Effects of parasitic sex-ratio distorters on host genetic structure in the Armadillidium vulgare-Wolbachia association

In the pill bug Armadillidium vulgare (Crustacea, Oniscidea), Wolbachia facilitates its spread through vertical transmission via the eggs by inducing feminization of genetic males. The spread of feminizing Wolbachia within and across populations is therefore expected to influence mitochondrial DNA (mtDNA) genetic structure by hitchhiking. To test this hypothesis, we analysed nuclear and mtDNA genetic structure, and Wolbachia prevalence in 13 populations of the pill bug host. Wolbachia prevalence (ranging from 0% to 100% of sampled females) was highly variable among populations. All three Wolbachia strains previously observed in A. vulgare were present (wVulC, wVulM and wVulP) with wVulC being the most prevalent (nine of 13 populations). The host showed a genetic structure on five microsatellite loci that is compatible with isolation by distance. The strong genetic structure observed on host mtDNA was correlated with Wolbachia prevalence: three mitotypes were in strong linkage disequilibrium with the three strains of Wolbachia. Neutrality tests showed that the mtDNA polymorphism is not neutral, and we thus suggest that this unusual pattern of mtDNA polymorphism found in A. vulgare was due to Wolbachia.     

Isolation and Characterization of Microsatellite Loci for the Isopod Crustacean Armadillidium vulgare and Transferability in Terrestrial Isopods

Armadillidium vulgare is a terrestrial isopod (Crustacea, Oniscidea) which harbors Wolbachia bacterial endosymbionts. A. vulgare is the major model for the study of Wolbachia-mediated feminization of genetic males in crustaceans. As a consequence of their impact on host sex determination mechanisms, Wolbachia endosymbionts are thought to significantly influence A. vulgare evolution on various grounds, including population genetic structure, diversity and reproduction strategies. To provide molecular tools for examining these questions, we isolated microsatellite loci through 454 pyrosequencing of a repeat-enriched A. vulgare genomic library. We selected 14 markers and developed three polymorphic microsatellite multiplex kits. We tested the kits on two A. vulgare natural populations and found high genetic variation, thereby making it possible to investigate the impact of Wolbachia endosymbionts on A. vulgare nuclear variation at unprecedented resolution. In addition, we tested the transferability of these kits by cross-species amplification in five other terrestrial isopod species harboring Wolbachia endosymbionts. The microsatellite loci showed good transferability in particular in Armadillidium nasatum and Chaetophiloscia elongata, for which these markers represent promising tools for future genetic studies.     

Population genetics of Wolbachia‐infected,parthenogenetic and uninfected,sexual populations of Tetrastichus coeruleus (Hymenoptera: Eulophidae)

Wolbachia are endosymbiotic bacteria known to manipulate the reproduction of their hosts. These manipulations are expected to have consequences on the population genetics of the host, such as heterozygosity levels, genetic diversity and gene flow. The parasitoid wasp Tetrastichus coeruleus has populations that are infected with parthenogenesis‐inducing Wolbachia and populations that are not infected. We studied the population genetics of T. coeruleus between and within Wolbachia‐infected and uninfected populations, using nuclear microsatellites and mitochondrial DNA. We expected reduced genetic diversity in both DNA types in infected populations. However, migration and gene flow could introduce new DNA variants into populations. We therefore paid special attention to individuals with unexpected (genetic) characteristics. Based on nuclear and mitochondrial DNA, two genetic clusters were evident: a thelytokous cluster containing all Wolbachia‐infected, parthenogenetic populations and an arrhenotokous cluster containing all uninfected, sexual populations. Nuclear and mitochondrial DNA did not exhibit concordant patterns of variation, although there was reduced genetic diversity in infected populations for both DNA types. Within the thelytokous cluster, there was nuclear DNA variation, but no mitochondrial DNA variation. This nuclear DNA variation may be explained by occasional sex between infected females and males, by horizontal transmission of Wolbachia, and/or by novel mutations. Several females from thelytokous populations were uninfected and/or heterozygous for microsatellite loci. These unexpected characteristics may be explained by migration, by inefficient transmission of Wolbachia, by horizontal transmission of Wolbachia, and/or by novel mutations. However, migration has not prevented the build‐up of considerable genetic differentiation between thelytokous and arrhenotokous populations.     

