Sydney Funnel-web Spider Atrax Robustus Research Articles

Australian funnel-web spiders evolved human-lethal δ-hexatoxins for defense against vertebrate predators

Australian funnel-web spiders are infamous for causing human fatalities, which are induced by venom peptides known as δ-hexatoxins (δ-HXTXs). Humans and other primates did not feature in the prey or predator spectrum during evolution of these spiders, and consequently the primate lethality of δ-HXTXs remains enigmatic. Funnel-web envenomations are mostly inflicted by male spiders that wander from their burrow in search of females during the mating season, which suggests a role for δ-HXTXs in self-defense since male spiders rarely feed during this period. Although 35 species of Australian funnel-web spiders have been described, only nine δ-HXTXs from four species have been characterized, resulting in a lack of understanding of the ecological roles and molecular evolution of δ-HXTXs. Here, by profiling venom-gland transcriptomes of 10 funnel-web species, we report 22 δ-HXTXs. Phylogenetic and evolutionary assessments reveal a remarkable sequence conservation of δ-HXTXs despite their deep evolutionary origin within funnel-web spiders, consistent with a defensive role. We demonstrate that δ-HXTX-Ar1a, the lethal toxin from the Sydney funnel-web spider Atrax robustus, induces pain in mice by inhibiting inactivation of voltage-gated sodium (NaV) channels involved in nociceptive signaling. δ-HXTX-Ar1a also inhibited inactivation of cockroach NaV channels and was insecticidal to sheep blowflies. Considering their algogenic effects in mice, potent insecticidal effects, and high levels of sequence conservation, we propose that the δ-HXTXs were repurposed from an initial insecticidal predatory function to a role in defending against nonhuman vertebrate predators by male spiders, with their lethal effects on humans being an unfortunate evolutionary coincidence.

Phylogenomic reclassification of the world\u2019s most venomous spiders (Mygalomorphae, Atracinae), with implications for venom evolution

Here we show that the most venomous spiders in the world are phylogenetically misplaced. Australian atracine spiders (family Hexathelidae), including the notorious Sydney funnel-web spider Atrax robustus, produce venom peptides that can kill people. Intriguingly, eastern Australian mouse spiders (family Actinopodidae) are also medically dangerous, possessing venom peptides strikingly similar to Atrax hexatoxins. Based on the standing morphology-based classification, mouse spiders are hypothesized distant relatives of atracines, having diverged over 200 million years ago. Using sequence-capture phylogenomics, we instead show convincingly that hexathelids are non-monophyletic, and that atracines are sister to actinopodids. Three new mygalomorph lineages are elevated to the family level, and a revised circumscription of Hexathelidae is presented. Re-writing this phylogenetic story has major implications for how we study venom evolution in these spiders, and potentially genuine consequences for antivenom development and bite treatment research. More generally, our research provides a textbook example of the applied importance of modern phylogenomic research.

The Lethal Toxin from Australian Funnel-Web Spiders Is Encoded by an Intronless Gene

Australian funnel-web spiders are generally considered the most dangerous spiders in the world, with envenomations from the Sydney funnel-web spider Atrax robustus resulting in at least 14 human fatalities prior to the introduction of an effective anti-venom in 1980. The clinical envenomation syndrome resulting from bites by Australian funnel-web spiders is due to a single 42-residue peptide known as δ-hexatoxin. This peptide delays the inactivation of voltage-gated sodium channels, which results in spontaneous repetitive firing and prolongation of action potentials, thereby causing massive neurotransmitter release from both somatic and autonomic nerve endings. Here we show that δ-hexatoxin from the Australian funnel-web spider Hadronyche versuta is produced from an intronless gene that encodes a prepropeptide that is post-translationally processed to yield the mature toxin. A limited sampling of genes encoding unrelated venom peptides from this spider indicated that they are all intronless. Thus, in distinct contrast to cone snails and scorpions, whose toxin genes contain introns, spiders may have developed a quite different genetic strategy for evolving their venom peptidome.

