One venom protein superfamily that is prevalent in snake venoms, with an incredibly vast range of activities, are the three-finger toxins (3FTxs). Three-finger toxins can be neurotoxic, acting as antagonists of nicotinic acetylcholine receptors (nAChRs) (Chang and Lee, 1963; Nirthanan and Gwee, 2004; Bourne et al., 2005), muscarinic acetylcholine receptors (mAChRs) (Karlsson et al., 2000; Chung et al., 2002), adrenergic receptors (Rajagopalan et al., 2007), GABA receptors (Rosso et al., 2015), or even bind to and alter the activation of ion channels (de Weille et al., 1991; Rivera-Torres et al., 2016; Yang et al., 2016). These toxins can also be anticoagulants, inhibiting platelet aggregation (Kini et al., 1988; McDowell et al., 1992). Regardless of activity, all 3FTxs maintain a conserved structural scaffold of three β-stranded loops, crosslinked by four disulfide bridges. This forms the “three-finger” arrangement, appearing like three fingers of a hand. Three-finger toxins are non-enzymatic, small proteins consisting of 60–85 amino acid residues, and occur either as monomers, which is most common, or as dimers (Kini and Doley, 2010).

Elapid venoms are the most well-known sources of 3FTxs (Fry et al., 2003b), but 3FTxs have been documented in the venoms of many RFS species (Fry et al., 2003a, 2008; Pawlak et al., 2006, 2009; Heyborne and Mackessy, 2013; Junqueira-de-Azevedo et al., 2016) and can make up large portions of these venoms, as much as 84–92% of the total venom composition (Pla et al., 2017a; Modahl et al., 2018b). Rear-fanged snake genera that have abundant proteins in the molecular mass range of 3FTxs include Boiga, Spilotes, Trimorphodon, Oxybelis, Leioheterodon, Psammophis, Rhamphiophis, and Thelotornis (Figure 2A). Of these snakes mentioned, 3FTxs have been sequenced and functionally characterized from the venoms of Boiga dendrophila (Lumsden et al., 2005; Pawlak et al., 2006), Boiga irregularis (Pawlak et al., 2009), Oxybelis fulgidus (Heyborne and Mackessy, 2013), and Spilotes sulphureus (Modahl et al., 2018b). Transcripts for 3FTx have been found in the venom gland, or in the venom, of Boiga cynodon, Boiga nigriceps, Trimorphodon biscutatus, Ahaetulla prasina, Leioheterodon madagascariensis, Thrasops jacksonii, and Psammophis mossambicus (Fry et al., 2008; Modahl and Mackessy, 2016; Dashevsky et al., 2018; Modahl et al., 2018a), among others.

Three-finger toxins were one of the first venom proteins characterized pharmacologically in RFS venoms and were reported to exhibit postsynaptic neurotoxicity, similar to elapid α-neurotoxins (Broaders et al., 1999; Fry et al., 2003a; Lumsden et al., 2005). Several 3FTxs from RFS venoms have been the only well-characterized venom proteins with taxon-specific toxicities, showing preferential binding to lizard or bird nAChRs, in direct relation to snake diet (Pawlak et al., 2006, 2009). Several elapid venoms have shown differential toxicity toward bird or rodent neuromuscular preparations (e.g., Hart et al., 2012, 2013), but these studies were on crude venoms and did not include toxicity assays on native prey species. Sequences and structures of 3FTxs isolated from RFS have an elongated N-terminal segment, and tend to be larger in size than observed in FFS venoms (Lumsden et al., 2007; Pawlak et al., 2009; Heyborne and Mackessy, 2013; Modahl et al., 2018b). It is currently unknown how this longer N-terminal region is involved in receptor binding.

Several of these 3FTxs are also N-terminally blocked by a pyroglutamic acid residue (Broaders et al., 1999; Fry et al., 2003a; Pawlak et al., 2006; Heyborne and Mackessy, 2013). This pyroglutamic acid blockage especially makes these proteins hard to sequence by Edman degradation, and obtaining venom protein transcript sequences has been the most successful approach to study 3FTx amino acid residue diversity in RFS (Fry et al., 2008; McGivern et al., 2014; Modahl and Mackessy, 2016; Pla et al., 2017a; Dashevsky et al., 2018; Modahl et al., 2018b). Rear-fanged snake 3FTxs demonstrate low amino acid sequence identity with 3FTxs from elapids (usually <50%), and all RFS 3FTxs are members of the “non-conventional” toxin classification, characterized by an additional fifth disulfide bond in the first loop (Nirthanan et al., 2003). This type of 3FTx also occurs in elapid venoms, where it was first characterized in cobra (Naja) venoms (Carlsson, 1975; Utkin et al., 2001), but they are the only 3FTx type present in RFS venoms. This suggests that the non-conventional cysteine pattern may be the more basal arrangement for 3FTxs, and provides insight into the evolution of 3FTxs (cf. Fry et al., 2003b). The incredible diversity of RFS 3FTxs with low identity to elapid 3FTxs, combined with the binding selectivity exhibited by some of these toxins, provides a database of proteins to explore how venom proteins target prey and adapt toxicity, and will be discussed more in another section below.

