Distinct regulatory networks control toxin gene expression in elapid and viperid snakes.

BACKGROUND: Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris). RESULTS: Although there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis. CONCLUSIONS: Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype.

Isolation and characterization of polymorphic microsatellite loci for the three Iberian vipers, Vipera aspis, V. Latastei and V. seoanei by Illumina MiSeq sequencing.

BACKGROUND: European vipers (genus Vipera) are a well-studied taxonomic group, but the low resolution of nuclear sanger-sequenced regions has precluded thorough studies at systematic, ecological, evolutionary and conservation levels. In this study, we developed novel microsatellite markers for the three Iberian vipers, Vipera aspis, V. latastei and V. seoanei, and assessed their polymorphism in north-central Iberian populations. METHODS AND RESULTS: Genomic libraries were developed for each species using an Illumina Miseq sequencing approach. From the 70 primer pairs initially tested, 48 amplified reliably and were polymorphic within species. Cross-species transferability was achieved for 31 microsatellites loci in the three target species and four additional loci that were transferable to one species only. The 48 loci amplified in average seven alleles, and detected average expected and observed heterozygosities of 0.7 and 0.55, in the three genotyped populations/species (26 V. aspis, 20 V. latastei and 10 V. seoanei). CONCLUSIONS: Our study provides a selection of 48 polymorphic microsatellite markers that will contribute significantly to current knowledge on genetic diversity, gene flow, population structure, demographic dynamics, systematics, reproduction and heritability in these species, and potentially in other congeneric taxa.

Hidden in the sand: Phylogenomics unravel an unexpected evolutionary history for the desert-adapted vipers of the genus Cerastes.

The desert vipers of the genus Cerastes are a small clade of medically important venomous snakes within the family Viperidae. According to published morphological and molecular studies, the group is comprised by four species: two morphologically similar and phylogenetically sister taxa, the African horned viper (Cerastes cerastes) and the Arabian horned viper (Cerastes gasperettii); a more distantly related species, the Saharan sand viper (Cerastes vipera), and the enigmatic Böhme's sand viper (Cerastes boehmei), only known from a single specimen in captivity allegedly captured in Central Tunisia. In this study, we sequenced one mitochondrial marker (COI) as well as genome-wide data (ddRAD sequencing) from 28 and 41 samples, respectively, covering the entire distribution range of the genus to explore the population genomics, phylogenomic relationships and introgression patterns within the genus Cerastes. Additionally, and to provide insights into the mode of diversification of the group, we carried out niche overlap analyses considering climatic and habitat variables. Both nuclear phylogenomic reconstructions and population structure analyses have unveiled an unexpected evolutionary history for the genus Cerastes, which sharply contradicts the morphological similarities and previously published mitochondrial approaches. Cerastes cerastes and C. vipera are recovered as sister taxa whilst C. gasperettii is a sister taxon to the clade formed by these two species. We found a relatively high niche overlap (OI > 0.7) in both climatic and habitat variables between C. cerastes and C. vipera, contradicting a potential scenario of sympatric speciation. These results are in line with the introgression found between the northwestern African populations of C. cerastes and C. vipera. Finally, our genomic data confirms the existence of a lineage of C. cerastes in Arabia. All these results highlight the importance of genome-wide data over few genetic markers to study the evolutionary history of species.

The Brazilian Atlantic Bushmaster Lachesis (Linnaeus, 1766) Mitogenome With Insights On Snake Evolution And Divergence (Serpentes: Viperidae: Crotalinae).

