© 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,327-330doi:10.1242/jeb.092833 Received 20June 2013;Accepted25September2013 § University, BowlingGreen,OH43403-0208,USA. BiologicalSciences, BowlingGreenState of *Present address:Department OH 45221-0006,USA. Cincinnati, Cincinnati, BiologicalSciences, University of of Department The larvaeof KEY WORDS:,Distanceestimation,Predator, Vision positioned photoreceptors. activation ofspecificsubsetstheirmany-tieredandpeculiarly distances, possiblymediatedbytheobject–distance-dependent likely employanunusualmechanismtoaccuratelydetermineprey ~4.5 larvae consistentlystruckourartificialtargetsfromadistanceof size bypresentingdifferent preysizes.Despitetheseconstraints, larvaesimplyapproachedtheirtargetsbasedonknownprey with anartificiallymovingprey, andexcludedthepossibilitythat stereopsis byocclusion,confoundedpossiblemotionparallaxcues are availabletothem.Specifically, weexcludedbilateralbinocular at artificialprey, evenifnoneofthesetypicaldistanceestimationcues demonstrated that from theimagesizeofknownobjects.Usingabehavioralassaywe from well-controlledtranslationalmovements(motionparallax)or are viewedfromdifferent angles,frominformationthatisobtained derivedistanceinformationfromdisparitiesofimagesthat are facedwithistoaccuratelyassessthedistanceoftheirprey. Most the divingbeetle One ofthebiggestchallengesthatpredators,suchaslarvae Kevin Bland*,NicholasP. Revetta Unilateral rangefindingindivingbeetlelarvae SHORT COMMUNICATION of Biological Sciences, Eastern Kentucky BiologicalSciences, Eastern University,of Richmond, KY40475,USA. INTRODUCTION ABSTRACT rarely beenconsidered.We herepresentevidencethatthelarvaeof situated atdifferent distances behindthelens,amechanismthathas differential activationofgroups ofphotoreceptorcellsthatare Specifically, distanceinformationcouldbederivedfromthe (Blest etal.,1981;CollettandHarkness,1982;Nagata2012). feature thatinprinciplecanbeusedtoobtainrange-finding cues of theirprincipaleyesconsistmanytiers,anorganizational the larvaeperformaballisticstriketocaptureprey. Theretinas 2007). Atcloserange,scanningmovementscease,andshortly after movements toscantheirfrontalvisualfield(Buschbecket al., slowly approachtheirtarget whileperformingdorso-ventralpivoting bring thepreyintovisualfieldoffourprincipaleyesandthen extremely narrowverticalvisualfields.Whilehunting,thelarvae a bifocallens(Stowasseretal.,2010)andmultipleretinaswith (Maksimovic etal.,2009;Mandapaka2006).Theseeyeshave organized, tubulareyes(E1andE2)oneachsideoftheirhead or midgelarvae.Theirheadsarecharacterizedbytwocomplexly successful visuallyguidedpredatorshuntingpreysuchasmosquito Author ([email protected]) for correspondence mm. Basedonthesefindingsweconcludethat marmoratus Thermonectus marmoratus T. marmoratus larvae continuetoaccuratelystrike ‡ Present address:Department ‡ (Coleoptera: ), , AnnetteStowasserandElkeK.Buschbeck Gray 1831arehighly T. marmoratus an unusualalternativemethod. distance oftheirtarget, wehereproposethattheselarvaeareusing excluded, butlarvaeneverthelesswereabletocorrectlygaugethe experimental designtypicalmechanismsofdistanceperceptionwere occlusion ofrelevanteyesononesidetheirhead.Asinour object distance,evenafterexcludingstereopticcuesthrough demonstrated thatlarvaemaintainedtheirabilitytoaccuratelygauge the sizeofpresentedprey. Inasecondsetofexperiments,we the case,larvaeshouldstrikefromdistancesthatareindependentof simply imagesize.Specifically, wehypothesizedthatifwere effective ingaugingthedistanceofatarget fromcues other than cues, wefirsttestedwhether a well-controlledartificialstimulusthatconfoundedmotionparallax dragonflies (Olberg etal.,2005).Usinganexperimentalarenawith locusts (Collett,1978;KralandPoteser, 1997;Sobel,1990)and as mantids(Kral,2012;PoteserandKral,1995;Rossel,1983), last twomechanismshasbeendemonstratedinmultiplespeciessuch parallax (forareview, seeSchwind, 1989).Theimportanceofthe of anobjectknownsize,fromstereopsisandmotion such anunusualrange-findingstrategy. deprived ofcommonlyknownmechanisms,andthuslikelyemploy T. marmoratus side ofthehead.Fig. principal eyesandaventraleye(seeMaterialsmethods)on one target. was significantlyshorterthantwicethestrikedistancetosmaller was notsignificantlydifferent fromthatforthesmallertarget, yetit experiment thiswasnotthecase:strikedistanceforlarger target 1A,however, showsthatinour from abouttwicethedistance.Fig. cue, onewouldexpectthemtostrikeatatarget oftwicethesize were usingtheabsoluteimagesizeastheirprimaryrange-finding distance ofdummypreyindependentlytheobjectsize.Iflarvae We firstaskedwhetherlarvaeareabletoaccuratelygaugethe further awayobjects. Thisdistancecueisparticularly effective ifan in closerobjectsmovingfaster acrossaphotoreceptorarraythan motion parallax,whichrelieson translationalmovementsthatresult severely limitedasdiscussedbelow. commonly usedrange-finding cueswereexcluded,oratleast gauge preydistance,even in anexperimentalsetupwhere significance valuesarebasedontwo-tailedStudent’s shorter thantwicethestrikingdistancetosmallerprey. All eyes-occluded animalstothelarger target remainedsignificantly difference wassmallandnot significant.Thestrikingdistanceofthe to strikeatthetarget from aslightlygreaterdistance,butthis of twicethesize,occludedaswellnon-occludedanimalstended compared withsham-treatedcontrols.Whenchallengedtargets in strikedistancefortheseunilaterallyblindedanimals, when RESULTS ANDDISCUSSION typicallyderivedistanceinformationfromtheimagesize To testforthenecessityof stereopsis,weoccludedthetwo One ofthemostprominentdistance visioncuesininsectsis Taken together, ourresultssuggestthatlarvaecouldaccurately are abletosuccessfullygaugedistancesevenwhen

