Utilising venom activity to infer dietary composition of the Kenyan horned viper (Bitis worthingtoni).

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.

Widespread and Differential Neurotoxicity in Venoms from the Bitis Genus of Viperid Snakes.

Research into the neurotoxic activity of venoms from species within the snake family Viperidae is relatively neglected compared with snakes in the Elapidae family. Previous studies into venoms from the Bitis genus of vipers have identified the presence of presynaptic phospholipase A2 neurotoxins in B. atropos and B. caudalis, as well as a postsynaptic phospholipase A2 in B. arietans. Yet, no studies have investigated how widespread neurotoxicity is across the Bitis genus or if they exhibit prey selectivity of their neurotoxins. Utilising a biolayer interferometry assay, we were able to assess the binding of crude venom from 14 species of Bitis to the neuromuscular α-1 nAChR orthosteric site across a wide range of vertebrate taxa mimotopes. Postsynaptic binding was seen for venoms from B. arietans, B. armata, B. atropos, B. caudalis, B. cornuta, B. peringueyi and B. rubida. To further explore the types of neurotoxins present, venoms from the representatives B. armata, B. caudalis, B. cornuta and B. rubida were additionally tested in the chick biventer cervicis nerve muscle preparation, which showed presynaptic and postsynaptic activity for B. caudalis and only presynaptic neurotoxicity for B. cornuta and B. rubida, with myotoxicity also evident for some species. These results, combined with the biolayer interferometry results, indicate complex neurotoxicity exerted by Bitis species, which varies dramatically by lineage tested upon. Our data also further support the importance of sampling across geographical localities, as significant intraspecific variation of postsynaptic neurotoxicity was reported across the different localities.

Varespladib (LY315920) neutralises phospholipase A2 mediated prothrombinase-inhibition induced by Bitis snake venoms.

Anticoagulant toxicity is a common function of venoms produced by species within the Bitis genus. Potent inhibition of the prothrombinase complex is an identified mechanism of action for the dwarf species B. cornuta and B. xeropaga, along with some localities of B. atropos and B. caudalis. Snake venom phospholipase A2 toxins that inhibit the prothrombinase complex have been identified in snake venom, including an isolated phospholipase A2 toxin from B. caudalis. Current research is investigating the ability of the drug varespladib to inhibit snake venom phospholipase A2 toxins and reduce their toxicity. In particular, varespladib is being investigated as a treatment that could be administered prior to hospital referral which is a major necessity for species such as those from the genus Bitis, due to envenomations often occurring in remote regions of Africa where antivenom is unavailable. Using previously validated coagulation assays, this study aimed to determine if the toxins responsible for inhibition of the prothrombinase complex in the venom of four Bitis species are phospholipase A2 toxins, and if varespladib is able to neutralise this anticoagulant activity. Our results demonstrate that varespladib strongly neutralises the prothrombinase-inhibiting effects of all venoms tested in this study, and that this prothrombinase-inhibiting mechanism of anticoagulant activity is driven by phospholipase A2 class toxins in these four species. This study extends previous reports demonstrating varespladib has broad efficacy for treatment of phospholipase A2 rich snake venoms, indicating it also inhibits their anticoagulant effects mediated by prothrombinase-inhibition.

Venomous Landmines: Clinical Implications of Extreme Coagulotoxic Diversification and Differential Neutralization by Antivenom of Venoms within the Viperid Snake Genus Bitis.

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.