Wolbachia与赤眼蜂互作及在种间水平转移的分子机制

Wolbachia species are intracellular symbionts that cause various reproductive alterations in their arthropod hosts. In some egg parasitoids of the Trichogramma genus, Wolbachia can induce thelytokous parthenogen (PI) phenotypes of their hosts, therefore parthenogenesis induction of a promising candidate which has not been infected by PI Wolbachia could reduce the cost of rearing and contribute to the mass production of this favorable insect. However, little is known about the molecular mechanisms underlying the Wolbachia – Trichogramma interaction. Microinjection techniques for transmissiom of Wolbachia from infected to uninfected species is an effective way to elucidate the interaction between Wolbachia and a new host and has been performed in many arthropod species. .In this project we plan to transfer a parthenogenesis-inducing（PI）Wolbachia endosymbiont derived from the parasitic wasp Trichogramma pretiosum to Trichogramma chilonis, a widely distributed and applied natural enemy in China. Firstly, different cell lines such as mosquito cell line or Drosophila cell line will be screened and compared for cultivation and maintenance of PI Wolbachia. Secondly, after the suitable injection titer of Wolbachia, the suitable stage and position of recipient wasps have been decided, the PI Wolbachia will be microinjected into uninfected T. chilonis, in which the original Wolbachia that could not induce parthenogenesis has been removed by an antibiotic treatment. Then, the fluorescence in situ hybridization (FISH) and quantitative RT-PCR will be used to detect the location, distribution, density and dynamics of Wolbachia PI in the recipient wasps. The phenotypes of Wolbachia in the recipient wasps (parthenogenesis-inducing) will be determined using the progeny sex ratio of unmated females of new hosts. Next, the transcriptomes of T. pretiosum in PI Wolbachia -infected vs. uninfected (PI Wolbachia has been removed by an antibiotic treatment) lines, T. chilonis Wolbachia-infected vs. uninfected and PI Wolbachia-tranfered lines are compared and analyzed. The differentially expressed genes possibly involved in the immune, defense and reproductive manipulation of the Trichogramma will be analyzed and discussed through bioinformatics methods and data analysis. At last, some important relative genes will be selected to validate their gene expression profiles obtained with the RNA-sequence analysis in both the infected and uninfected lines of two Trichogramma species using quantitative real-time PCR..The completion of this project will preliminary elucidate the molecular mechanism underlying Wolbachia - Trichogramma interaction, clarify the influencing factors contributing to successfully horizontal transmission of Wolbachia between host species, and finally provide us with the theoretical evidence and technologic support for genetically reproductive manipulation of commercialized Trichogramma products and promote biological control of insect pests in China.

Wolbachia是节肢动物胞内共生菌，具有多种调控宿主生殖方式的能力，但其与宿主互作的分子机理却知之甚少。本项目拟将诱导短管赤眼蜂孤雌产雌的Wolbachia菌株，在筛选出适合该菌株培养的细胞株基础上，通过微注射方法转染到螟黄赤眼蜂中，用荧光原位杂交和RT-PCR检测Wolbachia在新宿主内的分布及遗传稳定性。用转录组测序和表达谱分析方法，比较短管赤眼蜂Wolbachia感染和非感染系，螟黄赤眼蜂Wolbachia感染、非感染及转染系的基因差异表达，筛选出在Wolbachia-赤眼蜂互作中与免疫、防御及生殖调控相关的基因，用RT-PCR验证相关基因在赤眼蜂体内的表达动态，分析决定Wolbachia在新宿主内成功表型的相关基因。初步揭示Wolbachia-赤眼蜂互作的分子机理，明确影响Wolbachia在赤眼蜂种间水平转移的因素，为利用Wolbachia调控赤眼蜂的性比提供理论依据。

揭示Wolbachia与参与调控赤眼蜂性别的分子机制，可为利用Wolbachia控制赤眼蜂的蜂群性比提供理论依据和技术支持，对推动我国生物防治的发展具有重要的现实意义。.经亚致死剂量盐酸四环素处理短管赤眼蜂10代后，通过对连续3代孤雌后代单头追踪和与雄蜂杂交，我们获得了恢复两性生殖的短管赤眼蜂Wolbachia 非感染品系，并建立了稳定的实验室种群，为接下来的转录组测序和表达谱分析提供了材料保障。与孤雌生殖品系相比，短管赤眼蜂两性生殖恢复系的雌蜂寿命缩短、寄生卵量减少，种群参数值下降。随着产卵雌蜂年龄的增加，孤雌品系后代的雄蜂数量不断增加，母代雌蜂产卵后体内的Wolbachia滴度相比于产卵前都维持在比较低的水平，说明Wolbachia滴度与短管赤眼蜂的性别决定密切相关。抗生素处理孤雌品系短管赤眼蜂后还会产生同时具有雄性和雌性性状特征的嵌合体后代，嵌合体虽然具有雄性的触角但是生物学特性却与孤雌品系雌蜂相似，其体内Wolbachia的含量显著低于孤雌品系雌蜂，表明Wolbachia浓度不足是导致嵌合体的直接原因，这种性别变化与性别决定基因表达水平有关。.基于转录组的测序结果，短管赤眼蜂两性品系和孤雌品系雌蜂在成虫期共有差异表达基因813个，其中149个上调664个下调，共富集265条通路，主要富集通路有25条。qRT-PCR 结果表明，随机选取的9 个差异表达基因的表达趋势与 RNA-Seq转录组测序结果完全一致。该结果揭示了与赤眼蜂性别调控有潜在关联的基因和信号通路，为今后进一步研究主要关联基因及信号通路的功能以阐明Wolbachia参与调控赤眼蜂的性别机理奠定了必要的基础。.此外，我们在中国野外首次发现安荔赤眼蜂Trichogramma oleae，是完全感染Wolbachia的产雌孤雌生殖品系，为进一步探明Wolbachia与赤眼蜂的互作提供了新的研究材料。