Wolbachia in the spittlebug Prosapia ignipectus: Variable infection frequencies,but no apparent effect on host reproductive isolation

Animals serve as hosts for complex communities of microorganisms, including endosymbionts that live inside their cells. Wolbachia bacteria are perhaps the most common endosymbionts, manipulating host reproduction to propagate. Many Wolbachia cause cytoplasmic incompatibility (CI), which results in reduced egg hatch when uninfected females mate with infected males. Wolbachia that cause intense CI spread to high and relatively stable frequencies, while strains that cause weak or no CI tend to persist at intermediate, often variable, frequencies. Wolbachia could also contribute to host reproductive isolation (RI), although current support for such contributions is limited to a few systems. To test for Wolbachia frequency variation and effects on host RI, we sampled several local Prosapia ignipectus (Fitch) (Hemiptera: Cercopidae) spittlebug populations in the northeastern United States over two years, including closely juxtaposed Maine populations with different monomorphic color forms, “black” and “lined.” We discovered a group‐B Wolbachia (wPig) infecting P. ignipectus that diverged from group‐A Wolbachia—like model wMel and wRi strains in Drosophila—6 to 46 MYA. Populations of the sister species Prosapia bicincta (Say) from Hawaii and Florida are uninfected, suggesting that P. ignipectus acquired wPig after their initial divergence. wPig frequencies were generally high and variable among sites and between years. While phenotyping wPig effects on host reproduction is not currently feasible, the wPig genome contains three divergent sets of CI loci, consistent with high wPig frequencies. Finally, Maine monomorphic black and monomorphic lined populations of P. ignipectus share both wPig and mtDNA haplotypes, implying no apparent effect of wPig on the maintenance of this morphological contact zone. We hypothesize P. ignipectus acquired wPig horizontally as observed for many Drosophila species, and that significant CI and variable transmission produce high but variable wPig frequencies.     

Positive Selection and Horizontal Gene Transfer in the Genome of a Male-Killing Wolbachia

Wolbachia are a genus of widespread bacterial endosymbionts in which some strains can hijack or manipulate arthropod host reproduction. Male killing is one such manipulation in which these maternally transmitted bacteria benefit surviving daughters in part by removing competition with the sons for scarce resources. Despite previous findings of interesting genome features of microbial sex ratio distorters, the population genomics of male-killers remain largely uncharacterized. Here, we uncover several unique features of the genome and population genomics of four Arizonan populations of a male-killing Wolbachia strain, wInn, that infects mushroom-feeding Drosophila innubila. We first compared the wInn genome with other closely related Wolbachia genomes of Drosophila hosts in terms of genome content and confirm that the wInn genome is largely similar in overall gene content to the wMel strain infecting D. melanogaster. However, it also contains many unique genes and repetitive genetic elements that indicate lateral gene transfers between wInn and non-Drosophila eukaryotes. We also find that, in line with literature precedent, genes in the Wolbachia prophage and Octomom regions are under positive selection. Of all the genes under positive selection, many also show evidence of recent horizontal transfer among Wolbachia symbiont genomes. These dynamics of selection and horizontal gene transfer across the genomes of several Wolbachia strains and diverse host species may be important underlying factors in Wolbachia’s success as a male-killer of divergent host species.     

Male killing Spiroplasma protects Drosophila melanogaster against two parasitoid wasps

Maternally transmitted associations between endosymbiotic bacteria and insects are diverse and widespread in nature. Owing to imperfect vertical transmission, many heritable microbes have evolved compensational mechanisms to enhance their persistence in host lineages, such as manipulating host reproduction and conferring fitness benefits to host. Symbiont-mediated defense against natural enemies of hosts is increasingly recognized as an important mechanism by which endosymbionts enhance host fitness. Members of the genus Spiroplasma associated with distantly related Drosophila hosts are known to engage in either reproductive parasitism (i.e., male killing) or defense against natural enemies (the parasitic wasp Leptopilina heterotoma and a nematode). A male-killing strain of Spiroplasma (strain Melanogaster Sex Ratio Organism (MSRO)) co-occurs with Wolbachia (strain wMel) in certain wild populations of the model organism Drosophila melanogaster. We examined the effects of Spiroplasma MSRO and Wolbachia wMel on Drosophila survival against parasitism by two common wasps, Leptopilina heterotoma and Leptopilina boulardi, that differ in their host ranges and host evasion strategies. The results indicate that Spiroplasma MSRO prevents successful development of both wasps, and confers a small, albeit significant, increase in larva-to-adult survival of flies subjected to wasp attacks. We modeled the conditions under which defense can contribute to Spiroplasma persistence. Wolbachia also confers a weak, but significant, survival advantage to flies attacked by L. heterotoma. The host protective effects exhibited by Spiroplasma and Wolbachia are additive and may provide the conditions for such cotransmitted symbionts to become mutualists. Occurrence of Spiroplasma-mediated protection against distinct parasitoids in divergent Drosophila hosts suggests a general protection mechanism.     

