High-Voltage Toxin'Roll: Electrostatic Charge Repulsion as a Dynamic Venom Resistance Trait in Pythonid Snakes.

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.

The genetic regulatory architecture and epigenomic basis for age-related changes in rattlesnake venom.

Developmental phenotypic changes can evolve under selection imposed by age- and size-related ecological differences. Many of these changes occur through programmed alterations to gene expression patterns, but the molecular mechanisms and gene-regulatory networks underlying these adaptive changes remain poorly understood. Many venomous snakes, including the eastern diamondback rattlesnake (Crotalus adamanteus), undergo correlated changes in diet and venom expression as snakes grow larger with age, providing models for identifying mechanisms of timed expression changes that underlie adaptive life history traits. By combining a highly contiguous, chromosome-level genome assembly with measures of expression, chromatin accessibility, and histone modifications, we identified cis-regulatory elements and trans-regulatory factors controlling venom ontogeny in the venom glands of C. adamanteus. Ontogenetic expression changes were significantly correlated with epigenomic changes within genes, immediately adjacent to genes (e.g., promoters), and more distant from genes (e.g., enhancers). We identified 37 candidate transcription factors (TFs), with the vast majority being up-regulated in adults. The ontogenetic change is largely driven by an increase in the expression of TFs associated with growth signaling, transcriptional activation, and circadian rhythm/biological timing systems in adults with corresponding epigenomic changes near the differentially expressed venom genes. However, both expression activation and repression contributed to the composition of both adult and juvenile venoms, demonstrating the complexity and potential evolvability of gene regulation for this trait. Overall, given that age-based trait variation is common across the tree of life, we provide a framework for understanding gene-regulatory-network-driven life-history evolution more broadly.

ToxCodAn-Genome: an automated pipeline for toxin-gene annotation in genome assembly of venomous lineages.

BACKGROUND: The rapid development of sequencing technologies resulted in a wide expansion of genomics studies using venomous lineages. This facilitated research focusing on understanding the evolution of adaptive traits and the search for novel compounds that can be applied in agriculture and medicine. However, the toxin annotation of genomes is a laborious and time-consuming task, and no consensus pipeline is currently available. No computational tool currently exists to address the challenges specific to toxin annotation and to ensure the reproducibility of the process. RESULTS: Here, we present ToxCodAn-Genome, the first software designed to perform automated toxin annotation in genomes of venomous lineages. This pipeline was designed to retrieve the full-length coding sequences of toxins and to allow the detection of novel truncated paralogs and pseudogenes. We tested ToxCodAn-Genome using 12 genomes of venomous lineages and achieved high performance on recovering their current toxin annotations. This tool can be easily customized to allow improvements in the final toxin annotation set and can be expanded to virtually any venomous lineage. ToxCodAn-Genome is fast, allowing it to run on any personal computer, but it can also be executed in multicore mode, taking advantage of large high-performance servers. In addition, we provide a guide to direct future research in the venomics field to ensure a confident toxin annotation in the genome being studied. As a case study, we sequenced and annotated the toxin repertoire of Bothrops alternatus, which may facilitate future evolutionary and biomedical studies using vipers as models. CONCLUSIONS: ToxCodAn-Genome is suitable to perform toxin annotation in the genome of venomous species and may help to improve the reproducibility of further studies. ToxCodAn-Genome and the guide are freely available at https://github.com/pedronachtigall/ToxCodAn-Genome.

