Research Article Bacterial Infections across the Ants...

12
Hindawi Publishing Corporation Psyche Volume 2013, Article ID 936341, 11 pages http://dx.doi.org/10.1155/2013/936341 Research Article Bacterial Infections across the Ants: Frequency and Prevalence of Wolbachia, Spiroplasma, and Asaia Stefanie Kautz, 1 Benjamin E. R. Rubin, 1,2 and Corrie S. Moreau 1 1 Department of Zoology, Field Museum of Natural History, 1400 South Lake Shore Drive, Chicago, IL 60605, USA 2 Committee on Evolutionary Biology, University of Chicago, 1025 East 57th Street, Chicago, IL 60637, USA Correspondence should be addressed to Stefanie Kautz; skautz@fieldmuseum.org Received 21 February 2013; Accepted 30 May 2013 Academic Editor: David P. Hughes Copyright © 2013 Stefanie Kautz et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Bacterial endosymbionts are common across insects, but we oſten lack a deeper knowledge of their prevalence across most organisms. Next-generation sequencing approaches can characterize bacterial diversity associated with a host and at the same time facilitate the fast and simultaneous screening of infectious bacteria. In this study, we used 16S rRNA tag encoded amplicon pyrosequencing to survey bacterial communities of 310 samples representing 221 individuals, 176 colonies and 95 species of ants. We found three distinct endosymbiont groups—Wolbachia (Alphaproteobacteria: Rickettsiales), Spiroplasma (Firmicutes: Entomoplasmatales), and relatives of Asaia (Alphaproteobacteria: Rhodospirillales)—at different infection frequencies (at the ant species level: 22.1%, 28.4%, and 14.7%, resp.) and relative abundances within bacterial communities (1.0%–99.9%). Spiroplasma was particularly enriched in the ant genus Polyrhachis, while Asaia relatives were most prevalent in arboreal ants of the genus Pseudomyrmex. While Wolbachia and Spiroplasma have been surveyed in ants before, Asaia, an acetic acid bacterium capable of fixing atmospheric nitrogen, has received much less attention. Due to sporadic prevalence across all ant taxa investigated, we hypothesize facultative associations for all three bacterial genera. Infection patterns are discussed in relation to potential adaptation of specific bacteria in certain ant groups. 1. Introduction Recent studies have shown that insects are associated with a broad range of unrelated microbial taxa [1, 2]. ese interac- tions shape the ecology and evolution of hosts and bacterial symbionts and oſten heavily impact host biology [3, 4]. Congruent evolutionary histories between some symbiotic partners show the likely obligate nature of this relationship [5], while other associations occur sporadically and can vary both spatially and temporally [6]. Bacterial endosymbionts sometimes inhabit specialized host cells or structures [7, 8] and might even share metabolic pathways with their hosts [9], while others occur loosely in unspecific tissues or hemolymph [10]. Microbes associated with insects are extremely diverse and span-wide taxonomic groups, even within individual hosts. One of the best-characterized endosymbiont groups is comprised of insect-associated bacteria that increase the nutritive value of their hosts’ diets. ese bacteria are oſten highly specialized and coevolved associates, playing partic- ularly important roles in insects with nutritionally limited or deficient diets. Some well-known examples of such endosym- bionts include Buchnera aphidicola in aphids, which provide their hosts with essential amino acids lacking in the sugar- rich but nitrogen-poor phloem sap [11]. Other examples are the cospeciated and essential amino acid synthesizing Blochmannia endosymbionts of Camponotini ants [12, 13], nitrogen fixing taxa in the fungal gardens of the leaf-cutter ants [14], Wigglesworthia glossinidia, which provides vitamins that are lacking in the blood meals of its host, the tsetse fly [15], and the nitrogen-fixing microflora of termites [16, 17]. In ants, several recent studies have highlighted the importance of bacterial symbionts for nutrition, especially in ant taxa feeding low on the trophic scale [18–20]. Symbiotic bacteria can also play other beneficial roles by protecting insects from parasites and pathogens and thus

Transcript of Research Article Bacterial Infections across the Ants...

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Hindawi Publishing CorporationPsycheVolume 2013 Article ID 936341 11 pageshttpdxdoiorg1011552013936341

Research ArticleBacterial Infections across the Ants Frequency andPrevalence of Wolbachia Spiroplasma and Asaia

Stefanie Kautz1 Benjamin E R Rubin12 and Corrie S Moreau1

1 Department of Zoology Field Museum of Natural History 1400 South Lake Shore Drive Chicago IL 60605 USA2Committee on Evolutionary Biology University of Chicago 1025 East 57th Street Chicago IL 60637 USA

Correspondence should be addressed to Stefanie Kautz skautzfieldmuseumorg

Received 21 February 2013 Accepted 30 May 2013

Academic Editor David P Hughes

Copyright copy 2013 Stefanie Kautz et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Bacterial endosymbionts are common across insects but we often lack a deeper knowledge of their prevalence across mostorganisms Next-generation sequencing approaches can characterize bacterial diversity associated with a host and at the sametime facilitate the fast and simultaneous screening of infectious bacteria In this study we used 16S rRNA tag encoded ampliconpyrosequencing to survey bacterial communities of 310 samples representing 221 individuals 176 colonies and 95 species ofants We found three distinct endosymbiont groupsmdashWolbachia (Alphaproteobacteria Rickettsiales) Spiroplasma (FirmicutesEntomoplasmatales) and relatives of Asaia (Alphaproteobacteria Rhodospirillales)mdashat different infection frequencies (at the antspecies level 221 284 and 147 resp) and relative abundances within bacterial communities (10ndash999) Spiroplasmawas particularly enriched in the ant genus Polyrhachis while Asaia relatives were most prevalent in arboreal ants of the genusPseudomyrmex While Wolbachia and Spiroplasma have been surveyed in ants before Asaia an acetic acid bacterium capableof fixing atmospheric nitrogen has received much less attention Due to sporadic prevalence across all ant taxa investigated wehypothesize facultative associations for all three bacterial genera Infection patterns are discussed in relation to potential adaptationof specific bacteria in certain ant groups

1 Introduction

Recent studies have shown that insects are associated with abroad range of unrelated microbial taxa [1 2] These interac-tions shape the ecology and evolution of hosts and bacterialsymbionts and often heavily impact host biology [3 4]Congruent evolutionary histories between some symbioticpartners show the likely obligate nature of this relationship[5] while other associations occur sporadically and can varyboth spatially and temporally [6] Bacterial endosymbiontssometimes inhabit specialized host cells or structures [78] and might even share metabolic pathways with theirhosts [9] while others occur loosely in unspecific tissues orhemolymph [10]

Microbes associated with insects are extremely diverseand span-wide taxonomic groups even within individualhosts One of the best-characterized endosymbiont groupsis comprised of insect-associated bacteria that increase the

nutritive value of their hostsrsquo diets These bacteria are oftenhighly specialized and coevolved associates playing partic-ularly important roles in insects with nutritionally limited ordeficient diets Somewell-known examples of such endosym-bionts include Buchnera aphidicola in aphids which providetheir hosts with essential amino acids lacking in the sugar-rich but nitrogen-poor phloem sap [11] Other examplesare the cospeciated and essential amino acid synthesizingBlochmannia endosymbionts of Camponotini ants [12 13]nitrogen fixing taxa in the fungal gardens of the leaf-cutterants [14]Wigglesworthia glossinidiawhich provides vitaminsthat are lacking in the blood meals of its host the tsetse fly[15] and the nitrogen-fixingmicroflora of termites [16 17] Inants several recent studies have highlighted the importanceof bacterial symbionts for nutrition especially in ant taxafeeding low on the trophic scale [18ndash20]

Symbiotic bacteria can also play other beneficial rolesby protecting insects from parasites and pathogens and thus

2 Psyche

defending their hosts against natural enemies [4 7 21]For example Spiroplasma can convey increased resistance tonematode infections in Drosophila flies [22] and secondarysymbionts in aphids can confer resistance to parasitic wasps[23] Some insect-associated bacteria also contribute to nesthygiene [7] For example actinomycetes in the fungal gardensof leaf-cutter ants inhibit the growth of fungal pathogens butnot of mutualistic fungi [24] Actinomycetes are also foundin antennal glands of bee wolves and protect larvae in theirnests against infestation by pathogens [25] Othermutualisticbacteria can increase host tolerance to unfavorable abioticconditions such as temperature stress [26] or facilitate the useof novel hosts [27]

While the associations described above are typicallybeneficial to hosts many bacterial endosymbionts are detri-mental reproductive manipulators Wolbachia for examplecan cause cytoplasmic incompatibility parthenogenesismalekilling and male feminization [28] There are also exam-ples of Wolbachia which protect their host against RNAviruses thus acting as defensivemutualists [29] An estimated66 of insect species and about 30 of ant species havebeen reported to be facultatively infected with Wolbachia[30 31] Other less prevalent reproductive manipulators ininsects include Cardinium Arsenophonus and Spiroplasma[32 33] Spiroplasma although beneficial to hosts in somecases [22] can have various negative effects on their insecthosts including manipulation of sex ratios male killing andentomopathenogenicity [33ndash35]

Despite these fascinating findings our knowledge ofbacterial symbionts is based on a relatively small numberof organisms Thus we still know little about the identitiesand ecological or physiological functions of bacteria associ-ated with most animal groups [36] In-depth analyses andextensive surveys of the bacterial communities present in awide range of eukaryotic taxa are required to understandthe diversity and the function of microbial symbionts [37]Here we analyzed bacterial communities across the ants(Hymenoptera Formicidae) using 16S rRNA tag encodedamplicon pyrosequencing (454 pyrosequencing) to survey forinfection patterns with potential parasitic microbes Due totheir sporadic prevalence and unknown effects on host antbiology we refer to these microbes as infections In total wescreened 310 ant samples of 176 colonies from 95 ant speciesand encountered high prevalence of three bacterial groupsWolbachia Spiroplasma and Asaia

2 Materials and Methods

A total of 299 ant samples were subjected to 454 pyrose-quencing and combined with data from 11 samples analyzedby Ishak et al [38] that is for a total of 310 samples Allsamples represented 176 different colonies and 95 differentant species belonging to the generaCamponotus (Formicinae1 species) Cephalotes (Myrmicinae 7 species) Cremato-gaster (Myrmicinae 6 species) Myrmecia (Myrmeciinae2 species) Myrmecocystus (Formicinae 1 species) Oeco-phylla (Formicinae 1 species) Paraponera (Paraponerinae 1species)Polyrhachis (Formicinae 32 species)Pseudomyrmex

(Pseudomyrmecinae 36 species) Solenopsis (Myrmicinae2 species) and Tetraponera (Pseudomyrmecinae 6 species)DNA extractions were either prepared from entire ants orfrom dissected ant parts as described in Kautz et al [39] Acomplete list of samples used for this study can be found inSupplementary Table 1 (see Supplementary material availableonline at httpdxdoiorg1011552013936341)

21 454 Pyrosequencing To screen ant samples for over-all bacterial diversity bacterial tag-encoded FLX ampli-con pyrosequencing was performed by the Research andTesting Laboratory (Lubbock TX USA) as described byDowd et al [40] The 16S rRNA universal eubacterialprimers 28F (51015840-GAGTTTGATCITGGCTCAG) and 519R(51015840-GWATTACCGCGGCKGCTG) were used to amplifyapproximately 500 bp of the variable regions V1ndashV3

22 Bacterial 16S rRNA Data Processing and Analysis All16S rRNA pyrosequencing reads were quality controlled anddenoised using the QIIME v150 implementation of Ampli-conNoise v125 using default parameters [41] Chimeras wereremoved by Perseus a component of the AmpliconNoisepipeline [42] All the remaining reads were then clusteredinto operational taxonomic units (OTUs) at 97 sequencesimilarity using UCLUST [43] We used the longest sequencein a cluster as the representative sequence for that OTUSingletons that is OTUs with only one read in the entiredataset were removed We used the QIIME implementationof the Ribosomal Database Project [44] classifier trainedon the February 4 2011 release of the greengenes database[45] to classify OTUs at the level of bacterial orders Defaultsettings were used including a 08 confidence cutoff forclassifications

Our filtering approach recovered infections withWolbachia (Alphaproteobacteria Rickettsiales) Spiroplasma(Firmicutes Entomoplasmatales) and Asaia (Alpha-proteobacteria Rhodospirillales) All OTUs classified asRickettsiales Entomoplasmatales and Rhodospirillales thataccounted formore than one percent of reads within a samplewere considered as infections by the respective order andincluded in further analyses This cutoff also allowed us tocontrol the relatively high error rate of 454 pyrosequencingWe classified the sequences at the genus level using the RDPclassifier (see Supplementary Table 2 for results) All OTUsused in further analyses have been deposited in GenBank(accessions KF015767-KF015856 Supplementary Table 2)

We downloaded the closest relatives of each OTU fromGenBank Additionally we were interested in retrieving anyother sequence from GenBank of those three orders thatwere associated with ants and insects in general Thus wesearched for sequences using the search keywords ldquo16Srdquoand ldquosymbiontrdquo as well as the name of the respective orderGenBank sequences with 99 identity that were isolatedfrom the same source were considered duplicates and deletedfrom the dataset

23 Phylogenetic Tree Construction Sequences were com-piled and edited using Geneious v536 [46] The alignment

Psyche 3

was generated using the infernal secondary-structure-basedaligner of the ribosomal database project (RDP) [44] Weinferred a maximum likelihood phylogeny of the most com-mon OTUs and their GenBank relatives using the RAxML728 Black Box [47] on the CIPRES web portal [48] Themodel GTR+I+G was employed We then uploaded the mostlikely tree to the iToL website [49] to facilitate graphicalillustration of bacterial source ant subfamily and geographicregion for each sequence Trees with branch length andbootstrap support are provided as supplementary material(Figures S1ndashS3)

3 Results and Discussion

31 Wolbachia (Alphaproteobacteria Rickettsiales) In ourstudy 21 of 95 ant species had at least one individual infectedwithWolbachia (Table 1) Across all 304 samples from whichwe obtained data (Supplementary Table 1) we found 30Wolbachia OTUs Overall with 221 of infected speciesthis is a lower infection rate of Wolbachia across ants thanhas been reported before In an extensive compilation ofexisting data about 286 of ant species carried Wolbachiainfections [31] while a frequency of up to 50 had beenfound previously [50] This discrepancy from our study togeneral trends could be due to several reasons Often a speciesis counted as being infected with Wolbachia when just oneindividual carries this infection However not all individualsof a species or individuals from the same colony need to beinfected Thus discrepancies in infection rate across studiesmight merely be due to natural variation among individ-uals Also there is a strong bias in infection rate amongdifferent ant groups Species from the genera AcromyrmexFormica Solenopsis and Tetraponera are often infected withWolbachia while Dolichoderus and Leptogenys mostly lackinfection [51] For example in a screening of 24 Polyrhachisspecies 5 (208) were infected with Wolbachia [31] In thepresent study we found the genera Cephalotes (57) andSolenopsis (50) to have particularly high infection ratesTetraponera (333) and Polyrhachis (250) with intermedi-ate rates Crematogaster (167) and Pseudomyrmex (139)with rather low rates and no infections in the samplesof Camponotus Myrmecia Myrmecocystus Oecophylla andParaponera included here

Most studies that screen for Wolbachia use diagnos-tic approaches by conducting PCR with Wolbachia-specificprimers This is the most reliable means of Wolbachia detec-tion [51]However evenwhen using diagnostic PCR negativeresults can occur due to variations in the primer sequenceor low titers of the bacterial symbionts [52] In our study wefound high variability inWolbachia titers ranging from 103to 9736 (Supplementary Table 1) We used a 1 relativeabundance within a sample as the cutoff to control error ratesof 454 pyrosequencing which might also have led to lowerdetected infection rates among species

In addition to the 30 Wolbachia sequences obtained inthis study we downloaded sequence data from GenBank andcompiled a dataset of 111 taxa including the outgroup Rhi-zobium leguminosarum (Alpha-proteobacteria Rhizobiales)

The total alignment had a length of 1224 characters Four ant-specific clades of Wolbachia were recovered in the inferredtree (Figure 1 Figure S1) Ant clade 1 comprised Wolbachiathat was isolated from Australian Polyrhachis (6 sequences)as well as one sequence detected in Cephalotes variansfrom the Nearctic Ant clade 2 included mostly AustralianPolyrhachis (9 sequences) in addition to sequences foundin Nearctic Solenopsis and Neotropical Pseudomyrmex Antclade 3 exclusively contained sequences from EuropeanFormica species while ant clade 4 was the most diverseThis fourth clade comprised the majority of ant-associatedWolbachia sequences fromour dataset aswell as existingGen-Bank data and included the ant subfamilies DolichoderinaeEcitoninae Formicinae Myrmicinae Ponerinae and Pseu-domyrmecinae from the Afrotropics Nearctics Neotropicsand Palearctics Overall 68 out of 82 (829) ant-associatedWolbachia sequences clustered in ant-specific clades indi-cating a certain degree of host specialization Even thoughneither ant relatedness (subfamily) nor biogeographic region(continent) was a strong determinant for infection withsimilarWolbachia strains relatedWolbachia seemed to infectrelated hosts from the same geographic region to some extentA rather low degree of host specificity has previously beenreported forWolbachia across ants and butterflies while strictcospeciation between Wolbachia and its hosts has not beenfound [51 53]

Wolbachia are reported to be the most prevalent bacterialsymbionts across insects and ants [31] although infectionswith other bacterial groups were often more frequent inour present study Despite this ubiquity to date no studieshave been able to show the functional role of Wolbachia inants This is due to the difficulty of breeding most speciesof ants in the laboratory and thus we have to restrict ourknowledge to the correlations of Wolbachia infections withspecific host traits Wolbachia most commonly manipulatehost reproduction but in ants no such phenomena are known[51] In Formica truncorum Wolbachia infection leads to areduced production of sexuals although this could be dueto physiological costs rather than direct manipulation [54]However worker production is not affected and it has beensuggested thatWolbachiamight reduce the ability of workersto provide resources to alate development [51] Curing ofWolbachia infection within individuals has been observedwhich seems to be unique to ants but themechanisms behindthis phenomenon are not understood [54] Lastly ants oftenshow exceptionally high levels of coinfection with multipleWolbachia strains adding another layer of complexity to thispoorly understood symbiosis [51] It has been speculatedthat eusociality or haplodiploidy might have an impact onWolbachia infection [50 55] but such mechanisms havenever been confirmed Also there seems to be a weakcorrelation of Wolbachia infection with colony foundingmode as species that found new colonies independently areless frequently infected than species relying on dependentcolony founding [50] Speculations on effects of Wolbachiaon colony-founding behavior and colony structure have oftenbeen made as ants can show exceptional variations in thesetraits ranging from a single queen thatmated once tomultiplequeens andor multiple matings per queen [56ndash58]

4 Psyche

Table 1Wolbachia Spiroplasma and Asaia detected by 454 amplicon pyrosequencing across 310 ant samples

Ant genus and subfamily Speciesscreened

Individualsscreened

Coloniesscreened

Number (and percent) of infected speciesand number of individualscolonies

Wolbachia Spiroplasma Asaia

Camponotus (Formicinae) 1 1 1 0 1 (100)11 0

Cephalotes (Myrmicinae) 7 17 12 4 (571)44

2 (285)63

1 (143)11

Crematogaster (Myrmicinae) 6 6 6 1 (167)11 0 0

Myrmecia (Myrmeciinae) 2 3 3 0 0 0Myrmecocystus (Formicinae) 1 1 1 0 0 0Oecophylla (Formicinae) 1 1 1 0 0 0

Paraponera (Paraponerinae) 1 23 9 0 1 (100)22

1 (100)11

Polyrhachis (Formicinae) 32 64 60 8 (250)1010

15 (469)1515 0

Pseudomyrmex (Pseudomyrmecinae) 36 88 72 5 (139)55

5 (139)55

12 (333)1515

Solenopsis (Myrmicinae) 2 11 5 1 (50)11

2 (100)21 0

Tetraponera (Pseudomyrmecinae) 6 6 6 2 (333)22

1 (167)11 0

Total 95 221 176 21 (221) 27 (284) 14 (147)

32 Spiroplasma (Tenericutes Entomoplasmatales) A totalof 27 (284) ant species were infected with Spiroplasmarelatives (Mollicutes Entomoplasmatales) leading to one ofthe highest frequency estimates of this bacterial group acrossthe ants to date (Table 1) Previously an infection rate of62 across ant species had been reported and the infectionrates of approximately 6 were documented for ColeopteraDiptera Hymenoptera and Lepidoptera in general while231 of spiders (Araneae) carried Spiroplasma symbionts[31] There appears to be a strong bias towards certain groupsof ants that are more often associated with this group ofbacteria [31 59] The ant genus Polyrhachis showed a highinfection rate of 469 (15 of 32 species were infected) Thephenomenon of enriched Spiroplasma symbionts in this antgenus is in line with a study by Russell et al [31] and isparticularly interesting as ants of the tribe Camponotinito which Polyrhachis belong carry obligate Blochmanniaendosymbionts which are housed in specific bacteriocytesand provide essential amino acids to the ant host [12 13]Studying the prevalence of spiroplasmas in more generaof the Camponotini particularly the hyperdiverse genusCamponotus would reveal whether these bacteria are likelyto interact within their hosts Infections per species werehigh in Camponotus (11) Paraponera (11) and Solenopsis(22) However these values are not representative due tothe low number of species included Outside of Polyrhachisinfection rates were moderate in the better sampled generaCephalotes (27) Pseudomyrmex (536) and Tetraponera(16) No infection was detected in CrematogasterMyrmeciaMyrmecocystus and Oecophylla (Table 1) Again sampledspecies numbers were low for these ant genera so infectionfrequency can only be regarded as preliminary

An alignment of 175 taxa and 1311 characters wasgenerated including Selenomonas ruminantium (FirmicutesSelenomonadales) as an outgroup In this molecular phy-logeny three large ant-specific clades of spiroplasmaswere identified ant clade 1 that includes endosymbiontsof Cephalotes Solenopsis Tetraponera Pseudomyrmex andNeivamyrmex ant clade 2 that comprises spiroplasma-associates of the ant genera Polyrhachis Camponotus Pseu-domyrmex and Cephalotes and ant clade 3 which wasdominated by army ants (subfamilies Aenictinae Dorylinaeand Ecitoninae) (Figure 2) Additionally several small cladescontaining only ant-associated spiroplasmas were scatteredthroughout the phylogeny as well as several individual ant-associated OTUs Overall bioregion did not seem to be astrong predictor for relatedness among Spiroplasma sym-bionts (Figure 2 Figure S2)

Clade 3 which is dominated by army ants from theNew and Old World has been identified before [60] Inour analysis GenBank-derived Spiroplasma sequences thatwere isolated from the ant genera Odontomachus and Pachy-condyla also fell into this clade (Figure 2) Army ants arecharacterized by the ldquoarmy ant syndromerdquo of nomadismand group predation [61] Due to their specialized dietand a weak correlation of Entomoplasmatales infection withtrophic position a nutritive symbiosis between army ants andEntomoplasmatales has been suggested [60] Even thoughthis clade of Entomoplasmatales is highly dominated by armyants the association is not obligate as infection rates varywithrespect to species and individuals and the symbionts are notnecessary for host development and reproduction [60] AsEntomoplasmatales are generally absent in eggs and larvaehorizontal transmission is assumed

Psyche 5

Sitophilus oryzae AF035160 (rice weevil)

Form

icoxe

nus p

rova

nche

ri

GQ27

5125

(ant

)Co

lletes

inae

quali

s HM

1129

62 (b

ee)

Colle

tes in

aequ

alis H

M11

3014

(bee

)

Colle

tes in

aequ

alis H

M11

3081

(bee

)

Formica

aquilonia GU592784 (ant)

Formica cinerea GU592774 (ant)

Formica cinerea GU592776 (ant)

Formica cinerea GU592770 (ant)

Formica lagubris GU592789 (ant)

Formica cinerea GU592779 (ant)

Formica cinerea GU592777 (ant)

Formica cinerea GU592778 (ant)Formica lagubris GU592786 (ant)Formica polyctena GU592787 (ant)

Formica cinerea GU592781 (ant)

Formica rufa GU592782 (ant)

Formica cinerea GU592772 (ant)

Formica aquilonia GU592783 (ant)

Formica cinerea GU592771 (ant)

Formica cinerea GU592775 (ant)

Formica cinerea GU592780 (ant)

Formica cinerea GU592769 (ant)

Formica polyctena GU592788 (ant)

Formica aquilonia GU592785 (ant)

Formica

cinere

a GU592773 (ant)Philanthus gibbosu

s HM110630 (b

ee)

Form

ica oc

culta

GQ27

5105

(ant

)

Cardioc

ondy

la em

eryi G

Q2751

42 (a

nt)

Philan

thus g

ibbosu

s HM

1108

03 (b

ee)

Philan

thus g

ibbosu

s HM11

0645

(bee)

Odontoponera transversa GQ275132 (ant)

Phlebotomus papatasi WSU80584 (sand fly)

Anoplolepis gracilipes GQ275135 (ant)

Odontomachus clarus GQ275098 (ant)

Drosicha pinicola AB491204 (scale insect)Pheidologeton sp GQ275133 (ant)

Cimex lectularius AB475126 (bed bug)

Cimex lectularius AB475122 (bed bug)Myrmeleon mobilis DQ068830 (ant lion)

Gnamptogenys sp GQ275114 (ant)

Atta colombica HM559216 (ant)

Formica

wheeleri G

Q275103 (ant)

Heteroponera

microps G

Q275145 (ant)

Megalomyrmex latreillei G

Q275110 (ant)Azteca sp GQ275111 (ant)Myrmelachista sp GQ275113 (ant)

Labidus spininodis G

Q275115 (ant)

Pseudomyrmex tenuis BER112 (ant)

Pseudomyrmex PSW54 SK0066 (ant)

Pseudomyrmex filiformis BER114 (ant)

Tetraponera penzigi TP11 (ant)

Tetraponera penzigi KC137101 (ant)

Tetraponera penzigi KC137114 (ant)

Tetraponera penzigi KC137113 (ant)

Tetraponera penzigi KC137106 (ant)

Tetraponera penzigi KC137107 (ant)

Tetraponera penzigi KC137108 (ant)Tetraponera penzigi KC137109 (ant)

Dorymyrmex elegans GQ275124 (ant)Polyergus breviceps GQ275126 (ant)

Sten

amm

a sne

llingi

GQ27

5108

(ant

)

Cepha

lotes

sp SK

1555

(ant)

Cephalo

tes sp

SK15

31 (a

nt)

Pseudom

yrmex

filiform

is BER11

4 (an

t)

Pseudom

yrmex

tenuis B

ER112 (ant)

Pseu

domyr

mex sa

tanicu

s SK00

58 (a

nt)

Cremato

gaste

r sp

SK12

88 (a

nt)

Ceph

alotes

sp S

K152

1 (an

t)

Polyr

hach

is vi

ndex

GQ

2751

34 (a

nt)

Pseu

dom

yrm

ex ap

ache

GQ

2751

07 (a

nt)

Drosophila simulans N

R 074437 (fly)

Leptomyrm

ex sp GQ

275140 (ant)

Rediviva saetigera HM

111302 (bee)

Muscidifurax uniraptor W

OLPA

RTHA

(wasp)

Tetraponera sp SK1300 (ant)

Tetraponera penzigi KC137118 (ant)Redi

viva

saet

iger

a H

M11

1496

(bee

)

Redi

viva

saeti

gera

HM

1113

73 (b

ee)

Redi

viva

saeti

gera

HM

1114

06 (b

ee)

Dro

soph

ila si

mul

ans A

F468

032

(fly)

Redi

viva

saet

igera

HM

1115

63 (b

ee)

Rediviva saetigera HM

111367 (bee)

Rhytidoponera metallica GQ

275141 (ant)

Rediviva saetigera HM

111289 (bee)

Notoncus sp GQ275137 (ant)

Nysius expressus JQ726767 (stinkbug)

Polyrhachis schenckii CSM1035 (ant)

Polyrhachis tubifera CSM1108 (ant) So

lenop

sis ge

min

ata

alat

e B7

(ant

)

Polyr

hach

is sc

henk

ii CS

M10

35 (a

nt)

Pseu

dom

yrm

ex m

ixtec

us SK

0004

(ant

)Po

lyrha

chis

schoo

pae C

SM10

54 (a

nt)

Polyr

hach

is ru

fifem

ur C

SM07

40 (a

nt)

Poly

rhac

his t

hais

CSM

0746

(ant

)

Polyrhachis rufifemur CSM

0655 (ant)

Polyrhachis senilis CSM0798 (ant)

Polyrhachis sp CSM1019 (ant)

Bemisia tabaci JN

204502 (whitefly)

Kleidocerys resedae JQ726770 (catkin bug)

Polyrhachis schenkii CSM1035 (ant)

Polyrhachis senilis CSM1047 (ant)

Polyrhachis sp CSM1019 (ant)

Cephalotes varians CSM1280 (ant)

Polyrhachis tubifera CSM1108 (ant)

Polyrhachis thais CSM0746 (ant)

Polyrhachis monteithi CSM

1096 (ant)Culex pipiens WPU23709 (mosquito)

Kerria lacca JX669531 (scale insect)

Sitophilus oryzae SOMRRNA (rice weevil)

Rhizobium leguminosarum AB740503

Inner circle ant subfamilyDolichoderinaeEctatomminaeEcitoninaeFormicinae

MyrmicinaePonerinaePseudomyrmecinae

Middle circle bioregion

Afrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Ant clade 1

Ant clade 2A

nt clade 3

Ant c

lade 4

Figure 1 Phylogenetic tree ofWolbachia symbionts associated with ants and their closest relatives with sequence data available in GenBankAmaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shownThe host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) Yellow and red branches representbacteria isolated from ant hosts and other insect hosts respectively The inner circle shows ant subfamily and the outer circle refers tothe continent from which host organisms were collected Four ant-specific clades of Wolbachia symbionts are highlighted (Ant clades 1ndash4)Rhizobium leguminosarum was used as an outgroup

Even outside the army ants a certain degree of hostspecificity of Entomoplasmatales bacteria is evident fromour phylogeny and has been described for ants Drosophilaand other arthropod-associated spiroplasmas [60] In ourmolecular phylogeny clades 1 and 2 exclusively contained

ant-associated Entomoplasmatales (Figure 2) However bothclades contained symbionts from different ant subfamiliesand biogeographic regions indicating that neither phylogenynor geographic range drives the association with these sym-bionts and repeated environmental acquisition is common

6 Psyche

Laelapidae sp JF266582 (mite)

Neriene clathrata EU727102 (spider)

Tetragnatha montana EU

727105 (spider)

Meta m

engei EU727103 (spider)

Meta segm

entata EU727104 (spider)

Hepialus gonggaensis EU

344914 (moth)

Tipula oleracea EU727101 (cranefly)

Acyr

thos

ipho

n pi

sum

AB0

4826

3 (a

phid

)Ha

rmon

ia a

xyrid

is AJ

1324

12 (b

eetle

)

Aniso

sticta

nov

emde

cimpu

ncta

ta A

M08

7471

(bee

tle)

Spiro

plas

ma

sp A

Y837

731

(mos

quito

)

Laod

elpha

x stri

atell

us A

B553

862 (

plan

thop

per)

Spiro

plasm

a ixo

detis

GU5

8567

1

Tetra

nych

us ur

ticae

DQ91

0772

(spi

der m

ite)

Dana

us ch

rysip

pus A

J245

996 (

butte

rfly)

Dros

ophi

la an

anas

sae F

J657

247 (

fly)

Cicade

lla vi

ridis E

U7270

97 (le

afhop

per)

Notostir

a elon

gata E

U7270

98 (g

rass b

ug)

Agathem

era cla

razian

a JF26

6577

(phasm

id)

Antonina c

rawii A

B0300

22 (p

seudoco

ccid)

Derman

yssus

gallin

ae EU

6700

52 (p

oultr

y)

Polyr

hach

is tu

bifera

CSM

1108

(ant

)

Polyr

hach

is mon

teithi

CSM

0961

(ant

)

Polyr

hach

is sp

CSM

0983

(ant

)Fann

ia m

anica

ta A

Y569

829

(fly)

Cten

ocep

halid

es fe

lis (fl

ea)

Spiro

plas

ma

sp

JF26

6585

(spi

der)

Poly

rhac

his s

okol

ova

CSM

0854

-2 (a

nt)

Aran

eus d

iade

mat

us E

U72

7099

(spi

der)

Adal

ia b

ipun

ctat

a AJ

0067

75 (b

eetle

)

Chrysolina varians EU727100 (beetle)

Agathemera millepunctata JF266578 (phasmid)

Kerria lacca GU129148 (scale insect)

Trachymyrmex jamaicensis GQ275127 (ant)

Vollenhovia sp HM996753 (ant)

Vollenhovia sp GQ275136 (ant)Dorylus molestus HM996767 (ant)

Pheidole sp HM996747 (ant)

Pheidole soritis HM996754 (ant)

Spiroplasma platyhelix DQ860101

Spiroplasma platyhelix GU993266

Neivamyrmex carolinensis HM996770 (ant)

Neivamyrmex gibbatus HM996776 (ant)

Cephalotes varians JX990286 (ant)Tetraponera sp CSM

0841 (ant)

Solenopsis geminata SRR058848 A9 (ant)

Solenopsis geminata SRR058869 C4 (ant)

Cephalotes varians CSM1323 (ant gut)

Solenopsis geminata SRR058843 A4 (ant)

Cephalotes varians CSM1323 (ant head)

Cephalotes varians CSM1323 (ant leg)

Pseudomyrmex peperi SK1058 (ant)

Mesoplasm

a syrphidae AY231458 (fly)

Mesoplasm

a seiffertii AY351331 (flower)

Photuris pennsylvanica AY177627 (firefly)

Ento

mop

lasm

atal

es JN

5854

56

Myc

opla

sma

myc

oide

s AF0

8535

0

Capr

a hi

rcus

MPU

2605

5 (g

oat)

Ento

mop

lasm

a m

elaleu

cae D

Q51

4607

Myc

opla

sma

mon

odon

AF0

4219

4 (p

raw

ns)

Polyr

hach

is sc

hoop

ae C

SM06

26b

(ant

)

Cicin

dela

cam

pestr

is AF

0369

54 (b

eetle

)

Loph

omyr

mex

sp G

Q27

5146

(ant

)

Para

pone

ra cl

avata

SK12

49-3

(ant

mid

gut)

Para

pone

ra cl

avata

SK12

49-3

(ant

leg)

Ceph

alotes

sp S

K153

4 (an

t gas

ter)

Tetra

mor

ium

hisp

idum

GQ

2751

06 (a

nt)

Ocym

yrm

ex pi

card

i GQ2

7512

8 (an

t)

Pyra

ctom

ena

angu

lata

AY1

5787

1 (fi

refly

)

Spiro

plas

ma

sp J

F276

619 (

earth

worm

)

Ento

mop

lasm

a lu

civor

ax A

F547

212

Firmicu

tes FM252494 (so

il)

Cheliomyrm

ex sp HM996780 (an

t)

Nomamyrmex ese

nbecki H

M996768 (ant)

Nomamyrm

ex hartigii H

M996876 (ant)

Dorylus spininodis HM996755 (ant)Eciton burchellii HM996844 (ant)Dorylus sp HM996757 (ant)

Dorylus mayri HM996733 (ant)

Aenictus sp HM996772 (ant)

Aenictus dentatus HM996763 (ant)

Aenictus aratus HM996762 (ant)

Aenictus sp HM996765 (ant)

Dorylus gerstaeckeri HM996756 (ant)

Aenictus sp HM996764 (ant)

Aenictus cornutus HM996770 (ant)

Aenictus hottai HM996760 (ant)

Aenictus sp HM996775 (ant)

Aenictus cornutus HM996759 (ant)

Dorylus molestus HM996787 (ant)

Aenictus laeviceps HM996781 (ant)

Aenictus fergusoni HM996766 (ant)

Cheliomyrmex sp HM996778 (ant)

Aenictus sp GQ275130 (ant)

Odontomachus brunneus JQ957020 (ant)

Pachycondyla curvinodis JQ957018 (ant)

Odontomachus brunneus JQ957022 (ant)

Eciton lucanoides HM996758 (ant)

Eciton burchellii HM996844 (ant)

Nomamyrmex esenbecki HM996769 (ant)

Eciton vagans HM996761 (ant)

Eciton hamatum GQ275109 (ant)

Cheliomyrmex sp HM996872 (ant)

Cepha

lotes

sp SK

1531

(ant

)

Polyr

hach

is sok

olova

CSM

0854

-1 (a

nt)

Mesopla

sma l

actuc

ae A

F303

132

Calypto

myrmex

beccar

ii HM

9967

48 (a

nt)

Acromyrm

ex oct

ospinosu

s AF49

1879

(ant)

Neivam

yrmex

nigresc

ens (ant)

Photuris pennsylvanicus AY189134 (firefly)

Panorpa helena AY189125 (scorpion fly)

Alcides zodiaca HM

996749 (moth)

Spiroplasma sp AY189320

Culex annulus AY189130 (mosquito)

Spiroplasma sp AY189315

Liriodendron tulipifera AY189131 (tulip)

Entomoplasm

a melaleucae AY345990 (flower)

Aedes sollicitans AY189129 (mosquito)

Pachycondyla striata JQ957013 (ant)

Pachycondyla striata JQ957015 (ant)

Spiroplasma chrysopicola AY189127 (tabanid fly)

Spiroplasma syrphidicola AY189309 (fly)

Ixodes uriae EU727106 (tic

k)

Periplaneta americana JN653133 (cockroach)

Spiroplasma sp AY189313

Odontomachus brunneus JQ957019 (ant)

Drosophila hydei DQ412089 (fly)

Drosophila wheeleri FJ657225 (fly)

Drosophila aldrichi FJ657229 (fly)

Drosophila mojavensis FJ657219 (fly)

Polyrhachis sp CSM1056 (ant)

Polyrhachis sp CSM0785 (ant)

Polyrhachis ornata CSM0718 (ant)

Polyrhachis sp CSM0668 (ant)

Polyrhachis cupreata CSM1016 (ant)

Camponotus sp CSM0644 (ant)

Polyrhachis sp CSM0986 (ant)

Polyrhachis barretti CSM1121 (ant)

Cephalotes sp RA1052 (ant)

Polyrhachis sp CSM1030 (ant)

Polyrhachis sp CSM0998 (ant)

Pseudomyrmex gracilis BER125 (ant)

Pseudomyrmex apache SK0787 (ant)

Cephalotes varians CSM1280 (ant)

Pseudomyrmex PSW37 SK0076 (ant)

Polyrhachis sp CSM0796 (ant)

Pseudomyrmex major SK0990 (ant)

Chorthippus parallelus JN797639 (grasshopper)

Streblidae sp JF266584 (bat fly)

Spiroplasma sp EF151266

Pardosa lugubris EU727108 (spider)Spiroplasma melliferum AY325304

Apis mellifera JQ347516 (honeybee)

Spiroplasma citri X63781

Daucus carota HM124556 (carrot)

Lycaena arota HM996750 (butterfly)

Mesene phareus HM996752 (butterfly)Rhetus periander HM996751 (butterfly)

Myrmica incompleta HM996744 (ant)

Spiroplasma phoeniceum AY772395

Spiroplasma insolitum AY189133

Poritia sumatrae HM996745 (butterfly)

Spiroplasma sp DQ186642

Drosophila melanogaster FJ657180 (fly)

Drosophila willistoni SMERRNA11 (fly)

Drosophila melanogaster AJ631998 (fly)

Drosophila simulans FJ657181 (fly)

Trapezites eliena HM996746 (butterfly)

Anthidium florentinum EU727107 (bee)

Eurygaster

integrice

ps JQ914111 (sh

ield bug)

Panaeus vannamei DQ360069 (sh

rimp)

Eriocheir sinensis

DQ917753 (crab)Lasiu

s alien

us GQ275102 (an

t)

Rimica

ris ex

oculat

a AJ515720 (shrim

p)

Selenomonas ruminantium AB003379

Clade 1

Clade 2

Clad

e 3

Inner circle ant subfamily

DorylinaeEctatomminaeEcitoninae

FormicinaeMyrmicinaePonerinaePseudomyrmecinae

Middle circle bioregionAfrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Aenictinae

Figure 2 Phylogenetic tree of Spiroplasma-related ant symbionts and their closest relatives with sequence data available in GenBank Amaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shown The host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) The branch color refers to the sourcefrom which the bacteria were isolated with yellow representing ant hosts red other insect hosts blue vertebrates and green plants The innercircle refers to the ant subfamily and the outer circle refers to the continent from which samples were collected The three largest ant-specificclades of Spiroplasma symbionts are indicated (Clades 1ndash3) Selenomonas ruminantium was used as an outgroup

The infection with Spiroplasma seems to be systemic as wefound high titers of this bacterium in association with antguts heads and legs (Supplementary Table 1)

Entomoplasmatales can be pathogenic to plants andvertebrates [59 62] and have been isolated from variousinsect taxa including aphids ants bees beetles butterflies

fruit flies and horse flies [63ndash68] Mutualistic spiroplasmascan grant insects resistance to parasitic nematodes [22]and an increased ability to overwinter [69] Pathogenicphenotypes usually lead to insect death [34] and reproductivemanipulation includes altered sex ratios [33] andmale killing[35 70 71] In ants spiroplasmas have been surveyed and

Psyche 7

biocontrol potential has been hypothesized but their roleremains elusive to date [31 38 60] Functional studiesthat compare the performance of infected and uninfectedindividuals would improve our understanding of the role ofthese facultative symbionts

33 Asaia (Alphaproteobacteria Rhodospirillales) Of 95 antspecies 14 hosted bacteria related to Asaia (Alphapro-teobacteria Rhodospirillales) (Table 1) For these bacteriano previous surveys on their prevalence across the antshave been conducted We found a particularly high infectionrate of 333 (1236 species) in Pseudomyrmex In con-trast Asaia-related symbionts were lacking in CamponotusCrematogasterMyrmeciaMyrmecocystus PolyrhachisOeco-phylla and Solenopsis Low infection frequency was presentin Cephalotes (17) and Paraponera (11 species) (Table 1)The enrichment of Asaia symbionts in Pseudomyrmex isparticularly interesting as this ant genus is arboreal andcontains several obligate plant ants which exclusively feed onplant-derived food sources [58 72] However this bacterialgroup occurred facultatively in arboreal generalists and plantmutualists alike indicating that even if these symbionts aremore frequent in arboreal ormutualistic Pseudomyrmex antsthe association is not obligate

In total we obtained 25 Asaia-related OTUs in ourdataset Of these OTUs 21 were associated with Pseu-domyrmex 3 with Paraponera and 1 with Cephalotes Weinferred a maximum likelihood phylogeny of these OTUstheir closest GenBank relatives and other endosymbioticRhodospirillales bacteria fromGenBankThe total alignmentconsisted of 91 taxa and had a length of 1313 characters WeusedWolbachia pipientis (Alphaproteobacteria Rickettsiales)as an outgroup The phylogenetic tree shows three clades inwhich ant-associatedAsaiaOTUs cluster together (Figure 3)(1) a small clade with two Pseudomyrmex-associated OTUsand one Paraponera-associated OTU (2) a clade that appearsto be Hymenoptera specific containing the bulk of Pseu-domyrmex-associated OTUs a Formica-associated sequencefrom GenBank and bacteria isolated from several beespecies and (3) a clade comprised of many insect-associatedAsaia bacteria and five of our OTUs This last clade is par-ticularly interesting as it comprised several strains that wereisolated from different mosquito species as well as three ant-associated Asaia sequences from GenBank One sequence(JF514556) was isolated and cultivated from Tetraponerarufonigra in India [73] The nifH gene a gene associatedwith the fixation of atmospheric nitrogen has also beenfound in this bacterium (GenBank accession JF736510) andit has been experimentally shown that this strain is capableof fixing nitrogen in vitro suggesting possible nitrogen fixingattributes in its natural environment the ant body cavity[73] The two other sequences are cultivated bacteria fromCephalotes varians and were generated in the framework ofa previous study from our lab (GenBank accessions JX445137and JX445138) [39]

Bacteria from the family Acetobacteraceae are commonlyknown as ldquoacetic acid bacteriardquo and have the metaboliccapacity to oxidize ethanol to acetic acid [74] Asaia also

a member of the Acetobacteraceae however only weaklyoxidizes ethanol and shows higher rates of sugar oxidation[74] These bacteria are environmentally ubiquitous buthave also been found in association with insects such asbees [75 76] mosquitoes [77] Drosophila melanogaster [78]leafhoppers [79] and mealybugs [80] All these insects relyon sugar-rich and often nitrogen-limited diets and it hasbeen suggested that the bacteria function as nutritionalsymbionts Some acetic acid bacteria have the capacity tofix atmospheric nitrogen [73] However it remains entirelyspeculative whether this function can be retained in theinsect gut environment and whether these bacteria actuallycontribute to insect nitrogen metabolism or recycling [81]Interestingly neither acetic acid bacteria nor lactic acidbacteria are commonly found in the core gut microbiota ofarboreal Cephalotini ants an ant group with one of the mostthoroughly studied microbiomes [18 19 39] The metaboliccapacities of the core gut microbiota of the Cephalotiniconsisting of Burkholderiales Opitutales PseudomonadalesRhizobiales and Xanthomonadales might be redundant withthe role that acetic acid bacteria play in other insects

In Drosophila acetic acid bacteria are part of the normalcommensal bacterial gut community and can be involved inthe regulation of the innate immune system In healthy fliesa stable equilibrium of different gut microbes is maintainedPerturbation of the normal gut community which can becaused by a defective regulation of antimicrobial peptideleads to the dominance of the pathogenic commensal Glu-conobacter morbifer and ultimately to gut apoptosis [82]Potential other mechanisms by which acetic acid bacteriabenefit insect immunity are by decreasing the gut pHmakingit an unfavorable environment for pathogenic microorgan-isms or by competitive exclusion [81] However these aceticacid bacteria are not essential for the fitness and repro-duction of most insects as even in the well-studied Asaia-mosquito interaction experimental removal of bacteria hadno detectable negative impact on the host [81]

Several studies have been conducted to analyze themicrobial diversity associated with ants [18 19 39 60 83]However the symbiotic relationships of ants with Rhodospir-illales have rarely been observed In fact only two ant-associated Rhodospirillales sequences had been depositedin GenBank (GQ275104 from Formica occulta and JF514556from Tetraponera rufonigra) prior to work from our group[39] Clone libraries generated for theCephalotini ants [18 19]as well as tag-encoded amplicon data sets [38 39] are amongthe most extensive microbial data collections available forants to date and acetic acid bacteria were only sporadicallyassociated with the ant taxa that were investigated Thus theinteraction of Asaia relatives with ants is generally poorlyunderstood but due to the metabolic capacities of thesebacteria to utilize sugar-rich substrates and fix nitrogenthey might play an important nutritional role Particularlythey might be functionally important in the ant subfamilyPseudomyrmecinae in which they seem to be enriched asindicated by our present study

The phylogenetic history of ant-associated Rhodospiril-lales does not show host specificity and suggests likely acqui-sition from the environment (Figure 3) These observations

8 Psyche

Drosophila sp EU096229 (fruit fly)

Drosophila sp EU096235 (fruit fly)

Drosophila sp EU096233 (fruit fly)

Pseudomyrm

ex ejectus SK1181-3 (ant)

Paraponera clavata CS0513-2 (ant mouth)

Gluconobacter frateurii AB682075

Gluc

onob

acte

r fra

teur

ii A

B682

081Gluconobacter frateurii A

B682091

Pseudo

myrmex

ferru

gineu

s SK00

02-1

(ant)

Pseudomyrm

ex pallidus C

SM1333 (ant)

Pseudomyrmex spinicola SK0061-1 (ant)

Pseudomyrmex peperi SK1058-103-1 (ant)

Cephalotes atratus SK1465 (ant)

Xylocopa californica HM112403 (bee)

Caupolicana yarrowi HM109591 (bee)

Euglossa imperialis JQ389882 (bee)

BER145 (ant)Pseudomyrmex sp nr eduardi

Pseudomyrmex satanicus SK0058-1 (ant)Pseudomyrmex dendroicus BER121 (ant)

Pseudomyrmex dendroicus BER103 (ant)

Pseudomyrmex peperi

SK0007-1 (ant)Pseu

domyrm

ex nigroci

nctus

SK0060-1 (a

nt)Pseudom

yrmex

major

SK09

90-8-

3 (an

t gast

er)

Pseu

domyrm

ex de

ndroi

cus S

K1260

(ant)

Form

ica oc

culta

GQ27

5104

(ant

)

Alph

a pro

teoba

cteriu

m A

B513

363 (

flowe

r)

Para

pone

ra cl

avat

a CS

0513

-4 (a

nt m

outh

)Ca

lliop

sis su

balpi

nus H

M10

9230

(bee

)Ca

lliop

sis su

balpi

nus H

M10

9259

(bee

)

Pseu

dom

yrm

ex te

nuis

BER1

12 (a

nt)

Sacc

haric

occu

s sac

char

i AF1

2739

6 (m

ealy

bug)

Dro

soph

ila sp

EU

0962

34 (f

ruit

fly)

Atta colombica H

M557097 (fungus garden)

Aedes aegypti FJ372761 (mosquito)

Aedes aegypti FJ372770 (mosquito)

Anopheles maculipennis AM

404262 (mosquito)

Aedes albopictus JQ958879 (m

osquito)

Aedes albopictus JQ314055 (m

osquito)

Aedes albopictus JQ314027 (m

osquito)

Aedes albopictus F2f JX445138 (mosquito)

Aede

s aeg

ypti

FN81

4303

(mos

quito

)

Aede

s alb

opict

us F

1f JX

4451

37 (m

osqu

ito)

Aede

s alb

opict

us F

2l JX

4451

40 (m

osqu

ito)

Aede

s alb

opict

us F

1l JX

4451

39 (m

osqu

ito)

Aede

s aeg

ypti

FN81

4301

(mos

quito

)

Aede

s aeg

ypti

FN81

4300

(mos

quito

)

Aede

s aeg

ypti

FN29

7842

(mos

quito

)

Anoph

eles s

tephe

nsi A

M40

4261

(mos

quito

)

Aede

s aeg

ypti

FN29

7841

(mos

quito

)Asaia b

ogore

nsis A

B6820

04

Anoph

eles s

p HQ26

9803

(mos

quito

)

Tetrap

onera

rufon

igra JF

5145

56 (a

nt)

Pseudomyrmex elo

ngatus

CSM1373 (ant gut)Pseudomyrmex termitarius

SK1252-1 (ant)

Pseudomyrmex gracilis

CSM1374-2 (ant head)Anopheles gambiae FN814276 (mosquito)Pseudomyrmex ejectus SK1181-3 (ant)

Pseudomyrm

ex elongatus

CSM1373 (ant head)

Cephalotes va

rians JX

990151 (ant)

Anopheles stephensi FN814299 (mosquito)

Anopheles gambiae FN814277 (mosquito)

Anopheles stephensi FN814297 (mosquito)

Anopheles stephensi FN814296 (mosquito)

Cephalotes varians JX990161 (ant)

Lutzomyia longipalpis HE995765 (sand fly)

Anopheles gambiae FN821398 (mosquito)

Aedes aegypti FN297840 (mosquito)

Anopheles stephensi AM404260 (mosquito)

Asaia krungthepensis AB682130

Anopheles gambiae FJ816021 (mosquito)Anopheles gambiae FN814284 (mosquito)Anopheles gambiae FN814283 (mosquito)

Aedes albopictus FN814288 (mosquito)

Aedes albopictus FN814287 (mosquito)

Anopheles gambiae FN821397 (mosquito)

Anopheles gambiae FN814280 (mosquito)

Aedes albopictus FN814289 (mosquito)

Anopheles gambiae FN814285 (mosquito)

Anopheles gambiae FN814275 (mosquito)

Anopheles gambiae FN814281 (mosquito)

Anopheles gambiae FN814278 (mosquito)

Aedes albopictus FN814286 (mosquito)

Atta colombica HM556904 (fungus garden)

Saccharicoccus sacchari AF127400 (mealybug)

Saccharicoccus sacchari AF127408 (mealybug)

Saccharicoccus sacchari AF127401 (mealybug)

Pseudomyrmex ejectus SK1181-3 (ant)

Paraponera clavata CS0513-3 (ant mouth)

Pseudomyrm

ex termitarius SK1252-2 (ant)

Drosophila sp EU096231 (fruit fly)Wolbachia pipientis AY026912

Inner circle ant subfamilyFormicinaeMyrmicinaeParaponerinaePseudomyrmecinae

Middle circle bioregionAfrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Clade 1

Clade 2Cl

ade 3

Figure 3 Phylogenetic tree of Asaia-related symbionts associated with ants and closest relatives with sequence data available in GenBank Amaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shown The host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) The branch color refers to the sourcefromwhich the bacteria were isolated with yellow representing ant hosts and red other insect hostsThe inner circle refers to the ant subfamilyand the middle circle refers to the bioregion from which samples were collected The outer circle indicates three clades (Clades 1ndash3) whichcontained several ant-associated symbiontsWolbachia pipientis was used as an outgroup

indicate that Rhodospirillales aremost likely environmentallytransmitted and support the hypothesis that they are onlyfacultative associates of ants One clade of ant-associatedRhodospirillales was closely related to endosymbionts iso-lated from mosquitos (Figure 3) It has been experimentallyshown that mosquito-associated Asaia can successfully colo-nize leafhoppers further emphasizing the low-host specificityof this bacterial group [77]

4 Conclusion

Our broad bacterial screening approach has contributedto our understanding of the prevalence of ant-associatedmicrobes particularly with regard to their Wolbachia andSpiroplasma symbionts Furthermore we provide the firstextensive survey for ant-associated Asaia-related symbiontsWhile these symbionts of the order Rhodospirillales infect

Psyche 9

ants only sporadically some strains are capable of fixingatmospheric nitrogen and might retain this function inants Alternatively these bacteria might have an importantfunctional role for upgrading nitrogen-poor diets of someherbivorous ants which comprise the majority of all ant taxa[20] Even though we do not have experimental evidence ofthe role of most bacterial symbionts in ants previous studiesillustrate a broad variety of effects of these bacteria on insecthosts [4 7 9] Even a single group of microbes can havevery different effects on different hosts Our study shows thatdespite several extensive bacterial surveys across the ants thediversity and functional role of ant-associated microbes isfar from being fully understood and broad next generationsequencing approaches will provide a fast and cost-effectivetool to deepen our knowledge of the rare (and not so rare)biosphere

Acknowledgments

The authors thank Daniel Ballhorn and Tina Schroyer forhelpful comments and discussions Phil Ward kindly iden-tified Pseudomyrmex specimens This study was supportedin part by National Science Foundation DEB Grant no1050243 to Corrie S Moreau a Grainger Foundation grant toCorrie S Moreau a Negaunee Foundation grant to Corrie SMoreau and the Pritzker Laboratory forMolecular Evolutionand Systematics Stefanie Kautz acknowledges a postdoctoralfellowship from the German Academy of Sciences Leopold-ina (Grant no LPDS 2009-29) Benjamin E R Rubin wassupported in part by an NSF Graduate Research Fellowship

References

[1] H Koch D P Abrol J Li and P Schmid-Hempel ldquoDiversity ofevolutionary patterns of bacterial gut associates of corbiculatebeesrdquoMolecular Ecology vol 22 no 7 pp 2028ndash2044 2013

[2] R J Dillon and V M Dillon ldquoThe Gut bacteria of insectsnonpathogenic interactionsrdquoAnnual Review of Entomology vol49 pp 71ndash92 2004

[3] S Fraune and T C G Bosch ldquoWhy bacteria matter in animaldevelopment and evolutionrdquo BioEssays vol 32 no 7 pp 571ndash580 2010

[4] H Feldhaar ldquoBacterial symbionts as mediators of ecologicallyimportant traits of insect hostsrdquo Ecological Entomology vol 36no 5 pp 533ndash543 2011

[5] J M Urban and J R Cryan ldquoTwo ancient bacterialendosymbionts have coevolved with the planthoppers (InsectaHemiptera Fulgoroidea)rdquo BMC Evolutionary Biology vol 12article 87 2012

[6] H Toju and T Fukatsu ldquoDiversity and infection prevalence ofendosymbionts in natural populations of the chestnut weevilrelevance of local climate and host plantsrdquo Molecular Ecologyvol 20 no 4 pp 853ndash868 2011

[7] M Kaltenpoth ldquoActinobacteria as mutualists general health-care for insectsrdquo Trends inMicrobiology vol 17 no 12 pp 529ndash535 2009

[8] N A Moran J P McCutcheon and A Nakabachi ldquoGenomicsand evolution of heritable bacterial symbiontsrdquo Annual Reviewof Genetics vol 42 pp 165ndash190 2008

[9] J P McCutcheon and C D Von Dohlen ldquoAn interdependentmetabolic patchwork in the nested symbiosis of mealybugsrdquoCurrent Biology vol 21 no 16 pp 1366ndash1372 2011

