| The striped rice stem borer, Chilo suppressalis (Walker) (Lepidoptera:Pyralidae) is one of the most important boring insect pests on rice in China. This pest distributes widely in China and can also damage numerous other cultivated crops besides rice, such as wild rice shoots. The control of C. suppressalis is mainly relied on chemical insecticides in China. Long-term and extensive use of insecticides provided well selection pressure for C. suppressalis to develop different levels of resistance to common insecticides. To prolong the useful life of insecticides, effective and logical resistance management tactics are desiderated to establish. Research of molecular mechanism for insecticide resistance in C. suppressalis is very important for the development of resistance management tactics. In this paper, the resistance levels of different field populations of C. suppressalis to selected insecticides were monitored. The esterase genes (EST) were cloned and analyzed. This together with others finished the genomic analysis of the detoxification enzymes of C. Suppressalis, including cytochrome P450 genes (CYP) and glutathione-S transferase genes (GST). Then, the responses of all 167 cloned metabolic enzyme genes to the induction of Ethofenprox were studied. The expression of these detoxification enzyme genes in selected populations of C. suppressalis with different resistance level was compared. Our research results could be helpful to understand resistance status of C. suppressalis in China and to implement effective resistance management tactics. 1. Monitoring the resistance of Chilo suppressalis to EthofenproxFirstly, two bioassay methods were compared by resistance test with a laboratory susceptible strain and a field population of C. suppressalis. The results indicated that the toxin overlay method with neonates was simpler and more sensitive, could be used for monitoring insecticide resistance in field populations of C. suppressalis. Then, the resistance to Ethofenprox, Butene-fipronil, Avermectins and Triazophos among seven populations of C. suppressalis from different rice planting area of China was tested by using the overlay method. The results showed that most geographic populations developed high level of resistance to Triazophos (48.0?RR? 128.8), except that Wanzhou population was susceptible (1?RR?4.0). However, the populations tested were all susceptible to Butene-fipronil and Avermectins (0.1?RR? 2.7), when compared to the laboratory susceptible strain. Among geographic populations, there exists obvious difference in tolerance to Butene-fipronil. The reason needs further analysis. The resistance levels of different field populations to Ethofenprox were similar to Avermectin. Most populations were susceptible (6.6?RR?8.8), except for Wanzhou and Jinhua populations which showed low level of resistance (0.6?RR?3.8). Thus, a conclusion could be drawn that most populations were high resistance to the insecticide, Triazophos, with longer using history, and kept susceptibility to the newly introduced insecticides or the insecticides with unique mode of action. As agent effecting on sodium channel, Ethofenprox can be used to increase the diversity of insecticides, which should be helpful in delaying the onset of resistance development by insecticide rotation and mixture. However, Ethofenprox showed lower toxicity in laboratory bioassay. So, it should be tested with field trails before recommendation to use.2. Clonning and analysis of esterase genes in C. SuppressalisEsterase is an important member of the three major metabolic detoxification enzymes. Based on the existing work in the laboratory and the data of transcriptome and genome from C. suppressalis,28 new esterase genes were searched out and validated. Plus 17 EST genes verified by another laboratory member before,45 esterase genes from C. suppressalis were obtained. Through BlastX, we obtained the esterase genes from other species with highest similarity to the 45 esterase genes from C.suppressalis. The 27 EST genes which were longer than 350 amino acids were selected and used to construct phylogenetic tree with the genes from other insects. Further analysis found that 7 members of the 27 esterase genes had orthologous genes in Heliothis armigera and one in Helicoverpa assulta, indicating phylogenetic relations and fitness evolution. Through cooperation, total 167 detoxification enzyme genes of C. suppressalis were identified based on the transcriptomic and genomic analysis. This found the base for further study on the specific function of each detoxification enzyme gene in C. suppressalis.3. Expression induction of metabolic enzyme genes in C. suppressalis by EthofenproxMetabolic enzymes in insect can be induced by exogenous substances, hi this study, larvae of C. suppressalis were treated by sub-lethal dose, and the inductive effect of Ethofenprox on the expression of CYP, EST and GST genes was analyzed. Checking with semi-RT PCR, the expression of 16 CYP genes and 14 esterase genes were found up-regulated. Then, these 30 genes were checked by using QT-PCR, and 8 esterase genes and 3 CYP genes were confirmed to be induced by Ethofenprox, as their expression increased significantly and more than 2 times that of the control group. These genes are CsuEst3 (2.1 times), CsuEst4 (2.6 times), CsuEst7 (2.4 times), CsuEstl5 (3.0 times), CsuEst24 (2.2 times), CsuEst25 (2.1 times), CsuEst37 (4.2 times), CsuEst44 (3.0 times), CYP341 (4.5 times), CYP341B (2.5 times) and CYP304F13 (2.0 times). This study implied that these detoxification enzyme genes may play a role in detoxification metabolism of Ethofenprox.4. Expression of metabolic enzyme genes in populations of C. suppresslis with different levels of Ethofenprox resistanceAccording to the results of resistance monitoring, the field populations with different levels of resistance to Ethofenprox, Nanchang (RR=1.2) and Jinhua (RR=8.8), were selected and compared for their expression of metabolic enzyme genes with the laboratory susceptible strain as control. The expression of 45 esterase genes,96 CYP genes and 26 GST genes were checked first by semiRT-PCR and then by QT-PCR. However, no detoxification enzyme genes among these populations were found related to Ethofenprox resistance. The reason should be the lower level of resistance. On the other hand, the expression of detoxification enzyme genes could be interfered by many factors in this case, such as the genetic background of field populations. But this research found that CsuEstl4 and CYP6AB50 in both wild populations, Jinhua and Nanchang, were significantly over-expressed as compared with the susceptible strain. It needs further studies to see if these could be the mechanism for the resistance of field populations of C. suppressalis to other chemicals. |
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