Reproductive compensation favours male-killing Wolbachia in a live-bearing host

Wolbachia are maternally inherited, cellular endosymbionts that can enhance their fitness by biasing host sex ratio in favour of females. Male killing (MK) is an extreme form of sex-ratio manipulation that is selectively advantageous if the self-sacrifice of Wolbachia in males increases transmission through females. In live-bearing hosts, females typically produce more embryos than can be carried to term, and reproductive compensation through maternal resource reallocation from dead males to female embryos could increase the number of daughters born to infected females. Here, we report a new strain of MK Wolbachia (wCsc2) in the pseudoscorpion, Cordylochernes scorpioides, and present the first empirical evidence that reproductive compensation favours the killing of males in a viviparous host. Females infected with the wCsc2 strain produced 26 per cent more and significantly larger daughters than tetracycline-cured females. In contrast to the previously described wCsc1 MK Wolbachia strain in C. scorpioides, wCsc2 infection was not accompanied by an increase in the rate of spontaneous brood abortion. Characterization of the wCsc1 and wCsc2 strains by multi-locus sequence typing and by Wolbachia surface protein (wsp) gene sequencing indicates that the marked divergence between these two MK strains in their impact on host reproductive success, and hence in their potential to spread, has occurred in association with homologous recombination in the wsp gene.     

Oxidative Stress Correlates with Wolbachia-Mediated Antiviral Protection in Wolbachia-Drosophila Associations

Wolbachia mediates antiviral protection in insect hosts and is being developed as a potential biocontrol agent to reduce the spread of insect-vectored viruses. Definition of the molecular mechanism that generates protection is important for understanding the tripartite interaction between host insect, Wolbachia, and virus. Elevated oxidative stress was previously reported for a mosquito line experimentally infected with Wolbachia, suggesting that oxidative stress is important for Wolbachia-mediated antiviral protection. However, Wolbachia experimentally introduced into mosquitoes impacts a range of host fitness traits, some of which are unrelated to antiviral protection. To explore whether elevated oxidative stress is associated with antiviral protection in Wolbachia-infected insects, we analyzed oxidative stress of five Wolbachia-infected Drosophila lines. In flies infected with protective Wolbachia strains, hydrogen peroxide concentrations were 1.25- to 2-fold higher than those in paired fly lines cured of Wolbachia infection. In contrast, there was no difference in the hydrogen peroxide concentrations in flies infected with nonprotective Wolbachia strains compared to flies cured of Wolbachia infection. Using a Drosophila mutant that produces increased levels of hydrogen peroxide, we investigated whether flies with high levels of endogenous reactive oxygen species had altered responses to virus infection and found that flies with high levels of endogenous hydrogen peroxide were less susceptible to virus-induced mortality. Taken together, these results suggest that elevated oxidative stress correlates with Wolbachia-mediated antiviral protection in natural Drosophila hosts.     

Evolutionary Dynamics of wAu-Like Wolbachia Variants in Neotropical Drosophila spp.

Wolbachia bacteria are common intracellular symbionts of arthropods and have been extensively studied in Drosophila. Most research focuses on two Old Word hosts, Drosophila melanogaster and Drosophila simulans, and does not take into account that some of the Wolbachia associations in these species may have evolved only after their fast global expansion and after the exposure to Wolbachia of previously isolated habitats. Here we looked at Wolbachia of Neotropical Drosophila species. Seventy-one lines of 16 Neotropical Drosophila species sampled in different regions and at different time points were analyzed. Wolbachia is absent in lines of Drosophila willistoni collected before the 1970s, but more recent samples are infected with a strain designated wWil. Wolbachia is absent in all other species of the willistoni group. Polymorphic wWil-related strains were detected in some saltans group species, with D. septentriosaltans being coinfected with at least four variants. Based on wsp and ftsZ sequence data, wWil of D. willistoni is identical to wAu, a strain isolated from D. simulans, but can be discriminated when using a polymorphic minisatellite marker. In contrast to wAu, which infects both germ line and somatic tissues of D. simulans, wWil is found exclusively in the primordial germ line cells of D. willistoni embryos. We report on a pool of closely related Wolbachia strains in Neotropical Drosophila species as a potential source for the wAu strain in D. simulans. Possible evolutionary scenarios reconstructing the infection history of wAu-like Wolbachia in Neotropical Drosophila species and the Old World species D. simulans are discussed.     

