In-Depth Proteome Analysis Of Hypopharyngeal Glands In The Honeybee(Apis Mellifera)

The hypopharyngeal glands(HGs)of the honeybee workers are vital for secretion of pretentious food,royal jelly(RJ),for both the young larvae and queen bees.Despite efforts made to address the molecular basis underpinning the development and functionality of the HGs using proteomics,only small fraction of proteins were identified due to previous technical limitation of proteomics.This demands a comprehensive understanding of the molecular portraits to drive the gland development and function.We characterized and compared the HG proteome of newly emerged(NEBs),nurse(NBs)and forager(FBs)honeybee workers of Italian bees(ITBs)and high royal jelly producing honeybees(RJBs),a line of bees selected for increasing RJ production from ITBs in China for over 4 decades that can produce 10 folds more RJ than ITBs.The identified 5,751 proteins by removing 435 secreted proteins or RJ proteins in the HGs of both strains cover an unprecedented depth of proteome,representing ?38% the honeybee proteome.The functional groups enriched by the identified and up-regulated proteins across different ages of bees support the general function to underpin HGs' development,RJ and enzymatic production driven by protein synthesis and carbohydrate metabolism in both honeybee strains.However,the HGs of both honeybee stocks develop age-dependent proteome settings to coordinate the HG development and functions along with the age development.In NEBs,the identified and upregulated proteins enriched in functional groups related to protein synthesis,development regulation and protein folding are thought to promote the young HG development.In NBs,the identified and high abundant proteins enriched in functional groups associated with cytoskeleton,carbohydrate and fatty acid metabolism,and molecular transporter suggest their roles in maintaining the HG growth and promoting RJ secretion.In FBs,the identified and strongly expressed proteins enriched to functions implicated in carbohydrate metabolism,transport and catabolism are suggestive of their significances for nectar processing into honey,an agedependent task shift from brood nursing to enzymatic secretion.Notably,the observed wide array of different metabolic processes and pathways between RJBs and ITBs at different ages manifest the fact that both bee stocks have tailored distinct proteome programs to prime their HG development and functions through regulating protein synthesis and degradation activity that correspond to gland cell growth and RJ secretion.In NEBs of ITBs,higher number of identified and elevated proteins than RJBs related to protein synthesis and metabolism are supposed to the fact that the HG cell development trajectory may divergent at young age of adult bees.This difference is further profound in NBs of both bee lines.In NBs of RJBs,the significantly higher number of identified and up-regulated proteins than ITBs enriched in carbohydrate and fatty acid metabolism,protein biosynthesis and transport suggests that the function of HGs in RJBs is enhanced to produce more RJ than that in ITBs.In FBs of ITBs,higher number identified and strongly expressed proteins than RJBs implicated in protein synthesis and carbohydrate metabolism suggest that the HGs of ITBs have possibly higher maintenance strategy that enhance secretion of enzymes for nectar processing into honey.Our time-resolved comprehensive proteome data gain novel insight into the molecular details that underline the HG development and function,and the selection has reshaped the proteome programs in RJBs to prime the enhanced activity of HGs for the higher production of RJ.