Structural venomics reveals evolution of a complex venom by duplication and diversification of an ancient peptide-encoding gene.

Spiders are one of the most successful venomous animals, with more than 48,000 described species. Most spider venoms are dominated by cysteine-rich peptides with a diverse range of pharmacological activities. Some spider venoms contain thousands of unique peptides, but little is known about the mechanisms used to generate such complex chemical arsenals. We used an integrated transcriptomic, proteomic, and structural biology approach to demonstrate that the lethal Australian funnel-web spider produces 33 superfamilies of venom peptides and proteins. Twenty-six of the 33 superfamilies are disulfide-rich peptides, and we show that 15 of these are knottins that contribute >90% of the venom proteome. NMR analyses revealed that most of these disulfide-rich peptides are structurally related and range in complexity from simple to highly elaborated knottin domains, as well as double-knot toxins, that likely evolved from a single ancestral toxin gene.

An Integrated Proteomic and Transcriptomic Analysis Reveals the Venom Complexity of the Bullet Ant Paraponera clavata.

A critical hurdle in ant venom proteomic investigations is the lack of databases to comprehensively and specifically identify the sequence and function of venom proteins and peptides. To resolve this, we used venom gland transcriptomics to generate a sequence database that was used to assign the tandem mass spectrometry (MS) fragmentation spectra of venom peptides and proteins to specific transcripts. This was performed alongside a shotgun liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the venom to confirm that these assigned transcripts were expressed as proteins. Through the combined transcriptomic and proteomic investigation of Paraponera clavata venom, we identified four times the number of proteins previously identified using 2D-PAGE alone. In addition to this, by mining the transcriptomic data, we identified several novel peptide sequences for future pharmacological investigations, some of which conform with inhibitor cysteine knot motifs. These types of peptides have the potential to be developed into pharmaceutical or bioinsecticide peptides.

Craspedotropis gretathunbergae, a new species of Cyclophoridae (Gastropoda: Caenogastropoda), discovered and described on a field course to Kuala Belalong rainforest, Brunei.

BACKGROUND: Terrestrial Caenogastropoda form an important but threatened component of the Borneo tropical rainforest malacofauna, where the group is nearly as rich in species as the Stylommatophora. They are, however, more sensitive to drought, temperature extremes and forest degradation. NEW INFORMATION: On a field course at Kuala Belalong Field Studies Centre in Brunei Darussalam (Borneo), a new caenogastropod species, belonging to the genus Craspedotropis, was discovered by the course participants. The participants decided to name the species Craspedotropis gretathunbergae n. sp., in honour of the climate change activist Greta Thunberg, as caenogastropod land snails, such as this species, are likely to suffer because of climate change.

Venom Peptide Repertoire of the European Myrmicine Ant Manica rubida: Identification of Insecticidal Toxins.

Using an integrated transcriptomic and proteomic approach, we characterized the venom peptidome of the European red ant, Manica rubida. We identified 13 "myrmicitoxins" that share sequence similarities with previously identified ant venom peptides, one of them being identified as an EGF-like toxin likely resulting from a threonine residue modified by O-fucosylation. Furthermore, we conducted insecticidal assays of reversed-phase HPLC venom fractions on the blowfly Lucilia caesar, permitting us to identify six myrmicitoxins (i.e., U3-, U10-, U13-, U20-MYRTX-Mri1a, U10-MYRTX-Mri1b, and U10-MYRTX-Mri1c) with an insecticidal activity. Chemically synthesized U10-MYRTX-Mri1a, -Mri1b, -Mri1c, and U20-MYRTX-Mri1a irreversibly paralyzed blowflies at the highest doses tested (30-125 nmol·g-1). U13-MYRTX-Mri1a, the most potent neurotoxic peptide at 1 h, had reversible effects after 24 h (150 nmol·g-1). Finally, U3-MYRTX-Mri1a has no insecticidal activity, even at up to 55 nmol·g-1. Thus, M. rubida employs a paralytic venom rich in linear insecticidal peptides, which likely act by disrupting cell membranes.