ToxCodAn-Genome: an automated pipeline for toxin-gene annotation in genome assembly of venomous lineages

Background: The rapid development of sequencing technologies resulted in a wide expansion of genomics studies using venomous lineages. This facilitated research focusing on understanding the evolution of adaptive traits and the search for novel compounds that can be applied in agriculture and medicine. However, the toxin annotation of genomes is a laborious and time-consuming task, and no consensus pipeline is currently available. No computational tool currently exists to address the challenges specific to toxin annotation and to ensure the reproducibility of the process. Results: Here, we present ToxCodAn-Genome, the first software designed to perform automated toxin annotation in genomes of venomous lineages. This pipeline was designed to retrieve the full-length coding sequences of toxins and to allow the detection of novel truncated paralogs and pseudogenes. We tested ToxCodAn-Genome using 12 genomes of venomous lineages and achieved high performance on recovering their current toxin annotations. This tool can be easily customized to allow improvements in the final toxin annotation set and can be expanded to virtually any venomous lineage. ToxCodAn-Genome is fast, allowing it to run on any personal computer, but it can also be executed in multicore mode, taking advantage of large high-performance servers. In addition, we provide a guide to direct future research in the venomics field to ensure a confident toxin annotation in the genome being studied. As a case study, we sequenced and annotated the toxin repertoire of Bothrops alternatus, which may facilitate future evolutionary and biomedical studies using vipers as models. Conclusions: ToxCodAn-Genome is suitable to perform toxin annotation in the genome of venomous species and may help to improve the reproducibility of further studies. ToxCodAn-Genome and the guide are freely available at https://github.com/pedronachtigall/T oxCodAn-Genome.

Varying Modes of Selection Among Toxin Families in the Venoms of the Giant Desert Hairy Scorpions (Hadrurus).

Venoms are primarily believed to evolve under strong diversifying selection resulting from persistent coevolution between predator and prey. Recent research has challenged this hypothesis, proposing that venoms from younger venomous lineages (e.g., snakes and cone snails) are governed predominantly by diversifying selection, while venoms from older venomous lineages (e.g., centipedes, scorpions, and spiders) are under stronger purifying selection. However, most research in older lineages has tested selection at more diverse phylogenetic scales. Although these tests are important for evaluating broad macroevolutionary trends underlying venom evolution, they are less equipped to detect species-level evolutionary trends, which likely have large impacts on venom variation seen at more diverse phylogenetic scales. To test for selection among closely related species from an older venomous lineage, we generated high-throughput venom-gland transcriptomes and venom proteomes for four populations of Giant Desert Hairy Scorpions (Hadrurus), including three Hadrurus arizonensis populations and one Hadrurus spadix population. We detected significant episodic and pervasive diversifying selection across a highly abundant toxin family that likely has a major role in venom function ([Formula: see text]KTxs), providing a contrast to the stronger purifying selection identified from other studies on scorpion venoms. Conversely, we detected weak episodic diversifying and/or stronger purifying selection in four toxin families (non-disulfide bridged peptides, phospholipase A2s, scorpine-like antimicrobial peptides, and serine proteases), most of which were less abundant and likely have ancillary functional roles. Finally, although we detected several major toxin families at disproportionate transcriptomic and/or proteomic abundances, we did not identify significant sex-based variation in Hadrurus venoms.

The arylsulfatase- and phospholipase-rich venom of the plutoniumid centipede Theatops posticus.

Research on centipede venoms has led to the discovery of a diverse array of novel proteins and peptides, including those with homology to previously discovered toxin families (e.g., phospholipase A2s and pM12a metalloproteases) and novel toxin families not previously detected in venoms (e.g., ß-pore forming toxins and scoloptoxins). Most of this research has focused on centipedes in the order Scolopendromorpha, particularly those in the families Scolopendridae, Cryptopidae, and Scolopocryptopidae. To generate the first high-throughput venom characterization for a centipede in the scolopendromorph family Plutoniumidae, we performed venom-gland transcriptomics and venom proteomics on two Theatops posticus. We identified a total of 64 venom toxins, 60 of which were detected in both the venom-gland transcriptome and venom proteome and four of which were only detected transcriptomically. We detected a single highly abundant arylsulfatase B (ARSB) toxin, the first ARSB toxin identified from centipede venoms. As ARSBs have been detected in other venomous species (e.g., scorpions), ARSBs in T. posticus highlights a new case of convergent evolution across venoms. Theatops posticus venom also contained a much higher abundance and diversity of phospholipase A2 toxins compared to other characterized centipede venoms. Conversely, we detected other common centipedes toxins, such as CAPs and scoloptoxins, at relatively low abundances and diversities. Our observation of a diverse set of toxins from T. posticus venom, including those from novel toxin families, emphasizes the importance of studying unexplored centipede taxonomic groups and the continued potential of centipede venoms for novel toxin discovery and unraveling the molecular mechanisms underlying trait evolution.

