Infection density of Wolbachia and level of cytoplasmic incompatibility in the Mediterranean flour moth,Ephestia kuehniella

Wolbachia, a causative agent of various reproductive changes in arthropods, induces cytoplasmic incompatibility (CI) in the Mediterranean flour moth, Ephestia kuehniella. Two strains of E. kuehniella, Yokohama and Tsuchiura, harbor closely related Wolbachia, but the Yokohama strain expresses stronger CI than the Tsuchiura strain. A transinfected E. kuehniella strain that harbors the Wolbachia derived from the almond moth Cadra cautella, expresses weak CI at a similar level to the Tsuchiura strain. In the present study, we measured the Wolbachia density in the testis of the three E. kuehniella strains in order to examine the effects of bacterial strain and infection load on the expression of CI. When individuals of the same strain were compared, a correlation of bacterial density to CI level was observed. In addition, the Wolbachia density was higher in the Yokohama strain than the Tsuchiura strain in agreement with the CI levels expressed. However, this relationship did not hold in the comparison between the naturally infected and transinfected strains that carried phylogenetically distant Wolbachia.     

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.     

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.     

Coexistence of cytoplasmic incompatibility and male-killing-inducing endosymbionts, and their impact on host gene flow

Male-killing (MK) and cytoplasmic incompatibility (CI) inducing bacteria are among the most common endosymbionts of arthropods. Previous theoretical research has demonstrated that these two types of endosymbionts cannot stably coexist within a single unstructured host population if no doubly infected host individuals occur. Here, we analyse a model of two host subpopulations connected by migration. We demonstrate that coexistence of MK- and CI-inducing endosymbionts is possible if migration rates are sufficiently low. In particular, our results suggest that for coexistence to be possible, migration rates into the subpopulation infected predominantly with MK-inducing endosymbionts must be considerably low, while migration rates from the MK- to the CI-infected subpopulation can be very high. We also analyse how the presence of MK- and CI-inducing endosymbionts affects host gene flow between the two subpopulations. Employing the concept of the 'effective migration rate', we demonstrate that compared with an uninfected subdivided population, gene flow is increased towards the MK-infected island, but decreased towards the CI-infected island. We discuss our results with respect to the butterfly Hypolimnas bolina, in which infection polymorphism of CI- and MK-inducing Wolbachia has been reported across South-Pacific island populations.     

Intra-individual coexistence of a Wolbachia strain required for host oogenesis with two strains inducing cytoplasmic incompatibility in the wasp Asobara tabida

Cytoplasmically inherited symbiotic Wolbachia bacteria are known to induce a diversity of phenotypes on their numerous arthropod hosts including cytoplasmic incompatibility, male-killing, thelytokous parthenogenesis, and feminization. In the wasp Asobara tabida (Braconidae), in which all individuals harbor three genotypic Wolbachia strains (wAtab1, wAtab2 and wAtab3), the presence of Wolbachia is required for insect oogenesis. To elucidate the phenotype of each Wolbachia strain on host reproduction, especially on oogenesis, we established lines of A. tabida harboring different combinations of these three bacterial strains. We found that wAtab3 is essential for wasp oogenesis, whereas the two other strains, wAtabl and wAtab2, seem incapable to act on this function. Furthermore, interline crosses showed that strains wAtab1 and wAtab2 induce partial (about 78%) cytoplasmic incompatibility of the female mortality type. These results support the idea that bacterial genotype is a major factor determining the phenotype induced by Wolbachia on A. tabida hosts. We discuss the implications of these findings for current hypotheses regarding the evolutionary mechanisms by which females of A. tabida have become dependent on Wolbachia for oogenesis.     

Wolbachia-induced unidirectional cytoplasmic incompatibility and the stability of infection polymorphism in parapatric host populations

Wolbachia are intracellular, maternally inherited bacteria that are widespread among arthropods and commonly induce a reproductive incompatibility between infected male and uninfected female hosts known as unidirectional cytoplasmic incompatibility (CI). If infected and uninfected populations occur parapatrically, CI acts as a post-zygotic isolation barrier. We investigate the stability of such infection polymorphisms in a mathematical model with two populations linked by migration. We determine critical migration rates below which infected and uninfected populations can coexist. Analytical solutions of the critical migration rate are presented for mainland-island models. These serve as lower estimations for a more general model with two-way migration. The critical migration rate is positive if either Wolbachia causes a fecundity reduction in infected female hosts or its transmission is incomplete, and is highest for intermediate levels of CI. We discuss our results with respect to local adaptations of the Wolbachia host, speciation, and pest control.     

