A Study of Mosquito Behavior. An Experimental

The University of Notre Dame

A Study of Mosquito Behavior. An Experimental Laboratory Study of the Sexual Behavior of Aedes aegypti (Linnaeus) Author(s): Louis M. Roth Source: American Midland Naturalist, Vol. 40, No. 2 (Sep., 1948), pp. 265-352 Published by: The University of Notre Dame Stable URL: http://www.jstor.org/stable/2421604 . Accessed: 23/10/2013 04:58 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp

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The AmericanMidlandNaturalist PublishedBi-Monthlyby The University of NotreDame, NotreDame, Indiana

VOL. 40

SEPTEMBER,

1948

No. 2

A StudyofMosquitoBehavior' An Experimental Laboratory Studyof the SexualBehavior of Aedes aegypti(Linnaeus)2 LouisM. Roth Entomology Laboratory,U. S. ArmyQuartermaster Corps, Philadelphia45, Pa. CONTENTS Introduction -266 Acknowledgement 266 Experimentalmethods -266 268 Sexual behaviorof mosquitoes Historical-268 Normalmatingbehaviorand the attitudesassumedduringcopulation269 Lengthof time requiredfor copulation 272 Repeated copulationsof males272 The presenceof sperm in the spermathecae -274 The r6le of sound producedby the wingsof the femaleduringflightL275 Attemptedcopulationsbetweenaegyptimales, and betweenaeg_pti males and otherspeciesof Culici280 dac Rotationof the male genitaliaand - 282 age of male at firstcopulation Age of female at firstcopulation; "attractive"and "unattractive" females284 The "mating-state" 285 in malesIncreasingthe frequency of copulation 286 287 The r6le of themale antennae The r6le of themale legs294 Generaldiscussionof theattitudes of mosquitoes assumedduringcopula297 tion-

Generaldiscussionof thematingbehaviorof mosquitoes 306 The behaviorofmaleaegyptitosound 311 Historical-311 The matingresponseto sound311 Other reactionsto sound314 Tuning forkexperiments 314 The behaviorof males subjectedto an air currentand sound simul-315 taneously of The mechanism auditoryperception -315 Age of male at firstmating responseto sound-323 Preliminaryaudio oscillatorexperimentsto determinethe range of sound frequencieswhich will induce thematingresponse 325 Determination of theupperand lower limits of sound frequencies which will induce the matingresponse in virgin and non-virgin 327 males"Adaptation"of males to sound- 339 The behaviorof males of otherCulicidae to sound-341 The practical applicationof the behaviorof mosquitoes 343 to sound-344 Summaryand conclusions -348 References

1 The workdescribedin thispaper was done undera contract betweentheQuartermasterCorps, Army of the United States,and The Ohio State UniversityResearclh Foundation. 2 A thesissubmitted for the degreeof Doctor of Philosophy in partialfulfillment Columbus,Ohio. in theGraduateSchool of The Ohio State University,

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Introduction Manyof theobservations regarding mosquito matingbehavior havedealt withthefactors governing swarming, thesizeand positions of swarms(Howard, Dyar, and Knab, 1912,pp. 120-132;Marshall,1938, pp. 295-304), and whether or notthespeciesin questionwouldmatein a smallspaceor required a largeareaforcopulation ("stenogamy" and "eurygamy," Roubaud, 1932). Sexualbehavior otherthanthecopulatory act itselfhas beencalled 'epigamic and recent workon theAnopheles maculipennis grouphasshown thatthisepigamic behavior apparently is a specific and inheritable character (Bates,1941). Bates(1941) refers to thespecialflight of a singlemaleas a "sexualdance"andto a congregation ofsuchdancing malesas a "swarm." It is wellknownthatmosquitoes producean audiblehumduringflight whichmaybe so distinctive thatsomeworkers claimtheyare ableto identify thespeciesbythissoundalone.The literature contains manystatements assertingthatsoundplaysan important rolein theattraction of thesexesof mosquitoes;actually no one has provedthatthissoundwhich, at leastin part,is produced incidentally during flight, has anybiological significance. It has been supposed,thoughneverprovedexperimentally, thatJohnston's organwhich is greatly developed in thepedicelliof themaleantennae of culicidsserves an auditory function. The positionsassumedby mosquitoesduringcopulationhave been recorded by variousauthors,and Knab (1907b) correlated theseattitudes withtheextent of development of thefemaletarsalclaws. The present paperdescribes variousexperiments performed to investigate someof theaboveproblems. It wasprovedthatthemaleof Aedesaegypti is attracted to thefemale bythesoundsheproduces in flight; thattheantennae ofthemaleareauditory in function; andthatKnab'shypothesis thatthepositionassumedduring is dependent copulation uponthedegreeof development of thefemaletarsalclawsis invalidand wasbasedon theerroneous observationthatthe femalemosquitoclingsto the male.The last portionof the paperdealswiththebehavior of maleaegypti towards mechanically produced sound. mostof theexperiments Although wereperformed on A. aegypti, some laboratory observations on othermosquitoes, Culex pipiens L. and chiefly Anophelesquadrimaculatus Say are included. An attempt has beenmadeto interpret, in thelightof recentfindings, themanyobservations on mosquito behavior foundin theliterature, and also to pointoutproblems whichremain to be solved. ACKNOWLEDGEMENT

is indebted to Dr. ClarenceH. Kennedy, The writer Dr. VincentG. Dethier, Dr. Carl E. Venard,Dr. DwightM. DeLong,Dr. RalphH. Davidson,and othermembers of thelaboratory fortheirinterest andsuggestions giventhroughout theproblem. Appreto Mrs.EthelMelsheimer ciationis further expressed Millerand Mr. Joseph W. Enke forcritically reading themanuscript.

ExperimentalMethods colonies ofA. aegypti Laboratory andA. quadrimaculatus weremaintainled Laterin theworka colonyof Culexpipiensalso in theUniversity insectary.


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was established. Virginmosquitoes, whenwanted,wereobtainedby sexing pupaebysize (femalepupaearenoticeably largerthanmale) and rearing in

separatecages. Part of thisstudydeals withthe behaviorof mosquitoeswhich had variousstructures removedby operation,and the methodof anesthesia willnow be described.For the sake of claritythe varioustechniquesemployed are describedin the sectionof the paperwithwhicheach is concerned.

Operations wereperformed on mosquitoes, usingiridectomy and micro-

dissectingscissors.Mosquitoes were anesthetizedwith carbon dioxide after the method (with slightmodification)describedby Williams (1946). The apparatusis shownin Figure 1. A 3" diameterBuchnerfunnel (B) fitted into a woodenplate was substituted for the glass stage of the binoculardissectingmicroscope.A piece of wirescreen,on whichresteda small square of filterpaper,was fittedinto and raised a shortdistanceabove the bottomof the funnel.In orderto indicatethe rateof flowof the gas, the carbondioxide froma tank (not shownin Fig. 1) was bubbledthrougha gas washingbottle (C) containingwater,beforeenteringthe funnelthroughthe rubbertubing (TC). A "Y" tube (Y) was attachedto the rubbertube (TA) leading

TE~~~~~~~~~~~I

_

anesthesia Fig. 1.-Continuous apparatus. Abbreviations: A-aspirator(containing mosquitoes);B-Buchner funnel(with on squareof filter wirescreenand anesthetized mosquitoes paper); C-gas washing bottle(containing water); PC-pinchcockclamp; TA-rubber tubingleadingfrom of medicine at freeend, tankof CO2; TB-rubbertubewithglassportion dropper TC-rubbertubeleadingintoBuchnerfunnel;Yinserted in one endof aspirator; Y tube. Arrowindicates directioni of flowof CO2. (Photograph byW. E. Wagner.)


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fromthetankof carbondioxideso thatone branchled intothegas washing bottlewhiletheotherbranchled intoa rubber tube (TB) whichwas kept of a medicine clamp(PC). The glassportion closedbymeansof a pinchcock wasinserted TB. Mosquitoes fromcages in theendoftubing removed dropper theglassend of tubingTB by inserting withan aspirator wereanesthetized intoone endof theaspirator and opening thepinchcock clamp.The anesthewereplacedon thesquareof filter paperresting on thewire tizedmosquitoes clampdiverts thecarbon funnel. Closingthepinchcock screenin theBuchner so thatthemosquibottleandintothefunnel dioxidethrough thegaswashing The operations anesthesia. toessurrounded by thegas are undercontinuous on individual wereperformed mosquitoes placedon thesmallpieceof filter apparently to treatthewoundssinceclotting paper;it was foundunnecessary It is also probable and therewas littlelossof haemolymph. occurred rapidly thefinescissors thecutedgestogether and brought thatduringtheoperation helpedseal the wound.Males werekeptformorethan30 minutesunder The operations behavior. anesthesia showednormalmating and afterrecovery werekeptin thefunnelat required onlya fewsecondsand manymosquitoes and upontheywereremoved onetime;afterseveralindividuals wereoperated and testing. placedin a cage11" x 11" x 15" forobservation Sexual Behaviorof Mosquitoes HISTORICAL

one of theearliest Godeheude Riville(1760) madewhatwas probably of a speciesof mosquito which on themating behavior recorded observations Knab (1907a) quotedfromde Riville's Knab believedwas Aedes aegypti. of herbody,sat recognized bythelengthand stoutness article, "The female, of mosquitoes; in theordinary withherfront sheclungto thefabric position aboveherwings. and middlelegs,thetwohindlegselevatedin a half-circle The smaller size The male,on thecontrary, attitude. had assumeda different ofobtaining whodid not unionwiththefemale, ofhisbodyandthenecessity lefthimapparently lessat ease.His two herself, seeminclined to incommode frontlegsaloneheldhimto thetop of thecanopywhile greatly elongated withtheotherfourlegshe graspedthefemale."Goeldi (1905,p. 74) obcalopus3tendedto gatherin the malesof Stegomyia servedthat,indoors, objects, suchas a tableor or moreoverprominent groupsof fifteen or twenty otherfurniture, whilethe femalesflewall abouttheroom.When a female oneofthesegroupsof malesshewouldbe seizedbya malewhich approached to herfromthelowersideand permits herto carryhim ".... uniteshimself fora fewseconds(twoor threeonly),andthenagain witha slowheavyflight Goeldialsonotedthatthisspecieswillcopulatein a bottle separates himself." or breeding malewillcopulatewithmanyfemales; and cage;thatthesanme thattherearenottwodistinct swarms, oneof malesand theotherof females, forthesexesto keepseparate. thereis a tendency though the Howardet al. (1912,p. 275) recorded the observations concerning of aegypti begins madebyE. G. Hinds,whonotedthat". . . mating mating 3 Stegomgiacalopus,Aedes calopus,Slegomgiafasciala= Aedes aegypli.


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butthe whilestillflying, whenon thewing.It may,and usuallyis completed The comfemalefrequently alightsduringtheact and beforeits completion. to thisobserver, of a minute. According pleteact requires but the fraction to malesthanthosewhich femalespartlyor fullyfedseemmoreattractive occursbefore either sometimes, perhaps havenotfedat all. Copulation usually, ofa personsitting sexhasfed.Malesoftenalighton thedarkcoloredclothing quietlyandwatchtheirchanceto pounceupona femalecomingto feed.The 4 and 6 p. m. of copulation seemsto be between timeof thegreatest activity eggs A malemaymatewithseveralfemales. More thanone batchof fertile maybe laidwithout intervening fertiiization." NORMAL MATING BEHAVIOR AND THE ATTITUDES ASSUMED DURING COPULATION

in cages11" x 11" x 15" Observations weremadeon mosquitoes confined withcheesecloth sidesand glasstops.The floorsof thecageswerepainted theroleof observation. In orderto observe whiteon theinsideto facilitate thelegsof bothsexesduring copulation, a finethreadwasgluedto themesofromtheglasstop of a cage.A notumof a femaleand she was suspended withits stageremoved was placedon the glasstop dissecting microscope underhighmnagniaboveher.In thiswaycopulation couldbe observed directly The following of thematingbehavior of aegypti is based fication. description on observations copulations between females andinnumerable ofmanytethered free-flying mosquitoes.

on the In a cagecrowded withbothsexes,femalesare seen (1) resting ignorethem,(2) by maleswhichapparently sidesof the cage surrounded on thesidesof thecage,or (3) flying copulating withmaleswhileresting aboutthecageusuallywithoneor moremalesclinging to them.A cageconhourwithout wasobserved forone-half taining about25 malesand 25 females flewaboutand fewcopulathemosquitoes. Veryfewindividuals disturbing thesides a maleflying tionstookplace.Occasionally touching about,briefly of thecage,willsometimes toucha femaleand shetakesto flight, whereupon male he pursuesand seizesher.A femalein flight willpassclosebya resting A pairjoinedin flight often whichwilltaketo flight, seizeher,and copulate. As manyas 3 or to seizethesamefemale. attracts othermaleswhichattempt moremaleswillpursueor clingto a singlefemale.The maleis theaggressor If to gaina ventralattitude. attempts and afterseizingthefemalein flight severalmalesmanageto seizethesamefemaleshedropsto theflooror lands on thesideof thecage.The malewhichfirst attitude usually gainsa ventral The others stillclingto thefemale(or othermales) succeedsin copulating. to makeconnection trying aftershe lands,but flyawayafterunsuccessfully withthe female.

If theattitude whicha pairhas uponlandingon thesideof thecageis onewhichdoesnotpermit mating(i.e. themaleon top), themalewill (1) the to copulateby flexing (2) makesomeeffort flyawayand not return, to crawlunderthe female,or (3) the femaletakesto abdomenor trying himself to her.In thelatter casehe reorients flight withthemalestillclinging is completed in theair,or if thefemalelandsagain in flight and copulation


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whilethepairis at rest.In mostcasesthe is successful copulation generally to herand copufemalelandson thesideof thiecagewiththemaleclinging with lationis completed hereratherthan in flight.This is in agreement after copulatemostfrequentlv thataegypti Hiovanitz(1946) whoconcluded and at restis compartlyin flight thefemalecomesto rest,whilecopulation inflight is theleastcommon. monandcopulation (face-to-face), are belly-to-belly The male and femalewhilecopulating in the same direction (Fig. 36). The frontand withtheirheadspointing hishindlegs,initially, middlelegsof themaleareusedto seizethefemale, passiveduringthe takingno partin theprocess.The femaleis completely seizingprocessand doesnotholdthemaleto her.Whenthemaleseizesthe tarsiseizesherhindfemur oneofhisfront female attitude, andgainsa ventral nearitsbasewhilehisotherfronttarsuswillseizeherotherhindleg in the on beyondthebaseof thefemur(fromobservations sameplaceor somewhat attifemales).At othertimes,aftergaininga face-to-face seizureof tethered middle,or hind tudehis frontlegsgraspotherpartsof thefemale'sfront, legs.Althoughthefemaleis notalwaysseizedexactlyin thesamemanner, seizessomepartof herlegs. themaleiniitially and middlelegsof themaleare holof thefront The lasttarsalsegment lowedon one side (Figs. 17-19) and thatpartof the femaleleg whichis seizedfitsintothehollowwhereit is heldby thelargetarsalclawsof the themaleclingsto herwith by a thread, Whena femaleis suspended mnale. whena free-flying paircometo rest bothhisfront andmiddlelegs.However, usedin on thesideof thecage,thefront legsof themalearetheoneschiefly to clingto legsbackwards to hispartner; somemalesbendtheirfront clinging is accomplished. withthefemalegenitalia thesideofthecageafterconnection The middlelegsof themaleareusedto pushtheabdomenor hindlegsof thisis parthefemaleupwardsuntilcontactwiththe genitaliais effected; Once thegeniin makingconnection. ticularly trueif themalehas difficulty taliaarejoined,themiddlelegspushon thetibiaeor usuallythebaseof the raisedin theair; if a tarsiof the hindlegsof thefemaleand keepthemn to dislodgethemale. sheoftenuseshernindlegsin an attempt femaleresists, thetarsalclawsofthemnale's whenthepairis inthefacing Sometimes position of thefemale's wings.The hindlegs middlelegsarehookedoverthemargins ofthemalesometimes help,withhismiddlelegs,to pushthefemaleabdomen

irntothe properpositionfor connection.However,when connectionis accomplished,the hind legs of the male usuallyhang downwardsor rarelyare bent backwardand rest on the side of the cage. Once in the properpositon, the male flexeshis abdomenventraduntilhe connectswiththe female'sgenitalia; copulationis rapidand the male releasesthe femaleand fliesaway.The angle formedby the abdomenof the male withthe nearlyverticalabdonienof the femalevariesfroma moreor less V-shape to one of about 90 degreesif the male is in a low position.Copulationtakes place on the floorof the cage when the femaledrops with the male directlyunder her. Occasionallythe femalein landingon the side of the cage clingsto the cloth withonly one frontleg and copulationtakesplace withbothindividualsassuminga sidewise the nmalecopulateswhenslightlyoffto one side of the position.Somnetimes


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cagemalesareseenoccasionunderher.In a crowded femaleandnotdirectly at copulation females, ,ut no attempt allylandingon (or partlyon) resting or vibrates herwings.No maleswere is madeunlessthefemaletakesto flight copulation whileastridethefemale. cverseento accomplish whilein flight, aegypticopulating Goeldi(1905,Plate1, Fig. 3a) figured fromherbody The female's legsweredrawnextended position. ina face-to-face heshowedthemalein connecthemaleto her.However, andwerenotholding The oncmale thefemale. tionwiththefemale butnoneofitslegswereholding beyondthefemalemesonrotum. shownwasextended tarsalsegment prothoracic beyondthe Both pairsof his middleand hindlegs weredrawnextended themale Withnoneof themalelegsseizingthefemale, female'sabdomen. from or facein theoppositedirection actuallywouldhangheaddownward thefemale(see Figs.37, 38), and wouldnotbe in the positionshownin Goeldi'sdrawing (cf.Fig.36). theattito describe Lamb (1922) used theterms"pose"and "position" The poseis insects duringcopulation. tudesassumedbythesexesof different to pairing, i.e. at tiheinjustprevious theattitude adoptedbytheindividuals into thevagina.The positionis the stantat whichthe penisis inserted Thoughthe attitudeadoptedby the pairin the laterstagesof copuiation. so thathe is uppermost, seizesthefemaledorsally usuallyfirst maleaegypti that canbe made.Assuming before connection 1hem-ust attitude gaina ventral themaleclaspsthefemalegeniinsertion of thepenisoccursat themoment Lalia,thereis no changeof attitudeso thatthe position("belly-to-belly" as of Hardv,1944) is thesamre of Richards, 1927;"facing position" position the belly-to-belly pose ("verticalpose" of Lamb, the pose. In mosquitoes of the linearposesinceit is a modification 1922) is knownas themodified by linearpose (Lamb, 1922; Richards,1927). This can be demonstrated mosquitoes; a tubesaturated withetherfumesovera pairofcopulating placing if donequickly, the pairwillbecomeanesthetized aLndthemalewillrelease by theirgenitalia. his leg holdon thefemalebuttheymayremainattached withtheir so thatthe pairis joinedtailsto-tail Theymaythenbe extended The maleof somespecies, likeA. quadridirections. headsfacingin opposite initiates in themodified linearpose and afterconneccopulation maculatus, hislegholdandswings intothelinearposition(Fig. 38). tionis madereleases flewunderthefemaleand thatthemaleaegypti Banks(1908) reported claspedher frombeneath.Lamb (1922) statedthat if Bank'sstatement upside it wouldappearto involvethemale flying " . be takenliterally, whichmayindeedbe justpossible, of pairing, butoffers downat theinstant of It is possiblethat,at thefirstinstant mechanical difficulties. considerable namely, an ordinary is thesameas in Empisborealis, copula,thecondition F. V. P., . . ." (F. V. P. = femalevertical pose). Richards(1927,p. 335) thatthebelly-to-belly posemight on Lamb'sstatement suggested commenting if bothinsects flewnearlyperpendicularly and "Theywould be accomplished surfaces upsidedown."In most thenhavetheirventral flying apposedwithout in his initialseizureof thefemalegraspsherdorthemaleaegypti instances he rapidly to a ventral attitude changes Apparently, sallyandhe is uppermost.


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whileclingmg to theflying female. Thus it is notnecessary thatthemalefly upsidedownwhenhe firstseizesthefemale.Copulation was neveraccomplishedor initiated in thefemalevertical pose (i.e. withthefemaleabovethe maleand his dorsalsurfaceagainstherventer)as suggested by Lamb.The of pairalso mayflyperpendicularly as suggested byRichards. The abdomen thelonefemalein flight is heldnearlyhorizontally butwhensheis seizedby themalewhichgailisa ventral attitude herabdomen mayhangdownward so of bothmaleand femalearepractically thattheabdomens and perpendicular thepairflyaboutbriefly in thismanner. Hardy (1944) described thisas the "vertical poise." LENGTH OF TIME

REQUIRED FOR COPULATION

The length oftimethemaleclingsto thefemale inattempting andsuiccessfullycompleting copulation is variableand dependsupon (1) whether there is interference fromothermales,(2) howquickly thefemalecomesto rest,if copulation is not completed in flight, (3) howquicklythe malegainsthe ventral or notthefemaleresists pose,and (4) whether or submits. Thus the timerequired forthecomplete act,fromthemomen.of seizureto therelease ofthefemaleafterconnection, whenmorethanonemale usuallyis lengthened whenthefemaletendsto remainin pursuesalndclingsto a singlefemale, flight withthemaleniotmakingconnection, whenthiemaiedoes notgaina venter-to-venter attitude quickly, and whenthefemaleresists. Conversely, the timeis shortened whena maleseizesa femalewithno intervention fromother males,whenhe gainsa ventral posequickly, and whenthefemaledoes not resistand theycomplete copulation duringflight. or theuniting Actualcopulation, of themaleand femalegenitalia, is a 25 copulations weretimedfromthe veryrapidprocess.Usinga stop-watch, withthefemalegenitalia moment themalemadeconnection to themoment he releasedher,and it wasfoundthatthetimerequired variesfrom4 to 59 seconds,withan averageof about16 seconds.With repeatedcopulations a femaleresistsbyjerking awayherabdomenor pushingthemaleawaywith herhindlegs.This resistance, thoughshortening the timethegenitaliaare thetimethemaleclingsto thefemale, forhe makes joined,usuallylengthens to connect withhergenitalia whenshemanagesto pullher repeated attempts he releases her. abdomen awaybefore REPEATED COPULATIONS

OF MALES

Darticuwiththesameor different Maleswillcopulaterepeatedly females, the mosquitoes cometo rest.This larlyif the cage is agitatedwhenever disturbance inducesthemto flyand themalesimmediately pursuethefemales and seizethem.If a femaletakesto flight justas themaleleavesherafter is completed, thesamemalemaypursue,seize,and copulatewith copulation hera secondtime.One virginmaleplacedin a cagewith16 virginfemales


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matings 30 timesand attempted10 otherunsuccessful4 copulatedsuccessfully4 duringa 30-minuteperiod. Eleven virginmales copulated successfully50 times during 1 hour (unsuccessfulcopulationswere not counted) with a singlevirginfemale.In the above cases the cage was agitatedto induceflight. TABLE

I.-Results of Matinga Single Male with 17 VirginFemales over a Ten-Day Period.

Age of male (hours)

Numberand age of females

Remarks

70 (virgin)-.----------.-..7 (71 hours) ..............The matingsoccurredover a one hour period; the firstfour females that mated had two of their spermathecaefilled with sperm; the last three females matedhad emptyspermathecae. 89 (19 hourslater)-.

3 (81 hours)-

.

The matingsoccurredover a ten minute period; the first-mated femalehad spermin the middle only; theotherthree spermatheca femaleswere negativeforsperm.

4 (age unknown) 122 (33 hourslater)---------

168 (46 hours later).....

1 (6 days) -

240 (72 hourslater)----- 2 (8 days)-

.. .

The .... threematingsoccurredover a five minute period; the firstmatadfemalehad spermonly in her middle spermatheca; the other two females were completelynegativefor sperm.

well-filledwith Two spermathecae sperm. first female was lost; the The .. second was negativefor sperm.

Banks (1908, p. 246) observeda singlemale Stegomyiacopulatewith7 to 8 females.Low (Theobald, 1903, p. 3) noted that aftera pair copulate, the male pursuesanotherfemale,untilit may have fertilizedmanyin a day." Other reportson repeatedcopulationson the part of male aegypti alreadyhave been mentioned.However,the mereact of copulationdoes not has takenplace and the followingexperiindicatethatfecundation necessarily by how manyfemalescould be impregnated to determine mentwas performed a single male. One male was allowed to copulatewith a numberof virgin 4 Copulationwas consideredsuccessfulif themale succeededin makingconnection withthe female'sgenitaliaand the pair remainedjoined for a briefperiod; however,it does not implythatthe femalehas been thatmakingconnection shouldbe remembered if a male seized impregnated(see Table 1). Copulationwas consideredunsuccessful failed to make and clungto a femalebut.becauseof a faultypositionor her struggles, connection.


