New Insights into the Evolution of Wolbachia Infections in Filarial Nematodes Inferred from a Large Range of Screened Species

Background Wolbachia are intriguing symbiotic endobacteria with a peculiar host range that includes arthropods and a single nematode family, the Onchocercidae encompassing agents of filariases. This raises the question of the origin of infection in filariae. Wolbachia infect the female germline and the hypodermis. Some evidences lead to the theory that Wolbachia act as mutualist and coevolved with filariae from one infection event: their removal sterilizes female filariae; all the specimens of a positive species are infected; Wolbachia are vertically inherited; a few species lost the symbiont. However, most data on Wolbachia and filaria relationships derive from studies on few species of Onchocercinae and Dirofilariinae, from mammals. Methodology/Principal Findings We investigated the Wolbachia distribution testing 35 filarial species, including 28 species and 7 genera and/or subgenera newly screened, using PCR, immunohistochemical staining, whole mount fluorescent analysis, and cocladogenesis analysis. (i) Among the newly screened Onchocercinae from mammals eight species harbour Wolbachia but for some of them, bacteria are absent in the hypodermis, or in variable density. (ii) Wolbachia are not detected in the pathological model Monanema martini and in 8, upon 9, species of Cercopithifilaria. (iii) Supergroup F Wolbachia is identified in two newly screened Mansonella species and in Cercopithifilaria japonica. (iv) Type F Wolbachia infect the intestinal cells and somatic female genital tract. (v) Among Oswaldofilariinae, Waltonellinae and Splendidofilariinae, from saurian, anuran and bird respectively, Wolbachia are not detected. Conclusions/Significance The absence of Wolbachia in 63% of onchocercids, notably in the ancestral Oswaldofilariinae estimated 140 mya old, the diverse tissues or specimens distribution, and a recent lateral transfer in supergroup F Wolbachia, modify the current view on the role and evolution of the endosymbiont and their hosts. Further genomic analyses on some of the newly sampled species are welcomed to decipher the open questions.


