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Australian elapid snake venoms are uniquely procoagulant, utilizing blood clotting enzyme Factor Xa (FXa) as a toxin, which evolved as a basal trait in this clade. The subsequent recruitment of Factor Va (FVa) as a toxin occurred in the last common ancestor of taipans ( Oxyuranus species) and brown snakes ( Pseudonaja species). Factor II (prothrombin) activation has been stated as the primary mechanism for the lethal coagulopathy, but this hypothesis has never been tested. The additional activation of Factor VII (FVII) by Oxyuranus / Pseudonaja venoms has historically been considered as a minor, unimportant novelty. This study aimed to investigate the significance of toxic FVII activation relative to prothrombin activation by testing a wide taxonomical range of Australian elapid species with procoagulant venoms. The activation of FVII or prothrombin, with and without the Factor Va as a cofactor, was assessed, along with the structural changes involved in these processes. All procoagulant species could activate FVII, establishing this as a basal trait. In contrast, only some lineages could activate prothrombin, indicating that this is a derived trait. For species able to activate both zymogens, Factor VII was consistently more strongly activated than prothrombin. FVa was revealed as an essential cofactor for FVII activation, a mechanism previously undocumented. Species lacking FVa in their venom utilized endogenous plasma FVa to exert this activity. The ability of the human FXa:FVa complex to activate FVII was also revealed as a new feedback loop in the endogenous clotting cascade. Toxin sequence analyses identified structural changes essential for the derived trait of prothrombin activation. This study presents a paradigm shift in understanding how elapid venoms activate coagulation factors, highlighting the critical role of FVII activation in the pathophysiological effects upon the coagulation cascade produced by Australian elapid snake venoms. It also documented the novel use of Factor Va as a cofactor for FVII activation for both venom and endogenous forms of FXa. These findings are crucial for developing better antivenoms and treatments for snakebite victims and have broader implications for drug design and the treatment of coagulation disorders. The research also advances the evolutionary biology knowledge of snake venoms.

Jenna Crowe-Riddell and colleagues introduce sea snakes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Kissing bugs are known to produce anticoagulant venom that facilitates blood-feeding. However, it is unknown how this saliva evolved and if the venom produced by the entomophagous ancestors of kissing bugs would have helped or hindered the trophic shift. In this study, we show that venoms produced by extant predatory assassin bugs have strong anticoagulant properties mediated chiefly by proteolytic degradation of fibrinogen, and additionally contain anticoagulant disulfide-rich peptides. However, venom produced by predatory species also has pain-inducing and membrane-permeabilizing activities that would be maladaptive for blood-feeding, and which venom of the blood-feeding species lack. This study demonstrates that venom produced by the predatory ancestors of kissing bugs was exapted for the trophic switch to blood-feeding by virtue of its anticoagulant properties. Further adaptation to blood-feeding occurred by downregulation of venom toxins with proteolytic, cytolytic, and pain-inducing activities, and upregulation and neofunctionalization of toxins with anticoagulant activity independent of proteolysis., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

In the ongoing evolutionary arms race between predators and prey, adaptive innovations often trigger a reciprocal response. For instance, the emergence of α-neurotoxins in snake venom has driven prey species targeted by these snakes to evolve sophisticated defense mechanisms. This study zeroes in on the particular motifs within the orthosteric sites of post-synaptic nicotinic acetylcholine receptors (nAChR) that confer resistance to α-neurotoxins, often through structural alterations of nAChR. This research examined Australian agamid lizards, a primary prey group for Australian elapid snakes, which are subject to predatory selection pressures. We previously showed that Pogona vitticeps (Central bearded dragon) was resistant to α-neurotoxic snake venoms through a steric hindrance form resistance evolving within the nAChR orthosteric, specifically through the 187-189NVT motif resulting in the presence of N-glycosylation, with the branching carbohydrate chains impeding the binding by the neurotoxins. This adaptive trait is thought to be a compensatory mechanism for the lizard's limited escape capabilities. Despite the significance of this novel adaptation, the prevalence and evolutionary roots of such venom resistance in Australian agamids have not been thoroughly investigated. To fill this knowledge gap, we undertook a comprehensive sequencing analysis of the nAChR ligand-binding domain across the full taxonomical diversity of Australian agamid species. Our findings reveal that the N-glycosylation resistance mechanism is a trait unique to the Pogona genus and absent in other Australian agamids. This aligns with Pogona's distinctive morphology, which likely increases vulnerability to neurotoxic elapid snakes, thereby increasing selective pressures for resistance. In contrast, biolayer interferometry experiments with death adder (Acanthophis species) venoms did not indicate any resistance-related binding patterns in other agamids, suggesting a lack of similar resistance adaptations, consistent with these lineages either being fast-moving, covered with large defensive spines, or being arboreal. This research not only uncovers a novel α-neurotoxin resistance mechanism in Australian agamids but also highlights the complex dynamics of the predator-prey chemical arms race. It provides a deeper understanding of how evolutionary pressures shape the interactions between venomous snakes and their prey., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite a recent surge in high-throughput venom research that has enabled many species to be studied, some snake venoms remain understudied. The long-tailed rattlesnakes (Crotalus ericsmithi, C. lannomi, and C. stejnegeri) are one group where such research lags, largely owing to the rarity of these snakes and the hazardous areas, ripe with drug (marijuana and opium) production, they inhabit in Mexico. To fill this knowledge gap, we used multiple functional assays to examine the coagulotoxic (including across different plasma types), neurotoxic, and myotoxic activity of the venom of the long-tailed rattlesnakes. All crude venoms were shown to be potently anticoagulant on human plasma, which we discovered was not due to the destruction of fibrinogen, except for C. stejnegeri displaying minor fibrinogen destruction activity. All venoms exhibited anticoagulant activity on rat, avian, and amphibian plasmas, with C. ericsmithi being the most potent. We determined the mechanism of anticoagulant activity by C. ericsmithi and C. lannomi venoms to be phospholipid destruction and inhibition of multiple coagulation factors, leading to a net disruption of the clotting cascade. In the chick biventer assay, C. ericsmithi and C. lannomi did not exhibit neurotoxic activity but displayed potential weak myotoxic activity. BIRMEX® (Faboterápico Polivalente Antiviperino) antivenom was not effective in neutralising this venom effect. Overall, this study provides an in-depth investigation of venom function of understudied long-tailed rattlesnakes and provides a springboard for future venom and ecology research on the group., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Jason Jones (co-author) is affiliated with herp. mx - a non-profit dedicated to researching and conserving Mexican reptiles and amphibians. Wayne Hodgson (co-author) is on the editorial council for Toxicon. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Snakes in the family Elapidae largely produce venoms rich in three-finger toxins (3FTx) that bind to the α 1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specific mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specificity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan → serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey., (© 2024. The Author(s).)

The interplay between predator and prey has catalyzed the evolution of venom systems, with predators honing their venoms in response to the evolving resistance of prey. A previous study showed that the African varanid species Varanus exanthematicus has heightened resistance to snake venoms compared to the Australian species V. giganteus , V. komodoensis , and V. mertensi , likely due to increased predation by sympatric venomous snakes on V. exanthematicus . To understand venom resistance among varanid lizards, we analyzed the receptor site targeted by venoms in 27 varanid lizards, including 25 Australian varanids. The results indicate an active evolutionary arms race between Australian varanid lizards and sympatric neurotoxic elapid snakes. Large species preying on venomous snakes exhibit inherited neurotoxin resistance, a trait potentially linked to their predatory habits. Consistent with the 'use it or lose it' aspect of venom resistance, this trait was secondarily reduced in two lineages that had convergently evolved gigantism ( V. giganteus and the V. komodoensis / V. varius clade), suggestive of increased predatory success accompanying extreme size and also increased mechanical protection against envenomation due to larger scale osteoderms. Resistance was completely lost in the mangrove monitor V. indicus , consistent with venomous snakes not being common in their arboreal and aquatic niche. Conversely, dwarf varanids demonstrate a secondary loss at the base of the clade, with resistance subsequently re-evolving in the burrowing V. acanthurus / V. storri clade, suggesting an ongoing battle with neurotoxic predators. Intriguingly, within the V. acanthurus / V. storri clade, resistance was lost again in V. kingorum , which is morphologically and ecologically distinct from other members of this clade. Resistance was also re-evolved in V. glebopalma which is terrestrial in contrast to the arboreal/cliff dwelling niches occupied by the other members of its clade ( V. glebopalma , V. mitchelli , V. scalaris , V. tristis ). This 'Russian doll' pattern of venom resistance underscores the dynamic interaction between dwarf varanids and Australian neurotoxic elapid snakes. Our research, which included testing Acanthophis (death adder) venoms against varanid receptors as models for alpha-neurotoxic interactions, uncovered a fascinating instance of the Red Queen Hypothesis: some death adders have developed more potent toxins specifically targeting resistant varanids, a clear sign of the relentless predator-prey arms race. These results offer new insight into the complex dynamics of venom resistance and highlight the intricate ecological interactions that shape the natural world.

