Summary Life inside ant colonies is orchestrated with diverse pheromones, but it is not clear how ants perceive these social signals. It has been proposed that pheromone perception in ants evolved via expansions in the numbers of odorant receptors (ORs) and antennal lobe glomeruli. Here we generate the first mutant lines in the clonal raider ant, Ooceraea biroi, by disrupting orco, a gene required for the function of all ORs. We find that orco mutants exhibit severe deficiencies in social behavior and fitness, suggesting they are unable to perceive pheromones. Surprisingly, unlike in Drosophila melanogaster, orco mutant ants also lack most of the approximately 500 antennal lobe glomeruli found in wild-type ants. These results illustrate that ORs are essential for ant social organization and raise the possibility that, similar to mammals, receptor function is required for the development and/or maintenance of the highly complex olfactory processing areas in the ant brain.

Highlights d Ant odorant receptors show unique early expression and localization in pupae d Sensory neuron innervation is necessary for proper antennal lobe development d Sensory neuron ablation reveals latent antennal lobe structure in ants, but not flies d Insects with complex olfactory systems may employ a different developmental logic

Life inside ant colonies is orchestrated with a diverse set of pheromones, but it is not clear how ants perceive these social cues. It has been proposed that pheromone perception in ants evolved via expansions in the numbers of odorant receptors (ORs) and antennal lobe glomeruli. Here we generate the first mutant lines in ants by disrupting orco, a gene required for the function of all ORs. We find that orco mutants exhibit severe deficiencies in social behavior and fitness, suggesting that they are unable to perceive pheromones. Surprisingly, unlike in Drosophila melanogaster, orco mutant ants also lack most of the approximately 500 antennal lobe glomeruli found in wild-types. These results illustrate that ORs are essential for ant social organization, and raise the possibility that, similar to mammals, receptor function is required for the development and/or maintenance of the highly complex olfactory processing areas in the ant brain.

Oxytocin/vasopressin-related neuropeptides are highly conserved and play major roles in regulating social behavior across vertebrates. However, whether their insect orthologue, inotocin, regulates the behavior of social groups remains unknown. Here, we show that in the clonal raider ant Ooceraea biroi, individuals that perform tasks outside the nest have higher levels of inotocin in their brains than individuals of the same age that remain inside the nest. We also show that older ants, which spend more time outside the nest, have higher inotocin levels than younger ants. Inotocin thus correlates with the propensity to perform tasks outside the nest. Additionally, increasing inotocin pharmacologically increases the tendency of ants to leave the nest. However, this effect is contingent on age and social context. Pharmacologically treated older ants have a higher propensity to leave the nest only in the presence of larvae, whereas younger ants seem to do so only in the presence of pupae. Our results suggest that inotocin signaling plays an important role in modulating behaviors that correlate with age, such as social foraging, possibly by modulating behavioral response thresholds to specific social cues. Inotocin signaling thereby likely contributes to behavioral individuality and division of labor in ant societies.

Ants communicate via large arrays of pheromones and possess expanded, highly complex olfactory systems, with antennal lobes in the brain comprising ~500 glomeruli. This expansion implies that odors could activate hundreds of glomeruli, which would pose challenges for higher order processing. To study this problem, we generated the first transgenic ants, expressing the genetically encoded calcium indicator GCaMP6s in olfactory sensory neurons. Using two-photon imaging, we mapped complete glomerular responses to four ant alarm pheromones. Alarm pheromones robustly activated ≤6 glomeruli, and activity maps for the three pheromones inducing panic-alarm in our study species converged on a single glomerulus. These results demonstrate that, rather than using broadly tuned combinatorial encoding, ants employ precise, narrowly tuned, and stereotyped representation of alarm pheromone cues. The identification of a central sensory hub glomerulus for alarm behavior suggests that a simple neural architecture is sufficient to translate pheromone perception into behavioral outputs.