Abstract: 【Aim】 Circular RNA (circRNA) plays a key role in alternative splicing, transcription regulation and expression regulation of source genes. This study aims to analyze the quantity, variety, structural characteristics and function of circRNAs in the midgut of Apis mellifera ligustica workers, and to explore the regulatory function of circRNAs via constructing and analyzing regulatory networks. 【Methods】 A. m. ligustica workers were reared under laboratory conditions, and the midgut samples from 7- and 10-day-old workers were subjected to deep sequencing using circRNA-seq technology. CircRNAs were predicted from sequencing data after quality control using find_circ software. Source genes of these circRNAs were annotated to GO and KEGG databases to gain function and pathway annotations via BLAST. The target mRNAs of circRNAs and miRNAs were predicted with TargetFinder software, and the regulatory networks between circRNAs and miRNAs and between circRNAs, miRNAs and mRNAs were constructed and visualized using Cytoscape v.3.2.1software. The predicted circRNAs were validated by RT-PCR with the divergent and convergent primers designed. 【Results】 Sequencing of midgut samples from A. m. ligustica workers produced a mean of 136 463 071 clean reads, and for each sample over 136 779 122 anchor reads were obtained after removing rRNA. A total of 10 833 circRNAs were predicted, and their length ranged mainly from 15 to 1 000 nt. The types of these circRNAs were abundant, and the largest one was annotated to be exonic circRNA. The number of circRNAs distributed on chromosome 1 of Apis mellifera was the most and that distributed on chromosome 8 was the second. The source genes of circRNAs could be annotated to 45 GO terms including binding, cellular process, and cell, as well as 121 KEGG metabolic pathways including endocytosis, protein processing in endoplasmic reticulum, and ribosome, suggesting that circRNAs may play key roles in such biological processes as growth, development, metabolism, and cellular activity in the midgut of A. m. ligustica workers. Furthermore, the regulatory networks between circRNA and miRNA and between circRNA, miRNA and mRNA were constructed, and the analysis result demonstrated that partial circRNAs could bind microRNAs as competitive endogenous RNAs (ceRNAs). Finally, the true existence of randomly selected three circRNAs was validated by RT-PCR. 【Conclusion】 In this study, we predicted, analyzed, and verified circRNAs in the midgut of A. m. ligustica workers. Our findings provide the data of the quantity, variety, structural characteristics, roles, and regulatory network of circRNAs in the midgut, indicating that circRNAs can play roles in the developmental and immune defense processes in the midgut of A. m. ligustica workers through affecting source genes and acting as ceRNAs. This study lays a foundation for further studying the roles of circRNAs in the development and stress response of the midgut of A. m. ligustica.

Key words: Apis mellifera ligustica, midgut, bioinformatics, circRNAs, regulatory network