[10] C B Montllor A Maxmen and A H Purcell ldquoFacultative bac-terial endosymbionts benefit pea aphids Acyrthosiphon pisumunder heat stressrdquo Ecological Entomology vol 27 no 2 pp 189ndash195 2002

[11] A E Douglas ldquoThe nutritional physiology of aphidsrdquoAdvancesin Insect Physiology vol 31 pp 73ndash140 2003

[12] J J Wernegreen S N Kauppinen S G Brady and P SWard ldquoOne nutritional symbiosis begat another phylogeneticevidence that the ant tribe Camponotini acquired Blochmanniaby tending sap-feeding insectsrdquo BMC Evolutionary Biology vol9 no 1 article 292 2009

[13] H Feldhaar J StrakaM Krischke et al ldquoNutritional upgradingfor omnivorous carpenter ants by the endosymbiont Blochman-niardquo BMC Biology vol 5 article 48 2007

[14] A A Pinto-Tomas M A Anderson G Suen et al ldquoSymbioticnitrogen fixation in the fungus gardens of leaf-cutter antsrdquoScience vol 326 no 5956 pp 1120ndash1123 2009

[15] R Pais C Lohs Y Wu J Wang and S Aksoy ldquoThe obligatemutualist Wigglesworthia glossinidia influences reproductiondigestion and immunity processes of its host the tsetse flyrdquoApplied and Environmental Microbiology vol 74 no 19 pp5965ndash5974 2008

[16] J A Breznak ldquoIntestinal microbiota of termites and otherxylophagous insectsrdquo Annual Review of Microbiology vol 36pp 323ndash343 1982

[17] J A Breznak W J Brill J W Mertins and H C CoppelldquoNitrogen fixation in termitesrdquo Nature vol 244 no 5418 pp577ndash579 1973

[18] J A Russell C SMoreau BGoldman-HuertasM FujiwaraDJ Lohman andN E Pierce ldquoBacterial gut symbionts are tightlylinkedwith the evolution of herbivory in antsrdquoProceedings of theNational Academy of Sciences of theUnited States of America vol106 no 50 pp 21236ndash21241 2009

[19] K E Anderson J A Russell C S Moreau et al ldquoHighlysimilar microbial communities are shared among related andtrophically similar ant speciesrdquo Molecular Ecology vol 21 pp2282ndash2296 2012

[20] D W Davidson S C Cook R R Snelling and T H ChualdquoExplaining the abundance of ants in lowland tropical rainforestcanopiesrdquo Science vol 300 no 5621 pp 969ndash972 2003

[21] J C Brownlie and K N Johnson ldquoSymbiont-mediated protec-tion in insect hostsrdquo Trends in Microbiology vol 17 no 8 pp348ndash354 2009

[22] J Jaenike R Unckless S N Cockburn L M Boelio andS J Perlman ldquoAdaptation via symbiosis recent spread of aDrosophila defensive symbiontrdquo Science vol 329 no 5988 pp212ndash215 2012

[23] K M Oliver J A Russell N A Morant and M S HunterldquoFacultative bacterial symbionts in aphids confer resistance toparasitic waspsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 4 pp 1803ndash18072003

[24] C R Currie J A Scott R C Summerbell and D MallochldquoFungus-growing ants use antibiotic-producing bacteria tocontrol garden parasitesrdquo Nature vol 398 pp 701ndash704 1999

[25] M Kaltenpoth W Gottler G Herzner and E Strohm ldquoSym-biotic bacteria protect wasp larvae from fungal infestationrdquoCurrent Biology vol 15 no 5 pp 475ndash479 2005

10 Psyche

[26] H EDunbar A C CWilsonN R Ferguson andNAMoranldquoAphid thermal tolerance is governed by a point mutation inbacterial symbiontsrdquo PLoS Biology vol 5 no 5 pp 1006ndash10152007

[27] T Tsuchida R Koga S Matsumoto and T Fukatsu ldquoInterspe-cific symbiont transfection confers a novel ecological trait to therecipient insectrdquo Biology Letters vol 7 no 2 pp 245ndash248 2011

[28] R Stouthamer J A J Breeuwer and G D D Hurst ldquoWolbachiapipientis microbial manipulator of arthropod reproductionrdquoAnnual Review of Microbiology vol 53 pp 71ndash102 1999

[29] L M Hedges J C Brownlie S L OrsquoNeill and K N JohnsonldquoWolbachia and virus protection in insectsrdquo Science vol 322 no5902 p 702 2008

[30] K Hilgenboecker P Hammerstein P Schlattmann ATelschow and J H Werren ldquoHow many species are infectedwith Wolbachiamdasha statistical analysis of current datardquo FEMSMicrobiology Letters vol 281 no 2 pp 215ndash220 2008

[31] J A Russell C F Funaro Y M Giraldo et al ldquoA veritablemenagerie of heritable bacteria from ants butterflies andbeyond broadmolecular surveys and a systematic reviewrdquoPLoSONE vol 7 no 12 Article ID e51027 2012

[32] O Duron D Bouchon S Boutin et al ldquoThe diversity ofreproductive parasites among arthropods Wolbachia do notwalk alonerdquo BMC Biology vol 6 article 27 2008

[33] D L Williamson and D F Poulson ldquoSex ratio organisms(Spiroplasmas) of Drosophilardquo in The Mycoplasmas III Plantand Insect Mycoplasmas R F Whitcomb and J G Tully Edspp 175ndash208 Academic Press New York NY USA 1979

[34] J M Bove ldquoSpiroplasmas infectious agents of plants arthro-pods and vertebratesrdquoWiener Klinische Wochenschrift vol 109no 14-15 pp 604ndash612 1997

[35] M C Tinsley and M E N Majerus ldquoA new male-killingparasitism Spiroplasma bacteria infect the ladybird beetleAnisosticta novemdecimpunctata (Coleoptera Coccinellidae)rdquoParasitology vol 132 no 6 pp 757ndash765 2006

[36] A Dobson K D Lafferty A M Kuris R F Hechinger andW Jetz ldquoHomage to Linnaeus howmany parasites Howmanyhostsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 105 no 1 pp 11482ndash11489 2008

[37] S T Kelley and S Dobler ldquoComparative analysis of microbialdiversity in Longitarsus flea beetles (Coleoptera Chrysomeli-dae)rdquo Genetica vol 139 no 5 pp 541ndash550 2011

[38] H D Ishak R Plowes R Sen et al ldquoBacterial diversityin Solenopsis invicta and Solenopsis geminata ant coloniescharacterized by 16S amplicon 454 pyrosequencingrdquo MicrobialEcology vol 61 no 4 pp 821ndash831 2011

[39] S Kautz B E R Rubin J A Russell and C S Moreau ldquoSur-veying the microbiome of ants comparing 454 pyrosequencingwith traditionalmethods to uncover bacterial diversityrdquoAppliedand Environmental Microbiology vol 79 no 2 pp 525ndash5342013

[40] S E Dowd T R Callaway R D Wolcott et al ldquoEvaluationof the bacterial diversity in the feces of cattle using 16SrDNA bacterial tag-encoded FLX amplicon pyrosequencing(bTEFAP)rdquo BMCMicrobiology vol 8 article 125 2008

[41] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[42] C Quince A Lanzen R J Davenport and P J TurnbaughldquoRemoving noise from pyrosequenced ampliconsrdquo BMC Bioin-formatics vol 12 article 38 2011

[43] R C Edgar ldquoSearch and clustering orders of magnitude fasterthan BLASTrdquo Bioinformatics vol 26 no 19 pp 2460ndash24612010

[44] J R Cole QWang E Cardenas et al ldquoTheRibosomal databaseproject improved alignments and new tools for rRNA analysisrdquoNucleic Acids Research vol 37 no 1 pp D141ndashD145 2009

[45] T Z DeSantis P Hugenholtz N Larsen et al ldquoGreengenesa chimera-checked 16S rRNA gene database and workbenchcompatible with ARBrdquo Applied and Environmental Microbiol-ogy vol 72 no 7 pp 5069ndash5072 2006

[46] A J Drummond B Ashton S Buxton et al ldquoGeneious v5 4rdquo2011 httpwwwgeneiouscom

[47] A Stamatakis P Hoover and J Rougemont ldquoA rapid bootstrapalgorithm for the RAxML web serversrdquo Systematic Biology vol57 no 5 pp 758ndash771 2008

[48] M A Miller M T Holder R Vos et al ldquoThe CIPRES portalsrdquohttpwwwphyloorgsub sectionsportal

[49] I Letunic and P Bork ldquoInteractive Tree of Life v2 onlineannotation and display of phylogenetic treesmade easyrdquoNucleicAcids Research vol 39 no 2 pp W475ndashW478 2011

[50] T Wenseleers F Ito S van Borm R Huybrechts F Volckaertand J Billen ldquoWidespread occurrence of the micro-organismWolbachia in antsrdquo Proceedings of the Royal Society B vol 265no 1404 pp 1447ndash1452 1998

[51] J A Russell ldquoThe ants (Hymenoptera Formicidae) are uniqueand enigmatic hosts of prevalent Wolbachia (Alphaproteobac-teria) symbiontsrdquoMyrmecological News vol 16 pp 7ndash23 2012

[52] W Arthofer M Riegler D Schneider M Krammer W JMiller and C Stauffer ldquoHidden Wolbachia diversity in fieldpopulations of the European cherry fruit fly Rhagoletis cerasi(Diptera Tephritidae)rdquo Molecular Ecology vol 18 no 18 pp3816ndash3830 2009

[53] J A Russell B Goldman-Huertas C S Moreau et al ldquoSpecial-ization and geographic isolation among Wolbachia symbiontsfrom ants and lycaenid butterfliesrdquo Evolution vol 63 no 3 pp624ndash640 2009

[54] T Wenseleers L Sundstrom and J Billen ldquoDeleterious Wol-bachia in the ant Formica truncorumrdquo Proceedings of the RoyalSociety B vol 269 no 1491 pp 623ndash629 2002

[55] F Vavre F Fleury J Varaldi P Fouillet and M BouletreauldquoEvidence for female mortality in Wolbachia-mediated cyto-plasmic incompatibility in haplodiploid insects epidemiologicand evolutionary consequencesrdquo Evolution vol 54 no 1 pp191ndash200 2000

[56] J Heinze ldquoThe demise of the standard ant (HymenopteraFormicidae)rdquoMyrmecological News vol 11 pp 11ndash20 2008

[57] S Kautz S U Pauls D J Ballhorn H T Lumbsch andM HeilldquoPolygynous supercolonies of the acacia-ant Pseudomyrmexpeperi an inferior colony founderrdquo Molecular Ecology vol 18no 24 pp 5180ndash5194 2009

[58] S Kautz D J Ballhorn J Kroiss et al ldquoHost plant use bycompeting acacia-ants mutualists monopolize while parasitesshare hostsrdquo PLoS ONE vol 7 no 5 Article ID e37691 2012

[59] J Fletcher G A Schultz R E Davis C E Eastman and R MGoodman ldquoBrittle root disease of horseradish-evidence for anetiological role of Spiroplasma citrirdquo Phytopathology vol 71 pp1073ndash1080 1981

[60] C F Funaro D J C Kronauer C S Moreau B Goldman-Huertas N E Pierce and J A Russell ldquoArmy ants harbor ahost-specific clade of Entomoplasmatales bacteriardquo Applied andEnvironmental Microbiology vol 77 no 1 pp 346ndash350 2011

Psyche 11

[61] D J C Kronauer ldquoRecent advances in army ant biology(Hymenoptera Formicidae)rdquoMyrmecological News vol 12 pp51ndash65 2009

[62] F Thiaucourt and G Bolske ldquoContagious caprine pleurop-neumonia and other pulmonary mycoplasmoses of sheep andgoatsrdquo OIE Revue Scientifique et Technique vol 15 no 4 pp1397ndash1414 1996

[63] T Fukatsu T Tsuchida N Nikoh and R Koga ldquoSpiroplasmaSymbiont of the pea aphid Acyrthosiphon pisum (InsectaHomoptera)rdquo Applied and Environmental Microbiology vol 67no 3 pp 1284ndash1291 2001

[64] T S Haselkorn T A Markow and N A Moran ldquoMultipleintroductions of the Spiroplasma bacterial endosymbiont intoDrosophilardquo Molecular Ecology vol 18 no 6 pp 1294ndash13052009

[65] J G Lundgren R M Lehman and J Chee-Sanford ldquoPhysiol-ogy biochemistry and topology bacterial communities withindigestive tracts of ground beetles (Coleoptera Carabidae)rdquoAnnals of the Entomological Society of America vol 100 no 2pp 275ndash282 2007

[66] I Meeus V Vercruysse and G Smagghe ldquoMolecular detectionof Spiroplasma apis and Spiroplasmamelliferum in beesrdquo Journalof Invertebrate Pathology vol 109 no 1 pp 172ndash174 2012

[67] S van Borm J Billen and J J Boomsma ldquoThe diversity ofmicroorganisms associated with Acromyrmex leafcutter antsrdquoBMC Evolutionary Biology vol 2 article 9 2002

[68] J Wedincamp F E French R F Whitcomb and R B HenegerldquoLaboratory infection and release of Spiroplasma (Entomoplas-matales Spiroplasmataceae) from horse flies (Diptera Taban-idae)rdquo Journal of Entomological Science vol 32 no 4 pp 398ndash402 1997

[69] M A Ebbert and L R Nault ldquoImproved overwintering abilityin Dalbulus maidis (Homoptera Cicadellidae) vectors infectedwith Spiroplasma kunkelii (Mycoplasmatales Spiroplasmat-aceae)rdquo Environmental Entomology vol 23 no 3 pp 634ndash6441994

[70] F M Jiggins G D D Hurst C D Jiggins J H G V DSchulenburg and M E N Majerus ldquoThe butterfly Danauschrysippus is infected by a male-killing Spiroplasma bacteriumrdquoParasitology vol 120 no 5 pp 439ndash446 2000

[71] J H G V D Schulenburg G D D Hurst D Tetzlaff GE Booth I A Zakharov and M E N Majerus ldquoHistory ofinfection with different male-killing bacteria in the two-spotladybird beetle Adalia bipunctata revealed through mitochon-drial DNA sequence analysisrdquoGenetics vol 160 no 3 pp 1075ndash1086 2002

[72] S Kautz H T Lumbsch P S Ward and M Heil ldquoHowto prevent cheating a digestive specialization ties mutualisticplant-ants to their ant-plant partnersrdquo Evolution vol 63 no 4pp 839ndash853 2009

[73] N Samaddar A Paul S Chakravorty et al ldquoNitrogen fixationin Asaia sp (Family Acetobacteraceae)rdquo Current Microbiologyvol 63 no 2 pp 226ndash231 2011

[74] Y Ano H Toyama O Adachi and K Matsushita ldquoEnergymetabolism of a unique acetic acid bacteriumAsaia bogorensisthat lacks ethanol oxidation activityrdquo Bioscience Biotechnologyand Biochemistry vol 72 no 4 pp 989ndash997 2008

[75] V G Martinson B N Danforth R L Minckley O RueppellS Tingek and N A Moran ldquoA simple and distinctive micro-biota associated with honey bees and bumble beesrdquo MolecularEcology vol 20 no 3 pp 619ndash628 2011

[76] N A Moran A K Hansen J E Powell and Z L SabreeldquoDistinctive gut microbiota of honey bees assessed using deepsampling from individual worker beesrdquo PLoS ONE vol 7 no 4Article ID e36393 2012

[77] E Crotti C Damiani M Pajoro et al ldquoAsaia a versatile aceticacid bacterial symbiont capable of cross-colonizing insectsof phylogenetically distant genera and ordersrdquo EnvironmentalMicrobiology vol 11 no 12 pp 3252ndash3264 2009

[78] C R Cox and M S Gilmore ldquoNative microbial colonization ofDrosophilamelanogaster and its use as a model of Enterococcusfaecalis pathogenesisrdquo Infection and Immunity vol 75 no 4 pp1565ndash1576 2007

[79] M Marzorati A Alma L Sacchi et al ldquoA novel Bacteroidetessymbiont is localized in Scaphoideus titanus the insect vector ofFlavescence Doree in Vitis viniferardquo Applied and EnvironmentalMicrobiology vol 72 no 2 pp 1467ndash1475 2006

[80] N J Ashbolt and P A Inkerman ldquoAcetic acid bacterial biota ofthe pink sugar cane mealybug Saccharococcus sacchari and itsenvironsrdquo Applied and Environmental Microbiology vol 56 no3 pp 707ndash712 1990

[81] E Crotti A Rizzi B Chouaia et al ldquoAcetic acid bacteria newlyemerging symbionts of insectsrdquo Applied and EnvironmentalMicrobiology vol 76 no 21 pp 6963ndash6970 2010

[82] J Ryu S Kim H Lee et al ldquoInnate immune homeostasis bythe homeobox gene Caudal and commensal-gut mutualism inDrosophilardquo Science vol 319 no 5864 pp 777ndash782 2008

[83] S Stoll J Gadau R Gross and H Feldhaar ldquoBacterial micro-biota associated with ants of the genus Tetraponerardquo BiologicalJournal of the Linnean Society vol 90 no 3 pp 399ndash412 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

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International Journal of

Microbiology

Page 2: Research Article Bacterial Infections across the Ants ...downloads.hindawi.com/journals/psyche/2013/936341.pdf · Department of Zoology, Field Museum of Natural History, South Lake

2 Psyche

defending their hosts against natural enemies [4 7 21]For example Spiroplasma can convey increased resistance tonematode infections in Drosophila flies [22] and secondarysymbionts in aphids can confer resistance to parasitic wasps[23] Some insect-associated bacteria also contribute to nesthygiene [7] For example actinomycetes in the fungal gardensof leaf-cutter ants inhibit the growth of fungal pathogens butnot of mutualistic fungi [24] Actinomycetes are also foundin antennal glands of bee wolves and protect larvae in theirnests against infestation by pathogens [25] Othermutualisticbacteria can increase host tolerance to unfavorable abioticconditions such as temperature stress [26] or facilitate the useof novel hosts [27]

While the associations described above are typicallybeneficial to hosts many bacterial endosymbionts are detri-mental reproductive manipulators Wolbachia for examplecan cause cytoplasmic incompatibility parthenogenesismalekilling and male feminization [28] There are also exam-ples of Wolbachia which protect their host against RNAviruses thus acting as defensivemutualists [29] An estimated66 of insect species and about 30 of ant species havebeen reported to be facultatively infected with Wolbachia[30 31] Other less prevalent reproductive manipulators ininsects include Cardinium Arsenophonus and Spiroplasma[32 33] Spiroplasma although beneficial to hosts in somecases [22] can have various negative effects on their insecthosts including manipulation of sex ratios male killing andentomopathenogenicity [33ndash35]

Despite these fascinating findings our knowledge ofbacterial symbionts is based on a relatively small numberof organisms Thus we still know little about the identitiesand ecological or physiological functions of bacteria associ-ated with most animal groups [36] In-depth analyses andextensive surveys of the bacterial communities present in awide range of eukaryotic taxa are required to understandthe diversity and the function of microbial symbionts [37]Here we analyzed bacterial communities across the ants(Hymenoptera Formicidae) using 16S rRNA tag encodedamplicon pyrosequencing (454 pyrosequencing) to survey forinfection patterns with potential parasitic microbes Due totheir sporadic prevalence and unknown effects on host antbiology we refer to these microbes as infections In total wescreened 310 ant samples of 176 colonies from 95 ant speciesand encountered high prevalence of three bacterial groupsWolbachia Spiroplasma and Asaia

2 Materials and Methods

A total of 299 ant samples were subjected to 454 pyrose-quencing and combined with data from 11 samples analyzedby Ishak et al [38] that is for a total of 310 samples Allsamples represented 176 different colonies and 95 differentant species belonging to the generaCamponotus (Formicinae1 species) Cephalotes (Myrmicinae 7 species) Cremato-gaster (Myrmicinae 6 species) Myrmecia (Myrmeciinae2 species) Myrmecocystus (Formicinae 1 species) Oeco-phylla (Formicinae 1 species) Paraponera (Paraponerinae 1species)Polyrhachis (Formicinae 32 species)Pseudomyrmex

(Pseudomyrmecinae 36 species) Solenopsis (Myrmicinae2 species) and Tetraponera (Pseudomyrmecinae 6 species)DNA extractions were either prepared from entire ants orfrom dissected ant parts as described in Kautz et al [39] Acomplete list of samples used for this study can be found inSupplementary Table 1 (see Supplementary material availableonline at httpdxdoiorg1011552013936341)

21 454 Pyrosequencing To screen ant samples for over-all bacterial diversity bacterial tag-encoded FLX ampli-con pyrosequencing was performed by the Research andTesting Laboratory (Lubbock TX USA) as described byDowd et al [40] The 16S rRNA universal eubacterialprimers 28F (51015840-GAGTTTGATCITGGCTCAG) and 519R(51015840-GWATTACCGCGGCKGCTG) were used to amplifyapproximately 500 bp of the variable regions V1ndashV3

22 Bacterial 16S rRNA Data Processing and Analysis All16S rRNA pyrosequencing reads were quality controlled anddenoised using the QIIME v150 implementation of Ampli-conNoise v125 using default parameters [41] Chimeras wereremoved by Perseus a component of the AmpliconNoisepipeline [42] All the remaining reads were then clusteredinto operational taxonomic units (OTUs) at 97 sequencesimilarity using UCLUST [43] We used the longest sequencein a cluster as the representative sequence for that OTUSingletons that is OTUs with only one read in the entiredataset were removed We used the QIIME implementationof the Ribosomal Database Project [44] classifier trainedon the February 4 2011 release of the greengenes database[45] to classify OTUs at the level of bacterial orders Defaultsettings were used including a 08 confidence cutoff forclassifications

Our filtering approach recovered infections withWolbachia (Alphaproteobacteria Rickettsiales) Spiroplasma(Firmicutes Entomoplasmatales) and Asaia (Alpha-proteobacteria Rhodospirillales) All OTUs classified asRickettsiales Entomoplasmatales and Rhodospirillales thataccounted formore than one percent of reads within a samplewere considered as infections by the respective order andincluded in further analyses This cutoff also allowed us tocontrol the relatively high error rate of 454 pyrosequencingWe classified the sequences at the genus level using the RDPclassifier (see Supplementary Table 2 for results) All OTUsused in further analyses have been deposited in GenBank(accessions KF015767-KF015856 Supplementary Table 2)

We downloaded the closest relatives of each OTU fromGenBank Additionally we were interested in retrieving anyother sequence from GenBank of those three orders thatwere associated with ants and insects in general Thus wesearched for sequences using the search keywords ldquo16Srdquoand ldquosymbiontrdquo as well as the name of the respective orderGenBank sequences with 99 identity that were isolatedfrom the same source were considered duplicates and deletedfrom the dataset

23 Phylogenetic Tree Construction Sequences were com-piled and edited using Geneious v536 [46] The alignment

Psyche 3

was generated using the infernal secondary-structure-basedaligner of the ribosomal database project (RDP) [44] Weinferred a maximum likelihood phylogeny of the most com-mon OTUs and their GenBank relatives using the RAxML728 Black Box [47] on the CIPRES web portal [48] Themodel GTR+I+G was employed We then uploaded the mostlikely tree to the iToL website [49] to facilitate graphicalillustration of bacterial source ant subfamily and geographicregion for each sequence Trees with branch length andbootstrap support are provided as supplementary material(Figures S1ndashS3)

3 Results and Discussion

31 Wolbachia (Alphaproteobacteria Rickettsiales) In ourstudy 21 of 95 ant species had at least one individual infectedwithWolbachia (Table 1) Across all 304 samples from whichwe obtained data (Supplementary Table 1) we found 30Wolbachia OTUs Overall with 221 of infected speciesthis is a lower infection rate of Wolbachia across ants thanhas been reported before In an extensive compilation ofexisting data about 286 of ant species carried Wolbachiainfections [31] while a frequency of up to 50 had beenfound previously [50] This discrepancy from our study togeneral trends could be due to several reasons Often a speciesis counted as being infected with Wolbachia when just oneindividual carries this infection However not all individualsof a species or individuals from the same colony need to beinfected Thus discrepancies in infection rate across studiesmight merely be due to natural variation among individ-uals Also there is a strong bias in infection rate amongdifferent ant groups Species from the genera AcromyrmexFormica Solenopsis and Tetraponera are often infected withWolbachia while Dolichoderus and Leptogenys mostly lackinfection [51] For example in a screening of 24 Polyrhachisspecies 5 (208) were infected with Wolbachia [31] In thepresent study we found the genera Cephalotes (57) andSolenopsis (50) to have particularly high infection ratesTetraponera (333) and Polyrhachis (250) with intermedi-ate rates Crematogaster (167) and Pseudomyrmex (139)with rather low rates and no infections in the samplesof Camponotus Myrmecia Myrmecocystus Oecophylla andParaponera included here

Most studies that screen for Wolbachia use diagnos-tic approaches by conducting PCR with Wolbachia-specificprimers This is the most reliable means of Wolbachia detec-tion [51]However evenwhen using diagnostic PCR negativeresults can occur due to variations in the primer sequenceor low titers of the bacterial symbionts [52] In our study wefound high variability inWolbachia titers ranging from 103to 9736 (Supplementary Table 1) We used a 1 relativeabundance within a sample as the cutoff to control error ratesof 454 pyrosequencing which might also have led to lowerdetected infection rates among species

In addition to the 30 Wolbachia sequences obtained inthis study we downloaded sequence data from GenBank andcompiled a dataset of 111 taxa including the outgroup Rhi-zobium leguminosarum (Alpha-proteobacteria Rhizobiales)

The total alignment had a length of 1224 characters Four ant-specific clades of Wolbachia were recovered in the inferredtree (Figure 1 Figure S1) Ant clade 1 comprised Wolbachiathat was isolated from Australian Polyrhachis (6 sequences)as well as one sequence detected in Cephalotes variansfrom the Nearctic Ant clade 2 included mostly AustralianPolyrhachis (9 sequences) in addition to sequences foundin Nearctic Solenopsis and Neotropical Pseudomyrmex Antclade 3 exclusively contained sequences from EuropeanFormica species while ant clade 4 was the most diverseThis fourth clade comprised the majority of ant-associatedWolbachia sequences fromour dataset aswell as existingGen-Bank data and included the ant subfamilies DolichoderinaeEcitoninae Formicinae Myrmicinae Ponerinae and Pseu-domyrmecinae from the Afrotropics Nearctics Neotropicsand Palearctics Overall 68 out of 82 (829) ant-associatedWolbachia sequences clustered in ant-specific clades indi-cating a certain degree of host specialization Even thoughneither ant relatedness (subfamily) nor biogeographic region(continent) was a strong determinant for infection withsimilarWolbachia strains relatedWolbachia seemed to infectrelated hosts from the same geographic region to some extentA rather low degree of host specificity has previously beenreported forWolbachia across ants and butterflies while strictcospeciation between Wolbachia and its hosts has not beenfound [51 53]

Wolbachia are reported to be the most prevalent bacterialsymbionts across insects and ants [31] although infectionswith other bacterial groups were often more frequent inour present study Despite this ubiquity to date no studieshave been able to show the functional role of Wolbachia inants This is due to the difficulty of breeding most speciesof ants in the laboratory and thus we have to restrict ourknowledge to the correlations of Wolbachia infections withspecific host traits Wolbachia most commonly manipulatehost reproduction but in ants no such phenomena are known[51] In Formica truncorum Wolbachia infection leads to areduced production of sexuals although this could be dueto physiological costs rather than direct manipulation [54]However worker production is not affected and it has beensuggested thatWolbachiamight reduce the ability of workersto provide resources to alate development [51] Curing ofWolbachia infection within individuals has been observedwhich seems to be unique to ants but themechanisms behindthis phenomenon are not understood [54] Lastly ants oftenshow exceptionally high levels of coinfection with multipleWolbachia strains adding another layer of complexity to thispoorly understood symbiosis [51] It has been speculatedthat eusociality or haplodiploidy might have an impact onWolbachia infection [50 55] but such mechanisms havenever been confirmed Also there seems to be a weakcorrelation of Wolbachia infection with colony foundingmode as species that found new colonies independently areless frequently infected than species relying on dependentcolony founding [50] Speculations on effects of Wolbachiaon colony-founding behavior and colony structure have oftenbeen made as ants can show exceptional variations in thesetraits ranging from a single queen thatmated once tomultiplequeens andor multiple matings per queen [56ndash58]

4 Psyche

Table 1Wolbachia Spiroplasma and Asaia detected by 454 amplicon pyrosequencing across 310 ant samples

Ant genus and subfamily Speciesscreened

Individualsscreened

Coloniesscreened

Number (and percent) of infected speciesand number of individualscolonies

Wolbachia Spiroplasma Asaia

Camponotus (Formicinae) 1 1 1 0 1 (100)11 0

Cephalotes (Myrmicinae) 7 17 12 4 (571)44

2 (285)63

1 (143)11

Crematogaster (Myrmicinae) 6 6 6 1 (167)11 0 0

Myrmecia (Myrmeciinae) 2 3 3 0 0 0Myrmecocystus (Formicinae) 1 1 1 0 0 0Oecophylla (Formicinae) 1 1 1 0 0 0

Paraponera (Paraponerinae) 1 23 9 0 1 (100)22

1 (100)11

Polyrhachis (Formicinae) 32 64 60 8 (250)1010

15 (469)1515 0

Pseudomyrmex (Pseudomyrmecinae) 36 88 72 5 (139)55

5 (139)55

12 (333)1515

Solenopsis (Myrmicinae) 2 11 5 1 (50)11

2 (100)21 0

Tetraponera (Pseudomyrmecinae) 6 6 6 2 (333)22

1 (167)11 0

Total 95 221 176 21 (221) 27 (284) 14 (147)

32 Spiroplasma (Tenericutes Entomoplasmatales) A totalof 27 (284) ant species were infected with Spiroplasmarelatives (Mollicutes Entomoplasmatales) leading to one ofthe highest frequency estimates of this bacterial group acrossthe ants to date (Table 1) Previously an infection rate of62 across ant species had been reported and the infectionrates of approximately 6 were documented for ColeopteraDiptera Hymenoptera and Lepidoptera in general while231 of spiders (Araneae) carried Spiroplasma symbionts[31] There appears to be a strong bias towards certain groupsof ants that are more often associated with this group ofbacteria [31 59] The ant genus Polyrhachis showed a highinfection rate of 469 (15 of 32 species were infected) Thephenomenon of enriched Spiroplasma symbionts in this antgenus is in line with a study by Russell et al [31] and isparticularly interesting as ants of the tribe Camponotinito which Polyrhachis belong carry obligate Blochmanniaendosymbionts which are housed in specific bacteriocytesand provide essential amino acids to the ant host [12 13]Studying the prevalence of spiroplasmas in more generaof the Camponotini particularly the hyperdiverse genusCamponotus would reveal whether these bacteria are likelyto interact within their hosts Infections per species werehigh in Camponotus (11) Paraponera (11) and Solenopsis(22) However these values are not representative due tothe low number of species included Outside of Polyrhachisinfection rates were moderate in the better sampled generaCephalotes (27) Pseudomyrmex (536) and Tetraponera(16) No infection was detected in CrematogasterMyrmeciaMyrmecocystus and Oecophylla (Table 1) Again sampledspecies numbers were low for these ant genera so infectionfrequency can only be regarded as preliminary

An alignment of 175 taxa and 1311 characters wasgenerated including Selenomonas ruminantium (FirmicutesSelenomonadales) as an outgroup In this molecular phy-logeny three large ant-specific clades of spiroplasmaswere identified ant clade 1 that includes endosymbiontsof Cephalotes Solenopsis Tetraponera Pseudomyrmex andNeivamyrmex ant clade 2 that comprises spiroplasma-associates of the ant genera Polyrhachis Camponotus Pseu-domyrmex and Cephalotes and ant clade 3 which wasdominated by army ants (subfamilies Aenictinae Dorylinaeand Ecitoninae) (Figure 2) Additionally several small cladescontaining only ant-associated spiroplasmas were scatteredthroughout the phylogeny as well as several individual ant-associated OTUs Overall bioregion did not seem to be astrong predictor for relatedness among Spiroplasma sym-bionts (Figure 2 Figure S2)

Clade 3 which is dominated by army ants from theNew and Old World has been identified before [60] Inour analysis GenBank-derived Spiroplasma sequences thatwere isolated from the ant genera Odontomachus and Pachy-condyla also fell into this clade (Figure 2) Army ants arecharacterized by the ldquoarmy ant syndromerdquo of nomadismand group predation [61] Due to their specialized dietand a weak correlation of Entomoplasmatales infection withtrophic position a nutritive symbiosis between army ants andEntomoplasmatales has been suggested [60] Even thoughthis clade of Entomoplasmatales is highly dominated by armyants the association is not obligate as infection rates varywithrespect to species and individuals and the symbionts are notnecessary for host development and reproduction [60] AsEntomoplasmatales are generally absent in eggs and larvaehorizontal transmission is assumed

Psyche 5

Sitophilus oryzae AF035160 (rice weevil)

Form

icoxe

nus p

rova

nche

ri

GQ27

5125

(ant

)Co

lletes

inae

quali

s HM

1129

62 (b

ee)

Colle

tes in

aequ

alis H

M11

3014

(bee

)

Colle

tes in

aequ

alis H

M11

3081

(bee

)

Formica

aquilonia GU592784 (ant)

Formica cinerea GU592774 (ant)

Formica cinerea GU592776 (ant)

Formica cinerea GU592770 (ant)

Formica lagubris GU592789 (ant)

Formica cinerea GU592779 (ant)

Formica cinerea GU592777 (ant)

Formica cinerea GU592778 (ant)Formica lagubris GU592786 (ant)Formica polyctena GU592787 (ant)

Formica cinerea GU592781 (ant)

Formica rufa GU592782 (ant)

Formica cinerea GU592772 (ant)

Formica aquilonia GU592783 (ant)

Formica cinerea GU592771 (ant)

Formica cinerea GU592775 (ant)

Formica cinerea GU592780 (ant)

Formica cinerea GU592769 (ant)

Formica polyctena GU592788 (ant)

Formica aquilonia GU592785 (ant)

Formica

cinere

a GU592773 (ant)Philanthus gibbosu

s HM110630 (b

ee)

Form

ica oc

culta

GQ27

5105

(ant

)

Cardioc

ondy

la em

eryi G

Q2751

42 (a

nt)

Philan

thus g

ibbosu

s HM

1108

03 (b

ee)

Philan

thus g

ibbosu

s HM11

0645

(bee)

Odontoponera transversa GQ275132 (ant)

Phlebotomus papatasi WSU80584 (sand fly)

Anoplolepis gracilipes GQ275135 (ant)

Odontomachus clarus GQ275098 (ant)

Drosicha pinicola AB491204 (scale insect)Pheidologeton sp GQ275133 (ant)

Cimex lectularius AB475126 (bed bug)

Cimex lectularius AB475122 (bed bug)Myrmeleon mobilis DQ068830 (ant lion)

Gnamptogenys sp GQ275114 (ant)

Atta colombica HM559216 (ant)

Formica

wheeleri G

Q275103 (ant)

Heteroponera

microps G

Q275145 (ant)

Megalomyrmex latreillei G

Q275110 (ant)Azteca sp GQ275111 (ant)Myrmelachista sp GQ275113 (ant)

Labidus spininodis G

Q275115 (ant)

Pseudomyrmex tenuis BER112 (ant)

Pseudomyrmex PSW54 SK0066 (ant)

Pseudomyrmex filiformis BER114 (ant)

Tetraponera penzigi TP11 (ant)

Tetraponera penzigi KC137101 (ant)

Tetraponera penzigi KC137114 (ant)

Tetraponera penzigi KC137113 (ant)

Tetraponera penzigi KC137106 (ant)

Tetraponera penzigi KC137107 (ant)

Tetraponera penzigi KC137108 (ant)Tetraponera penzigi KC137109 (ant)

Dorymyrmex elegans GQ275124 (ant)Polyergus breviceps GQ275126 (ant)

Sten

amm

a sne

llingi

GQ27

5108

(ant

)

Cepha

lotes

sp SK

1555

(ant)

Cephalo

tes sp

SK15

31 (a

nt)

Pseudom

yrmex

filiform

is BER11

4 (an

t)

Pseudom

yrmex

tenuis B

ER112 (ant)

Pseu

domyr

mex sa

tanicu

s SK00

58 (a

nt)

Cremato

gaste

r sp

SK12

88 (a

nt)

Ceph

alotes

sp S

K152

1 (an

t)

Polyr

hach

is vi

ndex

GQ

2751

34 (a

nt)

Pseu

dom

yrm

ex ap

ache

GQ

2751

07 (a

nt)

Drosophila simulans N

R 074437 (fly)

Leptomyrm

ex sp GQ

275140 (ant)

Rediviva saetigera HM

111302 (bee)

Muscidifurax uniraptor W

OLPA

RTHA

(wasp)

Tetraponera sp SK1300 (ant)

Tetraponera penzigi KC137118 (ant)Redi

viva

saet

iger

a H

M11

1496

(bee

)

Redi

viva

saeti

gera

HM

1113

73 (b

ee)

Redi

viva

saeti

gera

HM

1114

06 (b

ee)

Dro

soph

ila si

mul

ans A

F468

032

(fly)

Redi

viva

saet

igera

HM

1115

63 (b

ee)

Rediviva saetigera HM

111367 (bee)

Rhytidoponera metallica GQ

275141 (ant)

Rediviva saetigera HM

111289 (bee)

Notoncus sp GQ275137 (ant)

Nysius expressus JQ726767 (stinkbug)

Polyrhachis schenckii CSM1035 (ant)

Polyrhachis tubifera CSM1108 (ant) So

lenop

sis ge

min

ata

alat

e B7

(ant

)

Polyr

hach

is sc

henk

ii CS

M10

35 (a

nt)

Pseu

dom

yrm

ex m

ixtec

us SK

0004

(ant

)Po

lyrha

chis

schoo

pae C

SM10

54 (a

nt)

Polyr

hach

is ru

fifem

ur C

SM07

40 (a

nt)

Poly

rhac

his t

hais

CSM

0746

(ant

)

Polyrhachis rufifemur CSM

0655 (ant)

Polyrhachis senilis CSM0798 (ant)

Polyrhachis sp CSM1019 (ant)

Bemisia tabaci JN

204502 (whitefly)

Kleidocerys resedae JQ726770 (catkin bug)

Polyrhachis schenkii CSM1035 (ant)

Polyrhachis senilis CSM1047 (ant)

Polyrhachis sp CSM1019 (ant)

Cephalotes varians CSM1280 (ant)

Polyrhachis tubifera CSM1108 (ant)

Polyrhachis thais CSM0746 (ant)

Polyrhachis monteithi CSM

1096 (ant)Culex pipiens WPU23709 (mosquito)

Kerria lacca JX669531 (scale insect)

Sitophilus oryzae SOMRRNA (rice weevil)

Rhizobium leguminosarum AB740503

Inner circle ant subfamilyDolichoderinaeEctatomminaeEcitoninaeFormicinae

MyrmicinaePonerinaePseudomyrmecinae

Middle circle bioregion

Afrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Ant clade 1

Ant clade 2A

nt clade 3

Ant c

lade 4

Figure 1 Phylogenetic tree ofWolbachia symbionts associated with ants and their closest relatives with sequence data available in GenBankAmaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shownThe host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) Yellow and red branches representbacteria isolated from ant hosts and other insect hosts respectively The inner circle shows ant subfamily and the outer circle refers tothe continent from which host organisms were collected Four ant-specific clades of Wolbachia symbionts are highlighted (Ant clades 1ndash4)Rhizobium leguminosarum was used as an outgroup

Even outside the army ants a certain degree of hostspecificity of Entomoplasmatales bacteria is evident fromour phylogeny and has been described for ants Drosophilaand other arthropod-associated spiroplasmas [60] In ourmolecular phylogeny clades 1 and 2 exclusively contained

ant-associated Entomoplasmatales (Figure 2) However bothclades contained symbionts from different ant subfamiliesand biogeographic regions indicating that neither phylogenynor geographic range drives the association with these sym-bionts and repeated environmental acquisition is common

6 Psyche

Laelapidae sp JF266582 (mite)

Neriene clathrata EU727102 (spider)

Tetragnatha montana EU

727105 (spider)

Meta m

engei EU727103 (spider)

Meta segm

entata EU727104 (spider)

Hepialus gonggaensis EU

344914 (moth)

Tipula oleracea EU727101 (cranefly)

Acyr

thos

ipho

n pi

sum

AB0

4826

3 (a

phid

)Ha

rmon

ia a

xyrid

is AJ

1324

12 (b

eetle

)

Aniso

sticta

nov

emde

cimpu

ncta

ta A

M08

7471

(bee

tle)

Spiro

plas

ma

sp A

Y837

731

(mos

quito

)

Laod

elpha

x stri

atell

us A

B553

862 (

plan

thop

per)

Spiro

plasm

a ixo

detis

GU5

8567

1

Tetra

nych

us ur

ticae

DQ91

0772

(spi

der m

ite)

Dana

us ch

rysip

pus A

J245

996 (

butte

rfly)

Dros

ophi

la an

anas

sae F

J657

247 (

fly)

Cicade

lla vi

ridis E

U7270

97 (le

afhop

per)

Notostir

a elon

gata E

U7270

98 (g

rass b

ug)

Agathem

era cla

razian

a JF26

6577

(phasm

id)

Antonina c

rawii A

B0300

22 (p

seudoco

ccid)

Derman

yssus

gallin

ae EU

6700

52 (p

oultr

y)

Polyr

hach

is tu

bifera

CSM

1108

(ant

)

Polyr

hach

is mon

teithi

CSM

0961

(ant

)

Polyr

hach

is sp

CSM

0983

(ant

)Fann

ia m

anica

ta A

Y569

829

(fly)

Cten

ocep

halid

es fe

lis (fl

ea)

Spiro

plas

ma

sp

JF26

6585

(spi

der)

Poly

rhac

his s

okol

ova

CSM

0854

-2 (a

nt)

Aran

eus d

iade

mat

us E

U72

7099

(spi

der)

Adal

ia b

ipun

ctat

a AJ

0067

75 (b

eetle

)

Chrysolina varians EU727100 (beetle)

Agathemera millepunctata JF266578 (phasmid)

Kerria lacca GU129148 (scale insect)

Trachymyrmex jamaicensis GQ275127 (ant)

Vollenhovia sp HM996753 (ant)

Vollenhovia sp GQ275136 (ant)Dorylus molestus HM996767 (ant)

Pheidole sp HM996747 (ant)

Pheidole soritis HM996754 (ant)

Spiroplasma platyhelix DQ860101

Spiroplasma platyhelix GU993266

Neivamyrmex carolinensis HM996770 (ant)

Neivamyrmex gibbatus HM996776 (ant)

Cephalotes varians JX990286 (ant)Tetraponera sp CSM

0841 (ant)

Solenopsis geminata SRR058848 A9 (ant)

Solenopsis geminata SRR058869 C4 (ant)

Cephalotes varians CSM1323 (ant gut)

Solenopsis geminata SRR058843 A4 (ant)

Cephalotes varians CSM1323 (ant head)

Cephalotes varians CSM1323 (ant leg)

Pseudomyrmex peperi SK1058 (ant)

Mesoplasm

a syrphidae AY231458 (fly)

Mesoplasm

a seiffertii AY351331 (flower)

Photuris pennsylvanica AY177627 (firefly)

Ento

mop

lasm

atal

es JN

5854

56

Myc

opla

sma

myc

oide

s AF0

8535

0

Capr

a hi

rcus

MPU

2605

5 (g

oat)

Ento

mop

lasm

a m

elaleu

cae D

Q51

4607

Myc

opla

sma

mon

odon

AF0

4219

4 (p

raw

ns)

Polyr

hach

is sc

hoop

ae C

SM06

26b

(ant

)

Cicin

dela

cam

pestr

is AF

0369

54 (b

eetle

)

Loph

omyr

mex

sp G

Q27

5146

(ant

)

Para

pone

ra cl

avata

SK12

49-3

(ant

mid

gut)

Para

pone

ra cl

avata

SK12

49-3

(ant

leg)

Ceph

alotes

sp S

K153

4 (an

t gas

ter)

Tetra

mor

ium

hisp

idum

GQ

2751

06 (a

nt)

Ocym

yrm

ex pi

card

i GQ2

7512

8 (an

t)

Pyra

ctom

ena

angu

lata

AY1

5787

1 (fi

refly

)

Spiro

plas

ma

sp J

F276

619 (

earth

worm

)

Ento

mop

lasm

a lu

civor

ax A

F547

212

Firmicu

tes FM252494 (so

il)

Cheliomyrm

ex sp HM996780 (an

t)

Nomamyrmex ese

nbecki H

M996768 (ant)

Nomamyrm

ex hartigii H

M996876 (ant)

Dorylus spininodis HM996755 (ant)Eciton burchellii HM996844 (ant)Dorylus sp HM996757 (ant)

Dorylus mayri HM996733 (ant)

Aenictus sp HM996772 (ant)

Aenictus dentatus HM996763 (ant)

Aenictus aratus HM996762 (ant)

Aenictus sp HM996765 (ant)

Dorylus gerstaeckeri HM996756 (ant)

Aenictus sp HM996764 (ant)

Aenictus cornutus HM996770 (ant)

Aenictus hottai HM996760 (ant)

Aenictus sp HM996775 (ant)

Aenictus cornutus HM996759 (ant)

Dorylus molestus HM996787 (ant)

Aenictus laeviceps HM996781 (ant)

Aenictus fergusoni HM996766 (ant)

Cheliomyrmex sp HM996778 (ant)

Aenictus sp GQ275130 (ant)

Odontomachus brunneus JQ957020 (ant)

Pachycondyla curvinodis JQ957018 (ant)

Odontomachus brunneus JQ957022 (ant)

Eciton lucanoides HM996758 (ant)

Eciton burchellii HM996844 (ant)

Nomamyrmex esenbecki HM996769 (ant)

Eciton vagans HM996761 (ant)

Eciton hamatum GQ275109 (ant)

Cheliomyrmex sp HM996872 (ant)

Cepha

lotes

sp SK

1531

(ant

)

Polyr

hach

is sok

olova

CSM

0854

-1 (a

nt)

Mesopla

sma l

actuc

ae A

F303

132

Calypto

myrmex

beccar

ii HM

9967

48 (a

nt)

Acromyrm

ex oct

ospinosu

s AF49

1879

(ant)

Neivam

yrmex

nigresc

ens (ant)

Photuris pennsylvanicus AY189134 (firefly)

Panorpa helena AY189125 (scorpion fly)

Alcides zodiaca HM

996749 (moth)

Spiroplasma sp AY189320

Culex annulus AY189130 (mosquito)

Spiroplasma sp AY189315

Liriodendron tulipifera AY189131 (tulip)

Entomoplasm

a melaleucae AY345990 (flower)

Aedes sollicitans AY189129 (mosquito)

Pachycondyla striata JQ957013 (ant)

Pachycondyla striata JQ957015 (ant)

Spiroplasma chrysopicola AY189127 (tabanid fly)

Spiroplasma syrphidicola AY189309 (fly)

Ixodes uriae EU727106 (tic

k)

Periplaneta americana JN653133 (cockroach)

Spiroplasma sp AY189313

Odontomachus brunneus JQ957019 (ant)

Drosophila hydei DQ412089 (fly)

Drosophila wheeleri FJ657225 (fly)

Drosophila aldrichi FJ657229 (fly)

Drosophila mojavensis FJ657219 (fly)

Polyrhachis sp CSM1056 (ant)

Polyrhachis sp CSM0785 (ant)

Polyrhachis ornata CSM0718 (ant)

Polyrhachis sp CSM0668 (ant)

Polyrhachis cupreata CSM1016 (ant)

Camponotus sp CSM0644 (ant)

Polyrhachis sp CSM0986 (ant)

Polyrhachis barretti CSM1121 (ant)

Cephalotes sp RA1052 (ant)

Polyrhachis sp CSM1030 (ant)

Polyrhachis sp CSM0998 (ant)

Pseudomyrmex gracilis BER125 (ant)

Pseudomyrmex apache SK0787 (ant)

Cephalotes varians CSM1280 (ant)

Pseudomyrmex PSW37 SK0076 (ant)

Polyrhachis sp CSM0796 (ant)

Pseudomyrmex major SK0990 (ant)

Chorthippus parallelus JN797639 (grasshopper)

Streblidae sp JF266584 (bat fly)

Spiroplasma sp EF151266

Pardosa lugubris EU727108 (spider)Spiroplasma melliferum AY325304

Apis mellifera JQ347516 (honeybee)

Spiroplasma citri X63781

Daucus carota HM124556 (carrot)

Lycaena arota HM996750 (butterfly)

Mesene phareus HM996752 (butterfly)Rhetus periander HM996751 (butterfly)

Myrmica incompleta HM996744 (ant)

Spiroplasma phoeniceum AY772395

Spiroplasma insolitum AY189133

Poritia sumatrae HM996745 (butterfly)

Spiroplasma sp DQ186642

Drosophila melanogaster FJ657180 (fly)

Drosophila willistoni SMERRNA11 (fly)

Drosophila melanogaster AJ631998 (fly)

Drosophila simulans FJ657181 (fly)

Trapezites eliena HM996746 (butterfly)

Anthidium florentinum EU727107 (bee)

Eurygaster

integrice

ps JQ914111 (sh

ield bug)

Panaeus vannamei DQ360069 (sh

rimp)

Eriocheir sinensis

DQ917753 (crab)Lasiu

s alien

us GQ275102 (an

t)

Rimica

ris ex

oculat

a AJ515720 (shrim

p)

Selenomonas ruminantium AB003379

Clade 1

Clade 2

Clad

e 3

Inner circle ant subfamily

DorylinaeEctatomminaeEcitoninae

FormicinaeMyrmicinaePonerinaePseudomyrmecinae

Middle circle bioregionAfrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Aenictinae

Figure 2 Phylogenetic tree of Spiroplasma-related ant symbionts and their closest relatives with sequence data available in GenBank Amaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shown The host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) The branch color refers to the sourcefrom which the bacteria were isolated with yellow representing ant hosts red other insect hosts blue vertebrates and green plants The innercircle refers to the ant subfamily and the outer circle refers to the continent from which samples were collected The three largest ant-specificclades of Spiroplasma symbionts are indicated (Clades 1ndash3) Selenomonas ruminantium was used as an outgroup

The infection with Spiroplasma seems to be systemic as wefound high titers of this bacterium in association with antguts heads and legs (Supplementary Table 1)

Entomoplasmatales can be pathogenic to plants andvertebrates [59 62] and have been isolated from variousinsect taxa including aphids ants bees beetles butterflies

fruit flies and horse flies [63ndash68] Mutualistic spiroplasmascan grant insects resistance to parasitic nematodes [22]and an increased ability to overwinter [69] Pathogenicphenotypes usually lead to insect death [34] and reproductivemanipulation includes altered sex ratios [33] andmale killing[35 70 71] In ants spiroplasmas have been surveyed and

Psyche 7

biocontrol potential has been hypothesized but their roleremains elusive to date [31 38 60] Functional studiesthat compare the performance of infected and uninfectedindividuals would improve our understanding of the role ofthese facultative symbionts

33 Asaia (Alphaproteobacteria Rhodospirillales) Of 95 antspecies 14 hosted bacteria related to Asaia (Alphapro-teobacteria Rhodospirillales) (Table 1) For these bacteriano previous surveys on their prevalence across the antshave been conducted We found a particularly high infectionrate of 333 (1236 species) in Pseudomyrmex In con-trast Asaia-related symbionts were lacking in CamponotusCrematogasterMyrmeciaMyrmecocystus PolyrhachisOeco-phylla and Solenopsis Low infection frequency was presentin Cephalotes (17) and Paraponera (11 species) (Table 1)The enrichment of Asaia symbionts in Pseudomyrmex isparticularly interesting as this ant genus is arboreal andcontains several obligate plant ants which exclusively feed onplant-derived food sources [58 72] However this bacterialgroup occurred facultatively in arboreal generalists and plantmutualists alike indicating that even if these symbionts aremore frequent in arboreal ormutualistic Pseudomyrmex antsthe association is not obligate

In total we obtained 25 Asaia-related OTUs in ourdataset Of these OTUs 21 were associated with Pseu-domyrmex 3 with Paraponera and 1 with Cephalotes Weinferred a maximum likelihood phylogeny of these OTUstheir closest GenBank relatives and other endosymbioticRhodospirillales bacteria fromGenBankThe total alignmentconsisted of 91 taxa and had a length of 1313 characters WeusedWolbachia pipientis (Alphaproteobacteria Rickettsiales)as an outgroup The phylogenetic tree shows three clades inwhich ant-associatedAsaiaOTUs cluster together (Figure 3)(1) a small clade with two Pseudomyrmex-associated OTUsand one Paraponera-associated OTU (2) a clade that appearsto be Hymenoptera specific containing the bulk of Pseu-domyrmex-associated OTUs a Formica-associated sequencefrom GenBank and bacteria isolated from several beespecies and (3) a clade comprised of many insect-associatedAsaia bacteria and five of our OTUs This last clade is par-ticularly interesting as it comprised several strains that wereisolated from different mosquito species as well as three ant-associated Asaia sequences from GenBank One sequence(JF514556) was isolated and cultivated from Tetraponerarufonigra in India [73] The nifH gene a gene associatedwith the fixation of atmospheric nitrogen has also beenfound in this bacterium (GenBank accession JF736510) andit has been experimentally shown that this strain is capableof fixing nitrogen in vitro suggesting possible nitrogen fixingattributes in its natural environment the ant body cavity[73] The two other sequences are cultivated bacteria fromCephalotes varians and were generated in the framework ofa previous study from our lab (GenBank accessions JX445137and JX445138) [39]

Bacteria from the family Acetobacteraceae are commonlyknown as ldquoacetic acid bacteriardquo and have the metaboliccapacity to oxidize ethanol to acetic acid [74] Asaia also

a member of the Acetobacteraceae however only weaklyoxidizes ethanol and shows higher rates of sugar oxidation[74] These bacteria are environmentally ubiquitous buthave also been found in association with insects such asbees [75 76] mosquitoes [77] Drosophila melanogaster [78]leafhoppers [79] and mealybugs [80] All these insects relyon sugar-rich and often nitrogen-limited diets and it hasbeen suggested that the bacteria function as nutritionalsymbionts Some acetic acid bacteria have the capacity tofix atmospheric nitrogen [73] However it remains entirelyspeculative whether this function can be retained in theinsect gut environment and whether these bacteria actuallycontribute to insect nitrogen metabolism or recycling [81]Interestingly neither acetic acid bacteria nor lactic acidbacteria are commonly found in the core gut microbiota ofarboreal Cephalotini ants an ant group with one of the mostthoroughly studied microbiomes [18 19 39] The metaboliccapacities of the core gut microbiota of the Cephalotiniconsisting of Burkholderiales Opitutales PseudomonadalesRhizobiales and Xanthomonadales might be redundant withthe role that acetic acid bacteria play in other insects

In Drosophila acetic acid bacteria are part of the normalcommensal bacterial gut community and can be involved inthe regulation of the innate immune system In healthy fliesa stable equilibrium of different gut microbes is maintainedPerturbation of the normal gut community which can becaused by a defective regulation of antimicrobial peptideleads to the dominance of the pathogenic commensal Glu-conobacter morbifer and ultimately to gut apoptosis [82]Potential other mechanisms by which acetic acid bacteriabenefit insect immunity are by decreasing the gut pHmakingit an unfavorable environment for pathogenic microorgan-isms or by competitive exclusion [81] However these aceticacid bacteria are not essential for the fitness and repro-duction of most insects as even in the well-studied Asaia-mosquito interaction experimental removal of bacteria hadno detectable negative impact on the host [81]

Several studies have been conducted to analyze themicrobial diversity associated with ants [18 19 39 60 83]However the symbiotic relationships of ants with Rhodospir-illales have rarely been observed In fact only two ant-associated Rhodospirillales sequences had been depositedin GenBank (GQ275104 from Formica occulta and JF514556from Tetraponera rufonigra) prior to work from our group[39] Clone libraries generated for theCephalotini ants [18 19]as well as tag-encoded amplicon data sets [38 39] are amongthe most extensive microbial data collections available forants to date and acetic acid bacteria were only sporadicallyassociated with the ant taxa that were investigated Thus theinteraction of Asaia relatives with ants is generally poorlyunderstood but due to the metabolic capacities of thesebacteria to utilize sugar-rich substrates and fix nitrogenthey might play an important nutritional role Particularlythey might be functionally important in the ant subfamilyPseudomyrmecinae in which they seem to be enriched asindicated by our present study

The phylogenetic history of ant-associated Rhodospiril-lales does not show host specificity and suggests likely acqui-sition from the environment (Figure 3) These observations

8 Psyche

Drosophila sp EU096229 (fruit fly)

Drosophila sp EU096235 (fruit fly)

Drosophila sp EU096233 (fruit fly)

Pseudomyrm

ex ejectus SK1181-3 (ant)

Paraponera clavata CS0513-2 (ant mouth)

Gluconobacter frateurii AB682075

Gluc

onob

acte

r fra

teur

ii A

B682

081Gluconobacter frateurii A

B682091

Pseudo

myrmex

ferru

gineu

s SK00

02-1

(ant)

