3006 Received 14April 2014;Accepted10June2014 *Author ([email protected]) for correspondence USA. BiologicalSciences, Vanderbilt University, of Department Nashville, TN37235, processes, insectsmustensure thatmoleculesrequiredforproper Because antennaeareintricatelyinvolvedinessentialphysiological al., 2009;Wang etal., 2009; Careyetal.,2010;Rinker2013). neurons controlsovipositionandhost-seekingbehaviors(Cator et and thereceptionofodorantthermalcuesbyantennalreceptor by theantennae’s Johnston’s organs influencescourtshipandmating, Merlin etal.,2009).Inmosquitoes,thedetectionofnear-field sound autumn migrationofmonarchbutterflies(HsuandWu, 2001; and containthetimingelementsofsuncompassthatdrives the The antennaealsomechanicallyassistduringthematingof fleas, tactile, thermalandauditorystimuli(ChapmanSimpson,2013). The antennaeofinsectsareinvolvedinthedetectionchemical, Antennal heart,Heart,Vessel KEY WORDS:Antenna,Hemocoel,Circulatoryphysiology, larger cavity, hemolymphtravelsuptheantennaat0.2 and returnstothehead.Becauseanarrowvesselemptiesinto end oftheappendage,itisdischargedintoantennalhemocoel has aneffective diameterof1–2 an antennalvesselthattraversesthelengthofappendageand that eachantennalAPOpropelshemolymphintotheantennathrough than theheart.Bymeansofhistologyandintravitalimaging,weshow Antennal APOscontractinsynchronyat1 antenna butventraltothemedial-mostregionofcompoundeyes. between thefronsandvertexinanareathatisdorsalto located oneachsideofthehead’s dorsalmidline.Theyaresituated show thatmosquitoespossesspairedantennalAPOsare antennae of structural mechanicsofhemolymphpropulsionthroughoutthe hemolymph intotheantennalspace.Thisstudycharacterizes evolved antennalaccessorypulsatileorgans(APOs)thatdrive appendages. To accomplishthis,mosquitoesandotherinsectshave ensure theefficient transportofmoleculesintoandoutthese seeking, matingandovipositionbehaviors.Thus,mosquitoesmust Mosquito antennaeprovidesensoryinputthatmodulateshost- Sushma BoppanaandJuliánF. Hillyer* hearts) inthemosquito mechanics ofantennalaccessorypulsatileorgans(auxiliary Hemolymph circulationininsectsensoryappendages:functional RESEARCH ARTICLE © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,3006-3014doi:10.1242/jeb.106708 characterization ofcirculatoryphysiologyinthemosquitoantennae. rates. Insummary, thisstudypresentsacomprehensivefunctional (triethylamine) increasesbothantennalAPOandheartcontraction treatment ofmosquitoeswiththeanestheticagentFlyNap reduces itsvelocityby75%asitreturnstothehead.Finally, INTRODUCTION ABSTRACT Anopheles gambiae μm. Whenhemolymphreachesthe . Usingintravitalvideoimaging,we Hz, whichis45%slower Anopheles gambiae mm s − 1 but malaria mosquito mechanics ofantennalaccessory pulsatileorgans inthe African 1993; RichterandHertel,1997; Predel,2001;Herteletal.,2012). these APOs(Herteletal.,1985;Pass1988;Lange al., of antennalhearts,illustratingthephysiologicalcomplexity of Neoptera) havedescribedthecontractileandneurohemalactivity several studiesinBlattodeaandotherPolyneoptera(lower analyses andnotfromactivevisualizationsofflow. Nevertheless, hemolymph throughtheantennaehasbeeninferredfromstructural understood. Thisisprimarilybecausethecirculation of hemolymph flowthroughtheseappendagesremainpoorly investigated inmultipleinsectorders,thefunctionalmechanics of Although thestructureofantennalAPOshasbeenthoroughly 1985; Pass,1991;SunandSchmidt,1997;Matus1999). means ofanantennalvessel(Clements,1956;Pass,1980; Pass, located intheheadthatforceshemolymphintoantennaeby variability acrosstaxa,buttheirgeneralplaninvolvesanampulla et al.,2013).AntennalAPOsshowremarkablestructural known asantennalhearts(Pass,2000;Passetal.,2006;Wirkner through theactivecontractilityofantennalAPOs,whicharealso Sláma, 2008;Anderecketal.,2010). 1999; Wasserthal, 1999;Gereben-KrennandPass,2000; such asthecerci(Wasserthal, 1980;Herteletal.,1985;Hustert, antennae, wings,head,ventralabdomen,andabdominalappendages et al.,2010).Specifically, theseAPOsdrivehemolymphthroughthe ventral extracardiacspace(Pass,2000;Passetal.,2006;Andereck hemolymph throughnarrow, dead-endappendagesandthroughthe auxiliary hearts)thateitherpropelorassistinthepropulsionof structurally independentaccessorypulsatileorgans (APOsor To overcomethislimitation,insectshaveevolvedaseriesof directional forcetodrivehemolymphallregionsoftheorganism. central bodycavity, thispumpdoesnotproducesufficient 2013). Althougheffective incirculatinghemolymphacrossthe (Jones, 1977;Wasserthal, 2007;Glennetal.,2010;Wirkner etal., is anatomicallydividedintoathoracicaortaandanabdominalheart extends fromtheinsect’s headtothe posterioroftheabdomenand circulation iscalledthedorsalvessel,whichamusculartubethat 2013; Klowden,2013).Themainpumpthatdriveshemolymph through anopenbodycavitycalledthehemocoel(Chapmanetal., to anotherisbycirculatingafluidmediumcalledhemolymph hormones, wasteandothermoleculesfromoneregionofthebody peripheral sensoryappendages. antennal functioningareefficiently transportedintoandoutofthese (Diptera: Culicidae: Culicinae),butphysiological dataonthese has beendescribedfortheyellow fevermosquito, (Diptera: Culicidae:Anophelinae). ThestructureofantennalAPOs The goalofthepresentstudywas tocharacterizethefunctional In mostinsectorders,hemolymphflowsintotheantennae The primarymannerbywhichinsectstransportnutrients, Anopheles gambiae Giles 1902sensustricto Aedes aegypti

