Fireflies Thwart Bat Attack with Multisensory Warnings


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Fireflies Thwart Bat Attack with Multisensory Warnings

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Title:
Fireflies Thwart Bat Attack with Multisensory Warnings
Series Title:
Ecology
Creator:
Leavell, Brian C.
Rubin, Juliette J.
McClure, Christopher J. W.
Miner, Krystie A.
Branham, Marc A.
Barber, Jesse R.
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English

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Subjects / Keywords:
Warning Signals ( local )
Bioluminescing Fireflies ( local )
Eptesicus Fuscus ( local )
Photinus Pyralis ( local )
Echolocating Bats ( local )
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serial ( sobekcm )

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Abstract:
Many defended animals prevent attacks by displaying warning signals that are highly conspicuous to their predators. We hypothesized that bioluminescing fireflies, widely known for their vibrant courtship signals, also advertise their noxiousness to echolocating bats. To test this postulate, we pit naïve big brown bats (Eptesicus fuscus) against chemically defended fireflies (Photinus pyralis) to examine whether and how these beetles transmit salient warnings to bats. We demonstrate that these nocturnal predators learn to avoid noxious fireflies using either vision or echolocation and that bats learn faster when integrating information from both sensory streams—providing fundamental evidence that multisensory integration increases the efficacy of warning signals in a natural predator-prey system. Our findings add support for a warning signal origin of firefly bioluminescence and suggest that bat predation may have driven evolution of firefly bioluminescence.
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Ecology, Vol. 4, no. 8 (2018-08-22).

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K26-05052 ( USFLDC: LOCAL DOI )
k26.5052 ( USFLDC: LOCAL Handle )

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ECOLOGYCopyright2018 TheAuthors,some rightsreserved; exclusivelicensee AmericanAssociation fortheAdvancement ofScience.Noclaimto originalU.S.Government Works.Distributed underaCreative CommonsAttribution NonCommercial License4.0(CCBY-NC).FirefliesthwartbatattackwithmultisensorywarningsBrianC.Leavell1*,JulietteJ.Rubin1,ChristopherJ.W.McClure1,2,KrystieA.Miner1, MarcA.Branham3,JesseR.Barber1Manydefendedanimalspreventattacksbydisplayingwarning signalsthatarehighlyconspi cuoustotheirpredators.We hypothesizedthatbioluminescingfireflie s,widelyknownfortheirvibrantcourtshipsignals,alsoadvertisetheirnoxiousnesstoecholocatingbats.Totestthispostulate,wepitnavebigbrownbats( Eptesicusfuscus)againstchemicallydefended fireflies( Photinuspyralis )toexaminewhetherandhowthesebeetlestransmitsalientwarningstobats.Wedemonstrate thatthesenocturnalpredatorslearntoav oidnoxiousfirefliesusingeithervision orecholocationandthatbatslearnfaster whenintegratinginformationfrombothsensorystreams — providingfundamentalevidence thatmultisensoryintegration increasestheefficacyofwarningsignals inanaturalpredator-preysystem.Ourfindingsaddsupportforawarningsignal originoffireflybioluminescenceandsuggestthatbatpreda tionmayhavedrivenevolution offireflybioluminescence. INTRODUCTIONPronouncedadvertisementofidentityandlocationisacommonstrategyofdefendedanimals(1 ).Thesebrazenpreywarnpredatorsofthe costofattackwithclearandsalientsignals.Vividpoisondartfrogswarn visuallyspecializedpredatorsoftoxinsstockpiledintheirskinglands ( 2 , 3 ),seaslugsreekodorstoalertolfactory-specialistpredatorsof