Solving the 'Brown snake paradox': in vitro characterisation of Australasian snake presynaptic neurotoxin activity

Pseudonaja textilis (Eastern Brown snake) and Oxyuranus scutellatus scutellatus (Coastal taipan) are clinically important Australian elapid snakes, whose potent venoms contain the presynaptic (β) neurotoxins, textilotoxin and taipoxin, respectively, and a number of postsynaptic neurotoxins. However, while taipan envenoming frequently results in neurotoxicity, Brown snake envenoming causes an isolated coagulopathy and neurotoxicity is rare. This phenomenon is called the 'Brown snake paradox'. This study compared the pharmacology of both venoms and their respective presynaptic neurotoxins to investigate this phenomenon. From size-exclusion high performance liquid chromatography (HPLC) analysis textilotoxin represents a significantly smaller proportion (5.7%) of P. textilis venom compared to taipoxin in O. s. scutellatus venom (20.4%). In the chick biventer cervicis nerve-muscle (CBCNM) preparation both venoms caused concentration-dependent neurotoxicity, with P. textilis venom being significantly more potent than O. s. scutellatus venom. Conversely, taipoxin was significantly more potent than textilotoxin when compared at the same concentration. Textilotoxin only partially contributed to the overall neurotoxicity of P. textilis venom, while taipoxin accounted for the majority of the neurotoxicity of O. s. scutellatus venom in the CBCNM preparation. Compared with taipoxin, textilotoxin is less potent and constitutes a smaller proportion of the venom. This is likely to be the reason for the absence of neurotoxicity in envenomed humans thus explaining the 'Brown snake paradox'.     

Intersexual variations in the pharmacological properties of Coremiocnemis tropix (Araneae, Theraphosidae) spider venom

This study aimed to determine the biochemical, insecticidal and neurotoxic properties of venom from both sexes of the Australian spider Coremiocnemis tropix (Araneae, Theraphosidae). Insecticidal properties were tested in crickets, while in vitro neurotoxicity was determined in an avian skeletal muscle preparation. Some intersexual differences in venom composition were identified by rp-HPLC and by LC-MS, but the majority of components were found in venoms of both sexes. Injecting the venom into crickets revealed that venom from male specimens was slightly more potent, while female venom induced more prominent effects in the chick biventer cervicis nerve-muscle preparation. The results from the chick assay suggest the presence of at least two vertebrate-active neurotoxins. A pre-synaptic neurotoxin may explain the reversible inhibition of muscle twitches and the unaffected response to nicotinic agonists at medium concentrations of female and medium to high concentrations of male venom. In addition, the presence of a neurotoxin that blocks post-synaptic nicotinic receptors might explain the irreversible inhibition of muscle twitches and the reduced response to nicotinic agonists at high concentrations (5-10 μg/ml) of venom from female specimens only.     

Identification and characterization of venom proteins of two solitary wasps, Eumenes pomiformis and Orancistrocerus drewseni

Secretory proteins were identified in the venoms of two solitary hunting wasps, Eumenes pomiformis and Orancistrocerus drewseni, by SDS-PAGE in conjunction with mass analysis. More than 30 protein bands (2-300 kDa) were detected from the crude venom of each wasp. With the aid of the previously constructed venom gland/sac-specific EST libraries, a total of 31 and 20 proteins were identified from 18 to 20 distinctive protein bands of E. pomiformis and O. drewseni venoms, respectively. Arginine kinase was the most predominant protein in both wasp venoms. Along with the full-length arginine kinase, a truncated form, which was known to have paralytic activity on a spider, was a common predominant protein in the two wasp venoms. Insulin/insulin-like peptide-binding protein was abundantly found only in E. pomiformis venom, which might be due to its unique behaviors of oviposition and provision. The presence of various immune response-related proteins and antioxidants suggested that wasps might use their venom to maintain prey fresh while feeding wasp larvae by protecting the prey from microbial invasion and physiological stresses. It seemed that some venom proteins are secreted into venom fluid from venom gland cells via exosomes, not by signal sequence-mediated transport processes.     

