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.     

A re-examination of Wolbachia-induced cytoplasmic incompatibility in California Drosophila simulans

Background

In California Drosophila simulans, the maternally inherited Riverside strain Wolbachia infection (wRi) provides a paradigm for rapid spread of Wolbachia in nature and rapid evolutionary change. wRi induces cytoplasmic incompatibility (CI), where crosses between infected males and uninfected females produce reduced egg-hatch. The three parameters governing wRi infection-frequency dynamics quantify: the fidelity of maternal transmission, the level of cytoplasmic incompatibility, and the relative fecundity of infected females. We last estimated these parameters in nature in 1993. Here we provide new estimates, under both field and laboratory conditions. Five years ago, we found that wRi had apparently evolved over 15 years to enhance the fecundity of infected females; here we examine whether CI intensity has also evolved.

Methodology/Principal Findings

New estimates using wild-caught flies indicate that the three key parameters have remained relatively stable since the early 1990s. As predicted by our three-parameter model using field-estimated parameter values, population infection frequencies remain about 93%. Despite this relative stability, laboratory data based on reciprocal crosses and introgression suggest that wRi may have evolved to produce less intense CI (i.e., higher egg hatch from incompatible crosses). In contrast, we find no evidence that D. simulans has evolved to lower the susceptibility of uninfected females to CI.

Conclusions/Significance

Evolution of wRi that reduces CI is consistent with counterintuitive theoretical predictions that within-population selection on CI-causing Wolbachia does not act to increase CI. Within taxa, CI is likely to evolve mainly via pleiotropic effects associated with the primary targets of selection on Wolbachia, i.e., host fecundity and transmission fidelity. Despite continuous, strong selection, D. simulans has not evolved appreciably to suppress CI. Our data demonstrate a lack of standing genetic variation for CI resistance in the host.     

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.     

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.     

Wolbachia Transfer from Rhagoletis cerasi to Drosophila simulans: Investigating the Outcomes of Host-Symbiont Coevolution

Wolbachia is an endosymbiont of diverse arthropod lineages that can induce various alterations of host reproduction for its own benefice. Cytoplasmic incompatibility (CI) is the most common phenomenon, which results in embryonic lethality when males that bear Wolbachia are mated with females that do not. In the cherry fruit fly, Rhagoletis cerasi, Wolbachia seems to be responsible for previously reported patterns of incompatibility between populations. Here we report on the artificial transfer of two Wolbachia variants (wCer1 and wCer2) from R. cerasi into Drosophila simulans, which was performed with two major goals in mind: first, to isolate wCer1 from wCer2 in order to individually test their respective abilities to induce CI in the new host; and, second, to test the theoretical prediction that recent Wolbachia-host associations should be characterized by high levels of CI, fitness costs to the new host, and inefficient transmission from mothers to offspring. wCer1 was unable to develop in the new host, resulting in its rapid loss after successful injection, while wCer2 was established in the new host. Transmission rates of wCer2 were low, and the infection showed negative fitness effects, consistent with our prediction, but CI levels were unexpectedly lower in the new host. Based on these parameter estimates, neither wCer1 nor wCer2 could be naturally maintained in D. simulans. The experiment thus suggests that natural Wolbachia transfer between species might be restricted by many factors, should the ecological barriers be bypassed.     

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.     

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.     

Variation in Antiviral Protection Mediated by Different Wolbachia Strains in Drosophila simulans

Drosophila C virus (DCV) is a natural pathogen of Drosophila and a useful model for studying antiviral defences. The Drosophila host is also commonly infected with the widespread endosymbiotic bacteria Wolbachia pipientis. When DCV coinfects Wolbachia-infected D. melanogaster, virus particles accumulate more slowly and virus induced mortality is substantially delayed. Considering that Wolbachia is estimated to infect up to two-thirds of all insect species, the observed protective effects of Wolbachia may extend to a range of both beneficial and pest insects, including insects that vector important viral diseases of humans, animals and plants. Currently, Wolbachia-mediated antiviral protection has only been described from a limited number of very closely related strains that infect D. melanogaster. We used D. simulans and its naturally occurring Wolbachia infections to test the generality of the Wolbachia-mediated antiviral protection. We generated paired D. simulans lines either uninfected or infected with five different Wolbachia strains. Each paired fly line was challenged with DCV and Flock House virus. Significant antiviral protection was seen for some but not all of the Wolbachia strain-fly line combinations tested. In some cases, protection from virus-induced mortality was associated with a delay in virus accumulation, but some Wolbachia-infected flies were tolerant to high titres of DCV. The Wolbachia strains that did protect occurred at comparatively high density within the flies and were most closely related to the D. melanogaster Wolbachia strain wMel. These results indicate that Wolbachia-mediated antiviral protection is not ubiquitous, a finding that is important for understanding the distribution of Wolbachia and virus in natural insect populations.     

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.     