Monitoring long-term evolutionary changes following Wolbachia introduction into a novel host: the Wolbachia popcorn infection in Drosophila simulans

Wolbachia may act as a biological control agent for pest management; in particular, the Wolbachia variant wMelPop (popcorn) shortens host longevity and may be useful for dengue suppression. However, long-term changes in the host and Wolbachia genomes can alter Wolbachia spread and/or host effects that suppress disease. Here, we investigate the phenotypic effects of wMelPop in a non-native host, Drosophila simulans, following artificial transinfection approximately 200 generations ago. Long-term rearing and maintenance of the bacteria were at 19°C in the original I-102 genetic background that was transinfected with the popcorn strain. The bacteria were then introgressed into three massbred backgrounds, and tetracycline was used to create uninfected sublines. The effect of wMelPop on longevity in this species appears to have changed; longevity was no longer reduced at 25°C in some nuclear backgrounds, reflecting different geographical origin, selection or drift, although the reduction was still evident for flies held at 30°C. Wolbachia influenced productivity and viability, and development time in some host backgrounds. These findings suggest that long-term attenuation of Wolbachia effects may compromise the effectiveness of this bacterium in pest control. They also emphasize the importance of host nuclear background on Wolbachia phenotypic effects.     

Effects of Wolbachia on fitness of the Mediterranean fruit fly (Diptera: Tephritidae)

Wolbachia pipientis is a widespread endosymbiont of insects and other arthropods exerting a wide range of biological effects on their hosts. A growing number of recent studies document the influence of Wolbachia on reproduction and lifespan of insect host species. However, little is known regarding effects of Wolbachia on the demographic traits of different host populations. Moreover, whether different Wolbachia strains exert different effects on fitness components of their hosts remains largely unknown. We studied the effects of (a) the Wolbachia strain wCer2 on fitness components of two laboratory lines of the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae) and (b) two different Wolbachia strains (wCer2 and wCer4) on one of the Mediterranean fruit fly lines. Wolbachia infection (wCer2) shortens the egg‐to‐adult developmental duration of both C. capitata lines, although it prolongs embryonic development. In one of the two lines, egg‐to‐adult mortality increased. Wolbachia infection shortens adult lifespan (to a different extent in males and females) and reduces female fecundity. The different Wolbachia strains differentially affect both immature mortality and developmental duration, and adult longevity and female fecundity. Our findings demonstrate both differential response of two C. capitata lines to Wolbachia infection and differential effects of two Wolbachia strains on the same Mediterranean fruit fly line. Practical and theoretical implications of our findings are discussed.     

Mutualism Breakdown by Amplification of Wolbachia Genes

Most insect species are associated with vertically transmitted endosymbionts. Because of the mode of transmission, the fitness of these symbionts is dependent on the fitness of the hosts. Therefore, these endosymbionts need to control their proliferation in order to minimize their cost for the host. The genetic bases and mechanisms of this regulation remain largely undetermined. The maternally inherited bacteria of the genus Wolbachia are the most common endosymbionts of insects, providing some of them with fitness benefits. In Drosophila melanogaster, Wolbachia wMelPop is a unique virulent variant that proliferates massively in the hosts and shortens their lifespan. The genetic bases of wMelPop virulence are unknown, and their identification would allow a better understanding of how Wolbachia levels are regulated. Here we show that amplification of a region containing eight Wolbachia genes, called Octomom, is responsible for wMelPop virulence. Using Drosophila lines selected for carrying Wolbachia with different Octomom copy numbers, we demonstrate that the number of Octomom copies determines Wolbachia titers and the strength of the lethal phenotype. Octomom amplification is unstable, and reversion of copy number to one reverts all the phenotypes. Our results provide a link between genotype and phenotype in Wolbachia and identify a genomic region regulating Wolbachia proliferation. We also prove that these bacteria can evolve rapidly. Rapid evolution by changes in gene copy number may be common in endosymbionts with a high number of mobile elements and other repeated regions. Understanding wMelPop pathogenicity and variability also allows researchers to better control and predict the outcome of releasing mosquitoes transinfected with this variant to block human vector-borne diseases. Our results show that transition from a mutualist to a pathogen may occur because of a single genomic change in the endosymbiont. This implies that there must be constant selection on endosymbionts to control their densities.     

