Bidirectional incompatibility among divergent Wolbachia and incompatibility level differences among closely related Wolbachia in Nasonia

Most insect groups harbor obligate bacterial symbionts from the alpha-proteobacterial genus Wolbachia. These bacteria alter insect reproduction in ways that enhance their cytoplasmic transmission. One of the most common alterations is cytoplasmic incompatibility (CI) - a post-fertilization modification of the paternal genome that renders embryos inviable or unable to complete diploid development in crosses between infected males and uninfected females or infected females harboring a different strain. The parasitic wasp species complex Nasonia (N. vitripennis, N. longicornis and N. giraulti) harbor at least six different Wolbachia that cause CI. Each species have double infections with a representative from both the A and B Wolbachia subgroups. CI relationships of the A and B Wolbachia of N. longicornis with those of N. giraulti and N. vitripennis are investigated here. We demonstrate that all pairwise crosses between the divergent A strains are bidirectionally incompatible. We were unable to characterize incompatibility between the B Wolbachia, but we establish that the B strain of N. longicornis induces no or very weak CI in comparison to the closely related B strain in N. giraulti that expresses complete CI. Taken together with previous studies, we show that independent acquisition of divergent A Wolbachia has resulted in three mutually incompatible strains, whereas codivergence of B Wolbachia in N. longicornis and N. giraulti is associated with differences in CI level. Understanding the diversity and evolution of new incompatibility strains will contribute to a fuller understanding of Wolbachia invasion dynamics and Wolbachia-assisted speciation in certain groups of insects.     

Widespread Wolbachia infection in terrestrial isopods and other crustaceans

Wolbachia bacteria are obligate intracellular alpha-Proteobacteria of arthropods and nematodes. Although widespread among isopod crustaceans, they have seldom been found in non-isopod crustacean species. Here, we report Wolbachia infection in fourteen new crustacean species. Our results extend the range of Wolbachia infections in terrestrial isopods and amphipods (class Malacostraca). We report the occurrence of two different Wolbachia strains in two host species (a terrestrial isopod and an amphipod). Moreover, the discovery of Wolbachia in the goose barnacle Lepas anatifera (subclass Thecostraca) establishes Wolbachia infection in class Maxillopoda. The new bacterial strains are closely related to B-supergroup Wolbachia strains previously reported from crustacean hosts. Our results suggest that Wolbachia infection may be much more widespread in crustaceans than previously thought. The presence of related Wolbachia strains in highly divergent crustacean hosts suggests that Wolbachia endosymbionts can naturally adapt to a wide range of crustacean hosts. Given the ability of isopod Wolbachia strains to induce feminization of genetic males or cytoplasmic incompatibility, we speculate that manipulation of crustacean-borne Wolbachia bacteria might represent potential tools for controlling crustacean species of commercial interest and crustacean or insect disease vectors.     

Wolbachia in the Culex pipiens group mosquitoes: introgression and superinfection

Wolbachia bacteria in mosquitoes induce cytoplasmic incompatibility (CI), where sperm from Wolbachia-infected males can produce inviable progeny. The wPip strain in the Culex pipiens group of mosquitoes produces a complexity of CI crossing types. Several factors are thought to be capable of influencing the expression of CI including Wolbachia strain type and host genotype. In this study, the unidirectional CI that occurs between 2 C. pipiens complex laboratory strains, Col and Mol, was further investigated by nuclear genotype introgression. The unidirectional CI between Col and Mol was not found to be influenced by host genetic background, in contrast to a previous introgression study carried out using bidirectionally incompatible C. pipiens group strains. A line containing both wPip strain variants superinfection was also generated by embryonic cytoplasmic transfer. The same crossing type as the parental Col strain was observed in the superinfected line. Quantitative polymerase chain reaction demonstrated a low density of the injected wPipMol variant in the superinfected line after 18 generations, which was considered likely to be responsible for the crossing patterns observed. The Wolbachia density was also shown to be lower in the parental Mol strain males compared with Col strain males, and no inverse relationship between WO phage and Wolbachia density could be detected.     

