Extensive Variation in the Activities of Pseudocerastes and Eristicophis Viper Venoms Suggests Divergent Envenoming Strategies Are Used for Prey Capture.

Snakes of the genera Pseudocerastes and Eristicophis (Viperidae: Viperinae) are known as the desert vipers due to their association with the arid environments of the Middle East. These species have received limited research attention and little is known about their venom or ecology. In this study, a comprehensive analysis of desert viper venoms was conducted by visualising the venom proteomes via gel electrophoresis and assessing the crude venoms for their cytotoxic, haemotoxic, and neurotoxic properties. Plasmas sourced from human, toad, and chicken were used as models to assess possible prey-linked venom activity. The venoms demonstrated substantial divergence in composition and bioactivity across all experiments. Pseudocerastes urarachnoides venom activated human coagulation factors X and prothrombin and demonstrated potent procoagulant activity in human, toad, and chicken plasmas, in stark contrast to the potent neurotoxic venom of P. fieldi. The venom of E. macmahonii also induced coagulation, though this did not appear to be via the activation of factor X or prothrombin. The coagulant properties of P. fieldi and P. persicus venoms varied among plasmas, demonstrating strong anticoagulant activity in the amphibian and human plasmas but no significant effect in that of bird. This is conjectured to reflect prey-specific toxin activity, though further ecological studies are required to confirm any dietary associations. This study reinforces the notion that phylogenetic relatedness of snakes cannot readily predict venom protein composition or function. The significant venom variation between these species raises serious concerns regarding antivenom paraspecificity. Future assessment of antivenom is crucial.

A Taxon-Specific and High-Throughput Method for Measuring Ligand Binding to Nicotinic Acetylcholine Receptors.

The binding of compounds to nicotinic acetylcholine receptors is of great interest in biomedical research. However, progress in this area is hampered by the lack of a high-throughput, cost-effective, and taxonomically flexible platform. Current methods are low-throughput, consume large quantities of sample, or are taxonomically limited in which targets can be tested. We describe a novel assay which utilizes a label-free bio-layer interferometry technology, in combination with adapted mimotope peptides, in order to measure ligand binding to the orthosteric site of nicotinic acetylcholine receptor alpha-subunits of diverse organisms. We validated the method by testing the evolutionary patterns of a generalist feeding species (Acanthophis antarcticus), a fish specialist species (Aipysurus laevis), and a snake specialist species (Ophiophagus hannah) for comparative binding to the orthosteric site of fish, amphibian, lizard, snake, bird, marsupial, and rodent alpha-1 nicotinic acetylcholine receptors. Binding patterns corresponded with diet, with the Acanthophis antarcticus not showing bias towards any particular lineage, while Aipysurus laevis showed selectivity for fish, and Ophiophagus hannah a selectivity for snake. To validate the biodiscovery potential of this method, we screened Acanthophis antarcticus and Tropidolaemus wagleri venom for binding to human alpha-1, alpha-2, alpha-3, alpha-4, alpha-5, alpha-6, alpha-7, alpha-9, and alpha-10. While A. antarcticus was broadly potent, T. wagleri showed very strong but selective binding, specifically to the alpha-1 target which would be evolutionarily selected for, as well as the alpha-5 target which is of major interest for drug design and development. Thus, we have shown that our novel method is broadly applicable for studies including evolutionary patterns of venom diversification, predicting potential neurotoxic effects in human envenomed patients, and searches for novel ligands of interest for laboratory tools and in drug design and development.

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.