Paranannizziopsis spp. Infection in Wild Vipers, Europe.

We describe the detection of Paranannizziopsis sp. fungus in a wild population of vipers in Europe. Fungal infections were severe, and 1 animal likely died from infection. Surveillance efforts are needed to better understand the threat of this pathogen to snake conservation.

Phylogenomic insights into the diversity and evolution of Palearctic vipers.

Despite decades of molecular research, phylogenetic relationships in Palearctic vipers (genus Vipera) still essentially rely on a few loci, such as mitochondrial barcoding genes. Here we examined the diversity and evolution of Vipera with ddRAD-seq data from 33 representative species and subspecies. Phylogenomic analyses of âˆ¼ 1.1 Mb recovered nine major clades corresponding to known species/species complexes which are generally consistent with the mitochondrial phylogeny, albeit with a few deep discrepancies that highlight past hybridization events. The most spectacular case is the Italian-endemic V. walser, which is grouped with the alpine genetic diversity of V. berus in the nuclear tree despite carrying a divergent mitogenome related to the Caucasian V. kaznakovi complex. Clustering analyses of SNPs suggest potential admixture between diverged Iberian taxa (V. aspis zinnikeri and V. seoanei), and confirm that the Anatolian V. pontica corresponds to occasional hybrids between V. (ammodytes) meridionalis and V. kaznakovi. Finally, all analyzed lineages of the V. berus complex (including V. walser and V. barani) form vast areas of admixture and may be delimited as subspecies. Our study sets grounds for future taxonomic and phylogeographic surveys on Palearctic vipers, a group of prime interest for toxinological, ecological, biogeographic and conservation research.

Isolation and characterization of polymorphic microsatellite loci for the three Iberian vipers, Vipera aspis, V. Latastei and V. seoanei by Illumina MiSeq sequencing.

BACKGROUND: European vipers (genus Vipera) are a well-studied taxonomic group, but the low resolution of nuclear sanger-sequenced regions has precluded thorough studies at systematic, ecological, evolutionary and conservation levels. In this study, we developed novel microsatellite markers for the three Iberian vipers, Vipera aspis, V. latastei and V. seoanei, and assessed their polymorphism in north-central Iberian populations. METHODS AND RESULTS: Genomic libraries were developed for each species using an Illumina Miseq sequencing approach. From the 70 primer pairs initially tested, 48 amplified reliably and were polymorphic within species. Cross-species transferability was achieved for 31 microsatellites loci in the three target species and four additional loci that were transferable to one species only. The 48 loci amplified in average seven alleles, and detected average expected and observed heterozygosities of 0.7 and 0.55, in the three genotyped populations/species (26 V. aspis, 20 V. latastei and 10 V. seoanei). CONCLUSIONS: Our study provides a selection of 48 polymorphic microsatellite markers that will contribute significantly to current knowledge on genetic diversity, gene flow, population structure, demographic dynamics, systematics, reproduction and heritability in these species, and potentially in other congeneric taxa.

Anaphylaxis as probable cause of death in a rare case of fatal Vipera berus bite.

This case report describes the death of a 52-year-old male who was bitten by a venomous snake, Vipera berus (common European adder), on his left wrist. Despite experiencing symptoms, the patient refused medical care and self-medicated with alcohol instead. He was later found dead in his residence. Autopsy and histological examination revealed evidence of an anaphylactic reaction in response to the snake bite, with additional findings of alcohol intoxication and other underlying medical conditions.

Comparison of cytotoxicity methods for studying Vipera ammodytes venom and the anticytotoxic potency of antivenom.

Introduction: Alternative in vitro tests that can be used instead of animal experiments are those that can most closely evaluate the biological activity of the drug of interest. For testing the potency of antivenom, these are the methods used to assess cytotoxicity. The aim of this study was to evaluate the most commonly used cytotoxicity methods for determining the protective potency of the antivenom Viekvin, which neutralizes Vipera ammodytes venom. Material and methods: The selected methods are based on different biological mechanisms: MTT assay, based on the activity of cell oxidoreductase enzymes; crystal violet staining, based on the degree of cell adhesion; trypan blue staining, based on cell membrane permeability, and propidium iodide staining, based on measurement of nucleic acids of dead cells. The pro-apoptotic effect of the venom was also determined with annexin V staining. Results: The IC50 value of V. ammodytes venom obtained by these methods was very similar, while the EC50 values differed significantly. Conclusions: We concluded that the choice of the method used to measure the anticytotoxic anti-venom potency depends on the immunogenicity of the venom components that cause cell death; for each venom/antivenom pair, it is necessary to select the appropriate assay separately, and at present, none of the standard cytotoxic methods can be universally applied to determine antivenom potency.