The Journal of Experimental Biology compound eyes. antenna’s scapeandpedicel,butventralto themedial-mostregionof the dorsalmidlineofmosquito.The APOsaredorsalandposteriortothe located intheheadbetweenfrons andthevertexinanareathatflanks antennal APOs(greenfluorescence insideredcircles).TheAPOsare Anopheles gambiae. Fig. contraction. we wereabletoidentifytheirlocationandmonitor fluorescent aggregatesthatforminandaroundeachantennalAPO, material Movie spatial shiftofthesefluorescentaggregates(supplementary the antennalAPOs,andcontractionofAPOsresultsina et al.,2010).Someofthesemicrospheresaggregateinandaround by thecontractileactionofheart(Anderecketal.,2010;Glenn injected intothehemocoelaredisseminatedtoallpartsofbody action oftheheart.Specifically, fluorescentmicrospheresthatare developed anovelfluorescence-basedmethodthatreliesonthe visualize theantennalAPOsinliveandintactmosquitoes,we for theirvisualizationusingbrightfieldillumination.Inorderto together withthelackoftranslucencyhead,doesnotallow Lao etal.,2013).However, thesmallsizeofantennalAPOs, al., 2010;Hillyeret2012;ChenandHillyer, 2013;Estévez- illuminating theabdomenusingtrans-brightfieldlight(Glennet Contractions ofthe antennal APOcontractionsrates. FlyNap (activeingredient:triethylamine)acceleratesbothheartand and theantennalvessel,(6)showthatanestheticagent the antennalspace,(5)describestructureofAPOs measured thedirectionalvelocityofhemolymphasittravelsacross (3) determinedtheeffective diameteroftheantennalvessel,(4) antennal APOscontractinamannerthatisindependentoftheheart, contraction rateofanophelineantennalAPOs,(2)showthatthe using novelfluorescence-basedmethodswe(1)determinedthe organs arelacking(Clements,1956;SunandSchmidt,1997).Here, RESEARCH ARTICLE vertex, and contract insynchrony vertex, andcontract located andthe Antennal APOsare between thefrons RESULTS

.Location oftheantennalaccessorypulsatileorgans(APOs)in 1.

1). Thus,byvisualizingtheoscillationsof Brightfield andfluorescenceoverlay showingthe APOs A. gambiae midline Dorsal heart canbeobservedby Flagellum Scape Pedicel Thorax Eye contractions (Fig. to detectanykindofcorrelationbetweenheartandantennalAPO P=0.196), andgraphicalanalysesofindividualcontractionsfailed APOs andtheheart(Fig. was nocorrelationbetweenthecontractionratesofantennal 1.808 aggregated inor aroundtheantennalAPOs,and thesemicrospheres hemocoel, weobservedthat2 than whenmicrospherestravel in theupwarddirection. movement, whilelinear, displaysmorelateralmobilityandisslower which onaveragecontractedat0.97 APOs thatdiffered by 59 antenna (supplementarymaterial Movie (toward thehead)along medial andlateralportionsofthe microspheres turnmediallyandbeginadownwardtrajectory mosquito. Uponreachingthedistalendofantenna, the portion oftheappendagethatisclosesttodorsalmidline the trajectory uptheantenna(awayfromhead)alonglateral the antennalspace.These1 revealed thattheyentertheantennalAPOsandarepropelled into fluorescent microspheresthathadbeeninjectedintothehemocoel Qualitative observationsofthemovement1 (supplementary materialMovie APOs, theheartofthesesamemosquitoeswasalsomonitored Immediately followingthephysiologicalrecordingofantennal not changeduringthecourseofeachrecording. remain thesame,andmechanicsofindividualcontractionsdo videos arebrokendownintosmallerintervals,thecontractionrates continuous overthecourseofeach60 Estévez-Lao etal.,2013),antennalAPOcontractionsarenormally direction approximately10timesperminute(Hillyeretal.,2012; the sametime.Finally, althoughtheheartreversescontraction pause simultaneouslyandthenreturntotheircontractingstateat (when thisoccurs,theyusuallystopfor2–3 Likewise, intheinstanceswherebothAPOsstopcontracting two accessoryheartsimmediatelyreturntocontractinginsync. the twoAPOscontractinharmony, whenanAPOskipsabeat,the propelling hemolymph(Fig. involves aweakcontractionthatislikelynoteffective in intensity profileoftheAPO,indicatingthatskippedbeat analysis oftheskipshowsaminimalbutobservablechangein mechanism ( two APOsareeitherlinkedorcontrolledbythesame revealed thattheAPOscontractinsynchrony, signifyingthatthe correlation left andrightantennalAPOs(Fig. There wasastrongcorrelationbetweenthecontractionratesof 0.976 and0.974 the leftandrightantennalAPOscontractatanaveragerateof eyes. Intravitalvideoimagingofthe antenna butventraltothemedial-mostregionofcompound vertex inanareathatisdorsalandposteriortothebaseof abut againstthecuticleandarelocatedbetweenfrons on eachsideofthehead’s dorsal midline ( antennal space velocityHemolymph changes asittravels the across notsynchronized are Antennal APOsandtheheart When differently sizedmicrosphereswereinjectedinto the Anopheles gambiae contractions Hz (Fig. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.106708 P i.3A,B).WhenoneAPOskipsabeat,graphical Fig. <0.0001), and78%ofthemosquitoessampledhad 2A; repeatedmeasuresANOVA P

min Hz, respectively(

3A,B,D,E). − 1 ). GraphicalanalysisofAPOcontractions possess twoantennalAPOsthatarelocated ≤ 3 3C). Insupportoftheobservationthat

μm microspherestravelinalinear 2D; contractions

2). IncontrasttotheantennalAPOs, μm diametermicrospheresrarely i.2A;pairedt Fig. R A. gambiae =0.165; Pearsoncorrelation

Hz, theheartcontractedat

s recording.Thatis,when

2B;

min

3). Thisdownward i.1). Fig. − R

1 s), thetwoAPOs head revealedthat =0.974; Pearson <0.0001). There (Fig. -test μm diameter These APOs P 2C; mean =0.8127). 3007

The Journal of Experimental Biology (mean ±s.d.,683±125and687±134 distances whenmovingintheupwardanddownwarddirections 4).Microsphereswere trackedfornearlyidentical appendage (Fig. was trackedtoquantitativelymeasurehemolymphflowwithinthis antennal space,themovementoftheseneutrallybuoyantparticles a velocityof197.0 P and 2 μmindiameter. effective circulatorydiameterofthemosquitoantennaisbetween1 became stuckwithinantennalstructures.Thesedatasuggestthatthe diameter microspheresattimesstoppedmoving,indicatingthatthey 1 diameter microsphereswasefficient andunimpeded,theflowof but only3067 3008 RESEARCH ARTICLE visualized bybrightfieldmicroscopy. Earlierworkin antennal hemocoel,mosquitoheadswereembedded,sectioned and through anantennalvesselandreturnstotheheada wider To confirmthehypothesis thathemolymphenterstheantennae APOs andintheantennalspace.However, whiletheflowof0.5 microspheres werealwaysobservedinandaroundtheantennal did notentertheantennalspace.Incontrast,1and0.5 microspheres averaged13,797 test Structure of theantennal APOsandtheantennalvessel of Structure and Schmidt,1997),weidentified in the descriptionsbySunandSchmidt asapointofreference(Sun Here, bymeansofacomparative anatomyapproachthatreliedon in culicinemosquitoes(Clements, 1956;SunandSchmidt,1997). extensively describedthestructural organization ofantennal APOs across theantennalspace. supports theantennalvesselhypothesisofhemolymphmovement hemolymph transitionsfrommovingupwardtodownward P chamber, orampulla,that includes aseptum-likestructure thathas structures describedin μm diametermicrospheresmetsomedegreeofresistance;1 <0.0001). Thisreductioninvelocityandmaximumacceleration as =0.8835), and,onaverage,microspheresmoveduptheantennaat No. of mosquitoes Contraction rate (Hz) Given that0.5 12 Left–right antennal APO contractionsmin 0 4 8 0 1 2 P –11 <0.0001). Furthermore,themaximumaccelerationof APO (left) CD AB Antennal

