Phosphoproteome Comparison Of The Hypopharyngeal Glands Of Honeybee (Apis Mellifera Ligustica) Workers Between Royal Jelly Bees And Italian Bees

Royal jelly(RJ)is the food which is used to feed young workers and drone larvae during the first three days and queens for their entire life.Royal jelly bees(Apis mellifera liguistica,Royal jelly bees,RJBs)is a strain genetically selected and bred from original Italian bees(Apis mellifera liguistica,Italian bees,ITBs)with a high yield of production of royal jelly(RJ).It is contributed to the industry of Chinese RJ,and up to now,over 90%of RJ in the world is produced from China.Hypopharyngeal gland(HGs)is an important gland of honeybees that produce RJ.However,there is still a need for more information regarding the molecular mechanism that underpins the biological basis of RJBs to produce RJs stronger than ITBs.For this purpose,we compared the acinus diameter and area of HGs,larval acceptance and RJ production rate between RJBs and ITBs.We analyzed the phosphoproteome profile that elucidates deep inside into molecular underpinnings HGs development,functionality,and the difference between the newly emerged bees(NEBs)and nurse bees(NBs)of RJBs and ITBs,which phosphopeptides of HGs were enriched by CAE-Ti-IMAC and SPE-Ti-IMAC methods.The main results were as follows:1.Our findings revealed that the larval acceptance rate and RJ production percentage were significantly higher in RJBs than ITBs colonies(p<0.05).The mean queen cell acceptance rate was72%in RJBs and 19%in ITBs.The mean RJ production was 54.86±7.88 g in RJBs colonies,while8.81±4.02 g in ITBs colonies.In addition,RJ production was 700.42±12.05 mg/cell in RJBs colonies,whereas 194.69±7.80 mg/cell in ITBs colonies2.The mean acinus diameter and area of HGs of RJBs workers were significantly larger than ITBs workers(p<0.05).In NEBs,the mean acinus diameter of HGs was 0.141±0.076 mm in RJBs and0.103±0.024 mm in ITBs.Similarly,the mean acinus area was greater in NEBs of RJBs than ITBs,which was 0.0062±0.002 mm~2and 0.0043±0.001 mm~2,respectively.In NBs,a significant difference was recorded in the acinus diameter and area of HGs of ITBs and RJBs(p<0.05).The mean acinus diameter of HGs was 0.229±0.041 mm in RJBs and 0.196±0.036 mm in ITBs.Similarly,the mean acinus area was more significant in RJBs than ITBs,which was 0.079±0.04 mm~2and 0.030±0.019mm~2(p<0.05),respectively.3.Phosphopeptides,phosphorylation sites,and phosphoproteins were discovered more in NEBs of RJBs than in the ITBs.Most proteins,such as heat shock protein 90 and eukaryotic translation initiation factor 3 subunit B were phosphorylated at 1 residue and 2 in NEBs of both bee stocks.In NEBs of RJBs,intracellular ribonucleoprotein complex,translation factor activity,RNA binding and m RNA surveillance pathway,translation initiation factor binding uniquely enriched GO terms.While ammonium transmembrane transporter,plasma membrane organization,translocon complex and Sec61translocon complex were uniquely enriched in ITBs,and carbamoyl-phosphate synthase(glutamine-hydrolzing)activity,intermediate filament,and intermediate filament cytoskeleton were shared enriched in HGs of NEBs of both bee stocks.4.The uniquely enriched functional groups in the NBs of RJBs were involved in the nucleoside phosphate metabolic process,amide biosynthetic process,translation elongation factor activity,NAD binding,proteasome complex,large ribosomal unity,unfolded protein binding,cytosol,r RNA binding,endoplasmic reticulum lumen,ligase activity,forming carbon-nitrogen bonds,and RNA binding.The uniquely enriched functional groups in the NBs of ITBs involved the carboxylic acid metabolic process,cytoplasmic transition,nuclear outer membrane-endoplasmic reticulum membrane network,and cellular amide metabolic process.Regarding NBs of RJBs and ITBs,the shared identified phosphoproteins were significantly enriched with functional groups such as glycolytic process,Golgi-associated vesicle,ribosome,translation factor activity,RNA binding,ribosomal subunit,Golgi vesicles transport,intramolecular oxidoreductase activity,and translational elongation.The highly activated ribosomal pathway,m RNA surveillance pathway,and Wnt signaling pathway play critical roles in large volumes of RJ production in NBs of RJBs.In HGs,both bee stocks showed age-dependent phosphoproteome setting to in-depth understanding of HG development and function across with age development.This research may show detailed phosphorylation of HGs and provide helpful information for understanding the biological activities of HGs development in NEBs and NBs from both bee stocks.Furthermore,it may contribute to understanding cellular processes in honeybee biology and other social insects.