Temporal
variation
of
vitellogenin
synthesis
in
Ectatomma
tuberculatum
(Formicidae:
Ectatomminae)
workers
Dihego
Oliveira
Azevedo
a,
Jose´ Cola
Zanuncio
a,
Jacques
Hubert
Charles
Delabie
b,
Jose´ Eduardo
Serra˜o
c,*
aDepartamentodeBiologiaAnimal,UniversidadeFederaldeVic¸osa,Vic¸osa,MG,Brazil b
Laborato´riodeMirmecologia,ConveˆnioCEPLAC-UESC,Itabuna,BA,Brazil
c
DepartamentodeBiologiaGeral,UniversidadeFederaldeVic¸osa,Av.P.H.Rolfss/n,36570-000Vic¸osa,MG,Brazil
1. Introduction
Vitellogenin is theprecursorof vitellin, a phospholipoglyco-proteinthatconstitutesthemajorfractionoftheeggyolkproteins in insects and is the main source of nutrients for the embryo (RaikhelandDhadialla,1992;TufailandTakeda,2008).Ininsects, theaminoacidsequenceofvitellogeninsisconservedatmanysites (Chenetal.,1997;TufailandTakeda,2008),althoughthenumber of genes that encode them varies in different species. In hemimetabolousinsects,onegeneispresentinBlattellagermanica (Blattaria) (Comas et al., 2000) and two genes in Leucophaea maderae(Blattaria)(Tufailetal.,2007).Forholometabolainsects, fivegeneswereidentifiedinAedesaegypti(Diptera)(Chenetal., 1994),oneinbothBombyxmori(Lepidoptera)(Yanoetal.,1994) andApismellifera(Hymenoptera)(Piulachsetal.,2003),andthree inSolenopsisinvicta(Hymenoptera)(TufailandTakeda,2008).
Vitellogeninismainlysynthesizedinthefatbodyoffemales, wheresingleormultiplepolypeptidesundergomodificationssuch as glycosylation, lipidification, phosphorylation, sulfation, and proteolytic cleavage (Tufail and Takeda, 2008). They are then released into the haemolymph as oligomeric proteins with molecular weights ranging from 300 to 600kDa (Tufail and
Takeda,2008;Wheeleretal.,1999).Theseproteinaggregatesare thentransferredtooocytesviareceptor-mediatedendocytosisand stored in theform of crystals, at which time they are termed vitellins(Giorgietal.,1999;RaikhelandDhadialla,1992).
Insocialinsects,theproductionofvitellogeninsisnotexclusive toqueens,thereproductivefemales,butalsooccursinthenon-or subfertile worker castes (Engels, 1974; Guidugli et al., 2005; Seehuusetal.,2006),andinthehoneybeeitwasevenfoundin males(Piulachsetal.,2003;Trensceketal.,1989).Workersofthe stinglessbeeFrieseomelittavariaaresterilebut produce vitello-geninconstitutivelythroughouttheirlife(Dallacquaetal.,2007). In several species of ants, workers may activate ovaries and produceunfertilizedeggs,termedtrophiceggs,thatareusedto feed the brood and the reproductive castes (Dietemann and
Peeters, 2000; Fe´ne´ron and Billen, 1996; Gobin et al., 1998;
Ho¨lldoblerandWilson,1990).Theproductionofvitellogeninby the non-reproductive castes suggests that it has functions in additiontosupplyingnutrientstotheembryo,whichhavebeen bettercharacterizedinbees(Amdametal.,2003).
