关键词: 西方蜜蜂, 蛹, 低温胁迫, 转录组, 差异表达基因, PDK1

Abstract: 【Aim】 This study aims to screen and analyze the differentially expressed genes (DEGs) across different pupal stages of Apis mellifera workers in response to low temperature stress at the transcriptomic level. 【Methods】 The 3-d-old post-capped prepupae, and 6- and 9-d-old post-capped pupae of A. mellifera workers were exposed to 20℃ for 4 h as the cold treatment groups (T), and those reared at 35℃ (their optimal developmental temperature) for 4 h as the control groups (CK). The DEGs in A. mellifera at the three developmental stages between the cold treatment groups and the control groups were screened by transcriptomic technology, followed by GO functional classification and KEGG pathway analysis. Subsequently, the expression profiles of 8, 6, and 5 DEGs from the 3-d-old post-capped prepupae, and 6- and 9-d-old post-capped pupae, respectively, were validated by RT-qPCR. 【Results】 There were 220, 50 and 26 DEGs between the 3-d-old post-capped prepupae and 6- and 9-d-old post-capped pupae exposed to low temperature and their control groups, respectively. GO functional classification of DEGs showed that the most enriched terms are related to metabolic process, cellular process, catalytic activity and binding. In addition, more DEGs in the 3-d-old post-capped prepupae were enriched in biological process regulation, cell part and organelle. KEGG pathway analysis showed that DEGs in A. mellifera at the three developmental stages between the cold treatment groups and the control groups were enriched in global and overview maps, amino acid metabolism, signal transduction, transport and catabolism. The expression of the shared DEG 3-phosphoinositide-dependent protein kinase 1 gene PDK1 in the 3-d-old post-capped prepupae and 6- and 9-d-old post-capped pupae exposed to low temperature was up-regulated and this gene was enriched in autophagy-animal, and mTOR and FoxO signaling pathways. In the 3-d-old post-capped prepupae in response to low temperature, the expression levels of insulin receptor substrate 1-B gene IRS1-B and Kruppel homolog 1 gene Kr-h1 were up-regulated, while those of the nuclear hormone receptor FTZ-F1 gene Ftz-F1 and ecdysis triggering hormone gene Eth were down-regulated significantly, suggesting that autophagy and ecdysis are inhibited to a greater degree in the 3-d-old post-capped prepupae in response to low temperature. The expression of tyrosine hydroxylase gene TyHyd related to tanning and immunity in the 6-d-old post-capped pupae exposed to low temperature was down-regulated. The number of DEGs identified between the 9-d-old post-capped pupae exposed to low temperature and the control group was the least, suggesting that the 9-d-old post-capped pupae are less affected by low temperature among the three pupal stages. 【Conclusion】 In this study, the DEGs in different developmental stages of A. mellifera pupa in response to low temperature were determined. The results show that most of DEGs are stage-specific, suggesting that different developmental stages of A. mellifera have different response mechanisms to low temperature. The function and mechanism of some shared and stage-specific DEGs are the key content to further study the low temperature response mechanism of honeybee, which provides basic data for exploring the molecular mechanism of the response of suggesting honeybee pupae to low temperature stress.

Key words:  Apis mellifera, pupae, low temperature stress, transcriptome, differentially expressed genes, PDK1