The process of pair formation mediated by substrate-borne vibrations in a small insect

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ContentslistsavailableatScienceDirect

Behavioural

Processes

j ou rn a l h o m epa ge : w w w . e l s e v i e r . c o m / l o c a t e / b e h a v p r o c

The

process

of

pair

formation

mediated

by

substrate-borne

vibrations

in

a

small

insect

Jernej

Polajnar

a,∗

_,

_Anna

_Eriksson

a,b

_,

_Marco

_Valerio

_Rossi

_Stacconi

a

_,

_Andrea

_Lucchi

b

_,

Gianfranco

Anfora

a

_,

_Meta

_{Virant-Doberlet}

c

_,

_Valerio

_Mazzoni

a

a_Research_and_Innovation_Centre,_Fondazione_Edmund_Mach,_Via_E._Mach_1,_San_Michele_all’_Adige,_Italy b_Department_of_Agriculture,_Food_and_Environment,_University_of_Pisa,_Via_del_Borghetto_80,_Pisa,_Italy c_Department_of_Entomology,_National_Institute_of_Biology,_Veˇcna_pot_111,_Ljubljana,_Slovenia

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received9April2014

Receivedinrevisedform15July2014 Accepted27July2014

Availableonline4August2014

Keywords: Scaphoideustitanus Matingbehaviour Identiﬁcation Localization Courtship Vibrationalcommunication

a

b

s

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Theabilitytoidentifyandlocateconspecificsdependsonreliabletransferofinformationbetweenemitter andreceiver.Foramajorityofplant-dwellinginsectscommunicatingwithsubstrate-bornevibrations, localizationofapotentialpartnermaybeadifficulttaskduetotheirsmallbodysizeandcomplex trans-missionpropertiesofplants.Inthepresentstudy,weusedtheleafhopperScaphoideustitanusasamodel toinvestigateduettingandmatesearchingassociatedwithpairformation.Studyingtheseinsectsona naturalsubstrate,weshowedthatthespatio-temporalstructureofavibrationalduetandtheperceived intensityofpartner’ssignalsinfluencethematingbehaviour.Identification,localizationandcourtship stageswereeachcharacterizedbyaspecificduetstructure.Inparticular,theduetstructurediffered insynchronizationbetweenmaleandfemalepulses,whichenablesidentificationofthepartner,while theswitchbetweenbehaviouralstageswasassociatedwiththemale-perceivedintensityofvibrational signals.Thissuggeststhatmalesobtaintheinformationabouttheirdistancefromthefemaleand opti-mizetheirstrategyaccordingly.Morebroadly,ourresultsshowthatevenininsectssmallerthan1cm, vibrationalsignalsprovidereliableinformationneededtofindamatingpartner.

1. Introduction

Substrate-bornevibrational signalling is an ancient

commu-nication channel that is widely used by both invertebrates

(Virant-Doberletand ˇCokl,2004;CocroftandRodriguez,2005)and

vertebrates(Hill,2008).Ininsectsalone,itisusedbyanestimated

195,000species(CocroftandRodriguez,2005),oftenexclusively,

buthassofarreceivedmuchlessattentionthanairbornesound

communication.

The ﬁrst step of mating sequences in sexually reproducing

insectsispairformationthatisachievedbyidentiﬁcationand

local-izationofapotentialpartnerinthehabitat(Alexanderetal.,1997).

Species-speciﬁcvibrationalsignalsusedinsexualcommunication

enableidentiﬁcationoftheemitterandprovidedirectional

infor-mation(e.g.Virant-Doberletetal.,2006;Hill,2008;Legendreetal.,

2012;DeGrootetal.,2012).Insomeinsectsthatrelyon

vibra-tionalcommunication,thesearchingforamatingpartnerhasbeen

describedas“trialanderror”(e.g.Gillham,1992)whileinothers,

∗ Correspondingauthor.Tel.:+390461615509. E-mailaddress:jernej.polajnar@fmach.it(J.Polajnar).

individualstravelledashorterpaththantheywouldduringpure

randomsearch(StewartandSandberg,2006;Legendreetal.,2012),

suggestingthattheyextracteddirectionalinformationfromsignals

themselves.

Plants are the most common signalling substrate for

invertebrates (Barth, 1998; ˇCokl and Virant-Doberlet, 2003;

CocroftandRodriguez,2005);however,theyarecomplex

struc-tures and due to signal degradation and frequency ﬁltering

duringtransmission(Michelsenetal.,1982;Barth,1998;Magal

et al., 2000; Cocroft et al., 2006), signals may be distorted in

thefrequencyand timedomains(Michelsenetal.,1982;Miklas

etal.,2001).Differencesinamplitudeandtimeofarrivalofthe

vibrational signal to spatially separated vibration receptors in

legsarethemostobviousdirectionalcuesthatinsects mayuse

(Virant-Doberletetal.,2006).Ininsects,mostvibrationreceptors

arelocatedinthelegs(Coklˇ etal.,2006)andthereforethesize(i.e.

maximallegspan)oftheinsectisanessentialfactorforcreating

timeoramplitudedifferenceslargeenoughtobedirectlyusedin

orientation(Virant-Doberletetal.,2006).Amplitudedifferencesat

distancesasshortas2cmarelargeenoughtobedetectedinthe

nervoussystemofinsects (Stritihetal.,2000; ˇCokletal.,2006).

Ontheotherhand,theintensitygradientonplants maynotbe

http://dx.doi.org/10.1016/j.beproc.2014.07.013

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areliablecueduetoamplitudeoscillationsofvibrationalsignals

during transmission (Michelsen et al., 1982; ˇCokl et al., 2007;

Polajnar et al., 2012) and the role of amplitude in orientation

behaviour is still under debate (Virant-Doberlet et al., 2006;

Mazzonietal., 2014).Furthermore,themajority ofinsects that

relyonvibrationalcommunicationaresmallerthan1cm.Inthis

case,derivingdirectionalcuesbydirectly comparingamplitude

ortimedifferencesbetweensensoryinputsmaynotbepossible

(Virant-Doberlet et al., 2006).Some small insects may instead

beable toextract directional information fromthe mechanical

responseof thewholebody (Cocroft etal.,2000),but solutions

havebeeninsufﬁcientlystudied.

Inthepresentwork,wedescribepairformation and

search-inginasmallplant-dwellinginsectforwhichobtainingdirectional

information may be difﬁcult. We used the Nearcticleafhopper

ScaphoideustitanusBall(Hemiptera:Cicadellidae),which

commu-nicateswithsubstrate-bornesignals(Mazzonietal.,2009a),asa

modelspecies.Thebodylengthofthisleafhopperisaround5mm,

withalegspanthatisprobablytoosmalltoenableorientationby

directcomparisonofsensoryinputs(Virant-Doberletetal.,2006).

Likeotherleafhoppers,S.titanusdoesnotrelyonchemicalsignals

(Claridge,1985;Mazzonietal.,2009a),whichallowedustofocus

onvibrationalcuesalone.

InS.titanus,themaleissearchingforthefemale,andmating

sequenceis alwaysinitiatedbythemaleemitting acalling

sig-nal(MCS)towhichthestationary femalesrespondwithpulses

emittedingapsbetweenthemalepulses(Mazzonietal.,2009a).

