A multi-species lipidomic analysis of viperid snake venoms from the Americas

Snake venoms are multilayered and chemically diverse mixtures with important ecological and medical relevance, particularly in many regions of the Americas. Although venom research has largely focused on proteinaceous toxins, venoms also contain low-molecular-weight components that remain poorly characterized. Among these, the lipidome represents one of the least explored layers of venom composition, and comparative information across species is limited. In this study, we applied an untargeted high-resolution mass spectrometry-based lipidomics approach to characterize and compare lipid signatures across nine viperid venoms representing eight American snake species from the genera Crotalus, Lachesis, and Bothrops. This multi-species analysis enabled the identification of both shared and divergent lipid features. Across all venoms, sphingolipids constituted a conserved and dominant lipid class, forming a common lipidomic core, while glycerophospholipids, glycerolipids, and fatty acid-derived lipids exhibited genus- and species-specific quantitative variation. Multivariate analyses revealed moderate but consistent lipidomic structuring associated with taxonomic grouping, with Bothrops and most Crotalus species clustering more closely, whereas Lachesis venoms displayed distinct lipidomic profiles. Supervised discrimination highlighted a restricted set of lipid species driving group separation, primarily through relative abundance differences rather than unique presence or absence. Notably, intra-genus variability was observed within Crotalus and between geographically distinct Lachesis muta populations. Together, these findings demonstrate that viperid venoms possess structured yet variable lipidomic landscapes that complement known proteomic diversity. This study expands the comparative framework of venom lipidomics and supports its integration with other omics approaches to achieve a more comprehensive understanding of venom composition and diversity.