the gut bacterial communities associated with lab-raised and field-collected ants of camponotus...

The Gut Bacterial Communities Associated with Lab-Raisedand Field-Collected Ants of Camponotus fragilis (Formicidae:Formicinae)

Hong He • Cong Wei • Diana E. Wheeler

Received: 22 January 2014 / Accepted: 20 February 2014

� Springer Science+Business Media New York 2014

Abstract Camponotus is the second largest ant genus and

known to harbor the primary endosymbiotic bacteria of the

genus Blochmannia. However, little is known about the

effect of diet and environment changes on the gut bacterial

communities of these ants. We investigated the intestinal

bacterial communities in the lab-raised and field-collected

ants of Camponotus fragilis which is found in the south-

western United States and northern reaches of Mexico. We

determined the difference of gut bacterial composition and

distribution among the crop, midgut, and hindgut of the

two types of colonies. Number of bacterial species varied

with the methods of detection and the source of the ants.

Lab-raised ants yielded 12 and 11 species using classical

microbial culture methods and small-subunit rRNA genes

(16S rRNAs) polymerase chain reaction-restriction frag-

ment-length polymorphism analysis, respectively. Field-

collected ants yielded just 4 and 1–3 species using the same

methods. Most gut bacterial species from the lab-raised

ants were unevenly distributed among the crop, midgut,

and hindgut, and each section had its own dominant

bacterial species. Acetobacter was the prominent bacteria

group in crop, accounting for about 55 % of the crop clone

library. Blochmannia was the dominant species in midgut,

nearly reaching 90 % of the midgut clone library. Pseu-

domonas aeruginosa dominated the hindgut, accounting

for over 98 % of the hindgut clone library. P. aeruginosa

was the only species common to all three sections. A

comparison between lab-raised and field-collected ants,

and comparison with other species, shows that gut bacterial

communities vary with local environment and diet. The

bacterial species identified here were most likely com-

mensals with little effect on their hosts or mild pathogens

deleterious to colony health.

Introduction

The alimentary canal of insect is a nutrient-rich ecological

niche in which various groups of microbes survive and

multiply, and this microbial community plays a crucial role

in the nutrition, development, survival, and reproduction of

insects [2, 5, 8, 12]. Insects not only harbor primary en-

dosymbionts which supply essential amino acids and

facilitate insect growth [3, 13] but also host many other

nonessential bacteria as secondary or facultative symbi-

onts, which can confer a fitness advantage in terms of diet,

heat tolerance, or resistance to pathogens and parasitoids

[6, 22, 33, 38, 41]. It is becoming increasingly apparent

that animals generally harbor multiple microbial taxa [18,

21, 24, 31, 32].

Ants are the most successful social insects on earth, and

they have complex interactions with a variety of organisms,

including bacteria that range from mutualistic to parasitic

[9, 14, 20, 25, 34]. Camponotus, the second largest genus

of ants with nearly 2,000 known species worldwide

H. He (&)

College of Forestry, Northwest A&F University,

Yangling 712100, Shaanxi, China

e-mail: [email protected]

C. Wei

Key Laboratory of Plant Protection Resources and Pest

Management, Ministry of Education, College of Plant

Protection, Northwest A&F University,

Yangling 712100, Shaanxi, China


D. E. Wheeler

Department of Entomology, University of Arizona, Tucson,

AZ 85721, USA


123

Curr Microbiol

DOI 10.1007/s00284-014-0586-8

(antweb.org), is known to harbor the primary endosymbi-

otic bacteria of the genus Blochmannia [42]. Blochmannia

is the best-studied bacterial mutualist in Formicidae, and it

has been detected in all Camponotus species screened to

date [10, 11, 16, 35, 36]. Most research on ant-microbe

associations in Camponotus has focused on Blochmannia.

Much less emphasis has been given to other gut microor-

ganisms and their potential impact on individuals and

colonies. Feldhaar et al. [16] investigated the presence of

additional gut microbiota in C. floridanus using TGGE

along with culturing methods and found 1 species [16].

However, He et al. [19] and Li et al. [30] found a few other

bacteria in the gut of C. japonicus with the 16S rRNA-

RFLP and polymerase chain reaction (PCR)-DGGE and

culturing method, specifically Serratia symbiotica (usually

known as a secondary endosymbiont of aphids), Fructo-

bacillus fructosus, and several other bacteria [19, 30].

Here, we investigate the gut microbes of another Camp-

onotus species. Camponotus fragilis (formerly considered

as part of the species C. festinatus) is typically found in the

southwestern United States and northern reaches of Mexico

[17]. Our field investigation found its adult workers feed

nocturnally on extrafloral nectar of Staghorn Cholla,

Cylindropuntia versicolor. Herein, we define and charac-

terize the gut bacterial communities of lab-raised and field-

collected ants of C. fragilis using the classical microbial

culturing method and a culture-independent molecular

technique (16S rRNA-RFLP).