Asuccessfulcopulationisprecededbya male–femalecourtship

duet (CrD),which can bedisrupted by a rival male emitting a

disturbancenoise (DN)and takingover theduet. Duetting

sys-temsarecommoninarthropodcommunication(Claridge,1985;

Bailey,2003;Virant-Doberletand ˇCokl,2004),ofteninvolving

com-plexinteractionswheresignallingismodiﬁedbytheperceptionof

thepartner’sreply(DeGrootetal.,2012;Mazzonietal.,2009a;

Rodríguezetal.,2012).Insuchasystem,repliesbythe

station-ary individual(usuallythe female) provideinformation needed

for localization by the searching partner, but alsoby potential

eavesdroppingcompetitors(Bailey,2003).Therefore,amaleshould

optimizetheprocessofgatheringthenecessaryinformationfrom

thefemalesignalsinordertoreduceboththeenergeticcostsand

competition.Toachievethis,amaleshouldperform(1)accurate

identiﬁcationand (2) rapid localization, and shouldonly begin

withmore complexand demandingcourtship after thesetasks

have been accomplished. The same general principle hasbeen

recognizedin numerousotheranimals before(Alexander etal.,

1997),buttheapparentmonomodalityofsexualcommunication

inleafhoppersandtheabilitytoaccuratelymeasuresignalsusing

laservibrometryallowustoidentifythecuesthatguidebehaviour

inthesestagesandtriggertransitionsbetweenthem.

Understand-ingthisprocessmaythenshedlightontheproblemofextracting

informationfromvibrationalsignalsbysmallinsects.

Inthepresentwork,wethereforetestedthefollowing

assump-tion:duringpairformation,sexualbehaviourprogressesthrough

differentstages thatare characterizedand triggeredbyspeciﬁc

vibrationalcues,favouringreliabilityofrecognitionandspeedof

localizationbeforetheonsetofthemostcomplexadvertisingstage

ofcourtship.TheprocessisfacilitatedbytheabilityofS.titanus

malestouseinformation in femalesignals tomake directional

decisionsanddetectfemaleproximitydespitetheirsmallbodysize.

2. Materialandmethods

2.1. Insects

Rearingof S.titanus fromeggto adultfollowedthemethod

describedinErikssonetal.(2011).Allexperimentsweredonewith

virginandsexuallymaturemalesandfemalesatleast8daysafter

theiremergence(Mazzonietal.,2009a).Eachleafhopperwastested

onlyonce.

2.2. Experimentalsetup

Weusedgrapevinecuttingswithtwodifferentgeometriesas

substrate.Ineachcasethebottomofthestemwasputinaglassvial

ﬁlledwithwatertopreventwitheringandthevialwasplacedonan

anti-vibrationtable(Astels.a.s.,Ivrea,Italy).Inonecasethecutting

hadtwoleaves(surface6cm×10cm)withpetiolesseparatedby

a10-cmlongstemsection(Fig.1A),whileintheotherthecutting

hadthreeleaveswithpetiolesseparatedby5-cmlongstemsections

(Fig.1B).Forthepurposeofanalysis,thecuttingsweredividedinto

sections,eachwithameasuringpointinthemiddle.Forcuttings

withtwoleaves,thosewere:(1)basalleaf,(2)basalpetiole,(3)

stembetweenthetwoleaves,(4)apicalpetioleand(5)apicalleaf

(Fig.1A).Forcuttingswiththreeleaves,thesectionlabellingwas

equivalent,butfollowedthepositionofmaleandfemale(Fig.1B).

Thecuttingswerereplacedwithequivalentlyshapedfreshonesas

theywilted.

To prevent the insects from escaping, the setup was

con-tainedwithina clearPlexiglasscylinder(50cm×30cm).Mating

behaviourwasobservedfor20minoruntilthemalereachedthe

female,whichevercameﬁrst.Theexperimentswereperformedat

23±1◦Cbetween5pmand9pmlocaltimetoobtainhighest

sex-ualactivityfromS.titanus(Mazzonietal.,2009a),exceptintests

1.2and1.4wherethetemperaturewas28±1◦C.

Movementwasrecorded witha CanonMV1 miniDVcamera

(50FPS).Vibrationalsignalswererecordedwithalaservibrometer

(OmetronVQ-500-D-V)anddigitizedwith48kHzsamplerateand

16-bitresolution,thenstoreddirectlyontoaharddrivethrough

aLANXIdataacquisitiondevice(BrüelandKjærSound&

Vibra-tionA/S,Nærum,Denmark).Thelaserbeamwasfocusedonasmall

pieceofareﬂectivetapegluedtoeachmeasuringpoint.

2.3. Dataanalysisandterminology

Spectralandtemporalparametersoftherecordedsignalswere

analyzed withPulse 14.0 (Brüel and Kjær) after applying Fast

FourierTransform(FFT)withwindowlengthof400samplesand

66.7%overlap.Theequipmentwascalibrated,whichenableddirect

measurementsoftheactualsubstratevelocity.

Theterminologyusedfordescriptionofvibrationalsignalsin

S.titanus followsMazzoniet al.(2009a).Vibrationalsignalsnot

previouslydescribedwerelabelledaccordingtotheirbehavioural

context.

2.4. Test1.Signalparametersinvolvedinpairformation

2.4.1. Test1.1.Male–femalesynchronyinthecourseofmating

behaviour

PairformationwasstudiedusingamaleandafemaleofS.titanus

(N=20pairs),eachplacedonadifferentleafofthesamegrapevine

cuttingwithtwoleaves.Vibrationalsignalswereregisteredfrom

themeasuringpointonthelaminaofthebasalleafwiththemale

(Fig.1A,point1).

Toanalyzethesynchronyofmale–femalepulseswithin

vibra-tionalduets,wemeasuredpulserepetitiontime(=period)inmale

signalinpresenceandabsenceoffemalereply(i.e.femalepulse),

andfemalepulselatency(theintervalbetweenonsetsofthemale

and the femalepulse). Each of these parameters wasanalyzed

throughout the whole male–female communication sequence,

fromthestarting positionwhen amale wasona differentleaf

thanafemale,throughthemale’ssearchingphasetohisarrival

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Fig.1.Experimentalsetupofthetwotests.InTest1(A),thelaserwaspointedtowardsareﬂectivetapeonthemaleleaflamina(ML,point1).Theotherpointsareasfollows: (2)maleleafpetiole,(3)stem,(4)femaleleafpetiole,and(5)femaleleaflamina.InTest2.2(B),8measuringpointsweredistributedalongthegrapevinecutting:6onthe leavesand2onthestem.Theleavesweremarkedasmaleleaf(ML),femaleleaf(FL)oremptyleaf(EL)accordingtotherespectivepositionsofmaleandfemaleineachtrial. Intheexampleshown,themalewasplacedonthelowerleaf,andthefemaleontheupperone.Thenumbersthereforerepresentthefollowing:(1)maleleaflamina,(2), maleleafpetiole,(3)stemfarfromthefemaleleaf,(4)emptyleafpetiole,(5)emptyleaflamina,(6)stemnearthefemaleleaf,(7)femaleleafpetiole,and(8)femaleleaf lamina.

theperiod,wecalculatedmaleresponsephase(MRP)andfemale

latencyphase(FLP)(Greenﬁeld,1994).TheMRPwasequivalentto:

(((T₋T)/T)_×360◦),whereTandTweretheaveragepulseperiod

inmalesignalinabsenceandinpresenceoffemalepulse,

respec-tively.TheFLPwasequivalentto:((femalepulselatency/T)×360◦).

