Abstract: 【Objective】The previous research results showed that insect trehalase (TRE) can regulate chitin metabolism and control the molting process. In this study, the brown planthopper (Nilaparvata lugens) molting process, the changes of the expression of chitin content and chitin synthase (CHS) and chtinase (Cht) genes were detected when TRE genes were knocked down by the way of RNAi, in order to explore the roles of different trehalase genes in the regulation of chitin metabolism in the epidermis.【Method】N. lugens fed in the lab was chosen as the experimental material, and RNAi technology was used to inhibit the single or two TRE genes’ expression by injection of double stranded RNA. The total RNA was extracted from the cuticle of N. lugens using the TRIzol^® reagent as instructed by manufacturer. First-stand cDNA was synthesized using the PrimeScript^TM RT reagent Kit with gDNA Eraser following the manufacturer’s instructions. And the effect of RNAi was firstly determined after 48 h of injection by quantitative real-time PCR (qRT-PCR). Secondly, the chitin content of N. lugens whole body was determined at 48 h using potassium hydroxide method qRT-PCR, and photos of the insects with molting difficulties were taken in the same time. In the last, the relative expression levels of CHS and Cht of N. lugens were detected by qRT-PCR, and the regulatory function on chitin metabolism of TRE was analyzed at the same time. 【Result】Compared with the injection of dsGFP which was used as a control group, the results showed that in other groups injected with dsRNA the chitin content of N. lugens was decreased significantly, in which the dsTRE1 mixed injection group and the Validamycin injection group showed a significant decrease, meanwhile its molting problems appeared at the same time. qRT-PCR results showed that the gene expression of individual TRE was inhibited at 48 h after one TRE dsRNA injection, and the other TRE expression was increased and indicated it has a complementary function. The expressions of TRE1-2 and TRE2 were decreased in all groups, and dsTRE1s also inhibited the expression of TRE2. Secondly, the obvious effects could be found when mixed dsTRE1 trehalase inhibitor Validamycin injected into N. lugens and TRE genes were decreased significantly. The expression level of CHS and its splicing variants had no obvious effect when every TRE genes’ expression was knocked down, while CHS1 and CHS1a expressions were significantly decreased at 48 h after Validamycin injection. The expression of CHS1 in the cuticle increased after dsTRE1-2 injection and the expression of CHS1a increased after injection of dsTRE1-2. Thirdly, the expression levels of Cht1 and Cht8 decreased or decreased significantly after four dsTRE and Validamycin injection. The expression levels of Cht2 and Cht5 increased significantly when dsTRE1 was injected, as well as Cht2 and Cht4 increased significantly while Cht1, Cht6 and Cht8 decreased after dsTRE1-2 injection and Cht2 expressed increased significantly while the expression of Cht1, Cht8 and Cht10 decreased at 48 h when TRE2 knocked down. In the same time, the expressions of Cht1 and Cht5 decreased significantly while Cht9 increased significantly at 48 h after dsTRE1s injection. In the last, about all of 10 chitinase genes’ expression decreased significantly or extremely significantly after Validamycin injection.【Conclusion】TRE can control the synthesis of chitin through the regulation of chitin metabolic pathways in N. lugens. The results of this study will provide a theoretical basis for developing and screening effective trehalase inhibitors to control N. lugens.

Key words: Nilaparvata lugens, RNA interference, trehalase, cuticle, chitin metabolism, quantitative real-time PCR (qRT-PCR)