Chemical cuticular signature of leafcutter ant Atta sexdens (Hymenoptera, Formicidae) worker subcastes

(1)

RevistaBrasileiradeEntomologia60 (2016)308–311

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

Chemical

cuticular

signature

of

leafcutter

ant

Atta

sexdens

(Hymenoptera,

Formicidae)

worker

subcastes

Lohan

Valadares

∗

,

Fabio

Santos

do

Nascimento

UniversidadedeSãoPaulo,DepartamentodeBiologia,LaboratóriodeComportamentoeEcologiadeInsetosSociais,RibeirãoPreto,SP,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24March2016

Accepted23June2016

Availableonline21July2016

Associateeditor:RodrigoGonc¸alves

Keywords: Attasexdens Cuticularhydrocarbons Leafcutterants Workersubcaste

a

b

s

t

r

a

c

t

Inleafcutterantsthedivisionoflabourisassociatedtoworkersizevariationclusteredinfoursubcastes. InthisworkweusedAttasexdensForel(1908)asamodeltotestthehypothesisthateachsubcaste expressesitsownchemicalsignaturecomprisedofcuticularlipids.Toassessit,weextracted epicu-ticularcompoundsbyusingnonpolarsolventhexaneandanalysedthesamplesinacombinedGas Chromatography–MassSpectrometer(GC–MS).Wefound24hydrocarbonswithcarbonchainsranging from19to39atomsmostofthemclassifiedaslinearandbranchedalkanes.Nocompoundoccurredinthe cuticleofspecificworkersubcaste,however,therelativeproportionpatternvariedgreatlyamongthem. Ourresultssuggestthatalthoughsubcasteshavesimilarchemicalsignatures,significantdifferencesin theirrelativeproportionsmayplayanimportantrolebetweennestmateandgroupidentification.

Introduction

Akeyfactorintheevolutionofleafcutterants(genusAttaand Acromyrmex)istheadaptationoftaskallocationsystembasedon workersize variation fittedprimarilyto theutilisationof fresh vegetationasfoodresourcetothesymbioticfungusthatthey culti-vate(Wilson,1980).InAttasexdens,thedistributionofactivities relatedtofoliar substratetreatmentand funguscultivation fol-lowanassembly-linefashionperformedbyatotaloffourworker subcastes;largeworkers(foragers)forage,cut,andtransportmore efficientlyleaffragments,whereassmallworkers(gardeners)tend toremaininsidethenestperformingactivitiesrelatedtofinal treat-mentandimplantingoffoliarsubstrateparticles(Della-Luciaetal., 1993;Wilson,1980).Intermediate-sizeworkers(generalists) per-formawidearrayoftasksfromfoliarsubstratetreatmenttobrood andqueencaring.Inaddition,majorworkers,alsoreferredasto soldiers,arevirtuallylimitedtodefense(Wilson,1980).

Althoughveryorganised,thedivisionoflabourinsocialinsects donotfollowacentralcontrol(Gordon,2015).Asingleindividual isabletoperformavarietyoftasks,buttheprobabilityofa leaf-cutterantworkertoperformasize-relatedtaskishigh(Wilson, 1980).Ithasbeenevidencedthatdivisionoflabourcanbe medi-atedbycuticularhydrocarbons(CHCs)thatactastaskdecisioncues (GreeneandGordon,2003).CHCsarespreadallovertheoutmost

∗ Correspondingauthor.

E-mail:valadareslohan@gmail.com(L.Valadares).

cuticlelayerofinsects,theepicuticle.Duetothegreatdiversityof thesesubstancesineusocialinsectscombinedwiththeir exoge-nousandendogenousorigins,theyevolvedtoplayanimportant roleinnestmaterecognitionandsocialorganisation(Greeneand Gordon,2003;Sturgis&Gordon,2012;Tannure-Nascimentoetal., 2007;VanderMeerandMorel,1998;Wilgenburgetal.,2011;).

