Wolbachia have made it twice: Hybrid introgression between two sister species of Eurema butterflies

Wolbachia, cytoplasmically inherited endosymbionts of arthropods, are known to hijack their host reproduction in various ways to increase their own vertical transmission. This may lead to the selective sweep of associated mitochondria, which can have a large impact on the evolution of mitochondrial lineages. In Japan, two different Wolbacahia strains (wCI and wFem) are found in two sister species of pierid butterflies, Eurema mandarina and Eurema hecabe. In both species, females infected with wCI (C females) produce offspring with a nearly 1:1 sex ratio, while females infected with both wCI and wFem (CF females) produce all‐female offspring. Previous studies have suggested the historical occurrence of hybrid introgression in C individuals between the two species. Furthermore, hybrid introgression in CF individuals is suggested by the distinct mitochondrial lineages between C females and CF females of E. mandarina. In this study, we performed phylogenetic analyses based on nuclear DNA and mitochondrial DNA markers of E. hecabe with previously published data on E. mandarina. We found that the nuclear DNA of this species significantly diverged from that of E. mandarina. By contrast, mitochondrial DNA haplotypes comprised two clades, mostly reflecting Wolbachia infection status rather than the individual species. Collectively, our results support the previously suggested occurrence of two independent historical events wherein the cytoplasms of CF females and C females moved between E. hecabe and E. mandarina through hybrid introgression.     

Reproductive compensation favours male-killing Wolbachia in a live-bearing host

Wolbachia are maternally inherited, cellular endosymbionts that can enhance their fitness by biasing host sex ratio in favour of females. Male killing (MK) is an extreme form of sex-ratio manipulation that is selectively advantageous if the self-sacrifice of Wolbachia in males increases transmission through females. In live-bearing hosts, females typically produce more embryos than can be carried to term, and reproductive compensation through maternal resource reallocation from dead males to female embryos could increase the number of daughters born to infected females. Here, we report a new strain of MK Wolbachia (wCsc2) in the pseudoscorpion, Cordylochernes scorpioides, and present the first empirical evidence that reproductive compensation favours the killing of males in a viviparous host. Females infected with the wCsc2 strain produced 26 per cent more and significantly larger daughters than tetracycline-cured females. In contrast to the previously described wCsc1 MK Wolbachia strain in C. scorpioides, wCsc2 infection was not accompanied by an increase in the rate of spontaneous brood abortion. Characterization of the wCsc1 and wCsc2 strains by multi-locus sequence typing and by Wolbachia surface protein (wsp) gene sequencing indicates that the marked divergence between these two MK strains in their impact on host reproductive success, and hence in their potential to spread, has occurred in association with homologous recombination in the wsp gene.     

The genetics and evolution of obligate reproductive parasitism in Trichogramma pretiosum infected with parthenogenesis-inducing Wolbachia

Parthenogenesis-inducing (PI) Wolbachia belong to a class of intracellular symbionts that distort the offspring sex ratio of their hosts toward a female bias. In many PI Wolbachia-infected species sex ratio distortion has reached its ultimate expression-fixation of infection and all-female populations. This is only possible with thelytokous PI symbionts as they provide an alternative form of reproduction and remove the requirement for males and sexual reproduction. Many populations fixed for PI Wolbachia infection have lost the ability to reproduce sexually, even when cured of the infection. We examine one such population in the species Trichogramma pretiosum. Through a series of backcrossing experiments with an uninfected Trichogramma pretiosum population we were able to show that the genetic basis for the loss of female sexual function could be explained by a dominant nuclear effect. Male sexual function had not been completely lost, though some deterioration of male sexual function was also evident when males from the infected population (created through antibiotic curing of infected females) were mated to uninfected females. We discuss the dynamics of sex ratio selection in PI Wolbachia-infected populations and the evolution of non-fertilizing mutations.     