Effect of drugs on the lethality in mice of the venoms and neurotoxins from sundry snakes

I investigated the efficacy of 10 drugs with respect to reducing the lethality in mice of the following venoms and their respective neurotoxins: Bungarus caeruleus venom; Bungarus multicinctus venom, alpha-bungarotoxin, beta-bungarotoxin; Crotalus durissus terrificus venom, crotoxin: Notechis scutatus scutatus venom; Oxyuranus scutellatus venom, taipoxin. The drugs diltiazem, nicergoline, primaquine, verapamil and vesamicol protected mice from the lethality of B. caeruleus venom, B. multicinctus venom, and/or beta-bungarotoxin. Dexamethasone provided protection from B. multicinctus venom, beta-bungarotoxin, crotoxin, O. scutellatus venom and taipoxin. Protective activity resided in amphiphilic drugs and correlated with the charge on the drug at physiological pH. Protection from lethality was maximal when the drugs were administered immediately after injection of the venom or toxin. Nifedipine, piracetam and reserpine provided no protection from any of the venoms or toxins tested.     

Isolation of subunits, alpha, beta and gamma of the complex taipoxin from the venom of Australian taipan snake (Oxyuranus s. scutellatus): characterization of beta taipoxin as a potent mitogen.

The venom of Australian taipan snake (Oxyuranus s. scutellatus) is extremely potent due to the presence of taipoxin. The intact complex molecule of taipoxin having molecular weight 45.6 kDa is composed of alpha, beta and gamma subunits. This report describes the high pressure liquid chromatography (HPLC) separation of alpha, beta (beta-1 and beta-2) and gamma subunits from taipan crude venom. The fractions containing the taipoxin subunits were further purified to obtain homogeneous proteins. The toxicity in mice showed the alpha subunit as most toxic, the gamma subunit as moderately toxic and the beta-1 and beta-2 subunits were nontoxic. The proteins beta-1 and beta-2 were found to be mitogenic having neurotrophic activity on PC12 cells in culture similar to nerve growth factor. Immunologically, alpha, beta-1, beta-2 and gamma subunits were found to be different, showing cross reactivity, and beta-1 and beta-2 were found to be identical for biological properties and molecular weight. Further characterization of unexpected mitogenic activity of beta subunits is underway.     

Full length nucleotide sequence of a factor V-like subunit of oscutarin from Oxyuranus scutellatus scutellatus (coastal Taipan).

An Oxyuranus scutellatus scutellatus venom gland cDNA expression library was screened with antivenom. Positive clones were isolated and their nucleotide sequences determined. The complete sequence for a Factor V-like component from the Taipan venom prothrombin activator, oscutarin (EC 3.4.21.60) (Walker, F.J., Owen, W.G., Esmon, C.T., 1980. Characterization of the prothrombin activator from the venom of Oxyuranus scutellatus scutellatus (Taipan venom). Biochemistry, 19(5), 1020-1023; Speijer, H.G.R., Zwall, J., Robert, F.A., Rosing, J., 1986. Prothrombin acitvation by an activator from the venom of Oxyuranus Scutellatus (Taipan Snake). J. Biol. Chem. 261, 13258-13267) was determined from the sequencing of antigenic cDNA clones. The cDNA sequence encoded a protein of 1460 amino acid residues, including a 30-residue signal peptide. This sequence shared 95% sequence similarity with the non-enzymic subunit of the prothrombin activator (pseutarin C) from brown snake (Pseudonaja textilis) venom. This sequence in turn has been reported to share high similarity with mammalian Factor V. Sequence comparisons indicated the size, charge and cleavage sites were conserved across the two species. This is the first nucleotide sequence of a Factor V-like component from Oxyuranus venom and the second sequence within Elapidae to be reported.     

The mass of venom injected by two elapidae: the taipan (Oxyuranus scutellatus) and the Australian tiger snake (Notechis scutatus)

Using an enzyme immunoassay technique, a new method for measuring, in vivo, the mass of venom injected during snake bite, is presented. The venom injected into mice (as prey) and the venom left on the skin surface during bites by the two Australian Elapidae, the Taipan (Oxyuranus scutellatus) and the Tiger Snake (Notechus scutatus) has been measured. Venom delivery patterns vary significantly between these two species. In the case of the Tiger Snake (a total of 45 bites studied) the mean mass of venom injected in a first bite was 12.7 mg (S.E. 3.4 mg, median 8.1 mg); an average mass of 0.8 mg (S.E. 0.4 mg, median 0.17 mg) was left on the skin surface. A second bite delivered by the same snake yielded a mean venom mass only 27% of the first. In the case of the Taipan (a total of 24 bites) the mean venom mass injected in the first bite was 20.8 mg (S.E. 6.4 mg); with an average of 0.9 mg (S.E. 0.5 mg) left on the skin surface. In contrast to the situation observed with Tiger Snakes, second and third bites delivered in a rapid sequence yielded increasing masses of venom. The mean delivered in the third of a sequence of three bites was 48.8 mg (S.E. 23.8 mg). The ranges of venom mass, by species and by the sequence number of the bite, are also presented. In 66 of the 69 experimental bites studied in this report, venom could be easily detected, the species identified, and the absolute mass of venom measured.     