Sequence Divergence in Venom Genes Within and Between Montane Pitviper (Viperidae: Crotalinae: Cerrophidion) Species is Driven by Mutation-Drift Equilibrium.

Snake venom can vary both among and within species. While some groups of New World pitvipers-such as rattlesnakes-have been well studied, very little is known about the venom of montane pitvipers (Cerrophidion) found across the Mesoamerican highlands. Compared to most well-studied rattlesnakes, which are widely distributed, the isolated montane populations of Cerrophidion may facilitate unique evolutionary trajectories and venom differentiation. Here, we describe the venom gland transcriptomes for populations of C. petlalcalensis, C. tzotzilorum, and C. godmani from Mexico, and a single individual of C. sasai from Costa Rica. We explore gene expression variation in Cerrophidion and sequence evolution of toxins within C. godmani specifically. Cerrophidion venom gland transcriptomes are composed primarily of snake venom metalloproteinases, phospholipase A[Formula: see text]s (PLA[Formula: see text]s), and snake venom serine proteases. Cerrophidion petlalcalensis shows little intraspecific variation; however, C. godmani and C. tzotzilorum differ significantly between geographically isolated populations. Interestingly, intraspecific variation was mostly attributed to expression variation as we did not detect signals of selection within C. godmani toxins. Additionally, we found PLA[Formula: see text]-like myotoxins in all species except C. petlalcalensis, and crotoxin-like PLA[Formula: see text]s in the southern population of C. godmani. Our results demonstrate significant intraspecific venom variation within C. godmani and C. tzotzilorum. The toxins of C. godmani show little evidence of directional selection where variation in toxin sequence is consistent with evolution under a model of mutation-drift equilibrium. Cerrophidion godmani individuals from the southern population may exhibit neurotoxic venom activity given the presence of crotoxin-like PLA[Formula: see text]s; however, further research is required to confirm this hypothesis.

RESUMEN: El veneno de las serpientes puede variar entre y dentro de las especies. Mientras algunos grupos de viperidos del Nuevo Mundo­como las cascabeles­han sido bien estudiadas, muy poco se sabe acerca del veneno de las nauyacas de frío (Cerrophidion) que se encuentran en las zonas altas de Mesoamérica. Comparadas con las extensamente estudiadas cascabeles, que estan ampliamente distribuidas, las poblaciones de Cerrophidion, aisladas en montañas, pueden poseer trayectorias evolutivas y diferenciación en su veneno unicos. En el presente trabajo, describimos el transcriptoma de las glándulas de veneno de poblaciones de C. petlalcalensis, C. tzotzilorum, y C. godmani de México, y un individuo de C. sasai de Costa Rica. Exploramos la variación en la expresión de toxinas en Cerrophidion y la evolución en las secuencias geneticas en C. godmani específicamente. El transcriptoma de la glándula de veneno de Cerrophidion esta compuesto principalmente de Metaloproteinasas de Veneno de Serpiente, Fosfolipasas A[Formula: see text] (PLA[Formula: see text]s), y Serin Proteasas de Veneno de Serpiente. Cerrophidion petlalcalensis presenta poca variación intraespecífica; sin embargo, los transcriptomas de la glandula de veneno de C. godmani y C. tzotzilorum difieren significativamente entre poblaciones geográficamente aisladas. Curiosamente, la variación intraespecífica estuvo atribuida principalmente a la expresión de las toxinas ya que no encontramos señales de selección en las toxinas de C. godmani. Adicionalmente, encontramos miotoxinas similares a PLA[Formula: see text] en todas las especies excepto C. petlalcalensis, y PLA[Formula: see text]s similares a crotoxina en la población sureña de C. godmani. Nuestros resultados demuestran la presencia de variacion intraespecífica presente en el veneno de C. godmani y C. tzotzilorum. Las toxinas de Cerrophidion godmani muestran poca evidencia de selección direccional, y la variación en la secuencias de las toxinas es consistente con evolucion bajo un modelo de equilibrio de mutación-deriva. Algunos individuos de C. godmani de la población del sur potencialmente tienen un veneno neurotóxico dada la presencia de PLA[Formula: see text]s similares a la crotoxina, sin embargo, se necesita más evidencia para corroborar esta hipótesis.