Materials and Methods

Source of Insects

C. fragilis colonies reared from founding queens collected

in Tucson, Arizona, USA in June of 2009. Colonies were

kept in plastic containers (20 9 20 9 10 cm) in climate

chambers (constant temperature of 30 �C) and were fed

twice a week with cockroaches (Nauphoeta cinerea),

honey water (1:1), and larvae of tobacco hornworms

(Manduca sexta). Both types of insects were raised in the

lab. Lab-raised ants used for analysis were taken from the

six-month-old colonies. The foraging workers of C. fragilis

in the field were collected from two sites in September of

2010, respectively. First colony was collected from Stag-

horn Chollas (C. versicolor) at night at the Saguaro

National Park East (Tucson, AZ), and second colony was

collected from Pajarita Mountains (Santa Cruz Co., AZ).

The foraging workers collected from Pajarita Mountains

were used just for 16S rRNA-RFLP analysis, because only

a small number of workers were collected. The workers

were transferred alive to an insulated cooler after being

collected and brought back to the lab for dissection.

Dissection of Ant Guts

Each worker of C. fragilis was first narcotized by placed on

ice for a few minutes, then externally sterilized with 70 %

ethanol and rinsed twice with sterilized water. The whole

gut was dissected out of the ant abdomen with sterilized

fine-tip forceps and washed twice with sterile 0.9 % NaCl

solution as soon as exposed, and then the crop, midgut, and

hindgut were carefully separated and placed in different

microcentrifuge tubes (1.5 ml) for DNA extraction and

bacteria culture.

Culturing Bacteria

Ten workers of the lab-raised ants and ten workers col-

lected from the Saguaro National Park East were dissected

for bacterial culturing, respectively. Every dissected crop,

midgut, and hindgut was transferred separately to an

individual microcentrifuge tube containing 50 ll 10 mM

PBS. Each sample was grounded and homogenized with a

sterile pellet pestle and then spread on plates of TSA

(Tryptic soy agar) and LB (Luria–Bertani agar) medium.

Plates were incubated in a growth chamber at 30 �C. After

2 days culturing, the different bacterial colonies were

chosen based on morphology (shape, elevation, surface,

size, opacity, pigmentation, etc.) for PCR amplification

using 16S rRNA primers (10F and 1507R), and subse-

quently sequenced for species identification. At least two

representatives of each kind of cultured bacterial colony

were sequenced.

Genomic DNA Extraction, Amplification,

and Restriction Fragment-Length Polymorphism

Analysis

DNA Extraction

Separately pooled crops, midguts, and hindguts of 10–20

workers of the lab-raised and field-collected colonies were

used for genome DNA extraction. Genomic DNA was

extracted using DNeasy� Blood & Tissue Kit (QIAGEN

Inc.) according to the manufacturer’s instructions.

PCR Amplification

The genome DNA samples were amplified for the 16S

rRNA gene with the universal primers 10F (50-AGTTT

GATCATGGCTCAGATTG-30) and 1507R (50-TACCTT

GTTACGACTTCACCCCAG-30). PCR was performed

using a PTC-200 Peltier Thermal Cycler (MJ Research).

The amplification was performed in 25 ll reactions system,

containing: 5 ll 59 TagMaster Buffer, 2.5 ll 109 Tag-

Master Buffer, 0.5 ll 10 mM dNTPMix, 0.5 ll of each

H. He et al.: The Gut Bacteria Associated with Camponotus fragilis

123

primer (10 mM/l), 0.25 ll Taq DNA polymerase (Master-

Taq Kit, 5 Prime Company), and 2 ll Temple DNA. The

amplification program consisted of initial denaturation at

94 �C for 3 min, followed by 40 cycles of denaturation

(1 min at 94 �C), annealing (1 min at 55 �C), and exten-

sion (2 min at 72 �C), followed by a final 10 min extension

at 72 �C.

Clone Libraries and RFLP Analysis

Genome DNA extracted from the sample of hindgut of

workers collected from Pajarita Mountains was very weak,

possibly due to low concentration of bacterial DNA in the

extraction, so we constructed only the other eight clone

libraries. Cloning was done using Invitrogen TOPO TA

cloning kit (Invitrogen Corporation, California, USA)

according to the manufacturer’s instructions, and intro-

duced into Escherichia coli cells for transformation. A

dilution series of transformed E. coli cells containing the

M13 plasmid with ligated 16S rRNA inserts were trans-

ferred to plates of Luria–Bertani agar amended with

50 mg/l kanamycin and 40 ll of 40 mg/ml X-gal. The

plates were incubated at 37 �C for 48 h. One hundred

forty-four of the white clones were picked randomly using

a toothpick into 180 ll liquid LB media in a 96-well plate

and cultured at 37 �C for 48 h, then clones lysates were

diluted 10-fold with sterile Tris-buffer (10 mM, pH 8.0)

and used as DNA templates for PCR amplification of the

insert with M13 vector primers to check if they were

transformed successfully.