–10

–9

–8

–7 –6 μm μm diametermicrospheresflowfreelyacrossthe

–5 –4–3 APO (right) μm

Antennal s –2

s when travelingdowntheantenna( –2 –1 A. aegypti − 1 and downtheantennaat51.7

0

1 23 μm Heart

s . Mainly, eachAPOcontainsa

4 56 –2 A. gambiae when movinguptheantenna μm, respectively; –1 Contraction rate of Contraction rate of heart (Hz) right APO (Hz) many ofthesame 0 1 2 3 0 1 2 012 012 A. aegypti μm diameter Contraction rateofleft APO (Hz) Contraction rateof APOs (Hz) μm

s t t − -test -test 1 μm μm has ( t - of mosquitoes anesthetized byexposureto coldtemperatures mosquitoes exposedtoFlyNap contracts15%fasterthantheheart APO andheartcontractionrates. mosquitoes witheithercoldor FlyNap andmeasuredbothantennal similar effect ontheantennalAPOs,weanesthetizedgroups of 2013). To testwhetherexposingmosquitoestoFlyNapinduces a acceleratory effect oftheneuropeptide CCAP (ChenandHillyer, increases heartcontractionratesandeliminatesthecardio- We havepreviouslyreported thatexposingmosquitoestoFlyNap hemocoel (Fig. to thehemocoelviamedialandlateralportionsofantennal where hemolymphisdischarged intotheantennalcavityandreturns The antennalvesselendsnearthedistalportionofflagellum, of theantennathatisclosesttodorsalmidlinemosquito. pedicel, enterstheflagellum,andextendsalonglateralportion vessel exitstheAPO,crossesJohnston’s organ locatedin the antenna bymeansofanantennalvessel(Fig. structure ofthesecells. employed inthisstudycouldnotproperlycapturetheshapeor However, itispossible,andevenlikely, thattheangleofsectioning resemble thecolumnarcellspreviouslydescribedinculicines. observed thattheanteriorofampullaattachestocellsdonot they propelhemolymphviapassivesystole.Interestingly, we type (SunandSchmidt,1997;Pass,2000;Passetal.,2006), antenna. Thus,mosquitoantennalAPOsareoftheampulladilator ampulla toamorecompactshape,forcinghemolymphintothe filling itwithhemolymph.Then,musclerelaxationreturnsthe Specifically, musclecontractionexpands(dilates)theampulla, attached tomusclethatdrivesantennalAPOcontractions. (Fig. body whereastheanteriorisattachedtocuticularepidermis Schmidt, 1997).Theposteriorofthechamberisattachedtoz- 5A)(Sunand been hypothesizedtofunctionasavalve(Fig. FlyNap increases antennal APO and heart contraction rates contraction antennal APOandheart increases FlyNap Observations ofthedorsalabdomen revealedthattheheartof Hemolymph thatenterstheampullaofanAPOisdriveninto 5A). Thez-bodyiscomposedofdensegroupscells,and The JournalofExperimentalBiology(2014)doi:10.1242/jeb.106708

5F). by briefexposuretocoldtemperature. mosquito ( (average ofleftandright)thehearteach between thecontractionratesofantennalAPOs antennal APOsofeachmosquito.(D)Correlation of contractionsperminutebetweentheleftandright each mosquito( contraction ratesoftheleftandrightantennalAPOs denote thes.e.m.(B)Correlationbetween APOs andtheheart.Samplesizeis63whiskers (A) Contractionratesoftheleftandrightantennal Fig.

.AntennalAPOandheartcontractionrates. 2. R =0.165). Allmosquitoeswereanesthetized R =0.974). (C)Difference inthenumber

5B–E). Theantennal

The Journal of Experimental Biology antennal APOs (P to FlyNapdidnotinducedifferent effects ontheheartand anesthetic agentandthepumping organ, indicatingthatexposure ( FlyNap contractedfasterthan pumpingorgans exposed to cold ( ANOVA, theheart wasfoundtocontractfasterthantheAPOs entire datasetwasanalyzedbytwo-wayrepeatedmeasures P APOs ofmosquitoesexposedtocold(Fig. exposed toFlyNapcontract29%faster(bothAPOs)than the revealed thattheleftandrightantennalAPOsofmosquitoes observations (ChenandHillyer, 2013).Observations ofthehead 6A; (Fig. RESEARCH ARTICLE E D B A 1 0 1 0 P P <0.0001 fortheleftandrightAPOs,respectively).When the <0.0001). However, therewasnointeractionbetweenthe <0.0001), andpumpingorgans (heartandAPOs)exposed to 62 03 34 32 30 28 26 62 03 34 32 30 28 26 t -test 02 02 P =0.9988). Time (s) =0.0018), whichisinlinewithourearlier 03 03 Time (s) Time (s) Time (s) 6A; 04 04 t -test C 0246810 P =0.0001 and 05 05 exposed toFlyNap (Fig. FlyNap anesthetic agent(cold APO andheartcontractionratesfollowingexposureto either P correlation left andrightantennalAPOsfollowingbothcold( ≤ exposed tocoldanesthesia hadAPOsthatdiffered by between antennalAPOs.Specifically, whereas77%ofthemosquitoes exhibited highervariabilityinthe difference incontractionsper minute APOs thatdidnotcontractinperfect synchrony, andtheseAPOs also However, a higherproportionofmosquitoesexposedtoFlyNaphad 3 <0.0001) anesthesia,buttherewasnocorrelationbetweenantennal There wasastrongcorrelationbetweenthecontractionratesof the

contractions 06 06 * R =0.092 andPearsoncorrelation The JournalofExperimentalBiology(2014)doi:10.1242/jeb.106708 P <0.0001) andFlyNap(R

min 0 0 − 1 , thisvaluewasonly68%for mosquitoes R

6C). Perhapsthis difference isduetothefact =0.092 andPearsoncorrelation acquired approximately1 D showthesamemosquitobutwere magnified inE.Videos usedtocreateA and a contractionandtheareashadedinDis mosquito showninA.Eachpeakrepresents representation ofheartcontractionsinthe contraction rates.(D,E)Graphical natural variabilityofantennalAPO Comparison ofBandCillustratesthe showing askippedbeat(asterisk). APO contractionsinadifferent mosquito, representation ofleft(top)andright(bottom) shaded inA ismagnifiedinB.(C)Graphical represents acontractionandthearea (bottom) APOsofamosquito.Eachpeak contractions oftheleft(top)andright (A,B) Graphicalrepresentationofthe antennal APOandheartcontractions. Fig. exposure tocoldtemperature. mosquitoes wereanesthetizedbybrief