Pseudomyrm

ex pallidus C

SM1333 (ant)

Pseudomyrmex spinicola SK0061-1 (ant)

Pseudomyrmex peperi SK1058-103-1 (ant)

Cephalotes atratus SK1465 (ant)

Xylocopa californica HM112403 (bee)

Caupolicana yarrowi HM109591 (bee)

Euglossa imperialis JQ389882 (bee)

BER145 (ant)Pseudomyrmex sp nr eduardi

Pseudomyrmex satanicus SK0058-1 (ant)Pseudomyrmex dendroicus BER121 (ant)

Pseudomyrmex dendroicus BER103 (ant)

Pseudomyrmex peperi

SK0007-1 (ant)Pseu

domyrm

ex nigroci

nctus

SK0060-1 (a

nt)Pseudom

yrmex

major

SK09

90-8-

3 (an

t gast

er)

Pseu

domyrm

ex de

ndroi

cus S

K1260

(ant)

Form

ica oc

culta

GQ27

5104

(ant

)

Alph

a pro

teoba

cteriu

m A

B513

363 (

flowe

r)

Para

pone

ra cl

avat

a CS

0513

-4 (a

nt m

outh

)Ca

lliop

sis su

balpi

nus H

M10

9230

(bee

)Ca

lliop

sis su

balpi

nus H

M10

9259

(bee

)

Pseu

dom

yrm

ex te

nuis

BER1

12 (a

nt)

Sacc

haric

occu

s sac

char

i AF1

2739

6 (m

ealy

bug)

Dro

soph

ila sp

EU

0962

34 (f

ruit

fly)

Atta colombica H

M557097 (fungus garden)

Aedes aegypti FJ372761 (mosquito)

Aedes aegypti FJ372770 (mosquito)

Anopheles maculipennis AM

404262 (mosquito)

Aedes albopictus JQ958879 (m

osquito)

Aedes albopictus JQ314055 (m

osquito)

Aedes albopictus JQ314027 (m

osquito)

Aedes albopictus F2f JX445138 (mosquito)

Aede

s aeg

ypti

FN81

4303

(mos

quito

)

Aede

s alb

opict

us F

1f JX

4451

37 (m

osqu

ito)

Aede

s alb

opict

us F

2l JX

4451

40 (m

osqu

ito)

Aede

s alb

opict

us F

1l JX

4451

39 (m

osqu

ito)

Aede

s aeg

ypti

FN81

4301

(mos

quito

)

Aede

s aeg

ypti

FN81

4300

(mos

quito

)

Aede

s aeg

ypti

FN29

7842

(mos

quito

)

Anoph

eles s

tephe

nsi A

M40

4261

(mos

quito

)

Aede

s aeg

ypti

FN29

7841

(mos

quito

)Asaia b

ogore

nsis A

B6820

04

Anoph

eles s

p HQ26

9803

(mos

quito

)

Tetrap

onera

rufon

igra JF

5145

56 (a

nt)

Pseudomyrmex elo

ngatus

CSM1373 (ant gut)Pseudomyrmex termitarius

SK1252-1 (ant)

Pseudomyrmex gracilis

CSM1374-2 (ant head)Anopheles gambiae FN814276 (mosquito)Pseudomyrmex ejectus SK1181-3 (ant)

Pseudomyrm

ex elongatus

CSM1373 (ant head)

Cephalotes va

rians JX

990151 (ant)

Anopheles stephensi FN814299 (mosquito)

Anopheles gambiae FN814277 (mosquito)

Anopheles stephensi FN814297 (mosquito)

Anopheles stephensi FN814296 (mosquito)

Cephalotes varians JX990161 (ant)

Lutzomyia longipalpis HE995765 (sand fly)

Anopheles gambiae FN821398 (mosquito)

Aedes aegypti FN297840 (mosquito)

Anopheles stephensi AM404260 (mosquito)

Asaia krungthepensis AB682130

Anopheles gambiae FJ816021 (mosquito)Anopheles gambiae FN814284 (mosquito)Anopheles gambiae FN814283 (mosquito)

Aedes albopictus FN814288 (mosquito)

Aedes albopictus FN814287 (mosquito)

Anopheles gambiae FN821397 (mosquito)

Anopheles gambiae FN814280 (mosquito)

Aedes albopictus FN814289 (mosquito)

Anopheles gambiae FN814285 (mosquito)

Anopheles gambiae FN814275 (mosquito)

Anopheles gambiae FN814281 (mosquito)

Anopheles gambiae FN814278 (mosquito)

Aedes albopictus FN814286 (mosquito)

Atta colombica HM556904 (fungus garden)

Saccharicoccus sacchari AF127400 (mealybug)

Saccharicoccus sacchari AF127408 (mealybug)

Saccharicoccus sacchari AF127401 (mealybug)

Pseudomyrmex ejectus SK1181-3 (ant)

Paraponera clavata CS0513-3 (ant mouth)

Pseudomyrm

ex termitarius SK1252-2 (ant)

Drosophila sp EU096231 (fruit fly)Wolbachia pipientis AY026912

Inner circle ant subfamilyFormicinaeMyrmicinaeParaponerinaePseudomyrmecinae

Middle circle bioregionAfrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Clade 1

Clade 2Cl

ade 3

Figure 3 Phylogenetic tree of Asaia-related symbionts associated with ants and closest relatives with sequence data available in GenBank Amaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shown The host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) The branch color refers to the sourcefromwhich the bacteria were isolated with yellow representing ant hosts and red other insect hostsThe inner circle refers to the ant subfamilyand the middle circle refers to the bioregion from which samples were collected The outer circle indicates three clades (Clades 1ndash3) whichcontained several ant-associated symbiontsWolbachia pipientis was used as an outgroup

indicate that Rhodospirillales aremost likely environmentallytransmitted and support the hypothesis that they are onlyfacultative associates of ants One clade of ant-associatedRhodospirillales was closely related to endosymbionts iso-lated from mosquitos (Figure 3) It has been experimentallyshown that mosquito-associated Asaia can successfully colo-nize leafhoppers further emphasizing the low-host specificityof this bacterial group [77]

4 Conclusion

Our broad bacterial screening approach has contributedto our understanding of the prevalence of ant-associatedmicrobes particularly with regard to their Wolbachia andSpiroplasma symbionts Furthermore we provide the firstextensive survey for ant-associated Asaia-related symbiontsWhile these symbionts of the order Rhodospirillales infect

Psyche 9

ants only sporadically some strains are capable of fixingatmospheric nitrogen and might retain this function inants Alternatively these bacteria might have an importantfunctional role for upgrading nitrogen-poor diets of someherbivorous ants which comprise the majority of all ant taxa[20] Even though we do not have experimental evidence ofthe role of most bacterial symbionts in ants previous studiesillustrate a broad variety of effects of these bacteria on insecthosts [4 7 9] Even a single group of microbes can havevery different effects on different hosts Our study shows thatdespite several extensive bacterial surveys across the ants thediversity and functional role of ant-associated microbes isfar from being fully understood and broad next generationsequencing approaches will provide a fast and cost-effectivetool to deepen our knowledge of the rare (and not so rare)biosphere

Acknowledgments

The authors thank Daniel Ballhorn and Tina Schroyer forhelpful comments and discussions Phil Ward kindly iden-tified Pseudomyrmex specimens This study was supportedin part by National Science Foundation DEB Grant no1050243 to Corrie S Moreau a Grainger Foundation grant toCorrie S Moreau a Negaunee Foundation grant to Corrie SMoreau and the Pritzker Laboratory forMolecular Evolutionand Systematics Stefanie Kautz acknowledges a postdoctoralfellowship from the German Academy of Sciences Leopold-ina (Grant no LPDS 2009-29) Benjamin E R Rubin wassupported in part by an NSF Graduate Research Fellowship

References

[1] H Koch D P Abrol J Li and P Schmid-Hempel ldquoDiversity ofevolutionary patterns of bacterial gut associates of corbiculatebeesrdquoMolecular Ecology vol 22 no 7 pp 2028ndash2044 2013

[2] R J Dillon and V M Dillon ldquoThe Gut bacteria of insectsnonpathogenic interactionsrdquoAnnual Review of Entomology vol49 pp 71ndash92 2004

[3] S Fraune and T C G Bosch ldquoWhy bacteria matter in animaldevelopment and evolutionrdquo BioEssays vol 32 no 7 pp 571ndash580 2010

[4] H Feldhaar ldquoBacterial symbionts as mediators of ecologicallyimportant traits of insect hostsrdquo Ecological Entomology vol 36no 5 pp 533ndash543 2011

[5] J M Urban and J R Cryan ldquoTwo ancient bacterialendosymbionts have coevolved with the planthoppers (InsectaHemiptera Fulgoroidea)rdquo BMC Evolutionary Biology vol 12article 87 2012

[6] H Toju and T Fukatsu ldquoDiversity and infection prevalence ofendosymbionts in natural populations of the chestnut weevilrelevance of local climate and host plantsrdquo Molecular Ecologyvol 20 no 4 pp 853ndash868 2011

[7] M Kaltenpoth ldquoActinobacteria as mutualists general health-care for insectsrdquo Trends inMicrobiology vol 17 no 12 pp 529ndash535 2009

[8] N A Moran J P McCutcheon and A Nakabachi ldquoGenomicsand evolution of heritable bacterial symbiontsrdquo Annual Reviewof Genetics vol 42 pp 165ndash190 2008

[9] J P McCutcheon and C D Von Dohlen ldquoAn interdependentmetabolic patchwork in the nested symbiosis of mealybugsrdquoCurrent Biology vol 21 no 16 pp 1366ndash1372 2011

[10] C B Montllor A Maxmen and A H Purcell ldquoFacultative bac-terial endosymbionts benefit pea aphids Acyrthosiphon pisumunder heat stressrdquo Ecological Entomology vol 27 no 2 pp 189ndash195 2002

[11] A E Douglas ldquoThe nutritional physiology of aphidsrdquoAdvancesin Insect Physiology vol 31 pp 73ndash140 2003

[12] J J Wernegreen S N Kauppinen S G Brady and P SWard ldquoOne nutritional symbiosis begat another phylogeneticevidence that the ant tribe Camponotini acquired Blochmanniaby tending sap-feeding insectsrdquo BMC Evolutionary Biology vol9 no 1 article 292 2009

[13] H Feldhaar J StrakaM Krischke et al ldquoNutritional upgradingfor omnivorous carpenter ants by the endosymbiont Blochman-niardquo BMC Biology vol 5 article 48 2007

[14] A A Pinto-Tomas M A Anderson G Suen et al ldquoSymbioticnitrogen fixation in the fungus gardens of leaf-cutter antsrdquoScience vol 326 no 5956 pp 1120ndash1123 2009

[15] R Pais C Lohs Y Wu J Wang and S Aksoy ldquoThe obligatemutualist Wigglesworthia glossinidia influences reproductiondigestion and immunity processes of its host the tsetse flyrdquoApplied and Environmental Microbiology vol 74 no 19 pp5965ndash5974 2008

[16] J A Breznak ldquoIntestinal microbiota of termites and otherxylophagous insectsrdquo Annual Review of Microbiology vol 36pp 323ndash343 1982

[17] J A Breznak W J Brill J W Mertins and H C CoppelldquoNitrogen fixation in termitesrdquo Nature vol 244 no 5418 pp577ndash579 1973

[18] J A Russell C SMoreau BGoldman-HuertasM FujiwaraDJ Lohman andN E Pierce ldquoBacterial gut symbionts are tightlylinkedwith the evolution of herbivory in antsrdquoProceedings of theNational Academy of Sciences of theUnited States of America vol106 no 50 pp 21236ndash21241 2009

[19] K E Anderson J A Russell C S Moreau et al ldquoHighlysimilar microbial communities are shared among related andtrophically similar ant speciesrdquo Molecular Ecology vol 21 pp2282ndash2296 2012

[20] D W Davidson S C Cook R R Snelling and T H ChualdquoExplaining the abundance of ants in lowland tropical rainforestcanopiesrdquo Science vol 300 no 5621 pp 969ndash972 2003

[21] J C Brownlie and K N Johnson ldquoSymbiont-mediated protec-tion in insect hostsrdquo Trends in Microbiology vol 17 no 8 pp348ndash354 2009

[22] J Jaenike R Unckless S N Cockburn L M Boelio andS J Perlman ldquoAdaptation via symbiosis recent spread of aDrosophila defensive symbiontrdquo Science vol 329 no 5988 pp212ndash215 2012

[23] K M Oliver J A Russell N A Morant and M S HunterldquoFacultative bacterial symbionts in aphids confer resistance toparasitic waspsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 4 pp 1803ndash18072003

[24] C R Currie J A Scott R C Summerbell and D MallochldquoFungus-growing ants use antibiotic-producing bacteria tocontrol garden parasitesrdquo Nature vol 398 pp 701ndash704 1999

[25] M Kaltenpoth W Gottler G Herzner and E Strohm ldquoSym-biotic bacteria protect wasp larvae from fungal infestationrdquoCurrent Biology vol 15 no 5 pp 475ndash479 2005

10 Psyche

[26] H EDunbar A C CWilsonN R Ferguson andNAMoranldquoAphid thermal tolerance is governed by a point mutation inbacterial symbiontsrdquo PLoS Biology vol 5 no 5 pp 1006ndash10152007

[27] T Tsuchida R Koga S Matsumoto and T Fukatsu ldquoInterspe-cific symbiont transfection confers a novel ecological trait to therecipient insectrdquo Biology Letters vol 7 no 2 pp 245ndash248 2011

[28] R Stouthamer J A J Breeuwer and G D D Hurst ldquoWolbachiapipientis microbial manipulator of arthropod reproductionrdquoAnnual Review of Microbiology vol 53 pp 71ndash102 1999

[29] L M Hedges J C Brownlie S L OrsquoNeill and K N JohnsonldquoWolbachia and virus protection in insectsrdquo Science vol 322 no5902 p 702 2008

[30] K Hilgenboecker P Hammerstein P Schlattmann ATelschow and J H Werren ldquoHow many species are infectedwith Wolbachiamdasha statistical analysis of current datardquo FEMSMicrobiology Letters vol 281 no 2 pp 215ndash220 2008

[31] J A Russell C F Funaro Y M Giraldo et al ldquoA veritablemenagerie of heritable bacteria from ants butterflies andbeyond broadmolecular surveys and a systematic reviewrdquoPLoSONE vol 7 no 12 Article ID e51027 2012

[32] O Duron D Bouchon S Boutin et al ldquoThe diversity ofreproductive parasites among arthropods Wolbachia do notwalk alonerdquo BMC Biology vol 6 article 27 2008

[33] D L Williamson and D F Poulson ldquoSex ratio organisms(Spiroplasmas) of Drosophilardquo in The Mycoplasmas III Plantand Insect Mycoplasmas R F Whitcomb and J G Tully Edspp 175ndash208 Academic Press New York NY USA 1979

[34] J M Bove ldquoSpiroplasmas infectious agents of plants arthro-pods and vertebratesrdquoWiener Klinische Wochenschrift vol 109no 14-15 pp 604ndash612 1997

[35] M C Tinsley and M E N Majerus ldquoA new male-killingparasitism Spiroplasma bacteria infect the ladybird beetleAnisosticta novemdecimpunctata (Coleoptera Coccinellidae)rdquoParasitology vol 132 no 6 pp 757ndash765 2006

[36] A Dobson K D Lafferty A M Kuris R F Hechinger andW Jetz ldquoHomage to Linnaeus howmany parasites Howmanyhostsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 105 no 1 pp 11482ndash11489 2008

[37] S T Kelley and S Dobler ldquoComparative analysis of microbialdiversity in Longitarsus flea beetles (Coleoptera Chrysomeli-dae)rdquo Genetica vol 139 no 5 pp 541ndash550 2011

[38] H D Ishak R Plowes R Sen et al ldquoBacterial diversityin Solenopsis invicta and Solenopsis geminata ant coloniescharacterized by 16S amplicon 454 pyrosequencingrdquo MicrobialEcology vol 61 no 4 pp 821ndash831 2011

[39] S Kautz B E R Rubin J A Russell and C S Moreau ldquoSur-veying the microbiome of ants comparing 454 pyrosequencingwith traditionalmethods to uncover bacterial diversityrdquoAppliedand Environmental Microbiology vol 79 no 2 pp 525ndash5342013

[40] S E Dowd T R Callaway R D Wolcott et al ldquoEvaluationof the bacterial diversity in the feces of cattle using 16SrDNA bacterial tag-encoded FLX amplicon pyrosequencing(bTEFAP)rdquo BMCMicrobiology vol 8 article 125 2008

[41] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[42] C Quince A Lanzen R J Davenport and P J TurnbaughldquoRemoving noise from pyrosequenced ampliconsrdquo BMC Bioin-formatics vol 12 article 38 2011

[43] R C Edgar ldquoSearch and clustering orders of magnitude fasterthan BLASTrdquo Bioinformatics vol 26 no 19 pp 2460ndash24612010

[44] J R Cole QWang E Cardenas et al ldquoTheRibosomal databaseproject improved alignments and new tools for rRNA analysisrdquoNucleic Acids Research vol 37 no 1 pp D141ndashD145 2009

[45] T Z DeSantis P Hugenholtz N Larsen et al ldquoGreengenesa chimera-checked 16S rRNA gene database and workbenchcompatible with ARBrdquo Applied and Environmental Microbiol-ogy vol 72 no 7 pp 5069ndash5072 2006

[46] A J Drummond B Ashton S Buxton et al ldquoGeneious v5 4rdquo2011 httpwwwgeneiouscom

[47] A Stamatakis P Hoover and J Rougemont ldquoA rapid bootstrapalgorithm for the RAxML web serversrdquo Systematic Biology vol57 no 5 pp 758ndash771 2008

[48] M A Miller M T Holder R Vos et al ldquoThe CIPRES portalsrdquohttpwwwphyloorgsub sectionsportal

[49] I Letunic and P Bork ldquoInteractive Tree of Life v2 onlineannotation and display of phylogenetic treesmade easyrdquoNucleicAcids Research vol 39 no 2 pp W475ndashW478 2011

[50] T Wenseleers F Ito S van Borm R Huybrechts F Volckaertand J Billen ldquoWidespread occurrence of the micro-organismWolbachia in antsrdquo Proceedings of the Royal Society B vol 265no 1404 pp 1447ndash1452 1998

[51] J A Russell ldquoThe ants (Hymenoptera Formicidae) are uniqueand enigmatic hosts of prevalent Wolbachia (Alphaproteobac-teria) symbiontsrdquoMyrmecological News vol 16 pp 7ndash23 2012

[52] W Arthofer M Riegler D Schneider M Krammer W JMiller and C Stauffer ldquoHidden Wolbachia diversity in fieldpopulations of the European cherry fruit fly Rhagoletis cerasi(Diptera Tephritidae)rdquo Molecular Ecology vol 18 no 18 pp3816ndash3830 2009

[53] J A Russell B Goldman-Huertas C S Moreau et al ldquoSpecial-ization and geographic isolation among Wolbachia symbiontsfrom ants and lycaenid butterfliesrdquo Evolution vol 63 no 3 pp624ndash640 2009

[54] T Wenseleers L Sundstrom and J Billen ldquoDeleterious Wol-bachia in the ant Formica truncorumrdquo Proceedings of the RoyalSociety B vol 269 no 1491 pp 623ndash629 2002

[55] F Vavre F Fleury J Varaldi P Fouillet and M BouletreauldquoEvidence for female mortality in Wolbachia-mediated cyto-plasmic incompatibility in haplodiploid insects epidemiologicand evolutionary consequencesrdquo Evolution vol 54 no 1 pp191ndash200 2000

[56] J Heinze ldquoThe demise of the standard ant (HymenopteraFormicidae)rdquoMyrmecological News vol 11 pp 11ndash20 2008

[57] S Kautz S U Pauls D J Ballhorn H T Lumbsch andM HeilldquoPolygynous supercolonies of the acacia-ant Pseudomyrmexpeperi an inferior colony founderrdquo Molecular Ecology vol 18no 24 pp 5180ndash5194 2009

[58] S Kautz D J Ballhorn J Kroiss et al ldquoHost plant use bycompeting acacia-ants mutualists monopolize while parasitesshare hostsrdquo PLoS ONE vol 7 no 5 Article ID e37691 2012

[59] J Fletcher G A Schultz R E Davis C E Eastman and R MGoodman ldquoBrittle root disease of horseradish-evidence for anetiological role of Spiroplasma citrirdquo Phytopathology vol 71 pp1073ndash1080 1981

[60] C F Funaro D J C Kronauer C S Moreau B Goldman-Huertas N E Pierce and J A Russell ldquoArmy ants harbor ahost-specific clade of Entomoplasmatales bacteriardquo Applied andEnvironmental Microbiology vol 77 no 1 pp 346ndash350 2011

Psyche 11

[61] D J C Kronauer ldquoRecent advances in army ant biology(Hymenoptera Formicidae)rdquoMyrmecological News vol 12 pp51ndash65 2009

[62] F Thiaucourt and G Bolske ldquoContagious caprine pleurop-neumonia and other pulmonary mycoplasmoses of sheep andgoatsrdquo OIE Revue Scientifique et Technique vol 15 no 4 pp1397ndash1414 1996

[63] T Fukatsu T Tsuchida N Nikoh and R Koga ldquoSpiroplasmaSymbiont of the pea aphid Acyrthosiphon pisum (InsectaHomoptera)rdquo Applied and Environmental Microbiology vol 67no 3 pp 1284ndash1291 2001

[64] T S Haselkorn T A Markow and N A Moran ldquoMultipleintroductions of the Spiroplasma bacterial endosymbiont intoDrosophilardquo Molecular Ecology vol 18 no 6 pp 1294ndash13052009

[65] J G Lundgren R M Lehman and J Chee-Sanford ldquoPhysiol-ogy biochemistry and topology bacterial communities withindigestive tracts of ground beetles (Coleoptera Carabidae)rdquoAnnals of the Entomological Society of America vol 100 no 2pp 275ndash282 2007

[66] I Meeus V Vercruysse and G Smagghe ldquoMolecular detectionof Spiroplasma apis and Spiroplasmamelliferum in beesrdquo Journalof Invertebrate Pathology vol 109 no 1 pp 172ndash174 2012

[67] S van Borm J Billen and J J Boomsma ldquoThe diversity ofmicroorganisms associated with Acromyrmex leafcutter antsrdquoBMC Evolutionary Biology vol 2 article 9 2002

[68] J Wedincamp F E French R F Whitcomb and R B HenegerldquoLaboratory infection and release of Spiroplasma (Entomoplas-matales Spiroplasmataceae) from horse flies (Diptera Taban-idae)rdquo Journal of Entomological Science vol 32 no 4 pp 398ndash402 1997

[69] M A Ebbert and L R Nault ldquoImproved overwintering abilityin Dalbulus maidis (Homoptera Cicadellidae) vectors infectedwith Spiroplasma kunkelii (Mycoplasmatales Spiroplasmat-aceae)rdquo Environmental Entomology vol 23 no 3 pp 634ndash6441994

[70] F M Jiggins G D D Hurst C D Jiggins J H G V DSchulenburg and M E N Majerus ldquoThe butterfly Danauschrysippus is infected by a male-killing Spiroplasma bacteriumrdquoParasitology vol 120 no 5 pp 439ndash446 2000

[71] J H G V D Schulenburg G D D Hurst D Tetzlaff GE Booth I A Zakharov and M E N Majerus ldquoHistory ofinfection with different male-killing bacteria in the two-spotladybird beetle Adalia bipunctata revealed through mitochon-drial DNA sequence analysisrdquoGenetics vol 160 no 3 pp 1075ndash1086 2002

[72] S Kautz H T Lumbsch P S Ward and M Heil ldquoHowto prevent cheating a digestive specialization ties mutualisticplant-ants to their ant-plant partnersrdquo Evolution vol 63 no 4pp 839ndash853 2009

[73] N Samaddar A Paul S Chakravorty et al ldquoNitrogen fixationin Asaia sp (Family Acetobacteraceae)rdquo Current Microbiologyvol 63 no 2 pp 226ndash231 2011

[74] Y Ano H Toyama O Adachi and K Matsushita ldquoEnergymetabolism of a unique acetic acid bacteriumAsaia bogorensisthat lacks ethanol oxidation activityrdquo Bioscience Biotechnologyand Biochemistry vol 72 no 4 pp 989ndash997 2008

[75] V G Martinson B N Danforth R L Minckley O RueppellS Tingek and N A Moran ldquoA simple and distinctive micro-biota associated with honey bees and bumble beesrdquo MolecularEcology vol 20 no 3 pp 619ndash628 2011

[76] N A Moran A K Hansen J E Powell and Z L SabreeldquoDistinctive gut microbiota of honey bees assessed using deepsampling from individual worker beesrdquo PLoS ONE vol 7 no 4Article ID e36393 2012

[77] E Crotti C Damiani M Pajoro et al ldquoAsaia a versatile aceticacid bacterial symbiont capable of cross-colonizing insectsof phylogenetically distant genera and ordersrdquo EnvironmentalMicrobiology vol 11 no 12 pp 3252ndash3264 2009

[78] C R Cox and M S Gilmore ldquoNative microbial colonization ofDrosophilamelanogaster and its use as a model of Enterococcusfaecalis pathogenesisrdquo Infection and Immunity vol 75 no 4 pp1565ndash1576 2007

[79] M Marzorati A Alma L Sacchi et al ldquoA novel Bacteroidetessymbiont is localized in Scaphoideus titanus the insect vector ofFlavescence Doree in Vitis viniferardquo Applied and EnvironmentalMicrobiology vol 72 no 2 pp 1467ndash1475 2006

[80] N J Ashbolt and P A Inkerman ldquoAcetic acid bacterial biota ofthe pink sugar cane mealybug Saccharococcus sacchari and itsenvironsrdquo Applied and Environmental Microbiology vol 56 no3 pp 707ndash712 1990

[81] E Crotti A Rizzi B Chouaia et al ldquoAcetic acid bacteria newlyemerging symbionts of insectsrdquo Applied and EnvironmentalMicrobiology vol 76 no 21 pp 6963ndash6970 2010

[82] J Ryu S Kim H Lee et al ldquoInnate immune homeostasis bythe homeobox gene Caudal and commensal-gut mutualism inDrosophilardquo Science vol 319 no 5864 pp 777ndash782 2008

[83] S Stoll J Gadau R Gross and H Feldhaar ldquoBacterial micro-biota associated with ants of the genus Tetraponerardquo BiologicalJournal of the Linnean Society vol 90 no 3 pp 399ndash412 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Volume 2014

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International Journal of

Microbiology

Page 3: Research Article Bacterial Infections across the Ants ...downloads.hindawi.com/journals/psyche/2013/936341.pdf · Department of Zoology, Field Museum of Natural History, South Lake

Psyche 3

was generated using the infernal secondary-structure-basedaligner of the ribosomal database project (RDP) [44] Weinferred a maximum likelihood phylogeny of the most com-mon OTUs and their GenBank relatives using the RAxML728 Black Box [47] on the CIPRES web portal [48] Themodel GTR+I+G was employed We then uploaded the mostlikely tree to the iToL website [49] to facilitate graphicalillustration of bacterial source ant subfamily and geographicregion for each sequence Trees with branch length andbootstrap support are provided as supplementary material(Figures S1ndashS3)

3 Results and Discussion

31 Wolbachia (Alphaproteobacteria Rickettsiales) In ourstudy 21 of 95 ant species had at least one individual infectedwithWolbachia (Table 1) Across all 304 samples from whichwe obtained data (Supplementary Table 1) we found 30Wolbachia OTUs Overall with 221 of infected speciesthis is a lower infection rate of Wolbachia across ants thanhas been reported before In an extensive compilation ofexisting data about 286 of ant species carried Wolbachiainfections [31] while a frequency of up to 50 had beenfound previously [50] This discrepancy from our study togeneral trends could be due to several reasons Often a speciesis counted as being infected with Wolbachia when just oneindividual carries this infection However not all individualsof a species or individuals from the same colony need to beinfected Thus discrepancies in infection rate across studiesmight merely be due to natural variation among individ-uals Also there is a strong bias in infection rate amongdifferent ant groups Species from the genera AcromyrmexFormica Solenopsis and Tetraponera are often infected withWolbachia while Dolichoderus and Leptogenys mostly lackinfection [51] For example in a screening of 24 Polyrhachisspecies 5 (208) were infected with Wolbachia [31] In thepresent study we found the genera Cephalotes (57) andSolenopsis (50) to have particularly high infection ratesTetraponera (333) and Polyrhachis (250) with intermedi-ate rates Crematogaster (167) and Pseudomyrmex (139)with rather low rates and no infections in the samplesof Camponotus Myrmecia Myrmecocystus Oecophylla andParaponera included here

Most studies that screen for Wolbachia use diagnos-tic approaches by conducting PCR with Wolbachia-specificprimers This is the most reliable means of Wolbachia detec-tion [51]However evenwhen using diagnostic PCR negativeresults can occur due to variations in the primer sequenceor low titers of the bacterial symbionts [52] In our study wefound high variability inWolbachia titers ranging from 103to 9736 (Supplementary Table 1) We used a 1 relativeabundance within a sample as the cutoff to control error ratesof 454 pyrosequencing which might also have led to lowerdetected infection rates among species

In addition to the 30 Wolbachia sequences obtained inthis study we downloaded sequence data from GenBank andcompiled a dataset of 111 taxa including the outgroup Rhi-zobium leguminosarum (Alpha-proteobacteria Rhizobiales)

The total alignment had a length of 1224 characters Four ant-specific clades of Wolbachia were recovered in the inferredtree (Figure 1 Figure S1) Ant clade 1 comprised Wolbachiathat was isolated from Australian Polyrhachis (6 sequences)as well as one sequence detected in Cephalotes variansfrom the Nearctic Ant clade 2 included mostly AustralianPolyrhachis (9 sequences) in addition to sequences foundin Nearctic Solenopsis and Neotropical Pseudomyrmex Antclade 3 exclusively contained sequences from EuropeanFormica species while ant clade 4 was the most diverseThis fourth clade comprised the majority of ant-associatedWolbachia sequences fromour dataset aswell as existingGen-Bank data and included the ant subfamilies DolichoderinaeEcitoninae Formicinae Myrmicinae Ponerinae and Pseu-domyrmecinae from the Afrotropics Nearctics Neotropicsand Palearctics Overall 68 out of 82 (829) ant-associatedWolbachia sequences clustered in ant-specific clades indi-cating a certain degree of host specialization Even thoughneither ant relatedness (subfamily) nor biogeographic region(continent) was a strong determinant for infection withsimilarWolbachia strains relatedWolbachia seemed to infectrelated hosts from the same geographic region to some extentA rather low degree of host specificity has previously beenreported forWolbachia across ants and butterflies while strictcospeciation between Wolbachia and its hosts has not beenfound [51 53]

Wolbachia are reported to be the most prevalent bacterialsymbionts across insects and ants [31] although infectionswith other bacterial groups were often more frequent inour present study Despite this ubiquity to date no studieshave been able to show the functional role of Wolbachia inants This is due to the difficulty of breeding most speciesof ants in the laboratory and thus we have to restrict ourknowledge to the correlations of Wolbachia infections withspecific host traits Wolbachia most commonly manipulatehost reproduction but in ants no such phenomena are known[51] In Formica truncorum Wolbachia infection leads to areduced production of sexuals although this could be dueto physiological costs rather than direct manipulation [54]However worker production is not affected and it has beensuggested thatWolbachiamight reduce the ability of workersto provide resources to alate development [51] Curing ofWolbachia infection within individuals has been observedwhich seems to be unique to ants but themechanisms behindthis phenomenon are not understood [54] Lastly ants oftenshow exceptionally high levels of coinfection with multipleWolbachia strains adding another layer of complexity to thispoorly understood symbiosis [51] It has been speculatedthat eusociality or haplodiploidy might have an impact onWolbachia infection [50 55] but such mechanisms havenever been confirmed Also there seems to be a weakcorrelation of Wolbachia infection with colony foundingmode as species that found new colonies independently areless frequently infected than species relying on dependentcolony founding [50] Speculations on effects of Wolbachiaon colony-founding behavior and colony structure have oftenbeen made as ants can show exceptional variations in thesetraits ranging from a single queen thatmated once tomultiplequeens andor multiple matings per queen [56ndash58]

4 Psyche

Table 1Wolbachia Spiroplasma and Asaia detected by 454 amplicon pyrosequencing across 310 ant samples

Ant genus and subfamily Speciesscreened

Individualsscreened

Coloniesscreened

Number (and percent) of infected speciesand number of individualscolonies

Wolbachia Spiroplasma Asaia

Camponotus (Formicinae) 1 1 1 0 1 (100)11 0

Cephalotes (Myrmicinae) 7 17 12 4 (571)44

2 (285)63

1 (143)11

Crematogaster (Myrmicinae) 6 6 6 1 (167)11 0 0

Myrmecia (Myrmeciinae) 2 3 3 0 0 0Myrmecocystus (Formicinae) 1 1 1 0 0 0Oecophylla (Formicinae) 1 1 1 0 0 0

Paraponera (Paraponerinae) 1 23 9 0 1 (100)22

1 (100)11

Polyrhachis (Formicinae) 32 64 60 8 (250)1010

15 (469)1515 0

Pseudomyrmex (Pseudomyrmecinae) 36 88 72 5 (139)55

5 (139)55

12 (333)1515

Solenopsis (Myrmicinae) 2 11 5 1 (50)11

2 (100)21 0

Tetraponera (Pseudomyrmecinae) 6 6 6 2 (333)22

1 (167)11 0

Total 95 221 176 21 (221) 27 (284) 14 (147)

32 Spiroplasma (Tenericutes Entomoplasmatales) A totalof 27 (284) ant species were infected with Spiroplasmarelatives (Mollicutes Entomoplasmatales) leading to one ofthe highest frequency estimates of this bacterial group acrossthe ants to date (Table 1) Previously an infection rate of62 across ant species had been reported and the infectionrates of approximately 6 were documented for ColeopteraDiptera Hymenoptera and Lepidoptera in general while231 of spiders (Araneae) carried Spiroplasma symbionts[31] There appears to be a strong bias towards certain groupsof ants that are more often associated with this group ofbacteria [31 59] The ant genus Polyrhachis showed a highinfection rate of 469 (15 of 32 species were infected) Thephenomenon of enriched Spiroplasma symbionts in this antgenus is in line with a study by Russell et al [31] and isparticularly interesting as ants of the tribe Camponotinito which Polyrhachis belong carry obligate Blochmanniaendosymbionts which are housed in specific bacteriocytesand provide essential amino acids to the ant host [12 13]Studying the prevalence of spiroplasmas in more generaof the Camponotini particularly the hyperdiverse genusCamponotus would reveal whether these bacteria are likelyto interact within their hosts Infections per species werehigh in Camponotus (11) Paraponera (11) and Solenopsis(22) However these values are not representative due tothe low number of species included Outside of Polyrhachisinfection rates were moderate in the better sampled generaCephalotes (27) Pseudomyrmex (536) and Tetraponera(16) No infection was detected in CrematogasterMyrmeciaMyrmecocystus and Oecophylla (Table 1) Again sampledspecies numbers were low for these ant genera so infectionfrequency can only be regarded as preliminary

An alignment of 175 taxa and 1311 characters wasgenerated including Selenomonas ruminantium (FirmicutesSelenomonadales) as an outgroup In this molecular phy-logeny three large ant-specific clades of spiroplasmaswere identified ant clade 1 that includes endosymbiontsof Cephalotes Solenopsis Tetraponera Pseudomyrmex andNeivamyrmex ant clade 2 that comprises spiroplasma-associates of the ant genera Polyrhachis Camponotus Pseu-domyrmex and Cephalotes and ant clade 3 which wasdominated by army ants (subfamilies Aenictinae Dorylinaeand Ecitoninae) (Figure 2) Additionally several small cladescontaining only ant-associated spiroplasmas were scatteredthroughout the phylogeny as well as several individual ant-associated OTUs Overall bioregion did not seem to be astrong predictor for relatedness among Spiroplasma sym-bionts (Figure 2 Figure S2)

Clade 3 which is dominated by army ants from theNew and Old World has been identified before [60] Inour analysis GenBank-derived Spiroplasma sequences thatwere isolated from the ant genera Odontomachus and Pachy-condyla also fell into this clade (Figure 2) Army ants arecharacterized by the ldquoarmy ant syndromerdquo of nomadismand group predation [61] Due to their specialized dietand a weak correlation of Entomoplasmatales infection withtrophic position a nutritive symbiosis between army ants andEntomoplasmatales has been suggested [60] Even thoughthis clade of Entomoplasmatales is highly dominated by armyants the association is not obligate as infection rates varywithrespect to species and individuals and the symbionts are notnecessary for host development and reproduction [60] AsEntomoplasmatales are generally absent in eggs and larvaehorizontal transmission is assumed

Psyche 5

Sitophilus oryzae AF035160 (rice weevil)

Form

icoxe

nus p

rova

nche

ri

GQ27

5125

(ant

)Co

lletes

inae

quali

s HM

1129

62 (b

ee)

Colle

tes in

aequ

alis H

M11

3014

(bee

)

Colle

tes in

aequ

alis H

M11

3081

(bee

)

Formica

aquilonia GU592784 (ant)

Formica cinerea GU592774 (ant)

Formica cinerea GU592776 (ant)

Formica cinerea GU592770 (ant)

Formica lagubris GU592789 (ant)

Formica cinerea GU592779 (ant)

Formica cinerea GU592777 (ant)

Formica cinerea GU592778 (ant)Formica lagubris GU592786 (ant)Formica polyctena GU592787 (ant)

Formica cinerea GU592781 (ant)

Formica rufa GU592782 (ant)

Formica cinerea GU592772 (ant)

Formica aquilonia GU592783 (ant)

Formica cinerea GU592771 (ant)

Formica cinerea GU592775 (ant)

Formica cinerea GU592780 (ant)

Formica cinerea GU592769 (ant)

Formica polyctena GU592788 (ant)

Formica aquilonia GU592785 (ant)

Formica

cinere

a GU592773 (ant)Philanthus gibbosu

s HM110630 (b

ee)

Form

ica oc

culta

GQ27

5105

(ant

)

Cardioc

ondy

la em

eryi G

Q2751

42 (a

nt)

Philan

thus g

ibbosu

s HM

1108

03 (b

ee)

Philan

thus g

ibbosu

s HM11

0645

(bee)

Odontoponera transversa GQ275132 (ant)

Phlebotomus papatasi WSU80584 (sand fly)

Anoplolepis gracilipes GQ275135 (ant)

Odontomachus clarus GQ275098 (ant)

Drosicha pinicola AB491204 (scale insect)Pheidologeton sp GQ275133 (ant)

Cimex lectularius AB475126 (bed bug)

Cimex lectularius AB475122 (bed bug)Myrmeleon mobilis DQ068830 (ant lion)

Gnamptogenys sp GQ275114 (ant)

Atta colombica HM559216 (ant)

Formica

wheeleri G

Q275103 (ant)

Heteroponera

microps G

Q275145 (ant)

Megalomyrmex latreillei G

Q275110 (ant)Azteca sp GQ275111 (ant)Myrmelachista sp GQ275113 (ant)

Labidus spininodis G

Q275115 (ant)

Pseudomyrmex tenuis BER112 (ant)

Pseudomyrmex PSW54 SK0066 (ant)

Pseudomyrmex filiformis BER114 (ant)

Tetraponera penzigi TP11 (ant)

Tetraponera penzigi KC137101 (ant)

Tetraponera penzigi KC137114 (ant)

Tetraponera penzigi KC137113 (ant)

Tetraponera penzigi KC137106 (ant)

Tetraponera penzigi KC137107 (ant)

Tetraponera penzigi KC137108 (ant)Tetraponera penzigi KC137109 (ant)

Dorymyrmex elegans GQ275124 (ant)Polyergus breviceps GQ275126 (ant)

Sten

amm

a sne

llingi

GQ27

5108

(ant

)

Cepha

lotes

sp SK

1555

(ant)

Cephalo

tes sp

SK15

31 (a

nt)

Pseudom

yrmex

filiform

is BER11

4 (an

t)

Pseudom

yrmex

tenuis B

ER112 (ant)

Pseu

domyr

mex sa

tanicu

s SK00

58 (a

nt)

Cremato

gaste

r sp

SK12

88 (a

nt)

Ceph

alotes

sp S

K152

1 (an

t)

Polyr

hach

is vi

ndex

GQ

2751

34 (a

nt)

Pseu

dom

yrm

ex ap

ache

GQ

2751

07 (a

nt)

Drosophila simulans N

R 074437 (fly)

Leptomyrm

ex sp GQ

275140 (ant)

Rediviva saetigera HM

111302 (bee)

Muscidifurax uniraptor W

OLPA

RTHA

(wasp)

Tetraponera sp SK1300 (ant)

Tetraponera penzigi KC137118 (ant)Redi

viva

saet

iger

a H

M11

1496

(bee

)

Redi

viva

saeti

gera

HM

1113

73 (b

ee)

Redi

viva

saeti

gera

HM

1114

06 (b

ee)

Dro

soph

ila si

mul

ans A

F468

032

(fly)

Redi

viva

saet

igera

HM

1115

63 (b

ee)

Rediviva saetigera HM

111367 (bee)

Rhytidoponera metallica GQ

275141 (ant)

Rediviva saetigera HM

111289 (bee)

Notoncus sp GQ275137 (ant)

Nysius expressus JQ726767 (stinkbug)

Polyrhachis schenckii CSM1035 (ant)

Polyrhachis tubifera CSM1108 (ant) So

lenop

sis ge

min

ata

alat

e B7

(ant

)

Polyr

hach

is sc

henk

ii CS

M10

35 (a

nt)

Pseu

dom

yrm

ex m

ixtec

us SK

0004

(ant

)Po

lyrha

chis

schoo

pae C

SM10

54 (a

nt)

Polyr

hach

is ru

fifem

ur C

SM07

40 (a

nt)

Poly

rhac

his t

hais

CSM

0746

(ant

)

Polyrhachis rufifemur CSM

0655 (ant)

Polyrhachis senilis CSM0798 (ant)

Polyrhachis sp CSM1019 (ant)

Bemisia tabaci JN

204502 (whitefly)

Kleidocerys resedae JQ726770 (catkin bug)

Polyrhachis schenkii CSM1035 (ant)

Polyrhachis senilis CSM1047 (ant)

Polyrhachis sp CSM1019 (ant)

Cephalotes varians CSM1280 (ant)

Polyrhachis tubifera CSM1108 (ant)

Polyrhachis thais CSM0746 (ant)

Polyrhachis monteithi CSM

1096 (ant)Culex pipiens WPU23709 (mosquito)

Kerria lacca JX669531 (scale insect)

Sitophilus oryzae SOMRRNA (rice weevil)

Rhizobium leguminosarum AB740503

Inner circle ant subfamilyDolichoderinaeEctatomminaeEcitoninaeFormicinae

MyrmicinaePonerinaePseudomyrmecinae

Middle circle bioregion

Afrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Ant clade 1

Ant clade 2A

nt clade 3

Ant c

lade 4

Figure 1 Phylogenetic tree ofWolbachia symbionts associated with ants and their closest relatives with sequence data available in GenBankAmaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shownThe host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) Yellow and red branches representbacteria isolated from ant hosts and other insect hosts respectively The inner circle shows ant subfamily and the outer circle refers tothe continent from which host organisms were collected Four ant-specific clades of Wolbachia symbionts are highlighted (Ant clades 1ndash4)Rhizobium leguminosarum was used as an outgroup

Even outside the army ants a certain degree of hostspecificity of Entomoplasmatales bacteria is evident fromour phylogeny and has been described for ants Drosophilaand other arthropod-associated spiroplasmas [60] In ourmolecular phylogeny clades 1 and 2 exclusively contained

ant-associated Entomoplasmatales (Figure 2) However bothclades contained symbionts from different ant subfamiliesand biogeographic regions indicating that neither phylogenynor geographic range drives the association with these sym-bionts and repeated environmental acquisition is common

6 Psyche

Laelapidae sp JF266582 (mite)

Neriene clathrata EU727102 (spider)

Tetragnatha montana EU

727105 (spider)

Meta m

engei EU727103 (spider)

Meta segm

entata EU727104 (spider)

Hepialus gonggaensis EU

344914 (moth)

Tipula oleracea EU727101 (cranefly)

Acyr

thos

ipho

n pi

sum

AB0

4826

3 (a

phid

)Ha

rmon

ia a

xyrid

is AJ

1324

12 (b

eetle

)

Aniso

sticta

nov

emde

cimpu

ncta

ta A

M08

7471

(bee

tle)

Spiro

plas

ma

sp A

Y837

731

(mos

quito

)

Laod

elpha

x stri

atell

us A

B553

862 (

plan

thop

per)

Spiro

plasm

a ixo

detis

GU5

8567

1

Tetra

nych

us ur

ticae

DQ91

0772

(spi

der m

ite)

Dana

us ch

rysip

pus A

J245

996 (

butte

rfly)

Dros

ophi

la an

anas

sae F

J657

247 (

fly)

Cicade

lla vi

ridis E

U7270

97 (le

afhop

per)

Notostir

a elon

gata E

U7270

98 (g

rass b

ug)

Agathem

era cla

razian

a JF26

6577

(phasm

id)

Antonina c

rawii A

B0300

22 (p

seudoco

ccid)

Derman

yssus

gallin

ae EU

6700

52 (p

oultr

y)

Polyr

hach

is tu

bifera

CSM

1108

(ant

)

Polyr

hach

is mon

teithi

CSM

0961

(ant

)

Polyr

hach

is sp

CSM

0983

(ant

)Fann

ia m

anica

ta A

Y569

829

(fly)

Cten

ocep

halid

es fe

lis (fl

ea)

Spiro

plas

ma

sp

JF26

6585

(spi

der)

Poly

rhac

his s

okol

ova

CSM

0854

-2 (a

nt)

Aran

eus d

iade

mat

us E

U72

7099

(spi

der)

Adal

ia b

ipun

ctat

a AJ

0067

75 (b

eetle

)

Chrysolina varians EU727100 (beetle)

Agathemera millepunctata JF266578 (phasmid)

Kerria lacca GU129148 (scale insect)

Trachymyrmex jamaicensis GQ275127 (ant)

Vollenhovia sp HM996753 (ant)

Vollenhovia sp GQ275136 (ant)Dorylus molestus HM996767 (ant)

Pheidole sp HM996747 (ant)

Pheidole soritis HM996754 (ant)

Spiroplasma platyhelix DQ860101

Spiroplasma platyhelix GU993266

Neivamyrmex carolinensis HM996770 (ant)

Neivamyrmex gibbatus HM996776 (ant)

Cephalotes varians JX990286 (ant)Tetraponera sp CSM

0841 (ant)

Solenopsis geminata SRR058848 A9 (ant)

Solenopsis geminata SRR058869 C4 (ant)

Cephalotes varians CSM1323 (ant gut)

Solenopsis geminata SRR058843 A4 (ant)

Cephalotes varians CSM1323 (ant head)

Cephalotes varians CSM1323 (ant leg)

Pseudomyrmex peperi SK1058 (ant)

Mesoplasm

a syrphidae AY231458 (fly)

Mesoplasm

a seiffertii AY351331 (flower)

Photuris pennsylvanica AY177627 (firefly)

Ento

mop

lasm

atal

es JN

5854

56

Myc

opla

sma

myc

oide

s AF0

8535

0

Capr

a hi

rcus

MPU

2605

5 (g

oat)

Ento

mop

lasm

a m

elaleu

cae D

Q51

4607

Myc

opla

sma

mon

odon

AF0

4219

4 (p

raw

ns)

Polyr

hach

is sc

hoop

ae C

SM06

26b

(ant

)

Cicin

dela

cam

pestr

is AF

0369

54 (b

eetle

)

Loph

omyr

mex

sp G

Q27

5146

(ant

)

Para

pone

ra cl

avata

SK12

49-3

(ant

mid

gut)

Para

pone

ra cl

avata

SK12

49-3

(ant

leg)

Ceph

alotes

sp S

K153

4 (an

t gas

ter)

Tetra

mor

ium

hisp

idum

GQ

2751

06 (a

nt)

Ocym

yrm

ex pi

card

i GQ2

7512

8 (an

t)

Pyra

ctom

ena

angu

lata

AY1

5787

1 (fi

refly

)

Spiro

plas

ma

sp J

F276

619 (

earth

worm

)

Ento

mop

lasm

a lu

civor

ax A

F547

212

Firmicu

tes FM252494 (so

il)

Cheliomyrm

ex sp HM996780 (an

t)

Nomamyrmex ese

nbecki H

M996768 (ant)

Nomamyrm

ex hartigii H

M996876 (ant)

Dorylus spininodis HM996755 (ant)Eciton burchellii HM996844 (ant)Dorylus sp HM996757 (ant)

Dorylus mayri HM996733 (ant)

Aenictus sp HM996772 (ant)

Aenictus dentatus HM996763 (ant)

Aenictus aratus HM996762 (ant)

Aenictus sp HM996765 (ant)

Dorylus gerstaeckeri HM996756 (ant)

Aenictus sp HM996764 (ant)

Aenictus cornutus HM996770 (ant)

Aenictus hottai HM996760 (ant)

Aenictus sp HM996775 (ant)

Aenictus cornutus HM996759 (ant)

Dorylus molestus HM996787 (ant)

Aenictus laeviceps HM996781 (ant)

Aenictus fergusoni HM996766 (ant)

Cheliomyrmex sp HM996778 (ant)

Aenictus sp GQ275130 (ant)

Odontomachus brunneus JQ957020 (ant)

Pachycondyla curvinodis JQ957018 (ant)

Odontomachus brunneus JQ957022 (ant)

Eciton lucanoides HM996758 (ant)

Eciton burchellii HM996844 (ant)

Nomamyrmex esenbecki HM996769 (ant)

Eciton vagans HM996761 (ant)

Eciton hamatum GQ275109 (ant)

Cheliomyrmex sp HM996872 (ant)

Cepha

lotes

sp SK

1531

(ant

)

Polyr

hach

is sok

olova

CSM

0854

-1 (a

nt)

Mesopla

sma l

actuc

ae A

F303

132

Calypto

myrmex

beccar

ii HM

9967

48 (a

nt)

Acromyrm

ex oct

ospinosu

s AF49

1879

(ant)

Neivam

yrmex

nigresc

ens (ant)

Photuris pennsylvanicus AY189134 (firefly)

Panorpa helena AY189125 (scorpion fly)

Alcides zodiaca HM

996749 (moth)

Spiroplasma sp AY189320

Culex annulus AY189130 (mosquito)

Spiroplasma sp AY189315

Liriodendron tulipifera AY189131 (tulip)

Entomoplasm

a melaleucae AY345990 (flower)

Aedes sollicitans AY189129 (mosquito)

Pachycondyla striata JQ957013 (ant)

Pachycondyla striata JQ957015 (ant)

Spiroplasma chrysopicola AY189127 (tabanid fly)

Spiroplasma syrphidicola AY189309 (fly)

Ixodes uriae EU727106 (tic

k)

Periplaneta americana JN653133 (cockroach)

Spiroplasma sp AY189313

Odontomachus brunneus JQ957019 (ant)

Drosophila hydei DQ412089 (fly)

Drosophila wheeleri FJ657225 (fly)

Drosophila aldrichi FJ657229 (fly)

Drosophila mojavensis FJ657219 (fly)

Polyrhachis sp CSM1056 (ant)

Polyrhachis sp CSM0785 (ant)

Polyrhachis ornata CSM0718 (ant)

Polyrhachis sp CSM0668 (ant)

Polyrhachis cupreata CSM1016 (ant)

Camponotus sp CSM0644 (ant)

Polyrhachis sp CSM0986 (ant)

Polyrhachis barretti CSM1121 (ant)

Cephalotes sp RA1052 (ant)

Polyrhachis sp CSM1030 (ant)

Polyrhachis sp CSM0998 (ant)

Pseudomyrmex gracilis BER125 (ant)

Pseudomyrmex apache SK0787 (ant)

Cephalotes varians CSM1280 (ant)

Pseudomyrmex PSW37 SK0076 (ant)

Polyrhachis sp CSM0796 (ant)

Pseudomyrmex major SK0990 (ant)

Chorthippus parallelus JN797639 (grasshopper)

Streblidae sp JF266584 (bat fly)

Spiroplasma sp EF151266

Pardosa lugubris EU727108 (spider)Spiroplasma melliferum AY325304

Apis mellifera JQ347516 (honeybee)

Spiroplasma citri X63781

Daucus carota HM124556 (carrot)

Lycaena arota HM996750 (butterfly)

Mesene phareus HM996752 (butterfly)Rhetus periander HM996751 (butterfly)

Myrmica incompleta HM996744 (ant)

Spiroplasma phoeniceum AY772395

Spiroplasma insolitum AY189133

Poritia sumatrae HM996745 (butterfly)

Spiroplasma sp DQ186642

Drosophila melanogaster FJ657180 (fly)

Drosophila willistoni SMERRNA11 (fly)

Drosophila melanogaster AJ631998 (fly)

Drosophila simulans FJ657181 (fly)

Trapezites eliena HM996746 (butterfly)

Anthidium florentinum EU727107 (bee)

Eurygaster

integrice

ps JQ914111 (sh

ield bug)

Panaeus vannamei DQ360069 (sh

rimp)

Eriocheir sinensis

DQ917753 (crab)Lasiu

s alien

us GQ275102 (an

t)

Rimica

ris ex

oculat

a AJ515720 (shrim

p)

Selenomonas ruminantium AB003379

Clade 1

Clade 2

Clad

e 3

Inner circle ant subfamily

DorylinaeEctatomminaeEcitoninae

FormicinaeMyrmicinaePonerinaePseudomyrmecinae

Middle circle bioregionAfrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Aenictinae

Figure 2 Phylogenetic tree of Spiroplasma-related ant symbionts and their closest relatives with sequence data available in GenBank Amaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shown The host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) The branch color refers to the sourcefrom which the bacteria were isolated with yellow representing ant hosts red other insect hosts blue vertebrates and green plants The innercircle refers to the ant subfamily and the outer circle refers to the continent from which samples were collected The three largest ant-specificclades of Spiroplasma symbionts are indicated (Clades 1ndash3) Selenomonas ruminantium was used as an outgroup

The infection with Spiroplasma seems to be systemic as wefound high titers of this bacterium in association with antguts heads and legs (Supplementary Table 1)

Entomoplasmatales can be pathogenic to plants andvertebrates [59 62] and have been isolated from variousinsect taxa including aphids ants bees beetles butterflies

fruit flies and horse flies [63ndash68] Mutualistic spiroplasmascan grant insects resistance to parasitic nematodes [22]and an increased ability to overwinter [69] Pathogenicphenotypes usually lead to insect death [34] and reproductivemanipulation includes altered sex ratios [33] andmale killing[35 70 71] In ants spiroplasmas have been surveyed and

Psyche 7

biocontrol potential has been hypothesized but their roleremains elusive to date [31 38 60] Functional studiesthat compare the performance of infected and uninfectedindividuals would improve our understanding of the role ofthese facultative symbionts

33 Asaia (Alphaproteobacteria Rhodospirillales) Of 95 antspecies 14 hosted bacteria related to Asaia (Alphapro-teobacteria Rhodospirillales) (Table 1) For these bacteriano previous surveys on their prevalence across the antshave been conducted We found a particularly high infectionrate of 333 (1236 species) in Pseudomyrmex In con-trast Asaia-related symbionts were lacking in CamponotusCrematogasterMyrmeciaMyrmecocystus PolyrhachisOeco-phylla and Solenopsis Low infection frequency was presentin Cephalotes (17) and Paraponera (11 species) (Table 1)The enrichment of Asaia symbionts in Pseudomyrmex isparticularly interesting as this ant genus is arboreal andcontains several obligate plant ants which exclusively feed onplant-derived food sources [58 72] However this bacterialgroup occurred facultatively in arboreal generalists and plantmutualists alike indicating that even if these symbionts aremore frequent in arboreal ormutualistic Pseudomyrmex antsthe association is not obligate

In total we obtained 25 Asaia-related OTUs in ourdataset Of these OTUs 21 were associated with Pseu-domyrmex 3 with Paraponera and 1 with Cephalotes Weinferred a maximum likelihood phylogeny of these OTUstheir closest GenBank relatives and other endosymbioticRhodospirillales bacteria fromGenBankThe total alignmentconsisted of 91 taxa and had a length of 1313 characters WeusedWolbachia pipientis (Alphaproteobacteria Rickettsiales)as an outgroup The phylogenetic tree shows three clades inwhich ant-associatedAsaiaOTUs cluster together (Figure 3)(1) a small clade with two Pseudomyrmex-associated OTUsand one Paraponera-associated OTU (2) a clade that appearsto be Hymenoptera specific containing the bulk of Pseu-domyrmex-associated OTUs a Formica-associated sequencefrom GenBank and bacteria isolated from several beespecies and (3) a clade comprised of many insect-associatedAsaia bacteria and five of our OTUs This last clade is par-ticularly interesting as it comprised several strains that wereisolated from different mosquito species as well as three ant-associated Asaia sequences from GenBank One sequence(JF514556) was isolated and cultivated from Tetraponerarufonigra in India [73] The nifH gene a gene associatedwith the fixation of atmospheric nitrogen has also beenfound in this bacterium (GenBank accession JF736510) andit has been experimentally shown that this strain is capableof fixing nitrogen in vitro suggesting possible nitrogen fixingattributes in its natural environment the ant body cavity[73] The two other sequences are cultivated bacteria fromCephalotes varians and were generated in the framework ofa previous study from our lab (GenBank accessions JX445137and JX445138) [39]

