Trends in the evolution of snake toxins underscored by an integrative omics approach to profile the venom of the colubrid phalotris mertensi

Only few studies on snake venomswere dedicated to deeply characterize the toxin secretion of animals from the Colubridae family, despite the fact that they represent the majority of snake diversity. As a consequence, some evolutionary trends observed in venom proteins that underpinned the evolutionary histories of snake toxins were based on data from a minor parcel of the clade. Here, we investigated the proteins of the totally unknown venom from Phalotrismertensi (Dipsadinae subfamily), in order to obtain a detailed profile of its toxins and to appreciate evolutionary tendencies occurring in colubrid venoms. By means of integrated omics and functional approaches, including RNAseq, Sanger sequencing, high-resolution proteomics, recombinant protein production, and enzymatic tests,we verified an active toxic secretion containing up to 21 types of proteins.Ahigh content of Kunitz-type proteins and C-type lectinswere observed, although several enzymatic components such asmetalloproteinases and an L-amino acid oxidasewere also present in the venom. Interestingly, an arguable venomcomponent of other species was demonstrated as a true venomprotein and named svLIPA (snake venom acid lipase). This finding indicates the importance of checking the actual protein occurrence across species before rejecting genes suggested to code for toxins, which are relevant for the discussion about the early evolution of reptile venoms.Moreover, trends in the evolution of some toxin classes, such as simplification of metalloproteinases and rearrangements of Kunitz andWap domains, parallel similar phenomena observed in other venomous snake families and provide a broader picture of toxin evolution.