Thevalueofresponsephasedelaywas:˛=MRP/FLP.˛=1indicated

adelayofanentirepulseperiod.Aonetailpairedt-testwasused

tocomparethedifferencebetweenTandT inordertoevaluate

whetherperiodincreasedduringeachbehaviouralstage.To

deter-minewhetherfemalepulselatencyand˛valuesdifferedamong

thebehaviouralstages,weperformedtheKruskal–Wallistest

fol-lowedbytheSteel-Dwasspairwisemultiplecomparisontest,using

statisticssoftwareKyPlot5.0(KyensLabInc.,Tokyo,Japan).

2.4.2. Test1.2.Iscourtshipinitiatedbythemaleorbythefemale?

Test1.1clearlyrevealedachangeofmalesignallingbehaviour

during the approach to a stationary female (see Section 3),

detectablebytheemissionofaharmonic“buzz”thatsigniﬁes

pro-gressionfromthelocation duettothecourtshipduet (Mazzoni

etal.,2009a).

Suchabehaviouralswitchinamalemaybeelicitedbya

signiﬁ-cantchangeinperceptionofthefemaleresponse;however,theﬁrst

questioniswhetherthechangeoffemalesignalsispassive,i.e.acue

createdbytransmissionpropertiesofthesubstratealone,oractive.

Thelatterwouldimplythatthestaticfemalereactstocuesabout

distanceinmalesignalsandmakesachangeinsomeparameter

ofherreplythatwaspreviouslyoverlookedincreatingthesignal

classiﬁcationdescribedinMazzonietal.(2009a).Toanswerthis

question,werecordedfemalesignalsatthesourceandcheckedif

thefemaleemitsaconstantsignalthroughoutthewholemale

mat-ingapproachorifthereisaneffectivevariationofanyparameter

inthefemaleresponsethatcouldelicitthemaletoprogressfrom

theLocationtotheCourtshipsignal.

WerecordedmatingbehaviourofS.titanuspairs(N=18)ona

grapevinecuttingwithtwoleaves(Fig.1A),thefemalebeingalways

placedontheapicalleaf.Malepositions duringeachtrialwere

noted,andthevibratoryemissionswererecordedfromthecentre

oftheapicalleafinclosevicinity(<5cm)tothefemale(Fig.1A,

point5).Upto12femalereplieswereanalyzedpermalelocation

pertrial(totaln=202),andallthemeasurementsfromeachplant

sectionwerepooled.Thenumberofanalyzedpulseswaslimited

topreventgivinglargerweighttotrialswheremalesearchtook

longer.SectionswerethencomparedusingtheKruskal–Wallistest

followedbytheSteel-Dwasspairwisemultiplecomparisontest.

2.4.3. Test1.3Courtshipbehaviourtrigger

Test1.2showedthatthefemalepulsesremainrelatively

con-stantthroughoutthepre-copulativephaseofthematingbehaviour

(seeSection3).Thisletusformulateafurtherhypothesisthatmales

switchfromlocationtocourtshipbehaviouraccordingtoacuein

thefemalereply,relatedtotheirownperceptionofpassivesignal

changesthatindicateproximityofthefemale.Wepredictedthat

iftheswitchinbehaviournormallyoccursatacertaindistance(or

relativepositionontheplant,i.e.occurrenceonthesameleaf)from

thefemale,weshoulddetectasigniﬁcantdifferenceinany

param-eter’svaluebetweenthemeasuringpointwherethechangeﬁrst

occurredandallmeasuringpointsfurtheraway.

Totestthishypothesis,amaleandafemale(N=30)wereputon

separateleavesofthegrapevinecuttingwiththreeleaves(Fig.1B).

Themaleandthefemalewererandomlyplacedoneitherofthe

basal,middleorapicalleaf,thusobtainingsixdifferent

combina-tions,andleaveswerelabelled“femaleleaf”,“emptyleaf”and“male

leaf”accordingly;therefore,maleshadtodistinguishnotonlythe

leafwiththefemalefromtheirstarting position,butalsofrom

another,emptyleaf.

Priortothestartofeachtrial,weusedaminishaker(Type4810;

BrüelandKjærSound&VibrationA/S,Nærum,Denmark)tovibrate

theplantwithplaybackofpre-recordedMCSinordertoinitiate

matingbehaviour.Thefemalerepliedtotheplaybackand, asa

resultofsuchduet,themalerespondedwithrivalry(DNsignal).

Theplaybackwasthenstoppedtoallowthemaletoestablishaduet

withthefemale.Locationofthemaleduringeachphaseofmating

behaviourwasdeterminedfromvideorecordingstosynchronize

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F re q ue ncy [H z]

A

m

pl

it

ude [

m

/s

]

0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 0 0 .0 05 0. 01 0. 015 0. 02 0. 025

Fig.2.Frequencyspectrumofa“typical”femalesignalwithfrequencyrangesusedforanalysis(delimitedbygreylines).Thelowestrange0–40Hzcompriseslow-frequency externalnoise,andwasexcludedfromanalysis.

Since females remainstationary(Mazzoniet al.,2009a), the

amplitudeoftheirvibrationalsignals,measuredasvibrational

sub-stratevelocity(␮m/s)anditsspectralcomponents(Hz,frequency

ranges), couldbemeasureddirectly at8 measuringpoints

dis-tributedonthegrapevinecuttingbymovingthelaserbeamduring

malesearch(Fig.1B).Thegoalwastorecordthesignalsatallthe

measuringpointsduringeachtrial,sowedidnotnecessarilyfollow

themale’smovementwiththelaserbeam.

Wecalculated signalamplitudebyaveragingallthespectral

componentswithintherange40–250Hz where themajorityof

acoustic energy was concentrated (21 frequency bins at given

FFTsettings;Fig.2).Thismeasure,whileabsolute,shouldstillbe

regardedasaproxyforthe“true”amplitudebecauseitisnotyet

certainwhichpropertyofabroad-bandsignaltheanimals

actu-allyrespondto.Additionally, we tested whetheranyparticular

frequencycomponentofasignal,includinglow-amplitudeones,

mightalso contributetodetection,by splittingthesignal’s

fre-quencyspectrumto10rangeswithonedominantorsubdominant

spectralpeakeach,rangingupto1kHz(Fig.2).Peakvelocityvalue

withineach ofthosefrequencyrangeswasaveragedfromthree

femalepulsespermeasuringpointpertrial.Totestforstatistical

differencesofthefemalepulseamplitudesandfrequencyranges

betweenthemeasuringpoints,weperformedtheKruskal–Wallis

testfollowedbytheSteel-Dwassmultiplecomparisontest.Finally,

withthehelpofthevideos,wetooknoteofthepositiononthe

plant(Fig.1B)wheretheswitchofmalebehaviourfromlocation

tocourtshipoccurred.Thus,weassociatedthespectralanalysisof

thefemalepulseateachmeasuringpointwiththeoccurrenceof

behaviouralswitchesontheplant.

2.4.4. Test1.4.Conﬁrmationofcourtshipbehaviourtriggerwith

playback

TofurtherconﬁrmtheresultsofTest1.3,weperformedan

addi-tionalplaybacktest.Weusedplaybacktoreducevariabilitycaused

bythemale–femaleinteraction,whichenabledustostudy

trans-missionalone.