Host genotype changes bidirectional to unidirectional cytoplasmic incompatibility in Nasonia longicornis

Wolbachia are the most abundant maternally inherited endosymbionts of insects and cause various reproductive alterations in their hosts. One such manipulation is cytoplasmic incompatibility (CI), which is a sperm-egg incompatibility typically resulting in zygotic death. Nasonia longicornis (Hymenoptera: Pteromalidae) has an A supergroup and two closely related B supergroup Wolbachia infections. The B supergroup bacteria co-diverged in this host genus. Both triple (wNlonAwNlonB1wNlonB2) and double infections (wNlonAwNlonB1, wNlonAwNlonB2) have been obtained from the field. In the present study, CI was determined among the three Wolbachia types in different host genetic backgrounds. Results show that host genetic background determines whether bidirectional CI or unidirectional CI occurs between the two closely related B group Wolbachia. Results show that the wNlonB1-infected males are bidirectionally incompatible with wNlonB2 in their 'native' nuclear genetic background, whereas wNlonB1 males are compatible with wNlonB2 in two other N. longicornis genetic backgrounds, resulting in unidirectional CI. In contrast, wNlonB2-infected males are incompatible with wNlonB1 females in all three host genetic backgrounds. These changes in incompatibility are not due to the loss of the bacteria. We hypothesize that a repressor gene for sperm modification by wNlonB1 is segregating in N. longicornis populations. The relevance of these findings to the potential role of Wolbachia in host-reproductive divergence and speciation is discussed.     

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.     

Detection of the Wolbachia protein WPIP0282 in mosquito spermathecae: Implications for cytoplasmic incompatibility

Cytoplasmic incompatibility (CI) is a conditional sterility induced by the bacterium Wolbachia pipientis that infects reproductive tissues in many arthropods. Although CI provides a potential tool to control insect vectors of arthropod-borne diseases, the molecular basis for CI induction is unknown. We hypothesized that a Wolbachia-encoded, CI-inducing factor would be enriched in sperm recovered from spermathecae of female mosquitoes. Using SDS-PAGE and mass spectrometry, we detected peptides from the 56 kDa hypothetical protein, encoded by wPip_0282, associated with sperm transferred to females by Wolbachia infected males. We also detected peptides from the same protein in Wolbachia infected ovaries. Homologs of wPip_0282 and the co-transcribed downstream gene, wPip_0283, occur as multiple divergent copies in genomes of CI-inducing strains of Wolbachia. The operon is located in a genomic context that includes mobile genetic elements. The absence of wPip_0282 and wPip_0283 homologs from genomes of Wolbachia in filarial nematodes, as well as other members of the Rickettsiales, suggests a role as a candidate CI effector.     

共生菌Wolbachia在宿主中诱导表达胞质不相容因子的研究进展

沃尔巴克氏体(Wolbachia)作为节肢动物的胞内共生菌,可以引起宿主产生雌性化、孤雌生殖、杀雄和胞质不相容性(cytoplasmic incompatibility, CI) 4种生殖表型。其中CI是最常见的现象,表现为受感染的雄性昆虫与未感染或感染不兼容Wolbachia的雌性昆虫交配时引起胚胎死亡;而雌性感染同种Wolbachia时胚胎能够正常发育。CI是由被称为CI因子(cifA和cifB)的Wolbachia基因对调控的。其中,CifB作为毒剂在雄性中表达诱导产生CI,而CifA作为解毒剂在雌性中表达拯救CI。本文综述了CI因子结构、功能和作用机制的研究,以期为未来利用Wolbachia和CI进行蚊媒疾病和农业虫害的防控奠定基础。     

Partial cytoplasmic incompatibility between two Australian populations of Drosophila melanogaster

Drosophila melanogaster (Meigen) females from stocks collected at Melbourne (latitude 37°S) show partial incompatibility when mated with males from stocks collected at Townsville (latitude 19°S) on the east coast of Australia. The reciprocal cross is compatible. Eggs have reduced hatchability in the incompatible cross. The incompatibility is maternally inherited over three generations. Compatibility can be restored by culturing Townsville flies on medium with tetracycline for one generation and by using 2-week-old Townsville males.

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Incompatibilité cytoplasmique partielle entre deux populations australiennes de Drosophila melanogaster

Résumé Les souches de D. melanogaster récoltées à Melbourne (37°S) et Townsville (19°S) sur la côte Est de l'Australie montrent une incompatibilité partielle lorsque les femelles Melbourne sont accouplées aux mâles Townsville. Une telle incompatibilité n'est décelée, ni dans les croisements intrapopulations, ni dans le croisement réciproque. Le taux d'éclosion des oeufs est réduit d'environ 30% dans le croisement incompatible, mais la viabilité des larves n'est pas modifiée. Les éléments, mâle et femelle, de ce système d'incompatibilité sont hérités maternellement pendant 3 générations de croisements en retour. La compatibilité peut être intégralement rétablie en cultivant pendant une génération la souche Townsville avec un régime contenant de la tétracycline, et partiellement rétablie en utilisant des mâles âgés de 2 semaines.