Introduction
The alpha proteobacteria Wolbachia (Rickettsiales) are present in two distinct zoological groups: the arthropods, where they are widespread [1], and the nematodes, where they are restricted to a single but notable family of parasites, the Onchocercidae [2,3]. They encompass the agents of human onchocerciasis and lymphatic filariases [4]. The zoological host range of Wolbachia raised a fundamental question on the origin of infection in the filarial nematodes [5,6]. Investigations performed during the past fifteen years on Wolbachia in filarial and arthropod hosts has led to establish a rather clear and complex picture of the taxonomic status of the bacterium, its distribution and phylogeny [7]. Several distinct bacterial lineages have been called supergroups [8], and, at this date, they are all attributed to the only valid recognized species Wolbachia pipientis. The taxonomy of this species is quite uncertain, and in the scientific literature the genus name Wolbachia has been widely used as a specific name. This is taxomically incorrect, but common in microbiology (where species concept is usually complicated) and in the present work we will follow this trend until new data will be made available for a proper taxonomic restructuring [9,10]. The supergroups are in majority distinct in arthropods and filariae: A, B, E, H, I, K are found in the arthropods; C, D and J in the nematodes [6,8,11]. However, the supergroup F is a relevant and very well supported exception, encompassing arthropod and filarial hosts (i.e. some insects such as termites and the human filariae of the genus Mansonella, [12][13][14][15]). Moreover, a newly discovered Wolbachia harboured by a plant parasitic nematode might represent a further supergroup [16], while the supergroup G [17] has been decommissioned due to the high probability of being characterised on the basis of an event of recombination [18].
Whereas the bacteria are mainly parasites in arthropods, usually acting as manipulators of reproduction [19][20][21], they are mutualistic in filariae [4,22]. These mechanisms may be diverse, considering that Wolbachia is not only present in the germline but also in a somatic tissue, the hypodermis (lateral chords) of both females and males [23][24][25]. The biological studies and the Wolbachia genome projects [26] allowed us to suppose that the bacteria may be essential in the biosynthesis of some molecules necessary for filarial host fertility and viability, such as heme, riboflavin or nucleotide synthesis. Biosynthetic pathways are currently analyzed to determine the components of the symbiotic relationships [27][28][29][30][31][32]. To date, the mutualistic partnership is targeted in treatments against filariases using antibiotics [33].
The spiruroid ancestors of filariae that have been screened so far are devoid of endobacteria [5,34]. The presence/absence of Wolbachia mapped on a filarial nematodes phylogenetic tree suggests that the bacteria may have possibly been acquired as a single event in the lineage leading to the onchocercid nematodes, followed by host-parasite co-evolution, assuming that Wolbachia in filariae was strictly vertically transmitted to the offspring through the infected female germline [3,35]. Analysis of supergroup F is changing this view, due to the presence of Wolbachia from both filariae and some insects.
Another potential discrepancy with respect to the suggestion of coevolution is provided by observations of the absence of Wolbachia in two filarial species within the onchocercid lineage: the human parasite Loa loa and the rodent parasite Acanthocheilonema viteae [3,36]. It has been suggested that for these host species, the endobacteria had been present but subsequently were lost during further evolution [5]. As a corollary, the loss of Wolbachia led to a further hypothesis that the bacterial genes essential to the host fitness might have been successfully transferred and expressed into the host genome. Although still subject of discussion [21], some support for this hypothesis derives from studies on lateral gene transfer, as shown with several insect and filarial hosts [37]. Remnants of Wolbachia-like gene sequences have been identified in the filarial host genomes of the endobacteria-free L. loa and A. viteae, with some of the transferred genes being transcribed [38]. The elimination of the bacteria might be an adaptive advantage because their antigens are inflammation inducers and contribute to filarial pathologies and immunological responses [39]. However, a recent study suggests that the bacteria might act as a decoy target for polynuclear neutrophils, preventing harmful effect of eosinophils on filariae [40]. Furthermore, a strain of Wolbachia that overreplicates in Aedes aegypti inhibits the development of Brugia malayi larvae and switches on a few important immune system genes [41][42][43]. Thus the limitation of the filarial infection may either be due to immune activation of the invertebrate host or/and the bacteria may outcompetes filariae for some metabolites.
In our previous study [5], it appeared that the number of endobacteria-free filarial species had been underestimated and that several species without Wolbachia detected were parasitic in lizards and frogs. Thus it was suggested that these filariae from reptiles and anurans diversified before the first bacterial invasion on the onchocercid lineage which had been tentatively dated to 110 mya [3,6]. Indeed the origin of the Oswaldofilariinae, parasitic in crocodiles and squamates, was hypothetically dated from the late Jurassic, at the beginning of Gondwanian dislocation, 140 mya [44,45]. However representatives of this subfamily had not yet been screened.
Until the present study, Wolbachia screening had been done in only about 10% of the 93 genera currently recognized in the Onchocercidae [46,47]. This is not surprising since the recovery of filariae from connective tissues, their main localization, is not easy. Our investigation was resolutely rooted in biodiversity, expecting that the exploration of a broader range of filarial species would contribute to decipher the history of the Wolbachia-filaria symbiosis. The recovery of materials from wild animals from several biomes was undertaken. The first oswaldofilarine, the first splendidofilarine (a parasite of birds), and several onchocercid genera from mammals have now been screened through PCR, as well as classic immunohistochemical staining and whole mount fluorescent analysis [21].
This study confirms that Wolbachia are not detected until now in the filarioid species parasitic in amphibians and reptiles. Several other features have emerged from this study: i) lateral hypodermal localization of Wolbachia is not obligatory in bacteria-positive filarial species; ii) new somatic tissue localizations of the bacteria are observed; iii) the number of Wolbachia-free filarial species is greater than expected among filariae of mammals; (iv) lastly, one secondary event of Wolbachia infection, also well supported by a formal cocladogenesis, likely took place in filariae in supergroup F.