The rarity of tool manufacture in wild parrots is surprising because they share key life-history traits with advanced tool-using species, including large brains, complex sociality and prolonged parental care. When it does occur, tool manufacture in parrots tends to be innovative, spontaneous and individually variable, but most cases have been in captivity. In the wild, only palm cockatoos ( Probosciger aterrimus ) have been observed using tools regularly. However, they are unusual because they use tools to enhance their displays rather than for foraging or self-maintenance. Males in northern Australia make two types of tool from sticks and seed pods, which they tap rhythmically against a tree during display. We analysed 256 sound tools retrieved from 70 display trees. Drumsticks (89% of tools) were used more often than seed pod tools; most males manufactured only drumsticks, but some made both types. Individual males differed significantly in the design of their drumsticks including the length, width and mass but we found no evidence that neighbours copied each other. We discuss the highly individualized preferences for sound tool design in context of the behavioural predispositions behind the rarity of tool manufacture in wild parrots.

Larvae of the genus Megalopyge (Lepidoptera: Zygaenoidea: Megalopygidae), known as asp or puss caterpillars, produce defensive venoms that cause severe pain. Here, we present the anatomy, chemistry, and mode of action of the venom systems of caterpillars of two megalopygid species, the Southern flannel moth Megalopyge opercularis and the black-waved flannel moth Megalopyge crispata . We show that megalopygid venom is produced in secretory cells that lie beneath the cuticle and are connected to the venom spines by canals. Megalopygid venoms consist of large aerolysin-like pore-forming toxins, which we have named megalysins, and a small number of peptides. The venom system differs markedly from those of previously studied venomous zygaenoids of the family Limacodidae, suggestive of an independent origin. Megalopygid venom potently activates mammalian sensory neurons via membrane permeabilization and induces sustained spontaneous pain behavior and paw swelling in mice. These bioactivities are ablated by treatment with heat, organic solvents, or proteases, indicating that they are mediated by larger proteins such as the megalysins. We show that the megalysins were recruited as venom toxins in the Megalopygidae following horizontal transfer of genes from bacteria to the ancestors of ditrysian Lepidoptera. Megalopygids have recruited aerolysin-like proteins as venom toxins convergently with centipedes, cnidarians, and fish. This study highlights the role of horizontal gene transfer in venom evolution.

Snakebite is a globally neglected tropical disease, with coagulation disturbances being the primary pathology of many deadly snake venoms. Age-related differences in human plasma have been abundantly reported, yet the effect that these differences pose regarding snakebite is largely unknown. We tested for differences in coagulotoxic effects (via clotting time) of multiple snake venoms upon healthy human adult (18+) and paediatric (median 3.3 years old) plasma in vivo and compared these effects to the time it takes the plasmas to clot without the addition of venom (the spontaneous clotting time). We tested venoms from 15 medically significant snake species (from 13 genera) from around the world with various mechanisms of coagulotoxic actions, across the three broad categories of procoagulant, pseudo-procoagulant, and anticoagulant, to identify any differences between the two plasmas in their relative pathophysiological vulnerability to snakebite. One procoagulant venom ( Daboia russelii , Russell's Viper) produced significantly greater potency on paediatric plasma compared with adult plasma. In contrast, the two anticoagulant venoms ( Pseudechis australis , Mulga Snake; and Bitis cornuta , Many-horned Adder) were significantly more potent on adult plasma. All other procoagulant venoms and all pseudo-procoagulant venoms displayed similar potency across both plasmas. Our preliminary results may inform future studies on the effect of snake venoms upon plasmas from different age demographics and hope to reduce the burden of snakebite upon society.

Evidence suggests that snakes can hear, but how snakes naturally respond to sound is still unclear. We conducted 304 controlled experiment trials on 19 snakes across five genera in a sound-proof room (4.9 x 4.9 m) at 27ºC, observing the effects of three sounds on individual snake behavior, compared to controls. We quantified eight snake behaviors (body movement, body freezing, head-flicks, tongue-flicks, hissing, periscoping, head fixation, lower jaw drop) in response to three sounds, which were filtered pink-noise within the following frequency ranges: 0-150Hz (sound 1, which produced ground vibrations, as measured by an accelerometer), 150-300Hz (sound 2, which did not produced ground vibrations), 300-450Hz (sound 3, which did not produced ground vibrations). All snake responses were strongly genus dependent. Only one genus (Aspidites, Woma Pythons) significantly increased their probability of movement in response to sound, but three other genera (Acanthophis (Death Adders), Oxyuranus (Taipans), and Pseudonaja (Brown Snakes)) were more likely to move away from sound, signaling potential avoidance behavior. Taipans significantly increased their likelihood of displaying defensive and cautious behaviors in response to sound, but three of the five genera exhibited significantly different types of behaviors in sound trials compared to the control. Our results highlight potential heritable behavioral responses of snakes to sound, clustered within genera. Our study illustrates the behavioral variability among different snake genera, and across sound frequencies, which contributes to our limited understanding of hearing and behavior in snakes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Zdenek et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Factor XIIa (FXIIa) is a promising target for developing new drugs that prevent thrombosis without causing bleeding complications. A native cyclotide (MCoTI-II) is gaining interest for engineering FXIIa-targeted anticoagulants as this peptide inhibits FXIIa but not other coagulation proteases. Here, we engineered the native biosynthetic cyclization loop of MCoTI-II (loop 6) to generate improved FXIIa inhibitors. Decreasing the loop length led to gains in potency up to 7.7-fold, with the most potent variant having five residues in loop 6 ( K i = 25 nM). We subsequently examined sequence changes within loop 6 and an adjacent loop, with substitutions at P4 and P2' producing a potent FXIIa inhibitor ( K i = 2 nM) that displayed more than 700-fold selectivity, was stable in human serum, and blocked the intrinsic coagulation pathway in human plasma. These findings demonstrate that engineering the biosynthetic cyclization loop can generate improved cyclotide variants, expanding their potential for drug discovery.

The targeting of specific prey by snake venom toxins is a fascinating aspect of molecular and ecological evolution. Neurotoxic targeting by elapid snakes dominates the literature in this regard; however, recent studies have revealed viper toxins also induce neurotoxic effect. While this effect is thought to primarily be driven by prey selectivity, no study has quantified the taxonomically specific neurotoxicity of the viper clade consisting of Daboia, Macrovipera, Montivipera, and Vipera genera. Here, we tested venom toxin binding from 28 species of vipers from the four genera on the alpha 1 neuronal nicotinic acetylcholine receptors (nAChRs) orthosteric sites of amphibian, avian, lizard, rodent, and human mimotopes (synthetic peptides) using the Octet HTX biolayer interferometry platform. Daboia siamensis and D. russelii had broad binding affinity towards all mimotopes, while D. palestinae had selectivity toward lizard. Macrovipera species, on the other hand, were observed to have a higher affinity for amphibian mimotopes except for M. schweizeri, which inclined more toward lizard mimotopes. All Montivipera and most Vipera species also had higher affinity toward lizard mimotopes. Vipera a. montandoni, V. latastei, V. nikolski, and V. transcaucasina had the least binding to any of the mimotopes of the study. While a wide range of affinity binding towards various mimotopes were observed within the clade, the lowest affinity occurred towards the human target. Daboia siamensis and Macrovipera lebetina exhibited the greatest affinity toward the human mimotope, albeit still the least targeted of the mimotopes within those species. Overlaying this toxin-targeting trait over phylogeny of this clade revealed multiple cases of amplification of this trait and several cases of secondary loss. Overall, our results reveal dynamic variation, amplification, and some secondary loss of the prey targeting trait by alpha-neurotoxins within the venoms of this clade, indicating evolutionary selection pressure shaping the basic biochemistry of these venoms. Our work illustrates the successful use of this biophysical assay to further research snake venom neurotoxins and emphasizes the risk of generalizing venom effects observed on laboratory animals to have similar effects on humans., (© 2022. The Author(s).)