Venom phenotype conservation suggests integrated specialization in a lizard-eating snake.

Biological specialization reduces the size of niche space while increasing efficiency in the use of available resources. Specialization often leads to phenotypic changes via natural selection aligning with niche space constraints. Commonly observed changes are in size, shape, behavior, and traits associated with feeding. One often selected trait for dietary specialization is venom, which, in snakes, often shows variation dependent on diet across and within species. The Neotropical Blunt-headed Treesnake (Imantodes cenchoa) is a highly specialized, rear-fanged, arboreal, lizard hunter that displays a long thin body, enlarged eyes, and a large Duvernoy's gland. However, toxin characterization of I. cenchoa has never been completed. Here, we use RNA-seq and mass spectrometry to assemble, annotate, and analyze the venom gland transcriptomes of four I. cenchoa from across their range. We find a lack of significant venom variation at the sequence and expression levels, suggesting venom conservation across the species. We propose this conservation provides evidence of a specialized venom repertoire, adapted to maximize efficiency of capturing and processing lizards. Importantly, this study provides the most complete venom gland transcriptomes of I. cenchoa and evidence of venom specialization in a rear-fanged snake, giving insight into selective pressures of venom across all snake species.

The remarkably enzyme-rich venom of the Big Bend Scorpion (Diplocentrus whitei).

Scorpion venoms have long been studied for their peptide discovery potential, with modern high-throughput venom-characterization techniques paving the way for the discovery of thousands of novel putative toxins. Research into these toxins has provided insight into the pathology and treatment of human diseases, even resulting in the development of one compound with Food and Drug Administration (FDA) approval. Although most of this research has focused on the toxins of scorpion species considered medically significant to humans, the venom of harmless scorpion species possess toxins that are homologous to those from medically significant species, indicating that harmless scorpion venoms may also serve as valuable sources of novel peptide variants. Furthermore, as harmless scorpions represent a vast majority of scorpion species diversity, and therefore venom toxin diversity, venoms from these species likely contain entirely new toxin classes. We sequenced the venom-gland transcriptome and venom proteome of two male Big Bend scorpions (Diplocentrus whitei), providing the first high-throughput venom characterization for a member of this genus. We identified a total of 82 toxins in the venom of D. whitei, 25 of which were identified in both the transcriptome and proteome, and 57 of which were only identified in the transcriptome. Furthermore, we identified a unique, enzyme-rich venom dominated by serine proteases and the first arylsulfatase B toxins identified in scorpions.

Evolutionary allometry and ecological correlates of fang length evolution in vipers.

