Effect of the presence of brood and fungus on the nest architecture and digging activity of Acromyrmex subterraneus Forel (Hymenoptera, Formicidae)

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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

Effect

of

the

presence

of

brood

and

fungus

on

the

nest

architecture

and

digging

activity

of

Acromyrmex

subterraneus

Forel

(Hymenoptera,

Formicidae)

Carlos

Magno

dos

Santos

a

_,

_Roberto

_da

_Silva

_Camargo

a,b,∗

_,

_Mariana

_Brugger

b

_,

_Luiz

_Carlos

_Forti

b

_,

Juliane

Floriano

Santos

Lopes

a,c

a_Universidade_Federal_de_Juiz_de_Fora,_Instituto_de_Ciências_Biológicas,_Programa_de_{Pós-graduac¸}_ão_em_{Comportamento}_e_Biologia_Animal,_Juiz_de_Fora,_MG,_Brazil b_Universidade_Estadual_Paulista,_Faculdade_de_Ciências_{Agronômicas,}_Departamento_de_Produc¸_ão_Vegetal,_Botucatu,_SP,_Brazil

c_Universidade_Federal_de_Juiz_de_Fora,_Instituto_de_Ciências_Biológicas,_Programa_de_{Pós-graduac¸}_ão_em_Ecologia,_Juiz_de_Fora,_MG,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29August2016 Accepted2December2016 Availableonline22December2016 Associateeditor:RodrigoFeitosa Keywords: Acromyrmexsubterraneus Diggingbehavior Leaf-cuttingants Nestbuilding Socialinsect

a

b

s

t

r

a

c

t

ThisstudyinvestigatedthestimulithattriggerdiggingbehaviorinAcromyrmexsubterraneusduringnest building.Thehypothesiswasthatthepresenceofthefungusgardenand/orbroodtriggerstheexcavation oftunnelsandchambers.Fortheexperiment,theexcavationrateofindividuallymarkedworkerskept inplasticcylindersfilledwithsoilwasrecorded.Fourtreatmentswereapplied:(1)30medium-sized workers,5gfungusgardenand30brooditems(larvaeandpupae);(2)30medium-sizedworkersand 5gfungusgarden;(3)30medium-sizedworkersand30brooditems;(4)30medium-sizedworkers withoutfungusandbrood.After24h,morphologicalparametersofneststructure(lengthandwidthof thechambersandtunnelsincm)andthevolumeofexcavatedsoilwererecorded.Incontrasttothe expectedfindings,nochangeinmorphologicalstructure,rateofexcavationbyworkers,orvolumeof excavatedsoilwasobservedbetweentreatments,exceptfortunnelwidth,whichwasgreater,whenno broodorfungusgardenwaspresent.Thus,theresultsdonotsupportthehypothesisthatthefungus gardenand/orbroodarelocalstimulifornestexcavationorthattheymoldtheinternalarchitectureof thenest.AlthoughthishypothesiswasconfirmedforAcromyrmexlundiiandAttasexdensrubropilosa, thesamedoesnotapplytoA.subterraneus.Thediggingbehaviorofworkersisprobablytheresultof adaptationduringnestbuildingindifferenthabitats.

Introduction

Theabilityofworkersofleaf-cuttingantstobuildnestsof com-plexarchitectureisanimportantecologicalaspecttobeconsidered (KleineidamandRoces,2000;Moreiraetal.,2004).Theadultnests ofleaf-cuttingantsofthegenusAcromyrmex,insteadofthoseofthe genusAttawhichconsistofthousandsofundergroundchambers (Camargoetal.,2013),areoftensmallerwithhighlyvariable num-beroffunguschamber.InA.molestansSantschi,1925forexample ithasbeenfoundruralnestswithjust1chamberwhereasinthe urbanareatheyhaveuptofourchambers(Lopesetal.,2011).InA. rugosus(Smith,1858)thenumberofchambersrangesfrom1to26 (Verzaetal.,2007)andinA.landolti(Forel,1885)from1to11(Silva Junioretal.,2013).ForallAcromyrmexandAttaspecieschambers

∗ Correspondingauthor.

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

areconnectedtoeachotherandtosurfacebytunnels(Camargo etal.,2013;SilvaJunioretal.,2013;Lopesetal.,2011;Verzaetal., 2007).

