# Endosymbiont diversity among sibling weevil species competing for the same resource

Adrien Merville, Samuel Venner, Hélène Henri, Agnès Vallier, Frédéric Menu, Fabrice Vavre, Abdelaziz Heddi, Marie-Claude Bel-Venner

► To cite this version:

Adrien Merville, Samuel Venner, Hélène Henri, Agnès Vallier, Frédéric Menu, et al. Endosymbiont diversity among sibling weevil species competing for the same resource. BMC Evolutionary Biology, 2013, 13, pp.28. 10.1186/1471-2148-13-28. hal-00850306

HAL Id: hal-00850306

https://inria.hal.science/hal-00850306

Submitted on 29 May 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Endosymbiont diversity among sibling weevil
species competing for the same resource

Adrien Merville<fnref n="1" />,<fnref n="2" />*, Samuel Venner<fnref n="1" />, Hélène Henri<fnref n="1" />, Agnès Vallier<fnref n="2" />, Frédéric Menu<fnref n="1" />, Fabrice Vavre<fnref n="1" />, Abdelaziz Heddi<fnref n="2" /> and Marie-Claude Bel-Venner<fnref n="1" />*

**Abstract**

**Background:** Whereas the impact of endosymbionts on the ecology of their hosts is well known in some insect species, the question of whether host communities are influenced by endosymbionts remains largely unanswered. Notably, the coexistence of host species competing with each other, which is expected to be stabilized by their ecological differences, could be facilitated by differences in their endosymbionts. Yet, the composition of endosymbiotic communities housed by natural communities of competing host species is still almost unknown. In this study, we started filling this gap by describing and comparing the bacterial endosymbiotic communities of four sibling weevil species (*Curculio spp.*) that compete with each other to lay eggs into oak acorns (*Quercus spp.*) and exhibit marked ecological differences.

**Results:** All four species housed the primary endosymbiont *Candidatus* Curculioniphilus buchneri, yet each of these had a clearly distinct community of secondary endosymbionts, including *Rickettsia*, *Spiroplasma*, and two *Wolbachia* strains. Notably, three weevil species harbored their own predominant facultative endosymbiont and possessed the remaining symbionts at a residual infection level.

**Conclusions:** The four competing species clearly harbor distinct endosymbiotic communities. We discuss how such endosymbiotic communities could spread and keep distinct in the four insect species, and how these symbionts might affect the organization and species richness of host communities.

**Keywords:** Endosymbiosis, Host community, Curculio, Oak weevil, Infection pattern, Niche partitioning, Field study

**Background**

The last decade has seen a growing number of studies
exploring microbial endosymbiosis among arthropods and
its implication for the evolution of the host species. The
impressive array and diversity of species harboring one or
more type(s) of endosymbionts raises the possibility that
such associations may play a predominant role in the
ecology and evolution of both partners. Most studies
that have considered symbiotic associations from the
perspective of the host support the view that heritable
symbionts offer their host an opportunity for rapid and
deep evolutionary changes and for greater adaptation to a
novel environment [1-3]. Symbionts are considered to be

a keystone in the diversification and expansion of the
ecological niche of their host [3-5]. Thus, endosymbionts
have been shown to assist their host in exploiting poor
nutritional resources, notably by complementing its nutri-
tion [6-8], often to the point that they become essential or
obligatory symbionts for the host. From the differences
observed between the endosymbiotic communities of
various host populations, it has been suggested that
symbionts may mediate the specialization of their hosts
on distinct resources and thus promote the emergence of
allopatric host races, which might subsequently lead to
speciation [9-15].

Mutualistic facultative symbionts are also able to pro-
vide their host with other skills, such as broadening the
range of temperatures they can tolerate [16], reinforcing
their ability to resist natural enemies [17-20], or increasing
their dispersal capacity [21-24]. These rapidly-acquired
capacities are likely to promote the expansion of the host
populations and the diversification of their habitat [1-5].

<footnote>* Correspondence: adrien.merville@univ-lyon1.fr; marie-claude.venner@univ-lyon1.fr</footnote>

<footnote n="1">¹Université de Lyon, Université Lyon 1, CNRS, UMR5558, Laboratoire de Biométrie et Biologie Evolutive, Villeurbanne, France</footnote>

<footnote n="2">²INSA-Lyon, UMR203 BF2I, INRA, Biologie Fonctionnelle Insectes et Interactions, Bat. L. Pasteur 20 ave A. Einstein, Villeurbanne, France</footnote>
Besides these mutualistic relationships, facultative endosymbionts are also known to manipulate the reproduction of the host. In response to selective pressures favoring their vertical transmission, many symbionts, such as *Wolbachia*, have been shown either to skew the sex-ratio of the offspring toward females or to induce sterilization of uninfected females [25-27]. Such effects can affect both the population dynamics and gene flow, notably by promoting the reproductive isolation of infected hosts.

While firm knowledge is accumulating about the
symbiont-mediated interactions between given host species
and their environments, the possibility that endosymbionts
may influence the organization of communities composed
of several competing host species has so far been
seldom investigated. The first empirical evidence of
an endosymbiont-mediated relationship between com-
peting host species has been recently provided from
artificial protist communities kept under highly-controlled
laboratory conditions. It was suggested that a green algal
symbiont is essential for competing protist species to
coexist, notably because the allochemicals and the nutri-
tional resource provided by this microorganism seem to
balance the competitive capacity of the hosts [28]. More-
over, the issue of the competition between two hymenop-
teran parasitoid species has been shown experimentally to
be tightly dependent on the presence of an inherited virus
in one of the insect species [29]. These findings highlight
the fact that the influence of microbial symbionts on the
organization of competing host communities may have
been considerably underestimated. The paucity of empir-
ical data about the role of endosymbionts on competing
host communities contrasts with the body of knowledge
that has accumulated about extracellular symbiosis that
has been shown to play a key role in the organization and
composition of plant communities [30-32]. Microbial
communities present in the soil often mediate nutrient
uptake of plants, and might facilitate the coexistence of
plant species in various ways, notably by allowing resource
partitioning (reviewed in [33]).

A first support to the hypothesis that endosymbiosis
does impact communities of insect species competing
with each other would be that endosymbiotic profiles vary
with respect to the host species. To study this aspect, we
investigated endosymbiosis in natural oak weevil commu-
nities composed of four *Curculio* species – *C. glandium*
(Marsham), *C. elephas* (Gyllenhal), *C. pellitus* (Boheman),
and *C. venosus* (Gravenhorst) (Coleoptera, Curculionidae).
These weevils coexist on the same individual oak trees
(*Quercus* spp.) in southern Europe [34-36], where they
compete for oak acorns that constitute the sole food
resource for larval development and that have been shown
to be highly limiting some years [37]. This biological
system is relevant for examining our proposal in that (i)
the four oak weevil species are expected to host

endosymbionts, like the few other *Curculio* species already
described [15,38] and (ii) they exhibit marked differences
in their life history traits despite their recent divergence
[36], suggesting that their coexistence is stabilized by
means of ecological niche partitioning [37].

