Modification of Male Courtship Motivation by Olfactory Habituation via the GABAA Receptor in ...

21 Pages·2015·1.53 MB·English

RESEARCHARTICLE
Modification of Male Courtship Motivation
by Olfactory Habituation via the GABA
A
Receptor in
Drosophila melanogaster
Shin-IchiroTachibana1,2¤,KazushigeTouhara3,4,AkiEjima1,3,4*
1 Career-PathPromotionUnitforYoungLifeScientists,KyotoUniversity,Kyoto,606–8501,Japan,
2 DepartmentofBiologyandGeosciences,GraduateSchoolofScience,OsakaCityUniversity,Osaka,
558–8585,Japan,3 DepartmentofAppliedBiologicalChemistry,GraduateSchoolofAgriculturalandLife
Sciences,TheUniversityofTokyo,Tokyo,113–8657,Japan,4 ERATOTouharaChemosensorySignal
Project,JST,TheUniversityofTokyo,Tokyo,113–8657,Japan
¤ CurrentAddress:DepartmentofMolecularandCellularParasitology,GraduateSchoolofMedicine,
JuntendoUniversity,Tokyo,113–8421,Japan
* [email protected]
Abstract
OPENACCESS
Amale-specificcomponent,11-cis-vaccenylacetate(cVA)worksasananti-aphrodisiac
Citation:TachibanaS-I,TouharaK,EjimaA(2015) pheromoneinDrosophilamelanogaster.ThepresenceofcVAonamalesuppressesthe
ModificationofMaleCourtshipMotivationby
courtshipmotivationofothermalesandcontributestosuppressionofmale-malehomosex-
OlfactoryHabituationviatheGABA Receptorin
A
ualcourtship,whiletheabsenceofcVAonafemalestimulatesthesexualmotivationof
Drosophilamelanogaster.PLoSONE10(8):
e0135186.doi:10.1371/journal.pone.0135186 nearbymalesandenhancesthemale-femaleinteraction.However,littleisknownhowa
maledistinguishesthepresenceorabsenceofcVAonatargetflyfromeitherself-produced
Editor:FredericMarion-Poll,AgroParisTech,
FRANCE cVAorsecondhandcVAfromothermalesinthevicinity.Inthisstudy,wedemonstratethat
maleflieshavekeensensitivitytocVA;therefore,thepresenceofanothermaleinthearea
Received:April3,2015
reducescourtshiptowardafemale.Thisreducedlevelofsexualmotivation,however,could
Accepted:July18,2015
beovercomebypretestodorexposureviaolfactoryhabituationtocVA.Real-timeimaging
Published:August7,2015
ofcVA-responsivesensoryneuronsusingtheneuralactivitysensorrevealedthatprolonged
Copyright:©2015Tachibanaetal.Thisisanopen exposuretocVAdecreasedthelevelsofcVAresponsesintheprimaryolfactorycenter.
accessarticledistributedunderthetermsofthe
PharmacologicalandgeneticscreeningrevealedthatsignaltransductionviaGABA recep-
CreativeCommonsAttributionLicense,whichpermits A
torscontributedtothisolfactoryhabituation.Wealsofoundthatthehabituationexperience
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare increasedthecopulationsuccessofwild-typemalesinagroup.Incontrast,transgenic
credited. males,inwhichGABAinputinasmallsubsetoflocalneuronswasblockedbyRNAi,failed
DataAvailabilityStatement:Allrelevantdataare toacquirethesexualadvantageconferredbyhabituation.Thus,weillustrateanovelphe-
withinthepaperanditsSupportingInformationfiles. nomenoninwhicholfactoryhabituationpositivelyaffectssexualcapabilityinacompetitive
Funding:ThisworkwasfundedbyJSPS(Japan environment.
SocietyforthePromotionofScience)KAKENHI
GrantNumbers22680026,23657055,25115008to
AE,andtheERATOTouharaChemosensorySignal
ProjectfromJSTtoKTandAE(https://www.jsps.go.
jp/english/index.html;http://www.jst.go.jp/EN/index.
