Received 13December 2001 Accepted 19February 2002 Publishedonline 20 May 2002

Regulation of reproduction ina queenlessant: aggression,pheromonesandreduction incon ict Virginie Cuvillier-Hot 1* ,Raghavendra Gadagkar 2,Christian Peeters 1 and Matthew Cobb 1 1Laboratoired’ Ecologie, CNRS UMR7625,Universite ´ Pierre-et-MarieCurie, 7quaiSaint Bernard,75005 Paris, France 2Centrefor Ecological Sciences, Indian Institute ofScience, Bangalore560 012, India In themonogynous queenless ant Diacammaceylonense ,thefuture reproductive (futuregamergate) isvery aggressive towardsinfertile workersduring theŽ rstdays of her adult life. Overt aggression disappears at about threeweeks, when the future gamergate begins tolay male-destinedeggs andis ready tomate. Over thesame period, her cuticular hydrocarbon proŽle alters, changing from achemical signature similar tothat ofasterile worker towardsthat ofa gamergate. In nature,these behavioural andchemical changes will coincidewith areductionin conict within thenest: faced with avirgin futuregamergate, infertile workershave aninterest in producing male-destinedeggs; however, once the gamergate producesfemale eggs,they have aninterest in rearing her offspring.This demonstrationof ashift from physical inhibition tochemical signalling is interpretedin termsof sociogenetictheory, therole ofcuticular hydrocarbonsas anindicator offertility in insectsand the fact that theregulation ofreproduction in Diacamma involves mechanismsredolent of both queenlessand queenright ant species. Keywords: reproductivecon ict; chemical signalling; cuticular hydrocarbons; Diacamma; gamergate

1. INTRODUCTION have apparently equivalent reproductivepotentials. How- ever,in many species,a single individual monopolizes In theeusocial Hymenoptera, reproductive conicts are reproduction.Dominance interactions regulate whichant particularly complex becausevirgin femalescan lay unfert- becomesthe ‘ gamergate’(i.e. inseminated and egg-laying ilized, male-destinedeggs, creating diverging interests worker) (Peeters1993). Only dominantworkers are sex- betweencolony members. However, when a mated repro- ually active andcopulate when a foreign male visits the ductiveis present, virgin relatives always beneŽt from rais- nest(Ito &Higashi 1991; Monnin& Peeters1998; Gobin ing her diploid (female) offspring (review in Bourke& et al. 2001). Betweenthe onset of sexual maturity and Franks 1995). The questionof how workers detect the mating, the‘ futuregamergate’ lays unfertilizedmale- presenceof a reproductiveindividual lies at theheart of destinedeggs, as other workerscould do. During this thebehavioural basesof sociality. period,a sharp conict thusexists between the future In many social insects,queens produce a pheromone gamergate andordinary workers.However, once the that ensuresthat workersdo not lay haploid eggs (Vargo gamergate beginsto produce female-destined eggs, the 1998). These‘ queenpheromones’ were long thought to conict is resolved:as in aqueenright species,ordinary act directly onthe physiology ofworkers, inhibiting egg workersmaximize their inclusiveŽ tnessby repressing their laying. However,Keller &Nonacs(1993) predictedthat ovarian activity andby rearing thefemale-destined eggs if queenpheromones were honest signals rather than producedby thereproductive. This article focuseson this physiological inhibitors, thenworkers should suppress change in reproductive conict. their ownreproduction when queens produce both sexes, In themonogynous queenless ant genus Diacamma, as butnot when queens produce only males.Such differen- in other queenlessant species,the future gamergate pro- tial repressionwould not occur if queenpheromones were ducesexclusively haploid eggs for avariable period of inhibitory. In mostant species,no such effect can be time. However,the genus Diacamma is uniquein that observedbecause queens always mate beforethey start monogyny is determinedby anirreversible behavioural laying eggs.However, in mostqueenless ants, future mechanism,which strongly reducescon ict between reproductivesstart tooviposit beforethey mate andthus workersand the reproductive. Workers eclosewith two have aninitial phaseof laying male-destinedeggs before tiny innervatedthoracic appendages(‘ gemmae’; Peeters& producing both femalesand males. This situationprovides Billen 1991; Gronenberg& Peeters1993), whichare auniqueopportunity for testingthe nature and effect of essentialfor mating (Fukumoto et al. 1989; Peeters& signals producedby thereproductive individual. Higashi 1989). Soonafter aworker ecloses,its gemmae In queenlessant species,workers have retainedthe are removedby thegamergate orfuturegamergate: these ability tomate andstore sperm (Peeters1991). All female mutilated workerscan never mate andproduce females, members ofthe colony are morphologically similar and although they retain their ability toproduce males. In Diacamma,thecon ict betweenthe gamergate, whopro- ducesfemale-destined eggs, and the rest of the workers, *Authorfor correspondence ([email protected]). whocannot, is thussimilar tothat in queenright species,

