Within-species diversity of Wolbachia-induced cytoplasmic incompatibility in haplodiploid insects

Wolbachia-induced cytoplasmic incompatibility (CI) can have two consequences in haplodiploid insects: fertilized eggs either die (female mortality, FM) or they develop into haploid males (male development, MD). Origin of this diversity remains poorly understood, but current hypotheses invoke variation in damage suffered by paternal chromosomes in incompatible eggs, thus intermediate CI types should be expected. Here, we show the existence of such a particular CI type. In the parasitoid wasp Leptopilina heterotoma, we compared CI effects in crosses involving lines derived from a single inbred line with various Wolbachia infection statuses (natural tri-infection, mono-infection, or no infection). Tri-infected males induce a FM CI type when crossed with either uninfected or mono-infected females. Crossing mono-infected males with uninfected females results in almost complete CI with both reduced offspring production, indicating partial mortality of fertilized eggs, and increased number of sons, showing haploid male development of others. Mono-infected males thus induce an intermediate Cl type when mated with uninfected females. The first evidence of this expected particular CI type demonstrates that no discontinuity separates MD and FM CI types, which appear to be end points of a phenotypic continuum. Second, different CI types can occur within a given species and even within offspring of a single pair. Third, phenotypic expression of the particular CI type induced by a given Wolbachia variant depends on other bacterial variants that co-infect the same tissues. These results support the idea that haplodiploids should be helpful in clarifying evolutionary pathways of insect-Wolbachia associations.     

Prevailing triple infection with Wolbachia in Callosobruchus chinensis (Coleoptera: Bruchidae)

Prevailing triple infection with three distinct Wolbachia strains was identified in Japanese populations of the adzuki bean beetle, Callosobruchus chinensis. When a polymerase chain reaction (PCR) assay was conducted using universal primers for ftsZ and wsp, Wolbachia was detected in all the individuals examined, 288 males and 334 females from nine Japanese populations. PCR-restriction fragment length polymorphism (RFLP) analysis of cloned wsp gene fragments from single insects revealed that three types of wsp sequences coexist in the insects. Molecular phylogenetic analysis of the wsp sequences unequivocally demonstrated that C. chinensis harbours three phylogenetically distinct Wolbachia, tentatively designated as wBruCon, wBruOri and wBruAus, respectively. Diagnostic PCR analysis using specific primers demonstrated that, of 175 males and 235 females from nine local populations, infection frequencies with wBruCon, wBruOri and wBruAus were 100%, 96.3% and 97.0%, respectively. As for the infection status of individuals, triple infection (93.7%) dominated over double infection (6.1%) and single infection (0.2%). The amounts of wBruCon, wBruOri and wBruAus in field-collected adult insects were analysed by using a quantitative PCR technique in terms of wsp gene copies per individual insect. Irrespective of original populations, wBruCon and wBruOri (107 -108 wsp copies/insect) were consistently greater in amount than wBruAus (106 -107 wsp copies/insect), suggesting that the population sizes of the three Wolbachia strains are controlled, although the mechanism is unknown. Mating experiments suggested that the three Wolbachia cause cytoplasmic incompatibility at different levels of intensity.     

Wolbachia与昆虫精卵细胞质不亲和

Wolbachia是广泛分布在昆虫体内的一类共生菌,能通过多种机制调节宿主的生殖方式,包括诱导宿主精卵细胞质不亲和(CI)、孤雌生殖、雌性化、杀雄等,其中细胞质不亲和为最普遍的表型,即感染Wolbachia的雄性和未感染或感染不同品系Wolbachia的雌性宿主交配后,受精卵不能正常发育,在胚胎期死亡。多数CI胚胎在第1次分裂时,来自父本的染色质浓缩缺陷,导致父本遗传物质无法正常分配到子细胞中,因而引起胚胎死亡。守门员模型认为,产生CI可能需要有两种因子,其中之一使得精子发生修饰改变,导致受精后雄性原核发育滞后。第2种因子可能与Wolbachia的原噬菌体有关,在胚胎发育后期导致胚胎死亡。近期的研究已发现,在Wolbachia感染的宿主中,一些与生殖细胞发生和繁殖相关基因的表达发生了显著改变,Wolbachia可能因此对宿主的生殖产生重大影响,进而导致CI的产生。本文主要综述了CI的细胞学表型、解释CI的模型及其分子机理,向读者展示一个小小的细菌是如何通过精妙的策略影响昆虫宿主的繁殖,从而实现其自身的生存和传播的。     

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.     

