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Intraspecific phenotypic variation can be used as a window into the ecological differences among individuals of a species and lead to a better understanding of adaptive evolution. Adaptive traits, such as venom, that play an important ecological role for a species are useful models for understanding the sources of intraspecific variation. Intraspecific studies on front-fanged venomous snakes have offered deeper insights into the diverse mechanisms and adaptations that support the effectiveness of venom across species. Despite the extensive research on front-fanged venomous snakes, rear-fanged snakes, representing two-thirds of all snake species, have been largely overlooked. To test for sex and age-based intraspecific venom variation, we sequenced the messenger RNA from the Duvernoy's gland of 9 male and 10 female Common Garter Snakes, Thamnophis sirtalis, of different sizes from a single location. Our data represent the most venom gland transcriptomes of any venomous snake species from a single location and represent the first Duvernoy's venom gland transcriptomes for Thamnophis sirtalis. We found four toxin families dominate the Thamnophis sirtalis transcriptome: Snake Venom Metalloproteinases (SVMPs), Three-finger toxins (3FTxs), Cysteine-Rich Secretory Proteins (CRISPs), and C-type lectins (CTLs). Thamnophis sirtalis exhibits a unique balance in toxin expression, with approximately 30% each of neurotoxic (3FTx-dominated) and enzymatic (SVMP-dominated) components. No other published RFS Duvernoy's gland transcriptome displays this ratio, rather they are dominated by one or the other. Additionally, venom expression varies with sex and size, with differences in toxin gene expression between males and females as they grow. Our study provides new insights on venom composition in a RFS species and highlights the amount of intraspecific variation possible among individuals from a single population., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Aaron J. Stewart reports financial support was provided by Southwestern Association of Naturalists. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Snake venoms are complex mixtures of toxic proteins that hold significant medical, pharmacological and evolutionary interest. To better understand the genetic diversity underlying snake venoms, we developed VenomCap, a novel exon-capture probe set targeting toxin-coding genes from a wide range of elapid snakes, with a particular focus on the ecologically diverse and medically important subfamily Hydrophiinae. We tested the capture success of VenomCap across 24 species, representing all major elapid lineages. We included snake phylogenomic probes in the VenomCap capture set, allowing us to compare capture performance between venom and phylogenomic loci and to infer elapid phylogenetic relationships. We demonstrated VenomCap's ability to recover exons from ~1500 target markers, representing a total of 24 known venom gene families, which includes the dominant gene families found in elapid venoms. We find that VenomCap's capture results are robust across all elapids sampled, and especially among hydrophiines, with respect to measures of target capture success (target loci matched, sensitivity, specificity and missing data). As a cost-effective and efficient alternative to full genome sequencing, VenomCap can dramatically accelerate the sequencing and analysis of venom gene families. Overall, our tool offers a model for genomic studies on snake venom gene diversity and evolution that can be expanded for comprehensive comparisons across the other families of venomous snakes., (© 2024 The Author(s). Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Although non-front fanged snakes account for almost two-thirds of snake diversity, most studies on venom composition and evolution focus exclusively on front-fanged species, which comprise most of the clinically relevant accidents. Comprehensive reports on venom composition of non-front fanged snakes are still scarce for several groups. In this study, we address such shortage of knowledge by providing new insights about the venom composition among species of Phalotris, a poorly studied Neotropical dipsadid genus. Phalotris are known for their specialized venom delivery system and toxic venoms, which can cause life-threatening accidents in humans. We evaluate the venom-gland transcriptome of Phalotris, comparing the following three South American species: P. reticulatus for the Araucaria Pine forests, P. lemniscatus for the Pampa grasslands, and P. mertensi for the Brazilian Cerrado. Our results indicate similar venom profiles, in which they share a high expression level of Kunitz-type inhibitors (KUNZ). On the other hand, comparative analyses revealed substantial differences in the expression levels of C-type lectins (CTL) and snake venom metalloproteinases (SVMP). The diverse set of SVMP and CTL isoforms shows signals of positive selection, and we also identified truncated forms of type III SVMPs, which resemble type II and type I SVMPs of viperids. Additionally, we identified a CNP precursor hosting a proline-rich region containing a BPP motif resembling those commonly detected in viperid venoms with hypotensive activity. Altogether, our results suggest an evolutionary history favoring high expression levels of few KUNZ isoforms in Phalotris venoms, contrasting with a highly diverse set of SVMP and CTL isoforms. Such diversity can be comparable with the venom variability observed in some viperids. Our findings highlight the extreme phenotypic diversity of non-front fanged snakes and the importance to allocate greater effort to study neglected groups of Colubroidea., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Pedro Nachtigall reports financial support was provided by State of Sao Paulo Research Foundation. Omar M. Entiauspe-Neto reports financial support was provided by State of Sao Paulo Research Foundation. Inacio Loiola Meirelles Junqueira de Azevedo reports financial support was provided by National Council for Scientific and Technological Development. Felipe Gobbi Grazziotin reports financial support was provided by National Council for Scientific and Technological Development. Felipe Gobbi Grazziotin reports financial support was provided by State of Sao Paulo Research Foundation. Inacio Loiola Meirelles Junqueira de Azevedo reports financial support was provided by State of Sao Paulo Research Foundation. Omar M. Entiauspe-Neto reports financial support was provided by Coordination of Higher Education Personnel Improvement. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The evolutionary interplay between predator and prey has significantly shaped the development of snake venom, a critical adaptation for subduing prey. This arms race has spurred the diversification of the components of venom and the corresponding emergence of resistance mechanisms in the prey and predators of venomous snakes. Our study investigates the molecular basis of venom resistance in pythons, focusing on electrostatic charge repulsion as a defense against α-neurotoxins binding to the alpha-1 subunit of the postsynaptic nicotinic acetylcholine receptor. Through phylogenetic and bioactivity analyses of orthosteric site sequences from various python species, we explore the prevalence and evolution of amino acid substitutions that confer resistance by electrostatic repulsion, which initially evolved in response to predatory pressure by Naja (cobra) species (which occurs across Africa and Asia). The small African species Python regius retains the two resistance-conferring lysines (positions 189 and 191) of the ancestral Python genus, conferring resistance to sympatric Naja venoms. This differed from the giant African species Python sebae , which has secondarily lost one of these lysines, potentially due to its rapid growth out of the prey size range of sympatric Naja species. In contrast, the two Asian species Python brongersmai (small) and Python bivittatus (giant) share an identical orthosteric site, which exhibits the highest degree of resistance, attributed to three lysine residues in the orthosteric sites. One of these lysines (at orthosteric position 195) evolved in the last common ancestor of these two species, which may reflect an adaptive response to increased predation pressures from the sympatric α-neurotoxic snake-eating genus Ophiophagus (King Cobras) in Asia. All these terrestrial Python species, however, were less neurotoxin-susceptible than pythons in other genera which have evolved under different predatory pressure as: the Asian species Malayopython reticulatus which is arboreal as neonates and juveniles before rapidly reaching sizes as terrestrial adults too large for sympatric Ophiophagus species to consider as prey; and the terrestrial Australian species Aspidites melanocephalus which occupies a niche, devoid of selection pressure from α-neurotoxic predatory snakes. Our findings underline the importance of positive selection in the evolution of venom resistance and suggest a complex evolutionary history involving both conserved traits and secondary evolution. This study enhances our understanding of the molecular adaptations that enable pythons to survive in environments laden with venomous threats and offers insights into the ongoing co-evolution between venomous snakes and their prey.

