Table Of ContentOdonatologica39(I):47-56 March I,2010
Matingexperienceaffecting malediscrimination
betweensexes andfemalemorphs in
Ischnura senegalensis(Rambur)
(Zygoptera: Coenagrionidae)
Y.Takahashi and M.Watanabe*
GraduateSchool ofLife andEnvironmental Sciences,UniversityofTsukuba,Tsukuba,
Ibaraki,305-8572,Japan
[email protected]
Received October16,2009/ReviewedandAcceptedNovember 25,2009
Ischnura senegalensis9 9 exhibit colourdimorphism,appearingasandromorphs
and gynomorphs.Binarychoice experimentsbetween sexesand morphswerecon-
ductedinthelaboratory.Virgind3 rearedseparatelyfrom9 9showednopreference
between sexesorbetweenmorphs,suggestingthatvirgin 33wereunable torecog-
nizepotentialmatesandhad noinnatematingpreferenceforaparticular 9morph.
Afterenclosurewith asingle9in asmallcage,33thathad experiencedcopulation
significantly preferredthesame 9morphwith whichtheyhadcopulated,while 33
that failedtocopulatewiththe 2showednopreference.The66 thathadexperienced
copulationsignificantlypreferred $ 2over33.Therefore,abilityofdStodiscrimi-
natebetween sexesand morphswasconfirmed bytheircopulationexperience.
INTRODUCTION
Differencesin reproductive interestsbetween sexes can lead to sexual conflict
overmating frequency(PARKER, 1979),potentiallycausing asexually antago-
nistic co-evolution(HOLLAND & RICE, 1998). GAVR1LETS& WAXMAN
(2002) suggested thatone ofthe outcomes insexually antagonistic coevolution
is femaledivergence withina population, e.g. femalepolymorphism. Inzygop-
terans, femalecolourpolymorphism involvesanandromorph, resembling con-
specific males,andgynomorphs, exhibiting differentcolourationfromthemales
(e.g. ROBERTSON, 1985).
■"Correspondingauthor:[email protected]
48 Y.Takahashi &M.Watanabe
Becausefrequencyoffemalemorphs inapopulation variesspatiallyandtem-
porally (BERGSTEN etal., 2001; CORDERO RIVERA & EGIDOPEREZ,
1998;CORDERO RIVERA&sAnCHEZ-GUILLEN,2007), formingasearch-
-image of the common femalemorph maybe advantageous formales if it in-
creases their searching efficiency (FINCKE, 1994). Males have to adjust their
mating preference tothechange inthemorphfrequency. Infemale-polymorphic
species, frequency-dependence inmalemating preference forfemalemorphs has
beenreported inIschnuraelegans(Vander Linden) (VANGOSSUM etal., 1999)
and in I. senegalensis (Rambur) (TAKAHASHI & WATANABE, 2009). Such
preferentialmatingattemptswithcommonfemalemorphsmightleadtonegative
frequency-dependent selection, which is apotential driving forceformaintain-
ing themultiplefemalemorphs withinapopulation (SVENSSON & ABBOTT,
2005).
Malesencounter variousinsects intheirhabitatthroughout theirlifespan, in-
cluding potential mates as well as unsuitableindividuals such as sexually im-
mature females, conspecific males or heterospecific individuals. Sinceattempts
to mate withunsuitableindividualsare costly interms of timewasted(STOKS
& DE BRUYN, 1998),males have to distinguish between potential matesand
unsuitableindividualsas wellas betweenmorphs inorderto increase theirmat-
ing success. In /. elegans males preferentially attempted to mate with sexually
,
mature femalesratherthanwithsexually immaturefemales(VAN GOSSUM et
al.,2001a) orconspecific males(VANGOSSUM etal., 2005).
In Enallagma civile (Elagen), a previous experience by males of encounter-
ing aparticular femalemorph increasedtheirmatingpreference forthatmorph
(MILLER& FINCKE, 1999).VAN GOSSUM etal.(2001b) showedthatI. el-
egansmales reversibly change theirmating preference basedon theexperience.
Although MILLER& FINCKE(2004)suggested thatan encounter experience
elicitedachange inthemales’matingpreferences, the matingpreferences might
beaffected by the type of interactions withthe individualsencountered.Inthe
presentstudy, weexaminedtheinnatemalematingpreference forfemalemorphs
andsexes,andclarifiedtheeffects of matingexperience on matingpreferences.
