Abstract: 【Aim】 The aim of this study is to clarify the regulatory effects of ame-miR-bantam on the expression of target genes USP and P300 by performing overexpression and knockdown of ame-miR-bantam in the larval gut of Apis mellifera ligustica workers through feeding method. 【Methods】 Overexpression and knockdown of ame-miR-bantam in tissues of the larval gut of the 4-6-day-old workers of A. m. ligustica were conducted by feeding mimic-ame-miR-bantam and inhibitor-ame-miR-bantam, and their corresponding negative controls mimic-NC-ame-miR-bantam and inhibitor-NC-ame-miR-bantam of ame-miR-bantam, respectively. Target genes of ame-miR-bantam were predicted and analyzed using bioinformatic software. RT-qPCR was used to detect the overexpression and knockdown effects of ame-miR-bantam and the relative expression levels of target genes USP and P300 in the larval gut of the 4-6-day-old workers of A. m. ligustica. 【Results】 Compared with feeding with mimic-NC-ame-miR-bantam, feeding with mimic-ame-miR-bantam resulted in significant up-regulation of ame-miR-bantam in the larval gut of the 4-6-day-old workers of A. m. ligustica. Compared with feeding with inhibitor-NC-ame-miR-bantam, feeding with inhibitor-ame-miR-bantam resulted in significant down-regulation of ame-miR-bantam in the larval gut of the 4-day-old workers, and down-regulation of ame-miR-bantam in the larval gut of the 5- and 6-day-old workers. ame-miR-bantam can target a total of 222 genes, which can be annotated to 35 GO terms such as cellular process and 160 KEGG pathways such as Wnt signaling pathway. After overexpression of ame-miR-bantam, the expression levels of USP and P300 in the larval gut of the 4-day-old workers were up-regulated, that of USP in the larval gut of the 5-day-old workers was down-regulated, that of P300 in the larval gut of the 5-day-old workers was significantly down-regulated, those of USP and P300 in the 6-day-old larval gut were significantly down-regulated as compared to those in the negative control group fed with mimic-NC-ame-miR-bantam. After knockdown of ame-miR-bantam, the expression levels of USP and P300 in the larval gut of the 4- and 5-day-old workers were up-regulated, that of USP in the larval gut of the 6-day-old workers was significantly up-regulated whereas that of P300 in the larval gut of the 6-day-old workers was up-regulated as compared with those in the negative control group fed with inhibitor-NC-ame-miR-bantam. 【Conclusion】 Effective overexpression and knockdown of ame-miR-bantam in the larval gut of A. m. ligustica workers can be achieved by feeding with mimic and inhibitor, respectively, and ame-miR-bantam in the larval gut may negatively regulates the expression of USP and P300.

Key words: Apis melliferae ligustica, microRNA, ame-miR-bantam, mimic, inhibitor, gut, development