A new species of Clavicornaltica (Coleoptera: Chrysomelidae), discovered and described on a field course to Kuala Belalong, Brunei.

BACKGROUND: Clavicornaltica is a genus of very small flea beetles living in the leaf litter layer of Asian forests, easily sampled with Winkler extraction. The genus is presumably very rich in species, but their taxonomy is hampered by their small size and morphological uniformity. NEW INFORMATION: On a 'taxon expedition'-style field course at Kuala Belalong Field Studies Centre in Brunei Darussalam (Borneo), a new species, Clavicornaltica belalongensis n. sp., was discovered and taxonomically treated by the course participants. We also present the first DNA barcodes for the genus.

High-Accuracy Mass Spectrometry Based Screening Method for the Discovery of Cysteine Containing Peptides in Animal Venoms and Toxins.

Venom and toxin samples derived from animal origins are a rich source of bioactive peptides. A high proportion of bioactive peptides that have been identified in venom contain one or more disulfide bridges, which are thought to stabilize tertiary structure, and therefore influence the peptides' specificity and activity. In this chapter, we describe a label-free mass spectrometry-based screening workflow specifically to detect peptides that contain inter- and intramolecular disulfide bonds, followed by elucidation of their primary structure. This method is based on the determination of the normalized isotope shift (NIS) and the normalized mass defect (NMD) of peptides, two parameters which are heavily influenced by the presence of sulfur in a peptide, where cysteines are the main contributing residues. Using ant defensive secretions as an example, we describe the initial fractionation of the venom on strong cation exchange followed by nanoflow HPLC and mass spectrometry. High resolution zoom scan spectra of high-abundance peptides are acquired, allowing an accurate determination of both monoisotopic and average mass, which are essential for calculation of NMD and NIS. Candidate peptides exhibiting relative low NMD and high NIS values are selected for targeted de novo sequencing. By fine-tuning the collision energy for optimal fragmentation of each selected precursor ions, the full sequence of several novel inter- and intramolecular disulfide bond containing ant defensive peptides can be established.

Discovery and molecular interaction studies of a highly stable, tarantula peptide modulator of acid-sensing ion channel 1.

Acute pharmacological inhibition of acid-sensing ion channel 1a (ASIC1a) is efficacious in rodent models in alleviating symptoms of neurological diseases such as stroke and multiple sclerosis. Thus, ASIC1a is a promising therapeutic target and selective ligands that modulate it are invaluable research tools and potential therapeutic leads. Spider venoms have provided an abundance of voltage-gated ion channel modulators, however, only one ASIC modulator (PcTx1) has so far been isolated from this source. Here we report the discovery, characterization, and chemical stability of a second spider venom peptide that potently modulates ASIC1a and ASIC1b, and investigate the molecular basis for its subtype selectivity. π-TRTX-Hm3a (Hm3a) is a 37-amino acid peptide isolated from Togo starburst tarantula (Heteroscodra maculata) venom with five amino acid substitutions compared to PcTx1, and is also three residues shorter at the C-terminus. Hm3a pH-dependently inhibited ASIC1a with an IC50 of 1-2 nM and potentiated ASIC1b with an EC50 of 46.5 nM, similar to PcTx1. Using ASIC1a to ASIC1b point mutants in rat ASIC1a revealed that Glu177 and Arg175 in the palm region opposite α-helix 5 play an important role in the Hm3a-ASIC1 interaction and contribute to the subtype-dependent effects of the peptide. Despite its high sequence similarity with PcTx1, Hm3a showed higher levels of stability over 48 h. Overall, Hm3a represents a potent, highly stable tool for the study of ASICs and will be particularly useful when stability in biological fluids is required, for example in long term in vitro cell-based assays and in vivo experiments. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.'