Snake venom metalloproteinases occur primarily in viperid venoms (Mackessy, 2010b), and similar to 3FTxs, are also a large multigene family, exhibiting a diversity of activities. These enzymes have effects including hemorrhage, coagulopathy, fibrinolysis, apoptosis, and the activation of factor X and prothrombin (Takeya et al., 1993). Many SVMPs function by degrading endothelial cell membrane components or target proteins involved in coagulation, such as fibrinogen or platelet receptors (Takeda et al., 2012). As part of the metzincin superfamily of proteinases, they are characterized by the presence of the Zn²⁺-binding motif HEXXHXXGXXH at the catalytic site. Snake venom metalloproteinases are closely related to mammalian ADAM (a disintegrin and metalloproteinase) and ADAMTS (ADAM with thrombospondin type-1 motif), but differ in domain organization (Fox and Serrano, 2005; Takeda et al., 2012). The SVMP P-I class has only a catalytic metalloproteinase domain present, P-IIs contain a metalloproteinase domain followed by a disintegrin domain, and P-IIIs have a metalloproteinase, disintegrin-like, and cysteine-rich domain (Hite et al., 1994; Fox and Serrano, 2005).

In some RFS venoms, SVMPs are the most abundant toxins, making up 62–70% of the total venom composition (Modahl et al., 2018a). SVMP-dominated venoms are found in RFS species such as A. prasina (Modahl et al., 2018a), B. portoricensis (Weldon and Mackessy, 2012; Modahl et al., 2018a), D. typus (Kamiguti et al., 2000; Pla et al., 2017b), Hydrodynastes gigas (Hill and Mackessy, 2000), Hypsiglena torquata (McGivern et al., 2014), Phalotris mertensi (Campos et al., 2016), Pseudoboa neuwiedii (Torres-Bonilla et al., 2018), Thamnodynastes strigatus (Ching et al., 2012), Thamnophis sirtalis (Perry et al., 2018), and several Philodryas species (Ching et al., 2006; Peichoto et al., 2012; Urra et al., 2015) (Figure 2B). Potent SVMPs are observed in the venoms of snakes from the genus Philodryas (Assakura et al., 1994; Rocha et al., 2006; Sánchez et al., 2014), and venoms from these species in particular have been commonly reported to induced hemorrhage, myonecrosis and edema (Peichoto et al., 2005; Nery et al., 2014; Sánchez et al., 2014; Oliveira et al., 2017). The proteolytic activity of Philodryas venoms is inhibited by metal chelators (Assakura et al., 1992; Acosta et al., 2003; Peichoto et al., 2005, 2012; Rocha and Furtado, 2007), suggesting that SVMPs are resulting in these clinical snakebite symptomologies; in some species, serine proteinases may also be involved (Assakura et al., 1994; Peichoto et al., 2005; Ching et al., 2006). In comparison to venom from the pit viper species Bothrops jararaca, proteinase activity was 25 times greater for P. baroni venom (Sánchez et al., 2014), and also greater for P. olfersii and P. patagoniensis venoms (Carreiro da Costa et al., 2008). This demonstrates that the SVMP enzymatic activity from RFS venoms can be even more impressive than some FFS species. Philodryas sp. have been noted to share similar SVMP epitopes with snakes of the genus Bothrops (Assakura et al., 1992; Tancioni et al., 2004; Carreiro da Costa et al., 2008) and antivenom produced from Bothrops venoms have been observed to neutralize the systemic envenomation effects of Philodryas (Rocha et al., 2006).

Several SVMPs from RFS venoms have been purified and characterized, including patagonfibrase from P. patagoniensis (Peichoto et al., 2007), alsophinase from Borikenophis (previously the genus Alsophis) portoricensis (Weldon and Mackessy, 2012), and several from P. olfersii (Assakura et al., 1994). These characterized SVMPs are most noted for fibrin(ogen)olytic activity, preferentially degrading the α-chain of fibrinogen over the β-chain, however selectivity was variable, but none degraded the γ-chain (Assakura et al., 1994; Peichoto et al., 2007; Weldon and Mackessy, 2012). Patagonfibrase also impaired platelet aggregation induced by collagen and ADP (Peichoto et al., 2007). Both patagonfibrase and alsophinase were shown to cause hemorrhage and edema in mice, once again suggestive of the significant role SVMPs play in producing the envenomation symptomologies from these snakes (Peichoto et al., 2007, 2011; Weldon and Mackessy, 2012).

