Surveying the Microbiome of Ants: Comparing 454 Pyrosequencing
with Traditional Methods To Uncover Bacterial Diversity
Stefanie Kautz,aBenjamin E. R. Rubin,a,bJacob A. Russell,cCorrie S. Moreaua
Field Museum of Natural History, Department of Zoology, Chicago, Illinois, USAa; University of Chicago, Committee on Evolutionary Biology, Chicago, Illinois, USAb; Drexel
University, Department of Biology, Philadelphia, Pennsylvania, USAc
turtle ant Cephalotes varians were Rhizobiales, Burkholderiales, Opitutales, Xanthomonadales, and Campylobacterales, as re-
identifiedfromthesegroupsclusteredwithinant-specificlineages,indicatingadeepcoevolutionaryhistoryof Cephalotes ants
ciated with Cephalotes varians is dominated by a few dozen bacterial lineages and that 454 sequencing is a cost-efficient tool to
organism, we know little about their diversity and prevalence
across most of the eukaryotic tree of life (1). At the very least, it is
known that among insects, bacterial endosymbionts are wide-
spread and often have a significant impact on their hosts’ biology
associated bacteria that upgrade their hosts’ diets. These bacteria
ticularly important roles in insects with nutritionally limited or
deficient diets. Some well-known examples of such insect-mi-
thia glossinidia in tsetse flies (6), and the diverse microflora of
and function of bacterial symbionts are being performed using a
limited range of model organisms. We therefore still know little
about the identities and significance of bacteria associated with
most animal groups (10, 11). Carefully analyzing the bacterial
communities present in a wide range of eukaryotic taxa is neces-
sary to understand the diversity and ecological function of such
symbionts. Unfortunately, about 99% of all bacteria are not cul-
independent methods (1).
to assess and analyze bacterial communities across the tree of life.
Sanger sequencing of the bacterial 16S rRNA gene has been used
successfully to characterize diversity (e.g., see references 12 and
13). Tag-encoded FLX amplicon pyrosequencing is increasingly
being used to characterize both bacterial communities (e.g., see
references 14 to 16) and fungal communities (e.g., see references
systems. Although they form facultative to obligate associa-
17 and 18) from various environments. Such studies characteriz-
ing the microbiomes of insects are facilitating the discovery of the
host-associated rare biosphere (19–22).
Ants are among the most abundant animal groups and often
(23, 24). In addition, based on biomass estimates, arboreal ants
appear to be much more abundant than their supposed prey or-
ganisms. Several hypotheses have been suggested as an explana-
tion for “Tobin’s ant-biomass paradox” (25), and it has been
shown using stable isotope analyses that ecologically dominant
feed on plant-derived resources (26). As plant-derived resources
(e.g., honeydew and extrafloral nectar) are usually poor in nitro-
biotic bacteria that recycle nitrogen waste or fix atmospheric ni-
are deprived of rich nitrogen sources, such as obligate plant ants,
other groups low on the trophic scale are prime candidates for
symbiotic nitrogen provisioning.
Turtle ants of the genus Cephalotes are exclusively arboreal, as
they nest inside twigs; Cephalotes varians nests primarily in man-
Received 11 October 2012 Accepted 30 October 2012
Published ahead of print 2 November 2012
Address correspondence to Stefanie Kautz, firstname.lastname@example.org.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
January 2013 Volume 79 Number 2Applied and Environmental Microbiologyp. 525–534aem.asm.org
such as extrafloral nectar or honeydew, and are considered pre-
dominantly herbivorous, as demonstrated in stable isotope anal-
yses (26). The hypothesized core gut microbiota of the turtle ant
species Cephalotes varians has been characterized using Sanger
sequencing of the 16S rRNA gene, and evidence of symbiotic bac-
communities from a variety of tissues and individuals. By com-
terial cultivation from live ants followed by PCR and Sanger se-
quencing of the 16S rRNA region, we provide a detailed survey of
the microbes associated with herbivorous turtle ants, while di-
rectly comparing methods and their abilities to characterize bac-
MATERIALS AND METHODS
Study species and sample preparation. In our study, we focused on
Cephalotes varians (Formicidae: Myrmicinae). Specimens were collected
in the Florida Keys and either stored in 95% ethanol immediately until
further analysis (colony CSM1280) or kept live for cultivation experi-
ments (colonies CSM1235, CSM1323, and CSM1396). Live ants were
maintained in the laboratory and supplied only with sterile 30% sucrose
and sterile water ad libitum until the time of dissection (more than 14
weeks) to eliminate bacteria that are obtained through the diet, leaving
behind the resident and symbiotic gut community. An overview of all
samples that were subjected to culture-independent analyses is given in
Table S1 in the supplemental material, while information on cultivated
bacteria is given in Table S2.
To assess bacterial diversity associated with specific host organs or
structures, the following protocols were implemented. In the lab, ants
ant specimen was taken out of the collection vial (CSM1280) or the nest
(CSM1235, CSM1323, and CSM1396) using sterile forceps and placed in
a petri dish with 96% ethanol for 2 min to surface sterilize the specimen.
Then, the specimen was transferred into a petri dish with sterile double-
distilled water (ddH2O) for 2 min and subsequently placed in a sterile
watchmaker glass under the stereomicroscope. The head was pulled off
and transferred into a 1.5-ml reaction tube; a single leg was removed and
put in a separate 1.5-ml reaction tube. The digestive tract was dissected
under sterile ddH2O by carefully removing the abdominal segments, be-
ginning at the posterior end. The individual parts of the digestive tract
(i.e., the crop, the midgut, and the hindgut) were carefully pulled apart,
without rupturing, using forceps. These were then individually placed in
1.5-ml collection tubes. Alternatively, the entire gut (crop, midgut, and
hindgut) was used. Between dissecting different individuals, forceps and
watchmaker glasses were washed with ddH2O and then with 10% bleach
before being sterilized under UV light for 10 min.
