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.     

Wolbachia transfer from Drosophila melanogaster into D. simulans: Host effect and cytoplasmic incompatibility relationships.

Wolbachia are maternally transmitted endocellular bacteria causing a reproductive incompatibility called cytoplasmic incompatibility (CI) in several arthropod species, including Drosophila. CI results in embryonic mortality in incompatible crosses. The only bacterial strain known to infect Drosophila melanogaster (wDm) was transferred from a D. melanogaster isofemale line into uninfected D. simulans isofemale lines by embryo microinjections. Males from the resulting transinfected lines induce >98% embryonic mortality when crossed with uninfected D. simulans females. In contrast, males from the donor D. melanogaster line induce only 18-32% CI on average when crossed with uninfected D. melanogaster females. Transinfected D. simulans lines do not differ from the D. melanogaster donor line in the Wolbachia load found in the embryo or in the total bacterial load of young males. However, >80% of cysts are infected by Wolbachia in the testes of young transinfected males, whereas only 8% of cysts are infected in young males from the D. melanogaster donor isofemale line. This difference might be caused by physiological differences between hosts, but it might also involve tissue-specific control of Wolbachia density by D. melanogaster. The wDm-transinfected D. simulans lines are unidirectionally incompatible with strains infected by the non-CI expressor Wolbachia strains wKi, wMau, or wAu, and they are bidirectionally incompatible with strains infected by the CI-expressor Wolbachia strains wHa or wNo. However, wDm-infected males do not induce CI toward females infected by the CI-expressor strain wRi, which is found in D. simulans continental populations, while wRi-infected males induce partial CI toward wDm-infected females. This peculiar asymmetrical pattern could reflect an ongoing divergence between the CI mechanisms of wRi and wDm. It would also confirm other results indicating that the factor responsible for CI induction in males is distinct from the factor responsible for CI rescue in females.     

Sorting out the effects of Wolbachia, genotype and inbreeding on life-history traits of a spider mite

Wolbachia bacteria manipulate host reproduction by inducing cytoplasmic incompatibility (CI) and sex ratio distortion. Wolbachia are transmitted from mother to offspring through the cytoplasm of the egg. Therefore, reproduction of Wolbachia is tightly coupled to reproduction of its host. Mathematical analysis predicts that in the course of evolution, traits that reduce the physiological costs of the infection will be selectively favored. For a Wolbachia-host system to evolve, traits under selection must have some genetic component and variation must be present in the population. We have previously established that highly inbred isofemale lines of the two-spotted spider mite Tetranychus urticae may differ regarding the effects of infection by Wolbachia, and that at least some of the traits affected had a genetic component. However, the effects measured could have been affected by the fact that the lines were severely inbred prior to the experiments. In this paper we attempt to distinguish between the effects of Wolbachia, isofemale line, and inbreeding. We show that Wolbachia did not affect longevity but infected females produced smaller clutch sizes, more daughter-biased sex ratios and had decreased F1 mortality; between-line variation was found for clutch size, F1 mortality and sex ratio; finally, inbreeding resulted in an overall reduction of clutch sizes, and a change in survival curves and mean longevity. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Overcoming cytoplasmic incompatibility in Drosophila.

The endocellular microbe Wolbachia pipientis infects a wide variety of invertebrate species, in which its presence is closely linked to a form of reproductive failure termed cytoplasmic incompatibility (CI). CI renders infected males unable to father offspring when mated to uninfected females. Because CI can dramatically affect fitness in natural populations, mechanisms that abate CI can have equally large impacts on fitness. We have discovered that repeated copulation by Wolbachia-infected male Drosophila simulans significantly diminishes CI. Repeated copulation does not prevent Wolbachia from populating developing spermatids, but may reduce the time during spermatogenesis when Wolbachia can express CI. This restoration of fertility in premated infected males could have important implications for Wolbachia transmission and persistence in nature and for its exploitation as an agent of biological pest control.     

