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Dynamic Wolbachia prevalence in Acromyrmex leaf-cutting ants:potential for a nutritional symbiosis
S. B. ANDERSEN*, M. BOYE� , D. R. NASH* & J. J. BOOMSMA*
*Centre for Social Evolution, Department of Biology, University of Copenhagen, Copenhagen, Denmark
�National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark
Introduction
Symbiotic interactions span the entire spectrum between
mutualism and parasitism, because the respective costs
and benefits for hosts and symbionts ultimately deter-
mine whether interactions become ‘win–win’ or ‘win–
lose’ (Bull, 1994; Herre et al., 1999). Vertical transmission
typically aligns the reproductive interests of host and
symbiont but this transmission mode is neither necessary
nor sufficient to keep a symbiotic interaction mutualistic.
For example, Wolbachia is usually a vertically transmitted
parasite with relatively high virulence (Werren et al.,
2008), whereas Termitomyces, the garden symbiont of
fungus-growing termites, is a horizontally transmitted
mutualist with an unusually stable commitment to its
hosts (Aanen et al., 2009). In addition, even vertically
transmitted mutualists are not permanently evolution-
arily stable, as some are known to have been lost over
time (Sachs & Simms, 2006).
Symbioses are increasingly known to involve more
than two partners (e.g. Palmer et al., 2010). This further
complicates the dynamics and selective forces that shape
the ultimate nature of these interactions, because coop-
eration and conflict in such multiple partnerships depend
on the interactions between symbionts in addition to
those between host and symbionts (Vautrin & Vavre,
2009; Telfer et al., 2010). Such interactions can have
either positive or negative effects on the host, but
typically require that symbionts have spatially and
temporally overlapping niches within hosts.
Communities of bacterial symbionts with complemen-
taryrolesmayproducestablemutualismswhenconfinedto
specifichostorgansortissues.Examplesarethegutpouches
of Tetraponera ants that contain multiple highly divergent
species of nitrogen-fixing bacteria (Van Borm et al., 2002)
and the bacteriomes of hemipteran sharpshooters (Homa-
lodisca coagulata) that contain two bacterial species supply-
ing amino acids and vitamins to the host (Wu et al., 2006).
However, when it comes to genetic variation among
symbionts with similar roles, diversity may be costly for
Correspondence: Sandra B. Andersen, Centre for Social Evolution,
Department of Biology, University of Copenhagen, Universitetsparken
15, DK-2100 Copenhagen, Denmark.
Tel.: +45 26209197; fax: +45 35 32 12 50; e-mail: [email protected]
ª 2 0 1 2 T H E A U T H O R S . J . E V O L . B I O L .
J O U R N A L O F E V O L U T I O N A R Y B I O L O G Y ª 2 0 1 2 E U R O P E A N S O C I E T Y F O R E V O L U T I O N A R Y B I O L O G Y 1
Keywords:
Acromyrmex ants;
fluorescence in situ hybridization;
gut bacteria;
symbiosis;
Wolbachia.
Abstract
Wolbachia are renowned as reproductive parasites, but their phenotypic effects
in eusocial insects are not well understood. We used a combination of qrt-PCR,
fluorescence in situ hybridization and laser scanning confocal microscopy to
evaluate the dynamics of Wolbachia infections in the leaf-cutting ant
Acromyrmex octospinosus across developmental stages of sterile workers. We
confirm that workers are infected with one or two widespread wsp genotypes
of Wolbachia, show that colony prevalence is always 100% and characterize
two rare recombinant genotypes. One dominant genotype is always present
and most abundant, whereas another only proliferates in adult workers of
some colonies and is barely detectable in larvae and pupae. An explanation
may be that Wolbachia genotypes compete for host resources in immature
stages while adult tissues provide substantially more niche space. Tissue-
specific prevalence of the two genotypes differs, with the rarer genotype being
over-represented in the adult foregut and thorax muscles. Both genotypes
occur extracellularly in the foregut, suggesting an unknown mutualistic
function in worker ant nutrition. Both genotypes are also abundant in the
faecal fluid of the ants, suggesting that they may have extended functional
phenotypes in the fungus garden that the ants manure with their own faeces.
doi: 10.1111/j.1420-9101.2012.02521.x
hosts, as within-host competition often selects for more
virulent parasites (Frank, 1996; Davies et al., 2002) or less
cooperative mutualists (Herre et al., 1999; Poulsen &
Boomsma, 2005).
Wolbachia are a-Proteobacteria that are intracellular
symbionts in many insects, mites and some nematodes
and crustaceans. They often affect host fitness as reproduc-
tive parasites by causing cytoplasmic incompatibility, as in
Drosophilaflies(Bourtziset al.,1996),Ephestiamoths(Lewis
et al., 2011) and Nasonia wasps (Tram & Sullivan, 2002).
Other Wolbachia cause host parthenogenesis as in Bryobia
mites (Weeks & Breeuwer, 2001), male-killing as in
ladybirdsandbutterflies (Hurst et al.,1999)or feminization
as in various isopods (Bouchon et al., 1998). However, in
otherassociations, thehosthasbecomedependentonthese
bacteria as nutritional mutualists or reproduction facilita-
tors (Pannebakker et al., 2007; Hosokawa et al., 2010). The
default Wolbachia transmission mode is vertical, from
mother to offspring, but host and symbiont phylogenies
often indicate that horizontal transmission occurs fre-
quently enough over evolutionary time to prevent co-
cladogenesis (Werren et al.,2008).Horizontal transmission
is most likely due to predator–prey and host–parasitoid
interactions (Vavre et al., 1999; Kittayapong et al., 2003).
