The trade-off between the transmission of chemical cues and parasites: behavioral interactions between leaf-cutting ant workers of different age classes

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REVISTA

BRASILEIRA

DE

Entomologia

AJournalonInsectDiversityandEvolution

w w w . r b e n t o m o l o g i a . c o m

Biology,

Ecology

and

Diversity

The

trade-off

between

the

transmission

of

chemical

cues

and

parasites:

behavioral

interactions

between

leaf-cutting

ant

workers

of

different

age

classes

Juliane

F.S.

Lopes

a,b

_,

_Roberto

_da

_Silva

_Camargo

a,∗

_,

_Luiz

_C.

_Forti

a

_,

_William

_O.H.

_Hughes

c

a_Universidade_Estadual_Paulista,_Faculdade_de_Ciências_{Agronômicas,}_Departamento_de_Produc¸_ão_Vegetal,_Laboratório_de_Insetos_{Sociais-Praga,}_Botucatu,_SP,_Brazil b_Universidade_Federal_de_Juiz_de_Fora,_Instituto_de_Ciências_Biológicas,_{Pós-Graduac¸}_ão_em_{Comportamento}_e_Biologia_Animal,_Juiz_de_Fora,_MG,_Brazil

c_University_of_Sussex,_School_of_Life_Sciences,_Brighton,_United_Kingdom

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received8August2016 Accepted2November2016 Availableonline23November2016 AssociateEditor:RodrigoFeitosa Keywords: Acromyrmex Agepolyethism Behavioralinteraction Chemicalstransfer

a

b

s

t

r

a

c

t

Socialanimalsarefacedwithanintriguingdilemma.Ontheonehand,interactionsbetweenindividuals areessentialtoexchangeinformationandtopromotecohesion,whileontheotherhandsuchinteractions carrywiththemtheriskofcatchingandtransmittingparasites.Thistrade-offisparticularlysignificantfor socialinsectsbecauselowwithin-colonygeneticdiversitymakestheircoloniespotentiallyvulnerable toparasiteswhilefrequentinteractionsareessentialtothedevelopmentofthecolonialodorprofile necessaryfornestmaterecognition.Hereweinvestigatewhethersocialinteractionsbetweenyoungand oldleaf-cuttingantworkersshowevidenceofthistrade-off.Wefindthatoldworkersengageinmore selfgroomingandmandibularscrapingthanyoungworkers,bothinkeepingwitholdworkershaving beenmoreexposedtoparasites.Incontrast,wefindthatyoungworkersengagedinmoreallogrooming thanoldworkers,whichseemslikelytohaveadifferentmotivationpossiblythetransferofrecognition cues.Furthermore,youngworkerstendedtoengageinallogroomingwithotheryoungworkers,although itwastheoldworkersthatweremostactiveandwithwhomallogroomingwouldseemlikelytooptimize informationorchemicalstransfer.Thissuggeststhatyoungworkersmaybeattemptingtominimizethe riskofparasitetransmissionduringtheirsocialinteractions.Althoughlimitedtobehavioraldata,these resultshintthatantworkersmaybesensitivetothetrade-offbetweenthetransmissionofrecognition cuesanddisease,andadjusttheirsocialinteractionsaccordingly.

Introduction

Interactionsarefundamental to theorganization ofall soci-eties.Eveninsimplesocieties,individualssharefoodresourcesor nestingsites,andexchangeinformationtomaintaingroup

cohe-sion(Wilson,1975; Krausand Ruxton,2002).Thetransmission

ofinformationbecomesstill moreimportantassocial complex-ity increases.Advanced societies,suchas those ofhumans and socialinsects,arecharacterizedbyadivisionoflaborthatis under-pinnedbyinformationtransfer.However,socialinteractionsalso carrywiththemariskofparasitetransmission,whichislargely dependentontheinteractionratebetweeninfectedandsusceptible hosts(AndersonandMay,1981;McCallumetal.,2001).Although

∗ Correspondingauthor.

E-mail:camargobotucatu@yahoo.com.br(R.S.Camargo).

thiseffectwilldependonthetransmissionmodeofparasites(Côte

andPoulin,1995),andmaybemitigatedbydecreasedinter-group

transmission(Wilsonetal.,2003),parasitesareneverthelessoften predictedtobeaparticularhazardforsocialanimals(Alexander,

1974;Freeland,1976;KrausandRuxton,2002).Socialinteractions

maythereforeinvolveatrade-offbetweentheneedtoexchange informationandtheriskofparasitetransmission.