This study presents the first complete mitogenome of the Brazilian Atlantic bushmaster Lachesis with insights into snake evolution. The total length was 17,177 bp, consisting of 13 PCGs, 22 tRNAs, two rRNAs and a duplicate control region (CRs). Almost all genes were encoded by the heavy-strand, except for the ND6 gene and eight tRNAs (tRNA-Gln, Ala, Asn, Cys, Tyr, Ser[TGA anticodon], Glu, Pro). Only ATG, ATA, and ATC were starting codons for protein-coding sequences. Stop codons mainly were TAA, AGA, AGG, and TAG; whereas ND1, ND3, and CYTB terminated with incomplete stop codons. Phylogeny retrieved Lachesis within the Crotalinae as the sister group of Agkistrodon; and the Lachesis+Agkistrodon clade as the sister group of (Sistrurus+Crotalus)+Bothrops. The tree supports Crotalinae, Viperinae, and Azemiopinae in the Viperidae family, being sister taxa of Colubridae+(Elapidae+Psammophiidae). The mean genetic distance across 15 snake families and 57 nucleotide sequences was 0.37. The overall mean value of genetic distance across the Crotalinae was 0.23, with Lachesis muta exhibiting the shortest distance of 0.2 with Agkistrodon piscivorus, Protobothrops dabieshanensis and P. flavoviridis and the greatest 0.25 with Gloydius blomhoffii, Trimeresurus albolabris, S. miliarius, and Deinagkistrodon acutus. The complete Atlantic L. muta mitogenome presented herein is only the third annotated mitogenome from more than 430 described Brazilian snake species.

Discovery of a new Hepatozoon species namely Hepatozoon viperoi sp. nov. in nose-horned vipers in Türkiye.

Although Hepatozoon spp. remains the most prevalent intracellular protozoa infecting snakes, it was reported only in a few snake species of the Colubridae family in Türkiye. Moreover, studies on these hemoparasites are not available in venomous nose-horned vipers from Türkiye. In this study, we investigated Hepatozoon spp. in three individual Vipera ammodytes using morphological and molecular methods. Our results were positive for intraerythrocytic Hepatozoon spp. gamonts in all three snakes, exhibiting low parasitemia. The microscopic findings were further confirmed through molecular data. A genus-specific PCR assay targeting the 18S rRNA gene region of Hepatozoon spp., was performed using HemoF/HemoR and Hep300/Hep900 primers. The obtained sequences were concatenated and used in phylogenetic analyses in comparison with different Hepatozoon species. Although our (OP377741) isolate was separated into a different branch, it was clustered with the isolates of H. massardi (KC342526), H. cevapii (KC342525), and H. annulatum (ON262426) from Brazilian snakes. Moreover, gene similarity and pair-wise distance between our isolate and other Hepatozoon species infecting snakes were found to be 89.30-98.63% and 0.009-0.077, respectively. Hence, we reported a new species of Hepatozoon, namely Hepatozoon viperoi sp. nov. infecting V. ammodytes. Since the literature does not indicate the existence of such a Hepatozoon species in V. ammodytes in different countries, our data may contribute to the expanding knowledge of Hepatozoon species in snakes, providing new insights into the biodiversity of the haemogregarine protozoan parasite.

Total-evidence phylogeny and evolutionary morphology of New World pitvipers (Serpentes: Viperidae: Crotalinae).

Crotalines (pitvipers) in the Americas are distributed from southern Canada to southern Argentina, and are represented by 13 genera and 163 species that constitute a monophyletic group. Their phylogenetic relationships have been assessed mostly based on DNA sequences, while morphological data have scarcely been used for phylogenetic inquiry. We present a total-evidence phylogeny of New World pitvipers, the most taxon/character comprehensive phylogeny to date. Our analysis includes all genera, morphological data from external morphology, cranial osteology and hemipenial morphology, and DNA sequences from mitochondrial and nuclear genes. We performed analyses with parsimony as an optimality criterion, using different schemes for character weighting. We evaluated the contribution of the different sources of characters to the phylogeny through analyses of reduced datasets and calculation of weighted homoplasy and retention indexes. We performed a morphological character analysis to identify synapomorphies for the main clades. In terms of biogeography, our results support a single colonization event of the Americas by pitvipers, and a cladogenetic event into a Neotropical clade and a North American/Neotropical clade. The results also shed light on the previously unstable position of some taxa, although they could not sufficiently resolve the position of Bothrops lojanus, which may lead to the paraphyly of either Bothrops or Bothrocophias. The morphological character analyses demonstrated that an important phylogenetic signal is contained in characters related to head scalation, the jaws and the dorsum of the skull, and allowed us to detect morphological convergences in external morphology associated with arboreality.

