Characterization of Wolbachia transfection efficiency by using microinjection of embryonic cytoplasm and embryo homogenate

Wolbachia spp. are intracellular alpha proteobacteria closely related to Rickettsia. The maternally inherited infections occur in a wide range of invertebrates, causing several reproductive abnormalities, including cytoplasmic incompatibility. The artificial transfer of Wolbachia between hosts (transfection) is used both for basic research examining the Wolbachia-host interaction and for applied strategies that use Wolbachia infections to affect harmful insect populations. Commonly employed transfection techniques use embryonic microinjection to transfer Wolbachia-infected embryo cytoplasm or embryo homogenate. Although microinjections of both embryonic cytoplasm and homogenate have been used successfully, their respective transfection efficiencies (rates of establishing stable germ line infections) have not been directly compared. Transfection efficiency may be affected by variation in Wolbachia quantity or quality within the donor embryos and/or the buffer types used in embryo homogenization. Here we have compared Wolbachia bacteria that originate from different embryonic regions for their competencies in establishing stable germ line infections. The following three buffers were compared for their abilities to maintain an appropriate in vitro environment for Wolbachia during homogenization and injection: phosphate-buffered saline, Drosophila Ringer's buffer, and a sucrose-phosphate-glutamate solution (SPG buffer). The results demonstrate that Wolbachia bacteria from both anterior and posterior embryo cytoplasms are competent for establishing infection, although differing survivorships of injected hosts were observed. Buffer comparison shows that embryos homogenized in SPG buffer yielded the highest transfection success. No difference was observed in transfection efficiencies when the posterior cytoplasm transfer and SPG-homogenized embryo techniques were compared. We discuss the results in relation to intra- and interspecific Wolbachia transfection and the future adaptation of the microinjection technique for additional insects.     

Uncovering Wolbachia Diversity upon Artificial Host Transfer

The common endosymbiotic Wolbachia bacteria influence arthropod hosts in multiple ways. They are mostly recognized for their manipulations of host reproduction, yet, more recent studies demonstrate that Wolbachia also impact host behavior, metabolic pathways and immunity. Besides their biological and evolutionary roles, Wolbachia are new potential biological control agents for pest and vector management. Importantly, Wolbachia-based control strategies require controlled symbiont transfer between host species and predictable outcomes of novel Wolbachia-host associations. Theoretically, this artificial horizontal transfer could inflict genetic changes within transferred Wolbachia populations. This could be facilitated through de novo mutations in the novel recipient host or changes of haplotype frequencies of polymorphic Wolbachia populations when transferred from donor to recipient hosts. Here we show that Wolbachia resident in the European cherry fruit fly, Rhagoletis cerasi, exhibit ancestral and cryptic sequence polymorphism in three symbiont genes, which are exposed upon microinjection into the new hosts Drosophila simulans and Ceratitis capitata. Our analyses of Wolbachia in microinjected D. simulans over 150 generations after microinjection uncovered infections with multiple Wolbachia strains in trans-infected lines that had previously been typed as single infections. This confirms the persistence of low-titer Wolbachia strains in microinjection experiments that had previously escaped standard detection techniques. Our study demonstrates that infections by multiple Wolbachia strains can shift in prevalence after artificial host transfer driven by either stochastic or selective processes. Trans-infection of Wolbachia can claim fitness costs in new hosts and we speculate that these costs may have driven the shifts of Wolbachia strains that we saw in our model system.     

