Solenodon genome reveals convergent evolution of venom in eulipotyphlan mammals.

Venom systems are key adaptations that have evolved throughout the tree of life and typically facilitate predation or defense. Despite venoms being model systems for studying a variety of evolutionary and physiological processes, many taxonomic groups remain understudied, including venomous mammals. Within the order Eulipotyphla, multiple shrew species and solenodons have oral venom systems. Despite morphological variation of their delivery systems, it remains unclear whether venom represents the ancestral state in this group or is the result of multiple independent origins. We investigated the origin and evolution of venom in eulipotyphlans by characterizing the venom system of the endangered Hispaniolan solenodon (Solenodon paradoxus). We constructed a genome to underpin proteomic identifications of solenodon venom toxins, before undertaking evolutionary analyses of those constituents, and functional assessments of the secreted venom. Our findings show that solenodon venom consists of multiple paralogous kallikrein 1 (KLK1) serine proteases, which cause hypotensive effects in vivo, and seem likely to have evolved to facilitate vertebrate prey capture. Comparative analyses provide convincing evidence that the oral venom systems of solenodons and shrews have evolved convergently, with the 4 independent origins of venom in eulipotyphlans outnumbering all other venom origins in mammals. We find that KLK1s have been independently coopted into the venom of shrews and solenodons following their divergence during the late Cretaceous, suggesting that evolutionary constraints may be acting on these genes. Consequently, our findings represent a striking example of convergent molecular evolution and demonstrate that distinct structural backgrounds can yield equivalent functions.

Three-Finger Toxin Diversification in the Venoms of Cat-Eye Snakes (Colubridae: Boiga).

The Asian genus Boiga (Colubridae) is among the better studied non-front-fanged snake lineages, because their bites have minor, but noticeable, effects on humans. Furthermore, B. irregularis has gained worldwide notoriety for successfully invading Guam and other nearby islands with drastic impacts on the local bird populations. One of the factors thought to allow B. irregularis to become such a noxious pest is irditoxin, a dimeric neurotoxin composed of two three-finger toxins (3FTx) joined by a covalent bond between two newly evolved cysteines. Irditoxin is highly toxic to diapsid (birds and reptiles) prey, but roughly 1000 × less potent to synapsids (mammals). Venom plays an important role in the ecology of all species of Boiga, but it remains unknown if any species besides B. irregularis produce irditoxin-like dimeric toxins. In this study, we use transcriptomic analyses of venom glands from five species [B. cynodon, B. dendrophila dendrophila, B. d. gemmicincta, B. irregularis (Brisbane population), B. irregularis (Sulawesi population), B. nigriceps, B. trigonata] and proteomic analyses of B. d. dendrophila and a representative of the sister genus Toxicodryas blandingii to investigate the evolutionary history of 3FTx within Boiga and its close relative. We found that 92.5% of Boiga 3FTx belong to a single clade which we refer to as denmotoxin-like because of the close relation between these toxins and the monomeric denmotoxin according to phylogenetic, sequence clustering, and protein similarity network analyses. We show for the first time that species beyond B. irregularis secrete 3FTx with additional cysteines in the same position as both the A and B subunits of irditoxin. Transcripts with the characteristic mutations are found in B. d. dendrophila, B. d. gemmicincta, B. irregularis (Brisbane population), B. irregularis (Sulawesi population), and B. nigriceps. These results are confirmed by proteomic analyses that show direct evidence of dimerization within the venom of B. d. dendrophila, but not T. blandingii. Our results also suggest the possibility of novel dimeric toxins in other genera such as Telescopus and Trimorphodon. All together, this suggests that the origin of these peculiar 3FTx is far earlier than was appreciated and their evolutionary history has been complex.

Viper Venom Botox: The Molecular Origin and Evolution of the Waglerin Peptides Used in Anti-Wrinkle Skin Cream.

The molecular origin of waglerin peptides has remained enigmatic despite their industrial application in skin cream products to paralyse facial muscles and thus reduce the incidence of wrinkles. Here we show that these neurotoxic peptides are the result of de novo evolution within the prepro region of the C-type natriuretic peptide gene in Tropidolaemus venoms, at a site distinct from the domain encoding for the natriuretic peptide. It is the same region that yielded the azemiopsin peptides from Azemiops feae, indicative of a close relationship of this toxin gene between these two genera. The precursor region for the molecular evolution is a biodiversity hotspot that has yielded other novel bioactive peptides with novel activities. We detail the diversity of components in this and other species in order to explore what characteristics enable it to be such a biodiscovery treasure trove. The unusual function of Tropidolaemus venoms may have been selected for due to evolutionary pressures brought about by a high likelihood of prey escape.

Squeezers and leaf-cutters: differential diversification and degeneration of the venom system in toxicoferan reptiles.