Loss of reproductive parasitism following transfer of male-killing Wolbachia to Drosophila melanogaster and Drosophila simulans

Wolbachia manipulate insect host biology through a variety of means that result in increased production of infected females, enhancing its own transmission. A Wolbachia strain (wInn) naturally infecting Drosophila innubila induces male killing, while native strains of D. melanogaster and D. simulans usually induce cytoplasmic incompatibility (CI). In this study, we transferred wInn to D. melanogaster and D. simulans by embryonic microinjection, expecting conservation of the male-killing phenotype to the novel hosts, which are more suitable for genetic analysis. In contrast to our expectations, there was no effect on offspring sex ratio. Furthermore, no CI was observed in the transinfected flies. Overall, transinfected D. melanogaster lines displayed lower transmission rate and lower densities of Wolbachia than transinfected D. simulans lines, in which established infections were transmitted with near-perfect fidelity. In D. simulans, strain wInn had no effect on fecundity and egg-to-adult development. Surprisingly, one of the two transinfected lines tested showed increased longevity. We discuss our results in the context of host-symbiont co-evolution and the potential of symbionts to invade novel host species.     

Superinfection of cytoplasmic incompatibility-inducing Wolbachia is not additive in Orius strigicollis (Hemiptera: Anthocoridae)

Cytoplasmic incompatibility (CI) allows the intracellular, maternally inherited bacterial symbiont Wolbachia to invade arthropod host populations by inducing infertility in crosses between infected males and uninfected females. The general pattern is consistent with a model of sperm modification, rescued only by egg cytoplasm infected with the same strain of symbiont. The predacious flower bug Orius strigicollis is superinfected with two strains of Wolbachia, wOus1 and wOus2. Typically, superinfections of CI Wolbachia are additive in their effects; superinfected males are incompatible with uninfected and singly infected females. In this study, we created an uninfected line, and lines singly infected with wOus1 and wOus2 by antibiotic treatment. Then, all possible crosses were conducted among the four lines. The results indicated that while wOus2 induces high levels of CI, wOus1 induces very weak or no CI, but can rescue CI caused by wOus2 to a limited extent. Levels of incompatibility in crosses with superinfected males did not show the expected pattern. In particular, superinfected males caused extremely weak CI when mated with either singly infected or uninfected females. An analysis of symbiont densities showed that wOus1 densities were significantly higher than wOus2 densities in superinfected males, and wOus2 densities were lower, but not significantly, in superinfected relative to singly infected males. These data lend qualified support for the hypothesis that wOus1 interferes with the ability of wOus2 to cause CI by suppressing wOus2 densities. To our knowledge, this is the first clear case of non-additive CI in a natural superinfection.     

Wolbachia‐induced feminisation newly found in Eurema hecabe,a sibling species of Eurema mandarina (Lepidoptera: Pieridae)

1. Complete feminisation of genetic males into functional females, a unique case among insects, is known in Eurema mandarina (former Eurema hecabe Y type) that are infected with two strains of Wolbachia, wCI^Em and wFem^Em. 2. Here, we newly found that a proportion of wild‐caught E. hecabe (former E. hecabe B type) produced only female offspring. Cytogenetic observations indicated that individuals of E. hecabe displaying the all‐female trait were genetically male (i.e. feminisation). 3. Multilocus sequence typing analyses demonstrated that the feminised individuals of E. hecabe were infected with two Wolbachia strains, wCI^Eh and wFem^Eh, that were indistinguishable from wCI^Em and wFem^Em, respectively. 4. Even identical strains of Wolbachia can be regulated differently depending on the host genetic background. Therefore, we compared the infection densities and vertical transmission efficiencies of Wolbachia between feminised E. mandarina and E. hecabe, but detected no significant differences in these traits. 5. The possible routes by which the two Wolbachia strains have transferred between E. mandarina and E. hecabe are discussed.     

Anatomical and functional analysis of domestication effects on the olfactory system of the silkmoth Bombyx mori

The silkmoth Bombyx mori is the main producer of silk worldwide and has furthermore become a model organism in biological research, especially concerning chemical communication. However, the impact domestication might have had on the silkmoth''s olfactory sense has not yet been investigated. Here, we show that the pheromone detection system in B. mori males when compared with their wild ancestors Bombyx mandarina seems to have been preserved, while the perception of environmental odorants in both sexes of domesticated silkmoths has been degraded. In females, this physiological impairment was mirrored by a clear reduction in olfactory sensillum numbers. Neurophysiological experiments with hybrids between wild and domesticated silkmoths suggest that the female W sex chromosome, so far known to have the sole function of determining femaleness, might be involved in the detection of environmental odorants. Moreover, the coding of odorants in the brain, which is usually similar among closely related moths, differs strikingly between B. mori and B. mandarina females. These results indicate that domestication has had a strong impact on odour detection and processing in the olfactory model species B. mori.     