A bacterial symbiont in the Bacteroidetes induces cytoplasmic incompatibility in the parasitoid wasp Encarsia pergandiella

Vertically transmitted symbionts of arthropods have been implicated in several reproductive manipulations of their hosts. These include cytoplasmic incompatibility (CI), parthenogenesis induction in haplodiploid species (PI), feminization and male killing. One symbiont lineage in the alpha-Proteobacteria, Wolbachia, is the only bacterium known to cause all of these effects, and has been thought to be unique in causing CI, in which the fecundity of uninfected females is reduced after mating with infected males. Here, we provide evidence that an undescribed symbiont in the Bacteroidetes group causes CI in a sexual population of the parasitic wasp Encarsia pergandiella. Wasps were crossed in all four possible combinations of infected and uninfected individuals. In the cross predicted to be incompatible, infected (I) males x uninfected (U) females, progeny production was severely reduced, with these females producing only 12.6% of the number of progeny in other crosses. The incompatibility observed in this haplodiploid species was the female mortality type; dissections showed that most progeny from the incompatible cross died as eggs. The 16S rDNA sequence of this symbiont is 99% identical to a parthenogenesis-inducing symbiont in other Encarsia, and 96% identical to a feminizing symbiont in haplodiploid Brevipalpus mites. Thus, this recently discovered symbiont lineage is capable of inducing three of the four principal manipulations of host reproduction known to be caused by Wolbachia.     

Natural interspecific and intraspecific horizontal transfer of parthenogenesis-inducing Wolbachia in Trichogramma wasps

The intracellular bacterium Wolbachia is one of the most common symbionts in arthropods and, because of its manipulative effects on host reproduction, is assumed to be an important factor in several evolutionary processes. These bacteria are mainly vertically transmitted from mother to daughter through the egg cytoplasm, and horizontal transmission is generally assumed to be rare. Here, we show natural inter- and intraspecific horizontal transfer of parthenogenesis-inducing Wolbachia between parasitoid wasps of the genus Trichogramma. Horizontal transfer was observed when infected and uninfected larvae shared the same host egg. This is the first report, to our knowledge, on interspecific horizontal transfer of Wolbachia between closely related sympatric species. Some originally uninfected immature wasps acquired Wolbachia while inside the host egg, but not all of these newly infected females exhibited the parthenogenesis phenotype. In general, intraspecific horizontal transfer was more successful than interspecific transfer. Wolbachia underwent vertical transmission in the new species but the infection tended to be lost within several generations. Our results have important implications for understanding the evolution of Wolbachia-host associations.     

Increased male mating rate in Drosophila is associated with Wolbachia infection

The maternally inherited bacterium Wolbachia pipientis infects 25-75% of arthropods and manipulates host reproduction to improve its transmission. One way Wolbachia achieves this is by inducing cytoplasmic incompatibility (CI), where crosses between infected males and uninfected females are inviable. Infected males suffer reduced fertility through CI and reduced sperm production. However, Wolbachia induce lower levels of CI in nonvirgin males. We examined the impact of Wolbachia on mating behaviour in male Drosophila melanogaster and D. simulans, which display varying levels of CI, and show that infected males mate at a higher rate than uninfected males in both species. This may serve to increase the spread of Wolbachia, or alternatively, may be a behavioural adaptation employed by males to reduce the level of CI. Mating at high rate restores reproductive compatibility with uninfected females resulting in higher male reproductive success thus promoting male promiscuity. Increased male mating rates also have implications for the transmission of Wolbachia.     

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.     

Can cytoplasmic incompatibility inducing Wolbachia promote the evolution of mate preferences?

The maternally inherited bacterium, Wolbachia pipientis, manipulates host reproduction by rendering uninfected females reproductively incompatible with infected males (cytoplasmic incompatibility, CI). Hosts may evolve mechanisms, such as mate preferences, to avoid fitness costs of Wolbachia infection. Despite the potential importance of mate choice for Wolbachia population dynamics, this possibility remains largely unexplored. Here we model the spread of an allele encoding female mate preference for uninfected males alongside the spread of CI inducing Wolbachia. Mate preferences can evolve but the spread of the preference allele depends on factors associated with both Wolbachia infection and the preference allele itself. Incomplete maternal transmission of Wolbachia, fitness costs and low CI, improve the spread of the preference allele and impact on the population dynamics of Wolbachia. In addition, mate preferences are found in infected individuals. These results have important consequences for the fate of Wolbachia and studies addressing mate preferences in infected populations.     

