Extent and complexity of RNA processing in the development of honey bee queen and worker castes revealed by Nanopore direct RNA sequencing

The distinct honey bee (Apis mellifera) worker and queen castes have become a model for the study of genomic mechanisms of phenotypic plasticity. Prior studies have explored differences in gene expression and methylation during development of the two castes, but thus far no study has performed a genome-wide analysis of differences in RNA processing. To address this here we performed a Nanopore-based direct RNA sequencing with exceptionally long reads to compare the mRNA transcripts between honey bee queen and workers at three points during their larval development. We found thousands of significantly differentially expressed isoforms (DEIs) between queen and worker larvae. Most DEIs contained alternative splicing, and many of them contained at least two types of alternative splicing patterns, indicating complex RNA processing in honey bee caste differentiation. We found a negative correlation between poly(A) length and DEI expression, suggesting that poly(A) tails participate in the regulation of isoform expression. Hundreds of isoforms uniquely expressed in either queens or workers during their larval development, and isoforms were expressed at different points in queen and worker larval development demonstrating a dynamic relationship between isoform expression and developmental mechanisms. These findings show the full complexity of RNA processing and transcript expression in honey bee phenotypic plasticity.