1B showsthatthereisnosignificantdifference § T. marmoratus larvae indeedare t -tests. 327

The Journal of Experimental Biology 328 ceased scanning shortlypriortostriking.Nevertheless, theywere monotonous portionofthetarget. Moreover, larvaefrequently fields frequentlywerefixated somewherealongthevertically videos suggeststhatshortlybefore striking,theirnarrowvisual and bottom)duringtheirvertical scans.Closeexaminationof leads toanearlyhomogeneous image(withedgesonlyatthetop shape ofourartificialpreyconsisted ofaverticalstreak,which between twoverticalimagepointsatanygiventime.Second, the organization allowseachtier todetectnomorethanthecontrast (see the verticalaxisthereareonlydorsalandventralphotoreceptors in depth(tiering)andalongthehorizontalplane.However, along resolution alongtheverticalaxis.Specifically, theirretinasextend fields (seeMandapakaetal.,2006),severelylimitingimage the principaleyesoftheseanimalshaveextremelynarrowvisual unlikely tohaveprovidednoteworthymotionparallaxcues. First, there areadditionalreasonswhytheseverticalmovements are movements havestrongrotatoryandsometranslatorycomponents, while hominginontheirprey(Buschbecketal.,2007),and these if presentedwithmovingobjects(forareview, seeKral,2012). parallax forstationaryobjects,useotherstrategiesrangefinding this reasonevenmantises,whicharewellknowntorelyonmotion on theretina,leadingtounresolvableambiguities.Presumablyfor because objectmovementelicitsconfoundingtranslationalpatterns ’s abilitytousemotionparallax (Schwind,1989).Thisis were inmotion,anditisknownthatsuchmotionimpairsan minimizing informationonrelativemovement.Second,ourtargets reasons. First,thearena’s background washomogeneous, cues whileapproachingtheirprey?Thisisunlikelyforthefollowing 2012). CouldT. marmoratus image translationcanleadtosystematicstrikedistanceerrors(Kral, horizontally priortostrikingastationarytarget. Exaggeratingtheir 2012). Forexample,aprayingmantiswilltranslateitshead frequently isthecaseforpeeringmovements(Collett,1978;Kral, viewing stationaryobjectsagainstastructuredbackground.This animal hastightcontroloveritsowntranslationalmovementswhile P target). Inbothexperimentstherewasnosignificantdifference (two-tailedStudent’s betweenthestrikingdistanceto largerandsmallertarget basedontheangularextentof illustrate twotimesthestrikingdistancetosmallertarget(thehypotheticaliflarvaewerestrikesolely Fig. SHORT COMMUNICATION on onesideoftheheadareoccluded(two-tailedStudent’s smaller thantwotimesthestrikingdistancetotarget(two-tailedStudent’s >0.05). N Although larvaeperformdorso-ventralscanningmovements