Genome-wide single nucleotide polymorphisms reveal recurrent waves of speciation in niche-pockets, in Europe's most venomous snake.

Within the Balkan Peninsula, topographic and climatic agents have promoted biodiversity and shaped the speciation history of many ectotherms. Here, we targeted an iconic European reptile, the nose-horned viper species-complex (Vipera ammodytes), and explored its spatial and temporal evolution. We (i) utilized genome-wide single nucleotide polymorphisms to infer genetic structure and build a time-calibrated species-tree, and (ii) applied species distribution modelling with niche-divergence tests among major phylogenomic clades. Geographically structured genetic diversity was found. Cycles of recurrent isolation and expansion during glacial-interglacial periods led to allopatric speciation and to secondary contacts and formation of multiple hybrid zones throughout the Balkan Peninsula. Deep divergence is still detected among populations separated by old and imminent biogeographical barriers (Pindos Mountain Range, the Cyclades islands, etc.), but in most cases speciation is incomplete. At the other end of the speciation continuum, we recognize two well-differentiated lineages, currently lacking any evidence of gene flow; one is distributed in the Northwestern Balkans and the other in the Southeastern Balkans, further expanding into Asia. Despite their split 5 million years ago, there is no evidence of ecological divergence, as speciation probably occurred in niche-pockets of analogous environments. These two lineages probably represent different species, while V. transcaucasiana does not merit species status. By comparing the genomic phylogenies to an updated mitochondrial one, we propose an evolutionary scenario that resolves all mitonuclear conflicts, according to which the history of the V. ammodytes species-complex was shaped by complex processes, including a major event of introgressive hybridization with asymmetric mitochondrial capture.

Rat Group IIA Secreted Phospholipase A2 Binds to Cytochrome c Oxidase and Inhibits Its Activity: A Possible Episode in the Development of Alzheimer's Disease.

Alzheimer's disease (AD), a progressive form of dementia, is characterized by the increased expression of secreted phospholipase A2 group IIA (GIIA) in the affected tissue and the dysfunction of neuronal mitochondria, similar to that induced by an orthologous GIIA from snake venom, ß-neurotoxic ammodytoxin (Atx), in the motor neurons. To advance our knowledge about the role of GIIA in AD, we studied the effect of rat GIIA on the neuronal mitochondria and compared it with that of the Atx. We produced recombinant rat GIIA (rGIIA) and its enzymatically inactive mutant, rGIIA(D49S), and demonstrated that they interact with the subunit II of cytochrome c oxidase (CCOX-II) as Atx. rGIIA and rGIIA(D49S) bound to this essential constituent of the respiratory chain complex with an approximately 100-fold lower affinity than Atx; nevertheless, both rGIIA molecules potently inhibited the CCOX activity in the isolated rat mitochondria. Like Atx, rGIIA was able to reach the mitochondria in the PC12 cells from the extracellular space, independent of its enzymatic activity. Consistently, the inhibition of the CCOX activity in the intact PC12 cells and in the rat's brain tissue sections was clearly demonstrated using rGIIA(D49S). Our results show that the effects of mammalian and snake venom ß-neurotoxic GIIA on the neuronal mitochondria have similar molecular backgrounds. They suggest that the elevated extracellular concentration of GIIA in the AD tissue drives the translocation of this enzyme into local neurons and their mitochondria to inhibit the activity of the CCOX in the respiratory chain. Consequently, the process of oxidative phosphorylation in the neurons is attenuated, eventually leading to their degeneration. Atx was thus revealed as a valuable molecular tool for further investigations of the role of GIIA in AD.

Extended Snake Venomics by Top-Down In-Source Decay: Investigating the Newly Discovered Anatolian Meadow Viper Subspecies, Vipera anatolica senliki.

