Profiling the succession of bacterial communities throughout the life stages of a higher termite

15 Pages·2015·0.49 MB·English

RESEARCHARTICLE
Profiling the Succession of Bacterial
Communities throughout the Life Stages of a
Nasutitermes arborum
Higher Termite
(Termitidae, Nasutitermitinae) Using 16S
rRNA Gene Pyrosequencing
MichelDiouf1*,VirginieRoy1,PhilippeMora1,SophieFrechault1,ThomasLefebvre2,
VincentHervé3,4,CorinneRouland-Lefèvre5,EdouardMiambi1
1 DépartementSOLéO,Institutd’EcologieetdesSciencesdel’EnvironnementdeParis(IEES),Université
ParisEstCréteil(U-PEC),BâtimentP4,61avenueduGénéraldeGaulle,94010,Créteil,France,2 YNSECT
BiotechnologyEnvironmentandAgro-Industry,Genopole-Campus3,4ruePierreFontaine,91058,Evry
CEDEX,France,3 INRA,InteractionsArbres–Microorganismes,UMR1136,F-54280,Champenoux,
France,4 UniversitédeLorraine,InteractionsArbres–Microorganismes,UMR1136,F-54500,Vandoeuvre-
lès-Nancy,France,5 UMR211–DépartementSOLéO,IEES,CentreIRDFranceNord,32avenueHenri
Varagnat,93143,Bondy,France
*[email protected]
OPENACCESS
Abstract
Citation:DioufM,RoyV,MoraP,FrechaultS,
LefebvreT,HervéV,etal.(2015)Profilingthe
Previoussurveysofthegutmicrobiotaoftermiteshavebeenlimitedtotheworkercaste.
SuccessionofBacterialCommunitiesthroughoutthe
LifeStagesofaHigherTermiteNasutitermes Termitegutmicrobiotahasbeenwelldocumentedoverthelastdecadesandconsists
arborum(Termitidae,Nasutitermitinae)Using16S mainlyoflineagesspecifictothegutmicrobiomewhicharemaintainedacrossgenerations.
rRNAGenePyrosequencing.PLoSONE10(10): Despitethisintimaterelationship,littleisknownofhowsymbiontsaretransmittedtoeach
e0140014.doi:10.1371/journal.pone.0140014
generationofthehost,especiallyinhighertermiteswhereproctodealfeedinghasnever
Editor:PedroL.Oliveira,UniversidadeFederaldo beenreported.Thebacterialsuccessionacrosslifestagesofthewood-feedinghigherter-
RiodeJaneiro,BRAZIL
miteNasutitermesarborumwascharacterizedby16SrRNAgenedeepsequencing.The
Received:March19,2015 microbialcommunityintheeggs,mainlyaffiliatedtoProteobacteriaandActinobacteria,
Accepted:September21,2015 wasmarkedlydifferentfromthecommunitiesinthefollowingdevelopmentalstages.Inthe
firstinstarandlastinstarlarvaeandworkercastetermites,ProteobacteriaandActinobac-
Published:October7,2015
teriawerelessabundantthanFirmicutes,Bacteroidetes,Spirochaetes,Fibrobacteresand
Copyright:©2015Dioufetal.Thisisanopen
thecandidatephylumTG3fromthelastinstarlarvae.Mostoftherepresentativesofthese
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits phyla(exceptFirmicutes)wereidentifiedastermite-gutspecificlineages,althoughtheirrel-
unrestricteduse,distribution,andreproductioninany ativeabundancesdiffered.Themostsalientdifferencebetweenlastinstarlarvaeand
medium,providedtheoriginalauthorandsourceare
workercastetermiteswastheveryhighproportionofSpirochaetes,mostofwhichwere
credited.
affiliatedtotheTreponemaIc,IaandIfsubclusters,inworkers.Theresultssuggestthatter-
DataAvailabilityStatement:Allrelevantdataare
mitesymbiontsarenottransmittedfrommothertooffspringbutbecomeestablishedbya
withinthepaperanditsSupportingInformationfiles.
gradualprocessallowingtheoffspringtohaveaccesstothebulkofthemicrobiotapriorto
Funding:Theauthorshavenosupportorfundingto
theemergenceofworkers,and,therefore,presumablythroughsocialexchangeswithnurs-
report.