Unravelling the Wolbachia evolutionary role: the reprogramming of the host genomic imprinting

Environmental factors can induce significant epigenetic changes that may also be inherited by future generations. The maternally inherited symbiont of arthropods Wolbachia pipientis is an excellent candidate as an ‘environmental’ factor promoting trans-generational epigenetic changes: by establishing intimate relationships with germ-line cells, epigenetic effects of Wolbachia symbiosis would be manifested as a ‘maternal effect’, in which infection of the mother modulates the offspring phenotype. In the leafhopper Zyginidia pullula, Wolbachia feminizes genetic males, leaving them as intersexes. With the exception of male chitinous structures that are present in the last abdominal segment, feminized males display phenotypic features that are typical of females. These include ovaries that range from a typical histological architecture to an altered structure. Methylation-sensitive random amplification of polymorphic DNA profiles show that they possess a female genomic imprint. On the other hand, some rare feminized males bear testes instead of ovaries. These specimens possess a Wolbachia density approximately four orders of magnitude lower than feminized males with ovaries and maintain a male genome—methylation pattern. Our results indicate that Wolbachia infection disrupts male imprinting, which dramatically influences the expression of genes involved in sex differentiation and development, and the alteration occurs only if Wolbachia exceeds a density threshold. Thus, a new Wolbachia''s role as an environmental evolutionary force, inducing epigenetic trans-generational changes, should now be considered.     

Rapid Sequential Spread of Two Wolbachia Variants in Drosophila simulans

The maternally inherited intracellular bacteria Wolbachia can manipulate host reproduction in various ways that foster frequency increases within and among host populations. Manipulations involving cytoplasmic incompatibility (CI), where matings between infected males and uninfected females produce non-viable embryos, are common in arthropods and produce a reproductive advantage for infected females. CI was associated with the spread of Wolbachia variant wRi in Californian populations of Drosophila simulans, which was interpreted as a bistable wave, in which local infection frequencies tend to increase only once the infection becomes sufficiently common to offset imperfect maternal transmission and infection costs. However, maternally inherited Wolbachia are expected to evolve towards mutualism, and they are known to increase host fitness by protecting against infectious microbes or increasing fecundity. We describe the sequential spread over approximately 20 years in natural populations of D. simulans on the east coast of Australia of two Wolbachia variants (wAu and wRi), only one of which causes significant CI, with wRi displacing wAu since 2004. Wolbachia and mtDNA frequency data and analyses suggest that these dynamics, as well as the earlier spread in California, are best understood as Fisherian waves of favourable variants, in which local spread tends to occur from arbitrarily low frequencies. We discuss implications for Wolbachia-host dynamics and coevolution and for applications of Wolbachia to disease control.     

Transfer of a parasitic sex factor to the nuclear genome of the host: A hypothesis on the evolution of sex-determining mechanisms in the terrestrial Isopod Armadillidium vulgare Latr.

In A. vulgare sex is usually determined either by a cytoplasmic feminizing factor (F symbiotic bacteria) or by another feminizing factor (f) which behaves like a mobile element of DNA and which seems to correspond to a fragment of bacterial DNA. By inhibiting the expression of male genes carried by the Z heterochromosome, these feminizing factors induce differentiation of neo-females [ZZ(+F) or ZZ(+f)]. Such a mechanism leads to the production of progenies whose sex ratio is highly female biased. In some populations in which F and/or f factors are present, genetic females (WZ) have disappeared and all individuals (males and females) are genetic males. However in other populations, cohabitation of ZZ(+f) neo-females and females in all points similar to genetic females is observed. Such a situation may be unstable and is not likely to be explainable only by migrations of individuals from distinct populations. Owing to certain types of crosses, in particular those which involve an artificial neo-male ( = female reversed into a functional male by an implant of androgenic gland) we show here that the f factor can be transmitted as a Mendelian gene. In these progenies Z_fZ females may appear: like WZ females, they breed broods whose sex ratio is unbiased. The hypothesis that the “F bacteria—A. vulgare” symbiosis may have led, after a complex co-evolutive process (F bacteria → f mobile element → insertion of f on Z heterochromosome), to the creation (from a male genotype) of a female genotype, is put forward. The consequences of such a phenomenon on the composition and the evolution of A. vulgare populations are examined.     

