Viper Species Research Articles

Comparative characterization of Viperidae snake venoms from Perú reveals two compositional patterns of phospholipase A2 expression.

Snake species within the Bothrops complex (sensu lato) are of medical relevance in Latin America, but knowledge on their venom characteristics is limited, or even unavailable, for some taxa. Perú harbors 17 species of pit vipers, within the genera Bothrops, Bothriechis, Bothrocophias, Porthidium, Crotalus, and Lachesis. This study compared the venoms of twelve species, through chromatographic and electrophoretic profiles, as well as proteolytic and phospholipase A2 (PLA2) activities. Also, proteomic profiles were analyzed for nine of the venoms using a shotgun approach. Results unveiled conspicuous differences in the expression of venom PLA2s among species, six of them presenting scarce levels as judged by RP-HPLC profiles. Since most species within the bothropoid lineage possess venoms with high to intermediate abundances of this protein family, our findings suggest the existence of a phenotypic duality in the expression of venom PLA2s within the Bothrops (sensu lato) complex. Bothrops barnetti and Bothrocophias andianus venoms, very scarce in PLA2s, were shown to lack significant myotoxic activity, highlighting that the observed variability in PLA2 expression bears toxicological correlations with effects attributed to these proteins. Finally, an attempt to identify phylogenetic relationships of bothropoid species from Perú presenting low- or high-PLA2 venom phenotypes showed an interspersed pattern, thus precluding a simple phylogenetic interpretation of this venom compositional dichotomy.

PLA2 Inhibitor Varespladib as an Alternative to the Antivenom Treatment for Bites from Nikolsky's Viper Vipera berus nikolskii.

Although envenoming by a small East European species of viper is rarely severe, and only exceptionally fatal, lack of specific antivenom stocks in a few areas within this region and possible severe side effects of antivenom application leave most bites to be treated only with antihistamines and supportive therapy. Varespladib is an effective inhibitor of snake phospholipase, and, as such, it could be considered as first-line therapy. The Nikolsky’s viper venom contains an extremely high concentration of phospholipase A2 (PLA2), responsible for the toxic effects of the venom, as well as minor amounts of other toxins. If Varespladib can successfully inhibit PLA2 activity, the Nikolsky’s viper could be one of the first venomous snakes having an antitoxin-specific treatment regimen. To assess that, Varespladib was administered alone subcutaneously to adult male CD-1 mice (8 mg/kg) and compared to mice exposed to Vipera berus nikolskii crude venom (8 mg/kg = 10 LD50) or a combination of Varespladib and the same amount of the venom. Experimental animals were monitored for the presence of envenoming symptoms and mortality for 48 h after injection. Eighty percent of mice receiving both Varespladib and venom survived, while 100% of the control group receiving venom alone died within 4 h. Experimental results are consistent with Varespladib acting as an effective antitoxin in the mouse model against Nikolsky’s viper venom. Further studies are needed under experimental conditions that more closely resemble natural envenoming (i.e., delayed administration).

Intercontinental test of constraint-breaking adaptations: Testing behavioural plasticity in the face of a predator with novel hunting strategies.

Constraint-breaking adaptations are evolutionary tools that provide a mechanism for incumbent-replacement between species filling similar ecological roles. In common-garden experiments, we exposed populations of two desert rodents to two different viper species, testing their ability to adjust to novel predators that use different hunting strategies. We aimed to understand whether both predators and prey with constraint-breaking adaptations actually manifest comparative advantage over their counterparts. We used convergent species from desert dunes in the Mojave Desert in North America, Merriam's kangaroo rat Dipodomys merriami and the sidewinder rattlesnake Crotalus cerastes, and from the Negev Desert in the Middle East, the greater Egyptian gerbil Gerbillus pyramidum and the Saharan horned viper Cerastes cerastes. Both Mojave species hold constraint-breaking adaptations in relation to their counterparts from the Negev. The rattlesnakes have heat sensing organs (pits) and the kangaroo rats have fur-lined cheek pouches that allow for greater foraging efficiency and food preservation. Using patch-usetheory, we evaluatedthe rodents' risk-assessment from each snake-separately, together and in combination with barn owls. Initially each rodent species foraged less in the presence of its familiar snake, but within a month both foraged less in the presence of the pit-viper (sidewinder). Our findings indicate a level of learning, and behavioural plasticity, in both rodents and ability to assess the risk from novel predators. The kangaroo rats were capable of harvesting far greater amounts of resources under the same conditions of elevated risk. However, the reason for their advantage may lie in bi-pedal agility and not only their ability collect food more efficiently.