ingworkers.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
PLOSONE|DOI:10.1371/journal.pone.0140014 October7,2015 1/15
BacterialCommunitiesthroughTermiteLifeStages
Introduction
Gutmicrobialsymbiontsenabletermitestoplayakeyroleinecosystemprocessessuchascarbon
andnitrogencycling.Termite-gutmicrobiotaisverydiverseandcomprisesmanyphylogenetic
lineagesthathavebeenextensivelydocumentedinrecentdecades[1].Previousstudieshave
reportedthepresenceofautochthonouslineagesthatarelittleaffectedbyoccasionalchangesof
foodorgeographiclocation.Specifictermitegutmicrobiotawasfoundinthecongenericsoil-
feedingspeciesC.ortognathusfromaKenyangrasslandandC.niokolensisfromaSenegalese
savannah[2].Thepresenceofgut-specificActinobacteriahasbeenreportedinthewood-feeding
termitespecies,NasutitermescornigerMotschulsky,collectedfromvariousgeographicalsites[3].
Astermitegutsymbiontshavecoevolvedwithtermites,theircommunitystructureisbasically
consistentwithinagenus[4].Inhighertermites,thegutofwood-feedingspeciesthatfeedonlig-
nocellulosecomponentsismainlycolonizedbySpirochaetesandmembersoftheFibrobacteres
phylumandrelatedcandidatephylumTG3whilethegutcommunityofsoil-feederswhichthrive
onnitrogen-richcomponentsisdominatedbyFirmicutes.Infungus-growingspecies,thedomi-
nantphylaareBacteroidetesandFirmicutes.Bothhostphylogenyanddietcanbeimportant
determinantsofthebacterialcommunitystructureintermiteguts[5–7]
Themaintenanceofsymbiosesthroughgenerationsofthehostsdependsonreliablesymbi-
onttransmission.Ithasbeenshownthatthemaintenanceofhost-specificflagellatesinlower
termiteswasachievedviaverticaltransmissionduringtrophallaxis[8].Littleisknownaboutthe
transmissionroutesofdigestivesymbiontsinhighertermitesandmosthypothesesarebasedon
transmissionprocessesinlowertermites.Recently,amolecularsurveyofthegutmicrobiomes
ofspecimensrepresentinghigherandlowertermitegenerashowedthatverticalinheritancewas
theprimaryforceshapingtermitegutmicrobiota[9].Asinmostpreviousstudiesontermitegut
microbiota,thismolecularsurveyfocusedonlyontheworkercaste.Nostudieshavesofarbeen
publishedonthegutcommunitiesoflarvalinstarsoronthesuccessivechangesinthegut
microbiotaduringthedevelopmentofhighertermites.Bymonitoringtheeggs,firstandsecond
instars(L1andL2),andadultworkersofawood-feedingtermitespeciesinacomparativeanaly-
sis,thepresentstudyspecificallyaddressesthequestionofwhetherthegutmicrobiotaofthelast
instarsoriginatesfrommaternaltransmissionthroughtheeggorearlyinoculationoftheoff-
spring.Eggswereincludedinthepresentstudytogiveacomprehensiveinsightintothesucces-
sivechangesofthemicrobiotaduringtermitedevelopmentanddeterminewhetherthegut
microbiotaoflaterinstarsoriginatesfrommaternaltransmissionthroughtheeggsorearlyinoc-
ulationoftheoffspring.Pyrosequencingofthe16SrRNAgenewasusedtocharacterizethevari-
ationinthetaxonomiccompositionofthebacterialcommunitiesassociatedwiththelifestages.
Toassesstheroleplayedbythetransmissionofsymbiontstooffspringinthecodiversification
ofthegutmicrobiotainahigherwood-feedingtermitehost,16SrRNAgenebasedclonelibrar-
iesgeneratinglongersequenceswereusedtodeterminethephylogeneticrelationshipsbetween
thesequencesofthemostdominantlineagesinthefinaldevelopmentstagesandthoseretrieved
fromthegutofallopatricNasutitermesspecies.Thepresentstudysetouttocharacterizechanges
andassessthedifferencesinmicrobialcommunitiesthroughoutthevariousdevelopmentstages
ofawood-feedingtermitespeciesNasutitermesarborum(Smeathman)togainabetterunder-
standingofthebacterialcolonizationofthegutbytheirsymbionts.
