube olfactometer set up containing compressed air tank leading to a charcoal filter (A) and then humidified by bubbling through dei onized water before being split into two glass chambers which held the sample volatiles (B).. ................................ ................................ ................................ ..................... 54 2 2 The wind tunnel used for the assays was constructed of plexiglass and consisted of a central chamber with arms attached on opposite sides (A). ........ 55 2 3 Layout of Immokalee, FL field trial for Summer 2012. 56 2 4 Layout of Wimauma, FL field trial for Summer 2012. 57 2 5 Layout of Immokalee, FL field trial for Fall 2012. ................................ .............. 58 2 6 Layout of Wimauma, FL field trial for Fall 2012. . ................................ .............. 59 2 7 Average number of pepper weevils caught per replicate over eight weeks by synthetic host plant volatile blends in summer field trial in Immokalee, FL (2012 ) . ................................ ................................ ................................ ............... 60 2 8 Average number of pepper weevils caught per replicate over eight weeks by synthetic host plant volatile blends summer field trial in Wimauma, FL (2012). ................................ ................................ ................................ ........................... 61 2 9 Average number of pepper weevils caught per replicate over eight weeks by synthetic host plant volatile blends in fall field trial in Immokalee, FL (2012). ... 62 2 10 Average number of pepper weevils caught per replicate over eight weeks by synthetic host plant volatile blends in fall field trial in Wimauma, FL (2012). . ... 63 3 1 Headspace col lections were done using glass, volatile collection chambers (A).. ................................ ................................ ................................ ..................... 75 3 2 The average amount (ng) of each compound in volatile headspace collections of pepper varieties, eggplant, and nightshad e hosts of pepper weevil (n=4).. ................................ ................................ ................................ ...... 76 3 3 The average percentage of each compound in volatile headspace collections of Bell pepper (n=4). ................................ ................................ ........................... 77 3 4 The average percentage of each compound in volatile headspace collections of Jalapeno pepper (n=4). ................................ ................................ .................. 78
9 3 5 The average percentage of each compound in volatile headspace collections of Serrano pepper (n=4). ................................ ................................ .................... 79 3 6 The average percentage of each compound in volatile headspace collections of Habanero pepper (n=4). ................................ ................................ ................. 80 3 7 The average percentage of each compound in volatile headspace collections of Tabasco pepper (n=4). ................................ ................................ ................... 81 3 8 The average percentage of each compound in volatile headspace collections of Eggplant (n=4). ................................ ................................ ............................... 82 3 9 The average percentage of each compound in volatile headspace collections of Nightshade (n=4). ................................ ................................ ........................... 83 3 10 The average percentage of each compound in volatile headspace collections of Pequin pepper (n=4) ................................ ................................ ....................... 84 3 11 The average percentage of each sesquiterpene in volatile headspace collections of Bell peppe r (n=4). ................................ ................................ ......... 85 3 12 The average percentage of each sesquiterpene in volatile headspace collections of Jalapeno pepper (n=4). ................................ ................................ . 86 3 13 The average percentage of each sesquiterpene in volatile headspace collections of Serrano pepper (n=4). ................................ ................................ ... 87 3 14 The average percentage of each sesquiterpene in volatile headspace collections of Habanero pepper (n=4). ................................ ............................... 88 3 15 The average percentage of each sesquiterpene in volatile headspace collections of Tabasco pepper (n=4). ................................ ................................ .. 89 3 16 The average percentage of each sesquiterpene in volatile headspace collections of Pequin pepper (n=4). ................................ ................................ .... 90 3 17 The average percentage of each sesquiterpene in volatile headspace c ollections of Eggplant (n=4). ................................ ................................ ............. 91 3 18 The average percentage of each sesquiterpene in volatile headspace collections of Nightshade (n=4). ................................ ................................ ......... 92
10 Abstract of Thes is Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science HOST PLANT VOLATILES FOR ATTRAC TING AND TRAPPING PEPPER WEEVIL By Casey Reed August 2014 Chai r: Heather J. McAuslane Major: Entomology and Nematology The objective of this research was to improve monitoring of pepper weevil, Anthonomus eugenii Cano, through addition of plant volatiles to traps baited with commercially available aggregation pheromone. Two appro aches were taken: 1) the use of volatile compounds from damaged, fruiting jalapeno pepper plants previously identified by gas chromatography electroantennographic detection as stimulatory to the antennae of male and female pepper weevils, and 2) h eadspace collections from several host plants to identify common sesquiterpenes to add to the most attractive volatile blend determined in the first approach . H ost plants included six pepper varieties, eggplant, and nightshade . Blends were tested in Y tube olfact ometer and wind tunnel assays and in baited traps deployed in the field. Only acetophenone was more attractive than air in a Y tube olfactometer when presented alone but two three component blends were marginally attractive: 1) Z 3 hexenyl acetate, Z 3 he xen 1 ol, and acetophenone, and 2) Z 3 hexenyl acetate, Z 3 hexen 1 ol, and 1 heptadecene. Addition of bisabolene, common in all of the host plants , to the blend of green leafy volatiles and 1 heptadecene, was
11 attractive to mated female we evils compared to air but blends containing sesquiterpenes, green leaf volatiles and acetophenone were not. In w ind tunnel assays mated females preferred a blend of Z 3 hexenyl acetate, Z 3 hexen 1 ol, and pheromone lure compared to the same blend with t he addition of acetophenone. Field trials set up in Wimauma and Immokalee, Florida, in summer and fall 2012 had six treatments; (1) control yellow sticky card ; (2) commercial pheromone lure with Z 3 hexenyl acetate, Z 3 hexen 1 ol and 1 heptadecene; (3) p heromone with Z 3 hexenyl acetate, Z 3 hexen 1 ol and acetophenone ; (4) Z 3 hexenyl acetate, Z 3 hexen 1 ol and 1 heptadecene blend; (5) Z 3 hexenyl acetate, Z 3 hexen 1 ol and acetophenone blend; and (6) pheromone alone . In the summer trials, the treatm ent of green leafy volatiles, 1 heptadecene, and pheromone lure (2) captured the highest average number of weevils. In the fall field trial in Immokalee more females were captured on the pheromone only trap and in Wimauma so few insects were captured that there were no statistical differences in trap capture for any treatment.
12 CHAPTER 1 LITERATURE REVIEW Economics of Pepper Production Pepper Production Pepper ( Capsicum spp.) production is a profitable business in the United States. Most peppers grown c ommercially in the United States are varieties of Capsicum annuum L. Tabasco pepper is a cultivar of Capsicum frutescens L., and is also very popular in the United States. The popularity of C. annuum is due to the numerous combinations of color and taste that have been selected for within the various species. The value of bell peppers in the United States was $38.90/cwt in 2011 and $33.73/cwt in 2012 (NASS 2013). The total value of bell peppers in the United States in 2012 was $627,540,000. California led the United States in total acres harvested in 2012 with 23,300 acres, followed by Florida with 18,000 acres. The total acreage of bell peppers harvested in the United States in 2012 was 55,500 which was 3,200 acres more than in 2011. Consumption of pe ppers in the United States has continued to rise over recent years. In 2005 the average person consumed 6.89 kg of peppers and in 2009 this number increased to 7.44 kg of peppers per person. Chile peppers saw the largest percentage growth in consumption increasing from 2.77 to 2.99 kg , while bell peppe r consumption increased from 4.17 to 4.45 kg (ERS 2011). Unfortunately, 52.5% of peppers consumed in the United States in 2010 were imported, due to difficulty in satisfying demand (ERS 2011). In 2013, impo rts of dried pepper were valued at $272,169,000 and imports of fresh/chilled bell peppers were valued at $931,863,000 which was an increase from the 2012 fresh/chilled bell pepper imports of $782,473,000
13 (ERS 2013). Mexico is the major source of fresh/chi lled bell peppers imported into the China accounts for the largest value of the dried pepper imports (24.9% based on the e values of exported bell peppers from the United States decreased in both dried and fresh/chilled bell pepper categories; dried bell peppers export values decreased from $10,226,000 in 2012 to $9,873,000 in 2013. With an increase in efficient pepper prod uction and reduction in fruit loss, the amount of imported fruit c ould decrease dramatically. Production of peppers is hindered by a variety of pests. There are numerous diseases which will affect peppers including bacterial spot, tobacco mosaic virus, an d wet rot (Pernezny and Kucharek 1999). Lepidopteran pests include fall ( Spodoptera frugiperda J.E. Smith) and beet ( Spodoptera exigua HÃ¼bner) armyworm. Broad mite ( Polyphagotarsonemus latus Banks), aphids (Aphididae), thrips (Thysanoptera), and leafmine rs ( Liriomyza sativae Blanchard) are all potential pests of pepper, especially in Florida. Pepper weevil, Anthonomous eugenii Cano, is the worst pest d ue to its long lived adult stage, its difficulty to control, and its ability to produce three to five ge nerations per growing season. Weevil damage can cause dramatic fruit drop, resulting in large yield losses. There is an additional relationship between weevil damage and an internal mold caused by Alternaria alternata (Fr.) Keissler (Bruton 1989), which further decreases the marketability of the fruit. Economic Importance of Pepper Weevil The pepper weevil is the most important pest of peppers on an economic basis. In Florida, peppers are most susceptible to pepper weevil damage during the winter and ear ly spring when there is little rainfall (Goff and Wilson 1937). Rapid infestation of
14 eggs and their short life cycle (Goff and Wilson 1937). Young peppers and flower buds may become infested immediately following blossom fall , causing fruit to drop in large numbers. Premature abscission of fruit is the most important cause of yield reduc tion (Eller 1995) but flower buds and full grown peppers have also been seen to dr op (Walker 1905); this can further decrease production yields. The infested fruit will turn yellow or red before falling to the ground (Mau and Kessing 1994), and, when opened, will present a black and decaying interior (Walker 1905). Infested fruit pods which do not fall from the plant will contain frass and decaying plant material making them unmarketable (Coudriet and Kishaba 1988). Feeding punctures will not affect dried pepper quality for market ; however , they will affect canned and fresh pepper qual ity (Mau and Kessing 1994). Taxonomy of the Pepper Weevil The pepper weevil, Anthonomus eugenii Cano, was described in 1894 from specimens found in Mexico (Burke and Woodruff 1980). The pepper weevil belongs to the order Coleoptera family Curculionidae, s ubfamily Anthonominae. There are over 500 species in the family Curculionidae in Florida alone (Anderson and Peck 1994), and 33 genera and 467 species in the subfamily Anthonominae in the world (Burke 1976). The pepper weevil belongs to the tribe Anthono mini and the genus Anthonomus , which is the largest genus within the tribe with 331 species described worldwide (Burke 1976) and 200 in North America (Triplehorn and Johnson 2005). The state of Florida contains over two dozen known Anthonomus species (O rien and Wibmer 1982). Many major agricultural pests, in addition to the pepper weevil, are included in this genus, such as
15 the boll weevil ( Anthonomus grandis grandis Boheman) and the strawberry blossom weevil ( Anthonomus rubi Herbst ). Life History of th e Pepper Weevil Adult Stage The adult pepper weevil is a brownish black colored beetle with gray and yellow scale like hairs. The beetle is approximately 3.2 mm in length with a long curving snout containing elbowed antennae emerging from its center and c hewing mouthparts at its end (Goff and Wilson 1937). Pepper weevils can be sexed by examining the eighth tergum where males have a notch and females do not. They can also can be sexed through observation of metatibial mucrones which on males are larger a nd more curved shorter and slender, appearing uncurved (Eller 1995). Exposure of male pepper weevils to carbon dioxide will cause the male to expose its aedeagus and is ano ther accurate method of determining the sex of weevils (Coudriet and Kishaba 1988). The average life span of adults is 79 days with one individual found to live 316 days (Goff and Wilson 1937). The weevil can have several generations annually in fields a nd up to eight in the lab (Elmore et al. 1934). The adult weevils will feed on leaves immediately following emergence before beginning to feed on flower buds and fruit as they become available (Burke 1976). If no fruit or buds become available , the weevil will continue to feed on leaves (Mau and Kessing 1994). Life Cycle and Development Pepper weevil mating occurs 2 3 days after emergence (Genung and Ozaki 1972). One mating is sufficient for the female weevil to possess fertility for life; however multipl e matings can occur (Elmore et al. 1934). Oviposition by female weevils can
16 begin approximately 4 days after emergence (Goff and Wilson 1937). In most female pepper weevils oviposition stops in winter and will resume in the spring (Mau and Kessing 1994). Eggs are laid in very small flower buds, flowers, and all stages of fruit (Walker 1905). The weevils prefer to oviposit in small jalapeno peppers between 10 14 mm in length with approximately 19.4 Â± 1.1 offspring emerging (Porter et al. 2007). Anthonom ine weevils usually only lay one egg in flower buds but will lay multiple eggs in fruit due to the larger host size (Burke 1976). The average oviposition period lasts 30 days with approximately 198 eggs laid (Goff and Wilson 1937). Females chew a deep hol e in the fruit up to their eyes and will then deposit an egg within 2 min. A brownish fluid is secreted to seal the hole (Goff and Wilson 1937) and creates an oviposition plug. The oviposition plugs act as a deterrent to other female pepper weevils that are looking for fruit in which to lay eggs (Addesso et al. 2007). Weevils can average six eggs laid per day (Genung and Ozaki 1972). Unfertilized eggs are deposited on the surface of the fruit even if a hole has already been made through feeding activity but, upon mating, a female weevil will make normal ovipositions inside the fruit (Goff and Wilson 1937). Pepper weevil eggs average 0.79 mm in diameter and are whitish cream in color with an ovoid shape when newly laid (Goff and Wilson 1937). The eggs w ill turn yellow and then brown with aging. The surface is smooth with no ornamentation (Toapanta et al. 2005). Eggs are laid most often at the base of the fruit near the stem, sometimes through the calyx. This placement may be due to the wound healing a bilities of the calyx or ease of drilling the initial hole (Toapanta et al. 2005). Eggs will hatch in 2 4 days (Walker 1905).