Male mating performance and cytoplasmic incompatibility in a wPip Wolbachia trans‐infected line of Aedes albopictus (Stegomyia albopicta)

Wolbachia pipientis Hertig (Rickettsiales: Rickettsiaceae) is a maternally inherited endosymbiont of a large number of insects and other arthropods that induces various effects on host reproductive biology. Among these, cytoplasmic incompatibility (CI) is a form of sterility induced in eggs produced by mating between infected males and females uninfected or infected by an incompatible Wolbachia strain. This phenomenon has been proposed as a potential way to produce functionally sterile males to be used in genetic control programmes. In this paper, we report on experiments carried out to evaluate the mating performances of males of an Aedes albopictus (Stegomyia albopicta) (Diptera: Culicidae) line (ARwP), harbouring a new Wolbachia infection [the wPip strain from Culex pipiens Linnaeus (Diptera: Culicidae)], in comparison with naturally infected males (SR line). ARwP males did not differ from SR males with regard to insemination capacity. Mating competitiveness did not differ significantly between lines in either laboratory or greenhouse conditions. Moreover, crosses with SR females were characterized by a 100% CI regardless of ARwP male age. All of these findings suggest that ARwP males may represent a very efficient tool for control programmes against Ae. albopictus based on the release of functionally sterile males.     

A DNA test to sex most birds

Birds are difficult to sex. Nestlings rarely show sex-linked morphology and we estimate that adult females appear identical to males in over 50% of the world's bird species. This problem can hinder both evolutionary studies and human-assisted breeding of birds. DNA-based sex identification provides a solution. We describe a test based on two conserved CHD (chromo-helicase-DNA-binding) genes that are located on the avian sex chromosomes of all birds, with the possible exception of the ratites (ostriches, etc.; Struthioniformes). The CHD-W gene is located on the W chromosome; therefore it is unique to females. The other gene, CHD-Z, is found on the Z chromosome and therefore occurs in both sexes (female, ZW; male, ZZ). The test employs PCR with a single set of primers. It amplifies homologous sections of both genes and incorporates introns whose lengths usually differ. When examined on a gel there is a single CHD-Z band in males but females have a second, distinctive CHD-W band.     

Pervasive effects of Wolbachia on host activity

Heritable symbionts have diverse effects on the physiology, reproduction and fitness of their hosts. Maternally transmitted Wolbachia are one of the most common endosymbionts in nature, infecting about half of all insect species. We test the hypothesis that Wolbachia alter host behaviour by assessing the effects of 14 different Wolbachia strains on the locomotor activity of nine Drosophila host species. We find that Wolbachia alter the activity of six different host genotypes, including all hosts in our assay infected with wRi-like Wolbachia strains (wRi, wSuz and wAur), which have rapidly spread among Drosophila species in about the last 14 000 years. While Wolbachia effects on host activity were common, the direction of these effects varied unpredictably and sometimes depended on host sex. We hypothesize that the prominent effects of wRi-like Wolbachia may be explained by patterns of Wolbachia titre and localization within host somatic tissues, particularly in the central nervous system. Our findings support the view that Wolbachia have wide-ranging effects on host behaviour. The fitness consequences of these behavioural modifications are important for understanding the evolution of host–symbiont interactions, including how Wolbachia spread within host populations.     

