Würzburg, Germany. Behavioral Physiology andSociobiology, Würzburg, D-97074 University of *Author ([email protected]) for correspondence Riverside, 3401Watkins Drive, Riverside, CA92521,USA. Complexity, Arizona State University, Tempe, AZ85287,USA. OX1 3PS, UK. © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,3229-3236doi:10.1242/jeb.106849 Received 25April 2014;Accepted18June2014 1 2009; Seeley, 1989;Wheeler, 1912).Extensivestudyoftheantsand and complexityintheeusocialinsects(HölldoblerWilson, group behaviortheyunderlie,havereachedespeciallygreatdiversity interactions (Sumpter, 2010).Communication systems,andthe specialized signalsthatgroupmembersexchangein these members. Understandingthisprocessrequiresdecoding the ways fromacomplexnetworkoflocalinteractionsamonggroup Ward etal.,2011). Collectivecognitionemerges innon-obvious than asolitaryanimalcoulddo(Biroetal.,2006;Sasaki 2013; make consensusdecisions,oftentimesmorerapidlyandaccurately across manyindividuals,groupscanassesstheirenvironmentand Marshall andFranks,2009).Bydistributingtheburdenofcognition process informationtoachievecollectivecognition(Couzin,2009; In manytaxa,fromslimemoldstohumans,groupscooperatively KEY WORDS:Alarmpheromone,Collectivedecision-making, selection performance. deterrent functioncanimproveanemigratingcolony’s nestsite leptothoracine .We discussthepossibilitythatthispheromone’s the firstdetailedinvestigationofchemicalcommunicationin nest andalsotoattractantswhenreleasedatthehomenest.Thisis dimethylpyrazine wassufficient toinducerejectionofamarkednew each oneinanestchoicebioassay. We foundthat2,5- identify candidatecompoundsfromthemandibularglandandtested colony. We usedcoupledgaschromatography/mass spectrometryto instead attractedtoit,presumablyrespondathreatthe same pheromonewaspresentedneartheants’ homenest,theywere potential newhome,presumablytoavoiddanger. Whenthe mandibular glandthatsignaledotherantstorejectthisnestasa and unabletomovefreely, shereleasedapheromonefromher on context.Whenanantwastetheredinsideunfamiliarnestsite not beenwellstudied.Herewereportanalarmpheromoneintheant responses todanger, butthefunctionalplasticityofthesesignalshas widespread inthesocialinsectsandcanelicitavarietyofbehavioral understanding theunderlyingmechanisms.Alarmsignalsare group members,decipheringcommunicationsystemsiscrucialto Because collectivecognitionemergesfromlocalsignalingamong Takao Sasaki rugatulus A context-dependentalarmsignalintheant RESEARCH ARTICLE INTRODUCTION ABSTRACT Department of Zoology, Oxford, SouthParks Road, Oxford of University of Department 2 School of Life Sciencesand Centerfor SocialDynamicsand School of 1,2, 4 Department of Entomology, California, of University of Department *, BertHölldobler that elicitstwodifferent behaviorsdepending 2,3 , JocelynG.Millar 3 Biocenter, 4 and StephenC.Pratt pharaonis alarm communicationinantswasonthepharaohant, an earlyreport(Goetsch,1953),thefirstthoroughstudyofchemical (Blum, 1969;CreweandFletcher, 1974;Maschwitz,1964).Besides nestmates tocombatapotentialdangerorwarnthemstayaway behavior iscloselyconnectedwithalarmsignalsthateitherrecruit of communicationisalarmsignaling.Insocialinsects,defensive successful foragersornestsitescouts,butanotherfundamentaltype 2001; Seeley, 1997;Visscher, 2007). 2009; PassinoandSeeley, 2006; Pratt,2005;SeeleyandBuhrman, properties (Franks,1989;HirshandGordon,2001;Marshalletal., content ofsignals,andhowtheycontributetoemergent colony bees hasrevealedmuchaboutthephysicalnatureandinformation 2002; Mallonet al.,2001;PrattandSumpter, 2006;Prattet al.,2002; choose asingleoptimalnestamong multipleoptions(Franksetal., (Möglich, 1978),andtheyshow remarkableabilitiestocollectively moves usingrecruitmentbytandem runningandcarryingofnestmates requires frequentemigrationsto newhomes.Theyorganize these live insmallcavities,suchasacorns androckcrevices,whosefragility small coloniestypicallycomprising 150–250workers.Theyusually functions ofahithertounknownalarmpheromoneinthemyrmicine affect behavioralresponsesto alarmpheromonesinants. particularly thoseassociatedwithcollectiveinformationprocessing, been giventospecifyinghowsocialandenvironmentalcontexts, first recognized50 space (Hölldobler, 1977).Althoughthis functional plasticitywas vary indifferent groupsand castesofsocieties,andintime 2006; HölldoblerandWilson, 2009).Theresponse behaviorcanalso (Bradshaw etal.,1975;Bradshaw1979;Fujiwara-Tsujii etal., accordingly elicitdifferent behavioralresponsesinreceivers blend ofglandularsecretionshavedifferent diffusion ratesand multi-component signals,wherebyindividualconstituentsofthe Morgan, 1979;Vander MeerandAlonso,1998). identified (Blum,1985;HölldoblerandWilson, 1990;Parryand (Buschinger andMaschwitz,1984)manycompoundshavebeen glands havebeendeterminedtobethesourcesofalarmpheromones 1969). Innumeroussubsequentinvestigations,variousexocrine the activecomponentinalarmpheromoneof heptanone (McGurketal.,1966),whichwasalsolateridentifiedas a threat.Thepheromoneof smelling substancefromthemandibularglandwhentheyperceive showed thattheworkerantsofthesespeciesdischarge astrong- (Butenandt etal.,1959)ontheleafcutterant harvester ant nature ofalarmcommunicationbyWilson (Wilson, 1958)onthe experimental investigationsoftheanatomicaloriginandchemical behavior whenanestmatewascrushednearby. Thefirst Most ofthisworkhasconcernedrecruitmentsignalsusedby The presentstudyreportsthefirstanalysisofcontext-specific In someantspecies,alarmpheromoneshavebeenrecognizedas Temnothorax rugatulus(Emery1895).Antsofthisgenusform (Sudd, 1957).Workers ofthisspeciesreactedwithescape Pogonomyrmex badius

years ago(Maschwitz,1964),littleattentionhas Temnothorax 2 P. badius was identifiedas4-methyl-3- and byButenandtetal. Atta sexdens A. sexdens Monomorium (Blum, further 3229