Results
The screening for Wolbachia was performed on 35 species (Table 1; specimens detailed in Figure S1 and Tables S1, S2), of which 28 are here examined for the first time and one recently by us [48]. These were the first representatives of Oswaldofilariinae, Piratuba scaffi from a lizard, and of Splendidofilariinae, Aproctella sp. 1 from passeriforms; two more species, Ochoterenella sp. 1 and O. royi, in Waltonellinae, a subfamily restricted to anurans; five genera of Onchocercinae parasitic in mammals, a species of Monanema, Mo. martini, parasitic in African murids and used during a decade as a model for onchocerciasis because of its skin-dwelling microfilariae [49]; several species of Cercopithifilaria, six from ruminants, one from a bear (all from Japan), and one species from an African porcupine; Loxodontofilaria, with the recently described Lo. caprini, recovered from a Japanese caprine bovid [50]; in the genus Mansonella, two of the six subgenera, Tetrapetalonema and Cutifilaria, with a species each, M. (T.) atelensis amazonae from a monkey, and M. (Cu.) perforata from a cervid; and the recently studied species of Litomosa from a South African bat (reported in [48]. The histoimmunostainings are presented according to the following genera: Litomosoides and Litomosa (Figure 1), Onchocerca and Loxodontofilaria (Figure 2), Cercopithifilaria japonica and Mansonella (Figure 3), other species of Cercopithifilaria ( Figure 4). Whole mount fluorescent analysis is presented on Figure 5.
The results (Table 1 and 2; Figures 1-7) can be summarised as following: 1. The presence or absence of Wolbachia were confirmed in species previously screened (Tables 2  and S2). Species previously studied and as expected confirmed positive are: Onchocerca volvulus (female worm from a human nodule, Cameroon), Dirofilaria repens (from an Italian patient), Litomosoides sigmodontis (2 females recovered from a wild Sigmodon hispidus, Venezuela) and a less commonly studied species, Dipetalonema gracile (a female recovered from Cebus olivaceus,  (Tables 2 and S2).
However, in four infected filarial species, Wolbachia were not detected in each specimen. These species were Lo. caprini, M. (Cu.) perforata, O. d. japonica and O. suzukii. The prevalence of Wolbachia varied from 50% to 66% in these species (9, 4, 8 and 4 samples, respectively, see Tables 2 and S2).  Figure 5A), but a few bacteria were observed in the second specimen ( Figure 5B).
4. Rare or novel tissue Wolbachia localizations were observed in Mansonella (Cutifilaria) perforata and Cercopithifilaria japonica. In M. (Cu.) perforata the somatic gonad was found positive once on sectioned worms and this was confirmed on a whole mounted worm; the epithelium lining the gonad harboured the bacteria, but not the external muscle layer ( Figure 5C). Moreover, bacteria were constantly found in the cells of the intestine wall, in both sectioned and whole mount material (Figures 3C-F; 5D). Wolbachia were also detected in the somatic gonad of C. japonica, in whole mounted worms, but not in sectioned worms ( Figure 5E); in this species, Wolbachia were not found in the intestinal cells.
5. Four types of Wolbachia were identified in the whole study. Interestingly, no events of recombination were found in 16S rDNA, dnaA, ftsZ and groEL genes of Wolbachia from the sequences of the filarial nematodes studied. According to the Wolbachia supergroups, four types of Wolbachia were identified in this study: bacteria harboured by Onchocerca, Dirofilaria and Loxodontofilaria species were assigned to supergroup C; bacteria harboured by L. sigmodontis and Litomosoides taylori were assigned to supergroup D; bacteria harboured by the species of the two subgenera of Mansonella (Tetrapetalonema and Cutifilaria) studied, as well as those harboured by Cercopithifilaria japonica were assigned to supergroup F; bacteria harboured by Dipetalonema gracile were assigned to supergroup J ( Figure 6).
6. Wolbachia was not detected in more than half of the newly screened species (Tables 2 and S2 7. Inconsistency between phylogenies of Wolbachia and filarial hosts was evidenced in the cocladogenetic analysis. The topologies of the trees of the infected filariae and their respective Wolbachia were found to have a significant level of similarity for all of the four tree metrics tested in Component, based on comparisons of the Wolbachia tree with 1000 random host trees (p,0.03 in all cases). The lnL scores of the filarial and Wolbachia ML trees based on the filarial dataset were 26189. 35 and 26207.14 respectively. A Shimodaira-Hasegawa test showed that the Wolbachia topology was significantly less likely (p = 0.035) than the filaria topology. The lnL scores of the Wolbachia and filarial ML trees based on the Wolbachia dataset were 210757.38 and 210950.85 respectively. A Shimodaira-Hasegawa test showed that the filaria tree was significantly less likely than the Wolbachia tree (p,0.001). Thus the null hypothesis that the host and parasite have strictly co-speciated was rejected. The major inconsistency was due to the newly screened Cercopithifilaria japonica and Mansonella (Cutifilaria) perforata ( Figure 7).
Another point of inconsistency in the phylogenies of hosts and symbionts regards the positioning of L. sigmodontis: while Wolbachia from this filarial is placed as the sister group of endosymbionts from lymphatic filariae (B. malayi, B. pahangi and W. bancrofti), the filaria itself is placed as a deeper branch in the filarial tree ( Figure 7). Indeed, based on morphological adult and larval criteria, Litomosoides is closer to lymphatic filariae than to Mansonella [45].