Although it has been established that all toxicoferan squamates share a common venomous ancestor, it has remained unclear whether the maxillary and mandibular venom glands are evolving on separate gene expression trajectories or if they remain under shared genetic control. We show that identical transcripts are simultaneously expressed not only in the mandibular and maxillary glands, but also in the enigmatic snake rictal gland. Toxin molecular frameworks recovered in this study were three-finger toxin (3FTx), CRiSP, crotamine (beta-defensin), cobra venom factor, cystatin, epididymal secretory protein, kunitz, L-amino acid oxidase, lectin, renin aspartate protease, veficolin, and vespryn. We also discovered a novel low-molecular weight disulfide bridged peptide class in pythonid snake glands. In the iguanian lizards, the most highly expressed are potentially antimicrobial in nature (crotamine (beta-defensin) and cystatin), with crotamine (beta-defensin) also the most diverse. However, a number of proteins characterized from anguimorph lizards and caenophidian snakes with hemotoxic or neurotoxic activities were recruited in the common toxicoferan ancestor and remain expressed, albeit in low levels, even in the iguanian lizards. In contrast, the henophidian snakes express 3FTx and lectin toxins as the dominant transcripts. Even in the constricting pythonid and boid snakes, where the glands are predominantly mucous-secreting, low-levels of toxin transcripts can be detected. Venom thus appears to play little role in feeding behavior of most iguanian lizards or the powerful constricting snakes, and the low levels of expression argue against a defensive role. However, clearly the incipient or secondarily atrophied venom systems of these taxa may be a source of novel compounds useful in drug design and discovery.

The sweet side of venom: Glycosylated prothrombin activating metalloproteases from Dispholidus typus (boomslang) and Thelotornis mossambicanus (twig snake).

Dispholidus typus and Thelotornis mossambicanus are closely related rear-fanged colubrid snakes that both possess strongly procoagulant venoms. However, despite similarities in overall venom biochemistry and resulting clinical manifestations, the underlying venom composition differs significantly between the two species. As a result, the only available antivenom-which is a monovalent antivenom for D. typus-has minimal cross reactivity with T. mossambicanus and is not a clinically viable option. It was hypothesised that this lack of cross reactivity is due to the additional large metalloprotease protein within T. mossambicanus venom, which may also be responsible for faster coagulation times. In this study, we found that T. mossambicanus venom is a more powerful activator of prothrombin than that of D. typus and that the SVMP transcripts from T. mossambicanus form a clade with those from D. typus. The sequences from D. typus and T. mossambicanus were highly similar in length, with the calculated molecular weights of the T. mossambicanus transcripts being significantly less than the molecular weights of some isoforms on the 1D SDS-PAGE gels. Analyses utilising degylcosylating enzymes revealed that T. mossambicanus SVMPs are glycosylated during post-translational modification, but that this does not lead to the different molecular weight bands observed in 1D SDS-PAGE gels. However, differences in glycosylation patterns may still explain some of the difference between the enzymatic activities and neutralization by antivenom that have been observed in these venoms. The results of this study provide new information regarding the treatment options for patients envenomated by T. mossambicanus as well as the evolution of these dangerous snakes.

Evolutionary Interpretations of Nicotinic Acetylcholine Receptor Targeting Venom Effects by a Clade of Asian Viperidae Snakes.

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.

Clinical implications of differential antivenom efficacy in neutralising coagulotoxicity produced by venoms from species within the arboreal viperid snake genus Trimeresurus.

Snake envenomation globally is attributed to an ever-increasing human population encroaching into snake territories. Responsible for many bites in Asia is the widespread genus Trimeresurus. While bites lead to haemorrhage, only a few species have had their venoms examined in detail. We found that Trimeresurus venom causes haemorrhaging by cleaving fibrinogen in a pseudo-procoagulation manner to produce weak, unstable, short-lived fibrin clots ultimately resulting in an overall anticoagulant effect due to fibrinogen depletion. The monovalent antivenom 'Thai Red Cross Green Pit Viper antivenin', varied in efficacy ranging from excellent neutralisation of T. albolabris venom through to T. gumprechti and T. mcgregori being poorly neutralised and T. hageni being unrecognised by the antivenom. While the results showing excellent neutralisation of some non-T. albolabris venoms (such as T. flavomaculaturs, T. fucatus, and T. macrops) needs to be confirmed with in vivo tests, conversely the antivenom failure T. hageni, and the very poor results against T. gumprechti and T. mcgregori, despite being conducted in the ideal scenario of preincubation of antivenom:venom, indicates that the likelihood of clinically relevant cross-reactivity for these species is low (T. gumprechti and T. mcgregori) to non-existent (T. hageni). These same latter three species were also not inhibited by the serine protease inhibitor AEBSF, suggesting that the toxins leading to a coagulotoxic effect in these species are non-serine proteases while in contrast T. albolabris coagulotoxicity was completely impeded by AEBSF, and thus driven by kallikrein-type serine proteases. There was a conspicuous lack of phylogenetic pattern in venom variation, with the most potent venoms (T. albolabris and T. hageni) being distant to each other on the organismal tree, and with the three most divergent and poorly neutralised venoms (T. gumprechti, T. hageni, and T. mcgregori) were also not each others closest relatives. This reinforces the paradigm that the fundamental dynamic evolution of venom results in organismal phylogeny being a poor predictor of venom potency or antivenom efficacy. This study provides a robust investigation on the differential venom effects from a wide range of Trimeresurus species on coagulation, highlighting differential fibrinogenolytic effects, while also investigating the relative antivenom neutralisation capabilities of the widely available Thai Red Cross Green Pit Viper antivenom. These results therefore have immediate, real-world implications for patients envenomed by Trimeresurus species.

