Independent Recruitment of Different Types of Phospholipases A2 to the Venoms of Caenophidian Snakes: The Rise of PLA₂-IIE within Pseudoboini (Dipsadidae)

Abstract

Snake venoms harbor a wide and diverse array of enzymatic and nonenzymatic toxic components, allowing them to exert myriad effects on their prey. However, they appear to trend toward a few optimal compositional scaffolds, dominated by four major toxin classes: SVMPs, SVSPs, 3FTxs, and PLA₂s. Nevertheless, the latter appears to be restricted to vipers and elapids, as it has never been reported as a major venom component in rear-fanged species. Here, by investigating the original transcriptomes from 19 species distributed in eight genera from the Pseudoboini tribe (Dipsadidae: Xenodontinae) and screening among seven additional tribes of Dipsadidae and three additional families of advanced snakes, we discovered that a novel type of venom PLA₂, resembling a PLA₂-IIE, has been recruited to the venom of some species of the Pseudoboini tribe, where it is a major component. Proteomic and functional analyses of these venoms further indicate that these PLA₂s play a relevant role in the venoms from this tribe. Moreover, we reconstructed the phylogeny of PLA₂s across different snake groups and show that different types of these toxins have been recruited in at least five independent events in caenophidian snakes. Additionally, we present the first compositional profiling of Pseudoboini venoms. Our results demonstrate how relevant phenotypic traits are convergently recruited by different means and from homologous and nonhomologous genes in phylogenetically and ecologically divergent snake groups, possibly optimizing venom composition to overcome diverse adaptative landscapes.

Introduction

Venomous animals and their toxins have been increasingly scrutinized by researchers from around the world in the last four decades (Fox and Serrano 2007; Fry 2015; Zhang 2015; Mackessy 2021). Studies aiming to understand and alleviate the epidemiological phenomenon of human envenomation by these animals were the main drivers of the toxinological sciences throughout most of human history (Prado-Franceschi and Hyslop 2002; Otero-Patiño 2009; Junqueira-de-Azevedo et al. 2016; Mackessy 2021; Sevilla-Sánchez et al. 2021). Among the great diversity of vertebrate and invertebrate venomous animals, snakes cause higher proportions of human accidents and deaths worldwide (Prado-Franceschi and Hyslop 2002; Chippaux 2015; Williams et al. 2019). Therefore, venoms from medically relevant snake species belonging to the Viperidae and Elapidae families, which are characterized by their front-fanged dentitions, are among the better-known animal secretions (Fry 2015; Mackessy 2021). However, most snake diversity lies elsewhere, mainly within the family Dipsadidae (superfamily: Colubroidea), which contains venom-producing species that have been historically neglected in toxinological studies due to their low medical relevance (Junqueira-de-Azevedo et al. 2016; Uetz et al. 2019; Zaher et al. 2019). These snakes exhibit a diverse array of ecological and morphological traits, which might be mirrored by equally varied venoms (Henderson 1982; de Oliveira et al. 2008; Barbo et al. 2011; Weinstein et al. 2011; Gaiarsa et al. 2013; Giraudo et al. 2014).

Recently, there has been an increased number of works that address venom-related questions for the abovementioned species that reveal highly complex venom compositions, which resemble the characteristics observed in vipers and elapids. Moreover, these works have revealed a series of new, poorly characterized venom components that appear to be found only in this group of snakes (de Oliveira et al. 2008; Weinstein et al. 2011; Campos et al. 2016; Junqueira-de-Azevedo et al. 2016; Modahl et al. 2018; Bayona-Serrano et al. 2020; Mackessy 2021). However, there is still a lack any kind of compositional or functional information about the venoms of most dipsadid genera (Junqueira-de-Azevedo et al. 2016; Barua and Mikheyev 2019; Bayona-Serrano et al. 2020). The species addressed thus far have indicated that proteolytic enzymes (e.g., zinc-dependent metalloproteinases), followed by cysteine-rich secretory proteins (CRiSPs) and C-type lectins (CTLs), tend to dominate in their venoms (Ching et al. 2012; Campos et al. 2016; Junqueira-de-Azevedo et al. 2016; Bayona-Serrano et al. 2020). Remarkably, phospholipases A₂ (PLA₂s), a common denominator in the venoms of front-fanged snakes and one of the major effectors of their toxic actions (e.g., myotoxicity, myonecrosis, lipid membrane damage, neurotoxicity, and prey immobilization), have not been assertively associated with venom features in any dipsadid (Junqueira-de-Azevedo et al. 2016; Barua and Mikheyev 2019).

PLA₂s catalyze the hydrolysis of glycerophospholipids at the sn-2 position, producing free fatty acids and lysophospholipids (Six and Dennis 2000; Huang et al. 2015). They are ubiquitous in vertebrates, where they fulfill several physiological roles and are divided into three main categories: secretory, cytosolic, and Ca²⁺-independent PLA₂s, depending on their cellular location and catalytic mechanism (Six and Dennis 2000). Snake venom PLA₂s belong to the secretory type, which has been classically divided into 11 different groups based on their primary structures and the tissues in which they are most commonly expressed (Six and Dennis 2000). Venom PLA₂s have been commonly reported in two front-fanged snake families: the Elapidae family possesses secretory PLA₂s from group I and the Viperidae family produces group IIA PLA₂s in its venoms (Ogawa et al. 1995; Jeyaseelan et al. 2000; Huang et al. 2015; Dowell et al. 2016). These enzymes are generally associated with some of the most severe symptoms observed in accidents involving front-fanged snakes that harbor them as a major toxin class (Gutiérrez et al. 2009; Tsai 2016; Pla et al. 2018; Tasoulis et al. 2020; Mackessy 2021). These enzymes have undergone accelerated evolution and genetic expansion in these front-fanged snakes families, where they represent a multigene family with several paralogs (Jeyaseelan et al. 2000; Yamaguchi et al. 2014; Dowell et al. 2016; Suranse et al. 2022).