Many compatible Wolbachia strains coexist within natural populations of Culex pipiens mosquito

Maternally inherited Wolbachia often manipulate the reproduction of arthropods to promote their transmission. In most species, Wolbachia exert a form of conditional sterility termed cytoplasmic incompatibility (CI), characterized by the death of embryos produced by the mating between individuals with incompatible Wolbachia infections. From a theoretical perspective, no stable coexistence of incompatible Wolbachia infections is expected within host populations and CI should induce the invasion of one strain or of a set of compatible strains. In this study, we investigated this prediction on CI dynamics in natural populations of the common house mosquito Culex pipiens. We surveyed the Wolbachia diversity and the expression of CI in breeding sites of the south of France between 1990 and 2005. We found that geographically close C. pipiens populations harbor considerable Wolbachia diversity, which is stably maintained over 15 years. We also observed a very low frequency of infertile clutches within each sampled site. Meanwhile, mating choice experiments conducted in laboratory conditions showed that assortative mating does not occur. Overall, this suggests that a large set of compatible Wolbachia strains are always locally dominant within mosquito populations thus, fitting with the theoretical expectations on CI dynamics.     

Wolbachia infections and superinfections in cytoplasmically incompatible populations of the European cherry fruit fly Rhagoletis cerasi (Diptera,Tephritidae)

Wolbachia is an obligately intracellular, maternally inherited bacterium which has been detected in many arthropods. Wolbachia infections disperse in host populations by mechanisms such as cytoplasmic incompatibility (CI). CI leads to embryonic mortality which occurs when infected males mate with uninfected females or females with a different Wolbachia strain. Populations of the European cherry fruit fly Rhagoletis cerasi (Diptera, Tephritidae) were found to be infected by two different Wolbachia strains, wCer1 and wCer2. Superinfections with both strains occurred throughout southern and central Europe and infections with wCer1 were found in northern, western and eastern Europe. Strong unidirectional CI between European populations of R. cerasi were first reported in the 1970s. From the conformity in the recent geographical distribution of the Wolbachia infections and the CI expression patterns found 25 years ago it was deduced that wCer2 potentially causes CI in R. cerasi. The comparison of the geographical distributions indicated that wCer1 + 2 must have spread into wCer1-infected populations in some areas. In other regions, a spread of wCer1 + 2 was probably prevented by dispersal barriers. There, a sharp transition from infected to superinfected populations suggested regional isolation between wCer1 and wCer1 + 2-infected populations.     

Evolution of Wolbachia-induced cytoplasmic incompatibility in Drosophila simulans and D. sechellia

Abstract.— The intracellular bacterium Wolbachia invades arthropod host populations through various mechanisms, the most common of which being cytoplasmic incompatibility (CI). CI involves elevated embryo mortality when infected males mate with uninfected females or females infected with different, incompatible Wolbachia strains. The present study focuses on this phenomenon in two Drosophila species: D. simulans and D. sechellia . Drosophila simulans populations are infected by several Wolbachia strains, including w Ha and w No. Drosophila sechellia is infected by only two Wolbachia : w Sh and w Sn. In both Drosophila species, double infections with Wolbachia are found. As indicated by several molecular markers, w Ha is closely related to w Sh, and w No to w Sn. Furthermore, the double infections in the two host species are associated with closely related mitochondrial haplotypes, namely si I (associated with w Ha and w No in D. simulans ) and se (associated with w Sh and w Sn in D. sechellia ). To test the theoretical prediction that Wolbachia compatibility types can diverge rapidly, we injected w Sh and w Sn into D. simulans , to compare their CI properties to those of their sister strains w Ha and w No, respectively, in the same host genetic background. We found that within each pair of sister strains CI levels were similar and that sister strains were fully compatible. We conclude that the short period for which the Wolbachia sister strains have been evolving separated from each other was not sufficient for their CI properties to diverge significantly.     

Incompatible insect technique: incompatible males from a Ceratitis capitata genetic sexing strain

Wolbachia are obligatory intracellular and maternally inherited bacteria that infect and spread through natural arthropod populations by inducing male-killing, feminization, parthenogenesis, and, most commonly, unidirectional and bidirectional cytoplasmic incompatibility (CI). Cytoplasmic incompatibility can be used to control natural populations of insect pests, in a way analogous to the Sterile Insect Technique (SIT), namely through the Incompatible Insect Technique (IIT). For the successful application of the IIT (based on a unidirectional CI approach) against a target species, it is essential that only males are released, as the release of females would lead to fertile matings between the released males and the released females and the establishment of a Wolbachia -carrying field population. In the present study, we describe a Wolbachia -infected line of the VIENNA 8 genetic sexing strain of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), that carries the selectable marker temperature sensitive lethal ( tsl ). We show that (1) transferred Wolbachia induce high levels of CI even after the temperature treatment required for the male-only production, and (2) the Wolbachia -infected genetic sexing C. capitata line can be used in cage population suppression experiments analogous to the SIT. We also discuss our results in a comparison between IIT and SIT, investigating whether irradiation and cytoplasmic factors can be combined toward the development of novel strategies for insect pest control.     

