Microbiomes of ant castes implicate new microbial roles in the

34 Pages·2011·2.23 MB·English

Microbiomes of ant castes implicate new
microbial roles in the fungus-growing ant
SUBJECTAREAS: Trachymyrmex septentrionalis
BIODIVERSITY
ENVIRONMENTAL
MICROBIOLOGY HeatherD.Ishak1,JessicaL.Miller1,RuchiraSen1,ScotE.Dowd2,EliMeyer1&UlrichG.Mueller1
ECOLOGY
EVOLUTION 1SectionofIntegrativeBiology,1UniversityStation(C0930),UniversityofTexasatAustin,Austin,TX78712,USA,2MedicalBiofilm
ResearchInstitute,4321MarshaSharpFreeway,Lubbock,TX79407,USA.
Received
31May2011 Fungus-growingantsemployseveraldefensesagainstdiseases,includingdisease-suppressingmicrobial
biofilmsontheirintegumentandinfungalgardens.Here,wecomparethephenologyofmicrobiomesin
Accepted naturalnestsofthetemperatefungus-growingantTrachymyrmexseptentrionalisusingculture-dependent
9December2011 isolationsandculture-independent16S-amplicon454-sequencing.454-sequencingrevealeddiverse
actinobacteriaassociatedwithants,includingmostprominentlySolirubrobacter(12.2–30.9%ofsequence
Published
reads),Pseudonocardia(3.5–42.0%),andMicrolunatus(0.4–10.8%).Bacterialabundancesremained
22December2011 relativelyconstantinmonthlysurveysthroughouttheannualactiveperiod(latewintertolatesummer),
exceptPseudonocardiaabundancedeclinedinfemalesduringthereproductivephase.Pseudonocardia
speciesfoundonantsarephylogeneticallydifferentfromthoseingardensandsoil,indicatingecological
separationofthesePseudonocardiatypes.BecausethepathogenEscovopsisisnotknowntoinfectgardens
Correspondenceand
ofT.septentrionalis,theant-associatedmicrobesdonotseemtofunctioninEscovopsissuppression,but
requestsformaterials
couldprotectagainstantdiseases,helpinnestsanitation,orserveunknownfunctions.
shouldbeaddressedto
H.D.I.(heatherishak@
gmail.com)
Fungus-growingants(Attini,Formicidae)dependonfungalgardenscultivatedforfood.Thecultivatedfungi
(Agaricales, Basidiomycotina) grow in the gardens alongside an assortment of secondary microbes that
interactmutualistically,commensally,orantagonisticallywiththecultivatedfungiandtheants1–6.Attine
gardensaresusceptibletodiseases, thebeststudiedofwhicharefilamentousfungiinthegenusEscovopsis7,8.
Attineworkersemployanumberofdefensesagainsttheirdiseases,includingmanagementofauxiliarymicrobes
(bacteria,yeasts)withpotentialdisease-suppressingfunctions2–4.
Actinomycete bacteria such as Pseudonocardia, Streptomyces, Amycolatopsis, Kribbella, Tsukamurella, and
Nocardioides have been isolated from lab-maintained attine ants and their gardens using culture-dependent
methods5,9–11.Oftheknownauxiliarymicrobes,Pseudonocardiabacteriahaveattractedthegreatestattentionfor
theirpossiblemutualisticrolesinsuppressionofdiseasesofgardensoroftheants.Pseudonocardiacanbeisolated
readily from microbial biofilms on the ant’s integument, and several ant-associated Pseudonocardia species
secretechemicalsinvitrothatinhibitthegrowthofEscovopsis,aswellasthegrowthofagreatdiversityofother
fungiandbacteria5,11–16.Typicalforactinomycetebacteria,Pseudonocardiabacteriapossesspolyenegeneclusters
regulatingthesynthesisofantifungalpolyketidecompounds17,andwhole-genomesequencinghasconfirmedthe
presenceofsuchpolyenegenesinPseudonocardiastrainsisolatedfromaleafcutterant14,18.
Ant-associated Pseudonocardia strains were initially thought to be antibiotically highly specialized and
primarilyverticallytransmittedbetweenantgenerations,givingrisetotheviewoflong-termco-evolutionary
interactions between ants and their Pseudonocardia associates8,19. Under the original co-evolutionary inter-
pretationoftheant-Escovopsis-Pseudonocardiaassociation,attineantswerebelievedtomanageintegumental
Pseudonocardiaasaspecificco-evolvedantibioticdefensetargetedagainstEscovopsisfungi,whichwerethought
nottoevolveresistancetoPseudonocardiaantibioticsduetosomeunknowndisadvantagesinaco-evolutionary
armsrace10,13,19.However,morecomprehensiverecentstudieshavebeguntoquestionthepostulatedtightant-
Pseudonocardiaco-evolution,suggestinginsteadfrequentrecruitmentofPseudonocardiafromenvironmental
sources(plantmaterial,soil)intotheintegumentalbiofilmsandpossiblehorizontaltransmissionofmicrobesby
malesduringmating5,9,15,18,20–22.
Westudyherethefungus-growingantTrachymyrmexseptentrionalistocharacterizeseasonalchangesinant-
bacteriaassociationsinundisturbedfieldcolonies.T.septentrionalisprovidesuniqueopportunitiestoexplorethe
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potential roles of auxiliary, secondary microbes in the attine ant- couldbefoundwithinasinglenest.Fournestshadtwomorphotypes
microbesymbiosis,fortwomainreasons.First,despitethefrequent and one nest had three morphotypes found onthe workers of the
presenceofwhitishaccretions(presumedactinomycetegrowth)on samenest(Table2).
theintegumentofT.septentrionalis,Escovopsis,aprevalentpathogen TheP.cf.carboxydivoransmorphotypesfellintofourcategories
infecting gardens of tropical attine ants, does not appear to infect (morphotype A–D; Supplementary Fig. S1). Morphotype A was
gardensofthetemperateT.septentrionalis21,23.Ourstudypopulation the most predominant type isolated from garden workers, outside
of T. septentrionalis was previously surveyed for non-cultivar fila- workers, reproductives, and gardens, although a large number of
mentousfungiandyeastsgrowingingardensalongsidethecultivated morphotype B was found associated with reproductive females in
fungus23.NoEscovopsis could beisolated fromany of16surveyed nests collected in July 2009 (Table 2). We were unable to culture
nests(4nestssurveyedeverythreemonthsoveranentireyear),nor Pseudonocardiabacteriafromanysoilsamplesduetorapidfungal
fromdozensofgardensoftwoothersympatricattinespecies(Atta contaminationofchitinisolationplates.