The Journal of Experimental Biology 3230 releasing anaversivepheromoneoralarmare parts often slowandwidelyopenedpointedtotheground.Obviously, behavior. Inthecontextofmarking,however, mandiblegapingis typically fasterandaimedatan‘enemy’ target duringaggressive on ourobservationalexperience,however, themandibleflaringis The mandibleopeningcanalsoindicateaggressivebehavior. Based from theirmandibularglands(supplementarymaterialMovie suggests thatthisbehaviorisassociatedwithreleaseofapheromone mandibles verywidewhilefacingtowardthenestfloor. This recordings showedthatthetetheredantsrepeatedlyopenedtheir pheromone thatsignalstootherantsrejectthenestsite.Video Fig. before amigrationstarted(two-tailedbinomialtest: consistent evenwhenthetetheredantswereremovedfromnest (two-tailed binomialtest: showed thatcolonieshaveastrongpreferencefortheemptynest between anestwithfivetetheredantsandanemptynest.Theresults during colonymigration.Coloniesweregivenabinarychoice them toreleaseapheromonethatsignalsotherantsrejectthenest We testedwhethertetheringantsinanunfamiliarnestsitecaused preliminary observations,wenotedthat nature andoriginofanysuchnegativesignalremainsunknown.In 2007; Stroeymeytetal.,2011; Stroeymeytetal.,2014).However, the colonies tocollectivelychoosethebestavailablesite(Franksetal., distinctive marksonundesirableneststhatenhancetheabilityof evidence suggeststhatnestsitescoutsof recruitment signal(Möglichetal.,1974).Inaddition,indirect leaders discharge secretionsfromthepoisonglandthatfunctionasa Temnothorax Sasaki etal.,2013).Theroleofchemicalcommunicationin RESEARCH ARTICLE nest siteselection. whether andhowthissignalfunctionsoutsidethecontextofcollective anatomical sourceanditschemicalidentity. We furtherexamined other antsfromchoosingthesiteand,ifso,todeterminesignal’s whether thesetetheredantsreleasedapheromonethatdiscouraged nestmates hadbeentetheredtothenestwall.We setouttotest reluctant tomoveintocandidatenestsitesinwhichsomeoftheir Experiment 1: tethered ants emit a deterrent signal antsemitadeterrent Experiment 1:tethered RESULTS Frequency Frequency 10 15 10 5 0 0 5 1B). Theseresultssuggestedthattetheredantsreleaseda A A Tethered ant Heads societies ispoorlyknown,otherthanthattandemrun P 008 i.1A).Thispatternremained =0.008; Fig. Alitrunks Empty T. rugatulus T. albipennis 10 5 0 15 10 0 5 B colonies seemed Tethered ant B may place Heads P =0.016;

1). five splitdecisions;two-tailedbinomialtest: binomial test: (eight inempty, fourinalitrunk,threesplitdecisions;two-tailed an emptynest.Coloniesshowednopreferenceforeitheralitrunks binary choicebetweenanestwithfivealitrunksorgastersand alitrunks andgasters.To excludethispossibility, wealsotesteda heads, butitmightinsteadbethecasethattheywereattractedto P significantly preferredovertheheadnest(two-tailedbinomialtest: gasters wereusedinsteadofalitrunks,thegasternestwas alitrunk nest(two-tailedbinomialtest: five crushedalitrunks,coloniesshowedastrongpreferenceforthe binary choicebetweenanestwithfivecrushedheadsand pheromone, wouldcauseantstorejectit.Whenpresentedwitha that markinganestwithcrushedheads,thusreleasingthe If thepheromoneoriginatesinmandibulargland,wepredicted behavior. of thesamebehavioralsyndromecloselyrelatedtoaggressive to identifycompoundsinantheads.TheGC/MSanalysesofvolatile Coupled gaschromatography/massspectrometry(GC/MS)was used for atleast14 from headssignaledantstorejectthenest,andthiseffect persisted P compounds wereappliedtothepapers(two-tailedbinomialtest: consistent evenwhenmigrationsstarted14 tailed binomialtest: hexane, coloniesstronglypreferredthehexane-treatednest(two- nest withahexaneextractoftheheadandtreatedonly by chemicalextractsoftheheads.Givenabinarychoicebetween the signal,wenexttestedwhethersameeffect couldbeproduced After theresultsofExperiment2indicatedheadassource Experiment 2:thesignaloriginates inthehead compounds containsmultiple Experiment 4:themandibulargland the head insolventExperiment 3:thesignalispresent extracts of <0.001; Fig. =0.049; Fig. These resultssuggestthattheantsrejectednestcontained Empty Gasters The JournalofExperimentalBiology(2014)doi:10.1242/jeb.106849

h. 3B). Theseresultsindicatethatchemicalcompounds 2B). P =0.38) orgasters(seveninempty, threeingaster, Colonies didnotsplitbetweenthenests. had beenremovedbeforethemigrationstarted(B). chose theemptynest(A),evenwhentetheredants with tetheredantsandanemptynest.Allcolonies Results ofabinarychoicebetweennest 1. Fig. not splitbetweenthenests. the gasternest(B)overheadnest. Coloniesdid gasters. with headsandanesteitheralitrunksor Resultsofabinarychoicebetweennest 2. Fig. P 001 i.3A).Thispatternremained <0.001; Fig. All colonieschosethealitrunknest(A)or P 001 i.2A).When <0.001; Fig. P =0.34).