Clinical implications of coagulotoxic variations in Mamushi (Viperidae: Gloydius) snake venoms.

Snake bite is currently one of the most neglected tropical diseases affecting much of the developing world. Asian pit vipers are responsible for a considerable amount of envenomations annually and bites can cause a multitude of clinical complications resulting from coagulopathic and neuropathic effects. While intense research has been undertaken for some species of Asian pit viper, functional coagulopathic effects have been neglected for others. We investigated their effects upon the human clotting cascade using venoms of four species of Gloydius and Ovophis okinavensis, a species closely to Gloydius. All species of included within this investigation displayed varying fibrinogenolytic effects, resulting in a net anticoagulant outcome. Gloydius saxatilis and Gloydius ussuriensis displayed the most variable effects from differing localities, sampled from Russia and Korea. As this Gloydius investigation includes some geographical variation, notable results indicate key variations of these species that point to possible limitations in antivenom cross-reactivities, which may have implications for the clinical care of victims envenomed by these snakes.

Habu coagulotoxicity: Clinical implications of the functional diversification of Protobothrops snake venoms upon blood clotting factors.

Venom can affect any part of the body reachable via the bloodstream. Toxins which specifically act upon the coagulation cascade do so either by anticoagulant or procoagulant mechanisms. Here we investigated the coagulotoxic effects of six species within the medically important pit viper genus Protobothrops (Habu) from the Chinese mainland and Japanese islands, a genus known to produce hemorrhagic shock in envenomed patients. Differential coagulotoxicity was revealed: P. jerdonii and P. mangshanensis produced an overall net anticoagulant effect through the pseudo-procoagulant clotting of fibrinogen; P. flavoviridis and P. tokarensis exhibit a strong anticoagulant activity through the destructive cleavage of fibrinogen; and while P. elegans and P. mucrosquamatus both cleaved the A-alpha and B-beta chains of fibrinogen they did not exhibit strong anticoagulant activity. These variations in coagulant properties were congruent with phylogeny, with the closest relatives exhibiting similar venom effects in their action upon fibrinogen. Ancestral state reconstruction indicated that anticoagulation mediated by pseudo-procoagulant cleavage of fibrinogen is the basal state, while anticoagulation produced by destructive cleavage of fibrinogen is the derived state within this genus. This is the first in depth study of its kind highlighting extreme enzymatic variability, functional diversification and clotting diversification within one genus surrounding one target site, governed by variability in co-factor dependency. The documentation that the same net overall function, anticoagulation, is mediated by differential underlying mechanics suggests limited antivenom cross-reactivity, although this must be tested in future work. These results add to the body of knowledge necessary to inform clinical management of the envenomed patient.

Basal but divergent: Clinical implications of differential coagulotoxicity in a clade of Asian vipers.

Envenomations by Asian pitvipers can induce multiple clinical complications resulting from coagulopathic and neuropathic effects. While intense research has been undertaken for some species, functional coagulopathic effects have been neglected. As these species' venoms affect the blood coagulation cascade we investigated their effects upon the human clotting cascade using venoms of species from the Azemiops, Calloselasma, Deinagkistrodon and Hypnale genera. Calloselasma rhodostoma, Deinagkistrodon acutus, and Hypnale hypnale produced net anticoagulant effects through pseudo-procoagulant clotting of fibrinogen, resulting in weak, unstable, transient fibrin clots. Tropidolaemus wagleri was only weakly pseudo-procoagulant, clotting fibrinogen with only a negligible net anticoagulant effect. Azemiops feae and Tropidolaemus subannulatus did not affect clotting. This is the first study to examine in a phylogenetic context the coagulotoxic effects of related genera of basal Asiatic pit-vipers. The results reveal substantial variation between sister genera, providing crucial information about clinical effects and implications for antivenom cross-reactivity.