Proteome of urticating setae of Ochrogaster lunifer, a processionary caterpillar of medical and veterinary importance, including primary structures of putative toxins.

Ochrogaster lunifer (Lepidoptera: Notodontidae) is an Australian processionary caterpillar with detachable urticating setae that have a defensive function. These true setae induce inflammation when they contact human skin, and equine foetal loss syndrome if they are accidentally ingested by gravid horses. We used transcriptomics and proteomics to identify proteins and peptides present in and on urticating setae, which may include toxins that contribute to inflammation and/or foetal loss syndromes. This process identified 37 putative toxins, including multiple homologues of the honeybee venom peptide secapin, and proteins with similarity to odorant binding proteins, arylphorins, and the insect immune modulator Diedel. This work identifies candidate molecules that may contribute to the adverse effects of processionary caterpillar setae on human and animal health.

Two Novel Mosquitocidal Peptides Isolated from the Venom of the Bahia Scarlet Tarantula (Lasiodora klugi).

Effective control of diseases transmitted by Aedes aegypti is primarily achieved through vector control by chemical insecticides. However, the emergence of insecticide resistance in A. aegypti undermines current control efforts. Arachnid venoms are rich in toxins with activity against dipteran insects and we therefore employed a panel of 41 spider and 9 scorpion venoms to screen for mosquitocidal toxins. Using an assay-guided fractionation approach, we isolated two peptides from the venom of the tarantula Lasiodora klugi with activity against adult A. aegypti. The isolated peptides were named U-TRTX-Lk1a and U-TRTX-Lk2a and comprised 41 and 49 residues with monoisotopic masses of 4687.02 Da and 5718.88 Da, respectively. U-TRTX-Lk1a exhibited an LD50 of 38.3 pmol/g when injected into A. aegypti and its modeled structure conformed to the inhibitor cystine knot motif. U-TRTX-Lk2a has an LD50 of 45.4 pmol/g against adult A. aegypti and its predicted structure conforms to the disulfide-directed ß-hairpin motif. These spider-venom peptides represent potential leads for the development of novel control agents for A. aegypti.

Horizontal gene transfer underlies the painful stings of asp caterpillars (Lepidoptera: Megalopygidae).

Larvae of the genus Megalopyge (Lepidoptera: Zygaenoidea: Megalopygidae), known as asp or puss caterpillars, produce defensive venoms that cause severe pain. Here, we present the anatomy, chemistry, and mode of action of the venom systems of caterpillars of two megalopygid species, the Southern flannel moth Megalopyge opercularis and the black-waved flannel moth Megalopyge crispata. We show that megalopygid venom is produced in secretory cells that lie beneath the cuticle and are connected to the venom spines by canals. Megalopygid venoms consist of large aerolysin-like pore-forming toxins, which we have named megalysins, and a small number of peptides. The venom system differs markedly from those of previously studied venomous zygaenoids of the family Limacodidae, suggestive of an independent origin. Megalopygid venom potently activates mammalian sensory neurons via membrane permeabilization and induces sustained spontaneous pain behavior and paw swelling in mice. These bioactivities are ablated by treatment with heat, organic solvents, or proteases, indicating that they are mediated by larger proteins such as the megalysins. We show that the megalysins were recruited as venom toxins in the Megalopygidae following horizontal transfer of genes from bacteria to the ancestors of ditrysian Lepidoptera. Megalopygids have recruited aerolysin-like proteins as venom toxins convergently with centipedes, cnidarians, and fish. This study highlights the role of horizontal gene transfer in venom evolution.

Ant venoms contain vertebrate-selective pain-causing sodium channel toxins.

Stings of certain ant species (Hymenoptera: Formicidae) can cause intense, long-lasting nociception. Here we show that the major contributors to these symptoms are venom peptides that modulate the activity of voltage-gated sodium (NaV) channels, reducing their voltage threshold for activation and inhibiting channel inactivation. These peptide toxins are likely vertebrate-selective, consistent with a primarily defensive function. They emerged early in the Formicidae lineage and may have been a pivotal factor in the expansion of ants.