Thistrade-offmaybeparticularlysigniﬁcantforsocialinsects. Social insect colonies are characterized by low within-colony geneticdiversity, which may greatlyfacilitate thetransmission and evolution of any parasite that enters a colony (Hamilton,

1987;Schmid-Hempel,1998;Boomsmaetal.,2005;Hughesand

Boomsma,2006;Morelos-Juárezetal.,2010).Socialinsectsdefend

themselves against this threat at several levels. Workers have effectiveindividualdefenses,consistingofselfgroomingtoremove parasitesfromtheircuticle,theprophylacticproductionof antibi-oticstomaketheircuticlesamorehostileenvironmentfor any parasites theyencounter, andan effectiveimmune response to http://dx.doi.org/10.1016/j.rbe.2016.11.002

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dealwithanyparasitesthatsuccessfullyinfectthem(Hughesetal.,

2002;Poulsenet al.,2002a;Rosengauset al.,2004;Baeret al.,

2005;Rosengausetal.,2007;Okunoetal.,2012).Atasecondlevel,

socialinsectsalsoengageingroup-leveldefenses,allogroomingone anothertoremove parasites,possiblyexchangingantibiotic sec-retions,anddealinghygienicallywithwastematerial(Rosengaus

etal.,1998;Botetal.,2001;Hughesetal.,2002).Atathirdlevel,

social insect colonies are subdivided both architecturally,with nestsoftenconsistingofmultiplechambers,anddemographically, withyoungerindividualstendingtobelocatedatthecenterofnests andolderindividualstowardtheexterior(HölldoblerandWilson,

1990;Robinson,1992).Inaddition,hazardoustaskssuchaswaste

managementareoftenpartitionedsuchthatthoseindividualsmost exposedtothehazardhaveleastinteractionwiththeremainder ofthecolony(HartandRatnieks,2001,2002).Modelssuggestthat thecombinationofthisstructuringwithgroup-levelandindividual defensemechanismsiskeytoparasitetransmissionwithinsocial insectcoloniesbeingminimized(NaugandCamazine,2002;Pie

etal.,2004;Feffermanetal.,2007).

Whileparasitesselectforsocialinsectstominimizetheirsocial interactions,theneedforinformationtransferconverselyrequires thesetobefrequentto,inparticular,developthenecessarycuesto allownestmaterecognition.Althoughotherpheromonesmayplay asecondaryroleinsometaxa(Hughesetal.,2001;Hernandezetal., 2006),theprimarycuesusedfornestmaterecognitionarenormally non-volatilecuticular hydrocarbons(CHCs) (Lahav et al., 1999;

HowardandBlomquist,2005;GuerrieriandD’Ettorre,2008;Martin

etal.,2008).IndividualstransfertheseCHCsbetweenoneanother byallogroomingortrophallaxistoproduceacolony-speciﬁcblend or‘gestalt’odorwhichallowsnestmatestobedistinguishedfrom non-nestmates(Sorokeretal., 1995;Boulayetal., 2000;Lenoir

et al., 2001; Soroker et al., 2003) so CHCs constitutes a great

sourceofinformationandwhentheyaretransferrednotjustthe gestaltodorisobtainedbutsotheinformationaboutindividual colonyorigin. Colonyspeciﬁc odors that maintaincolony insu-laritybyrejectinganyindividualthatcarriesadifferentodorare wellknowninsocial insects (Breed,1983; Wilson, 1971). Indi-vidualseclosewithanindividually-distinctiveproﬁleofcuticular hydrocarbonsandacquirethegestaltodorrapidlyoverthedays followingeclosion(Moreletal.,1988;Dahbietal.,1998;Kaibetal.,

2000;Cuvillier-Hotetal.,2001).Individualscontinueto

biosynthe-sizecuticularhydrocarbonsthroughouttheirlife,socontinually needto interactwithnestmates tomaintain and tocontribute totheirgestaltcolonyodorformationandavoidredevelopingan individually-distinctiveproﬁle (Boulay et al., 2000; Boulay and

Lenoir,2001).

Aswithothergroup-livinganimals,socialinsectworkers there-foreneed tobalancetheneedforsocial interactionstotransfer chemicalcueswiththeriskofthesameinteractionstransmitting parasites.Hereweinvestigatewhethersomesocialbehaviorsin leaf-cuttingantsshowevidenceofsuchatrade-off.Fourbehaviors wereexamined:allogrooming,selfgrooming,mandibularscraping andantennation.