A new species of green pit vipers of the genus Trimeresurus Lacépède, 1804 (Reptilia, Serpentes, Viperidae) from western Arunachal Pradesh, India

A new species of green pit vipers of the genus Trimeresurus Lacépède, 1804 is described from the lowlands of western Arunachal Pradesh state of India. The new species, Trimeresurus salazar, is a member of the subgenus Trimeresurus, a relationship deduced contingent on two mitochondrial genes, 16S and ND4, and recovered as sister to Trimeresurus septentrionalis Kramer, 1977. The new species differs from the latter in bearing an orange to reddish stripe running from the lower border of the eye to the posterior part of the head in males, higher number of pterygoid and dentary teeth, and a short, bilobed hemipenis. Description of the new species and T. arunachalensis Captain, Deepak, Pandit, Bhatt & Athreya, 2019 from northeastern India in a span of less than one year highlights the need for dedicated surveys to document biodiversity across northeastern India.

Evaluating taxonomic inflation: towards evidence-based species delimitation in Eurasian vipers (Serpentes: Viperinae)

Abstract The designation of taxonomic units has important implications for the understanding and conservation of biodiversity. Eurasian vipers are a monophyletic group of viperid snakes (Serpentes, Viperinae), currently comprising four genera (Daboia, Macrovipera, Montivipera and Vipera) and up to 40 species. Taxonomic units have been described using a wide variety of methods and criteria, and consequently, considerable controversy still surrounds the validity of some currently listed species. In order to promote a consensus- and evidence-based taxonomy of Eurasian vipers, we analysed published mitochondrial and nuclear DNA sequences for this group to reconstruct phylogenetic relationships among currently recognized viper species. We also compiled information on external morphology to assess their morphological distinctiveness. Phylogenetic inference based on mtDNA sequences shows contrasting levels of divergence across genera and species and identifies several instances of non-monophyly in described species. Nuclear DNA sequences show extremely low levels of genetic variation, with a widespread pattern of allele sharing among distant species, and even among genera. Revision of morphological data shows that most species designations rely on scalation traits that overlap extensively among species of the same genus. Based on our combined assessment, we recognize 15 taxa as valid species, three taxa which likely represent species complexes, 17 taxa of doubtful validity as species, and five taxa for which species status is maintained but further research is highly recommended to assess taxonomic arrangements. We stress the need to implement integrative taxonomic approaches for the recognition of evidence-based taxonomic units in Eurasian vipers.

Embryonic development of skull bones in the Sahara horned viper (Cerastes cerastes), with new insights into structures related to the basicranium and braincase roof.

Ontogenetic studies are crucial for understanding functional morphology, origin and adaptation of skulls in vertebrates. However, very few studies have so far released complete embryonic series focusing on skull embryonic development in species showing diverse and extreme cranial morphologies such as snakes. The wide distribution and unique reproductive and ecological behaviors of venomous vipers, including the heterogeneity in breeding and egg incubation periods in oviparous species, make this group an excellent new model for studying the diversity of skull developmental processes in snakes. Here we present the first complete description of osteocranium development in a viperine snake, Cerastes cerastes, using detailed analysis of the ossification pattern of individual bones across different embryonic stages based on high-resolution micro-computed tomography data. Particularly, we describe in detail the development of the laterosphenoid from its dorsal and ventral components, dividing the trigeminal foramen into maxillary and mandibular foramina. Furthermore, our data help clarify some controversy concerning the presence and/or origin of structures related to the snake basicranium and braincase roof. For example, our detailed description of supraoccipital development suggests that this bone derived, at least in part, from the tectum posterius, although the involvement of the tectum synoticum cannot be totally excluded. Similarly, the epiotic centers of supraoccipital ossification are confirmed during braincase development, and the ancestral lacrimal bone primordium is observed as a ventral element at the early stages of prefrontal development. Finally, our embryonic C. cerastes data highlight a plausible asymmetry in snake skull development, mostly occurring in the basicranium region, but further investigations of embryonic samples and viper species would be required to confirm such phenomenon.