The evolution of endosymbiont density in doubly infected host species

Multiple infection of individual hosts with several species or strains of maternally inherited endosymbionts is commonly observed in animals, especially insects. Here, we address theoretically the effect of co-infection on the optimal density of the endosymbionts in doubly infected hosts. Our analysis is based on the observation that a maternally inherited double infection is only stable if doubly infected females produce more doubly infected daughters than singly infected or uninfected females produce daughters. We consider both a general model and a model involving two endosymbionts inducing bidirectional cytoplasmic incompatibility (CI). We demonstrate that the optimal replication rate of endosymbionts in doubly infected hosts can be expected to be similar to or below the optimal replication rate in singly infected hosts. This is in contrast to some theoretical predictions for horizontally transmitted parasites and stems from the two strains of endosymbionts having coupled fitness. We discuss our results with respect to recent empirical results on endosymbiont densities, the evolution of CI-inducing bacteria and, more generally, the evolution of cooperation through direct fitness benefits.     

Wolbachia transmission in a naturally bi-infected Drosophila simulans strain from New-Caledonia

Wolbachia are maternally-transmitted endocellular bacteria infecting several arthropod species. In order to study the possibility of Wolbachia segregation in a naturally bi-infected host, isofemale lines from a bi-infected Drosophila simulans (Sturtevant) strain from Nouméa (New Caledonia) were backcrossed using uninfected males carrying the same nuclear background. Uninfected males were used to avoid the cytoplasmic incompatibility syndrome (CI) associated with the presence of Wolbachia in males. Each line was established using a female infected simultaneously by the two different Wolbachia variants wHa and wNo. The backcross led to some individuals carrying only one type of infection being recovered among the progeny of the bi-infected foundress females. Rarely, uninfected individuals were also recovered. Isolated for the first time in its natural host, wNo exhibited a significantly weaker CI phenotype than the isolated wHa variant. Infection fate when backcross conditions were relaxed varied depending on rearing conditions of the host. Under favourable conditions, the infection was generally maintained, while it was frequently lost under unfavourable conditions. This result probably reflects the direct fitness dependence of the symbiont on its host.     

Molecular evidence for multiple infections of a new subgroup of Wolbachia in the European raspberry beetle Byturus tomentosus

Wolbachia, a group of maternally inherited intracellular parasitic bacteria, alter host reproduction, including the induction of thelytokous parthenogenesis, feminization of genetic males, son killing and, most commonly, the induction of cytoplasmic incompatibility (CI), in a diverse array of arthropods. CI can result in infertility and has attracted attention because of its potential in biological control and as an agent in speciation. Although there has been some analysis of overall infection rates in arthropods and within individual insect orders, there has been little exploration of within-species variation. In this study, primers specific for the ftsZ gene of Wolbachia were used to amplify it from different geographical samples of the European raspberry beetle (Byturus tomentosus), confirming the presence of Wolbachia. More than 99% of UK individuals were found to be infected with Wolbachia and 97% of these B. tomentosus beetles harboured multiple infections. Preliminary analysis of B. tomentosus beetles from continental European populations revealed a lower level of infection (24%) than those from the UK. Phylogenetic analysis using the ftsZ DNA sequences places Wolbachia from B. tomentosus into a new clade (Abt) within the A division, with some revisions to the existing Wolbachia phylogeny.     

