Male-Killing Spiroplasma Induces Sex-Specific Cell Death via Host Apoptotic Pathway

Some symbiotic bacteria cause remarkable reproductive phenotypes like cytoplasmic incompatibility and male-killing in their host insects. Molecular and cellular mechanisms underlying these symbiont-induced reproductive pathologies are of great interest but poorly understood. In this study, Drosophila melanogaster and its native Spiroplasma symbiont strain MSRO were investigated as to how the host''s molecular, cellular and morphogenetic pathways are involved in the symbiont-induced male-killing during embryogenesis. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining, anti-cleaved-Caspase-3 antibody staining, and apoptosis-deficient mutant analysis unequivocally demonstrated that the host''s apoptotic pathway is involved in Spiroplasma-induced male-specific embryonic cell death. Double-staining with TUNEL and an antibody recognizing epidermal marker showed that embryonic epithelium is the main target of Spiroplasma-induced male-specific apoptosis. Immunostaining with antibodies against markers of differentiated and precursor neural cells visualized severe neural defects specifically in Spiroplasma-infected male embryos as reported in previous studies. However, few TUNEL signals were detected in the degenerate nervous tissues of male embryos, and the Spiroplasma-induced neural defects in male embryos were not suppressed in an apoptosis-deficient host mutant. These results suggest the possibility that the apoptosis-dependent epidermal cell death and the apoptosis-independent neural malformation may represent different mechanisms underlying the Spiroplasma-induced male-killing. Despite the male-specific progressive embryonic abnormality, Spiroplasma titers remained almost constant throughout the observed stages of embryonic development and across male and female embryos. Strikingly, a few Spiroplasma-infected embryos exhibited gynandromorphism, wherein apoptotic cell death was restricted to male cells. These observations suggest that neither quantity nor proliferation of Spiroplasma cells but some Spiroplasma-derived factor(s) may be responsible for the expression of the male-killing phenotype.     

Low Temperature Reveals Genetic Variability Against Male-Killing Spiroplasma in Drosophila melanogaster Natural Populations

Spiroplasma endosymbionts are maternally inherited microorganisms which infect many arthropod species. In some Drosophila species, it acts as a reproductive manipulator, spreading in populations by killing the sons of infected mothers. Distinct Drosophila melanogaster populations from Brazil exhibit variable male-killing Spiroplasma prevalences. In this study, we investigated the presence of variability for the male-killing phenotype among Drosophila and/or Spiroplasma strains and verified if it correlates with the endosymbiont prevalence in natural populations. For that, we analyzed the male-killing expression when Spiroplasma strains from different populations were transferred to a standard D. melanogaster line (Canton-S) and when a common Spiroplasma strain was transferred to different wild-caught D. melanogaster lines, both at optimal and challenging temperatures for the bacteria. No variation was observed in the male-killing phenotype induced by different Spiroplasma strains. No phenotypic variability among fly lines was detected at optimal temperature (23 °C), as well. Conversely, significant variation in the male-killing expression was revealed among D. melanogaster lines at 18.5 °C, probably caused by imperfect transmission of the endosymbiont. Distinct lines differed in their average sex ratios as well as in the pattern of male-killing expression as the infected females aged. Greater variation occurred among lines from one locality, although there was no clear correlation between the male-killing intensity and the endosymbiont prevalence in each population. Imperfect transmission or male killing may also occur in the field, thus helping to explain the low or intermediate prevalences reported in nature. We discuss the implications of our results for the dynamics of male-killing Spiroplasma in natural populations.     