Introduction
html).
11-cis-vaccenylacetate(cVA)isavolatilemalepheromonecomponentofthefruitflyDrosoph-
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. ilamelanogaster.Itisamale-specificlipid,synthesizedintheejaculatoryduct[1,2],anda
PLOSONE|DOI:10.1371/journal.pone.0135186 August7,2015 1/21
OlfactoryHabituationinDrosophila
singlematuremalepossesses200ngto2μgcVAonitscuticularsurface[3,4].Thepresenceor
absenceofcVAisusedforsexdiscriminationandcontrolsreproductivebehaviorsofmales
andfemales[5].cVAisananti-aphrodisiacforamaleandinhibitscourtship[4–8],whilecVA
emittedbyacourtingmaleisanaphrodisiacforafemaleandenhancescopulationreceptivity
[8].cVAisalsoknowntotriggerintraspecificaggregation[3]andmale-maleaggression[9,
10].
Theanti-aphrodisiacfunctionofcVAnotonlycontributestosuppressingmale-malehomo-
sexualcourtship[8],butalsohelpsamalediscriminateavirginfemalefromamatedfemale.
PreviouslywefoundthatcVAistransferredtoafemaleduringcopulationwithotherseminal
fluidcomponentsandreducessexualattractivenessofthematedfemaleduetotheslight
amountofcVAthatshepresents[4].Massspectrometryanalysisofthecuticularsurfaces
revealedthattheaverageamountofcVAonasinglematedfemale24hoursaftermatingwas
9–200ng[4,6,11,12].Sinceamatedfemalewillnotacceptasecondcourtingmaleandavoids
furthercopulation,itisfeasibleforamaletojudgethepartner'smatingstatusbythepresence
orabsenceofcVAandadjusthiscourtshipactivityaccordingly.However,ithasbeenunclear
howmalefliesdistinguishpresenceorabsenceofatinyamountofcVAonthefemaletargetfly
fromothernearbymalesinacomplexolfactoryenvironmentandmakeanappropriatecourt-
shipdecision.
IninsectsincludingDrosophila,odorsareinitiallyreceivedbyodorantreceptors(ORs)
expressedonthesurfaceofolfactoryreceptorneurons(ORNs)locatedontheantennae.ORNs
expressinganygivenORprojecttheiraxonstoaspecificglomerulusintheantennallobe(AL),
theprimaryolfactorycenter.Theseaxonsformsynapticcontactswithprojectionneurons
(PNs),whichsendaxonstothemushroombody,andlocalneurons(LNs),whichmakecon-
nectionsbetweenmultipleglomeruli[13].Thusfar,twoORs,Or65aandOr67d,havebeen
identifiedascVAreceptors[14].Amongthese,Or67dfunctionwasshowntoberequiredfor
theacuteaggressionresponsetocVA,whilethechronicfunctionofOr65aneuronswascrucial
forsocialsuppressionofaggression[10].ChronicsuppressionofOr65aneuronactivity,but
notOr67dactivity,alsoresultedinanimpairedcVAresponseforcourtshipinhibition[4].
Conversely,thelackofOr67dreceptorissufficienttoblocktheacutecontrolofcourtship
behavior[8].Recently,Or65aandOr67dreceptorswerefoundtohavedistinctrequirements
foranodor-bindingproteinfortheiractivities,whichcouldbebiasedbyhighconcentrations
ofcVA[15],indicatingfunctionalsubdivisionofthesetworeceptors.
Inthisstudy,weinvestigatedhowmalesrespondtocVAinavariableolfactoryenvironment
andhowthisleadstoadaptivecourtshipdecisions.First,wefoundthatthepresenceofanother
maleorcVAintheareareducesthecourtshipmotivationofamaletowardafemale.Thisinhi-
bitioncouldbeovercomebypretestodorexposureandolfactoryhabituationtocVAormale
odor.Real-timeimagingofOr67dORNs,whichhavehighcVAsensitivity,usingtheneural
activitymarkerGCaMP,revealedthatprolongedexposuretocVAintheenvironment
decreasedthelevelsofcVAresponsesintheAL.Pharmacologicalandgeneticscreeningindi-
catedthatGABA receptorsonasmallsubsetofLNscontributedtoolfactoryhabituation.We
A
alsodiscoveredthathabituationallowedwild-typemalestogainasexualadvantageinacom-
petitivecourtshipenvironmentthatGABA mutantmalesfailedtoacquire.