Many host species have also been found to carry multiple
Wolbachia strains, and in some cases, these strains reside in
different tissues (e.g. Ijichi et al., 2002).
Although a number of thorough case studies have
clarified the phenotypic effects of Wolbachia infections in
models of solitary invertebrates, rather little progress has
been made in understanding the phenotypic effects of
similar infections in eusocial insects. Surveys have shown
that a wide range of termites are infected, but that
eusocial wasps and bees are rarely hosts (Lo & Evans,
2007; Russell, 2012). Many ants are also known to
harbour Wolbachia, but prevalence varies considerably
between species, between colonies in populations and
between castes within colonies (Wenseleers et al., 1998;
Russell et al., 2009; Russell, 2012). Although some of
these differences may be caused by the screening meth-
ods employed, this variation in prevalence is perhaps not
surprising given the substantial ecological differences
between the ants studied, and suggests that the fitness
effects of Wolbachia differ between hosts. A general
negative effect of infection has been suggested by the
finding that Wolbachia have been frequently lost in
invasive ants compared to their native sister populations
or species (Shoemaker et al., 2000; Reuter et al., 2004;
Cremer et al., 2008), consistent with an enemy-release
explanation of the success of invasive species. A potential
reduction in host fitness by Wolbachia infection was also
found in Formica truncorum (Wenseleers et al., 2002), but
no Wolbachia-related sex ratio biasing occurred in the
same and another Formica species (Keller et al., 2001;
Wenseleers et al., 2002), so our understanding of the
impact of Wolbachia infections for ant hosts remains
enigmatic.
In the present study, we use a novel combination of
techniques to assess how genotype-specific Wolbachia
prevalence varies across different life stages of sterile
workers of the fungus-growing ant Acromyrmex octospino-
sus. The ants live in a well-studied multitrophic symbiosis
involving among others a basidiomycete fungus, reared
as a crop in underground chambers, and antibiotic-
producing bacteria (Currie et al., 2003). Earlier studies of
this ant have indicated that most workers are infected
with Wolbachia (Van Borm et al., 2001; Frost et al., 2010)
and often by multiple strains (Van Borm et al., 2003).
However, the questions of where the strains are located
and in what relative densities have not previously been
addressed. We therefore investigated the potential for
interaction between Wolbachia genotypes within hosts
and the possible consequences of such interactions for
host fitness. We hypothesized that if genotypes co-occur
within tissues, they would interact either synergistically
or (more likely) antagonistically, and that densities
would be affected by the nature of such interactions.
It has been suggested that a decrease in bacterial
prevalence with age, as found in workers of some ant
species (Wenseleers et al., 2002), could be adaptive when
these ants are unable to vertically transmit Wolbachia to
the next generation. In Acromyrmex, worker reproduction
is negligible in colonies with a healthy queen and likely
to remain insignificant when a colony becomes orphaned
(Dijkstra & Boomsma, 2007), which is probably similar in
the Formica ants studied by Wenseleers et al. (2002). We
would thus expect a decline in Wolbachia prevalence with
worker age in Acromyrmex if the bacteria would be
reproductive parasites. We used quantitative real-time
PCR (qrt-PCR) and fluorescence in situ hybridization
(FISH) to measure the density of Wolbachia symbionts
and the distribution of bacteria among host tissues.
We found an increase in bacterial density with worker
age, and after establishing that considerable concentra-
tions of Wolbachia are associated with the ant gut, we
used laser scanning confocal microscopy to document
this in more detail. Our visualizations of bacteria in ant
tissues revealed an unexpected extracellular presence of
Wolbachia in the ant gut. These findings corroborate the
idea that Wolbachia in Acromyrmex no longer expresses
parasitic phenotypes and suggest that these bacteria have
obtained novel roles as mutualists in the fungus-growing
ant symbiosis.
Methods
DNA extraction, sequencing and quantitative PCR
Acromyrmex octospinosus colonies werecollected in Gamboa,
Panama, in the period 2004–2010 (Table 1). DNA was
extracted from whole individuals after crushing them with
aplasticpestleandfromdissectedtissues(DNeasybloodand
tissuekit;Qiagen,Hilden,Germany).Thewspprimers from
Zhou et al. (1998), targeting a surface protein, were used to
2 S. B. ANDERSEN ET AL.
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amplify c. 560 bp of Wolbachia DNA. The PCR product
wascloned(TOPOTAcloningkit; Invitrogen,Carlsbad,CA,
USA), and 10–23 clones from a single adult worker from six
field-collected (Nærum, Denmark) colonies (102 clones in
total) were sequenced by Eurofins MWG Operons (Ebers-
berg, Germany). Two dominant genotypes were identified,
which were identical to strains previously sequenced from
ants (Shoemaker et al., 2000; Van Borm et al., 2001, 2003).
The sequences were translated to amino acids (http://
web.expasy.org/translate/), and the four hypervariable
regions (HVRs) of wsp (Baldo et al., 2005) were identified
using the Wolbachia MLST website (http://pubmlst.org/
wolbachia/; Jolley et al., 2004). Following Shoemaker et al.