The contribution of PLA₂s to the venoms of rear-fanged snakes is less evident, and their activity, abundance, structural diversity, and evolution might be underestimated (Huang and Mackessy 2004; Fry et al. 2012; Fry 2015; Junqueira-de-Azevedo et al. 2016; Pla et al. 2017; Torres-Bonilla et al. 2018; Mackessy et al. 2020). In 2004, a protein showing PLA₂ catalytic activity and high similarity at its N-terminus to PLA₂-IA from sea snakes was isolated from the venom of the colubrid, Trimorphodon lambda (Huang and Mackessy 2004). However, a few other rear-fanged species were shown to have a different type of PLA₂ in their venoms, PLA₂-IIE, which commonly occurs in the human and mouse brain/heart/uterus and serves physiological functions (Six and Dennis 2000; Suzuki et al. 2000; Mackessy 2021). Transcripts for PLA₂-IIE have been detected at low levels in the venom glands of several snakes, although they never appear to be as preponderant as their I or IIA counterparts found in elapids or vipers, respectively (Fry et al. 2012; Yamaguchi et al. 2014; Pla et al. 2017). Dispholidus typus and Oxyrhopus guibei, species from two completely different families (i.e.., Colubridae and Dipsadidae, respectively), have the highest expression levels of PLA₂-IIE transcripts among rear-fanged snakes reported thus far (Junqueira-de-Azevedo et al. 2016; Pla et al. 2017). The former is a colubrid that is notorious for its potent venom, being involved in human casualties, including the renowned case of Karl Patterson Schmidt (Pla et al. 2017). On the other hand, O. guibei, a dipsadid belonging to the Pseudoboini tribe, is a docile species with a scarce record of human accidents. In 2018, Torres-Bonilla et al. studied the enzymatic actions of the venom of the pseudoboine Pseudoboa neuwiedii and found that it had PLA₂ activity levels similar to those observed in viper species. Subsequent proteomic analyses of the venom of P. neuwiedii identified peptides belonging to the PLA₂s of group II (Torres-Bonilla et al. 2018). This finding, along with a previous report of PLA₂-IIE transcripts being expressed in the venom gland of O. guibei, hinted that PLA₂s might be a relevant venom component in the tribe Pseudoboini.

In this work, we elucidated the occurrence of PLA₂s in the venoms of the Dipsadidae family by scrutinizing the venoms and venom glands of the Pseudoboini tribe and additional outgroups through transcriptomic, proteomic, and functional approaches. We reconstructed the evolutionary history of PLA₂-IIE in snakes and discuss the possible recruitment and duplication events that turned this family of proteins into a major player in dozens of rear-fanged species. Moreover, we contrast our findings with previously reported PLA₂s from other snake families and infer that multiple recruitment events have shaped the dispersion of these toxins across caenophidian snakes. Additionally, the data regarding other toxins of the Pseudoboini tribe represent a substantial addition to the poor knowledge of the venom compositions of rear-fanged snakes and bring new research opportunities for the exploration of colubroid venoms.

Results

Venom Compositional Profile of the Pseudoboini Tribe: A PLA₂-IIE–Rich Group of Snakes

To establish the phylogenetic relationships of species within the Pseudoboini tribe, which is associated with their venom profiles, we used our assembled transcriptomic data to build a data set of 2,161 conserved loci and used them to reconstruct the phylogenetic relationships of the tribe (supplementary fig. S1, Supplementary Material online). The full mitochondrial genomes of the individuals were also recovered using the MITGARD approach and used to reconstruct the phylogenetic relationships of the tribe (supplementary fig. S2, Supplementary Material online) (Nachtigall, Grazziotin, et al. 2021). Both trees showed similar relationships, but we adopted the tree based on the conserved loci to draw the illustrative tree shown in figure 1 due to its higher node support values.

Fig. 1.

Compositional profiles of venom-related transcripts in the Pseudoboini tribe. The phylogenetic tree was adapted from ZAHER et al. 2019 for general relationships outside of the tribe and was derived from our phylogenetic analyses for relationships within the tribe (supplementary fig. S1, Supplementary Material online). Bars represent the average amount of each annotated toxin type in the species. Pie plots to the right are from a representative species for each group. Note the elevated amount of PLA₂ present in the Clelia-like group.

Venom gland transcriptome (VGT) annotation of species from this tribe uncovered a wide array of toxin classes, both enzymatic and nonenzymatic, with a varying number of putative paralogs and expression levels (fig. 1 and supplementary table S1, Supplementary Material online). Snake venom metalloproteinases from the P-III subtype (SVMP P-III) were a dominant component in all species of the tribe. Despite this protease dominance, we observed great compositional variations, especially regarding CRiSPs and PLA₂s. Based on its toxin expression profiles, the tribe could be divided into two main groups: the Oxyrhopus-like group and the Clelia-like group (fig. 1).

The Oxyrhopus-like group contains species from the genera Oxyrhopus and Siphlophis that possess VGTs dominated by SVMP-PIII and CRiSPs, with lower expressions of CTLs, natriuretic peptides (CNP), snake endogenous matrix metalloproteinases (seMMP-9), and PLA₂-IIE. On the other hand, the Clelia-like group contains the genera Mussurana, Phimophis, Rhachidelus, Pseudoboa, Boiruna, and Clelia. These genera form a monophyletic group (supplementary fig. S1, Supplementary Material online) and showed similar expression levels of minor toxins but an overall higher proportion of PLA₂-IIE, which was the dominant toxin class in some species. Within the Clelia-like group, the genera Pseudoboa, Boiruna, and Clelia showed the highest expression levels of PLA₂-IIE and a PCA confirmed that species belonging to these genera cluster closer together and away from other species of the tribe and are more compositionally related (supplementary fig. S3, Supplementary Material online).

Most species from the Oxyrhopus-like group retained only one PLA₂-IIE transcript, the exception being Oxyrhopus clathratus, which retained two PLA₂-IIE transcripts after curation of their VGT (supplementary table S1, Supplementary Material online). On the other hand, all species in the Clelia-like group possess two different PLA₂-IIE transcripts that show radically different expression levels, one very highly expressed and the other lowly expressed. When looking at the primary amino acid structure of the PLA₂-IIE-derived proteins retained for species of the tribe, we determined that all PLA₂-IIEs from the Clelia-like group encoded shorter proteins, which lack a portion of the C-terminal that is present in some of the Oxyrhopus group-derived proteins and is the typical structure in endogenous PLA₂-IIEs from other snake groups (fig. 2A).

