Monophyly of Wolbachia pipientis genomes within Drosophila melanogaster: geographic structuring,titre variation and host effects across five populations

Wolbachia pipientis is one of the most widely studied endosymbionts today, yet we know little about its short‐term adaptation and evolution. Here, using a set of 91 inbred Drosophila melanogaster lines from five populations, we explore patterns of diversity and recent evolution in the Wolbachia strain wMel. Within the D. melanogaster lines, we identify six major mitochondrial clades and four wMel clades. Concordant with past studies, the Wolbachia haplotypes contain an overall low level of nucleotide diversity, yet they still display geographic structuring. Using Bayesian analysis informed with demographic estimates of colonization times, we estimate that all extant D. melanogaster mitochondrial haplotypes coalesce to a Wolbachia‐infected ancestor approximately 2200 years ago. Finally, we measure wMel titre within the infected flies and find that titre varies across populations, an effect attributable to host genetic factors. This demonstration of local phenotypic divergence suggests that intraspecific host genetic variation plays a key role in shaping this model symbiotic system.     

A wMel Wolbachia variant in Aedes aegypti from field-collected Drosophila melanogaster with increased phenotypic stability under heat stress

Mosquito-borne diseases remain a major cause of morbidity and mortality. Population replacement strategies involving the wMel strain of Wolbachia are being used widely to control mosquito-borne diseases. However, these strategies may be influenced by temperature because wMel is vulnerable to heat. wMel infections in Drosophila melanogaster are genetically diverse, but few transinfections of wMel variants have been generated in Aedes aegypti. Here, we successfully transferred a wMel variant (termed wMelM) originating from a field-collected D. melanogaster into Ae. aegypti. The new wMelM variant (clade I) is genetically distinct from the original wMel transinfection (clade III), and there are no genomic differences between wMelM in its original and transinfected host. We compared wMelM with wMel in its effects on host fitness, temperature tolerance, Wolbachia density, vector competence, cytoplasmic incompatibility and maternal transmission under heat stress in a controlled background. wMelM showed a higher heat tolerance than wMel, likely due to higher overall densities within the mosquito. Both wMel variants had minimal host fitness costs, complete cytoplasmic incompatibility and maternal transmission, and dengue virus blocking under laboratory conditions. Our results highlight phenotypic differences between Wolbachia variants and wMelM shows potential as an alternative strain in areas with strong seasonal temperature fluctuations.     

The Wolbachia protein TomO interacts with a host RNA to induce polarization defects in Drosophila oocytes

Wolbachia is an endosymbiont prevalent in arthropods. To maximize its transmission thorough the female germline, Wolbachia induces in infected hosts male‐to‐female transformation, male killing, parthenogenesis, and cytoplasmic incompatibility, depending on the host species and Wolbachia strain involved. However, the molecular mechanisms underlying these host manipulations by Wolbachia remain largely unknown. The Wolbachia strain wMel, an inhabitant of Drosophila melanogaster, impairs host oogenesis only when transplanted into a heterologous host, for example, Drosophila simulans. We found that egg polarity defects induced by wMel infection in D. simulans can be recapitulated in the natural host D. melanogaster by transgenic overexpression of a variant of the Wolbachia protein Toxic manipulator of oogenesis (TomO), TomO_wMel^?HS, in the female germline. RNA immunoprecipitation assays demonstrated that TomO physically associates with orb mRNA, which, as a result, fails to interact with the translation repressor Cup. This leads to precocious translation of Orb, a posterior determinant, and thereby to the misspecification of oocytes and accompanying polarity defects. We propose that the ability of TomO to bind to orb mRNA might provide a means for Wolbachia to enter the oocyte located at the posterior end of the egg chamber, thereby accomplishing secure maternal transmission thorough the female germline.     