Venom composition and bioactive RF-amide peptide toxins of the saddleback caterpillar, Acharia stimulea (Lepidoptera: Limacodidae).

Limacodidae is a family of lepidopteran insects comprising >1500 species. More than half of these species produce pain-inducing defensive venoms in the larval stage, but little is known about their venom toxins. Recently, we characterised proteinaceous toxins from the Australian limacodid caterpillar Doratifera vulnerans, but it is unknown if the venom of this species is typical of other Limacodidae. Here, we use single animal transcriptomics and venom proteomics to investigate the venom of an iconic limacodid, the North American saddleback caterpillar Acharia stimulea. We identified 65 venom polypeptides, grouped into 31 different families. Neurohormones, knottins, and homologues of the immune signaller Diedel make up the majority of A.stimulea venom, indicating strong similarities to D. vulnerans venom, despite the large geographic separation of these caterpillars. One notable difference is the presence of RF-amide peptide toxins in A. stimulea venom. Synthetic versions of one of these RF-amide toxins potently activated the human neuropeptide FF1 receptor, displayed insecticidal activity when injected into Drosophila melanogaster, and moderately inhibited larval development of the parasitic nematode Haemonchus contortus. This study provides insights into the evolution and activity of venom toxins in Limacodidae, and provides a platform for future structure-function characterisation of A.stimulea peptide toxins.

The Predatory Stink Bug Arma custos (Hemiptera: Pentatomidae) Produces a Complex Proteinaceous Venom to Overcome Caterpillar Prey.

Predatory stink bugs capture prey by injecting salivary venom from their venom glands using specialized stylets. Understanding venom function has been impeded by a scarcity of knowledge of their venom composition. We therefore examined the proteinaceous components of the salivary venom of the predatory stink bug Arma custos (Fabricius, 1794) (Hemiptera: Pentatomidae). We used gland extracts and venoms from fifth-instar nymphs or adult females to perform shotgun proteomics combined with venom gland transcriptomics. We found that the venom of A. custos comprised a complex suite of over a hundred individual proteins, including oxidoreductases, transferases, hydrolases, ligases, protease inhibitors, and recognition, transport and binding proteins. Besides the uncharacterized proteins, hydrolases such as venom serine proteases, cathepsins, phospholipase A2, phosphatases, nucleases, alpha-amylases, and chitinases constitute the most abundant protein families. However, salivary proteins shared by and unique to other predatory heteropterans were not detected in the A. custos venom. Injection of the proteinaceous (>3 kDa) venom fraction of A. custos gland extracts or venom into its prey, the larvae of the oriental armyworm Mythimna separata (Walker, 1865), revealed insecticidal activity against lepidopterans. Our data expand the knowledge of heteropteran salivary proteins and suggest predatory asopine bugs as a novel source for bioinsecticides.

Intra-colony venom diversity contributes to maintaining eusociality in a cooperatively breeding ant.

BACKGROUND: Eusociality is widely considered to evolve through kin selection, where the reproductive success of an individual's close relative is favored at the expense of its own. High genetic relatedness is thus considered a prerequisite for eusociality. While ants are textbook examples of eusocial animals, not all ants form colonies of closely related individuals. One such example is the ectatommine ant Rhytidoponera metallica, which predominantly forms queen-less colonies that have such a low intra-colony relatedness that they have been proposed to represent a transient, unstable form of eusociality. However, R. metallica is among the most abundant and widespread ants on the Australian continent. This apparent contradiction provides an example of how inclusive fitness may not by itself explain the maintenance of eusociality and raises the question of what other selective advantages maintain the eusocial lifestyle of this species. RESULTS: We provide a comprehensive portrait of the venom of R. metallica and show that the colony-wide venom consists of an exceptionally high diversity of functionally distinct toxins for an ant. These toxins have evolved under strong positive selection, which is normally expected to reduce genetic variance. Yet, R. metallica exhibits remarkable intra-colony variation, with workers sharing only a relatively small proportion of toxins in their venoms. This variation is not due to the presence of chemical castes, but has a genetic foundation that is at least in part explained by toxin allelic diversity. CONCLUSIONS: Taken together, our results suggest that the toxin diversity contained in R. metallica colonies may be maintained by a form of group selection that selects for colonies that can exploit more resources and defend against a wider range of predators. We propose that increased intra-colony genetic variance resulting from low kinship may itself provide a selective advantage in the form of an expanded pharmacological venom repertoire. These findings provide an example of how group selection on adaptive phenotypes may contribute to maintaining eusociality where a prerequisite for kin selection is diminished.

