Biochemical And Molecular Mechanisms For Imidacloprid Resistance In Laodelphax Striatellus (Fallen)

The small brown planthopper (SBPH), Laodelphax striatellus (Fallen), belongs to Delphacidae, Hemiptera, and distributes an extensive geographic range in Asia and the temperate zone of Europe where from the Philippines to Siberia. It infests important crops including rice, maize and wheat not only by nymphs and adults, but also transmission of plant virus. In recent years, population density and its damage intensity increased dramatically in Jiangsu, Zhejiang, Anhui and Shandong, which posed a serious threat to the trait of high and stable yield of the crop. Currently, control of L.striatellus relies mainly on chemical insecticides, while the development of resistance of L.striatellus to insecticides made chemical control face some problems. Therefore, insecticide resistance management strategies must be developed to prevent further increase in resistance and obtain higher control efficacy of L.striatellus. Lacking of the knowledge of resistance mechanism is the barrier for implementation of resistance management and efficient control. In this paper, the resistant strain was selected in laboratory and efforts have been made to declare its biochemical and molecular mechanism of resistance to imidacloprid. The results obtained are summarized as follows.1. The resistance selection and biochemical mechanism of imidacloprid resistance in L. striatellusBased on a field population of small brown planthopper collected from Jianhu in2010, an imidacloprid resistant strain (JH-R) with13.99-fold resistance and a susceptible strain (JH-S) were developed after24generations selection. The biochemical mechanisms of imidacloprid resistance in L.striatellus were first studied by analysis of detoxifying enzyme activity. The activities of3detoxification enzymes (Estersae, GST and MFO) in NJ-S and NJ-R strains were determined. The results showed that MFO activity of JH-R was1.88times of that in JH-S, significantly different between two strains. The GST activity was not significantly different between resistant and susceptible strains (0.96times). Though esterase activity in resistant strain was well higher (1.19times), but not significant statistically. To verify the results of enzyme activity assay, synergism experiments with TPP, DEM and PBO were carried out. All the three synergists displayed no obvious synergy effect on imidacloprid in JH-S strain, with synergism ratio below1.20. But in JH-R strain, except for the glutathione inhibitor DEM (1.11), the oxidase inhibitor PBO showed1.70-fold synergism and the esterase inhibitor TPP showed1.31-fold, both played obvious synergy effect. All aboved results together show that enhenced oxidase activity plays key role, esterase activity plays minor role, but the GST has no obvious effect in the metabolic resistance of L.striatellus to imidacloprid.2. The expression of Cytochrome P450genes in resistant and susceptible strains of L. striatellusThe specific primers for57P450genes were designed to analyze their relative expression levels in JH-S and JH-R of L.striatellus. The results obtained though semi-quantitative PCR showed that4P450genes, CYP4C72, CYP4C71v2, CYP6AY3v2and CYP353Dlv2had higher levels of relative mRNA expression in resistant strain. Then real-time quantitative PCR confirmed that CYP4C72, CYP4C71v2, CYP6AY3v2and CYP353Dlv2genes had1.67-,7.96-,8.94-,1.82-fold over-expression, respectively, when compared with those corresponding genes in JH-S strain, which was significant different in statistics and consistent with the results of semi-quatitative PCR. All of these suggested that overexpression of CYP4C71v2, CYP4C72, CYP6AY3v2, CYP353Dlv2might play important roles in conferring imidacloprid resistances of L.striatellus. Furthermore, semi-quatitative PCR was found a fast and effective way for high-throughout screening the metabolic detoxification enzyme genes.3. The induction effect of imidacloprid on different Cytochrome P450genes of L. striatellusThe semi-quantitative PCR was used to analyze the relative expression levels of the cytochrome P450genes following the inducing treatment with imidacloprid in JH-S strains of L.striatellus. The results showed that mRNA expression of CYP4C71, CYP4C71v2and CYP439Alv3genes reached a peak at about4h after the treatment with LD25dosage of imidacloprid. Then, the real-time quantitative PCR was used to confirm the inducibility of CYP4C71, CYP4C71v2and CYP439Alv3genes in different strains of L.striatellus, HX, JH, JH-S and JH-R. The results showed that all the three P450genes in various strains could be induced by imidacloprid, and the most obvious induction occurred in the resistant strain. These results suggested that CYP4C71, CYP4C71v2and CYP439Alv3might play important roles in the detoxification metabolism of imidacloprid in L.striatellus.