PCR products from positive clones using M13 vector

primer were digested with EcoRI and HaeIII restriction

endonucleases (Fermentas Life Science, USA) according to

the manufacturer’s specifications. The restriction PCR

fragments were separated by 2 % agarose gel electropho-

resis in 19 TE buffer at 4 V/cm. The gels were stained

with ethidium bromide and visualized under UV light. The

restriction profiles of isolates were compared and visually

grouped. One to three representative clones for each unique

RFLP profiles were sequenced.

Nucleotide Sequencing and Phylogenetic Analysis

All sequencing was conducted at the University of Arizona

DNA Sequencing Facility. All clones and isolates have

been sequenced in both forward and reverse directions

using 16S rRNA primers (10F and 1507R), and the

sequences were manually corrected and spliced using

Chromas Lite 2.01 (Technelysium Pty Ltd, Australia). In

order to assign each sequence to the correct taxonomical

affiliation, sequences were blasted both in GenBank and

Ribosomal Database Project (http://rdp.cme.msu.edu/) to

find their best matched relatives. All sequences we

obtained and their best matched sequences were aligned

using Clustal X2.1 [27]. Aligned sequences were added to

construct Maximum Likelihood tree in MEGA 5 [37].

Taxonomic descriptions were determined based on the

position of each aligned sequence in the phylogenetic tree,

and 97 % sequence identity was used as a decision crite-

rion of bacterial species. Diversity indices were calculated

using software SPADE (http://chao.stat.nthu.edu.tw/blog/

software-download/spade/), and rarefaction curves were

conducted using Analytic Rarefaction 1.3 (http://strata.uga.

edu/software/anRareReasme.html) to gauge the adequacy

of sampling.

The 16S rRNA gene sequences of cultured bacteria and

clones from lab-raised colony are available under accession

numbers JN846883 to JN846917 and JN846918 to

JN846929, respectively. The 16S rRNA gene sequences of

cultured bacteria and clones from colony of the Saguaro

National Park East are available under accession numbers

JN904079 to JN904082 and JN904008 to JN904032,

respectively. The 16S rRNA gene sequences of clones from

colony of Pajarita Mountains are available under accession

numbers JN904033 to JN904059.

Results

The Cultured Bacteria from the Guts of Lab-Raised

Ants of C. fragilis

The 12 cultured bacterial strains isolated from the gut of lab-

raised ants of C. fragilis are presented as in Table 1. All of

them belong to the Firmicutes and the Proteobacteria, and

represent seven genera: Pseudomonas, Stenotrophomonas,

Enhydrobacter, Roseomonas, Caulobacterales, Bacillus,

and Staphylococcus. The sequences of all bacterial species

were at least 99 % identity to known sequences in the data-

base of GenBank. The location of the bacterial species across

the three gut sections (crop, midgut, and hindgut) showed

some differences. Pseudomonas aeruginosa, Stenotropho-

monas sp., and Staphylococcus epidermidis are found in all

sections, Pseudomonas boreopolis, Staphylococcus hominis,

and Staphylococcus capitisi were isolated only from the

crop, Enhydrobacter aerosaccus, Enhydrobacter sp., and

Staphylococcus sp. only from the midgut, and Roseomonas

genomospecies and Caulobacterales sp. only from the

hindgut.

A Maximum Likelihood phylogenetic tree was con-

structed using the sequences of the 12 bacteria strains as

well as their best match sequences from GenBank (Fig. 1).

The strains clustered in three clear groups, the Gamma

Proteobacteria, Alpha Proteobacteria, and Bacillus which

are supported by bootstrap values of 99, 100 and 86 %,

respectively.


123

http://rdp.cme.msu.edu/

http://chao.stat.nthu.edu.tw/blog/software-download/spade/

http://chao.stat.nthu.edu.tw/blog/software-download/spade/

http://strata.uga.edu/software/anRareReasme.html

http://strata.uga.edu/software/anRareReasme.html

The 16S rRNA-RFLP Analysis for the Gut Bacteria

of Lab-Raised Ants of C. fragilis

PCR–RFLP analysis for the bacterial composition and

distribution in the guts of the lab-raised ants found 30

distinct restriction profiles. One to three clones of each

profile were selected, and total 63 clones were sequenced,

yielding 11 bacterial species (Table 2). These identified

bacteria are all Proteobacteria and represent eight genera:

Acetobacter, Gluconacetobacter, Pseudomonas, Bloch-

mannia, Enterobacter, Proteus, Escherichia, and Steno-

trophomonas. In addition, one uncultured bacterial species

could not be identified by sequence homology.

In addition, the bacterial composition and distribution of

the bacteria showed clear differences across the three gut

sections. The crop had a rich bacterial community of seven

species. Acetobacter was the most abundant group in the

crop clone library [accounting for about 55.2 % (48/87)],

and P. aeruginosa was second [accounting for 31.0 %

(27/87)]. Blochmannia festinatus was by far the most

abundant bacteria in the midgut [accounting for about

89.8 % (97/108)]. The hindgut had three bacterial species,

and P. aeruginosa, the only species found in all the three

gut sections, was the dominant species in the hindgut clone

library [accounting for 97.7 % (85/87)].