Polyandry and non-random fertilisation maintain long-term genetic diversity in an isolated island population of adders (Vipera berus).

Conservation genetic theory suggests that small and isolated populations should be subject to reduced genetic diversity i.e., heterozygosity and allelic diversity. Our 34 years study of an isolated island population of adders (Vipera berus) in southern Sweden challenges this notion. Despite a lack of gene flow and a yearly mean estimated reproductive adult population size of only 65 adult adders (range 12-171), the population maintains high levels of heterozygosity and allelic diversity similar to that observed in two mainland populations. Even a 14-year major "bottleneck" i.e., a reduction in adult adder numbers, encompassing at least four adder generations, did not result in any reduction in the island adders' heterozygosity and allelic diversity. Female adders are polyandrous, and fertilisation is non-random, which our empirical data and modelling suggest are underpinning the maintenance of the population's high level of heterozygosity. Our empirical results and subsequent modelling suggest that the positive genetic effects of polyandry in combination with non-random fertilisation, often overlooked in conservation genetic analyses, deserve greater consideration when predicting long-term survival of small and isolated populations.

De Novo Genome Assembly Highlights the Role of Lineage-Specific Gene Duplications in the Evolution of Venom in Fea's Viper (Azemiops feae).

Despite the medical significance to humans and important ecological roles filled by vipers, few high-quality genomic resources exist for these snakes outside of a few genera of pitvipers. Here we sequence, assemble, and annotate the genome of Fea's Viper (Azemiops feae). This taxon is distributed in East Asia and belongs to a monotypic subfamily, sister to the pitvipers. The newly sequenced genome resulted in a 1.56 Gb assembly, a contig N50 of 1.59 Mb, with 97.6% of the genome assembly in contigs >50 Kb, and a BUSCO completeness of 92.4%. We found that A. feae venom is primarily composed of phospholipase A2 (PLA2) proteins expressed by genes that likely arose from lineage-specific PLA2 gene duplications. Additionally, we show that renin, an enzyme associated with blood pressure regulation in mammals and known from the venoms of two viper species including A. feae, is expressed in the venom gland at comparative levels to known toxins and is present in the venom proteome. The cooption of this gene as a toxin may be more widespread in viperids than currently known. To investigate the historical population demographics of A. feae, we performed coalescent-based analyses and determined that the effective population size has remained stable over the last 100 kyr. This suggests Quaternary glacial cycles likely had minimal influence on the demographic history of A. feae. This newly assembled genome will be an important resource for studying the genomic basis of phenotypic evolution and understanding the diversification of venom toxin gene families.

A new cryptic species of green pit viper of the genus Trimeresurus Lacépède, 1804 (Serpentes, Viperidae) from northeast India.

A new cryptic species of green pit viper is described from northeast India, based on specimens collected from the state of Mizoram and Meghalaya. The new species is a member of the subgenus Viridovipera and is sister to Trimeresurus medoensis based on molecular data for mitochondrial cytochrome b gene, whereas resembles Trimeresurus gumprechti morphologically. A combination of characters helps delimit the new species from its congeners. Description of the new species highlights the need for dedicated surveys across northeast India to document its reptilian diversity, as this represents the third new species of the genus to be described in the past three years.

What's in a mass?

This short essay pretends to make the reader reflect on the concept of biological mass and on the added value that the determination of this molecular property of a protein brings to the interpretation of evolutionary and translational snake venomics research. Starting from the premise that the amino acid sequence is the most distinctive primary molecular characteristics of any protein, the thesis underlying the first part of this essay is that the isotopic distribution of a protein's molecular mass serves to unambiguously differentiate it from any other of an organism's proteome. In the second part of the essay, we discuss examples of collaborative projects among our laboratories, where mass profiling of snake venom PLA2 across conspecific populations played a key role revealing dispersal routes that determined the current phylogeographic pattern of the species.

Substrate thermal properties influence ventral brightness evolution in ectotherms.

