Spatial versus spatio-temporal approaches for studying metacommunities: a multi-taxon analysis in Mediterranean and tropical temporary ponds.

Prior research on metacommunities has largely focused on snapshot surveys, often overlooking temporal dynamics. In this study, our aim was to compare the insights obtained from metacommunity analyses based on a spatial approach repeated over time, with a spatio-temporal approach that consolidates all data into a single model. We empirically assessed the influence of temporal variation in the environment and spatial connectivity on the structure of metacommunities in tropical and Mediterranean temporary ponds. Employing a standardized methodology across both regions, we surveyed multiple freshwater taxa in three time periods within the same hydrological year from multiple temporary ponds in each region. To evaluate how environmental, spatial and temporal influences vary between the two approaches, we used nonlinear variation partitioning analyses based on generalized additive models. Overall, this study underscores the importance of adopting spatio-temporal analytics to better understand the processes shaping metacommunities. While the spatial approach suggested that environmental factors had a greater influence, our spatio-temporal analysis revealed that spatial connectivity was the primary driver influencing metacommunity structure in both regions. Temporal effects were equally important as environmental effects, suggesting a significant role of ecological succession in metacommunity structure.

Venom-microbiomics of eight species of Neotropical spiders from the Theraphosidae family.

AIM: Tarantulas are one of the largest predatory arthropods in tropical regions. Tarantulas though not lethal to humans, their venomous bite kills small animals and insect upon which they prey. To understand the abiotic and biotic components involved in Neotropical tarantula bites, we conducted a venom-microbiomics study in eight species from Costa Rica. METHODS AND RESULTS: We determined that the toxin profiles of tarantula venom are highly diverse using shotgun proteomics; the most frequently encountered toxins were ω-Ap2 toxin, neprilysin-1, and several teraphotoxins. Through culture-independent and culture-dependent methods, we determined the microbiota present in the venom and excreta to evaluate the presence of pathogens that could contribute to primary infections in animals, including humans. The presence of opportunistic pathogens with hemolytic activity was observed, with a prominence of Stenotrophomonas in the venoms. Other bacteria found in venoms and excreta with hemolytic activity included members of the genera Serratia, Bacillus, Acinetobacter, Microbacterium, and Morganella. CONCLUSIONS: Our data shed light on the venom- and gut-microbiome associated with Neotropical tarantulas. This information may be useful for treating bites from these arthropods in both humans and farm animals, while also providing insight into the toxins and biodiversity of this little-explored microenvironment.

Sequence Divergence in Venom Genes Within and Between Montane Pitviper (Viperidae: Crotalinae: Cerrophidion) Species is Driven by Mutation-Drift Equilibrium.

Snake venom can vary both among and within species. While some groups of New World pitvipers-such as rattlesnakes-have been well studied, very little is known about the venom of montane pitvipers (Cerrophidion) found across the Mesoamerican highlands. Compared to most well-studied rattlesnakes, which are widely distributed, the isolated montane populations of Cerrophidion may facilitate unique evolutionary trajectories and venom differentiation. Here, we describe the venom gland transcriptomes for populations of C. petlalcalensis, C. tzotzilorum, and C. godmani from Mexico, and a single individual of C. sasai from Costa Rica. We explore gene expression variation in Cerrophidion and sequence evolution of toxins within C. godmani specifically. Cerrophidion venom gland transcriptomes are composed primarily of snake venom metalloproteinases, phospholipase A[Formula: see text]s (PLA[Formula: see text]s), and snake venom serine proteases. Cerrophidion petlalcalensis shows little intraspecific variation; however, C. godmani and C. tzotzilorum differ significantly between geographically isolated populations. Interestingly, intraspecific variation was mostly attributed to expression variation as we did not detect signals of selection within C. godmani toxins. Additionally, we found PLA[Formula: see text]-like myotoxins in all species except C. petlalcalensis, and crotoxin-like PLA[Formula: see text]s in the southern population of C. godmani. Our results demonstrate significant intraspecific venom variation within C. godmani and C. tzotzilorum. The toxins of C. godmani show little evidence of directional selection where variation in toxin sequence is consistent with evolution under a model of mutation-drift equilibrium. Cerrophidion godmani individuals from the southern population may exhibit neurotoxic venom activity given the presence of crotoxin-like PLA[Formula: see text]s; however, further research is required to confirm this hypothesis.

