Investigating the Regulation of Hypopharyngeal Gland Activity in Honeybees Undergoing Harsh Winter Conditions through RNA-seq Transcriptomics Combined with iTRAQ-based Comparative Proteomics. Apis mellifera

The honeybee model organism, Apis mellifera carnica, is a hymenopteran with a remarkable ability to withstand cold winters by instinctively entering into hibernation in the hive. Hibernation is characterized by a significant reduction in the metabolic rate, triggered by a drop in the ambient temperature and a shortage of reserves. It provides a token cue that initiates an alternative developmental program that leads to dormancy and switches social behaviors. The age-dependent changes in the generic proteins and substance composition of royal jelly (RJ) have been well documented. However, the molecular mechanism of RJ secretion and the biological consequences of regulation by the hypopharyngeal gland (HG) according to the season remain to be addressed. In this study, large-scale transcriptomic and proteomic approaches were used to detect statistically significant changes in the honeybee proteome and to explore the mechanistic basis for the HG regulation that accompanies hibernation in the winter and arousal in the spring. HG activity was associated with hundreds of gene and protein expression changes in the winter-spring cycle. A systematic study of HG activity and secretion identified 21,441 genes and 2,156 proteins at three time points (i.e., A, winter bees; B, spring bees; and C, retired bees). Of these, 737 genes and 698 proteins were qualified and compared to reveal differential expression patterns, including 197 up-regulated and 291 down-regulated DEGs in group A vs. B, 118 up-regulated and 259 down-regulated DEGs in group A vs. C, and 106 up-regulated and 53 down-regulated DEGs in group B vs. C, respectively. The results were validated by qPCR.Honeybee HG activity was associated with multiple genes regulation.The results present the first global pictures of the regulation of HG activity in winter bee. Overall design: The 3 samples at given time (A,winter bee; B,spring bee; and C, retired bee) are collected to reveal the mechanism of the regulation of HG activity and RJ secretion, the differentially expressed genes involved in the Metabolic process and protein processing in endoplasmic reticulum related DEGs were identified and the shifts appeared to show dormancy-induced transcriptional changes across the winter. 30 pooled heads of each samples were pooled as one biologiacl repeat.