Molecular evidence for single Wolbachia infections among geographic strains of the flour beetle Tribolium confusum.

Infections with the rickettsial microorganism Wolbachia are cytoplasmically inherited and occur in a wide range of insect species and several other arthropods. Wolbachia infection often results in unidirectional cytoplasmic incompatibility (CI): crosses between infected males and uninfected females are incompatible and show a reduction of progeny or complete inviability. Unidirectional CI can also occur when males harbouring two incompatible Wolbachia strains are crossed with females infected with only one of the two strains. In the flour beetle Tribolium confusum, Wolbachia infections are of particular interest because of the severity of incompatibility. Typically, no progeny results from the incompatible cross, whereas only partial incompatibility is observed in most other hosts. Werren et al. (1995a) reported that Wolbachia infections in T. confusum consist of two bacterial strains belonging to distinct phylogenic groups, based on PCR amplification and sequence analysis of the bacterial cell division gene ftsZ. However, Fialho & Stevens (1996) showed that eight strains of T. confusum were infected with a single and common incompatibility type. Here we report analysis of the ftsZ gene by specific PCR amplification. Diagnostic restriction enzyme assays revealed no evidence of double infections in 11 geographic strains of T. confusum, including the strain examined by Werren et al. (1995a). Further, sequence analysis of the Wolbachia ftsZ gene and an internal transcribed spacer (ITS) region in two of these strains displayed no nucleotide variation or evidence of polymorphisms. Results suggest that T. confusum is infected with B-group Wolbachia only.     

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

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

Wolbachia affects oviposition and mating behaviour of its spider mite host

Wolbachia bacteria are transmitted from mother to offspring via the cytoplasm of the egg. When mated to males infected with Wolbachia bacteria, uninfected females produce unviable offspring, a phenomenon called cytoplasmic incompatibility (CI). Current theory predicts that ‘sterilization’ of uninfected females by infected males confers a fitness advantage to Wolbachia in infected females. When the infection is above a threshold frequency in a panmictic population, CI reduces the fitness of uninfected females below that of infected females and, consequently, the proportion of infected hosts increases. CI is a mechanism that benefits the bacteria but, apparently, not the host. The host could benefit from avoiding incompatible mates. Parasite load and disease resistance are known to be involved in mate choice. Can Wolbachia also be implicated in reproductive behaviour? We used the two‐spotted spider mite – Wolbachia symbiosis to address this question. Our results suggest that uninfected females preferably mate to uninfected males while infected females aggregate their offspring, thereby promoting sib mating. Our data agrees with other results that hosts of Wolbachia do not necessarily behave as innocent bystanders – host mechanisms that avoid CI can evolve.     

Evolution and invasion dynamics of multiple infections with Wolbachia investigated using matrix based models

Endosymbiotic bacteria are often transmitted vertically from one host generation to the next via oocytes cytoplasm. The generally small number of colonizing bacteria in the oocytes leads to a bottleneck at each generation, resulting in genetic homogenization of the symbiotic population. Nevertheless, in many of the species infected by Wolbachia (maternally transmitted bacteria), individuals do sometimes simultaneously harbor several bacterial strains, owing to the fact that Wolbachia induces cytoplasmic incompatibility (CI) that maintains multiple infections. CI occurs in crosses in which the male is infected by at least one Wolbachia strain that the female lacks, and consequently it favors individuals with the greatest symbiotic diversity. CI results in death of offspring in diploid species. In haplodiploid individuals, unfertilized eggs hatch normally into males and fertilized ones, which would lead to females, either die (female mortality type: FM) or develop into males (male development type: MD). Until now, only one theoretical study, restricted to diploid species, has investigated the associations where multiple CI-inducing Wolbachia co-exist, and explored the conditions under which multiple infections can spread. The consequences of double infections on Wolbachia maintenance in host populations, and the selective pressures to which it is subjected have not yet been analysed. Here, we have re-written a model previously developed for single infection in matrix form, which allows easy extension to multiple infections and introduction of mutant strains. We show that (i) the CI type has a strong influence on invasiveness and maintenance of multiple infections; (ii) double infection lowers the invasion threshold of less competitive strains that hitch-hike with their companion strain; (iii) when multiple infections occur, as in single infections, the strains selected are those which maximize the production of infected offspring; and (iv) for the MD CI type, invasion of mutant strains can carry the whole infection to extinction.     

