Regulating Effects Of Trehalase And Its Inhibitor (Validamycin) On The Trehalose And Chitin Metabolism In Nilaparvata Lusens

Trehalase (TRE), an enzyme that hydrolyzes trehalose to yield two glucose molecules, which can be divided into two types, soluble trehalase (TRE1) and membrane-bond trehalase (TRE2). The activity of trehalase can be competitively inhibited by combining with trehalase inhibitors at the active site of the amino acid of trehalase, which can further affect the related metabolism processes in insect. In this paper, Nilaparvata lugens was used as research object and Validamycin, a kind of trehalase inhibitor with better effect, was chosen to explore the regulating effect of trehalase and its inhibitor on the trehalose and chitin metabolic in N. lugens. The main contents and results of this study are followed:Firstly, the regulating effects of TRE on the related genes in chitin synthesis and degradation pathway in N. lugens were studied using RNAi technology. The expression of partial chitinase, CHS1 and two different variable transcripts (CHS1a and CHS1b) and other key genes chitin synthesis pathway decreased in dsTRE injected groups, and the genes regulated by TRE1-1, TRE 1-2 and TRE2 were different. These results indicated that the expression of the key genes in chitin metabolic pathway in N. lugens can be regulated by TRE, and the functions of the three different TREs in chitin metabolism were various. In addition, CHS1, CHS1a, CHS1b and 12 Cht genes expression were significantly decreased after; the expression of GFAT, GNNPNA, UAP, CHS1b and 7 or 8 Cht genes dropped after TPS1 and TPS2 RNAi for 72 h. The results showed that the expression of related genes in chitin metabolic pathway was inhibited when trehalose synthesis pathway was disturbed.Secondly, the injected concentrations of Validamycin were preliminarily screened. According to the screening results, five appropriate concentration of Validamycin were set to microinject the five instar larvae of N. lugens to inhibit the activity of trehalase, which were 0.1,0.5,1,5,10 μg/μL. And the inhibiting effect was evaluated after injected for 48 h, taking the excessive buffer, trehalose and glucose injetcted groups as controls. The results showed that there appeared certain mortality and deformity rates after Validamycin being injected for 48 hours. The mortality and deformity rates increased as the injected concentration of Validamycin rose to the maximum of 56.67% and 37.78% at the injection concentration of 10 μg/μL, respectively. There appeared certain deformity and mortality rate after N. lugens was injected with Validamycin, in which mainly included three kinds of malformation: molting deformity, wing abnormality, molting and wing abnormality. The degree of each deformity was different, and the rates of molting deformity and molting and wing abnormality were lager.Thirdly, the effect of Validamycin on the trehalose metabolic pathway after N. lugens was injected for 48 hours was explored. The results are followed:the activity of soluble and membrane-bound trehalase all decreased significantly after injection for 48 h, indicating that the activity of trehalase was effectively inhibited by Validamycin; the content of trehalose was significantly increased, while the content of glucose and glycogen decreased significantly. The results also showed that the activity of trehalase was effectively inhibited when of Validamycin was above 0.1 μg/μL, and the mortality of N. lugens increased with the increasing the injection of Validamycin, indicating that higher density Validamycin can more effective to control the pests.Finally, the malformations and death of N. lugens were discovered which resulted from the directed regulating effects of Validamycin on chitin metabolic pathway. The results after N. lugens injected for 48 hours are followed:compared with control, the expression of HK, G6PI, GFAT, GNPNA, PGM, UAP, CHS (including its variable exon genes, CHS1a and CHS1b) and seven chitinase genes were reduced; the content of chitin was declined with the increasing of Validamycin injected concentration. These results indicated that the activity of the two kinds of trehalase can be inhibited by Validamycin, thereby further regulating chitin synthesis and degradation, and resulted in deformity and death of N. lugens. In addition, there appeared certain mortality and deformity rate after excessive trehalose or glucose being injected for 48 hours. And compared with sterile water injected group, the expression of HK, GFAT, GNPNA, PGM, UAP, CHS and 9 or 4 chitinase genes were decreased and the chitin content dropped. The results indicated that excessive trehalose or glucose can regulate the chitin metabolism in N. lugens.In summary, the expression of the key genes in the chitin metabolism in N. lugens can be regulated by trehalase and its inhibitor (Validamycin), so that the chitin content was decreased and abnormal deformities, such as molting deformity and wing deformity, and even death were discovered.