Discussion
As expected, the screening of a broader and more diversified set of species samples, by a combination of PCR and gene sequencing, immunohistological staining and whole mount fluorescent analysis,  revealed new information about Wolbachia biology and evolution: novel tissue localizations, strong evidence for recent transfers between unrelated filarial species, and a larger number of species that do not harbour Wolbachia. In addition, the occurrence of Wolbachia in some members of a species and its absence in others raises questions about the evolution of its obligate requirement [51].

New filarial tissues infected with Wolbachia
It is clear that tissues other than the female germline and the hypodermal lateral chords may be infected with Wolbachia. One of these infected tissues is the somatic gonad (epithelial layer), once briefly reported previously [52]. This Wolbachia localization was evidenced in Mansonella (Cutifilaria) perforata and Cercopithifilaria japonica (Table 2; Figures 5C,E). Interestingly, they are both members of the supergroup F of Wolbachia.
The real novelty is the Wolbachia tissue localization in the intestinal wall; it was only observed in M. (Cu.) perforata, but in all sectioned and whole mount samples ( Figures 3C-F, 5D).
These divergent localizations suggest a more complex and diversified relationship between the bacteria and filariae. They also raise the question of how and when Wolbachia bacteria reach the appropriate filarial host tissues. It is likely that it is an early event, since it was shown in Brugia malayi an asymmetric distribution of bacteria in the egg followed by a preferential segregation in defined blastomeres [21].

Recent capture of Wolbachia type F suggested by
Cercopithifilaria japonica, parasite of the Japanese bear The species screened in this study generally confirm the previously identified types of Wolbachia in the Onchocercidae ( Figure 6; see for instance [9]). The newly screened Loxodontofilaria is placed among the species of Onchocerca [53]. In addition, endosymbionts from this filaria belong to Wolbachia supergroup C ( Figure 6). There is a major phylogenetic congruence discrepancy between Wolbachia and their hosts and it occurs in the genus Cercopithifilaria and the supergroup F of Wolbachia. One African and seven Japanese species of Cercopithifilaria have no Wolbachia, while one species in Japan is Wolbachia positive. The filarial hosts belong to a well-supported genus, Cercopithifilaria as evidenced by adult morphology [45,54], larval morphology [55], 12S rDNA gene sequences [53,56], the transmission by hard ticks and the skin-dwelling microfilariae [45]. A parsimonious interpretation of the Wolbachia screening is that a single acquisition event took place in C. japonica. This hypothesis is supported by the co-cladogenetic analyses (Figure 7).
The supergroup F is intriguing as it is presently the only Wolbachia type infecting both insects and onchocercid nematodes [7]. The Wolbachia supergroup F contains the species of Mansonella  [58], has not been screened for Wolbachia but it likely harbours the type F Wolbachia. The bacterial host switching might have occurred between the two filarial parasites of the bear, perhaps via an oral infection route. Indeed filariae, despite their apparent small mouth, can ingest particles from their environment, such as red blood cells [59] and larger bodies, such as microfilariae released in the coelomic cavities of the filarial host [60].

Absence of Wolbachia
The filarial species in which Wolbachia were not detected appeared more numerous than it was thought, based upon previous observations. In [34], the percentage of negative species was 10.5% (2 negative among 19), in [5] with a larger sample, it was 37%. In this study, it is twice more elevated, 63%. It has to be emphasized that the negative results were not due to DNA degradations or bad extractions because all of the Wolbachia PCR negative samples gave positive amplifications using filarial nematode specific primers. However, we must take into account the fact that, in a few species, Wolbachia were not detected in all the specimens. This can partly be explained in species which do not harbour Wolbachia in the lateral chords, or at very low density, as Onchocerca dewittei japonica (Figures 5A,B) and Loxodontofilaria caprini (Figures 2H,I). In M. (Cu.) perforata, the bacteria are in the intestine wall of the female worms ( Figures 3C,D, 5D) and any part of female body would be Wolbachia positive, but this is not the case (Tables 2, S1 and S2). Thus, in M. (Cu.) perforata, presence/absence of Wolbachia may occur, as suggested by [15] for another Mansonella species, M. (Esslingeria) perstans from humans. It is interesting to note that in both of these cases, the Wolbachia supergroup is F. Further, research performed on deeply studied filariae, such as Brugia malayi, have also shown that the amount of Wolbachia carried by a worm may vary greatly over time and be stage-dependent [21]. This dynamic probably has little impact when considering developing larval stages, because these are transient and the chance of recovering them in the wild is extremely low. More interesting is the observation that the female worms recovered in the wild are not all fully gravid in some species ( Figure 2F). This was not the case of filariae from frogs, lizards, bats, birds, but was the case from some parasites of Japanese ungulates. It is worth to note that we paid great attention to the part of worm sampled to ensure that the germline was screened in all species.

