Activity-based Proteome Profiling Of Hypopharyngeal Gland And Brain Reveals The Molecular Basis Of Extreme Reproductive Investments That Induce Colony-level High Royal Jelly-producing Bee Syndrome In Honeybee(Apis Mellifera Ligustica)

Reproductive investment is one of the most important variables in life history that affect all aspects of life.Colonies of honeybees rely on the collaboration between a reproductive queen and her functionally sterile workers,who together determine reproductive investment.In honeybee(Apis mellifera),the reproductive investment includes alloparenting female‐destined larvae into new queens via the provision of royal jelly(RJ)by nurse bees(NBs).RJ is a critical food that determines whether fertilized eggs develop into queen bees or worker bees in the early larval stages.The genetic selection of a high RJproducing strain of bees(RJBs)from Italian bees(ITBs)has been conducted for over four decades in China since the 1980 s.As a result,the commercial production of RJ has increased this reproductive investment to a great extent due to artificial selection.Compared to ITBs,the selected RJBs can now produce 10-fold RJ,which a colony could produce 10 kg at least in a year.RJBs are the most important producers of RJ in the world and produce > 90% of the total output with an annual market value of >$2.5 billion.The reproductive phenotype of RJBs is unprecedented among social and solitary organisms,and further studies of the RJBs at all organizational levels could determine general causes and consequences of the development of extreme reproductive investments.With technological advancements in proteomics,the mechanism underpinning the high RJ production by RJBs has been explored to new depths in the last decades.Despite the various studies so far,functional states of proteins in their native proteome to exactly pinpoint the molecular basis of enhanced RJ production of RJBs are still limited.To gain novel insight into the molecular basis of the behavior of enhanced RJ production in RJBs compared to ITBs,we used activity-based protein profiling(ABPP)combined with mass-spectrometry-based proteomics to generate a deep portrait of hypopharyngeal gland(HG)(Chapter 2)and Brain(Chapter 3)protein activity between the two strains.Most RJ proteins are secreted by HGs of the NBs.Our data discover distinct molecular landscapes that regulate the activity of HG between the two strains,and its activity corresponding to age-specific tasks.In HG of RJB NBs,pathways involved in protein synthesis and lipid transporter activity are functionally induced to promote the enhanced RJ secretion compared to ITBs.In Hg of RJB FBs,highly activated proteins involved in carbohydrate metabolism and energy production are induced compared to ITBs to support the high nutritional requirement of the colony through efficient foraging and the supply of nutrients.The results reveal specific molecular changes in the HG of NBs that may change their behavior in alloparental care to induce colony-level RJB syndrome.Our findings gain novel insight into the molecular basis of how altruistic castes optimize the colony’s reproductive success via alloparenting female-destined larvae into new queens via efficient provision of RJ.Furthermore,the distinct molecular landscapes that regulate the brain activity between the two strains,as well as its activity corresponding to age-specific tasks are investigated.The honeybee’s brain is a plastic organ that changes its physiological properties due to internal and external stimuli in order to perform certain tasks depending on age.In brain of RJB nurses,highly regulated brain-active proteins were induced to optimize efficient energy production,protein metabolism and signaling so that brain cells function properly,presumably to support the increased RJ secretion of RJBs.In addition,pathways such as ribosome,phototransduction,and cell redox homeostasis were uniquely enriched compared to that of the ITBs suggesting their involvement in enhanced RJ secretion.While,in brain of RJB foragers,the highly regulated brain-active proteins that are involved in carbohydrate metabolism and energy production were functionally induced to support the colony’s high nutritional requirements through efficient foraging.RJBs reshapes the functional states of the brain by activating specific protein in their native proteome to optimize the improved performance of RJ secretion.Moreover,the active brain proteomes in nurse bees and forager bees showed various proteome programs that consolidated agespecific biological missions.Our findings provide a novel understanding of the neurological basis that consolidates the high RJ output in RJBs.To our knowledge,this is the first work that applied ABPP on honeybee HG and brain.The honeybee HG study gains insight into the molecular basis for behavioral changes in the regulation of reproductive investments through alloparental care in social insects.While the honeybee brain study is making substantial progress in understanding the molecular mechanism that consolidates the high RJ output in RJBs.