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.

Ontogenetic change in the venom composition of one Mexican black-tailed rattlesnake (Crotalus molossus nigrescens) from Durango, Mexico.

To corroborate the ontogenetic shift in the venom composition of the Mexican Black-tailed Rattlesnake (Crotalus molossus nigrescens) previously reported through the census approach, we evaluated the shift in the protein profile, lethality, and proteolytic and phospholipase activities of four venom samples obtained in 2015, 2018, 2019, and 2021 from one C. m. nigrescens individual (CMN06) collected in Durango, Mexico. We demonstrated that the venom of C. m. nigrescens changed from a myotoxin-rich venom to a phospholipase A2 and snake venom metalloproteinase-rich venom. Additionally, the proteolytic and phospholipase activities increased with age, but the lethality decreased approximately three times.

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.

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.

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.

Characterization of the venom and external morphology of a natural hybrid between Crotalus atrox and Crotalus mictlantecuhtli.

Here we report, for the first time, a natural hybrid between Crotalus atrox and C. mictlantecuhtli based on intermediate characteristics of the external morphology and venom. Morphologically, the individual had characteristics of both parent species. The hybrid's venom exhibited an intermediate composition including the presence of crotoxin which has never been documented in C. atrox but is well documented in C. mictlantecuhtli. The hybrid's venom was highly toxic and showed an intermediate proteolytic activity between the parental species. The two Mexican antivenoms were able to neutralize the hybrid's venom's lethality.

Intraspecific venom variation of Mexican West Coast Rattlesnakes (Crotalus basiliscus) and its implications for antivenom production.

Intraspecific variation in snake venoms has been widely documented worldwide. However, there are few studies on this subject in Mexico. Venom characterization studies provide important data used to predict clinical syndromes, to evaluate the efficacy of antivenoms and, in some cases, to improve immunogenic mixtures in the production of antivenoms. In the present work, we evaluated the intraspecific venom variation of Crotalus basiliscus, a rattlesnake of medical importance and whose venom is used in the immunization of horses to produce one of the Mexican antivenoms. Our results demonstrate that there is variation in biological and biochemical activities among adult venoms and that there is an ontogenetic change from juvenile to adult venoms. Juvenile venoms were more lethal and had higher percentages of crotamine and crotoxin, while adult venoms had higher percentages of snake venom metalloproteases (SVMPs). Additionally, we documented crotoxin-like PLA2 variation in which specimens from Zacatecas, Sinaloa and Michoacán (except 1) lacked the neurotoxin, while the rest of the venoms had it. Finally, we evaluated the efficacy of three lots of Birmex antivenom and all three were able to neutralize the lethality of four representative venoms but were not able to neutralize crotamine. We also observed significant differences in the LD50 values neutralized per vial among the different lots. Based on these results, we recommend including venoms containing crotamine in the production of antivenom for a better immunogenic mixture and to improve the homogeneity of lots.

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.

Características para aquisição de presas formam a interface fenotípica das interações predador-presa. Em predadores venenosos, a variação morfológica no aparelho de entrega de veneno, como presas e ferrões, pode ser otimizada para despachar presas. Aqui, determinamos como uma única dimensão dos sistemas de injeção de veneno evolui em resposta à variação no tamanho, condições climáticas e ecologia alimentar de cobras viperídeos. Medimos o comprimento das presas em mais de 1.900 espécimes de museus representando 199 espécies de víboras (55% das espécies reconhecidas). Encontramos tanto o sinal filogenético quanto a variação dentro do clado no comprimento relativo das presas entre as víboras, sugerindo tendências taxonômicas gerais e potencial divergência adaptativa no comprimento das presas. Recuperamos alometria evolutiva positiva e pouca alometria estática no comprimento das presas. Presas proporcionalmente mais longas evoluíram em espécies maiores, o que pode facilitar a injeção de veneno em presas mais volumosas. Por fim, aproveitamos os dados climáticos e de dieta para avaliar os correlatos globais do comprimento das presas. Descobrimos que os modelos de evolução do comprimento das presas são melhorados através da inclusão de temperatura e dieta, particularmente na medida em que as dietas são dietas pesadas para mamíferos. Essas descobertas demonstram como a variação adaptativa pode surgir entre os componentes de sistemas complexos de captura de presas.

Duvernoy's Gland Transcriptomics of the Plains Black-Headed Snake, Tantilla nigriceps (Squamata, Colubridae): Unearthing the Venom of Small Rear-Fanged Snakes.

