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. Full article

While snake venoms have been the subject of intense study, comparatively little work has been done on lizard venoms. In this study, we have examined the structural and functional diversification of anguimorph lizard venoms and associated toxins, and related these results to dentition and predatory ecology. Venom composition was shown to be highly variable across the 20 species of Heloderma, Lanthanotus, and Varanus included in our study. While kallikrein enzymes were ubiquitous, they were also a particularly multifunctional toxin type, with differential activities on enzyme substrates and also ability to degrade alpha or beta chains of fibrinogen that reflects structural variability. Examination of other toxin types also revealed similar variability in their presence and activity levels. The high level of venom chemistry variation in varanid lizards compared to that of helodermatid lizards suggests that venom may be subject to different selection pressures in these two families. These results not only contribute to our understanding of venom evolution but also reveal anguimorph lizard venoms to be rich sources of novel bioactive molecules with potential as drug design and development lead compounds. Full article

Venom gland transcriptomes and proteomes of six Micrurus taxa (M. corallinus, M. lemniscatus carvalhoi, M. lemniscatus lemniscatus, M. paraensis, M. spixii spixii, and M. surinamensis) were investigated, providing the most comprehensive, quantitative data on Micrurus venom composition to date, and more than tripling the number of Micrurus venom protein sequences previously available. The six venomes differ dramatically. All are dominated by 2–6 toxin classes that account for 91–99% of the toxin transcripts. The M. s. spixii venome is compositionally the simplest. In it, three-finger toxins (3FTxs) and phospholipases A₂ (PLA₂s) comprise >99% of the toxin transcripts, which include only four additional toxin families at levels ≥0.1%. Micrurus l. lemniscatus venom is the most complex, with at least 17 toxin families. However, in each venome, multiple structural subclasses of 3FTXs and PLA₂s are present. These almost certainly differ in pharmacology as well. All venoms also contain phospholipase B and vascular endothelial growth factors. Minor components (0.1–2.0%) are found in all venoms except that of M. s. spixii. Other toxin families are present in all six venoms at trace levels (<0.005%). Minor and trace venom components differ in each venom. Numerous novel toxin chemistries include 3FTxs with previously unknown 8- and 10-cysteine arrangements, resulting in new 3D structures and target specificities. 9-cysteine toxins raise the possibility of covalent, homodimeric 3FTxs or heterodimeric toxins with unknown pharmacologies. Probable muscarinic sequences may be reptile-specific homologs that promote hypotension via vascular mAChRs. The first complete sequences are presented for 3FTxs putatively responsible for liberating glutamate from rat brain synaptosomes. Micrurus C-type lectin-like proteins may have 6–9 cysteine residues and may be monomers, or homo- or heterodimers of unknown pharmacology. Novel KSPIs, 3× longer than any seen previously, appear to have arisen in three species by gene duplication and fusion. Four species have transcripts homologous to the nociceptive toxin, (MitTx) α-subunit, but all six species had homologs to the β-subunit. The first non-neurotoxic, non-catalytic elapid phospholipase A₂s are reported. All are probably myonecrotic. Phylogenetic analysis indicates that the six taxa diverged 15–35 million years ago and that they split from their last common ancestor with Old World elapines nearly 55 million years ago. Given their early diversification, many cryptic micrurine taxa are anticipated. Full article

Theraphosid spiders (tarantulas) are venomous arthropods found in most tropical and subtropical regions of the world. Tarantula venoms are a complex cocktail of toxins with potential use as pharmacological tools, drugs and bioinsecticides. Although numerous toxins have been isolated from tarantula venoms, little research has been carried out on the venom of Australian tarantulas. We therefore investigated the venom profile of the Australian theraphosid spider Phlogius crassipes and examined whether there are ontogenetic changes in venom composition. Spiders were divided into four ontogenic groups according to cephalothorax length, then the venom composition of each group was examined using gel electrophoresis and mass spectrometry. We found that the venom of P. crassipes changes continuously during development and throughout adulthood. Our data highlight the need to investigate the venom of organisms over the course of their lives to uncover and understand the changing functions of venom and the full range of toxins expressed. This in turn should lead to a deeper understanding of the organism’s ecology and enhance the potential for biodiscovery. Full article

