Wolbachia requirement for oogenesis: occurrence within the genus Asobara (Hymenoptera, Braconidae) and evidence for intraspecific variation in A. tabida

Wolbachia are symbiotic bacteria that induce a diversity of phenotypes on their numerous invertebrate host species. In the wasp Asobara tabida (Braconidae), each individual harbours three Wolbachia strains: wAtab3, which is required for host oogenesis, and wAtab1 and wAtab2, that do not have this function but induce cytoplasmic incompatibility. In this study, we surveyed and identified Wolbachia strains in four additional Asobara species. We detected Wolbachia in one of these species, but both the identity (based on wsp gene) and prevalence of the Wolbachia detected in natural population indicate that this host species is not dependent on Wolbachia for oogenesis. We also compared A. tabida lines of different geographical origin for their dependence on Wolbachia. All individuals from 16 A. tabida lines proved to be infected by the three Wolbachia strains wAtab1, wAtab2 and wAtab3, but, interestingly, we found variation among lines in the degree to which females were dependent on Wolbachia to produce their oocytes. In three lines, aposymbiotic females (cured from the three Wolbachia strains by antibiotics) can produce some oocytes. However, these aposymbiotic females produce fewer and smaller oocytes than symbiotic ones, and the larvae they produce die before full development. Thus, depending on which nuclear genotype they have, A. tabida females depend on Wolbachia either because they fail to produce any oocyte or because the few oocytes they do produce generate unviable offspring. We discuss the implications of these findings for the understanding of the physiological and genetic deficiency of aposymbiotic females.     

Multiple infections and diversity of cytoplasmic incompatibility in a haplodiploid species

Cytoplasmic incompatibility (CI) is a sperm-egg incompatibility commonly induced by the intracellular endosymbiont bacterium Wolbachia that, in diploid species, results in embryo mortality. In haplodiploid species, two types of CI exist depending on whether the incompatible fertilized eggs develop into males (male development (MD)) or abort (female mortality (FM)). CI allows multiple infections to be maintained in host populations, and thus allows interactions to occur between co-infecting strains. In Leptopilina heterotoma, three Wolbachia strains coexist naturally (wLhet1, wLhet2, wLhet3). When these three strains are all present, they induce a CI of FM type, whereas wLhet1 alone expresses a CI phenotype intermediate between MD and FM. Here, we compare CI effects in crosses involving insect lines sharing the same nuclear background, but harboring different mixtures of strains. Mating experiments showed that: (i) wLhet2 and wLhet3 also induce an intermediate CI when acting alone, and show a bidirectional incompatibility; (ii) there is no interaction between the co-infecting strains in CI expression; (iii) the diversity of Wolbachia present within a male host influences the expression of CI: an increase in the number of strains is correlated with a decrease in the proportion of the MD type, which is also correlated with an increase in bacterial density. All these data suggest that the CI of FM type results from a stronger effect than the MD type, which conflicts with the conventional hypotheses used to explain CI diversity in haplodiploids, and could provide some new information about CI mechanisms in insects.     

Fitness advantage and cytoplasmic incompatibility in Wolbachia single- and superinfected Aedes albopictus

Wolbachia are obligate, maternally inherited, intracellular bacteria that infect numerous insects and other invertebrates. Wolbachia infections have evolved multiple mechanisms to manipulate host reproduction and facilitate invasion of naive host populations. One such mechanism is cytoplasmic incompatibility (CI) that occurs in many insect species, including Aedes albopictus (Asian tiger mosquito). The multiple Wolbachia infections that occur naturally in A. albopictus make this mosquito a useful system in which to study CI. Here, experiments employ mosquito strains that have been introgressed to provide genetically similar strains that harbor differing Wolbachia infection types. Cytoplasmic incompatibility levels, host longevity, egg hatch rates, and fecundity are examined. Crossing results demonstrate a pattern of additive unidirectional cytoplasmic incompatibility. Furthermore, relative to uninfected females, infected females are at a reproductive advantage due to both cytoplasmic incompatibility and a fitness increase associated with Wolbachia infection. In contrast, no fitness difference was observed in comparisons of single- and superinfected females. We discuss the observed results in regard to the evolution of the Wolbachia/A. albopictus symbiosis and the observed pattern of Wolbachia infection in natural populations.     