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.     

Fitness advantage and cytoplasmic incompatibility in Wolbachia single- and superinfected Aedes albopictus

Wolbachia are obligate, maternally inherited, intracellular bacteria that infect numerous insects and other invertebrates. Wolbachia infections have evolved multiple mechanisms to manipulate host reproduction and facilitate invasion of naive host populations. One such mechanism is cytoplasmic incompatibility (CI) that occurs in many insect species, including Aedes albopictus (Asian tiger mosquito). The multiple Wolbachia infections that occur naturally in A. albopictus make this mosquito a useful system in which to study CI. Here, experiments employ mosquito strains that have been introgressed to provide genetically similar strains that harbor differing Wolbachia infection types. Cytoplasmic incompatibility levels, host longevity, egg hatch rates, and fecundity are examined. Crossing results demonstrate a pattern of additive unidirectional cytoplasmic incompatibility. Furthermore, relative to uninfected females, infected females are at a reproductive advantage due to both cytoplasmic incompatibility and a fitness increase associated with Wolbachia infection. In contrast, no fitness difference was observed in comparisons of single- and superinfected females. We discuss the observed results in regard to the evolution of the Wolbachia/A. albopictus symbiosis and the observed pattern of Wolbachia infection in natural populations.     

Wolbachia distribution and cytoplasmic incompatibility based on a survey of 42 spider mite species (Acari: Tetranychidae) in Japan

Wolbachia are a group of maternally inherited bacteria that infect a wide range of arthropods. Wolbachia infections are known to result in the expression of various abnormal reproductive phenotypes, the best known being cytoplasmic incompatibility. The first systematic survey of 42 spider mite species in Japan revealed that seven species (16.7%) were infected with Wolbachia. Wolbachia in the spider mites were grouped into three subgroups in supergroup B by phylogenetic analyses of the wsp gene. Most spider mites did not show cytoplasmic incompatibility when infected males were crossed with uninfected females. However, all infected populations of Panonychus mori and Oligonychus gotohi (five and four populations, respectively) possessed modification-positive strains of Wolbachia, and the cytoplasmic incompatibility decreased egg hatchability and female ratio of the spider mites. Thus, some Wolbachia strains cause sex ratio distortion in their hosts.     

<Emphasis Type="Italic">Wolbachia</Emphasis> Are Present in Southern African Scorpions and Cluster with Supergroup F

The presence and distribution of the intracellular bacteria Wolbachia in the arthropod subphylum Chelicerata (including class Arachnida) has not been extensively explored. Here we report the discovery of Wolbachia in scorpions. Five strains found in host species of the genus Opistophthalmus (Southern African burrowing scorpions) have been characterized by Multilocus Sequence Typing and by Wolbachia Surface Protein. Phylogenetic analyses indicate clustering in the supergroup F and a high genetic relatedness among all scorpion strains as a result of a potential transmission within the host genus. The F-group is an uncommon lineage compared to the A and B supergroups, although it is present in a broad range of hosts (including insects, filarial nematodes, and now arachnids) and across a large geographical area (e.g., North America, Africa, Europe, and Australia). It also shows no evidence of recombination and has a significantly higher genetic diversity than supergroup A and B. Overall, this pattern suggests an older radiation of F-strains with respect to A and B-strains, followed by limited horizontal transmission across host genera and reduced genetic flux among strains. A more extensive sampling of supergroup F-strains is required to confirm this scenario.     

Variability and expression of ankyrin domain genes in Wolbachia variants infecting the mosquito Culex pipiens

Wolbachia strains are maternally inherited endosymbiotic bacteria that infect many arthropod species and have evolved several different ways of manipulating their hosts, the most frequent way being cytoplasmic incompatibility (CI). CI leads to embryo death in crosses between infected males and uninfected females as well as in crosses between individuals infected by incompatible Wolbachia strains. The mosquito Culex pipiens exhibits the highest crossing type variability reported so far. Our crossing data support the notion that CI might be driven by at least two distinct genetic units that control the CI functions independently in males and females. Although the molecular basis of CI remains unknown, proteins with ankyrin (ANK) domains represent promising candidates since they might interact with a wide range of host proteins. Here we searched for sequence variability in the 58 ANK genes carried in the genomes of Wolbachia variants infecting Culex pipiens. Only five ANK genes were polymorphic in the genomes of incompatible Wolbachia variants, and none correlated with the CI pattern obtained with 15 mosquito strains (representing 14 Wolbachia variants). Further analysis of ANK gene expression evidenced host- and sex-dependent variations, which did not improve the correlation. Taken together, these data do not support the direct implication of ANK genes in CI determinism.     