ThecuttingforthistestwasthesameasforTest1.2(Fig.1A).

Again,thefemalewasalwaysplacedontheapicalleaf.A

represen-tativemalecallingsignal(withintherangeofvariabilitydescribed

inMazzonietal.,2009a)wasplayedbacktofemales(N=16)using

astaticloudspeakerplacedparalleltotheemptybasalleaf,atthe

distanceof3cm. Theamplitudeofresultingvibrationsnearthe

femalewasadjustedbeforehandtoalevelnaturallyexperienced

byfemaleswhenlisteningtoamalesignallingfromadifferentleaf,

asdeterminedfromrecordingsmadeinTest1.2.Aloudspeakerwas

usedforstimulationinordernottoinﬂuencesignaltransmission

nearthepointofexcitation.Eachfemale’sresponseswererecorded

byalaservibrometeratalltherecordingpoints.

Weexaminedresponselatencyandtwospectralparameters:

peakamplitude,andfrequencyofthedominantspectralpeakin

eachsignal’sfrequencyspectrum.Onlyrelativepeakamplitudein

dBwasconsideredbecausetheabsolutevalueswereofless

impor-tanceforthepurposeofthistest.Valueswerecomparedbetween

plantsectionswiththeKruskal–Wallistestfollowedbythe

Steel-Dwasspairwisemultiplecomparisontest.

2.5. Test2.DirectionalityinS.titanus

Totestthehypothesisthatmalesmake directionaldecisions

onthebasisoffemalereply,videorecordingstakenfromTest1.1

werealsoanalyzedandthemale’sdirectionalchoicesannotated.

Threeparameters were evaluated.First, we annotated whether

themale started towalktowards thefemale(1), if yes,it was

recordedasarightdecision,ifnot,asawrongdecision.Whenmales

reversedthedirection(2),itwasannotatedasawrongdecisionif

itturnedawayfromthefemaleandrightifturnedtowardsher.

Whenmalesreachedafork betweenstem andleaftherightor

wrongdecisionsatthebranchingpoint(3)werealsoannotated.

Toevaluateifmaleswereabletomakedirectionaldecisionsother

thanbychance,thenumbersofrightandwrongdecisionswere

compared inaone-tailedt-testfordependentsamplesforeach

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Fig.3.FlowchartwiththebehaviouralstepsofmaleScaphoideustitanuswhen searchingforafemaleonagrapevineplant.MCS,malecallingsignal;DN, disturb-ancenoise;IdD,identiﬁcationduet;LoD,localizationduet;CrD,courtshipduet;♀ rep,femalereply;Rec,recognition;♀leaf,arrivalatthefemaleleaf;Rdir,right decision;Inv,inversionofdirection.

3. Results

3.1. Test1.PairformationinS.titanus

3.1.1. Test1.1.Male–femalesynchronyinthecourseofmating

behaviour

ThemainstepsofthematingbehaviourofS.titanusare

summa-rizedinFig.3.AsdescribedpreviouslyinMazzonietal.(2009a),in

alltrials(N=20)malesinitiatedvibrationalcommunicationwith

emissionofamalecallingsignal(MCS).Whenfemaleswerenot

responding(N=4),maleseitherremainedstationaryorexhibited

“call-ﬂy”behaviour.Whenfemalesresponded(N=16),mostmales

(N=13)emittedpulsetrainswithanirregularrhythmandwith

anincreasedpulseperiod(Table1).Thecalculatedmaleresponse

phasedelay(˛)was0.85(Table1),whichindicatesthatfemale

responseresetstheemissionofmalepulseforalmostacomplete

pulseperiod.Such delayedexchangeof maleandfemalepulses

wastermedidentiﬁcationduet(IdD,Fig.4A)and wasobserved

onlywhenamaleandafemalewereplacedonseparateleaves.

DuringIdD, maleswalkedrandomlyontheleaf.Seven females

alsoemittedshortpulsetrains(m±SD=3.03±1.28pulses,

dura-tion=0.89±0.61s,n=31)inreplytothemalesignal.Asaresult,

maleseitherwalkedrandomlyand calledagain,oremitted

dis-turbancesignals(DN)(Mazzonietal.,2009a).

FollowingIdD,males(N=13)movedtowardsthepetioleand

walkedtostemandtowardstheleafhostingthefemale.In this

stagefemalereplyhadasmallbutsigniﬁcanteffectonthepulse

periodinmalesignal(˛=0.23)(Table1).Thisphaseofmale–female

vibrationalinteractionwasnamedlocalizationduet(LoD;Fig.4B)

Fig.4. Oscillogramof(A)identificationduet(IdD),(B)localizationduet(LoD)and(C) courtshipduet(CrD).Recordingsweremadewiththelaseronthelowerleafhosting themale.(A)Malecallingsignal(MCS)thatprogressesintoanidentificationduet (IdD),(B)twosections(S1andS2)formingthelocalizationduetwithnoisecaused bythemalewalkingfollowingtheduet.(C)S1andS2ofamalecourtshipphrase thattogetherwithfemalepulsesconstituteCrD.Asterisks(*)indicatefemalepulses; opencircles()malepulses;filledcircles(䊉)theS2singlemalepulseofsection two;hashes(#)malepulseoftypetwo(Mazzonietal.,2009a).Themalepulseperiod isTandT_,_in_absence_or_presence_of_the_female_reply,_{respectively.}

and wasrecorded fromthebeginning of thedirectionalsearch

untilreachingthefemaleleaf.LoDwascomposedoftwosections

repeatedcontinuously. In section 1,males werestationary and

emittedshortseries ofpulses. Insection 2,maleswalkedfor a

fewcentimetresbeforestoppingandoftenemittedasinglestrong

pulse.Femalesweresometimesobservedtoemitmultiplepulse

trains(n=6)(3.6±1.39pulses, duration=1.09±0.62s)afterthe

lastmale pulse.Themale behaviouralresponsetothemultiple

femaletrainswaseitheradirectionalsearchfollowedbyanother

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Table1

˛(maleresponsephasedelay)valuesfromScaphoideustitanusidentiﬁcation,localization(section1)andcourtshipduet(section1andsection2).

n N T/T(s) Flatency(s) ˛ Identiﬁcationduet 10 7 0.43±0.02/0.68±0.04 *** (t=22.4,df=9) 0.30±0.03 (25.5)b 0.85±0.17 (34.7)b

Localizationduet(section1) 10 7 0.44±0.01/0.50±0.07 ** (t=3.61,df=9) 0.29±0.01 (24.6)b 0.23±0.27 (12.7)a CrDduet(section1) 10 7 0.42±0.04/0.50±0.05 *** (t=6.4,df=9) 0.23±0.01 (9.1)a 0.37±0.22 (19.7)a CrDduet(section2) 10 7 0.62±0.06/0.69±0.08 *** (t=6.9,df=9) 0.29±0.01 (22.8)ab 0.26±0.11 (14.9)a

MRP:maleresponsephase;FLP:femalelatencyphase;T,T:averagepulseperiodinmalesignalinabsenceandinpresenceoffemalepulse,respectively;IdD:identiﬁcation duet;LoD:locationduet;CrD:courtshipduet.Meanvalues±SDofT,T,femalelatency˛obtainedfromMRPandFLPofIdDandLoDrecordedfrommalesandfemaleson differentleavesaswellasfromCrD(S1andS2)ofmalesandfemalesonthesameleaf.Thenumberofinsectpairstestedforeachsignal(n)andthenumberofanalyzedsignals perpair(N)areshown.DifferentlettersinthesamecolumnindicatesigniﬁcantdifferenceafterKruskal–Wallistest(rankmeanisgiveninbrackets;Flatency:X2₌_13.0,

df=3,P<0.01;˛:X2₌_21.5,_df₌_3,_P_<_0.001),_followed_by_Steel-Dwass_pairwise_comparison_test. **_P_<_0.01_after_one_tailed_paired_t-test.

