In-depth Proteome of the Hypopharyngeal Glands of Honeybee Workers Reveals Highly Activated Protein and Energy Metabolism in Priming the Secretion of Royal Jelly
2019; Elsevier BV; Volume: 18; Issue: 4 Linguagem: Inglês
10.1074/mcp.ra118.001257
ISSN1535-9484
AutoresHan Hu, Gebreamlak Bezabih, Feng Mao, Qiaohong Wei, Xufeng Zhang, Fan Wu, Lifeng Meng, Fang Yu, Bin Han, Chuan Ma, Jianke Li,
Tópico(s)Plant and animal studies
ResumoRoyal jelly (RJ) is a secretion of the hypopharyngeal glands (HGs) of honeybee workers. High royal jelly producing bees (RJBs), a stock of honeybees selected from Italian bees (ITBs), have developed a stronger ability to produce RJ than ITBs. However, the mechanism underpinning the high RJ-producing performance in RJBs is still poorly understood. We have comprehensively characterized and compared the proteome across the life span of worker bees between the ITBs and RJBs. Our data uncover distinct molecular landscapes that regulate the gland ontogeny and activity corresponding with age-specific tasks. Nurse bees (NBs) have a well-developed acini morphology and cytoskeleton of secretory cells in HGs to prime the gland activities of RJ secretion. In RJB NBs, pathways involved in protein synthesis and energy metabolism are functionally induced to cement the enhanced RJ secretion compared with ITBs. In behavior-manipulated RJB NBs, the strongly expressed proteins implicated in protein synthesis and energy metabolism further demonstrate their critical roles in the regulation of RJ secretion. Our findings provide a novel understanding of the mechanism consolidating the high RJ-output in RJBs. Royal jelly (RJ) is a secretion of the hypopharyngeal glands (HGs) of honeybee workers. High royal jelly producing bees (RJBs), a stock of honeybees selected from Italian bees (ITBs), have developed a stronger ability to produce RJ than ITBs. However, the mechanism underpinning the high RJ-producing performance in RJBs is still poorly understood. We have comprehensively characterized and compared the proteome across the life span of worker bees between the ITBs and RJBs. Our data uncover distinct molecular landscapes that regulate the gland ontogeny and activity corresponding with age-specific tasks. Nurse bees (NBs) have a well-developed acini morphology and cytoskeleton of secretory cells in HGs to prime the gland activities of RJ secretion. In RJB NBs, pathways involved in protein synthesis and energy metabolism are functionally induced to cement the enhanced RJ secretion compared with ITBs. In behavior-manipulated RJB NBs, the strongly expressed proteins implicated in protein synthesis and energy metabolism further demonstrate their critical roles in the regulation of RJ secretion. Our findings provide a novel understanding of the mechanism consolidating the high RJ-output in RJBs. One of the most important characteristics of honeybees (Apis mellifera) in colonies, is the age-dependent role change, known as age polyethism (1Ohashi K. Sasaki M. Sasagawa H. Nakamura J. Natori S. Kubo T. Functional flexibility of the honey bee hypopharyngeal gland in a dequeened colony.Zoolog. Sci. 2000; 17: 1089-1094Crossref PubMed Scopus (41) Google Scholar, 2Winston M.L. The Biology of the Honey Bee. Harvard University Press, Cambridge1987Google Scholar). The exocrine hypopharyngeal glands (HGs) 1The abbreviations used are:HGhypopharyngeal glandsRJroyal jellyITBsItalian beesRJBshigh royal jelly producing beesMGsmandibular glandsNEBsnewly emerged beesNBsnurse beesFBsforager beesWBwestern-blottingRNsreversed nurse beesCFscontrol forager beesEOethyl oleatePNsprolonged nurse beesIHCimmunohistochemistryMRJP1major royal jelly protein 1RpL60S ribosomal proteinRpS40S ribosomal proteinHex110hexamerin 110LamLamin Dm0MDHMalate dehydrogenaseUGP2UTP-glucose-1-phosphate uridylyltransferasegltAcitrate synthase10-HDA10-hydroxy-2-decenoic acid2-DEtwo-dimensional electrophoresis-basedMSMass spectrometryDAPI4′,6-diamidino-2-phenylindoleERendoplasmic reticulumPCAprincipal component analysisGOGene OntologyFDRfalse discovery rateQMPqueen mandibular pheromoneDTTdithiothreitolTICtotal ion currentDAPI4′, 6-diamidino-2-phenylindole. 