Male killing Spiroplasma protects Drosophila melanogaster against two parasitoid wasps

Maternally transmitted associations between endosymbiotic bacteria and insects are diverse and widespread in nature. Owing to imperfect vertical transmission, many heritable microbes have evolved compensational mechanisms to enhance their persistence in host lineages, such as manipulating host reproduction and conferring fitness benefits to host. Symbiont-mediated defense against natural enemies of hosts is increasingly recognized as an important mechanism by which endosymbionts enhance host fitness. Members of the genus Spiroplasma associated with distantly related Drosophila hosts are known to engage in either reproductive parasitism (i.e., male killing) or defense against natural enemies (the parasitic wasp Leptopilina heterotoma and a nematode). A male-killing strain of Spiroplasma (strain Melanogaster Sex Ratio Organism (MSRO)) co-occurs with Wolbachia (strain wMel) in certain wild populations of the model organism Drosophila melanogaster. We examined the effects of Spiroplasma MSRO and Wolbachia wMel on Drosophila survival against parasitism by two common wasps, Leptopilina heterotoma and Leptopilina boulardi, that differ in their host ranges and host evasion strategies. The results indicate that Spiroplasma MSRO prevents successful development of both wasps, and confers a small, albeit significant, increase in larva-to-adult survival of flies subjected to wasp attacks. We modeled the conditions under which defense can contribute to Spiroplasma persistence. Wolbachia also confers a weak, but significant, survival advantage to flies attacked by L. heterotoma. The host protective effects exhibited by Spiroplasma and Wolbachia are additive and may provide the conditions for such cotransmitted symbionts to become mutualists. Occurrence of Spiroplasma-mediated protection against distinct parasitoids in divergent Drosophila hosts suggests a general protection mechanism.     

Population Dynamics of Male-Killing and Non-Male-Killing Spiroplasmas in Drosophila melanogaster

The endosymbiotic bacteria Spiroplasma spp. are vertically transmitted through female hosts and are known to cause selective death of male offspring in insects. One strain of spiroplasma, NSRO, causes male killing in Drosophila species, and a non-male-killing variant of NSRO, designated NSRO-A, has been isolated. It is not known why NSRO-A does not kill males. In an attempt to understand the mechanism of male killing, we investigated the population dynamics of NSRO and NSRO-A throughout the developmental course of the laboratory host Drosophila melanogaster by using a quantitative PCR technique. In the early development of the host insect, the titers of NSRO were significantly higher than those of NSRO-A at the first- and second-instar stages, whereas at the egg, third-instar, and pupal stages, the titers of the two spiroplasmas were almost the same. Upon adult emergence, the titers of the two spiroplasmas were similar, around 2 × 10⁸ dnaA copy equivalents. However, throughout host aging, the two spiroplasmas showed strikingly different population growth patterns. The titers of NSRO increased exponentially for 3 weeks, attained a peak value of around 4 × 10⁹ dnaA copy equivalents per insect, and then decreased. In contrast, the titers of NSRO-A were almost constant throughout the adult portion of the life cycle. In adult females, consequently, the titer of NSRO was significantly higher than the titer of NSRO-A except for a short period just after emergence. Although infection of adult females with NSRO resulted in almost 100% male killing, production of some male offspring was observed within 4 days after emergence when the titers of NSRO were as low as those of NSRO-A. Based on these results, we proposed a threshold density hypothesis for the expression of male killing caused by the spiroplasma. The extents of the bottleneck in the vertical transmission through host generations were estimated to be 5 × 10⁻⁵ for NSRO and 3 × 10⁻⁴ for NSRO-A.     

Infection densities of three Spiroplasma strains in the host Drosophila melanogaster

Maternally transmitted endosymbiotic bacteria of the genus Spiroplasma associate with numerous insect species, including the genus Drosophila. Among the Spiroplasma strains associated with Drosophila, several manipulate their host??s reproduction by killing the male offspring of the infected females. Although the male-killing mechanism is not well understood, previous studies of non-native strains transferred to D. melanogaster (strain Oregon-R) indicate that the male-killing strain achieves higher densities than two non-male-killing strains. Whether this pattern of higher male-killing strain densities occurs in other host-symbiont strain combinations is not known. Herein, we used quantitative PCR to examine infection densities of one non-male-killing strain native to D. hydei (Hyd1), and two male-killing strains; one native to D. nebulosa (NSRO), and one native to D. melanogaster (MSRO; recently discovered), upon artificial transfer to D. melanogaster (strain Canton-S). Infection densities were examined at four weekly intervals in adult flies, across three consecutive generations following artificial transfer. Infection densities of the non-male-killing strain were significantly lower than those of the two male killers immediately after adult emergence. At later time points, however, the non-male-killing strain (Hyd1) is capable of proliferating to densities similar to those of the two male-killing strains (NSRO and MSRO) in D. melanogaster (Canton-S). We also examined the effect of co-infection by the heritable bacterium Wolbachia, on Spiroplasma densities and male-killing ability. Wolbachia had little to no effect of Spiroplasma densities, but the male-killing ability of MSRO was lower in the presence of Wolbachia. Generation post-infection had little effect on Spiroplasma densities, but affected the male-killing ability.     

