Abstract: 【Aim】This study aims to identify the humoral immune pathway-associated genes and their splice variants in the larval guts of Apis cerana cerana, so as to provide the resource and basis for the molecular cloning and functional study of genes and splice variants related to the humoral immune pathways in A. c. cerana. 【Methods】All of the identified full-length transcripts based on previous nanopore sequencing data of the gut transcriptome of A. c. cerana worker larvae were respectively aligned to the KEGG and Nr databases to screen out the genes and their splice variants related to the humoral immune signaling pathway by Blast. The identified humoral immune signaling pathway-associated genes were mapped to the A. cerana reference genome (version number: ACSNU-2.0) to optimize the structure of annotated genes using GffCompare software. Astalavista software was used to identify the types of alternative splicing (AS) events of the humoral immune signaling pathway-associated genes, followed by calculation of the numbers of different types of AS events. RT-PCR was employed to verify the authenticity of AS events. TAPIS pipeline software was utilized to predict the alternative polyadenylation (APA) sites contained in the genes related to the humoral immune signaling pathways, and the motifs of 50 bp upstream of the APA sites were identified by MEME software. The authenticity of the APA site was validated through 3′RACE. 【Results】 A total of 23 genes and 135 splice variants related to JNK-p38-MAPK signaling pathway, 21 genes and 130 splice variants related to Toll signaling pathway, 12 genes and 53 splice variants related to JAK/STAT signaling pathway, and 10 genes and 46 splice variants related to IMD signaling pathway were identified. The 5′UTRs of 18 genes related to the humoral immune pathways on the reference genome of A. cerana were extended, the 3′UTRs of 19 genes were extended, and the 5′UTRs and 3′UTRs of 8 genes were simultaneously extended. A total of 60 AS events of 16 genes related to humoral immune signaling pathways were identified, of which the most abundant type was alternative 5′splice site (A5SS) (26 events). The authenticity of the AS events of the randomly selected three genes was confirmed by RT-PCR. A total of 47 genes associated with humoral immune signaling pathways were detected to contain one or more APA sites, of which the genes containing one APA site were the largest group. Three consensus sequences were identified upstream of the APA sites: ATATA WAWWTATATRWATNYD, HVVNVNNDBDNBHNDDDNWNNNWNNHH and BSHDVWDRDBDDDKK NVWDRDKHHVKHDVNNVWDNDBHWHB. The results of 3′RACE confirmed the authenticity of the APA sites contained in two randomly selected genes. 【Conclusion】 For the first time, 66 genes and 364 splice variants related to humoral immune signaling pathways of A. c. cerana larval gut were identified. The structures of 29 genes annotated to humoral immune pathways on the A. cerana reference genome were optimized. Sixty AS events and 139 APA sites associated with humoral immune were discovered. The authenticity of AS events and APA sites of humoral immune genes was confirmed.

Key words: Apis cerana cerana, nanopore sequencing, humoral immune pathway, gene structure optimization, alternative splicing, alternative polyadenylation