Stonefish toxin defines an ancient branch of the perforin-like superfamily.

The lethal factor in stonefish venom is stonustoxin (SNTX), a heterodimeric cytolytic protein that induces cardiovascular collapse in humans and native predators. Here, using X-ray crystallography, we make the unexpected finding that SNTX is a pore-forming member of an ancient branch of the Membrane Attack Complex-Perforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. SNTX comprises two homologous subunits (α and ß), each of which comprises an N-terminal pore-forming MACPF/CDC domain, a central focal adhesion-targeting domain, a thioredoxin domain, and a C-terminal tripartite motif family-like PRY SPla and the RYanodine Receptor immune recognition domain. Crucially, the structure reveals that the two MACPF domains are in complex with one another and arranged into a stable early prepore-like assembly. These data provide long sought after near-atomic resolution insights into how MACPF/CDC proteins assemble into prepores on the surface of membranes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs are distributed throughout eukaryotic life and play a broader, possibly immune-related function outside venom.

A central role for venom in predation by Varanus komodoensis (Komodo Dragon) and the extinct giant Varanus (Megalania) priscus.

The predatory ecology of Varanus komodoensis (Komodo Dragon) has been a subject of long-standing interest and considerable conjecture. Here, we investigate the roles and potential interplay between cranial mechanics, toxic bacteria, and venom. Our analyses point to the presence of a sophisticated combined-arsenal killing apparatus. We find that the lightweight skull is relatively poorly adapted to generate high bite forces but better adapted to resist high pulling loads. We reject the popular notion regarding toxic bacteria utilization. Instead, we demonstrate that the effects of deep wounds inflicted are potentiated through venom with toxic activities including anticoagulation and shock induction. Anatomical comparisons of V. komodoensis with V. (Megalania) priscus fossils suggest that the closely related extinct giant was the largest venomous animal to have ever lived.

An in vivo comparison of the efficacy of CSL box jellyfish antivenom with antibodies raised against nematocyst-derived Chironex fleckeri venom.

Although CSL box jellyfish antivenom (AV) remains the primary treatment for Chironex fleckeri envenoming, there has been considerable debate regarding its clinical effectiveness. Animal studies have shown that AV is largely ineffective in preventing C. fleckeri-induced cardiovascular collapse. This study examined the effectiveness of CSL box jellyfish AV (ovine IgG), raised against 'milked' venom, and polyclonal rabbit IgG antibodies (Ab) raised against nematocyst-derived venom. A venom dose of 30microg/kg, i.v., which causes an initial presser response (34+/-5mmHg; n=7) followed by cardiovascular collapse, was used in all experiments. A bolus dose of AV (3000U/kg, i.v.) or Ab (12mg; i.e. an equivalent protein 'load' to 3000U/kg AV), administered 15min prior to a bolus dose of venom, did not significantly attenuate the effects of venom. The venom response was also not significantly attenuated when AV (3000U/kg) was given as a bolus dose 10-60min prior to venom infusion. However, when the venom was incubated with either AV (3000U/kg) or Ab (12mg) for 3h prior to infusion, the effect of the venom was almost abolished. The results of this study demonstrate that antibodies raised against both 'milked' and nematocyst-derived venom are able to neutralise the cardiovascular collapse produced by the venom. However, large amounts of AV are required and must be preincubated with the venom to be protective. This indicates a very rapid action of the toxin(s) and that AV is unlikely to be clinically effective because it cannot be administered early enough.

An examination of the cardiovascular effects of an 'Irukandji' jellyfish, Alatina nr mordens.

Irukandji syndrome is usually characterized by delayed severe abdominal, back and chest pain associated with autonomic effects including diaphoresis, hypertension and, in severe cases, myocardial injury and pulmonary oedema. It is most often associated with envenoming by the jellyfish Carukia barnesi, but a number of other jellyfish, including Alatina mordens, are now known to produce Irukandji syndrome. In the present study, nematocyst-derived venom from A. nr mordens (150-250 microg/kg, i.v.) produced a long-lasting pressor effect in anaesthetised rats. This pressor response (250 microg/kg, i.v.) was significantly inhibited by prior administration of the alpha-adrenoceptor antagonist prazosin (200 microg/kg, i.v.) but not by CSL box jellyfish antivenom (300 U/kg, i.v.). A. nr mordens venom 250 microg/kg (i.v.) caused marked increases in plasma adrenaline and noradrenaline concentrations following administration in anaesthetised rats. The venom did not contain appreciable amounts of either adrenaline or noradrenaline. A. nr mordens venom (25 microg/ml) produced a contractile response in rat electrically stimulated vas deferens which was markedly reduced in tissues pre-treated with reserpine (0.1mM) or guanethidine (0.1mM). Sodium dodecyl sulphate (SDS)-PAGE analysis showed that A. nr mordens venom is comprised of multiple protein bands ranging from 10 to 200 kDa. Western blot analysis using CSL box jellyfish antivenom indicated several antigenic proteins in A. nr mordens venom, however, it did not detect all proteins present in the venom. This study characterizes the in vitro and in vivo effects of A. nr mordens venom and indicates that the cardiovascular effects are at least partially mediated by endogenous catecholamine release.

