摘要Caste differentiation in social hymenopterans is an intriguing example of phenotypic plasticity.However,the co-ordination among gene regulatory factors to mediate caste differentiation remains inconclusive.In this study,we determined the role of gene regulation and related epigenetic processes in pre-imaginal caste differentiation in the primitively eusocial bumblebee Bombus terrestris.By combining RNA-Seq data from Illumina and PacBio and accurately quantifying methylation at whole-genomic base pair resolution,we found that queens,workers,and drones mainly differentiate in gene expression but not in alternative splicing and DNA methylation.Gynes are the most distinct with the lowest global level of whole-genomic methylation and with the largest number of caste-specific transcripts and alternative splicing events.By contrast,workers exhibit few uniquely expressed or alternatively spliced genes.Moreover,several genes involved in hormone and neurotransmitter metabolism are related to caste differentiation,whereas several neuropeptides are linked with sex differentiation.Despite little genome-wide association among differential gene expression,splicing,and differential DNA methylation,the overlapped gene ontology(GO)terms point to nutrition-related activity.Therefore,variations in gene regulation correlate with the behavioral differences among castes and highlight the specialization of toolkit genes in bumblebee gynes at the beginning of the adult stage.