Are the habitat niches of female green pit vipers Cryptelytrops macrops and Viridovipera vogeli partitioned by vertical stratification?

We used telemetry to track the movements of the females of two sympatric green pit viper species, Cryptelytrops macrops and Viridovipera vogeli, in north-eastern Thailand. This has generated information on their home ranges, lengths of displacements, and vertical niche partitioning. Five female vipers were tracked simultaneously within the same general habitat for 78 days in the cold, dry season for a mean of 113.6 ± 5.38 (range: 101 - 129) fixes. The data show that V. vogeli had larger home ranges and greater displacements than C. macrops (0.49 ± 0.02 and 0.26 ± 0.07 ha, 38.01 ± 9.72 and 25.12 ± 2.40 m; respectively). Interestingly, V. vogeli selected arboreal sites significantly more frequently (mean 53.0 ± 14.4 % of observations, range 38.6 - 67.5) than sympatric C. macrops (mean 23.4 ± 13.2 %, range 5.3 - 49.2). We cautiously suggest that vertical stratification of these two sympatric vipers may be one factor facilitating the co-existence of otherwise morphologically similar species.

Clinical implications of coagulotoxic variations in Mamushi (Viperidae: Gloydius) snake venoms

Snake bite is currently one of the most neglected tropical diseases affecting much of the developing world. Asian pit vipers are responsible for a considerable amount of envenomations annually and bites can cause a multitude of clinical complications resulting from coagulopathic and neuropathic effects. While intense research has been undertaken for some species of Asian pit viper, functional coagulopathic effects have been neglected for others. We investigated their effects upon the human clotting cascade using venoms of four species of Gloydius and Ovophis okinavensis, a species closely to Gloydius. All species of included within this investigation displayed varying fibrinogenolytic effects, resulting in a net anticoagulant outcome. Gloydius saxatilis and Gloydius ussuriensis displayed the most variable effects from differing localities, sampled from Russia and Korea. As this Gloydius investigation includes some geographical variation, notable results indicate key variations of these species that point to possible limitations in antivenom cross-reactivities, which may have implications for the clinical care of victims envenomed by these snakes.

Carbon monoxide inhibits the anticoagulant activity of Mojave rattlesnake venoms type A and B.

The Mojave rattlesnake is a unique species of pit viper that expresses either a highly potent phospholipase A2 (PLA2)-dependent neurotoxin containing venom nearly devoid of fibrinogenolytic metalloproteinases (venom type A) or a hemotoxic venom with a high percentage of metalloproteinases and PLA2 without any neurotoxin present (venom type B) depending on its geographical location in the Southwestern United States and Mexico. Given that PLA2 have been demonstrated to affect coagulation, it was hypothesized that the anticoagulant effects of both type A and B venoms could be assessed by thrombelastography, and determination made if these venoms were heme modulated. Both venom types were exposed to carbon monoxide releasing molecule-2 or its inactivated molecule (0 or 100µM) in isolation and then placed in human plasma with consequent coagulation kinetics assessed by thrombelastography. It was determined that type A venom was twice as potent as an anticoagulant compared to type B venom, and that both venoms were inhibited by carbon monoxide releasing molecule-2 but not its inactivated molecule. Given the lack of proteolytic activity of type A venom and the dependence of neurotoxicity on PLA2 activity, it may be possible that carbon monoxide could inhibit neurotoxicity based on inhibition of PLA2 anticoagulant activity. These data may serve as the rationale for extension of these observations into animal models to determine if CO may inhibit not just hemotoxic venom, but also PLA2-dependent neurotoxic venom.