Background: Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris)., Results: Although there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis., Conclusions: Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype., (© 2024. The Author(s).)

Snake venoms harbor a wide and diverse array of enzymatic and nonenzymatic toxic components, allowing them to exert myriad effects on their prey. However, they appear to trend toward a few optimal compositional scaffolds, dominated by four major toxin classes: SVMPs, SVSPs, 3FTxs, and PLA2s. Nevertheless, the latter appears to be restricted to vipers and elapids, as it has never been reported as a major venom component in rear-fanged species. Here, by investigating the original transcriptomes from 19 species distributed in eight genera from the Pseudoboini tribe (Dipsadidae: Xenodontinae) and screening among seven additional tribes of Dipsadidae and three additional families of advanced snakes, we discovered that a novel type of venom PLA2, resembling a PLA2-IIE, has been recruited to the venom of some species of the Pseudoboini tribe, where it is a major component. Proteomic and functional analyses of these venoms further indicate that these PLA2s play a relevant role in the venoms from this tribe. Moreover, we reconstructed the phylogeny of PLA2s across different snake groups and show that different types of these toxins have been recruited in at least five independent events in caenophidian snakes. Additionally, we present the first compositional profiling of Pseudoboini venoms. Our results demonstrate how relevant phenotypic traits are convergently recruited by different means and from homologous and nonhomologous genes in phylogenetically and ecologically divergent snake groups, possibly optimizing venom composition to overcome diverse adaptative landscapes., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Background: Snake venoms are trophic adaptations that represent an ideal model to examine the evolutionary factors that shape polymorphic traits under strong natural selection. Venom compositional variation is substantial within and among venomous snake species. However, the forces shaping this phenotypic complexity, as well as the potential integrated roles of biotic and abiotic factors, have received little attention. Here, we investigate geographic variation in venom composition in a wide-ranging rattlesnake (Crotalus viridis viridis) and contextualize this variation by investigating dietary, phylogenetic, and environmental variables that covary with venom., Results: Using shotgun proteomics, venom biochemical profiling, and lethality assays, we identify 2 distinct divergent phenotypes that characterize major axes of venom variation in this species: a myotoxin-rich phenotype and a snake venom metalloprotease (SVMP)-rich phenotype. We find that dietary availability and temperature-related abiotic factors are correlated with geographic trends in venom composition., Conclusions: Our findings highlight the potential for snake venoms to vary extensively within species, for this variation to be driven by biotic and abiotic factors, and for the importance of integrating biotic and abiotic variation for understanding complex trait evolution. Links between venom variation and variation in biotic and abiotic factors indicate that venom variation likely results from substantial geographic variation in selection regimes that determine the efficacy of venom phenotypes across populations and snake species. Our results highlight the cascading influence of abiotic factors on biotic factors that ultimately shape venom phenotype, providing evidence for a central role of local selection as a key driver of venom variation., (© 2023. The Author(s).)

The ubiquitous cellular heterogeneity underlying many organism-level phenotypes raises questions about what factors drive this heterogeneity and how these complex heterogeneous systems evolve. Here, we use single-cell expression data from a Prairie rattlesnake (Crotalus viridis) venom gland to evaluate hypotheses for signaling networks underlying snake venom regulation and the degree to which different venom gene families have evolutionarily recruited distinct regulatory architectures. Our findings suggest that snake venom regulatory systems have evolutionarily co-opted trans-regulatory factors from extracellular signal-regulated kinase and unfolded protein response pathways that specifically coordinate expression of distinct venom toxins in a phased sequence across a single population of secretory cells. This pattern of co-option results in extensive cell-to-cell variation in venom gene expression, even between tandemly duplicated paralogs, suggesting this regulatory architecture has evolved to circumvent cellular constraints. While the exact nature of such constraints remains an open question, we propose that such regulatory heterogeneity may circumvent steric constraints on chromatin, cellular physiological constraints (e.g., endoplasmic reticulum stress or negative protein-protein interactions), or a combination of these. Regardless of the precise nature of these constraints, this example suggests that, in some cases, dynamic cellular constraints may impose previously unappreciated secondary constraints on the evolution of gene regulatory networks that favors heterogeneous expression., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

For decades, studies of snake venoms focused on the venom-ome-specific toxins (VSTs). VSTs are dominant soluble proteins believed to contribute to the main venomous effects and emerged into gene clusters for fast adaptation and diversification of snake venoms. However, the conserved minor venom components, such as snake venom phosphodiesterase (svPDE), remain largely unexplored. Here, we focus on svPDE by genomic and transcriptomic analysis across snake clades and demonstrate that soluble svPDE is co-opted from the ancestral membrane-attached ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase 3) gene by replacing the original 5' exon with the exon encoding a signal peptide. Notably, the exons, promoters, and transcription/translation starts have been replaced multiple times during snake evolution, suggesting the evolutionary necessity of svPDE. The structural and biochemical analyses also show that svPDE shares the similar functions with ENPP family, suggesting its perturbation to the purinergic signaling and insulin transduction in venomous effects., Competing Interests: CP, CL, IL, KC, YL, HC, WW No competing interests declared, (© 2023, Pan et al.)

Crotamine, myotoxin a and homologs are short peptides that often comprise major fractions of rattlesnake venoms and have been extensively studied for their bioactive properties. These toxins are thought to be important for rapidly immobilizing mammalian prey and are implicated in serious, and sometimes fatal, responses to envenomation in humans. While high quality reference genomes for multiple venomous snakes are available, the loci that encode myotoxins have not been successfully assembled in any existing genome assembly. Here, we integrate new and existing genomic and transcriptomic data from the Prairie Rattlesnake (Crotalus viridis viridis) to reconstruct, characterize, and infer the chromosomal locations of myotoxin-encoding loci. We integrate long-read transcriptomics (Pacific Bioscience's Iso-Seq) and short-read RNA-seq to infer gene sequence diversity and characterize patterns of myotoxin and paralogous β-defensin expression across multiple tissues. We also identify two long non-coding RNA sequences which both encode functional myotoxins, demonstrating a newly discovered source of venom coding sequence diversity. We also integrate long-range mate-pair chromatin contact data and linked-read sequencing to infer the structure and chromosomal locations of the three myotoxin-like loci. Further, we conclude that the venom-associated myotoxin is located on chromosome 1 and is adjacent to non-venom paralogs. Consistent with this locus contributing to venom composition, we find evidence that the promoter of this gene is selectively open in venom gland tissue and contains transcription factor binding sites implicated in broad trans-regulatory pathways that regulate snake venoms. This study provides the best genomic reconstruction of myotoxin loci to date and raises questions about the physiological roles and interplay between myotoxin and related genes, as well as the genomic origins of snake venom variation., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The origin of snake venom involved duplication and recruitment of non-venom genes into venom systems. Several studies have predicted that directional positive selection has governed this process. Venom composition varies substantially across snake species and venom phenotypes are locally adapted to prey, leading to coevolutionary interactions between predator and prey. Venom origins and contemporary snake venom evolution may therefore be driven by fundamentally different selection regimes, yet investigations of population-level patterns of selection have been limited. Here, we use whole-genome data from 68 rattlesnakes to test hypotheses about the factors that drive genomic diversity and differentiation in major venom gene regions. We show that selection has resulted in long-term maintenance of genetic diversity within and between species in multiple venom gene families. Our findings are inconsistent with a dominant role of directional positive selection and instead support a role of long-term balancing selection in shaping venom evolution. We also detect rapid decay of linkage disequilibrium due to high recombination rates in venom regions, suggesting that venom genes have reduced selective interference with nearby loci, including other venom paralogues. Our results provide an example of long-term balancing selection that drives trans-species polymorphism and help to explain how snake venom keeps pace with prey resistance., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Many venomous animals express toxins that show extraordinary levels of variation both within and among species. In snakes, most studies of venom variation focus on front-fanged species in the families Viperidae and Elapidae, even though rear-fanged snakes in other families vary along the same ecological axes important to venom evolution. Here we characterized venom gland transcriptomes from 19 snakes across two dipsadine rear-fanged genera ( Leptodeira and Helicops , Colubridae) and two front-fanged genera ( Bothrops , Viperidae; Micrurus , Elapidae). We compared patterns of composition, variation, and diversity in venom transcripts within and among all four genera. Venom gland transcriptomes of rear-fanged Helicops and Leptodeira and front-fanged Micrurus are each dominated by expression of single toxin families (C-type lectins, snake venom metalloproteinase, and phospholipase A2, respectively), unlike highly diverse front-fanged Bothrops venoms. In addition, expression patterns of congeners are much more similar to each other than they are to species from other genera. These results illustrate the repeatability of simple venom profiles in rear-fanged snakes and the potential for relatively constrained venom composition within genera.