For DNA extraction prior to 454 pyrosequencing and cloning, one
Qiagen tungsten carbide bead was placed into each tube to break up the
cell wall material of bacteria associated with the dissected ant body parts
using the Qiagen TissueLyser (20 s at 30 rpm · s?1). To extract total
ing protocol B for insects. The final elution was performed with ddH2O.
We extracted DNA of 21 samples for pyrosequencing, of which 13 were
also subjected to cloning and Sanger sequencing.
454 pyrosequencing. Bacterial tag-encoded titanium amplicon pyro-
sequencing (bTEFAP) was performed by the Research and Testing Labo-
ratory (Lubbock, TX) as described by Dowd et al. (34). The 16S rRNA
universal eubacterial primers 28F (5=-GAGTTTGATCNTGGCTCAG)
and 519R (5=-GTNTTACNGCGGCKGCTG) were used to amplify ap-
in the present study were multiplexed with samples not included in this
study and were part of two separate quarter plates of different 454 runs.
PCR, cloning, and Sanger sequencing. For Sanger sequencing of
clone libraries, 16S rRNA fragments were amplified from genomic DNA
with universal bacterial primers 9Fa (5=-GAGTTTGATCITIGCTCAG-
rogen TopoTA cloning kit (vector 150 pCR2.1), using One Shot chemi-
cally competent E. coli cells for transformation and blue-white colony
screening on LB plates with ampicillin and carbenicillin. We picked 24 to
110 white colonies from each cloning reaction. PCR of cloned products
plasmids. An agarose gel (1% high melt) stained with ethidium bromide
was run to verify insert size.
PCR products that had the target length were cleaned using ExoSap
before cycle sequencing using the BigDye Terminator reaction kit (ABI
PRISM, Applied Biosystems, Foster City, CA). Sequencing primers were
the PCR primers 9Fa and 1513R, along with internal primers 319F, 559F,
953F, 559R, and 1072R (30). Sequence products were precipitated and
loaded on an ABI 3730 (Applied Biosystems) automatic sequencer. Se-
quence fragments obtained were assembled with Geneious Pro 5.4 (35)
and ambiguities manually corrected. Newly generated 16S rRNA se-
et al. (27) (n ? 304 previously generated sequences).
Primers used in the present study. Different primers were used for
cloning and Sanger sequencing and for 454 sequencing (as described
above). Using RDP probe match (36), the first 10 bases of the forward
primer 9Fa (cloning and Sanger sequencing) were an exact match to
allowing one mismatch, while the forward primer 28F (454) was an exact
match to 87.83% and to 98.64% with one mismatch. The 1513R reverse
primer (cloning and Sanger sequencing) exactly matched 95.50% of da-
tabased sequences (102,442/107,267) and 99.33% with one mismatch,
while the 519R (454) primer exactly matched 94.2% (1,749,896/
1,857,322) and 99.13% with one mismatch.
Cultivation of bacteria. For cultivation of live bacteria, dissected ant
body parts were placed in 500 ?l of liquid lysogeny broth (LB) medium
and homogenized with a micropestle. Using sterile technique, 200 ?l of
the homogenate was plated onto LB agar plates. Cultures were incubated
at 25°C and checked daily for bacterial growth. Morphologically unique
colonies from every plate were transferred to new petri dishes, and DNA
using 16S rRNA primers 9Fa and 1513R, and PCR products were se-
Bacterial 16S rRNA data processing and analysis. All 16S rRNA
demultiplexed reads, and then filtered them according to Phred quality
scores. Quality criteria were a minimum sequence length of 200 bp, a
maximum sequence length of 1,000 bp, and a minimum average quality
score of 25. Using stringent quality control parameters, we allowed no
ambiguous bases or mismatches in the primer sequence and no barcode
errors, and a maximum homopolymer length of 6 bp. Sequence reads
were then clustered into operational taxonomic units (OTUs) at 97%
a cluster as the representative sequence for that OTU. Singletons, i.e.,
ras were excluded using ChimeraSlayer (39). The closest BLAST hits for
some sequences were from different orders, suggesting that chimeric se-
quences remained. Therefore, we also used the “BLAST fragments”
method as implemented in QIIME to remove chimeras. We conducted a
the addition of sequences of all ant-associated bacteria from a previous
study (30). The addition of these sequences decreased the number of
OTUs that were determined to be chimeric. Bacterial taxonomic classifi-
cations of our sequences were obtained by BLAST searching against the
SILVA database. Sequences were aligned against the greengenes database
Kautz et al.
aem.asm.orgApplied and Environmental Microbiology
(http://greengenes.lbl.gov/cgi-bin/nph-index.cgi) using PyNAST (40)
and filtered using the greengenes lanemask. The final alignment was used
to construct phylogenetic trees using the default settings in FastTree2
(41). We summarized the proportions of identified taxa in each sample
and calculated the amount of bacterial diversity shared between samples
using the weighted UniFrac metric (42, 43) as implemented in QIIME.