(2000), we called the two dominant genotypes ‘WSinvic-
taA’ and ‘WSinvictaB’. Specific primers for thesegenotypes
weredesigned(wspaF:5¢-GAAAACTGCTGTGAATGGTC-3¢,wspa R: 5¢-TCCTCCTTTGTCTTTCTC-3¢; wspb F: 5¢-GAAA-
ACTGCTGTGAATGGTC-3¢, wspb R: 5¢-ATTKCAGCATCG-
TCTTTARCT-3¢) to amplify 167–170 bp, and the specificity
of the primers was checked with direct sequencing.
The primers amplified a region where WSinvictaB was
100% identical to the other nondominant genotypes (see
Results), and it was thus not possible to quantify the
presence of these apparently rare additional genotypes
any further. However, the primers for this short region
were chosen as they enabled the identification of the
two dominant genotypes with high accuracy while ampli-
fying a region short enough to be employed in quantitative
real-time PCR (qrt-PCR, see below).
For the analysis of the distribution of Wolbachia geno-
types across different individuals, castes and colonies DNA
was extracted from eight colonies sampled in the field and
from six colonies reared under laboratory conditions for
> 7 months (no colonies were sampled both in the field
and in the laboratory). From each colony, eight entire large
larvae, pupae and adult workers were sampled (see Table 1
for colony ID and exact sample number). Field colonies
were sampled after the annual mating flight, when they
were not producing sexuals, to ensure that the large larvae
were immature large workers. For the analysis of Wolba-
chia genotype distributions across worker tissues, DNA was
extracted from dissected thoracic muscle tissues, from
three different parts of the gut and from faecal droplets of
eight ants from a single laboratory-reared colony (Ao492).
Absolute wsp copy numbers were quantified by quantita-
tive real-time PCR (qrt-PCR) with the genotype-specific
primers using SYBR Premix Ex Taq (Takara Bio Inc.,
St Germain en Laye, France) on the Mx3000P system
(Stratagene, Santa Clara, CA, USA). Reactions took place
in a final volume of 20.5 lL containing 10 lL buffer,
8.8 lL ddH2O, 0.4 lL of each primer (10 lMM), 0.4 lL ROX
standard and 0.5 lL template DNA. Bacterial measure-
ments were standardized with qrt-PCR of the single-copy
ant gene, elongation factor 1a (primers EF-1a f: 5¢ AC-
GGAAGCTCTGCCCGGTGA-3¢ EF-1a r: 5¢-TGGCAGTCA-
AGCACTGGCGT-3¢), providing an estimate of host cell
number, under the assumption that bacterial and ant DNA
were preserved and extracted equally well between castes
and independent of storage method (in ethanol at )20 �Cvs. freshly collected). All PCRs consisted of a 2-min
denaturation step at 95 �C, 35 cycles of 95 �C for 30 s,
52 �C for 30 s and 72 �C for 30 s, followed by a
dissociation curve analysis. All samples were replicated
in the same run, and the mean was used for analysis.
Each run also included three negative controls with no
added template. The initial template concentration was
calculated from a standard curve with PCR product in
tenfold dilutions of known concentration, as quantified
by nanodrop.
Cross-sectioning and embedding
Larvae (n = 4), pupae (n = 2) and workers (n = 8) from
colonies Ao49a, Ao491 and Ao496 were fixed and embed-
ded following the protocol of Kulzer Technovit 8100
(Heraeus Kulzer, Wehrheim, Germany). Tissues were cut
Table 1 Collection data for the colonies of Acromyrmex octospinosus that were used for estimating Wolbachia abundance by qrt-PCR.
Colony ID
Sample size
Lab ⁄ field
Date
Infection statusLarvae Pupae Workers Collection Sampling
Ao273 8 8 8 Lab May 2004 December 2010 Double
Ao346 5 8 8 Lab May 2007 December 2010 Double
Ao367 8 8 8 Lab May 2008 December 2010 Double
Ao404 16 16 16 Lab May 2009 December 2010 Double
Ao431 8 8 8 Lab May 2009 December 2010 Double
Ao471 8 7 8 Field April 2010 May 2010 Single (B)
Ao482 4 8 8 Field May 2010 May 2010 Double
Ao483 8 8 8 Field May 2010 May20 10 Double
Ao491 7 8 8 Field May 2010 May 2010 Single (B)
Ao49a 8 8 8 Field May 2010 May 2010 Single (B)
Ao492 8 8 8 Lab May 2010 December 2010 Double
Ao493 8 8 8 Field May 2010 May 2010 Double
Ao496 8 8 8 Field May 2010 May 2010 Double
AoClay 8 8 8 Field May 2010 May 2010 Double
Wolbachia in Acromyrmex ants 3
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to allow the penetration of the fixative (2% paraformalde-
hyde in phosphate buffer, pH 7.4) for < 4 h followed by
overnight washing in PBS pH 7.4 at 4 �C. The tissues were
dehydrated in 100% acetone for 1 h at 4 �C and infiltrated
with Technovit 8100 solution for 6–10 h at 4 �C, followed
by transfer to the embedding solution, agitation for 5 min
and transfer to a plastic mould. Moulds were sealed with
plastic foil and left to harden on ice at 4 �C overnight. The
tissue blocks were cut with a glass knife and sections
attached to superfrost plus slides (Menzel-Glaser, Ger-
many) by heating for 15 min. For whole-mount laser
scanningconfocalmicroscopy,eggswerecollectedfromthe
fungus garden of an isolated laying queen (n = 5, Ao492)
and ant guts were dissected out in fixative, fixed for > 4 h
and washed in PBS (Ao492, n = 10).