Diversity analysis of the three gut sections showed that

crop has significantly higher Shannon index and lower

Simpson index than midgut and hindgut (95 % confi-

dence) (Table 3). Coverage C of the three clone libraries

from lab-raised workers are more than 0.9 (Table 3).

Moreover, the rarefaction curves for these three libraries

also reached plateaus at 3 % difference between sequen-

ces (95 % confidence) (Fig. 2). These suggest that the

number of clones sampled was sufficient to provide an

accurate estimation of gut bacterial diversity in lab-raised

workers.

A Maximum Likelihood phylogenetic tree was con-

structed using the 35 sequences of clones isolated from the

guts of lab-raised ants of C. fragilis and their best matched

sequences downloaded from GenBank (Fig. 3). All the 35

clones were clustered into four major groups: Acetobact-

eraceae (9 clones), Xanthomonadaceae (1 clone), Pseudo-

monadaceae (12 clones), and Enterobacteriaceae (13

clones).

Table 1 The composition and distribution of cultured bacterial strains isolated from the guts of lab-raised ants of C. fragilis

Identification from closest match in GenBank

(Accession No. and classification status)

% Identity to

closest match

The representative strains

and their GenBank accession No.

Distribution of strains

Crop Midgut Hindgut

Pseudomonas aeruginosa (HQ844513.1)

(Proteobacteria)

100 Strain HH07Hmix2a (JN846924) ? ? ?

Stenotrophomonas sp. (GQ381282.1)

(Proteobacteria)

99 Strain 1-m1-1 (JN846918) ? ? ?

Pseudomonas boreopolis (AJ864722.1)

(Proteobacteria)

99 Strain 12bC1-4 (JN846925) ? / /

Enhydrobacter aerosaccus (FN386747.1)

(Proteobacteria)

99 Strain 4-m12-3 (JN846927) / ? /

Enhydrobacter sp. (EU305591.1) 98 Strain 14b-m12-2 (JN846928) / ? /

Roseomonas genomospecies 5 (AF533356.1)

(Proteobacteria)

100 Strain HH10H133 (JN846926) / / ?

Uncultured Caulobacterales (HM799001.1)

(Proteobacteria)

99 Strain 8-H1-4 (JN846929) / / ?

Bacillus sp. (EU584551.1)

(Firmicutes)

100 Strain EE10m15 (JN846923) ? ? /

Staphylococcus epidermidis (JF769744.1)

(Firmicutes)

100 Strain GG10H17 (JN846922) ? ? ?

S. hominis (HQ908730.1) 99 Strain 3-m2-1 (JN846921) ? / /

S. capitis (NR_027519.1) 99 Strain EE09C102 (JN846919) ? / /

S. sp. (HQ331102.1) 100 Strain DD10C161 (JN846920) / ? /

Number of bacterial species 7 7 5

?, present in the sample; /, undetected in the sample


123

Fig. 1 The ML phylogenetic tree based on 16S rRNA gene

sequences of cultured bacteria belonging to the gut bacteria of lab-

raised colony of C. fragilis, including selected database sequences.

The tree was generated using the Maximum Likely with 1,000

bootstrap method and Tamura-Nei model in MEGA5 software.

Isolated bacteria in our research are listed in boldface type followed

by GenBank accession numbers. The scale bar represents 0.05

substitutions per nucleotide position


123

The Cultured Gut Bacteria and 16S rRNA-RFLP

Analysis for the Field-Collected Ants of C. fragilis

Just four gut bacterial strains were isolated from the

workers collected from the Saguaro National Park East

(Table 4). They all belong to the Firmicutes and include

two genera (Bacillus and Staphylococcus) which were also

found in the guts of lab-raised ants. In addition, only four

different RFLP profiles were found using 16S rRNA-RFLP

method. In order to make sure all gut bacteria in the field-

Table 2 The bacterial composition and distribution in the guts of lab-raised ants of C. fragilis based on the 16S rRNA-RFLP analysis



% Identity to

closest match

The representative clones and their

GenBank accession No.

