Tohoku University, Kawauchi 41,Aoba-ku,Sendai,980-8576,Japan. 1150 Received 17August 2013;Accepted27November2013 1 which asnaildrivesitslovedart(s)intomatingpartner (i.e. in simultaneoushermaphrodites.Dartshootingisabehaviourduring one ofthebestknownexamplesconspicuousreproductive traits evolution oftheirreproductivebehaviours. is stillunclearhowimportantsexualselectionhasbeenfor the relatively limitedliteratureonsexualselectioninhermaphrodites, it 1998; Koeneetal.,2005;Chase,2007).However, becauseofthe reported inhermaphroditicanimals(e.g.MichielsandNewman, 2006). Also,variousconspicuousreproductivebehaviourshave been in specieswithseparatesexes(Charnov, 1979;MichielsandKoene, selection aresignificantinsimultaneoushermaphrodites,asthey are Theoretical studieshavesuggestedthattheinfluencesofsexual Anthes, 2010;Leonard,2013;NakaderaandKoene,2013). on sexualselectioninsuchhermaphroditicorganisms (reviewedin nature. Inthelastfewdecades,therehasbeenincreasingattention Simultaneous hermaphroditismisacommonreproductivesystemin Pulmonate landsnails,Simultaneoushermaphrodites KEY WORDS:Matemanipulation,Spermdigestion,Allohormone, used thebrowngardensnail because almostallthepreviousstudiesoneffect ofthemucus However, thegeneralityofthiseffect ofthedart’s mucusisunclear, thereby decreasesthenumberofspermdigestedbyrecipient. influences thespermrecipientviamucuscoveringdartand has previouslybeenshownthatthespermdonorphysiologically land snailspeciesstabamatingpartnerwithmucus-coateddart.It behaviour, theshootingofso-calledlovedarts.Duringmating,such Several taxaofpulmonatelandsnailsexhibitaconspicuousmating Kazuki Kimura dart-shooting snailspeciesfromdifferent families Common effect ofthemucus transferredduringmatingintwo RESEARCH ARTICLE © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,1150-1153 doi:10.1242/jeb.095935 *Author ([email protected]) for correspondence Boelelaan 1085,1081HVAmsterdam, The Netherlands. andLife Sciences, VUUniversity, Earth EcologicalScience, Faculty of De of INTRODUCTION ABSTRACT a crucialroleintheevolutionofdartanditsmucus. the sameorganandthatinhibitionofspermdigestionhasplayed various dart-bearingspeciesthemucusfromdartglandstargets gametolytic organ,asfoundinC.aspersum.Thisindicatesthat mucus hadaphysiologicaleffect andloweredtheaccessibilityof aspersum compared ourfindingswiththeresultsofpreviousworkusing peliomphala physiological effect ofthedartmucus,weexaminedthisin its mucus,isstillopentodebate.To testthecommonalityof mucus effect ontherecipientandevolutionofdartitself, Helicidae. Therefore,therelationshipbetweenacquisitionof Department of Environmental Life Sciences, Graduate School of Life Sciences, Environmental Life Sciences, Graduate School of of Department Here, wefocusontheshootingofso-calledlovedarts,which is . Ourexperimentsshowedthatin , aspeciesfromtheBradybaenidaefamily, and 1, *, SatoshiChiba Cornu aspersum 1 and JorisM.Koene E. peliomphala from thefamily 2 Department , thedart Euhadra C. 2 the bursacopulatrixin conformation ofthefemalereproductivesystemandaccessibility to experiment, weinvestigatedtheeffect ofthedartmucuson Bradybaenidae, justasitdoesin digestion ofdonatedsperminaspeciesthefamily shooting andthedartmucususingvariousdart-bearingspecies. investigate thegeneralityandvariationsinfunctionof dart conspicuous reproductivetraithasevolved,itisimportant to bearing landsnailspecies.To betterunderstandhowandwhythis limited understandingofthegeneralitytheseeffects amongdart- Although theeffects ofdartshootinghavebeenwellstudied,thereis quaesita, whichbelongstotheBradybaenidae(Kimuraetal.,2013). shooting, asuppressionofrematingbydartrecipients,inEuhadra 2002). Moreover, amorerecentstudyrevealednoveleffect