2526 Received 28January 2014;Accepted7April2014 *Author ([email protected]) for correspondence Germany. BiologyandBiotechnology,Ruhr University Bochum, Faculty of D-44780Bochum, cuticle transfersthevibrationto thesubgenualorgan (Howse, 1964). the dorsalblood-spaceoftibia.Whenlegisvibrated, the subgenual organ, achordotonal organ inthelegs.Theorgan liesin these vibrations.Thevibrationsenseorgan ofthetermitesis (Hager andKirchner, 2013).Nestmatesare extremelysensitiveto ground. Thenumberofpulsespersignalvariesbetweenfiveand 22 pulse arisesfromasingletapofthetermite’s headagainstthe their headsagainstthesubstrateandcreateapulsedvibration. Each Hewitt, 1990).Whensoldiersareattacked,theytendtodrum with et al.,1978;HueyandPianka,1981;Redford,1984;Kok and of predatorsarepotentialdangertoforagingtermites(Longhurst long-distance communication(HagerandKirchner, 2013).A variety promote vibrationalcommunicationandareusedaschannels for to theirforagingareas.Thegallerieshavephysicalfeaturesthat builds longsubterraneangallerieswhichleadoutwardsfromthenest natalensis natalensis KEY WORDS:Orientation,,Vibration, Macrotermes and vibrotropotaxisonsolidsubstratesininsects. demonstrating forthefirsttimeperceptionoftime-of-arrivaldelays a significantpositivetropotaxistotheplatformstimulatedearlier, Delays asshort0.2 difference intime-of-arrivalisthedirectionalcueusedfororientation. and rightsidesofthebodywithatimedelay, weshowthatthe Using twomovableplatformsallowingustovibratethelegsofleft nest material,soldiersareabletolocalisethesourceofvibration. towards thesourceofsignal.Inarenaexperimentsonnatural in thecontextofcolonydefence.Infield,soldiersarerecruited show thatM.natalensis leading toanamplificationandpropagationofthesignal.Herewe retreat intothenest.Soldiersinvicinitystarttodrumthemselves, substrate andcreateapulsedvibration.Workers respondbyafast attacked bypredators,theytendtodrumwiththeirheadsagainstthe signals transmittedalongsubterraneangalleries.Whensoldiersare Macrotermes natalensis to beprocessedbyinsectnervoussystems.Thetermite difference betweenreceptorystructuresarethoughttobetoosmall signals onsolidsubstrates,becausetime-of-arrivalandamplitude insects areabletoperceivedirectionalinformationfromvibratory insects tolocalisethesourceofvibrations,itisstillunclearhow Although severalbehaviouralstudiesdemonstratetheabilityof Felix A.Hager*andWolfgang H.Kirchner natalensis Directional vibrationsensinginthetermite RESEARCH ARTICLE © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,2526-2530doi:10.1242/jeb.103184 INTRODUCTION ABSTRACT ms aresufficient tobedetected.Soldiersshow makes useofadirectionalvibrationsensing communicates usingvibrationaldrumming (Haviland) (Isoptera:) natalensis Virant-Doberlet etal.,2006).Herewedemonstratethat be processedintheircentralnervoussystem( orientation, iftimeand/oramplitudedifferences arelarge enoughto suitable foradirectionalvibrationsensingandvibrotropotactic insects therearevibrationreceptorsinallsixlegs,whichwouldbe time delaysasshort0.2 of 4 ms,andthesandscorpion salei the signalsarrivingatlegsarewanderingspider of vibrationscanbeperceivedthroughsimultaneouscomparisons which ithasbeenclearlydemonstratedthatthedirectionofasource Virant-Doberlet etal.,2006;Hill,2009).Theonlyarthropodsin information abouttheunderlyingmechanisms(forreviews,see ability ofinsectstolocalisethesourcevibration,butthereislittle of vibrationoutthevibrationalsignals. the termitestoextractinformationaboutdirectionofsource direction ortheother. Inthiscontextitwouldbeadvantageousfor alarm signals,ithastomakeadecisionwalkeitherinone 2013). Ifatermitesomewhereinthevastgallerysystemdetects reamplifying (Röhrigetal.,1999;HagerandKirchner, to asociallong-distancecommunicationthroughchainsofsignal- themselves, therebyamplifyingtheintensityofsignal,leading In responsetodrummingnestmates,soldiersalsostartdrum After sometime, termitesappearatthenestsurface andstarttoclose behaviour inM.natalensis Röhrig etal.,1999;Hertel 2011). Bothstimulielicitdrumming system andvibrationsofthenest material(Connétableetal.,1999; nests. Thisisassociatedwith both aflowofairintothegallery aardvarks andpangolins,with theirpowerfulfrontlegs,canopen conical baseoftermitemounds. Specialisedpredators,suchas surface, predatorattacksweresimulatedbydrillingholesinto the To ensurethatdirectlyobservable termiteswerelocatedatthenest substrate-borne alarm signals at the mound signalsat thetermite alarm substrate-borne to andsoldiers workers termite of response Directional RESULTS orientation. substrate-borne wavesbetweenthelegsareusedasacue for objective wastoinvestigatewhethertimedelaysofthearriving waves inthetermitenest(HagerandKirchner, 2013),thethird orientation. Becauseofthelowpropagationvelocitysurface substrate-borne alarmsignalsprovideadirectionalcueused for alarm signals.Thesecondobjectivewastoinvestigatewhether show different behaviouralstrategiesinresponsetovibrational vulnerable. Therefore,weassumedthatworkersandsoldierswould morphologically wellprotected.Incontrast,workersaremuchmore signals. With theirstrongheadcapsuleandmandibles,soldiersare workers andsoldiersofM.natalensis To date,morethan40behaviouralstudieshavedemonstratedthe The firstaimofthisstudywastoinvestigatetheresponse (Hergenröder andBarth,1983),whichcandetecttimedelays soldiers solvethetaskaswellsandscorpions. Macrotermes within 1