Thecompletestructureoftheundergroundnestfromboth gen-eraisobtainedbythesimplediggingbehaviorofworkers,which remove soilparticleswiththeirmandibles(Cassillet al.,2002). Thusnestsofleaf-cuttingantsaretheresultofthecoordinated andself-organizedactivityofspecializedworkers.Nestbuilding representsabehavioral evolutioninanattempttooptimizethe protectionagainstpredatorsandvitalmaintenanceofthecolony, regulatingtheentryandexitofgases,temperatureandhumidity insidethenestandthusensuringgooddevelopmentofthebrood andsymbionticfungus(Bollazzietal.,2008;BollazziandRoces, 2010a,b,c).

Duringexcavation,workersshouldbeinvolvedintheactivity inacoordinatedmannerandshouldrespondactivelytostimuli. Aftercommencementoftheexcavationprocess,nestmatesshould be recruited to the digging site. This recruitment is probably

http://dx.doi.org/10.1016/j.rbe.2016.12.002

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controlledbyapositivefeedbackmechanism,whichisactivated bypheromones(PielströmandRoces,2012;CamargoandForti, 2014).Thephysicalcontactbetweennestmatesalsoincreasesat diggingsitesbecauseofthehigherdensityofantsatthesesites,a factorthatseemstoinﬂuenceandtriggertheexcavationprocess withahighdensityofants(Römerand Roces,2015).Thesocial organizationofleaf-cuttingantsitselfproducesacontextinwhich workersshouldrespondnotonlytotheirindividualnecessities, butalsototheneedsofthewholecolony.Inthisrespect, work-ersadjustthesizeoftheirnestsaccordingtothepopulationsize ofthecolony(RasséandDeneubourg,2001),aswellastothe vol-umeofthesymbionticfungus(FröhleandRoces,2009;Camargo etal.,2013).Alsoclimaticvariables,assoiltemperatureand inter-nalnesthumidity,actasshort-termregulatorybuildingstimulifor Acromyrmex(BollazziandRoces,2010c).

Theinvestigationoftheexcavationprocessprovidesan under-standingofthebehavioralpatternsthatarefundamentalforthe social organization of leaf-cuttingants. Thesepatterns forman importantbasisforecologicalstudiesonthebehavioraldynamics thatregulatestheorganizationofcollectiveactivitiesineusocial insects.

CamargoandForti(2014)determinedthestimuliforexcavation inAttasexdensrubropilosa(Linnaeus,1758)andconcludedthatthe presenceofbroodandsymbionticfungusisanimportantstimulus forworkerstoimplementtasksinvolvedinnestbuilding,i.e.,tasks associatedwiththeprotectionofthebroodandsymbionticfungus. Furthermore,Camargoetal.(2013)concludedthatthesymbiontic fungusisablueprint fortheconstructionoftunnelsand cham-bers.However,itremainsunknownwhetherandhowmuchthese factorsactasstimulusforotherleaf-cuttingantspecies.

Theinternalcomplexityanddepthofnestsvarywidelyin leaf-cuttingants.Somenestsareshallowandothersaredeeper,andthe numberofchamberscontainingfungusvaries(Gonc¸alves,1961; Lopesetal.,2011).ThethreesubspeciesofA.subterraneus(Forel, 1893)buildneststhatareundergroundorpartiallyunderground inmostcasesandlocatedneartherootsystemofplants(Bondar, 1923apudDellaLucia,2011;Gonc¸alves,1961;Andrade,2002). Fur-thermore,there are descriptions ofyoungnests ofAcromyrmex subterraneusbrunneus (Forel, 1912), whichconsist externallyof loosesoilandinternallyofasinglechambercontainingthefungus (Camargoetal.,2004).

Toinvestigatethestimulithatdeterminenestarchitectureand thediggingactivityofworkers,thepresentstudyevaluatedthe physicalparametersofA.subterraneusnestsbuiltunderlaboratory conditionsinwhichthepresenceofthefungusand/orbroodwas manipulated.Thepremisewasthatthefungusgardenand/orbrood arestimuliforexcavationanddirectlyinﬂuencenestmorphometry andworkeractivity.

Materialandmethods

Studiedspecies

SixteenA.subterraneuscoloniesmaintainedsince2012inthe LaboratoryofMyrmecology,UniversidadeFederaldeJuizdeFora (UFJF),JuizdeFora,MinasGerais,Brazil,wereused.Antspecies identiﬁcationwasmadeaccordingtoFortietal.(2006).Thecolonies aremaintainedina closedsystemconsistingofthree compart-mentsinterconnectedbyclearplastic tubing,whichcorrespond tothefunguschamber,foraging arena,and wastechamber.The coloniesaremaintainedundercontrolledconditionsof tempera-ture(26◦C)andhumidity(70%)andreceivedAcalyphawilkesiana (Euphorbiaceae)leavesdaily.

