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.

Augmented anandamide signalling in the substantia nigra pars reticulata mediates panicolytic-like effects in mice confronted by Crotalus durissus terrificus pit vipers.

RATIONALE: The endocannabinoid modulation of fear and anxiety due to the on-demand synthesis and degradation is supported by a large body of research. Although it has been proposed that anandamide (AEA) in the substantia nigra pars reticulata (SNpr) seems to be important for the organisation of innate fear-related behaviours, a role for endogenous AEA has yet to be clarified. METHODS: Mice were treated with the fatty acid amide hydrolase (FAAH) selective inhibitor URB597 at different concentrations (0.01, 0.1, 1 nmol/0.1 µL) in the SNpr and confronted by rattlesnakes (Crotalus durissus terrificus). The most effective dose of URB597 (1 nmol) was also preceded by microinjections of the CB1 receptor antagonist AM251 (0.1 nmol) into the SNpr, and mice were then confronted by the venomous snake. RESULTS: URB597 (0.1 and 1 nmol) in the SNpr decreased the expression of defensive behaviours such as defensive attention, escape, and time spent inside the burrow of mice confronted by rattlesnakes. Moreover, pretreatment of SNpr with AM251 suppressed these antiaversive effects of URB597 in this midbrain structure. CONCLUSION: Overall, these data clearly indicate that the panicolytic consequences of endogenous AEA enhancement in the SNpr are mediated by CB1 receptor signalling.

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.

Cytosolic phospholipase A2-α participates in lipid body formation and PGE2 release in human neutrophils stimulated with an L-amino acid oxidase from Calloselasma rhodostoma venom.

Cr-LAAO, an L-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, has been demonstrated as a potent stimulus for neutrophil activation and inflammatory mediator production. However, the mechanisms involved in Cr-LAAO induced neutrophil activation has not been well characterized. Here we investigated the mechanisms involved in Cr-LAAO-induced lipid body (also known as lipid droplet) biogenesis and eicosanoid formation in human neutrophils. Using microarray analysis, we show for the first time that Cr-LAAO plays a role in the up-regulation of the expression of genes involved in lipid signalling and metabolism. Those include different members of phospholipase A2, mostly cytosolic phospholipase A2-α (cPLA2-α); and enzymes involved in prostaglandin synthesis including cyclooxygenases 2 (COX-2), and prostaglandin E synthase (PTGES). In addition, genes involved in lipid droplet formation, including perilipin 2 and 3 (PLIN 2 and 3) and diacylglycerol acyltransferase 1 (DGAT1), were also upregulated. Furthermore, increased phosphorylation of cPLA2-α, lipid droplet biogenesis and PGE2 synthesis were observed in human neutrophils stimulated with Cr-LAAO. Treatment with cPLA2-α inhibitor (CAY10650) or DGAT-1 inhibitor (A922500) suppressed lipid droplets formation and PGE2 secretion. In conclusion, we demonstrate for the first time the effects of Cr-LAAO to regulate neutrophil lipid metabolism and signalling.

Novel Treatment Strategy for Patients with Venom-Induced Consumptive Coagulopathy from a Pit Viper Bite.

Pit viper venom commonly causes venom-induced consumptive coagulopathy (VICC), which can be complicated by life-threatening hemorrhage. VICC has a complex pathophysiology affecting multiple steps of the coagulation pathway. Early detection of VICC is challenging because conventional blood tests such as prothrombin time (PT) and activated partial thromboplastin time (aPTT) are unreliable for early-stage monitoring of VICC progress. As the effects on the coagulation cascade may differ, even in the same species, the traditional coagulation pathways cannot fully explain the mechanisms involved in VICC or may be too slow to have any clinical utility. Antivenom should be promptly administered to neutralize the lethal toxins, although its efficacy remains controversial. Transfusion, including fresh frozen plasma, cryoprecipitate, and specific clotting factors, has also been performed in patients with bleeding. The effectiveness of viscoelastic monitoring in the treatment of VICC remains poorly understood. The development of VICC can be clarified using thromboelastography (TEG), which shows the procoagulant and anticoagulant effects of snake venom. Therefore, we believe that TEG may be able to be used to guide hemostatic resuscitation in victims of VICC. Here, we aim to discuss the advantages of TEG by comparing it with traditional coagulation tests and propose potential treatment options for VICC.

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.