stingingnematocysts( 4 ),andtigermothsproducesonar-triggered burstsofultrasoundtowarnecholocatingbatsofnoxioustaste( 5 , 6 ). Giventhedemonstratedefficacyofunisensorywarnings,whydodefendedanimalsroutinelyintegratemultiplesensorychannelsintheir displays?Arecentframeworkoffunctionalhypotheses,adaptedfrom workoncomplexcommunicationsignals( 7 ),suggestsmanywaysin whichmultisensorywarningsi gnalsmaybeadvantageous( 8 ).Experimentalevidencefromartificialandseminaturalparadigmspointsto severalfunctions,suchasenhancedavoidancelearning( 9 ).Yet,demonstrationoftheseadvantagesinnaturalpredator-preysystemsis lacking,despitetheirpotentiallyp rofoundimplicationsforthetempo andmodeofspeciationandextinction( 10 ). Here,wepresentevidencethattoxic,bioluminescentfireflies (Lampyridae)transmitmultisensorywarningsignalstoecholocating bats.Predatorsacrosstaxashowintenseaversionstothesenoxious beetles( 11– 17 ),somedyingwithinanhourofingestion,likelyfrompoisonoussteroidalpyrones(lucibufagins)( 18 ).Previouswork( 15 , 19 )and ourownpalatabilityexperimentsindicatethat Photinus spp.firefliesare markedlydistastefultobats(n =3bats;7fireflies,completerejection;see movieS1forstrongaversivebehavi orssuchasrapidheadshakingand coughing).Itisthenunsurprisingthatfirefliesarealmostentirelyabsentfrombatdiets( 15 , 20 )[thesingledocumentedexception( 21 )is disputedbecauseofmethodologicalissues( 22 )],despiteabundant opportunitiesforpredatorandpreytointeract( 15 , 22 ).Yet,itisunknownwhetherandhowbatsdiscriminateprofitablepreyfrom fireflyfoe.RESULTSANDDISCUSSIONTofirsttestourhypothesisthatfireflieswarnbatsoftheirchemicaldefense,wepitfree-flying,bioluminescingfireflies( Photinuspyralis ) againstthreenavebigbrownbats( Eptesicusfuscus )inadark,anechoic flightroomfor1to4daysandfilmedtheirinteractionsusingthree high-speedcameras.Bayesianmixed-effectslogisticregressionofbat capturebehaviordemonstratesstrongsupportforlearnedavoidance ofthesefireflies[intact b control b = 4.691;95%credibleinterval (CI), 8.53to 1.969;Fig.1andtableS1].Allbatscapturedatleast onefireflyonthefirstnightofpresentation(range,1to3;threetofour firefliespresentedpernight,comprising25%oftotalpreyforall experiments)andsubsequentlydroppedthebeetle.Overjustafew nights,thebatsinourexperimentlearnedtoavoidthesechemically protectedanimals(movieS2).Controlpalatable,nonbioluminescent scarabbeetles,andpyralidmoths( 75%presentationratethroughout) pitagainstbatsduringthesameforagingnightswereconsistently captured(98.7%capturesuccess; n =242;Fig.1A).Sinceatleastthe 19thcentury,naturalistshavehypothesizedthatfireflieswarnbatsof their “ offensiveness ” ( 23, 24),andhere,weprovidedefinitiveevidence. Asanadditionaltest,wepittwobat sthatlearnedtoavoidfireflies against “ darkened ” firefliesthatwereunabletoproducelightforanotheronetotwonights.Wethoroughlypaintedfireflybioluminescing organs,includingadjacentabdominalsegments,toblockalllightproduction.Onebatcapturedalldarkenedfireflies( n =2)andcontrols( n = 2),demonstratingthatbioluminescencewasusedforavoidance learningbythisbat.Thisobservationisinlinewithmountingevidence thatbatsintegrateecholocationwithvisionwhenforaging( 25, 26)and thatinsectivorousbatretinascontainphotoreceptorssensitivetothe dominantwavelengthsoffireflybioluminescence( 27 , 28 ).Asecond batavoidedalldarkenedfireflies( n =3)andcapturedallcontrols( n = 6),indicatingthatanothersourceofinformationwassufficientforthis battodiscriminatenoxiousfireflies. Totestwhetherbatscanlearntodiscriminatefireflieswithoutbioluminescence,wepitdarkenedfirefl iesagainstfouradditionalnavebig brownbatsfor1to4days.Thesebatslearnedtoavoiddarkenedfireflies (darkened b control b = 2.306;95%CI, 5.783to 