Distribution and Evolutionary Impact of Wolbachia on Butterfly Hosts

Wolbachia are maternally inherited endosymbiotic alpha-proteobacteria found in terrestrial arthropods and filarial nematodes. They are transmitted vertically through host cytoplasm and alter host biology by inducing various reproductive alterations, like feminization, parthenogenesis, male killing (MK) and cytoplasmic incompatibility. In butterflies, some effects especially MK and sperm-egg incompatibility are well established. All these effects skew the sex ratio towards female and subsequently favor the vertical transmission of Wolbachia. Some of the insects are also infected with multiple Wolbachia strains which may results in some complex phenomenon. In the present review the potential of Wolbachia for promoting evolutionary changes in its hosts with emphasis on recent advances in interactions of butterfly–Wolbachia is discussed. In addition to this, strain diversity of Wolbachia and its effects on various butterfly hosts are also highlighted.     

Diversifying selection and host adaptation in two endosymbiont genomes

Background  

The endosymbiont Wolbachia pipientis infects a broad range of arthropod and filarial nematode hosts. These diverse associations form an attractive model for understanding host:symbiont coevolution. Wolbachia 's ubiquity and ability to dramatically alter host reproductive biology also form the foundation of research strategies aimed at controlling insect pests and vector-borne disease. The Wolbachia strains that infect nematodes are phylogenetically distinct, strictly vertically transmitted, and required by their hosts for growth and reproduction. Insects in contrast form more fluid associations with Wolbachia. In these taxa, host populations are most often polymorphic for infection, horizontal transmission occurs between distantly related hosts, and direct fitness effects on hosts are mild. Despite extensive interest in the Wolbachia system for many years, relatively little is known about the molecular mechanisms that mediate its varied interactions with different hosts. We have compared the genomes of the Wolbachia that infect Drosophila melanogaster, w Mel and the nematode Brugia malayi, w Bm to that of an outgroup Anaplasma marginale to identify genes that have experienced diversifying selection in the Wolbachia lineages. The goal of the study was to identify likely molecular mechanisms of the symbiosis and to understand the nature of the diverse association across different hosts.     

Wolbachia modifies thermal preference in Drosophila melanogaster

Environmental variation can have profound and direct effects on fitness, fecundity, and host–symbiont interactions. Replication rates of microbes within arthropod hosts, for example, are correlated with incubation temperature but less is known about the influence of host–symbiont dynamics on environmental preference. Hence, we conducted thermal preference (T_p) assays and tested if infection status and genetic variation in endosymbiont bacterium Wolbachia affected temperature choice of Drosophila melanogaster. We demonstrate that isogenic flies infected with Wolbachia preferred lower temperatures compared with uninfected Drosophila. Moreover, T_p varied with respect to three investigated Wolbachia variants (wMel, wMelCS, and wMelPop). While uninfected individuals preferred 24.4°C, we found significant shifts of −1.2°C in wMel- and −4°C in flies infected either with wMelCS or wMelPop. We, therefore, postulate that Wolbachia-associated T_p variation within a host species might represent a behavioural accommodation to host–symbiont interactions and trigger behavioural self-medication and bacterial titre regulation by the host.     

wFlu: Characterization and Evaluation of a Native Wolbachia from the Mosquito Aedes fluviatilis as a Potential Vector Control Agent