The thermal environment can affect the evolution of morpho-behavioral adaptations of ectotherms. Heat is transferred from substrates to organisms by conduction and reflected radiation. Because brightness influences the degree of heat absorption, substrates could affect the evolution of integumentary optical properties. Here, we show that vipers (Squamata:Viperidae) inhabiting hot, highly radiative and superficially conductive substrates have evolved bright ventra for efficient heat transfer. We analyzed the brightness of 4161 publicly available images from 126 species, and we found that substrate type, alongside latitude and body mass, strongly influences ventral brightness. Substrate type also significantly affects dorsal brightness, but this is associated with different selective forces: activity-pattern and altitude. Ancestral estimation analysis suggests that the ancestral ventral condition was likely moderately bright and, following divergence events, some species convergently increased their brightness. Vipers diversified during the Miocene and the enhancement of ventral brightness may have facilitated the exploitation of arid grounds. We provide evidence that integument brightness can impact the behavioral ecology of ectotherms.

Genetic rescue restores long-term viability of an isolated population of adders (Vipera berus).

Climate change is regarded as a major threat to global biodiversity [1]. However, another key driver of declines in biodiversity during the last century has been, and still is, the devastating impact of anthropogenic habitat destruction [2]. Human degradation of natural habitats has resulted in large, formerly homogeneous areas becoming exceedingly isolated and fragmented, resulting in reduced genetic diversity and a concomitant increased vulnerability to pathogens [3] and increased risk of inbreeding [4]. In order to restore genetic diversity in small isolated or fragmented populations, genetic rescue - that is, an intervention in which unrelated individuals are brought into a population, leading to introduction of novel alleles - has been shown to reduce the deleterious effects of inbreeding [4,5].

Thermal melanism explains macroevolutionary variation of dorsal pigmentation in Eurasian vipers.

Colouration may endorse thermoregulatory and antipredatory functions in snakes. The thermal melanism hypothesis predicts that dark-coloured individuals are ecologically favoured in cool climates. However, the loss of aposematic and cryptic colourations may imply high predation for melanistic snakes. Here, we used the monophyletic group of Eurasian vipers (subfamily Viperinae) to test whether an increase in the extent of dark area inside the characteristic zigzag dorsal pattern is associated to colder environments. We measured two colouration traits in zigzag-patterned individuals (number of dorsal marks and weighted pigmentation index) and used a phylogenetic comparative approach to explore macroevolutionary patterns of dorsal pigmentation and test whether its extent is associated to ecogeographic characteristics of lineages' ranges. Phylogenetically-naïve and phylogenetically-informed analyses yielded a significant association between the degree of pigmentation of the zigzag pattern and environmental variables such as solar radiation, elevation and latitude. The degree of pigmentation of the zigzag pattern is highlighted as an adaptive trait that matches range attributes mirroring cold environments irrespective of the phylogeny. These results constitute the first large-scale evidence supporting the thermal melanism hypothesis in snakes, opening new avenues of inquiry for the mechanisms that shape the evolution of colour phenotypes.

Genetic and demographic vulnerability of adder populations: Results of a genetic study in mainland Britain.

Genetic factors are often overlooked in conservation planning, despite their importance in small isolated populations. We used mitochondrial and microsatellite markers to investigate population genetics of the adder (Vipera berus) in southern Britain, where numbers are declining. We found no evidence for loss of heterozygosity in any of the populations studied. Genetic diversity was comparable across sites, in line with published levels for mainland Europe. However, further analysis revealed a striking level of relatedness. Genetic networks constructed from inferred first degree relationships suggested a high proportion of individuals to be related at a level equivalent to that of half-siblings, with rare inferred full-sib dyads. These patterns of relatedness can be attributed to the high philopatry and low vagility of adders, which creates high local relatedness, in combination with the polyandrous breeding system in the adder, which may offset the risk of inbreeding in closed populations. We suggest that reliance on standard genetic indicators of inbreeding and diversity may underestimate demographic and genetic factors that make adder populations vulnerable to extirpation. We stress the importance of an integrated genetic and demographic approach in the conservation of adders, and other taxa of similar ecology.