Traits for prey acquisition form the phenotypic interface of predator-prey interactions. In venomous predators, morphological variation in venom delivery apparatus like fangs and stingers may be optimized for dispatching prey. Here, we determine how a single dimension of venom injection systems evolves in response to variation in the size, climatic conditions and dietary ecology of viperid snakes. We measured fang length in more than 1900 museum specimens representing 199 viper species (55% of recognized species). We find both phylogenetic signal and within-clade variation in relative fang length across vipers suggesting both general taxonomic trends and potential adaptive divergence in fang length. We recover positive evolutionary allometry and little static allometry in fang length. Proportionally longer fangs have evolved in larger species, which may facilitate venom injection in more voluminous prey. Finally, we leverage climatic and diet data to assess the global correlates of fang length. We find that models of fang length evolution are improved through the inclusion of both temperature and diet, particularly the extent to which diets are mammal-heavy diets. These findings demonstrate how adaptive variation can emerge among components of complex prey capture systems.

De Novo Genome Assembly Highlights the Role of Lineage-Specific Gene Duplications in the Evolution of Venom in Fea's Viper (Azemiops feae).

Despite the medical significance to humans and important ecological roles filled by vipers, few high-quality genomic resources exist for these snakes outside of a few genera of pitvipers. Here we sequence, assemble, and annotate the genome of Fea's Viper (Azemiops feae). This taxon is distributed in East Asia and belongs to a monotypic subfamily, sister to the pitvipers. The newly sequenced genome resulted in a 1.56 Gb assembly, a contig N50 of 1.59 Mb, with 97.6% of the genome assembly in contigs >50 Kb, and a BUSCO completeness of 92.4%. We found that A. feae venom is primarily composed of phospholipase A2 (PLA2) proteins expressed by genes that likely arose from lineage-specific PLA2 gene duplications. Additionally, we show that renin, an enzyme associated with blood pressure regulation in mammals and known from the venoms of two viper species including A. feae, is expressed in the venom gland at comparative levels to known toxins and is present in the venom proteome. The cooption of this gene as a toxin may be more widespread in viperids than currently known. To investigate the historical population demographics of A. feae, we performed coalescent-based analyses and determined that the effective population size has remained stable over the last 100 kyr. This suggests Quaternary glacial cycles likely had minimal influence on the demographic history of A. feae. This newly assembled genome will be an important resource for studying the genomic basis of phenotypic evolution and understanding the diversification of venom toxin gene families.

Venom Gene Sequence Diversity and Expression Jointly Shape Diet Adaptation in Pitvipers.

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.

Contrasting patterns of venom regeneration in a centipede (Scolopendra viridis) and a scorpion (Centruroides hentzi).

As biochemical traits with clear fitness consequences, venoms serve a critical ecological role for the animals that produce them. Understanding how venoms are maintained and regenerated after use will, therefore, provide valuable insight into the ecology of venomous animals. Furthermore, most studies on venomous organisms often require removing animals from the wild and waiting extended periods of time between venom extractions. Uncovering the patterns of venom regeneration across different species will likely lead to the development of more efficient venom extraction protocols, reducing both experimental time and the number of animals required. Using reversed-phase high-performance liquid chromatography, we identified asynchronous regeneration of venom protein component abundances in the centipede Scolopendra viridis, but found no evidence for asynchronous venom regeneration in the scorpion Centruroides hentzi. We also observed high levels of intraspecific venom variation in C. hentzi, emphasizing the importance of testing for intraspecific venom variation in studies evaluating the synchronicity of venom regeneration. Although the regeneration of relative venom protein component abundances is an asynchronous process in S. viridis, we provide evidence that the presence-absence of major venom components is not an asynchronous process and suggest that studies relying on just the presence-absence of individual proteins (e.g. bioprospecting, drug discovery) could use catch-and-release methods of venom extraction to reduce the number of animals removed from the wild.

An integrative view of the toxic potential of Conophis lineatus (Dipsadidae: Xenodontinae), a medically relevant rear-fanged snake.

