Ovary single-nucleus transcriptomics reveal follicle cell maturation as a driver of reproductive hierarchy in bumblebees.

The regulation of reproductive division of labor in eusocial insects is pivotal for the evolution of social behavior and the maintenance of eusociality. Primitively eusocial bumblebee workers retain reproductive totipotency, with dominant workers capable of ovarian activation and egg-laying. Here, we investigated the cellular and molecular basis of reproductive hierarchy in Bombus terrestris by constructing a single-nucleus transcriptomic atlas of the ovary in queenless bumblebee groups. Using single-nucleus RNA sequencing, we profiled ovarian cell types and revealed that α-worker bees possess more mature follicle cells, which are essential for ovarian development. Differential maturation of follicle cells, particularly at the vitellogenic stage, emerges as a key regulatory node in this process. More mature follicle cells promote the production of growth factors that activate PDK1. This activation subsequently induces AKT phosphorylation and downstream signaling. As a result, the levels of 20-hydroxyecdysone are elevated in dominant α-workers. By demonstrating how follicle cell maturation and signaling drive reproductive activation, our findings link cellular physiology to social organization and provide new insight into the molecular mechanisms underlying the evolution of eusociality.