Abstract: 【Aim】 To offer a scientific basis for further study on the regulatory function and mechanism of lncRNA16999 in Apis mellifera ligustica by analyzing the regulatory mode and role of lncRNA16999 and detecting the expression patterns of lncRNA16999 in various developmental stages and tissues of A. mellifera ligustica workers. 【Methods】 According to the genomic position of the parental gene of lncRNA16999 on the chromosome of A. mellifera drone adult, the protein-coding genes located within 10 kb upstream and downstream were predicted for cis-acting analysis and annotated in GO and KEGG datasets. Lnc Tar software was used to predict the target mRNA of lncRNA16999 for trans-acting analysis. Miranda, RNAhybrid and TargetScan software were used to respectively predict lncRNA16999-targeted miRNAs and miRNA-targeted mRNAs, and the lncRNA16999-involved competitive endogenous RNA (ceRNA) regulatory network was constructed based on the target relationships, followed by GO and KEGG database annotation of target mRNAs. The expression levels of lncRNA16999 in the antenna, venom gland, brain, midgut, fat body, cuticle and hypopharyngeal gland of the newly emerged adult workers were detected by RT-PCR. RT-qPCR was employed to detect the relative expression levels of lncRNA16999 in the worker egg, larva, prepupa and pupa and various day-old adult workers, and the antenna, venom gland, brain, midgut, fat body, cuticle and hypopharyngeal gland of the newly emerged adult workers. 【Results】 LncRNA16999 potentially regulated seven upstream and downstream genes, involving 41 GO terms and one KEGG pathway. There was a positive correlation between lncRNA16999 and one co-expressed mRNA. LncRNA16999 was found to target 35 miRNAs and further target 84 mRNAs, forming a complex ceRNA regulatory network. These target mRNAs were annotated to 23 functional terms such as biological processes and metabolic processes as well as 11 pathways such as endocytosis and metabolic pathway. The target fragments with expected size were successfully amplified in the aforementioned seven tissues. There was no significant difference in the expression level of lncRNA16999 between larva and prepupa, however, the expression levels of lncRNA16999 in larva and prepupa were significantly lower than that in the egg. The expression level of lncRNA16999 in the pupa was the highest and significantly higher than those in the egg, larva and prepupa. In addition, the expression levels of lncRNA16999 in the 6- and 18-day-old adult workers was significantly lower than that in the 1-day-old adult workers. The expression levels of lncRNA16999 in worker’s brain and fat body were similar to that in the cuticle but significantly lower than that in the antenna. Additionally, the expression level of lncRNA16999 in the venom gland was significantly higher than that in the antennae. 【Conclusion】 lncRNA16999 plays potential regulatory roles in A. mellifera ligustica through cis-acting, trans-acting and ceRNA network. LncRNA16999 was dynamically and differentially expressed in various developmental stages and tissues of A. mellifera ligustica workers.

Key words: Apis mellifera ligustica, long non-coding RNA; cis-acting; trans-acting, endogenous competitive RNA, spatiotemporal expression profile