In A. mellifera workers, variation in their production of vitellogenin is related to their permanence inside the colony andtheonsetofforagingflights(MarcoAntoˆnioetal.,2008;Nelson etal.,2007).Productionofvitellogeninalsoincreasesthelongevity ofqueenswhen comparedtoworkersby reducingtheir rateof agingthroughresistancetooxidativestress(Coronaetal.,2007; Seehuus etal.,2006).Vitellogeninshave importantfunctionsin
A R T I C L E I N F O Articlehistory:
Received3February2011
Receivedinrevisedform14April2011 Accepted15April2011 Keywords: Vitellogenin Vitellin Agepolyethism Trophicegg Fatbody Ant A B S T R A C T
WorkersoftheantspeciesEctatommatuberculatum(Ectatomminae)haveactiveovariesandlayeggs thatareeatenbythequeenandlarvae(trophiceggs).Vitellogeninsarethemainproteinsfoundinthe eggsofinsectsandareasourceofnutrients.Theaimofthisstudywastocharacterizetheperiodof vitellogeninproductioninworkersofE.tuberculatum.Thevitellogeninwasidentifiedfromqueenand workereggsbySDS-PAGE.Anti-vitellogeninantibodieswereobtainedandusedtodetectthisproteinin thefatbodyandhaemolymphofworkersatdifferentages.VitellogeninfromE.tuberculatumconsistsof twopolypeptidesof31and156kDa.Intheeggsofqueens,the156kDapolypeptideiscleavedintotwo subunitsof36and123kDa.Theanalysisofthehaemolymphofworkersshowedthatthesecretionof vitellogeninvarieswithage.Thesecretionisinitiatedaroundthefifthdayafteremergence,withpeak productionfromdays20to60,andstopsaroundday100.Thevariationinproductionisrelatedtothe differentactivitiesperformedbytheworkerswithinthecolony,suggestingthatvitellogeninmayhave animportantroleinmaintainingagepolyethism.
ß2011ElsevierLtd.
*Correspondingauthor.Tel.:+553138991301;fax:+553138992549. E-mailaddress:jeserrao@ufv.br(J.E.Serra˜o).
ContentslistsavailableatScienceDirect
Journal
of
Insect
Physiology
j our na l ho me pa ge : w ww . e l se v i e r . com / l oca t e / j i ns phy s
0022-1910 ß2011ElsevierLtd. doi:10.1016/j.jinsphys.2011.04.015
Open access under the Elsevier OA license.
somaticmaintenanceandintheimmunesystemofbees(Amdam et al.,2004; Seehuus etal., 2006), and are partof the insulin/ insulin-likesignalingpathway,whichregulatesgrowth,aging,and reproductioninvertebratesandinvertebrates(Coronaetal.,2007). TheantspeciesEctatommatuberculatum(Ectatomminae)forms coloniesofupto400workersandoneormorequeens(Horaetal., 2005).Theworkershavethesamesizeandaremorphologically differentfromqueens,andperformdifferenttasksinthecolony accordingtotheirage(Fe´ne´ronandBillen,1996;Fe´ne´ronetal., 1996).Theworkersalsohaveactiveovariesthatproducetrophic
eggs (Fe´ne´ron and Billen, 1996; Hora et al., 2007) and the
developmentoftheirovariesisrelatedtotheirage(Fe´ne´ronetal., 1996). Therefore, the production of vitellogenin is related to nourishingcolonymembersandpossiblytothedifferentactivities performedbyworkers.Inthisworkwetestthehypothesisthatthe periodofvitellogeninproductionislinkedtointranidalactivitiesin agepolyetismofE.tuberculatumworkers.Wefindthatvitellogenin isproducedwhenworkersareinsidethenestactinginbroodcare and ceasing when workers are in activities out of the nest, suggestingthatthisproteincanbeusedasanutrientsupplement sincetheeggsproducedbyworkersaretrophiceggsthatareused forqueenandbroodfeeding.
2. Materialsandmethods 2.1. Insects
FiveE.tuberculatumcolonieswereprovidedbytheLaboratory of Myrmecology at the Cocoa Research Centre (CEPLAC) in Itabuna,Brazil.Theantswerekeptinartificialcoloniesbuiltin plastic cages (18cm25cm) and filled with plaster. The colonieswereconnectedbytubestoothercages(10cm10cm) cm) without plaster that were used as foraging areas. All colonies were polygynous, containing two to five queens and morethan30workersinadditiontothebrood.Thecolonieswere maintained at 2628C and fed every two days with Tenebrio molitor (Coleoptera: Tenebrionidae) larvae, honey, and waterad libitum.Inordertoobtainantswithknownages,newlyemerged workersweremarkedwithanenamelpaintdotonthethoraxand returnedtotheircolonies.