Proc.R. Soc.Lond. B (2002) 269, 1295–1300 1295 Ó 2002 TheRoyal Society DOI10.1098/ rspb.2002.1991 1296V. Cuvillier-Hot andothers Queenlessant reproduction andfar lessthan in ‘classic’ queenlessants, in whichdomi- determinedby regularlychecking for fresh eggs inthe colonies; nantindividuals retain their ability tomate andcan we alsoobserved oviposition on anumberof occasions.In each replace thegamergate. The situationin Diacamma, com- group, we recordedthe Ž rst occurrenceof ‘sexualcalling ’: this bining aspectsof both queenright andqueenless species, conspicuousbehaviour, duringwhich the futuregamergate rubs makesit possibleto explore whetherthe regulatory mech- herhind legs on hergaster, continuesdaily and ceasesimmedi- anismsinvolved in thereproductive division oflabour vary atelyafter mating (Peeters et al. 1992;Nakata et al. 1998). Dur- according tothe intensity of the con  ict. ing the periodof behaviouralobservations, futuregamergates Studies of Diacamma specieshave suggestedthat there werenot allowedto encounterforeign males and thus remained are differentmodes of regulation at differentmoments in virgin,arti Ž ciallyprolonging the periodof potentialcon  ict thelife ofthegamergate. In D. australe,thefuture gamer- betweenthe futuregamergate and the restof the workers.We gate persistentlyharasses nestmate workers, whereas alsostudied two colonieswith agamergate (onewild-caught, gamergates (that is,fertilized reproductives)are not and the other matedin the laboratory) ( n = 10observations of aggressive, suggesting that mating isassociated with a 30mineach). The gamergates that had beenstudied were also change in regulatory mechanism (Peeters& Higashi dissectedto verifythat theirspermathecae were Ž lled with 1989). However,this studydid not examine changesin mobilesperm. thefuture gamergate ’sbehaviour betweeneclosion and mating. (c) Chemical analyses In Diacamma sp.from Japan, inhibition ofworker egg Cuticularhydrocarbon pro Ž lesof livingants weremeasured laying requiresdirect contact with thegamergate, suggest- with direct-contactsolid-phase microextraction(SPME) and gas ing that sheproduces chemical signals (Tsuji et al. 1999). chromatography. Unlikedestructive solvent extraction, SPME It is possiblethat acontactpheromone, perhaps composed allowsrepeated observations of liveinsects. A polydimethylsilox- ofcuticular hydrocarbons,is involved, asis apparently the ane SUPELCO Ž brewas rubbed for 2minagainst the interseg- casein other queenlessants (Peeters et al. 1999; Liebig et mentalmembranes between the sixth and eighth abdominal al. 2000). In D.ceylonense ,thereis astriking correlation tergites,after which the ant was returnedto hernest (Cuvillier- betweencuticular hydrocarbon pro Ž lesand ovarian Hot et al. (2001)provide full details of chemicalanalysis). activity (Cuvillier-Hot et al. 2001), suggesting that ants Elevenfuture gamergates (10of which werestudied may usevariations in theblend of hydrocarbonsto reveal behaviourally)were measured either in the few days after emerg- thefertility ofindividuals. ence (0–4 days old; n = 5;FG1 group), orat severalweeks old In order tosee whether there is a relation betweenregu- (25–66 days old; n = 9;FG2 group); threefuture gamergates latory mechanism,fertility, mating statusand the nature werethus measuredtwice. Two futuregamergates matedin the andintensity of intracolony con  ict,we measuredchanges laboratory (onewas usedfor behaviouralobservations, see in behaviour andcuticular hydrocarbonsin D.ceylonense above);they wereeach measured three times after mating, at futuregamergates betweeneclosion and mating. various ages. Inthe analyses,we includeddata fromCuvillier- Hot et al. (2001)for other functionalgroups inthe colony,i.e. gamergates (Gg, n = 13),young callows(C1, 0 –4 days old, 2. MATERIALAND METHODS n = 17),nurse workers (C3,16 –42 days old, n = 11)and foragers (a) Samples (F, n = 33).Future gamergates belongingto the FG2group were Fivewhole coloniesof D.ceylonense (231 ± 102adult workers approximately the sameage as the C3workers (orwere even percolony) were collected from Bangalore (southern India) in older);however, C3 workers wereall completely infertile, October1998 and Apriland June2000. All workers wereindi- whereas allFG2 ants had begun to lay eggs. Two futuregamer- viduallymarked with paint, includingthose that eclosedin the gates (outof 16)did not performsexual calling, yet they had laboratory. In Diacamma, the gamergate orfuture gamergate can begun to lay eggs. They weresubsequently found deadoutside berecognized visually because she aloneretains her gemmae. the nests, aged 21and 36days old.In the days precedingtheir The wild-caught gamergate was removedfrom four colonies. death, mutilatedworkers had frequentlyattacked them.These Afterremoval of the gamergate, the Ž rst worker to eclosein each two individualswere excluded from the chemicalanalysis (see of these coloniestook onthe rank of futuregamergate, because § 4). therewas no reproductivepresent to removeher gemmae (this The correctedareas of19identi Ž edhydrocarbon peaks were iswhat occursnaturally following the death of the gamergate or usedas variablesto performa discriminantanalysis. These peaks colony Ž ssion).To increaseour sample size of futuregamer- includedthe 16peaks usedin Cuvillier-Hot et al. (2001), gates, the two largest colonieswere divided into three groups of together with two alkanes(n-C26 and n-C30)and an alkene 50–100mutilated workers together with cocoonsand younger (n-C27:1)chosen on a heuristicbasis. Each of these three brood; 16future gamergates werethus studied. additionalpeaks makesup lessthan 2%ofthe cuticularhydro- carbons. The discriminantanalysis clearlyseparated the three