Male-killing Wolbachia in a flour beetle

The bacteria in the genus Wolbachia are cytoplasmically inherited symbionts of arthropods. Infection often causes profound changes in host reproduction, enhancing bacterial transmission and spread in a population. The reproductive alterations known to result from Wolbachia infection include cytoplasmic incompatibility (CI), parthenogenesis, feminization of genetic males, fecundity enhancement, male killing and, perhaps, lethality Here, we report male killing in a third insect, the black flour beetle Tribolium madens, based on highly female-biased sex ratios of progeny from females infected with Wolbachia. The bias is cytoplasmic in nature as shown by repeated backcrossing of infected females with males of a naturally uninfected strain. Infection also lowers the egg hatch rates significantly to approximately half of those observed for uninfected females. Treatment of the host with antibiotics eliminated infection, reverted the sex ratio to unbiased levels and increased the percentage hatch. Typically Wolbachia infection is transmitted from mother to progeny, regardless of the sex of the progeny; however, infected T. madens males are never found. Virgin females are sterile, suggesting that the sex-ratio distortion in T. madens results from embryonic male killing rather than parthenogenesis. Based on DNA sequence data, the male-killing strain of Wolbachia in T. madens was indistinguishable from the CI-inducing Wolbachia in Tribolium confusum, a closely related beetle. Our findings suggest that host symbiont interaction effects may play an important role in the induction of Wolbachia reproductive phenotypes.     

Phylogenetic evidence for horizontal transmission of Wolbachia in host-parasitoid associations

Endosymbiotic Wolbachia infect a number of arthropod species in which they can affect the reproductive system. While maternally transmitted, unlike mitochondria their molecular phylogeny does not parallel that of their hosts. This strongly suggests horizontal transmission among species, the mechanisms of which remain unknown. Such transfers require intimate between-species relationships, and thus host-parasite associations are outstandingly appropriate for study. Here, we demonstrate that hymenopteran parasitoids of frugivorous Drosophila species are especially susceptible to Wolbachia infection. Of the five common European species, four proved to be infected; furthermore, multiple infections are common, with one species being doubly infected and two triply infected (first report). Phylogenetic statuses of the Wolbachia infecting the different species of the community have been studied using the gene wsp, a highly variable gene recently described. This study reveals exciting similarities between the Wolbachia variants found in parasitoids and their hosts. These arguments strongly support the hypothesis of frequent natural Wolbachia transfers into other species and open a new field for genetic exchanges among species, especially in host-parasitoid associations.     

Influence of aging on cytoplasmic incompatibility, sperm modification and Wolbachia density in Culex pipiens mosquitoes

Wolbachia are maternally inherited endocellular bacteria, widespread in invertebrates and capable of altering several aspects of host reproduction. Cytoplasmic incompatibility (CI) is commonly found in arthropods and induces hatching failure of eggs from crosses between Wolbachia-infected males and uninfected females (or females infected by incompatible strains). Several factors such as bacterial and host genotypes or bacterial density contribute to CI strength and it has been proposed, mostly from Drosophila data, that older males have a lower Wolbachia load in testes which, thus, induces a lighter CI. Here, we challenge this hypothesis using different incompatible Culex pipiens mosquito strains and show that CI persists at the same intensity throughout the mosquito life span. Embryos from incompatible crosses showed even distributions of abortive phenotypes over time, suggesting that host ageing does not reduce the sperm-modification induced by Wolbachia. CI remained constant when sperm was placed in the spermathecae of incompatible females, indicating that sperm modification is also stable over time. The capacity of infected females to rescue CI was independent of age. Last, the density of Wolbachia in whole testes was highly strain-dependent and increased dramatically with age. Taken together, these data stress the peculiarity of the C.pipiens/Wolbachia interaction and suggest that the bacterial dosage model should be rejected in the case of this association.     

Wolbachia infection lowers fertile sperm transfer in a moth

The endosymbiotic bacterium Wolbachia pipientis manipulates host reproduction by rendering infected males reproductively incompatible with uninfected females (cytoplasmic incompatibility; CI). CI is believed to occur as a result of Wolbachia-induced modifications to sperm during maturation, which prevent infected sperm from initiating successful zygote development when fertilizing uninfected females' eggs. However, the mechanism by which CI occurs has been little studied outside the genus Drosophila. Here, we show that in the sperm heteromorphic Mediterranean flour moth, Ephestia kuehniella, infected males transfer fewer fertile sperm at mating than uninfected males. In contrast, non-fertile apyrene sperm are not affected. This indicates that Wolbachia may only affect fertile sperm production and highlights the potential of the Lepidoptera as a model for examining the mechanism by which Wolbachia induces CI in insects.     