2.2. Haemolymphextraction
Haemolymphsampleswerecollectedfrommatedandactive egglayingqueensofunknownageandworkers2,5,10,15,20,30, 60,and100daysold.Theworkerswerecryo-anesthetized(08C) anddecapitated,whilequeenshadanincisionmadeintheirthorax with a sterile entomological pin. Between 0.5 and 0.75
m
L of haemolymphwascollectedfromeachantbymicrocapillary.The queenswereputbackintheircoloniesoforiginafterextraction. Thecollectedhaemolymphwasaddedtoatubecontaining20m
L ofTris–HCl(0.05M,pH7.2)with15%(v/v)ofproteaseinhibitor cocktail[4-(2-aminoethyl)benzenesulfonylfluoride(AEBSF),E-64, bestatin,leupeptin,aprotinin,andsodiumEDTA(Sigma-Aldrich)]. 2.3. VitellogeninsamplesE. tuberculatum vitellogenin and/or vitellin samples were obtainedfromnewlylaidqueeneggsandmatureoocytesdissected fromworkers’ovaries.
Eggsandoocytesweremaceratedin0.05MTris–HClbuffer,pH 7.2,containing15%(v/v)ofproteaseinhibitorcocktail(Sigma).The extracts were centrifuged at 9300g for 10min and the supernatant was collected. The soluble proteins present in the extractswerequantifiedaccordingtoBradford(1976)usingbovine serumalbuminasastandard.
2.4. SDS-PAGE
Thehaemolymphsamplesand eggextractsfromthequeens and workers were subjected to electrophoresis on a 12% polyacrylamidegelcontainingsodiumdodecylsulfate(SDS-PAGE) (Laemmli,1970)inordertoassesstheproteinprofiles.Thesamples weredilutedtoaratioof1:2(v/v)insamplebuffer[20%(v/v)of 10% SDS,12.5% (v/v)0.5MTris–HClpH 6.8,25% (v/v)glycerol, 0.01%(w/v)bromophenolblue,5%(v/v)
b
-mercaptoetanol],boiled for4min,andrunonthegel.Weused5m
gofproteinfromegg extractsand5m
L ofdilutedhaemolymphsamples.Thegelwas stainedwithaCoomassiebluesolution(2%blueCoomassieG250, 10% acetic acid, 47.5% ethanol). The molecular weights of the proteinsweredeterminedwithastandardcurvebasedonalinear regression between the log of molecular weight of standard proteins (PromegaTM Broad Range Protein Molecular Weight Marker)andtheirrf-values.2.5. Antibodyproduction
Thetwomajorvitellinproteinsidentifiedinqueeneggson SDS-PAGEwereisolatedandusedintheproductionofanti-vitellogenin antibodies.
