Severe Systemic Envenomation Following Vipera berus Bite Managed with ViperaTAb Antivenom.

Bites by the European adder (Vipera berus) are a rare medical emergency in the UK with 20 to 50% of an estimated 50 to 200 cases per year necessitating treatment with antivenom. We present a case demonstrating both severe systemic and local effects necessitating out of hospital resuscitation, vasopressor support, and prolonged rehabilitation. Hemodynamic stability was restored promptly after administration of ViperaTAb antivenom, the first published case of its use in the UK.

Thrombotic Microangiopathy, Hemolytic Uremic Syndrome, and Thrombotic Thrombocytopenic Purpura Following Hump-nosed Pit Viper (Genus: Hypnale) Envenoming in Sri Lanka.

Thrombotic microangiopathy (TMA), which includes the spectrum of hemolytic uremic syndrome and thrombotic thrombocytopenic purpura, is an uncommon complication of hump-nosed pit viper envenomation. We describe 4 cases of TMA following hump-nosed pit viper (Hypnale spp) bites in Sri Lanka. The first case is a typical TMA that spontaneously resolved with supportive treatments. The second and third cases are related to hemolytic uremic syndrome complicated with acute kidney injury that required hemodialysis. The fourth case is thrombotic thrombocytopenic purpura associated with acute kidney injury that required hemodialysis and therapeutic plasma exchange. For each patient we describe the circumstances of the bite, clinical features, laboratory findings, and management.

Isolation and characterization of an anti-leishmanial disintegrin from Cerastes cerastes venom.

Investigating new antimicrobial and antiparasitic components from Viperidae venoms represents an alternative therapeutic strategy. In this study, we report the characterization of a disintegrin isolated from Cerastes cerastes venom, exhibiting antiparasitic activity on Leishmania infantum promastigotes. Indeed, isolated disintegrin, referred to Disintegrin_Cc, induced 84.75% of parasiticidal activity and deep morphological alterations on the parasites. SDS-PAGE analysis indicated that this disintegrin was homogenous. This dimeric disintegrin of 14,193.97 Da contains an RGD domain and four intramolecular disulfide bridges. It presents a high percentage of identity with other related snake disintegrins. Predicted 3D structure indicated that this peptide shares partial homology with well-known active antimicrobial peptides. Disintegrin_Cc inhibited 80% of arachidonic acid-induced platelet aggregation. The obtained results suggest that the isolated molecule plays a dual role as a disintegrin and as an anti-leishmanial compound. This component could be useful as a drug in the treatment of leishmaniasis.

Arboreality constrains morphological evolution but not species diversification in vipers.

An increase in ecological opportunities, either through changes in the environment or acquisition of new traits, is frequently associated with an increase in species and morphological diversification. However, it is possible that certain ecological settings might prevent lineages from diversifying. Arboreality evolved multiple times in vipers, making them ideal organisms for exploring how potentially new ecological opportunities affect their morphology and speciation regimes. Arboreal snakes are frequently suggested to have a very specialized morphology, and being too large, too small, too heavy, or having short tails might be challenging for them. Using trait-evolution models, we show that arboreal vipers are evolving towards intermediate body sizes, with longer tails and more slender bodies than terrestrial vipers. Arboreality strongly constrains body size and circumference evolution in vipers, while terrestrial lineages are evolving towards a broader range of morphological variants. Trait-dependent diversification models, however, suggest similar speciation rates between microhabitats. Thus, we show that arboreality might constrain morphological evolution but not necessarily affect the rates at which lineages generate new species.

Oxidative stress in a capital breeder (Vipera aspis) facing pregnancy and water constraints.

The physiological mechanisms underlying the 'cost of reproduction' remain under debate, though oxidative stress has emerged as a potential candidate. The 'oxidative cost of reproduction' has received considerable attention with regards to food and antioxidant availability; however, the limitation of water availability has thus far been neglected. In this study, we experimentally examined the combined effect of pregnancy and water deprivation on oxidative status in a viviparous snake (Vipera aspis), a species naturally exposed to periods of water and food deprivation. We predicted a cumulative effect of pregnancy and dehydration on oxidative stress levels. Our results support the occurrence of an oxidative cost of reproduction as we found higher oxidative damage levels in pregnant females than in non-reproductive individuals, despite an up-regulation of antioxidant defences. Surprisingly, water deprivation was associated with an up-regulation of antioxidant defences, and did not increase oxidative damage, either alone or in combination with reproduction.

Water deprivation increases maternal corticosterone levels and enhances offspring growth in the snake Vipera aspis.