Immunosuppressive, antimicrobial and insecticidal activities of inhibitor cystine knot peptides produced by teratocytes of the endoparasitoid wasp Cotesia flavipes (Hymenoptera: Braconidae).

Teratocytes are specialized cells released by parasitoid wasps into their hosts. They are known for producing regulatory molecules that aid the development of immature parasitoids. We have recently reported the primary structures of cystine-rich peptides, including some containing inhibitor cystine knot (ICK) motifs, produced by teratocytes of the parasitoid Cotesia flavipes (Hymenoptera: Braconidae). ICKs are known for their stability and diverse biological functions. In this study, we produced four putative ICK peptides from the teratocytes of C. flavipes using solid-phase peptide synthesis or recombinant expression in E. coli, and investigated their functions on host immune modulation as well their potential to impair the development of two lepidopterans after ingestion of the peptides. In addition, the peptides were assayed against pathogens and human cells. The peptides did not influence total hemocyte count but suppressed cellular immunity, detectable as a reduction of hemocyte encapsulation (CftICK-I, CftICK-II, CftICK-III) and spread indexes (CftICK-IV) in the host. None of the peptides influenced the activities of prophenoloxidase and phenoloxidase in the hemolymph of larval Diatraea saccharalis (Lepidoptera: Crambidae). CftICK-I and CftICK-II with previously unknown function showed antifungal activity against Candida albicans but were non-toxic to human cells. CftICK-I, CftICK-II, and CftICK-III increased larval mortality and reduced leaf consumption of D. saccharalis, a permissive host for C. flavipes. The CftICK-III also increased larval mortality and reduced leaf consumption of Spodoptera frugiperda (Lepidoptera: Noctuidae), a non-permissive host for C. flavipes. This study highlights biological functions and biotechnological potential of ICK peptides from the teratocytes of C. flavipes.

Venom composition and pain-causing toxins of the Australian great carpenter bee Xylocopa aruana.

Most species of bee are capable of delivering a defensive sting which is often painful. A solitary lifestyle is the ancestral state of bees and most extant species are solitary, but information on bee venoms comes predominantly from studies on eusocial species. In this study we investigated the venom composition of the Australian great carpenter bee, Xylocopa aruana Ritsema, 1876. We show that the venom is relatively simple, composed mainly of one small amphipathic peptide (XYTX1-Xa1a), with lesser amounts of an apamin homologue (XYTX2-Xa2a) and a venom phospholipase-A2 (PLA2). XYTX1-Xa1a is homologous to, and shares a similar mode-of-action to melittin and the bombilitins, the major components of the venoms of the eusocial Apis mellifera (Western honeybee) and Bombus spp. (bumblebee), respectively. XYTX1-Xa1a and melittin directly activate mammalian sensory neurons and cause spontaneous pain behaviours in vivo, effects which are potentiated in the presence of venom PLA2. The apamin-like peptide XYTX2-Xa2a was a relatively weak blocker of small conductance calcium-activated potassium (KCa) channels and, like A. mellifera apamin and mast cell-degranulating peptide, did not contribute to pain behaviours in mice. While the composition and mode-of-action of the venom of X. aruana are similar to that of A. mellifera, the greater potency, on mammalian sensory neurons, of the major pain-causing component in A. mellifera venom may represent an adaptation to the distinct defensive pressures on eusocial Apidae.