***_P_<_0.001_after_one_tailed_paired_t-test.

communicationwithanIdD(N=4),however,inthelattercases,the

re-identiﬁcationwaslimitedtoexchangeofafewpulsesbetween

maleandfemale–characterizedby˛valuecloseto1–that

imme-diatelyprogressedintoaLoD.ThedurationsofIdDandLoDwere

similar(Table2).

Whenthemalearrivedattheleafhostingthefemale,courtship

duet(CrD)wasestablished(N=13)(Fig.4C).During CrD,males

emittedpulsesataregularrhythmandfemalereplyhadagaina

smalleffectonthepulseperiodinthemalesignal.Thephasedelays

duringtwosectionsoftheCrD weresimilartoonedetermined

forLoDandsigniﬁcantlylowerthaninIdD(Kruskal–Wallistest:

X2₌_13.0,_df₌_3,_P_<_0.01)₍_Table₁_)._The_female_pulse_latency_was

constantthroughoutallstages ofmale–female vibrational

inter-action, withvalues signiﬁcantlylower only in section1 of CrD

(Kruskal–Wallis:X2₌_21.5,_df₌_3,_P_<_0.001).

3.1.2. Test1.2.Iscourtshipinitiatedbythemaleorbythefemale?

Measurements from the apical leaf revealed no consistent

changesinfemalereplyduringthecourseofthemaleapproach.

Outoftheinitial18pairstested,N=18setsofmeasurementswere

obtainedwiththemale callingfromthebasal leaf(initialmale

position),N=6fromthebasalpetiole,N=10fromthestem,N=12

fromtheapical petiole,and N=10 fromthe apicalleaf.Allthe

malesswitchedtoCrDafterreachingtheapicalleaflamina,atthe

distanceoflessthan10cmfromthefemale.Asigniﬁcant

differ-enceinthedominantfrequencyoffemalesignalswasmeasured

onlywhenmaleswerecallingfromthebasalpetioleandthe

api-calleaf(Kruskal–Wallis:X2₌_13.9, _df₌_4,_P₌_0.0078)._Conversely,

femaledominantpeak amplitudewassigniﬁcantlyhigherwhen

maleswerecalling fromtheapical petiolethanfrom thebasal

leaf(Kruskal–Wallis:X2₌_14.0,_df₌_4,_P₌_0.007)._Female_response

latencydecreasedfrom210±23ms(maleonthebasalleaf,n=80)

to182±19ms(maleonthestem,n=22),thenremainedstable,

with184±26ms(n=37)whenthemalereachedtheapicalleaf

(Kruskal–Wallis: X2₌_44.2, _df₌_4, _P_<_0.001), _however, _the

Steel-Dwasstestonlyshowedsigniﬁcantdifferencebetweenthebasal

leafandthethreesectionsclosesttothefemale,i.e.stem,apical

petiole,andapicalleaf(Steel-Dwass,P<0.001).

3.1.3. Test1.3.Courtshipbehaviourtrigger

Becausefemaleresponselatencychangedonlyafterthestartof

CrDinTest1.1andbecausewedidnotmeasureanychangesin

femalereplyconsistentwithbehaviourinTest1.2,wefocusedon

spectralparametersandamplitudeoffemalesignalsasperceived

bythemale.

Mostenergyofthefemalesignalwasconcentratedinthe

cho-senfrequencyrange40–250Hz.Amplitude(expressedasaverage

velocity of spectral bins withinthis range) of thefemale reply

perceivedbythemalealongthegrapevinecuttingissummarized

inFig.5.Therewasnostatisticaldifferenceinmeasuredamplitude

offemalesignalsbetweenmalelamina,malepetiole,empty

lam-ina,emptypetioleandstem,whereastheamplitudeleveloffemale

pulsewassigniﬁcantlyincreasedwhenperceivedatthefemaleleaf

petioleandfemaleleaflamina(Kruskal–Wallistest:X2₌_400,_df₌_6,

P<0.001;Fig.5,Table3).Twenty-ﬁveoutof27 courtshipduets

startedonthesectionoftheplantclosesttothefemale,most

com-monlyonthepetiole.Intheothertwocasesthecourtshipduet

startedonthestem,butitwasalwaysnearthefemaleleafpetiole.

Nodifferenceindominantfrequencywasfoundbetween

sec-tions (Kruskal–Wallis test: X2₌_9.72, _df₌_6, _P₌_0.137; _Table ₃_).

Whenpeakfrequencieswithinindividualfrequencyranges(Fig.2)

ofthefemalereplywerecompared,uptothreerecordingpoints

differed signiﬁcantly from the rest (Kruskal–Wallis test: df=6,

P<0.05;Table3),nevertheless,differenceswereneverrelatedto

Table2

DescriptivestatisticsondurationofScaphoideustitanusidentiﬁcationandlocalizationduets. Duration(s)

IdD(singletrain) LoD(section1) Identiﬁcation(wholestage) Localization(wholestage)

Mean±SD 11.46±7.86 3.74±1.56 106.36±70.98 83.45±108.97

Max 32.96 6.92 236.96 393.11

Min 1.96 1.28 21.15 4.14

N 16 15 15 16

Mean±SD,maxandminvaluesofdurationareshownbothforsingletrainsandwholelengthofidentiﬁcationduet(IdD)andlocalizationduet(LoD,section1).Nindicates thenumberofinsectpairsanalyzed.

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ma le petiole n = 7 2 ma le le af n = 6 9 em pty lea f n = 7 5 em pty pe tio le n = 7 5 s te m n = 7 8 fe ma le p etio le n = 7 8 fe ma le le af n = 6 7 ap ic a l pe tiole n = 4 5 ap ic a l lea f n = 4 5

thre e -le a f se tup (Te st 1 .3 ) tw o-le af s e tup (Te st 1 .4 ) 0 0.1 0.2 0 .3 0 .4 0.5 A ve ra g e v e lo c ity [ m m /s ] d *** NS c b a a a a ***

Fig.5.Box-plotsofaveragespectralvelocityvaluesintherange40–250Hz,measuredfromfemalesignalsonbothkindsofexperimentalsubstrates.Differentlettersfor valuesinthethree-leafsetup(Fig.1B,Test2.2)indicatestatisticalsigniﬁcanceafterKruskal–WallistestfollowedbySteel-Dwassmultiplecomparisontest.Sectionwhere theswitchfrommalelocalizationtocourtshipbehaviouroccurredismarkedwithgrey(seealsoFig.4).Valuesthatcorrespondtothebehaviouralswitchmatchthevalues obtainedonatwo-leafsetup(Fig.1A,Test2.1),alsocorrespondingtothebehaviouralswitch,butatadifferentsection(Mann–WhitneytestwithBonferronicorrection,see textfordetails).

Table3

Spectralpropertiesoffemalerepliesasperceivedbythemaleatdifferentsectionsonthecutting(Fig.1B).