Effect of Bitis gabonica and Dendroaspis angusticeps snake venoms on apoptosis-related genes in human thymic epithelial cells

Certain environmental toxins permanently damage the thymic epithelium, accelerate immune senescence and trigger secondary immune pathologies. However, the exact underlying cellular mechanisms and pathways of permanent immune intoxication remain unknown. The aim of the present study was to demonstrate gene expressional changes of apoptosis-related cellular pathways in human thymic epithelial cells following exposure to snake venom from Bitis gabonica and Dendroaspis angusticeps. Methods: Snake venoms were characterized by analytical methods including reversed phase high-performance liquid chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, then applied on human thymic epithelial cells (1889c) for 24 h at 10 μg/mL (as used in previous TaqMan Array study). Gene expressional changes restricted to apoptosis were assayed by TaqMan Array (Human Apoptosis Plate). Results: The most prominent gene expressional changes were shown by CASP5 (≈ 2.5 million-fold, confirmed by dedicated quantitative polymerase chain reaction) and CARD9 (0.016-fold) for B. gabonica, and BIRC7 (6.46-fold) and CASP1 (0.30-fold) for D. angusticeps. Conclusion: The observed apoptotic environment suggests that pyroptosis may be the dominant pathway through which B. gabonica and D. angusticeps snake venoms trigger thymic epithelial apoptosis following envenomation.(AU)

Calibrating the tree of vipers under the fossilized birth-death model.

Scaling evolutionary trees to time is essential for understanding the origins of clades. Recently developed methods allow including the entire fossil record known for the group of interest and eliminated the need for specifying prior distributions for node ages. Here we apply the fossilized birth-death (FBD) approach to reconstruct the diversification timeline of the viperines (subfamily Viperinae). Viperinae are an Old World snake subfamily comprising 102 species from 13 genera. The fossil record of vipers is fairly rich and well assignable to clades due to the unique vertebral and fang morphology. We use an unprecedented sampling of 83 modern species and 13 genetic markers in combination with 197 fossils representing 28 extinct taxa to reconstruct a time-calibrated phylogeny of the Viperinae. Our results suggest a late Eocene-early Oligocene origin with several diversification events following soon after the group's establishment. The age estimates inferred with the FBD model correspond to those from previous studies that were based on node dating but FBD provides notably narrower credible intervals around the node ages. Viperines comprise two African and an Eurasian clade, but the ancestral origin of the subfamily is ambiguous. The most parsimonious scenarios require two transoceanic dispersals over the Tethys Sea during the Oligocene.

Comprehensive Study of the Proteome and Transcriptome of the Venom of the Most Venomous European Viper: Discovery of a New Subclass of Ancestral Snake Venom Metalloproteinase Precursor-Derived Proteins.

The nose-horned viper, its nominotypical subspecies Vipera ammodytes ammodytes ( Vaa), in particular, is, medically, one of the most relevant snakes in Europe. The local and systemic clinical manifestations of poisoning by the venom of this snake are the result of the pathophysiological effects inflicted by enzymatic and nonenzymatic venom components acting, most prominently, on the blood, cardiovascular, and nerve systems. This venom is a very complex mixture of pharmacologically active proteins and peptides. To help improve the current antivenom therapy toward higher specificity and efficiency and to assist drug discovery, we have constructed, by combining transcriptomic and proteomic analyses, the most comprehensive library yet of the Vaa venom proteins and peptides. Sequence analysis of the venom gland cDNA library has revealed the presence of messages encoding 12 types of polypeptide precursors. The most abundant are those for metalloproteinase inhibitors (MPis), bradykinin-potentiating peptides (BPPs), and natriuretic peptides (NPs) (all three on a single precursor), snake C-type lectin-like proteins (snaclecs), serine proteases (SVSPs), P-II and P-III metalloproteinases (SVMPs), secreted phospholipases A2 (sPLA2s), and disintegrins (Dis). These constitute >88% of the venom transcriptome. At the protein level, 57 venom proteins belonging to 16 different protein families have been identified and, with SVSPs, sPLA2s, snaclecs, and SVMPs, comprise ∼80% of all venom proteins. Peptides detected in the venom include NPs, BPPs, and inhibitors of SVSPs and SVMPs. Of particular interest, a transcript coding for a protein similar to P-III SVMPs but lacking the MP domain was also found at the protein level in the venom. The existence of such proteins, also supported by finding similar venom gland transcripts in related snake species, has been demonstrated for the first time, justifying the proposal of a new P-IIIe subclass of ancestral SVMP precursor-derived proteins.