Fluorescence in situ hybridization
Tissue sections were treated with lysozyme (5 mg mL)1)
for 30 min at 37 �C to increase cell permeabilization
(Moter & Gobel, 2000) and dehydrated for 3 min each in
50%, 70% and 100% ethanol prior to hybridization.
Slides were hybridized with a 16S rRNA-targeted probe
specific for Wolbachia and labelled with Cy3 (Wol:
5¢-CTAACCCGCCTACGCGCC-3¢, from Eurofins MWG
Operons) overnight at 46 �C. This was carried out in
100 lL hybridization buffer (100 mMM Tris pH 7.2, 0.9 MM
NaCl, 0.1% sodium dodecyl sulphate) with 5 ng lL)1
probe in a Sequenza slide rack (Thermo Shandon, Cheshire,
UK). As a negative control, a Cy3-labelled probe target-
ing the spirochaete bacteria Treponema sp. was used (S-S-
Trep DDKL 12-432: 5¢-CATCTCAAGGTCATTCCC-3¢).Slides were then washed with preheated (46 �C) hybrid-
ization buffer for 3 · 3 min followed by wash with
preheated (46 �C) washing buffer (100 mMM Tris pH 7.2,
0.9 MM NaCl) for 3 · 3 min. Finally, the slides were rinsed
in water, air-dried and mounted with Vectashield (Vector
Laboratories Inc., Burlingame, CA, USA) for epifluores-
cence microscopy using an Axioimager M1 epifluores-
cence microscope. Images were obtained using an
AxioCAM MRm version 3 FireWire monochrome camera
(Carl Zeiss, Oberkochen, Germany).
Gut dissections and ant eggs were treated with lysozyme,
dehydrated, hybridized and washed as above in an Eppen-
dorf tube and mounted on slides with Vectashield contain-
ingDAPI(DAPIstainshostnucleiblue,andit is thuspossible
to infer whether bacteria are intra- or extracellularly
located). These slides were observed and photographed
using a Zeiss LSM 710 laser scanning confocal microscope
equipped with ZEN 2009EN 2009 software. After some editing, the
images were further processed to adjust contrast and crop
irrelevant parts using Photoshop CS3 for Mac.
Live ⁄ dead bacterial staining
To evaluate the occurrence of bacteria in the faecal fluid
of the ants, a droplet of c. 0.5 lL was deposited on a
microscope slide by squeezing the ant gaster with forceps
(as described in Schiøtt et al., 2010). The bacteria were
stained with the BacLight L 13152 live ⁄ dead stain
(Molecular Probes Inc., Life Technologies Europe,
Naerum, Denmark), staining live bacteria green (Syto-9
probe) and dead bacteria (i.e. cells with a compromised
membrane) red (propidium iodide); 0.5 lL of each stain
was added to each fresh faecal droplet, and slides were
sealed with a cover slide and incubated in the dark for
15 min, after which slides were analysed using the
Axioimager M1 epifluorescence microscope (n = 5).
Results
Identification of Wsp genotypes using the HVR typingsystem
Previously, Wolbachia phylogenies were primarily based
on the highly variable Wsp gene, but this gene later
turned out to be unsuitable for inferring phylogenetic
relationships, because of its high divergence and recom-
bination rate (Baldo et al., 2005, 2010). However, Wsp
remains a useful marker for identifying different strains
and allowed us to identify four different Wolbachia wsp
genotypes from the six screened colonies. Two were
identical to the WSinvictaA and WSinvictaB strains
found in Solenopsis invicta (GenBank accession number
AF243435 and AF243436, Shoemaker et al., 2000) and in
three Panamanian Acromyrmex species (Van Borm et al.,
2003). All colonies carried the WSinvictaB genotype,
whereas only some had the WSinvictaA genotype. 3.9%
of the sequences were different with colonies Ao493 and
Ao483, each yielding an additional genotype (GenBank
accession number JQ414026 and JQ414027) that was
98–99% similar to a previously identified genotype in
A. octospinosus (GenBank accession number AF472561.1).
A new genotype was obtained from colony Ao496
(GenBank accession number JQ414028), showing 90%
similarity to other sequences in GenBank.
The Wsp gene consists of four HVRs, each with
multiple alleles that have been numbered, alternating
with conserved sequences. Recombination typically takes
place between the four regions, and HVR typing is a
useful way of identifying recombination points (Baldo
et al., 2005). All genotypes were thus further character-
ized with the HVR system. WSinvictaA of A. octospinosus
contained the elements 42-43-198-25, and WSinvictaB
had the elements 21-21-25-21. The other three geno-
types turned out to be chimeras of WSinvictaA and B and
had HVRs 42-43-25-21 (found in colony Ao496) and 42-
21-25-21 (found in colony Ao493 and Ao492, the
sequences from each colony were slightly different but
translated to the same protein sequence). The fact that
recombination was localized between the HVRs, as
previously reported for other strains, confirms that these
genotypes are true chimeras and not simply the result of
sequencing errors.
4 S. B. ANDERSEN ET AL.
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qrt-PCR
All individuals from all colonies were found to be
infected with Wolbachia. qrt-PCR showed that WSinvic-
taB was dominant in all individuals at all life stages
(Fig. 1). In three of the field-collected colonies, this was
the only genotype found in measurable amounts, except
for two adult workers from one colony that also carried
WSinvictaA. This colony (Ao471) had been kept in the
laboratory at the field site in Gamboa, Panama, for
> 1 month, which may have enhanced the expression of
WSinvictaA (see below). In the remaining colonies, all
adult individuals carried both genotypes.