Fig. 2.

(A) Multiple sequence alignment of PLA₂-IIEs from Pseudoboini and other snakes. Structural features are indicated by closed boxes. The aspartate residue, typical in catalytically active PLA2s, is highlighted in yellow. The asterisks indicate Pseudoboini PLA2-IIE with the C-terminal extension. The accession numbers for the sequences from other snakes are gi_698375631, gi_384110785, XM_015820366, MN831292, XM_039367457, gi_1147529007, gi_25140376, and KX211996. (B) Protein sequence alignment of the highly and lowly expressed transcripts from representative species of the Clelia-like group. Dots indicate conserved amino acids between aligned sequences. Peptides identified by proteomic analyses are highlighted with closed boxes. Heterogeneous regions found across the different proteins are indicated with light-blue shading. The end of the signal peptide is marked by a red cross above residue 24. Total spectrum counts, unique peptides, and coverages of mature proteins are indicated for identified proteins.

However, O. clathratus had both the long and short forms. Multiple sequence alignments of full-length PLA₂-IIE transcripts revealed that this shorter C-terminus is caused by deletions of 30 and 21 bp in Pseudoboini and in the colubrid D. typus, respectively, while all other PLA₂-IIEs from the analyzed snakes possess a longer C-terminus (supplementary fig. S4, Supplementary Material online). Moreover, when comparing the primary structures from representative sequences of the highly and weakly expressed transcripts from the genera Pseudoboa, Boiruna, and Clelia (Clelia-like group), we determined that they possess three different heterogeneous portions between them, even though their signal peptides and active sites were similar (fig. 2B).

Proteomic analyses not only confirmed the occurrence of PLA₂-IIE in the venoms of Pseudoboa nigra, Boiruna sertaneja, and Clelia equatoriana but also indicated that it was a major constituent of these venoms (fig. 2B and supplementary fig. S5, Supplementary Material online). Moreover, the predicted protein from the highly expressed transcript in the venom glands was detected in all three species, harboring a higher proportion of mass spectra than the protein from the weakly expressed transcript, even when both were identified as occurring in B. sertaneja (supplementary table S2, Supplementary Material online). In Clelia clelia and P. nigra, only the highly expressed form was detected in the proteome. Other major venom components reported in the VGTs of the tribe, such as SVMPs, CRiSPs, and seMMP-9, were also confirmed to be present in the venom of these three species (fig. 3 and supplementary table S2, Supplementary Material online). Moreover, the abundances of identified venom toxins estimated in Scaffold 5 followed the same compositional trends that we observed in VGTs, with SVMPs and PLA₂-IIE being dominant toxins in C. equatoriana and B. sertaneja (supplementary fig. S5, Supplementary Material Online).

Fig. 3.

Three-dimensional alignments of a B. jararacussu PLA₂-IIA with different PLA₂ forms from vipers and colubrid species. The table below displays the RMSD scores for each pairwise alignment. Yellow tones indicate better (lower) scores.

The 3D structures of a highly expressed PLA₂-IIE from P. nigra, used as a representative from the Clelia-like group; the longer PLA₂-IIE from O. guibei, a PLA₂-IIE from D. typus; a PLA₂-IIE from Crotalus adamanteus; and a noncatalytic PLA₂-IIA from Bothrops jararaca, were predicted with RoseTTAFold (Hiranuma et al. 2021). Comparisons between these structures and the 3D crystal structure of a catalytically active PLA₂-IIA from Bothrops jararacussu revealed that some of the shorter PLA₂-IIE forms obtained from the Clelia-like group have better RMSD scores across all aligned pairs than the PLA₂-IIE forms obtained from other viper species (fig. 3).

PLA₂-IIEs Inside and Outside of the Pseudoboini Tribe

The elevated expression levels of PLA₂-IIE found within the Pseudoboini tribe led us to wonder if these were a unique characteristic of this group within dipsadids or if PLA₂s were also dominant in other tribes. We gathered previously generated snake venom transcriptomic data for representative species of seven additional tribes of Dipsadidae and for the available species of Colubridae, Elapidae, and Viperidae and screened for PLA₂-IIE–like sequences, as we performed in a previous work (Bayona-Serrano et al. 2020). The analysis indicated that the elevated expression levels of PLA₂-IIE in venom glands are likely exclusive to the Pseudoboini tribe within Dipsadidae (supplementary fig. S6, Supplementary Material online), suggesting that, when present in other groups, the PLA₂-IIE transcript may correspond to the endophysiological protein. A phylogenetic tree reconstruction performed using PLA₂ sequences from elapids, vipers, colubroids, and other vertebrates revealed that the PLA₂s recovered from Pseudoboini species are nested within the type IIE of PLA₂s from other vertebrates. PLA₂-IIE is the sister group of PLA₂-IIA found on viper venoms (fig. 4 and supplementary figs. S7, S8, S9, and S10, Supplementary Material online). Moreover, PLA₂-IIEs recovered from the Oxyrhopus group separate themselves from those of the Clelia-like group. Interestingly, within the Clelia-like group, the highly and weakly expressed transcripts formed separate independent groups. An orthology analysis performed with OrthoFinder clustered all assembled PLA₂-IIEs from Pseudoboini within a single orthogroup, which was probably due to their overall sequence similarity.

Fig. 4.

Phylogenetic reconstruction of the assembled PLA₂-IIE from Pseudoboini with PLA₂s from other snakes using 1,000 ultrafast bootstrap replicates. Assembled PLA₂s from Pseudoboini cluster within the IIE subgroup, which is the sister group of PLA₂s-IIA from vipers. Black crosses within the Pseudoboini tribe indicate sequences without the C-terminal extension commonly seen in endogenous PLA₂-IIEs from other snakes. A schematic representation of multiple sequence alignments of full transcripts of PLA₂-IIEs across sampled species exhibiting the variation of the C-terminal arrangement is shown to the right. The C-terminal region of the coding sequence is highlighted in blue. The 3′UTR of the full transcript is highlighted in green. D. typus (Colubridae), some Oxyrhopus, and all species from the Clelia-like group present a deletion before the stop codon, as indicated by red crosses.

