Snake venoms are complex secretions of various enzymes, peptides and small organic molecules that have diverse modes of action on both prey and accidental human bite victims [1,2,3,4,5,6]. Though targeting a wide array of physiological processes and targets, the proteins that make up snake venom come from relatively few protein families. This is due to core protein scaffolds being maintained with accelerated rates of evolution at the secondary through quaternary levels facilitating the evolution of multiple derived functions within each protein family [3,7,8,9]. The use of mass spectrometry in venom proteomics [7,10,11,12,13,14,15,16,17,18,19] and venom gland transcriptome analysis [9,20,21,22,23,24,25,26,27,28] has improved knowledge of venom composition tremendously. Variation in venom profiles has been shown interspecifically [7,13,18,19,29,30,31,32,33,34] and intraspecifically, with intraspecific differences found among geographic locales [12,35,36,37,38,39,40,41,42,43], between sexes [36,37,44,45], and between age classes [10,15,46,47,48,49,50,51,52]. Some authors have argued that diversity in venom composition is the result of neutral evolutionary processes and is not subject to natural selection [53,54], whereas others have argued that adaptation has been driven, by strong natural selection, to specific prey species [19,29,36,37,55,56,57,58,59,60].

New World pitvipers are believed to be descendants of an ancestral Asian pitviper species, by way of a single New World colonization event, via the Bering land bridge [61,62,63,64]. In regards to the current study particular interest is given to the members of the genera Bothriechis and Bothrops that have independently evolved to occupy arboreal niches. That Bothriechis is a distinctive genus from the “bothropoid” genera [61] has been asserted by multiple authors [61,62,63,64,65,66,67]; however, phylogenetic relationships within the bothropoids, in particular the validity of Bothriopsis, have proven more difficult due to limited sampling of taxa. Numerous studies have presented evidence of the paraphyly of Bothrops with respect to Bothriopsis [61,62,63,64,65,66,67,68,69,70,71,72] leading some authors to advocate for the synonymizing of Bothriopsis and Bothrocophias with Bothrops [70,72] or Bothriechis [73], while others have maintained that Bothriopsis and Bothrocophias should remain distinct [61,62,65,74,75] (Figure 1). Regardless of taxonomical divisions preferred, it is clear that the arboreal specialization has occurred upon two separate occasions.

Recent studies utilizing a combined morphological/molecular approach and increased sampling of taxa have disagreed on generic assignment as well. Fenwick et al. (2009), sampling 90% of the known “bothropoid” taxa and utilizing both morphological characters and mtDNA sequence data found support for several monophyletic clades within Bothrops (sensu lato) and proposed taxonomic revisions, including recognition of Bothriopsis, based on the distinct species groups recovered [76]. Carrasco et al. (2012) [77] incorporated additional morphological characters not utilized by Fenwick et al. (2009) [76] in addition to previously published molecular sequences and ecological characters. Citing possible paraphyly of Bothrops with respect to Bothriopsis (with the inclusion of Bothrops sanctaecrucis), they proposed following the recommendation of Salomão et al. (1997) [70] and Wüster et al. (2002) [72] and synonymized Bothriopsis with Bothrops [77]. We have followed the proposals of Salomão et al. (1997) [70] and Wüster et al. (2002) [72] in recognizing the bothropoids as a single genus, Bothrops, as the weight of current evidence (cited above) favors this classification.

Despite the medical significance of pitviper envenomations in the New World [78,79,80,81,82,83], the majority of toxinological research published to date has been on North American members of the genus Crotalus [14,25,26,35,38,39,40,41,42,43,49,50,82,83,84,85,86,87,88,89,90], and the larger members of the genus Bothrops [10,91,92,93,94,95,96,97,98,99,100,101,102,103,104]. While investigations into Bothriechis, in particular B. schlegelii, venom [31,105,106,107] and some of the smaller bothropoid pitvipers [108,109,110,111] have occurred, knowledge gaps remain on the proteomic make up of the venom of the majority of New World pitviper species. This knowledge gap is unfortunate as snake venoms have proved valuable sources of investigational ligands [112,113,114,115,116] and potential therapeutics [117,118,119,120,121,122,123,124] due to the high degree of target specificity exhibited by their constituent toxins. Bothrops and Bothriechis are both medically relevant species across their range and together are responsible for a large number of severe envenomations [78,79,80,81,125,126]. American pitviper venoms are typically rich in two types of enzymes: Snake venom metalloprotease (SVMP) and phospholipase A₂ (PLA₂) [3,42,127]. Due to variance in retention of ancestral domains, SVMPs span a broad range of molecular weights from ~30–70 kDa and exhibit a variety of activities. Structural diversity within SVMPs is the basis for the development of a variety of symptoms in victims of envenomation including; haemorrhage, edema, hypotension, hypovolemia, inflammation, and necrosis [128,129]. The realities of inter/intraspecific venom variation necessitate a thorough understanding of species venom proteomes as the information allows for improved clinical treatment of envenomations by way of informing antivenom selection for heterologous venoms [127,130]. Determining a species proteome is often the first step of antivenomic studies undertaken to confirm antivenom neutralization efficacy of species/populations/age classes [12,84,131] and determination of toxins unable to be neutralized is valuable information not only for clinicians in charge of managing envenomations but also for future antivenom production.