Systematic position of Wolbachia negative filariae
It is clear that there is a need to increase the number of screened specimens for more solid results. However, in several cases, the global results are impressive and the distribution of the Wolbachia negative species does not appear random. As a matter of fact, at present, the species parasitic in frogs and lizards are negative for Wolbachia if they are Waltonellinae (three species of Ochoterenella), Oswaldofilariinae (one species of Piratuba), or Dirofilariinae (two species of Foleyella). It was also shown here that the first screened species parasitic in birds, an Aproctella in the Splendidofilariinae, was Wolbachia negative (7 females screened with PCR, from two passeriform species, totalising 7 specimens hosts). Wolbachia were not detected also in several species of Onchocercinae from mammals. The first is Litomosa chiropterorum, from an African bat (8 specimens screened from 8 Miniopterus schreibersi), an unexpected observation since the single Litomosa species screened previously, Li. westi from a North American rodent Geomyoidea, was Wolbachia positive [5].
The second species is Monanema martini (8 females screened from 8 murid specimens). The absence of the bacteria is an important feature, considering that this filaria was used, in the past, as a model of onchocerciasis, a true limit due to the important role played by the bacteria in the pathology of the disease [39,[61][62][63]. The absence of Wolbachia could explain partly the weak ocular pathology induced in the murid host [49]. Severe or mild ocular onchocerciases have been related to a strain-dependent variation of density of Wolbachia per filarial genome [64].
Other species in which Wolbachia were not detected belong to the genus Cercopithifilaria; 8 out of 9 species were Wolbachia-negative (Table 2; Figures 4; 5F).
Two hypotheses might be taken into account to explain the absence of Wolbachia. In the first case, Wolbachia could be considered to be present and then were subsequently lost [38]. This could have occurred before the mutually dependent symbiosis between symbiont and host developed. The second hypothesis is that the bacteria were not yet acquired within that filarial group.
The first hypothesis seems to possibly explain some of the observed cases, such as Litomosoides yutajensis (5 samples: 2 males previously and now 3 more females), a species without Wolbachia, in contrast with 5 congeneric species infected with Wolbachia. In the cases of Acantocheilonema viteae and Onchocerca flexuosa [38], the absence of Wolbachia appears to be a secondary loss, because some genes of the bacteria were incorporated in the filarial genomes. However, it is not clear whether this loss occurred before symbiosis was established or not. This is of interest because if loss was after symbiotic establishment, perhaps some of the genes incorporated into the host genome were those required by the host now provided by Wolbachia, but the extent of this event is still to be understood.
The hypothesis that the Wolbachia negative species might never have been infected, might be considered for the filarial groups that are supposed to be ''ancient'' such as the Oswaldofilariinae [44,45,65]. Estimation of dates of divergence has been proposed for some groups of nematodes based on molecular phylogenetic analyses [66]. In filariae, data from morphology, biology, geographic distribution, host range and palaeontology led to the proposal that the Oswaldofilariinae emerged during the late Jurassic, at the beginning of the Gondwanian break up, 140 mya [44,45]. This is before the hypothesized ancestral acquisition of a Wolbachia by an onchocercid [3]; it follows that the absence of Wolbachia in Oswaldofilariinae could be primitive.
Foleyella is another parasite of saurians, which appears to have no Wolbachia; it is presently placed in the Dirofilariinae, which includes the Wolbachia positive Dirofilaria, but this systematic position needs to be revised because the characters of the infective larvae are very distinct [55]. A solid phylogeny of Onchocercidae linking traditional and molecular data is needed and warrants further investigations.
The subfamily Setariinae, parasitic in ungulates and in which Wolbachia were not detected, as first shown by [67], also deserves a comment. Based on larval morphology, it has been hypothesized that it evolved separately from the other onchocercids and derived from a group of spirurid Habronematinae [65]. Until now no spirurids have been found infected with Wolbachia [5,34].
Did the bacterial infection never occur, or was the useful part of Wolbachia genome incorporated in the host genome and subsequently the bacteria eliminated, to reply to some adverse constraint? Further genomic analyses will solve this question concerning the absence of Wolbachia in ancestral filariae.
At present, the features observed on tissue or specimen distribution, and a very probable recent lateral transfer, suggest complex evolutionary dynamics of interactions between the symbionts, their host filariae and the nematode hosts. In the sampling studied, which will be further enlarged, it may become possible to use some defined species to decipher these questions.
