Abstract

While high-throughput sequencing and bioinformatic predictions have identified numerous circRNAs in honeybees, research into their functional roles and molecular mechanisms remains limited. This study aims to characterize the regulatory functions of a previously identified circRNA (novel_circ_002651, ac2651) and its key target miRNA (ace-miR-6001-y), in eastern honeybee worker larvae responding to infection by A. apis, a causative fungal pathogen for chalkbrood disease. Here, RT-qPCR detection showed that the expression of ac2651 was significantly down-regulated in the 6-d-old larval gut, while the ace-miR-6001-y expression exhibited an opposite trend. RNAi-based interference of ac2651 significantly increased the expression of both host immune genes and fungal proliferation-associated genes. Additionally, silencing ac2651 resulted in a reduction of host survival rate and a significant elevation of chalkbrood incidence. Overexpression and knockdown experiments demonstrated that ace-miR-6001-y negatively regulated the expression of dorsal in the larval guts and affected the expression of mat1-2-1, Ste11-like, and Htf in A. apis. Dual-luciferase reporter assay confirmed direct interactions between ac2651 and ace-miR-6001-y as well as between ace-miR-6001-y and Ac14-3-3ζ. It was validated that simultaneous ac2651 silencing and ace-miR-6001-y knockdown reversed the upregulation of Ac14-3-3ζ induced by ace-miR-6001-y knockdown alone. These results delineate a novel regulatory axis wherein ac2651 sponges ace-miR-6001-y to alleviate its repression of Ac14-3-3ζ, thereby activating the host immune defense against the A. apis infection. Our findings not only offer novel insights into the interaction between honeybee larvae and fungal pathogen but also provide promising biomarkers and targets for the diagnosis and treatment of chalkbrood.