.Graphicalrepresentationof 3. =0.901; Pearsoncorrelation P =0.683; Fig. R

min apart.All =0.966; Pearson P =0.683; 6B,D). 3009

The Journal of Experimental Biology 58 3010 RESEARCH ARTICLE Fig. orders (Pass,2000;Passetal.,2006;Hertel2012).Common Antennal circulatoryorgans havebeenidentifiedinnumerousinsect contraction rates(mean75 that mosquitoesexposedtoFlyNaphavehigherantennalAPO were anesthetizedbybriefexposuretocoldtemperature. size is63ineachdirectionandwhiskersdenotethes.e.m.Allmosquitoes and downward(returningtothehead)withinantennalspace.Sample 0.5 μm diametermicrospheresastheytravelupward(awayfromthehead) across theantennalspace. Fig. DISCUSSION the dorsalmidline of themosquito).Scalebarapplies to allmicrographs. A,anterior;M, medial(toward and theantennalhemocoel, hemolymphvelocityisfour timeshigherintheupwarddirection thanitisinthedownwarddirection. released intoawiderantennalhemocoel, orcavity, anditflowsbacktoward thehead.Becauseofdifference oftheantennal vessel betweenthediameters oftheappendageitis APO locatedintheheadpropelshemolymph intotheantennabymeansofanantennalvessel.Whenhemolymph reachesthedistalend toscale).Anantennal hemocoel. (F)Modelofhemolymph flowacrosstheantennalspace,basedondataandhistological (structuresarenotdrawn showing theantennalvesseland antennalhemocoel.(D,E)Cross-sectionoftwodifferent andtheantennal antennaeshowingtheantennalvessel and antennashowingtheantennalvessel extendingacrosstheJohnston’s ofanantenna organandthroughtheantennalhemocoel.(C)Coronalsection APO andtravelsupthescapepedicel containingtheJohnston’s sectionofthe head organ(JO)andthroughtheantennalhemocoel(AH).(B)Histological vessel (AV)an ampulla(Am;chamber)thatisattachedanteriorlytothecuticularepidermis andposteriorlytothez-body(ZB).Anantennal extendsfromthe contractions

.StructureoftheantennalAPOsandvessel. 5. Velocity and maximumaccelerationofhemolymphasittravels 4.

Velocity (µm s–1) 100 150 200 50 0 AB Upward min JO DE ABC − 1

AH Downward for cold). ZB Velocity (A)andmaximumacceleration(B)of AH

contractions APO

Max. acceleration (µm s–2) 10,000 15,000 AV 5000

min 0 Am M A

− Upward 1 for FlyNapversus JO

Downward AV (A) HistologicalsectionoftheheadandantennashowinganantennalAPO thatcontains AH A. gambiae histological experimentsshowedthatthestructuralorganization of of hemolymphpropulsionacrosstheantennalspace.First, presented hereinareinagreementwiththis‘passivesystole’ model means ofanantennalvessel.Thestructuralandfunctionaldata to amorecompactsize,drivinghemolymphintotheantennaby head intotheAPO.Whenmusclerelaxes,ampullareturns of thismuscleexpandstheampulla,forcinghemolymphfrom the aorta(Clements,1956;SunandSchmidt,1997).Contraction as asyncytialbody),whichisattachedtomusclethatextends described anampullathatisattachedtoaz-body(alsoreferred space. functional mechanicsofhemolymphpropulsionacrosstheantennal ampulla dilatortypeanddescribeforthefirsttimereal-time approach toconfirmthatthemosquitoantennalAPOsareof the presentstudy, weuseda correlative structuralandfunctional ampulla dilatortype(Clements,1956;SunandSchmidt,1997).In classified theirantennalcirculatoryorgans asmembersofthe al., 2006).Structuralanalysesintheculicinemosquito where musclecontractiondilatestheampulla(Pass,2000;Passet muscle contractioncompressestheampulla,andpulsatileorgans can begroupedintonon-pulsatileorgans, pulsatileorgans where taxa. Ninedifferent typeshavebeenstructurallydescribed,andthese circulatory organs displayremarkablestructuralvariabilityacross that leadshemolymphintotheantenna.However, antennal amongst antennalcirculatoryorgans istheirfunctionasaconduit Earlier structuralanalysesontheantennalAPOsof AH AM AV AH AV The JournalofExperimentalBiology(2014)doi:10.1242/jeb.106708 and 50 µm A. aegypti A M F hemocoel Scape & Antennal pedicel antennal APOsissimilar. Second, Downward flow Upward flow Flagellum Antennal vessel Antennal APO A M A. aegypti A. aegypti

The Journal of Experimental Biology hemolymph inside theheartandinotherregions oftheabdomen we havepreviouslyusedtomeasure thedirectionalvelocityof across theantennalspace.Specifically, byadaptingmethodologies direct visualizationandmeasurement ofhemolymphflowvelocity visualization ofAPOcontractions. Uniquetothepresentworkis has beeninferredfromacombination ofstructuralanalysesandthe dorsal vessel(Herteletal.,2012). rate issignificantlyslowerthanthe1.28 are notsynchronized,andthatthe0.45 and theheartofAmericancockroach, Nevertheless, oneofthesestudiesshowedthattheantennalAPOs is distantlyrelatedtomosquitoes(Trautwein etal.,2012). 1985; Langeetal.,1993;Hertel2012),agroupofinsects that these studieshavelargely focused onPolyneoptera(Herteletal., studies havereportedabsoluteantennalAPOcontractionrates, and Comparison ofthesefindingswithearlierreportsisdifficult, asfew synchronized withoneanother, arenotsynchronized withtheheart. contracts at~1.8 through theantennalvesselisunidirectional. visualizations offlowshowednoevidencebackflow: also resultsinbackflowfromtheantennalvessel.Ourfunctional ampulla, besidesdrivinghemolymphfromtheheadintoAPO, Schmidt (SunandSchmidt,1997)proposedthatdilationofthe key respect.Basedontheirstructuralobservations,Sunand differs fromearlierdescriptionsofmosquitoantennalAPOsinone However, ourmodelofhemolymphflowacrosstheantennalspace vessel andreturnstotheheadviaawiderantennalhemocoel. that hemolymphenterstheappendagethroughanarrowantennal systole). Lastly, intravitalvideoimagingoftheantennashowed and drivinghemolymphintotheantenna(compressivemovement; returns theAPOtoitsoriginalposition,contractingampulla movement; diastole),whichisfollowedbyaspatialshiftthat expands theampullaandinflatesitwithhemolymph(dilatory contraction ismanifestedbyaspatialshifttowardtheaortathat intravital videoimagingoftheheadshowedthateachAPO RESEARCH ARTICLE No. of mosquitoes Contraction rate (Hz) To date,thepathofhemolymphflowacrossinsectantennae Mosquito antennalAPOscontractat~1 Left–right antennalAPOcontractionsmin 0 1 2 0 2 4 6 0 2 4 6 –12 AB C APO (left) Antennal

–10 –8–6–4 FlyNap Cold FlyNap Cold APO (right)