All experiments, procedures and ethical issues were conformed to the competent national ethical bodies: Venezuelian animals were captured according to Licencia con Fines Cientificos Nu 2192 dated June 18, 2007 and Contrato Marco Acceso Recursos Genéticos Nu 33/2007 both granted by Ministerio del Ambiente de la Republica Bolivariana de Venezuela. Japanese serows, sika deer, bears, and wild boars were killed by hunters who have an individual permit to kill wild animals in accordance with the conservation and control policies of the Ministry of the Environment of Japan. Italian samples were collected by veterinarians and physicians and no permits were necessary. African rodents and agama do not belong to protected species and were obtained from local hunters. African mountain reedbuck, plain zebra, gemsbok and porcupine were also obtained from local hunters. Bats from South African bats have been collected for previous studies [48] in which no permits were necessary. Brazilian birds were donated by the Brazilian Institute of the Environment and Renewable Resources (IBAMA), Region of Juiz de Fora, Minas Gerais for the laboratory of Taxonomia and Ecology of Helminths of the Department of the Zoology.
Many of the filariae from large mammals were extracted from the subconnective tissue, dermis, or tendons of limbs. Host animals kept at 4uC were sent to the laboratory after they were killed. Afterwards the parts of the animals were dissected to collect living filarioids for Wolbachia study. From frogs, lizards, birds, bats and rodents filariae were generally recovered immediately after host death. Samples used for positive and negative PCR controls were laboratory strains.
Species identification of the specimens was done with morphological studies performed by several of us (OB, SU, RG, SL, MD). Some species are not yet named, but all are under study, and morphological analysis and sequencing of coxI gene in this study and in [53] showed that they represent distinct molecular entities. Co-infection of a host specimen by several congeneric or non-congeneric filarial species was rather frequent in large mammals, but in a few cases, the same filarial species was recovered from two host species. The supraspecific levels of taxonomy followed the systematic works of [46,73,74] and more recent studies for some taxa: [65,71,75] Figure S1, Tables S1 and S2.
Filarial worms were fixed and kept in absolute alcohol at 4uC for PCR analyses, in 4% PFA (paraformaldehyde) for overnight at 4uC for immunohistochemical staining and whole mount fluorescent analysis. In many cases, filarial specimens were cut into anterior (a), median (m) and posterior (p) parts, which were fixed for the different analysis approaches. Since Wolbachia are transmitted by female filariae, almost all of the studies were based on female worms; male specimens were rarely examined, and we analysed this sex alone in a single case, Di. gracile (Table S3).
Molecular screening for Wolbachia on filarial nematodes PCR screening for Wolbachia was conducted according to [5,12], using general Wolbachia primers for 16S rDNA (99f and 994r [76]), originally designed to work on Wolbachia from the supergroups A and B, and primers for 16S rDNA (16SWolbF and 16SWolbR3), originally designed to work on Wolbachia from the supergroups A-D [12], but whose target sites are also conserved in Wolbachia from supergroups E and F [8].
PCRs were performed in a 20 ml final volume under the following conditions: 1x buffer (containing 1.5 mM MgCl 2 , Eppendorf TM ), 0.2 mM of each dNTP, 1 mM of each primer, and 0.5 U of Taq DNA Polymerase (Eppendorf TM ). The thermal profile used was: 94uC 45 sec, 52uC 45 sec, and 72uC 90 sec for 40 cycles.
When the PCRs were negative under the above PCR conditions, a nested-PCR approach was implemented in order to improve the sensitivity of the PCR screening [5,77]. The first PCR was performed using the general eubacterial primer 27F [78] combined with 16SWolbR3; PCR volumes and conditions were as above. Five ml were visualised on a 1.5% w/v agarose gel and one ml of the first PCR was diluted 1/10 and 1/100 in water, and then both used as templates in a second PCR, performed using primers W-EF and W-ER [70]. W-ER and W-EF recognize sites that are conserved in supergroups E-F and that are internal to the primers used in the first PCR. PCR conditions for this amplification were as described in [79].
Of samples remaining negative after the two PCRs approaches described above, PCRs with primers 16SWolbF and 16SWolbR3 were performed varying the following parameters: MgCl 2 concentrations at 2.5, 4 and 6 mM and annealing temperatures of 52uC +/25uC. DNA preparations from filarial species harbouring Wolbachia (D. immitis and Brugia pahangi) and from a Wolbachia-infected strain of mosquitoes (Culex pipiens) [5] were included in the screening as positive controls. DNA preparations from a filarial species not harbouring Wolbachia (A. viteae) [5] were included as negative controls.
Of the samples positive for PCR screening homologous to Wolbachia 16S rDNA, dnaA, ftsZ and groEL were also amplified using the primers described in [5,80]  Amplifications were performed in 20-50 ml volumes. In all cases, in order to ascertain the DNA conditions before Wolbachia screenings and to confirm morphological identification, coxI amplification was performed as described in [12].