Silence of the killers: discovery of male-killing suppression in a rearing strain of the small brown planthopper,Laodelphax striatellus

According to evolutionary theory, sex ratio distortions caused by reproductive parasites such as Wolbachia and Spiroplasma are predicted to be rapidly normalized by the emergence of host nuclear suppressors. However, such processes in the evolutionary arms race are difficult to observe because sex ratio biases will be promptly hidden and become superficially unrecognizable. The evolution of genetic suppressors has been reported in just two insect species so far. In the small brown planthopper, Laodelphax striatellus, female-biases caused by Spiroplasma, which is a ‘late’ male-killer, have been found in some populations. During the continuous rearing of L. striatellus, we noted that a rearing strain had a 1 : 1 sex ratio even though it harboured Spiroplasma. Through introgression crossing experiments with a strain lacking suppressors, we revealed that the L. striatellus strain had the zygotic male-killing suppressor acting as a dominant trait. The male-killing phenotype was hidden by the suppressor even though Spiroplasma retained its male-killing ability. This is the first study to demonstrate the existence of a late male-killing suppressor and its mode of inheritance. Our results, together with those of previous studies, suggest that the inheritance modes of male-killing suppressors are similar regardless of insect order or early or late male killing.     

Mating rates and the prevalence of male‐killing Spiroplasma in Harmonia axyridis (Coleoptera: Coccinellidae)

Maternally inherited bacteria that kill male but not female hosts during embryogenesis have been widely reported in invertebrates. Harmonia axyridis is one of the species infected by male‐killing Spiroplasma. The presence of male‐killers in host populations can lead to the occurrence of extremely female‐biased sex ratios. Furthermore, infected females may have fewer chances to mate if males can discriminate between infected and uninfected females and prefer the latter. Although there have been many investigations of male‐killer infection rates in H. axyridis, little is known about the influence of host mating on male‐killer infection dynamics. We investigated copulation rates and changes in infection frequency in a wild population of H. axyridis in western Japan. Almost all infected females collected each year laid fertilized eggs and had therefore mated. Mean infection rates of females collected each year were 13% in 2003, 15% in 2012 and 23% in 2013. Statistical analysis showed that neither the copulation rate nor the infection rate differed significantly among years. These results suggest that the infection rate of H. axyridis with male‐killing Spiroplasma is kept approximately constant and that there is no difference in the chance of mating with infected and uninfected females.     

Fitness effects of Wolbachia and Spiroplasma in Drosophila melanogaster

Maternally inherited endosymbionts that manipulate the reproduction of their insect host are very common. Aside from the reproductive manipulation they produce, the fitness of these symbionts depends in part on the direct impact they have on the female host. Although this parameter has commonly been investigated for single infections, it has much more rarely been established in dual infections. We here establish the direct effect of infection with two different symbionts exhibiting different reproductive manipulation phenotypes, both alone and in combination, in the fruit fly Drosophila melanogaster. This species carries a cytoplasmic incompatibility inducing Wolbachia and a male-killing Spiroplasma, occurring as single or double (co-) infections in natural populations. We assessed direct fitness effects of these bacteria on their host, by comparing larval competitiveness and adult fecundity of uninfected, Wolbachia, Spiroplasma and Wolbachia–Spiroplasma co-infected females. We found no effect of infection status on the fitness of females for both estimates, that is, no evidence of any benefits or costs to either single or co-infection. This leads to the conclusion that both bacteria probably have other sources of benefits to persist in D. melanogaster populations, either by means of their reproductive manipulations (fitness compensation from male death in Spiroplasma infection and cytoplasmic incompatibility in Wolbachia infection) or by positive fitness interactions on other fitness components.     