Inleafcutterants,somestudieshavecharacterisedthe cuticu-larcompositionofworkers,mostofthemassociatingtheinfluence ofleafsubstrateontheirCHCprofilesandnestmate recognition (Lambardietal.,2004;Marinhoetal.,2008;MartinandMacConnel, 1970;Valadaresetal.,2015).However,tothebestofour knowl-edge, theassociation between worker CHC profileand itsfour physicalsubcasteremainsunclear.Withinthiscontext,we hypoth-esisedthat eachsubcasteof leafcutterantgenusAttaexpresses itsownchemicalsignaturecomprisedofcuticularhydrocarbons. Totestit,weusedA.sexdensasamodel,whichisabroadly dis-tributedspeciesintheNeotropicalecosystemsandconsideredto beoneofthemajorherbivoresofthisregion(Forti&Boaretto,1997; HölldoblerandWilson,1990).

Materialandmethods

Colonies

Thepresentstudywasconductedwithtwolaboratory popula-tions(Colony1andColony2)collectedfromlocationsthatwere atleast50kmapartinRibeirãoPreto,SãoPauloState,Brazil, dur-ingtheirinitialphase (approximately3monthsold).Theywere

http://dx.doi.org/10.1016/j.rbe.2016.06.008

(2)

L.Valadares,F.S.Nascimento/RevistaBrasileiradeEntomologia60(2016)308–311 309

keptinartiﬁcialconditions,withtemperatureandhumiditykept at approximately 25◦C and 75%, respectively, until the age of twoyearswhentheexperimentwasconducted.Atthetime,the colonieshadapproximately5Loffungusgarden.Wealsocollected individualsundernaturalconditions(Colony3)fromanest char-acterisedbyabigamountofvoluminous,dismounted,excavated soilwithapproximately2m2_.

Subcasteidentiﬁcation

Beforethechemicalprocedureswecollectedandmeasuredthe headwidth(HW)of50workers.Aftermeasurements,the individu-alswererank-orderedinrowsanditwerechosenworkerswithHW around1.0mm,1.4mm,2.2mmand>3.0mm,respectively classi-fiedasgardeners,generalists,foragers,andsoldiers(Wilson,1980). Thespecimenswerethenfixedwithanentomologicalpinwitha subcasteidentificationlabelandusedasreferencetocollect indi-vidualsforchemicalanalyses.

Chemicalanalyses

Forchemicalanalysisweused15individualspersubcasefor eachcolony.Cuticularwaxeswereextractedbysubjectingeach individualtoanappropriatevolumeofthenonpolarsolvent hex-anefor2min.Inordertoobtainwellconcentratedsamples,we previouslydeterminedthebestvolumeofhexanetoextractthe compoundsofthesmallestsubcaste(gardener). Then,wemade acorrelationbetweenthesolventvolumeusedingardenersand theHWoftheothersubcastes.Thus,wesubjectedworkersin50, 100,130,and200␮Lofhexaneforeachoneoftherespective sub-castes:gardeners,generalists,foragersandsoldiers.Thesamples wereranina combinedGasChromatography–Mass spectrome-tryGC–MS(Shimadzu,modelQP2010plus)equippedwithsilica capillarycolumnandheliumascarriergasat1mL/min.Theoven temperaturewasinitiallysetto150◦C,increasing3◦C/minuntilit reached280◦C(maintainedfor10min)andagain10◦C/minuntil reach300◦C(maintainedfor15min).

Theidentiﬁcationofthecompoundswasbasedontheirmass spectra and with the aid of a standard solution of synthetic hydrocarbons(SigmaChemical Co.), aswell asWiley and NIST Librarydatabase.Theidentiﬁcationofthepositionofalkene dou-blebounds wasmadethroughderivatisation methodof hexane extractsfrom20foragerspersubcaste,withDimethylDisulphide (DMDS) (Carlson,1989).The extractswere driedwithnitrogen andre-suspended in200␮Lof hexane.Subsequently, 200␮Lof DMDS(Sigma–Aldrich)and100␮Lofiodinesolution(dissolved indiethylether,6%p/v)wereadded.Thevialswerethenpurged withnitrogen,closed, and agitatedat ambienttemperature for 24h.Thereafter,themixturewasdilutedinhexaneand5%sodium thiosulfatesolution,therebyextractingtheorganicphase,which wassubsequentlydriedwithsodiumsulphateandanalysedinthe GC–MS.