Malepetiole Maleleaf(lamina) Emptypetiole Emptyleaf(lamina) Stem Femalepetiole Femaleleaf(lamina)

Dominantfrequency(Hz) 105±49 106±44 95±42 102±42 107±57 109±52 122±52

Avg.velocitywithin40–250Hz(␮m/s) 3.5±2.5 3.7±3.9 3.3±2.9 3.0±2.5 5.6±4.3 9.6±7.6 17.1±11.7 Peakfrequencieswithinspectralranges(Hz)(Fig.5)

40–100Hz 71±20 75±20 68±19 73±19 69±21 73±20 80±23 110–150Hz 137±15 133±14 140±16 139±14 134±16 138±17 134±14 160–200Hz 184±14 190±17 182±15 182±15 183±14 177±10 181±11 210–250Hz 229±14 235±16 228±13 236±15 233±14 232±14 236±16 260–300Hz 283±15 282±15 288±15 286±15 282±15 284±16 283±15 310–350Hz 336±16 333±14 335±16 334±17 336±15 334±15 330±13 360–400Hz 387±16 388±17 387±17 387±16 387±15 388±16 385±17 410–550Hz 488±50 473±54 466±49 451±43 468±49 475±57 459±48 560–700Hz 614±41 632±41 627±46 638±40 619±41 615±43 610±36 710–1000Hz 815±86 805±83 814±79 824±81 824±88 776±76 804±81 Peakvelocitieswithinspectralranges(␮m/s)(Fig.5)

40–100Hz 8.8±8.6 9.2±9.4 8.7±8.5 7.2±6.0 16.2±15.9 23.9±18.1 33.3±20.5 110–150Hz 7.6±11.0 6.2±4.9 6.2±6.5 6.7±7.0 10.5±9.0 19.2±17.0 35.3±32.9 160–200Hz 5.1±7.5 4.0±2.9 4.3±4.0 3.2±3.5 7.5±7.5 12.7±11.8 27.4±30.5 210–250Hz 2.9±5.1 2.0±2.2 1.5±1.6 1.4±1.9 3.4±4.2 4.2±4.0 11.1±12.5 260–300Hz 1.5±2.1 1.0±1.1 0.9±0.8 1.0±1.1 1.6±1.5 2.4±2.8 6.8±7.1 310–350Hz 0.6±0.6 0.7±0.9 0.6±0.6 0.5±0.7 1.0±1.3 1.4±1.3 4.6±4.8 360–400Hz 0.5±0.4 0.4±0.3 0.4±0.4 0.5±0.9 0.9±1.7 1.1±1.2 3.0±4.4 410–550Hz 0.4±0.5 0.7±0.8 0.4±0.6 0.4±0.7 0.9±1.7 1.0±0.9 2.1±2.6 560–700Hz 0.3±0.3 0.5±0.6 0.3±0.4 0.2±0.3 0.5±0.7 0.7±0.8 1.2±1.7 710–1000Hz 0.2±0.2 0.3±0.3 0.2±0.4 0.2±0.2 0.4±0.6 0.4±0.5 0.6±0.9 n 72 69 75 75 78 78 70

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thedistancefromthefemale.Ontheamplitudeaxis,peak

ampli-tudewithin thefollowing individual frequency rangeschanged

consistentlywiththedistancefromthefemale(i.e.signiﬁcant

dif-ferencebetweenthefemalepetioleandthestemsectionclosest

to the female leaf, sections further away with lower

ampli-tude): 110–150Hz, 160–200Hz, 310–350Hz, 360–400Hz, and

410–550Hz(Kruskal–Wallistest:df=6,P<0.05;Table3).

3.1.4. Test1.4.Conﬁrmationofcourtshipbehaviourtriggerwith

playback

Measurementsoffemalerepliesrecordedfromdifferentpoints

ontheplantrevealedchangescausedbytransmissionalongthe

substrate.Thestimuluselicitedrepliesbyeachfemale(N=16),so

weanalyzedallthefemalesignals(exceptthefemalepulsetrains)

fromalltherecordingpointsineachtrial(totaln=643).The

base-linelatencyvalue,asmeasuredontheapicalleafclosesttothe

female(216±38ms,n=147),didnotincreasesigniﬁcantlyuntilthe

basalleafat232_±37ms,n=147(Steel-Dwass,P<0.001;Fig.6A).A

small,butstatisticallysigniﬁcant,differenceoccurredonlybetween

theapicalpetioleand thestem (Steel-Dwass,P<0.05).Relative

amplitudeofthedominantpeakdecreasedconsistently,withallthe

differencesbetweenpointssigniﬁcant(Kruskal–Wallis,X2₌_325.4,

df=4,P<0.001;Fig.6B),exceptbetweenthestemandthebasal

petiole,andbetweenthebasal petioleandthebasalleaf

(Steel-Dwass,P=NS).Dominantfrequencydifferedsigniﬁcantlybetween

recordingpoints(Kruskal–Wallis,X2₌_99.6,_df₌_4,_P_<_0.001),_but_no

consistentchangesrelatedtothedistancefromtheemitterwas

observed: frequenciesat basal petiole,apicalpetioleandapical

leafwerenotstatisticallydifferent,butallwerehigherthanthe

frequencyatthebasalleaf,whichwasinturnhigherthanthe

fre-quencyatthestem(Steel-Dwass,P<0.01;Fig.6C).

Tovalidatetheaveragesubstratevelocityatwhichtheswitch

inbehaviourwasobservedinTest1.3,wecombinedbehavioural

datafromTest1.2andrecordingsfromTest1.4,bothobtainedon

acuttingwithtwoleaves(Fig.1A).Forthispurpose, the

ampli-tudewasexpressedastheaveragevelocityinthefrequencyrange

40–250Hz,thesameasinTest1.3,andcomparedbetweentests

usingtheMann–WhitneytestwithBonferronicorrection.Average

velocityincreasedfrom0.049±0.037(n=45)atfemalepetioleto

0.124±0.101(n=45)atfemaleleaflamina(Mann–Whitneytest,

W=321,P<0.001)(Fig.5).Averagevelocityatthefemaleleaf

lam-inaon thecuttingwith two leaveswas notdifferent fromthe

averagevelocityatthefemalepetioleonthecuttingwiththree

leaves(Mann–Whitneytest,W=1416,P=0.08),whileitwas

sig-niﬁcantlyhigherthanatthestemonthecuttingwiththreeleaves

(Mann–Whitneytest,W=781,P<0.001)(Fig.5).Measured

differ-encesbetweenplantsectionsforotheranalyzedparametersdid

notcorrespondtothebehaviouralswitchinTest1.2.

3.2. Test2.Malesareabletomakedirectionaldecisions

Thenumberofrightorwrongdirectionaldecisionsmadeby

malesmovingtowardsafemaleafterafemaleresponseisshown

inFig.7.Signiﬁcantlymoredecisionsweretowardsthefemale

(t-test:t=12.72,df=27,P<0.001)andwhenreversingthedirection,

signiﬁcantlymoremalesmadeacorrectratherthanwrong

direc-tionaldecision(t-test:t=4.72,df=27,P<0.001).Ontheotherhand,

nodifferencebetweencorrectandwrongdirectionaldecisionswas

observedatbranchingpoints(t-test:t=1.43, df=27,P=0.32).A

male thatturned inthewrong directionmade onaveragetwo

(1.8±1.4)additionalmovesinthisdirectionbeforeturningaround.

