椰扁甲啮小蜂毒液特异蛋白及其调控两种入侵甲虫的分子机理

In the system of invasive pest Octodonta nipae Maulik/Brontispa longissima Gestro and their endoparasitic wasp Tetrastichus brontispae Ferrière, the types and functions of pupa hemocyte in O. nipae will be studied with the technology of physiological ecology and molecular biology, to reveal invasive characteristics of invasive pest from the angle of celluar immunity. Kinds of parasitic factors and their timeliness will be analyzed to determine kinds of protein existed in venom and their regulations on host immune system. Changes of the number of total hemocyte cells (THC), and their extension, cytophagy and encapsulation in O. nipae pupa parasitized by T. brontispae will be studied, in order to clarify responses of the key types of hemocyte and their physiological mechanism of the hemocyte immune response. The correlations among antioxidase, phenoloxidases (PO) and ROS after the parasitism by T. brontispae will be investigated to expound the response of O. nipae parasitism by T. brontispae in the immune reactivity and its effect to the function such as humoral melanization. Also, this research will uncover the physiological and biochemistry process in the host which caused by the parasitic wasp for further explaining the mechanism that T. brontispae damages the host humoral immunity. This research will not only attribute to further analysis about the immune interaction between T. brontispae and host to enhance the nursing of parasitic wasp and improve the natural control effect in the field, but also provide a new idea and approach for the development of new-type pest inhibitor which takes the insect immune system as a target.

本项目以椰扁甲啮小蜂-水椰八角铁甲/椰心叶甲系统为对象，在明确毒液是椰扁甲啮小蜂的主要寄生因子的基础上，从细胞免疫和体液免疫角度，分析不同寄主饲养的椰扁甲啮小蜂毒液对不同寄主免疫抑制的效能及其差异；综合应用二代测序、蛋白质组学及生物信息学的方法明确不同寄主上饲养的椰扁甲啮小蜂毒液特异蛋白组分及其表达差异；分析原核表达特异毒液蛋白抑制寄主细胞免疫及体液免疫反应的能力，探讨其抑制寄主酶的级联反应和关键受体基因表达，揭示椰扁甲啮小蜂特异毒液蛋白调控不同寄主的分子机理等。该研究不仅从寄生蜂毒液特异蛋白表达的视角，揭示入侵害虫的适应性变异机制，进一步丰富和完善入侵生物学的研究体系，而且有助于深入剖析椰扁甲啮小蜂与不同寄主的免疫互作效应和多样化的寄生策略，加强寄生蜂保育和提高田间自然控害效果，为开发以昆虫免疫系统为靶标的新型害虫抑制剂等提供新的思路和途径。

椰扁甲啮小蜂可同时寄生椰心叶甲和水椰八角铁甲，但在两寄主上的适应性存在显著差异。因此，分析椰扁甲啮小蜂针对不同寄主的寄生策略差异，探讨其毒液组分以及其调控两种入侵甲虫的分子机理等，对于揭示寄生蜂毒液特异蛋白的作用效应及其与寄主之间的相互适应性变异具有重要的意义。本项目以椰扁甲啮小蜂-水椰八角铁甲/椰心叶甲系统为对象，在明确毒液是椰扁甲啮小蜂的主要寄生因子的基础上，1）从细胞免疫和体液免疫角度，分析不同寄主饲养的椰扁甲啮小蜂（铁甲蜂和叶甲蜂）毒液对不同寄主免疫抑制的效能及其差异，以铁甲为寄主时，毒液和卵巢液都能起作用，其中毒液对寄主的免疫抑制具有重要作用，而以叶甲为寄主时，卵巢液对寄主的免疫抑制占主导作用。2）综合应用二代测序、蛋白质组学及生物信息学的方法明确了不同品系啮小蜂毒液特异蛋白组分及其表达差异，其差异主要富集在调控寄主黑化反应和脂肪体代谢的通路上，这种差异可能与调控寄主脂肪和免疫反应相关。3）分析了原核表达特异毒液蛋白抑制寄主细胞免疫及体液免疫反应的能力，探讨其抑制寄主酶的级联反应和关键受体基因表达，调控寄主黑化的毒液蛋白Tbserpin6与寄主铁甲蛹的Onerpin6的结构以及底物结合位点相似，能与铁甲的原酚氧化酶激活蛋白酶（OnPPAF1）发生互作进而抑制寄主的酚氧化酶激活通路抑制黑化反应；调控寄主包囊的毒液蛋白Tb-CRT、Tb-RhoGAP可抑制寄主钙网蛋白OnCRT、整合蛋白OnINT和OnRac1基因的表达从而抑制寄主血细胞的包囊反应，TbCRT和TbRhoGAP与寄主叶甲的互作更强，更容易调控寄主叶甲血淋巴蛋白，因而椰扁甲啮小蜂在寄主叶甲上的适应性更强。该研究从寄生蜂毒液特异蛋白表达的视角，揭示入侵害虫的适应性变异机制，明确了椰扁甲啮小蜂与不同寄主的免疫互作效应和多样化的寄生策略，为开发以昆虫免疫系统为靶标的新型害虫抑制剂等提供新的思路和途径。