Comparative analysis of the high molecular mass subproteomes of eight Bothrops snake venoms

Snake venoms are extremely active biological secretions composed primarily of various classes of enzymes. The genus Bothrops comprises various pit viper species that represent the most medically significant taxa in Central and South America, accounting for more human envenomations and fatalities than any other snakes in the region. Venom proteomes of many Bothrops species have been well-characterized but investigations have focused almost exclusively on proteins smaller than 100 kDa despite expression of larger components being documented in several Bothrops venoms. This study sought to achieve detailed identification of major components in the high molecular mass subproteome of venoms from eight Bothrops species (B. brazili, B. cotiara, B. insularis, B. jararaca, B. jararacussu, B. leucurus, B. moojeni and B. neuwiedi). Enzymes such as metalloproteinases and L-amino acid oxidases were the most prominent components identified in the first size-exclusion chromatography fractions of these venoms. Minor components also identified in the first peaks included 5′-nucleotidase, aminopeptidase, phosphodiesterase, and phospholipases A2 and B. Most of these components disappeared in electrophoretic profiles under reducing conditions, suggesting that they may be composed of more than one polypeptide chain. A significant shift in the molecular masses of these protein bands was observed following enzymatic N-deglycosylation, indicating that they may contain N-glycans. Furthermore, none of the identified high molecular mass proteins were shared by all eight species, revealing a high level of interspecific variability among these venom components.

The complete mitochondrial genome of Echis coloratus (painted carpet viper)

The painted carpet viper (Echis coloratus) is a venomous viper species endemic to the Middle East and Egypt, occurs in rocky deserts, from sea level to altitudes as high as 2500 m (8200 ft). Herein, we describe the first snake assembly mtDNA in Echis genera, which contains 13 protein-coding genes (PCGs), two rRNA genes (12S rRNA and 16S rRNA), 22 transfer RNA (tRNA) genes, and one control region (D-loop). The complete mitochondrial genome sequence provided here would be useful for further understanding the evolution of Echis and conservation genetics of E. coloratus.

Molecular and morphological analyses have revealed a new species of blunt-nosed viper of the genus Macrovipera in Iran

Molecular and morphological analyses have revealed a new species of blunt-nosed viper of the genus Macrovipera in Iran

Phylogenetic patterns in learning and decision making in pit vipers (Viperidae: Crotalinae)

Learning is a key behavioural adaptation allowing animals to respond to complex and changing environments. Although the field of animal behaviour has seen an increase in the taxonomic breadth of learning studies in recent decades, investigations within an explicit, broad phylogenetic framework are rare, curtailing our understanding of the evolution of learning and advanced cognition. Pit vipers (Viperidae: Crotalinae) represent a particularly interesting taxon in which to study patterns in learning as they are widely studied, have had learning documented in many species, occupy a range of ecological niches and possess vast and varied natural history traits. We investigated the latency to decision in a laboratory thermal maze in 13 species of pit viper (7 species of rattlesnakes and 6 species of nonrattlesnake pit viper) and one true viper species (the puff adder, Bitis arietans (Viperidae: Viperinae), as an outgroup comparison). Relative to the other pit vipers, rattlesnakes uniformly and quickly (after only one trial) decreased their latency to respond to thermal stress; their latency remained low and uniform across the remaining 11 trials. All other pit viper species failed to modify their behaviour across trials and maintained a consistent latency across all 12 trials, as did the puff adder. This pattern could reflect differential learning abilities between rattlesnake and nonrattlesnake pit vipers. Our results underscore the advantages of incorporating a broad phylogenetic perspective when investigating learning and comparative cognition.