Antennal Hz. ContractionsofantennalAPOs,while

–2

0

2

4

Heart 6

8

10

Hz antennalAPOcontraction

Hz contractionrateofthe –1 Periplaneta americana

Hz whereastheheart Contraction rate of Contraction rate of heart (Hz) right APO (Hz) 0 1 2 3 0 1 2 012 012 D Contraction rate of left APO left (Hz) of rate Contraction Contraction rate of APOs of rate (Hz) Contraction FlyNap Cold FlyNap Cold , antennae at197 we showedthathemolymphtypicallytravelsupthemosquito (Andereck etal.,2010;GlennEstévez-Lao2013), 2014), andtwo ofthesestudiesdetecteddifferences incardiac Matsuo, 2008;Chomaetal.,2010; Tsai etal.,2011; Tang etal., Wessells andBodmer, 2004;Lo etal.,2007;Neckameyerand activity in Hillyer, 2013). FlyNapiscommonlyusedinstudiesassessingheart cardioacceleratory effect oftheneurohormoneCCAP (Chenand (triethylamine) increasesheart contraction ratesandeliminatesthe hemocytes. significantly smallerthanthe~10 effective diameteroftheantennal vesselis1–2 2013): trackingofdifferently sizedmicrospheresshowedthatthe are seldom,ifever, observedinsidetheantennae (KingandHillyer, from thecontractilepump.Thismodelalsoexplainswhyhemocytes acceleration ispredictedtodecreaseasmicrospheresmove away than particlesreturningthroughawidercavity. Similarly, maximum a narrowantennalvesselarepredictedtotravelfasterandstraighter model ofhemolymphpropulsion.Thatis,microspheresmoving up when travelingdowntheantenna,supportsantennalvessel traveling uptheantennabutexperienceslightlateralmovements observation thatmicrospheresmoveinalinearmannerwhen from goingupwardtodownward,togetherwiththe maximum accelerationby74%and54%,respectively, astheyshift Specifically, thefindingthatmicrospheresreducetheirvelocityand model ofhemolymphpropulsionacrosstheantennalspace. hemolymph flowdataprovidefunctionalevidencesupportingour abdominal cavity(Anderecketal.,2010). the downwarddirectionis~75%slowerthanflowvelocityin velocity intheextracardiacabdomen,butantennalflow hemolymph velocityintheupwarddirectioniscomparabletoflow et al.,2010;Estévez-Lao2013).Furthermore,antennal magnitude slowerthanhemolymphflowthroughtheheart(Glenn Hemolymph velocitythroughtheantennaisonetotwoordersof Finally, werecentlyshowedthatexposingmosquitoestoFlyNap In additiontoprovidingdirectinformationonflow, these The JournalofExperimentalBiology(2014)doi:10.1242/jeb.106708 Drosophila melanogaster μm mosquito followingcold( (average ofleftandright)thehearteach between thecontractionratesofantennalAPOs following coldorFlyNapanesthesia.(D)Correlation left andrightantennalAPOsofeachmosquito (C) Difference incontractionsperminutebetweenthe ( antennal APOsofeachmosquitofollowingcold between thecontractionratesofleftandright 22 andwhiskersdenotethes.e.m.(B)Correlation anesthesia. Foreachanestheticagent,samplesizeis antennal APOsandtheheartfollowingcoldorFlyNap anesthesia. following coldorFlyNap(triethylamine) Fig. ( R R

=0.966) orFlyNap( =0.361) anesthesia. s −

.AntennalAPOandheartcontractionrates 6. 1 and downtheantennaat52 (A) Contractionratesoftheleftandright μm diameterofcirculating R (Paternostro etal.,2001; =0.901) anesthesia. R =0.092) orFlyNap μm, whichis μm 3011

s − 1 .

The Journal of Experimental Biology performed at27°Cand75%relativehumidity, undera12 post-eclosion. photoperiod. Allexperimentswereperformedonadultfemalesat4or 5 3012 RESEARCH ARTICLE employed thisform ofchemicalanesthesia. in thisstudywas cold,andonlyexperimentsexplicitly labeled‘FlyNap’ several hours(ChenandHillyer, 2013).Thedefault anestheticagentused anesthesia (activeingredient:triethylamine) knocksdownmosquitoesfor anesthesia knocksdownmosquitoes forseveralminuteswhereasFlyNap transferred toaPetridishheld at roomtemperatureuntiluse.Cold the vesselfor30 (Carolina BiologicalSupply, Burlington,NC,USA)wasintroducedinto enclosure andanabsorbentwandthathadbeenloadedwithFlyNap FlyNap-based anesthesia,mosquitoeswereplacedinacovered60 then transferredtoaPetridishthatwasplacedovericeuntiluse. For based anesthesia,mosquitoeswereexposedto anesthesia asdescribedelsewhere(ChenandHillyer, 2013).Forcold- Mosquitoes wereanesthetizedusingcold-basedorFlyNap-based 10% sucrosesolution fed amixtureofkoifoodandyeast,uponemergence adultswerefeda (Estévez-Lao etal.,2013).Briefly, eggswerehatchedinwater, larvaewere Anopheles gambiae blood feeding. processes requiredforpathogentransmission,reproductionand presented hereinmayalsobegintoshednewlightonmosquito Wang etal.,2009;Carey2010;Rinker2013),thedata involved inmatingandhost-seekingbehaviors(Catoretal.,2009; mosquito antennaeprovidesensoryinputthatdrivesprocesses pathogens inthehemocoel(KingandHillyer, 2012),andthat 2010). Giventhathemolymphcurrentsinfluencethebiologyof insect thatisasignificantpestanddiseasevector(Beckeretal., sensory appendages.Moreover, thisstudyalsofocusedonan understanding ofthebiologyhemolymphpropulsionacross fundamental entomologicalquestionsandexpandsour al., 2012).Thepresentstudyprovidescomprehensiveanswersto the mostunderstudiedofinsectphysiologicalsystems(Harrisonet Hillyer etal.,2014). and Lange,2008;Walker etal.,2009;Estévez-Lao2013; including mosquitoes(Orchardetal.,2001;Dulcis2005;Ejaz and FMRFamide-relatedpeptides,areproducedbymostarthropods, Predel etal.,2010),othercardiomyotropicpeptides,suchasCCAP produced byeither and Lange,2008)contractionrates.Althoughproctolinisnot 2012) andheart(Zorniketal.,1999;Sliwowska2001;Ejaz antennal APO(Herteletal.,1985;Lange1993;Hertel 2001). Second,insomeinsectstheneuropeptideproctolinmodulates capable ofproducingmyotropicfactors(Passetal.,1988;Predel, evidence. First,antennalAPOshaveneurohemalfunctionandare stimuli. Thishypothesisisalsosupportedbytwoadditionallinesof Klowden, 2013),aremodulatedbysimilarneuralorhormonal (Hertel etal.,1985;RichterandHertel,1997;Chapman2013; hypothesis thattheantennalAPOsandheart,whilemyogenic physiology. Moreover, ourFlyNap-basedfindingsalsosupportthe FlyNap shouldbeavoidedinstudiesassessingcirculatory antennal APOandheartcontractionratesfurthercautionsthat et al.,2011). OurfindingthatexposuretoFlyNapincreasesboth with othermethodsofinsectrestraint(Paternostroetal.,2001;Tsai function whentheeffect ofFlyNap-basedanesthesiawascompared Mosquito anesthesia andmaintenance Mosquito rearing MATERIALS ANDMETHODS There islittledoubtthattheinsectcirculatorysystemamong

