Nest architecture development of grass-cutting ants

REVISTA

BRASILEIRA

DE

Entomologia

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

Biology,

Ecology

and

Diversity

Nest

architecture

development

of

grass-cutting

ants

Luiz

Carlos

Forti

_,

_Aldenise

_Alves

_Moreira

_,

_Roberto

_da

_Silva

_Camargo

_,

_Nadia

_Caldato

_,

Maria

Aparecida

Castellani

a_{UniversidadeEstadualPaulista(UNESP),FaculdadedeCiênciasAgronômicas,Botucatu,SP,Brazil}

b_{UniversidadeEstadualdoSudoestedaBahia(UESB),DepartamentodeFitotecniaeZootecnia,LaboratóriosdeEntomologia,VitóriadaConquista,BA,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received27June2017 Accepted18October2017 Availableonline16November2017 AssociateEditor:JeffreySosa-Calvo Keywords:

Grass-cuttingants Nest

Socialinsects

a

b

s

t

r

a

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Attabisphaericaisaspeciesofgrass-cuttingantscommonlyfoundintheCerradobiome.TheBrazilian Cerrado(savanna)biomecovers2millionkmrepresenting23%oftheareaofthecountry.Itisanancient biomewithrichbiodiversity,estimatedat160,000speciesofplants,fungiandanimals.However,little isknownabouttheirnestarchitecturedevelopment.Thisstudyinvestigatedthearchitectureoffourteen A.bisphaericanestsfromBotucatu,SãoPaulo,Brazil.Moldsweremadeofthenestsbyfillingthemwith cementtoallowbettervisualizationofinternalstructuressuchaschambersandtunnels.Afterexcavation, thedepthanddimensions(length,width,andheight)ofthechambersweremeasured.Asexpected,there wasalateraldevelopmentinthenestsandincreaseinthenumberofchambersovertime.Resultsshowed thatinnestswithanestimatedageof14months,theaveragedepthwas1.6±0.4m;forthosewith18 monthsitwas2.2±0.7mandat28monthsitwas2.5±0.7m.Thenumberofchambersvariedfrom4to 7in28-monthnests,2to4in18-monthnests,andfrom2to3in14-monthnests.Withrespecttothe dimensionsoftheinternaltunnels,therewerevariationsintheiraveragewidth,increasingwithtime. Thefunguschamberswerelocatedbeneaththelargestmoundofloosesoil.Thisstudycontributestoa betterunderstandingofthesofarunknownnestarchitecturedevelopmentofA.bisphaericagrass-cutting ants.

©2017SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Thenestsofleaf-cuttingantsconsistofanexternallyvisiblepart, amoundofloosesoil,whichischaracterizedbyhavingalarge num-berofholesthatleadtotheinternalpart,withthelatterbeingmade upoftunnelsofvariousdiametersandshapes,whichallowthe traf-ficoftheantsandinterconnectholeswithchambers.Thetunnels thatreachthesurfaceoftheloosesoilofthenestopenthrough holesthataretheexitsofthelandfilltunnels,alongwhichtheants transportthesoilremovedfromtheirexcavations.Theforaging tunnels,alongwhichworkerscutleaves,opentotheoutsideofthe nestinholes(Gonc¸alves,1964)usuallylocatedoutsidethelimits oftheloosesoilareaatvariabledistancesfromthelatter.

ThemoundofloosesoilofAttanestsisoneofthemost rel-evantaspects,sinceitisacharacteristicinitiallyobservedinthe fieldforidentificationofspecies,assmallorlargevariationsmay occurdependingonthespecies.AccordingtoMariconi(1970),ina generalway,Attasexdens,Attalaevigata,AttabisphaericaandAtta

∗ Correspondingauthor.

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

cephalotesnestspresentasoilmoundonly.Ontheotherhand,Atta capiguaraalsoshowsacertainnumberofsecondaryareas.