Bacteria from the family Acetobacteraceae are commonlyknown as ldquoacetic acid bacteriardquo and have the metaboliccapacity to oxidize ethanol to acetic acid [74] Asaia also

a member of the Acetobacteraceae however only weaklyoxidizes ethanol and shows higher rates of sugar oxidation[74] These bacteria are environmentally ubiquitous buthave also been found in association with insects such asbees [75 76] mosquitoes [77] Drosophila melanogaster [78]leafhoppers [79] and mealybugs [80] All these insects relyon sugar-rich and often nitrogen-limited diets and it hasbeen suggested that the bacteria function as nutritionalsymbionts Some acetic acid bacteria have the capacity tofix atmospheric nitrogen [73] However it remains entirelyspeculative whether this function can be retained in theinsect gut environment and whether these bacteria actuallycontribute to insect nitrogen metabolism or recycling [81]Interestingly neither acetic acid bacteria nor lactic acidbacteria are commonly found in the core gut microbiota ofarboreal Cephalotini ants an ant group with one of the mostthoroughly studied microbiomes [18 19 39] The metaboliccapacities of the core gut microbiota of the Cephalotiniconsisting of Burkholderiales Opitutales PseudomonadalesRhizobiales and Xanthomonadales might be redundant withthe role that acetic acid bacteria play in other insects

In Drosophila acetic acid bacteria are part of the normalcommensal bacterial gut community and can be involved inthe regulation of the innate immune system In healthy fliesa stable equilibrium of different gut microbes is maintainedPerturbation of the normal gut community which can becaused by a defective regulation of antimicrobial peptideleads to the dominance of the pathogenic commensal Glu-conobacter morbifer and ultimately to gut apoptosis [82]Potential other mechanisms by which acetic acid bacteriabenefit insect immunity are by decreasing the gut pHmakingit an unfavorable environment for pathogenic microorgan-isms or by competitive exclusion [81] However these aceticacid bacteria are not essential for the fitness and repro-duction of most insects as even in the well-studied Asaia-mosquito interaction experimental removal of bacteria hadno detectable negative impact on the host [81]

Several studies have been conducted to analyze themicrobial diversity associated with ants [18 19 39 60 83]However the symbiotic relationships of ants with Rhodospir-illales have rarely been observed In fact only two ant-associated Rhodospirillales sequences had been depositedin GenBank (GQ275104 from Formica occulta and JF514556from Tetraponera rufonigra) prior to work from our group[39] Clone libraries generated for theCephalotini ants [18 19]as well as tag-encoded amplicon data sets [38 39] are amongthe most extensive microbial data collections available forants to date and acetic acid bacteria were only sporadicallyassociated with the ant taxa that were investigated Thus theinteraction of Asaia relatives with ants is generally poorlyunderstood but due to the metabolic capacities of thesebacteria to utilize sugar-rich substrates and fix nitrogenthey might play an important nutritional role Particularlythey might be functionally important in the ant subfamilyPseudomyrmecinae in which they seem to be enriched asindicated by our present study

The phylogenetic history of ant-associated Rhodospiril-lales does not show host specificity and suggests likely acqui-sition from the environment (Figure 3) These observations

8 Psyche

Drosophila sp EU096229 (fruit fly)

Drosophila sp EU096235 (fruit fly)

Drosophila sp EU096233 (fruit fly)

Pseudomyrm

ex ejectus SK1181-3 (ant)

Paraponera clavata CS0513-2 (ant mouth)

Gluconobacter frateurii AB682075

Gluc

onob

acte

r fra

teur

ii A

B682

081Gluconobacter frateurii A

B682091

Pseudo

myrmex

ferru

gineu

s SK00

02-1

(ant)

Pseudomyrm

ex pallidus C

SM1333 (ant)

Pseudomyrmex spinicola SK0061-1 (ant)

Pseudomyrmex peperi SK1058-103-1 (ant)

Cephalotes atratus SK1465 (ant)

Xylocopa californica HM112403 (bee)

Caupolicana yarrowi HM109591 (bee)

Euglossa imperialis JQ389882 (bee)

BER145 (ant)Pseudomyrmex sp nr eduardi

Pseudomyrmex satanicus SK0058-1 (ant)Pseudomyrmex dendroicus BER121 (ant)

Pseudomyrmex dendroicus BER103 (ant)

Pseudomyrmex peperi

SK0007-1 (ant)Pseu

domyrm

ex nigroci

nctus

SK0060-1 (a

nt)Pseudom

yrmex

major

SK09

90-8-

3 (an

t gast

er)

Pseu

domyrm

ex de

ndroi

cus S

K1260

(ant)

Form

ica oc

culta

GQ27

5104

(ant

)

Alph

a pro

teoba

cteriu

m A

B513

363 (

flowe

r)

Para

pone

ra cl

avat

a CS

0513

-4 (a

nt m

outh

)Ca

lliop

sis su

balpi

nus H

M10

9230

(bee

)Ca

lliop

sis su

balpi

nus H

M10

9259

(bee

)

Pseu

dom

yrm

ex te

nuis

BER1

12 (a

nt)

Sacc

haric

occu

s sac

char

i AF1

2739

6 (m

ealy

bug)

Dro

soph

ila sp

EU

0962

34 (f

ruit

fly)

Atta colombica H

M557097 (fungus garden)

Aedes aegypti FJ372761 (mosquito)

Aedes aegypti FJ372770 (mosquito)

Anopheles maculipennis AM

404262 (mosquito)

Aedes albopictus JQ958879 (m

osquito)

Aedes albopictus JQ314055 (m

osquito)

Aedes albopictus JQ314027 (m

osquito)

Aedes albopictus F2f JX445138 (mosquito)

Aede

s aeg

ypti

FN81

4303

(mos

quito

)

Aede

s alb

opict

us F

1f JX

4451

37 (m

osqu

ito)

Aede

s alb

opict

us F

2l JX

4451

40 (m

osqu

ito)

Aede

s alb

opict

us F

1l JX

4451

39 (m

osqu

ito)

Aede

s aeg

ypti

FN81

4301

(mos

quito

)

Aede

s aeg

ypti

FN81

4300

(mos

quito

)

Aede

s aeg

ypti

FN29

7842

(mos

quito

)

Anoph

eles s

tephe

nsi A

M40

4261

(mos

quito

)

Aede

s aeg

ypti

FN29

7841

(mos

quito

)Asaia b

ogore

nsis A

B6820

04

Anoph

eles s

p HQ26

9803

(mos

quito

)

Tetrap

onera

rufon

igra JF

5145

56 (a

nt)

Pseudomyrmex elo

ngatus

CSM1373 (ant gut)Pseudomyrmex termitarius

SK1252-1 (ant)

Pseudomyrmex gracilis

CSM1374-2 (ant head)Anopheles gambiae FN814276 (mosquito)Pseudomyrmex ejectus SK1181-3 (ant)

Pseudomyrm

ex elongatus

CSM1373 (ant head)

Cephalotes va

rians JX

990151 (ant)

Anopheles stephensi FN814299 (mosquito)

Anopheles gambiae FN814277 (mosquito)

Anopheles stephensi FN814297 (mosquito)

Anopheles stephensi FN814296 (mosquito)

Cephalotes varians JX990161 (ant)

Lutzomyia longipalpis HE995765 (sand fly)

Anopheles gambiae FN821398 (mosquito)

Aedes aegypti FN297840 (mosquito)

Anopheles stephensi AM404260 (mosquito)

Asaia krungthepensis AB682130

Anopheles gambiae FJ816021 (mosquito)Anopheles gambiae FN814284 (mosquito)Anopheles gambiae FN814283 (mosquito)

Aedes albopictus FN814288 (mosquito)

Aedes albopictus FN814287 (mosquito)

Anopheles gambiae FN821397 (mosquito)

Anopheles gambiae FN814280 (mosquito)

Aedes albopictus FN814289 (mosquito)

Anopheles gambiae FN814285 (mosquito)

Anopheles gambiae FN814275 (mosquito)

Anopheles gambiae FN814281 (mosquito)

Anopheles gambiae FN814278 (mosquito)

Aedes albopictus FN814286 (mosquito)

Atta colombica HM556904 (fungus garden)

Saccharicoccus sacchari AF127400 (mealybug)

Saccharicoccus sacchari AF127408 (mealybug)

Saccharicoccus sacchari AF127401 (mealybug)

Pseudomyrmex ejectus SK1181-3 (ant)

Paraponera clavata CS0513-3 (ant mouth)

Pseudomyrm

ex termitarius SK1252-2 (ant)

Drosophila sp EU096231 (fruit fly)Wolbachia pipientis AY026912

Inner circle ant subfamilyFormicinaeMyrmicinaeParaponerinaePseudomyrmecinae

Middle circle bioregionAfrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Clade 1

Clade 2Cl

ade 3

Figure 3 Phylogenetic tree of Asaia-related symbionts associated with ants and closest relatives with sequence data available in GenBank Amaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shown The host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) The branch color refers to the sourcefromwhich the bacteria were isolated with yellow representing ant hosts and red other insect hostsThe inner circle refers to the ant subfamilyand the middle circle refers to the bioregion from which samples were collected The outer circle indicates three clades (Clades 1ndash3) whichcontained several ant-associated symbiontsWolbachia pipientis was used as an outgroup

indicate that Rhodospirillales aremost likely environmentallytransmitted and support the hypothesis that they are onlyfacultative associates of ants One clade of ant-associatedRhodospirillales was closely related to endosymbionts iso-lated from mosquitos (Figure 3) It has been experimentallyshown that mosquito-associated Asaia can successfully colo-nize leafhoppers further emphasizing the low-host specificityof this bacterial group [77]

4 Conclusion

Our broad bacterial screening approach has contributedto our understanding of the prevalence of ant-associatedmicrobes particularly with regard to their Wolbachia andSpiroplasma symbionts Furthermore we provide the firstextensive survey for ant-associated Asaia-related symbiontsWhile these symbionts of the order Rhodospirillales infect

Psyche 9

ants only sporadically some strains are capable of fixingatmospheric nitrogen and might retain this function inants Alternatively these bacteria might have an importantfunctional role for upgrading nitrogen-poor diets of someherbivorous ants which comprise the majority of all ant taxa[20] Even though we do not have experimental evidence ofthe role of most bacterial symbionts in ants previous studiesillustrate a broad variety of effects of these bacteria on insecthosts [4 7 9] Even a single group of microbes can havevery different effects on different hosts Our study shows thatdespite several extensive bacterial surveys across the ants thediversity and functional role of ant-associated microbes isfar from being fully understood and broad next generationsequencing approaches will provide a fast and cost-effectivetool to deepen our knowledge of the rare (and not so rare)biosphere

Acknowledgments

The authors thank Daniel Ballhorn and Tina Schroyer forhelpful comments and discussions Phil Ward kindly iden-tified Pseudomyrmex specimens This study was supportedin part by National Science Foundation DEB Grant no1050243 to Corrie S Moreau a Grainger Foundation grant toCorrie S Moreau a Negaunee Foundation grant to Corrie SMoreau and the Pritzker Laboratory forMolecular Evolutionand Systematics Stefanie Kautz acknowledges a postdoctoralfellowship from the German Academy of Sciences Leopold-ina (Grant no LPDS 2009-29) Benjamin E R Rubin wassupported in part by an NSF Graduate Research Fellowship

References

[1] H Koch D P Abrol J Li and P Schmid-Hempel ldquoDiversity ofevolutionary patterns of bacterial gut associates of corbiculatebeesrdquoMolecular Ecology vol 22 no 7 pp 2028ndash2044 2013

[2] R J Dillon and V M Dillon ldquoThe Gut bacteria of insectsnonpathogenic interactionsrdquoAnnual Review of Entomology vol49 pp 71ndash92 2004

[3] S Fraune and T C G Bosch ldquoWhy bacteria matter in animaldevelopment and evolutionrdquo BioEssays vol 32 no 7 pp 571ndash580 2010

[4] H Feldhaar ldquoBacterial symbionts as mediators of ecologicallyimportant traits of insect hostsrdquo Ecological Entomology vol 36no 5 pp 533ndash543 2011

[5] J M Urban and J R Cryan ldquoTwo ancient bacterialendosymbionts have coevolved with the planthoppers (InsectaHemiptera Fulgoroidea)rdquo BMC Evolutionary Biology vol 12article 87 2012

[6] H Toju and T Fukatsu ldquoDiversity and infection prevalence ofendosymbionts in natural populations of the chestnut weevilrelevance of local climate and host plantsrdquo Molecular Ecologyvol 20 no 4 pp 853ndash868 2011

[7] M Kaltenpoth ldquoActinobacteria as mutualists general health-care for insectsrdquo Trends inMicrobiology vol 17 no 12 pp 529ndash535 2009

[8] N A Moran J P McCutcheon and A Nakabachi ldquoGenomicsand evolution of heritable bacterial symbiontsrdquo Annual Reviewof Genetics vol 42 pp 165ndash190 2008

[9] J P McCutcheon and C D Von Dohlen ldquoAn interdependentmetabolic patchwork in the nested symbiosis of mealybugsrdquoCurrent Biology vol 21 no 16 pp 1366ndash1372 2011

[10] C B Montllor A Maxmen and A H Purcell ldquoFacultative bac-terial endosymbionts benefit pea aphids Acyrthosiphon pisumunder heat stressrdquo Ecological Entomology vol 27 no 2 pp 189ndash195 2002

[11] A E Douglas ldquoThe nutritional physiology of aphidsrdquoAdvancesin Insect Physiology vol 31 pp 73ndash140 2003

[12] J J Wernegreen S N Kauppinen S G Brady and P SWard ldquoOne nutritional symbiosis begat another phylogeneticevidence that the ant tribe Camponotini acquired Blochmanniaby tending sap-feeding insectsrdquo BMC Evolutionary Biology vol9 no 1 article 292 2009

[13] H Feldhaar J StrakaM Krischke et al ldquoNutritional upgradingfor omnivorous carpenter ants by the endosymbiont Blochman-niardquo BMC Biology vol 5 article 48 2007

[14] A A Pinto-Tomas M A Anderson G Suen et al ldquoSymbioticnitrogen fixation in the fungus gardens of leaf-cutter antsrdquoScience vol 326 no 5956 pp 1120ndash1123 2009

[15] R Pais C Lohs Y Wu J Wang and S Aksoy ldquoThe obligatemutualist Wigglesworthia glossinidia influences reproductiondigestion and immunity processes of its host the tsetse flyrdquoApplied and Environmental Microbiology vol 74 no 19 pp5965ndash5974 2008

[16] J A Breznak ldquoIntestinal microbiota of termites and otherxylophagous insectsrdquo Annual Review of Microbiology vol 36pp 323ndash343 1982

[17] J A Breznak W J Brill J W Mertins and H C CoppelldquoNitrogen fixation in termitesrdquo Nature vol 244 no 5418 pp577ndash579 1973

[18] J A Russell C SMoreau BGoldman-HuertasM FujiwaraDJ Lohman andN E Pierce ldquoBacterial gut symbionts are tightlylinkedwith the evolution of herbivory in antsrdquoProceedings of theNational Academy of Sciences of theUnited States of America vol106 no 50 pp 21236ndash21241 2009

[19] K E Anderson J A Russell C S Moreau et al ldquoHighlysimilar microbial communities are shared among related andtrophically similar ant speciesrdquo Molecular Ecology vol 21 pp2282ndash2296 2012

[20] D W Davidson S C Cook R R Snelling and T H ChualdquoExplaining the abundance of ants in lowland tropical rainforestcanopiesrdquo Science vol 300 no 5621 pp 969ndash972 2003

[21] J C Brownlie and K N Johnson ldquoSymbiont-mediated protec-tion in insect hostsrdquo Trends in Microbiology vol 17 no 8 pp348ndash354 2009

[22] J Jaenike R Unckless S N Cockburn L M Boelio andS J Perlman ldquoAdaptation via symbiosis recent spread of aDrosophila defensive symbiontrdquo Science vol 329 no 5988 pp212ndash215 2012

[23] K M Oliver J A Russell N A Morant and M S HunterldquoFacultative bacterial symbionts in aphids confer resistance toparasitic waspsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 4 pp 1803ndash18072003

[24] C R Currie J A Scott R C Summerbell and D MallochldquoFungus-growing ants use antibiotic-producing bacteria tocontrol garden parasitesrdquo Nature vol 398 pp 701ndash704 1999

[25] M Kaltenpoth W Gottler G Herzner and E Strohm ldquoSym-biotic bacteria protect wasp larvae from fungal infestationrdquoCurrent Biology vol 15 no 5 pp 475ndash479 2005

10 Psyche

[26] H EDunbar A C CWilsonN R Ferguson andNAMoranldquoAphid thermal tolerance is governed by a point mutation inbacterial symbiontsrdquo PLoS Biology vol 5 no 5 pp 1006ndash10152007

[27] T Tsuchida R Koga S Matsumoto and T Fukatsu ldquoInterspe-cific symbiont transfection confers a novel ecological trait to therecipient insectrdquo Biology Letters vol 7 no 2 pp 245ndash248 2011

[28] R Stouthamer J A J Breeuwer and G D D Hurst ldquoWolbachiapipientis microbial manipulator of arthropod reproductionrdquoAnnual Review of Microbiology vol 53 pp 71ndash102 1999

[29] L M Hedges J C Brownlie S L OrsquoNeill and K N JohnsonldquoWolbachia and virus protection in insectsrdquo Science vol 322 no5902 p 702 2008

[30] K Hilgenboecker P Hammerstein P Schlattmann ATelschow and J H Werren ldquoHow many species are infectedwith Wolbachiamdasha statistical analysis of current datardquo FEMSMicrobiology Letters vol 281 no 2 pp 215ndash220 2008

[31] J A Russell C F Funaro Y M Giraldo et al ldquoA veritablemenagerie of heritable bacteria from ants butterflies andbeyond broadmolecular surveys and a systematic reviewrdquoPLoSONE vol 7 no 12 Article ID e51027 2012

[32] O Duron D Bouchon S Boutin et al ldquoThe diversity ofreproductive parasites among arthropods Wolbachia do notwalk alonerdquo BMC Biology vol 6 article 27 2008

[33] D L Williamson and D F Poulson ldquoSex ratio organisms(Spiroplasmas) of Drosophilardquo in The Mycoplasmas III Plantand Insect Mycoplasmas R F Whitcomb and J G Tully Edspp 175ndash208 Academic Press New York NY USA 1979

[34] J M Bove ldquoSpiroplasmas infectious agents of plants arthro-pods and vertebratesrdquoWiener Klinische Wochenschrift vol 109no 14-15 pp 604ndash612 1997

[35] M C Tinsley and M E N Majerus ldquoA new male-killingparasitism Spiroplasma bacteria infect the ladybird beetleAnisosticta novemdecimpunctata (Coleoptera Coccinellidae)rdquoParasitology vol 132 no 6 pp 757ndash765 2006

[36] A Dobson K D Lafferty A M Kuris R F Hechinger andW Jetz ldquoHomage to Linnaeus howmany parasites Howmanyhostsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 105 no 1 pp 11482ndash11489 2008

[37] S T Kelley and S Dobler ldquoComparative analysis of microbialdiversity in Longitarsus flea beetles (Coleoptera Chrysomeli-dae)rdquo Genetica vol 139 no 5 pp 541ndash550 2011

[38] H D Ishak R Plowes R Sen et al ldquoBacterial diversityin Solenopsis invicta and Solenopsis geminata ant coloniescharacterized by 16S amplicon 454 pyrosequencingrdquo MicrobialEcology vol 61 no 4 pp 821ndash831 2011

[39] S Kautz B E R Rubin J A Russell and C S Moreau ldquoSur-veying the microbiome of ants comparing 454 pyrosequencingwith traditionalmethods to uncover bacterial diversityrdquoAppliedand Environmental Microbiology vol 79 no 2 pp 525ndash5342013

[40] S E Dowd T R Callaway R D Wolcott et al ldquoEvaluationof the bacterial diversity in the feces of cattle using 16SrDNA bacterial tag-encoded FLX amplicon pyrosequencing(bTEFAP)rdquo BMCMicrobiology vol 8 article 125 2008

[41] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[42] C Quince A Lanzen R J Davenport and P J TurnbaughldquoRemoving noise from pyrosequenced ampliconsrdquo BMC Bioin-formatics vol 12 article 38 2011

[43] R C Edgar ldquoSearch and clustering orders of magnitude fasterthan BLASTrdquo Bioinformatics vol 26 no 19 pp 2460ndash24612010

[44] J R Cole QWang E Cardenas et al ldquoTheRibosomal databaseproject improved alignments and new tools for rRNA analysisrdquoNucleic Acids Research vol 37 no 1 pp D141ndashD145 2009

[45] T Z DeSantis P Hugenholtz N Larsen et al ldquoGreengenesa chimera-checked 16S rRNA gene database and workbenchcompatible with ARBrdquo Applied and Environmental Microbiol-ogy vol 72 no 7 pp 5069ndash5072 2006

[46] A J Drummond B Ashton S Buxton et al ldquoGeneious v5 4rdquo2011 httpwwwgeneiouscom

[47] A Stamatakis P Hoover and J Rougemont ldquoA rapid bootstrapalgorithm for the RAxML web serversrdquo Systematic Biology vol57 no 5 pp 758ndash771 2008

[48] M A Miller M T Holder R Vos et al ldquoThe CIPRES portalsrdquohttpwwwphyloorgsub sectionsportal

[49] I Letunic and P Bork ldquoInteractive Tree of Life v2 onlineannotation and display of phylogenetic treesmade easyrdquoNucleicAcids Research vol 39 no 2 pp W475ndashW478 2011

[50] T Wenseleers F Ito S van Borm R Huybrechts F Volckaertand J Billen ldquoWidespread occurrence of the micro-organismWolbachia in antsrdquo Proceedings of the Royal Society B vol 265no 1404 pp 1447ndash1452 1998

[51] J A Russell ldquoThe ants (Hymenoptera Formicidae) are uniqueand enigmatic hosts of prevalent Wolbachia (Alphaproteobac-teria) symbiontsrdquoMyrmecological News vol 16 pp 7ndash23 2012

[52] W Arthofer M Riegler D Schneider M Krammer W JMiller and C Stauffer ldquoHidden Wolbachia diversity in fieldpopulations of the European cherry fruit fly Rhagoletis cerasi(Diptera Tephritidae)rdquo Molecular Ecology vol 18 no 18 pp3816ndash3830 2009

[53] J A Russell B Goldman-Huertas C S Moreau et al ldquoSpecial-ization and geographic isolation among Wolbachia symbiontsfrom ants and lycaenid butterfliesrdquo Evolution vol 63 no 3 pp624ndash640 2009

[54] T Wenseleers L Sundstrom and J Billen ldquoDeleterious Wol-bachia in the ant Formica truncorumrdquo Proceedings of the RoyalSociety B vol 269 no 1491 pp 623ndash629 2002

[55] F Vavre F Fleury J Varaldi P Fouillet and M BouletreauldquoEvidence for female mortality in Wolbachia-mediated cyto-plasmic incompatibility in haplodiploid insects epidemiologicand evolutionary consequencesrdquo Evolution vol 54 no 1 pp191ndash200 2000

[56] J Heinze ldquoThe demise of the standard ant (HymenopteraFormicidae)rdquoMyrmecological News vol 11 pp 11ndash20 2008

[57] S Kautz S U Pauls D J Ballhorn H T Lumbsch andM HeilldquoPolygynous supercolonies of the acacia-ant Pseudomyrmexpeperi an inferior colony founderrdquo Molecular Ecology vol 18no 24 pp 5180ndash5194 2009

[58] S Kautz D J Ballhorn J Kroiss et al ldquoHost plant use bycompeting acacia-ants mutualists monopolize while parasitesshare hostsrdquo PLoS ONE vol 7 no 5 Article ID e37691 2012

[59] J Fletcher G A Schultz R E Davis C E Eastman and R MGoodman ldquoBrittle root disease of horseradish-evidence for anetiological role of Spiroplasma citrirdquo Phytopathology vol 71 pp1073ndash1080 1981

[60] C F Funaro D J C Kronauer C S Moreau B Goldman-Huertas N E Pierce and J A Russell ldquoArmy ants harbor ahost-specific clade of Entomoplasmatales bacteriardquo Applied andEnvironmental Microbiology vol 77 no 1 pp 346ndash350 2011

Psyche 11

[61] D J C Kronauer ldquoRecent advances in army ant biology(Hymenoptera Formicidae)rdquoMyrmecological News vol 12 pp51ndash65 2009

[62] F Thiaucourt and G Bolske ldquoContagious caprine pleurop-neumonia and other pulmonary mycoplasmoses of sheep andgoatsrdquo OIE Revue Scientifique et Technique vol 15 no 4 pp1397ndash1414 1996

[63] T Fukatsu T Tsuchida N Nikoh and R Koga ldquoSpiroplasmaSymbiont of the pea aphid Acyrthosiphon pisum (InsectaHomoptera)rdquo Applied and Environmental Microbiology vol 67no 3 pp 1284ndash1291 2001

[64] T S Haselkorn T A Markow and N A Moran ldquoMultipleintroductions of the Spiroplasma bacterial endosymbiont intoDrosophilardquo Molecular Ecology vol 18 no 6 pp 1294ndash13052009

[65] J G Lundgren R M Lehman and J Chee-Sanford ldquoPhysiol-ogy biochemistry and topology bacterial communities withindigestive tracts of ground beetles (Coleoptera Carabidae)rdquoAnnals of the Entomological Society of America vol 100 no 2pp 275ndash282 2007

[66] I Meeus V Vercruysse and G Smagghe ldquoMolecular detectionof Spiroplasma apis and Spiroplasmamelliferum in beesrdquo Journalof Invertebrate Pathology vol 109 no 1 pp 172ndash174 2012

[67] S van Borm J Billen and J J Boomsma ldquoThe diversity ofmicroorganisms associated with Acromyrmex leafcutter antsrdquoBMC Evolutionary Biology vol 2 article 9 2002

[68] J Wedincamp F E French R F Whitcomb and R B HenegerldquoLaboratory infection and release of Spiroplasma (Entomoplas-matales Spiroplasmataceae) from horse flies (Diptera Taban-idae)rdquo Journal of Entomological Science vol 32 no 4 pp 398ndash402 1997

[69] M A Ebbert and L R Nault ldquoImproved overwintering abilityin Dalbulus maidis (Homoptera Cicadellidae) vectors infectedwith Spiroplasma kunkelii (Mycoplasmatales Spiroplasmat-aceae)rdquo Environmental Entomology vol 23 no 3 pp 634ndash6441994

[70] F M Jiggins G D D Hurst C D Jiggins J H G V DSchulenburg and M E N Majerus ldquoThe butterfly Danauschrysippus is infected by a male-killing Spiroplasma bacteriumrdquoParasitology vol 120 no 5 pp 439ndash446 2000

[71] J H G V D Schulenburg G D D Hurst D Tetzlaff GE Booth I A Zakharov and M E N Majerus ldquoHistory ofinfection with different male-killing bacteria in the two-spotladybird beetle Adalia bipunctata revealed through mitochon-drial DNA sequence analysisrdquoGenetics vol 160 no 3 pp 1075ndash1086 2002

[72] S Kautz H T Lumbsch P S Ward and M Heil ldquoHowto prevent cheating a digestive specialization ties mutualisticplant-ants to their ant-plant partnersrdquo Evolution vol 63 no 4pp 839ndash853 2009

[73] N Samaddar A Paul S Chakravorty et al ldquoNitrogen fixationin Asaia sp (Family Acetobacteraceae)rdquo Current Microbiologyvol 63 no 2 pp 226ndash231 2011

[74] Y Ano H Toyama O Adachi and K Matsushita ldquoEnergymetabolism of a unique acetic acid bacteriumAsaia bogorensisthat lacks ethanol oxidation activityrdquo Bioscience Biotechnologyand Biochemistry vol 72 no 4 pp 989ndash997 2008

[75] V G Martinson B N Danforth R L Minckley O RueppellS Tingek and N A Moran ldquoA simple and distinctive micro-biota associated with honey bees and bumble beesrdquo MolecularEcology vol 20 no 3 pp 619ndash628 2011

[76] N A Moran A K Hansen J E Powell and Z L SabreeldquoDistinctive gut microbiota of honey bees assessed using deepsampling from individual worker beesrdquo PLoS ONE vol 7 no 4Article ID e36393 2012

[77] E Crotti C Damiani M Pajoro et al ldquoAsaia a versatile aceticacid bacterial symbiont capable of cross-colonizing insectsof phylogenetically distant genera and ordersrdquo EnvironmentalMicrobiology vol 11 no 12 pp 3252ndash3264 2009

[78] C R Cox and M S Gilmore ldquoNative microbial colonization ofDrosophilamelanogaster and its use as a model of Enterococcusfaecalis pathogenesisrdquo Infection and Immunity vol 75 no 4 pp1565ndash1576 2007

[79] M Marzorati A Alma L Sacchi et al ldquoA novel Bacteroidetessymbiont is localized in Scaphoideus titanus the insect vector ofFlavescence Doree in Vitis viniferardquo Applied and EnvironmentalMicrobiology vol 72 no 2 pp 1467ndash1475 2006

[80] N J Ashbolt and P A Inkerman ldquoAcetic acid bacterial biota ofthe pink sugar cane mealybug Saccharococcus sacchari and itsenvironsrdquo Applied and Environmental Microbiology vol 56 no3 pp 707ndash712 1990

[81] E Crotti A Rizzi B Chouaia et al ldquoAcetic acid bacteria newlyemerging symbionts of insectsrdquo Applied and EnvironmentalMicrobiology vol 76 no 21 pp 6963ndash6970 2010

[82] J Ryu S Kim H Lee et al ldquoInnate immune homeostasis bythe homeobox gene Caudal and commensal-gut mutualism inDrosophilardquo Science vol 319 no 5864 pp 777ndash782 2008

[83] S Stoll J Gadau R Gross and H Feldhaar ldquoBacterial micro-biota associated with ants of the genus Tetraponerardquo BiologicalJournal of the Linnean Society vol 90 no 3 pp 399ndash412 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 4: Research Article Bacterial Infections across the Ants ...downloads.hindawi.com/journals/psyche/2013/936341.pdf · Department of Zoology, Field Museum of Natural History, South Lake

4 Psyche

Table 1Wolbachia Spiroplasma and Asaia detected by 454 amplicon pyrosequencing across 310 ant samples

Ant genus and subfamily Speciesscreened

Individualsscreened

Coloniesscreened

Number (and percent) of infected speciesand number of individualscolonies

Wolbachia Spiroplasma Asaia

Camponotus (Formicinae) 1 1 1 0 1 (100)11 0

Cephalotes (Myrmicinae) 7 17 12 4 (571)44

2 (285)63

1 (143)11

Crematogaster (Myrmicinae) 6 6 6 1 (167)11 0 0

Myrmecia (Myrmeciinae) 2 3 3 0 0 0Myrmecocystus (Formicinae) 1 1 1 0 0 0Oecophylla (Formicinae) 1 1 1 0 0 0

Paraponera (Paraponerinae) 1 23 9 0 1 (100)22

1 (100)11

Polyrhachis (Formicinae) 32 64 60 8 (250)1010

15 (469)1515 0

Pseudomyrmex (Pseudomyrmecinae) 36 88 72 5 (139)55

5 (139)55

12 (333)1515

Solenopsis (Myrmicinae) 2 11 5 1 (50)11

2 (100)21 0

Tetraponera (Pseudomyrmecinae) 6 6 6 2 (333)22

1 (167)11 0

Total 95 221 176 21 (221) 27 (284) 14 (147)

32 Spiroplasma (Tenericutes Entomoplasmatales) A totalof 27 (284) ant species were infected with Spiroplasmarelatives (Mollicutes Entomoplasmatales) leading to one ofthe highest frequency estimates of this bacterial group acrossthe ants to date (Table 1) Previously an infection rate of62 across ant species had been reported and the infectionrates of approximately 6 were documented for ColeopteraDiptera Hymenoptera and Lepidoptera in general while231 of spiders (Araneae) carried Spiroplasma symbionts[31] There appears to be a strong bias towards certain groupsof ants that are more often associated with this group ofbacteria [31 59] The ant genus Polyrhachis showed a highinfection rate of 469 (15 of 32 species were infected) Thephenomenon of enriched Spiroplasma symbionts in this antgenus is in line with a study by Russell et al [31] and isparticularly interesting as ants of the tribe Camponotinito which Polyrhachis belong carry obligate Blochmanniaendosymbionts which are housed in specific bacteriocytesand provide essential amino acids to the ant host [12 13]Studying the prevalence of spiroplasmas in more generaof the Camponotini particularly the hyperdiverse genusCamponotus would reveal whether these bacteria are likelyto interact within their hosts Infections per species werehigh in Camponotus (11) Paraponera (11) and Solenopsis(22) However these values are not representative due tothe low number of species included Outside of Polyrhachisinfection rates were moderate in the better sampled generaCephalotes (27) Pseudomyrmex (536) and Tetraponera(16) No infection was detected in CrematogasterMyrmeciaMyrmecocystus and Oecophylla (Table 1) Again sampledspecies numbers were low for these ant genera so infectionfrequency can only be regarded as preliminary

An alignment of 175 taxa and 1311 characters wasgenerated including Selenomonas ruminantium (FirmicutesSelenomonadales) as an outgroup In this molecular phy-logeny three large ant-specific clades of spiroplasmaswere identified ant clade 1 that includes endosymbiontsof Cephalotes Solenopsis Tetraponera Pseudomyrmex andNeivamyrmex ant clade 2 that comprises spiroplasma-associates of the ant genera Polyrhachis Camponotus Pseu-domyrmex and Cephalotes and ant clade 3 which wasdominated by army ants (subfamilies Aenictinae Dorylinaeand Ecitoninae) (Figure 2) Additionally several small cladescontaining only ant-associated spiroplasmas were scatteredthroughout the phylogeny as well as several individual ant-associated OTUs Overall bioregion did not seem to be astrong predictor for relatedness among Spiroplasma sym-bionts (Figure 2 Figure S2)

Clade 3 which is dominated by army ants from theNew and Old World has been identified before [60] Inour analysis GenBank-derived Spiroplasma sequences thatwere isolated from the ant genera Odontomachus and Pachy-condyla also fell into this clade (Figure 2) Army ants arecharacterized by the ldquoarmy ant syndromerdquo of nomadismand group predation [61] Due to their specialized dietand a weak correlation of Entomoplasmatales infection withtrophic position a nutritive symbiosis between army ants andEntomoplasmatales has been suggested [60] Even thoughthis clade of Entomoplasmatales is highly dominated by armyants the association is not obligate as infection rates varywithrespect to species and individuals and the symbionts are notnecessary for host development and reproduction [60] AsEntomoplasmatales are generally absent in eggs and larvaehorizontal transmission is assumed

Psyche 5

Sitophilus oryzae AF035160 (rice weevil)

Form

icoxe

nus p

rova

nche

ri

GQ27

5125

(ant

)Co

lletes

inae

quali

s HM

1129

62 (b

ee)

Colle

tes in

aequ

alis H

M11

3014

(bee

)

Colle

tes in

aequ

alis H

M11

3081

(bee

)

Formica

aquilonia GU592784 (ant)

Formica cinerea GU592774 (ant)

Formica cinerea GU592776 (ant)

Formica cinerea GU592770 (ant)

Formica lagubris GU592789 (ant)

Formica cinerea GU592779 (ant)

Formica cinerea GU592777 (ant)

Formica cinerea GU592778 (ant)Formica lagubris GU592786 (ant)Formica polyctena GU592787 (ant)

Formica cinerea GU592781 (ant)

Formica rufa GU592782 (ant)

Formica cinerea GU592772 (ant)

Formica aquilonia GU592783 (ant)

Formica cinerea GU592771 (ant)

Formica cinerea GU592775 (ant)

Formica cinerea GU592780 (ant)

Formica cinerea GU592769 (ant)

Formica polyctena GU592788 (ant)

Formica aquilonia GU592785 (ant)

Formica

cinere

a GU592773 (ant)Philanthus gibbosu

s HM110630 (b

ee)

Form

ica oc

culta

GQ27

5105

(ant

)

Cardioc

ondy

la em

eryi G

Q2751

42 (a

nt)

Philan

thus g

ibbosu

s HM

1108

03 (b

ee)

Philan

thus g

ibbosu

s HM11

0645

(bee)

Odontoponera transversa GQ275132 (ant)

Phlebotomus papatasi WSU80584 (sand fly)

Anoplolepis gracilipes GQ275135 (ant)

Odontomachus clarus GQ275098 (ant)

Drosicha pinicola AB491204 (scale insect)Pheidologeton sp GQ275133 (ant)

Cimex lectularius AB475126 (bed bug)

Cimex lectularius AB475122 (bed bug)Myrmeleon mobilis DQ068830 (ant lion)

Gnamptogenys sp GQ275114 (ant)

Atta colombica HM559216 (ant)

Formica

wheeleri G

Q275103 (ant)

Heteroponera

microps G

Q275145 (ant)

Megalomyrmex latreillei G

Q275110 (ant)Azteca sp GQ275111 (ant)Myrmelachista sp GQ275113 (ant)

Labidus spininodis G

Q275115 (ant)

Pseudomyrmex tenuis BER112 (ant)

Pseudomyrmex PSW54 SK0066 (ant)

Pseudomyrmex filiformis BER114 (ant)

Tetraponera penzigi TP11 (ant)

Tetraponera penzigi KC137101 (ant)

Tetraponera penzigi KC137114 (ant)

Tetraponera penzigi KC137113 (ant)

Tetraponera penzigi KC137106 (ant)

Tetraponera penzigi KC137107 (ant)

Tetraponera penzigi KC137108 (ant)Tetraponera penzigi KC137109 (ant)

Dorymyrmex elegans GQ275124 (ant)Polyergus breviceps GQ275126 (ant)

Sten

amm

a sne

llingi

GQ27

5108

(ant

)

Cepha

lotes

sp SK

1555

(ant)

Cephalo

tes sp

SK15

31 (a

nt)

Pseudom

yrmex

filiform

is BER11

4 (an

t)

Pseudom

yrmex

tenuis B

ER112 (ant)

Pseu

domyr

mex sa

tanicu

s SK00

58 (a

nt)

Cremato

gaste

r sp

SK12

88 (a

nt)

Ceph

alotes

sp S

K152

1 (an

t)

Polyr

hach

is vi

ndex

GQ

2751

34 (a

nt)

Pseu

dom

yrm

ex ap

ache

GQ

2751

07 (a

nt)

Drosophila simulans N

R 074437 (fly)

Leptomyrm

ex sp GQ

275140 (ant)

Rediviva saetigera HM

111302 (bee)

Muscidifurax uniraptor W

OLPA

RTHA

(wasp)

Tetraponera sp SK1300 (ant)

Tetraponera penzigi KC137118 (ant)Redi

viva

saet

iger

a H

M11

1496

(bee

)

Redi

viva

saeti

gera

HM

1113

73 (b

ee)

Redi

viva

saeti

gera

HM

1114

06 (b

ee)

Dro

soph

ila si

mul

ans A

F468

032

(fly)

Redi

viva

saet

igera

HM

1115

63 (b

ee)

Rediviva saetigera HM

111367 (bee)

Rhytidoponera metallica GQ

275141 (ant)

Rediviva saetigera HM

111289 (bee)

Notoncus sp GQ275137 (ant)

Nysius expressus JQ726767 (stinkbug)

Polyrhachis schenckii CSM1035 (ant)

Polyrhachis tubifera CSM1108 (ant) So

lenop

sis ge

min

ata

alat

e B7

(ant

)

Polyr

hach

is sc

henk

ii CS

M10

35 (a

nt)

Pseu

dom

yrm

ex m

ixtec

us SK

0004

(ant

)Po

lyrha

chis

schoo

pae C

SM10

54 (a

nt)

Polyr

hach

is ru

fifem

ur C

SM07

40 (a

nt)

Poly

rhac

his t

hais

CSM

0746

(ant

)

Polyrhachis rufifemur CSM

0655 (ant)

Polyrhachis senilis CSM0798 (ant)

Polyrhachis sp CSM1019 (ant)

Bemisia tabaci JN

204502 (whitefly)

Kleidocerys resedae JQ726770 (catkin bug)

Polyrhachis schenkii CSM1035 (ant)

Polyrhachis senilis CSM1047 (ant)

Polyrhachis sp CSM1019 (ant)

Cephalotes varians CSM1280 (ant)

Polyrhachis tubifera CSM1108 (ant)

Polyrhachis thais CSM0746 (ant)

Polyrhachis monteithi CSM

1096 (ant)Culex pipiens WPU23709 (mosquito)

Kerria lacca JX669531 (scale insect)

Sitophilus oryzae SOMRRNA (rice weevil)

Rhizobium leguminosarum AB740503

Inner circle ant subfamilyDolichoderinaeEctatomminaeEcitoninaeFormicinae

MyrmicinaePonerinaePseudomyrmecinae

Middle circle bioregion

Afrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Ant clade 1

Ant clade 2A

nt clade 3

Ant c

lade 4

Figure 1 Phylogenetic tree ofWolbachia symbionts associated with ants and their closest relatives with sequence data available in GenBankAmaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shownThe host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) Yellow and red branches representbacteria isolated from ant hosts and other insect hosts respectively The inner circle shows ant subfamily and the outer circle refers tothe continent from which host organisms were collected Four ant-specific clades of Wolbachia symbionts are highlighted (Ant clades 1ndash4)Rhizobium leguminosarum was used as an outgroup

Even outside the army ants a certain degree of hostspecificity of Entomoplasmatales bacteria is evident fromour phylogeny and has been described for ants Drosophilaand other arthropod-associated spiroplasmas [60] In ourmolecular phylogeny clades 1 and 2 exclusively contained

ant-associated Entomoplasmatales (Figure 2) However bothclades contained symbionts from different ant subfamiliesand biogeographic regions indicating that neither phylogenynor geographic range drives the association with these sym-bionts and repeated environmental acquisition is common

6 Psyche

Laelapidae sp JF266582 (mite)

Neriene clathrata EU727102 (spider)

Tetragnatha montana EU

727105 (spider)

Meta m

engei EU727103 (spider)

Meta segm

entata EU727104 (spider)

Hepialus gonggaensis EU

344914 (moth)

Tipula oleracea EU727101 (cranefly)

Acyr

thos

ipho

n pi

sum

AB0

4826

3 (a

phid

)Ha

rmon

ia a

xyrid

is AJ

1324

12 (b

eetle

)

Aniso

sticta

nov

emde

cimpu

ncta

ta A

M08

7471

(bee

tle)

Spiro

plas

ma

sp A

Y837

731

(mos

quito

)

Laod

elpha

x stri

atell

us A

B553

862 (

plan

thop

per)

Spiro

plasm

a ixo

detis

GU5

8567

1

Tetra

nych

us ur

ticae

DQ91

0772

(spi

der m

ite)

Dana

us ch

rysip

pus A

J245

996 (

butte

rfly)

Dros

ophi

la an

anas

sae F

J657

247 (

fly)

Cicade

lla vi

ridis E

U7270

97 (le

afhop

per)

Notostir

a elon

gata E

U7270

98 (g

rass b

ug)

Agathem

era cla

razian

a JF26

6577

(phasm

id)

Antonina c

rawii A

B0300

22 (p

seudoco

ccid)

Derman

yssus

gallin

ae EU

6700

52 (p

oultr

y)

Polyr

hach

is tu

bifera

CSM

1108

(ant

)

Polyr

hach

is mon

teithi

CSM

0961

(ant

)

Polyr

hach

is sp

CSM

0983

(ant

)Fann

ia m

anica

ta A

Y569

829

(fly)

Cten

ocep

halid

es fe

lis (fl

ea)

Spiro

plas

ma

sp

JF26

6585

(spi

der)

Poly

rhac

his s

okol

ova

CSM

0854

-2 (a

nt)

Aran

eus d

iade

mat

us E

U72

7099

(spi

der)

Adal

ia b

ipun

ctat

a AJ

0067

75 (b

eetle

)

Chrysolina varians EU727100 (beetle)

Agathemera millepunctata JF266578 (phasmid)

Kerria lacca GU129148 (scale insect)

Trachymyrmex jamaicensis GQ275127 (ant)

Vollenhovia sp HM996753 (ant)

Vollenhovia sp GQ275136 (ant)Dorylus molestus HM996767 (ant)

Pheidole sp HM996747 (ant)

Pheidole soritis HM996754 (ant)

Spiroplasma platyhelix DQ860101

Spiroplasma platyhelix GU993266

Neivamyrmex carolinensis HM996770 (ant)

Neivamyrmex gibbatus HM996776 (ant)

Cephalotes varians JX990286 (ant)Tetraponera sp CSM

0841 (ant)

Solenopsis geminata SRR058848 A9 (ant)

Solenopsis geminata SRR058869 C4 (ant)

Cephalotes varians CSM1323 (ant gut)

Solenopsis geminata SRR058843 A4 (ant)

Cephalotes varians CSM1323 (ant head)

Cephalotes varians CSM1323 (ant leg)

Pseudomyrmex peperi SK1058 (ant)

Mesoplasm

a syrphidae AY231458 (fly)

Mesoplasm

a seiffertii AY351331 (flower)

Photuris pennsylvanica AY177627 (firefly)

Ento

mop

lasm

atal

es JN

5854

56

Myc

opla

sma

myc

oide

s AF0

8535

0

Capr

a hi

rcus

MPU

2605

5 (g

oat)

Ento

mop

lasm

a m

elaleu

cae D

Q51

4607

Myc

opla

sma

mon

odon

AF0

4219

4 (p

raw

ns)

Polyr

hach

is sc

hoop

ae C

SM06

26b

(ant

)

Cicin

dela

cam

pestr

is AF

0369

54 (b

eetle

)

Loph

omyr

mex

sp G

Q27

5146

(ant

)

Para

pone

ra cl

avata

SK12

49-3

(ant

mid

gut)

Para

pone

ra cl

avata

SK12

49-3

(ant

leg)

Ceph

alotes

sp S

K153

4 (an

t gas

ter)

Tetra

mor

ium

hisp

idum

GQ

2751

06 (a

nt)

Ocym

yrm

ex pi

card

i GQ2

7512

8 (an

t)

Pyra

ctom

ena

angu

lata

AY1

5787

1 (fi

refly

)

Spiro

plas

ma

sp J

F276

619 (

earth

worm

)

Ento

mop

lasm

a lu

civor

ax A

F547

212

Firmicu

tes FM252494 (so

il)

Cheliomyrm

ex sp HM996780 (an

t)

Nomamyrmex ese

nbecki H

M996768 (ant)

Nomamyrm

ex hartigii H

M996876 (ant)

Dorylus spininodis HM996755 (ant)Eciton burchellii HM996844 (ant)Dorylus sp HM996757 (ant)

Dorylus mayri HM996733 (ant)

Aenictus sp HM996772 (ant)

Aenictus dentatus HM996763 (ant)

Aenictus aratus HM996762 (ant)

Aenictus sp HM996765 (ant)

Dorylus gerstaeckeri HM996756 (ant)

Aenictus sp HM996764 (ant)

Aenictus cornutus HM996770 (ant)

Aenictus hottai HM996760 (ant)

Aenictus sp HM996775 (ant)

Aenictus cornutus HM996759 (ant)

Dorylus molestus HM996787 (ant)

Aenictus laeviceps HM996781 (ant)

Aenictus fergusoni HM996766 (ant)

Cheliomyrmex sp HM996778 (ant)

Aenictus sp GQ275130 (ant)

Odontomachus brunneus JQ957020 (ant)

Pachycondyla curvinodis JQ957018 (ant)

Odontomachus brunneus JQ957022 (ant)

Eciton lucanoides HM996758 (ant)

Eciton burchellii HM996844 (ant)

Nomamyrmex esenbecki HM996769 (ant)

Eciton vagans HM996761 (ant)

Eciton hamatum GQ275109 (ant)

Cheliomyrmex sp HM996872 (ant)

Cepha

lotes

sp SK

1531

(ant

)

Polyr

hach

is sok

olova

CSM

0854

-1 (a

nt)

Mesopla

sma l

actuc

ae A

F303

132

Calypto

myrmex

beccar

ii HM

9967

48 (a

nt)

Acromyrm

ex oct

ospinosu

s AF49

1879

(ant)

Neivam

yrmex

nigresc

ens (ant)

Photuris pennsylvanicus AY189134 (firefly)

Panorpa helena AY189125 (scorpion fly)

Alcides zodiaca HM

996749 (moth)

Spiroplasma sp AY189320

Culex annulus AY189130 (mosquito)

Spiroplasma sp AY189315

Liriodendron tulipifera AY189131 (tulip)

Entomoplasm

a melaleucae AY345990 (flower)

Aedes sollicitans AY189129 (mosquito)

Pachycondyla striata JQ957013 (ant)

Pachycondyla striata JQ957015 (ant)

Spiroplasma chrysopicola AY189127 (tabanid fly)

Spiroplasma syrphidicola AY189309 (fly)

Ixodes uriae EU727106 (tic

k)

Periplaneta americana JN653133 (cockroach)

Spiroplasma sp AY189313

Odontomachus brunneus JQ957019 (ant)

Drosophila hydei DQ412089 (fly)

Drosophila wheeleri FJ657225 (fly)

Drosophila aldrichi FJ657229 (fly)

Drosophila mojavensis FJ657219 (fly)

Polyrhachis sp CSM1056 (ant)

Polyrhachis sp CSM0785 (ant)

Polyrhachis ornata CSM0718 (ant)

Polyrhachis sp CSM0668 (ant)

Polyrhachis cupreata CSM1016 (ant)

Camponotus sp CSM0644 (ant)

Polyrhachis sp CSM0986 (ant)

Polyrhachis barretti CSM1121 (ant)

Cephalotes sp RA1052 (ant)

Polyrhachis sp CSM1030 (ant)

Polyrhachis sp CSM0998 (ant)

Pseudomyrmex gracilis BER125 (ant)

Pseudomyrmex apache SK0787 (ant)

Cephalotes varians CSM1280 (ant)

Pseudomyrmex PSW37 SK0076 (ant)

Polyrhachis sp CSM0796 (ant)

Pseudomyrmex major SK0990 (ant)

Chorthippus parallelus JN797639 (grasshopper)

Streblidae sp JF266584 (bat fly)

Spiroplasma sp EF151266

Pardosa lugubris EU727108 (spider)Spiroplasma melliferum AY325304

Apis mellifera JQ347516 (honeybee)

Spiroplasma citri X63781

Daucus carota HM124556 (carrot)

Lycaena arota HM996750 (butterfly)

Mesene phareus HM996752 (butterfly)Rhetus periander HM996751 (butterfly)

Myrmica incompleta HM996744 (ant)

Spiroplasma phoeniceum AY772395

Spiroplasma insolitum AY189133

Poritia sumatrae HM996745 (butterfly)

Spiroplasma sp DQ186642

Drosophila melanogaster FJ657180 (fly)

Drosophila willistoni SMERRNA11 (fly)

Drosophila melanogaster AJ631998 (fly)

Drosophila simulans FJ657181 (fly)

Trapezites eliena HM996746 (butterfly)

Anthidium florentinum EU727107 (bee)

Eurygaster

integrice

ps JQ914111 (sh

ield bug)

Panaeus vannamei DQ360069 (sh

rimp)

Eriocheir sinensis

DQ917753 (crab)Lasiu

s alien

us GQ275102 (an

t)

Rimica

ris ex

oculat

a AJ515720 (shrim

p)

Selenomonas ruminantium AB003379

Clade 1

Clade 2

Clad

e 3

Inner circle ant subfamily

DorylinaeEctatomminaeEcitoninae

FormicinaeMyrmicinaePonerinaePseudomyrmecinae

Middle circle bioregionAfrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Aenictinae

Figure 2 Phylogenetic tree of Spiroplasma-related ant symbionts and their closest relatives with sequence data available in GenBank Amaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shown The host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) The branch color refers to the sourcefrom which the bacteria were isolated with yellow representing ant hosts red other insect hosts blue vertebrates and green plants The innercircle refers to the ant subfamily and the outer circle refers to the continent from which samples were collected The three largest ant-specificclades of Spiroplasma symbionts are indicated (Clades 1ndash3) Selenomonas ruminantium was used as an outgroup

The infection with Spiroplasma seems to be systemic as wefound high titers of this bacterium in association with antguts heads and legs (Supplementary Table 1)

Entomoplasmatales can be pathogenic to plants andvertebrates [59 62] and have been isolated from variousinsect taxa including aphids ants bees beetles butterflies

fruit flies and horse flies [63ndash68] Mutualistic spiroplasmascan grant insects resistance to parasitic nematodes [22]and an increased ability to overwinter [69] Pathogenicphenotypes usually lead to insect death [34] and reproductivemanipulation includes altered sex ratios [33] andmale killing[35 70 71] In ants spiroplasmas have been surveyed and

Psyche 7

biocontrol potential has been hypothesized but their roleremains elusive to date [31 38 60] Functional studiesthat compare the performance of infected and uninfectedindividuals would improve our understanding of the role ofthese facultative symbionts

33 Asaia (Alphaproteobacteria Rhodospirillales) Of 95 antspecies 14 hosted bacteria related to Asaia (Alphapro-teobacteria Rhodospirillales) (Table 1) For these bacteriano previous surveys on their prevalence across the antshave been conducted We found a particularly high infectionrate of 333 (1236 species) in Pseudomyrmex In con-trast Asaia-related symbionts were lacking in CamponotusCrematogasterMyrmeciaMyrmecocystus PolyrhachisOeco-phylla and Solenopsis Low infection frequency was presentin Cephalotes (17) and Paraponera (11 species) (Table 1)The enrichment of Asaia symbionts in Pseudomyrmex isparticularly interesting as this ant genus is arboreal andcontains several obligate plant ants which exclusively feed onplant-derived food sources [58 72] However this bacterialgroup occurred facultatively in arboreal generalists and plantmutualists alike indicating that even if these symbionts aremore frequent in arboreal ormutualistic Pseudomyrmex antsthe association is not obligate

In total we obtained 25 Asaia-related OTUs in ourdataset Of these OTUs 21 were associated with Pseu-domyrmex 3 with Paraponera and 1 with Cephalotes Weinferred a maximum likelihood phylogeny of these OTUstheir closest GenBank relatives and other endosymbioticRhodospirillales bacteria fromGenBankThe total alignmentconsisted of 91 taxa and had a length of 1313 characters WeusedWolbachia pipientis (Alphaproteobacteria Rickettsiales)as an outgroup The phylogenetic tree shows three clades inwhich ant-associatedAsaiaOTUs cluster together (Figure 3)(1) a small clade with two Pseudomyrmex-associated OTUsand one Paraponera-associated OTU (2) a clade that appearsto be Hymenoptera specific containing the bulk of Pseu-domyrmex-associated OTUs a Formica-associated sequencefrom GenBank and bacteria isolated from several beespecies and (3) a clade comprised of many insect-associatedAsaia bacteria and five of our OTUs This last clade is par-ticularly interesting as it comprised several strains that wereisolated from different mosquito species as well as three ant-associated Asaia sequences from GenBank One sequence(JF514556) was isolated and cultivated from Tetraponerarufonigra in India [73] The nifH gene a gene associatedwith the fixation of atmospheric nitrogen has also beenfound in this bacterium (GenBank accession JF736510) andit has been experimentally shown that this strain is capableof fixing nitrogen in vitro suggesting possible nitrogen fixingattributes in its natural environment the ant body cavity[73] The two other sequences are cultivated bacteria fromCephalotes varians and were generated in the framework ofa previous study from our lab (GenBank accessions JX445137and JX445138) [39]

Bacteria from the family Acetobacteraceae are commonlyknown as ldquoacetic acid bacteriardquo and have the metaboliccapacity to oxidize ethanol to acetic acid [74] Asaia also

a member of the Acetobacteraceae however only weaklyoxidizes ethanol and shows higher rates of sugar oxidation[74] These bacteria are environmentally ubiquitous buthave also been found in association with insects such asbees [75 76] mosquitoes [77] Drosophila melanogaster [78]leafhoppers [79] and mealybugs [80] All these insects relyon sugar-rich and often nitrogen-limited diets and it hasbeen suggested that the bacteria function as nutritionalsymbionts Some acetic acid bacteria have the capacity tofix atmospheric nitrogen [73] However it remains entirelyspeculative whether this function can be retained in theinsect gut environment and whether these bacteria actuallycontribute to insect nitrogen metabolism or recycling [81]Interestingly neither acetic acid bacteria nor lactic acidbacteria are commonly found in the core gut microbiota ofarboreal Cephalotini ants an ant group with one of the mostthoroughly studied microbiomes [18 19 39] The metaboliccapacities of the core gut microbiota of the Cephalotiniconsisting of Burkholderiales Opitutales PseudomonadalesRhizobiales and Xanthomonadales might be redundant withthe role that acetic acid bacteria play in other insects