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wereremoved thefemales overa periodof 10 days.Aftercopulation females If sperm and examined. weredissected fromthecageand theirspermathecae areshownin The results fecundated. wereconsidered thefemales wcrepresent Table I. FromtheTable it willbe seenthatovera 10-dayperiod,one male 6 of them(one in impregnating females succeeded with17 virgin copulating thata malewillconwasnotdissected).It is apparent of thematedfemales intothefemaleand the tinueto copulatethoughno spermare introduced witha femaledoes not implythatshe has been factthata malecopulated the Also,we can concludethatafterseveralinitialcopulations impregnated. aftera varyare produced butmorespermatozoa of sperm, maleridshimself 10 thatonemaleaegypti fertilized oftime.Bacot(1916) indicated inglength periodduringwhichthespermremained and themaximum outof 21 females ofthefemalewas62 days;thefemalein aliveand activein thespermathecae butnonewerefertile. of eggsafterthisperiod, batches laidfurther question THE

PRESENCE OF SPERM IN THE SPERMATHECAE

spherical, of aegypti femalesconsistof 3 sclerotized, The spermathecae The central withinthe eighthsegment. lyingventrally rescrvoirs pigmented is largerthanthe 2 lateraloneswhichare of equal size. Each spermatheca ductleading ductwhichunitesintoa common hasitsowncolorless reservoir of theoviduct).Duringoviposiintotheatrium(expandeddistalextremity aiongtheductsand each travelfromthespermathecae tionthespermatozoa theatrium(Marshall,1938). The presas it passesthrough eggis fertilized are apparently and bloodin thestomach enceof spermin thespermathecae in aegypti (McGregor, of ovulation forthe inception theonlyprerequisites thebloodmea; mustprecede thatfertilization 1931). Macfie(1915) belicved before eggswillbe laid.Gordon(1922) showedthata femalewhichhas not do nothatch. Ihada bloodmealmaylayeggswhich 11daysold,andwhichhadbeenwithmalesfromthetimeof Ten females In all individuals examined. and theirspermathecae weredissected emergence, one contained sperm;thesewerethelargecenitral cnly2 of the3 reservoiri thecompound Witha strong lightthrough lateralspermatheca. -anda smaller filledwithspermwereseento be distinctly the2 spermathecae microscope, werc However,all 3 spermathecae moreopaquethanthe emptyreservoir. or absenceofsDerm. ofthepresence in orderto makecertain crushed snd thefollowing experieggsafterone copulation Femalcswilllayfertile everbecamefilledwith ifall 3 spermathecac to determine mentwasperformed virgin werematedonce,eachwitha different virginfemales sperm.Fourtecn forsperm1/2 to and examined wereremoved male,and theirspermathecae hadno sperm wereas follows:2 females The results 1 hoursafter copulation. and one 8 showedonlythelargecentral spermatheca in anyof thereservoirs; filledwithsperm,the thirdbeingempty;of the other4 lateralreservoir werefilledwhilethe thirdsmall 2 of each of theirspermathecae females, 9 In a secondexperiment, mayhavehad a fewspermpresent. spermatheca in a cage containing manymalesand after wereplacedindividually females and examined dissected andtheirspermathecae twicewereremoved copulating


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maybavehad mating. Of the9, 2 females to 1I/2 hoursafter three-quarters of spermin 1 of the2 smallspermabutthenumber spermin all 3 reservoirs spermwereoriginally to decidewhether thecaewasso smallthatitwasdifficult theycame fromthe other2 crushed or whether presentin thisreservoir and 1 lateralspermahad onlythecentral The other7 females spermathecae. 1 of With1 or 2 copulations thethirdbeingempty. thecafilledwithsperm, doesnotbecomefilledwithsperm.The signifigenerally thelateralreservoirs is notknown. cance,ifany,northereasonforthisphenomenon THE

ROLE OF SOUND PRODUCED BY THE WINGS OF THE FEMALE DURING FLIGHT

forone flying closeby a resting a femalein flight Males can recognize maleofteninducedhimto leavethesideof thecageand pursueher.Appara restof aegypti. Although behavior in themating entlyodoris nota factor bymales,somebeingso closethattheytouch ingfemalemaybe surrounded copulafemales seento inducea maleto attempt her,neverwereanyresting to wereindifferent females matedwithflying tion.Also,maleswhichreadily thatodor evidence The mostconvincing thecrushed bodiesof thesefemales. in locating thefemalewasshownbythefactthatmaleaegypti is nota factor mating soundsbutalso showa characteristic bycertain arenotonlyattracted (see p. 311). to thesesoundfrequencies response themale,and andattracting in activating Flightofthefemaleis important whichwillpermit himto assumea positionduringflight also in permitting was bythefemalein flight of thesoundproduced The importance copulation. A veryfinewirewas loopedaround in thefollowing manner. demonstrated or gluedto her the neckof a femaleor a pieceof threadwas shellacked in themiddleof a cagefullof males.The cage thoraxandshewassuspended to inducethemalesto fly.As longas thefemalehungfrom wasthenagitated themalesremained indifferent herwings, vibrating thewireor thread without a malewouldland on thefemalebut no and flewabouther.Occasionally motionwingsremained wasmadeto copulateas longas thefemale's attempt less. However,as soon as she beganto vibrateherwings,the malesflying to copulate.Motionalonewas seizedherand attempted nearbyimmediately to the suspended malesremained indifferent sinceflying of littleimportance herwingsweremotionless) thoughthethreadto whichshe female(provided wasswungto and froamongthem.The wireor threadholding wasattached the femalewas also loopedarounda glassrod,insteadof beingsuspended males in closeproxiniity to theresting fromthecage,and thefemalebrought to copulateuntilshevibrated themalesdid notattempt results; withsimilar to of male mosquitoes herwings.Lutz (1924,p. 337) notedno attraction thespecies in a widemouthed jar. He did notindicate confined females flying and it is quitepossiblethatLutz was dealingwitha form,likeC. observed The malesof these whichmatesperiodically. pipiensor A. quadrimaculatus, becomesexuallyactiveat certainperiodsof theday but at all mosquitoes othertimestheyignorethefemales. producedsoundsin twoways,one by Landoisbelievedthatmosquitoes


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the movement of thewingsand the otherby air beingforcedout through thoracic spiracles(Howardet al. 1912,p. 114). The latterviewhas since organon a stridulating beendisproven. Shipleyand Wilson(1902) described maculipenntis whichtheyconsidered thebasalpartof thewingsof Anopheles organ.Theyquotedtheworkof Perez sound-producing to be theprincipal whoshowedexperimentally thatthestigmata takeno partin theproduction thebuzzingis due ofsoundandthat". . . in bothDipteraandHymenoptera of thewingwhich to twocauses,one,thevibrations due to thearticulation constitutes thetruehumand theotherthebeatingof thewingsagainstthe Bellesme(quotedbyShipley moreor lessmodifies thefirst." air,whoseeffect theworkof Perezand notedthatof thetwo and Wilson,1902) confirmed onedeepandoneacute,thelatteris usuallytheoctaveof the soundsemitted, organat thebase former. Shipleyand Wilsonbelievedthatthestridulating (1911) also pernote.Pemberton of thewingswasresponsible forthehigher whichconfirmed thefactthatthebuzzingof the wing formed experiments of themagainstan adjoinor a striking bases,in Dipteraand Hymenoptera, thesound.Kahn,Celestin, and Offenof thebodywallproduced ingportion fromthe mosquitowere hauser(1945) determined thatsoundsemanating thewingswhenat rest,and (3) (2) bybeating produced(1) duringflight, the hindtarsiagainstthe wings.Pure "bird-like" soundswere by rubbing Shipleyand Wilson (1902) but theiroriginwas not determined. recorded secof themosquito mayfunction thepossibility thatthehalteres suggested a sound;thehalteres forthe"birdin producing maybe responsible ondarily byKahnet al. like"soundsrecorded It is apparent thatthe soundsproduced by femaleaegyptiwhileat rest sincemalesignore females in mating and attempt areofno significance resting or vibrates herwings.Banks to copulateonlywhenthe femaleis in flight of aegypticonfinedin small jars (1908, p. 246) statedthatspecimens ". . . havebeenseento copulatewhilethefemalehangsfromthegauzecovherfromtheventralsurface." eringthevessel,themalealwaysapproaching themaleinitiated canbe takenliterally, whilethe If thisstatement copulation wasa ,rnale seento initiate femalewasat rest.Neverin ourobservations copufemale.Kahnet al. (1945) inducedmalesto turntheir lationwitha resting of a recorded in thedirection femalesoundbutthey antennae and hypopygia did notstatewhatfemale"call"wasusedas thestimulus. Femaledegyptiwithabouta thirdoftheouterendofeachwingamputated withthem.Femalewings werecapableof flight and malesreadilycopulated fromtheirbasesand thesemosat variousdistances werecutoff,bilaterally, in a cagecontaining males.Males wereattracted to quitoesweresuspended whichhad onlysmallstumpsof thewingsvibratwithfemales and copulated also to femalesvibrating bothwingsbut lacking ing.Males wereattracted weresuspended in the cage since The halterectomized halteres. mosquitoes theirwingsmalesimmediately theycouldnot fly,and whentheyvibrated Males ignored females. wingless seizedthemandcopulated. offmoreand moreof Nuttalland Shipley(1902) foundthatby cutting


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thewingsofA. maculipennis thesounddecreased in volumeandthenoterose progressively. Whenthewingswerecutoffquitecloselya veryhighpitched noteof slightintensity remained. The soundsproducedby thecutwingsof in pitchfromthosemadebynormal aegypti aredifferent females during flight. The factthatmalescopulatereadilywithfemalespossessing onlya partof theirwingsindicates that(1) themalesare capableof beingstimulated by varying pitches produced bythefemalein flight, or (2) themalesare being stimulated bythesoundproduced bya stridulating organlocatedat thebase of thefemale's wings.Proofthatthemaleresponds to variouspitches willbe shownin thenextsection(see p. 282) andunderthestudyof thebehavior of malesto mechanically produced sounds(see p. 326). Few workers haveattempted to determine thefrequencies of the sounds produced bymosquitoes butit is generally knownthatthesounddiffers with thespeciesand thatthemalesin flight producea soundwhichis distinctly inpitchthanthatmadebythefemales higher ofthesamespecies.Nuttalland Shipley(1902) believedthatthisdifference in thesoundsof thesexeswas due to thefactthatthemalewingsweremarkedly narrower than and shorter thoseofthefemale. Weber(1906), Banks(1908,p. 248), Kahnet al. (1945), and othershavebeenableto recognize'different speciesof mosquitoes by the soundstheyproduced in flight. Kahnet al. successfully recorded thesounds givenoffby A. aegypti, A. quadrimaculatus, A. aIbopictus, and C. pipiens; thesesoundswerefaintly audibleor completely inaudibleto thehumanear and rangedfrom250 to 1500cyclespersecond.NuttallandShipley(1902), withtheaidoftuning forks, determined thefrequencies ofthesoundsproduced by themalesand femalesof A. maculipennis in flight. Theyfoundthatin bothsexesthepitchof soundwas higherwhenthemosquitoes h-lad fed,the greater themealthehigher thenote.One unfedfemalegavean abnormally low noteof about175 vibrations; threeothersproduced soundsfromabout 240 to 270vibrations persecond.Threeunfedmalesgavenotescorresponding to 880 vibrations thoughovertones wereheard.Goeldi(1905) usinga zither and tuningforksconcluded thatthesoundmadeby femaleaegyptiin flight wasoneof480 vibrations whilethatofthemalewas880 cycles.He indicated also thattheextent of dilation of theabdomen withfoodinfluenced thepitch of thesoundproduced. Chadwick(1939a) poinited out the factthatmatching an insect'sflight soundwiththetoneof an instrument of knownfrequency (i.e. tuningfork, etc.) is inaccurate since"The relation between thepitchof an insect'stone of itswingmotionis stillnotcompletely and thefrequency understood, and in manycasesit appearslikelythatharmonics maybe mistaken forthefundamental.For thisreason,and becauseof difficulties to be introduced tlhought notmuchattention bytheDopplereffect, has beengivento thismethodby laterworkers." Cihadwick theuse of theEdgerton in-troduced for stroboscope thestudyof insectflight and employed thisinstrument in determining the inDrosophila(1939b,1943;seealsoReedetal. 1942). ofwing-beat frequency In thepresent of soundsmadebymosquitoes-,were work,thefrequencies not determined butsoundrecordings weremadeof flying individual males


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ofA. aegypti andfemales The pitchvariednotonlv andA. quadrimaculatus. withthe sex, species,and extentof feeding, but also withage. Recently emerged malesand femalesproduced soundsin flight whichweredecidedly lowerin pitchthanolderindividuals. The femalewiththeunusually lownote of about175cycles, mentioned byNuttalland Shipley, hadprobably recently emerged. The recordings indicated thatthe pitchof soundproduced by an is variable. individual mosquito during flight The majority of observations thematingof mosquitoes concerning indicatesthatcopulation is initiated in flight generally and it is probablethatin thesespeciessoundplaysan important role in matingbehavior. However, thereareseveralinstances in theliterature whichindicate thatsoundis ootor in thecopulation maynotbe significant of certain speciesof mosquitoes. The New ZealandspeciesOpifexfuscusHutton,originally as a tipulid described (Hutton,1902) was latershownto be a culicinemosquitoby Edwards (1921). Edwards(1921) and Miller(1922) described certain morphological peculiarities of themale,mainlyconnected withseconldary sex characters, and of possiblebiological significance. The male antennae are sparsely feathered and verysimilarto thefemale's;his frontpairof legsare short,stout,and withverylong,simpletarsalclaws.The remarkable matingbehavior ot this speciesdeparts fromthatknownto occurin othermosquitoes. markedly Kirk (1923) wasthefirst to describe thisbehavior and hisobservations weresummarizedlaterbyEdwards(1926). Briefly, thematingbehavior is as follows: The malesof Opifexreston thesurface ofpoolsandwithin 10 to 20 minutes afteremergence, seekout,and bymeansof theiran-lforceps, seizea mature thepupalskinandworkshisanalforceps pupa.The maleruptures backwards, theslit.The imagogradually extending emerges withtwoor threeabdominal ofthemalewithin segments thepupalskin.If theemerging imagois a female. connection is madebeforeherabdomen is quiteclearof theskin,and when emergence is complete is concluded copulation in theend-to-end the position, mosquitoes on thewater. resting The actlasts3 to 7 minutes andwasobserved. to lastoverhalfan hour.The proboscis and front clawsof themaleareused a pupais seized.Femaleswhichemerge tc wardoffrivalmalesafter unaided, as a ruleareunmolested thatthesoundproduced bymales.It is apparent by thefemaleof Opifexduring flight playsno rolein mating behavior. A. G. Richards (1941,p. 213) briefly thecopulation described of a stenogamicautogenous strainof Culexpipiensand notedthatthemalerestedon thesideof theaquaria". . . and thefemaleseemedto be theaggressor since she flewaroundthemaleand eventually cameto reston topof him.Copulationensued,themaleappearing passivethroughout theentireperformance." Tate and Vincent(1936,p. 136) foundtheopposite to be thecase forthey notedthatmalesof theEnglishautogenous raceof C. pipienssettledon and forcedtheirway beneathresting females, copulation takingplace withthe females at rest.In either wasinitiated case,ifcopulation withoneofthesexes at restthenthesoundproducedduringflight was notthematingstimulus. theautogenous jobling(1937) rearedseveralspeciesof mosquitoes, including race of pipiens,in completedarkness. This specieswas able to producea


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(pp. 473-474)stated,"The male and Jobling in darkness secondgelneration to it either it is attracted therefore darkness; in complete cannotseethefemale or bybothof these bythescent,or thesoundwhichthefemalemayproduce, factors." on thematingof Anopheles and Hill's (1940) observations Camboumac raisea similarquestion.When the malesof this atroparvus maculipennis and females, speciesbecomeactivetheyflyalongthewall,"attack"resting whcnthemalemomentarily attack, "The preliminary matewithsomeofth-em. the latteris whether determines touchesthebackof the female,apparently thelatter If so, themalemovesaroundand up underthefemale, receptive. or half-flying of theformer by flying to the efforts herself accommodating howthemale to imagine stillagainstthewall."It is difficult withtheforelegs her back.However,if his touching the femaleby motnentarily recognizes of her vibration themomentary herinducesherto flyor "half-fly," touching to inducethemaieto copulate.This also wouldbe sufficient wingsprobably raceof on theautogenous observations maybe thecasein Tate andVincent's pipiens.

offers someindication of themaleantennae thatthestructure It is possible of different species.Edwards(1941, p. 35) though of thematingbehavior did recordthe of Harpagomyia the matingbehavior actuallynot observing thenon-plumose female,". factthatmalesmayhoverarounda resting in theair of themalesuggestthatmatingswarmsare notformed antennae as in mostothermosquitoes."Amongour mosquitofauna,at least two moreor lesssimilarin structure specieshavetheirmaleand femaleantennae cancer in thatthemaleantennae TheseareDeinocerites plumose. aresparsely are of Deinocerites lowiiTheob.The maleantennae Theob.and Uranotaenia ofsensillahairson all theflagellar butthepresence feathered notonlysparsely Littleis knownabout to thefemale-s. makethemstillmoresimilar segments of this on thebiology of D. cancer.Thoughobservations themating behavior et al., 1946, byKnab (1906b), Duffy(Carpenter specieshavebeenrecorded p. 277), and Grabhamand Low (Theobald,1903,p. 279), the attitudes inflight wasnotrecorded. wasinitiated ornotcopulation andwhether assumed The observations made by Fisk (1941) on the behaviorof Deinocerites during mating wasinitiated whether spanius(D. andK.) alsodidnotindicate copulating Pratt(1946) collected described. norwasthematinig position flight on thesuror floating on Lemnafronds vegetation pairsof U. lowiiresting was whether copulation places.It was not indicated faceof theirbreeding or at rest. flight during initiated .

.

have mosquitomatingbehavior concerning of observations The majority Aedes,and Culex.Paine(1934) notedoneinstance beenmadeon Anopheles, and tookplace whichlastedtwominutes of coitionof Megarhinus splendens of sevbehavior themating Hovanitz(1946) described withthepairsettled. nothing Practically mosquitoes. Haemogogus eralspeciesof SouthAmerican of othergenera. behavior i< knownofthemating


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ATTEMPTED COPULATIONS BETWEEN AEGYPTI MALES, AND BETWEFN AEGYPTI MALES AND OTHER SPECIES OF CULICIDAE

Occasionallymale aegypti wereseento attemptcopulationwithothermales. However,this was not a commonoccurrencewhen both sexes of about the same age werein a cage. When large numbersof males were isolatedfrom females,the frequencywith whichmales seized othersof the same sex was

increased greatly. One cagecontaining severalhundred virginmaleswas dis-

turbedforhalfan hourand duringthisperiod59 pairsof maleswerecounted as theydroppedto the floorof thecage and attemptedto copulate (those that did not drop to the floorwerenot counted). In mostinstancesthe aggressive male would flexhis abdomenin a definiteeffortto nmake connectionwithhis male partner.When the males werenot inducedto flyby agitatingthe cage thenumberof attempted thischiefly copulationswas less thantheabove figure, beingdue to the factthatthemosquitoestendto reston the sides of the cage. The same males in whichthe above 59 attemptedmatingswerenoticed,were observedfor one-halfhour withonly an initialjarringof the cage. Only 11 attemptedcopulationstook place and theseoccurredduringthe first10 minutes afterthe cage had been movedon the table. Comparatively fewmosquitoes werein flightwhichindicatedthattheseattemptedmatingswerenot due to accidentalcollisionsin mid-airwhichmightbe possiblewhenmanymosquitoes wereflying. While observinga cage containingvirginmales only,mostly5 days old, an unusuallylarge numberof attemptedcopulationswas seen to take place. Two days before,a beakercontainingseveralhundredmale pupae had been placed in the cage and the adults had begunto emerge.Upon removingthe struggling pairs,it was foundthatin almostall cases one was a youngmale onlyrecently emerged(shownby the factthatthe genitaliawererotatedonly slightlyor not at all). The followingexperiments wereperformed.Eighteen non-virgin males,3 days old, wereplaced in a cage and disturbedfor 15 minutes. During this periodonly one attemptat copulationwas observed.Four youngmalesthegenitaliaof whichhad notyetbegunto rotatewereintroduced into this cage and the mosquitoeswere disturbed.Immediatelythe young males wereseized by the old ones whichattemptedto copulate.The cage was disturbedfor 15 minutesduringwhichtime 98 attemptedcopulationswere made by the old malesupon the youngones; the youngmales could be recog12 virgin nized by theirsluggishwalk and flight.In the second experiment, males,4 days old, 2 virginfemales,4 days old, and 2 males,2 hoursold, were placed in a cage togetherand the mosquitoesweredisturbedfor 15 minutes. There were55 attempted copulationsbetweenthe 4-day-oldmalesand females (these werenot dividedinto successfuland unsuccessful copulations)and 49 males. Thus it is obattemptedcopulationsbetweenthe 4-dayand 2-hour-old vious that old males cannotdifferentiate betweenveryyoungmales and old females,bothbeingabout equally"attractive." Usually an old male would attemptto copulate with a young male whereasseveralmales would pursue a female. The sound of the male mosquitoin flightis muchmorehigh-pitched than


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thefemale.The pitchof thesoundof a maleis largely dependent uponthe of vibrations number persecondof itswings.The flight of recently emerged malesis veryslowandthepitchofsoundproduced bythesemalesis distinctly lowerthanthatmadeby oldermales.The difference between the pitchof malesis clearly soundmadebyold andrecently emerged heardin a veryquiet room.Sincethesoundof thefemaleis theexternal stimulus whichinduces malematingbehavior, the inability of sexuallyactivemalesto differentiate between youngmalesand old femalesis due to thefactthatthesoundof theyoungmaleis reducedto a frequency whichresembles or falls thefemale, within therangeof soundwhichactsas a mating stimulus. Sinceaegypti malesrespondto a rangeof soundrather thanto a fixed stimulus required to pitch,and sincesoundappearsto be theonlyexternal :nducemating, thentheoretically malesof aegypti shouldattempt to copulate withotherspeciesof mosquitoes if thesoundmadeby themfallswithina stimulating range.The behavior of aegyptimalestowardsseveralspeciesof in thefield, wasobserved in thelaboratory. Culicidaecollected Variousspecies of mosquitoes wereintroduced intoan 11" x 11" x 15" cageandtheirrelative "attractivelness" was determined by observing whether or not male aegypti to copulatewiththem,afterthemosquitoes wouldattempt weredisturbed and aresummarized in thefollowing inducedto fly.The findings table: Species 1. Anopheles quadrimaculatusSay

2. Aniophelespunclipennis(Say),,,,,,. 3. Culex restuansTheob................. ... 4. Culex pipiens L.

5. Aedes trivitallus(Ccq.) -Females

Behaviorof male aegypli . ,,,,,,Malesand femalespursuedby virginand non-virginaegypli. Attemptsto seize the male anophelinewhichproducesa soundof higherpitchthanthe femaleappearedto be less persistent thanattempts to seize the female. The aegypli male had difficulty in seizing the female quadrimactulatus which has verylong legs,but if he did manageto cling to her a definiteeffortwas made to clasp her genitalia.This was demonstrated a tetheredfemalethelegs of by suspending whichhad been amputatedat the apices of the femoraso that she could not struggle with the males when theyseized her. Males and femalesvery attractiveto 2,V2 day-old virgins. Females appeared to be littleor not at all

attractive.

Males very attractive to 4I,2-day-old virgins.Engorgedfemale pipienswere attractiveto 232-day-oldvirginmales; nonengorgedfemales appeared less attractive than male pipiens to 7-day-old virgin aegypli males (feeding raises the pitchof soundproducedby flyingfemales). virgins.

very attractiveto 412-day-old


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6. Aedes vexans (Meig.)-,

Males and femalesvery attractiveto 7day-old virgins.

7.Psorophorahorrida(D. and K.) -

Females very attractiveto 4t 2-day-old virgins.

8.Psorophoraciliala (Fab.) -

Virgin aegypli males, 2?/2 days old were indifferent to a femaleof thisculicine.