ms (BrownellandFarley, 1979).In Paruroctonus mesaensis,detecting s (HagerandKirchner, 2013). to substrate-bornealarm Č okl etal.,2006; Cupiennius M.

The Journal of Experimental Biology preference (** whiskers indicate minima andmaxima.Asterisksindicate asignificantside termites). Boxesshowfirstandthird quartiles;boldlinesindicatemedians; vibrational signals(whitebar),termites donotshowsidepreferences( the otherside( significantly longeratthearenaside facingthesourceofvibrationthanon arena. Fig. and toasignificantincreaseinthenumberofsoldiers( the numberofworkers(Wilcoxon signed-ranktest, of substrate-bornedrummingsignalsledtoasignificantdecreasein recruited towardsthesourceofsignalatnestsurface.Playback mound, termiteworkersfledtowardsthenest,whereassoldierswere response toartificialvibrationalstimuliatthesurfaceoftermite termites atthenestsurfaceusedinsubsequentexperiment.In the holeinnestwall.Thisresultspresenceofobservable leads toasignificantdecreaseinthenumberofworkers( without playbackofavibrationalsignal. experiments withavibrationalalarmstimulusandcontrol (white bars)ofMacrotermesnatalensis Fig. RESEARCH ARTICLE and toasignificantincreaseinthenumberofsoldiers( vibrational stimuli,noeffects werefound(workers: n the numberofworkers( the nestsurface.Incontrolexperimentsthereisnosignificantdifference in soldiers: changes inthenumberoftermitesatnestsurface(** truncated (1.5timesinterquartilerange).Asterisksindicatesignificant Boxes showfirstandthirdquartiles;boldlinesindicatemedians;whiskersare 4)a h etsrae(i.1).Incontrolexperimentswithout =45) atthenestsurface(Fig.

.Preference indexofindividualsoldiersM.natalensis 2. Preferenceindexofworkers(greybars)andmajorsoldiers 1. If vibrationalalarmsignalsarepresented (greybar),termitesstay

P Directional response at the mound =0.132,

P (preference index) n <0.01). –1.0 –0.5 =134 termites).Incontrolexperiments withoutplaybackof 0.5 1.0

Directional response in an arena 0 n =25).