Enzymatic Assays

Some Pseudoboini venoms tested for PLA₂ activity showed activities comparable to those of viper venoms (fig. 5). Comparisons were made among four different groups: venoms from viper species known to possess PLA₂ activity, venoms from dipsadids shown to be devoid of PLA₂s, venoms from species from the Oxyrhopus group, and venoms from species from the Clelia-like group. We observed significant differences between the PLA₂ activities found in species from the Clelia-like group and species from the Oxyrhopus group. The latter were statistically equal to venom from other dipsadids. For the comparison between activity levels from viper venoms and venoms from the Clelia-like group, we were unable to find statistically significant differences, although crude venoms from C. Clelia and B. sertaneja presented higher enzymatic activity values than the crude venom of B. jararacussu, which is known to be one of the most PLA₂-rich species of snake (Freitas-De-sousa et al. 2020) (fig. 5).

Fig. 5.

PLA₂ enzymatic activities measured for Pseudoboini venoms. Each color represents a different group of samples. Asterisks indicate significant differences between groups. SEM is plotted for each bar.

Discussion

The phylogenetic analyses allowed us to reconstruct a robust topology for the sampled species of the Pseudoboini tribe and take advantage of the multigene approach that high-throughput transcriptomics generate. Our results allowed us to resolve some internal relationships within the tribe, which clustered the genus Rhachidelus along with the monophyletic group formed by Pseudoboa, Clelia, and Boiruna (Zaher et al. 2019) (supplementary figs. S1 and S2, Supplementary Material online).

The venoms from the Pseudoboini tribe showed the same protease-rich profiles observed in most dipsadids (Junqueira-de-Azevedo et al. 2016; Bayona-Serrano et al. 2020). SVMPs remain a major toxin class in the venoms of these snakes, and elevated levels of CTLs, CRiSPs, and seMMP-9 point to them as the usual players in Dipsadidae venoms. However, we noticed unusually high proportions of PLA₂s in this tribe, both in their VGTs and proteomes. The PLA₂ amounts were particularly high in the Clelia-like group, in which the PLA₂-IIE expression levels ranged from ∼1% to ∼27% of whole transcriptomes (fig. 1 and supplementary table S1, Supplementary Material online). These high expression levels were found to be exclusive to the Pseudoboini tribe, at least among the species sampled in our screening (supplementary fig. S6, Supplementary Material online). All other analyzed tribes within the family had TPM values near zero for PLA₂-like contigs. Regarding other snake families (e.g., Colubridae, Elapidae, and Viperidae) that were screened, we also found almost null expressions of PLA₂-IIE–like contigs. However, previous works have reported PLA₂s from both type IA and IIE as venom components in some species of the Colubridae family (Huang and Mackessy 2004; Fry et al. 2012; Pla et al. 2017; Mackessy et al. 2020). This indicates that there might be other PLA₂s hidden in specific snake groups. An in-depth sampling of venom-producing colubroids is needed to truly determine their occurrence across all advanced snake clades.

Functional analyses of Pseudoboini venoms revealed high PLA₂ activities, which were comparable to those of viper venoms, in species from the Clelia-like group (fig. 5). These findings, along with the fact that a highly expressed PLA₂-IIE was found in the analyzed venom proteomes of the group, suggest that this form is responsible for the catalytic PLA₂ activity observed. Previous works had already reported high PLA₂ activity in the venom of P. neuwiedii (Torres-Bonilla et al. 2017, 2018). However, proteomic analyses of the venom of that species identified an ∼14–15-kDa SDS–PAGE band as PLA₂-IIA. In that work, the identified spectra were searched against the UniProtKB/Swiss-Prot database, which found several peptides that matched a myotoxic noncatalytic PLA₂-IIA from Bothrops moojeni. However, all PLA₂s we recovered for the tribe, including P. neuwiedii, were catalytically active and belonged to the IIE subgroup of PLA₂s. Therefore, we attribute the previous identification of PLA₂-IIA in the venom of P. neuwiedii to the general sequence similarity between PLA₂-IIA and PLA₂-IIE and to the lack of representative PLA₂-IIE from Pseudoboini in the databases used in that work (Yamaguchi et al. 2014), which hindered the correct identification of the PLA₂ subtype. It is worth highlighting the considerable PLA₂ activity that we measured for Phimophis guerini, since this species had the lowest proportion of PLA₂-IIE in its VGT within the Clelia-like group (fig. 1). Increased sampling within the genus is needed to confirm whether a highly expressed PLA₂-IIE transcript occurs within its VGTs or if the PLA₂ activity observed is mediated by other means. Species from the Oxyrhopus-like group showed no significant differences from the venoms obtained from dipsadids known to be devoid of PLA₂s, in agreement with the low PLA₂-IIE expressions we found in their transcriptomes.

PLA₂ enzymes have been predominantly reported as venom components of front-fanged snakes from the Viperidae family, harboring PLA₂-IIA, and the Elapidae family, harboring PLA₂-IA and PLA₂-IB (Jeyaseelan et al. 2000; Dowell et al. 2016). Interestingly, the genetic organization of the PLA₂-II locus in vipers and other vertebrates suggests that PLA₂s from group IID, which are the evolutionary precursor of all PLA₂-IIA from vipers, were derived from an ancestral duplication of the PLA₂-IIE gene followed by sequential gene duplication and diversification within Viperidae, originating venom PLA₂-IIA toxins (Yamaguchi et al. 2014; Dowell et al. 2016; Koludarov et al. 2019; Suranse et al. 2022). Moreover, genomic data have revealed the presence of exonic debris from the PLA₂-IIE gene spread downstream from the PLA₂-IIE gene in vipers, indicating plausible duplication and pseudogenization events (Dowell et al. 2016; Koludarov et al. 2019). To determine whether these duplications occurred before the diversification of vipers, we analyzed the available genomes of the colubrids Thamnophis sirtalis (NCBI accession number NW_013659820.1) and Pantherophis guttatus (NCBI accession number NW_023010753.1). We did not find exonic debris for the PLA₂-IIE gene in those species, indicating that the possible duplication of the PLA₂-IIE gene took place after viper diversification and that it does not represent a basal trait in advanced snakes. Therefore, the finding of two types of PLA₂-IIE transcripts showing structural and quantitative differences in some Pseudoboini species reinforces the hypothesis that the PLA₂-IIE gene has undergone at least one duplication event within the tribe.

