Abstract: 【Aim】The Italian honey bee (Apis mellifera ligustica) is a subspecies of the western honeybee, Apis mellifera, which has excellent production performance. This study aims to reveal the molecular mechanism underlying metamorphosis during pupal stage regulated by three microRNAs (miRNAs) ame-miR-13b, ame-miR-100 and ame-miR-bantam using molecular biological approaches.【Methods】Stem-loop RT-PCR was used to confirm the true expression of ame-miR-13b, ame-miR-100 and ame-miR-bantam in A. m. ligustica workers at the pupal stage. RT-qPCR was performed to survey the expression profiles of ame-miR-13b, ame-miR-100, ame-miR-bantam and their target mRNAs during the pupal development process of A. m. ligustica worker. Related bioinformatic software was used to predict target mRNAs of ame-miR-13b, ame-miR-100 and ame-miRbantam in A. m. ligustica workers at the pupal stage, to construct and analyze miRNA-mRNA regulation network, and to conduct annotation of target mRNAs in databases.【Results】The results revealed that ame-miR-13b, ame-miR-100 and ame-miR-bantam were truly expressed during the pupal development process of A. m. ligustica worker, and the overall expression displayed an up-regulation trend. The three miRNAs mentioned above could respectively target 850, 136 and 506 mRNAs, and complex regulatory network formed among these miRNAs and their target mRNAs,which could be respectively annotated to 32, 24 and 33 GO terms, and 204, 114 and 171 KEGG pathways.【Conclusion】ame-miR-13b, ame-miR-100 and ame-miR-bantam potentially regulate the expression of ecdysone gene, yellow gene and genes related to Hippo, FoxO, Wnt, Jak-STAT and Notch signaling pathways, further affecting metamorphosis during the pupal development process of A. m. ligustica worker.

Key words: Apis mellifera ligustica, pupal stage, metamorphosis, microRNA, expression profile, regulatory network