The Venom Composition of the Snake Tribe Philodryadini: 'Omic' Techniques Reveal Intergeneric Variability among South American Racers.

Snakes of the Philodryadini tribe are included in the Dipsadidae family, which is a diverse group of rear-fanged snakes widespread in different ecological conditions, including habitats and diet. However, little is known about the composition and effects of their venoms despite their relevance for understanding the evolution of these snakes or even their impact on the occasional cases of human envenoming. In this study, we integrated venom gland transcriptomics, venom proteomics and functional assays to characterize the venoms from eight species of the Philodryadini tribe, which includes the genus Philodryas, Chlorosoma and Xenoxybelis. The most abundant components identified in the venoms were snake venom metalloproteinases (SVMPs), cysteine-rich secretory proteins (CRISPs), C-type lectins (CTLs), snake endogenous matrix metalloproteinases type 9 (seMMP-9) and snake venom serinoproteinases (SVSPs). These protein families showed a variable expression profile in each genus. SVMPs were the most abundant components in Philodryas, while seMMP-9 and CRISPs were the most expressed in Chlorosoma and Xenoxybelis, respectively. Lineage-specific differences in venom composition were also observed among Philodryas species, whereas P. olfersii presented the highest amount of SVSPs and P. agassizii was the only species to express significant amounts of 3FTx. The variability observed in venom composition was confirmed by the venom functional assays. Philodryas species presented the highest SVMP activity, whereas Chlorosoma species showed higher levels of gelatin activity, which may correlate to the seMMP-9 enzymes. The variability observed in the composition of these venoms may be related to the tribe phylogeny and influenced by their diets. In the presented study, we expanded the set of venomics studies of the Philodryadini tribe, which paves new roads for further studies on the evolution and ecology of Dipsadidae snakes.

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

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.

The venom composition of the snake tribe Philodryadini: ‘Omic’ techniques reveal intergeneric variability among South American racers

Snakes of the Philodryadini tribe are included in the Dipsadidae family, which is a diverse group of rear-fanged snakes widespread in different ecological conditions, including habitats and diet. However, little is known about the composition and effects of their venoms despite their relevance for understanding the evolution of these snakes or even their impact on the occasional cases of human envenoming. In this study, we integrated venom gland transcriptomics, venom proteomics and functional assays to characterize the venoms from eight species of the Philodryadini tribe, which includes the genus Philodryas, Chlorosoma and Xenoxybelis. The most abundant components identified in the venoms were snake venom metalloproteinases (SVMPs), cysteine-rich secretory proteins (CRISPs), C-type lectins (CTLs), snake endogenous matrix metalloproteinases type 9 (seMMP-9) and snake venom serinoproteinases (SVSPs). These protein families showed a variable expression profile in each genus. SVMPs were the most abundant components in Philodryas, while seMMP-9 and CRISPs were the most expressed in Chlorosoma and Xenoxybelis, respectively. Lineage-specific differences in venom composition were also observed among Philodryas species, whereas P. olfersii presented the highest amount of SVSPs and P. agassizii was the only species to express significant amounts of 3FTx. The variability observed in venom composition was confirmed by the venom functional assays. Philodryas species presented the highest SVMP activity, whereas Chlorosoma species showed higher levels of gelatin activity, which may correlate to the seMMP-9 enzymes. The variability observed in the composition of these venoms may be related to the tribe phylogeny and influenced by their diets. In the presented study, we expanded the set of venomics studies of the Philodryadini tribe, which paves new roads for further studies on the evolution and ecology of Dipsadidae snakes.

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

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.

Independent recruitment of different types of Phospholipases A2 to the venoms of Caenophidian snakes: the rise of PLA2-IIE within Pseudoboini (Dipsadidae)

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.

Caracterização de adaptações do repertório molecular de serpentes das tribos Xenodontini e Pseudoboini relacionadas à sua ecologia de predação

Among the wide spectrum of food ecologies, some groups of snakes have become ophiophagous, feeding mainly on other snakes. Due to the relationship between diet and venom composition, these ophiophagous groups probably needed to develop specific toxins targeting snakes and some type of resistance to the venoms of the snakes they preyed on, associating these characteristics with the development of ophiophagy. In the Neotropics, more precisely in the Dipsadidae family, there are several ophiophagous species. The genus Erythrolamprus (Tribe Xenodontini) and the genera Clelia and Boiruna (Tribe Pseudoboini) contain species that are specialists in snakes. However, due to their relatively low medical relevance, they have been poorly studied and there is scarce information about their venoms and possible resistance factors or mechanisms. In the present work, the toxin composition of the venom gland transcriptome of 8 genera and 19 species of the tribe Pseudoboini and 3 genera and 8 species of the tribe Xenodontini was analyzed. In addition to being the first compositional analysis of the venom of the two tribes, a new type of enzymatic toxins exclusive to the Dipsadidae family (the svMMPs) was identified and characterized in the analyzed samples, as well as high expression levels of a type of phospholipases A2 in some species of the Pseudoboini tribe was detected. On the other hand, comparisons between liver transcriptomes and plasma proteomes of ophiophagous and non-ophiophagous species of the tribe Pseudoboini allowed identifying high expression levels of several types of toxin inhibitors. However, the occurrence of overexpression of these inhibitors in ophiophagous species was not detected. The analyzes revealed, in a broad scale, the evolutionary novelties present in the compositional profile of the venoms of the two tribes and verified the occurrence of plasma toxin inhibitors in the analyzed species.