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

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

DNA Content Differences Between Male and Female Chicken (Gallus gallus domesticus) Nuclei and Z and W Chromosomes Resolved by Image Cytometry

Chicken red blood cells (CRBCs) are widely used as standards for DNA content determination. Cytogenetic data have shown that the Z sex chromosome is approximately twice as large as the W, so that the DNA content differs to some extent between male (ZZ) and female (ZW) chickens. Despite this fact, male and female CRBCs have been indiscriminately used in absolute genome size determination. Our work was conducted to verify whether the DNA content differences between male and female Gallus gallus domesticus “Leghorn” nuclei and ZZ/ZW chromosomes can be resolved by image cytometry (ICM). Air-dried smears stained by Feulgen reaction were used for nuclei analysis. Chicken metaphase spreads upon Feulgen staining were analyzed for obtaining quantitative information on the Z and W chromosomes. Before each capture session, we conducted quality control of the ICM instrumentation. Our results from nuclear measurements showed that the 2C value is 0.09 pg higher in males than in females. In chromosomes, we found that the Z chromosome shows 200% more DNA content than does the W chromosome. ICM demonstrated resolution power to discriminate low DNA content differences in genomes. We suggest prudence in the general use of CRBC 2C values as standards in comparative cytometric analysis. (J Histochem Cytochem 58:229–235, 2010)     

The Effect of Virus-Blocking Wolbachia on Male Competitiveness of the Dengue Vector Mosquito,Aedes aegypti

Background

The bacterial endosymbiont Wolbachia blocks the transmission of dengue virus by its vector mosquito Aedes aegypti, and is currently being evaluated for control of dengue outbreaks. Wolbachia induces cytoplasmic incompatibility (CI) that results in the developmental failure of offspring in the cross between Wolbachia-infected males and uninfected females. This increases the relative success of infected females in the population, thereby enhancing the spread of the beneficial bacterium. However, Wolbachia spread via CI will only be feasible if infected males are sufficiently competitive in obtaining a mate under field conditions. We tested the effect of Wolbachia on the competitiveness of A. aegypti males under semi-field conditions.

Methodology/Principal Findings

In a series of experiments we exposed uninfected females to Wolbachia-infected and uninfected males simultaneously. We scored the competitiveness of infected males according to the proportion of females producing non-viable eggs due to incompatibility. We found that infected males were equally successful to uninfected males in securing a mate within experimental tents and semi-field cages. This was true for males infected by the benign wMel Wolbachia strain, but also for males infected by the virulent wMelPop (popcorn) strain. By manipulating male size we found that larger males had a higher success than smaller underfed males in the semi-field cages, regardless of their infection status.

Conclusions/Significance

The results indicate that Wolbachia infection does not reduce the competitiveness of A. aegypti males. Moreover, the body size effect suggests a potential advantage for lab-reared Wolbachia-males during a field release episode, due to their better nutrition and larger size. This may promote Wolbachia spread via CI in wild mosquito populations and underscores its potential use for disease control.     

Evolutionary transition to XY sex chromosomes associated with Y-linked duplication of a male hormone gene in a terrestrial isopod

Sex chromosomes are highly variable in some taxonomic groups, but the evolutionary mechanisms underlying this diversity are not well understood. In terrestrial isopod crustaceans, evolutionary turnovers in sex chromosomes are frequent, possibly caused by Wolbachia, a vertically-transmitted endosymbiont causing male-to-female sex reversal. Here, we use surgical manipulations and genetic crosses, plus genome sequencing, to examine sex chromosomes in the terrestrial isopod Trachelipus rathkei. Although an earlier cytogenetics study suggested a ZZ/ZW sex chromosome system in this species, we surprisingly find multiple lines of evidence that in our study population, sex is determined by an XX/XY system. Consistent with a recent evolutionary origin for this XX/XY system, the putative male-specific region of the genome is small. The genome shows evidence of Y-linked duplications of the gene encoding the androgenic gland hormone, a major component of male sexual differentiation in isopods. Our analyses also uncover sequences horizontally acquired from past Wolbachia infections, consistent with the hypothesis that Wolbachia may have interfered with the evolution of sex determination in T. rathkei. Overall, these results provide evidence for the co-occurrence of multiple sex chromosome systems within T. rathkei, further highlighting the relevance of terrestrial isopods as models for the study of sex chromosome evolution.Subject terms: Evolutionary genetics, Genome evolution     

A new marker based on the avian spindlin gene that is able to sex most birds,including species problematic to sex with CHD markers