Pharmacological characterisation of the highly NaV1.7 selective spider venom peptide Pn3a.

Human genetic studies have implicated the voltage-gated sodium channel NaV1.7 as a therapeutic target for the treatment of pain. A novel peptide, µ-theraphotoxin-Pn3a, isolated from venom of the tarantula Pamphobeteus nigricolor, potently inhibits NaV1.7 (IC50 0.9 nM) with at least 40-1000-fold selectivity over all other NaV subtypes. Despite on-target activity in small-diameter dorsal root ganglia, spinal slices, and in a mouse model of pain induced by NaV1.7 activation, Pn3a alone displayed no analgesic activity in formalin-, carrageenan- or FCA-induced pain in rodents when administered systemically. A broad lack of analgesic activity was also found for the selective NaV1.7 inhibitors PF-04856264 and phlotoxin 1. However, when administered with subtherapeutic doses of opioids or the enkephalinase inhibitor thiorphan, these subtype-selective NaV1.7 inhibitors produced profound analgesia. Our results suggest that in these inflammatory models, acute administration of peripherally restricted NaV1.7 inhibitors can only produce analgesia when administered in combination with an opioid.

Combined Peptidomic and Proteomic Analysis of Electrically Stimulated and Manually Dissected Venom from the South American Bullet Ant Paraponera clavata.

Ants have evolved venoms rich in peptides and proteins used for predation, defense, and communication. However, they remain extremely understudied due to the minimal amount of venom secreted by each ant. The present study investigated the differences in the proteome and peptidome of the venom from the bullet ant, Paraponera clavata. Venom samples were collected from a single colony either by manual venom gland dissection or by electrical stimulation and were compared using proteomic methods. Venom proteins were separated by 2D-PAGE and identified by nanoLC-ESI-QTOF MS/MS. Venom peptides were initially separated using C18 reversed-phase high-performance liquid chromatography, then analyzed by MALDI-TOF MS. The proteomic analysis revealed numerous proteins that could be assigned a biological function (total 94), mainly as toxins, or roles in cell regulation and transport. This investigation found that ca. 73% of the proteins were common to venoms collected by the two methods. The peptidomic analysis revealed a large number of peptides (total 309) but with <20% shared by the two collection methods. There was also a marked difference between venoms obtained by venom gland dissection from different ant colonies. These findings demonstrate the rich composition and variability of P. clavata venom.

Isolation and characterization of a structurally unique ß-hairpin venom peptide from the predatory ant Anochetus emarginatus.

BACKGROUND: Most ant venoms consist predominantly of small linear peptides, although some contain disulfide-linked peptides as minor components. However, in striking contrast to other ant species, some Anochetus venoms are composed primarily of disulfide-rich peptides. In this study, we investigated the venom of the ant Anochetus emarginatus with the aim of exploring these novel disulfide-rich peptides. METHODS: The venom peptidome was initially investigated using a combination of reversed-phase HPLC and mass spectrometry, then the amino acid sequences of the major peptides were determined using a combination of Edman degradation and de novo MS/MS sequencing. We focused on one of these peptides, U1-PONTX-Ae1a (Ae1a), because of its novel sequence, which we predicted would form a novel 3D fold. Ae1a was chemically synthesized using Fmoc chemistry and its 3D structure was elucidated using NMR spectroscopy. The peptide was then tested for insecticidal activity and its effect on a range of human ion channels. RESULTS: Seven peptides named poneritoxins (PONTXs) were isolated and sequenced. The three-dimensional structure of synthetic Ae1a revealed a novel, compact scaffold in which a C-terminal ß-hairpin is connected to the N-terminal region via two disulfide bonds. Synthetic Ae1a reversibly paralyzed blowflies and inhibited human L-type voltage-gated calcium channels (CaV1). CONCLUSIONS: Poneritoxins from Anochetus emarginatus venom are a novel class of toxins that are structurally unique among animal venoms. GENERAL SIGNIFICANCE: This study demonstrates that Anochetus ant venoms are a rich source of novel ion channel modulating peptides, some of which might be useful leads for the development of biopesticides.