Statisticalanalysis

Multivariateanalysiswasconductedwithasinglefactor Per-Manovausing9999permutations.Compoundsthatwerepresent inlessthaneightindividualspergroup,aswellascompounds con-tributinglessthan0.5%tothetotalcompoundswereexcludedfrom analysis.Posthocpairwisetestswereconductedtocomparethe CHCproﬁlesofworkersubcastes.Canonicalanalysiswasperformed toclassifyindividualsintheirgroups.PrincipalCoordinateanalysis wasconductedtodemonstratethemaincompoundsresponsible forsigniﬁcantdiscretisationofthegroups.Thesecalculationswere

0.4 0.2 –0.2 –0.4 –0.4 –0.2 0 0.2 0 CAP3 CAP1 Subcastes Gardener Generalist Forager Soldier

Fig.1. ResultofCanonicalAnalysisofPrincipleCoordinatescarriedoutwith

cutic-ularhydrocarbonsoffourAttasexdenssubcastes,Colony1.

carriedoutusingthestatisticalsoftwarePrimerversion6(Primer-e Ltd).

Results

TheepicuticularextractsofA.sexdensworkerspresentedatotal of24hydrocarbonswithcarbonchainsrangingfrom19to39atoms, mostofthemclassifiedasbranchedalkanes(61.2%)andlinear alka-nes(38.1%),butalsoalowproportionofalkeneswasfound(0.68%) (Table1).Thechemicalprofilesofindividualsweresignificantly dif-ferentaccordingtotheircolonies(PerMANOVA,Pseudo-F=48.537, p=0.0001).EuclideancalculationsinadditiontoCentroidDistance calculationsclassifiedindividualscollectedundernatural condi-tions(Colony3)asthemostchemicallydifferent(Table2).

AsigniﬁcantvariationonCHCworkerproﬁlewasfoundin rela-tiontotheirsubcastes(PerManova,Pseudo-F=25.568,p=0.0001,

Figs.1–3).AccordingtoPrincipalCoordinateAnalysis(PCO)the maincompoundsresponsibleforsigniﬁcantseparationofsubcastes weretwobranchedalkanes(4,8,12-TriMeC36,7,11-DiMeC39)and twolinearalkanes(C25H52,C27H56).Thesecompoundswereamong themostabundantones(Table1).Nocompoundoccurred exclu-sivelyinthecuticleofworkers.However,theconcentrationpattern variedgreatlyinthesubcastes,whichsuggestssimilarchemical signaturesastothevarietyofcompoundsbutwithgreat differ-encesintheirrelativeproportions(Fig.4).Trimethylalkaneswere moreconcentratedinforagers,makingup35%oftotalcompounds, whilelinearalkanesweregenerallymoreconcentratedonboth gar-denersandsoldiers.Overall,generalistspresentedahighrelative concentrationofbothclassesofcompounds,andinterestingly,they presentedall24identiﬁedhydrocarbons(Table1;Fig.4).

Discussion

Thisstudydemonstratesthatthereisavariationinthe cutic-ularhydrocarboncompositionofA.sexdensworkersinrelationto itsfoursubcastes.Thisvariationseemstobemoreinﬂuencedby theconcentrationproﬁleofmostabundanthydrocarbonsthanto

(3)

310 L.Valadares,F.S.Nascimento/RevistaBrasileiradeEntomologia60(2016)308–311

Table1

Relativeproportions(Mean±standarddeviation)ofcuticularhydrocarbonsoffourAttasexdenssubcastes,colonies1,2,and3.