9.Psorophoraconfinnis(Lynch-Arr.)

to 6-day-oldvirgins; Males veryattractive than males femalesappeared less attractive to 7-day-oldvirginmale aegypli.

willattempt tableshowsthatmaleaegypti oftheforegoing An examination of sex,thesoundof whichmade to copulatewithanyCulicidaeregardless stimulus. The sound fallswithin therangewhichactsas a mating flight during fallsoutsideof thisstimofP. ciliataapparently bythelargefemale produced bymaleaegypti. rangeso thatsheis unmolested ulating F., Merodonequestris a malesyrphid, Richards(1927,p. 316) observed malesbehavedin a similar Eristalis honey-bee; to matewitha worker attempt ". . . any insectwhichhas a bee-like hum,suchas they pursuing manner, .. ." ThoughGruhl(1924) believed theseactsdueto errors makethemselves, in thisbeof sight,Richards suggested thatsoundwas equallyimportant showing thatsoundis thematingstimulus experiments havior.The present young foraegyptimalesand thatsexuallyactivemalescannotdifferentiate inflight is lowerthanthatofoldermales) males(thepitchofwhich produced indicate old malesand females, between butcandistinguish fromold females, forthisbehavior. thatsoundis thechief,if not theonlyfactorresponsible the factthatodorplaysno partin the All of theseobservations emphasize to a single of aegypti andalsothatthemaledoesnotrespond sexualbehavior bya rangeof soundfrequencies. fixedpitchbutis capableofbeingstimulated ROTATION OF THE MALE GENITALIA AND AGE OF MALE AT FIRST COPULATION

malestendto reston thesurfaceof thewaterfora few At emergence, flyto thesidesof thecagewheretheyremain afterwards andshortly minutes are said to havea "hypopygium forsometime.Male mosquitoes quiescent 1922) or "terminaliainverted"(Hardy, 1944). inversum"(Feuerborn, and showedthatin inversion in mosquitoes (1915) mentioned Christophers so thateventuof 180degrees a rotation undergo maleCulicidaethegenitalia to the seventhoccupya positiondiametrically posterior ally the segments The rotationin the oppositethatwhichtheyfirstoccupiedat emergence. 24-48hours tookplacewithin studiedby Christophers speciesof mosquitoes rotation afteremergence. Marshalland Staley(1932) foundthathypopygial had whentheair in themosquito mid-gut reached completion to 180 degrees Marshall (1938, p. 289) been expelledinto the oesophagealdiverticula. have observations laboratory statedthat"In thecaseof at leastmanyspecies, of thehypothatthemalesare able to mateas soon as therotation slhown


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pygiumis completed-aprocesswhichtakesfromtwelveto twenty-four hours." male aegyptiwereisolatedat emergence Ninety-two and examinedat intervals to determine thelengthof timerequiredforrotation of the genitalia.At variousintervals someof thesemaleswereintroduced intoa cage femalesto determine at whattimeafteremergence containing copulation wouldoccur.It was foundthat (1) rotation couldbe clockwise or counter clockwise, and (2) therateof rotation was quitevariablebetween different males(at roomtemperature). Rotation(Figs. 2-5) was first 1 to noticeable 4 hoursafteremergence and in some individuals the genitalia had rotated about90 degreesas earlyas 5 hoursafteremergence. theratewas However, not uniform and rotation oftenceased for hoursafterthe genitaliahad reacheda certainangle.Most of the malescopulatedbetween 20 and 24 hoursafteremergence and in theseindividuals rotation was completed at this time.However, somecopulated between 15 and 19 hoursafteremergence and in thesetherotation also waspractically complete. Apparently complete rotationwasnotnecessary beforemaleswouldattempt to copulate.Manymales witha slightly less than180 degreerotation wereable to copulatesuccesswas successful whenconnection fully,assuming copulation withthe female genitalia was made;severalmalesthe genitalia of whichhad rotatedabout

t---7

T

_

_--v

---1

L

T

X

~~ ~~~~~~7T--- -_

D--?__esT

Figs.2-5. Drawings of theterminal abdominal segments of maleAedesaegypli showing different in rotation stages of thegenitalia. 2. Shortly afteremergence, no notethemembranous areas(M) of thebasistyles (B) andtheabsence rotation; of thelateral palescalesontheeighth 3. Genitalia rotated about45 degrees; the tergite; membranous areaoftheright is nowinvisible basistyle from indicates above;thearrow thedirection ofrotation; 4. Genitalia rotated 90 degrees. 'fhelateral sideofthebasiis nowseenfrom style as is oneof thelateral above, palespots(8LS) of theeighth abdominal sternite; 5. Genitalia completely rotated 180degrees; theoriginal ventral surfaces ofthebasistyles arenowdorsal andthemembranous areasarenolonger visible from thedorsal surface; theeighth sternite (8S) liesinthetergite position anditstwo lateral orlessinlinewiththose aremore palespots onthepreceding segments. (Drawings outlined from photographs; theabdominal segments shown inFigs.2 and3 areslightly swollen andremain so fora short while after emergence.) Abbreviations: B-basistyle; D-dististyle; M-membranous region of thebasistyle;7T-seventh abdominal tergite; 8T-eighthabdominal tergite; 7LT-lateral palespotontheseventh abdominal tergite; 8LS-lateralpalespotontheeighth abdominalsternite; sternite. 8S-eighth


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135 degreesattempted copulation and thoughmostof themwereunsuccessfulone maledid claspthefemalegenitalia and remained joinedforseveral seconds.Onlya fewindividuals required morethan25 hoursforrotation to be completed. It willbe recalled thatOpifexfuscusmaleswereobserved copulating10 to 20 minutesafteremergence (Kirk,1923) and thisraisesthe questionas to whether or not theirgenitaliahad rotatedwithinthisshort period. Although themalegenitaliamustundergoa nearly180 degreerotation beforecopulation can be successfully it was evidentthatmales completed, to matebeforetheirgenitalia wouldattempt had rotated sufficiently to insure successful copulation. Induction of mating on thepartof themaleapparently is dependent of theorganswhichreceive uponthecondition themating stimuius (i.e., the soundof thefemalein flight)and thesestructures are the antennae (see pp.293 and321). thegenitalia ofbothsexesarenecessary Obviously forsuccessful copulation and,in themale,mustundergorotation; however, theremovalof thegenitalia does notaffect thematingbehavior pattern. Non-virgin males,3 days old, whichhad theirabdominal segments posterior to theseventhremoved, readily attempted to copulatewitheither normalfemaLes or females whichhad theirgenitalsegments removed. AGE OF FEMALE AT FIRST COPULATION;

"ATTRACTIVE"

AND "UNATTRACTIVE" FEMALES

Afteremerging fromthepupalskin,femalesalso reston thesurfaceof waterfora shortwhile.Soon afterwards theyflyto thesidesof thecageand remainquiescentforseveralhours.Nineteenfemaleswereisolatedjust at emergence and placedin small1" x 3" screenwirecages.Femalegenitalia do rotation.At variousintervals thesefemaleswereliberated fora notunciergc 10-minute males. periodintoan 11" x 11" x 15" cagecontaining 15,4-day-old The mosquitoes weremadeto flybyjarring thecageat intervals. The earliest 50 minutes afteremergence copulations occurred (4 females).Most of th! tookplaceabout105 to 145minutes afterthefemales matings had emerged. Malesignored 2, 3-hour-old females. It wasapparent thatveryyoungfemales werenotas attractive as olderones.Malesignored a veryyoungfemaleflying withone nearlytheirowll age amongthemwhiletheywouldmateinstantly or older.Two examples can be noted:malesattempted 6 times copulation 2 unsuccessful) witha 2-hour (4 successful, old femaleduringa 10-minute 7 24 times(17 successful, whilethesamemalesattempted period, copulation withan olderfemaleduringa similar Another unsuccessful) female exposure. afterheremergence placedwithmales135 minutes copulatedonlyonce in 17 timessuccessfully 10 minutes whilethesamemalescopulated andattempted 10 moreunsuccessful matings duringthesameperiodwithan olderfemale. In bothoftheabovecasesthemosquitoes weremadeto flybyjarring thecage. whichinducesthemaleto copulateis thesoundproduced The stimulus With thisin mindthe relative unattractiveness by thefemaleduringfflight.


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emerged The flightof the recently of theseyoungfemalesis explainable. The sound likethatof themale,is veryslowand of shortduration. female, is lowerin pitchthanthatprobytheveryyoungfemalein flight produced can be easilyheardin a veryquiet ducedby olderfemales;the difference thepitchof thissoundis belowtherangewhichwill roomand apparently stimulate themaleto copulate. thatpartlyor fullyfed Hinds (Howardet al. 1912,p. 275) observed Sevto malesthanunfedindividuals. seemedmoreattractive females aegypti wereperformed usingvirginfemalesof thesameage,some eralexperiments and still others fedon honeysolution, sinceemergence, of whichwerestarved othersfedon blood.Thesefemaleswereabout2 daysold whenintroduced males.It was foundthatstarved and madeto flyaboutin a cagecontaining of thanfemalestheabdomens duringflight, wereno less attractive, females withabdomens withhoneysolution;thatindividuals whichweredistended withblood,themealhavingbeentakenabout12 hoursprior stilldistended than to thefemales beingexposedto themales,also wereno moreattractive afterhavinghad a immediately whenmosquitoes However, starvedfemales. few, blood meal were placed with males,' copulationswere comparatively femaleswerereluctantto flyalthoughthe cage was becausetheserecently-fed violentlyagitated.Even whenmade to flythe flightwas of veryshortduration, the femalesmovingslowlyand heavilyfromone restingplace to the next.Finlay (Howard et al. 1912, p. 260) observedthat aftera blood meal, aegypti ". . . is very sluggish and flies with difficulty;it seeks a hiding place

whereit remainsquiescentduringthe processof digestion."Thus it is most had a blood femaleswhichrecently unlikelythat,in natureand the laboratory, (1921) notedthatmalesof Aedes (Ochmeal willcopulate.Wesenberg-Lund le otatus) fletcheri copulatedwithfemalesthe abdomensof whichwere distended with blood which,because of its black color, apparentlyhad been takensome days earlier. due to a in attraction It is probablethatHinds was observinga difference in age of the females.The partiallyor fullyfed femalesin Hinds' difference may have been olderthan the unfedindividualswhichmay have observatiolns emergedrecently.The sounds of the femalevarywiththe extentof feeding but apparentlystillfall withinthe rangewhichwill act as a matingstimulus. THE "MATING-STATE" IN MALES

newlyemergedmales were placed in a cage and afterthey Twenty-five wereabout 25 hoursold, 10 femaleswereintroduced.Copulationtook place The cage was observedand agitatedseveraltimeseveryday (day immediately. and tight) to determinehow long the males would continueto copulatc. Over a 10-dayperiod (until the last male died) copulationsoccurredwheneverthemosquitoesweremade to fly.Thus oncemaleswerecapableof mating and began to do so, theyremainedin a constant"matingstate"throughout theirlife-timeand copulated readilyat any time,day or night,when the properstimuluswas supplied. Males show a definitemating responseto producedsound long beforetheyattemptto or are capable of mechanically


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copulating; apparently youngmalesmaybe in a mating-state thoughtheydo notattempt to copulate withfemales(see p. 324). INCREASING THE FREQUENCY

OF COPULATION

Howardet al. (1912,p. 275) quotedtheworksof Frenchinvestigators ofaegypti. "When whoclaimedthattemperature thesexualactivity influenced thetemperature risesabove250 C. (770 F.) themalesbecomeveryactive butrarely Whenthetemperature and thefemales escapefertilization. ranges 200 and250 C. (680-770 F.) fertilization is stilltheruleandthe between proportion of unfertilized females is verysmall,butbelowthistheproportion of unfertilized in temperature." females increases rapidly withthereduction We havesecnin thepresent studythatoncedegypti malesreacha state wherein theyare capableof copulating, usuallyabout15 to 24 hoursafter emergence, theycontinue to matewhenever femalesconfined withthemare madeto flyin thecage.The stimulus whichinducesthemalesto copulate is thesoundof thefemaleduringflight. Therefore anyfactorwhichinduces of increasethe frequency bothmalesand femalesto flywillautomatically observations withregardto copulation. Rudolfs(1922) madethe following behavior of Aedessollicitans at 'various thae and Aedescantator temperatures. At 170 C. theywereinactiveand at 200 C. theycrawledaboutwhenthe tubescontaining themwereshaken;at 250 and 300 theywerelivelyand active;at 350 theyflewaboutwithveryshortrestperiods;at 370 and 38' theyflewconstantly andat 390 theyflewwithshortlestperiods;between 40? and 460 themosquitoes becamelessactive,quiescent, and finally died.Aedes to showthegreatest aegypti hasbeenreported activity at about280 C. (Howatd et al. 1912,p. 271), temperatures beyond39? C. beingfatalwhilebelow 15? and 160 C. femalesbecamesluggish.Gutzevich(1931) statedthat 28? C. was theoptimum temperature foraegypti whileAragao (1939) believedthat270 to 320 C. was the optimum.High temperature does not increasesexualactivity itselfbut by its thermo-kinetic5 actioninducesthe to fly,thesoundof thefemaleincidentally mosquitoes activating themales, to copulate. whichattempt -Merelyagitating a cagecontaining aegyptialso inicreases "sexualactivity"' becausebothsexesaremadeto fly.Tate andVincent(1936) foundthesame to be trueof Culexfortheynotedthattheslightest disturbance whichcauses theadultsof autogenous C. pipiensto flyinducespairing."For instance, if a cage of recently emerged adultsis brought intothelightfromdarkness, pairingbeginsactively and females maybe surrounded by fiveor six males to pair.The sameresultarisesfromtappingthecageor blowing trying into is essentially it." Thisbehavior thesameas thatof aegypti. Hovanitz(1946) in hisstudyon Haemogogus mosquitoes notedit was difficult to distinguisl between matingactivity and generalflight activity aiid that"In thecagesit 5 Fracnkeland Gunn (1940) definekinesisas undirectedlocomotory reactionsin whichthe speed of movement dependsupon the intensity of the stimulation. The prefix indicatesthephysicalnature-qfthestimulus.


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thatshowedthe largest was the specieswiththe greatest generalactivity frequency of copulations and tlhose withverylittleactivity thatcouldnotbe matedat all." THE

ROLE OF THE MALE ANTENNAE

of Structure of themaleantenna.-Themaleantenna(Figs.6-8) consists a scape (segment 1), pedicel(torus;segment 2), and 13 flagellar segments, The scapeis a narrowirregular makinga totalof 15 antennalsegments. to thegreatly-enlarged sclerotizcd ringconnected bya membrane pedicel(Fig. 6A). Child(1894) described thehistology of theorganfoundin thepedicel of Nemocera(i.e. Culex and Corethra)and namedthe structure after Johnston (1855) whofirst suggested itssensory funccion. Sectionsweremadeof theheadsof newlyemerged malesand it aegypti6 was foundthat Johnston's organin this speciesis verysimilarto that cf the males of Culex pipiensand Corethraculiciformis Degeer, describedby Child (see Howard et al. 1912, pp. 29-33 and Plate 1). Figure6 is a diagrammatic representation of a longitudinalsectionthroughJohnston'sorgan.Briefly, the consistsof numeroussensecells (SC) withtheirdistalends attached structure to sclerotizedprocesses(SP) of a circularplate (P) whicharisesfromthe base of the thirdantennalsegment;the sense cells are arrangedaround the antennalnerve (AN) and are innervatedby sensoryfibersfromthis nerve. The distinguishing featureof chordotonalor sound leceptiveorgans (sensilla scolopophora) is the presenceof peg-shaped"sense-rods"or scolops at the apex of each sense cell and thoughJohnston's organis generallyclassedwith phonoreceptors, its sense rods are not typicallyscolopoid (Snodgrass,1935, p. 528). TIhe first11 flagellarsegmentsare shortenedthoughthe firstis somewhat longerthan the following10 segments(Fig. 6A). Each of the 11 flagellar segmentshas a subapicalor nearlymedialwhorlof numerouslong fibri'laeor hairs wllichdiffersomewhatin lengthon each segmentand becomeshorter towardstheapex of the antennae.The fibrillaeextendin all directions, dorsally, ventrally,and laterally;they form nearly a completering around the segmentusually with a small gap betweenthe hairs on one surfaceof th2 antenna(Fig. 7). The firstflagellarsegmentfitsintothe hollowof the pediceland is diarkly pigmentedexcept for a small irregularcolorlessarea below the subapical whorl. The pigmentedarea bears scales, minutespicules,and several long setae,whichare shorterthan the fibrillaeformingthe whorls.Th2 apical portion of the segmentdirectlyabove the whorlis pigmentedand spicular.The secondand thirdflagellarsegments(antennalsegments4 and 5) are similar, each withan irregularpigmentedbasal spicularringwhichis narrowerthan that found on the firstflagellarsegment;a colorlebsarea below the whorls bearsa fewspiculesor finehairsand the portionabove the whorlsis spicular 6 Fixed in Carnoy's; cut at 5As; stainedwith trioxyhaematin and Van Giesen's; some sectionswere also stainedwithDelafield'shaematoxylin.


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4 to 11 aremoreor lesssimilarwiththe and pigmented. Flagellarsegments in widthtowards Each segments decreasing slightly theapexof theantenna. of thesesegments hasa narrow, regular, basalpigmented ring,withthenearly colorless portion abovetheringbearing a fewsmallspicules.An apicalpigof The regionof insertion mented spicular ringliesaboveeachof thewhorls. The twelfth thewhorls on eachsegment is slightly enlarged. and thirteenth flagellar segments (antennalsegments 14 and 15, Fig. 8) differ markedly ones.Theyare considerably fromthepreceding elongated and thewhorlof is inserted on an expanded areaabovethebaseof the fibrillae of thetwelfth joint;a smallcleararea is foundbelowthewhorlas wellas a verynarrow The regionbeyondthewhorlis pigmented ringat thebaseof thesegment. and possessesnumerous darklypigmented longsetaeand manythin-walled fromdistinct The terminal sensilla arising depressions. segment is moreloosely thantheothers. Its whorl, whichconsists articulated usuallyof 8 longfibrillae,

0

3AS A~~~~N

p

Fig. of the antennaof male Aedes aegspti. Diagrammaticdrawingof a 6.-Parts longitudinal sectionthrough the first, second,and partof thethirdsegments, showingthe

structureof Johnston'sorgan within the pedicel. (See Fig. 6A-8 for abbreviations.)


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First four antennal Figs. 6A-8. Parts of the antenna of male Aede.saegypti.-A. segments; 7. Fourth and fifth antennal segments showing the slight gap between the fibrillae; the full length of the hairs is not indicated; 8. Terminal three antennal segments. (All figuresdrawn to same scale.) SC-sense cells; SP-sclerotized Abbrev7iations: AN-antennal nerve; P-plate; antennal segment process of plate; lAS-first antennal segment (scape); 2AS-second (pedicel; torus); 3AS-third antennal segment (first flagellar segment); 4AS-fourth antennal segment; 5AS-fifth antennal segment; 13AS-thirteenth antennal segment; 14AS-fourteenth antennal segment; 1 5A5fifteenth antennal segment.


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Figs. 9-13. Heads of differentaged males of Aedes aiegypti.9. Dorsal view of head shortly after emergence, most of the antennal fibrillae are recumbent; 10. Lateral view of head of a male of about the same age as the one shown in Fig. 9, note the, 1 wset-feather-like appearance of the antenna due to the recumbent hairs;11 Lateral view of head, older than the ,one in Fig. 10, showing the fibrillae beginning to extend away from the shaft; 12. Dorsal view of head about 24 hours after emergence. Most of the fibrillae on the basal half of the antennae are well separated from the shafts; some of the fibrsllae on the apical portions still c1ing together and are more or less recumbent; 13. Lateral view of head about 2 days after emergence. All of the antennal


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is completely The segment pigmented nearthebaseofthesegment. Asinserted exceptforthesmallareabelowthewhorl,and is coveredwithmanysmall thin-walled sensilla;exceptforthebasalwhorl and theirattached depressions of fibrillae and a singleseta at the apex,otherlong setaewhichare fairly one.The areabsentfromtheterminal segment on thepenultimate numerous becauseof the irregular aredistinctly of thelasttwosegments lateralmargins similar The lasttwosegments of themaleantennae aresomewhat depressions. are of the female'sin thattheirsurfaces segments to thoseof theflagellar of the twosegments sensilla.The terminal thin-walled withnumerous covered organs. maybe chemoreceptive sensilla, possibly maleantennae, bearing hairsforming thewhorlstendto In recently malesthefibrillar emerged a wetandlie recumbent againsttheshafts, givingtheantennae sticktogether (Figs. 9-10). As themalesbecomeolderthesefibrilappearance feather-like malesmostof the lae extendawayfromtheshafts(Fig. 11). In 24-hour-old while fullyextended hairson thebasal halfof the antennaeare practically and clingtogethei thoseon theapicalhalfmaystillbe moreor lessrecumbent hairsusuallyare (Fig. 12). In males2 or moredaysold all of theantennal theyremainso extendedaway fromthe shafts(Fig. 13) and generally in someold malesseveralof thelifetime of theinsect;however, throughout The the whorlson the apicalhalfof theantennae may.be partlyreclined. are curvedand theirtipspointmoreor lesstowards theapexof the fibrillae antennae.

mating-state and copulate in a constant The factthatmaleaegypti remain the at anytimeaftertheyarecapableof perceiving and repeatedly frequently opportunity to studythebean excellent offers soundof thefemalein flight, possibly usedin mating, whichhavevariousstructures, haviorof individuals themalesaregivenan opportunfromtheoperation Afterrecovery removed. bycountto do so is determined and theirability ityto copulatewithfemales A ratioof 1 copulations.7 and unsuccessful ing the numberof successful to determine femaleto 5 malesin a cage11" x 11" x 15" wasfoundsufficient on the experiments the behavior of theseoperatedmales.In the following

antennae,and in the later ones on the legs, 15 males usually were used in and, unless otherwisestated,3 femaleswereintroducedone each experiment or more timesat different intervalsafterthe males had been operatedupon. copulationswerecounted,usually The numberof successfuland unsuccessful period,withthe cage beingagitatedto induceflightwhenduringa half-hour everthe mosquitoescame to rest. was performedto determinethe beThe followingseriesof experiments haviortowardsfemales,of male aegyptilackingparts or all of the antennal flagellarsegments. 7 See footnote 4, page 273. fibrillaeare extendedaway from the shaft (cf. Fig. 10). (Photographsof heads mountedon minuten pins; themouthparts have been removedfromthe specimensshown in Figs. 9-12.) Abbreviation:P-pedicel.


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Experiment 1.-To determine the behaviorof maleslackingthe last two antennal segments. differ markedly As has beenpointedout,thelasttwoantennal segments fromtheothers.These segments wereremovedfrombothantennaeof 15 virgin males.Someof thelonghairsfoundsubapically on theothersegments Three females wereunavoidably severedor removed duringthe operation. for2, wereintroduced intothecageat 16 and 32 hoursaftertheoperation, trials.The results half-hour wereas follows: 16-houririal.-8 successful;10 unsuccessful copulations. 32-hourtrial.-42 successful;34 unsuccessful copulations.

Conclusion. Maleslacking thelast2 antennal stillcanlocatethe segments femalein flight. removal Experiment 2.-To determine thebehavior ofmalesafterthebilateral of 9 flagellarsegments.

In 24-hour-old malesmostof the fibrillae on the basal halfof the antennae alreadyare extendedwhilemanyof thoseon the apical half still are moreor less recumbentand tend to stick together'(Fig. 12). However,these males and clinging locate femalesreadilythoughone would imaginethatrecumbent hairs are not capable of beingset into motionby the sound stimulusof the female (see p. 324). That thesemales can locate the femalewithoutthe use of thesehairswas shownin the following experiment. Nine flagellarsegmentswereremovedfromboth antennaeof 15, 3-dayold, non-virgin males. The remainsof the antennaeconsistedof a scape, pedicel,and 4 flagellarsegmentsand theirattachedfibrillae; sinceit was imposof the remaining not siblenot to cut or removesome of the fibrillae segments, all of the males had the same numberof hairsremaining. Three femaleswere introducedinto the cage for 2, half-hourtrialsat 6 and 30 hours afterthe operation.At the 6-hour trial there were 5 successfuland 2 unsuccessful copulations;at the 30-hourtrialtherewere 11 successfuland 17 unsuccessful matings. Conclusions.Males lackingall but the scape, pecdicel, and 4 flagellarsegof bothantennaestillare capable of locatingand seizingthe femalein -ments flight. Experiment3.-To determinethe behaviorof males afterunilateralremoval of 13 flagellarsegments. The flagellarsegments,cut close to the pedicel,fromone antennawere removedfromeach of 15 virginmales.The malesweretestedat 6, 21, 25, and 45 hoursafterthe operation.The resultsof these4, half-hourtrialswere as follows: 6-hourtrial.-Only a few attempts were made to seize the femalebut none were successful. at copulation. 21-houririal.-3 unsuccessful attempts 3 femaleswere used and therewere2 unsuc25-hourtrial.-For thefirst15 minutes,


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cessful attemptsat copulation; twelve more females were introduced making a ratio of 1 male to 1 female and during the second 15 minutes there were 6 unsuccessful and 4 successful copulations. 45-hour trial.-9 unsuccessful and 11 successful copulations.

Duringthe6, 21,and45-hour trials, 3 females wereused. thattheability ofthemaleto seizethefemale Conclusions. It is apparent is markedly of theflagellar of one antenna. reducedbytheremoval segments removal 4.--To determine of malesafterbilateral Experiment thebehavior of all theflagellar segments. The flagellar segments ofbothantennae wereremoved closeto thepedicelli from19 virginmales.Afterrecovery thesemaleswerereluctant to flyand evenwhenthecagewas agitated theywouldclingto thesides.Whenflight to disturb, did occurit was of shortduration. the males Thoughdifficult walkedabout.Sincemaleslacking of oneantenna made theflagellar segments fewattempts 19 females to copulate, wereintroduced intothecageso thatthe

ratioof malesto femaleswas equal. Two trialswererun24 and 54 hoursafter the operation.No attemptsto copulateweremade,the malescompletely ignoring the females. As a control,15 malesof thesame age as thoseused in Experiments 3 and 4, duringa half-hour periodwith3 femalesmade 67 attemptsto copulate,34 of whichweresuccessful. Conclusions.The male antennaeof aegypti are the organsused to locate the femalein flight.The relativeinactivity of flagellectomized males and the difficulty encounteredin disturbingthem indicatethat the antennaeperform a generalstimulatory function.This behavioris comparableto thatof Rhodnius (Wigglesworthand Gillett,1934) whichentera state of "torpor"after amputationof theirantennae.It was concludedthat one of the functionsof the antennaeof Rhodniuswas a kineticone whichmaintainedthe nervous systemin a responsiveconditionso that it would react readilyto stimuli. This same kineticfunctionapparentlyis found in the male antennaeof aegypti. Some observations on the behaviorof Anophelesquadrimaculatus.-Observationson A. quadrimaculatus weremade withmosquitoesconfinedin a cage ll" x 11" x 15", paintedwhiteon the inside. Copulationswere infrequent and occurredonly periodically.During the day bothsexes of this anopheline wereseen restingon thesidesof the cage or the femalesflewabout unmolested by the males. The male antennaeare of particularinterestfor the whorlsof bairscan be retracted(Fig.14) or extendedawayfromthe shafts(Figs. 15-16; cf.aegyptip. 291). During the day whentllemales are quiescent,theirantennal fibrillae remainflattened againstthe shafts(Fig. 14). However,on several occasions,particularly at night,males with extendedfibrillae(Figs. 15-16) wereseen flyingabout the cage and attempting to mate withfemales.In one maleswereseento beginto copulateat 6 p. m. and,the cage underobservation, followingmorningat 7 a.m., males withtheirfibrillaeextendedwere still to mate..Notall maleswiththeirfibrillae attempting extendedwereattempting


294


40 (2)

againsttheshaftswere butno maleswiththesehairsflattened to copulate, everseento pursuefemales. and between cyclically malesreacha mating-stage Thus quadrimaculatus of the and willnotcopulate.The extension theseperiodstheyignorefemales thattheyare readyto is a good indication male alitennalhairsapparently long bothsexesof thisspeciesis observed copulateand if a cagecontaining to copulatewith willbe seenattempting fibrillae enough, maleswithextended willpursuefemale maleswhicharein a mating-state females. Quadrimaculatus intothecage. whenthelatter areintroduced acgypti and Shipley(1901, of A. maculipennis, Nuittall theantennae Concerning usuallycarried thedaytime, that"The captivemales,during p. 460) observed thehairsbeingextended oftheantennae, thehairscloselyappliedto theshafts On oneoccasion (as in PlateI.) whentheybeganto flyaboutin theevening. this hairsafterbeingfedsugarandwater, theirantennal threemalesextended whentheywereactive.One of them,on comingto rest, beingin theevening These writers did not flattened downthe hairsagain withhis fore-legs." withregardto mating,of the to explainthe possiblesignificance attempt withtheirantennaI extension of the antenna!hairs.Male quaddrimaculatus on sugarsolutions. feeding fibrillae havebeenobserved recumbent, THE ROLE OF THE MALE LEGS

themalelegsplayan important and genitalia, In addition to theantennae thatthe male Lamb (1922, p. 10) suggested partin mosquitocopulation. sincenon-virgin butthisis unfounded havesomerolein mating, palpspossibly removed readily malesseveraldaysold,whichhadtheirpalpsand proboscides takeno part The palpsand proboscis and copulated. in flight locatedfemales of aegypti. behavior in themating the matingbeto determine wereperformed The following experiments was The methodof testing l)aviorof maleslackingvarioustarsalsegments. similar

to thatused in the precedingantennalexperiments.

tarsectomy thebehavior ofmalesafterunilateral l.-To determine Experiment ofthefront legs. males. leg fromeachof 15 virgin fromone front The tarsuswasremoved andattempted 38 timessuccessfully hourslaterthesemalescopulated Fourteen a half-hour period. with3 females during matings 37 unsuccessful afterthe removalof one Conclusion.Males catncopulatesuccessfully tarsus. front the behaviorof males lackingboth front 2.-To determine Experiment of thefront tarsi. tarsi,or thelastsegments The malesused in Experiment 1, alreadylackingone fronttarsus,had EighteenhoursLaterthe malescopulated theirotherfronttarsusremoved. to mate. attempts only3 timesbutmade100unsuccessful successfuliy



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295

using15 othervirginmales.Threefemales was repeated The experiment and therewere49 at 11 and 28 hoursafterthe operation wereintroduced copulations trialand 74 unsuccessful duringthefirst copulations unsuccessful occurred duringboth30-minute copulations duringthesecond.No successful pose.His middle themalefailedto gaina vertical trials.Almostinvariably hindlegsbutwhenshecameto reston the to seizethefemale's legsmanaged beyond on top,offto oneside,orhanging sideofthecagethemalewaseither withhisback fromherlegs.Oftenwhilein thelastposition, stillsuspended upwardso thateventuagainstthecage,he wouldflexthetipof hisabdomen and his ally he was on top of the femalewithhis head facingposteriorly low whensuspended in a regionaboveherwings.Sometimes genitalsegments froma tail-to-tail theheads themaleattempted attitude, copulation enough,

'4 _........ .. ...............

