Missiles of Mass Disruption: Composition and Glandular Origin of Venom Used as a Projectile Defensive Weapon by the Assassin Bug Platymeris rhadamanthus.

Assassin bugs (Reduviidae) produce venoms that are insecticidal, and which induce pain in predators, but the composition and function of their individual venom components is poorly understood. We report findings on the venom system of the red-spotted assassin bug Platymeris rhadamanthus, a large species of African origin that is unique in propelling venom as a projectile weapon when threatened. We performed RNA sequencing experiments on venom glands (separate transcriptomes of the posterior main gland, PMG, and the anterior main gland, AMG), and proteomic experiments on venom that was either defensively propelled or collected from the proboscis in response to electrostimulation. We resolved a venom proteome comprising 166 polypeptides. Both defensively propelled venom and most venom samples collected in response to electrostimulation show a protein profile similar to the predicted secretory products of the PMG, with a smaller contribution from the AMG. Pooled venom samples induce calcium influx via membrane lysis when applied to mammalian neuronal cells, consistent with their ability to cause pain when propelled into the eyes or mucus membranes of potential predators. The same venom induces rapid paralysis and death when injected into fruit flies. These data suggest that the cytolytic, insecticidal venom used by reduviids to capture prey is also a highly effective defensive weapon when propelled at predators.

NaV1.1 inhibition can reduce visceral hypersensitivity.

Functional bowel disorder patients can suffer from chronic abdominal pain, likely due to visceral hypersensitivity to mechanical stimuli. As there is only a limited understanding of the basis of chronic visceral hypersensitivity (CVH), drug-based management strategies are ill defined, vary considerably, and include NSAIDs, opioids, and even anticonvulsants. We previously reported that the 1.1 subtype of the voltage-gated sodium (NaV; NaV1.1) channel family regulates the excitability of sensory nerve fibers that transmit a mechanical pain message to the spinal cord. Herein, we investigated whether this channel subtype also underlies the abdominal pain that occurs with CVH. We demonstrate that NaV1.1 is functionally upregulated under CVH conditions and that inhibiting channel function reduces mechanical pain in 3 mechanistically distinct mouse models of chronic pain. In particular, we use a small molecule to show that selective NaV1.1 inhibition (a) decreases sodium currents in colon-innervating dorsal root ganglion neurons, (b) reduces colonic nociceptor mechanical responses, and (c) normalizes the enhanced visceromotor response to distension observed in 2 mouse models of irritable bowel syndrome. These results provide support for a relationship between NaV1.1 and chronic abdominal pain associated with functional bowel disorders.

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects.

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, and the majority of extant heteropterans retain this trophic strategy. Some heteropterans have transitioned to feeding on vertebrate blood (such as the kissing bugs, Triatominae; and bed bugs, Cimicidae) while others have reverted to feeding on plants (most Pentatomomorpha). However, with the exception of saliva used by kissing bugs to facilitate blood-feeding, little is known about heteropteran venoms compared to the venoms of spiders, scorpions and snakes. One obstacle to the characterization of heteropteran venom toxins is the structure and function of the venom/labial glands, which are both morphologically complex and perform multiple biological roles (defense, prey capture, and extra-oral digestion). In this article, we describe three methods we have successfully used to collect heteropteran venoms. First, we present electrostimulation as a convenient way to collect venom that is often lethal when injected into prey animals, and which obviates contamination by glandular tissue. Second, we show that gentle harassment of animals is sufficient to produce venom extrusion from the proboscis and/or venom spitting in some groups of heteropterans. Third, we describe methods to harvest venom toxins by dissection of anaesthetized animals to obtain the venom glands. This method is complementary to other methods, as it may allow harvesting of toxins from taxa in which electrostimulation and harassment are ineffective. These protocols will enable researchers to harvest toxins from heteropteran insects for structure-function characterization and possible applications in medicine and agriculture.

The assassin bug Pristhesancus plagipennis produces two distinct venoms in separate gland lumens.

The assassin bug venom system plays diverse roles in prey capture, defence and extra-oral digestion, but it is poorly characterised, partly due to its anatomical complexity. Here we demonstrate that this complexity results from numerous adaptations that enable assassin bugs to modulate the composition of their venom in a context-dependent manner. Gland reconstructions from multimodal imaging reveal three distinct venom gland lumens: the anterior main gland (AMG); posterior main gland (PMG); and accessory gland (AG). Transcriptomic and proteomic experiments demonstrate that the AMG and PMG produce and accumulate distinct sets of venom proteins and peptides. PMG venom, which can be elicited by electrostimulation, potently paralyses and kills prey insects. In contrast, AMG venom elicited by harassment does not paralyse prey insects, suggesting a defensive role. Our data suggest that assassin bugs produce offensive and defensive venoms in anatomically distinct glands, an evolutionary adaptation that, to our knowledge, has not been described for any other venomous animal.

Centipede venoms as a source of drug leads.

INTRODUCTION: Centipedes are one of the oldest and most successful lineages of venomous terrestrial predators. Despite their use for centuries in traditional medicine, centipede venoms remain poorly studied. However, recent work indicates that centipede venoms are highly complex chemical arsenals that are rich in disulfide-constrained peptides that have novel pharmacology and three-dimensional structure. Areas covered: This review summarizes what is currently known about centipede venom proteins, with a focus on disulfide-rich peptides that have novel or unexpected pharmacology that might be useful from a therapeutic perspective. The authors also highlight the remarkable diversity of constrained three-dimensional peptide scaffolds present in these venoms that might be useful for bioengineering of drug leads. Expert opinion: Like most arthropod predators, centipede venoms are rich in peptides that target neuronal ion channels and receptors, but it is also becoming increasingly apparent that many of these peptides have novel or unexpected pharmacological properties with potential applications in drug discovery and development.

