In-depth Proteome of the Hypopharyngeal Glands of Honeybee Workers Reveals Highly Activated Protein and Energy Metabolism in Priming the Secretion of Royal Jelly.
ABSTRACT: 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.
Project description:Royal jelly (RJ) is a proteinaceous secretion of the hypopharyngeal glands (HGs) in the head of honeybee workers. It is a critical food for queen bees and young larvae that decides the fate of fertilized eggs in developing into either queen bees or worker bees during the early larval stages. RJ is also widely used in humans for health promotion as agent, such as antibacterial, antioxidant, and antiaging properties. To increase RJ yields, a stock of high RJ producing honeybees (RJBs) has genetically selected from Italian honeybees (ITBs) in China since 1980s. To date, one colony of RJBs can produce more than 10 kg of RJ per year, a yield that is 10-times greater than that produced by a colony of ITBs. To elucidate the mechanism of the enforced gland performance in producing RJ in RJBs, the spatio-temporal HG proteomes of newly emerged bees, nurse bees, and forager bees, were compared between the ITBs and RJBs. Proteins in the critical pathways that are implicated in the secretory activity of RJ in HGs are validated biochemically and biologically by manipulating the NBs into extended nursing periods and the FBs to revert into NBs. This will provide a novel mechanistic insight into the HGs achieving an enhanced biological mission of producing the valuable bee-product, RJ.
Project description:We characterized and compared hemolymph proteome of Royal Jelly bees (RJbs), a stock selected for increasing RJ output from Italian bees (ITbs) and ITbs across the larval and adult ages. Unprecedented depth of proteome was attained by identifying 3394 hemolymph proteins in both bee lines. The proteome supports the general function of hemolymph to drive development and immunity across different phases in both bees. However, age-specific proteome settings have adapted to prime the distinct physiology for larvae and adult bees. In larvae, proteome are thought to drive the temporal immunity, rapid organogenesis, and reorganization of larval structures. In adults, proteome play key roles to prompt tissues development and immune defense in NEBs, glands maturity in NBs and carbohydrate energy production in FBs. Comparing the proteome between the same aged larval and adult samples, RJbs and ITbs have tailored distinct hemolymph proteome programs to drive their physiology. Particularly, in day 4 larvae and NBs, a large number of highly abundant proteins enriched in protein synthesis and energy metabolism in RJbs relative to ITbs imply that RJb larvae and NBs have reprogrammed their proteome to initiate different developmental trajectory and high RJ secretion in response to the enhanced RJ production by selection. Our hitherto depth of proteome coverage gains novel sight on molecular details in driving hemolymph function and high RJ production by RJbs.
Project description:Transcriptome sequencing has become the main methodology for analyzing the relationship between genes and characteristics of interests, particularly those associated with diseases and economic traits. Because of its functional superiority, commercial royal jelly (RJ) and its production are major areas of focus in the field of apiculture. Multiple lines of evidence have demonstrated that many factors affect RJ output by activating or inhibiting various target genes and signaling pathways to augment their efficient replication. The coding sequences made available by the Honey Bee Genome Sequencing Consortium have permitted a pathway-based approach for investigating the development of the hypopharyngeal glands (HGs). In the present study, 3573941, 3562730, 3551541, 3524453, and 3615558 clean reads were obtained from the HGs of five full-sister honey bee samples using Solexa RNA sequencing technology. These reads were then assembled into 18378, 17785, 17065, 17105, and 17995 unigenes, respectively, and aligned to the DFCI Honey Bee Gene Index database. The differentially expressed genes (DEGs) data were also correlated with detailed morphological data for HGs acini. The results identify areas that warrant further study, including those that can be used to improve honey bee breeding techniques and help ensure stable yields of RJ with high quality traits. The 5 samples at given time (d3, d6, d9, d12, d16 after adult worker bees emergence from the comb) are in the critical stage of the RJ secretion and HGs developments indicated (triggered) the further caste differentiation (worker bees and queen) and task switch (nurse bees and foragers). 30 pooled heads of each samples were
Project description:Apis mellifera and Apis cerana are two sibling species of Apidae. Apis cerana is adept at collecting sporadic nectar in mountain and forest region and exhibits stiffer hardiness and acarid resistance as a result of natural selection, whereas Apis mellifera has the advantage of producing royal jelly. To identify differentially expressed genes (DEGs) that affect the development of hypopharyngeal gland (HG) and/or the secretion of royal jelly between these two honeybee species, we performed a digital gene expression (DGE) analysis of the HGs of these two species at three developmental stages (newly emerged worker, nurse and forager).Twelve DGE-tag libraries were constructed and sequenced using the total RNA extracted from the HGs of newly emerged workers, nurses, and foragers of Apis mellifera and Apis cerana. Finally, a total of 1482 genes in Apis mellifera and 1313 in Apis cerana were found to exhibit an expression difference among the three developmental stages. A total of 1417 DEGs were identified between these two species. Of these, 623, 1072, and 462 genes showed an expression difference at the newly emerged worker, nurse, and forager stages, respectively. The nurse stage exhibited the highest number of DEGs between these two species and most of these were found to be up-regulated in Apis mellifera. These results suggest that the higher yield of royal jelly in Apis mellifera may be due to the higher expression level of these DEGs.In this study, we investigated the DEGs between the HGs of two sibling honeybee species (Apis mellifera and Apis cerana). Our results indicated that the gene expression difference was associated with the difference in the royal jelly yield between these two species. These results provide an important clue for clarifying the mechanisms underlying hypopharyngeal gland development and the production of royal jelly.