Circulating glucocorticoid (GC) levels may increase as a result of reproductive effort or in response to unpredictable events. However, GC secretion can vary with the availability of vital trophic resources such as energy. While water represents another critical resource, the impact of water deprivation on GC secretion during reproduction has not yet been thoroughly investigated. Here, we examined the effects of water deprivation on plasma corticosterone (CORT) concentrations of female aspic vipers (Vipera aspis), and determined the impacts of water deprivation on offspring traits. We exposed both pregnant and non-reproductive females to a 20-day water deprivation and compared their pre- and post-deprivation CORT levels with those of control females. At the end of the treatment, only water-deprived pregnant females showed a significant increase in CORT levels. In pregnant females, changes in baseline CORT level were correlated with changes in female hydration state. Changes in baseline CORT levels were also negatively influenced by maternal reproductive effort in pregnant control females, while such a relationship was not apparent in pregnant water-deprived females. Finally, we found that offspring from water-deprived females had higher growth rates than offspring from control females. Offspring growth was also positively correlated with changes in both maternal osmolality and baseline CORT levels. Together, our results suggest that dehydration increases maternal CORT levels, which may subsequently influence offspring development. Further long-term field studies are therefore required to assess whether there is an adaptive significance of this response.

Are glucocorticoids good indicators of pregnancy constraints in a capital breeder?

Pregnancy imposes a substantial energetic demand on the mother (i.e. metabolic costs of pregnancy) and is often associated with modified maternal behavior and increased physical burdens that make females more vulnerable to predation. The Hypothalamic-Pituitary-Adrenal axis plays a fundamental role in reproduction through hormonal control of energy regulation and parental care. Therefore, evaluating the changes in baseline and stress-induced glucocorticoid levels in response to pregnancy may provide a robust tool to assess not only the constraints of gestation but also the way females may adjust to these constraints. In this study, we measured baseline and stress-induced corticosterone (CORT) concentrations in pregnant and non-pregnant aspic vipers (Vipera aspis), which are capital breeders. We also measured muscle condition (tail width) and locomotion performance (traction force) because these are robust proxies of protein mobilization associated with fasting. Baseline CORT concentration increased significantly over time in pregnant females, while they were lower and stable in non-reproductive females. Pregnant females had lower muscle condition at the onset of the study and tail width was negatively correlated with CORT concentration in this group. Contrary to our prediction, the stress response was not attenuated in pregnant females, but was proportional to baseline CORT concentration. Our results suggest that baseline CORT variations are closely related to energy constraints and structural protein mobilization in this capital breeder.

Effects of mild wintering conditions on body mass and corticosterone levels in a temperate reptile, the aspic viper (Vipera aspis).

Temperate ectotherms are expected to benefit from climate change (e.g., increased activity time), but the impacts of climate warming during the winter have mostly been overlooked. Milder winters are expected to decrease body condition upon emergence, and thus to affect crucial life-history traits, such as survival and reproduction. Mild winter temperature could also trigger a state of chronic physiological stress due to inadequate thermal conditions that preclude both dormancy and activity. We tested these hypotheses on a typical temperate ectothermic vertebrate, the aspic viper (Vipera aspis). We simulated different wintering conditions for three groups of aspic vipers (cold: ~6 °C, mild: ~14 °C and no wintering: ~24 °C) during a one month long period. We found that mild wintering conditions induced a marked decrease in body condition, and provoked an alteration of some hormonal mechanisms involved in emergence. Such effects are likely to bear ultimate consequences on reproduction, and thus population persistence. We emphasize that future studies should incorporate the critical, albeit neglected, winter season when assessing the potential impacts of global changes on ectotherms.

Comparative Studies of Structural and Functional Properties of Snake Venom Metalloproteinases.

Snake venom metalloproteinases (SVMPs) induces local and systemic effects on patients suffering from snakebite, degrading extracellular matrix (ECM) proteins such as collagen, gelatin, elastin, laminin, fibronectin, nidogen (entactin), and thrombospondin that cause local hemorrhage and tissue damage. They cleave or activate coagulation factors such as fibrinogen, fibrin, prothrombin, factor V, factor IX, factor X and protein C that bring about systemic coagulopathy. SVMPs and their truncated forms cleave or interfere with platelet adhesive proteins such as vWF, fibrinogen and collagen, and cleave or interfere with platelet receptors such as GPVI, alpha2beta1, GPIb, GPIX, and GPIIbIIIa that result in platelet aggregation defect. SVMPs induce cancer cell line to form morphological changes and apoptosis in vitro concordant with skin necrosis after snakebite in some cases. These local effects caused by SVMPs have no certain treatments, even with commercial anti-venom. SVMPs researches are focusing on their inhibitors, measurement and replacement of blood coagulation factor defects, or anti-cancer drug.