A genetic test of the mechanism of Wolbachia-induced cytoplasmic incompatibility in Drosophila

Cytoplasmic bacteria of the genus Wolbachia are best known as the cause of cytoplasmic incompatibility (CI): many uninfected eggs fertilized by Wolbachia-modified sperm from infected males die as embryos. In contrast, eggs of infected females rescue modified sperm and develop normally. Although Wolbachia cause CI in at least five insect orders, the mechanism of CI remains poorly understood. Here I test whether the target of Wolbachia-induced sperm modification is the male pronucleus (e.g., DNA or pronuclear proteins) or some extranuclear factor from the sperm required for embryonic development (e.g., the paternal centrosome). I distinguish between these hypotheses by crossing gynogenetic Drosophila melanogaster females to infected males. Gynogenetic females produce diploid eggs whose normal development requires no male pronucleus but still depends on extranuclear paternal factors. I show that when gynogenetic females are crossed to infected males, uniparental progeny with maternally derived chromosomes result. This finding shows that Wolbachia impair the male pronucleus but no extranuclear component of the sperm.     

Wolbachia and nuclear-nuclear interactions contribute to reproductive incompatibility in the spider mite Panonychus mori (Acari: Tetranychidae)

Maternally transmitted bacteria of the genus Wolbachia are obligate, intracellular symbionts that are responsible for cytoplasmic incompatibility in a wide range of arthropods such as insects and mites. Spider mites often show uni- and bidirectional incompatibilities among populations with and without Wolbachia. Therefore, we surveyed the presence of Wolbachia by PCR and then conducted crossing experiments among 25 populations of Panonychus mori to determine how Wolbachia are related to the incompatibility in this species. Five out of the 25 populations were infected with Wolbachia. These five populations were treated with an antibiotic (rifampicin) to eliminate Wolbachia. We carried out round-robin crosses among 20 Wolbachia-uninfected populations, five infected populations and five rifampicin-treated populations (30 x 30=900 crosses in total). Incompatibility among P. mori populations was caused by Wolbachia infection, nuclear-cytoplasmic interactions or nuclear-nuclear interactions. Wolbachia-mediated incompatibility was observed in crosses between uninfected females and infected males or between females and males harboring different Wolbachia strains. Nuclear-cytoplasmic interactions may be responsible for the unidirectional incompatibility in crosses between the two northern populations and one of the southern populations. Bidirectional incompatibility caused by nuclear-nuclear interactions was observed in 99 combinations of interpopulation crosses (99/300=0.33). Although no geographical trends were detected in the distribution of bidirectionally compatible populations, the results reveal a genetic divergence among P. mori populations.     

Characterization of non-cytoplasmic incompatibility inducing Wolbachia in two continental African populations of Drosophila simulans

Wolbachia is an endocellular bacterium infecting arthropods and nematodes. In arthropods, it invades host populations through various mechanisms, affecting host reproduction, the most common of which being cytoplasmic incompatibility (CI). CI is an embryonic mortality occurring when infected males mate with uninfected females or females infected by a different Wolbachia strain. This phenomenon is observed in Drosophila simulans, an intensively studied Wolbachia host, harbouring at least five distinct bacterial strains. In this study, we investigate various aspects of the Wolbachia infections occurring in two continental African populations of D. simulans: CI phenotype, phylogenetic position based on the wsp gene and associated mitochondrial haplotype. From the East African population (Tanzania), we show that (i) the siIII mitochondrial haplotype occurs in continental populations, which was unexpected based on the current views of D. simulans biogeography, (ii) the wKi strain (that rescues from CI while being unable to induce it) is very closely related to the CI-inducing strain wNo, (iii) wKi and wNo might not derive from a unique infection event, and (iv) wKi is likely to represent the same entity as the previously described wMa variant. In the West African population (Cameroon), the Wolbachia infection was found identical to the previously described wAu, which does not induce CI. This finding supports the view that wAu might be an ancient infection in D. simulans.     