Composition and toxicity of venom produced by araneophagous white-tailed spiders (Lamponidae: Lampona sp.).

Prey-specialised spiders are adapted to capture specific prey items, including dangerous prey. The venoms of specialists are often prey-specific and less complex than those of generalists, but their venom composition has not been studied in detail. Here, we investigated the venom of the prey-specialised white-tailed spiders (Lamponidae: Lampona), which utilise specialised morphological and behavioural adaptations to capture spider prey. We analysed the venom composition using proteo-transcriptomics and taxon-specific toxicity using venom bioassays. Our analysis identified 208 putative toxin sequences, comprising 103 peptides < 10 kDa and 105 proteins > 10 kDa. Most peptides belonged to one of two families characterised by scaffolds containing eight or ten cysteine residues. Toxin-like proteins showed similarity to galectins, leucine-rich repeat proteins, trypsins and neprilysins. The venom of Lampona was shown to be more potent against the preferred spider prey than against alternative cricket prey. In contrast, the venom of a related generalist was similarly potent against both prey types. These data provide insights into the molecular adaptations of venoms produced by prey-specialised spiders.

Untangling interactions between Bitis vipers and their prey using coagulotoxicity against diverse vertebrate plasmas.

Venom is a key evolutionary innovation which plays a primary role in prey subjugation by venomous snakes. However, while there is a growing body of literature indicating the composition and activity of snake venoms is under strong natural selection driven by differences in prey physiology, the majority of studies have historically focussed on the activity of snake venoms with regards only towards human or mammalian physiologies. This study aimed to use clotting assays measuring both time and strength of clotting to characterise the coagulotoxic activity of venoms from a taxonomically, morphologically, and ecologically diverse range of Bitis spp. of viperid snakes upon the plasma of model species: amphibian (Cane Toad, Rhinella marina); lizard (Blue-tongue Skink, Tiliqua scincoides); avian (Domestic Chicken, Gallus gallus); and rodent (Brown Rat, Rattus norvegicus). Significant variation in coagulotoxic activity across the different plasmas was observed between species and compared to the known affects upon human plasma. Bitis caudalis was notable in being active on all four plasmas, but in extremely divergent manners: accelerating clotting times and producing strong, stable clots upon amphibian plasma (consistent with true procoagulation); accelerating clotting time but producing weak, unstable clots upon lizard plasma (consistent with pseudo-procoagulation); delaying avian clotting time beyond machine maximum reading time (strong anticoagulation consistent with either inhibition of clotting enzymes or total destruction of fibrinogen, or both); and delaying clotting of rodent plasma (consistent with inhibition of clotting enzymes) and with only weak clots formed (consistent with destruction of fibrinogen). In contrast, the sister species B. peringueyi and B. schneideri displayed activity only upon the lizard plasma, slightly accelerating the clotting times to produce weak, unstable clots (consistent with pseudo-procoagulation). The other dwarf species, B. cornuta, displayed strong anticoagulation upon avian and rodent plasmas, delaying clotting beyond the machine maximum reading time (strong anticoagulation consistent with either inhibition of clotting enzymes or total destruction of fibrinogen, or both). In contrast, the giant species studied (B. gabonica) showed only a very weak pseudo-procoagulant activity upon lizard plasma. The wide range of variation seen within this study highlights the importance of studying venom activity on relevant models when making conclusions about the ecological role of venoms and the extreme limitation in extrapolating animal results to predict potential human clinical effects.

Proteotranscriptomics reveals the secretory dynamics of teratocytes, regulators of parasitization by an endoparasitoid wasp.