Venom is a dynamic trait that has contributed to the success of numerous organismal lineages. Predominantly composed of proteins, these complex cocktails are deployed for predation and/or self-defence. Many non-toxic physiological proteins have been convergently and recurrently recruited by venomous animals into their toxin arsenal. Phospholipase A 2 (PLA 2 ) is one such protein and features in the venoms of many organisms across the animal kingdom, including snakes of the families Elapidae and Viperidae. Understanding the evolutionary history of this superfamily would therefore provide insight into the origin and diversification of venom toxins and the evolution of novelty more broadly. The literature is replete with studies that have identified diversifying selection as the sole influence on PLA 2 evolution. However, these studies have largely neglected the structural/functional constraints on PLA 2 s, and the ecology and evolutionary histories of the diverse snake lineages that produce them. By considering these crucial factors and employing evolutionary analyses integrated with a schema for the classification of PLA 2 s, we uncovered lineage-specific differences in selection regimes. Thus, our work provides novel insights into the evolution of this major snake venom toxin superfamily and underscores the importance of considering the influence of evolutionary and ecological contexts on molecular evolution.

Understanding how regulatory mechanisms evolve is critical for understanding the processes that give rise to novel phenotypes. Snake venom systems represent a valuable and tractable model for testing hypotheses related to the evolution of novel regulatory networks, yet the regulatory mechanisms underlying venom production remain poorly understood. Here, we use functional genomics approaches to investigate venom regulatory architecture in the prairie rattlesnake and identify cis -regulatory sequences (enhancers and promoters), trans -regulatory transcription factors, and integrated signaling cascades involved in the regulation of snake venom genes. We find evidence that two conserved vertebrate pathways, the extracellular signal-regulated kinase and unfolded protein response pathways, were co-opted to regulate snake venom. In one large venom gene family (snake venom serine proteases), this co-option was likely facilitated by the activity of transposable elements. Patterns of snake venom gene enhancer conservation, in some cases spanning 50 million yr of lineage divergence, highlight early origins and subsequent lineage-specific adaptations that have accompanied the evolution of venom regulatory architecture. We also identify features of chromatin structure involved in venom regulation, including topologically associated domains and CTCF loops that underscore the potential importance of novel chromatin structure to coevolve when duplicated genes evolve new regulatory control. Our findings provide a model for understanding how novel regulatory systems may evolve through a combination of genomic processes, including tandem duplication of genes and regulatory sequences, cis -regulatory sequence seeding by transposable elements, and diverse transcriptional regulatory proteins controlled by a co-opted regulatory cascade., (© 2022 Perry et al.; Published by Cold Spring Harbor Laboratory Press.)

Understanding the joint roles of protein sequence variation and differential expression during adaptive evolution is a fundamental, yet largely unrealized goal of evolutionary biology. Here, we use phylogenetic path analysis to analyze a comprehensive venom-gland transcriptome dataset spanning three genera of pitvipers to identify the functional genetic basis of a key adaptation (venom complexity) linked to diet breadth (DB). The analysis of gene-family-specific patterns reveals that, for genes encoding two of the most important venom proteins (snake venom metalloproteases and snake venom serine proteases), there are direct, positive relationships between sequence diversity (SD), expression diversity (ED), and increased DB. Further analysis of gene-family diversification for these proteins showed no constraint on how individual lineages achieved toxin gene SD in terms of the patterns of paralog diversification. In contrast, another major venom protein family (PLA2s) showed no relationship between venom molecular diversity and DB. Additional analyses suggest that other molecular mechanisms-such as higher absolute levels of expression-are responsible for diet adaptation involving these venom proteins. Broadly, our findings argue that functional diversity generated through sequence and expression variations jointly determine adaptation in the key components of pitviper venoms, which mediate complex molecular interactions between the snakes and their prey., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Snake venoms represent a danger to human health, but also a gold mine of bioactive proteins that can be harnessed for drug discovery purposes. The evolution of snakes and their venom has been studied for decades, particularly via traditional morphological and basic genetic methods alongside venom proteomics. However, while the field of genomics has matured rapidly over the past 2 decades, owing to the development of next-generation sequencing technologies, snake genomics remains in its infancy. Here, we provide an overview of the state of the art in snake genomics and discuss its potential implications for studying venom evolution and toxinology. On the basis of current knowledge, gene duplication and positive selection are key mechanisms in the neofunctionalization of snake venom proteins. This makes snake venoms important evolutionary drivers that explain the remarkable venom diversification and adaptive variation observed in these reptiles. Gene duplication and neofunctionalization have also generated a large number of repeat sequences in snake genomes that pose a significant challenge to DNA sequencing, resulting in the need for substantial computational resources and longer sequencing read length for high-quality genome assembly. Fortunately, owing to constantly improving sequencing technologies and computational tools, we are now able to explore the molecular mechanisms of snake venom evolution in unprecedented detail. Such novel insights have the potential to affect the design and development of antivenoms and possibly other drugs, as well as provide new fundamental knowledge on snake biology and evolution., (© The Author(s) 2022. Published by Oxford University Press GigaScience.)

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.