(b) Observations groups Gg, Fand C1(Wilk ’s l = 0.011;approx. F38,84 = 18.85; The futuregamergates wereobserved at irregularintervals p , 0.00001; 100% correct classi Ž cation; Statistica software), fromthe timeof eclosionuntil they wereseveral weeks old,after which arerepresented by theircentroids. All the other groups which they weredissected to measuretheir ovarian develop- wereplotted on the graph as ‘illustrativeindividuals ’ without ment,using the ad-hoc scaleof Cuvillier-Hot et al. (2001).Dur- beinginvolved in the analysis. ing each30 minobservation period,we notedall aggressive eventsinitiated by the futuregamergate (antennalboxing fol- 3. RESULTS lowedby crouchingof the target individualand forward lunges with biting).Mutilation attempts directedat callowworkers In the Ž rstdays of their adult life, futuregamergates werenot consideredin this study. The onset of egg laying was frequentlypatrolled andbehaved aggressively towards

Proc.R. Soc.Lond. B (2002) Queenlessant reproduction V.Cuvillier-Hot andothers 1297

70 (a) is signiŽ cant(Page test,standardized F = 2.634, p , 0.005).

s 60 Futuregamergates began toperform sexual calling at t c

a ca.17 daysold (median value; range 10 –33; n = 9) and

e

v 50

i began egg laying at ca.19 daysold (median value; range s s

e 6–41; n = 11). Dissectionsindicated that all thefuture r

g 40

g gamergates over 24 daysof age ( n = 11) had ovaries with a

f mature eggs (state4 according toCuvillier-Hot et al. o

30 r

e (2001)). Matureeggs canbe present when the future b

m 20 gamergate isonly 10 daysold. There isthus a clear nega- u

n tive correlation betweenovarian activity andaggressive 10 behaviour: very young futuregamergates cannotproduce eggs andthey attack nestmates,whereas this aggression stopsaround the time that egg-laying begins. 0 10 20 30 40 50 Animportant degreeof variability in theinitial level of aggression wasobserved ( Ž gure 1a,b).This canbe (b) accountedfor by colonydifferences produced by thevary- s t

c ing periodsbetween the experimental removal ofthe a

e previous reproductivefrom thecolony and the appearance v i s

s ofa newfuture gamergate following eclosionof the Ž rst e r

g cocoon (0–44 days;median = 6). It takes ca. 13 days

g 40 * a

(median value, n = 5) beforethe ovaries oforphanedmuti- f o

r 30 lated workersbecome fully active; asa result,in somecol- e

b onies,mutilated workershad startedto lay male-destined m

u 20 * eggs beforethe new future gamergate eclosed.On six n

n occasions,we observed a worker oviposit in thepresence a 10 e *

m n.s. ofa futuregamergate. This situationis unusual, and all 0 suchovipositions occurredwhen the future gamergate was 0–9 gamergates 10– 1920 –2930–49 lessthan 5daysold; in twocases, she immediately ate the age (days) egg. Dissections( n = 87) conŽ rmed that mutilated work- ersgenerally have inactive ovaries (state1 or 2; Cuvillier- Figure 1. (a)Number of aggressive acts(antennal boxing and biting) performed byfuture gamergates(FG) atdifferent Hot et al. 2001). ages. Eachsolid square corresponds to asingle 30 min Wehave previously shownthat thegamergates