Abstract

Inhibition of the lnc6140 in A. apis affects the chalkbrood occurrence in honeybee larvae. • Lnc6140 positively regulates the milR5658-x–ATPase axis, promoting infection and proliferation of A. apis. • Lnc6140 and milR5658-x are effective targets for the control of chalkbrood. Chalkbrood is a fungal disease caused by the infection of honeybee larvae by Ascosphaera apis . Currently, effective methods for controlling chalkbrood are lacking. Fungal non-coding RNAs play a critical role in enhancing infectivity and pathogenicity, making them potential high-efficacy targets for suppressing A. apis infection. Based on full-length transcriptome data comparing spores and mycelium of A. apis , and the midgut of honeybees ( Apis mellifera ) infected by A. apis , a specifically expressed long non-coding RNA 6140 (lnc6140) in A. apis was identified. The study demonstrated that lnc6140 in A. apis positively regulates ATPase expression through milR5658-x, thereby influencing pathogen infectivity and chalkbrood development. In this study, after A. apis infection, the expression levels of lnc6140, milR5658-x, and ATPase increased over the infection period. Interference with lnc6140 suppressed the expression of genes associated with A. apis infection and proliferation— Chi3 , GriF , AdmB , Pkia , Ste11 , and Dmap1 —and significantly inhibited chalkbrood incidence. Dual-luciferase assays demonstrated that lnc6140 positively regulates the expression of milR5658-x, and milR5658-x positively regulates the expression of ATPase in A. apis . Similarly, reducing the RNA level of milR5658-x also significantly inhibited chalkbrood incidence. Concurrent reduction of lnc6140 and milR5658-x levels suppressed ATPase expression and markedly decreased chalkbrood occurrence. The result indicated that lnc6140 of A. apis plays a key role in pathogen infection by regulating the milR5658-x– ATPase axis. Suppressing lnc6140 and milR5658-x of A. apis may serve as a cruc