In Drosophila acetic acid bacteria are part of the normalcommensal bacterial gut community and can be involved inthe regulation of the innate immune system In healthy fliesa stable equilibrium of different gut microbes is maintainedPerturbation of the normal gut community which can becaused by a defective regulation of antimicrobial peptideleads to the dominance of the pathogenic commensal Glu-conobacter morbifer and ultimately to gut apoptosis [82]Potential other mechanisms by which acetic acid bacteriabenefit insect immunity are by decreasing the gut pHmakingit an unfavorable environment for pathogenic microorgan-isms or by competitive exclusion [81] However these aceticacid bacteria are not essential for the fitness and repro-duction of most insects as even in the well-studied Asaia-mosquito interaction experimental removal of bacteria hadno detectable negative impact on the host [81]

Several studies have been conducted to analyze themicrobial diversity associated with ants [18 19 39 60 83]However the symbiotic relationships of ants with Rhodospir-illales have rarely been observed In fact only two ant-associated Rhodospirillales sequences had been depositedin GenBank (GQ275104 from Formica occulta and JF514556from Tetraponera rufonigra) prior to work from our group[39] Clone libraries generated for theCephalotini ants [18 19]as well as tag-encoded amplicon data sets [38 39] are amongthe most extensive microbial data collections available forants to date and acetic acid bacteria were only sporadicallyassociated with the ant taxa that were investigated Thus theinteraction of Asaia relatives with ants is generally poorlyunderstood but due to the metabolic capacities of thesebacteria to utilize sugar-rich substrates and fix nitrogenthey might play an important nutritional role Particularlythey might be functionally important in the ant subfamilyPseudomyrmecinae in which they seem to be enriched asindicated by our present study

The phylogenetic history of ant-associated Rhodospiril-lales does not show host specificity and suggests likely acqui-sition from the environment (Figure 3) These observations

8 Psyche

Drosophila sp EU096229 (fruit fly)

Drosophila sp EU096235 (fruit fly)

Drosophila sp EU096233 (fruit fly)

Pseudomyrm

ex ejectus SK1181-3 (ant)

Paraponera clavata CS0513-2 (ant mouth)

Gluconobacter frateurii AB682075

Gluc

onob

acte

r fra

teur

ii A

B682

081Gluconobacter frateurii A

B682091

Pseudo

myrmex

ferru

gineu

s SK00

02-1

(ant)

Pseudomyrm

ex pallidus C

SM1333 (ant)

Pseudomyrmex spinicola SK0061-1 (ant)

Pseudomyrmex peperi SK1058-103-1 (ant)

Cephalotes atratus SK1465 (ant)

Xylocopa californica HM112403 (bee)

Caupolicana yarrowi HM109591 (bee)

Euglossa imperialis JQ389882 (bee)

BER145 (ant)Pseudomyrmex sp nr eduardi

Pseudomyrmex satanicus SK0058-1 (ant)Pseudomyrmex dendroicus BER121 (ant)

Pseudomyrmex dendroicus BER103 (ant)

Pseudomyrmex peperi

SK0007-1 (ant)Pseu

domyrm

ex nigroci

nctus

SK0060-1 (a

nt)Pseudom

yrmex

major

SK09

90-8-

3 (an

t gast

er)

Pseu

domyrm

ex de

ndroi

cus S

K1260

(ant)

Form

ica oc

culta

GQ27

5104

(ant

)

Alph

a pro

teoba

cteriu

m A

B513

363 (

flowe

r)

Para

pone

ra cl

avat

a CS

0513

-4 (a

nt m

outh

)Ca

lliop

sis su

balpi

nus H

M10

9230

(bee

)Ca

lliop

sis su

balpi

nus H

M10

9259

(bee

)

Pseu

dom

yrm

ex te

nuis

BER1

12 (a

nt)

Sacc

haric

occu

s sac

char

i AF1

2739

6 (m

ealy

bug)

Dro

soph

ila sp

EU

0962

34 (f

ruit

fly)

Atta colombica H

M557097 (fungus garden)

Aedes aegypti FJ372761 (mosquito)

Aedes aegypti FJ372770 (mosquito)

Anopheles maculipennis AM

404262 (mosquito)

Aedes albopictus JQ958879 (m

osquito)

Aedes albopictus JQ314055 (m

osquito)

Aedes albopictus JQ314027 (m

osquito)

Aedes albopictus F2f JX445138 (mosquito)

Aede

s aeg

ypti

FN81

4303

(mos

quito

)

Aede

s alb

opict

us F

1f JX

4451

37 (m

osqu

ito)

Aede

s alb

opict

us F

2l JX

4451

40 (m

osqu

ito)

Aede

s alb

opict

us F

1l JX

4451

39 (m

osqu

ito)

Aede

s aeg

ypti

FN81

4301

(mos

quito

)

Aede

s aeg

ypti

FN81

4300

(mos

quito

)

Aede

s aeg

ypti

FN29

7842

(mos

quito

)

Anoph

eles s

tephe

nsi A

M40

4261

(mos

quito

)

Aede

s aeg

ypti

FN29

7841

(mos

quito

)Asaia b

ogore

nsis A

B6820

04

Anoph

eles s

p HQ26

9803

(mos

quito

)

Tetrap

onera

rufon

igra JF

5145

56 (a

nt)

Pseudomyrmex elo

ngatus

CSM1373 (ant gut)Pseudomyrmex termitarius

SK1252-1 (ant)

Pseudomyrmex gracilis

CSM1374-2 (ant head)Anopheles gambiae FN814276 (mosquito)Pseudomyrmex ejectus SK1181-3 (ant)

Pseudomyrm

ex elongatus

CSM1373 (ant head)

Cephalotes va

rians JX

990151 (ant)

Anopheles stephensi FN814299 (mosquito)

Anopheles gambiae FN814277 (mosquito)

Anopheles stephensi FN814297 (mosquito)

Anopheles stephensi FN814296 (mosquito)

Cephalotes varians JX990161 (ant)

Lutzomyia longipalpis HE995765 (sand fly)

Anopheles gambiae FN821398 (mosquito)

Aedes aegypti FN297840 (mosquito)

Anopheles stephensi AM404260 (mosquito)

Asaia krungthepensis AB682130

Anopheles gambiae FJ816021 (mosquito)Anopheles gambiae FN814284 (mosquito)Anopheles gambiae FN814283 (mosquito)

Aedes albopictus FN814288 (mosquito)

Aedes albopictus FN814287 (mosquito)

Anopheles gambiae FN821397 (mosquito)

Anopheles gambiae FN814280 (mosquito)

Aedes albopictus FN814289 (mosquito)

Anopheles gambiae FN814285 (mosquito)

Anopheles gambiae FN814275 (mosquito)

Anopheles gambiae FN814281 (mosquito)

Anopheles gambiae FN814278 (mosquito)

Aedes albopictus FN814286 (mosquito)

Atta colombica HM556904 (fungus garden)

Saccharicoccus sacchari AF127400 (mealybug)

Saccharicoccus sacchari AF127408 (mealybug)

Saccharicoccus sacchari AF127401 (mealybug)

Pseudomyrmex ejectus SK1181-3 (ant)

Paraponera clavata CS0513-3 (ant mouth)

Pseudomyrm

ex termitarius SK1252-2 (ant)

Drosophila sp EU096231 (fruit fly)Wolbachia pipientis AY026912

Inner circle ant subfamilyFormicinaeMyrmicinaeParaponerinaePseudomyrmecinae

Middle circle bioregionAfrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Clade 1

Clade 2Cl

ade 3

Figure 3 Phylogenetic tree of Asaia-related symbionts associated with ants and closest relatives with sequence data available in GenBank Amaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shown The host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) The branch color refers to the sourcefromwhich the bacteria were isolated with yellow representing ant hosts and red other insect hostsThe inner circle refers to the ant subfamilyand the middle circle refers to the bioregion from which samples were collected The outer circle indicates three clades (Clades 1ndash3) whichcontained several ant-associated symbiontsWolbachia pipientis was used as an outgroup

indicate that Rhodospirillales aremost likely environmentallytransmitted and support the hypothesis that they are onlyfacultative associates of ants One clade of ant-associatedRhodospirillales was closely related to endosymbionts iso-lated from mosquitos (Figure 3) It has been experimentallyshown that mosquito-associated Asaia can successfully colo-nize leafhoppers further emphasizing the low-host specificityof this bacterial group [77]

4 Conclusion

Our broad bacterial screening approach has contributedto our understanding of the prevalence of ant-associatedmicrobes particularly with regard to their Wolbachia andSpiroplasma symbionts Furthermore we provide the firstextensive survey for ant-associated Asaia-related symbiontsWhile these symbionts of the order Rhodospirillales infect

Psyche 9

ants only sporadically some strains are capable of fixingatmospheric nitrogen and might retain this function inants Alternatively these bacteria might have an importantfunctional role for upgrading nitrogen-poor diets of someherbivorous ants which comprise the majority of all ant taxa[20] Even though we do not have experimental evidence ofthe role of most bacterial symbionts in ants previous studiesillustrate a broad variety of effects of these bacteria on insecthosts [4 7 9] Even a single group of microbes can havevery different effects on different hosts Our study shows thatdespite several extensive bacterial surveys across the ants thediversity and functional role of ant-associated microbes isfar from being fully understood and broad next generationsequencing approaches will provide a fast and cost-effectivetool to deepen our knowledge of the rare (and not so rare)biosphere

Acknowledgments

The authors thank Daniel Ballhorn and Tina Schroyer forhelpful comments and discussions Phil Ward kindly iden-tified Pseudomyrmex specimens This study was supportedin part by National Science Foundation DEB Grant no1050243 to Corrie S Moreau a Grainger Foundation grant toCorrie S Moreau a Negaunee Foundation grant to Corrie SMoreau and the Pritzker Laboratory forMolecular Evolutionand Systematics Stefanie Kautz acknowledges a postdoctoralfellowship from the German Academy of Sciences Leopold-ina (Grant no LPDS 2009-29) Benjamin E R Rubin wassupported in part by an NSF Graduate Research Fellowship

References

[1] H Koch D P Abrol J Li and P Schmid-Hempel ldquoDiversity ofevolutionary patterns of bacterial gut associates of corbiculatebeesrdquoMolecular Ecology vol 22 no 7 pp 2028ndash2044 2013

[2] R J Dillon and V M Dillon ldquoThe Gut bacteria of insectsnonpathogenic interactionsrdquoAnnual Review of Entomology vol49 pp 71ndash92 2004

[3] S Fraune and T C G Bosch ldquoWhy bacteria matter in animaldevelopment and evolutionrdquo BioEssays vol 32 no 7 pp 571ndash580 2010

[4] H Feldhaar ldquoBacterial symbionts as mediators of ecologicallyimportant traits of insect hostsrdquo Ecological Entomology vol 36no 5 pp 533ndash543 2011

[5] J M Urban and J R Cryan ldquoTwo ancient bacterialendosymbionts have coevolved with the planthoppers (InsectaHemiptera Fulgoroidea)rdquo BMC Evolutionary Biology vol 12article 87 2012

[6] H Toju and T Fukatsu ldquoDiversity and infection prevalence ofendosymbionts in natural populations of the chestnut weevilrelevance of local climate and host plantsrdquo Molecular Ecologyvol 20 no 4 pp 853ndash868 2011

[7] M Kaltenpoth ldquoActinobacteria as mutualists general health-care for insectsrdquo Trends inMicrobiology vol 17 no 12 pp 529ndash535 2009

[8] N A Moran J P McCutcheon and A Nakabachi ldquoGenomicsand evolution of heritable bacterial symbiontsrdquo Annual Reviewof Genetics vol 42 pp 165ndash190 2008

[9] J P McCutcheon and C D Von Dohlen ldquoAn interdependentmetabolic patchwork in the nested symbiosis of mealybugsrdquoCurrent Biology vol 21 no 16 pp 1366ndash1372 2011

[10] C B Montllor A Maxmen and A H Purcell ldquoFacultative bac-terial endosymbionts benefit pea aphids Acyrthosiphon pisumunder heat stressrdquo Ecological Entomology vol 27 no 2 pp 189ndash195 2002

[11] A E Douglas ldquoThe nutritional physiology of aphidsrdquoAdvancesin Insect Physiology vol 31 pp 73ndash140 2003

[12] J J Wernegreen S N Kauppinen S G Brady and P SWard ldquoOne nutritional symbiosis begat another phylogeneticevidence that the ant tribe Camponotini acquired Blochmanniaby tending sap-feeding insectsrdquo BMC Evolutionary Biology vol9 no 1 article 292 2009

[13] H Feldhaar J StrakaM Krischke et al ldquoNutritional upgradingfor omnivorous carpenter ants by the endosymbiont Blochman-niardquo BMC Biology vol 5 article 48 2007

[14] A A Pinto-Tomas M A Anderson G Suen et al ldquoSymbioticnitrogen fixation in the fungus gardens of leaf-cutter antsrdquoScience vol 326 no 5956 pp 1120ndash1123 2009

[15] R Pais C Lohs Y Wu J Wang and S Aksoy ldquoThe obligatemutualist Wigglesworthia glossinidia influences reproductiondigestion and immunity processes of its host the tsetse flyrdquoApplied and Environmental Microbiology vol 74 no 19 pp5965ndash5974 2008

[16] J A Breznak ldquoIntestinal microbiota of termites and otherxylophagous insectsrdquo Annual Review of Microbiology vol 36pp 323ndash343 1982

[17] J A Breznak W J Brill J W Mertins and H C CoppelldquoNitrogen fixation in termitesrdquo Nature vol 244 no 5418 pp577ndash579 1973

[18] J A Russell C SMoreau BGoldman-HuertasM FujiwaraDJ Lohman andN E Pierce ldquoBacterial gut symbionts are tightlylinkedwith the evolution of herbivory in antsrdquoProceedings of theNational Academy of Sciences of theUnited States of America vol106 no 50 pp 21236ndash21241 2009

[19] K E Anderson J A Russell C S Moreau et al ldquoHighlysimilar microbial communities are shared among related andtrophically similar ant speciesrdquo Molecular Ecology vol 21 pp2282ndash2296 2012

[20] D W Davidson S C Cook R R Snelling and T H ChualdquoExplaining the abundance of ants in lowland tropical rainforestcanopiesrdquo Science vol 300 no 5621 pp 969ndash972 2003

[21] J C Brownlie and K N Johnson ldquoSymbiont-mediated protec-tion in insect hostsrdquo Trends in Microbiology vol 17 no 8 pp348ndash354 2009

[22] J Jaenike R Unckless S N Cockburn L M Boelio andS J Perlman ldquoAdaptation via symbiosis recent spread of aDrosophila defensive symbiontrdquo Science vol 329 no 5988 pp212ndash215 2012

[23] K M Oliver J A Russell N A Morant and M S HunterldquoFacultative bacterial symbionts in aphids confer resistance toparasitic waspsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 4 pp 1803ndash18072003

[24] C R Currie J A Scott R C Summerbell and D MallochldquoFungus-growing ants use antibiotic-producing bacteria tocontrol garden parasitesrdquo Nature vol 398 pp 701ndash704 1999

[25] M Kaltenpoth W Gottler G Herzner and E Strohm ldquoSym-biotic bacteria protect wasp larvae from fungal infestationrdquoCurrent Biology vol 15 no 5 pp 475ndash479 2005

10 Psyche

[26] H EDunbar A C CWilsonN R Ferguson andNAMoranldquoAphid thermal tolerance is governed by a point mutation inbacterial symbiontsrdquo PLoS Biology vol 5 no 5 pp 1006ndash10152007

[27] T Tsuchida R Koga S Matsumoto and T Fukatsu ldquoInterspe-cific symbiont transfection confers a novel ecological trait to therecipient insectrdquo Biology Letters vol 7 no 2 pp 245ndash248 2011

[28] R Stouthamer J A J Breeuwer and G D D Hurst ldquoWolbachiapipientis microbial manipulator of arthropod reproductionrdquoAnnual Review of Microbiology vol 53 pp 71ndash102 1999

[29] L M Hedges J C Brownlie S L OrsquoNeill and K N JohnsonldquoWolbachia and virus protection in insectsrdquo Science vol 322 no5902 p 702 2008

[30] K Hilgenboecker P Hammerstein P Schlattmann ATelschow and J H Werren ldquoHow many species are infectedwith Wolbachiamdasha statistical analysis of current datardquo FEMSMicrobiology Letters vol 281 no 2 pp 215ndash220 2008

[31] J A Russell C F Funaro Y M Giraldo et al ldquoA veritablemenagerie of heritable bacteria from ants butterflies andbeyond broadmolecular surveys and a systematic reviewrdquoPLoSONE vol 7 no 12 Article ID e51027 2012

[32] O Duron D Bouchon S Boutin et al ldquoThe diversity ofreproductive parasites among arthropods Wolbachia do notwalk alonerdquo BMC Biology vol 6 article 27 2008

[33] D L Williamson and D F Poulson ldquoSex ratio organisms(Spiroplasmas) of Drosophilardquo in The Mycoplasmas III Plantand Insect Mycoplasmas R F Whitcomb and J G Tully Edspp 175ndash208 Academic Press New York NY USA 1979

[34] J M Bove ldquoSpiroplasmas infectious agents of plants arthro-pods and vertebratesrdquoWiener Klinische Wochenschrift vol 109no 14-15 pp 604ndash612 1997

[35] M C Tinsley and M E N Majerus ldquoA new male-killingparasitism Spiroplasma bacteria infect the ladybird beetleAnisosticta novemdecimpunctata (Coleoptera Coccinellidae)rdquoParasitology vol 132 no 6 pp 757ndash765 2006

[36] A Dobson K D Lafferty A M Kuris R F Hechinger andW Jetz ldquoHomage to Linnaeus howmany parasites Howmanyhostsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 105 no 1 pp 11482ndash11489 2008

[37] S T Kelley and S Dobler ldquoComparative analysis of microbialdiversity in Longitarsus flea beetles (Coleoptera Chrysomeli-dae)rdquo Genetica vol 139 no 5 pp 541ndash550 2011

[38] H D Ishak R Plowes R Sen et al ldquoBacterial diversityin Solenopsis invicta and Solenopsis geminata ant coloniescharacterized by 16S amplicon 454 pyrosequencingrdquo MicrobialEcology vol 61 no 4 pp 821ndash831 2011

[39] S Kautz B E R Rubin J A Russell and C S Moreau ldquoSur-veying the microbiome of ants comparing 454 pyrosequencingwith traditionalmethods to uncover bacterial diversityrdquoAppliedand Environmental Microbiology vol 79 no 2 pp 525ndash5342013

[40] S E Dowd T R Callaway R D Wolcott et al ldquoEvaluationof the bacterial diversity in the feces of cattle using 16SrDNA bacterial tag-encoded FLX amplicon pyrosequencing(bTEFAP)rdquo BMCMicrobiology vol 8 article 125 2008

[41] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[42] C Quince A Lanzen R J Davenport and P J TurnbaughldquoRemoving noise from pyrosequenced ampliconsrdquo BMC Bioin-formatics vol 12 article 38 2011

[43] R C Edgar ldquoSearch and clustering orders of magnitude fasterthan BLASTrdquo Bioinformatics vol 26 no 19 pp 2460ndash24612010

[44] J R Cole QWang E Cardenas et al ldquoTheRibosomal databaseproject improved alignments and new tools for rRNA analysisrdquoNucleic Acids Research vol 37 no 1 pp D141ndashD145 2009

[45] T Z DeSantis P Hugenholtz N Larsen et al ldquoGreengenesa chimera-checked 16S rRNA gene database and workbenchcompatible with ARBrdquo Applied and Environmental Microbiol-ogy vol 72 no 7 pp 5069ndash5072 2006

[46] A J Drummond B Ashton S Buxton et al ldquoGeneious v5 4rdquo2011 httpwwwgeneiouscom

[47] A Stamatakis P Hoover and J Rougemont ldquoA rapid bootstrapalgorithm for the RAxML web serversrdquo Systematic Biology vol57 no 5 pp 758ndash771 2008

[48] M A Miller M T Holder R Vos et al ldquoThe CIPRES portalsrdquohttpwwwphyloorgsub sectionsportal

[49] I Letunic and P Bork ldquoInteractive Tree of Life v2 onlineannotation and display of phylogenetic treesmade easyrdquoNucleicAcids Research vol 39 no 2 pp W475ndashW478 2011

[50] T Wenseleers F Ito S van Borm R Huybrechts F Volckaertand J Billen ldquoWidespread occurrence of the micro-organismWolbachia in antsrdquo Proceedings of the Royal Society B vol 265no 1404 pp 1447ndash1452 1998

[51] J A Russell ldquoThe ants (Hymenoptera Formicidae) are uniqueand enigmatic hosts of prevalent Wolbachia (Alphaproteobac-teria) symbiontsrdquoMyrmecological News vol 16 pp 7ndash23 2012

[52] W Arthofer M Riegler D Schneider M Krammer W JMiller and C Stauffer ldquoHidden Wolbachia diversity in fieldpopulations of the European cherry fruit fly Rhagoletis cerasi(Diptera Tephritidae)rdquo Molecular Ecology vol 18 no 18 pp3816ndash3830 2009

[53] J A Russell B Goldman-Huertas C S Moreau et al ldquoSpecial-ization and geographic isolation among Wolbachia symbiontsfrom ants and lycaenid butterfliesrdquo Evolution vol 63 no 3 pp624ndash640 2009

[54] T Wenseleers L Sundstrom and J Billen ldquoDeleterious Wol-bachia in the ant Formica truncorumrdquo Proceedings of the RoyalSociety B vol 269 no 1491 pp 623ndash629 2002

[55] F Vavre F Fleury J Varaldi P Fouillet and M BouletreauldquoEvidence for female mortality in Wolbachia-mediated cyto-plasmic incompatibility in haplodiploid insects epidemiologicand evolutionary consequencesrdquo Evolution vol 54 no 1 pp191ndash200 2000

[56] J Heinze ldquoThe demise of the standard ant (HymenopteraFormicidae)rdquoMyrmecological News vol 11 pp 11ndash20 2008

[57] S Kautz S U Pauls D J Ballhorn H T Lumbsch andM HeilldquoPolygynous supercolonies of the acacia-ant Pseudomyrmexpeperi an inferior colony founderrdquo Molecular Ecology vol 18no 24 pp 5180ndash5194 2009

[58] S Kautz D J Ballhorn J Kroiss et al ldquoHost plant use bycompeting acacia-ants mutualists monopolize while parasitesshare hostsrdquo PLoS ONE vol 7 no 5 Article ID e37691 2012

[59] J Fletcher G A Schultz R E Davis C E Eastman and R MGoodman ldquoBrittle root disease of horseradish-evidence for anetiological role of Spiroplasma citrirdquo Phytopathology vol 71 pp1073ndash1080 1981

[60] C F Funaro D J C Kronauer C S Moreau B Goldman-Huertas N E Pierce and J A Russell ldquoArmy ants harbor ahost-specific clade of Entomoplasmatales bacteriardquo Applied andEnvironmental Microbiology vol 77 no 1 pp 346ndash350 2011

Psyche 11

[61] D J C Kronauer ldquoRecent advances in army ant biology(Hymenoptera Formicidae)rdquoMyrmecological News vol 12 pp51ndash65 2009

[62] F Thiaucourt and G Bolske ldquoContagious caprine pleurop-neumonia and other pulmonary mycoplasmoses of sheep andgoatsrdquo OIE Revue Scientifique et Technique vol 15 no 4 pp1397ndash1414 1996

[63] T Fukatsu T Tsuchida N Nikoh and R Koga ldquoSpiroplasmaSymbiont of the pea aphid Acyrthosiphon pisum (InsectaHomoptera)rdquo Applied and Environmental Microbiology vol 67no 3 pp 1284ndash1291 2001

[64] T S Haselkorn T A Markow and N A Moran ldquoMultipleintroductions of the Spiroplasma bacterial endosymbiont intoDrosophilardquo Molecular Ecology vol 18 no 6 pp 1294ndash13052009

[65] J G Lundgren R M Lehman and J Chee-Sanford ldquoPhysiol-ogy biochemistry and topology bacterial communities withindigestive tracts of ground beetles (Coleoptera Carabidae)rdquoAnnals of the Entomological Society of America vol 100 no 2pp 275ndash282 2007

[66] I Meeus V Vercruysse and G Smagghe ldquoMolecular detectionof Spiroplasma apis and Spiroplasmamelliferum in beesrdquo Journalof Invertebrate Pathology vol 109 no 1 pp 172ndash174 2012

[67] S van Borm J Billen and J J Boomsma ldquoThe diversity ofmicroorganisms associated with Acromyrmex leafcutter antsrdquoBMC Evolutionary Biology vol 2 article 9 2002

[68] J Wedincamp F E French R F Whitcomb and R B HenegerldquoLaboratory infection and release of Spiroplasma (Entomoplas-matales Spiroplasmataceae) from horse flies (Diptera Taban-idae)rdquo Journal of Entomological Science vol 32 no 4 pp 398ndash402 1997

[69] M A Ebbert and L R Nault ldquoImproved overwintering abilityin Dalbulus maidis (Homoptera Cicadellidae) vectors infectedwith Spiroplasma kunkelii (Mycoplasmatales Spiroplasmat-aceae)rdquo Environmental Entomology vol 23 no 3 pp 634ndash6441994

[70] F M Jiggins G D D Hurst C D Jiggins J H G V DSchulenburg and M E N Majerus ldquoThe butterfly Danauschrysippus is infected by a male-killing Spiroplasma bacteriumrdquoParasitology vol 120 no 5 pp 439ndash446 2000

[71] J H G V D Schulenburg G D D Hurst D Tetzlaff GE Booth I A Zakharov and M E N Majerus ldquoHistory ofinfection with different male-killing bacteria in the two-spotladybird beetle Adalia bipunctata revealed through mitochon-drial DNA sequence analysisrdquoGenetics vol 160 no 3 pp 1075ndash1086 2002

[72] S Kautz H T Lumbsch P S Ward and M Heil ldquoHowto prevent cheating a digestive specialization ties mutualisticplant-ants to their ant-plant partnersrdquo Evolution vol 63 no 4pp 839ndash853 2009

[73] N Samaddar A Paul S Chakravorty et al ldquoNitrogen fixationin Asaia sp (Family Acetobacteraceae)rdquo Current Microbiologyvol 63 no 2 pp 226ndash231 2011

[74] Y Ano H Toyama O Adachi and K Matsushita ldquoEnergymetabolism of a unique acetic acid bacteriumAsaia bogorensisthat lacks ethanol oxidation activityrdquo Bioscience Biotechnologyand Biochemistry vol 72 no 4 pp 989ndash997 2008

[75] V G Martinson B N Danforth R L Minckley O RueppellS Tingek and N A Moran ldquoA simple and distinctive micro-biota associated with honey bees and bumble beesrdquo MolecularEcology vol 20 no 3 pp 619ndash628 2011

[76] N A Moran A K Hansen J E Powell and Z L SabreeldquoDistinctive gut microbiota of honey bees assessed using deepsampling from individual worker beesrdquo PLoS ONE vol 7 no 4Article ID e36393 2012

[77] E Crotti C Damiani M Pajoro et al ldquoAsaia a versatile aceticacid bacterial symbiont capable of cross-colonizing insectsof phylogenetically distant genera and ordersrdquo EnvironmentalMicrobiology vol 11 no 12 pp 3252ndash3264 2009

[78] C R Cox and M S Gilmore ldquoNative microbial colonization ofDrosophilamelanogaster and its use as a model of Enterococcusfaecalis pathogenesisrdquo Infection and Immunity vol 75 no 4 pp1565ndash1576 2007

[79] M Marzorati A Alma L Sacchi et al ldquoA novel Bacteroidetessymbiont is localized in Scaphoideus titanus the insect vector ofFlavescence Doree in Vitis viniferardquo Applied and EnvironmentalMicrobiology vol 72 no 2 pp 1467ndash1475 2006

[80] N J Ashbolt and P A Inkerman ldquoAcetic acid bacterial biota ofthe pink sugar cane mealybug Saccharococcus sacchari and itsenvironsrdquo Applied and Environmental Microbiology vol 56 no3 pp 707ndash712 1990

[81] E Crotti A Rizzi B Chouaia et al ldquoAcetic acid bacteria newlyemerging symbionts of insectsrdquo Applied and EnvironmentalMicrobiology vol 76 no 21 pp 6963ndash6970 2010

[82] J Ryu S Kim H Lee et al ldquoInnate immune homeostasis bythe homeobox gene Caudal and commensal-gut mutualism inDrosophilardquo Science vol 319 no 5864 pp 777ndash782 2008

[83] S Stoll J Gadau R Gross and H Feldhaar ldquoBacterial micro-biota associated with ants of the genus Tetraponerardquo BiologicalJournal of the Linnean Society vol 90 no 3 pp 399ndash412 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 5: Research Article Bacterial Infections across the Ants ...downloads.hindawi.com/journals/psyche/2013/936341.pdf · Department of Zoology, Field Museum of Natural History, South Lake

Psyche 5

Sitophilus oryzae AF035160 (rice weevil)

Form

icoxe

nus p

rova

nche

ri

GQ27

5125

(ant

)Co

lletes

inae

quali

s HM

1129

62 (b

ee)

Colle

tes in

aequ

alis H

M11

3014

(bee

)

Colle

tes in

aequ

alis H

M11

3081

(bee

)

Formica

aquilonia GU592784 (ant)

Formica cinerea GU592774 (ant)

Formica cinerea GU592776 (ant)

Formica cinerea GU592770 (ant)

Formica lagubris GU592789 (ant)

Formica cinerea GU592779 (ant)

Formica cinerea GU592777 (ant)

Formica cinerea GU592778 (ant)Formica lagubris GU592786 (ant)Formica polyctena GU592787 (ant)

Formica cinerea GU592781 (ant)

Formica rufa GU592782 (ant)

Formica cinerea GU592772 (ant)

Formica aquilonia GU592783 (ant)

Formica cinerea GU592771 (ant)

Formica cinerea GU592775 (ant)

Formica cinerea GU592780 (ant)

Formica cinerea GU592769 (ant)

Formica polyctena GU592788 (ant)

Formica aquilonia GU592785 (ant)

Formica

cinere

a GU592773 (ant)Philanthus gibbosu

s HM110630 (b

ee)

Form

ica oc

culta

GQ27

5105

(ant

)

Cardioc

ondy

la em

eryi G

Q2751

42 (a

nt)

Philan

thus g

ibbosu

s HM

1108

03 (b

ee)

Philan

thus g

ibbosu

s HM11

0645

(bee)

Odontoponera transversa GQ275132 (ant)

Phlebotomus papatasi WSU80584 (sand fly)

Anoplolepis gracilipes GQ275135 (ant)

Odontomachus clarus GQ275098 (ant)

Drosicha pinicola AB491204 (scale insect)Pheidologeton sp GQ275133 (ant)

Cimex lectularius AB475126 (bed bug)

Cimex lectularius AB475122 (bed bug)Myrmeleon mobilis DQ068830 (ant lion)

Gnamptogenys sp GQ275114 (ant)

Atta colombica HM559216 (ant)

Formica

wheeleri G

Q275103 (ant)

Heteroponera

microps G

Q275145 (ant)

Megalomyrmex latreillei G

Q275110 (ant)Azteca sp GQ275111 (ant)Myrmelachista sp GQ275113 (ant)

Labidus spininodis G

Q275115 (ant)

Pseudomyrmex tenuis BER112 (ant)

Pseudomyrmex PSW54 SK0066 (ant)

Pseudomyrmex filiformis BER114 (ant)

Tetraponera penzigi TP11 (ant)

Tetraponera penzigi KC137101 (ant)

Tetraponera penzigi KC137114 (ant)

Tetraponera penzigi KC137113 (ant)

Tetraponera penzigi KC137106 (ant)

Tetraponera penzigi KC137107 (ant)

Tetraponera penzigi KC137108 (ant)Tetraponera penzigi KC137109 (ant)

Dorymyrmex elegans GQ275124 (ant)Polyergus breviceps GQ275126 (ant)

Sten

amm

a sne

llingi

GQ27

5108

(ant

)

Cepha

lotes

sp SK

1555

(ant)

Cephalo

tes sp

SK15

31 (a

nt)

Pseudom

yrmex

filiform

is BER11

4 (an

t)

Pseudom

yrmex

tenuis B

ER112 (ant)

Pseu

domyr

mex sa

tanicu

s SK00

58 (a

nt)

Cremato

gaste

r sp

SK12

88 (a

nt)

Ceph

alotes

sp S

K152

1 (an

t)

Polyr

hach

is vi

ndex

GQ

2751

34 (a

nt)

Pseu

dom

yrm

ex ap

ache

GQ

2751

07 (a

nt)

Drosophila simulans N

R 074437 (fly)

Leptomyrm

ex sp GQ

275140 (ant)

Rediviva saetigera HM

111302 (bee)

Muscidifurax uniraptor W

OLPA

RTHA

(wasp)

Tetraponera sp SK1300 (ant)

Tetraponera penzigi KC137118 (ant)Redi

viva

saet

iger

a H

M11

1496

(bee

)

Redi

viva

saeti

gera

HM

1113

73 (b

ee)

Redi

viva

saeti

gera

HM

1114

06 (b

ee)

Dro

soph

ila si

mul

ans A

F468

032

(fly)

Redi

viva

saet

igera

HM

1115

63 (b

ee)

Rediviva saetigera HM

111367 (bee)

Rhytidoponera metallica GQ

275141 (ant)

Rediviva saetigera HM

111289 (bee)

Notoncus sp GQ275137 (ant)

Nysius expressus JQ726767 (stinkbug)

Polyrhachis schenckii CSM1035 (ant)

Polyrhachis tubifera CSM1108 (ant) So

lenop

sis ge

min

ata

alat

e B7

(ant

)

Polyr

hach

is sc

henk

ii CS

M10

35 (a

nt)

Pseu

dom

yrm

ex m

ixtec

us SK

0004

(ant

)Po

lyrha

chis

schoo

pae C

SM10

54 (a

nt)

Polyr

hach

is ru

fifem

ur C

SM07

40 (a

nt)

Poly

rhac

his t

hais

CSM

0746

(ant

)

Polyrhachis rufifemur CSM

0655 (ant)

Polyrhachis senilis CSM0798 (ant)

Polyrhachis sp CSM1019 (ant)

Bemisia tabaci JN

204502 (whitefly)

Kleidocerys resedae JQ726770 (catkin bug)

Polyrhachis schenkii CSM1035 (ant)

Polyrhachis senilis CSM1047 (ant)

Polyrhachis sp CSM1019 (ant)

Cephalotes varians CSM1280 (ant)

Polyrhachis tubifera CSM1108 (ant)

Polyrhachis thais CSM0746 (ant)

Polyrhachis monteithi CSM

1096 (ant)Culex pipiens WPU23709 (mosquito)

Kerria lacca JX669531 (scale insect)

Sitophilus oryzae SOMRRNA (rice weevil)

Rhizobium leguminosarum AB740503

Inner circle ant subfamilyDolichoderinaeEctatomminaeEcitoninaeFormicinae

MyrmicinaePonerinaePseudomyrmecinae

Middle circle bioregion

Afrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Ant clade 1

Ant clade 2A

nt clade 3

Ant c

lade 4

Figure 1 Phylogenetic tree ofWolbachia symbionts associated with ants and their closest relatives with sequence data available in GenBankAmaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shownThe host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) Yellow and red branches representbacteria isolated from ant hosts and other insect hosts respectively The inner circle shows ant subfamily and the outer circle refers tothe continent from which host organisms were collected Four ant-specific clades of Wolbachia symbionts are highlighted (Ant clades 1ndash4)Rhizobium leguminosarum was used as an outgroup

Even outside the army ants a certain degree of hostspecificity of Entomoplasmatales bacteria is evident fromour phylogeny and has been described for ants Drosophilaand other arthropod-associated spiroplasmas [60] In ourmolecular phylogeny clades 1 and 2 exclusively contained

ant-associated Entomoplasmatales (Figure 2) However bothclades contained symbionts from different ant subfamiliesand biogeographic regions indicating that neither phylogenynor geographic range drives the association with these sym-bionts and repeated environmental acquisition is common

6 Psyche

Laelapidae sp JF266582 (mite)

Neriene clathrata EU727102 (spider)

Tetragnatha montana EU

727105 (spider)

Meta m

engei EU727103 (spider)

Meta segm

entata EU727104 (spider)

Hepialus gonggaensis EU

344914 (moth)

Tipula oleracea EU727101 (cranefly)

Acyr

thos

ipho

n pi

sum

AB0

4826

3 (a

phid

)Ha

rmon

ia a

xyrid

is AJ

1324

12 (b

eetle

)

Aniso

sticta

nov

emde

cimpu

ncta

ta A

M08

7471

(bee

tle)

Spiro

plas

ma

sp A

Y837

731

(mos

quito

)

Laod

elpha

x stri

atell

us A

B553

862 (

plan

thop

per)

Spiro

plasm

a ixo

detis

GU5

8567

1

Tetra

nych

us ur

ticae

DQ91

0772

(spi

der m

ite)

Dana

us ch

rysip

pus A

J245

996 (

butte

rfly)

Dros

ophi

la an

anas

sae F

J657

247 (

fly)

Cicade

lla vi

ridis E

U7270

97 (le

afhop

per)

Notostir

a elon

gata E

U7270

98 (g

rass b

ug)

Agathem

era cla

razian

a JF26

6577

(phasm

id)

Antonina c

rawii A

B0300

22 (p

seudoco

ccid)

Derman

yssus

gallin

ae EU

6700

52 (p

oultr

y)

Polyr

hach

is tu

bifera

CSM

1108

(ant

)

Polyr

hach

is mon

teithi

CSM

0961

(ant

)

Polyr

hach

is sp

CSM

0983

(ant

)Fann

ia m

anica

ta A

Y569

829

(fly)

Cten

ocep

halid

es fe

lis (fl

ea)

Spiro

plas

ma

sp

JF26

6585

(spi

der)

Poly

rhac

his s

okol

ova

CSM

0854

-2 (a

nt)

Aran

eus d

iade

mat

us E

U72

7099

(spi

der)

Adal

ia b

ipun

ctat

a AJ

0067

75 (b

eetle

)

Chrysolina varians EU727100 (beetle)

Agathemera millepunctata JF266578 (phasmid)

Kerria lacca GU129148 (scale insect)

Trachymyrmex jamaicensis GQ275127 (ant)

Vollenhovia sp HM996753 (ant)

Vollenhovia sp GQ275136 (ant)Dorylus molestus HM996767 (ant)

Pheidole sp HM996747 (ant)

Pheidole soritis HM996754 (ant)

Spiroplasma platyhelix DQ860101

Spiroplasma platyhelix GU993266

Neivamyrmex carolinensis HM996770 (ant)

Neivamyrmex gibbatus HM996776 (ant)

Cephalotes varians JX990286 (ant)Tetraponera sp CSM

0841 (ant)

Solenopsis geminata SRR058848 A9 (ant)

Solenopsis geminata SRR058869 C4 (ant)

Cephalotes varians CSM1323 (ant gut)

Solenopsis geminata SRR058843 A4 (ant)

Cephalotes varians CSM1323 (ant head)

Cephalotes varians CSM1323 (ant leg)

Pseudomyrmex peperi SK1058 (ant)

Mesoplasm

a syrphidae AY231458 (fly)

Mesoplasm

a seiffertii AY351331 (flower)

Photuris pennsylvanica AY177627 (firefly)

Ento

mop

lasm

atal

es JN

5854

56

Myc

opla

sma

myc

oide

s AF0

8535

0

Capr

a hi

rcus

MPU

2605

5 (g

oat)

Ento

mop

lasm

a m

elaleu

cae D

Q51

4607

Myc

opla

sma

mon

odon

AF0

4219

4 (p

raw

ns)

Polyr

hach

is sc

hoop

ae C

SM06

26b

(ant

)

Cicin

dela

cam

pestr

is AF

0369

54 (b

eetle

)

Loph

omyr

mex

sp G

Q27

5146

(ant

)

Para

pone

ra cl

avata

SK12

49-3

(ant

mid

gut)

Para

pone

ra cl

avata

SK12

49-3

(ant

leg)

Ceph

alotes

sp S

K153

4 (an

t gas

ter)

Tetra

mor

ium

hisp

idum

GQ

2751

06 (a

nt)

Ocym

yrm

ex pi

card

i GQ2

7512

8 (an

t)

Pyra

ctom

ena

angu

lata

AY1

5787

1 (fi

refly

)

Spiro

plas

ma

sp J

F276

619 (

earth

worm

)

Ento

mop

lasm

a lu

civor

ax A

F547

212

Firmicu

tes FM252494 (so

il)

Cheliomyrm

ex sp HM996780 (an

t)

Nomamyrmex ese

nbecki H

M996768 (ant)

Nomamyrm

ex hartigii H

M996876 (ant)

Dorylus spininodis HM996755 (ant)Eciton burchellii HM996844 (ant)Dorylus sp HM996757 (ant)

Dorylus mayri HM996733 (ant)

Aenictus sp HM996772 (ant)

Aenictus dentatus HM996763 (ant)

Aenictus aratus HM996762 (ant)

Aenictus sp HM996765 (ant)

Dorylus gerstaeckeri HM996756 (ant)

Aenictus sp HM996764 (ant)

Aenictus cornutus HM996770 (ant)

Aenictus hottai HM996760 (ant)

Aenictus sp HM996775 (ant)

Aenictus cornutus HM996759 (ant)

Dorylus molestus HM996787 (ant)

Aenictus laeviceps HM996781 (ant)

Aenictus fergusoni HM996766 (ant)

Cheliomyrmex sp HM996778 (ant)

Aenictus sp GQ275130 (ant)

Odontomachus brunneus JQ957020 (ant)

Pachycondyla curvinodis JQ957018 (ant)

Odontomachus brunneus JQ957022 (ant)

Eciton lucanoides HM996758 (ant)

Eciton burchellii HM996844 (ant)

Nomamyrmex esenbecki HM996769 (ant)

Eciton vagans HM996761 (ant)

Eciton hamatum GQ275109 (ant)

Cheliomyrmex sp HM996872 (ant)

Cepha

lotes

sp SK

1531

(ant

)

Polyr

hach

is sok

olova

CSM

0854

-1 (a

nt)

Mesopla

sma l

actuc

ae A

F303

132

Calypto

myrmex

beccar

ii HM

9967

48 (a

nt)

Acromyrm

ex oct

ospinosu

s AF49

1879

(ant)

Neivam

yrmex

nigresc

ens (ant)

Photuris pennsylvanicus AY189134 (firefly)

Panorpa helena AY189125 (scorpion fly)

Alcides zodiaca HM

996749 (moth)

Spiroplasma sp AY189320

Culex annulus AY189130 (mosquito)

Spiroplasma sp AY189315

Liriodendron tulipifera AY189131 (tulip)

Entomoplasm

a melaleucae AY345990 (flower)

Aedes sollicitans AY189129 (mosquito)

Pachycondyla striata JQ957013 (ant)

Pachycondyla striata JQ957015 (ant)

Spiroplasma chrysopicola AY189127 (tabanid fly)

Spiroplasma syrphidicola AY189309 (fly)

Ixodes uriae EU727106 (tic

k)

Periplaneta americana JN653133 (cockroach)

Spiroplasma sp AY189313

Odontomachus brunneus JQ957019 (ant)

Drosophila hydei DQ412089 (fly)

Drosophila wheeleri FJ657225 (fly)

Drosophila aldrichi FJ657229 (fly)

Drosophila mojavensis FJ657219 (fly)

Polyrhachis sp CSM1056 (ant)

Polyrhachis sp CSM0785 (ant)

Polyrhachis ornata CSM0718 (ant)

Polyrhachis sp CSM0668 (ant)

Polyrhachis cupreata CSM1016 (ant)

Camponotus sp CSM0644 (ant)

Polyrhachis sp CSM0986 (ant)

Polyrhachis barretti CSM1121 (ant)

Cephalotes sp RA1052 (ant)

Polyrhachis sp CSM1030 (ant)

Polyrhachis sp CSM0998 (ant)

Pseudomyrmex gracilis BER125 (ant)

Pseudomyrmex apache SK0787 (ant)

Cephalotes varians CSM1280 (ant)

Pseudomyrmex PSW37 SK0076 (ant)

Polyrhachis sp CSM0796 (ant)

Pseudomyrmex major SK0990 (ant)

Chorthippus parallelus JN797639 (grasshopper)

Streblidae sp JF266584 (bat fly)

Spiroplasma sp EF151266

Pardosa lugubris EU727108 (spider)Spiroplasma melliferum AY325304

Apis mellifera JQ347516 (honeybee)

Spiroplasma citri X63781

Daucus carota HM124556 (carrot)

Lycaena arota HM996750 (butterfly)

Mesene phareus HM996752 (butterfly)Rhetus periander HM996751 (butterfly)

Myrmica incompleta HM996744 (ant)

Spiroplasma phoeniceum AY772395

Spiroplasma insolitum AY189133

Poritia sumatrae HM996745 (butterfly)

Spiroplasma sp DQ186642

Drosophila melanogaster FJ657180 (fly)

Drosophila willistoni SMERRNA11 (fly)

Drosophila melanogaster AJ631998 (fly)

Drosophila simulans FJ657181 (fly)

Trapezites eliena HM996746 (butterfly)

Anthidium florentinum EU727107 (bee)

Eurygaster

integrice

ps JQ914111 (sh

ield bug)

Panaeus vannamei DQ360069 (sh

rimp)

Eriocheir sinensis

DQ917753 (crab)Lasiu

s alien

us GQ275102 (an

t)

Rimica

ris ex

oculat

a AJ515720 (shrim

p)

Selenomonas ruminantium AB003379

Clade 1

Clade 2

Clad

e 3

Inner circle ant subfamily

DorylinaeEctatomminaeEcitoninae

FormicinaeMyrmicinaePonerinaePseudomyrmecinae

Middle circle bioregionAfrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Aenictinae

Figure 2 Phylogenetic tree of Spiroplasma-related ant symbionts and their closest relatives with sequence data available in GenBank Amaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shown The host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) The branch color refers to the sourcefrom which the bacteria were isolated with yellow representing ant hosts red other insect hosts blue vertebrates and green plants The innercircle refers to the ant subfamily and the outer circle refers to the continent from which samples were collected The three largest ant-specificclades of Spiroplasma symbionts are indicated (Clades 1ndash3) Selenomonas ruminantium was used as an outgroup

The infection with Spiroplasma seems to be systemic as wefound high titers of this bacterium in association with antguts heads and legs (Supplementary Table 1)

Entomoplasmatales can be pathogenic to plants andvertebrates [59 62] and have been isolated from variousinsect taxa including aphids ants bees beetles butterflies

fruit flies and horse flies [63ndash68] Mutualistic spiroplasmascan grant insects resistance to parasitic nematodes [22]and an increased ability to overwinter [69] Pathogenicphenotypes usually lead to insect death [34] and reproductivemanipulation includes altered sex ratios [33] andmale killing[35 70 71] In ants spiroplasmas have been surveyed and

Psyche 7

biocontrol potential has been hypothesized but their roleremains elusive to date [31 38 60] Functional studiesthat compare the performance of infected and uninfectedindividuals would improve our understanding of the role ofthese facultative symbionts

33 Asaia (Alphaproteobacteria Rhodospirillales) Of 95 antspecies 14 hosted bacteria related to Asaia (Alphapro-teobacteria Rhodospirillales) (Table 1) For these bacteriano previous surveys on their prevalence across the antshave been conducted We found a particularly high infectionrate of 333 (1236 species) in Pseudomyrmex In con-trast Asaia-related symbionts were lacking in CamponotusCrematogasterMyrmeciaMyrmecocystus PolyrhachisOeco-phylla and Solenopsis Low infection frequency was presentin Cephalotes (17) and Paraponera (11 species) (Table 1)The enrichment of Asaia symbionts in Pseudomyrmex isparticularly interesting as this ant genus is arboreal andcontains several obligate plant ants which exclusively feed onplant-derived food sources [58 72] However this bacterialgroup occurred facultatively in arboreal generalists and plantmutualists alike indicating that even if these symbionts aremore frequent in arboreal ormutualistic Pseudomyrmex antsthe association is not obligate

In total we obtained 25 Asaia-related OTUs in ourdataset Of these OTUs 21 were associated with Pseu-domyrmex 3 with Paraponera and 1 with Cephalotes Weinferred a maximum likelihood phylogeny of these OTUstheir closest GenBank relatives and other endosymbioticRhodospirillales bacteria fromGenBankThe total alignmentconsisted of 91 taxa and had a length of 1313 characters WeusedWolbachia pipientis (Alphaproteobacteria Rickettsiales)as an outgroup The phylogenetic tree shows three clades inwhich ant-associatedAsaiaOTUs cluster together (Figure 3)(1) a small clade with two Pseudomyrmex-associated OTUsand one Paraponera-associated OTU (2) a clade that appearsto be Hymenoptera specific containing the bulk of Pseu-domyrmex-associated OTUs a Formica-associated sequencefrom GenBank and bacteria isolated from several beespecies and (3) a clade comprised of many insect-associatedAsaia bacteria and five of our OTUs This last clade is par-ticularly interesting as it comprised several strains that wereisolated from different mosquito species as well as three ant-associated Asaia sequences from GenBank One sequence(JF514556) was isolated and cultivated from Tetraponerarufonigra in India [73] The nifH gene a gene associatedwith the fixation of atmospheric nitrogen has also beenfound in this bacterium (GenBank accession JF736510) andit has been experimentally shown that this strain is capableof fixing nitrogen in vitro suggesting possible nitrogen fixingattributes in its natural environment the ant body cavity[73] The two other sequences are cultivated bacteria fromCephalotes varians and were generated in the framework ofa previous study from our lab (GenBank accessions JX445137and JX445138) [39]

Bacteria from the family Acetobacteraceae are commonlyknown as ldquoacetic acid bacteriardquo and have the metaboliccapacity to oxidize ethanol to acetic acid [74] Asaia also

a member of the Acetobacteraceae however only weaklyoxidizes ethanol and shows higher rates of sugar oxidation[74] These bacteria are environmentally ubiquitous buthave also been found in association with insects such asbees [75 76] mosquitoes [77] Drosophila melanogaster [78]leafhoppers [79] and mealybugs [80] All these insects relyon sugar-rich and often nitrogen-limited diets and it hasbeen suggested that the bacteria function as nutritionalsymbionts Some acetic acid bacteria have the capacity tofix atmospheric nitrogen [73] However it remains entirelyspeculative whether this function can be retained in theinsect gut environment and whether these bacteria actuallycontribute to insect nitrogen metabolism or recycling [81]Interestingly neither acetic acid bacteria nor lactic acidbacteria are commonly found in the core gut microbiota ofarboreal Cephalotini ants an ant group with one of the mostthoroughly studied microbiomes [18 19 39] The metaboliccapacities of the core gut microbiota of the Cephalotiniconsisting of Burkholderiales Opitutales PseudomonadalesRhizobiales and Xanthomonadales might be redundant withthe role that acetic acid bacteria play in other insects

In Drosophila acetic acid bacteria are part of the normalcommensal bacterial gut community and can be involved inthe regulation of the innate immune system In healthy fliesa stable equilibrium of different gut microbes is maintainedPerturbation of the normal gut community which can becaused by a defective regulation of antimicrobial peptideleads to the dominance of the pathogenic commensal Glu-conobacter morbifer and ultimately to gut apoptosis [82]Potential other mechanisms by which acetic acid bacteriabenefit insect immunity are by decreasing the gut pHmakingit an unfavorable environment for pathogenic microorgan-isms or by competitive exclusion [81] However these aceticacid bacteria are not essential for the fitness and repro-duction of most insects as even in the well-studied Asaia-mosquito interaction experimental removal of bacteria hadno detectable negative impact on the host [81]

Several studies have been conducted to analyze themicrobial diversity associated with ants [18 19 39 60 83]However the symbiotic relationships of ants with Rhodospir-illales have rarely been observed In fact only two ant-associated Rhodospirillales sequences had been depositedin GenBank (GQ275104 from Formica occulta and JF514556from Tetraponera rufonigra) prior to work from our group[39] Clone libraries generated for theCephalotini ants [18 19]as well as tag-encoded amplicon data sets [38 39] are amongthe most extensive microbial data collections available forants to date and acetic acid bacteria were only sporadicallyassociated with the ant taxa that were investigated Thus theinteraction of Asaia relatives with ants is generally poorlyunderstood but due to the metabolic capacities of thesebacteria to utilize sugar-rich substrates and fix nitrogenthey might play an important nutritional role Particularlythey might be functionally important in the ant subfamilyPseudomyrmecinae in which they seem to be enriched asindicated by our present study

The phylogenetic history of ant-associated Rhodospiril-lales does not show host specificity and suggests likely acqui-sition from the environment (Figure 3) These observations

8 Psyche

Drosophila sp EU096229 (fruit fly)

Drosophila sp EU096235 (fruit fly)

Drosophila sp EU096233 (fruit fly)

Pseudomyrm

ex ejectus SK1181-3 (ant)

Paraponera clavata CS0513-2 (ant mouth)

Gluconobacter frateurii AB682075

Gluc

onob

acte

r fra

teur

ii A

B682

081Gluconobacter frateurii A

B682091

Pseudo

myrmex

ferru

gineu

s SK00

02-1

(ant)

Pseudomyrm

ex pallidus C

SM1333 (ant)

Pseudomyrmex spinicola SK0061-1 (ant)

Pseudomyrmex peperi SK1058-103-1 (ant)

Cephalotes atratus SK1465 (ant)

Xylocopa californica HM112403 (bee)

Caupolicana yarrowi HM109591 (bee)

Euglossa imperialis JQ389882 (bee)

BER145 (ant)Pseudomyrmex sp nr eduardi

Pseudomyrmex satanicus SK0058-1 (ant)Pseudomyrmex dendroicus BER121 (ant)

Pseudomyrmex dendroicus BER103 (ant)

Pseudomyrmex peperi

SK0007-1 (ant)Pseu

domyrm

ex nigroci

nctus

SK0060-1 (a

nt)Pseudom

yrmex

major

SK09

90-8-

3 (an

t gast

er)

Pseu

domyrm

ex de

ndroi

cus S

K1260

(ant)

Form

ica oc

culta

GQ27

5104

(ant

)

Alph

a pro

teoba

cteriu

m A

B513

363 (

flowe

r)

Para

pone

ra cl

avat

a CS

0513

-4 (a

nt m

outh

)Ca

lliop

sis su

balpi

nus H

M10

9230

(bee

)Ca

lliop

sis su

balpi

nus H

M10

9259

(bee

)

Pseu

dom

yrm

ex te

nuis

BER1

12 (a

nt)

Sacc

haric

occu

s sac

char

i AF1

2739

6 (m

ealy

bug)

Dro

soph

ila sp

EU

0962

34 (f

ruit

fly)

Atta colombica H

M557097 (fungus garden)

Aedes aegypti FJ372761 (mosquito)

Aedes aegypti FJ372770 (mosquito)

Anopheles maculipennis AM

404262 (mosquito)

Aedes albopictus JQ958879 (m

osquito)

Aedes albopictus JQ314055 (m

osquito)

Aedes albopictus JQ314027 (m

osquito)

Aedes albopictus F2f JX445138 (mosquito)

Aede

s aeg

ypti

FN81

4303

(mos

quito

)

Aede

s alb

opict

us F

1f JX

4451

37 (m

osqu

ito)

Aede

s alb

opict

us F

2l JX

4451

40 (m

osqu

ito)

Aede

s alb

opict

us F

1l JX

4451

39 (m

osqu

ito)

Aede

s aeg

ypti

FN81

4301

(mos

quito

)

Aede

s aeg

ypti

FN81

4300

(mos

quito

)

Aede

s aeg

ypti

FN29

7842

(mos

quito

)

Anoph

eles s

tephe

nsi A

M40

4261

(mos

quito

)

Aede

s aeg

ypti

FN29

7841

(mos

quito

)Asaia b

ogore

nsis A

B6820

04

Anoph

eles s

p HQ26

9803

(mos

quito

)

Tetrap

onera

rufon

igra JF

5145

56 (a

nt)

Pseudomyrmex elo

ngatus

CSM1373 (ant gut)Pseudomyrmex termitarius

SK1252-1 (ant)

Pseudomyrmex gracilis

CSM1374-2 (ant head)Anopheles gambiae FN814276 (mosquito)Pseudomyrmex ejectus SK1181-3 (ant)

Pseudomyrm

ex elongatus

CSM1373 (ant head)

Cephalotes va

rians JX

990151 (ant)

Anopheles stephensi FN814299 (mosquito)

Anopheles gambiae FN814277 (mosquito)

Anopheles stephensi FN814297 (mosquito)

Anopheles stephensi FN814296 (mosquito)