AllogroomingistheprincipalmechanismbywhichCHCsare transferredbetween leaf-cuttingants becausethis species only rarelyengageintrophallaxis(Moreiraetal.,2006).Alsoitisan important mechanism of diseaseresistance, but carries with it theriskofdiseasetransmission(Rosengausetal.,1998;Hughes

etal.,2002;Feffermanetal.,2007).Althoughallogroomingmay

alsoservetoincreaseimmunity(Tranielloetal.,2002;Ugelvigand

Cremer,2007),itseemsprobablethatthisbeneﬁtwillnormallybe

outweighedbytheriskofinfection.Selfgroomingbothplaysarole indiseasedefenseremovingfungalsporesandothercontaminants fromthecuticle(Hughesetal.,2002)andinthedevelopmentof thegestaltodor.Mandibularscrapingmostprobablyhasahygienic function in which the effective action is in one direction and

transfersdirtmaterialthathasaccumulatedonthemandiblesfrom thecleanedtothecleaningpart(Lopesetal.,2005).Antennation wasmeasuredasageneralmeasureofsocialactivity.Wecompared allfourbehaviorsbetweenyoungandoldworkers.

Youngworkersaremostvaluabletothecolonybecauseoftheir long expected life. Their tasks are generally limited towithin, and often at thecenter of the nest (Robinson, 1992; Camargo etal.,2007),sotheymaybelessexposedtoparasitesthanolder workerswhichengageinmovingsoil,wastemanagementor for-aging(Schmid-Hempel,1998;Boomsmaetal.,2005).Thiscontrast in exposure is likely to be particularly strong for leaf-cutting antsbecauseyoungworkersresideexclusivelywithinthefungus garden.Theontogeneticdifferences leadtoseveralhypotheses: mandibularscraping willbemorecommoninoldworkersthan youngworkersbecausetheformeraremorelikelytobe contami-natedwithsoilorfooddebris;selfgroomingwillbemorecommon inoldworkersthanyoungworkersbecausethetasksoftheformer (soilmoving,wastemanagement,foraging)carrymoreriskof par-asiteexposure;allogroomingwillalsobemore frequentforold ratherthanyoungworkersforthesamereason,andinaddition willtendtobewithinagecategoriesinordertolimittransmission bydividinginteractionnetworks(NaugandCamazine,2002;Pie

etal.,2004;Feffermanetal.,2007).Incontrast,information

trans-ferwouldpredictsimilarratesofselfgroomingandallogrooming foroldandyoungworkersbecausebothgroupsneedtodevelop andmaintaintheirCHCproﬁle.Furthermore,information trans-ferwouldbeoptimizedbyallogroomingbetweenagecategories inordertopreventthemdevelopingdistinctCHCproﬁleswhich wouldinterferenestmaterecognition.

Materialandmethods

The studywas conducted withtwo colonies of Acromyrmex crassispinus,collectedatJaguariaíva-PR(24◦32S;49◦57W),and two colonies of Acromyrmex rugosus, collected at Botucatu-SP (22◦5220S; 48◦2637W), both locations being in southeast Brazil. A. crassispinus is found mainly in pine forests and uses monocotyledonousasfungussubstratewhileA.rugosusoccursin grassland and disturbed habitatusing dicotyledonousplants as substrate(Verzaetal.,2007).Thetwospeciesotherwisehavevery similarecologyandlife-history,withbothnestingwithinthesoil andreachingasimilarsizeatmaturity withone ortwofungus gardens.Here,theywerechosenforeasyaccessandrearing capa-bilityofthesespecies.Also,bothspeciesshowtheclassicpattern ofalloethism forAcromyrmex,witha continuoussize-frequency distributionaroundtwomodes,normallytermedsmallworkers and largeworkers(Wetterer,1999; Hugheset al., 2003).Small Acromyrmexworkersresidewithinthefungusgardenwherethey careforthebroodandfungus.Largeworkersalsoremainwithin thefungusgardenwhileyoung,wheretheyserveasasubstratefor theculturingofantimicrobialbacteriatoprotectthefungusgarden

(Currieetal.,1999,2003;Poulsenetal.,2002b).Mediumandlarge

old-agedworkersworkoutsidethefungusgardenandnest, forag-ingforvegetation,removingwasteandbuildingthenest(Camargo

etal.,2007).

Here,we consideredthatyoungworkers(lightbrowncolor) were1weekold,andoldworkers(darkbrowncolor)weremore than3and4weeksold.Thisassumptionisbasedonstudyfrom

Camargoetal.(2007),whereleaf-cuttingantsstarttoparticipate

inactivitiesoutsidethenestby3or4weeksofage,anddeﬁnedas “outsideworkers”.Thusallofthemshouldbeolderthan1month. Ontheotherhandyoungworkerasdeﬁnedas“insideworkers”and allofthemshouldbeyoungerthan1week.

