HIVE MIND, INDIVIDUAL BRAINS: USING SPATIAL TRANSCRIPTOMICS TO EXPLORE DIFFERENCES IN GENE EXPRESSION AND CELL TYPE COMPOSITION ACROSS BRAIN REGIONS IN QUEEN AND WORKER BUMBLE BEES

Abstract

Eusocial insects offer a unique system for studying how a shared genome can produce different biological forms. In bumble bees (Bombus impatiens), queens and workers differ in physiology and development despite arising from the same genome, making them a useful model for examining how molecular and cellu- lar differences are organized in the brain. Although previous work has identi- fied caste-specific differences in gene expression, much less is known about how these differences are arranged spatially within brain tissue. In this study, we gen- erate a whole-brain, three-dimensional spatial transcriptomic atlas of queen and worker bumble bees by integrating single-nucleus RNA sequencing (snRNA-seq) with spatial transcriptomics. Spatial data were collected across serial brain sections and aligned to recon- struct a 3D representation of each brain. Clustering of gene expression identi- fied distinct groups of cells, which were then mapped to reference cell types from snRNA-seq data, allowing cell types to be assigned while keeping their spatial po- sitions. Comparing queens and workers shows that differences in gene expres- sion and cell type composition are not evenly distributed, but instead vary across brain regions. Differential expression analysis also highlights genes such as Tk (Tachykinin) that show caste-specific patterning. Overall, this work presents the first three-dimensional spatial transcriptomic atlas of a social insect brain and provides a foundation for understanding how molecular and cellular differences are organized within a shared neural structure.