Sexual selection in an isopod with Wolbachia‐induced sex reversal: males prefer real females

A variety of genetic elements encode traits beneficial to their own transmission. Despite their ‘selfish’ behaviour, most of these elements are often found at relatively low frequencies in host populations. This is the case of intracytoplasmic Wolbachia bacteria hosted by the isopod Armadillidium vulgare that distort the host sex ratio towards females by feminizing the genetic males they infect. Here we tested the hypothesis that sexual selection against Wolbachia‐infected females could maintain a polymorphism of the infection in populations. The infected neo‐females (feminized males) have lower mating rates and received less sperm relative to uninfected females. Males exhibited an active choice: they interacted more with uninfected females and made more mating attempts. A female behavioural difference was also observed in response to male mating attempts: infected neo‐females more often exhibited behaviours that stop the mating sequence. The difference in mating rate was significant only when males could choose between the two female types. This process could maintain a polymorphism of the infection in populations. Genetic females experimentally infected with Wolbachia are not exposed to the same sexual selection pressure, so the infection alone cannot explain these differences.     

Host tissues as microhabitats for Wolbachia and quantitative insights into the bacterial community in terrestrial isopods

Animal–bacterial symbioses are highly dynamic in terms of multipartite interactions, both between the host and its symbionts as well as between the different bacteria constituting the symbiotic community. These interactions will be reflected by the titres of the individual bacterial taxa, for example via host regulation of bacterial loads or competition for resources between symbionts. Moreover, different host tissues represent heterogeneous microhabitats for bacteria, meaning that host‐associated bacteria might establish tissue‐specific bacterial communities. Wolbachia are widespread endosymbiotic bacteria, infecting a large number of arthropods and filarial nematodes. However, relatively little is known regarding direct interactions between Wolbachia and other bacteria. This study represents the first quantitative investigation of tissue‐specific Wolbachia–microbiota interactions in the terrestrial isopod Armadillidium vulgare. To this end, we obtained a more complete picture of the Wolbachia distribution patterns across all major host tissues, integrating all three feminizing Wolbachia strains (wVulM, wVulC, wVulP) identified to date in this host. Interestingly, the different Wolbachia strains exhibited strain‐specific tissue distribution patterns, with wVulM reaching lower titres in most tissues. These patterns were consistent across different host genetic backgrounds and might reflect different co‐evolutionary histories between the Wolbachia strains and A. vulgare. Moreover, Wolbachia‐infected females carried higher total bacterial loads in several, but not all, tissues, irrespective of the Wolbachia strain. Taken together, this quantitative approach indicates that Wolbachia is part of a potentially more diverse bacterial community, as exemplified by the presence of highly abundant bacterial taxa in the midgut caeca of several A. vulgare 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.     

Males prefer virgin females,even if parasitized,in the terrestrial isopod Armadillidium vulgare

In many species, males increase their reproductive success by choosing high‐quality females. In natural populations, they interact with both virgin and mated females, which can store sperm in their spermatheca. Therefore, males elaborate strategies to avoid sperm competition. In the terrestrial isopod Armadillidium vulgare, females can store sperm and produce several clutches. Moreover, this species can be parasitized by Wolbachia, which feminizes genetic males, transforming them into functional females. Our study compared attractiveness and mate choice when a male is exposed to both virgin and experienced females (i.e., females who have produced offspring and rested for 6 months), with or without Wolbachia. Our results revealed that males are more attracted to virgin females than experienced females, even if these virgin females are parasitized. Moreover, the chemical analysis highlighted different odors in females according to their reproductive and infection (Wolbachia‐free or vertically Wolbachia‐infected) status. Males attempted copulation more frequently and for longer with virgin females, even if Wolbachia‐infected, while experienced females refused further copulation. The evolutionary consequences of both male choice and female resistance on their fitness are discussed in this study.     