Wolbachia Infections in Drosophila Melanogaster and D. Simulans: Polymorphism and Levels of Cytoplasmic Incompatibility

Wolbachia are endosymbiotic bacteria, widespread in terrestrial Arthropods. They are mainly transmitted vertically, from mothers to offspring and induce various alterations of their hosts' sexuality and reproduction, the most commonly reported phenomenon being Cytoplasmic Incompatibility (CI), observed in Drosophila melanogaster and D. simulans. Basically, CI results in a more or less intense embryonic mortality, occurring in crosses between males infected by Wolbachia and uninfected females. In D. simulans, Wolbachia and CI were observed in 1986. Since then, this host species has become a model system for investigating the polymorphism of Wolbachia infections and CI. In this review we describe the different Wolbachia infections currently known to occur in D. melanogaster and D. simulans. The two species are highly contrasting with regard to symbiotic diversity: while five Wolbachia variants have been described in D. simulans natural populations, D. melanogaster seems to harbor one Wolbachia variant only. Another marked difference between these two Drosophila species is their permissiveness with regard to CI, which seems to be fully expressed in D. simulans but partially or totally repressed in D. melanogaster, demonstrating the involvement of host factors in the control of CI levels. The potential of the two host species regarding the understanding of CI and its evolution is also discussed.     

Evidence for female mortality in Wolbachia-mediated cytoplasmic incompatibility in haplodiploid insects: epidemiologic and evolutionary consequences

Abstract.— Until now, only two Wolbachia-mediated cytoplasmic incompatibility (CI) types have been described in haplodiploid species, the first in Nasonia (Insect) and the second in Tetranychus (Acari). They both induce a malebiased sex ratio in the incompatible cross. In Nasonia, CI does not reduce fertility since incompatible eggs develop as haploid males, whereas in Tetranychus CI leads to a partial mortality of incompatible eggs, thus reducing the fertility of females. Here, we study Wolbachia infection in a Drosophila parasitoid, Leptopilina heterotoma (Hymenoptera: Figitidae). A survey of Wolbachia infection shows that all natural populations tested are totally infected. Crosses between infected males and cured females show complete incompatibility: almost no females are produced. Moreover, incompatible eggs die early during their development, unlike Nasonia. This early death allows the parasitized Drosophila larva to achieve its development and to emerge. Thus, uninfected females crossed with infected males have reduced offspring production consisting only of males. Evidence of this CI type in insects demonstrates that the difference in CI types of Nasonia and Tetranychus is not due to specific factors of insects or acari. Using theoretical models, we compare the invasion processes of different strategies of Wolbachia: CI in diploid species, the two CI types in haplodiploid species, and parthenogenesis (the classical effect in haplodiploid species). Models show that CI in haplodiploid species is less efficient than in diploid ones. However, the Leptopilina type is advantageous compared to the Nasonia type. Parthenogenesis may be more or less advantageous, depending on the infection cost and on the proportion of fertilized eggs. Finally, we can propose different processes of Wolbachia strategy evolution in haplodiploid species from Nasonia CI type to Leptopilina CI type or parthenogenesis.     

Bidirectional incompatibility among divergent Wolbachia and incompatibility level differences among closely related Wolbachia in Nasonia

Most insect groups harbor obligate bacterial symbionts from the alpha-proteobacterial genus Wolbachia. These bacteria alter insect reproduction in ways that enhance their cytoplasmic transmission. One of the most common alterations is cytoplasmic incompatibility (CI) - a post-fertilization modification of the paternal genome that renders embryos inviable or unable to complete diploid development in crosses between infected males and uninfected females or infected females harboring a different strain. The parasitic wasp species complex Nasonia (N. vitripennis, N. longicornis and N. giraulti) harbor at least six different Wolbachia that cause CI. Each species have double infections with a representative from both the A and B Wolbachia subgroups. CI relationships of the A and B Wolbachia of N. longicornis with those of N. giraulti and N. vitripennis are investigated here. We demonstrate that all pairwise crosses between the divergent A strains are bidirectionally incompatible. We were unable to characterize incompatibility between the B Wolbachia, but we establish that the B strain of N. longicornis induces no or very weak CI in comparison to the closely related B strain in N. giraulti that expresses complete CI. Taken together with previous studies, we show that independent acquisition of divergent A Wolbachia has resulted in three mutually incompatible strains, whereas codivergence of B Wolbachia in N. longicornis and N. giraulti is associated with differences in CI level. Understanding the diversity and evolution of new incompatibility strains will contribute to a fuller understanding of Wolbachia invasion dynamics and Wolbachia-assisted speciation in certain groups of insects.     