and period of observation of asingle FG( n = 14). Apolynomial infertile workersof D.ceylonense differin their blendsof regression curve Ž tsthe decreasing trend. Crosses refer to cuticular hydrocarbons,and that thecuticular pro Ž le of thetwo FGs thatnever performed sexual calling and were young infertile workerschanges with age tobecome ident- Ž nally found dead outside thenest (see § 2). Therange of ical tothat offoragers (Cuvillier-Hot et al. 2001). In the theage of onset of sexual calling (continuous line) and of presentstudy, using 19 hydrocarbon peaks(see § 2), gas egg laying (dashed line) of thesefuture gamergatesis given chromatography data indicatedthat thecuticular pro Ž les in theupper part of the Ž gure. Thecentral vertical line on ofyoung futuregamergates (FG1) are similar tothose of eachbar indicates themedian value. All individuals remained virgin throughout theexperimental period. young callows(C1 group; MANOVA, F = 0.73, p, n.s., (b) Data from (a)presented asthe mean number of Jump software;see Ž gure 2). Futuregamergates that had aggressive actsby future gamergates( n = 16) atdifferent ages begunlaying eggs (FG2) had strikingly differentpro Ž les compared withthe behaviour of gamergates( n = 2, 10 h of tothose of infertile workersof the same age (C3).Finally, observation). Error barsshow the standard error. Future theontogeny of the cuticular pro Ž le oftwoyoung gamer- gamergateswere grouped according to agefor statistical gates mated in thelaboratory showsa drift towardsthe analysis. 0–9 days, n = 15 observation periods; 10 –19 days, group ofolder gamergates ( Ž gure 2b).Notethat theFG2 n = 8; 20–29 days, n = 9; 30–49 days, n = 5. When an FG group wasnot discriminated from theC1 group whenonly wasobserved for several periods in agiven agegroup, the the16 largest peaksused by Cuvillier-Hot (2001) mean value of her scores wasused. Asterisksshow signi Ž cant et al. differences in comparisons between thegamergate group and wereconsidered (data notshown) —this canbe taken to eachage group of future gamergates( p , 0.005), as indicatethe importance ofsmall peaksin thecuticular sig- measured bypermutation tests. Corresponding changesin nal. From acharacteristic cuticular pro Ž le commonto all ovarian development withage are shown in theupper part of callow workers(with or withoutgemmae), thepro Ž le of the Ž gure (only one ovary shown). afuturegamergate becomessimilar tothat ofagamergate. In parallel, her aggressivenessdeclines to virtually zero. Futuregamergates andgamergates differin fertility: the cuticleof the gamergates wassampled soonafter Ž eld col- almost every worker they encountered( Ž gure 1a,b). The lection,when they exhibit maximum ratesof egg laying latter crouchedand never resisted. This aggression (threeeggs per day);in contrast,future gamergates (FG2) decreasedprogressively from ca.10 daysold, reaching the had only recentlybegun to oviposit (oneegg per day). level typical ofa gamergate (meanvalue = 0.4, n = 10 This probably explains thedifference between FG2 and observations)after ca.30 days.This declinein aggression thegamergate group.

