Resistance Monitoring To Insecticides In Chilo Suppressalis (Walker) And Cloning And Expression Analysis Of Ryanodine Receptor Genes In Sesamia Inferens (Walker)

The rice stem borer,Chilo suppressalis(Walker)(Lepidoptera:Pyrlidae)and the pink stem borer,Sesamia inferens(Walker)(Lepidoptera:Noctuidae)are important pests in rice fields,widely distributed in China's main rice-growing area.C.suppressalis damage more serious than S.inferens,and the latter usually occurr at peripheral part of paddy field.For a long period,chemical control remains a major measure for controlling the rice stem borer,the chemicals mainly include emamectin benzoate,abamectin,chlorpyrifos,triazophos,monosultap and the two diamide insecticides(chlorantraniliprole and flubendiamide).The diamides are the major chemical to control rice stem borers since 2008.However,because of long-term unreasonable application of insecticides,C.suppressalis have become resistant to many insecticides in most of geographical areas.In order to learn about the resistance development in C.suppressalis,we monitored the resistance of seven used insecticides in C.suppressalis.Then we also study the cross-resistance among the diamides for extending the usable life of this insecticides and making it play a better role in controlling the rice stem borer.In addition,we tested the sensibility of pink stem borer to chlorantraniliprole and flubendiamide for understanding the field control effects of the diamides insecticides.Ryanodine receptors is the main target of diamide insecticides,considering that there are no researches about the molecular mechanisms of diamide insecticides resistance in S.inferens,we studied on cloning and mRNA expression analysis of ryanodine receptor genes in S.inferens,which is helpful to provide the basis for further understanding of the mechanisms of resistance to diamide insecticides.1.Resistance monitoring to seven insecticides in C.suppressalisIn this study,resistance to chlorantraniliprole,flubendiamide emamectin benzoate,abamectin,chlorpyrifos,triazophos and monosultap of 59 C.suppressalis populations collected from 31 districts,10 provinces was monitored by the topical application and rice seedling dipping method in 2013-2015.The result showed susceptibility of the C.suppressalis field populations have obvious geographical differences,the resisitance of the rice stem borer from Zhejiang,Jiangxi and Hunan province were generally high,and the rice stem borer from Jiangsu,Shandong and Liaoning province were relatively susceptible,and in Liaoning province,the rice stem borer were all susceptible to the seven insecticides.In 2013-2015,there is a high resistant population to flubendiamide in Cangnan,Zhejiang province was found(138.4-fold),and twenty-one populations also developed low to medium level of resistance to flubendiamide;Yuyao,Cangnan,Xiangshan populations from Zhejiang province and Nancheng population from Jiangxi province showed medium level of resistance to hlorantraniliprole(10.9-77.6-fold),the other ten populations also were low level(5.7-8.9-fold).Comparing the data in 2013-2015,we found some populations from Zhejiang and Jiangxi province showed relative high resistane to chlorantraniliprole and flubendiamide,and Hanjiang,Yizhen and Huizhou populations from Jiangsu and Anhui provinces were relative susceptible to the two insecticides.Moreover,in order to study the difference between resistance levels to 97.3%chlorantraniliprole TC and 20%chlorantraniliprole SC,the resistance ratios to the two formulations in Yuyao and Xiangshan resistance populations were compared.The result showed higher resistance ratio to 20%chlorantraniliprole SC.In 2014-2015,most populations still remained susceptible or decrease in susceptibility to emamectin benzoate,abamectin and monosultap,which accounted for 78.2%,76.9%and 88.9%of monitoring populations.While Xiangshan and Jinhua populations from Zhejiang province,Nanchang and Shanggao populations from Jiangxi province and Dong'an population from Hunan province developed low to medium level of resistance to emamectin benzoate(5.6-22.5-fold);And low to medium level of resistance to abamectin was appeared in Cangnan and Yuyao populations from Zhejiang province,Nanchang and Shanggao populations from Jiangxi province and Dong'an and Youxian populations from Hunan province;Only Huizhou population from Anhui province,Dong'an population from Hunan province and Linyi population from Shandong province exhibited low to medium level of resistance to monosultap,and Shanggao,Cangnan population from Zhejiang province.Seven populations from Cangnan,Jinhua,Xiangshan,Zhejiang province,Shanggao populations,Jiangxi province and Dong'an,Hunan province showed medium level(11.2-67.6-fold).But to triazophos,Dong'an populations from Hunan province and Jinhua populations from Zhejiang province were already reached high level of resistance(216.8-and 209.9-fold).2.Cross-resistance to the diamides in C.suppressalisFor defining if there is cross-resistance among cyantraniliprole,SYP-9080,cyhalodiamide and chlorantraniliprole,flubendiamide,we determined the resistance of the high resistance(27.6 and 133.6-fold to flubendiamide and chlorantraniliprole respectively)C.suppressalis population collected from carried on research in Yuyao and Xiangshan 2014.The results showed the two populations have exhibited moderate or high level resistance to cyantraniliprole,SYP-9080 and cyantraniliprole(30.3-fold-120.7-fold).This indicated strong cross-resistance among cyantraniliprole,SYP-9080,cyhalodiamide and chlorantraniliprole,flubendiamide.3.Sensibility of S.inferens to chlorantraniliprole and flubendiamideIn 2014,subsceptitibility to chlorantraniliprole and flubendiamide of three S.inferens populations from Nanjing,Tongzhou populations from Jiangsu and Yuhang population from Zhejiang province was assayed by rice seedling dipping method.The result showed the toxicitiy of the two insecticides is is significantly different,susceptibility of S.inferens populations to flubendiamide were higher than chlorantraniliprole.But the susceptibility of S.inferens populations in different areas was no difference to the same insecticide.4.Cloning and expression analysis of ryanodine receptor genes in S.inferensFull-length RyR cDNAs were cloned from S.inferens using RT-PCR and RACE,and named as SiRyR,The composite NIRyR contains an ORF of 15428bp encoding 5118 amino acid residues,which shares 97%and 96%overall identity with Helicoverpa armigera and Spodoptera exigua respectively,and shares lower overall identity with Caenorhabditis elegans,Mus musculus,Homo sapiens(43%-50%)And by building phylogenetic relationships among SiRyR and other 21 species RyRs,we also found SiRyR showed close relatives to HaRyR and SeRyR.Prediction of SiRyR protein structure domain,there were seven kinds of protein structure domain,which included transmembrane domains,MIR domains,RIH domains,RIH-associated domain,SPRY domains,RyR domains and Identity helices in the family of ion transport proteins.In addition,The mRNA levels of SiRyR in different developmental stages and different body parts were analyzed using qRT-PCR.The result revealed that ryanodine receptor were expressed throughout the whole life cycle and the different body parts of S.inferens,and The relative expression levels of SiRyR in male adult was the highest among the whole life cycle.As in different body parts in S.inferens,thorax had the highest expression level.