A
Results
Presenceofanothermalesuppressesmalecourtship
Todeterminetheanti-aphrodisiacpotencyofcVA,weperformedabehavioralassayusinga
double-layeredchamber(Fig1A)inwhichanaïvemalehousedintheuppercellwasexposed
tovariousamountsofcVAinthelowercellunderthemeshbarrierandexaminedforits
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Fig1.MalecourtshipissuppressedbythepresenceofcVAoranothermale.(A)Schematicoftheassaysusingthedouble-layeredchamberfor
measuringmalecourtshipbehavior.Theuppercellofthechamberhousedatestmaleforodortrainingandcourtshiptestingandthelowercellbelowthe
meshbarriercontainedcVAoradecapitatedmaleasanodorsource.Forpretestodortraining,atestmalewashousedintheuppercellwithout(Mock)or
with(Trained)anothermaleflyinthelowercellfor60minutes.Itwasthentransferredtoatestchambertobetestedforthecourtshipleveltowarda
decapitatedvirginfemaleasacourtshippartnerinthepresence/absenceofcVAoradecapitatedmaleinthelowercell.Asacontrol,anunexperiencedmale
(Naïve)wastested.Fliesandthefilterpaperarenottoscaleinthecartoon.(B)CourtshipindexofmalefliesexposedtovariousamountofexogenouscVA.
cVAconcentrationof(cid:1)0.2ngsuppressedcourtshipbehaviorofthetestmales.(C)Courtshipindexunderavarietyofodorsourcesbetweenthemockand
trainedmales.Thecourtshipbehavioroftheuntrainedmockmaleswassuppressedbythepresenceof200ngcVAcomparedwiththepresenceofhexane
solvent(Sol)oradecapitatedmale(M)comparedwithnomale(-)inthelowercell.Incontrast,thetrainedmaleshowedundistinguishedlevelsofcourtship
behaviorregardlessofthepresenceofthedecapitatedmale(M)orcVAafterthemale-odorexperience.(D)Thetrainingeffectwassustainedfor20minutes
afterthetrainingbutnot30minutes.ThedatawithnointervalweredivertedfromCasreference.(E)Theodorexperiencefor30minutesledtoolfactory
habituation,while15minutesor20minuteswereinsufficienttoproducethiseffect.(F)ChronicexposuretocVAalsoledtoodorhabituation.Thetestmales
wereindividuallyrearedinatesttubecontainingaglasscapillaryfilledwith2μgofcVAin15μlhexaneor15μlhexanealonefor5days.Hexanewas
allowedtoevaporatefor2minutesbeforeintroductionintothetube.InB-F,barsindicatethemean±SEM.SignificantdifferencesaredenotedbylettersinB
andD(TukeyHSDtest).*,P<0.05;ns,notsignificantinC,E,andF(Student'st-test).
doi:10.1371/journal.pone.0135186.g001
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OlfactoryHabituationinDrosophila
behaviortowardavirginfemaleasacourtshiptargethousedintheuppercell.Wefoundthat
virginfemaleswithnooraslittleas0.02ngcVAelicitedhighlevelsofcourtshipbehaviorfrom
themales,whilethepresenceofcVAoverthemeshbarrierdecreasedthecourtshiplevelofthe
males(Fig1B).Inthepreviousstudies,150ngwasdemonstratedtobetheminimumcVAnec-
essaryforcourtshipsuppression[4,7,8,12,16–19].Inourassaysystem,0.2ngcVAwas
enoughtocauseasignificantsuppressionofcourtshipbehavior(Fig1B),demonstratingthe
keensensitivityofmalestocVA.Thepowerofsuppressionwasconsistent,asshownbynosig-
nificantdifferencesbetweencourtshiplevelsinthepresenceofvariousamountsofcVAexcept
0.02ng(P>0.05,Fig1B).WealsoconfirmedthatthepresenceofnotonlysyntheticcVAbut
alsoanothermaleasanodorsourceinthelowercellofthechamberreducedcourtshipbehav-
iorofthetestmale(Fig1Cmock).