Diversifying selection and color-biased dispersal in the asp viper.

BACKGROUND: The presence of intraspecific color polymorphism can have multiple impacts on the ecology of a species; as a consequence, particular color morphs may be strongly selected for in a given habitat type. For example, the asp viper (Vipera aspis) shows a high level of color polymorphism. A blotched morph (cryptic) is common throughout its range (central and western Europe), while a melanistic morph is frequently found in montane populations, presumably for thermoregulatory reasons. Besides, rare atypical uniformly colored individuals are known here and there. Nevertheless, we found in a restricted treeless area of the French Alps, a population containing a high proportion (>50%) of such specimens. The aim of the study is to bring insight into the presence and function of this color morph by (i) studying the genetic structure of these populations using nine microsatellite markers, and testing for (ii) a potential local diversifying selection and (iii) differences in dispersal capacity between blotched and non-blotched vipers. RESULTS: Our genetic analyses support the occurrence of local diversifying selection for the non-blotched phenotype. In addition, we found significant color-biased dispersal, blotched individuals dispersing more than atypical individuals. CONCLUSION: We hypothesize that, in this population, the non-blotched phenotype possess an advantage over the typical one, a phenomenon possibly due to a better background matching ability in a more open habitat. In addition, color-biased dispersal might be partly associated with the observed local diversifying selection, as it can affect the genetic structure of populations, and hence the distribution of color morphs.

Embryonic water uptake during pregnancy is stage- and fecundity-dependent in the snake Vipera aspis.

Water is a crucial resource that can profoundly impact the biology of terrestrial organisms. Early life stages are particularly sensitive to hydric constraints because water uptake is an important component of embryonic development. While amniotic eggs constitute a key innovation to terrestrial life, many vertebrates are viviparous wherein the mother must be the source of water for her developing embryos. Since most viviparous squamates are lecithotrophic (i.e., energy is supplied to the offspring as yolk deposited into pre-ovulated follicles), water is the predominant resource allocated from the mother to the offspring during development. Contrary to energy that can be stored (e.g., as fat reserves), water typically cannot be acquired in advance. Therefore, the embryos' need for water can impose significant constraints on the pregnant female. We detailed water flux during pregnancy in a viviparous snake, the aspic viper (Vipera aspis). We found that embryonic water uptake occurred mostly during the second half of pregnancy-a period dominated by somatic growth. We also found that, somewhat unexpectedly, changes in female plasma osmolality were negatively related to fecundity. This latter result suggests that water consumption by the female is especially important for large litter sizes, and thus may suggest an important sensitivity of reproductive females to environmental water availability.

Thermoregulation and microhabitat choice in the polymorphic asp viper (Vipera aspis).

In ectotherms, thermoregulation strongly depends on environmental conditions, as well as on intrinsic factors, such as skin colour. Indeed, due to its physical properties, melanin pigments allow melanistic morphs to benefit of a more efficient thermoregulation compared to non-melanistic ones. Despite thermal benefits of melanism have often been highlighted under experimental conditions, such field data remain scarce. In this study, we investigated the influence of colouration on body temperature and microhabitat choice in a montane population of colour polymorphic asp viper (Vipera aspis) characterized by a strong presence of melanism (64%). Results highlighted a difference in internal body temperature, but only within gravid females, with melanistic individuals having higher body temperatures compared to non-melanistic ones. No differences were found when considering both sexes. We also showed that melanistic and non-melanistic vipers were found in different microhabitat types, i.e. melanistic snakes used areas marked by a scarcer sun exposure and by higher vegetation cover compared to non-melanistic ones. This result has important implications. Indeed, besides providing a possible explanation for the lack of difference in body temperature (except for gravid females), it confirms that melanistic individuals can potentially use their efficient thermoregulation in order to inhabit less exposed and thermally unfavourable microhabitats.

Demographic consequences of climate change and land cover help explain a history of extirpations and range contraction in a declining snake species.

