Multiple rescue factors within a Wolbachia strain

Wolbachia-induced cytoplasmic incompatibility (CI) is expressed when infected males are crossed with either uninfected females or females infected with Wolbachia of different CI specificity. In diploid insects, CI results in embryonic mortality, apparently due to the the loss of the paternal set of chromosomes, usually during the first mitotic division. The molecular basis of CI has not been determined yet; however, several lines of evidence suggest that Wolbachia exhibits two distinct sex-dependent functions: in males, Wolbachia somehow "imprints" the paternal chromosomes during spermatogenesis (mod function), whereas in females, the presence of the same Wolbachia strain(s) is able to restore embryonic viability (resc function). On the basis of the ability of Wolbachia to induce the modification and/or rescue functions in a given host, each bacterial strain can be classified as belonging in one of the four following categories: mod(+) resc(+), mod(-) resc(+), mod(-) resc(-), and mod(+) resc(-). A so-called "suicide" mod(+) resc(-) strain has not been found in nature yet. Here, a combination of embryonic cytoplasmic injections and introgression experiments was used to transfer nine evolutionary, distantly related Wolbachia strains (wYak, wTei, wSan, wRi, wMel, wHa, wAu, wNo, and wMa) into the same host background, that of Drosophila simulans (STCP strain), a highly permissive host for CI expression. We initially characterized the modification and rescue properties of the Wolbachia strains wYak, wTei, and wSan, naturally present in the yakuba complex, upon their transfer into D. simulans. Confocal microscopy and multilocus sequencing typing (MLST) analysis were also employed for the evaluation of the CI properties. We also tested the compatibility relationships of wYak, wTei, and wSan with all other Wolbachia infections. So far, the cytoplasmic incompatibility properties of different Wolbachia variants are explained assuming a single pair of modification and rescue factors specific to each variant. This study shows that a given Wolbachia variant can possess multiple rescue determinants corresponding to different CI systems. In addition, our results: (a) suggest that wTei appears to behave in D. simulans as a suicide mod(+) resc(-) strain, (b) unravel unique CI properties, and (c) provide a framework to understand the diversity and the evolution of new CI-compatibility types.     

On the mod resc model and the evolution of Wolbachia compatibility types.

Cytoplasmic incompatibility (CI) is induced by the endocellular bacterium Wolbachia. It results in an embryonic mortality occurring when infected males mate with uninfected females. The mechanism involved is currently unknown, but the mod resc model allows interpretation of all observations made so far. It postulates the existence of two bacterial functions: modification (mod) and rescue (resc). The mod function acts in the males' germline, before Wolbachia are shed from maturing sperm. If sperm is affected by mod, zygote development will fail unless resc is expressed in the egg. Interestingly, CI is also observed in crosses between infected males and infected females when the two partners bear different Wolbachia strains, demonstrating that mod and resc interact in a specific manner: Two Wolbachia strains are compatible with each other only if they harbor the same compatibility type. Here we focus on the evolutionary process involved in the emergence of new compatibility types from ancestral ones. We argue that new compatibility types are likely to evolve under a wider range of conditions than previously thought, through a two-step process. First, new mod variants can arise by mutation and spread by drift. This is possible because mod is expressed in males and Wolbachia is transmitted by females. Second, once such a mod variant achieves a certain frequency, it can create the conditions for the deterministic invasion of a new resc variant, allowing the invasion of a new mod resc pair. Furthermore, we show that a stable polymorphism might be maintained in natural populations, allowing the long-term existence of "suicidal" Wolbachia strains.     

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.     

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.     

Multiple Wolbachia determinants control the evolution of cytoplasmic incompatibilities in Culex pipiens mosquito populations

Wolbachia are maternally inherited endosymbionts that can invade arthropod populations through manipulation of their reproduction. In mosquitoes, Wolbachia induce embryonic death, known as cytoplasmic incompatibility (CI), whenever infected males mate with females either uninfected or infected with an incompatible strain. Although genetic determinants of CI are unknown, a functional model involving the so-called mod and resc factors has been proposed. Natural populations of Culex pipiens mosquito display a complex CI relationship pattern associated with the highest Wolbachia (wPip) genetic polymorphism reported so far. We show here that C. pipiens populations from La Réunion, a geographically isolated island in the southwest of the Indian Ocean, are infected with genetically closely related wPip strains. Crossing experiments reveal that these Wolbachia are all mutually compatible. However, crosses with genetically more distant wPip strains indicate that Wolbachia strains from La Réunion belong to at least five distinct incompatibility groups (or crossing types). These incompatibility properties which are strictly independent from the nuclear background, formally establish that in C. pipiens, CI is controlled by several Wolbachia mod/resc factors.     

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.     