. . . . ......

....... . ........, ,s;:

D

}~~~~~~~~~~~~~1 ....... j ,.j;i .,.... ;. .... . . ..;............ ;, ;........ i............. .. ......

...;....

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. .... .......

.. ......

Figs. 14-16. Heads of malesof Anophelesquadrimaculatus.14. Dorsal view showagainstthe shafts; note the thickenedappearance ing the antennalfibrillaerecumbent of the shafts(cf. Fig. 15). Some of the hairs are extended; thisoccurredafterthe insecthad been anesthetized;15. Dorsal view showingthe antennalfibrillaeextended away fromthe shaftsgivingthe antennaea plumoseappearance.Some of the hairson the apical half of the left antennaare somewhatrecumbent;16. Lateral view of the same head shownin Fig. 15. (Photographsof heads mountedon minutenpins; the have been removedfromall specimens.) mouthparts


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40 (2)

themalenever in oppositedirections. of bothindividuals Hkowever, pointing in making connection froman opposedattitude. succeeded males,were legsof 15,3-day-old frombothfront The lasttarsalsegments 89 unsuccessful removed.Duringone trial 12 hoursafterthe operation, Therewereno successful weremade,with3 females. attempts at copulation of thesemaleswas similarto thoselackingboth The behavior topulations. tarsi. entire front of thefrotnt Conclusions. It is apparent thatthelasttarsalsegments legs butthescsegfortheinitialseizureof thefemalein flight, arenotnecessary forsuccessposenecessary mentsarevitalforthemaleto assumethevertical fulcopulation. of maleslackingthemiddletarsi. 3.-To determine thebehavior Experiment and 2 trialswere maleswererernoved The middletarsiof 15, 3-day-old results: withthefollowing made6 and27 hoursaftertheoperation, copulations. 66 unsuccessful copulations; 6-hourtrial.-1 successful copulations. 27-hour 39 unsuccessful trial.-27successful copulations;

was due to thefact in matingapparently Duringthefirsttrial,difficulty was "shaken"offjustbefore ableto seizethefemale, thatthemale,although or afterthepaircameto rest.Evenmaleswhichmanagedto gaina ventral was one which oftenwereunsuccessful in copulating if theattitude attitude, matabdomen. Severalof thesuccessful ofthefemale's required manipulation which ingsoccurred on thefloorof thecagewherethemalewasin a position withthe female merelyrequiredraisinghis abdomento makeconnection genitalia.

Conclusion.Males lackingthemiddletarsistillarecapableof copulating successfully. 4.-To determine thebehavior of maleslackingthehindtarsi. Experiment and onetrialwasrun The hindtarsiof 15,3-day-old maleswereremoved and 55 successful copula5 hoursaftertheoperation. unsuccessful Forty-five period.These maleseasilyseizedthe tionsoccurred duringthe30-minute posein spiteof theabsence femalein flight and readily assumedthevertical ofthehindtarsi. (see Conclusion.The hindlegsof themaletakelittlepartin copulation p. 270). 5.-To determine thebehavior of malesaftertarsectomy of the Experiment middleandhindlegs. from15 virginmalesand their The middleand hindtarsiwereremoved was observed 8 and 22 hoursafterthe operation. behavior towardsfemales wereas follows: The results 8-hourtrial.- 14 successful copulations; 45 unsuccessftilcopulations. 22-hour copulations. trial.-IO successfulcopulations;52 unsuccessftil


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to thoseobtainedin Experiment The results are comparable Conclusions. sinceit is evident 3 afterremoval of themiddletarsi.This is to be expected The total fromExperiment 4 thatthehindlegsplaylittlepartin copulation. again,thatthe frontlegs alone are indicates, copulations of 24 successful pose. thevertical andareusedin gaining thefemale capableofseizing of maleslackingthelastsegments 6.-To determine thebehavior Experiment andmiddletarsi. ofthefront maleswere andmiddletarsiof 15,3-day-old ofthefront Thlelastsegment In spiteof and one trialwas made 18 hoursafterthe operation. removed in seizinga not ohe malewas successful attempts, tepeatedand numerous femalein flight. of the frontand middlelegs are Conclusion.The last tarsalsegments forthemaleto\seize thefemaleandto assumethevertical necessary absolutely forcopulation. posenecessary OF THE ATTITUDES OF MOSQUITOES GENERAL DISCUSSION ASSUMED DURIN.G COPULATION

in on themaletarsimaybe summarized The resultsof theexperiments table: thefollowing Type of operation 1) Removal of one fronttarsus2) Removal of bothfronttarsi-Male

Behav,iortosaardsfemale Can copulatesuccessfully. is capable of seizing the female but generallycannotcopulate because of his inabilityto gain a verticalpose.

3) Removal of the last segmentof both fronttarsi-Similar

to 2) above.

4) Removal of both middletarsi-

Can copulatesuccessfully.

5) Removal of both hind tarsi-Can

6) Removalof bothmiddleand hind tarsi-Can of 7) Removal of the last segments both frontand middle ta-si-is

copulate successfully.

copulatesuccessfully. Male cannotseize the femaleand therefore unable to copulate.

The abilityof maleaegyptito seizeand hold the femaleis due to the of thelasttarsal innermargin tarsalclawsand thehollowed greatly enlarged segments of his frontand middlelegs (Figs. 17-19).Thus thepartof the femalefemur, tibia,or tarsuswhichis seizedbythemalefitsintothisdepresof herappendage and holdit sion,and hisclawsareclaspedoverthemargin firmly in place.The hindtarsi(Fig. 20) are not at all suitableforseizing alongtheinner becauseof thesmallclawsand theabsenceof theindentation of thelasttarsalsegment. Forthisreasonde Riville's earlyobservation margin



298

40 (2)

20

21

22

~~~8

j7

19

AEGYPTI

I8 MM

27

28

~~25

24

26

29 QUADRIMACULATUS

34 4,,3

30

31

'4

a

32

3 PIPIENS

Figs. 17-35. Last tarsal segmentsand tarsal claws. 17-23: Aedes aegypti.-17. Male, prothoracic;18. Male, prothoracic(tarsal claws retracted); 19. Male, mesothoracic;20. Male, metathoracic;21. Female, prothoracic;22. Female, mesothoracic; 24-29: Anophelesquadrimaculatus.-24.Male, prothoracic: 23. Female, metathoracic. 25. Male, mesothoracic;26. Male, metathoracic; 27. Female, prothoracic;28. Female,


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(see p. 268) thatthemaleusedthemiddleand hindlegsforgrasping the femaleis no doubtan error. He mayhavemistaken thepushing actionof the or legsforoneofgrasping. malehindlegsagainstthefemaleabdomen to In mating, of themaledependsuponhisability theprimary orientation from perceive and determine thedirection of thesoundatimulus emanating tliefemalein flight. Once thishas beenaccomplisbed, withthe aid of his antennae, andhe hassuccessfully pursued andseizedher,a secondorientation For if themaleseizesthefemalein otherthana venter-tobecomes necessary. he mustreorient himself beforecopulaventer attitude, as generally happens, tioncanibe successfully completed. This secondorientation, whichtakesplace whilethemaleclingsto thefemaie, is largely accomplis4ed withtheaid ofthe fronttarsiforwhentheseareremoved themale,in almostall cases,can no longergainthevertical posealthough stillcapableor clinging to thefemale withhis middlelegs.The behavior of thesemaleslackingthefronttarsiis also goodindirect thatthenormalmaleinitially evidence seizesthefemalein a dorsalposition himself andthenreorients whileclinging to her.The middle legsappearto function largely in theinitialseizureof thefemale, thoughthe frontlegsarealso usedin thisact.That successful copulations can occurin theabsenceof themiddleand hindtarsiindicates legscan perthatthefront to someextent, form, boththefunction of seizingthefemalein flight and in gaininga vertical pose.The tarsalclawsof themiddlelegsare largebutnot toothed(Fig. 19). The tarsalclawsof thefront legsarestillmoredeveloped by reasonof theirgreater lengthand toothedcondition(Figs. 17-18). This structural difference mayexplainwhymalescan copulatelackingtheirmiddle andhindtarsi,butnotwhenthegreater-developed front tarsiareremoved. Knab (1907b) compared theattitudes takenby thesexesof Anopheles punctipennis Say during copulation withthatassumed byotherspecies, chiefly Culexpipiens.He stated,"It seemsremarkable thatthe attitude takenby in copulation, Anopheles endto endandfacing in opposite directions, is identicalwiththatof Culexpipiens. Thisremarkable when agreement is significant thestructure of thetarsalclawsis considered. In bot;hformsit is practically thesameandall theclawsof thefemalearesimple.In bothcasesthelegsare notmadeuseofin thecopulatory actitselfbutonlyin thepreliminary scuffle. Dr. H. G. Dyar has foundthatCulisetaconsobrinus (_ Culisetainornata Will.), anotherformwithsimpleclawsand quitea distinct generictype, takesthesamepositionin copulation. It is fairto assumethatthisis the in all theforms in whichthefemalehassimpleclaws.On modeof copulation theotherhandit appearsthatin thoseforms in whichthefemalehas toothed in copulation is faceto face,thepairclasping clawstheposition each other. The writer canconfirm Goeldi'sobservation thatStegomyia calopuscopulates ir.thismanner. Dr. Dyar has observed the same methodof copulation in mesothoracic;29. Female, metathoracic. 30-35: Culex pip.iens.-30. Male, prothoracic (dottedline showsclaws retracted);31. Male, mesothoracic;32. Male, metathoracic; 33. Female, prothoracic;34. Female, mesothoracic;35. Female, metathoracic.(All drawingsoutlinedwiththeaid of cameralucida.)


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Aedesvaripalpus Coq. One cannotavoidtheinference thatthedifference in thestructure oftheclawsis closelycorrelated withthemodeof copulation. It wouldappearthenthatthestructure oftheclawsis a purely oneand adaptive therefore itcannotbe considered ofthedeepclassificatory significance attributed to it by Mr. Coquillet."Aedes spencerii Theob.and Aedes fitchii Felt and Knab (1908) stated,they Young copulatefaceto-face and of the former, grasp". . . eachotherwiththeirlonglegs,thefemalein theupperposition, themalebackdownward." Wesenberg-Lund (1921) notedthatthemaleof Aedesfletcheri thefemnale "dances"closeto theground, theleafon quitting which sheis resting, and whenthesexesmeettheygrasp". . . eachother's fore and middlelegs." In thisspeciescopulation also takesplace in the facing

position.

Earlierin thispaperit waspointedoutthatthefemalelegswereextended and usuallyheldoutstretched whena awayfromthebody,duringcopulation; joinedpair landedon theside of the cage the femaleused her frontand middlelegsto clingto theclothwhileherhindlegswerepushedupwardby theactionof themale'smiddlelegs.Directobservation through a binocular microscope placedabovethetethered females showedthatthemaleseizedthe female,her legs takingno partin theaction.Banks (1908, p. 246) also observed thatthemaleseizedtheflying female". . . withhis foreand mid feet."He recorded no observations to theeffect thauthefemalegraspedthe male.Final proofthatthefemaletarsalclawsare not used in matingwas obtained byremoving thelasttarsalsegments fromall thelegsof 10 females. Males,introduced intothecage,easilyseizedtheseoperated females, clinging to themin flight andcopulating. The onlydifficulty occurred whenthefemales triedto landon thesidesof thecage.Removalof theirtarsalclawsprevented themfrom gaining a holdon theclothsideandtheyinvariably fellto thefloor wherecopulation usuallywas completed withthe femalesastridethe male (i.e. venter-to-venter). When the last tarsalsegments fromthe frontand middlelegsonlywereremoved fromfemales, theresults weremoreor lessthe sameexceptthatin somecasesthefemales managed to clingawkwardly to the side of thecage withtheirhindtarsalclawsand copulation was completed there. It is understandable thatan observer witnessing pairingin thefield,with thenakedeye,couldbe misledto believethatthefemaleused herlegsto clingto the male.Again Howard,Dyar and Knab (1912, p. 121) stated that"The attitude assumedduringthecopulatory actdiffers according to the structure of theclawsof thefemale.In theforms withsimpleclaws(Culex, Anopheles)theposition is endto end,thepairfacingin oppositedirections. The forms in whichthefemaleclawsaretoothed copulatefaceto face,claspingeachotherwiththeirclaws."Duringcoitusthefemaleaegyptidoes not claspthemaleto her.PerhapsKnab was correct in his assumption thatall formsin whichfemales havesimpleclawscopulateend-to-end whilethosein whichtheclawsare toothed copulateface-to-face. However,thisdifference in at leastin aegypti, attitude, cannotbe attributed to thefactthatthefemale can claspthemaleto herbecauseof hertoothed claws,forherlegs,remain.


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aboutwiththemale,merely whileflying serveas structures ingoutstretched to statethatface-to-face to whichthe male can cling.It mightbe correct of mosquitoes in whichthetarsalclawsof copulation is possibly characteristic is characteristic of mosquithefemalearetoothed, and end-to-end copulation withthe toesin whichthe femaleshavesimpleclaws,withno correlation function of theseclaws. An examinationof the femaletarsiof aegypti(Figs. 21L23) revealsthat the last tarsalsegmentsof her frontand middlelegs (Figs. 21-22) are not at all suitableforclasping(cf. Figs. 17-19) in spite of the toothedcharacterof the claws. These claws are small and the marginsof the last segmentare practicallyparallel;even whenthe toothedclaws are retractedto theirfullest extenttheyare actuallyno more useful for claspingthe male than are the simple claws found in Anopheles quadrimaculatus(Figs. 27-28) or Culex pipiens (Figs. 33-34) females.That a toothedtarsal claw conditionis not .ctuallynecessaryforseizingis seen in the factthatmale aegyptiwhichhave theirfronit tarsiremovedcan stillgraspthe female (thoughtheycannotgain a ventralposition) withtheirmiddletarsi,thoughthe claws of the middleleg are long and simple. Boyd, Cain, and Mulrennan(1935) photographed a pair of quadrimaculatus in the end-to-end positionkilled duringcopulationand stated"Copulation occurredin flight,the individualsassumingan end-to-endpositionand settlingin pairs to the floorof the cage (Fig. 7)." In our 11" x 11" x 15" cages, males would become active and begin to fly in the evenings,a behaviorpresumablycomparableto the swarmingor quadrimaculatusin the field (see Freeborn,1923). Hackett and Bates (1936), Rao and Russell (1938) and othershave observedswarmingof Anophelesin insectarycages. In the cases of copulationseen in the laboratory, the male quadrimaculatus in seizing and clingingto the femalelong appearedto have great difficulty enoughfor him to clasp her genitalia.He seized her in flight,assumed the modifiedlinearpose, but once connectionwas made withher genitaliathe male releasedhis leg hold and in so doing sometimesdroppedto a pendant positionfor an instant(Fig. 37); he then passed to the opposed or linear position (Fig. 38) withthe heads of the two facingin oppositedirections. Sometimesthe pair flewabout withthe male hanginghead downwards.Copulationwas of shortdurationand could be completed(1) in the air, (2) with the femaleclingingto the side of the cage, the male hanginghead downward, or (3) on thefloorof thecage withthemosquitoesrestingon theirlegs,joined in oppositedirectiors.Aftercopulationwas comby theirgenitalia,and facingpleted,themale releasedthe femaleand flewaway. as reportedby Cambournacand Hill The matingpositionsof atroparvus, (1940), wereverysimilarto that of quadrimaculatus exceptthat the former "attacked"restingfemales.Once the male atroparvusmade connectionwith the femalehe releasedhis hold aftertwo or threesecondsand ". . . the two If the pair is on the remained5 or 6 seconds in a straightline, tail-to-tail. the insectsare quiet and the male hangsstraightdown wall insteadof in flight, withthe wingsoutspread."


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of Aedesaegypti theattitudes assumedduringcopulation If onecompares position (Fig. 38) theface-to-face quddrimaculatus (Fig. 36) and Anopheles with positionof thelattercan be correlated and theend-to-end of theformer of thelasttarsalsegments and clawsof thefront theextentof development ofthe thelasttarsalsegment and middlelegsofthemale.In quadrimaculatus as poorlydevelopedas thatof the hind middleleg (Fig. 25) is practically smalland theinnermargin leg (Fig. 26), withtheclawsbeingcomparatively foundon thefrontleg. Only the lackingthedepression of thelastsegment male'sfronttarsi(Fig. 24) are suitableforseizingand holdingthefemale. of themiddletarsal Thus onecan easilyreasonthatthereduceddevelopment to seizeand clingto the reducedhisability clawsof themalecorrespondingly an adaptation in theend-to-end position, resulted femaleand thiseventually withthefemaledid not requiretheuse of the whichaftertheinitialscuffle developed malelegsat all. We mayalso recallthatbecauseof theirgreatly

38 of mosquitoes.positions themating showing drawings Figs.36-38.Diagrammatic 36. Aedesaeggptiin copula,thefemaleat reston thesideof thecage,thepairin the quadrimacuface-to-face (thelegsof themaleare blackened);37. Anopheles position position;38. Anophelesquadripendant lilausin copulawiththemalein a temporary position. withthepairin theend-to-end maculatus in thefinalstagesof copulation


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fronttarsal claws, aegyptimales are capable of copulatingeven aftertheir middle tarsi have been removed.However, removalof the middle tatsi of aegyptidid not resultin that speciesassuminga tail-to-tail positionduring

copulation.

There are certainobservations in the literature whichmake it ratherdifficult to formulatea generalhypothesisregardingthe correlationof mating attitudesand the structure of the male claws. If we accept Knab's (1906a) observations that Culex pipiensassumesa tail-to-tail positionduringcopula. tion we possiblyhave an objectionto the hypothesis since in this speciesthe frontand middletarsiare bothabout equallydevelopedand suitedforseizing (Figs. 30-31). However,theretracted fronttarsalclaw of male aegyptireaches almostto the base of the last tarsalsegment(Fig. 18) whilethat of Culcx pipiensdoes not (Fig. 30, dotted lincs). This mightmake the aegyptilegs moreefficient thanthepipienslegs in seizingand holding.Regardingthe copulation of C. pipiens,Wesenberg-Lund(1921) stated "I nevercould directly observethatthe matingpositionwas end-to-end, as indicatedby Knab (1912, p. 122); the insectswerealwayscurledup in a ball and verysoon reachedthe grass. Here the act was accomplishedin the course of a few seconds,after whichthemosquitoesflewaway." A laboratory colonyof Culex pipienswas establishedfromlarvaeco'lected in the field. Reared adults were kept in a cage 24" x 24" x 30" lined with whitecheeseclothto facilitateobservation. Feedingand matingwas cyclicalin this speciesand occurredearlyin the morning.At first,femaleswould only feedon thearmbetween3 a. m. and 8 a. m. Copulationswereobserved[between 6 a. m. and 8 a. m. and a few attemptedcopulationstook place at 3 a. m. The mosquitoesweremoreor less inactiveat othertimesof the day; thatis, thefemalesdid not takeblood and themaleswouldnot copulate.Afterseverai generations, femaleswould feed on blood more readilyduringthe day and laterin theevening,and maleswereseen to attemptcopulationabout 11 p. m. Unlike quadrimaculatus males,pipiensmaleswereobservedwiththeirantennal Gbrillaeremaining extendedduringtheirsexuallyinactiveperiods;like aegypti, the males of pipienswereneverseen withall theirantennalfibrillaeflattened againstthe shaftsonce thesehairshad becomeextended. Copulationof pipiens,in our laboratorycolony,was alwaysinitiatedon the wing,the male beingthe aggressorand seizingthe femalein flight.The male appearedto be less adept at seizingthe femalethan was aegypti;many of pipiens'attemptswereunsuccessful. The male seized the female,assumed a verticalpose, and thenclasped her genitalia.They would flyabout in the facingpositionfor a briefperiod and then the maitewould release his leg hold and hang head downwardwhileclaspingthe femalegenitalia,and thus be carriedby her (similarto quadrimaculatus shownin Fig. 37). Copulation was completedin flight,the male releasingthe femaleaftera few seconds. In manyinstancespairswould flyabout joined face-to-face and thenseparate; however,the actionwas so transientthat it was difficult to determineif connectionwas made duringthe act. If a femalecame to restwiththe male clinging to her in a face-to-face attitude,he would releaseher and flyaway. If,


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connectionhad been made and the male was in an opposed position lhowever, suspendedfromthe female'sgenitaliawhenshe came to rest,the pair would remainattachedby theirgenitaliafora briefperioduntilcopulationwas completed. Apparently,beforecopulationcould be successful,the male had to make connectionwhilethe pair was in flightand beforethe femalecame to rest.Bates (1941, p. 167) found anophelinesbehaved in a similarmanner. small space since viable Successfulcopulationtook place in a comparatively egg raftswere obtainedfromblood-fedpipiens kept with males in a cage 11" x 11" x 15". of Culex molestusForskai (C. Marshall (1938) rearedmanygenerations pipiensmolestusForskal;Jobling,1938) and foundthatpairingof the adults positionwas oftenconcludedin thoughinvariablyinitiatedin the face-to-face the end-to-end one. ApparentlyMarshall refersto the factthatthe pair may forawhilebeforeassumingthe opposedposiflyabout joined,venter-to-venter on pipiens. tion.This behavioris similarto our observations may be said to initiatecopulationin a face-to-face Even quadrimaculatus attitudesince connectionis firstmade withthe pair flyingin a verticalpose. However,the opposed positionis assumed practicallythe instantconnection is made; thisdiffers somewhatfrompipienswherethe pair may flyabout for awhile in the facingpositionbeforeassumingthe end-to-endone. Rao and Russell (1938, p. 400) described2 cases of pairingof Anophel'esannularisin which ". . . both members of the pair faced the same direction, towards the

withtips of abdowind,one beingabove and the otherbelow,legs interlocked, men in contact."They noted a similarbehavioron the part of Anopheles siephensiin a cage colonyalthoughthis speciesalso was observedto pair in the end-to-end position.The writersdid not indicatewhetheror not theysaw the individualswhichcopulatedin the facingpositionseparatewithoutactually one. assumingan end-to-end In discussingthe positionsassumedduringcopulationit is importantthat one sees the completeact and differentiates betweenthe end-to-endposition taken duringactual copulationand an end-to-endpositionwhichsometimes may be takenbriefly as the sexestryto separateaftercopulationis completed. The writernoted on severaloccasionsthat aftermating,pairs of aegypti,in to freethemselves, momentarily assumedthe linearposition.Knab attempting thecopulationof A. fitchii states"Union takesplace 'faceto (1908) describing face,'the pair flyingobliquelyupwardforseveralyards.Then the pair would eitherseparatepromptlyor swingout end to end and struggleto disengage In thislattercase bothof themcould be seen to jerkviolentlyand themselves. to freethemselves, and the pair would slowlyrise,but rapidlyin theirefforts make no appreciableprogressin eitherdirection."An observernotingonly of the copulatoryact mightbe led to believethat in specie3 the termination behavingin the above manner,the linear or end-to-endpositionis the one one seeing only the initiationof the assumedduringcopulation;conversely, act in thoseformswhichcopulateend-to-end would be led to believethatthe for thatparticularspecies.Aedes aegyptinorfacingpositionis characteristic remainsonly position;A. quadrimaculatus mallycopulatein the face-to-face


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in thefacing thegreater momentarily position, partoftheactbeingperformed fallsintoa classbetween in theopposedposition;Culexpipiensapparently theabovetwospeciesoftenspending partofthetimeinbothpositions. thecopulation of a stenogamic A. G. Richards(1941) described autogencameto reston topof ous strainof Culexpipiensandnotedthatthefemales quiescent males,copulation takingplace withthe male appearingpassive throughout theact. If takenliterally, Richards' description givestheimpres withthesexesin thetypical femalevertical sionthatcopulation occurred pose, thatis,thefemaleastridethemalewithhis dorsalsurface againstherventer. the rotation of the male genitalia. This seemshighlyunlikelyconsidering Copulationin the femaleverticalpose occursin insectsin whichthe male hlypopygium is normal(i.e. no rotation; see 0. W. Richards, 1927,p. 336). In a personalcommunication the fact (1946) A. G. Richards emphasized thathisobservations couldnotbe compared withKnab'ssinceonewasa field studywhiletheotherwasmadein thelaboratory. in hisletter Richards stated, to a certain extent abnormal in thesense "Matingin thesecaseswasprobably thatrelatively wereusedand accordingly smallcontainers thedata maynot thebehavior to be foundin naturewhereunlimited reflect spaceis available." on ourcolonyof pipiensundoubtedly The observations deal witha different sincethefemalesrequired a bloodmealforeggracethanthatof Richards' laying(anautogenous). themating Concerning behavior of an Englishautogenousraceofpipiens, Tate andVincent(1936,p. 136) notedthatthemales themand copulated while settledon resting forcedtheirwaybeneath females, thefemales wereat rest.Thesesamewriters thatthebehavior of the observed raceofpipiensdiffered Englishanautogenous fromtheautogenous in thatthe malesswarmed. Whenfemales entered theswarmtheywerepursued bymales andpairing beganin theair.Males of thisstraindid no attempt to pairwith evenwhentheylitnearoruponthem. resting females, U. lowiias copulating Pratt(1946) recorded in theend-to-end position U. cookiRootmating thefactthatT. H. Aitkenobserved and alsomentioned end-to-end in a smalllantern chimney. The twotarsalclawson eachlegofthe femalelowiiaresmallandsimple(Figs.42-44) andnotsuitedforseizingthe male. The tarsalclawson the prothoracic and metathoracic legs of the male (Figs.39, 41) are somewhat similarto thoseof the'emale.However, hismiddletarsus(Fig.40) bearsa single, large,curved tarsalclawwhichwhen flexedbackreachesalmostto thebase of thelasttarsalsegment. Thus the malesof lowiiaresimilar to malesof quadrimaculatus in thatonlyonepairof legsis suitedforseizingand holding thefemale;however, in quadrimaculatus it is thefront pairoflegswhichis modified. It is obviousfromthisdiscussion thatthereis stillmuchto be learned aboutthemating and behavior of mosquitoes positions and thatcarefuland detailedobservations are neededto correct variousconflicting reports found in theliterature as wellas to add newknowledge. If myhypothesis is true, namely,thatthe positionsassumedby mosquitoes duringcopulation have evolvedlargelyas a resultof changesin thestructute of thelasttarsalsegof thefrontand middlelegsof the male,thenadditional mnents studiesof


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correlated withmale in variousgeneraand species, mating positions mosquito results. shouldyieldinteresting tarsalstructure, GENERAL DISCUSSION

OF THE MATING BEHAVIOR OF MOSQUITOES

of male Untilnowthisstudyhas dealtmainlywiththematingbehavior

of thestudy The remainder stimulus. aegyptiusingthefemaleas themating

soundstimuli. of malesto artificially produced withthebehavior dealschiefly of someof the observations already It is feltthatthepossiblesignificance on sound. thanat theendofthesection hererather notedshouldbe discussed as ". . (a) Kahnet al. (1945) wroteof varioussoundsof mosquitoes ofdanger,(c) callsof angerand othersounds calls,(b) callswarning mating thisinterpreBeach(1945) rightfully criticized functional." thataresimilarly of lower ". . . thedangers of interpretinig thereactions tationandemphasized of humanbeings." characteristic experiences speciesin termsof psychological by soundproduced thatthereis no specific indicate observations The present as a "mating call,"unlessit be anysound thefemalewhichcanbe interpreted severalhoursafteremergence. beginning she happensto makein flight, Femaleswhichareplacedin a cagewithmalesandaremadeto flycontinually tendto resistthe mnating so thatthemalescopulatewiththemrepeatedly, females, by theresisting of thesemales. Yet thesoundsproduced attempts to attract themales. continue flight, during which to a singlefixedpitchbutto frequencies The maledoesnotrespond in male resulting mayvaryoversomerange(see p. 326). Thoughpossibly theirresponse to a rangeof to copulatewithotherculicids, aegyptiattempting of forthe speciesforit insuresthe "attractiveness" sound is "fortunate" frequencies femaleaegyptiwhichmayproducesoundsof different different of factors suchas size,extentof distension uncontrollable becauseof certain

39

~~~~~42

40

41

44

and tarsalclaws of Uranotaenialomii.-39. Male, Figs. 39-44. Last tarsalsegments

42. Female,protlhoracic; 41. Male,metathoracic; 40. Male,mesothoracic; prothoracic; outlined withthe 44. Female,metathoracic. 43. Female,mesothoracic; (All drawings aid of cameralucida.)


