Venenos de serpentes do gênero Bothrops: impacto da glicosilação na complexidade dos proteomas e função de toxinas / Bothrops snake venoms: impact of glycosylation on the complexity of proteomes and toxins function

A variabilidade estrutural é uma característica das proteínas de venenos de serpentes, e a glicosilação é uma das principais modificações pós-traducionais que contribui para a diversificação de seus proteomas. Recentes estudos de nosso grupo demonstraram que venenos do gênero Bothrops são marcadamente definidos pelo seu conteúdo de glicoproteínas, e que a maioria das estruturas de N-glicanos dos tipos híbrido e complexo identificados em oito venenos deste gênero contêm unidades de ácido siálico. Em paralelo, em glicoproteínas do veneno de B. cotiara foi identificada a presença de uma estrutura de N-acetilglicosamina bissecada. Assim, com o objetivo de investigar a variação do conteúdo de glicoproteínas, assim como os mecanismos envolvidos na geração dos diferentes venenos de Bothrops, neste estudo foram analisados comparativamente os glicoproteomas de nove venenos do gênero Bothrops (B. atrox, B. cotiara, B. erythromelas, B. fonsecai, B. insularis, B. jararaca, B. jararacussu, B. moojeni e B. neuwiedi). As abordagens glicoproteômicas envolveram cromatografia de afinidade e ensaio de pull-down utilizando, respectivamente, as lectinas SNA (aglutinina de Sambucus nigra) e MAL I (lectina de Maackia amurensis), que mostram afinidade por unidades de ácido siálico nas posições, respectivamente, α2,6 e α2,3; e cromatografia de afinidade com a lectina PHA-E (eritroaglutinina de Phaseolus vulgaris), que reconhece N-acetilglicosamina bissecada. Ainda, eletroforese de proteínas, blot de lectina, e identificação de proteínas por espectrometria de massas foram empregadas para caracterizar os glicoproteomas. As lectinas geraram frações dos venenos enriquecidas de diferentes componentes, onde as principais classes de glicoproteínas identificadas foram metaloprotease, serinoprotease, e L-amino ácido oxidase, além de outras enzimas pouco abundantes nos venenos. Os diferentes conteúdos de proteínas reconhecidas por essas lectinas, com especificidades distintas, ressaltaram novos aspectos da variabilidade dos subproteomas de glicoproteínas desses venenos, dependendo da espécie. Ainda, considerando que metaloproteases e serinoproteases são componentes abundantes nesses venenos e fundamentais no quadro de envenenamento botrópico, e que estas enzimas contêm diversos sítios de glicosilação, o papel das unidades de ácido siálico na atividade proteolítica das mesmas foi avaliado. Assim, a remoção enzimática de ácido siálico (i) alterou o padrão de gelatinólise em zimografia da maioria dos venenos, (ii) diminuiu a atividade proteolítica de alguns venenos sobre o fibrinogênio e a atividade coagulante do plasma humano de todos os venenos, e (iii) alterou o perfil de hidrólise de proteínas plasmáticas pelo veneno de B. jararaca, indicando que este carboidrato pode desempenhar um papel na interação das proteases com seus substratos proteicos. Em contraste, o perfil da atividade amidolítica dos venenos não se alterou após a remoção de ácido siálico e incubação com o substrato Bz-Arg-pNA, indicando que ácido siálico não é essencial em N-glicanos de serinoproteases atuando sobre substratos não proteicos. Em conjunto, esses resultados expandem o conhecimento sobre a variabilidade de proteomas de venenos do gênero Bothrops e apontam a importância das cadeias de carboidratos contendo ácido siálico nas atividades enzimáticas das proteases desses venenos