Most traditional research on snake venoms has focused on front-fanged snake families (Viperidae, Elapidae, and Atractaspididae). However, venom is now generally accepted as being a much more broadly possessed trait within snakes, including species traditionally considered harmless. Unfortunately, due to historical inertia and methodological challenges, the toxin repertoires of non-front-fanged snake families (e.g., Colubridae, Dipsadidae, and Natricidae) have been heavily neglected despite the knowledge of numerous species capable of inflicting medically relevant envenomations. Integrating proteomic data for validation, we perform a de novo assembly and analysis of the Duvernoy's venom gland transcriptome of the Central American Road Guarder (Dipsadidae: Xenodontinae: Conophis lineatus), a species known for its potent bite. We identified 28 putative toxin transcripts from 13 toxin families in the Duvernoy's venom gland transcriptome, comprising 63.7% of total transcriptome expression. In addition to ubiquitous snake toxin families, we proteomically confirmed several atypical venom components. The most highly expressed toxins (55.6% of total toxin expression) were recently described snake venom matrix metalloproteases (svMMPs), with 48.0% of svMMP expression contributable to a novel svMMP isoform. We investigate the evolution of the new svMMP isoform in the context of rear-fanged snakes using phylogenetics. Finally, we examine the morphology of the venom apparatus using µCT and explore how the venom relates to autecology and the highly hemorrhagic effects seen in human envenomations. Importantly, we provide the most complete venom characterization of this medically relevant snake species to date, producing insights into the effects and evolution of its venom, and point to future research directions to better understand the venoms of 'harmless' non-front-fanged snakes.

Varying Intensities of Introgression Obscure Incipient Venom-Associated Speciation in the Timber Rattlesnake (Crotalus horridus).

Ecologically divergent selection can lead to the evolution of reproductive isolation through the process of ecological speciation, but the balance of responsible evolutionary forces is often obscured by an inadequate assessment of demographic history and the genetics of traits under selection. Snake venoms have emerged as a system for studying the genetic basis of adaptation because of their genetic tractability and contributions to fitness, and speciation in venomous snakes can be associated with ecological diversification such as dietary shifts and corresponding venom changes. Here, we explored the neurotoxic (type A)-hemotoxic (type B) venom dichotomy and the potential for ecological speciation among Timber Rattlesnake (Crotalus horridus) populations. Previous work identified the genetic basis of this phenotypic difference, enabling us to characterize the roles geography, history, ecology, selection, and chance play in determining when and why new species emerge or are absorbed. We identified significant genetic, proteomic, morphological, and ecological/environmental differences at smaller spatial scales, suggestive of incipient ecological speciation between type A and type B C. horridus. Range-wide analyses, however, rejected the reciprocal monophyly of venom type, indicative of varying intensities of introgression and a lack of reproductive isolation across the range. Given that we have now established the phenotypic distributions and ecological niche models of type A and B populations, genome-wide data are needed and capable of determining whether type A and type B C. horridus represent distinct, reproductively isolated lineages due to incipient ecological speciation or differentiated populations within a single species.

Identification and Characterization of Novel Proteins from Arizona Bark Scorpion Venom That Inhibit Nav1.8, a Voltage-Gated Sodium Channel Regulator of Pain Signaling.

The voltage-gated sodium channel Nav1.8 is linked to neuropathic and inflammatory pain, highlighting the potential to serve as a drug target. However, the biophysical mechanisms that regulate Nav1.8 activation and inactivation gating are not completely understood. Progress has been hindered by a lack of biochemical tools for examining Nav1.8 gating mechanisms. Arizona bark scorpion (Centruroides sculpturatus) venom proteins inhibit Nav1.8 and block pain in grasshopper mice (Onychomys torridus). These proteins provide tools for examining Nav1.8 structure-activity relationships. To identify proteins that inhibit Nav1.8 activity, venom samples were fractioned using liquid chromatography (reversed-phase and ion exchange). A recombinant Nav1.8 clone expressed in ND7/23 cells was used to identify subfractions that inhibited Nav1.8 Na+ current. Mass-spectrometry-based bottom-up proteomic analyses identified unique peptides from inhibitory subfractions. A search of the peptides against the AZ bark scorpion venom gland transcriptome revealed four novel proteins between 40 and 60% conserved with venom proteins from scorpions in four genera (Centruroides, Parabuthus, Androctonus, and Tityus). Ranging from 63 to 82 amino acids, each primary structure includes eight cysteines and a "CXCE" motif, where X = an aromatic residue (tryptophan, tyrosine, or phenylalanine). Electrophysiology data demonstrated that the inhibitory effects of bioactive subfractions can be removed by hyperpolarizing the channels, suggesting that proteins may function as gating modifiers as opposed to pore blockers.