Infection with endosymbiotic Spiroplasma disrupts tsetse (Glossina fuscipes fuscipes) metabolic and reproductive homeostasis

Tsetse flies (Glossina spp.) house a population-dependent assortment of microorganisms that can include pathogenic African trypanosomes and maternally transmitted endosymbiotic bacteria, the latter of which mediate numerous aspects of their host’s metabolic, reproductive, and immune physiologies. One of these endosymbionts, Spiroplasma, was recently discovered to reside within multiple tissues of field captured and laboratory colonized tsetse flies grouped in the Palpalis subgenera. In various arthropods, Spiroplasma induces reproductive abnormalities and pathogen protective phenotypes. In tsetse, Spiroplasma infections also induce a protective phenotype by enhancing the fly’s resistance to infection with trypanosomes. However, the potential impact of Spiroplasma on tsetse’s viviparous reproductive physiology remains unknown. Herein we employed high-throughput RNA sequencing and laboratory-based functional assays to better characterize the association between Spiroplasma and the metabolic and reproductive physiologies of G. fuscipes fuscipes (Gff), a prominent vector of human disease. Using field-captured Gff, we discovered that Spiroplasma infection induces changes of sex-biased gene expression in reproductive tissues that may be critical for tsetse’s reproductive fitness. Using a Gff lab line composed of individuals heterogeneously infected with Spiroplasma, we observed that the bacterium and tsetse host compete for finite nutrients, which negatively impact female fecundity by increasing the length of intrauterine larval development. Additionally, we found that when males are infected with Spiroplasma, the motility of their sperm is compromised following transfer to the female spermatheca. As such, Spiroplasma infections appear to adversely impact male reproductive fitness by decreasing the competitiveness of their sperm. Finally, we determined that the bacterium is maternally transmitted to intrauterine larva at a high frequency, while paternal transmission was also noted in a small number of matings. Taken together, our findings indicate that Spiroplasma exerts a negative impact on tsetse fecundity, an outcome that could be exploited for reducing tsetse population size and thus disease transmission.     

Asymmetrical Interactions between Wolbachia and Spiroplasma Endosymbionts Coexisting in the Same Insect Host

We investigated the interactions between the endosymbionts Wolbachia pipientis strain wMel and Spiroplasma sp. strain NSRO coinfecting the host insect Drosophila melanogaster. By making use of antibiotic therapy, temperature stress, and hemolymph microinjection, we established the following strains in the same host genetic background: the SW strain, infected with both Spiroplasma and Wolbachia; the S strain, infected with Spiroplasma only; and the W strain, infected with Wolbachia only. The infection dynamics of the symbionts in these strains were monitored by quantitative PCR during host development. The infection densities of Spiroplasma exhibited no significant differences between the SW and S strains throughout the developmental course. In contrast, the infection densities of Wolbachia were significantly lower in the SW strain than in the W strain at the pupal and young adult stages. These results indicated that the interactions between the coinfecting symbionts were asymmetrical, i.e., Spiroplasma organisms negatively affected the population of Wolbachia organisms, while Wolbachia organisms did not influence the population of Spiroplasma organisms. In the host body, the symbionts exhibited their own tissue tropisms: among the tissues examined, Spiroplasma was the most abundant in the ovaries, while Wolbachia showed the highest density in Malpighian tubules. Strikingly, basically no Wolbachia organisms were detected in hemolymph, the principal location of Spiroplasma. These results suggest that different host tissues act as distinct microhabitats for the symbionts and that the lytic process in host metamorphosis might be involved in the asymmetrical interactions between the coinfecting symbionts.     

