BmMBNL Knockout Disrupts Molting of Silkworm, Bombyx mori, Through Inhibiting Expressions of Chitin Synthetase and Chitinase

Insects periodically undergo molting to replace their old epidermis for growth and morphological adaptation. Muscleblind-like (MBNL) is a multifunctional protein involved in miRNA processing. To explore the role of miRNA processing in insect molting, we characterized BmMBNL in Bombyx mori. BmMBNL exhibited spatiotemporal expression across tissues and developmental stages, with the highest levels in the epidermis and upregulation during molting. CRISPR/Cas9-mediated knockout of BmMBNL (BmMBNL-KO) caused molting defects at the 3rd and 4th instar larvae, resulting in reduced size and eventual lethality. Moreover, BmMBNL-KO individuals showed a thinner chitin layer between the new and old epidermises. RT-PCR and transcriptome analyses revealed upregulation of miR-1, miR-71, miR-263, miR-8-5p, and miR-2a-3p, which suppressed chitinase expression, ultimately leading to molting failure. This study provides novel insights into the potential correlation between BmMBNL, miRNA abundance and insect molting in Bombyx mori.