Background: The explosive radiation and diversification of the advanced snakes (superfamily Colubroidea) was associated with changes in all aspects of the shared venom system. Morphological changes included the partitioning of the mixed ancestral glands into two discrete glands devoted for production of venom or mucous respectively, as well as changes in the location, size and structural elements of the venom-delivering teeth. Evidence also exists for homology among venom gland toxins expressed across the advanced snakes. However, despite the evolutionary novelty of snake venoms, in-depth toxin molecular evolutionary history reconstructions have been mostly limited to those types present in only two front-fanged snake families, Elapidae and Viperidae. To have a broader understanding of toxins shared among extant snakes, here we first sequenced the transcriptomes of eight taxonomically diverse rear-fanged species and four key viperid species and analysed major toxin types shared across the advanced snakes., Results: Transcriptomes were constructed for the following families and species: Colubridae - Helicops leopardinus, Heterodon nasicus, Rhabdophis subminiatus; Homalopsidae - Homalopsis buccata; Lamprophiidae - Malpolon monspessulanus, Psammophis schokari, Psammophis subtaeniatus, Rhamphiophis oxyrhynchus; and Viperidae - Bitis atropos, Pseudocerastes urarachnoides, Tropidolaeumus subannulatus, Vipera transcaucasiana. These sequences were combined with those from available databases of other species in order to facilitate a robust reconstruction of the molecular evolutionary history of the key toxin classes present in the venom of the last common ancestor of the advanced snakes, and thus present across the full diversity of colubroid snake venoms. In addition to differential rates of evolution in toxin classes between the snake lineages, these analyses revealed multiple instances of previously unknown instances of structural and functional convergences. Structural convergences included: the evolution of new cysteines to form heteromeric complexes, such as within kunitz peptides (the beta-bungarotoxin trait evolving on at least two occasions) and within SVMP enzymes (the P-IIId trait evolving on at least three occasions); and the C-terminal tail evolving on two separate occasions within the C-type natriuretic peptides, to create structural and functional analogues of the ANP/BNP tailed condition. Also shown was that the de novo evolution of new post-translationally liberated toxin families within the natriuretic peptide gene propeptide region occurred on at least five occasions, with novel functions ranging from induction of hypotension to post-synaptic neurotoxicity. Functional convergences included the following: multiple occasions of SVMP neofunctionalised in procoagulant venoms into activators of the clotting factors prothrombin and Factor X; multiple instances in procoagulant venoms where kunitz peptides were neofunctionalised into inhibitors of the clot destroying enzyme plasmin, thereby prolonging the half-life of the clots formed by the clotting activating enzymatic toxins; and multiple occasions of kunitz peptides neofunctionalised into neurotoxins acting on presynaptic targets, including twice just within Bungarus venoms., Conclusions: We found novel convergences in both structural and functional evolution of snake toxins. These results provide a detailed roadmap for future work to elucidate predator-prey evolutionary arms races, ascertain differential clinical pathologies, as well as documenting rich biodiscovery resources for lead compounds in the drug design and discovery pipeline., (© 2021. The Author(s).)

Motivation: Next-generation sequencing has become exceedingly common and has transformed our ability to explore nonmodel systems. In particular, transcriptomics has facilitated the study of venom and evolution of toxins in venomous lineages; however, many challenges remain. Primarily, annotation of toxins in the transcriptome is a laborious and time-consuming task. Current annotation software often fails to predict the correct coding sequence and overestimates the number of toxins present in the transcriptome. Here, we present ToxCodAn, a python script designed to perform precise annotation of snake venom gland transcriptomes. We test ToxCodAn with a set of previously curated transcriptomes and compare the results to other annotators. In addition, we provide a guide for venom gland transcriptomics to facilitate future research and use Bothrops alternatus as a case study for ToxCodAn and our guide., Results: Our analysis reveals that ToxCodAn provides precise annotation of toxins present in the transcriptome of venom glands of snakes. Comparison with other annotators demonstrates that ToxCodAn has better performance with regard to run time ($>20x$ faster), coding sequence prediction ($>3x$ more accurate) and the number of toxins predicted (generating $>4x$ less false positives). In this sense, ToxCodAn is a valuable resource for toxin annotation. The ToxCodAn framework can be expanded in the future to work with other venomous lineages and detect novel toxins., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The venom of the Deinagkistrodon acutus snake is composed of numerous bioactive proteins and peptides. In this study, we report the antithrombotic and anticoagulant activities of one of such proteins, herein known as SLPC. This novel protein was isolated and purified via multi-gel chromatography. Its amino acid sequence, structure and function were then determined. This protein was found to exhibit defibration, anticoagulation and general antithrombotic effects based on the results of both in vitro and in vivo studies. Based on same studies, it was found to cleave the α, β, γ chains of fibrinogen and generally improved antiplatelet aggregation and blood rheology. A metabolomic insight of the antithrombotic effects of SLPC was found to be mainly linked to perturbations in the synthesis of unsaturated fatty acids, glycerophospholipid metabolism, arachidonic acid metabolism and other metabolic pathways. In summary, the novel protein SLPC, elicits its antithrombotic effects via degradation of fibrinogen and regulation of various thrombogenic factors in multiple metabolic pathways., (Copyright © 2021. Published by Elsevier Masson SAS.)

Venom is a key adaptive innovation in snakes, and how nonvenom genes were co-opted to become part of the toxin arsenal is a significant evolutionary question. While this process has been investigated through the phylogenetic reconstruction of toxin sequences, evidence provided by the genomic context of toxin genes remains less explored. To investigate the process of toxin recruitment, we sequenced the genome of Bothrops jararaca , a clinically relevant pitviper. In addition to producing a road map with canonical structures of genes encoding 12 toxin families, we inferred most of the ancestral genes for their loci. We found evidence that 1) snake venom metalloproteinases (SVMPs) and phospholipases A 2 (PLA2) have expanded in genomic proximity to their nonvenomous ancestors; 2) serine proteinases arose by co-opting a local gene that also gave rise to lizard gilatoxins and then expanded; 3) the bradykinin-potentiating peptides originated from a C-type natriuretic peptide gene backbone; and 4) VEGF-F was co-opted from a PGF-like gene and not from VEGF-A. We evaluated two scenarios for the original recruitment of nontoxin genes for snake venom: 1) in locus ancestral gene duplication and 2) in locus ancestral gene direct co-option. The first explains the origins of two important toxins (SVMP and PLA2), while the second explains the emergence of a greater number of venom components. Overall, our results support the idea of a locally assembled venom arsenal in which the most clinically relevant toxin families expanded through posterior gene duplications, regardless of whether they originated by duplication or gene co-option., Competing Interests: The authors declare no competing interest.

The role of natural selection in the evolution of trait complexity can be characterized by testing hypothesized links between complex forms and their functions across species. Predatory venoms are composed of multiple proteins that collectively function to incapacitate prey. Venom complexity fluctuates over evolutionary timescales, with apparent increases and decreases in complexity, and yet the causes of this variation are unclear. We tested alternative hypotheses linking venom complexity and ecological sources of selection from diet in the largest clade of front-fanged venomous snakes in North America: the rattlesnakes, copperheads, cantils, and cottonmouths. We generated independent transcriptomic and proteomic measures of venom complexity and collated several natural history studies to quantify dietary variation. We then constructed genome-scale phylogenies for these snakes for comparative analyses. Strikingly, prey phylogenetic diversity was more strongly correlated to venom complexity than was overall prey species diversity, specifically implicating prey species' divergence, rather than the number of lineages alone, in the evolution of complexity. Prey phylogenetic diversity further predicted transcriptomic complexity of three of the four largest gene families in viper venom, showing that complexity evolution is a concerted response among many independent gene families. We suggest that the phylogenetic diversity of prey measures functionally relevant divergence in the targets of venom, a claim supported by sequence diversity in the coagulation cascade targets of venom. Our results support the general concept that the diversity of species in an ecological community is more important than their overall number in determining evolutionary patterns in predator trait complexity.

