Functional and Proteomic Insights into Aculeata Venoms.

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.

The Toxicological Intersection between Allergen and Toxin: A Structural Comparison of the Cat Dander Allergenic Protein Fel d1 and the Slow Loris Brachial Gland Secretion Protein.

Slow lorises are enigmatic animal that represent the only venomous primate lineage. Their defensive secretions have received little attention. In this study we determined the full length sequence of the protein secreted by their unique brachial glands. The full length sequences displayed homology to the main allergenic protein present in cat dander. We thus compared the molecular features of the slow loris brachial gland protein and the cat dander allergen protein, showing remarkable similarities between them. Thus we postulate that allergenic proteins play a role in the slow loris defensive arsenal. These results shed light on these neglected, novel animals.

Entomo-venomics: The evolution, biology and biochemistry of insect venoms.

The insects are a hyperdiverse class containing more species than all other animal groups combined-and many employ venom to capture prey, deter predators and micro-organisms, or facilitate parasitism or extra-oral digestion. However, with the exception of those made by Hymenoptera (wasps, ants and bees), little is known about insect venoms. Here, we review the current literature on insects that use venom for prey capture and predator deterrence, finding evidence for fourteen independent origins of venom usage among insects, mostly among the hyperdiverse holometabolan orders. Many lineages, including the True Bugs (Heteroptera), robber flies (Asilidae), and larvae of many Neuroptera, Coleoptera and Diptera, use mouthpart-associated venoms to paralyse and pre-digest prey during hunting. In contrast, some Hymenoptera and larval Lepidoptera, and one species of beetle, use non-mouthpart structures to inject venom in order to cause pain to deter potential predators. Several recently published insect venom proteomes indicate molecular convergence between insects and other venomous animal groups, with all insect venoms studied so far being potently bioactive cocktails containing both peptides and larger proteins, including novel peptide and protein families. This review summarises the current state of the field of entomo-venomics.

Scratching the Surface of an Itch: Molecular Evolution of Aculeata Venom Allergens.

Hymenopteran insects are infamous for their sting, and their ability to cause severe anaphylaxis and in some cases death. This allergic reaction is a result of allergens present in the venom. Hymenopterans have many common venom allergens, the most widespread of which include phospholipase A1, phospholipase A2, acid phosphatase, hyaluronidase, serine protease and antigen 5. While there have been studies that look at the phylogenetic histories of allergens within closely related species, to our knowledge, this is the first study using evolutionary analyses to compare across Hymenoptera the types of selection that are occurring on allergens. This research examined the publicly available sequences of six different groups of allergens and found that allergens had diverged and formed closely related clades which share greater sequence similarities. We also analysed the patterns of selection and found that they are predominately under the influence of negative selection.

Rattling the border wall: Pathophysiological implications of functional and proteomic venom variation between Mexican and US subspecies of the desert rattlesnake Crotalus scutulatus.

While some US populations of the Mohave rattlesnake (Crotalus scutulatus scutulatus) are infamous for being potently neurotoxic, the Mexican subspecies C. s. salvini (Huamantlan rattlesnake) has been largely unstudied beyond crude lethality testing upon mice. In this study we show that at least some populations of this snake are as potently neurotoxic as its northern cousin. Testing of the Mexican antivenom Antivipmyn showed a complete lack of neutralisation for the neurotoxic effects of C. s. salvini venom, while the neurotoxic effects of the US subspecies C. s. scutulatus were time-delayed but ultimately not eliminated. These results document unrecognised potent neurological effects of a Mexican snake and highlight the medical importance of this subspecies, a finding augmented by the ineffectiveness of the Antivipmyn antivenom. These results also influence our understanding of the venom evolution of Crotalus scutulatus, suggesting that neurotoxicity is the ancestral feature of this species, with the US populations which lack neurotoxicity being derived states.

The Evolution of Fangs, Venom, and Mimicry Systems in Blenny Fishes.