Pretestpheromoneexperiencechangesbehavioralresponse
ThiskeensensitivitytocVAraisesthequestionabouthowamaleflydistinguishesatiny
amountofexogenouscVAonamatedfemalefromitsself-producedcVAandhowamale
maintainshighcourtshipinthepresenceofmalecompetitors.Onepotentialmechanismis
habituation,definedasadecrementofbehavioralresponsetoastimulusafterrepeatedexpo-
sures,thatallowsananimaltofilteroutconstantstimuliandfocusonnoveland/orvariant
stimuli.
TodetermineifthecVAresponsesinmalecourtshiphabituated,wepreexposedmalesto
theodorofanothermalefor60minutesandtestedtheirsubsequentcourtshipresponsesto
cVAormaleodor(Fig1A).Asopposedtothecontrolmalesthatwereleftaloneinachamber
andshowedcourtshipsuppressioninthepresenceofeithermaleodororcVA(Fig1Cmock),
thetrainedmalesthathadbeenexposedtomaleodorretainedhighcourtshipactivityregard-
lessofthepresenceofamaleorcVA(Fig1Ctrained).Thecourtshiplatencies,definedasthe
timedelaybetweenpairingandfirstcourtshipperformanceofthemale,weresignificantly
delayedincontrolmalesinthepresenceofanothermaleorcVAwhilethetrainedmalestarted
courtshipwithnodelay(S1Fig).Sincetheexperimentswereperformedunderdimredlights,a
conditioninwhichthefliescannotusevisualcues,thisimpliesthatthemalesreliedonolfac-
tiontonoticethepresenceofthetargetfemaleandinitiatecourtship.Theseresultssuggestthat
olfactoryprocessingwasaffectedbythemale-odorhabituationexperience.
Thebehavioralhabituationtomaleodorremainedatleast20minutesaftertheodortrain-
ing,butby30minutesmaleshadrecoveredfromthepre-exposureexperienceandagain
showedreducedlevelsofcourtshiptofemalespairedwithmaleodor,equivalenttothatofthe
controlmockmales(Fig1D).Weconfirmedthat30minutesoftrainingwitheithermaleodor
or200ngofcVAwasenoughtochangethebehavioralresponsestotheconcurrentpresenceof
maleodororcVA(Fig1EandS2Fig).WealsofoundthatchroniccVAexposurefor5to6
daysachievedthesameeffect(Fig1F).However,15or20minutesofexposurewasnotenough
toeffectivelymodifytheinhibitioninoursystem.