Scorpion venom is a complex combinatorial library of peptides and proteins with multiple biological functions. A combination of transcriptomic and proteomic techniques has revealed its enormous molecular diversity, as identified by the presence of a large number of ion channel-targeted neurotoxins with different folds, membrane-active antimicrobial peptides, proteases, and protease inhibitors. Although the biodiversity of scorpion venom has long been known, how it arises remains unsolved. In this work, we analyzed the exon-intron structures of an array of scorpion venom protein-encoding genes and unexpectedly found that nearly all of these genes possess a phase-1 intron (one intron located between the first and second nucleotides of a codon) near the cleavage site of a signal sequence despite their mature peptides remarkably differ. This observation matches a theory of exon shuffling in the origin of new genes and suggests that recruitment of different folds into scorpion venom might be achieved via shuffling between body protein-coding genes and ancestral venom gland-specific genes that presumably contributed tissue-specific regulatory elements and secretory signal sequences. Full article

Sea anemones (Cnidaria, Anthozoa, and Actiniaria) use toxic peptides to incapacitate and immobilize prey and to deter potential predators. Their toxin arsenal is complex, targeting a variety of functionally important protein complexes and macromolecules involved in cellular homeostasis. Among these, actinoporins are one of the better characterized toxins; these venom proteins form a pore in cellular membranes containing sphingomyelin. We used a combined bioinformatic and phylogenetic approach to investigate how actinoporins have evolved across three superfamilies of sea anemones (Actinioidea, Metridioidea, and Actinostoloidea). Our analysis identified 90 candidate actinoporins across 20 species. We also found clusters of six actinoporin-like genes in five species of sea anemone (Nematostella vectensis, Stomphia coccinea, Epiactis japonica, Heteractis crispa, and Diadumene leucolena); these actinoporin-like sequences resembled actinoporins but have a higher sequence similarity with toxins from fungi, cone snails, and Hydra. Comparative analysis of the candidate actinoporins highlighted variable and conserved regions within actinoporins that may pertain to functional variation. Although multiple residues are involved in initiating sphingomyelin recognition and membrane binding, there is a high rate of replacement for a specific tryptophan with leucine (W112L) and other hydrophobic residues. Residues thought to be involved with oligomerization were variable, while those forming the phosphocholine (POC) binding site and the N-terminal region involved with cell membrane penetration were highly conserved. Full article

The “function debate” in the philosophy of biology and the “venom debate” in the science of toxinology are conceptually related. Venom systems are complex multifunctional traits that have evolved independently numerous times throughout the animal kingdom. No single concept of function, amongst those popularly defended, appears adequate to describe these systems in all their evolutionary contexts and extant variations. As such, a pluralistic view of function, previously defended by some philosophers of biology, is most appropriate. Venom systems, like many other functional traits, exist in nature as points on a continuum and the boundaries between “venomous” and “non-venomous” species may not always be clearly defined. This paper includes a brief overview of the concept of function, followed by in-depth discussion of its application to venom systems. A sound understanding of function may aid in moving the venom debate forward. Similarly, consideration of a complex functional trait such as venom may be of interest to philosophers of biology. Full article

Central and South American pitvipers, belonging to the genera Bothrops and Bothriechis, have independently evolved arboreal tendencies. Little is known regarding the composition and activity of their venoms. In order to close this knowledge gap, venom proteomics and toxin activity of species of Bothriechis, and Bothrops (including Bothriopsis) were investigated through established analytical methods. A combination of proteomics and bioactivity techniques was used to demonstrate a similar diversification of venom composition between large and small species within Bothriechis and Bothriopsis. Increasing our understanding of the evolution of complex venom cocktails may facilitate future biodiscoveries. Full article