RESUMEN: El veneno de las serpientes puede variar entre y dentro de las especies. Mientras algunos grupos de viperidos del Nuevo Mundo­como las cascabeles­han sido bien estudiadas, muy poco se sabe acerca del veneno de las nauyacas de frío (Cerrophidion) que se encuentran en las zonas altas de Mesoamérica. Comparadas con las extensamente estudiadas cascabeles, que estan ampliamente distribuidas, las poblaciones de Cerrophidion, aisladas en montañas, pueden poseer trayectorias evolutivas y diferenciación en su veneno unicos. En el presente trabajo, describimos el transcriptoma de las glándulas de veneno de poblaciones de C. petlalcalensis, C. tzotzilorum, y C. godmani de México, y un individuo de C. sasai de Costa Rica. Exploramos la variación en la expresión de toxinas en Cerrophidion y la evolución en las secuencias geneticas en C. godmani específicamente. El transcriptoma de la glándula de veneno de Cerrophidion esta compuesto principalmente de Metaloproteinasas de Veneno de Serpiente, Fosfolipasas A[Formula: see text] (PLA[Formula: see text]s), y Serin Proteasas de Veneno de Serpiente. Cerrophidion petlalcalensis presenta poca variación intraespecífica; sin embargo, los transcriptomas de la glandula de veneno de C. godmani y C. tzotzilorum difieren significativamente entre poblaciones geográficamente aisladas. Curiosamente, la variación intraespecífica estuvo atribuida principalmente a la expresión de las toxinas ya que no encontramos señales de selección en las toxinas de C. godmani. Adicionalmente, encontramos miotoxinas similares a PLA[Formula: see text] en todas las especies excepto C. petlalcalensis, y PLA[Formula: see text]s similares a crotoxina en la población sureña de C. godmani. Nuestros resultados demuestran la presencia de variacion intraespecífica presente en el veneno de C. godmani y C. tzotzilorum. Las toxinas de Cerrophidion godmani muestran poca evidencia de selección direccional, y la variación en la secuencias de las toxinas es consistente con evolucion bajo un modelo de equilibrio de mutación-deriva. Algunos individuos de C. godmani de la población del sur potencialmente tienen un veneno neurotóxico dada la presencia de PLA[Formula: see text]s similares a la crotoxina, sin embargo, se necesita más evidencia para corroborar esta hipótesis.

Trait differentiation and modular toxin expression in palm-pitvipers.

BACKGROUND: Modularity is the tendency for systems to organize into semi-independent units and can be a key to the evolution and diversification of complex biological systems. Snake venoms are highly variable modular systems that exhibit extreme diversification even across very short time scales. One well-studied venom phenotype dichotomy is a trade-off between neurotoxicity versus hemotoxicity that occurs through the high expression of a heterodimeric neurotoxic phospholipase A2 (PLA2) or snake venom metalloproteinases (SVMPs). We tested whether the variation in these venom phenotypes could occur via variation in regulatory sub-modules through comparative venom gland transcriptomics of representative Black-Speckled Palm-Pitvipers (Bothriechis nigroviridis) and Talamancan Palm-Pitvipers (B. nubestris). RESULTS: We assembled 1517 coding sequences, including 43 toxins for B. nigroviridis and 1787 coding sequences including 42 toxins for B. nubestris. The venom gland transcriptomes were extremely divergent between these two species with one B. nigroviridis exhibiting a primarily neurotoxic pattern of expression, both B. nubestris expressing primarily hemorrhagic toxins, and a second B. nigroviridis exhibiting a mixed expression phenotype. Weighted gene coexpression analyses identified six submodules of transcript expression variation, one of which was highly associated with SVMPs and a second which contained both subunits of the neurotoxic PLA2 complex. The sub-module association of these toxins suggest common regulatory pathways underlie the variation in their expression and is consistent with known patterns of inheritance of similar haplotypes in other species. We also find evidence that module associated toxin families show fewer gene duplications and transcript losses between species, but module association did not appear to affect sequence diversification. CONCLUSION: Sub-modular regulation of expression likely contributes to the diversification of venom phenotypes within and among species and underscores the role of modularity in facilitating rapid evolution of complex traits.

Integrated "omics" profiling indicates that miRNAs are modulators of the ontogenetic venom composition shift in the Central American rattlesnake, Crotalus simus simus.