Althoughmostspeciespresentasinglemound,thereare vari-ationsintheformofsoildepositionbytheantsandinthechoice ofthesitetobuildthesenests.TheA. sexdensandA. cephalotes speciesbuildtheirnestspreferablyinshadyplaces,andland depo-sitionoccursirregularly,throughtheformationofsmallmounds ofsoil,withtheshapeofsmallvolcanoeslocatedaroundtheholes abovethemoundofloosesoil.InA.laevigata,depositionoccursin aregularway,beingconcentratedandconvex,andnestsare con-structedinbothsunnyandshadyplaces(Pereira-da-Silva,1975; Moreiraetal.,2004a).

Normally,theloosesoilsareontheclusterofchambers. Accord-ingtoEidmann(1932),Autuori(1942),Jacoby (1950),Mariconi andPaivaCastro(1960),PaivaCastroetal.(1961),Amante(1967), Gonc¸alves (1967), Pereira-da-Silva (1975), Jonkman (1980a,b), Pretto(1996)andMoreiraetal.(2004a,b),A.bisphaerica,A.sexdens, A.laevigataandAttavollenweideriantsbuildtheirchambersinthe underground,intheprojectionofloosesoil.Therefore,theloose soilistheantcolony’score,while,accordingtoAmante(1967)and Forti(1985),theA.capiguaraspeciesconstructsitsnestina differ-entway–theapparentcoredoesnotcoincidewiththerealone, andhugegarbagechamberscanbefoundundertheapparentcore. https://doi.org/10.1016/j.rbe.2017.10.002

0085-5626/©2017SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Chambersvaryinshape,locationanddimensions,dependingon thespeciesandinsomecasesoftherole.StudiesonA. vollenwei-dericarriedoutbyBonetto(1959),Carvalho(1976)andJonkman (1980b)indicatedthatlivingchambershadanovalshapewithaflat bottomandthatgarbagechambershadaconicalshape.ForA. sex-dens,Jacoby(1950)concludedthatthechamberstructureissimple, varyingfromasphericalenclosureoranellipsoidonewithclosed dome,whileGonc¸alves(1964)foundsphericalchamberswithaflat bottom.

ThechambersofA.sexdensnestsstudiedbyGonc¸alves(1964) presenteddiameterbetween20and 40cm, whilePretto(1996) found chambers withan averageheight of 18.31cm, width of 24.25cmandlengthof24.24cm.ForA.capiguara,Mariconietal. (1961)foundsmallormediumchambersmeasuringapproximately 21cminheightby27cm,15cmand17cminwidth,and17cmand 16cminlength.

ThechambersofA.laevigata,inPaivaCastroetal.(1961),were mostlylocatedbetween0.50and1.80mdeep,while Pereira-da-Silva(1975)foundthemonthesurface(includingintheloosesoil area)upto6mdeep,andMoreiraetal.(2004a)upto7mdeep.Thus, dependingprobablyonloosesoilarea,soiltype,speciesstudiedand thecolony’smicroclimaticneeds,depthmayvary.

Asfordepthoflocationofthechambers,Bonetto(1959)found chambersfrom0.70to3.0m,concentratedinthecenterofthenest ofA.vollenweideri.InstudiesonA.capiguara,Mariconietal.(1961) observedthatthechamberswereconcentratedbetween1.0and 1.60mdeep.InA.sexdens,Gonc¸alves(1964)foundalargernumber oftheminthecenteroftheanthillandbetween1and2mdeep, whilePretto(1996)foundonly22%upto1m,withtheothersbeing belowthis.

InstudiesonnestarchitectureofA.cephalotes,Staheland Gei-jskes(1939)foundatotalof373chambers,ofwhich344contained fungus.ForA. sexdens,a totalof 1027chamberswerefoundby Autuori(1942),whichgeneratedadensityof8.52chambersm−3, while Pretto (1996) obtained densities of 1.75; 2.43, 2.66 and 4.17chambersm−3forthefournestsstudied.

