miR-34 regulates cuticle pigmentation by targeting Bm-iAANAT and Bmserpin3 in Bombyx mori

Pigmentation is an orchestrated process involved in cuticle melanization and sclerotization in insects, and plays a critical role in maintaining the structural integrity and functional completeness of the insect cuticle. Although the cascade reactions underlying pigmentation have been extensively studied, our understanding of the involvement of miRNA in this process remains limited. Here, we investigate the role of conserved microRNA-34 (miR-34) in regulating cuticular colouration in the silkworm, bombyx mori. Overexpression of miR-34 led to pronounced melanization in the larval abdomen. Mechanistically, LC/MS analysis revealed that miR-34 overexpression alters the epidermal amino acid composition, with a particularly notable increase in tyrosine and dopamine content. Enzyme activity assays confirmed the activation of phenoloxidase (PO). Through experimental validation, we identified two key target genes of miR-34, including Bm-iAANAT (a critical gene in melanin synthesis) and Bmserpin3 (a regulator of the serine protease cascade). This study uncovers a previously unreported miRNA mediated regulatory mechanism in insects and systematically elucidates how miR-34 coordinately modulates pigmentation through distinct molecular pathways, providing novel theoretical insights into this field.