Structural variability is a feature of snake venom proteins, and glycosylation is one of the main post-translational modifications that contributes to the diversification of venom proteomes. Recent studies by our group have shown that Bothrops venoms are markedly defined by their glycoprotein content, and that most hybrid and complex N-glycan structures identified in eight venoms of this genus contain sialic acid units. In parallel, the presence of a bisected N-acetylglucosamine structure was identified in B. cotiara venom glycoproteins. Thus, with the aim of investigating the variation in the content of glycoproteins, as well as the mechanisms involved in the generation of different Bothrops venoms, in this study the glycoproteomes of nine Bothrops venoms (B. atrox, B. cotiara, B. erythromelas, B. fonsecai, B. insularis, B. jararaca, B. jararacussu, B. moojeni e B. neuwiedi) were comparatively analyzed. The glycoproteomic approaches involved affinity chromatography and pulldown using, respectively, the lectins SNA (Sambucus nigra agglutinin) and MAL I (Maackia amurensis lectin), which show affinity for sialic acid units at positions, respectively, α2,6 and α2,3, and affinity chromatography with PHA-E (Phaseolus vulgaris erythroagglutinin), which recognizes bisected N-acetylglucosamine. In addition, protein electrophoresis, lectin blot, and protein identification by mass spectrometry were employed for glycoproteome characterization. The lectins generated venom fractions enriched with different components, where the main classes of glycoproteins identified were metalloprotease, serine protease, and L-amino acid oxidase, in addition to other low abundant enzymes. The different contents of proteins recognized by these lectins of distinct specificities highlighted new aspects of the variability of the glycoprotein subproteomes of these venoms, depending on the species. Furthermore, considering that metalloproteases and serine proteases are abundant components of these venoms and essential in Bothrops envenomation, and that these enzymes contain several glycosylation sites, the role of sialic acid units in their proteolytic activities was evaluated. Thus, enzymatic removal of sialic acid (i) altered the pattern of gelatinolysis in zymography of most venoms, (ii) decreased the proteolytic activity of some venoms on fibrinogen and the clotting activity of human plasma of all venoms, and (iii) altered the hydrolysis profile of plasma proteins by B. jararaca venom, indicating that this carbohydrate may play a role in the interaction of proteases with their protein substrates. In contrast, the profile of amidolytic activity of the venoms did not change after removal of sialic acid and incubation with the substrate Bz-Arg-pNA, indicating that sialic acid is not essential in N-glycans of serine proteases acting on small substrates. Together, these results expand the knowledge about the variability of proteomes of Bothrops venoms and point to the importance of carbohydrate chains containing sialic acid in the enzymatic activities of venom proteases

The role of vicariance and dispersal on the temporal range dynamics of forest vipers in the Neotropical region.

The emergence of the diagonal of open/dry vegetations, including Chaco, Cerrado and Caatinga, is suggested to have acted as a dispersal barrier for terrestrial organisms by fragmenting a single large forest that existed in South America into the present Atlantic and Amazon forests. Here we tested the hypothesis that the expansion of the South American diagonal of open/dry landscapes acted as a vicariant process for forest lanceheads of the genus Bothrops, by analyzing the temporal range dynamics of those snakes. We estimated ancestral geographic ranges of the focal lancehead clade and its sister clade using a Bayesian dated phylogeny and the BioGeoBEARS package. We compared nine Maximum Likelihood models to infer ancestral range probabilities and their related biogeographic processes. The best fitting models (DECTS and DIVALIKETS) recovered the ancestor of our focal clade in the Amazon biogeographic region of northwestern South America. Vicariant processes in two different subclades resulted in disjunct geographic distributions in the Amazon and the Atlantic Forest. Dispersal processes must have occurred mostly within the Amazon and the Atlantic Forest and not between them. Our results suggest the fragmentation of a single ancient large forest into the Atlantic and Amazon forests acting as a driver of vicariant processes for the snake lineage studied, highlighting the importance of the diagonal of open/dry landscapes in shaping distribution patterns of terrestrial biota in South America.

Tracking the recruitment and evolution of snake toxins using the evolutionary context provided by the Bothrops jararaca genome.

Venom is a key adaptive innovation in snakes, and how nonvenom genes were co-opted to become part of the toxin arsenal is a significant evolutionary question. While this process has been investigated through the phylogenetic reconstruction of toxin sequences, evidence provided by the genomic context of toxin genes remains less explored. To investigate the process of toxin recruitment, we sequenced the genome of Bothrops jararaca, a clinically relevant pitviper. In addition to producing a road map with canonical structures of genes encoding 12 toxin families, we inferred most of the ancestral genes for their loci. We found evidence that 1) snake venom metalloproteinases (SVMPs) and phospholipases A2 (PLA2) have expanded in genomic proximity to their nonvenomous ancestors; 2) serine proteinases arose by co-opting a local gene that also gave rise to lizard gilatoxins and then expanded; 3) the bradykinin-potentiating peptides originated from a C-type natriuretic peptide gene backbone; and 4) VEGF-F was co-opted from a PGF-like gene and not from VEGF-A. We evaluated two scenarios for the original recruitment of nontoxin genes for snake venom: 1) in locus ancestral gene duplication and 2) in locus ancestral gene direct co-option. The first explains the origins of two important toxins (SVMP and PLA2), while the second explains the emergence of a greater number of venom components. Overall, our results support the idea of a locally assembled venom arsenal in which the most clinically relevant toxin families expanded through posterior gene duplications, regardless of whether they originated by duplication or gene co-option.

