Venom peptides are key to the evolutionary success of most venomous animals, including stinging ants. The stingless ant subfamily Formicinae is thought to rely solely on formic acid in their venoms, despite early, unsubstantiated hints of proteinaceous venom constituents. Here, we show that carpenter ants, the largest formicine genus, produce a diverse venom peptide arsenal. Mass spectrometry and transcriptomics across eight geographically distant species uncovered 35 peptides, formicitoxins, belonging to two gene families. Several formicitoxins display antifungal activity. Application to pupae delays fungal outgrowth, suggesting a role of the peptides in colony hygiene. Genome analyses revealed that formicitoxins are widespread but variable across the Formicinae. Our findings overturn the paradigm of formicine venoms as simple acid sprays and establish them as a source of bioactive peptides.