Gene duplication is a known trigger of accelerated evolution (Ohno 1971; True and Carroll 2002) that is observed in many venom proteins (Ogawa et al. 1995, 2005; Kini and Doley 2010; Vonk et al. 2013; Dowell et al. 2016; Lomonte et al. 2016; Tadokoro et al. 2020). The two types of PLA₂-IIE found in Pseudoboini differ not only in their sequence substitutions but also in the small deletions on their C-terminal portions (fig. 2A). Previous works have noted that even though the primary structures of IIA and IIE PLA₂s are similar, the C-terminal tails are distinct between them, with PLA₂-IIE having a longer C-terminus (Yamaguchi et al. 2014). Interestingly, a shorter C-terminal deletion was also present in the PLA₂-IIE contig reported for the colubrid D. typus, which has moderate levels of PLA₂ expression in its venom glands (∼2.75% of whole transcriptome) (Pla et al. 2017). The C-terminal deletion, which shortens the primary structure of the PLA₂-IIE protein, observed only in D. typus and in all species from the Clelia-like group, constitutes a convergent event in rear-fanged snake groups displaying increased expression levels of this protein type in their venom glands. The role and relevance of this deletion are still not fully understood, but it might indicate a trend toward a more compact IIA-like structural scaffold. The 3D alignments favor this trend, as the PLA₂-IIE from the Clelia-like group with a shortened C-terminus showed better alignment scores toward the structure of a viper PLA₂-IIA than the PLA₂-IIEs from other vipers, which do not possess the C-terminal deletion (fig. 2A and fig. 3). However, as these 3D alignments were made with predicted structures and the Armstrong error estimate of the models, calculated by RoseTTAFold, always increased toward the C-terminal portion, it is hard to assertively link this deletion to a trend toward a more IIA-like structure.

The phylogenetic reconstruction of PLA₂s showed that PLA₂-IIEs from Pseudoboini form a sister clade to PLA₂-IIAs from vipers. Within the Pseudoboini tribe, the weakly expressed PLA₂-IIE from O. occipitalis possessing the longer C-terminal, was the most basal protein, resembling the PLA₂-IIE scaffold found outside of the tribe. Within the Clelia-like group, PLA₂-IIE is organized into two separate clades, one containing the highly expressed transcripts and the other containing the weakly expressed transcripts, both harboring the C-terminal deletion (fig. 4). The highly expressed form was found more consistently in the venom proteome of the analyzed genera, with more spectral counts, indicating a higher relative abundance of the protein. We also found that the two clades of sequences had three heterogeneous portions on their primary structures. The implications of these differences are not clear, but it would be expected that the different residues of the highly expressed form contribute to the overall enzymatic efficiency of the protein in the venom.

Outside Pseudoboini, PLA₂-IIE sequences can be retrieved from many snake taxa, mostly from genome annotations or PCR products amplified from various tissues, including the venom glands (Fry et al. 2012; Yamaguchi et al. 2014). There is no strong evidence, however, of PLA₂-IIE being a relevant venom component in other snake families, with the sole exception of the colubrid, D. typus (Pla et al. 2017). In this case, combined transcriptomic and proteomic analyses identified PLA₂-IIE among the top three most abundant toxins in the venom. On the other hand, our phylogenetic analysis indicated that the peculiar PLA₂ proteins reported in the colubrid genus, Trimorphodon, are not PLA₂-IIE but belong to the PLA₂-I type, as had been previously reported (Fry et al. 2008) (fig. 4). The evolutionary history of the PLA₂ gene family in rear-fanged snakes appears to be rather complex, as some species possess PLA₂-IA–like proteins (e.g., T. lambda), while others exhibit PLA₂-IIE–like proteins (e.g., D. typus and most Pseudoboini species) (Pla et al. 2017; Mackessy et al. 2020).

The genetic scaffold of the PLA₂-II gene cluster is highly conserved in humans, mice, birds, and snakes (Huang et al. 2015). The triplet organization of the locus, with the OTUD3 gene, followed by the PLA₂-IIE gene and then the PLA₂-IID cluster, is mostly maintained in these groups (Huang et al. 2015; Dowell et al. 2016; Suranse et al. 2022). A PLA₂-IID gene is assumed to be ancestrally recruited in vipers and co-opted into a venom protein, resulting in the modern PLA₂-IIA observed in viper venoms (Yamaguchi et al. 2014; Dowell et al. 2016; Koludarov et al. 2019). This recruitment was followed by sequential gene duplication and accelerated evolution, marked by diverse substitutions at the catalytic site, ultimately generating a noncatalytic (K49) form in some vipers (Huang et al. 2015; Dowell et al. 2016; Suranse et al. 2022). However, this genetic expansion of the PLA₂-IID cluster, derived from the PLA₂-IIA venom forms found in vipers, has not yet been observed in any other group of advanced snakes. On the other hand, less information is known regarding the genetic scaffolding and evolutionary history of PLA₂-I from elapids. These PLA₂s are structurally divided into group IB, commonly found in mammalian pancreases but also reported in the venoms of some elapid snakes (Armugam et al. 2004; Mackessy 2021), and group IA, which is found almost exclusively in elapid venoms and lacks the “pancreatic loop” characteristic of group IB (Jeyaseelan et al. 2000; Huang and Mackessy 2004; Mackessy 2021). PLA₂-I are placed in a different genomic locus, and their phylogenetic reconstruction indicates that their diversification was genus specific and influenced by the ecology and evolutionary history of each lineage (Jeyaseelan et al. 2000). Genomic data from rear-fanged species expressing PLA₂s in their venom are needed to reveal the genomic organization of the PLA₂-I and PLA₂-II gene loci and determine if they are in fact undergoing similar genetic processes as the ones observed in vipers and elapids.