     

High prevalence of Wolbachia infection does not explain unidirectional cytoplasmic incompatibility of Altica flea beetles

1. Wolbachia are intracellular bacteria found in a wide range of arthropods that can impact reproductive isolation of their hosts. Previous studies showed unidirectional cytoplasmic incompatibility (CI) and high infection rates by Wolbachia in the closely related leaf beetles Altica fragariae and Altica viridicyanea; however, whether this reproductive isolation was induced by Wolbachia remains unclear. 2. This study estimated the prevalence of Wolbachia in Altica beetles, assessed genetic diversity of Wolbachia strains infecting these beetles, and tested whether Wolbachia-induced CI explains reproductive isolation of A. fragariae and A. viridicyanea. 3. The results show that all of the 11 tested Altica species were infected by Wolbachia, and the infection rate was as high as 97.0% (n = 235). Multi-strain infections were common, being found in 10 of the 11 species tested and accounting for 23.0% of all screened specimens. In total, 35 Wolbachia strains were identified based on 208 wsp sequences obtained. Although a majority of A. fragariae and A. viridicyanea individuals from the Beijing population were infected with only one strain each, multi-strain infections did occur in both species. Antibiotic curing experiments did not change hatching success in either inter- or intraspecific crosses of A. fragariae and A. viridicyanea, indicating that these Wolbachia strains do not induce CI. These results were further corroborated by the lack of the Wolbachia cifB gene, which is responsible for causing CI. 4. These findings suggest that high prevalence of Wolbachia infection is unlikely to contribute to reproductive isolation and speciation in this system.     

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

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

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.     

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.     

<Emphasis Type="Italic">Wolbachia</Emphasis>-induced cytoplasmic incompatibility in Japanese populations of <Emphasis Type="Italic">Tetranychus urticae</Emphasis> (Acari: Tetranychidae)

Intracellular bacteria of the genus Wolbachia (alpha Proteobacteria) induce cytoplasmic incompatibility (CI) in many arthropod species, including spider mites, but not all Wolbachia cause CI. In spider mites CI becomes apparent by a reduced egg hatchability and a lower daughter:son ratio: CI in haplodiploid organisms in general was expected to produce all-male offspring or a male-biased sex ratio without any death of eggs. In a previous study of Japanese populations of Tetranychus urticae, two out of three green-form populations tested were infected with non-CI Wolbachia strains, whereas none of six red-form populations harbored Wolbachia. As the survey of Wolbachia infection in T. urticae is still fragmentary in Japan, we checked Wolbachia infection in thirty green-form populations and 29 red-form populations collected from a wide range of Japanese islands. For Wolbachia-infected populations, we tested the effects of Wolbachia on the reproductive traits and determined the phylogenetic relationships of the different strains of Wolbachia. All but one green-form populations were infected with Wolbachia and all strains belonged to the subgroup Ori when the wsp gene was used to determine the phylogenetic relationships of different strains of Wolbachia. Six out of 29 red-form populations harbored Wolbachia and the infected strains belonged to the subgroups Ori and Bugs. Twenty-four of 29 infected green-form populations and five of six infected red-form populations induced CI among the hosts. Thus, CI-Wolbachia strains are widespread in Japan, and no geographical trend was observed in the CI-Wolbachia. Although three red-form populations harbored other intracellular bacteria Cardinium, they did not affect host reproduction.     

Pervasive associations between Cybaeus spiders and the bacterial symbiont Cardinium

Cardinium is a recently discovered maternally transmitted bacterial endosymbiont in the Bacteroidetes that has thus far been documented in five arthropod orders. While its effects on his hosts are largely unknown, a few strains have been shown to manipulate host reproduction in parasitic wasps and in mites, either by transforming males into females, or by causing mating incompatibilities between infected males and uninfected males. Cardinium has recently been reported to be widespread in spiders, and in this study, we document pervasive infections in Cybaeus spiders, which are some of the most abundant yet understudied spiders in the understory of moist Western North American forests. 12/20 species, as well as 96% of individuals in a local population of Cybaeus signifer were infected. Phylogenetic analysis revealed three closely related symbiont haplotypes within Cybaeus. Haplotypes clustered within geographically close species, suggesting that horizontal transmission might be quite high in this symbiont lineage.     

Monitoring long-term evolutionary changes following Wolbachia introduction into a novel host: the Wolbachia popcorn infection in Drosophila simulans

Wolbachia may act as a biological control agent for pest management; in particular, the Wolbachia variant wMelPop (popcorn) shortens host longevity and may be useful for dengue suppression. However, long-term changes in the host and Wolbachia genomes can alter Wolbachia spread and/or host effects that suppress disease. Here, we investigate the phenotypic effects of wMelPop in a non-native host, Drosophila simulans, following artificial transinfection approximately 200 generations ago. Long-term rearing and maintenance of the bacteria were at 19°C in the original I-102 genetic background that was transinfected with the popcorn strain. The bacteria were then introgressed into three massbred backgrounds, and tetracycline was used to create uninfected sublines. The effect of wMelPop on longevity in this species appears to have changed; longevity was no longer reduced at 25°C in some nuclear backgrounds, reflecting different geographical origin, selection or drift, although the reduction was still evident for flies held at 30°C. Wolbachia influenced productivity and viability, and development time in some host backgrounds. These findings suggest that long-term attenuation of Wolbachia effects may compromise the effectiveness of this bacterium in pest control. They also emphasize the importance of host nuclear background on Wolbachia phenotypic effects.