Based on the prevalence differences in the WSinvictaA
and WSinvictaB genotypes, colonies were divided into
three categories: field-collected single-infected (FS,
n = 3), field-collected double-infected (FD, n = 5) and
laboratory-reared double-infected (LD, n = 6). No labo-
ratory-reared colonies showed single infection. The
differences in bacterial densities were analysed in JMP
9.0.2 for Mac OSX using a repeated-measures ANOVAANOVA, as
individuals collected from the same colony could not be
regarded as independent. There was considerable be-
tween-colony variation in standardized bacterial densi-
ties within colonies and castes, with outliers apparent in
many combinations, so the geometric mean density per
caste per colony was analysed, as this showed the most
homogenous variance of all measures examined. Cate-
gory was included as the between-subject effect, and
caste and the category-by-caste interaction were in-
cluded as within-subject effects. Post hoc testing was by
paired or unpaired t-tests for within- and between-
subject effects respectively, with Bonferroni correction
based on the total number of tests carried out. Overall,
there was an increase in total bacterial number with
developmental stage, with the bacterial density being
significantly higher in pupae than in larvae and signif-
icantly higher in workers than in pupae (F2,10 = 273.4,
P < 0.0001). There was also a significant caste-by-cate-
gory interaction, due to somewhat different development
of bacteria in the different categories (F4,20 = 4.68,
P £ 0.05). In single-infected field colonies, the bacterial
density did not vary significantly between castes. In
double-infected field colonies, the increase was signifi-
cant between all castes, whereas it was only significant
between larvae and pupae and larvae and adults in
laboratory-reared double-infected colonies. There was a
significant difference in the total number of bacteria
between categories, with laboratory-reared colonies
contained slightly higher densities at all life stages
(F2,11 = 5.46, P £ 0.05).
Looking at WSinvictaB only, the overall pattern was
bacterial density increasing from the larval to the pupal
stage and remaining at this high level in the adults
(F2,10 = 206.2, P < 0.0001). There was no significant
caste-by-category interaction, showing that this pattern
was the same in each category (F4,20 = 1.36, P = 0.284),
and the difference between categories did not quite reach
significance (F2,11 = 3.21, P = 0.08; Fig. 1).
The highest prevalence of the (nondominant) WSin-
victaA genotype was found in adult workers of FD and
LD colonies, where they reached a mean of 29%
Larvae WorkersPupae
Wol
bach
ia d
ensi
ty
0
1
2
3
4
5
6
7
8
0
1
2
3
4
5
6
7
8 WSinvictaA
WSinvictaB
Field collected single infected individualsField collected double infected individualsLab collected double infected individuals
∗∗∗
∗
∗ ∗
∗
∗
A
C
B
Fig. 1 The density of Wolbachia bacteria in three different life stages
(larvae, pupae and workers) in three different colony categories
(Field-collected double-infected, field-collected single-infected and
laboratory-reared double-infected individuals). Each bar represents
the number of Wolbachia cells per host cell (wsp copies), divided by
the copy number of the host gene EF-1a estimating the total number
of host cells, as measured with qrt-PCR (top panel = WSinvictaA
genotype; bottom panel = WSinvictaB genotype). For each caste in
each colony, the geometric mean ratio was calculated and presented
as ± SE. WSinvictaB dominates in all life stages, and WSinvictaA
only proliferates in adults of the double-infected colonies. The
differences in bacterial density were tested with Bonferroni-cor-
rected paired t-tests, following repeated-measures ANOVAANOVA of the
differences between castes (larvae, pupae, adults: within-subject
effect), sampling categories (field single-infected, field
double-infected and laboratory double-infected: between-subject
effect) and their interaction. The symbols (* and **) indicate
significant differences between castes within categories (e.g. for
WSinvictaA, the workers of field-collected double-infected colonies
had a significantly higher bacterial density than larvae and pupae).
The letters (A, B and C) indicate significant overall differences
between castes, where an increase in bacterial density was found
between larvae and pupae and between pupae and workers for
WSinvictaA but not for WSinvictaB.
Wolbachia in Acromyrmex ants 5
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(± 0.015 SE) of the total bacteria. In the FS colonies,
WSinvictaA was not present in measureable amounts,
and abundances in the immatures of FD colonies were
only slightly (not significantly) higher. The LD colonies
carried significantly higher amounts of WSinvictaA in
the pupal stages. There were thus significant differences
between castes (F2,10 = 58.49, P < 0.0001) and between
sampling categories (F2,11 = 30.30, P < 0.0001), and also
the interaction between these factors was significant
(F4,20 = 11.35, P < 0.0001; Fig. 1).
The bacterial estimates obtained from dissections of
different tissue types were not standardized with host
gene copy number, as the majority of the bacteria were
found to be extracellular (see below). As the variance in
proportions was very different across tissues, with the
faecal droplet material in particular containing either
high or low proportions of WSinvictaA (Fig. 2), they
were compared pairwise using the Steel–Dwass nonpara-
metric test (Day & Quinn, 1989). The proportions of
WSinvictaA were significantly higher in the crop and the
muscle tissues (44%) compared to the rest (Z = )3.31 to
3.31, P £ 0.05), whereas the rectum (37%) contained
significantly more than the whole ant (26%; Z = )3.31,
P £ 0.05) and the midgut (24%; Z = 2.90, P £ 0.05).
Because of the high variance in the WSinvictaA propor-
tion in the faecal droplets, the mean proportion in these
(29%) was not significantly different from the other
tissues.
