Snake venomics of the Brazilian pitvipers Bothrops cotiara and Bothrops fonsecai. Identification of taxonomy markers

Abstract

We report the proteomic characterization of venom of the pitvipers Bothrops cotiara and Bothrops fonsecai. Crude venoms were fractionated by reverse-phase HPLC, followed by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and CID-MS/MS. Each venom contained around 30 proteins in the range of 7–110 kDa belonging to only 8 (B. cotiara) and 9 (B. fonsecai) families which may target the hemostatic system, albeit distinctly distributed among the two species. B. cotiara and B. fonsecai share medium-sized disintegrins, disintegrin-like/cysteine-rich (DC) fragments, snake venom vascular endothelial growth factor, cysteine-rich secretory proteins, serine proteinases, C-type lectins, l-amino acid oxidase, and Zn²⁺-dependent metalloproteinases. In addition, B. fonsecai expresses a high abundance PLA₂ molecule (13,890 Da), whereas PLA₂ molecules were not detected in B. cotiara's venom. This stricking finding is in line with previous biochemical analyses showing the absence of phospholipasic activity in the venom of B. cotiara. The potential adaptive significance of the lack of PLA₂ molecules is enigmatic, and alternative explanations are discussed. B. fonsecai is morphologically extremely similar to B. cotiara. Our comparative proteomic analysis shows that compositional differences between their venoms can be employed as a taxonomy signature for unambiguous species identification independently of geographic origin and morphological characteristics.

Introduction

Venoms represent the critical innovation in ophidian evolution that allowed advanced snakes to transition from a mechanical (constriction) to a chemical (venom) means of subduing and digesting prey larger than themselves, and as such, venom proteins have multiple functions including immobilizing, paralyzing, killing and digesting prey. Venomous snakes of the Viperidae family produce a complex mixture of distinct proteins in specialized venom glands located in the upper jaw, which they inject into prey using fangs [1], [2]. Analysis of venom proteins has consistently shown high levels of intra and interspecific variation [3] and there are a small but increasing number of studies that strongly support the idea that this variation reflects local adaptation for feeding on different prey ([4], [5], [6] and references cited). Diet and diversifying selection pressure to maintain high levels of variation in venom genes through accelerated evolution have played a central role in the adaptive radiation of snakes [4], [5], [7]. Beside the evolutionary and ecological importance of venoms, accidental envenomation by snakebite constitutes a highly relevant public health hazard in Central and South America which has an impact in terms of mortality and morbidity [8], [9], [10]. Viperid venoms contain proteins that interfere with the coagulation cascade, the normal haemostatic system, and tissue repair [11], [12], [13]. Without treatment, the fatality rate is estimated to be about 10%, but with an appropriate antivenom therapy it can be reduced below 0.5%, although the incidence of sequelaes remains high [14].

Although snakebites can be deadly, studies on snake venom toxins offer a number of potential benefits for basic research, clinical diagnosis, development of new research tools and drugs of potential clinical use [15], [16], [17], [18], [19]. A thorough characterization of the venom proteomes of medically important species might contribute to a deeper understanding of the biology, ecology and pathophysiology of envenoming by these snakes, and would also serve as a starting point for studying structure–function correlations of individual toxins. In addition, knowledge of the relative contributions of different venom toxin families to the composition of the venoms might be relevant for generating immunization protocols that elicit the production of toxin-specific antibodies showing greater specificity and effectiveness than conventional antivenoms raised by immunizing horses with whole venom through selection of candidate epitopes for structure-based design of antitoxin antibodies [20], [21].

Members of genus Bothrops are responsible for more fatalities in the Americas than any other group of venomous snakes [9], [10], [14]. 19 Bothrops species are represented in the Brazilian herpetofauna. In this country, 28,597 snakebites were reported in 2005, of which 87.5% were related to Bothrops species (http://portal.saude.gov.br/portal/arquivos/pdf/situacao.pdf).

Bothropoid pitvipers comprise a diverse and widespread assemblage of venomous snakes which originated by divergence from a New World founding species that colonized Central America sometime during the Miocene (23–10 Mya ago) and produced the common ancestor of both the North American group (Agkistrodon, Crotalus, Sistrurus) and the Neotropical pitvipers [14], [22]. Four Old World genera (Protobothrops, Ovophis, Trimeresurus, and Gloydius) have been variously estimated as the sister group to the New World clade [23]. The Central American-based ancestral taxon may have undergone a relatively rapid and prolific radiation expanding northward into North America and southward into South America long before extensive uplifting occurred during the Pliocene and Pleistocene [14]. Currently, the genus Bothrops (subfamily Crotalinae of Viperidae) comprises 32 (http://www.reptile-database.org) or 37 species [14] of primarily South and Central American pitvipers, commonly referred as lanceheads. Except for southwestern South America, the extreme highlands of the Andes, and southernmost Patagonia, this genus is widely distributed in tropical Latin America, from northeastern Mexico to Argentina, and the southern parts of the lower Caribbean islands [14]. Bothrops are diverse in their morphology and natural history, and represent a particularly interesting group because of the wide array of habitat types they inhabit, such as lowland evergreen forests, montane semideciduous forests, savannas, and montane open formations.

Bothrops (sensu lato) is a paraphyletic clade composed of at least five separate lineages [14], [23]. B. cotiara and B. fonsecai form with B. itapetiningae and B. alternatus the alternatus group characterized by large size, one internasal on each side of the head, and 9–14 intersupraocular scales. Bothrops cotiara [24], named after the Tupi word “quatiara” meaning “painted” is a moderately heavy-bodied snake (adult length usually 0.7–1.0 m) whose habitat include the Araucaria forests of southern Brazil in the states of São Paulo, Paraná, Santa Catarina and Rio Grande do Sul. It is also found sporadically in northeastern Argentina in the province of Misiones (Fig. 1), with a vertical distribution from sea level to at least 1800 m. Bothrops fonsecai [25], Fonseca's lancehead (a patronym honoring Flávio da Fonseca, former Director of the Laboratory of Parasitology at the Instituto Butantan), is also a medium to large-sized, heavy-bodied pitviper. B. fonsecai is endemic to Southeastern Brazil (northeastern São Paulo, southern Rio de Janeiro and extreme southern Minas Gerais). Its elevational distribution ranges from 1000 to 1600 m. Both species are mammal specialists. Mammal specialization is synapomorphic in the subclade of the alternatus group including B. alternatus, B. cotiara, and B. fonsecai [26]. B. fonsecai is morphologically extremely similar to B. cotiara (Fig. 1) although they are not sympatric and B. cotiara has more black pigment on the intracephalic scales and the posterior portion of the postorbital stripe has a hook-like configuration [14], [27].

Data on the clinics of envenomations by B. cotiara and B. fonsecai bites are scarce [28], in part due to their very specialized habitats (Araucaria angustifolia pine forests on highlands) that separate them ecologically from humans. In addition, both snakes are catalogued as near threatened to extinction species [29], [30] due to deforestation by clear-cutting over vast areas. Forest reptiles are very susceptible to microclimate changes, and large tracts of Araucaria forest in Brazil are today restricted to a few isolated remnants in national and state parks. Here, we report a detailed proteomic characterization of the venoms of these two endangered Brazilian Bothrops species. Our comparative proteomic analysis shows that compositional differences between the venoms of B. cotiara and B. fonsecai can be employed as taxonomy markers for unambiguous species identification independently of geographic origin and morphological characteristics.