Comparisons of Protein and Peptide Complexity in Poneroid and Formicoid Ant Venoms.

Animal venom peptides are currently being developed as novel drugs and bioinsecticides. Because ants use venoms for defense and predation, venomous ants represent an untapped source of potential bioactive toxins. This study compared the protein and peptide components of the poneroid ants Neoponera commutata, Neoponera apicalis, and Odontomachus hastatus and the formicoid ants Ectatomma tuberculatum, Ectatomma brunneum, and Myrmecia gulosa. 1D and 2D PAGE revealed venom proteins in the mass range <10 to >250 kDa. NanoLC-ESI-QTOF MS/MS analysis of tryptic peptides revealed the presence of common venom proteins and also many undescribed proteins. RP-HPLC separation followed by MALDI-TOF MS of the venom peptides also revealed considerable heterogeneity. It was found that the venoms contained between 144 and 1032 peptides with 5-95% of peptides in the ranges 1-4 and 1-8 kDa for poneroid and formicoid ants, respectively. By employing the reducing MALDI matrix 1,5-diaminonapthalene, up to 28 disulfide-bonded peptides were also identified in each of the venoms. In particular, the mass range of peptides from poneroid ants is lower than peptides from other venoms, indicating possible novel structures and pharmacologies. These results indicate that ant venoms represent an enormous, untapped source of novel therapeutic and bioinsecticide leads.

The Biochemical Toxin Arsenal from Ant Venoms.

Ants (Formicidae) represent a taxonomically diverse group of hymenopterans with over 13,000 extant species, the majority of which inject or spray secretions from a venom gland. The evolutionary success of ants is mostly due to their unique eusociality that has permitted them to develop complex collaborative strategies, partly involving their venom secretions, to defend their nest against predators, microbial pathogens, ant competitors, and to hunt prey. Activities of ant venom include paralytic, cytolytic, haemolytic, allergenic, pro-inflammatory, insecticidal, antimicrobial, and pain-producing pharmacologic activities, while non-toxic functions include roles in chemical communication involving trail and sex pheromones, deterrents, and aggregators. While these diverse activities in ant venoms have until now been largely understudied due to the small venom yield from ants, modern analytical and venomic techniques are beginning to reveal the diversity of toxin structure and function. As such, ant venoms are distinct from other venomous animals, not only rich in linear, dimeric and disulfide-bonded peptides and bioactive proteins, but also other volatile and non-volatile compounds such as alkaloids and hydrocarbons. The present review details the unique structures and pharmacologies of known ant venom proteinaceous and alkaloidal toxins and their potential as a source of novel bioinsecticides and therapeutic agents.

The complexity and structural diversity of ant venom peptidomes is revealed by mass spectrometry profiling.

RATIONALE: Compared with other animal venoms, ant venoms remain little explored. Ants have evolved complex venoms to rapidly immobilize arthropod prey and to protect their colonies from predators and pathogens. Many ants have retained peptide-rich venoms that are similar to those of other arthropod groups. METHODS: With the goal of conducting a broad and comprehensive survey of ant venom peptide diversity, we investigated the peptide composition of venoms from 82 stinging ant species from nine subfamilies using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). We also conducted an in-depth investigation of eight venoms using reversed-phase high-performance liquid chromatography (RP-HPLC) separation coupled with offline MALDI-TOFMS. RESULTS: Our results reveal that the peptide compositions of ant venom peptidomes from both poneroid and formicoid ant clades comprise hundreds of small peptides (<4 kDa), while large peptides (>4 kDa) are also present in the venom of formicoids. Chemical reduction revealed the presence of disulfide-linked peptides in most ant subfamilies, including peptides structured by one, two or three disulfide bonds as well as dimeric peptides reticulated by three disulfide bonds. CONCLUSIONS: The biochemical complexity of ant venoms, associated with an enormous ecological and taxonomic diversity, suggests that stinging ant venoms constitute a promising source of bioactive molecules that could be exploited in the search for novel drug and biopesticide leads.