We provide an exhaustive description of the endosym-
biotic meta-community harbored by the four oak weevil
species in two distinct natural communities. Using a
correlative and a quantitative approach, we found that
endosymbiont communities differed across the four
*Curculio* species, while being consistent within each
species. We discuss the mechanisms possibly accounting
for the contrasted infection patterns observed across these
sibling species and the ways by which endosymbiosis may
contribute to structuring communities of insect species
competing with each other.

Methods

Study system

Four weevil species of the genus *Curculio* (*C. glandium*, *C. elephas*, *C. pellitus* and *C. venosus*) coexist on oak trees (*Quercus* spp.) in southern Europe [34]. In all four species, females lay eggs during summer within oak acorns, where larvae achieve their development before self-extracting and burrowing into the soil. Fully mature larvae then enter diapause during variable time periods -i.e., they spend from one to four years underground-, depending on the species or even on individuals within one species [37].

Insect sampling

Our study was conducted on two natural communities
found in two sites in southern France (site 1: N45° 35’;
E5° 01’; site 2: N45° 45’; E5° 16’). Adult weevils were
live-trapped on oak trees at each site weekly throughout
one breeding season (June to September 2009) by beating
branches with a wooden stick [39]. Insects were collected
on a white sheet laid under the tree and their species
was identified from morphological traits [34].

DNA extraction

Ovaries of 391 adult females caught on one of the two
study sites and belonging to one of four weevil species
(see Table 1) were dissected in buffer A (KCL 25 mM,
MgCl2 10 mM, Saccharose 250 mM, Tris–HCl 35 mM;
pH = 7.5) and homogenized in 500µL of buffer STE (NaCl
100 mM, Tris–HCl 10 mM and EDTA 1 mM, pH = 8).
These dissected tissue samples were individually subjected
to DNA extraction by adding proteinase K (120 mg/L)
and SDS (sodium dodecyl sulfate: 0.5% m/v), and were
then incubated for 2 h at 55°C. RNA contamination was
removed with the addition of RNaseA (60 mg/µL) for 1 h
at 37°C. DNA from each sample was then purified with
phenol-chloroform extraction and precipitated using
<table><caption>**Table 1 Number of adult males and females of each weevil species collected at the two sites**</caption><thead><tr><th rowspan="2">Species</th><th colspan="2">Females</th><th colspan="2">Males</th></tr><tr><th>Site 1</th><th>Site 2</th><th>Site 1</th><th>Site 2</th></tr></thead><tbody><tr><td><i>C. elephas</i></td><td>74</td><td>38</td><td>25</td><td>21</td></tr><tr><td><i>C. glandium</i></td><td>76</td><td>40</td><td>26</td><td>29</td></tr><tr><td><i>C. pellitus</i></td><td>78</td><td>7</td><td>36</td><td>7</td></tr><tr><td><i>C. venosus</i></td><td>64</td><td>14</td><td>15</td><td>12</td></tr></tbody></table>

isoamelic alcohol [40]. In addition, 171 adult males (Table 1) were automatically crushed individually with stainless steel beads shaken 20s at 20Hz (Tissue Lyser, Qiagen) [41] and their total DNA was extracted using NucleoSpin Tissue Kit (Macherey-Nagel).

## Characterization of the endosymbiotic lineages and phylogenetic analyses

To get an overview of the endosymbionts harbored by the four *Curculio* species, bacterial 16S rRNA amplification was performed individually on the ovaries of 95 females among which 40 *C. elephas* (24 from Site 1 and 16 from Site 2) and 20 females of each of the three remaining species (*C. glandium*, *C. pellitus* and *C. venosus*) comprising 10 females per site, except for *C. pellitus* from the site 2 for which only 5 females could be sampled. A 1.5 kb fragment was amplified using eubacterial universal primers (Table 2). Reactions were carried out in a 50µL final volume consisting of 2.5 units of Taq DNA polymerase (UptiTherm, Interchim, France), 2 mM MgCl2, 0.8 mM

dNTPs, 0.6 µM primers, and 200 ng DNA template. The polymerase chain reaction (PCR) started at 95°C for 5 min and was followed by 26 cycles, each of these running 95°C for 30s, 53°C for 1 min and 72°C for 2 min; the final step of the reaction was 7 min at 72°C. The PCR products were then pooled into eight groups, one per species and per site. Each of these pools was then subjected to cloning. In a first step, ten clones per pool were randomly selected and sequenced (Additional file 1; Beckman Coulter Genomics, Grenoble, France). Identifying these sequences revealed a predominance of three endosymbiont types (among which, mostly *Curculioniphilus buchneri*, and to a lesser extent, *Wolbachia* and *Rickettsia*; see below). Consequently, in a second step at least 130 clones were grown up for each pool (Additional file 1), and each clone was screened by diagnostic PCR using the specific primers corresponding to the three dominant bacterial types (Table 2). Clones that were grown successfully but that could be amplified by none of these three diagnostic PCRs (i.e., *Curculioniphilus buchneri*, *Wolbachia* and *Rickettsia*) were then sequenced (30 clones; see Additional file 1).

We characterized the bacteria hosted by *Curculio* females by performing phylogenetic analyses using the sequences obtained in this study together with the most similar ones found by Blast in Genbank. Multiple sequences were aligned using MUSCLE software [46]. The appropriate model of evolution was estimated with *jmodeltest* [47] for each set of sequences considered. Phylogenetic analyses were performed using maximum likelihood (ML) inference with *Phyml* v3.0.1 [48]. The

