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Venoms have evolved >100 times in all major animal groups, and their components, known as toxins, have been fine-tuned over millions of years into highly effective biochemical weapons. There are many outstanding questions on the evolution of toxin arsenals, such as how venom genes originate, how venom contributes to the fitness of venomous species, and which modifications at the genomic, transcriptomic, and protein level drive their evolution. These questions have received particularly little attention outside of snakes, cone snails, spiders, and scorpions. Venom compounds have further become a source of inspiration for translational research using their diverse bioactivities for various applications. We highlight here recent advances and new strategies in modern venomics and discuss how recent technological innovations and multi-omic methods dramatically improve research on venomous animals. The study of genomes and their modifications through CRISPR and knockdown technologies will increase our understanding of how toxins evolve and which functions they have in the different ontogenetic stages during the development of venomous animals. Mass spectrometry imaging combined with spatial transcriptomics, in situ hybridization techniques, and modern computer tomography gives us further insights into the spatial distribution of toxins in the venom system and the function of the venom apparatus. All these evolutionary and biological insights contribute to more efficiently identify venom compounds, which can then be synthesized or produced in adapted expression systems to test their bioactivity. Finally, we critically discuss recent agrochemical, pharmaceutical, therapeutic, and diagnostic (so-called translational) aspects of venoms from which humans benefit., (© The Author(s) 2022. Published by Oxford University Press GigaScience.)

Animal venoms are renowned for their toxicity, biochemical complexity, and as a source of compounds with potential applications in medicine, agriculture, and industry. Polypeptides underlie much of the pharmacology of animal venoms, and elucidating these arsenals of polypeptide toxins-known as the venom proteome or venome-is an important step in venom research. Proteomics is used for the identification of venom toxins, determination of their primary structure including post-translational modifications, as well as investigations into the physiology underlying their production and delivery. Advances in proteomics and adjacent technologies has led to a recent upsurge in publications reporting venom proteomes. Improved mass spectrometers, better proteomic workflows, and the integration of next-generation sequencing of venom-gland transcriptomes and venomous animal genomes allow quicker and more accurate profiling of venom proteomes with greatly reduced starting material. Technologies such as imaging mass spectrometry are revealing additional insights into the mechanism, location, and kinetics of venom toxin production. However, these numerous new developments may be overwhelming for researchers designing venom proteome studies. Here, the field of venom proteomics is reviewed and some practical solutions for simplifying mass spectrometry workflows to study animal venoms are offered., (© 2020 Wiley-VCH GmbH.)

A critical hurdle in ant venom proteomic investigations is the lack of databases to comprehensively and specifically identify the sequence and function of venom proteins and peptides. To resolve this, we used venom gland transcriptomics to generate a sequence database that was used to assign the tandem mass spectrometry (MS) fragmentation spectra of venom peptides and proteins to specific transcripts. This was performed alongside a shotgun liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the venom to confirm that these assigned transcripts were expressed as proteins. Through the combined transcriptomic and proteomic investigation of Paraponera clavata venom, we identified four times the number of proteins previously identified using 2D-PAGE alone. In addition to this, by mining the transcriptomic data, we identified several novel peptide sequences for future pharmacological investigations, some of which conform with inhibitor cysteine knot motifs. These types of peptides have the potential to be developed into pharmaceutical or bioinsecticide peptides.

