A simple protocol to obtain highly pure Wolbachia endosymbiont DNA for genome sequencing

Most genome sequencing projects using intracellular bacteria face difficulties in obtaining sufficient bacterial DNA free of host contamination. We have developed a simple and rapid protocol to isolate endosymbiont DNA virtually free from fly and mosquito host DNA. We purified DNA from six Wolbachia strains in preparation for genome sequencing using this method, and achieved up to 97% pure Wolbachia sequence, even after using frozen insects. This is a significant improvement for future Wolbachia and other endosymbiont genome projects.     

EFFECTS OF PARTHENOGENESIS AND GEOGRAPHIC ISOLATION ON FEMALE SEXUAL TRAITS IN A PARASITOID WASP

Population divergence in sexual traits is affected by different selection pressures, depending on the mode of reproduction. In allopatric sexual populations, aspects of sexual behavior may diverge due to sexual selection. In parthenogenetic populations, loss‐of‐function mutations in genes involved in sexual functionality may be selectively neutral or favored by selection. We assess to what extent these processes have contributed to divergence in female sexual traits in the parasitoid wasp Leptopilina clavipes in which some populations are infected with parthenogenesis‐inducing Wolbachia bacteria. We find evidence consistent with both hypotheses. Both arrhenotokous males and males derived from thelytokous strains preferred to court females from their own population. This suggests that these populations had already evolved population‐specific mating preferences when the latter became parthenogenetic. Thelytokous females did not store sperm efficiently and fertilized very few of their eggs. The nonfertility of thelytokous females was due to mutations in the wasp genome, which must be an effect of mutation accumulation under thelytoky. Divergence in female sexual traits of these two allopatric populations has thus been molded by different forces: independent male/female coevolution while both populations were still sexual, followed by female‐only evolution after one population switched to parthenogenesis.     

Genetic distinctness of parthenogenetic forms of European Polydrusus weevils of the subgenus Scythodrusus

Abstract Among eight species of Polydrusus weevils which belong to subgenus Scythodrusus, at least two possess parthenogenetic forms: P. (S.) inustus and P. (S.) pilifer. Both of these species consist of dioecious populations in the Caspian area and of parthenogenetic populations in Eastern Europe (P. (S.) inustus), the Caucasus region (both species) and Middle Asia (P. (S.) pilifer). The origin of parthenogenesis in this subgenus is unresolved; however some data suggest that the parthenogenetic forms are of hybrid ancestry. The genetic distinctness of parthenogenetic Scythodrusus was assessed on the basis of COII, ITS2, EF1‐α and Wolbachiawsp, 16S ribosomal DNA, ftsZ and hcpA sequence comparisons. Both taxa turned out to be monophyletic for all markers, which is an evidence against hybridization of their dioecious ancestors. On the other hand, a high frequency of heterozygous P. (S.) inustus females suggests an origin resulting from hybridization between genetically distinct dioecious representatives of this species. Very similar strains of Wolbachia supergroup A were found in both species, indicating that they have been either inherited from a common ancestor or were transmitted between parthenogenetic Scythodrusus weevils and probably spread randomly across their ranges.     

Cytological detection of Wolbachia in squashed and paraffin embedded insect tissues

Abstract

Wolbachia localization in situ is essential for accurate analysis of the infection and its consequences. Whole cell hybridization is proposed as an easy and rapid method for detecting Wolbachia cells in paraffin embedded tissues or testis squashes of Chorthippus parallelus (Orthoptera). Wolbachia is found in whole gonads and other adjacent tissues. A higher bacterial density, however, is observed in ovarioles and testis. Small independent bacteria with strictly cytoplasmic distribution are displayed. Bacterial density differences among individuals are also revealed.     