Project description:Royal jelly proteins (MRJPs) of the honeybee bear several open questions. One of them is their expression in tissues other than the hypopharyngeal glands (HGs), the site of royal jelly production. The sole MRJP-like gene of the bumblebee, Bombus terrestris (BtRJPL), represents a pre-diversification stage of the MRJP gene evolution in bees. Here we investigate the expression of BtRJPL in the HGs and the brain of bumblebees. Comparison of the HGs of bumblebees and honeybees revealed striking differences in their morphology with respect to sex- and caste-specific appearance, number of cells per acinus, and filamentous actin (F-actin) rings. At the cellular level, we found a temporary F-actin-covered meshwork in the secretory cells, which suggests a role for actin in the biogenesis of the end apparatus in HGs. Using immunohistochemical localization, we show that BtRJPL is expressed in the bumblebee brain, predominantly in the Kenyon cells of the mushroom bodies, the site of sensory integration in insects, and in the optic lobes. Our data suggest that a dual gland-brain function preceded the multiplication of MRJPs in the honeybee lineage. In the course of the honeybee evolution, HGs dramatically changed their morphology in order to serve a food-producing function.
Project description:The honey bee hive environment contains a rich microbial community that differs according to niche. Acetobacteraceae Alpha 2.2 (Alpha 2.2) bacteria are present in the food stores, the forager crop, and larvae but at negligible levels in the nurse and forager midgut and hindgut. We first sought to determine the source of Alpha 2.2 in young larvae by assaying the diversity of microbes in nurse crops, hypopharyngeal glands (HGs), and royal jelly (RJ). Amplicon-based pyrosequencing showed that Alpha 2.2 bacteria occupy each of these environments along with a variety of other bacteria, including Lactobacillus kunkeei. RJ and the crop contained fewer bacteria than the HGs, suggesting that these tissues are rather selective environments. Phylogenetic analyses showed that honey bee-derived Alpha 2.2 bacteria are specific to bees that "nurse" the hive's developing brood with HG secretions and are distinct from the Saccharibacter-type bacteria found in bees that provision their young differently, such as with a pollen ball coated in crop-derived contents. Acetobacteraceae can form symbiotic relationships with insects, so we next tested whether Alpha 2.2 increased larval fitness. We cultured 44 Alpha 2.2 strains from young larvae that grouped into nine distinct clades. Three isolates from these nine clades flourished in royal jelly, and one isolate increased larval survival in vitro. We conclude that Alpha 2.2 bacteria are not gut bacteria but are prolific in the crop-HG-RJ-larva niche, passed to the developing brood through nurse worker feeding behavior. We propose the name Parasaccharibacter apium for this bacterial symbiont of bees in the genus Apis.