s. FollowingexposuretoFlyNap, mosquitoeswere (G3 strain)wererearedasdescribedelsewhere dlibitum ad A. gambiae . Mosquitorearingandmaintenancewere or A. aegypti − 20°C for60 (Riehle etal.,2002;

h light:12 s, andwere

h dark

days cm 3 antennal APOs,allvaluesweremultipliedby microspheres onthesurfaceofheart(KingandHillyer, 2012).Asforthe oscillations ofperiostialhemocytesthathadphagocytosedfluorescent heart contractionswererenderedbymonitoringthespatialfluorescence of theabdominalheart(Glennetal.,2010).Graphicalrepresentations such thateachpeak(notvalley)marksacontraction. were analyzedpertreatment. difference betweencoldanesthesiaandFlyNapanesthesia,22mosquitoes 63 mosquitoeswereincludedintheanalysis.Forexperimenttesting APO contractionswereevenlydistributedacrosstime.Forcoldanesthesia, al., 2012;ChenandHillyer, 2013;Estévez-Laoetal.,2013),andantennal contraction direction(Anderecketal.,2010;GlennHillyer heart rateasrhythmicityinmosquitoesdoesnotchangewith the posteriorabdomen)directions,inthisstudyweonlymeasuredtotal between contractinginanterograde(towardthehead)andretrograde a contraction.Finally, althoughthemosquitoheartperiodicallyalternates the selectedarea.Forvisualizationease,allvaluesweremultipliedby changed aseachcontractionshiftedthefluorescentaggregateoutofandinto each antennalAPOandthenquantifyinghowthesumlightintensity rendered usingNISElementsbyselectingtheareaimmediatelysurrounding Graphical representationsofindividualantennalAPOcontractionswere oscillation ofthefluorescentaggregatesassociatedwitheachAPO. head. Absolutecontractionratesweredeterminedbymanuallycountingthe movement offluorescentaggregatesintheanterior–dorsalportion 10 following videoacquisitionparameterswereused:3×3binning,4×gainand imaged twice:oncefortheantennalAPOsandheart.The was acquired.Thisprotocolyieldedpaireddata,whereeachmosquito was imagedthroughthedorsalabdomen,andanother60 of thehead,focusingonbothantennalAPOs,wasacquired.Then,heart fluorescence (usinganeutraldensityfilterof4),and60 3–7 Germany) andNikonAdvancedResearchNISElementssoftware.At Photometrics CoolSNAP HQ2camera(Roper Scientific,Ottobrunn, with aNikonIntensilightC-HGFIfluorescenceilluminationunit, the slidewastransferredtoaNikon90icompoundmicroscopeequipped slide. ThemosquitoheadwasrestedontopofaflatpieceParafilmand M (PechineyPlasticPackagingCompany, Chicago,IL,USA)onaglass the mosquitowasplaceddorsalsideupbetweentworolledstripsofParafilm in PBS(pH carboxylate-modified microspheres(MolecularProbes,Eugene,OR,USA) ~0.1 μl of0.2%solids1mdiameteryellow–greenfluorescent(505/515) stereomicroscope (Nikon,Tokyo, Japan)andintrathoracicallyinjectedwith Each mosquitowasanesthetized,visualizedusingaSMZ645 they moveddown theantenna(towardhead). antenna (awayfrom thehead)andthreemicrospheres weretrackedwhile for eachmosquito,threemicrospheres weretrackedwhilemovingupthe al., 2010;Glennet2010).A totalof 21mosquitoeswereassayed,and maximum accelerationofeachmicrosphere werecalculated(Anderecket flowed upordowntheantennae.Then, thedistancetraveled,velocityand quantitatively trackthetrajectory of neutraldensitymicrospheresasthey manual featureoftheObjectTracker moduleofNIS-Elementswasusedto contractions, anda60 using thesameset-upandacquisitionparametersusedtomonitor APO by twoadditionalstripsofParafilm.Oneantennapermosquitowasimaged antennae lyingflatonastripofParafilm,andthelegswingsheld down PBS. Mosquitoeswerepositionedonaslideventralsideupwith both yellow–green fluorescent(505/515)carboxylate-modifiedmicrospheres in anesthetized andinjectedwith~0.1 To trackhemolymphflowwithintheantenna,mosquitoes were cold and theheart pulsatile organs antennalaccessory of Intravital videoimaging acceleration flow hemolymph distance,velocity and of Measurement Heart contractionsweremeasuredbycountingthewave-like The contractionofantennalAPOswasmeasuredbyvisualizingthe ms exposurewithnodelay. min post-injection,themosquitowasilluminatedwithlow-level 7.0). Thelegsandwingswereseveredwitharazorblade, The JournalofExperimentalBiology(2014)doi:10.1242/jeb.106708

s low-levelfluorescencevideowasrecorded.The μl of0.02%solids0.5mdiameter − 1 suchthateachpeakmarks