Male Killing and Incomplete Inheritance of a Novel <Emphasis Type="Italic">Spiroplasma</Emphasis> in the Moth <Emphasis Type="Italic">Ostrinia zaguliaevi</Emphasis>

Bacteria of the genus Spiroplasma are widely found in plants and arthropods. Some of the maternally transmitted Spiroplasma endosymbionts in arthropods are known to kill young male hosts (male killing). Here, we describe a new case of Spiroplasma-induced male killing in a moth, Ostrinia zaguliaevi. The all-female trait caused by Spiroplasma was maternally inherited for more than 11 generations but was spontaneously lost in several lineages. Antibiotic treatment eliminated the Spiroplasma infection and restored the 1:1 sex ratio. The survival rates and presence/absence of the W chromosome in the embryonic and larval stages of O. zaguliaevi showed that males were selectively killed, exclusively during late embryogenesis in all-female broods. Based on phylogenetic analyses of 16S rRNA, dnaA and rpoB gene sequences, the causative bacteria were identified as Spiroplasma belonging to the tick symbiont Spiroplasma ixodetis clade. Electron microscopy confirmed bacterial structures in the follicle cells and follicular sheath of adult females. Although many congeneric Ostrinia moths harbor another sex ratio-distorting bacterium (Wolbachia), only O. zaguliaevi harbors Spiroplasma.     

Female and male genetic contributions to post-mating immune defence in female Drosophila melanogaster

Post-mating reduction in immune defence is common in female insects, and a trade-off between mating and immunity could affect the evolution of immunity. In this work, we tested the capacity of virgin and mated female Drosophila melanogaster to defend against infection by four bacterial pathogens. We found that female D. melanogaster suffer post-mating immunosuppression in a pathogen-dependent manner. The effect of mating was seen after infection with two bacterial pathogens (Providencia rettgeri and Providencia alcalifaciens), though not after infection with two other bacteria (Enterococcus faecalis and Pseudomonas aeruginosa). We then asked whether the evolution of post-mating immunosuppression is primarily a ‘female’ or ‘male’ trait by assaying for genetic variation among females for the degree of post-mating immune suppression they experience and among males for the level of post-mating immunosuppression they elicit in their mates. We also assayed for an interaction between male and female genotypes to test the specific hypothesis that the evolution of a trade-off between mating and immune defence in females might be being driven by sexual conflict. We found that females, but not males, harbour significant genetic variation for post-mating immunosuppression, and we did not detect an interaction between female and male genotypes. We thus conclude that post-mating immune depression is predominantly a ‘female’ trait, and find no evidence that it is evolving under sexual conflict.     

The pathology of embryo death caused by the male-killing Spiroplasma bacterium in Drosophila nebulosa

Background

Inherited bacteria that kill male offspring, male-killers, are known to be common in insects, but little is understood about the mechanisms used by male-killing bacteria to kill males. In this paper we describe the tempo and changes that occur during male-killing by Spiroplasma bacteria in the host Drosophila nebulosa.

Results

Spiroplasma infected D. nebulosa males were developmentally retarded from 6–8 h into embryonic development at 25°C, and arrested at between stages 12 and 13 of embryogenesis (10–12 h). Dying males were characterized by a failure to form segments, and ultimately disintegration of the normal oval embryonic shape. Prior to death, dying males exhibited widespread apoptosis, as testified by TUNEL staining.

Conclusion

The Spiroplasma kills male Drosophila in a narrow developmental period, shortly after the formation of the host dosage compensation complex that is required for male-killing. Male death is preceded by widespread apoptosis, but it is uncertain if this is primary or secondary apoptosis.     