texana,Cyphomyrmexwheeleri23).Moreover,anothersurveycover- Intheculture-dependentscreens,Pseudonocardia,Amycolatopsis,
ingthefullrangeofT.septentrionalis(Texas,Illinois,Florida,New Kribbella,andStreptomyceswereisolatedingreaternumbersfrom
York)hadfailedtofindanyEscovopsisinfectioningardens21.The samplescollectedinJuneandJulythaninanyothermonth(Table3),
presenceofbiofilmsontheintegumentofT.septentrionalis inthe with Amycolatopsis and Kribbella isolations almost completely
apparent absence of Escovopsis therefore suggested possible novel restrictedtoJuneandJuly.Streptomyceswasisolatedinallmonths
functionsofthemicrobialaccretionsontheintegumentofT.septen- (except January) and in all sample types (Table 3). The culture-
trionalis(e.g.,protectionoftheantsagainstpredators,entomopatho- dependentscreenyieldeddifferentresultsthantheparallelculture-
genic diseases, or parasites such as mites; protection against other independentscreen(Table3),confirmingexpectedisolationbiases
gardendiseases). oftheminimum-carbonchitinmedium5,22,26.
A second reason T. septentrionalis represents a unique study
system is that this temperate ant exhibits life-history features that Culture-independentscreens.454-sequencingBLASTresultswere
permit testing for seasonal patterns of microbial association and analyzedbytheclosestoperationaltaxonomiclevel(Supplementary
abundance. Gardens are collapsed in winter (sometimes to small Table S1) and by genus in a forced-genus BLAST match (Supple-
fragments),thenarereactivatedinspringbyworkers,andgardens mentaryTableS2)toestimateaveragerelativesequence-abundances
reachthelargestsizesthroughoutearlysummerwhenalatesemerge by sample type. Rarefaction analyses at a 97% sequence-similarity
andfeedongardensuntilrainsstimulatematingflights24,25.Seasonal criterionrevealedthatmostofthegardenworkers,outsideworkers,
correlationsbetweentheabundanceofmicrobialassociates,garden andreproductives(males,females)wereadequatelysampled(species-
size,workeractivity,andproductionofreproductivescantherefore accumulationcurvesreachedanasymptotewith2000–5000sequences
providecluesforthepossiblerolesofthemicrobialassociatesinthe sampled; Supplementary Fig.S2). However, chamber soil, excavated
temperateT.septentrionalis,whereassuchcorrelationsarelessobvi- soil, and most garden samples were undersampled (Supplementary
ousinthecomparativelystable,tropicalant-microbesymbioses. Fig. S2). As expected, soil samples require a far greater sequencing
To generate insights into such phenological changes in ant- depth to evaluate bacterial diversities.Garden samples proved more
associatedbacterialmicrobiomes,weperformedculture-dependent difficult to sequence, resulting in lower sequence yields, with an
isolationandculture-independent16S-amplicon454-sequencingof averageof2,661sequencespergardenascomparedtoanaverageof
T. septentrionalis nests collected between January and September 4,007sequencesfromallothersampletypes.Additionally,sequences
2009. No study to date has quantified the relative abundance of obtained from garden samples also had fewer reliable BLAST hits,
bacteriainnatural,field-collectedattinenests. and many ofthese sequences thereforeremained unidentified (Sup-
plementaryTablesS1andS2).
Results Weusedanunweightedprincipalcoordinateanalysis(PCoA)in
Culture-dependent screens. Culture-dependent isolations iden- Fast Unifrac to visualize the differences between microbial com-
tified eleven genera from the order Actinomycetales among 914 munitiesforeachofoursampletypes(Fig.1).Bacterialcommunities
bacterial colonies isolated from garden workers, outside workers, associatedwithsoilsamplesweresignificantlydifferentthantheant-
reproductives, garden, and soil samples (Table 1). Isolates were associated bacterial communities (R250.21, adonis test P,0.01).
morphotyped(by colorand growthmorphologyonPDAmedium), Chamber soil samples clustered more tightly together than the
and264representativeisolateswereidentifiedthroughpartial16S- excavated soil (Fig. 1), but both soils had overlapping microbial
sequencing. Pseudonocardia, Kribbella, Amycolatopsis, and Strepto- community compositions. Some garden samples clustered with
mycesaccountedfor98%(n5890)oftheisolatedcolonies(Table1). soilsamples,whereasothergardensamplesseparatedoutdistinctly
RareisolatesincludedthegeneraActinomadura,Actinomycetospora, (Fig.1).
Nocardioides, Nocardia, Nonomuraea, and Sphaerisporangium. To Thecombinedantsampleshadanaverageof88(1/226)bac-
test whether bacterial growth was specific to ant body segments terialgeneraidentifiedpernest(SupplementaryTableS3).Soilsam-
(head,mesosoma,metasoma),wealsostreakedthesesegmentsindi- ples contained significantly more bacterial genera (average of 259
vidually on chitin medium. We found P. cf. carboxydivorans and (1/250)generapernest;t-testt515.641,df524,P,0.001).Ants
Streptomyces on all body segments of workers, males, and females sharedanaverageof65%oftheirbacterialgenerawiththosefoundin
(Table 1). Additionally, Amycolatopsis was found on all body seg- soil;however,thesesharedbacterialgenerahadtypicallylowabund-
mentsofmales. ance (i.e., abundant genera were typically not shared). Differences
SequencedPseudonocardiafromtheculture-dependentisolations between bacterial compositions of ants and soil are clearly evi-
had identical partial 16S-sequences, which were identified as dentwhencomparedattheordertaxonomiclevel(Supplementary
Pseudonocardia cf. carboxydivorans via BLASTn (99% identity to Fig.S3).