<table><caption>**Table 2 Primers used for PCR diagnostic**</caption><thead><tr><th>Target Species</th><th>Target gene</th><th>Primer name</th><th>Primer sequence (5'-3')</th><th>Product size (kb)</th><th>Temp.* (°C)</th><th>Refs.<sup>†</sup></th></tr></thead><tbody><tr><td>Host<br/>(Curculio sp.)</td><td>CytB rRNA</td><td>CBJ-10933</td><td>TATGTACTACCATGAGGACAAATATC</td><td>0.5</td><td>45</td><td>[42]</td></tr><tr><td></td><td>CBN-11367</td><td>ATTACACCTCCTAATTTATTAGGAAT</td><td></td><td></td><td></td><td>[42]</td></tr><tr><td>Eubacterial Universal Primers</td><td>16S rRNA</td><td>008For<br/>1487Rev</td><td>AGA GTT TGA TCA TGG CTC AG<br/>TAC CTT GTT ACG ACT TCA CC</td><td>1.5</td><td>53</td><td>[43]<br/>(**)</td></tr><tr><td>Candidatus Curculioniphilus buchneri</td><td>16S rRNA</td><td>16S-F<br/>16S-R</td><td>AGAGATCTGGAGGAATATCA<br/>CACTAAAGCATCTCTGCTAAAT</td><td>0.4</td><td>52</td><td>(*)<br/>(*)</td></tr><tr><td></td><td>GroEL</td><td>GroEL 2 F<br/>GroEL 2R</td><td>ATG GGB GCT CAA ATG GTK AAA<br/>CTCTTTCATTTCAACTTCNGTBGCA</td><td>0.9</td><td>55</td><td>[38]<br/>[38]</td></tr><tr><td>Wolbachia</td><td>16S rRNA</td><td>W-Spec F<br/>W-Spec R</td><td>CATACTATTCGAAGGGATAG<br/>AGCTTCGAGTGAAACCAATTC</td><td>0.4</td><td>60</td><td>[44]<br/>[44]</td></tr><tr><td>Rickettsia</td><td>16S rRNA</td><td>RbF<br/>RbR</td><td>GCTCAGAACGAACGCTATC<br/>GAAGGAAAGCATCTCTGC</td><td>0.9</td><td>58</td><td>[12]<br/>[12]</td></tr><tr><td>Spiroplasma</td><td>ITS and 16S rRNA</td><td>Spixo-16S F<br/>Spixo-16S R</td><td>TTAGGGGCTCAACCCCTAACC<br/>TCTGGCATTGCCAACTCTC</td><td>0.8</td><td>52</td><td>[45]<br/>[45]</td></tr><tr><td>Sodalis</td><td>16S rRNA</td><td>Sodalis 370 F<br/>16SSod590R</td><td>CGRTRGCGTTAAYAGCGC<br/>AACAGACCGCCTGCGTACG</td><td>0.2</td><td>55</td><td>[38]<br/>[38]</td></tr></tbody></table>

<footnote>*Annealing temperature. †References: (*) this study; (**) nucleotides numbering from Escherichia coli, GenBank accession number J01859.</footnote>
models selected were GTR + G for *Rickettsia* (16S, 873 sites) as well as for *Spiroplasma* (16S + ITS, 1405 sites), *HKY85 + I* for *Serratia* (16S, 503 sites), *K80 + I* for *Wolbachia* (16S, 340 sites). The robustness of the nodes was assessed with 100 bootstrap replicates. Additionally, Bayesian phylogenies were performed with MrBayes v3.1.2 [49] using appropriate parameters leading to convergence between two runs. We then compared topologies obtained with the two methods using approximate unbiased (AU) [50] and Shimodaira and Hasegawa (SH) [51] tests available in the CONSEL program package [52]. No significant difference was detected between the two topologies for any of the bacteria.

To examine the possible co-diversification between *Curculioniphilus buchneri*, considered as the primary symbiont, and its hosts, we used the method previously described to build the phylogenies based on *COI* gene sequences retrieved from Genbank for the weevil phylogeny, and on two distinct markers, the *16S rRNA* sequence and the *GroEL* gene, for the bacterial phylogeny. Nine host-symbiont pairs were included in the analysis, among which the four *Curculio* species studied here, the hazelnut weevil *C. nucum* that we collected in a French population near Lyon, and four Japanese Curculio species whose primary symbiont has already been described (*C. camelliae*, *C. sikkimensis*, *C. robustus* and *C. dentipes*; [38].) The models selected were *TPM2 + I* for *C. buchneri* (16S, 267 sites), *GTR + I + G* for *Curculioniphilus buchneri* (*GroEL* gene, 915 sites) and for *Curculio* spp. (*COI* gene, 375 sites). For *Curculioniphilus buchneri* bacteria, the congruence of the *16S rRNA* and the *GroEL* topologies was rejected by the reciprocal AU and SH tests (AU: *P* = 0.004; SH: *P* = 0.014) probably because the *16S* topology was weakly resolved, which precluded the concatenation of both sequences. Then, we tested the global congruence between the symbiont and host phylogenies, using *16S* and *GroEL* gene for the bacterial tree separately, with the CopyCat program (The Cophylogenetic Analysis Tool, version 2.00.02 [53]). After checking the occurrence of global co-diversification, we further tested the association of each of the nine individual host-symbiont pairs using the *COI* and the *GroEL* phylogeny that provided the best resolved bacterial tree. Patristic distances were used as input matrix and the AxParafit program was run with 999 permutations [54].

## Diagnostic PCR

After checking the quality of the DNA of each insect sample (by amplifying a 487 bp fragment of the *cytochrome b* gene of the insect; Table 2), 562 adult weevils (males or females from one of the four species; see Table 1) were screened for each of the five endosymbionts found in the first part of the study using their specific primers (i.e., *C. buchneri*, *Wolbachia*, *Rickettsia*, *Spiroplasma* and *Sodalis*; Table 2). Although diagnostic PCR might fail to detect an

endosymbiont that might be too rare in the host individual, we considered in subsequent analyses that the insect was free of this bacterium. To check whether the successful PCRs were actually specific of the targeted symbiont, two DNA amplicons randomly selected per symbiont and per weevil species were sequenced. To discriminate between the two possible *Wolbachia* strains detected in our samples (see Results), we performed a Restriction Fragment Length Polymorphism following the PCR assay based on the *16 s rRNA* sequence (PCR-RFLP) on each *Wolbachia*-infected weevil. For that purpose, 17 µl of each PCR product was digested at 37°C for 3 h with 5U of *AluI* (Fermentas, Villebons/Y., France) and was separated on a 2% agarose gel electrophoresis during 1 h at 100 V. One DNA sample from each *Wolbachia* strain and each host species was randomly selected and sequenced to check the accuracy of the bacterial lineage identified by PCR-RFLP.

## Data analysis

In each weevil species, we used a generalized linear model (GLM) fitted with a binomial error and a logit-link function to test whether the probability for an insect to be infected by the predominant facultative endosymbiont depended on its sex, on the locality it came from, or both. The best-fitted model was selected for each endosymbiont according to the AIC criterion. Further, the effects of each variable selected in the model and the possible interactions between variables were tested by an analysis of deviance. All the analyses were performed with the R software v.2.12.0 (http://www.r-project.org/).