Knottins are topologically complex peptides that are stabilised by a cystine knot and have exceptionally diverse functions, including protease inhibition. However, approaches for tuning their activity in situ are limited. Here, we demonstrate separate approaches for tuning the activity of knottin protease inhibitors using light or streptavidin. We show that the inhibitory activity and selectivity of an engineered knottin can be controlled with light by activating a second mode of action that switches the inhibitor ON against new targets. Guided by a knottin library screen, we also identify a position in the inhibitor's binding loop that permits insertion of a biotin tag without impairing activity. Using streptavidin, biotinylated knottins with nanomolar affinity can be switched OFF in activity assays, and the anticoagulant activity of a factor XIIa inhibitor can be rapidly switched OFF in human plasma. Our findings expand the scope of engineered knottins for precisely controlling protein function., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Despite coagulotoxicity being a primary weapon for prey capture by Bothrops species (lancehead pit vipers) and coagulopathy being a major lethal clinical effect, a genus-wide comparison has not been undertaken. To fill this knowledge gap, we used thromboelastography to compare 37 venoms, from across the full range of geography, taxonomy, and ecology, for their action upon whole plasma and isolated fibrinogen. Potent procoagulant toxicity was shown to be the main venom effect of most of the species tested. However, the most basal species ( B. pictus ) was strongly anticoagulant; this is consistent with procoagulant toxicity being a novel trait that evolved within Bothrops subsequent to their split from anticoagulant American pit vipers. Intriguingly, two of the arboreal species studied ( B. bilineatus and B. taeniatus ) lacked procoagulant venom, suggesting differential evolutionary selection pressures. Notably, some terrestrial species have secondarily lost the procoagulant venom trait: the Mogi Mirim, Brazil locality of B. alternatus ; San Andres, Mexico locality of B. asper ; B. diporus ; and the São Roque of B. jararaca . Direct action on fibrinogen was extremely variable; this is consistent with previous hypotheses regarding it being evolutionary decoupled due to procoagulant toxicity being the primary prey-capture weapon. However, human patients live long enough for fibrinogen depletion to be clinically significant. The extreme variability may be reflective of antivenom variability, with these results thereby providing a foundation for such future work of clinical relevance. Similarly, the venom diversification trends relative to ecological niche will also be useful for integration with natural history data, to reconstruct the evolutionary pressures shaping the venoms of these fascinating snakes.

Major variations in venom composition can occur between juvenile and adult venomous snakes. However, due to logistical constraints, antivenoms are produced using adult venoms in immunising mixtures, possibly resulting in limited neutralisation of juvenile snake venoms. Daboia russelii is one of the leading causes of snakebite death across South Asia. Its venom is potently procoagulant, causing stroke in prey animals but causing in humans consumptive coagulopathy-a net anticoagulant state-and sometimes death resulting from hemorrhage. In this in vitro study, we compared the venom activity of-and antivenom efficacy against-six 2-week-old D. russelii relative to that of their parents. Using a coagulation analyser, we quantified the relative coagulotoxicity of these venoms in human, avian, and amphibian plasma. The overall potency on human plasma was similar across all adult and neonate venoms, and SII (Serum Institute of India) antivenom was equipotent in neutralising these coagulotoxic effects. In addition, all venoms were also similar in their action upon avian plasma. In contrast, the neonate venoms were more potent on amphibian plasma, suggesting amphibians make up a larger proportion of neonate diet than adult diet. A similar venom potency in human and avian plasmas but varying selectivity for amphibian plasma suggests ontogenetic differences in toxin isoforms within the factor X or factor V activating classes, thereby providing a testable hypothesis for future transcriptomics work. By providing insights into the functional venom differences between adult and neonate D. russelii venoms, we hope to inform clinical treatment of patients envenomated by this deadly species and to shed new light on the natural history of these extremely medically important snakes., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cyclotides are plant-derived peptides with complex structures shaped by their head-to-tail cyclic backbone and cystine knot core. These structural features underpin the native bioactivities of cyclotides, as well as their beneficial properties as pharmaceutical leads, including high proteolytic stability and cell permeability. However, their inherent structural complexity presents a challenge for cyclotide engineering, particularly for accessing libraries of sufficient chemical diversity to design potent and selective cyclotide variants. Here, we report a strategy using mRNA display enabling us to select potent cyclotide-based FXIIa inhibitors from a library comprising more than 10 12 members based on the cyclotide scaffold of Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II). The most potent and selective inhibitor, cMCoFx1, has a pM inhibitory constant toward FXIIa with greater than three orders of magnitude selectivity over related serine proteases, realizing specific inhibition of the intrinsic coagulation pathway. The cocrystal structure of cMCoFx1 and FXIIa revealed interactions at several positions across the contact interface that conveyed high affinity binding, highlighting that such cyclotides are attractive cystine knot scaffolds for therapeutic development.

The use of venom in predation exerts a corresponding selection pressure for the evolution of venom resistance. One of the mechanisms related to venom resistance in animals (predators or prey of snakes) is the presence of molecules in the blood that can bind venom toxins, and inhibit their pharmacological effects. One such toxin type are venom phospholipase A 2 s (PLA 2 s), which have diverse effects including anticoagulant, myotoxic, and neurotoxic activities. BoaγPLI isolated from the blood of Boa constrictor has been previously shown to inhibit venom PLA 2 s that induced myotoxic and edematogenic activities. Recently, in addition to its previously described and very potent neurotoxic effect, the venoms of American coral snakes (Micrurus species) have been shown to have anticoagulant activity via PLA 2 toxins. As coral snakes eat other snakes as a major part of their diet, neonate Boas could be susceptible to predation by this sympatric species. Thus, this work aimed to ascertain if BoaγPLI provided a protective effect against the anticoagulant toxicity of venom from the model species Micrurus laticollaris in addition to its ability shown previously against other toxin types. Using a STA R Max coagulation analyser robot to measure the effect upon clotting time, and TEG5000 thromboelastographers to measure the effect upon clot strength, we evaluated the ability of BoaγPLI to inhibit M. laticollaris venom. Our results indicate that BoaγPLI is efficient at inhibiting the M. laticollaris anticoagulant effect, reducing the time of coagulation (restoring them closer to non-venom control values) and increasing the clot strength (restoring them closer to non-venom control values). These findings demonstrate that endogenous PLA 2 inhibitors in the blood of non-venomous snakes are multi-functional and provide broad resistance against a myriad of venom PLA 2 -driven toxic effects including coagulotoxicity, myotoxicity, and neurotoxicity. This novel form of resistance could be evidence of selective pressures caused by predation from venomous snakes and stresses the need for field-based research aimed to expand our understanding of the evolutionary dynamics of such chemical arms race., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

African spitting cobras are unique among cobras for their potent anticoagulant venom activity arising from strong inhibition of Factor Xa. This anticoagulant effect is exerted by venom phospholipase A 2 (Group I PLA 2 ) toxins whose activity contributes to the lethality of these species. This anticoagulant toxicity is particularly problematic as it is not neutralized by current antivenoms. Previous work demonstrated this trait for Naja mossambica, N. nigricincta , N. nigricollis , and N. pallida. The present work builds upon previous research by testing across the full taxonomical range of African spitting cobras, demonstrating that N. ashei , N. katiensis , and N. nubiae are also potently anticoagulant through the inhibition of Factor Xa, and therefore the amplification of potent anticoagulant activity occurred at the base of the African spitting cobra radiation. Previous work demonstrated that the enzyme-inhibitor varespladib was able to neutralize this toxic action for N. mossambica, N. nigricincta , N. nigricollis , and N. pallida venoms. The current work demonstrates that varespladib was also able to neutralize N. ashei , N. katiensis , and N. nubiae . Thus varespladib is shown to have broad utility across the full range of African spitting cobras. In addition, we examined the cross-reactivity of the metalloprotease inhibitor prinomastat, which had been previously intriguingly indicated as being capable of neutralizing viperid venom PLA 2 (Group II PLA 2 ). In this study prinomastat inhibited the FXa-inhibiting PLA 2 toxins of all the African spitting cobras at the same concentration at which it has been shown to inhibit metalloproteases, and thus was comparably effective in its cross-reactivity. In addition we showed that the metalloprotease-inhibitor marimastat was also able to cross-neutralize PLA 2 but less effectively than prinomastat. Due to logistical (cold-chain requirement) and efficacy (cross-reactivity across snake species) limitations of traditional antivenoms, particularly in developing countries where snakebite is most common, these small molecule inhibitors (SMIs) might hold great promise as initial, field-based, treatments for snakebite envenoming as well as addressing fundamental limitations of antivenom in the clinical setting where certain toxin effects are unneutralized., Competing Interests: Authors ML and RC are employed by the company Ophirex. However, the company had no input in experimental design or reviewing of results before publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MBV declared a past co-authorship with the authors CZ and BF to the handling Editor., (Copyright © 2021 Chowdhury, Lewin, Zdenek, Carter and Fry.)