17 The pepper weevil larva is cylindrical and curved in shape. It is legless with a yellowish brown head containing dark brown mout hparts and a grayish white body. The body of the larva contains 12 segments, each possessing short hairs (Goff and Wilson 1937). The larval period lasts from 6 9 days with three instars (Goff and Wilson 1937). If eggs are laid in the flower buds the lar va eats the pollen sacs and if laid in the pepper fruit the larva will eat the seeds and parts of the central core (Watson 1935). The last larval stage forms a definite cell from frass and decaying material from inside the fruit (Walker 1905) which causes the cell to be darker in color and brittle while the larva pupates inside (Goff and Wilson 1937). The pupa is 3.12 3.97 mm in length and all white at first. The snout , antennae, and legs are folded underneath the body with the wings folded against the si des. The head, prothorax, and abdomen have short hairs (Goff and Wilson 1937). The head also contains dark brown eyes (Walker 1905). The pupal stage lasts 4 days in the pepper weevil (Goff and Wilson 1937) with the time to adult emergence significantly shorter in small peppers than large (Porter et al. 2007). The newly eclosed light yellow weevil emerges into the interior of the pepper and remains there until a hardened black exoskeleton is formed. Once hardened the weevil cuts through the fruit pod wa ll (Walker 1905). For rearing of the pepper weevil in a laboratory setting it was found that 30Â°C is the best temperature for population increase and results in 3.1 eggs/day laid and a development time from oviposition to adult emergence of 12.9 days (Toap anta et al. 2005). The degree days for development were 256.4 on jalapeno pepper with a lower developmental threshold of 9.6Â°C (Toapanta et al. 2005). Devices have been
18 developed to harvest eggs for efficient laboratory rearing practices. Female weevils readily lay eggs o n polyurethane spheres, 1 cm in diameter, covered in pepper leaves followed by parafilm and hung from the tops of cages as well as parafilm balls surrounding leaves of eggplant, nightshade, potato, and Nicotiana alata (jasmine tobacco) ( Calderon Limon et al. 2002 , Addesso et al. 2009 ). Ecology of the Pepper Weevil Distribution The p epper weevil is native to Mexico and can also be found in Hawaii, California, Arizona, New Mexico, Texas, Florida, Central America, and the Caribbean basin (Wa lker 1905, Watson 1935, Goff and Wilson 1937, Fullaway and Krauss 1945, Genung and Ozaki 1972, Patrock and Schuster 1992). The weevil was seen for the first time in Texas in October 1903 (Walker 1905) and first reported in southern California in 1923 (Gof f and Wilson 1937). In February 1933 the pepper weevil was first discovered in Hawaii on Oahu and it has since spread to the other islands (Fullaway and Krauss 1945). The first Floridian pepper weevil specimens were sent to an experiment station in April 1935 for identification (Goff and Wilson 1937). Weevils can fly; however, they are mainly distributed from one place to another by being carried on young plants, field boxes, and equipment (Watson 1935). They have also been found at various ti mes in Geor gia, South Carolina, North Carolina, Virginia, New Jersey, and as far north as British Columbia; however, infestations of weevils in these regions died out due to low winter temperatures (Boswell et al. 1964 , Sparks et al. 2007, Schultz and Kuhar 2008 ). Hosts Host plants of the pepper weevil include members of the Capsicum and Solanum genera, including S. americanum var. nodiflorum (Jacq.) Edmonds (American black
19 nightshade), S. pseudogracile Heiser (glowing nightshade), S. eleagnifolium Cav. ( silverleaf nightshade), and S. carolinense L. (horse nettle). In California , wild nightshades act as the main overwintering host for the pepper weevil (Watson 1935) and in Florida, American black nightshade acts as an oversummering host (Patrock and Schuster 1987). The populations of pepper weevil on nightshade during the pepper growing off season allow for reinfestation of fields the following year (Patrock and Schuster 1987). Pepper weevils were found to oviposit in Solanum melongena L. (eggplant) (Genung and zoa ki 1972); however it was found to not be efficient in carrying weevils through the summer season in Florida due to the larvae not completing development (Goff and Wilson 1937) . N o difference was found in developmental time of weevils growing in peppers ve rsus nightshade fruit; however, the dry weights of weevils from bell pepper were significantly greater than those from other potential hosts (Patrock and Schuster 1992). Host plant resistance to pepper weevil was investigated in jalapeno, cayenne, bell, pi miento, Tabasco, Serrano, yellow, long chile, and cherry peppers. Results indicated that the weevils do not exhibit preferences among those pepper types (Berdegue et al. 1994). Various cultivars of C. annuum were evaluated for potential resistance against variety had the largest number of infested and uninfested fruit as well as the largest number of flow ers/plant and volume of foliage (Seal and Bondari 1999). Although the percentage of pepper weevil infested fruits (Seal and Bondari 1999). Pratt (1907) noted
20 that t C. annuum . In La Paz, Baja California Sur, wild solanaceous fruit were collected to observe their potential as alternate hosts during periods of no pepper production. Solanum hindsianum Benth. (mariola) was the only plant to produce pepper weevil specimens, while Datura discolor Bernth. (toloache), Physalis crassifolia Benth. (wild tomatillo), Nicotiana glauca Graham (Don Juan), and Lycium brevipes Benth. (frutilla) were free of pepper weevils (Aguilar and Servin 2002). These plants could possibly be used to surround growing fields to minimize the overwintering or oversummering of pepper weevils in alternative hosts. Host Plant Location The pepper weevil relies on several sources of stimuli to locate their hosts. Visual orientation is an important component in the host location process and is often used in conjunction with olfaction (Prokopy and Owens 1983). Boll weevil and apple blossom weevil, Anthonomus pomorum L. , have shown color preferen ces for green and blue (Hollingsworth et al. 1964, Hausmann et al. 2004). Pepper weevils have been found to be attracted to yellow sticky traps, preferably placed 10 60 cm above the ground and with approximately 300 cmÂ² of surface area (Riley and Schuster 1994). Pepper weevils were also found to be more likely to be caught between the hours of 1200 1400 h and the ratio of females to males on traps was higher than on plants inspected. However, in a study involving the location of weevils on various plant structures it was found that the number of weevils on exposed terminal buds was higher in the morning than at other times of the day (Riley et a l. 1992). In other Anthonomus weevils i t has been found that male and female boll weevils ( Anthonomus grandis Bo heman) are highly responsive to green leaf volatile s (GLV), m ost of which are six carbon saturated and monounsaturated primary alcohols ,
21 aldehydes, and acetates (Dickens 1984). Gas chromatography electrophysiological studies indicate that strawberry blosso m weevil ( Anthonomus rubi Herbst) can detect plant volatiles. These compounds included terpenoids, aromatic and aliphatic esters, alcohols, and aldehydes with some able to stimulate feeding in the weevils (Bichao et al. 2005). Another weevil known to res pond to volatile compounds is the cabbage seed weevil, Ceutorhynchus assimilis Paykull. These weevils are attracted to isothiocyanates and nitriles, both of which are metabolites of glucosinolates, and the GLV (Z) 3 hexen 1 ol and methyl salicylate which are emitted from a wide range of plant families (Bartlet et al. 1997). The apple blossom weevil is able to detect differences in plant volatiles between apple cultivars (Kalinova et al. 2000). Pepper weevils are attracted to the volatiles released from ho st plants such as, Jalapeno pepper, American black nightshade, and eggplant, and nonhost plants, lima bean, presented in a Y tube olfactometer. Ten day old mated males and females preferred the odor of host plants, while virgin females were attracted to b oth host and nonhost plants (Addesso and McAuslane 2009). The mated females preferred pepper volatiles over nightshade, mated males preferred pepper over eggplant, and both preferred nightshade over eggplant volatiles (Addesso and McAuslane 2009). The sa me study also found that weevils can orient themselves in a wind tunnel without the use of visual or pheromone stimuli. Virgin females and mated females differed in their attraction to plant volatiles (Addesso and McAuslane 2009), indicating that two vola tile odor blends may need to be synthesized in order to maximize weevil capture in volatile baited traps in the field. In another study, it was found that mated weevils preferred headspace volatiles of fruiting and flowering damaged pepper plants over und amaged
22 plants when given the choice in a Y tube olfactometer (Addesso et al. 2010). The weevils also preferred active feeding damage over old feeding damage. In a final experiment, using a four choice olfactometer, to determine the relative attractivenes s of volatiles released from damaged plants and the male produced aggregation pheromone, it was found that mated females showed no preference for plants damaged by male feeding over female feeding; however, virgin females and males preferred the plants dam aged by male feeding , which contained the male produced aggregation pheromone along with the plant volatiles (Addesso et al. 2010). This is further indication that different volatiles baited traps may need to be developed to monitor and c ontrol virgin an d mated females in the field. To determine which compounds released by damaged pepper plants may be responsible for weevil attraction, headspace extractions from damaged pepper plants were separated into four fractions using preparative gas chromatography and assayed in Y tube olfactometer tests to indicate which fractions were most attractive to male and female weevils (McNeill et al. unpublished data). Fractions one (GLV ocimene ) and four ( sesquiterpenes ) were found to be the most attract ive fractions for both males and females. Using gas chromatography electroantennographic detection, it was found that seven compounds stimulated both males and females, most notably (E, E) 4, 8, 12 trimethyl 1, 3, 7, 11 ocimene, Z 3 hexen 1 ol, and E 3 tetradecene (McNeill et al. unpublished data). These data form the basis for the choice of volatile compounds to be further tested in synthetic blends of volatiles for developing lures for attraction of pepper weevil.
23 Aggregation P heromone The use of aggregation pheromones in traps can be highly effective in detecting small crop infestations early in the season before major populations of insects develop. If the aggregation pheromone is very effective and reliably indicates low pop ulation levels, insecticide applications can be targeted to specific areas of the crop where the The first aggregation pheromone isolated for an Anthonomus species weevil was the male produced pheromone of the boll weevil ( Anthonomus grandis grandis Boheman ) (Tumlinson et al. 1969). Male boll weevils have been shown to release pheromone immediately following location of a host plant and beginning to feed (Dickens 1984). The boll weevil pheromone consists of (+) cis 2 isopropenyl 1 methylcyclobutaneethanol, cis 3,3 dimethyl cyclohexaneethanol, cis 3,3 dimethyl cyclohexaneacetaldehyde, and trans 3,3 dimethyl cyclohexaneacetaldehyde, which are called Grandlures I, II, III, and IV respectively . Boll weevil pheromone traps are now sold as ISCAlure Grandis and incorporate the four Grandlure compounds (ISCA Technologies, Riverside, CA) . Strawberry blossom weevil has also been investigated for aggregation pheromone and three male specific compounds have been ident ified (Innocenzi et al. 2001): (Z) 2 (3,3 dimethylcyclohexylidene)ethanol, ( cis ) 1 methyl 2 (1 methylethenyl)cyclobutaneethanol, and 2 (1 methylethenyl) 5 m ethyl 4 hexen 1 ol (lavandulol). T he first two components are also included in the boll weevil aggregation pheromone used in trapping (I nnocenzi et al. 200 1). Aggregation pheromones for the cranberry weevil ( Anthonomus musculus Say ) were discovered through headspace volatile collections of adults feeding on blueberry or cranberry flower buds (Szendrei et al. 2011). Originally the weevils were
24 highly attracted to field trial traps baited with pepper weevil pheromone indicating there may be similarities in the pheromone blends. Three male specific compounds were identified: cis 3,3 dimethyl cyclohexaneethanol (Grandlure II), cis 3,3 dimethyl cyclohexaneacetaldehyde (Grandlure III), trans 3,3 dimethyl cyclohexaneacetaldehyde (Grandlure IV) (Szendrei et al. 2011). Early research into pepper weevil produced attrac tants found that males produced an airborne attractant, but it only elicited responses from females. In laboratory assays male baited traps caught significantly more females than female baited and control traps. Field experiments demonstrated the same re sponse. Dichloromethane extracts of male weevils also elicited a response from female pepper weevils; however, 40 male equivalents were needed to gain the same response as five live males (Coudriet and Kishaba 1988). It was then found that male pepper we evil baited boll weevil traps attracted significantly more adult weevils than the control and female pepper weevil baited traps. Also, the same number of male and female weevils was consistently attracted to the male baited traps each week. T his could ha ve been due to the inherent attractiveness of boll weevil traps to pepper weevil due to their yellow color; however, the results suggested a male produced pheromone acting as a sex attractant and aggregation pheromone (Patrock et al. 1992). The components of the male produced aggregation pheromone were identified as (Z) 2 (3, 3 dimethylcyclohexylidene) ethanol, (E) 2 (3, 3 dimethylcyclohexylidene) ethanol, (Z) (3, 3 dimethylcyclohexylidene acetaldehyde, geranic acid and geraniol, and males release approxim developed commercial formulations of pheromone failed to release geranic acid properly and this component
