Interactions of juvenile hormone, 20-hydroxyecdysone, developmental genes, and miRNAs during pupal development in Apis mellifera

Insect development is primarily controlled by juvenile hormone (JH) and 20-hydroxyecdysone (20E), which regulate gene cascades leading to changes in phenotype, physiology, and behavior. Besides these hormones, microRNAs play a crucial role in insect development by regulating gene expression at the post-transcriptional level. To advance the molecular understanding of holometabolous developmental events, we investigate the pupal phase in the honeybee, Apis mellifera. In this study, we assessed the expression profiles of genes components of JH and 20E cascades - Usp, ftz-f1, EcR, Met, Chd64, InR-2, Kr-h1 and Tai - as well as the microRNAs miRNA-34 and miRNA-281 during pupal development of A. mellifera. We then analyzed the impact of JH and 20E treatments on the expression of these developmental genes and their putative regulators, the microRNAs. Overall, the selected genes and miRNAs remained stable or were downregulated following 20E treatment, while treatments with JH, upregulated most of our candidate developmental genes and microRNAs. Notably, the expression profile of Met, an intracellular receptor of JH, showed a strong correlation with fluctuations in 20E titers during pupal development. Furthermore, a computational analysis, followed by experimental assays, points to both miR-34 and miR-281 as potential regulators of pupal development in A. mellifera. This study paves the way for a better understanding of how JH and 20E hormones interact with developmental genes and microRNAs (miR-34 and miR-281) to regulate pupal development in honeybees, elucidating a piece of this complex network of interactions.