Is snake venom activity influenced by size? This is a long-standing question that can have important consequences for the treatment of snake envenomation. Ontogenetic shifts in venom composition are a well-documented characteristic of numerous snake species. Although snake venoms can cause a range of pathophysiological disturbances, establishing the coagulotoxic profiles related to such shifts is a justified approach because coagulotoxicity can be deadly, and its neutralisation is a challenge for current antivenom therapy. Thus, we aimed to assess the coagulotoxicity patterns on plasma and fibrinogen produced by B othrops jararacussu venoms from individuals of different sizes and sex, and the neutralisation potential of SAB (anti bothropic serum produced by Butantan Institute). The use of a metalloproteinase inhibitor (Prinomastat) and a serine proteinase inhibitor (AEBSF) enabled us to determine the toxin class responsible for the observed coagulopathy: activity on plasma was found to be metalloprotease driven, while the activity on fibrinogen is serine protease driven. To further explore differences in venom activity, the activation of Factor X and prothrombin as a function of snake size was also evaluated. All the venoms exhibited a potent procoagulant effect upon plasma and were less potent in their pseudo-procoagulant clotting effect upon fibrinogen. On human plasma, the venoms from smaller snakes produced more rapid clotting than the larger ones. In contrast, the venom activity on fibrinogen had no relation with size or sex. The difference in procoagulant potency was correlated with the bigger snakes being proportionally better neutralized by antivenom due to the lower levels of procoagulant toxins, than the smaller. Thus, while the antivenom ultimately neutralized the venoms, proportionally more would be needed for an equal mass of venom from a small snake than a large one. Similarly, the neutralisation by SAB of the pseudo-procoagulant clotting effects was also correlated with relative potency, with the smaller and bigger snakes being neutralized proportional to potency, but with no correlation to size. Thromboelastography (TEG) tests on human and toad plasma revealed that small snakes' venoms acted quicker than large snakes' venom on both plasmas, with the action upon amphibian plasma consistent with smaller snakes taking a larger proportion of anuran prey than adults. Altogether, the ontogenetic differences regarding coagulotoxic potency and corresponding impact upon relative antivenom neutralisation of snakes with different sizes were shown, underscoring the medical importance of investigating ontogenetic changes in order to provide data crucial for evidence-based design of clinical management strategies., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bites from helodermatid lizards can cause pain, paresthesia, paralysis, and tachycardia, as well as other symptoms consistent with neurotoxicity. Furthermore, in vitro studies have shown that Heloderma horridum venom inhibits ion flux and blocks the electrical stimulation of skeletal muscles. Helodermatids have long been considered the only venomous lizards, but a large body of robust evidence has demonstrated venom to be a basal trait of Anguimorpha. This clade includes varanid lizards, whose bites have been reported to cause anticoagulation, pain, and occasionally paralysis and tachycardia. Despite the evolutionary novelty of these lizard venoms, their neuromuscular targets have yet to be identified, even for the iconic helodermatid lizards. Therefore, to fill this knowledge gap, the venoms of three Heloderma species ( H. exasperatum , H. horridum and H. suspectum ) and two Varanus species ( V. salvadorii and V. varius ) were investigated using Gallus gallus chick biventer cervicis nerve-muscle preparations and biolayer interferometry assays for binding to mammalian ion channels. Incubation with Heloderma venoms caused the reduction in nerve-mediated muscle twitches post initial response of avian skeletal muscle tissue preparation assays suggesting voltage-gated sodium (Na V ) channel binding. Congruent with the flaccid paralysis inducing blockage of electrical stimulation in the skeletal muscle preparations, the biolayer interferometry tests with Heloderma suspectum venom revealed binding to the S3-S4 loop within voltage-sensing domain IV of the skeletal muscle channel subtype, Na V 1.4. Consistent with tachycardia reported in clinical cases, the venom also bound to voltage-sensing domain IV of the cardiac smooth muscle calcium channel, Ca V 1.2. While Varanus varius venom did not have discernable effects in the avian tissue preparation assay at the concentration tested, in the biointerferometry assay both V. varius and V. salvadorii bound to voltage-sensing domain IV of both Na V 1.4 and Ca V 1.2, similar to H. suspectum venom. The ability of varanid venoms to bind to mammalian ion channels but not to the avian tissue preparation suggests prey-selective actions, as did the differential potency within the Heloderma venoms for avian versus mammalian pathophysiological targets. This study thus presents the detailed characterization of Heloderma venom ion channel neurotoxicity and offers the first evidence of varanid lizard venom neurotoxicity. In addition, the data not only provide information useful to understanding the clinical effects produced by envenomations, but also reveal their utility as physiological probes, and underscore the potential utility of neglected venomous lineages in the drug design and development pipeline.

What factors influence the evolution of a heavily selected functional trait in a diverse clade? This study adopts rattlesnakes as a model group to investigate the evolutionary history of venom coagulotoxicity in the wider context of phylogenetics, natural history, and biology. Venom-induced clotting of human plasma and fibrinogen was determined and mapped onto the rattlesnake phylogenetic tree to reconstruct the evolution of coagulotoxicity across the group. Our results indicate that venom phenotype is often independent of phylogenetic relationships in rattlesnakes, suggesting the importance of diet and/or other environmental variables in driving venom evolution. Moreover, the striking inter- and intraspecific variability in venom activity on human blood highlights the considerable variability faced by physicians treating envenomation. This study is the most comprehensive effort to date to describe and characterize the evolutionary and biological aspects of coagulotoxins in rattlesnake venom. Further research at finer taxonomic levels is recommended to elucidate patterns of variation within species and lineages., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Palearctic vipers are medically significant snakes in the genera Daboia, Macrovipera, Montivipera , and Vipera which occur throughout Europe, Central Asia, Near and Middle East. While the ancestral condition is that of a small-bodied, lowland species, extensive diversification has occurred in body size, and niche specialization. Using 27 venom samples and a panel of in vitro coagulation assays, we evaluated the relative coagulotoxic potency of Palearctic viper venoms and compared their neutralization by three antivenoms (Insoserp Europe, VIPERFAV and ViperaTAb) and two metalloprotease inhibitors (prinomastat and DMPS). We show that variation in morphology parallels variation in the Factor X activating procoagulant toxicity, with the three convergent evolutions of larger body sizes ( Daboia genus, Macrovipera genus, and Vipera ammodytes uniquely within the Vipera genus) were each accompanied by a significant increase in procoagulant potency. In contrast, the two convergent evolutions of high altitude specialization (the Montivipera genus and Vipera latastei uniquely within the Vipera genus) were each accompanied by a shift away from procoagulant action, with the Montivipera species being particularly potently anticoagulant. Inoserp Europe and VIPERFAV antivenoms were both effective against a broad range of Vipera species, with Inoserp able to neutralize additional species relative to VIPERFAV, reflective of its more complex antivenom immunization mixture. In contrast, ViperaTAb was extremely potent in neutralizing V. berus but, reflective of this being a monovalent antivenom, it was not effective against other Vipera species. The enzyme inhibitor prinomastat efficiently neutralized the metalloprotease-driven Factor X activation of the procoagulant venoms. In contrast, DMPS (2,3-dimercapto-1-propanesulfonic acid), which as been suggested as another potential treatment option in the absence of antivenom, DMPS failed against all venoms tested. Overall, our results highlight the evolutionary variations within Palearctic vipers and help to inform clinical management of viper envenomation., Competing Interests: ML was employed by the company Ophirex, MA by Micropharm, and RS by Inosan Biopharma, all of which made products tested in this manuscript. However, the companies had no input in experimental design or reviewing of results before publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chowdhury, Zdenek, Lewin, Carter, Jagar, Ostanek, Harjen, Aldridge, Soria, Haw and Fry.)