Distribution of clones

Crop Midgut Hindgut

Acetobacter aceti (JF718428.1)

(Proteobacteria, Acetobacteraceae)

92 AA019A1 (JN846883) 40 / /

92 FF039H12 (JN846884)

91 HH129A3 (JN846885)

91 CC119D3 (JN846886)

92 HH019H7 (JN846887) 4 / /

91 EE039E12 (JN846889) 1 / /

91 DD019H3 (JN846890) 3 / /

Gluconacetobacter sp. (AB778532.1)

(Proteobacteria, Acetobacteraceae)

90 DD067E5 (JN846888) / / 1

89 AA039D11 (JN846914) 4 / /

Pseudomonas aeruginosa (JN412064.1)

(Proteobacteria, Pseudomonadaceae)

99 GG029H10 (JN846905) 1 / /

99 BB039G11 (JN846916) 1 / /

99 CC029D9 (JN846896) 25 / /

99 DD039D12 (JN846899)

99 BB048G2 (JN846897) / 1 /

99 CC058B10 (JN846893) / 5 /

100 FF117H14 (JN846895) / / 84

99 BB067A2 (JN846891)

99 DD127F14 (JN846892)

99 EE117A9 (JN846894)

99 DD117A4 (JN846900)

97 CC067D5 (JN846903) / / 1

Pseudomonas sp. (JX122841.1) 96 EE128F13 (JN846902) / 1 /

Pseudomonas sp. (FJ493141.1) 94 FF019B7 (JN846901) 3 / /

Blochmannia festinatus (AY196851.1)

(Proteobacteria, Enterobacteriaceae)

98 FF048B8 (JN846909) / 1 /

98 AA058G8 (JN846910) / 1 /

98 HH118G14 (JN846907) / 93 /

98 BB058E9 (JN846906)

98 FF058E13 (JN846911) / 1 /

94 AA048C1 (JN846917) / 1 /

Enterobacter gergoviae (NR_024641.1)


99 BB029H8 (JN846913) 2 / /

Proteus vulgaris (JN092605.1)


99 FF029G10 (JN846912) 2 / /

Escherichia sp. (JN626182.1)


94 DD048H6 (JN846908) / 2 /

Stenotrophomonas sp. (GQ381282.1)

(Proteobacteria, Xanthomonadaceae)

100 AA029D8 (JN846915) 1 / /

Uncultured bacterium (FM995936.1) 93 HH048F8 (JN846898) / 1 /

94 CC048B6 (JN846904) / 1 1

The total number of clones in each clone library 87 108 87

/, undetected in the sample


123

collected ants could be detected, clones of the four profiles

and 27 other randomly-chosen clones were sequenced and

yielded 31 different sequences. The blast result showed that

all of them were B. festinatus. Similarly, most clones iso-

lated from the workers collected from Pajarita Mountains

were Blochmannia festinates, except two clones found in

the crop that were identified as Herbaspirillum sp. and

Pseudoxanthomonas sp., respectively (Table 5).

In contrast to the lab-raised ants, B. festinatus of the

field-collected colony was distributed not only in the

midgut, but also in the crop and hindgut.

Discussion

DNA-based analysis identified exclusively Proteobacteria

in the guts of both the lab-raised and field-collected ants of

C. fragilis. Some bacteria belonging to Firmicutes were

also isolated from media, but they were not detected by

PCR–RFLP (Tables 1, 2, 4, 5). Identification of Firmicutes

by culture but not by DNA analysis is not unusual and has

been observed in several other systems, including Tetrap-

onera ants [39], red imported fire ants [29], gypsy moths

[4], and southern pine beetles [40]. This phenomenon

likely reflects the low abundance of Firmicutes in the

community but could also be due to biases introduced by

DNA extraction, PCR amplification, cloning, or culturing

methods.

Distribution and Condition of Blochmannia

Not surprisingly, a substantial amount of Blochmannia was

detected in both lab-reared and field-collected ants.

Blochmannia is a mutualist of the genus Camponotus and

has been found in all species tested. These bacteria are

generally described as living in specialized bacteriocyte

cells that are intercalated among midgut cells [36]. In the

case of field-collected ants, however, Blochmannia were

also found in the crop and hindgut, suggesting that they

may also invade gut tissue outside the midgut.

Bacterial Diversity

Gut bacterial diversity overall was notably greater in lab-

raised ants (23 species of bacteria) compared to field-col-

lected ones (7 species of bacteria). Only bacteria of

Bacillus, Staphylococcus, and Blochmannia were shared by

the two groups. A low number of bacterial species in field-

collected ants was reported previously in Camponotus

japonicus, in which only three species were detected with

both 16S rRNA-RFLP method and the culturing method

[19, 30], and one species of Pseudomonas was found again

with DGGE method [30]. The comparative distribution in

C. fragilis suggests that there is no core gut microbiota,

such as that identified in honey bees [32]. The presence of a

core microbiota would suggest that the microbes may have

a beneficial function. It seems most likely that the bacterial

species identified here were commensals with little effect

on their hosts or mild pathogens deleterious to colony

health.

