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.     

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.     

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.     

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.     

Population differentiation and Wolbachia phylogeny in mosquitoes of the Aedes scutellaris group

Mosquito species of the Aedes (Stegomyia) scutellaris (Walker) group (Diptera: Culicidae) are distributed across many islands of the South Pacific and include major regional vectors of filariasis, such as Aedes polynesiensis (Marks). Analysis of populations of Ae. polynesiensis at the extremes of its range, from Fiji and from Moorea, French Polynesia, using the rDNA ITS2 (internal transcribed spacer 2) region and six microsatellite markers showed considerable genetic differentiation between them (F(ST) = 0.298-0.357). Phylogenetic analysis of the Wolbachia endosymbionts in three members of the complex revealed that based on the wsp gene they are all very similar and belong to the Mel subgroup of the A clade, closely related to the Wolbachia strain present in the gall wasp Callyrhytis glandium (Giraud) (Hymenoptera: Cynipidae). By contrast they are only distantly related to the A-clade Wolbachia in Aedes albopictus (Skuse), a species closely allied to the Ae. scutellaris group. There was very low differentiation between the Wolbachia in the Moorea and Fiji populations of Ae. polynesiensis.     

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。     

Increased male mating rate in Drosophila is associated with Wolbachia infection

The maternally inherited bacterium Wolbachia pipientis infects 25-75% of arthropods and manipulates host reproduction to improve its transmission. One way Wolbachia achieves this is by inducing cytoplasmic incompatibility (CI), where crosses between infected males and uninfected females are inviable. Infected males suffer reduced fertility through CI and reduced sperm production. However, Wolbachia induce lower levels of CI in nonvirgin males. We examined the impact of Wolbachia on mating behaviour in male Drosophila melanogaster and D. simulans, which display varying levels of CI, and show that infected males mate at a higher rate than uninfected males in both species. This may serve to increase the spread of Wolbachia, or alternatively, may be a behavioural adaptation employed by males to reduce the level of CI. Mating at high rate restores reproductive compatibility with uninfected females resulting in higher male reproductive success thus promoting male promiscuity. Increased male mating rates also have implications for the transmission of Wolbachia.     

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.     

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。     

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.     

Filarial susceptibility and effects of Wolbachia in Aedes pseudoscutellaris mosquitoes

The mosquito Aedes pseudoscutellaris (Theobald), a member of the Aedes (Stegomyia) scutellaris complex (Diptera: Culicidae), is an important vector of subperiodic Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae), causing human lymphatic filariasis, on South Pacific islands. Maternal inheritance of filarial susceptibility in the complex has previously been asserted, and larval tetracycline treatment reduced susceptibility; the maternally inherited Wolbachia in these mosquitoes were suggested to be responsible. To investigate the relationship of these two factors, we eliminated Wolbachia from a strain of Ae. pseudoscutellaris by tetracycline treatment, and tested filarial susceptibility of the adult female mosquitoes using Brugia pahangi (Edeson & Buckley). Filarial susceptibility was not significantly different in Wolbachia-free and infected lines of Ae. pseudoscutellaris, suggesting that the Wolbachia in these mosquitoes do not influence vector competence. Crosses between Wolbachia-infected males and uninfected females of Ae. pseudoscutellaris showed cytoplasmic incompatibility (CI), i.e. no eggs hatched, unaffected by larval crowding or restricted nutrient availability, whereas these factors are known to affect CI in Drosophila simulans. Reciprocal crosses between Ae. pseudoscutellaris and Ae. katherinensis Woodhill produced no progeny, even when both parents were Wolbachia-free, suggesting that nuclear factors are responsible for this interspecific sterility.     

Evidence of natural Wolbachia infections and molecular identification of field populations of Culex pipiens complex (Diptera: Culicidae) mosquitoes in western Turkey

Establishing reliable risk projection information about the distribution pattern of members of the Culex pipiens complex is of particular interest, as these mosquitoes are competent vectors for certain disease‐causing pathogens. Wolbachia, a maternally inherited bacterial symbiont, are distributed in various arthropod species and can induce cytoplasmic incompatibility, i.e., reduced egg hatch, in certain crosses. It is being considered as a tool for population control of mosquito disease vectors. The Aegean region is characterized by highly populated, rural, and agricultural areas and is also on the route of the migratory birds. In this study, a fragment of the 658 bp of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene, which includes the barcode region, was employed to differentiate Cx. pipiens complex species found in this region. Moreover, for the first time, the prevalence of Wolbachia endobacteria in these natural populations was examined using PCR amplification of a specific wsp gene. Our results revealed a widespread (more than 90%, n=121) presence of the highly efficient West Nile virus vector Cx. quinquefasciatus in the region. We also found that Wolbachia infection is widespread; the average prevalence was 62% in populations throughout the region. This study provided valuable information about the composition of Cx. pipiens complex mosquitoes and the prevalence of Wolbachia infection in these populations in the Aegean region. This information will be helpful in tracking mosquito‐borne diseases and designing and implementing Wolbachia‐based control strategies in the region.     

