Male age,host effects and the weak expression or non-expression of cytoplasmic incompatibility in Drosophila strains infected by maternally transmitted Wolbachia

In Drosophila melanogaster, the maternally inherited endocellular microbe Wolbachia causes cytoplasmic incompatibility (CI) in crosses between infected males and uninfected females. CI results in a reduction in the number of eggs that hatch. The level of CI expression in this species has been reported as varying from partial (a few eggs fail to hatch) to nonexistent (all eggs hatch). We show that male age in this host species has a large impact on the level of CI exhibited and explains much of this variability. Strong CI is apparent when young males are used in crosses. CI declines rapidly with male age, particularly when males are repeatedly mated. Wolbachia from a Canton S line that was previously reported as not causing CI does in fact induce CI when young males are used in crosses, albeit at a weaker level than in other D. melanogaster strains. The strain differences in CI expression are due to host background effects rather than differences in Wolbachia strains. These results highlight the importance of undertaking crosses with a range of male ages and nuclear backgrounds before ascribing particular host phenotypes to Wolbachia strains.     

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.     

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.     

The distribution and proliferation of the intracellular bacteria Wolbachia during spermatogenesis in Drosophila.

Wolbachia is a cytoplasmically inherited alpha-proteobacterium found in a wide range of host arthropod and nematode taxa. Wolbachia infection in Drosophila is closely associated with the expression of a unique form of post-fertilization lethality termed cytoplasmic incompatibility (CI). This form of incompatibility is only expressed by infected males suggesting that Wolbachia exerts its effect during spermatogenesis. The growth and distribution of Wolbachia throughout sperm development in individual spermatocysts and elongating sperm bundles is described. Wolbachia growth within a developing cyst seems to begin during the pre-meiotic spermatocyte growth phase with the majority of bacteria accumulating during cyst elongation. Wolbachia are predominantly localized in the proximal end of the immature cyst, opposite the spermatid nuclei, and throughout development there appears little movement of Wolbachia between spermatids via the connecting cytoplasmic bridges. The overall number of new cysts infected as well as the number of spermatids/cysts infected seems to decrease with age and corresponds to the previously documented drop in CI with age. In contrast, in one CI expressing line of Drosophila melanogaster, fewer cysts are infected and a much greater degree of variation in numbers is observed between spermatids. Furthermore, the initiation and extent of the fastest period of Wolbachia growth in the D. melanogaster strain lags behind that of Drosophila simulans. The possible implications on the as yet unexplained mechanism of CI are discussed.     

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。     

Evidence for a global Wolbachia replacement in Drosophila melanogaster

Wolbachia are maternally inherited intracellular alpha-Proteobacteria found in numerous arthropod and filarial nematode species. They influence the biology of their hosts in many ways. In some cases, they act as obligate mutualists and are required for the normal development and reproduction of the host. They are best known, however, for the various reproductive parasitism traits that they can generate in infected hosts. These include cytoplasmic incompatibility (CI) between individuals of different infection status, the parthenogenetic production of females, the selective killing of male embryos, and the feminization of genetic males. Wolbachia infections of Drosophila melanogaster are extremely common in both wild populations and long-term laboratory stocks. Utilizing the newly completed genome sequence of Wolbachia pipientis wMel, we have identified a number of polymorphic markers that can be used to discriminate among five different Wolbachia variants within what was previously thought to be the single clonal infection of D. melanogaster. Analysis of long-term lab stocks together with wild-caught flies indicates that one of these variants has replaced the others globally within the last century. This is the first report of a global replacement of a Wolbachia strain in an insect host species. The sweep is at odds with current theory that cannot explain how Wolbachia can invade this host species given the observed cytoplasmic incompatibility characteristics of Wolbachia infections in D. melanogaster in the field.     

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.     

Wolbachia-mediated sperm modification is dependent on the host genotype in Drosophila.

Estimates of Wolbachia density in the eggs, testes and whole flies of drosophilid hosts have been unable to predict the lack of cytoplasmic incompatibility (CI) expression in so-called mod(-) variants. Consequently, the working hypothesis has been that CI expression, although related to Wolbachia density, is also governed by unknown factors that are influenced by both host and bacterial genomes. Here, we compare the behaviour of the mod(-) over-replicating Wolbachia popcorn strain in its native Drosophila melanogaster host to the same strain transinfected into a novel host, namely Drosophila simulans. We report that (i) the popcorn strain is a close relative of other D. melanogaster infections, (ii) the mod(-) status of popcorn in D. melanogaster appears to result from its inability to colonize sperm bundles, (iii) popcorn is present in the bundles in D. simulans and induces strong CI expression, which demonstrates that the bacterial strain does not lack the genetic machinery for inducing CI and that there is host-species-specific control over Wolbachia tissue tropism, and (iv) infection of sperm bundles by the mod(-) D. simulans wCof strain indicates that there are several independent routes by which a strain can be a CI non-expressor.     