Venomous Snakebites in Canada: A National Review of Patient Epidemiology and Antivenom Usage

There are 3 pit viper species in Canada. Limited Canadian literature exists on the epidemiology of venomous snakebites and the treatment patterns with antivenom. This study described the epidemiology, the utilization of antivenom, and estimated expenditures due to forfeited antivenom for pit viper envenomations in Canada. A retrospective review of the Health Canada Special Access Program records to generate descriptive statistics. Data are presented as mean±SD (range), as appropriate. The geographic distribution of Canadian pit viper species is presented. There were 99 envenomations reported in Canada from January 2009 to December 2015. The number of envenomations per year was 14±6 (6-21). CroFab and Antivipmyn are used in Canada to treat envenomations. The number of vials for patient treatment was 17±12 (3-66) and 16±9 (6-42) for CroFab and Antivipmyn, respectively. Antivenom stock usage for patient treatment varied across the country with provincial means reported for British Columbia (33%), Alberta (37%), Saskatchewan (27%), and Ontario (71%). The costs incurred secondary to forfeited stock where estimated as: $1,280,000 USD in British Columbia, $255,000 in Alberta, $60,000 in Saskatchewan, and $0 in Ontario. The absolute number of annual envenomations is small and the 3 Crotalinae species are limited to relatively narrow geographic areas in British Columbia, Alberta, Saskatchewan, and Ontario. The utilization of antivenom in the treatment of patients revealed that regions where the western and prairie rattlesnake reside forfeited a substantial amount of antivenom from 2009 to 2015. Organizations responsible for maintaining antivenom supplies on a provincial or regional level could use these data to guide antidote stocking and reduce costs.

Structural basis of phospholipase A2-like myotoxin inhibition by chicoric acid, a novel potent inhibitor of ophidian toxins

BackgroundSpecific compounds found in vegetal species have been demonstrated to be efficient inhibitors of snake toxins, such as phospholipase A2-like (PLA2-like) proteins. These particular proteins, present in several species of vipers (Viperidae), induce a severe local myotoxic effect in prey and human victims, and this effect is often not efficiently neutralized by the regular serum therapy. PLA2-like proteins have been functionally and structurally studied since the early 1990s; however, a comprehensive molecular mechanism was proposed only recently. MethodsMyographic and histological techniques were used to evaluate the inhibitory effect of chicoric acid (CA) against BthTX-I myotoxin. Isothermal titration calorimetry assays were used to measure the affinity between the inhibitor and the toxin. X-ray crystallography was used to reveal details of this interaction. ResultsCA prevented the blockade of indirectly evoked muscle contraction and inhibited muscle damage induced by BthTX-I. The inhibitor binds to the toxin with the highest affinity measured for a natural compound in calorimetric assays. The crystal structure and molecular dynamics simulations demonstrated that CA binds at the entrance of the hydrophobic channel of the toxin and binds to one of the clusters that participates in membrane disruption. ConclusionsCA prevents the myotoxic activity of the toxin, preventing its activation by simultaneous binding with two critical regions. General significanceCA is a potential myotoxic inhibitor to other PLA2-like proteins and a possible candidate to complement serum therapy.

The Role of Feeding Specialization on Post-Prandial Metabolic Rate in Snakes of the Genus Bothrops.

Feeding specialization is a recurrent issue in the evolution of snakes and is sometimes associated to morphological and/or behavioral adaptations that improve snake performance to exploit a particular food type. Despite its importance for animal fitness, the role of physiological traits has been much less studied than morphological and behavioral traits in the evolution of feeding specialization in snakes. In this context, the energetic cost of post-prandial period is an important physiological factor due to the remarkable effect on the snake energy budget. We collected data on post-prandial metabolic rate (SDA) in five species of pit vipers from the genus Bothrops with different degrees of mammal feeding specialization to test the hypothesis that feeding specialist species have lower energy costs during the digestion of their regular food item when compared to species with a more generalist diet. Our results support this hypothesis and suggest that ontogenetic changes in diet can be accompanied by changes in energy cost of the digestion process.

A novel pentameric phospholipase A2 myotoxin (PophPLA2) from the venom of the pit viper Porthidium ophryomegas

The first toxin isolated from the venomous pit viper Porthidium ophryomegas is a basic pentameric phospholipase A2 (PophPLA2). Elucidation of its amino acid sequence showed that it belongs to the group IIA of secreted PLA2s, with the presence of all 14 conserved cysteine positions. The toxin displayed catalytic activity, in agreement with the presence of Asp49 in its sequence of 121 residues. SDS-PAGE analysis revealed that this toxin is pentameric in non-reducing conditions, a structural organization that has not been described for any viperid PLA2. PophPLA2 displayed moderate myotoxic (in vivo) and cytotoxic (in vitro) activities, as well as anticoagulant activity on human plasma (in vitro). PophPLA2 was not lethal, and did not induce signs of toxicity or distress in mice, when administered intravenously at a dose of up to 100 μg (5.9 μg/g). The toxin showed highest sequence identity with other PLA2s from the venoms of ancestral Asian pit viper species.