Species within the viperid genus Macrovipera are some of the most dangerous snakes in the Eurasian region, injecting copious amounts of potent venom. Despite their medical importance, the pathophysiological actions of their venoms have been neglected. Particularly poorly known are the coagulotoxic effects and thus the underlying mechanisms of lethal coagulopathy. In order to fill this knowledge gap, we ascertained the effects of venom upon human plasma for Macrovipera lebetina cernovi, M. l. lebetina, M. l. obtusa, M. l. turanica, and M. schweizeri using diverse coagulation analysing protocols. All five were extremely potent in their ability to promote clotting but varied in their relative activation of Factor X, being equipotent in this study to the venom of the better studied, and lethal, species Daboia russelii. The Insoserp European viper antivenom was shown to be highly effective against all the Macrovipera venoms, but performed poorly against the D. russelii venom. Reciprocally, while Daboia antivenoms performed well against D. russelii venom, they failed against Macrovipera venom. Thus despite the two genera sharing a venom phenotype (Factor X activation) driven by the same toxin type (P-IIId snake venom metalloproteases), the surface biochemistries of the toxins differed significantly enough to impede antivenom cross- neutralization. The differences in venom biochemistry were reflected in coagulation co-factor dependence. While both genera were absolutely dependent upon calcium for the activation of Factor X, dependence upon phospholipid varied. The Macrovipera venoms had low levels of dependence upon phospholipid while the Daboia venom was three times more dependent upon phospholipid for the activation of Factor X. This suggests that the sites on the molecular surface responsible for phospholipid dependence, are the same differential sites that prevent inter-genera antivenom cross- neutralization. Due to cold-chain requirements, antivenoms may not be stocked in rural settings where the need is at the greatest. Thus we tested the efficacy of enzyme inhibitor Prinomastat as a field-deployable treatment to stabilise patients while being transported to antivenom stocks, and showed that it was extremely effective in blocking the Factor X activating pathophysiological actions. Marimastat however was less effective. These results thus not only shed light on the coagulopathic mechanisms of Macrovipera venoms, but also provide data critical for evidence-based design of snakebite management strategies., Competing Interests: Declaration of Competing Interest The authors declare that they have no other known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.’, (Copyright © 2021 Elsevier B.V. All rights reserved.)

Rattlesnakes are a diverse clade of pit vipers (snake family Viperidae, subfamily Crotalinae) that consists of numerous medically significant species. We used validated in vitro assays measuring venom-induced clotting time and strength of any clots formed in human plasma and fibrinogen to assess the coagulotoxic activity of the four medically relevant Mexican rattlesnake species Crotalus culminatus, C. mictlantecuhtli, C. molossus , and C. tzabcan . We report the first evidence of true procoagulant activity by Neotropical rattlesnake venom in Crotalus culminatus . This species presented a strong ontogenetic coagulotoxicity dichotomy: neonates were strongly procoagulant via Factor X activation, whereas adults were pseudo-procoagulant in that they converted fibrinogen into weak, unstable fibrin clots that rapidly broke down, thereby likely contributing to net anticoagulation through fibrinogen depletion. The other species did not activate clotting factors or display an ontogenetic dichotomy, but depleted fibrinogen levels by cleaving fibrinogen either in a destructive (non-clotting) manner or via a pseudo-procoagulant mechanism. We also assessed the neutralization of these venoms by available antivenom and enzyme-inhibitors to provide knowledge for the design of evidence-based treatment strategies for envenomated patients. One of the most frequently used Mexican antivenoms (Bioclon Antivipmyn®) failed to neutralize the potent procoagulant toxic action of neonate C. culminatus venom, highlighting limitations in snakebite treatment for this species. However, the metalloprotease inhibitor Prinomastat substantially thwarted the procoagulant venom activity, while 2,3-dimercapto-1-propanesulfonic acid (DMPS) was much less effective. These results confirm that venom-induced Factor X activation (a procoagulant action) is driven by metalloproteases, while also suggesting Prinomastat as a more promising potential adjunct treatment than DMPS for this species (with the caveat that in vivo studies are necessary to confirm this potential clinical use). Conversely, the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) inhibited the direct fibrinogen cleaving actions of C. mictlantecuhtli venom, thereby revealing that the pseudo-procoagulant action is driven by kallikrein-type serine proteases. Thus, this differential ontogenetic variation in coagulotoxicity patterns poses intriguing questions. Our results underscore the need for further research into Mexican rattlesnake venom activity, and also highlights potential limitations of current antivenom treatments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Seneci, Zdenek, Chowdhury, Rodrigues, Neri-Castro, Bénard-Valle, Alagón and Fry.)

Bitis are well known for being some of the most commonly encountered and medically important snake species in all of Africa. While the majority of species possess potently anticoagulant venom, only B. worthingtoni is known to possess procoagulant venom. Although known to be the basal species within the genus, B. worthingtoni is an almost completely unstudied species with even basic dietary information lacking. This study investigated various aspects of the unique procoagulant effects of B. worthingtoni venom. Coagulation assays determined the primary procoagulant effect to be driven by Factor X activating snake venom metalloprotease toxins. In addition to acting upon the mammalian blood clotting cascade, B. worthingtoni venom was also shown to clot amphibian plasma. As previous studies have shown differences in clotting factors between amphibian and mammalian plasmas, individual enzymes in snake venoms acting on plasma clotting factors can be taxon-selective. As venoms evolve under purifying selection pressures, this suggests that the procoagulant snake venom metalloprotease toxins present in B. worthingtoni have likely been retained from a recent common ancestor shared with the related amphibian-feeding Proatheris superciliaris, and that both amphibians and mammals represent a substantial proportion of B. worthingtoni current diet. Thus, taxon-specific actions of venoms may have utility in inferring dietary composition for rare or difficult to study species. An important caveat is that to validate this hypothesis field studies investigating the dietary ecology of B. worthingtoni must be conducted, as well as further investigations of its venom composition to reconstruct the molecular evolutionary history of the toxins present., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Snakebite is a neglected tropical disease with a massive global burden of injury and death. The best current treatments, antivenoms, are plagued by a number of logistical issues that limit supply and access in remote or poor regions. We explore the anticoagulant properties of venoms from the genus Micrurus (coral snakes), which have been largely unstudied, as well as the effectiveness of antivenom and a small-molecule phospholipase inhibitor-varespladib-at counteracting these effects. Our in vitro results suggest that these venoms likely interfere with the formation or function of the prothrombinase complex. We find that the anticoagulant potency varies widely across the genus and is especially pronounced in M. laticollaris. This variation does not appear to correspond to previously described patterns regarding the relative expression of the three-finger toxin and phospholipase A 2 (PLA 2 ) toxin families within the venoms of this genus. The coral snake antivenom Coralmyn, is largely unable to ameliorate these effects except for M. ibiboboca. Varespladib on the other hand completely abolished the anticoagulant activity of every venom. This is consistent with the growing body of results showing that varespladib may be an effective treatment for a wide range of toxicity caused by PLA 2 toxins from many different snake species. Varespladib is a particularly attractive candidate to help alleviate the burden of snakebite because it is an approved drug that possesses several logistical advantages over antivenom including temperature stability and oral availability., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The toxin composition of snake venoms and, thus, their functional activity, can vary between and within species. Intraspecific venom variation across a species' geographic range is a major concern for antivenom treatment of envenomations, particularly for countries like French Guiana that lack a locally produced antivenom. Bothrops asper and Bothrops atrox are the most medically significant species of snakes in Latin America, both producing a variety of clinical manifestations, including systemic bleeding. These pathophysiological actions are due to the activation by the venom of the blood clotting factors Factor X and prothrombin, thereby causing severe consumptive coagulopathy. Both species are extremely wide-ranging, and previous studies have shown their venoms to exhibit regional venom variation. In this study, we investigate the differential coagulotoxic effects on human plasma of six venoms (four B. asper and two B. atrox samples) from different geographic locations, spanning from Mexico to Peru. We assessed how the venom variation of these venom samples affects neutralisation by five regionally available antivenoms: Antivipmyn, Antivipmyn-Tri, PoliVal-ICP, Bothrofav, and Soro Antibotrópico (SAB). The results revealed both inter- and intraspecific variations in the clotting activity of the venoms. These variations in turn resulted in significant variation in antivenom efficacy against the coagulotoxic effects of these venoms. Due to variations in the venoms used in the antivenom production process, antivenoms differed in their species-specific or geographical neutralisation capacity. Some antivenoms (PoliVal-ICP, Bothrofav, and SAB) showed species-specific patterns of neutralisation, while another antivenom (Antivipmyn) showed geographic-specific patterns of neutralisation. This study adds to current knowledge of Bothrops venoms and also illustrates the importance of considering evolutionary biology when developing antivenoms. Therefore, these results have tangible, real-world implications by aiding evidence-based design of antivenoms for treatment of the envenomed patient. We stress that these in vitro studies must be backed by future in vivo studies and clinical trials before therapeutic guidelines are issued regarding specific antivenom use in a clinical setting.