Toxic weaponry in the form of venom and poison has evolved in most groups of animals, including all four major lineages of tetrapods. Moreover, the evolution of such traits has been linked to several key aspects of the biology of toxic animals including life-history and diversification. Despite this, attempts to investigate the macroevolutionary patterns underlying such weaponry are lacking. In this study we analyse patterns of venom and poison evolution across reptiles, amphibians, mammals, and birds using a suite of phylogenetic comparative methods. We find that each major lineage has a characteristic pattern of trait evolution, but mammals and reptiles evolve under a surprisingly similar regime, whilst that of amphibians appears to be particularly distinct and highly contrasting compared to other groups. Our results also suggest that the mechanism of toxin acquisition may be an important distinction in such evolutionary patterns; the evolution of biosynthesis is far less dynamic than that of sequestration of toxins from the diet. Finally, contrary to the situation in amphibians, other tetrapod groups show an association between the evolution of toxic weaponry and higher diversification rates. Taken together, our study provides the first broad-scale analysis of macroevolutionary patterns of venom and poison throughout tetrapods. Full article

Venomous snakes often display extensive variation in venom composition both between and within species. However, the mechanisms underlying the distribution of different toxins and venom types among populations and taxa remain insufficiently known. Rattlesnakes (Crotalus, Sistrurus) display extreme inter- and intraspecific variation in venom composition, centered particularly on the presence or absence of presynaptically neurotoxic phospholipases A₂ such as Mojave toxin (MTX). Interspecific hybridization has been invoked as a mechanism to explain the distribution of these toxins across rattlesnakes, with the implicit assumption that they are adaptively advantageous. Here, we test the potential of adaptive hybridization as a mechanism for venom evolution by assessing the distribution of genes encoding the acidic and basic subunits of Mojave toxin across a hybrid zone between MTX-positive Crotalus scutulatus and MTX-negative C. viridis in southwestern New Mexico, USA. Analyses of morphology, mitochondrial and single copy-nuclear genes document extensive admixture within a narrow hybrid zone. The genes encoding the two MTX subunits are strictly linked, and found in most hybrids and backcrossed individuals, but not in C. viridis away from the hybrid zone. Presence of the genes is invariably associated with presence of the corresponding toxin in the venom. We conclude that introgression of highly lethal neurotoxins through hybridization is not necessarily favored by natural selection in rattlesnakes, and that even extensive hybridization may not lead to introgression of these genes into another species. Full article

Venom delivery systems (VDS) are common in the animal kingdom, but rare amongst mammals. New definitions of venom allow us to reconsider its diversity amongst mammals by reviewing the VDS of Chiroptera, Eulipotyphla, Monotremata, and Primates. All orders use modified anterior dentition as the venom delivery apparatus, except Monotremata, which possesses a crural system. The venom gland in most taxa is a modified submaxillary salivary gland. In Primates, the saliva is activated when combined with brachial gland exudate. In Monotremata, the crural spur contains the venom duct. Venom functions include feeding, intraspecific competition, anti-predator defense and parasite defense. Including mammals in discussion of venom evolution could prove vital in our understanding protein functioning in mammals and provide a new avenue for biomedical and therapeutic applications and drug discovery. Full article

Along with food and drug allergic reactions, a Hymenoptera insect Sting (Apoidea, Vespidae, Formicidae) is one of the most common causes of anaphylaxis worldwide. Diagnoses of Hymenoptera venom allergy (HVA) and specific immunotherapy (SIT) have been based on the use of crude venom extracts. However, the incidence of cross-reactivity and low levels of sensibility during diagnosis, as well as the occurrence of nonspecific sensitization and undesired side effects during SIT, encourage the search for novel allergenic materials. Recombinant allergens are an interesting approach to improve allergy diagnosis and SIT because they circumvent major problems associated with the use of crude venom. Production of recombinant allergens depends on the profound molecular characterization of the natural counterpart by combining some “omics” approaches with high-throughput screening techniques and the selection of an appropriate system for heterologous expression. To date, several clinically relevant allergens and novel venom toxins have been identified, cloned and characterized, enabling a better understanding of the whole allergenic and envenoming processes. Here, we review recent findings on identification, molecular characterization and recombinant expression of Hymenoptera venom allergens and on the evaluation of these heterologous proteins as valuable tools for tackling remaining pitfalls on HVA diagnosis and immunotherapy. Full article