Influences of two coexisting endosymbionts,CI‐inducing Wolbachia and male‐killing Spiroplasma,on the performance of their host Laodelphax striatellus (Hemiptera: Delphacidae)

The small brown planthopper Laodelphax striatellus (Hemiptera: Delphacidae) is reported to have the endosymbiont Wolbachia, which shows a strong cytoplasmic incompatibility (CI) between infected males and uninfected females. In the 2000s, female‐biased L. striatellus populations were found in Taiwan, and this sex ratio distortion was the result of male‐killing induced by the infection of another endosymbiont, Spiroplasma. Spiroplasma infection is considered to negatively affect both L. striatellus and Wolbachia because the male‐killing halves the offspring of L. striatellus and hinders the spread of Wolbachia infection via CI. Spiroplasma could have traits that increase the fitness of infected L. striatellus and/or coexisting organisms because the coinfection rates of Wolbachia and Spiroplasma were rather high in some areas. In this study, we investigated the influences of the infection of these two endosymbionts on the development, reproduction, and insecticide resistance of L. striatellus in the laboratory. Our results show that the single‐infection state of Spiroplasma had a negative influence on the fertility of L. striatellus, while the double‐infection state had no significant influence. At late nymphal and adult stages, the abundance of Spiroplasma was lower in the double‐infection state than in the single‐infection state. In the double‐infection state, the reduction of Spiroplasma density may be caused by competition between the two endosymbionts, and the negative influence of Spiroplasma on the fertility of host may be relieved. The resistance of L. striatellus to four insecticides was compared among different infection states of endosymbionts, but Spiroplasma infection did not contribute to increase insecticide resistance. Because positive influences of Spiroplasma infection were not found in terms of the development, reproduction, and insecticide resistance of L. striatellus, other factors improving the fitness of Spiroplasma‐infected L. striatellus may be related to the high frequency of double infection in some L. striatellus populations.     

Male‐killer dynamics in Danaus chrysippus (L.) (Lepidoptera: Nymphalidae) in East Africa

Within certain regions in East Africa, the butterfly Danaus chrysippus (L.) shows female‐biased population sex ratio, because of the production by some females of all‐female broods, as a result of infection by maternally inherited, male‐killing bacterium of the genus Spiroplasma. In this study, we describe a 3‐year field survey for the population dynamics of the male‐killing Spiroplasma in D. chrysippus in four independent localities, namely Uganda, Ghana, Sudan and Madagascar. The prevalence of the bacterium was found to show extensive variations at multiple scales among different sites, in various countries, seasons and years. A novel, selection‐based hypothesis was suggested to explain the high variability of male‐killer prevalence over space and time, based on the existence of an adaptive link between larval food‐plant density and the magnitude of resource reallocation fitness advantage for the male‐killer.     

Wolbachia infection in the Korean endemic firefly,Luciola unmunsana (Coleoptera: Lampyridae)

Wolbachia is one of the most prevalent endosymbiontic bacteria of arthropods. The bacterium induces sex ratio distortions in various host insects through processes such as cytoplasmic incompatibility, feminization, male killing, and parthenogenesis. We investigated if the Korean endemic firefly, Luciola unmunsana was infected with the bacterium because the species had an abnormal sex ratio in the field. The results show that some individuals are infected with the bacterium. Phylogenetic analyses showed that the bacterial strain infecting the firefly is closely related to strains that infect phylogenetically distant hosts.     

Macroevolutionary persistence of heritable endosymbionts: acquisition,retention and expression of adaptive phenotypes in Spiroplasma

The phylogenetic incongruence between insects and their facultative maternally transmitted endosymbionts indicates that these infections are generally short‐lived evolutionarily. Therefore, long‐term persistence of many endosymbionts must depend on their ability to colonize and spread within new host species. At least 17 species of Drosophila are infected with endosymbiotic Spiroplasma that have various phenotypic effects. We transinfected five strains of Spiroplasma from three divergent clades into Drosophila neotestacea to test their capacity to spread in a novel host. A strain that causes male killing in Drosophila melanogaster (its native host) also does so in D. neotestacea, even though these host species diverged 40–60 mya. A strain native to D. neotestacea (designated sNeo) and the two other strains of the poulsonii clade of Spiroplasma confer resistance to wasp parasitism, suggesting that this trait may be ancestral within this clade of Spiroplasma. Conversely, no strain other than sNeo conferred resistance to the sterilizing effects of nematode parasitism, suggesting that nematode resistance is a recently derived condition. The apparent addition of nematode resistance to a Spiroplasma lineage that already confers resistance to wasp parasitism suggests endosymbionts can increase the repertoire of traits conducive to their spread. The capacity of an endosymbiont to undergo maternal transmission and express adaptive phenotypes in novel hosts, without requiring a period of host–symbiont co‐evolution, enables the spread of such symbionts immediately after the colonization of a new host. This could be critical for the macroevolutionary persistence of facultative endosymbionts whose sojourn times within individual host species are relatively brief.     