Exploring the evolution of Wolbachia compatibility types: a simulation approach

Wolbachia-induced cytoplasmic incompatibility (CI) is observed when males bearing the bacterium mate with uninfected females or with females bearing a different Wolbachia variant; in such crosses, paternal chromosomes are lost at the first embryonic mitosis, most often resulting in developmental arrest. The molecular basis of CI is currently unknown, but it is useful to distinguish conceptually the male and female sides of this phenomenon: in males, Wolbachia must do something, before it is shed from maturing sperm, that will disrupt paternal chromosomes functionality [this is usually termed "the modification (mod) function"]; in females, Wolbachia must somehow restore embryonic viability, through what is usually called "the rescue (resc) function." The occurrence of CI in crosses between males and females bearing different Wolbachia variants demonstrates that the mod and resc functions interact in a specific manner: different mod resc pairs make different compatibility types. We are interested in the evolutionary process allowing the diversification of compatibility types. In an earlier model, based on the main assumption that the mod and resc functions can mutate independently, we have shown that compatibility types can evolve through a two-step process, the first involving drift on mod variations and the second involving selection on resc variations. This previous study has highlighted the need for simulation-based models that would include the effects of nondeterministic evolutionary forces. This study is based on a simulation program fulfilling this condition, allowing us to follow the evolution of compatibility types under mutation, drift, and selection. Most importantly, simulations suggest that in the frame of our model, the evolution of compatibility types is likely to be a gradual process, with new compatibility types remaining partially compatible with ancestral ones.     

Wolbachia transmission in a naturally bi-infected Drosophila simulans strain from New-Caledonia

Wolbachia are maternally-transmitted endocellular bacteria infecting several arthropod species. In order to study the possibility of Wolbachia segregation in a naturally bi-infected host, isofemale lines from a bi-infected Drosophila simulans (Sturtevant) strain from Nouméa (New Caledonia) were backcrossed using uninfected males carrying the same nuclear background. Uninfected males were used to avoid the cytoplasmic incompatibility syndrome (CI) associated with the presence of Wolbachia in males. Each line was established using a female infected simultaneously by the two different Wolbachia variants wHa and wNo. The backcross led to some individuals carrying only one type of infection being recovered among the progeny of the bi-infected foundress females. Rarely, uninfected individuals were also recovered. Isolated for the first time in its natural host, wNo exhibited a significantly weaker CI phenotype than the isolated wHa variant. Infection fate when backcross conditions were relaxed varied depending on rearing conditions of the host. Under favourable conditions, the infection was generally maintained, while it was frequently lost under unfavourable conditions. This result probably reflects the direct fitness dependence of the symbiont on its host.     

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.     

Mutualistic Wolbachia infection in Aedes albopictus: accelerating cytoplasmic drive

Maternally inherited rickettsial symbionts of the genus Wolbachia occur commonly in arthropods, often behaving as reproductive parasites by manipulating host reproduction to enhance the vertical transmission of infections. One manipulation is cytoplasmic incompatibility (CI), which causes a significant reduction in brood hatch and promotes the spread of the maternally inherited Wolbachia infection into the host population (i.e., cytoplasmic drive). Here, we have examined a Wolbachia superinfection in the mosquito Aedes albopictus and found the infection to be associated with both cytoplasmic incompatibility and increased host fecundity. Relative to uninfected females, infected females live longer, produce more eggs, and have higher hatching rates in compatible crosses. A model describing Wolbachia infection dynamics predicts that increased fecundity will accelerate cytoplasmic drive rates. To test this hypothesis, we used population cages to examine the rate at which Wolbachia invades an uninfected Ae. albopictus population. The observed cytoplasmic drive rates were consistent with model predictions for a CI-inducing Wolbachia infection that increases host fecundity. We discuss the relevance of these results to both the evolution of Wolbachia symbioses and proposed applied strategies for the use of Wolbachia infections to drive desired transgenes through natural populations (i.e., population replacement strategies).     

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.     

Population dynamics of noncytoplasmic incompatibility-inducing Wolbachia in Nilaparvata lugens and its effects on host adult life span and female fitness

Wolbachia are bacteria that live intracellularly in a wide variety of arthropods. They are maternally inherited and can affect both reproduction and fitness of its host. When infected males mate with uninfected females or females infected by a different Wolbachia strain, there is often a failure of karyogamy, which is usually attributed to cytoplasmic incompatibility (CI). We measured the strength of CI induced by Wolbachia and the fitness effects in three Chinese populations of the brown planthopper Nilaparvata lugens from Hainan, Yunnan, and Guangxi provinces, respectively. No evidence for CI was found in any of the populations, whereas an enhanced fecundity and shortened longevity were observed only in the Hainan population. The infection density was significantly higher in the Hainan population than in the Guangxi population. The Wolbachia strain infecting the three populations appeared to be the same based on the nucleotide sequence of the wsp gene. Therefore, the variable effects of Wolbachia on host fitness seem to be the result of differences in the host genetic background and Wolbachia infection density. The ability of the non-CI-inducing Wolbachia to maintain themselves in their hosts may be attributed to their positive effects on host fecundity and efficient maternal transmission.     

