Author:
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
Abstract
MiRNAs are critical regulators of numerous physiological and pathological processes. Ascosphaera apis exclusively infects bee larvae and causes chalkbrood disease. However, the function and mechanism of miRNAs in the bee larval response to A. apis infection is poorly understood. Here, ame-miR-34, a previously predicted miRNA involved in the response of Apis mellifera larvae to A. apis invasion, 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 of A. mellifera. 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 of A. apis-infected 4-, 5-, and 6-day-old larvae, indicative of the remarkable suppression of host ame-miR-34 due to A. apis infection. 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 inhibitor-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 inhibitor-miR-34 group were significantly increased, indicating the involvement of ame-miR-34 in modulating larval body weight. Furthermore, the expression levels of both hsp and abct in the guts of A. 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 the A. apis-infected 4-, 5-, and 6-day-old larval guts were significantly downregulated. Together, the results demonstrated that effective overexpression and knockdown of ame-miR-34 in both noninfected and A. apis-infected A. mellifera larval guts could be achieved by the feeding method, and ame-miR-34 exerted a regulatory function in the host immune response to A. apis invasion through positive regulation of the expression of hsp and abct. 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 in A. mellifera larval weight and immune response. Additionally, the results of this study may provide a promising molecular target for the treatment of chalkbrood disease.
Funder
National Natural Science Foundation of China
China Agriculture Research System
Master Supervisor Team Fund of Fujian Agriculture and Forestry University
Scientific Research Project of College of Animal Sciences (College of Bee Science) of Fujian Agriculture and Forestry University
Undergraduate Innovation and Entrepreneurship Training Program of Fujian province
Subject
Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Computer Science Applications,Spectroscopy,Molecular Biology,General Medicine,Catalysis
Reference58 articles.
1. Honey Bee (Apis mellifera) Immunity;Morfin;Vet. Clin. North Am. Food Anim. Pract.,2021
2. Zeng, Z.J. (2017). Apiculture, China Agriculture Press. [3rd ed.]. (In Chinese).
3. Chalkbrood disease in honey bees;Aronstein;J. Invertebr. Pathol.,2010
4. miRBase: microRNA sequences, targets and gene nomenclature;Grocock;Nucleic Acids Res.,2006
5. miRNAs regulated by estrogens, tamoxifen, and endocrine disruptors and their downstream gene targets;Klinge;Mol. Cell. Endocrinol.,2015