This study aims to identify and analyze the genes and full-length transcripts related to humoral immune pathways in Apis mellifera ligustica using the obtained Nanopore long-read sequencing data, providing resource and basis for further functional study. All of the identified full-length transcripts in A. m. ligustica were aligned to the Nr database via BLAST tool, screening genes and isoforms related to humoral immune pathways. Structures of annotated genes were optimized by comparing full-length transcripts with annotated transcripts in the A. mellifera reference genome (Amel_HAv3.1) using gffcompare software. The TAPIS pipeline was applied to predict and analyze alternative polyadenylation (APA) sites of relevant genes, followed by identification of motifs upstream of APA sites by MEME software. Astalavista software was used to identify alternative splicing (AS) events, while visualization of isoforms' structures was performed by IGV browser. The authenticity of AS events was verified using RT-PCR. Specifically, 26 genes and 41 isoforms associated with Toll signaling pathway, 9 genes and 11 isoforms related to Imd signaling pathway, 18 genes and 21 isoforms relative to JNK-MAPK-p38 signaling pathway, 9 genes and 13 splices linked to JAK-STAT signaling pathway were identified, respectively. A total of 20 genes annotated to humoral immune pathways in the reference genome of A. mellifera were structurally optimized, including: 5 and 15 genes with positive and negative chain structure optimization, respectively; 13 genes with 5'-end extensions and 7 with 3'-end extensions, including two genes (LOC724728 and LOC411861) extended at both ends. Additionally: 69 AS events of humoral immune pathway-associated genes were identified, including: 2 alternative 3' splice sites (A3SS), 17 intron retention (IR), 14 alternative 5' splice sites (A5SS) and 36 exon skipping (ES). RT-qPCR results were indicative of the expected sizes of target fragments, confirming the authenticity of randomly selected 6 AS events. 40 genes related to humoral immune pathway were discovered to have one or more APA sites. Multiple motifs were identified upstream of the APA sites, and the consensus sequence was GGWRRWRTHAARHTWKSYGAYTTTGGTRTWTCNGSDHRMHTDRY HGMWWS. APA sites of two genes were validated by 3' RACE. We identified 62 genes and 86 splices relative to the humoral immune pathways of A. m. ligustica, and optimized structures of 20 related genes in A. mellifera, discovered 69 AS events and 106 APA sites and confirmed the authenticity of their AS events and APA sites.

The objective of this work is to enrich the information about Apis cerana synaptic vesicle glycoprotein 2B protein (AcSV2B) and provide a basis for further functional dissection, by analyzing the physicochemical property and molecular characteristics of the AcSV2B and detecting the expression profile of AcSV2B gene in different tissues and developmental stages of worker bees. The expression and sequence authenticity of AcSV2B in A. cerana worker bees were verified by Stem-loop RT-PCR and Sanger sequencing. Relevant bioinformatic software was employed to predict the physicochemical property, signal peptide, phosphorylation site, secondary structure and tertiary structure of AcSV2B, to identify and compare structural domains and conserved motifs in SV2B among A. cerana and other species followed by construction and investigation of the phylogenetic tree. Real-time quantitative PCR (RT-qPCR) was utilized to detect the relative expression level of AcSV2B in various tissues (antenna, midgut, brain, venom gland, epidermis, hypopharyngeal gland and fat body), as well as in different developmental stages (egg, larva, prepupa, pupa and adult) of worker bees, and worker adults at different day-old. AcSV2B was confirmed to be genuinely present in the worker bees of A. cerana, which was consistent with the predicted results. AcSV2B contained 3814 nucleotides, encoding 671 amino acids. The molecular weight of AcSV2B was approximately 72.87 ku, the molecular formula was C₃₃₁₇H₅₁₆₂N₈₄₂O₉₄₀S₃₁, the aliphatic index was 102.32, and the theoretical pI was 6.02. AcSV2B included 24 serine phosphorylation sites and 11 transmembrane domains, but there were no typical signal peptides. AcSV2B was mainly distributed in the endoplasmic reticulum. Additionally, AcSV2B included 1 structural domain and 3 conserved motifs. SV2B from A. cerana, Apis florea and Apis mellifera were clustered into a clade on the phylogenetic tree. Furthermore, the relative expression level of AcSV2B was the highest in brain and significantly higher (P < 0.05) than that in antenna, midgut, venomous gland, epididymis, hypopharyngeal gland and fat body; the relative expression level of AcSV2B was the highest in 8-day-old prepupa and significantly higher (P < 0.05) than that in egg, 3-day-old larva, and 7-day-old prepupa; the relative expression level of AcSV2B was the highest in 18-day-old adult and significantly higher (P < 0.05) than that in 1-, 2-, 6- and 12-day-old adults. AcSV2B is predicted to be a hydrophilic protein, transmembrane protein and intracellular protein. The SV2B of A. cerana, A. florea and A. mellifera were highly conserved and homologous, and AcSV2B was highly expressed in brain, 8-day-old prepupal and 18-day-old adult.