s intravitalvideo s intravitalvideo − 1

The Journal of Experimental Biology http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.106708/-/DC1 Supplementary materialavailableonlineat decision topublish,orpreparationofthismanuscript. 1051636. TheNSFhadnoroleinstudydesign,datacollectionandanalysis, 1051636 andIOS-1257936toJ.F.H., includinganREUsupplementtoIOS- This researchwasfundedbyUSNationalScienceFoundation(NSF)grantsIOS- experiments, analyzedthedata,andwrotemanuscript. J.F.H. conceivedthestudy. J.F.H. andS.B.designedperformedthe The authorsdeclarenocompetingfinancialinterests. 4% formaldehydein0.1 Mosquitoes weredecapitated,andtheirheadsfixedbyimmersionin RESEARCH ARTICLE Nikon DS-Fi1high-definitioncolorCCDcamera(Glennetal.,2010). and thesectionswereimagedusing90imicroscopeconnectedtoa were mountedusingPoly-Mount(PolysciencesInc.,Warrington, PA, USA), on glassslides,andstainedwithAzureII.Theslidesweredried,coverslips knife onaSorvallJB-4microtome(Sorvall,Newtown,CT, USA),placed holders andwax.Serialsectionsof2.5 embedded inpolyethylenemoldingtraysanaerobicallysealedwithblock JB4-Plus resin(ElectronMicroscopySciences,Hatfield,PA, USA),and Specimens weredehydratedthroughagradedethanolseries,infiltratedwith ile,J . sée-a,T . ukosr .J n loh V. A. Aluoch, and L.J. Funkhouser, T. Y., Estévez-Lao, J.F., Hillyer, neek .W,Kn,J .adHlyr J.F. Hillyer, and J.G. King, J.W., Andereck, References Supplementary material Funding Author contributions Competing interests Histology arsn .F,Wos .A n oet,S.P. Roberts, and H.A. Woods, J.F., Harrison, J.F. Hillyer, and J.G. King, J.D., Glenn, G. Pass, and B.A. Gereben-Krenn, J.F. Hillyer, and W. Chen, M.T. Siva-Jothy, and A.E. Douglas, R.F., Chapman, S.J. Simpson, and R. R.F. Hoy, Chapman, and L.C. Harrington, B.J., Arthur, L.J., A. Cator, Kaiser, and M. Madon, C., Dahl, C., Boase, M., Zgomba, D., Petric, N., Becker, etl . as .adPnln H. Penzlin, and G. Pass, W., Hertel, ae,A . ag . u .Y,Zibl .J n alo,J.R. Carlson, and L.J. Zwiebel, C.Y., Su, G., Wang, A.F., Carey, sée-a,T . oc,D . oegr .W n ile,J.F. Hillyer, and H.W. Honegger, D.S., Boyce, T. Y., Estévez-Lao, A.B. Lange, and J. A. Ejaz, Ewer, and R.B. Levine, D., Dulcis, A. N. Clements, G.J. Tearney, and B.E. Bouma, B.J., Vakoc, M.J., Suter, M.A., Choma, Hertel, heart physiology. corazonin Anopheles gambiae immunohistochemical study. system oflowerNeoptera:a comparativepharmacologicaland 213 mosquito heartanditsfunctioninbidirectionalhemolymphtransport. hemocoel. abdomen potentiatesextracardiacretrogradehemolymphpropulsioninthemosquito Ephemeroptera). appendages inprimitiveinsects(Hexapoda:Archaeognatha,Zygentoma and Simpson andA.E.Douglas),pp.107-131.Cambridge:CambridgeUniversityPress. blood andtheimmunesystem.In University Press. Function 1079. reception inthemalariamosquito (2010). antennal heartof Environmental PhysiologyofInsects gambiae Cardioacceleratory functionoftheneurohormoneCCAP inthemosquito Opt. Drosophila melanogaster Heart wallvelocimetryandexogenouscontrast-basedcardiacflowimagingin PLoS ONE mosquitoes andeliminatesthecardioacceleratoryeffect oftheneuropeptideCCAP. convergence inthelovesongsofdenguevectormosquito. Baculum extradentatum Drosophila Diptera. Physiol. , 541-550. 15, 056020. . epr,S n cet M. Eckert, and S. Neupert, W., Mosquitoes andTheirControl. Q. J.Microsc.Sci. 31, 563-572. . J.Exp.Biol. (ed. S.J.SimpsonandA.E.Douglas),pp.3-14.Cambridge:Cambridge PLoS ONE cardiac function. 8 , e70414. (1956). Theantennalpulsatingorgansofmosquitoesandother Insect Mol.Biol. Acta Zool. Periplaneta americana 216 5 . Peptides , e12943. (2008). Peptidergiccontroloftheheartstickinsect, , 601-613. using Doppleropticalcoherencetomography. (2013). FlyNap(triethylamine)increasestheheartrateof 97, 429-433. 81, 285-292. J. Neurobiol. Physiol. Entomol.