Population structure in relation to host-plant ecology and Wolbachia infestation in the comma butterfly

Experimental work on Polygonia c‐album, a temperate polyphagous butterfly species, has shown that Swedish, Belgian, Norwegian and Estonian females are generalists with respect to host‐plant preference, whereas females from UK and Spain are specialized on Urticaceae. Female preference is known to have a strong genetic component. We test whether the specialist and generalist populations form respective genetic clusters using data from mitochondrial sequences and 10 microsatellite loci. Results do not support this hypothesis, suggesting that the specialist and generalist traits have evolved more than once independently. Mitochondrial DNA variation suggests a rapid expansion scenario, with a single widespread haplotype occurring in high frequency, whereas microsatellite data indicate strong differentiation of the Moroccan population. Based on a comparison of polymorphism in the mitochondrial data and sequences from a nuclear gene, we show that the diversity in the former is significantly less than that expected under neutral evolution. Furthermore, we found that almost all butterfly samples were infected with a single strain of Wolbachia, a maternally inherited bacterium. We reason that indirect selection on the mitochondrial genome mediated by a recent sweep of Wolbachia infection has depleted variability in the mitochondrial sequences. We also surmise that P. c‐album could have expanded out of a single glacial refugium and colonized Morocco recently.     

Microsatellite and Wolbachia analysis in Rhagoletis cerasi natural populations: population structuring and multiple infections

Rhagoletis cerasi (Diptera: Tephritidae) is a major pest of sweet and sour cherries in Europe and parts of Asia. Despite its economic significance, there is a lack of studies on the genetic structure of R. cerasi populations. Elucidating the genetic structure of insects of economic importance is crucial for developing phenological‐predictive models and environmental friendly control methods. All natural populations of R. cerasi have been found to harbor the endosymbiont Wolbachia pipientis, which widely affects multiple biological traits contributing to the evolution of its hosts, and has been suggested as a tool for the biological control of insect pests and disease vectors. In the current study, the analysis of 18 R. cerasi populations collected in Greece, Germany, and Russia using 13 microsatellite markers revealed structuring of R. cerasi natural populations, even at close geographic range. We also analyzed the Wolbachia infection status of these populations using 16S rRNA‐, MLST‐ and wsp‐based approaches. All 244 individuals screened were positive for Wolbachia. Our results suggest the fixation of the wCer1 strain in Greece while wCer2, wCer4, wCer5, and probably other uncharacterized strains were also detected in multiply infected individuals. The role of Wolbachia and its potential extended phenotypes needs a thorough investigation in R. cerasi. Our data suggest an involvement of this symbiont in the observed restriction in the gene flow in addition to a number of different ecological factors.     

An elusive endosymbiont: Does Wolbachia occur naturally in Aedes aegypti?

Wolbachia are maternally inherited endosymbiotic bacteria found within many insect species. Aedes mosquitoes experimentally infected with Wolbachia are being released into the field for Aedes‐borne disease control. These Wolbachia infections induce cytoplasmic incompatibility which is used to suppress populations through incompatible matings or replace populations through the reproductive advantage provided by this mechanism. However, the presence of naturally occurring Wolbachia in target populations could interfere with both population replacement and suppression programs depending on the compatibility patterns between strains. Aedes aegypti were thought to not harbor Wolbachia naturally but several recent studies have detected Wolbachia in natural populations of this mosquito. We therefore review the evidence for natural Wolbachia infections in A. aegypti to date and discuss limitations of these studies. We draw on research from other mosquito species to outline the potential implications of natural Wolbachia infections in A. aegypti for disease control. To validate previous reports, we obtained a laboratory population of A. aegypti from New Mexico, USA, that harbors a natural Wolbachia infection, and we conducted field surveys in Kuala Lumpur, Malaysia, where a natural Wolbachia infection has also been reported. However, we were unable to detect Wolbachia in both the laboratory and field populations. Because the presence of naturally occurring Wolbachia in A. aegypti could have profound implications for Wolbachia‐based disease control programs, it is important to continue to accurately assess the Wolbachia status of target Aedes populations.     