Similarity of samples was visualized for the weighted UniFrac analysis
using principal coordinate analysis plots. Rarefaction curves were gener-
(clones) using mothur, version 1.15.0 (44). Rarefaction curves were first
individual ant if it had been dissected into different parts, pooled parts of
the gut, pooled all samples per colony, and pooled all samples (excluding
In order to identify the most prevalent bacteria and their closest rela-
tives, we clustered 500 random reads from each 454 sample into OTUs
(97% similarity) and subjected the clusters to a BLAST search against
GenBank. This subset of reads was selected to control for unequal read
numbers. Colony CSM1323 was excluded, as it happened to be infected
with Spiroplasma; see Results for details. We then inferred a maximum
likelihood phylogeny of the most common OTUs and their GenBank
relatives using the RAxML (45) on the CIPRES web portal (46). We in-
cluded the largest clusters that cumulatively accounted for 90% of read
numbers generated by 454 sequencing and also the largest clusters that
accounted for 90% of sequences generated through cloning. We then
uploaded the most likely tree to the iToL website (47) to facilitate graph-
ical illustration of bacterial habitat (i.e., host, other free-living environ-
ments), bacterial order, and the methods used to obtain the given se-
We subsequently conducted three distinct analyses on our generated
we addressed within-colony variation using colonies CSM1280 and
CSM1323, which were each represented by six or more DNA extractions
from each of three and four individuals per colony, respectively. (ii) Sec-
ond, we directly compared results of 454 sequencing to those from clon-
sequencing to evaluate the bacterial communities prevalent in different
Within-colony analyses using 454 pyrosequencing. From colony
CSM1280, we prepared nine samples. We used one entire ant worker
and CSM1280 individual 3) into four parts: the crop, the head, the
midgut, and the hindgut. For colony CSM1323, DNA was extracted from
as well as three guts, three heads, and three legs derived from three work-
Chao1 estimator, the Shannon index of diversity, and Simpson’s index as
implemented in QIIME. As OTU number and estimated diversity in-
creased with read number, we repeated the assessment of alpha diversity
We used the RDP library comparison tool (36) to test for pairwise signif-
icant differences between communities and visualized differences be-
tween all samples using weighted UniFrac PCoA.
Comparing bacterial diversity methods. 454 pyrosequencing and
cloning were used to sample communities from the same DNA extrac-
obtained from cloned samples was much lower than from 454 pyrose-
quencing, we rarefied to 17, which was the lowest number of quality se-
3-legs; see Table S1 in the supplemental material). Alpha diversity mea-
sures were subsequently inferred exactly as described above.
To compare our three methods to characterize bacterial diversity on
the level of OTUs (97% similarity), we combined all 454 reads with all
Sanger sequences of clone libraries and cultivated bacteria and clustered
these into OTUs. Consecutively, we excluded singletons and removed
chimeric OTUs as described above. We then determined which OTUs
contained (i) only 454 reads, (ii) only Sanger sequences of clone libraries,
(iii) only Sanger sequences of cultivated bacteria, (iv) 454 reads and
Sanger sequenced clones, (v) 454 reads and Sanger-sequenced cultivated
bacteria, (vi) Sanger-sequenced clones and Sanger-sequenced cultivated
bacteria, and, lastly, (vii) data retrieved with all three methods. We deter-
mined the taxonomy of OTUs by BLAST searching against the SILVA
database as described above.
Assessing differences between ant tissues. In order to test whether
454 sequence data set. Due to a potential pathogenic infection of colony
CSM1323 with a Spiroplasma sp. (order Entomoplasmatales), these sam-
ples were excluded from the analyses, as almost all sequences returned
distances were used for PCoA analysis, and all analyses were carried out
using QIIME. Prior to assessing alpha and beta diversity measures, sam-
ples were rarefied to 400, which corresponded to the lowest number of
quality reads obtained from any individual sample in the third data set.
Nucleotide sequence accession numbers. 454 data are found in
GenBank’s Short Read Archive under accession number SRA05997,
while accession numbers of cloned and cultivated sequences have been
deposited in GenBank under accession numbers JQ254320 to JQ254364,
to JX990334 (see Table S2 in the supplemental material).
Analysis of 454 pyrosequencing data. A total of 144,679 16S
the samples yielded no data (CSM1235 leg and CSM1396 head),
possibly due to a low concentration of bacterial DNA in the ex-
tractions. Reads clustered into 980 OTUs, of which 77 and 329
ments method, respectively. Of the remaining OTUs, 290 were
singletons. All chimeras and singletons were removed before fur-
remained after quality control and chimera detection. The num-
ber of reads after quality control per sample ranged from 448 to
11,807 (mean ? standard error [SE], 5,034.5 ? 829.9). Three
samples had fewer than 2,000 reads (see Table S1 in the supple-
Pyrosequencing revealed the presence of at least 19 bacterial
orders associated with herbivorous Cephalotes varians turtle ants:
Acidithiobacillales, Actinobacteriales, Alteromonadales, Burkhold-
eriales, Campylobacterales, Chromatiales, Enterobacteriales, Flavo-
bacteriales, Hydrogenophilales, Lactobacillales, Oceanospirillales,
Opitutales (previously referred to as Verrucomicrobiales ),
Pseudomonadales, Rhizobiales, Rhodobacterales, Rhodocyclales,
tutales (24.3%), Burkholderiales (9.4%), Campylobacterales (9.3%),
The nonparametric Chao1 estimator (48) predicted that the
number of OTUs ranged from 8 to 180 across all samples (see
Table S1 in the supplemental material). One single OTU (at 97%
sequence similarity) accounted for an average of 29.0% of all 454
reads and was a relative of an ant-associated Bartonella sp. (Rhi-
zobiales). Relatives of an ant-associated Opitutus sp. (Opitutales)
454 Pyrosequencing of Ant Bacteria
January 2013 Volume 79 Number 2aem.asm.org 527
ter (Pseudomonadales) relative (14.3%), which was not detected
through cloning (Table 1) and was not related to other ant-asso-
ciated bacteria (Fig. 1). In fact, this OTU dominated the nongut
samples CSM1235 head and CSM1396 leg, which had not been
Acrobacter sp. relative (Campylobacterales [7.3%]), an uncultured
gammaproteobacterium (4.1%), another Bartonella sp. relative
(Rhizobiales [4.1%]), an Alcaligenaceae bacterium (Burkholderia-
les [2.0%]), and an uncultured Xanthomonadaceae bacterium
(Xanthomonadales [1.6%]) were among the most abundant
OTUs, and all related to previously described ant-associated taxa.
Characteristics of the most prevalent OTUs accounting for about
90% of all reads and their closest GenBank relatives are summa-
rized in Table 1.
