Wolbachia segregation dynamics and levels of cytoplasmic incompatibility in Drosophila sechellia

In Drosophila sechellia, the endocellular bacterium Wolbachia induces cytoplasmic incompatibility (CI): in crosses involving infected males, a partial or complete embryonic mortality occurs unless the female bears the same Wolbachia. D. sechellia is known to harbour two Wolbachia variants, namely wSh and wSn, closely related to wHa and wNo, respectively, two strains infecting the populations of D. simulans from the Seychelles archipelago and New Caledonia. Strikingly, the two species show similar infection patterns: in D. sechellia, wSh can be present on its own or in double infection with wSn, but individuals carrying wSn only do not occur; in D. simulans, wHa can be present on its own or in double infection with wNo, but individuals carrying wNo only do not occur, or occur at very low frequency. Previous experiments on D. simulans showed that lines singly infected by wNo can be obtained by segregation, and stably maintained. Here we investigate this issue in D. sechellia through an 18 generation experiment, and show that wSn and wSh singly infected lines can arise by segregation. Using singly infected lines obtained in this experiment, we estimate the CI intensities of wSh and wSn in D. sechellia, and compare these to the CI intensities of the same Wolbachia injected into D. simulans. Our results do not suggest any consistent effect of the host species on the CI induced by wSh. On the contrary, it seems that wSn expression is repressed by host factors in D. sechellia.     

Dynamics of double and single Wolbachia infections in Drosophila simulans from New Caledonia

The bacterial symbiont Wolbachia can cause cytoplasmic incompatibility in Drosophila simulans flies: if an infected male mates with an uninfected female, or a female with a different strain of Wolbachia, there can be a dramatic reduction in the number of viable eggs produced. Here we explore the dynamics associated with double and single Wolbachia infections in New Caledonia. Doubly infected females were compatible with all males in the population, explaining the high proportion of doubly infected flies. In this study, males that carry only wHa or wNo infections showed reduced incompatibility when mated to uninfected females, compared with previous reports. These data suggest that either the DNA of these bacterial isolates have diverged from those previously collected, or the genetic background of the host has lead to a reduction in the phenotype of incompatibility. Mitochondrial sequence polymorphism at two sites within the host genome was assayed to investigate population structure related to infection types. There was no correlation between sequence polymorphism and infection type suggesting that double infections are the stable type, with singly infected and uninfected flies arising from stochastic segregation of bacterial strains. Finally, we discuss the nomenclature of Wolbachia strain designation.     

Expression of cytoplasmic incompatibility in Drosophila simulans and its impact on infection frequencies and distribution of Wolbachia pipientis

The aim of this study is to examine the expression of cytoplasmic incompatibility and investigate the distribution and population frequencies of Wolbachia pipientis strains in Drosophila simulans. Nucleotide sequence data from 16S rDNA and a Wolbachia surface protein coding sequence and cytoplasmic incompatibility assays identify four distinct Wolbachia strains: wHa, wRi, wMa, and wAu. The levels of cytoplasmic incompatibility between six lines carrying these strains of bacteria and three control lines without bacteria are characterized. Flies infected with wHa and wRi are bidirectionally incompatible, and males that carry either strain can only successfully produce normal numbers of offspring with females carrying the same bacterial strain. Males infected with wAu do not express incompatibility. Males infected with the wMa strain express intermediate incompatibility when mated to females with no bacteria and no incompatibility with females with any other Wolbachia strain. We conduct polymerase chain reaction/restriction fragment length polymorphism assays to distinguish the strain of Wolbachia and the mitochondrial haplotype to survey populations for each type and associations between them. Drosophila simulans is known to have three major mitochondrial haplotypes (siI, sill, and siIII) and two subtypes (siIIA and siIIB). All infected lines of the sil haplotype carry wHa, wNo, or both; wMa and wNo are closely related and it is not clear whether they are distinct strains or variants of the same strain. Infected lines with the silIA haplotype harbor wRi and the siIIB haplotype carries wAu. The wMa infection is found in siIII haplotype lines. The phenotypic expression of cytoplasmic incompatibility and its relation to between-population differences in frequencies of Wolbachia infection are discussed.     

Variability within the Seychelles Cytoplasmic Incompatibility System in Drosophila Simulans

In Drosophila simulans, we described a cytoplasmic incompatibility (CI) system (Seychelles) restricted to insular populations that harbor the mitochondrial type SiI. Since then, these populations have been shown to be heterogeneous, some being infected by one Wolbachia genetic variant only (wHa), while others are infected simultaneously by wHa and by another variant (wNo) always found in association with wHa. We have experimentally obtained two D. simulans strains only infected by the wNo variant. This variant determines its own cytoplasmic incompatibility type. In particular, the cross between wNo-bearing flies and wHa-bearing ones is bidirectionally incompatible. The Seychelles CI type, stricto sensu, is distinguished by being determined by the simultaneous presence of two Wolbachia variants that we found to be mutually incompatible. In addition, we observed incomplete maternal transmission of the Wolbachia.     

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.     

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.     

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.     

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.     