MaterialsandMethods
Termitesampling
Ahigherwood-feedingtermitespecies,N.arborum(Smeathman)wasusedinthisstudy.The
genusNasutitermesDudley,1890isakeytaxonwidespreadinalltropicalareas,with243
PLOSONE|DOI:10.1371/journal.pone.0140014 October7,2015 2/15
BacterialCommunitiesthroughTermiteLifeStages
speciescurrentlydescribed.Nasutitermesspeciesarewood-feedingtermitesandtheirpost-
embryonicdevelopmenthasbeenwellestablished.AnestofN.arborum(Termitidae,Nasuti-
termitinae)wascollectedintheRepublicoftheCongo(Brazzaville),intheMayombeforest
withinUNESCO-ManandtheBiosphereReserve,inthedistrictofMvouti(4°14’S,12°26’E),
withthepermissionofDieudonnéMBOUMBA,theAdministrativeHeadofthedistrictof
Mvouti(Congo).Thisfieldstudydidnotinvolveendangeredorprotectedspecies.Thenest
waskeptinatermitariumattheInstitutdeRecherchepourleDéveloppement(IRD)inPointe
Noire(Congo).Termiteswereidentifiedonthebasisofmorphologicalcriteriaandincompari-
sonwithlaboratorycollectionsbyDrAlainRobert,entomologistandtermiteexpert.Thenest
waspartiallybrokentocollecttheeggs,larvaeandadultworkers.Thetwolarvalinstarswere
distinguishedusingthestandardmorphometriccriteriafortheNasutitermesgenus[10,11].
DNAextraction
Triplicatesamplesof50eggs,30individualsofeachlarvalstageand25workerswereused.The
eggsweresurface-sterilizedbydippingthemtwicein70%ethanolfor1min,followedbyfive
rinsesinsterilewater.Thewholegutoftermitelarvaeandworkerswasremovedaseptically
usingfinesterileforcepsunderabacteriologicalhoodandpooledforeachreplicatein1.5ml
sterilemicrotubes.
Gutsandeggswerefirstcrushedusingapolypropylenemicropestlein1.5mlmicrotubes.
TheDNAwasthenextractedusingtheDNeasyBlood&TissueKit(QIAGEN,QIAGEN
GmbH,D.40724Hilden,Germany)inaccordancewithmanufacturer’sinstructions.Thefinal
DNAconcentrationwasdeterminedbyelectrophoresisina1%agarosegel,withaquantitative
DNAladder(HighDNAMassLadder,Invitrogen,5791VanAllenWayCarlbad,CA92008)
forcomparison.Priortopyrosequencinganalyses,theDNAconcentrationofthesampleswas
quantifiedphotometricallyusingaND–1000Spectrophotometer(NanoDropproducts,Wil-
mington,USA).
Pyrosequencingandprocessingof16SrRNAgenesequence
ThepurifiedDNAfromthetriplicateswaspooledforpyrosequencing.Aliquotswithsimilar
DNAconcentrationsweresenttotheResearchandTestingLaboratory(Lubbock,TX,USA)
forsequencing.ADNAfragmentspanningtheV1-V3variableregionsofthe16SrRNAgenes
wasamplifiedusingtheeubacterialprimers28Fand519RandpyrosequencedusingaRoche
454FLXpyrosequencer.TheresultingsequenceswereanalyzedusingMothurv.1.33.3[12].
PyrosequencingreadswereprocessedusingthemethoddescribedbySchloss[13].Sequencing
errorwasreducedusinganimplementationoftheAmpliconNoisealgorithmandlow-quality
sequenceswereremoved(minimumlength200bp,allowing1mismatchtothebarcode,2mis-
matchestotheprimer,andhomopolymersnolongerthan8bp).Sequenceswerethentrimmed
tokeeponlyhighqualityreads(Q(cid:1)35).Chimeraswereremovedusingthechimera.uchime
mothurcommand.Singletonswereincludedintheanalysis.Sequenceswerealignedandclassi-
fiedaccordingtotheSILVAbacterialSSUreferencedatabasev.102[14].Theywerethen
assignedtogenus-levelphylotypesusingthenaiveBayesclassifierimplementedinMothurand
clusteredintooperationaltaxonomicunits(OTU)usingtheaverageneighboralgorithmanda
sequenceidentitycutoffof97%.TheGreengenesdatabase[15],andtheDictDbreferencedata-
basev.2.3[6]wereusedforthetaxonomicassignmentofOTUs.ThesharedOTUs(97%
sequenceidentity)betweenlifestages(eggs,L2larvaeandworkers)weredeterminedusingthe
VenndiagrampackageimplementedinMothur.TheVenny2.0.2softwarewasusedtoplot
resultingdata.RelativeabundanceofeachgroupofOTUswasaddedmanually.Sequencesare
availableinNCBISequenceReadArchiveunderBioSampleaccessionIDSAMN03114856and
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BacterialCommunitiesthroughTermiteLifeStages
SAMN03114878toSAMN03114880associatedwithBioProjectsPRJNA270400-PRJNA270403.