ofdart belongs totheHelicidaejustlike no influenceonspermstoragein reported that,contrarytotheresultsin Chase, 2001;ChaseandBlanchard,2006).However, ithasbeen paternity ofthespermdonor(Landolfaetal.,2001;Rogersand injection ofonlythemucus,resultedingreaterspermstorageand follow-up studiesfoundthatsuccessfuldartshooting,orexperimental delay digestionofdonatedspermandpromotestorage.Indeed, proposed thattheconformationalchangeandperistalsiswould Helicidae, inparticular research ondartshootinghasbeendonespeciesfromthe land snails(DavisonandMordan,2007).Nonetheless,mostofthe species areknownfromatleastninefamiliesofstylommatophoran aragonite, andareevertedusingamusculardartsac.Dart-bearing objects, madeofacrystallineformcalciumcarbonatecalled through itspartner’s bodywall).Suchdartsarehardandsharp the twotreatments ( position (oviductorbursatract) weresignificantlydifferent between Fisher’s exacttestshowedthattheproportionsoftransferred four snailsitwasfoundinboth thebursatractandoviduct. 1A), whileintheremaining in thebursatractandcopulatrix (Fig. of 15snailsinjectedwiththecontrol, thecolouredsalinewasfound 1B).Incontrast,in11 saline wastransferredintothe oviduct (Fig. In allofthe15snailsinjectedwithmucus,AlcianBlue-coloured transferred intothisbursatractdiverticulumin off fromthetracttobursacopulatrix.Asspermatophoreis in thebursatractdiverticulum,whichisadead-endductbranching Chase, 1998).Furthermore,themucusinducesperistalticmovements copulatrix, whichisagametolyticorgan, isclosedoff (Koene and reproductive systemandthat,asaresult,theroutetobursa that thismucuscausesconformationalchangesinthefemale (Adamo andChase,1990).Ithassubsequentlybeendemonstrated from theglandsassociatedwithdartintorecipient’s blood experiment hasshownthatpenetrationofthedarttransfersmucus RESULTS In thisstudy, wetestedthehypothesisthatdartmucusinhibits P <0.01). Cornu aspersum Euhadra peliomphala Arianta arbustorum,whichalso C. aspersum C. aspersum.Inalaboratory C. aspersum . ForC.aspersum (Pfeiffer 1850). C. aspersum,itwas (Bojat andHaase, , dartshootinghas , asurgical

The Journal of Experimental Biology recipient. In donated sperminthefemale reproductive systemofthesperm accessibility ofthebursacopulatrixprobablyaffects thefateof peliomphala. Regardlessofthemechanism,changein findings suggestthatsimilarconformationalchangesoccur in 1998). Althoughfurtherexperimentsareneeded,ourcurrent reproductive systemaftertheadditionofmucus(KoeneandChase, Chase reportedconformationalchangesoccurringin the not seemtobeanunnaturalcondition. 1017 gml mg (1.0–1.75 peliomphala, theexperimentalconcentrationsofmucus examine theamountofmucustransferredviadartshooting in species. However, althoughfurtherexperimentsareneededto aspersum dart mucustransferredintoarecipienthasbeeninvestigatedin also interruptedwhenthemucusisinjected.Whileamountof indicates thattransferfromtheoviductintobursacopulatrixis mucus. However, thechangeinaccessibilityofbursacopulatrix was difficult toinjectintotheoviductwithoutpresenceof the dartmucusoracontrolinjection,becausecolouredsaline oviduct (thespermatophore-receivingorgan) undertheinfluenceof 1998). Inourexperimentwedidnotinvestigatetransferfromthe similar waytothatreportedfor of objectsintothatgametolyticorgan inourstudyspeciesa to theoviduct.Thisresultindicatesthatmucusinterruptstransfer the injectedcolouredsalinewastakenupfrombursacopulatrix system, weshowthatthedart’s mucus alteredtheorgan bywhich endemic Japaneselandsnail first physiologicalexperimentsusingadifferent species,the aspersum was, uptonow, largely basedononespeciesofgardensnail, Our knowledgeaboutthefunctionoflovedartinlandsnails RESEARCH ARTICLE (Lind, 1973); therefore, onlythespermthat