Thementionedstudieswerecarriedoutinadultnests,but lit-tleisknownaboutthegrowthofthecolonyand,consequently,of thenest.Thepresentstudydescribednestarchitecture develop-mentinthreeperiods:estimatedagesof14,18and28monthsof A.bisphaericagrass-cuttingants.

Materialandmethods Studiedarea

The study was conducted at Santana Farm (20◦5046S; 48◦262W), locatednear UNESP’s LageadoExperimental Farm, Botucatu,SãoPaulo,Brazil,inaCerradofragment.

Ageofnests

Twothousandfounderqueenswereobservedimmediatelyafter thenuptialflighton4October2008attheLageadoExperimental Farm,FCA/UNESP,Botucatu,Brazil,after,theystartedtodigtheir nests,wemarkedindividually allnestswithwoodwhitestake. Afterthat,fourteennestswereexcavatedforarchitecturestudy. Studiednests

Fourteennestswereusedinthisstudy–withapproximately 14months(4nests),eighteenmonths(5nests)andtwenty-eight months(5nests)afternestfoundation.

Thesizeofthenestswasconfirmedbymappingtwoorthree activeentranceholesnearthenests.Theholesweremappedusing

plastic strawbaitsofdifferentcolorsand cuttypes(totaling48 typesofbaits).

Thestrawswerecutinto3–4mmpiecesand immersedina solutionofconcentratedorangejuicecontainingsugar(forminga syrup).Thestrawfragmentswereremovedwithsievesand trans-ferredtotrayswhereamixtureofcitruspulpandsugarcaneleaf powderwasadded.Thepieceswerecarefullymovedinsidethese trayssothemixtureimpregnatedwellonthestrawsandwashighly attractivetotheants.Thestrawsweredriedfor24hatroom tem-perature.Afterthisperiod,thebaitswerestoredinplasticbags identifiedwithnumbersbycolorandtypeofstraw tofacilitate handlinginthefield.

Inthefield,theplasticstrawbaitswereplacedintheactiveholes nearthenests.Thenumbercorrespondingtoeachtypeofbaitwas markedonthestake.After24h,themoundofloosesoiltowhich eachstrawwasreturnedbytheantswasidentifiedandthetype andcolorfound,whichindicatewhichentranceholesbelongto eachnest,wererecorded.

Internalarchitecture

Thenestsweremoldedusingamixtureof5kgof cementin 10Lofwater,whichwaspouredintoallopenholesabovethesoil moundofthenestandintotheholesspreadonthesoilsurface (foragingholes)asdescribedbyMoreiraetal.(2004a,b).The water-cementmixturewaspouredintotheholesusingaluminumfunnels. Excavationstarted7daysafterapplicationofthecement.

Priortoexcavation,theexternalareaofthenestwasdetermined bythetraditionalmethod,whichconsistsofmeasuringthegreatest widthandgreatestlengthoftheareacomprisingallmoundsof loosesoil.Twonylonstringswerethenstretchedoverthenestarea, formingtwoorthogonalaxes(x;y),whosecentercorrespondedto thecenterofthenestarea.Theseaxeswereusedtoidentifythe chambersandtunnels.

Theareasofthenestsfilledwithcementwereexcavatedusing smallmanualtoolstoavoidtheirdestruction.A0.70-mwideand 1-mdeepditchwasopenedaroundthenestareaandexcavationwas performedfromtheoutsidetotheinside.Theditchwasdeepened untilcompleteappearanceofthechambers,whichwerenumbered forsubsequentmeasurement.Afterexcavation,thechambersand tunnelsweremeasuredandphotographed.Thefollowing param-eterswereobtained:dimension(width,height,andlength),depth fromthesoilsurface,andpositionontheorthogonalaxes(x,y). Themean,standarddeviationandrangeofthemeasurementswere calculatedforstatisticalanalysis.Thevariablesstudiedwere com-paredbetweennestsatthe3differentages,bymeansofANOVA, at5%ofsignificance.