In this study we investigate the venom proteomes of arboreal members of the Bothriechis and Bothrops genera that have independently invaded the tree canopies of South America [7,31,64,71,77,107]. In both genera variation in adult size has been documented, with Bothriechis aurifer, Bothriechis lateralis and Bothriechis marchi typically exceeding Bothriechis schlegelli in adult size, while Bothrops bilineata is a much smaller species than Bothrops taeniata. For comparison, we have included two terrestrial Bothrops species that also differ markedly in maximum adult size, with Bothrops asper reaching adult sizes in excess of twice that of Bothrops neuwiedi bolivianus. While B. lateralis and B. schlegelli have been proteomically previously profiled, as has Bothriechis nigroviridis [31,132], their venom variation has not been considered in relation to size and prey preference.

Shotgun mass spectrometry recovered proteins of known pitviper toxin types (Table 1) in agreement with previous proteomic [10,11,13,14,30,32,83,84,92] and transcriptomic analyses [22,24,29,59,77,89,90,93,95,97,103].

1D gel analysis revealed greater complexity in all venoms than indicated by the shotgun results. The venoms of the larger Bothriechis species (B. aurifer, B. lateralis and B. marchi) contained more P-III, P-II and P-I SVMP than the venom of the smaller sized species B. schlegelii (Figure 2). In contrast, B. schlegelii venom contained more PLA₂ (lower molecular weights of synovial PLA₂ 12–15 kDa). A similar correlation between body size and SVMP/PLA₂ content was found in arboreal Bothrops. However this trend was reversed in the terrestrial Bothrops. These results were complemented by the 2D gel analyses (Figure 3).

The relative straightforward proteomics trends were not reflected by the most complex differences in venom composition revealed by the bioactivity testing, which is consistent with venoms being complex mixtures of bioactive substances. While B. aurifer was indeed much more potent than B. schlegelli in a fluorescence-based metalloprotease activity assay (Figure 4), and B. lateralis also more active, B. marchi was only weakly active, being equipotent to B. schlegelli. This result was despite B. marchi having very similar 1D and 2D gel patterns to the more potent B. lateralis, especially in the known metalloprotease regions. It must be noted that these trends are of course specific only to this particular substrate and that other assays (such as the gel based zymography) may present differential results. Indeed, further investigation of metalloprotease activity yielded results that contrasted those gathered with the aforementioned analyses (Figure 4). Zymography gels with a casein substrate revealed active P-III SVMP in all four Bothriechis venoms in the 75 kDa region (Figure 2). The region of digestion in the casein zymography gel (Figure 5) corresponds not with the 50 kDa heavy bands seen in the 1D gels (Figure 2), but a 75 kDa region above that, with the 50 kDa 1D bands clearly evident in the zymography gels below the zone of digestion. This suggests that this digestive activity is due to the presence of heavily glycosylated enzymes that are resistant to staining, while the well-stained 50 kDa serine protease bands do not have this type of activity. In contrast, zymography gels with gelatin as the substrate produced bands of digestion in the Bothriechis venoms at the 50 kDa region (Figure 5), except for B. aurifer despite it having seemingly homologous serine protease band in the same region.

The arboreal Bothrops venoms were both weakly active on the casein substrate zymography gel (Figure 5). However, these two venoms differed sharply in the gelatin substrate zymography gel, with B. taeniata having two discrete zones of digestion, one of which was similar to that of the Bothriechis species, while the other was higher in molecular weight (Figure 5). Neither arboreal Bothrops venom, however, displayed significant activity in the fluorescence based assay. Both terrestrial Bothrops species were only weakly active in either the casein or gelatin (Figure 5) substrate zymography gels. However both were active in the fluorescence based metalloprotease assay, with B. asper displaying high levels of activity.