swimoutofthe receiving organ, theyarerapidlyandpermanentlyimmobilised copulatrix byperistalticwaves occurringinthespermatophore- Chiba, 2013).Oncethesperm aretransferredintothebursa from theoviducttobursacopulatrix aftermating(Kimuraand the dartin DISCUSSION For theunderlyingmechanismofpositionchange,Koeneand (e.g. ChaseandBlanchard,2006).Here,wepresentthe (Koene andChase,1998),itisunknownforourstudy C. aspersum E. peliomphala –1 ) weresimilartothoseinthesnailsstabbedwith (Koene andChase,1998)and,therefore,do , thedonatedspermatophoreis moved E. peliomphala.Usingitsreproductive C. aspersum (Koene andChase, C. C. E. E. Euhadra quaesita,whichiscloselyrelatedto that thedartmucushasalong-termeffect intherecipient unclear howlongthischangelasts.However, ithasbeenrevealed found thatthechangeinaccessibilityisrapidlyinduced,itstill the spermathecaofrecipient.Inpresentstudy, whilewe is thoughttoresultinanincreasethenumberofspermthatreach Therefore, inhibitionofremovalthespermandspermatophore before removalofthespermatophorecancontributetofertilisation. spermatophore intotheallospermstorageorgan (thespermatheca) C. aspersum shooter, andsubsequentlyfertilisation,in by themucuspromotesstorageofspermdonateddart expected thatthelowaccessibilityofbursacopulatrixinduced hofmatingin within 8 it hasalsobeenreportedthatspermswimoutofthespermatophore copulatrix butadelayinremating(Kimuraetal.,2013).Moreover, although thiseffect wasnotachangeintheaccessibilityofbursa the recipientvia thedartmucusiswidespread inthedart-bearing suggests thatthephysiologicalcontrol ofthereproductivesystem broad taxonomicgroupsareneeded toconfirmthis,ourfinding and Baur, 2000). Therefore,althoughfurtherinvestigationsusing (Baminger andHaase,1999;Baminger andHaase,2000;Locher has alsobeensuggestedthatsexual selectionisweakinthisspecies and Haase,2002),itseemstobe anexceptionalexamplebecauseit arbustorum inconsistent findinghasbeenreportedinthehelicidsnail generality oftheeffect among theHelicoidea.Althoughan dart-bearing speciesprovidesimportantinformationon the commonality ofthephysiologicaleffect ofthedartmucusintwo 2013). Regardlessoftheirevolutionaryorigin,however, the peliomphala min.Moreover, mating eventanditusuallylastsmorethan10 during courtship, Whereas Chiba, 2006),althoughtheyseemmorphologicallyrathersimilar. because theyhavedifferent strategies ofdartshooting(Koeneand helicid andbradybaenidsnailspeciesmayhavedifferent origins evolved onceormultipletimes(Wade etal.,2007).Theseorgans of it isstillcontroversialwhetherthelovedartsandmucushave Among theHelicoidea,includingHelicidaeandBradybaenidae, C. aspersum The JournalofExperimentalBiology(2014)doi:10.1242/jeb.095935 that dartshootinghasnoeffect onspermstorage(Bojat . uses itsdartduringcopulation(KimuraandChiba, E. peliomphala stabs amatingpartneroncewiththeirdart E. quaesita stabs multipletimesinasingle (Emura, 1932).Therefore,itis bursa copulatrix;OD,oviduct. mucus extract.V, vaginalduct;BC, individual injectedwiththedart saline solution.(B)Forthe individual thatwasinjectedwith Euhadra peliomphala.(A)Forthe coloured salineinjectedinto Thepositionofthe 1. Fig. E. peliomphala E. peliomphala 1151 as in A. E. ,