Theexperimentalcoloniesweremaintainedinthelaboratoryat 24±1◦CandRH80±10%onadietofLigustrumsp.andAcalypha

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0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 Fr e q u en cy ob se rv e d 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 F req ue nc y a n ten na ti o n s Young x young Young x old Old x old Old Young

b

a

Fig.1.Overallactivitylevels.Mean±s.e.frequenciesthatyoungandoldantswere:(a)observedengaginginoneofthefocalbehaviorsand(b)observedengagedin antennationwithanotherant,duringa15sobservationperiod.

sp.Thecolonieseachhadasingle500mLfungusgardenhoused inaplasticcontainer,thebottomofwhichwascoveredwitha 1-cmlayerofplastertomaintainthehumidityofthefungusgarden. Eachcolonyhadaconnectiontoaforagingarena,madeoutofglass, andacontainerinwhichtheantsdepositedwaste.Youngandold workerswereidentiﬁedbytheirlightanddarkcuticularcoloration andcapturedinsidethefungusgardenchamberandforagingarena, respectively.ThecuticleofAcromyrmexworkersdarkens progres-sivelyastheyagefromalighttoverydarkbrowncolor(Armitage

andBoomsma,2010).Whilecuticularcolorationonlyprovidesan

approximatemeasureofage,itdoesthereforeallowyoungandold workerstobeveryreliablydistinguished.

Foreachcolony,50largeworkers(1.3–1.7cm bodylengthin A.crassispinusand1.0–1.3cmbodylengthinA.rugosus)ofeach agecategoryhadadifferentcoloredpaintdotappliedtotheir tho-raxestofacilitatesubsequent identiﬁcationand wereplaced in a lidded glassexperimentalarena (square of 225cm2_)._Colored pens (EddingTM,Edding International GmbH,Ahrensburg, Ger-many)wereusedtolabeltheworkersbecauseoftheirexcellent adherence,quickdryingandgoodvisibility(Camargoetal.,2007). Thelidsoftheexperimentalarenaweredemarkedintoagridof9 squares.Theantswereleftfor5mintobecomeaccustomedtothe arena,whichwasthenvideoedusingamicrocamera(Paciﬁc-Co) for45min.Subsequently,thefrequencyofbehavioralinteractions betweenyoungandoldworkersinthearenawascounted,aswell, betweensameagegroups. Fourfocalbehaviors wererecorded: (1)antennation,(2)selfgrooming,(3)mandibularscrapingand(4) allogrooming.The ages(youngor old)ofantsinvolved andthe directionofeachbehaviorwasalsorecorded.Thebehaviorsineach

square werecountedfor a15speriod, atintervalsof45s.Grid squareswereobserved ina randomorder,withthesetofnine squaresmakingupthearenabeingrecorded13 timesover the courseofthe45min.Thedatasetforeacharenathusconsistedof ninereplicatecounts(squares),eachlasting15s,withtherebeing 13repeatedmeasuresovertimeofeachsetofsquares.Thedata werelog(x+1)transformedandanalyzedwithrepeated-measures analysesofvariancefollowedbypairedt-testwithp-value adjust-mentbyBenjamini–Yekutielimethod.

Results

Overall,oldworkerswereobservedengaginginthefourfocal behaviorssigniﬁcantlymoreoftenthanyoungworkers(F1,64=194, p<0.0001; Fig. 1a), even though the same numbers of each category were present in the arenas. The number of anten-nations involving old workers, either with other old workers (p=0.02)orwithyoungworkers(p=0.041),wasalsosigniﬁcantly higherthanforantennationsbetweenyoungworkers(F1,64=5.36, p=0.006; Fig. 1b). Selfgrooming was observed almost twiceas oftenbyoldworkersasbyyoungworkers(F1,64=56.3,p<0.0001;

Fig.2a).Mandibularscrapingwasarelativelyinfrequent behav-iorthatconsequentlydidnotdiffersigniﬁcantlybetweentheage groups(F1,64=0.528,p=0.591),althoughitwasobserved approx-imatelytwiceasoftenbetweenoldworkersthaninvolvingyoung workers (Fig. 2b). Finally, the frequency of allogrooming dif-feredsigniﬁcantlybetweentheagegroups(F3,128=6.7,p=0.0003). Allogroomingwasmostcommonbetweenyoungworkers,andwas approximatelyequallyfrequentbetweenoldworkersasbetween

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Frequency selfgrooming 0 0.005 0.01 0.015 0.02 0.025 0.03

Frequency mandible scrapping

Young x young Young x old Old x old Old Young

b

a

Fig.2.Mean±s.e.frequenciesthatyoungandoldantswere:(a)observedselfgroomingand(b)observedengagedinmandiblescrapingwithanotherant,duringa15s observationperiod.