The venoms of small rear-fanged snakes (RFS) remain largely unexplored, despite increased recognition of their importance in understanding venom evolution more broadly. Sequencing the transcriptome of venom-producing glands has greatly increased the ability of researchers to examine and characterize the toxin repertoire of small taxa with low venom yields. Here, we use RNA-seq to characterize the Duvernoy's gland transcriptome of the Plains Black-headed Snake, Tantilla nigriceps, a small, semi-fossorial colubrid that feeds on a variety of potentially dangerous arthropods including centipedes and spiders. We generated transcriptomes of six individuals from three localities in order to both characterize the toxin expression of this species for the first time, and to look for initial evidence of venom variation in the species. Three toxin families-three-finger neurotoxins (3FTxs), cysteine-rich secretory proteins (CRISPs), and snake venom metalloproteinases (SVMPIIIs)-dominated the transcriptome of T. nigriceps; 3FTx themselves were the dominant toxin family in most individuals, accounting for as much as 86.4% of an individual's toxin expression. Variation in toxin expression between individuals was also noted, with two specimens exhibiting higher relative expression of c-type lectins than any other sample (8.7-11.9% compared to <1%), and another expressed CRISPs higher than any other toxin. This study provides the first Duvernoy's gland transcriptomes of any species of Tantilla, and one of the few transcriptomic studies of RFS not predicated on a single individual. This initial characterization demonstrates the need for further study of toxin expression variation in this species, as well as the need for further exploration of small RFS venoms.

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.

The Tiger Rattlesnake genome reveals a complex genotype underlying a simple venom phenotype.

Variation in gene regulation is ubiquitous, yet identifying the mechanisms producing such variation, especially for complex traits, is challenging. Snake venoms provide a model system for studying the phenotypic impacts of regulatory variation in complex traits because of their genetic tractability. Here, we sequence the genome of the Tiger Rattlesnake, which possesses the simplest and most toxic venom of any rattlesnake species, to determine whether the simple venom phenotype is the result of a simple genotype through gene loss or a complex genotype mediated through regulatory mechanisms. We generate the most contiguous snake-genome assembly to date and use this genome to show that gene loss, chromatin accessibility, and methylation levels all contribute to the production of the simplest, most toxic rattlesnake venom. We provide the most complete characterization of the venom gene-regulatory network to date and identify key mechanisms mediating phenotypic variation across a polygenic regulatory network.

Genomic Adaptations to Salinity Resist Gene Flow in the Evolution of Floridian Watersnakes.

The migration-selection balance often governs the evolution of lineages, and speciation with gene flow is now considered common across the tree of life. Ecological speciation is a process that can facilitate divergence despite gene flow due to strong selective pressures caused by ecological differences; however, the exact traits under selection are often unknown. The transition from freshwater to saltwater habitats provides strong selection targeting traits with osmoregulatory function. Several lineages of North American watersnakes (Nerodia spp.) are known to occur in saltwater habitat and represent a useful system for studying speciation by providing an opportunity to investigate gene flow and evaluate how species boundaries are maintained or degraded. We use double digest restriction-site associated DNA sequencing to characterize the migration-selection balance and test for evidence of ecological divergence within the Nerodia fasciata-clarkii complex in Florida. We find evidence of high intraspecific gene flow with a pattern of isolation-by-distance underlying subspecific lineages. However, we identify genetic structure indicative of reduced gene flow between inland and coastal lineages suggesting divergence due to isolation-by-environment. This pattern is consistent with observed environmental differences where the amount of admixture decreases with increased salinity. Furthermore, we identify significantly enriched terms related to osmoregulatory function among a set of candidate loci, including several genes that have been previously implicated in adaptation to salinity stress. Collectively, our results demonstrate that ecological differences, likely driven by salinity, cause strong divergent selection which promotes divergence in the N. fasciata-clarkii complex despite significant gene flow.

Phylogenetically diverse diets favor more complex venoms in North American pitvipers

The role of natural selection in the evolution of trait complex-ity can be characterized by testing hypothesized links betweencomplex forms and their functions across species. Predatory ven-oms are composed of multiple proteins that collectively function toincapacitate prey. Venom complexity fluctuates over evolutionarytimescales, with apparent increases and decreases in complexity,and yet the causes of this variation are unclear. We tested alterna-tive hypotheses linking venom complexity and ecological sourcesof selection from diet in the largest clade of front-fanged ven-omous snakes in North America: the rattlesnakes, copperheads,cantils, and cottonmouths. We generated independent transcrip-tomic and proteomic measures of venom complexity and collatedseveral natural history studies to quantify dietary variation. Wethen constructed genome-scale phylogenies for these snakes forcomparative analyses. Strikingly, prey phylogenetic diversity wasmore strongly correlated to venom complexity than was overallprey species diversity, specifically implicating prey species’ diver-gence, rather than the number of lineages alone, in the evolutionof complexity. Prey phylogenetic diversity further predicted tran-scriptomic complexity of three of the four largest gene familiesin viper venom, showing that complexity evolution is a concertedresponse among many independent gene families. We suggest thatthe phylogenetic diversity of prey measures functionally relevantdivergence in the targets of venom, a claim supported by sequencediversity in the coagulation cascade targets of venom. Our resultssupport the general concept that the diversity of species in an eco-logical community is more important than their overall number indetermining evolutionary patterns in predator trait complexity.