Oral venom systems evolved multiple times in numerous vertebrates enabling the exploitation of unique predatory niches. Yet how and when they evolved remains poorly understood. Up to now, most research on venom evolution has focused strictly on the toxins. However, using toxins present in modern day animals to trace the origin of the venom system is difficult, since they tend to evolve rapidly, show complex patterns of expression, and were incorporated into the venom arsenal relatively recently. Here we focus on gene regulatory networks associated with the production of toxins in snakes, rather than the toxins themselves. We found that overall venom gland gene expression was surprisingly well conserved when compared to salivary glands of other amniotes. We characterized the "metavenom network," a network of ∼3,000 nonsecreted housekeeping genes that are strongly coexpressed with the toxins, and are primarily involved in protein folding and modification. Conserved across amniotes, this network was coopted for venom evolution by exaptation of existing members and the recruitment of new toxin genes. For instance, starting from this common molecular foundation, Heloderma lizards, shrews, and solenodon, evolved venoms in parallel by overexpression of kallikreins, which were common in ancestral saliva and induce vasodilation when injected, causing circulatory shock. Derived venoms, such as those of snakes, incorporated novel toxins, though still rely on hypotension for prey immobilization. These similarities suggest repeated cooption of shared molecular machinery for the evolution of oral venom in mammals and reptiles, blurring the line between truly venomous animals and their ancestors., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

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.

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Geographical variation of animal venom is common among venomous animals. This kind of intraspecific variation based on geographical location mainly concerned from envenomation cases and brought new problems in animal venom studies, including venom components regulatory mechanisms, differentiation of venom activities, and clinical treatment methods. At present, food is considered as the most related factor influencing venom development. Related research defined the variational venomous animal species by the comparison of venom components and activities in snakes, jellyfish, scorpions, cone snails, ants, parasitoid wasps, spiders and toads. In snake venom studies, researchers found that antivenom effectiveness was variated to different located venom samples. As described in some snake venom research, developing region-specific antivenom is the development trend. The difficulties of developing region-specific antivenom and theoretical solutions have been discussed. This review summarized biological studies of animal venom geographical variation by species, compared venom components and major biological activities of the vary venom from the same species, and listed the basic methods in comparing venom protein compositions and major toxicity differences to provide a comprehensive reference., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

While SARS-CoV-2 uses angiotensin converting enzyme 2 (ACE2) as the receptor for cell entry, it is important to examine other potential interactions between the virus and other cell receptors. Based on the clinical observation of low prevalence of smoking among hospitalized COVID-19 patients, we examined and identified a "toxin-like" amino acid (aa) sequence in the Receptor Binding Domain of the Spike Glycoprotein of SARS-CoV-2 (aa 375-390), which is homologous to a sequence of the Neurotoxin homolog NL1, one of the many snake venom toxins that are known to interact with nicotinic acetylcholine receptors (nAChRs). We present the 3D structural location of this "toxin-like" sequence on the Spike Glycoprotein and the superposition of the modelled structure of the Neurotoxin homolog NL1 and the SARS-CoV-2 Spike Glycoprotein. We also performed computational molecular modelling and docking experiments using 3D structures of the SARS-CoV-2 Spike Glycoprotein and the extracellular domain of the nAChR α9 subunit. We identified a main interaction between the aa 381-386 of the SARS-CoV-2 Spike Glycoprotein and the aa 189-192 of the extracellular domain of the nAChR α9 subunit, a region which forms the core of the "toxin-binding site" of the nAChRs. The mode of interaction is very similar to the interaction between the α9 nAChR and α-bungarotoxin. A similar interaction was observed between the pentameric α7 AChR chimera and SARS-CoV-2 Spike Glycoprotein. The findings raise the possibility that SARS-CoV-2 may interact with nAChRs, supporting the hypothesis of dysregulation of the nicotinic cholinergic system being implicated in the pathophysiology of COVID-19. Nicotine and other nicotinic cholinergic agonists may protect nAChRs and thus have therapeutic value in COVID-19 patients.

The genetic origins of novelty are a central interest of evolutionary biology. Most new proteins evolve from preexisting proteins but the evolutionary path from ancestral gene to novel protein is challenging to trace, and therefore the requirements for and order of coding sequence changes, expression changes, or gene duplication are not clear. Snake venoms are important novel traits that are comprised of toxins derived from several distinct protein families, but the genomic and evolutionary origins of most venom components are not understood. Here, we have traced the origin and diversification of one prominent family, the snake venom metalloproteinases (SVMPs) that play key roles in subduing prey in many vipers. Genomic analyses of several rattlesnake ( Crotalus ) species revealed the SVMP family massively expanded from a single, deeply conserved adam28 disintegrin and metalloproteinase gene, to as many as 31 tandem genes in the Western Diamondback rattlesnake ( Crotalus atrox ) through a number of single gene and multigene duplication events. Furthermore, we identified a series of stepwise intragenic deletions that occurred at different times in the course of gene family expansion and gave rise to the three major classes of secreted SVMP toxins by sequential removal of a membrane-tethering domain, the cysteine-rich domain, and a disintegrin domain, respectively. Finally, we show that gene deletion has further shaped the SVMP complex within rattlesnakes, creating both fusion genes and substantially reduced gene complexes. These results indicate that gene duplication and intragenic deletion played essential roles in the origin and diversification of these novel biochemical weapons., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The CAP protein superfamily (Cysteine-rich secretory proteins (CRISPs), Antigen 5 (Ag5), and Pathogenesis-related 1 (PR-1) proteins) is widely distributed, but for toxinologists, snake venom CRISPs are the most familiar members. Although CRISPs are found in the majority of venoms, very few of these proteins have been functionally characterized, but those that have been exhibit diverse activities. Snake venom CRISPs (svCRISPs) inhibit ion channels and the growth of new blood vessels (angiogenesis). They also increase vascular permeability and promote inflammatory responses (leukocyte and neutrophil infiltration). Interestingly, CRISPs in lamprey buccal gland secretions also manifest some of these activities, suggesting an evolutionarily conserved function. As we strive to better understand the functions that CRISPs serve in venoms, it is worth considering the broad range of CRISP physiological activities throughout the animal kingdom. In this review, we summarize those activities, known crystal structures and sequence alignments, and we discuss predicted functional sites. CRISPs may not be lethal or major components of venoms, but given their almost ubiquitous occurrence in venoms and the accelerated evolution of svCRISP genes, these venom proteins are likely to have functions worth investigating.