Only SVMPs of the P-III class has been reported in RFS (Saviola et al., 2014; Junqueira-de-Azevedo et al., 2016), with the exception of one truncated SVMP of a new class (Campos et al., 2016). This new type of SVMP was identified in the RFS P. mertensi, in which a P-III SVMP was discovered to be truncated in the middle of the disintegrin-like domain by a nonsense mutation in the gene; the resulting truncated SVMP was also observed in the venom (Campos et al., 2016). Rear-fanged SVMPs appear to have such nucleotide mutations frequently; within the venom gland transcriptome of B. portoricensis, a nucleotide substitution resulted in the elimination of the conserved stop codon and produced SVMP transcripts with a 9-residue extension at the C-terminus (Modahl et al., 2018a). P-IIIs were the first class of SVMPs recruited as a venom toxin (Casewell et al., 2011), and these RFS toxin genes and transcripts provide insight into the evolution of SVMPs and where alterations in ancestral gene sequences occurred, allowing the domains of this superfamily of proteins to diversify.

Cysteine-rich secretory proteins are common in many reptile venoms (Mackessy, 2002; Sunagar et al., 2012), but very little is currently known about their exact biological targets as venom components. These non-enzymatic proteins all share a conserved 16 cysteine residue pattern, forming eight disulfide bonds (Mackessy and Heyborne, 2010). CRiSPs lack proteolytic, hemorrhage and coagulant activity (Lodovicho et al., 2017). The few snake venom CRiSPs that have been characterized have been found to inhibit various ion channels (Nobile et al., 1996; Brown et al., 1999; Yamazaki et al., 2002; Wang et al., 2006) or induce inflammation, activating the complement system (Lodovicho et al., 2017).

CRiSPs occur in a wide range of RFS venoms (Hill and Mackessy, 2000; Peichoto et al., 2012), and are likely at least present at the transcript level for all species (Junqueira-de-Azevedo et al., 2016). Few CRiSPs have been purified and characterized from RFS venoms, but the CRiSP patagonin was purified from the venom of Philodryas patagoniensis and demonstrated necrotic activity toward murine gastrocnemius muscle when injected intramuscularly at doses of 43 μg and greater (Peichoto et al., 2009). It was suggested that patagonin was potentially binding to ion channels. Patagonin had no effect on the aggregation of human platelets and no proteolytic activity toward azocasein or fibrinogen (Peichoto et al., 2009). Helicopsin, a CRiSP from the RFS Helicops angulatus, was found have robust neurotoxic activity, causing respiratory paralysis in mice (Estrella et al., 2011). However, tigrin, a CRiSP isolated from the venom of RFS R. tigrinus tigrinus, was found to have no effect on high potassium or caffeine-induced contraction of helical strips of endothelium-free rat-tail arterial smooth muscle (Yamazaki et al., 2002), suggesting a lack of any neurotoxicity. The biological roles of CRiSPs in venoms still remains a bit unclear, but given the wide occurrence of these proteins in venoms, their conserved sequence and cysteine scaffold, and moderate-high levels of abundance in RFS venoms (often one of the most abundant venom components; Pla et al., 2017a; Modahl et al., 2018a), they likely serve important biological roles.

Other proteins that have been found in RFS venom proteomes include serine proteinases, phospholipase A₂s (type IA), acetylcholinesterases, and C-type lectins. Serine proteinases dominate many viper venoms (Mackessy, 2010b) and are responsible for both promoting and inhibiting blood coagulation, via activation of coagulation factors to induction of platelet aggregation or direct action on fibrinogen (Braud et al., 2000). In RFS venoms, serine proteinases have been identified in the venoms of P. olfersii (Assakura et al., 1994; Ching et al., 2006), P. patagoniensis (Peichoto et al., 2005), and P. mertensi (Campos et al., 2016), but overall these enzymes appear to be uncommon in this group of snakes. Phospholipase A₂s (type IA) are also uncommon in RFS venoms, but for some species, such as T. biscutatus lambda, PLA₂ enzymatic activity is detectable from crude venom and a PLA₂ has even been purified from this venom (Hill and Mackessy, 2000; Huang and Mackessy, 2004). Front-fanged snake venoms usually contain many PLA₂ isoforms and this large venom protein superfamily has a wide range of pharmacological effects, including neurotoxicity, myotoxic, cardiotoxic, anticoagulant, hemolytic, and hypotensive activities (Kini, 2003). PLA₂ enzymes from RFS appear to be more similar to PLA₂ sequences from elapid venoms (Huang and Mackessy, 2004; Fry et al., 2008), and perhaps serve a predigestive or prey capture role in RFS venoms, but they are not an abundant venom component. Acetylcholinesterase is another enzyme that has been detected at low levels in some RFS venoms, such as Boiga species (Broaders and Ryan, 1997; Hill and Mackessy, 2000) and Leptophis ahaetulla marginatus (Sánchez et al., 2018), but it is also not broadly distributed in RFS venoms (Mackessy, 2002; Junqueira-de-Azevedo et al., 2016).