Fortheexperiments,30workerswererandomlyselectedfrom eachcolony.Allworkersselectedfortheexperimentbelongedto

themedium size class, witha head width rangingfrom 1.2 to 1.6mm.

Allindividualsweremarkedwithauniquecolorcombinationon thepronotumtoidentifyeachworker.Edding®markerswereused forthispurposebecauseoftheirexcellentadhesion,rapiddrying, andgoodvisibility(Camargoetal.,2007).Workerswereplacedin plasticcontainerswhoseborderswerecoveredwithneutraltalcto preventescapeuntildryingoftheink,andafter24htransferred toanothercontainerputovertheexcavationcylinderwherethey remaineduntilthetimeofﬁlming.

Portionsofthesymbionticfunguswerealsoremovedfromthe samecoloniesusingafungalmassof5gperexcavationcylinder. Unmarkedworkers,whichmayhavebeenremovedtogetherwith thefungalportion,werereturnedtothecolony.Thelarvaeand pupaeusedintheexperimentwereremovedfromthesamecolony. Hence,theexcavationcylinderscontainedelements(workers, sym-bionticfungus,andbrood)fromthesamecolony.

Observationcylinders

Excavationcylinders,eachmeasuring25cminheightand10cm indiameter,wereﬁlledwithlatosolcollectedatthecampusofUFJF. Thesoilwasremovedfromadepthof60cmandpreviouslysieved. Consideringthevolumeoftheexcavationcylinder,thesoilwas weighedtoobtainadensityof1.6/cm3_according_to_Camargo_et_al.

(2011).

A250-mLplasticcontainerwasplacedaboveeachcylinderfor observation.Thecontainerhadaholeatthebottomtopermitdirect contactwiththesoiltobeexcavated.Amonitoringcamerawas positionedperpendiculartothecontainertorecordtheactivities ofworkersfor24h.

Stimuliforexcavationbyworkers

Fourtreatments,eachconsistingoffourrepetitionsappliedto differentcolonies,whichvariedintermsofthestimulifor excava-tion,wereapplied:

TreatmentFB:30workers,5gfungusgardenand30brooditems (larvaeorpupae);

TreatmentFG:30workersand5gfungusgarden;

TreatmentLP:30workersand30brooditems(larvaeorpupae); TreatmentWK:30workerswithoutfungusgardenandbrood. Afterﬁlming,theexcavatedsoilfoundabovetheexcavation cylin-derwasweighedforthedeterminationofwetweightandthen driedinanoven(70◦C)for72hforthedeterminationofdryweight (g).

Diggingratebyworkers

Theﬁrst10minofeachhourwereevaluatedperrepetitions, recordingtheindividualrateofdiggingactivity(transportofthe soilpellet).

Tunnelarchitecture

Themethodofmodelingantnestswithcementusedbysome authorsfacilitatesvisualizationoftheshapeofchambersand tun-nels and permits toobtainan overall idea of nest architecture (Moreira,2001).Thus,attheendoftheexcavationperiodand ﬁlm-ing,thetunnelsandchamberswereﬁlledwithliquidplasterata plaster–waterproportionof3:2(Fig.1).Aftertheplasterhaddried, theexcavationcylinderswerecutwithastylusandthesoilwas removed,thusobtainingplastermoldsofthestructureofthe exca-vatednest.Thesemoldswereusedtomeasurethelengthandwidth

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Fig.1.PlastermoldofanestexcavatedbyAcromyrmexsubterraneusworkers.(A)Plastermoldoftunnels;(B)amoldedanddriednestreadytoberemoved;(C)labeledand measuredstructure.

ofthetunnelsandchambersandtoquantifythenumberofentrance holes.

Statisticalanalysis

Theeffectsofthetreatmentsonthearchitectureofemerging structuresanddiggingactivitywereevaluatedusinggeneralized linearmodels(GLM).The responsevariables were wetand dry weightof excavatedsoil, length and width of the tunnels and chamber,numberoftunnels,chambersandentranceholes, and excavationrate.Treatmentwasconsideredtheexplanatory vari-able.TheanalyseswereperformedusingtheRi3863.1.1program (RCoreTeam,2015).

