| The hypopharyngeal gland , sited in the head of the honeybee workers, is a paired of long tuberous organ connected to many acini, can secrete a proteinaceous substance named royal jelly which is fed to larvae, queens and drones and plays a key role in honeybee nutrition and caste differentiation. Royal jelly is becoming more and more popular for its nutritional, health, and pharmacological functions.High royal jelly producing bees (RJb) (Apis mellifera L.) which is selected from native Italian bees (ITb) (Apis mellifera L.) for increased royal jelly production in 1980's, has become the best royal jelly producer in the world. But the mechanism for its high royal jelly production still keeps unclear. To better understanding the characteristic during the development of hypophargngeal gland, the hypopharyngeal gland was studied by the proteomic approach.The 1, 3, 6, 12, 15 and 20-days-old honeybee workers were selected in this study according to their age-related function polymorphism. The results of scanning electron microscopy showed that the acini diameters of hypopharyngeal gland of RJb were larger than that of ITb on each development stage, respectively. And the acini diameter both reached the highest level during 6-12 days.With the combination of two-dimensional gel electrophoresis, mass spectrometry, and protein engine identification tools that were applied to protein search engines and biological network software, a total of 175, 189, 209, 190, 168 and 151 protein spots were detected in the protein profiles on each development stage of ITb. Proteins were identified by MALDI-TOF/MS and MALDI-TOF-TOF/MS and the differentially expressed proteins were classified based on their biological functions and analyzed with hierarchical cluster software.The results showed that, among the 78 differentially expressed proteins, 34 proteins were up-regulated in the early development stage (1-3 days after eclosion), 9 proteins were involved in metabolism of carbohydrate and energy, 10 proteins were related to protein biosynthesis and 6, 3, 3, 1 and 2 proteins were assigned to protein folding, development regulation, antioxidant, transporter, and cytoskeleton, respectively. It was very interesting to identified major royal jelly proteins on 1-day-old hypopharyngeal gland.Thirty-one proteins were up-regulated on the medial stage (6-12 days), including 3 proteins involved in metabolism of carbohydrate and energy, 4 proteins related to protein folding, 1 protein for protein biosynthesis, 2 proteins for development regulation, 2 proteins for antioxidant, 1 protein acted as cytoskeleton, and 18 major royal jelly proteins.Thirteen proteins were up-regulated in the late stage (15-20 days), including 7 major royal jelly proteins and glucose oxidase,α-glycosidase andα-amylse. The comparative proteomic analysis between RJb and ITb showed that the amounts of proteins showed up-regulated trends in the hypopharyngeal of RJb on each development stage were much higher than that of the ITb, including proteins related to metabolism of carbohydrate and energy, protein biosynthesis, transporter, antioxidant, cytoskeleton, respectively.Real-time PCR analysis showed that the mRNA coded for major royal jelly protein 1, 2, 3 were expressed since the eclosion of honeybee workers. The expression trends between protein level and gene level were difference.The results indicate that the acini differentiation of RJb is faster than that of ITb, the volume and acini diameter are becoming big during the development of the hypopharyngeal gland.The protein abundance analysis certified that the workers can secrete royal jelly since eclosion, the hypopharyngeal glands are becoming more active and reach to the peak level on 6-12 days during the development. In order to ensure the high amounts of secretion, proteins related to metabolism of carbohydrate and energy, protein biosynthesis, development regulation, antioxidant, cytoskeleton and transporter were expressed more in early and medial development period than that of late stage. The proteins up-regulated on the late development stage were enzymes involved in the convertion of honey. This gives an explanation for the development of hypopharyngeal gland in protein level for the first time.The comparative proteomic analysis between RJb and ITb indicated that the protein abundance of major royal jelly proteins were higher than that of ITb. So the higher royal jelly production and higher protein abundance of major royal jelly proteins might be an evolution strategy to provide the balance nutrition for the development of larvae. For the purpose of high royal jelly production, proteins involving in energy metabolism and cytoskeleton proteins are in higher expression level for the cell differentiation and growth of hypopharyngeal gland, and proteins related to protein biosynthesis and protein folding are expressed more to ensure the synthesis and secretion of large amounts of major royal jelly proteins and the development of hypopharyngeal gland itself at the same time. Proteins with antioxidant functions are expressed in higher level to protect the gland from the oxidative damage occurred during the metabolism of the gland. For the first time we supply an explanation for the mechanism of high royal jelly production for high royal jelly producing bees from molecular level.Biological network analysis of all the identified proteins and differentially regulated proteins showed that 35 proteins were the key node proteins, these key node proteins probably play an important role in the development of hypopharyngeal gland and provide us some target proteins for further genetically manipulation of the hypopharyngeal gland.
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