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-firme upland forest and várzea) 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 PLA2s and LAAOs. However, when distribution of particular isoforms was analyzed by either method, the venom from várzea snakes showed a decrease in hemorrhagic SVMPs and an increase in SVSPs, and procoagulant SVMPs and PLA2s. 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 silico 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 (várzea 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.

Snake population venomics and antivenomics of Bothropsatrox: Paedomorphism along its transamazonian dispersaland implications of geographic venom variability on snakebite management

We describe two geographically differentiated venomphenotypes across the wide distributionrange of Bothrops atrox, fromthe ColombianMagdalena Medio Valley through Puerto Ayacuchoand El Paují, in the Venezuelan States ofAmazonas and Orinoquia, respectively, and São Bentoin the Brazilian State of Maranhão. Colombian and Venezuelan venoms show an ontogenetictoxin profile phenotype whereas Brazilian venoms exhibit paedomorphic phenotypes.Venoms from each of the 16 localities sampled contain both population-specific toxins andproteins shared by neighboring B. atrox populations.Mapping themolecular similarity betweenconspecific populations onto a physical map of B. atrox range provides clues for tracingdispersal routes that account for the current biogeographic distribution of the species. Theproteomic pattern is consistent with a model of southeast and southwest dispersal andallopatric fragmentation northern of the Amazon Basin, and trans-Amazonian expansionthrough the Andean Corridor and across the Amazon river between Monte Alegre andSantarém. An antivenomic approach applied to assess the efficacy towards B. atrox venoms oftwo antivenomsraised in Costa Rica and Brazil using Bothrops venomsdifferent than B. atrox inthe immunization mixtures showed that both antivenoms immunodepleted very efficientlythe major toxins (PIII-SVMPs, serine proteinases, CRISP, LAO) of paedomorphic venoms fromPuerto Ayacucho (Venezuelan Amazonia) through São Bento, but had impaired reactivitytowards PLA2 and P-I SVMP molecules abundantly present in ontogenetic venoms. The degreeofimmunodepletion achieved suggests that each of these antivenomsmay be effective againstenvenomations by paedomorphic, and some ontogenetic, B. atrox venoms.