MITGARD: an automated pipeline for mitochondrial genome assembly in eukaryotic species using RNA-seq data.

MOTIVATION: Over the past decade, the field of next-generation sequencing (NGS) has seen dramatic advances in methods and a decrease in costs. Consequently, a large expansion of data has been generated by NGS, most of which have originated from RNA-sequencing (RNA-seq) experiments. Because mitochondrial genes are expressed in most eukaryotic cells, mitochondrial mRNA sequences are usually co-sequenced within the target transcriptome, generating data that are commonly underused or discarded. Here, we present MITGARD, an automated pipeline that reliably recovers the mitochondrial genome from RNA-seq data from various sources. The pipeline identifies mitochondrial sequence reads based on a phylogenetically related reference, assembles them into contigs, and extracts a complete mtDNA for the target species. RESULTS: We demonstrate that MITGARD can reconstruct the mitochondrial genomes of several species throughout the tree of life. We noticed that MITGARD can recover the mitogenomes in different sequencing schemes and even in a scenario of low-sequencing depth. Moreover, we showed that the use of references from congeneric species diverging up to 30 million years ago (MYA) from the target species is sufficient to recover the entire mitogenome, whereas the use of species diverging between 30 and 60 MYA allows the recovery of most mitochondrial genes. Additionally, we provide a case study with original data in which we estimate a phylogenetic tree of snakes from the genus Bothrops, further demonstrating that MITGARD is suitable for use on biodiversity projects. MITGARD is then a valuable tool to obtain high-quality information for studies focusing on the phylogenetic and evolutionary aspects of eukaryotes and provides data for easily identifying a sample using barcoding, and to check for cross-contamination using third-party tools.

Extractivism of palm tree fruits: A risky activity because of snakebites in the state of Acre, Western Brazilian Amazon.

INTRODUCTION: We present here the risk from snakebites because of palm extractivism in western Amazonia. METHODS: The data were extracted from a cross-sectional sample study, from January 2016 to April 2018, at the Juruá Regional Hospital of Cruzeiro do Sul (Acre). RESULTS: There were 14 Bothrops incidents involving males, most of them occurring during the harvesting of "açaí" (Euterpe precatoria). CONCLUSIONS: During the harvesting of "açaí," there is the risk of the presence of B. atrox on the ground near the palm tree, and of B. bilineatus at the top of the palm tree.

Extractivism of palm tree fruits: A risky activity because of snakebites in the state of Acre, Western Brazilian Amazon

Abstract INTRODUCTION: We present here the risk from snakebites because of palm extractivism in western Amazonia. METHODS: The data were extracted from a cross-sectional sample study, from January 2016 to April 2018, at the Juruá Regional Hospital of Cruzeiro do Sul (Acre). RESULTS: There were 14 Bothrops incidents involving males, most of them occurring during the harvesting of "açaí" (Euterpe precatoria). CONCLUSIONS: During the harvesting of "açaí," there is the risk of the presence of B. atrox on the ground near the palm tree, and of B. bilineatus at the top of the palm tree.

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.

Phylogeography of the Central American lancehead Bothrops asper (SERPENTES: VIPERIDAE).

The uplift and final connection of the Central American land bridge is considered the major event that allowed biotic exchange between vertebrate lineages of northern and southern origin in the New World. However, given the complex tectonics that shaped Middle America, there is still substantial controversy over details of this geographical reconnection, and its role in determining biogeographic patterns in the region. Here, we examine the phylogeography of Bothrops asper, a widely distributed pitviper in Middle America and northwestern South America, in an attempt to evaluate how the final Isthmian uplift and other biogeographical boundaries in the region influenced genealogical lineage divergence in this species. We examined sequence data from two mitochondrial genes (MT-CYB and MT-ND4) from 111 specimens of B. asper, representing 70 localities throughout the species' distribution. We reconstructed phylogeographic patterns using maximum likelihood and Bayesian methods and estimated divergence time using the Bayesian relaxed clock method. Within the nominal species, an early split led to two divergent lineages of B. asper: one includes five phylogroups distributed in Caribbean Middle America and southwestern Ecuador, and the other comprises five other groups scattered in the Pacific slope of Isthmian Central America and northwestern South America. Our results provide evidence of a complex transition that involves at least two dispersal events into Middle America during the final closure of the Isthmus.