(preference index) n =25 experiments)andsoldiers( –1.0 –0.5 1.0 0.5 Alarm signal ** 0 Alarm signal ** ** at thenestsurfacein Playback ofdrummingsignals Control Control n =45 experiments)at P n n <0.01). =25 experiments). =45 experiments) P P <0.001, =0.569, P in an <0.005, n n n =30 =45) =25; termites didnotshowasidepreference( 2).Incontrolexperimentswithoutavibrationalstimulus, Fig. significant withtimedelaysof0.2 effect couldnotbesupportedstatistically. However, theeffect was n nests ofM.natalensis , vibrationalalarm signalsarepropagatedwith solve thetaskofdirectionalorientation fortermitesoldiers:inthe those detectedby (Brownell andFarley, 1979)and used fororientationarethearachnids for whichithasbeendirectlyshownthattimeofarrivalisthe cue are usedfortropotacticorientationbyaninsect.Theonlyarthropods Here wereportthattime-of-arrivaldelaysofthevibrationalsignals ( 3).Inexperimentswithsmall timedifferences of0.09 first (Fig. delays, termitesshowedatendencytoturntheplatformvibrated vibrates first.We testedfourdifferent timedelays. Foralltestedtime vibrating ashortmomentlaterturnedtowardstheplatformthat platform andwiththelegsofotherbodysideona Termites standingwiththelegsofonebodysideonavibrating vibrational stimulus(Wilcoxon signed-ranktest, stayed significantlylongeratthearenasidefacingsourceofa source ofvibrationalpulses.Termite soldiersorientedtowardsand laboratory onnaturalnestmaterialfortheirabilitytolocalisethe Single majorsoldiersweretestedinarenaexperimentsthe platforms arevibratedwithasmalltimedelayof0.09 time delays,soldierstendtoturntheplatformvibratedearlier. Whenthe vibrational stimuligeneratedwithtwomovableplatforms. Fig. ( detected bythescorpion delays detectedbythetermites areassmallthetimedelays been conductedwithinsects(Virant-Doberlet etal.,2006).The time vibrate legswithdifferent amplitudes ortimedelayshavesofarnot Barth, 1983).Similarexperimentsusingtwomovableplatforms that borne alarm signals alarm borne tosubstrate- soldiers of response Individual directional DISCUSSION differences basedontime-of-arrival soldiers of response Directional information aboutthedirectionofsourcevibration. short as0.2 ( When theplatformsvibratedwithtimedelaysof0.20 Asterisks indicatesignificantsidepreferences(* n n χ =86) and0.29 =49), orientationtowardstheplatformvibratingfirstwasnotsignificant. =26) termitesturnsignificantlymoreoftentotheplatformvibratedfirst. 2 The temporalresolutionofthe systemseemstobesufficient to =0.61,

.Preferenceindexof 3. P <0.43, Directional response on two The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103184

ms weresufficient tobedetected andtoprovide platforms (preference index) 0.1 0.2 0.3 0.4 ms (χ 0 C. salei. n =26) and0.16 2 =3.846, M. natalensis 0.09 P. mesaensis P Time delay(ms) <0.05, Cupiennius salei

ms (two-tailedχ 0.16 ms (χ in relationtothetimedelayof n 2 and 20timessmallerthan P =0.18, =26). Time differences as P Paruroctonus mesaensis =0.588, <0.05; **P 0.20 **

ms (n ms (n P <0.67, (Hergenröder and P 2 n =86) or0.29 =26) or0.16 =7.86, <0.005, <0.01). =30). 0.29 * For alltested n =49), the P <0.005, n =134;