0.302;Fig.1), althoughtheylearnedataslowerra teandtoalesserdegreethanthose pitagainstfree-flying,bioluminescingfireflies(intact b darkened b = 2.385;95%CI, 5.222to 0.023;Fig.1).Howwerebatsabletodifferentiatecontrolpreyfromdarkenedfireflies?Olfactoryinformationisa possibili ty.However,hereandinthreeotherstudies( 15 , 17, 19 )that performedpalatabilitytestsbypresentingnonflyingbatswithchemicallydefendedfireflies,thesepredatorsshowednoabilitytousesmell torejectthesenoxiousbeetles,insteadwaitingtorejectuntiltastingthe prey.Determiningwhetherbatsusevolatilecomponentsoffireflychemistrytoavoidfirefliesinshort-durat ion,high-speedae rialinteractions 1DepartmentofBiologicalSciences,BoiseStateUniversity,Boise,ID83725,USA.2ThePeregrineFund,Boise,ID83709,USA.3DepartmentofEntomologyand Nematology,UniversityofFlorida,Gainesville,FL32611,USA. *Presentaddress:DepartmentofBiologicalSciences,PurdueUniversity,WestLafayette, IN47906,USA. Correspondingauthor.Email:jessebarber@boisestate.edu SCIENCEADVANCES | RESEARCHARTICLELeavell etal ., Sci.Adv. 2018; 4 :eaat660122August2018 1of6 on April 7, 2020 http://advances.sciencemag.org/ Downloaded from

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requiresadditionalwork.Insectivorousbatshavesomeofthesmallest olfactoryorgansinmammalsandinvestsignificantlylessinolfactory tissuesthanbatswithfrugivorousormixed(frugivorousandinsectivorous)diets( 29 ).Understandingtheroleofolf actioninbat-insectinteractionsisanimportantfrontier. Alternatively,batsmayhavelearn edtoavoiddarkenedfirefliesby extractingacousticinformation.Batsmightuseecho-derivedshape, size,andtexture( 30 , 31 ),theamplitudeandspectralmodulationimposedbywingbeatrateonthereturningechostream( 32, 33 ),oroverall three-dimensionalflightpathstoassociatefireflieswithnoxiousness. Firefliesflywiththenonchalanceofachemicallyprotectedinsectina slow,nonerraticflightpath.Poulton( 34 )reportedthistypeofmovementbehaviorin1890toaccompanyv isualwarningsignalsindiurnal moths,andithassincebeenreportedinchemicallyprotectedbutterflies ( 35)andnocturnaltigermoths( 36 ).Previouslaboratoryworkwitha rotatingdevicemimickingaflutteringinsecthasshownthatbigbrown batscandiscriminatethedifferentwingbeatratesintherangeofthe insectswepresentedtobatshere(meanSE;firefly,63.31.9Hz, n =4;scarab,93.31.9Hz, n =4;pyralidmoth,44.3.8Hz,n =4; seeMaterialsandMethods)( 37 , 38 ).Wingbeatrateshavebeenimplicated asacomponentofwarningsignalsinsystemswithvisuallyguidedpredators.Convergenceinwingbeatrateswithinaposematicmimicry groupsoccursinheliconiinebutterflies( 39, 40),aswellasindamselfly mimicsofithomiinebutterflies( 41 ).Consideringtheprevalenceof chemicallyprotectedflyinginsects( 8 ),therolesofflight-basedwarning signals,transducedbyvision,echolocation,orpassivelisteningarefertile groundsforfutureresearch,particularlyasitappliestomultisensory signaling. Tofurtherparsetherolesofbioluminescentandechoicinformation inavoidancelearning,wepresentedimmobilefirefliestoonebatona monofilamenttether,renderingthebeetlesflightlessbutpreserving theirabilitytoproducetheirbioluminescentdisplays.Wegentlypushed thesetetheredpreyintoaswinging motiontostimulatelightproduction.Asacontrolforthisexperiment,wealsopresentedsize-matched, flightlesspyralidmothsandscara bbeetlesinthesamependulummotiononthetether.Thebatlearnedtoavoidthesefireflies(tethered b control b = 2.085;95%CI, 4.412to 0.088;Fig.1),althoughtoalesser degreethanfree-flying,bioluminescingfireflies(intact b tethered b, 2.607;95%CI, 6.333to0.228;96.2%probabilitythatdifferencebetweentreatmentsisnot0;Fig.1B)butcomparabletofree-flying, darkenedfireflies(tethered b darkened b =0.222;95%CI, 2.006 