Resultsanddiscussion

As expected, there was a lateral development in the nests and increase in the number of chambers over time (ANOVA, F2;13=16.96,p<0.001),thatis,thecolonygrowingasafunctionof time.However,A.bisphaericanestsofdifferentagesdidnotpresent majorchangesindepth(ANOVA,F2;13=1.35,p=0.2973).

Morphologically,thechambersofA.bisphaericanestsstudied werelocatedintheprojectionoftheloosesoilmound,asalready reportedbyAmante(1967)andMoreiraetal.(2004b).OtherAtta speciesalsoconstructtheirchambersintheprojectionofloose soilmound,including:A.sexdens(Eidmann,1932;Autuori,1942; Jacoby,1950;Amante,1967;Gonc¸alves,1967;Pretto,1996),A. vol-lenweideri(Jonkman,1980b)andA.laevigata(MariconiandPaiva Castro,1960;PaivaCastroetal.,1961;Pereira-da-Silva,1975; Mor-eiraet al.,2004a).However,in coloniesof A. capiguara,fungus chambers arelocated outside theapparentcore,distanced and

Fig.1.DetailsoftheinternalarchitectureofAttabisphaericanests,withapproximate ageof14months(A),18months(B)and28months(C).Botucatu,SP,2014.

distributedlaterallybelowgroundlevel,andgarbagechambersare locatedbelowthiscore(Amante,1967;Fortietal.,2017).

Thedeterminationoftheplaceofhighestconcentrationof fun-gus chambers hasbeen a relevant factor in theapplication of measuresforAttacontrol,becausewhenthelimitsofloosesoil areacoincideswiththeareaofhighestconcentrationofchambers inthenest,controlismucheasier(Moreiraetal.,2004a).

Abouttheinternalstructureofthenests,itwasobservedthat amongnestsofdifferentagestherewasnogreatvariationindepth (Fig.1,Table1);however,alateralgrowthwasobserved(Fig.1),as alreadyreportedbyMoreiraetal.(2004b)inmaturenests.

Innestswithanestimatedageof14months,theaveragedepth was1.6_±0.4m,rangingfrom1.2to2.1m,whileforthosewith18 months,itwas2.2±0.7mandrangedfrom1.4to2.9m.For 18-monthnests,theaveragedepthwas2.5±0.7,rangingfrom1.3to

Table1

Numberofchambersandtotaldepthofthenestsandrespectivedataonmean, modeandstandarddeviation(S*),maximumandminimumnumberofchambers byestimatedageofAttabisphaericanests,Botucatu,SP,2014.

Nests Estimatedage(months) Chambers(No) Nesttotaldepth(m) I 14 3 1.8 II 2 2.1 III 2 1.2 IV 3 1.2 Mean 2.5 1.6 Mode 3.0 1.2 S* 0.6 0.4 Maximum 3.0 2.1 Minimum 2.0 1.2 V 18 3 1.5 VI 3 2.9 VII 2 1.4 VIII 3 2.6 IX 4 2.5 Mean 3.0 2.2 Mode 3.0 – S* 0.7 0.7 Maximum 4.0 2.9 Minimum 2.0 1.4 X 28 4 1.3 XI 7 2.9 XII 7 2.6 XIII 5 2.8 XIV 7 2.8 Mean 6.0 2.5 Mode 7.0 – S* 1.4 0.7 Maximum 7.0 2.9 Minimum 4.0 1.3

2.9m(Table1).AsobservedinA.bisphaerica,chamberscontaining fungusgardenwithfreshlyincorporatedplantfragmentsarefound nearthesoilsurface.Thisfactsuggestsanadaptationofthespecies towithstandvariationsintemperatureandsoilhumidity,sinceit usuallybuildsitsnestsinopenareaswithplentyofsunlight;even deepsoilschambersarenotlocatedatgreatdepths.Ittherefore seemsthatnestdepthisnotlimitedbythegroundwater table (Moreiraetal.,2004b).Ontheotherhand,duetothecomplexityof thenests,antschoosethebestlocationwithinthenestintermsof temperatureandhumiditygradientstoreareggs,larvaeandpupae, aswellasforthesymbioticfungusandallbiotainvolved(Moreira etal.,2004b).