IntensityofcVAodorstimulusdeterminesOr67dORNsresponselevels
TodeterminehowcVA-sensitiveORNsrespondtovariousamountsofcVAandhowORN
responsesaredynamicallycontrolled,wefirstperformedreal-timeimagingusingtheCa+-sen-
sitivefluorescentmarker(GCaMP3.0)expressedincVA-responsiveOr67dORNs[8,20]to
examinedose-dependentneuralactivityuponcVAstimulation.TheneuralresponsesofORNs
andPNstocVAhavebeenmonitoredbyeitherelectrophysiologyorinvivoimagingusingrela-
tivelyhighconcentrationsofcVA:0.1–10%[21],1%[22],1–100%[23],0.1–100%[24],or
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OlfactoryHabituationinDrosophila
Fig2.Dose-dependentcVAresponseanddesensitizationbyprolongedstimulationinOr67dORNs.(A)Odorstimulationsystemwithinvivoimaging
ofneuralresponsesintheDA1glomeruli(seeMethodsandS3Fig).NeuralresponsestocVAweremonitoredwithafluorescentmicroscopeuponodor
stimulation.cVAwasappliedonthetoothpickofthecartridgeandanairpufffromtheairnozzledeliveredthevolatilizedcVAtotheantennaeofthefly.(B)
RepresentativefluorescentchangesevokedbyodorstimulationofvariousamountsofcVA(0to100pginthiscase)inamalefly.(C)Peakfluorescent
changesinresponsetocVAandalivemale.Peakvaluesofthetransientfluorescentchangeinresponsetoodorstimulationareshownasboxplots
representing25th,50th,and75thpercentileswithwhiskersfromminimumtomaximumandcircles(mean).Alogisticcurvewasplottedbasedonmean
valuesofthepeakfluorescentchangesineachamountofcVA(seeMethods).EC is27.5%in22.3pgcVA.Odorstimulationusingalivemaleisshownin
50
S4Fig.(D)Representativeneuralresponsesto10pgcVAoftheOr67dORNsbefore(Naïve)andaftercontinuousexposure(AfterCE)to200ngcVA(blue)
orhexanesolventcontrol(red)for15minutes.Duringthe15minutesCE,theresponseto200ngcVAwasdecreasedandthereaftertheacuteresponseto
10pgcVAalsodecreased.(E)Peakresponsesto10pgcVAbefore(Naïve),immediatelyafter(AfterCE),and30minutesafterthecontinuousexposure(30
minutesafterCE).
doi:10.1371/journal.pone.0135186.g002
1–100%[8].Intheseexperiments,thefinalodorconcentrationaftertravellingthroughthe
deliverytubeswasunclear.
Inthisstudy,wedevelopedanovelreal-timeimagingset-uptomonitorcalciumresponses
toawiderangeofodoramountspresentedinfrontoftheobjectmale(Fig2AandS3A–S3C
Fig).Thestimulantodorwasappliedonthetipoftoothpickpositioned25mminfrontofthe
subjectmale,andagentleairpuffwasdeliveredfor2secondsfromanairnozzletip5mm
fromthetoothpicktip.ResponsesweredirectlymonitoredintheDA1glomerulusofthebrain
wherethecVA-responsiveOr67dORNsprojecttheiraxons[8,25,26](S3DandS3EFig).The
intensityofGCaMPfluorescenceincreasedaftercVAdeliveryviatheairpuffandreturnedto
thebasallevelafterturningofftheairpuffwithlessthana2-seconddelay(Fig2B).Application
of10pgcVAwasenoughtoleadtoasignificantincreaseinpeakfluorescencecomparedwith
thesolvent-only0pgcontrol(Fig2C).Thelevelofneuralresponseincreasedastheamountof
appliedcVAincreasedandreachedaplateauat100pgcVA(Fig2C).Thisindicatesthatodor
strengthistransmittedastheintensityoftheneuralresponsesoftheORNsintheAL.Asaref-
erence,wealsomonitoredtheresponsetotheodorderivedfromalivingwing-amputatedmale
fixedonthetoothpick(S4Fig).Themeanfluorescencechangeuponmaleodorstimulation
was6.6%,whichiscomparabletotheintensityof0to10pgcVA(Fig2C).
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OlfactoryHabituationinDrosophila
ContinuouscVAexposurecausesdesensitizationoftheOr67dORN
response
WethenexaminedhowtheneuralresponsesofOr67dORNswereaffectedbycontinuous
exposuretocVAusingreal-timeimaging.Afterabrief10pgcVAstimulation,thesubjectfly
received200ngcVAcontinuouslyfor15minutesandwastestedforresponseto10pgcVA.
Duringcontinuousexposure(CE,Fig2D),fluorescentintensityintheDA1glomerulusgradu-
allydecreasedovertimeandfinallyreachedthebasallevel,whichwasmatchedbysolvent-only
exposureat15minutes.AfterthecontinuouscVAexposure,theintensityofacuteresponseto
10pgcVAdeclinedbyabout58%comparedwiththatobservedbeforeCEandabout59%com-
paredwiththatobservedwiththesolventcontrol(Fig2E),clearlyshowingthedesensitization
phenomenon.Thisreducedresponsestartedtorecover30minutesafterthecVAexposureter-
minatedwithabout25%increaseintheintensity(Fig2E).