Cross-reactivity and inhibition myotoxic effects induced by Bothrops snake venoms using specific polyclonal anti-BnSP7 antibodies.

Polyclonal antibodies raised in Balb-c mice against BnSP-7, a Lys-49 phospholipase A2, were used to measure cross reactivity against other snake venoms. Using ELISA, these antibodies were able to recognize PLA2s isoforms present in venoms of botropic snakes at 1:6400, 1:3200 and 1:100 ratios (w/w). These antibodies highly recognized proteins of low molecular weight (∼14,000) from crude snake venom Bp and Bm by Western Blotting. PLA2 these venoms, by alignment of primary structures demonstrated high identity with BnSP-7 PLA2, especially in the C-terminal region. However, the crude snake venom Bd and Bj, showed low recognition. The PLA2 activity of Bothrops pauloensis, Bothrops moojeni venoms or BpPLA2-TXI was inhibited significantly when anti-BnSP-7 antibodies were incubated at 1:10 and 1:20 ratios (venoms or toxin:anti-BnSP-7, w/w), respectively. The myotoxic effect induced by the same venoms was also reduced significantly at 1:1, 1:10 and 1:20 ratios, by decreased creatine kinase levels. The anti-PLA2 polyclonal antibodies effectively recognized PLA2s from Bothrops pauloensis and Bothrops moojeni venoms, and neutralized specific catalytic and myotoxic activity.

An overview of Bothrops erythromelas venom.

This review discusses studies on the venom of Bothrops erythromelas published over the past 36 years. During this period, many contributions have been made to understand the venomous snake, its venom, and its experimental and clinical effects better. The following chronological overview is based on 29 articles that were published between 1979 and 2015, with emphasis on diverse areas. The complexity of this task demands an integration of multidisciplinary research tools to study toxinology. This science is in need of renewed conceptual and experimental platforms aimed at obtaining a profound understanding of the highly complex pathophysiology of snakebite envenoming and toxins isolated from snakes.

An overview of Bothrops erythromelas venom

Abstract This review discusses studies on the venom of Bothrops erythromelas published over the past 36 years. During this period, many contributions have been made to understand the venomous snake, its venom, and its experimental and clinical effects better. The following chronological overview is based on 29 articles that were published between 1979 and 2015, with emphasis on diverse areas. The complexity of this task demands an integration of multidisciplinary research tools to study toxinology. This science is in need of renewed conceptual and experimental platforms aimed at obtaining a profound understanding of the highly complex pathophysiology of snakebite envenoming and toxins isolated from snakes.

Canopy Venom: Proteomic Comparison among New World Arboreal Pit-Viper Venoms.

Central and South American pitvipers, belonging to the genera Bothrops and Bothriechis, have independently evolved arboreal tendencies. Little is known regarding the composition and activity of their venoms. In order to close this knowledge gap, venom proteomics and toxin activity of species of Bothriechis, and Bothrops (including Bothriopsis) were investigated through established analytical methods. A combination of proteomics and bioactivity techniques was used to demonstrate a similar diversification of venom composition between large and small species within Bothriechis and Bothriopsis. Increasing our understanding of the evolution of complex venom cocktails may facilitate future biodiscoveries.

Another new and threatened species of lancehead genus Bothrops (Serpentes, Viperidae) from Ilha dos Franceses, Southeastern Brazil.

A new insular species of the genus Bothrops is described from Ilha dos Franceses, a small island off the coast of Espírito Santo State, in southeastern Brazil. The new species differs from mainland populations of B. jararaca mainly by its small size, relative longer tail, relative smaller head length, and relative larger eyes. The new species is distinguished from B. alcatraz, B. insularis and B. otavioi by the higher number of ventral and subcaudal scales, relative longer tail and smaller head. The new species is highly abundant on the island, being nocturnal, semiarboreal, and feeding on small lizards and centipeds. Due its unique and restricted area of occurrence, declining quality of habitat, and constant use of the island for tourism, the new species may be considered as critically endangered.