h afterchemical

The Journal of Experimental Biology RESEARCH ARTICLE with anA areartifactsfrom theSPMEdevice. geranyl acetone;(9)unknown;(10) unknown; (11) unknown.Peaksmarked (4) 2-phenethylalcohol;(5)decanal; (6)nonanoicacid;(7)undecanal;(8) Peak identification:(1)2,5-dimethylpyrazine; (2)benzylalcohol;(3)nonanal; mlclosed vial(top),andcontrolSPMEcollectionfromanemptyvial. 1.5 (SPME) collectionofvolatilesfrom 25crushedmandibularglandsina Total ion chromatogramsfromasolidphasemicroextraction 4. Fig. nonanal nest(two-tailedbinomialtest: (two-tailed binomialtest: to choosethehexane-treatednestonlywhenotherhadDMP a nesttreatedonlywithhexane.Antsweresignificantlymorelikely solutions ofoneeightcompoundsidentifiedinExperiment4and We testedaseriesofbinarychoicesbetweennestwithhexane compounds DMP, benzylalcoholand2-phenethylalcohol. human skinodors,althoughthisisclearlynotthecasefor aldehydes arecommoncontaminants(seeFig. It isalsoworthnotingthatsomeofthesecomponentssuchasthe no extendedbioassayserieswerecarriedoutwiththesesubstances. elicited anydetectablebehavioralresponsesfromtestants,andso undecanal) withthesecompounds.Noneofcompounds (for nonanalanddecanal)orpilottestsgeranylacetone secretions. We thereforealsoconductedeitherfullbioassayseries 7) andgeranylacetone(peak8)arepartofthemandibulargland several othercompounds,suchasnonanal(peak3),undecanal postpharyngeal glandorothersources,wecannotbecertainwhether ants withoutriskingsomecontaminationwithsecretionsfromthe extremely difficult todissectthemandibularglandsofthesetiny alcohol (peak2)and2-phenethyl4).Becauseitis the mandibularglands:2,5-dimethylpyrazine(DMP;peak1),benzyl 4)andfoundthreecompoundsthatwereclearlyderivedfrom (Fig. we comparedtheseresultswithparallelanalysesofemptyvials substances. To distinguishglandularcompoundsfromcontaminants, phase microextraction(SPME)revealedthepresenceofseveral compounds collectedfromdissectedmandibularglandsbysolid Experiment 5: DMP induces rejection of anestsite of Experiment 5:DMPinducesrejection Abundance Frequency .070 .01.01.01.017.00 15.00 13.00 11.00 9.00 7.00 5.00 0 10 15 20 0 5 1 A extract Head 2 3 4 Hexane A Retention time(min) 5 P <0.01). Theyalsotendedtorejectthe 6 7 A Split P =0.10). Whenthesolutions 8 A 15 10 20 0 5

4), forexamplefrom B extract Head A 9 10 11 Hexane the compoundcanstillbedetectedbyantsafter14 entities sothattheappliedDMP dissipatesslowly, andresiduesof is possiblyduetothefactthatthesenestsarerelativelyclosed long-lasting effect ofDMP (which is quitevolatile)insidetestnests showsthesummaryofthesetests.The 1 in Experiment3.Table binomial test: 5 above thebiologicallyrelevantamount.Furthermore,effect of the mandibulargland,itisuncertainwhetherthistinydoseator because wewereunabletomeasuretheactualamountofDMP in each filterpaper;two-tailedbinomialtest: with DMP evenwhenitwasaslow0.5 tailed binomialtest: for nonanal(two-tailedbinomialtest: elicited thesebehaviors(Fig. behaviors. Surprisingly, averylowdoseofDMP (0.5 confirming thatDMP isthesemiochemicalmediatingthese repelled themwhentheywereawayfromhome(Table between 5 5)andthuscoulddistinguishdifferent DMP doses,atleast (Fig. nest withthehigherdoseofDMP (two-tailedbinomialtest: with 0.5 DMP bypresentingachoicebetweennestwith5 of nonanalandDMP weredilutedto5 norecords yetexistastowhetheralarm pheromonesare et al.,1978;WheelerandBlum, 1973).Nevertheless,formanyant 1973; Duffield etal.,1976;HölldoblerandTaylor, 1983;Longhurst communication (BillenandMorgan, 1998;Duffield andBlum, Ponerinae andMyrmeciinae,which typicallydonotemploymass found eveninphylogenetically less derivedsubfamilies,suchasthe Chemical alarmsignalsareubiquitous intheFormicidae.Theyare intact head:itattractedantsthatwereinahomenest(Fig. that themandibularglandwassourceofpheromone. glands hadbeenremoveddidnotelicitthesebehaviors,indicating contexts. Furthermore,crushedheadsfromwhichthemandibular glands elicitedresponsessimilartothosebytheheadin both 2).Ourpreliminarytestshowedthatdissectedmandibular (Table from theirhomenest,itwasmoreoftenrejectedthanthecontrols 6).Alternatively, whentheheadwaspresentedtoantsaway (Fig. entrance, itattractedsignificantlymoreantsthandidthecontrols context. Whenaheadwascrushedandpresentednearthehomenest we testedwhetheritwouldelicitadifferent behaviorinanother Once weidentifiedDMP asthesignalresponsiblefornestrejection, DISCUSSION at released thehomenest when Experiment 6:thesignalinducesattractiontoentrance

ppm DMP (~25 We furthertestedwhethertheantsweresensitivetodoseof Presentation ofDMP elicitedthesamepatternsofresponsesas Split ppm DMP. Theresultssuggestedthattheantsrejected and 0.5 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.106849 P <0.01), consistentwiththeresultsofextractedheads binomial test: more likelytochoosethehexanenest(B)(two-tailed compounds wereapplied,coloniesstillsignificantly (A). Evenwhenmigrationsstarted14 hexane only. Allcolonieschosethehexane-treatednest a hexaneextractofheadsandnesttreatedwith Results ofabinarychoicebetweennestwith 3. Fig. ng) seemedtopersistevenafter14 ppm. P <0.01). Surprisingly, antsrejected thenest