Wolbachia and genetic variability in the birdnest blowfly Protocalliphora sialia

Wolbachia are widespread cytoplasmically inherited bacteria that induce various reproductive alterations in host arthropods, including cytoplasmic incompatibility (CI), an incompatibility between sperm and egg that typically results in embryonic death. CI has been invoked as a possible mechanism for reproductive isolation and speciation in arthropods, by restricting gene flow and promoting maintenance (and evolution) of genetic divergence between populations. Here we investigate patterns of Wolbachia infection and nuclear and mitochondrial differentiation in geographical populations of the birdnest blowfly Protocalliphora sialia. Blowflies in western North America are infected with two A-group Wolbachia, with some individuals singly and others doubly infected. Individuals in eastern North America mostly show single infections with a B-group Wolbachia. Populations in the Midwest are polymorphic for infections and show A- or B-group infection. There is a low level of mitochondrial divergence and perfect concordance of mitochondrial haplotype with infection type, suggesting that two Wolbachia-associated selective sweeps of the mitochondrion have occurred in this species. Amplified fragment length polymorphism analysis of nuclear genetic variation shows genetic differentiation between the eastern-Midwestern and western populations. Both Midwestern and eastern flies infected with A-Wolbachia show eastern nuclear genetic profiles. Current results therefore suggest that Wolbachia has not acted as a major barrier to gene flow between western and eastern-Midwestern populations, although some genetic differentiation between A-Wolbachia infected and B-Wolbachia infected individuals in eastern-Midwestern populations cannot be ruled out.     

Between- and within-host species selection on cytoplasmic incompatibility-inducing Wolbachia in haplodiploids

Abstract The most common effect of the endosymbiont Wolbachia is cytoplasmic incompatibility (CI), a form of postzygotic reproductive isolation that occurs in crosses where the male is infected by at least one Wolbachia strain that the female lacks. We revisited two puzzling features of Wolbachia biology: how Wolbachia can invade a new species and spread among populations, and how the association, once established in a host species, can evolve, with emphasis on the possible process of infection loss. These questions are particularly relevant in haplodiploid species, where males develop from unfertilized eggs, and females from fertilized eggs. When CI occurs in such species, fertilized eggs either die (female mortality type: FM), or develop into males (male development type: MD), raising one more question: how transition among CI types is possible. We reached the following conclusions: (1) the FM type is a better invader and should be retained preferentially after a new host is captured; (2) given the assumptions of the models, FM and MD types are selected on neither the bacterial side nor the host side; (3) selective pressures acting on both partners are more or less congruent in the FM type, but divergent in the MD type; (4) host and symbiont evolution can drive infection to extinction for all CI types, but the MD type is more susceptible to the phenomenon; and (5) under realistic conditions, transition from MD to FM type is possible. Finally, all these results suggest that the FM type should be more frequent than the MD type, which is consistent with the results obtained so far in haplodiploids.     

Widespread Wolbachia infection in terrestrial isopods and other crustaceans

Wolbachia bacteria are obligate intracellular alpha-Proteobacteria of arthropods and nematodes. Although widespread among isopod crustaceans, they have seldom been found in non-isopod crustacean species. Here, we report Wolbachia infection in fourteen new crustacean species. Our results extend the range of Wolbachia infections in terrestrial isopods and amphipods (class Malacostraca). We report the occurrence of two different Wolbachia strains in two host species (a terrestrial isopod and an amphipod). Moreover, the discovery of Wolbachia in the goose barnacle Lepas anatifera (subclass Thecostraca) establishes Wolbachia infection in class Maxillopoda. The new bacterial strains are closely related to B-supergroup Wolbachia strains previously reported from crustacean hosts. Our results suggest that Wolbachia infection may be much more widespread in crustaceans than previously thought. The presence of related Wolbachia strains in highly divergent crustacean hosts suggests that Wolbachia endosymbionts can naturally adapt to a wide range of crustacean hosts. Given the ability of isopod Wolbachia strains to induce feminization of genetic males or cytoplasmic incompatibility, we speculate that manipulation of crustacean-borne Wolbachia bacteria might represent potential tools for controlling crustacean species of commercial interest and crustacean or insect disease vectors.     