Sequencing conditions
coxI sequencing was performed as described in [12]. dnaA, ftsZ and groEL sequencing were performed as described in [5,80]. From the Wolbachia PCR positive samples, almost the full length of the 16S rDNA gene of Wolbachia was sequenced using primers 27F and 16SWolbR3. The amplifications obtained (about 1400 bp) were gel-purified (using the QIAquickH PCR Purification Kit, Qiagen) and directly sequenced using ABI technology. The sequences obtained have been deposited in the EMBL Data Library. The dnaA, groEL and ftsZ sequences were not obtained from all the taxa included in this study mainly caused by the scarcity of certain specimens, and amplification/sequencing problems for some of the species examined. Where DNA of the host was amplified, the PCR product was purified as above and directly sequenced using ABI technology.
A list of the sequences including accession numbers is available in Table S2.

Immunohistochemical staining of worm sections
Immunohistochemical staining was performed according to [24,48]. A rabbit polyclonal antiserum raised against the WSP of the Wolbachia from B. pahangi (designed by [81]) has been used to stain samples of 13 filarial species (see Tables S2 and S3). After paraffin inclusion, 4 mm sections were obtained and placed on Silane coated glass slides (3-aminpropyltriethoxysilane) and then kept at 63uC overnight, to avoid sections detaching from slides. To verify that the 63uC overnight did not alter the specific epitopes, Wolbachia positive L. sigmodontis laboratory strain were incubated at both 63uC and room temperature overnight and then stained: no significant differences in the staining were observed. Negative controls were carried out by omitting the primary antibody.
Fixed female worms were divided in three main parts, posterior (p), median (c) and anterior (a) in order to observe the different regions of the genital tract. Transverse sections were made at different levels of each part, and few of them were stained with hemalun-eosin for anatomical identification. Lateral cuticular internal crests were identified to orient the worm section; hypodermal chords extend above and on the side of the crests. The filarial species used for histology are opisthodelphic and the initial part of the ovaries is in the posterior part of the worm; the distal region of the ovary is composed of a cytoplasmic axis, the rachis [82,83], and an outer cytoplasmic Figure 6. Phylogeny of Wolbachia based on 16S rDNA gene sequences. Names at the terminal nodes are those of the host species (with the exceptions of the outgroup species). A-J are the supergroups names according to [1,3,8,14]. The tree has been obtained by Bayesian inference of phylogeny, using MrBayes 2.01; numbers at the nodes are the posterior probability values. Species in bold are those newly found positive in the screening for Wolbachia performed in this study. Accession numbers are given for the sequences present in the databases. doi:10.1371/journal.pone.0020843.g006 layer with the nuclei of oogonia and oocytes (in the text we will refer to these states as oocytes, and germline to describe the whole production of the gonad). An epithelial layer and an outer muscle layer surround the gonad, both referred to as somatic gonad in the text [25]. Uteri occupy almost the whole body and are found in median and anterior part of worms. The different uterine contents are ovulae, spermatozoa, divided eggs and microfilariae. Eggs were identified as aborted by hemalun-eosin staining when divided eggs were eosinophilic and nuclei not discernible. The laboratory strain of Litomosoides sigmodontis, which has been shown in several studies to harbour Wolbachia [84][85][86] was used as a positive reference for Wolbachia immunostaining.