Bothrops (lance-head pit vipers) venoms are rich in weaponised metalloprotease enzymes (SVMP). These toxic enzymes are structurally diverse and functionally versatile. Potent coagulotoxicity is particularly important for prey capture (via stroke-induction) and relevant to human clinical cases (due to consumption of clotting factors including the critical depletion of fibrinogen). In this study, three distinct isoforms of P-III class SVMPs (IC, IIB and IIC), isolated from Bothrops neuwiedi venom, were evaluated for their differential capacities to affect hemostasis of prey and human plasma. Furthermore, we tested the relative antivenom neutralisation of effects upon human plasma. The toxic enzymes displayed differential procoagulant potency between plasma types, and clinically relevant antivenom efficacy variations were observed. Of particular importance was the confirmation the antivenom performed better against prothrombin activating toxins than Factor X activating toxins, which is likely due to the greater prevalence of the former in the immunising venoms used for antivenom production. This is clinically relevant as the enzymes displayed differential potency in this regard, with one (IC) in particular being extremely potent in activating Factor X and thus was correspondingly poorly neutralised. This study broadens the current understanding about the adaptive role of the SVMPs, as well as highlights how the functional diversity of SVMP isoforms can influence clinical outcomes. Key Contribution: Our findings shed light upon the hemorrhagic and coagulotoxic effects of three SVMPs of the P-III class, as well as the coagulotoxic effects of SVMPs on human, avian and amphibian plasmas. Antivenom neutralised prothrombin-activating isoforms better than Factor X activating isoforms., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The evolution of an aquatic lifestyle from land dwelling venomous elapids is a radical ecological modification, bringing about many evolutionary changes from morphology to diet. Diet is an important ecological facet which can play a key role in regulating functional traits such as venom composition and prey-specific targeting of venom. In addition to predating upon novel prey (e.g., fish, fish eggs and invertebrates), the venoms of aquatic elapids also face the challenge of increased prey-escape potential in the aquatic environment. Thus, despite the independent radiation into an aquatic niche on four separate occasions, the venoms of aquatic elapids are evolving under convergent selection pressures. Utilising a biolayer interferometry binding assay, this study set out to elucidate whether crude venoms from representative aquatic elapids were target-specific to the orthosteric site of postsynaptic nicotinic acetylcholine receptor mimotopes of fish compared to other terrestrial prey types. Representatives of the four aquatic lineages were: aquatic coral snakes representative was Micrurus surinamensis ;, sea kraits representative was Laticauda colubrina; sea snakes representatives were two Aipysurus spp. and eight Hydrophis spp; and water cobras representative was Naja annulata . No prey-specific differences in crude venom binding were observed from any species tested, except for Aipysurus laevis, which showed slight evidence of prey-potency differences. For Hydrophis caerulescens , H. peronii , H. schistosus and M. surinamensis , there was a lack of binding to the orthosteric site of any target lineage. Subsequent testing on the in vitro chick-biventer cervicis muscle preparation suggested that, while the venoms of these species bound postsynaptically, they bound to allosteric sites rather than orthosteric. Allosteric binding is potentially a weaker but faster-acting form of neurotoxicity and we hypothesise that the switch to allosteric binding is likely due to selection pressures related to prey-escape potential. This research has potentially opened up the possibility of a new functional class of toxins which have never been assessed previously while shedding light on the selection pressures shaping venom evolution.

Snakebite is a common occurrence for pet cats and dogs worldwide and can be fatal. In Australia the eastern brown snake (Pseudonaja textilis) is responsible for an estimated 76% of reported snakebite cases to domestic pets nationally each year, with the primary pathology being venom-induced consumptive coagulopathy. While only 31% of dogs survive P. textilis bites without antivenom, cats are twice as likely to survive bites (66%). Even with antivenom treatment, cats have a significantly higher survival rate. The reason behind this disparity is unclear. Using a coagulation analyser (Stago STA R Max), we tested the relative procoagulant effects of P. textilis venom-as well as 10 additional procoagulant venoms found around the world-on cat and dog plasma in vitro, as well as on human plasma for comparison. All venoms acted faster upon dog plasma than cat or human, indicating that dogs would likely enter coagulopathic states sooner, and are thus more vulnerable to procoagulant snake venoms. The spontaneous clotting time (recalcified plasma with no venom added) was also substantially faster in dogs than in cats, suggesting that the naturally faster clotting blood of dogs predisposes them to being more vulnerable to procoagulant snake venoms. This is consistent with clinical records showing more rapid onset of symptoms and lethal effects in dogs than cats. Several behavioural differences between cats and dogs are also highly likely to disproportionately negatively affect prognosis in dogs. Thus, compared to cats, dogs require earlier snakebite first-aid and antivenom to prevent the onset of lethal venom effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ecological variability among closely related species provides an opportunity for evolutionary comparative studies. Therefore, to investigate the origin and evolution of neurotoxicity in Asian viperid snakes, we tested the venoms of Azemiops feae, Calloselasma rhodostoma, Deinagkistrodon acutus, Tropidolaeums subannulatus, and T. wagleri for their relative specificity and potency upon the amphibian, lizard, bird, rodent, and human α-1 (neuromuscular) nicotinic acetylcholine receptors. We utilised a biolayer interferometry assay to test the binding affinity of these pit viper venoms to orthosteric mimotopes of nicotinic acetylcholine receptors binding region from a diversity of potential prey types. The Tropidolaemus venoms were much more potent than the other species tested, which is consistent with the greater prey escape potential in arboreal niches. Intriguingly, the venom of C. rhodostoma showed neurotoxic binding to the α-1 mimotopes, a feature not known previously for this species. The lack of prior knowledge of neurotoxicity in this species is consistent with our results due to the bias in rodent studies and human bite reports, whilst this venom had a greater binding affinity toward amphibian and diapsid α-1 targets. The other large terrestrial species, D. acutus, did not display any meaningful levels of neurotoxicity. These results demonstrate that whilst small peptide neurotoxins are a basal trait of these snakes, it has been independently amplified on two separate occasions, once in Azemiops and again in Tropidolaemus, and with Calloselasma representing a third possible amplification of this trait. These results also point to broader sources of novel neuroactive peptides with the potential for use as lead compounds in drug design and discovery.

Contralaterally positioned maxillary (upper jaw) venom glands in snakes are mechanically independent, being able to discharge venom from either gland separately. This has led some studies to test venom function and composition of each contralaterally positioned venom gland to investigate any differences. However, the data on the subject to-date derives from limited sample sizes, appearing somewhat contradictory, and thus still remains inconclusive. Here, we tested samples obtained from the left and right venom glands of four N. siamensis specimens for their relative binding to the orthosteric site of amphibian, lizard, snake, bird, and rodent alpha-1 nicotinic acetylcholine receptors. We also show the relative proteomic patterns displayed by reversed phase liquid chromatography - mass spectrometry. Our results indicate that three of the venom gland sets showed no difference in both functional binding and composition, whilst one venom gland set showed a slight difference in functional binding (but not in specificity patterns between prey types) or venom composition. We hypothesise that these differences in functional binding may be due to one gland having previously ejected venom at some time prior to venom extraction, whilst its contralateral counterpart did not. This might cause the differential rate of toxin replenishment to be unequal between glands, thus instigating the difference in potency, likely due to uneven toxin proportions between glands at the time of venom extraction. These results demonstrate that the separate venom producing glands in snakes remain under the same genetic control elements and produce identical venom components., Competing Interests: The authors declare no conflicts of interest., (© 2020 The Author(s).)