Eastern Indigo Snakes Exhibit an Innate Response to Pit Viper Scent and an Ontogenetic Shift in Their Response to Mouse Scent

Abstract The ability to discriminate among potential prey is foundational for predators making foraging decisions, as well as for the evolution of food preferences. Eastern Indigo Snakes (Drymarchon couperi; EIS) are federally threatened and considered dietary generalists, but qualitative evidence indicates that snakes are consumed more often than other prey types. In this study, we used a repeated-measures tongue-flick assay to investigate EIS response to prey scent experimentally, and made inferences about potential preferences. Hatchling EIS responded more strongly to scents from snakes as compared to their response to scents from mice or controls. Moreover, hatchling EIS discriminated between the scents of representative colubrid and crotaline snakes, exhibiting a stronger response to the latter. A follow-up experiment using scents from three different species of pit vipers indicated that hatchling EIS did not discriminate among scents from members of this snake subfamily. After a year in captivity in...

Biochemical and pharmacological characterization of Trimersurus malabaricus snake venom.

Trimeresurus malabaricus is a venomous pit viper species endemic to southwestern part of India. In earlier reports, we have shown that envenomation by T. malabaricus venom leading to strong local tissue damage but the mechanism of action is not clearly revealed. Local tissue damage affected by T. malabaricus venom is of great importance since the poison has serious systemic effects including death in the case of multiple attacks. The present study details the major manifestations of T. malabaricus venom and the induction of local tissue damage, which suggests that most toxins are present in the form of hydrolytic enzymes. Hydrolytic activity of the enzymes was measured and the data indicated that protease and phospholipase A2 activity was high which is responsible for local tissue damage. Furthermore, the role of hydrolytic enzymes in the induction of pathological events such as hemorrhage, edema, myotoxicity, and blood coagulation examination were assessed through animal models.

SEROPREVALENCE AND MOLECULAR CHARACTERIZATION OF FERLAVIRUS IN CAPTIVE VIPERS OF COSTA RICA.

Ferlaviruses (FV, previously referred to as ophidian paramyxoviruses, OPMV), are enveloped viruses with a negative-strand RNA genome, affecting snakes in captivity worldwide. Infection is characterized by respiratory and nervous clinical signs and carries high mortality rates, but no specific treatment or vaccine is currently available. Costa Rica has 16 species of vipers, found in captivity in collections essential for antivenom production, reintroduction, and public education. FV circulation in these populations was previously unknown, and the risk of introducing the viruses into naïve collections or free-ranging populations exists if the virus's presence is confirmed. The objective of this study was to determine seroprevalence and FV shedding in 150 samples from captive vipers in nine collections across Costa Rica. A hemagglutination inhibition (HI) assay was performed to determine the antibody titer against two Ferlavirus strains, Bush viper virus (BV) and Neotropical virus (NT), and reverse-transcriptase polymerase chain reaction (RT-PCR) and sequencing to determine virus secretion in cloacal swabs. Ferlavirus strains were replicated in Vero cells, and chicken anti-FV polyclonal antibodies were produced and used as a positive control serum for the HI. Results demonstrate that seroprevalence of anti-FV antibodies in viper serum was 26.6% (n = 40) for the BV strain and 30% (n = 45) for the NT strain in the population tested. Furthermore, molecular characterization of FV group A was possible by sequencing the virus recovered from three cloacal swabs, demonstrating circulation of FV in one collection. This study demonstrates for the first time serological evidence of FV exposure and infection in vipers in captivity in Costa Rica, and suggests cross reactivity between antibodies against both strains. Appropriate biosafety measures could prevent the spread of FV between and within collections of reptiles in the country.