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0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 Frequency allogrooming Young to young Young to old Old to young Old to old

Fig.3. Mean±s.e.frequenciesthatyoungandoldantswereobservedgivingor receivingallogroomingduringa15sobservationperiod.

youngandoldworkers(Fig.3).Allogroomingbetweenyoungand oldworkerswassimilarlylikelytoinvolveeachagegroupasthe giverorreceiveroftheinteraction.Accordingly,youngworkers engagedinallogroomingsigniﬁcantlymoreoftenthanoldworkers (youngworkerinteractionsvs.oldworkerinteractions:F1,64=4.43, p=0.039),inspiteofbeinglessactiveoverall.Youngworkerswere morelikelytoengageinallogroomingwithotheryoungworkers thanwitholdworkers(F1,64=4.87,p=0.031),whereasoldworkers weresimilarlylikelytoengageinallogroomingwiththedifferent agegroups(F1,64=0.638,p=0.428).Notrophallaxiswasobserved betweenanyoftheantsduringtheexperiment.

Discussion

Inspiteofthehighlyartiﬁcialexperimentalset-up,we never-thelessfoundclearevidenceofanontogeneticdifferenceinsocial interactions.TheseresultsshowusthatAcromyrmexworkers there-foreneed tobalancetheneedforsocial interactionstotransfer chemicalcuesrelatedtonestmaterecognitionwiththeriskofthe sameinteractionstransmitparasites.

Oldworkers,whichwereingeneralmoreactive,selfgroomed moreoftenthanyoungworkers,andengagedinmoremandibular scraping.However,youngworkersallogroomedmoreoftenthan oldworkers,inspiteoftheiroverallloweractivity.Furthermore, allogroomingbyyoungworkerswasnon-random,being preferen-tiallydirectedatotheryoungworkers.

Selfgroomingremovesfungalsporesand othercontaminants fromthecuticle(Hughesetal., 2002), while mandibular scrap-ingismostprobablytoremovematerialthathasaccumulatedon themandibles(Lopesetal.,2005).Oldworkersarelikelytohave beenmostexposedtoparasitesandothermicrobesbecausethey carryout allthehazardoustasks, suchas foraging,soil moving andwastemanagement(Schmid-Hempel,1998;Boomsmaetal.,

2005;Camargoetal.,2007).Selfgroomingmayalsobeimportant

inblendingrecognitioncues,butthiswouldpredictsimilarrates bothforyoungandoldworkers.Itthereforeseemsmostlikelythat thehigherratesofselfgroomingandmandibularscrapingobserved inoldworkersareforhygienicreasons.Morelos-Juárezetal.(2010)

showthatleaf-cuttingantsareengagedinproactiveself-cleaning behaviortoremoveundetectedmicroorganismandpreventthem fromcontaminatingthevulnerablefungalcrop.

Explainingthehigherratesofallogroominginyoungworkersis moreproblematic.Possiblythelevelofinteractionneededbyyoung workerstodeveloptheirgestaltproﬁleisgreaterthanthelevel neededbyoldworkerstomaintainit(Moreletal.,1988;Dahbietal.,

1998;Boulayetal.,2000;Kaibetal.,2000;BoulayandLenoir,2001;

Cuvillier-Hotetal.,2001).Alternatively,it maybenot somuch

thatyoungworkershavehighrates ofallogrooming,but rather

thatoldworkershavereducedrates,perhapsinordertoreduce theriskofthemtransmittingparasitestonestmates(Rosengaus

etal.,1998;Hughesetal.,2002;Feffermanetal.,2007).Thefact

thatyoungworkerspreferentiallyallogroomedwithotheryoung workersisintriguing.Ifallogroomingwasgovernedsolelyby infor-mationtransferthenitseemsprobablethatallogroomingwould tendtobebetweenagecategoriesbecausethiswouldoptimizethe developmentofa singlecolony-level gestaltodor.Similarly,the greateractivityofoldworkersmeansmostallogroomingshould havebeenwitholdworkersifinteractionswererandom.Finally,if allogroomingwasdrivensolelybytherequirementsofthe receiv-ing individual, then this again would predict that old workers shouldreceivemostallogroominggiventhattheirtasksarelikely toinvolvegreaterexposuretoparasites(Schmid-Hempel,1998;

Boomsmaetal.,2005),andgiventhattheirhigherratesof

self-groomingandmandibularscraping supportthis.Youngworkers thereforeengagedinallogroomingnotwiththosethatwould opti-mize information transfer,nor themost activeindividuals, nor thosemostinneedofcleaning,butrather thosethatpresented theleastriskfromparasites.Thissuggeststhattheyaresensitive totheriskofparasitetransmissionandadjusttheirsocial interac-tionstominimizethisrisk.Anotheralternativeexplanationisthat youngworkersengagedinhigherlevelsofallogromingwithintheir classbecauseoftheyareinalearningprocessofthecolonyodor. Alsotheirownchemicalswillbeanintegralpartofthegestaltodor ofthecolony.Inthisstudywehaveonlyinvestigatedbehavioral interactions.Whilepreviousstudieshavedemonstratedthatsuch interactionscantransmitbothinformation,intheformofCHCs