Artificial transfer of a thelytoky-inducing <Emphasis Type="Italic">Wolbachia</Emphasis> endosymbiont between strains of the endoparasitoid wasp <Emphasis Type="Italic">Asobara japonica</Emphasis> (Hymenoptera: Braconidae)

Infection with Wolbachia is known to induce diploidization of haploid eggs and enables the production of females from unfertilized eggs. Although there have been several attempts to achieve the artificial horizontal transfer of thelytoky-inducing Wolbachia in parasitoid wasps, the artificial induction of thelytoky has generally been unsuccessful. In this study, we used two strains of Asobara japonica as study materials—one infected with thelytoky-inducing Wolbachia and the other not. We investigated methods of artificially inducing thelytoky by transferring thelytoky-inducing Wolbachia from wasps of the infected strain (the donor wasps) to wasps that had been cured of Wolbachia and to wasps of the uninfected strain (the recipient wasps). To examine the efficiencies of various methods of transfection, we compared the survival and infection rates of recipient wasps that received microinjections at the pupal and adult stages and in different body parts. We also examined the infection rate of the recipients due to cannibalism of Wolbachia-infected pupae. Among those methods, only microinjection at the adult stage resulted in the successful artificial horizontal transfer of Wolbachia, and some of the Wolbachia-infected wasps showed incomplete thelytoky. A low Wolbachia titer in the artificially infected wasps may explain why the thelytoky was incomplete.     

Bidirectional Cytoplasmic Incompatibility Induced by Cross-Order Transfection of Wolbachia: Implications for Control of the Host Population

Wolbachia are widespread endosymbionts in arthropods and some nematodes. This genus of bacteria is known to manipulate host reproduction by inducing cytoplasmic incompatibility (CI). This important phenotype is implicated in the control of host populations since Wolbachia can suppress host populations through the induction of CI in a way similar to the sterile insect technique. Here, we identified a candidate CI-inducing Wolbachia strain from the parasitic wasp Scleroderma guani (wSguBJ) by sequencing and phylogenetic analysis. This Wolbachia strain was then isolated, purified, and artificially transfected into the new whitefly host Bemisia tabaci through nymphal microinjection. Infection frequency monitoring by molecular detection showed that 60–80 % of the offspring from transfected whitefly populations was infected with wSguBJ six generations after the transfer. Laboratory rearing experiments indicated that the artificial transfection caused no significant difference in the numbers of offspring between the transfected and naturally infected populations and had no significant detrimental effects on the development of transfected males, although the development of transfected females was delayed. Reciprocal crossings revealed that bidirectional CI was induced between the transfected and naturally infected whiteflies. These data indicated that the cross-order transfer of the heterologous Wolbachia strain by nymphal microinjection was successful. Mass release of the transfected males that could stably carry the heterologous Wolbachia without significant compromise of fecundity/development may provide an alternative approach to control of host populations.     

Are ecological communities the seat of endosymbiont horizontal transfer and diversification? A case study with soil arthropod community

Maternally inherited endosymbionts of arthropods are one of the most abundant and diverse group of bacteria. These bacterial endosymbionts also show extensive horizontal transfer to taxonomically unrelated hosts and widespread recombination in their genomes. Such horizontal transfers can be enhanced when different arthropod hosts come in contact like in an ecological community. Higher rates of horizontal transfer can also increase the probability of recombination between endosymbionts, as they now share the same host cytoplasm. However, reports of community‐wide endosymbiont data are rare as most studies choose few host taxa and specific ecological interactions among the hosts. To better understand endosymbiont spread within host populations, we investigated the incidence, diversity, extent of horizontal transfer, and recombination of three endosymbionts (Wolbachia, Cardinium, and Arsenophonus) in a specific soil arthropod community. Wolbachia strains were characterized with MLST genes whereas 16S rRNA gene was used for Cardinium and Arsenophonus. Among 3,509 individual host arthropods, belonging to 390 morphospecies, 12.05% were infected with Wolbachia, 2.82% with Cardinium and 2.05% with Arsenophonus. Phylogenetic incongruence between host and endosymbiont indicated extensive horizontal transfer of endosymbionts within this community. Three cases of recombination between Wolbachia supergroups and eight incidences of within‐supergroup recombination were also found. Statistical tests of similarity indicated supergroup A Wolbachia and Cardinium show a pattern consistent with extensive horizontal transfer within the community but not for supergroup B Wolbachia and Arsenophonus. We highlight the importance of extensive community‐wide studies for a better understanding of the spread of endosymbionts across global arthropod communities.     