Diversity of peptide toxins from stinging ant venoms.

Ants (Hymenoptera: Formicidae) represent a taxonomically diverse group of arthropods comprising nearly 13,000 extant species. Sixteen ant subfamilies have individuals that possess a stinger and use their venom for purposes such as a defence against predators, competitors and microbial pathogens, for predation, as well as for social communication. They exhibit a range of activities including antimicrobial, haemolytic, cytolytic, paralytic, insecticidal and pain-producing pharmacologies. While ant venoms are known to be rich in alkaloids and hydrocarbons, ant venoms rich in peptides are becoming more common, yet remain understudied. Recent advances in mass spectrometry techniques have begun to reveal the true complexity of ant venom peptide composition. In the few venoms explored thus far, most peptide toxins appear to occur as small polycationic linear toxins, with antibacterial properties and insecticidal activity. Unlike other venomous animals, a number of ant venoms also contain a range of homodimeric and heterodimeric peptides with one or two interchain disulfide bonds possessing pore-forming, allergenic and paralytic actions. However, ant venoms seem to have only a small number of monomeric disulfide-linked peptides. The present review details the structure and pharmacology of known ant venom peptide toxins and their potential as a source of novel bioinsecticides and therapeutic agents.

Venom toxicity and composition in three Pseudomyrmex ant species having different nesting modes.

We aimed to determine whether the nesting habits of ants have influenced their venom toxicity and composition. We focused on the genus Pseudomyrmex (Pseudomyrmecinae) comprising terrestrial and arboreal species, and, among the latter, plant-ants that are obligate inhabitants of myrmecophytes (i.e., plants sheltering ants in hollow structures). Contrary to our hypothesis, the venom of the ground-dwelling species, Pseudomyrmex termitarius, was as efficacious in paralyzing prey as the venoms of the arboreal and the plant-ant species, Pseudomyrmex penetrator and Pseudomyrmex gracilis. The lethal potency of P. termitarius venom was equipotent with that of P. gracilis whereas the venom of P. penetrator was less potent. The MALDI-TOF MS analysis of each HPLC fraction of the venoms showed that P. termitarius venom is composed of 87 linear peptides, while both P. gracilis and P. penetrator venoms (23 and 26 peptides, respectively) possess peptides with disulfide bonds. Furthermore, P. penetrator venom contains three hetero- and homodimeric peptides consisting of two short peptidic chains linked together by two interchain disulfide bonds. The large number of peptides in P. termitarius venom is likely related to the large diversity of potential prey plus the antibacterial peptides required for nesting in the ground. Whereas predation involves only the prey and predator, P. penetrator venom has evolved in an environment where trees, defoliating insects, browsing mammals and ants live in equilibrium, likely explaining the diversity of the peptide structures.

Elucidation of the unexplored biodiversity of ant venom peptidomes via MALDI-TOF mass spectrometry and its application for chemotaxonomy.