Project description:Most of the proteins contained in royal jelly (RJ) are secreted from the hypopharyngeal glands (HG) of young bees. Although generic protein composition of RJ has been investigated, little is known about how age-dependent changes on HG secretion affect RJ composition and their biological consequences. In this study, we identified differentially expressed proteins (DEPs) during HG development by using the isobaric tag for relative and absolute quantification (iTRAQ) labeling technique. This proteomic method increases the potential for new protein discovery by improving the identification of low quantity proteins.A total of 1282 proteins were identified from five age groups of worker bees, 284 of which were differentially expressed. 43 (15.1%) of the DEPs were identified for the first time. Comparison of samples at day 6, 9, 12, and 16 of development relative to day 3 led to the unambiguous identification of 112, 117, 127, and 127 DEPs, respectively. The majority of these DEPs were up-regulated in the older worker groups, indicating a substantial change in the pattern of proteins expressed after 3 days. DEPs were identified among all the age groups, suggesting that changes in protein expression during HG ontogeny are concomitant with different states of worker development. A total of 649 proteins were mapped to canonical signaling pathways found in the Kyoto Encyclopedia of Genes and Genomes (KEGG), which were preferentially associated with metabolism and biosynthesis of secondary metabolites. More than 10 key high-abundance proteins were involved in signaling pathways related to ribosome function and protein processing in the endoplasmic reticulum. The results were validated by qPCR.Our approach demonstrates that HG experienced important changes in protein expression during its ontogenic development, which supports the secretion of proteins involved in diverse functions in adult workers beyond its traditional role in royal jelly production.
Project description:Transcriptome sequencing has become the main methodology for analyzing the relationship between genes and characteristics of interests, particularly those associated with diseases and economic traits. Because of its role of functional food for humans, commercial royal jelly (RJ) and its production are major research focuses in the field of apiculture. Multiple lines of evidence have demonstrated that many factors affect RJ output by activating or inhibiting various target genes and signaling pathways. Available coding sequences from the Honey Bee Genome Sequencing Consortium have permitted a pathway-based approach for investigating the development of the hypopharyngeal glands (HGs). In the present study, 3573941, 3562730, 3551541, 3524453, and 3615558 clean reads were obtained from the HGs of five full-sister honey bee samples using Solexa RNA sequencing technology. These reads were then assembled into 18378, 17785, 17065, 17105, and 17995 unigenes, respectively, and aligned to the DFCI Honey Bee Gene Index database. The differentially expressed genes (DEGs) data were also correlated with detailed morphological data for HGs acini.
Project description:Tears are secreted from the lacrimal gland (LG), a dysfunction in which induces dry eye, resulting in ocular discomfort and visual impairment. Honey bee products are used as a nutritional source in daily life and medicine; however, little is known about their effects on dry eye. The aim of the present study was to investigate the effects of honey bee products on tear secretion capacity in dry eye. We selected raw honey, propolis, royal jelly (RJ), pollen, or larva from commercially available honey bee products. Tear secretion capacity was evaluated following the oral administration of each honey bee product in a rat blink-suppressed dry eye model. Changes in tear secretion, LG ATP content, and LG mitochondrial levels were measured. RJ restored the tear secretion capacity and decrease in LG ATP content and mitochondrial levels to the largest extent. Royal jelly can be used as a preventative intervention for dry eye by managing tear secretion capacity in the LG.
Project description:The honeybee model organism, Apis mellifera carnica, is a hymenopteran with a remarkable ability to withstand cold winters by instinctively entering into hibernation in the hive. Hibernation is characterized by a significant reduction in the metabolic rate, triggered by a drop in the ambient temperature and a shortage of reserves. It provides a token cue that initiates an alternative developmental program that leads to dormancy and switches social behaviors. The age-dependent changes in the generic proteins and substance composition of royal jelly (RJ) have been well documented. However, the molecular mechanism of RJ secretion and the biological consequences of regulation by the hypopharyngeal gland (HG) according to the season remain to be addressed. In this study, large-scale transcriptomic and proteomic approaches were used to detect statistically significant changes in the honeybee proteome and to explore the mechanistic basis for the HG regulation that accompanies hibernation in the winter and arousal in the spring. HG activity was associated with hundreds of gene and protein expression changes in the winter-spring cycle. A systematic study of HG activity and secretion identified 21,441 genes and 2,156 proteins at three time points (i.e., A, winter bees; B, spring bees; and C, retired bees). Of these, 737 genes and 698 proteins were qualified and compared to reveal differential expression patterns, including 197 up-regulated and 291 down-regulated DEGs in group A vs. B, 118 up-regulated and 259 down-regulated DEGs in group A vs. C, and 106 up-regulated and 53 down-regulated DEGs in group B vs. C, respectively. The results were validated by qPCR.Honeybee HG activity was associated with multiple genes regulation.The results present the first global pictures of the regulation of HG activity in winter bee. Overall design: The 3 samples at given time (A,winter bee; B,spring bee; and C, retired bee) are collected to reveal the mechanism of the regulation of HG activity and RJ secretion, the differentially expressed genes involved in the Metabolic process and protein processing in endoplasmic reticulum related DEGs were identified and the shifts appeared to show dormancy-induced transcriptional changes across the winter. 30 pooled heads of each samples were pooled as one biologiacl repeat.