25 was necessary for fullest response by pepper weevils (Eller et al. 1994). A dual lure system was de veloped (TRE8420 + 8462) and is active for up to 5 weeks in the field (Bottenberg and Lingren 1998). These lures are currently used for monitoring pepper weevils as well as detecting thresholds of one weevil before insecticide application. Pepper weevils have been found to be attracted to yellow sticky traps . These same sticky traps, when baited with pheromone caught twice as many weevils as control unbaited traps, with some catching almost 20 times more (Eller et al. 1994). Early in the season when ther e is low food availability and virgin females are looking to mate, the attraction of pepper weevils to pheromone baited traps is high. This attraction decreases midseason with increased food availability and as the number of mated females increases, who a re now looking for places to oviposit. Traps again become increasingly attractive at the end of the season as plants die and weevils search for food (Eller et al. 1994). Management Techniques Cultural The most common method of cultural control for pepper weevils is the destruction of fallen fruit (Fullaway and Krauss 1945). It is not cost effective to collect and destroy fallen fruit if pesticides are being used, but this tactic could be valuable if insecticides are not used (Andrews et al. 1986). Plowin g under old plants and destruction of wild hosts, such as wild nightshade, is another cultural method of control (Watson 1935, Mau and Kessing 1994). If the entire field is not plowed under following harvest, weevils will become a major pest and possibly destroy the entire crop during the next season (Goff and Wilson 1937). This applies to all fields within a community, not solely one farm, as the weevils can spread (Goff and Wilson 1937). Scars on the
26 surface of the fruit will indicate weevil ovipositio n and feedings, and the absence of blooms will indicate a weevil infestation within a crop (Walker 1905). Chemical Chemical control of pepper weevil is the most commonly used control method. Larvae and pupae of the pepper weevil are protected within the f ruit from externally applied chemicals. Therefore, insecticide application must be targeted and directed toward adult weevils (Eller et al. 1994). A long residual material or close interval applications must be used in order to control the infestation. In the case of extreme infestations, chemical controls will not be effective enough (Genung and Ozaki 1972). Aguilar and Servin noted in 1995 that despite heavy pesticide applications, pepper weevil and its parasitoid Catolaccus hunteri Crawford were reco vered indicating that pesticide resistance had developed in both species (Aguilar and Servin 2000).The amount of pesticides used in various growing regions is very high. In Sinaloa, Mexico it was noted during a study from 2001 2003 that the standard crop production practices of the region included nine insecticide applications during the first growing season and 13.5 insecticide sprays during the second (Cortez et al. 2005). A variety of pesticides is currently being used to treat crops against pepper wee vil damage and infestation. Over 16 different chemicals are approved for use on pepper weevil in the state of Florida ( Santos et al. 2013 ), many with limiting factors; for example, Actara (thiamethoxam) is toxic to bees and can only be used up to 11 oz pe r season and Ambush 25W (permethrin) can only be applied to bell peppers. Biological Biological control, in particular the use of parasitoids, has been explored for the control of pepper weevil. The parasitoid Bracon vestiticida (Vier.) was released in
27 Ha waii; however no specimens were recovered (Mau and Kessing 1994). Catolaccus hunteri Crawford is currently the most abundant parasitoid attacking the larvae of pepper weevil in Florida (Schuster 2007). In 1995, C. hunteri was found in Baja California Su r, Mexico as a primary parasitoid of pepper weevil. Of the parasitoids collected 75% were C. hunteri with other parasitoids found from the families Eulophidae (11%), Braconidae (4%), Ichneumonidae (4%), and Chalcididae, Eurytomidae, and Diapriidae (2%) (A guilar and Servin 2000). Five parasitoid species associated with pepper weevil were identified in Sinaloa, Mexico and included C. hunteri , as well as Eupelmus sp., Urosigalphus sp., Bracon sp., and Eurytoma sp. (Cortez et al. 2005). Parasitoids collected from the Pacific and Atlantic coasts in Mexico belonged to previously identified genera, but also included two new species, Triaspis eugenii (Wharton & Lopez Martinez) and Ceratoneura sp. (Rodri g uez Leyva et al. 2007). Triaspis eugenii is capable of para sitizing the eggs of the pepper weevil; however, its location of hosts is dependent upon the presence of oviposition plugs and commonly superparasitizes in laboratory rearing systems (Rodriguez Leyva 2006). It has been found that weekly releases of C. hunt eri result in fewer weevil infested fruit compared to areas where no parasitoids were released (Schuster 2007). Parasitism ranges from 5 35% with the parasitoid acting primarily on third instars in flower buds and smaller fruit (Riley and Schuster 1992). Parasitoids could not be found in fruit with a diameter greater than 2.5 cm, possibly due to the thickness of the fruit and Solanum spp. nightshade hosts possess small fruit and would act as an ideal release point of C. hunteri
28 experiment in which C. hunteri was released when the weevils inhabit the American black nightshade and during the regular pepper growin g season, the number of infested pepper fruit was decreased by 50% and the number of uninfested fruit increased by 150% compared to fields where C. hunteri was not released (Schuster 2007). Behavioral Manipulation with Semiochemicals Push pull strategies o f pest management involve manipulation of the pest (the push) and attract the insect to another location where the pests can be removed (the pull) (Cook et al. 2007) . The main goals of a push pull strategy are to maximize efficiency and sustainability and minimize negative environmental effects (Cook et al. 2007). Semiochemicals are chemical compounds that are produced by one organism and perceived by a second with a subsequent effect on the et al. 2007). Sometimes, host plants will release semiochemicals as volatiles and they may either act as repellents that reduce future feeding or as attractants to beneficial insects (Cook et al. 2007) . Plant volatiles can also be used to attract pest insects and these types of chemicals can be utilized in attracting the pests to traps. These types of compounds are often utilized in a push pull system. No push pull management strategy has been develop ed for an Anthonomus species; however, a n attract and kill device was investigated for the boll weevil, using a mixture of Grandlure, feeding stimulants, and toxicant (McKibben et al. 1990). These baited traps were placed in the field and were not affecte d by rain so could last for upwards of 6 weeks (McKibben et al. 1990). The traps were very efficient because the toxicant was so deadly that only a short exposure was necessary to kill the insect. Both male and female boll weevils were attracted to the G LV complex consisting of six -
29 carbon or longer saturated and monounsaturated primary alcohols and the aldehyde heptanal (Dickens 1984). A push pull strategy was investigated for the German cockroach, Blattella germanica L., with great success in managing p opulations (Nalyanya et al. 2000). Male and female roaches were found nearest areas treated with attractant and furthest away from deterrent treated surfaces, and mortality was greatest in areas baited with insecticide near attractant treated surfaces (Na lyanya et al. 2000). Push pull management of the Colorado potato beetle ( L eptinotarsa decemlineata (Say) . ) has been investigated. An attractant blend of three compounds was used to draw beetles to trap crops bordering potato fields and resulted in reduce d insecticide usage (Martel et al. 2005). Subsequent field studies conducted using a neem based antifeedant and the previous trap crop method produced a significant shift in the spatial distribution of the beetles within the fields, demonstrating an effec tive push pull technique (Martel et al. 2006). Our overall research goal is to develop a push pull management technique for use against the pepper weevil. Two components are necessary in order to develop a successful management plan using this idea: a pus h that will deter the weevils from the pepper crops, and a pull, which will attract the weevils to a trap or bait, away from the crop. The push currently being developed is an oviposition deterring pheromone which is derived from the oviposition plugs of female pepper weevils (Addesso et al. 2007). This pheromone could be used to push weevils away from pepper plants. Alternatively, insecticides that deter oviposition on plants could be used. The organic approved insecticides that have been field tested and found to deter oviposition are azadirachtin, Chenopodium , diatomaceous earth, and clay (Addesso et al. 2014 ). The pull aspect will
30 involve the use of aggregation pheromone and plant volatiles or just plant volatiles alone to attract the insects. My r esearch goal was to investigate the use of host plant volatiles in increasing the attractiveness of commercially available pheromone baited traps. Research Objectives Pepper production is a highly profitable business within the United States. Individual c onsumption of peppers, including bell and chile peppers, has continued to rise over the years with an increase in importation of peppers to meet domestic demands. Pepper growing efficiency is greatly hindered by the pepper weevil, which causes considerabl e crop loss and decrease in marketability due to poor pepper appearance and contamination. My main research objectives are: 1. To develop a blend of pepper volatiles that is attractive to pepper weevils in short range assays, and 2. To compare attraction of pe pper weevils in field tests to pheromone baited traps and to pheromone baited traps with the addition of attractive pepper volatile blends. Those volatiles found to be active from GC EAD assays were explored in Chapter 2 and the common volatiles from vario us pepper varieties and pepper weevil host plants were investigated in Chapter 3.
31 CHAPTER 2 INVESTIGATION OF EAG ACTIVE PEPPER PLANT VOLATILES AS ATTRACTANTS FOR PEPPER WEEVIL Introduction Pepper weevil, Anthonomus eugenii Cano, is the most severe pest o f pepper in Florida . Chemical control of pepper weevil is the most commonly used control method; however, larvae and pupae of the pepper weevil are protected within the fruit from externally applied chemicals. Therefore, insecticide application must be t argeted and directed toward adult weevils (Eller et al. 1994). A long residual material or close interval applications must be used in order to control the infestation. In the case of extreme infestations, chemical controls will not be effective enough ( Genung and Ozaki 1972). With concerns increasing regarding the use of chemical control agents it has become necessary to develop means of monitoring insect populations in order to increase the efficiency and specificity of insecticide application. A dual lure system containing the male pepper weevil aggregation pheromone has been developed (TRE8420 + 8462) for monitoring pepper weevils as well as detecting thresholds before insecticide application and is active for up to 5 weeks in the field (Bottenberg an d Lingren 1998). Improvement to this lure could be made through the addition of host plant volatile compounds. For example , in dual choice olfactometer experiments , females of the boll weevil, Anthonomus grandis grandis Boheman , were more attracted to a combination of the aggregation pheromone (Grandlure) and the host bisabolol than to the host plant volatile alone. However, n o significant difference was found when the combination of the pheromone and volatile w as test ed against the pher omone alone (Dickens 1986) , possibly because the pheromone/volatile
32 . EAG studies were conducted with various green leaf volatiles from the boll weevil host plant and although some were found to elicit a response, only E 2 hexen 1 ol improved capture of weevils in pheromone traps (Dickens 1989). It has been found that the banana weevil, Cosmopolites sordidus (Germar) , is attracted to the combination of host plant volatiles with its aggregation pheromone (Cosmolure+) in various bioassays in the laboratory (Tinzaara et al. 2003). Olfactometer assays conducted with the palm weevil, Rhynchophorus palmarum (L.) , showed higher attraction to the male pheromone with the a ddition of the host plant volatile acetoin than to the pheromone alone . Field trials showed that traps baited with some form of plant volatile and pheromone caught ten times more weevils than traps containing the pheromone alone (Said et al. 2005). Ten day old mated male and female pepper weevils preferred the odor of host plants (Jalapeno pepper, American black nightshade, and eggplant) to purified air, while virgin females were attracted to both host and nonhost (lima bean) plants when presented in a Y tu be olfactometer (Addesso and McAuslane 2009) . T wo volatile odor blends may need to be synthesized in order to maximize weevil capture in volatile baited traps in the field due to virgin and mated female pepper weevils differing in their attraction to vola tiles (Addesso and McAuslane 2009) . It was also found that mated weevils preferred headspace volatiles of fruiting and flowering damaged pepper plants over undamaged plants as well as active feeding damage over old feeding damage when given the choice in a Y tube olfactometer (Addesso et al. 2010). U si ng a four choice olfactometer, it was found that virgin females and males preferred the plants damaged by male feeding, which contained the aggregation pheromone along with the
33 plant volatiles , while mated f emales did not prefer male over female produced feeding damage (Addesso et al. 2010). Headspace extractions from damaged fruiting pepper plants were separated to determine the compounds released that may be responsible for weevil attraction. F our fraction s were created using preparative gas chromatography and assayed in Y tube dual choice tests to indicate which fractions were most attractive to male and female weevils (McNeill et al. , unpublished data). Fractions one and four were found to be the most at tractive fractions for both males and female s. Fraction one contained green leaf v ocimene, and fraction four contained various sesquiterpenes . Using gas chromatography electroantennographic detection, it was found that seven compounds stimulated both males and females, (E, E) 4, 8, 12 trimethyl 1, 3, 7, 11 trid ocimene, Z 3 hexen 1 ol, E 3 pinene, Z 3 hexenyl acetate, and 1 heptadecene (McNeill et al. unpublished data). These volatile compounds were selected as potentially useful for developing lures for field attraction of p epper weevil. The overall objective of this research was to improve the effectiveness of the commonly used aggregation pheromone with the addition of plant volatiles. First, the attractiveness of the host plant volatiles found to be stimulatory through GC EAD analysis was analyzed using Y tube olfactometer studies. Once the attractive compounds had been identified, blends were created for additional testing within the Y tube. Wind tunnel studies and field trials were conducted to determine the attractive ness of traps baited with host plant volatile blends and the commercial pheromone lure.