Each putative vitellogenin protein was used as antigen for immunizationofthreerabbitsuptothreemonthsold.Intheinitial immunization a total of 1mg of protein mixed with Freund’s completeadjuvant(v/v)wasinjectedsubcutaneously.Thesecond andthirdboosterimmunizationswereperformed30and60days afterthefirst,each of themusinga totalof0.25mg ofprotein mixedwithincompleteFreund’sadjuvant(v/v).Therabbitswere bled 30 days after the third immunization and the serum containingtheantibodieswasobtainedandstoredat208C. 2.6. Westernblotting
Haemolymphsamplesandeggextractsweresubjectedto SDS-PAGE as describedabove. The gelwasincubated for 20min in transferbuffer(0.58% Trisbase,0.28% glycine,0.037% SDS,20% methanol),followedbytransferoftheproteinstoa0.2
m
mpore size nitrocellulose membrane (Sigma–Aldrich, cat. N7892-5EA). Themembranewasincubatedovernightatroomtemperaturewith 5%non-fatdriedmilkin0.1MTBSbuffer,pH8.0,containing0.1% Tween-20(TBST),washedinTBST,andincubatedfor2hatroom temperaturewiththeanti-vitellogeninantibodiesdiluted1:1500 in TBSTwith2.5% non-fatdriedmilk.Thenegativecontrolwas donebyincubatingsamplesofqueeneggextractswithrabbit pre-immuneserumdiluted1%inTBSTwith2.5%non-fatdriedmilk. ThemembranewaswashedinTBST,incubatedwithanti-rabbit IgGconjugatedtohorseradishperoxidase(Sigma-Aldrich)diluted 1:4000 in TBST with 2.5% non-fat dried milk for 2h at room temperature,washed,andrevealedwithDAB/H2O2solution(0.1% 3-30-diaminobenzidinein 50mMTris–HCl, pH7.6, 2.5%of 0.3%nickelchlorideinH2O,0.1%H2O2). 2.7. Immunohistochemistry
Samplesoffatbodyfromworkersaged30daysweredissected, fixed in Zamboni’s solution(Stefanini et al., 1967)for 30min, dehydratedinalcohol,andembeddedinLRWhiteresin.Sections 5
m
mthickweretreatedwith1%phenylhydrazinein0.1MPBS,pH 7.4,for30min,washedinPBS,andincubatedwith2%(v/v)normal rabbitseruminPBSfor20min.Thesectionswerethenincubated with anti-vitellogeninantibody diluted1:500 in PBS for 2h at 378C, washed in PBS and incubated with anti-rabbit IgG conjugated to horseradish peroxidase (Sigma-Aldrich) diluted 1:1000inPBSfor2hat378C.ThesectionswerewashedinPBSand0.05MTris–HCl,pH7.6, revealedwithDAB/H2O2,and counter-stainedwithhematoxyline.
2.8. Immunofluorescence
Samplesoffatbodyandoocyteswereobtainedbydissectionof workersaged30days.ThesamplesweretransferredtoZamboni’s fixativesolution(Stefaninietal.,1967)for30minandwashedwith 0.1MPBS,pH7.4,with0.1%Tween-20 (PBST).Theywerethen incubatedinasolutionof1.5%bovineserumalbumininPBSTfor 10minat378C,washedinPBST,andincubatedin2%normalrabbit seruminPBSTfor20minat378C.Thesampleswerewashedagain in PBST and incubated overnight at 48Cwith anti-vitellogenin antibodydiluted1:500inPBST.Then,thesampleswereincubated for2hat378Cwithanti-rabbitIgGconjugatedwithFITC (Sigma-Aldrich)diluted 1:100 in PBST.The samples weremounted on microscopeslidesina50%sucrosesolutionandviewedundera fluorescentmicroscope(OlympusBX-50)withanexcitationfilter of495–530nm.Thenegativecontrolwasperformedbyomitting theanti-vitellogeninantibody.
2.9. NativePAGE
Egg extracts from queens and workers and haemolymph samplesfrom workers with 30 days of agewere subjected to discontinuousnativePAGE(Davis,1964)inordertocomparethe nativevitellinsandvitellogenins.Samplescontaining2.5–5
m
gof proteinswerediluted1:2(v/v)insamplebuffer[12.5%(v/v)0.5M Tris–HClpH6.8,30%(v/v)glycerol,0.01%(w/v)bromophenolblue] andappliedontoa7%polyacrylamidegel.Aftertheelectrophoresis thegelwasprefixedovernightwithmethanolaceticsolution[10% (v/v)methanol,10%(v/v) glacialaceticacid],fixed for 1hwith 12.5%formaldehydesolutionandstainedwithammoniacalsilver nitratesolution[0.15% (w/v)silver nitrate, 0.05% (w/v)sodium hydroxideand2.5%(v/v)ammoniumhydroxide].3. Results
SDS-PAGEanalysiscomparingeggproteinsfromE.tuberculatum queensandworkersshoweddifferencesbetweenthe castes.The eggsoftheworkerscontainedtwomajorproteinswithmolecular weightsof31and156kDa,whiletheeggsofqueenshadeightmajor proteins,fourofwhich(31,36,123,and156kDa)appearedwith greaterintensitythantheothers(81,86,96,and101kDa)(Fig.1).