METHODS
STUDYSPECIES— Ischnurasenegalensis(Rambur)femalesexhibit colourdimorphism,witha
conspicuousblueandromorphandabrown gynomorph,thoughmales aremonomorphicwithgreen
thoraciccolour. Femalemorphswerecontrolled by asingleautosomallocushavingtwoalleles,with
expressionlimitedtofemales(Y.Takahashi&M.Watanabe,unpubl.data),asinthecaseofI.demorsa
(Hagen)(JOHNSON,1964)andI.damulaCalvert (JOHNSON, 1966).Bothsexesstayatthewater’s
edgealldaylong.Copulationsstart in earlymorningand terminate aroundnoon(TAKAHASHI
&WATANABE,2009).Femalesoviposit aloneduringtheafternoon withoutcopulation,although
malesmakemanyattemptstomatewiththem.
PREPARATIONOFTESTSPECIMENS — Inordertoobtaintestspecimens,artificialovipo-
sitionby femalescollected fromthefieldwas conductedinthelaboratory.Sexuallymaturefemales
MatingexperienceinmaleIschnura senegalensis 49
wereputindividuallyintopetri dishes(ip90mm)forseveraldayswithawetfilter paperasanovipo-
sitionsubstrate. Eggslaidonthe filterpaper werekeptinapetri dishfilled withwateratroom tem-
perature, and mosthatched 10-11days afteroviposition.Thefirstinstarlarvaeweremovedtolarge
plastic containers (8x12 cm,height 5 cm)with asubmergedpolypropylenemesh (5x5 cm,weave
size Imm)astheirperchingsites.Theywerefedonlive brineshrimps(Altemiasp.).Whentheyhad
grown toapproximately3mm inbody length,theywereplacedindividuallyinplastic bottles(ip3.5
cm, height5.8 cm)in orderto avoid cannibalism. Medium-sized larvae(>5mm)werefedforthree
months onlive Tubifexspp. astheir main fooduntil emergence. Atwig about 10cm inlengthwas
providedineach bottleasasupportfortheemergence ofthefinal instarlarva.
Anindividual identification codewasmarkedontherighthindwingofeachadultwith afinefelt-
-tippedpen afterthewingshadhardened. Thesexeswereseparatedinrespectiveflyingcages(40x40x50
cm)with wooden framescovered by polypropylenemesh (weavesize 1 mm).Less thanten adults
werereared ineachcage,inordertodecrease therisk ofcannibalism. Theadultswerefedonfruit
flies,Drosophilaspp.,whichhad beencultured. Toincrease thehumidityaswell astosupplywater,
wemistedinside ofthecage withwater2-3 timesperday.
PROCEDUREOFBINARY CHOICE EXPERIMENT - Fivetonine days old sexuallyma-
tureadults ofbothsexeswereused asthetest specimensinthebinarychoiceexperiment.Theywere
alreadysexuallymature,judgingfromthebody colour and thewingtransparency. Since themale’s
matechoicebehaviour mightbe affectedbymaterefusal behaviour aswell asbyunknown female
matechoicebehaviour,itwasnecessarytouseimmobile specimensin thebinarychoiceexperiment.
Sexuallymature virgin adultsreared inflyingcages werekilled bybeingplacedinafreezer fortwo
minutes. Afterthat,theirabdomenswerestraightened,and theirwingswerekeptclosed. Beforethe
experiment,two specimenswerepinnedwith amicro-needle (<p0.18mm)ontheupper frameinthe
experimentalcage(30x30x30cm wooden framescoveredbypolypropylenemesh,weavesize 1mm)
intheperchingposture.Toevaluatethemale’s responseto eachcolourmorph,bothtypesofspeci-
menwerepinnednexttoeach other 3cmapart.Thesiteofeachspecimenwaschangedineverytrial.
Sincethebodycolouration ofthefreshlykilledspecimensdeterioratedby theevening,newspecimens
werepreparedforeachexperimental day.