(Sorokeretal.,1995,2003;Boulayetal.,2000;Lenoiretal.,2001),

and parasites (Rosengaus et al., 1998; Schmid-Hempel, 1998;

Hughesetal.,2002;Boomsmaetal.,2005;Feffermanetal.,2007),

wedidnotattempttoquantifyeither here,sotheimplications ofourstudymustremainsomewhatspeculative.Nevertheless,it seemscertainthatthetrade-offbetweeninformationtransferand parasitetransmissionisrealandmaybeofparticularimportancein socialinsectcolonieswiththeirlowgeneticdiversityandhighneed forinformationtransfer.Thebehavioralresultswehavepresented heresuggestthatleaf-cuttingantsmaybesensitivetothistrade-off. Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest. Acknowledgements

WearegratefultoFundaçãodeAmparoàPesquisadoEstado deSãoPaulo(FAPESP)forfinancialsupportandforthefellowships grantedtotheauthors(grants07/07091-0and07/04010-0).LCF wastherecipientofagrantfromConselhoNacionalde Desenvolvi-mentoCientificoeTecnológico(grant301718/2013-0).

References

Alexander,R.D.,1974.Theevolutionofsocialbehavior.Ann.Rev.Ecol.Syst.5, 324–383.

Anderson,R.M.,May,R.M.,1981.Thepopulationdynamicsofmicroparasitesand theirinvertebratehosts.Philos.Trans.R.Soc.Lond.B291,451–524.

Armitage,S.A.O.,Boomsma,J.J.,2010.Theeffectsofageandsocialinteractionson innateimmunityinaleaf-cuttingant.J.InsectPhysiol.56,780–787.

Baer,B.,Krug,A.,Boomsma,J.J.,Hughes,W.O.H.,2005.Examinationoftheimmune responsesofmalesandworkersoftheleaf-cuttingantAcromyrmexechinatior andtheeffectofinfection.InsectesSoc.52,298.

Boomsma,J.J.,Schmid-Hempel,P.,Hughes,W.O.H.,2005.Lifehistoriesandparasite pressureacrossthemajorgroupsofsocialinsects.In:Fellowes,M.D.E.,Holloway, G.J.,Rolff,J.(Eds.),InsectEvolutionaryEcology.CABIPublishing,Wallingford,pp. 139–175.

Bot,A.N.M.,Currie,C.R.,Hart,A.G.,Boomsma,J.J.,2001.Wastemanagementin leaf-cuttingants.Ethol.Ecol.Evol.13,225–237.

(5)

Boulay,R.,Lenoir,A.,2001.Socialisolationofmatureworkersaffectsnestmate recognitionintheantCamponotusfellah.Behav.Process.55,67–73.

Boulay,R.,Hefetz,A.,Soroker,V.,Lenoir,A.,2000.Camponotusfellahcolony integra-tion:workerindividualitynecessitatesfrequenthydrocarbonexchanges.Anim. Behav.59,1127–1133.

Breed,M.D.,1983.Nestmaterecognitioninhoneybees.Anim.Behav.31,86–91. Camargo,R.S., Forti,L.C.,Lopes,J.F.S.,Andrade, A.P.P.,Otatti,A.L.T.,2007. Age

polyethismintheleaf-cuttingantAcromyrmexsubterraneusbrunneusForel,1911 (Hymenoptera:Formicidae).J.Appl.Entomol.131,139–145.

Côte,I.M.,Poulin,R.,1995.Parasitismandgroupsizeinsocialanimals:a meta-analysis.Behav.Ecol.6,159–165.

Currie,C.R.,Bot,A.N.M.,Boomsma,J.J.,2003.Experimentalevidenceofa tripar-titemutualism:bacteriaprotectantfungusgardensfromspecializedparasites. Oikos101,91–102.

Currie,C.R.,Scott,J.A.,Summerbell,R.C.,Malloch,D.,1999.Fungus-growingantsuse antibiotic-producingbacteriatocontrolgardenparasites.Nature398,701–704. Cuvillier-Hot,V.,Cobb,M.,Malosse,C.,Peeters,C.,2001.Sex,ageandovarianactivity affectcuticularhydrocarbonsinDiacammaceylonense,aqueenlessant.J.Insect Physiol.47,485–493.

Dahbi,A.,Cerda,X.,Lenoir,A.,1998.Ontogenyofcolonialhydrocarbonlabelincallow workersoftheantCataglyphisiberica.C.R.Acad.Sci.III321,395–402. Fefferman,N.H.,Traniello,J.F.A.,Rosengaus,R.B.,Calleri,D.V.,2007.Disease

pre-ventionandresistanceinsocialinsects:modelingthesurvivalconsequencesof immunity,hygienicbehavior,andcolonyorganization.Behav.Ecol.Sociobiol. 61,565–577.