Phylogenomics of the Reproductive Parasite Wolbachia pipientis wMel: A Streamlined Genome Overrun by Mobile Genetic Elements

The complete sequence of the 1,267,782 bp genome of Wolbachia pipientis wMel, an obligate intracellular bacteria of Drosophila melanogaster, has been determined. Wolbachia, which are found in a variety of invertebrate species, are of great interest due to their diverse interactions with different hosts, which range from many forms of reproductive parasitism to mutualistic symbioses. Analysis of the wMel genome, in particular phylogenomic comparisons with other intracellular bacteria, has revealed many insights into the biology and evolution of wMel and Wolbachia in general. For example, the wMel genome is unique among sequenced obligate intracellular species in both being highly streamlined and containing very high levels of repetitive DNA and mobile DNA elements. This observation, coupled with multiple evolutionary reconstructions, suggests that natural selection is somewhat inefficient in wMel, most likely owing to the occurrence of repeated population bottlenecks. Genome analysis predicts many metabolic differences with the closely related Rickettsia species, including the presence of intact glycolysis and purine synthesis, which may compensate for an inability to obtain ATP directly from its host, as Rickettsia can. Other discoveries include the apparent inability of wMel to synthesize lipopolysaccharide and the presence of the most genes encoding proteins with ankyrin repeat domains of any prokaryotic genome yet sequenced. Despite the ability of wMel to infect the germline of its host, we find no evidence for either recent lateral gene transfer between wMel and D. melanogaster or older transfers between Wolbachia and any host. Evolutionary analysis further supports the hypothesis that mitochondria share a common ancestor with the α-Proteobacteria, but shows little support for the grouping of mitochondria with species in the order Rickettsiales. With the availability of the complete genomes of both species and excellent genetic tools for the host, the wMel–D. melanogaster symbiosis is now an ideal system for studying the biology and evolution of Wolbachia infections.     

Phylogenomics of the Reproductive Parasite Wolbachia pipientis wMel: A Streamlined Genome Overrun by Mobile Genetic Elements

The complete sequence of the 1,267,782 bp genome of Wolbachia pipientis wMel, an obligate intracellular bacteria of Drosophila melanogaster, has been determined. Wolbachia, which are found in a variety of invertebrate species, are of great interest due to their diverse interactions with different hosts, which range from many forms of reproductive parasitism to mutualistic symbioses. Analysis of the wMel genome, in particular phylogenomic comparisons with other intracellular bacteria, has revealed many insights into the biology and evolution of wMel and Wolbachia in general. For example, the wMel genome is unique among sequenced obligate intracellular species in both being highly streamlined and containing very high levels of repetitive DNA and mobile DNA elements. This observation, coupled with multiple evolutionary reconstructions, suggests that natural selection is somewhat inefficient in wMel, most likely owing to the occurrence of repeated population bottlenecks. Genome analysis predicts many metabolic differences with the closely related Rickettsia species, including the presence of intact glycolysis and purine synthesis, which may compensate for an inability to obtain ATP directly from its host, as Rickettsia can. Other discoveries include the apparent inability of wMel to synthesize lipopolysaccharide and the presence of the most genes encoding proteins with ankyrin repeat domains of any prokaryotic genome yet sequenced. Despite the ability of wMel to infect the germline of its host, we find no evidence for either recent lateral gene transfer between wMel and D. melanogaster or older transfers between Wolbachia and any host. Evolutionary analysis further supports the hypothesis that mitochondria share a common ancestor with the α-Proteobacteria, but shows little support for the grouping of mitochondria with species in the order Rickettsiales. With the availability of the complete genomes of both species and excellent genetic tools for the host, the wMel–D. melanogaster symbiosis is now an ideal system for studying the biology and evolution of Wolbachia infections.     

Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements

The complete sequence of the 1,267,782 bp genome of Wolbachia pipientis wMel, an obligate intracellular bacteria of Drosophila melanogaster, has been determined. Wolbachia, which are found in a variety of invertebrate species, are of great interest due to their diverse interactions with different hosts, which range from many forms of reproductive parasitism to mutualistic symbioses. Analysis of the wMel genome, in particular phylogenomic comparisons with other intracellular bacteria, has revealed many insights into the biology and evolution of wMel and Wolbachia in general. For example, the wMel genome is unique among sequenced obligate intracellular species in both being highly streamlined and containing very high levels of repetitive DNA and mobile DNA elements. This observation, coupled with multiple evolutionary reconstructions, suggests that natural selection is somewhat inefficient in wMel, most likely owing to the occurrence of repeated population bottlenecks. Genome analysis predicts many metabolic differences with the closely related Rickettsia species, including the presence of intact glycolysis and purine synthesis, which may compensate for an inability to obtain ATP directly from its host, as Rickettsia can. Other discoveries include the apparent inability of wMel to synthesize lipopolysaccharide and the presence of the most genes encoding proteins with ankyrin repeat domains of any prokaryotic genome yet sequenced. Despite the ability of wMel to infect the germline of its host, we find no evidence for either recent lateral gene transfer between wMel and D. melanogaster or older transfers between Wolbachia and any host. Evolutionary analysis further supports the hypothesis that mitochondria share a common ancestor with the α-Proteobacteria, but shows little support for the grouping of mitochondria with species in the order Rickettsiales. With the availability of the complete genomes of both species and excellent genetic tools for the host, the wMel–D. melanogaster symbiosis is now an ideal system for studying the biology and evolution of Wolbachia infections.     