The ω-atracotoxins: Selective blockers of insect M-LVA and HVA calcium channels

The ω-atracotoxins (ω-ACTX) are a family of arthropod-selective peptide neurotoxins from Australian funnel-web spider venoms (Hexathelidae: Atracinae) that are candidates for development as biopesticides. We isolated a 37-residue insect-selective neurotoxin, ω-ACTX-Ar1a, from the venom of the Sydney funnel-web spider Atrax robustus, with high homology to several previously characterized members of the ω-ACTX-1 family. The peptide induced potent excitatory symptoms, followed by flaccid paralysis leading to death, in acute toxicity tests in house crickets . Using isolated smooth and skeletal nerve-muscle preparations, the toxin was shown to lack overt vertebrate toxicity at concentrations up to 1 μM. To further characterize the target of the ω-ACTXs, voltage-clamp analysis using the whole-cell patch-clamp technique was undertaken using cockroach dorsal unpaired median neurons. It is shown here for the first time that ω-ACTX-Ar1a, and its homolog ω-ACTX-Hv1a from Hadronyche versuta, reversibly block both mid–low- (M-LVA) and high-voltage-activated (HVA) insect calcium channel (Ca v) currents. This block occurred in the absence of alterations in the voltage-dependence of Ca v channel activation, and was voltage-independent, suggesting that ω-ACTX-1 family toxins are pore blockers rather than gating modifiers. At a concentration of 1 μM ω-ACTX-Ar1a failed to significantly affect global K v channel currents. However, 1 μM ω-ACTX-Ar1a caused a modest 18% block of insect Na v channel currents, similar to the minor block of Na v channels reported for other insect Ca v channel blockers such as ω-agatoxin IVA. These findings validate both M-LVA and HVA Ca v channels as potential targets for insecticides.

Isolation of δ-missulenatoxin-Mb1a, the major vertebrate-active spider δ-toxin from the venom of Missulena bradleyi (Actinopodidae)

The present study describes the isolation and pharmacological characterisation of the neurotoxin δ-missulenatoxin-Mb1a (δ-MSTX-Mb1a) from the venom of the male Australian eastern mouse spider, Missulena bradleyi. This toxin was isolated using reverse-phase high-performance liquid chromatography and was subsequently shown to cause an increase in resting tension, muscle fasciculation and a decrease in indirect twitch tension in a chick biventer cervicis nerve-muscle bioassay. Interestingly, these effects were neutralised by antivenom raised against the venom of the Sydney funnel-web spider Atrax robustus. Subsequent whole-cell patch-clamp electrophysiology on rat dorsal root ganglion neurones revealed that δ-MSTX-Mb1a caused a reduction in peak tetrodotoxin (TTX)-sensitive sodium current, a slowing of sodium current inactivation and a hyperpolarising shift in the voltage at half-maximal activation. In addition, δ-MSTX-Mb1a failed to affect TTX-resistant sodium currents. Subsequent Edman degradation revealed a 42-residue peptide with unusual N- and C-terminal cysteines and a cysteine triplet (Cys14-16). This toxin was highly homologous to a family of δ-atracotoxins (δ-ACTX) from Australian funnel-web spiders including conservation of all eight cysteine residues. In addition to actions on sodium channel gating and kinetics to δ-ACTX, δ-MSTX-Mb1a caused significant insect toxicity at doses up to 2000 pmol/g. δ-MSTX-Mb1a therefore provides evidence of a highly conserved spider δ-toxin from a phylogenetically distinct spider family that has not undergone significant modification.

Characterisation of the effects of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, on sodium channel activation and inactivation.