Dentre o amplo espectro de ecologias alimentares, alguns grupos de serpentes tornaram-se ofiófagos, alimentando-se principalmente de outras serpentes. Devido à relação entre a dieta e a composição do veneno, esses grupos ofiófagos provavelmente precisaram desenvolver toxinas específicas para serpentes e algum tipo de resistência aos venenos das serpentes que predavam, associando essas características ao desenvolvimento da ofiofagia. Nos neotrópicos, mais precisamente na família Dipsadidae, ocorrem diversas espécies ofiófagas. O gênero Erythrolamprus (Tribo Xenodontini) e os gêneros Clelia e Boiruna (Tribo Pseudoboini) contêm espécies especialistas em serpentes. No entanto, devido à sua relevância médica relativamente baixa, foram pouco estudados e existem poucas informações sobre seus venenos e possíveis fatores ou mecanismos de resistência. No presente trabalho foi analisada a composição de toxinas do transcriptoma da glândula de veneno de 8 gêneros e 19 espécies da tribo Pseudoboini e de 3 gêneros e 8 espécies da tribo Xenodontini. Além de ser a primeira análise composicional do veneno das duas tribos, permitiu a identificação e caracterização de um novo tipo de toxinas enzimáticas exclusivo da família Dipsadidae (as svMMPs), assim como revelou níveis de expressão elevados de um tipo de fosfolipases A2 em algumas espécies da tribo Pseudoboini. Por outro lado, as comparações entre os transcriptomas do fígado e os proteomas do plasma de espécies ofiófagas e não ofiófagas da tribo Pseudoboini indicaram níveis de expressão elevados de vários tipos de inibidores de toxinas. Porém, não foi detectada a ocorrência de sobre expressão destes inibidores nas espécies ofiófagas. As análises revelaram, em escala abrangente, as novidades evolutivas presentes no perfil composicional dos venenos das duas tribos e verificaram a ocorrência de inibidores de toxinas plasmáticos nas espécies analisadas.

An integrative view of the toxic potential of Conophis lineatus (Dipsadidae: Xenodontinae), a medically relevant rear-fanged snake.

Most traditional research on snake venoms has focused on front-fanged snake families (Viperidae, Elapidae, and Atractaspididae). However, venom is now generally accepted as being a much more broadly possessed trait within snakes, including species traditionally considered harmless. Unfortunately, due to historical inertia and methodological challenges, the toxin repertoires of non-front-fanged snake families (e.g., Colubridae, Dipsadidae, and Natricidae) have been heavily neglected despite the knowledge of numerous species capable of inflicting medically relevant envenomations. Integrating proteomic data for validation, we perform a de novo assembly and analysis of the Duvernoy's venom gland transcriptome of the Central American Road Guarder (Dipsadidae: Xenodontinae: Conophis lineatus), a species known for its potent bite. We identified 28 putative toxin transcripts from 13 toxin families in the Duvernoy's venom gland transcriptome, comprising 63.7% of total transcriptome expression. In addition to ubiquitous snake toxin families, we proteomically confirmed several atypical venom components. The most highly expressed toxins (55.6% of total toxin expression) were recently described snake venom matrix metalloproteases (svMMPs), with 48.0% of svMMP expression contributable to a novel svMMP isoform. We investigate the evolution of the new svMMP isoform in the context of rear-fanged snakes using phylogenetics. Finally, we examine the morphology of the venom apparatus using µCT and explore how the venom relates to autecology and the highly hemorrhagic effects seen in human envenomations. Importantly, we provide the most complete venom characterization of this medically relevant snake species to date, producing insights into the effects and evolution of its venom, and point to future research directions to better understand the venoms of 'harmless' non-front-fanged snakes.