EVOLUTION OF INCOMPATIBILITY-INDUCING MICROBES AND THEIR HOSTS

In many insect species, males infected with microbes related to Wolbachia pipientis are “incompatible” with uninfected females. Crosses between infected males and uninfected females produce significantly fewer adult progeny than the other three possible crosses. The incompatibility-inducing microbes are usually maternally transmitted. Thus, incompatibility tends to confer a reproductive advantage on infected females in polymorphic populations, allowing these infections to spread. This paper analyzes selection on parasite and host genes that affect such incompatibility systems. Selection among parasite variants does not act directly on the level of incompatibility with uninfected females. In fact, selection favors rare parasite variants that increase the production of infected progeny by infected mothers, even if these variants reduce incompatibility with uninfected females. However, productivity-reducing parasites that cause partial incompatibility with hosts harboring alternative variants can be favored once they become sufficiently abundant locally. Thus, they may spread spatially by a process analogous to the spread of underdominant chromosome rearrangements. The dynamics of modifier alleles in the host are more difficult to predict, because such alleles will occur in both infected and uninfected individuals. Nevertheless, the relative fecundity of infected females compared to uninfected females, the efficiency of maternal transmission and the mutual compatibility of infected individuals all tend to increase under within-population selection on both host and parasite genes. In addition, selection on host genes favors increased compatibility between infected males and uninfected females. Although vertical transmission tends to harmonize host and parasite evolution, competition among parasite variants will tend to maintain incompatibility.     

沃尔巴克氏菌调控小茶尺蠖和灰茶尺蠖杂交卵孵化

【目的】明确沃尔巴克氏菌Wolbachia对茶尺蠖两近缘种杂交卵孵化的影响及相关机制。【方法】使用2.5 mg/mL四环素浸液处理1 min后的新鲜茶树叶片连续饲喂灰茶尺蠖Ectropis grisescens幼虫3代,建立不携带Wolbachia的灰茶尺蠖种群。通过携带和不携带Wolbachia的灰茶尺蠖成虫分别与小茶尺蠖E.obliqua成虫进行杂交,探究Wolbachia对二者杂交卵孵化的影响。同时利用iTRAQ技术检测并分析携带和不携带Wolbachia的灰茶尺蠖精子蛋白的差异。【结果】与自然携带Wolbachia的灰茶尺蠖相比,去除Wolbachia后的灰茶尺蠖与小茶尺蠖杂交卵孵化率从3.92%显著提高到56.20%,表明Wolbachia诱导胞质不亲合。精子蛋白差异分析显示,携带和不携带Wolbachia的灰茶尺蠖的精子蛋白中,共有128个蛋白显著差异表达,其中KEGG数据库注释到45个差异蛋白富集到鞘脂类代谢、唾液分泌、溶酶体、鞘脂信号通路和鞘糖脂生物合成等106个通路中,其中鞘脂类代谢通路中与神经酰胺合成相关的神经酰胺酶和磷酸酯酶显著上调表达。【结论】共生菌Wolbac...     

The Wolbachia protein TomO interacts with a host RNA to induce polarization defects in Drosophila oocytes

Wolbachia is an endosymbiont prevalent in arthropods. To maximize its transmission thorough the female germline, Wolbachia induces in infected hosts male‐to‐female transformation, male killing, parthenogenesis, and cytoplasmic incompatibility, depending on the host species and Wolbachia strain involved. However, the molecular mechanisms underlying these host manipulations by Wolbachia remain largely unknown. The Wolbachia strain wMel, an inhabitant of Drosophila melanogaster, impairs host oogenesis only when transplanted into a heterologous host, for example, Drosophila simulans. We found that egg polarity defects induced by wMel infection in D. simulans can be recapitulated in the natural host D. melanogaster by transgenic overexpression of a variant of the Wolbachia protein Toxic manipulator of oogenesis (TomO), TomO_wMel^?HS, in the female germline. RNA immunoprecipitation assays demonstrated that TomO physically associates with orb mRNA, which, as a result, fails to interact with the translation repressor Cup. This leads to precocious translation of Orb, a posterior determinant, and thereby to the misspecification of oocytes and accompanying polarity defects. We propose that the ability of TomO to bind to orb mRNA might provide a means for Wolbachia to enter the oocyte located at the posterior end of the egg chamber, thereby accomplishing secure maternal transmission thorough the female germline.     