The Estuarine stonefish (Synanceia horrida) is recognised as one of the most venomous fish species in the world but the overall venom composition has yet to be investigated using in-depth transcriptomic and proteomic methods. To date, known venom components are restricted to a hyaluronidase and a large, pore-forming toxin known as Stonustoxin (SNTX). Transcriptomic sequencing of the venom gland resulted in over 170,000 contigs with only 0.4% that were homologous to putative venom proteins. Integration of the transcriptomic data with proteomic data from the S. horrida venom confirmed the hyaluronidase and SNTX to be present, together with several other protein families including major contributions from C-type lectins. Other protein families observed included peroxiredoxin and several minor protein families such as Golgi-associated plant pathogenesis related proteins, tissue pathway factor inhibitors, and Kazal-type serine protease inhibitors that, although not putative venom proteins, may contribute to the venom's adverse effects. BIOLOGICAL SIGNIFICANCE: Proteomic analysis of milked Synanceia horrida venom, paired with transcriptomic analysis of the venom gland tissue revealed for the first time the composition of one of the world's most dangerous fish venoms. The results demonstrate that the venom is relatively less complex compared to other well-studied venomous animals with a number of unique proteins not previously found in animal venoms., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Introduction: Animal venoms are complex chemical arsenals. Most venoms are rich in bioactive peptides with proven potential as research tools, drug leads and drugs. Areas covered: We review recent advances in venom-peptide discovery, particularly the adoption of combined transcriptomic/proteomic approaches for the exploration of venom composition. Expert commentary: Advances in transcriptomics and proteomics have dramatically altered the manner and rate of venom-peptide discovery. The increasing trend towards a toxin-driven approach, as opposed to traditional target-based screening of venoms, is likely to expedite the discovery of venom-peptides with novel structures and new and unanticipated mechanisms of action. At the same time, these advances will drive the development of higher-throughput approaches for target identification. Taken together, these approaches should enhance our understanding of the natural ecological function of venom peptides and increase the rate of identification of novel venom-derived pharmacological tools, drug leads and drugs.

For over a century, venom samples from wild snakes have been collected and stored around the world. However, the quality of storage conditions for "vintage" venoms has rarely been assessed. The goal of this study was to determine whether such historical venom samples are still biochemically and pharmacologically viable for research purposes, or if new sample efforts are needed. In total, 52 samples spanning 5 genera and 13 species with regional variants of some species (e.g., 14 different populations of Notechis scutatus) were analysed by a combined proteomic and pharmacological approach to determine protein structural stability and bioactivity. When venoms were not exposed to air during storage, the proteomic results were virtually indistinguishable from that of fresh venom and bioactivity was equivalent or only slightly reduced. By contrast, a sample of Acanthophis antarcticus venom that was exposed to air (due to a loss of integrity of the rubber stopper) suffered significant degradation as evidenced by the proteomics profile. Interestingly, the neurotoxicity of this sample was nearly the same as fresh venom, indicating that degradation may have occurred in the free N- or C-terminus chains of the proteins, rather than at the tips of loops where the functional residues are located. These results suggest that these and other vintage venom collections may be of continuing value in toxin research. This is particularly important as many snake species worldwide are declining due to habitat destruction or modification. For some venoms (such as N. scutatus from Babel Island, Flinders Island, King Island and St. Francis Island) these were the first analyses ever conducted and these vintage samples may represent the only venom ever collected from these unique island forms of tiger snakes. Such vintage venoms may therefore represent the last remaining stocks of some local populations and thus are precious resources. These venoms also have significant historical value as the Oxyuranus venoms analysed include samples from the first coastal taipan (Oxyuranus scutellatus) collected for antivenom production (the snake that killed the collector Kevin Budden), as well as samples from the first Oxyuranus microlepidotus specimen collected after the species' rediscovery in 1976. These results demonstrate that with proper storage techniques, venom samples can retain structural and pharmacological stability. This article is part of a Special Issue entitled: Proteomics of non-model organisms., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Aculeate hymenopterans use their venom for a variety of different purposes. The venom of solitary aculeates paralyze and preserve prey without killing it, whereas social aculeates utilize their venom in defence of their colony. These distinct applications of venom suggest that its components and their functions are also likely to differ. This study investigates a range of solitary and social species across Aculeata. We combined electrophoretic, mass spectrometric, and transcriptomic techniques to characterize the compositions of venoms from an incredibly diverse taxon. In addition, in vitro assays shed light on their biological activities. Although there were many common components identified in the venoms of species with different social behavior, there were also significant variations in the presence and activity of enzymes such as phospholipase A2s and serine proteases and the cytotoxicity of the venoms. Social aculeate venom showed higher presence of peptides that cause damage and pain in victims. The venom-gland transcriptome from the European honeybee (Apis mellifera) contained highly conserved toxins which match those identified by previous investigations. In contrast, venoms from less-studied taxa returned limited results from our proteomic databases, suggesting that they contain unique toxins. [ABSTRACT FROM AUTHOR]