Host genotype determines cytoplasmic incompatibility type in the haplodiploid genus Nasonia

In haplodiploid species, Wolbachia-induced cytoplasmic incompatibility (CI) can be expressed in one of two ways: as a "conversion" of diploid fertilized eggs into haploid males or as embryonic mortality. Here we describe CI-type variation within the parasitic wasp genus Nasonia and genetically analyze the basis of this variation. We reach four main conclusions: (i) CI is expressed primarily as conversion in N. vitripennis, but as embryonic mortality in the sibling species N. giraulti and N. longicornis; (ii) the difference in CI type between N. giraulti (mortality) and N. vitripennis (conversion) is determined by host nuclear genotype rather than by Wolbachia differences; (iii) N. vitripennis "conversion genes" are recessive in hybrid females; and (iv) a difference in CI level between the sibling species N. giraulti and N. longicornis is due to the different Wolbachia infections in the species rather than to the host genotype. These results show that host nuclear genes can influence the type of CI present in a species. On the basis of these findings, we propose a model for how different CI types evolve in haplodiploids due to selection on nuclear genes modifying CI.     

Influence of aging on cytoplasmic incompatibility, sperm modification and Wolbachia density in Culex pipiens mosquitoes

Wolbachia are maternally inherited endocellular bacteria, widespread in invertebrates and capable of altering several aspects of host reproduction. Cytoplasmic incompatibility (CI) is commonly found in arthropods and induces hatching failure of eggs from crosses between Wolbachia-infected males and uninfected females (or females infected by incompatible strains). Several factors such as bacterial and host genotypes or bacterial density contribute to CI strength and it has been proposed, mostly from Drosophila data, that older males have a lower Wolbachia load in testes which, thus, induces a lighter CI. Here, we challenge this hypothesis using different incompatible Culex pipiens mosquito strains and show that CI persists at the same intensity throughout the mosquito life span. Embryos from incompatible crosses showed even distributions of abortive phenotypes over time, suggesting that host ageing does not reduce the sperm-modification induced by Wolbachia. CI remained constant when sperm was placed in the spermathecae of incompatible females, indicating that sperm modification is also stable over time. The capacity of infected females to rescue CI was independent of age. Last, the density of Wolbachia in whole testes was highly strain-dependent and increased dramatically with age. Taken together, these data stress the peculiarity of the C.pipiens/Wolbachia interaction and suggest that the bacterial dosage model should be rejected in the case of this association.     

Multiple infections and diversity of cytoplasmic incompatibility in a haplodiploid species

Cytoplasmic incompatibility (CI) is a sperm-egg incompatibility commonly induced by the intracellular endosymbiont bacterium Wolbachia that, in diploid species, results in embryo mortality. In haplodiploid species, two types of CI exist depending on whether the incompatible fertilized eggs develop into males (male development (MD)) or abort (female mortality (FM)). CI allows multiple infections to be maintained in host populations, and thus allows interactions to occur between co-infecting strains. In Leptopilina heterotoma, three Wolbachia strains coexist naturally (wLhet1, wLhet2, wLhet3). When these three strains are all present, they induce a CI of FM type, whereas wLhet1 alone expresses a CI phenotype intermediate between MD and FM. Here, we compare CI effects in crosses involving insect lines sharing the same nuclear background, but harboring different mixtures of strains. Mating experiments showed that: (i) wLhet2 and wLhet3 also induce an intermediate CI when acting alone, and show a bidirectional incompatibility; (ii) there is no interaction between the co-infecting strains in CI expression; (iii) the diversity of Wolbachia present within a male host influences the expression of CI: an increase in the number of strains is correlated with a decrease in the proportion of the MD type, which is also correlated with an increase in bacterial density. All these data suggest that the CI of FM type results from a stronger effect than the MD type, which conflicts with the conventional hypotheses used to explain CI diversity in haplodiploids, and could provide some new information about CI mechanisms in insects.