C-type lectins (CTL) do appear to be a ubiquitous component of RFS venoms, and they are consistently present as transcripts in venom glands of RFS species (Junqueira-de-Azevedo et al., 2016). CTLs are non-enzymatic proteins capable of binding reversibly and non-covalently to carbohydrates, inducing hemagglutination by binding surface glycoconjugates on erythrocytes (Lu et al., 2005; Morita, 2005). Lectins have been isolated from many natural sources, and are more common components of viper than elapid venoms (Mackessy, 2010b). For FFS species, CTL abundance appears to be variable in snake venoms (Durban et al., 2011) and has only been detected at a transcript level in the venom gland of some species (e.g., Vonk et al., 2013). A CTL from the RFS Cerberus rynchops shared 79% sequence identity to a CTL from the elapid Bungarus multicinctus (OmPraba et al., 2010), and CTL transcripts from the venom gland of P. olfersii were also found to be more similar to those of elapids than viperids (Ching et al., 2006). All lectins have a carbohydrate recognition domain responsible for the glycan interaction activity, and the EPD triplet and conserved calcium-binding region indicated mannose specificity for the CTL discovered in venom from the RFS C. rynchops. Interestingly, CTLs in RFS have various glycan binding motifs, including EPD, QPD, EPN, RPS, QVE, and EPK (Fry et al., 2008; Junqueira-de-Azevedo et al., 2016). The variability in binding motifs in various FFS species suggests that these genes have greatly diversified in colubroids and might provide different functionalities in RFS venoms.

A new venom protein family, named veficolins (venom ficolins), was discovered in the venom of C. rynchops (OmPraba et al., 2010), one of only two RFS species of the family Homalopsidae that has been studied. OmPraba et al. used a combined transcriptomic and proteomic approach to characterize C. rynchops venom, confirming both veficolin gene expression in the venom gland and its presence in the venom. Transcripts for veficolins have been identified in the venom glands of other RFS species (Fry et al., 2012b; Junqueira-de-Azevedo et al., 2016; Pla et al., 2017a; Modahl et al., 2018b), but C. rynchops venom has been the only species reported with these toxins in the venom proteome (OmPraba et al., 2010). Veficolins share amino acid sequence similarities to mammalian ficolins with collagen-like and fibrinogen-like domains. It is possible that these venom proteins can induce platelet aggregation and/or initiate complement activation when delivered into prey (OmPraba et al., 2010), aiding in prey capture. However, the two veficolins from C. rynchops venom have yet to be experimentally characterized.

Snake venom matrix metalloproteinases (svMMPs) were first identified as a new venom protein present in the venom of RFS R. tigrinus tigrinus (Komori et al., 2006). Transcripts for svMMPs appear to be abundant in the venom glands of Dipsadinae RFS, including T. strigatus (Ching et al., 2012) and Erythrolamprus miliaris (Junqueira-de-Azevedo et al., 2016), and they are closely related to but functionally distinct from SVMPs. The expression of svMMPs in the venom gland of T. strigatus was considerably higher than that of SVMPs: 46.2–8.2%, respectively (Ching et al., 2012). Snake venom matrix metalloproteinases were also detected in the 2-D electrophoresis venom profile of P. mertensi (Campos et al., 2016). RFS svMMP genes cluster with a single MMP-9 ancestral gene, regardless of the presence or absence of ancillary domains (Junqueira-de-Azevedo et al., 2016), suggestive of a separate recruitment event relative to SVMPs. Matrix metalloproteinases are also zinc-dependent enzymes that degrade extracellular matrix proteins, such as collagens, elastin, proteoglycans, and laminins (Ra and Parks, 2007). These enzymes have been well-recognized for their roles in many physiological and pathological processes, including organ growth, wound healing, bone remodeling, immunity modulation, tumor invasion, and metastasis (Vu and Werb, 2000; Parks et al., 2004; Page-McCaw et al., 2007). They likely serve tissue-degrading roles in RFS venoms.