2527 ms ms

ms

The Journal of Experimental Biology direction sensinghasbeenstudiedby orientation basedontheanalysisoftime-of-arrivaldelays. body sizeofmostinsectsissufficient foravibrotropotactic Doberlet etal.,2006;Hill,2009).Theyalsodemonstratethatthe behavioural studiesinvariousinsects(Cocroftetal.,2000;Virant- detected byinsects.Ourresultsconfirmindirectevidencefrom arrival differences ofvibrationalsignalsonsolidsubstratescanbe natural conditionstheresolutionmightevenbebetter. of magnitudeasthetimelag0.12 directional orientationinarenaexperiments.Thisisthesameorder differences asshort0.2 temporal resolution ofvibrotropotacticreactions inthesand vibrational signalshaspreviously exclusivelybeenusedtostudythe differences betweenthelegsareusedtolocalisesources of present studytoshowdirectlyand unequivocallythattime-of-arrival Hedwig, 2010;Masonetal., 2001).Theapproachusedinthe hundred microsecondstoa few milliseconds(Schöneichand inducing latencydifferences inthemanageablerangeof some amplitude differences, orneuronally throughbinauralinteractions mechanically intheperipherybytranslatingtimedelays in However, insectauditoryorgans solvetheproblemeither interaural timedelaysareapproximately50 fly al., 1978),andtheyareevenlessinsmallerinsectssuchasthe tiny interaural timedifferences are intherangeof0.01 even smallerthanthosedetectedbyourtermites.Inlocusts, the Larsen, 2008). explanation fordirectionalityinsmallanimals(Michelsen and coupled), pressuredifference receptionhasbecomethestandard exception ofthefly encode directionalinformation(MasonandFaure,2004).With the interaural differences inresponserateorlatency, orboth,could sound sourcesgenerateintensitydifferences atthetwoears,sothat including temporalcodingfordirectionalinformation.Directional acoustic parametersmayberepresentedinreceptorresponses, Robert, 1996).Severalmechanismshavebeenidentifiedbywhich sources. Thiscontinuumalsoincludestympanalhearing(Hoyand mechanoreceptor-based communicationsystemfordetectingsignal legs (Wiese, 1974). backswimmers areabletodetectphasedifferences betweentheir surface. Atthelowfrequenciesofwatersurfacewaves, glauca, whichcandetectthedirectionofwavesonwater sensing onsolidsurfacesissolvedbythebackswimmer 1985). 5 size, receptorsindifferent legsareatadistanceofapproximately receptors fromseverallegs.Becauseofthelocust’s relativelylarge the ventralcordlevelbyintegratinginputsfromvibratory the presentedvibrationalstimulus.Directionalprocessingoccursat ventral cordneuronsdependsonthedirectionandtimedelayof They showedin platform vibratedearlier, evenwithtimelagsof0.09 movable platformsshowedatendencyofsoldierstoturntowardsthe of approximately0.12 of theotherbodysideisapproximately16 The distancebetweenthefrontlegofonebodysideandhind 2528 RESEARCH ARTICLE a velocityofapproximately130

cm, leadingtoatimedelaybetween0.4and4 On aphysiologicallevel,thetemporalresolutionofvibrational This isthefirstbehaviouralstudydemonstratingthattime-of- auditorysystemshavetodealwithtimedelaysthat are Vibration sensingisatoneendofacontinuumintheinsectan A taskthatisinsomerespectscomparable todirectionalvibration O. ochracea.Asthisfly’s eardrumsarelessthan0.5 Locusta migratoria Ormia ochracea(theeardrumsaremechanically

ms betweenlegs.Ourexperimentsusingtwo ms weresufficient togenerateasignificant

m

ms calculatedabove.Undermore s − 1 Č that theresponsepatternof (Hager andKirchner, 2013). okl etal.(