1The abbreviations used are:HGhypopharyngeal glandsRJroyal jellyITBsItalian beesRJBshigh royal jelly producing beesMGsmandibular glandsNEBsnewly emerged beesNBsnurse beesFBsforager beesWBwestern-blottingRNsreversed nurse beesCFscontrol forager beesEOethyl oleatePNsprolonged nurse beesIHCimmunohistochemistryMRJP1major royal jelly protein 1RpL60S ribosomal proteinRpS40S ribosomal proteinHex110hexamerin 110LamLamin Dm0MDHMalate dehydrogenaseUGP2UTP-glucose-1-phosphate uridylyltransferasegltAcitrate synthase10-HDA10-hydroxy-2-decenoic acid2-DEtwo-dimensional electrophoresis-basedMSMass spectrometryDAPI4′,6-diamidino-2-phenylindoleERendoplasmic reticulumPCAprincipal component analysisGOGene OntologyFDRfalse discovery rateQMPqueen mandibular pheromoneDTTdithiothreitolTICtotal ion currentDAPI4′, 6-diamidino-2-phenylindole. are one of the organs that are subject to age polyethism to achieve their biological mission of secreting larval food: royal jelly (RJ). HGs are coiled in the sides of the head and deliver the proteinaceous secretory product, RJ, via a large collecting duct to the hypopharynx (3Deseyn J. Billen J. Age-dependent morphology and ultrastructure of the hypopharyngeal gland of Apis mellifera workers (Hymenoptera, Apidae).Apidologie. 2005; 36: 49-57Crossref Scopus (105) Google Scholar, 4Michener C.D. The social behavior of the bee: a comparative study. Harvard University Press, Cambridge1974Google Scholar, 5Seeley T.D. The Wisdom of the Hive.Ilo Working Papers. 1995; 9: 5-31Google Scholar). Once the worker bees emerge from their cells in the honeycombs, hereafter referred to as newly emerged bees (NEBs, < 24 h after eclosion), the HGs are formed but not fully developed (6Liu H. Wang Z.L. Zhou L.B. Zeng Z.J. Quantitative analysis of the genes affecting development of the hypopharyngeal gland in honey bees (Apis mellifera L.).Sociobiology. 2015; 62: 412-416Crossref Scopus (4) Google Scholar). 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For instance, an enhanced level of neuropeptides is implicated in regulating water homeostasis, brood pheromone recognition, foraging capacity, and pollen collection in RJBs to regulate the behavior of RJ secretion (35Han B. Fang Y. Feng M. Hu H. Qi Y. Huo X. Meng L. Wu B. Li J. Quantitative neuropeptidome analysis reveals neuropeptides are correlated with social behavior regulation of the honeybee workers.J. Proteome Res. 2015; 14: 4382-4393Crossref PubMed Scopus (40) Google Scholar). The activities of phosphatidylinositol signaling and arachidonic acid metabolism in RJB NBs are elevated to increase olfactory sensation in response to larval pheromone stimulation (33Han B. Fang Y. Feng M. Hu H. Hao Y. Ma C. Huo X. Meng L. Zhang X. Wu F. Li J. Brain membrane proteome and phosphoproteome reveal molecular basis associating with nursing and foraging behaviors of honeybee workers.J. Proteome Res. 2017; 16: 3646-3663Crossref PubMed Scopus (21) Google Scholar). The selective enhancement of the mandibular glands of RJBs on the activity of pathways related to lipid synthesis to maintain a proper proportion of 10-hydroxy-2-decenoic acid (10-HAD), a vital fatty acid in RJ for larval nutrition, fits the increased RJ production (30Huo X. Wu B. Feng M. Han B. Fang Y. Hao Y. Meng L. Wubie A.J. Fan P. Hu H. Qi Y. Li J. Proteomic Analysis Reveals the Molecular Underpinnings of Mandibular Gland Development and Lipid Metabolism in Two Lines of Honeybees (Apis mellifera ligustica).J. Proteome Res. 2016; 15: 3342-3357Crossref PubMed Scopus (27) Google Scholar). Furthermore, RJBs have reshaped their hemolymph proteome by providing surplus protein and energy molecules to prime augmented RJ outputs (32Ararso Z. Ma C. Qi Y. Feng M. Han B. Hu H. Meng L. Li J. Proteome Comparisons between Hemolymph of Two Honeybee Strains (Apis mellifera ligustica) Reveal Divergent Molecular Basis in Driving Hemolymph Function and High Royal Jelly Secretion.J. Proteome Res. 2018; 17: 402-419Crossref PubMed Scopus (16) Google Scholar). Despite the importance of HGs for RJ production, only one study has been done that compares the proteome differences between ITBs and RJBs, by using two-dimensional electrophoresis-based (2-DE) proteomics (9Li J.K. Feng M. Desalegn B. Fang Y. Zheng A.J. Proteome comparison of hypopharyngeal gland development between Italian and royal jelly producing worker honeybees (Apis mellifera L.).J. Proteome Res. 2010; 9: 6578-6594Crossref PubMed Scopus (59) Google Scholar). Because of technical limitations, only a small fraction of proteins (116 proteins) were identified in HGs, thus more in-depth understanding of the molecular basis of high RJ production in RJBs is required. Therefore, the aim of this work was to investigate and compare the age-specific HG proteome of NEBs, NBs, and FBs between ITBs and RJBs by using cutting-edge high-resolution MS-based proteomics. The functions of proteins in the critical pathways that are implicated in the secretory activity of RJ in HGs had been validated by two behavioral experiments, through