Snake venoms are primarily composed of proteins and peptides, and these toxins have developed high selectivity to their biological targets. This makes venoms interesting for exploration into protein evolution and structure-function relationships. A single venom protein superfamily can exhibit a variety of pharmacological effects; these variations in activity originate from differences in functional sites, domains, posttranslational modifications, and the formations of toxin complexes. In this review, we discuss examples of how the major venom protein superfamilies have diversified, as well as how newer technologies in the omics fields, such as genomics, transcriptomics, and proteomics, can be used to characterize both known and unknown toxins.Because toxins are bioactive molecules with a rich diversity of activities, they can be useful as therapeutic and diagnostic agents, and successful examples of toxin applications in these areas are also reviewed. With the current rapid pace of technology, snake venom research and its applications will only continue to expand.

Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

The Harderian gland is a cephalic structure, widely distributed among vertebrates. In snakes, the Harderian gland is anatomically connected to the vomeronasal organ via the nasolacrimal duct, and in some species can be larger than the eyes. The function of the Harderian gland remains elusive, but it has been proposed to play a role in the production of saliva, pheromones, thermoregulatory lipids and growth factors, among others. Here, we have profiled the transcriptomes of the Harderian glands of three non-front-fanged colubroid snakes from Cuba: Caraiba andreae (Cuban Lesser Racer); Cubophis cantherigerus (Cuban Racer); and Tretanorhinus variabilis (Caribbean Water Snake), using Illumina HiSeq2000 100 bp paired-end. In addition to ribosomal and non-characterized proteins, the most abundant transcripts encode putative transport/binding, lipocalin/lipocalin-like, and bactericidal/permeability-increasing-like proteins. Transcripts coding for putative canonical toxins described in venomous snakes were also identified. This transcriptional profile suggests a more complex function than previously recognized for this enigmatic organ., (Copyright © 2018. Published by Elsevier Inc.)

Snakebite envenomation is considered a neglected tropical disease, although it also occurs outside the tropics. In this work, we analyzed the literature on Philodryas species in Chile ( Philodryas chamissonis , P. simonsii , and P. tachymenoides ) from 1834 to 2019, searching for epidemiological, clinical, and molecular aspects of envenomation. Ninety-one percent of the studies found regarded taxonomy, ecology, and natural history, suggesting that snakebites and venom toxins are a neglected issue in Chile. All snakebite cases reported and toxicological studies concerned the species Philodryas chamissonis . Using 185 distributional records from the literature and museum collections for this species, we show for the first time that the reported snakebite cases correlate with human population density, occurring in the Valparaiso and Metropolitan regions in Central Chile. The reduced number of snakebite cases, which were previously considered as having a low incidence in Chile, may be a consequence of under-reported cases, probably due to the inadequate publication or scarce research on this issue. Absence of information about official pharmacological treatment, post-envenoming sequels, clinical management of particular patient groups (e.g., with non-communicable diseases, pregnant women, and the elderly) was also detected. In conclusion, despite having over 185 years of literature on Chilean snakes, knowledge on the envenomation of Philodryas genus remains scarce, seriously affecting adequate medical handling during an ophidic accident. This review highlights the need to develop deep research in this area and urgent improvements to the management of this disease in Chile., Competing Interests: The authors declare no conflict of interest.

Agkistrodon acutus is a traditional Chinese herb medicine which has immunological regulation,anti-tumor,anti-inflammatory and analgesic effects,which is mainly used for the treatment of rheumatoid arthritis,ankylosing spondylitis,sjogren's syndrome and tumors. In order to excavate more important functional genes from A. acutus,the transcriptome of the venom gland was sequenced by the Illumina Hi Seq 4000,and 32 862 unigenes were assembled. Among them,26 589 unigenes were mapped to least one public database. 2 695 unigenes were annotated and assigned to 62 TF families,and 5 920 SSR loci were identified. The majority of mapped unigenes was from Protobothrops mucrosquamatus in the NR database,which revealed their closest homology. Three secretory phospholipase A_2 with different amino acid sequences showed similar spatial structures and all had well-conserved active sites. The 3 D structural models of C-type lectin showed conserved glycosylation binding sites( Asn45). This study will lay the foundation for the further study of the function of snake venom protein,and promoting the development and utilization of genome resources from A. acutus.

Snake venoms are complex protein mixtures with different biological activities that can act in both their preys and human victims. Many of these proteins play a role in prey capture and in the digestive process of these animals. It is known that some snakes are resistant to the toxicity of their own venom by mechanisms not yet fully elucidated. However, it was observed in the Laboratory of Herpetology of Instituto Butantan that some Bothrops moojeni individuals injured by the same snake species showed mortalities caused by envenoming effects. This study analyzed the biochemical composition of 13 venom and plasma samples from Bothrops moojeni specimens to assess differences in their protein composition. Application of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed distinct venom protein profiles, but very homogeneous plasma profiles. Western Blotting (WB) was performed with plasma samples, which were submitted to incubation with the respective venom. Some individuals showed an immunorecognized band zone around 25 kDa, indicating interaction between the same individual plasma and venom proteins. Crossed-WB assay using non-self-plasma and venom showed that this variability is due to venom protein composition instead of plasma composition. These venoms presented higher caseinolytic, collagenolytic and coagulant activities than the venoms without these regions recognized by WB. Mass spectrometry analyses performed on two individuals revealed that these individuals present, in addition to higher protein concentrations, other exclusive proteins in their composition. When these same two samples were tested in vivo, the results also showed higher lethality in these venoms, but lower hemorrhagic activity than in the venoms without these regions recognized by WB. In conclusion, some Bothrops moojeni specimens differ in venom composition, which may have implications in envenomation. Moreover, the high individual venom variability found in this species demonstrates the importance to work with individual analyses in studies involving intraspecific venom variability and venom evolution., Competing Interests: The authors have declared that no competing interests exist.

Snakebite is a major medical concern in many parts of the world with metalloproteases playing important roles in the pathological effects of Viperidae venoms, including local tissue damage, hemorrhage, and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloprotease from Bothrops jararaca venom, induces local hemorrhage and targets extracellular matrix (ECM) components, including collagens and proteoglycans, and plasma proteins. However, the full substrate repertoire of this metalloprotease is unknown. We report positional proteomic studies identifying >2000 N-termini, including neo-N-termini of HF3 cleavage sites in mouse embryonic fibroblast secretome proteins. Terminal amine isotopic labeling of substrates (TAILS) analysis identified a preference for Leu at the P1' position among candidate HF3 substrates including proteins of the ECM and focal adhesions and the cysteine protease inhibitor cystatin-C. Interestingly, 190 unique peptides matched to annotated cleavage sites in the TopFIND N-termini database, suggesting that these cleavages occurred at a site prone to cleavage or might have been generated by other proteases activated upon incubation with HF3, including caspases-3 and -7, cathepsins D and E, granzyme B, and MMPs 2 and 9. Using Proteomic identification of cleavage site specificity (PICS), a tryptic library derived from THP-1 monocytic cells was used as HF3 substrates for identifying protease cleavage sites and sequence preferences in peptides. A total of 799 unique cleavage sites were detected and, in accordance with TAILS analysis using native secreted protein substrates of MEF cells, revealed a clear preference for Leu at P1'. Taken together, these results greatly expand the known substrate degradome of HF3 and reveal potential new targets, which may serve as a basis to better elucidate the complex pathophysiology of snake envenomation.