.Secondinstarlarvaetypicallystrikefrom~4.5 1. i.2D, Fig.

indicates thenumberoflarvaethatweretested. Striking distance (mm) 10 12 14 0 2 4 6 8 inset). Therefore,withineachprincipaleye,this A ml agtSmalltarget×2 Small target N=11 larvae thereforeobtainmotionparallax NS Large target N=8

mm, regardlessofthesizetarget. P=0.001 t -test, eyesoccluded N=11 P Harkness, 1982). calculation (Eqn axis. Makingsomeassumptions,thiscanbequantified by a information, evenifimagescouldberesolvedsufficiently alongthis eyes areverycloselypositionedandhencewouldgainlimited depth be triangulationbetweenthedorsalandventraleye.However, these on eachsideofthehead,wealsoneedtoaskwhetherthere could sides oftheheadisnotnecessary. Aslarvaehavetwoprincipaleyes head areoccluded,suggestingthattriangulationbetweenthe two can strikefromconsistentdistancesevenifeyesononesideof the 1989). Oursecondexperiment(Fig. relies onsystematicimagedifferences betweentwoeyes(Schwind, alternative strategies. conceivable thatsuchconstrainshavedriventheevolutionof perturbations mayprovidemajorconstraintsinthatregard.Itis homogeneous backgroundandlimitedcontroloverwater conditions. However, frequentlymovingprey, anearly unclear towhatextentsuchcuesplayaroleundernatural obtained sufficient motionparallaxcuesinoursetup.Itremains provide utilizabledistancecues. translational patternsontheretinaandhenceareunlikelyto and E.K.B.,personalobservation),whichwouldnotresultin horizontal movementstendtobelimitedtrackingtheprey(A.S. (Buschbeck etal.,2007).Duringpreyapproach,incontrast, at relativelylarge distanceswhiletheanimalorientstoprey provide motionparallaxcues,becausetheyareprimarilyobserved and 2).Horizontalmovementsofthelarvaearealsounlikelyto target continuedtomove(seesupplementarymaterialMovies able tomaintainasteadydistanceduringthistime,evenwhilethe Here, target distance inordertobedistinguishable. Ifthepreferredstrike =0.0003, control Another importantdistancecueforinsectsisstereopsis,which Taken together, itishighlyunlikelythatlarvaecouldhave t -test, P 10 12 14 0 2 4 6 8 Δ′d B =0.001). (B)Similarresultsareobservedwhentheprincipaleyes is theminimumdistancethatan objectmustbefroma Small target N=6 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.092833 P NS Control (A) Thestrikingdistancetothelargertargetissignificantly Eyes occluded =0.00004). Inbothgraphs,thecolumnsonright

1) describedbyCollettandHarkness(Collett N=16 NS Δ′d Δ′d closer further NS N=10 ag agtSmalltarget×2 Large target = s = s 2 NS α′ /(a 2 α′ /(a P=0.0003 N=13 1B) demonstratedthatlarvae + s – s P=0.00008 α′) α′)

N=6 , N=16 t -test, (1)

1

The Journal of Experimental Biology larval eyeorganization, weproposethatthemostlikelyexplanation what otherdistancecueswereavailabletothem.Giventheelaborate distance-measuring mechanismswerenotavailable,wehavetoask the verticalaxis. highly limitstheinformationthatcouldhavebeenobtainedalong experimental designincombinationwiththelarvae’s anatomy failures. Inaddition,asdiscussedintheprevioussection,our distances between2.8and11 distance of4.5 SHORT COMMUNICATION distal photoreceptors. limited extentoftheretinainvertical directionaswellthetieringof schematic diagramofasagittalsection oftheprincipaleyes,illustrating calculate thestrikedistancewithin 3Dspace.Theinsetdepictsa shown) weredeterminedfromdirect andmirroredvideoframes,usedto center oftheirheads.(D)Three-dimensional coordinatesofkeypoints(as opaque nailpolishwasapplied.(C) Control animalswerepaintedinthe (A) Arena.(B)To occludetheprincipaleyesonleftsideofhead, Fig.2. Experimentalsetupofbeetlelarvaeattackinganartificialtarget. Δ′d determined frompreliminaryphysiology)is0.026 1.5 deg proximalphotoreceptorverticalacceptanceanglethatwas unpublished histology) distance As larvaewereabletogaugedistance,evenwhencommon further D BC A Dorsal receptors 5 mm =6.5 s Ventral receptors is 4.5 mm andΔ′d Mirror