Consequences of Wolbachia transmission process on the infection dynamics

In some species displaying Wolbachia-induced cytoplasmic incompatibility, the intensity of incompatibility depends on the density of symbionts in both parents. Although modalities of the transmission process are poorly known, it appears that the density of Wolbachia within the offspring of a female is variable and is correlated with that of the mother. Assuming that the infection level of an host is a continuous trait, we examine some theoretical consequences of the Wolbachia transmission process on the evolution of the infection level within a population. The hypotheses of this model concern two main points: the transmission of Wolbachia is affected by stochastic processes and a deterministic bias, and the bacterial load of the parents of a cross affects their compatibility relationships. It is shown that the variance of the number of bacteria transmitted induced by the stochastic processes tends to counteract the effect of bacterial curing on the dynamics of infection. A general consequence of the model is that the extinction of Wolbachia is possible even if there is strong incompatibility and no selective disadvantage for the host to bear the bacteria. The model indicates that the evolution of bacterial mutants does not depend on the level of incompatibility they induce, but that mutants with higher transmission variance can be selected for. Moreover, the mean infection level of the host population increases in the presence of such bacteria.     

Endosymbiotic Wolbachia bacteria as biological control tools of disease vectors and pests

Wolbachia bacteria are common cytoplasmic symbionts of insects, mites and filarial nematodes. They can alter the reproduction of their hosts. The symbiont could be eliminated, transferred or used through genetic alteration to take advantage or remove their possible influences on pests and/or natural enemies. Their extensive effects on reproduction and host fitness have made Wolbachia the subject of growing attention as a potential biocontrol agent. Here, we summarize the relations of Wolbachia in the control of disease vectors and pests. Furthermore, the drawbacks of these bacteria are also discussed.     

我国尖音库蚊复合组蚊虫的杂交及其与Wolbachia感染的关系

为了了解我国尖音库蚊复合组蚊虫间杂交卵的不孵化现象和明确该现象与共生微生物Wolbachia感染的关系,对该复合组实验室种群4个亚种进行了笼内杂交和抗生素处理后的杂交。试验表明: 在复合组蚊虫中骚扰库蚊Culex pipiens molestus与淡色库蚊Cx. Pipiens pallens、致倦库蚊Cx. Ipiens quinquefasciatus与尖音库蚊Cx. Pipiens pipiens之间存在有单向胞质不融合现象,骚扰库蚊的雄虫与尖音库蚊、致倦库蚊和淡色库蚊的雌虫杂交卵的孵化率分别为0.06%、0.46%和0.19%;该胞质不融合现象可以通过抗生素处理而消除,处理后骚扰库蚊雄虫与其余3个亚种雌虫F₃杂交卵的孵化率均有提高,分别为89.49%（t=3.90×10^-28＜t_0.01=2.704）、23.39%（t=9.15×10^-7＜t_0.01=2.660_）和22.27%（t=5.08×10^-4＜t_0.01=2.750）,并可因抗生素处理而产生新的不融合类型。     

Experimental evidence of Wolbachia introgressive acquisition between terrestrial isopod subspecies

Wolbachia are the most widespread endosymbiotic bacteria in animals. In many arthropod host species, they manipulate reproduction via several mechanisms that favor their maternal transmission to offspring. Among them, cytoplasmic incompatibility (CI) promotes the spread of the symbiont by specifically decreasing the fertility of crosses involving infected males and uninfected females, via embryo mortality. These differences in reproductive efficiency may select for the avoidance of incompatible mating, a process called reinforcement, and thus contribute to population divergence. In the terrestrial isopod Porcellio dilatatus, the Wolbachia wPet strain infecting the subspecies P. d. petiti induces unidirectional CI with uninfected individuals of the subspecies P. d. dilatatus. To study the consequences of CI on P. d. dilatatus and P. d. petiti hybridization, mitochondrial haplotypes and Wolbachia infection dynamics, we used population cages seeded with different proportions of the 2 subspecies in which we monitored these genetic parameters 5 and 7 years after the initial setup. Analysis of microsatellite markers allowed evaluating the degree of hybridization between individuals of the 2 subspecies. These markers revealed an increase in P. d. dilatatus nuclear genetic signature in all mixed cages, reflecting an asymmetry in hybridization. Hybridization led to the introgressive acquisition of Wolbachia and mitochondrial haplotype from P. d. petiti into nuclear genomes dominated by alleles of P. d. dilatatus. We discuss these results with regards to Wolbachia effects on their host (CI and putative fitness cost), and to a possible reinforcement that may have led to assortative mating, as possible factors contributing to the observed results.     

