Abstract: 【Aim】To investigate the change in expression patterns of genes and the cold resistance mechanisms of the western honey bee (Apis mellifera) during the overwintering period, so as to provide better guidance for overwintering management of bee colonies. 【Methods】 Differentially expressed genes (DEGs) in four subspecies of A. mellifera, including A. m. caucasica, A. m. mellifera, A. m. ligustica and A. m. carnica during the overwintering period were identified and compared using transcriptomics and subsequently subjected to GO and KEGG enrichment analyses. The reliability of transcriptome sequencing results was verified by qRT-PCR. 【Results】 Changes in the expression levels of DEGs in four subspecies of A. mellifera possessed distinctive characteristics during the overwintering period. The expression levels of DEGs changed actively in A. m. ligustica from the initial overwintering period to the middle overwintering period, and in A. m. carnica and A. m. mellifera from the middle overwintering period to the anaphase overwintering period. For A. m. caucasica, the expression levels of DEGs were relatively stable throughout the overwintering period. There were differences in the expression patterns of DEGs in the four subspecies of A. mellifera at the initial overwintering period. The expression levels of genes of cuticular protein 22, vitellogenin, odorant-binding protein 14, heat shock protein 70, and cytochrome P4509e2 in A. m. caucasica and A. m. mellifera were higher than those in A. m. ligustica and A. m. carnica. The expression levels of genes of chitinase, defensin 1, retinol dehydrogenase and cytochrome b5 in A. m. ligustica were higher. The expression levels of DEGs in A. m. carnica were lower. According to the expression trend of DEGs, the expression levels of 
heat shock protein and ATP synthase subunit genes in A. m. ligustica and A. m. carnica were upregulated, and those of cuticular protein 22, vitellogenin, and cytochrome b/c genes in A. m. caucasica and A. m. mellifera were downregulated during overwintering. The results of enrichment analysis by GO and KEGG indicated that the DEGs in A. m. ligustica, A. m. carnica and A. m. caucasica from the initial overwintering period to the middle overwintering period were enriched in the redox processes and metabolic pathways of glycolysis/gluconeogenesis, oxidative phosphorylation and TCA cycle pathways, and the enriched DEGs in A. m. ligustica and A. m. carnica were upregulated and those in A. m. caucasica downregulated. The DEGs in A. m. mellifera were mainly enriched in amino acid metabolic pathway during the overwintering period and were involved in the Hippo, FoxO and Hedgehog signaling pathways. The qRT-PCR validation result was the same as the expression trend of RNA-Seq, indicating that the transcriptome sequencing data are reliable.【Conclusion】 The cold resistance mechanisms of the four subspecies of A. mellifera were preliminarily determined in this study, indicating that glycogenolysis decomposition in A. m. ligustica and A. m. carnica is enhanced during overwintering period to provide energy, whereas A. m. caucasica decreases the decomposition of glycogen before overwintering, suggesting that the cold resistance mechanisms in A. m. caucasica and A. m. mellifera may be the same by accumulating cold resistance substances in the body before overwintering and releasing energy gradually during the overwintering period.

Key words: Apis mellifera, transcriptome, overwintering, cold resistance, differentially expressed genes, functional annotation