On the ubiquity and phylogeny of Wolbachia in lice

Wolbachia are intracellular bacteria that occur in an estimated 20% of arthropod species. They are of broad interest because they profoundly affect the reproductive fitness of diverse host taxa. Here we document the apparent ubiquity and diversity of Wolbachia in the insect orders Anoplura (sucking lice) and Mallophaga (chewing lice), by detecting single or multiple infections in each of 25 tested populations of lice, representing 19 species from 15 genera spanning eight taxonomic families. Phylogenetic analyses indicate a high diversity of Wolbachia in lice, as evidenced by the identification of 39 unique strains. Some of these strains are apparently unique to lice, whereas others are similar to strains that infect other insect taxa. Wolbachia are transmitted from infected females to their offspring via egg cytoplasm, such that similar species of lice are predicted to have similar strains of Wolbachia. This predicted pattern is not supported in the current study and may reflect multiple events of recent horizontal transmission between host species. At present, there is no known mechanism that would allow for this latter mode of transmission to and within species of lice.     

Variability and expression of ankyrin domain genes in Wolbachia variants infecting the mosquito Culex pipiens

Wolbachia strains are maternally inherited endosymbiotic bacteria that infect many arthropod species and have evolved several different ways of manipulating their hosts, the most frequent way being cytoplasmic incompatibility (CI). CI leads to embryo death in crosses between infected males and uninfected females as well as in crosses between individuals infected by incompatible Wolbachia strains. The mosquito Culex pipiens exhibits the highest crossing type variability reported so far. Our crossing data support the notion that CI might be driven by at least two distinct genetic units that control the CI functions independently in males and females. Although the molecular basis of CI remains unknown, proteins with ankyrin (ANK) domains represent promising candidates since they might interact with a wide range of host proteins. Here we searched for sequence variability in the 58 ANK genes carried in the genomes of Wolbachia variants infecting Culex pipiens. Only five ANK genes were polymorphic in the genomes of incompatible Wolbachia variants, and none correlated with the CI pattern obtained with 15 mosquito strains (representing 14 Wolbachia variants). Further analysis of ANK gene expression evidenced host- and sex-dependent variations, which did not improve the correlation. Taken together, these data do not support the direct implication of ANK genes in CI determinism.     

Wolbachia affects oviposition and mating behaviour of its spider mite host

Wolbachia bacteria are transmitted from mother to offspring via the cytoplasm of the egg. When mated to males infected with Wolbachia bacteria, uninfected females produce unviable offspring, a phenomenon called cytoplasmic incompatibility (CI). Current theory predicts that ‘sterilization’ of uninfected females by infected males confers a fitness advantage to Wolbachia in infected females. When the infection is above a threshold frequency in a panmictic population, CI reduces the fitness of uninfected females below that of infected females and, consequently, the proportion of infected hosts increases. CI is a mechanism that benefits the bacteria but, apparently, not the host. The host could benefit from avoiding incompatible mates. Parasite load and disease resistance are known to be involved in mate choice. Can Wolbachia also be implicated in reproductive behaviour? We used the two‐spotted spider mite – Wolbachia symbiosis to address this question. Our results suggest that uninfected females preferably mate to uninfected males while infected females aggregate their offspring, thereby promoting sib mating. Our data agrees with other results that hosts of Wolbachia do not necessarily behave as innocent bystanders – host mechanisms that avoid CI can evolve.     

Molecular evidence for single Wolbachia infections among geographic strains of the flour beetle Tribolium confusum.

Infections with the rickettsial microorganism Wolbachia are cytoplasmically inherited and occur in a wide range of insect species and several other arthropods. Wolbachia infection often results in unidirectional cytoplasmic incompatibility (CI): crosses between infected males and uninfected females are incompatible and show a reduction of progeny or complete inviability. Unidirectional CI can also occur when males harbouring two incompatible Wolbachia strains are crossed with females infected with only one of the two strains. In the flour beetle Tribolium confusum, Wolbachia infections are of particular interest because of the severity of incompatibility. Typically, no progeny results from the incompatible cross, whereas only partial incompatibility is observed in most other hosts. Werren et al. (1995a) reported that Wolbachia infections in T. confusum consist of two bacterial strains belonging to distinct phylogenic groups, based on PCR amplification and sequence analysis of the bacterial cell division gene ftsZ. However, Fialho & Stevens (1996) showed that eight strains of T. confusum were infected with a single and common incompatibility type. Here we report analysis of the ftsZ gene by specific PCR amplification. Diagnostic restriction enzyme assays revealed no evidence of double infections in 11 geographic strains of T. confusum, including the strain examined by Werren et al. (1995a). Further, sequence analysis of the Wolbachia ftsZ gene and an internal transcribed spacer (ITS) region in two of these strains displayed no nucleotide variation or evidence of polymorphisms. Results suggest that T. confusum is infected with B-group Wolbachia only.     

