Terminal Filament Bundling Underlies the Development of Ovariole Number Dimorphism Between Queen and Worker Ants

Reproductive division of labour is a key feature underlying the remarkable success of ants, where a reproductive queen caste and non-reproductive worker caste develop from a totipotent egg or larva. The number of ovarioles that develop after the egg or larva is determined to be a queen or worker caste ultimately determines their reproductive potential. Yet since very early descriptions of ant larval ovaries made by Ezhikov (1923) and Brian (1950s), the developmental basis of ovariole number dimorphism in ants has received little attention. Here, we revisit their work in light of more recent knowledge from Drosophila melanogaster, where terminal filaments (TFs) in the larval ovary determine ovariole number on a one-to-one basis. Ezhikov and Brian observed that queen and worker larval ovaries develop the same number of TFs, but in the worker caste, they 'bundle' together to form a single ovariole. Using in situ hybridisation chain reaction (HCR) on multiple queen and worker larval stages, we confirm this bundling process is present in two derived ant species with ovariole dimorphism, but is absent in a species that lacks it. Our findings therefore suggest that this terminal filament bundling reflects a mechanism evolved within ants to regulate differences in worker ovariole number between castes independently of overall number of terminal filaments. This work sheds fresh light on historical ant research and sets up future work investigating the origins and regulation of this putative mechanism.