Venomics and antivenomics of Bothrops erythromelas from five geographic populations within the Caatinga ecoregion of northeastern Brazil.

The Caatinga lancehead, Bothrops erythromelas, is a medically relevant species, responsible for most of the snakebite accidents in most parts of its distribution range in northeastern Brazil. The spectrum and geographic variability of its venom toxins were investigated applying a venomics approach to venom pools from five geographic areas within the Caatinga ecoregion. Despite its wide habitat, populations of B. erythromelas from Ceará, Pernambuco, Juazeiro, Paraiba, and Ilha de Itaparica exhibit highly conserved venom proteomes. Mirroring their compositional conservation, the five geographic venom pools also showed qualitatively and quantitatively overlapping antivenomic profiles against antivenoms generated in Vital Brazil (BR) and Clodomiro Picado (CR) Institutes, using different venoms in the immunization mixtures. The paraspecificity exhibited by the Brazilian SAB and the Costa Rican BCL antivenoms against venom toxins from B. erythromelas indicates large immunoreactive epitope conservation across genus Bothrops during the last ~14 million years, thus offering promise for the possibility of generating a broad-spectrum bothropic antivenom. Biological Significance Accidental snakebite envenomings represent an important public health hazard in Brazil. Ninety per cent of the yearly estimated 20-30,000 snakebite accidents are caused by species of the Bothrops genus. Bothrops erythromelas, a small, moderately stocky terrestrial venomous snake, is responsible for most of the snakebite accidents in its broad distribution range in the Caatinga, a large ecoregion in northeastern Brazil. To gain a deeper insight into the spectrum of medically important toxins present in the venom of the Caatinga lancehead, we applied a venomics approach to define the proteome and geographic variability of adult B. erythromelas venoms from five geographic regions. Although intraspecific compositional variation between venoms among specimens from different geographic regions has long been appreciated by herpetologists and toxinologists as a general feature of highly adaptable and widely distributed snake species, the five B. erythromelas populations investigated exhibit highly conserved venom proteomes. The overall toxin profile of the Caatinga lancehead's venom explains the local and systemic effects observed in envenomations by B. erythromelas. The five geographic venom pools sampled also showed qualitatively and quantitatively overlapping antivenomic profiles against antivenoms generated using different bothropic venoms in the immunization mixtures. The large immunoreactive epitope conservation across genus Bothrops offers promise for the generation of a broad-spectrum bothropic antivenom.

Preliminary assessment of Hedychium coronarium essential oil on fibrinogenolytic and coagulant activity induced by Bothrops and Lachesis snake venoms

The search for new inhibitors of snake venom toxins is essential to complement or even replace traditional antivenom therapy, especially in relation to compounds that neutralize the local effects of envenomations. Besides their possible use as alternative to traditional antivenom therapy, some plant species possess bioactive secondary metabolites including essential oils, which can be extracted from weeds that are considered substantial problems for agriculture, such as Hedychium coronarium. The essential oils of leaves and rhizomes from H. coronarium were extracted by hydrodistillation, and their potential inhibitory effects on the coagulant and fibrinogenolytic activities induced by the venoms of Lachesis muta, Bothrops atrox and Bothrops moojeni were analyzed. Citrated human plasma was used to evaluate the clotting time whereas changes in fibrinogen molecules were visualized by electrophoresis in polyacrylamide gel. The experimental design used for testing coagulation inhibition was randomized in a 3 × 2 factorial arrangement (concentration × essential oils), with three replications. The essential oils were compared since they were extracted from different organs of the same botanical species, H. coronarium. The results suggest that the oils interact with venom proteases and plasma constituents, since all oils evaluated, when previously incubated with venoms, were able to inhibit the clotting effect, with less inhibition when oils and plasma were preincubated prior to the addition of venoms. Thus, after extensive characterization of their pharmacological and toxicological effects, the essential oils can be used as an alternative to complement serum therapy, especially considering that these plant metabolites generally do not require specific formulations and may be used topically immediately after extraction.

Mitochondrial genome of Protobothrops dabieshanensis (Squamata: Viperidae: Crotalinae).