Infection density of Wolbachia and level of cytoplasmic incompatibility in the Mediterranean flour moth,Ephestia kuehniella

Wolbachia, a causative agent of various reproductive changes in arthropods, induces cytoplasmic incompatibility (CI) in the Mediterranean flour moth, Ephestia kuehniella. Two strains of E. kuehniella, Yokohama and Tsuchiura, harbor closely related Wolbachia, but the Yokohama strain expresses stronger CI than the Tsuchiura strain. A transinfected E. kuehniella strain that harbors the Wolbachia derived from the almond moth Cadra cautella, expresses weak CI at a similar level to the Tsuchiura strain. In the present study, we measured the Wolbachia density in the testis of the three E. kuehniella strains in order to examine the effects of bacterial strain and infection load on the expression of CI. When individuals of the same strain were compared, a correlation of bacterial density to CI level was observed. In addition, the Wolbachia density was higher in the Yokohama strain than the Tsuchiura strain in agreement with the CI levels expressed. However, this relationship did not hold in the comparison between the naturally infected and transinfected strains that carried phylogenetically distant Wolbachia.     

Many compatible Wolbachia strains coexist within natural populations of Culex pipiens mosquito

Maternally inherited Wolbachia often manipulate the reproduction of arthropods to promote their transmission. In most species, Wolbachia exert a form of conditional sterility termed cytoplasmic incompatibility (CI), characterized by the death of embryos produced by the mating between individuals with incompatible Wolbachia infections. From a theoretical perspective, no stable coexistence of incompatible Wolbachia infections is expected within host populations and CI should induce the invasion of one strain or of a set of compatible strains. In this study, we investigated this prediction on CI dynamics in natural populations of the common house mosquito Culex pipiens. We surveyed the Wolbachia diversity and the expression of CI in breeding sites of the south of France between 1990 and 2005. We found that geographically close C. pipiens populations harbor considerable Wolbachia diversity, which is stably maintained over 15 years. We also observed a very low frequency of infertile clutches within each sampled site. Meanwhile, mating choice experiments conducted in laboratory conditions showed that assortative mating does not occur. Overall, this suggests that a large set of compatible Wolbachia strains are always locally dominant within mosquito populations thus, fitting with the theoretical expectations on CI dynamics.     

Transposable element polymorphism of Wolbachia in the mosquito Culex pipiens: evidence of genetic diversity, superinfection and recombination

Wolbachia is a group of maternally inherited endosymbiotic bacteria that infect and induce cytoplasmic incompatibility (CI) in a wide range of arthropods. In contrast to other species, the mosquito Culex pipiens displays an extremely high number of CI types suggesting differential infection by multiple Wolbachia strains. Attempts so far failed to detect Wolbachia polymorphism that might explain this high level of CI diversity found in C. pipiens populations. Here, we establish that Wolbachia infection is near to or at fixation in worldwide populations of the C. pipiens complex. Wolbachia polymorphism was addressed by sequence analysis of the Tr1 gene, a unique transposable element of the IS5 family, which allowed the identification of five C. pipiens Wolbachia strains, differing either by nucleotide substitution, presence or absence pattern, or insertion site. Sequence analysis also showed that recombination, transposition and superinfection occurred at very low frequencies. Analysis of the geographical distributions of each Wolbachia strain among C. pipiens populations indicated a strong worldwide differentiation independent from mosquito subspecies type, except in the UK. The availability of this polymorphic marker now opens the way to investigate evolution of Wolbachia populations and CI dynamics, in particular in regions where multiple crossing types coexist among C. pipiens populations.     