Mitochondrial DNA polymorphism and feminizing sex factors dynamics in a natural population of Armadillidium vulgare (Crustacea,Isopoda)

Sex determination in Armadillidium vulgare may be under the control of two parasitic sex factors that reverse genetic males into functional neo-females. The first feminizing factor (F) is a Wolbachia and the other (f) is probably a sequence of the F bacterial DNA unstably integrated into the host genome. Both of these feminizing factors are mainly maternally transmitted. Here we investigate the mitochondrial DNA polymorphism of wild iso-female lineages harbouring either F or f. Among the four haplotypes present in the population, two were the f-harbouring lineages, while two were common to the F- and f-harbouring lineages. This result suggests that there has been an introgression of the f factor into lineages infected by F Wolbachia. Based on previous data, we propose two different ways to account for such introgression. Given the particular dynamics of feminizing factors (f-harbouring lineages increase in populations at the expense of F-harbouring lineages), such an introgression should prevent the replacement of F-linked mitochondrial types by f-linked mitochondrial types in wild populations.     

Wolbachia infection in natural populations of Dictyophara europaea,an alternative vector of grapevine Flavescence dorée phytoplasma: effects and interactions

The European lantern fly, Dictyophara europaea, is an alternative vector of the Flavescence dorée phytoplasma (FDp) disease of grapevine in European vineyards, enabling infection initiation from wild reservoir compartment (Clematis vitalba). Heretofore recorded rate of D. europaea FDp‐infection has been very low (3%), making it less epidemiologically significant than would be expected based on reservoir plant infection rate (30%). In this study we present findings on a heavily FDp‐infected D. europaea population (>60%), on the natural Wolbachia infection of populations with low FDp‐infection rates (DeWo+) and on Wolbachia absence in highly FDp‐infected population (DeWo?). We examine several possible causes underlying the differences in vector infection rates: (a) population genetic characteristics of D. europaea and correlation with Wolbachia strain wEur natural infections, (b) Wolbachia effects on fitness components of DeWo+ laboratory colony and (c) rate of reservoir plant FDp‐infection and differences in FDp genotypes harboured by low and highly infected vector populations. The vector genetic diversity level was found to be lower in DeWo+ than in uninfected individuals and to exhibit a different evolution of fixed haplotypes. All DeWo+ populations were infected with the same strain of wEur. The FDp was found to be genetically diversified (five genotypes) but had no relation to infection rates. We did not find evidence of fitness upgrades with regard to Wolbachia infection status. Although more experimentation is needed, it seems that Wolbachia confers protection against FDp or is in competition with FDp according to the observed correlations: low FDp‐infected vector populations are infected with Wolbachia and vice versa.     

Population structure of the mosquito Aedes aegypti (Stegomyia aegypti) in Pakistan

Eleven microsatellite markers were used to determine the genetic population structure and spread of Aedes aegypti (Stegomyia aegypti) (Diptera: Culicidae) in Pakistan using mosquitoes collected from 13 different cities. There is a single genetic cluster of Ae. aegypti in Pakistan with a pattern of isolation by distance within the population. The low level of isolation by distance suggests the long‐range passive dispersal of this mosquito, which may be facilitated by the tyre trade in Pakistan. A decrease in genetic diversity from south to north suggests a recent spread of this mosquito from Karachi. A strong negative correlation between genetic distance and the quality of road connections shows that populations in cities connected by better road networks are less differentiated, which suggests the human‐aided passive dispersal of Ae. aegypti in Pakistan. Dispersal on a large spatial scale may facilitate the strategy of introducing transgenic Ae. aegypti or intracellular bacteria such as Wolbachia to control the spread of dengue disease in Pakistan, but it also emphasizes the need for simple measures to control container breeding sites.     