Cephalotes varians JX990161 (ant)

Lutzomyia longipalpis HE995765 (sand fly)

Anopheles gambiae FN821398 (mosquito)

Aedes aegypti FN297840 (mosquito)

Anopheles stephensi AM404260 (mosquito)

Asaia krungthepensis AB682130

Anopheles gambiae FJ816021 (mosquito)Anopheles gambiae FN814284 (mosquito)Anopheles gambiae FN814283 (mosquito)

Aedes albopictus FN814288 (mosquito)

Aedes albopictus FN814287 (mosquito)

Anopheles gambiae FN821397 (mosquito)

Anopheles gambiae FN814280 (mosquito)

Aedes albopictus FN814289 (mosquito)

Anopheles gambiae FN814285 (mosquito)

Anopheles gambiae FN814275 (mosquito)

Anopheles gambiae FN814281 (mosquito)

Anopheles gambiae FN814278 (mosquito)

Aedes albopictus FN814286 (mosquito)

Atta colombica HM556904 (fungus garden)

Saccharicoccus sacchari AF127400 (mealybug)

Saccharicoccus sacchari AF127408 (mealybug)

Saccharicoccus sacchari AF127401 (mealybug)

Pseudomyrmex ejectus SK1181-3 (ant)

Paraponera clavata CS0513-3 (ant mouth)

Pseudomyrm

ex termitarius SK1252-2 (ant)

Drosophila sp EU096231 (fruit fly)Wolbachia pipientis AY026912

Inner circle ant subfamilyFormicinaeMyrmicinaeParaponerinaePseudomyrmecinae

Middle circle bioregionAfrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Clade 1

Clade 2Cl

ade 3

Figure 3 Phylogenetic tree of Asaia-related symbionts associated with ants and closest relatives with sequence data available in GenBank Amaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shown The host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) The branch color refers to the sourcefromwhich the bacteria were isolated with yellow representing ant hosts and red other insect hostsThe inner circle refers to the ant subfamilyand the middle circle refers to the bioregion from which samples were collected The outer circle indicates three clades (Clades 1ndash3) whichcontained several ant-associated symbiontsWolbachia pipientis was used as an outgroup

indicate that Rhodospirillales aremost likely environmentallytransmitted and support the hypothesis that they are onlyfacultative associates of ants One clade of ant-associatedRhodospirillales was closely related to endosymbionts iso-lated from mosquitos (Figure 3) It has been experimentallyshown that mosquito-associated Asaia can successfully colo-nize leafhoppers further emphasizing the low-host specificityof this bacterial group [77]

4 Conclusion

Our broad bacterial screening approach has contributedto our understanding of the prevalence of ant-associatedmicrobes particularly with regard to their Wolbachia andSpiroplasma symbionts Furthermore we provide the firstextensive survey for ant-associated Asaia-related symbiontsWhile these symbionts of the order Rhodospirillales infect

Psyche 9

ants only sporadically some strains are capable of fixingatmospheric nitrogen and might retain this function inants Alternatively these bacteria might have an importantfunctional role for upgrading nitrogen-poor diets of someherbivorous ants which comprise the majority of all ant taxa[20] Even though we do not have experimental evidence ofthe role of most bacterial symbionts in ants previous studiesillustrate a broad variety of effects of these bacteria on insecthosts [4 7 9] Even a single group of microbes can havevery different effects on different hosts Our study shows thatdespite several extensive bacterial surveys across the ants thediversity and functional role of ant-associated microbes isfar from being fully understood and broad next generationsequencing approaches will provide a fast and cost-effectivetool to deepen our knowledge of the rare (and not so rare)biosphere

Acknowledgments

The authors thank Daniel Ballhorn and Tina Schroyer forhelpful comments and discussions Phil Ward kindly iden-tified Pseudomyrmex specimens This study was supportedin part by National Science Foundation DEB Grant no1050243 to Corrie S Moreau a Grainger Foundation grant toCorrie S Moreau a Negaunee Foundation grant to Corrie SMoreau and the Pritzker Laboratory forMolecular Evolutionand Systematics Stefanie Kautz acknowledges a postdoctoralfellowship from the German Academy of Sciences Leopold-ina (Grant no LPDS 2009-29) Benjamin E R Rubin wassupported in part by an NSF Graduate Research Fellowship

References

[1] H Koch D P Abrol J Li and P Schmid-Hempel ldquoDiversity ofevolutionary patterns of bacterial gut associates of corbiculatebeesrdquoMolecular Ecology vol 22 no 7 pp 2028ndash2044 2013

[2] R J Dillon and V M Dillon ldquoThe Gut bacteria of insectsnonpathogenic interactionsrdquoAnnual Review of Entomology vol49 pp 71ndash92 2004

[3] S Fraune and T C G Bosch ldquoWhy bacteria matter in animaldevelopment and evolutionrdquo BioEssays vol 32 no 7 pp 571ndash580 2010

[4] H Feldhaar ldquoBacterial symbionts as mediators of ecologicallyimportant traits of insect hostsrdquo Ecological Entomology vol 36no 5 pp 533ndash543 2011

[5] J M Urban and J R Cryan ldquoTwo ancient bacterialendosymbionts have coevolved with the planthoppers (InsectaHemiptera Fulgoroidea)rdquo BMC Evolutionary Biology vol 12article 87 2012

[6] H Toju and T Fukatsu ldquoDiversity and infection prevalence ofendosymbionts in natural populations of the chestnut weevilrelevance of local climate and host plantsrdquo Molecular Ecologyvol 20 no 4 pp 853ndash868 2011

[7] M Kaltenpoth ldquoActinobacteria as mutualists general health-care for insectsrdquo Trends inMicrobiology vol 17 no 12 pp 529ndash535 2009

[8] N A Moran J P McCutcheon and A Nakabachi ldquoGenomicsand evolution of heritable bacterial symbiontsrdquo Annual Reviewof Genetics vol 42 pp 165ndash190 2008

[9] J P McCutcheon and C D Von Dohlen ldquoAn interdependentmetabolic patchwork in the nested symbiosis of mealybugsrdquoCurrent Biology vol 21 no 16 pp 1366ndash1372 2011

[10] C B Montllor A Maxmen and A H Purcell ldquoFacultative bac-terial endosymbionts benefit pea aphids Acyrthosiphon pisumunder heat stressrdquo Ecological Entomology vol 27 no 2 pp 189ndash195 2002

[11] A E Douglas ldquoThe nutritional physiology of aphidsrdquoAdvancesin Insect Physiology vol 31 pp 73ndash140 2003

[12] J J Wernegreen S N Kauppinen S G Brady and P SWard ldquoOne nutritional symbiosis begat another phylogeneticevidence that the ant tribe Camponotini acquired Blochmanniaby tending sap-feeding insectsrdquo BMC Evolutionary Biology vol9 no 1 article 292 2009

[13] H Feldhaar J StrakaM Krischke et al ldquoNutritional upgradingfor omnivorous carpenter ants by the endosymbiont Blochman-niardquo BMC Biology vol 5 article 48 2007

[14] A A Pinto-Tomas M A Anderson G Suen et al ldquoSymbioticnitrogen fixation in the fungus gardens of leaf-cutter antsrdquoScience vol 326 no 5956 pp 1120ndash1123 2009

[15] R Pais C Lohs Y Wu J Wang and S Aksoy ldquoThe obligatemutualist Wigglesworthia glossinidia influences reproductiondigestion and immunity processes of its host the tsetse flyrdquoApplied and Environmental Microbiology vol 74 no 19 pp5965ndash5974 2008

[16] J A Breznak ldquoIntestinal microbiota of termites and otherxylophagous insectsrdquo Annual Review of Microbiology vol 36pp 323ndash343 1982

[17] J A Breznak W J Brill J W Mertins and H C CoppelldquoNitrogen fixation in termitesrdquo Nature vol 244 no 5418 pp577ndash579 1973

[18] J A Russell C SMoreau BGoldman-HuertasM FujiwaraDJ Lohman andN E Pierce ldquoBacterial gut symbionts are tightlylinkedwith the evolution of herbivory in antsrdquoProceedings of theNational Academy of Sciences of theUnited States of America vol106 no 50 pp 21236ndash21241 2009

[19] K E Anderson J A Russell C S Moreau et al ldquoHighlysimilar microbial communities are shared among related andtrophically similar ant speciesrdquo Molecular Ecology vol 21 pp2282ndash2296 2012

[20] D W Davidson S C Cook R R Snelling and T H ChualdquoExplaining the abundance of ants in lowland tropical rainforestcanopiesrdquo Science vol 300 no 5621 pp 969ndash972 2003

[21] J C Brownlie and K N Johnson ldquoSymbiont-mediated protec-tion in insect hostsrdquo Trends in Microbiology vol 17 no 8 pp348ndash354 2009

[22] J Jaenike R Unckless S N Cockburn L M Boelio andS J Perlman ldquoAdaptation via symbiosis recent spread of aDrosophila defensive symbiontrdquo Science vol 329 no 5988 pp212ndash215 2012

[23] K M Oliver J A Russell N A Morant and M S HunterldquoFacultative bacterial symbionts in aphids confer resistance toparasitic waspsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 4 pp 1803ndash18072003

[24] C R Currie J A Scott R C Summerbell and D MallochldquoFungus-growing ants use antibiotic-producing bacteria tocontrol garden parasitesrdquo Nature vol 398 pp 701ndash704 1999

[25] M Kaltenpoth W Gottler G Herzner and E Strohm ldquoSym-biotic bacteria protect wasp larvae from fungal infestationrdquoCurrent Biology vol 15 no 5 pp 475ndash479 2005

10 Psyche

[26] H EDunbar A C CWilsonN R Ferguson andNAMoranldquoAphid thermal tolerance is governed by a point mutation inbacterial symbiontsrdquo PLoS Biology vol 5 no 5 pp 1006ndash10152007

[27] T Tsuchida R Koga S Matsumoto and T Fukatsu ldquoInterspe-cific symbiont transfection confers a novel ecological trait to therecipient insectrdquo Biology Letters vol 7 no 2 pp 245ndash248 2011

[28] R Stouthamer J A J Breeuwer and G D D Hurst ldquoWolbachiapipientis microbial manipulator of arthropod reproductionrdquoAnnual Review of Microbiology vol 53 pp 71ndash102 1999

[29] L M Hedges J C Brownlie S L OrsquoNeill and K N JohnsonldquoWolbachia and virus protection in insectsrdquo Science vol 322 no5902 p 702 2008

[30] K Hilgenboecker P Hammerstein P Schlattmann ATelschow and J H Werren ldquoHow many species are infectedwith Wolbachiamdasha statistical analysis of current datardquo FEMSMicrobiology Letters vol 281 no 2 pp 215ndash220 2008

[31] J A Russell C F Funaro Y M Giraldo et al ldquoA veritablemenagerie of heritable bacteria from ants butterflies andbeyond broadmolecular surveys and a systematic reviewrdquoPLoSONE vol 7 no 12 Article ID e51027 2012

[32] O Duron D Bouchon S Boutin et al ldquoThe diversity ofreproductive parasites among arthropods Wolbachia do notwalk alonerdquo BMC Biology vol 6 article 27 2008

[33] D L Williamson and D F Poulson ldquoSex ratio organisms(Spiroplasmas) of Drosophilardquo in The Mycoplasmas III Plantand Insect Mycoplasmas R F Whitcomb and J G Tully Edspp 175ndash208 Academic Press New York NY USA 1979

[34] J M Bove ldquoSpiroplasmas infectious agents of plants arthro-pods and vertebratesrdquoWiener Klinische Wochenschrift vol 109no 14-15 pp 604ndash612 1997

[35] M C Tinsley and M E N Majerus ldquoA new male-killingparasitism Spiroplasma bacteria infect the ladybird beetleAnisosticta novemdecimpunctata (Coleoptera Coccinellidae)rdquoParasitology vol 132 no 6 pp 757ndash765 2006

[36] A Dobson K D Lafferty A M Kuris R F Hechinger andW Jetz ldquoHomage to Linnaeus howmany parasites Howmanyhostsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 105 no 1 pp 11482ndash11489 2008

[37] S T Kelley and S Dobler ldquoComparative analysis of microbialdiversity in Longitarsus flea beetles (Coleoptera Chrysomeli-dae)rdquo Genetica vol 139 no 5 pp 541ndash550 2011

[38] H D Ishak R Plowes R Sen et al ldquoBacterial diversityin Solenopsis invicta and Solenopsis geminata ant coloniescharacterized by 16S amplicon 454 pyrosequencingrdquo MicrobialEcology vol 61 no 4 pp 821ndash831 2011

[39] S Kautz B E R Rubin J A Russell and C S Moreau ldquoSur-veying the microbiome of ants comparing 454 pyrosequencingwith traditionalmethods to uncover bacterial diversityrdquoAppliedand Environmental Microbiology vol 79 no 2 pp 525ndash5342013

[40] S E Dowd T R Callaway R D Wolcott et al ldquoEvaluationof the bacterial diversity in the feces of cattle using 16SrDNA bacterial tag-encoded FLX amplicon pyrosequencing(bTEFAP)rdquo BMCMicrobiology vol 8 article 125 2008

[41] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[42] C Quince A Lanzen R J Davenport and P J TurnbaughldquoRemoving noise from pyrosequenced ampliconsrdquo BMC Bioin-formatics vol 12 article 38 2011

[43] R C Edgar ldquoSearch and clustering orders of magnitude fasterthan BLASTrdquo Bioinformatics vol 26 no 19 pp 2460ndash24612010

[44] J R Cole QWang E Cardenas et al ldquoTheRibosomal databaseproject improved alignments and new tools for rRNA analysisrdquoNucleic Acids Research vol 37 no 1 pp D141ndashD145 2009

[45] T Z DeSantis P Hugenholtz N Larsen et al ldquoGreengenesa chimera-checked 16S rRNA gene database and workbenchcompatible with ARBrdquo Applied and Environmental Microbiol-ogy vol 72 no 7 pp 5069ndash5072 2006

[46] A J Drummond B Ashton S Buxton et al ldquoGeneious v5 4rdquo2011 httpwwwgeneiouscom

[47] A Stamatakis P Hoover and J Rougemont ldquoA rapid bootstrapalgorithm for the RAxML web serversrdquo Systematic Biology vol57 no 5 pp 758ndash771 2008

[48] M A Miller M T Holder R Vos et al ldquoThe CIPRES portalsrdquohttpwwwphyloorgsub sectionsportal

[49] I Letunic and P Bork ldquoInteractive Tree of Life v2 onlineannotation and display of phylogenetic treesmade easyrdquoNucleicAcids Research vol 39 no 2 pp W475ndashW478 2011

[50] T Wenseleers F Ito S van Borm R Huybrechts F Volckaertand J Billen ldquoWidespread occurrence of the micro-organismWolbachia in antsrdquo Proceedings of the Royal Society B vol 265no 1404 pp 1447ndash1452 1998

[51] J A Russell ldquoThe ants (Hymenoptera Formicidae) are uniqueand enigmatic hosts of prevalent Wolbachia (Alphaproteobac-teria) symbiontsrdquoMyrmecological News vol 16 pp 7ndash23 2012

[52] W Arthofer M Riegler D Schneider M Krammer W JMiller and C Stauffer ldquoHidden Wolbachia diversity in fieldpopulations of the European cherry fruit fly Rhagoletis cerasi(Diptera Tephritidae)rdquo Molecular Ecology vol 18 no 18 pp3816ndash3830 2009

[53] J A Russell B Goldman-Huertas C S Moreau et al ldquoSpecial-ization and geographic isolation among Wolbachia symbiontsfrom ants and lycaenid butterfliesrdquo Evolution vol 63 no 3 pp624ndash640 2009

[54] T Wenseleers L Sundstrom and J Billen ldquoDeleterious Wol-bachia in the ant Formica truncorumrdquo Proceedings of the RoyalSociety B vol 269 no 1491 pp 623ndash629 2002

[55] F Vavre F Fleury J Varaldi P Fouillet and M BouletreauldquoEvidence for female mortality in Wolbachia-mediated cyto-plasmic incompatibility in haplodiploid insects epidemiologicand evolutionary consequencesrdquo Evolution vol 54 no 1 pp191ndash200 2000

[56] J Heinze ldquoThe demise of the standard ant (HymenopteraFormicidae)rdquoMyrmecological News vol 11 pp 11ndash20 2008

[57] S Kautz S U Pauls D J Ballhorn H T Lumbsch andM HeilldquoPolygynous supercolonies of the acacia-ant Pseudomyrmexpeperi an inferior colony founderrdquo Molecular Ecology vol 18no 24 pp 5180ndash5194 2009

[58] S Kautz D J Ballhorn J Kroiss et al ldquoHost plant use bycompeting acacia-ants mutualists monopolize while parasitesshare hostsrdquo PLoS ONE vol 7 no 5 Article ID e37691 2012

[59] J Fletcher G A Schultz R E Davis C E Eastman and R MGoodman ldquoBrittle root disease of horseradish-evidence for anetiological role of Spiroplasma citrirdquo Phytopathology vol 71 pp1073ndash1080 1981

[60] C F Funaro D J C Kronauer C S Moreau B Goldman-Huertas N E Pierce and J A Russell ldquoArmy ants harbor ahost-specific clade of Entomoplasmatales bacteriardquo Applied andEnvironmental Microbiology vol 77 no 1 pp 346ndash350 2011

Psyche 11

[61] D J C Kronauer ldquoRecent advances in army ant biology(Hymenoptera Formicidae)rdquoMyrmecological News vol 12 pp51ndash65 2009

[62] F Thiaucourt and G Bolske ldquoContagious caprine pleurop-neumonia and other pulmonary mycoplasmoses of sheep andgoatsrdquo OIE Revue Scientifique et Technique vol 15 no 4 pp1397ndash1414 1996

[63] T Fukatsu T Tsuchida N Nikoh and R Koga ldquoSpiroplasmaSymbiont of the pea aphid Acyrthosiphon pisum (InsectaHomoptera)rdquo Applied and Environmental Microbiology vol 67no 3 pp 1284ndash1291 2001

[64] T S Haselkorn T A Markow and N A Moran ldquoMultipleintroductions of the Spiroplasma bacterial endosymbiont intoDrosophilardquo Molecular Ecology vol 18 no 6 pp 1294ndash13052009

[65] J G Lundgren R M Lehman and J Chee-Sanford ldquoPhysiol-ogy biochemistry and topology bacterial communities withindigestive tracts of ground beetles (Coleoptera Carabidae)rdquoAnnals of the Entomological Society of America vol 100 no 2pp 275ndash282 2007

[66] I Meeus V Vercruysse and G Smagghe ldquoMolecular detectionof Spiroplasma apis and Spiroplasmamelliferum in beesrdquo Journalof Invertebrate Pathology vol 109 no 1 pp 172ndash174 2012

[67] S van Borm J Billen and J J Boomsma ldquoThe diversity ofmicroorganisms associated with Acromyrmex leafcutter antsrdquoBMC Evolutionary Biology vol 2 article 9 2002

[68] J Wedincamp F E French R F Whitcomb and R B HenegerldquoLaboratory infection and release of Spiroplasma (Entomoplas-matales Spiroplasmataceae) from horse flies (Diptera Taban-idae)rdquo Journal of Entomological Science vol 32 no 4 pp 398ndash402 1997

[69] M A Ebbert and L R Nault ldquoImproved overwintering abilityin Dalbulus maidis (Homoptera Cicadellidae) vectors infectedwith Spiroplasma kunkelii (Mycoplasmatales Spiroplasmat-aceae)rdquo Environmental Entomology vol 23 no 3 pp 634ndash6441994

[70] F M Jiggins G D D Hurst C D Jiggins J H G V DSchulenburg and M E N Majerus ldquoThe butterfly Danauschrysippus is infected by a male-killing Spiroplasma bacteriumrdquoParasitology vol 120 no 5 pp 439ndash446 2000

[71] J H G V D Schulenburg G D D Hurst D Tetzlaff GE Booth I A Zakharov and M E N Majerus ldquoHistory ofinfection with different male-killing bacteria in the two-spotladybird beetle Adalia bipunctata revealed through mitochon-drial DNA sequence analysisrdquoGenetics vol 160 no 3 pp 1075ndash1086 2002

[72] S Kautz H T Lumbsch P S Ward and M Heil ldquoHowto prevent cheating a digestive specialization ties mutualisticplant-ants to their ant-plant partnersrdquo Evolution vol 63 no 4pp 839ndash853 2009

[73] N Samaddar A Paul S Chakravorty et al ldquoNitrogen fixationin Asaia sp (Family Acetobacteraceae)rdquo Current Microbiologyvol 63 no 2 pp 226ndash231 2011

[74] Y Ano H Toyama O Adachi and K Matsushita ldquoEnergymetabolism of a unique acetic acid bacteriumAsaia bogorensisthat lacks ethanol oxidation activityrdquo Bioscience Biotechnologyand Biochemistry vol 72 no 4 pp 989ndash997 2008

[75] V G Martinson B N Danforth R L Minckley O RueppellS Tingek and N A Moran ldquoA simple and distinctive micro-biota associated with honey bees and bumble beesrdquo MolecularEcology vol 20 no 3 pp 619ndash628 2011

[76] N A Moran A K Hansen J E Powell and Z L SabreeldquoDistinctive gut microbiota of honey bees assessed using deepsampling from individual worker beesrdquo PLoS ONE vol 7 no 4Article ID e36393 2012

[77] E Crotti C Damiani M Pajoro et al ldquoAsaia a versatile aceticacid bacterial symbiont capable of cross-colonizing insectsof phylogenetically distant genera and ordersrdquo EnvironmentalMicrobiology vol 11 no 12 pp 3252ndash3264 2009

[78] C R Cox and M S Gilmore ldquoNative microbial colonization ofDrosophilamelanogaster and its use as a model of Enterococcusfaecalis pathogenesisrdquo Infection and Immunity vol 75 no 4 pp1565ndash1576 2007

[79] M Marzorati A Alma L Sacchi et al ldquoA novel Bacteroidetessymbiont is localized in Scaphoideus titanus the insect vector ofFlavescence Doree in Vitis viniferardquo Applied and EnvironmentalMicrobiology vol 72 no 2 pp 1467ndash1475 2006

[80] N J Ashbolt and P A Inkerman ldquoAcetic acid bacterial biota ofthe pink sugar cane mealybug Saccharococcus sacchari and itsenvironsrdquo Applied and Environmental Microbiology vol 56 no3 pp 707ndash712 1990

[81] E Crotti A Rizzi B Chouaia et al ldquoAcetic acid bacteria newlyemerging symbionts of insectsrdquo Applied and EnvironmentalMicrobiology vol 76 no 21 pp 6963ndash6970 2010

[82] J Ryu S Kim H Lee et al ldquoInnate immune homeostasis bythe homeobox gene Caudal and commensal-gut mutualism inDrosophilardquo Science vol 319 no 5864 pp 777ndash782 2008

[83] S Stoll J Gadau R Gross and H Feldhaar ldquoBacterial micro-biota associated with ants of the genus Tetraponerardquo BiologicalJournal of the Linnean Society vol 90 no 3 pp 399ndash412 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 6: Research Article Bacterial Infections across the Ants ...downloads.hindawi.com/journals/psyche/2013/936341.pdf · Department of Zoology, Field Museum of Natural History, South Lake

6 Psyche

Laelapidae sp JF266582 (mite)

Neriene clathrata EU727102 (spider)

Tetragnatha montana EU

727105 (spider)

Meta m

engei EU727103 (spider)

Meta segm

entata EU727104 (spider)

Hepialus gonggaensis EU

344914 (moth)

Tipula oleracea EU727101 (cranefly)

Acyr

thos

ipho

n pi

sum

AB0

4826

3 (a

phid

)Ha

rmon

ia a

xyrid

is AJ

1324

12 (b

eetle

)

Aniso

sticta

nov

emde

cimpu

ncta

ta A

M08

7471

(bee

tle)

Spiro

plas

ma

sp A

Y837

731

(mos

quito

)

Laod

elpha

x stri

atell

us A

B553

862 (

plan

thop

per)

Spiro

plasm

a ixo

detis

GU5

8567

1

Tetra

nych

us ur

ticae

DQ91

0772

(spi

der m

ite)

Dana

us ch

rysip

pus A

J245

996 (

butte

rfly)

Dros

ophi

la an

anas

sae F

J657

247 (

fly)

Cicade

lla vi

ridis E

U7270

97 (le

afhop

per)

Notostir

a elon

gata E

U7270

98 (g

rass b

ug)

Agathem

era cla

razian

a JF26

6577

(phasm

id)

Antonina c

rawii A

B0300

22 (p

seudoco

ccid)

Derman

yssus

gallin

ae EU

6700

52 (p

oultr

y)

Polyr

hach

is tu

bifera

CSM

1108

(ant

)

Polyr

hach

is mon

teithi

CSM

0961

(ant

)

Polyr

hach

is sp

CSM

0983

(ant

)Fann

ia m

anica

ta A

Y569

829

(fly)

Cten

ocep

halid

es fe

lis (fl

ea)

Spiro

plas

ma

sp

JF26

6585

(spi

der)

Poly

rhac

his s

okol

ova

CSM

0854

-2 (a

nt)

Aran

eus d

iade

mat

us E

U72

7099

(spi

der)

Adal

ia b

ipun

ctat

a AJ

0067

75 (b

eetle

)

Chrysolina varians EU727100 (beetle)

Agathemera millepunctata JF266578 (phasmid)

Kerria lacca GU129148 (scale insect)

Trachymyrmex jamaicensis GQ275127 (ant)

Vollenhovia sp HM996753 (ant)

Vollenhovia sp GQ275136 (ant)Dorylus molestus HM996767 (ant)

Pheidole sp HM996747 (ant)

Pheidole soritis HM996754 (ant)

Spiroplasma platyhelix DQ860101

Spiroplasma platyhelix GU993266

Neivamyrmex carolinensis HM996770 (ant)

Neivamyrmex gibbatus HM996776 (ant)

Cephalotes varians JX990286 (ant)Tetraponera sp CSM

0841 (ant)

Solenopsis geminata SRR058848 A9 (ant)

Solenopsis geminata SRR058869 C4 (ant)

Cephalotes varians CSM1323 (ant gut)

Solenopsis geminata SRR058843 A4 (ant)

Cephalotes varians CSM1323 (ant head)

Cephalotes varians CSM1323 (ant leg)

Pseudomyrmex peperi SK1058 (ant)

Mesoplasm

a syrphidae AY231458 (fly)

Mesoplasm

a seiffertii AY351331 (flower)

Photuris pennsylvanica AY177627 (firefly)

Ento

mop

lasm

atal

es JN

5854

56

Myc

opla

sma

myc

oide

s AF0

8535

0

Capr

a hi

rcus

MPU

2605

5 (g

oat)

Ento

mop

lasm

a m

elaleu

cae D

Q51

4607

Myc

opla

sma

mon

odon

AF0

4219

4 (p

raw

ns)

Polyr

hach

is sc

hoop

ae C

SM06

26b

(ant

)

Cicin

dela

cam

pestr

is AF

0369

54 (b

eetle

)

Loph

omyr

mex

sp G

Q27

5146

(ant

)

Para

pone

ra cl

avata

SK12

49-3

(ant

mid

gut)

Para

pone

ra cl

avata

SK12

49-3

(ant

leg)

Ceph

alotes

sp S

K153

4 (an

t gas

ter)

Tetra

mor

ium

hisp

idum

GQ

2751

06 (a

nt)

Ocym

yrm

ex pi

card

i GQ2

7512

8 (an

t)

Pyra

ctom

ena

angu

lata

AY1

5787

1 (fi

refly

)

Spiro

plas

ma

sp J

F276

619 (

earth

worm

)

Ento

mop

lasm

a lu

civor

ax A

F547

212

Firmicu

tes FM252494 (so

il)

Cheliomyrm

ex sp HM996780 (an

t)

Nomamyrmex ese

nbecki H

M996768 (ant)

Nomamyrm

ex hartigii H

M996876 (ant)

Dorylus spininodis HM996755 (ant)Eciton burchellii HM996844 (ant)Dorylus sp HM996757 (ant)

Dorylus mayri HM996733 (ant)

Aenictus sp HM996772 (ant)

Aenictus dentatus HM996763 (ant)

Aenictus aratus HM996762 (ant)

Aenictus sp HM996765 (ant)

Dorylus gerstaeckeri HM996756 (ant)

Aenictus sp HM996764 (ant)

Aenictus cornutus HM996770 (ant)

Aenictus hottai HM996760 (ant)

Aenictus sp HM996775 (ant)

Aenictus cornutus HM996759 (ant)

Dorylus molestus HM996787 (ant)

Aenictus laeviceps HM996781 (ant)

Aenictus fergusoni HM996766 (ant)

Cheliomyrmex sp HM996778 (ant)

Aenictus sp GQ275130 (ant)

Odontomachus brunneus JQ957020 (ant)

Pachycondyla curvinodis JQ957018 (ant)

Odontomachus brunneus JQ957022 (ant)

Eciton lucanoides HM996758 (ant)

Eciton burchellii HM996844 (ant)

Nomamyrmex esenbecki HM996769 (ant)

Eciton vagans HM996761 (ant)

Eciton hamatum GQ275109 (ant)

Cheliomyrmex sp HM996872 (ant)

Cepha

lotes

sp SK

1531

(ant

)

Polyr

hach

is sok

olova

CSM

0854

-1 (a

nt)

Mesopla

sma l

actuc

ae A

F303

132

Calypto

myrmex

beccar

ii HM

9967

48 (a

nt)

Acromyrm

ex oct

ospinosu

s AF49

1879

(ant)

Neivam

yrmex

nigresc

ens (ant)

Photuris pennsylvanicus AY189134 (firefly)

Panorpa helena AY189125 (scorpion fly)

Alcides zodiaca HM

996749 (moth)

Spiroplasma sp AY189320

Culex annulus AY189130 (mosquito)

Spiroplasma sp AY189315

Liriodendron tulipifera AY189131 (tulip)

Entomoplasm

a melaleucae AY345990 (flower)

Aedes sollicitans AY189129 (mosquito)

Pachycondyla striata JQ957013 (ant)

Pachycondyla striata JQ957015 (ant)

Spiroplasma chrysopicola AY189127 (tabanid fly)

Spiroplasma syrphidicola AY189309 (fly)

Ixodes uriae EU727106 (tic

k)

Periplaneta americana JN653133 (cockroach)

Spiroplasma sp AY189313

Odontomachus brunneus JQ957019 (ant)

Drosophila hydei DQ412089 (fly)

Drosophila wheeleri FJ657225 (fly)

Drosophila aldrichi FJ657229 (fly)

Drosophila mojavensis FJ657219 (fly)

Polyrhachis sp CSM1056 (ant)

Polyrhachis sp CSM0785 (ant)

Polyrhachis ornata CSM0718 (ant)

Polyrhachis sp CSM0668 (ant)

Polyrhachis cupreata CSM1016 (ant)

Camponotus sp CSM0644 (ant)

Polyrhachis sp CSM0986 (ant)

Polyrhachis barretti CSM1121 (ant)

Cephalotes sp RA1052 (ant)

Polyrhachis sp CSM1030 (ant)

Polyrhachis sp CSM0998 (ant)

Pseudomyrmex gracilis BER125 (ant)

Pseudomyrmex apache SK0787 (ant)

Cephalotes varians CSM1280 (ant)

Pseudomyrmex PSW37 SK0076 (ant)

Polyrhachis sp CSM0796 (ant)

Pseudomyrmex major SK0990 (ant)

Chorthippus parallelus JN797639 (grasshopper)

Streblidae sp JF266584 (bat fly)

Spiroplasma sp EF151266

Pardosa lugubris EU727108 (spider)Spiroplasma melliferum AY325304

Apis mellifera JQ347516 (honeybee)

Spiroplasma citri X63781

Daucus carota HM124556 (carrot)

Lycaena arota HM996750 (butterfly)

Mesene phareus HM996752 (butterfly)Rhetus periander HM996751 (butterfly)

Myrmica incompleta HM996744 (ant)

Spiroplasma phoeniceum AY772395

Spiroplasma insolitum AY189133

Poritia sumatrae HM996745 (butterfly)

Spiroplasma sp DQ186642

Drosophila melanogaster FJ657180 (fly)

Drosophila willistoni SMERRNA11 (fly)

Drosophila melanogaster AJ631998 (fly)

Drosophila simulans FJ657181 (fly)

Trapezites eliena HM996746 (butterfly)

Anthidium florentinum EU727107 (bee)

Eurygaster

integrice

ps JQ914111 (sh

ield bug)

Panaeus vannamei DQ360069 (sh

rimp)

Eriocheir sinensis

DQ917753 (crab)Lasiu

s alien

us GQ275102 (an

t)

Rimica

ris ex

oculat

a AJ515720 (shrim

p)

Selenomonas ruminantium AB003379

Clade 1

Clade 2

Clad

e 3

Inner circle ant subfamily

DorylinaeEctatomminaeEcitoninae

FormicinaeMyrmicinaePonerinaePseudomyrmecinae

Middle circle bioregionAfrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Aenictinae

Figure 2 Phylogenetic tree of Spiroplasma-related ant symbionts and their closest relatives with sequence data available in GenBank Amaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shown The host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) The branch color refers to the sourcefrom which the bacteria were isolated with yellow representing ant hosts red other insect hosts blue vertebrates and green plants The innercircle refers to the ant subfamily and the outer circle refers to the continent from which samples were collected The three largest ant-specificclades of Spiroplasma symbionts are indicated (Clades 1ndash3) Selenomonas ruminantium was used as an outgroup

The infection with Spiroplasma seems to be systemic as wefound high titers of this bacterium in association with antguts heads and legs (Supplementary Table 1)

Entomoplasmatales can be pathogenic to plants andvertebrates [59 62] and have been isolated from variousinsect taxa including aphids ants bees beetles butterflies

fruit flies and horse flies [63ndash68] Mutualistic spiroplasmascan grant insects resistance to parasitic nematodes [22]and an increased ability to overwinter [69] Pathogenicphenotypes usually lead to insect death [34] and reproductivemanipulation includes altered sex ratios [33] andmale killing[35 70 71] In ants spiroplasmas have been surveyed and

Psyche 7

biocontrol potential has been hypothesized but their roleremains elusive to date [31 38 60] Functional studiesthat compare the performance of infected and uninfectedindividuals would improve our understanding of the role ofthese facultative symbionts

33 Asaia (Alphaproteobacteria Rhodospirillales) Of 95 antspecies 14 hosted bacteria related to Asaia (Alphapro-teobacteria Rhodospirillales) (Table 1) For these bacteriano previous surveys on their prevalence across the antshave been conducted We found a particularly high infectionrate of 333 (1236 species) in Pseudomyrmex In con-trast Asaia-related symbionts were lacking in CamponotusCrematogasterMyrmeciaMyrmecocystus PolyrhachisOeco-phylla and Solenopsis Low infection frequency was presentin Cephalotes (17) and Paraponera (11 species) (Table 1)The enrichment of Asaia symbionts in Pseudomyrmex isparticularly interesting as this ant genus is arboreal andcontains several obligate plant ants which exclusively feed onplant-derived food sources [58 72] However this bacterialgroup occurred facultatively in arboreal generalists and plantmutualists alike indicating that even if these symbionts aremore frequent in arboreal ormutualistic Pseudomyrmex antsthe association is not obligate

In total we obtained 25 Asaia-related OTUs in ourdataset Of these OTUs 21 were associated with Pseu-domyrmex 3 with Paraponera and 1 with Cephalotes Weinferred a maximum likelihood phylogeny of these OTUstheir closest GenBank relatives and other endosymbioticRhodospirillales bacteria fromGenBankThe total alignmentconsisted of 91 taxa and had a length of 1313 characters WeusedWolbachia pipientis (Alphaproteobacteria Rickettsiales)as an outgroup The phylogenetic tree shows three clades inwhich ant-associatedAsaiaOTUs cluster together (Figure 3)(1) a small clade with two Pseudomyrmex-associated OTUsand one Paraponera-associated OTU (2) a clade that appearsto be Hymenoptera specific containing the bulk of Pseu-domyrmex-associated OTUs a Formica-associated sequencefrom GenBank and bacteria isolated from several beespecies and (3) a clade comprised of many insect-associatedAsaia bacteria and five of our OTUs This last clade is par-ticularly interesting as it comprised several strains that wereisolated from different mosquito species as well as three ant-associated Asaia sequences from GenBank One sequence(JF514556) was isolated and cultivated from Tetraponerarufonigra in India [73] The nifH gene a gene associatedwith the fixation of atmospheric nitrogen has also beenfound in this bacterium (GenBank accession JF736510) andit has been experimentally shown that this strain is capableof fixing nitrogen in vitro suggesting possible nitrogen fixingattributes in its natural environment the ant body cavity[73] The two other sequences are cultivated bacteria fromCephalotes varians and were generated in the framework ofa previous study from our lab (GenBank accessions JX445137and JX445138) [39]

Bacteria from the family Acetobacteraceae are commonlyknown as ldquoacetic acid bacteriardquo and have the metaboliccapacity to oxidize ethanol to acetic acid [74] Asaia also

a member of the Acetobacteraceae however only weaklyoxidizes ethanol and shows higher rates of sugar oxidation[74] These bacteria are environmentally ubiquitous buthave also been found in association with insects such asbees [75 76] mosquitoes [77] Drosophila melanogaster [78]leafhoppers [79] and mealybugs [80] All these insects relyon sugar-rich and often nitrogen-limited diets and it hasbeen suggested that the bacteria function as nutritionalsymbionts Some acetic acid bacteria have the capacity tofix atmospheric nitrogen [73] However it remains entirelyspeculative whether this function can be retained in theinsect gut environment and whether these bacteria actuallycontribute to insect nitrogen metabolism or recycling [81]Interestingly neither acetic acid bacteria nor lactic acidbacteria are commonly found in the core gut microbiota ofarboreal Cephalotini ants an ant group with one of the mostthoroughly studied microbiomes [18 19 39] The metaboliccapacities of the core gut microbiota of the Cephalotiniconsisting of Burkholderiales Opitutales PseudomonadalesRhizobiales and Xanthomonadales might be redundant withthe role that acetic acid bacteria play in other insects

In Drosophila acetic acid bacteria are part of the normalcommensal bacterial gut community and can be involved inthe regulation of the innate immune system In healthy fliesa stable equilibrium of different gut microbes is maintainedPerturbation of the normal gut community which can becaused by a defective regulation of antimicrobial peptideleads to the dominance of the pathogenic commensal Glu-conobacter morbifer and ultimately to gut apoptosis [82]Potential other mechanisms by which acetic acid bacteriabenefit insect immunity are by decreasing the gut pHmakingit an unfavorable environment for pathogenic microorgan-isms or by competitive exclusion [81] However these aceticacid bacteria are not essential for the fitness and repro-duction of most insects as even in the well-studied Asaia-mosquito interaction experimental removal of bacteria hadno detectable negative impact on the host [81]

Several studies have been conducted to analyze themicrobial diversity associated with ants [18 19 39 60 83]However the symbiotic relationships of ants with Rhodospir-illales have rarely been observed In fact only two ant-associated Rhodospirillales sequences had been depositedin GenBank (GQ275104 from Formica occulta and JF514556from Tetraponera rufonigra) prior to work from our group[39] Clone libraries generated for theCephalotini ants [18 19]as well as tag-encoded amplicon data sets [38 39] are amongthe most extensive microbial data collections available forants to date and acetic acid bacteria were only sporadicallyassociated with the ant taxa that were investigated Thus theinteraction of Asaia relatives with ants is generally poorlyunderstood but due to the metabolic capacities of thesebacteria to utilize sugar-rich substrates and fix nitrogenthey might play an important nutritional role Particularlythey might be functionally important in the ant subfamilyPseudomyrmecinae in which they seem to be enriched asindicated by our present study

The phylogenetic history of ant-associated Rhodospiril-lales does not show host specificity and suggests likely acqui-sition from the environment (Figure 3) These observations

8 Psyche

Drosophila sp EU096229 (fruit fly)

Drosophila sp EU096235 (fruit fly)

Drosophila sp EU096233 (fruit fly)

Pseudomyrm

ex ejectus SK1181-3 (ant)

Paraponera clavata CS0513-2 (ant mouth)

Gluconobacter frateurii AB682075

Gluc

onob

acte

r fra

teur

ii A

B682

081Gluconobacter frateurii A

B682091

Pseudo

myrmex

ferru

gineu

s SK00

02-1

(ant)

Pseudomyrm

ex pallidus C

SM1333 (ant)

Pseudomyrmex spinicola SK0061-1 (ant)

Pseudomyrmex peperi SK1058-103-1 (ant)

Cephalotes atratus SK1465 (ant)

Xylocopa californica HM112403 (bee)

Caupolicana yarrowi HM109591 (bee)

Euglossa imperialis JQ389882 (bee)

BER145 (ant)Pseudomyrmex sp nr eduardi

Pseudomyrmex satanicus SK0058-1 (ant)Pseudomyrmex dendroicus BER121 (ant)

Pseudomyrmex dendroicus BER103 (ant)

Pseudomyrmex peperi

SK0007-1 (ant)Pseu

domyrm

ex nigroci

nctus

SK0060-1 (a

nt)Pseudom

yrmex

major

SK09

90-8-

3 (an

t gast

er)

Pseu

domyrm

ex de

ndroi

cus S

K1260

(ant)

Form

ica oc

culta

GQ27

5104

(ant

)

Alph

a pro

teoba

cteriu

m A

B513

363 (

flowe

r)

Para

pone

ra cl

avat

a CS

0513

-4 (a

nt m

outh

)Ca

lliop

sis su

balpi

nus H

M10

9230

(bee

)Ca

lliop

sis su

balpi

nus H

M10

9259

(bee

)

Pseu

dom

yrm

ex te

nuis

BER1

12 (a

nt)

Sacc

haric

occu

s sac

char

i AF1

2739

6 (m

ealy

bug)

Dro

soph

ila sp

EU

0962

34 (f

ruit

fly)

Atta colombica H

M557097 (fungus garden)

Aedes aegypti FJ372761 (mosquito)

Aedes aegypti FJ372770 (mosquito)

Anopheles maculipennis AM

404262 (mosquito)

Aedes albopictus JQ958879 (m

osquito)

Aedes albopictus JQ314055 (m

osquito)

Aedes albopictus JQ314027 (m

osquito)

Aedes albopictus F2f JX445138 (mosquito)

Aede

s aeg

ypti

FN81

4303

(mos

quito

)

Aede

s alb

opict

us F

1f JX

4451

37 (m

osqu

ito)

Aede

s alb

opict

us F

2l JX

4451

40 (m

osqu

ito)

Aede

s alb

opict

us F

1l JX

4451

39 (m

osqu

ito)

Aede

s aeg

ypti

FN81

4301

(mos

quito

)

Aede

s aeg

ypti

FN81

4300

(mos

quito

)

Aede

s aeg

ypti

FN29

7842

(mos

quito

)

Anoph

eles s

tephe

nsi A

M40

4261

(mos

quito

)

Aede

s aeg

ypti

FN29

7841

(mos

quito

)Asaia b

ogore

nsis A

B6820

04

Anoph

eles s

p HQ26

9803

(mos

quito

)

Tetrap

onera

rufon

igra JF

5145

56 (a

nt)

Pseudomyrmex elo

ngatus

CSM1373 (ant gut)Pseudomyrmex termitarius

SK1252-1 (ant)

Pseudomyrmex gracilis

CSM1374-2 (ant head)Anopheles gambiae FN814276 (mosquito)Pseudomyrmex ejectus SK1181-3 (ant)

Pseudomyrm

ex elongatus

CSM1373 (ant head)

Cephalotes va

rians JX

990151 (ant)

Anopheles stephensi FN814299 (mosquito)

Anopheles gambiae FN814277 (mosquito)

Anopheles stephensi FN814297 (mosquito)

Anopheles stephensi FN814296 (mosquito)

Cephalotes varians JX990161 (ant)

Lutzomyia longipalpis HE995765 (sand fly)

Anopheles gambiae FN821398 (mosquito)

Aedes aegypti FN297840 (mosquito)

Anopheles stephensi AM404260 (mosquito)

Asaia krungthepensis AB682130

Anopheles gambiae FJ816021 (mosquito)Anopheles gambiae FN814284 (mosquito)Anopheles gambiae FN814283 (mosquito)

Aedes albopictus FN814288 (mosquito)

Aedes albopictus FN814287 (mosquito)

Anopheles gambiae FN821397 (mosquito)

Anopheles gambiae FN814280 (mosquito)

Aedes albopictus FN814289 (mosquito)

Anopheles gambiae FN814285 (mosquito)

Anopheles gambiae FN814275 (mosquito)

Anopheles gambiae FN814281 (mosquito)

Anopheles gambiae FN814278 (mosquito)

Aedes albopictus FN814286 (mosquito)

Atta colombica HM556904 (fungus garden)

Saccharicoccus sacchari AF127400 (mealybug)

Saccharicoccus sacchari AF127408 (mealybug)

Saccharicoccus sacchari AF127401 (mealybug)

Pseudomyrmex ejectus SK1181-3 (ant)

Paraponera clavata CS0513-3 (ant mouth)

Pseudomyrm

ex termitarius SK1252-2 (ant)

Drosophila sp EU096231 (fruit fly)Wolbachia pipientis AY026912

Inner circle ant subfamilyFormicinaeMyrmicinaeParaponerinaePseudomyrmecinae

Middle circle bioregionAfrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Clade 1

Clade 2Cl

ade 3

Figure 3 Phylogenetic tree of Asaia-related symbionts associated with ants and closest relatives with sequence data available in GenBank Amaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shown The host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) The branch color refers to the sourcefromwhich the bacteria were isolated with yellow representing ant hosts and red other insect hostsThe inner circle refers to the ant subfamilyand the middle circle refers to the bioregion from which samples were collected The outer circle indicates three clades (Clades 1ndash3) whichcontained several ant-associated symbiontsWolbachia pipientis was used as an outgroup

indicate that Rhodospirillales aremost likely environmentallytransmitted and support the hypothesis that they are onlyfacultative associates of ants One clade of ant-associatedRhodospirillales was closely related to endosymbionts iso-lated from mosquitos (Figure 3) It has been experimentallyshown that mosquito-associated Asaia can successfully colo-nize leafhoppers further emphasizing the low-host specificityof this bacterial group [77]

4 Conclusion

Our broad bacterial screening approach has contributedto our understanding of the prevalence of ant-associatedmicrobes particularly with regard to their Wolbachia andSpiroplasma symbionts Furthermore we provide the firstextensive survey for ant-associated Asaia-related symbiontsWhile these symbionts of the order Rhodospirillales infect

Psyche 9

ants only sporadically some strains are capable of fixingatmospheric nitrogen and might retain this function inants Alternatively these bacteria might have an importantfunctional role for upgrading nitrogen-poor diets of someherbivorous ants which comprise the majority of all ant taxa[20] Even though we do not have experimental evidence ofthe role of most bacterial symbionts in ants previous studiesillustrate a broad variety of effects of these bacteria on insecthosts [4 7 9] Even a single group of microbes can havevery different effects on different hosts Our study shows thatdespite several extensive bacterial surveys across the ants thediversity and functional role of ant-associated microbes isfar from being fully understood and broad next generationsequencing approaches will provide a fast and cost-effectivetool to deepen our knowledge of the rare (and not so rare)biosphere

Acknowledgments

The authors thank Daniel Ballhorn and Tina Schroyer forhelpful comments and discussions Phil Ward kindly iden-tified Pseudomyrmex specimens This study was supportedin part by National Science Foundation DEB Grant no1050243 to Corrie S Moreau a Grainger Foundation grant toCorrie S Moreau a Negaunee Foundation grant to Corrie SMoreau and the Pritzker Laboratory forMolecular Evolutionand Systematics Stefanie Kautz acknowledges a postdoctoralfellowship from the German Academy of Sciences Leopold-ina (Grant no LPDS 2009-29) Benjamin E R Rubin wassupported in part by an NSF Graduate Research Fellowship

References

[1] H Koch D P Abrol J Li and P Schmid-Hempel ldquoDiversity ofevolutionary patterns of bacterial gut associates of corbiculatebeesrdquoMolecular Ecology vol 22 no 7 pp 2028ndash2044 2013

[2] R J Dillon and V M Dillon ldquoThe Gut bacteria of insectsnonpathogenic interactionsrdquoAnnual Review of Entomology vol49 pp 71ndash92 2004

[3] S Fraune and T C G Bosch ldquoWhy bacteria matter in animaldevelopment and evolutionrdquo BioEssays vol 32 no 7 pp 571ndash580 2010

[4] H Feldhaar ldquoBacterial symbionts as mediators of ecologicallyimportant traits of insect hostsrdquo Ecological Entomology vol 36no 5 pp 533ndash543 2011

[5] J M Urban and J R Cryan ldquoTwo ancient bacterialendosymbionts have coevolved with the planthoppers (InsectaHemiptera Fulgoroidea)rdquo BMC Evolutionary Biology vol 12article 87 2012

[6] H Toju and T Fukatsu ldquoDiversity and infection prevalence ofendosymbionts in natural populations of the chestnut weevilrelevance of local climate and host plantsrdquo Molecular Ecologyvol 20 no 4 pp 853ndash868 2011

[7] M Kaltenpoth ldquoActinobacteria as mutualists general health-care for insectsrdquo Trends inMicrobiology vol 17 no 12 pp 529ndash535 2009

[8] N A Moran J P McCutcheon and A Nakabachi ldquoGenomicsand evolution of heritable bacterial symbiontsrdquo Annual Reviewof Genetics vol 42 pp 165ndash190 2008

[9] J P McCutcheon and C D Von Dohlen ldquoAn interdependentmetabolic patchwork in the nested symbiosis of mealybugsrdquoCurrent Biology vol 21 no 16 pp 1366ndash1372 2011

[10] C B Montllor A Maxmen and A H Purcell ldquoFacultative bac-terial endosymbionts benefit pea aphids Acyrthosiphon pisumunder heat stressrdquo Ecological Entomology vol 27 no 2 pp 189ndash195 2002

[11] A E Douglas ldquoThe nutritional physiology of aphidsrdquoAdvancesin Insect Physiology vol 31 pp 73ndash140 2003

[12] J J Wernegreen S N Kauppinen S G Brady and P SWard ldquoOne nutritional symbiosis begat another phylogeneticevidence that the ant tribe Camponotini acquired Blochmanniaby tending sap-feeding insectsrdquo BMC Evolutionary Biology vol9 no 1 article 292 2009

[13] H Feldhaar J StrakaM Krischke et al ldquoNutritional upgradingfor omnivorous carpenter ants by the endosymbiont Blochman-niardquo BMC Biology vol 5 article 48 2007

[14] A A Pinto-Tomas M A Anderson G Suen et al ldquoSymbioticnitrogen fixation in the fungus gardens of leaf-cutter antsrdquoScience vol 326 no 5956 pp 1120ndash1123 2009

[15] R Pais C Lohs Y Wu J Wang and S Aksoy ldquoThe obligatemutualist Wigglesworthia glossinidia influences reproductiondigestion and immunity processes of its host the tsetse flyrdquoApplied and Environmental Microbiology vol 74 no 19 pp5965ndash5974 2008

[16] J A Breznak ldquoIntestinal microbiota of termites and otherxylophagous insectsrdquo Annual Review of Microbiology vol 36pp 323ndash343 1982

[17] J A Breznak W J Brill J W Mertins and H C CoppelldquoNitrogen fixation in termitesrdquo Nature vol 244 no 5418 pp577ndash579 1973

[18] J A Russell C SMoreau BGoldman-HuertasM FujiwaraDJ Lohman andN E Pierce ldquoBacterial gut symbionts are tightlylinkedwith the evolution of herbivory in antsrdquoProceedings of theNational Academy of Sciences of theUnited States of America vol106 no 50 pp 21236ndash21241 2009

[19] K E Anderson J A Russell C S Moreau et al ldquoHighlysimilar microbial communities are shared among related andtrophically similar ant speciesrdquo Molecular Ecology vol 21 pp2282ndash2296 2012

[20] D W Davidson S C Cook R R Snelling and T H ChualdquoExplaining the abundance of ants in lowland tropical rainforestcanopiesrdquo Science vol 300 no 5621 pp 969ndash972 2003

[21] J C Brownlie and K N Johnson ldquoSymbiont-mediated protec-tion in insect hostsrdquo Trends in Microbiology vol 17 no 8 pp348ndash354 2009

[22] J Jaenike R Unckless S N Cockburn L M Boelio andS J Perlman ldquoAdaptation via symbiosis recent spread of aDrosophila defensive symbiontrdquo Science vol 329 no 5988 pp212ndash215 2012

[23] K M Oliver J A Russell N A Morant and M S HunterldquoFacultative bacterial symbionts in aphids confer resistance toparasitic waspsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 4 pp 1803ndash18072003

[24] C R Currie J A Scott R C Summerbell and D MallochldquoFungus-growing ants use antibiotic-producing bacteria tocontrol garden parasitesrdquo Nature vol 398 pp 701ndash704 1999

[25] M Kaltenpoth W Gottler G Herzner and E Strohm ldquoSym-biotic bacteria protect wasp larvae from fungal infestationrdquoCurrent Biology vol 15 no 5 pp 475ndash479 2005

10 Psyche

[26] H EDunbar A C CWilsonN R Ferguson andNAMoranldquoAphid thermal tolerance is governed by a point mutation inbacterial symbiontsrdquo PLoS Biology vol 5 no 5 pp 1006ndash10152007

[27] T Tsuchida R Koga S Matsumoto and T Fukatsu ldquoInterspe-cific symbiont transfection confers a novel ecological trait to therecipient insectrdquo Biology Letters vol 7 no 2 pp 245ndash248 2011

[28] R Stouthamer J A J Breeuwer and G D D Hurst ldquoWolbachiapipientis microbial manipulator of arthropod reproductionrdquoAnnual Review of Microbiology vol 53 pp 71ndash102 1999

[29] L M Hedges J C Brownlie S L OrsquoNeill and K N JohnsonldquoWolbachia and virus protection in insectsrdquo Science vol 322 no5902 p 702 2008

[30] K Hilgenboecker P Hammerstein P Schlattmann ATelschow and J H Werren ldquoHow many species are infectedwith Wolbachiamdasha statistical analysis of current datardquo FEMSMicrobiology Letters vol 281 no 2 pp 215ndash220 2008

[31] J A Russell C F Funaro Y M Giraldo et al ldquoA veritablemenagerie of heritable bacteria from ants butterflies andbeyond broadmolecular surveys and a systematic reviewrdquoPLoSONE vol 7 no 12 Article ID e51027 2012

[32] O Duron D Bouchon S Boutin et al ldquoThe diversity ofreproductive parasites among arthropods Wolbachia do notwalk alonerdquo BMC Biology vol 6 article 27 2008

[33] D L Williamson and D F Poulson ldquoSex ratio organisms(Spiroplasmas) of Drosophilardquo in The Mycoplasmas III Plantand Insect Mycoplasmas R F Whitcomb and J G Tully Edspp 175ndash208 Academic Press New York NY USA 1979

[34] J M Bove ldquoSpiroplasmas infectious agents of plants arthro-pods and vertebratesrdquoWiener Klinische Wochenschrift vol 109no 14-15 pp 604ndash612 1997

[35] M C Tinsley and M E N Majerus ldquoA new male-killingparasitism Spiroplasma bacteria infect the ladybird beetleAnisosticta novemdecimpunctata (Coleoptera Coccinellidae)rdquoParasitology vol 132 no 6 pp 757ndash765 2006

[36] A Dobson K D Lafferty A M Kuris R F Hechinger andW Jetz ldquoHomage to Linnaeus howmany parasites Howmanyhostsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 105 no 1 pp 11482ndash11489 2008

[37] S T Kelley and S Dobler ldquoComparative analysis of microbialdiversity in Longitarsus flea beetles (Coleoptera Chrysomeli-dae)rdquo Genetica vol 139 no 5 pp 541ndash550 2011

[38] H D Ishak R Plowes R Sen et al ldquoBacterial diversityin Solenopsis invicta and Solenopsis geminata ant coloniescharacterized by 16S amplicon 454 pyrosequencingrdquo MicrobialEcology vol 61 no 4 pp 821ndash831 2011

[39] S Kautz B E R Rubin J A Russell and C S Moreau ldquoSur-veying the microbiome of ants comparing 454 pyrosequencingwith traditionalmethods to uncover bacterial diversityrdquoAppliedand Environmental Microbiology vol 79 no 2 pp 525ndash5342013

[40] S E Dowd T R Callaway R D Wolcott et al ldquoEvaluationof the bacterial diversity in the feces of cattle using 16SrDNA bacterial tag-encoded FLX amplicon pyrosequencing(bTEFAP)rdquo BMCMicrobiology vol 8 article 125 2008

[41] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[42] C Quince A Lanzen R J Davenport and P J TurnbaughldquoRemoving noise from pyrosequenced ampliconsrdquo BMC Bioin-formatics vol 12 article 38 2011

[43] R C Edgar ldquoSearch and clustering orders of magnitude fasterthan BLASTrdquo Bioinformatics vol 26 no 19 pp 2460ndash24612010