KovatsIndex Compound Gardener(n=45) Generalist(n=45) Forager(n=45) Soldier(n=45)

Z-9-C19 0.35±0.77 1.39±2.29 1613 Z,Z-C23:2 0.51±0.87 0.26±0.57 2500 n-C25 12.7±5.7 9.95±3.95 6.91±2.28 13.3±7.29 2600 n-C26 0.78±.53 0.75±0.46 0.48±0.38 1.69±0.81 2700 n-C27 14±4.51 13.6±4.41 10±3.87 16.2±6.75 2900 n-C29 3.58±1.89 2.97±1.61 2.21±1.9 2.89±3.13 3034 9-MeC30 0.48±0.52 0.53±0.56 0.44±0.66 0.19±0.34 3060 7,11-DiMeC30 8.33±1.6 6.69±2.17 6.17±2.69 8.14±2.2 3100 n-C31 1.26±0.54 1.09±0.41 0.74±0.93 0.69±0.61 3134 9-MeC31 5.72±1.3 4.32±1.02 3.55±1.51 5.66±1.5 3165 3,7,11-TriMeC31 2.81±3.68 2.06±3.11 1.49±1.78 1.53±2.21 3234 9-MeC32 10.2±2.85 8.8±3.39 6.23±3.68 5.81±1.78 3250 4,8,12-TriMeC32 16.8±3.15 14.3±4.66 11.1±5.22 12±3.48 3300 n-C33 2.78±3.11 2.58±3.69 2.31±3.34 1.77±2.7 3310 Unknown 4.64±3.86 3.48±2.57 1.87±1.41 2.65±2.11 3330 9-MeC33 6.02±1.22 5.09±1.28 3.62±1.27 4.78±1.49 3368 3,7,11-TriMeC33 0.17±0.34 0.3±0.45 3.25±3.19 3400 n-C34 0.89±0.61 0.87±0.35 0.33±0.53 0.39±0.41 3424 Unknown 0.6±0.69 0.77±1.07 0.32±0.33 0.29±0.45 3445 4,8,12-TriMeC34 4.39±1.09 4.15±1.55 3.03±1.3 3.34±1.1 3500 n-C35 0.1±0.22 0.4±0.32 0.39±0.5 3570 3,7,11-TriMeC35 1.37±1.57 3.22±3.09 1.78±2.02 3753 4,8,12-TriMeC36 2.45±2.7 8.2±4.57 17.4±6.77 6.85±4.1 3965 7,11-DiMeC39 5.22±3.72 15.2±7.87 3.87±2.13 Table2

Chemicaldistance,centroiddistance,andpairwisetestscarriedoutwiththecuticularhydrocarbonproﬁlesoffourA.sexdenssubcastes.

Colonycombinations Chemicaldistance Centroiddistance Pairwisetest(pvalue)

Colony1vs.Colony2 5.57 3.24 0.0001

SubcasteCombinations Chemicaldistance Centroiddistance Pairwisetest(pvalue)

Gardenervs.Generalist 5.22 2.99 0.0001 Gardenervs.Forager 6.96 5.54 0.0001 Gardenervs.Soldier 5.21 3.05 0.0001 Generalistvs.Forager 5.51 3.05 0.0001 Generalistvs.Soldier 4.78 1.62 0.0025 Foragervs.Soldier 5.68 3.45 0.0001 0.2 _Subcastes Gardener Generalist Forager Soldier 0.1 0 –0.1 CAP1 CAP2 –0.2 –0.3 –0.3 –0.2 –0.1 0 0.1 0.2 0.3

Fig.2.ResultofCanonicalAnalysisofPrincipleCoordinatescarriedoutwith cutic-ularhydrocarbonsoffourAttasexdenssubcastes,Colony2.

thequalitativedifferences,becausenosubstanceoccurred exclu-sivelyinthecuticle ofanyworker groups.Similarresultswere foundin the weaver ant Camponotus textor where only a sin-glecompoundoccurredexclusivelyinthecuticleofdifferentsize

0.3 0.2 0.1 0 –0.1 –0.2 –0.3 –0.2 –0.1 0 0.1 CAP1 CAP2 Subcastes Gardener Generalist Forager Soldier 0.2 0.3

Fig.3. ResultofCanonicalAnalysisofPrincipleCoordinatescarriedoutwith cutic-ularhydrocarbonsoffourAttasexdenssubcastes,Colony3.