Experimental evolution reveals habitat‐specific fitness dynamics among Wolbachia clades in Drosophila melanogaster

The diversity and infection dynamics of the endosymbiont Wolbachia can be influenced by many factors, such as transmission rate, cytoplasmic incompatibility, environment, selection and genetic drift. The interplay of these factors in natural populations can result in heterogeneous infection patterns with substantial differences between populations and strains. The causes of these heterogeneities are not yet understood, partly due to the complexity of natural environments. We present experimental evolution as a new approach to study Wolbachia infection dynamics in replicate populations exposed to a controlled environment. A natural Drosophila melanogaster population infected with strains of Wolbachia belonging to different clades evolved in two laboratory environments (hot and cold) for 1.5 years. In both treatments, the rate of Wolbachia infection increased until fixation. In the hot environment, the relative frequency of different Wolbachia clades remained stable over 37 generations. In the cold environment, however, we observed marked changes in the composition of the Wolbachia population: within 15 generations, one Wolbachia clade increased more than 50% in frequency, whereas the other two clades decreased in frequency, resulting in the loss of one clade. The frequency change was highly reproducible not only among replicates, but also when flies that evolved for 42 generations in the hot environment were transferred to the cold environment. These results document how environmental factors can affect the composition of Wolbachia in D. melanogaster. The high reproducibility of the pattern suggests that experimental evolution studies can efficiently determine the functional basis of habitat‐specific fitness among Wolbachia strains.     

First report on Wolbachia endosymbiosis in freshwater Aphelocheirus aestivalis (Heteroptera: Aphelocheiridae) and its potential impact on genetic diversity of host

Alphaproteobacteria Wolbachia have been described as endosymbionts of approximately half of all aquatic insect species. These bacteria might affect not only reproduction but also the genetic diversity of its hosts. In the present study we identified Wolbachia endosymbiosis in freshwater true bug Aphelocheirus aestivalis F., 1794 (Heteroptera: Aphelocheiridae). Despite the fact that A. aestivalis is widely distributed in Europe, it occurs rather locally, often in isolated populations. Taking into account that Wolbachia, close relationships and past demographic phenomena could affect the genetic diversity of its host, we analyzed mitochondrial (COI and 16S) and nuclear (internal transcribed spacer 2) markers determined for A. aestivalis individuals collected from five populations. Moreover, we compared obtained COI sequences with those deposited in GenBank. Analyses revealed low genetic differentiation among samples tested, as well as low variation among determined COI sequences and those downloaded from the database. Although Wolbachia infection could correlate with decreasing mitochondrial diversity of its host, we suggest that low genetic variation observed in tested A. aestivalis samples (at both mitochondrial and nuclear levels) is a result of populations’ close relationships, past demographic phenomena or is characteristic for this species. Detailed analysis of the wsp gene fragment revealed two distinct strains of Wolbachia infecting A. aestivalis. Both of them belong to supergroup A, also found in other arthropods.     

Molecular characterization of <Emphasis Type="Italic">Psyttalia lounsburyi</Emphasis>, a candidate biocontrol agent of the olive fruit fly,and its <Emphasis Type="Italic">Wolbachia</Emphasis> symbionts as a pre-requisite for future intraspecific hybridization

Numerous arthropod species are genetically differentiated across their distribution area. Diversifying the geographical origins of a biocontrol agent species can be used to favour their perennial establishment by the sampling of pre-adapted genotypes and/or the production of new genotypes through hybridization. Hybridization can be nevertheless challenged by reproductive isolations induced by some common microbial endosymbionts. In this study, we aimed at characterizing (i) the genetic diversity of six populations of Psyttalia lounsburyi (Hymenoptera: Braconidae), a candidate biocontrol agent of the olive fruit fly Bactrocera oleae (Diptera: Tephritidae) and (ii) the diversity of their Wolbachia endosymbionts. Both mitochondrial and microsatellite markers evidence clustering between the South African population and several Kenyan/Namibian populations. The survey of the Wolbachia also distinguished two main variants with a spatial heterogeneity in the infection status. All these results are discussed in the context of the use of these P. lounsburyi populations for hybridization and further field releases.     