to3.534;Fig.1B).Thus,althoughunisensoryfireflydisplaysproduced avoidancelearning,theadditionofflightorbioluminescencetoeither unisensorydisplaypotentiatedthis effect.Toourknowledge,thisisthe firstexampleofamultisensorywarn ing displayenhancingavoidance learninginanaturalpr edator-preysystem. Usingecholocationasawindowintoreal-timeinformationprocessing,wealsoinvestigatedsonarbehaviorbybatsduringtheseexperiments.Momentsbeforeacaptureattempt,bigbrownbatsactively updatetheiracousticscenesbyemittingsonarcriesatarapidrate. Thefirstpartofthis “ buzz ” behavior(buzzI)giveswaytobuzzIIjust beforecapture,whereinrapidcryr atecontinues,butthefundamental frequencyofthecriesisloweredbyanoctave.Bothhighcallrateand loweredfrequencyareputativeadaptationsfortrackingmovingprey fromshortdistances( 42 ).Here,batsshortenedtheirbuzzIIdurations astheygainedexperiencedwithfree-flying,bioluminescingfireflies (intact b control b = 0.063;95%CI, 0.125to 0.004;Fig.2,Aand B,andtableS2).Duringnights3and4,batsoftendidnotbuzzthese fireflies(Fig.2C).BatsfacingdarkenedortetheredfirefliesdidnotreducebuzzIIdurationovertimeandcontinuedextractingadditional informationfromthesepreythroughoutourexperiments(darkened b control b = 0.025;95%CI, 0.073to0.022;tethered b control b = 0.021;95%CI, 0.106to0.065;Fig.2,AtoC).Inaddition,during interactionswithallprey,batsdidnotaltertheirbuzzIdurations(fig.S1 andtableS3).Thesesonardatarein forceourbehavioralfindingsthat preysignalingthroughmultiplesensesenhancespredatordiscriminationlearninginthissystem. Fig.1.Batslearntoavoidunisensoryandmultisensoryfireflywarnings. ( A )Probabilitythatbatscapturefireflytreatmentsandcontrolpreyoversuccessive nights.Line,mean;shading,95%CI.( B )Comparisonsofavoida ncelearningbetween preytypes(depictedassilhouettesonright;scarabbeetlesilhouetterepresents pooledscarabbeetleandpyralidmothcontroldata).Batslearntoavoidallfirefly treatments.Avoidancelearningisdefined hereasaslopelessthanzeroincomparisonsoffireflytreatmentsandscarabcontro ls.Forcomparisonsoffireflytreatments, negativevaluesindicatefasteravoidancelearningofpreyshownonleft.Thereisno differenceinlearningratesbetweenunisensory(tetheredversusdarkened)treatments.Intactfirefliesemittingmultisensorywarningsinducefasteravoidance learningcomparedtoeitherunisensorytreatment.Circle,median;thickline,68% CI(whichis1SDunderanormaldistribution);thinline,95%CI. SCIENCEADVANCES | RESEARCHARTICLELeavell etal ., Sci.Adv. 2018; 4 :eaat660122August2018 2of6 on April 7, 2020 http://advances.sciencemag.org/ Downloaded from

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Thedatawepresentheretriangulateontoaclearaposematicsignalingrelationshipbetweenbatsand fireflies.Paststudiesandanecdotal observationshavedescribedbatsthataresometimesattractedtoartificialfirefly-likeluresandevenattackfireflies( 15, 43).Inlightofourfindings,wecontendthatthesedatamayre flect(i)observationsofjuvenile, inexperiencedbats;(ii)artificialluresthatdonotaccuratelyrecreatethe echoicandvisualpropertiesofrealfir efliesandthusinitiatebatexploratorybehavior;orperhaps,(iii)bats peciesthatdifferintheirsensory abilities(44 )ortoleranceoffireflychemicaldefenses( 6 ). Ourdataalsorevealintriguingaspectsofbatsensorybiology.For batswithafrequency-modulatedsonarstrategy,suchasthebigbrown batsstudiedhere,theuseofecho-derivedinformationinanecologically relevantpreydiscriminationtask isanovelobservationthatexpands ourunderstandingofbatperception.Further,weprovideevidencethat visualaposematicsignalsaresufficientfortransmittingwarningsof chemicaldefensetobats,openingupthepossibilitythatotherprotected insects(forexample,tigermoths)us ethissensorychanneltocommunicate withthesenocturnalpredators.Ourexp