Abstract: 【Aim】 PIWI-interacting RNAs (piRNAs) play a key regulatory role in crucial biological processes such as development and immunity in insects. The objective of this study is to enrich the information of Apis mellifera piRNAs and to provide a basis for further exploration of the molecular mechanism underlying differentially expressed piRNA (DEpiRNA) regulation of the development of the midgut of A. m. ligustica workers. 【Methods】 piRNAs were predicated and analyzed based on previously gained small RNA (sRNA) omics data from the midguts of the 7-day-old (Am7) and 10-day-old (Am10) workers of A. m. ligustica. The sRNA data after quality control was mapped to the reference genome of A. mellifera, the mapped tags were then further mapped to database to filter out small non-coding RNAs (ncRNAs) such as rRNAs and tRNAs, and the identification of piRNAs according to their length characteristics followed. The expression levels of piRNAs were calculated and normalized using tags per million (TPM) arithmetic. DEpiRNAs in the Am7 vs Am10 comparison group were screened out according to the standard of |log₂ fold change|≥1 and P≤0.05. Target mRNAs of DEpiRNAs were predicated with related software, and GO and KEGG annotation of DEpiRNAs was performed. The DEpiRNA-mRNA regulatory networks were visualized with Cytoscape software. The expression of six randomly selected piRNAs shared in Am7 and Am10 was verified by Stem-loop RT-PCR, and the expression trends of six randomly selected DEpiRNAs were validated by RT-qPCR. 【Results】 In total, we identified 596 piRNAs from the midgut of A. m. ligustica workers. The length distribution of the above-mentioned piRNAs was among 24-33 nt, and there was obvious difference in the bias of the first base of piRNAs in different lengths within Am7 and Am10 groups. Additionally, we identified 41 DEpiRNAs in the Am7 vs Am10 comparison group, in which piR-ame-11093, piR-ame-111451,piR-ame-190949 and piR-ame-932156 could target 1 195, 1 018, 4 040 and 1 063 mRNAs, respectively. These target mRNAs could be respectively annotated to 45 functional terms and 45 pathways. Stem-loop RT-PCR results verified the true expression of six piRNAs (piR-ame-1084826, piR-ame-11093, piR-ame-14476, piR-ame-24995, piR-ame-39500 and piR-ame-774987). RT-qPCR results exhibited that the expression trends of six DEpiRNAs (piR-ame-1084826, piR-ame-11093, piR-ame-14476, piR-ame-24995, piR-ame-39500 and piR-ame-774987) were in accordance with those in the sequencing data, confirming the reliability of sRNA-seq data and the authenticity of differential expression trends of piRNAs in this study. 【Conclusion】 In this study, a total of 596 piRNAs have been identified from the midgut of A. m. ligustica workers, with a length distribution of 24-33 nt. The bias of the first base of piRNAs with various length distribution ranges has apparent differences, and piR-ame-11093, piR-ame-1111451, piR-ame-190949 and piR-ame-932156 are potentially involved in the developmental process of the midgut of A. m. ligustica workers via regulation of target gene expression.

Key words: Apis mellifera; Apis mellifera ligustica, non-coding RNA, piRNA, midgut