ThenestsofA.bisphaericastudiedpresentedchambersofvaried forms(Fig.2),asalreadyobservedbyMoreiraetal.(2004b), Pereira-da-Silva(1975),andMoreiraetal.(2004a)forA.laevigata.Itwas observed,however,thatthesphericalshapewaspredominantfor thespeciesstudied,asconcludedbyMoreiraetal.(2004b)forthe samespecies,andMoreiraetal.(2004a)forA.laevigata.

Thenumberofchambersvariedfrom4to7in28-monthnests, from2to4in18-monthonesandfrom2to3in14-monthnests (Table1,Fig.1).

The width, height, and length dimensions of the chambers (Table2)werevariable,showingsomedifferencesbetweennests, alreadyobservedbyJonkman(1980b)whenanalyzingthe aver-ageheightofA.vollenweiderichambers,attributingthisdifference tocolonyage,andbyMoreiraetal.(2004a,b),whoobserved varia-tionsinchamberdimensionsbetweenA.laevigataandA.bisphaerica nests.Variationsinthedimensionsofchambersofthesamespecies mayderivefromthenest’sneedtoincreasefunguscultivation,due tocolonygrowth.

WhencomparedtothedimensionsofA.bisphaericanest cham-bersstudied,variationswerefoundastoaveragewidth,height andlength.FornestsfromItoIV,withanapproximateageof14

Fig.2.DetailofthefungalchamberofAttabisphaericaindifferentforms(AandB).Botucatu,SP,2014.

Table2

Mode,mean,standarddeviation(S*),andmaximumandminimumvaluesofdimensions(width,heightandlength)ofAttabisphaericanestchambers,Botucatu,SP,2014.

Nests Width(cm) Height(cm) Length(cm)

Mode Mean S* Maximum Minimum Mode Mean S* Maximum Minimum Mode Mean S* Maximum Minimum I ne 7.3 1.5 9 6 9 8.3 1.2 9 7 11 10.0 1.7 11 8 II ne 14.0 1.4 15 13 ne 14.0 1.4 15 13 ne 14.5 2.1 16 13 III ne 10.5 2.1 12 9 9 9.0 0.0 9 9 ne 11.5 0.7 12 11 IV 8 8.3 0.6 9 8 8 8.3 0.6 9 8 ne 9.3 1.5 11 8 V ne 9.0 2.6 11 6 ne 9.3 2.1 11 7 ne 12.0 4.6 17 8 VI ne 12.7 2.1 15 11 12 10.7 2.3 12 8 15 14.0 1.7 15 12 VII 10 10.0 0.0 10 10 10 10.0 0.0 10 10 ne 13.0 4.2 16 10 VIII ne 11.0 2.6 13 8 12 10.7 2.3 12 8 ne 11.7 4.0 16 8 IX 8 10.0 4.7 17 7 ne 11.5 5.8 20 7 ne 11.8 6.9 22 7 X ne 7.5 3.1 11 4 ne 9.0 3.9 14 5 ne 9.5 4.8 16 5 XI 13 13.3 4.2 19 7 ne 15.4 4.4 21 8 18 15.4 4.8 21 7 XII ne 12.9 3.8 18 8 10 10.9 2.5 15 7 ne 13.4 3.5 18 8 XIII 11 11.8 2.9 16 8 13 14.4 2.8 19 12 ne 14.2 3.4 18 9 XIV 11 11.6 4.5 16 3 11 10.6 4.4 16 3 15 13.9 4.1 20 8 S*,standarddeviation;ne,non-existentrepeatedvalues.