Ineachpyrotaglibrary,therelativeabundanceofanOTUisthepercentageofreadsincludedin
thisOTUwithrespecttothetotalnumberofreads.Therelativeabundanceofeachtaxonina
givensampleisthesumofabundancesofalltheOTUsincluded.
Clonelibrariesandphylogeneticanalysis
Thenear-full-length16SrRNAgeneswereamplifiedusingthe27F/1390Rprimers[16].PCR
reactionswereperformedin25μLreactionmixturescontaining15.2μlTaqpolymeraseMaster
Mix(Qiagen),0.25μMofeachprimerand50ngoftemplateDNAasdescribedbyThongaram
1
etal[16]usingaPrimeElitethermalcycler(TECHNE ,BibbyScientificLimited,Stafford-
shire,UK).Ampliconsweregel-purifiedusingtheGFXTMPurificationKit(GEHealthcare,
Buckinghamshire,UK)andclonedintothepCR4-TOPOvectorusingtheTOPOTAcloning
kit(Invitrogen),followingthemanufacturer’sinstructions.Whitecloneswerescreenedforthe
presenceoftheexpectedinsertbyPCRamplificationusingthevector-specificprimersM13F-
20/M13-26R.Foreachsample,70positiveclonesweredouble-sequencedatBeckmanCoulter
Genomics(Takeley,CM226TAEssexUnitedKingdom).Thesequenceswerequalitychecked
andprocessedinthesamewayasforpyrotagsequences.Aphylogeneticanalysiswasper-
formedusingOTUsclassifiedwithinthemostrepresentativephylum(Spirochaetes).Anucleo-
tideBlastsearchwasrunandthefiveclosestsequencesimportedforeachOTU.Other
Spirochaetes16SrRNAgenesequenceswererandomlyimported.Theredundantsequences
wereremovedandthesequenceswerealignedusingSILVASINA(www.arb-silva.de/aligner/)
withthereferencedatasetSILVASSURefNR[17].Thealignmentwasimprovedmanuallyby
removingconservedgapsandambiguouslyalignedregions.Thephylogenetictreewasrecon-
structedusingtheMaximumLikelihoodmethodimplementedinMEGAv6.0[18]andthe
Tamura-Neimodelwith1000bootstraps.
Results
Analysisofpyrosequencingdataandtaxonomicclassification
454pyrosequencingproducedatotalof33351rawpyrotagreadsforthefoursamples.After
qualityfilteringandremovalofchimera,18310highqualityreadswereretrieved.Theseranged
from1470to5990sequencespersample(Table1)clusteredat97%sequenceidentityinto78
bacterialOTUs.TheOTUsaffiliatedtoWolbachiaspp.covered77.2%ofreadsfromeggs,
98.6%inL1larvae,53%inL2Larvaeandlessthan1%inworkers.AsWolbachiaspp.areendo-
symbiontsandnotdigestivesymbiontsperseinIsoptera,allrepresentativeWolbachiaOTUs
weresubsequentlyexcludedfromanalysis.Consequently,asonly50non-Wolbachiareads
remainedfromL1larvae,thisdevelopmentstagewasnotincludedfordownstreamanalysis.
Fortheremainingstages(eggs,L2larvaeandworkers),inordertoavoidbiasattachedtoan
unbalancedlibrarysize,thedatawerenormalizedbasedontheeggsizelibrarythathadthe
lowernumberofsequences(1367).Thesamenumberofsequenceswas,therefore,randomly
subsampledinL2larvaeandworkers.Theresultingsequencesfromthesethreestageswere
assignedto687OTUsunequallydistributeddependingonthestage.TheDictDBdatabase[6]
providedthebesttaxonomicassignmentofOTUsandclassifiedtheminto18bacterialphyla.