Proc.R. Soc.Lond. B (2002) 1298V. Cuvillier-Hot andothers Queenlessant reproduction

12 (a) ferenttypes of con  ict within thenest. In the Ž rst weeks 10 ofadult life, thefuture gamergate cannotlay eggs ormate; during this period,she uses aggression tosubduethe other 8 workers,all ofwhom could produce male-destined eggs ) 6 C1

% andare thusin con  ict with her. 2 . 4

7 With sexual maturity, whichin naturewill berapidly 3 ( 2 followedby mating andthe production of female-destined 2

t eggs,one level ofcon  ict within thenest is resolved:ordi- o 0 o r nary workersnow have aninterest in rearing thegamer- –2 F Gg gate’sfemale offspring.At this stage,the gamergate stops –4 being aggressive andproduces chemical signals that re  ect –6 her reproductive state.The shift from physical inhibition tochemical signalling that wehave demonstratedhere thuscoincides with areductionin within-colonycon  ict. 12 (b) ArtiŽ cially inducedinterqueen con  ictsin polygynous ant 10 speciesshow changes between aggressive andchemical

) 8 controlthat are associatedwith differencesin reproductive %

2 44 activity (Ortius &Heinze1999). Our studyshows the . 6 C1 7

3 importance ofnaturally occurring developmental changes (

4 2

in theregulation ofreproduction, in relation tochanging t 2 65 o

o 79 genetic con ictsat thecolony level. r 0 49 78 98 It might beexpected that, becausethe decisive –2 F reductionin geneticcon  ict within thecolony in fact Gg –4 occurswhen the gamergate producesfemale-destined eggs following mating, ordinary workersshould be able to –6 detectthis eventthrough aputative ‘fertilization signal ’, –6 –4 –2 0 2 4 6 8 10 which is associatedwith sexual calling, thepresence of a root 1 (62.8 %) foreign male in thecolony, mating or eventhe presence Figure 2. Ontogeny of future gamergate cuticular offertilized eggs.However, by experimentally prolonging hydrocarbon pro Ž les. (a)Future gamergatesthat have begun thedelay betweenthe onset of sexual calling andthe pro- to layeggs (FG2, open diamonds) compared withfuture ductionof female-destined eggs, we have shownthat the gamergatesthat have yet to layeggs (FG1, open triangles) stimulusthat leads D.ceylonense workersto repress their and nurses (C3, open squares) of thesame age as FG2. fertility isan honest signal that thereproductive individual Adiscriminant analysisusing gamergates(Gg), foragers (F) producesbefore and after mating. This signal re  ects her and young callows (C1, 0 –4daysold) (Cuvillier-Hot et al. fertility rather than her matedstatus. The factthat, in a 2001) wasused asa framework, asrepresented bythe numberof cases, egg-laying futuregamergates ceased centroids of thedistributions of thesethree groups; theFG1, being aggressive beforethe onset of sexual calling (see FG2 and C3 datawere superimposed on this. FG1, infertile Ž gure 1a)canbe taken as proof that thereis no ‘fertiliz- future gamergates(0 –4 days old, n = 5); C1, young callows ation signal ’ in this species.It shouldbe noted ( Ž gure 1a) (0–4 days old, n = 17); FG2, fertile future gamergates (25–66 days old, n = 9); C3, nurses (16 –42 days old, n = 11); that thepresence of a fertility signal doesnot necessarily foragers (. 42 days old, n = 21); Gg, gamergates( n = 13). andimmediately meanthat eggs are being produced:some (b)Individual changesin thecuticular hydrocarbon pro Ž les individuals clearly usechemical signalling rather than of two FGs thatwere inseminated in thelaboratory. Each aggression, buthave yet tobegin egg laying. Weinterpret individual wasmeasured atthree different times after this asmeaning that theprocesses underlying thefertility mating. Thenumber next to eachof thethree points refers signal are more rapidly effectivethan thoseinvolved in egg to theage, in days, of theFG when themeasure wastaken. production,even though they both have thesame physio- Arrows showthe direction of change withtime. logical starting point. Usingfertility, rather than fertilization, asan indicator ofthedegree of con  ict within thecolony would have the 4. DISCUSSION advantage ofalso providing directinformation about the After acolonyof D.ceylonense is orphaned,the Ž rst quality ofthe reproductive in termsof productivity. How- worker toemerge becomesthe future gamergate. Sheis ever,if thefuture gamergate werenot to mate, mutilated initially very aggressive towardsher nestmates,but this workerswould no longer bene Ž tfrom remaining sterile aggression decreasesdramatically after 3 –4 weeks (Ž gure 1). butwould nevertheless be unable to detectthis situation. The aggression shownby young futuregamergates is Although thereare no Ž elddata onthe duration ofthe exceptional becauseant callowsare normally timid (this period betweenthe onset of sexual maturity andmating, is also trueof mutilated D.ceylonense callows). it seemshighly improbable that this period will bevery It is signiŽ cantthat thetemporal change in thebehav- long. The stability ofthe system, and the very factthat iour ofthe future gamergate andthe development of a Diacamma workerscannot detect whether a sexually gamergate-like cuticular pro Ž le coincidewith theonset of mature futuregamergate has matedor not,suggests that egg laying andof sexual calling (sexual maturity). Wesug- theselection pressure is extremely weak.Furthermore, gestthat thetwo phases in her behaviour correspondto males are producedthroughout theyear (R.Gadagkar and differentunderlying physiological statesand relate todif- C.Peeters,unpublished data): for afuturegamergate, the

