坡柳皂苷对大菜粉蝶幼虫拒食作用机制研究

The good antifeeding effect of Dononaea viscosa saponins on a variety of lepidoptera pests shall be confirmed and the part of the body where it is intended to work shall be inferred by symptom observation after pesticide application to the test insect, based on which the project is to study its inhibiting effect on the gustation sensilla and the feeding of the test insect, the activity of the cell stimulation after the experiment of the larvae of Pieris brassicae by the electric mirror, the electrophysiology, the biochemical analysis and so on. Then the changes of the induction of detoxication enzymes and the content of the γ- antinobutryric acid of the test insect in its excitement shall be analysized. Accordingly the inhibitory effects and modes of the evoked responses of various taste receptors by the Dononaea viscosa saponins shall be identified, which determines whether its antifeedant effect is related to the systems of the detoxication enzymes and the content of the γ-antinobutryric acid. The study shall help to reveal the antifeeding mechanism of the Dononaea viscosa saponins, which may enrich the mechanism of action of the natural insect antifeedants. The theory of feeding selection of the insect shall be also supplied. Ultimately it shall lay the foundation for the developemnt and utilization of the Dononaea viscosa saponins.

坡柳是一种需要防除的恶性杂草，项目组已从中分离鉴定出具新结构的主要拒食活性成分皂苷A，优化了坡柳(总)皂苷提取工艺和建立了其主成分皂苷A含量测定方法。在后续确定坡柳皂苷对多种鳞翅目害虫具有良好拒食活性以及药后对试虫行为症状观察推测其作用部位的基础上，本申请项目拟以大菜粉蝶幼虫为试虫，利用电镜、电生理学、生化分析等技术研究坡柳皂苷作用后，对试虫味觉感受器的抑制以及对其取食抑制素细胞和刺激素细胞的活性；比较分析中肠解毒酶系以及兴奋期试虫γ-氨基丁酸等含量的变化，从而明确坡柳皂苷对各种味觉感受器诱发反应的抑制作用及方式，确定其拒食作用是否与影响γ-氨基丁酸的神经传导和解毒酶系有关。本项目的研究结果将有助于揭示坡柳皂苷拒食作用机制，丰富天然昆虫拒食剂的作用机理，充实昆虫取食选择行为的理论体系，最终为坡柳皂苷的开发利用奠定基础。

使用大菜粉蝶幼虫等作为模式昆虫，首先调查坡柳皂苷怎样调节试虫的摄食反应。我们假设坡柳皂苷通过（1）摄食反应和（2）摄食后反应引起拒食作用，得到的研究结果与两个假设一致。即在短期摄食试验中，坡柳皂苷抑制摄食和中和了取食刺激物的影响，电生理结果表明，坡柳皂苷直接刺激诱导了试虫中、侧栓锥的取食抑制素感受细胞，并调节外周蔗糖和肌醇感受细胞的活性；继而引发神经递质γ-氨基丁酸介导的神经冲动传导。2在长期摄食实验中，随着时间的延长，坡柳皂苷抑制摄食能力被削弱，酶学分析表明，坡柳皂苷在试虫体内积累到24 h后，虫体内羧酸酯酶和谷胱甘肽-s转移酶被激活，18 h-24 h内坡柳皂苷在试虫体内达到临界点，细胞色素p450被激活，试虫开始分解坡柳皂苷。同时，我们从坡柳中分离鉴定出7个化合物，其中坡柳皂苷B经检索为新化合物，且拒食活性与坡柳皂苷A相当。该系列试验的开展有助于揭示坡柳皂苷拒食作用机制，丰富植物源昆虫拒食剂的作用机理。充实昆虫取食选择行为的理论体系，为坡柳皂苷的开发利用奠定基础。