Associative olfactory learning in Agraulis vanillae (L.) (Lepidoptera, Nymphalidae: behavioral and physiological aspects

67 Pages·2001·2.6 MB·English

by  Kroutov, Vadim

ASSOCIATIVEOLFACTORYLEARNINGINAGRAULIS
VANILLAE(L.)(LEPIDOPTERA,NYMPHALIDAE):
BEHAVIORALANDPHYSIOLOGICALASPECTS
By
VADIMKROUTOV
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
2001
ACKNOWLEDGEMENTS
IamextremelygratefultoDr.ThomasC.Emmelforhissupportandsupervision
duringthe6yearsofmyresearchattheUF.IthankDr.M.S.Mayerfordiscussinginsect
olfactionandsensoryphysiologywithme,andforhisdirectionofthefirstpartofmy
research.IamalsogratefultoDrs.J.L.Nation,F.SlanskyandJ.F.Andersonforserving
onmysupervisorycommitteeandforallthehelpandadvicetheygaveme.
IthankDr.R.L.Reepforcollaboratingwithmeontheinvestigationofthebutterfly
brainmorphology;Dr.T.FukudaforgrantingmeaccesstohislaboratoryattheUSDA;
Dr.R.T.KennedyandJ.McKenzieforcapillaryliquidchromatographyanalysisofbrain
samples,Dr.M.E.Bittermanforreviewingthefirstpartofmythesis;Dr.V.Chewand
GalinJonesforhelpwithstatisticalanalysis;ScottWhittakerandDrs.G.Erdos,L.Green
andN.Aptsiauriforhelpingmeinmyimmunologicalventures.
ii
TABLEOFCONTENTS
page
ACKNOWLEDGMENTS
ii
ABSTRACT iv
GENERALINTRODUCTION
1
CHAPTER1:OLFACTORYCONDITIONINGOFAgraulisvanillae 3
Introduction 3
Materialsandmethods 4
Results 8
Discussion 17
CHAPTER2:EXPERIENCE-RELATEDMORPHOLOGICALANDCHEMICAL
CHANGESINTHEBRAINOFAgraulisvanillae 24
Introduction 24
Materialsandmethods 29
Results 34
Discussion 40
CONCLUSIONS 51
REFERENCES 54
BIOGRAPHICALSKETCH 60
iii
AbstractofDissertationPresentedtotheGraduateSchool
oftheUniversityofFloridainPartialFulfillmentofthe
RequirementsfortheDegreeofDoctorofPhilosophy
ASSOCIATIVEOLFACTORYLEARNINGINAGRAULIS
VANILLAE(L.)(LEPIDOPTERA,NYMPHALIDAE):
BEHAVIORALANDPHYSIOLOGICALASPECTS
By
VADIMKROUTOV
August2001
Chair:ThomasC.Emmel
MajorDepartment:EntomologyandNematology
Theassociativelearningcapacityalongwiththemorphologicalandchemicalchanges
inthebrainduringtheacquisitionofexperiencewereinvestigatedinmaleandfemale
nymphalidbutterflies,Agraulisvanillae. Bothmalesandfemaleswereconditionedto
chemicalstimuliofamylacetateandbutylacetate,butonlymaleswereconditionedto
host-plantvolatileemissions,althoughmyelectroantennogramrecordingsdemonstrate
thatbothsexesofAgrauliscandetecthost-plantaromaaswellasbothacetates. More
femalesthanmaleswereconditionedtoamylandbutylacetates. Femalebutterflies
rearedinthelaboratorygenerallyexhibitedahigherpercentageofconditionalresponses
thanthosecollectedinthefield. Thenumberofconditionalresponsesonthel"dayof
experimentswassignificantlysmallerthanontheensuing2-7days.Electroantennogram
recordingsshowedthatAgraulisbutterfliesrespondtotheodorsemittedfromthe
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abdominalglandsoftheoppositesex.Odorfromvirginalfemalesdidnotelicit
electroantennogramresponseinmales.
InthebrainofAgraulisvanillae,thesizeofneuropilinvolvedintheprocessingof
olfactoryinformationwasfoundtodependonthebutterfly'sexperience.Butterflies
collectedinnaturehaveolfactoryglomeruliandmushroombodycalycesoflarger
relativesizethandobutterfliesrearedandkeptinthelaboratoryinisolationfromnormal
environmentalstimuli.TheoppositewasfoundfortherelativevolumeoftheKenyon
cellsregioninfemales,thisregionbeingsmallerinbutterfliescollectedinthefield.No
sizedifferencewasfoundintheopticlobesorthecentralbodyineithermalesorfemales.