Comparative Studies of the Venom of a New Taipan Species,Oxyuranus temporalis,with Other Members of Its Genus

Taipans are highly venomous Australo-Papuan elapids. A new species of taipan, the Western Desert Taipan (Oxyuranus temporalis), has been discovered with two specimens housed in captivity at the Adelaide Zoo. This study is the first investigation of O. temporalis venom and seeks to characterise and compare the neurotoxicity, lethality and biochemical properties of O. temporalis venom with other taipan venoms. Analysis of O. temporalis venom using size-exclusion and reverse-phase HPLC indicated a markedly simplified “profile” compared to other taipan venoms. SDS-PAGE and agarose gel electrophoresis analysis also indicated a relatively simple composition. Murine LD₅₀ studies showed that O. temporalis venom is less lethal than O. microlepidotus venom. Venoms were tested in vitro, using the chick biventer cervicis nerve-muscle preparation. Based on t₉₀ values, O. temporalis venom is highly neurotoxic abolishing indirect twitches far more rapidly than other taipan venoms. O. temporalis venom also abolished responses to exogenous acetylcholine and carbachol, indicating the presence of postsynaptic neurotoxins. Prior administration of CSL Taipan antivenom (CSL Limited) neutralised the inhibitory effects of all taipan venoms. The results of this study suggest that the venom of the O. temporalis is highly neurotoxic in vitro and may contain procoagulant toxins, making this snake potentially dangerous to humans.     

Venom lethality and diet: Differential responses of natural prey and model organisms to the venom of the saw-scaled vipers (Echis)

The composition of snake venoms shows a high degree of variation at all taxonomic levels, and natural selection for diet has been implicated as a potential cause. Saw-scaled vipers (Echis) provide a good model for studying this phenomenon. The venoms of arthropod feeding species of Echis are significantly more toxic to natural scorpion prey than those of species which feed predominantly upon vertebrate prey. Although testing venom activity on natural prey is important for our understanding of the evolution of venom, natural prey species are often difficult to obtain in sufficient numbers for toxinological work. In order to test the viability of using cheaper and more easily available model organisms for toxicity assessments in evolutionary research, and the extent to which toxicity of arthropod-eating Echis venoms is increased to arthropods in general or targeted to certain groups, we conducted median lethal dosage (LD₅₀) and time to death trials using the desert locust (Schistocerca gregaria) as a model arthropod, rarely consumed by wild Echis. The venoms of arthropod specialist Echis were found to be significantly more toxic to locusts than the venom of a vertebrate feeding outgroup (Bitis arietans), and one arthropod specialist venom was found to be more toxic than those species which feed upon arthropods infrequently or not at all. The venoms of arthropod specialists were also found to cause death and incapacitation faster than the vertebrate feeding outgroup. Despite some similarity of trends, there are considerable differences between the response of natural prey (scorpions) and a model arthropod (locust) to the venoms of Echis species. This suggests that when possible, natural prey rather than convenient model organisms should be used to gain an understanding of the functional significance of variation in venom composition in snakes.     

Costs of venom production in the common death adder (Acanthophis antarcticus)

The utilization of venom in predatory and defensive contexts is associated with benefits regarding minimization of energetic expenditure on hunting, maximization of success in prey acquisition and avoidance of injury from dangerous prey and aggressors. Multiple characteristics suggest that venom is quite expensive to produce, thereby creating a tradeoff between advantages and disadvantages associated with its possession. The metabolic costs of venom production have rarely been studied and no information on the detailed metabolic processes during venom replenishment exists. Where costs of venom production have been studied they are often not in context with other components of the energy budget of the study organism. Using flow-through respirometry, we examined changes in metabolic rate in the Australian elapid Acanthophis antarcticus after venom expenditure and feeding as well as during preparation for shedding to establish a comparison of the magnitude of energetic expenditure during venom replenishment and other common physiological processes. We also defined the temporal pattern of metabolic processes during venom replenishment at a higher resolution than has previously been attempted in snakes. Our results suggest that total costs of venom replenishment are relatively small when compared to costs of digestion and shedding. We conclude that, in spite of the manifold factors suggesting a high cost of venom in snakes, its production is less energetically costly than often assumed. Until further research can clarify the reasons for this more caution should therefore be applied when assuming that costs of venom production exert strong selection pressures on the ecology, behavior and evolution of venomous taxa.     

