非寄主气味调控云南切梢小蠹产卵的作用和机理

Tomicus yunnanensis is one of the most economically important beetles in southwestern China, which causes serious damage to Pinus yunanensis. Our preliminarily studies showed that volatiles emitted from non-host plants efficiently suppress the reproduction of this pest. However, the active volatiles are not identified and the suppression mechanism remains to be elucidated. Thus, this project will conduct the following contents using Celtis yunnanensis and Cupressus torulosa as non-host materials: analyzing the active odor compositions with the ability to suppress the reproduction of P. yunanensis from the non-hosts; and investigation the effects of the active non-hosts odors on the fecundity, growth and development, accumulation of nutrients related to oviposition, yolk protein synthesis, and gene expression of vitellogenesis of this pest. The results will illustrate the mechanism of pest population changes that is regulated by non-host odors at the nutrient metabolism, growth and development, and reproduction levels. The project will provide a new way for revealing the mechanism of how mixed forest control the pests, and are useful for designing novel pest control strategies.

云南切梢小蠹（Tomicus yunnanensis）是西南地区最重要的森林害虫之一，造成云南松大面积死亡。前期研究发现非寄主气味物质能有效减少该虫产卵数量，但是尚不清楚何种气味物质影响产卵，也不知影响产卵的机理。基于此，本申请项目拟选用能有效抑制产卵的非寄主植物滇朴、藏柏作为材料，提取挥发性成分，分析能有效抑制该小蠹产卵的气味物质；研究活性成分作为胁迫因子影响云南切梢小蠹产卵、幼虫生长发育、营养物质积累、卵黄蛋白合成、卵黄蛋白相关基因表达等。通过这些研究试图从害虫营养代谢、生长发育和繁殖角度揭示非寄主调控害虫种群的机理，为混交林控制害虫的机理研究提供新思路，为害虫调控提供新技术和新方法。

本项目通过GC-EAG和生物检测从非寄主旱冬瓜、滇朴气味成分中筛选出能有效抑制产卵的气味单体化合物4个，分别是2-己烯醛、3-己烯醛、3-己烯醇、4-乙基苯乙酮。基于构建的转录组数据库，共鉴定获得卵黄原蛋白及其受体基因序列各4条，云南切梢小蠹卵黄原蛋白基因为：comp9075_c0、comp303908_c0、comp239614_c0、comp86306_c1；云南切梢小蠹卵黄原蛋白受体基因为：comp309118_c0、comp53752_c0、comp2044_c0、comp78496_c0。旱冬瓜和滇朴气味干扰30d后，云南切梢小蠹卵和幼虫数量减少56.4%、64.6%，卵孵化率减少50.0%和57.0%，60d后幼虫化蛹率比对照低39%和41%，90d后蛹羽化率比对照降低33%和38%。旱冬瓜和滇朴气味干扰后，幼虫体长比对照短5.8%和9.4%。反转录之后进行荧光定量PCR显示。与对照组相比滇朴和旱冬瓜叶片提取物干扰处理20 d、 40 d后，滇朴组Vg和VgR基因的转录水平分别为对照的13.43%和47.08%；旱冬瓜组的Vg和VgR基因的转录水平分别对照组的80.30%和69.42%。非寄主气味干扰后卵巢发育受到明显影响，卵巢小管体积缩小36-65%。精子异性率超过63%。ELISA测定表明非寄主旱冬瓜干扰40d后，卵巢中卵黄蛋白含量减少34.9%； SDS-PAGE聚凝胶电泳结果表明非寄主植物干扰不会造成云南切梢小蠹卵黄蛋白的亚基组成发生改变；小蠹虫的卵黄蛋白亚基组成分子量分别是: 99KD、94KD、38KD、15KD。