months,theaveragewidthrangedfrom7.3±1.5to14±1.4cm; theaverageheightwas8.3±0.6to14±1.4cm,andtheaverage lengthwas9.3±1.5to14.5±2.1cm.FornestsVtoIX,with approx-imateageof18months,theaveragewidthrangedfrom9.0±2.6 to12.7±2.1cm,theaverageheightwas9.3±2.1to11.5±5.8cm, andtheaveragelengthwas11.7±4to14.0±1.7cm.Asfornests

X to XIV, withan approximate age of 28 months, the average

widthrangedfrom7.5±3.1to13.3±4.2cm,withaverageheight of 9.0±3.9 to15. 4±4.4cm and averagelength of 9.5±4.8 to 15.4±4.8cm(Table2).However,therewasnosignificant differ-encein chamber width (ANOVA, F2;13=0.24, p=0.7870), height (ANOVA,F2;13=0.93,p=0.5772)andlength(ANOVA,F2;13=0.96, p=0.5881)betweenthe3agesstudied.

ThefirstchambersofA.bisphaericawerelocatedatsmalldepths (between13and20cm);possibly,thisspeciesdoesnotneedvery stableconditionsoftemperatureandhumidity,sincethesenests occurinopenareaswithalotofinsolationandinsoilsthatretain littlewater.Inspeciesthatnestinshadyareas,suchasA.sexdens, temperaturerangesfrom21to29◦Candhumidityissometimes around50%(Eidmann,1932).

Concerninginternaltunneldimensions,variationswerefound inaveragewidthandheight.FornestsItoIV,withapproximate age of 14 months, the average width ranged from1.5±0.2 to 1.9±0.6cmandtheaverageheightwas1.4±0.2to1.6±0.3cm.For nestsVtoIX,withanapproximateageof18months,theaverage widthvariedfrom1.6±0.8to2.8±1.9cmandtheaverageheight was1.3±0.2to2.6±0.9cm.AsfornestsXtoXIV,withan approx-imateageof28months,theaveragewidthrangedfrom3.4±1.7 to2±0.6cmandtheaverageheightwas1.8±0.3to2.6±1.6cm (Table3).Significantdifferencewasfoundastothewidth(ANOVA, F2;13=13.40,p<0.05)ofthetunnels betweenthedifferentages studied.Thesamewasnotobservedfortheheightofthetunnels

(ANOVA,F2;13=0.2143,p=0.8117).Probably,thisdifferencefound inthewidthofthetunnelsoccurredwiththegrowthofthecolony and,consequently,oftheflowofworkersinsidethenest.

ThemeasuresfoundbyPaivaCastroetal.(1961)forinternal tun-nelsinA.laevigatawere3.5cmwideand2.8cmhigh.Perhaps,the differenceinthewidthofthetunnelsbetweenthedifferentspecies isduetothegreaterorlesserflowofworkersinsidethem,which arewidenedwiththegrowthofthenest.InstudiesonAttanests bytheprocessofcementmolding,Jacoby(1938,1950)detected theexistenceofseveraltypesoftunnelswithdifferentpurposes.In additiontonarrowtunnelsthatservedascommunicationbetween largertunnels,circulartunnels,moreorlessperpendicular,with 3–4cmindiameter,werefound,aswellasellipticaltunnels,usually horizontal,withadiameterof10–12cm.

TherearesomereportsontunnelsinsideA. sexdensnestsin StahelandGeijskes(1939),whichfoundthepresenceoftunnels descendingtoward thewater table, havingarole inthesupply ofwatertothecolony.Thestudyalsoobservedthemovementof smallpuffsofsmokeinjectedintoholesabovethenest,showingthe existenceofaprimitiveventilationsysteminintactnests.Access tothechambers,asperobservationsbyBonetto(1959),forA. vol-lenweideri,ispossiblethroughtubulargalleriesofdiametervarying between5and8cm.