GenBankaccessionFJ532384).Theonlyexceptionswerethreeiso- 454-sequencingrevealed19bacterialgenerafrequentlyassociated
lates from garden samples in June, which were closely related to withT.septentrionalisants(eachofthesegenerahadaminimumof
Pseudonocardia spinosispora (696 bp at 98% identity to GenBank 1%sequencereadsaveraged acrossallsamplesfromallantcastes;
accessionNR_025367).AlthoughalltheisolatesidentifiedasP.cf. Table4).Elevenofthese19generabelongtotheclassActinobacteria,
carboxydivorans had identical 16S-sequences, we found four dis- including most prominently Solirubrobacter (order Solirubrobac-
tinct morphotypes among these isolates (Supplementary Fig. S1). terales), Microlunatus, Pseudonococardia, Aeromicrobium, Phyci-
Interestingly, more than one P. cf. carboxydivorans morphotype coccus,andAgrococcus(allintheorderActinomycetales;Table4).
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withculture-dependentmethods.Wholeants,garden,chambersoil,TrachymyrmexseptentrionalisTable1|Bacteriafromelevenactinomycetegeneraisolatedfromthefungus-growingantandexcavatedsoilwereplatedasmaceratedbuffersuspensionsonminimum-carbonchitinmediumfavoringgrowthofautotrophicbacteria.Antswerescreenedwhole,butthethreemainbodysegmentsofants(head,mesosoma,andmetasoma)werealsostreakedseparatelyonchitinmedium.Bacterialcountspersampletypeneedtobeinterpretedwithcautionbecausesamplingeffortcouldnotbecompletelystandardizedacrossallsampletypes.andwereisolatedinallexceptonesampletypeandfromallthreebodyPseudonocardiaStreptomycessegments.Emptycellsindicateanisolationcountofzero.SeeSupplementaryMethodsfordetailsonthecombined16S-sequencingandmorphotypingapproachtoidentifybacteriatogenus samplesTrachymyrmexseptentrionalisActinomycetegeneraisolatedfrom UnclassifiedPseudonocardiaStreptomycesKribbellaAmycolatopsisNocardiaNocardioidesActinomaduraActinomycetosporaNonomuraeaSphaerisporangiumactinobacteriumSampleType#Samples Gardenworkerwholeant3111348291452head16163mesosoma161031metasoma1681Outsideworkerwholeant291247146231head15307mesosoma1556111metasoma15252Malewholeant21211head21321mesosoma2963metasoma21142Reproductivewholeant6106332femalehead5136mesosoma5111metasoma5205Garden2829281232Excavatedsoil308121Chambersoil303341922 Totalnumberofisolates60917969338822211identifiedtothisgenus Fractionofthisgenus66.619.67.53.60.90.90.20.20.20.10.1amongallbacteriaidentified %ofsamplesidentified20.541.931.966.787.537.55050100100100by16Ssequencing %ofsamplesidentified79.558.168.133.312.562.55050000bymorphotypingonly
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arepresentedasthenumberofisolatesofP.cf.carboxydivorans54-sequencesfoundperantcasteandthenumberofdifferentaparticularsampletype(e.g.,reproductiveant)wasnotfoundinntheculture-independentscreen.InourcustomPseudonocardiaashitintheBLASTofeveryantsample;seealsoBLAST-results Culture-IndependentScreen Distinct#ofPseudonocardiastrainspernest454-sequencesPseudonocardia#of outsidegardenoutsideworkerworkerworkerfemalemale nananananananananana3029nana17416nana2183147nana612467nana415736nana4na2011nana259100nana266108nana1132182nana1155128nana34696nana24659nana1233204nana32218144nana5436357nana352299nana112456nana2273125308118067nanana544028637293521na2nanananana8384729na215250nana22331531513na7184171110na22476167nana282181681na6 verage290.32118.52172510462.92
strainsfoundpernestof.Theculture-dependentcountsPseudonocardiaTrachymyrmexseptentrionalisTable2|NumberofmorphotypeA–Dfoundinaparticularnest.Theculture-independentcountslistthetotalnumberof4Pseudonocardiastrainsamongthesesequences.Emptycellsindicateabsenceof,and‘‘na’’indicatesthatPseudonocardiaPseudonocardiaanestatthetimeofcollection.Becauseofanoversight,nestsJLM090322-04andJLM090630-02BwereonlyscreenediBLAST,themajorityofthesequenceswereidentifiedas(i.e.,sequenceaccessionFJ490549wP.cf.carboxydivoransidentifyingclade-3inSupplementaryTableS5).Pseudonocardia Culture-DependentScreen CulturedMorphotypesP.cf.carboxydivorans NestID(AbbreviatednestID)MonthABCD gardenoutsideoutsidegardenoutsidegardenworkerworkermalefemaleworkerworkerworkerfemaleworker JLM090131-01Jan2nananaJLM090131-03Jan1nananaJLM090207-01(J0201)FebnananaJLM090322-01(J8-12)MarnananaJLM090322-2A(J0302A)MarnananaJLM090322-2B(J0302B)MarnananaJLM090322-03(J0303)MarnananaJLM090322-04(J0304)MarnananananananananaJLM090405-01(J0401)Apr116nananaJLM090405-02(J0402)Apr3nananaJLM090405-03(J0403)Apr73nananaJLM090405-04(J0404)Apr31nananaJLM090528-01(J0501)May11nana23na1JLM090528-02(J0502)May77nananaJLM090528-03(J0503)MaynananaJLM090528-04(J0504)May39nananaJLM090627-01(J0601)Jun10naJLM090627-02(J0602)Jun55nanaJLM090627-03(J0603)Jun13nanaJLM090630-02(J1-J7)Jun6253524JLM090630-02B(J0630-02)JunnananananananananaJLM090630-03(J0630-03)Jun109na17RS090713-01Jul33nanaJLM090713-01(J0701)Jul2528na27JLM090713-02(J0702)Jul833nanaJLM090713-04(J0704)Julna456JLM090830-01(J0801)Aug2947na22JLM090830-02(J0802)Augnana5JLM090830-03(J0803)Aug6na A