Two obvious sources of the bacteria are surfaces in the

ants’ surrounding environment and their food. Lab and

field environments are very different and would have dif-

ferent microbes available. Lab nests are soil free and

consist of plastic boxes and glass tubes, while field colonies

nest in natural substrates of wood and/or soil. In the lab,

ants were provided a diet of dilute honey and corpses of 2

types of lab-reared insects. Field colonies are likely to have

a more diverse diet, one typical of Camponotus ants in

general that includes insect honeydew and many types of

protein from scavenging or small prey [17]. In C. japoni-

cus, two of all species of bacteria identified could easily

have been obtained in their diet [19, 30]. Specifically,

Serratia symbiotica, a mutualist bacterium of aphids, could

have been obtained by taking aphids as prey or by taking

aphids’ honeydew, and F. fructosus, which grows well on

D-fructose and D-glucose solutions from flowers and fruit

[15], maybe been derived from plant nectar, generally an

important part of the diet of Camponotus ants [17]. The

Table 3 Diversity indexes of the 16S rRNA gene clone libraries

constructed from different gut sections of lab-raised ants (95 %

confidence interval)

Gut

section

Number of

clones (N)

Species

richness

Shannon

index

Simpson

index

Coverage

C

Crop 87 7 1.174 0.398 0.989

Midgut 108 5 0.809 0.448 0.991

Hindgut 87 3 0.125 0.954 0.977

Fig. 2 Rarefaction analyses of 16S rRNA gene libraries constructed

from crop, midgut and hindgut of lab-raised colony of C. fragilis


123


123

importance of both nest site and diet to bacterial compo-

sition has also been shown for gut bacteria of the fire ant

Solenopsis invicta [28, 29] and some other ant species from

different trophic niches [1].

Bacterial Distribution and Importance of the Crop

The gut bacterial composition and richness showed sig-

nificant differences in the crop, midgut, and hindgut.

Acetobacter and P. aeruginosa dominated the crop,

Blochmannia the midgut, and P. aeruginosa the hindgut.

Acetobacter was found exclusively in the crop of the lab-

raised ants. The genus is characterized by the ability to

convert ethanol to acetic acid in the presence of oxygen [7],

and it has not been reported previously in ants. Some

Acetobacter species have symbiotic relationships with

insects, e.g., honey bees, the endoparasitoid wasp Asobara

tabida, the fruit fly Drosophila melanogaster, the olive

fruit fly Bactrocera oleae, and the mosquito Anopheles

stephensi [7, 26]. In the case of C. fragilis, however,

Acetobacter species may have been a contaminant of the

honey water that was a mainstay of their lab diet.

Pseudomonas aeruginosa was detected in all three sec-

tions of the gut and was especially common in the crop and

hindgut. P. aeruginosa is a common pathogenic bacterium

of insects and is known to colonize many natural and

artificial environments [23]. Behar [2] showed that exper-

imental inoculations with high levels of P. aeruginosa can

b Fig. 3 The ML phylogenetic tree based on 16S rRNA gene

sequences of representative clones belonging to the gut bacteria of

lab-raised colony of C. fragilis, including selected database

sequences. The tree was generated using the Maximum Likely

method with 1,000 bootstrap method and General Time Reversible

model in MEGA5 software. Bootstrap values above 50 % are

indicated. The scale bar represents 0.05 substitutions per nucleotide

position. Representative clones are listed in boldface type followed by

GenBank accession numbers, and clones with asterisk were from the

crop, clones with check mark were from the midgut, clones with black

circles were from the hindgut

Table 4 The composition and distribution of cultured bacterial strains isolated from the guts of field-collected ants of C. fragilis



% Identity to

closest match

GenBank accession

No. of strains

Distribution of strains

Crop Midgut Hindgut

Bacillus sp. (JN208185.1)

(Firmicutes)

100 JN904079 / / ?

Staphylococcus capitis (JN644490.1)

(Firmicutes)

100 JN904080 ? / ?

Staphylococcus sp. (JF923461.1)

(Firmicutes)

100 JN904081 ? / /

Staphylococcus sp. (JF899874.1)

(Firmicutes)

100 JN904082 / ? /

Number of bacterial species 2 1 2

?, present in the sample; /, undetected in the sample

Table 5 The bacterial composition and distribution in the guts of field-collected ants of C. fragilis based on the 16S rRNA-RFLP analysis

Source of

workers



% Identity to

closest match

GenBank Accession No. of

the representative clones

Distribution of clones

Crop Midgut Hindgut

First field-

collected

ants

Blochmannia festinatus

(AY196851.1)

(Proteobacteria)

98 JN904009–JN904031 96 95 49

The total number of clones in each clone library 96 95 49

Second field-

collected

ants

Herbaspirillum sp. (FN386764.1)

(Proteobacteria)

99 JN904033–JN904059 1 / –

Pseudoxanthomonas sp. (GU908487.2)

(Proteobacteria)

98 1 / –

Blochmannia festinatus 98 135 152 –

The total number of clones in each clone library 137 152

/, undetected in the sample; –, no detected


123

reduce the longevity of the Mediterranean fruit fly Ceratitis

capitata. The high levels of P. aeruginosa in ants from the

lab-raised colony and the absence of it in field-collected

ants suggest that the infection is a laboratory artifact. It

may be harmful to laboratory colonies.