which the worker bees were manipulated to extended nursing periods or the FBs were forced to revert into NBs. This work provides a novel insight into the mechanism with which the HGs achieve enhanced production of the valuable bee-product, RJ. The colonies used for sampling the HGs of ITBs and RJBs (Apis mellifera ligustica) were kept at the Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing. The queens of the ITBs were from Bologna, Italy, and the queens of RJBs were from Zhejiang Province, China, where the RJBs were selected and maintained in a breeding station. All colonies were managed with almost identical population structure, brood pattern, and food during the nectar flow of chaste berry (Vitexnegundo L.) in June. The workflow of the investigation and comparison of the HG proteome during 3 stages of worker bees between ITBs and RJBs is shown in Fig. 1. A total of 34 colonies (17 colonies from each strain) received similar management practices to attain comparable colony size for measuring RJ production. The collection method of RJ was performed as previously described, with minor modifications (9Li J.K. Feng M. Desalegn B. Fang Y. Zheng A.J. Proteome comparison of hypopharyngeal gland development between Italian and royal jelly producing worker honeybees (Apis mellifera L.).J. Proteome Res. 2010; 9: 6578-6594Crossref PubMed Scopus (59) Google Scholar, 22Li J.K. Zhang L. Zhong B.X. Cheng S.L. How royal jelly maintains its quality within the colony.Am. Bee J. 2005; 145: 736-738Google Scholar). The young worker larvae (> 24 h old) were grafted into to the cell cups, the accepted larvae were fed royal jelly by nurse bees, and the unaccepted larvae (if there were) were removed by worker bees. After around 72 h of larval grafting, the accepted queen cell numbers were counted, and RJ was collected from each colony. The rate of queen cell acceptance is the accepted cell number divided by the total grafted cell number, 128. RJ of each colony was sampled five times, and the samples were weighed with a digital scale (Mettler Toledo, Columbus, OH; accurate to 0.001 g). After the RJ production survey, five colonies of RJBs with the highest RJ outputs and five colonies of ITBs with the lowest production were selected for HG sampling. Honeybees from five colonies per time point were collected and pooled as one sample. For each time point (at NEBs, NBs, and FBs), at least 100 bees were sampled from each colony. And triplicates were produced in each sample for LC-MS/MS analysis. The expressional profile of regulated proteins between different samples in all proteomics was created by the PEAKS software using an un-centered Pearson correlation and average linkage. All proteomics and western-blotting (WB) data were evaluated with Student's t test, and a p value of < 0.05 was significant. Graph was represented by the mean and S.D. Parts of NEBs (< 24 h) were sampled when they emerged from sealed brood in an incubator, and others were then marked on their thoraxes with paint dots and returned to the colonies for further development. During the following 8–10 days, the marked bees were collected as NBs when they were observed to extend their head into the cells containing the young larvae. The FBs, carrying pollen loads, were collected at the hive entrance after 15 days of emergence. The HGs were dissected in cold PBS containing protease inhibitors (Roche; Indianapolis) with the help of a binocular microscope, and the samples were then pooled based on age and bee colony and stored at −80 °C for later analysis. To exclude the effect of age on the expression of candidate proteins and validate that the biological functions of the proteins are related to secretory behavior of RJ rather than age regulation, two social manipulation experiments to regulate behavioral biology of honeybees were performed twice, in order to reveal the proteins' functions in the regulation of RJ secretory biology. The two experimental strategies were performed as shown in supplemental Fig. S1. The purpose of experiment 1 (supplemental Fig. S1A) was to collect normal NBs, FBs, and reversed nurse bees (RNs) according to the previously described protocol (43Baker N. Wolschin F. Amdam G.V. Age-related learning deficits can be reversible in honeybees Apis mellifera.Exp. Gerontol. 2012; 47: 764-772Crossref PubMed Scopus (37) Google Scholar, 44Amdam G.V. Aase A.L. Seehuus S.C. Kim Fondrk M. Norberg K. Hartfelder K. Social reversal of immunosenescence in honey bee workers.Exp. Gerontol. 2005; 40: 939-947Crossref PubMed
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