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Batesian mimicry, in which harmless species (mimics) deter predators by deceitfully imitating the warning signals of noxious species (models), generates striking cases of phenotypic convergence that are classic examples of evolution by natural selection. However, mimicry of venomous coral snakes has remained controversial because of unresolved conflict between the predictions of mimicry theory and empirical patterns in the distribution and abundance of snakes. Here we integrate distributional, phenotypic and phylogenetic data across all New World snake species to demonstrate that shifts to mimetic coloration in nonvenomous snakes are highly correlated with coral snakes in both space and time, providing overwhelming support for Batesian mimicry. We also find that bidirectional transitions between mimetic and cryptic coloration are unexpectedly frequent over both long- and short-time scales, challenging traditional views of mimicry as a stable evolutionary ‘end point’ and suggesting that insect and snake mimicry may have different evolutionary dynamics. Toxic and venomous species often have conspicuous warning colouration that is mimicked by harmless species. Here, Davis Rabosky et al . combine phylogenetic and biogeographic analyses to reveal that mimicry of venomous coral snakes has been a major driver of snake colour evolution in the New World.

Gene expression changes contribute to complex trait variations in both individuals and populations. However, the evolution of gene expression underlying complex traits over macroevolutionary timescales remains poorly understood. Snake venoms are proteinaceous cocktails where the expression of each toxin can be quantified and mapped to a distinct genomic locus and traced for millions of years. Using a phylogenetic generalized linear mixed model, we analyzed expression data of toxin genes from 52 snake species spanning the 3 venomous snake families and estimated phylogenetic covariance, which acts as a measure of evolutionary constraint. We find that evolution of toxin combinations is not constrained. However, although all combinations are in principle possible, the actual dimensionality of phylomorphic space is low, with envenomation strategies focused around only four major toxin families: metalloproteases, three-finger toxins, serine proteases, and phospholipases A2. Although most extant snakes prioritize either a single or a combination of major toxin families, they are repeatedly recruited and lost. We find that over macroevolutionary timescales, the venom phenotypes were not shaped by phylogenetic constraints, which include important microevolutionary constraints such as epistasis and pleiotropy, but more likely by ecological filtering that permits a small number of optimal solutions. As a result, phenotypic optima were repeatedly attained by distantly related species. These results indicate that venoms evolve by selection on biochemistry of prey envenomation, which permit diversity through parallelism, and impose strong limits, since only a few of the theoretically possible strategies seem to work well and are observed in extant snakes.

Background Advances in DNA sequencing and proteomics have facilitated quantitative comparisons of snake venom composition. Most studies have employed one approach or the other. Here, both Illumina cDNA sequencing and LC/MS were used to compare the transcriptomes and proteomes of two pit vipers, Protobothrops flavoviridis and Ovophis okinavensis , which differ greatly in their biology. Results Sequencing of venom gland cDNA produced 104,830 transcripts. The Protobothrops transcriptome contained transcripts for 103 venom-related proteins, while the Ovophis transcriptome contained 95. In both, transcript abundances spanned six orders of magnitude. Mass spectrometry identified peptides from 100% of transcripts that occurred at higher than contaminant (e.g. human keratin) levels, including a number of proteins never before sequenced from snakes. These transcriptomes reveal fundamentally different envenomation strategies. Adult Protobothrops venom promotes hemorrhage, hypotension, incoagulable blood, and prey digestion, consistent with mammalian predation. Ovophis venom composition is less readily interpreted, owing to insufficient pharmacological data for venom serine and metalloproteases, which comprise more than 97.3% of Ovophis transcripts, but only 38.0% of Protobothrops transcripts. Ovophis venom apparently represents a hybrid strategy optimized for frogs and small mammals. Conclusions This study illustrates the power of cDNA sequencing combined with MS profiling. The former quantifies transcript composition, allowing detection of novel proteins, but cannot indicate which proteins are actually secreted, as does MS. We show, for the first time, that transcript and peptide abundances are correlated. This means that MS can be used for quantitative, non-invasive venom profiling, which will be beneficial for studies of endangered species.

Novel phenotypes are commonly associated with gene duplications and neofunctionalization, less documented are the cases of phenotypic maintenance through the recruitment of novel genes. Proteolysis is the primary toxic character of many snake venoms, and ADAM metalloproteinases, named snake venom metalloproteinases (SVMPs), are largely recognized as the major effectors of this phenotype. However, by investigating original transcriptomes from 58 species of advanced snakes (Caenophidia) across their phylogeny, we discovered that a different enzyme, matrix metalloproteinase (MMP), is actually the dominant venom component in three tribes (Tachymenini, Xenodontini, and Conophiini) of rear-fanged snakes (Dipsadidae). Proteomic and functional analyses of these venoms further indicate that MMPs are likely playing an “SVMP-like” function in the proteolytic phenotype. A detailed look into the venom-specific sequences revealed a new highly expressed MMP subtype, named snake venom MMP (svMMP), which originated independently on at least three occasions from an endogenous MMP-9. We further show that by losing ancillary noncatalytic domains present in its ancestors, svMMPs followed an evolutionary path toward a simplified structure during their expansion in the genomes, thus paralleling what has been proposed for the evolution of their Viperidae counterparts, the SVMPs. Moreover, we inferred an inverse relationship between the expression of svMMPs and SVMPs along the evolutionary history of Xenodontinae, pointing out that one type of enzyme may be substituting for the other, whereas the general (metallo)proteolytic phenotype is maintained. These results provide rare evidence on how relevant phenotypic traits can be optimized via natural selection on nonhomologous genes, yielding alternate biochemical components.

