Alternative Splicing and Alternative Polyadenylation-Regulated Cold Stress Response of Apis cerana

Bees, as individual poikilotherms capable of group homeothermy, possess temperature tolerance and regulatory mechanisms crucial for biodiversity conservation. The Asian honeybee (Apis cerana), a key pollinator for alpine and winter-flowering plants, exhibits superior cold tolerance over Apis mellifera, making it a dominant model species for insect cold resistance studies. While extensive research has delved into the behavioral and physiological facets of honeybee cold resistance, the post-transcriptional regulation in response to cold stress remains understudied. In this study, full-length transcriptome data obtained from PacBio and Illumina sequencing identified 25,443 events of alternative splicing (AS) and 2821 genes of alternative polyadenylation (APA) involved in the cold stress response of honeybees. GO and KEGG functional annotations were performed on the differential AS and APA between the cold stress (T vs. CK) and cold tolerance (TR vs. CK) groups in A. cerana workers of different ages, (3 days (Ac3d), 10 days (Ac10d), and 21 days (Ac21d) old), and the results indicated that these differentially expressed AS (DASs) and differentially expressed APAs (DAPAs) implicate signal transduction, energy metabolism, and oxidative metabolism. Venn analysis, expression clustering, and trend analysis highlight the roles of DASs in regulating high-stress proteins like HSP70 and BAG, with increasing expression levels, while DAPAs are mainly associated with signal transduction and energy metabolism, including genes like CUBN and UGCG, with gene expression levels initially decreasing and then increasing. We verified the isoforms of three DASs and the polyadenylation sites of three DAPAs using RT-PCR and 3 ' RACE. In conclusion, the post-transcriptional regulation of AS and APA in A. cerana may respond to chilling stress by participating in the regulation of different metabolic pathways and antifreeze proteins. The results of this study have reference value for the study of the molecular mechanisms of insect cold resistance and provide clues for the development of molecular breeding targets for cold-resistant strains of bees.