Stable isotopic composition of Bothrops atrox (common lancehead) in four distinct land covers in eastern Amazonia

Since consumers reflect the isotopic composition of an assimilated diet, stable isotopes can be a useful tool to address the feeding ecology of tropical snakes. This is the first study reporting carbon and nitrogen stable isotopic composition of Bothrops atrox (Linnaeus, 1758) living in different landscapes located in the lower Amazon river, encompassing four main natural landscapes of the Amazon: old-growth forests, várzeas (flooded forests), savannas, and pastures. Our null hypothesis is that the δ13C of forest specimens of B.atrox is more negative because forests are dominated by C3 plants, while C4 plants are common in the other landscapes. On the other hand, δ15N of forest specimens should be more positive, since the δ15N of old-growth forests are higher than plants of savanna, várzea, and pastures. Confirming our hypothesis, the δ13C of B. atrox scales of the Tapajós National Forest was approximate −25‰ to −24‰, increased to approximately −23.5‰ to −23.0‰ in the savanna and pasture, and to −21‰ in the várzea, showing an increased contribution of C4-derived carbon. Some specimens of B. atrox had δ15N as high as 18‰, which is much higher than the average δ15N of the snake's prey (7‰), confirming the apex position of B. atrox in the Amazon region. The δ15N values of the forest specimens were 5‰ higher than the savanna specimens, and this difference decreased to 3‰ between the forest and the pasture, and the várzea specimens. Finally, there were not large differences between δ15N values of livers and scales in any of the landscapes, suggesting a constant diet through time, and reinforcing the possibility of the use of snake's scale as a less invasive and non-lethal tissue to analyze.

Evidence for snake venom plasticity in a long-term study with individual captive Bothrops atrox

Variability in snake venom composition has been frequently reported and correlated to the adaptability of snakes to environmental conditions. Previous studies report plasticity for the venom phenotype. However, these observations are not conclusive, as the results were based on pooled venoms, which present high individual variability. Here we tested the hypothesis of plasticity by influence of confinement and single diet type in the venom composition of 13 adult specimens of Bothrops atrox snakes, maintained under captivity for more than three years. Individual variability in venom composition was observed in samples extracted just after the capture of the snakes. However, composition was conserved in venoms periodically extracted from nine specimens, which presented low variability restricted to the less abundant components. In a second group, composed of four snakes, drastic changes were observed in the venom samples extracted at different periods, mostly related to snake venom metalloproteinases (SVMPs), the core function toxins of B. atrox venom, which occurred approximately between 400 and 500 days in captivity. These data show plasticity in the venom phenotype during the lifetime of adult snakes maintained under captive conditions. Causes or functional consequences involved in the phenotype modification require further investigations.

Evidence for snake venom plasticity in a long-term study with individual captive Bothrops atrox

Variability in snake venom composition has been frequently reported and correlated to the adaptability of snakes to environmental conditions. Previous studies report plasticity for the venom phenotype. However, these observations are not conclusive, as the results were based on pooled venoms, which present high individual variability. Here we tested the hypothesis of plasticity by influence of confinement and single diet type in the venom composition of 13 adult specimens of Bothrops atrox snakes, maintained under captivity for more than three years. Individual variability in venom composition was observed in samples extracted just after the capture of the snakes. However, composition was conserved in venoms periodically extracted from nine specimens, which presented low variability restricted to the less abundant components. In a second group, composed of four snakes, drastic changes were observed in the venom samples extracted at different periods, mostly related to snake venom metalloproteinases (SVMPs), the core function toxins of B. atrox venom, which occurred approximately between 400 and 500 days in captivity. These data show plasticity in the venom phenotype during the lifetime of adult snakes maintained under captive conditions. Causes or functional consequences involved in the phenotype modification require further investigations.