In vivo and in vitro cardiovascular effects of Papuan taipan (Oxyuranus scutellatus) venom: Exploring "sudden collapse"

'Sudden collapse' following envenoming by some Australasian elapids is a poorly understood cause of mortality. We have previously shown that Oxyuranus scutellatus venom causes cardiovascular collapse in anaesthetized rats. Prior administration of a sub lethal dose of venom attenuated the response to subsequent administration of higher (lethal) venom doses. In this study, we investigated the possible mechanisms mediating this 'protective effect'. Papuan taipan venom (5μg/kg, i.v.) produced a small transient hypotension in anaesthetized rats, while 10μg/kg resulted in a 73±12% decrease in arterial pressure. Venom (20μg/kg or 50μg/kg) produced cardiovascular collapse in all animals tested (n=12). Cardiovascular collapse by 50μg/kg venom was prevented by prior administration of 'priming' doses of venom (5, 10 and 20μg/kg). Also, prior administration of indomethacin (30mg/kg, i.v.) or heparin (300units/kg, i.v.) prevented sudden collapse induced by venom (20μg/kg). Venom was without effect in isolated hearts indicating that a direct cardiac effect was unlikely to be responsible for 'sudden collapse'. Venom induced endothelium-dependent and -independent relaxation in pre-contracted rat mesenteric artery rings which was inhibited by indomethacin, IbTx and Rp-8-CPT-cAMPs. This relaxation was markedly reduced upon second exposure. Our results indicate that cardiovascular collapse induced by O. scutellatus venom may be due to a combination of release of dilator autacoids and to direct relaxation of vascular smooth muscle involving the cAMP/protein kinase A cascade. Further work will involve identification of the venom component(s) responsible for this action and may provide insight into the management of envenomed patients.     

Biological properties of the venom from the scorpionfish (Scorpaena plumieri) and purification of a gelatinolytic protease.

In this work we describe some biological properties and a partial biochemical characterization of the Scorpanea plumieri crude venom. The fresh venom induced a decrease in blood pressure, cardiac and respiratory frequency, and exhibited hemorrhagic, hemolytic and proteolytic activities. The LD(50) (i.v. mouse) was 0.28 mg/kg. The pharmacological activities were found to be very unstable and this fact could be associated with proteolytic activity. Enzymes which hydrolyze casein and gelatin were found in this venom. A gelatinolytic protease (Sp-GP) was purified to homogeneity from S. plumieri venom through a combination of three chromatographic steps: gel filtration on Sephacryl S-200; ion exchange on DEAE-cellulose and reverse-phase/HPLC on a Vydac C4 column. The purified protease was approximately 2% of the whole protein in the soluble crude venom. The molecular mass of the Sp-GP scorpionfish gelatinase estimated by SDS-PAGE was around 80,000 Da under reducing conditions and 72,000 Da under non-reducing conditions. Attempts to determine the N-terminal sequence by automatic Edman degradation were unsuccessful, probably due to blockage of the N-terminal group. Gelatinolytic activity was optimal at pH 7-8. This is the first report of the isolation and characterization of a scorpionfish venom protease.     

General biochemical and immunological characteristics of the venom from Peruvian scorpion Hadruroides lunatus

This communication describes the general biochemical properties and some immunological characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic to mice, the LD₅₀ determined was 0.1 mg/kg and 21.55 mg/kg when the venom was injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector.     

Channel-forming activity in the venom of the cockroach-hunting wasp, Ampulex compressa.

The parasitoid solitary wasp Ampulex compressa uses the cockroach Periplaneta americana as a food supply for its larvae. To subdue its prey, the wasp injects a venom cocktail into the brain of the cockroach. We investigated channel activity of A. compressa venom by collecting venom and incorporating it into a planar lipid bilayer. The venom, reconstituted into the bilayer, showed ion channel activity, forming a fast-fluctuating channel with a small conductance of 20+/-0.1pS, with no voltage sensitivity. These channels were not observed when the venom was digested with proteases before application to the bilayer, but were not affected by exposure to protease after their incorporation into the bilayer, indicating that the active venom component is a peptide. The channels were found to be cation selective with similar selectivity for the monovalent cations K(+), Li(+) and Na(+), but showed high selectivity against anions (Cl(-)) and divalent cations (Ca(2+) and Mg(2+)). This study is the first demonstration and biophysical characterization of channel activity in the venom of A. compressa. The possible functional significance of this channel activity is discussed in light of the unusual nature of the effects of this wasp venom on the behavior of its prey.     