Venom systems have evolved on multiple occasions across the animal kingdom, and they can act as key adaptations to protect animals from predators [1]. Consequently, venomous animals serve as models for a rich source of mimicry types, as non-venomous species benefit from reductions in predation risk by mimicking the coloration, body shape, and/or movement of toxic counterparts [2-5]. The frequent evolution of such deceitful imitations provides notable examples of phenotypic convergence and are often invoked as classic exemplars of evolution by natural selection. Here, we investigate the evolution of fangs, venom, and mimetic relationships in reef fishes from the tribe Nemophini (fangblennies). Comparative morphological analyses reveal that enlarged canine teeth (fangs) originated at the base of the Nemophini radiation and have enabled a micropredatory feeding strategy in non-venomous Plagiotremus spp. Subsequently, the evolution of deep anterior grooves and their coupling to venom secretory tissue provide Meiacanthus spp. with toxic venom that they effectively employ for defense. We find that fangblenny venom contains a number of toxic components that have been independently recruited into other animal venoms, some of which cause toxicity via interactions with opioid receptors, and result in a multifunctional biochemical phenotype that exerts potent hypotensive effects. The evolution of fangblenny venom has seemingly led to phenotypic convergence via the formation of a diverse array of mimetic relationships that provide protective (Batesian mimicry) and predatory (aggressive mimicry) benefits to other fishes [2, 6]. Our results further our understanding of how novel morphological and biochemical adaptations stimulate ecological interactions in the natural world.

From Marine Venoms to Drugs: Efficiently Supported by a Combination of Transcriptomics and Proteomics.

The potential of marine natural products to become new drugs is vast; however, research is still in its infancy. The chemical and biological diversity of marine toxins is immeasurable and as such an extraordinary resource for the discovery of new drugs. With the rapid development of next-generation sequencing (NGS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), it has been much easier and faster to identify more toxins and predict their functions with bioinformatics pipelines, which pave the way for novel drug developments. Here we provide an overview of related bioinformatics pipelines that have been supported by a combination of transcriptomics and proteomics for identification and function prediction of novel marine toxins.

The Cardiovascular and Neurotoxic Effects of the Venoms of Six Bony and Cartilaginous Fish Species.

Fish venoms are often poorly studied, in part due to the difficulty in obtaining, extracting, and storing them. In this study, we characterize the cardiovascular and neurotoxic effects of the venoms from the following six species of fish: the cartilaginous stingrays Neotrygon kuhlii and Himantura toshi, and the bony fish Platycephalus fucus, Girella tricuspidata, Mugil cephalus, and Dentex tumifrons. All venoms (10-100 µg/kg, i.v.), except G. tricuspidata and P. fuscus, induced a biphasic response on mean arterial pressure (MAP) in the anesthetised rat. P. fucus venom exhibited a hypotensive response, while venom from G. tricuspidata displayed a single depressor response. All venoms induced cardiovascular collapse at 200 µg/kg, i.v. The in vitro neurotoxic effects of venom were examined using the chick biventer cervicis nerve-muscle (CBCNM) preparation. N. kuhlii, H. toshi, and P. fucus venoms caused concentration-dependent inhibition of indirect twitches in the CBCNM preparation. These three venoms also inhibited responses to exogenous acetylcholine (ACh) and carbachol (CCh), but not potassium chloride (KCl), indicating a post-synaptic mode of action. Venom from G. tricuspidata, M. cephalus, and D. tumifrons had no significant effect on indirect twitches or agonist responses in the CBCNM. Our results demonstrate that envenoming by these species of fish may result in moderate cardiovascular and/or neurotoxic effects. Future studies aimed at identifying the molecules responsible for these effects could uncover potentially novel lead compounds for future pharmaceuticals, in addition to generating new knowledge about the evolutionary relationships between venomous animals.

Canopy Venom: Proteomic Comparison among New World Arboreal Pit-Viper Venoms.

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.

Extreme venom variation in Middle Eastern vipers: a proteomics comparison of Eristicophis macmahonii, Pseudocerastes fieldi and Pseudocerastes persicus.