mol : genestructure,expression andeffect onmosquito Anopheles gambiae 21, 343-355. 29, 214-225. l . NewYork, NY: Oxford UniversityPress. − The Insects:StructureandFunction (1985). Electrophysiologicalinvestigation ofthe 1 (2013). Head.In New York, NY: Springer-Verlag. (2012). Proctolinintheantennalcirculatory phosphate buffer (pH (2005). RoleoftheneuropeptideCCAP in (2000). Circulatoryorgansofabdominal 64, 259-274. μm thicknesswerecutusingaglass and itsreactionstoproctolin. 37, 160-170. (2010). Structuralmechanicsofthe (2010). Contractionoftheventral . Nature The Insects:Structureand (2013). Circulatorysystem, (2012). 464 Science , 66-71. (2009). Harmonic 7.0) for2 (2010). Odorant Ecological and J. Exp.Biol. 323 J. Biomed. Anopheles (ed. S.J. J. Insect , 1077- (2012). (2013). (2010). h. T akr .J,Ppianu .adHle-y,L. Holden-Dye, and S. Papaioannou, R.J., Walker, si .T,Cag .Y,Le .K,Ci .T,Yn,K . i,L . u .T and J.T. Wu, L.Y., Lin, K.M., Yang, T. T., Chi, C.K., Lee, F. Y., Chang, M.T., Tsai, Pass, ig .G n ile,J.F. Hillyer, and J.G. King, as . pr,G,Arcl,H,Buan .adPnln H. Penzlin, and E. Baumann, H., Agricola, G., Sperk, G., Pass, aensr,G,Vgoa . atc,D . mn,J . culc,A .and A.D. McCulloch, J.H., Omens, D.U., Bartsch, C., Vignola, G., Paternostro, ag . un . omr . u .adOor K. Ocorr, and X. Wu, R., Bodmer, W., Yuan, M., Tang, utr,R. Hustert, L.E. delaParte, and T. Y. Estévez-Lao, J.F., Hillyer, ig .G n ile,J.F. Hillyer, and J.G. King, as G. Pass, S.M. Reppert, and R.J. Gegear, C., Merlin, G. Pass, and S. M. Matus, Frasch, and S. Sénatore, S., Zaffran, P. C., Lo, B. Stay, and K. Chan, A.B., Lange, A.J. Klowden, as . eee-rn,B . el .adPat J. Plant, and M. Merl, B.A., Gereben-Krenn, G., Pass, G. Pass, u,B .adShit J.M. Schmidt, and B.D. Sun, s,M .adW,W. J. Wu, and M.H. Hsu, oe,J.C. Jones, as G. Pass, W. G. Bendena, and A.B. Lange, I., Orchard, H. Matsuo, and W. S. Neckameyer, lm,K. Sláma, R. Predel, lwwk,J,Rsnk,G n äsl D.R. Nässel, and G. Rosinski, J., Sliwowska, W. K., Russell, M.R., Brown, J.W., Crim, S.F., Garczynski, S., Neupert, R., Predel, ikr .C,Pts .J,Zo,X,Sh . oa,A n wee,L.J. Zwiebel, and A. Rokas, E., Suh, X., Zhou, R.J., Pitts, D.C., Rinker, ihe .A,Grznk,S . rm .W,Hl,C .adBon M.R. W. Brown, Hertel, and and M. C.A. Richter, Hill, J.W., Crim, S.F., Garczynski, M.A., Riehle, aten .D,Wemn,B . etl . jr .M n ets D.K. Yeates, and K.M. Kjer, R., Beutel, B.M., Wiegmann, M.D., rautwein, the differentiation ofintracardiacvalvesin and RFamide-likepeptides inmetazoa. 618. mutant Drosophilaewithopticalcoherence tomography. C. Yang, Entomol. Advances ininsectphylogenyatthe dawn ofthepostgenomicera. cockroaches (Blattodea,Insecta). phylogenetic aspects. (Hymenoptera: Apidae)andotherHymenoptera:functionalmorphology mosquito circulatoryandimmunesystems. Biol. and sessileimmunecellsinmosquitoes:hemocytemitosisfollowinginfection. a morphologicaldatamatrix. relationships oftheordersinHexapoda:contributionsfromcirculatoryorgansfor Biol. in aneurohemalareawithintheantennalheartofAmericancockroach. Rev. Entomol. 110 Hexapoda: functionalmorphologyandevolutionaryimplications. ed J.C. Reed, aegypti ( behaviors inthecatflea(Siphonaptera:Pulicidae). Gen. Comp.Endocrinol. FMRFamide containingpeptidesontheheartofmosquito Neuroscience maturity, andreproductivestatusinresponsetostress Thomas. Zoomorphology coordinate suncompassorientationinmigratorymonarchbutterflies. metamorphosis. gene isrequiredforremodelingofthedevelopingadultheartandmidgutduring Arch. InsectBiochem.Physiol. antennal pulsatileorganandhindgutmuscleinthecockroach, 365-413. Boston,MA:AcademicPress. Circ. Res. Reviews neuroendocrine system(coelopulse)ofterrestrialinsects. morphology. Peptides tachykinin-related neuropeptidesandproctolinintwocoleopteraninsectspecies. 91-96. the cockchaferbeetle Physiol. a multifunctionalfamilyofstructurallyrelatedneuropeptidesininsects. 1700-1704. sensitivities in Blood meal-inducedchangestoantennaltranscriptomeprofilesrevealshiftsin odor an,J,Rsel .H n aha,R.J. Nachman, and D.H. mosquito Russell, J., Kahnt, Neuropeptides andpeptidehormonesin Periplaneta americana Schistocerca gregaria , 145-164. 11 135 G. (2000). Accessorypulsatileorgans:evolutionaryinnovationsininsects. , 55. (1980). Theanatomyandultrastructureoftheantennalcirculatoryorgansin (Linnaeus). 28, 267-329. , 495-498. (1985). Grossandfinestructureoftheantennalcirculatoryorganin 1 (1991). AntennalcirculatoryorgansinOnychophora,Myriapodaand (2001). Peptidergicneurohemalsystemofaninsect:massspectrometric 22, 209-217. 57, 449-468. Aedes aegypti (1999). Accessoryhemolymphpumpinthemesothoraciclegsoflocusts, , 39-80. 88, 1053-1058. (2008). Extracardiachaemocoelicpulsationsandtheautonomic (1977). (2001). Age-associatedcardiacdysfunctionin The JournalofExperimentalBiology(2014)doi:10.1242/jeb.106708 J. Comp.Neurol. (2011). Observationsofcardiacbeatingbehaviors ofwild-typeand (2013). Circulatorysystems.In 156 Anopheles gambiae 45, 495-518. 96, 77-89. Dev. Biol. , 841-856. Can. J.Zool. The CirculatorySystemofInsects Int. J.InsectMorphol. (L.) (Blattodea:Blattidae). Forskal) (Orthoptera,Acrididae). . J.ProteomeRes. 202 Melolontha melolontha (1997). Contributionstophysiologyoftheantenna-heartin (1999). Antennalcirculatoryorganof 311, 287-296. (2013). Spatialandtemporalinvivoanalysisofcirculating (2001). Off-host observationsofmatingandpostmating , 15-25. Arthropod Syst.Phylogeny 436 (1997). Thestructureoftheantennalheart 24, 79-92. (2012). Infection-inducedinteractionbetweenthe 75, 444-458. J. Morphol. , 363-375. . Proc.Natl.Acad.Sci.USA (2008). Distinctneuralcircuitsreflectsex,sexual (1993). Effect ofallatostatinandproctolinon Invert. Neurosci. npee gambiae Anopheles Drosophila PLoS Pathog. 9 28, 97-109. , 2006-2015. 185 Physiological SystemsinInsects (2001). FMRFamide-relatedpeptides: (2001). Cardioacceleratoryactionof (2009). Antennalcircadianclocks J. InsectPhysiol. , 255-268. J. Med.Entomol. (2010). Neuropeptidomicsofthe L. (Coleoptera,Scarabaeidae). (2009). A reviewofFMRFamide- . Genesis (2007). The (2014). Theroleofpygopusin . Springfield,IL:CharlesC. 64, 165-203. (2014). Myotropiceffects of 9 8 Int. J.InsectMorphol. , 111-153. Drosophila melanogaster Drosophila melanogaster , e1003058. J. Biophotonics . Science 110 52, 19-28. (2006). Phylogenetic Terrestrial Arthropod Anopheles gambiae Diploptera punctata (1988). Octopamine , 8260-8265. 43, 1015-1021. Apis mellifera Drosophila 38, 352-360. Zoomorphology 298 Science Annu. Rev. Adv. Insect , 172-175. 4 (2012). (2013). (2002). J. Exp. 3013 Aedes , 610- Annu. Hand BMC , pp. 325 28, L. . . . . ,

The Journal of Experimental Biology asrhl L.T. Wasserthal, asrhl L.T. Wasserthal, 3014 RESEARCH ARTICLE ag . i,Y . u . wn .W,Pts .J,VnLo,J . akn W. Takken, J.J., Van Loon, R.J., Pitts, H.W., Kwon, T., Lu, Y. T., Qiu, G., Wang, 111-129. in hemolymphtransportandtrachealventilation. pulsatile organintheblowfly machaon Comp. Physiol. 974. expressed inthermosensitivesensillaoffemaleantennae. L.J. Zwiebel, and L. revealedbycontactthermographyandphotocellmeasurements. 139 (1999). Functionalmorphologyoftheheartandanewcephalic (1980). Oscillatinghemolymphcirculationinthebutterfly , 145-163. (2009). AnophelesgambiaeTRPA1 isaheat-activatedchannel Calliphora vicina (Diptera: Calliphoridae)andtheirroles Int. J.InsectMorphol.Embryol. Eur. J.Neurosci. 30, 967- Papilio 28, J. onk . ase,K n ihl,R. Nichols, and K. Paisley, E., Zornik, G. Pass, and M. Togel, C.S., Wirkner, L.T. Wasserthal, esls .J n omr R. Bodmer, and R.J. Wessells, in Drosophila valves and‘venous’ channels. circulation intheanteriorbodymediatedbyanewlydiscoveredpairofostial modulate Minelli, G.BoxshallandFusco),pp.343-391.Heidelberg:Springer. Arthropod BiologyandEvolution:Molecules,Development,Morphology Drosophila The JournalofExperimentalBiology(2014)doi:10.1242/jeb.106708 . Biotechniques (2007). heart rate.Peptides Drosophila 37, 58-60,62,64passim. J. Exp.Biol. (2004). Screeningassaysforheartfunctionmutants flies combineperiodicheartbeatreversalwitha (2013). Thearthropodcirculatorysystem.In (1999). Neuraltransmittersandapeptide 20, 45-51. 210 , 3707-3719. (ed. A.

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