In-depth Brain Phosphoproteome Study Reveals Neurobiological Underpinnings For Nurse And Forager Honeybee Workers (Apis Mellifera Ligustica)

The transition from nurse bee to forager bee worker is key to accomplish age-dependent tasks in response to pre-established genetic programs. There is a lack of knowledge about how the neurobiological activities during protein phosphorylation in the brains of honeybee workers align with their age-related task performances. The phosphoproteome in the brains of nurse and forager honeybee workers was characterized using immobilized titanium ion affinity chromatography (Ti4+-IMAC) phosphopeptide enrichment, shotgun proteome, label-free quantitation, and bioinformatics. Of the 1,242 phosphoproteins identified in the nurse and forager bees’brains,58.9%(732) were shared by both age groups as a central set of phosphoproteins (core phosphoproteome) while 510 (41.1%) proteins were differentially expressed between both. In the core phosphoproteome, the phosphorylated proteins were involved in a wide spectrum of biological functions, metabolic pathways and kinase activities, indicating their pivotal roles to drive the neurobiological activities, the cognition, and decision making of the worker bee brain during adult life.However, the nurse and forager bees expressed their own specific phosphoproteome, pathway signatures, and kinases in their brains to support performance of defined tasks via different neural activities. The more strongly represented phosphoproteins in the nurse bees’brains were intensively involved in the biological pathways of the phosphatidylinositol signaling system, wnt signaling, phototransduction, and kinases of Cyclin-dependent kinase2_Cyclin-dependent kinase3 (CDK2CDK3), P38 mitogen-activated protein kinases (p38), Casein kinase (CK2), and cdc2-like kinase (CLK), suggesting their vital roles in the regulation of biological processes, signal transduction, and olfactory learning processes to make possible the mission of nurse bees through the brain maturation and the development of cerebral structures. While the phosphoproteins in the forager bees’brains were highly implicated in brain maintenance, response to stimulus & stress, and signal transduction. They were intensively involved in the biological pathways of the phosphatidylinositol signaling system, phototransduction, wnt signaling, and glycerophospholipid metabolism, as well as kinase families of CDK2_CDK3, p38, CK2, CLK, Protein kinase C (PKC), and Protein kinase A (PKA). These proteins, pathways and kinases mainly serve in nerve cell differentiation, cell proliferation regulation, phototransduction pathway, and wnt signaling pathway regulation, indicating their pivotal roles in responding to sequences of environmental signals and information, and performing signal transduction activity to sufficiently support the guarding and foraging activities as forager bees.Our work is the first in-depth and comprehensive phosphoproteome report on honeybee brains and provides novel insights into the molecular details of phosphoproteins that tune protein function to the needs of different aged honeybee workers. These data provide a firm basis for future mechanistic research to better understand the neurobiological roles of targeted proteins in the honeybee brain.