Abstract

Chalkbrood disease, caused by Ascosphaera apis , threatens Apis cerana larvae. However, miRNA-mediated antifungal regulation remains poorly defined. Here, we characterized ace-miR-3759-y in A. apis -challenged A. cerana larvae. Stem-loop RT-PCR and Sanger sequencing confirmed its gut expression. Target prediction identified 146 candidate mRNAs, and dual-luciferase assays verified AcDorsal 1 and AcARL 4 C as direct targets, significantly suppressing reporter activity ( P < 0.01). During infection, ace-miR-3759-y was upregulated ( P = 0.0008), whereas AcDorsal 1 was downregulated ( P = 0.0011). Inhibiting ace-miR-3759-y reduced its expression ( P < 0.0001), relieved target repression, enhanced reactive oxygen species (ROS) accumulation ( P = 0.0006), tended to increase antimicrobial peptide (AMP) gene expression, and decreased fungal ADM-B and chsD expression. Functionally, ace-miR-3759-y inhibition reduced cumulative mortality and chalkbrood incidence (both P < 0.0001), whereas AcARL 4 C silencing suppressed AMP genes and aggravated disease. These findings reveal a dual-target miRNA module and provide a potential RNAi-based target for chalkbrood control.