Royal Jelly (RJ) is a natural substance produced by honeybees, serving not only as nutrition for bee brood and queens but also as a functional food due to its health-promoting properties. Despite its well-known broad-spectrum antibacterial activity, the precise molecular mechanism underlying its antibacterial action has remained elusive. In this study, we investigated the impact of RJ on the bacteria model MG1655 at its half-maximal inhibitory concentration, employing LC–MS/MS to analyze proteomic changes. The differentially expressed proteins were found to primarily contribute to the suppression of gene expression processes, specifically transcription and translation, disrupting nutrition and energy metabolism, and inducing oxidative stress. Notably, RJ treatment led to a marked inhibition of superoxide dismutase and catalase activities, resulting in heightened oxidative damage and lipid peroxidation. Furthermore, through a protein–protein interaction network analysis using the STRING database, we identified CRP and IHF as crucial host regulators responsive to RJ. These regulators were found to play a pivotal role in suppressing essential hub genes associated with energy production and antioxidant capabilities. Our findings significantly contribute to the understanding of RJ's antibacterial mechanism, highlighting its potential as a natural alternative to conventional antibiotics. The identification of CRP and IHF as central players highlights the intricate regulatory networks involved in RJ's action, offering new targets for developing innovative antimicrobial strategies.

The division of labor is a dominant characteristic of honeybees and is accompanied by behavioral specialization and cognitive enhancement. As the central nervous system to control the labor-specific behaviors of honeybee, the brain is richest in lipid in terms of both diversity and abundance. In this study, an in-depth LC-MS/MS-based lipidomic method was applied to systematically characterize the brain lipid compositions of worker bees with three labor stages: newly emerged bee (NEB), nurse bee (NB), and forager bee (FB). A total number of 337 lipid species that assigned to 20 lipid classes were analyzed. The association of the brain lipidomes with the division of labors was suggested by the results of both the unsupervised and supervised multivariate pattern recognition analysis. More than 68% of the identified lipid species were found to be significantly changed in at least one comparison between NEB, NB, and FB. A total of 81 lipid species were identified as the potential labor-featured molecules with VIP > 1 and p-adj < 0.05. The labor-featured lipids of FA(18:2), FA(18:3), FA(26:0), PC(18:0_18:3), PS(18:1_18:1), SM(d38:1), CoQ10, and CoQ9, as well as their interactions with 12 behavior-related genes, including AmEST-6, AmFABP, AmE75, AmDGAT2, AmLSD1, AmNPC1, AmABCA1, AmNMDAR1, AmHTT, AmNOS, etc., were revealed by the further IPA analysis. These findings demonstrate for the first time that the brain lipidomes of worker bees are associated with the stable differences in their labors, which help understand the function of brain lipids on the labor-dependent behaviors of honeybees.

10-Hydroxy-2-decenoic acid (10-HDA), a major fatty acid (FA) component of royal jelly, is synthesized in the mandibular glands (MGs) of worker honeybees. Despite its well-documented nutritional and therapeutic significance, the biosynthetic pathway and regulatory mechanisms of 10-HDA production remain largely unresolved. In this study, the molecular basis of 10-HDA biosynthesis and regulation in the MGs of newly emerged bees (NEBs), nurse bees (NBs), and forager bees (FBs) were investigated using RNA sequencing and weighted gene co-expression network analysis (WGCNA). A five-step biosynthetic pathway for 10-HDA was proposed, and cross-species analysis of Apis mellifera and A. cerana revealed the conserved expression patterns of 15 key enzymes involved. Functional validation via RNA interference (RNAi) demonstrated that knockdown of acyl-CoA Delta(11) desaturase (d11ds, LOC551527), a key enzyme in FA desaturation, led to a 50% reduction in 10-HDA levels. Protein–protein interaction (PPI) network analysis further identified transcriptional regulators Kay and Drep-2 as potential modulators of 10-HDA metabolism. This study provides the first comprehensive mechanistic model of 10-HDA biosynthesis in honeybee MGs and highlights the labor-specific regulation of FA metabolism. These findings offer promising genetic targets for improving the royal jelly quality through genetic technology.

Target of rapamycin complex 1 (TORC1) is a master regulator of cell metabolism, and its dysregulation has been linked to an array of pathologies, including cancer and age-related diseases. Nprl3, a component of GTPase-activating protein towards Rags complex 1 (GATOR1), inhibits TORC1 activity under nutrient scarcity status. The nprl3 mutant exhibits some metabolic defects due to hyper TORC1 activity in Drosophila. Royal jelly (RJ) is a honeybee-secreted product and plays an essential role in caste differentiation that requires TORC1 activity. RJ is also used as a health-benefit food for its potential roles on antioxidant and anti-aging. In this study, nprl3-mutant flies were used to measure the effect of RJ on metabolic modulation. Interestingly, RJ feeding significantly increased survival and decreased TORC1 activity in the nprl3 mutant. RJ feeding also ameliorated the abnormal reactive oxygen species (ROS) levels and energy status in the nprl3 mutant. The proteins in RJ were characterized to be the essential components in increasing nprl3 mutant viability. These findings suggest that RJ modulates some metabolic defects associated with elevated TORC1 activity and that the nprl3-mutant fly might be a useful tool for investigating the bioactive components of RJ in vivo.