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.

O misterioso perfil tóxico da tribo Philodryadini: Técnicas ‘ômicas’ revelam variabilidade nos venenos destas serpentes

Philodryadini snakes belong to one of the most diverse families in the world and are the main opisthoglyphic snakes involved in human envenomation. These snakes play a fundamental ecological role, with most of them having a generalist feeding habit, but on the other hand, the species Philodryas agassizii feeds exclusively on arthropods. Despite all the diversity found in this group, there are still few studies on the composition and variability of the venoms of this tribe. Thus, this work aims to carry out the biochemical and functional characterization of the venom of five species of the genus Philodryas (P. patagoniensis, P. olfersii, P. nattereri, P. mattogrossensis e P. agassizii), two species of the genus Chlorosoma (C. viridissimum, previously known as Philodryas viridissima and C. laticeps, formerly known as Philodryas laticeps) and a species of the genus Xenoxybelis (X. argenteus), through transcriptomic analysis combined with proteomics and functional analysis of venoms through enzymatic assays. The most abundant components identified in the venoms were Snake Venom Metalloproteinases (SVMPs), Cysteine-rich Secretory Proteins (CRISPs) and C-type Lectins (CTLs), Snake Endogenous Matrix Metalloproteinases type 9 (seMMP-9) and Snake Venom Serinoproteinases (SVSPs). However, these protein families showed quantitative variability and a different expression profile in each analyzed genus. SVMPs were the most abundant components in Philodryas, while seMMP-9 were the most expressed in Chlorosoma and CRISPs in Xenoxybelis. Furthermore, we can observe variability within the same genus, where P. olfersii presented a greater amount of SVSPs than the other species of the genus. The composition of the venoms also reflected differences in the functional assays, as expected, only P. olfersii showed proteolytic activity for the substrate of SVSPs, while the other Philodryas were the most active on the substrate of SVMPs. Chlorosoma species showed higher activity in gelatin degradation with activity only partially inhibited by EDTA. The gel bands with the greatest degradation were identified by mass spectrometry and indicated the presence of seMMP-9, confirming the hypothesis that the proteolytic character of these proteins is maintained. Thus, the results indicate that within the Philodryadini tribe the Philodryas, Chlorosoma and Xenoxybelis genera are not distinguished only by morphological characteristics, but also by the biochemical composition of their venoms. In addition, we describe here for the first time the complete transcriptome of 7 species of the Philodryadini tribe and the proteome of 5 individuals of different species of this tribe.

As serpentes da tribo Philodryadini pertencem a uma das famílias mais diversas do mundo e são as principais serpentes opistóglifas envolvidas em envenenamentos humanos. Estas serpentes desempenham papel ecológico fundamental, sendo a maioria de hábito alimentar generalista, mas em contrapartida, a espécie Philodryas agassizii se alimenta exclusivamente de artrópodes. Apesar de toda a diversidade encontrada neste grupo, ainda há poucos estudos sobre a composição e variabilidade dos venenos desta tribo. Assim, este trabalho visa realizar a caracterização bioquímica e funcional do veneno de cinco espécies do gênero Philodryas (P. patagoniensis, P. olfersii, P. nattereri, P. mattogrossensis e P. agassizii), duas espécies do gênero Chlorosoma (C. viridissimum, anteriormente nomeada como Philodryas viridissima e C. laticeps, anteriormente conhecida como Philodryas laticeps) e uma espécie do gênero Xenoxybelis (X. argenteus), através da análise transcriptômica combinada à proteômica e análise funcional dos venenos através de ensaios enzimáticos. Os componentes mais abundantes identificados nos venenos foram as Metaloproteinases de Veneno de Serpente (SVMPs), Proteínas Secretoras Ricas em Cisteína (CRISPs) e Lectinas do tipo C (CTLs), Metaloproteinases de Matriz do tipo 9 Endógenas de Serpente (seMMP-9) e Serinoproteinases de Veneno de Serpente (SVSPs). No entanto, essas famílias de proteínas apresentaram variabilidade quantitativa e um perfil de expressão diferente em cada gênero analisado. As SVMPs foram os componentes mais abundantes em Philodryas, enquanto que as seMMP-9 foram as mais expressas em Chlorosoma e as CRISPs em Xenoxybelis. Além disso, podemos observar variabilidade dentro do mesmo gênero, onde P. olfersii apresentou uma maior quantidade de SVSPs que as outras espécies do gênero. A composição dos venenos também refletiu diferenças nos ensaios funcionais, como já era esperado, apenas P. olfersii apresentou atividade proteolítica para o substrato de SVSPs, enquanto que as demais Philodryas foram as mais ativas no substrato de SVMPs. As espécies de Chlorosoma apresentaram maior atividade na degradação da gelatina com atividade apenas parcialmente inibida pelo EDTA. As bandas do gel com maior degradação foram identificadas por espectrometria de massas e indicaram a presença de seMMP-9, confirmando a hipótese de que o caráter proteolítico destas proteínas é mantido. Dessa forma, os resultados indicam que dentro da tribo Philodryadini os gêneros Philodryas, Chlorosoma e Xenoxybelis não se distinguem apenas por características morfológicas, mas também pela composição bioquímica de seus venenos. Além disso, descrevemos aqui pela primeira vez o transcriptoma completo de 7 espécies da tribo Philodryadini e o proteoma de 5 indivíduos de espécies diferentes dessa tribo.