The Journal of Experimental Biology the spermrecipientisnowknownfortwospecies,onefrom the accessibility ofthegametolyticorgan inthereproductivesystemof order tounderstandthegeneraleffects ofthedartmucus. 1152 RESEARCH ARTICLE tested in generality oftheeffect, however, isunknownandhasalsonotbeen 2013). Thisrematingsuppressionwasreportedfor effect: suppressionofremating bymatingpartners(Kimuraetal., digesting organ, itwasrecently shownthatdartshootinghasanother sperm digestion. accessibility totheorgan maybeacommonstrategyagainstsuch possess agametolyticorgan (Michiels,1998),loweringofthe because manyhermaphroditicanimals,includingsuchlandsnails, evolution ofdartshootinginpulmonatelandsnails.Moreover, of thedonatedspermbyrecipienthasanimportantrolein C. aspersum species. Thecommonalityofthemucuseffect in h lopr trg ra.Saebr 0mm. the allospermstorageorgan.Scalebar, 10 (BC), oviduct(OD)andspermatheca(S)areshown.DSincludesalovedart;MGsecretesthemucuscoveringBCis the gametolyticorgan;Sis Reproductivesystemof 2. Fig. In conclusion,theeffect of thedartmucusinlowering Besides thereportedeffect ontheaccessibilityofsperm- C. aspersum.Therefore,thisremainstoberesolvedin also suggeststhatselectionforavoidingthedigestion E. peliomphala . Dart sac(DS),mucusglands(MG),genitalatrium(GA),vaginalduct(V),bursatract(BT),copulatrix E. peliomphala E. quaesita.The and stab eachotherrepeatedlywiththeirdart.Atthistime,theyalsoreciprocally copulation butpriortospermatophoreexchange,thetwomatingpartners min.During diverticulum. Copulationofthisspecieslasts100–160 Contrary toC.aspersum while thespermathecastoressomeofallosperm(receivedsperm). that digeststheexcessofspermaswellcasingspermatophore, is piercedintoamatingpartner. Thebursacopulatrixisagametolyticorgan glands produceandstorethesecretionthatcoatslovedartwhen dart 3),while themucus spermatheca. Thedartsaccontainsalove(Fig. glands, genitalatrium,vagina,bursatract,copulatrix,oviduct and 2,includesadartsac,mucus study. Itsreproductivesystem,showninFig. We usedthesimultaneouslyhermaphroditiclandsnail between spermdonorsandrecipients. shooting, hasevolvedthroughaconflictoverspermdigestion idea thatthisconspicuousreproductivetraitoflandsnails,dart Bradybaenidae andonefromtheHelicidae.Thisresultsupports Study species MATERIALS ANDMETHODS The JournalofExperimentalBiology(2014)doi:10.1242/jeb.095935 , ourstudyspeciesdoesnothavethebursatract section. Scalebar, 100 mm.(B) Thecross- Scale bar, 1 E. peliomphala.(A)Thesideview. photographs ofthelovedart 3.Electronmicroscopic Fig. E. peliomphala μ m. in this

The Journal of Experimental Biology uu ln xrc ece .–.5m ml mg mucus glandextractreaches1.0–1.75 mlin blood is2.0 al., 1958),ourpreliminaryinvestigationsuggestedthatthemeanvolumeof mass withouttheshellinourstudyspecies,as accessibility. suggested thattheamountofcolouredsalineinjectedwasadequatetoassess minoftheinjection.Ourpreliminaryinvestigation individuals within1 position ofthissalinewassubsequentlyexaminedbydissectingthe coloured withAlcianBluewasinjectedthroughtheinsertedtubeand mlofsaline mgofthemucusglandextract.Then,~0.05 contained 2.0–3.5 mlofthissupernatant diameter), imitatingasuccessfuldartshooting;0.1 mm atrium (wherethedartofamatingpartnerstabs)withsyringe(0.45 ml)wasthenhypodermicallyinjectedaroundthegenital mucus glands(0.1 (for C.aspersum)(Chiarandini,1964).Thesupernatantoftheextract mlofsalinesolution glands wasmadeinahand-heldhomogeniserwith0.5 glands weredissectedoutofanotheradultsnailandanextractthese with thislengthofinsertionwereachedthevaginalduct( mmofthetube was inside.Ourpreliminaryinvestigationrevealedthat ~7 mmdiameter)wascarefullyinsertedintothegenitalatriumuntil (0.50 (to maintaintheanimal’s hydrostaticpressure)andasmallplastictube the skinunderbackofshell.Subsequently, thesnailwasleftintact xeiet okpaea es weeksaftercollection. experiments tookplaceatleast8 the influenceofmatingsalreadyexperiencedinfield,laboratory the studyanddraftedmanuscript. analyses, anddraftedthemanuscript.J.M.K.S.C.participatedindesign of K.K. designedthestudy, carriedoutthephysiologicalexperimentsanddata The authorsdeclarenocompetingfinancialinterests. improving themanuscript. for collectingmaterials.We