Fluorescence in situ hybridization
The FISH analyses showed bacterial colonization of
multiple tissue types. In the ant eggs, the bacterial
density was highest around one pole (Fig. S1). In larvae,
the dominant fat body cells were carrying many bacteria
and the gut tissue also housed some (Fig. 3a, b). In the
pupae, the ant cells are diversifying into more tissue
types, which were widely infected (e.g. muscle fibres and
fat cells, data not shown). This was also the case in the
adults, where particularly the muscle cells, fat body and
gut tissue harboured many bacteria (Fig. 3c, d). Histology
showed a large amount of Wolbachia occurring extracel-
lularly in the crop part of the gut (in six of eight
individuals, Fig. 3c), and this was confirmed by confocal
microscopy of whole guts (in ten of ten individuals,
Fig. 4). These extracellular bacteria were to a lesser
extent also seen in the midgut (Fig. S2). No clear
identification of Wolbachia in the rectum could be made
because of strong autofluorescence of the tissues. As a
negative control for unspecific hybridization, a probe
specific to the bacterium Treponema sp. was used. This
showed some unspecific hybridization to the part of the
gut leading to the crop and the ileum connecting the
midgut and rectum, so hybridization to these tissues by
the Wolbachia probe was ignored, as it was possibly
unspecific.
The live ⁄ dead bacterial staining of faecal droplets
showed a high density of living bacteria, but it was not
possible with the applied methods to confirm how many
of these were Wolbachia.
Discussion
Wolbachia prevalence and diversity
We found that all individuals and all life stages and
colonies were infected with Wolbachia, and vertical
symbiont transmission was confirmed by the visualiza-
tion of Wolbachia in ant eggs (Fig. S1). All else equal, this
should imply that also larvae should predictably have
detectable Wolbachia infection rates, but reported worker
prevalence has been surprisingly variable even in the
same ant sampled from the same area (Van Borm et al.,
2001; mean infection rate of individuals of 40%; Frost
et al., 2010, 81%). The explanation for this may be
technical rather than biological, because qrt-PCR allows
for a higher level of detection and the amplification of a
shorter DNA fragment (c. 170 bp in the present study vs.
783 bp by Van Borm et al., 2001), which ensures that
even slightly fragmented DNA is amplified.
The two dominant Wolbachia wsp genotypes that we
found have been observed in other ants as well
0.0
0.2
0.4
0.6
0.8
1.0
Pro
port
ion
WSi
nvic
taA
Tissue
Crop Midgut RectumThoracicmuscle Whole ant
Faecaldroplet
midgut rectum
crop
A
AB
B
C
A
C
faecaldroplet
Fig. 2 Box plots showing the proportion of WSinvictaA Wolbachia in
thoracic muscle tissue, three different parts of the ant gut (crop,
midgut and rectum) and faecal droplets in comparison with whole
ant samples. The bacterial load of WsinvictaA and WSinvistaB was
measured by qrt-PCR of individual samples. Central lines are median
proportions, whereas boxes run from the lower 25% to the upper
75% quartile and whiskers connect the extremes of the ranges.
Letters indicate group-level differences following the pairwise Steel–
Dwass method (see text for details). The insert provides a schematic
overview of the ant gut and its surroundings, with the crop being
connected to the midgut where the Malpighian tubules attach, and
the ileum connecting the midgut to the rectum.
6 S. B. ANDERSEN ET AL.
ª 2 0 1 2 T H E A U T H O R S . J . E V O L . B I O L . d o i : 1 0 . 1 1 1 1 / j . 1 4 2 0 - 9 1 0 1 . 2 0 1 2 . 0 2 5 2 1 . x
J O U R N A L O F E V O L U T I O N A R Y B I O L O G Y ª 2 0 1 2 E U R O P E A N S O C I E T Y F O R E V O L U T I O N A R Y B I O L O G Y
(Shoemaker et al., 2000; Van Borm et al., 2003) and are
also very similar to strains from beetles and spiders (e.g.
Sintupachee et al., 2006). WSinvictaB was found in all
colonies of A. octospinosus, whereas WSinvictaA only
occurred in some colonies and never alone. There were
thus colony-level differences in genotype diversity, as
either all or no individuals within a colony carried
WSinvictaA. The presence of the WSinvictaA genotype
was not correlated with sampling site, indicating that
geographic clustering in the sample population is
unlikely (data not shown). However, no good estimates
of colony age and colony size upon collection were
available, so we cannot directly evaluate whether these
variables, which may be important for the development
of bacterial infections, had any effect.
In the colonies harbouring both genotypes, we iden-
tified two recombinant genotypes. Although the preva-
lence of these was not assessed by qrt-PCR for all life
stages, the low frequencies in three adult ants for which
cloning estimates were available suggest that they were
rare. Our HVR typing further indicated that these
chimera genotypes arose by recombination of WSinvic-
taA and WSinvictaB between the first and second HVR,
and the second and third HVR, respectively. Recombi-
nation may thus be rather frequent but although these
recombinant genotypes may persist in the population,
they do not appear to be particularly successful. The very
occurrence of within-host recombination shows that
some degree of interaction between WSinvictaA and
WSinvictaB takes place. Wsp is a major outer surface
protein of Wolbachia and has been suggested to mediate
the contact with the host cells via its two transmembrane
regions that likely interact with the host immune system
(Braig et al., 1998; Bazzocchi et al., 2007), so that
(a) (b)
(c) (d)
Fig. 3 Fluorescence in situ hybridization of
Wolbachia (stained red) in ant tissues.