The rise of integrative taxonomy, a multi-criteria approach used in characterizing species, fosters the development of new tools facilitating species delimitation. Mass spectrometric (MS) analysis of venom peptides from venomous animals has previously been demonstrated to be a valid method for identifying species. Here we aimed to develop a rapid chemotaxonomic tool for identifying ants based on venom peptide mass fingerprinting. The study focused on the biodiversity of ponerine ants (Hymenoptera: Formicidae: Ponerinae) in French Guiana. Initial experiments optimized the use of automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to determine variations in the mass profiles of ant venoms using several MALDI matrices and additives. Data were then analyzed via a hierarchical cluster analysis to classify the venoms of 17 ant species. In addition, phylogenetic relationships were assessed and were highly correlated with methods using DNA sequencing of the mitochondrial gene cytochrome c oxidase subunit 1. By combining a molecular genetics approach with this chemotaxonomic approach, we were able to improve the accuracy of the taxonomic findings to reveal cryptic ant species within species complexes. This chemotaxonomic tool can therefore contribute to more rapid species identification and more accurate taxonomies. BIOLOGICAL SIGNIFICANCE: This is the first extensive study concerning the peptide analysis of the venom of both Pachycondyla and Odontomachus ants. We studied the venoms of 17 ant species from French Guiana that permitted us to fine-tune the venom analysis of ponerine ants via MALDI-TOF mass spectrometry. We explored the peptidomes of crude ant venom and demonstrated that venom peptides can be used in the identification of ant species. In addition, the application of this novel chemotaxonomic method combined with a parallel genetic approach using COI sequencing permitted us to reveal the presence of cryptic ants within both the Pachycondyla apicalis and Pachycondyla stigma species complexes. This adds a new dimension to the search for means of exploiting the enormous biodiversity of venomous ants as a source for novel therapeutic drugs or biopesticides. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Characterization of monomeric and multimeric snake neurotoxins and other bioactive proteins from the venom of the lethal Australian common copperhead (Austrelaps superbus).

Envenomation by Australian copperheads results mainly in muscle paralysis largely attributed to the presence of postsynaptic α-neurotoxins. However, poorly reversible neurotoxic effects suggest that these venoms may contain snake presynaptic phospholipase A2 neurotoxins (SPANs) that irreversibly inhibit neurotransmitter release. Using size-exclusion liquid chromatography, the present study isolated the first multimeric SPAN complex from the venom of the Australian common copperhead, Austrelaps superbus. The multimeric SPAN P-elapitoxin-As1a (P-EPTX-As1a) along with two novel monomeric SPANs and a new postsynaptic α-neurotoxin were then pharmacologically characterized using the chick biventer cervicis nerve-muscle preparation. All SPANs inhibited nerve-evoked twitch contractions at the neuromuscular junction without inhibiting contractile responses to cholinergic agonists or KCl. These actions are consistent with a prejunctional action to inhibit neurotransmitter release, without direct myotoxicity. Furthermore, the multimeric P-EPTX-As1a caused tetanic 'fade' in muscle tension under high frequency nerve stimulation, and produced a triphasic alteration to neurotransmitter release. These actions have been previously noted with other multimeric SPAN complexes such as taipoxin. Moreover, the neurotoxic α-subunit of P-EPTX-As1a shows high homology to taipoxin α-chain. Several other coagulopathic and myotoxic high mass proteins including a class PIII snake venom metalloproteinase, C-type lectin, l-amino acid oxidase, acetylcholinesterase and phospholipase B were also identified that may contribute to the overall toxicity of A. superbus venom. In conclusion, clinicians should be aware that early antivenom intervention might be necessary to prevent the onset of irreversible presynaptic neurotoxicity caused by multimeric and monomeric SPANs and that A. superbus venom is potentially capable of producing coagulopathic and myotoxic effects.

Unravelling the complex venom landscapes of lethal Australian funnel-web spiders (Hexathelidae: Atracinae) using LC-MALDI-TOF mass spectrometry.