Toinvestigateinnatematingpreferenceformorphsandsexes,abinarychoiceexperimentwascon-
ductedusingavirginmalethatwasrearedseparatelyfromfemales afteremergence. Inthemorning
(07:00-09:00),themalewasintroduced intoanexperimentalcageinwhichapairconsistingofanan-
dromorphand gynomorph,anandromorphandmale,oragynomorphand malewaspinned.Each
binarychoiceexperimentwasstoppedwhenthe male‘attacked’ onespecimen,i.e.,when it dashed
toafemaleand tried toformatandem withit.Themales’ ‘attacking’behaviour inthe direction of
apinnedspecimenwasjudgedasanindicator ofthemale’spreference.Ifamale hadchosen neither
specimenwithin fiveminutes,themalewasdiscardedfrom theexperiment.
To determinetheeffectofthecopulationexperienceofthemale onmatingpreferenceformorphs
or sexes,asinglevirginmale wasintroduced into theexperimentalcage with asingle sexuallyma-
turefemale,eitheranandromorphoragynomorph,inthemorning.Themale’smatingattemptsand
copulationbehaviour inthecagewereobserved duringtheenclosure periodforabout4hours.Just
afterremoval ofthecoexistingfemaleatnoon,thebinarychoiceexperimentbetween andromorph
and gynomorph,andromorphandmale,orgynomorphandmale wasconductedforthemales.
STATISTICALTEST— Outcomes ofbinarychoiceexperimentswereanalyzedbyperforming
binominal tests. Differences and changesin preferenceofmales wereanalyzed bythe Chi-square
tests. The programused wasthe Rversion2.7.0(R DevelopmentCoreTeam,2008).
50 Y.Takahashi&M.Watanabe
RESULTS
MATING PREFERENCEOFVIRGINMALES
Ineachbinary choiceexperiment, most males ‘attacked’the killed specimen
presented as soonas they wereintroducedintotheexperimental cage.Themales
grasped and tried to form atandemwith the specimen. Some males hovered
aroundthe specimens justbeforethey ‘attacked’them. Suchbehavioursuggest-
edthatthemalesrecognized thepaired specimens simultaneously andthatthey
wereableto choosefreely oneofthespecimens inthisexperiment.
Inthebinary choiceexperiment betweenandromorph andgynomorph, every
virginmaleimmediately attackedto matewiththefemalespecimen. Virgin males
thatwereoffspringoftheandromorphs exhibitednopreferenceforfemalemorphs
(binomial test,P=0.215) (Tab. I). Alsono preference was foundinvirginmales
thatwere offspring of gynomorphs (binomial test,P=1.0); nordidtheoffspring
ofunknownfemalemorphs (binomial test, P=1.0). Theselectivity was not sig-
nificantlydifferentbetweenmalesderivedfromandromorphs andthosederived
fromgynomorphs (x2=0.739, P=0.390), indicatingthatvirginmales didnot dis-
tinguish betweenmorphs irrespective oftheirmaternalmorph.
AsshowninTableII,virginmalesexhibitednopreferencebetweenmaleandan-
dromorph(binomialtest,P=0.860) andbetweenmaleandgynomorph(binomial
test,P=0.122). Everymaleattempted to matewiththespecimens presented irre-
spective of sex,and formedatandembyattaching theiranalappendages to the
pronotumofthe specimen. Theseresults indicatedthatvirgin malescouldnot
discriminatepotentialmates fromnonspecific males.
DISCRIMINATIONBETWEENFEMALE MORPHS
Whenvirginmaleswereenclosedwithasingle virgin or amatedfemaleinthe
morning, they repeatedly tried to mate with it,and sometimes grasped it. Few
matedfemalesaccepted the male mating attemptthroughout the enclosurepe-
riod,showing amaterefusaldisplay, i.e.fluttering theirwings andbending their
abdomens.On theotherhand,most virgin femalesaccepted themalesaftersev-
TableI
Binarychoiceexperimentbetween anandromorphand gynomorphfor virginmales
(P-valuesrefertobinomial tests)
Maternal morph No. males that No. malesthat
P
ofmalestested chose andromorph chosegynomorph
Andromorph 12 20 0.215
Gynomorph 9 9 1.000
Unknown 37 36 1.000
MatingexperienceinmaleIschnurasenegalensis 51
Table II
Binary choice experiment between anandromorph and gynomorphfor virginmale
(P-valuesrefertobinomial tests)
No.males that No. malesthat
P
chose male chosefemale
Between male
and andromorph 17 15 0.860
Betweenmale
and gynomorph 18 9 0.122
eral mating attempts. Eachcopulation lasted for about threehours and termi-
natedaroundnoon.Figure 1 showsthe outcomeoftheenclosurejustbeforethe
binary choiceexperimentsbetween anandromorph andgynomorph. Outof22
malesthatwereenclosedwithanandromorph, ten malessucceededincopulat-
ing,and 12males failedto copulate. Outof36malesthatwereenclosedwitha
gynomorph, 24 malessucceeded incopulating, and 12malesfailedto copulate.