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.     

Asymmetric Wolbachia Segregation during Early Brugia malayi Embryogenesis Determines Its Distribution in Adult Host Tissues

Wolbachia are required for filarial nematode survival and fertility and contribute to the immune responses associated with human filarial diseases. Here we developed whole-mount immunofluorescence techniques to characterize Wolbachia somatic and germline transmission patterns and tissue distribution in Brugia malayi, a nematode responsible for lymphatic filariasis. In the initial embryonic divisions, Wolbachia segregate asymmetrically such that they occupy only a small subset of cells in the developing embryo, facilitating their concentration in the adult hypodermal chords and female germline. Wolbachia are not found in male reproductive tissues and the absence of Wolbachia from embryonic germline precursors in half of the embryos indicates Wolbachia loss from the male germline may occur in early embryogenesis. Wolbachia rely on fusion of hypodermal cells to populate adult chords. Finally, we detect Wolbachia in the secretory canal lumen suggesting living worms may release bacteria and/or their products into their host.     

Tracing horizontal Wolbachia movements among bees (Anthophila): a combined approach using multilocus sequence typing data and host phylogeny

The endosymbiotic bacterium Wolbachia enhances its spread via vertical transmission by generating reproductive effects in its hosts, most notably cytoplasmic incompatibility (CI). Additionally, frequent interspecific horizontal transfer is evident from a lack of phylogenetic congruence between Wolbachia and its hosts. The mechanisms of this lateral transfer are largely unclear. To identify potential pathways of Wolbachia movements, we performed multilocus sequence typing of Wolbachia strains from bees (Anthophila). Using a host phylogeny and ecological data, we tested various models of horizontal endosymbiont transmission. In general, Wolbachia strains seem to be randomly distributed among bee hosts. Kleptoparasite‐host associations among bees as well as other ecological links could not be supported as sole basis for the spread of Wolbachia. However, cophylogenetic analyses and divergence time estimations suggest that Wolbachia may persist within a host lineage over considerable timescales and that strictly vertical transmission and subsequent random loss of infections across lineages may have had a greater impact on Wolbachia strain distribution than previously estimated. Although general conclusions about Wolbachia movements among arthropod hosts cannot be made, we present a framework by which precise assumptions about shared evolutionary histories of Wolbachia and a host taxon can be modelled and tested.     

Loss of reproductive parasitism following transfer of male-killing Wolbachia to Drosophila melanogaster and Drosophila simulans

Wolbachia manipulate insect host biology through a variety of means that result in increased production of infected females, enhancing its own transmission. A Wolbachia strain (wInn) naturally infecting Drosophila innubila induces male killing, while native strains of D. melanogaster and D. simulans usually induce cytoplasmic incompatibility (CI). In this study, we transferred wInn to D. melanogaster and D. simulans by embryonic microinjection, expecting conservation of the male-killing phenotype to the novel hosts, which are more suitable for genetic analysis. In contrast to our expectations, there was no effect on offspring sex ratio. Furthermore, no CI was observed in the transinfected flies. Overall, transinfected D. melanogaster lines displayed lower transmission rate and lower densities of Wolbachia than transinfected D. simulans lines, in which established infections were transmitted with near-perfect fidelity. In D. simulans, strain wInn had no effect on fecundity and egg-to-adult development. Surprisingly, one of the two transinfected lines tested showed increased longevity. We discuss our results in the context of host-symbiont co-evolution and the potential of symbionts to invade novel host species.     