mm thereforewouldbeindistinguishablefrom mm, theinterocularseparation(basedonour Eye spot a 1 mm is 0.2 closer Strike point

45 deg mm, presumablyleadingtofrequent =1.7 mm and Corner 3 mm. Thepreferredstriking α′ (correspondingtothe surface Water Corner 1 Corner 2 Motor

radians then 1 mm

Camera eyes werestilloccluded. results (datanotshown).Aftereachtrialitwasconfirmedthatthelarvae’s included inthedataanalysis,thoughsucceedingstrikesyieldedsimilar individual, onlythefirstsuccessfulstrikeatnovelartificialtarget was determined fromtheconsecutiveframe.To avoidlearningeffects, foreach cluster (Fig. corners oftheartificialtarget, aswellthepositionofcentereye immediately precededthestrikewasusedtorecordpositionof calculated aftercalibrationoftheimagestopreysize.Theframethat points thatwerevisibleinbothperspectives.Actualdistances point ontheartificialtarget wasobtainedfrom3Dcoordinatesofseveral from a45 on iceandheadregionsweredriedwithcotton. (Fig. a naturalhuntingposition.Shamcontrolswerepaintedontopofthehead under themicroscope.OcclusionofventralE3wasnecessarytomaintain horizontal circles(~1 motor-driven dummyprey, thatwasnoveltothelarvae,movedinsmall given 15 Slightly starvedsecond-instarlarvaeweretransferredintothetestarenaand Thermonectus marmoratus been proposedforanyinsect. the bestofourknowledge,acomparablemechanismhasnotyet focused withintheretinasandretinaltiersoftheirprincipaleyes.To distance informationunilaterallyfromthedetailsofhowimagesare investigation, wehereproposethat mechanism. Whilethedetailsofmechanismneedfurther considered essentialforthedescribeddistance-estimation tubular, andtheytoocontainadistinctlytieredretinathatis parallel, becausetheprincipaleyesofjumpingspidersaresimilarly jumping spider(Nagataetal.,2012),aparticularlyintriguing Recently, anovelmonocular distancesensorwasdescribedina of different retinatiersofoneorboththetwoprincipaleyes. is amechanismthatreliesonspecificactivationofphotoreceptors http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.092833/-/DC1 Supplementary materialavailableonline at IOS0545978 andIOS1050754toE.K.B. This workwassupportedbytheNationalScienceFoundationundergrants and E.K.B.draftedrevisedthemanuscript. K.B. performedtheanalysis,A.S.modeledstereopsisandeditedmanuscript, All authorsdevelopedtheconcept,K.B.andN.P.R. performedtheexperiments, The authorsdeclarenocompetingfinancialinterests. Zoo andBotanicalGardenforSunburstDivingBeetles. We thankShannonWerner forbeetlecare,andRandyMorgantheCincinnati (test animals;Fig. Hansen, Farmingdale,NY, USA)sothatE1toE3wereunilaterallyoccluded painted 1 were accustomedtohuntpriortheexperiment.Someanimals dimensions arecompatiblewithpreythattheselarvaenaturallyhuntand and 5 dummy preyconsistedofaverticalblackrodthatwaseither0.36 ls,A . ade .C,MItr,P n ilas D.S. Williams, and P. McIntyre, R.C., Hardie, A.D., Blest, MATERIALS ANDMETHODS References Supplementary material Funding Author contributions Competing interests Acknowledgements eyes ofajumpingspider. sensitivities ofidentified receptorsandthefunctionofretinal tieringintheprincipal The animals’ huntingbehaviorwasfilmeddirectlyandasareflection 2C). Priortoapplicationofnailpolish,animalsweretransientlycooled mm long,ortwicethosedimensions.Thisorientationandthese mins toacclimatepriora15

deg mirror. Theabsolutedistanceofthelarvaleyestostrike- day priortotestingwithopaquenailpolish(Chrome,Sally 2D). Thestriketrajectoryandpointonthetarget were The JournalofExperimentalBiology(2014)doi:10.1242/jeb.092833