(4)

L.Valadares,F.S.Nascimento/RevistaBrasileiradeEntomologia60(2016)308–311 311 70 0 10 20 30 Relativ e propor tion, % n-C₂₅ n-C₂₇ Compounds 4,8,12-TriMeC₃₆ 7,11-DiMeC₃₉ 40 50 60 Nurser Generalist Forager Solider

Fig.4.DistributionpatternoffourcuticularhydrocarbonsamongAttasexdens workersubcastes.

workers(Camposet al.,2012).AccordingtoWilson(1980),the mostspecialisedworkersinA. sexdenshaveheadcapsulewidth less than1.2mm and largerthan2.4mm, excludinggeneralists withitsmorphologicaltraitsmorelikelytothebasalAttinespecies. Interestingly,ouranalysesusingthreedifferentcoloniesindicated generalistsasthemostchemicallydifferentsubcaste(Table2).This maybeexplainedbecausethisworkergrouphasanintermediate sizeandperformsawidearrayofactivitiesinsideandoutsidethe nest.Inourresults,thepresenceofthreetrimethyalkanesthatwas alsoreportedbyMartinandMacConnel(1970)forthesamespecies reﬂectthemostderivedpositionofgenusAttainsidethe fungus-growingAttinitribe(SchultzandBrady,2008).Indeed,thepresence ofmethylalkanesanddimethylalkanesisanancestraltraitinants, whereasthepresenceoftrimethyalkanesseemstobeaderivedone (Wilgenburgetal.,2011).Furthermore,branchedhydrocarbonsare likelyspecies-speciﬁccompoundsinvolved innestmate recogni-tion(MartinandDrijfhout,2009),andlinearhydrocarbonsseems tobemorerelatedtoprotectionagainstparasitesanddesiccation (BlomquistandBagnéres,2010).

Inadditiontotheinfluenceofinternalfactorsontaskallocation (e.g.,worker age,body size, and genetic factors), we hypothe-sisedthatinAttasexdensthedifferencesfoundinworkerchemical cuticularprofilesmayactastaskdecisioncues.Pheromonesthat determinetheglobalsituationofa colonyseemstobeinvolved intaskdecisioninsocialinsects,asdemonstratedintheharvest antPogonomyrmexbarbatuswherecolonyorganisationarisesfrom theresponsesofindividualstolocalcues,andindividualsregulate foragingusinginteractionsmediatedbyCHCsbetweenreturning andoutgoingforagers(GreeneandGordon,2003).Furtherstudies approachingtheinfluenceofabioticfactors,suchastemperature andhumidity,aswellasinvestigationsonthedirectevidenceof CHCsonnestmaterecognitionandtaskallocationarerequiredto abetterunderstandingontheirreliabilityaschemicalcuesin leaf-cutterants.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

TheauthorsthankNationalCouncilofScientificand Technolog-icalDevelopment(CNPq)andFundaçãodeAmparoàPesquisado EstadodeSãoPaulo(Fapesp2010/10027-5)forfinancialsupport. Theauthorsalsothanktwoanonymousrefereesfortheirvaluable suggestionsonthepreviousversionofthismanuscript.

References

Blomquist,G.J.,Bagnéres,A.G.,2010.Structureandanalysisofinsecthydrocarbons.

In:Blomquist,G.J.(Ed.),InsectHydrocarbons.CambridgeUniversityPress, Cam-bridge,pp.19–34.