Does the venom of Trimeresurus albolabris (white-lipped pit viper) differ between neonate and adults? This species is responsible for most snakebites within south and southeast Asia, yet it is unknown whether ontogenetic variation in venom composition occurs in this species, or how this might affect antivenom efficacy. Using a coagulation analyser robot, we examined the anticoagulant activity of T. albolabris venom from eight individuals across multiple age classes. We then compared the efficacy of Thai Red Cross Green Pit Viper Antivenom across these age classes. Venoms from all age classes were equally potent in their pseudo-procoagulant, fibrinogenolytic activity, in that fibrinogen was cleaved to form weak, unstable fibrin clots that rapidly broke down, thus resulting in a net anticoagulant state. Similarly, this coagulotoxic activity was well neutralised by antivenom across all venoms. Given that coagulotoxicity is the primary serious pathology in T. albolabris envenomations, we conclude that Thai Red Cross Green Tree Pit Viper Antivenom is a valid treatment for envenomations by this species, regardless of age or sex of the offending snake. These results are relevant for clinical treatment of envenomations by T. albolabris., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Venoms from Pseudechis species (Australian black snakes) within the Elapidae family are rich in anticoagulant PLA 2 toxins, with the exception of one species (P. porphyriacus) that possesses procoagulant mutated forms of the clotting enzyme Factor Xa. Previously the mechanism of action of the PLA 2 toxins' anticoagulant toxicity was said to be due to inhibition of Factor Xa, but this statement was evidence free. We conducted a series of anticoagulation assays to elucidate the mechanism of anticoagulant action produced by P. australis venom. Our results revealed that, rather than targeting FXa, the PLA 2 toxins inhibited the prothrombinase complex, with FVa-alone or as part of the prothrombinase complex-as the primary target; but with significant thrombin inhibition also noted. In contrast, FXa, and other factors inhibited only to a lesser degree were minor targets. We quantified coagulotoxic effects upon human plasma caused by all nine anticoagulant Pseudechis species, including nine localities of P. australis across Australia, and found similar anticoagulant potency across all Pseudechis species, with greater potency in P. australis and the undescribed Pseudechis species in the NT. In addition, the northern localities and eastern of P. australis were significantly more potent than the central, western, and southern localities. All anticoagulant venoms responded well to Black Snake Antivenom, except P. colletti which was poorly neutralised by Black Snake Antivenom and also Tiger Snake Antivenom (the prescribed antivenom for this species). However, we found LY315920 (trade name: Varespladib), a small molecule inhibitor of PLA 2 proteins, exhibited strong potency against P. colletti venom. Thus, Varespladib may be a clinically viable treatment for anticoagulant toxicity exerted by this species that is not neutralised by available antivenoms. Our results provide insights into coagulotoxic venom function, and suggest future in vivo work be conducted to progress the development of a cheaper, first-line treatment option to treat PLA 2 -rich snake venoms globally., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Prey-selective venoms and toxins have been documented across only a few species of snakes. The lack of research in this area has been due to the absence of suitably flexible testing platforms. In order to test more species for prey specificity of their venom, we used an innovative taxonomically flexible, high-throughput biolayer interferometry approach to ascertain the relative binding of 29 α-neurotoxic venoms from African and Asian elapid representatives (26 Naja spp., Aspidelaps scutatus , Elapsoidea boulengeri, and four locales of Ophiophagus hannah ) to the alpha-1 nicotinic acetylcholine receptor orthosteric (active) site for amphibian, lizard, snake, bird, and rodent targets. Our results detected prey-selective, intraspecific, and geographical differences of α-neurotoxic binding. The results also suggest that crude venom that shows prey selectivity is likely driven by the proportions of prey-specific α-neurotoxins with differential selectivity within the crude venom. Our results also suggest that since the α-neurotoxic prey targeting does not always account for the full dietary breadth of a species, other toxin classes with a different pathophysiological function likely play an equally important role in prey immobilisation of the crude venom depending on the prey type envenomated. The use of this innovative and taxonomically flexible diverse assay in functional venom testing can be key in attempting to understanding the evolution and ecology of α-neurotoxic snake venoms, as well as opening up biochemical and pharmacological avenues to explore other venom effects.

Envenomations are complex medical emergencies that can have a range of symptoms and sequelae. The only specific, scientifically-validated treatment for envenomation is antivenom administration, which is designed to alleviate venom effects. A paucity of efficacy testing exists for numerous antivenoms worldwide, and understanding venom effects and venom potency can help identify antivenom improvement options. Some spider venoms can produce debilitating injuries or even death, yet have been largely neglected in venom and antivenom studies because of the low venom yields. Coagulation disturbances have been particularly under studied due to difficulties in working with blood and the coagulation cascade. These circumstances have resulted in suboptimal spider bite treatment for medically significant spider genera such as Loxosceles and Sicarius . This study identifies and quantifies the anticoagulant effects produced by venoms of three Loxoscles species ( L. reclusa , L. boneti, and L. laeta ) and that of Sicarius terrosus . We showed that the venoms of all studied species are able to cleave the fibrinogen Aα-chain with varying degrees of potency, with L. reclusa and S. terrosus venom cleaving the Aα-chain most rapidly. Thromboelastography analysis revealed that only L. reclusa venom is able to reduce clot strength, thereby presumably causing anticoagulant effects in the patient. Using the same thromboelastography assays, antivenom efficacy tests revealed that the commonly used Loxoscles- specific SMase D recombinant based antivenom failed to neutralize the anticoagulant effects produced by Loxosceles venom. This study demonstrates the fibrinogenolytic activity of Loxosceles and Sicarius venom and the neutralization failure of Loxosceles antivenom, thus providing impetus for antivenom improvement., Competing Interests: The authors declare no conflict of interest.

Australian elapid snakes are some of the most venomous snakes in the world and are unique among venomous snakes in having mutated forms of the blood clotting factor X in an activated form (FXa) as a key venom component. In human bite victims, an overdose of this activated clotting enzyme results in the systemic consumption of fibrinogen due to the large amounts of endogenous thrombin generated by the conversion of prothrombin to thrombin by venom FXa. Within Australian elapids, such procoagulant venom is currently known from the tiger snake clade (Hoplocephalus, Notechis, Paroplocephalus, and Tropidechis species), brown/taipan (Oxyuranus and Pseudonaja species) clade, and the red-bellied black snake Pseudechis porphyriacus. We used a STA-R Max coagulation analyser and TEG5000 thromboelastographers to test 47 Australian elapid venoms from 19 genera against human plasma in vitro. In addition to activity being confirmed in the two clades above, FXa-driven potent procoagulant activity was found in four additional genera (Cryptophis, Demansia, Hemiaspis, and Suta). Ontogenetic changes in procoagulant function was also identified as a feature of Suta punctata venom. Phylogenetic analysis of FX sequences confirmed that snake venom FXa toxins evolved only once, that the potency of these toxins against human plasma has increased in a stepwise fashion, and that multiple convergent amplifications of procoagulant activity within Australian elapid snakes have occurred. Cofactor dependence tests revealed all procoagulant venoms in our study, except those of the tiger snake clade, to be highly calcium-dependent, whereas phospholipid dependence was less of a feature but still displayed significant variation between venoms. Antivenom testing using CSL Tiger Snake Antivenom showed broad but differential cross-reactivity against procoagulant venoms, with P. porphyriacus and S. punctata extremely well neutralised but with Cryptophis, Demansia, and Hemiaspis less well-neutralised. The relative variation was not in accordance to genetic relatedness of the species used in antivenom production (Notechis scutatus), which underscores a fundamental principle that the rapid evolution characteristic of venoms results in organismal phylogeny being a poor predictor of antivenom efficacy. Our results have direct and immediate implications for the design of clinical management plans in the event of snakebite by such lesser known Australian elapid snake species that have been revealed in this study to be as potent as the better studied, and proven lethal, species., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