mm, leadingtotimelags

ns (Masonetal.,2001). Č

ms (Č okl etal.,1985). ms (Mörchenet

ms, andtime

Notonecta mm apart, okl etal., day ofcollection. containers untilusedinexperiments.Allexperimentswereconductedonthe soldiers forthelaboratoryexperiments.Soldiersweremaintainedinplastic 1.5 m. Termite moundswereopenedattheirconicalbasetocollectmajor total ofsixcolonies Witwatersrand inMpumalanga,SouthAfrica(25°34 29°26′ peak amplitudeofthenestmaterialwascalibratedto0.5 preference, indices( ( workers werecountedseparatelybeforethedrummingstimuluswasgiven material drieswithinminutes,ensuringaverytightcoupling.Soldiersand surface 0.05 2013). Theaccelerometerwasmountedwithwetnestmaterialonthe vibrational alarmsignalsproducedbyM.natalensis connected tothecomputer. Thisamplitudeisinthenaturalrangeof using anaccelerometer(B&K4381)andacharge amplifier(B&K2635) When soldiersof Soutpansberg Mountainrange,LimpopoProvince,SouthAfrica(23°02 The experimentswerecarriedoutattheLajumaResearchCentrein All experimentswereperformedwithtermitesofthespecies in whichdirectionalresponsestovibrationshavebeenreported. experimental approachforcomparativestudieswithotherinsecttaxa the taskaswellsandscorpion,itwillbeinterestingtousethis scorpion. Nowthatweknowatleastoneinsectspeciessolves substrate (0.4×0.28×0.18 the sourceofvibrationalpulses.We usedapieceofnaturalnestmaterial as Single majorsoldiersweretestedin thelaboratoryfortheirabilitytolocalise coupled tothenest0.1 drumming signalsweregeneratedbyavibrationexcitermechanically started 10 (diameter=8 Predator attacksatthetermitemoundweresimulatedbydrillingholes Naerum, Denmark). sound card(Tascam US-144)andavibrationexciter(Brüel&Kjaer4810, signals weregeneratedbyanotebookcomputerconnectedtoanexternal silence betweenburstswererepeatedthreetimes.Artificialdrumming pulses withapulserepetitionrateof11 The artificialdrummingsignalsusedinallthreeexperimentsweresquare (Gridi-Papp, 2007)andRavenPro(BioacousticResearchProgram,2011). synthesised andanalysedvibrationalstimuliusingthesoftwareSoundRuler against thesubstrateandtherebycreateavibrationalalarmsignal.We given (t Soldiers andworkerswerecountedseparatelybeforethefakestimulus was attached tothetermitemound,butnovibrationalstimuliwereoffered. of drummingsignalswerealsoconducted.Thevibrationexciterwas also at thesametermitemoundwerecarriedoutleast2 selected controlexperiments.Subsequentexperiments/controlexperiments colony wastestedin10to12experimentsandsixsevenrandomly experiments wereconductedatfourdifferent termitemounds.Each ‘after’ thevibrationalstimulus.Forty-fiveexperimentsand25control analysis. Thetworelatedgroupsare‘numberofworkers/soldiersbefore’ and workers by:x Artificial drumming signals drumming Artificial Termites MATERIALS ANDMETHODS nest material signalsonapieceof drumming toartificial soldiers Reaction of mound signalsat thetermite alarm substrate-borne to andsoldiers workers termite of response Directional used torestrictthetermites’ locomotoryactivity. At10 a distanceof1 the rightofarena, vibrationexciters(B&K4810) wereattachedviatwo t 1 ) and1 E), andatthePullenFarmfieldstationofUniversity 1 ) and1 min afterthetreatment( min afterthefirsttermiteappearedatsurface.Artificial

cm) intotheconicalbaseoftermitemounds.Theexperiments The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103184 m fromthecouplingpointofvibrationexciter. Wet nest i =

mm abovethesubstratetoexclude mechanicalcouplingwas t 2 – min afterthecontroltreatment( t M. natalensis 1 / t 2 x + i ) werecalculatedseparatelyformajorsoldiersand

m fromtheedgeofdrilledhole.Thevibrational t M. natalensis 1 . Wilcoxon signed-ranktestwasusedforstatistical m). A lidofaPetridish(diameter=5.5 are disturbed,theydrumwiththeirheads t 2 ). Controlexperimentswithoutplayback with amoundheightbetween1and

