Abstract: 【Aim】 The objective of this study is to unravel the regulatory role of ame-miR-14 in the developmental process of the larval guts of Apis mellifera ligustica workers.【Methods】 The expression and sequence authenticity of ame-miR-14 in the 6-day-old larval guts of A. m. ligustica workers were proved by Stem-loop RT-PCR and Sanger sequencing, respectively. Overexpression and knockdown of ame-miR-14 were conducted by feeding its corresponding mimic (mimic-ame-miR-14) and inhibitor (inhibitor-ame-miR-14), and their corresponding negative controls mimic-NC and inhibitor-NC, respectively, and then RT-qPCR was used to detect the expression levels of ame-miR-14 in the 4-6-day-old larval guts of A. m. ligustica workers. Bioinformatic software was used to predict and analyze the target genes of ame-miR-14. RT-qPCR was employed to detect the relative expression levels of the target genes FoxO and Hedgehog in the 4-6-day-old larval guts after overexpression and knockdown of ame-miR-14.【Results】 ame-miR-14 truly exists and is expressed in the 6-day-old larval guts of A. m. ligustica workers. The expression levels of ame-miR-14 in the 4-6-day-old larval guts of A. m. ligustica workers fed with mimic-ame-miR-14 were significantly up-regulated as compared to those fed with mimic-NC. The expression levels of ame-miR-14 in the 4-6-day-old larval guts of A. m. ligustica workers fed with inhibitor-ame-miR-14 were significantly down-regulated as compared to those fed with inhibitor-NC. In total, ame-miR-14 can target 309 genes, which could be annotated to 45 KEGG pathways and 36 GO terms. Further analysis showed that ame-miR-14 can target 14 genes associated with growth and development and have potential targeting relationship with target genes FoxO and Hedgehog. After overexpression of ame-miR-14, the expression level of FoxO in the 4dayold larval gut of the mimic-ame-miR-14 group was down-regulated and those in the 5- and 6-day-old larval guts significantly down-regulated as compared to those in the mimic-NC group. After knockdown of ame-miR-14, the expression level of FoxO in the 4-day-old larval gut of the inhibitor-ame-miR-14 group was down-regulated, while that in the 5-day-old larval gut was significantly up-regulated and that in the 6-day-old larval gut was up-regulated as compared to those in the inhibitor-NC group. After overexpression of ame-miR-14, the expression levels of Hedgehog in the 4-6-day-old larval guts of the mimic-ame-miR-14 group were significantly down-regulated in comparison with those in the mimic-NC group, whereas those in the 4-6-day-old larval guts of the inhibitor-ame-miR-14 group were up-regulated in comparison with those in the inhibitor-NC group. 【Conclusion】ame-miR-14 truly exists and is expressed in the larval guts of A. m. ligustica workers. Effective overexpression and knockdown of ame-miR-14 in the larval guts of A. m. ligustica workers can be achieved by feeding mimic and inhibitor, respectively. ame-miR-14 potentially participates in regulation of the larval gut development by negatively regulating the expression of FoxO and Hedgehog.

Key words: Apis melliferae ligustica, larva, gut, ame-miR-14, target gene, regulation, development