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bacteriafoundinculture-dependentandculture-independentmonthlysurveysintheolunatusellindicatesabsence;cellswith‘‘na’’indicatethatasamplewasnotavailableforscreening.mplesintheculture-independentscreens.,,,andPseudonocardiaAmycolatopsisKribbelladentscreens.Culture-independentscreensfailedtodetectinantsamples,whereasKribbellaectedintheculture-independentscreensbecauseitisactuallyrare,butitgrowswellontheandwerefoundabundantlyintheculture-independentscreens,olirubrobacterMicrolunatus umisolation)Culture-IndependentScreen(454sequencing) JulAugSepJanFebMarAprMayJunJulAug 43301544533 111111111111111111111111111111111111111111nanananana1nanananananananana11111111111111111111nanananana1nanananananananana111111111111nananananananananananananana111111111111111111111111111111111111111111111111nanananana1nanananananananana1111111111111111111111111111111111111nanananana1nanananananananana1111111111111111111111111111nanananana1nanananananananana
,,,,andPseudonocardiaAmycolatopsisKribbellaStreptomyces,SolirubrobacterMicrTable3|1fungus-growingant.Thesignindicatespresence;anemptycTrachymyrmexseptentrionalisThepresenceofthespecificbacterialgeneralistedwasconsistentacrossthemonthlysaweremostfrequentlyisolatedinthemonthsofJuneandJulyintheculture-depenStreptomycesculture-dependentscreensreadilydetected.ItispossiblethatwasnotdetKribbellaKribbellaminimum-carbonchitinmediumfavoringautotrophicbacteria.Incontrastto,bothKribbellaSbutwereneverisolatedintheculture-dependentscreens Culture-DependentScreen(chitin-medi BacteriumCultureSampleTypeJanFebMarAprMayJun #nestsscreened214445 ExcavatedSoilPseudonocardiaChamberSoil1Garden1111GardenWorker111OutsideWorker1ReproductiveFemalenanananana1MalenananananaExcavatedSoilAmycolatopsis11ChamberSoilGarden1GardenWorker1OutsideWorker1ReproductiveFemalenanananana1MalenananananaExcavatedSoilKribbella1ChamberSoilGarden11GardenWorker1OutsideWorkerReproductiveFemalenananananaMalenanananana111ExcavatedSoilStreptomyces1111ChamberSoil1Garden1111GardenWorker11OutsideWorker1ReproductiveFemalenanananana1MalenananananaExcavatedSoilSolirubrobacterChamberSoilGardenGardenWorkerOutsideWorkerReproductiveFemalenananananaMalenananananaExcavatedSoilMicrolunatusChamberSoilGardenGardenWorkerOutsideWorkerReproductiveFemalenananananaMalenanananana
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Figure1|FastUniFracunweightedprincipalcoordinateanalysis(PCoA)forTrachymyrmexseptentrionalisants,gardens,soilsamples,andcontrol
ants(Pheidole). ThePCoAusesaphylogeneticapproachtocomparethewholemicrobialcommunitiesbetweensamples.Thepolygoncontainsallant
samplesexceptforonemaleant;thelattergroupednearthegardenandPheidolesamples.Thechamber-soilandexcavated-soilsamplesclusterloosely
together,butthegardensamplesrangewidely,withseveralsamplesgroupingmostlyamongthesoils;despitecarefulexcavation,gardensmayhave
becomecontaminatedwithsoildustthatspreadtogardensaccidentallybyagitatedworkers.
The two actinobacterial orders Actinomycetales and Solirubro- Average percent of sequence reads of Pseudonocardia on repro-
bacteralescomprisealmost70%ofthetotalbacterialsequencereads ductive females collected in June was initially high (68%), but
identifiedfromT.septentrionalisants(Table4,SupplementaryFig. declined by August (10%). Dealate females collected on 30-Aug
S3). Other bacterial genera (e.g., Bacillus, Burkholderia, Coryne- (i.e., females that lost their wings and had failed to leave the nest
bacterium, Mesoplasma, Spiroplasma) were detected occasionally for a mating flight) showed a low percentage of Pseudonocardia
as abundant sequence reads, but this was the case in only a few sequence reads, but showed increased percentage sequence-reads
individual ants. Overall, the 19 common genera comprise more of other bacteria commonly found on workers (Solirubrobacter,
than90%ofallthebacterialsequencereadsidentifiedforworkers Microlunatus,Phycicoccus,andAeromicrobium).
and female reproductives (Table 4). Reproductive males also had
abundantsequencereadsofComamonas,Escherichia,andPropioni- Pseudonocardia.WefoundPseudonocardiasequencesineachofthe
bacterium bacteria (Supplemental Table S2), but not consistently antsamplesofgardenworkers(n525),outsideworkers(n525),and
so in all males. The significance of the male-associated bacteria is male and female reproductives (n512). In the custom BLAST of
unclearbecauseonlythreemaleswerescreened. 26,965 Pseudonocardia 454-sequences, we found 14 distinct
As a quality check of the 454-BLAST reference assignments Pseudonocardia strains among the garden workers, 14 distinct
generated from the Medical Biofilm’s reference database, we also strains among outside workers, and 6 distinct strains among
used the Ribosomal Database Classifier (RDC) to assign identities reproductiveants(SupplementaryTableS5).Thesestrainscovered
toobserved454-sequences27.Thecomparisonbetweenthebacterial much of the known Pseudonocardia diversity, including repre-
assignments derived from the two databases revealed comparable sentativesfrom7ofthe10knownPseudonocardiaclades(Supple-
resultsforthecommonbacteriafoundinantsamples.Specifically, mentaryTableS5;Fig.2from22).Eightypercentofthenests(n525)
SolirubrobacterandPseudonocardiahad98%and93%identicalhits hadmorethanonePseudonocardiastrainonworkerspernest,with
frombothreferencedatabases.Thesoilandgardensamplesshowed anaverageof2.9strainsonworkersfromthesamenest(hereastrain
lesssimilaritybetweenthetwoBLASTdatabases,suggestingcaution referstoauniquePseudonocardiasequence;Table2;seeMethods).
in the interpretation of the relative bacterial abundances in these However, most of the strains were rare with the exception of one
samples. A sub-set of the RDC results is presented in Supple- predominant Pseudonocardia strain from clade 3 (the so-called P.