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withfood,etc.If malesresponded onlvto a singlefixedpitcl1, theabdomen whichin turnwould femaleswouldbe diminished thenumberof attractive of females.Thereare severalmethods reducethe numberof impregnated mechanisms whichrestrict randommatingbetween species isolating biological femalemosquitoes (Mayr,1942,p. 247). The soundproducedby flying an isolating factor sinceit servesas thematconceivably couldbe considered ing stimulusforthe male.However,it is apparentfromthe behaviorof sincethemaleresponds aegyptithatsoundplayslittlepartin sexualisolation to copulatewithanymosquito andwillattempt to a widerangeof frequencies therangewhichwillactas a mating fallswithin thesoundof whichin flight is concerned sexualisolation mustbe accomplished stimulus. As faras aegypti etc.). However,it is not impos(mechanical, genetic, in someothermannerto a rangeof soundwhichis considerably siblethatsomespeciesmayrespond narrower thanthatof aegypti and in theseformssoundcouldplaya partin mating-state throughin a constant Thataegypti malesremain sexualisolation. with outtheirlifetime, oncetheyhavereachedthatstate,and materepeatedly not onlyincreases the chancesfor manyfemalesif giventheopportunity, butmorethanone femalecan be impregnated bya females to be fecundated singlemale. Bothmalesand femalestendto remainquietforsometimeafteremersuggest in flight, severalpossiblebenefits is initiated gence.Sincecopulation forthisdelayin flying. themselves Undoubtedly t-hisrestperiodgivesthe havedetermined to harden.Severalinvestgators exoskeleton an opportunity at whichfertilization occurs.Howardet al. theapproximate age of aegypti (1912) and Putnamand Shannon(1934) claimedthatbothsexesmate Gutzevich within a fewhoursafteremergence. (1931) statedthatat 280 C., Seatonand Lumsden fertilization usuallyoccurs2 to 3 daysafteremergence. as theage withgreater frequency occurred (1941) foundthat"Fertilization on thefirst nightand beingexceptional increased, of thefemalemosquitoes Fromthepresent studyit is appartheruleon thethirdnightaftereclosion." ent that the timeof successful copulationdependsupon threeimportant factors. and thisoccursseveral First,the femalemustbecome"attractive" at whichtimethesoundproduced by herin flight hoursafterheremergence reachesa pitchwhichwillserveas a matingstimulasforthemale.Though afteremergence afterbeinginducedto fly, 50 minutes somefemales copulated willoccurlater, copulations it is probable thatif thecageis leftundisturbed The flight or sincethefemales tendto restforseveralhoursafteremerging. no benefit femalesoffers in matingsincethepitchof the recently emerged is belowtherangewhichwill mosquitoes bytheseslow-flying soundproduced activemalesto copulate. stimulate sexually factors in successful concern The secondand thirdimportant copulation rotateto about1800 buttheantennae themale.Not onlymusttlhegenitalia to a condition of thefibrillae wherethey mustalso "mature" bytheextension It is probable thesoundof thefemalein flight. that, arecapableofperceiving untilthis lattercondition is the male remainsquiescent unlessdisturbed, before havefoundthataegypti malesoftenemerge Someinvestigators reached.


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maleswereusuallya females.Bacot (1916) notedthattheearlyemerging thanthefemales, in theirdevelopment andGordon(1922) found dayquicker of malesthanfemalesemerged duringthefirst number thata muchgreater Shannonand Putnam(1934) in theirlaboratory fewdays of emergences. was thattheaverageemergence periodof females observed studyof aegypti are of all theseworkers 14 hourslongerthanthatof males.The observations thatbythetime in thelightof thepresent studyfortheyindicate interesting themale'santennae have thefemalebeginsto flyand becomes"attractive," can be perceived and his genitalia reacheda statewherethesoundstimulus can be successful (usuallyabout15 sufficiently so thatcopulation has rotated to 24 hoursafteremergence). malesare quicklyseizedby "old"8 malesbecausethe Recently emerged fallswithinthe soundspectrum by a youngmalein flight soundproduced a sexually activemaleto copulate. Theseoldermalescannot whichstimulates betweenthe sex of veryyoungmales and several-hours-old differentiate untilsometimeafteremergence fromflight Thus refraining avoid. females. of old malesuponyoungones;it was notedthat theattempted copulations wererepeated oneormorelegsof the often, whentheseattempted copulations youngmale wouldbe pulledoffduringthestruggle.By thetimea male thesoundhe makesin flight is sufficiently highin beginsto flyvoluntarily, old malesand therefore pitchto be beyondtherangewhichwillstimulate he is unmolested. (Lang, 1920;Howardet al., 1912; Hearle, 1926,and Severalworkers others)believedthat,at leastin thosespecieswhichswarm,the femaleis C. pipiens,Wesenberg-Lund (1921) wrote attracted to themale.Concerning thatfemalesflyin a straight lineintotheswarmand "It was as if bysome to flydirectly intotheswarmof dancing wereforced magicpowertheinsects theyweredirectedby the soundissuingfromthese males.Undoubtedly on thematingbehavior observations males."Bates (1941) in his laboratory maleswere of anopheline swarming mosquitoes foundthat". . . laboratory on females, only and matingoccurred not observed to exertany attraction In aegypti themaleis theaggressor whena femalechancedintotheswarm." No evidence wasfoundin thepresent andis attracted bythefemalein flight. in any way by the male, studiesto indicatethatthe femaleis attracted aboutin a cageand a resting malesmaybe flying femalemaytake although Somepreliminary to flight indicate that experiments and be seizedin mid-air. to soundsthatfallwithin tnefemaledoesnotreactvisibly(i.e. is attracted) a femalereaches a mating-state therangemadebymales.Whether comparable it is impossible to separate to thatin themalecannotbe said;apparently any fromgeneralactivity sincethefemaleis passiveand possiblesexualactivity similarto thatof themale.Copulation does notshowanymatingbehavior willnotoccuruntilthefemalebeginsto flyand it is notknownwhether the to flyis sexualornutritional. whichfirst Whether stimulus inducesthefemale Ornotthefemale is attracted bythesoundofthemaieis stillopento question. 8 The term"old" is used to designatemales withtheirantennalfibrillaeextended, the sound of the femalein flight. and therebycapable of perceiving


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Afterrepeated femalesbecomemoreand morereluctant matings, to fly themalebypushing himawaywithherhindlegs, andifseizedwillalsoresist or byjerking whenhe is aboutto makeconnection. awayherabdomen This reluctance to flyandmateon thepartofthefemaleafterrepeated copulations may partlybe due to fatigue(certainly the malesare not fatiguedafter repeatedcopulations), or in a sensemayactuallybe a formof "coyness." Richards(1927) suggested thatrepeated copulations wouldnotonlyexhaust in general)butalso mightl theindividuals (i.e. insects exposethejoinedpairs to theirenemies. He also notedthatin somespeciescoyness is onlyexhibited whenthespermatheca is full. It is not knownhowmanytimesa femaleaegyptiwillcopulateunder normalconditions, butit is undoubtedly muchlessthantheforcedpolyandry lecordedin thisexperimental laboratory study.Onlyone copulation is necessaryforfertileeggsto be laid (Hinds)!; normally onlytwo of the three spermathecae arefilledafteroneor twocopulations, andthismaybe sufficient to inducecoyness in femaleaegypti. Gibson(1945,p. 267) foundthatonce thefemales ofthechironomid Spaniotoma minima Meigenhadcopulated they didnotriseagainintotheswarmof males,butremained on thefloor resting of the cage. It is not knownwhether repeatedcopulations are in any way lharmful to femaleaegypti. Whena maleseizesa femaleand shecontinues to flyabout,othermalesare immediately attracted' and will "mob" the pair ttying to copulate. In manyinstances aftera maleseizesa femaleshequickly landson thesideof thecageand copulation is completed whileat rest,thus preventing anyinterference fromothermales. It wouldappearthatwithspeciesthatswarm in largenumbers in thefield, predators shouldbe an important factor in survival. Howardet al. (1912,pp. 165,171,177,179) givevariousrecords of dragonflies, wasps,birds,andbats on adultmosquitoes. Gruhl(1927), quotedby Gibson(1945), befeeding lievedthat contrary to expectation, swarmers enjoyeda high degreeof immunity frompredators and he thought thatthisfactwouldact as a sustaining principle, tending to fixthehabit,onceit hadbeenacquired. However, Banks(1919) noteddragonfles in and out of swarming darting anophelines, and Rao and Russell(1938) notedthe appearance of bats simultaneouslv withswarmsof A. annularis, whichattackedmosquitoes and otherinsects at thesametime.Predators areof no imporatnce swarming in thelaboratory is chiefly a housemosquito and thoughAedesaegypti withcopulation often severalworkers havefoundnaturalenemies takingplaceindoors, of thismosquito.Bacot (1916) considered toads,lizards,spiders,ants,scorpions, and possiblyyoungMantidaeto be destroyers of aegypti. Gordan(1922) conthatboththegeckoand a jumpingspiderreadily cludedfromexperiments S. calopusand C. fatigans destroyed andowingto theirwidedistribution were ofsomeimportance in limiting probably thenumbers of mosquitoes occurring That thecopulation irnhumanhabitations. of aegyptiis rapidand themale releases thefemaleas soonas theactis completed woulddecrease thechances of a joinedpairbeingdestroyed byotheranimals. It is interesting to speculateupontheevolution of theepigamic mating


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of mosquitoes. The remarkable of Opifexmightwellreprebehavior behavior formosquitoes, whereapparently the sentone of themostprimitive recorded As thebesoundproduced by thefemalein flight playsno rolein mating. in flight, Gibson haviorevolvedto whereit occurred the speciesswarmed. (1945, p. 293) in his reviewon swarming of Chironomidae suggested that swarming led ". . . to moreeasymeeting of thesexesthrough visionor possiblysound."In thosespeciesin whichswarming occurs,themalesgenerally congregate periodically, apparently theresultof an internal rhythm. Gibson or hormonic suggested thatsomeformof nervous changeoccursat thistime. state.Fromtheepigamic inactive At othertimesthemalesare in a sexually to themaculipennis swarmof thevariousspeciesbelonging behavior complex, withthisbehavior ingappearsto be theolderformof behavior, apparently of swarms evolving awayfromthe formation towardsthe individual dance, to no danceat all. Thusthebehavior of A. atroparvus, whichmay andfinally matewithor without swarming, led Cambournac and Hill (1940) to write that". . . theevening has largelylost its functional dance,foratroparvus, character, although theformof theritualis stillpreserved." Russelland Rao (1942) alsofoundthatswarming wasnotobligatory forthemating ofAnopheles culicifacies, copulating pairsbeingseenin theabsenceof swarms; these tookplaceat aboutthesametimein theevening matings and alwaysoccurred Anflight. Knab (1906a) stated". . within theCulicidae, thereis notonlya marked in themodeof swarming of different diversity species,but thatthereare manyformsthatdo notswarmat all. In thecase of thealmostexclusively house-inhabiting Stegomyia calopus,as Goeldihas shown,thereare no such The malescongregate as in Culexproper. in littlegroupsof 15 or 20, swarms overthecorner of a cabinetor otherprominent hlovering object,and pounce thatcomewithin uponthefemales range.I canadd thatcopulation frequently of evensuchweakswarms." takesplacewhenthereis no evidence The nresent nas also seenon severaloccasions writer pairsof aegypti flying freely in the laboratory, joinedin theair,withno othermosquitoes flying about.According to Bates (1941, p. 169) thematingof aegyptigenerally is precededby a on thepartof themale.However, sexualflight thepresent studyshowsthat is notobligatory thisflight (in cages11" x 11" x 15") or a necessarv preformating.It is possiblethatthe behaviorof male aegyptihas requisite to thatshowntoday.Gibson evolvedfroma formwhichoriginally swarmed thata changeof habit,likethefrequenting (1945,p. 292) suggested of small of individuals enclosures of thesamespeciesmayresultin bya largelnumber ". . . divorced fromtheactualmating thedancebecoming as a preludeto its totalsuppression." Finallywithregardto theattitudes assumedduringcopulation it is suglinearposition(Fig. 36) and;thelinearposition(Fig. gestedthatthemodified fromchangesor modifications in thelast tarsalsegments 38) haveresulted andmiddlelegs. and clawsofthemale'sfront


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311

The Behaviorof Male aegyptito Sound HISTORICAL

It has longbeenknownthatmosquitoes reactto sounds.Child (1894), Ross (1901), OstenSacken(1901), Laurence(1902), Knab (1907b), Cambournacand Hill (1940), and Bates (1941) observed thatvarioussounds alteredthebehavior of mosquitoswarms. Thesesounds,whichincludedthe of a locomotive, voice,whistle soundof oarson water,a pistolshot,or the soundsfrommusicalinstruments, wouldattractor throwthe swarmsinto someconfusion. In mostof theseinstances theswarms weremales,although notall of theobservers indicated wlhich sexwasinvolved. Nuttalland Shipley (1902) quotedGrassias believing were thatpersons engagedin conversation moreliableto be bittenbyAnopheles thanwhensilent;however thiswasno proofthatthefemalewasattracted bythesoundof thevoice. Sir Hiram Maxim (1901) whileerecting an electric in lighting system New York in 1878 observed thatone of thedynamos usedgaveoffa note whichwas practically constant and malemosquitoes to enterthe attempted box fromwhichthe soundwas beingemitted. No femalemosquitoes were Maximsuggested thattheantennae wereauditory attracted. organsand the malesmistook thesoundfromthedynamoas thebuzzingof thefemale.He also notedthatstriking a tuningfork,whichcloselyresembled thesoundor tlhefemale, wouldcausea malemosquito 20 feetawayto ". . . turn within about,facethemusic,anderectthetwolittlefeathers on hishead.. Mr. A. De P. Weaver,an electrical engineer, recorded observations similar to Maxim'sand informed Howard(1901,p. 15) that". . . whileengagedin in harmonic someexperiments in whicha musicalnoteof a certain telegraphy, pitchwasproduced byelectrical means,he wasamazedto findthatwhenthe notewas raisedto a certain number of vibrations persecond,all mosquitoes, notonlyin theroomwheretheapparatus was,butalso fromotherpartsand fromoutside, neartheapparatus wouldcongregate and wouldbe precipitated fromtheairwithastonishing force, striking theirbodiesagainst theapparatus." Kahn and his co-workers (1945) made the interesting observation that ". . . whenthecall of a femaleis transmitted to twoor threemalesunder thecircumscribed spaceof a smalltest-tube, it has beenobserved underthe thattheantennae inicroscope and hypopygium of themalewillturntoward thedirection fromwhence thesoundis beingtransmitted." This "turning" of the hypopygium was apparently the firstobservation thatthe male is not merely attracted by certain soundsbut actuallyshowsa matingresponse to thesesoundfrequencies. THE MATING RESPONSE TO SOUND

of maleaegyptito varioussoundswas studiedwithboth The behavior The malematingresponse, tuningforksand an audiooscillator.9 to sounds can be separated of different intoat leastfourdistinct frequencies, behavior patterns. 9 Model 200-A, Hewlett-PackardCo., Palo Alto, Calif.


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and Flightmaybe consideredthefirstresponseto propersound frequencies in the followingmanner.A cage fullof virginmales sevcan be demonstrated eral days old is placed about a foot or more away froma loud speaker'0 connectedto the audio oscillator.The frequencydial of the oscillatoris adjusted to producea sound of 450 cycleswith the amplifierdial reading zero. The oscillatoris turnedon. When all the males in the cage come to so thatthe sound becomesaudidial is turnedmomentarily rest,the amplifier makingthe ble. Many of the restingmales will take to flight.By alternately sound audible and thensubliminal,the majorityof males will be inducedto fly.A vibratingtuningfork (480 vps, etc.) whenmoved quicklyalong the males,also inducesmanyindividualsto fly. outsideof a cage containing The second responseto sound is attraction to the sourceof the stimulus. When a cage containingmanyvirginmale aegyptiis agitatedto induceflight. and a 480 tuningforkis struckand held near the outside,the males are attractedin largenumbersto the sound source (Figs 45-46). Often in their "frenzy"theyseize one anotherand attemptto copulate.When thetuningfork is struckinsidethe cage and held amongthe flyingmales,theyare attracted to the vibratingprongsof the fork.Males also followthe soundingtuLning forkwhenit is waved about,indicatingthatthe mosquitoesare respondingto aerial vibrationsand not to the vibrationsof a substrateon whichtheymight tuningfork. be resting.Males do not followa moving,non-vibrating Once the males are attractedtheyseize the cloth of the cage near the soundingfork,usingtheirfrontand middlelegs.At thaesame timetheirwings continueto vibrateand as theyclingto the cloththeirbodiesshakeviolently; it appearsas thoughthe males are being shakenby the sound superficially vibrations.This thirdbehavioris comparableto seizingthe femalein flight will be called the seizingresponse.It is possiblethat the rapid and therefore vibrationof themale wingsafterseizingthe female,lessensthe burdenof the latterand helpsto maintainthepair in flight.If the tuningforkis strucknear restingmalesor if the oscillatoris used so thatthe sound emanatingfromthe speakeris diffused,the seizingresponsesometimesis given fromthe lesting positionwithoutthe initialflight. The fourthbehaviorpatternconsistsin flexingthe abdomenventradso that the genitaliatouchthe clothof the cage. This usuallyis done afterthe wingscome to restthoughsome individualsflexthe abdomenwhilethe wings to by Kahn is the one referred This last responseapparently are stillvibrating. to the finalstage in copulationwhenthe et al. as "turning"and corresponds male makesconnectionwiththe female.The flexingof the abdomenwill be to as theclaspingresponse. Whetheror not spermis ejaculatedduring referred a claspingresponseevoked by mechanicallyproducedsound was not investigated. The seizingresponsecan be elicitedwithoutthe follow-upclaspingresponse 10 Utah 3PW1; speaker; 21V2watts.

Utah-Radio ProductsCo., Chicago; 312" permanentmagnet


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and theclaspingresponsesometimesis inducedwitho-ut the preliminary seizing response.When males flyto the sourceof sound the seizingresponsealmost invariablyis given. If males respond froma restingpositionwithoutfirst takingto flight, eitherthe seizing,clasping,or bothresponsesare given.After repeatedor continuousexposureto sound,the claspingresponsemay be given withoutthe initialseizingresponse.After a seizingresponseis evoked,the abdomenmay be flexedventradand back severaltimesso thatmorethan one claspingresponseis elicitedby thesound of a singfetone,evenafterthesound is discontinued.

A typicalmatingresponse (i.e. attraction, seizing,and clasping) can be elicitedfromvirginmales by the propersound of the voice.Laurence (1902)

