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 Bold and the Beautiful: a Neurotoxicity Comparison of New World Coral Snakes in the Micruroides and Micrurus Genera and Relative Neutralization by Antivenom.

Coral snake envenomations are well characterized to be lethally neurotoxic. Despite this, few multispecies, neurotoxicity and antivenom efficacy comparisons have been undertaken and only for the Micrurus genus; Micruroides has remained entirely uninvestigated. As the USA's supplier of antivenom has currently stopped production, alternative sources need to be explored. The Mexican manufacturer Bioclon uses species genetically related to USA species, thus we investigated the efficacy against Micrurus fulvius (eastern coral snake), the main species responsible for lethal envenomations in the USA as well as additional species from the Americas. The use of Coralmyn® coral snake antivenom was effective in neutralizing the neurotoxic effects exhibited by the venom of M. fulvius but was ineffective against the venoms of Micrurus tener, Micrurus spixii, Micrurus pyrrhocryptus, and Micruroides euryxanthus. Our results suggest that the Mexican antivenom may be clinically useful for the treatment of M. fulvius in the USA but may be of only limited efficacy against the other species studied.

How the Cobra Got Its Flesh-Eating Venom: Cytotoxicity as a Defensive Innovation and Its Co-Evolution with Hooding, Aposematic Marking, and Spitting.

The cytotoxicity of the venom of 25 species of Old World elapid snake was tested and compared with the morphological and behavioural adaptations of hooding and spitting. We determined that, contrary to previous assumptions, the venoms of spitting species are not consistently more cytotoxic than those of closely related non-spitting species. While this correlation between spitting and non-spitting was found among African cobras, it was not present among Asian cobras. On the other hand, a consistent positive correlation was observed between cytotoxicity and utilisation of the defensive hooding display that cobras are famous for. Hooding and spitting are widely regarded as defensive adaptations, but it has hitherto been uncertain whether cytotoxicity serves a defensive purpose or is somehow useful in prey subjugation. The results of this study suggest that cytotoxicity evolved primarily as a defensive innovation and that it has co-evolved twice alongside hooding behavior: once in the Hemachatus + Naja and again independently in the king cobras (Ophiophagus). There was a significant increase of cytotoxicity in the Asian Naja linked to the evolution of bold aposematic hood markings, reinforcing the link between hooding and the evolution of defensive cytotoxic venoms. In parallel, lineages with increased cytotoxicity but lacking bold hood patterns evolved aposematic markers in the form of high contrast body banding. The results also indicate that, secondary to the evolution of venom rich in cytotoxins, spitting has evolved three times independently: once within the African Naja, once within the Asian Naja, and once in the Hemachatus genus. The evolution of cytotoxic venom thus appears to facilitate the evolution of defensive spitting behaviour. In contrast, a secondary loss of cytotoxicity and reduction of the hood occurred in the water cobra Naja annulata, which possesses streamlined neurotoxic venom similar to that of other aquatic elapid snakes (e.g., hydrophiine sea snakes). The results of this study make an important contribution to our growing understanding of the selection pressures shaping the evolution of snake venom and its constituent toxins. The data also aid in elucidating the relationship between these selection pressures and the medical impact of human snakebite in the developing world, as cytotoxic cobras cause considerable morbidity including loss-of-function injuries that result in economic and social burdens in the tropics of Asia and sub-Saharan Africa.

The Snake with the Scorpion's Sting: Novel Three-Finger Toxin Sodium Channel Activators from the Venom of the Long-Glanded Blue Coral Snake (Calliophis bivirgatus).