Contrasting effects of Wolbachia on cytoplasmic incompatibility and fecundity in the haplodiploid mite Tetranychus urticae

Recent studies on Wolbachia‐induced incompatibility in haplodiploid insects and mites have revealed a diversity of cytoplasmic incompatibility (CI) patterns among host species. Here, we report intraspecific diversity in CI expression among four strains of the arrhenotokous mite Tetranychus urticae and in T. turkestani. Variability of CI expression within T. urticae ranged from no CI to complete CI, and included either female embryonic mortality or male conversion types of CI. A fecundity cost attributed to the infection with the high‐CI Wolbachia strain was the highest ever recorded for Wolbachia (?80 to ?100% decrease). Sequence polymorphism at a 550‐bp‐portion of Wolbachia wsp gene revealed two clusters distant by 21%, one of which included three Wolbachia strains infecting mite populations sampled from the same host‐plant species, but showing distinct CI patterns. These data are discussed in the light of theoretical predictions on the evolutionary pathways followed in this symbiotic interaction.     

Reproductive effects and geographical distributions of two Wolbachia strains infecting the Neotropical beetle, Chelymorpha alternans Boh. (Chrysomelidae, Cassidinae)

Wolbachia are maternally inherited endocellular bacteria known to alter insect host reproduction to facilitate their own transmission. Multiple Wolbachia infections are more common in tropical than temperate insects but few studies have investigated their dynamics in field populations. The beetle, Chelymorpha alternans, found throughout the Isthmus of Panama, is infected with two strains of Wolbachia, wCalt1 (99.2% of beetles) and wCalt2 (53%). Populations infected solely by the wCalt1 strain were limited to western Pacific Panama, whereas populations outside this region were either polymorphic for single (wCalt1) and double infections (wCalt1 + wCalt2) or consisted entirely of double infections. The wCalt2 strain was not found as a single infection in the wild. Both strains caused cytoplasmic incompatibility (CI). The wCalt1 strain caused weak CI (approximately 20%) and the double infection induced moderate CI (approximately 70-90%) in crosses with uninfected beetles. The wCalt1 strain rescued about 75% of eggs fertilized by sperm from wCalt2 males. Based on the relationships of beetle mtDNA and infection status, maternal transmission, and repeated population sampling we determined that the double infection invaded C. alternans populations about 100,000 years ago and that the wCalt2 strain appears to be declining in some populations, possibly due to environmental factors. This may be the first study to demonstrate an association between widespread strain loss and environmental factors in the field.     

Naturally occurring single and double infection with Wolbachia strains in the butterfly Eurema hecabe: transmission efficiencies and population density dynamics of each Wolbachia strain

Wolbachia belonging to Alphaproteobacteria are transovarially transmitted bacteria responsible for reproductive alterations in a wide range of arthropods. In natural populations of the butterfly Eurema hecabe, there are two different types of Wolbachia-infected individuals. Individuals singly infected with Wolbachia strain wHecCI exhibit strong cytoplasmic incompatibility, whereas those doubly infected with wHecCI and wHecFem exhibit feminization. Here, we examined the infection frequencies and population densities of each Wolbachia strain in different host tissues (ovary, testis, fat body, midgut, Malpighian tubule and leg), and the cost of infection in offspring produced by single-infected and double-infected mothers of E. hecabe. The vertical transmission rate of wHecCI was nearly 100%, and that of wHecFem was c. 80%. The wHecCI densities were 10(3)-10(4)-fold higher than the wHecFem densities. In most tissues, the wHecCI densities were significantly higher in offspring of single-infected mothers than in offspring of double-infected mothers. In offspring of double-infected mothers, however, the wHecCI densities were not affected by the presence of wHecFem, suggesting a lack of interaction between the wHecCI and wHecFem densities. The offspring development time was dependent on the infection status of the mothers. These results imply that the maternal infection status affects the Wolbachia densities and fitness of offspring.     