BACKGROUND: Understanding the processes that drive the evolution of snake venom is a topic of great research interest in molecular and evolutionary toxinology. Recent studies suggest that ontogenetic changes in venom composition are genetically controlled rather than environmentally induced. However, the molecular mechanisms underlying these changes remain elusive. Here we have explored the basis and level of regulation of the ontogenetic shift in the venom composition of the Central American rattlesnake, Crotalus s. simus using a combined proteomics and transcriptomics approach. RESULTS: Proteomic analysis showed that the ontogenetic shift in the venom composition of C. s. simus is essentially characterized by a gradual reduction in the expression of serine proteinases and PLA2 molecules, particularly crotoxin, a ß-neurotoxic heterodimeric PLA2, concominantly with an increment of PI and PIII metalloproteinases at age 9-18 months. Comparison of the transcriptional activity of the venom glands of neonate and adult C. s. simus specimens indicated that their transcriptomes exhibit indistinguisable toxin family profiles, suggesting that the elusive mechanism by which shared transcriptomes generate divergent venom phenotypes may operate post-transcriptionally. Specifically, miRNAs with frequency count of 1000 or greater exhibited an uneven distribution between the newborn and adult datasets. Of note, 590 copies of a miRNA targeting crotoxin B-subunit was exclusively found in the transcriptome of the adult snake, whereas 1185 copies of a miRNA complementary to a PIII-SVMP mRNA was uniquely present in the newborn dataset. These results support the view that age-dependent changes in the concentration of miRNA modulating the transition from a crotoxin-rich to a SVMP-rich venom from birth through adulthood can potentially explain what is observed in the proteomic analysis of the ontogenetic changes in the venom composition of C. s. simus. CONCLUSIONS: Existing snake venom toxins are the result of early recruitment events in the Toxicofera clade of reptiles by which ordinary genes were duplicated, and the new genes selectively expressed in the venom gland and amplified to multigene families with extensive neofunctionalization throughout the approximately 112-125 million years of ophidian evolution. Our findings support the view that understanding the phenotypic diversity of snake venoms requires a deep knowledge of the mechanisms regulating the transcriptional and translational activity of the venom gland. Our results suggest a functional role for miRNAs. The impact of specific miRNAs in the modulation of venom composition, and the integration of the mechanisms responsible for the generation of these miRNAs in the evolutionary landscape of the snake's venom gland, are further challenges for future research.

[Distribution, surface and protected area of palm-swamps in Costa Rica and Nicaragua]. / Distribución, superficie y área protegida de humedales dominados por pantanos de palmas (Arecaceae) en Costa Rica y Nicaragua.

In Central America, palm swamps are known collectively as yolillales. These wetlands are usually dominated by the raffia palm Raphia taedigera, but also by the royal palm Manicaria saccifera and -in lower extensions- by the American oil palm Elaeis oleifera. The yolillales tend to be poor in woody species and are characteristic of regions with high rainfall and extensive hydroperiods, so they remain flooded most of the year. The dominance of large raffia palm leaves in the canopy, allow these environments to be distinguishable in aerial photographs, which consequently has helped to map them along most of their distribution. However, while maps depicting yolillales are available, the extent of their surface area, perimeter and connectivity remains poorly understood. This is particularly true for yolillales in Costa Rica and Nicaragua, countries that share a good proportion of palm dominated swaps in the Rio San Juan Basin. In addition, it is not known the actual area of these environments that is under any category of protection according to the conservation systems of both countries. As a first step to catalog yolillal wetlands in Costa Rica and Nicaragua, this paper evaluates cartographic maps to delineate yolillales in the region. A subsample of yolillales mapped in this study were visited and we geo-referenced them and evaluate the extent and condition of the swamp. A total of 110 883.2ha are classified as yolillales in Nicaragua, equivalent to 22% of wetland surface area recorded for that country (excluding the Cocibolca and Xolothn Lakes). In Costa Rica, 53 931.3ha are covered by these palm dominated swamps, which represent 16.24% of the total surface area covered by wetlands. About 47% of the area covered by yolillales in Nicaragua is under some category of protection, the largest extensions protected by Cerro Silva, Laguna Tale Sulumas and Indio Maiz Nature Reserves. In Costa Rica, 55.5% of the area covered by yolillal is located within protected areas, mainly the Tortuguero National Park, Barra del Colorado Wildlife Refuge and the Sierpe-Thrraba National Wetland. Therefore, in both countries, about half the area covered by these wetlands is not protected by their systems of protection of wilderness areas.