34 Materials and Methods Insects The insects used in this study were originally collected from south Florida in the spring of 2004 near the city of Clewiston. A laborator y colony was established and maintained at the University of Florida, Gainesville. In order to ensure colony health and genetic diversity, additional field collections were made in the fall of 2005, 2006, and the summer of 2011 from Immokalee, Bradenton, and Wimauma, Florida. The laboratory settings for the colony were a 14:10 light: dark photoperiod and 27Â°C with 30% R.H. The pepper weevils were raised on Jalapeno peppers for food and oviposition. Two colony boxes (31 cm x 31 cm x 31 cm, sealed plexigl ass with a cloth sleeve opening on one side) were maintained at all times and were started 1 month apart. Gravid females older than 10 d old were transferred out of colony boxes into oviposition cups made from 250 mL, 8.5 cm diameter waxed cardboard cans with screened lids (The Fonda Group, Inc., Union, NJ). Females were removed from oviposition cups after 3 weeks and placed in the oldest colony box. The oldest box was discarded after 1 month. Weevils were sexed by observation of metatibial mucrones whi ch on males are larger and more curved than those of females (Eller 1995). Plants The Jalapeno pepper plants and fruit used in the experiments were started from seed (Trade Winds Fruit, Windsor, CA) at the USDA, Agricultural Research Station greenhouses in Gainesville and then moved to the University of Florida in Gainesville. They were grown in a mixture of Metromix 200 and 500 in 11.5 L (28 cm x 23 cm) pots. They were maintained inside a greenhouse during colder months (November March) and outdoors in warmer months. Pepper plants were fertilized twice per week using
35 Tracite Foliar 20 20 20 (Helena Chemical Company, Collierville, TN) at a rate of 6 g/L of water applied to the soil. Fruit that were used for colony maintenance were approximately 2.5 cm in length with smooth skin and had not begun to toughen or ripen. Y Tube Olfactometer Assays Y tube olfactometer bioassays (Fig ure 2 1) were conducted using two olfactometers (Analytical Research Systems, Gainesville, FL) connected with corrugated Teflon tub ing. Compressed air was purified by passing through a charcoal filter (Fig ure 2 1A) and then humidified by bubbling through deionized water before being split into two glass chambers (26 cm x 3 cm) which held the sample volatiles (Fig ure 2 1B) . The air flows were then split into four different air flows, each regulated by a flowmeter (Manostat, New York, NY) at 250 ml/min, which were attached to the arms of glass Y tubes (12 cm common tube, 10 cm arms, 2.5 cm internal diameter). The Y tubes were held at a 60 enclosure (46 cm Ã— 28 cm Ã— 42 cm) to prevent visual disturbances (Fig ure 2 1C) . Fluorescent lighting (four 85 W Sylvania cool white bulbs, 70 cm above table) was used within the bioassay room to ligh t the assembly. Room conditions were maintained at 25Â° 27Â°C and 40% R.H. Insects were inserted at the base of the Y tube and allowed 10 min to make a choice between the two arms of the olfactometer. A choice was registered when the weevil crossed over th e ridge between the glass pieces connecting the Y tube to the Teflon tubing, approximately 7 cm up the arm from the common arm. Assays were conducted until 50 insects were registered as making a choice. The glass Y tubes were rotated 180Â° after every fiv e insects to account for weevil phototaxis and variation in light intensity. Volatile samples were applied to filter papers (Millipore absorbent pads,
36 3.7 cm in diameter, Tullagreen, Carrigtwohill, Co. Cork, I reland ) cut in half. A 10 quantity of mine ral oil (Publix Brand, Lakeland, FL) was first placed on the disks, and then 5 ÂµL samples at 100 x dilutions were added , unless specified differently. The individual compounds tested for initial attraction were the seven mutually attractive compounds foun d by GC EAD as well as ethyl acetophenone and E 2 hexenal which were stimulatory to female weevils in the GC EAD, and E E farnesene which was found to be stimulatory to males. Based on the results of a preliminary testing of 20 weevils, acetophenone, 1 heptadecene, and the green leafy volatiles (GLV), Z 3 hexenyl acetate and Z 3 hexen 1 ol , were further assayed. Although ethyl acetophenone was found to be stimulatory in the GC EAD assay and found in the host plant volatiles, attraction in the Y tube ass ay could only be achieved with acetophenone, which was being investigated for its presence in pepper weevil oviposition plugs (Addesso, unpublished data). Based on the results of these tests two blends were tested : 1) Z 3 hexenyl acetate, Z 3 hexen 1 ol, and acetophenone , and 2) Z 3 hexenyl acetate, Z 3 hexen 1 ol, and 1 heptadecene with 5 ÂµL at a 100 x dilution. Additional blends made of four compounds with 5 ÂµL at a 100 x dilution were tested including, Z 3 hexenyl acetate, Z 3 hexen 1 ol, 1 heptadecene , and E 2 hexenal ; Z 3 hexenyl acetate, Z 3 hexen 1 ol, 1 heptadecene, and acetophenone ; and Z 3 hexenyl acetate, Z 3 hexen 1 ol, acetophenone, and ocimene. Control filter papers were treated with 10 ÂµL mineral oil and 5 ÂµL methylene chloride . New sample s were created and tested every 30 min . Mated males and females were at least 10 d old. Virgin insects were removed from emergence boxes and sexed immediately, then maintained separately with
37 peppers until they were at least 10 d old. All insects were s tarved overnight before the assay with access to water . Data was analyzed using a dual choice Chi Squared analysis in Excel. The expected values were calculated to indicate no difference between the treatment and control for each compound and blend tested . The p value set for significance was 0.05. Wind Tunnel Dual Choice Assays The wind tunnel used for the assay s was constructed of plexiglass and consisted of a central chamber (27 cm Ã— 48 cm Ã— 27 cm) with arms attached on opposite sides (31 cm Ã— 122 cm Ã— 31 cm) (Fig ure 2 2A) . The end of each arm was screened to allow airflow to be pulled toward the center chamber from both directions past and over sources of volatiles placed at each end (Fig ure 2 2B). A vacuum type system was used to create the airflow (41.3 9 m per min) through an opening (26 cm x 26 cm) covered by screen in the back of the central chamber (Fig ure 2 2C and D) . Airflow was measured using the Heavy Duty CFM CMM Thermo Anemometer (Extech Instruments, Model HD 300). Assays were conducted by placin g weevils in the central chamber and recording their position 6 h later. Choice was indicated by capture of weevils on yellow sticky cards (15 cm x 15 cm , Trece Incorporated, Adair, OK) , propped up at an angle at either end of the dual choice wind tunnel, or by location of the weevils more than halfway up the arm of the tunnel. Twenty weevils were released at a time and four replicates were (to delay volatilization) ; six replicates were done wit response after 6 h was scored as: missing, no choice made, or choice made for one stimulus or the other. Mated females were tested with a choice of a blend containing Z -
38 3 hexenyl acetate, Z 3 hexen 1 ol, a cetophenone, and pheromone lure versus the same blend without acetophenone . Volatile blends were prepared by placing appropriate amounts of oleic acid and each blend volatile into 0.6 mL polypropylene microcentrifuge tubes (Fisherbrand, Pittsb urg h , PA). Each volatile was placed in a separate tube and attached to the appropriate sticky card by pressing the tube upright and open against the sticky portion of the card. A 27 gauge needle was used to punch a hole in the top of each tube. Pepper we evil pheromone lures (Trece Incorporated, Adair, OK) were attached to the sticky cards by placing them inside holes punched in the non sticky area of the card. Lures were used for an entire set of blend repetitions due to their ability to last up to 6 wee ks in the field. Pheromone lures were stored in the freezer wrapped in aluminum foil between uses. Data was analyzed using a dual choice Chi Squared analysis in Excel. The expected values were calculated to indicate no difference between the two treatmen t s . Any weevils not found or which made no choice were not considered in the calculations. The P value set for significance was 0.05. Field Trials Fields used were located at the Gulf Coast Research and Education Center in Wimauma and the Southwest Flori da Research and Education Center in Immokalee, Florida and were maintained by Research and Education Center staff. In Immokalee rep licate s were established at various distances from a row of actively growing jalapeno pepper plants for the summer 2012 tria l. Traps in Rep licate 1 and Replicate 2 were placed along a 128 m transect 104 m and 207 m from the row of pepper plants, respectively. Traps in Replicate 3 and Replicate 4 were placed along a 152 m transect,
39 856 m and 960 m from the row of pepper plants, respectively. All traps were spaced 24 29 m apart (Fig ure 2 3) . In Wimauma a crop of dying bell pepper plants w as used as the potential source of weevils for the summer 2012 study. The field was 30 m by 91 m with 0.30 m between plants and one 3 m gap eve ry 6 m. Traps were placed along the four edges of the field; Rep licate s 1 and 4 were approximately 18 m from the edge of the pepper crop and Replicates 2 and 3 were approximately 9 m from the pepper crop (Fig ure 2 4). In the Fall 2012 trial in Immokalee , Re plicates 1 and 2 were placed on one side of the active jalapeno pepper row at 91 m and 3 m away , respectively. Replicates 3 and 4 were placed on the opposite side of the row of peppers at 103 and 207 m away , respectively (Fig ure 2 5) . For the fall 2012 tri al at the Wimauma location , traps were placed every 30 m along the outside border of an actively growing jalapeno pepper crop. Replicates 1, 2, and 4 were placed along the edges of the field closest to ditches containing weeds and potential oversummering sights for pepper weevil. Replicate 3 was placed on the opposite side of a ditch in a field with no active crops growing (Fig ure 2 6) . Sticky traps (30 cm x 15 cm, Trece Incorp orated , Adair, O K ) were attached to 2.13 m wooden tomato stakes and driven 30 cm into the ground. Traps were placed in the field on the first day of planting and replaced every week when data were recorded from each trap. In the summer 2012 trial in Wimauma, traps were placed in the field during a senescing crop period. There were si x treatments; 1) a control yellow sticky trap , 2) the commercial pheromone lure with Z 3 hexenyl acet ate, Z 3 hexenol and 1 heptadecene added, 3) the commercial pheromone lure with Z 3 hexenyl acetate, Z 3 hex enol and acetophenone added, 4) a Z 3 hexenyl a cetate, Z 3 hexe nol and 1 -
40 heptadecene blend, 5) a Z 3 hexenyl acetate, Z 3 hexenol and acetophenone blend, and 6) the commercial pheromone lure. Treatments were randomized every week. Volatiles were prepared with 50 uL of each component of the blend and t he appropriate 1:1 ratio of mineral oil. Blends were placed in a sealed 0.6 mL eppendorf tube mixed in a vortex before being shipped to the field test location in a Styrofoam cooler with ice packs. Once the samples arrived at the test location the tubes were punctured with a size 27 needle and attached to the sticky cards by inserting through the premade holes or b y using a single hole punch to create holes for holding all components of the treatment. Volatile samples were changed weekly and any pheromon e lures changed every four weeks. Traps were wrapped in cellophane or plastic bags cut lengthwise and mailed to the Gainesville lab for analysis and evaluation. Weevils were removed from the trap and stored in ethanol before and /or after being evaluated f or sex. At the end of the season plants were tilled under and both research locations left one row of jalapeno peppers in order to maintain a population of weevils . Data was analyzed using SAS version 9.3 (SAS Institute Inc., Cary, NC) with a PROC MIXED analysis using weeks as a repeated measures. The variables analyzed were week, treatment , replicate, and week by treatment interaction. Treatment means were separated using a Tukey Kramer adjustment to the least squared means test. Results Y Tube Olfac tometer Assays M ated female and mated male weevils were significantly attracted to acetophenone at 1000x dilution in the Y tube olfactometer. However, no other compounds showed attraction when tested singly, including Z 3 hexen 1 ol, Z 3 hexenyl acetate, and 1 heptadecene attracted mated or virgin females at the 100x concentration
41 tested (Table 2 1). Of t he t hree compound blends tested , only the GLV mixture of Z 3 hexen 1 ol and Z 3 hexenyl acetate with acetophenone and the GLV mixture with the addition o f 1 heptadecene were marginally attractive (P=0.08) (Table 2 2). None of the four component blends tested in the Y tube were attractive (Table 2 3). Wind Tunnel Dual Choice Assays T he GLV mixture with the pheromone lure was significantly more attractive to mated female weevils acetophenone and th < 0.05 ) when tested in the wind tunnel. The GLV mixture with the pheromone lure had a total of 25 weevils (out of 120, released 20 per r eplicate) register as making this choice, with an average of 4.16 weevils selecting this end of the wind tunnel. The GLV mixture with the addition of acetophenone and the pheromone lure had a total of 7 weevils (out of 120, released 20 per replicate) selec t it with an average of 1.16 weevils select this trap per replicate. In this experiment, 32 weevils did not make a choice, 55 weevils could not be found and one weevil was found dead. there was no difference in pheromone lure had a total of five insects (out of 80 released) make a choice with an average of 1.25 insects per rep licate and the GLV mixture with the addition of acetophenone and the pheromone lure averaged 2.75 insects per replicate with a total of 11 insects. During this experiment, 47 weevils did not make a choice, 16 weevils could not be found, and one weevil was found dead. Field Trials In the summer 2012 field trial in Immokalee there were significant differences among the treatments in capture of total weevils and capture of female weevils (Table
42 2 4) but not in capture of male weevils. The t reatment of GLV, 1 heptadecene, and the pheromone lure captured statistically more total weevils than the GLV and acetophenone baited trap (adjusted Tukey Kramer test, P= 0.0202) and the control sticky trap (adjusted Tukey Kramer test, P=0.022) (which did not capture any wee vils), but the other treatments did not differ from each other (Fig ure 2 7). No weevils were caught after August 14. In the summer 2012 trial in Wimauma, the GLV, 1 heptadecene, and pheromone lure treatment also captured the highest number of wee vils over t he trial period (2.57 Â± 1.10 weevils per week ) (Table 2 5; Fig ure 2 8), significantly more than any of the other treatments which did not differ from each other (Table 2 5). This was also the treatment that caught more female weevils than any of the other t raps (Table 2 5). There was no difference in trap capture of male weevils. There were no captures on the control sticky cards, or the GLV and 1 heptadecene blend treatment during the entire trial period (Fig ure 2 8). In the fall 2012 field trial in Immok alee there were statistical differences in treatments for capture of female weevils only (Table 2 6; Fig u re 2 9). Significantly more females were captured on the pheromone only trap than on the other traps which did not differ from each other. The treatmen t blends of GLV and acetophenone, GLV and 1 heptadecene, and the control sticky card did not capture any weevils. In the f all 2012 field trial in Wimauma so few insects were captured that there were no statistical differences in trap capture for any treatm ent (Table 2 7). Only the treatments of GLV, acetophenone and pheromone, GLV and acetophenone, and GLV, 1 heptadecene, and pheromone caught any weevils at all (Fig ure 2 10).
43 Discussion While many volatiles were shown to be EAG active (McNeill, unpubl. data), when tested alone they were not all found to be behaviorally attractive at the concentrations tested. The EAG response of the weevils is not indicative of an attractive or repellent quality, but is merely an indicator that the insect could detect the vol atile. Therefore, compounds may have been repellents. These compounds could also be attractive at different concentrations than were assayed or are effective attractants when included in blends with other compounds. Early studies with the Colorado potat o beetle illustrated this concept through experiments in which the individual synthetic compounds did not illicit an attractive response, but the entire plant smell was attractive (Visser and Ave 1978). Also, the addition of the synthetic compounds to the potato host plant treatment resulted in a repellent response. Research with the black bean aphid, Aphis fabae Scopoli , supports the idea that the overall blend composition and ratios of compounds within the blends are critical for behavioral activity (We bster et al 2010). In that study, each individual component from the host volatile blend that was identified was tested in a four choice olfactometer at the concentrations that were present in the overall blend. Ten of the compounds elicited negative beh avioral responses, two elicited no response, and only Z 3 hexen 1 ol and 1 hexanol elicited a positive response. Nine compounds from the group with a negative behavioral response when tested individually were combined into a blend and tested again, this t ime attracting the black bean aphid. Acetophenone was attractive to mated female and mated male pepper weevils when tested alone in the Y tube assay. Mated females were also shown to be marginally attracted to a mixture of Z 3 hexenyl acetate, Z 3 hexen 1 ol, and acetophenone as well as a mixture of Z 3 hexenyl acetate, Z 3 hexen 1 ol, and 1 -
44 heptadecene when assayed in the Y t ube. Initial wind tunnel assays showed the mixture of Z 3 hexenyl acetate and Z 3 hexen 1 ol with a pheromone lure to be more attra ctive that the same mixture with the addition of acetophenone with a pheromone lure . Acetophenone has the potential to aid in attractiveness of blends in a small arena; however, in a larger bioassay arena like the wind tunnel it did not. The blend which works best in the field or large arena setting is the blend that should be used for improving the current pheromone lure . In field trials, the Z 3 hexenyl acetate, Z 3 hexen 1 ol, and 1 heptadecene alongside the pheromone lure were the most effective in c apturing weevils compared to the other blends tested. Low numbers of weevils were caught on the traps in the field trials, with two weevils being the highest number of weevils caught on a trap over a period of a week. The number of weevils caught in the traps would need to be much larger in order to evaluate trap effectiveness, but the current data would allow for the traps to be minor indicators of weevil movement into a field. During the Summer 2012 field trials, at least one treatment containing volat iles was more effective at capturing weevils than the current pheromone lure. The Wimauma, FL field trial had a moderately significant difference between the traps with volatiles and the pheromone trap acting alone. Very little can be concluded from the Fall field trials due to the fact that so few weevils were caught on any of the traps. It may be necessary to use different blends at different times of the year when attempting to monitor weevil populations. In the summer, when most pepper crops are dy ing or being plowed under, female and male weevils are searching for a host for a source of food and the treatment of GLV , 1 heptadecene, and pheromone was the most attractive. However, during the fall, when there is typically a healthy pepper crop
45 growin g, the pheromone lure may work better for monitoring due to weevils searching for mates and not for a limited supply of host material. Investigation into attractants for the cranberry weevil ( Anthonomus musculus Say) led to identification of four host plan t volatiles that elicited an antennal response (Szendrei et al. 2009). Potential pheromone compounds produced by male cranberry weevils were identified and various pheromone compounds and host plant volatiles were field tested in traps (Szendrei et al. 20 11). Addition of the green leaf volatiles Z 3 hexenyl acetate and hexyl acetate into their traps baited with the pheromone yielded no increase in attraction, only an increase in the ratio of female to male cranberry weevils caught. The attraction of app le blossom weevil ( Anthonomous pomorum L.) to volatiles released over multiple phenological stages of apple has been tested (Piskorski and Dorn 2010). There were differences identified in the ratios of compounds collected at each stage as well as the exac t number of compounds that were identified. Bioassays of a synthetic blend released by apple trees at the main colonization period of the apple blossom indicated that an artificial blend was as attractive as a living plant (Piskorski and Dorn 2010). Mated pepper weevils prefer damaged flowering and fruiting plants over undamaged plants when tested in a Y tube olfactometer (Addesso et al. 2010). Weevils also preferred active feeding over old feeding damage and active feeding on fruiting plants over floweri ng plants. Female pepper weevils specifically preferred 48 h of feeding damage over feeding damage only for 1 h. It is essential that the life stage of the host plant as well as the ecological stage of the insect be taken into consideration when designin g a trap for the interruption of a biological process and signaling.