Millions of years of evolution have fine-tuned the ability of venom peptides to rapidly incapacitate both prey and potential predators. Toxicofera reptiles are characterized by serous-secreting mandibular or maxillary glands with heightened levels of protein expression. These glands are the core anatomical components of the toxicoferan venom system, which exists in myriad points along an evolutionary continuum. Neofunctionalisation of toxins is facilitated by positive selection at functional hotspots on the ancestral protein and venom proteins have undergone dynamic diversification in helodermatid and varanid lizards as well as advanced snakes. A spectacular point on the venom system continuum is the long-glanded blue coral snake (Calliophis bivirgatus), a specialist feeder that preys on fast moving, venomous snakes which have both a high likelihood of prey escape but also represent significant danger to the predator itself. The maxillary venom glands of C. bivirgatus extend one quarter of the snake's body length and nestle within the rib cavity. Despite the snake's notoriety its venom has remained largely unstudied. Here we show that the venom uniquely produces spastic paralysis, in contrast to the flaccid paralysis typically produced by neurotoxic snake venoms. The toxin responsible, which we have called calliotoxin (δ-elapitoxin-Cb1a), is a three-finger toxin (3FTx). Calliotoxin shifts the voltage-dependence of NaV1.4 activation to more hyperpolarised potentials, inhibits inactivation, and produces large ramp currents, consistent with its profound effects on contractile force in an isolated skeletal muscle preparation. Voltage-gated sodium channels (NaV) are a particularly attractive pharmacological target as they are involved in almost all physiological processes including action potential generation and conduction. Accordingly, venom peptides that interfere with NaV function provide a key defensive and predatory advantage to a range of invertebrate venomous species including cone snails, scorpions, spiders, and anemones. Enhanced activation or delayed inactivation of sodium channels by toxins is associated with the extremely rapid onset of tetanic/excitatory paralysis in envenomed prey animals. A strong selection pressure exists for the evolution of such toxins where there is a high chance of prey escape. However, despite their prevalence in other venomous species, toxins causing delay of sodium channel inhibition have never previously been described in vertebrate venoms. Here we show that NaV modulators, convergent with those of invertebrates, have evolved in the venom of the long-glanded coral snake. Calliotoxin represents a functionally novel class of 3FTx and a structurally novel class of NaV toxins that will provide significant insights into the pharmacology and physiology of NaV. The toxin represents a remarkable case of functional convergence between invertebrate and vertebrate venom systems in response to similar selection pressures. These results underscore the dynamic evolution of the Toxicofera reptile system and reinforces the value of using evolution as a roadmap for biodiscovery.

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.

Venom proteomic characterization and relative antivenom neutralization of two medically important Pakistani elapid snakes (Bungarus sindanus and Naja naja).

Intra- and interspecific variation in venom composition has been shown to have a major effect upon the efficacy of antivenoms. Due to the absence of domestically produced antivenoms, Pakistan is wholly reliant upon antivenoms produced in other countries, such as India. However, the efficacy of these antivenoms in neutralising the venoms of Pakistani snakes has not been ascertained. This is symptomatic of the general state of toxicological research in this country, which has a myriad of highly toxic and medically important venomous animals. Thus, there is a dire need for knowledge regarding the fundamental proteomics of these venoms and applied knowledge of the relative efficacy of foreign antivenoms. Here we present the results of our proteomic research on two medically important snakes of Pakistan: Bungarus sindanus and Naja naja. Indian Polyvalent Antivenom (Bharat Serums and Vaccines Ltd), which is currently marketed for use in Pakistan, was completely ineffective against either Pakistani species. In addition to the expected pre- and post-synaptic neurotoxic activity, the venom of the Pakistan population of N. naja was shown to be quite divergent from other populations of this species in being potently myotoxic. These results highlight the importance of studying divergent species and isolated populations, where the same data not only elucidates clinical problems in need of immediate attention, but also uncovers sources for novel toxins with potentially useful activities. BIOLOGICAL SIGNIFICANCE: Pakistan Bungarus sindanus and Naja naja venoms are differentially complex. Naja naja is potently myotoxic. Neither venom is neutralized by Indian antivenom. These results have direct implications for the treatment of envenomed patients in Pakistan. The unusually myotoxic effects of Naja naja demonstrates the value of studying remote populations for biodiscovery.

Differential evolution and neofunctionalization of snake venom metalloprotease domains.

Snake venom metalloproteases (SVMP) are composed of five domains: signal peptide, propeptide, metalloprotease, disintegrin, and cysteine-rich. Secreted toxins are typically combinatorial variations of the latter three domains. The SVMP-encoding genes of Psammophis mossambicus venom are unique in containing only the signal and propeptide domains. We show that the Psammophis SVMP propeptide evolves rapidly and is subject to a high degree of positive selection. Unlike Psammophis, some species of Echis express both the typical multidomain and the unusual monodomain (propeptide only) SVMP, with the result that a lower level of variation is exerted upon the latter. We showed that most mutations in the multidomain Echis SVMP occurred in the protease domain responsible for proteolytic and hemorrhagic activities. The cysteine-rich and disintegrin-like domains, which are putatively responsible for making the P-III SVMPs more potent than the P-I and P-II forms, accumulate the remaining variation. Thus, the binding sites on the molecule's surface are evolving rapidly whereas the core remains relatively conserved. Bioassays conducted on two post-translationally cleaved novel proline-rich peptides from the P. mossambicus propeptide domain showed them to have been neofunctionalized for specific inhibition of mammalian a7 neuronal nicotinic acetylcholine receptors. We show that the proline rich postsynaptic specific neurotoxic peptides from Azemiops feae are the result of convergent evolution within the precursor region of the C-type natriuretic peptide instead of the SVMP. The results of this study reinforce the value of studying obscure venoms for biodiscovery of novel investigational ligands.