The phylum Cnidaria is the oldest extant venomous group and is defined by the presence of nematocysts, specialized organelles responsible for venom production and delivery. Although toxin peptides and the cells housing nematocysts are distributed across the entire animal, nematocyte and venom profiles have been shown to differ across morphological structures in actiniarians. In this study, we explore the relationship between patterns of toxin expression and the ecological roles of discrete anatomical structures in Telmatactis stephensoni. Specifically, using a combination of proteomic and transcriptomic approaches, we examined whether there is a direct correlation between the functional similarity of regions and the similarity of their associated toxin expression profiles. We report that the regionalization of toxin production is consistent with the partitioning of the ecological roles of venom across envenomating structures, and that three major functional regions are present in T. stephensoni: tentacles, epidermis and gastrodermis. Additionally, we find that most structures that serve similar functions not only have comparable putative toxin profiles but also similar nematocyst types. There was no overlap in the putative toxins identified using proteomics and transcriptomics, but the expression patterns of specific milked venom peptides were conserved across RNA‐sequencing and mass spectrometry imaging data sets. Furthermore, based on our data, it appears that acontia of T. stephensoni may be transcriptionally inactive and only mature nematocysts are present in the distal portions of the threads. Overall, we find that the venom profile of different anatomical regions in sea anemones varies according to its ecological functions. [ABSTRACT FROM AUTHOR]

Spiders are one of the most successful venomous animals, with more than 48,000 described species. Most spider venoms are dominated by cysteine-rich peptides with a diverse range of pharmacological activities. Some spider venoms contain thousands of unique peptides, but little is known about the mechanisms used to generate such complex chemical arsenals. We used an integrated transcriptomic, proteomic, and structural biology approach to demonstrate that the lethal Australian funnel-web spider produces 33 superfamilies of venom peptides and proteins. Twentysix of the 33 superfamilies are disulfide-rich peptides, and we show that 15 of these are knottins that contribute >90% of the venom proteome. NMR analyses revealed that most of these disulfide-rich peptides are structurally related and range in complexity from simple to highly elaborated knottin domains, as well as double-knot toxins, that likely evolved from a single ancestral toxin gene. [ABSTRACT FROM AUTHOR]

Centipedes are among the most ancient groups of venomous predatory arthropods. Extant species belong to five orders, but our understanding of the composition and evolution of centipede venoms is based almost exclusively on one order, Scolopendromorpha. To gain a broader and less biased understanding we performed a comparative proteotranscriptomic analysis of centipede venoms from all five orders, including the first venom profiles for the orders Lithobiomorpha, Craterostigmomorpha, and Geophilomorpha. Our results reveal an astonishing structural diversity of venom components, with 93 phylogenetically distinct protein and peptide families. Proteomically-annotated gene trees of these putative toxin families show that centipede venom composition is highly dynamic across macroevolutionary timescales, with numerous gene duplications as well as functional recruitments and losses of toxin gene families. Strikingly, not a single family is found in the venoms of representatives of all five orders, with 67 families being unique for single orders. Ancestral state reconstructions reveal that centipede venom originated as a simple cocktail comprising just four toxin families, with very little compositional evolution happening during the approximately 50 My before the living orders had diverged. Venom complexity then increased in parallel within the orders, with scolopendromorphs evolving particularly complex venoms. Our results show that even venoms composed of toxins evolving under the strong constraint of negative selection can have striking evolutionary plasticity on the compositional level. We show that the functional recruitments and losses of toxin families that shape centipede venom arsenals are not concentrated early in their evolutionary history, but happen frequently throughout. [ABSTRACT FROM AUTHOR]

