Abstract: 【Objective】The principal biological function of worker bees’ mandibular glands is to secrete fatty acids to provide the nutrition for the colony and participate in synthesis of alarm pheromone. High royal jelly producing bees (RJBs, Apis mellifera liguatica) and Italian bees (ITBs, Apis mellifera liguatica) are both major honeybee species in China, but the mechanism of regulating the development and function of mandibular glands by phosphoproteome is not reported yet. The objective of this study is to compare the differences of mandibular glands of phosphoproteome, reveal the mechanism of the regulation of mandibular glands by phosphoproteome and fatty acids metabolism.【Method】The mandibular glands of newly emerged, nurse, and foragers of RJBs and ITBs were collected, then after, mandibular gland proteins were extracted and digested by an enzyme. Phosphopeptides were enriched by IMAC (immobilized metal-affinity chromatography) and desalted by using Zip-tip C18 columns. Peptides from each of the samples were analyzed by LC-MS/MS (liquid chromatography-mass/mass). Furthermore, MaxQuant and Perseus bioinformatics tools were used to evaluate phosphoproteome quality. These bioinformatics tools were further utilized for principal component and profiling hierarchical clustering quantification analyses. In addition, PEAKS software was employed for protein quantification and quality analyses. Finally, biological processes and KEGG metabolic pathways in mandibular glands of RJB and ITB were compared. Phosphosites were determined and motifs were predicted by using Scaffold PTM bioinformatics tool.【Result】 The numbers of identified phosphoproteins of RJBs (2 225, 1 922, 2 159) were significantly higher than those of 1 740, 1 592, 1 682 in ITBs, illustrating that RJBs’ mandibular gland phosphorylation regulation network was likely more complicated than ITBs’. Although the phosphorylation process in mandibular glands of RJBs was significantly different from that in ITBs, a similar phosphoproteomes profiling across the gland development suggested that the phosphoproteins at distinct stages in RJBs and ITBs performed similar biological function to ensure the gland development and secretion activity. the glands of each stage in RJBs and ITBs had significant phosphoproteome differences, of which nurse bees were the most significantly diverged. Eighty-seven phosphoproteins were highly abundant in RJBs, and most of them were mainly related to energy and fatty acid metabolic process. These indicated the key roles of fatty acid metabolism in boosting the ability of fatty acid synthesis (including 10-HDA) in mandibular glands to prime the quantity of 10-HDA in royal jelly alongside the increased royal jelly production, which was essential for providing qualitative nutrition for the survival of population. Forty-one highly abundant phosphoproteins in ITBs were mainly related to energy metabolism. Three kinds of motifs were detected at each stage of glands in both RJBs and ITBs: acidic, basic and pro-directed. The unique kinase family recognized by motifs in nurse bees of RJBs was associated with cell proliferation, which might support gland morphological development and related to increased secretion capacity of RJBs.【Conclusion】RJBs and ITBs have shaped different phosphoproteome signatures to maintain mandibular gland development and function at each stage. The most profound divergence occurs during the nurse stage, which RJBs may increase the ability of fatty acid synthesis to ensure the basic function of royal jelly. The data help us gain a novel understanding of the molecular underpinnings to drive enhanced high royal jelly production in RJBs at the level of protein modification.

Key words: high producing royal jelly bee, Italian bee, mandibular gland, phosphoproteome