Venoms of the viperid sister genera Eristicophis and Pseudocerastes are poorly studied despite their anecdotal reputation for producing severe or even lethal envenomations. This is due in part to the remote and politically unstable regions that they occupy. All species contained are sit and wait ambush feeders. Thus, this study examined their venoms through proteomics techniques in order to establish if this feeding ecology, and putatively low levels of gene flow, have resulted in significant variations in venom profile. The techniques indeed revealed extreme venom variation. This has immediate implications as only one antivenom is made (using the venom of Pseudocerastes persicus) yet the proteomic variation suggests that it would be of only limited use for the other species, even the sister species Pseudocerastes fieldi. The high degree of variation however also points toward these species being rich resources for novel compounds which may have use as lead molecules in drug design and development. BIOLOGICAL SIGNIFICANCE: These results show extreme venom variation between these closely related snakes. These results have direct implications for the treatment of the envenomed patient.

A ray of venom: Combined proteomic and transcriptomic investigation of fish venom composition using barb tissue from the blue-spotted stingray (Neotrygon kuhlii).

Fish venoms remain almost completely unstudied despite the large number of species. In part this is due to the inherent nature of fish venoms, in that they are highly sensitive to heat, pH, lyophilisation, storage and repeated freeze-thawing. They are also heavily contaminated with mucus, which makes proteomic study difficult. Here we describe a novel protein-handling protocol to remove mucus contamination, utilising ammonium sulphate and acetone precipitation. We validated this approach using barb venom gland tissue protein extract from the blue-spotted stingray Neotrygon kuhlii. We analysed the protein extract using 1D and 2D gels with LC-MS/MS sequencing. Protein annotation was underpinned by a venom gland transcriptome. The composition of our N. kuhlii venom sample revealed a variety of protein types that are completely novel to animal venom systems. Notably, none of the detected proteins exhibited similarity to the few toxin components previously characterised from fish venoms, including those found in other stingrays. Putative venom toxins identified here included cystatin, peroxiredoxin and galectin. Our study represents the first combined survey of gene and protein composition from the venom apparatus of any fish and our novel protein handling method will aid the future characterisation of toxins from other unstudied venomous fish lineages. BIOLOGICAL SIGNIFICANCE: These results show an efficient manner for removing mucus from fish venoms. These results are the first insights into the evolution of proteins present on stingrayvenom barbs.

Vintage venoms: proteomic and pharmacological stability of snake venoms stored for up to eight decades.

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.

Proteomic comparison of Hypnale hypnale (hump-nosed pit-viper) and Calloselasma rhodostoma (Malayan pit-viper) venoms.

Treatment of Hypnale hypnale bites with commercial antivenoms, even those raised against its sister taxon Calloselasma rhodostoma, has never been clinically successful. As these two genera have been separated for 20million years, we tested to see whether significant variations in venom had accumulated during this long period of evolutionary divergence, and thus could be responsible for the failure of antivenom. Proteomic analyses of C. rhodostoma and H. hypnale venom were performed using 1D and 2D PAGE as well as 2D-DIGE. C. rhodostoma venom was diverse containing large amounts of Disintegrin, Kallikrein, l-amino acid oxidase, Lectin, phospholipase A2 (acidic, basic and neutral) and Snake Venom Metalloprotease. In contrast, while H. hypnale also contained a wide range of toxin types, the venom was overwhelmingly dominated by two molecular weight forms of basic PLA2. 2D-DIGE (2-D Fluorescence Difference Gel Electrophoresis analysis) showed that even when a particular toxin class was shared between the two venoms, there were significant molecular weights or isoelectric point differences. This proteomic difference explains the past treatment failures with C. rhodostoma antivenom and highlights the need for a H. hypnale specific antivenom. BIOLOGICAL SIGNIFICANCE: These results have direct implications for the treatment of envenomed patients in Sri Lanka. The unusual venom profile of Hypnale hypnale underscores the biodiscovery potential of novel snake venoms.