Unraveling The Molecular Underpinnings Of Embryonic Development Of Honeybee Worker And Drone(Apis Mellifera Ligustica)Using Proteomics And Phosphoproteomics

In honeybee?Apis mellifera L.?society,worker and drone bees make a great contribution to the social organization and survival of honeybee populations.Derived from complete genetic makeup,the worker?diploid?and drone?haploid?have evolved quite distinct development trajectory and social behaviors in the honeybee community.Embryogenesis is an important period during which the body plan of adult honeybee is formed.Although molecular underpinnings that drives the embryogenesis of worker and drone bee has been conducted by the approach of two-dimensional electrophoresis?2-DE?,the identification of 105 proteins block the further understand of honeybee embryogenesis to some extent.With the technologies of mass spectrum progress,the sensitivity,accuracy,resolution and the identification capacity improved significantly,about 20 times more than 2-DE proteome,which is of crucial importance for elucidating the molecular underpinnings of honeybee embryogenesis.As an important and ubiquitous post-translational modification,phosphorylation is one of the most critical regulatory mechanisms of protein functions,which has been widely applied to various fields of life science research.Unraveling the molecular underpinnings of embryonic development of honeybee worker and drone using proteomics and phosphoproteomics,basing on the high resolution mass spectrometry,is still a gap area to be filled.Towards this goal,the proteome and phosphoproteome of worker and drone will be analyzed and compared using shotgun proteomics,label-free quantitation,IMAC?Immobilized metal ion affinity chromatography?and TiO₂ phosphoprotein enrichments and bioinformatics.Totally 3443 proteins were identified in the 3 day stages of worker and drone,which increasing the proteome coverage of the honey bee embryo by about 30-fold.Although embryos at different developmental stages have their specific signatures,the core proteome consist of protein synthesis,metabolic energy,development and molecular transporter related proteins play the center role in driving embryogenesis.Besides,to coordinate and modulate developmental events,the young?<24 h?embryo strongly expressed proteins involved in carbohydrate metabolism and fatty acid degradation indicates their roles in offering metabolic energy for zygote cleaving and blastoderm formation.The 48-72 h aged embryoenhancedexpressionofproteinsandactivatedpathwaysimplicatedin transcriptional-translational machinery,morphogenesis and cytoskeleton,are suggestive of its ability to provide the building blocks for the rudimentary organs.Moreover,drone adopts a distinct strategy to respond to the large body size.The morphogenetic occurrence in the drone embryo is earlier than that in the worker;the strengthened cytoskeletal and antioxidant system of drone are to match the physiological needs of embryonic development.RNAi showed the 24-48hr aged embryo induced knockdown gene expression of Rp L36 successfully.Large-scale phosphoproteome profiling in the embryos of worker and drone identified 6342phosphorylation sites in 2438 phosphoproteins,by employing IMAC and TiO₂ enrichment of phosphopeptides,which were complementary advantages of phosphopeptide enrichment method.Then the site-specific kinase-substrate relations were computationally predicted for constructing kinase-substrate phosphorylation networks?KSPNs?,totally 91 kinases were involved in the regulation of honeybee embryonic development,comprising the largest,most in-depth phosphorylation dataset in the honeybee embryo currently.Phosphorylation regulated the biological pathways intensively,such as transcription,morphogenesis,cell cycle control/apoptosis,cytoskeleton,and signal transduction.Meanwhile the increased expression of kinases CDK,MAPK,RSK promote the processes of cell division,protein systhesis,tissue differentiation,playing an important role in the establishment of organ rudiments.However,the profiles of the phosphorylate regulation are different depending on the developmental stages.The young embryos?24hr?regulated the processes of energy metabolism,signal transduction and tissue differentiation by the kinases PKAs,providing molecular block and energy to cell proliferation and blastoderm formation.At the middle-late stages?48-72hr?,the kinase subfamily DYRK and CDK play roles in the regulation of gene transcription,protein systhesis and differentiation of germ layers.Simulataneously,the actived signaling pathways foxO and wnt,contribute crucial roles in the key processes of antioxidant stress,germ layer development,organogenesis and dorsal closure.As the proteome of honeybee embryos,it show divergences between the phosphorylatd regulations on the embryo of worker and drone,which result in the differences of physical development by kinases triggering.At the early stage of embryos,the negative regulation of cell proliferation of mnb,besides the inhibitory effect on the processes of gene transcription and cytoskeleton caused by the phosphorylation of MAPK3 site T564,indicate worker embryo is still in its early development stage.While the fully activated CDK12 accelerated the mature of mRNAs,then transfer to ribosomes translating into protein by the activated RNA transport pathway,the evidences show drone embryo has launched the large-scale protein synthesis.The middle aged woker embryo imposed restrictions on the cell cycle for the inactivation of CDK1,because of the phosphorylation on Y15.Similarly,the highly phosphorylated raf prevent the signaling pathways from activation involved in the formation of gastrula,imaginal disc and dorsal-ventral axis.Whereas DCAMKL of highly active and launch of the wnt signaling pathway push the massive formation of brain and other organs in drone embryo to a new high.At the late stage of embryo,the foxO pathways provide the energy for embryogenesis and protect embryo form oxidant stress.Furthermore,the activation of notch,hippo,wnt and hedgehog pathways,with the addition of highly actived CDK9,CDK12,mnb and SLK,mark the peaktime of cell growth,transcription,translation and organ structure.Nevertheless,the negative effect on cytoskeleton organization,epidermal neural differentiation,cell migration and cell cycle caused by the phosphorylation on Abl site S637,indicate the rudiment organs have been completed fundamentally.The high-activity myo3 contributes to the processes of blastokinesis and dorsal closure.The RNAi technology platform built in this study provides an academic and practical foundation for the functional research of key protein.The identified biological pathways,key node proteins,KSPNs,kinase activity information,subcellular and chromosomal location data,allow for further functional analysis and genetic manipulation for both the honey bee embryos and other eusocial insects.