Characterization Of Two Trehalases In Nilaparvata Lugens (St(?)l) And The Inhibitory Effect Of Xenobiotics Against Trehalase

Nilaparvata lugens (Stal) (brown planthopper, BPH) is a long distance migratory rice pest in Asia. Trehalose is the main blood sugar in the haemolymph of insects. As major energy storage used by insects for flight, trehalose plays a key role in the energy providing. When it is transported to various tissues through haemolymph, it can be hydrolyzed by trehalases into glucose and utilized by cells. Trehalose in haemolymph also can be converted into glycerol, sorbitol and polyols, which can protect proteins and cellular membranes from inactivation or denaturation caused by different stress conditions.For the improvement of BPH monitoring and control, two forms (soluble and membrane-bound) of trehalase and the corresponding genes (N1Tre-1 and N1Tre-2) were identified from the brown planthopper, Nilaparvata lugens. Comparison of trehalase activity and gene mRNA level at different developmental stages were both assayed, several potential trehalase inhibitors were also tested. Some valuable breakthroughs had been achieved, which can promote the study of new trehalase inhibitors in insects.1. Molecular cloning and characterization of two forms of trehalaseWe obtained the full-length cDNAs encoding two forms of trehalase in BPH by RT-PCR and RACE technology. Both N1Tre-1 and N1Tre-2 contain trehalase signature motifs, and N1Tre-2 contains a putative transmembrane domain. The amino acid sequence of N1Tre-1 shows an identity of 52% and 49% with Acyrthosiphon pisum and Pimpla hypochondriaca trehalase-1 (soluble trehalase), respectively. The amino acid sequence of N1Tre-2 shows an identity of 64% and 63% with Apis mellifera and Acyrthosiphon pisum trehalase-2 (membrane-bound trehalase), respectively.2. Developmental changes of trehalase activities and the regulation by 20ESoluble and membrane-bound trehalase activities were measured after the application of different doses of 20E. In macropterous females, a small increase in soluble trehalase activity was observed. However, no such increase occurred in membrane-bound trehalase.3. Developmental expression patterns and the regulation by 20E of two trehalase genesThe developmental expression of the two trehalase genes was compared, with or without 20E application. Without the treatment of 20E, the mRNA level of N1Tre-1 was much higher than NlTre-2 in all samples, and the mRNA level of N1Tre-1 and NlTre-2 kept stable since the ecdysis of the 5th instar macropterous female nymph. When 8 ng 20E/insect was applied (soon after ecdysis), an increase was found in the mRNA levels of N1tre-1 from the 4th day after ecdysis onward, while no increase of N1Tre-2 mRNA level was observed under the same conditions.4. The inhibitory effect of several xenobiotics against trehalaseSeveral potential trehalase inhibitors were tested at a dose of 50μM. Membrane-bound trehalase was inhibited to different contents, while no effects were found on soluble trehalase. At the dose of 50μM, both IPP03 and IPP04 showed a good inhibition against membrane-bound trehalase with the inhibitory rate of 72.49% and 79.20%, respectively. The IC50 value of IPP03 and IPP04 was 20.32μM and 18.21μM.Our study may have a positive effect on the reserch progress in insect trehalose metabolism and may pave a way for pesticide screening based on trehalase inhibitiors. The research objective will be beneficial both to the research on insect physiology and insecticide development.