Molecular mechanisms underlying intraspecific variation in snake venom

Elucidating the molecular mechanisms underlying snake venom variability provides important clues for understanding how the biological functions of this powerful toxic arsenal evolve. We analyzed in detail individual transcripts and venom protein isoforms produced by five specimens of a venomous snake (Bothrops atrox) from two nearby but genetically distinct populations from the Brazilian Amazon rainforest which show functional similarities in venom properties. Individual variation was observed among the venoms of these specimens, but the overall abundance of each general toxin family was conserved both in transcript and in venom protein levels. However, when expression of independent paralogues was analyzed, remarkable differences were observed within and among each toxin group, both between individuals and between populations. Transcripts for functionally essential venom proteins ("core function" proteins) were highly expressed in all specimens and showed similar transcription/translation rates. In contrast, other paralogues ("adaptive" proteins) showed lower expression levels and the toxins they coded for varied among different individuals. These results provide support for the inferences that (a) expression and translational differences play a greater role in defining adaptive variation in venom phenotypes than does sequence variation in protein coding genes and (b) convergent adaptive venom phenotypes can be generated through different molecular mechanisms. Significance: Analysis of individual transcripts and venom protein isoforms produced by specimens of a venomous snake (Bothrops atrox), from the Brazilian Amazon rainforest, revealed that transcriptional and translational mechanisms contribute to venom phenotypic variation. Our finding of evidence for high expression of toxin proteins with conserved function supports the hypothesis that the venom phenotype consists of two kinds of proteins: conserved "core function" proteins that provide essential functional activities with broader relevance and less conserved "adaptive" proteins that vary in expression and may permit customization of protein function. These observations allowed us to suggest that genetic mechanisms controlling venom variability are not restricted to selection of gene copies or mutations in structural genes but also to selection of the mechanisms controlling gene expression, contributing to the plasticity of this important phenotype for venomous snakes.

Recent lineage diversification in a venomous snake through dispersal across the Amazon River

Identifying the evolutionary and ecological mechanisms that drive lineage diversification in the species-rich tropics is of broad interest to evolutionary biologists. Here, we use phylogeographical and demographic analyses of genome-scale RADseq data to assess the impact of a large geographical feature, the Amazon River, on lineage formation in a venomous pitviper, Bothrops atrox. We compared genetic differentiation in samples from four sites near Santarem, Brazil, that spanned the Amazon and represented major habitat types. A species delimitation analysis identified each population as a distinct evolutionary lineage while a species tree analysis with populations as taxa revealed a phylogenetic tree consistent with dispersal across the Amazon from north to south. Phylogenetic analyses of mitochondrial DNA variation confirmed this pattern and suggest that all lineages originated during the mid- to late Pleistocene. Historical demographic analyses support a population model of lineage formation through isolation between lineages with low ongoing migration between large populations and reject a model of differentiation through isolation by distance alone. The results provide a rare example of a phylogeographical pattern demonstrating dispersal over evolutionary timescales across a large tropical river and suggest a role for the Amazon River as a driver of in situ divergence both by impeding (but not preventing) gene flow and through parapatric differentiation along an ecological gradient.

Molecular mechanisms underlying intraspecific variation in snake venom

Elucidating the molecular mechanisms underlying snake venom variability provides important clues for understanding how the biological functions of this powerful toxic arsenal evolve. We analyzed in detail individual transcripts and venom protein isoforms produced by five specimens of a venomous snake (Bothrops atrox) from two nearby but genetically distinct populations from the Brazilian Amazon rainforest which show functional similarities in venom properties. Individual variation was observed among the venoms of these specimens, but the overall abundance of each general toxin family was conserved both in transcript and in venom protein levels. However, when expression of independent paralogues was analyzed, remarkable differences were observed within and among each toxin group, both between individuals and between populations. Transcripts for functionally essential venom proteins ("core function" proteins) were highly expressed in all specimens and showed similar transcription/translation rates. In contrast, other paralogues ("adaptive" proteins) showed lower expression levels and the toxins they coded for varied among different individuals. These results provide support for the inferences that (a) expression and translational differences play a greater role in defining adaptive variation in venom phenotypes than does sequence variation in protein coding genes and (b) convergent adaptive venom phenotypes can be generated through different molecular mechanisms. Significance: Analysis of individual transcripts and venom protein isoforms produced by specimens of a venomous snake (Bothrops atrox), from the Brazilian Amazon rainforest, revealed that transcriptional and translational mechanisms contribute to venom phenotypic variation. Our finding of evidence for high expression of toxin proteins with conserved function supports the hypothesis that the venom phenotype consists of two kinds of proteins: conserved "core function" proteins that provide essential functional activities with broader relevance and less conserved "adaptive" proteins that vary in expression and may permit customization of protein function. These observations allowed us to suggest that genetic mechanisms controlling venom variability are not restricted to selection of gene copies or mutations in structural genes but also to selection of the mechanisms controlling gene expression, contributing to the plasticity of this important phenotype for venomous snakes.

