Abstract

Chalkbrood fungus Ascosphaera apis (A. apis) produces miRNA-like small RNAs (milRNAs) that may modulate honey bee (Apis mellifera) larval biology through cross-kingdom RNA interference (ckRNAi). We identified 380 , 106 , 107, and 110 highly expressed milRNAs (HmilRNAs) in pure spores and infected larval guts at days 4, 5, and 6, respectively. Eighteen HmilRNAs were conserved across all groups, forming a complex regulatory network predicted to target 188 host genes (6,057 mRNAs) associated with critical signaling pathways, including Wnt, Hippo, mTOR, MAPK, and Toll/Imd. Among these, aap-milR-11980-x was validated to directly target the ecdysone-inducible gene E 75 and the transcriptional repressor tramtrack ( TTK ). Dual-luciferase assays confirmed that aap-milR-11980-x represses these targets via specific binding to their 3′-UTR. In vivo modulation of aap-milR-11980-x in 6-day-old larval guts using agomirs and antagomirs successfully altered E 75, TTK , and antimicrobial peptide (AMP) transcript levels. However, despite significant molecular regulation, no statistically significant difference in larval survival was observed between treatment groups ( P > 0.05). This discrepancy suggests that while aap-milR-11980-x effectively rewires host gene expression, its primary function may be to induce sub-lethal physiological changes, such as developmental delay or immune modulation, rather than acute lethality. These findings indicate that A. apis HmilRNAs fine-tune host cellular responses, potentially to extend the temporal window for fungal development or resource acquisition, highlighting a sophisticated layer of molecular manipulation in the A. apis -honey bee interaction.