P 6). =0.049). P

ppm, theeffect disappeared =1), butnotforDMP (two- ppm (~2.5 P <0.01). However,

h afterchemical

ppm andanest ng ofDMP on

h (two-tailed

h.

ppm) still P

=0.07) 6) but 3231

2),

The Journal of Experimental Biology Nonanal Nonanal 2-Phenylethanol ,-iehlyaie(M)50 2,5-Dimethylpyrazine (DMP) Decanal DMP DMP DMP Benzyl alcohol Benzyl acetate different recipients.Forexample, insomeantspeciesyoungworkers alarm pheromoneoriginatinginthemandibulargland. 1998). To ourknowledgethisisthefirstreportofitsfunctionasan pheromone originatingfromthepoisongland(BillenandMorgan, myrmicine species.However, itistypicallyusedasatrail identified hereasanalarmpheromone,isalsoknowninother males andfemalesexuals(Vander Meeretal.,2010).DMP, dimethylpyrazine originatinginthemandibularglandsofworkers, a pyrazineasanalarmpheromone,specifically2-ethyl-3,6- only thefireant al., 1978;WheelerandBlum,1973).Amongthemyrmicineants, DMP 3232 0.5 dimethylpyrazine (DMP). ng)]of2,5- ppm(2.5 ng)and0.5 ppm(25.0 concentrations [5 Resultsofabinarychoicebetweennests withdifferent 5. Fig. brunneus part ofanalarmpheromoneintheponerinespecies 2,5-dimethyl-3-isopentylpyrazine havebeenreportedtobeatleast other antspecies.Forexample,2-ethyl-3,5-dimethylpyrazineand Pyrazines havebeenpreviouslyreportedasalarmpheromonesin behavioral bioassaysidentifiedDMP asanalarmpheromone. (formerly investigation ofalarmcommunicationinthegenus group defenseisunlikely(Maschwitz,1964). genera, whichhaveverysmallcolonysizes,becauseamassive alarm pheromonesmightbeabsentinspeciessuchasthosethese Temnothorax used. Thecloselyrelatedmyrmicinegenera Experiment 4.DMP wastheonlychemicalthatclearlyelicitedrejectionresponsesfromtestants. inthehead One nestalwayswastreatedwithhexaneasacontrol;theotheroneofchemicalcompoundsthatwereidentified Chemical compound RESEARCH ARTICLE Benzaldehyde DMP Table Alarm pheromonesmayhavedifferent behavioraleffects on Our presentstudyisthefirstdemonstrationandin-depth ppm nestoverthe 5

Frequency 1. A seriesofbinarynestchoicebioassays evaluatingcandidatealarmpheromones 15 10 20 0 5 and ). Chemicalanalysescombinedwith belong tothisgroup.Ithasevenbeensuggestedthat Odontomachus hastatus Solenopsis invicta 5 ppm

ppm nest(two-tailed binomialtest: There wasatrendtowardscolonies choosingthe 50 Immediately 5 Immediately 50 Immediately 50 50 Immediately 50 ocnrto pm nuto feirto etcmon eaecnrlSplit Hexanecontrol Test compound Inductionofemigration Concentration (ppm) 50 Immediately 0.5 Immediately 1 Immediately 5 Immediately 0.1 Immediately Experimental design 5 After 0.5 ppm has previouslybeenshowntouse , respectively(Longhurstet Leptothorax Split P Odontomachus =0.07). Temnothorax Immediately Immediately Immediately 14

and h consistent withthepatternMaschwitzdescribed.When investigation ofcontext-specificresponses.Ourfindingsare nest. Inthesubsequent50 close tothenest,butescapebehaviorwhenemittedfarfrom species, alarmsignalsreleaseaggressivebehaviorwhendischarged Maschwitz (Maschwitz,1964)showedthat,forsomehymenopteran specificity ofresponses.Inthefirstthoroughresearchonthistopic, is wellappreciated,littleattentionhasbeengiventothecontext (Duffield etal.,1976). 2,5-dimethyl-3-isopentylpyrazine fromtheirmandibularglands frantically evacuatetheareawhennestmatesreleasealarmsignal example, workersoftheponerineant with smallcolonies,incontrast,mayreactverydifferently. For buried nestmate(Wilson, 1958;Wilson andBossert,1963).Species behavior orperformdigginginanattempttorescuea heptanone. Athighconcentrations,theyeithershowaggressive to lowconcentrationsoftheiralarmpheromone,4-methyl-3- Pogonomyrmex vary amongdifferent species.Intheharvesterantgenus (Maschwitz, 1964;alsoseeHölldobler, 1977).Reactionsmayalso older workersmoveoutandexhibitaggressivebehavior respond toalarmpheromonesbyretreatingintothenest,whereas concentration was higherthan0.5 attracted toahexanecontrol.DMP significantlyattractedants whenthe index iscalculatedasthenumberof antsattractedtoDMP minus thenumber concentrations ofDMP whenpresentedinthehomenest. Theattraction Numberofantsattractedtocrushedheads anddifferent 6. Fig.