Based on our findings and previous literature reports, we can hypothesize at least three distinct events of recruitment and restriction of PLA₂-like toxins into the venom glands of rear-fanged snakes (fig. 6). PLA₂s from Group I, which are commonly found in elapid venoms, are apparently recruited to the venom glands of the genus Trimorphodon, which has been shown to possess PLA₂-IA–like proteins in its VGTs and proteomes (Huang and Mackessy 2004; Mackessy et al. 2020). This is the first and only record of a non-elapid snake genus harboring PLA₂-I as a venom protein and might indicate that a duplication of the endogenous PLA₂-IB gene, followed by sequential mutations toward an IA-like structure, occurred exclusively in this genus within Colubridae. On the other hand, PLA₂s from group II appear to have been recruited into the venom glands of two separate families of rear-fanged snakes. These enzymes are arranged in a well-characterized cluster that is conserved in various vertebrate lineages and are known to be dominant toxins in vipers, where the PLA₂-IIA gene, evolutionarily derived from PLA₂-IID, has undergone several duplication/loss events. However, a different type of PLA₂-II, PLA₂-IIE, was recruited to the venom arsenal of some species of rear-fanged snakes within the Pseudoboini tribe (Dipsadidae) and the genus Dispholidus (Fry et al. 2012; Junqueira-de-Azevedo et al. 2016; Pla et al. 2017). We hypothesize that the PLA₂-IIE gene suffered at least one event of duplication and shortening of the C-terminal tail after the Pseudoboini diversification from other Dipsadidae and this gene was recruited to the venom gland during the radiation of the Clelia-like group. A parallel recruitment of the PLA₂-IIE gene occurred in Colubridae, specifically in the genus Dispholidus. The order in which these events took place and whether or not they occurred similarly or simultaneously in both groups is still a matter of investigation.

Fig. 6.

Hypothetical genomic expansion events within each PLA₂ type among different snake families. Each colored box represents a gene. Physiological copies are assumed to be present in all snake groups. The open box shown in the Clelia-like group indicates the uncertainty of the physiological PLA₂-IIE gene structure, as we could not find a transcript without the C-terminal deletion in the sampled species. Genomic data are needed to reveal the true arrangement of the PLA₂-IIE gene in that group.

Moreover, these independent recruitments of PLA₂-IIE and structural changes parallel the better-known evolutionary trajectories of group PLA₂-I in Elapidae and group PLA₂-IIA in Viperidae. Examples of independent recruitment of similar genes to become toxins in different snake groups are now becoming frequent and seem to indicate a trend toward the selection of a few optimal scaffolds to exert toxic functions in snake venom (Campos et al. 2016; Barua and Mikheyev 2019; Bayona-Serrano et al. 2020). The recruitment of PLA₂-IIE to the venom glands of Pseudoboini represents a prime example of this trend.

In summary, although PLA₂s are widespread venom components of several venomous snakes, we suggest that PLA₂s became part of the venoms of Caenophidian snakes on at least five occasions and their appearance is not likely to be a basal trait selected early upon the divergence of the group. The PLA₂-IIE gene was recruited and restricted to venoms of the rear-fanged families Colubridae (at least on D. typus) and Dipsadidae (Pseudoboini tribe) in two independent events, mirroring the recruitment and expansion of the PLA₂-IIA gene in the Viperidae family (a third event). The PLA₂-I gene, on the other hand, was apparently selected independently in both in Elapidae (fourth event) and in Trimorphodon, a specific Colubridae genus (fifth event). Since PLA₂s are associated with some of the major toxic phenotypes of snake venoms, causing a wide array of effects, including cytotoxicity, myotoxicity, neurotoxicity, and many others, it is not surprising that these proteins have been selected multiple times during snake evolution. Nevertheless, the reiterated recruitment of nonvenom PLA₂ genes in different snake families indicates that the intrinsic features of the PLA₂ scaffold make it a valuable asset to effectively impose toxicity in different ecological contexts. Increased genomic sampling of rear-fanged snakes, along with increased functional and structural information from colubroid PLA₂s, is still needed to shed light upon the complex evolutionary history of these toxins within snakes.

Materials and Methods

Collection and Storage of Samples

Specimens from eight genera and 19 species (IBAMA authorization 57585–1 and MAATE authorization MAE-DNB-CM-2019–0115) were collected during a series of field trips to different localities in Brazil and Ecuador. Venom samples were extracted using pilocarpin on sedated individuals as described in previous works (Mackessy et al. 2006). Four days after extraction, the venom glands and other tissues were surgically collected and stored in RNAlater at −80 °C.

RNA Extraction and Analysis

Tissues were pulverized in a Precellys 24 homogenizer, and RNA was extracted with TRIzol (Invitrogen) following the modification of the method described by Chomczynski (1987) based on the use of guanidine isothiocyanate followed by phenolic extraction (Chomzynski 1987). Total RNA was quantified by the Quant-iTTM RiboGreen RNA reagent and kit (Invitrogen, Life Technologies Corp.). Quality control of the extracted RNA was then performed in an Agilent 2100 Bioanalyzer using an Agilent RNA 6000 Nano Kit to verify the integrity of total RNA through band discrimination corresponding to fractions 18S and 28S of total RNA. All procedures involving RNA were performed with RNase-free tubes and filter tips and using water treated with diethylpyrocarbonate (DEPC, Sigma). The general RNA integrity number (RIN) obtained for analyzed samples is available in supplementary figure S11, Supplementary Material online.

cDNA Library Construction and Sequencing

Libraries were prepared for each individual sample. One microgram of total RNA was used with an Illumina TruSeq Stranded RNA HT kit consisting of TruSeq Stranded RNA HT/cDNA Synthesis PCR, TruSeq Stranded RNA HT/Adapter Plate Box, and TruSeq Stranded HT mRNA. Fragment size distributions were evaluated by microfluidic gel electrophoresis in a Bioanalyzer device (Agilent 2100) using an Agilent DNA 1000 Kit according to the manufacturer's protocol. Quantification of each library was then performed by real-time PCR using a KAPA SYBR FAST Universal qPCR Kit, according to the manufacturer's protocol, using the StepOnePlus Real-Time PCR System. Aliquots of each cDNA library were diluted to a concentration of 2 nM. Next, a pool of all samples, 5 μl of each library, was prepared and the concentration of the pool was again determined by real-time PCR. The cDNA libraries were sequenced on an Illumina HiSeq 1500 System in Rapid Run mode using a paired-end flow cell for 300 cycles of 2*151 bp.