Survival of Wolbachia pipientis in Cell-Free Medium

Wolbachia pipientis is an obligate intracellular bacterium found in a wide range of invertebrate taxa. While over ecological timescales Wolbachia infections are maintained by strict maternal inheritance, horizontal transfer events are common over evolutionary time. To be horizontally transferred between organisms, Wolbachia bacteria must pass through and survive an extracellular phase. We used BacLight live-dead staining, PCR, and fluorescence in situ hybridization to assess the ability for purified Wolbachia bacteria to survive in cell-free media. We found that purified Wolbachia bacteria were able to survive extracellularly for up to 1 week with no decrease in viability. While no replication was observed in the extracellular phase, purified Wolbachia bacteria were able to reinvade cells and establish stable infections at all time points. The ability of Wolbachia bacteria to survive outside host cells may increase the probability of successful horizontal transfer and the exploitation of new ecological niches. Our development of methods to purify and maintain viable Wolbachia bacteria from cultured cells will be useful for other researchers studying Wolbachia biology.     

Review of treatment methods to remove Wolbachia bacteria from arthropods

Wolbachia are intracellular bacteria that infect numerous and diverse arthropod species including economic pests of crops and disease vectors affecting livestock and humans. Research on these symbionts has identified profound effects of Wolbachia on their hosts with possible application in pest control. Such research often requires methods to cure infections. To facilitate future research on these bacteria, we reviewed the Wolbachia literature to summarize results of 110 studies spanning 62 taxa that report on treatment methods and outcomes. Application of tetracycline in diet is the most common method and is typically successful. Rifampicin is secondarily used, and may be successful when tetracycline is not. Elevated temperatures can be used to eliminate infections, but is not often used. Rearing hosts under crowded conditions or starvation has been shown to reduce Wolbachia titre which affects maternal transmission. Application of treatment methods has a number of considerations with possible implications for the interpretation of data. This review is intended to alert the reader to treatment options and potential non-target effects.     

Successful horizontal transfer of Wolbachia symbionts between Trichogramma wasps

Rickettsial symbionts of the genus Wolbachia, harboured by many arthropod species, are implicated in feminization, cytoplasmic incompatibility and parthenogenesis phenomena. These symbionts induce thelytokous parthenogenesis in some egg parasitoids of the Trichogramma genus. In our study of these minute wasps, purified Wolbachia from an infected species, T. pretiosum, were transferred by microinjection into in vitro developed pupae of an uninfected species, T. dendrolimi. We believe this to be the first successful transfer of Wolbachia in parasitoids. The presence or absence of Wolbachia was determined using DAPI staining, PCR and ftsZ gene sequencing. An ftsZ gene fragment from microinjected T. dendrolimi was shown to be identical to that of T. pretiosum, confirming that transfer was successful. Wolbachia were still present in the recipient species 26 generations after the transfer, although only partial induction of thelytoky was observed. Therefore, in Trichogramma, density of symbionts or symbiont–host interactions may be involved in the expression of parthenogenesis. The successful transfer of the symbiont within the Trichogrammatidae, a group of insects used in biological control strategies, could increase their agronomic importance by manipulating their reproductive system.     

The transmission and effects of Wolbachia bacteria in parasitoids

Wolbachia bacteria are obligatory intracellular parasites of arthropods and have been detected in about 70 species of parasitic wasps and three parasitoid flies. Wolbachia are transmitted cytoplasmically (maternally) and modify host reproduction in different ways to enhance their own transmission: parthenogenesis induction (PI), cytoplasmic incompatibility (CI), or feminization (F) of genetic males. Only PI and CI are known in parasitoids. PI-Wolbachia cause thelytoky in otherwise arrhenotokous parasitoids by generating diploid (rather than haploid) unfertilized wasp eggs. CI-Wolbachia cause incompatibility of crosses between infected males and uninfected females because the paternally derived chromosomes fail to decondense and are destroyed after syngamy. More complex situations arise when hosts harbor multiple infections, which can lead to bidirectional incompatibility and may be involved in parasitoid speciation. The relative fitness of infected and uninfected hosts is important to the population dynamics of Wolbachia, and more data are needed. Evolutionary conflict should be common between host genes, Wolbachia genes, and other "selfish" genetic elements. Wolbachia-specific PCR primers are now available for several genes with different rates of evolution. These primers will permit rapid screening in future studies of spatial and temporal patterns of single and multiple infection. Molecular phylogenies show that CI- and PI-Wolbachia do not form discrete clades. In combination with experimental transfection data, this result suggests that host reproductive alterations depend on the interaction between attributes of both Wolbachia and host. Moreover, Wolbachia isolates from closely related hosts do not usually cluster together, and phylogenies suggest that Wolbachia may have radiated after their arthropod hosts. Both results support considerable horizontal transmission of Wolbachia between host species over evolutionary time. Natural horizontal transmisson between parasitoids and their hosts, or with entomoparasitic nematodes or ectoparasitic mites, remains a tantalizing but equivocal possibility. Received: November 27, 1998 / Accepted: January 15, 1999     