Venomics research is being revolutionized by the increased use of sensitive -omics techniques to identify venom toxins and their transcripts in both well studied and neglected venomous taxa. The study of neglected venomous taxa is necessary both for understanding the full diversity of venom systems that have evolved in the animal kingdom, and to robustly answer fundamental questions about the biology and evolution of venoms without the distorting effect that can result from the current bias introduced by some heavily studied taxa. In this review we draw the outlines of a roadmap into the diversity of poorly studied and understood venomous and putatively venomous invertebrates, which together represent tens of thousands of unique venoms. The main groups we discuss are crustaceans, flies, centipedes, non-spider and non-scorpion arachnids, annelids, molluscs, platyhelminths, nemerteans, and echinoderms. We review what is known about the morphology of the venom systems in these groups, the composition of their venoms, and the bioactivities of the venoms to provide researchers with an entry into a large and scattered literature. We conclude with a short discussion of some important methodological aspects that have come to light with the recent use of new -omics techniques in the study of venoms. Full article

The genes encoding group IIE phospholipase A₂, abbreviated as IIE PLA₂, and its 5' and 3' flanking regions of Crotalinae snakes such as Protobothrops flavoviridis, P. tokarensis, P. elegans, and Ovophis okinavensis, were found and sequenced. The genes consisted of four exons and three introns and coded for 22 or 24 amino acid residues of the signal peptides and 134 amino acid residues of the mature proteins. These IIE PLA₂s show high similarity to those from mammals and Colubridae snakes. The high expression level of IIE PLA₂s in Crotalinae venom glands suggests that they should work as venomous proteins. The blast analysis indicated that the gene encoding OTUD3, which is ovarian tumor domain-containing protein 3, is located in the 3' downstream of IIE PLA₂ gene. Moreover, a group IIA PLA₂ gene was found in the 5' upstream of IIE PLA₂ gene linked to the OTUD3 gene (OTUD3) in the P. flavoviridis genome. It became evident that the specified arrangement of IIA PLA₂ gene, IIE PLA₂ gene, and OTUD3 in this order is common in the genomes of humans to snakes. The present finding that the genes encoding various secretory PLA₂s form a cluster in the genomes of humans to birds is closely related to the previous finding that six venom PLA₂ isozyme genes are densely clustered in the so-called NIS-1 fragment of the P. flavoviridis genome. It is also suggested that venom IIA PLA₂ genes may be evolutionarily derived from the IIE PLA₂ gene. Full article

The monotremes (platypuses and echidnas) represent one of only four extant venomous mammalian lineages. Until recently, monotreme venom was poorly understood. However, the availability of the platypus genome and increasingly sophisticated genomic tools has allowed us to characterize platypus toxins, and provides a means of reconstructing the evolutionary history of monotreme venom. Here we review the physiology of platypus and echidna crural (venom) systems as well as pharmacological and genomic studies of monotreme toxins. Further, we synthesize current ideas about the evolution of the venom system, which in the platypus is likely to have been retained from a venomous ancestor, whilst being lost in the echidnas. We also outline several research directions and outstanding questions that would be productive to address in future research. An improved characterization of mammalian venoms will not only yield new toxins with potential therapeutic uses, but will also aid in our understanding of the way that this unusual trait evolves. Full article

Elapid snake venom is a highly valuable, but till now mainly unexplored, source of pharmacologically important peptides. We analyzed the peptide fractions with molecular masses up to 10 kDa of two elapid snake venoms—that of the African cobra, N. m. mossambica (genus Naja), and the Peninsula tiger snake, N. scutatus, from Kangaroo Island (genus Notechis). A combination of chromatographic methods was used to isolate the peptides, which were characterized by combining complimentary mass spectrometric techniques. Comparative analysis of the peptide compositions of two venoms showed specificity at the genus level. Three-finger (3-F) cytotoxins, bradykinin-potentiating peptides (BPPs) and a bradykinin inhibitor were isolated from the Naja venom. 3-F neurotoxins, Kunitz/basic pancreatic trypsin inhibitor (BPTI)-type inhibitors and a natriuretic peptide were identified in the N. venom. The inhibiting activity of the peptides was confirmed in vitro with a selected array of proteases. Cytotoxin 1 (P01467) from the Naja venom might be involved in the disturbance of cellular processes by inhibiting the cell 20S-proteasome. A high degree of similarity between BPPs from elapid and viperid snake venoms was observed, suggesting that these molecules play a key role in snake venoms and also indicating that these peptides were recruited into the snake venom prior to the evolutionary divergence of the snakes. Full article