Evolution of Wolbachia cytoplasmic incompatibility types

The success of obligate endosymbiotic Wolbachia infections in insects is due in part to cytoplasmic incompatibility (CI), whereby Wolbachia bacteria manipulate host reproduction to promote their invasion and persistence within insect populations. The observed diversity of CI types raises the question of what the evolutionary pathways are by which a new CI type can evolve from an ancestral type. Prior evolutionary models assume that Wolbachia exists within a host individual as a clonal infection. While endosymbiotic theory predicts a general trend toward clonality, Wolbachia provides an exception in which there is selection to maintain diversity. Here, evolutionary trajectories are discussed that assume that a novel Wolbachia variant will co-exist with the original infection type within a host individual as a superinfection. Relative to prior models, this assumption relaxes requirements and allows additional pathways for the evolution of novel CI types. In addition to describing changes in the Wolbachia infection frequency associated with the hypothesized evolutionary events, the predicted impact of novel CI variants on the host population is also described. This impact, resulting from discordant evolutionary interests of symbiont and host, is discussed as a possible cause of Wolbachia loss from the host population or host population extinction. The latter is also discussed as the basis for an applied strategy for the suppression of insect pest populations. Model predictions are discussed relative to a recently published Wolbachia genome sequence and prior characterization of CI in naturally and artificially infected insects.     

昆虫共生细菌Wolbachia的研究进展

Wolbachia是广泛分布在节肢动物生殖组织的一类细胞质遗传的细菌,它们能通过多种机制调节寄生的生殖,包括诱导细胞质不亲和,孤雌生殖和遗传上的雄性雌性化,目前对这些微生物的研究已取了可喜的进展,通过主要综述Wolbachia的分布,功能,系统发育,多样性及时空动态,也讨论了将来的研究方向     

Stable coexistence of incompatible Wolbachia along a narrow contact zone in mosquito field populations

In arthropods, the intracellular bacteria Wolbachia often induce cytoplasmic incompatibility (CI) between sperm and egg, which causes conditional embryonic death and promotes the spatial spread of Wolbachia infections into host populations. The ability of Wolbachia to spread in natural populations through CI has attracted attention for using these bacteria in vector‐borne disease control. The dynamics of incompatible Wolbachia infections have been deeply investigated theoretically, whereas in natural populations, there are only few examples described, especially among incompatible infected hosts. Here, we have surveyed the distribution of two molecular Wolbachia strains (wPip11 and wPip31) infecting the mosquito Culex pipiens in Tunisia. We delineated a clear spatial structure of both infections, with a sharp contact zone separating their distribution areas. Crossing experiments with isofemale lines from different localities showed three crossing types: wPip11‐infected males always sterilize wPip31‐infected females; however, while most wPip31‐infected males were compatible with wPip11‐infected females, a few completely sterilize them. The wPip11 strain was thus expected to spread, but temporal dynamics over 7 years of monitoring shows the stability of the contact zone. We examined which factors may contribute to the observed stability, both theoretically and empirically. Population cage experiments, field samples and modelling did not support significant impacts of local adaptation or assortative mating on the stability of wPip infection structure. By contrast, low dispersal probability and metapopulation dynamics in the host Cx. pipiens probably play major roles. This study highlights the need of understanding CI dynamics in natural populations to design effective and sustainable Wolbachia‐based control strategies.     

Transposable element polymorphism of Wolbachia in the mosquito Culex pipiens: evidence of genetic diversity, superinfection and recombination

Wolbachia is a group of maternally inherited endosymbiotic bacteria that infect and induce cytoplasmic incompatibility (CI) in a wide range of arthropods. In contrast to other species, the mosquito Culex pipiens displays an extremely high number of CI types suggesting differential infection by multiple Wolbachia strains. Attempts so far failed to detect Wolbachia polymorphism that might explain this high level of CI diversity found in C. pipiens populations. Here, we establish that Wolbachia infection is near to or at fixation in worldwide populations of the C. pipiens complex. Wolbachia polymorphism was addressed by sequence analysis of the Tr1 gene, a unique transposable element of the IS5 family, which allowed the identification of five C. pipiens Wolbachia strains, differing either by nucleotide substitution, presence or absence pattern, or insertion site. Sequence analysis also showed that recombination, transposition and superinfection occurred at very low frequencies. Analysis of the geographical distributions of each Wolbachia strain among C. pipiens populations indicated a strong worldwide differentiation independent from mosquito subspecies type, except in the UK. The availability of this polymorphic marker now opens the way to investigate evolution of Wolbachia populations and CI dynamics, in particular in regions where multiple crossing types coexist among C. pipiens populations.