Results

Architectureofemergingstructures

Nests composed of up to four tunnels were observed, but thenumberoftunnelsdidnotdiffersigniﬁcantlybetween treat-ments(GLM:F=1.41;d.f.=3;p=0.19).Theneststhatpossessed fourtunnels wereobserved intreatment WK,which contained only workers. There was also no signiﬁcant difference in the numberofentranceholes(GLM:F=35.04;d.f.=3;p=0.69).Only

oneoftherepetitionssubmittedtotreatmentLPhadsevenentrance holes.Thelengthofthetunnelsdidnotdiffersigniﬁcantlybetween treatments(GLM:F=28.30;d.f.=3;p=0.44).However,the treat-mentsexertedasigniﬁcanteffectontunnelwidth(GLM:F=539.02; d.f.=3;p=0.04),withtheobservationofwidertunnelsintreatment WK(Table1).

No signiﬁcant difference in the number of chambers was observed between treatments (GLM: F=68.17; GL=3;p=0.22), highlightingtheoccurrenceofsevenchambersinonerepetitionof treatmentLP.Therewasalsonosigniﬁcantdifferenceinthelength (GLM:F=52.65;d.f.=3;p=0.42)orwidthofthechambers(GLM: F=117.73;d.f.=3;p=0.43).

Diggingactivity

Thedryweightofexcavatedsoildidnotdifferbetween treat-ments(GLM:F=31.86;d.f.=3;p=0.35).Thesamewasobservedfor thewetweightofexcavatedsoil(GLM:F=74.14;d.f.=3;p=0.26) (Fig.2AandB).

Diggingactivityalsodidnotvarysigniﬁcantlyasafunctionof treatment(GLM:F=0.40;d.f.=3;p=0.74)ortime(GLM:F=1.07; d.f.=3;p=0.37).Thisactivityappearedtobehigher intheﬁrst 12h.Peakactivitywasobservedbetweenthe8thand12thhour intreatmentLP(Fig.3).

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Table1

Rangeofthenumberofentranceholes,numberandsizeoftunnelsandchambersexcavatedbyAcromyrmexsubterraneusworkers.

Treatment Entrancehole(n) Tunnels(n) Chambers(n) Tunnellength(cm) Tunnelwidth(cm) Chamberlength(cm) Chamberwidth(cm)

FP 1–3 1–3 1–5 6.0–23.0 0.8–1.0 1.0–2.0 1.0–5.0 FG 1–3 1–3 1–3 2.5–6.0 0.7–2.0 0.7–3.0 0.7–3.0 LP 1–7 1–2 1–7 3.0–20.0 0.5–1.0 0.5–3.0 0.5–4.5 WK 1–4 1–4 1–3 1.0–1.5 1.0–1.5 1.5–2.0 1.5–3.3 FB FG LP WK 01 0 2 0 3 0 4 0 Dr y s o il (g ) FB FG LP WK 01 0 2 0 3 0 4 0 We t so il (g )

Fig.2.Dryandwetmass(g)ofexcavatedsoilaccordingtothetreatment.

Discussion

Theresultsshowedthatthefungusgardenorbrooddidnotact asastimulusforexcavationinA.subterraneus.Thisﬁndingisin

contrasttothestudyofCamargoandForti(2014)onAttasexdens rubropilosa,inwhichthefungusgardenandbroodservedasstimuli forworkerstoexcavatechambersandtunnels.

AlthoughRömerandRoces(2014,2015)hadreportedaneffect ofthesestimuliinAcromyrmexlundii,theyadditionallyobserved thattheworkersofthisspeciesareabletouseavailablespaceinthe nestsinsteadofexcavatingnewgalleries.Thesameseemstoapply toA.subterraneus,consideringthesimilarmeasurementsofnest structureandsimilardiggingactivity,regardlessofthepresenceof thefungusorbrood.Thisopportunisticbehaviorinnestbuilding, inwhichpreexistingemptyspacesareused,hasbeenreportedfor Acromyrmexsubterraneusmolestans(Lopesetal.,2011).

Inaﬁrstinstance,onemayspeculatethatthefactofkeepingthe numberofindividualsconstantinalltreatmentsledtoasimilar rateofexcavationinalltreatments.Thesamenumberofworkers permitsthesamerateofcontactsbetweenindividuals,withthe contactrateactingasastimulusforworkers(RömerandRoces, 2015).Inthepresentstudy,thisfactorservedasasimilarstimulus inalltreatments.AccordingtoBuhletal.(2004),boththeincrease andthedecreaseinexcavationratearerelatedtotheperception ofsignalsbytheindividuals.Thisfacthasalsobeenanalyzedby

PielströmandRoces(2012)whosuggestedstridulationasatrigger torecruitnestmatesfordiggingactivity.

