| The rice stem borer, Chilo suppressalis (Walker) (Lepidoptera:Crambidae) is one of the most damaging rice pests in Asia, Middle East and Southern Europe. Currently, the control of C. suppressalis mainly depends on chemical pesticides, but with the unreasonable use of chemical pesticides, C. suppressalis has developed resistance to conventional insecticides including triazophos, monosultap, fipronil. The novel anthranilic diamide insecticide, chlorantraniliprole (RynaxypyrTM), was discovered and developed commercially by Dupont Crop Protection, and showed high insecticidal activity against lepidopteran insects, Since its introduction in China in 2008, chlorantraniliprole has been widely used to control several lepidopteran pests including C. suppressalis. The objective of the present study was to evaluate the sublethal effects of chlorantraniliprole on development and reproduction as well as thermotolerance of C. suppressalis. 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.Sublethal concentrations of chlorantraniliprole (LC10 and LC30) of chlorantraniliprole against the third instar larvae were determined via the diet incorporation method, and the sublethal effects of chlorantraniliprole on development and reproduction of C. suppressalis were investigated. The results showed that the development durations of larvae treated with LC10 (16.15 days) and LC30 (17.14 days) were significantly delayed compared with the control (14.80 days). The pupae duration was not affected by LC10 treatment (7.05 days), but significantly prolonged after exposure to LC30 (9.03 days) when compared with the control (6.66 days). While no significant difference of female adult longevity was observed between treatments and control, male adult longevity was significantly shortened after treatments with both LC10 and LC30. The mean weight of larvae treated with LC10 (5.72 mg) and LC30 (3.40 mg) was significantly lower than the untreated control (12.47 mg). The mean weight of male pupae in treatment groups of LC10 (46.74 mg) and LC30 (44.94 mg) was significantly lower than the control (50.49 mg). Similarly, the mean weight of female pupae in treatment group of LC30 (49.31 mg) was significantly lower than the control (59.79 mg), but no significant difference in female pupal weight was observed between LC10 treatment and control. The pupation rates in treatment groups of LC10 (41.30%) and LC30 (23.98%) were significantly lower than the control (71.86%), but adult emergence rate was not significantly affected by LC10 and LC30 treatments. The LC10 and LC30 chlorantraniliprole significantly decreased number of eggs laid per female by 32.18% and 52.94%, respectively, but the sex ratio of adult C. suppressalis as well as hatching rate of the eggs was not significantly different from that of control individuals.RT-PCR and RACE were used to amplify the full length CsVg cDNA from C. suppressalis. Compilation of the cDNA clones resulted in a 5,732 bp contiguous sequence containing a 5,373 bp ORF. The encoded 1,790 amino acid residues predict a protein with a calculated molecular mass of 204.63 kDa. Analysis of the CsVg amino acid sequence reveals a multi-domain protein organization including a Vitellogenin_N domain (pfam01347) at positions 32-742, a DUF1943 domain (pfam09172) at positions 775-1056, and a VWD domain (pfam000940) at positions 1460-1626. The cleavage signal RVRR was found in the CsVg near the NH2-terminus within amino acid positions 362-365. Two polyserine regions were located at positions 334-345 and 387-397, respectively. An amino acid sequence alignment shows that CsVg shares high amino acid identity with other lepidopteran Vg homologues including CmVg in Cnaphalocrocis medinalis (AEM75020,59%), HaVg in Helicoverpa armigera (AFV40972,52%), and SIVg in Spodoptera litura (ABU68426,50%). Additionally, in the COOH-terminal region, the DGXR motif (1591-1594) and GL/ICG motif (1608-1611) followed by six cysteine residues were highly conserved among CsVg and other lepidopteran Vgs. To gain understanding into the developmental expression of CsVg in C. suppressalis, the mRNA levels of this gene was analyzed using RT-qPCR at various female pupal and adult developmental stages of C. suppressali. The developmental expression pattern revealed that CsVg was expressed at all developmental stages tested. The mRNA expression levels of CsVg gradually increased during pupal development, reached peak in female adults 12h after emergence, and significantly decreased in female adults 24h after emergence. Compared with control, expression levels of CsVg mRNA after exposure to LCio and LC30 chlorantraniliprole decreased by 42.52% and 47.84%, respectively. Significantly decreased expression was also observed in female 24-h-old adults after exposure to LC30 of chlorantraniliprole, while there is no significant difference between control and LC10 treatment.The sublethal effects of chlorantraniliprole on thermotolerance of C. suppressalis was studied, and the result revealed that after treated with LT50, the larvae exposed to LC10 and LC30 concentrations of Chlorantraniliprole showed significantly higher mortality compared with control. Further analysis of mRNA expression of heat shock protein genes and arginine kinase gene of C. suppressalis showed that, after the treatment of 42?, the relative expressions of hsp60 and hsp19.8 in larvae exposed to LC10 and LC30 concentrations of Chlorantraniliprole significantly increased, the relative expressions of hsp60 in treatment groups of LC10 and LC30 were 2.05 and 2.13 times higher than that in control treatment, respectively, and the relative expressions of hspl9.8 in treatment groups of LC10 and LC30 were 1.92 and 2.56 times higher than that in control treatment, respectively. When exposed to LC10 concentration of Chlorantraniliprole, the relative expressions of hsp70 and hsp21.7a were 2.27 and 2.24times higher than that in control treatment, respectively. There were no significantly differences in the expression levels of hsp90, hsp21.7b, hsp21.4 and hsp21.5 compared with the control. Additionally, the expression levels of CsAg mRNA after exposure to LC10 and LC30 chlorantraniliprole decreased by 33.12% and 62.01%, respectively, compared with the control. |
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