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Additional file 4: Figure S1. Activity of Rm1a and Rm4a. Figure S2. Cytotoxicity of R. metallica aculeatoxin peptides. Figure S3. Gene structures of some ant aculeatoxins. Figure S4. Presence of aculeatoxin alleles among workers from the same R. metallica colony. Figure S5. Phylogeny of R. metallica aculeatoxins estimated from full-length or only signal- and propeptide domains. Table S4. Recombination analysis of R. metallica clades 1–3.Table S5. Primers used to amplify aculeatoxins from R. metallica genomic DNA.

Additional file 2: Table S1. Peptides and proteins identified in the pooled venom of R. metallica. Peptide and protein names and corresponding encoding contig id are provided for each, along with the best BLAST hit against UniProtKB, evidence supporting their presence in the venom, mature molecular weight, expression level (TPM), and the sequence of the mature domain and full prepropeptide. Complete (1) evidence includes identification of the complete mature peptide sequence from either the bottom-up or top-down proteomic experiments, while “partial” includes only partial identification from the bottom-up experiment.

Venoms have evolved >100 times in all major animal groups, and their components, known as toxins, have been fine-tuned over millions of years into highly effective biochemical weapons. There are many outstanding questions on the evolution of toxin arsenals, such as how venom genes originate, how venom contributes to the fitness of venomous species, and which modifications at the genomic, transcriptomic, and protein level drive their evolution. These questions have received particularly little attention outside of snakes, cone snails, spiders, and scorpions. Venom compounds have further become a source of inspiration for translational research using their diverse bioactivities for various applications. We highlight here recent advances and new strategies in modern venomics and discuss how recent technological innovations and multi-omic methods dramatically improve research on venomous animals. The study of genomes and their modifications through CRISPR and knockdown technologies will increase our understanding of how toxins evolve and which functions they have in the different ontogenetic stages during the development of venomous animals. Mass spectrometry imaging combined with spatial transcriptomics, in situ hybridization techniques, and modern computer tomography gives us further insights into the spatial distribution of toxins in the venom system and the function of the venom apparatus. All these evolutionary and biological insights contribute to more efficiently identify venom compounds, which can then be synthesized or produced in adapted expression systems to test their bioactivity. Finally, we critically discuss recent agrochemical, pharmaceutical, therapeutic, and diagnostic (so-called translational) aspects of venoms from which humans benefit., This work is funded by the European Cooperation in Science and Technology (COST, www.cost.eu) and based upon work from the COST Action CA19144 – European Venom Network (EUVEN, see https://euven-network.eu/). This review is an outcome of EUVEN Working Group 2 (“Best practices and innovative tools in venomics”) led by B.M.v.R. As coordinator of the group Animal Venomics until end 2021 at the Institute for Insectbiotechnology, JLU Giessen, B.M.v.R. acknowledges the Centre for Translational Biodiv Danish Council for Independent Researchersity Genomics (LOEWE-TBG) in the programme “LOEWE – Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz” of Hesse's Ministry of Higher Education, Research, and the Arts. B.M.v.R. and I.K. further acknowledge funding on venom research by the German Science Foundation to B.M.v.R. (DFG RE3454/6-1). A.C., A.V., and G.Z. were supported by the European Union's Horizon 2020 Research and Innovation program through Marie Sklodowska-Curie Individual Fellowships (grant agreements No. A.C.: 896849, A.V.: 841576, and G.Z.: 845674). M.P.I. is supported by the TALENTO Program by the Regional Madrid Government (2018-T1/BIO-11262). T.H.'s venom research is funded by the DFG projects 271522021 and 413120531. L.E. was supported by grant No. 7017-00288 from the (Technology and Production Sciences). N.I. acknowledges funding on venom research by the Research Fund of Nevsehir Haci Bektas Veli University (project Nos. ABAP20F28, BAP18F26). M.I.K. and A.P. acknowledge support from GSRT National Research Infrastructure structural funding project INSPIRED (MIS 5002550). G.A. acknowledges support from the Slovenian Research Agency grants P1-0391, J4-8225, and J4-2547. G.G. acknowledges support from the Institute for Medical Research and Occupational Health, Zagreb, Croatia. E.A.B.U. is supported by a Norwegian Research Council FRIPRO-YRT Fellowship No. 287462.

Additional file 1: Fig. S1. Normalised across-tissue averaged spectrum across the full acquired range of m/z. Fig. S2. The experimental setup for collecting spat venom. Fig. S3. A summary of the weight of spat and milked venom collected from the spitting cobras used in this study. Fig. S4. Profiles of spat and milked venoms measured via cation exchange chromatography and RP-HPLC. Fig. S5. Coagulation profiles of spitting cobra venoms on citrated bovine plasma. Fig. S6. Cell viability assays. Fig. S7. Enzymatic phospholipase assay absorbance curves. Fig. S8. Snake venom metalloproteinase assay fluorescence curves. Fig. S9. PLA2 is evenly distributed through the length of the venom gland of N. nigricollis. Table S1. Three finger toxins identified by mass spectrometry imaging. Table S2. Spatial correlation and genetic distances of three finger toxins identified by mass spectrometry imaging of N. nigricollis venom gland.

16 páginas, 7 figuras. Snakebite envenoming is a neglected tropical disease that may claim over 100,000 human lives annually worldwide. Snakebite occurs as the result of an interaction between a human and a snake that elicits either a defensive response from the snake or, more rarely, a feeding response as the result of mistaken identity. Snakebite envenoming is therefore a biological and, more specifically, an ecological problem. Snake venom itself is often described as a "cocktail", as it is a heterogenous mixture of molecules including the toxins (which are typically proteinaceous) responsible for the pathophysiological consequences of envenoming. The primary function of venom in snake ecology is pre-subjugation, with defensive deployment of the secretion typically considered a secondary function. The particular composition of any given venom cocktail is shaped by evolutionary forces that include phylogenetic constraints associated with the snake's lineage and adaptive responses to the snake's ecological context, including the taxa it preys upon and by which it is predated upon. In the present article, we describe how conceptual frameworks from ecology and evolutionary biology can enter into a mutually enlightening relationship with clinical toxinology by enabling the consideration of snakebite envenoming from an "ecological stance". We detail the insights that may emerge from such a perspective and highlight the ways in which the high-fidelity descriptive knowledge emerging from applications of -omics era technologies - "venomics" and "antivenomics" - can combine with evolutionary explanations to deliver a detailed understanding of this multifactorial health crisis. Studies by JJC’s research group cited in this review were partially funded by grants from the Ministerio de Ciencia e Innovacion, ´ Madrid, Spain (BMC 2004-01432, BFU 2007-61563, BFU 2010-173730, BFU 2013-42833-P, and BFU 2017-89103-P). JJC wants to acknowledge and heartly thank all the researchers and collaborators of these projects who contributed laboratory work and numerous hours of scientific discussions. EABU was supported by a Norwegian Research Council FRIPROYRT Fellowship no. 287462. TNWJ was supported by National Health and Medical Research Grant 13/093/002 AVRU. KS was supported by DBT / Wellcome Trust India Alliance Fellowship (IA/I/19/2/504647). Support by Vicerrectoría de Investigacion ´ (University of Costa Rica) to work performed at Instituto Clodomiro Picado is also gratefully acknowledged.