Neutralizing potency and immunochemical evaluation of an anti-Crotalus mictlantecuhtli experimental serum.

Snakebite in Mexico is commonly treated with an antivenom which uses Bothrops asper and Crotalus simus venoms as immunogens. Current taxonomic recommendations for the C. simus species complex suggest a novel endemic species from Mexico: Crotalus mictlantecuhtli. The aim of this report was to evaluate the immunogenic properties of C. mictlantecuhtli venom and its potential to generate polyclonal antibodies capable of neutralizing other pitviper venoms. We generated an experimental anti-Crotalus mictlantecuhtli serum, using the rabbit model, to test recognition and neutralizing capacity against the homologous venom as well as venoms from C. atrox, C.basiliscus, C. durissus terrificus, C. scutulatus salvini, C. tzabcan and Ophryacus sphenophrys. Pre-incubation neutralization experiments using our experimental serum showed positive results against venoms containing crotoxin, while venoms from two non-neurotoxic pit-vipers were not neutralized. Rescue experiments in mice showed that, when intravenously injected (i.v.), C. mictlantecuhtli venom is not neutralized by a maximum dose of Antivipmyn® and the experimental serum after 5 min of envenomation, albeit mice envenomated intraperitoneally (i.p.) and rescued i.v. with Antivipmyn® survived even at 50 min after envenomation. Our results highlight the importance of using the highly neurotoxic C. mictlantecuhtli venom to increase antivenom effectiveness against Mexican neurotoxic pitvipers.

Trait differentiation and modular toxin expression in palm-pitvipers.

BACKGROUND: Modularity is the tendency for systems to organize into semi-independent units and can be a key to the evolution and diversification of complex biological systems. Snake venoms are highly variable modular systems that exhibit extreme diversification even across very short time scales. One well-studied venom phenotype dichotomy is a trade-off between neurotoxicity versus hemotoxicity that occurs through the high expression of a heterodimeric neurotoxic phospholipase A2 (PLA2) or snake venom metalloproteinases (SVMPs). We tested whether the variation in these venom phenotypes could occur via variation in regulatory sub-modules through comparative venom gland transcriptomics of representative Black-Speckled Palm-Pitvipers (Bothriechis nigroviridis) and Talamancan Palm-Pitvipers (B. nubestris). RESULTS: We assembled 1517 coding sequences, including 43 toxins for B. nigroviridis and 1787 coding sequences including 42 toxins for B. nubestris. The venom gland transcriptomes were extremely divergent between these two species with one B. nigroviridis exhibiting a primarily neurotoxic pattern of expression, both B. nubestris expressing primarily hemorrhagic toxins, and a second B. nigroviridis exhibiting a mixed expression phenotype. Weighted gene coexpression analyses identified six submodules of transcript expression variation, one of which was highly associated with SVMPs and a second which contained both subunits of the neurotoxic PLA2 complex. The sub-module association of these toxins suggest common regulatory pathways underlie the variation in their expression and is consistent with known patterns of inheritance of similar haplotypes in other species. We also find evidence that module associated toxin families show fewer gene duplications and transcript losses between species, but module association did not appear to affect sequence diversification. CONCLUSION: Sub-modular regulation of expression likely contributes to the diversification of venom phenotypes within and among species and underscores the role of modularity in facilitating rapid evolution of complex traits.

Intraspecific sequence and gene expression variation contribute little to venom diversity in sidewinder rattlesnakes ( Crotalus cerastes).

Traits can evolve rapidly through changes in gene expression or protein-coding sequences. However, these forms of genetic variation can be correlated and changes to one can influence the other. As a result, we might expect traits lacking differential expression to preferentially evolve through changes in protein sequences or morphological adaptation. Given the lack of differential expression across the distribution of sidewinder rattlesnakes ( Crotalus cerastes), we tested this hypothesis by comparing the coding regions of genes expressed in the venom gland transcriptomes and fang morphology. We calculated Tajima's D and FST across four populations comparing toxin and nontoxin loci. Overall, we found little evidence of directional selection or differentiation between populations, suggesting that changes to protein sequences do not underlie the evolution of sidewinder venom or that toxins are under extremely variant selection pressures. Although low-expression toxins do not have higher sequence divergence between populations, they do have more standing variation on which selection can act. Additionally, we found significant differences in fang length among populations. The lack of differential expression and sequence divergence suggests sidewinders-given their generalist diet, moderate gene flow and environmental variation-are under stabilizing selection which functions to maintain a generalist phenotype. Overall, we demonstrate the importance of examining the relationship between gene expression and protein-coding changes to understand the evolution of complex traits.