Recently, another new venom protein, snake venom acid lipase (svLIPA) was identified in the venom of the RFS P. mertensi (Campos et al., 2016). The venom gland transcriptome of this species revealed a highly expressed transcript coding for a lysosomal acid lipase protein; P. mertensi venom also exhibited lipase activity (Campos et al., 2016), and the recombinantly expressed svLIPA transcript produced a functional protein. Campos et al. supported their identification of svLIPA as a new venom protein based on its presence in P. mertensi venom, presence in the venom of the FFS Micrurus corallinus (Correa-Netto et al., 2011), high transcript abundance in the venom gland of P. mertensi in comparison to other body tissues, and previous reports of its transcription in the oral glands of other snakes (Hargreaves et al., 2014). Acid lipases hydrolyze cholesteryl esters and triglycerides to free cholesterol and fatty acids, aiding in cell metabolism and immunity (Gomaraschi et al., 2019). As with svMMPs, lipases could serve predigestive roles in RFS venoms.

Crude venoms from several RFS species show drastic toxicity differences depending on the model organism used for lethal dose (LD₅₀) experiments (Mackessy, 2002). Philodryas patagoniensis venom was tested on pigeons (Columba livia domestica), guinea pigs (Cavia porcellus), rabbits (Oryctolagus cuniculus), and frogs (Leptodactylus sp.), and pigeons were the most sensitive to this venom (Martins, 1907). Boiga irregularis venom LD₅₀ values were determined for domestic chickens (Gallus domesticus), geckos (Hemidactylus sp.), skinks (Carlia sp.), and mice (Mus musculus), and it was found that crude venom was much more toxic to birds and lizards than to mammals (Table 1) (Mackessy et al., 2006). Venom from S. sulphureus was also more toxic toward geckos (Hemidactylus sp.) than mice (M. musculus), even at 22-fold higher mass-adjusted doses in mice (Table 1) (Modahl et al., 2018b). This impressive toxicity in geckos of S. sulphureus venom is equivalent to that observed for venom from the highly toxic FFS Naja kaouthia (Table 1). The enhanced toxicity of crude venom toward different taxa has led to explorations into these venoms to discover components responsible. In most cases, 3FTxs have been identified to be the taxon-specific targeting toxins in these venoms, first characterized in the venoms of RFS species in the genus Boiga, commonly referred to as cat snakes.

From B. dendrophila venom, two 3FTxs have been characterized with postsynaptic neurotoxicity, boigatoxin-A (Lumsden et al., 2005) and denmotoxin, B. dendrophila monomeric toxin (Pawlak et al., 2006). Purified denmotoxin was found to bind to postsynaptic nAChRs in chick muscle preparations 100-fold more readily in comparison to mice nAChRs. This is also consistent with the lack of B. dendrophila crude venom toxic effects after injections into mice of doses up to 20 μg/g (Pawlak et al., 2006). Boigatoxin-A shows 78% identity to denmotoxin, but was only tested on chick neuromuscular junctions, so it is unknown if this toxin would display taxa targeting specificity. For the venom of B. irregularis, taxon-specific toxicity was found to be a result of the 3FTx complex irditoxin, B. irregularis dimeric toxin (Pawlak et al., 2009). Irditoxin inhibited postsynaptic nAChRs in chick biventer cervicis muscle preparations, but was three orders of magnitude less effective at the mammalian neuromuscular junction. This corresponds with the in vivo toxicity of irditoxin, which was nontoxic in mammals at doses up to 25 μg/g, but has an LD₅₀ value of 0.55 μg/g in Hemidactylus geckos and 0.22 μg/g in chickens (G. domesticus) (Table 1; Pawlak et al., 2009). Irditoxin was the first identified covalently linked 3FTx heterodimeric complex; previous 3FTx complexes were observed to be non-covalent homodimers, examples including κ-bungarotoxins (Dewan et al., 1994). Both of these rear-fanged cat snake species, B. dendrophila and B. irregularis, are arboreal snakes with diets primarily of birds and lizards, especially as juveniles (Greene, 1989). Therefore, the 3FTx taxon-specific toxicity appears to be correlated to prey items these snakes commonly eat.