erimentsprovideempiricalsupport forseveralfunctionalhypothesesdevelopedformultisensorywarnings ( 8 ).Regardingsignalcontent,bioluminescenceandechoiccomponents separatelygeneratedavoidancelearning,indicatingthattheylikelyprovide redundantinformationofpreydefensetobats.Thedeterrencecapabilityof individualsensorycomponentsmaybeparticularlyimportantforfirefly survivalifbatsinteractwithfirefliesbetweenflashes[whichare0.4to 8.0sapartfor Photinus spp.;( 45, 46 )]orifthelightenvironmentreduces theflash ’ scontrastagainstthebackground.Theseredundantunisensory componentsmayalsobeseparatelytargetingnotonlydistincttaxawith divergentsensorysystems[forexample,( 47 )]butalsointraspecificperceptualvariationwithinbats,aswehavedocumentedhere( 7 ).Itisclear thattheintegrationofvisualandechoicsignalcomponentsbyfireflies enhancesavoidancelearningbybats,resultinginlearningratescomparable tothoseelicitedbyultrasonicallyadvertising,distastefultigermoths( 5 , 6 ). Theevolutionaryimplicationsofourdataarenontrivial.Larvalbioluminescenceisashared,derivedcharacteristicofallfireflies,andthere isexperimental( 12 , 14 )evidencethatthese “ glow-worms ” advertise chemicaldefensestopredators.Inaddition,currentphylogenies Fig.2.Batsonarbehavior. ( A )Thedurationofbats ’ finalecholocationphase(buzzII)presentedagainstsuccessi venightsofinteractionswithd ifferentfireflytreatments. Line,mean;shading,95%CI.( B )Between-preycomparisonsofchangesinbuzzIIdurations.Lowervalues indicategreaterreductionofbuzzdurationovertimeforpreyonleft. Comparedwiththecontrol,batsreducedtheirbuzzIIdurationastheylearn edtoavoidintactfireflies.Interactionswithdarkenedortetheredfire fliesdidnotaffectbuzzII durations.( C )Examplespectrogramsofecholocationbehaviordu ringthelastnightofinteractionswitheachfireflytreatment.BatsenteredbuzzIIduringinter actionswith tetheredanddarkenedfireflies,butnotforinteractionswithintactfirefly.Eachspectrogramdepictsthefinal1sofaninteraction.Pseudocolor ationillustratesintensity gradientswithinasinglespectrogrambuti snotstandardizedacrossspectrograms. SCIENCEADVANCES | RESEARCHARTICLELeavell etal ., Sci.Adv. 2018; 4 :eaat660122August2018 3of6 on April 7, 2020 http://advances.sciencemag.org/ Downloaded from

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supportthehypothesisthatadultsofth eancestralfireflylineagedidnot usebioluminescenceinsexualcommunication,insteadrelyingonpheromonesformating( 48 , 49).Mostfirefliesthatuseonlypheromonesfor sexualcommunicationarediurnal( 48 ).Itfollowsthatasancestral lineagesoffirefliesshiftedtonocturnalactivity,theyexperiencedheavy selectivepressurefrombatsandlikelyotherpredators[forexample, spiders( 16 )],whichdrovetheevolutionofadultbioluminescence. Wepostulatethateachtimebioluminescencehasevolved[atleastsix timesinfireflies( 49 )withatleasttwoadditionaloriginsinnonfirefly beetles],selectivepressuretobroadcastwarningsofchemicaldefense initiallyshapedthesignal,whichthenwassecondarilyco-optedfor mating.ArecentdatinganalysisofColeoptera( 50 )placestheorigin offirefliesat~75millionyearsago(MYA),justbeforetheemergence ofbats~65MYA( 51).Forfirefliesonthewinginthenightsky,the predatorstoavoidhavebeen,andcontinuetobe,bats.Wepredictthat atime-calibratedfireflyphylogenywillrevealthatbioluminescence emergedwithbatsandthatbatsmayhaveinventedfireflies.MATERIALSANDMETHODSBehavioralexperiments Wemist-nettedeightbigbrownbats( E.fuscus )insouthwestIdahounderIdahoDepartmentofFishan dGamepermit#110615.Allbatswere housedandcaredforatBoiseStateUni versityfollowingtheInstitutionalAnimalCareandUseCommittee protocol#006-AC14-014andcare protocolsestablishedbyLollarandSchmidt-French(52).Thelightregimewas10-hourdark/14-hourlight.Beforebehavioraltrials,we