Attraction index Although thediversityofbehaviorselicitedbyalarmpheromones 10 15 –5 20 0 5 Heads 5 2 7 5 6 10 6 13 2 8 3 10 2 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.106849 *** , whichhavelarge colonies,oldworkersareattracted p . p 0.05ppm 0.1ppm 5 ppm **

years, therehasbeenlittlefurther ppm. **P 7 18 9 10 13 11 6 19 Choice 18 21 16 8 13 0.5 ppm *** <0.01; *** Hypoponera opacior P <0.001. 0 4 3 2 3 1 8 7 0 1 1 2 5 <0.01 0.77 0.63 0.10 0.33 1.00 0.38 1.00 <0.01 0.02 <0.01 0.81 <0.01 P

The Journal of Experimental Biology RESEARCH ARTICLE attainment ofconsensus onasinglesite,andmay alsoenhancethe recruitment tocompetingsites. Thismayservetospeedthe to recruitingthesitetheyhave found,usestopsignalstoinhibit collective choiceofanewnest site.Successfulscouts,inaddition foragers, isalsousedbynest sitescoutsduringacolony’s (Seeley etal.,2012).Thestop signal,notedaboveforitsuseby context ofdecision-making was recentlyfoundinhoneybees identified. A muchclearerexampleofnegativesignalinginthe 1998). However, noneoftheseproposed pheromoneshavebeen 1992; Robinsonetal.,2005;2008;Stout al., locking acolonyintosuboptimalchoice(GiurfaandNúñez, prevent thestrongpositivefeedbackofmassrecruitment from 1998). Theoreticalmodelspredictthatsuchrepellentsignals can 1992; Robinsonetal.,2005;2008;Stout al., from foraginginareasoflow-qualityfood(GiurfaandNúñez, suggests thatscoutsapplyrepellentpheromonestodeternestmates 2009; Seeley, 1995;Sumpter, 2010).Inafewspecies,evidence forces onthebestavailablefoodsource(HölldoblerandWilson, positive feedbackfrommassrecruitmenttoconcentrateforaging of acolony’s collectivedecision-making. Manyspeciesrelyon as awholetoavoiddanger(Blum,1985). it doesnotleadtorescueofthesignaler, butinsteadhelpsthecolony avoid movingthere.Thiseffect canbeconsideredaltruisticbecause tethered withinasitereleasesignalthatinducestheirnestmatesto 2010). Ourstudysimilarlyshowedthat perhaps toreducethecolony’s exposure todangerousareas(Nieh, recruitment toafoodsourcewheretheyhadbeenbrieflytrapped, vibrational communication–thestopsignaltosuppress appreciated. Honeybeeforagershavebeenfoundtouseaformof the roleofnegativefeedbackhas,untilrecently, beenless Jeanson etal.,2012;Sumpter, 2010;SumpterandPratt,2009),but making hasbeenextensivelyinvestigated(Camazineetal.,2003; whether thecompoundactuallyelicitsentrance-closingbehavior. preliminary, andfurtherinvestigationwillberequiredtoshow defensive purposes(Aleksievetal.,2007).Theseobservationsare and debristoreduceentrancesizeorevencloseitentirelyfor control. Thisisconsistentwithpreviousfindingsthattheyusesoil Movie attempted toclosethenestentrance(supplementarymaterial Video recordingsofpilottestssuggestthattheseworkersthen number ofworkersinsidethenestmovedtowardsentrance. the alarmsignalwasinsteadpresentedatnestentrance,alarge far fromtheirnest,theyexhibitedescapebehavior. Incontrast,when Temnothorax the hexanecontrols. compound. Eachantwastestedonlyonce.Allstatisticalcomparisonsareto value representsthenumberofantsthatavoided/attractedchemical hexane aslongtheconcentrationofDMP washigherthan0.1 were farfromtheirhomenest,theymorelikelytoavoidDMP than Ant behaviorwascategorizedaseither‘avoidance’ or‘attraction’.Whenants Attraction Avoidance Chemical compound ed 0085 0.003 8.57 0 6 10 4 5 Heads Hexane home nest Table 0.05 0.1 0.5

p M .90.019 5.49 1 9 ppm DMP Negative signalsmayalsocontributetothespeedoraccuracy The importanceofpositivefeedbacktocollectivedecision- p M .90.019 0.003 5.49 8.57 1 0 9 10 ppm DMP ppm DMP p M .20.64 0.22 7 3 ppm DMP

2. EffectsofcrushedheadsandDMP onantsfarfromthe 2), behaviorthatwasnotseenonexposuretoahexane workers perceivedthealarmpheromoneinarena Temnothorax χ 2

ppm. Each workers P emigration. how scoutsemitandrespondtothissignalduringcolony testing thisideamustawaitdetailedobservationsonwhetherand as anintegralpartoftheirdecision-makingstrategy. However, ants tothenest.We speculatethat marked nest,perhapscausinghertorefrainfromrecruitingother accomplish thisbyalteringthebehaviorofascoutthatenters 2014; T.S. and B.H.,personalobservation).Thesignalcould the colony’s probabilityofmoving tothenest(Stroeymeytetal., actually repelantsfromenteringamarkednest,butinsteadreduces 2005; Robinsonetal.,2008;Stout1998),thesignaldoesnot negative pheromones(GiurfaandNúñez,1992;Robinsonetal., have identifiedin determined, butitmaybethesameasalarmpheromonethatwe Stroeymeyt etal.,2011). Thenatureofthissignalhasnotbeen emigrations (Franksetal.,2007;Stroeymeyt2014; albipennis decisions remainsuncertain.Indirectevidenceindicatesthat nest choicestrategy, butthe potentialroleofinhibitionfortheir between decisionspeedandaccuracy(Marshalletal.,2009). populations allowsthemtomakeastatisticallyoptimaltrade-off numbers). Modelssuggestthatmutualinhibitionbetweenthe population firstcrossesathreshold(ofeitherneuralactivityorant evidence forcompetingoptions;adecisionismadewhichever In bothsystems,populations(ofeitherneuronsorants)accumulate Passino etal.,2008;SeeleyandBuhrman,2001;Visscher, 2007). decision-making systemsintheprimatebrain(Hofstadter, 1999; is remarkablysimilartoinhibitorypathwaysinanalogous and accuracy. Indeed,therole ofthesesignalsinnestsitechoice colony’s abilitytooptimizethetrade-off betweendecisionspeed size forbettervisualization. enlarged drawingatright).Thestrings areshownthickerthantheiractual the nestcavityusingasilkthreadthat waswrappedaroundthepetiole(see through thecenterofroof.InExperiment 1,fiveantsweretetheredwithin the nestweremadeofglassmicroscope slides.Anentranceholewasdrilled wood slatwithacircularholedrilled throughitscenter. Theroofandfloorof 7.Nest designandanttethering. Fig. nest waseitherahole(Ø=3.2 7).Theentranceofthehome drilled throughthecenterofglassroof(Fig. through themiddleofslat,andaroundentrancehole(Ø=2 microscope slides(50×75 from abalsawoodslat(2.4 experiments carriedoutinlaboratoryarenas.Eachcandidatenestwasmade We evaluatedpheromoneeffects inthecontextofnestsiteselection Nest designs MATERIALS ANDMETHODS Nest cavity Emigrating Roof Entrance The JournalofExperimentalBiology(2014)doi:10.1242/jeb.106849 leave adeterrentsignalinlow-qualitynestsduring Temnothorax Glass floor T. rugatulus.Inbothcases,unlikeotherreported mm). A circularcavity(38

