Correlation between ontogenetic dietary shifts and venom variation in Australian brown snakes ( Pseudonaja )

Venom is a key evolutionary trait, as evidenced by its widespread convergent evolution across the animal kingdom. In an escalating prey-predator arms race, venoms evolve rapidly to guarantee predatory or defensive success. Variation in venom composition is ubiquitous among snakes. Here, we tested variation in venom activity on substrates relevant to blood coagulation among Pseudonaja (brown snake) species, Australian elapids responsible for the majority of medically important human envenomations in Australia. A functional approach was employed to elucidate interspecific variation in venom activity in all nine currently recognised species of Pseudonaja. Fluorometric enzymatic activity assays were performed to test variation in whole venom procoagulant activity among species. Analyses confirmed the previously documented ontogenetic shift from non-coagulopathic venom in juveniles to coagulopathic venom as adults, except for the case of P. modesta, which retains non-coagulopathic venom as an adult. These shifts in venom activity correlate with documented ontogenetic shifts in diet among brown snakes from specialisation on reptilian prey as juveniles (and throughout the life cycle of P. modesta), to a more generalised diet in adults that includes mammals. The results of this study bring to light findings relevant to both clinical and evolutionary toxinology

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

Enter the Dragon: The Dynamic and Multifunctional Evolution of Anguimorpha Lizard Venoms

While snake venoms have been the subject of intense study, comparatively little work has been done on lizard venoms. In this study, we have examined the structural and functional diversification of anguimorph lizard venoms and associated toxins, and related these results to dentition and predatory ecology. Venom composition was shown to be highly variable across the 20 species of Heloderma, Lanthanotus, and Varanus included in our study. While kallikrein enzymes were ubiquitous, they were also a particularly multifunctional toxin type, with differential activities on enzyme substrates and also ability to degrade alpha or beta chains of fibrinogen that reflects structural variability. Examination of other toxin types also revealed similar variability in their presence and activity levels. The high level of venom chemistry variation in varanid lizards compared to that of helodermatid lizards suggests that venom may be subject to different selection pressures in these two families. These results not only contribute to our understanding of venom evolution but also reveal anguimorph lizard venoms to be rich sources of novel bioactive molecules with potential as drug design and development lead compounds

Venom down under: Dynamic evolution of Australian elapid snake toxins

Despite the unparalleled diversity of venomous snakes in Australia, research has concentrated on a handful of medically significant species and even of these very few toxins have been fully sequenced. In this study, venom gland transcriptomes were sequenced from eleven species of small Australian elapid snakes, from eleven genera, spanning a broad phylogenetic range. The particularly large number of sequences obtained for three-finger toxin (3FTx) peptides allowed for robust reconstructions of their dynamic molecular evolutionary histories. We demonstrated that each species preferentially favoured different types of α-neurotoxic 3FTx, probably as a result of differing feeding ecologies. The three forms of α-neurotoxin [Type I (also known as (aka): short-chain), Type II (aka: long-chain) and Type III] not only adopted differential rates of evolution, but have also conserved a diversity of residues, presumably to potentiate prey-specific toxicity. Despite these differences, the different α-neurotoxin types were shown to accumulate mutations in similar regions of the protein, largely in the loops and structurally unimportant regions, highlighting the significant role of focal mutagenesis. We theorize that this phenomenon not only affects toxin potency or specificity, but also generates necessary variation for preventing/delaying prey animals from acquiring venom-resistance. This study also recovered the first full-length sequences for multimeric phospholipase A2 PLA2) ‘taipoxin/paradoxin’ subunits from non-Oxyuranus species, confirming the early recruitment of this extremely potent neurotoxin complex to the venom arsenal of Australian elapid snakes. We also recovered the first natriuretic peptides from an elapid that lack the derived C-terminal tail and resemble the plesiotypic form (ancestral character state) found in viper venoms. This provides supporting evidence for a single early recruitment of natriuretic peptides into snake venoms. Novel forms of kunitz and waprin peptides were recovered, including dual domain kunitz-kunitz precursors and the first kunitz-waprin hybrid precursors from elapid snakes. The novel sequences recovered in this study reveal that the huge diversity of unstudied venomous Australian snakes are of considerable interest not only for the investigation of venom and whole organism evolution but also represent an untapped bioresource in the search for novel compounds for use in drug design and development.Timothy N. W. Jackson , Kartik Sunagar, Eivind A. B. Undheim, Ivan Koludarov, Angelo H. C. Chan, Kate Sanders, Syed A. Ali, Iwan Hendrikx, Nathan Dunstan and Bryan G. Fr