mentaryTableS4. nitrificans/alni/carboxydivorans clade22), which received 99.7% of
the Pseudonocardia BLAST hits (Supplementary Table S5). Soils
Phenologyofant-associatedbacterialmicrobiomes.Theculture- contained a greater diversity of Pseudonocardia strains (chamber
independent screen captured the bacterial microbiomes of T. soil and excavated soil revealed, respectively, 45 and 44 strains,
septentrionalis nests from late winter to late summer, covering whichwereprimarilyfromclades 6–10;Supplementary TableS5).
reactivation of the fungal gardens in spring, emergence of repro- Compared to allBLAST results, Pseudonocardia was found at low
ductivesinearlysummer,andtheperiodofpost-matingflightsin percentsequencereadsinthesoils,comprisingonly1.33%(chamber
late summer. Outside and garden workers showed few changes in soil)and1.05%(excavatedsoil)ofthetotalsequencesobtained.
theirbacterialprofilesfromwintertosummer,withtheexceptionof
Spiroplasmabacteria,whichwereobservedonlyinMarchandJune Discussion
(Fig. 2). However, in late summer (30-Aug) the percent sequence Thisstudyisthefirsttocomparenaturalmicrobiomesoffield-collected
reads of Pseudonocardia increased in both worker types, but fungus-growing ants using both culture-dependent and culture-
especially in garden workers (from 5% in July to 34% in August). independent methods. We find that the ant- and garden-associated
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samples(outsideworkers,accordingtotheassignedcyofassociationbetween Male 12.05%/.06(2/3)12.02%/.04(1/3)120.83%/1.44(1/3)121.16%/1.89(2/3)120.36%/.62(1/3)12/.44(1/3).25%0%120.05%/0.10(1/3)127.35%/21.73(3/3)122.19%/19.26(3/3)120.03%/0.06(1/3)120.08%/0.09(2/3)124.80%/8.31(1/3)120.08%/0.14(1/3)120.68%/1.18(1/3)129.95%/14.25(3/3)0%0%0%
ntn)en 11
aost
T.septentrionalisstandarddeviatimeasureofconsiened. uctiveFemale 12/5.03(8/9)12/8.93(8/9)0%12/0.82(6/9)12/4.07(9/9)12/0.21(4/9)12/5.55(7/9)12/.96(6/9)12/29.00(9/9)12/11.92(9/9)12/1.28(8/9)210.08(2/9)/12/5.30(4/9)12/6.62(2/9)12/2.35(7/9)12/0.92(6/9)0%0%12/0.03(2/9)
dent16S-amplicon454-sequencingin12ence-readsperantsampletype(/ced-genusBLAST-identification).Asaenuspertotalnumberofsamplesscre OutsideWorkerReprod 128.24%/5.01(25/25)3.94%127.15%/3.15(25/25)10.75%120.17%/.35(9/25)120.62%/1.45(13/25)0.81%120.10%/4.23(25/25)4.00%121.46%/2.65(22/25)0.12%128.14%/4.09(25/25)4.58%121.29%/0.85(25/25)0.93%123.52%/2.55(25/25)41.99%129.21%/10.17(25/25)15.47%122.95%/2.01(25/25)1.06%121.46%/7.11(10/25)0.04%120.14%/0.40(9/25)2.54%120.08%/.14%(12/25)3.33%128.32%/5.35(25/25)2.05%120.12%/0.16(20/25)0.52%121.49%/1.91(17/25)121.54%/6.96(3/25)127.28%/19.83(6/25)0.01%
generaestimatedbyculture-indepenpresentstheaveragepercentsequTableS2forassignmentsintheforontainingtherespectivebacterialg GardenWorker 126.37%/6.05(24/25)127.42%/4.06(25/25)121.90%/7.00(12/25)121.05%/3.38(13/25)1210.76%/4.62(25/25)1121.23%/2.49(21/25)126.82%/3.43(24/25)121.72%/1.44(25/25)127.05%/17.15(25/25)1230.93%/11.08(25/25)2122.66%/1.8(25/25)120.11%/0.27(10/25)120.24%/0.44(14/25)120.08%/0.17(11/25)128.69%/5.70(25/25)120.18%/0.35(15/25)121.35%/1.91(19/25)121.80%/6.32(2/25)21/9.75(4/25)2.59%
ofcommonbacterialales).TheinformationT(seeSupplementarynumberofsamplesc Class ActinobacteriaActinobacteriaActinobacteriaActinobacteriaActinobacteriaActinobacteriaActinobacteriaActinobacteriaActinobacteriaActinobacteriaActinobacteriaBacilliBacteroidiaBetaproteobacteriaBetaproteobacteriaGammaproteobacteriaGammaproteobacteriaMollicutesMollicutes
dsmAShe
ercentsequence-reamalereproductives,heforced-genusBLnparenthesesgivet Order ActinomycetalesActinomycetalesActinomycetalesActinomycetalesActinomycetalesActinomycetalesActinomycetalesActinomycetalesActinomycetalesSolirubrobacteralesActinomycetalesBacillalesBacteroidalesBurkholderialesBurkholderialesPseudomonadalesXanthomonadalesEntomoplasmatalesEntomoplasmatales
Table4|Averagepgardenworkers,fereferencegenusintsamples,numbersi Genus AeromicrobiumAgrococcusCorynebacteriumDermacoccusMicrolunatusNocardiodesPhycicoccusPonticoccusPseudonocardiaSolirubrobacterunknowngenusBacillusBacteriodesBurkholderiaDerxiaPseudomonasXanthomonasMesoplasmaSpiroplasma
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Figure2|PhenologyofcommonbacterialgeneraassociatedwithT.septentrionalisgardenworkers,outsideworkers,andreproductiveantsas
determinedby16S-amplicon454-sequencing.Percentsequence-readsforthecommonbacteriafortheantcasteswereaveragedbymonth.Reproductive
malesarenotincludedinthisfigurebecausemaleswereonlycollectedinJune.Arrowsindicatethemonthofimportantlifehistorystagesintheannual
reproductivecycleofT.septentrionalis.Gardenandoutsideworkersshowedrelativelyconstantbacterial-communityprofiles,exceptforincreasesof
SpiroplasmainMarchandPseudonocardiainAugust.Reproductivefemalesshowedinitially(June)ahighpercentageofsequence-readsfor
Pseudonocardia,butpercentageofPseudonocardiasequence-readdecreasedinfemalesthathadnotdispersedfromthenestbylateAugust.
microbiomes of T. septentrionalis ants are complex communities Pseudonocardiaspeciesonworkersofasingleantnest,corroborate
of bacteria, including most prominently the actinobacterial genera for field colonies the finding of complex integumental biofilm
Solirubrobacter, Microlunatus, Pseudonococardia, Aeromicrobium, communities that were first reported for laboratory colonies of T.