Hz. Burstsof10pulseswith1

t (Hager andKirchner, days later. ′ 2 S, 31°10′

). Forvisualisation cm totheleftand

m

s − M. natalensis. 2 peak to

cm) keptat E), usinga ′ S, s

The Journal of Experimental Biology as describedbeforewith were testedonlyonce.Forvisualisationpreference,indicescalculated arena. Termites fromfourmoundswereusedinthisexperiment.Termites measure thetimetermitespentinright-orleft-handpartof 30 the arenathroughatube.Thetermite’s locomotoryactivitywasobservedfor right sideofthearena.Theexperimentwasstartedwhentermiteentered nest surface.Vibrational alarmstimuliweregiveneitherontheleftor exciters. Theaccelerometersweremountedwithwetnestmaterialonthe computer. Theamplitudeofthedrummingsignalwas0.5 two accelerometers(B&K4381),charge amplifiers(B&K2635)anda with 0.5 filter papersontheplatforms,twopiecesofpaperweremoistened two piecestoensuremechanicalisolation.To ensureabetteradhesion ofthe were coatedwithfilterpaper(diameter=7 Petri dish.To preventtermitesfromslipping,thebottomsof platforms 4).Fivemajorsoldierswereplacedinthearenaunder platforms (Fig. connected tothecomputer0.01 two accelerometers(B&K4381)andcharge amplifiers(B&K2635) analyses, thevideo wasstoppedoneframe (video recordingat experimenter was notawarewhichplatformside vibrated first.Fordata stimuli viatheaudiochannelof camcorder. Duringvideoanalysis,the HDC-SD20). Thevideorecording wassynchronisedwiththevibrational were selected. video recordingsandonlycaseswhere alllegswereappropriatelypositioned by anexperimenterwatchingtermites inrealtime;datawereanalysedfrom aware whichplatformvibratedfirst.Thevibrationalstimuliweretriggered platform thatvibratedfirstwaschosenrandomly. Theexperimenterwasnot 5).The vibrated inexactlythesameway, exceptforashorttimedelay(Fig. measured atthebottomofplatforms;bothplatformswerecalibrated and amplitude ofthenestmaterialwascalibrated(0.5 drumming signalsweregeneratedasdescribedbefore.Thevibrationalpeak (diameter=5.5 prevent termitesfromleavingtheplatforms,aPetridishlid of 1 coupled tothebottomoftwosquarePVCplatforms(15×15×0.5 a personalcomputerandtwovibrationexciters(B&K4810)mechanically laboratory usingartificialdrummingsignalsasdescribedabove,inducedby soldiers ofM.natalensis.Thedetectiontimedelayswasstudiedinthe We measuredvibratorydirectionalityusingwalkingresponsesofmajor PVC rods(150 video. termite’s positionontheplatformaccurately, experimentswererecordedon the legsofotherbodysideonplatform.To determinethe the experimentalsetup,withlegsofonebodysideonplatformand vibrational stimuluswastriggeredbyanobserverwhenthetermitestraddled on anaccelerometer(B&K4381)andavibrationexciter2635).(B)The a groupoffivetermitesoldiersunderPetridish.Theplatformsaremounted vibrational directionalsensing. Fig. RESEARCH ARTICLE the vibrationfacingarenaside’ and‘time(s)onthevibrationfarside’. Wilcoxon signed-rank testwasused.Thetworelatedgroupsare‘time(s)on t Soldiers ontwo movableSoldiers platforms 2 A =time (s)onthevibrationfacingarenaside.Forstatisticalanalysis, The termite’s reactionwasvideorecorded usingacamcorder(Panasonic s. ThePetridishwasmarkedwithalineinthemiddlesothatwecould

.Experimentalapertureforthebehaviouralmeasurementof 4. mm widthensuredmechanicalisolationbetweentheplatforms.To ml watereach.Theamplitudesofthestimuliweredeterminedusing

cm) wasmountedupside-down1 mm long,10 t 1 =time (s)onthevibrationfararenasideand mm diameter)tothesubstrate.Artificialtermite