Taxon-specific 3FTxs have also been isolated in two New World species, O. fulgidus (Heyborne and Mackessy, 2013) and S. sulphureus (Modahl et al., 2018b). Fulgimotoxin, O. fulgidus monomeric toxin, has an LD₅₀ of 0.28 μg/g in Anolis lizards, but was found to be nontoxic to mice at mass-adjusted doses more than 15 times the observed lizard LD₅₀ (Heyborne and Mackessy, 2013). Oxybelis fulgidus is an arboreal snake that has a diet of birds and lizards (Robert, 1982), supporting the link between taxon-specific 3FTxs and snake diet. Sulditoxin, S. sulphureus dimeric toxin, was identified as the lizard-specific 3FTx complex in S. sulphureus venom, and has a LD₅₀ value of 0.22 μg/g in Hemidactylus geckos, but is non-toxic to mammals up to doses of 5 μg/g (Table 1) (Modahl et al., 2018b). Sulditoxin was the second heterodimeric 3FTx complex discovered in RFS venoms, and interestingly shares the same cysteine residue pattern as irditoxin, likely using the same additional cysteines in the first and second loops (residues 17 and 42, respectively) to form an intermolecular disulfide linkage of the two subunits. A second taxon-specific toxin exists in S. sulphureus venom, but this toxin demonstrated specific toxicity toward mammals instead of lizards. This mammal-specific toxin was found to be a monomer and was named sulmotoxin 1, S. sulphureus monomeric toxin (Table 1; Modahl et al., 2018b). The third most abundant 3FTx in S. sulphureus venom, sulmotoxin 2, did not show taxon specificity. Spilotes sulphureus are arboreal snakes that are generalist predators, feeding on birds, lizards, amphibians, and small mammals (Andrade et al., 2017). The venom of S. sulphureus has two taxa specific 3FTxs present, and this correlates with its diet, which includes a diversity of prey.

Gene trees of RFS 3FTx sequences suggest that taxon-specific targeting convergently evolved in these separate snake species, at least for the dimeric complexes (irditoxin and sulditoxin), because even though these sequences share the same cysteine pattern, they cluster separately (Modahl et al., 2018b); and the two irditoxin subunit sequences are not common to all species within the Boiga genus (Dashevsky et al., 2018). It is also possible that these genes could have been lost in some Boiga species, which has been suggested for adaptive PLA₂s in the rattlesnake clade (Dowell et al., 2016). Until genome sequences are obtained, the evolutionary histories of these genes are difficult to assess, especially given the challenges with assembling de novo venom gland transcriptomes. Currently, most toxin sequences are obtained from venom gland NGS, and assemblers that are commonly used struggle to assemble toxin genes because of the multitude of similar isoforms and high expression levels of these toxins (Macrander et al., 2015; Holding et al., 2018; Modahl et al., 2019).

Selection analyses have revealed conflicting trends in RSF 3FTx evolution, where positive selection was reported previously (Sunagar et al., 2013), and it has since been observed that selection is variable for different 3FTx clades (Dashevsky et al., 2018). Some RFS 3FTxs are evolving under negative selection, especially sequences of subunits that form the dimeric complexes, where residues that are responsible for subunit interactions must be maintained (Dashevsky et al., 2018). With the current shortage of 3FTx sequences in databases from RFS species and absence of genomes, it is difficult to fully recognize the evolutionary complexities for this group toxins. However, by continuing to study these sequences from phylogenetically diverse species that are converging on the same trophic adaptation, it is possible to begin to explore how a venom protein with a conserved structure, like 3FTxs, can evolve specific activities and alter its mechanism of action. These toxins are therefore ideal to examine protein structure-function relationships.

When lizard-specific 3FTx sequences from RFS species were aligned, conserved motifs were observed in the central loop, where residues critical to nAChR receptor binding are found for FFS 3FTxs erabutoxin-a (Pillet et al., 1993) and α-cobratoxin (Antil et al., 1999). These conserved sequences were identified as CYTLY and WAVK for residues 34–38 and 46–49, respectively (Heyborne and Mackessy, 2013). These two motifs were hypothesized to be involved in toxin specificity to lizard nAChRs. Sulmotoxin 1, which is mammal specific, exhibits CYNLY, a threonine to asparagine substitution, and WTVK, an alanine to threonine substitution, motifs in this region (Modahl et al., 2018b), supporting this hypothesis, because sulmotoxin 1 lacks lizard toxicity. The size of the central loops between sulmotoxin, denmotoxin, and fulgimotoxin is also variable (Figure 3A), with longer central loops observed in lizard-specific 3FTxs, in addition to a proline in this region, followed by a negatively charged amino acid (either an aspartic acid or glutamic acid, or both; Figure 3B). Site-directed mutagenesis studies have yet to be performed for these taxa-specific 3FTxs and would help to elucidate the residues responsible for taxon-specific receptor binding. Key residues involved in receptor interactions could also be different than what is known for elapid 3FTxs, and identification of novel receptor-ligand interactions could also provide insight into how covalent 3FTx complexes (irditoxin or sulditoxin) interact with nAChRs.

