The peptide toxins in the venoms of small invertebrates such as stinging ants have rarely been studied due to the limited amount of venom available per individual. Recently, comprehensive venom studies were conducted on three ants species: Myrmecia gulosa (1), Odontomachus monticola (2) and Tetramorium bicarinatum (3). These venomics studies showed it is now possible to reveone of them al the whole molecular diversity of peptide toxins in small venomous species including ants. Our research group have therefore embarked on the study of several phylogenetic subfamilies of ants in order to improve our understanding about peptide toxins diversification. Thus, we undertook venomic studies of the close related ant subfamilies, Pseudomyrmecinae and Myrmicinae since their venom composition is still poorly investigated despite their important ecological and specific diversity. The present study shows the venom peptidome characterization of the ant species: Tetraponera aethiops (Pseudomyrmecinae) and Manica rubida (Myrmicinae). We used a venomics methodology including (i) peptidomics, in which the venom peptides are sequenced through a de novo mass spectrometry approach and Edman degradation and (ii) transcriptomics, based on the data mining of the sequenced transcriptome of the venom glands. Both M. rubida and T. aethiops venom peptidomes showed about ten peptide precursors. Interestingly, besides presenting high homology with those from M. gulosa and O. monticola, all of them could be classified into the precursors superfamilies described in the peptidome of T. bicarinatum. These toxin precursors encoded for mature peptides, which were mainly linear for M. rubida, and dimeric for T. aethiops. Most of them were predicted to be membrane disruptors according to their sequences and structures. Altogether, those results provide the first comparison of molecular diversity between myrmicine and pseudomyrmecine ants venoms and novel insights into peptide toxin evolution. Future functional investigations of these novel peptides could link them to useful pharmacological properties with an eye toward drug discovery. References: 1. Robinson, S. D. et al. A comprehensive portrait of the venom of the giant red bull ant, Myrmecia gulosa, reveals a hyperdiverse hymenopteran toxin gene family. Sci. Adv. (2018). 2. Tani, N. et al. Mass Spectrometry Analysis and Biological Characterization of the Predatory Ant Odontomachus monticola Venom and Venom Sac Components. Toxins (Basel). 11, 50 (2019). 3. Touchard, A. et al. Deciphering the molecular diversity of an ant venom peptidome through a venomics approach. J. Proteome Res. acs.jproteome.8b00452 (2018). doi:10.1021/acs.jproteome.8b00452