Gene expression is more strongly associated with behavioural specialization than with age or fertility in ant workers.

The ecological success of social insects is based on division of labour, not only between queens and workers, but also among workers. Whether a worker tends the brood or forages is influenced by age, fertility and nutritional status, with brood carers being younger, more fecund and more corpulent. Here, we experimentally disentangle behavioural specialization from age and fertility in Temnothorax longispinosus ant workers and analyse how these parameters are linked to whole-body gene expression. A total of 3,644 genes were associated with behavioural specialization which is ten times more than associated with age and 50 times more than associated with fertility. Brood carers were characterized by an upregulation of three Vitellogenin (Vg) genes, one of which, Vg-like A, was the most differentially expressed gene that was recently shown experimentally to control the switch from brood to worker care. The expression of Conventional Vg was unlinked to behavioural specialization, age or fertility, which contrasts to studies on bees and some ants. Diversity in Vg/Vg-like copy number and expression bias across ants supports subfunctionalization of Vg genes and indicates that some regulatory mechanisms of division of labour diverged in different ant lineages. Simulations revealed that our experimental dissociation of co-varying factors reduced transcriptomic noise, suggesting that confounding factors could potentially explain inconsistencies across transcriptomic studies of behavioural specialization in ants. Thus, our study reveals that worker gene expression is mainly linked to the worker's function for the colony and provides novel insights into the evolution of sociality in ants.