Herein, we report on the venom proteome of Vipera anatolica senliki, a recently discovered and hitherto unexplored subspecies of the critically endangered Anatolian meadow viper endemic to the Antalya Province of Turkey. Integrative venomics, including venom gland transcriptomics as well as complementary bottom-up and top-down proteomics analyses, were applied to fully characterize the venom of V. a. senliki. Furthermore, the classical top-down venomics approach was extended to elucidate the venom proteome by an alternative in-source decay (ISD) proteomics workflow using the reducing matrix 1,5-diaminonaphthalene. Top-down ISD proteomics allows for disulfide bond counting and effective de novo sequencing-based identification of high-molecular-weight venom constituents, both of which are difficult to achieve by commonly established top-down approaches. Venom gland transcriptome analysis identified 96 toxin transcript annotations from 18 toxin families. Relative quantitative snake venomics revealed snake venom metalloproteinases (42.9%) as the most abundant protein family, followed by several less dominant toxin families. Online mass profiling and top-down venomics provide a detailed insight into the venom proteome of V. a. senliki and facilitate a comparative analysis of venom variability for the closely related subspecies, Vipera anatolica anatolica.

Comprehensive Study of the Proteome and Transcriptome of the Venom of the Most Venomous European Viper: Discovery of a New Subclass of Ancestral Snake Venom Metalloproteinase Precursor-Derived Proteins.

The nose-horned viper, its nominotypical subspecies Vipera ammodytes ammodytes ( Vaa), in particular, is, medically, one of the most relevant snakes in Europe. The local and systemic clinical manifestations of poisoning by the venom of this snake are the result of the pathophysiological effects inflicted by enzymatic and nonenzymatic venom components acting, most prominently, on the blood, cardiovascular, and nerve systems. This venom is a very complex mixture of pharmacologically active proteins and peptides. To help improve the current antivenom therapy toward higher specificity and efficiency and to assist drug discovery, we have constructed, by combining transcriptomic and proteomic analyses, the most comprehensive library yet of the Vaa venom proteins and peptides. Sequence analysis of the venom gland cDNA library has revealed the presence of messages encoding 12 types of polypeptide precursors. The most abundant are those for metalloproteinase inhibitors (MPis), bradykinin-potentiating peptides (BPPs), and natriuretic peptides (NPs) (all three on a single precursor), snake C-type lectin-like proteins (snaclecs), serine proteases (SVSPs), P-II and P-III metalloproteinases (SVMPs), secreted phospholipases A2 (sPLA2s), and disintegrins (Dis). These constitute >88% of the venom transcriptome. At the protein level, 57 venom proteins belonging to 16 different protein families have been identified and, with SVSPs, sPLA2s, snaclecs, and SVMPs, comprise ∼80% of all venom proteins. Peptides detected in the venom include NPs, BPPs, and inhibitors of SVSPs and SVMPs. Of particular interest, a transcript coding for a protein similar to P-III SVMPs but lacking the MP domain was also found at the protein level in the venom. The existence of such proteins, also supported by finding similar venom gland transcripts in related snake species, has been demonstrated for the first time, justifying the proposal of a new P-IIIe subclass of ancestral SVMP precursor-derived proteins.

Anticancer and antibacterial effects of Iranian viper (Vipera latifii) venom; an in-vitro study.

Viper venom contains antibacterial and cytotoxic components. The aim of this study was to identify and evaluate the antimicrobial and cytotoxic properties of the crude venom of Vipera latifii (V. latifii). Lyophilized venom of V. latifii was quantified by Bradford method and its antibacterial activity (6.25-400 µg/ml) was assessed using the MTT, MIC, Disc diffusion, and Well diffusion assays. Also, its cytotoxic activity was investigated using MTT reduction, Neutral uptake, and Comet assay on human liver cancer (HepG2) cell line. Crude venom showed antibacterial effects against Bacillus subtilis and Staphylococcus aureus, but was not effective on Escherichia coli. Also, the crude venom showed apoptotic and necrotic effects on human liver cancer cells. The venom of V. latifii can inhibit the growth of bacteria and cancer cells. These findings suggest that this may be a potential source of molecules with antibacterial and anticancer characteristics.