[Amphibians and reptiles in the swamps dominated by the palm Raphia taedigera (Arecaceae) in northeastern Costa Rica]. / Anfibios y reptiles de los pantanos dominados per la palma de yolillo Raphia taedigera (Arecaceae) en el noreste de Costa Rica.

The herpetofauna that inhabits Caribbean Costa Rica has received considerable attention in the last two decades. This assemblage includes a total of 141 species of reptiles and 95 amphibians mostly distributed in tropical wet and moist lowland forests. While most information available came from primary and secondary forest sites, little is known about the amphibians and reptiles that inhabit more open habitats, such as wetlands and swamps. For instances, swaps dominated by the yolillo palm Raphia taedigera extend through much of the northeastern Caribbean coast of Costa Rica and eastern Nicaragua, but information about the herpetological community that uses such environments remains practically unknown. This situation reflects the little research conducted in such inhospitable environments. Here, we report the results of an intensive survey conducted to assess the herpetological community that inhabit R. taedigera palm-swamps. A total of 14 species of amphibians and 17 of reptiles have been recorded from these swamps. Amphibians and reptiles that inhabit yolillo swamps have wide distributions along much of Middle America and are considered common species throughout their range. In general, yolillo swamps are poor environments for herpetofauna: richness of reptiles and amphibians is almost two times higher in the adjacent forest than in the palm dominated swamps. Furthermore, most species observed in this swamps can be considered habitat generalists that are well adapted to the extreme conditions imposed by the changes in hydroperiods, reduce understory cover, low tree diversity and simple forest architecture of these environments. Despite similarities in the herpetofauna, it is clear that not all forest species use yolillo habitat, a characteristic that is discussed in terms of physical stress driven by the prolonged hydroperiod and reduced leaflitter in the ground, as these features drive habitat structure and herpetofaunal complexity. Our list of species using yolillo swamps is not definitive, and is likely to grow as more studies are conducted in this unexplored environment.

[Use and conservation of palm swamps Raphia taedigera (Arecaceae) in the Area de Conservación Tortuguero, Costa Rica]. / Uso y conservación de humedales de yolillo Raphia taedigera (Arecaceae) en el aea de cConservación Tortuguero, Costa Rica.

The swamps dominated by raffia palm Raphia taedigera are conspicuous environments in the Tortuguero floodplains and in other wet regions along the Caribbean and Pacific coasts of Costa Rica. However, these environments have been little studied and are exposed to numerous threats, most importantly their replacement by agricultural activities or pastureland. In this paper, we describe some applications and uses of the raffia palms and other palms that are common in these flooded swamps. We also describe the efforts that have been made in Costa Rica for the protection or raffia-dominated swamps, through the environmental law frame of the country and the establishment of a protection system based on wilderness areas under different categories of protection. We discuss issues relevant to the future of these environments in the regions where they are distributed.

Half a century of research on Bothrops asper venom variation: biological and biomedical implications.

Snake venoms are a complex biological mixture of proteins with or without enzymatic activity, peptides, and nucleotides, among other components. It is produced in specialized secretory glands located in the maxillary region, being the result of millions of years of evolution and whose biological functions are defense, immobilization, and digestion of prey. Venoms present intraspecific (i.e., individual, ontogenetic, geographical) and interspecific (i.e., between sympatric and allopatric species) variation, and the study of this variability has become the focus of toxinological research. Bothrops asper is responsible for highest incidence, morbimortality and severe cases of envenoming in Mesoamerica and northern South America. Given its clinical importance, its venom has been characterized and compared qualitatively and quantitatively across the species range. More than 50 years of research show that B. asper venom is endowed with an interesting intraspecific variability. Knowing this variation has allowed advances in the elucidation of the biological role of the venom, a better understanding of the clinical signs and symptoms in patients envenomed by B asper, the immunological implications in the context of antivenoms production, and the generation of new ideas that could be useful to solve different biological and evolutionary questions of one of the venomous snakes with the greatest distribution and strongest public health impact in Latin America.

Proteomic profile of the venom of three dark-colored Tityus (Scorpiones: Buthidae) from the tropical rainforests of Costa Rica.

