Molecular Metabolic Mechanism For Monosultap Resistance In Chilo Suppressalis

The striped rice stem borer, Chilo suppressalis (Walker) (Lepidoptera:Crambidae) is an important insect pest of rice in China. It had outbroken from the end of last century to 2005. Since then, the damages of rice stem borer occured continuously in medium level each year, which had a serious impact on China’s yield of rice. The control of C. suppressalis is mostly relied on chemical insecticides. As the limitation of insecticides applying on rice, few insecticides were available and usually intensively used. This accelerated the development of insecticide resistance in C. suppressalis, which made the field control even difficult.Monosultap, a kind of nereistoxin insecticide, can combine with nACHR in insects, which blocks signal transmission in nervous system and results in insect death. With good selective and systematic character, monosultap is environment friendly and high toxic to lepidopteran pests, especially for rice borers. It has been used as the major insecticide for borer control since putting into commercials. Though monosultap resistance out-broke regionally at the end of last century, this insecticide is still effectively used in most centre, western and northern China. Moreover, there was no new nereistoxin insecticide commercialized. Thus, study on the molecular mechanism for monosultap resisrance in C. suppressalis has a great significance.Insecticide resistance in arthropods has been shown evolved by three main mechanisms, the enhanced production of metabolic enzymes, which can break down or bind to the insecticide, structural changes in the target protein, which make it less sensitive to the insecticide, and modification of insect cuticular proteins, which leads to a slower penetration of the insecticide entering into the insect. Three metabolic enzyme families mainly involved in the metabolic resistance are cytochrome P450 monooxygenase (P450), esterases (EST) and glutathione S-transferases (GST). In order to find out the metabolic enzyme genes which might be implicated in monosultap resistance in C. suppressalis, the resistance of C. suppressalis collected from different geographic populations was firstly monitored. Then,26 GST genes and other main metabolic enzymes genes in C. suppressalis were cloned by cooperation. At the same time, sublethal dose of monosultap was used to induct P450, EST and GST genes which were possibly involved in monosultap detoxification in C. suppressalis. Finally, the gene expression differences were compared at the transcriptional level among different populations. The results are showed as following which founded a base for study on the molecular mechanism for monosultap resistance in C. suppressalis.1. Monitoring of insecticide resistance in field populations of C. suppressalis.Firstly, this study tyied to select a better method for monitoring the insecticide resistance in C. suppressalis by comparison of the topical application treatment and diet surface overlay bioassay with a laboratory susceptible strain and Nanchang field population. The result showed that these two methods were consistent to make order of various populations according their resistance level. However, diet surface overlay bioassay was more sensitive, and more obvious results could be obtained with it. Then, with the diet surface overlay bioassay, the resistance of C. suppressalis collected from various areas of China was monitored. The population of Yangzhou was found susceptible to monosultap in 2012(RR 3.8). But the other populations monitored in 2012, Nanchang, Jinhua, Wenzhou and Hexian, all showed moderate resistance to monosultap in 2012 (RR 13.2-29.5). In 2013, among 7 populations, only the populations from Wanzhou and Yangzhou showed low resistance to monosultap(RR 9.0,7.7), and the other 5 populations from Guilin, Xiangtan, Nanchang, Jinhua and Wuhan had all moderate level of resistance (RR 13.5-21.7). Profenofos resistance ranged from moderate to high level in all populations tested (RR 33.3-86.5) except for Wanzhou (RR 0.9). The populations from Wanzhou, Wuhan and Guilin were susceptible to chlorpyrifos (RR<1.4); the other populations had low resistance to chlorpyrifos (RR 3.6-8.8). It was found that most populations were susceptible to a new insecticide chlorantraniliprole(RR 1.0-1.9), only Jinhua and Nanchang showed low resistance (RR 9.4,5.3).2. Cloning and homology analysis of GST genes in C. suppressalis.Glutathione S-transferase is a supergene family, which is widely distributed in creatures, and is an important detoxification enzyme. More than one hundred of GST genes have been detected in over 20 insects. The GST genes are sorted into several classes, such as Delta, Epsilon, Omega, Sigma, Theta and Zeta. Of which, delta and epsilon GST classes are unique in insect. In this study, the GST genes in C. suppressalis were first searched from the transcriptome and genome data. Then,26 GST genes from C. suppressalis were successfully cloned. The similarity of GST sequences from C. suppressalis and other species were analysised, including Danaus plexippus, Bombyx mori, Helicoverpa assulta, Papilio xuthus and Manduca sexta. The result showed that among 26 GSTs from C. suppressalis,23 belong to Delta(6), Epsilon(6),Omega(4), Sigma(1), Theta(4) and Zeta(2) classes, and the rest 3 belong to unclassified. Besides, phylogenetic relationship of GSTs between C. suppressalis and Bombyx mori was also analyzed.3. The induction effect of monosultap on metabolic enzyme genes of C.suppressalisIn this chapter, the 4 instar larvae of C.suppressalis were treated with sublethal dose of monosultap, and the changes in the expression of their metabolic enzyme genes (P450, EST and GST) were checked to see which gene could be induced at transcriptome level. Firstly, the relative expression levels of the 96 P450 genes,45 EST genes and 26 GST genes were screened by the semi-quantitative PCR. The result showed that 14 P450 genes, 4 EST genes and 2 GST genes were up-regulated. Then, the real-time quantitative PCR (RT-PCR) was used to confirm the inducibility of the 19 metabolic enzyme genes in C. suppressalis. Similar with semi-quantitative PCR, RT-PCR indicated that all the 19 metabolic enzyme genes expressed more in the treated larvae. But it confirmed that only 13 of them were significantly induced, and to more than 2 folds expression. They are:P450 (CYP341, CYP9A52, CYP340, CYP338A1, CYP341B, CYP4G92, CYP321F1, CYP304F13, CYP6AB13 and CYP6AB46), and EST (CsEst4, CsEst42 and CsEst44). While, only one GST gene named CsGST1 was significantly induced, but only to 1.38 folds. These implied that these up-regulated genes might be responsible for the detoxification metabolism of monosultap in C. suppressalis.4. Expression of P450, EST and GST genes in different populations of C. suppressalis.To explore the metabolic mechanism of monosultap, the expression of P450, EST and GST genes in populations from Yangzhou (RR=7.7), Nanchang (RR=13.5) and Jinhua (RR=19.0) were examined. Firstly, the relative expression levels of the 96 P450 genes,45 EST genes and 26 GST genes were screened by the semi-quantitative PCR. The result showed that there were obvious expression differences in 7 P450 genes,7 EST genes and 2 GST genes among different strains. Secondly, these genes were examined by RT-PCR to confirm if they had statistical difference among these three strains of C.suppressalis. The result showed that 12 genes had significant difference in transcriptional level:6 P450 genes(CYP4L27, CYP304F13, CYP354A, CYP6AB13, CYP9A68 and CYP9A69),5 EST genes(CsEST14, CsEST25, CsEST37, CsEST4 and CsEST44)and 1 GST gene (CsGSTT). Besides, CYP9A68 showed 9.8 folds over-expression in Jinghua population and CsEST37 showed 9.2 folds over-expression in Nanchang population as compared with Yangzhou population. Discussion came to the conclusion that the higher detoxification ability of P450 and EST might played important role in the resistant field population of C. suppressalis.