Haemolymphsamplesfromworkersofdifferentagesdisplayed differentproteinpatterns.ProteinswithMWsof43,84,89,and 195kDaoccurredinsamplesfromworkersofallages(Fig.1).The haemolymphofworkersaged2and5dayshada120kDaprotein thatwasnotfoundinworkersofotherages.Haemolymphfrom workers with 10 days of age showed small quantities of the proteinsofMWs31and156kDapresentinworkeroocytes(Fig.1). Theseproteinsalsoappearedinthehaemolymphofworkersaged 15,20,30,and60days.Fromtheageof20days,allantsexpressed theproteinsof38,71,and135kDa.Workers100daysofagedid not show the proteins of 31 and 156kDa (Fig. 1). In the haemolymph of queens, proteins of 85, 135, 156 and 195kDa appearedin greaterquantity,whilethe31 and43kDaproteins wereslightlydetected(Fig.1).
Toverifythepresenceofvitellogenininantsofdifferentages, thetwomostabundantproteinsineggsofqueens(Fig.1)were isolatedandusedtoimmunizerabbitsfor antibodyproduction. The antibodies obtained to proteins of 123 and 156kDa were termedvg1andvg2,respectively.
Immunolocalizationtestswereperformedtoprovideindirect evidencethattheisolatedproteinsmaycorrespondtovitellogenin. Immunohistochemistry (Fig. 2A–C) and immunofluorescence (Fig. 2D–F) showed positive reactions for antibodies vg1 and vg2infatbodycellsandoocytes.Thefatbodywascharacterizedby largecellsthathadacentralnucleusandmanyvacuoles(Fig.2A– C). The immunostained granules were found around these vacuoles and were clusteredat the cellperiphery (Fig.2A and B).Intheoocytes,thepositivegranuleswereobservedthroughout theooplasm(Fig.2E).
Theantibodiesvg1andvg2wereusedinWesternblotanalysis ofeggextractsfromqueensandworkers,whilethehaemolymph sampleswereanalyzedusingonlythevg2antibody.Theanalysis showedthatbothantibodiesreactedpositivelytotheproteinsof 123and 156kDa andalso forsmallerunspecific fragmentation products(Fig.3).Analysisofthehaemolymphshowedapositive reaction to a protein of 156kDa in samples from queens and workersaged5,10,15,20,30,and60days(Fig.4).Anincreasein theintensityofthereactionwasobtainedinsamplesfromworkers aged20and30days.Theintensitydecreasedat60daysofagebut itwashigherthanat10and15daysofage.Workersaged2and100 dayshadnopositivereactionsfortheanti-vitellogeninantibody (Fig.4).Neitherthe120kDaproteinfoundinworkersaged2and5 daysnortheproteinof135kDafromsamplesofworkersaged20– 100daysreactedpositivelytothevg2antibody(Fig.4).
Thenativevitellinsfromqueensandworkerswerecompared andtheireggsshowedonemainproteinwiththesamesize(Fig.5). Inthehaemolymphfrom30daysoldworkersasimilarproteinwas alsoidentified(Fig.5).
4. Discussion
IneggsfromqueensofantspeciesinthefamiliesMyrmicinae, PonerinaeandEctatomminaethevitellinisformedbytwoormore proteins(Lewisetal.,2001;Wheeleretal.,1999).Ourresultsshow thattheeggsofE.tuberculatumqueenshavevitellinsthatconsistof fourmajorproteins,whileintheeggsofworkersonlytwoofthem arethevitellins. Inqueens,thesefour proteinsjoin toforman oligomericproteinwhichinitsnativeformhasamolecularweight between400and500kDaestimatedbasedondatafromWheeler etal.(1999).Intheeggsofworkers,the156and31kDavitellins formanoligomericproteinwiththesamemolecularweightofthe nativevitellinfoundinqueens.