Recent changes in phenotype and patterns of host specialization in Wolbachia bacteria

Wolbachia are a genus of bacterial symbionts that are known to manipulate the reproduction of their arthropod hosts, both by distorting the host sex ratio and by inducing cytoplasmic incompatibility. Previous work has suggested that some Wolbachia clades specialize in particular host taxa, but others are diverse. Furthermore, the frequency with which related strains change in phenotype is unknown. We have examined these issues for Wolbachia bacteria from Acraea butterflies, where different interactions are known in different host species. We found that bacteria from Acraea butterflies mostly cluster together in several different clades on the bacterial phylogeny, implying specialization of particular strains on these host taxa. We also observed that bacterial strains with different phenotypic effects on their hosts commonly shared identical gene sequences at two different loci. This suggests both that the phenotypes of the strains have changed recently between sex ratio distortion and cytoplasmic incompatibility, and that host specialization is not related to the bacterial phenotype, as suggested from previous data. We also analysed published data from other arthropod taxa, and found that the Wolbachia infections of the majority of arthropod genera tend to cluster together on the bacterial phylogeny. Therefore, we conclude that Wolbachia is most likely to move horizontally between closely related hosts, perhaps because of a combination of shared vectors for transmission and physiological specialization of the bacteria on those hosts.     

Wolbachia属共生细菌及其对节肢动物生殖活动的调控作用

Wolbachia属是广泛分布于节肢动物生殖组织内的一类共生细菌。这些共生菌通过卵的细胞质传播并参与多种调控其宿主生殖活动的机制,包括:诱导生殖不亲和、诱导孤雌生殖、雌性化、雄性致死和调节繁殖力。Wolbachia被认为与性别决定、共生关系和物种形成等重要生物学问题密切相关,是探索这些研究领域的新线索。而且Wolbachia可作为特定的载体对其宿主种群进行遗传调控,如增强寄生蜂在害虫生物防治中的作用,控制线虫引起的疾病传播。该文综述了Wolbachia的形态学及存在部位、基因组结构、系统发育、种的命名、水平传递和Wolbachia对其宿主生殖活动的调控作用,并分析了Wolbachia研究的科学意义和发展趋势,以期引起我国生物学家对Wolbachia研究的注意和快速切入。     

Wolbachia in leafcutter ants: a widespread symbiont that may induce male killing or incompatible matings

Wolbachia is a maternally inherited bacterium that manipulates host reproduction by inducing cytoplasmic incompatibility (CI), parthenogenesis or male killing (MK). Here, we report on a screening of seven leafcutter ant species of the genera Atta and Acromyrmex. Using Wolbachia‐specific polymerase chain reaction (PCR) primers we show that all species are infected, usually by double A + B strain infections. For Acromyrmex echinatior and A. octospinosus, a screening across all castes shows that gynes (prospective queens) have higher infection rates than workers and males. The low infection rate of workers suggests that workers lose their infection during development. This we interpret as adaptive, because a heritable symbiont does not benefit from being present in sterile workers. Both CI and MK could potentially account for the low infection rate of males. Formal theoretical models show greater support for the MK scenario in the free living species A. echinatior and A. octospinosus but indicate that Wolbachia in the social parasite A. insinuator may cause CI, supporting a scenario of sympatric speciation of the social parasite. We conclude that Wolbachia represents a previously unrecognized source of reproductive conflict in leafcutter ant colonies.     

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 infection and incompatibility dynamics in experimental selection lines

High and low levels of Wolbachia-induced cytoplasmic incompatibility (CI) were selected for in the parasitic wasp Nasonia vitripennis, in the single-infected strain Ti277. After nine generations of selection, males from lines selected for high incompatibility level (HI lines) were significantly more incompatible with uninfected females (AsymC) than the maternal strain. The reverse response, a full compatibility with AsymC, was observed in eight out of 12 lines selected for low incompatibility (LO lines), correlated with loss of Wolbachia infection. Bacterial density estimates in the eggs of some HI lines increased significantly. The procedure for line maintenance resulted in introgression of AsymC nuclear genome into the Ti277 background. Significant changes of CI level and bacterial density due to the introgression were also observed in the control lines, possibly reflecting an effect of host genotype on bacterial density and CI. After selection had been relaxed for six generations, bacterial density in the five high-infected HI lines declined back to a level comparable to the other lines. The data are consistent with the ‘bacterial dosage’ model, but with an upper threshold of bacterial infection above which there is no correlation between infection level and CI level. We further investigate the maternal transmission of bacterial density by a mother–daughter regression on bacterial density. The pattern observed is consistent with a density dependent regulation of bacterial numbers around an ‘equilibrium’ density, independent of any effects of CI. The equilibrium value is likely to be determined by both bacterial strain and host genotype.