46 Due to the lack of success in developing a highly attractive blend from the GC EAG active compounds during the above pepper weevil trials a different approach was taken to identify additi onal compounds which may be included in the blends that were found to be moderately attractive. Thus, in Chapter 3, the volatiles released by six varieties of pepper and two non pepper host plants were identified and blends developed based on common volat iles within those host plants.
47 Table 2 1. Response of adult pepper weevils to single host plant volatiles in a Y tube dual choice olfactometer when presented with a choice between the compound and purified air. (P< 0.05). Plant volatile Sex Treatment Air Chi s quare P value Acetophenone Mated Females 42 8 23.12 < 0.05 Virgin Females 25 25 0 1 Mated Males 40 10 18 < 0.05 Z 3 hexen 1 ol Mated Females 22 28 0.72 0.40 Virgin Females 26 24 0.08 0.78 Z 3 hexenyl acetate Mated Females 23 28 0.52 0.48 Vi rgin Females 26 24 0.08 0.78 1 heptadecene Mated Females 29 21 1.28 0.26 Virgin Females 26 23 0.18 0.65
48 Table 2 2. Response of adult pepper weevils to 3 component blends in a Y tube olfactometer when presented with a choice between the blend and purif ied air. GLV indicates Z 3 hexen 1 ol and Z 3 hexenyl acetate. (P<0.05). Plant volatiles Sex Treatment Air Chi s quare P v alue GLV + a cetophenone Mated Females 41 27 2.882 0.08 Mated Males 28 19 1.724 0.19 GLV + 1 heptadecene Mated Females 50 34 3.048 0 .08 Virgin Females 7 7 0 1 Mated Males 34 25 1.372 0.08
49 Table 2 3. Response of adult pepper weevils to 4 component blends in a Y tube olfactometer when presented with a choice between the blend and purified air. GLV indicates Z 3 hexen 1 ol and Z 3 hexenyl acetate. (P<0.05). Experiment Sex Treatment Air Chi s quare P v alue GLV + 1 heptadecene + E 2 hexenal Mated Females 6 7 0.076 0.78 GLV + 1 heptadecene + a cetophenone Mated Females 16 12 0.572 0.45 Virgin Females 18 19 0.027 0.87 Mated Males 18 20 0.106 0.74 GLV + a cetophenone + o cimene Mated Females 1 3 1 0.32 Virgin Females 3 5 0.5 0.48
50 Table 2 4. ANOVA results for pepper weevils captured on traps in Immokalee field trial for Summer 2012. Variable Num DF Den DF F P value Total weevils Week 7 13 6 1. 82 0.0 875 Treatment 5 13 6 3. 33 0.00 7 2 Replicate 3 13 6 1.36 0.2565 Week x treatment 35 13 6 1.2 7 0.1 696 Female weevils Week 7 13 6 1.01 0.4 259 Treatment 5 13 6 3.0 8 0.01 16 Replicate 3 13 6 0.95 0.4195 Week x treatment 35 13 6 1.2 6 0 .1 740 Male weevils Week 7 1 36 1. 71 0.11 09 Treatment 5 13 6 1. 80 0.1 163 Replicate 3 13 6 1.42 0.2390 Week x treatment 35 139 0.99 0.4983
51 Table 2 5. ANOVA results for pepper weevils captured on traps in Wimauma field trial for Summer 2012. Variable Num DF Den DF F P value Total weevils Week 6 12 1 4. 09 0.000 9 Treatment 5 12 1 7. 72 <0.0001 Replicate 3 12 1 0.17 0.9155 Week x treatment 30 12 1 2. 15 0.001 9 Female weevils Week 6 12 1 1.9 4 0.07 9 7 Treatment 5 12 1 5.7 0 <0.0001 Replicate 3 12 1 0 .54 0.6588 Week x treatment 30 12 1 1.5 1 0.0 633 Male weevils Week 6 12 1 1.9 1 0.0 850 Treatment 5 12 1 1. 87 0. 1049 Replicate 3 12 1 0.20 0.8933 Week x treatment 30 12 1 1. 77 0.01 58
52 Table 2 6. ANOVA results for pepper weevils captured on traps in Imm okalee field trial for Fall 2012. Variable Num DF Den DF F P value Total weevils Week 7 14 0 2.8 3 0.00 88 Treatment 5 14 0 2. 06 0.0 743 Replicate 3 14 0 0.31 0.8215 Week x treatment 35 14 0 1.1 0 0.3 439 Female weevils Week 7 14 0 2.5 4 0.01 73 Treatm ent 5 14 0 5.9 4 <0.0001 Replicate 3 14 0 1.01 0.3916 Week x treatment 35 14 0 2.5 6 <0.0001 Male weevils Week 7 14 0 1. 49 0.1 745 Treatment 5 14 0 1.4 4 0.2 151 Replicate 3 14 0 0.44 0.7275 Week x treatment 35 14 0 0.7 7 0.8 196
53 Table 2 7. ANOVA results f or pepper weevils captured on traps in Wimauma field trial for Fall 2012. Variable Num DF Den DF F P value Total weevils Week 7 1 39 0.5 5 0.79 37 Treatment 5 1 39 0.9 7 0.43 93 Replicate 3 1 39 0.65 0.5826 Week x treatment 35 1 39 0.98 0.5 122 Female wee vils Week 7 1 39 0.5 5 0.79 37 Treatment 5 1 39 0.9 7 0.43 93 Replicate 3 1 39 0.65 0.5826 Week x treatment 35 1 39 0.98 0.5 122 Male weevils No weevils captured
54 Figure 2 1. Casey Michelle Reed. Y tube Olfactometer. April 13, 2014. Gainesville. Y tube olfactometer set up containing compressed air tank leading to a cha rcoal filter (A) and then humidified by bubbling through deionized water before being split into two glass chambers which held the sample volatiles (B). Air flows were split into four different air flows, each regulated by a flowmeter which were attached t o the arms of glass Y tubes. The Y tubes were held at prevent visual disturbances. Blue tape indicates the treatment vessel and airflow (C).
55 Figure 2 2. Casey Michelle Reed. Wind tunnel . April 13, 2014. Gainesville . The wind tunnel used f or the assays was constructed of plexiglass and consisted of a central chamber with arms attached on opposite sides (A). The end of each arm was screened to allow airflow to be pulled toward the central chamber from both directions past and over sources of volatiles placed at each end (B). A vacuum type system was used to create the airflow through an opening covered by screen in the back of the central chamber. Weevils were inserted in the center via the door in the front of the wind tunnel (C). The back of the wind tunnel central chamber with the vacuum type system that was used to create the airflow through an opening covered by screen is shown (D).
56 Figure 2 3. Layout of Immokalee, FL field trial for Summer 2012. The red lines indicate the trap lines , and the green line indicates the row of actively growing peppers .
57 Figure 2 4. Layout of Wimauma, FL field trial for Summer 2012. The red lines indicate the trap lines, and the green lines indicate the row of actively growing peppers.
58 Figure 2 5. Layout of Immokalee, FL field trial for Fall 2012. The red lines indicate the traps lines, the green line indicates the row of actively growing peppers, and the purple area indicates an area with jalapeno and eggplants actively growing.
59 Figure 2 6. La yout of Wimauma, FL field trial for Fall 2012. The red lines indicate the traps lines, and the green line indicates the row of actively growing peppers .
60 Figure 2 7. Average number of pepper weevils caught per replicate over eight weeks by synthetic ho st plant volatile blends in summer field trial in Immokalee, FL (2012). GLV indicates Z 3 hexen 1 ol and Z 3 hexenyl acetate. PHE indicates pheromone lure. Error bars indicate +1 SEM . 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Average Number of Pepper Weevils Caught per Replicate GLV + Acetophenone + PHE GLV + Acetophenone GLV + 1-heptadecene GLV + 1-heptadecene + PHE PHE Control Sticky Card
61 Figure 2 8. Average number of pepper weevils caught per replicate over eight weeks by synthetic host plant volatile blends summer field trial in Wimauma, FL (2012). GLV indicates Z 3 hexen 1 ol and Z 3 hexenyl acetate. PHE indicates pheromone lure. Error bars indicate +1 SEM. 0 0.5 1 1.5 2 2.5 3 Average Number of Pepper Weevils Caught per Replicate GLV + Acetophenone + PHE GLV + Acetophenone GLV + 1-heptadecene GLV + 1-heptadecene + PHE PHE Control Sticky Card
62 Figure 2 9. Average number of pepper wee vils caught per replicate over eight weeks by synthetic host plant volatile blends in fall field trial in Immokalee, FL (2012). GLV indicates Z 3 hexen 1 ol and Z 3 hexenyl acetate. PHE indicates pheromone lure. Error bars indicate +1 SEM. 0 0.2 0.4 0.6 0.8 1 1.2 Average Number of Pepper Weevils Caught per Replicate GLV + Acetophenone + PHE GLV + Acetophenone GLV + 1-heptadecene GLV + 1-heptadecene + PHE PHE Control Sticky Card
63 Figure 2 10 . Average number of pepper weevils caught per replicate over eight weeks by synthetic host plant volatile blends in fall field trial in Wimauma, FL (2012). GLV indicates Z 3 hexen 1 ol and Z 3 hexenyl acetate. PHE indicates pheromone lure. Error bars ind icate +1 SEM . 0 0.1 0.2 0.3 0.4 0.5 0.6 Average Number of Pepper Weevils Caught per Replicate GLV + Acetophenone + PHE GLV + Acetophenone GLV + 1-heptadecene GLV + 1-heptadecene + PHE PHE Control Sticky Card
64 CHAPTER 3 INVESTIGATION OF COMMONALITY IN VOLATILES AMONG WEEVIL HOST PLANTS Introduction The pepper weevil is attracted to American black nightshade and eggplant as well as Jalapeno pepper in Y tube assays (Addesso et al. 2009). Attract ion to different pepper varieties by pepper weevil has never been experimentally shown; however many pepper varieties have been found infested with pepper weevil in field settings (Elmore et al. 1934, Burke and Woodruff 1980, Patrock and Schuster 1992, Berdeg ue et al. 1994, Seal and Bondari 1999). Analysis of the headspace volatiles released by different species and varieties of peppers and other non pepper hosts may reveal common volatiles which are pote ntially useful as attractants. Cotton essential oil was isolated and fractionated to investigate the attractiveness of individual host volatile compounds and blends of compounds to the boll weevil (Minyard et al. 1969). It was found that individual compounds of one fraction were attractive when tested individ ually, but not when combined. These compounds were the bisabolol was moderately attractive to the boll weevil, but is difficult to purchase commercially which discourages its use in trapping and management methods. It was concluded that attraction of insects to plant volatiles will require multi component blends with as many as 10 compounds contributing to the attraction. Essential oils were isolated from known boll weev il overwintering leaf trash sources and volatile compounds were identified (Hedin et al. 2000). Blends were created and tested in the field; however, none was effective. A sesquiterpene blend consisting of ca ryophyllene, longifoline, and humulene added to the Grandlure pheromone lure was shown to be
65 attractive; however, due to potential presence of caryophyllene in the female produced sex pheromone (Hedin et al. 1979) the results could have been due to m ale Research was conducted on additional components which may be added to the previously tested pepper weevil host plant volatile blends to improve their attractiveness . Investigation i nto the addition of sesquiterpenes to the blends was conducted through analysis of headspace collections of various pepper weevil host plants. Components of each collection were analyzed and a comparison between the concentrations and ratios of the differ ent components in each host were made, looking for both similarities and differences. The host plants selected for analysis were six varieties of pepper (jalapeno, bell, habanero, pequin, serrano, and tabasco), and eggplant, and nightshade, based upon pre vious readings and documentation of overwintering and oversummering capabilities and potentials. Sesquiterpenes were selected for consideration based on availability and presence in hosts . The blends considered for the addition of sesquiterpenes were the Z 3 hexenyl acetate, Z 3 hexen 1 ol, and acetophenone blend as well as the Z 3 hexenyl acetate, Z 3 hexen 1 ol, and 1 heptadecene blend. Materials and Methods Plants The Jalapeno pepper plants and fruit used in the experiments were started from seed (Trad e Winds Fruit, Windsor, CA) at the USDA, Agricultural Research Station greenhouses in Gainesville, FL and then moved to the Department of Entomology & Nematology, University of Florida , Gainesville. They were grown in a mixture of Metromix 200 and 500 in 11.5 L (28 cm x 23 cm) pots. They were maintained inside a
66 greenhouse during colder months (November March) and outdoors in warmer months. Pepper plants were fertilized twice per week using Tracite Foliar 20 20 20 (Helena Chemical Company, Collierville, TN) at a rate of 6 g/L of water applied to the soil. Fruit that were used for colony maintenance were approximately 2.5 cm in length with smooth skin and had not begun to toughen or ripen. For the host plant variety experiments, seeds of C . frutescens (Ta basco), C. annum (Pequin Chile, Serrano Huasteco, Bell California Wonder ) and C. chinense (Orange Habanero) were obtained from Trade Winds Fruit (Windsor, CA). Solanum nigr u m ( Black nightshade ) was obtained from wild patches at the Entomology and Nemato logy Department, University of Florida and Solanum melongena ( eggplant ) ( Ichiban ) was obtained from Alachua County Feed a nd Seed (Gainesville, FL). All plants were grown to similar size and amounts of foliage and fruit (2.5 cm target length) before bein g analyzed for headspace volatiles. Four plants of each variety were analyzed. Insects The insects used in this study were originally collected from south Florida in the spring of 2004 near the city of Clewiston, Florida. A laboratory colony was establish ed and maintained at the University of Florida, Gainesville. In order to ensure colony health and genetic diversity, additional field collections were made in the fall of 2005, 2006, and the summer of 2011 from Immokalee, Bradenton, and Wimauma, Florida. The laboratory settings for the Gainesville colony were a 14:10 light: dark photoperiod and 27Â°C with 30% R.H. The pepper weevils were raised on Jalapeno peppers for food and oviposition. Two colony boxes (31 cm x 31 cm x 31 cm, sealed plexiglass with c loth opening on one side tied) were maintained at all times and were started 1 month