Proc.R. Soc.Lond. B (2002) Queenlessant reproduction V.Cuvillier-Hot andothers 1299 limiting factor controlling therapidity ofmating will eggs,and queue to replace her assoon as her ef Ž ciency merely bethe probability that aforeign male locatesher declines,either in termsof fertility orability todominate. nest. In thesespecies, in whichcon  ict isparticularly strong The premature deathof two future gamergates that at thetop ofthe hierarchy (Monnin& Ratnieks2001), neverperformed sexual calling (see § 2), and the regulation ofreproduction relies onboth behavioural aggression they weresubjected to, may provide uswith interactionsand chemical communication.For instance, someclues about thefactors that wouldprevent workers in Dinoponeraquadriceps, thegamergate continuesphysical from becoming thevictims ofsuch a fertility-based recog- interactions,mainly with thesecond-ranking worker who nition system.We do not know how the workers detected has ahigh probability ofreplacing her (andsubsequently thesenon-calling futuregamergates, which diedaged 21 mating) (Monnin& Peeters1999). Suchbehaviour would and36 days.Age wasclearly nota factor:some future make nosense in Diacamma species,in whichthe key con- gamergates wereobserved for uptothreemonths, during  ict betweenthe reproductive and the workers is irreversi- which time they continuedto perform sexual calling and bly settledat eclosion,via themutilation ofthe callow thelow level ofaggression in their coloniesremained worker’sgemmae by thegamergate or thefuture gamer- unchanged,with noneof the mutilated workerslaying gate. Aslong asthe gamergate ’sfertility isadequate, muti- eggs. lated workersshould self-regulate their oogenesiswith no Variations in cuticular hydrocarbonsare reliable mark- needfor aggressive interactions.As we have shown,this ersof ovarian activity in other ponerineants and in some is thesituation for themajority ofthe time in a wasps(Monnin et al. 1998; Liebig et al. 2000; Sledge et Diacamma colony. al. 2001). Experimental manipulation ofsocial statuslead- The existenceof a fertility signal madeup of cuticular ing topredictable changesin cuticular pro Ž lesprovides hydrocarbonsas a regulator ofsocial peace,generally evenmore convincingdata ofthe link betweenovarian coupledwith aggression, may bewidespread in primitively activity andcuticular hydrocarbons.Our cuticular meas- organized insectsocieties (Peeters et al. 1999; Liebig et ures of D.ceylonense futuregamergates showthat their al. 2000; Sledge et al. 2001). Diacamma speciesappear proŽ le developsinto one similar tothat ofa gamergate. torepresent a uniquesituation among queenlessants: the Wesuggest that, assoon as the future gamergate ’s cuticu- regulation ofreproduction is irreversible (asin queenright lar proŽ le reveals that sheis suf Ž ciently fecund,nestmates species),and the role ofaggression by thereproductive is recognizeher egg-laying statusthrough this signal and minimal compared with that ofchemical communication consequentlysuppress their ownovarian activity. Asa basedon an honest signal that reveals thefertility ofthe result,sexually mature futuregamergates nolonger need reproductive.It seemspossible that, in queenright species tobehave aggressively towardstheir nestmates. also,queen pheromones will