OBJECTIVE: We aimed to elucidate the potential differences in the venom peptide sequences of three Tityus species from Costa Rican rainforests: T. jaimei, T. championi and T. dedoslargos, compared to T. cf. asthenes from Colombia, which could explain the low level of scorpionism in Costa Rica, evidenced by the lack of epidemiological data. METHODOLOGY: We applied venom proteomics of peptides purified by RP-HPLC and compared the obtained sequences from venoms of these Tityus species to the sequences previously identified from Tityus inhabiting other Central and South American regions. RESULTS: Venom proteome analysis evidences that most of the putative peptide toxins identified in Costa Rican dark-colored Tityus are very similar to those present in other T. (Atreus) from the region. CONCLUSIONS: Our study suggests that, in the case of potential envenomation by Tityus in Costa Rica, the same level of toxicity should be observed, compared to other cases caused by members of the subgenus from other geographical localities. On the other hand, compared to countries with more accelerated urban expansion, Costa Rican Tityus still inhabit secondary rainforests and do not commonly share the same spaces with humans, so the lack of epidemiological evidence of medical emergencies caused by envenoming by this scorpion group could be more related to ecological and demographic factors and less attributed to the characteristics of the venom.

Amphibian diversity across three adjacent ecosystems in Área de Conservación Guanacaste, Costa Rica.

Amphibians are the most threatened species-rich vertebrate group, with species extinctions and population declines occurring globally, even in protected and seemingly pristine habitats. These 'enigmatic declines' are generated by climate change and infectious diseases. However, the consequences of these declines are undocumented as no baseline ecological data exists for most affected areas. Like other neotropical countries, Costa Rica, including Área de Conservación Guanacaste (ACG) in north-western Costa Rica, experienced rapid amphibian population declines and apparent extinctions during the past three decades. To delineate amphibian diversity patterns within ACG, a large-scale comparison of multiple sites and habitats was conducted. Distance and time constrained visual encounter surveys characterised species richness at five sites-Murciélago (dry forest), Santa Rosa (dry forest), Maritza (mid-elevation dry-rain forest intersect), San Gerardo (rainforest) and Cacao (cloud forest). Furthermore, species-richness patterns for Cacao were compared with historic data from 1987-8, before amphibians declined in the area. Rainforests had the highest species richness, with triple the species of their dry forest counterparts. A decline of 45% (20 to 11 species) in amphibian species richness was encountered when comparing historic and contemporary data for Cacao. Conservation efforts sometimes focus on increasing the resilience of protected areas, by increasing their range of ecosystems. In this sense ACG is unique containing many tropical ecosystems compressed in a small geographic space, all protected and recognised as a UNESCO world heritage site. It thus provides an extraordinary platform to understand changes, past and present, and the resilience of tropical ecosystems and assemblages, or lack thereof, to climate change.

Inconsistent response of taxonomic groups to space and environment in mediterranean and tropical pond metacommunities.

The metacommunity concept provides a theoretical framework that aims at explaining organism distributions by a combination of environmental filtering, dispersal, and drift. However, few works have attempted a multitaxon approach and even fewer have compared two distant biogeographical regions using the same methodology. We tested the expectation that temperate (mediterranean-climate) pond metacommunities would be more influenced by environmental and spatial processes than tropical ones, because of stronger environmental gradients and a greater isolation of waterbodies. However, the pattern should be different among groups of organisms depending on their dispersal abilities. We surveyed 30 tropical and 32 mediterranean temporary ponds from Costa Rica and Spain, respectively, and obtained data on 49 environmental variables. We characterized the biological communities of bacteria and archaea (from the water column and the sediments), phytoplankton, zooplankton, benthic invertebrates, amphibians and birds, and estimated the relative role of space and environment on metacommunity organization for each group and region, by means of variation partitioning using generalized additive models. Purely environmental effects were important in both tropical and mediterranean ponds, but stronger in the latter, probably due to their larger limnological heterogeneity. Spatially correlated environment and pure spatial effects were greater in the tropics, related to higher climatic heterogeneity and dispersal processes (e.g., restriction, surplus) acting at different scales. The variability between taxonomic groups in the contribution of spatial and environmental factors to metacommunity variation was very wide, but higher in active, compared with passive, dispersers. Higher environmental effects were observed in mediterranean passive dispersers, and higher spatial effects in tropical passive dispersers. The unexplained variation was larger in the tropical setting, suggesting a higher role for stochastic processes, unmeasured environmental factors, or biotic interactions in the tropics, although this difference affected some actively dispersing groups (insects and birds) more than passive dispersers. These results, despite our limitations in comparing only two regions, provide support, for a wide variety of aquatic organisms, for the classic view of stronger abiotic niche constraints in temperate areas compared with the tropics. The heterogeneous response of taxonomic groups between regions also points to a stronger influence of regional context than organism adaptations on metacommunity organization.