We have developed a new marker (Z43B) that can be successfully used to identify the sex of most birds (69%), including species difficult or impossible to sex with other markers. We utilized the zebra finch Taeniopygia guttata EST microsatellite sequence (CK309496) which displays sequence homology to the 5′ untranslated region (UTR) of the avian spindlin gene. This gene is known to be present on the Z and W chromosomes. To maximize cross‐species utility, the primer set was designed from a consensus sequence created from homologs of CK309496 that were isolated from multiple distantly related species. Both the forward and reverse primer sequences were 100% identical to 14 avian species, including the Z chromosome of eight species and the chicken Gallus gallus W chromosome, as well as the saltwater crocodile Crocodylus porosus. The Z43B primer set was assessed by genotyping individuals of known sex belonging to 61 non‐ratite species and a single ratite. The Z and W amplicons differed in size making it possible to distinguish between males (ZZ) and females (ZW) for the majority (69%) of non‐ratite species tested, comprising 10 orders of birds. We predict that this marker will be useful for obtaining sex‐typing data for ca 6,869 species of birds (69% of non‐ratites but not galliforms). A wide range of species could be sex‐typed including passerines, shorebirds, eagles, falcons, bee‐eaters, cranes, shags, parrots, penguins, ducks, and a ratite species, the brown kiwi, Apteryx australis. Those species sexed include species impossible or problematic to sex‐type with other markers (magpie, albatross, petrel, eagle, falcon, crane, and penguin species). Zoo Biol. 35:533–545, 2016. © 2016 The Authors. Zoo Biology published by Wiley Periodicals, Inc.     

Rapid evolution of Wolbachia incompatibility types

In most insects, the endosymbiont Wolbachia induces cytoplasmic incompatibility (CI), an embryonic mortality observed when infected males mate either with uninfected females or with females infected by an incompatible Wolbachia strain. Although the molecular mechanism of CI remains elusive, it is classically viewed as a modification–rescue model, in which a Wolbachia mod function disables the reproductive success of the sperm of infected males, unless eggs are infected and express a compatible resc function. The extent to which the modification–rescue model can predict highly complex CI pattern remains a challenging issue. Here, we show the rapid evolution of the mod–resc system in the Culex pipiens mosquito. We have surveyed four incompatible laboratory isofemale lines over 50 generations and observed in two of them that CI has evolved from complete to partial incompatibility (i.e. the production of a mixture of compatible and incompatible clutches). Emergence of the new CI types depends only on Wolbachia determinants and can be simply explained by the gain of new resc functions. Evolution of CI types in Cx. pipiens thus appears as a gradual process, in which one or several resc functions can coexist in the same individual host in addition to the ones involved in the self-compatibility. Our data identified CI as a very dynamic process. We suggest that ancestral and mutant Wolbachia expressing distinct resc functions can co-infect individual hosts, opening the possibility for the mod functions to evolve subsequently. This gives a first clue towards the understanding of how Wolbachia reached highly complex CI pattern in host populations.     

Multiple paternity in a wild population of Armadillidium vulgare: influence of infection with Wolbachia?

Female multiple mating has been extensively studied to understand how nonobvious benefits, generally thought to be of genetic nature, could overcome heavy costs such as an increased risk of infection during mating. However, the impact of infection itself on multiple mating has rarely been addressed. The interaction between the bacterium Wolbachia and its terrestrial crustacean host, Armadillidium vulgare, is a relevant model to investigate this question. In this association, Wolbachia is able to turn genetic males into functional females (i.e. feminization), thereby distorting the sex ratio and decreasing the number of available males at the population scale. Moreover, in A. vulgare, females have been shown to mate multiply under laboratory conditions and males prefer uninfected females over infected ones. Additionally, different Wolbachia strains are known to infect A. vulgare and these strains differ in their transmission rate and virulence. All these elements suggest a potential impact of different Wolbachia strains on multiple mating. To investigate this assumption, we collected gravid females in a wild A. vulgare population harbouring both uninfected females and females infected with one of two different Wolbachia strains (wVulM and wVulC) and performed paternity analyses on the obtained broods using microsatellite markers. We demonstrate that (i) multiple paternity is common in this wild population of A. vulgare, with a mean number of fathers of 4.48 ± 1.24 per brood and (ii) females infected with wVulC produced broods with a lower multiple paternity level compared with females infected with wVulM and uninfected ones. This work improves our knowledge of the impact of infections on reproductive strategies.