Envenomations by venomous snakes have major public health implications on a global scale. Despite its medical importance, snakebite has long been a neglected tropical disease by both governments and medical science. Many aspects of the resulting pathophysiology have been largely under-investigated. Most research on snake venom has focused on the neurological effects, with coagulotoxicity being relatively neglected, especially for venoms in the Elapidae snake family. In order to fill the knowledge gap regarding the coagulotoxic effects of elapid snake venoms, we performed functional activity tests to determine the fibrinogenolytic activity of 29 African and Asian elapid venoms across eight genera. The results of this study revealed that destructive (non-clotting) fibrinogenolytic activity is widespread across the African and Asian elapids. This trait evolved independently twice: once in the Hemachatus/Naja last common ancestor and again in Ophiophagus. Further, within Naja this trait was amplified on several independent occasions and possibly explains some of the clinical symptoms produced by these species. Species within the Hemachatus/Naja with fibrinogenolytic activity only cleaved the Aα-chain of fibrinogen, whereas Ophiophagus venoms degraded both the Aα- and the Bβ-chain of fibrinogen. All other lineages tested in this study lacked significant fibrinogenolytic effects. Our systematic research across Afro-Asian elapid snake venoms helps shed light on the various molecular mechanisms that are involved in coagulotoxicity within Elapidae., (Copyright © 2019. Published by Elsevier Ltd.)

Snakebite is a neglected tropical disease that disproportionately affects the poor. Antivenom is the only specific and effective treatment for snakebite, but its distribution is severely limited by several factors, including the prohibitive cost of some products. Papua New Guinea (PNG) is a snakebite hotspot but the high costs of Australian antivenoms (thousands of dollars per treatment) makes it unaffordable in PNG. A more economical taipan antivenom has recently been developed at the Instituto Clodomiro Picado (ICP) in Costa Rica for PNG and is currently undergoing clinical trials for the treatment of envenomations by coastal taipans (Oxyuranus scutellatus). In addition to potentially having the capacity to neutralise the effects of envenomations of non-PNG taipans, this antivenom may have the capacity to neutralise coagulotoxins in venom from closely related brown snakes (Pseudonaja spp.) also found in PNG. Consequently, we investigated the cross-reactivity of taipan antivenom across the venoms of all Oxyuranus and Pseudonaja species. In addition, to ascertain differences in venom biochemistry that influence variation in antivenom efficacy, we tested for relative cofactor dependence. We found that the new ICP taipan antivenom exhibited high selectivity for Oxyuranus venoms and only low to moderate cross-reactivity with any Pseudonaja venoms. Consistent with this genus level distinction in antivenom efficacy were fundamental differences in the venom biochemistry. Not only were the Pseudonaja venoms significantly more procoagulant, but they were also much less dependent upon the cofactors calcium and phospholipid. There was a strong correlation between antivenom efficacy, clotting time and cofactor dependence. This study sheds light on the structure-function relationships of the procoagulant toxins within these venoms and may have important clinical implications including for the design of next-generation antivenoms., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The genus Bitis 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 Bitis (B. arietans and again in the common ancestor of the clade consisting of B. gabonica, B. nasicornis, B. parviocula and B. rhinoceros ) induce an array of debilitating effects including anticoagulation, hemorrhagic shock and cytotoxicity, whilst the dwarf species B. atropos 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 B. worthingtoni, suggesting anticoagulant venom is a derived trait within the Bitis 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 Bitis and the action of inhibiting the prothrombinase complex, which is present in a clade of dwarf Bitis . Antivenom studies revealed that while the procoagulant effects of B. worthingtoni 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 Bitis genus and that clinical management strategies are limited for the procoagulant effects of B. worthingtoni , 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.

The functional activities of Anguimorpha lizard venoms have received less attention compared to serpent lineages. Bite victims of varanid lizards often report persistent bleeding exceeding that expected for the mechanical damage of the bite. Research to date has identified the blockage of platelet aggregation as one bleeding-inducing activity, and destructive cleavage of fibrinogen as another. However, the ability of the venoms to prevent clot formation has not been directly investigated. Using a thromboelastograph (TEG5000), clot strength was measured after incubating human fibrinogen with Heloderma and Varanus lizard venoms. Clot strengths were found to be highly variable, with the most potent effects produced by incubation with Varanus venoms from the Odatria and Euprepriosaurus clades. The most fibrinogenolytically active venoms belonged to arboreal species and therefore prey escape potential is likely a strong evolutionary selection pressure. The results are also consistent with reports of profusive bleeding from bites from other notably fibrinogenolytic species, such as V. giganteus . Our results provide evidence in favour of the predatory role of venom in varanid lizards, thus shedding light on the evolution of venom in reptiles and revealing potential new sources of bioactive molecules useful as lead compounds in drug design and development.

Due to their potent coagulotoxicity, Australian elapid venoms are unique relative to non-Australian members of the Elapidae snake family. The majority of Australian elapids possess potent procoagulant venom, while only a few species have been identified as possessing anticoagulant venoms. The majority of research to-date has concentrated on large species with range distributions overlapping major city centres, such as brown snakes (Pseudonaja spp.) and taipans (Oxyuranus spp.). We investigated the venom from the poorly studied genus Denisonia and documented anticoagulant activities that were differentially potent on amphibian, avian, and human plasmas. Both species were potently anticoagulant upon amphibian plasma, consistent with these snakes preying upon frogs as their primary food source. While D. devisi was only relatively weakly active on avian and human plasma, D. maculata was potently anticoagulant to amphibian, avian, and human plasma. The mechanism of anticoagulant action was determined to be the inhibition of prothrombin activation by Factor Xa by blocking the formation of the prothrombinase complex. Fractionation of D. maculata venom followed by MS sequencing revealed that the toxins responsible were Group I phospholipase A 2 . As no antivenom is produced for this species or its near relatives, we examined the ability of Seqirus Australian snake polyvalent antivenom to neutralise the anticoagulant effects, with this antivenom shown to be effective. These results contribute to the body of knowledge regarding adaptive evolution of venom, revealing a unique taxon-specific anticoagulant effect for D. devisi venom. These results also reveal the potential effects and mechanisms behind envenomation by the potently acting D. maculata venom on human plasma, while the discovery of the efficacy of an available antivenom provides information crucial to the design of snakebite management strategies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Snakebite is a global tropical disease that has long had huge implications for human health and well-being. Despite its long-standing medical importance, it has been the most neglected of tropical diseases. Reflective of this is that many aspects of the pathology have been underinvestigated. Snakebite by species in the Elapidae family is typically characterised by neurotoxic effects that result in flaccid paralysis. Thus, while clinically significant disturbances to the coagulation cascade have been reported, the bulk of the research to date has focused upon neurotoxins. In order to fill the knowledge gap regarding the coagulotoxic effects of elapid snake venoms, we screened 30 African and Asian venoms across eight genera using in vitro anticoagulant assays to determine the relative inhibition of the coagulation function of thrombin and the inhibition of the formation of the prothrombinase complex through competitive binding to a nonenzymatic site on Factor Xa (FXa), thereby preventing FXa from binding to Factor Va (FVa). It was revealed that African spitting cobras were the only species that were potent inhibitors of either clotting factor, but with Factor Xa inhibited at 12 times the levels of thrombin inhibition. This is consistent with at least one death on record due to hemorrhage following African spitting cobra envenomation. To determine the efficacy of antivenom in neutralising the anticoagulant venom effects, for the African spitting cobras we repeated the same 8-point dilution series with the addition of antivenom and observed the shift in the area under the curve, which revealed that the antivenom performed extremely poorly against the coagulotoxic venom effects of all species. However, additional tests with the phospholipase A₂ inhibitor LY315920 (trade name: varespladib) demonstrated a powerful neutralisation action against the coagulotoxic actions of the African spitting cobra venoms. Our research has important implications for the clinical treatment of cobra snakebites and also sheds light on the molecular mechanisms involved in coagulotoxicity within Naja . As the most coagulotoxic species are also those that produce characteristic extreme local tissue damage, future research should investigate potential synergistic actions between anticoagulant toxins and cytotoxins., Competing Interests: The authors declare no conflict of interest.