4?~~~. i~~~~

~

i

~4Z

%

ri

9..,,.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

~~~~~~~~~~~~~~~~~~~~~~ .4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Figs. 45, 46.

Attractionof male -

Aedes

~

aegipf,

to

the sound of a tuning fork(480 .

~~ ~ ~ ~ ~~~u.~~~-

s;i~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~A of male Aedes aegyplito the soundof a tuningfork(480 Figs. 45 46. Attraction vps).-45. The outlineof the tuningforkcan be seen throughthe cloth side of the cage. Practicallyall of the males are showngivingthe seizingresponse(vibratingthe wingswhile seizingthe clothwiththe frontand middle legs); 46. The arrow points to a male givingthe-claspingresponse(flexingthe abdomenventrad). (Photographby E. R. Willis.)


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bythesoundof thevoiceandstated,". . . as faras my inosquitoes attracted memoryservesme, the sound thatwas most in favourwas a continuous'oo' pitchedin a mediumkey,and thensuddenlychangedto a lower'ah,' drawn out in the same way. Most vivid is the sudden rise in the volume of their noise, and the equally sudden attackon the face as theypromptlyanswered the invitation." OTHER

REACTIONS TO SOUND

Male aegyptinot only give a matingresponseto sound but also reactto by (1) rubbingthe antennaewiththe frontlegs, (2) rubcertainfrequencies bingthe hind legs together,(3) rubbingthe abdomenor wingswiththe hind legs, (4) jerkingthe body or hind legs slightly,(5) sudden flight,(6) sudor (8) continuousactivity(flyingor denlycomingto rest, (7) immobility, walking). In generalthese are consideredby Warden et al. (1940, p. 642) to sound and are formsof insectbehaviormanifestations as themostprimitive The presentpaper deals groupedunder the generalterm"shock-reactions." chieflywiththe matingresponseof aegypti males, inducedby varioussound frequencies. TUNING

FORK EXPERIMENTS

whichsounds The followingavailabletuningforkswereused to determine would inducethematingresponse:256C, 288D, 320E, 341.3F, 384G, 426.7A, 480B, 512C, 1024C, 2048C, 4096C.11 Eitherthe seizingor claspingresponse, or both,wereconsideredcriteriaformatingbehavior. Mosquitoeswereexposedto the forksfora fewseconds,or untilthe sound died out. Tests showedthatthe males respondto the sounds of forksranging to the sounds of any of the from256 to 512 cyclesbut remainindifferent above forkswith frequenciesgreaterthan 512 vibrationsper second. \hen When the responseis negative, positive,the responseis usuallyinstantaneous. males,more themale failsto respondas longas thesound is audible.Fifty-five than 24 hoursold, wereplaced (severalat one time) in an 11" x 11" x 15" forksstartingwiththe lowest (256) werestruckand cage and the different Leld on theoutsideof thecage neartherestingmosquitoes.If theresponsewas positive,the male was removedfromthe cage and was not subjectedto any of the higherfrequencyforks,since in generalmales respondedto aI the whichstimulatedthem,up to 512 vps. If sounds above the lowestfrequency the responsewas negative,the next higherforkwas used until a mating responsewas elicited.The 512 and 480 forksinducedan immediatemating response,but since all the males testedrespondedto frequenciesbelow 512 and 480, these two forkswerenot used in thesetests.The resultswiththe 55 males wereas follows: 7 respondedto 256, 5 to 288, 22 to 320, 12 to 341.3, 8 to 384, and 1 to 426. between320 and 512 vps,oftenbeing Most malesrespondedto frequencies attractedfromsome distanceaway and followingthe forkwhenit was moved. Co.; all others I1I The 1024 and 2048 forkswere obtainedfromCentralScientific were made by RiversideLaboratories,Geneva, Ill.


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response fromtheinsects. produced an immediate Striking theforkslightly attracted to thesoundsof the256 and288 forksas Maleswerenotso readily and these Later,100 and 128C forkswereobtained, theywereto theothers. fromvirgin malesseveraldaysold. induceda mating response THE BEHAVIOR OF MALES SUBJECTED TO AN AIR CURRENT AND SOUND

SIMULTANEOUSLY

inhibited theresponse Minnich(1925) foundthata continuous aircurrent as beingdue to one thisbehavior of Vanessalarvaeto sound.He explained theresponse to another formof stimulation. formofstimulation inhibiting in front ofthebreeze Whena cagefullofmaleaegypti wasplaceddirectly quicklycameto reston thesidesand floor of an electric fan,themosquitoes of thecage.As soonas thefanwasshutoffand thespeedof theaircurrent themosquitoes tookto flight. A cageofmaleswasplaceda fewfeet decreased in front of airpassingthrough thecage ofa fanand thespeedof thecurrent Two airspeedswereused;one withthe was measured withan anemometer. ofthecage,nearest thefan,at a rateofabout2.8 thefront airpassing through milesperhourand through thebackof thecageat about2.3 mph;theother at about3.5 mphandthrough withtheairpassingthrough thefront theback ofthecageat about2.6 mph.The loudspeakerconnected to theaudiooscil(400-600cycles)were latorwas placedin thecage and variousfrequencies oniand themaleshad cometo rest(tuning soundedafterthefanwasturned to giveboththeseizingand clasping forkswerealso used). Males continued in spiteof thebreezeand flewto thesourceof sound.However, responses to thetuningforkthatcouldbe obtained therewasnotthegreatattraction of airfroma fanwasblowing thecage.Whenmale whenno current through wereexposedto soundand an aircurrent simultaneously, themating aegypti was response elicitedby a tuningforkor soundsfroman oscillator-speaker to clingto thecage (remainat rest)whenstimuthantheresponse stronger latedbyan aircurrent. thatMinnich'sobservations are not It shouldbe pointedout,however, on aegypto. Withregard to Vane3experiments quitecomparable to thepresent whiletheeffect of sa theaircurrent causedthelegsto clingto thesubstrata thesamelarvato releaseitslegholdfromthesubstrata. In this soundinduced to aircurrent opposable. However, and soundweredirectly casetheresponse withaegypti theaircurrent some causedthemalesto cometo restand though malesflewto thesourceof sound(tuningfork)to givethematingresponse in spiteof theaircurrent, fromthe response manygavea seizingor clasping to flight. malescangivea mating without Sinceaegypti taking resting position thebehavior responses to an air current position, response fromtheresting andsoundarenotnecessarily opposable. THE

MECHANISM

OF AUDITORY

PERCEPTION

no experimental evidence thefunction and mcdeof conceming Although theorganhas actionof Johnston's organin theCulicidaehas beenobtained,


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generally been considered one of "hearing."12 As earlyas 1855,Johnston concludedfrommorphological evidencethatthe pedicelof the antennaof Culexwasauditory in function. LaterMayer(1874) observed thesympathetic vibration of themaleantennal fibrillae to thesoundsof varioustuningforks, and he concluded thatJohnston was correct in believing thatthisstructure to Eltringham female.According detectsthesoundproducedby the flying (1933, p. 106) thesensory function of Johnston's organwas quiteobscure and stillopento question. Wardenet al. (1935,p. 315) wrote". . . thereis is a to support thecontetntion verylittleevidence thattheorganof Johnston soundreceptor. Its widedistribution and its constant positionin thesecond thatit is a deviceforregistering themovements of antennal segment suggest theflagellum of theantenna." Wigglesworth (1939,p. 136) stated,"Judging of the fromitsstructure, Johnston's organmustbe stimulated bymovements flagellum of the anteinaat its base." The lack of experimental evidence concerning thefunction andmodeof actionof thisorganwassummed up by Pumphrey (1940,p. 129) whowrote,". . . , thename'chordotonal organ' has come to be construed in a structural sense. ratherthana functional thepresence of suchan organ(Johnston's Nevertheless, organ)in thebase of theantennae ofmostinsects has beentakento implyan auditory function for theseappendages. The experimental evidencefor sucha viewis very The observations of Mayerquotedin theIntroduction meagre. do notrecessarilyimplythatJohnston's organis involved, forthelonghairson theantento sound nae ofmalegnatsareprobably directly innervated andmayrespond in a waysimilar to otherlongsensory hairs.Fromtheworkof Hollick(comto theSocietyforExperimental nmunication Biology,1939) it appearsthatin to displacement cycloraphous DipteraJohnston's organresponds of theantenna byaircurrents andplaysan important partin flight in mediating thereflex adjustment of thewing-beat to theappropriate formfora givenair velocity. According to Eggers(1926a), thesameorganin Gyrinus responds to changes in curvature of the watersurface.Johnston's organis, therefore, obviouslv rathera labilestructure and thepossibility of its mediating soundreception in someinsectscan by no meansbe ruledout. The subjectneedsfurther investigation." Thereareessentially twomethods ofstudying hearing in insects. One is to observe thebehavior of insects to variousmechanically-produced soundsor the ofthespeciesstudied(Turner,1914;Turner soundsmadebyotherindividuals and Schwarz,1914;Minnich,1925,1936,1937;Abbot,1927; Baier,1930, is to record actioncurrents in auditory andmanyothers).The secondmethod to acoustical nervesin response stimulation (Wever,1935;Weverand Bray, 1933;Pumphrey andRawdon-Smith, 1936a,b, c, 1937). The following experiof maleaegyptitowardsfemalesand the soundsor mentson thebehavior to testthevalidity of thehypotheses tuningforkswereperformed concerning thefunction andmodeof actionof Johnston's organ.Duringthetests,when12 The term"hearing"is used in thebroad sense to indicatea responseto vibratory stimuliby the aid of a phonoreceptor.


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themosquitoes thecage containing everthemaleswereexposedto females, to inducetheinsects to fly. wasdisturbed malesto the of antennectomized thebehavior Experiment 1.-To determine forks. soundsof tuning It has been shownearlier(see p. 293) thatmales whichhad their closeto thepedicelliwereindifferent segments removed flagellar antennal to

47

48

49 .0? MM.

50

51

52

Figs. 47-52. Heads of male aegyptishowingthe various antennaloperationsperorgan.-47. Flagelthe function and mode of actionof Johnston's formedto determine extending beycndthe lai segments removedleavingsmall portionsof the thirdsegments pedicelli; 48. Flagellar segmentsremovedand the pedicelli,remainsof the thirdsegments,and partsof the eyes coveredwithshellac; 49. Shellac placed at the base of the antennaecoveringthe pedicelli,segmentsthreeand four,and parts of the eyes; 50. Tips of bothantennaeweightedwith drops of shellac; 51. Weightedportionsof theantennaein Fig. 50 amputated;52. Practicallyall of the antennalfibrillaeremoved leavingtheantennalshaftsnearlybare. Abbreviations:E-eye; P-pedicel; S-shellac.


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to determine if similarly females. The following experiment was performed operated maleswouldbe indifferent to thesoundsof tuning forks. ofbothantennae closeto thepedicelThe flagellar segments wereremoved, li, from10 males;onlysmallstumpsof thefirstflagellar segments and no thesemales fibrillae, remained (Fig. 47). Exposed14 hoursaftertheoperation wereactivated(shock-reactions) by the soundsof the 480 and 512 tuning forks.Onlyone malegavea matingresponse; apparently theothers, by the wereableto perceive thesounds.The experiment factthattheytookto flight, wasrepeated males.Thesewereexposedto thesouncd using18 newly emerged of the480 fork, 42 hourslater.None gavethemating response, butgenerally All of thesemaleshad eitherthefirst or partof the wereactivated to flight. of the pedicelli, firstflagellar with segments protruding beyondthemargins no fibrillae remaining. Fifteenmaleshad theirflagellar segments removedand the remaining stumpsand pedicellicoveredwithshellac(Fig. 48); partsof theeyeswere also unavoidably afterthe operation, covered. Tested20, 46, and 54 h-ours to thesoundsof the480 and 512 forks. theyremained completely indifferent Conclusion. Maleswhichhavetheirantennae eliminated byremoval ofthe of the remainswithshellacfail to givea and covering fiagellar segments mating response to soundsoftuning forks. Experiment 2.-To determine thebehavior of malesto femalesand tuning forksoundsafter shellacwasplacedat thebaseoftheantennae. A smalldropof shellacwas placed,by meansof a micro-pipette, at the baseoftheantennae (Fig. 49) ofeachof 20 non-virgin 7-day-old males.This operation cemented thefirst flagellar segments to thepedicelliand prevented theshaftsfromvibrating at theirbases.The remainder of thesegments and theirattached fibrillae werefreeto vibrate. The maleswereexposedto the soundof a 480 tuningfork12 hourslater.Seventeen maleswereindifferent to sound;threemalesgavea mating response. Theselast3 maleswereexaminedundera binocular microscope and eachwasfoundto havethehollowof one pedicelincompletely one shaftwas filledwithshellac,so thatapparently at itsbase. capableof vibrating The experiment wasrepeated using20, 3-day-old non-virgin males.When to thesoundof the480 exposed4 and 17 hourslater,theywereall indifferent fork.Thesemaleswerealso indifferent to 50 females introduced intothecage 4 hoursaftertheapplication of shellac.All themilesused in theseexperito thetuningforksoundpriorto theapplicamentsgavea mating response tionof shellac. to flying Conclusions. Males areindifferent females and to thesoundsof shaftsare prevented frommovingat their a tuningforkaftertheantennal bases. 3.-To determine the behavior of malesto femalesand tuning Experiment thetipsoftheantennae forksoundsafterweighting withshellac.


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weighted maleshadoneoftheirantennae non-virgin 3-day-old Twenty-five, Thesemaleswere witha dropof shellacplacedat thetip of theflagellum. males exposedto thesoundsofa 480 fork,19 and 28 hourslater.Twenty-two The.e malesappearedto give threefailedto respond. response; gavea mating thannormalindividuals. response a lessvigorous A smallblobof shellacwasplacedon thetips,or apicalthirdor half,of not males.The fibrillae non-virgin bothantennae(Fig. 50) of 52, 3-day-old werefree segments flagellar and thefirst extended byshellacremained covered of the480 forkat at theirbases.Thesemaleswereexposedto thevibrations of shellac.Only 2 malesgavea 12, 19, and 24 hoursaftertheapplication and one was foundto haveonlya smallamountof shellac matingresponse theothermale withtheotherantenna)on one of theantennae; (compared had2 largedropsofshellacat thetipsof bothantennae. and theporAt the12 and 19 hourtrials,27 of themaleswereremoved, tionsof theantennaebearingthe shellacwereamputated(Fig. 51). The antennal variedfrom5 to 9. Exposedto the segments of remaining number portions, of theweighted to 5 hoursafterremoval fork,one-half 480 tuning and onehad died. onefailedto respond response; 25 malesgavea mating and antennae, Afterthe24-hourtrial,20 of the maleshavingweighted whichfailedto respondto sound,wereplacedin a cagewith5 femalesfor The males to thefemales. indifferent 15 minutes. The maleswerecompletely wereampuof theantennae portions and theshellacked werethenremoved partsof the tated.Threehourslaterthesesamemales,lackingtheweighted at whichtimetheywereattracted wereagainexposedto 5 females, antennae, to copulate. and attempted with weighted Conclusions. Males whichhavethetipsof theirantennae to femalesand sound.Males respondto femalesand shellacare indifferent Weighting of theantennae areamputated. portions sound,aftertheweighted at their theshaftsfrommoving prevents apparently thetipsof theantennae bases. thebehavior of malesto femalesand tuning 4.-To determine Experiment oftheantennal fibrillae. forksoundsafterremoval fibrillae all of theirantennal removed Five maleswhichhad practically witha camel'shairbrush,leavingthe shaftsnearlybare (Fig. 52), were by the 480 and 512 forksand usuallygave the seizing readilyactivated Whenthefork whentested19 and 45 hoursaftertheoperation. response

was sounded insidethe cage and held near the males, theywould flyto the vibrating prongs.

was repeatedwith22 males,less than one-day-old.These The experiment wereexposedto the 480 tuningforkat 39, 43, 50 and 66 hoursafterremoval of the fibrillae.Seventeengave a matingresponse.A seizing responsewas usuallygiven,thoughsome individualsalso gave a claspingresponse.Forty12 of the males whichhad given rninehoursafterthe startof the experiment,


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40 females. a positiveresponse to soundwereplacedin a cage containing oneof Duringthe15-minute trial,only3 attempts to copulatewereobserved, wasexamwhichwassuccessful. The malewhichhad succeeded in copulating inedundera binocular microscope and was foundto have severalantennal on thefirst fibrillae remaining and secondflagellar segments. Malesrespond to thesoundsof a tuningforkafterremoval Conclusions. vibraof practically all of theantennal fibrillae. thesympathetic Apparently thesensecells shaftalone,to intense tionof theantennal sound,stimulated indifference of thesemalesto females in Johnston's organ.The comparative as beingdue to thegreater of a tuning forksound maybe explained intensity as compared fibrillae to thatproduced bythefemalein flight. The antennal serveas an "amplifier" in thesensethattheyareset intovibramaypossibly soundof theflying female. Lutz (1924,pp. 336-337), tionbythelessintense of on Mayer'stuningforkexperiments, commenting statedthattheintensity at a distance thesoundof a female". . . especially greatenoughto makeit in guiding a desirable factor themale,is so slight thatthesoundfroma tuning forkon a resonant boxplacednearand turnedtowardtheantennawouldbe likethatof an artillery battlein comparison." . Discussion.-Maleaegyptishowa definite attraction to certainsounds, and Wever'sstatement (Howard,1901) thatmosquitoes (speciesnot indisounds certain cated)wouldbe "precipitated" againstan apparatus producing is quiteapplicable to thebehavior of aegypti malesto thesoundsof various are so drawnto the forkthatthe response tuningforks.The mosquitoes The path reminds one of theinevitable attraction of ironfilings to a magnet. line;thetypeof takenbytheflying maleappearsto be moreor lessa straight of orientation wouldpossiblyfitFraenkeland Gunn's (1940) description of intropotaxis whoseessential features are ". . . simultaneous comparison on the two sides,and orientation tensities to the balancethus according thesympathetic vibration of theantennal hairsof Culex struck." Concerning to the soundsof a tuningfork,Mayer (1874, p. 586) statedthat"If a is at right so thatitswave-front sonorous wavefallsuponone of thesefibrils of thepulsesin thewavea,rein and hencethedirection anglesto thefibril, of thefibril's thelattercannotbe set in vibration; thedirection butif length, in thewaveare brought thevibrations moreand moreto bearathwart the fibrilit willvibrate withamplitudes increasing untilit reachesits maximum whenthewave-front swingof co-vibration, is parallelto itslengthand thereforethedirection oftheimpulses on thewaveareat right anglesto thefibril." the Fromhisobservations he concluded thatthesoundof thefemalevibrated moreforcibly fibrillae thanthoseoftheotherandthemosquito ofoneanteinna hisbodyin thedirection of thatantenna turned thefibrils ofwhichweremost thevibrations Whenthemalebrought of eachantennato equality affected. hisbodyfacedin thedirection of theradiation of intensity, of thesound,and hedirected hisflight accordingly. Mayerfurther concluded thatthemalecould to within5 degreesof thedirection of the female.Mayer's guidehimself and Johnston's beforehim,was essentially a tropohypothetical explanation, tacticone. However,in the presentexperiments, it was foundthatmales


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lacking one antenna wouldflyto thevibrating prongsof a tuning fork,when theforkwasstruck and heldinsidethecageamongtheflying insects. These males did not show continuos unilaterally flagellectomized turning movements(circusmovements, Fraenkel and Gunn,1940,p. 77) whichare supof tropotactic posedlycharacteristic responses. However,it will be recalled thattheability ofthemaleto seizea female in flight is greatly reduced bythe removal ofall theflagellar segments fromoneofhisantennae (see p. 293). Tropotactic responses usuallyare associatedwiththe termphototaxis (Fraenkeland Gunn,1940). Snodgrass(1928) usedthetermphonotropism of insects forthereaction to sound.In keeping withmorerecent terminology, is hereby of certain phonotaxis usedto indicate theinfluence soundfrequencies on themovement ofmalesofaegypti. Fulton(1928,p. 553) statedthatMayer'stuningforkexperiments did notactuallyprovethatmosquitoes hearwiththeirantennae anymore thanit couldbe provedthata pianohearswhenitswiresaresetintosympatheticvibration by soundwaves.The mosquitogave no indication thatit of sound."Wardenet al. (1935,p. 313) also stated perceived anysensation thatMayer'sworkwas ". . . of smallvalue,unlesssupported by evidence frombehavior." The foregoing behaviorexperiments, summarized in Table II, supportMayer'shypothesis the hypothesis and confirm thatJohnston's an assumption organis auralin function, previously basedon morphological evidence. The experiments also provethatthemalesperceive vibratory stimuli in theliterature. as had beenhypothesized That is, theantennal or fibrillae in response whorlsof hairs,vibrating to certain soundfrequencies, causethe shaftto vibrate.The movement on antennal of theflagellum of theantenna itsbasestimulates thesensecellsattached to sclerotized processes of thecirculal plateat thebaseof thefirst flagellar segment (Fig. 6) and theresulting impulsetravelsalongtheantennal nerveto thebrain.The antennal fibrillae are not inlervated as suggested apparently directly by Pumphrey(1940) sincemalesgenerally failto respondto soundwhenthemajority of fibrillae arefreeto vibrate butmovement of theantennal shaftat itsbaseis prevented 2 and3, pp. 318-319). (see Experiments Howardet al. (1912, p. 35) statedthat"Our ownobservations of the habitsofmosquitoes leadus to doubtthattheantennae, if auditory at all,are so in theirprimary function. The foregoing hypotheses are founded uponthe thatthemaleseeksthe* assumption femaleand is attracted by hersong.In factsuchis notthecase.At leastin thoseforms in whiich theantennae of the malearemosthighlyspecialized themales'swarm'and thefemaleseeksthe male.In ourexperience thesongof thefemaledoesnotattract themale."It has beenpointedout (see p. 308) thatthereis no experimental proofthat thefemaleis attracted bymaleswarms. The soundof femaleaegypti is the chief,if nottheonly,extetnal stimulus thatwillinducematingbehavior on thepartof males,andwhenexposedto certain mechanical soundwaves,these maleswillshowa characteristic matingresponse whichis comparable to the copulating behavior withthefemale.Althoughthe femalehas a Johnston's organ,it is notas welldevelopedas thatof themale.The modeof attach-


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(in CulexandCorethra, mentofthesensecellsto thebaseofthethirdsegment

would be see Howard et al., 1912, Plate 1, Fig. 7) indicatesthatthe structure in respondingto movementsof the flagellumat the base. much less efficient fromthe audio Female aegypti exposedto a wide rangeof sound frequencies showedno unusualbehaviorotherthansome shock-reactions. oscillator-speaker TABLE II.-Summary of the Behavior of Aedes aeg_pti Males, under Various Conditions,Towards Female aegyptiand to the Sound of Tuning Forks Experimental (480 and 512 vps).

Type of antennal operation

To female

Behavior To tuningfork

1. Normal male (Fig. 13)

Seized femalein flightand copulated

Gave eithera seizingor clasping r e s p o n s e oI both

2. Flagellar segmentsremovedclose to thepedicelli,or below the apex of the first flagellar segments, leavingno fibrillae(Fig. 47)

Ignored the females

Rarely gave a matina responsebut some were activatedto flight

3. Complete antennectomy;flagellar segmentsXemoved and pedicelli stumpsof the first and remaining flagellar segmentscovered with shellac (Fig. 48)

Not e x p os e d, since males in 2, ab o v e, ignored females

Unresponsive

4. Shellac placed at the base of the antennae,completelycoveringthe basal flagellarsegments and pediof the shaftsand celli; reinainder fibrillaewere freeto vibrate(Fig. 49)

Ignored the females

Unresponsive

5. a) Tip of antennaweightedwith drop of shellac

Not exposed to females

Generally gave a mating response

b) Tips or about upper thirdof both ailtennae weightedwith shellac (Fig. 50)

Ignored the females

Generally did not re spond(may dependupon weight or amount of shellac applied)

c) Weightedportionsof antennae in b), above, amputated(Fig. 51)

Attractedto females and copulated

Gave a m a ti n g response

6. Practically all of the antennal fibrillaeremoved,leavingthe antenisal shafts nearly bare (Fig. 52)

G e n e ra I I y ignored the females

Usually gave a mating response; some males s h o w directional response, flying to the vibratingprongs of the

Co-k


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Child(1894) believed thatthefunction ofJohnston's organwasoriginally theperception of tactilestimuliand afterthestructure becamemorehighly developedit also servedforthe perception of soundwaves.Howardet al. (1912) did notdenythattheantennae werealso in a rudimentary wayauditoryin function. Theyclaimedthatthough". . . mosquitoes reactto sound.waves,it is by no meansclearthattheycan perceive themas such.If the sound-impressions are received through theantennae, as theorganization of theseappendages seemsto indicate, it is stillto be doubtedthatthereis a differentiation of theimpressions produced bysound-waves and otherstimuli, suchas a current of air,or between soundsof different pitch."Mosquitoes orientto air currents, and aegyptimaleswillcometo restquicklyin a cage whenexposedto thecurrent froman electric fan.Undoubtedly theantennae aredeflected bygrossairmovements butit cannotbe saidforcertain thatthe malescometo restas a resultof theantennae beingset intomotionbythe air current. it has beenassumedthatmosquitoes Although orientthemselves to air currents by meansof theirantennae, Kennedy(1939) foundevidence to indicate thatin femaleaegyptivisionplaysan important rolein up wind orientation. However,maledegypti respondto certainsoundfrequencies bv givinga mating responise, yetthesesamemalesmayreactto otherfrequencies a shock-reaction bygiving (i.e. jerking thehindlegstogeththebody,rubbing er, etc.). Thesedifferences in behavior implysome-formof discrimination, and thismaybe accomplished by different numbersof sensecells of Johnston's organbeingstimulated.Adrian (1928, p. 31) concerning nervefiberimpulses

stated that ". .

.

the only way in which the message can be made to vary at all

is by a variationin the total numberof the impulsesand in the frequency withwhichtheyoccur." The deflectionor movementof the flagellumof the antennaof aegypti males may varyin responseto different sound frequencies and air currents, and thisin turncould varythe typeof responsein the antennal nerve.Pumphreyand Rawdon-Smith(1936b) showedthat the responses of the cercalnerveof the cockroachvariedwiththe frequency of the auditory stimulus.The same authors(1936b, c) foundthatthe cerciof the cockroach wereverysensitiveto movements of the air sinceactioncurrents wererecorded from the cercal nerve after exposing the cerci to a ". . . light puff of air." The

actioncurrentrecordedfromthe cercalnerveafterstimulating the receptorby air movementswas different fromthe impulsesrecordedafterstimulating the organwithsound. In 1939, Pumphreyand Rawdon-Smith advanceda theory to explain "frequencydiscrimination" in insectspossessingtympanicorgans. Final proofthatJohnston's organof male aegypti is actuallycapableof distinsound guishingbetweenan air currentand sound, and betweendifferent frequencies, can be obtainedby recordingaction currentsfromthe antennal nerveaftersubjectingthe antennaeto thesevariousstimuli. AGE OF MALE AT FIRST MATING RESPONSE

TO SOUND

It was shownearlierthatmale degyptimay attemptto copulatewhentheir genitaliahave rotatedabout 135 degrees.These attemptsare generallyunsuccessfulbecause a rotationof about 180 degreesis n-ecessary beforethe male can make connectionwith the female.The followingexperimentwas per-