Parasitoid wasps have evolved sophisticated mechanisms of host regulation that establish a favorable environment for the development of immature parasitoids. While maternal venom and symbiotic virus-like particles are well-known mechanisms of host regulation, another less-studied mechanism is the secretion of host regulation factors by cells called teratocytes, extra-embryonic cells released during parasitoid larval eclosion. Consequently, identification and characterization of teratocyte secretory products has not been reported in detail for any parasitoid wasp. We aimed to analyze teratocyte secretory products released into hemolymph of the larval sugarcane borer Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) by its biological control agent, the koinobiont endoparasitoid wasp Cotesia flavipes Cameron, 1891 (Hymenoptera: Braconidae). Teratocytes were released upon eclosion of parasitoid larvae four days after parasitization (DAP) and increased in number and size until six DAP. Total D. saccharalis hemocyte viability was reduced immediately after parasitization until DAP 2, while total hemocyte count was lower from the third DAP, and phenoloxidase and lysozyme activity were disrupted compared to non-parasitized controls. To examine the secretory products of teratocytes, we generated a teratocyte transcriptome and compared its in silico translated open reading frames to mass spectra obtained from hemolymph from parasitized and unparasitized hosts. This led to the identification of 57 polypeptides secreted by teratocytes, the abundance of which we tracked over 0-10 DAP. Abundant teratocyte products included proteins similar to bracovirus proteins and multiple disulfide-rich peptides. Most teratocyte products accumulated in hemolymph, reaching their highest concentrations immediately before parasitoid pupation. Our results provide insights into host regulation by teratocytes and reveal molecules that may be useful in biotechnology.

A pain-causing and paralytic ant venom glycopeptide.

Ants (Hymenoptera: Formicidae) are familiar inhabitants of most terrestrial environments. Although we are aware of the ability of many species to sting, knowledge of ant venom chemistry remains limited. Herein, we describe the discovery and characterization of an O-linked glycopeptide (Mg7a) as a major component of the venom of the ant Myrmecia gulosa. Electron transfer dissociation and higher-energy collisional dissociation tandem mass spectrometry were used to localize three α-N-acetylgalactosaminyl residues (α-GalNAc) present on the 63-residue peptide. To allow for functional studies, we synthesized the full-length glycosylated peptide via solid-phase peptide synthesis, combined with diselenide-selenoester ligation-deselenization chemistry. We show that Mg7a is paralytic and lethal to insects, and triggers pain behavior and inflammation in mammals, which it achieves through a membrane-targeting mode of action. Deglycosylation of Mg7a renders it insoluble in aqueous solution, suggesting a key solubilizing role of the O-glycans.

Venom composition of the endoparasitoid wasp Cotesia flavipes (Hymenoptera: Braconidae) and functional characterization of a major venom peptide.

Endoparasitoid wasps use complex biochemical arsenals to suppress the normal humoral and cellular immune responses of their hosts in order to transform them into a suitable environment for development of their eggs and larvae. Venom injected during oviposition is a key component of this arsenal, but the functions of individual venom toxins are still poorly understood. Furthermore, there has been little investigation of the potential biotechnological use of these venom toxins, for example for control of agricultural pests. The endoparasitoid Cotesia flavipes (Hymenoptera: Braconidae) is a biocontrol agent reared in biofactories and released extensively in Brazil to control the sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae). The objectives of this work were to reveal venom components produced by C. flavipes and explore the function of a major venom peptide, Cf4. Using a combined proteomic/transcriptomic approach, we identified 38 putative venom toxins including both linear and disulfide-rich peptides, hydrolases, protease inhibitors, apolipophorins, lipid-binding proteins, and proteins of the odorant binding families. Because of its high abundance in the venom, we selected Cf4, a 33-residue peptide with three disulfide bonds, for synthesis and further characterization. We found that synthetic Cf4 reduced the capacity of D. saccharalis hemocytes to encapsulate foreign bodies without any effect on phenoloxidase activity, consistent with a role in disruption of the cellular host immune response. Feeding leaves coated with Cf4 to neonate D. saccharalis resulted in increased mortality and significantly reduced feeding compared to caterpillars fed untreated leaves, indicating that Cf4 is a potential candidate for insect pest control through ingestion. This study adds to our knowledge of endoparasitoid wasp venoms composition, host regulation mechanisms and their biotechnological potential for pest management.

Multipurpose peptides: The venoms of Amazonian stinging ants contain anthelmintic ponericins with diverse predatory and defensive activities.

