Convergent evolution provides insights into the selective drivers underlying evolutionary change. Snake venoms, with a direct genetic basis and clearly defined functional phenotype, provide a model system for exploring the repeated evolution of adaptations. While snakes use venom primarily for predation, and venom composition often reflects diet specificity, three lineages of cobras have independently evolved the ability to spit venom at adversaries. Using gene, protein, and functional analyses, we show that the three spitting lineages possess venoms characterized by an up-regulation of phospholipase A2 (PLA2) toxins, which potentiate the action of preexisting venom cytotoxins to activate mammalian sensory neurons and cause enhanced pain. These repeated independent changes provide a fascinating example of convergent evolution across multiple phenotypic levels driven by selection for defense.

The mambas (genus Dendroaspis) comprise five especially notorious medically important venomous snakes endemic to sub-Saharan Africa. Their highly potent venoms comprise a high diversity of pharmacologically active peptides, including extremely rapid-acting neurotoxins. Previous studies on mamba venoms have focused on the biochemical and pharmacological characterisation of their most relevant toxins to rationalize the common neurological and neuromuscular symptoms of envenomings caused by these species, but there has been little work on overall venom composition or comparisons between them. Only very recently an overview of the composition of the venom of two Dendroaspis species, D. angusticeps and D. polylepis, has been unveiled through venomics approaches. Here we present the first genus-wide transcriptomic-proteomic analysis of mamba venom composition. The transcriptomic analyses described in this paper have contributed 29 (D. polylepis), 23 (D. angusticeps), 40 (D. viridis), 25 (D. j. jamesoni) and 21 (D. j. kaimosae), novel full-length toxin sequences to the non-redundant Dendroaspis sequence database. The mamba genus-wide venomic analysis demonstrated that major D. polylepis venom components are Kunitz-fold family toxins. This feature is unique in relation to the relatively conserved three-finger toxin (3FTx)-dominated venom compositions of the green mambas. Venom variation was interpreted in the context of dietary variation due to the divergent terrestrial ecology of D. polylepis compared to the arboreal niche occupied by all other mambas. Additionally, the degree of cross-reactivity conservation of mamba venoms was assessed by antivenomics against a panel of commercial antivenoms generated for the sub-Saharan Africa market. This study provides a genus-wide overview to infer which available antivenoms may be capable of neutralising human envenomings caused by mambas, irrespective of the species responsible. The information gathered in this study lays the foundations for rationalising the pharmacological profiles of mamba venoms at locus resolution. This understanding will contribute to the generation of a safer and more efficacious pan-Dendroaspis therapeutic antivenom against any mamba envenomation.

Highlightsd Snake venom gland cells can be cultured as adult-stem-cellbased organoids d Organoids contain proliferating progenitors and various venom-producing cells d Regional and cellular heterogeneity of venom components is maintained in culture

Snakebite is a neglected tropical disease that causes 138,000 deaths each year. Neurotoxic snake venoms contain small neurotoxins, including three-finger toxins (3FTxs), which can cause rapid paralysis in snakebite victims by blocking postsynaptic transmission via nicotinic acetylcholine receptors (nAChRs). These toxins are typically weakly immunogenic and thus are often not effectively targeted by current polyclonal antivenom therapies. We investigated whether nAChR mimics, also known as acetylcholine binding proteins (AChBPs), could effectively capture 3FTxs and therefore be developed as a novel class of snake-generic therapeutics for combatting neurotoxic envenoming. First, we identified the binding specificities of 3FTx from various medically important elapid snake venoms to nAChR using two recombinant nAChR mimics: the AChBP from Lymnaea stagnalis and a humanized neuronal α7 version (α7-AChBP). We next characterized these AChBP-bound and unbound fractions using SDS-PAGE and mass spectrometry. Interestingly, both mimics effectively captured long-chain 3FTxs from multiple snake species but largely failed to capture the highly related short-chain 3FTxs, suggesting a high level of binding specificity. We next investigated whether nAChR mimics could be used as snakebite therapeutics. We showed that while α7-AChBP alone did not protect against Naja haje (Egyptian cobra) venom lethality in vivo , it significantly prolonged survival times when coadministered with a nonprotective dose of antivenom. Thus, nAChR mimics are capable of neutralizing specific venom toxins and may be useful adjunct therapeutics for improving the safety and affordability of existing snakebite treatments by reducing therapeutic doses. Our findings justify exploring the future development of AChBPs as potential snakebite treatments.

Snakebite is a neglected tropical disease that results in a variety of systemic and local pathologies in envenomed victims and is responsible for around 138,000 deaths every year. Many snake venoms cause severe coagulopathy that makes victims vulnerable to suffering life-threating haemorrhage. The mechanisms of action of coagulopathic snake venom toxins are diverse and can result in both anticoagulant and procoagulant effects. However, because snake venoms consist of a mixture of numerous protein and peptide components, high throughput characterizations of specific target bioactives is challenging. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that perturb coagulation. To do so, we used a high-throughput screening approach consisting of a miniaturised plasma coagulation assay in combination with a venom nanofractionation approach. Twenty snake venoms were first separated using reversed-phase liquid chromatography, and a post-column split allowed a small fraction to be analyzed with mass spectrometry, while the larger fraction was collected and dispensed onto 384-well plates. After fraction collection, any solvent present in the wells was removed by means of freeze-drying, after which it was possible to perform a plasma coagulation assay in order to detect coagulopathic activity. Our results demonstrate that many snake venoms simultaneously contain both procoagulant and anticoagulant bioactives that contribute to coagulopathy. In-depth identification analysis from seven medically-important venoms, via mass spectrometry and nanoLC-MS/MS, revealed that phospholipase A 2 toxins are frequently identified in anticoagulant venom fractions, while serine protease and metalloproteinase toxins are often associated with procoagulant bioactivities. The nanofractionation and proteomics approach applied herein seems likely to be a valuable tool for the rational development of next-generation snakebite treatments by facilitating the rapid identification and fractionation of coagulopathic toxins, thereby enabling PLOS NEGLECTED TROPICAL DISEASES

Convergent evolution provides unparalleled insights into the selective drivers underlying evolutionary change. While snakes use venom primarily for predation, and venom composition often reflects diet specificity, three lineages of spitting cobras have independently evolved the ability to use venom as a defensive projectile. Using gene, protein and functional analyses, we show that the three spitting lineages possess venom characterized by an upregulation of PLA2 toxins, which potentiate the action of venom cytotoxins to activate mammalian sensory neurons and cause enhanced pain. These repeated independent changes provide a fascinating example of convergent evolution across multiple phenotypic levels driven by exaptations. Notably, the timing of their origins suggests that defensive venom spitting may have evolved in response to the emergence of bipedal hominids in Africa and Asia.

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