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.     

Cytoplasmic incompatibility and sperm cyst infection in different Drosophila-Wolbachia associations

Wolbachia are a group of maternally transmitted obligatory intracellular alpha-proteobacteria that infect a wide range of arthropod and nematode species. Wolbachia infection in Drosophila in most cases is associated with the induction of cytoplasmic incompatibility (CI), manifested as embryonic lethality of offspring in a cross between infected males and uninfected females. While the molecular basis of CI is still unknown, it has been suggested that two bacterial functions are involved: mod (for modification) modifies the sperm during spermatogenesis and resc (for rescue) acts in the female germline and/or in early embryos, neutralizing the modification. There is considerable variation in the level of incompatibility in different Wolbachia/host interactions. We examine the relationship between the levels of CI in a number of naturally infected and transinfected Drosophila hosts and the percentage of Wolbachia-infected sperm cysts. Our results indicate the presence of two main groups of Drosophila-Wolbachia associations: group I, which exhibits a positive correlation between CI levels and the percentage of infected sperm cysts (mod(+) phenotype), and group II, which does not express CI (mod(-) phenotype) irrespective of the infection status of the sperm cysts. Group II can be further divided into two subgroups: The first one contains associations with high numbers of heavily Wolbachia-infected sperm cysts while in the second one, Wolbachia is rarely detected in sperm cysts, being mostly present in somatic cells. We conclude that there are three requirements for the expression of CI in a host-Wolbachia association: (a) Wolbachia has to be able to modify sperm (mod(+) genotype), (b) Wolbachia has to infect sperm cysts, and (c) Wolbachia has to be harbored by a permissive host.     

Effects of Wolbachia on sperm maturation and architecture in Drosophila simulans Riverside

Wolbachia is an intracellular obligate symbiont, that is relatively common in insects and also found in some nematodes. Cytoplasmic incompatibility (CI) is the most commonly expressed form, of several sex altering phenotypes caused by this rickettsial-like bacterium. CI is induced when infected males mate with uninfected females, and is likely the result of bacterial-induced modification of sperm grown in a Wolbachia-infected environment. Several studies have explored the dynamics of Wolbachia bacteria during sperm development in Drosophila. This study confirms and extends these earlier investigations of Wolbachia's distribution and proliferation in male germ cell lines. We examined Wolbachia population dynamics during testis development of Drosophila simulans (Riverside) by studying their distribution during the early mitotic divisions of secondary spermatogonial and subsequent meiotic cyst cells. Wolbachia are found in lower concentration in spermatogonial than in spermatocyte cells. Cytoplasmically incompatible crosses result in low levels of viable embryos despite the occurrence of fairly high levels of uninfected cysts. During meiotic divisions Wolbachia organize themselves at the poles during prophase and telophase but arrange themselves in equatorial bands during metaphase and anaphase. Moreover, during meiosis Wolbachia are asymmetrically divided between some daughter cells. There is no strong relationship between the fusome and Wolbachia and we have not found evidence that bacteria cross the ring canals. Wolbachia were observed at the distal and proximal sides of individualization complexes. Multiple altered sperm structures were observed during the process of individualization of infected sperm.     

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.     

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 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 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.     

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.     

Offsetting effects of Wolbachia infection and heat shock on sperm production in Drosophila simulans: analyses of fecundity, fertility and accessory gland proteins

Infection in Drosophila simulans with the endocellular symbiont Wolbachia pipientis results in egg lethality caused by failure to properly initiate diploid development (cytoplasmic incompatibility, CI). The relationship between Wolbachia infection and reproductive factors influencing male fitness has not been well examined. Here we compare infected and uninfected strains of D. simulans for (1) sperm production, (2) male fertility, and (3) the transfer and processing of two accessory gland proteins, Acp26Aa or Acp36De. Infected males produced significantly fewer sperm cysts than uninfected males over the first 10 days of adult life, and infected males, under varied mating conditions, had lower fertility compared to uninfected males. This fertility effect was due to neither differences between infected and uninfected males in the transfer and subsequent processing of accessory gland proteins by females nor to the presence of Wolbachia in mature sperm. We found that heat shock, which is known to decrease CI expression, increases sperm production to a greater extent in infected compared to uninfected males, suggesting a possible link between sperm production and heat shock. Given these results, the roles Wolbachia and heat shock play in mediating male gamete production may be important parameters for understanding the dynamics of infection in natural populations.     