TABLE 1 The most common OTUs (at 97% identity level) associated with Cephalotes varians ants as discovered by 454 sequencinga
Cluster no. Cluster sizeCumulative % Bacterial order
Three closest GenBank
accession no.% identityCloning Cultivation
01 1392 29.0 Rhizobiales Uncultured Rhizobiales
03 68863.4 PseudomonadalesNo No
04350 70.7 CampylobacteralesYes No
07 9680.9Burkholderiales YesNo
1069 85.3Xanthomonadales YesNo
1247 87.4 BurkholderialesNoNo
1337 88.2 FlavobacterialesYes No
1436 88.9Burkholderiales YesNo
1533 89.6 XanthomonadalesYesNo
aA total of 400 reads were randomly selected for each of 13 C. varians samples. Colony CSM1323 was excluded, as it was infected with Enteroplasmatales bacteria, which strongly
biased the data toward this group of bacteria alone. This table provides the cluster size, cumulative percentage, the three top BLAST hits, GenBank accession numbers, percent
identity, and the detection of the same OTUs through cloning or cultivation. Sixteen OTUs accounted for about 90% of bacterial diversity. BLAST searches were performed against
the NCBI GenBank database on 12 October 2011.
Kautz et al.
aem.asm.org Applied and Environmental Microbiology
A large proportion of the sequences (average of 92.9% of all
reads) obtained from ant colony CSM1323 were from the genus
Spiroplasma. Given the known life histories of Spiroplasma across
the arthropods (49–51), we concluded that this colony was in-
fected by the pathogen and we excluded this colony from further
Within-colony analyses using 454 pyrosequencing. For ant
colony CSM1280, we obtained 1,199 to 6,539 reads per sample
across nine samples after quality control (see Table S1 in the sup-
plemental material). On average, Rhizobiales accounted for
(10.46%), Campylobacterales (10.33%), an unclassified gamma-
proteobacterium (5.66%), and Xanthomonadales (5.24%). The
samples CSM1280 crop-2 and CSM1280 midgut-2 stood out, as
both had extremely low alpha diversity (see Table S1), with each
being dominated by a single bacterial order—Rhizobiales
(99.02%) and Campylobacterales (71.58%), respectively (Fig. 2).
rarefaction to 1,000 reads. In a PCoA based on weighted UniFrac
distances, all samples clustered together except CSM1280 midgut-2.
The two head samples grouped with the two crop samples along
the first and second axes. The samples from the entire worker
(CSM1280 wk-1) clustered loosely with the samples from the
hindguts and midguts along the first axis. Rhizobiales bacteria
to the high abundance of Opitutales for mid- and hindguts as well
as Burkholderiales in three of four samples (Fig. 3).
Read numbers retrieved for colony CSM1323 were high and
ranged from 3,229 to 11,850 (Fig. 2; see also Table S1 in the sup-
plemental material). The highest diversity of bacterial communi-
with 88 and 89 OTUs present and the Chao1 estimator equaling
120.0 and 132.0, respectively (after rarefaction to 3,000). Samples
prepared from the head and legs had lower diversity than gut
40.71 to 45.12 (see Table S1). The bacterial communities discov-
colony CSM1323, although this is not surprising considering the
high Spiroplasma infection in this sample. In a PCoA plot, the
samples prepared from the gut clustered together and were sepa-
rated from the nongut tissues (Fig. 2).
Comparing 454 sequencing with cloning. After quality con-
rRNA sequences from the cloning and Sanger sequencing ap-
proach, with the number of clones analyzed ranging from 17 to
103 per sample (mean ? SE, 35.38 ? 6.72) (see Table S1 in the
supplemental material). In pairwise comparisons, results ob-
tained through cloning were qualitatively similar to results ob-
tained from 454 sequencing across all samples for each sample
obtained by the two methods from the same sample in the PCoA
plots (Fig. 3). However, it should be noted that samples also
most prevalent OTUs as generated by 454 sequencing and by cloning. For each method, we clustered reads (454 sequencing) or sequences (cloning) at 97%
CSM1323 was excluded from these analyses due to an infection with Entomoplasmatales bacteria. Please note the misplacement of Campylobacterales, which
should group with other Proteobacteria.
454 Pyrosequencing of Ant Bacteria
January 2013 Volume 79 Number 2aem.asm.org 529
of Xanthomonadales (9 of 9 libraries) and Burkholderiales (5 of 7
libraries) in clone-based data sets, while Rhizobiales (9 of 10) and
sequence libraries (Fig. 2), although we had far less coverage for
that the differences were significant for all nine Xanthomonadales
comparisons, for one Rhizobiales comparison (CSM1280 wk-1), for
two Burkholderiales comparisons (CSM1235 gut-1 and CSM1280
crop-3), and for three Opitutales comparisons (CSM1235 gut-1,
Across all 454 libraries, 16 OTUs accounted for 90% of reads,
inferred a phylogenetic tree of these most prevalent OTUs from
these two methods and their GenBank relatives and found that
eight of the dominant OTUs were found using both methods,
including two Burkolderiales, two Xanthomonadales, one unclas-
teriales, and one Campylobacterales OTU (Fig. 1). Using 454 se-
quencing, additional OTUs, which were in the pool of OTUs
accounting for 90% of reads, belonged to the Burkholderiales, an
uncultured Pseudomonadales bacterium, Flavobacteriales, and
Opitutales. In total, 445 OTUs were detected in 454 libraries that
were not recovered in any of the clone libraries. Using cloning,
three additional OTUs were detected in clone libraries, but not in
454 libraries. A total of 34 OTUs were shared between the two
Within the Burkholderiales, Xanthomonadales, an unclassified
FIG 3 PCoA analysis of bacterial communities from samples that were sub-
jected to 454 sequencing and cloning. Positions of the bacterial communities
for each species along the two first principal coordinate axes are illustrated,
outlines illustrate results obtained through cloning and Sanger sequencing.