Phycicoccus, and Agrococcus (Table 4; Supplementary Table S2). septentrionalis5 and that have recently been documented for other
The consistent association of diverse actinobacterial bacteria with attine ants14,18. The presence of multiple actinobacterial lineages
T. septentrionalis, as well as the consistent association of multiple suggests that either the ants are readily colonized by a far greater
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diversity of transient, commensal actinobacteria than previously accidental(e.g.,inertintegumentalaccretionsmayaccidentallyaccu-
realized12,15,28,orthattheantsaccumulatecomplexbiofilmsthatmay mulate autotrophic microbes) or may serve one or several of the
include actinobacteria with beneficial complementary or synergistic functionsmentionedabove.Nevertheless,thedocumentedprevalence
properties2,5,11,14,16,18,20,29,30. ofP.cf.carboxydivoransinboththeculture-dependentandculture-
AnunweightedUnifracanalysisrevealedthatdifferentantcastes independent screens suggest that P. cf. carboxydivorans likely oc-
carrysimilarbacterialcommunities(Figure1),exceptthattheper- cupies an important ecological role in the microbial ecology of T.
centageofPseudonocardiasequencereadsonreproductivefemales septentrionalis.
early in the mating season appears to exceed the corresponding Pseudonocardiacanbereadilyisolatedfromtheheadandmeta-
Pseudonocardia percentages on nestmate workers collected at the somaoftheantsinadditiontothemesosoma(propleuralthoracic
sametime(Figure2).However,thisdifferenceinpercentsequence plates; Table 1). This finding contradicts the current belief that
reads may be confounded by the difference in body size between Pseudonocardia occurs primarily on the mesosoma of attine ants
castes and by the different number of individuals pooled in each andthatPseudonocardiabiomassonantscorrelateswiththeamount
caste sample. Moreover, 454-sequencing-read abundance patterns of whitish integumental accretion that is visible on the propleural
needtobeinterpretedwithcautionbecauseofpossibleamplification thoracic plates in tropical Trachymyrmex13. The occurrence of
biases31. Pseudonocardia on multiple body segments (Table 1) could imply
Whilebetween-castecomparisonsarecomplicatedbysizediffer- that the extent of visible, whitish accretions on the integument
encesandpossibleamplificationbiases,bacterialcomparisonswithin of attine ants therefore may not be a reliable indicator of Pseu-
acasteatdifferenttimepointscouldbelesssubjecttosuchcomplica- donocardiaabundance.
tions.Forexample,femalereproductivescollectedinJuneshoweda Of all Pseudonocardia species identified, P. cf. carboxydivorans
high percentage of Pseudonocardia sequence reads, but sequence was the dominant species found in culture-dependent isolations
percentagesdeclinedduringthenexttwomonths(Figure2).Bylate (99.5%) and culture-independent screens (99.7%), consistent with
summer, female reproductives that failed to leave the nest (likely recentreports15,22thatP.cf.carboxydivoransandcloselyrelativesare
becauseofaseveredroughtin2009thatreducedopportunitiesfor mostfrequentlyisolatedfromTrachymyrmexandAcromyrmexants
mating flights) had shed their wings. These dealate females had inthetropics.SequencereadsassignedtoP.cf.carboxydivoranswith
bacterial read-abundances similar to outside and garden workers. 100% sequence identity could be further subcategorized into four
The seasonal change in percent sequence reads suggests either a distinct morphotypes (Supplementary Fig. S1). All four of these
decrease of Pseudonocardia on the integument of dealate females, P. cf. carboxydivorans morphotypes occurred on T. septentrionalis
orincreasedprevalenceofothermicrobialtypesontheintegument, workers. In some cases, we observed the presence of two distinct
orboth. P. cf. carboxydivorans morphotypes on ants from the same nest
Throughouttheseasons,bothgardenandoutsideworkersshowed (Table2),suggestingcrypticdiversityontheantintegumentthatis
constant proportions of sequence reads for the common bacterial notdistinguishedby16Ssequencing.Becausethiscrypticdiversity
genera,withtheexceptionofanincreaseofPseudonocardiasequence mayrepresentbiochemicalvariantsarisingthroughmutationwithin
readsinlateAugust.Thislate-seasonincreasewasmorepronounced the nest, it will be important in future screens to isolate multiple
in garden workers than outside workers (Figure 2). Future studies strains(perhapsdozensorhundredsstrains)tocharacterizethetrue
could test whether a caste-specific late-season Pseudonocardia in- biochemical and metabolic diversity of Pseudonocardia associates,
crease could be related to the accumulation of specific diseases or contraryto theprevailing beliefthat identificationofoneorafew
enemies(e.g.,mites,predators)thatmayincreasinglyimpacttheants isolates adequately characterizes the Pseudonocardia associates
ortheirgardensinlatesummer. of an ant nest10,15. Moreover, it appears that the diversity of
Ifcomponentsofthemicrobiomesaremutualistsoftheanthost, Pseudonocardiacoexistingonasingleantworkerdoesnotbecome
asislikelytruefortropicalattineants3,5,18,29,32,33,theseputative,bene- apparent until microbes are subcultured and allowed to grow for
ficialmicrobescouldservedistinctfunctionsindifferentantcastesof sufficient time. Specifically, the morphologically homogenous
T. septentrionalis. We observed high sequence reads of Pseudono- growth on chitin plates [e.g., Electronic supplementary material
cardiain male and female reproductives in June, whereas workers S1b-d in15] appears to be insufficient evidence that only one
hadseveralcommonactinobacteriainadditiontoPseudonocardia; Pseudonocardia type grows on the ants. In fact, we found in our
this could implicate functional differences of bacterial mutualists isolations that bacteria from several actinomycete genera can be
betweencastes,orreflectdifferentialbacterialacquisitionresulting intermixedindeceptively‘‘homogenous’’cultures.