沃尔巴克氏体在中国三种稻飞虱中的感染

用PCR方法检测了采集于不同地域稻田的3种稻飞虱共生菌沃尔巴克氏体(Wolbachia)的感染,发现灰飞虱Laodelphax striatellus、褐飞虱Nilaparvata lugens、白背飞虱Sogatella furcifera为沃尔巴克氏体所感染。克隆了编码沃尔巴克氏体外膜蛋白质的wsp基因并进行了序列测定。对wsp的RFLP分析证实了这些飞虱为单一沃尔巴克氏体感染。研究了灰飞虱中沃尔巴克氏体所诱导的胞质不相容性及其在不同地域灰飞虱中的分布。还发现能寄生于上述3种飞虱的稻虱红螯蜂也受同种沃尔巴克氏体感染。沃尔巴克氏体可能通过这种寄生蜂在不同昆虫间横向传播。     

Single and Double Infections with Wolbachia in the Parasitic Wasp Nasonia Vitripennis: Effects on Compatibility

Wolbachia are cytoplasmically inherited bacteria responsible for reproductive incompatibility in a wide range of insects. There has been little exploration, however, of within species Wolbachia polymorphisms and their effects on compatibility. Here we show that some strains of the parasitic wasp Nasonia vitripennis are infected with two distinct bacterial strains (A and B) whereas others are singly infected (A or B). Double and single infections are confirmed by both PCR amplification and Southern analysis of genomic DNA. Furthermore, it is shown that prolonged larval diapause (the overwintering stage of the wasp) of a double-infected strain can lead to stochastic loss of one or both bacterial strains. After diapause of a double-infected line, sublines were produced with AB, A only, B only or no Wolbachia. A and B sublines are bidirectionally incompatible, whereas males from AB lines are unidirectionally incompatible with females of A and B sublines. Results therefore show rapid development of bidirectional incompatibility within a species due to segregation of associated symbiotic bacteria.     

Occasional males in parthenogenetic populations of Asobara japonica (Hymenoptera: Braconidae): low Wolbachia titer or incomplete coadaptation?

Wolbachia are endosymbiotic bacteria known to manipulate the reproduction of their hosts. Some populations of the parasitoid wasp Asobara japonica are infected with Wolbachia and reproduce parthenogenetically, while other populations are not infected and reproduce sexually. Wolbachia-infected A. japonica females regularly produce small numbers of male offspring. Because all females in the field are infected and infected females are not capable of sexual reproduction, male production seems to be maladaptive. We investigated why these females nevertheless produce males. We tested three hypotheses: high rearing temperatures could result in higher offspring sex ratios (more males), low Wolbachia titer of the mother could lead to higher offspring sex ratios and/or the Wolbachia infection is of relatively recent origin and not enough time has passed to allow complete coadaptation between Wolbachia and host. In all, 33% of the Wolbachia-infected females produced males and 56% of these males were also infected with Wolbachia. Neither offspring sex ratio nor male infection frequency was significantly affected by rearing temperature or Wolbachia concentration of the mother. The mitochondrial DNA sequence of one of the uninfected populations was identical to that of two of the infected populations. Therefore, the initial Wolbachia infection of A. japonica must have occurred recently. Mitochondrial sequence variation among the infected populations suggests that the spread of Wolbachia through the host populations involved horizontal transmission. We conclude that the occasional male production by Wolbachia-infected females is most likely a maladaptive side effect of incomplete coevolution between symbiont and host in this relatively young infection.     

Evolution of Wolbachia-induced cytoplasmic incompatibility in Drosophila simulans and D. sechellia

Abstract.— The intracellular bacterium Wolbachia invades arthropod host populations through various mechanisms, the most common of which being cytoplasmic incompatibility (CI). CI involves elevated embryo mortality when infected males mate with uninfected females or females infected with different, incompatible Wolbachia strains. The present study focuses on this phenomenon in two Drosophila species: D. simulans and D. sechellia . Drosophila simulans populations are infected by several Wolbachia strains, including w Ha and w No. Drosophila sechellia is infected by only two Wolbachia : w Sh and w Sn. In both Drosophila species, double infections with Wolbachia are found. As indicated by several molecular markers, w Ha is closely related to w Sh, and w No to w Sn. Furthermore, the double infections in the two host species are associated with closely related mitochondrial haplotypes, namely si I (associated with w Ha and w No in D. simulans ) and se (associated with w Sh and w Sn in D. sechellia ). To test the theoretical prediction that Wolbachia compatibility types can diverge rapidly, we injected w Sh and w Sn into D. simulans , to compare their CI properties to those of their sister strains w Ha and w No, respectively, in the same host genetic background. We found that within each pair of sister strains CI levels were similar and that sister strains were fully compatible. We conclude that the short period for which the Wolbachia sister strains have been evolving separated from each other was not sufficient for their CI properties to diverge significantly.