Unveiling underestimated species diversity within the Central American Coralsnake, a medically important complex of venomous taxa.

Coralsnakes of the genus Micrurus are a diverse group of venomous snakes ranging from the southern United States to southern South America. Much uncertainty remains over the genus diversity, and understanding Micrurus systematics is of medical importance. In particular, the widespread Micrurus nigrocinctus spans from Mexico throughout Central America and into Colombia, with a number of described subspecies. This study provides new insights into the phylogenetic relationships within M. nigrocinctus by examining sequence data from a broad sampling of specimens from Mexico, Guatemala, Honduras, Nicaragua, Costa Rica, and Panama. The recovered phylogenetic relationships suggest that M. nigrocinctus is a species complex originating in the Pliocene and composed of at least three distinct species-level lineages. In addition, recovery of highly divergent clades supports the elevation of some currently recognized subspecies to the full species rank while others may require synonymization.

Proteomic and toxicological analysis of the venom of Micrurus yatesi and its neutralization by an antivenom.

Coralsnakes belong to the family Elapidae and possess venoms which are lethal to humans and can be grouped based on the predominance of either three finger toxins (3FTxs) or phospholipases A2 (PLA2s). A proteomic and toxicological analysis of the venom of the coralsnake Micrurus yatesi was performed. This species, distributed in southeastern Costa Rica, was formerly considered a subspecies of M. alleni. Results showed that this venom is PLA2-rich, in contrast with the previously studied venom of Micrurus alleni. Toxicological evaluation of the venom, in accordance with proteomic data, revealed that it has a markedly higher in vitro PLA2 activity upon a synthetic substrate than M. alleni. The evaluation of in vivo myotoxicity in CD-1 mice using histological evaluation and plasma creatine kinase release also showed that M. yatesi venom caused muscle damage. A commercial equine antivenom prepared using the venom of Micrurus nigrocinctus displayed a similar recognition of the venoms of M. yatesi and M. nigrocinctus by enzyme immunoassay. This antivenom also immunorecognized the main fractions of the venom of M. yatesi and was able to neutralize its lethal effect in a murine model.

Morphology and seasonality of the sexual segment of the kidney in genus Bothrops snakes.

In Squamata, the sexual segment of the male kidney (SSK) produces secretory granules that are transmitted to the female tract during copulation. Here, we investigate the morphology of SSK in various species of the pitviper-genus Bothrops, a medically relevant clade of Neotropical snakes, using histology, histochemistry, as well as scanning and transmission electron microscopy. We also evaluated the relationship of SSK hypertrophy with spermatogenesis and storage of sperm. The SSK in Bothrops consists of tall columnar epithelial cells filled with electron-dense secretory granules. These granules are apparently released into the lumen by an apocrine secretory process. In general, the ultrastructural morphology of SSK in Bothrops is similar to those described for other pitvipers in the genus Agkistrodon. In most Bothrops males, the SSK produces carbohydrates and proteins, and occurs in all lobes of the kidney, occupying most of the kidney mass. In contrast, intersexual females (with male copulatory organs) of Bothrops insularis had a smaller SSK diameter than males and did not show hypertrophy. The seasonal development of SSK in Bothrops species seems to be driven by environmental factors rather than phylogeny, since sister species exhibited periods of hypertrophy at different months. In most Bothrops males, hypertrophy occurs at various seasons of the year (spring, summer, autumn), even in winter, and seasonal changes in SSK do not necessarily accompany spermatogenesis.

Profiling the venom gland transcriptomes of Costa Rican snakes by 454 pyrosequencing.