The two proteins found in thegreatest amounts in the egg extracts of E.tuberculatum were used for antibody production becausethevitellinsmakeupthelargestfractionofproteinsfound in theeggs of insects (Raikhel and Dhadialla, 1992; Tufailand Fig.1.SDS-PAGEshowingtheproteinprofilefromeggsandhaemolymphofE.
tuberuclatumqueens andworkers. Theputative vitelinsare indicatedby the numbers1(156kDa),2(123kDa),3(36kDa)and4(31kDa).Thevg1andvg2 antibodiesweremadeusingtheproteins1and2.Arrowsshowsproteinweakly stained.S,standartmolecularweightfollowedbytheirrespectivevaluesinkDa. Queenegg(Qe).Workeregg(We).Queenhaemolymph(Qh).Workerhaemolymph with2(2d),5(5d),10(10d),15(15d),20(20d),30(30d),60(60d)and100(100d) daysofage.
Takeda,2008). Theimmunolocalizationtestsshowedthat these proteins occur in fat body cells, the main production site of vitellogenins.Since vitellogenins are the precursorsof vitellins (Chapman,1998),ourresultsconfirmthatthesetwoproteinsare actuallyvitellincompounds.
Comparingthevitellinsofthequeenandworkereggswiththe vitellogenins from their haemolymph revealed that only the proteins of 31 and 156kDa were shared, suggesting that the vitellogenin circulating in the haemolymph of E. tuberculatum consistsofonlythesetwoproteins.Also,thepresenceofanative proteininworker’shaemolymphwithsimilarsizeofthenative
vitellins found in queen and worker eggs indicates that the vitellogeninformsaproteincomplexinthehaemolymphsimilarto thevitellinsfoundin theeggs.The proteinsof 36and 123kDa present in the eggs of queens may be products of additional cleavage of the 156kDa protein, which is supported by the occurrenceofcross-reactivitybetweenantibodiesvg1andvg2to theproteinsof123and156kDa.Moreover,thehaemolymphofthe queensshowsonlytheproteinsof31and156kDa.InB.germanica, vitellogeninsof160kDaarecleavedintosubunitsof50and95kDa afterinternalizationintheoocyte(Wojchowskietal.,1986).
Thedifferenceinvitellinprocessingfoundbetweenqueenand workereggsofE.tuberculatummaybeduetheabsenceofactive proteasesinworker’seggsortheabsenceoftheiractivationinside thetrophiceggs,whichhavenoembryonicdevelopment(Giorgi etal.,1999;KhilaandAbouheif,2008).Inthecourseofembryonic development, the vitellin peptides undergo specific cleavages inside the oocyte resulting in new smaller peptides. These cleavagesoccurdueproteasesassociatedwiththeyolkgranules, thatmaybesynthesizedinsidetheoocyteorextraovarially,and activatedduringtheembryonicdevelopment(Giorgietal.,1999). Thelackofimmunoreactivityofthevg2antibodyagainstthe 36kDa fragment may be due to low immunogenicity of this
Fig.2.VitellogenininorgansofthirtydaysoldE.tuberculatumworkers.(AandB)Immunohistochemistryinfatbodycellsshowingpositivestaininggranules(arrows).(C) Immunohistochemistrynegativecontrol.Thadarkgranulesareartefactsofthecouterstainprocedurewithhematoxilyn.Bar=20mm(lightmicroscope).Thevg1 anti-vitellogeninantibodywasusedinA,whilethevg2antibodywasusedin(B).Immunofluorescenceinfatbodycells(D)andoocytes(E).Thepositivestainingisevidencedby thestronggreenish,asindicatedbyarrows.(F)Immunofluorescencenegativecontrolinfatbodycells.Bars=50mm(fluorescencemicroscope).Thevg2antibodywasusedin (D)andthevg1antibodywasusedin(E).c,Tergite’scuticle;fb,fatbodycells;m,musclecells;o,oenocytes.arrowheads,nucleioffatbodycells.