Molecular basis of the remarkable species selectivity of an insecticidal sodium channel toxin from the African spider Augacephalus ezendami.

The inexorable decline in the armament of registered chemical insecticides has stimulated research into environmentally-friendly alternatives. Insecticidal spider-venom peptides are promising candidates for bioinsecticide development but it is challenging to find peptides that are specific for targeted pests. In the present study, we isolated an insecticidal peptide (Ae1a) from venom of the African spider Augacephalus ezendami (family Theraphosidae). Injection of Ae1a into sheep blowflies (Lucilia cuprina) induced rapid but reversible paralysis. In striking contrast, Ae1a was lethal to closely related fruit flies (Drosophila melanogaster) but induced no adverse effects in the recalcitrant lepidopteran pest Helicoverpa armigera. Electrophysiological experiments revealed that Ae1a potently inhibits the voltage-gated sodium channel BgNaV1 from the German cockroach Blattella germanica by shifting the threshold for channel activation to more depolarized potentials. In contrast, Ae1a failed to significantly affect sodium currents in dorsal unpaired median neurons from the American cockroach Periplaneta americana. We show that Ae1a interacts with the domain II voltage sensor and that sensitivity to the toxin is conferred by natural sequence variations in the S1-S2 loop of domain II. The phyletic specificity of Ae1a provides crucial information for development of sodium channel insecticides that target key insect pests without harming beneficial species.

A tarantula-venom peptide antagonizes the TRPA1 nociceptor ion channel by binding to the S1-S4 gating domain.

BACKGROUND: The venoms of predators have been an excellent source of diverse highly specific peptides targeting ion channels. Here we describe the first known peptide antagonist of the nociceptor ion channel transient receptor potential ankyrin 1 (TRPA1). RESULTS: We constructed a recombinant cDNA library encoding ∼100 diverse GPI-anchored peptide toxins (t-toxins) derived from spider venoms and screened this library by coexpression in Xenopus oocytes with TRPA1. This screen resulted in identification of protoxin-I (ProTx-I), a 35-residue peptide from the venom of the Peruvian green-velvet tarantula, Thrixopelma pruriens, as the first known high-affinity peptide TRPA1 antagonist. ProTx-I was previously identified as an antagonist of voltage-gated sodium (NaV) channels. We constructed a t-toxin library of ProTx-I alanine-scanning mutants and screened this library against NaV1.2 and TRPA1. This revealed distinct partially overlapping surfaces of ProTx-I by which it binds to these two ion channels. Importantly, this mutagenesis yielded two novel ProTx-I variants that are only active against either TRPA1or NaV1.2. By testing its activity against chimeric channels, we identified the extracellular loops of the TRPA1 S1-S4 gating domain as the ProTx-I binding site. CONCLUSIONS: These studies establish our approach, which we term "toxineering," as a generally applicable method for isolation of novel ion channel modifiers and design of ion channel modifiers with altered specificity. They also suggest that ProTx-I will be a valuable pharmacological reagent for addressing biophysical mechanisms of TRPA1 gating and the physiology of TRPA1 function in nociceptors, as well as for potential clinical application in the context of pain and inflammation.

Cloning and activity of a novel α-latrotoxin from red-back spider venom.

The venom of the European black widow spider Latrodectus tredecimguttatus (Theridiidae) contains several high molecular mass (110-140 kDa) neurotoxins that induce neurotransmitter exocytosis. These include a vertebrate-specific α-latrotoxin (α-LTX-Lt1a) responsible for the clinical symptoms of latrodectism and numerous insect-specific latroinsectoxins (LITs). In contrast, little is known about the expression of these toxins in other Latrodectus species despite the fact that envenomation by these spiders induces a similar clinical syndrome. Here we report highly conserved α-LTX, α-LIT and δ-LIT sequence tags in Latrodectus mactans, Latrodectus hesperus and Latrodectus hasselti venoms using tandem mass spectrometry, following bioassay-guided separation of venoms by liquid chromatography. Despite this sequence similarity, we show that the anti-α-LTX monoclonal antibody 4C4.1, raised against α-LTX-Lt1a, fails to neutralize the neurotoxicity of all other Latrodectus venoms tested in an isolated chick biventer cervicis nerve-muscle bioassay. This suggests that there are important structural differences between α-LTXs in theridiid spider venoms. We therefore cloned and sequenced the α-LTX from the Australian red-back spider L. hasselti (α-LTX-Lh1a). The deduced amino acid sequence of the mature α-LTX-Lh1a comprises 1180 residues (∼132kDa) with ∼93% sequence identity with α-LTX-Lt1a. α-LTX-Lh1a is composed of an N-terminal domain and a central region containing 22 ankyrin-like repeats. The presence of two furin cleavage sites, conserved with α-LTX-Lt1a, indicates that α-LTX-Lh1a is derived from the proteolytic cleavage of an N-terminal signal peptide and C-terminal propeptide region. However, we show that α-LTX-Lh1a has key substitutions in the 4C4.1 epitope that explains the lack of binding of the monoclonal antibody.