Thegroupof30individualsusedintheexperimentwas con-sidered sufﬁcient to excavate the nest, with workers digging throughoutthe24-hperiod(Fig.3).Despitethelackofa signif-icantdifferenceover time,theexcavationrateexhibitedamore

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lineardistributionintreatmentsFBandFG,whilethedistribution wasmoreexponentialintreatmentLP,withtheobservationofa markedincreasefollowedbyadecline.Thesamepatternhasbeen observedbyFröhleandRoces(2009)forAcromyrmexlundiiandby

Camargoetal.(2013)forAttasexdensrubropilosa.Incontrast,this variationwaslesspronouncedintreatmentWK.

Adifferencecouldalsobeobservedinthefirst12h(Fig.2).Ifthe excavationperiodwereshorter,i.e.,only12h,differencesin struc-tureandexcavationratecouldhavebeendetectedsinceahigher activityisobservedinthefirst12h.Itisthereforeassumedthat, duringaperiodof24h,thedifferencesbecamesimilarforall treat-ments,i.e.,associatingboth issues,30workerswereefficientin diggingduringtheobservationperiod.

Thearchitectureofemergingstructuresdidnotshowsigniﬁcant variationsbetweentreatments,withtheobservationofasimilarity ofthesestructures.AnexceptionwasobservedintreatmentWK, whichexhibitedwidertunnelsandcouldbeconsideredthecontrol oftheexperiment.Theabsenceofbroodandfungusseemstohave alteredthediggingbehaviorofworkers,whichbuiltwidertunnels intheabsenceofothertaskstobeperformed.Thisfactisclearly supportedbythe“foraging-for-work”theory(Tofts,1993),which proposesthatworkersofleaf-cuttingantstendtoactivelysearch forwork.Intheabsenceofstimuli,theantsmayhavesearchedfor work,intensifyingtheenlargementoftunnels.Fromthispointof view,itwouldthenbecomenecessarytodecidetoimplement dig-gingasataskduetotheabsenceofothertaskstobeperformed. Incontrasttotheproposalofthisstudy,inthesituationobserved speciﬁcallyinthecontroltreatment,itappearsthatthestimulus forexcavationwasexactlytheabsenceofbroodand fungus,in agreementwiththetheoryofTofts(1993).

Withrespecttotheassumption thatbrood andfungus trig-gerexcavationinA.subterraneus,nosignificantdifferencesinthe rateofexcavationwereobservedbetweentreatments.However,an increaseintheexcavationratewasseenintreatmentLP, suggest-ingaroleofthebroodasastimulusforexcavation.Thepresence ofbroodmayhaveexertedapositiveeffectonworkerdensityat thesite,stimulatingexcavation,sincetheaggregationofworkers atacertainlocationtriggerstheexcavationprocess(Römerand Roces,2015).Workersexerttheirfunctiontomaintainstabilityand toensurethesuccessofcolonygrowth.Larvaeandpupae repre-sentfutureworkersandanintimaterelationshipexistsbetween adultworkersandtheirimmaturesiblingsinsuchawaythatthe formerareresponsibleforthecareofthelatter,constantly seek-ingconditionsthatprovideprotectionandensuretheirintegrity andfulldevelopment.Theprioritywouldthusbetheexecutionof actionsthatensurethesuccessofthefuturegeneration.Bollazzi andRoces(2002)addthatthebroodisalwaysrelocatedfirst. Fol-lowingthislineofreasoning,RömerandRoces(2015)identifiedin abehavioralstudythatdiggingactivitywasmoreintenseatsites containingbroodcomparedtothosewherethebroodwasabsent. Analyzingtheissuefromaprioritypointofviewinnestbuilding, theprioritywouldbetheexecutionofactionsthatensurethe suc-cessofthefuturegeneration.Althoughthesymbionticfungusis theonlyfoodsourceofthecolony,inthiscaseitdoesnot repre-sentanimmediateprioritythatwouldjustifytheinvestmentof energyinnestbuilding,atleastduringthe24hevaluatedinthe presentexperiment.Afterbroodrelocation,thepossiblenext pri-oritystepcouldbethetransportofthefungustoguaranteefood resources.

Inconclusion,thepresenceofbroodorfungusdidnotserve asastimulusforexcavation,althoughthisbehavioralpatternhas beenobservedinotherleaf-cuttingantssuchasAcromyrmexlundii (RömerandRoces,2015)andAttasexdensrubropilosa(Camargoand Forti,2014).ThisobservationhighlightsthefactthatA.subterraneus workersdonotadheretothisbehavioralpattern,probablybecause diggingbehavioristheresultofadaptationduringnestbuildingin

differenthabitats.However,furtherstudiesareneededtoanswer thisquestion.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest.

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