Insecticide Resistance Monitoring Of Spodoptera Frugiperda And Sublethal Effects Of Chlorantraniliprole

Spodoptera frugiperda(J.E.Smith)exhibits the characteristics of wide host range,high reproductive and migratory capacity.Since its invasion into China in 2019,S.frugiperda has caused serious damage to crops in China.Insecticides can quickly and effectively control S.frugiperda,but the large use of insecticides makes S.frugiperda rapidly develop resistance.Therefore,it is urgent to monitor the resistance status of S.frugiperda to commonly used insecticides and screen effective control agents.Chlorantraniliprole is a relatively new anthranilic diamide insecticide,which acts on the ryanodine receptor(RyR)of insects and has excellent insecticidal activity against lepidopteran pests.In this paper,the RyR gene was cloned from S.frugiperda,and the resistance monitoring of field populations of S.frugiperda in Jiangsu Province was carried out.On this basis,the sublethal effect of chlorantraniliprole on S.frugiperda and its potential mechanism were studied.The S.frugiperda RyR gene SfRyR was cloned by RT-PCR.The open reading frame(ORF)of SfRyR was 15246 bp,encoding 5081 amino acid residues.The predicted protein molecular weight was 574.65 kDa and the isoelectric point was 5.43.Amino acid sequence alignment showed that SfRyR had 95%identity with RyR of Cnaphalocrocis medinalis,79%identity with RyR of Bemisia tabaci and 78%identity with RyR of Drosophila melanogaster.Phylogenetic analysis also showed that SfRyR had the closest relationship with other lepidopteran insects.The susceptibility baseline of S.frugiperda to seven insecticides was established using diet incorporation method.It was found that the toxicity of seven insecticides to S.frugiperda was different.Among them,emamectin benzoate had the highest toxicity with the LC50 of 0.013 mg/L,and lambda-cyhalothrin had the lowest toxicity with the of LC50 of 45.633 mg/L.The LC50 values of lufenuron,chlorfenapyr,chlorantraniliprole,cyantraniliprole and indoxacarb were 1.134 mg/L,1.473 mg/L,4.350 mg/L,4.875 mg/L and 6.339 mg/L,respectively.The resistance levels of S.frugiperda field populations to seven insecticides were monitored in Hanjiang,Jurong,Rudong,Xinghua,Dafeng,Suining,Zhangjiagang,Changshu and Liuhe of Jiangsu Province.The results showed that the field populations in different regions had no obvious resistance to chlorantraniliprole,cyantraniliprole,indoxacarb,lambdacyhalothrin and lufenuron.S.frugiperda population in Jurong had a low level of resistance to emamectin benzoate,with a resistance ratio of 7.77-fold,whereas other populations were relatively susceptible to emamectin benzoate.The resistance ratios of S.frugiperda to chlorfenapyr were 6.28-fold and 5.08-fold in Jurong and Xinghua populationj s,respectively.Other populations were relatively susceptible to chlorfenapyr.The effects of sublethal concentrations of chlorantraniliprole(LC10,0.021 mg/L and LC30,0.494 mg/L)on the growth,development and reproduction of S.frugiperda were studied using diet incorporation method.The results showed that the weight of larvae treated with sublethal concentrations of chlorantraniliprole decreased significantly,and the development time of larvae in the LC 10 and LC30 treatment groups increased significantly compared with the control group(12.34 days),prolonged by 3.65 days and 5.74 days,respectively.LC10 and LC30 treatments resulted in significantly lower male pupal weight(207.28 mg and 208.29 mg)compared with the control group(232.23 mg),and female pupal weight(197.65 mg)in the LC30 treatment group was also significantly lower than the control group(226.70 mg).Compared with the control group,the pupal stage of LC30 treatment group was significantly prolonged.In addition,the pupation rate(71.26%)and eclosion rate(86.43%)of the LC30 treatment group were significantly lower than those of the control group(pupation rate 92.34%,eclosion rate 94.97%).The average fecundity of female adults in LC10 and LC30 treatment groups decreased by 15.68%and 32.70%,respectively,compared with the control group(1369 eggs).Similarly,the egg hatching rate of LC30 treatment group was 15.49%lower than that of control group(88.8%),whereas there was no significant difference between LC10 treatment group and control group.The juvenile hormone esterase gene SfJHE and ribosomal protein S6 kinase gene SfS6K1 were cloned by RT-PCR.The open reading frame(ORF)of SfJHE was 1683 bp in length,encoding 560 amino acid residues containing five highly conserved motifs RF,DQ,GQSAG,E and GxxHxxD/E.The predicted molecular weight was 63.13 kDa and the isoelectric point was 8.45.The ORF length of SfS6K1 was 1362 bp,and the encoded 453 amino acid residues had a conserved kinase domain and an extension domain.The predicted molecular weight was 60.94 kDa and the isoelectric point was 5.69.Amino acid sequence alignment showed that SfS6K1 had 87.3%,74.9%and 67.6%identities with PxS6K1 in Plutella xylostella、DmS6K1 in Drosophila melanogaster and NIS6K1 in Nilaparvata lugens,respectively.Phylogenetic analysis showed that SJS6K1 was clustered with other Lepidopteran S6K1s.The effects of LC30 chlorantraniliprole treatment on juvenile hormone(JH)titer and SfJHE mRNA expression level of S.frugiperda were analyzed by HPLC/MS-MS and RT-qPCR.The results showed that compared with the control group,the JH titer of the LC30 treatment group increased after 24 h and 48 h of treatment,but there was no significant difference,while the JH titer after 72 h of LC30 treatment was significantly increased by 0.94 times compared with the control group.RT-qPCR analysis showed that the mRNA expression levels of SfJHE were significantly down-regulated by 49.14%and 50.13%at 48 h and 72 h after LC30 chlorantraniliprole treatment.The effects of sublethal concentrations of chlorantraniliprole on the mRNA expression levels of SJS6K1 gene and the vitellogenin gene SfVg in S.frugiperda were further analyzed by RT-qPCR.It was found that the relative expression levels of SfS6K1 mRNA were downregulated by 45.85%,21.57%and 44.75%,and the relative expression levels of SfVg mRNA were significantly down-regulated by 56.04%,68.64%and 34.55%at 24 h,48 h and 72 h after adult emergence,respectively.In addition,the treatment of female adults with S6K inhibitor PF-4708671 also led to a significant decrease in egg production and the mRNA expression level of SfVg.Taken together,in this paper,the insecticide resistance levels of S.frugiperda field populations in different regions of Jiangsu Province was monitored,the sublethal effects of chlorantraniliprole on the growth and reproduction of S.frugiperda were studied,and the putative mechanisms was explored.The results have applied implications for the resistance management and rational control of S.frugiperda.