## Results

### Endosymbiotic communities in the oak weevil communities

On the basis of 1,358 *16S rRNA* clones derived from the ovaries of females of the four oak weevil species collected at the two study sites, ten distinct bacterial lineages were identified (Additional file 1): *Curculioniphilus buchneri*, *Wolbachia*, *Rickettsia*, *Spiroplasma*, *Sodalis*, *Serratia*, *Xenorhabdus*, *Brevundimonas*, *Erwinia* and *Escherichia*. *Xenorhabdus*, *Brevundimonas* and *Erwinia* are known to be entomopathogenic and/or free-living bacteria. The identified strain of *Escherichia* corresponds to the vector used for the cloning step. The phylogenetic analysis of *Serratia* reveals that it is part of a monophyletic group of nonsymbiotic bacteria that are distant from those known as symbionts (data not shown). Furthermore, this bacterium was shown to infect very few insect individuals at any site and irrespective of the weevil species, suggesting that the *Serratia* strain identified in this study was likely to be an insect pathogen: we discarded it from further analysis. A single clone among 1,358 ones was identified as a *Sodalis*-like endosymbiont (97,77% of similarity with the *Sodalis* strain found in *C. sikkimensis*). It
was detected in the pool of *C. elephas* females sampled at the site 2, and could be a *posteriori* detected by means of individual PCR diagnostic in one of the 16 females of this pool. We could not detect this symbiont in any of the 562 oak weevils individually screened with the same method. Consequently, we conducted phylogenetic analyses on the five endosymbionts identified (i.e. *Curculioniphilus*, *Wolbachia* strains 1 and 2, *Rickettsia*, *Spiroplasma*) (Figures 1 and 2).

For the *Curculioniphilus* endosymbiont, four distinct sequences were obtained, one for each weevil species. We found statistical evidence of global co-diversification between weevil hosts and their primary symbiont on each dataset (P = 0.002 for COI-GroEL comparison, P = 0.005 for COI-16S comparison). In addition, the association of seven of the nine host-symbiont pairs individually tested

was found significant (see Figure 1). The insignificant associations corresponded to *Curculio elephas* and *C. dentipes* with their symbionts, and could be explained by their weakly supported positions in the phylogenetic trees generated either by bootstrap or by Bayesian posterior probabilities. This finding suggests co-diversification between *Curculioniphilus* and its weevil hosts and implies that this endosymbiont has been acquired by a common ancestor of *Curculio* spp. and subsequently strictly vertically transmitted. The *Wolbachia* 16 s rRNA sequences revealed at least two distinct strains. The corresponding phylogeny (Figure 2A) indicates that one of them belongs to the *Wolbachia* supergroup A while the other one seems rather positioned at the edge of the *Wolbachia* supergroup B, according to the way *Wolbachia* sequences are annotated in Genbank. While we detected *Rickettsia* in all four

<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /><figcaption>**Figure 2** (See legend on next page.)</figcaption></figure>
(See figure on previous page.)

**Figure 2 Phylogenies of the facultative endosymbionts identified in *Curculio* spp.** (A) *Wolbachia* (340 bp unambiguously-aligned nucleotide sites, 16S rRNA sequences) is shown with indication of the supergroups A and B based on the annotation of sequences found in Genbank; (B) *Rickettsia* (873 sites, 16S rRNA sequences); (C) *Spiroplasma* (1405 sites, ITS and 16S rRNA sequences). The Bayesian trees are shown, as the Maximum-likelihood trees (not shown) exhibit substantially the same topology (see Methods). The bootstrap values for the maximum-likelihood analysis (100 replicates) and the Bayesian posterior probabilities are shown above and below the nodes only if greater than 50 and 0.50, respectively. The accession numbers of the nucleotide sequences are shown in brackets. Sequences obtained from this study are indicated by asterisks. The name of each bacterial sequence corresponds to that of its host. Bacteria reported as non-endosymbiotic ones in the literature are underlined.

weevil species, we failed to discriminate them from
one host species to the next. The phylogenetic analysis
(Figure 2B) suggests that this bacterium is closely related
to the *Kytorhinus sharpianus* endosymbiont (Coleoptera:
Bruchidae, [55]). Finally, 3.33% and less than 1% of
the bacterial clones derived from *C. elephas* and *C.
glandium* females contained *Spiroplasma*, respectively:
this bacterium was allied to the *Curculio sikkimensis*
endosymbiont (Figure 2C, [15]).

Individual and multiple infection status of the oak weevils

We have screened *Curculioniphilus* as well as the three
other endosymbionts in each of 562 weevils collected
(Table 1). *Curculioniphilus* is highly prevalent in adults
from the four species irrespective of the sex or the study
site (it was found in 90.5% *C. elephas*, 84.2% *C. glandium*,
98.4% *C. pellitus* and 97.1% *C. venosus*; see Figure 3). The
few uninfected individuals were essentially males (37 out
of 44 uninfected individuals). Besides this so-called pri-
mary symbiont, three weevil species were shown to house
their own predominant facultative symbiont (see Figure 3):
97.1% of 171 *C. glandium* individuals were infected by the
*Wolbachia* strain belonging to the supergroup A, 98.5% of
128 *C. pellitus* individuals were infected by the *Wolbachia*
strain at the basis of the supergroup B, and 77.1% of 105
*C. venosus* individuals hosted *Rickettsia*. In none of these
species did the prevalence of the predominant secondary
symbiont significantly vary according to either the sex or
the study site (Table 3). A significant effect of the sex-
locality interaction was statistically detected, however, on
the prevalence of the supergroup A *Wolbachia* strain in
*C. glandium*. This effect seems to be due to the unequal
infection rate observed mostly in males between the two
sites (infected males: 87% (site 1, n = 26) vs 100% (site 2, n
= 29); infected females: 100% (site 1, n = 76) vs 95% (site 2,
n = 40); Figure 3). This finding might stem from an unba-
lanced distribution of aged males in the two sites sampled
(see discussion), and seems to be of low relevance owing
to the small sample size of males as compared with that of
females. In the two oak weevil communities studied,
*C. elephas* differed from the three other species in
that it lacked a predominant symbiont and exhibited
*Spiroplasma* in addition to the three other secondary
symbionts, all four endosymbionts being detected at a

residual frequency. The three other weevil species, in
addition to their predominant secondary symbiont, also
harbored the other endosymbionts -except Spiroplasma-
at a residual frequency. Considering the individual host
level, multiple infections commonly occurred in the four
species, mostly due to the co-occurrence of the primary
symbiont and the predominant secondary symbiont
(Figure 3). Interestingly, the two strains of Wolbachia
were never detected simultaneously in any individual
despite their marginal co-occurrence within the same
species and study site.