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.     

Sperm chromatin remodelling and Wolbachia-induced cytoplasmic incompatibility in Drosophila.

Wolbachia pipientis is an obligate bacterial endosymbiont, which has successfully invaded approximately 20% of all insect species by manipulating their normal developmental patterns. Wolbachia-induced phenotypes include parthenogenesis, male killing, and, most notably, cytoplasmic incompatibility. In the future these phenotypes might be useful in controlling or modifying insect populations but this will depend on our understanding of the basic molecular processes underlying insect fertilization and development. Wolbachia-infected Drosophila simulans express high levels of cytoplasmic incompatibility in which the sperm nucleus is modified and does not form a normal male pronucleus when fertilizing eggs from uninfected females. The sperm modification is somehow rescued in eggs infected with the same strain of Wolbachia. Thus, D. simulans has become an excellent model organism for investigating the manner in which endosymbionts can alter reproductive programs in insect hosts. This paper reviews the current knowledge of Drosophila early development and particularly sperm function. Developmental mutations in Drosophila that are known to affect sperm function will also be discussed.incompatibility.     

Wolbachia infection reduces sperm competitive ability in an insect

The maternally inherited bacterium Wolbachia pipientis imposes significant fitness costs on its hosts. One such cost is decreased sperm production resulting in reduced fertility of male Drosophila simulans infected with cytoplasmic incompatibility (CI) inducing Wolbachia. We tested the hypothesis that Wolbachia infection affects sperm competitive ability and found that Wolbachia infection is indeed associated with reduced success in sperm competition in non-virgin males. In the second male role, infected males sired 71% of the offspring whereas uninfected males sired 82% of offspring. This is the first empirical evidence indicating that Wolbachia infection deleteriously affects sperm competition and raises the possibility that polyandrous females can utilize differential sperm competitive ability to bias the paternity of broods and avoid the selfish manipulations of Wolbachia. This suggests a relationship between Wolbachia infection and host reproductive strategies. These findings also have important consequences for Wolbachia population dynamics because the transmission advantage of Wolbachia is likely to be undermined by sperm competition.     

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.     

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.     

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.     

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 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.     

Wolbachia plays no role in the one-way reproductive incompatibility between the hybridizing field crickets Gryllus firmus and G. pennsylvanicus

Wolbachia are cytoplasmically inherited alpha-proteobacteria that can cause cytoplasmic incompatibility (CI) in insects. This incompatibility between sperm and egg is evident when uninfected females mate with infected males. Wolbachia-driven reproductive incompatibilities are of special interest because they may play a role in speciation. However, the presence of Wolbachia does not always imply incompatibility. The field crickets Gryllus firmus and G. pennsylvanicus exhibit a very clear unidirectional incompatibility and have been cited as a possible example of Wolbachia-induced CI. Here, we conduct curing experiments, intra- and interspecific crosses, cytological examination of Wolbachia in testes and Wolbachia quantifications through real-time PCR. All of our data strongly suggest that Wolbachia are not involved in the reproductive incompatibility between G. firmus and G. pennsylvanicus.     

Natural Wolbachia infections in the Drosophila yakuba species complex do not induce cytoplasmic incompatibility but fully rescue the wRi modification

In this study, we report data about the presence of Wolbachia in Drosophila yakuba, D. teissieri, and D. santomea. Wolbachia strains were characterized using their wsp gene sequence and cytoplasmic incompatibility assays. All three species were found infected with Wolbachia bacteria closely related to the wAu strain, found so far in D. simulans natural populations, and were unable to induce cytoplasmic incompatibility. We injected wRi, a CI-inducing strain naturally infecting D. simulans, into the three species and the established transinfected lines exhibited high levels of CI, suggesting that absence of CI expression is a property of the Wolbachia strain naturally present or that CI is specifically repressed by the host. We also tested the relationship between the natural infection and wRi and found that it fully rescues the wRi modification. This result was unexpected, considering the significant evolutionary divergence between the two Wolbachia strains.