frombehavioraldifferences.Bacterialassociatescouldpotentiallybe Theculture-independent16S-sequencingfoundotherPseudono-
used by male and female reproductives for antibiotic protection cardiastrainsbesidesP.cf.carboxydivoransassociatedwithT.sep-
againstentomopathogenicdiseases,orinthecaseoffemales,protec- tentrionalisants.Onaverage, gardenworkersandoutside workers
tionofthenestenvironmentorthegardenafternestestablishment. from the same nest carried one P. cf. carboxydivorans strain and
In contrast, garden and outside workers may accumulate a more twoadditionalPseudonocardiastrainspernest(Table2).Although
diversified array of bacteria for protection against a diverse set of these additional Pseudonocardia strains were rare (as judged by
harmful microbes encountered during foraging and gardening. sequence abundance in the 454-screens), these Pseudonocardia
Overall, the roles of any beneficial microbes within the nest could strainsspannedessentiallythefulldiversitywithinthegenusPseu-
be diverse, including suppression of ant diseases34–37; general donocardia[SupplementaryTableS522].Thissuggeststhatdifferent
sanitation of the nest environment (e.g., chamber walls); suppres- Pseudonocardiastrainscouldcompetewitheachotherforresources,
sion of mites, nematodes, or thieving fungivores in a nest38; or perhaps evolving features that confer competitive advantages but
detoxification39. thataresimultaneouslydetrimentaltotheanthosts5,40.
BecausegardensofthetemperateattineantT.septentrionalisare Finally,wefoundagreatdiversityofPseudonocardiastrainsinthe
rarelyorneverinfectedwiththepathogenEscovopsis,theactinobac- soilsamples,butasalowpercentageofoverall454-sequencereads(45
teria(e.g.,Pseudonocardia)carriedbyT.septentrionalisareunlikelyto differentPseudonocardiastrainsamong2,067Pseudonocardiasequences
representaspecificdefenseagainstEscovopsis.Thissuggeststhateither from 202,908 total sequences characterized for soil). Detection of
ant-associatedPseudonocardiacanoccupyrolesdifferentthanspecific Pseudonocardia in soil samples couldbe more difficult than inant
defense against Escovopsis, or that the integumental Pseudonocardia samples because soil samples may be more difficult to extract,
may serve no beneficial function (at least in some attine ant popu- contain more PCR inhibitors, or facilitate primer competition41.
lations). High Pseudonocardia loads on T. septentrionalis may be Pseudonocardia were more difficult to detect in culture-dependent
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screensofsoilbecause,unliketheplatesforantandgardenisolation, the finding of cryptic Pseudonocardia diversity (i.e., several P. cf.
soil-isolationplatesweremorereadilyovergrownwithfungi.Thus, carboxydivorans morphotypes coexisting on workers of the same
estimatesofPseudonocardiaprevalenceinsoilandantnestscannot nest)callsformorecarefulcharacterizationoftheant-andgarden-
be directly compared in our culture-dependent screens. The 45 associatedmicrobiomesinfuturestudies,andforcontinuedrethink-
strains from the culture-independent analysis covered the full ing of the interaction complexity inherent in such diversity [e.g.,
knownPseudonocardiadiversity,butthemajorityofPseudonocardia possible competition between bacteria occupying the same niche
from soil belonged to clades 6–10 as defined by22 (Supplementary ontheantintegument5,40,43].
TableS5).Only16sequencesfromthesoilsamplesbelongedtoclade Complexinteractionswithinhost-associatedmicrobiomesdonot
3(theP.cf.carboxydivoransclade),suggestingthateithercoloniza- necessarily rule out overall beneficial contributions of microbes to
tion of the ant integument by Pseudonocardia from soil is highly hostfitness18,43,butresearchintosuchcomplexassociationsfacesa
selective [i.e., symbiont-acquisition is highly selective; sensu18,42,43], seriesofchallenges:therolesofsymbioticmicrobesarelikelydiverse
or soil is an unlikely source for environmental acquisition of andcontext-dependent;therolescanchangerapidlyoverevolution-
Pseudonocardia. Instead, plant material, which frequently contains ary time; strongly interacting host-microbe associations may be
endophytic Pseudonocardia closely related to P. cf. carboxydivor- difficulttodistinguishfromunimportanttransientassociations;sig-
ans22, maybe a more likelyexternalsourceof Pseudonocardia col- nificantcontributionofaparticularmicrobetohostfitnesscannot
onizingtheantintegument. readilybeinferredfrommicrobeabundance;andstrongly-interacting
Overall, bacterial communities of ants and soil are easily differ- hostsandmicrobesoftenlackthefootprintsofevolutionarymodi-
entiatedfromeachother(Fig.1,SupplementaryFig.S3).Bacterial ficationthatdocumentco-evolutionaryinterplay.Apluralisticcon-
communities of gardens showed some similarity with soil com- ceptualapproachacknowledgingdiversefunctionsofmicrobiomesis
munities (Fig. 1), possibly reflecting the fact that ants forage for thereforemostlikelytoadvanceunderstandingoftheattine-associated
gardensubstrateontheground.Duringoursamplecollections,gar- microbes37.
densweremoredifficulttocollectthantheants,andthereforewe
cannotcompletelyruleoutoccasionalcontaminationofgardensby
Methods
accidental soil fallout during excavation (such contamination may
SampleCollection.EverymonthbetweenJanuaryandSeptember2009,wesearched
alsoapplytothesinglemalewhosebacterialcommunityclustered
forT.septentrionalisnestsalonga200metertrailsegmentatStengl‘‘LostPines’’
amonggardensandsoil;Fig.1). BiologicalStation,Smithville,BastropCounty,Texas(GPSN30.086,W97.168,
454-sequencing revealed some previously unknown and poten- elevation:145m;seeSupplementaryMethods).Wecollectedants,fungalgardens,soil
tially important bacterial associates such as Solirubrobacter (order fromthenestchamber,andworker-excavatedsoilfromfourT.septentrionalisnests
everymonth,exceptinJanuary(2nests),February(1nest),andSeptember(3nests).