Vipers of the Middle East: A Rich Source of Bioactive Molecules.

Snake venom serves as a tool of defense against threat and helps in prey digestion. It consists of a mixture of enzymes, such as phospholipase A2, metalloproteases, and l-amino acid oxidase, and toxins, including neurotoxins and cytotoxins. Beside their toxicity, venom components possess many pharmacological effects and have been used to design drugs and as biomarkers of diseases. Viperidae is one family of venomous snakes that is found nearly worldwide. However, three main vipers exist in the Middle Eastern region: Montivipera bornmuelleri, Macrovipera lebetina, and Vipera (Daboia) palaestinae. The venoms of these vipers have been the subject of many studies and are considered as a promising source of bioactive molecules. In this review, we present an overview of these three vipers, with a special focus on their venom composition as well as their biological activities, and we discuss further frameworks for the exploration of each venom.

Proteome and Peptidome of Vipera berus berus Venom.

Snake venom is a rich source of peptides and proteins with a wide range of actions. Many of the venom components are currently being tested for their usefulness in the treatment of many diseases ranging from neurological and cardiovascular to cancer. It is also important to constantly search for new proteins and peptides with properties not yet described. The venom of Vipera berus berus has hemolytic, proteolytic and cytotoxic properties, but its exact composition and the factors responsible for these properties are not known. Therefore, an attempt was made to identify proteins and peptides derived from this species venom by using high resolution two-dimensional electrophoresis and MALDI ToF/ToF mass spectrometry. A total of 11 protein classes have been identified mainly proteases but also l-amino acid oxidases, C-type lectin like proteins, cysteine-rich venom proteins and phospholipases A2 and 4 peptides of molecular weight less than 1500 Da. Most of the identified proteins are responsible for the highly hemotoxic properties of the venom. Presence of venom phospholipases A2 and l-amino acid oxidases cause moderate neuro-, myo- and cytotoxicity. All successfully identified peptides belong to the bradykinin-potentiating peptides family. The mass spectrometry data are available via ProteomeXchange with identifier PXD004958.

Mitochondrial phylogeny shows multiple independent ecological transitions and northern dispersion despite of Pleistocene glaciations in meadow and steppe vipers (Vipera ursinii and Vipera renardi).

The phylogeny and historical demography of small Eurasian vipers of the Vipera ursinii and V. renardi complexes were studied using mitochondrial DNA sequences analysed with Bayesian inference, Maximum Likelihood and Maximum Parsimony approaches, and mismatch distributions. Diversification in the group resulted from an initial dispersion in the later Pliocene - Pleistocene in two directions: north-westwards via the Balkans (V. ursinii complex) and north-eastwards from Asia Minor via the Caucasus (V. renardi complex). An independent, comparatively recent transition occurred from montane habitats to lowland grasslands in different mitochondrial lineages during the Late Pleistocene, when representatives of the both complexes had reached lowland steppes to the north. Effective population size showed clear signs of rapid growth in eastern V. renardi, triggered by colonization of vast lowland steppes, but in western V. ursinii complex grew during the Last Glaciation and experienced stabilization in Holocene. Expansion and population growth in lowland lineages of V. renardi was not strongly affected by Pleistocene climatic oscillations, when cold, dry conditions could have favoured species living in open grasslands. The high diversity of closely related haplotypes in the Caucasus and Tien-Shan could have resulted from repetitive expansion-constriction-isolation events in montane regions during Pleistocene climate fluctuations. The mitochondrial phylogeny pattern conflicts with the current taxonomy.

Snake (Vipera berus) bite: The cause of severe anaphylactic shock and hepatocellular injury.

Vipera berus bites lead to a variety of clinical manifestations. Local swelling, coagulopathy, nephrotoxicity, cardiac effects and myotoxicity are known to be associated with envenoming by a viper bite. Although a variety of clinical manifestations have been reported in viper bite cases, anaphylactic reactions and liver injury events have not been described. We report a unique case of an anaphylaxis and transitional liver cell injury due to a Vipera berus bite in the case of a 58-year-old man with no past history suggestive of allergy and liver disease. These observations need to be further explored with laboratory studies to identify the venom components which could have pre-disposed the patient to the development of these complications.

