ame-miR-34 modulates the larval body weight and immune response ofApis melliferaworkers toAscosphara apisinvasion
Authors: Wu Ying , Guo Yilong , Fan Xiaoxue , Zhao Haodong , Zhang Yiqiong , Guo Sijia , Jing Xin , Liu Zhitan , Feng Peilin , Liu Xiaoyu , Zou Peiyuan , Li Qiming , Na Zhihao , Zhang Kuihao , Chen Dafu , Guo Rui 
Published: November 2022

MiRNAs are critical regulators of numerous physiological and pathological processes.Ascosphaera apisexclusively infects bee larvae and causes chalkbrood disease. However, the function and mechanism of miRNAs in the bee larval response toA. apisinfection is poorly understood. Here, ame-miR-34, a previously predicted miRNA involved in the response ofApis mellifera ligusticalarvae toA. apisinvasion, was subjected to molecular validation, and overexpression and knockdown were then conducted to explore the regulatory functions of ame-miR-34 in larval body weight and immune response. Stem-loop RT–PCR and Sanger sequencing confirmed the authenticity of ame-miR-34 in the larval gut ofA. m. ligustica. RT–qPCR results demonstrated that compared with that in the uninfected larval guts, the expression level of ame-miR-34 was significantly downregulated (P< 0.001) in the guts ofA. apis-infected 4-, 5-, and 6-day-old larvae, indicative of the remarkable suppression of host ame-miR-34 due toA. apisinfection. In comparison with the corresponding negative control (NC) groups, the expression level of ame-miR-34 in the larval guts in the mimic-miR-34 group was significantly upregulated (P< 0.001), while that in the inhibi- tor-miR-34 group was significantly downregulated (P< 0.01). Similarly, effective overexpression and knockdown of ame-miR-34 were achieved. In addition, the body weights of 5- and 6-day-old larvae were significantly increased compared with those in the mimic-NC group; the weights of 5-day-old larvae in the inhibitor-miR-34 group were significantly decreased in comparison with those in the inhibitor-NC group, while the weights of 4- and 6-day-old larvae in the inhibi- tor-miR-34 group were significantly increased, indicating the involvement of ame-miR-34 in modulating larval body weight. Furthermore, the expression levels of bothhspandabctin the guts ofA. apis-infected 4-, 5- and 6-day-old larvae were significantly upregulated after ame-miR-34 overexpression. In contrast, after ame-miR-34 knockdown, the expression levels of the aforementioned two key genes in theA. apis-infected 4-, 5- and 6-day-old larval guts were significantly downregu- lated. Together, the results demonstrated that effective overexpression and knockdown of ame-miR-34 in both noninfected andA. apis-infectedA. m. ligusticalarval guts could be achieved by the feeding method, and ame-miR-34 exerted a regulatory function in the host immune response toA. apisinvasion through positive regulation of the expression ofhspandabct. Our findings not only provide a valuable reference for the functional investigation of bee larval miRNAs but also reveal the regulatory role of ame-miR-34 inA. melliferalarval weight and immune response. Additionally, the results of this study may provide a promising molecular target for the treatment of chalkbrood disease.