Abstract: 【Aim】 This study aims to analyze the possible coping mechanism and the adverse effects in response to low temperature stress during the middle pupal stage of Apis mellifera workers. 【Methods】 The 8-d-capped broods of A. mellifera workers were exposed to cold temperature (20℃) for 96 h (T), with those unexposed to cold stress used as the 
control (CK), and then subjected to transcriptomic sequencing (RNA-seq) and differentially expressed gene (DEG) screening. In addition, GO classification and KEGG pathway analyses of DEGs were performed. The expression levels of  five randomly selected DEGs were verified by RT-qPCR. 【Results】 A total of 1 101 DEGs were detected in the 8-d-capped brood workers of A. mellifera exposed to cold temperature (20℃) for 96 h. GO classification showed that the GO terms with the largest number of DEGs were metabolism process (142 DEGs) and cellular process (142 DEGs), followed by binding (131 DEGs), catalytic activity (120 DEGs) and single-organism process (118 DEGs). The KEGG pathway analysis result showed that DEGs were significantly enriched in peroxisome pathway, D-glutamine metabolic pathway, D-glutamate metabolic pathway, and glutathione metabolic pathway, which are related to oxidative damage. In addition, DEGs were also significantly enriched in insect hormone metabolism pathway, mTOR signaling pathway and FoxO pathway related to hormone regulation. The results of RNA-Seq and RT-qPCR of the five randomly selected DEGs were consistent. 【Conclusion】 In this study, it has been found that the mid-staged pupae of A. mellifera cope with low temperature via regulating endogenous hormones, slowing down or stopping the development. In the pupae oxidative damage may accumulate by reducing the expression of antioxidant enzymes in low temperature stress, consequently causing detrimental effects on honeybees after emergence. The results of this study are helpful to understand the coping 
mechanism to low temperature in insects with narrow temperature ecological amplitude during developmental stage and the adverse effects of low temperature on them.

Key words: Apis mellifera, mid-staged pupae, low-temperature stress, differentially expressed genes, oxidative damage