There is currently considerable interest and practical progress in using the endosymbiotic bacteria Wolbachia as a vector control agent for human vector-borne diseases. Such vector control strategies may require the introduction of multiple, different Wolbachia strains into target vector populations, necessitating the identification and characterization of appropriate endosymbiont variants. Here, we report preliminary characterization of wFlu, a native Wolbachia from the neotropical mosquito Aedes fluviatilis, and evaluate its potential as a vector control agent by confirming its ability to cause cytoplasmic incompatibility, and measuring its effect on three parameters determining host fitness (survival, fecundity and fertility), as well as vector competence (susceptibility) for pathogen infection. Using an aposymbiotic strain of Ae. fluviatilis cured of its native Wolbachia by antibiotic treatment, we show that in its natural host wFlu causes incomplete, but high levels of, unidirectional cytoplasmic incompatibility, has high rates of maternal transmission, and no detectable fitness costs, indicating a high capacity to rapidly spread through host populations. However, wFlu does not inhibit, and even enhances, oocyst infection with the avian malaria parasite Plasmodium gallinaceum. The stage- and sex-specific density of wFlu was relatively low, and with limited tissue distribution, consistent with the lack of virulence and pathogen interference/symbiont-mediated protection observed. Unexpectedly, the density of wFlu was also shown to be specifically-reduced in the ovaries after bloodfeeding Ae. fluviatilis. Overall, our observations indicate that the Wolbachia strain wFlu has the potential to be used as a vector control agent, and suggests that appreciable mutualistic coevolution has occurred between this endosymbiont and its natural host. Future work will be needed to determine whether wFlu has virulent host effects and/or exhibits pathogen interference when artificially-transfected to the novel mosquito hosts that are the vectors of human pathogens.     

New Insight into Wolbachia Epidemiology: Its Varying Incidence During the Host Life Cycle Can Alter Bacteria Spread

Wolbachia is an obligate endosymbiont whose spread depends mainly on its capacity to alter host reproduction by, for instance, cytoplasmic incompatibility. Several mathematical models have been developed to explain the dynamics of bacterial spread, because of its applied interest. However, some aspects of the host’s and bacterium’s biology have not been considered in modelling: for instance, changes in Wolbachia proportions during the host’s life cycle have been observed in several species, including Drosophila sp., Nasonia sp. and Aedes sp. (Diptera), but also in the grasshopper Chorthippus parallelus (Orthoptera), the species studied in this article. These changes influence the proportion of incompatible crosses and, consequently, infection prevalence in subsequent generations. In this paper, we are interested in ascertaining whether these changes in the infection proportions during the host’s life cycle can influence the dynamics of the spread of these bacteria. We have examined its consequences using a mathematical model to predict the evolution of Wolbachia infection frequencies. The simulations were validated by experimental field data from C. parallelus. The main outcome is that those changes above mentioned might affect long-term infection spread, with possible consequences for the current distribution of Wolbachia and the way it affects its host’s reproduction.     

Rapid Sequential Spread of Two Wolbachia Variants in Drosophila simulans

The maternally inherited intracellular bacteria Wolbachia can manipulate host reproduction in various ways that foster frequency increases within and among host populations. Manipulations involving cytoplasmic incompatibility (CI), where matings between infected males and uninfected females produce non-viable embryos, are common in arthropods and produce a reproductive advantage for infected females. CI was associated with the spread of Wolbachia variant wRi in Californian populations of Drosophila simulans, which was interpreted as a bistable wave, in which local infection frequencies tend to increase only once the infection becomes sufficiently common to offset imperfect maternal transmission and infection costs. However, maternally inherited Wolbachia are expected to evolve towards mutualism, and they are known to increase host fitness by protecting against infectious microbes or increasing fecundity. We describe the sequential spread over approximately 20 years in natural populations of D. simulans on the east coast of Australia of two Wolbachia variants (wAu and wRi), only one of which causes significant CI, with wRi displacing wAu since 2004. Wolbachia and mtDNA frequency data and analyses suggest that these dynamics, as well as the earlier spread in California, are best understood as Fisherian waves of favourable variants, in which local spread tends to occur from arbitrarily low frequencies. We discuss implications for Wolbachia-host dynamics and coevolution and for applications of Wolbachia to disease control.     

Depletion of host cell riboflavin reduces Wolbachia levels in cultured mosquito cells

Wolbachia is an obligate intracellular alphaproteobacterium that occurs in arthropod and nematode hosts. Wolbachia presumably provides a fitness benefit to its hosts, but the basis for its retention and spread in host populations remains unclear. Wolbachia genomes retain biosynthetic pathways for some vitamins, and the possibility that these vitamins benefit host cells provides a potential means of selecting for Wolbachia-infected cell lines. To explore whether riboflavin produced by Wolbachia is available to its host cell, we established that growth of uninfected C7-10 mosquito cells decreases in riboflavin-depleted culture medium. A well-studied inhibitor of riboflavin uptake, lumiflavin, further inhibits growth of uninfected C7-10 cells with an LC₅₀ of approximately 12 μg/ml. Growth of C/wStr1 mosquito cells, infected with Wolbachia from the planthopper, Laodelphax striatellus, was enhanced in medium containing low levels of lumiflavin, but Wolbachia levels decreased. Lumiflavin-enhanced growth thus resembled the improved growth that accompanies treatment with antibiotics that deplete Wolbachia, rather than a metabolic advantage provided by the Wolbachia infection. We used the polymerase chain reaction to validate the decrease in Wolbachia abundance and evaluated our results in the context of a proteomic analysis in which we detected nearly 800 wStr proteins. Our data indicate that Wolbachia converts riboflavin to FMN and FAD for its own metabolic needs, and does not provide a source of riboflavin for its host cell.     