Discussion

This study thoroughly describes the endosymbiotic com-
munities hosted simultaneously by four weevil sibling
species that coexist, compete with each other for a limiting
resource (i.e., oak acorns as egg-laying sites) [37,56,57] and
display contrasted ecological traits [37,58]. We found that
while all four species hosted the same primary endosym-
biont, they harbored markedly distinct communities of
secondary endosymbionts. Even if our results are strictly
correlative, since we did not investigate the actual role
of each symbiont on its host, this finding is compatible
with our proposal that stable coexistence of host species
competing with each other, which is expected to be
ensured by their ecological differences, might be mediated
by their endosymbionts.

*Curculioniphilus buchneri* was identified in all four weevil species using an *a priori*-free method based on 16S rRNA cloning and sequencing. This symbiont is known to be part of a distinct clade of Gammaproteobacteria, and is hosted by as many as 9 *Curculio* species. These insects feed on achenes or capsules and are specialist of perennial plants, including Theaceae (*Camellia* spp.) and Fagaceae (*Corylus*, *Castanea* and *Quercus* spp.) [36,38]. The congruence observed between the phylogenies of *C. buchneri* and that of their host (Figure 1) suggests that this symbiont has been ancestrally acquired [4], which sustains its primary status. *C. buchneri* was found at a very high infection rate in the weevils screened in this study, and the few insects that did not carry *C. buchneri* were almost exclusively males (Figure 3). As primary endosymbionts are most often involved in complementing the host's diet, this finding suggests that this bacterium might have
<figure><img src="image_4.png" /><figcaption>**Figure 3** Individual multiple infection status observed in two French communities of acorn weevils. Five different endosymbionts have been detected in the four *Curculio* species studied: (Cb) Blue - *Candidatus* Curculioniphilus buchneri, (Ri) Red -*Rickettsia*, (Sp) Green-*Spiroplasma*, (WA) Pink - *Wolbachia* strain A and (WB) Gray - *Wolbachia* strain B. The geographical origin and the sex of the weevils are indicated on the left of the figure. The number of individuals tested is shown at the top of each graph. Horizontal reading informs us about the proportion of individuals sharing a given infection status. The prevalence of a given bacteria in a sample (of a given sex, site, species) is obtained by summing vertically the corresponding color.</figcaption></figure>
<table><caption>**Table 3 Probability for a predominant facultative symbiont to infect an individual host as a function of its sex and of the study site**</caption><thead><tr><th>Variable</th><th>Deviance</th><th>df</th><th>P</th></tr></thead><tbody><tr><td><em>Rickettsia (C. venosus)</em></td><td></td><td></td><td></td></tr><tr><td>Site</td><td>0.31589</td><td>1</td><td>0.57409</td></tr><tr><td>Sex</td><td>0.66578</td><td>1</td><td>0.41453</td></tr><tr><td>Site:Sex</td><td>2.75129</td><td>1</td><td>0.09718</td></tr><tr><td><em>Wolbachia A (C. glandium)</em></td><td></td><td></td><td></td></tr><tr><td>Site</td><td>0.0003</td><td>1</td><td>0.98704</td></tr><tr><td>Sex</td><td>1.746</td><td>1</td><td>0.18635</td></tr><tr><td>Site:Sex</td><td>8.9503</td><td>1</td><td><strong>0.00277</strong></td></tr><tr><td><em>Wolbachia B (C. pellitus)</em></td><td></td><td></td><td></td></tr><tr><td>Site</td><td>1.9356</td><td>1</td><td>0.16415</td></tr><tr><td>Sex</td><td>0.0747</td><td>1</td><td>0.78467</td></tr><tr><td>Site:Sex</td><td>3.7132</td><td>1</td><td>0.05398</td></tr></tbody></table>

Each analysis was performed on the weevil species shown in brackets. For each bacterium, the best-fitted generalized linear model (logistic regression) is shown, and was selected on the basis of the Akaike Information Criterion (AIC =117.2 for *Rickettsia*, 42.48 for *Wolbachia A* and 22.88 for *Wolbachia B*). The P-values for respective explanatory variables were obtained with chi-square statistics.

been lost during adulthood by a few males and hence, that it might be essential to the larval rather than the adult stage [59].

Three of the secondary symbionts (two distinct clades of *Wolbachia* and one *Rickettsia*) were commonly detected in all four species, whereas the fourth one, *Spiroplasma*, was found exclusively in *C. elephas*. This pattern resembles that of an endosymbiotic metacommunity, in which the endosymbiotic communities of each host population/species comprise all the bacterial types and are connected to one another by means of horizontal transfers [2,4,60-65]. Horizontal transfers have been reported to occur between parasitoid species sharing the same host [65], and are likely to occur between the four weevil species under study here, because of the concomitant presence of larvae from several species occasionally feeding on the same individual acorns (unpublished results). Evidence of such transfers remains to be established, however, notably through the analysis of more variable molecular sequences of the symbionts.

While at first sight the set of secondary endosymbionts detected was found to be identical across the four weevil species, a finer-tuned, quantitative analysis revealed that these ones had clearly distinct endosymbiotic communities. The four insect species differed from one another in that, with the exception of *C. elephas*, each had its own major secondary symbiont. Two questions remain to be addressed: (i) why not all symbionts have spread to all four weevil species, and (ii) why different symbionts predominate in the different host species? Multiple infections are likely to be counter-selected since at the individual

host level, competition between endosymbionts for the resource, might lead to competitive exclusion and/or to increased cost for the host [66,67]. Although we observed the presence of a particular endosymbiont correlating with a weevil species, we cannot infer at this time its actual impact on host phenotype. However, assuming that symbionts have spread in response to selective pressures, two alternatives can be envisaged, depending on whether each of the major secondary symbionts engages in a mutualist interaction with its host or manipulates its reproduction [2,4].

First, mutualistic endosymbionts provide their hosts with higher benefits than costs and are expected to invade the population rapidly [5,20]. The net benefit for a host to house a given endosymbiont might greatly depend on its physiological or ecological requirements. These needs might differ from one host species to the other, notably when these ones interact in different ways with the same environment [11,64]. In the oak weevil communities, the marked ecological differences observed between the four species [37,58] would coincide with unequal probability for a given symbiont to spread. Conversely, we cannot rule out that the marked ecological differences between the four weevil sibling species might be due to the major symbionts having a distinct impact on their host. Hence, the different symbionts might facilitate the partitioning of ecological niches between their hosts by providing them with expanded or new skills, thereby possibly leading to unequal nutritive assistance [6,7,68-71], dispersal capacity [21-24], or ability to resist natural enemies among host species [17-20,72].