Developing conservation strategies for threatened species increasingly requires understanding vulnerabilities to climate change, in terms of both demographic sensitivities to climatic and other environmental factors, and exposure to variability in those factors over time and space. We conducted a range-wide, spatially explicit climate change vulnerability assessment for Eastern Massasauga (Sistrurus catenatus), a declining endemic species in a region showing strong environmental change. Using active season and winter adult survival estimates derived from 17 data sets throughout the species' range, we identified demographic sensitivities to winter drought, maximum precipitation during the summer, and the proportion of the surrounding landscape dominated by agricultural and urban land cover. Each of these factors was negatively associated with active season adult survival rates in binomial generalized linear models. We then used these relationships to back-cast adult survival with dynamic climate variables from 1950 to 2008 using spatially explicit demographic models. Demographic models for 189 population locations predicted known extant and extirpated populations well (AUC = 0.75), and models based on climate and land cover variables were superior to models incorporating either of those effects independently. These results suggest that increasing frequencies and severities of extreme events, including drought and flooding, have been important drivers of the long-term spatiotemporal variation in a demographic rate. We provide evidence that this variation reflects nonadaptive sensitivity to climatic stressors, which are contributing to long-term demographic decline and range contraction for a species of high-conservation concern. Range-wide demographic modeling facilitated an understanding of spatial shifts in climatic suitability and exposure, allowing the identification of important climate refugia for a dispersal-limited species. Climate change vulnerability assessment provides a framework for linking demographic and distributional dynamics to environmental change, and can thereby provide unique information for conservation planning and management.

Influence of climate on the presence of colour polymorphism in two montane reptile species.

The coloration of ectotherms plays an important role in thermoregulation processes. Dark individuals should heat up faster and be able to reach a higher body temperature than light individuals and should therefore have benefits in cool areas. In central Europe, montane local populations of adder (Vipera berus) and asp viper (Vipera aspis) exhibit a varying proportion of melanistic individuals. We tested whether the presence of melanistic V. aspis and V. berus could be explained by climatic conditions. We measured the climatic niche position and breadth of monomorphic (including strictly patterned individuals) and polymorphic local populations, calculated their niche overlap and tested for niche equivalency and similarity. In accordance with expectations, niche overlap between polymorphic local populations of both species is high, and even higher than that of polymorphic versus monomorphic montane local populations of V. aspis, suggesting a predominant role of melanism in determining the niche of ectothermic vertebrates. However, unexpectedly, the niche of polymorphic local populations of both species is narrower than that of monomorphic ones, indicating that colour polymorphism does not always enable the exploitation of a greater variability of resources, at least at the intraspecific level. Overall, our results suggest that melanism might be present only when the thermoregulatory benefit is higher than the cost of predation.

Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography.

In order to perform their function, proteins frequently interact with other proteins. Various methods are used to reveal protein interacting partners, and affinity chromatography is one of them. Snake venom is composed mostly of proteins, and various protein complexes in the venom have been found to exhibit higher toxicity levels than respective components separately. Complexes can modulate envenomation activity of a venom and/or potentiate its effect. Our previous data indicate that the most toxic components of the Vipera ammodytes ammodytes (Vaa) venom isolated so far-ammodytoxins (Atxs)-are contributing to the venom's toxicity only moderately; therefore, we aimed to explore whether they have some interacting partner(s) potentiating toxicity. For screening of possible interactions, immuno-affinity chromatography combined with identification by mass spectrometry was used. Various chemistries (epoxy, carbonyldiimidazole, ethylenediamine) as well as protein G functionality were used to immobilize antibodies on monolith support, a Convective Interaction Media disk. Monoliths have been demonstrated to better suit the separation of large biomolecules. Using such approach, several proteins were indicated as potential Atx-binding proteins. Among these, the interaction of Atxs with a Kunitz-type inhibitor was confirmed by far-Western dot-blot and surface plasmon resonance measurement. It can be concluded that affinity chromatography on monolithic columns combined with mass spectrometry identification is a successful approach for screening of protein interactions and it resulted with detection of the interaction of Atx with Kunitz-type inhibitor in Vaa venom for the first time.

Stage dependence of phenotypical and phenological maternal effects: insight into squamate reptile reproductive strategies.

Enhanced thermal conditions have been credited as a driving force for the evolution of viviparity, particularly in squamate reptiles, among which it has independently evolved more than 100 times. However, maternal thermoregulation is also a critical component of reproduction in oviparous squamates, for which considerable embryonic development occurs prior to oviposition. When carrying eggs, oviparous mothers modify thermoregulation in a manner similar to that of pregnant females. To further understand the role of temperature in influencing reproductive strategies, it is critical that we elucidate the degree to which thermal sensitivity varies across developmental stages. We studied stage-dependent embryonic sensitivity in a viviparous snake, the aspic viper (Vipera aspis). We manipulated female body temperature at different stages of pregnancy-early development, early embryonic growth, and late embryonic growth-by imposing two contrasting daily thermal cycles that mimicked reproductive (warm) and nonreproductive (cool) female temperature profiles. Thermal sensitivity of offspring phenotype was stage dependent, with offspring quality more negatively affected when exposure to cool temperatures occurred early in development. In contrast, developmental rate was slowed by the cooler cycle, independent of the timing of the exposure. Given the more persistent effect on phenology, phenological effects likely provide a greater driving force for complete embryonic retention (i.e., viviparity).