The crop is a thin-walled sac in which ingested food can

be stored. Its anterior position makes it the primary col-

lection site for ingested microbes and enables its contents

to be shared by regurgitation with nestmates, including

larvae and the queen [20]. We have shown that the crop

contains the greatest bacterial diversity of the 3 gut sec-

tions. More work is needed to investigate the bacterial

diversity in ants’ crop, in particular to clarify how they

contribute to colony health.

Acknowledgments We are grateful to N. Buck (Department of

Entomology, University of Arizona, Tucson, USA) for his helpful

technical support, and to P. Rodrigues (Department of Entomology,

University of Arizona, Tucson, USA) for helping with the collection

of ants in the field. H. He was supported, in part, by the National

Natural Science Foundation of China (No. 31070342) and Special

Foundation for Key Project of Northwest A&F University

(PY200903) to pursue research related to this paper at the University

of Arizona. C. Wei was supported, in part, by the Program for

Changjiang Scholars and Innovative Research Team in the Univer-

sities of China (IRT1035) to pursue research related to this paper at

the University of Arizona. D. Wheeler was supported by NSF Grant

0604067.

References

1. 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

2. Behar A, Yuval B, Jurkevitch E (2008) Gut bacterial communi-

ties in the Mediterranean fruit fly (Ceratitis capitata) and their

impact on host longevity. J Insect Physiol 54:1377–1383

3. Boursaux-Eude C, Gross R (2000) New insights into symbiotic

associations between ants and bacteria. Res Microbiol 151:513–519

4. Broderick NA, Raffa KF, Goodman RM, Handelsman J (2004)

Census of the bacterial community of the gypsy moth larval

midgut by using culturing and culture-independent methods.

Appl Environ Microbiol 70:293–300

5. Buchner P (1965) Endosymbiosis of animals with plant micro-

organisms. Wiley, New York

6. Burke GR, Normark BB, Favret C, Moran NA (2009) Evolution

and diversity of facultative symbionts from the aphid subfamily

Lachninae. Appl Environ Microbiol 75:5328–5335

7. Crotti E, Rizzi A, Chouaia B, Ricci I, Favia G, Alma A, Sacchi L,

Bourtzis K, Mandrioli M, Cherif A, Bandi C, Daffonchio D

(2010) Acetic acid bacteria, newly emerging symbionts of

insects. Appl Environ Microbiol 76:6963–6970

8. Dale C, Moran NA (2006) Molecular interactions between bac-

terial symbionts and their hosts. Cell 126:453–465

9. Davidson DW, Cook SC, Snelling RR, Chua TH (2003)

Explaining the abundance of ants in lowland tropical rain forest

canopies. Science 300:969–972

10. Degnan PH, Lazarus AB, Brock CD, Wernegreen JJ (2004) Host-

symbiont stability and fast evolutionary rates in an ant-bacterium

association: cospeciation of Camponotus species and their en-

dosymbionts, Candidatus Blochmannia. Syst Biol 53:95–110

11. de Souza DJ, Bezier A, Depoix D, Drezen J-M, Lenoir A (2009)