Atroxlysin-III, A Metalloproteinase from the Venom of the Peruvian Pit Viper Snake Bothrops atrox (Jergón) Induces Glycoprotein VI Shedding and Impairs Platelet Function.

Atroxlysin-III (Atr-III) was purified from the venom of Bothrops atrox. This 56-kDa protein bears N-linked glycoconjugates and is a P-III hemorrhagic metalloproteinase. Its cDNA-deduced amino acid sequence reveals a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domain. Its identity with bothropasin and jararhagin from Bothrops jararaca is 97% and 95%, respectively. Its enzymatic activity is metal ion-dependent. The divalent cations, Mg2+ and Ca2+, enhance its activity, whereas excess Zn2+ inhibits it. Chemical modification of the Zn2+-complexing histidine residues within the active site by using diethylpyrocarbonate (DEPC) inactivates it. Atr-III degrades plasma fibronectin, type I-collagen, and mainly the α-chains of fibrinogen and fibrin. The von Willebrand factor (vWF) A1-domain, which harbors the binding site for GPIb, is not hydrolyzed. Platelets interact with collagen via receptors for collagen, glycoprotein VI (GPVI), and α2ß1 integrin. Neither the α2ß1 integrin nor its collagen-binding A-domain is fragmented by Atr-III. In contrast, Atr-III cleaves glycoprotein VI (GPVI) into a soluble ~55-kDa fragment (sGPVI). Thereby, it inhibits aggregation of platelets which had been stimulated by convulxin, a GPVI agonist. Selectively, Atr-III targets GPVI antagonistically and thus contributes to the antithrombotic effect of envenomation by Bothrops atrox.

Molecular interactions between p-coumaric acid and snake venom toxins.

A large number of natural compounds, such as phenolic compounds, have been scientifically evaluated in the search for enzyme inhibitors. The interactions between the phenolic compound p-coumaric acid and the enzymes present in snake venoms (used as research tools) were evaluated in vitro and in silico. The p-coumaric acid was able to inhibit 31% of the phospholipase activity induced by Bothrops alternatus venom, 27% of the hemolytic activity induced by B. moojeni, 62.5% of the thrombolytic activity induced by B. jararacussu, and approximately 27% of the activity thrombosis induced by Crotalus durissus terrificus. Previous incubation of p-coumaric acid with the venoms of B. atrox and B. jararacussu increased the coagulation time by 2.18 and 2.16-fold, respectively. The activity of serine proteases in B. atrox and B. jararacussu venoms was reduced by 60% and 66.34%, respectively. Computational chemistry analyses suggests the specific binding of p-coumaric acid to the active site of proteases through hydrogen and hydrophobic interactions. The phenolic compound evaluated in this work has great potential in therapeutic use to both prevent and treat hemostatic alterations, because the venom proteins inhibited by the p-coumaric acid have high homology with human proteins that have a fundamental role in several pathologies.

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.

Venomics of Tropidolaemus wagleri, the sexually dimorphic temple pit viper: Unveiling a deeply conserved atypical toxin arsenal.

Tropidolaemus wagleri (temple pit viper) is a medically important snake in Southeast Asia. It displays distinct sexual dimorphism and prey specificity, however its venomics and inter-sex venom variation have not been thoroughly investigated. Applying reverse-phase HPLC, we demonstrated that the venom profiles were not significantly affected by sex and geographical locality (Peninsular Malaya, insular Penang, insular Sumatra) of the snakes. Essentially, venoms of both sexes share comparable intravenous median lethal dose (LD50) (0.56-0.63 µg/g) and cause neurotoxic envenomation in mice. LCMS/MS identified six waglerin forms as the predominant lethal principles, comprising 38.2% of total venom proteins. Fourteen other toxin-protein families identified include phospholipase A2, serine proteinase, snaclec and metalloproteinase. In mice, HPLC fractions containing these proteins showed insignificant contribution to the overall venom lethality. Besides, the unique elution pattern of approximately 34.5% of non-lethal, low molecular mass proteins (3-5 kDa) on HPLC could be potential biomarker for this primitive crotalid species. Together, the study unveiled the venom proteome of T. wagleri that is atypical among many pit vipers as it comprises abundant neurotoxic peptides (waglerins) but little hemotoxic proteinases. The findings also revealed that the venom is relatively well conserved intraspecifically despite the drastic morphological differences between sexes.