Solirubrobacterales),whichcomprisedapproximatelyathirdofthe
Allsampleswerecollectedintotwosterilevials,onevialwith1mLof100%ethanolfor
totalsequencereadsfromtheantsamples.Solirubrobacterhasprev- culture-independent454-pyrosequencinganalysesofbacterialcommunity
iouslybeenreportedinagriculturalsoils,soilcrust,andearthworm composition,andasecondvialwithsalinebuffersolutionforculture-dependent
burrows44–46,butthebiologyofthesebacteriaisotherwiseunknown. isolationofactinomycetebacteria.
A prior study also found Solirubrobacter in one lab colony of T. Wecollectedfromallnestsso-called‘‘outsideworkers’’(exitingorincoming
extranidalantscollectedfromthenestmoundbeforenestexcavation),‘‘garden
septentrionalis5. Independent screening of the internal (e.g., gut)
workers’’(intranidalantscollectedfromthesurfaceofthegarden),and,whenpresent
andexternalmicrobiomeswillbeneededtoidentifytheexactloca- (June–August),reproductivefemalesandmales.Thegreatmajorityoffemale
tion of Solirubrobacter associated with T. septentrionalis ants. In reproductiveswerecollectedaswingedindividualsinthenest(pre-matingflights),
addition, future culture-dependent surveys of attine integumental butafewfemalesencounteredlateinthematingseason(August)hadshedtheirwings
insidetheirnatalnests.Weassumedthatsuchdealatefemalesfoundlateintheseason
microbiomes need to improve isolation methods, which failed so
hadfailedtoleavethenestforamatingflight,andwereintheprocessofgradually
far to reveal the abundant presence of Solirubrobacter and Micro- assumingaworker-likerole;suchcastetransitionhasbeenobservedinanumberof
lunatusonanyattineantusingaminimum-carbonchitinmedium attinespecies38.Whenpossible,fiveantswerecollectedforeachworkersample,butin
forscreening(Table3). somecasesfewerantshadtobecollectedbecausecoloniesweretoosmall,orshowed
littleornoforagingactivityatthetimeofcollecting.Samplesofreproductivefemales
Additional bacterial associates of the ants were Bacillus,
ormaleswerepooledwithonetothreeindividualspervial.Afewlivegardenworkers,
Burkholderia, Corynebacterium, Mesoplasma, and Spiroplasma,
outsideworkers,males,andfemalereproductiveswerealsocollectedinempty,sterile
whichweredetectedasahighpercentageofsequencereadsonlyin vialsandanalyzedforthebody-segment-specificpresenceofactinomycetebacteria
afewantsamples.Thissuggeststhatthesebacteriamaybepathogens usingculture-dependentisolation(i.e.,targetingbacteriaonthehead,mesosoma,or
thatinfectonlytheoddant,orthesebacteriaareacquiredbyants metasoma).
Eachnestwasexcavatedbyfirstdigginga40–50cmdeepholeat,15cmdistance
onlyunderuniquecircumstances,suchasauniqueforaginghistory.
fromtheentrancetunnel,andthencarefullyexcavatinglaterallyuntilagarden
Both Spiroplasma and the closely related Mesoplasma are known chamberwasfound.Gardenchamberswerecarefullyaccessedfromthesideto
insect pathogens, but thefrequency ofSpiroplasma in ants is rela- minimizefalloutofsoilthatwouldcontaminatethegarden,butsubtle,inadvertent
tivelyunknown47–50,whereasMesoplasmaandotherEntoplasmatales contaminationwithsoilmayhaveoccurredinsomegardensamples.A,1cm3
fragmentofuncontaminatedgardenwascollectedfromthechamberandplaced
bacteriahavebeenfoundindiverseantlineages51.Futureresearch
immediatelyintovialswithethanolorbuffer.Additionally,,5mgofsoilwascol-
shouldelucidatetherolesofthesebacterialassociatesinthebiology lectedfromthechamberwallbyscrapinga1–2mmthicklayeroffthewall(chamber
ofT.septentrionalis. soil),and,5mgfreshlydepositedsoilexcavatewascollectedfromtheedgeofthe
While culture-dependent methods capture some of the overall nestmoundwheresuchexcavatehadbeendepositedbyworkers(excavatedsoil).All
soilandgardensampleswerecollectedwithflame-sterilizedspatulasandforceps,
actinobacterialdiversityassociatedwithT.septentrionalis,ourstudy
whichwereallowedtocoolbeforeeachcollection.Aftercollection,thechamberswere
showsthatthetraditionalminimum-carbonchitinmediumappears
carefullyclosedandtheexcavatedholewasrefilledwithsoilsothatthenestwould
insufficient to adequately characterize dominant actinobacterial survive.Forcomparison,wealsoincludedfoursamplesofnon-attineworkers
associates(e.g.,Solirubrobacter,Microlunatus)andfavorgrowthof (Pheidolesp.)collectedabout50metersdistantfromtheT.septentrionaliscollection
specific bacteria (e.g., Pseudonocardia, Amycolatopsis, Kribbella; site(n53samplesofPheidoleincluding3–5workers,July)ornexttoaT.septen-
trionalisnest(n51samplesof3Pheidoleworkers,August).
Table3).Theobserveddifferencesbetweenculture-dependentand
ThesamplescollectedinJanuaryandSeptember2009wereanalyzedonlywith
culture-independentscreensareundoubtedlyduetoisolationbiases culture-dependentmethods,butallothersampleswereanalyzedwithbothculture-
known for culture-dependent methods5,22,26. While 16S-amplicon dependentandculture-independentmethods.Samplesinsalinebuffersolutionand
454-sequencingreflectsmoreaccuratelythetotalbacterialcommun- liveantsfromthefieldwereprocessedviaculture-dependentmethodsinthelabon
itycomposition,greatersequencingdepthsareneededtocharacter- thesamecollectionday.Ethanol-preservedsampleswerestoredat280uCand
shippedattheendofthestudyasasinglebatchtotheBiofilmsInstituteinLubbock,
izethediversityofrarebacterialassociates.Moreover,thefindingof
TXfor454-pyrosequencingof16SrDNAampliconsfrombacteria,followingthe
severalPseudonocardiaspeciesonworkersofthesameantnest,and methodsof5,47,52.
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