Variation in lethality and effects of two Australian chirodropid jellyfish venoms in fish.

The North Queensland chirodropid box jellyfish Chironex fleckeri and Chiropsalmus sp. share similar nematocyst composition and the same prey of Acetes australis shrimps in their early medusa stages; however, as C. fleckeri individuals reach larger size, the animals add fish to their diet and their complement of nematocyst types changes, allowing larger doses of venom to be delivered to prey. This study demonstrated that the venoms of the two species differ as well: despite similar effects previously documented in crustacean prey models, the two had widely different cardiac and lethal effects in fish, with C. fleckeri being substantially more potent in its ability to cause death. Comparisons between the venom delivery abilities of the two species showed that the change in nematocysts of C. fleckeri cannot alone account for its ontogenetic shift to prey fish; instead, its prey ecology clearly necessitates it having venom capable of acting efficiently to cause death in fish. Although this venom is almost certainly produced at greater metabolic cost to the animal than the less-lethal venom of Chiropsalmus sp., owing to its greater molecular protein complexity, it confers the advantage of increased caloric intake from fish prey, facilitating larger size and potentially greater reproductive output of C. fleckeri over Chiropsalmus sp.     

Isolation and physiological characterization of taicatoxin, a complex toxin with specific effects on calcium channels.

Taicatoxin is a new complex oligomeric toxin that was isolated from the venom of the Australian taipan snake Oxyuranus scutellatus scutellatus. It is composed of three different molecular entities: an alpha-neurotoxin-like peptide of mol. wt 8000, a neurotoxic phospholipase of mol. wt of 16,000 and a serine protease inhibitor of mol. wt 7000, linked by non-covalent bonds, at an approximate stoichiometry of 1:1:4. The most active form of the complex was isolated by ion exchange chromatography through DE-Cellulose followed by two steps of CM-Cellulose chromatography at pH 4.7 and pH 6.0, respectively. At this stage the complex migrates as a single component in beta-alanine-acetate-urea gel electrophoresis and is very toxic to mice (1 or 2 micrograms of the complex protein kills a mouse of 20 g within 2 hr). It blocks the high threshold calcium channel current of excitable membranes in heart and does not affect the low threshold calcium channel current. The block occurs at a site that is accessible extracellularly but not intracellularly. The block is selective for calcium channels, reversible, does not affect single channel conductance but only changes channel gating, and is voltage dependent with higher affinity for inactivated channels. The phospholipase activity of the complex toxin can be separated by affinity-chromatography using a phospholipid analog (PC-Sepharose). The resulting complex contains only alpha-neurotoxin and protease inhibitor and is still capable of blocking calcium channels, although with less potency than the native oligomeric form. Sephadex G-50 gel filtration chromatography in the presence of high salt (1M NaCl) at alkaline pH (8.2), separates the alpha-neurotoxin-like peptide from the protease inhibitor, but at this stage the resulting peptides lose physiological activity towards the calcium channels. The amino acid sequence of the protease inhibitor was determined by automatic Edman degradation. The alpha-neurotoxin-like peptide and two isosubunits displaying phospholipase activity were sequenced at the N-terminal part of the molecule.     

Sphingomyelinase D in sicariid spider venom is a potent insecticidal toxin

Spider venoms have evolved over hundreds of millions of years with a primary role of immobilizing prey. Sphingomyelinase D (SMase D) and homologs in the SicTox gene family are the most abundantly expressed toxic protein in venoms of Loxosceles and Sicarius spiders (Sicariidae). While SMase D is well known to cause dermonecrotic lesions in mammals, little work has investigated the bioactivity of this enzyme in its presumed natural role of immobilizing insect prey. We expressed and purified recombinant SMase D from Loxosceles arizonica (Laz-SMase D) and compared its enzymatic and insecticidal activity to that of crude venom. SMase D enzymatic activities of purified protein and crude venom from the same species were indistinguishable. In addition, SMase D and crude venom have comparable and high potency in immobilization assays on crickets. These data indicate that SMase D is a potent insecticidal toxin, the role for which it presumably evolved.