Within- and between-population variation for Wolbachia-induced reproductive incompatibility in a haplodiploid mite

Wolbachia pipientis is a bacterium that induces cytoplasmic incompatibility (CI), the phenomenon in which infected males are reproductively incompatible with uninfected females. CI spreads in a population of hosts because it reduces the fitness of uninfected females relative to infected females. CI encompasses two steps: modification (mod) of sperm of infected males and rescuing (resc) of these chromosomes by Wolbachia in the egg. Infections associated with CI have mod+ resa+ phenotypes. However, mod- resc+ phenotypes also exist; these do not result in CI. Assuming mod/resc phenotypes are properties of the symbiont, theory predicts that mod- resc+ infections can only spread in a host population where a mod+ resc+ infection already occurs. A mod- resc+ infection spreads if the cost it imposes on the infected females is lower than the cost inflicted by the resident (mod+ resc+) infection. Furthermore, introduction of a mod- Wolbachia eventually drives infection to extinction. The uninfected population that results can be recolonized by a CI-causing Wolbachia. Here, we investigated whether variability for induction of CI was present in two Tetranychus urticae populations. In one population all isofemale lines tested were mod-. In the other, mod+ resc+ and mod- resc+ isofemale lines coexisted. We found no evidence for a cost difference to females expressing either type (mod-/-). Infections in the two populations could not be distinguished based on sequences of two Wolbachia genes. We consider the possibility that mod- is a host effect through a population dynamics model. A mod- host allele leads to infection extinction in the absence of fecundity differences. Furthermore, the uninfected population that results is immune to reestablishment of the (same) CI-causing Wolbachia.     

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.     

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.     

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.     

Interspecific transfer of Wolbachia between two lepidopteran insects expressing cytoplasmic incompatibility: a Wolbachia variant naturally infecting Cadra cautella causes male killing in Ephestia kuehniella

Wolbachia is known as the causative agent of various reproductive alterations in arthropods. The almond moth Cadra cautella is doubly infected with A- and B-group Wolbachia and expresses complete cytoplasmic incompatibility (CI). The Mediterranean flour moth Ephestia kuehniella carries A-group Wolbachia and expresses partial CI. In the present study, the Wolbachia in C. cautella was transferred to E. kuehniella from which the original Wolbachia had been removed. We obtained transfected lines of three different infection states: single infection with A, single infection with B, and double infection with A and B. The doubly transfected lines and those transfected with only A produced exclusively female progeny. Two lines of evidence suggested that the sex ratio distortion was due to male killing. First, reduced egg hatch rate was observed. Second, removal of the Wolbachia from the transfected lines resulted in the recovery of a normal sex ratio of approximately 1:1. The occurrence of male killing following transfection showed that host factors influence the determination of the reproductive phenotype caused by Wolbachia. The transfected E. kuehniella males carrying exclusively B-group Wolbachia expressed partial incompatibility when crossed with the uninfected females. In addition, the transfected lines were bidirectionally incompatible with the naturally infected strain, which was the first demonstration of bidirectional CI in a lepidopteran.     

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.     

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 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 在花蓟马中的感染及其与寄主线粒体DNA多样性的关系

【目的】Wolbachia 是一种广泛存在于节肢动物中的胞内共生细菌,影响寄主的生物学特性。花蓟马 Frankliniella intonsa (Trybom)是重要的害虫,对农作物及园林植物造成危害。本研究旨在明确 Wolbachia 在花蓟马中的感染情况,并分析其与寄主线粒体DNA多样性的关系。【方法】采集中国境内26个花蓟马自然种群,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;利用线粒体 COI 分子标记研究花蓟马的遗传分化及遗传多样性;通过比较感染和未感染 Wolbachia 个体 COI 数据,探究 Wolbachia 多样性与寄主线粒体DNA多样性之间的关系。【结果】花蓟马中 Wolbachia 的感染率为0%~60%,共检测到5种 Wolbachia 株系（wFint1,wFint2,wFint3,wFint4及wFint5）,均属于B大组且形成一个单系群。Wolbachia感染情况与这些花蓟马种群（除CC, GZ, TA和TY, N<5）的线粒体DNA多样性相关,表现为不感染 Wolbachia 的种群中线粒体DNA单倍型多样性（Hd）与核苷酸多样性（Pi）均高于感染 Wolbachia 的种群,且 Wolbachia 感染率与 Hd 呈显著负相关（ P <0.05）。AMOVA分析表明花蓟马线粒体DNA遗传分化与Wolbachia 感染情况有关。【结论】 Wolbachia 可能在侵染花蓟马种群后出现遗传分化;Wolbachia 感染与寄主线粒体DNA多样性有关。     

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-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.     

Effects on male fitness of removing Wolbachia infections from the mosquito Aedes albopictus

Abstract.  Cytoplasmic incompatibility (CI) induced by maternally inherited Wolbachia bacteria is a potential tool for the suppression of insect pest species with appropriate patterns of infection. The Asian tiger mosquito Aedes albopictus (Skuse) (Diptera: Culicidae) is known to be infected by two strains of Wolbachia pipientis Hertig (Rickettsiales: Rickettsiaceae), w Alb A and w Alb B, throughout its geographical distribution. This infection pattern theoretically restricts the application of CI-based control strategies. However, Wolbachia can be horizontally transferred using embryonic microinjection to generate incompatible transfected lines harbouring a single new strain of Wolbachia. In order to assess the feasibility of this approach, the effects of Wolbachia removal on mosquito fitness need to be clearly evaluated as the removal of natural superinfection is an inescapable step of this approach. Previous research has shown that uninfected females, produced by antibiotic treatment, showed a decrease in fitness compared with those infected with Wolbachia. In this study, the effect of Wolbachia removal on male fitness was investigated. Longevity and reproductive potential (mating competitiveness and sperm capacity) were assessed in both laboratory cages and greenhouses. No differences were observed between uninfected and infected males with respect to longevity, mating rate, sperm capacity and mating competitiveness in either laboratory conditions or greenhouses. The preservation of fitness in males of Ae. albopictus deprived of natural Wolbachia infection is discussed in relation to the development of incompatible insect technique suppression strategies. Finally, the potential application of aposymbiotic males in mark–release–recapture studies is suggested.