Host Adaptation of a Wolbachia Strain after Long-Term Serial Passage in Mosquito Cell Lines

The horizontal transfer of the bacterium Wolbachia pipientis between invertebrate hosts hinges on the ability of Wolbachia to adapt to new intracellular environments. The experimental transfer of Wolbachia between distantly related host species often results in the loss of infection, presumably due to an inability of Wolbachia to adapt quickly to the new host. To examine the process of adaptation to a novel host, we transferred a life-shortening Wolbachia strain, wMelPop, from the fruit fly Drosophila melanogaster into a cell line derived from the mosquito Aedes albopictus. After long-term serial passage in this cell line, we transferred the mosquito-adapted wMelPop into cell lines derived from two other mosquito species, Aedes aegypti and Anopheles gambiae. After a prolonged period of serial passage in mosquito cell lines, wMelPop was reintroduced into its native host, D. melanogaster, by embryonic microinjection. The cell line-adapted wMelPop strains were characterized by a loss of infectivity when reintroduced into the original host, grew to decreased densities, and had reduced abilities to cause life-shortening infection and cytoplasmic incompatibility compared to the original strain. We interpret these shifts in phenotype as evidence for genetic adaptation to the mosquito intracellular environment. The use of cell lines to preadapt Wolbachia to novel hosts is suggested as a possible strategy to improve the success of transinfection in novel target insect species.     

Symbiont strain is the main determinant of variation in Wolbachia‐mediated protection against viruses across Drosophila species

Wolbachia is a common heritable bacterial symbiont in insects. Its evolutionary success lies in the diverse phenotypic effects it has on its hosts coupled to its propensity to move between host species over evolutionary timescales. In a survey of natural host–symbiont associations in a range of Drosophila species, we found that 10 of 16 Wolbachia strains protected their hosts against viral infection. By moving Wolbachia strains between host species, we found that the symbiont genome had a much greater influence on the level of antiviral protection than the host genome. The reason for this was that the level of protection depended on the density of the symbiont in host tissues, and Wolbachia rather than the host‐controlled density. The finding that virus resistance and symbiont density are largely under the control of symbiont genes in this system has important implications both for the evolution of these traits and for public health programmes using Wolbachia to prevent mosquitoes from transmitting disease.     

Comparative Genomics of Wolbachia and the Bacterial Species Concept

The importance of host-specialization to speciation processes in obligate host-associated bacteria is well known, as is also the ability of recombination to generate cohesion in bacterial populations. However, whether divergent strains of highly recombining intracellular bacteria, such as Wolbachia, can maintain their genetic distinctness when infecting the same host is not known. We first developed a protocol for the genome sequencing of uncultivable endosymbionts. Using this method, we have sequenced the complete genomes of the Wolbachia strains wHa and wNo, which occur as natural double infections in Drosophila simulans populations on the Seychelles and in New Caledonia. Taxonomically, wHa belong to supergroup A and wNo to supergroup B. A comparative genomics study including additional strains supported the supergroup classification scheme and revealed 24 and 33 group-specific genes, putatively involved in host-adaptation processes. Recombination frequencies were high for strains of the same supergroup despite different host-preference patterns, leading to genomic cohesion. The inferred recombination fragments for strains of different supergroups were of short sizes, and the genomes of the co-infecting Wolbachia strains wHa and wNo were not more similar to each other and did not share more genes than other A- and B-group strains that infect different hosts. We conclude that Wolbachia strains of supergroup A and B represent genetically distinct clades, and that strains of different supergroups can co-exist in the same arthropod host without converging into the same species. This suggests that the supergroups are irreversibly separated and that barriers other than host-specialization are able to maintain distinct clades in recombining endosymbiont populations. Acquiring a good knowledge of the barriers to genetic exchange in Wolbachia will advance our understanding of how endosymbiont communities are constructed from vertically and horizontally transmitted genes.