Despite the unparalleled diversity of venomous snakes in Australia, research has concentrated on a handful of medically significant species and even of these very few toxins have been fully sequenced. In this study, venom gland transcriptomes were sequenced from eleven species of small Australian elapid snakes, from eleven genera, spanning a broad phylogenetic range. The particularly large number of sequences obtained for three-finger toxin (3FTx) peptides allowed for robust reconstructions of their dynamic molecular evolutionary histories. We demonstrated that each species preferentially favoured different types of α-neurotoxic 3FTx, probably as a result of differing feeding ecologies. The three forms of α-neurotoxin [Type I (also known as (aka): short-chain), Type II (aka: long-chain) and Type III] not only adopted differential rates of evolution, but have also conserved a diversity of residues, presumably to potentiate prey-specific toxicity. Despite these differences, the different α-neurotoxin types were shown to accumulate mutations in similar regions of the protein, largely in the loops and structurally unimportant regions, highlighting the significant role of focal mutagenesis. We theorize that this phenomenon not only affects toxin potency or specificity, but also generates necessary variation for preventing/delaying prey animals from acquiring venom-resistance. This study also recovered the first full-length sequences for multimeric phospholipase A₂ (PLA₂) ‘taipoxin/paradoxin’ subunits from non-Oxyuranus species, confirming the early recruitment of this extremely potent neurotoxin complex to the venom arsenal of Australian elapid snakes. We also recovered the first natriuretic peptides from an elapid that lack the derived C-terminal tail and resemble the plesiotypic form (ancestral character state) found in viper venoms. This provides supporting evidence for a single early recruitment of natriuretic peptides into snake venoms. Novel forms of kunitz and waprin peptides were recovered, including dual domain kunitz-kunitz precursors and the first kunitz-waprin hybrid precursors from elapid snakes. The novel sequences recovered in this study reveal that the huge diversity of unstudied venomous Australian snakes are of considerable interest not only for the investigation of venom and whole organism evolution but also represent an untapped bioresource in the search for novel compounds for use in drug design and development. Full article

Although known for their potent venom and ability to prey upon both invertebrate and vertebrate species, the Barychelidae spider family has been entirely neglected by toxinologists. In striking contrast, the sister family Theraphosidae (commonly known as tarantulas), which last shared a most recent common ancestor with Barychelidae over 200 million years ago, has received much attention, accounting for 25% of all the described spider toxins while representing only 2% of all spider species. In this study, we evaluated for the first time the venom arsenal of a barychelid spider, Trittame loki, using transcriptomic, proteomic, and bioinformatic methods. The venom was revealed to be dominated by extremely diverse inhibitor cystine knot (ICK)/knottin peptides, accounting for 42 of the 46 full-length toxin precursors recovered in the transcriptomic sequencing. In addition to documenting differential rates of evolution adopted by different ICK/knottin toxin lineages, we discovered homologues with completely novel cysteine skeletal architecture. Moreover, acetylcholinesterase and neprilysin were revealed for the first time as part of the spider-venom arsenal and CAP (CRiSP/Allergen/PR-1) were identified for the first time in mygalomorph spider venoms. These results not only highlight the extent of venom diversification in this neglected ancient spider lineage, but also reinforce the idea that unique venomous lineages are rich pools of novel biomolecules that may have significant applied uses as therapeutics and/or insecticides. Full article