Second, the probability for reproductive parasites to successfully invade their host heavily relies on the efficiency of their transmission from mother to offspring. In each of the four weevil species, males and females were frequently and equally infected, which makes male-killing or feminization processes unlikely. For instance, the infection patterns observed would rather be compatible with cytoplasmic incompatibility (CI) [25-27]. Notably, the two strains of *Wolbachia* identified on the basis of 16 s rRNA, that are found to be the major secondary symbionts of *C. pellitus* and *C. glandium*, show almost complete mutual exclusion at the individual host level (Figure 3), suggesting mutual bidirectional CI. Should this mechanism actually occur, these *Wolbachia* strains would have been prevented from invading *C. elephas* and *C. venosus* populations because of their too low infection rate (reviewed in [73]). Interestingly, endosymbiont-mediated CI has theoretically been shown to facilitate and reinforce reproductive isolation and speciation [25], and such endosymbiont-mediated species enrichment would therefore be expected particularly in communities composed of sibling host species. However, empirical evidence for this mechanism is still scarce and much debated [73-77].
Conclusions

Our findings raise the question of whether intracellular
micro-organisms might contribute to the structure of
natural communities of host species competing with each
other, either by accelerating their ecological divergence or
by reinforcing their reproductive isolation, possibly lea-
ding to speciation. Exploring this proposal calls for both
theoretical development and empirical investigations
describing the endosymbiotic communities housed by
various arthropod communities composed of sibling and
competing host species. It should then be possible to test
the predictions that communities harboring diverse
secondary symbionts have greater species richness and in-
clude more closely related species than those deprived of
symbionts. It would be worth running experiments in
parallel to elucidate the actual impact endosymbionts have
on their host, which still remains largely unknown in most
natural systems.

Data accessibility

DNA sequences have been deposited at Genbank with
the accession numbers AB514497-AB514504, AB507714-
AB507716, and JN100059-JN10090. Phylogenetic trees have
been deposited at TreeBase (http://purl.org/phylo/treebase/
phylows/study/TB2:S13815).

Additional file

**Additional file 1: Identification of the clones sequenced in the four weevil species.** To identify the endosymbiotic diversity, clones were sequenced in adult females belonging to the four weevil species and live-trapped in two sites (see Methods). We described the best hit blast of each clone and the ratio of the number of similar nucleotides to the total number of nucleotides (called Identities).

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

This study is part of the PhD thesis of AM who is focused on the implication of endosymbionts on the structure of sibling insect communities. MCBV and AH supervised this PhD at the interface between community ecology and endosymbiosis interactions. MCBV and SV suggested the exploration of endosymbiotic communities within competing species, supervised and contributed to the ecological research. FM provided his experience about weevil ecology and contributed to the experimental scheduling. AH and FV gave their expertise in the interactions existing between insects and their bacterial endosymbionts. AV conducted the fine dissections of the female ovaries and molecular analyses from DNA purification, cloning, up to sequencing. HH contributed to the methodological setting up of diagnostic PCRs and supervised the phylogenetic analyses. All authors read and approved the final manuscript.

Authors' information

MCBV, SV and FM are focused on evolutionary and community ecology. FV, AH, HH and AV have interest in associations between insects and bacteria endosymbionts. This work is part of the PhD of AM who develops an integrative research project linking ecological and symbiotic aspects.

Acknowledgements

We thank P.F. Pélisson, B. Rey and F. Débias for their help on insect
community surveys, and S. Balmand, J. Blugeot and C. Vincent-Monégat for
their contribution to the molecular and phylogenetic analyses. We are also
grateful to D. Charif for her statistical advices and we thank two anonymous
reviewers that helped us improving an earlier draft of the manuscript. This
work was funded by the French National Research Agency (ANR: CoCoReCo
project JC09-470585), by the CNRS, and by Envirhonalp ('Sante' et
Environnement' Environment Cluster).

Received: 3 September 2012 Accepted: 30 January 2013
Published: 4 February 2013

**References**

1. Feldhaar H, Gross R: Immune reactions of insects on bacterial pathogens and mutualists. *Microbes Infect* 2008, 10:1082-1088.

2. Clark EL, Karley AJ, Hubbard SF: Insect endosymbionts: manipulators of insect herbivore trophic interactions? *Protoplasma* 2010, 244:25-51.

3. Ferrari J, Vavre F: Bacterial symbionts in insects or the story of communities affecting communities. *Phil Trans Biol Sci* 2011, 366:1389-1400.

4. Moran NA, McCutcheon JP, Nakabachi A: Genomics and evolution of heritable bacterial symbionts. *Annu Rev Genet* 2008, 42:165-190.

5. Himler AG, Adachi-Hagimori T, Bergen JE, Kozuch A, Kelly SE, Tabashnik BE, Chiel E, Duckworth VE, Dennehy TJ, Zchori-Fein E, Hunter MS: Rapid spread of a bacterial symbiont in an invasive whitefly is driven by fitness benefits and female bias. *Science* 2011, 332:254-256.

6. Douglas AE: Nutritional interactions in insect-microbial symbioses: aphids and their symbiotic bacteria *Buchnera*. *Annu Rev Entomol* 1998, 43:17-37.

7. McCutcheon JP, McDonald BR, Moran NA: Convergent evolution of metabolic roles in bacterial co-symbionts of insects. *Proc Natl Acad Sci U S A* 2009, 106:15394-15399.

8. Lamelas A, Gosalbes MJ, Moya A, Latorre A: New clues about the evolutionary history of metabolic losses in bacterial endosymbionts, provided by the genome of *buchnera aphidicola* from the aphid *cinara tujafilina*. *Appl Environ Microbiol* 2011, 77:4446-4454.

9. Leonardo TE, Muir GT: Facultative symbionts are associated with host plant specialization in pea aphid populations. *Proc R Soc B* 2003, 270:S209-S212.

10. Simon J-C, Carré S, Boutin M, Prunier-Leterme N, Sabater-Mun B, Latorre A, Bournville R: Host-based divergence in populations of the pea aphid: insights from nuclear markers and the prevalence of facultative symbionts. *Proc R Soc B* 2003, 270:1703-1712.

11. Tsuchida T, Koga R, Fukatsu T: Host plant specialization governed by facultative symbiont. *Science* 1989, **2004**:303.

12. Gottlieb Y, Ghanim M, Chiel E, Gerling D, Portnoy V, Steinberg S, Tzuri G, Horowitz AR, Belausov E, Mozes-daube N, Kontsedalov S, Gershon M, Gal S, Katzir N, Zchori-fein E: Identification and localization of a *rickettsia* sp. In *bemisia tabaci* (homoptera: aleyrodidae). *Appl Environ Microbiol* 2006, 72:3646-3652.

13. Chiel E, Gottlieb Y, Zchori-Fein E, Mozes-Daube N, Katzir N, Inbar M, Ghanim M: Biotype-dependent secondary symbiont communities in sympatric populations of *bemisia tabaci*. *Bull Entomol Res* 2007, 97:407-413.