turnout to beat least partly Although bioassay data showingthat theants use basedon honest fertility signals, whichprovide workers cuticular hydrocarbonsfor recognition donot yet exist, with theessential information they needin order torepress chemical communicationof ovarian activity has been their reproductiveactivity andthus maximize their demonstrated(Tsuji et al. 1999) andvariations in cuticu- inclusive Ž tness. lar hydrocarbonsare areliable indicator ofovarian activity, at least for human researchers.If thesignals pro- Theauthors thankS. Ganeshand R.Karthik (Indian Institute ducedby themature futuregamergate are cuticular hydro- of Science, Bangalore) for excavating most of the Diacamma carbons,our data suggestthat relatively small peaksmay nests. Thisstudy was Ž nanced bythe Indo-French Centre for have akey role. In ourprevious study(Cuvillier-Hot et al. thePromotion of Advanced Research (IFCPAR/CEFIPRA) (joint grant to R.G. and C.P.). K.Tsujiand two anonymous 2001), weanalyseddifferences between D.ceylonense ants referees are thanked for their comments on aprevious version using16 peaks.In this study,although these16 peaksdid of thisarticle. discriminate FG2 andC3, they werenot able todiscrimi- natethe FG2 andC1 groups.To obtain asatisfactory dis- crimination ofthese two groups, we had toinclude three REFERENCES additional peaks(n-C26, n-C27:1 andn-C30), which together make upless than 2% ofthe total cuticular Bourke, A.F.G.&Franks, N.R.1995 Social evolution in ants . Princeton University Press. hydrocarbons.Any selection of peaks is, by de Ž nition, Cuvillier-Hot, V., Cobb, M., Malosse, C.&Peeters, C.2001 heuristic—thecuticle of D.ceylonense carries upto 76 Sex, ageand ovarian activityaffect cuticular hydrocarbons identiŽ edsubstances(Cuvillier-Hot et al. 2001), some,or in Diacamma ceylonense ,aqueenless ant. J.Insect Physiol. 47, all, ofwhichare presumably involved in protectionagainst 485–493. desiccation,as well asdistinguishing species,sex, social Fukumoto, Y., Abe, T.&Taki,A. 1989 Anovel form of col- statusand colony membership. The factthat minor peaks ony organization in the ‘queenless’ ant Diacamma rugosum . are apparently involved in distinguishing social groups is Physiol. Ecol. Jpn 26, 55–61. notsurprising: theprinciple ofminor substancescontribu- Gobin, B., Billen, J.&Peeters, C.2001 Dominance interac- ting decisively toachemical bouquetis well known.Fur- tions regulate worker mating in thepolygynous ponerine ant thermore, surprisesmay lurk in eventhe most well studied Gnamptogenys menadensis . Ethology 107, 495–508. Gronenberg, W.&Peeters, C.1993 Central projections of the ofchemical systems:small levels ofhitherto ignored sub- sensory hairs on thegemma of theant Diacamma: substrate stanceson the cuticle of female Drosophilamelanogaster for behavioural modulation? Cell Tissue Res. 273, 401–415. have recentlybeen shown to have afundamentalrole in Ito, F.&Higashi, S.1991 Alinear dominance hierarchy reg- mating in this species(Savarit et al. 1999). ulating reproduction and polyethism of thequeenless ant In other queenlessants, dominant workers can replace Pachycondyla sublaevis . Naturwissenschaften 78, 80–82. thegamergate asthe sole producer of female-destined Keller, L.&Nonacs, P.1993 Therole of queen pheromones