安荔赤眼蜂在中国野外首次发现及其体内Wolbachia的检测

【目的】为丰富赤眼蜂Trichogramma的种类资源,明确野外新采集的一种赤眼蜂的种类,探明该赤眼蜂所感染Wolbachia的类型。【方法】采用挂米蛾Corcyra cephalonica卵卡法在华南农业大学树木园诱集到两批赤眼蜂,通过形态鉴定和PCR扩增ITS2序列并测序分析的分子鉴定手段对野外采集的赤眼蜂材料进行种类鉴定;通过PCR扩增Wolbachia的外膜蛋白基因(wsp)序列,检测赤眼蜂体内Wolbachia的感染情况;通过PCR扩增wsp序列和多位点序列分型(multilocus sequence typing, MLST)对检测到的赤眼蜂体内的Wolbachia进行同源性分析。【结果】所诱集到的两批赤眼蜂均被鉴定为安荔赤眼蜂Trichogramma oleae Voegelé & Pointel,体内Wolbachia的感染率达100%。该Wolbachia株系与安荔赤眼蜂(前南斯拉夫品系)、短管赤眼蜂T. pretiosum(乌拉圭品系)以及T. deion(荷兰品系)体内Wolbachia亲缘关系较近,属于B超组Sib亚组,对应MLST序列型为ST486。【结论】安荔赤眼蜂T. oleae为中国野外首次发现,是完全感染Wolbachia的产雌孤雌生殖品系。本研究为害虫生物防治提供了一种新的天敌种类资源,并为进一步探明Wolbachia与赤眼蜂的互作提供了研究材料。     

Diversity of Wolbachia isolated from the Cubitermes sp. affinis subarquatus complex of species (Termitidae), revealed by multigene phylogenies

Wolbachia are endosymbiotic bacteria that may alter the reproductive mechanisms of arthropod hosts. Eusocial termites provide considerable scope for Wolbachia studies owing to their ancient origin, their great diversity and their considerable ecological, biological and behavioral plasticity. This article describes the phylogenetic distribution of Wolbachia infecting termites of the Cubitermes genus, which are particularly abundant soil-feeders in equatorial Africa. Fourteen colonies of the Cubitermes sp. affinis subarquatus complex of species were screened using five bacterial genes (wsp, ftsZ, coxA, fbpA and 16S rRNA genes) and a striking diversity of Wolbachia strains was identified within these closely related species. In the host complex, three Wolbachia variants were found that were not in the super groups usually reported for termites (F and H), each infecting one or two Cubitermes species.     

Distribution of the bacterial symbiont Cardinium in arthropods

Abstract 'Candidatus Cardinium', a recently described bacterium from the Bacteroidetes group, is involved in diverse reproduction alterations of its arthropod hosts, including cytoplasmic incompatibility, parthenogenesis and feminization. To estimate the incidence rate of Cardinium and explore the limits of its host range, 99 insect and mite species were screened, using primers designed to amplify a portion of Cardinium 16S ribosomal DNA (rDNA). These arthropods were also screened for the presence of the better-known reproductive manipulator, Wolbachia. Six per cent of the species screened tested positive for Cardinium, compared with 24% positive for Wolbachia. Of the 85 insects screened, Cardinium was found in four parasitic wasp species and one armoured scale insect. Of the 14 mite species examined, one predatory mite was found to carry the symbiont. A phylogenetic analysis of all known Cardinium 16S rDNA sequences shows that distantly related arthropods can harbour closely related symbionts, a pattern typical of horizontal transmission. However, closely related Cardinium were found to cluster among closely related hosts, suggesting host specialization and horizontal transmission among closely related hosts. Finally, the primers used revealed the presence of a second lineage of Bacteroidetes symbionts, not related to Cardinium, in two insect species. This second symbiont lineage is closely allied with other arthropod symbionts, such as Blattabacterium, the primary symbionts of cockroaches, and male-killing symbionts of ladybird beetles. The combined data suggest the presence of a diverse assemblage of arthropod-associated Bacteroidetes bacteria that are likely to strongly influence their hosts' biology.