Immunocytochemicalexperimentsrevealedthepresenceofthefour
neurotransmitters:histamine,glutamate,serotoninandGABAinthebrainofAgraulis
vanillae.CapillaryLiquidChromatographyshowedadifferenceinthecontentofanother
neurotransmitter-candidate,aspartate,between"naive"and"experienced"femalesonly.It
alsomeasuredthecontentofneurotransmitter-candidates:GABA,glutamate,
noradrenaline,dopamine,serotonin,taurineandP-alanine.Glutamate,aspartateand
taurinewerefoundtohavethehighestconcentrationofalltheanalyzedcompounds.
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GENERALINTRODUCTION
InthisstudyIinvestigatevariousaspectsofmorphological,physiologicaland
behavioralphenomenaoflearningandsensoryinformationprocessinginthebutterfly
Agraulisvanillae,andpresenttheminthecontextofthebiologyofthisspecies.
ThebutterflyAgraulisvanillaeisacommonspeciesinFloridaandinmostofthe
USA.ItbelongstothemainlytropicalsubfamilyHeliconiinaeofthefamily
Nymphalidae.InFloridathisspecieshasseveralgenerationsayear,andadultsandlarvae
canbecollectedalmostallyeararound.Thehostplantsofthisbutterflyaredifferent
speciesofPassiflora,PassifloraincarnatabeingthemainhostplantinGainesvillearea.
TheubiquityofAgraulisandtheeasewithwhichitcanberearedinthelaboratorymake
thisspecieshighlysuitableforexperimentalpurposes.Whatmakesthisbutterfly
especiallyattractiveasanobjectofbehavioralandphysiologicalstudiesisthatitsadults
arelong-lived(3-5weeks)and,astypicaloftheHeliconiinaeingeneral,possessavery
complexbehavior.Theyhavehighlydevelopedsensoryorgans,employedincourtship
andlocationofsuitableovipositionsitesandnectarsources.
himyresearchIusedAgraulisvanillaeasamodelforthestudyofbehavioral,
physiologicalandmorphologicalprinciplesinvolvedinolfactionandlearningin
Lepidoptera.Theexistingknowledgeoflearningandmechanismsofmemoryformation
ininsectsislargelybasedontheworkonbeesandafewotherspecieswithwell-known
complexsocialcommunicationandbehavior.Toofewspecieshavebeenstudiedwith
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regardtomorphologyofthebrainregionsinvolved,orsuspectedtobeinvolved,in
processingofsensoryinformation,andtheneurophysiologicalevents,whichtakeplace
inthoseregions,tofullyunderstandtheextentofvariabilitysocharacteristicofthese
phenomenainkisecta.
IinvestigateifAgraulisvanillaeiscapableofassociativeolfactorylearning,and,
therefore,ifitcanadjustitsbehaviortochangesinitsenvironment.Theresponsetothese
changes,presentedtotheinsectintheformofchangingintensitiesofexternalstimuli,is
thefunctionofinsect'sbrain.Thisstudyreportsontheeffectofthebutterfly'sindividual
experiencewithitschangingenvironmentonthemorphologyandneurophysiologyofits
brain.
CHAPTER
1
OLFACTORYCONDITIONINGOFTHEBUTTERFLYAgraulisvanillae
Introduction
Itisdifficulttooverestimatetheimportanceofbehavioralflexibilitytoalong-lived
animal,whichhastorespondtomultiplestimuliwithinacomplexandvariable
environment.Forexample,stimuliassociatedwithfoodandreproductionmaychange
rapidlythroughoutabutterfly'slifespan.Behavioralflexibilitymayvaryconsiderably
betweendifferentspeciesandevenindividualsofthesamespecies.Onewouldexpectthe
specificreproductivesuccessofalong-livedinsectthatencountershighlyvariable
conditionsduringitslifetimetohingeuponitsindividualexperience.Asaconsequence,
theabilitytosuccessfullycopewithproblemsimposedbyachangingenvironmentcanbe
enhancedthroughassociationsbasedonexperience.Theacquisitionofnewinformation
throughindividualreiteration,leadingtoaspecificmodificationofbehavior,islearning
(Grier,1984).