[44] J R Cole QWang E Cardenas et al ldquoTheRibosomal databaseproject improved alignments and new tools for rRNA analysisrdquoNucleic Acids Research vol 37 no 1 pp D141ndashD145 2009

[45] T Z DeSantis P Hugenholtz N Larsen et al ldquoGreengenesa chimera-checked 16S rRNA gene database and workbenchcompatible with ARBrdquo Applied and Environmental Microbiol-ogy vol 72 no 7 pp 5069ndash5072 2006

[46] A J Drummond B Ashton S Buxton et al ldquoGeneious v5 4rdquo2011 httpwwwgeneiouscom

[47] A Stamatakis P Hoover and J Rougemont ldquoA rapid bootstrapalgorithm for the RAxML web serversrdquo Systematic Biology vol57 no 5 pp 758ndash771 2008

[48] M A Miller M T Holder R Vos et al ldquoThe CIPRES portalsrdquohttpwwwphyloorgsub sectionsportal

[49] I Letunic and P Bork ldquoInteractive Tree of Life v2 onlineannotation and display of phylogenetic treesmade easyrdquoNucleicAcids Research vol 39 no 2 pp W475ndashW478 2011

[50] T Wenseleers F Ito S van Borm R Huybrechts F Volckaertand J Billen ldquoWidespread occurrence of the micro-organismWolbachia in antsrdquo Proceedings of the Royal Society B vol 265no 1404 pp 1447ndash1452 1998

[51] J A Russell ldquoThe ants (Hymenoptera Formicidae) are uniqueand enigmatic hosts of prevalent Wolbachia (Alphaproteobac-teria) symbiontsrdquoMyrmecological News vol 16 pp 7ndash23 2012

[52] W Arthofer M Riegler D Schneider M Krammer W JMiller and C Stauffer ldquoHidden Wolbachia diversity in fieldpopulations of the European cherry fruit fly Rhagoletis cerasi(Diptera Tephritidae)rdquo Molecular Ecology vol 18 no 18 pp3816ndash3830 2009

[53] J A Russell B Goldman-Huertas C S Moreau et al ldquoSpecial-ization and geographic isolation among Wolbachia symbiontsfrom ants and lycaenid butterfliesrdquo Evolution vol 63 no 3 pp624ndash640 2009

[54] T Wenseleers L Sundstrom and J Billen ldquoDeleterious Wol-bachia in the ant Formica truncorumrdquo Proceedings of the RoyalSociety B vol 269 no 1491 pp 623ndash629 2002

[55] F Vavre F Fleury J Varaldi P Fouillet and M BouletreauldquoEvidence for female mortality in Wolbachia-mediated cyto-plasmic incompatibility in haplodiploid insects epidemiologicand evolutionary consequencesrdquo Evolution vol 54 no 1 pp191ndash200 2000

[56] J Heinze ldquoThe demise of the standard ant (HymenopteraFormicidae)rdquoMyrmecological News vol 11 pp 11ndash20 2008

[57] S Kautz S U Pauls D J Ballhorn H T Lumbsch andM HeilldquoPolygynous supercolonies of the acacia-ant Pseudomyrmexpeperi an inferior colony founderrdquo Molecular Ecology vol 18no 24 pp 5180ndash5194 2009

[58] S Kautz D J Ballhorn J Kroiss et al ldquoHost plant use bycompeting acacia-ants mutualists monopolize while parasitesshare hostsrdquo PLoS ONE vol 7 no 5 Article ID e37691 2012

[59] J Fletcher G A Schultz R E Davis C E Eastman and R MGoodman ldquoBrittle root disease of horseradish-evidence for anetiological role of Spiroplasma citrirdquo Phytopathology vol 71 pp1073ndash1080 1981

[60] C F Funaro D J C Kronauer C S Moreau B Goldman-Huertas N E Pierce and J A Russell ldquoArmy ants harbor ahost-specific clade of Entomoplasmatales bacteriardquo Applied andEnvironmental Microbiology vol 77 no 1 pp 346ndash350 2011

Psyche 11

[61] D J C Kronauer ldquoRecent advances in army ant biology(Hymenoptera Formicidae)rdquoMyrmecological News vol 12 pp51ndash65 2009

[62] F Thiaucourt and G Bolske ldquoContagious caprine pleurop-neumonia and other pulmonary mycoplasmoses of sheep andgoatsrdquo OIE Revue Scientifique et Technique vol 15 no 4 pp1397ndash1414 1996

[63] T Fukatsu T Tsuchida N Nikoh and R Koga ldquoSpiroplasmaSymbiont of the pea aphid Acyrthosiphon pisum (InsectaHomoptera)rdquo Applied and Environmental Microbiology vol 67no 3 pp 1284ndash1291 2001

[64] T S Haselkorn T A Markow and N A Moran ldquoMultipleintroductions of the Spiroplasma bacterial endosymbiont intoDrosophilardquo Molecular Ecology vol 18 no 6 pp 1294ndash13052009

[65] J G Lundgren R M Lehman and J Chee-Sanford ldquoPhysiol-ogy biochemistry and topology bacterial communities withindigestive tracts of ground beetles (Coleoptera Carabidae)rdquoAnnals of the Entomological Society of America vol 100 no 2pp 275ndash282 2007

[66] I Meeus V Vercruysse and G Smagghe ldquoMolecular detectionof Spiroplasma apis and Spiroplasmamelliferum in beesrdquo Journalof Invertebrate Pathology vol 109 no 1 pp 172ndash174 2012

[67] S van Borm J Billen and J J Boomsma ldquoThe diversity ofmicroorganisms associated with Acromyrmex leafcutter antsrdquoBMC Evolutionary Biology vol 2 article 9 2002

[68] J Wedincamp F E French R F Whitcomb and R B HenegerldquoLaboratory infection and release of Spiroplasma (Entomoplas-matales Spiroplasmataceae) from horse flies (Diptera Taban-idae)rdquo Journal of Entomological Science vol 32 no 4 pp 398ndash402 1997

[69] M A Ebbert and L R Nault ldquoImproved overwintering abilityin Dalbulus maidis (Homoptera Cicadellidae) vectors infectedwith Spiroplasma kunkelii (Mycoplasmatales Spiroplasmat-aceae)rdquo Environmental Entomology vol 23 no 3 pp 634ndash6441994

[70] F M Jiggins G D D Hurst C D Jiggins J H G V DSchulenburg and M E N Majerus ldquoThe butterfly Danauschrysippus is infected by a male-killing Spiroplasma bacteriumrdquoParasitology vol 120 no 5 pp 439ndash446 2000

[71] J H G V D Schulenburg G D D Hurst D Tetzlaff GE Booth I A Zakharov and M E N Majerus ldquoHistory ofinfection with different male-killing bacteria in the two-spotladybird beetle Adalia bipunctata revealed through mitochon-drial DNA sequence analysisrdquoGenetics vol 160 no 3 pp 1075ndash1086 2002

[72] S Kautz H T Lumbsch P S Ward and M Heil ldquoHowto prevent cheating a digestive specialization ties mutualisticplant-ants to their ant-plant partnersrdquo Evolution vol 63 no 4pp 839ndash853 2009

[73] N Samaddar A Paul S Chakravorty et al ldquoNitrogen fixationin Asaia sp (Family Acetobacteraceae)rdquo Current Microbiologyvol 63 no 2 pp 226ndash231 2011

[74] Y Ano H Toyama O Adachi and K Matsushita ldquoEnergymetabolism of a unique acetic acid bacteriumAsaia bogorensisthat lacks ethanol oxidation activityrdquo Bioscience Biotechnologyand Biochemistry vol 72 no 4 pp 989ndash997 2008

[75] V G Martinson B N Danforth R L Minckley O RueppellS Tingek and N A Moran ldquoA simple and distinctive micro-biota associated with honey bees and bumble beesrdquo MolecularEcology vol 20 no 3 pp 619ndash628 2011

[76] N A Moran A K Hansen J E Powell and Z L SabreeldquoDistinctive gut microbiota of honey bees assessed using deepsampling from individual worker beesrdquo PLoS ONE vol 7 no 4Article ID e36393 2012

[77] E Crotti C Damiani M Pajoro et al ldquoAsaia a versatile aceticacid bacterial symbiont capable of cross-colonizing insectsof phylogenetically distant genera and ordersrdquo EnvironmentalMicrobiology vol 11 no 12 pp 3252ndash3264 2009

[78] C R Cox and M S Gilmore ldquoNative microbial colonization ofDrosophilamelanogaster and its use as a model of Enterococcusfaecalis pathogenesisrdquo Infection and Immunity vol 75 no 4 pp1565ndash1576 2007

[79] M Marzorati A Alma L Sacchi et al ldquoA novel Bacteroidetessymbiont is localized in Scaphoideus titanus the insect vector ofFlavescence Doree in Vitis viniferardquo Applied and EnvironmentalMicrobiology vol 72 no 2 pp 1467ndash1475 2006

[80] N J Ashbolt and P A Inkerman ldquoAcetic acid bacterial biota ofthe pink sugar cane mealybug Saccharococcus sacchari and itsenvironsrdquo Applied and Environmental Microbiology vol 56 no3 pp 707ndash712 1990

[81] E Crotti A Rizzi B Chouaia et al ldquoAcetic acid bacteria newlyemerging symbionts of insectsrdquo Applied and EnvironmentalMicrobiology vol 76 no 21 pp 6963ndash6970 2010

[82] J Ryu S Kim H Lee et al ldquoInnate immune homeostasis bythe homeobox gene Caudal and commensal-gut mutualism inDrosophilardquo Science vol 319 no 5864 pp 777ndash782 2008

[83] S Stoll J Gadau R Gross and H Feldhaar ldquoBacterial micro-biota associated with ants of the genus Tetraponerardquo BiologicalJournal of the Linnean Society vol 90 no 3 pp 399ndash412 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Volume 2014

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International Journal of

Microbiology

Page 7: Research Article Bacterial Infections across the Ants ...downloads.hindawi.com/journals/psyche/2013/936341.pdf · Department of Zoology, Field Museum of Natural History, South Lake

Psyche 7

biocontrol potential has been hypothesized but their roleremains elusive to date [31 38 60] Functional studiesthat compare the performance of infected and uninfectedindividuals would improve our understanding of the role ofthese facultative symbionts

33 Asaia (Alphaproteobacteria Rhodospirillales) Of 95 antspecies 14 hosted bacteria related to Asaia (Alphapro-teobacteria Rhodospirillales) (Table 1) For these bacteriano previous surveys on their prevalence across the antshave been conducted We found a particularly high infectionrate of 333 (1236 species) in Pseudomyrmex In con-trast Asaia-related symbionts were lacking in CamponotusCrematogasterMyrmeciaMyrmecocystus PolyrhachisOeco-phylla and Solenopsis Low infection frequency was presentin Cephalotes (17) and Paraponera (11 species) (Table 1)The enrichment of Asaia symbionts in Pseudomyrmex isparticularly interesting as this ant genus is arboreal andcontains several obligate plant ants which exclusively feed onplant-derived food sources [58 72] However this bacterialgroup occurred facultatively in arboreal generalists and plantmutualists alike indicating that even if these symbionts aremore frequent in arboreal ormutualistic Pseudomyrmex antsthe association is not obligate

In total we obtained 25 Asaia-related OTUs in ourdataset Of these OTUs 21 were associated with Pseu-domyrmex 3 with Paraponera and 1 with Cephalotes Weinferred a maximum likelihood phylogeny of these OTUstheir closest GenBank relatives and other endosymbioticRhodospirillales bacteria fromGenBankThe total alignmentconsisted of 91 taxa and had a length of 1313 characters WeusedWolbachia pipientis (Alphaproteobacteria Rickettsiales)as an outgroup The phylogenetic tree shows three clades inwhich ant-associatedAsaiaOTUs cluster together (Figure 3)(1) a small clade with two Pseudomyrmex-associated OTUsand one Paraponera-associated OTU (2) a clade that appearsto be Hymenoptera specific containing the bulk of Pseu-domyrmex-associated OTUs a Formica-associated sequencefrom GenBank and bacteria isolated from several beespecies and (3) a clade comprised of many insect-associatedAsaia bacteria and five of our OTUs This last clade is par-ticularly interesting as it comprised several strains that wereisolated from different mosquito species as well as three ant-associated Asaia sequences from GenBank One sequence(JF514556) was isolated and cultivated from Tetraponerarufonigra in India [73] The nifH gene a gene associatedwith the fixation of atmospheric nitrogen has also beenfound in this bacterium (GenBank accession JF736510) andit has been experimentally shown that this strain is capableof fixing nitrogen in vitro suggesting possible nitrogen fixingattributes in its natural environment the ant body cavity[73] The two other sequences are cultivated bacteria fromCephalotes varians and were generated in the framework ofa previous study from our lab (GenBank accessions JX445137and JX445138) [39]

Bacteria from the family Acetobacteraceae are commonlyknown as ldquoacetic acid bacteriardquo and have the metaboliccapacity to oxidize ethanol to acetic acid [74] Asaia also

a member of the Acetobacteraceae however only weaklyoxidizes ethanol and shows higher rates of sugar oxidation[74] These bacteria are environmentally ubiquitous buthave also been found in association with insects such asbees [75 76] mosquitoes [77] Drosophila melanogaster [78]leafhoppers [79] and mealybugs [80] All these insects relyon sugar-rich and often nitrogen-limited diets and it hasbeen suggested that the bacteria function as nutritionalsymbionts Some acetic acid bacteria have the capacity tofix atmospheric nitrogen [73] However it remains entirelyspeculative whether this function can be retained in theinsect gut environment and whether these bacteria actuallycontribute to insect nitrogen metabolism or recycling [81]Interestingly neither acetic acid bacteria nor lactic acidbacteria are commonly found in the core gut microbiota ofarboreal Cephalotini ants an ant group with one of the mostthoroughly studied microbiomes [18 19 39] The metaboliccapacities of the core gut microbiota of the Cephalotiniconsisting of Burkholderiales Opitutales PseudomonadalesRhizobiales and Xanthomonadales might be redundant withthe role that acetic acid bacteria play in other insects

In Drosophila acetic acid bacteria are part of the normalcommensal bacterial gut community and can be involved inthe regulation of the innate immune system In healthy fliesa stable equilibrium of different gut microbes is maintainedPerturbation of the normal gut community which can becaused by a defective regulation of antimicrobial peptideleads to the dominance of the pathogenic commensal Glu-conobacter morbifer and ultimately to gut apoptosis [82]Potential other mechanisms by which acetic acid bacteriabenefit insect immunity are by decreasing the gut pHmakingit an unfavorable environment for pathogenic microorgan-isms or by competitive exclusion [81] However these aceticacid bacteria are not essential for the fitness and repro-duction of most insects as even in the well-studied Asaia-mosquito interaction experimental removal of bacteria hadno detectable negative impact on the host [81]

Several studies have been conducted to analyze themicrobial diversity associated with ants [18 19 39 60 83]However the symbiotic relationships of ants with Rhodospir-illales have rarely been observed In fact only two ant-associated Rhodospirillales sequences had been depositedin GenBank (GQ275104 from Formica occulta and JF514556from Tetraponera rufonigra) prior to work from our group[39] Clone libraries generated for theCephalotini ants [18 19]as well as tag-encoded amplicon data sets [38 39] are amongthe most extensive microbial data collections available forants to date and acetic acid bacteria were only sporadicallyassociated with the ant taxa that were investigated Thus theinteraction of Asaia relatives with ants is generally poorlyunderstood but due to the metabolic capacities of thesebacteria to utilize sugar-rich substrates and fix nitrogenthey might play an important nutritional role Particularlythey might be functionally important in the ant subfamilyPseudomyrmecinae in which they seem to be enriched asindicated by our present study

The phylogenetic history of ant-associated Rhodospiril-lales does not show host specificity and suggests likely acqui-sition from the environment (Figure 3) These observations

8 Psyche

Drosophila sp EU096229 (fruit fly)

Drosophila sp EU096235 (fruit fly)

Drosophila sp EU096233 (fruit fly)

Pseudomyrm

ex ejectus SK1181-3 (ant)

Paraponera clavata CS0513-2 (ant mouth)

Gluconobacter frateurii AB682075

Gluc

onob

acte

r fra

teur

ii A

B682

081Gluconobacter frateurii A

B682091

Pseudo

myrmex

ferru

gineu

s SK00

02-1

(ant)

Pseudomyrm

ex pallidus C

SM1333 (ant)

Pseudomyrmex spinicola SK0061-1 (ant)

Pseudomyrmex peperi SK1058-103-1 (ant)

Cephalotes atratus SK1465 (ant)

Xylocopa californica HM112403 (bee)

Caupolicana yarrowi HM109591 (bee)

Euglossa imperialis JQ389882 (bee)

BER145 (ant)Pseudomyrmex sp nr eduardi

Pseudomyrmex satanicus SK0058-1 (ant)Pseudomyrmex dendroicus BER121 (ant)

Pseudomyrmex dendroicus BER103 (ant)

Pseudomyrmex peperi

SK0007-1 (ant)Pseu

domyrm

ex nigroci

nctus

SK0060-1 (a

nt)Pseudom

yrmex

major

SK09

90-8-

3 (an

t gast

er)

Pseu

domyrm

ex de

ndroi

cus S

K1260

(ant)

Form

ica oc

culta

GQ27

5104

(ant

)

Alph

a pro

teoba

cteriu

m A

B513

363 (

flowe

r)

Para

pone

ra cl

avat

a CS

0513

-4 (a

nt m

outh

)Ca

lliop

sis su

balpi

nus H

M10

9230

(bee

)Ca

lliop

sis su

balpi

nus H

M10

9259

(bee

)

Pseu

dom

yrm

ex te

nuis

BER1

12 (a

nt)

Sacc

haric

occu

s sac

char

i AF1

2739

6 (m

ealy

bug)

Dro

soph

ila sp

EU

0962

34 (f

ruit

fly)

Atta colombica H

M557097 (fungus garden)

Aedes aegypti FJ372761 (mosquito)

Aedes aegypti FJ372770 (mosquito)

Anopheles maculipennis AM

404262 (mosquito)

Aedes albopictus JQ958879 (m

osquito)

Aedes albopictus JQ314055 (m

osquito)

Aedes albopictus JQ314027 (m

osquito)

Aedes albopictus F2f JX445138 (mosquito)

Aede

s aeg

ypti

FN81

4303

(mos

quito

)

Aede

s alb

opict

us F

1f JX

4451

37 (m

osqu

ito)

Aede

s alb

opict

us F

2l JX

4451

40 (m

osqu

ito)

Aede

s alb

opict

us F

1l JX

4451

39 (m

osqu

ito)

Aede

s aeg

ypti

FN81

4301

(mos

quito

)

Aede

s aeg

ypti

FN81

4300

(mos

quito

)

Aede

s aeg

ypti

FN29

7842

(mos

quito

)

Anoph

eles s

tephe

nsi A

M40

4261

(mos

quito

)

Aede

s aeg

ypti

FN29

7841

(mos

quito

)Asaia b

ogore

nsis A

B6820

04

Anoph

eles s

p HQ26

9803

(mos

quito

)

Tetrap

onera

rufon

igra JF

5145

56 (a

nt)

Pseudomyrmex elo

ngatus

CSM1373 (ant gut)Pseudomyrmex termitarius

SK1252-1 (ant)

Pseudomyrmex gracilis

CSM1374-2 (ant head)Anopheles gambiae FN814276 (mosquito)Pseudomyrmex ejectus SK1181-3 (ant)

Pseudomyrm

ex elongatus

CSM1373 (ant head)

Cephalotes va

rians JX

990151 (ant)

Anopheles stephensi FN814299 (mosquito)

Anopheles gambiae FN814277 (mosquito)

Anopheles stephensi FN814297 (mosquito)

Anopheles stephensi FN814296 (mosquito)

Cephalotes varians JX990161 (ant)

Lutzomyia longipalpis HE995765 (sand fly)

Anopheles gambiae FN821398 (mosquito)

Aedes aegypti FN297840 (mosquito)

Anopheles stephensi AM404260 (mosquito)

Asaia krungthepensis AB682130

Anopheles gambiae FJ816021 (mosquito)Anopheles gambiae FN814284 (mosquito)Anopheles gambiae FN814283 (mosquito)

Aedes albopictus FN814288 (mosquito)

Aedes albopictus FN814287 (mosquito)

Anopheles gambiae FN821397 (mosquito)

Anopheles gambiae FN814280 (mosquito)

Aedes albopictus FN814289 (mosquito)

Anopheles gambiae FN814285 (mosquito)

Anopheles gambiae FN814275 (mosquito)

Anopheles gambiae FN814281 (mosquito)

Anopheles gambiae FN814278 (mosquito)

Aedes albopictus FN814286 (mosquito)

Atta colombica HM556904 (fungus garden)

Saccharicoccus sacchari AF127400 (mealybug)

Saccharicoccus sacchari AF127408 (mealybug)

Saccharicoccus sacchari AF127401 (mealybug)

Pseudomyrmex ejectus SK1181-3 (ant)

Paraponera clavata CS0513-3 (ant mouth)

Pseudomyrm

ex termitarius SK1252-2 (ant)

Drosophila sp EU096231 (fruit fly)Wolbachia pipientis AY026912

Inner circle ant subfamilyFormicinaeMyrmicinaeParaponerinaePseudomyrmecinae

Middle circle bioregionAfrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Clade 1

Clade 2Cl

ade 3

Figure 3 Phylogenetic tree of Asaia-related symbionts associated with ants and closest relatives with sequence data available in GenBank Amaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shown The host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) The branch color refers to the sourcefromwhich the bacteria were isolated with yellow representing ant hosts and red other insect hostsThe inner circle refers to the ant subfamilyand the middle circle refers to the bioregion from which samples were collected The outer circle indicates three clades (Clades 1ndash3) whichcontained several ant-associated symbiontsWolbachia pipientis was used as an outgroup

indicate that Rhodospirillales aremost likely environmentallytransmitted and support the hypothesis that they are onlyfacultative associates of ants One clade of ant-associatedRhodospirillales was closely related to endosymbionts iso-lated from mosquitos (Figure 3) It has been experimentallyshown that mosquito-associated Asaia can successfully colo-nize leafhoppers further emphasizing the low-host specificityof this bacterial group [77]

4 Conclusion

Our broad bacterial screening approach has contributedto our understanding of the prevalence of ant-associatedmicrobes particularly with regard to their Wolbachia andSpiroplasma symbionts Furthermore we provide the firstextensive survey for ant-associated Asaia-related symbiontsWhile these symbionts of the order Rhodospirillales infect

Psyche 9

ants only sporadically some strains are capable of fixingatmospheric nitrogen and might retain this function inants Alternatively these bacteria might have an importantfunctional role for upgrading nitrogen-poor diets of someherbivorous ants which comprise the majority of all ant taxa[20] Even though we do not have experimental evidence ofthe role of most bacterial symbionts in ants previous studiesillustrate a broad variety of effects of these bacteria on insecthosts [4 7 9] Even a single group of microbes can havevery different effects on different hosts Our study shows thatdespite several extensive bacterial surveys across the ants thediversity and functional role of ant-associated microbes isfar from being fully understood and broad next generationsequencing approaches will provide a fast and cost-effectivetool to deepen our knowledge of the rare (and not so rare)biosphere

Acknowledgments

The authors thank Daniel Ballhorn and Tina Schroyer forhelpful comments and discussions Phil Ward kindly iden-tified Pseudomyrmex specimens This study was supportedin part by National Science Foundation DEB Grant no1050243 to Corrie S Moreau a Grainger Foundation grant toCorrie S Moreau a Negaunee Foundation grant to Corrie SMoreau and the Pritzker Laboratory forMolecular Evolutionand Systematics Stefanie Kautz acknowledges a postdoctoralfellowship from the German Academy of Sciences Leopold-ina (Grant no LPDS 2009-29) Benjamin E R Rubin wassupported in part by an NSF Graduate Research Fellowship

References

[1] H Koch D P Abrol J Li and P Schmid-Hempel ldquoDiversity ofevolutionary patterns of bacterial gut associates of corbiculatebeesrdquoMolecular Ecology vol 22 no 7 pp 2028ndash2044 2013

[2] R J Dillon and V M Dillon ldquoThe Gut bacteria of insectsnonpathogenic interactionsrdquoAnnual Review of Entomology vol49 pp 71ndash92 2004

[3] S Fraune and T C G Bosch ldquoWhy bacteria matter in animaldevelopment and evolutionrdquo BioEssays vol 32 no 7 pp 571ndash580 2010

[4] H Feldhaar ldquoBacterial symbionts as mediators of ecologicallyimportant traits of insect hostsrdquo Ecological Entomology vol 36no 5 pp 533ndash543 2011

[5] J M Urban and J R Cryan ldquoTwo ancient bacterialendosymbionts have coevolved with the planthoppers (InsectaHemiptera Fulgoroidea)rdquo BMC Evolutionary Biology vol 12article 87 2012

[6] H Toju and T Fukatsu ldquoDiversity and infection prevalence ofendosymbionts in natural populations of the chestnut weevilrelevance of local climate and host plantsrdquo Molecular Ecologyvol 20 no 4 pp 853ndash868 2011

[7] M Kaltenpoth ldquoActinobacteria as mutualists general health-care for insectsrdquo Trends inMicrobiology vol 17 no 12 pp 529ndash535 2009

[8] N A Moran J P McCutcheon and A Nakabachi ldquoGenomicsand evolution of heritable bacterial symbiontsrdquo Annual Reviewof Genetics vol 42 pp 165ndash190 2008

[9] J P McCutcheon and C D Von Dohlen ldquoAn interdependentmetabolic patchwork in the nested symbiosis of mealybugsrdquoCurrent Biology vol 21 no 16 pp 1366ndash1372 2011

[10] C B Montllor A Maxmen and A H Purcell ldquoFacultative bac-terial endosymbionts benefit pea aphids Acyrthosiphon pisumunder heat stressrdquo Ecological Entomology vol 27 no 2 pp 189ndash195 2002

[11] A E Douglas ldquoThe nutritional physiology of aphidsrdquoAdvancesin Insect Physiology vol 31 pp 73ndash140 2003

[12] J J Wernegreen S N Kauppinen S G Brady and P SWard ldquoOne nutritional symbiosis begat another phylogeneticevidence that the ant tribe Camponotini acquired Blochmanniaby tending sap-feeding insectsrdquo BMC Evolutionary Biology vol9 no 1 article 292 2009

[13] H Feldhaar J StrakaM Krischke et al ldquoNutritional upgradingfor omnivorous carpenter ants by the endosymbiont Blochman-niardquo BMC Biology vol 5 article 48 2007

[14] A A Pinto-Tomas M A Anderson G Suen et al ldquoSymbioticnitrogen fixation in the fungus gardens of leaf-cutter antsrdquoScience vol 326 no 5956 pp 1120ndash1123 2009

[15] R Pais C Lohs Y Wu J Wang and S Aksoy ldquoThe obligatemutualist Wigglesworthia glossinidia influences reproductiondigestion and immunity processes of its host the tsetse flyrdquoApplied and Environmental Microbiology vol 74 no 19 pp5965ndash5974 2008

[16] J A Breznak ldquoIntestinal microbiota of termites and otherxylophagous insectsrdquo Annual Review of Microbiology vol 36pp 323ndash343 1982

[17] J A Breznak W J Brill J W Mertins and H C CoppelldquoNitrogen fixation in termitesrdquo Nature vol 244 no 5418 pp577ndash579 1973

[18] J A Russell C SMoreau BGoldman-HuertasM FujiwaraDJ Lohman andN E Pierce ldquoBacterial gut symbionts are tightlylinkedwith the evolution of herbivory in antsrdquoProceedings of theNational Academy of Sciences of theUnited States of America vol106 no 50 pp 21236ndash21241 2009

[19] K E Anderson J A Russell C S Moreau et al ldquoHighlysimilar microbial communities are shared among related andtrophically similar ant speciesrdquo Molecular Ecology vol 21 pp2282ndash2296 2012

[20] D W Davidson S C Cook R R Snelling and T H ChualdquoExplaining the abundance of ants in lowland tropical rainforestcanopiesrdquo Science vol 300 no 5621 pp 969ndash972 2003

[21] J C Brownlie and K N Johnson ldquoSymbiont-mediated protec-tion in insect hostsrdquo Trends in Microbiology vol 17 no 8 pp348ndash354 2009

[22] J Jaenike R Unckless S N Cockburn L M Boelio andS J Perlman ldquoAdaptation via symbiosis recent spread of aDrosophila defensive symbiontrdquo Science vol 329 no 5988 pp212ndash215 2012

[23] K M Oliver J A Russell N A Morant and M S HunterldquoFacultative bacterial symbionts in aphids confer resistance toparasitic waspsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 4 pp 1803ndash18072003

[24] C R Currie J A Scott R C Summerbell and D MallochldquoFungus-growing ants use antibiotic-producing bacteria tocontrol garden parasitesrdquo Nature vol 398 pp 701ndash704 1999

[25] M Kaltenpoth W Gottler G Herzner and E Strohm ldquoSym-biotic bacteria protect wasp larvae from fungal infestationrdquoCurrent Biology vol 15 no 5 pp 475ndash479 2005

10 Psyche

[26] H EDunbar A C CWilsonN R Ferguson andNAMoranldquoAphid thermal tolerance is governed by a point mutation inbacterial symbiontsrdquo PLoS Biology vol 5 no 5 pp 1006ndash10152007

[27] T Tsuchida R Koga S Matsumoto and T Fukatsu ldquoInterspe-cific symbiont transfection confers a novel ecological trait to therecipient insectrdquo Biology Letters vol 7 no 2 pp 245ndash248 2011

[28] R Stouthamer J A J Breeuwer and G D D Hurst ldquoWolbachiapipientis microbial manipulator of arthropod reproductionrdquoAnnual Review of Microbiology vol 53 pp 71ndash102 1999

[29] L M Hedges J C Brownlie S L OrsquoNeill and K N JohnsonldquoWolbachia and virus protection in insectsrdquo Science vol 322 no5902 p 702 2008

[30] K Hilgenboecker P Hammerstein P Schlattmann ATelschow and J H Werren ldquoHow many species are infectedwith Wolbachiamdasha statistical analysis of current datardquo FEMSMicrobiology Letters vol 281 no 2 pp 215ndash220 2008

[31] J A Russell C F Funaro Y M Giraldo et al ldquoA veritablemenagerie of heritable bacteria from ants butterflies andbeyond broadmolecular surveys and a systematic reviewrdquoPLoSONE vol 7 no 12 Article ID e51027 2012

[32] O Duron D Bouchon S Boutin et al ldquoThe diversity ofreproductive parasites among arthropods Wolbachia do notwalk alonerdquo BMC Biology vol 6 article 27 2008

[33] D L Williamson and D F Poulson ldquoSex ratio organisms(Spiroplasmas) of Drosophilardquo in The Mycoplasmas III Plantand Insect Mycoplasmas R F Whitcomb and J G Tully Edspp 175ndash208 Academic Press New York NY USA 1979

[34] J M Bove ldquoSpiroplasmas infectious agents of plants arthro-pods and vertebratesrdquoWiener Klinische Wochenschrift vol 109no 14-15 pp 604ndash612 1997

[35] M C Tinsley and M E N Majerus ldquoA new male-killingparasitism Spiroplasma bacteria infect the ladybird beetleAnisosticta novemdecimpunctata (Coleoptera Coccinellidae)rdquoParasitology vol 132 no 6 pp 757ndash765 2006

[36] A Dobson K D Lafferty A M Kuris R F Hechinger andW Jetz ldquoHomage to Linnaeus howmany parasites Howmanyhostsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 105 no 1 pp 11482ndash11489 2008

[37] S T Kelley and S Dobler ldquoComparative analysis of microbialdiversity in Longitarsus flea beetles (Coleoptera Chrysomeli-dae)rdquo Genetica vol 139 no 5 pp 541ndash550 2011

[38] H D Ishak R Plowes R Sen et al ldquoBacterial diversityin Solenopsis invicta and Solenopsis geminata ant coloniescharacterized by 16S amplicon 454 pyrosequencingrdquo MicrobialEcology vol 61 no 4 pp 821ndash831 2011

[39] S Kautz B E R Rubin J A Russell and C S Moreau ldquoSur-veying the microbiome of ants comparing 454 pyrosequencingwith traditionalmethods to uncover bacterial diversityrdquoAppliedand Environmental Microbiology vol 79 no 2 pp 525ndash5342013

[40] S E Dowd T R Callaway R D Wolcott et al ldquoEvaluationof the bacterial diversity in the feces of cattle using 16SrDNA bacterial tag-encoded FLX amplicon pyrosequencing(bTEFAP)rdquo BMCMicrobiology vol 8 article 125 2008

[41] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[42] C Quince A Lanzen R J Davenport and P J TurnbaughldquoRemoving noise from pyrosequenced ampliconsrdquo BMC Bioin-formatics vol 12 article 38 2011

[43] R C Edgar ldquoSearch and clustering orders of magnitude fasterthan BLASTrdquo Bioinformatics vol 26 no 19 pp 2460ndash24612010

[44] J R Cole QWang E Cardenas et al ldquoTheRibosomal databaseproject improved alignments and new tools for rRNA analysisrdquoNucleic Acids Research vol 37 no 1 pp D141ndashD145 2009

[45] T Z DeSantis P Hugenholtz N Larsen et al ldquoGreengenesa chimera-checked 16S rRNA gene database and workbenchcompatible with ARBrdquo Applied and Environmental Microbiol-ogy vol 72 no 7 pp 5069ndash5072 2006

[46] A J Drummond B Ashton S Buxton et al ldquoGeneious v5 4rdquo2011 httpwwwgeneiouscom

[47] A Stamatakis P Hoover and J Rougemont ldquoA rapid bootstrapalgorithm for the RAxML web serversrdquo Systematic Biology vol57 no 5 pp 758ndash771 2008

[48] M A Miller M T Holder R Vos et al ldquoThe CIPRES portalsrdquohttpwwwphyloorgsub sectionsportal

[49] I Letunic and P Bork ldquoInteractive Tree of Life v2 onlineannotation and display of phylogenetic treesmade easyrdquoNucleicAcids Research vol 39 no 2 pp W475ndashW478 2011

[50] T Wenseleers F Ito S van Borm R Huybrechts F Volckaertand J Billen ldquoWidespread occurrence of the micro-organismWolbachia in antsrdquo Proceedings of the Royal Society B vol 265no 1404 pp 1447ndash1452 1998

[51] J A Russell ldquoThe ants (Hymenoptera Formicidae) are uniqueand enigmatic hosts of prevalent Wolbachia (Alphaproteobac-teria) symbiontsrdquoMyrmecological News vol 16 pp 7ndash23 2012

[52] W Arthofer M Riegler D Schneider M Krammer W JMiller and C Stauffer ldquoHidden Wolbachia diversity in fieldpopulations of the European cherry fruit fly Rhagoletis cerasi(Diptera Tephritidae)rdquo Molecular Ecology vol 18 no 18 pp3816ndash3830 2009

[53] J A Russell B Goldman-Huertas C S Moreau et al ldquoSpecial-ization and geographic isolation among Wolbachia symbiontsfrom ants and lycaenid butterfliesrdquo Evolution vol 63 no 3 pp624ndash640 2009

[54] T Wenseleers L Sundstrom and J Billen ldquoDeleterious Wol-bachia in the ant Formica truncorumrdquo Proceedings of the RoyalSociety B vol 269 no 1491 pp 623ndash629 2002

[55] F Vavre F Fleury J Varaldi P Fouillet and M BouletreauldquoEvidence for female mortality in Wolbachia-mediated cyto-plasmic incompatibility in haplodiploid insects epidemiologicand evolutionary consequencesrdquo Evolution vol 54 no 1 pp191ndash200 2000

[56] J Heinze ldquoThe demise of the standard ant (HymenopteraFormicidae)rdquoMyrmecological News vol 11 pp 11ndash20 2008

[57] S Kautz S U Pauls D J Ballhorn H T Lumbsch andM HeilldquoPolygynous supercolonies of the acacia-ant Pseudomyrmexpeperi an inferior colony founderrdquo Molecular Ecology vol 18no 24 pp 5180ndash5194 2009

[58] S Kautz D J Ballhorn J Kroiss et al ldquoHost plant use bycompeting acacia-ants mutualists monopolize while parasitesshare hostsrdquo PLoS ONE vol 7 no 5 Article ID e37691 2012

[59] J Fletcher G A Schultz R E Davis C E Eastman and R MGoodman ldquoBrittle root disease of horseradish-evidence for anetiological role of Spiroplasma citrirdquo Phytopathology vol 71 pp1073ndash1080 1981

[60] C F Funaro D J C Kronauer C S Moreau B Goldman-Huertas N E Pierce and J A Russell ldquoArmy ants harbor ahost-specific clade of Entomoplasmatales bacteriardquo Applied andEnvironmental Microbiology vol 77 no 1 pp 346ndash350 2011

Psyche 11

[61] D J C Kronauer ldquoRecent advances in army ant biology(Hymenoptera Formicidae)rdquoMyrmecological News vol 12 pp51ndash65 2009

[62] F Thiaucourt and G Bolske ldquoContagious caprine pleurop-neumonia and other pulmonary mycoplasmoses of sheep andgoatsrdquo OIE Revue Scientifique et Technique vol 15 no 4 pp1397ndash1414 1996

[63] T Fukatsu T Tsuchida N Nikoh and R Koga ldquoSpiroplasmaSymbiont of the pea aphid Acyrthosiphon pisum (InsectaHomoptera)rdquo Applied and Environmental Microbiology vol 67no 3 pp 1284ndash1291 2001

[64] T S Haselkorn T A Markow and N A Moran ldquoMultipleintroductions of the Spiroplasma bacterial endosymbiont intoDrosophilardquo Molecular Ecology vol 18 no 6 pp 1294ndash13052009

[65] J G Lundgren R M Lehman and J Chee-Sanford ldquoPhysiol-ogy biochemistry and topology bacterial communities withindigestive tracts of ground beetles (Coleoptera Carabidae)rdquoAnnals of the Entomological Society of America vol 100 no 2pp 275ndash282 2007

[66] I Meeus V Vercruysse and G Smagghe ldquoMolecular detectionof Spiroplasma apis and Spiroplasmamelliferum in beesrdquo Journalof Invertebrate Pathology vol 109 no 1 pp 172ndash174 2012

[67] S van Borm J Billen and J J Boomsma ldquoThe diversity ofmicroorganisms associated with Acromyrmex leafcutter antsrdquoBMC Evolutionary Biology vol 2 article 9 2002

[68] J Wedincamp F E French R F Whitcomb and R B HenegerldquoLaboratory infection and release of Spiroplasma (Entomoplas-matales Spiroplasmataceae) from horse flies (Diptera Taban-idae)rdquo Journal of Entomological Science vol 32 no 4 pp 398ndash402 1997

[69] M A Ebbert and L R Nault ldquoImproved overwintering abilityin Dalbulus maidis (Homoptera Cicadellidae) vectors infectedwith Spiroplasma kunkelii (Mycoplasmatales Spiroplasmat-aceae)rdquo Environmental Entomology vol 23 no 3 pp 634ndash6441994

[70] F M Jiggins G D D Hurst C D Jiggins J H G V DSchulenburg and M E N Majerus ldquoThe butterfly Danauschrysippus is infected by a male-killing Spiroplasma bacteriumrdquoParasitology vol 120 no 5 pp 439ndash446 2000

[71] J H G V D Schulenburg G D D Hurst D Tetzlaff GE Booth I A Zakharov and M E N Majerus ldquoHistory ofinfection with different male-killing bacteria in the two-spotladybird beetle Adalia bipunctata revealed through mitochon-drial DNA sequence analysisrdquoGenetics vol 160 no 3 pp 1075ndash1086 2002

[72] S Kautz H T Lumbsch P S Ward and M Heil ldquoHowto prevent cheating a digestive specialization ties mutualisticplant-ants to their ant-plant partnersrdquo Evolution vol 63 no 4pp 839ndash853 2009

[73] N Samaddar A Paul S Chakravorty et al ldquoNitrogen fixationin Asaia sp (Family Acetobacteraceae)rdquo Current Microbiologyvol 63 no 2 pp 226ndash231 2011

[74] Y Ano H Toyama O Adachi and K Matsushita ldquoEnergymetabolism of a unique acetic acid bacteriumAsaia bogorensisthat lacks ethanol oxidation activityrdquo Bioscience Biotechnologyand Biochemistry vol 72 no 4 pp 989ndash997 2008

[75] V G Martinson B N Danforth R L Minckley O RueppellS Tingek and N A Moran ldquoA simple and distinctive micro-biota associated with honey bees and bumble beesrdquo MolecularEcology vol 20 no 3 pp 619ndash628 2011

[76] N A Moran A K Hansen J E Powell and Z L SabreeldquoDistinctive gut microbiota of honey bees assessed using deepsampling from individual worker beesrdquo PLoS ONE vol 7 no 4Article ID e36393 2012

[77] E Crotti C Damiani M Pajoro et al ldquoAsaia a versatile aceticacid bacterial symbiont capable of cross-colonizing insectsof phylogenetically distant genera and ordersrdquo EnvironmentalMicrobiology vol 11 no 12 pp 3252ndash3264 2009

[78] C R Cox and M S Gilmore ldquoNative microbial colonization ofDrosophilamelanogaster and its use as a model of Enterococcusfaecalis pathogenesisrdquo Infection and Immunity vol 75 no 4 pp1565ndash1576 2007

[79] M Marzorati A Alma L Sacchi et al ldquoA novel Bacteroidetessymbiont is localized in Scaphoideus titanus the insect vector ofFlavescence Doree in Vitis viniferardquo Applied and EnvironmentalMicrobiology vol 72 no 2 pp 1467ndash1475 2006

[80] N J Ashbolt and P A Inkerman ldquoAcetic acid bacterial biota ofthe pink sugar cane mealybug Saccharococcus sacchari and itsenvironsrdquo Applied and Environmental Microbiology vol 56 no3 pp 707ndash712 1990

[81] E Crotti A Rizzi B Chouaia et al ldquoAcetic acid bacteria newlyemerging symbionts of insectsrdquo Applied and EnvironmentalMicrobiology vol 76 no 21 pp 6963ndash6970 2010

[82] J Ryu S Kim H Lee et al ldquoInnate immune homeostasis bythe homeobox gene Caudal and commensal-gut mutualism inDrosophilardquo Science vol 319 no 5864 pp 777ndash782 2008

[83] S Stoll J Gadau R Gross and H Feldhaar ldquoBacterial micro-biota associated with ants of the genus Tetraponerardquo BiologicalJournal of the Linnean Society vol 90 no 3 pp 399ndash412 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 8: Research Article Bacterial Infections across the Ants ...downloads.hindawi.com/journals/psyche/2013/936341.pdf · Department of Zoology, Field Museum of Natural History, South Lake

8 Psyche

Drosophila sp EU096229 (fruit fly)

Drosophila sp EU096235 (fruit fly)

Drosophila sp EU096233 (fruit fly)

Pseudomyrm

ex ejectus SK1181-3 (ant)

Paraponera clavata CS0513-2 (ant mouth)

Gluconobacter frateurii AB682075

Gluc

onob

acte

r fra

teur

ii A

B682

081Gluconobacter frateurii A

B682091

Pseudo

myrmex

ferru

gineu

s SK00

02-1

(ant)

Pseudomyrm

ex pallidus C

SM1333 (ant)

Pseudomyrmex spinicola SK0061-1 (ant)

Pseudomyrmex peperi SK1058-103-1 (ant)

Cephalotes atratus SK1465 (ant)

Xylocopa californica HM112403 (bee)

Caupolicana yarrowi HM109591 (bee)

Euglossa imperialis JQ389882 (bee)

BER145 (ant)Pseudomyrmex sp nr eduardi

Pseudomyrmex satanicus SK0058-1 (ant)Pseudomyrmex dendroicus BER121 (ant)

Pseudomyrmex dendroicus BER103 (ant)

Pseudomyrmex peperi

SK0007-1 (ant)Pseu

domyrm

ex nigroci

nctus

SK0060-1 (a

nt)Pseudom

yrmex

major

SK09

90-8-

3 (an

t gast

er)

Pseu

domyrm

ex de

ndroi

cus S

K1260

(ant)

Form

ica oc

culta

GQ27

5104

(ant

)

Alph

a pro

teoba

cteriu

m A

B513

363 (

flowe

r)

Para

pone

ra cl

avat

a CS

0513

-4 (a

nt m

outh

)Ca

lliop

sis su

balpi

nus H

M10

9230

(bee

)Ca

lliop

sis su

balpi

nus H

M10

9259

(bee

)

Pseu

dom

yrm

ex te

nuis

BER1

12 (a

nt)

Sacc

haric

occu

s sac

char

i AF1

2739

6 (m

ealy

bug)

Dro

soph

ila sp

EU

0962

34 (f

ruit

fly)

Atta colombica H

M557097 (fungus garden)

Aedes aegypti FJ372761 (mosquito)

Aedes aegypti FJ372770 (mosquito)

Anopheles maculipennis AM

404262 (mosquito)

Aedes albopictus JQ958879 (m

osquito)

Aedes albopictus JQ314055 (m

osquito)

Aedes albopictus JQ314027 (m

osquito)

Aedes albopictus F2f JX445138 (mosquito)

Aede

s aeg

ypti

FN81

4303

(mos

quito

)

Aede

s alb

opict

us F

1f JX

4451

37 (m

osqu

ito)

Aede

s alb

opict

us F

2l JX

4451

40 (m

osqu

ito)

Aede

s alb

opict

us F

1l JX

4451

39 (m

osqu

ito)

Aede

s aeg

ypti

FN81

4301

(mos

quito

)

Aede

s aeg

ypti

FN81

4300

(mos

quito

)

Aede

s aeg

ypti

FN29

7842

(mos

quito

)

Anoph

eles s

tephe

nsi A

M40

4261

(mos

quito

)

Aede

s aeg

ypti

FN29

7841

(mos

quito

)Asaia b

ogore

nsis A

B6820

04

Anoph

eles s

p HQ26

9803

(mos

quito

)

Tetrap

onera

rufon

igra JF

5145

56 (a

nt)

Pseudomyrmex elo

ngatus

CSM1373 (ant gut)Pseudomyrmex termitarius

SK1252-1 (ant)

Pseudomyrmex gracilis

CSM1374-2 (ant head)Anopheles gambiae FN814276 (mosquito)Pseudomyrmex ejectus SK1181-3 (ant)

Pseudomyrm

ex elongatus

CSM1373 (ant head)

Cephalotes va

rians JX

990151 (ant)

Anopheles stephensi FN814299 (mosquito)

Anopheles gambiae FN814277 (mosquito)

Anopheles stephensi FN814297 (mosquito)

Anopheles stephensi FN814296 (mosquito)

Cephalotes varians JX990161 (ant)

Lutzomyia longipalpis HE995765 (sand fly)

Anopheles gambiae FN821398 (mosquito)

Aedes aegypti FN297840 (mosquito)

Anopheles stephensi AM404260 (mosquito)

Asaia krungthepensis AB682130

Anopheles gambiae FJ816021 (mosquito)Anopheles gambiae FN814284 (mosquito)Anopheles gambiae FN814283 (mosquito)

Aedes albopictus FN814288 (mosquito)

Aedes albopictus FN814287 (mosquito)

Anopheles gambiae FN821397 (mosquito)

Anopheles gambiae FN814280 (mosquito)

Aedes albopictus FN814289 (mosquito)

Anopheles gambiae FN814285 (mosquito)

Anopheles gambiae FN814275 (mosquito)

Anopheles gambiae FN814281 (mosquito)

Anopheles gambiae FN814278 (mosquito)

Aedes albopictus FN814286 (mosquito)

Atta colombica HM556904 (fungus garden)

Saccharicoccus sacchari AF127400 (mealybug)

Saccharicoccus sacchari AF127408 (mealybug)

Saccharicoccus sacchari AF127401 (mealybug)

Pseudomyrmex ejectus SK1181-3 (ant)

Paraponera clavata CS0513-3 (ant mouth)

Pseudomyrm

ex termitarius SK1252-2 (ant)

Drosophila sp EU096231 (fruit fly)Wolbachia pipientis AY026912

Inner circle ant subfamilyFormicinaeMyrmicinaeParaponerinaePseudomyrmecinae

Middle circle bioregionAfrotropic

IndomalayaAustralasia

Palearctic

NearcticNeotropicOceania

Madagascar

Clade 1

Clade 2Cl

ade 3

Figure 3 Phylogenetic tree of Asaia-related symbionts associated with ants and closest relatives with sequence data available in GenBank Amaximum likelihood phylogeny of the 16S rRNA region of bacterial symbionts is shown The host name is given together with the GenBankaccession number (GenBank sequences) or collection code (sequences generated in the present study) The branch color refers to the sourcefromwhich the bacteria were isolated with yellow representing ant hosts and red other insect hostsThe inner circle refers to the ant subfamilyand the middle circle refers to the bioregion from which samples were collected The outer circle indicates three clades (Clades 1ndash3) whichcontained several ant-associated symbiontsWolbachia pipientis was used as an outgroup

indicate that Rhodospirillales aremost likely environmentallytransmitted and support the hypothesis that they are onlyfacultative associates of ants One clade of ant-associatedRhodospirillales was closely related to endosymbionts iso-lated from mosquitos (Figure 3) It has been experimentallyshown that mosquito-associated Asaia can successfully colo-nize leafhoppers further emphasizing the low-host specificityof this bacterial group [77]

4 Conclusion

Our broad bacterial screening approach has contributedto our understanding of the prevalence of ant-associatedmicrobes particularly with regard to their Wolbachia andSpiroplasma symbionts Furthermore we provide the firstextensive survey for ant-associated Asaia-related symbiontsWhile these symbionts of the order Rhodospirillales infect

Psyche 9

ants only sporadically some strains are capable of fixingatmospheric nitrogen and might retain this function inants Alternatively these bacteria might have an importantfunctional role for upgrading nitrogen-poor diets of someherbivorous ants which comprise the majority of all ant taxa[20] Even though we do not have experimental evidence ofthe role of most bacterial symbionts in ants previous studiesillustrate a broad variety of effects of these bacteria on insecthosts [4 7 9] Even a single group of microbes can havevery different effects on different hosts Our study shows thatdespite several extensive bacterial surveys across the ants thediversity and functional role of ant-associated microbes isfar from being fully understood and broad next generationsequencing approaches will provide a fast and cost-effectivetool to deepen our knowledge of the rare (and not so rare)biosphere

Acknowledgments

The authors thank Daniel Ballhorn and Tina Schroyer forhelpful comments and discussions Phil Ward kindly iden-tified Pseudomyrmex specimens This study was supportedin part by National Science Foundation DEB Grant no1050243 to Corrie S Moreau a Grainger Foundation grant toCorrie S Moreau a Negaunee Foundation grant to Corrie SMoreau and the Pritzker Laboratory forMolecular Evolutionand Systematics Stefanie Kautz acknowledges a postdoctoralfellowship from the German Academy of Sciences Leopold-ina (Grant no LPDS 2009-29) Benjamin E R Rubin wassupported in part by an NSF Graduate Research Fellowship

References

[1] H Koch D P Abrol J Li and P Schmid-Hempel ldquoDiversity ofevolutionary patterns of bacterial gut associates of corbiculatebeesrdquoMolecular Ecology vol 22 no 7 pp 2028ndash2044 2013

[2] R J Dillon and V M Dillon ldquoThe Gut bacteria of insectsnonpathogenic interactionsrdquoAnnual Review of Entomology vol49 pp 71ndash92 2004

[3] S Fraune and T C G Bosch ldquoWhy bacteria matter in animaldevelopment and evolutionrdquo BioEssays vol 32 no 7 pp 571ndash580 2010

[4] H Feldhaar ldquoBacterial symbionts as mediators of ecologicallyimportant traits of insect hostsrdquo Ecological Entomology vol 36no 5 pp 533ndash543 2011

[5] J M Urban and J R Cryan ldquoTwo ancient bacterialendosymbionts have coevolved with the planthoppers (InsectaHemiptera Fulgoroidea)rdquo BMC Evolutionary Biology vol 12article 87 2012

[6] H Toju and T Fukatsu ldquoDiversity and infection prevalence ofendosymbionts in natural populations of the chestnut weevilrelevance of local climate and host plantsrdquo Molecular Ecologyvol 20 no 4 pp 853ndash868 2011

[7] M Kaltenpoth ldquoActinobacteria as mutualists general health-care for insectsrdquo Trends inMicrobiology vol 17 no 12 pp 529ndash535 2009

[8] N A Moran J P McCutcheon and A Nakabachi ldquoGenomicsand evolution of heritable bacterial symbiontsrdquo Annual Reviewof Genetics vol 42 pp 165ndash190 2008

[9] J P McCutcheon and C D Von Dohlen ldquoAn interdependentmetabolic patchwork in the nested symbiosis of mealybugsrdquoCurrent Biology vol 21 no 16 pp 1366ndash1372 2011

[10] C B Montllor A Maxmen and A H Purcell ldquoFacultative bac-terial endosymbionts benefit pea aphids Acyrthosiphon pisumunder heat stressrdquo Ecological Entomology vol 27 no 2 pp 189ndash195 2002

[11] A E Douglas ldquoThe nutritional physiology of aphidsrdquoAdvancesin Insect Physiology vol 31 pp 73ndash140 2003

[12] J J Wernegreen S N Kauppinen S G Brady and P SWard ldquoOne nutritional symbiosis begat another phylogeneticevidence that the ant tribe Camponotini acquired Blochmanniaby tending sap-feeding insectsrdquo BMC Evolutionary Biology vol9 no 1 article 292 2009

[13] H Feldhaar J StrakaM Krischke et al ldquoNutritional upgradingfor omnivorous carpenter ants by the endosymbiont Blochman-niardquo BMC Biology vol 5 article 48 2007

[14] A A Pinto-Tomas M A Anderson G Suen et al ldquoSymbioticnitrogen fixation in the fungus gardens of leaf-cutter antsrdquoScience vol 326 no 5956 pp 1120ndash1123 2009

[15] R Pais C Lohs Y Wu J Wang and S Aksoy ldquoThe obligatemutualist Wigglesworthia glossinidia influences reproductiondigestion and immunity processes of its host the tsetse flyrdquoApplied and Environmental Microbiology vol 74 no 19 pp5965ndash5974 2008

[16] J A Breznak ldquoIntestinal microbiota of termites and otherxylophagous insectsrdquo Annual Review of Microbiology vol 36pp 323ndash343 1982

[17] J A Breznak W J Brill J W Mertins and H C CoppelldquoNitrogen fixation in termitesrdquo Nature vol 244 no 5418 pp577ndash579 1973

[18] J A Russell C SMoreau BGoldman-HuertasM FujiwaraDJ Lohman andN E Pierce ldquoBacterial gut symbionts are tightlylinkedwith the evolution of herbivory in antsrdquoProceedings of theNational Academy of Sciences of theUnited States of America vol106 no 50 pp 21236ndash21241 2009

[19] K E Anderson J A Russell C S Moreau et al ldquoHighlysimilar microbial communities are shared among related andtrophically similar ant speciesrdquo Molecular Ecology vol 21 pp2282ndash2296 2012

[20] D W Davidson S C Cook R R Snelling and T H ChualdquoExplaining the abundance of ants in lowland tropical rainforestcanopiesrdquo Science vol 300 no 5621 pp 969ndash972 2003

[21] J C Brownlie and K N Johnson ldquoSymbiont-mediated protec-tion in insect hostsrdquo Trends in Microbiology vol 17 no 8 pp348ndash354 2009

[22] J Jaenike R Unckless S N Cockburn L M Boelio andS J Perlman ldquoAdaptation via symbiosis recent spread of aDrosophila defensive symbiontrdquo Science vol 329 no 5988 pp212ndash215 2012

[23] K M Oliver J A Russell N A Morant and M S HunterldquoFacultative bacterial symbionts in aphids confer resistance toparasitic waspsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 4 pp 1803ndash18072003

[24] C R Currie J A Scott R C Summerbell and D MallochldquoFungus-growing ants use antibiotic-producing bacteria tocontrol garden parasitesrdquo Nature vol 398 pp 701ndash704 1999

[25] M Kaltenpoth W Gottler G Herzner and E Strohm ldquoSym-biotic bacteria protect wasp larvae from fungal infestationrdquoCurrent Biology vol 15 no 5 pp 475ndash479 2005

10 Psyche

[26] H EDunbar A C CWilsonN R Ferguson andNAMoranldquoAphid thermal tolerance is governed by a point mutation inbacterial symbiontsrdquo PLoS Biology vol 5 no 5 pp 1006ndash10152007

[27] T Tsuchida R Koga S Matsumoto and T Fukatsu ldquoInterspe-cific symbiont transfection confers a novel ecological trait to therecipient insectrdquo Biology Letters vol 7 no 2 pp 245ndash248 2011

[28] R Stouthamer J A J Breeuwer and G D D Hurst ldquoWolbachiapipientis microbial manipulator of arthropod reproductionrdquoAnnual Review of Microbiology vol 53 pp 71ndash102 1999