14. Gueguen G, Vavre F, Gnankine O, Peterschmitt M, Charif D, Chiel E, Gottlieb Y, Ghanim M, Zchori-Fein E, Fleury F: Endosymbiont metacommunities, mtDNA diversity and the evolution of the *bemisia tabaci* (hemiptera: aleyrodidae) species complex. *Mol Ecol* 2010, 19:4365-4378.

15. Toju H, Fukatsu T: Diversity and infection prevalence of endosymbionts in natural populations of the chestnut weevil: relevance of local climate and host plants. *Mol Ecol* 2010, 20:853-868.

16. Montllor CB, Maxmen A, Purcell AH: Facultative bacterial endosymbionts benefit pea aphids *acyrthosiphon pisum* under heat stress. *Ecol Entomol* 2002, 27:189-195.

17. Oliver KM, Russell JA, Moran NA, Hunter MS: Facultative bacterial symbionts in aphids confer resistance to parasitic wasps. *Proc Natl Acad Sci U S A* 2003, 100:1803-1807.

18. Haine ER, Moret Y, Siva-jothy MT, Rolff J: Antimicrobial defense and persistent infection in insects. *Science* 2008, 322:1257-1259.

19. Brownlie JC, Johnson KN: Symbiont-mediated protection in insect hosts. *Trends Microbiol* 2009, **17**:348-354.
20. Jaenike J, Unckless R, Cockburn SN, Boelio LM, Perlman SJ: Adaptation via symbiosis: recent spread of a *Drosophila* defensive symbiont. *Science* 2010, **329**:212–215.

21. Grenier A-M, Nardon C, Nardon P: The role of symbiotes in flight activity of *Sitophilus weevils*. *Entomol Exp Appl* 1994, **70**:201–208.

22. Heddi A, Grenier AM, Khatchadourian C, Charles H, Nardon P: Four intracellular genomes direct weevil biology: nuclear, mitochondrial, principal endosymbiont, and *Wolbachia*. *Proc Natl Acad Sci U S A* 1999, **96**:6814–6819.

23. Leonardo TE, Mondor EB: Symbiont modifies host life-history traits that affect gene flow. *Proc R Soc B* 2006, **273**:1079–1084.

24. Goodacre SL, Martin OY, Bonte D, Hutchings L, Woolley C, Ibrahim K, Thomas CFG, Hewitt GM: Microbial modification of host long-distance dispersal capacity. *BMC Biol* 2009, **7**:32.

25. Bordenstein SR: Symbiosis and the origin of species. In *Insect Symbiosis*. Edited by Bourtzis K, Miller TA. Boca Raton, FL: CRC Press; 2003:283–304.

26. Werren JH, Baldo L, Clark ME: *Wolbachia*: master manipulators of invertebrate biology. *Nat Rev Microbiol* 2008, **6**:741–751.

27. Merçot H, Poinson D: Infection by *Wolbachia*: from passengers to residents. *C R Biol* 2009, **332**:284–297.

28. Müller JP, Hauzy C, Hulot FD: Ingredients for protist coexistence: competition, endosymbiosis and a pinch of biochemical interactions. *J Anim Ecol* 2011, **81**:222–232.

29. Patot S, Allemand R, Fleury F, Varaldi J: An inherited virus influences the coexistence of parasitoid species through behaviour manipulation. *Ecol Lett* 2012, **15**:603–610.

30. Harnett DC, Wilson GWT: Mycorrhizae influence plant community structure and diversity in tallgrass prairie. *Ecology* 1999, **80**:1187–1195.

31. Reynolds HL, Packer A, Bever JD, Clay K: Grassroots ecology: plant-microbe-soil interactions as drivers of plant community structure and dynamics. *Ecology* 2003, **84**:2281–2291.

32. Van der Heijden MGA, Bardgett RD, Straalen MV: The unseen majority: soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems. *Ecol Lett* 2006, **11**:296–310.

33. Bever JD, Dickie IA, Facelli E, Facelli JM, Klironomos J, Moora M, Rillig MC, Stock WD, Tibbett M, Zobel M: Rooting theories of plant community ecology in microbial interactions. *Trends Ecol Evol* 2010, **25**:468–478.

34. Hoffmann A: *Faune de France Coléoptères Curculionides* (Deuxième partie). Paris, France: Fédération Française des Sociétés de Sciences Naturelles; 1954.

35. Coutin R: Original characteristics of the evolving cycles of some European weevil species: *Curculio elephas Gyll., C. nucum L., C. glandium Marsh., C. venosus Grav. and C. villosus F*. *Memoires de la Societe royale belge d'Entomologie* 1992, **35**:259–266.

36. Hughes J, Vogler AP: The phylogeny of acorn weevils (genus *curculio*) from mitochondrial and nuclear DNA sequences: the problem of incomplete data. *Mol Phylogenet Evol* 2004, **32**:601–615.

37. Venner S, Pélisson PF, Bel-Venner MC, Débias F, Rajon E, Menu F: Coexistence of insect species competing for a pulsed resource: toward a unified theory of biodiversity in fluctuating environments. *Public Library of Science One* 2011, **6**:e18039.

38. Toju H, Hosokawa T, Koga R, Nikoh N, Meng XY, Kimura N, Fukatsu T: "Candidatus curculioniphilus buchneri," a novel clade of bacterial endocellular symbionts from weevils of the genus *curculio*. *Appl Environ Microbiol* 2010, **76**:275–282.

39. Schauff ME: *Collecting and preserving insects and mites: tools and techniques*. Washington: USDA Misc Publ n°1443; Museum of Natural History; 1986.

40. Sambrook J, Russell DW: *Molecular cloning: a laboratory manual*. 3rd edition. New York, NY: Cold Spring Harbor Laboratory Press; 2001.

41. Pélisson PF, Henri H, Bel-Venner MC, Allemand R, Merville A, Menu F, Venner S: Identification at the larval stage of four *curculio* species coexisting on oak trees using PCR-RFLP. *Entomol Exp Appl* 2011, **138**:77–82.

42. Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P: Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymers chain reaction primers. *Ann Entomol Soc Am* 1994, **87**:651–701.

43. Lefèvre C, Charles H, Vallier A, Delobel B, Farrell B, Heddi A: Endosymbiont phylogenesis in the dryophthoridae weevils: evidence for bacterial replacement. *Mol Biol Evol* 2004, **21**:965–973.

44. Werren JH, Windsor DM: *Wolbachia* infection frequencies in insects: evidence of a global equilibrium? *Proc R Soc B* 2000, **267**:1277–1285.

45. Duron O: Never completely trust a model: insights from cytoplasmic incompatibility beyond *wolbachia-drosophila* interactions. *Heredity* 2008, **101**:473–474.

46. Edgar RC: MUSCLE: a multiple sequence alignment method with reduced time and space complexity. *BMC Bioinforma* 2004, **5**:1–19.