trainedbatstohuntgreaterwaxmoths(Galleriamellonella )suspended fromamonofilamenttether.Wild,malefireflies( P.pyralis )were collectedinLehighCounty,PAandDouglasCounty,KS,whilecontrol scarabbeetles( Callistethusmarginatus and Dyscinetusmorator)were collectedinAlachuaCounty,FLinJunetoJuly2015.Bigbrownbats occurthroughoutNorthAmericaandareconsidered “ nonmigratory ” ( 53),whilefirefliesthatflashasadultsaregenerallyonlyfoundeastof theRockyMountains.Wethusassumedthatthebatsusedinthese experimentswerenavetoflashing,noxiousfireflies. Weconductedbat-fireflybehavioralexperimentsinasound-attenuated flightroom(6.85.63.9m)illuminatedbyWildlifeEngineeringinfraredlight-emittingdiodearraysandred-filteredlighting.Torecord bat-preyinteractions,wecapturedstreamingvideofromthreehighspeed,infrared-sensitivecameras[Baslerscout,100framespersecond (fps)]usingaNationalInstrumentsPCIe-8235GigEVisionframegrabberandacustomizedLabVIEWprogram.Simultaneously,werecorded echolocationbehaviorusingfourultrasonicAvisoftmicrophones[three CM16,dB(Z),20to140kHz;oneUS GElectretUltrasoundMicrophone,9dB(Z),20to120kHz]connectedtoafour-channelAvisoft UltraSoundGate416H(samplingat300kHz)viaXLRcablesand recordingtoadesktopcomputer runningAvisoft-RECORDER software.Wemountedmicrophonesontheceilinginthecenterofthe room,placingeachCM16microphone1.5mapartfromandencircling thecentralUSGElectretmic.Forallinteractions,wesynchronizedaudio andvideorecordingsbytriggering bothwithaNationalInstruments 9402digitalI/Omodule. Wepresented P.pyralis firefliestobatsinoneofthreetreatments:(i) intact,free-flying,andflashing( n =3bats);(ii)intact,free-flying,and nonflashing( n =4bats);and(iii)intact,tethered,andflashing( n = 1bat).Tooccludebioluminescencefortreatment#2,wepaintedallbioluminescentabdominaltergaandad jacentterga,blackorred(Testors EnamelPaints “ GIBlack” and “ RedCherry, ” respectively).Bothcolored paintseffectivelyblockedbioluminescenttransmittance.Wealso presentedpaintedscarabs( n =2)tobatstocontrolfortheeffectofpaint. Onceitwasapparentthatpaintedscarabswerecaughtandingested withvigor,wethenpresentedunpai ntedscarabstominimizebatexposuretotheenamelpaints.Flightcontrolswerewaxmoths,scarab beetles,oracombinationofthetwo.Free-flyingpreywerehand-released, whiletetheredinsectsweresuspendedfroman~75-cm-longmonofilamentlineattachedtotheceiling.Toavoidcontaminatingcontrol prey,experimenters ’ handsandthetetherwerewashedfollowingcontactwitheachfirefly.Atrialbeganoncewereleasedthepreyandwe allowedbatstohuntfor1minafterrelease.Weassessedcapturebehaviorduringexperimentsandconfirmedcapturesbyreviewingrecordings ofallinteractionsfromallthreecameras. Echolocationanalysis WeanalyzedaudiofilesusingAvisoft-SASLabProsoftware.Weinspectedallfouraudiochannels,beginning900msbeforetheselected interaction,andanalyzedthechannelwiththehighestsignal-to-noise ratio[following( 54 )].Sincefree-flightinteractionsoccurredatunpredictablelocationsaroundtheroo m,recordingsofbatsecholocating faroroff-axisfromthemicrophonesresultedinlowsignal-to-noiseratiosandwerethusdiscardedfromtheanalysis.Whenallecholocation c allsofaninteractionweredistinctfrombackgroundnoise,wemanuallyextractedbuzzIandbuzzIIdur ationsfromtheoscillogram.Wedid notassessfrequencycomponentsofthecalls,asthevariablepositioning ofthebatsinrelationtothemicropho neswouldlikelyleadtoinaccurate frequencydataduetotheeffectsofoff-axisdirectionalityandatmosphericattenuation. Wingbeatrates Todeterminepreywingbeatrates,wemanuallycountedthenumberof wingbeatsovera0.1-to0.5-sinterva lusinghigh-speedvideorecordings offlyingprey(Edgertonic,monochro me,500fps,19201080resolution). Palatabilityexperiment