mm) onthecenterofrooforaslit(2 colonies followaremarkablysimilar

mm thick)sandwichedbetweenglass Temnothorax Nests wereconstructedfromabalsa Balsa sheet 1 cm

mm diameter)wascut ants mayuseDMP

mm) was 3233

mm) T.

The Journal of Experimental Biology which tetheredantswereabsentduringthemigration.Theprocedurewas contacts withthetetheredants,wealsoconductedanotherexperiment,in as a‘split’ decision. colony’s choice.Ifthiscriterionwasnotachieved,thechoicerecorded members, includingallqueensandbrooditems,wedesignatedthatasthe nest tooneofthetarget sites.Ifonesitecontainedmorethan90%ofcolony inducing themigration.Ineverytrial,allantsmovedentirelyfromhome (Bhatkar andWhitcomb,1970;Sasakietal.,2013). capped withcottonandanagar-based dietthatwasrefreshedweekly Fluon-coated walls.Eachboxwasprovidedwithawater-filled plastictube 3234 methods fordetails). Thearenasizewas20×20 identical indesign butcontaineddifferent materials (seeMaterialsand Colonies wereallowedtochoosebetween twotargetnests,whichwere the homenest,fromwhichroof was removedtoinducemigration. Experimentalarenafornestchoicetests. 8. Fig. migration. target nests.Finally, theroofofhomenestwasremovedtoinduce was thenplacedagainstthecenterofwalloppositetolocation home nestcontainingthecolonyfromwhichtetheredantsweretaken 8).The placed adjacenttooneanotheragainstwallofthetestarena(Fig. a nestthathadfivestringsbutnoants.Thesetwotarget nestswerefirst each other(Fig. ants fromthesamecolonyweretetheredinnest,equidistant with adhesivetapebetweenthefloorglassandbalsasheet.Fiveworker (Haight, 2012).Thelengthofeachstringwas~2 America, Prescott,ONT, Canada)tiedaroundthepetioleusingaknottyer Ants weretetheredwithastringofsilk(partno.7.091,LouetNorth as thosedescribedabove.Nestswerekeptinaplasticbox(11×11 populations rangingfrom18to~300.Eachcolonywashousedinanestsuch queen, withworkerpopulationsrangingfrom121to280andbrood collected inthePinalMountainsnearGlobe,Arizona.Allhadatleastone used onlyonceineachexperimentexceptforExperiment5.Colonieswere A total126coloniesof chemical marksthattheantsmayhaveleft. experiment, theexperimentalarenawascleanedwithethanoltoremoveany coated withFluontopreventtheantsfromescaping.Beforeeach washing inacommercialdishwasher. Thewallsofexperimentalarenaswere fresh foreachexperimentandneverreused.Glassslideswerereusedafter cut outofthesidenest(Sasakietal.,2013).Balsaslatsweremade RESEARCH ARTICLE Experiment 1: do tethered ants release apheromone? antsrelease Experiment 1:dotethered Subjects To excludethepossibilitythatantsavoidednestasaresultofdirect The colony’s choicewasassayedbyrecordingthesiteoccupied12 Colonies weregivenabinarychoicebetweennestwithtetheredantsand

7). 17 cm Home nest Temnothorax rugatulus Target nests

cm and1 were used.Eachcolonywas 17 cm

cm withonesidefastened Colonies initiallylivedin 2 cm

cm inheight.