m fromthecouplingpointsofvibration (A) Diagramoftwomovableplatformswith

cm). Thefilterpaperwascutinto 270 deg B

m

s

− mm abovethetwo 2 peak topeak)using 180 deg

0 deg m

s − 2 peak to

cm). A gap 90 deg before witht tested. Forvisualisationpreferenceindiceswerecalculatedasdescribed soldiers wereplacedunderthePetridish.Overall,32termitegroups The termitegroupsweretestedinupto20trials,andthenfivenew avoid habituationeffects, stimuliwereoffered atintervals ofatleast2 estimate theinter-individual within-colonyvarianceoftheresponse.To Röhrig etal.,1999;HagerandKirchner, 2013),noattemptsweremadeto studies oftermiteresponsestovibrationalstimuli(Kirchneretal.,1994; were tested.Thetermitesnotindividuallymarked;asinprevious platform thatvibratedfirst.Termites fromtwodifferent termitemounds reaction towardsthesourceofvibrationwhentermiteturnedto within 1 to oneoftheplatformswhentermiteenteredparticularplatform side wereincontactwiththeotherplatform.Itwasregardedasareaction body sidewereincontactwithoneplatformandthetarsiofother was usedtoselectthetestsinwhichthreetarsioftermite’s one for deviationfromexpectedvalues. turns totheplatformvibratingfirst.Chi-squareanalysiswasusedtest (DAAD). This studywassupportedbygrantsof theGermanAcademicExchangeService manuscript. W.H.K. supervisedexperimentsanddataanalysis. the study. F.A.H. carriedouttheexperiments,analyseddataandwrote This studyispartofthePhDthesisF.A.H. Bothauthorsconceivedanddesigned The authorsdeclarenocompetingfinancialinterests. allowing ustousethefieldstationPullenFarmforourprojects. and EnvironmentalScience,UniversityoftheWitwatersrand,Johannesburg, for conduct researchattheLajumaResearchCentre,andSchoolofAnimal, Plant We thankRhetaandIanGaigherfortheirhospitalitytheopportunityto 25 amplitude ofthevibrationalpulsesis0.5 beneath oneofthemovableplatforms.Thepulserepetitionrateis11 Fig. spectrogram ofthepulsegroupshowninA (band-passfilter10 Bioacoustic Research Program References Funding Author contributions Competing interests Acknowledgements indicate thetimedelayof0.29 first (a)andontheplatformvibratingashortmomentlater(b).Redbars (C) Oscillogramofsignalsrecordedsimultaneouslyontheplatformvibrating

Software Relative Amplitude

frames –2

.Artificialdrummingsignals.(A)Oscillogramof10pulsesmeasured 5. amplitude (m s ) 0.2 0.4 –1 b a 0 0 1 0 0 0 s ofthestartdrummingstimulus.Itwasregardedasa C B A (Version 1.4). Ithaca,NY: TheCornell LabofOrnithology. s − 1 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103184 1 ) beforethevibrationalsignalwasgiven.Thefrozenimage =termite turnstotheplatformvibratinglaterand 0.1 1 0.2 2 0.3 3

ms betweenthevibrationalstimuli. 1 (2011). Frequency (kHz) 0.4 4 Time (ms) Time (s) Raven Pro:Interactive SoundAnalysis