Centipede venoms have emerged as a rich source of novel bioactive compounds. However, most centipede species are commonly considered too small for venom extraction and transcriptomics is likely to be an attractive way of probing the molecular diversity of these venoms. Examining the venom composition of Scolopendra subspinipes, we test the accuracy of this approach. We compared the proteomically determined venom profile with four common toxin transcriptomic toxin annotation approaches: BLAST search against toxins in UniProt, lineage-specific toxins, or species-specific toxins and comparative expression analyses of venom and non-venom producing tissues. This demonstrated that even toxin annotation based on lineage-specific homology searches is prone to substantial errors compared to a proteomic approach. However, combined comparative transcriptomics and phylogenetic analysis of putative toxin families substantially improves annotation accuracy. Furthermore, comparison of the venom composition of S. subspinipes with the closely related S. subspinipes mutilans revealed a surprising lack of overlap. This first insight into the intraspecific venom variability of centipedes contrasts the sequence conservation expected from previous findings that centipede toxins evolve under strong negative selection. Our results highlight the importance of proteomic data in studies of even comparably well-characterized venoms and warrants caution when sourcing venom from centipedes of unknown origin. [ABSTRACT FROM AUTHOR]

Predatory robber flies (Diptera, Asilidae) have been suspected to be venomous due to their ability to overpower well-defended prey. However, details of their venom composition and toxin arsenal remained unknown. Here, we provide a detailed characterization of the venom system of robber flies through the application of comparative transcriptomics, proteomics and functional morphology. Our results reveal asilid venoms to be dominated by peptides and non-enzymatic proteins, and that the majority of components in the crude venom is represented by just ten toxin families, which we have named Asilidin1-10. Contrary to what might be expected for a liquid-feeding predator, the venoms of robber flies appear to be rich in novel peptides, rather than enzymes with a putative pre-digestive role. The novelty of these peptides suggests that the robber fly venom system evolved independently from hematophagous dipterans and other pancrustaceans. Indeed, six Asilidins match no other venom proteins, while three represent known examples of peptide scaffolds convergently recruited to a toxic function. Of these, members of Asilidin1 closely resemble cysteine inhibitor knot peptides (ICK), of which neurotoxic variants occur in cone snails, assassin bugs, scorpions and spiders. Synthesis of one of these putative ICKs, U-Asilidin1-Mar1a, followed by toxicity assays against an ecologically relevant prey model revealed that one of these likely plays a role as a neurotoxin involved in the immobilization of prey. Our results are fundamental to address these insights further and to understand processes that drive venom evolution in dipterans as well as other arthropods. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This review examines the current state of knowledge regarding toxins from anthozoans (sea anemones, coral, zoanthids, corallimorphs, sea pens and tube anemones). We provide an overview of venom from phylum Cnidaria and review the diversity of venom composition between the two major clades (Medusozoa and Anthozoa). We highlight that the functional and ecological context of venom has implications for the temporal and spatial expression of protein and peptide toxins within class Anthozoa. Understanding the nuances in the regulation of venom arsenals has been made possible by recent advances in analytical technologies that allow characterisation of the spatial distributions of toxins. Furthermore, anthozoans are unique in that ecological roles can be assigned using tissue expression data, thereby circumventing some of the challenges related to pharmacological screening. [ABSTRACT FROM AUTHOR]

Assassin bugs (Reduviidae) produce venoms that are insecticidal, and which induce pain in predators, but the composition and function of their individual venom components is poorly understood. We report findings on the venom system of the red-spotted assassin bug Platymeris rhadamanthus, a large species of African origin that is unique in propelling venom as a projectile weapon when threatened. We performed RNA sequencing experiments on venom glands (separate transcriptomes of the posterior main gland, PMG, and the anterior main gland, AMG), and proteomic experiments on venom that was either defensively propelled or collected from the proboscis in response to electrostimulation. We resolved a venom proteome comprising 166 polypeptides. Both defensively propelled venom and most venom samples collected in response to electrostimulation show a protein profile similar to the predicted secretory products of the PMG, with a smaller contribution from the AMG. Pooled venom samples induce calcium influx via membrane lysis when applied to mammalian neuronal cells, consistent with their ability to cause pain when propelled into the eyes or mucus membranes of potential predators. The same venom induces rapid paralysis and death when injected into fruit flies. These data suggest that the cytolytic, insecticidal venom used by reduviids to capture prey is also a highly effective defensive weapon when propelled at predators. [ABSTRACT FROM AUTHOR]

The article focuses on findings of a study on use of imaging mass spectrometry to study peptide toxin distribution in Australian sea anemones, and mentions application of a venomics strategy that combines transcriptomics and proteomics with MALDI-IMS.