Transcriptome Assembly and Annotation

To assemble the venom transcriptomes of the samples, we checked and removed cross-contamination using an in-house script (Hofmann et al. 2018) that compares sequences from other libraries within the sequencing pool and then trimmed the sequencing adaptors using TrimGalore (Krueger 2015). We merged our reads using PEAR software (Zhang et al. 2014) by taking advantage of the common overlap on the 3′ ends that characterizes paired-end short reads (Rokyta et al. 2012) and used those longer merged reads as an input for our assembly. We ran all the assemblies in a standardized way using five different assemblers with different k-mer values and assembly parameters (Trinity: k-mer 31; rnaSPADES: k-mer 31, 75, and 127; Extender: default, overlap 150, and seed size 2000; SeqMan Ngen: k-mer 21; and Bridger: k-mer 30) (Grabherr et al. 2011; Rokyta et al. 2012; Chang et al. 2015; Holding et al. 2018; Bushmanova et al. 2019). Then, we performed toxin annotation using ToxCodan (Nachtigall, Rautsaw, et al. 2021) against a curated data set of toxin sequences. Annotated toxin transcripts were manually reviewed and used to purge toxic-like contigs from the Trinity assembly of each individual. Then, both annotated toxin sequences and the remaining nontoxin Trinity contigs were combined to obtain a complete VGT of each individual, in which the toxin transcripts were curated. The coding sequences from nontoxin-purged contigs were predicted using CodAn with the full vertebrate model (Nachtigall, Kashiwabara, et al. 2021) and annotated by Blast searches against NCBI nr and PFAM following the ToxCodan pipeline available online (Nachtigall, Rautsaw, et al. 2021). The expression levels of each individual transcript were estimated using RSEM software (Li and Dewey 2011) after mapping the merged reads from each sample using Bowtie2 and were measured in transcripts per million (TPM) (Bankar et al. 2015).

Proteomic Analyses

Analyses by reversed-phased nano chromatography coupled to tandem mass spectrometry analyses of the venoms from three species were performed by the Florida State University College of Medicine Translational laboratory and by the Laboratory of Toxinology (FIOCRUZ, Rio de Janeiro), as detailed in the supplementary methods, Supplementary material online. Protein identifications of the obtained spectra were performed using MASCOT (Matrix Science, London, UK; version 2.6.2) and X! Tandem (The GPM, thegpm.org, last accessed August 3, 2020; version X! Tandem Alanine [2017.2.1.4]) as the search engine. We considered a 99% and 95% threshold for protein and peptide identification, respectively. Custom-generated FASTA databases containing curated sequences of identified toxins for each specimen and translated protein sequences from the assembled transcriptome (Trinity contigs) for the species were used as a database for spectral identification, as detailed in the supplementary methods, Supplementary material online. To quantify the estimated abundance of each toxin class, we normalized the total spectra of all identified proteins using the Normalized Spectral Abundance Factor (NSAF) as implemented in Scaffold 5 (Zybailov et al. 2006).

Venom Variation and Complexity within the Tribe

We transformed the expression data using the log-rate (center log-ratio [clr]) transformation method (Egozcue et al. 2003; Filzmoser et al. 2009) and applied the functions implemented in the robCompositions package (Templ et al. 2011) in the R environment. We used the clr transformation for visualization purposes, as it takes the simplex data into real space while retaining the individual identities of each toxin class. With these transformed values, we performed a principal component analysis (PCA) to evaluate the toxin compositions of the sampled Pseudoboini species. We used the prcomp function from the stats package in R version 4.1.0 (Team 2015). Then, we separated the poorly represented toxins (i.e., with average expressions of less than 1% of the total toxins) and grouped them into a category called “OtherToxins,” which was compared with the main toxins of the tribe. The graph was plotted using the ggplot package (Wickham 2016), and different colors were assigned for each analyzed species.

Phylogenetic and Evolutionary Analyses of PLA₂s in Snakes

We screened for PLA₂-IIE-like contigs among four different snake families and seven additional tribes within Dipsadidae using an approach similar to that of Bayona-Serrano et al. 2020 (Bayona-Serrano et al. 2020). Briefly, we performed BlastN searches using de novo–assembled contigs from Trinity against the curated database of PLA₂-IIE–like sequences obtained herein. The expression of each individual contig was calculated using RSEM (Li and Dewey 2011) by mapping the reads from each sample using Bowtie2. Expressions were estimated in TPM (Wagner et al. 2012, 2013). Afterward, PLA₂-IIE–like contigs were identified and their expression values were added to obtain an approximate value for PLA₂-IIE participation in each individual transcriptome. To better understand the phylogenetic history of the PLA₂-IIE gene, we used the annotated PLA₂-IIE sequences from our sampled individuals and combined them with the PLA₂s from other publicly available vertebrates. The final nucleotide data set was then aligned through its corresponding translated amino acid sequences using the MUSCLE algorithm (Edgar 2004), with 20 iterations and default parameters in Geneious v.2020.0.5 software. Phylogenetic tree inference was then carried out using IQ-Tree2 (Minh et al. 2020) by combining the substitution model estimation with ModelFinder, a tree search with 1,000 replicates of ultrafast bootstrap and implementing the Shimodaira–Hasegawa approximate likelihood ratio test (SH-aLRT) and following the command recommended by the software developers. Moreover, we performed three additional tree searches using ultrafast bootstrap with 5,000 replicates, a nonparametric bootstrap with 1,000 replicates, both implemented in IQ-Tree2 (Minh et al. 2020) and a Bayesian approach in Mr. Bayes (Ronquist et al. 2012) using the nexus block available in the supplementary methods, Supplementary material online. Trees were visualized and edited using the iTol online platform (Letunic and Bork 2016). Orthology analyses were carried out for PLA₂-IIE transcripts recovered from the tribe with OrthoFinder v2.4.0 to identify possible duplication events within the tribe (Emms and Kelly 2019). An inflation parameter of 0.5 was used.

