| The rice stem borer,Chilo suppressalis(Walker)(Lepidoptera:Crambidae)is considered as the major pest of rice in paddy fields of Asia,Middle East and Southern Europe.In China,due to large scale cultivation of hybrid rice,the intensity of C.suppressalis damage has increased dramatically.The chemical insecticides have played a prominent role in the control of C.suppressalis,however,as a consequence of continuous exposure to insecticides,C.suppressalis has developed high levels of resistance against several classes of insecticides,including organochlorines,organophosphates(OPs),nereistoxin analogues,and fipronil.The anthranilic diamide insecticide,chlorantraniliprole(RynaxypyrTM),was discovered and developed commercially by Dupont Crop Protection.It is a highly potent activator of insect ryanodine receptors(RyRs)and shows exceptional selectivity for insect RyRs compared to mammalian RyRs.Due to its low mammalian toxicity and high insecticidal activity against lepidopteran insects,chlorantraniliprole has been widely used to control several lepidopteran pests including C.suppressalis since its introduction in China in 2008.In our previous study,exposure of third instar larvae of C.suppressalis to sublethal concentrations(LC10 and LC30)of chlorantraniliprole significantly extended the larvae and pupae duration,and decreased the pupation rates and fecundity.On the basis of these results,the objective of the present study was to evaluate the sublethal effects of chlorantraniliprole on the levels of juvenile hormone(JH)and molting hormone(MH)in C.suppressalis.The sublethal effects-associated potential mechanisms were also studied.The results might provide an important basis for the understanding of the mechanisms underling the sublethal effects of insecticides as well as for the rational use of Chlorantraniliprole.The effects of sublethal concentrations of chlorantraniliprole on the levels of JH and MH in C.suppressalis were studied using Enzyme Linked Immunosorbent Assay.The results showed that the JH levels in LC10 and LC30 treatment groups were significantly higher than the control group,while lower MH levels were observed in LC10 and LC30 treatment groups compared with the control group.At 72h after treatment of third instar larvae,the JH levels in LC10 and LC30 treatment groups elevated by 21.01%and 41.38%,respectively,when compared with the control group.The JH levels in the first,third and fifth day of femal pupae in LC10 treatment groups increased by 21.24%,12.63%and 7.54%,respectively,while that in LC30 treatment groups increased by 26.74%,20.12%and 14.31%,respectively,when compared with the control group.In female adults Oh,12h and 24h after emergence,the LC10 treatment resulted in increase of JH by 18.62%,50.2%and 51.57%,respectively,while the LC30 treatment resulted in increase of JH by 16.29%,49.66%and 45.41%,respectively,when compared with the control.On the other hand,at 72h after treatment of third instar larvae,the MH levels in LC30 treatment groups decreased by 11.25%compared with the control group.The MH levels in the first,third and fifth day of femal pupae in LC10 treatment groups decreased by 19.22%,21.31%and 17.07%,respectively,while that in LC30 treatment groups decreased by 13.89%,27.56%and 29.17%,respectively,when compared with the control group.In female adults Oh,12h and 24h after emergence,the LC10 treatment resulted in decrease of MH by 24.25%,15.26%and 33.83%,respectively,while the LC30 treatment resulted in decrease of MH by 27.85%,23.25%and 35.94%,respectively,when compared with the control.These results suggested that sublethal concentrations of chlorantraniliprole may disrupt the endocrine system and balance between JH and MH in C.suppressalis.Full-length cDNA sequences of JH biosynthesis relative genes including CsJHAMT.CsFPPS1、CsFPPS2 were cloned by RT-PCR and RACE.Sequence analysis showed that the 828bp ORF of CsJHAMT encoded 275 amino acid residues,while CsFPPSl and CsFPPS2 contain a 1287bp ORF and a 1161bp ORF encoding 428 and 386 amino acid residues,respectively.An amino acid sequence alignment shows that CsJHAMT shares the greatest amino acid identity with DpJHAMT in Danaus plexippus(55.64%)and is 36.69%and 36.24%identical in pairwise comparisons with TcJHAMT in Tribolium castaneum and DmJHAMT in Drosophila melanogaster,respectively.The deduced CsFPPS1 amino acid sequence showed 80.61%,51.64%and 47.32%identities to BmFPPS1 in Bombyx mori、TcFPPS1 in T.castaneum and DmFPPS1in D.melanogaste,respectively,while CsFPPS2 shared 48.12%and 49.10%amino acid identities with CfFPPS2 in Choristoneura fumiferana and BmFPPS3 in Bombyx mori,respectively.The real-time qPCR analysis revealed that compared with the control,the mRNA expression levels of CsJhamt、CsFPPS1 and CsFPPS2 had an increasing tendency,among which,the mRNA expression levels of CsJhamt in larvae of LC30 treatment group and the mRNA expression levels of CsFPPS2 in the fifth day femal pupae of LC10 and LC30 treatment groups as well as in female adults 12h after emergence in LC30 treatment group were significantly higher than that in control group.These results suggested that sublethal concentrations of chlorantraniliprole may affect the expression of JH biosynthesis genes,and in turn result in the elevation of JH level.RT-PCR was used to amplify the full length cDNA of ecdysteroid biosynthesis relative genes including CsPhm(CsCYP306A1)CsShd(CsCYP314A1)and CsSpo(CsCYP307A1).Sequence analysis showed that the 1617bp ORF of CsPhm encoded a protein of 538 amino acid,the 1551bp ORF of CsShd encoded a protein of 516 amino acid,while CsSpo contains a 1620bp ORF encoding a protein of 539 amino acid.An amino acid sequence alignment shows that CsPhm shares the greatest amino acid identity with S’lCYP306A1 in Spodoptera littoralis(79.59%)and is 36.22%and 36.47%identical in pairwise comparisons with TcCYP306A1 in T.castaneum and DmCYP306A1 in D.melanogaster,respectively.The deduced CsShd amino acid sequence showed 80.61%,51.64%and 47.32%identities to MsCYP314A1 in Manduca sexta.DmCYP314A1 in D.melanogaster and TcCYP314A1 in T.castaneum,respectively.CsSpo shared 79.11%.78.56%and 49.37%amino acid identities with HaCYP307A1 in Helicoverpa armigera,SlCYP307A1 in S.littoralis and DmCYP307A1 in D.melanogaster,respectively.The real-time qPCR analysis revealed that the mRNA expression level of CsPhm in the fifth day femal pupae of LC30 treatment group and the mRNA expression level of CsSpo in larval 72h after LC30 treatment were significantly higher than that in control group.However,no significant difference in the mRNA expression level was observed between control and other treatment groups.Thus the mechanisms involved in the decrease of MH by sublethal concentrations of chlorantraniliprole remain to be further studied. |
