Unmated Queens Show Worker-Like Behaviour and Gene Expression in Polygynous Colonies of the Ant Stigmatomma pallipes

Insect societies show a reproductive division of labor between egg-laying queens and workers that fulfil all non-reproductive tasks. Polygyny, the coexistence of several queens in a colony, has evolved multiple times in social insects. Although queens in polygynous colonies are often assumed to have similar reproductive outputs, they may actually show variation in terms of physiology and behaviour. However, little is known on the mechanistic basis of such variation. Here, we used the ant Stigmatomma pallipes to investigate: (i) whether nestmate queens from polygynous colonies differed in mobility, behaviour, and gene expression; and (ii) whether this variation was explained by their mating status. We used individual tracking and behavioural scoring to identify two types of queens: low-mobility (LM) queens that behaved like monogynous queens and high-mobility (HM) queens with worker-like behaviour. Dissections of reproductive organs revealed that only one queen per colony was mated and reproductively active, consistently corresponding to the LM category. As a complementary approach, we performed transcriptomic-based analyses of brains and fat bodies, finding that mated LM-polygynous queens had similar transcriptomic profiles to monogynous queens, while unmated HM-polygynous queens resembled workers. Transcriptomic differences between LM- and HM-polygynous queens were primarily associated with processes related to protein synthesis, transcription, and neural activity. Our finding that unmated queens in polygynous S. pallipes colonies exhibit worker-like behaviour and gene expression challenges traditional views of queen specialization at different biological levels and provides new insights into reproductive division of labor in insect societies.