Shelter availability, stress level and digestive performance in the aspic viper.

The lack of shelter can perturb behaviors, increase stress level and thus alter physiological performance (e.g. digestive, immune or reproductive functions). Although intuitive, such potential impacts of lack of shelter remain poorly documented. We manipulated shelter availability and environmental and physiological variables (i.e. access to a heat source, predator attack, feeding status) in a viviparous snake, and assessed sun-basking behavior, digestive performance (i.e. digestive transit time, crude estimate of assimilation, regurgitation rate) and plasma corticosterone levels (a proxy of stress level). Shelter deprivation provoked a strong increase in sun-basking behavior and thus elevated body temperature, even in unfed individuals for which energy savings would have been otherwise beneficial. The lack of heat was detrimental to digestive performance; simulated predator attacks worsened the situation and entailed a further deterioration of digestion. The combination of the lack of shelter with cool ambient temperatures markedly elevated basal corticosterone level and was associated with low digestive performance. This hormonal effect was absent when only one negative factor was involved, suggesting a threshold response. Overall, our results revealed important non-linear cascading impacts of shelter availability on stress-hormone levels, behaviors and physiological performance. These results infer that shelter availability is important for laboratory studies, captive husbandry and possibly conservation plans.

Impact of cool versus warm temperatures on gestation in the aspic viper (Vipera aspis).

Previous experimental data suggested that digestion and growth rates are not impaired under cool constant temperature (23°C) in a viviparous snake (Vipera aspis). These results challenged the widespread notion that both elevated temperatures (e.g. 30°C) and temperature fluctuations are required for digestion and growth in temperate climate reptiles. Here, we investigated the impact of constant cool temperatures on another physiological performance that is crucial to population persistence: gestation. At the time when reproductive females were midway through vitellogenesis, we placed ten reproductive and two non-reproductive female aspic vipers at each of two contrasted constant temperature conditions: cool (23°C) versus warm (28°C). Sixty percent of the females placed at 28°C gave birth to healthy offspring, suggesting that constant warm body temperatures were compatible with normal offspring production. Conversely, none of the cool females gave birth to healthy offspring. A blister disease affected exclusively cool pregnant females. Apparently, the combination of cool temperatures plus gestation was too challenging for such females. Our results suggest that reproduction is more thermally sensitive than digestion or growth, indeed gestation faltered under moderately cool thermal constraints. This sensitivity could be a crucial factor determining the capacity of this species to colonize different habitats.

Testing the morphological constraint hypothesis of tail length in the sexually dimorphic Cerastes vipera and new perspectives.

The morphological constraint hypothesis (MCH) states that, in snakes, males typically have relatively longer tails than females to accommodate the hemipenes and retractor muscles. To date, most studies testing the MCH have been interspecific and results have been equivocal. We tested the MCH intraspecifically on Cerastes vipera, a species with a relatively short tail and suitable for testing the MCH. The relative tail length and length of the hemipenes pocket in Cerastes vipera were measured in preserved museum-maintained males (n = 35) and in free-ranging males and females (n = 277). Males exhibited relatively longer tails than females, which was explained fully by the length of the hemipenes pocket. The relatively short tail of C. vipera presents a constraint to the reproductive structures in males, as the length of the hemipenes pocket occupies a greater proportion in shorter- than longer-tailed individuals. This is the first report presenting these intraspecific findings in support of the MCH. Whether these relations are widespread among snake families, within Viperidae, or specifically within C. vipera warrants further studies.

Molecular convergence of infrared vision in snakes.

It has been discovered that the transient receptor potential ankyrin 1 (TRPA1) proteins of Boidae (boas), Pythonidae (pythons), and Crotalinae (pit vipers) are used to detect infrared radiation, but the molecular mechanism for detecting the infrared radiation is unknown. Here, relating the amino acid substitutions in their TRPA1 proteins and the functional differentiations, we propose that three parallel amino acid changes (L330M, Q391H, and S434T) are responsible for the development of infrared vision in the three groups of snakes. Protein modeling shows that the three amino acid changes alter the structures of the central region of their ankyrin repeats.