Experimental Manipulation of Corticosterone Does Not Affect Venom Composition or Functional Activity in Free-Ranging Rattlesnakes.

AbstractVenom is an integral feeding trait in many animal species. Although venom often varies ontogenetically, little is known about the proximate physiological mediators of venom variation within individuals. The glucocorticoid hormone corticosterone (CORT) can alter the transcription and activation of proteins, including homologues of snake venom components such as snake venom metalloproteinases (SVMPs) and phospholipase A2 (PLA2). CORT is endogenously produced by snakes, varies seasonally and also in response to stress, and is a candidate endogenous mediator of changes in venom composition and functional activity. Here, we tested the hypothesis that CORT induces changes in snake venom by sampling the venom of wild adult rattlesnakes before and after they were treated with either empty (control) or CORT-filled (treatment) Silastic implants. We measured longitudinal changes in whole-venom composition, whole-venom total protein content, and enzymatic activity of SVMP and PLA2 components of venom. We also assessed the within-individual repeatability of venom components. Despite successfully elevating plasma CORT in the treatment group, we found no effect of CORT treatment or average plasma CORT level on any venom variables measured. Except for total protein content, venom components were highly repeatable within individuals ([Formula: see text]). Our results indicate that the effects of CORT, a hormone commonly associated with stress and metabolic functions, in adult rattlesnake venom are negligible. Our findings bode well for venom researchers and biomedical applications that rely on the consistency of venoms produced from potentially stressed individuals and provide an experimental framework for future studies of proximate mediators of venom variation across an individual's life span.

The Chemosensory Repertoire of the Eastern Diamondback Rattlesnake (Crotalus adamanteus) Reveals Complementary Genetics of Olfactory and Vomeronasal-Type Receptors.

Pitviper sensory perception incorporates diverse stimuli through the integration of trichromatic color vision, bifocal heat-sensing, and dual-system chemoperception. Chemoperception, or olfaction, is mediated by chemoreceptors in the olfactory bulb and the vomeronasal organ, but the true genomic complexity of the gene families and their relative contributions is unknown. A full genomic accounting of pitviper chemoperception directly complements our current understanding of their venoms by generating a more complete polyphenic representation of their predatory arsenal. To characterize the genetic repertoire of pitviper chemoperception, we analyzed a full-genome assembly for Crotalus adamanteus, the eastern diamondback rattlesnake. We identified hundreds of genes encoding both olfactory receptors (ORs; 362 full-length genes) and type-2 vomeronasal receptors (V2Rs; 430 full-length genes). Many chemoreceptor genes are organized into large tandem repeat arrays. Comparative analysis of V2R orthologs across squamates demonstrates how gene array expansion and contraction underlies the evolution of the chemoreceptor repertoire, which likely reflects shifts in life history traits. Chromosomal assignments of chemosensory genes identified sex chromosome specific chemoreceptor genes, providing gene candidates underlying observed sex-specific chemosensory-based behaviors. We detected widespread episodic evolution in the extracellular, ligand-binding domains of both ORs and V2Rs, suggesting the diversification of chemoreceptors is driven by transient periods of positive selection. We provide a robust genetic framework for studying pitviper chemosensory ecology and evolution.

Phylogenetically diverse diets favor more complex venoms in North American pitvipers.

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.

ToxCodAn: a new toxin annotator and guide to venom gland transcriptomics.

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.