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.

Size Matters: An Evaluation of the Molecular Basis of Ontogenetic Modifications in the Composition of Bothrops jararacussu Snake Venom.

Ontogenetic changes in venom composition have been described in Bothrops snakes, but only a few studies have attempted to identify the targeted paralogues or the molecular mechanisms involved in modifications of gene expression during ontogeny. In this study, we decoded B. jararacussu venom gland transcripts from six specimens of varying sizes and analyzed the variability in the composition of independent venom proteomes from 19 individuals. We identified 125 distinct putative toxin transcripts, and of these, 73 were detected in venom proteomes and only 10 were involved in the ontogenetic changes. Ontogenetic variability was linearly related to snake size and did not correspond to the maturation of the reproductive stage. Changes in the transcriptome were highly predictive of changes in the venom proteome. The basic myotoxic phospholipases A2 (PLA2s) were the most abundant components in larger snakes, while in venoms from smaller snakes, PIII-class SVMPs were the major components. The snake venom metalloproteinases (SVMPs) identified corresponded to novel sequences and conferred higher pro-coagulant and hemorrhagic functions to the venom of small snakes. The mechanisms modulating venom variability are predominantly related to transcriptional events and may consist of an advantage of higher hematotoxicity and more efficient predatory function in the venom from small snakes.

Size matters: an evaluation of the molecular basis of ontogenetic modifications in the composition of Bothrops jararacussu snake venom

Ontogenetic changes in venom composition have been described in Bothrops snakes, but only a few studies have attempted to identify the targeted paralogues or the molecular mechanisms involved in modifications of gene expression during ontogeny. In this study, we decoded B. jararacussu venom gland transcripts from six specimens of varying sizes and analyzed the variability in the composition of independent venom proteomes from 19 individuals. We identified 125 distinct putative toxin transcripts, and of these, 73 were detected in venom proteomes and only 10 were involved in the ontogenetic changes. Ontogenetic variability was linearly related to snake size and did not correspond to the maturation of the reproductive stage. Changes in the transcriptome were highly predictive of changes in the venom proteome. The basic myotoxic phospholipases A2 (PLA2s) were the most abundant components in larger snakes, while in venoms from smaller snakes, PIII-class SVMPs were the major components. The snake venom metalloproteinases (SVMPs) identified corresponded to novel sequences and conferred higher pro-coagulant and hemorrhagic functions to the venom of small snakes. The mechanisms modulating venom variability are predominantly related to transcriptional events and may consist of an advantage of higher hematotoxicity and more efficient predatory function in the venom from small snakes.