(a) Larval gut showing some hybridization.
(b) A larval fat body with many Wolbachia
around cell nuclei. (c) Crop (foregut) of an
adult large worker ant with extracellular
Wolbachia in the lumen. (d) Muscle fibres of
an adult worker with some intracellular
concentrations of Wolbachia. Scale bar 50 lm
(a) and 20 lm (b–d).
Fig. 4 Extracellular Wolbachia bacteria in the crop of the ant gut,
a highly flexible sac that is slightly deflated so it appears somewhat
folded. The original LSCM image shows the 3D structure of the crop
containing Wolbachia bacteria (stained red). The central image
provided here is a typical horizontal optical section, whereas the two
flanking images represent the vertical optical sections. Scale bar
50 lm.
Wolbachia in Acromyrmex ants 7
ª 2 0 1 2 T H E A U T H O R S . J . E V O L . B I O L . d o i : 1 0 . 1 1 1 1 / j . 1 4 2 0 - 9 1 0 1 . 2 0 1 2 . 0 2 5 2 1 . x
J O U R N A L O F E V O L U T I O N A R Y B I O L O G Y ª 2 0 1 2 E U R O P E A N S O C I E T Y F O R E V O L U T I O N A R Y B I O L O G Y
recombination may affect these recognition processes.
Recombination between Wolbachia wsp genotypes has
previously been found in other host species, including
ants (Reuter & Keller, 2003).
Bacterial density increases with host age
We found an increase in the bacterial load with age,
suggesting that Wolbachia thrive in the mature workers.
Adult worker ants of the species F. truncorum were
previously found to have lower infection rates than
immatures (Wenseleers et al., 2002), which generated
the hypothesis that workers may lose infection for
reasons that are adaptive for the bacteria, because they
are evolutionary dead ends for a reproductive parasite.
This appears not to be the case for A. octospinosus. For the
dominant WSinvictaB genotype, the increase in density
occurs between the larval and the pupal stage and
prevalence stays at this level in adults, equivalent to what
has been found in the Adzuki bean beetle, where
Wolbachia is a confirmed reproductive parasite (Ijichi
et al., 2002).
The increase in bacterial load could reflect the
appearance of new tissue types that the bacteria are
able to invade after metamorphosis (see below). In most
host–symbiont interactions, whether parasitic or mutu-
alistic, the host has a clear interest in controlling
bacterial growth and dispersal (Login et al., 2011). In
Drosophila, the ability to do so appears connected to life
stage–specific expression of immunity genes (Samakovlis
et al., 1990). In the eusocial honeybee Apis mellifera,
phenoloxidase activity (a measure of immune defence)
was low in both larvae and pupae, most likely because
alternative social immunity mechanisms provide effi-
cient protection of brood (Wilson-Rich et al., 2008). Such
a down-regulation of the individual immune defence
could be of importance for the ability of vertically
transmitted symbionts to grow in the immature stages of
social insects. If this is also the case for A. octospinosus, it
may partly explain the increase in Wolbachia density
with host age, suggesting that the bacteria mostly grow
and disperse when host control mechanisms are
constrained.
Niche segregation of bacterial symbionts
Our results (Fig. 1) indicate that WSinvictaA proliferates
mainly in adult individuals. However, when comparing
the single- and double-infected field colonies, there is a
suggestion of WSinvictaA proliferation in adult workers
being associated with lower WSinvictaB prevalence in
the larval and pupal stages. This could reflect some form
of scramble competition between WSinvictaA and WSin-
victaB bacteria in the immature developmental stages. In
this hypothetical scenario, the initial degree of domi-
nance of WSinvictaB would then determine the available
niche space for the other genotype, so that individuals
where WSinvictaA remains under the detection limit in
the immature stages will only be able to grow very few of
them as adults (the observed pattern of single-infected
field colonies; Fig. 1). However, when the WSinvictaA
for some reason becomes more abundant already in the
immature stages (so that immature field individuals are
scored as double-infected), they are much more likely to
proliferate further in adult workers.
A competitive scenario as outlined above would be
most likely when bacterial genotypes interact in the same
host tissues during the larval and pupal stages but, at
least partly, segregate into different tissue types in adult
workers. Such tissue tropism of Wolbachia strains has
previously been observed in Adzuki bean beetles (Ijichi
et al., 2002). As our FISH results showed a high density of
bacteria in the muscle fibres and the gut, we further
measured the distribution of Wolbachia genotypes in
these tissues. The qrt-PCRs of specific tissue types showed
that WSinvictaA was significantly more abundant in
muscle fibres and in the crop of the gut, relative to later
stages in the digestive process (midgut and rectum) and
the whole ant (Fig. 2). The muscle tissue is only fully
developed in the adult ants, and the adult gut is very
distinct from the larval gut, being more complex and
divided into sections varying morphologically and in pH,
enzyme activity and retention time of contents (Erthal
et al., 2004). This corroborates the notion that, although
overlapping, the adult Wolbachia niches are somewhat
distinct and that they are unlikely to be differentiated
earlier in development. However, the substantial overlap
in Wolbachia niches within hosts also raises the possibility
that these genotypes may have different functional roles
in adult ants.