Wolbachia in the Flesh: Symbiont Intensities in Germ-Line and Somatic Tissues Challenge the Conventional View of Wolbachia Transmission Routes

Symbionts can substantially affect the evolution and ecology of their hosts. The investigation of the tissue-specific distribution of symbionts (tissue tropism) can provide important insight into host-symbiont interactions. Among other things, it can help to discern the importance of specific transmission routes and potential phenotypic effects. The intracellular bacterial symbiont Wolbachia has been described as the greatest ever panzootic, due to the wide array of arthropods that it infects. Being primarily vertically transmitted, it is expected that the transmission of Wolbachia would be enhanced by focusing infection in the reproductive tissues. In social insect hosts, this tropism would logically extend to reproductive rather than sterile castes, since the latter constitute a dead-end for vertically transmission. Here, we show that Wolbachia are not focused on reproductive tissues of eusocial insects, and that non-reproductive tissues of queens and workers of the ant Acromyrmex echinatior, harbour substantial infections. In particular, the comparatively high intensities of Wolbachia in the haemolymph, fat body, and faeces, suggest potential for horizontal transmission via parasitoids and the faecal-oral route, or a role for Wolbachia modulating the immune response of this host. It may be that somatic tissues and castes are not the evolutionary dead-end for Wolbachia that is commonly thought.     

Passage of Wolbachia pipientis through Mutant Drosophila melanogaster Induces Phenotypic and Genomic Changes

Wolbachia pipientis is a nearly ubiquitous, maternally transmitted bacterium that infects the germ line of insect hosts. Estimates are that Wolbachia infects 40 to 60% of insect species on the planet, making it one of the most prevalent infections on Earth. However, we know surprisingly little about the molecular mechanisms used by Wolbachia to infect its hosts. We passaged Wolbachia through normally restrictive Drosophila melanogaster hosts, bottlenecking Wolbachia through stochastic segregation while simultaneously selecting for mutants that could recolonize these previously restrictive hosts. Here, we show that Wolbachia alters its behavior when passaged through heterozygous mutant flies. After only three generations, Wolbachia was able to colonize the previously restrictive hosts at control titers. Additionally, the Wolbachia organisms passaged through heterozygous mutant D. melanogaster alter their pattern of tissue-specific Wsp protein production, suggesting a behavioral response to the host genotype. Using whole-genome resequencing, we identified the mutations accumulated by these lineages of Wolbachia and confirmed the existence and persistence of the mutations through clone library Sanger sequencing. Our results suggest that Wolbachia can quickly adapt to new host contexts, with genomic mutants arising after only two generations.     

Wolbachia sp. (Rickettsiales: Rickettsiaceae) a symbiont of the almond moth,Ephestia cautella: Ultrastructure and influence on host fertility

Wolbachia sp. is a maternally inherited symbiont of the almond moth, Ephestia cautella. It is transmitted through the cytoplasm of the egg and occurs normally in the gonads of all stages of the moth. The symbiont is responsible for reproductive cytoplasmic incompatibility between crosses of experimental laboratory strains of aposymbiotic female moths and symbiotic (normal) males. Although female moths were inseminated in laboratory tests, their eggs failed to hatch and exhibited no signs of embryonic development. The reciprocal cross, i.e., symbiotic female months × aposymbiotic males, produced normal progeny.The ultrastructure of Wolbachia was studied in sections of E. cautella larval testes. Symbionts, minute rod-shaped structures, were abundant in the cytoplasm of hypertrophied spermatids. There was no indication of deleterious influence of symbionts on sperm production or activity. Strains of Wolbachia occur in allopatric populations of insects where they may function as a genetic isolation mechanism. Microorganismal reproductive incompatibility has been suggested as a possible approach for insect control.     