4. Discussion

PairformationinS.titanusstartswithidentiﬁcationofthe

mat-ingpartnerandcontinueswitha localizationstageuntila ﬁnal

courtshipstagebeforecopulation.Ingeneral,signalsshouldﬁrst

informthereceiveraboutthesender’sidentity(who?),thenthe

quality(what?)andthelocation(where?)(Pollack,2000).

4.1. Male–femalesynchrony

OurresultsindicatethattheﬁrstactofpairformationinS.titanus

ismaleidentiﬁcationofaconspeciﬁcfemalethroughastrict

syn-chronizationwithhisownpulses.Femalereplyhastoarrivewithin

aspeciﬁctime window,likeinseveralotherleafhopperspecies

(Nuhardiyatiand Bailey,2005;Derlinket al.,2014),and should

notoverlapwiththenextmalepulse,becauseoverlappingwould

bemistakenbybothpartnersforadisruptivesignalemittedbya

rivalmale(Mazzonietal.,2009b);however,whileitwas

previ-ouslythoughtthatfemalepulsesareemittedonlyin-betweenthe

malepulses(Mazzonietal.,2009a),wealsofoundinthepresent

studythatfemalepulsesmaybeemittedaspulsetrains,mostoften

afterthelastmalepulse.Suchmultiplerepliesoccurredwhenthe

malewasidentifyingorlocatingthefemalefromdistantplantparts,

whichmayrepresentfemaleadaptationforincreasing

detectabil-ityand/ortraceability(i.e.serialredundancy).Malecallingsong

pulsesareemittedwithregularrhythm,indicatingthattheyare

generatedbyanendogenousoscillator.Femalesdonotreplytoall

malepulses(Mazzonietal.,2009a),suggestingthattheylistenout

foreachmalepulseandreply(ornot)toit.

Resettingofthemaleendogenousoscillatorbythecentral

ner-voussystemiscomparabletosignalinteractionsamongchorusing

males(Greenﬁeld,1994,2005);however,S.titanusmalesdonot

formchoruses,engaginginsteadinrivalbehaviourifacompeting

maleisdetectedinthevicinity(Mazzonietal.,2009b).Thechange

inrhythmcouldinthiscasehelpthemaletodistinguishthefemale

signalfromconspeciﬁcmaleemissionsintheﬁrststageofmating

behaviour,whenrecognitionhasnotyetbeenachieved.Without

achangeinrhythm,twomalesemittingMCSoutofphasemight

mistakeeachotherforaconspeciﬁcfemale,whileifareply

trigg-ersprolongationofthepulseperiod,overlappingwillnecessarily

ensue.

4.2. DirectionalityinS.titanus

Theeffectoffemalereplyonmalepulseperiodatlaterstages

wassmall,suggestingthatmaterecognitionisthemainfunctionof

pulseperiodresettinginthecallingphase.Thisobservation

indi-catesacomplexandsituation-dependentneuronalcontrolofsignal

productioninmales.Afteridentiﬁcationhasbeenachieved,

accu-racyofrecognitionbecamesecondaryandspeedwasthekey,with

maleLoDsignalsthreetimesshorterthanIdDonaverage.

Asnotedbeforeinthisandotherspeciesofsmallplant-dwelling

insects(Mazzonietal.,2009a,2010;Legendreetal.,2012),males

interrupt walkingboutswithcalls afterwhich directional

deci-sions are made.Although plants constitutecomplex structures

withbranchingpoints,leavesandstems,ofwoodyandgreen

tis-sues, males of S. titanus were ableto make correct directional

decisionswhenwalkingtowardsastationaryfemale,seemingly

basedonacontinuousprocessofevaluatingtheperceived

infor-mation.Accordingtoourresults,S.titanusmalesareabletoextract

directionalinformationfromfemalereplyduringthesearch,since

signiﬁcantlymoremaleswalkedtowardsthefemales;however,

malesmademanymistakesatbranchingpoints.Malesofthelarger

stinkbugNezaraviridula(Heteroptera:Pentatomidae)(size1cm),

whichcanorientreliablyatbranchingpoints,stopandstretchtheir

legsbetweenbranches,thusextendingthelegspan(Coklˇ etal.,

1999).WeneverobservedsuchbehaviourinS.titanus.Instead,we

showedthatmalesareabletocorrectthedirectionaftertheyhad

madeawrongdecision,despitetheirshortlegspan.The

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0 0 .1 0 .2 0 .3 0 .4 Lat enc y [ s ] b a sa l (m a le ) le a f n = 1 4 8 b a sa l (m a le ) p e tio le n = 1 2 6

a p ica l (fem a le ) p e tio le n = 1 3 5 a p ica l (fem a le ) le a f n = 1 4 8 ste m n = 8 6 0 1 0 R e la ti v e peak am pl it ude [ d B ] 2 0 3 0 4 0 5 0 6 0 7 0 0 1 0 0 2 0 0 3 0 0 4 0 0 D o mi nant f re q uenc y [ H z] A B C c a b a b a b c c b a b a d c a c b c b a sa l (m a le ) le a f n = 1 4 8 b a sa l (m a le ) p e tio le n = 1 2 6

a p ica l (fem a le ) p e tio le n = 1 3 5 a p ica l (fem a le ) le a f n = 1 4 8 ste m n = 8 6 b a sa l (m a le ) le a f n = 1 4 8 b a sa l (m a le ) p e tio le n = 1 2 6

a p ica l (fem a le ) p e tio le n = 1 3 5

a p ica l (fem a le ) le a f n = 1 4 8 ste m

n = 8 6

Fig.6.Changesinfemaleresponsecausedbytransmissionalongthesubstrate.(A)Latency,(B)relativeamplitudeofthedominantspectralpeakindBand(C)frequencyof thedominantspectralpeak.DifferentlettersindicatestatisticalsigniﬁcancesafterKruskal–WallistestfollowedbySteel-Dwassmultiplecomparisontest.Sectionwherethe switchfrommalelocalizationtocourtshipbehaviouroccurredismarkedwithgrey.

twomovesawayfromthefemalebeforereversingtheirdirection.

Thisindicatesaperceptionofadirectionalcueeveninthe

orien-tationofsuchsmallinsects, perhapsbycomparingtherelevant

parameterofthefemaleresponsebetweenconsecutivelocations

wheretheystoptosignal.Decisionsbyamaleweremadeashe

walkedaftereveryidentiﬁedfemaleresponse,whileinabsenceof

femalereply,heremainedstationary.Suchasearchtacticwould

requireshort-term memoryfor comparison of signals between

neighbouringlocations,butnotthecapacityfordirect stereoor

multi-channel comparison, bearing more resemblanceto

trian-gulatingsearchbehaviourofsomebeetlesandstoneﬂieson2-D

surfaces(AbbottandStewart,1993;Goulsonetal.,1994)thanto

directorientationofthemorecloselyrelatedPentatomidbugs(Coklˇ

etal.,1999).