Acquisitionanduseofnewinformationininsectsarewellknownfi-omstudiesof
honeybeebehavior.Learninghasbeendemonstratedbyconditioningexperimentsin
whichbeesweretrainedtoextendtheirproboscisinresponsetoapresentationofvarious
odors(Frings,1944;Kuwabara,1957;VareschiandKaissling,1970;Bittermanetal.,
1983;Menzel,1993;Bitterman,1996).However,beesarenottheonlyinsectscapableof
learning.Trainingexperimentsdemonstratethatvariousinsectscanlearn,including
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cockroaches(GatesandAllee,1933),grainbeetles(Cherkashinetal,1968),fruitflies
(Murphy,1967;DuerrandQuinn,1982),ants(Hoagland,1931;Schneirla,1941),and
wasps(Thorpe,1939;Shafir,1996).Inrecentconditioningexperiments,twonoctuid
mothspecies,HeliothisvirescensandSpodopteralittoralis,acquiredaproboscis
extensionreflextofloralodors,thusdemonstratingthecapacitytolearn(Hartlieb,1996;
Fanetal.,1997).
Theprecedingdiscussionshowsthatlearning,orbehavioralplasticity,iswidespread
intheInsecta.Forbutterfliesafewstudieshavedemonstratedthatsomespeciesuse
visualcuestoassociateahost-plant'sleaf-shapewithitschemicalconstituents{Battus
philenor[Papilionidae];Papaj,1986)andflowercolorationwithanectarreward{B.
philenor;Weiss,1997;mdAgraulisvanillae[Nymphalidae];Weiss,1995).
hithisstudyIinvestigatedthelearningcapabilityofAgraulisbymeansofclassical
conditioningexperiments,withtwofloralodorsandonehost-plantodorasconditional
stimuli.
MaterialsandMethods
Insects
MaleandfemaleAgraulisvanillaeadultsandlarvaewerecollectedintheNatural
TeachingAreaneartheDepartmentofEntomologyandNematologyoftheUniversityof
Florida,Gainesville,Florida,throughoutaperiodoftwoyears.Larvaewerefedinthe
laboratoryonPassifloraincamata(L.),which,amongotherPassifloraspecies,isthe
naturallocalhostforAgraulis.Theplantswereobtainedevery2daysfromthesamearea
wherelarvaewerecollected.AlthoughwildadultAgrauliswereofindeterminateage,
—
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onlythosehavingafreshappearancewereused.Hereafter,thesewillbereferredtoas
"wild"butterflies.Laboratory-rearedbutterflieswereusedonthe2nddayaftereclosion
andwillbereferredtoas"reared".
ThemainpartoftheconditioningexperimentswasdoneduringAugust-October,
1996and1997,duringthepeakofabundanceofAgraulisinthearea.Additional
experimentswithhost-plantodorweredoneinSeptember2000.Experimentswere
performedinthelaboratory(25°C,65%relativehumidity,L:D16h:8h).Butterflieswere
keptovernightinthesamelaboratoryroomin25X25X25cmscreencages.Theywere
givenwatereveryday.
StimulusProcedures
Threeconditionalstimuli(CS)wereused:amylacetate(AA),butylacetate(BA)
(bothchemicalsfromAldrichChemicalCompany,Inc.,Milwaukee,WI),andthehost-
plantodor.TenmicrolitersofAAand20microlitersofBAwerepipettedontoIcm^
Whatman#1filterpapers(additionalBAwasusedtooffsetthedifferenceinairborne
concentrationbetweenamylandbutylacetatesduetothedifferenceintheirvolatility).
Thehost-plantstimuluswasarolled-up6cm^pieceofPassifloraleafthatwasmacerated
withforceps.TheAA,BAorleafwasinsertedinthewideendofaPasteurpipette,the
tipofwhichwasbrokentoadiameterof6mm.Theodorwasblownovertheinsect's
antennae5timesbypressingarubberbulbattachedtothepipette.
Toaccountforpossibleinhibitionoftheproboscisextensionreflexbydeterrents
emittedbydamagedleaf,experimentswithdirectdeliveryofhost-plantodorfroman
intactplantwereperformed.Theexperimentalprotocolwasasfollows.ApottedP.
incarnataplantwasplacedinanairtightplasticbag.Atubecarryingastreamofair,