All metalloproteinases that have been characterized from RFS venoms are of the P-III class and commonly exhibit fibrinogenolytic activity, with rapid degradation of the α-chain of fibrinogen and lower activity toward β- and γ-chains (Assakura et al., 1992; Peichoto et al., 2007; Weldon and Mackessy, 2012). Several SVMPs purified from the venom of P. olfersii degraded the α-chain of fibrinogen, with some also hydrolyzing the β-chain, but with differences in selectivity and rate (Assakura et al., 1994). In the venom of P. patagoniensis, the isolated SVMP named patagonfibrase was found to be the venom component responsible for the hydrolysis of the α-chain of fibrinogen, but not the β-chain (Peichoto et al., 2007), and for B. portoricensis, the SVMP alsophinase was found to hydrolyzed the α-subunit of fibrinogen almost instantly, and slightly degraded the β-chain after a 60 min incubation (Weldon and Mackessy, 2012). Although no individual SVMPs have been purified from the venom of A. prasina, there are SVMPs in this venom that act at a more rapid rate than those from the venom of B. portoricensis, hydrolyzing both α- and β-chains of fibrinogen (Figure 4A) even faster than what is observed with crude Prairie Rattlesnake (Crotalus viridis viridis) venom (Figure 4B; Modahl et al., 2018a). SVMPs of the P-III class are also found in venoms of elapid snakes but do not typically degrade the β-chain of fibrinogen and only target the α-chain (Evans, 1981; Guo et al., 2007; Sun and Bao, 2010). These studies demonstrate the underappreciated potency and functional diversity of SVMPs in RFS venoms.

Isoforms of SVMP P-IIIs have been observed to have distinct coagulation mechanisms in humans, birds, and small rodents, with some isoforms more specialized for rats or chickens (Bernardoni et al., 2014). There have been several studies that suggest SVMPs from RFS venoms might also have taxon selectivity. Higher molecular mass proteins isolated from the venom of P. olfersii were found to produce an irreversible blockade in chick biventer cervicis preparations, but had no effect on mouse phrenic nerve-diaphragm preparations (Prado-Franceschi et al., 1996). A separate study of P. olfersii venom revealed that all large molecular mass protein spots on a 2-D gel were SVMPs (Ching et al., 2006). Venom from P. patagoniensis, induced a time-dependent neuromuscular blockade of chick biventer cervicis preparations, but had no effect of mouse phrenic nerve-diaphragm preparations (Carreiro da Costa et al., 2008). Although not currently tested, SVMPs in this venom might also be responsible for the taxon-specific neuromuscular effects, given that this is a closely related species and that the venom lacks 3FTxs (Harvey and Mackessy, unpubl. obs.). An SVMP-dominated pit viper venom (from Bothrops insularis) was shown to have differential effects on mouse phrenic nerve diaphragm and chick biventer cervicis muscle preparations (Cogo et al., 1993). In FFS venoms, variations in SVMP P-III isoform expression have been observed in rattlesnake populations that are locally adapted to prey (Margres et al., 2017), and a similar relationship between SVMP variants and prey targeting has been hypothesized for Bothrops neuwiedi venom SVMPs (Bernardoni et al., 2014). More work in this area is needed to determine if SVMPs from RFS venoms are indeed taxon-specific venom components and linked to snake diet. Dietary studies show that for Philodryas species, amphibians and lizards are the primary prey taken (Marques and Hartmann, 2005; López and Giraudo, 2008) and isolated SVMPs have yet to be evaluated on these taxa. In addition, it would be important to determine what molecular interactions mediate SVMP taxon-specific toxicity.

SVMPs from RFS could therefore provide interesting models for future work focused on structure-function relationships. How do some SVMPs degrade both α- and β-chains while others do not? How can SVMPs be selective to prey type? Venoms from RFS species are ideal for addressing these questions because these venoms are overall less complex. For instance, vipers have several different classes of SVMPs, as well as serine proteinases that can also degrade fibrinogen, making it difficult to study specific toxin activity without multiple purification steps. Because of the abundance and extensive occurrence of P-III SVMPs in RFS venoms, these proteins can provide insight into the evolution of this toxin family and the biological roles that SVMPs provide in venoms.