Abstract Protobothrops dabieshanensis is new species snake which is only distributed in Dabie Mountain of Anhui China at present. In this study, the complete mitochondrial genome of P. dabieshanensis (AHU 2013-EE-Hp01) had been determined. The circle genome with the 17,193 bp total length contained 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 control regions. Overall base composition of the complete mtDNA was 33.20% A, 25.13% T, 29.22% C and 12.45% G. All the genes in P. dabieshanensis were distributed on the H-strand, except for the ND6 subunit gene and eight tRNA genes which were encoded on the L-strand.

Proteomic and functional profiling of the venom of Bothrops ayerbei from Cauca, Colombia, reveals striking interspecific variation with Bothrops asper venom.

Bothrops ayerbei, a pitviper inhabiting the Patía River's basin (Valle Alto del Río Patía) in the Southwestern Department of Cauca, Colombia, was considered as a variant form of Bothrops asper prior to being proposed as a new species in 2010, on the basis of subtle morphological differences. This study reports the proteomic and functional profiling of B. ayerbei venom. Its most striking feature is an almost complete absence (0.7%) of phospholipases A2 (PLA2), which is in contrast to the high proportion of these enzymes (25.3%) in the venom of B. asper from Cauca, as well as in other species of Bothrops. The predominant proteins in B. ayerbei venom are metalloproteinases (53.7%), in agreement with its higher hemorrhagic and lethal activities compared to B. asper venom. Moreover, the negligible content of PLA2s in B. ayerbei venom correlates with its weaker myotoxic effect, in contrast to B. asper venom, here shown to contain abundant Asp49- and Lys49-type PLA2s responsible for its strong myotoxic activity. Other components identified in B. ayerbei venom include bradykinin-potentiating-like peptides and proteins belonging to the C-type lectin/lectin-like, serine proteinase, l-amino acid oxidase, disintegrin, cysteine-rich secretory protein, nerve growth factor, and phosphodiesterase families. The venom composition of B. ayerbei resembles that of neonate specimens of B. asper, which shows a predominance of metalloproteinases, with only low amounts of PLA2s. Therefore, the present findings suggest that the expression of venom proteins in B. ayerbei, in contrast to B. asper, might retain a marked 'paedomorphic' condition. Altogether, the proteomic and toxicological characterization of the venom of B. ayerbei here reported argues in favor of its taxonomical separation from B. asper in Cauca, Colombia. BIOLOGICAL SIGNIFICANCE: B. ayerbei, a pitviper found in Cauca, Colombia, had been considered as a variant form of B. asper, but was recently described as a new species on the basis of subtle morphological differences. Our study provides the first detailed proteomic and functional analysis of the venom of B. ayerbei, revealing striking interspecific variation from B. asper, thus arguing in favor of their taxonomical separation. In addition, the observed composition of the venom of B. ayerbei correlates well with its functional and toxicological properties, helping to predict the main clinical manifestations in envenomings by this species, which inflicts a considerable number of snakebites in the Southwestern regions of Colombia.

Comparison of phylogeny, venom composition and neutralization by antivenom in diverse species of bothrops complex.

In Latin America, Bothrops snakes account for most snake bites in humans, and the recommended treatment is administration of multispecific Bothrops antivenom (SAB--soro antibotrópico). However, Bothrops snakes are very diverse with regard to their venom composition, which raises the issue of which venoms should be used as immunizing antigens for the production of pan-specific Bothrops antivenoms. In this study, we simultaneously compared the composition and reactivity with SAB of venoms collected from six species of snakes, distributed in pairs from three distinct phylogenetic clades: Bothrops, Bothropoides and Rhinocerophis. We also evaluated the neutralization of Bothrops atrox venom, which is the species responsible for most snake bites in the Amazon region, but not included in the immunization antigen mixture used to produce SAB. Using mass spectrometric and chromatographic approaches, we observed a lack of similarity in protein composition between the venoms from closely related snakes and a high similarity between the venoms of phylogenetically more distant snakes, suggesting little connection between taxonomic position and venom composition. P-III snake venom metalloproteinases (SVMPs) are the most antigenic toxins in the venoms of snakes from the Bothrops complex, whereas class P-I SVMPs, snake venom serine proteinases and phospholipases A2 reacted with antibodies in lower levels. Low molecular size toxins, such as disintegrins and bradykinin-potentiating peptides, were poorly antigenic. Toxins from the same protein family showed antigenic cross-reactivity among venoms from different species; SAB was efficient in neutralizing the B. atrox venom major toxins. Thus, we suggest that it is possible to obtain pan-specific effective antivenoms for Bothrops envenomations through immunization with venoms from only a few species of snakes, if these venoms contain protein classes that are representative of all species to which the antivenom is targeted.

