A reference brain for the clonal raider ant

Ants exhibit remarkable collective and social behaviors, such as alloparental care,¹ chemical communication,² homing,³ and cooperative group hygiene.⁴ The clonal raider ant Ooceraea biroi is especially well-suited for investigating the neuronal and genetic underpinnings of these behaviors.⁵ Unlike most ant species, O. biroi lacks a queen caste. Instead, colonies consist entirely of workers that reproduce in synchrony via parthenogenesis, giving rise to age-matched cohorts of clonally identical offspring.^6,7 This unique life history enables precise experimental control over age, genotype, and colony composition. These features have also facilitated the introduction of genetically encoded calcium indicators into O. biroi, enabling in vivo two-photon imaging to investigate the neural basis of social behaviors.⁸ Despite its promise as a neuroscience model, the structure of the clonal raider ant brain has not been systematically characterized, and a representative reference brain does not exist. To address this gap, we imaged the brains of 40 age-matched, genetically identical individuals with confocal microscopy and, using 3D groupwise registration, generated the first reference brain. We introduce a registration pipeline to align brains to this reference, facilitating the comparison of anatomical features across labeling experiments with high spatial precision. Unexpectedly, we discovered extensive interindividual variability across our collection of brain samples. This raises the possibility that the behavioral division of labor in O. biroi is linked to individual differences in brain structure. This work provides a powerful resource for the social insect neuroscience community and reveals novel features of clonal raider ant neurobiology that may influence social behaviors and colony function.