Loss of cytoplasmic incompatibility and minimal fecundity effects explain relatively low Wolbachia frequencies in Drosophila mauritiana

Maternally transmitted Wolbachia bacteria infect about half of all insect species. Many Wolbachia cause cytoplasmic incompatibility (CI) and reduced egg hatch when uninfected females mate with infected males. Although CI produces a frequency‐dependent fitness advantage that leads to high equilibrium Wolbachia frequencies, it does not aid Wolbachia spread from low frequencies. Indeed, the fitness advantages that produce initial Wolbachia spread and maintain non‐CI Wolbachia remain elusive. wMau Wolbachia infecting Drosophila mauritiana do not cause CI, despite being very similar to CI‐causing wNo from Drosophila simulans (0.068% sequence divergence over 682,494 bp), suggesting recent CI loss. Using draft wMau genomes, we identify a deletion in a CI‐associated gene, consistent with theory predicting that selection within host lineages does not act to increase or maintain CI. In the laboratory, wMau shows near‐perfect maternal transmission; but we find no significant effect on host fecundity, in contrast to published data. Intermediate wMau frequencies on the island of Mauritius are consistent with a balance between unidentified small, positive fitness effects and imperfect maternal transmission. Our phylogenomic analyses suggest that group‐B Wolbachia, including wMau and wPip, diverged from group‐A Wolbachia, such as wMel and wRi, 6–46 million years ago, more recently than previously estimated.     

The Wolbachia genome of Brugia malayi: endosymbiont evolution within a human pathogenic nematode

Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachia's principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease.     

The Wolbachia Genome of Brugia malayi: Endosymbiont Evolution within a Human Pathogenic Nematode

Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachia's principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease.     

Fecundity as one of possible factors contributing to the dominance of the wMel genotype of Wolbachia in natural populations of Drosophila melanogaster

Alphaproteobacteria of the genus Wolbachia are common intracellular endosymbionts of a variety of insects. Their successful spread over a vast range of host taxa is often attributed to selective advantages conferred by the bacteria to infected individuals. Among the known diversity of Wolbachia pipientis infecting Drosophila melanogaster, a single genotype, wMel, within the wMel strain has been found to dominate over other genotypes world-wide. Genotyping of D. melanogaster wild populations from Ukraine reveals a relatively high frequency of the wMel genotype, although 31 % flies from an Uman’ population are infected with the rare genotype wMelCS. We demonstrate that wMelCS-infected females have lower fecundity compared to wMel-infected flies, which might be the cause of wMel prevalence in D. melanogaster populations. We report no difference in the bacterial transmission rate between these two bacterial genotypes. However, we observed an association between transmission fidelity of Wolbachia and genotype of D. melanogaster indicating that Wolbachia-host relationships in this case are more complex. Furthermore our study reveals fluctuations in Wolbachia infection rates in wMel-infected populations.     

Life‐history traits and physiological limits of the alpine fly Drosophila nigrosparsa (Diptera: Drosophilidae): A comparative study

Interspecific variation in life‐history traits and physiological limits can be linked to the environmental conditions species experience, including climatic conditions. As alpine environments are particularly vulnerable under climate change, we focus on the montane‐alpine fly Drosophila nigrosparsa. Here, we characterized some of its life‐history traits and physiological limits and compared these with those of other drosophilids, namely Drosophila hydei, Drosophila melanogaster, and Drosophila obscura. We assayed oviposition rate, longevity, productivity, development time, larval competitiveness, starvation resistance, and heat and cold tolerance. Compared with the other species assayed, D. nigrosparsa is less fecund, relatively long‐living, starvation susceptible, cold adapted, and surprisingly well heat adapted. These life‐history characteristics provide insights into invertebrate adaptations to alpine conditions which may evolve under ongoing climate change.     