BACKGROUND: A long term research goal of venomics, of applied importance for improving current antivenom therapy, but also for drug discovery, is to understand the pharmacological potential of venoms. Individually or combined, proteomic and transcriptomic studies have demonstrated their feasibility to explore in depth the molecular diversity of venoms. In the absence of genome sequence, transcriptomes represent also valuable searchable databases for proteomic projects. RESULTS: The venom gland transcriptomes of 8 Costa Rican taxa from 5 genera (Crotalus, Bothrops, Atropoides, Cerrophidion, and Bothriechis) of pitvipers were investigated using high-throughput 454 pyrosequencing. 100,394 out of 330,010 masked reads produced significant hits in the available databases. 5.165,220 nucleotides (8.27%) were masked by RepeatMasker, the vast majority of which corresponding to class I (retroelements) and class II (DNA transposons) mobile elements. BLAST hits included 79,991 matches to entries of the taxonomic suborder Serpentes, of which 62,433 displayed similarity to documented venom proteins. Strong discrepancies between the transcriptome-computed and the proteome-gathered toxin compositions were obvious at first sight. Although the reasons underlaying this discrepancy are elusive, since no clear trend within or between species is apparent, the data indicate that individual mRNA species may be translationally controlled in a species-dependent manner. The minimum number of genes from each toxin family transcribed into the venom gland transcriptome of each species was calculated from multiple alignments of reads matched to a full-length reference sequence of each toxin family. Reads encoding ORF regions of Kazal-type inhibitor-like proteins were uniquely found in Bothriechis schlegelii and B. lateralis transcriptomes, suggesting a genus-specific recruitment event during the early-Middle Miocene. A transcriptome-based cladogram supports the large divergence between A. mexicanus and A. picadoi, and a closer kinship between A. mexicanus and C. godmani. CONCLUSIONS: Our comparative next-generation sequencing (NGS) analysis reveals taxon-specific trends governing the formulation of the venom arsenal. Knowledge of the venom proteome provides hints on the translation efficiency of toxin-coding transcripts, contributing thereby to a more accurate interpretation of the transcriptome. The application of NGS to the analysis of snake venom transcriptomes, may represent the tool for opening the door to systems venomics.

Venomics of the poorly studied hognosed pitvipers Porthidium arcosae and Porthidium volcanicum.

We report the first proteomics analyses of the venoms of two poorly studied snakes, the Manabi hognosed pitviper Porthidium arcosae endemic to the western coastal province of Manabí (Ecuador), and the Costa Rican hognosed pitviper P. volcanicum with distribution restricted to South Pacific Costa Rica and western Panamá. These venom proteomes share a conserved compositional pattern reported in four other congeneric species within the clade of South American Porthidium species, P. nasutum, P. lansbergii, P. ophryomegas, and P. porrasi. The paraspecific immunorecognition profile of antivenoms produced in Costa Rica (ICP polyvalent), Perú (Instituto Nacional de Salud) and Brazil (soro antibotrópico pentavalente, SAB, from Instituto Butantan) against the venom of P. arcosae was investigated through a third-generation antivenomics approach. The maximal venom-binding capacities of the investigated antivenoms were 97.1 mg, 21.8 mg, and 25.7 mg of P. arcosae venom proteins per gram of SAB, ICP, and INS-PERU antibody molecules, respectively, which translate into 28.4 mg, 13.1 mg, and 15.2 mg of total venom proteins bound per vial of SAB, ICP, and INS-PERU AV. The antivenomics results suggest that 21.8%, 7.8% and 6.1% of the SAB, ICP, and INS-PERU antibody molecules recognized P. arcosae venom toxins. The SAB antivenom neutralized P. arcosae venom's lethality in mice with an ED50 of 31.3 mgV/g SAB AV. This preclinical neutralization paraspecificity points to Brazilian SAB as a promising candidate for the treatment of envenomings by Ecuadorian P. arcosae. BIOLOGICAL SIGNIFICANCE: Assessing the preclinical efficacy profile of antivenoms against homologous and heterologous medically relevant snake venoms represents an important goal towards defining the biogeographic range of their clinical utility. This is particularly relevant in regions, such as Mesoamerica, where a small number of pharmaceutical companies produce antivenoms against the venoms of a small number of species of maximum medical relevance among the local rich herpetofauna, leaving a wide range of snakes of secondary medical relevance, but also causing life-threatening human envenomings without nominal clinical coverage. This work is part of a larger project aiming at mapping the immunological characteristics of antivenoms generated in Latin American countries towards venoms of such poorly studied snakes of the local and neighboring countries' herpetofauna. Here we report the proteomics characterization of the Manabi hognosed pitviper Porthidium arcosae endemic to the western coastal province of Manabí (Ecuador), and the Costa Rican hognosed pitviper P. volcanicum with distribution restricted to southwestern Costa Rica, the antivenomics assessment of three bothropoid commercial antivenoms produced in Costa Rica, Perú, and Brazil against the venom components of P. arcosae, and the in vivo capacity of the Brazilian soro antibotrópico pentavalente (SAB) from Instituto Butantan to neutralize the murine lethality of P. arcosae venom. The preclinical paraspecific ED50 of 31.3 mg of P. arcosae venom per gram of antivenom points to Brazilian SAB as a promising candidate for the treatment of envenomings by the Manabi hognosed pitviper P. arcosae.