cm) with

h after was temperatureprogrammedfrom10°Cfor1 30 mode, withaninjectortemperatureof250°C.TheGCwasfitteda thermally desorbedintheinjectorportofGC/MSfor30 were replicatedwithtwosetsofdissected glands. volatiles. Thevolatileswerethenanalyzed asdescribedabove.Theanalyses a needle,andtheSPMEfiberwas insertedthroughtheholetocollect glass screw-capvialwithaTeflon septum.Theseptumwaspuncturedwith 9)andplacedina1 from theheadsoffreeze-killedworkers(Fig. were fromthemandibularglands,approximately35glandsdissected from AldrichChemicalCo.(Milwaukee,WI,USA). the crushedbodiesminusheads.Authenticstandardswerepurchased with thoseofauthenticstandards.Analogousanalyseswereconducted on identifications wereconfirmedbymatchingmassspectraandretention times identified bymatcheswiththeNISmassspectraldatabase, and USA), withelectronimpactionization(70 to a5975Cmassselectivedetector(AgilentTechnologies, Wilmington, DE, 280°C, hold20 recording thesiteoccupied12 these nests,asinExperiment1(Fig. colony fromwhichtheantsweretakenwastheninducedtochoosebetween nest receivedasimilarfilterpapermarkedwith5 paper (~1×1 (www.hamiltoncompany.com) toapply5 crushed withawoodenapplicatorstick.After3 Twenty headsfromthesamecolonywereplacedin100 the emigrationwasinduced14 pheromone wouldpersistovertime,werepeatedtheexperiment,exceptthat these nests,asinExperiment1(Fig. from whichthecrushedantsweretakenwastheninducedtochoosebetween similarly crushedeitherfivealitrunksorgastersinanothernest.Thecolony with awoodenapplicatorsticktoreleaseanypotentialpheromones.We placed fiveheadsinanest,equidistantfromoneanother, andcrushedthem forceps toseparateeachant’s headandgasterfromitsalitrunk.We then We freeze-killedfiveworkerants fromthesamecolonyandusedfine described above. 65 EOS RebelT2i;www.usa.canon.com) withamacrolens(CanonMP-E additionally filmedtetheredantsusingahigh-resolutioncamera(Canon induced. in thenestfor3 identical totheonedescribedaboveexceptthattetheredantswereleft to 50 All eightcompoundsidentifiedfrom themandibularglandwerefirstdiluted exposed totheheadspacevolatilesfor45 and aftercooling,thefiberwasinsertedintocoveredvialleft was cleanedbythermaldesorptioninaGCinjectorportat250°Cfor5 tightly coveredwithaluminumfoil.A polydimethylsiloxaneSPMEfiber were crushedwithaflat-bottomedglassrod,andthetopofvialwas of approximately50headsweretransferredto1.5 Riverside, ondryice.Afterthawing,theantsweredecapitated,andgroups Ants werefreeze-killedandshippedtotheUniversityofCalifornia, of heads? of inachemical present extract Experiment 3:isthepheromone thehead? comefrom Experiment 2:doesthepheromone Experiment 5:testingcandidate chemical compounds gland substancesinthemandibular Experiment 4:identification of criteria asinExperiment1. emigration usingthesamecriteriaasinExperiment1. choice wasassayedbyrecordingthesiteoccupied12 To confirmthatcompoundsfoundinthevolatilesfromcrushed heads To closelyobservethebehaviorofantsreleasingpheromone,we m×0.25 mm f/2.81-5xmacrolens).Theantsweretetheredinthesameway ppm inhexane,or evenlowerifa50 mm IDDB-5column(J&W Scientific,Folsom,CA,USA),and cm), whichwasthenplacedinastandardnest(Fig. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.106849 min. Analyseswereconductedwitha6890NGCinterfaced h andthenremovedimmediatelybeforethemigrationwas

h afterinducingtheemigrationusingsame

h aftercrushingthebodyparts.Thecolony’s

8). Thecolony’s choicewasassayedby

8). To testwhethertheeffect ofalarm μl ofthissolutiontoasmallfilter

eV). Compoundsweretentatively

min. Theloadedfiberwasthen

ppm dilutionelicited aneffect. h, weusedaglasssyringe

min, then10°C ml glassvials.Theheads μl ofpurehexane.The h afterinducingthe μl hexaneand

s insplitless

7). Another

ml tapered

min

− min, 1 to

The Journal of Experimental Biology ant wastestedonlyonce. (walking towardsthestick).Theorderoftestswasrandomized,and each categorized aseitheravoidance(walkingawayfromthestick)orattraction inside thehomenest,anda The statisticalpackageR(v. 2.9.0)wasusedforallanalyses. investigating avoidanceofDMP awayfromthehomenestinExperiment6. RESEARCH ARTICLE We thank KevinHaightforteachingushow to maketheknottyer. A two-tailed Experiments 1,2,3and5.Splitcolonies werenotincludedintheanalyses. We testednestsitepreferencesusingatwo-tailedbinomialtestin that wereatleast10 DMP wasfirstappliedtoastick.We thenslowlypresentedthesticktoants when theywerenotinthehomenest.Similartoprevioustest,ahead or stick andatreatedwithhexaneascontrols. mandibular glandhadbeenremoved.Finally, wepresentedanuntreated we alsopresentedadissectedmandibularglandandheadfromwhich the purchased fromSigma-Aldrich(StLouis,MO,USA). was randomized,andatleast45 already bytheentrancewhenstickwasintroduced.Theorderoftests completely crossedthislinewascounted).We didnotcountantsthatwere entrance wasplacedonthecomputerscreenandanumberofantswho home nestmovedtowardstheentrance(i.e.1 nest. Thereactionwasmeasuredbycountinghowmanyantswithinthe solution] toastick.Thestickwasthenslowlypresentedneartheant’s home 5 ppm (10.0ng),0.5(1.00.1(200pg)or0.05(100 We crushedaheadwithwoodenapplicatorstickorappliedDMP [2 Langridge etal.,2008). previous migrationsonthecurrentmigration(Langridgeetal.,2004; elapsed betweenexperimentsonagivencolony, toavoidanyinfluenceof colony experiencedthesamecompoundmorethanonce.Atleast10 of 69colonieswereused,andallusedthreeorfourtimes,butno inducing theemigrationusingsamecriteriaasinExperiment1.A total induced tochoosebetweenthesenests,asinExperiment1(Fig. hexane toafilterpaperandplaceditinanidenticalnest.A colonywasthen paper andplaceditinthestandardnest(Fig. As inExperiment3,weapplied5 pulling amandiblewithfineforceps. Dissected mandibulargland.Theglandwasremovedbycarefully 9. Fig. Acknowledgments Statistical analysis contexts?different in behaviors elicitdifferent Experiment 6:doesthepheromone We furthertestedhowtheantsrespondedtosamealarmpheromone To confirmthatthesourceofpheromonewasmandibulargland, The colony’s choicewasassayedbyrecordingthesiteoccupied12 t -test wasusedforinvestigatingattraction releasedbyDMP

cm awayfromtheirhomenest.Theirreactionwas χ 2 test ofpartialindependencewasused for

μl ofonethesesolutionstoasmallfilter min elapsedbetweentests.TheDMP was Mandibular gland

7). We alsoapplied5 0.5 mm

cm markfromthe

8).