Whole mount fluorescent analysis
Worms were cut with a razor blade to expose the different tissues to RNAse A (15 mg/mL, Sigma) in rotating tubes overnight at 4C. They were rinsed in PBS, and incubated with a fluorochrome-conjugated Phalloidin (atto2488 Phalloidin, Fluka, at 10 nanomolar) overnight to stain F-actin, followed by a Propidium Iodide (Molecular Probes, 1.0 mg/mL solution) incubation for DNA staining for 20 minutes in PBS (1:50) and a 5 minutes wash. Tissues were mounted in Vectashield (Vector Laboratories) [25]. The species analyzed were C. crassa, C. japonica, Lo. caprini, M. (Cu.) perforata and O. d. japonica (Table S1).

Phylogenetic reconstruction
The bacterial 16S rDNA, dnaA, groEL and ftsZ sequences and filarial 12S rDNA and coxI sequences generated were aligned with the sequences available in the databases (for the ribosomal genes according to their secondary structures) using ClustalX2 [87].
The alignments were analysed using Maximum Likelihood (ML) and Bayesian Inference of phylogeny (BI) methods. The appropriate model of sequence evolution for ML and BI was estimated via likelihood ratio test using Modeltest 3.7 [88]: the model selected for the filarial concatenated dataset was HKY+G, while the model for the Wolbachia concatenated dataset was GTR+I+G. Phylogenetic analyses were performed using PAUP* 4.0 b10 [89] and MrBayes [90]. In addition, a phylogenetic tree was inferred with a GTR+C 4 nucleotide substitution model in a Bayesian framework using MrBayes version 3.0 [90]. Two independent runs were performed, each using 1 million steps with four chains sampling every 100 steps. The first 10% of the trees were removed and posterior probabilities were calculated from these post-burnin trees.

Test for recombination
Four Wolbachia alignments (relative to the genes 16S rDNA, ftsZ, groEL and dnaA) were screened for presence of recombination events by using a set of nonparametric detection programs: RDP, GENECONV, Bootscan, MaxChi, Chimaera, SisScan and LARD [91][92][93][94][95][96][97]. The first six programs search for putative recombination breakpoints in a set of aligned DNA sequences and are implemented in RDP3 software package, whilst LARD checks signals detected by other methods [92]. Sequences were automasked for optimal recombination detection. General recombination settings were as follows: sequences were considered linear Figure 7. Cocladogenesis of Wolbachia and filarial nematodes based on representatives of the species studied. For these species sequences for nematode coxI and 12S rDNA (phylogeny on the left) and bacterial 16S rDNA (phylogeny on the right) were available. Dotted lines have been added to emphasize major discrepancies between filarial and Wolbachia trees. For further details, see paragraph 7 in the Results section. doi:10.1371/journal.pone.0020843.g007 and the highest acceptable P-Value was set to 0.01 and overlapping signals were disentangled.
Method specific options were as follow: MaxChi and Chimaera were run with a variable window size; Bootscan and SisScan were forced for exploratory screening. Successive phases of refining analysis and manual tests were performed where needed.

Cocladogenesis analysis
To test for congruence, phylogenies for the hosts (based on a concatenated dataset of coxI and 12S rDNA) and for the bacterium (based on a concatenated dataset of 16S rDNA, ftsZ, dnaA and groEL) were compared with two methods. The first tested whether there was a greater than random correspondence between reconstructed nodes for host and symbiont. This was performed in Component (R. Page, University of Glasgow, UK) using the ''compare tree with'' function, with 1,000 randomized trees and the four available tree-comparison metrics (partition; triplets; quartets, nearest neighbour interchange). The second test examined the null hypothesis that the endosymbionts have undergone cocladogenesis with their hosts. ML trees for each dataset were first estimated using the successive approximation method [89]. Then, the scores for each of these ML trees based on the host dataset were compared using the [98] test. This was repeated using the parasite dataset. A single uninfected filarial (T. callipaeda) and a non-filarial Wolbachia (from C. quinquefasciatus) were used as outgroups. Figure S1 Position of the genera screened in the present study indicated on a schematic representation of a key of the onchocercid subfamilies, based on morphological characters (following [46]). Total number of genera per subfamily listed. *Genus screened for the first time. ** Two subgenera in Mansonella.

(DOC)
Table S1 Details of material studied with PCR, immunostaining assays (IHS) and whole mount fluorescent analysis (fluo). The scheme follows the classification as in Tables 1 and 2. Species, genus and subgenus, subfamily are in bold characters when newly screened; specimens ids are in bold characters when female worms; m = male; f = female; a = anterior part; c = central part; p = posterior part; *150 infective larvae; + = Wolbachia positive specimens. (DOC)