GABAinputisrequiredforneuraldesensitizationofOr67dORNs
Wenextperformedpharmacologicalscreeningtoidentifytheneurotransmitter(s)involvedin
theneuraldesensitizationdrivenbycVAexposure.Asacontrolforthedrugapplicationinto
thesalinebathsurroundingtheexposedflybrain(S5AFig),weexchangedsalinealoneand
foundthatbathexchangeitselfdecreasedtheresponsesto10pgcVA(S5BFig,Dosedbefore
CE).AfterthecontinuouscVAexposure,theresponsewasfurtherdecreasedwhiletherewas
nochangedetectedafterthesolventexposure(S5BFig,AfterCE).TheamplitudeoftheCE
effect,apartfromthegeneraleffectofdosingtreatment,wascalculatedbysubtractionofthe
valueofrelativefluorescentchange"beforeCE"fromthat"afterCE"(S5Fig)ineachindividual
andsummarizedinFig3.Intheflythatreceivedbathexchangewithnodrug,the"CEeffect"
valueaftercVACE(blue)wassignificantlysmallerthanthatafterthesolventexposure(red),
demonstratingthatthecVACEledtotheneuraldesensitizationalsointhiscontrolfly.
WithintheAL,LNsaremainlyGABAergicandglutamatergic[27–31],afewarecholinergic
[32,33],andafewmorearedopaminergic[30].Conversely,ORNsandPNsarecholinergic
[34,35]andthepresenceand/orfunctionofseveralotherneurotransmittersandneuropep-
tides,e.g.,serotonin,tachykinin,andsmallneuropeptideF(sNPF),hasalsobeenindicated
[36–38].Inouractivityanalysis,theapplicationoffiveneurotransmitterantagonistsshowed
significanteffectsintheresponsetocVAafterCE(Fig3,P<0.0001;d.f.=1inCEcondition,
P<0.05;d.f.=5ininteractionwiththeapplicationofdrugsandCEconditionbytwo-way
ANOVA).Oncloserinspectionintheeffectofeachdrug,theapplicationofGABAantagonists,
picrotoxinagainsttheGABA receptor,andCGP54626againsttheGABA receptorresulted
A B
intheunaffectedcVAresponsesaftercVAexposure,indicatingtheimpairedactionoftheneu-
raldesensitization.Itshouldbenoted,however,thattheresponsechangesaftereachCEinthe
presenceofthesedrugsremainedatthesamelevelsasthoseinthecontrol(P>0.05byStu-
dent’st-testwithHolm'scorrectionwithineachCEcondition),implyingthealternativepossi-
bilitythattheresponsevariationsdrivenbytheapplicationofthesedrugs(S5CandS5DFig)
indirectlydiminishedthecVACEeffect.Therefore,weperformedthefurtheranalysisusing
genetictoolstoconfirmthefunctionofGABAintheneuraldesensitizationinthefollowing
section.Ontheotherhand,thoughtheinvolvementofglutamateanddopamineforinforma-
tionprocessingwithintheALhasbeenindicated[30,31],antagonistsagainstN-methyl-D-
aspartate(NMDA)(MK801),anddopamine(butaclamol)receptorsdidnotaffecttheneural
desensitization.Meanwhile,theinhibitionofacetylcholineinputsbymecamylaminehadthe
generalstimulatoryeffect,whichgraduallyincreasedthebasalresponselevelsoftheneurons
(S5EFig)andconsequentlyincreasedthe"CEeffect"valueinthesolvent-exposedcontrolfly
(Fig3,P<0.05comparedtothatofno-drugcontrolbyStudent’st-testwithHolm's
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OlfactoryHabituationinDrosophila
Fig3.GABAinputisrequiredforneuraldesensitizationofOr67dORNs.Effectsoffiveneurotransmitterantagonistsontheneuraldesensitizationdriven
bytheodorexposurewereexamined.TheresponsechangesaftereachCEwerecalculatedbysubtractionofvaluesofrelativefluorescentchangeof
"DosedbeforeCE"fromthat"afterCE"inindividualflies(S5B–S5GFig).Picrotoxin,CGP54626,Mecamylamine,MK801,andButaclamolwereusedas
antagonistsofGABA ,GABA ,Nicotinicacetylcholine,NMDA,anddopaminereceptors,respectively.Boxplotsrepresent25th,50th,and75thpercentiles
A B
withwhiskersfromminimumtomaximumandmeanwithcircles.Significantdifferencesaredenotedby*,P<0.05;ns,notsignificant(Student'st-test).