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.     

Wolbachia infection decreased the resistance of Drosophila to lead

Background

The heavy metal lead has been shown to be associated with a genotoxic risk. Drosophila melanogaster is a model organism commonly utilized in genetic toxicology testing. The endosymbionts — Wolbachia are now very common in both wild populations and laboratory stocks of Drosophila. Wolbachia may induce resistance to pathogenic viruses, filarial nematodes and Plasmodium in fruit fly and mosquito hosts. However the effect of Wolbachia infection on the resistance of their hosts to heavy metal is unknown.

Methodology/Principal Findings

Manipulating the lead content in the diet of Drosophila melanogaster, we found that lead consumption had no different effects on developmental time between Wolbachia-infected (Dmel wMel) and –uninfected (Dmel T) flies. While in Pb-contaminated medium, significantly reduced amount of pupae and adults of Dmel wMel were emerged, and Dmel wMel adults had significantly shorter longevity than that of Dmel T flies. Lead infusion in diet resulted in significantly decreased superoxide dismutase (SOD) activity in Dmel T flies (P<0.05), but not in Dmel wMel flies. Correspondingly, lead cultures induced a 10.8 fold increase in malonaldehyde (MDA) contents in Dmel T larvae (P<0.05). While in Dmel wMel larvae, it resulted in only a 1.3 fold increase. By quantitative RT-PCR, we showed that lead infused medium caused significantly increased expression level of relish and CecA2 genes in Dmel T flies (P<0.01). Lead cultures did not change dramatically the expression of these genes in Dmel wMel flies.

Conclusions/Significance

These results suggest that Wolbachia infection decreased the resistance of Drosophila to lead likely by limiting the production of peroxides resulted from lead, thus being unable to activate the immunological pathway in the host to prevent them from lead damage. This represents a novel Wolbachia–host interaction and provides information that researchers working on Drosophila toxicology should take in consideration the presence of Wolbachia in the stocks they are analyzing.     

Transinfection and growth discrepancy of Drosophila Wolbachia strain wMel in cell lines of the mosquito Aedes albopictus

Aim: The Wolbachia strain wMel can protect Drosophila melanogaster against pathogenic RNA viruses. To analyse the potential of this inhibitory effect against arboviruses vectorized by these mosquitoes, we here first transinfected the Aedes albopictus Aa23 and C6/36 cell lines with the Wolbachia strain wMel and then monitored their infection dynamics. Methods and Results: Wolbachia strain wMel was transferred into A. albopictus Aa23 and C6/36 cell lines using the shell vial technique. The presence of the bacterium in the transinfected cells was monitored by quantitative PCR and fluorescence in situ hybridization. Bacteria could be detected in the cytoplasm of both the Aa23 and C6/36 cell lines. However, the dynamics and stability of the bacterial infection differed depending on the initial cell background. The Aa23 cell line, which had been treated with a tetracycline antibiotic 2 years previously to eliminate its natural Wolbachia wAlbB‐infecting strain, lost the introduced Wolbachia wMel strain after 12 passages postinfection. In contrast, the C6/36 cell line, which had originally been aposymbiotic, displayed a stable infection with Wolbachia wMel. The bacterial density in C6/36 was greater than that of the A. albopictus RML12 cell line from which the wMel strain had originated. Conclusions: Transient or persistent transinfection of A. albopictus Aa23 and C6/36 cell lines with Wolbachia wMel strain was achieved. The results indicate the influence of the genetic background of mosquito cells in maintaining Wolbachia originating from a distant dipteral host. Significance and Impact of the Study: The cell model built here can now be used to investigate the viral inhibitory effect of the Wolbachia wMel strain against arboviruses such as dengue and chikungunya, which are transmitted by the mosquito A. albopictus.     