Transinfection of the olive fruit fly Bactrocera oleae with Wolbachia: towards a symbiont‐based population control strategy

The olive fruit fly Bactrocera oleae is responsible for worldwide economic damage. In this report, we describe the first B. oleae lines transinfected with the Wolbachia strain wCer2, an endosymbiont of the cherry fruit fly Rhagoletis cerasi. Immunostaining followed by confocal microscopy, detects high numbers of Wolbachia in embryos as well as in ovarioles and sperm from individuals of both transinfected lines. wCer2 was uniformly distributed in B. oleae egg chambers and the cortex of preblastoderm embryos. Wolbachia is known to manipulate host reproduction with several strategies, one of which is cytoplasmic incompatibility (CI), resulting in embryonic mortality in incompatible crosses. Wolbachia was found to induce complete CI in the novel host, suggesting that symbiont‐based approaches can be used as novel environmentally friendly tools for the control of natural olive fruit fly populations.     

Wolbachia affect behavior and possibly reproductive compatibility but not thermoresistance,fecundity, and morphology in a novel transinfected host,Drosophila nigrosparsa

Wolbachia, intracellular endosymbionts, are estimated to infect about half of all arthropod species. These bacteria manipulate their hosts in various ways for their maximum benefits. The rising global temperature may accelerate species migration, and thus, horizontal transfer of Wolbachia may occur across species previously not in contact. We transinfected and then cured the alpine fly Drosophila nigrosparsa with Wolbachia strain wMel to study its effects on this species. We found low Wolbachia titer, possibly cytoplasmic incompatibility, and an increase in locomotion of both infected larvae and adults compared with cured ones. However, no change in fecundity, no impact on heat and cold tolerance, and no change in wing morphology were observed. Although Wolbachia increased locomotor activities in this species, we conclude that D. nigrosparsa may not benefit from the infection. Still, D. nigrosparsa can serve as a host for Wolbachia because vertical transmission is possible but may not be as high as in the native host of wMel, Drosophila melanogaster.     

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.     

Wolbachia-induced cytoplasmic incompatibility is associated with decreased Hira expression in male Drosophila

Background

Wolbachia are obligate endosymbiotic bacteria that infect numerous species of arthropods and nematodes. Wolbachia can induce several reproductive phenotypes in their insect hosts including feminization, male-killing, parthenogenesis and cytoplasmic incompatibility (CI). CI is the most common phenotype and occurs when Wolbachia-infected males mate with uninfected females resulting in no or very low numbers of viable offspring. However, matings between males and females infected with the same strain of Wolbachia result in viable progeny. Despite substantial scientific effort, the molecular mechanisms underlying CI are currently unknown.

Methodology/Principal Findings

Gene expression studies were undertaken in Drosophila melanogaster and D. simulans which display differential levels of CI using quantitative RT-PCR. We show that Hira expression is correlated with the induction of CI and occurs in a sex-specific manner. Hira expression is significantly lower in males which induce strong CI when compared to males inducing no CI or Wolbachia-uninfected males. A reduction in Hira expression is also observed in 1-day-old males that induce stronger CI compared to 5-day-old males that induce weak or no CI. In addition, Hira mutated D. melanogaster males mated to uninfected females result in significantly decreased hatch rates comparing with uninfected crosses. Interestingly, wMel-infected females may rescue the hatch rates. An obvious CI phenotype with chromatin bridges are observed in the early embryo resulting from Hira mutant fertilization, which strongly mimics the defects associated with CI.

Conclusions/Significance

Our results suggest Wolbachia-induced CI in Drosophila occurs due to a reduction in Hira expression in Wolbachia-infected males leading to detrimental effects on sperm fertility resulting in embryo lethality. These results may help determine the underlying mechanism of CI and provide further insight in to the important role Hira plays in the interaction of Wolbachia and its insect 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.     

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.     

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.     

Population Genomics of the Wolbachia Endosymbiont in Drosophila melanogaster

Wolbachia are maternally inherited symbiotic bacteria, commonly found in arthropods, which are able to manipulate the reproduction of their host in order to maximise their transmission. The evolutionary history of endosymbionts like Wolbachia can be revealed by integrating information on infection status in natural populations with patterns of sequence variation in Wolbachia and host mitochondrial genomes. Here we use whole-genome resequencing data from 290 lines of Drosophila melanogaster from North America, Europe, and Africa to predict Wolbachia infection status, estimate relative cytoplasmic genome copy number, and reconstruct Wolbachia and mitochondrial genome sequences. Overall, 63% of Drosophila strains were predicted to be infected with Wolbachia by our in silico analysis pipeline, which shows 99% concordance with infection status determined by diagnostic PCR. Complete Wolbachia and mitochondrial genomes show congruent phylogenies, consistent with strict vertical transmission through the maternal cytoplasm and imperfect transmission of Wolbachia. Bayesian phylogenetic analysis reveals that the most recent common ancestor of all Wolbachia and mitochondrial genomes in D. melanogaster dates to around 8,000 years ago. We find evidence for a recent global replacement of ancestral Wolbachia and mtDNA lineages, but our data suggest that the derived wMel lineage arose several thousand years ago, not in the 20th century as previously proposed. Our data also provide evidence that this global replacement event is incomplete and is likely to be one of several similar incomplete replacement events that have occurred since the out-of-Africa migration that allowed D. melanogaster to colonize worldwide habitats. This study provides a complete genomic analysis of the evolutionary mode and temporal dynamics of the D. melanogaster–Wolbachia symbiosis, as well as important resources for further analyses of the impact of Wolbachia on host biology.     

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.     

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.