Weassessedthepalatabilityof P.pyralis tothreebats( n =7totalinteractions)alongwithscarabandwaxmothcontrols( n =8totalinteractions;movieS1).Toavoidcontaminatingcontrolinsects,wepresented preytoeachbatwithforceps,whichwewashedfollowingcontactwith fireflies.Wescoredpalatabilityfrom0to100%inaccordancewith methodsoutlinedbyHristovandConner( 55).Weaddedthefollowing scoresbasedonhowmuchofthepreywasconsumed:head,1;thorax,2; abdomen,3.Foreachprofferedinsect,thesummedscorewasthen dividedby6andmultipliedby100toob tainthepalatabilitypercentage. Statisticalanalyses WeusedgeneralizedlinearmixedmodelsfitunderaBayesian frameworktoexaminedifferencesbetweenslopesoftreatmentgroups inrelationtonightoftheexperiment(56 , 57).Todetermin edifferences betweenslopesoftreatments,weusedmodelsincludingfixedmain effectsoftreatmentandnightofexperimentandtheirinteraction. Wesubtractedsamplesfromposteriordistributionsofslopesofeach treatmenttoobtainposteriordistribu tionsofdifferencesbetweenslopes oftreatments.Wedetermineddifferenceswhen95%CIsfromtheresultingdistributiondidnotintersectzero.Forfurtherinference,wealso examinedtheprobabilitythatagivenparameterwasdifferentfrom0. Wepooleddatafromallcontrols(scarabbeetlesandpyralidmoths)for thebehavioralanalysisandusedscarabbeetlesforthecontrolinthe echolocationanalysis. SCIENCEADVANCES | RESEARCHARTICLELeavell etal ., Sci.Adv. 2018; 4 :eaat660122August2018 4of6 on April 7, 2020 http://advances.sciencemag.org/ Downloaded from

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Allmodelsincludedrandominterceptsandslopesforindividual bats.Weusedabinomialdistributionandlogitlinkformodeling capturerateandanormaldistributionandidentitylinkformodeling durationofecholocationphases(b uzzIandbuzzII).Weimplemented themodelinJAGS(JustAnotherGibbsSampler)version4.2.0( 58 ) usingthejagsUIpackageversion1.4.4( 59)andRversion3.2.3( 60). Weranthreechainsfor50,000iterationsafter10,000burn-ins andathinningrateof2,yielding60,000drawsfromthejointposterior.Weusedstandardweaklyinformativepriors( 57 )andvisually assessedtraceplotsandusedt heGelman-Rubinstatistic( 61 )tocheck forconvergence.Allmodelshadgoodmixingofchainsand ^ R < 1 : 1for allparameters. SUPPLEMENTARYMATERIALSSupplementarymaterialforthisarticleisavailableathttp://advances.sciencemag.org/cgi/ content/full/4/8/eaat6601/DC1 Fig.S1.ComparisonsofthechangeinbuzzIdurationbetweenfireflytreatmentsandcontrol scarabbeetles. MovieS1.Fireflypalatability. MovieS2.Fireflyavoidance. 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Fireflies thwart bat attack with multisensory warnings Brian C. Leavell, Juliette J. Rubin, Christopher J. W. McClure, Krystie A. Miner, Marc A. Branham and Jesse R. Barber DOI: 10.1126/sciadv.aat6601 (8), eaat6601. 4 Sci Adv ARTICLE TOOLS http://advances.sciencemag.org/content/4/8/eaat6601 MATERIALS SUPPLEMENTARY http://advances.sciencemag.org/content/suppl/2018/08/20/4.8.eaat6601.DC1 REFERENCES http://advances.sciencemag.org/content/4/8/eaat6601#BIBL This article cites 51 articles, 6 of which you can access for free PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions Terms of Service Use of this article is subject to the is a registered trademark of AAAS. Science Advances York Avenue NW, Washington, DC 20005. The title (ISSN 2375-2548) is published by the American Association for the Advancement of Science, 1200 New Science Advances License 4.0 (CC BY-NC). Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial Copyright 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of on April 7, 2020 http://advances.sciencemag.org/ Downloaded from


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