Wolbachia do not live by reproductive manipulation alone: infection polymorphism in Drosophila suzukii and D. subpulchrella

Drosophila suzukii recently invaded North America and Europe. Populations in Hawaii, California, New York and Nova Scotia are polymorphic for Wolbachia, typically with <20% infection frequency. The Wolbachia in D. suzukii, denoted wSuz, is closely related to wRi, the variant prevalent in continental populations of D. simulans. wSuz is also nearly identical to Wolbachia found in D. subpulchrella, plausibly D. suzukii's sister species. This suggests vertical Wolbachia transmission through cladogenesis (‘cladogenic transmission’). The widespread occurrence of 7–20% infection frequencies indicates a stable polymorphism. wSuz is imperfectly maternally transmitted, with wild infected females producing on average 5–10% uninfected progeny. As expected from its low frequency, wSuz produces no cytoplasmic incompatibility (CI), that is, no increased embryo mortality when infected males mate with uninfected females, and no appreciable sex‐ratio distortion. The persistence of wSuz despite imperfect maternal transmission suggests positive fitness effects. Assuming a balance between selection and imperfect transmission, we expect a fitness advantage on the order of 20%. Unexpectedly, Wolbachia‐infected females produce fewer progeny than do uninfected females. We do not yet understand the maintenance of wSuz in D. suzukii. The absence of detectable CI in D. suzukii and D. subpulchrella makes it unlikely that CI‐based mechanisms could be used to control this species without transinfection using novel Wolbachia. Contrary to their reputation as horizontally transmitted reproductive parasites, many Wolbachia infections are acquired through introgression or cladogenesis and many cause no appreciable reproductive manipulation. Such infections, likely to be mutualistic, may be central to understanding the pervasiveness of Wolbachia among arthropods.     

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.     

Genome comparisons indicate recent transfer of wRi‐like Wolbachia between sister species Drosophila suzukii and D. subpulchrella

Wolbachia endosymbionts may be acquired by horizontal transfer, by introgression through hybridization between closely related species, or by cladogenic retention during speciation. All three modes of acquisition have been demonstrated, but their relative frequency is largely unknown. Drosophila suzukii and its sister species D. subpulchrella harbor Wolbachia, denoted wSuz and wSpc, very closely related to wRi, identified in California populations of D. simulans. However, these variants differ in their induced phenotypes: wRi causes significant cytoplasmic incompatibility (CI) in D. simulans, but CI has not been detected in D. suzukii or D. subpulchrella. Our draft genomes of wSuz and wSpc contain full‐length copies of 703 of the 734 single‐copy genes found in wRi. Over these coding sequences, wSuz and wSpc differ by only 0.004% (i.e., 28 of 704,883 bp); they are sisters relative to wRi, from which each differs by 0.014%–0.015%. Using published data from D. melanogaster, Nasonia wasps and Nomada bees to calibrate relative rates of Wolbachia versus host nuclear divergence, we conclude that wSuz and wSpc are too similar—by at least a factor of 100—to be plausible candidates for cladogenic transmission. These three wRi‐like Wolbachia, which differ in CI phenotype in their native hosts, have different numbers of orthologs of genes postulated to contribute to CI; and the CI loci differ at several nucleotides that may account for the CI difference. We discuss the general problem of distinguishing alternative modes of Wolbachia acquisition, focusing on the difficulties posed by limited knowledge of variation in absolute and relative rates of molecular evolution for host nuclear genomes, mitochondria, and Wolbachia.     

Enhancing Nanos expression via the bacterial TomO protein is a conserved strategy used by the symbiont Wolbachia to fuel germ stem cell maintenance in infected Drosophila females

The toxic manipulator of oogenesis (TomO) protein has been identified in the wMel strain of Wolbachia that symbioses with the vinegar fly Drosophila melanogaster, as a protein that affects host reproduction. TomO protects germ stem cells (GSCs) from degeneration, which otherwise occurs in ovaries of host females that are mutant for the gene Sex‐lethal (Sxl). We isolated the TomO homologs from wPip, a Wolbachia strain from the mosquito Culex quinquefasciatus. One of the homologs, TomO_wPip1, exerted the GSC rescue activity in fly Sxl mutants when lacking its hydrophobic stretches. The GSC‐rescuing action of the TomO_wPip1 variant was ascribable to its abilities to associate with Nanos (nos) mRNA and to enhance Nos protein expression. The analysis of structure–activity relationships with TomO homologs and TomO deletion variants revealed distinct modules in the protein that are each dedicated to different functions, i.e., subcellular localization, nos mRNA binding or Nos expression enhancement. We propose that modular reshuffling is the basis for structural and functional diversification of TomO protein members.