The episodic nature of natural selection and the accumulation of extreme sequence divergence in venom-encoding genes over long periods of evolutionary time can obscure the signature of positive Darwinian selection. Recognition of the true biocomplexity is further hampered by the limited taxon selection, with easy to obtain or medically important species typically being the subject of intense venom research, relative to the actual taxonomical diversity in nature. This holds true for scorpions, which are one of the most ancient terrestrial venomous animal lineages. The family Buthidae that includes all the medically significant species has been intensely investigated around the globe, while almost completely ignoring the remaining non-buthid families. Australian scorpion lineages, for instance, have been completely neglected, with only a single scorpion species (Urodacus yaschenkoi) having its venom transcriptome sequenced. Hence, the lack of venom composition and toxin sequence information from an entire continent’s worth of scorpions has impeded our understanding of the molecular evolution of scorpion venom. The molecular origin, phylogenetic relationships and evolutionary histories of most scorpion toxin scaffolds remain enigmatic. In this study, we have sequenced venom gland transcriptomes of a wide taxonomical diversity of scorpions from Australia, including buthid and non-buthid representatives. Using state-of-art molecular evolutionary analyses, we show that a majority of CSα/β toxin scaffolds have experienced episodic influence of positive selection, while most non-CSα/β linear toxins evolve under the extreme influence of negative selection. For the first time, we have unraveled the molecular origin of the major scorpion toxin scaffolds, such as scorpion venom single von Willebrand factor C-domain peptides (SV-SVC), inhibitor cystine knot (ICK), disulphide-directed beta-hairpin (DDH), bradykinin potentiating peptides (BPP), linear non-disulphide bridged peptides and antimicrobial peptides (AMP). We have thus demonstrated that even neglected lineages of scorpions are a rich pool of novel biochemical components, which have evolved over millions of years to target specific ion channels in prey animals, and as a result, possess tremendous implications in therapeutics. Full article

Three-finger toxins (3FTx) represent one of the most abundantly secreted and potently toxic components of colubrid (Colubridae), elapid (Elapidae) and psammophid (Psammophiinae subfamily of the Lamprophidae) snake venom arsenal. Despite their conserved structural similarity, they perform a diversity of biological functions. Although they are theorised to undergo adaptive evolution, the underlying diversification mechanisms remain elusive. Here, we report the molecular evolution of different 3FTx functional forms and show that positively selected point mutations have driven the rapid evolution and diversification of 3FTx. These diversification events not only correlate with the evolution of advanced venom delivery systems (VDS) in Caenophidia, but in particular the explosive diversification of the clade subsequent to the evolution of a high pressure, hollow-fanged VDS in elapids, highlighting the significant role of these toxins in the evolution of advanced snakes. We show that Type I, II and III α-neurotoxins have evolved with extreme rapidity under the influence of positive selection. We also show that novel Oxyuranus/Pseudonaja Type II forms lacking the apotypic loop-2 stabilising cysteine doublet characteristic of Type II forms are not phylogenetically basal in relation to other Type IIs as previously thought, but are the result of secondary loss of these apotypic cysteines on at least three separate occasions. Not all 3FTxs have evolved rapidly: κ-neurotoxins, which form non-covalently associated heterodimers, have experienced a relatively weaker influence of diversifying selection; while cytotoxic 3FTx, with their functional sites, dispersed over 40% of the molecular surface, have been extremely constrained by negative selection. We show that the a previous theory of 3FTx molecular evolution (termed ASSET) is evolutionarily implausible and cannot account for the considerable variation observed in very short segments of 3FTx. Instead, we propose a theory of Rapid Accumulation of Variations in Exposed Residues (RAVER) to illustrate the significance of point mutations, guided by focal mutagenesis and positive selection in the evolution and diversification of 3FTx. Full article

Although snake venoms have been the subject of intense research, primarily because of their tremendous potential as a bioresource for design and development of therapeutic compounds, some specific groups of snakes, such as the genus Atractaspis, have been completely neglected. To date only limited number of toxins, such as sarafotoxins have been well characterized from this lineage. In order to investigate the molecular diversity of venom from Atractaspis aterrima—the slender burrowing asp, we utilized a high-throughput transcriptomic approach completed with an original bioinformatics analysis pipeline. Surprisingly, we found that Sarafotoxins do not constitute the major ingredient of the transcriptomic cocktail; rather a large number of previously well-characterized snake venom-components were identified. Notably, we recovered a large diversity of three-finger toxins (3FTxs), which were found to have evolved under the significant influence of positive selection. From the normalized and non-normalized transcriptome libraries, we were able to evaluate the relative abundance of the different toxin groups, uncover rare transcripts, and gain new insight into the transcriptomic machinery. In addition to previously characterized toxin families, we were able to detect numerous highly-transcribed compounds that possess all the key features of venom-components and may constitute new classes of toxins. Full article