Hybridization at an ecotone: ecological and genetic barriers between three Iberian vipers.

The formation of stable genetic boundaries between emerging species is often diagnosed by reduced hybrid fitness relative to parental taxa. This reduced fitness can arise from endogenous and/or exogenous barriers to gene flow. Although detecting exogenous barriers in nature is difficult, we can estimate the role of ecological divergence in driving species boundaries by integrating molecular and ecological niche modelling tools. Here, we focus on a three-way secondary contact zone between three viper species (Vipera aspis, V. latastei and V. seoanei) to test for the contribution of ecological divergence to the development of reproductive barriers at several species traits (morphology, nuclear DNA and mitochondrial DNA). Both the nuclear and mitochondrial data show that all taxa are genetically distinct and that the sister species V. aspis and V. latastei hybridize frequently and backcross over several generations. We find that the three taxa have diverged ecologically and meet at a hybrid zone coincident with a steep ecotone between the Atlantic and Mediterranean biogeographical provinces. Integrating landscape and genetic approaches, we show that hybridization is spatially restricted to habitats that are suboptimal for parental taxa. Together, these results suggest that niche separation and adaptation to an ecological gradient confer an important barrier to gene flow among taxa that have not achieved complete reproductive isolation.

Resembling a viper: implications of mimicry for conservation of the endangered smooth snake.

The phenomenon of Batesian mimicry, where a palatable animal gains protection against predation by resembling an unpalatable model, has been a core interest of evolutionary biologists for 150 years. An extensive range of studies has focused on revealing mechanistic aspects of mimicry (shared education and generalization of predators) and the evolutionary dynamics of mimicry systems (co-operation vs. conflict) and revealed that protective mimicry is widespread and is important for individual fitness. However, according to our knowledge, there are no case studies where mimicry theories have been applied to conservation of mimetic species. Theoretically, mimicry affects, for example, frequency dependency of predator avoidance learning and human induced mortality. We examined the case of the protected, endangered, nonvenomous smooth snake (Coronella austriaca) that mimics the nonprotected venomous adder (Vipera berus), both of which occur in the Åland archipelago, Finland. To quantify the added predation risk on smooth snakes caused by the rarity of vipers, we calculated risk estimates from experimental data. Resemblance of vipers enhances survival of smooth snakes against bird predation because many predators avoid touching venomous vipers. Mimetic resemblance is however disadvantageous against human predators, who kill venomous vipers and accidentally kill endangered, protected smooth snakes. We found that the effective population size of the adders in Åland is very low relative to its smooth snake mimic (28.93 and 41.35, respectively).Because Batesian mimicry is advantageous for the mimic only if model species exist in sufficiently high numbers, it is likely that the conservation program for smooth snakes will fail if adders continue to be destroyed. Understanding the population consequences of mimetic species may be crucial to the success of endangered species conservation. We suggest that when a Batesian mimic requires protection, conservation planners should not ignore the model species (or co-mimic in Mullerian mimicry rings) even if it is not itself endangered.

A Guide to the Clinical Management of Vipera Snakebite in Italy.

The genus Vipera encompasses most species of medically significant venomous snakes of Europe, with Italy harbouring four of them. Envenomation by European vipers can result in severe consequences, but underreporting and the absence of standardised clinical protocols hinder effective snakebite management. This study provides an updated, detailed set of guidelines for the management and treatment of Vipera snakebite tailored for Italian clinicians. It includes taxonomic keys for snake identification, insights into viper venom composition, and recommendations for clinical management. Emphasis is placed on quick and reliable identification of medically relevant snake species, along with appropriate first aid measures. Criteria for antivenom administration are outlined, as well as indications on managing potential side effects. While the protocol is specific to Italy, its methodology can potentially be adapted for other European countries, depending on local resources. The promotion of comprehensive data collection and collaboration among Poison Control Centres is advocated to optimise envenomation management protocols and improve the reporting of epidemiological data concerning snakebite at the country level.

Venom-induced myocarditis: An unusual case attributable to Vipera aspis bite.