Palaeosymbiosis Revealed by Genomic Fossils of Wolbachia in a Strongyloidean Nematode

Wolbachia are common endosymbionts of terrestrial arthropods, and are also found in nematodes: the animal-parasitic filaria, and the plant-parasite Radopholus similis. Lateral transfer of Wolbachia DNA to the host genome is common. We generated a draft genome sequence for the strongyloidean nematode parasite Dictyocaulus viviparus, the cattle lungworm. In the assembly, we identified nearly 1 Mb of sequence with similarity to Wolbachia. The fragments were unlikely to derive from a live Wolbachia infection: most were short, and the genes were disabled through inactivating mutations. Many fragments were co-assembled with definitively nematode-derived sequence. We found limited evidence of expression of the Wolbachia-derived genes. The D. viviparus Wolbachia genes were most similar to filarial strains and strains from the host-promiscuous clade F. We conclude that D. viviparus was infected by Wolbachia in the past, and that clade F-like symbionts may have been the source of filarial Wolbachia infections.     

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.     

A Review of Bacteria-Animal Lateral Gene Transfer May Inform Our Understanding of Diseases like Cancer

Lateral gene transfer (LGT) from bacteria to animals occurs more frequently than was appreciated prior to the advent of genome sequencing. In 2007, LGT from bacterial Wolbachia endosymbionts was detected in ∼33% of the sequenced arthropod genomes using a bioinformatic approach. Today, Wolbachia/host LGT is thought to be widespread and many other cases of bacteria-animal LGT have been described. In insects, LGT may be more frequently associated with endosymbionts that colonize germ cells and germ stem cells, like Wolbachia endosymbionts. We speculate that LGT may occur from bacteria to a wide variety of eukaryotes, but only becomes vertically inherited when it occurs in germ cells. As such, LGT may happen routinely in somatic cells but never become inherited or fixed in the population. Lack of inheritance of such mutations greatly decreases our ability to detect them. In this review, we propose that such noninherited bacterial DNA integration into chromosomes in human somatic cells could induce mutations leading to cancer or autoimmune diseases in a manner analogous to mobile elements and viral integrations.     

Bacteria Endosymbiont,Wolbachia, Promotes Parasitism of Parasitoid Wasp Asobara japonica

Wolbachia is the most widespread endosymbiotic bacterium that manipulates reproduction of its arthropod hosts to enhance its own spread throughout host populations. Infection with Wolbachia causes complete parthenogenetic reproduction in many Hymenoptera, producing only female offspring. The mechanism of such reproductive manipulation by Wolbachia has been extensively studied. However, the effects of Wolbachia symbiosis on behavioral traits of the hosts are scarcely investigated. The parasitoid wasp Asobara japonica is an ideal insect to investigate this because symbiotic and aposymbiotic strains are available: Wolbachia-infected Tokyo (TK) and noninfected Iriomote (IR) strains originally collected on the main island and southwest islands of Japan, respectively. We compared the oviposition behaviors of the two strains and found that TK strain females parasitized Drosophila melanogaster larvae more actively than the IR strain, especially during the first two days after eclosion. Removing Wolbachia from the TK strain wasps by treatment with tetracycline or rifampicin decreased their parasitism activity to the level of the IR strain. Morphological and behavioral analyses of both strain wasps showed that Wolbachia endosymbionts do not affect development of the host female reproductive tract and eggs, but do enhance host-searching ability of female wasps. These results suggest the possibility that Wolbachia endosymbionts may promote their diffusion and persistence in the host A. japonica population not only at least partly by parthenogenesis but also by enhancement of oviposition frequency of the host females.