In the face of increasing drug resistance, the development of new anthelmintics is critical for controlling nematodes that parasitise livestock. Although hymenopteran venom toxins have attracted attention for applications in agriculture and medicine, few studies have explored their potential as anthelmintics. Here we assessed hymenopteran venoms as a possible source of new anthelmintic compounds by screening a panel of ten hymenopteran venoms against Haemonchus contortus, a major pathogenic nematode of ruminants. Using bioassay-guided fractionation coupled with liquid chromatography-tandem mass spectrometry, we identified four novel anthelmintic peptides (ponericins) from the venom of the neotropical ant Neoponera commutata and the previously described ponericin M-PONTX-Na1b from Neoponera apicalis venom. These peptides inhibit H. contortus development with IC50 values of 2.8-5.6 µM. Circular dichroism spectropolarimetry indicated that the ponericins are unstructured in aqueous solution but adopt α-helical conformations in lipid mimetic environments. We show that the ponericins induce non-specific membrane perturbation, which confers broad-spectrum antimicrobial, insecticidal, cytotoxic, hemolytic, and algogenic activities, with activity across all assays typically correlated. We also show for the first time that ponericins induce spontaneous pain behaviour when injected in mice. We propose that the broad-spectrum activity of the ponericins enables them to play both a predatory and defensive role in neoponeran ants, consistent with their high abundance in venom. This study reveals a broader functionality for ponericins than previously assumed, and highlights both the opportunities and challenges in pursuing ant venom peptides as potential therapeutics.

Venom chemistry underlying the painful stings of velvet ants (Hymenoptera: Mutillidae).

Velvet ants (Hymenoptera: Mutillidae) are a family of solitary parasitoid wasps that are renowned for their painful stings. We explored the chemistry underlying the stings of mutillid wasps of the genus Dasymutilla Ashmead. Detailed analyses of the venom composition of five species revealed that they are composed primarily of peptides. We found that two kinds of mutillid venom peptide appear to be primarily responsible for the painful effects of envenomation. These same peptides also have defensive utility against invertebrates, since they were able to incapacitate and kill honeybees. Both act directly on cell membranes where they directly increase ion conductivity. The defensive venom peptides of Dasymutilla bear a striking similarity, in structure and mode of action, to those of the ant Myrmecia gulosa (Fabricius), suggesting either retention of ancestral toxins, or convergence driven by similar life histories and defensive selection pressures. Finally, we propose that other highly expressed Dasymutilla venom peptides may play a role in parasitisation, possible in delay or arrest of host development. This study represents the first detailed account of the composition and function of the venoms of the Mutillidae.

Production, composition, and mode of action of the painful defensive venom produced by a limacodid caterpillar, Doratifera vulnerans.

Venoms have evolved independently several times in Lepidoptera. Limacodidae is a family with worldwide distribution, many of which are venomous in the larval stage, but the composition and mode of action of their venom is unknown. Here, we use imaging technologies, transcriptomics, proteomics, and functional assays to provide a holistic picture of the venom system of a limacodid caterpillar, Doratifera vulnerans Contrary to dogma that defensive venoms are simple in composition, D. vulnerans produces a complex venom containing 151 proteinaceous toxins spanning 59 families, most of which are peptides <10 kDa. Three of the most abundant families of venom peptides (vulnericins) are 1) analogs of the adipokinetic hormone/corazonin-related neuropeptide, some of which are picomolar agonists of the endogenous insect receptor; 2) linear cationic peptides derived from cecropin, an insect innate immune peptide that kills bacteria and parasites by disrupting cell membranes; and 3) disulfide-rich knottins similar to those that dominate spider venoms. Using venom fractionation and a suite of synthetic venom peptides, we demonstrate that the cecropin-like peptides are responsible for the dominant pain effect observed in mammalian in vitro and in vivo nociception assays and therefore are likely to cause pain after natural envenomations by D. vulnerans Our data reveal convergent molecular evolution between limacodids, hymenopterans, and arachnids and demonstrate that lepidopteran venoms are an untapped source of novel bioactive peptides.

