The role of dopaminergic neuronal clusters in governing division of labor in ants

Reproductive division of labor is one of the most prominent features of social insects. Yet, the neural mechanisms that govern this division and the associated behavioral differentiation among castes remain obscure. In this study, we systematically characterized the anatomical features of dopamine neurons in the brain of Monomorium pharaonis and compared the cell number and spatial distribution of these neurons across castes. We identified 17 anatomically distinct clusters of dopamine neurons in the ant brain, with cell numbers varying from ∼322 to ∼431 across castes. The major dopamine clusters are located in brain regions analogous to those in flies, with 2 clusters, PAL and PPL1 exhibiting significantly higher cell numbers in ants than in flies. Notably, 4 clusters, DAM, D1, DPL, and PPL2, showed remarkable variation in cell numbers across castes. Using single-cell transcriptomics, we identified specific molecular markers for subdividing dopamine neurons. We validated the expression of multiple neuropeptide genes in specific dopamine clusters. In particular, we found that PPL2 cluster can be further divided into 2 subclusters, PPL2a and PPL2b, which are partially labeled by the peptide gene Nplp1. PPL2b neurons, characterized by larger cell bodies, and present only in unmated queens and males, are absent in mature queens and workers. These neurons are located adjacent to Nlg2-expressing lobular neurons, which are also absent in workers and may play a role in regulating mating behaviors. Our findings provide a foundation for further investigation into the neural mechanism underlying division of labor and caste-specific behaviors in ant.