Ecology,Wolbachia infection frequency and mode of reproduction in the parasitoid wasp Tetrastichus coeruleus (Hymenoptera: Eulophidae)

Whereas sexual reproduction may facilitate adaptation to complex environments with many biotic interactions, simplified environments are expected to favour asexual reproduction. In agreement with this, recent studies on invertebrates have shown a prevalence of asexual species in agricultural (simplified) but not in natural (complex) environments. We investigated whether the same correlation between reproductive mode and habitat can be found in different populations within one species. The parasitoid wasp Tetrastichus coeruleus forms an ideal model to test this question, since it occurs both in natural and agricultural environments. Further, we investigated whether Wolbachia infection caused parthenogenesis in female‐biased populations. In contrast to the general pattern, in Dutch and French natural areas, we found Wolbachia‐infected, highly female‐biased populations that reproduce parthenogenetically. In contrast, populations on Dutch agricultural fields were not infected with Wolbachia, showed higher frequencies of males and reproduced sexually. However, we also found a female‐only, Wolbachia‐infected population on agricultural fields in north‐eastern United States. All Wolbachia‐infected populations were infected with the same Wolbachia strain. At this moment, we do not have a convincing explanation for this deviation from the general pattern of ecology and reproductive mode. It may be that asparagus agricultural fields differ from other crop fields in ways that favour sexual reproduction. Alternatively, Wolbachia may manipulate life history traits in its host, resulting in different fitness pay‐offs in different habitats. The fixation of Wolbachia in the United States populations (where the species was introduced) may be due to founder effect and lack of uninfected, sexual source populations.     

Vertical and horizontal transmission drive bacterial invasion

A huge variety of Arthropod species is infected with endosymbiotic Wolbachia bacteria that manipulate their host’s reproduction to invade populations. In addition to vertical transmission from mother to offspring through the egg cytoplasm, it has been demonstrated through phylogenetic analyses and natural transfer experiments that horizontal transmission of Wolbachia (i.e. contagion) can occur between Arthropod hosts. More recently, factors influencing horizontal transfer have also been explored. While it is clear that horizontal transmission between species plays a major role in the evolutionary history of Wolbachia infections among insects, its role in the spread of a new infection through a host population, notably through within‐species transfers, remained unknown. In this issue of Molecular Ecology, Kraaijeveld et al. (2011) present the first evidence that horizontal transmission played a key role in the early spread of parthenogenesis‐inducing Wolbachia through the parasitoid wasp Leptopilina clavipes. To support their finding, the authors studied genetic variation in three types of markers, including host nuclear DNA, mitochondrial DNA and Wolbachia DNA. Specifically, they examined potential associations between their diversity patterns. No diversity was detected in Wolbachia genes, indicating that a single Wolbachia strain must have infected and spread through L. clavipes. In addition, a correlation between substantial variation in mitochondrial and nuclear genotypes suggested that horizontal transmission played an important role in the current clonal genetic variation in this wasp. Such horizontal transmission could be facilitated by a specific host ecology (e.g. parasitoid wasps sharing the same host resource) and potentially impact co‐evolution between host and symbiont.     

Wolbachia affects survival to different oxidative stressors dependent upon the genetic background in Drosophila melanogaster

It is well known that the rate of ageing varies among individuals dependent on the genetic background. In the present study, we explore how Wolbachia infection (a common insect endosymbiont bacterium) and oxidative stress interact in ageing with respect to two different genetic backgrounds of Drosophila melanogaster. Naturally infected and cured lines of Drosophila are challenged with either paraquat or l ‐arginine to generate two different types of oxidative stress. We first observe that removing Wolbachia infection shortens the lifespan in one genetic background but not in the other. Wolbachia infection only makes one of the genetic lines more sensitive to paraquat. However, only the line unaffected by Wolbachia in the paraquat treatment is protected by Wolbachia from l ‐arginine induced stress. Hence, Wolbachia is modifying free radical defence via two different mechanisms dependent on the genetic background. This supports the idea that factors that can govern ageing (infection and oxidative stress) are not universal, but are specific to the genetic make‐up of an individual.