Proc.R. Soc.Lond. B (2002) 1300V. Cuvillier-Hot andothers Queenlessant reproduction

in social insects: queen control or queen signal? Anim. withor without queens. In Queen numberand sociality in Behav. 45, 787–794. insects (ed. L.Keller), pp. 234 –261. Oxford University Press. Liebig, J., Peeters, C., Oldham, N.J., Marksta¨dter, C. & Peeters, C.&Billen, J.1991 Anovel exocrine gland inside the Ho¨lldobler, B.2000 Are variations in cuticular hydrocarbons thoracic appendages( ‘gemmae’)of thequeenless ant of queens and workers areliable signal of fertility in theant Diacamma australe . Experientia 47, 229–231. Harpegnathos saltator ? Proc.Natl Acad.Sci. USA 97, 4124– Peeters, C.&Higashi, S.1989 Reproductive dominance con- 4131. trolled bymutilation in thequeenless ant Diacamma australe . Monnin, T.&Peeters, C.1998 Monogyny and theregulation Naturwissenschaften 76, 177–180. of worker mating in thequeenless ant Dinoponera quadriceps . Peeters, C.,Billen, J.&Ho¨lldobler, B.1992 Alternative domi- Anim.Behav. 55, 299–306. nance mechanismsregulating monogyny in thequeenless ant Monnin, T.&Peeters, C.1999 Dominance hierarchy and genus Diacamma. Naturwissenschaften 79, 572–573. reproductive con  icts among subordinates in amonogynous Peeters, C.,Monnin, T.&Malosse, C.1999 Cuticular hydro- queenless ant. Behav. Ecol. 10, 323–332. carbons correlated withreproductive statusin aqueenless Monnin, T.&Ratnieks, F.L.2001 Policing in queenless ant. Proc.R. Soc. Lond. B 266, 1323–1327. ponerine ants. Behav. Ecol. Sociobiol. 50, 97–108. Savarit, F., Sureau, G., Cobb, M.&Ferveur, J.-F. 1999 Gen- Monnin, T., Malosse, C.&Peeters, C.1998 Solid-phase etic elimination of known pheromones reveals thefunda- microextraction and cuticular hydrocarbon differences mental chemical basesof mating and isolation in Drosophila. related to reproductive activityin thequeenless ant Proc.Natl Acad.Sci. USA 96, 9015–9020. Dinoponera quadriceps . J.Chem. Ecol. 24, 473–490. Sledge, M.F., Boscaro, F.&Turillazi, S.2001 Cuticular Nakata,K., Tsuji, K., Ho¨lldobler, B.&Taki,A. 1998 Sexual hydrocarbons and reproductive statusin thesocial wasp calling byworkers using themetatibial glands in theant, Polistes dominulus . Behav. Ecol. Sociobiol. 49, 401–409. Diacamma sp., from Japan(Hymenoptera: Formicidae). Tsuji, K., Egashira, K.&Ho¨lldobler, B.1999 Regulation of J.Insect Behav. 11, 869–877. worker reproduction bydirect physicalcontacts in theant Ortius, D.&Heinze, J.1999 Fertility signalling in queens of Diacamma sp. from Japan. Anim.Behav. 58, 337–343. aNorth American ant. Behav. Ecol. Sociobiol. 45, 151–159. Vargo, E.L.1998 Primer pheromones in ants. In Pheromone Peeters, C.1991 Theoccurrence of sexual reproduction communication in social insects—ants, wasps, bees and termites among ant workers. Biol. J.Linn.Soc. 44, 141–152. (ed. R.K.Vander Meer, M.D.Breed, M.L.Winston & Peeters, C.1993 Monogyny and polygyny in ponerine ants K.E.Espelie), pp. 293 –313. Boulder, CO:WestviewPress.

Proc.R. Soc.Lond. B (2002)