Note the distinct clustering of samples prepared from colony CSM1323 (gray
roplasma sp.), and also the clustering of the samples prepared from the hind-
guts of colony CSM1280 (1280 hind-2 and 1280 hind-3), as well as of the
midguts of colony CSM1280 (1280 mid-2 and 1280 mid-3). “Hind” refers to
the hindgut, “mid” refers to the midgut, and “wk” refers to samples prepared
from whole worker extractions. Results are based on weighted UniFrac
FIG 2 Comparison of bacterial communities in Cephalotes varians ants detected through 454 pyrosequencing (upper rows) and cloning (lower rows). Samples
“other.” “Unclass. gamma” refers to an unclassified gammaproteobacterium that clustered with the Pseudomonadales bacteria.
Kautz et al.
aem.asm.org Applied and Environmental Microbiology
gammaproteobacterium, Pseudomonadales, Rhizobiales, Sphingo-
bacteriales, and Opitutales, OTUs from this study and GenBank
(i.e., top BLAST hits) grouped within ant-specific lineages, sug-
gesting a long coevolutionary history and specialized role of the
microbes. In the case of the Rhizobiales, we detected a clade of
ant-specific OTUs that was sister to a clade of bee-specific OTUs
were able to detect a total of 12 OTUs belonging to the bacterial
genera Asaia, Brachybacterium, Bacillus, Enterobacter, Lysinibacil-
lus, Microbacterium, Paenibacillus, Pantoea, Pseudomonas, Rah-
and Serratia) were also detected using 454 sequencing and were
represented by 2, 45, 2, and 1 reads of the 95,656 reads across all
pect potential bacterial symbionts that might upgrade the ants’
on insects (52–54), and thus, we dissected various ant body parts
to characterize their bacterial communities. The highest diversity
estimate was found for the one entire worker analyzed. Of the
nongut tissues, bacterial diversity associated with heads was
since the mouth is connected to the digestive tract. Due to the
small sample size, this finding should be considered preliminary.
It should be pointed out that the average read number was lower
for the nongut samples than for the gut samples. However, even
after rarefying all samples to 400 reads in this data set, we still
found lower OTU numbers and Chao1 estimator values for non-
terial communities associated with gut tissues had significantly
higher diversity than nongut tissues but were dominated by a
characteristic microbiome of Rhizobiales, Opitutales, Burkhold-
eriales, and Xanthomonadales. Samples prepared from the head
and leg, but in part also from one of the crop and one of the
midgut samples, often showed a different community composi-
abundant OTU in other samples dominated the samples
ples cluster closely together (see Fig. S3 in the supplemental ma-
Many host-associated microbes provide essential functions to
their hosts, and we are only beginning to characterize the micro-
tera: Formicidae) are one of the most abundant animal groups
(23, 25), and microbial symbionts may have facilitated their evo-
lution and diversification (29, 55). Recent studies have found a
range of putative symbionts in different ant lineages (22, 27, 28,
30, 49), and next-generation sequencing techniques are facilitat-
ing the endeavor to characterize the microbiome of such arthro-
rRNA amplicon 454 pyrosequencing, Sanger-sequenced clone li-
braries, and bacterial cultivation, but it also shows some surpris-
ing similarities. Alpha diversity measures were higher with the
next-generation sequencing approach than with cloning (see Fig.
S1 in the supplemental material). These differences are to be ex-
ing. The cultivation approach uncovered 12 OTUs in total, none
of which were revealed with cloning, while 4 were detected in 454
At the level of bacterial orders, results obtained through clon-
ing and 454 sequencing were qualitatively similar despite the use
of different primers, but they also showed significant systematic
les, Rhizobiales, and Opitutales (Fig. 3; see also Fig. S2 in the sup-
primers might have caused these differences. While universal 16S
ing exceptions can be present in different bacterial lineages (56).
Engelbrektson et al. (20) showed that primer choice affected am-
plification in their study due to mismatched templates and noted
that variations in templates need to be accounted for by degener-
ate primers. The primers used for 454 sequencing as well as our
cloning primers have degenerate bases and yield similar numbers
when using RDP’s probe match tool (36), revealing comparable
In contrast to the culture-independent methods, we found a
total of only 12 OTUs using cultivation approaches belonging to
the orders Actinomycetales (genera Brachybacterium, Microbacte-
rium, and Streptomyces), Bacillales (genera Bacillus, Lysinibacillus,
and Paenibacillus), Enterobacteriales (genera Enterobacter, Pan-
toea, Rahnella, and Serratia), Pseudomonadales (genus Pseudomo-
nas), and Rhodospirillales (genus Asaia) (see Table S2 in the sup-
plemental material). The bacteria detected through cultivation
were found only in low titers or not at all when using cultivation-
bias toward bacteria that are able to grow on the media provided.
This is not surprising, since about 99% of all bacteria are not
cultivable (1), while on the other hand, some bacterial strains are
easily cultured even though they comprise only a small portion of
the bacterial diversity in a given sample. For example, E. coli is
often used as a marker for fecal contamination using cultivation
methods but usually comprises less than 1% of the fecal bacterial
community (34). Developing reproducible cultivation protocols
for bacteria that are abundant in their natural habitats is still a
challenge, and future studies should consider using a broad range
functional and manipulative assays (see, for example, reference
29), it is clear that advances in this field will greatly expand our
understanding of the roles of bacteria in general.
Previous studies have indicated that bacterial symbionts are
localized in the gut of Cephalotes spp. (57). The ileum—the ante-
rior region of the hindgut—possesses structural adaptations har-
in association with microvilli in the midgut (60, 61). In Tetrapon-
symbiotic bacteria (62, 63). In accordance, we found the bacterial
the communities in nongut tissues. These gut communities are
highly stable, as they persist in colonies exclusively reared on su-
crose (reference 30 and the present study). While entire workers,
the entire gut (i.e., the crop, midgut, and hindgut), and the hind-
gut had similar microbial communities, symbiont compositions
454 Pyrosequencing of Ant Bacteria
January 2013 Volume 79 Number 2 aem.asm.org 531
but not in a second ant worker (CSM1280 individual 2), whose
community was strongly dominated by Campylobacterales. Sam-
ples prepared from the crop were dominated by Rhizobiales bac-
bacterial flora consisting of Rhizobiales, Opitutales, Burkholderia-
les, and Xanthomonadales in a second sample (CSM1280 crop-3).