Campos,M.C.G.C.,Campos,M.L.G.,Turatti,I.C.,Nascimento,F.S.,2012.Cuticular

hydrocarbonvariationofCastesandSexintheWeaverAntCamponotustextor (Hymenoptera:Formicidae).Sociobiology59,1025–1035.

Carlson,D.A.,1989.Dimethyldisulﬁdederivatesoflongchainalkenes,alkadienes,

andalkatrienesforgaschromatography/massspectrometry.Anal.Chem.61, 1564–1571.

Della-Lucia,T.M.C.,Folwer,H.G.,Araújo,M.S.,1993.Castasdeformigascortadeiras.

In:Della-Lucia,T.M.C.(Ed.),AsFormigasCortadeiras.FolhadeVic¸osa,Vic¸osa, pp.43–53.

Forti,L.C.,Boaretto,M.A.C.,1997.FormigasCortadeiras:Biologia.Ecologia,Danose

Controle,Botucatu.

Gordon,M.,2015.Fromdivisionoflabortocollectivebehaviorofsocialinsects.

Behav.Ecol.Sociobiol.,1–8.

Greene,M.J.,Gordon,D.M.,2003.Cuticularhydrocarbonsinformtaskdecisions.

Nature423,423–432.

Hölldobler,B.,Wilson,E.O.,1990.TheAnts.BelknapPressofHarvardUniversity

Press,Cambridge.

Lambardi,D.,Chegia,B.,Turilazzi,S.,Boomsma,J.J.,2004.Diet-inducedaggression

amongcoloniesoftheleafcutterantAcromyrmexechinatiorFore1(Hymenoptera Formicidae).REDIA,LXXXVII21,219–221.

Marinho,G.C.S.,Della-Lucia,M.T.C., Ribeiro,M.M.R.,Magalhães,S.T.V.,Guedes,

R.N.C.,Jham,G.N.,2008.Interferenceofb-Eudesmolinnestmaterecognition

inAttasexdensrubropilosa(Hymenoptera:Formicidae).Bull.Entomol.Res.98, 467–473.

Martin,S.,Drijfhout,F.,2009.Areviewofantcuticularhydrocarbons.J.Chem.Ecol.

32,1151–1161.

Martin,M.M.,MacConnel,J.G.,1970.ThealkanesofAttacolombica.Tetrahedron26,

307–319.

Schultz,T.R.,Brady,S.G.,2008.Majorevolutionarytransitionsinantagriculture.

Proc.Natl.Acad.Sci.U.S.A.14,5435–5440.

Sturgis,S.J.,Gordon,M.D.,2012.Nestmaterecognitioninants(Hymenoptera:

Formi-cidae):areview.Myrmecol.News16,101–110.

Tannure-Nascimento,I.C.,Nascimento,F.S.,Turatti,I.C.,Lopes,N.P.,Trigo,J.R.,Zucchi,

R.,2007.Colonymembershipisreﬂectedbyvariationsincuticular

hydrocar-bonproﬁleinaneotropicalpaperwasp,Polistessatan(Hymenoptera,Vespidae). Genet.Mol.Res.6,390–396.

Valadares,L.,Nascimento,D.,Nascimento,F.S.,2015.FoliarSubstrateAffects

Cutic-ularHydrocarbonProﬁlesandIntraspeciﬁcAggressionintheLeafcutterAntAtta sexdens.Insects6,141–151.

VanderMeer,R.K.,Morel,L.,1998.Nestmaterecognitioninants.In:VanderMeer,

R.K.,Breed,M.,Wintson,M.,Espelie,K.E.(Eds.),PheromoneCommunicationin SocialInsects.WestviewPress.

Wilgenburg,E.,Symonds,M.R.E.,Elgar,M.A.,2011.Evolutionofcuticular

hydrocar-bondiversityinants.J.Evol.Biol.24,1188–1198.

Wilson,E.O.,1980.Casteanddivisionoflaborinleaf-cutterants(Hymenoptera,

Formicidae:Atta).I:TheoverallpatterninA.Sexdens.Behav.Ecol.Sociobiol.7, 143–156.