Crouching Tiger, Hidden Protein: Searching for Insecticidal Toxins in Venom of the Red Tiger Assassin Bug (Havinthus rufovarius).

Assassin bugs are venomous insects that prey on other arthropods. Their venom has lethal, paralytic, and liquifying effects when injected into prey, but the toxins responsible for these effects are unknown. To identify bioactive assassin bug toxins, venom was harvested from the red tiger assassin bug (Havinthus rufovarius), an Australian species whose venom has not previously been characterised. The venom was fractionated using reversed-phase high-performance liquid chromatography, and four fractions were found to cause paralysis and death when injected into sheep blowflies (Lucilia cuprina). The amino acid sequences of the major proteins in two of these fractions were elucidated by comparing liquid chromatography/tandem mass spectrometry data with a translated venom-gland transcriptome. The most abundant components were identified as a solitary 12.8 kDa CUB (complement C1r/C1s, Uegf, Bmp1) domain protein and a 9.5 kDa cystatin. CUB domains are present in multidomain proteins with diverse functions, including insect proteases. Although solitary CUB domain proteins have been reported to exist in other heteropteran venoms, such as that of the bee killer assassin bug Pristhesancus plagipennis, their function is unknown, and they have not previously been reported as lethal or paralysis-inducing. Cystatins occur in the venoms of spiders and snakes, but again with an unknown function. Reduction and alkylation experiments revealed that the H. rufovarius venom cystatin featured five cysteine residues, one of which featured a free sulfhydryl group. These data suggest that solitary CUB domain proteins and/or cystatins may contribute to the insecticidal activity of assassin bug venom.

Two for the Price of One: Heterobivalent Ligand Design Targeting Two Binding Sites on Voltage-Gated Sodium Channels Slows Ligand Dissociation and Enhances Potency.

Voltage-gated sodium (NaV) channels are pore-forming transmembrane proteins that play essential roles in excitable cells, and they are key targets for antiepileptic, antiarrhythmic, and analgesic drugs. We implemented a heterobivalent design strategy to modulate the potency, selectivity, and binding kinetics of NaV channel ligands. We conjugated µ-conotoxin KIIIA, which occludes the pore of the NaV channels, to an analogue of huwentoxin-IV, a spider-venom peptide that allosterically modulates channel gating. Bioorthogonal hydrazide and copper-assisted azide-alkyne cycloaddition conjugation chemistries were employed to generate heterobivalent ligands using polyethylene glycol linkers spanning 40-120 Å. The ligand with an 80 Å linker had the most pronounced bivalent effects, with a significantly slower dissociation rate and 4-24-fold higher potency compared to those of the monovalent peptides for the human NaV1.4 channel. This study highlights the power of heterobivalent ligand design and expands the repertoire of pharmacological probes for exploring the function of NaV channels.

The evolutionary dynamics of venom toxins made by insects and other animals.

Animal venoms are recognised as unique biological systems in which to study molecular evolution. Venom use has evolved numerous times among the insects, and insects today use venom to capture prey, defend themselves from predators, or to subdue and modulate host responses during parasitism. However, little is known about most insect venom toxins or the mode and tempo by which they evolve. Here, I review the evolutionary dynamics of insect venom toxins, and argue that insects offer many opportunities to examine novel aspects of toxin evolution. The key questions addressed are: How do venomous animals evolve from non-venomous animals, and how does this path effect the composition and pharmacology of the venom? What genetic processes (gene duplication, co-option, neofunctionalisation) are most important in toxin evolution? What kinds of selection pressures are acting on toxin-encoding genes and their cognate targets in envenomated animals? The emerging evidence highlights that venom composition and pharmacology adapts quickly in response to changing selection pressures resulting from new ecological interactions, and that such evolution occurs through a stunning variety of genetic mechanisms. Insects offer many opportunities to investigate the evolutionary dynamics of venom toxins due to their evolutionary history rich in venom-related adaptations, and their quick generation time and suitability for culture in the laboratory.