三种新型化合物对草地贪夜蛾海藻糖与几丁质代谢及生长发育的影响

【目的】几丁质是昆虫外骨骼和围食膜的主要成分,其合成始于海藻糖酶（trehalase,TRE）,终于几丁质合成酶（chitin synthase,CHS）,蜕皮与外表皮重塑过程则需要依靠几丁质酶（chitinase,CHT）完成。本研究通过注射3种新型化合物,检测草地贪夜蛾（Spodoptera frugiperda）体内海藻糖酶和几丁质酶活性、相关基因表达量并观察其生长发育,验证新型化合物对海藻糖酶及几丁质酶的抑制效果,对效果显著的化合物进行筛选,探究其调控草地贪夜蛾生长发育的机理。【方法】利用显微注射法向草地贪夜蛾3龄幼虫分别注射丁烯内酯类化合物ZK-I-21、ZK-I-23和胡椒碱类似物ZK-PI-4。注射后48 h检测其海藻糖酶活性、几丁质酶活性及相关糖含量的变化情况,并利用实时荧光定量PCR（qRT-PCR）在分子水平上测定4个关键基因（SfTRE1、SfTRE2、SfCHS2、SfCHT）的相对表达量;观察注射后草地贪夜蛾幼虫至成虫期间的表型变化,记录发育过程中的死亡率及畸形情况。【结果】与对照组相比,注射ZK-I-21和ZK-PI-4后草地贪夜蛾膜结合型海藻糖酶活性分别为极显著下降（P<0.01）和显著下降（P<0.05）。qRT-PCR检测结果显示,注射ZK-I-21后SfTRE1表达量极显著上升,SfCHT表达量极显著下降;注射ZK-I-23后SfTRE1表达量极显著下降,SfCHT表达量极显著上升;注射ZK-PI-4后SfCHT表达量极显著下降。发育历期观察结果显示,ZK-I-21使草地贪夜蛾6龄幼虫发育历期极显著延长,同时蛹重变轻、蛹长变短;ZK-I-23使5龄和6龄幼虫长度显著缩短;ZK-PI-4使成虫羽化率显著降低。3种抑制剂均可紊乱草地贪夜蛾的海藻糖代谢进而紊乱几丁质代谢,造成草地贪夜蛾蜕皮困难甚至死亡。【结论】ZK-I-21与ZK-PI-4是膜结合型海藻糖酶抑制化合物,ZK-I-23可抑制可溶性海藻糖酶基因的表达,3种化合物均通过影响海藻糖代谢过程进而导致几丁质代谢的紊乱,导致昆虫蜕皮困难、畸形、生长发育受影响。以上结果可为将来利用新型抑制剂调控害虫生长发育从而防控害虫提供理论依据,为绿色、高效杀虫剂的研发提供支持。

Effect of Three Novel Compounds on Trehalose and Chitin Metabolism and Development of Spodoptera frugiperda

【Objective】Chitin is the main component of insect exoskeleton and peritrophic membrane. Its synthesis begins with trehalase and ends with chitin synthase. The process of molting and epidermal remodeling needs to be completed by chitinase. In this study, three novel compounds were injected to detect the activities of trehalase and chitinase, the expression levels of related genes in Spodoptera frugiperda, and their growth and development were also observed. This study aims to verify the inhibitory effects of novel compounds on trehalase and chitinase, screen the compounds with obvious effects, and to explore the mechanism of their regulation on the growth and development of S. frugiperda.【Method】Microinjection method was used to inject butenolactone analogues ZK-I-21, ZK-I-23 and piperine analogue ZK-PI-4 into the 3rd instar larvae of S. frugiperda. The changes of trehalase activity, chitinase activity and related sugar content were detected 48 h after injection, and the relative expression levels of SfTRE1, SfTRE2, SfCHS2 and SfCHT were measured at the molecular level by quantitative real-time PCR (qRT-PCR). The phenotypic changes of S. frugiperda were observed during the process from larvae to adults after injection. Besides, the mortality and deformities during developmental period were recorded.【Result】Compared with the control group, the membrane-bound trehalase activity of S. frugiperda decreased significantly after ZK-I-21 (P<0.01) and ZK-PI-4 (P<0.05) injection. The expression level of SfTRE1 was significantly increased and SfCHT was significantly decreased after ZK-I-21 injection. After ZK-I-23 injection, the expression level of SfTRE1 was significantly decreased, and the expression level of SfCHT was significantly increased. The expression level of SfCHT was significantly decreased after ZK-PI-4 injection. The observation results of developmental duration showed that ZK-I-21 significantly prolonged the developmental period of 6th instar larvae of S. frugiperda, and at the same time, the pupa weight became lighter and the pupa length became shorter. In addition, ZK-I-23 significantly shortened the length of 5th and 6th instar larvae, and ZK-PI-4 caused a significant reduction in adult emergence rate. All the three inhibitors could disrupt trehalose metabolism of S. frugiperda and then disrupt chitin metabolism, resulting in difficulties in molting and even death.【Conclusion】ZK-I-21 and ZK-PI-4 are membrane-bound trehalase inhibitor compounds, and ZK-I-23 can inhibit the expression of soluble trehalase gene. The three compounds all lead to the disturbances of chitin metabolism by affecting the process of trehalose metabolism, which results in difficulties in molting, deformities, and impaired growth and development of insects. The above results can provide a theoretical basis for the future use of novel inhibitors to regulate the growth of pests and thus control them, and support to promote the development of green and efficient pesticides.