SupplytunnelsofA.laevigatastudiedbyMoreiraetal.(2004a), moldedwithcementinsomeportions,alsohadanellipticalsection, withaveragewidthof14.12cmandaverageheightof2cm. Maxi-mumandminimumwidthsandheightswere24and3cmand9.5 and1cm,respectively,withthesupplyholesinterconnected.They reachedasingletunnelthatwenttotheareawiththehighest con-centrationofchambers,withaveragedepthof55cmfromground level,maximumof73cmandminimumof27cm.Itwasobserved that,asthetunnelapproachedtheroyalcoreofthenest,thedepth

Table3

Mode,mean,standarddeviation(S*),andmaximumandminimumvaluesofthedimensions(widthandheight)oftheinternaltunnelsofAttabisphaericanests.Botucatu, SP,2014.

Nests Width(cm) Height(cm)

Mode Mean S* Maximum Minimum Mode Mean S* Maximum Minimum I 1.6 1.7 0.5 2.9 1.3 1.3 1.6 0.3 2.3 1.3 II 1.6 1.9 0.6 2.9 1.0 1.6 2.2 0.9 4.5 1.4 III 1.5 1.5 0.4 2.3 1.1 1.5 1.4 0.3 1.8 1.0 IV 1.5 1.5 0.2 7.0 1.2 1.5 1.4 0.2 1.7 1.2 V 1.8 1.7 0.3 2.5 1.4 1.7 1.7 0.2 2.0 1.4 VI 2.0 2.8 1.9 7.0 1.4 1.7 2.6 0.9 4.0 1.7 VII 1.4 1.6 0.8 3.7 1.0 1.5 1.3 0.2 1.7 1.1 VIII 1.7 1.7 0.4 2.7 1.2 1.7 1.7 0.3 2.2 1.2 IX 1.2 1.9 1.2 4.6 1.2 1.1 1.6 0.5 2.4 1.1 X 1.7 2.0 0.6 3.2 1.2 ne 2.1 0.6 3.2 1.2 XI 2.0 2.4 0.6 3.7 1.9 1.9 1.9 0.2 2.4 1.5 XII 4.2 3.4 1.7 6.2 1.6 ne 2.6 1.6 6.7 0.9 XIII 1.9 2.4 0.9 4.5 1.4 1.8 1.8 0.3 2.4 1.5 XIV ne 2.3 1.5 6.0 0.9 ne 1.2 0.7 3.1 0.9 S*,standarddeviation;ne,non-existentrepeatedvalues;n=1.

increased,andthenestsometimesreachedadepthgreaterthan 1.20m,afactobservedbyPretto(1996)fortunnelsofA.sexdens. Mainremarks

ThenestsofA.bisphaericaofdifferentagesdidnotshowgreat changesindepth,probablyduetoastabilityinthetemperature andhumidityofthesoil,propitiousforthegooddevelopmentof thecolony.

Alateraldevelopmentinthenestsandanincreaseinthenumber ofchambersovertimewerefound,probablyduetothegrowthof thecolony(populationandfungusgarden)asawhole.

Thewidthofthetunnelsincreasedovertime,probablydueto thehigherflowofworkers.

Authorscontributions

Conceived and designed the experiments: Luiz Carlos Forti, Aldenise Alves Moreira. Performed the experiments: Aldenise AlvesMoreira.Analyzedthedata:LuizCarlosForti,AldeniseAlves Moreira.Contributedwithreagents/materials/analysistools:Luiz CarlosForti.Wrotethepaper:LuizCarlosForti,RobertodaSilva Camargo,AldeniseAlvesMoreira,MariaAparecidaCastellani,Nadia Caldato.

Conflictsofinterest

Theauthorsdeclarenoconflictofinterest. Acknowledgements

LCFwastherecipientofafellowshipgrantedbytheNational Council for Scientific and Technological Development [Con-selhoNacionaldeDesenvolvimentoCientíficoeTecnológico](Grant 301718/2013-0).

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