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formedto determinehow soon afteremergencethe male will show a mrating response(eitherseizing,clasping,or both) to the soundsof tuningforks. Ninetymalesisolatedat emergence wereexposedto soundsof tuningforks at variousintervals;the forkswere struckand held near the mosquitoes.Six malesgave positiveresponsesto the 288 and 320 forksas earlyas 5 hoursand 20 minutesafteremergence;12 individualsrespondedbetween5 hoursand 20 minutesand 11 hoursafteremergence.Practicallyall of the othersresponded pbout 14 hours 40 minutesor later,to the 480 and 512 forks.The early responseswere obtainedfrommales the genitaliaof whichhad rotatedless than 90 degrees.Most of the males whichrespondedto the sounds of the tuningforksabout 15 hoursor less afteremergencedid not have completely rotatedgeniitalia. Also, manywould not attemptto copulatewhenplaced in a cage containingfemales.This responseto the tuningforkand indifference to the femalemay be explainedby the factthat the sound of the flyingfemale is much less intense,and more transientthan that of a tuningforkbeing soundedand held nearthe male. Thus tihemating-state or conditionwhenthe male will show a seizingor claspingresponse(matingbehlavior)may be reachedlong beforehe is either physicallycapable of copulatingsuccessfully because of incompletely rotated genitaliaor beforehe will even attemptto copulatewithfemales.If the eighth abdominalsegmentof the male is ligatedwitha finethread,beforelittleor no rotationhas takenplace,the genitalsegmentsare preventedfromrotating. This was done to 6 males about 4 to 5 hoursafteremergence, and 15 hours later all gave matingresponsesto the 320 and 426.7 vps. tuningforks.The male matingresponseelicitedby a female in flightapparentlyis correlated withor dependentupon the conditionof his antennalflbrillae, ratherthanthe rotationof his genitalia.In youngmales,less than 5 hoursold, the antennal fibrillaecling together,are flattenedagainst the shafts,and apparentlyare of covibratingeven to the intensesound of a tuningfork. It is irncapable possiblethatsome internalchangetakesplace or perhapsthe antennalshafts harden;older males whichhad nearlyall theirfibrillaeremoved,leavingthe slhafts practically bare,gave a matingresponseto the sound of a tuningfork (see p. 320). As the male ages, the antennalfibrillae,beginningwith the ones on the basal segments,becomeextendedand therebycapable of being set intosympathetic vibrationby sound stimuli.Though some males less than 15 hours old may respondto tuningforksounds,they often ignoreflying femalesprcsumablybecause the femalesound is much less intensethan the sound of a vibratingfork,and not enoughof the male antennalfibrillaeare extendedto perceivetheslightsoundmade by the femalein flight. It is generallyacceptedthatsexual behaviorapparentlyis uninfluenced by the gonads (Wigglesworth,1939, p. 383). Bates (1939) found that the Fa males of the atroparvusx maculipennis cross,even whenrenderedcompletely sterilebecauseof atrophiedgonads,formedin a swarmand attempted to mate whenplaced in a small cage. From thishe concluded(1941, p. 170) thatthe sexualbehaviorpatternis probably". . . not dependenton the functioning of the primarysexual organs,and thatit may have a complexgeneticbasis,since


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thecharacters of theF1 seemto differ fromtheparental characters and may represent a blend." PRELIMINARYAUDIO OSCILLATOR EXPERIMENTS TO DETERMINE THE RANGE OF SOUND FREQUENCIES WHICH WILL INDUCE THE MATING RESPONSE

The previoustuningforkexperiments did not determine the limitsof soundwhichwillinducethe matingresponsein maleaegypti. Not enough forkswereused and puretoneswerenot produced. A tuningforkgivesa singlepuretoneonlywhenstruck soft gently at theproper placewitha fairly hammer;otherwise overtones are produced,and theseare not necessarily harmonic (Saunders,1936,p. 286). No attempt in anyof thetuningfork experiments wasmadeto producea puretone,theforksusuallybeingstruck at theirtipswitha resulting veryintense sound.To determine theupperand lowerlimitsof soundfrequencies whichwillinducethematingresponse, an audiooscillator wasused,forwiththisinstrument a widerangeof soundfrequencies couldbe explored andrelatively puretoneswereproduced. The following preliminary experiments wereundertaken to givethewriter someideaas to thelowerandupperlimitsof soundfrequencies whichwould in aegypti inducethemating response males.The audiooscillator washooked up to the3I2" loudspeaker. Two methods wereusedwiththesepreliminary studies.Withthefirst, thespeaker, whichwasbuiltintoa smallwoodenbox, wasplacednextto onesideontheoutsideofa cagecontaining severalhundred virginmalesmorethan24 hoursold. The speakerwas leftstationary or movedbackand forthfromone end of thecage to the other.With this methodthesoundwas notconcentrated at one pointbutwas somewhat difthecageso thatcomparatively to fusedthroughout fewmaleswereattracted thesource;mostof theindividuals responded wherever theyhappenedto be in thecage.Withthesecondmethod, resting a glasstube1?/4"in diameter was fitted intothespeakerand thewholeboxwas wrapped in severallayers of absorbent cottonso thatthesoundwhenemitted was moreconcentrated neartheopening of thetube.The speaker wasmovedaboutand placedwith theopening oftheattached tubeneartheresting mosquitoes. Withthismethod therewas someattraction, arLdtheproperfrequency and malesflewfroma shortdistance seizingtheclothnextto theopening of thetube.The attraction obtained withthespeaker attached to theaudiooscillator wasneverso marked as thatobtained withtuningforks, thisapparently beingdue to thefactthat a tuning fork,in comparison to thespeaker, produced a moreor lessconcentratedpointsourceof sound.The fullrangeof the oscillator, from35 to 35,000cyclespersecondwas explored, thefrequencies beingchangedat few It is notknownwhether secondintervals. thespeakerused was able to procducetheveryhighfrequencies and it is doubtful thatsoundsabove5,000 cycleswereof desirable purityand intensity. However,themalesresponded to ratherlow frequencies (about150 to 750 cycles)so thatthehigherfrequenciesdid nothaveto be tested.Severaltestswererun,first with starting the lowestfrequency withthe highestand and working up, thenstarting down. working


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withthelowestand working Starting thelowestfrequency upwards, which induceda matingresponse was 150 cycles.However,fewmosquitoes gavea positive to soundsbetween150 and 300 cycles.When exposedto response soundswhosefrequencies rangedfrom100to 300 vibrations persecond,many malesshowedtheusualshock-reactions suchas (1) a slighttwitching of the abdomen,(2) cleaning theantennae withtheirfrontlegs,(3) wavingtheir Ihindlegs up and downor rubbing themtogether, etc. A largenumberof malesgavethemating to frequencies response between 350 and 650 cycles. Startingwiththe highest frequencies and working the first downwards, soundto inducemanymalesto respond was650 cycleswit..someindividuals responding to 750 and700 vibrations persecond. Aftera groupof mosquitoes wasexposedto certain frequencies (i.e. 300600) some individuals responded to lowerfrequencies whichtheyignored initially. Thus in one trialwhilenIo matingresponse was givento the first soundsof 200-250cycles,some maleslaterresponded to thesefrequencies afterreacting to higherfrequencies. Also someindividuals whichfailedto respondto 700-750vibrations initially, to thesewhentheywere responded soundeda secondtime,afterthemaleshad beenmadeto respond to frequenciesbetween 600 and 400 cycles.Sincetherewas a comparatively briefsilent periodbetweenthesoundings,it was possiblethatthe responseto one frequency was carriedoverto the soundingof another.

Conclusions. From these preliminary trialsit is seen that the limitsof whichwill induce the matingresponsein male aegypti are sound frequencies about 150 and 750 cycles.In thenextsectionit willbe shownthattheselimits are influenced or non-virginity by age and virginity of the males.

LOUD SPEAKER

OBSERVATION

CAGE

53 AUDIO

OSOLLATOR

REDCTION

GEAR

Fig. 53.-Apparatus used for determining the limitsof sound frequencies which will inducethematingresponsein male Aedes aegypti.


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DETERMINATION OF THE UPPER AND LOWER LIMITS OF SOUND FREQUENCIES WHICH WILL INDUCE THE MATING RESPONSE IN VIRGIN AND NON-VIRGIN MALES

Method.-The previouspreliminary experiments indicatedthatthe mating responsecould be elicitedfrommale aegyptiby sound frequenciesranging from150 to 750 vibrations per second.It was also noted thattherewas some variationbetweendifferent males in theirresponseto this range of sound frequencies. For the followingexperiments an apparatuswas devisedwherebymosquitoes could be exposedto knownfrequenciesof sound froma speakerconnectedto an audio oscillator,sounded for definitelengthsof time at fixed and reguiarintervals.The apparatus(Fig. 53) consistedof an electricstirrer connectedby a flexiblecouplingto a reducinggear havingan attachedarm whichwhen rotatingwould make and break the contactwith the oscillator. By varyingthe speed of the stirrer, or usingarms of different lengthson the reducinggear, frequenciescould be sounded for desiredlengthsof time at desiredintervals.Individualmosquitoeswere placed in a small (3" x 3" x 1") cloth,wire-framed cage with celluloidsides for observation.The cage was then placed up againstthe circularopeningof the box containingthe loud speakerand the oscillatorwas turnedon. Withithe observation cage in this positionthe mosquitoescould be about '/2 to 2 inches away fromthe speakerat the momentof exposureto the sound. The electricstirrer produced an audiblehum in operationbut males apparently did not reactin any wayto this sound. The behaviorof the mosquitoesto the various frequencieswas observedand noted; only the seizingand claspingresponse,or both if given, wereconsideredcriteriaforthe matingresponse.All ctherreactionsto various frequencies, suchas rubbingthe antennaewiththe frontlegs,rubbingthe hind legs together,rubbingthe abdomen or wings with the hind legs, a slight jerkingmotionof the abdomen,suddenlycomingto rest,immobility or continuousactivity, althoughindicativeof a responseto sound, werenot considered. The followingtestsdetermined only the limitsforinducingthe mating responseand not the limitsof hearing. To determinethe upper and lowersound limitsfor inducingthe mating response,individualmale aegyptiwere subjectedto frequencies rangingfrom 100 to 800 vibrationsper second.For the upperlimiteach male was exposed to frequencies startingwith800 cycles.Each frequency was sounded 3 times, each time for 3 secondsat 6-secondintervals.If the male failed to respond duringthese 3 trials,the frequencywas lowered50 cycles,duringthe third, 6-secondsilentinterval,to 750 cyclesand so on untila positiveresponsewas obtained.After responding,the male was returnedto the stock cage. If a male failedto respondby the time350 cycleswas reached,he was countedas llot responding.The lowerlimitwas determinedin the same way; however, the frequencies used startedwith 100 cycles,proceedingupwardsby 50 cycle increasesuntil a positiveresponsewas obtained.If a male failedto respond by the time600 cycleswas reached,he was countedas not responding.


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cageat one time;the Usuallyfrom1 to 3 maleswerein theobservation othermalesstillto be testedwerekeptoutsidetheroomuntilexposedto the fromall partsof theobserMales responded soundsof theoscillator-speaker. vationcage (facingor withtheirbacksor sidesto thespeaker)thoughsome wouldreoriof a frequency to theinitialsounding notresponding individuals when samefrequency to hl-e and respond or walking byflying entthemselves soundeda secondorthirdtime. of the Various Scund Frequencieswhen the TABLE III.-Approximate Intensities AmplifierDial of the OscillatorRead 100. These were the Frequenciesand Intensities used to Determinethe Upper and Lower Limitsof Sound which would Induce the MatingResponsein Virginand Non-virginMale aegypti. Sound frequencies

in Intensities decibels

103.8 150O 112.6 200113.2 250300--- -- - - -- - ---- -- -- - -- - - 111.0 108.3 400 109.9 450 500----------------------- 108.6 550-----------------------108 .5 600-----------------------109 .9 650----------------------- 110.7 700-----------------------107. 1 106.8 750 106.1 800-

oftheoscillator control All thefrequencies weresoundedwiththeamplifier A was producedat its fullestintensity. set at 100,so thateach frequency of the different the intensities was used to determine soundlevelmeter13 shownin Table in decibels, expressed readings used.The intensity frequencies weremade in a sincereadings approximations III, can onlybe considered were darkroom,wherethe mosquitoes small photographic comparatively of the varioussoundsagainstthe walls exposedto sound.Reverberations on the soundlevelmeter.Eventhepositionof the thereadings influenced theresulting of themeterinfluenced to themicrophone observer withrespect weredetermined frequencies The intensities of thedifferent readings. intensity room.In bothteststhecenter in boththedarkroomand a largerlaboratory of themeterwas 2?2 inchesawayfromtheloudspeaker. of themicrophone shownin Table III are basecdon an averageof five The intensity readings takenin thelargerlaboratory readings Intensity foreachfrequency. readings lowerthanthosemade insidethedarkroomwherethe roomwereslightly 6 inches werealsotakenwiththemicrophone Readings weretested. mosquitoes werelowerbut registered awayfromthe loud speakerand the intensities 13 Type 759-A, General Radio Co., Cambridge,Mass.


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the samegeneralcurveobtainedwhenthe readingsin Table III followed controlof theoscillator It can be seenthatwiththeamplifier weregraphed. in intensity. Though variedsomewhat frequencies reading100,thedifferent wereused, bytheapparatus intensities capableof beingproduced thegreatest to soundsthatwerebarelyaudible. malesresponded mosand non-virgin for6 agegroupsofvirgin The upperandlowerlimits In somecases the same in the mannerdescribed. quitoesweredetermined long wereusedfor2 or 3 age groupsbutbecause.f thecomparatively nmales to soundsat one age did notinflutheexposure thegroups, between interval at a laterage. The resultsobtainedwiththesame encetheinsect'sbehavior periodsin theirliveswereessentiallv malesexposedto soundsat different similarto thoseobtainedwithsameaged malesexposedto soundsforthe first time. Determinations of thelimitsof virginmalesweremadefromindividuals shownin Tables to sound.The results untilexposed fromfemales keptisolated fortheupperand 100positive responses IV andVI arebasedon 100positive limitsforeachage group;thusthe forthelowersoundfrequency responses limitsforail virginage groupsare basedon a totalof 1200malesgivinga limits, soundfrequency of non-virgin In thedetermination matingresponse. wereexposedto sound. themaleswerekeptwithfemalesuntilthe former wereusuallyagitatedseveraltimesa thesemosquitoes The cagescontaining The age groupstestedwerethesameas copulations. day,to insurerepeated forvirginmales.The resultsshownin TablesV and VII are basedon 50 forthelowerlimits responses fortheupperand 50 positive responses positive groups. forall non-virgin foreachage group,givinga totalof 600 responses malesfailingto respondis also shown of virginand non-virgin The number in TablesIV-VII. wereloweredor the limitsthe soundfrequencies Since in determining to a frequlency a malecouldhaveresponded theoretically taisedby50 cycles, whichfailedto inducea response thelastsoundfrequency between anywhere For themaleto givea matingresponse. whichstimulated and thefrequency maleswhichfailedto respond to 800 cyclesbutdid respondto 750 example, 750 and800 sincetheseinter between to anyfrequency couldhaverespotnded shownin Tables soundswerenotused.Forthisreasonthefrequencies mediate IV-VII and Figs.54-57arenottheactualsoundsused (see Table III) but of tested.Thus in thedetermination of thefrequencies thecenters represent to 750 to 800 butdid respond maleswhichdid notrespond theupperlimits, 750 to 775 cycles,thecenterpointbetween wereassumedto haveresponded to 700 to 800 and 750 butresponded 800; maleswhichfailedto respond arnd to 725 cycles,etc. Individualsthat as responding cycleswereconsidered to 825 cyclessince as responding designated to 800 werearbitrarily responded testsno maleswereexposedto soundsabove800 vibrations in thefollowing to thelowerlimits, maleswhichfailedto respond persecond.In determining to 125 100 cyclesbutdid respondto 150 wereassumedto haveresponded to 100 werearbitrarily as designated cycles,etc. Males whichresponded since100vibrations to 75 cycles, persecondwasusedas a starting responding point.


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limitswhichwillinducethemating Uppersoundfrequency in virresponse ginandnon-virgin males.-TableIV showsthatineachvirgin agegroupa highi percentage of malesresponded to certain withfewerindividuals frequencies, responding to soundsaboveand belowthesefrequencies. The meanfrequency forthe15-17-hour-old maleswas 531 cycles,forthe24-26-hour group623.5 cyclesand forthe4 oldergroups,709.5,701.5,721 and 702.5cyclesrespectively.The differences between themeanlimitsof the4 oldestgroupswere smallas compared to therisein meanupperlimitbetween the15-17and 2426 age groups,and the24-26-and 48-50-hour groupsso thatthedata from the4 oldestgroupswerecombined (Table IV, age group48-309)to give2 mrain shifts in theupperlimit,withincrease in age. This is clearlyshownin Fig. 54. In the15-17-hour occursabout525 and groupthepeakof response 575 cycles, in the24-26-hour groupthepeakis 625 cycles, whilein the48-309 groupthepeakis about675 and 725 cycles.Withincrease in age themales to higher respond frequencies. Table V showsthe resultsobtainedin the determination of the upper limitsof soundfrequencies whichinducedthematingresponse in non-virgin males.The results arecomparable to thoseobtained withvirgin males,and by thedataof the4 oldestgroupswe find,as withvirgin combining males,that thereare2 mainchangesin thelimits, clearly shownin Fig. 55. However, it is evident thattheupperlimitdoesnotriseas highin non-virign malesas it doesin thevirgin forthenon-virgin 15-17group groups;thepeakof response is 525 cycles, forthe24-26groupabout575 and 625 cyclesand forthe48309 agegroupabout625 and675 cycles(cf.Figs.54 and 55). Lowersoundfrequency limitswhichwillinducethe matingresponsein virginand non-virgin males.-TableVI showsthe resultsobtainedforthe determination of thelowerlimitof virginmales.Here,too,we finda shift in thelowerlimitwithincrease in age.By combining thegroupswhosemean frequency limitsare comparatively slightly different, we find2 maindrops. Thesedropsdo notoccursimultaneously withtherisein theupperlimit,but takeplacebetween the48-50-and 63-73-hour groupsand between the 157176and 203-309age groups.Figure56 showsthatexceptforthe203-309age group,thedropin thelowerlimitis notso markedas therisein theupper limitof virginmales(cf. Figs.54 and 56). The curveforthelowerlimit does not moveuniformly to tlhelowerfrequencies withincreased age. The in thenumber dropis apparently dueto an increase ofindividuals responding to frequencies below275 cycles.Thusin the15-50-hour-old malesonly2.99%o to frequencies responded below275 cycles,16.5% of the 63-176-hour-old malesresponded to frequencies below275, while59% of the 203-309age groupgavea mating response to frequencies below275 vibrations persecond. The greatest dropin thelowerlimitoccursin theoldestmalestested. Table VII givesthe resultsobtainedin thedetermination of the lower limitof non-virgin males.Here,too,thereis a dropin thelimit,withan increase in age,whichoccurslargely between the48-50-and63-73-hour groups and the63-73and 157-176age groups.The changein soundlimitsforthe combined groupsis shownin Fig. 57, and it can be seenthatthe dropis partly due to a moreor lessuniform shiftof thecurveto thelowetfrequen-


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ciesand partly to a smallincrease in thenumber of malesresponding to frequenciesbelow275 cycles.Only2.6% of the15-50-hour-old malesresponded to frequencies below275, 6% of the63-73-hour-old malesresponded to frequenciesbelow275 cycleswhile20%oof the157-309age groupsresponded to soundsbelow275 cycles.It is clearthatthedropin thelowerlimitof the oldestnon-virgin malestestedis not as markedas thatof virginindividuals (cf.Figs.56 and 57). AGEINHOURS

40;

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Figs. 54, 55.-54. Graph showing the upper limits of sound frequencies which induced the mating response in three age groups of virgin aegjipti males. The age group 48-309 representsthe combined data of the four oldest groups tested, 48-50, 63-73, 157-

176,and 203-309.(Data fromTable IV); 55. Graphshowing theupperlimitsof soundfrequencies whichinducedthemating in threeage groups of non-virgin response male aegypti(males keptwithfemalesuntilexposedto sound). The age group48-309

represents thecombined data of groups48-50,63-73,157-176,and 203-309.(Data fromTable V)


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334

THE

AMERICAN

MIDLAND

40 (2)

NATURALIST

based on Discussion.-Figure 58 shows the range of sound frequencies, the mean upper and lower limits,which induced the matingresponsein 6

male aegypti.One pointis age groupsof virginand non-virgin different

It is thatwithincreased apparentand commonto bothvirginsand non-virgins.

AGE IN HOURS 40

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Figs. 56, 57.-56. Graph showingthe lower limitsof sound frequencieswhichinduced thematingresponsein threeage groupsof virginmaleaegypti. The age group15-50 the combineddata of the 15-17, 24-26, and 48-50 groups,and the 63-176 represents thecombineddata of the63-73 and 157-176groups.(Data fromTable grouprepresents whichinducedthe mnatVI); 57. Graph showingthe lowerlimitsof soundfrequencies male aegypti (males kept withfemales ing responsein threeage groupsof non-virgin the combineddata of the 15-17, 24-26, untiltested).The age group 15-50 represents the combineddata of the 157-176 and and 48-50 groups;the-157-309 grouprepresents 203-309 age groups.(Data fromTable VII)


1948


335

age thereis a shiftin boththeupperand lowerlimits,so thatthemales respond to a greater overallrangeofsound.It is also apparent thattheupper limitsof the4 oldestgroupsof non-virgins did notriseas highas thecomparableage groupsof virgins, nordid thenon-virgin lowerlimitof theoldest groupdropas lowas theoldestvirgin group.Thustheoverallrangeofsound (i.e. difference between themeanupperand lowerlimits)whichinducesthe in non-virgins is narrower virgin matingresponse thanthatwhichstimulates males.This is clearlyshownin Fig. 59. The youngest virgin groupresponds to a meanrangeof onlyabout150 cyclesand thisrangeincreases to about group 425 cyclesin the 203-309-hour-old males.The youngest non-virgin to a meanrangeof about125 cycles, withtheoldestgrouprespondresponds ingto a meanrangeof about310 cycles. The soundlimitdeterminations of different agedmalesshowthata sound frequency whichactsas a matingstimulusat one age does not necessarily themaleat an earlierage. For examplea 48-hour-old stimulate virginmale to 775cycleswhileone26 or 17 hoursold willnot;or a 26-hourmayrespond to 675 cycleswhilea 17-hour-old malewillnot.Adrian old malewillrespond (1928,pp. 18, 19,20) defined a stimulus as ". . . anychangein theenvironmentof an excitable if sufficiently willexcitethetissue, tissuewhich, intense, The stimulus is thusthe i.e.,willcauseit to displayitscharacteristic activity. stimulation external change, andit maybe ineffective-the maynothavebeen intense enoughto excite.. . The stimulus mustbe .a rapidchangeof condiof 775 tions,and a gradualchangewillnotdo." Sincethesoundstimulus to a 48-hour-old or oldermalewas cycleswhichactedas a matingstimulus thesameas thesoundwhichfailedto stimulate a 17-or 26-hour-old male,the lackofresponse in thecondition waseither dueto a difference of thereceptor in nervous Thereis a difference organor to a changein thecentral system. the external appearance of theantennae, withage,whichcan be correlated withthedifference of different It willbe in thereception soundfrequencies. against recalled thaton emergence theantennal hairsof themaleareflattened of vibrating to various theshafts(Figs.9, 10) and apparently are incapable in age thesehairsbecomeextended awayfromthe frequencies. Withincrease 24 hoursafteremergence, thoseon shafts. Not all of thesehairsareextended theapicalhalfstillremaining moreor less recumbent together and clinging (Fig. 12). As moreand moreof thesehairsbecomeextendedthe male After48 hourspractically all of tespondsto higherand higherfrequencies. that thehairsareextended fromtheshafts(Fig. 13). It is perhaps significant the2 mainshiftsin theupperlimitsoccur(1) beforeall of the antennal fibrillae are extended and (2) afterthehairsbecomeextended. Age groups in theirupper littlefluctuation olderthan48 hoursshowedcomparatively of aegyptibecomeextended theyremain limits.Once the antennalfibrillae moreor lessso throughout theirlifetime. someold maleswereseen However, on theapicalhalfof theantennae withsomeof thefibrillae partlyrecumbent accountforsomeof thefluctuation in theupperlimits and thismaypossibly of the4 oldestgroupstested.The correlation between the extension of the antennal fibrillae limitsindicates and therisein theuppersoundfrequency


336 203-309

THE

AMERICAN

40 (2)

NATURALIST

MIDLAND

_

58

157-176

58

s~

0. 63-73 ,48-50

24-26 5=VIRGIN MALES MALES 'NON-VIRGIN

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based on mean Figs. 58, 59.-58. Graph showingthe rangeof sound frequencies, upper and lower limits,whichinducedthe matingresponsein six different age groups of virginand non-virgin(males kept with femalesuntil tested) male aegypti. (Data fromTables IV-VII); 59. Graphshowingthemean rangeof sound (difference between the mean upper and lower limits),expressedin numbersof cycles,whichinducedthe matingresponsein six different age groupsof virginand non-virgin(males kept with femalesuntil tested) male aegypti. (Data fromTables IV-VII)


1948

ROTH:


337

on theapicalhalfof theantennae thatthefibrillae covibrate to the higher or a greater number of vibrating frequencies, hairsarerequired to deflect the flagellum forthe higherfrequencies sufficiently to exciteJohnston's organ. No conclusive wereperformed to determine experiments thelimitsof males lacking various numbers ofantennal segments. The shiftin thelowersoundfrequency limitsapparently cannotbe correof theantennal latedwiththeextension fibrillae sincethegreatest droptakes placelong afterall of the antennal hairsbecomeextended awayfromthe shafts. It is possiblethattheuppersoundfrequency limitsof non-virgin males do notriseas highas virgins becauseof someformof conditioning byrepeatedlycopulating withfemales. It is notmerely lossof virginity butcontinuous exposure to femaleswhichapparently affects theupperlimits.It was noted thatnon-virgin malestakenfromverycrowdedcagescontaining bothsexes, in the determination to lowerfrequencies generally of the upper responded limits(and oftenfailedto respond at all), thannon-virgin malestakenfrom less crowded cageswherepresumably fewercopulations tookplace.Isolation fromfemales of non-virgins also raisedtheupperlimitsof themales.Two examples can be noted.Twenty-five non-virgin malesabout96 hoursold and keptwithfemales fromemergence untilexposedto soundsgavea meanupper limitof 601 cycles;thesemaleswereisolatedfromfemales and 48 hourslater tlhemeanupperlimitfor20 of theseindividuals was 705 cycles.In aniother 25 non-virgin experiment, males,47-48hoursold and keptwithfemales from untilexposedto thesoundsof theoscillator-speaker, emergence gavea mean upperlimitof 611 cycles;theywerethenisolatedfromfemalesand their limitsdetermined 38 hourslaterat whichtimethemeanhad risento 693 cycles. A parallelism maybe drawnbetween theresponse of virginmalesto a greater rangeof soundthannon-virgins of thesameage, withthe feeding behavior of somepolyphagous larvae.Brues (1946, p. 113) pointsout the factthatthoughgipsymothlarvaepreferoak foliage,"The lack of their preferred foodand consequent hunger leadsthemto feedon less acceptable species,and finally as foodgrowsscarcer theywillshowlessand less choice, to the needlesof whitepinewhichwillservethe sometimes eventurning largerlarvae,although thenewlyhatched broodareunableto utilizethepine of virginmaleaegypti whichhavebeenisolatedfrom foliage."The behavior forsometimemaybe theresultof a formof "hunger" females so thatthese malesrespond to a greater "sex-starved" indirangeof soundthannon-virgin malesmaybe said to show"lesschoice"as to thesound viduals.The virgin wavewithwhichtheyattempt to copulate. Figure60 compares thepercentage of virginand non-virgin maleswhich to soundin thedetermination of theupperandlowerlimits. failedto respond A greater malesfailto respond number ofnon-virgin to soundthando virgin males(non-virgin maleswhichfailto respondto soundoftencopulatewhen placedin a cagecontaining of females).Forsomereason,a higher percentage