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.     

Population biology of cytoplasmic incompatibility: maintenance and spread of Cardinium symbionts in a parasitic wasp

Bacteria that cause cytoplasmic incompatibility (CI) are perhaps the most widespread parasites of arthropods. CI symbionts cause reproductive failure when infected males mate with females that are either uninfected or infected with a different, incompatible strain. Until recently, CI was known to be caused only by the alpha-proteobacterium Wolbachia. Here we present the first study of the population biology of Cardinium, a recently discovered symbiont in the Bacteroidetes that causes CI in the parasitic wasp Encarsia pergandiella (Hymenoptera: Aphelinidae). Cardinium occurs at high frequency ( approximately 92%) in the field. Using wasps that were recently collected in the field, we measured parameters that are crucial for understanding how CI spreads and is maintained in its host. CI Cardinium exhibits near-perfect rates of maternal transmission, causes a strong reduction in viable offspring in incompatible crosses, and induces a high fecundity cost, with infected females producing 18% fewer offspring in the first 4 days of reproduction. We found no evidence for paternal transmission or horizontal transmission of CI Cardinium through parasitism of an infected conspecific. No evidence for cryptic parthenogenesis in infected females was found, nor was sex allocation influenced by infection. We incorporated our laboratory estimates into a model of CI dynamics. The model predicts a high stable equilibrium, similar to what we observed in the field. Interestingly, our model also predicts a high threshold frequency of CI invasion (20% for males and 24% for females), below which the infection is expected to be lost. We consider how this threshold may be overcome, focusing in particular on the sensitivity of CI models to fecundity costs. Overall our results suggest that the factors governing the dynamics of CI Wolbachia and Cardinium are strikingly similar.     

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.     

A Wolbachia-associated fitness benefit depends on genetic background in Drosophila simulans

The alpha-proteobacteria Wolbachia infect a number of insect species and influence host reproduction to favour the spread of infected females through a population. The fitness effect of this infection is important in understanding the spread and maintenance of Wolbachia within and among host populations. However, a full elucidation of fitness effect requires careful control of host genetic background. Here, I transferred a single clone of Wolbachia (the wHa strain) into three genetically distinct isofemale lines of the fly Drosophila simulans using microinjection methodology. These lines carried one of the three described mitochondrial haplogroups (siI, siII or siIII) and differ in nuclear genome as well. Population cage assays showed that wHa-infected siIII flies enjoyed a dramatic fitness benefit compared to uninfected siIII. In contrast, wHa did not affect the fitness of siI or siII flies. This study points to the importance of host-by-symbiont interaction terms that may play an important role in organismal-fitness.     

Wolbachia在山楂双叶螨中的感染及对寄主生殖的影响

【目的】共生菌Wolbachia在多种叶螨寄主中引起细胞质不亲和及适合度改变,影响寄主的生物学特性。山楂双叶螨Amphitetranychus viennensis是重要的果树害螨,常暴发成灾。本研究旨在明确Wolbachia在山楂双叶螨中的感染情况及对寄主生殖的影响。【方法】采集自然种群的山楂双叶螨,运用多位点序列分型技术（multilocus sequence typing, MLST）对其体内Wolbachia感染率及株系进行分析;通过杂交试验及生物学观察,分析感染Wolbachia对山楂双叶螨单雌产卵量、后代孵化率、性比及死亡率的影响。【结果】山楂双叶螨自然种群感染一种株系的Wolbachia （wVie）,该Wolbachia株系与小黑花椿象Orius strigicollis和丽蝇蛹集金小蜂Nasonia vitripennis中的Wolbachia株系亲缘关系较近,而与叶螨属Tetranychus叶螨感染的Wolbachia株系亲缘关系较远。Wolbachia与4种分化较小的线粒体单倍型相关联。Wolbachia感染雌虫与不感染雌虫产卵量没有显著差异（P>0.05）。不感染雌虫与感染雄虫交配,卵孵化率显著低于其他杂交组合 (P<0.05),但孵化率仍达近75%。各交配组合的后代性比及死亡率变化不明显（P>0.05）。【结论】Wolbachia在山楂双叶螨种群中的侵染历史较短,对山楂双叶螨的产卵力、后代性比、死亡率没有影响。Wolbachia在山楂双叶螨中诱导产生弱的CI表型。     

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.