Enzymatic Assay for PLA₂ Activity

Venom PLA₂ activities were assayed in 96-well plates using 4-nitro-3- (octanoyloxy) benzoic acid (NOB) as substrate in 0.1 M Tris-HCl, pH 8, containing 0.01 M Ca2+ as reaction buffer for 30 min at 37 °C. The standard assay mixture contained 200 μl of buffer, 20 μl of substrate, and 20 μl of venom (1 µg/µl) in a final volume of 240 μl. After adding the venom, reactions were run in a SpectraMax 340 plate reader for 30 min at 37 °C, with the absorbance changes read at 425 nm. Venoms from nine species of Pseudoboini were tested. Crude venoms from the viper B. jararacussu and the CB subunit from the crotoxin of the rattlesnake, Crotalus durissus, were used as positive controls since they are recognized to have high PLA₂ activities (Freitas-De-sousa et al. 2020; Montoni et al. 2020). Venoms from colubrid snakes, Philodryas olfersii and Thamnodynastes chaquensis, were used as negative controls, since the venoms of these genera were reported to have low or no PLA₂ activity (Diaz et al. 2004; Ching et al. 2006; <bibref rid=“b14”>Correia et al. 2010, 2012; Zelanis et al. 2010; ). The obtained absorbance values were plotted for each sample, and the standard errors of the mean (SEM) were calculated for samples for which we had enough venom to run duplicate tests. To determine if there were significant differences among the groups, the nonparametric Kruskal–Wallis test was used. Multiple comparisons between the different groups were made through the nonparametric Wilcoxon test. All statistical tests were performed using R software version 4.1.0. Groups were considered significantly different if they had P < 0.05.

Structural Analysis of the PLA₂-IIE from Pseudoboini

To understand the structural differences between PLA₂-IIEs from Pseudoboini and other snakes and the venom PLA₂-IIA from vipers, we aligned the primary structures of the assembled PLA₂-IIE to PLA₂-IIA/IIE sequences from other publicly available snakes. We used the MUSCLE algorithm (Edgar 2004), with 20 iterations and default parameters in Geneious v.2020.0.5 software. Then, to see how those differences in primary structure might affect the 3D organization of each protein, we predicted the 3D structures of PLA₂-IIE from Pseudoboini and other snake species using the RoseTTAFold method implemented in the Robetta protein structure prediction server (Hiranuma et al. 2021). Predicted protein structures were only considered for further analyses if they had a predicted local distance difference test (l-DDT) higher than 0.80. We downloaded the crystal structure of a catalytically active PLA₂-IIA from B. jararacussu (UniProt code 1ZL7) and aligned it against our predicted models using the Matchmaker function available on ChimeraX 1.3 software. A fraction parameter of 1 was used to prioritize secondary structure over residue composition. The root-mean-square deviations of atomic positions (RMSD) of each alignment were used to estimate how well each of the models adjusted to the IIA structure.

Phylotranscriptomic Analyses

First, we checked for putative sample contamination by assembling the mitochondrial sequences from each sample using MITGARD (v1.2) (Nachtigall, Grazziotin, et al. 2021) with the Imantodes cenchoa mitochondrial genome as reference (GenBank accession number EU728586.1). MITGARD is a tool that recovers the mitochondrial genome from RNA sequencing (RNA-seq) data by using a reference as bait to retrieve the mitochondrial reads and use it to assemble the mitogenome. We annotate the assembled mitogenomes using MitoZ (v2.4) (Meng et al. 2019). Then, we used the assembled and annotated mitochondrial sequences to compare with previously obtained mitochondrial sequences of Pseudoboini species to validate the species identity. We also used 15 annotated mitochondrial genes (i.e., two ribosomal and 13 protein coding) to infer a phylogenetic tree for each gene separately. To do this, we aligned their sequences using MAFFT (v7.310) (Katoh and Standley 2014), trimmed the alignments using trimAl (v1.2) (Capella-Gutiérrez et al. 2009) with the “-automated1” parameter, and built trees using IQ-TREE (v2.0.3) (Minh et al. 2020). Branches with Bootstrap values lower than or equal to 95 were removed from each mitochondrial gene tree using the Newick Utilities package (v1.6) (Junier and Zdobnov 2010), and the final consensus tree was generated using the coalescent approach implemented in Astral (v5.15.4) (Mirarab et al. 2014).

Then, we employed the software BUSCO (v5.2.2) (Manni et al. 2021), which infers measurements of genome and transcriptome completeness based on evolutionary informed expectations of gene content through the use of sets of lineage-specific sets benchmarking universal single-copy orthologs. We used the “aves_odb10” set (total of 8,338 genes in the BUSCO set) that represents the set with closer relationship to snakes among all other BUSCO sets and allowed the recovery of a higher number of nuclear loci to be used in the tree inference. We retrieved a total of 5,359 loci and filtered it to only keep loci containing at least 15 samples to avoid bias related to missing data, which resulted in a final set containing 2,161 loci. We aligned each locus separately in the final set using MAFFT (v7.310) (Katoh and Standley 2014) with the parameters “–auto” and “–adjustdirectionaccurately.” The alignments were cleaned using CIAlign (v1.0.14) (Tumescheit et al. 2022), with the following parameters “–remove_divergent –remove_divergent_minperc 0.80 –remove_insertions –crop_ends –remove_short.” The alignments were trimmed using trimAl (v1.2) (Capella-Gutiérrez et al. 2009) with the “-strictplus” parameter. The trimmed alignments were used to infer the phylogenetic trees for each locus using IQ-TREE (v2.0.3) (Minh et al. 2020). Then, branches with Bootstrap values lower than or equal to 95 were removed from each locus tree using the Newick Utilities package (v1.6) (Junier and Zdobnov 2010) and the final consensus tree was generated using the coalescent approach implemented in Astral (v5.15.4) (Mirarab et al. 2014).

Supplementary Material

Supplementary data are available at Molecular Biology and Evolution online.

Data availability

Raw transcriptomic data are available at NCBI's GenBank under Bioproject accession number PRJNA625548. Curated sequences (CDS) for all toxin transcripts generated in this work are available in supplementary table S1, Supplementary Material online, organized per species. Additional supplementary information (i.e., RAW proteomic data and multiple sequence alignments for phylogenetic analyses are available in a Figshare project [accessible at https://figshare.com/projects/Independent_recruitments_of_different_types_of_phospholipases_A2_to_the_venom_of_Caenophidian_snakes_the_rise_of_PLA2-IIE_within_Pseudoboini_Dipsadidae_/162772]).