All dissections were carried out with great care, and samples were
discarded when tissues were disrupted, reducing the likelihood of
sample cross-contamination. Even though leakage of gut com-
partments when performing dissections is possible, this should
only lead to slight deviations, as the concentration of bacteria in
Russell et al. (30), ant-specific bacterial lineages of the Burkhold-
eriales, Pseudomonadales, Rhizobiales, Verrucomicrobiales (classi-
fied as Opitutales in our present study), and Xanthomonadales
were found only in gut-derived tissues and absent from nongut
tissues based on diagnostic PCR. The clade-specific primers used
found the highest diversity of microbes in samples derived from
colony CSM1280 (Fig. S1), despite lower read numbers on the
individual and colony level than those of colony CSM1323 (see
Fig. S1 in the supplemental material). CSM1280 was the only
onies had been kept on a sterile diet for several weeks. This sug-
gests that field-caught colonies are associated with transient mi-
crobes that are not part of the stable community. However, larger
other reasons for this finding. At the same time, the gut commu-
nity of samples from colony CSM1280 had a large proportion of
unclassified gammaproteobacterium, which characterize the core
gut microbiota of the Cephalotini (27).
ever, due to the known metabolic capacities of the bacterial lin-
eages consistently found in turtle ants, nutritional supplementa-
atmospheric nitrogen in their ant hosts, but acetylene assays were
not able to support this idea under experimental conditions (30).
Members of the Opitutales can reduce nitrate to nitrite, and it has
ant host (27). They could also ferment sugars to propionate and
acetate (64), which then might serve as the substrate for other
symbionts such as the Xanthomonadales (27). Burkholderiales are
extremely diverse, and so are the metabolic capacities known for
while others can produce antibiotics against fungi. Such func-
tional roles remain speculative to date, and a detailed discussion
can be found in the work of Anderson et al. (27).
One colony investigated—CSM1323—was infected with Spi-
roplasma, which is not uncommon in ants (49). Likely the infec-
tion was systemic, as this bacterium accounted for an average of
this bacterium at similarly high abundances. Although Spiro-
plasma has been shown to protect Drosophila neotestacea against
the sterilizing effects of a parasitic nematode (65), we have no
understanding of the impact of this bacterium in ants. We can
only speculate that this Spiroplasma occasionally infects ants as a
pathogen (50), although in the lab, no negative effects were ob-
served within our infected colony. The Spiroplasma bacterium
sequenced from Cephalotes varians was closely related to spiro-
plasma strain likely has the tendency to colonize ants and other
biota of the Cephalotini (27, 30) (Fig. 2).
with ants but also across the tree of life still remains a challenging
endeavor. For herbivorous turtle ants, we found that gut tissues
had significantly higher diversity than nongut tissues and that
many of the OTUs clustered within ant-specific lineages, indicat-
ing a deep coevolutionary history of Cephalotes ants and their
associated microbes. Pyrosequencing recovered 445 rare OTUs
ity filtering, suggesting that this method will facilitate the discov-
ery of the host-associated rare biosphere. The next-generation
techniques explored here are a great tool to characterize many
previously underexplored communities and shed light on the di-
versity of host-associated microbiomes.
grant 1050360 to J.A.R., a Grainger Foundation grant to C.S.M., a Ne-
gaunee Foundation grant to C.S.M, and the Pritzker Laboratory for Mo-
lecular Evolution and Systematics. Alexandra Gray and NSF REU intern
doctoral fellowship from the German Academic Exchange Service
(Deutscher Akademischer Austauschdienst [DAAD]) and from the Ger-
man Academy of Sciences Leopoldina (grant LPDS 2009-29). B.E.R.R.
was supported in part by an NSF Graduate Research Fellowship.
1. Rappé MS, Giovannoni SJ. 2003. The uncultured microbial majority.
Annu. Rev. Microbiol. 57:369–394.
2. Douglas AE. 2011. Lessons from studying insect symbioses. Cell Host
3. Feldhaar H. 2011. Bacterial symbionts as mediators of ecologically im-
portant traits of insect hosts. Ecol. Entomol. 36:533–543.
4. Douglas AE. 2003. The nutritional physiology of aphids. Adv. Insect
5. Feldhaar H, Straka J, Krischke M, Berthold K, Stoll S, Mueller MJ,
Gross R. 2007. Nutritional upgrading for omnivorous carpenter ants by
the endosymbiont Blochmannia. BMC Biol. 5:48.
6. Pais R, Lohs C, Wu Y, Wang JW, Aksoy S. 2008. The obligate mutualist
7. Breznak JA, Bill WJ, Mertins JW, Coppel HC. 1973. Nitrogen fixation in
termites. Nature 244:577–579.
8. Ohkuma M. 2001. Symbiosis within the gut microbial community of
termites. RIKEN Rev. 41:69–72.
9. Ohkuma M, Noda S, Usami R, Horikoshi K, Kudo T. 1996. Diversity of
nitrogen fixation genes in the symbiotic intestinal microflora of the ter-
mite Reticulitermes speratus. Appl. Environ. Microbiol. 62:2747–2752.
10. Dobson A, Lafferty KD, Kuris AM, Hechinger RF, Jetz W. 2008.
Homage to Linnaeus: how many parasites? How many hosts? Proc. Natl.
Acad. Sci. U. S. A. 105:11482–11489.