338

THE

AMERICAN

MIDLAND

40 (2)

NATURALIST

virginsand non-virgins failedto respondduringthe determinations of the lowerlimitsthanduringthe determinations of the upperlimits.Only .5% of virgin males48 hoursand olderfailedto respond in thedetermination of theupperlimits.Theseindividuals areveryresponsive to thesoundsusedin theupperfrequency limitswhichinducea mating determining response.

lF-

-VIRGINMALES V__ -NON-VIRGINMALES

55_ 50

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LOWER

48-309 15-26 AGE GROUPS

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thedifference in thepercentage of virginand non-virgin Fig. 60.-Graph showing of theupperand to soundin thedetermination maleaegyptiwhichfailedto respond whichinduceda matingresponse. lowerlimitsof soundfrequencies The non-virgins untiltheformer wereexposedto sound.The age group15-26 werekeptwithfemales data of the 15-17and 24-26-hour-old the combined represents males; the 48-309 dataof the48-50,63-73,157-176,and203-309groups.(Data groupis thecombined fromTables IV-VII)


1948

ROTH: SEXUAL BEHAVIORIN MOSQUITOES "ADAPTATION"

OF MALES

339

TO SOUND

Minnich (1925) found that he could "fatigue" Vanessa larvae tc one soundfrequency and theninducethe insectto respond(i.e. throwthe anterior erndof the bodydorsallyor dorsolaterally)by changingto anotherfrequency. He explainedthis as being due to different hairs reactingto specificsound vibrations. Male aegyptiwhenrepeatedlyexposedto soundsof one frequency also cease givinga matingresponsebut beginto respondagain whenthe sound frequencyis changed.It is possiblethat the cessationof response,aftel the male is exposed to a constalitsound stimulusis not due to fatigue,but to adaptation.Hoagland (1936) definedsensoryadaptation". . . as a reversible reductionin theabilityof a sensorycell,underthe actionof a constantZtimulus, to initiatenerve impulses."Recoveryof a nervefromfatigueis much slowerthan fromadaptationand, in contrastto adaptation,is facilitatedby the presenceof oxygen.The presentexperiments do not determinewhether the cessationof a matingresponseto a repeatedstimulusis due to adaptation or fatigue.Adrian (1928, p. 68) statedthatadaptation". . . is a muchmore potentfactorin causingthe declinein frequency." The failureof male aegypti to give a matingresponse,afterbeingsubjectedto continuousor intermittent sound stimuli,willbe called adaptation. When male aegyptiwere exposed to sounds fromthe audio oscillatorspeaker,it was foundthatthe insectcould be adapted to one frequency and thenmade to respondagain withouta prolongedrestperiod,by changingthe frequencyof the sound stimulus.When subjectedto continuousstimulation the male respondsvigorouslyat first,but then the responsebecomeserratic and weak until he fails to respondat all. For example,a 12-day-oldvirgin male subjectedto continuousstimulation of 600 cyclesrespondedforabout 2 minutes.After no responsewas given for one minute,the frequencywas loweredto 400 cyclesand the male began to respondagain and continuedto do so sporadicallyfor5 minutes.Althoughmales can be adapted muchmore quickly by continuousthan intermittent stimulation,their responseis not uniformand it is difficult to decide when adaptationtakes place. For this reasonin the followingexperiments the males weresubjectedto soundsmade for3 secondsat 6-secondintervals, and some testswererun withsoundsmade for3 secondsat 2-secondintervalsand 10 secondsat 5-secondintervals.The individualmosquitoeswereexposedto thesoundsin t.hesame manneras those males whichwere used in determining the upper and lowerlimitsof sound whichwould inducethematingresponse. The claspingresponseto one soundingof a frequency may consistof one or more distinctflexingsof the abdomen.Sometimesthe responseis a brief flexingof the abdomenfollowedby a returnto the normalposition,or the male keeps his abdomenflexedventradforseveralscconds.In the following testsone or moreseizingresponses,and one or moreclaspingresponsestegardless of the lengtnof timethe abdomenwas kept in the flexedposition,were


40 (2)


340

foreachtrial.14Onlyseizing response considered as oneseizingand clasping frequency of the particular givenduringthe sounding or claspingresponses as between thesoundings wereconsidered or duringthebriefsilentintervals adaptedwhenhe failedto positiveforone trial.A male was considered When this frequency. of a particular soundings respondto 10 consecutive withno intervening restperiodotherthan waschanged thefrequency occurred (i.e. 2, 5, or 6 seconds). between soundings thefixedsilentinterval ofSubjecting MalestoSoundFrequentheResults aegypti TABLEVIII.-Showing Fin a LongSeriesof Trials.C-ClaspingResponse; ciesof400 and600cycles, SC-SeizingandCiaspina S-SeizingResponse; Malein Flight;N-No Response; at6Sounded for3 Seconds Males12DaysOld.EachFrequency Virgin Responses. 100. Control ofOscillator Reading Amplifier SecondIntervals. MaleNo. 2

MaleNo. I

400 Cycles (Continued) (Continued) 600Cycles (Continued) 7 SC

2 SC

IC

1 SC

3C

I SC 2C 3 SC 6C 1 SC 1C IF lSC IF 3 SC IC

11 SC 1C 4 SC IC 2 SC 1C 2F IS 7 SC IC 8 SC

IC 2 SC 3C 4 SC IC 5 SC 3S IC 3SC IF 5C

3 SC 34 C 1 SC 2N IS IN 3C IS 3 SC IC IN

IC 1SC IN 2C IN 1C IN IS 3C 1S IN

IC 2 SC 1C 1S 2C IF 3C 2 SC 1C 2 SC 1C 4SC IC 3 SC IC 1 SC 1C

8 SC IC 1N CC I SC 3C 3 SC IC 3 SC 2C 8 SC IC 5SC IC 5SC 2C 2 SC

IC

1 SC

I C

I C

1 SC

1SC

IC 2 SC 1N IC 3SC Ic 2SC 7C 1F 1 SC 1C 2SC IC ISC IC (discontinued; not after adapted 207trials)

IC

4 C

IN 8C 1 SC 11C 2N IS IC 3N 1 SC 1C 1N

1 SC

IC

1SC 3C 1 SC 3C IS 3C 1SC 2C not (discontinued; after adapted 122trials)

14 A singlesoundingof a frequencyfollowedby its silentintervalrepresents one trial.


1948

RoTH: SEXUAL BEHAVIORIN MOSQUITOES

341

frequento adaptto a particular of timerequired Malesvaryin thelength 2, 12day-oldvirginmalesto cy.Table VIII showstheresultsof exposing overa longseriesof trials.Male number1 400 and 600 cyclesrespectively, was not adaptedto 400 cyclesafter207 trialsat whichtimethetestswere was obtainedafter110 response In thiscase thefirstnegative discontinued. Male number2 exposedto 600 cycleswas not adapted positiveresponses. In thiscase was discontinued. after122 trialsat whichtimetheexperiment responses. after40 positive occurred response negative thefirst and then malesto one frequency of adapting Table IX showstheresults 3 becameadaptedto 400 cycles Male number frequency. to another changing to 500 cyclesafter54 trials,and to 600 cyclesafter68 trials. after39 trials, (44 of negative responses It can be seenthatthismalegavea highnumber adaptedto 600 cyclesper second.Male number4, N's) beforebecoming of adaptedto 500 cycles,failedto respondto 5 soundings afterbecoming TablesVIII and to thatfrequency. response givingthefirst 600 cyclesbefore ofonly4 of thedozenmaleste3ted. IX showtheresults nerveof tactilehairsof (1936) was ableto adapttheafferent Pumphrey that witha fineglassneedle.It is possible thereceptor theroachbydeflecting numbers of antennal vibratedifferent soundfrequencies irl aegyptidifferent whichin turncausetheantennalshaftto deflector vibrateat its fibrillae organmay Certainsensecellsof Johnston's or lesserextent. baseto a greater whenexposedto soundsof 400 oftheantenna bythedeflection be stimulated When cyclesandafterthesecellsbecomeadaptedthemaleceasesto respond. of the shafts vibrations is changedto 500 cycles,differential the frequency until and themaleagainresponds tesultsin othersensecellsbeingstimulated on youngmalesabout25 thesecellsbecomeadapted.Sometestsperformed intervals) fortensecondsat five-second eachfrequency hoursold (sounding thatwhenadaptedto 400 cyclestheywillnotrespondto 500 and indicated withthecondition be correlated 600 vibrations persecond.Thismaypossibly away are extended as notall of thefibrillae hairsinasmuch of theantennal at thistime. fromtheshafts The-Behaviorof Males of OtherCulicidae to Sound by (determined whichare in a mating-state Males of A. quardimaculatus to copulatewitl fibrillae and theirattempts of theantennal the extension freto tuningforkswiththefollowing response females)mayshowa mating quencies:320, 341.3,384, 426.7,and 480 vps. Tbe responseis somewhat similarto thatgivenby aegyptimalesthoughusuallyit is notso vigorous. or both. malesgiveeithertheseizingor claspingresponse, Quadrimaculatus casemaybe noted.A malewas inducedto givetheclasping One interesting in claspingtheclothof the succeeded and in so doinghis genitalia response cage; he thenreleasedhis hold withhis frontlegs and leanedbackwards is comparable to thereleaseofthe up byhishindlegs.Thisresponse propped is made.It has previously beensuglcgholdon thefemaleonceconnection fromthereducis an adaptation resulting position gestedthattheend-to-end


342

THE AMERICAN

MIDLAND

40 (2)

NATURALIST

TABLE IX.-Results of Adaptina aeg9pli Males to the Sounds of Three Different Frequenciesover a Long Series of Trials. C-Clasping Response; F-Male in Flight; N-No Response; S-Seizing Response; SC-Seizing and ClaspingResponses.Virgin Males 12 Days Old. Each FrequencySounded for 3 Seconds at 6-Second Intervals. Controlof OscillatorReading 100. Amplifier

Male No. 3 400 cycles 2 SC 4 S 1 SC 2 C 1 SC 7 C 1 SC 3 C I N 10 C 2 N 3 C I N IC 10 N (adapted after 39 trials) 500 cycles IC 1 SC 7C I N 3 C 1N 2 C 1 SC I C

1s

I N 3 C 1N 3C 1 SC I N 10 C 1N 4C I N 7C 1S 1 SC

10 N

(adapted after 54 trials)

(continued) 600 cycles IC 1S 3N IC 2N IF IS IF 2 N 2S IN 2 C 1SC 1C 1N t 9 N 2 C 6 N IC 7N 2C I N 2 C 7N 1C 3 N 1C 2 N 2C 10 N (adapted after 68 trials)

Male No. 4

400 cycles 3 SC 3 C 7 N 2 S 2 SC IC 4 SC I N IC I N 1 SC 3 N 2 C 1 SC 1C 3 N 1C 1S 1 SC 1N 3C I SC 3C I N 2 C 10 N (adapted after 50 trials) 500 cycles

(continued) 1 SC 2 N 1S IC 1 SC 2 C 3 N IC I N 2 C 1N IC 2 N 15 SC 2 C 8 SC iON (adapted after 78 trials) 600 cycles 5 N 11 C I SC 4 C (discontinued;not adapted after 21 trials)

1 SC 8 C 1 SC IC 1 SC 6 C 1S 1 SC 2C I SC 9C 1S

IC


1948


343

forhimto cling tionof thetarsalclawsof themalemakingit moredifficult sinceonlythe fronttarsiare suitedforseizingand holding. to thefemale, thefemalegenitalia thatclasping It wouldappearfromtheaboveobservation maybe thestimulus whichinduces themaleto releasehisfront leghold. results afterattemptNuttalland Shipley(1902,p. 77) obtained negative of electromagnetically excited tuningforksuponmale ingto studytheeffects due to themaleshaving A. maculipennis and theybelieved thiswaspossibly The present withfemales. beenconfined forsomedaysin smallboxestogether males to tuningforkswereobtained fromquadrimaculatus positive responses to whichwereconfined withfemalesin thesamecage and wereattempting butwhichhad their copulatewiththemor frommalesisolatedfromfemales antennal fibrillae extended andwereactive.It is likelvthatNuttalland Shipley'snegative resultsweredue to thefactthatthemalesexposedto sound thecondiwerenotin a mating-state. did notindicate vibrations Thesewriters the notrealizing theirtests,againapparently tionofthemaleantennae during of theantennal hairs.It is also possiblethatthe of theextension significance thespecieswith whichwouldstimulate soundstheyusedwerenotfrequencies Bates (1941) observed thatA. superpictus swarms whichtheyexperimented. cage.Here it is did not respondto tuningforks, soundedin a room-sized a -:angewhichwouldstimupossible thatthefrequencies usedwerenotwithin latethisspecies,or theforksmaynothavebeenstruckcloseenoughto the to be effective. swarms confinnis (Lynch-Arr.) Four-and 6-day-old rearedmalesof Psorophora The antennal hairs wereexposedto thesoundsoftheaudiooscillator-speaker. of theseindividuals weremoreor less recumbent and clungtogether against to sounds, the shafts.In general, the malestestedwereveryunresponsive 260 and 310 vibrations per second. responding onlyto frequencies between It is probable thattherangeof soundto whichmalesof thisspeciesrespond in thosemalestheantennal of whichare all extended. is greater fibrillae In malesis lessvigorous thanthat comparison, thematingresponse of confinnis wasgiven.The rapidvibration of shownbyaegypti. Onlya clasping response the wingsand body-shaking characteristic of the aegyptiresponsewas not observed. to matewithfemales(i.e. were Culexpipiens maleswhichwereattempting response, or both,whenexposed in a mating-state), gavea seizingorclasping to thesoundsofthetuning forks 288,320,341.3,and384 vps. The PracticalApplicationof the Behavior of Mosquitoesto Sound and also devisingan electrocuting apparatus, Weaver Using fly-paper or killthemosquitoes in the (Howard,1901) wasableto attract andcapture a particular note.The soundfrequency, species,and sex vicinity bysounding of themosquitoes werenotindicated. Laurence(1902,)recalled that attracted as a childhe and his friends wouldattract mosquitoes (speciesand sex not by hand). indicated)by tnesoundof thevoiceand killthem(presumably


344


40 (2)

thattheknowledge NuttallandShipley(1902) believed gainedfroma studv be ultimately oftheeffect ofsoundon variousspeciesofCulicidae". . . might thevarioussoundsproput to practical use." Kahnet al. (1945) recorded ducedby severalspeciesof culicidswiththe intention of reproducing these A recentpopularmagazine15indisoundsto attract and killthemosquitoes. to leaveforAfrica, wherehe willtry catedthatDr. Kahnwas". . . preparing to trapand destroy backto themsome disease-bearing mosquitoes byplaying oftheirownlovecalls." recordings Thereis no questionthataegyptimalesare attracted by certainsound to be attracted theirlifetime once frequencies and willcontinue throughout thley arecapableof perceiving sound.However, malesof quadrimaculatius and pipiens,and probably thoseof otherspecieswhichswarm,are indifferent to soundsunlesstheyare in a mating-state and thisoccursonlyperiodically. Evenmalesof theselasttwospecieswhichare attempting to copulatewith females(in thelaboratory) appearto be lessrespotisive thanaegyptito the soundsof tuningforksin thesensethata massresponse(attraction) to a stimulating wasnotobserved. soundfreqeuncy Actuallyit wouldbe unnecessaryto playback"lovecalls,"as suggested males by Kahnet al., to attract sincethey(i.e. aegypti)respond to a rather widerange .ofsoundfrequencies. Thoughtheliterature suggests thatfemales of sonme speciesof mosquitoes no evidence areattracted wasfoundin thepresent byand entermaleswarms, to indicate thatfemales of aegypti are attracted experiments bythesoundof themales.Thereis no experimental thatfemalesare proofin theliterature If soundis usedas a meansofattractattracted bythesoundsofmaleculicids. all indications ing and killingmosquitoes, are thatmaleswouldbe the sex It should be that our mosquito-borne remembered diseasesare destroyed. of thespecies.It is conceivable transmitted byfemales thatdestroying a large ofmaleswoulddecrease percentage thenumber of impregnated females(probeforemating)and overa periodof time videdthe malesweredestroyed wouldresultin the population beingreducedto a pointwherethe species wouldbe medically unimportant. However,untilfurther study,it appears thata killingdeviceemploying unlikely soundas an attractant wouldbe a moreefficient or economical thanthepresent substitute methods of mosquito control. Summaryand Conclusions Unlessotherwise stated,thefollowing statements concern Aedes aegypti. 1. Copulation is initiated duringflight. The maleusuallyseizesthefemale fromabove,reorients himself, and assumesthemodified linearpose.Copulationis concluded in themodified linearposition. The act maybe completed thepairin flight or usuallyafterthefemalecomesto restwiththemale wvith to her.The pairremains stillclinging joinedin coitubriefly, connection lasting about4 to 59 seconds. 15 "Hot Plattersby Mosquitoes,"an anonymous notein Science Illustrated, 2, No. 5 (May, 1947), p. 55.


1948

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females.One 2. Males will copulaterepeatedlywiththe same or different male may fecundatemorethanone femalebut the factthata niale has copuA female lated witha femaledoes not implythatshe has beelnimpregnated. whichhas copulatedonce or twiceusuallyhas only two of her threesperma thecaefilledwithsperm. 3. The sound producedby the femaleis the externalstimuluswhichattractsthe male and induceshim to copulate. Odor and sounds otherthan play no role in matingbehavior. thosemade by the femalein flightapparently agitatAny factorwhichcauses both sexes to fly (e.g. increasedtemperature, of copulations. increasethe frequency ing the cage,etc.) will automatically 4. The male respondsto a rangeof sound frequenciesratherthan to a Male aegyptiattemptto copulatewithmales and femalesof singlefrequency. speciesof Culicidae because the sounds made by thesemos severaldifferent range.Sexuallyactivemales cannotdifferenquitoes fall withina stimulating tiatebetweenrecentlyemergedmales and old femalessince the sounds made by veryyoungmales in flightfall withinthe spectrumwhichwill inducecopulation. 5. The male palps and proboscistakeno partin copulation. 6. The male antennaeare the organsused to locatethe femalein flight.At and clingtogetheragainst the male antennalfibrillaeare recumbent emergence the shafts.The fibrillae becomeextendedas the insectages. Usually all of the fibrillaeare extendedabout 48 hours after emergence.Once the antennal of male aegyptibecomeextendedtheyremainmoreor less so throughfibrillae out thelifetimeof the insect. 7. The male genitaliamust undergoabout a 180 degreerotationbefore copulationcan be successful.However,males may attemptto copulatewhen theirgenitaliahave rotatedonly about 135 degreesthoughthese attempts Males may give because of mechanicaldifficulties. generallyare unsuccessful a typicalmatingresponseto the soundsof a tuningforkas earlyas 5 hours long beforetheyare physicallycapable of copu20 minutesafteremergence, latingdue to incompleterotationof the genitalia.Male matingbehaviorelicited by a femalein flightis not correlatedwithrotationof the genitalia. 8. The inductionof male matingbehaviorapparentlyis dependentupon the conditionof theantennae.The male attemptsto copulatewhena sufficient numberof its antennalfibrillaeare extendedmakingpossiblethe perception of the sound of the flyingfemale.This occursabout 15 to 24 hours after so that has rotatedsufficiently emergenceand by this time his hypopygium Since matingis initiatedin flight copulationcan be accomplishedsuccessfully. and the stimulusis the sound of the flyingfemale,copulationwill occuronly at leastseveralhoursafteremergence. whenthefemalebeginsto flyvoluntarily By this timeshe becomes"attractive"because the sound made in flightfalls withinthe rangewhichwill stimulatethe male. Very young femalescan be made to flyunmolestedin a cage containingsexuallyactivemales since the


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soundproduced by a recently emerged femalein flight fallsoutsideof the rangewhichwillinducethemaleto copulate.Whether thestimulus which first induces thefemale to flyis sexualornutritional (i.e. thesearchforfood) was not determined. No evidencewas foundto indicatethatthe femaleis attracted to themale.Investigators have shownthatthe averageemergence periodof aegyptifemalesis about14 hourslongerthanthatof themales. Thusbythetimethefemalebeginsto flythemalesarereadyto copulateby reasonoftheir"matured" antennae and rotated genitalia. 9. Once male aegyptibeginto copulate (i.e. 15 to 24 hours afteremergence), theycontinueto do so at any timeof the day, untildeath,whenever stimulatedby flyilng females.These males may be said to be in a constant mating-state throughout theirlives. The males of Anophelesquadrimaculatus mate cyclicallyand betweenmatingperiodsignorefemales.During inactive periodsthe antennalfibrillaeof male quadrimaculatuslie recumbentcagainst the shafts.When thesemales are sexuallyactive,theirantennalfibrillacare extendedaway fromthe shafts.The males of Culex pipiensalso mate periodically; however,unlikequadrimaculatus, the antennalfibrillaeof sexuallyinactivemale pipiensremainextendedaway fromthe shafts. 10. The frontand middle tarsi of the male are structurally suited for seizingand holdingthe female.The middletarsiand clawsappearto function chieflyin the initialseizure.The frontlegs are used in bothseizingthe female and in gainingthe modifiedlinear pose requiredfor copulation.The male hind tarsi are not suited for seizing and take comparatively littlepart in mating. 11. Exceptforflight, the femaleis passivewhilebeingseized by the male, and her legs remainingoutstretched serveas structures to whichthe male can cling.The femaletarsiare not suitedforseizingin .pite of the toothedcondition of the claws, and she does not cling to the male or hold him to her duringcopulation.Normal male aegypticopulatereadilywithfemaleswhich have all theirlast tarsalsegmentsremoved. 12. The hypothesis is suggestedthat the face-to-face and end-to-end positionsassumedby different speciesof mosquitoesmightbe correlatedwiththe degree of developmentof the last tarsal segmentsand claws of the male's frontand middle legs. A. quadrimaculatus males withonly one pair of legs suited for seizing,initiatecopulationin the modifiedlinearpose, releasethe femalealmostimmediately aftermakingconnection,and swingout into the linearposition.The attitudesof C. pipiensappear to representa transitional statewherethe pair may remainjoined forawhilein the face-to-face position and finallyconcludescopulationin the opposed position.In this speciesboth the frontand middle male tarsi are fairlywell fittedfor seizing.The front and middlelegs of male aegyptiare well suitedforseizingand thisspecieswas neverseen to copulateend-toLend (sometimesan opposedpositionis assumed whilethe pair triesto separateaftercompletingcopulation). The fronttarsi of aegyptiappearto be fittedbetterforseizingthan the same legs of pipiens.


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mayhaveevolved behavior of aegypti 13. It is suggested thatthemating swarmed to thatshowri today,wherethemales froma specieswhichoriginally a preliminary and maycopulateevenwithout generally do notformswarms "dance." elicitedby of aegyptimaleswhether 14. The instinctive matingbehavior produced soundsis basicallysimilar. a femalemosquitoor by mechanically depending uponthesourceof thestimulus, Thesecoordinated reflex actions, maybe tabulated as follows: Source of stimulus A. Female in flight

B. Tuningfork(of

proper frequency)

Behavior of male 1. Flight (if male is at rest) 2. Attraction 3. Seizure of female 4. Clasping the femalegenitalia 1. Flight(if male is at rest)

2. Attraction

3. Seizing response 4. Claspingresponse

The seizingresponse elicitedby thesoundsof a tuningforkor audio consistsof clinging withthefrontand middlelegsto the oscillator-speaker The clothofthecage,nearthesoundsource,and vibrating thewingsrapidly. whichusuallyfollows consists of flexing theseizingresponse clasping response theabdomen touches thesideof thecage. ventrad so thatthegenitalia by givingso-called 15. Males also respondto certainsoundfrequencies

'shock-reactions."

is presented thatin to confirm thehypotheses 16. Experimental evidence and thatthemodeof action CulicidaeJohnston's organis auralin function of thisstructure is thatdescribed in theliterature. werepreThesehypotheses evidence. viouslybasedsolelyon morphological 17. The limitsof soundfrequencies whichwillinducea matingresponse fromaegyptimalesareabout100and 800 cyclespersecond.The limitsvary or non-virginity ofthemales.Bothvirgin andnonwiththeageandvirginity withage, the shiftsoccurring virginmalesrespondto higherfrequencies, These largely between 17 and24 hours,and24 and48 hoursafteremergence. withtheextension of theantennal shiftsin theupperlimitcan be correlated of moreand morefibrillae fibrillae. themaleis able to Withtheextension The lowerlimitof soundfrequencies which respondto higherfrequencies. invirgins aftermalesareisolated willinducea mating dropsmarkedly response in age,bothvirginand non fromfemales for203-309hours.Withincrease malesrespond to a widerrangeof sound,buttherangeof non-virgins, virgin is narrower thanthatof virginmaleskept whichhavecopulatedrepeatedly, Malescanbe "adapted"to onesoundfrequency so that isolatedfromfemales. aftercontinuous stimulation Theycan be theyfailto givea matingresponse. thefrequency of thesoundstimulus. Posmadeto respond againbychanging


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forthephenomenon sibleexplanations of "adaptation" and the response to a widerrangeofsoundwithincrease in age arediscussed. 18. A. quadrimaculatus and C. pipienswhenin a mating-state, givea mating response to thesoundsoftuning forks. Quadrimaculatus malesrespond to the320,341.3,384,and 426.7forks. Pipiensmalesgivea mating response to soundsfromthe288,320,341.3,and 384 tuning forks. Psorophora confinnismalesgivea mating response to oscillator-speaker soundsbetween 260 and 310 cyclesper second (these males did not have all theirantennalfibrillae

fullyextended). The limitsofsoundfrequencies whichwillinducethenmating in theabovethreespeciesofmosquitoes response werenotdetermined. 19. No evidencewas foundthatfemaleaegyptiare attracted to sounds though to certain theymaygiveshock-reactions frequencies. 20. Withrespect to mating, thefunctions of themaleantennae are (1) to perceive thesoundof theflying female,(2) to locatethedirection of the soundsource, and (3) to discriminate between certain soundfrequencies since somesoundsmayinduceshock-reactions whileothers actas a mating stimulus. 21. At present it seemsunlikely thatkillingadultmosquitoes withtheaid of a sound-attractant devicewouldbe moreefficient and economical thanthe control. current methods of mosquito REFERENCES C. E. 1927-The reactionof Datana larvae to sounds.Psyche 34:129-133. E. D. 1928-The basis of sensation.The actionof the senseorgans.W. W. Nortonand Co. Inc. N. Y. 122 pp. ARAGAo,H. DE BEAUREPAIRE 1939-Mosquitoes and yellow fevervirus. Mem. Inst. Oswaldo Cruz 34:565-581. undertakenfor the investigaticn BACOT, A. W. 1916-Report of the entomological forthe year,August1914 to July1915. Rept. Yellow Fever Comm. commission (West Africa), London 3:1-191. (Summarizedin Rev. App. Ent. 4(B): 161-164, 1916). and hearingof to the physiologyof the stridulation BAIER, L. J. 1930-Contribution insects.Zo6l. Jahrb.,Abt. Allg. Zool. u. Physiol.47:151-248. BANKS, C. S. 1908-Biology of PhilippineCulicidae. Philip. Jour.Sci. 3(A) :235-258. 1919-The swarmingof anophelinemosquitoes.Ibid. 15:283-288. with Anopheles maculipennis.Amer. BATES, M. 1939-Hybridization experiments Jour.Hyg. 29(C) :1-6. on the sexual behaviorof anophelinemosquitoes. 1941-Laboratoryobservations Jour.Exp. Zool. 86:153-173. Science 101:610-611. BEACH, F. A. 1945-"Angry" mosquitoes. BoYD, M. F., CAIN, T. L., AND J. A. MULRENNAN 1935-The insectaryrearingof Amer. Jour.Trop. Med. 15:385-402. Anophelesquadrimaculatus. BRUES, C. T. 1946-Insect dietary;an accountof the food habitsof insects.Harvard Univ. Pre-s. Cambridge,Mass. 466 pp. 1940-Observationson the swarmingof CAMBOURNAC, F. J. C., AND R. B. HILL Amer. Jour.Trop. Med. 20:133-140. var. afroparvus. Anophelesmaculipenni`s CARPENTER, S. J., MIDDLEKAUFF, W. W., AND R. W. CHAMBERLAIN 1946-The mosABBOTT, ADRIAN,


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