Royal jelly, a highly nutritious food for bee larvae and the queen bee in a bee colony, is also a bee product that has been consumed by humans for centuries due to its purported health benefits1,2,3,4. The pharmaceutical properties of royal jelly have been extensively studied in both animal models and humans2,5,6,7. While the mechanisms of action remain under investigation, royal jelly has attracted considerable interest from researchers worldwide8,9. The increasing demand, driven by an aging population, has intensified the focus on royal jelly quality. Major royal jelly protein 1 (MRJP1) and 10-hydroxy-2-decenoic acid (10-HDA) are the most abundant protein and fatty acid, respectively, in royal jelly, and both serve as critical quality markers10.

The paired hypopharyngeal gland (HPG) and mandibular gland, both located in the heads of honey bees (Apis mellifera), are involved in the production of royal jelly11,12. The HPG comprises numerous tiny, interconnected glandular units known as acini. These acini consist of secretory cells that synthesize and secrete royal jelly, which is transported via the glandular lumen and collecting duct to the mouthparts12,13. HPG activity is age-dependent, with peak activity typically occurring between the 6th and 12th days after the emergence of worker bees14,15,16,17,18. A comprehensive proteome study revealed that highly activated protein and energy metabolism in the HPG are responsible for royal jelly production19. Moreover, dietary factors, including bee pollen and protein supplements, significantly affect the developmental size and activity of the HPG17,18,20,21.

The mandibular glands are located at the base of each mandible22. These glands are primarily involved in synthesizing and secreting lipid-based compounds that contribute to the composition of royal jelly, including the most abundant fatty acid, 10-HDA23. Through a comparative transcriptomic analysis of the mandibular glands between worker and queen bees, several CYP450 genes have been identified to be involved in the putative three-step biosynthesis pathway of 10-HDA, that is, in fatty acid hydroxylation, fatty acid chain shortening, and modification of the end hydroxy group24,25. Among these, CYP450 6AS8 was confirmed as a key enzyme in 10-HDA biosynthesis through RNAi knockdown experiments26, is believed to catalyze fatty acid hydroxylation in this pathway24. Dietary factors, such as high-protein diets and zinc supplementation, have been shown to enhance mandibular gland development27,28.

Taiwan’s tropical and subtropical climate fosters a diverse array of nectariferous plants. Beekeepers in Taiwan manage approximately 160,000 A. mellifera colonies, producing at least 700 tons of royal jelly annually, as per records provided by the Taiwan Beekeeping Association. Five predominant bee pollen types annually collected by local beekeepers include rape (Brassica napus, Bn), beggartick (Bidens pilosa var. radiata, Bp), tea tree (Camellia sinensis, Cs), nutgall tree (Rhus chinensis var. roxburghii, Rc), and corn (Zea mays, Zm)29. Given the prevalence of royal jelly production alongside bee pollen harvesting, and our understanding of these bee pollen types’ nutritional composition, this study investigated the influence of bee pollen diets on HPG development in newly emerged bees. We further examined the impact of these diets on the expression of mrjp1 and cyp450 6AS8, genes encoding key proteins involved in royal jelly biosynthesis. While royal jelly production is closely tied to bee nutrition, studying this relationship within a colony is challenging due to numerous confounding factors, including diet composition, worker bee population, and environmental conditions (humidity levels and pheromones). To isolate the effects of bee pollen on royal jelly quality and quantity, we aim to establish an in vitro royal jelly production system. By confining bees in a controlled laboratory environment, we can effectively mitigate the influence of colony-level factors. These works lay the groundwork for a comprehensive understanding of bee nutrition and royal jelly production.

Bee pollen consumption of caged bees

To evaluate bee pollen preference, 30 newly emerged bees were confined in cages containing a nest foundation, one of six bee pollen types (Bn, Rc, Cs, Bp, Zm, or a pollen mix), and a 50% sucrose solution. Cumulative bee pollen consumption was recorded over six days. As shown in Supplementary Fig. 1A, the total bee pollen consumption did not vary significantly among the different bee pollen types (ANOVA, F = 2.511, df1 = 5, df2 = 18, P = 0.0682). However, after correcting for evaporative loss, Rc had the highest average consumption (2.0 g), followed by Bn (1.8 g), Cs (1.6 g), Bp and mixed pollen (1.4 g each), and Zm (1.0 g). On an individual basis, bees consumed an average of 10.2, 11.0, 8.9, 7.6, 5.5, and 7.5 mg of the respective pollen types per day (ANOVA, F = 2.511, df1 = 5, df2 = 18, P = 0.0682) (Supplementary Fig. 1B). Notably, bee pollen consumption in Rc was twice that of Zm, this difference was not statistically significant, likely due to the high variability in bee pollen consumption among individual bees.