2B). Theopaquenessofthenailpolishwasconfirmed

cm diameter)at~12 J. Comp.Physiol.A larvae wereoffspring ofourlaboratorycolony. T. marmoratus 145

min testperiod,duringwhicha revolutions , 227-239.

min are abletoderive (1981). Thespectral − 1 (Fig.

mm wide 2A). The 329

The Journal of Experimental Biology rl K. Kral, olt,T .adHrns,L.I.K. Harkness, and T. S. Collett, T. S. Collett, adpk,K,Mra,R .adBshek E.K. Buschbeck, and R.C. Morgan, K., Mandapaka, E.K. Buschbeck, and T. A. Cook, S., Maksimovic, M. Poteser, and K. Kral, 330 SHORT COMMUNICATION ucbc,E . bt,S .adMra,R.C. Morgan, and S.J. Sbita, E.K., Buschbeck, Cambridge, MA:MIT Press. Visual Behavior Entomol. hroetsmarmoratus Thermonectus opsin-encoding mRNAsinthetieredlarvalretinasofsunburstdivingbeetle locusts andmantids. information. 982. Dytiscidae) enlargesvisualfieldpriortopreycapture. larvae ofthepredaciousdivingbeetle, but onlytwelveeyes:ananatomicalanalysisofthelarvalvisualsystemdiving (2012). Thefunctionalsignificanceofmantispeeringbehaviour. 109 (1978). Peering–locustbehaviorpatternforobtainingmotionparallax J. Exp.Biol. , 295-301. (ed. D.Ingle,M.A.GoodaleandR.J.Mansfield),pp.111-176. J. InsectBehav. (1997). Motionparallaxasasourceofdistanceinformationin 76, 237-241. (Coleoptera: Dytiscidae). (1982). Depthvisioninanimals.In 10, 145-163. hroetsmarmoratus Thermonectus J. Exp.Biol. (2007). Scanningbehaviorby J. Comp.Physiol.A (2009). Spatialdistributionof (2006). Twenty-eight 212 , 3781-3794. (Coleoptera: Analysis of 193 Eur. J. , 973- aaa . oaai . skmt,H,Sei . sn,K,Sihd,Y., Shichida, K., Isono, S., Saeki, H., Tsukamoto, M., Koyanagi, T., Nagata, oe,E.C. Sobel, cwn,R. Schwind, S. Rossel, M.P. Loosemore, and C.E. Bessette, J.L., Fox, A.H., Worthington, R.M., Olberg, twse,A,Rppr,A,Lye .E,Mra,R .adBshek E.K. Buschbeck, and R.C. Morgan, J.E., Layne, A., Rapaport, A., Stowasser, oee,M n rl K. Kral, and M. Poteser, Vision ouaa . ioht,M,Aiaa .adTrkt,A. Terakita, and K. Arikawa, M., Kinoshita, perception fromimagedefocusinajumpingspider. F., Tokunaga, 166-181. beetle Physiol. A 2127-2137. targets inpraying-mantis.Anindexoftheusemotionparallax. Comp. Physiol.A (2005). Preysizeselectionanddistanceestimationinforagingadultdragonflies. (2010). Biologicalbifocallenseswithimageseparation. (ed. D.G.StavengaandR.C.Hardie),pp.425-444.Berlin:Springer. hroetsmarmoratus Thermonectus (1983). Binocularstereopsisinaninsect. 167 (1990). Thelocust’s useofmotionparallaxtomeasuredistance. (1989). Sizeanddistanceperceptionincompoundeyes.In The JournalofExperimentalBiology(2014)doi:10.1242/jeb.092833 , 579-588. 191 , 791-797. (1995). Visual distancediscriminationbetweenstationary (Coleoptera: Dytiscidae). Nature Science Curr. Biol. 302 335 , 821-822. J. Comp.Neurol. , 469-471. 20, 1482-1486. J. Exp.Biol. (2012). Depth Facets of J. Comp. 497 198 J. , ,

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