Molecular Responses Of The Larvae Of The Rice Stem Borer, Chilo Suppressalis Walker(Lepidoptera:Pyralidae) To Thermal Stress

The stem borer, Chilo suppressalis (Walker)(Lepidoptera:Pyralidae) is one of the most important pests of rice. This pest has been widely distributed in all rice fields of China, which is constantly adapting to its surrounding environment. In this paper, Hsps which is closely associated with Ch. suppressalis tolerance to thermal stress was studied. We measured the generation of reactive oxygen species (ROS), apoptosis rate in larvae haemocytes, antioxidant enzymes activities in the larval bodies of Ch. suppressalis caused by thermal stress, and further explored their protection mechanisms to oxidative stress and physiological adaptation to thermal stress tolerance. The main results are as follows:(1) In this research, RT-PCR and RACE (Rapid amplification of cDNA ends) methods were used for the cloning of full length cDNA encoding HSP60and HSP70from haemocytes of Ch. suppressalis. The full length cDNA of HSP60obtained2142bp, containing an ORF of1719bp, encoding572amino acid residues, with a5’UTR of158bp and a3’UTR of265bp. The length cDNA of HSP70obtained2102bp, containing an ORF of1959bp, encoding652amino acid residues, with a5’UTR of81bp and a3’UTR of62bp. Cluster analysis confirmed that HSP60and HSP70cDNA sequence shared high identity with the reported sequences from other insects. Also18sRNA gene of Ch. suppressalis was cloned as housegene in real-time quantitative PCR.(2) To investigate whether HSPs mRNA in Ch. suppressalis responds to thermal stress, the expression levels of HSP60、HSP70and HSP90mRNA in larvae haemocytes across temperature gradients from33-39℃were analysed by real-time quantitative PCR. The results indicated that thermal stress significantly elevated the HSPs gene expression levels. The temperatures for maximal induction of HSP60mRNA expression in the larvae haemocytes was at36-36℃. The temperatures for maximal induction of HSP70gene expression was at33-36℃, and there were no significant difference for HSP70gene expression at33-39℃.(3) In the present study, Flow Cytometry (FCM) was for the first time introduced to monitor HSPs expression in hemolymph cells of insect. HSP60, HSP70and HSP90expression in the larvae haemocytes of Ch. suppressalis was observed by using Flow cytometry. These results revealed that thermal stress significantly induced HSPs synthesis in larvae haemocytes, and the expression profiles of HSPs at the mRNA and protein levels are highly in agreement with each other, suggesting that their transcription and translation may be regulated in response to thermal stress.(4) ROS generation and apoptosis rate in larvae haemocytes, antioxidant enzymes activities in the larval bodies were measured, and HSPs along with antioxidant enzymes work together for cellular defense against thermal stress in Chilo suppressalis was analysed. These results indicated that thermal stress was accompanied by oxidative stress in which reactive oxygen species (ROS) were produced in haemocytes. A positive correlation was drawn between HSPs expression and ROS generation in these groups. However, the exposures to the target temperatures did not lead to any significant apoptosis differences in the larvae haemocytes. The increase of HSPs expression simultaneously with intensification antioxidant enzymes activity tempted us to speculate that both the defense systems work together for their greater resistance to thermal stress, which would be sufficient to counteract cellular damage induced by thermal stress.