arealsogratefultotwoanonymousrefereesfor this study, C.LevesqueandS.M.Karsfortechnicalassistance,Yokoyama We expressoursinceregratitude toD.MiuraandY. Moriiforhelpfulcommentson l mmol Adult snailswereanaesthetisedwithaninjectionof50 calcium These snailswerefedcucumberscoatedwithpowderincludingproteinsand relative humidity. Allsnailsweremaintainedunderthesamephotoperiod. ml)at22°Cand~65% Japan, andkeptindividuallyinplasticpots(450 secretions. visual inspectionoftheglandsrevealedthatthesealwayscontainedstored synthesise itsmucusthroughoutthisperiod.Inourpreliminaryinvestigation, in thewild.Therefore, peliomphala donates aspermatophoreintothebursatractdiverticulum.Matingsof oviduct ofamatingpartner(KimuraandChiba,2013),while insert theirpenises. RESEARCH ARTICLE The samplesizeofeachtreatmentwas15snails. mucus extractwasinjectedwereconductedforanothergroupofadultsnails. mlsalineinsteadof experimental injection.Asacontrol,trialsinwhich0.1 Author contributions Competing interests Acknowledgements organ Accessibility tothegametolytic Given thatthebloodvolumeisestimatedtobe40.3%ofwetbody Adult snailswerecollectedinthesummerof2010and2011 inSendai, dlibitum ad are observedthroughouttheiractiveperiod(i.e.exceptwinter) E. peliomphala n h osn a lae vr weeks.To exclude and thehousingwascleanedevery2 Euhadra peliomphala E. peliomphala ( N =8). Therefore,theconcentrationof is assumedtohavethedartand donates aspermatophoreintothe –1 in theindividualsafter Achatina fulica N =5). Themucus − C. aspersum 1 MgCl (Martin et 2 via E. Sports, Science&Technology (MEXT),Japan. management adaptingtoglobalchange)oftheMinistryEducation,Culture, This studywasfinanciallysupportedbyTheGlobalCOEProgramJ03(Ecosystem dm,S .adCae R. Chase, and S.A. Adamo, References Funding aigr .adHae M. Haase, and H. Baminger, nhs N. Anthes, ohr .adBu,B. Baur, and R. Locher, H. Lind, J.L. Leonard, adla .A,Gen .M n hs,R. Chase, and D.M. Green, M.A., Landolfa, aigr .adHae M. Haase, and H. Baminger, oa,N .adHae M. Haase, and N.C. Bojat, aaea .adKee J.M. Koene, and Y. Nakadera, ihes .K n on,J.M. Koene, and N.K. Michiels, N.K. Michiels, atn .W,Hrio,F . utn .J n twr,D.M. Stewart, and M.J. Huston, F. M., Harrison, A.W., Martin, haadn,D.J. Chiarandini, K.C. Blanchard, and R. Chase, hs,R. Chase, E.L. Charnov, ae .M,Hdlt . aio,A,Ngs .adMra,P. B. Mordan, and F. Naggs, A., Davison, C., Hudelot, C.M., Wade, R. Chase, and D. Rogers, ihes .K n emn L.J. Newman, and N.K. Michiels, aio,A n odn P. Mordan, and A. Davison, mr,S. Emura, iua .adCia S. Chiba, and K. Kimura, iua . hby,K n hb,S. Chiba, and K. Shibuya, K., Kimura, on,J .adCae R. Chase, and J.M. Koene, Koene, on,J .adCia S. Chiba, and J.M. Koene, a pheromonethatdecreasescourtshipduration. Zool. spermatozoa inthegenitaltractof Stylommatophora). paternal reproductivesuccessinthesnail Sociobiol. influences spermuptakeintheearthworm caused bysexualselection? hermaphroditic landsnail snail amount ofspermstoredinpopulationsthesimultaneouslyhermaphroditicland Kappeler), pp329-357.Berlin:Springer-Verlag. hermaphrodites. In volumes ofsomerepresentativemolluscs. simultaneously hermaphroditiclandsnail Curr. Zool. Bull. hermaphroditic landsnail, snail gastropods: conceptualandempiricalapproaches. Nature hermaphrodites morethaningonochorists. A. P. Møller),pp219-254.London:AcademicPress. hermaphrodites. In 53-60. increased spermcompetitionrisk. increase paternity. Natl. Acad.Sci.USA special emphasisontheCamaenidae. phylogeny ofthehelicoidlandsnails(Pulmonata:Stylommatophora:Helicoidea), with stylommatophoran landsnailsandslugs. 1518. Zool. uar peliomphala Euhadra suppresses subsequentmatingsindartedindividuals. 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