| Cotesia chilonis（Matsumura）,the dominant parasitic wasp of the rice pest,Chilo suppressalis（Walker）,belongs to Hymenoptera,Braconidae,Cotesia.Temperature is one of the critical factors that affects insect growth and development and determines the number and distribution of insect populations.With the onset of global warming,Temperature affects not only pests but also natural enemies.Insects can actively adopt various survival strategies to avoid and adapt to the maladaptive temperature when they are exposed to adverse temperatures.For example,changing the progress of growth and development,reproduction mode,regulating temperature-responsive genes and so on.Heat shock transcription factor protein-mediated regulation of heat shock proteins in response to adverse temperature stress is considered to be the main process by which organisms respond to adverse temperature.In addition,studies have also found that there are other genes related to the main pathways in the organism are also involved in the regulation.Therefore,in this study,we consider the influence of temperature stress on C.chilonis to explore the effect of high temperature stress on its growth,development and reproduction,to clarify and explore the high temperature response characteristics of C.chilonis,Meanwhile,temperatureinduced genes were cloned and their expression patterns and functions were explored and analyzed.And we tried to establish the RNAi technology system of C.chilonis and initially revealed the function of Caspase-1.The main results are as follows:1.According to the previous research results,36℃ was selected to simulate the field temperature that C.chilonis may encounter in nature.The research showed that high temperature inhibited growth and development.The developmental duration of C.chilonis was generally shortened in response to high temperatures,the body length and head width of C.chilonis larvae were reduced,cocoon numbers were slightly reduced at 36℃,while the emergence rate of C.chilonis was lower,Although the number of males,females and adults declined in the 36℃ treatment relative to 27℃,the ratio of females to males increased at 36℃.2.According to the transcriptomic data,the full-length cDNA sequences of five temperature-induced genes were isolated and cloned from C.chilonis,including one sHSP,one HSF,two IAPs and one Caspase,and named Cchsp11.0,Cchsf,Cciap1,Cciap and Cccaspase-1,respectively.Their complete cDNA sequences were 508,2073,1404,1184 and 831 bp,including 306,1875,1155,951 and 585 bp open reading frames,encoding 101,624,384,316 and 194 amino acids,respectively.Genomic validation found that the three genes all contained no introns.CcHSP11.0 contained a chaperonin cpn10 signature motif but lack the typical α-crystalline domains,CcHSF possessed DBD,HR-A/B,HR-C and CTAD domains.Both CcIAP1 and CcIAP contain two BIR domains;however,only CcIAP1 contains a RING domain.CcCaspase-1 contains a highly conserved amino acid motif QACQG.Phylogenetic tree analysis found that CcHSP11.0,CcHSF,CcIAP1,CcIAP and CcCaspase-1 had high relationship to other Hymenopteran insects.3.The expression patterns of Cchsp11.0,Cchsf,Cciap1,Cciap and Cccaspase-1 were analyzed by RT-qPCR technique.The results found that Cchsp11.0 and Cchsf only could could be induced by low temperature,and the relative expression levels reached highest at6℃,while Cciap1,Cciap and Cccaspase-1 could be induced by high and low temperature,and the relative expression levels reached highest at-6 and 30 ℃.Exposed to continuous low temperature at-6℃ and high temperature at 36℃,the relative mRNA expression of Cchsp11.0 and Cchsf were both reached the highest after 2 h,and the expression pattern of them were positively correlated during temperature stress.Overall,the induction of Cchsf was lower than Cchsp11.0 at low temperatures,whereas the opposite was true at high temperatures.the relative mRNA expression of Cciap1,Cciap and Cccaspase-1 were significantly upregulated at 4 h and reached the highest,indicating the expression of five proteins were closely related to temperature.The highest expression level of five temperature-induced genes was in 1st instar larvae of C.chilonis at 27℃ while that in 3rd instar larvae of C.chilonis at 36℃,indicating high temperature would stimulate C.chilonis and lead to an earlier response to temperature at different developmental stages.And the expression levels of Cciap1,Cciap and Cccaspase-1 were significantly increased in 2nd instar larvae,showing that the activity of Caspase in insects is regulated by IAPs.4.We constructed the RNAi technology system and successfully silenced the expression of Cccaspase-1 in C.chilonis.The discovery of silencing Cccaspase-1 by microinjection interfered with different pupal stages of C.chilonis showed that the silencing efficiency was 37.86%,36.05%,36.40%,41.34%and 35.39%,respectively.And the silencing efficiency in 72-96 h（4 days）was the highest;the results of nanomaterial immersion showed that the nanomaterial/dsCsIAP1/detergent solution successfully penetrated the body wall cavity of C.suppressalis.And the silencing efficiency of Cccaspase-1 expression was 45.63%.There is little difference in silencing efficiency between the two methods.We selected 4-day pupal stage with the highest silencing efficiency for IAP detection by microinjection and nanomaterial immersion.The results showed that whether by microinjection or nanomaterial immersion,the expression levels of Cciap1 and Cciap were significantly up-regulated in C.chilonis after interfering with Cccaspase-1.The total number of offspring,the emergence rate of pupae on day 1-4 and the number of cocoons were also significantly reduced,indicating that and the absence of CcCaspase-1 had a significant effect on the expression of Cciaps. |
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