Inthebinarychoiceexperiment intheafternoonfollowingtheenclosure, males
‘attacked’thedeadspecimen ofeach morphas soonas theywereintroducedinto
the cage,except fourtestedmales, probably dueto handling damage orweari-
ness. Malesthathadsucceededincopulating withanandromorph subsequently
tendedto chooseandromorphs andthiswas significant(binomialtest,P=0.008)
(Fig. 1). Similarly malesthatsucceeded incopulating witha gynomorph tended
to choose gynomorphs, again significatnly (binomial test, P=0.007). Flowever,
malesthathadfailedto copulatewithanandromorph oragynomorph intheen-
closureperiod showednopreferencebetweenmorphs intheafternoon(binomial
test,P=1.0andP=0.754, respectively).
DISCRIMINATIONBETWEENSEXES
Binary choiceexperimentsbetweenthesexesaftertheenclosureinthemorning
wereconductedformalesthathadsucceededincopulating withvirgin females.In
thebinarychoiceexperiment betweenafemaleandromorphandmale,malesthat
hadcopulated withandromorphs significantly chosethe andromorph (binomial
test,P=0.003) (Fig. 2).On theotherhand, inthe binary choiceexperiment be-
tween afemalegynomorph andmale,althoughmalesthathadexperienced cop-
ulationwitha gynomorph wereapt to prefer to matewithgynomorphs, nosig-
nificantdifferencewas found(binomial test, P=0.308). Theproportion ofmales
thatchosea gynomorph amongtheexperienced males(62.5%) was significantly
higher than thatamong thevirgin males (33.3%) (x2=4.339, P=0.037), indicat-
ing thatmaleswereableto improve theirprecision ofmaterecognition through
theircopulation experience.
52 Y.Takahashi &M.Watanabe
DISCUSSION
JOHNSON (1975) suggested that males ofI. demorsaand I. damulahaving
an andromorphic genotypeandmaleshaving a gynomorphic genotypeinnately
had amating preference for andromorphs and forgynomorphs, respectively. If
mating preference of males depends on theirown genotype,thepreference was
partially affected by maternalgenotypes. Inthe present study, however, males
showed no mating preference irrespective of maternalmorphs, suggesting that
no virgin males haveinnate preference fora particularfemalemorph, as inthe
caseofEnallagma spp. (FINCKE etal., 2007). Inaddition, no virginmales dis-
Fig. 1,Male preferenceforfemalemorphsinthebinarychoice experimentsfollowingenclosurewith
afemale. Numerals intheboxes indicatethenumberofmalesthatwereenclosedwith afemale and
theresultsoftheirmatingattempt.Nrepresents the numberofmales thatchose thefemale morph
in thebinarychoiceexperiments. P-values werecalculated usingbinomial tests.
MatingexperienceinmaleIschnurasenegalensis 53
tinguished conspecific males from females, indicating thatvirgin malescannot
recognize sexes andmorphs.
FINCKE(1994)showedthatmaleslearnedtorecognize potentialmatesthrough
theirpriorexperience. WhenI. elegans males hadbeenenclosedwitha particu-
larfemalemorph,themaleschanged theirmatingpreference in accordancewith
this previous enclosureexperience (VANGOSSUM etal., 2001b). MILLER&
FINCKE (1999) suggested thatmalesswitchedtheirmating preference withthe
frequency offemalemorphs encountered.Flowever, in I.senegalensis, exposure
to aparticular femalemorph withoutcopulation didnotaffectsubsequent mat-
ing behaviour, while malesthat experienced copulation preferred to mate with
themorphwithwhichthey hadpreviouslymated.Therefore, ourresultsrevealed
that copulation experience elicited achange in the matingpreference of I. sen-
egalensis males, butencounters didnot. In thepresent study, somemales failed
Fig.2.Malepreferenceforfemalemorphsinthebinarychoiceexperiments followingenclosurewith
afemale that involved successfulmating.Numerals in theboxes indicatethenumber ofmales that
hadmatedwith afemaleofeachmorph.Nrepresentsthenumber ofmales thatchosethespecimen
presentedinthebinary choiceexperiments.P-valueswerecalculated usingbinomial tests.