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Phospholipase A₂ (PLA₂) is a major component in snake venoms and it is found in many different isoforms. To identify transcripts encoding different PLA₂ isoforms, we developed a simple, rapid procedure. Total RNA was extracted from the venoms of three cottonmouth snakes and two diamondback rattlesnakes, and further reverse-transcribed into complementary DNA (cDNA). Using one pair of cottonmouth PLA₂-specific primers and a Reverse Transcription Polymerase Chain Reaction (RT-PCR) technique, we identified 27 unique full-length PLA₂ transcripts, including nine sequences identical to the previously documented ones in the database and one novel GIII-like PLA₂. Two common transcripts respectively encoding Asp49 and Lys49 PLA₂ isoforms were identified in all three cottonmouth venoms, that contain more PLA₂ transcripts than the diamondback rattlesnake venoms. The placement of cloned PLA₂ transcripts in snake venom PLA₂s was further discussed by phylogenetic analysis. The procedure developed in this study paves the way for accelerated acquisition of transcriptome data on any other venom toxin families. The results obtained are crucial for insight into the structure and function of PLA₂ isoforms for scientific and potential therapeutic purposes., Competing Interests: The authors declare no conflict of interest.

DNA barcoding is a simple technique used to develop a large-scale system of classification that is broadly applicable across a wide variety of taxa. DNA-based analysis of snake venoms can provide a system of classification independent of currently accepted taxonomic relationships by generating DNA barcodes specific to each venom sample. DNA purification from dried snake venoms has previously required large amounts of starting material, has resulted in low yields and inconsistent amplification, and was possible with front-fanged snakes only. Here, we present a modified DNA extraction protocol applied to venoms of both front- and rear-fanged snakes that requires significantly less starting material (1 mg) and yields sufficient amounts of DNA for successful PCR amplification of regions commonly used for DNA barcoding. [Formula: see text].

Snake venom α-neurotoxins potently inhibit rodent nicotinic acetylcholine receptors (nAChRs), but their activity on human receptors and their role in human paralysis from snakebite remain unclear. We demonstrate that two short-chain α-neurotoxins (SαNTx) functionally inhibit human muscle-type nAChR, but are markedly more reversible than against rat receptors. In contrast, two long-chain α-neurotoxins (LαNTx) show no species differences in potency or reversibility. Mutant studies identified two key residues accounting for this. Proteomic and clinical data suggest that paralysis in human snakebites is not associated with SαNTx, but with LαNTx, such as in cobras. Neuromuscular blockade produced by both subclasses of α-neurotoxins was reversed by antivenom in rat nerve-muscle preparations, supporting its effectiveness in human post-synaptic paralysis.

Although there were a lot of weakly active animal toxins in the venoms, their values and applications are still mysterious, such as BF9, which is a Kunitz-type toxin isolated from the venom of the elapid snake Bungarus fasciatus. Here, we used BF9 to be a molecular scaffold, and engineered eight BF9-derived peptides by changing P1 site Asn17 of BF9, such as BF9-N17Y and BF9-N17T designed from the polar subfamily, BF9-N17L and BF9-N17G designed from the Non-polar subfamily, BF9-N17D designed from acidic subfamily, and BF9-N17H, BF9-N17K and BF9-N17R designed from basic subfamily. Through enzyme inhibitor experiment assays, we found a potent and selective chymotrypsin inhibitor BF9-N17Y, a potent and selective coagulation factor XIa inhibitor BF9-N17H, and two highly potent coagulation factor XIa inhibitors BF9-N17K and BF9-N17. APTT and PT assays further showed that BF9-N17H, BF9-N17K and BF9-N17R were three novel anticoagulants with selectively intrinsic coagulation pathway inhibitory activity. Considering that natural weakly active animal toxins are also a huge peptide resource, our present work might open a new window about pharmacological applications of weakly active animal toxins, which might be good templates for potent and selective molecular probe and lead drug designs., (Copyright © 2018. Published by Elsevier B.V.)

Three-finger toxins (TFTs) are well-recognized non-enzymatic venom proteins found in snakes. However, although TFTs exhibit accelerated evolution, the drivers of this evolution remain poorly understood. The structural complexes between long-chain α-neurotoxins, a subfamily of TFTs, and their nicotinic acetylcholine receptor targets have been determined in previous research, providing an opportunity to address such questions. In the current study, we observed several previously identified positively selected sites (PSSs) and the highly variable C-terminal loop of these toxins at the toxin/receptor interface. Of interest, analysis of the molecular adaptation of the toxin-recognition regions in the corresponding receptors provided no statistical evidence for positive selection. However, these regions accumulated abundant amino acid variations in the receptors from the prey of snakes, suggesting that accelerated substitution of TFTs could be a consequence of adaptation to these variations. To the best of our knowledge, this atypical evolution, initially discovered in scorpions, is reported in snake toxins for the first time and may be applicable for the evolution of toxins from other venomous animals.

We developed a bacterial expression system to produce a recombinant disintegrin, vicrostatin (VCN), whose structure is based on a natural disintegrin isolated from southern copperhead snake venom. Our goal is to develop VCN for potential clinical translation as an anti-cancer agent. VCN is a peptide of 69 amino acids with a single tyrosine residue. We have employed VCN as integrin-targeted radionuclide therapy (brachytherapy) for treatment of glioblastoma (GBM, glioma). GBM is a deadly brain cancer that doesn't discriminate between sexes and knows no age limit. We established that the tyrosine residue in VCN can be radioiodinated with full retention of bioactivity. 131 I-VCN was utilized for integrin-targeted radionuclide therapy using mouse models of glioma. The combination of radioiodinated VCN plus temozolomide (a DNA alkylating agent) significantly prolonged survival of glioma-bearing mice. We also obtained similar results using an immunocompetent mouse model and a murine glioma cell line. In summary, as demonstrated in studies reported here we have shown that VCN as targeted radionuclide therapy for GBM has significant translational potential for therapy of this deadly disease.

High-throughput technologies were used to identify venom gland toxin expression and to characterize the venom proteomes of two rear-fanged snakes, Ahaetulla prasina (Asian Green Vine Snake) and Borikenophis portoricensis (Puerto Rican Racer). Sixty-nine complete toxin-coding transcripts from 12 venom protein superfamilies (A. prasina) and 50 complete coding transcripts from 11 venom protein superfamilies (B. portoricensis) were identified in the venom glands. However, only 18% (A. prasina) and 32% (B. portoricensis) of the translated protein isoforms were detected in the proteome of these venoms. Both venom gland transcriptomes and venom proteomes were dominated by P-III metalloproteinases. Three-finger toxins, cysteine-rich secretory proteins, and C-type lectins were present in moderate amounts, but other protein superfamilies showed very low abundances. Venoms contained metalloproteinase activity comparable to viperid snake venom levels, but other common venom enzymes were absent or present at negligible levels. Western blot analysis showed metalloproteinase and cysteine-rich secretory protein epitopes shared with the highly venomous Boomslang (Dispholidus typus). The abundance of metalloproteinases emphasizes the important trophic role of these toxins. Comprehensive, transcriptome-informed definition of proteomes and functional characterization of venom proteins in rear-fanged snake families help to elucidate toxin evolution and provide models for protein structure-function analyses., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Venom proteins evolve rapidly, and as a trophic adaptation are excellent models for predator-prey evolutionary studies. The key to a deeper understanding of venom evolution is an integrated approach, combining prey assays with analysis of venom gene expression and venom phenotype. Here, we use such an approach to study venom evolution in the Amazon puffing snake, Spilotes sulphureus , a generalist feeder. We identify two novel three-finger toxins: sulditoxin and sulmotoxin 1. These new toxins are not only two of the most abundant venom proteins, but are also functionally intriguing, displaying distinct prey-specific toxicities. Sulditoxin is highly toxic towards lizard prey, but is non-toxic towards mammalian prey, even at greater than 22-fold higher dosage. By contrast, sulmotoxin 1 exhibits the reverse trend. Furthermore, evolutionary analysis and structural modelling show highest sequence variability in the central loop of these proteins, probably driving taxon-specific toxicity. This is, to our knowledge, the first case in which a bimodal and contrasting pattern of toxicity has been shown for proteins in the venom of a single snake in relation to diet. Our study is an example of how toxin gene neofunctionalization can result in a venom system dominated by one protein superfamily and still exhibit flexibility in prey capture efficacy., (© 2018 The Author(s).)