Fig.3.WesternblottinginE.tuberculatumqueenandworker’seggswith anti-vitellogeninantibodies.Thevg1andvg2antibodiesshowedpositivereactionwith the123and156kDaproteins.Theseproteinsdidnotreactwiththepre-immune serum.Notetheunspecificpositivereactionshowedbyminorproteins.S,standart molecularweightproteinpatternwithrespectivemassesinkDa.Qvg1,queen’segg proteinsreactedwithvg1antibody;Qvg2,queen’seggproteinsreactedwithvg2 antibody;Wvg2,worker’seggproteinsstainedbyvg2antibody.Qc–queen’segg proteinsincubatedwithrabitpre-immuneserum(negativecontrol).
Fig.4.WesternblottinginhaemolymphsamplesofE.tuberculatumqueen(Qh)and workerwith02(2d),05(5d),10(10d),15(15d),20(20d),30(30d),60(60d)and100 (100d)daysofagestainedwithvg2antibody.Qe,Queeneggproteinsshowingthe 123and156kDavitellinsstainedwiththevg2antibody.
proteinportionorbecausethereisasmallfractionofantibodiesin thepolyclonalserumraisedagainstthe156kDaproteinthatbind tothe36kDafragment.InA.melliferaworkers,the180kDa full-lengthvitellogeniniscleavedintwodistinctfragmentsinthefat body, being one small N-terminal of 40kDa and one large C-terminal of 150kDa, and the antibodies produced against the 180kDa vitellogenin fail in recognize the 40kDa fragment (Havukainenetal.,2011).The36kDaproteinofE.tuberculatum queeneggs wasalsoused asan immunogen, but theantibody obtained wasunsatisfactory. The small proteins present in the queeneggextractsthatreactedunspecificallywiththevg1andvg2 antibodiesmaybeartefactsoftheextractionprocess.
About some other proteinspresentin the haemolymphof E. tuberculatum, the 195kDa and 80kDa may be lipophorin and hexamerin subunits, respectively, like described for some ants
(Martı´nez et al., 2000; Wheeler and Buck, 1995; Wheeler and
Martı´nez,1995).Thelipophorinsareimportantforlipidtransport, while the hexamerins may have functions in nutrient storage, hormone carriers, immune protection and cuticle formation (Burmester,1999).The120kDaproteinfoundinthehaemolymph ofE.tuberculatum workers with 2 and 5days of age maybe a hexamerinremainingfromthepupalstagesthatisdepletedfrom thehaemolymphduringthefirstdaysofadultlifespan,likelyfound fora110kDahexamerininotherants(WheelerandBuck,1995).
Our resultsindicatethat theproductionof vitellogeninin E. tuberculatumisrelatedtotheageoftheworkersandtheovarian cycledescribedbyFe´ne´ron andBillen(1996).Our datashowed thatvitellogeninappearsinthehaemolymphofworkersaround thefifth day afteremergence, beingsecreted in quantities not detectablebySDS-PAGE.Theageatwhichtheovariesofworkersof E.tuberculatumbegintobeactivatedisvariable,sinceattheendof thefirstweekafteradultemergenceworkerscanbefoundthat eitherhaveovarioleswithoutfolliclesandonlyundifferentiated cells or ovarioles with oocytes in the early accumulation of vitellogenin(Fe´ne´ron and Billen,1996). Vitellogeninproduction remainslowuntilthesecondweekafteremergence.Atthe20th dayitispresentinlargeamountsinthehaemolymph,atwhich timetheworkershave developing oocytes(Fe´ne´ron and Billen, 1996). The vitellogenin is still present in the haemolymph of workersaged30and60days,which istheperiodofmaximum developmentofoocytesandsubsequentoviposition(Fe´ne´ronand
Billen,1996).Oocytescanstillbefoundinworkers90daysold,but theovariesarealreadyintheregressionstage(Fe´ne´ronandBillen, 1996). The vitellogenin is not present in the haemolymph of workers with more than 100 days of age, during which time workershavedegeneratedovaries(Fe´ne´ronandBillen,1996).