Interpreting the infection patterns of double-infected
laboratory-reared colonies as being consistent with geno-
type competition would imply that resource constraints
somehow affect field colonies more than laboratory-
reared colonies. This is reasonable, as laboratory-reared
colonies were being fed regularly with a standard selec-
tion of Danish bramble leaves (Rubus sp.), experienced no
predation or other hazards while foraging and generally
had large and thriving fungus gardens while living under
stable humidity and temperature regimes. All of these
laboratory-reared colonies were double-infected and also
harboured slightly higher densities of WSinvictaA in the
immature life stages compared to the field-sampled
colonies (Fig. 1). In addition, the total bacterial number
in the laboratory-reared colonies was slightly higher than
that found in the field, suggesting that the bacteria thrive
when their hosts experience laboratory conditions. The
finding of two double-infected workers in an otherwise
single-infected colony in the field seems consistent with
this interpretation, as this was the only colony that had
been kept for more than a month in the field laboratory in
Panama under ad libitum resource conditions before
ant samples were collected (Ao471). The presence of
WSinvictaA in measurable quantities early on could thus
8 S. B. ANDERSEN ET AL.
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J O U R N A L O F E V O L U T I O N A R Y B I O L O G Y ª 2 0 1 2 E U R O P E A N S O C I E T Y F O R E V O L U T I O N A R Y B I O L O G Y
be dependent on colony resource condition, which in the
field may be correlated with colony size.
Are Wolbachia new mutualists in the attinefungus-farming symbiosis?
The FISH data surprisingly showed that Wolbachia
bacteria are abundant in the lumen of the adult
worker gut (Figs 3 and 4). Although Wolbachia has
been found in the gut tissue (Dobson et al., 1999; Ijichi
et al., 2002), an extracellular location is highly unusual
and has to our knowledge not been documented before
(but see Fischer et al., 2011 showing the occasional
appearance of extracellular Wolbachia close to ovarian
tissue in nematodes). However, this observation is
consistent with WSinvictaA and WSinvictaB being
present in the faecal droplets of Acromyrmex, as we
would not expect intracellular bacteria to be excreted
with gut bacteria (Fig. 2). The faecal droplets contained
a large amount of viable bacteria, and positive DNA-
level evidence suggests that at least part of these
bacteria were Wolbachia. This combined result therefore
indicates that Wolbachia cells are not harmed by
digestive gut enzymes, consistent with this environ-
ment being their natural niche.
The faecal droplets have unique functions in the
fungus-growing ants. They contain proteins from the
fungal garden, which are ingested by the ants but pass
undigested through the gut to assist decomposition in
newly established fungus garden (Schiøtt et al., 2010).
They also play a role in the recognition and elimination
of genetically different fungal cultivars that workers may
collect (Poulsen & Boomsma, 2005). The various adap-
tive functions of the faecal droplets to the ant–fungal
symbiosis suggest that there is strong selective pressure
on the gut environment and the composition of faecal
fluid. This and the atypical location of Wolbachia in the
gut lumen and faecal droplets suggest that Wolbachia in
A. octospinosus may have a mutualistic nutritional role in
the ant–fungus cultivation symbiosis. In addition, the use
of the faecal droplets by the ants in the fungus garden
may represent a novel mechanism for Wolbachia to be
horizontally transmitted, at least between colony mem-
bers within the same nest. This would then suggest that
the sterile workers are not a complete evolutionary dead
end for Wolbachia, but a useful reservoir to help making
sure that all reproductives of the colony are infected
before they disperse.
The recent finding of a beneficial role of Wolbachia
symbionts in the Western rootworm, larvae of Diabrotica
virgifera virgifera, causing the down-regulation of defence
compounds in the plants that they feed on (Barr et al.,
2010), offers an intriguing possible analogue to our
present results. Similar to the herbivorous beetle larva,
the alliance of leaf-cutting ants and their fungus garden
symbionts also faces challenges from secondary plant
defences. Recent work (Schiøtt et al., 2010) has shown
that the fungal symbionts of leaf-cutting ants have
convergently evolved an entire set of pectinases that
are normally only found in pathogenic fungi that attack
live plant hosts, and also these enzymes pass the ant gut
unharmed. It therefore seems highly worthwhile to
further explore the functional role of Wolbachia in
Acromyrmex, both in the worker guts and in the faecal
fluid where the bacteria interact with the multiple
microorganisms that are now known from attine ant
fungus gardens (Pinto-Tomas et al., 2009). We note that
recent work has also suggested that plant defences may
not only be chemical, but also biotic, as leaf-substrate
choice by the ants is affected by the endophytic commu-
nity of the leaves (Van Bael et al., 2009; Bittleston et al.,
2010). Further studies along these lines will also have the
potential to elucidate why only some colonies carry the
WSinvictaA genotype in measurable amounts.
Acknowledgments
We would like to thank Joanna Amenuvor and Annie
Ravn Pedersen (DTU) and Lisbeth Haugkrogh and Aase
Jespersen (KU) for advice concerning histology and FISH,
Morten Schiøtt, Henrik de Fine Licht and Tom Gilbert
(KU) for advice on qrt-PCR and Panagiotis Sapountzis for
comments on the manuscript. SBA was funded by a PhD
Scholarship from the Science Faculty of the University of
Copenhagen, and SBA, DRN and JJB were supported by
the Danish National Research Foundation.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1 Wolbachia bacteria in eggs of Acromyrmex
octospinosus.
Figure S2 Extracellular Wolbachia in the ant midgut.
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provides supporting information supplied by the authors.
Such materials are peer-reviewed and may be re-
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Data deposited at Dryad: doi: 10.5061/dryad.7fq7d558
Received 21 November 2011; revised 23 January 2012; accepted 24
March 2012
Wolbachia in Acromyrmex ants 11
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