[The toxicity of venom of Bothrops (Rhinocerophris) alternatus in different areas of Cordoba State in Argentina]. / Caracterización tóxica del veneno de Bothrops (Rhinocerophis) alternatus de diferentes regiones de la provincia de Córdoba (Argentina).

Snake venoms can show biochemical and toxicological variability even in specimens from the same specie. The geographical localization of the snakes is one of the factors that can influence those variations. By these reasons the venom from specimens of Bothrops (Rhinocerophis) alternatus ("crucera", "yararágrande"), one of the snakes of highest medical importance in Argentina, from three different regions of Córdoba was studied. Lehtal potency, hemorrhagic, coagulant on plasma and thrombin like activities as well as the electrophoretic patterns of venom from snakes of Calamuchita, Traslasierras and the East of the province were determined. The venom from the snakes of the three regions showed the characteristic activities of the venom of the majority of Bothrops, causing hemorrhage, hemostatic disturbances acting on plasma or directly on fibrinogen with a "thrombin like activity". The different samples were very similar regarding their biochemical characteristics and toxic potencies at difference of previous observations on venoms from the same specie in different regions of other provinces fro Argentina. Bivalent antivenom, the one used by the Provincial Ministry of Health to treat the bothropic accidents, neutralized in all the cases the toxic activities of the venom in very similar range of neutralizing potency.

Los venenos serpientes pueden presentar variabilidad en sus características bioquímicas y toxicológicas aún en ejemplares de una misma especie. La localización geográfica de los ejemplares es uno de los factores que puede influenciar estas variaciones. Por este motivo se estudió el veneno de ejemplares Bothrops (Rhinocerophis) alternatus ("crucera", "yarará grande"), una de las serpientes de mayor importancia médica de Argentina, de tres regiones diferentes de Córdoba. Se estudió la potencia letal, la actividad hemorrágica, coagulante en plasma y trombina símil y el patrón electroforético de ejemplares de Calamuchita, Traslasierras y del Este de la provincia. El veneno de las serpientes de las tres regiones presentó las actividades características de los venenos de la mayoría de las Bothrops, causando hemorragias y alteraciones en el sistema hemostático, afectando tanto al plasma como actuando directamente sobre el fibrinógeno mediante una actividad trombina símil. Las distintas muestras fueron muy similares en cuanto a sus características bioquímicas ya sus potencias tóxicas, a diferencia de lo observado con veneno de ejemplaresB. alternatusde distintas regiones de otras provincias de la Argentina. El antiveneno Bivalente utilizado por el Ministerio de Salud de Córdoba para tratar los accidentes por Bothrops neutralizó, en todos los casos las actividades tóxicas ensayadas en rangos de potencias neutralizantes muy similares.

Caracterización tóxica del veneno de Bothrops (Rhinocerophis) alternatus de diferentes regiones de la provincia de Córdoba (Argentina) / [The toxicity of venom of Bothrops (Rhinocerophris) alternatus in different areas of Cordoba State in Argentina].

Snake venoms can show biochemical and toxicological variability even in specimens from the same specie. The geographical localization of the snakes is one of the factors that can influence those variations. By these reasons the venom from specimens of Bothrops (Rhinocerophis) alternatus ("crucera", "yararágrande"), one of the snakes of highest medical importance in Argentina, from three different regions of Córdoba was studied. Lehtal potency, hemorrhagic, coagulant on plasma and thrombin like activities as well as the electrophoretic patterns of venom from snakes of Calamuchita, Traslasierras and the East of the province were determined. The venom from the snakes of the three regions showed the characteristic activities of the venom of the majority of Bothrops, causing hemorrhage, hemostatic disturbances acting on plasma or directly on fibrinogen with a "thrombin like activity". The different samples were very similar regarding their biochemical characteristics and toxic potencies at difference of previous observations on venoms from the same specie in different regions of other provinces fro Argentina. Bivalent antivenom, the one used by the Provincial Ministry of Health to treat the bothropic accidents, neutralized in all the cases the toxic activities of the venom in very similar range of neutralizing potency.