47. Posada D: *jModelTest: phylogenetic model averaging*. *Mol Biol Evol* 2008, **25**:1253–1256.

48. Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. *Syst Biol* 2003, **52**:696–704.

49. Ronquist F, Huelsenbeck JP: *MrBayes 3: Bayesian phylogenetic inference under mixed models*. *Bioinformatics* 2003, **19**:1572–1574.

50. Shimodaira H, Hasegawa M: Multiple comparisons of loglikelihoods with applications to phylogenetic inference. *Mol Biol Evol* 1999, **16**:1114–1116.

51. Shimodaira H: An approximately unbiased test of phylogenetic tree selection. *Syst Biol* 2002, **51**:492–508.

52. Shimodaira H, Hasegawa M: CONSEL: for assessing the confidence of phylogenetic tree selection. *Bioinformatics* 2001, **17**:1246–1247.

53. Meier-Kolthoff JP, Auch AF, Huson DH, Göker M: COPYCAT: cophylogenetic analysis tool. *Bioinformatics* 2007, **7**:898–900.

54. Legendre P, Desdevises Y, Bazin E: A statistical test for host-parasite coevolution. *Syst Biol* 2002, **51**:217–234.

55. Fukatsu T, Shimada M: Molecular characterization of *rickettsia* sp in a bruchid beetle *kytorhinus sharpianus*. *Appl Entomol Zool* 1999, **34**:391–397.

56. Sork VL, Bramble J, Sexton O: Ecology of mast-fruiting in three species of North American deciduous oaks. *Ecology* 1993, **74**:528–541.

57. Leibold MA, McPeek MA: Coexistence of the niche and neutral perspectives in community ecology. *Ecology* 2006, **87**:1399–1410.

58. Pélisson PF, Bel-Venner MC, Rey B, Burgevin L, Martineau F, Fourel F, Lecuyer C, Menu F, Venner S: Contrasted breeding strategies in four sympatric sibling insect species: when a proovigenic and capital breeder copes with a stochastic environment. *Funct Ecol* 2012, **26**:198–206.

59. Mansour K: On the so-called symbiotic relationship between coleopterous insect and intracellular microorganisms. *Q J Microsc Sci* 1934, **77**:255–272.

60. Boyle L, O'Neill SL, Robertson HM, Karr TL: Interspecific and intraspecific horizontal transfer of *wolbachia* in *drosophila*. *Science* 1993, **260**:1796–1799.

61. Sandström JP, Russell JA, White JP, Moran NA: Independent origins and horizontal transfer of bacterial symbionts of aphids. *Mol Ecol* 2001, **10**:217–228.

62. Russell JA, Latorre A, Sabater-Muñoz B, Moya A, Moran NA: Side-stepping secondary symbionts: widespread horizontal transfer across and beyond the Aphidoidea. *Mol Ecol* 2003, **12**:1061–1075.

63. Russell JA, Moran NA: Horizontal transfer of bacterial symbionts: heritability and fitness effects in a novel aphid host. *Society* 2005, **71**:7987–7994.

64. Tsuchida T, Koga R, Horikawa M, Tsunoda T, Maoka T, Matsumoto S, Simon JC, Fukatsu T: Symbiotic bacterium modifies aphid body color. *Science* 2010, **330**:1102–1104.

65. Huigens ME, de Almeida RP, Boons PAH, Luck RF, Stouthamer R: Natural interspecific and intraspecific horizontal transfer of parthenogenesis-inducing *wolbachia* in *trichogramma* wasps. *Proc Roy Soc Lond B Biol Sci* 2004, **271**:509–515.

66. Mouton L, Dedeine F, Henri H, Boulétéreau M, Profizi N, Vavre F: Virulence, multiple infections and regulation of symbiotic population in the *wolbachia-asobara tabida* symbiosis. *Genetics* 2004, **168**:181–189.

67. Oliver KM, Moran NA, Hunter MS: Costs and benefits of a superinfection of facultative symbionts in aphids. *Proc R Soc B* 2006, **273**:1273–1280.

68. Dowd PF: Insect fungal symbionts: a promising source of detoxifying enzymes. *J Ind Microbiol Biotechnol* 1992, **9**:149–161.

69. Tokuda G, Watanabe H: Hidden cellulases in termites: revision of an old hypothesis. *Biol Lett* 2007, **3**:336–339.

70. Brownlie JC, Cass BN, Riegler M, Witsenburg JJ, Iturbe-Ormaetxe I, McGraw EA, O'Neill SL: Evidence for metabolic provisioning by a common invertebrate endosymbiont, *wolbachia pipiensis*, during periods of nutritional stress. *PLoS Pathog* 2009, **5**:e1000368.

71. Carpenter KJ, Horak A, Keeling PJ: Phylogenetic position and morphology of spirotriichosomidae (parabasalasia): new evidence from *leptospronympha* of *cryptocercus punctulatus*. *Protist* 2010, **161**:122–132.

72. Oliver KM, Moran NA, Hunter MS: Variation in resistance to parasitism in aphids is due to symbionts not host genotype. *Proc Natl Acad Sci U S A* 2005, **102**:12795–12800.
73. Engelstädter J, Telschow A: Cytoplasmic incompatibility and host population structure. *Heredity* 2009, **103**:196-207.

74. Shoemaker DD, Katju V, Jaenike J: *Wolbachia* and the evolution of reproductive isolation between *drosophila recens* and *drosophila subquinaria*. *Evolution* 1999, **53**:1157-1164.

75. Marshall JL: The *allonemobius-wolbachia* host-endosymbiont system: evidence for rapid speciation and against reproductive isolation driven by cytoplasmic incompatibility. *Evolution* 2004, **58**:2409-2425.

76. Telschow A, Hammerstein P, Werren JH: The effect of *wolbachia* versus genetic incompatibilities on reinforcement and speciation. *Evolution* 2005, **59**:1607-1619.

77. Jaenike J, Dyer KA, Cornish C, Minhas MS: Asymmetrical reinforcement and *wolbachia* infection in *drosophila*. *PLoS Pathog* 2006, **4**:e325.

doi:10.1186/1471-2148-13-28

Cite this article as: Merville *et al*.: Endosymbiont diversity among sibling weevil species competing for the same resource. *BMC Evolutionary Biology* 2013 **13**:28.

Submit your next manuscript to BioMed Central
and take full advantage of:

* Convenient online submission

* Thorough peer review

* No space constraints or color figure charges

* Immediate publication on acceptance

* Inclusion in PubMed, CAS, Scopus and Google Scholar

* Research which is freely available for redistribution

Submit your manuscript at
www.biomedcentral.com/submit

BioMed Central