Blochmannia endosymbionts improve colony growth and

immune defence in the ant Camponotus fellah. BMC Microbiol

9:29

12. Dillon RJ, Dillon VM (2004) The gut bacteria of insects: non-

pathogenic interactions. Annu Rev Entomol 49:71–92

13. Douglas AE (1998) Nutritional interactions in insect-microbial

symbioses: aphids and their symbiotic bacteria Buchnera. Annu

Rev Entomol 43:17–37

14. Eilmus S, Heil M (2009) Bacterial associates of arboreal ants and

their putative functions in an obligate ant–plant mutualism. Appl

Environ Microbiol 75:4324–4332

15. Endo A, Futagawa-Endo Y, Dicks LMT (2009) Isolation and

characterization of fructophilic lactic acid bacteria from fructose-

rich niches. Syst Appl Microbiol 32:593–600

16. Feldhaar H, Straka J, Krischke M, Berthold K, Stoll S, Mueller

MJ, Gross R (2007) Nutritional upgrading for omnivorous car-

penter ants by the endosymbiont Blochmannia. BMC Biol 5:48

17. Hansen LD, Klotz JH (2005) Carpenter ants of the United States

and Canada. Cornell University Press, New York, pp 144–146

18. Haynes S, Darby AC, Daniell TJ, Webster G, van Veen FJF,

Godfray HCJ, Prosser JI, Douglas AE (2003) Diversity of bac-

teria associated with natural aphid populations. Appl Environ

Microbiol 69:7216–7223

19. He H, Chen YY, Zhang YL, Wei C (2011) Bacteria associated

with gut lumen of Camponotus japonicus Mayr. Environ Entomol

40:1405–1409

20. Holldobler B, Wilson EO (1990) The ants. Belknap Press, Har-

vard University Press, Cambridge

21. Hooper LV, Gordon JI (2001) Commensal host-bacterial rela-

tionships in the gut. Science 292:1115–1118

22. Jaenike J, Unckless R, Cockburn SN, Boelio LM, Perlman SJ

(2010) Adaptation via symbiosis: recent spread of a Drosophila

defensive symbiont. Science 329:212–215

23. Jander G, Rahme LG, Ausubel FM (2000) Positive correlation

between virulence of Pseudomonas aeruginosa mutants in mice

and insects. J Bacteriol 182:3843–3845

24. Jeyaprakash A, Hoy MA, Allsopp MH (2003) Bacterial diversity

in worker adults of Apis mellifera capensis and Apis mellifera

scutellata (Insecta: Hymenoptera) assessed using 16S rRNA

sequences. J Invertebr Pathol 84:96–103

25. Kautz S, Rubin BER, Russell JA, Moreau CS (2013) Surveying

the microbiome of ants: comparing 454 pyrosequencing with

traditional methods to uncover bacterial diversity. Appl Environ


26. Kounatidis I, Crotti E, Sapountzis P, Sacchi L, Rizzi A, Chouasia

B, Bandi C, Alma A, Daffonchio D, Mavragani-Tsipidou P,

Bourtzis K (2009) Acetobacter tropicalis is a major symbiont of

the olive fruit fly (Bactrocera oleae). Appl Environ Microbiol

75:3281–3288

27. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan

PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R,

Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and

clustal X version 2.0. Bioinformatics 23:2947–2948

28. Lee AH, Hooper-Bui L (2012) The origin and in situ identifica-

tion of uncultured gut bacteria in fourth-instar larvae of the red

imported fire ant, Solenopsis invicta (Hymenoptera: Formicidae).

Sociobiology 59:27–48

29. Lee AH, Husseneder C, Hooper-Bui L (2008) Culture-indepen-

dent identification of gut bacteria in fourth-instar red imported

fire ant, Solenopsis invicta Buren, larvae. J Invert Path 98:20–33

30. Li X-P, Nan X-N, Wei C, He H (2012) The gut bacteria asso-

ciated with Camponotus japonicas Mayr with culture-dependent

and DGGE methods. Curr Microbiol 65:610–616


123

31. Lilburn TG, Schmidt TM, Breznak JA (1999) Phylogenetic

diversity of termite gut spirochaetes. Environ Microbiol 1:331–345

32. Martinson VG, Danforth BN, Minckley RL, Rueppell O, Tingek

S, Moran NA (2011) A simple and distinctive microbiota asso-

ciated with honey bees and bumble bees. Mol Ecol 20:619–628

33. Rio RVM, Wu YN, Filardo G, Aksoy S (2006) Dynamics of

multiple symbiont density regulation during host development:

tsetse fly and its microbial flora. Proc R Soc B 273:805–814

34. Russell JA, Moreau CS, Goldman-Huertas B, Fujiwara M, Loh-

man DJ, Pierce NE (2009) Bacterial gut symbionts are tightly

linked with the evolution of herbivory in ants. Proc Natl Acad Sci

106:21236–21241

35. Sauer C, Dudaczek D, Holldobler B, Gross R (2002) Tissue

localization of the endosymbiotic bacterium ‘‘Candidatus

Blochmannia floridanus’’ in adults and larvae of the carpenter ant

Camponotus floridanus. Appl Environ Microbiol 68:4187–4193

36. Schroder D, Deppisch H, Obermayer M, Krohne G, Stackebrandt

E, Holldobler B, Goebel W, Gross R (1996) Intracellular endo-

symbiotic bacteria of Camponotus species (carpenter ants): sys-

tematic, evolution and ultrastructural characterization. Mol


37. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S

(2011) MEGA5: molecular evolutionary genetics analysis using

maximum likelihood, evolutionary distance, and maximum par-

simony methods. Mol Biol Evol. doi:10.1093/molbev/msr121

38. Thao ML, Clark MA, Baumann L, Brennan EB, Moran NA,

Baumann P (2000) Secondary endosymbionts of psyllids have

been acquired multiple times. Curr Microbiol 41:300–304

39. van Borm S, Buschinger A, Boomsma JJ, Billen J (2002) Tet-

raponera ants have gut symbionts related to nitrogen-fixing root-

nodule bacteria. Proc R Soc Lond B 269:2023–2027

40. Vasanthakumar A, Delalibera I, Handelsman JJ, Klepzig KD,

Schloss PD, Raffa KF (2006) Characterization of gut-associated

bacteria in larvae and adults of the southern pine beetle, Dend-

roctonus frontalis Zimmermann. Environ Entomol 35:1710–1717

41. Werren JH, Windsor DM (2000) Wolbachia infection frequencies

in insects: evidence of a global equilibrium? Proc R Soc Lond B

267:1277–1285

42. Zientz E, Feldhaar H, Stoll S, Gross R (2005) Insights into the

microbial world associated with ants. Arch Microbiol

184:199–206


123

http://dx.doi.org/10.1093/molbev/msr121

the gut bacterial communities associated with lab-raised and field-collected ants of camponotus...

Documents