MostoftheOTUsfellwithinthefollowingphyla:Proteobacteria(31.46%),Spirochaetes
(27.94%),Bacteroidetes(15.44%),Firmicutes(7.27%),Actinobacteria(6.02%),Fibrobacteres
(4.71%),CandidatephylumTG3(4.54%),Acidobacteria(0.98%)andSynergistetes(0.29%).
TheremainingOTUsfellwithin9minorphyla,eachaccountingforlessthan1%ofthereads
withfewerthan5OTUsperstage:Cyanobacteria,Caldiserica,CandidatedivisionSR1,Chlor-
obi,Chloroflexi,Deferribacteres,Elusimicrobia,Planctomycetes,andVerrucomicrobia.The
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BacterialCommunitiesthroughTermiteLifeStages
Table1. SamplingdepthandnumberoftaxaatOTUlevel(at97%identity),atgenuslevelandphylumlevelby16SrRNAgenepyrosequencing
fromthevariouslifestagesofN.arborum.Foreggs,L2larvaeandworkers,thenumbersoftaxaarebasedonasub-sampleof1367reads.
Eggs L1larvae L2Larvae Workers Total
Samplingdepth
Qualityfilteredreads 5990 3678 7172 1470 18310
Non-Wolbachiareads 1367 50 3371 1458 6246
Taxonomiccomposition
Numberofphylum-leveltaxa 7 7* 13 17 18**
Numberofgenus-leveltaxa 58 19* 93 97 178
NumberofuniqueOTUs(97%identity) 72 24* 297 357 687**
*ForL1larvae,theyarebasedon50sequencesexcludingWolbachia.Theyaregivenforillustrationonlyandarenotincludedinthetotalforthe
taxonomiccomposition,whichreferstouniquetaxa.
**Forthetaxonomiccomposition,thetotalnumberoftaxaatthephylum,genusandOTUlevelsisthesumoftheindividualtaxafromthethreepyrotag
libraries,takingaccountofthetaxacommontodifferentstages.
doi:10.1371/journal.pone.0140014.t001
numberofOTUsintheeggswasmorethan5timeslowerthaninthegutofL2larvaeand
workers.Furthermore,theworkersandL2larvaehadfarmoregenusandphylumleveltaxa,
suggestinganoverallhigherbacterialdiversityinthelastdevelopmentalstagesthanineggs.
Changesinthebacterialcommunitystructurethroughoutlifestagesof
N.arborum
Thebacterialcommunitycompositiondependedonthelifestagesofthetermites.Theeggs
weredominantlycolonizedbyProteobacteria(86.25%ofreads)andActinobacteria(8.41%of
reads)(Fig1).Moreover,thesetwophylaaccountedfornearly70%ofOTUsretrievedfrom
thisstage.WithinProteobacteria,themostabundantOTUsfellintotheBurkholderiaceae
(Betaproteobacteria)andXanthomonadaceae(Gammaproteobacteria)families(Fig2andS1
Table).TheremainingOTUswereaffiliatedtolessabundantphyla,noneofwhichaccounted
formorethan1.5%ofreadswiththeexceptionofFirmicutes.ThegutofL1larvaeweredomi-
nantlycolonizedbyOTUsclusteredwithWolbachia.AfterexcludingtheseOTUsindown-
streamanalyses,theremainingsequencesfellprimarilywithinFirmicutes(32%ofreads),
Bacteroidetes(24%),Proteobacteria(20%)andSpirochaetes(14%).OfFirmicutes,themost
abundantsequenceswereaffiliatedtothegeneraStreptococcus(Lactobacillales)andPaenibacil-
lus(Bacillales)whileBacteroidetesweremainlyrepresentedbytheM2PB4-65termitegroup,
Alkaliflexus(Marinilabiaceae)andPaludibacter(Porphyromonadaceae).Inthelastlarval
stage,Bacteroidetesweremoreabundant(36.58%ofreadsandmorethan20%ofOTUs)and
werefromtheM2PB4-65Termitegroupaccountingfor26.12%ofreads.BesidesBacteroidetes,
Spirochaetes(21.51%)mainlyfromTreponemaIcluster,Fibrobacteres(12.00%)especially
fromtheTermitesubclustersIandIIandtheCandidatephylumTG3(10.75%)weremore
abundantthaninpreviousstages.Intheworkercaste,themajorityofreadsfellwithinthe
samephylaasforL2larvae,butwithamarkedrelativeincreaseintheabundanceofSpiro-
chaetes(61.45%ofreadsand39%ofOTUs).
