Investigation Of Molecular Basis Associating With Division Of Labor And High Royal Jelly Yields By Analyzing Brain Neuropeptidome,Membrane Proteome And Membrane Phosphoproteome

The honeybee(Apis mellifera)has a wide variety of social behaviors,in which the brain plays fundamental roles in processing sensory clues and alters its morphology and functional dynamics to fulfill behavioral demands.In the brain,neuropeptides are vital important in orchestrating development,metabolism,reproduction,mating and foraging,acting as neurotransmitters,neuromodulators,and neurohormones.Moreover,the brain cells are highly specialized to process and transmit information using electrical and chemical signals.In this context,great deals of membrane proteins are employed for physiological requirements of the brain,such as receptor proteins,ion channel proteins,and transporter proteins.Protein phosphorylation plays comprehensive and precise regulation roles in protein function and signal transduction.However,much knowledge of how neuropeptidome,membrane proteome and membrane phosphoproteome regulate social behaviors is still largely unknown.To bridge the cap,in-depth comparisons of the brain neuropeptidome,membrane proteome and membrane phosphoproteome over four time points during age-related polyethism of honeybee workers were performed on two strains of bees,the Italian bee(Apis mellifera ligustica,ITB)and the high royal jelly producing bee(Apis mellifera ligustica,RJB).In the neuropeptidome analysis,158 nonredundant neuropeptides derived from 22 protein precursors were identified,of which 77 were previously unreported,expanding the honeybee neuropeptidome coverage to an unprecedented depth.The fact that 14 identical neuropeptide precursors changed their expression levels during the division of labor in both the ITB and RJB indicates they are highly related to task transition of honeybee workers.These observations further suggest the two lines of bees employ a similar neuropeptidome modification to tune their respective physiology of age polyethism via regulating excretory system,circadian clock system,and so forth.Noticeably,the enhanced level of neuropeptides implicated in regulating water homeostasis,brood pheromone recognition,foraging capacity,and pollen collection in RJB signify the fact that neuropeptides are also involved in the regulation of RJ secretion.The brain is a vital organ in regulating complex social behaviors of honeybees including learning and memory.Brain membrane proteins are significantly involved in 10 KEGG pathways,including oxidative phosphorylation,protein processing in the ER,phagosome,and SNARE interactions in vesicular transport.Having the highest pathway coverage in oxidative phosphorylation indicates its key role in supplying metabolic energy required for neural activity.The high enriched pathway of protein processing in the ER signifies the vital role of the ER in synthetization and modification of neuro-secretory peptides and neuronal proteins.In younger worker bees,the neuron construction and neural circuit refinement are the basic requirements of the neonatal nervous system,the up-regulated proteins implicated in the metabolism of carbohydrates,nucleosides,and lipids are thought to satisfy the high metabolic rate and to stimulate brain cell development.In NBs and FBs,to satisfy the demand of intensive information processing in hive and field works,the enriched oxidative phosphorylation,SNARE interactions in vesicular transport,and neuroactive ligand-receptor interaction pathways are supposed to underpin signal transduction.Furthermore,RJB has adapted distinct membrane proteome signatures in regulating the behaviors to cement the performance of RJ secretion.The up-regulated proteins related to SNARE interactions in vesicular transport,endocytosis,and the wnt signaling pathway in the brain of RJB NBs and FBs are important for the release and absorption of signal molecules to increase nerve sensitivity for facilitating larvae pheromone and flower odor recognition.Brain phosphoproteome analysis suggests MAPKs,CDKs,and CK2 are potentially the major kinase families in honeybee brains that modulate the phosphorylation network of membrane proteins in their respective signaling pathways.In younger bees,the upregulated phosphoproteins related to development and differentiation,and cell membrane adhesion are thought to stimulate the development of neuron and to support the maturation of synapsis.In older bees,the strongly activated phosphoproteins related to ion transport and signal transduction suggest that phosphorylation plays vital roles in driving neural activities.Notably,the upregulated phosphoproteins associated with efficient neurotransmitter transmission and recycling in RJB indicate their key roles in supporting elevated RJ yields.The intensive brain neuropeptidomic,membrane proteomic and membrane phosphoproteomic analyses of honeybee workers provide novel perspectives of neuromodulation.The identified key node neuropeptides,membrane proteins,membrane phosphoproteins,and the significantly enriched pathways add a valuable resource for further functional investigation of honeybee neurobiology as well as of the neurobiology of other insects.