Venom characterization of the three species of Ophryacus and proteomic profiling of O. sphenophrys unveils Sphenotoxin, a novel Crotoxin-like heterodimeric ß-neurotoxin.

Venoms of the three species of Ophryacus (O. sphenophrys, O. smaragdinus, and O. undulatus), a viperid genus endemic to Mexico, were analyzed for the first time in the present work. The three venoms lacked procoagulant activity on human plasma, but induced hemorrhage and were highly lethal to mice. These venoms also displayed proteolytic and phospholipase A2 activities in vitro. The venom of O. sphenophrys was the most lethal and caused hind-limb paralysis in mice. Proteomic profiling of O. sphenophrys venom showed a predominance of metalloproteinase (34.9%), phospholipase A2 (24.8%) and serine protease (17.1%) in its composition. Strikingly, within its PLA2 components, 12.9% corresponded to a Crotoxin-like heterodimer, here named Sphenotoxin, which was not found in the other two species of Ophryacus. Sphenotoxin, like Crotoxin, is composed of non-covalently bound A and B subunits. Partial amino acid sequence was obtained for Sphenotoxin B and was similar (78-89%) to other subunits described. The mouse i.v. LD50 of Sphenotoxin at 1:1 M radio was 0.16 µg/g. Also, like Crotoxin, Sphenotoxin induced a potent neuromuscular blockade in the phrenic nerve-diaphragm preparation. Ophryacus is the fifth genus and O. sphenophrys the third non-rattlesnake species shown to contain a novel Crotoxin-like heterodimeric ß-neurotoxin. BIOLOGICAL SIGNIFICANCE: Ophryacus is an endemic genus of semi-arboreal pitvipers from Mexico that includes three species with restricted distributions. Little is known about the natural history of these species and nothing is known about the properties of their venoms. Research on these species' venoms could generate relevant information regarding venom composition of Mexican pitvipers. Additionally, research into the presence of neurotoxic Crotoxin-like molecules outside of rattlesnakes (genera Crotalus and Sistrurus) has identified this molecule in several new genera. Knowing which genera and species possess neurotoxic components is important to fully understand the repercussions of snakebites, the interaction with prey and predators, and the origin, evolution, and phylogenetic distribution of Crotoxin-like molecules during the evolutionary history of pitvipers. Our study expands current knowledge regarding venom's compositions and function from Mexican pitvipers, providing a comparative venom characterization of major activities in the three Ophryacus species. Additionally, the discovery and characterization of a novel Crotoxin-like molecule, here named Sphenotoxin, in O. sphenophrys, and the detailed protein composition of O. sphenophrys venom supports the hypotheses that Crotoxin-like -ß-neurotoxins are more widespread than initially thought.

Ontogenetic Change in the Venom of Mexican Black-Tailed Rattlesnakes (Crotalus molossus nigrescens).

Ontogenetic changes in venom composition have important ecological implications due the relevance of venom in prey acquisition and defense. Additionally, intraspecific venom variation has direct medical consequences for the treatment of snakebite. However, ontogenetic changes are not well documented in most species. The Mexican Black-tailed Rattlesnake (Crotalus molossus nigrescens) is large-bodied and broadly distributed in Mexico. To document venom variation and test for ontogenetic changes in venom composition, we obtained venom samples from twenty-seven C. m. nigrescens with different total body lengths (TBL) from eight states in Mexico. The primary components in the venom were detected by reverse-phase HPLC, western blot, and mass spectrometry. In addition, we evaluated the biochemical (proteolytic, coagulant and fibrinogenolytic activities) and biological (LD50 and hemorrhagic activity) activities of the venoms. Finally, we tested for recognition and neutralization of Mexican antivenoms against venoms of juvenile and adult snakes. We detected clear ontogenetic venom variation in C. m. nigrescens. Venoms from younger snakes contained more crotamine-like myotoxins and snake venom serine proteinases than venoms from older snakes; however, an increase of snake venom metalloproteinases was detected in venoms of larger snakes. Venoms from juvenile snakes were, in general, more toxic and procoagulant than venoms from adults; however, adult venoms were more proteolytic. Most of the venoms analyzed were hemorrhagic. Importantly, Mexican antivenoms had difficulties recognizing low molecular mass proteins (<12 kDa) of venoms from both juvenile and adult snakes. The antivenoms did not neutralize the crotamine effect caused by the venom of juveniles. Thus, we suggest that Mexican antivenoms would have difficulty neutralizing some human envenomations and, therefore, it may be necessary improve the immunization mixture in Mexican antivenoms to account for low molecular mass proteins, like myotoxins.