The present study investigates the actions of robustoxin (atracotoxin-Ar1) purified from the venom of the male Sydney funnel-web spider Atrax robustus on sodium channel gating. Using whole-cell patch-clamp techniques the study assessed the actions of robustoxin on tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) sodium currents in rat dorsal root ganglion cells. Similar to the closely related funnel-web spider toxin versutoxin (delta-atracotoxin-Hv1) from Hadronyche versuta, robustoxin had no effect on TTX-R sodium currents but exerted potent effects on TTX-S sodium currents. The main action of robustoxin was a concentration-dependent slowing or removal of TTX-S sodium current inactivation. This steady-state current was maintained during long-lasting depolarisations at all test potentials. Robustoxin (30 nM) also caused a 13-mV hyperpolarising shift in the voltage midpoint of steady-state sodium channel inactivation (h infinity) leading to a reduced peak current at a holding potential of -80 mV. Moreover there was a steady-state or non-inactivating component present (18% of maximal sodium current) at prepulse potentials that normally inactivate all TTX-S sodium channels (more depolarised than -40 mV). In addition robustoxin produced a significant increase in the repriming kinetics of the sodium channel when channels returned to the resting state following activation. This increase in the rate of recovery of sodium current appears to explain the use-dependent effects on peak sodium current amplitude at high stimulation frequencies. Finally 30 nM robustoxin caused an 11-mV hyperpolarising shift in the voltage dependence of the channel but did not markedly modify tail current kinetics. These actions suggest that robustoxin inhibits conversion of the open state to the inactivated state of TTX-S sodium channels, thus allowing a fraction of the sodium current to remain at membrane potentials at which inactivation is normally complete. Given the recent reclassification of funnel-web spider toxins as atracotoxins, robustoxin should henceforth be known as delta-atracotoxin-Ar1 to reflect this main action on sodium channel inactivation. These present results further support the hypothesis that funnel-web spider toxins interact with neurotoxin receptor site 3 to slow channel inactivation in a manner similar to that of alpha-scorpion and sea anemone toxins.

Solution structure of robustoxin, the lethal neurotoxin from the funnel-web spider Atrax robustus

The solution structure of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, has been determined from 2D 1H NMR data. Robustoxin is a polypeptide of 42 residues cross-linked by four disulphide bonds, the connectivities of which were determined from NMR data and trial structure calculations to be 1–15, 8–20, 14–31 and 16–42 (a 1–4/2–6/3–7/5–8 pattern). The structure consists of a small three-stranded, anti-parallel β-sheet and a series of interlocking γ-turns at the C-terminus. It also contains a cystine knot, thus placing it in the inhibitor cystine knot motif family of structures, which includes the ω-conotoxins and a number of plant and animal toxins and protease inhibitors. Robustoxin contains three distinct charged patches on its surface, and an extended loop that includes several aromatic and non-polar residues. Both of these structural features may play a role in its binding to the voltage-gated sodium channel.

Actions of the crude venom of the Sydney funnel-web spider. Atrax robustus on autonomic neuromuscular transmission.

1 The effects on mammalian autonomic neuromuscular transmission of the crude venom of the female Sydney funnel-web spider Atrax robustus, have been investigated.2 At doses of 10 mug/ml or lower the indirectly elicited twitch-like responses of the rat anococcygeus preparation were inhibited. At doses greater than 10 mug/ml there was an initial reduction in the twitch-like response followed by a sustained contracture of the tissue.3 The long-lasting contracture caused by the venom was abolished by the application of phentolamine. It was virtually non-existent in muscle preparations isolated from reserpine-treated rats.4 In the presence of tetrodotoxin the contracture was smaller and less well maintained than in its absence.5 The venom caused a small reduction in the amplitude of the indirectly elicited twitch-like response of the longitudinal muscle of the guinea-pig ileum, followed by an increase in the tone of the preparation. The increase in tone was maintained for several minutes and was rapidly abolished by the application of atropine. The presence of venom did not affect control responses to either histamine or acetylcholine.6 Inhibitory transmission in the rat anococcygeus preparation was unaffected by the venom.7 The neurally-mediated twitch-like responses of both guinea-pig and rat vas deferens were inhibited by the venom at doses below 10 mug/ml. At higher doses the inhibition was accompanied by spontaneous contractions, and at doses in excess of 100 mug/ml the inhibition of twitch-like responses was transient and was followed by a potentiation of the motor response and extensive spontaneous activity. The preparation became quiescent 20 min after the application of venom and the evoked response was abolished after 60 min.8 The venom had qualitatively similar effects on motor transmission in the human vas deferens as on the rat and guinea-pig preparations. However, the human preparations were 50 to 100 times more sensitive to the effects of the venom.