Wolbachia distribution and cytoplasmic incompatibility during sperm development: the cyst as the basic cellular unit of CI expression

The growth and distribution of the intracellular microbe Wolbachia pipientis during spermatogenesis in several different host/symbiont genetic combinations in Drosophila melanogaster and Drosophila simulans is described. Considerable intra- and inter-strain variation in Wolbachia density and tissue distribution was observed. Wolbachia were found inside spermatocytes and spermatids or within the somatic cyst cells surrounding the germ cells. Some strains displayed both tissue distributions. High rates of cytoplasmic incompatibility (CI) are correlated with high levels of Wolbachia only when spermatocytes and/or spermatids harbor the microbe. Wolbachia infection of somatic cyst cells, although sometimes present at high levels, did not result in significant CI expression. CI-inducing Wolbachia strains within D. simulans showed no distinguishable differences in distribution or density within infected spermatids. To dissect the relative contribution of host and symbiont to the expression of CI, Wolbachia from various host strains known to exhibit varying levels of CI were introgressed into new uninfected host genetic backgrounds. These introgression experiments confirm that the mod(+)/mod(-) phenotype is an intrinsic Wolbachia trait and is not determined by host factors. The level of sperm modification in those lines harboring Wolbachia capable of modifying sperm, however, is influenced by host genetic background. These results form the basis of the Wolbachia Infected Spermatocyte/Spermatid Hypothesis (WISSH). According to WISSH, Wolbachia infection in spermatocytes and then spermatids during sperm development is required for CI expression.     

Variable fitness effects of Wolbachia infection in Drosophila melanogaster

Maternally inherited Wolbachia bacteria are extremely widespread among insects and their presence is usually associated with parasitic modifications of host fitness. Wolbachia pipientis infects Drosophila melanogaster populations from all continents, but their persistence in this species occurs despite any strong parasitic effects. Here, we have investigated the symbiosis between Wolbachia and D. melanogaster and found that Wolbachia infection can have significant survival and fecundity effects. Relative to uninfected flies, infected females from three fly strains showed enhanced survival or fecundity associated with Wolbachia infection, one strain showed both and one strain responded positively to Wolbachia removal. We found no difference in egg hatch rates (cytoplasmic incompatibility) for crosses between infected males and uninfected females, although there were fecundity differences. Females from this cross consistently produced fewer eggs than infected females and these fecundity differences could promote the spread of infection just like cytoplasmic incompatibility. More surprising, we found that infected females often had the greatest fecundity when mated to uninfected males. This could also promote the spread of Wolbachia infection, though here the fitness benefits would also help to spread infection when Wolbachia are rare. We suggest that variable fitness effects, in both sexes, and which interact strongly with the genetic background of the host, could increase cytoplasmic drive rates in some genotypes and help explain the widespread persistence of Wolbachia bacteria in D. melanogaster populations. These interactions may further explain why many D. melanogaster populations are polymorphic for Wolbachia infection. We discuss our results in the context of host-symbiont co-evolution.     

Wolbachia effects in Drosophila melanogaster: in search of fitness benefits

Insect endosymbionts often influence host nutrition but these effects have not been comprehensively investigated in Wolbachia endosymbionts that are widespread in insects. Using strains of Drosophila melanogaster with the wMel Wolbachia infection, we showed that Wolbachia did not influence adult starvation resistance. Wolbachia also had no effect on larval development time or the size of emerging adults from a low nutrition medium. While Wolbachia may influence the expression of heat shock proteins, we found that there was no effect on adult heat resistance when tested in terms of survival or virility following heat stress. The absence of nutrition or stress effects suggests that other processes maintain wMel frequencies in natural populations of Drosophila melanogaster.     

The effects of host age,host nuclear background and temperature on phenotypic effects of the virulent Wolbachia strain popcorn in Drosophila melanogaster

Because of their obligate endosymbiotic nature, Wolbachia strains by necessity are defined by their phenotypic effects upon their host. Nevertheless, studies on the influence of host background and environmental conditions upon the manifestation of Wolbachia effects are relatively uncommon. Here we examine the behavior of the overreplicating Wolbachia strain popcorn in four different Drosophila melanogaster backgrounds at two temperatures. Unlike other strains of Wolbachia in Drosophila, popcorn has a major fitness impact upon its hosts. The rapid proliferation of popcorn causes cells to rupture, resulting in the premature death of adult hosts. Apart from this effect, we found that popcorn delayed development time, and host background influenced both this trait and the rate of mortality associated with infection. Temperature influenced the impact of popcorn upon host mortality, with no reduction in life span occurring in flies reared at 19 degrees. No effect upon fecundity was found. Contrary to earlier reports, popcorn induced high levels of incompatibility when young males were used in tests, and CI levels declined rapidly with male age. The population dynamics of popcorn-type infections will therefore depend on environmental temperature, host background, and the age structure of the population.     

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.     

