Dynamic Wolbachia prevalence in Acromyrmex leaf-cutting ants: potential for a nutritional symbiosis

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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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

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





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)




AoClay 8 8 8 Field May 2010 May 2010 Double

Wolbachia in Acromyrmex ants 3



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.




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.




(± 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.




(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.




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




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.

As a service to our authors and readers, this journal

provides supporting information supplied by the authors.

Such materials are peer-reviewed and may be re-

organized for online delivery, but are not copy-edited

or typeset. Technical support issues arising from support-

ing information (other than missing files) should be

addressed to the authors.

Data deposited at Dryad: doi: 10.5061/dryad.7fq7d558

Received 21 November 2011; revised 23 January 2012; accepted 24

March 2012




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Dynamic Wolbachia prevalence in Acromyrmex leaf-cutting ants: potential for a nutritional symbiosis