h after μl of μl of

days ufed .M,Bu,M .adWelr J.W. N.R. Franks, Wheeler, and M.S. Blum, R.M., Duffield, rdhw .W . ae,R n os,P. E. Howse, and R. Baker, P. E. J.W. S., Howse, Bradshaw, and R. Baker, J.W. S., Bradshaw, M.S. Blum, M.S. Blum, Franks, ufed .M n lm M.S. Blum, and R.M. Duffield, D. Fletcher, and R.M. Crewe, Sneyd,J.,Theraulaz,G.and I.D. N., Couzin, Franks, and J. Deneubourg, S., Camazine, M. Lindauer, and B. Linzen, A., Butenandt, ucigr .adMshiz U. Maschwitz, and A. Buschinger, io . upe,D .T,Mae .adGifr,T. Guilford, and J. Meade, D.J.T., Sumpter, D., Biro, http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.106849/-/DC1 Supplementary materialavailableonlineat and byfundsofArizonaStateUniversitytoB.H. This workwassupportedbytheNationalScienceFoundation(awardno.1012029) the paper:T.S., B.H.,J.G.M.andS.C.P. experiments: T.S., B.H.andJ.G.M.Analyzeddata:T.S., J.G.M.andS.C.P. Wrote Conceived anddesignedtheexperiments:T.S., B.H.andS.C.P. Performedthe The authorsdeclarenocompetingfinancialinterests. rns .R,Hoe,J . onas . uet .J,Hyad .L and A.L. Hayward, P. J., Aukett, A., Dornhaus, J.W., Hooper, N.R., Franks, lkiv .S,SnoaFak,A .adFak,N.R. Franks, and A.B. Sendova-Franks, A.S., Aleksiev, iln .adMra,E.D. Morgan, and J. Billen, W. H. Whitcomb, and A. Bhatkar, is,A n odn D. Gordon, and A. Hirsh, R. Yamaoka, and M. Mizunami, T., Takeda, N., Yamagata, N., Fujiwara-Tsujii, References Supplementary material Funding Author contributions Competing interests osatr D.R. Hofstadter, K.L. Haight, W. Goetsch, J.A. Núñez, and M. Giurfa, ölolr .adWlo,E.O. Wilson, and B. Hölldobler, ölolr .adTyo,R.W. Taylor, and B. Hölldobler, B. Hölldobler, 54, 439-440. pheromones inprimitiveantswithsmallcolonialunits. 66, 1357. function in Oecophylla longinoda. pheromones inthemandibularglandsofmajorworkersAfricanweaverant, pheromones oftheweaverant. Gilbert), pp.193-224.NewYork, NY: PergamonPress. Behaviour, Biochemistry andPharmacology: leadership inpigeonhoming. secretion of 43. Princeton UniversityPress. Bonabeau, E. Microsc. Morphol.Exp. Mandibeldrüse derBlattschneiderameise pp. 95-150.NewYork, NY: Praeger. mechanisms inants.In ants. Fla.Entomol. M. L.Winston),pp.3-33.Boulder, CO:Westview Press. Wasps, BeesandTermites sources andsecretions.In 274 S.M. Berghoff, . Philos.Trans. R.Soc.B Information flow, opinionpolling andcollectiveintelligenceinhouse-huntingsocial the antTemnothorax albipennis Math. Artif.Intell. 1574. obscuripes (2006). Behavioralresponsestothealarmpheromoneofant Harvard University. workers ofJerdon’s jumpingant, Portig. visited flowers. Nothomyrmecia macrops University Press. Communicate , 1505-1509. .R,Pat .C,Mlo,E . rto,N .adSmtr D.J. Sumpter, and N.F. Britton, E.B., Mallon, S.C., Pratt, N. R., (1969). Alarmpheromones. Neoponera villosa (: Formicidae). (1953). (2009). Collectivecognitioninanimalgroups. (1989). Armyants:acollectiveintelligence. Paltothyreus tarsatus The JournalofExperimentalBiology(2014)doi:10.1242/jeb.106849 (1985). Alarmpheromones.In (2012). Patternsofvenomproductionandtemporalpolyethismin (1977). CommunicationinsocialHymenoptera. In (ed. T. A.Sebeok),pp.418-470.Bloomington, IN;London:Indiana Oecologia (1999). (2003). (2007). Reconnaissanceandlatentlearninginants. 31, 199-221. 53, 229-232. Vergleichende BiologiederInsektenstaaten Physiol. Entomol. Gödel, Escher, Bach 48, 13-19. Defensive MechanismsinSocialInsect Self-organization inBiologicalSystems (2001). Distributedproblemsolvinginsocial insects. Clark. InsectesSoc. 89, 113-117. (ed. R.K.Vander Meer, M.D.Breed,K.E.Espelieand (1992). Honeybeesmarkwithscentandrejectrecently Curr. Biol. (1974). Ponerineantsecretions:themandibulargland (1998). Pheromonecommunicationinsocialinsects: Pheromone CommunicationinSocialInsectsAnts, (Hymenoptera: Formicidae). . Anim.Behav. Nature (1990). 357 Harpegnathos saltator (1970). Artificialdietforrearingvariousspeciesof Fabr. J.Entomol.Soc.SouthAfrica (1973). 4-Methyl-3-heptanone:identificationand (1983). A behavioralstudyoftheprimitiveant , 1567-1583. Annu. Rev. Entomol. (1984). Defensivebehavioranddefensive 258 The Ants.Cambridge,MA:Belknap Pressof 16, 2123-2128. Zoolog. Sci. 4 Atta sexdensrubropilosa , 15-25. , 230-231. . NewYork, NY: Basic Books. 30, 384-401. (1959). ÜbereinenDuftstoff ausder 74, 567-575. Vol. 9(ed.G.A.KerkutandL.I. Comprehensive InsectPhysiology, 23, 353-358. (1979). Multicomponentalarm (1975). Multicomponentalarm Am. Sci. (2006). Fromcompromiseto (1976). Alkylpyrazinealarm . Comp. Biochem.Physiol.B J. InsectPhysiol. Trends Cogn.Sci. (2007). Nest‘moulting’ in 14, 57-80. Ann. Entomol.Soc.Am. (ed. H.R.Hermann), . Leipzig:Geestund 77, 138-145. Forel. . Princeton,NJ: Proc. Biol.Sci. 37, 291-298. How Camponotus Arch. Anat. 58, 1568- 13, 36- (2002). 3235 Ann.

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