ExperimentalprocedureincludingthedrugapplicationisreferredinS5Fig.
doi:10.1371/journal.pone.0135186.g003
correction),whilethedesensitizationwasnotaffected(P<0.05byStudent’st-testwithHolm's
correction,comparisonbetweentwoCEconditions).Altogether,itwassuggestedthatthecVA
CEdesensitizedtheolfactoryneuronsindependentlyofactionsofglutamate,dopamineor
acetylcholine.
cVAolfactoryhabituationrequiresGABAinputsintoLNs
ToconfirmtheinvolvementofGABAintheplasticcontrolofodorsensitivityandalsodeter-
minetheolfactoryneuronsthatreceivedGABAinputfortheaction,weperformedgenetic
screeningusingRNAi.ExpressingRNAiagainsttheGABA receptorRdlunderthecontrolof
A
variousGAL4drivers,wesearchedforconditionsinwhichmaleodorsensitivitywasuninflu-
encedbyhabituation.Usingapan-neuraldriver(Fig4A),twoORNdrivers(Fig4B),fiveLN
drivers(Fig4C),andthreePNdrivers(Fig4D),theexpressionofRdlwassuppressedbyRdl-
RNAianditseffectonthecourtshipresponsesafterhabituationtrainingwasexamined.Then
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Fig4.TheGABA receptorinLNsisinvolvedinthebehavioralhabituationtocVA.Thedefectof
A
behavioralhabituationtocVAwasanalyzedbyinterferenceoftheexpressionofGABA receptorsinamajor
A
classofolfactoryneuronsusingpan-neuronal(A),ORNs(B),LNs(C),andPNs(D)drivers.Themaleflies
werealltrainedwiththemaleodorfor60minutes,andthencourtshipresponsestothefemaleswithout(-M)
orwith(+M)maleodorinthelowercellofthechamberweremeasured(seeFig1A).Barsindicatemean
±SEM.*,P<0.05;ns,notsignificantfromtheStudent'st-testwithHolm'scorrection.
doi:10.1371/journal.pone.0135186.g004
wefoundthattheRdlsuppressionbytheLNdriverMz97ledtoimpairedhabituationas
shownbythesuppressedcourtshipinthepresenceofmaleodorevenaftermaleodortraining
(P<0.05Student'st-testwithHolm'scorrection,comparisonbetweentwotestconditions;Fig
4C).TheMz97strainmarks3–4AL-associatedneuronswhosecelltypesarenotidentifiedyet
[39].ThisnullificationofhabituationtrainingwasnotfoundinmalesexpressingtheRNAi
constructswithanyotherdrivers(Fig4),indicatingthatGABAinputsactingthroughGABA
A
receptoronaspecificsubsetofLNsmarkedbyMz97playanimportantroleinolfactoryhabit-
uationtocVA.