OTUscommontothevariouslifestages
OnlytwoOTUs,accountingforlessthan1%inallthepyrotaglibraries,werecommontothe
eggs,L2larvaeandworkercastetermitesofNasutitermesarborum(Fig3).ThesewereBacter-
oidetesaffiliatedtotheM2PB4-61Termitegroup(Rikenellaceae)andthehighertermiteaclus-
ter(Rs-E47termitegroup)respectively(S2Table).ThreeOTUswerefoundintheeggsandL2
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BacterialCommunitiesthroughTermiteLifeStages
Fig1.Phylum-leveldistributionofbacterialtaxafrompyrotaglibrariesbasedonthe16SrRNAgene
fromeggsandthegutsofthefirstinstar(L1larvae),secondinstar(L2larvae)andworkersofN.
arborum.Foreachstage,theinnerpiechartcorrespondstotherelativeabundanceofreadsaffiliatedto
eachphylum.TheouterpiechartcorrespondstothenumberofOTUsforeachphylumasafractionofthe
totalnumberofOTUs.*ThechartsforL1larvaearebasedon50sequencesexcludingWolbachiaunlikethe
chartsforeggs,L2larvaeandworkersstageswhicharebasedonsubsamplesof1367reads.“Minorphyla”
correspondtothepoolofallphylawith<1%ofreadsand(cid:3)5OTUsinwellsequencedlibraries(eggs,L2
larvae,workers).
doi:10.1371/journal.pone.0140014.g001
larvaeandnotinworkers.TheywerefromthegeneraStaphylococcus(Staphylococcaceae,Fir-
micutes),Massilia–6(Oxalobacteraceae,Betaproteobacteria)andSubclusterVb(ClusterVb,
Bacteroidetes)andtogetheraccountedforabout0.5%ofreadsineachofthesestages.Sixteen
OTUswerefoundintheeggsandworkercastetermitesbutwerenotfoundinL2Larvae.
ThesecommonOTUsaccountedfor6.3%and18.9%ofreadsineggsandworkersrespectively.
TheywereprimarilyFirmicutes(5OTUs,1.76%orreadsfromeggs),Actinobacteria(4OTUs,
2.71%)andProteobacteria(4OTUs,1.39%).16OTUswerefoundinbothL2larvaeandwork-
ersbutnotinotherstages.TherewasahigherproportionofthesecommonOTUsinL2larvae
(31.4%ofreads),mainlyfromBacteroidetes(10OTUs,24.36%ofreads)especiallyfromthe
M2PB4-65termitegroup,fromTreponemaIcclusterofSpirochetes(2OTUs,5.78%ofreads
inL2larvae)andfromFirmicutes(3OTUs,0.58%ofreadsinL2larvae).Theseresultsindicate
thattherearesignificantlyfewerOTUsincommonbetweendevelopmentalstagesthanOTUs
foundonlyinonestage,whichrepresentedupto92.4%ofreads(or70.53%ofOTUs)from
eggs,68.4%reads(or92.92%ofOTUs)fromL2larvae,and74.8%ofreads(90.47%ofOTUs)
fromtheworkercaste.
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BacterialCommunitiesthroughTermiteLifeStages
Fig2.Relativeabundancesofthegenus-levelbacterialgroupsineggs(E),thefirstinstarlarvae(L1),thesecondinstarlarvae(L2)andtheworker
caste(W)ofN.arborum.Onlygenera>0.5%inatleastonestagearepresented.Theremaininggeneraareincludedin“Others”.Thedetailedclassification
withallthetaxonomiclevelsisprovidedintheinteractivespreadsheet(S1Table).*TherelativeabundancesfortheL1larvalstagearebasedon50
sequencesexcludingWolbachia.
doi:10.1371/journal.pone.0140014.g002
PhylogeneticrelationshipbetweenallopatricNasutitermesspecies
Owingtothelongerreads,the16SrRNAgene-basedclonelibraryallowedustocarryouta
comparativeanalysisofthebacterialcommunitiesatahigherlevelofphylogeneticresolution.
