Ectopic expression of structurally similar major royal jelly proteins reveals their distinct functions in Drosophila

Royal jelly (RJ), secreted by the hypopharyngeal and mandibular glands of young worker bees, is rich in proteins, 80%‒90% of which are major royal jelly proteins (MRJPs). While MRJPs from RJ have been shown to exhibit specific biological functions and are also expressed in neuronal cells, their roles in vivo remain poorly understood. The aim of this study was to elucidate the functional roles of individual MRJPs (MRJP1‒9) in vivo by ectopically expressing them in Drosophila neurons in a binary expression system using fluorescent proteins as controls. Transcriptome sequencing revealed that although MRJP1‒9 share similar tertiary structures, their overexpression affects distinct gene sets. MRJP1, MRJP2, MRJP3, MRJP5, and MRJP7 induced more differentially expressed genes (DEGs), while MRJP4, MRJP6, MRJP8, and MRJP9 induced fewer such genes. Weighted gene co-expression network analysis (WGCNA) and gene set enrichment analysis (GSEA) revealed that MRJP1, MRJP2, MRJP3, MRJP5, and MRJP7 regulated overlapping gene sets, including an estradiol-responsive set, and activated cell proliferation pathways. MRJP6 lacked any significant gene set enrichment, while MRJP8 and MRJP9 modulated similar sets. Notably, the neuron-specific overexpression of MRJP1, MRJP2, MRJP3, and MRJP5 in Drosophila showed activated cell proliferation-related pathways and increased body sizes, highlighting their functional diversity and context-dependent effects. These findings expand our understanding of the functional roles of MRJPs and provide a foundation for further exploring their biological significance in honeybees and beyond.