11. May RM. 1988. How many species are there on Earth? Science 241:1441–
12. Eckburg Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M,
Kautz et al.
aem.asm.org Applied and Environmental Microbiology
Gill SR, Nelson KE, Relman DA. 2005. Diversity of the human intestinal
microbial flora. Science 308:1635–1638.
13. Hugenholz P, Goebel BM, Pace NR. 1998. Impact of culture-
sity. J. Bacteriol. 18:4765–4774.
14. Charlson ES, Chen J, Custers-Allen R, Bittinger K, Li H, Sinha R,
Hwang J, Bushman FD, Collman RG. 2010. Disordered microbial com-
munities in the upper respiratory tract of cigarette smokers. PLoS One
15. De Filippo C, Cavalieri D, Di Paola M, Ramazzotti M, Poullet JB,
Massart S, Collini S, Pieraccini G, Lionetti P. 2010. Impact of diet in
shaping gut microbiota revealed by a comparative study in children from
Europe and rural Africa. Proc. Natl. Acad. Sci. U. S. A. 107:14691–14696.
16. Wu GD, Lewis JD, Hoffmann C, Chen YY, Knight R, Bittinger K,
Hwang J, Chen J, Berkowsky R, Nessel L, Li H, Bushman FD. 2010.
Sampling and pyrosequencing methods for characterizing bacterial com-
munities in the human gut using 16S sequence tags. BMC Microbiol.
17. Jumpponen A, Jones KL. 2010. Seasonally dynamic fungal communities
in the Quercus macrocarpa phyllosphere differ between urban and nonur-
ban environments. New Phytol. 186:496–513.
18. Jumpponen A, Jones KL, Mattox D, Yaege C. 2010. Massively parallel
454-sequencing of fungal communities in Quercus spp. ectomycorrhizas
19. Andreotti R, Pérez de León AA, Dowd SE, Guerrero FD, Bendele KG,
Scoles GA. 2011. Assessment of bacterial diversity in the cattle tick Rhipi-
cephalus (Boophilus) microplus through tag-encoded pyrosequencing.
BMC Microbiol. 11:6.
20. Engelbrektson A, Kunin V, Wrighton KC, Zvenigorodsky Chen NF,
Ochman H, Hugenholtz P. 2010. Experimental factors affecting PCR-
21. Hirsch J, Strohmeier S, Pfannkuchen M, Reineke A. 2012. Assessment of
bacterial endosymbiont diversity in Otiorhynchus spp. (Coleoptera: Cur-
culionidae) larvae using a multitag pyrosequencing approach. BMC Mi-
22. Ishak HD, Plowes R, Sen R, Kellner K, Meyer E, Estrada DA, Dowd SE,
Mueller UG. 2011. Bacterial diversity in Solenopsis invicta and Solenopsis
Microb. Ecol. 61:821–831.
23. Davidson DW, Patrell-Kim L. 1996. Tropical ants: why so abundant?, p
127–140. In Gibson AC (ed), Neotropical biodiversity and conservation.
24. Hölldobler B, Wilson EO. 1990. The ants. Springer, Berlin, Germany.
25. Tobin JE. 1995. Ecology and diversity of tropical forest canopy ants, p
Press, San Diego, CA.
26. Davidson DW, Cook SC, Snelling RR, Chua TH. 2003. Explaining the
abundance of ants in lowland tropical rainforest canopies. Science 300:
27. Anderson KE, Russell JA, Moreau CS, Kautz S, Sullam KE, Hu Y,
Basinger U, Mott BM, Buck N, Wheeler DE. 2012. Highly similar
microbial communities are shared among related and trophically similar
ant species. Mol. Ecol. 21:2282–2296.
28. Eilmus S, Heil M. 2009. Bacterial associates of arboreal ants and their
putative functions in an obligate ant-plant mutualism. Appl. Environ.
29. Pinto-Tomás AA, Anderson M, Suen G, Stevenson D, Chu F, Cleland
W, Weimer P, Currie C. 2009. Symbiotic nitrogen fixation in the fungus
gardens of leaf-cutter ants. Science 326:1120–1123.
30. Russell JA, Moreau CS, Goldman-Huertas B, Fujiwara M, Lohman DJ,
Pierce NE. 2009. Bacterial gut symbionts are tightly linked with the evo-
lution of herbivory in ants. Proc. Natl. Acad. Sci. U. S. A. 106:21236–
31. Stoll S, Gadau J, Gross R, Feldhaar H. 2007. Bacterial microbiota asso-
ciated with ants of the genus Tetraponera. Biol. J. Linn. Soc. 90:399–412.
32. Kautz S, Lumbsch HT, Ward PS, Heil M. 2009. How to prevent cheating:
a digestive specialization ties mutualistic plant-ants to their ant-plant
partners. Evolution 63:839–853.
33. Kautz S, Pauls SU, Ballhorn DJ, Lumbsch HT, Heil M. 2009. Polygy-
nous supercolonies of the acacia-ant Pseudomyrmex peperi, an inferior
colony founder. Mol. Ecol. 18:5180–5194.
34. Dowd SE, Callaway TR, Wolcott RD, Sun Y, McKeehan T, Hagevoort
RG, Edrington TS. 2008. Evaluation of the bacterial diversity in the feces
of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrose-
quencing (bTEFAP). BMC Microbiol. 8:125.
35. Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Duran C,
Field M, Heled J, Kearse M, Markowitz S, Moir R, Stones-Havas S,
Sturrock S, Thierer T, Wilson A. 2011. Geneious v5.4. Biomatters Ltd.,
Auckland, New Zealand.
36. Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-
Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM. 2009.
The Ribosomal Database Project: improved alignments and new tools for
rRNA analysis. Nucleic Acids Res. 37:D141–D145.
37. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD,
Costello EK, Fierer N, Peña AG, Goodrich JK, Gordon JI, Huttley GA,
Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D,
Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters
WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R. 2010. QIIME
allows analysis of high-throughput community sequencing data. Nat.