Evolutionary history of burrowing asps (Lamprophiidae: Atractaspidinae) with emphasis on fang evolution and prey selection.

Atractaspidines are poorly studied, fossorial snakes that are found throughout Africa and western Asia, including the Middle East. We employed concatenated gene-tree analyses and divergence dating approaches to investigate evolutionary relationships and biogeographic patterns of atractaspidines with a multi-locus data set consisting of three mitochondrial (16S, cyt b, and ND4) and two nuclear genes (c-mos and RAG1). We sampled 91 individuals from both atractaspidine genera (Atractaspis and Homoroselaps). Additionally, we used ancestral-state reconstructions to investigate fang and diet evolution within Atractaspidinae and its sister lineage (Aparallactinae). Our results indicated that current classification of atractaspidines underestimates diversity within the group. Diversification occurred predominantly between the Miocene and Pliocene. Ancestral-state reconstructions suggest that snake dentition in these taxa might be highly plastic within relatively short periods of time to facilitate adaptations to dynamic foraging and life-history strategies.

Multilocus phylogeography of the brown-spotted pitviper Protobothrops mucrosquamatus (Reptilia: Serpentes: Viperidae) sheds a new light on the diversification pattern in Asia.

Understanding the influence of geographical events and climate changes on genetic diversity is essential in explaining current patterns of genetic structure and geographic distribution of organisms. We inferred phylogenetic relationships, investigated historical demography, explored the evolutionary history, and clarified intraspecific taxonomy of Protobothrops mucrosquamatus, which is one of the commonest and most wide-ranging Asian pitvipers. A total of 184 samples from 54 localities were sequenced and analyzed for two mitochondrial gene fragments and two nuclear genes. Phylogenetic reconstruction based on mtDNA sequences revealed the existence of a minimum of five geographically structured and well-supported lineages within P. mucrosquamatus. Based on the mtDNA gene tree, and the geographic relationship between populations allied by matrilineal lineages, a complex longitudinal and latitudinal diversification pattern was uncovered in P. mucrosquamatus. The estimated date of the origin of the species (about 5.3 Ma) and divergence of the intraspecific lineages match the rapid uplifting of Qinghai-Xizang Plateau, and is also consistent with those of some other co-distributed Asian pitvipers. Formation of the two island lineages (Taiwan and Hainan) was generally congruent with the first isolation of the islands, but the two lineages showed different relationships with the continental Asian populations in comparison with some other pitvipers. Population historical demographic analyses, based on three methods, showed that all lineages have experienced slight population expansion in and around the Dali Glacial. Tests of intraspecific taxonomy indicated that no cryptic taxon is present within this widely distributed snake.

Comparative Profiling of Three Atheris Snake Venoms: A. squamigera, A. nitschei and A. chlorechis.

A proteomic and transcriptomic comparative analysis of the venoms of three Atheris species (A. squamigera, A. nitschei and A. chlorechis) was carried out by size exclusion liquid chromatography, gel electrophoresis, mass spectrometry, and mRNA sequencing. The improved proteomic profiling utilised in this work was combined with transcript studies, advancing our insights into venom composition, protein distribution and inter-species variation among the three bush vipers. Crude venoms of all three samples contained at least 10-20 protein components, ranging in size from ≤ 3 to > 98 kDa. Both approaches yielded converging overall information, pointing to phospholipases, disintegrins, serine proteases and metalloproteases as the major toxin classes, which are likely to explain the local and systemic symptoms observed in envenomation by Atheris genus. Being considered as the main factors involved in the distinct venom-induced pathologies, these identified snake venom proteins are of particular interest in terms of understanding their physiological and biological function as well as for their contribution in potential medical treatments.