54 Y.Takahashi&M.Watanabe
tocopulateaftertandemformationduetofemalerejection. Effectofthetandem
formationwithoutcopulation on mating preference needsto beinvestigated.
CORDERO RIVERA & SAnCHEZ-GUILLEN (2007) indicated that the
matingfrequency ofandromorphs was lowerthan thatof gynomorphs inI. el-
egans. ROBERTSON (1985) suggested thatI. ramburi(Selys) malespotentially
preferred gynomorphs because andromorphs resemble conspecific males, and
thusthemaleshavedifficultyrecognizing anandromorph asapotentialmatedue
to male-mimiccolouration.Elowever, inCoenagrion puella(L.), boththecolour
andpatternofandromorphs differedfromthoseofconspecific males(JOOP et
ah, 2007), indicating that the malesmight be able to distinguish andromorphs
fromconspecific males.FINCKE et al.(2007) alsoreported thatthe reflectance
spectrum ofthethorax inandromorphs differedfrom that inmales forE. ebri-
umandE. hageni (Walsh). Inthepresent study, experienced males distinguished
femalesfrommalesirrespective of themorphs ofthefemales,indicatingthatthe
colourandpatternaloneoftheandromorphs didnotcause themtobemistaken
formalesinI.senegalensis.
Inthe field, E. civile(MILLER & FINCKE, 2004) and /. senegalensis males
(TAKAHASHI & WATANABE,2009) had no mating preference in the early
morning,suggesting thattemporal biasofmales’mating preference disappeared
overnight. Becausethematingpreference ofthemalesconformedwiththeircop-
ulationexperience, wildmalesmightchange theirmating preference inresponse
to theirday-to-daycopulation experience. Thismatingpreference couldhelp the
maleseasily distinguishindividualswhoare suitableformatingfromindividuals
whoare not. Because mating attempts withunsuitableindividualsare probably
costlyintermsoftimewasted (STOKS& DE BRUYN, 1998),learnedmaterec-
ognition depending oncopulation experience mightincreasethemating success
ofthemales.
In I. senegalensis, most females in the field accepted copulation during the
morning (TAKAHASHI& WATANABE,2009). If males matedrandomly in
themorning withfemalesofeithermorphwithnopreference, thenthemajority
ofthemaleswouldmate withthecommon morph inthe population. Therefore,
the proportion ofmales that prefer each femalemorph in the afternoonmust
coincidewith theproportion ofmorphs inthe femalepopulation.Thissuggests
thatchanges in thepreference on thebasisofcopulation experience mightlead
to thefrequency-dependent prevalence ofmalematingpreference foreachmorph
(TAKAHASHI & WATANABE.2009).
InI. senegalensis, femalesforageandoviposit aloneafter dailycopulation ac-
tivity, i.e., intheafternoon. Themales tried to matewith foraging andoviposit-
ing females, but fewfemalesaccepted thecopulation (SAWADA, 1999).Theat-
temptby males to mate inthe afternoon mightinterferewith thebehaviourof
femalesof searching for oviposition sites (CORDOBA-AGUILAR, 1993) or
withovipositing itself(FINCKE, 1987). Becausemales thatharassed thecom-
MatingexperienceinmaleIschnurasenegalensis 55
monmorphwouldoutnumbermalesthatharassedtherare morph, thecommon
morphsweremorefrequently harassedby malesencountered(TAKAHASHI &
WATANABE, 2010). This suggests thatthe common morph might havetheir
reproductive success decreasedmorethantherare morphs. InI. elegans, females
ofeach morphexhibiteda negative frequency-dependence intheirreproductive
success (SVENSSON etah,2005). Therefore, inI.senegalensis, experience-based
behaviouralchange inindividualmalesmightdrivemaintenanceoffemalemulti-
plemorphs withinapopulation undernegative frequency-dependent selection.
ACKNOWLEDGEMENT
Y.T.wassupportedby Research FellowshipsoftheJapanSocietyforthe Promotion ofScience
forYoungScientists (20-104).
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