The in vitro vascular effects of two chirodropid (Chironex fleckeri and Chiropsella bronzie) venoms.

Clinical observations suggest a primary cardiotoxic role in fatal Chironex fleckeri stings. The limited research available indicates that Chiropsella bronzie venom acts in a similar manner although appears to be less potent. The aim of the present study was to elucidate the vascular effects of C. fleckeri and C. bronzie venoms using rat isolated aorta. Both venoms produced a sustained contraction of endothelium-denuded aorta which was not significantly affected by prazosin or box jellyfish antivenom. Felodipine significantly reduced the contractile response to C. fleckeri venom but not C. bronzie venom. Both venoms produced an initial relaxation (Phase 1), followed by a sustained contraction (Phase 2), in pre-contracted endothelium-intact aorta. Removal of the endothelium significantly inhibited both phases of the response. NOLA significantly inhibited Phase 1, but not Phase 2, of the response to both venoms. Atropine, HOE 140 or BQ 123 did not have any significant inhibitory effect on either phase. In conclusion, neither C. fleckeri nor C. bronzie venoms appear to contain components with activity at alpha(1)-adrenoceptors. Antivenom was ineffective in reversing the effects of the venom suggesting it is incapable of completely neutralising nematocyst-derived venom. Determining the mechanism of action of these venoms will allow for the development of better treatment strategies.

Venom physiology and composition in a litter of Common Death Adders (Acanthophis antarcticus) and their parents.

Metabolic expenditure has been shown to increase abruptly in several snake species directly after venom expenditure, while the later stages of venom replenishment seem to involve minor costs. This study examines the dependence of increases in metabolic rate following venom expenditure on the stage of venom replenishment that the venom producing tissue is in at the time of venom extraction in the Common Death Adder, Acanthophis antarcticus. Potential changes in venom composition during venom replenishment are also explored to elucidate whether replenishment is achieved via low rates of synthesis of all venom components or by non-parallel protein production, i.e. initial production of some venom components and subsequent synthesis of others. The results of this study indicate that venom expenditure is followed by a sudden increase in metabolic rate when snakes have previously not expended venom for at least two days, suggesting that repetitive venom expenditure does not further increase the activity of venom gland tissue in this initial time period but that a second upregulation occurs when the tissue is past the initial activation stage. In addition, venom composition appears to remain constant during replenishment within an individual, while substantial variations can be observed even between siblings.

A pharmacological and biochemical examination of the geographical variation of Chironex fleckeri venom.

Chironex fleckeri (box jellyfish) are found in the northern tropical waters of Australia. Although C. fleckeri have a wide geographical distribution and are able to swim large distances, adults tend to stay in small restricted areas. Clinical data shows that deaths from envenoming have not been recorded in Western Australia, yet numerous fatalities have occurred in Northern Territory and Queensland waters. One explanation for this discrepancy is a geographical variation in venom composition. This study examined the pharmacological and biochemical profiles of C. fleckeri venom from different geographical locations and seasons. Venoms were screened for cytotoxicity using a rat aortic smooth muscle cell line (A7r5). While all venoms caused concentration-dependent cytotoxicity, differences were seen in the potency of venoms from Mission Beach and Weipa, when collected in different seasons, as indicated by IC(50) values. Similarly venoms collected within the same season, from different locations around Australia, displayed marked differences in venom composition as shown by size exclusion HPLC and SDS-PAGE profiles which indicated the absence or reduced quantity of 'peaks' in some venoms. Based on IC(50) data obtained from the cell assay, the effects of the most potent (i.e. from Weipa in 2006) and the least potent (i.e. from Broome in 2007) venoms were examined in anesthetised rats. Both venoms at 10 microg/kg (i.v.) caused a transient hypertensive phase followed by cardiovascular collapse. However, at 4 microg/kg (i.v.) venom from Weipa 2006 caused a transient hypertensive phase followed by a transient decrease in MAP while venom from Broome 2007 only caused a small transient increase in MAP. This study demonstrates that there is considerable geographical variation in the composition of C. fleckeri venoms which is most distinct between specimens from western and eastern Australia and may explain the geographical variation in reported deaths.