Venomic research, powered by techniques adapted from proteomics, transcriptomics, and genomics, seeks to unravel the diversity and complexity of venom through which knowledge can be applied in the treatment of envenoming, biodiscovery, and conservation. Snake venom proteomics is most extensively studied, but the methods varied widely, creating a massive amount of information which complicates data comparison and interpretation. Advancement in mass spectrometry technology, accompanied by growing databases and sophisticated bioinformatic tools, has overcome earlier limitations of protein identification. The progress, however, remains challenged by limited accessibility to samples, non-standardized quantitative methods, and biased interpretation of -omic data. Next-generation sequencing (NGS) technologies enable high-throughput venom-gland transcriptomics and genomics, complementing venom proteomics by providing deeper insights into the structural diversity, differential expression, regulation and functional interaction of the toxin genes. Venomic tissue sampling is, however, difficult due to strict regulations on wildlife use and transfer of biological materials in some countries. Limited resources for techniques and funding are among other pertinent issues that impede the progress of venomics, particularly in less developed regions and for neglected species. Genuine collaboration between international researchers, due recognition of regional experts by global organizations (e.g., WHO), and improved distribution of research support, should be embraced.

More than 400,000 people each year suffer adverse effects following bites from venomous snakes. However, snake venom is also a rich source of bioactive molecules with known or potential therapeutic applications. Manually ‘milking’ snakes is the most common method to obtain venom. Safer alternative methods to produce venom would facilitate the production of both antivenom and novel therapeutics. This protocol describes the generation, maintenance and selected applications of snake venom gland organoids. Snake venom gland organoids are 3D culture models that can be derived within days from embryonic or adult venom gland tissues from several snake species and can be maintained long-term (we have cultured some organoids for more than 2 years). We have successfully used the protocol with glands from late-stage embryos and recently deceased adult snakes. The cellular heterogeneity of the venom gland is maintained in the organoids, and cell type composition can be controlled through changes in media composition. We describe in detail how to derive and grow the organoids, how to dissociate them into single cells, and how to cryopreserve and differentiate them into toxin-producing organoids. We also provide guidance on useful downstream assays, specifically quantitative real-time PCR, bulk and single-cell RNA sequencing, immunofluorescence, immunohistochemistry, fluorescence in situ hybridization, scanning and transmission electron microscopy and genetic engineering. This stepwise protocol can be performed in any laboratory with tissue culture equipment and enables studies of venom production, differentiation and cellular heterogeneity. This protocol describes the generation, maintenance and applications of snake venom gland organoids. These organoids can be derived within days from embryonic or adult venom gland tissues from various snake species.

Snake venom evolution is typically considered to be predominantly driven by diet-related selection pressures. Most evidence for this is based on lethality to prey and non-prey species and on the identification of prey specific toxins. Since the broad toxicological activities (e.g., neurotoxicity, coagulotoxicity, etc.) sit at the interface between molecular toxinology and lethality, these classes of activity may act as a key mediator in coevolutionary interactions between snakes and their prey. Indeed, some recent work has suggested that variation in these functional activities may be related to diet as well, but previous studies have been limited in geographic and/or taxonomic scope. In this paper, we take a phylogenetic comparative approach to investigate relationships between diet and toxicological activity classes on a global scale across caenophidian snakes, using the clinically oriented database at toxinology.com. We generally find little support for specific prey types selecting for particular toxicological effects except that reptile-feeders are more likely to be neurotoxic. We find some support for endothermic prey (with higher metabolic rates) influencing toxic activities, but differently from previous suggestions in the literature. More broadly, we find strong support for a general effect of increased diversity of prey on the diversity of toxicological effects of snake venom. Hence, we provide evidence that selection pressures on the toxicological activities of snake venom has largely been driven by prey diversity rather than specific types of prey. These results complement and extend previous work to suggest that specific matching of venom characteristics to prey may occur at the molecular level and translate into venom lethality, but the functional link between those two is not constrained to a particular toxicological route.