m 0.5 5

s − 2 peak topeak.(B)Frequency 0.6 6 2 0.7 7 0.8 8

Hz to3 t 2 0.9 =termite 9

Hz. The kHz). 2529

min. 10 3 1

The Journal of Experimental Biology 2530 RESEARCH ARTICLE rwel .adFre,R.D. Farley, and P. Brownell, orf,R . iu .D,Hy .R n ie,R.N. Miles, and R. Hoy, T. D., Tieu, R.B., Cocroft, Č Č ontbe . oet . ofal,F n odra,C. Bordereau, and F. Bouffault, A., Robert, S., Connétable, egnöe,R n at,F. G. Barth, and R. Hergenröder, etl . asah .adPar,R. Plarre, and A. Hanspach, H., Hertel, W. H. Kirchner, and F. A. Hager, il P. S.M. Hill, M. Gridi-Papp, os,P. E. Howse, k,A,Ot,C n amig K. Kalmring, and C. Otto, A., okl, k,A,Vrn-oelt .adZorović and M. Virant-Doberlet, A., okl, nocturnal scorpion, Comp. Physiol.A Membracidae: Umboniacrassicornis). mechanical responsetosubstratevibrationinatreehopper(Hemiptera: signals intheventralnervecordof 52. Drosopoulos andM.F. Claridge),pp.71-80.London:Taylor &Francis. vibratory communicationofbugs.In 152 do thesensoryinputsfromeightlegsinteractinorientation? 3256. signals intwosympatrichighertermitespecies: americana to physicalstimuli. drywood andsubterraneantermites(Isoptera:KalotermitidaeRhinotermitidae) the termitesMacrotermesnatalensis Available at:http://soundruler.sourceforge.net. militaris the termite,Zootermopsisangusticollis source? , 361-371. (, Macrotermitinae). Naturwissenschaften (2009). Howdoanimalsusesubstrate-bornevibrationsasaninformation L. J.InsectPhysiol. (1964). Aninvestigationintothemodeofactionsubgenualorganin (2007). 131 J. InsectBehav. Paruroctonus mesaensis , 31-38. SoundRuler: AcousticAnalysisforResearchandTeaching 96, 1355-1371. 10, 409-424. (2013). Vibrational long-distancecommunicationin (1979). Orientationtovibrationsinsandbythe (1983). Vibratory signalsandspiderbehavior:how 24, 106-115. Locusta migratoria (1985). Theprocessingofdirectionalvibratory and J. Comp.Physiol.A J. InsectBehav. Insect SoundsandCommunication Emerson, andinthecockroach, (2011). Differences inalarmresponses ,M.(2006). Senseorgansinvolvedinthe Odontotermes : mechanismsoftargetlocalization. Pseudacanthotermes spiniger . 12, 329-342. J. Comp.Physiol.A (2000). Directionalityinthe sp. 186 J. Exp.Biol. (1999). Vibratory alarm , 695-705. J. Comp.Physiol.A Periplaneta 216 156 (ed. S. , 3249- and , 45- P. J. . icnr .H,Boce,I n at,J. Tautz, and I. Broecker, W. H., Kirchner, E.R. Pianka, and R. Huey, o,R .adRbr,D. Robert, and R. Hoy, ihle,A n asn O.N. Larsen, and A. Michelsen, R. Hoy, and M.L. Oshinsky, A.C., Mason, P. A. Faure, and A.C. Mason, T. G. Wood, and R.A. Johnson, C., Longhurst, Kok, O.B.andHewitt,P. H.(1990).Birdandmammalpredatorsoftheharvester is,K. Wiese, M., Virant-Doberlet, cöec,S n ewg B. R.H. Hedwig, and S. Leuthold, Schöneich, and W. H. Kirchner, A., Röhrig, K.H. Redford, öce,A,Renane,J n cwrzof,J. Schwartzkopff, and J. Rheinlaender, A., Mörchen, in amicroscaleauditorysystem. Afr. J.Sci. the damp-woodtermite Ecology 41, 433-450. Comp. Physiol. auditory nervefibers. Biomim. Microsc. Res.Tech. Guinea savanna. foetens termite S. DrosopoulosandM.F. Claridge),pp.81-97.London:Taylor &Francis. orientation: frombehaviourtophysiology. In steering inthecricket( Termitidae). communication intheAfricanfungus-growingtermitegenus mouse (Oxymycterusroberti Hodotermes mossambicus (Fabr.) (Hymenoptera:Formicidae)ontermitesintheNigerianSouthern 62, 991-999. (1974). ThemechanoreceptivesystemofpreylocalizationinNotonecta. 3 , 011001. 86, 34-37. Insectes Soc. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103184 (1984). Mammalianpredationontermites:testswiththeburrowing 92, 317-325. Oecologia Č 63, 338-350. k,A n Zorović and A. okl, Naturwissenschaften Gryllus bimaculatus Zootermopsis nevadensis (1996). Tympanal hearingininsects. 46, 71-77. ) anditsprey. Oecologia 32, 101-107. (1981). Ecologicalconsequencesofforagingmode. (2010). Hyperacutedirectionalhearingandphonotactic (2004). Thephysiologyofinsectauditoryafferents. (2008). Pressuredifference receivingears. Nature (Hagen) insemi-aridregionsofSouthAfrica. 410 ,M.(2006). Useofsubstratevibrationsfor (1994). Vibrational alarmcommunicationin deGeer). PLoSONE 65, 656-657. , 686-690. Insect SoundsandCommunication (2001). Hyperacutedirectionalhearing hso.Entomol . Physiol. (1978). Predationby 65, 145-152. (1978). Latencyshiftininsects (1999). Vibrational alarm Macrotermes 5 Annu. Rev. Entomol. , e15141. . 19187-190. Megaponera (Isoptera, Bioinspir. (ed. S. J.

The Journal of Experimental Biology