Thisapproachgenerated280sequencesfromthefoursamples(eggs,L1,L2andadultworkers)
including253highqualityreads.Togainaninsightintotheroleofthesymbionttransmission
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BacterialCommunitiesthroughTermiteLifeStages
Fig3.VenndiagramofbacterialOTUs(at97%identity)commontothepyrosequencinglibrariesfor
eggs(E),L2larvae(L2)andworkers(W)ofN.arborum.NumbersinboldindicatethenumberofOTUs
andnumbersinitalicstherelativeabundanceofthecorrespondingOTUsineachstagewheretheyare
detected.DetailsofthecommonOTUsaregiveninS2Table.
doi:10.1371/journal.pone.0140014.g003
routeinthecodiversificationofthegutmicrobiotawiththetermitehost,aphylogeneticanaly-
siswascarriedoutontheSpirochates-relatedOTUs,thedominantlineagefromadultworkers
ofN.arborumincomparisonwithpreviouslypublishedsequencesavailableindatabases.Most
oftheSpirochaetes-relatedOTUsclusteredwiththosepreviouslyretrievedfromthegutofallo-
patricNasutitermesspecies(Fig4).
Discussion
Thereisstilllittleinformationonthecolonizationofthetermitegutbymicroorganismssince
mostpreviousstudiesofthegutmicrobiotahavebeenlimitedtotheworkercaste.Thisisthe
firstmolecularsurveyofthemicrobialcompositionineggsandlarvalstagesoftermitesand
providesinformationonwhetherthegutmicrobiotaoflaterinstarsoriginatesfromtransovar-
iantransmissionthroughtheeggsorfromearlyinoculationoftheoffspring.
Thedetectionofbacteriafromtheeggsraisedthequestionwhetherthesebacteriawere
locatedwithintheegg.Theeggsweresurface-sterilizedin70%ethanoltocheckthehypothesis
thattermitesymbiontsaretransmittedtransovariallytotheeggs.Unfortunately,thelackofan
untreatedcontrolmadeitimpossibletodrawanyconclusionsonthispoint.Althoughsome
bacterialocatedonthesurfaceoftheeggswerecertainlylysedbythe70%ethanolandwashed
awaybysterilewater,thepresenceofpersistentcontaminationbybacteriaorDNAshouldnot
beruledout[19].Thepresenceofbacteriainsidetheeggwasnotunexpectedastermiteeggs
havetinychannelsthroughtheouterliningoftheeggscalledmicropyles[20].Theeffectiveness
ofsterilizingthesurfaceoftheeggsusing70%ethanolinthisstudycannotbeassessedanditis
notpossible,therefore,toconcludewhetherthebacteriadetectedwerelocatedonthesurface
orinsidetheeggs.TotalDNArecoveryfromtheeggswasofthesameorderofmagnitudeasin
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BacterialCommunitiesthroughTermiteLifeStages
Fig4.PhylogenetictreeillustratingthepositionofSpirochaetes-relatedOTUsfrom16SrRNAgene
libraries.ThetreewasconstructedusingtheMaximumLikelihood-methodimplementedinMEGAversion
6.0andtheTamura-Neimodel(analysis,1000bootstraps).Onlybootstrapvalues>50%arepresented.
ClonesfromthisstudyarereferredtoasGLandGLS(fromthesecondinstarlarvae)andGO(fromworkers)
andnumbersinbracketscorrespondtoNCBIaccessionnumbersofsequences.*indicatessequences
undergoingNCBIsubmission.Branchesingreenareentirelymadeofsequencesfromtermites.
doi:10.1371/journal.pone.0140014.g004
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BacterialCommunitiesthroughTermiteLifeStages
workersbut13–15foldhigherthaninL1andL2larvalstages.Althoughtheconcentrationof
DNAwasadjustedtobeequalforallsamplespriortopyrosequencinganalyses,thisquantifica-
tiononlyprovidedinformationonthepoolofDNAextractedfromeggtissue,Wolbachiaand
othermicroorganismsfoundinthesample.Thisstudydidnotspecificallyquantifymicrobial
DNAorthebacterialcolonizationdensity.Lowbacterialdensitycanhaveasignificanteffect
onmicrobialprofilingbypyrosequencinganalyses[21,22].Therefore,changesinrelativeabun-
dance,especiallyineggsandfirstinstarL1,shouldbeconsideredwithcautionsincethedomi-
nanceofWolbachiaatthesehostdevelopmentalstagesmeansthatthecontributionofgut
bacteriatothetotalDNAintheextractswasverysmall.