Thesecretionofvitellogeninisanotherfactortobeconsideredin the maintenance of age polyethismin E. tuberculatum. In most speciesofants,theyoungworkersstarttheirtasksinsidethecolony andwhenolderperformothertasksoutsidethecolony(Ho¨lldobler andWilson,1990).InE.tuberculatum,theworkershavethissame patternofactivity,withagradualprogressionofinsidecolonytasks tooutsidecolonytasksastheyage(ChampalbertandLachaud,1990; Fe´ne´ronetal.,1996).Inthefirstweekofadultlife,theworkersare involvedinreconnaissanceactivitiesdirectedtowardsnestmates andbroodandarenotperforminganyspecificfunctioninsidethe colony.Fromthesecondweek,theworkersdirecttheircaretothe eggs,larvaeandpupae,whichmaybeconsiderednursingactivities. Ataround30days,theworkers’mainactivityremainsthecareof brood, but they begin non-specific activities like exploring the colony.Theworkersaround90 daysofage performthetasksof cleaning and maintaining the colony, guarding and foraging (ChampalbertandLachaud,1990;Fe´ne´ronetal.,1996).Ourresults showthatvitellogeninisnotproducedduringthestabilizationofthe socialinteractionsofnewlyemergedworkers.Insteaditssynthesis beginswhenworkersstartbroodcareactivities,ismaintainedwhile workersactasnursesandendswhenworkersbegintoforage.
The interruption of vitellogenin synthesis in workers of E. tuberculatumataround100daysofagemaybeatriggerforthe beginningofoutsidecolonyactivities.Workerantsthatperform activitiesoutsideofthecolonyaremoreatriskofdeathbyexternal factorsthanthosewhoremainwithinthecolony.Therefore,they showgreaterratesofagingandashorterlifespan(Chapuisatand
Keller, 2002; Keller and Genoud, 1999). In the weaver ant
Oecophyllasmaragdina(Formicidae:Formicinae)theminor work-ersthat remainwithinthecolonyare subjecttolowerrates of extrinsicmortalityandshowedgreaterlongevitythanthecasteof major workers, responsible for activities outside the colony (Chapuisat and Keller, 2002). In A. mellifera, the inhibition of vitellogenin production causes the workers to begin foraging flightsearlier,anactivitycharacteristicofolderworkers(Marco Antoˆnioetal.,2008).Thereducedlongevityofindividualsathigher risk of extrinsic mortality saves energy and resources for the colony since it is more advantageous to invest resources into somaticmaintenanceoforganismsthathaveagreaterchanceof survival(ChapuisatandKeller,2002).
Basedonthistheory,theonsetofforagingactivitiesofworker antsandbeesislinkedtoadeclineoftheirphysiologicalfunctions andtoanincreasedchanceofextrinsicmortality.Theinterruption oftheproductionofvitellogeninnegativelyaffectsaninsect’sbody sinceitmaycompromiseitsimmunityandresistancetooxidative stress(Amdametal., 2004;Coronaetal.,2007),both ofwhich promoteaging(Mulleretal.,2007).
5. Conclusion
Inconclusion,theproductionofvitellogeninbyworkersofE. tuberculatumisagedependentandrelatedtotheperformanceof varioustasksbythiscaste.
Acknowledgements
The authors thank the Brazilian research agencies Program PRONEX-FAPESB-CNPq, projectPNX0011/2009, CNPqand FAPE-MIGforfinancialsupportandtheMicroscopyandMicroanalysis ResearchCenteroftheUniversidadeFederaldeVic¸osafortechnical assistance.
Fig.5.NativediscontinuousPAGEshowingthe vitellinsfromE.tuberculatum queens(Qe)andworkers (We)eggs andthe vitellogenin(arrow)in workers haemolymph(Wh).Themolecularweightofvitellininqueeneggswasestimated between400and500kDa(Wheeleretal.,1999).
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