38. Edgar RC. 2010. Search and clustering orders of magnitude faster than
BLAST. Bioinformatics 26:2460–2461.
39. Haas BJ, Gevers D, Earl AM, Feldgarden M, Ward DV, Giannoukos G,
Ciulla D, Tabbaa D, Highlander SK, Sodergren E, Methé B, DeSantis
TZ, The Human Microbiome Consortium, Petrosino JF, Knight R,
Birren BW. 2011. Chimeric 16S rRNA sequence formation and detection
in Sanger and 454-pyrosequenced PCR amplicons. Genome Res. 21:494–
40. Caporaso JG, Bittinger K, Bushman FD, DeSantis TZ, Andersen GL,
Knight R. 2010. PyNAST: a flexible tool for aligning sequences to a tem-
plate alignment. Bioinformatics 26:266–267.
41. Price MN, Dehal PS, Arkin AP. 2010. FastTree 2. Approximately maxi-
mum-likelihood trees for large alignments. PLoS One 5:e9490. doi:10
42. Lozupone C, Knight R. 2005. UniFrac: a new phylogenetic method for
comparing microbial communities. Appl. Environ. Microbiol. 71:8228–
43. Lozupone CA, Hamady M, Kelley ST, Knight R. 2007. Quantitative and
qualitative beta diversity measures lead to different insights into factors
that structure microbial communities. Appl. Environ. Microbiol. 73:
44. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB,
Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B,
Thallinger GG, Van Horn DJ, Weber CF. 2009. Introducing mothur:
open-source, platform-independent, community-supported software for
45. Stamatakis A, Hoover P, Rougemont J. 2008. A rapid bootstrap algo-
rithm for the RAxML web servers. Syst. Biol. 57:758–771.
46. Miller MA, Holder MT, Vos R, Liebowitz T, Chan L, Hoover P,
Warnow T. 2012. The CIPRES Science Gateway V. 3.1. http://www.phylo
.org/sub_sections/portal. Accessed 1 November 2011.
display of phylogenetic trees made easy. Nucleic Acids Res. 39:W475–
48. Chao A, Chazdon RL, Colwell RK, Shen T-J. 2005. A new statistical
approach for assessing similarity of species composition with incidence
and abundance data. Ecol. Lett. 8:148–159.
49. Funaro CF, Kronauer DJ, Moreau CS, Goldman-Huertas B, Pierce NE,
Russell JA. 2011. Army ants harbor a host-specific clade of Entomoplas-
matales bacteria. Appl. Environ. Microbiol. 77:346–350.
50. Bové JM. 1997. Spiroplasmas: infectious agents of plants, arthropods and
vertebrates. Wien. Klin. Wochenschr. 109:604–612.
51. Mouches C, Bové JM, Albisetti J. 1984. Pathogenicity of Spiroplasma apis
and other spiroplasmas for honey-bees in southwestern France. Ann. Mi-
crobiol. (Paris) 135A:151–155.
52. Chen XA, Li S, Aksoy S. 1999. Concordant evolution of a symbiont with
its host insect species: molecular phylogeny of genus Glossina and its bac-
teriome-associated endosymbiont, Wigglesworthia glossinidia. J. Mol.
53. Hosokawa T, Kikuchi Y, Nikoh N, Shimada M, Fukatsu T. 2006. Strict
bacteria. PLoS Biol. 4:1841–1851.
54. Login FH, Balmand S, Vallier A, Vincent-Monégat C, Vigneron A,
454 Pyrosequencing of Ant Bacteria
January 2013 Volume 79 Number 2aem.asm.org 533
Weiss-Gayet M, Rochat D, Heddi A. 2011. Antimicrobial peptides keep Download full-text
insect endosymbionts under control. Science 334:362–365.
55. Moreau CS, Bell CD, Vila R, Archibald SB, Pierce NE. 2006. Phylogeny
56. Hugenholtz P, Goebel BM. 2001. The polymerase chain reaction as a tool
to investigate microbial diversity in environmental samples. In Rochelle
PA (ed), Environmental molecular microbiology: protocols and applica-
tions. Horizon Scientific Press, Norfolk, England.
57. Roche RK, Wheeler DE. 1997. Morphological specializations of the di-
gestive tract of Zacryptocerus rohweri. J. Morphol. 234:253–262.
58. Bution ML, Caetano FH. 2008. Ileum of the Cephalotes ants: a specialized
structure to harbor symbiont microorganisms. Micron 39:897–909.
59. Bution ML, Caetano FH. 2010. Symbiotic bacteria and the structural
specializations in the ileum of Cephalotes ants. Micron 41:373–381.
60. Bution ML, Caetano FH. 2010. The midgut of Cephalotes ants (Formi-
cidae: Myrmicinae): ultrastructure of the epithelium and symbiotic bac-
teria. Micron 41:448–454.
61. Bution ML, Bresil C, Destéfano RH, Tango MF, da Silveira WD,
Paulino LC, Caetano FH, Solferini VN. 2010. Molecular and ultra-
62. Billen J, Buschinger A. 2000. Morphology and ultrastructure of a special-
Pseudomyrmecinae). Arthropod Struct. Dev. 29:259–266.
63. van Borm S, Buschinger A, Boomsma JJ, Billen J. 2002. Tetraponera ants
have gut symbionts related to nitrogen-fixing root-nodule bacteria. Proc.
R. Soc. Lond. 296:2023–2027.
64. Chin K, Janssen PH. 2002. Propionate formation by Opitutus terrae in
pure culture and in mixed culture with a hydrogenotrophic methanogen
and implications for carbon fluxes in anoxic rice paddy soil. Appl. Envi-
ron. Microbiol. 68:2089–2092.
65. Jaenike J, Unckless R, Cockburn SN, Boelio LM, Perlman SJ. 2010.
Adaptation via symbiosis: recent spread of a Drosophila defensive symbi-
ont. Science 329:212–215.
Kautz et al.
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