**1. Introduction**

Snake venoms are complex mixtures of toxic components belonging to multipleprotein families [1], each of which expresses several isoforms that are present in the venoms in different proportions [2]. The concentration of each isoform present in the venoms is highly variable and modulates venom function [3] and, as a consequence, the complexity

Del-Rei, T.H.M.; Rocha, M.M.T.; Mourão, R.H.V.; Chalkidis, H.M.; Prezoto, B.; Gibbs, H.L.; Moura-da-Silva, A.M. Individual Variability in *Bothrops atrox* Snakes Collected from Different Habitats in the Brazilian Amazon: New Findings on Venom Composition and Functionality. *Toxins* **2021**, *13*, 814. https://doi.org/10.3390/ toxins13110814

**Citation:** Sousa, L.F.; Holding, M.L.;

Received: 13 September 2021 Accepted: 10 November 2021 Published: 18 November 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of the venoms is associated with differences in their toxicity to a wide range of prey [4]. In addition, differences in venom composition are argued to be an adaptation that has evolved to facilitate the capture and digestion of prey and evasion of predators and is observed across all taxonomic levels, particularly in species with wide distributions [5,6].

*Bothrops atrox* (common lancehead) is a terrestrial pitviper widely distributed across the Amazon, from tropical lowlands to the rainforest of northern South America east of the Andes [7], where it occupies different types of habitats. Variability is widely reported in the composition of *B. atrox* venom and has been associated with ontogeny [8–10], geographical distribution [11–14], and environmental characteristics [15]. This species is responsible for most of the human envenomations in the Amazon region [16]. In envenomed patients, the intraspecific variability in *B. atrox* venom composition may hamper the patients' prognosis as venom isoforms are involved in distinct clinical symptoms [17]. In some cases, functionally relevant isoforms present in high levels in venoms of a particular group of snakes may show lower reactivity with antivenoms and reduce the effectiveness of the treatment of some victims of snakebite [15,18].

Genetic differences among populations may correspond to the expression of different venom isoforms with distinct or similar functions [2,14]. However, venom variability also occurs among specimens from the same geographical areas [2] and may occur during the life span of individuals as ontogenetic variation related to an increase in body size, allowing capture and digestion of larger prey [19,20].

Large rivers in the Amazon basin contain ecologically diverse habitats and these have been associated with diversification in specific groups of vertebrates, such as birds [21]. In the west of Pará State, Brazil, the Amazon River is wide and has distinct habitats, such as upland forests on either bank and floodplain habitats. These represent distinct habitats that could generate diversification in venom phenotypes. For the last few years, our studies have been focusing on the variability in venom composition in this particular region of the Brazilian Amazon. Snakes have been collected at forest, pasture, and floodplain areas on both banks of the Amazon river. Using genome-scale RADseq data, we showed an interesting pattern of gene dispersal, suggesting 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 [22]. The transcriptomes of the venom glands from the snakes collected in the northern or southern banks of the river denoted the same pattern of transcripts regarding the major toxin groups, but with the expression of different alleles or paralogs in snakes from the northern or the southern banks [2]. Using the comprehensive transcriptomic annotations described above, we compared the phenotype of pooled venoms from *B. atrox* snakes collected forest, pasture, or floodplain habitats by comparing the major toxic activities with data from free-label proteomics of the whole pools of venoms and the identification of relevant isoforms separated from these pools by RP-HPLC chromatographies, also by the proteomics of each fraction [15]. We observed two predominant phenotypes: pooled venoms from the forest, pasture, and degraded areas were more hemorrhagic, while the venom pool from snakes collected at the floodplain was more procoagulant. However, these analyses used pooled venom samples and so the level and significance of venom variation at the level of the individual snake are not understood. This information could help clarify the relative importance of habitat compared to individual variation as modulators of the venom variability observed in our previous report [15].

Here, using individual venom samples extracted from the snakes collected at the same habitats, and the proteomics information obtained previously, we investigated the hypothesis that the heterogeneity in venom composition could be higher in more unsettled environments. We found that the variability in the composition and functional activities is higher within individual venoms from snakes collected at the floodplain habitat, an extremely dynamic environment subjected to drastic seasonal changes, thus supporting our initial assumption. Moreover, our findings provide a deeper view of the main toxins and biological activities related to the individual venom variability within these *B. atrox*

groups and suggest that the functional diversity of the venoms appears to be relevant to the ability of these snakes to persist in highly variable unsettled environments, such as the floodplain habitat.

#### **2. Results**
