Comprehensive Genomic Analysis Reveals Novel Transposable Element-Derived MicroRNA Regulating Caste Differentiation in Honeybees

The honeybee (Apis mellifera) is a highly social insect whose caste differentiation is regulated by epigenetic mechanisms, representing a classic example of phenotypic plasticity in social insects. Although the importance of transposable elements (TEs) in epigenetic research is well recognized, their specific role in honeybee caste differentiation has not been fully explored. This study reveals a novel regulatory mechanism where the microRNA (miRNA) ame-mir-3721-3p, derived from ApME (Apis miniature inverted-repeat TEs), suppresses DNA methyltransferase gene DNMT3, promoting queen-like development in honeybee larvae. Genome-wide analysis identified 43 ApME elements in Apis, with ApMETm15 being particularly abundant and species-specific. These elements gave rise to 6 miRNAs, including ame-mir-3721-3p which showed notable regulatory potential. Target gene prediction and luciferase reporter assays confirmed that ame-mir-3721-3p binds to and suppresses DNMT3 expression. Spatiotemporal expression analysis indicated that ame-mir-3721-3p is significantly upregulated during the critical L3 larval stage, exhibiting a similar expression pattern to DNMT3. Larval feeding experiments with agomir demonstrated that ame-mir-3721-3p suppresses DNMT3 expression and significantly impacts the expression of genes related to the juvenile hormone and ecdysone pathways. Further physiological evidence showed that when larvae were treated with agomir-3721 during the critical caste differentiation window (L3-L4 stage), the emerging adult bees exhibited increased body size, doubled ovarian area, and significantly higher frequency of ovary development, with significant upregulation of ovarian-specific marker genes. These findings provide direct evidence for ame-mir-3721-3p's role in promoting queen-like developmental trajectories during caste differentiation, uncovering a new regulatory pathway in honeybee development and offering insights into epigenetic mechanisms in social insects.