Recent lineage diversification in a venomous snake through dispersal across the Amazon River

Identifying the evolutionary and ecological mechanisms that drive lineage diversification in the species-rich tropics is of broad interest to evolutionary biologists. Here, we use phylogeographical and demographic analyses of genome-scale RADseq data to assess the impact of a large geographical feature, the Amazon River, on lineage formation in a venomous pitviper, Bothrops atrox. We compared genetic differentiation in samples from four sites near Santarem, Brazil, that spanned the Amazon and represented major habitat types. A species delimitation analysis identified each population as a distinct evolutionary lineage while a species tree analysis with populations as taxa revealed a phylogenetic tree consistent with dispersal across the Amazon from north to south. Phylogenetic analyses of mitochondrial DNA variation confirmed this pattern and suggest that all lineages originated during the mid- to late Pleistocene. Historical demographic analyses support a population model of lineage formation through isolation between lineages with low ongoing migration between large populations and reject a model of differentiation through isolation by distance alone. The results provide a rare example of a phylogeographical pattern demonstrating dispersal over evolutionary timescales across a large tropical river and suggest a role for the Amazon River as a driver of in situ divergence both by impeding (but not preventing) gene flow and through parapatric differentiation along an ecological gradient.

Functional proteomic analyses of Bothrops atrox venom reveals phenotypes associated with habitat variation in the Amazon

Venom variability is commonly reported for venomous snakes including Bothrops atrox. Here, we compared the composition of venoms from B. atrox snakes collected at Amazonian conserved habitats (terra-flrme upland forest and vcirzea) and human modified areas (pasture and degraded areas). Venom samples were submitted to shotgun proteomic analysis as a whole or compared after fractionation by reversed-phase chromatography. Whole venom proteomes revealed a similar composition among the venoms with predominance of SVMPs, CTLs, and SVSPs and intermediate amounts of PLA(2)s and LAAOs. However, when distribution of particular isoforms was analyzed by either method, the venom from varzea snakes showed a decrease in hemorrhagic SVMPs and an increase in SVSPs, and procoagulant SVMPs and PLA(2)s. These differences were validated by experimental approaches including both enzymatic and in vivo assays, and indicated restrictions in respect to antivenom efficacy to variable components. Thus, proteomic analysis at the isoform level combined to in silica prediction of functional properties may indicate venom biological activity. These results also suggest that the prevalence of functionally distinct isoforms contributes to the variability of the venoms and could reflect the adaptation of B. atrox to distinct prey communities in different Amazon habitats. Biological significance: In this report, we compared isoforms present in venoms from snakes collected at different Amazonian habitats. By means of a species venom gland transcriptome and the in silico functional prediction of each isoform, we were able to predict the principal venom activities in vitro and in animal models. We also showed remarkable differences in the venom pools from snakes collected at the floodplain (varzea habitat) compared to other habitats. Not only was this venom less hemorrhagic and more procoagulant, when compared to the venom pools from the other three habitats studied, but also this enhanced procoagulant activity was not efficiently neutralized by Bothrops antivenom. Thus, using a functional proteomic approach, we highlighted intraspecific differences in B. atrox venom that could impact both in the ecology of snakes but also in the treatment of snake bite patients in the region.