Venomous bites are medical emergencies that may result in life-threatening clinical effects. Cardiovascular complications are uncommon but they can be dangerous if not early detected and treated. Cerebral and myocardial infarction are described; myocarditis as consequence of viper envenomation in humans are very rare, almost anedoctal. We present the case of a 33-year-old man, working as keeper in a reptile zoo, who arrived after a viper bite of the Vipera aspis species, on the left wrist. The patient presented with clouded sensorium, edema of the lips and tongue, rapidly worsened with angioedema, and the need for oro-tracheal intubation; severe thrombocytopenia and anemia were treated with transfusions of platelet, plasma and red blood cells. The left hand and arm worsened, with compartment syndrome, treated with surgical fasciotomy. From a cardiological point of view, the patient presented a sudden drop in blood pressure, electrocardiographic anterior and infero-lateral ST depression, pericardial effusion and hypokinesia of the interventricular septum on echocardiography, and a significant increase in troponin T. Cardiac magnetic resonance imaging confirmed the myocarditis, with the presence of septal and anterior intramyocardial edema in T2 weighted sequences, with prolonged T2 time at T2 mapping analysis, without late gadolinium enhancement areas. Cardiological and general clinical conditions gradually improved only after the antivenom was administred. This is one of the rare cases of viper bite myocarditis with echocardiographic and magnetic resonance imaging documentation in Europe; it emphasizes the importance of identifying uncommon complications of venomous snake-bites and the prompt administration of antivenom, even though snake bites are less frequent at our latitudes.

Alpine viper in changing climate: thermal ecology and prospects of a cold-adapted reptile in the warming Mediterranean.

In a rapidly changing thermal environment, reptiles are primarily dependent on in situ adaptation because of their limited ability to disperse and the restricted opportunity to shift their ranges. However, the rapid pace of climate change may surpass these adaptation capabilities or elevate energy expenditures. Therefore, understanding the variability in thermal traits at both individual and population scales is crucial, offering insights into reptiles' vulnerability to climate change. We studied the thermal ecology of the endangered Greek meadow viper (Vipera graeca), an endemic venomous snake of fragmented alpine-subalpine meadows above 1600 m of the Pindos mountain range in Greece and Albania, to assess its susceptibility to anticipated changes in the alpine thermal environment. We measured preferred body temperature in artificial thermal gradient, field body temperatures of 74 individuals in five populations encompassing the entire geographic range of the species, and collected data on the available of temperatures for thermoregulation. We found that the preferred body temperature (Tp) differed only between the northernmost and the southernmost populations and increased with female body size but did not depend on sex or the gravidity status of females. Tp increased with latitude but was unaffected by the phylogenetic position of the populations. We also found high accuracy of thermoregulation in V. graeca populations and variation in the thermal quality of habitats throughout the range. The overall effectiveness of thermoregulation was high, indicating that V. graeca successfully achieves its target temperatures and exploits the thermal landscape. Current climatic conditions limit the activity period by an estimated 1278 h per year, which is expected to increase considerably under future climate scenarios. Restricted time available for thermoregulation, foraging and reproduction will represent a serious threat to the fitness of individuals and the persistence of populations in addition to habitat loss due to mining, tourism or skiing and habitat degradation due to overgrazing in the shrinking mountaintop habitats of V. graeca.

A longitudinal study of the blood and urine metabolome of Vipera berus envenomated dogs.

Envenomation of dogs by the common European adder (Vipera berus) is associated with high morbidity. The cytotoxic venom of Vipera berus contains enzymes with the potential to cause acute kidney injury, among other insults, however robust biomarkers for such effects are lacking. A prospective observational follow-up study of naturally envenomated dogs and controls was conducted to fill knowledge gaps regarding canine Vipera berus envenomation, attempt to identify novel biomarkers of envenomation and related kidney injury, and elucidate potential long-term effects. Blood and urine samples were analyzed with a global metabolomics approach using liquid chromatography-mass spectrometry, uncovering numerous features significantly different between cases and controls. After data processing and feature annotation, eight features in blood and 24 features in urine were investigated in order to elucidate their biological relevance. Several of these are associated with AKI, while some may also originate from disturbed fatty acid ß-oxidation and soft tissue damage. A metabolite found in both blood and a venom reference sample may represent identification of a venom component in case dogs. Our findings suggest that envenomated dogs treated according to current best practice are unlikely to suffer permanent injury.