Spider venoms represent vast sources of bioactive molecules whose diversity remains largely unknown. Indeed, only a small subset of species have been studied out of the ~43,000 extant spider species. The present study investigated inter- and intra-species venom complexity in 18 samples collected from a variety of lethal Australian funnel-web spiders (Mygalomorphae: Hexathelidae: Atracinae) using C4 reversed-phase separation coupled to offline MALDI-TOF mass spectrometry (LC-MALDI-TOF MS). An in-depth investigation focusing on four atracine venoms (male Illawarra wisharti, male and female Hadronyche cerberea, and female Hadronyche infensa Toowoomba) revealed, on average, ~800 peptides in female venoms while male venoms contained ~400 peptides, distributed across most HPLC fractions. This is significantly higher than previous estimates of peptide expression in mygalomorph venoms. These venoms also showed distinct intersexual as well as intra- and inter-species variation in peptide masses. Construction of both 3D and 2D contour plots revealed that peptide mass distributions in all 18 venoms were centered around the 3200-5400m/z range and to a lesser extent the 6600-8200m/z range, consistent with previously described hexatoxins. These findings highlight the extensive diversity of peptide toxins in Australian funnel-web spider venoms that that can be exploited as novel therapeutic and biopesticide lead molecules. BIOLOGICAL SIGNIFICANCE: In the present study we describe the complexity of 18 venoms from lethal Australian funnel-web spiders using LC-MALDI-TOF MS. The study includes an in-depth investigation, focusing on four venoms, that revealed the presence of ~800 peptides in female venoms and ~400 peptides in male venoms. This is significantly higher than previous estimates of peptide expression in spider venoms. By constructing both 3D and 2D contour plots we were also able to reveal the distinct intersexual as well as intra- and inter-species variation in venom peptide masses. We show that peptide mass distributions in all 18 venoms were centered around the 3200-5400 m/z range and to a lesser extent the 6600-8200 m/z range, consistent with the small number of previously described hexatoxins from these spiders. These findings highlight the extensive diversity of peptide toxins in Australian funnel-web spider venoms that that can be exploited as novel therapeutic and biopesticide lead molecules. The present study has greatly expanded our understanding of peptide variety and complexity in these lethal mygalomorph spiders. Specifically it highlights both the utility of LC-MALDI-TOF in spider taxonomy and the massive combinatorial peptide libraries that spider venoms offer the pharmaceutical and agrochemical industry.

Pharmacological characterization of α-elapitoxin-Al2a from the venom of the Australian pygmy copperhead (Austrelaps labialis): an atypical long-chain α-neurotoxin with only weak affinity for α7 nicotinic receptors.

Despite the in vivo lethality of venom, neurotoxicity has not previously been considered a significant complication of envenoming by the Australian pygmy copperhead (Austrelaps labialis). However, recent evidence has emerged demonstrating that this venom contains potent presynaptic and postsynaptic neurotoxicity. The present study describes the isolation and pharmacological characterization of the first postsynaptic neurotoxin, α-EPTX-Al2a, from the venom of A. labialis. α-EPTX-Al2a (8072.77 Da) caused a concentration-dependent block of twitch contractions and a complete block of responses to cholinergic agonists in the chick biventer cervicis nerve-muscle preparation. This action is consistent with postjunctional neurotoxicity. Monovalent tiger snake antivenom prevented the onset of neurotoxicity if applied prior to toxin administration, but was only able to partially reverse neurotoxicity once muscle paralysis had developed. α-EPTX-Al2a produced a potent pseudo-irreversible antagonism of chick muscle nicotinic acetylcholine receptors (nAChRs), with an estimated pA(2) value of 7.902 (K(B) = 12.5 nM). Interestingly, the toxin only produced a modest block of neuronal α7 nAChRs, with an IC(50) of 1.2 µM, and failed to inhibit ganglionic α3ß2/α3ß4 nAChRs in a fluorescence-based FLIPR assay using SH-SY5Y cells. α-EPTX-Al2a contained 75 amino acid residues with five disulfide bonds that had significant homology to classical long-chain α-neurotoxins. While α-EPTX-Al2a retains most pharmacophore residues critical for binding to muscle-type (α1)(2)ßγδ nAChRs it lacks the key Ala(28) and Arg(36) residues important for α7 nAChR affinity. Given that A. labialis venom contains both irreversible presynaptic and postsynaptic neurotoxins, clinicians need to be aware of potential neurotoxic complications associated with pygmy copperhead envenomation.