Mutual enlightenment: A toolbox of concepts and methods for integrating evolutionary and clinical toxinology via snake venomics and the contextual stance.

Snakebite envenoming is a neglected tropical disease that may claim over 100,000 human lives annually worldwide. Snakebite occurs as the result of an interaction between a human and a snake that elicits either a defensive response from the snake or, more rarely, a feeding response as the result of mistaken identity. Snakebite envenoming is therefore a biological and, more specifically, an ecological problem. Snake venom itself is often described as a "cocktail", as it is a heterogenous mixture of molecules including the toxins (which are typically proteinaceous) responsible for the pathophysiological consequences of envenoming. The primary function of venom in snake ecology is pre-subjugation, with defensive deployment of the secretion typically considered a secondary function. The particular composition of any given venom cocktail is shaped by evolutionary forces that include phylogenetic constraints associated with the snake's lineage and adaptive responses to the snake's ecological context, including the taxa it preys upon and by which it is predated upon. In the present article, we describe how conceptual frameworks from ecology and evolutionary biology can enter into a mutually enlightening relationship with clinical toxinology by enabling the consideration of snakebite envenoming from an "ecological stance". We detail the insights that may emerge from such a perspective and highlight the ways in which the high-fidelity descriptive knowledge emerging from applications of -omics era technologies - "venomics" and "antivenomics" - can combine with evolutionary explanations to deliver a detailed understanding of this multifactorial health crisis.

Promoting co-existence between humans and venomous snakes through increasing the herpetological knowledge base.

Snakebite incidence at least partly depends on the biology of the snakes involved. However, studies of snake biology have been largely neglected in favour of anthropic factors, with the exception of taxonomy, which has been recognised for some decades to affect the design of antivenoms. Despite this, within-species venom variation and the unpredictability of the correlation with antivenom cross-reactivity has continued to be problematic. Meanwhile, other aspects of snake biology, including behaviour, spatial ecology and activity patterns, distribution, and population demography, which can contribute to snakebite mitigation and prevention, remain underfunded and understudied. Here, we review the literature relevant to these aspects of snakebite and illustrate how demographic, spatial, and behavioural studies can improve our understanding of why snakebites occur and provide evidence for prevention strategies. We identify the large gaps that remain to be filled and urge that, in the future, data and relevant metadata be shared openly via public data repositories so that studies can be properly replicated and data used in future meta-analyses.

New insights into snakebite epidemiology in Costa Rica: A retrospective evaluation of medical records.

Continuous monitoring of the snakebite envenoming allows elucidating factors that affect its incidence at spatial and temporal scales, and is a great tool to evaluate the proper management of snakebite in health centers. To determine if there have been changes over time in snakebite epidemiology in Costa Rica, we conducted a retrospective study using medical records from six hospitals for the years 2012-2013. A total of 475 snakebite patients were treated at the selected hospital during this period. Most bites occurred during the rainy season and primarily affected young men, mainly farm workers and schoolchildren. About 55% of bites occur in peri-domiciliary environments, although its prevalence varies geographically. Bothrops asper generates the vast majority of envenoming in the country, which is why the main local symptoms registered are edema, pain, and bleeding disorders. The time elapsed until treatment did not explain the degree of severity at admission. However, complications were observed more frequently in patients who took longer to receive treatment. The primary complications were bacterial infections, whereas kidney failure and compartment syndrome documented at very low frequencies. Only one death was recorded, reflecting the low fatality rate exhibited in the country. Hospital treatment included the rapid administration of antivenom and complementary treatment of antibiotics, analgesics, and antihistamines. The application of the latter as prophylactic does not seem to prevent the appearance of mild early adverse reactions, registered in 22.5% of the cases. Morbidity and mortality rates from snakebite have continued to decrease in the country, as a result of the efforts that Costa Rica has made to improve its public health system. Among those efforts, the creation of primary care centers (EBAIS) has reduced the time to treatment in many regions of the country. The Costa Rican experience of using antivenom in primary health care centers and maintaining good medical records could be considered for application in other countries where snakebite is a major health problem.