67 apart. Gravid females older than 10 d old were transferred out of colony boxes into oviposition cups made from 250 mL, 8.5 cm diameter waxed cardboard cans with screened lids (The Fonda Group, Inc., Union, NJ). Females were removed from oviposition cups after 3 weeks and placed in the oldest colony box. The oldest box was discarded after 1 month. Weevils were sexed by observation of metatibial mucrones which on males ar e larger and more curved than those of females (Eller 1995). Headspace Collection and Volatile Analysis Headspace col lections were done using glass volatile collection chambers (Fig ure 3 1A) . Five female weevils , at least 10 days old, were allowed to feed on the plants for 48 h, with netting covering the top of the c hamber to allow for plant respiration (Fig ure 3 1B) . Plants were sealed into the chambers using guillotine boards and cotton swabs around the trunk of the plant (Fig ure 3 1C) . After 48 h of feedi ng, glass tops were added, replacing the netting. A ir flow was directed into absorbent cartridges filled with 50 mg Super Q, 80 100 mesh filters (Alltech , State College, PA) for collection of plant volatiles for 6 h. Vacuum valves were attached to the filters to pull air through at 0.25 L/min , while charcoal purified air was introduced via the glass top at 3 L/min . Volatiles were extracted from the column using 150 ÂµL of methylene chloride (Honeywell Burdick and Jackson, M u skegon, MI) and placed in the freezer ( 80Â°C) until analysis . For analysis, 4 ÂµL of nonanyl acetate was a dded as an internal standard at a concentration of 100 ng/ÂµL. Volatiles were analyzed using gas chromatography for quantification and gas chromatography mass spectrometry for comp arison of spectra with a spectral li brary and authentic chemicals. Volatile samples were analyzed through direct injection by GC MS in EI mode. Samples (1 ÂµL ) were injected into a methyl silicone column (HP1, 30m x 250 Âµm x 0.25 Âµm film thickness; Agilen t Technologies,
68 Santa Clara, CA). The injector and column were kept at 35 Â°C for 1 min and then temperature programmed at 10Â°C/min to 230Â°C. The He carrier gas flow rate was 1.3 mL/min (constant flow). Spectral library search was performed using a flora l scent database compiled at the Department of Chemical Ecology, GÃ¶teborg, Sweden, the Adams2 terpenoid/natural product library (Allured Corporation), and the NIST05 library. Behavioral Analysis Sesquiterpenes were selected for behavioral assay because pre vious research indicated that a preparative GC fraction containing sesquiterpenes attracted weevils in a Y tube olfactometer (McNeill unpubl. data). Compounds which occurred in every host plant were given prime consideration. Choice of sesquiterpenes too k into consideration which compounds are commonly occurring in other plant insect interactions as well as compounds which are available for purchase at an economical price. If compounds were not available for purchase, consideration was taken regarding th e ease of generating the compounds from natural or artificial sources cheaply and efficiently. The bisabolene. elemene was purchased b isabolene sample was purified via a silver nitrate column from a sample isolated at the USDA ARS in Gainesville, FL. Y tube olfactometer bioassays were conducted using two olfactometers (Analytical Research Systems, Gainesville, FL) connected with corrug ated Teflon tubing. Compressed air was purified by passing through a charcoal filter and then humidified by bubbling through deionized water before being split into two glass chambers (26 cm x 3 cm) which held the sample volatiles. The air flows were the n split into four different air flows, each regulated by a flowmeter (Manostat, New York, NY) at
69 250 ml/min, which were attached to the arms of glass Y tubes (12 cm common tube, 10 cm arms, 2.5 cm internal diameter). Th e Y horizontal inside an open top cardboard enclosure (46 cm Ã— 28 cm Ã— 42 cm) to prevent visual disturbances. Fluorescent lighting (four 85 W Sylvania cool white bulbs, 70 cm above table) was used within the bioass ay room to light the assembly. Room conditions were maintained at 25Â° 27Â°C and 40% R.H. Insects were inserted at the base of the Y tube and allowed 10 min to make a choice between the two arms of the olfactometer (one arm with a test volatile blend and on e with a solvent control) . A choice was registered when the weevil crossed over the ridge between the glass pieces connecting the Y tube to the Teflon tubing, approximately 7 cm up the arm from the common arm. Assays were conducted until 50 insects were registered as making a choice. The glass Y tubes were rotated 180Â° every five runs to account for weevil phototaxis and variation in light intensity. Volatile samples were applied to filter papers (Millipore absorbent pads, 3.7 cm in diameter, Tullagreen , Carrigtwohill, Co. Cork, IRL) cut in half. A 10 (Publix Brand, Lakeland, FL) was first placed on the disks, and then 10 ÂµL samples at various dilutions were added. The three component blends found to be moderately attractive from previous bioassays (GLV with acetophenone and GLV with 1 heptadecene) were considered for the addition of the two primary sesquiterpenes. Blends were assayed at 10,000x, 1,000x, and 100x dilutions with methylene chloride at a 1:1 ratio. Control filte r papers were trea ted with 10 ÂµL mineral oil and 10 ÂµL methylene chloride. New samples were created and tested every half hour. Mated females were
70 at least 10 d old. All insects were starved overnight , with access to water, before the assay. Data was an alyzed using a dual choice Chi Squared analysis in Excel. The expected values were calculated to indicate no difference between the treatment and control for each compound and blend tested. The P value set for significance was 0.05. Results Based on the total amount of headspace volatiles collected from each plant species, habanero and serrano were the most fragrant pepper varieties and bell was the least (Fig ure 3 2, last line of table). Jalapeno was the third least fragrant headspace collection. The sesq uiterpenes considered for behavioral analysis were compounds 15 37. The compounds highlighted in green were those that were considered after initial investigation into availability from natural sources or through purchase, as well as occurring within a ma elemene and elemene compounds that are highlighted in yellow were considered to be the same compound that was partially fractioned during GC MS analysis. Figure 3 3 A H illustrates the percentages of each compou nd in the total headspace collection of each host plant species. A majority of the compounds are green leafy volatiles with smaller percentages of the blend consisting of sesquiterpenes. The goal was to utilize this smaller subset of volatiles to improve and improve pepper weevil attraction. Figure 3 4 A H illustrates the percentages of each sesquiterpene in the total headspace collection of each potential host plant. Bell, Jalapeno, Tabasco, and Pequin each had 17 different ses quiterpenes detected in their headspace collections. Serrano contained the least with 11 different sesquiterpenes, followed by Habanero with
71 13 different compounds. The overwintering and oversummering plants of eggplant and nightshade each contained 15 d ifferent compounds detected. Trideca te traene was one of the most abundant volatiles in comparison to the other sesquiterepenes and was found in all host species but with a very low concentration in eggplant. Bell and funebrene , respectively , as their most abundant sesquiterpene; tridecatetraene was the most abundant sesquiterpene in all other host plants. Habanero contained the highest amount of tridecatetraene in its blend with 27054 Â± 11864 ng. There is no commercial source of tridecatetraene and the small amount of synthesized product at the USDA ARS CMAVE laboratory was not sufficient for bioassays. bisabolene were chosen for addition to the blend for Y tube olfactometer testing first. Elemenes and bisabolenes are commonly found in plant floral blends and also in the pheromone blends of other insects. The sesquiterpene blend of GLV, 1 heptadecene, bisabolene at 100x dilution was significantly attractive to mated females when tested in the Y tube olfactometer (Table 3 1). None of the other blends or dilutions tested was attractive. Discussion There are many difficultie s involved in developing synthetic volatile blends to assist in monitoring and trapping of agricultural pests. The first sources of error or variation in analysis of headspace plant volatiles could be the choice of appropriate collection technique and pro per handling of the plants. Some variation in headspace sampling between varieties and even between samples of the same variety could have occurred due to different amounts of plant material being inserted into the glass cylinders. In addition, no contro l extraction was made to illustrate potential background odor. If the volatile collection containers were not thoroughly cleaned, or possibly used
72 after initial cleaning without a follow up cleaning, various compounds could have influence d the GC MS analy sis and indicated fragments of compounds or compounds that would not typically have been present or in the correct quantities. A control extraction of empty PET bags was done during headspace analysis of Petasites paradoxus and Adenostyles alliariae under various feeding treatments by the leaf beetle Oreina cacaliae Schrank in order to remove this potential source of error (Kalberer et al., 2001). When incorporating sesquiterpenes into host plant volatile blends for trapping, there can be many issues wit h acquiring the proper sesquiterpenes due to cost or abundance. Oftentimes the entire success of the blend may be dependent on a select group of compounds. During analysis of the compounds attractive to the boll weevil in cotton bud essential oil, it was bisabolol was greatly caryophyllene oxide (Minyard et al. 1969). The bisabolol alone compared to 0.80 with the addition of caryophyllene oxide. Research with the emerald ash borer ( Agrilus planipennis Fairmaire) has shown that baiting with Phoebe oil will more than double the number of beetles caught in a trap compared to baiting with Manuka oil, even though the two oils d iffer only in the presence of one to two sesquiterpenes (Crook et al. 2008). F urther testing of different concentrations and ratios of the attractive green leaf volatiles and sesquiterpenes from pepper weevil host plants could yield a more attractive synth etic blend to use in baiting traps. Bell and Jalapeno are common host plants for pepper weevil in Florida, each containing 17 different sesquiterpenes, and the
73 overwintering and oversummering hosts of eggplant and nightshade contained 15 different sesquit erpenes. Differences in these four blends may be useful in identifying which compounds mated female pepper weevils are attracted to as they move into host plant fields. The addition of trideca te traene (TMTT) to the tested blends should be considered due to its presence in all of the plants analyzed and with the large concentration found in Jalapeno . TMTT is also known as a common stress compound in plants. The compound was available via the USDA ARS in Gainesville, FL; however, recommendations for starti bisabolene were made because of the cost and time involved in isolating the TMTT sample. E bergamotene is present in each host plant that was analyzed, as well as santalene, curcumene, and iso italicene. These compou nds would need to be isolated from various essential oils and purified in order to have a samp le available for bioassays. The blend created could be added to traps alone or alongside the currently available commercial pheromone lure. Minyard et al. (1969) recommended developing a lure for the boll weevil at a higher than ideal concentration to account for evaporation Experimentation on loading amounts and release rates would be necessary once an attractive blend has been formulated.
74 Table 3 1. Response of adult mated female pepper weevils to host plant volatile blends in a Y tube dual choice olfactometer when presented with a choice between the blend and purified air. Blend/dilu tion Treatment Air Chi Square P Value GLV + 1 heptadecene + elemene + bisabolene 10,000x 29 21 1.28 0.26 1,000x 30 20 2 0.16 100x 36 14 9.68 <0.05 GLV + acetophenone + elemene + bisabolene 10,000x 26 24 0.08 0.78 1,000x 21 29 1.28 0.26 100x 27 23 0.32 0.57
75 . Figure 3 1. Casey Michelle Reed. Headspace collections. April 13, 2014. Gainesville. Headspace collections wer e done using glass, volatile collection chambers (A). Collections were done with five female weevils at least 10 days old being allowed to feed on the plants for 48 h, with netting cover the top of the container to allow for plant respiration (B). Plants were sealed into the chambers using guillotine boards and cotton swabs around the trunk of the plant (C) .