Itwasexpectedthattermite-specificbacteriawouldcolonizetheembryosorthegutoflar-
vaeatanearlystage,thuspreventingtheestablishmentofenvironmentallineagesthatwould
weakenthepartnerfidelity,asisthecaseforsomeinsects[23].ApartfromWolbachia(77.7%
ofreadsineggs),ubiquitousaerobicbacteriamainlyaffiliatedtoProteobacteriaandActinobac-
teriawerepredominantintheeggsofN.arborum.ThedominanceofWolbachiaintheeggsas
wellinthefirstlarvaeandlastinstarisconsistentwithapreviousstudystressingtheabundance
ofthesematernallytransmittedendosymbiontsintissuesofimmaturestagessuchaslarvae
andpre-soldiers[24].Itisnotclearwhethertheotherbacteriafoundintheeggswerealsover-
ticallytransmitted.Togiveabetterestimateoftermite-associatedsymbionts,adistinctionwas
drawnbetweenOTUsmatchingexclusivelytermite-specificphylotypesandOTUsmatching
free-livingstrains(S1Fig).Thedominantphylaintheeggs,ProteobacteriaandActinobacteria,
consistedmainlyoffree-livinglineages.OnlytwoOTUsaffiliatedtothecluster–1(Rs-K70ter-
mitegroup,Deltaproteobacteria)andaccountingforlessthan0.5%inthisstageweregut-spe-
cificsymbionts,oneofwhichwasalsofoundinalldevelopmentalstages.Fourteenothergut-
specificOTUsamountingtonotmorethan2.5%mainlyfromSpirochaetes(Treponemaand
Uncultured1Spirochaetaceae)andBacteroidetes(SubclusterVb,Rs-E47termitegroup,
M2PB4-61termitegroup)werefoundintheeggs.Overall,thesefindingssuggestthateggsare
predominantlycolonizedbybacteriaacquiredfromtheenvironment.Thepresenceofafew
gut-specificOTUsintheeggsmaysimplyresultfromoccasionaltransfersbyworkers,either
duringthefrequentdisplacementsviatheirsalivarium,orduringegggroomingbylicking.
Transferduringegg-layingbythequeenmightalsobeasourceofbacterialcontamination.The
importanceofthesebehavioraltraitsintermiteswasshownbytheincreaseineggsizeasa
resultofsalivatransfer,andthefailureofunlickedeggstohatch[25].Itispossiblethatthese
interactionsdepositgut-derivedsymbiontsonthesurfaceofeggs,followedbytheingestionof
thesebacteriabylarvae.Inthiscase,surfacesterilizationmayhaveunderestimatedthegut
derivedbacteriaintheeggs.Thesurfaceoftheeggswassterilizedontheassumptionthatitwas
notasuitableenvironmentforthesurvivalofmosttermite-gutsymbionts,mostofwhichare
anaerobicbacteria.
ThehighproportionofProteobacteriaandActinobacteriacouldhaveafunctionalsignifi-
canceastheirdefensiveroleineggsorlarvaehasbeenreportedinsoilandmarineinvertebrates
[26–30].ThelargelydominantphylotypeintheeggswasaffiliatedtoBurkholderia(Betapro-
teobacteria)someofwhosemembershavebeenreportedtoprotectleaf-cuttingantsagainst
entomopathogenicfungi[31].ThefunctionsofProteobacteriaandActinobacteriaintermite
eggshavenotyetbeendocumented.
Inthefirstinstarlarvae,therewasarelativeincreaseofFirmicutes,Bacteroidetes,Spiro-
chaetesandtheemergenceofFibrobacteresinadditiontoActinobacteriaandProteobacteria
detectedintheeggs,indicatingthediversificationofthecommunityatthisdevelopmental
stage.Ofaparticularinterestwastherelativeincreaseinspecifictermite-gutOTUs,covering
upto32%ofreads,mostofwhichwerecloselyrelatedtoTreponemasubclustersIcandIa(Spi-
rochaetes),Alkaliflexus,PaludibacterandTannerella(Bacteroidetes).Inthelastinstarlarvae,
PLOSONE|DOI:10.1371/journal.pone.0140014 October7,2015 10/15