High Temperatures Eliminate Wolbachia, a Cytoplasmic Incompatibility Inducing Endosymbiont, From the Two-spotted Spider Mite

Wolbachia can induce cytoplasmic incompatibility (CI) in the arrhenotokous two-spotted spider mite between uninfected females and infected males. Cytoplasmic incompatibility is expressed through a male-biased sex ratio and a low hatchability, and can be suppressed by removing Wolbachia from spider mites reared on a diet with antibiotics. Here we investigated whether heat-treatment can elimate Wolbachia from infected mites. Using a PCR assay with a Wolbachia-specific primer pair (ftsZ), and by standard crosses, we were able to show that 71 per cent of the mites had lost the Wolbachia infection after rearing the infected population at 32 ± 0.5 °C for four generations. The infection could be completely removed when mites were reared at 32 ± 0.5 °C for six generations. Curing through high temperatures could be one of the reasons why mixed infected/uninfected populations occur in the field. An additional consequence of rearing mites at 32 ± 0.5 °C was the shortened development time. The effect of environmental temperature on the abundance of Wolbachia and possible behavioural consequences for the spider mite are discussed.     

On the evolution of cytoplasmic incompatibility in haplodiploid species

The most enigmatic sexual manipulation by Wolbachia endosymbionts is cytoplasmic incompatibility (CI): infected males are reproductively incompatible with uninfected females. In this paper, we extend the theory on population dynamics and evolution of CI, with emphasis on haplodiploid species. First, we focus on the problem of the threshold to invasion of the Wolbachia infection in a population. Simulations of the dynamics of infection in small populations show that it does not suffice to assume invasion by drift alone (or demographic "accident"). We propose several promising alternatives that may facilitate invasion of Wolbachia in uninfected populations: sex-ratio effects, meta population structure, and other fitness-compensating effects. Including sex-ratio effects of Wolbachia allows invasion whenever infected females produce more infected daughters than uninfected females produce uninfected daughters. Several studies on haplodiploid species suggest the presence of such sex-ratio effects. The simple metapopulation model we analyzed predicts that, given that infecteds are better "invaders," uninfecteds must be better "colonizers" to maintain coexistence of infected and uninfected patches. This condition seems more feasible for species that suffer local extinction due to predation (or parasitization) than for species that suffer local extinction due to overexploiting their resource(s). Finally, we analyze the evolution of CI in haplodiploids once a population has been infected. Evolution does not depend on the type of CI (female mortality or male production), but hinges solely on decreasing the fitness cost and/or increasing the transmission efficiency. Our models offer new perspectives for increasing our understanding of the population and evolutionary dynamics of CI.     

我国三地区黑腹果蝇中Wolbachia的系统发育关系及其对宿主生殖的影响

【目的】Wolbachia 是广泛存在于节肢动物和丝状线虫体内的一类共生菌, 能够以多种方式对宿主产生影响。精卵细胞质不亲和（CI）是其引起的最普遍的表型, 即感染Wolbachia的雄性宿主与未感染或感染不同品系的雌性宿主交配后, 不能产生后代或后代极少, 而感染同品系Wolbachia的雌雄宿主交配后则能正常产生后代。我们前期研究发现, 湖北武汉、 云南六库和天津3个地区黑腹果蝇Drosophila melanogaster被Wolbachia感染。本研究旨在明确这3个地区黑腹果蝇中Wolbachia的系统发育关系及其对宿主生殖的影响。【方法】利用Clustal X软件对Wolbachia的wsp基因序列进行比对, 利用MEGA软件构建系统发育树。采用多位点序列分型（MLST）的方法对Wolbachia进行分型。通过区内交配和区之间杂交的方式研究不同地区黑腹果蝇体内Wolbachia 的关系及其对果蝇生殖的影响。【结果】湖北武汉、 云南六库和天津3个地区黑腹果蝇中感染的Wolbachia都是属于A大组的Mel亚群。这3个地区果蝇感染的Wolbachia的序列类型（ST）不同, Wolbachia之间存在一定的差异。湖北武汉和天津果蝇中的Wolbachia能引起强烈的CI表型, 而云南六库果蝇中的Wolbachia引起的CI强度相对较弱。武汉果蝇中Wolbachia不能完全挽救天津果蝇中Wolbachia引起的CI表型, 而天津果蝇中Wolbachia也不能完全挽救武汉果蝇中Wolbachia引起的CI表型。【结论】武汉和天津地区黑腹果蝇中的Wolbachia可能距离较远。Wolbachia的长期共生可能对黑腹果蝇的进化产生了一定的影响, 湖北武汉与云南六库的黑腹果蝇中感染的Wolbachia属于不同的序列类型, 这2个地区的黑腹果蝇已发生了一定的分歧, 产生了一定的生殖隔离。