Predatory strategy, venom vs. constriction, can drive venom evolution and snake behavior, favoring the evolution of venom that allows for a greater diversity and larger prey to be taken with minimum prey retaliation, and transitioning away from reliance on constriction (Savitsky, 1980). Boiga irregularis has the lizard-specific 3FTx complex irditoxin (Table 1) in abundance within its venom, and lizard prey is bitten and held by these snakes (Mackessy et al., 2006). However, mammalian prey is constricted by B. irregularis, and this is likely because this venom lacks mammal-specific toxins, as the crude venom murine LD₅₀ value is >30 μg/g (Table 1) (Mackessy et al., 2006). Spilotes sulphureus venom has two taxon-specific 3FTxs, one that is lizard-specific and one that is mammal-specific (Table 1), and both are abundant in the venom; constriction is not observed for S. sulphureus (Boos, 2001; Modahl et al., 2018b). Larger mammalian prey are metabolically more favorable to consume than smaller lizard prey, and sulmotoxin, the mammal-specific 3FTx, appears to be a more recent adaptation facilitating mammalian prey capture. In the case of the FFS snake Naja kaouthia, a species that also does not use constriction, this highly toxic snake has one main 3FTx, α-cobratoxin, that is lethal toward both mammal and lizard prey (Table 1; Modahl et al., 2016). These examples demonstrate two different venom evolutionary strategies to target a greater diversity of prey, one venom with a single 3FTx that has evolved lethal toxicity to many prey species, and a second venom in which toxin gene duplication and neofunctionalization has resulted in separate 3FTxs that are selectively toxic toward different prey types. Again, toxin evolution in rear-fanged snake venoms can provide key insights into venom evolution and predatory strategies, as these snakes vary greatly in predatory behavior and types of prey consumed.

When atypical prey items are taken, venom proteins with unique activities may be discovered. This has been the case for several FFS species, such as snakes that consume other snakes. The King Cobra, Ophiophagus hannah, is a species that consumes other snakes and was found to have a toxin from a new venom protein superfamily, ohanin, in its venom (Pung et al., 2005). The Long-glanded Coral Snake, Calliophis bivirgatus, also a snake-eating snake, was found to have a 3FTx with unusual activity toward sodium ion channels (Yang et al., 2016). Snakes of the genus Bungarus, which also commonly feed on other snakes, exhibit unique 3FTx homodimer complexes (κ-bungarotoxins; Dewan et al., 1994). Rear-fang snakes have even more dietary specialists, including species that feed on scorpions, spiders or centipedes, and so it is expected that novel toxins remain to be discovered in venoms of this diverse group of colubroid snakes.

Therefore, it is likely that venoms from RFS species potentially have many new and currently undiscovered potent toxins. Experimental studies have found that only about half of the total venom expended by the RFS B. irregularis is delivered into the viscera of prey, and the other half remains embedded in the skin (Hayes et al., 1993). But even with these lower yields delivered into prey tissue, effects are still observed as prey that is removed without being consumed by the snake may become sluggish and eventually die after many minutes or several hours (Hayes et al., 1993). In combination with a less rapid venom delivery system, these snakes also generally have less complex venom (Mackessy, 2010b; Peichoto et al., 2012), resulting in venom toxins that are optimized for specific types of prey. Venoms from most species of RFS are uncharacterized, and because of historic biases, most LD₅₀ work utilizes domestic murine models due to availability of mice, genetic uniformity and presumed closer applicability to humans. However, by excluding other models, such as lizards, other vertebrates and invertebrates, one can overlook the biological potency of specialized venom proteins that may have been selected for toxic effects on non-mammalian prey (cf. Richards et al., 2012; Smiley-Walters et al., 2018).

Metalloproteinases in rattlesnake venoms have been suggested to facilitate efficient digestion of prey at suboptimal temperatures or when large prey are consumed (Mackessy, 1988, 2010a); however, several studies have indicated that envenomation does not increase digestive efficacy (McCue, 2007; Chu et al., 2009). In the case of many rattlesnake species, the extent of SVMP activity in a venom is negatively correlated with overall venom toxicity (Mackessy, 2010a). This has led to rattlesnake venoms being characterized as type I (SVMP-dominated venoms that are less toxic) or type II (venom that is more toxic but with low metalloproteinase activity; Mackessy, 2010a). A similar dichotomy is found in many RFS venoms, where a venom is either dominated by toxic 3FTxs or enzymatic SVMPs (McGivern et al., 2014). For the RFS venoms that are heavily dominated by SVMPs, these venom components likely also aid in prey predigestion, as similar fibrinogenolytic activity is observed for SVMPs from both rattlesnakes and RFS species (Figure 4; Modahl et al., 2018a). Studying RFS venoms can reveal parallels to trends seen in FFS venoms, emphasizing the important of specific venom proteins in facilitating prey handling in diverse species of venomous snakes, regardless of the venom delivery system.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.