Venom-gland transcriptomics is a key tool in the study of the evolution, ecology, function, and pharmacology of animal venoms. In particular, gene-expression variation and coding sequences gained through transcriptomics provide key information for explaining functional venom variation over both ecological and evolutionary timescales. The accuracy and usefulness of inferences made through transcriptomics, however, is limited by the accuracy of the transcriptome assembly, which is a bioinformatic problem with several possible solutions. Several methods have been employed to assemble venom-gland transcriptomes, with the Trinity assembler being the most commonly applied among them. Although previous evidence of variation in performance among assembly software exists, particularly regarding recovery of difficult-to-assemble multigene families such as snake venom metalloproteinases, much work to date still employs a single assembly method. We evaluated the performance of several commonly used de novo assembly methods for the recovery of both nontoxin transcripts and complete, high-quality venom-gene transcripts across eleven snake and four scorpion transcriptomes. We varied k -mer sizes used by some assemblers to evaluate the impact of k -mer length on transcript recovery. We showed that the recovery of nontoxin transcripts and toxin transcripts is best accomplished through different assembly software, with SDT at smaller k -mer lengths and Trinity being best for nontoxin recovery and a combination of SeqMan NGen and a seed-and-extend approach implemented in Extender as the best means of recovering a complete set of toxin transcripts. In particular, Extender was the only means tested capable of assembling multiple isoforms of the diverse snake venom metalloproteinase family, while traditional approaches such as Trinity recovered at most one metalloproteinase transcript. Our work demonstrated that traditional metrics of assembly performance are not predictive of performance in the recovery of complete and high quality toxin genes. Instead, effective venom-gland transcriptomic studies should combine and quality-filter the results of several assemblers with varying algorithmic strategies.

The Brown Treesnake (Boiga irregularis) is an arboreal, nocturnal, rear-fanged venomous snake native to northern and eastern regions of Australia, Papua New Guinea and the Solomon Islands. It was inadvertently introduced onto the island of Guam during the late 1940's to early 1950's, and it has caused massive declines and extirpations of the native bird, lizard, and mammal populations. In the current study, we report the characterization of the venom proteome of an adult and a neonate B. irregularis specimens from Guam by a combination of venom gland transcriptomic and venomic analyses. Venom gland transcriptomic analysis of an adult individual identified toxins belonging to 18 protein families, with three-finger toxin isoforms being the most abundantly expressed transcripts, comprising 94% of all venom protein transcript reads. Transcripts for PIII-metalloproteinases, C-type lectins, cysteine-rich secretory proteins, acetylcholinesterases, natriuretic peptides, ficolins, phospholipase A 2 (PLA 2 ) inhibitors, PLA 2 s, vascular endothelial growth factors, Kunitz-type protease inhibitors, cystatins, phospholipase Bs, cobra venom factors, waprins, SVMP inhibitors, matrix metalloproteinases, and hyaluronidases were also identified, albeit, at very low abundances ranging from 0.05% to 1.7% of the transcriptome. The venom proteomes of neonate and adult B. irregularis were also both overwhelmingly (78 and 84%, respectively) dominated by monomeric and dimeric 3FTxs, followed by moderately abundant (21% (N) and 13% (A)) CRISPs, low abundance (1% (N), 3% (A)) PIII-SVMPs, and very low abundance (<0.01%) PLA 2 and SVMP inhibitors. The differences in relative toxin abundances identified between neonate and adult snakes likely correlates to shifts in prey preference between the two age classes, from nearly-exclusively lizards to lizards, birds and small mammals. Immunoaffinity antivenomics with experimentally designed rabbit anti-Brown Treesnake (anti-BTS) venom IgGs against homologous venom from adult snakes demonstrated that CRISPs, PIII-SVMPs, and 60-70% of 3FTxs were effectively immunocaptured. Western blot analysis showed that all venom proteins were recognized by anti-BTS IgGs, and cross-reactivity with other rear-fanged snake venoms was also observed. Incubation of anti-BTS venom IgGs with crude B. irregularis venom resulted in a significant decrease in proteolytic (SVMP) activity against azocasein. These results provide the first comparative venomic and anti-venomic analysis of neonate and adult B. irregularis from Guam, further highlighting evolutionary trends in venom composition among rear-fanged venomous snakes., Significance Paragraph: The Brown Treesnake (Boiga irregularis) has caused extensive ecological and economic damage to the island of Guam where it has become a classic example of the negative impacts of invasive species. In the current study, we report the first combined transcriptomic and proteomic analysis of B. irregularis venom of Guam origin. The transcriptome of an adult snake contained toxin sequences belonging to 18 protein families, with three-finger toxin (3FTx) isoforms being the most abundant and representing 94% of all venom protein transcript reads. Our bottom-up and top-down venomic analyses confirmed that 3FTxs are the major components of B. irregularis venom, and a comparative analysis of neonate and adult venoms demonstrate a clear ontogenetic shift in toxin abundance, likely driven by dietary variation between the two age classes. Second-generation antivenomics and Western blot analysis using purified anti-Brown Treesnake rabbit serum IgGs (anti-BTS IgGs) showed strong immunoreactivity toward B. irregularis venom. Interestingly, our anti-BTS IgGs did not cross-react with 3FTxs found in several other rear-fanged snake venoms, or against 3FTxs in the venom of the elapid Ophiophagus hannah, indicating that epitopes in these 3FTx molecules are quite distinct., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Research involving snake venom has gradually surpassed the simple discovery of new molecules using purification and structural characterization processes, and extended to the identification of their molecular targets and the evaluation of their therapeutic potential. Nevertheless, this only became possible due to constant progress in experimental biology and protein purification approaches., Objective: This review aims to discuss the main components of snake venoms that have been investigated for biotechnological purposes, and to discover how these promising biomolecules were obtained with the satisfactory degree of purity that have enabled such studies. Advances in purification technologies of various snake venom molecules have allowed for important discoveries of proteins and peptides with different biomedical and biotechnological applications., Result and Conclusion: It is believed that significant experimental and computational advances will arise in similar proportions in the coming years that will allow researchers to map the molecular regions responsible for their pharmacological actions, their respective mechanisms of action and their cell targets., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)