Fossilized venom: the unusually conserved venom profiles of heloderma species (beaded lizards and gila monsters).

Research into snake venoms has revealed extensive variation at all taxonomic levels. Lizard venoms, however, have received scant research attention in general, and no studies of intraclade variation in lizard venom composition have been attempted to date. Despite their iconic status and proven usefulness in drug design and discovery, highly venomous helodermatid lizards (gila monsters and beaded lizards) have remained neglected by toxinological research. Proteomic comparisons of venoms of three helodermatid lizards in this study has unravelled an unusual similarity in venom-composition, despite the long evolutionary time (~30 million years) separating H. suspectum from the other two species included in this study (H. exasperatum and H. horridum). Moreover, several genes encoding the major helodermatid toxins appeared to be extremely well-conserved under the influence of negative selection (but with these results regarded as preliminary due to the scarcity of available sequences). While the feeding ecologies of all species of helodermatid lizard are broadly similar, there are significant morphological differences between species, which impact upon relative niche occupation

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

Endless forms most beautiful: the evolution of ophidian oral glands, including the venom system, and the use of appropriate terminology for homologous structures

The differentiated serous-secreting dental glands of caenophidian snakes are diverse in form despite their developmental homology. This variation makes the elucidation of their evolutionary history a complex task. In addition, some authors identify as many as ten discrete types/subtypes of ophidian oral gland. Over the past decade and a half, molecular systematics and toxinology have deepened our understanding of the evolution of these fascinating and occasionally enigmatic structures. This paper includes a comprehensive examination of ophidian oral gland structure and (where possible) function, as well as new data on rictal glands and their associated anatomy. Following this, appropriate use of terminology, especially that pertaining to homologous structures (including the controversial “venom gland” vs “Duvernoy’s gland” debate), is considered. An interpretation of the evolutionary history of the ophidian venom system, drawing on recent results from molecular systematics, toxinology and palaeontology, concludes the paper

Venomous landmines: Clinical implications of extreme coagulotoxic diversification and differential neutralization by antivenom of venoms within the viperid snake genus Bitis

The genus comprises 17 snake species that inhabit Africa and the Arabian Peninsula. They are responsible for a significant proportion of snakebites in the region. The venoms of the two independent lineages of giant and again in the common ancestor of the clade consisting of and ) induce an array of debilitating effects including anticoagulation, hemorrhagic shock and cytotoxicity, whilst the dwarf species is known to have strong neurotoxic effects. However, the venom effects of the other species within the genus have not been explored in detail. A series of coagulation assays were implemented to assess the coagulotoxic venom effects of fourteen species within the genus. This study identified procoagulant venom as the ancestral condition, retained only by the basal dwarf species suggesting anticoagulant venom is a derived trait within the genus and has been secondarily amplified on at least four occasions. A wide range of anticoagulant mechanisms were identified, such as coagulant and destructive activities upon fibrinogen in both giant and dwarf and the action of inhibiting the prothrombinase complex, which is present in a clade of dwarf . Antivenom studies revealed that while the procoagulant effects of were poorly neutralized, and thus a cause for concern, the differential mechanisms of anticoagulation in other species were all well neutralized. Thus, this study concludes there is a wide range of coagulotoxic mechanisms which have evolved within the genus and that clinical management strategies are limited for the procoagulant effects of , but that anticoagulant effects of other species are readily treated by the South African polyvalent antivenom. These results therefore have direct, real-work implications for the treatment of envenomed patients

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