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.

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.