Two Strains of Male-Killing Wolbachia in a Ladybird,Coccinella undecimpunctata,from a Hot Climate

Ladybirds are a hot-spot for the invasion of male-killing bacteria. These maternally inherited endosymbionts cause the death of male host embryos, to the benefit of female sibling hosts and the bacteria that they contain. Previous studies have shown that high temperatures can eradicate male-killers from ladybirds, leaving the host free from infection. Here we report the discovery of two maternally inherited sex ratio distorters in populations of a coccinellid, Coccinella undecimpunctata, from a hot lowland region of the Middle East. DNA sequence analysis indicates that the male killing is the result of infection by Wolbachia, that the trait is tetracycline sensitive, and that two distinct strains of Wolbachia co-occur within one beetle population. We discuss the implications of these findings for theories of male-killing and suggest avenues for future field-work on this system.     

The Bacterial Symbiont Wolbachia Induces Resistance to RNA Viral Infections in Drosophila melanogaster

Wolbachia are vertically transmitted, obligatory intracellular bacteria that infect a great number of species of arthropods and nematodes. In insects, they are mainly known for disrupting the reproductive biology of their hosts in order to increase their transmission through the female germline. In Drosophila melanogaster, however, a strong and consistent effect of Wolbachia infection has not been found. Here we report that a bacterial infection renders D. melanogaster more resistant to Drosophila C virus, reducing the load of viruses in infected flies. We identify these resistance-inducing bacteria as Wolbachia. Furthermore, we show that Wolbachia also increases resistance of Drosophila to two other RNA virus infections (Nora virus and Flock House virus) but not to a DNA virus infection (Insect Iridescent Virus 6). These results identify a new major factor regulating D. melanogaster resistance to infection by RNA viruses and contribute to the idea that the response of a host to a particular pathogen also depends on its interactions with other microorganisms. This is also, to our knowledge, the first report of a strong beneficial effect of Wolbachia infection in D. melanogaster. The induced resistance to natural viral pathogens may explain Wolbachia prevalence in natural populations and represents a novel Wolbachia–host interaction.     

Misdirection of dosage compensation underlies bidirectional sex-specific death in Wolbachia-infected Ostrinia scapulalis

Endosymbiotic bacteria of the genus Wolbachia often manipulate the reproductive system of their hosts to propagate themselves in host populations. Ostrinia scapulalis moths infected with Wolbachia (wSca) produce female-only progeny (sex chromosomes: ZW), whereas females cured of the infection by antibiotic treatment produce male-only progeny (ZZ). The occurrence of female- and male-only progeny has been attributed to the specific death of the opposite sex during embryonic and larval development. In this bidirectional sex-specific lethality, embryos destined to die express a phenotypic sex opposite to their genotypic sex. On the basis of these findings, we suggested that wSca carries a genetic factor that feminizes the male host, the W chromosome of the host has lost its feminizing function, and discordance between the genotypic and phenotypic sexes underlies this sex-specific death. In the present study, we examined whether the failure of dosage compensation was responsible for this sex-specific mortality. Quantitative PCRs showed that Z-linked gene expression levels in embryos destined to die were not properly dosage compensated; they were approximately two-fold higher in the male progeny of wSca-infected females and approximately two-fold lower in the female progeny of infected-and-cured females. These results support our hypothesis that misdirection of dosage compensation underlies the sex-specific death.