Protein expression is a major link in the genotype–phenotype relationship, and processes affecting protein abundances, such as rates of transcription and translation, could contribute to phenotypic evolution if they generate heritable variation. Recent work has suggested that mRNA abundances do not accurately predict final protein abundances, which would imply that post-transcriptional regulatory processes contribute significantly to phenotypes. Post-transcriptional processes also appear to buffer changes in transcriptional patterns as species diverge, suggesting that the transcriptional changes have little or no effect on the phenotypes undergoing study. We tested for concordance between mRNA and protein expression levels in snake venoms by means of mRNA-seq and quantitative mass spectrometry for 11 snakes representing 10 species, six genera, and three families. In contrast to most previous work, we found high correlations between venom gland transcriptomes and venom proteomes for 10 of our 11 comparisons. We tested for protein-level buffering of transcriptional changes during species divergence by comparing the difference between transcript abundance and protein abundance for three pairs of species and one intraspecific pair. We found no evidence for buffering during divergence of our three species pairs but did find evidence for protein-level buffering for our single intraspecific comparison, suggesting that buffering, if present, was a transient phenomenon in venom divergence. Our results demonstrated that post-transcriptional mechanisms did not contribute significantly to phenotypic evolution in venoms and suggest a more prominent and direct role for cis-regulatory evolution in phenotypic variation, particularly for snake venoms.

Three-finger toxins (3FTx) represent one of the most abundantly secreted and potently toxic components of colubrid (Colubridae), elapid (Elapidae) and psammophid (Psammophiinae subfamily of the Lamprophidae) snake venom arsenal. Despite their conserved structural similarity, they perform a diversity of biological functions. Although they are theorised to undergo adaptive evolution, the underlying diversification mechanisms remain elusive. Here, we report the molecular evolution of different 3FTx functional forms and show that positively selected point mutations have driven the rapid evolution and diversification of 3FTx. These diversification events not only correlate with the evolution of advanced venom delivery systems (VDS) in Caenophidia, but in particular the explosive diversification of the clade subsequent to the evolution of a high pressure, hollow-fanged VDS in elapids, highlighting the significant role of these toxins in the evolution of advanced snakes. We show that Type I, II and III α-neurotoxins have evolved with extreme rapidity under the influence of positive selection. We also show that novel Oxyuranus/Pseudonaja Type II forms lacking the apotypic loop-2 stabilising cysteine doublet characteristic of Type II forms are not phylogenetically basal in relation to other Type IIs as previously thought, but are the result of secondary loss of these apotypic cysteines on at least three separate occasions. Not all 3FTxs have evolved rapidly: κ-neurotoxins, which form non-covalently associated heterodimers, have experienced a relatively weaker influence of diversifying selection; while cytotoxic 3FTx, with their functional sites, dispersed over 40% of the molecular surface, have been extremely constrained by negative selection. We show that the a previous theory of 3FTx molecular evolution (termed ASSET) is evolutionarily implausible and cannot account for the considerable variation observed in very short segments of 3FTx. Instead, we propose a theory of Rapid Accumulation of Variations in Exposed Residues (RAVER) to illustrate the significance of point mutations, guided by focal mutagenesis and positive selection in the evolution and diversification of 3FTx.

Background A long term research goal of venomics, of applied importance for improving current antivenom therapy, but also for drug discovery, is to understand the pharmacological potential of venoms. Individually or combined, proteomic and transcriptomic studies have demonstrated their feasibility to explore in depth the molecular diversity of venoms. In the absence of genome sequence, transcriptomes represent also valuable searchable databases for proteomic projects. Results The venom gland transcriptomes of 8 Costa Rican taxa from 5 genera ( Crotalus , Bothrops , Atropoides , Cerrophidion , and Bothriechis ) of pitvipers were investigated using high-throughput 454 pyrosequencing. 100,394 out of 330,010 masked reads produced significant hits in the available databases. 5.165,220 nucleotides (8.27%) were masked by RepeatMasker, the vast majority of which corresponding to class I (retroelements) and class II (DNA transposons) mobile elements. BLAST hits included 79,991 matches to entries of the taxonomic suborder Serpentes , of which 62,433 displayed similarity to documented venom proteins. Strong discrepancies between the transcriptome-computed and the proteome-gathered toxin compositions were obvious at first sight. Although the reasons underlaying this discrepancy are elusive, since no clear trend within or between species is apparent, the data indicate that individual mRNA species may be translationally controlled in a species-dependent manner. The minimum number of genes from each toxin family transcribed into the venom gland transcriptome of each species was calculated from multiple alignments of reads matched to a full-length reference sequence of each toxin family. Reads encoding ORF regions of Kazal-type inhibitor-like proteins were uniquely found in Bothriechis schlegelii and B. lateralis transcriptomes, suggesting a genus-specific recruitment event during the early-Middle Miocene. A transcriptome-based cladogram supports the large divergence between A. mexicanus and A. picadoi , and a closer kinship between A. mexicanus and C. godmani . Conclusions Our comparative next-generation sequencing (NGS) analysis reveals taxon-specific trends governing the formulation of the venom arsenal. Knowledge of the venom proteome provides hints on the translation efficiency of toxin-coding transcripts, contributing thereby to a more accurate interpretation of the transcriptome. The application of NGS to the analysis of snake venom transcriptomes, may represent the tool for opening the door to systems venomics.