PLA₂ activity levels, however, were more congruent with the relative presence of venom components in the corresponding range (12–15 kDa), with B. aurifer and B. marchi having only weak activity while B. lateralis and B. schlegelli were very active (Figure 6). This is consistent with the latter two species having darker staining bands at ~12–15 kDa than the former two species. Thus envenomations by B. lateralis and B. schlegelli would be expected to produce more myotoxicity than envenomations by B. aurifer and B. marchi. Despite Bothrops bilineata and B. taeniata both containing abundant PLA₂, they were only weakly active in this region, suggesting that myotoxicity would not be as significant a complication but that other non-enzymatic PLA₂ activities (e.g., antiplatelet aggregation or neurotoxicity) may be more potent in these two species. Similarly, despite both being rich in PLA₂, the two terrestrial Bothrops species displayed significantly different levels of PLA₂ enzymatic activity. Bothrops asper displayed high levels of PLA₂ enzymatic activity, consistent with myotoxic envenomation effects, while Bothrops neuwiedi bolivianus was much less potent in this regard.

For the Bothrops species tested, SVMP levels are correlated with consumed prey class percentages, reported by Martins et al. (2002) [132], with species containing higher percentage of anurans in their diet (B. bilineata and B. taeniata) possessing lower levels of SVMP activity than species that contained non-volant mammals as a higher percentage of their diet (B. asper and B. neuwiendi). Pitviper venoms have been dichotomously classified into type 1 (proteolytic or “tenderizer”) and type II (more toxic) venoms [133], as anurans have a high surface area to volume ratio and skin lacking tough dermal scales, it is unlikely that strong selection pressures for venoms with high metalloprotease activity exist in species that contain anurans as a large percentage of their diet as the need for such cleavage enzymes is reduced. The increased levels of SVMP activity for non-volant mammal generalist B. asper (59.4% mammals) in regards to B. neuwiedi (93.1% mammals) may be due to size class of mammal taken, as pitvipers are gape-limited predators that consume their prey whole [134,135,136]. Pitviper SVL (snout vent length) in the North American pitviper, Crotalus atrox, was found as the best predictor of overall maximum gape [137]. Although B. asper is recorded as consuming less percentage of mammals than B. neuwiedi, B. asper obtains an average SVL length roughly twice that of B. neuwiedi [131], suggesting the requirement for increased amounts of SVMP, as larger prey requires increased levels of SVMP which impairs homeostasis during predation. In addition our venom samples were taken from adult individuals displaying the predicted adult requirement of toxin activity. Other studies have documented that pitvipers consume prey species with lower surface area to volume ratios as they grow [50,132,138,139] and the lytic activities of SVMP may aid in predigestion of acquired prey. A detailed dietary analysis including prey species/size would be necessary in order to determine if B. asper does indeed consume larger prey/ prey of lower surface area to volume ratio than B. neuwiedi.

Detailed dietary studies for members of Bothriechis are lacking. As a whole the genus appears to be a generalist with an ontogenetic shift toward mammalian prey, with B. lateralis and B. schlegelii even documented as occasionally predating on bats and small birds (Campbell and Lamar 2004 and references therein) [131]. We detected increased levels of SVMP activity for B. aurifer in relation to other members of Bothriechis. This increased level of SVMP activity in combination with Campbell and Lamar (2004) [131] noting that the snake is frequently encountered on the ground is suggestive that B. aurifer may contain a higher percentage of non-volant mammals in its diet than the other members of Bothriechis tested. In contrast to high activity levels of SVMP recorded in B. aurifer, B. schleglii and B. lateralis both present higher levels of sPLA₂ activity in relation to other members of Bothriechis. The increased levels of sPLA₂ activity present in the venom profiles of B. lateralis and B. schleglii is suggestive of their arboreal encounters with volant animals. As sPLA₂ is a neurotoxin affecting the nervous system, recruiting such a toxin would aid in quickly immobilizing flying prey whilst ambushing from a tree branch high up in the forest canopy, therefore reducing the risk of escape and injury.

Detailed knowledge of venom components present in Bothrops and Bothriechis venoms has the ability to improve treatment for those receiving an envenomation [12,15,84,126,130,140,141]. This is an important task as the two genera are responsible for a considerable number of bites [78,79,80,81,82,83] and knowledge of inter and intraspecific venom variation is crucial for optimizing neutralization capabilities of antivenoms cf. [84,126].

Further research into the identification and activity of toxins present in the venoms of snakes within this clade will be conducted in order to elucidate the selection pressures that have shaped these fine-tuned weapon systems. This research will hopefully contribute towards filling existing knowledge gaps concerning the evolution of venom composition and activity in the Viperidae.