76 Compound Number Compound Name Bell Jalapeno Serrano Habanero Tabasco Pequin Eggplant Nightshade 1 e hexenal 0 Â± 0 0 Â± 0 0 Â± 0 0 Â± 0 0 Â± 0 0 Â± 0 0 Â± 0 0 Â± 0 2 Z 3 hexen 1 ol 0.81 Â±0.81 12 Â± 3 459 Â± 197 1429 Â± 1287 126 Â± 31 104 Â± 72 47 Â± 30 85 Â± 52 3 Alpha pinene 60 Â± 29 51 Â± 20 2631 Â± 1328 3813 Â± 3375 407 Â± 85 532 Â± 99 201 Â± 31 689 Â± 163 4 Camphene 54 Â± 8 60 Â± 12 13204 Â± 2904 32743 Â± 28649 3782 Â± 835 4469 Â± 1118 1830 Â± 421 6949 Â± 1642 5 Beta pinene 0 Â± 0 0 Â± 0 1174 Â± 357 3525 Â± 3172 404 Â± 82 325 Â± 156 1546 Â± 1256 461 Â± 104 6 Myrcene 49 Â± 41 87Â± 56 1767 Â± 507 4335 Â± 3765 596 Â± 120 500 Â± 174 2818 Â± 2333 663 Â± 224 7 Z 3 hexenyl acetate 3 Â± 1 163Â± 74 7 9843 Â± 15078 201131 Â± 176103 22585 Â± 5298 26349 Â± 6369 13633 Â± 3870 37849 Â± 10962 8 P cumene 0 Â± 0 140 Â± 214 6978 Â± 1616 14386 Â± 12629 1699 Â± 393 1952 Â± 362 1218 Â± 213 2546 Â± 804 9 Limonen 154 Â± 15 16315Â± 15 14501 Â± 5261 26332 Â± 23033 3111 Â± 682 3283 Â± 987 1535 Â± 265 4514 Â± 1283 10 E Beta Ocimene 24583 Â± 11954 14337 Â± 6581 25567 Â± 16673 4754 Â± 3158 4213 Â± 440 937 Â± 502 530 Â± 81 437 Â± 291 11 Linalool 9 Â± 1 0 Â± 0 0 Â± 0 01576 Â± 1576 0 Â± 0 0 Â± 0 1121 Â± 1133 3609 Â± 3609 12 G terpinene 5 Â± 2 3 Â± 0.2 5576 Â± 1248 13860 Â± 12123 1590 Â± 345 1854 Â± 448 893 Â± 223 2358 Â± 674 13 Nonatriene 0 Â± 0 9914 Â± 3294 9797 Â± 6319 154529 Â± 77509 26178 Â± 3058 4602 Â± 2131 588 Â± 128 2830 Â± 1253 14 Methyl Salicylate 3 Â± 1 509 Â± 139 0 Â± 0 0 Â± 0 0 Â± 0 0 Â± 0 0 Â± 0 0 Â± 0 15 E bergam otene 5 Â± 4 324 Â± 121 17 Â± 10 606 Â± 238 131 Â± 28 92 Â± 49 4 Â± 2 6 Â± 1 16 Beta elemene 0.1 Â± 0.1 0 Â± 0 184 Â± 117 6027 Â± 2530 1344 Â± 270 936 Â± 501 43 Â± 21 76 Â± 21 17 Alpha zingiberene 32 Â± 31 462 Â± 228 0 Â± 0 0 Â± 0 23 Â± 9 0 Â± 0 0 Â± 0 0 Â± 0 18 Alpha santale ne 4 Â± 4 29 Â± 29 12 Â± 12 36 Â± 21 4804 Â± 1378 3 Â± 1 1 Â± 1 88 Â± 44 19 Beta caryophyllene 0.78 Â± 0.45 0 Â± 0 28 Â± 15 129 Â± 68 5873 Â± 1848 6 Â± 4 4 Â± 4 118 Â± 41 20 Bergamotene 2 Â± 2 66 Â± 11 0 Â± 0 0 Â± 0 0 Â± 0 0 Â± 0 0 Â± 0 0 Â± 0 21 Beta funebrene 0 Â± 0 267 Â± 90 4 Â± 2 0 Â± 0 128 Â± 57 14 Â± 9 8789 Â± 6007 0 Â± 0 22 Beta farnesene 17 Â± 9 710 Â± 145 0 Â± 0 76 Â± 63 0 Â± 0 9 Â± 6 4 Â± 4 8 Â± 8 23 Alpha caryophelene 0 Â± 0 0 Â± 0 28 Â± 21 140 Â± 85 2773 Â± 769 81 Â± 44 309 Â± 164 109 Â± 18 24 Alpha curcumene 12 Â± 6 175 Â± 63 12 Â± 12 17 Â± 10 79 Â± 12 8 Â± 5 5 Â± 3 2 Â± 1 25 Iso italicene 103 Â± 47 1465 Â± 533 74 Â± 74 184 Â± 106 2199 Â± 558 112 Â± 75 51 Â± 36 27 Â± 27 26 Zingeberene 3 Â± 1 53 Â± 24 0 Â± 0 0 Â± 0 8 Â± 8 6 Â± 4 0 Â± 0 0 Â± 0 27 Beta selinene 0 Â± 0 0 Â± 0 35 Â± 21 0 Â± 0 1256 Â± 264 1 Â± 1 1 Â± 1 22 Â± 12 28 Alpha farnesene 1 Â± 1 562 Â± 157 0 Â± 0 0 Â± 0 0 Â± 0 0 Â± 0 0 Â± 0 10 Â± 10 29 Elemene 226 Â± 101 2453 Â± 331 89 Â± 79 3233 Â± 2571 1260 Â± 337 227 Â± 154 161 Â± 58 48 Â± 24 30 Beta Bisabolene 350 Â± 165 943 Â± 118 83 Â± 102 5061 Â± 3267 1403 Â± 346 179 Â± 135 163 Â± 108 20 Â± 21 31 Z bisabolene 2 Â± 2 29 Â± 8 0 Â± 0 0 Â± 0 2 Â± 1 0 Â± 0 0 Â± 0 0 Â± 0 32 Beta sesquiphellandrene 9 Â± 5 221 Â± 76 0 Â± 0 37 Â± 25 2295 Â± 478 12 Â± 7 214 Â± 114 15 Â± 8 33 Beta cadinene 13 Â± 2 5 Â± 2 0 Â± 0 40 Â± 29 2451 Â± 519 18 Â± 10 291 Â± 164 22 Â± 6 34 Nerolidol 1 Â± 0 867 Â± 167 0 Â± 0 641 Â± 289 496 Â± 159 66 Â± 34 28 Â± 20 125 Â± 22 35 tridecatetraene 250 Â± 17 10659 Â± 1760 573 Â± 340 27054 Â± 11864 23015 Â± 4750 5213 Â± 2765 41 Â± 19 7985 Â± 1414 Total Volatiles 25955 Â± 12174 42339 Â± 11629 162645 Â± 45904 50 3997 Â± 336518 114245 Â± 13940 51902 Â± 13922 33840 Â± 8051 71685 Â± 20077 Figure 3 2. The average amount (ng) of each compound in volatile headspace collections of pepper varieties, eggplant, and nightshade hosts of pepper weevil (n=4). Compounds highlighte d in green occurred within a majority of the host plants analyzed and we elemene and elemene compounds that are highlighted in yellow were considered to be the same compound that was partially fractioned during GC MS analysis.
77 Figure 3 3 . The average percentage of each compound in volatile headspace collections of Bell pepper (n=4).
78 Figure 3 4 . The average percentage of each compound in volatile headspace collections of Jalapeno pepper (n=4) .
79 Figure 3 5 . The average percentage of each compound in volatile h eadspace collections of Serrano pepper (n=4).
80 Figure 3 6 . The average percentage of each compound in volatile headspace collections of Habanero pepper (n=4).
81 Figure 3 7 . The average percentage of each compound in volatile headspace collections of Tab asco pepper (n=4).
82 Figure 3 8 . The average percentage of each compound in volatile headspace collections of Eggplant (n=4).
83 Figure 3 9 . The average percentage of each compound in volatile headspace collections of Nightshade (n=4).
84 Figure 3 10 . Th e average percentage of each compound in volatile headspace collections of Pequin pepper (n=4)
85 Figure 3 11 . The average percentage of each sesquiterpene in volatile headspace collections of Bell pepper ( n=4 ) .
86 Figure 3 12 . The average percentage of ea ch sesquiterpene in volatile headspace collections of Jalapeno pepper (n=4).
87 Figure 3 13 . The average percentage of each sesquiterpene in volatile headspace collections of Serrano pepper (n=4).
88 Figure 3 14 . The average percentage of each sesquiterpen e in volatile headspace collections of Habanero pepper (n=4).
89 Figure 3 15 . The average percentage of each sesquiterpene in volatile headspace collections of Tabasco pepper (n=4).
90 Figure 3 16 . The average percentage of each sesquiterpene in volatile h eadspace collections of Pequin pepper (n=4).
91 Figure 3 17. The average percentage of each sesquiterpene in volatile headspace collections of Eggplant (n=4).
92 Figure 3 18 . The average percentage of each sesquiterpene in volatile headspace collections of Nightshade (n=4).
93 CHAPTER 4 CONCLUSIONS While many volatiles were shown to be EAG active, when tested alone, they were not all found to be behaviorally attractive at the concentrations tested. These compounds could potentially be attractive at different concentrations than were assayed or are effective attractants when included in blends with other compounds. Acetophenone was attractive to mated female and mated male pepper weevils when tested alone in the Y tube olfactometer assay. Mated females were also shown to be marginally attracted to a mixture of Z 3 hexenyl acetate, Z 3 hexen 1 ol, and acetophenone as well as a mixture of Z 3 hexenyl acetate, Z 3 hexen 1 ol, and 1 heptadecene when assayed in the Y t ube olfactometer . Initial wind tunnel assays showed the mixture of Z 3 hexenyl acetate and Z 3 hexen 1 ol with a pheromone lure to be more attractive that the same mixture with the addition of acetophenone in combination with a pheromone lure . In field trials, the Z 3 hexenyl acetate, Z 3 hexen 1 ol , and 1 heptadecene in combination with the pheromone lure showed to be most effective in comparison to the other blends tested. The number of weevils caught in the traps would need to be much larger in order to evaluate trap effectiveness, but the curren t data would support the use of the traps to be minor indicators of weevil movement into a field. With the exception of Wimauma, FL in the fall 2012 field trial , in which the treatment of GLV, acetophenone, and pheromone caught the most weevils, the treat ments of GLV s, 1 heptadecene, pheromone lure and the pheromone lure alone caught the most total number of weevils as well as the most male and female weevils. Over a season i t may be necessary to use various blends to monitor weevil populations. In the su mmer, when most pepper crops are dying or being plowed under,
94 female and male weevils are searching for a host for a source of food and the treatment of GLV s, 1 heptadecene, and pheromone was the most attractive. However, during the fall, when there is ty pically a healthy pepper crop growing, the pheromone lure may work better for monitoring due to weevils searching for mates and not for a limited supply of host material. It may be difficult for the host plant volatile lure to compete with the abundance o f host plants. Future field trials should utilize an entire growing season with traps being deployed two weeks or more before the first day of planting and maintained until the following growing season. There are many difficulties involved in developing s ynthetic volatile blends to assist in monitoring and trapping of agricultural pests. With further testing altering the concentrations and ratios of the attractive green leaf volatiles and sesquiterpenes from pepper weevil host plants, a synthetic blend co uld be created for addition to field traps. The method of selecting the most attractive blend could be altered. Research with the apple blossom weevil and its attractiveness to apple trees during their pre oviposition stage used an elimination method of selecting the most attractive blend (Collatz and Dorn 2013). A synthetic blend was created mimicking the composition of the silver tip stage in apple twigs and then assayed eliminating one component at a time and comparing the results to maintain previous attractiveness. In this way, a six component blend was created that maintained the attractiveness of the original twelve component blend and that also reduced the cost for using the lure in the field. This method could be employed with the pepper weevil and jalapeno headspace collection; however, since the original jalapeno headspace includes a larger number of compounds in comparison to the apple blend, an elimination procedure could take a long period of time. Additional
95 compounds to consider in the f inal attractant blend would be TMTT, due to its prominence in the Jalapeno headspace collection, E curcumene, and iso italicene, due to their presence in all host plants that were analyzed. These compounds were initially consid ered for use; however, they will need to be isolated from various essential oils and other sources and then purified. When extracting headspace volatiles, handling of plants must be consistent. It may be necessary to quantify the amount of plant material that is inserted into the glass cylinders and keep it the same for all host plant varieties. The mass of each plant sample could be analyzed and used for comparison between the different host plant varieties. Also, a control extraction should be done to determine the background odor within the cylinder. The presence of acetophenone in oviposition plugs may have an effect on the attraction of mated female weevils to host plant volatile blends which contain acetophenone. As the blend is improved with the a ddition of sesquiterpenes the presence of acetophenone may trigger the mated female weevils to gravitate towards volatiles which do not indicate the presence of oviposition plugs. This is due to mated female weevils attempting to locate oviposition sites and oviposition plugs have the potential of indicating that oviposition has already been achieved by another female weevil. The presence of acetophenone may also indicate to mated female pepper weevils the presence of fruit in which to oviposit and would therefore attract them to the volatile lure. When developing semiochemical lures it is important to consider the biological status of the insect in order to properly capitalize on the potential attractant option.
96 LIST OF REFERENCES Addesso, K.M., H.J. McA uslane, P.A. Stansly, and D.J. Schuster. 2007. Host marking by female pepper weevils, Anthonomous eugenii. Entomol. Exp. Appl. 125: 269 276. Addesso, K.M. and H.J. McAuslane. 2009. Pepper weevil attraction to volatiles from host and nonhost plants. Environ . Entomol. 38:1 9. Addesso, K.M., H.J. McAuslane, P.A. Stansly, F. Slansky, and D.J. Shuster. 2009. Artificial substrates for oviposition and larval development of the pepper weevil (Coleoptera: Curculionidae). J. Econ. Entomol. 102: 257 264. Addesso, K.M. , H.J. McAuslane, and H.T. Alborn. 2010. Attraction of pepper weevil to volatiles from damaged pepper plants. Entomol. Exp. Appl. 138: 1 11. Addesso, K.M., P.A. Stansly, B. C. Kostyk, and H.J. McAuslane. 2014. Organic treatments for control of pepper weevi l (Coleoptera: Curculionidae). Fla. Entomol. (in press). Aguilar, R. and Servin R. 2000. First record of Catolaccus hunteri , a parasitoid of Anthonomous eugenii , in Baja California Sur, Mexico. Southwest. Entomol. 25:151 152. Aguilar, R. and Servin R. 2002 . Alternate wild host of the pepper weevil, Anthonomus eugenii Cano in Baja California Sur, Mexico. Southwest. Entomol. 25: 153 154. Anderson, R.S. and S.B. Peck. 1994. Origin and biogeography of the weevils of southern Florida (Coleoptera: Curculionidae). Can. Entomol. 126: 819 839. Andrews, K.L., A. Rueda, G. Gandini, S. Evans, A. Aranga, and M. Avedillo. 1986. A supervised control programme for the pepper weevil, Anthonomous eugenii Cano, in Honduras, Central America. Trop. Pest Manage. 32:1 4. Bartlet, E., M.M. Blight, P. Lane, and I.H. Williams. 1997. The responses of the cabbage seed weevil Ceutorhynchus assimilis to volatile compounds from oilseed rape in a linear track olfactometer. Entomol. Exp. Appl. 85: 257 262. Berdegue, M., M.K. Harris, D.W. Ril ey, and B. Villalon. 1994. Host plant resistance on pepper to the pepper weevil, Anthonomous eugenii Cano. Southwest. Entomol. 19: 265 271. BichÃ£o, H., A.K. Borg Karlson, A. Wibe, J. Araujo, and H. Mustaparta. 2005. Molecular receptive ranges of olfactory receptor neurons responding selectively to terpenoids, aliphatic green leaf volatiles and aromatic compounds, in the strawberry blossom weevil Anthonomous rubi. Chemoecology 15: 211 226.