IntheleafhopperGraminellanigrifrons,searchingisfacilitated

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Fig.7.Totalnumber(m±SD)ofright(towardsfemale)andwrongdirectionaldecisionsmadebymalesafterreceivingfemaleresponsealongagrapevinecutting(left). Directionaldecisionsatbranchingpoints(middle).Thenumberofmalesmakingarightorwrongdecisionafterchangingdirection(right).Asterisks(***)indicatestatistical signiﬁcancesafterone-tailpairedt-test(P<0.001).

perchingonthetopoftheplant,enableslocalizationevenwithout

theneedtoextractexactdirectionalinformationfromvibrational

signals(HuntandNault,1991).Weobservednosuchpreferencein

S.titanus.Directionaldecisionsatpetiole-stemcrossingsappeared

randomandmalesfoundfemalesperchedonleavesbelowtheir

startinglocationwithoutdifﬁculty,furtherconﬁrmingthat

vibra-tionalsignalsaloneprovidetheinformationaboutbothidentity

andlocationinthisspecies.

4.3. Courtshipbehaviourtrigger

Thechange in behaviouras males progressedfrom

identiﬁ-cation tolocalization and ultimately tocourtship suggests that

amale isaware ofwhetherthefemaleisin closeproximityor

not. Accordingtoourresults, female responseis all-or-nothing

andsignalchangesduetotransmissionthroughtheplantactas

atriggerformalecourtshipbehaviour.Signiﬁcantlyhigherfemale

signalamplitudewasdetectedonor nearthefemaleleaf

com-paredtoothersectionsof theplant.We demonstratedthatthe

switchtocourtshipbehaviouroccurredwhentheaveragesubstrate

velocityoffemalesignals,asmeasuredfromcalibratedfrequency

spectraofindividualsignals,increasedtoapproximately10␮m/s.

Behaviouralswitchcorrespondedtotheamplitudethreshold,while

exact geometrywasnot important.Thisis supportedby

previ-ousresultswhenpairswereplacedonthesameleafatthestart

(Mazzoni etal., 2009a).In sucha situation,malesdidnot

per-formneitherIdDnorLoD,andMCSimmediatelyprogressedinto

acourtship duet.It couldbebeneﬁcialtorestraintheemission

ofcourtshipsignals,whichareenrichedwithadditionalelements

(twopulsetypesandthebuzz,Mazzonietal., 2009a)and thus

likelymoreenergeticallydemanding,tothestagewhenafemaleis

alreadynearby.ThecomplexityoftheCrDcontrastswiththe

rela-tivesimplicityoftheLoD,whichisformedbyonlyonetypeofpulse

andisshorterindurationthanbothIdDandCRS.

In thepresent study, noneof themales expressed“call-ﬂy”

behaviouraftertheduetwasestablished,whichmaybeduetothe

smallsizeoftheexperimentalsubstrate.The“call-ﬂy”behaviour

isusuallyassociatedwithastrategytoincreasesignallingspace

(Gwynne,1987;HuntandNault,1991)andtheamplitudeinour

experimentswasprobablyhighenoughforthemalestoperforma

morelocalizedsearchbywalking,unlikeinthecaseofinter-plant

communication(Erikssonetal.,2011).

Previousworksuggestedthatfrequency-dependentattenuation

mayprovidemorereliableinformationaboutdistancethantotal

amplitude(Barthet al.,1988).We foundseveralindividual

fre-quencycomponentswhosepeakamplitudechangereﬂectedthe

changeintheaveragevelocityofthewholerange40–250Hz.For

thisreason,theymighthavearoleasaproximitycue,perhaps

com-plementingthechangeinamplitudeofthewholesignal,whichis

themostlikelytriggertoswitchlocalizationstageintocourtship;

however,whilewefoundsigniﬁcantchangesonthefrequencyaxis,

nopatternconsistentwithbehaviourordistancefromtheemitter

wasobvious,neitherinthedominantfrequency,northe

frequen-ciesofindividualsubdominantpeaks.Initiallywealsoconsidered

femaleresponselatencyasapotentiallyrelevantparameter,but

while latency increase wasconsistent withdistance, we found

noabruptsigniﬁcantchangeinthisparameterbetweensections

wheretheswitchinbehaviouroccurred,sowedidnotconsiderit

further.Hence,amplitudeistheparameterwebelievethemales

use,asopposedtoresponselatencyandfrequencystructureofa

femalereply.

Polajnar et al. (2012) previously demonstrated that due to

resonance,amplitudeisanunreliablecuefor assessingdistance

fromtheemitterinspeciesthatusealmostpure-tonesignals.In

broad-bandsignals,ontheotherhand,amplitudeoscillations

aver-ageoutbetweenfrequencycomponentsandleadtomonotonous

attenuationwithdistance,asalsoobservedinthepresentstudy.

Unpredictablepropagationofpure-tonesignalsbecauseof

reso-nancemaytherefore bean additionalreasonwhytheemission

of aharmonic “buzz”asanelementof male vibrationalsignals

(Mazzonietal.,2009a)isrestrictedtotheﬁnalstage,emittedwhen

themaleisalreadyclosetothefemale.Anotherconfoundingfactor

iseccentricityofstemmotionwhereperceivedsignalamplitude

mightdependontheanglerelativetotheemitter(McNettetal.,

2006),butthisisonlynoticeableveryclosetoanemitterstanding

onapetiole.

While visualor chemical cuesmayalsobe involved in

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lesslikely possibilities.Our videorecordingsshowthat females

werenotvisible fromthepetioleof thefemaleleaf(i.e. where

most courtship duets started). Until now there has been no

evidencethatchemicalcommunicationplaysarolein

reproduc-tivebehaviour of leafhoppers (Claridge, 1985), and adults rely

exclusivelyonsubstrate-bornevibrationsforintraspeciﬁc

commu-nication(Virant-Doberletand ˇCokl,2004;Mazzonietal.,2009a).

TheantennaeofS.titanusadultsinparticularhaveareduced

num-berofolfactorysensillae(StacconiandRomani,2012)andonlythe

nymphshavebeenshowntouseolfactionforrecognitionofthe

hostplant(Mazzonietal.,2009c).

Tosummarize,pairformationinleafhoppersisadynamic

pro-cesswhere the identiﬁcation stage seems to be optimized for

reliability and the localization stage for speed, while energetic

demandsmayberationalizedbystartingthecostliestandmost

complexadvertisingstageonlyaftertheﬁrsttwotaskshavebeen

accomplished.Secondly,leafhoppermalesareabletointerpret

rel-evantinformationcontainedinfemalesignals’perceivedamplitude

andtemporalsynchronywithmales’ownsignals,therefore

utiliz-ingnotonlyfemalereplyperse,butalsotransmissionproperties

ofthesubstratetoguidetheirbehaviour.

It is theauthors’belief thatbehaviour in ourchosen model

speciesmay,becauseofitssimplicity,providefurtherinsightsin

theinsectmatingprocess,eitherthroughobservationorthrough

manipulationofsignals(seealsoMazzonietal.,2009b;Eriksson

etal., 2012).Buildingupon thesebasicinsights, similarly

opti-mizedstrategiesformaterecognitionandlocalizationshouldalso

besearchedforinotherspeciesofanimals.

Acknowledgments

WethankElisabettaLeonardelli,MatteoGraiffandLuca

Nico-lettiforrearingofinsects,andDr.GiovannaFlaimforimproving

the English. This work was supported by the European Union

SeventhFramework Programme(grant agreementno.265865),

AutonomousProvinceof Trento(Accordo di Programma 2010),

Slovenian Research Agency (P1-0255, J1-2181), Pisa University

(FondidiAteneo)andCBC-EuropeLtd.,Milano,Italy.

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