Freeland,W.J.,1976.Pathogensandtheevolutionofprimatesociality.Biotropica8, 12–24.

Guerrieri,F.J.,D’Ettorre,P., 2008.Themandibleopeningresponse:quantifying aggressionelicitedbychemicalcuesinants.J.Exp.Biol.211,1109–1113. Hamilton,W.D.,1987.Kinship,recognition,disease,andintelligence:constraints

ofsocialevolution.In:Ito,Y.,Brown,J.L.,Kirkkawa,J.(Eds.),AnimalSocieties: TheoriesandFacts.JapanScientiﬁcSocietiesPress,Tokyo,pp.81–100. Hart,A.G.,Ratnieks,F.L.W.,2001.Taskpartitioning,divisionoflabourandnest

com-partmentalisationcollectivelyisolatehazardouswasteintheleafcuttingantAtta cephalotes.Behav.Ecol.Sociobiol.49,387–392.

Hart,A.G.,Ratnieks,F.L.W.,2002.Wastemanagementintheleaf-cuttingantAtta colombica.Behav.Ecol.Sociobiol.13,224–231.

Hernandez,J.V.,Goitia,W.,Osio,A.,Cabrera,A.,Lopez,H.,Sainz,C.,Jaffe,K.,2006. Leaf-cutterantspecies(Hymenoptera:Atta)differinthetypesofcuesusedto differentiatebetweenselfandothers.Anim.Behav.71,945–952.

Hölldobler,B.,Wilson,E.O.,1990.TheAnts.BelknapPress,Cambridge.

Howard,R.W.,Blomquist,G.J.,2005.Ecological,behavioral,andbiochemicalaspects ofinsecthydrocarbons.Annu.Rev.Entomol.50,371–393.

Hughes,W.O.H.,Boomsma,J.J.,2006.Doesgeneticdiversityhinderparasite evolu-tioninsocialinsectcolonies?J.Evol.Biol.19,132–143.

Hughes,W.O.H.,Howse,P.E.,Goulson,D.,2001.Mandibularglandchemistryof grass-cuttingants:species,caste,andcolonyvariation.J.Chem.Ecol.27,109–124. Hughes,W.O.H.,Eilenberg,J.,Boomsma,J.J.,2002.Trade-offsingroupliving:

trans-missionanddiseaseresistanceinleaf-cuttingants.Proc.R.Soc.Lond.B269, 1811–1819.

Hughes,W.O.H.,Sumner,S.,VanBorm,S.,Boomsma,J.J.,2003.Workercaste poly-morphismhasageneticbasisinAcromyrmexleaf-cuttingants.Proc.Natl.Acad. Sci.U.S.A.100,9394–9397.

Kaib,M.,Eisermann,B.,Schoeters,E.,Billen,J.,Franke,S.,Francke,W.,2000. Task-relatedvariationofpostpharyngealandcuticularhydrocarboncompositionsin theantMyrmicariaeumenoides.J.Comp.Physiol.A186,939–948.

Kraus,J.,Ruxton,G.D.,2002.LivinginGroups.OxfordUniversityPress,Oxford. Lahav,S.,Soroker,V.,Hefetz,A.,VanderMeer,R.K.,1999.Directbehavioralevidence

forhydrocarbonsasantrecognitiondiscriminators.Naturwissenschaften86, 246–249.

Lenoir,A.,Hefetz,A.,Simon,T.,Soroker,V.,2001.Comparativedynamicsofgestalt odourformationintwoantspeciesCamponotusfellahandAphaenogastersenilis (Hymenoptera:Formicidae).Physiol.Entomol.26,275–283.

Lopes,J.F.S.,Hughes,W.O.H.,Camargo,R.S.,Forti,L.C.,2005.Larvalisolationand broodcareinAcromyrmexleaf-cuttingants.InsectesSoc.52,333.

Martin,S.J.,Vitikainen,E.,Helanterä,H.,Drijfhout,F.P.,2008.Chemicalbasisof nest-matediscriminationintheantFormicaexsecta.Proc.R.Soc.Lond.B275, 1271–1278.

McCallum,H.,Barlow,N.,Hone,J.,2001.Howshouldpathogentransmissionbe modelled?TREE16,295–300.

Moreira,D.,Erthal,M.,Carrera,M.,Silva,C.,Samuels,R.,2006.Oraltrophallaxis inadultleaf-cuttingantsAcromyrmexsubterraneussubterraneus(Hymenoptera, Formicidae).InsectesSoc.53,345–348.