[29] L M Hedges J C Brownlie S L OrsquoNeill and K N JohnsonldquoWolbachia and virus protection in insectsrdquo Science vol 322 no5902 p 702 2008

[30] K Hilgenboecker P Hammerstein P Schlattmann ATelschow and J H Werren ldquoHow many species are infectedwith Wolbachiamdasha statistical analysis of current datardquo FEMSMicrobiology Letters vol 281 no 2 pp 215ndash220 2008

[31] J A Russell C F Funaro Y M Giraldo et al ldquoA veritablemenagerie of heritable bacteria from ants butterflies andbeyond broadmolecular surveys and a systematic reviewrdquoPLoSONE vol 7 no 12 Article ID e51027 2012

[32] O Duron D Bouchon S Boutin et al ldquoThe diversity ofreproductive parasites among arthropods Wolbachia do notwalk alonerdquo BMC Biology vol 6 article 27 2008

[33] D L Williamson and D F Poulson ldquoSex ratio organisms(Spiroplasmas) of Drosophilardquo in The Mycoplasmas III Plantand Insect Mycoplasmas R F Whitcomb and J G Tully Edspp 175ndash208 Academic Press New York NY USA 1979

[34] J M Bove ldquoSpiroplasmas infectious agents of plants arthro-pods and vertebratesrdquoWiener Klinische Wochenschrift vol 109no 14-15 pp 604ndash612 1997

[35] M C Tinsley and M E N Majerus ldquoA new male-killingparasitism Spiroplasma bacteria infect the ladybird beetleAnisosticta novemdecimpunctata (Coleoptera Coccinellidae)rdquoParasitology vol 132 no 6 pp 757ndash765 2006

[36] A Dobson K D Lafferty A M Kuris R F Hechinger andW Jetz ldquoHomage to Linnaeus howmany parasites Howmanyhostsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 105 no 1 pp 11482ndash11489 2008

[37] S T Kelley and S Dobler ldquoComparative analysis of microbialdiversity in Longitarsus flea beetles (Coleoptera Chrysomeli-dae)rdquo Genetica vol 139 no 5 pp 541ndash550 2011

[38] H D Ishak R Plowes R Sen et al ldquoBacterial diversityin Solenopsis invicta and Solenopsis geminata ant coloniescharacterized by 16S amplicon 454 pyrosequencingrdquo MicrobialEcology vol 61 no 4 pp 821ndash831 2011

[39] S Kautz B E R Rubin J A Russell and C S Moreau ldquoSur-veying the microbiome of ants comparing 454 pyrosequencingwith traditionalmethods to uncover bacterial diversityrdquoAppliedand Environmental Microbiology vol 79 no 2 pp 525ndash5342013

[40] S E Dowd T R Callaway R D Wolcott et al ldquoEvaluationof the bacterial diversity in the feces of cattle using 16SrDNA bacterial tag-encoded FLX amplicon pyrosequencing(bTEFAP)rdquo BMCMicrobiology vol 8 article 125 2008

[41] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[42] C Quince A Lanzen R J Davenport and P J TurnbaughldquoRemoving noise from pyrosequenced ampliconsrdquo BMC Bioin-formatics vol 12 article 38 2011

[43] R C Edgar ldquoSearch and clustering orders of magnitude fasterthan BLASTrdquo Bioinformatics vol 26 no 19 pp 2460ndash24612010

[44] J R Cole QWang E Cardenas et al ldquoTheRibosomal databaseproject improved alignments and new tools for rRNA analysisrdquoNucleic Acids Research vol 37 no 1 pp D141ndashD145 2009

[45] T Z DeSantis P Hugenholtz N Larsen et al ldquoGreengenesa chimera-checked 16S rRNA gene database and workbenchcompatible with ARBrdquo Applied and Environmental Microbiol-ogy vol 72 no 7 pp 5069ndash5072 2006

[46] A J Drummond B Ashton S Buxton et al ldquoGeneious v5 4rdquo2011 httpwwwgeneiouscom

[47] A Stamatakis P Hoover and J Rougemont ldquoA rapid bootstrapalgorithm for the RAxML web serversrdquo Systematic Biology vol57 no 5 pp 758ndash771 2008

[48] M A Miller M T Holder R Vos et al ldquoThe CIPRES portalsrdquohttpwwwphyloorgsub sectionsportal

[49] I Letunic and P Bork ldquoInteractive Tree of Life v2 onlineannotation and display of phylogenetic treesmade easyrdquoNucleicAcids Research vol 39 no 2 pp W475ndashW478 2011

[50] T Wenseleers F Ito S van Borm R Huybrechts F Volckaertand J Billen ldquoWidespread occurrence of the micro-organismWolbachia in antsrdquo Proceedings of the Royal Society B vol 265no 1404 pp 1447ndash1452 1998

[51] J A Russell ldquoThe ants (Hymenoptera Formicidae) are uniqueand enigmatic hosts of prevalent Wolbachia (Alphaproteobac-teria) symbiontsrdquoMyrmecological News vol 16 pp 7ndash23 2012

[52] W Arthofer M Riegler D Schneider M Krammer W JMiller and C Stauffer ldquoHidden Wolbachia diversity in fieldpopulations of the European cherry fruit fly Rhagoletis cerasi(Diptera Tephritidae)rdquo Molecular Ecology vol 18 no 18 pp3816ndash3830 2009

[53] J A Russell B Goldman-Huertas C S Moreau et al ldquoSpecial-ization and geographic isolation among Wolbachia symbiontsfrom ants and lycaenid butterfliesrdquo Evolution vol 63 no 3 pp624ndash640 2009

[54] T Wenseleers L Sundstrom and J Billen ldquoDeleterious Wol-bachia in the ant Formica truncorumrdquo Proceedings of the RoyalSociety B vol 269 no 1491 pp 623ndash629 2002

[55] F Vavre F Fleury J Varaldi P Fouillet and M BouletreauldquoEvidence for female mortality in Wolbachia-mediated cyto-plasmic incompatibility in haplodiploid insects epidemiologicand evolutionary consequencesrdquo Evolution vol 54 no 1 pp191ndash200 2000

[56] J Heinze ldquoThe demise of the standard ant (HymenopteraFormicidae)rdquoMyrmecological News vol 11 pp 11ndash20 2008

[57] S Kautz S U Pauls D J Ballhorn H T Lumbsch andM HeilldquoPolygynous supercolonies of the acacia-ant Pseudomyrmexpeperi an inferior colony founderrdquo Molecular Ecology vol 18no 24 pp 5180ndash5194 2009

[58] S Kautz D J Ballhorn J Kroiss et al ldquoHost plant use bycompeting acacia-ants mutualists monopolize while parasitesshare hostsrdquo PLoS ONE vol 7 no 5 Article ID e37691 2012

[59] J Fletcher G A Schultz R E Davis C E Eastman and R MGoodman ldquoBrittle root disease of horseradish-evidence for anetiological role of Spiroplasma citrirdquo Phytopathology vol 71 pp1073ndash1080 1981

[60] C F Funaro D J C Kronauer C S Moreau B Goldman-Huertas N E Pierce and J A Russell ldquoArmy ants harbor ahost-specific clade of Entomoplasmatales bacteriardquo Applied andEnvironmental Microbiology vol 77 no 1 pp 346ndash350 2011

Psyche 11

[61] D J C Kronauer ldquoRecent advances in army ant biology(Hymenoptera Formicidae)rdquoMyrmecological News vol 12 pp51ndash65 2009

[62] F Thiaucourt and G Bolske ldquoContagious caprine pleurop-neumonia and other pulmonary mycoplasmoses of sheep andgoatsrdquo OIE Revue Scientifique et Technique vol 15 no 4 pp1397ndash1414 1996

[63] T Fukatsu T Tsuchida N Nikoh and R Koga ldquoSpiroplasmaSymbiont of the pea aphid Acyrthosiphon pisum (InsectaHomoptera)rdquo Applied and Environmental Microbiology vol 67no 3 pp 1284ndash1291 2001

[64] T S Haselkorn T A Markow and N A Moran ldquoMultipleintroductions of the Spiroplasma bacterial endosymbiont intoDrosophilardquo Molecular Ecology vol 18 no 6 pp 1294ndash13052009

[65] J G Lundgren R M Lehman and J Chee-Sanford ldquoPhysiol-ogy biochemistry and topology bacterial communities withindigestive tracts of ground beetles (Coleoptera Carabidae)rdquoAnnals of the Entomological Society of America vol 100 no 2pp 275ndash282 2007

[66] I Meeus V Vercruysse and G Smagghe ldquoMolecular detectionof Spiroplasma apis and Spiroplasmamelliferum in beesrdquo Journalof Invertebrate Pathology vol 109 no 1 pp 172ndash174 2012

[67] S van Borm J Billen and J J Boomsma ldquoThe diversity ofmicroorganisms associated with Acromyrmex leafcutter antsrdquoBMC Evolutionary Biology vol 2 article 9 2002

[68] J Wedincamp F E French R F Whitcomb and R B HenegerldquoLaboratory infection and release of Spiroplasma (Entomoplas-matales Spiroplasmataceae) from horse flies (Diptera Taban-idae)rdquo Journal of Entomological Science vol 32 no 4 pp 398ndash402 1997

[69] M A Ebbert and L R Nault ldquoImproved overwintering abilityin Dalbulus maidis (Homoptera Cicadellidae) vectors infectedwith Spiroplasma kunkelii (Mycoplasmatales Spiroplasmat-aceae)rdquo Environmental Entomology vol 23 no 3 pp 634ndash6441994

[70] F M Jiggins G D D Hurst C D Jiggins J H G V DSchulenburg and M E N Majerus ldquoThe butterfly Danauschrysippus is infected by a male-killing Spiroplasma bacteriumrdquoParasitology vol 120 no 5 pp 439ndash446 2000

[71] J H G V D Schulenburg G D D Hurst D Tetzlaff GE Booth I A Zakharov and M E N Majerus ldquoHistory ofinfection with different male-killing bacteria in the two-spotladybird beetle Adalia bipunctata revealed through mitochon-drial DNA sequence analysisrdquoGenetics vol 160 no 3 pp 1075ndash1086 2002

[72] S Kautz H T Lumbsch P S Ward and M Heil ldquoHowto prevent cheating a digestive specialization ties mutualisticplant-ants to their ant-plant partnersrdquo Evolution vol 63 no 4pp 839ndash853 2009

[73] N Samaddar A Paul S Chakravorty et al ldquoNitrogen fixationin Asaia sp (Family Acetobacteraceae)rdquo Current Microbiologyvol 63 no 2 pp 226ndash231 2011

[74] Y Ano H Toyama O Adachi and K Matsushita ldquoEnergymetabolism of a unique acetic acid bacteriumAsaia bogorensisthat lacks ethanol oxidation activityrdquo Bioscience Biotechnologyand Biochemistry vol 72 no 4 pp 989ndash997 2008

[75] V G Martinson B N Danforth R L Minckley O RueppellS Tingek and N A Moran ldquoA simple and distinctive micro-biota associated with honey bees and bumble beesrdquo MolecularEcology vol 20 no 3 pp 619ndash628 2011

[76] N A Moran A K Hansen J E Powell and Z L SabreeldquoDistinctive gut microbiota of honey bees assessed using deepsampling from individual worker beesrdquo PLoS ONE vol 7 no 4Article ID e36393 2012

[77] E Crotti C Damiani M Pajoro et al ldquoAsaia a versatile aceticacid bacterial symbiont capable of cross-colonizing insectsof phylogenetically distant genera and ordersrdquo EnvironmentalMicrobiology vol 11 no 12 pp 3252ndash3264 2009

[78] C R Cox and M S Gilmore ldquoNative microbial colonization ofDrosophilamelanogaster and its use as a model of Enterococcusfaecalis pathogenesisrdquo Infection and Immunity vol 75 no 4 pp1565ndash1576 2007

[79] M Marzorati A Alma L Sacchi et al ldquoA novel Bacteroidetessymbiont is localized in Scaphoideus titanus the insect vector ofFlavescence Doree in Vitis viniferardquo Applied and EnvironmentalMicrobiology vol 72 no 2 pp 1467ndash1475 2006

[80] N J Ashbolt and P A Inkerman ldquoAcetic acid bacterial biota ofthe pink sugar cane mealybug Saccharococcus sacchari and itsenvironsrdquo Applied and Environmental Microbiology vol 56 no3 pp 707ndash712 1990

[81] E Crotti A Rizzi B Chouaia et al ldquoAcetic acid bacteria newlyemerging symbionts of insectsrdquo Applied and EnvironmentalMicrobiology vol 76 no 21 pp 6963ndash6970 2010

[82] J Ryu S Kim H Lee et al ldquoInnate immune homeostasis bythe homeobox gene Caudal and commensal-gut mutualism inDrosophilardquo Science vol 319 no 5864 pp 777ndash782 2008

[83] S Stoll J Gadau R Gross and H Feldhaar ldquoBacterial micro-biota associated with ants of the genus Tetraponerardquo BiologicalJournal of the Linnean Society vol 90 no 3 pp 399ndash412 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 9: Research Article Bacterial Infections across the Ants ...downloads.hindawi.com/journals/psyche/2013/936341.pdf · Department of Zoology, Field Museum of Natural History, South Lake

Psyche 9

ants only sporadically some strains are capable of fixingatmospheric nitrogen and might retain this function inants Alternatively these bacteria might have an importantfunctional role for upgrading nitrogen-poor diets of someherbivorous ants which comprise the majority of all ant taxa[20] Even though we do not have experimental evidence ofthe role of most bacterial symbionts in ants previous studiesillustrate a broad variety of effects of these bacteria on insecthosts [4 7 9] Even a single group of microbes can havevery different effects on different hosts Our study shows thatdespite several extensive bacterial surveys across the ants thediversity and functional role of ant-associated microbes isfar from being fully understood and broad next generationsequencing approaches will provide a fast and cost-effectivetool to deepen our knowledge of the rare (and not so rare)biosphere

Acknowledgments

The authors thank Daniel Ballhorn and Tina Schroyer forhelpful comments and discussions Phil Ward kindly iden-tified Pseudomyrmex specimens This study was supportedin part by National Science Foundation DEB Grant no1050243 to Corrie S Moreau a Grainger Foundation grant toCorrie S Moreau a Negaunee Foundation grant to Corrie SMoreau and the Pritzker Laboratory forMolecular Evolutionand Systematics Stefanie Kautz acknowledges a postdoctoralfellowship from the German Academy of Sciences Leopold-ina (Grant no LPDS 2009-29) Benjamin E R Rubin wassupported in part by an NSF Graduate Research Fellowship

References

[1] H Koch D P Abrol J Li and P Schmid-Hempel ldquoDiversity ofevolutionary patterns of bacterial gut associates of corbiculatebeesrdquoMolecular Ecology vol 22 no 7 pp 2028ndash2044 2013

[2] R J Dillon and V M Dillon ldquoThe Gut bacteria of insectsnonpathogenic interactionsrdquoAnnual Review of Entomology vol49 pp 71ndash92 2004

[3] S Fraune and T C G Bosch ldquoWhy bacteria matter in animaldevelopment and evolutionrdquo BioEssays vol 32 no 7 pp 571ndash580 2010

[4] H Feldhaar ldquoBacterial symbionts as mediators of ecologicallyimportant traits of insect hostsrdquo Ecological Entomology vol 36no 5 pp 533ndash543 2011

[5] J M Urban and J R Cryan ldquoTwo ancient bacterialendosymbionts have coevolved with the planthoppers (InsectaHemiptera Fulgoroidea)rdquo BMC Evolutionary Biology vol 12article 87 2012

[6] H Toju and T Fukatsu ldquoDiversity and infection prevalence ofendosymbionts in natural populations of the chestnut weevilrelevance of local climate and host plantsrdquo Molecular Ecologyvol 20 no 4 pp 853ndash868 2011

[7] M Kaltenpoth ldquoActinobacteria as mutualists general health-care for insectsrdquo Trends inMicrobiology vol 17 no 12 pp 529ndash535 2009

[8] N A Moran J P McCutcheon and A Nakabachi ldquoGenomicsand evolution of heritable bacterial symbiontsrdquo Annual Reviewof Genetics vol 42 pp 165ndash190 2008

[9] J P McCutcheon and C D Von Dohlen ldquoAn interdependentmetabolic patchwork in the nested symbiosis of mealybugsrdquoCurrent Biology vol 21 no 16 pp 1366ndash1372 2011

[10] C B Montllor A Maxmen and A H Purcell ldquoFacultative bac-terial endosymbionts benefit pea aphids Acyrthosiphon pisumunder heat stressrdquo Ecological Entomology vol 27 no 2 pp 189ndash195 2002

[11] A E Douglas ldquoThe nutritional physiology of aphidsrdquoAdvancesin Insect Physiology vol 31 pp 73ndash140 2003

[12] J J Wernegreen S N Kauppinen S G Brady and P SWard ldquoOne nutritional symbiosis begat another phylogeneticevidence that the ant tribe Camponotini acquired Blochmanniaby tending sap-feeding insectsrdquo BMC Evolutionary Biology vol9 no 1 article 292 2009

[13] H Feldhaar J StrakaM Krischke et al ldquoNutritional upgradingfor omnivorous carpenter ants by the endosymbiont Blochman-niardquo BMC Biology vol 5 article 48 2007

[14] A A Pinto-Tomas M A Anderson G Suen et al ldquoSymbioticnitrogen fixation in the fungus gardens of leaf-cutter antsrdquoScience vol 326 no 5956 pp 1120ndash1123 2009

[15] R Pais C Lohs Y Wu J Wang and S Aksoy ldquoThe obligatemutualist Wigglesworthia glossinidia influences reproductiondigestion and immunity processes of its host the tsetse flyrdquoApplied and Environmental Microbiology vol 74 no 19 pp5965ndash5974 2008

[16] J A Breznak ldquoIntestinal microbiota of termites and otherxylophagous insectsrdquo Annual Review of Microbiology vol 36pp 323ndash343 1982

[17] J A Breznak W J Brill J W Mertins and H C CoppelldquoNitrogen fixation in termitesrdquo Nature vol 244 no 5418 pp577ndash579 1973

[18] J A Russell C SMoreau BGoldman-HuertasM FujiwaraDJ Lohman andN E Pierce ldquoBacterial gut symbionts are tightlylinkedwith the evolution of herbivory in antsrdquoProceedings of theNational Academy of Sciences of theUnited States of America vol106 no 50 pp 21236ndash21241 2009

[19] K E Anderson J A Russell C S Moreau et al ldquoHighlysimilar microbial communities are shared among related andtrophically similar ant speciesrdquo Molecular Ecology vol 21 pp2282ndash2296 2012

[20] D W Davidson S C Cook R R Snelling and T H ChualdquoExplaining the abundance of ants in lowland tropical rainforestcanopiesrdquo Science vol 300 no 5621 pp 969ndash972 2003

[21] J C Brownlie and K N Johnson ldquoSymbiont-mediated protec-tion in insect hostsrdquo Trends in Microbiology vol 17 no 8 pp348ndash354 2009

[22] J Jaenike R Unckless S N Cockburn L M Boelio andS J Perlman ldquoAdaptation via symbiosis recent spread of aDrosophila defensive symbiontrdquo Science vol 329 no 5988 pp212ndash215 2012

[23] K M Oliver J A Russell N A Morant and M S HunterldquoFacultative bacterial symbionts in aphids confer resistance toparasitic waspsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 4 pp 1803ndash18072003

[24] C R Currie J A Scott R C Summerbell and D MallochldquoFungus-growing ants use antibiotic-producing bacteria tocontrol garden parasitesrdquo Nature vol 398 pp 701ndash704 1999

[25] M Kaltenpoth W Gottler G Herzner and E Strohm ldquoSym-biotic bacteria protect wasp larvae from fungal infestationrdquoCurrent Biology vol 15 no 5 pp 475ndash479 2005

10 Psyche

[26] H EDunbar A C CWilsonN R Ferguson andNAMoranldquoAphid thermal tolerance is governed by a point mutation inbacterial symbiontsrdquo PLoS Biology vol 5 no 5 pp 1006ndash10152007

[27] T Tsuchida R Koga S Matsumoto and T Fukatsu ldquoInterspe-cific symbiont transfection confers a novel ecological trait to therecipient insectrdquo Biology Letters vol 7 no 2 pp 245ndash248 2011

[28] R Stouthamer J A J Breeuwer and G D D Hurst ldquoWolbachiapipientis microbial manipulator of arthropod reproductionrdquoAnnual Review of Microbiology vol 53 pp 71ndash102 1999

[29] L M Hedges J C Brownlie S L OrsquoNeill and K N JohnsonldquoWolbachia and virus protection in insectsrdquo Science vol 322 no5902 p 702 2008

[30] K Hilgenboecker P Hammerstein P Schlattmann ATelschow and J H Werren ldquoHow many species are infectedwith Wolbachiamdasha statistical analysis of current datardquo FEMSMicrobiology Letters vol 281 no 2 pp 215ndash220 2008

[31] J A Russell C F Funaro Y M Giraldo et al ldquoA veritablemenagerie of heritable bacteria from ants butterflies andbeyond broadmolecular surveys and a systematic reviewrdquoPLoSONE vol 7 no 12 Article ID e51027 2012

[32] O Duron D Bouchon S Boutin et al ldquoThe diversity ofreproductive parasites among arthropods Wolbachia do notwalk alonerdquo BMC Biology vol 6 article 27 2008

[33] D L Williamson and D F Poulson ldquoSex ratio organisms(Spiroplasmas) of Drosophilardquo in The Mycoplasmas III Plantand Insect Mycoplasmas R F Whitcomb and J G Tully Edspp 175ndash208 Academic Press New York NY USA 1979

[34] J M Bove ldquoSpiroplasmas infectious agents of plants arthro-pods and vertebratesrdquoWiener Klinische Wochenschrift vol 109no 14-15 pp 604ndash612 1997

[35] M C Tinsley and M E N Majerus ldquoA new male-killingparasitism Spiroplasma bacteria infect the ladybird beetleAnisosticta novemdecimpunctata (Coleoptera Coccinellidae)rdquoParasitology vol 132 no 6 pp 757ndash765 2006

[36] A Dobson K D Lafferty A M Kuris R F Hechinger andW Jetz ldquoHomage to Linnaeus howmany parasites Howmanyhostsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 105 no 1 pp 11482ndash11489 2008

[37] S T Kelley and S Dobler ldquoComparative analysis of microbialdiversity in Longitarsus flea beetles (Coleoptera Chrysomeli-dae)rdquo Genetica vol 139 no 5 pp 541ndash550 2011

[38] H D Ishak R Plowes R Sen et al ldquoBacterial diversityin Solenopsis invicta and Solenopsis geminata ant coloniescharacterized by 16S amplicon 454 pyrosequencingrdquo MicrobialEcology vol 61 no 4 pp 821ndash831 2011

[39] S Kautz B E R Rubin J A Russell and C S Moreau ldquoSur-veying the microbiome of ants comparing 454 pyrosequencingwith traditionalmethods to uncover bacterial diversityrdquoAppliedand Environmental Microbiology vol 79 no 2 pp 525ndash5342013

[40] S E Dowd T R Callaway R D Wolcott et al ldquoEvaluationof the bacterial diversity in the feces of cattle using 16SrDNA bacterial tag-encoded FLX amplicon pyrosequencing(bTEFAP)rdquo BMCMicrobiology vol 8 article 125 2008

[41] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[42] C Quince A Lanzen R J Davenport and P J TurnbaughldquoRemoving noise from pyrosequenced ampliconsrdquo BMC Bioin-formatics vol 12 article 38 2011

[43] R C Edgar ldquoSearch and clustering orders of magnitude fasterthan BLASTrdquo Bioinformatics vol 26 no 19 pp 2460ndash24612010

[44] J R Cole QWang E Cardenas et al ldquoTheRibosomal databaseproject improved alignments and new tools for rRNA analysisrdquoNucleic Acids Research vol 37 no 1 pp D141ndashD145 2009

[45] T Z DeSantis P Hugenholtz N Larsen et al ldquoGreengenesa chimera-checked 16S rRNA gene database and workbenchcompatible with ARBrdquo Applied and Environmental Microbiol-ogy vol 72 no 7 pp 5069ndash5072 2006

[46] A J Drummond B Ashton S Buxton et al ldquoGeneious v5 4rdquo2011 httpwwwgeneiouscom

[47] A Stamatakis P Hoover and J Rougemont ldquoA rapid bootstrapalgorithm for the RAxML web serversrdquo Systematic Biology vol57 no 5 pp 758ndash771 2008

[48] M A Miller M T Holder R Vos et al ldquoThe CIPRES portalsrdquohttpwwwphyloorgsub sectionsportal

[49] I Letunic and P Bork ldquoInteractive Tree of Life v2 onlineannotation and display of phylogenetic treesmade easyrdquoNucleicAcids Research vol 39 no 2 pp W475ndashW478 2011

[50] T Wenseleers F Ito S van Borm R Huybrechts F Volckaertand J Billen ldquoWidespread occurrence of the micro-organismWolbachia in antsrdquo Proceedings of the Royal Society B vol 265no 1404 pp 1447ndash1452 1998

[51] J A Russell ldquoThe ants (Hymenoptera Formicidae) are uniqueand enigmatic hosts of prevalent Wolbachia (Alphaproteobac-teria) symbiontsrdquoMyrmecological News vol 16 pp 7ndash23 2012

[52] W Arthofer M Riegler D Schneider M Krammer W JMiller and C Stauffer ldquoHidden Wolbachia diversity in fieldpopulations of the European cherry fruit fly Rhagoletis cerasi(Diptera Tephritidae)rdquo Molecular Ecology vol 18 no 18 pp3816ndash3830 2009

[53] J A Russell B Goldman-Huertas C S Moreau et al ldquoSpecial-ization and geographic isolation among Wolbachia symbiontsfrom ants and lycaenid butterfliesrdquo Evolution vol 63 no 3 pp624ndash640 2009

[54] T Wenseleers L Sundstrom and J Billen ldquoDeleterious Wol-bachia in the ant Formica truncorumrdquo Proceedings of the RoyalSociety B vol 269 no 1491 pp 623ndash629 2002

[55] F Vavre F Fleury J Varaldi P Fouillet and M BouletreauldquoEvidence for female mortality in Wolbachia-mediated cyto-plasmic incompatibility in haplodiploid insects epidemiologicand evolutionary consequencesrdquo Evolution vol 54 no 1 pp191ndash200 2000

[56] J Heinze ldquoThe demise of the standard ant (HymenopteraFormicidae)rdquoMyrmecological News vol 11 pp 11ndash20 2008

[57] S Kautz S U Pauls D J Ballhorn H T Lumbsch andM HeilldquoPolygynous supercolonies of the acacia-ant Pseudomyrmexpeperi an inferior colony founderrdquo Molecular Ecology vol 18no 24 pp 5180ndash5194 2009

[58] S Kautz D J Ballhorn J Kroiss et al ldquoHost plant use bycompeting acacia-ants mutualists monopolize while parasitesshare hostsrdquo PLoS ONE vol 7 no 5 Article ID e37691 2012

[59] J Fletcher G A Schultz R E Davis C E Eastman and R MGoodman ldquoBrittle root disease of horseradish-evidence for anetiological role of Spiroplasma citrirdquo Phytopathology vol 71 pp1073ndash1080 1981

[60] C F Funaro D J C Kronauer C S Moreau B Goldman-Huertas N E Pierce and J A Russell ldquoArmy ants harbor ahost-specific clade of Entomoplasmatales bacteriardquo Applied andEnvironmental Microbiology vol 77 no 1 pp 346ndash350 2011

Psyche 11

[61] D J C Kronauer ldquoRecent advances in army ant biology(Hymenoptera Formicidae)rdquoMyrmecological News vol 12 pp51ndash65 2009

[62] F Thiaucourt and G Bolske ldquoContagious caprine pleurop-neumonia and other pulmonary mycoplasmoses of sheep andgoatsrdquo OIE Revue Scientifique et Technique vol 15 no 4 pp1397ndash1414 1996

[63] T Fukatsu T Tsuchida N Nikoh and R Koga ldquoSpiroplasmaSymbiont of the pea aphid Acyrthosiphon pisum (InsectaHomoptera)rdquo Applied and Environmental Microbiology vol 67no 3 pp 1284ndash1291 2001

[64] T S Haselkorn T A Markow and N A Moran ldquoMultipleintroductions of the Spiroplasma bacterial endosymbiont intoDrosophilardquo Molecular Ecology vol 18 no 6 pp 1294ndash13052009

[65] J G Lundgren R M Lehman and J Chee-Sanford ldquoPhysiol-ogy biochemistry and topology bacterial communities withindigestive tracts of ground beetles (Coleoptera Carabidae)rdquoAnnals of the Entomological Society of America vol 100 no 2pp 275ndash282 2007

[66] I Meeus V Vercruysse and G Smagghe ldquoMolecular detectionof Spiroplasma apis and Spiroplasmamelliferum in beesrdquo Journalof Invertebrate Pathology vol 109 no 1 pp 172ndash174 2012

[67] S van Borm J Billen and J J Boomsma ldquoThe diversity ofmicroorganisms associated with Acromyrmex leafcutter antsrdquoBMC Evolutionary Biology vol 2 article 9 2002

[68] J Wedincamp F E French R F Whitcomb and R B HenegerldquoLaboratory infection and release of Spiroplasma (Entomoplas-matales Spiroplasmataceae) from horse flies (Diptera Taban-idae)rdquo Journal of Entomological Science vol 32 no 4 pp 398ndash402 1997

[69] M A Ebbert and L R Nault ldquoImproved overwintering abilityin Dalbulus maidis (Homoptera Cicadellidae) vectors infectedwith Spiroplasma kunkelii (Mycoplasmatales Spiroplasmat-aceae)rdquo Environmental Entomology vol 23 no 3 pp 634ndash6441994

[70] F M Jiggins G D D Hurst C D Jiggins J H G V DSchulenburg and M E N Majerus ldquoThe butterfly Danauschrysippus is infected by a male-killing Spiroplasma bacteriumrdquoParasitology vol 120 no 5 pp 439ndash446 2000

[71] J H G V D Schulenburg G D D Hurst D Tetzlaff GE Booth I A Zakharov and M E N Majerus ldquoHistory ofinfection with different male-killing bacteria in the two-spotladybird beetle Adalia bipunctata revealed through mitochon-drial DNA sequence analysisrdquoGenetics vol 160 no 3 pp 1075ndash1086 2002

[72] S Kautz H T Lumbsch P S Ward and M Heil ldquoHowto prevent cheating a digestive specialization ties mutualisticplant-ants to their ant-plant partnersrdquo Evolution vol 63 no 4pp 839ndash853 2009

[73] N Samaddar A Paul S Chakravorty et al ldquoNitrogen fixationin Asaia sp (Family Acetobacteraceae)rdquo Current Microbiologyvol 63 no 2 pp 226ndash231 2011

[74] Y Ano H Toyama O Adachi and K Matsushita ldquoEnergymetabolism of a unique acetic acid bacteriumAsaia bogorensisthat lacks ethanol oxidation activityrdquo Bioscience Biotechnologyand Biochemistry vol 72 no 4 pp 989ndash997 2008

[75] V G Martinson B N Danforth R L Minckley O RueppellS Tingek and N A Moran ldquoA simple and distinctive micro-biota associated with honey bees and bumble beesrdquo MolecularEcology vol 20 no 3 pp 619ndash628 2011

[76] N A Moran A K Hansen J E Powell and Z L SabreeldquoDistinctive gut microbiota of honey bees assessed using deepsampling from individual worker beesrdquo PLoS ONE vol 7 no 4Article ID e36393 2012

[77] E Crotti C Damiani M Pajoro et al ldquoAsaia a versatile aceticacid bacterial symbiont capable of cross-colonizing insectsof phylogenetically distant genera and ordersrdquo EnvironmentalMicrobiology vol 11 no 12 pp 3252ndash3264 2009

[78] C R Cox and M S Gilmore ldquoNative microbial colonization ofDrosophilamelanogaster and its use as a model of Enterococcusfaecalis pathogenesisrdquo Infection and Immunity vol 75 no 4 pp1565ndash1576 2007

[79] M Marzorati A Alma L Sacchi et al ldquoA novel Bacteroidetessymbiont is localized in Scaphoideus titanus the insect vector ofFlavescence Doree in Vitis viniferardquo Applied and EnvironmentalMicrobiology vol 72 no 2 pp 1467ndash1475 2006

[80] N J Ashbolt and P A Inkerman ldquoAcetic acid bacterial biota ofthe pink sugar cane mealybug Saccharococcus sacchari and itsenvironsrdquo Applied and Environmental Microbiology vol 56 no3 pp 707ndash712 1990

[81] E Crotti A Rizzi B Chouaia et al ldquoAcetic acid bacteria newlyemerging symbionts of insectsrdquo Applied and EnvironmentalMicrobiology vol 76 no 21 pp 6963ndash6970 2010

[82] J Ryu S Kim H Lee et al ldquoInnate immune homeostasis bythe homeobox gene Caudal and commensal-gut mutualism inDrosophilardquo Science vol 319 no 5864 pp 777ndash782 2008

[83] S Stoll J Gadau R Gross and H Feldhaar ldquoBacterial micro-biota associated with ants of the genus Tetraponerardquo BiologicalJournal of the Linnean Society vol 90 no 3 pp 399ndash412 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 10: Research Article Bacterial Infections across the Ants ...downloads.hindawi.com/journals/psyche/2013/936341.pdf · Department of Zoology, Field Museum of Natural History, South Lake

10 Psyche

[26] H EDunbar A C CWilsonN R Ferguson andNAMoranldquoAphid thermal tolerance is governed by a point mutation inbacterial symbiontsrdquo PLoS Biology vol 5 no 5 pp 1006ndash10152007

[27] T Tsuchida R Koga S Matsumoto and T Fukatsu ldquoInterspe-cific symbiont transfection confers a novel ecological trait to therecipient insectrdquo Biology Letters vol 7 no 2 pp 245ndash248 2011

[28] R Stouthamer J A J Breeuwer and G D D Hurst ldquoWolbachiapipientis microbial manipulator of arthropod reproductionrdquoAnnual Review of Microbiology vol 53 pp 71ndash102 1999

[29] L M Hedges J C Brownlie S L OrsquoNeill and K N JohnsonldquoWolbachia and virus protection in insectsrdquo Science vol 322 no5902 p 702 2008

[30] K Hilgenboecker P Hammerstein P Schlattmann ATelschow and J H Werren ldquoHow many species are infectedwith Wolbachiamdasha statistical analysis of current datardquo FEMSMicrobiology Letters vol 281 no 2 pp 215ndash220 2008

[31] J A Russell C F Funaro Y M Giraldo et al ldquoA veritablemenagerie of heritable bacteria from ants butterflies andbeyond broadmolecular surveys and a systematic reviewrdquoPLoSONE vol 7 no 12 Article ID e51027 2012

[32] O Duron D Bouchon S Boutin et al ldquoThe diversity ofreproductive parasites among arthropods Wolbachia do notwalk alonerdquo BMC Biology vol 6 article 27 2008

[33] D L Williamson and D F Poulson ldquoSex ratio organisms(Spiroplasmas) of Drosophilardquo in The Mycoplasmas III Plantand Insect Mycoplasmas R F Whitcomb and J G Tully Edspp 175ndash208 Academic Press New York NY USA 1979

[34] J M Bove ldquoSpiroplasmas infectious agents of plants arthro-pods and vertebratesrdquoWiener Klinische Wochenschrift vol 109no 14-15 pp 604ndash612 1997

[35] M C Tinsley and M E N Majerus ldquoA new male-killingparasitism Spiroplasma bacteria infect the ladybird beetleAnisosticta novemdecimpunctata (Coleoptera Coccinellidae)rdquoParasitology vol 132 no 6 pp 757ndash765 2006

[36] A Dobson K D Lafferty A M Kuris R F Hechinger andW Jetz ldquoHomage to Linnaeus howmany parasites Howmanyhostsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 105 no 1 pp 11482ndash11489 2008

[37] S T Kelley and S Dobler ldquoComparative analysis of microbialdiversity in Longitarsus flea beetles (Coleoptera Chrysomeli-dae)rdquo Genetica vol 139 no 5 pp 541ndash550 2011

[38] H D Ishak R Plowes R Sen et al ldquoBacterial diversityin Solenopsis invicta and Solenopsis geminata ant coloniescharacterized by 16S amplicon 454 pyrosequencingrdquo MicrobialEcology vol 61 no 4 pp 821ndash831 2011

[39] S Kautz B E R Rubin J A Russell and C S Moreau ldquoSur-veying the microbiome of ants comparing 454 pyrosequencingwith traditionalmethods to uncover bacterial diversityrdquoAppliedand Environmental Microbiology vol 79 no 2 pp 525ndash5342013

[40] S E Dowd T R Callaway R D Wolcott et al ldquoEvaluationof the bacterial diversity in the feces of cattle using 16SrDNA bacterial tag-encoded FLX amplicon pyrosequencing(bTEFAP)rdquo BMCMicrobiology vol 8 article 125 2008

[41] J G Caporaso J Kuczynski J Stombaugh et al ldquoQIIMEallows analysis of high-throughput community sequencingdatardquo Nature Methods vol 7 no 5 pp 335ndash336 2010

[42] C Quince A Lanzen R J Davenport and P J TurnbaughldquoRemoving noise from pyrosequenced ampliconsrdquo BMC Bioin-formatics vol 12 article 38 2011

[43] R C Edgar ldquoSearch and clustering orders of magnitude fasterthan BLASTrdquo Bioinformatics vol 26 no 19 pp 2460ndash24612010

[44] J R Cole QWang E Cardenas et al ldquoTheRibosomal databaseproject improved alignments and new tools for rRNA analysisrdquoNucleic Acids Research vol 37 no 1 pp D141ndashD145 2009

[45] T Z DeSantis P Hugenholtz N Larsen et al ldquoGreengenesa chimera-checked 16S rRNA gene database and workbenchcompatible with ARBrdquo Applied and Environmental Microbiol-ogy vol 72 no 7 pp 5069ndash5072 2006

[46] A J Drummond B Ashton S Buxton et al ldquoGeneious v5 4rdquo2011 httpwwwgeneiouscom

[47] A Stamatakis P Hoover and J Rougemont ldquoA rapid bootstrapalgorithm for the RAxML web serversrdquo Systematic Biology vol57 no 5 pp 758ndash771 2008

[48] M A Miller M T Holder R Vos et al ldquoThe CIPRES portalsrdquohttpwwwphyloorgsub sectionsportal

[49] I Letunic and P Bork ldquoInteractive Tree of Life v2 onlineannotation and display of phylogenetic treesmade easyrdquoNucleicAcids Research vol 39 no 2 pp W475ndashW478 2011

[50] T Wenseleers F Ito S van Borm R Huybrechts F Volckaertand J Billen ldquoWidespread occurrence of the micro-organismWolbachia in antsrdquo Proceedings of the Royal Society B vol 265no 1404 pp 1447ndash1452 1998

[51] J A Russell ldquoThe ants (Hymenoptera Formicidae) are uniqueand enigmatic hosts of prevalent Wolbachia (Alphaproteobac-teria) symbiontsrdquoMyrmecological News vol 16 pp 7ndash23 2012

[52] W Arthofer M Riegler D Schneider M Krammer W JMiller and C Stauffer ldquoHidden Wolbachia diversity in fieldpopulations of the European cherry fruit fly Rhagoletis cerasi(Diptera Tephritidae)rdquo Molecular Ecology vol 18 no 18 pp3816ndash3830 2009

[53] J A Russell B Goldman-Huertas C S Moreau et al ldquoSpecial-ization and geographic isolation among Wolbachia symbiontsfrom ants and lycaenid butterfliesrdquo Evolution vol 63 no 3 pp624ndash640 2009

[54] T Wenseleers L Sundstrom and J Billen ldquoDeleterious Wol-bachia in the ant Formica truncorumrdquo Proceedings of the RoyalSociety B vol 269 no 1491 pp 623ndash629 2002

[55] F Vavre F Fleury J Varaldi P Fouillet and M BouletreauldquoEvidence for female mortality in Wolbachia-mediated cyto-plasmic incompatibility in haplodiploid insects epidemiologicand evolutionary consequencesrdquo Evolution vol 54 no 1 pp191ndash200 2000

[56] J Heinze ldquoThe demise of the standard ant (HymenopteraFormicidae)rdquoMyrmecological News vol 11 pp 11ndash20 2008

[57] S Kautz S U Pauls D J Ballhorn H T Lumbsch andM HeilldquoPolygynous supercolonies of the acacia-ant Pseudomyrmexpeperi an inferior colony founderrdquo Molecular Ecology vol 18no 24 pp 5180ndash5194 2009

[58] S Kautz D J Ballhorn J Kroiss et al ldquoHost plant use bycompeting acacia-ants mutualists monopolize while parasitesshare hostsrdquo PLoS ONE vol 7 no 5 Article ID e37691 2012

[59] J Fletcher G A Schultz R E Davis C E Eastman and R MGoodman ldquoBrittle root disease of horseradish-evidence for anetiological role of Spiroplasma citrirdquo Phytopathology vol 71 pp1073ndash1080 1981

[60] C F Funaro D J C Kronauer C S Moreau B Goldman-Huertas N E Pierce and J A Russell ldquoArmy ants harbor ahost-specific clade of Entomoplasmatales bacteriardquo Applied andEnvironmental Microbiology vol 77 no 1 pp 346ndash350 2011

Psyche 11

[61] D J C Kronauer ldquoRecent advances in army ant biology(Hymenoptera Formicidae)rdquoMyrmecological News vol 12 pp51ndash65 2009

[62] F Thiaucourt and G Bolske ldquoContagious caprine pleurop-neumonia and other pulmonary mycoplasmoses of sheep andgoatsrdquo OIE Revue Scientifique et Technique vol 15 no 4 pp1397ndash1414 1996

[63] T Fukatsu T Tsuchida N Nikoh and R Koga ldquoSpiroplasmaSymbiont of the pea aphid Acyrthosiphon pisum (InsectaHomoptera)rdquo Applied and Environmental Microbiology vol 67no 3 pp 1284ndash1291 2001

[64] T S Haselkorn T A Markow and N A Moran ldquoMultipleintroductions of the Spiroplasma bacterial endosymbiont intoDrosophilardquo Molecular Ecology vol 18 no 6 pp 1294ndash13052009

[65] J G Lundgren R M Lehman and J Chee-Sanford ldquoPhysiol-ogy biochemistry and topology bacterial communities withindigestive tracts of ground beetles (Coleoptera Carabidae)rdquoAnnals of the Entomological Society of America vol 100 no 2pp 275ndash282 2007

[66] I Meeus V Vercruysse and G Smagghe ldquoMolecular detectionof Spiroplasma apis and Spiroplasmamelliferum in beesrdquo Journalof Invertebrate Pathology vol 109 no 1 pp 172ndash174 2012

[67] S van Borm J Billen and J J Boomsma ldquoThe diversity ofmicroorganisms associated with Acromyrmex leafcutter antsrdquoBMC Evolutionary Biology vol 2 article 9 2002

[68] J Wedincamp F E French R F Whitcomb and R B HenegerldquoLaboratory infection and release of Spiroplasma (Entomoplas-matales Spiroplasmataceae) from horse flies (Diptera Taban-idae)rdquo Journal of Entomological Science vol 32 no 4 pp 398ndash402 1997

[69] M A Ebbert and L R Nault ldquoImproved overwintering abilityin Dalbulus maidis (Homoptera Cicadellidae) vectors infectedwith Spiroplasma kunkelii (Mycoplasmatales Spiroplasmat-aceae)rdquo Environmental Entomology vol 23 no 3 pp 634ndash6441994

[70] F M Jiggins G D D Hurst C D Jiggins J H G V DSchulenburg and M E N Majerus ldquoThe butterfly Danauschrysippus is infected by a male-killing Spiroplasma bacteriumrdquoParasitology vol 120 no 5 pp 439ndash446 2000

[71] J H G V D Schulenburg G D D Hurst D Tetzlaff GE Booth I A Zakharov and M E N Majerus ldquoHistory ofinfection with different male-killing bacteria in the two-spotladybird beetle Adalia bipunctata revealed through mitochon-drial DNA sequence analysisrdquoGenetics vol 160 no 3 pp 1075ndash1086 2002

[72] S Kautz H T Lumbsch P S Ward and M Heil ldquoHowto prevent cheating a digestive specialization ties mutualisticplant-ants to their ant-plant partnersrdquo Evolution vol 63 no 4pp 839ndash853 2009

[73] N Samaddar A Paul S Chakravorty et al ldquoNitrogen fixationin Asaia sp (Family Acetobacteraceae)rdquo Current Microbiologyvol 63 no 2 pp 226ndash231 2011

[74] Y Ano H Toyama O Adachi and K Matsushita ldquoEnergymetabolism of a unique acetic acid bacteriumAsaia bogorensisthat lacks ethanol oxidation activityrdquo Bioscience Biotechnologyand Biochemistry vol 72 no 4 pp 989ndash997 2008

[75] V G Martinson B N Danforth R L Minckley O RueppellS Tingek and N A Moran ldquoA simple and distinctive micro-biota associated with honey bees and bumble beesrdquo MolecularEcology vol 20 no 3 pp 619ndash628 2011

[76] N A Moran A K Hansen J E Powell and Z L SabreeldquoDistinctive gut microbiota of honey bees assessed using deepsampling from individual worker beesrdquo PLoS ONE vol 7 no 4Article ID e36393 2012

[77] E Crotti C Damiani M Pajoro et al ldquoAsaia a versatile aceticacid bacterial symbiont capable of cross-colonizing insectsof phylogenetically distant genera and ordersrdquo EnvironmentalMicrobiology vol 11 no 12 pp 3252ndash3264 2009

[78] C R Cox and M S Gilmore ldquoNative microbial colonization ofDrosophilamelanogaster and its use as a model of Enterococcusfaecalis pathogenesisrdquo Infection and Immunity vol 75 no 4 pp1565ndash1576 2007

[79] M Marzorati A Alma L Sacchi et al ldquoA novel Bacteroidetessymbiont is localized in Scaphoideus titanus the insect vector ofFlavescence Doree in Vitis viniferardquo Applied and EnvironmentalMicrobiology vol 72 no 2 pp 1467ndash1475 2006

[80] N J Ashbolt and P A Inkerman ldquoAcetic acid bacterial biota ofthe pink sugar cane mealybug Saccharococcus sacchari and itsenvironsrdquo Applied and Environmental Microbiology vol 56 no3 pp 707ndash712 1990

[81] E Crotti A Rizzi B Chouaia et al ldquoAcetic acid bacteria newlyemerging symbionts of insectsrdquo Applied and EnvironmentalMicrobiology vol 76 no 21 pp 6963ndash6970 2010

[82] J Ryu S Kim H Lee et al ldquoInnate immune homeostasis bythe homeobox gene Caudal and commensal-gut mutualism inDrosophilardquo Science vol 319 no 5864 pp 777ndash782 2008

[83] S Stoll J Gadau R Gross and H Feldhaar ldquoBacterial micro-biota associated with ants of the genus Tetraponerardquo BiologicalJournal of the Linnean Society vol 90 no 3 pp 399ndash412 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 11: Research Article Bacterial Infections across the Ants ...downloads.hindawi.com/journals/psyche/2013/936341.pdf · Department of Zoology, Field Museum of Natural History, South Lake

Psyche 11

[61] D J C Kronauer ldquoRecent advances in army ant biology(Hymenoptera Formicidae)rdquoMyrmecological News vol 12 pp51ndash65 2009

[62] F Thiaucourt and G Bolske ldquoContagious caprine pleurop-neumonia and other pulmonary mycoplasmoses of sheep andgoatsrdquo OIE Revue Scientifique et Technique vol 15 no 4 pp1397ndash1414 1996

[63] T Fukatsu T Tsuchida N Nikoh and R Koga ldquoSpiroplasmaSymbiont of the pea aphid Acyrthosiphon pisum (InsectaHomoptera)rdquo Applied and Environmental Microbiology vol 67no 3 pp 1284ndash1291 2001

[64] T S Haselkorn T A Markow and N A Moran ldquoMultipleintroductions of the Spiroplasma bacterial endosymbiont intoDrosophilardquo Molecular Ecology vol 18 no 6 pp 1294ndash13052009

[65] J G Lundgren R M Lehman and J Chee-Sanford ldquoPhysiol-ogy biochemistry and topology bacterial communities withindigestive tracts of ground beetles (Coleoptera Carabidae)rdquoAnnals of the Entomological Society of America vol 100 no 2pp 275ndash282 2007

[66] I Meeus V Vercruysse and G Smagghe ldquoMolecular detectionof Spiroplasma apis and Spiroplasmamelliferum in beesrdquo Journalof Invertebrate Pathology vol 109 no 1 pp 172ndash174 2012

[67] S van Borm J Billen and J J Boomsma ldquoThe diversity ofmicroorganisms associated with Acromyrmex leafcutter antsrdquoBMC Evolutionary Biology vol 2 article 9 2002

[68] J Wedincamp F E French R F Whitcomb and R B HenegerldquoLaboratory infection and release of Spiroplasma (Entomoplas-matales Spiroplasmataceae) from horse flies (Diptera Taban-idae)rdquo Journal of Entomological Science vol 32 no 4 pp 398ndash402 1997

[69] M A Ebbert and L R Nault ldquoImproved overwintering abilityin Dalbulus maidis (Homoptera Cicadellidae) vectors infectedwith Spiroplasma kunkelii (Mycoplasmatales Spiroplasmat-aceae)rdquo Environmental Entomology vol 23 no 3 pp 634ndash6441994

[70] F M Jiggins G D D Hurst C D Jiggins J H G V DSchulenburg and M E N Majerus ldquoThe butterfly Danauschrysippus is infected by a male-killing Spiroplasma bacteriumrdquoParasitology vol 120 no 5 pp 439ndash446 2000

[71] J H G V D Schulenburg G D D Hurst D Tetzlaff GE Booth I A Zakharov and M E N Majerus ldquoHistory ofinfection with different male-killing bacteria in the two-spotladybird beetle Adalia bipunctata revealed through mitochon-drial DNA sequence analysisrdquoGenetics vol 160 no 3 pp 1075ndash1086 2002

[72] S Kautz H T Lumbsch P S Ward and M Heil ldquoHowto prevent cheating a digestive specialization ties mutualisticplant-ants to their ant-plant partnersrdquo Evolution vol 63 no 4pp 839ndash853 2009

[73] N Samaddar A Paul S Chakravorty et al ldquoNitrogen fixationin Asaia sp (Family Acetobacteraceae)rdquo Current Microbiologyvol 63 no 2 pp 226ndash231 2011

[74] Y Ano H Toyama O Adachi and K Matsushita ldquoEnergymetabolism of a unique acetic acid bacteriumAsaia bogorensisthat lacks ethanol oxidation activityrdquo Bioscience Biotechnologyand Biochemistry vol 72 no 4 pp 989ndash997 2008

[75] V G Martinson B N Danforth R L Minckley O RueppellS Tingek and N A Moran ldquoA simple and distinctive micro-biota associated with honey bees and bumble beesrdquo MolecularEcology vol 20 no 3 pp 619ndash628 2011

[76] N A Moran A K Hansen J E Powell and Z L SabreeldquoDistinctive gut microbiota of honey bees assessed using deepsampling from individual worker beesrdquo PLoS ONE vol 7 no 4Article ID e36393 2012

[77] E Crotti C Damiani M Pajoro et al ldquoAsaia a versatile aceticacid bacterial symbiont capable of cross-colonizing insectsof phylogenetically distant genera and ordersrdquo EnvironmentalMicrobiology vol 11 no 12 pp 3252ndash3264 2009

[78] C R Cox and M S Gilmore ldquoNative microbial colonization ofDrosophilamelanogaster and its use as a model of Enterococcusfaecalis pathogenesisrdquo Infection and Immunity vol 75 no 4 pp1565ndash1576 2007

[79] M Marzorati A Alma L Sacchi et al ldquoA novel Bacteroidetessymbiont is localized in Scaphoideus titanus the insect vector ofFlavescence Doree in Vitis viniferardquo Applied and EnvironmentalMicrobiology vol 72 no 2 pp 1467ndash1475 2006

[80] N J Ashbolt and P A Inkerman ldquoAcetic acid bacterial biota ofthe pink sugar cane mealybug Saccharococcus sacchari and itsenvironsrdquo Applied and Environmental Microbiology vol 56 no3 pp 707ndash712 1990

[81] E Crotti A Rizzi B Chouaia et al ldquoAcetic acid bacteria newlyemerging symbionts of insectsrdquo Applied and EnvironmentalMicrobiology vol 76 no 21 pp 6963ndash6970 2010

[82] J Ryu S Kim H Lee et al ldquoInnate immune homeostasis bythe homeobox gene Caudal and commensal-gut mutualism inDrosophilardquo Science vol 319 no 5864 pp 777ndash782 2008

[83] S Stoll J Gadau R Gross and H Feldhaar ldquoBacterial micro-biota associated with ants of the genus Tetraponerardquo BiologicalJournal of the Linnean Society vol 90 no 3 pp 399ndash412 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 12: Research Article Bacterial Infections across the Ants ...downloads.hindawi.com/journals/psyche/2013/936341.pdf · Department of Zoology, Field Museum of Natural History, South Lake

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology


Y (COLEOPTERA, CHRYSOMELIDAE, BY Insect A …downloads.hindawi.com/journals/psyche/1994/080931.pdfcollection of Museumof Comparative Zoology (MCZ), Harvard University, USA. Holotypes

Y (COLEOPTERA, CHRYSOMELIDAE, BY Insect A …downloads.hindawi.com/journals/psyche/1994/080931.pdfcollection of Museumof Comparative Zoology (MCZ), Harvard University, USA. Holotypes

OF NOMENCLATURE CONNECTED ANT of Hym.,downloads.hindawi.com/journals/psyche/1916/047506.pdfLasius for the reception of ten species of ants, but this use of the name is invalid since

OF NOMENCLATURE CONNECTED ANT of Hym.,downloads.hindawi.com/journals/psyche/1916/047506.pdfLasius for the reception of ten species of ants, but this use of the name is invalid since

Psyche Tests

Psyche Tests

Psyche Nursing

Psyche Nursing

Psyche Report

Psyche Report

Ants and ants based routing

Ants and ants based routing

psyche + cupid

psyche + cupid

PSYCHE - Hindawi Publishing Corporationdownloads.hindawi.com/journals/psyche/1907/014536.pdf · PSYCHE BIOLOGICALNOTES ONTHE COLORADOPOTATO BEETLE, LEP- TINOTARSA DECEMLINEATA (SAY),

PSYCHE - Hindawi Publishing Corporationdownloads.hindawi.com/journals/psyche/1907/014536.pdf · PSYCHE BIOLOGICALNOTES ONTHE COLORADOPOTATO BEETLE, LEP- TINOTARSA DECEMLINEATA (SAY),

Psyche Questions

Psyche Questions

Psyche Handouts

Psyche Handouts

Http:// ants/ ants/ .

Http:// ants/ ants/ .

Systems Psyche: Its Structure, Operation and Possible ...the psyche! “The psyche is mind!” “The psyche is feelings!” “The psyche is consciousness!” “The psyche is self!”

Systems Psyche: Its Structure, Operation and Possible ...the psyche! “The psyche is mind!” “The psyche is feelings!” “The psyche is consciousness!” “The psyche is self!”

Psyche Notes

Psyche Notes

STEATODA (THERIDIIDAE), A SPIDER FEEDS ON HARVESTER ANTS…downloads.hindawi.com/journals/psyche/1970/052793.pdf · 2019-08-01 · 1970] 8o 7o H61ldobler Steatoda 205 6o z 50 0 3o

STEATODA (THERIDIIDAE), A SPIDER FEEDS ON HARVESTER ANTS…downloads.hindawi.com/journals/psyche/1970/052793.pdf · 2019-08-01 · 1970] 8o 7o H61ldobler Steatoda 205 6o z 50 0 3o

Ifc Psyche

Ifc Psyche

Psyche - core.ac.uk

Psyche - core.ac.uk

Psyche Exam

Psyche Exam

BETWEENANT THE (EUPHORBIACEAE) · 2019. 8. 1. · 20 Psyche [Vol. 102 In this paperwereport acase ofdirect interference competition, in which ants rob another ant species of the seeds

BETWEENANT THE (EUPHORBIACEAE) · 2019. 8. 1. · 20 Psyche [Vol. 102 In this paperwereport acase ofdirect interference competition, in which ants rob another ant species of the seeds

PSYCHE - CORE

PSYCHE - CORE

PSYCHE - Hindawi Publishing Corporationdownloads.hindawi.com/journals/psyche/2000/097127.pdf · PSYCHE Vol. 103 2000 No. 1-2 ANTSFROMNORTHWESTERNCHINA (HYMENOPTERA,FORMICIDAE) BYCEDRICCOLLINGWOOD

PSYCHE - Hindawi Publishing Corporationdownloads.hindawi.com/journals/psyche/2000/097127.pdf · PSYCHE Vol. 103 2000 No. 1-2 ANTSFROMNORTHWESTERNCHINA (HYMENOPTERA,FORMICIDAE) BYCEDRICCOLLINGWOOD