We have examined sequences from the ligand-binding domain of the nicotinic acetyl choline receptor (nAChR) in 148 vertebrate species. We are in interested in this receptor because the α-neurotoxins... Show moreWe have examined sequences from the ligand-binding domain of the nicotinic acetyl choline receptor (nAChR) in 148 vertebrate species. We are in interested in this receptor because the α-neurotoxins of many venomous snakes binds to this receptor in its location at the neuromuscular junction in all vertebrates. Furthermore, some animals have evolved resistance to snake venoms and show modifications in the ligand binding domain of the nAChR which inhibit the binding of snake α-neurotoxins. Our analysis has shown that numerous vertebrate species, most of which were not previously known to possess α-neurotoxin resistance, do actually contain resistance-related modifications. These modifications are present in most of the taxa in our dataset, with the unexpected exclusion of the birds. It was particularly surprising to us that the snake-specialist predatory birds Circaetus pectoralis (black-chested snake eagle) and Sagittarius serpentarius (secretary bird) did not possess resistance modifications. There were also relatively few resistance-related mutations within the mammals. By contrast, there were multiple convergent evolutions of the well-characterised N-glycosylation motif within the squamate reptiles—particularly the snakes. We also identified a number of sites under positive selection, such as mutations to the proline subsite. Future functional testing will be needed to validate that these modifications do indeed confer resistance. To provide functional confirmation that resistance-related modifications do indeed reduce susceptibility to toxins, we used developmental bioassays. These assays showed that two species possessing resistance-related modifications of the nAChR (stickleback and bearded dragon) were less susceptible to the toxic effects of cobra venom than two species that lacked such modifications (zebrafish and chicken). In summary, we demonstrate that the range of mechanisms along with the phylogenetic distribution of resistance to snake α-neurotoxin appears to be more extensive than was previously appreciated. It also shows strong evidence of the convergent evolution of the same resistance mutations in independent linages. Our findings also support the notion that the mutations we have identified in this thesis may represent adaptive change in response to selective pressures exerted by α-neurotoxic snake venoms in an evolutionary arms race. Thus, we conclude that the evolutionary arms race between predator and prey appears to be a pervasive feature of the trophic interactions surrounding venomous snakes, which is shaping the molecular evolution of the nAChR in the vertebrates. Show less
Snake envenomation globally is attributed to an ever-increasing human population encroaching into snake territories. Responsible for many bites in Asia is the widespread genus Trimeresurus. While... Show moreSnake envenomation globally is attributed to an ever-increasing human population encroaching into snake territories. Responsible for many bites in Asia is the widespread genus Trimeresurus. While bites lead to haemorrhage, only a few species have had their venoms examined in detail. We found that Trimeresurus venom causes haemorrhaging by cleaving fibrinogen in a pseudo-procoagulation manner to produce weak, unstable, short-lived fibrin clots ultimately resulting in an overall anticoagulant effect due to fibrinogen depletion. The monovalent antivenom `Thai Red Cross Green Pit Viper antivenin', varied in efficacy ranging from excellent neutralisation of T. albolabris venom through to T. gumprechti and T. mcgregori being poorly neutralised and T. hageni being unrecognised by the antivenom. While the results showing excellent neutralisation of some non-T. albolabris venoms (such as T. flavomaculaturs, T. fucatus, and T. macrops) needs to be confirmed with in vivo tests, conversely the antivenom failure T. hageni, and the very poor results against T. gumprechti and T. mcgregori, despite being conducted in the ideal scenario of preincubation of antivenom:venom, indicates that the likelihood of clinically relevant cross-reactivity for these species is low (T. gumprechti and T. mcgregori) to non-existent (T. hageni). These same latter three species were also not inhibited by the serine protease inhibitor AEBSF, suggesting that the toxins leading to a coagulotoxic effect in these species are non-serine proteases while in contrast T. albolabris coagulotoxicity was completely impeded by AEBSF, and thus driven by kallikrein-type serine proteases. There was a conspicuous lack of phylogenetic pattern in venom variation, with the most potent venoms (T. albolabris and T. hageni) being distant to each other on the organismal tree, and with the three most divergent and poorly neutralised venoms (T. gumprechti, T. hageni, and T. mcgregori) were also not each others closest relatives. This reinforces the paradigm that the fundamental dynamic evolution of venom results in organismal phylogeny being a poor predictor of venom potency or antivenom efficacy. This study provides a robust investigation on the differential venom effects from a wide range of Trimeresurus species on coagulation, highlighting differential fibrinogenolytic effects, while also investigating the relative antivenom neutralisation capabilities of the widely available Thai Red Cross Green Pit Viper antivenom. These results therefore have immediate, real-world implications for patients envenomed by Trimeresurus species. Show less
In this thesis I have shown that snakes have undergone multiple changes in their genome and embryonic development that has provided them with the variation to which natural selection could act.... Show moreIn this thesis I have shown that snakes have undergone multiple changes in their genome and embryonic development that has provided them with the variation to which natural selection could act. This thesis provides evidence for the variable mechanisms of venom gene evolution, which presumably is much more flexible than previously thought. But it also underscores the potential use of the many different types of snake venom toxins that could be screened for use against human disorders. And most of all, I hope I have contributed towards the fact that snakes are just an incredibly interesting group of vertebrates from both the perspective of ecology and life-style, as well as from a genomic and molecular perspective. They are, and will always be, my first and true love. Show less