Olfactoryhabituationprovidesasexualadvantageincompetitive
courtshipsituations
Lastly,todetermineiftherewasareproductivebenefitofmale-odorhabituation,wemeasured
copulationsuccessofmaleswithorwithouthabituationtraining.Inacompetitivesituation,in
whichatestmalewaspairedwithafemaleandanothermale,thetrainedwild-typemales
showedgreatercopulationsuccesscomparedwiththatofcontrolmales(Fig5A).Copulation
latency,whichisthedelayintimeuntilcopulationinitiation,wassignificantlysmallerforthe
trainedmale(Fig5B),suggestingthepretestexperienceprovidedthemalessomesexualadvan-
tagesinthecompetitivecourtshipsituationinwhichthemalemeetsapotentialmatingpartner
inthepresenceofasexualcompetitor.Incontrast,thetransgenicmalesinwhichGABAinput
intotheLNswasblockedbyRdl-RNAishowednodifferenceincopulationsuccessandcourt-
shiplatencyregardlessofthepreviousodorexperience(Fig5Cand5D),indicatingnosexual
benefitacquiredinthesemutantmales.Thus,thehabituationfunctioniscrucialfortheir
reproductivesuccess.
Discussion
Biologicalbenefitofolfactoryhabituation
Habituationisdefinedasdecrementofbehavioralresponsetoaniterativestimulusandiscon-
sideredasthesimplestformoflearningandmemory;itallowsanimalstofilteroutirrelevant
stimuliofdiminishingrelevanceandfocusselectivelyonimportantstimuli[40–44].
InDrosophilamelanogaster,habituationhasbeenstudiedingenetic,molecular,andneural
controlsofescape-relatedbehaviors,e.g.,jump,startle,legmovement,orescapeuponstrong
odorstimulation[28,45–47].However,thebiologicalrelevanceofhabituationfortheanimal
intermsofsurvivalandreproductiveprobabilityhasreceivedlessattention.Inthisstudy,we
demonstratedthattheolfactoryhabituationtomale-odororcVA,acourtshipinhibitorypher-
omone,allowsamaletomaintainahighlevelofsexualmotivationandthereforeincreasethe
probabilityofcopulationsuccessinacompetitiveenvironment(Fig5).Thisisthefirstreport
ofhabituationpositivelycontributingtosexualadvantageinasemi-naturalexperimental
condition.
WesuspectthatcVAhabituationprovidesbenefittothemaleinthewildsincetheamount
ofcVApresentintheenvironmentisvariable.Anewlyemergedimmaturemalecontainsno
detectablecVA,but4hourslater,smallamountsofcVA(20ng)appearandgraduallyincrease
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Fig5.Odorhabituationprovidessexualadvantage.Thenumbersofsuccessfulcopulations,copulation
latency,andtimelagbetweenpairingandcopulation,weremeasuredinthemalesofwild-type(A,B)andthe
GABA receptormutant,Mz97/Rdl-RNAi(C,D).Duringthe30-minuteobservation,thetrainedwild-type
A
malesshowedahighratioofcopulationsuccess(A)anddecreasedlatency(B)comparedwiththatofthe
controlmockmales.Conversely,theGABA receptormutantmales,inwhichGABAinputintoasubsetof
A
LNswasblockedbyRdl-RNAi,showedundistinguishedlevelsofcopulationsuccessandlatencyinthe
competitivecourtshipcondition(C,D).*,P<0.05;ns,notsignificantfromtheStudent'st-test.
doi:10.1371/journal.pone.0135186.g005
toreachasmuchas2.9μgat4weeksofage[3].TheamountofcVAonthematedfemalevaries
dependingontheintervalaftercopulation[6].Therearealsostrain-dependentdifferences
foundinwild-caughtmales[48].Undersuchvariableodorconditionsincludingitsownsmell,
plasticityincVAsensitivitycouldprovideareasonablebenefittotheanimalinmakingadap-
tivebehavioralchoices.
HabituationtocVAinfluencedmalebehavioronlyinthecontextoffemalecourtship—it
didnotincreasemale-malehomosexualcourtshipbehavior(S6Fig).Althoughthepresenceof
cVAhasbeenconsideredtocontributetosexdiscrimination,itisnottheonlyanti-aphrodisiac
factor,andother"maleness"factors,includingbehavioralpattern[49]orgustatoryinformation
[50,51],couldcompensateforthereducedlevelsofodorsensitivitycausedbyhabituation.
PLOSONE|DOI:10.1371/journal.pone.0135186 August7,2015 10/21