97 Boswell, V.R., S.P. Doolittle, L.M. Pultz, A.L. Taylor, L.L. Danielson, and R.E. Campbell. 1964. Pepper Production. USDA, ARS, Agricultural Information Bulletin 276. Bottenburg, H. and B. Lingren. 1998. Field performance of a new pepper weevil pheromone fo rmulation. Proc. Fla. State Hortic. Soc. 111: 48 50. Bruton, B.D. 1989. Relationships between pepper weevil and internal mold of sweet pepper. Plant Dis. 73:170 173. Burke, H.R. 1976. Bionomics of the Anthonomine weevils. Annu. Rev. Entomol. 21: 283 303. B urke, H.R. and R.E. Woodruff. 1980. The pepper weevil ( Anthonomus eugenii Cano) in Florida (Coleoptera: Curculionidae). Florida Department of Agriculture and Consumer Services, Entomology Circular DPI No. 219. Calderon Limon, B.A., J.L. Garcia Hernandez, a nd E. Troyo Dieguez. 2002. Technique for oviposition of the pepper weevil (Coleoptera: Curculionidae) to obtain massive colonies in the laboratory. Fol. Entomol. Mex. 41: 249 251. Collatz, J. and S. Dorn. 2013. A host plant derived volatile blend to attrac t the apple blossom weevil Anthonomus pomorum the essential volatiles include a repellent constituent. Pest Manag. Sci. 69: 1092 1098. Cook, S.M., Z.R. Khan, and J.A. Pickett. 2007. The use of push pull strategies in integrated pest management. Annu. Rev. Entomol. 52: 375 400. Cortez, E.M., E.D. Cabanillas, and D.B. Armenta. 2005. Parasitoides y parasitismo natural del picudo del chile Anthonomous eugenii en el norte de Sinaloa, Mexico. Southwest. Entomol. 30: 181 190. Coudriet, D.L. and A.N. Kishaba.1988. Bioassay procedure for an attractant of the pepper weevil (Coleoptera: Curculionidae). J. Econ. Entomol. 81: 1499 1502. Crook, D.J., A. Khrimian, J. A. Francese, I. Fraser, T.M. Poland, A.J. Sawyer, and V.C. Mastro. 2008. Development of a host based semio chemical lure for trapping emerald ash borer Agrilus planipennis (Coleoptera: Buprestidae). Environ. Entomol. 37: 356 365. Dickens, J.C. 1984. Olfaction in the boll weevil, Anthonomous grandis boh. (Coleoptera: Curculionidae): electroantennogram studies. J. Chem. Ecol. 10: 1759 1785. Dickens, JC. 1986. Orientation of boll weevil, Anthonomous grandis BOH. (Coleoptera: Curculionidae, to pheromone and volatile host compound in the laboratory. J. Chem. Ecol. 12:91 98. Dickens, JC. 1989. Green leaf volatiles en hance aggregation pheromone of the boll weevil, Anthonomus grandis . Entomol. Exp. Appl. 52: 191 203.
98 Eller, F.J., R.J. Bartelt, B.S. Sasha, D.J. Schuster, D.G. Riley, P.A. Stansly, T.F. Mueller, K.D. Schuler, B. Johnson, J.H. Davis, and C.A. Sutherland. 19 94. Aggregation pheromone for the pepper weevil Anthonomus eugenii Cano (Coleoptera: Curculionidae): identification of field activity. J. Chem. Ecol. 20: 1537 1555. Eller, F.J. 1995. A previously unknown sexual character for the pepper weevil (Coleoptera: Curculionidae). Fla. Entomol. 78:180 183. Elmore, J.C., A.C. Davis, and R.E. Campbell. 1934. The pepper weevil. USDA Technical Bulletin 310. (ERS) Economic Research Service. 2011. Vegetables and Melons Yearbook Data. USDA. Beltsville, MD. (ERS) Economic Re search Service. 2013. Vegetables 2012 Summary. USDA. Beltsville, MD. Fullaway, D.T., and N.L.H. Kraus. 1945. Common Insects of Hawaii. Tongg Publishing Company, Honolulu, Hawaii. Genung, W.G., and H.Y. Ozaki. 1972. Pepper weevil on the Florida East Coast. University of Florida, EREC Belle Glade Mimeo Report, EV 2. 150. Goff, C.C., and J.W. Wilson. 1937. The pepper weevil. Florida Agricultural Experimental Station Bulletin 310. Hausmann, C., J. Samietz, and S. Dorn. 2004. Visual orientation of overwintered A nthonomous pomorum (Coleoptera: Curculionidae). Environ. Entomol. 33: 1410 1415. Hedin, P.A., G.H. McKibben, E.B. Mitchell, and W.L. Johnson. 1979. Identification and field evaluation of the compounds comprising the sex pheromone of the female boll weevil. J. Chem. Ecol. 5: 617 627. Hedin, P.A., G.H. McKibben, and D.A. Dollar. 2000. Role of ground trash volatiles in the selection of hibernation sites by boll weevils. J. Agric. Food Chem. 48: 3673 3676. Hollingsworth, J.P., P.L. Wright and D.A. Lindquist. 19 64. Spectral response characteristics of the boll weevil. J. Econ. Entomol. 57: 38 41. Innocenzi, P.J., D.R. Hall, and J.V. Cross. 2001. Components of male aggregation pheromone of strawberry blossom weevil, Anthonomus rubi Herbst. (Coleoptera: Curculionid ae). J. Chem. Ecol. 27: 1203 1217. Kalberer, N.M., T.C.J. Turlings, and M. Bahier. 2001. Attraction of a leaf beetle ( Oreina cacaliae ) to damaged host plants. J. Chem. Ecol. 27: 647 661.
99 2000. Can chemical cues from blossom buds influence cultivar preference in the apple blossom weevil ( Anthonomus pomorum )? Entomol. Exp. Appl. 95:47 52. Martel J.W., A.R Alford., and J.C. Dickens 2005. Synthetic host volatiles increase efficacy of trap cro pping for management of Colorado potato beetle, Leptinotarsa decemlineata (Say). Agric. For. Entomol . 7:79 86. Martel J.W. 2006. Development of semiochemical based strategies for management of Colorado potato beetle, Leptinotarsa decemlineata (Say). Ph.D. dissertation, University of Maine, Orono. Mau, R.F., and J.L. Kessing. 1994. Extension office online publication. Anthonomus emigratella (Busck). University of Hawaii. McKibben, G.H., J.W. Smith, and W.L. McGovern. 1990. Design of an attract and kill devic e for the boll weevil (Coleoptera: Curculionidae). J. Entomol. Sci. 25: 581 586. Minyard, J.P., D.D. Hardee, R.C. Gueldner, A.C. Thompson, G. Wiygul, and P.A. Hedin. 1969. Constituents of the cotton bud; compounds attractive to the boll weevil. J. Agr. Foo d Chem. 17:1093 1097. Nalyanya, G., C.B. Moore, and C. Schal. 2000. Integration of repellents, attractants, and (Dictyoptera: Blattellidae) populations. J. Med. Entomol. 37: 427 433. (NAS S) National Agricultural Statistics Service. 2013. Agricultural Statistics 2013. Vegetables 2012 Summary. 34 35. America, Central America and the West Indies (Coleoptera: Cur culionidae). Memoir 34. Amer. Entomol. Instit. Patrock, R.J., and D.J. Schuster. 1987. Field survey for the pepper weevil, Anthonomous eugenii , on nightshade. Proc. Fla. State Hort. Soc. 100: 217 220. Patrock, R.J., and D.J. Schuster. 1992. Feeding, ovipos ition, and development of the pepper weevil ( Anthonomous eugenii Cano), on selected species of Solanaceae. Trop. Pest Manage. 38:65 69. Patrock, R.J., D.J. Schuster, and E.J. Mitchell. 1992. Field evidence for an attractant produced by the male pepper weev il (Coleoptera: Curculionidae) Fla. Entomol. 75: 138 144. Pernezny, K. and T. Kucharek. 1999. Some common diseases of pepper in Florida. Circular for the Florida Cooperative Extension Service. IFAS. 946.
100 Piskorski, R. and S. Dorn. 2010. Early season headsp ace v olatiles from apple and their effect on the apple bossom weevil Anthonomus pomorum . Chem. Biodivers. 7:2254 2260. Porter, P., B.E. Lewis, R. Scanlon, and L. Murray. 2007. Pepper weevil infestation of cv. Early jalapeno peppers of different size classe s. Southwest. Entomol. 32: 1 6. Pratt, F.C. 1907. Papers on the cotton boll weevil and related and associated insects. Notes on the pepper weevil. USDA Bureau of Entomology Bulletin 63: 55 58. Prokopy, R.J. and E.D. Owens. 1983. Visual detection of plants by herbivorous insects. Annu. Rev. Entomol. 28:337 364. Riley, D.G. and D.J. Schuster. 1992. The occurrence of Catolaccus hunteri , a parasitoid of Anthonomous eugenii , in insecticide treated bell pepper. Southwest. Entomol. 17: 71 72. Riley, D.G., and D.J. Schuster. 1994. Pepper weevil adult response to colored sticky traps in pepper fields. Southwest. Entomol. 19: 93 107. Riley, D.G., D.J. Schuster, and C.S. Barfield. 1992. Sampling and dispersion of pepper weevil (Coleoptera: Curculionidae) adults. Enviro n. Entomol. 21: 1013 1021. Rodriguez Leyva, E. 2006. Life history of Triaspis eugenii Wharton and Lopez Martinez (Hymenoptera: Braconidae) and evaluation of its potential for biological control of pepper weevil Anthonomus eugenii Cano (Coleoptera: Curculio nidae). Ph. D. dissertation. University of Florida, Gainesville. Rodriguez Leyva, E., P.A. Stansly, D.J. Schuster, and E. Bravo Mosqueda. 2007. Diversity and distribution of parasitoids of Anthonomous eugenii (Coleoptera: Curculionidae) from Mexico and pro spects for biological control. Fla. Entomol. 90: 693 702. Said, I., M. Renou, J. Morin, J. Ferreira, and D. Rochat. 2005. Interactions between acetoin, a plant volatile, and pheromone in Rhynchophorus palmarum : behavioral and olfactory neuron responses. J. Chem. Ecol. 31: 1789 1804. Santos, B.M., E.J. McAvoy, M. Ozores Hampton, P.J. Dittmar, G.E. Vallad, S.E. Webb, and S.M. Olson 2013. Pepper production, pp. 121 132. In B.M. Santos and G.E. Vallad (eds.), Vegetable Production Handbook for Florida. Institute of Food and Agricultural Sciences, Gainesville, FL. http://edis.ifas.ufl.edu/pdffiles/CV/CV29200.pdf. Schultz, P.B. and T.P. Kuhar. 2008. First record of pepper weevil infestation in Virginia. Online. Plant Health Progress doi: 10.1094/PHP 2008 0118 01 BR .
101 Schuster, D.J. 2007. Suppression of Anthonomous eugenii (Coleoptera: Curculionidae) pepper fruit infestation with releases of Catolaccus hunteri (Hymenoptera: Pteromalidae). Biocontrol Sci. Techn. 17: 345 351. Seal, D.R. and K. Bondari. 1999. Evaluation of various cultivars of pepper for resistance against pepper weevil (Coleoptera: Curculionidae). Proc. Fla. State Hort. Soc. 112: 342 345. Sparks, S., D. Riley, D. Langston, S. Culpepper, T. Kelley, A.P. Keinath, and J.P. Smith. 2007. Pest management strat egic plan for pepper in Georgia and South Carolina. In Proceedings, 2007 Tomato PMSP Workshop, 5 6 July 2007 Savannah, GA. Szendrei, Z., E. Malo, L. Stekinski, and C. Rodriguez Saona. 2009. Response of cranberry weevil (Coleoptera: Curculionidae) to host p lant volatiles. Environ. Entomol. 38: 861 869. Szendrei, Z., A. Averill, H. Alborn, and C. Rodriguez Saona. 2011. Identification and field evaluation of attractants for the cranberry weevil, Anthonomus musculus Say. J. Chem. Ecol. 37:387 397. Tinzaara, W., M. Dicke, A. Van Huis, J.J.A. Van Loon, C.S. Gold. 2003. Different bioassays for investigating orientation response of the banana weevil, Cosmopolites sordidus , show additive effects of host plant volatiles and a synthetic male produced aggregation pherom one. Entomol. Exp. Appl. 106:169 175. Toapanta, M.A., D.J. Schuster, and P.A. Stansly. 2005. Development and life history of Anthonomous eugenii (Coleopteran: Curculionidae) at constant temperatures. Environ. Entomol. 34: 999 1008. Triplehorn, C.A. and N.F of Insects (7 th edn). Thomson Brooks/Coke, Belmont, CA. Tumlinson, J.H., D.D. Hardee, R.C. Gueldner, A.C. Thompson, P.A. Hedin, and J.P. Minyard. 1969. Sex pheromones produced by male boll weev il: isolation, identification, and synthesis. Science 166:1010 1012. Vasquez, E., D. Dean, D. Schuster, and P. Van Etten. 2005. A laboratory method for rearing Catolaccus hunteri (Hymenoptera: Pteromalidae), a parasitoid of the pepper weevil (Coleoptera: C urculionidae). Fla. Entomol. 88: 191 194. Visser, J.H. and D.A. Ave. 1978. General green leaf volatiles in the olfactory orientation of the Colorado Beetle, Leptinotarsa decemlineata . Entomol. Exp.Appl. 24:538 549. Walker, C.M. 1905. Miscellaneous results of the work of the bureau of entomology. VIII. The pepper weevil. ( Anthonomus aeneotinctus Champ.). United States Department of Agricultural Bureau of Entomology Bulletin 54:43 48.
102 Watson, J.R. 1935. The pepper weevil in Florida. Bulletin 479 Agricultural Experiment Station Florida 2 pp. Webster, B., T. Bruse, J. Pickett, and J. Hardie. 2010. Volatiles functioning as host cues in a blend become nonhost cues when presented alone to the black bean aphid. Anim. Behav.79: 451 457.
103 BIOGRAPHICAL SKETCH Casey Reed was born in San Diego, CA and traveled throughout the United avy. Casey earned her Bachelor of Science degree in e ntomology and nematology at the University of the Florida with a focus on the Pre Professional track and minor in n utritional s ciences. She currently resides in Gainesville, FL and hopes to pursue a career in the private sector working with agricultural education and production.