Morel,L.,Meer,R.K.,Lavine,B.K.,1988.Ontogenyofnestmaterecognitioncues intheredcarpenterant(Camponotusﬂoridanus).Behav.Ecol.Sociobiol.22, 175–183.

Morelos-Juárez,C.,Walker,T.N.,Lopes,J.F.S.,Hughes,W.O.H.,2010.Antfarmers practiceproactivepersonalhygienetoprotecttheirfunguscrop.Curr.Biol.20, 553–554.

Naug,D.,Camazine,S.,2002.Theroleofcolonyorganizationonpathogen transmis-sioninsocialinsects.J.Theor.Biol.215,427–439.

Okuno,M.,Tsuji,K.,Sato,H.,Fujisaki,K.,2012.Plasticityofgroomingbehavioragainst entomopathogenicfungusMetarhiziumanisopliaeintheantLasiusjaponicus.J. Ethol.30,23–27.

Pie,M.R.,Rosengaus,R.B.,Traniello,J.F.A.,2004.Nestarchitecture,activitypattern, workerdensityandthedynamicsofdiseasetransmissioninsocialinsects.J. Theor.Biol.226,45–51.

Poulsen,M.,Bot,A.N.M.,Nielsen,M.G.,Boomsma,J.J.,2002a.Experimentalevidence forthecostsandhygienicsigniﬁcanceoftheantibioticmetapleuralgland secre-tioninleaf-cuttingants.Behav.Ecol.Sociobiol.52,151–157.

Poulsen,M.,Bot,A.N.M.,Currie,C.R.,Boomsma,J.J.,2002b.Mutualisticbacteriaand apossibletrade-offbetweenalternativedefencemechanismsinAcromyrmex leaf-cuttingants.InsectesSoc.49,15–19.

Robinson,G.E.,1992.Regulationofdivisionoflaborininsectsocieties.Annu.Rev. Entomol.37,637–665.

Rosengaus,R.B.,Maxmen,A.B.,Coates,L.E.,Traniello,J.F.A.,1998.Disease resis-tance:abeneﬁtofsocialityinthedampwoodtermiteZootermopsisangusticollis (Isoptera:Termopsidae).Behav.Ecol.Sociobiol.44,125–134.

Rosengaus,R.B.,Traniello,J.F.A.,Lefebvre,M.L.,Maxmen,A.B.,2004.Fungistatic activityofthesternalglandsecretionofthedampwoodtermiteZootermopsis angusticollis.InsectesSoc.51,259–264.

Rosengaus,R.B.,Tara,C.,Katerina,G.,James,F.A.T.,2007.Inducibleimmuneproteins inthedampwoodtermiteZootermopsisangusticollis.Naturwissenschaften94, 25–33.

Schmid-Hempel,P.,1998.ParasitesinSocialInsects.PrincetonUniversityPress, Princeton.

Soroker,V.,Vienne,C.,Hefetz,A.,1995.Hydrocarbondynamicswithinandbetween nestmatesinCataglyphisniger(Hymenoptera:Formicidae).J.Chem.Ecol.21, 365–378.

Soroker,V.,Lucas,C.,Simon,T.,Fresneau,D.,Durand,J.L.,Hefetz,A.,2003. Hydro-carbondistributionandcolonyodourhomogenisationinPachycondylaapicalis. InsectesSoc.50,212–217.

Traniello,J.F.A.,Rosengaus,R.B.,Savoie,K.,2002.Thedevelopmentofimmunityina socialinsect:evidenceforthegroupfacilitationofdiseaseresistance.Proc.Natl. Acad.Sci.U.S.A.99,6838–6842.

Ugelvig,L.V.,Cremer,S.,2007.Socialprophylaxis:groupinteractionpromotes col-lectiveimmunityinantcolonies.Curr.Biol.17,1967–1971.

Verza,S.,Forti,L.C.,Lopes,J.F.S.,Hughes,W.O.H.,2007.Nestarchitectureofthe leaf-cuttingantAcromyrmexrugosusrugosus.InsectesSoc.54,303–309.

Wetterer,J.K.,1999.Theecologyandevolutionofworkersize-distributionin leaf-cuttingants(Hymenoptera:Formicidae).Sociobiology34,119–144.

Wilson,E.O.,1971.TheInsectSocieties.BelknapPress,Cambridge.

Wilson,E.O.,1975.Sociobiology:TheNewSynthesis.BelknapPressofHarvard Uni-versityPress,Harvard.

Wilson,K.,Knell,R.,Boots,M.,Koch-Osborne,J.,2003.Grouplivingandinvestment inimmunedefence:aninterspeciﬁcanalysis.J.Anim.Ecol.72,133–143.