An integrative view of the toxic potential of Conophis lineatus (Dipsadidae: Xenodontinae), a medically relevant rear-fanged snake

Most traditional research on snake venoms has focused on front-fanged snake families (Viperidae, Elapidae, and Atractaspididae). However, venom is now generally accepted as being a much more broadly possessed trait within snakes, including species traditionally considered harmless. Unfortunately, due to historical inertia and methodological challenges, the toxin repertoires of non-front-fanged snake families (e.g., Colubridae, Dipsadidae, and Natricidae) have been heavily neglected despite the knowledge of numerous species capable of inflicting medically relevant envenomations. Integrating proteomic data for validation, we perform a de novo assembly and analysis of the Duvernoy's venom gland transcriptome of the Central American Road Guarder (Dipsadidae: Xenodontinae: Conophis lineatus), a species known for its potent bite. We identified 28 putative toxin transcripts from 13 toxin families in the Duvernoy's venom gland transcriptome, comprising 63.7% of total transcriptome expression. In addition to ubiquitous snake toxin families, we proteomically confirmed several atypical venom components. The most highly expressed toxins (55.6% of total toxin expression) were recently described snake venom matrix metalloproteases (svMMPs), with 48.0% of svMMP expression contributable to a novel svMMP isoform. We investigate the evolution of the new svMMP isoform in the context of rear-fanged snakes using phylogenetics. Finally, we examine the morphology of the venom apparatus using μCT and explore how the venom relates to autecology and the highly hemorrhagic effects seen in human envenomations. Importantly, we provide the most complete venom characterization of this medically relevant snake species to date, producing insights into the effects and evolution of its venom, and point to future research directions to better understand the venoms of ‘harmless’ non-front-fanged snakes.

Replacement and Parallel Simplification of Nonhomologous Proteinases Maintain Venom Phenotypes in Rear-Fanged Snakes.

Novel phenotypes are commonly associated with gene duplications and neofunctionalization, less documented are the cases of phenotypic maintenance through the recruitment of novel genes. Proteolysis is the primary toxic character of many snake venoms, and ADAM metalloproteinases, named snake venom metalloproteinases (SVMPs), are largely recognized as the major effectors of this phenotype. However, by investigating original transcriptomes from 58 species of advanced snakes (Caenophidia) across their phylogeny, we discovered that a different enzyme, matrix metalloproteinase (MMP), is actually the dominant venom component in three tribes (Tachymenini, Xenodontini, and Conophiini) of rear-fanged snakes (Dipsadidae). Proteomic and functional analyses of these venoms further indicate that MMPs are likely playing an "SVMP-like" function in the proteolytic phenotype. A detailed look into the venom-specific sequences revealed a new highly expressed MMP subtype, named snake venom MMP (svMMP), which originated independently on at least three occasions from an endogenous MMP-9. We further show that by losing ancillary noncatalytic domains present in its ancestors, svMMPs followed an evolutionary path toward a simplified structure during their expansion in the genomes, thus paralleling what has been proposed for the evolution of their Viperidae counterparts, the SVMPs. Moreover, we inferred an inverse relationship between the expression of svMMPs and SVMPs along the evolutionary history of Xenodontinae, pointing out that one type of enzyme may be substituting for the other, whereas the general (metallo)proteolytic phenotype is maintained. These results provide rare evidence on how relevant phenotypic traits can be optimized via natural selection on nonhomologous genes, yielding alternate biochemical components.

Replacement and parallel simplification of non-homologous proteinases maintain venom phenotypes in rear-fanged snakes

Novel phenotypes are commonly associated with gene duplications and neofunctionalization, less documented are the cases of phenotypic maintenance through the recruitment of novel genes. Proteolysis is the primary toxic character of many snake venoms, and ADAM metalloproteinases, named Snake Venom Metalloproteinases (SVMPs), are largely recognized as the major effectors of this phenotype. However, by investigating original transcriptomes from 58 species of advanced snakes (Caenophidia) across their phylogeny, we discovered that a different enzyme, matrix metalloproteinase (MMP), is actually the dominant venom component in three tribes (Tachymenini, Xenodontini, and Conophiini) of rear-fanged snakes (Dipsadidae). Proteomic and functional analyses of these venoms further indicate that MMPs are likely playing an ‘SVMP-like’ function in the proteolytic phenotype. A detailed look into the venom-specific sequences revealed a new highly expressed MMP subtype, named snake venom MMP (svMMP), which originated independently on at least three occasions from an endogenous MMP-9. We further show that by losing ancillary non-catalytic domains present in its ancestors, svMMPs followed an evolutionary path toward a simplified structure during their expansion in the genomes, thus paralleling what has been proposed for the evolution of their Viperidae counterparts, the SVMPs. Moreover, we inferred an inverse relationship between the expression of svMMPs and SVMPs along the evolutionary history of Xenodontinae, pointing out that one type of enzyme may be substituting for the other, while the general (metallo)proteolytic phenotype is maintained. These results provide rare evidence on how relevant phenotypic traits can be optimized via natural selection on non-homologous genes, yielding alternate biochemical components.