Mechanisms promoting the long-term persistence of a Wolbachia infection in a laboratory-adapted population of Drosophila melanogaster

Intracellular bacteria of the genus Wolbachia are widespread endosymbionts across diverse insect taxa. Despite this prevalence, our understanding of how Wolbachia persists within populations is not well understood. Cytoplasmic incompatibility (CI) appears to be an important phenotype maintaining Wolbachia in many insects, but it is believed to be too weak to maintain Wolbachia in Drosophila melanogaster, suggesting that Wolbachia must also have other effects on this species. Here we estimate the net selective effect of Wolbachia on its host in a laboratory-adapted population of D. melanogaster, to determine the mechanisms leading to its persistence in the laboratory environment. We found i) no significant effects of Wolbachia infection on female egg-to-adult survival or adult fitness, ii) no reduced juvenile survival in males, iii) substantial levels of CI, and iv) a vertical transmission rate of Wolbachia higher than 99%. The fitness of cured females was, however, severely reduced (a decline of 37%) due to CI in offspring. Taken together these findings indicate that Wolbachia is maintained in our laboratory environment due to a combination of a nearly perfect transmission rate and substantial CI. Our results show that there would be strong selection against females losing their infection and producing progeny free from Wolbachia.     

黑腹果蝇Mst84Db敲降引起的父本缺陷类似Wolbachia诱导的细胞质不亲和

【目的】Wolbachia 是广泛存在于昆虫体内的一类通过母系传递的共生菌,能够通过多种方式影响宿主的生殖。细胞质不亲和（CI）是Wolbachia 引起的最普遍的一种表型,即感染Wolbachia的雄性和未感染的雌性宿主交配后,胚胎发育停滞于早期阶段。但目前有关CI的分子机理还不清楚。本研究组前期实验表明,Wolbachia感染引起黑腹果蝇Drosophila melanogaster 3龄幼虫精巢中Mst84Db基因的表达显著下调。本研究的目的是进一步研究Mst84Db与CI的关系。【方法】我们体外合成了Mst84Db的双链RNA（dsRNA）,注射雄性果蝇,将注射过的雄果蝇与野生雌果蝇交配,检测其繁殖力。基因表达采用定量RT-PCR方法进行检测。胚胎表型采用DAPI染色进行分析。【结果】注射dsRNA 24 h后,Mst84Db基因的表达水平发生显著下调。注射后72 h,基因表达下调幅度最大。与对照组相比,基因敲降后的雄蝇繁殖能力显著下降,与雌果蝇交配后胚胎孵化率显著低于对照组,这与Wolbachia诱导的CI现象类似。未孵化胚胎的表皮上没有体节出现,说明胚胎停滞于发育的早期。部分胚胎细胞核分裂不同步,且有染色质间桥出现,这也与CI胚胎中的细胞学表型一致。【结论】Wolbachia感染可能抑制果蝇精子发生过程中Mst84Db基因的表达,从而使精子失去正常功能,最终导致与雌性果蝇交配后,胚胎发育停滞,并最终死亡。Mst84Db基因在雄性果蝇中表达下调可能是产生CI的重要原因之一。     

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.     

Nonrandom Wolbachia infection status of Drosophila melanogaster strains with different mtDNA haplotypes

Wolbachia are maternally inherited bacteria, which typically spread in the host population by inducing cytoplasmic incompatibility (CI). In Drosophila melanogaster, Wolbachia is quite common but CI is variable, with most of the studies reporting low levels of CI. Surveying mitochondrial DNA (mtDNA) variation and infection status in a worldwide D. melanogaster collection, we found that the Wolbachia infection was not randomly distributed among flies with different mtDNA haplotypes. This preferential infection of some mtDNA haplotypes could be caused by a recent spread of mtDNA haplotypes associated with the infection. The comparison of contemporary D. melanogaster samples with lines collected more than 50 years ago shows that indeed one haplotype with a high incidence of Wolbachia infection has increased in frequency. Consistent with this observation, we found that the acquisition of a Wolbachia infection in a population from Crete was accompanied with an almost complete mtDNA replacement, with the Wolbachia-associated haplotype becoming abundant. Although it is difficult to identify the evolutionary forces causing the global increase of wMel, the parallel sweep of Wolbachia and an mtDNA haplotype suggests a fitness advantage of the Wolbachia infection.     

Induced paternal effects mimic cytoplasmic incompatibility in Drosophila

Wolbachia is an intracellular microbe found in a wide diversity of arthropod and filarial nematode hosts. In arthropods these common bacteria are reproductive parasites that manipulate central elements of their host's reproduction to increase their own maternal transmission in one of several ways. Cytoplasmic incompatibility (CI) is one such manipulation where sperm are somehow modified in infected males and this modification must be rescued by the presence of the same bacterial strain in the egg for normal development to proceed. The molecular mechanisms involved in the expression of CI are unknown. Here we show that Wolbachia infection results in increased mRNA and protein expression of the Drosophila simulans nonmuscle myosin II gene zipper. Induced overexpression of zipper in Wolbachia-free transgenic D. melanogaster males results in paternal-effect lethality that mimics the fertilization defects associated with CI. Likewise, overexpression of the tumor suppressor gene, lethal giant larvae [l(2)gl], results in egg lethality and a CI phenotype. Stoichiometric levels of zipper and l(2)gl are required for proper segregation of cellular determinants during neuroblast stem cell division. Taken together these results form the basis of a working hypothesis whereby Wolbachia induces paternal effects in sperm by manipulating the expression of key regulators of cytoskeletal activity during spermatogenesis.     

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.