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.

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.

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.

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.

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.

Deadly Proteomes: A Practical Guide to Proteotranscriptomics of Animal Venoms.

Animal venoms are renowned for their toxicity, biochemical complexity, and as a source of compounds with potential applications in medicine, agriculture, and industry. Polypeptides underlie much of the pharmacology of animal venoms, and elucidating these arsenals of polypeptide toxins-known as the venom proteome or venome-is an important step in venom research. Proteomics is used for the identification of venom toxins, determination of their primary structure including post-translational modifications, as well as investigations into the physiology underlying their production and delivery. Advances in proteomics and adjacent technologies has led to a recent upsurge in publications reporting venom proteomes. Improved mass spectrometers, better proteomic workflows, and the integration of next-generation sequencing of venom-gland transcriptomes and venomous animal genomes allow quicker and more accurate profiling of venom proteomes with greatly reduced starting material. Technologies such as imaging mass spectrometry are revealing additional insights into the mechanism, location, and kinetics of venom toxin production. However, these numerous new developments may be overwhelming for researchers designing venom proteome studies. Here, the field of venom proteomics is reviewed and some practical solutions for simplifying mass spectrometry workflows to study animal venoms are offered.

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.

Giant fish-killing water bug reveals ancient and dynamic venom evolution in Heteroptera.

True Bugs (Insecta: Heteroptera) produce venom or saliva with diverse bioactivities depending on their feeding strategies. However, little is known about the molecular evolution of the venom toxins underlying these biological activities. We examined venom of the giant fish-killing water bug Lethocerus distinctifemur (Insecta: Belostomatidae) using infrared spectroscopy, transcriptomics, and proteomics. We report 132 venom proteins including putative enzymes, cytolytic toxins, and antimicrobial peptides. Over 73% (96 proteins) showed homology to venom proteins from assassin bugs (Reduviidae), including 21% (28 proteins from seven families) not known from other sources. These data suggest that numerous protein families were recruited into venom and diversified rapidly following the switch from phytophagy to predation by ancestral heteropterans, and then were retained over > 200 my of evolution. In contrast, trophic switches to blood-feeding (e.g. in Triatominae and Cimicidae) or reversions to plant-feeding (e.g., in Pentatomomorpha) were accompanied by rapid changes in the composition of venom/saliva, including the loss of many protein families.

Melt With This Kiss: Paralyzing and Liquefying Venom of The Assassin Bug Pristhesancus plagipennis (Hemiptera: Reduviidae).

Assassin bugs (Hemiptera: Heteroptera: Reduviidae) are venomous insects, most of which prey on invertebrates. Assassin bug venom has features in common with venoms from other animals, such as paralyzing and lethal activity when injected, and a molecular composition that includes disulfide-rich peptide neurotoxins. Uniquely, this venom also has strong liquefying activity that has been hypothesized to facilitate feeding through the narrow channel of the proboscis-a structure inherited from sap- and phloem-feeding phytophagous hemipterans and adapted during the evolution of Heteroptera into a fang and feeding structure. However, further understanding of the function of assassin bug venom is impeded by the lack of proteomic studies detailing its molecular composition.By using a combined transcriptomic/proteomic approach, we show that the venom proteome of the harpactorine assassin bug Pristhesancus plagipennis includes a complex suite of >100 proteins comprising disulfide-rich peptides, CUB domain proteins, cystatins, putative cytolytic toxins, triabin-like protein, odorant-binding protein, S1 proteases, catabolic enzymes, putative nutrient-binding proteins, plus eight families of proteins without homology to characterized proteins. S1 proteases, CUB domain proteins, putative cytolytic toxins, and other novel proteins in the 10-16-kDa mass range, were the most abundant venom components. Thus, in addition to putative neurotoxins, assassin bug venom includes a high proportion of enzymatic and cytolytic venom components likely to be well suited to tissue liquefaction. Our results also provide insight into the trophic switch to blood-feeding by the kissing bugs (Reduviidae: Triatominae). Although some protein families such as triabins occur in the venoms of both predaceous and blood-feeding reduviids, the composition of venoms produced by these two groups is revealed to differ markedly. These results provide insights into the venom evolution in the insect suborder Heteroptera.

More than one way to spin a crystallite: multiple trajectories through liquid crystallinity to solid silk.

Arthropods face several key challenges in processing concentrated feedstocks of proteins (silk dope) into solid, semi-crystalline silk fibres. Strikingly, independently evolved lineages of silk-producing organisms have converged on the use of liquid crystal intermediates (mesophases) to reduce the viscosity of silk dope and assist the formation of supramolecular structure. However, the exact nature of the liquid-crystal-forming-units (mesogens) in silk dope, and the relationship between liquid crystallinity, protein structure and silk processing is yet to be fully elucidated. In this review, we focus on emerging differences in this area between the canonical silks containing extended-ß-sheets made by silkworms and spiders, and 'non-canonical' silks made by other insect taxa in which the final crystallites are coiled-coils, collagen helices or cross-ß-sheets. We compared the amino acid sequences and processing of natural, regenerated and recombinant silk proteins, finding that canonical and non-canonical silk proteins show marked differences in length, architecture, amino acid content and protein folding. Canonical silk proteins are long, flexible in solution and amphipathic; these features allow them both to form large, micelle-like mesogens in solution, and to transition to a crystallite-containing form due to mechanical deformation near the liquid-solid transition. By contrast, non-canonical silk proteins are short and have rod or lath-like structures that are well suited to act both as mesogens and as crystallites without a major intervening phase transition. Given many non-canonical silk proteins can be produced at high yield in E. coli, and that mesophase formation is a versatile way to direct numerous kinds of supramolecular structure, further elucidation of the natural processing of non-canonical silk proteins may to lead to new developments in the production of advanced protein materials.

Convergently-evolved structural anomalies in the coiled coil domains of insect silk proteins.

The use of coiled coil proteins as the basis of silk materials is an engineering solution that has evolved convergently in at least five insect lineages-the stinging hymenopterans (ants, bees, hornets), argid sawflies, fleas, lacewings, and praying mantises-and persisted throughout large radiations of these insect families. These coiled coil silk proteins share a characteristic distinct from other coiled coil proteins, in that they are fabricated into solid materials after accumulating as highly concentrated solutions within dedicated glands. Here, we relate the amino acid sequences of these proteins to the secondary and tertiary structural information available from biophysical methods such as X-ray scattering, nuclear magnetic resonance and Raman spectroscopy. We investigate conserved and convergently evolved features within these proteins and compare these to the features of classic coiled coil proteins including tropomyosin and leucine zippers. Our analysis finds that the coiled coil domains of insect silk proteins have several common structural anomalies including a high prevalence of alanine residues in core positions. These atypical features of the coiled coil fibrous proteins - which likely produce deviations from canonical coiled-coil structure - likely exist due to selection pressures related to the process of silk fabrication and the final function of the proteins.

A comparison of convergently evolved insect silks that share ß-sheet molecular structure.

Raspy crickets produce silk webs that are used to build shelters. These webs have been found to consist of both fiber and film components. Raman spectra obtained from both components were found to be very similar for a given species. The protein structure of the fibers and films produced by both species was predominately ß-sheet with lesser amounts of ß-turns, unordered and α-helical protein also detected. The orientation of the ß-sheet backbone in the fiber was determined to be parallel to the fiber axis. Compared to cocoon and dragline silk the orientation distribution exhibits a significant randomly orientated protein component. Amino acid analysis confirmed the presence of glycine, serine, and alanine in both species, which are known to form antiparallel ß-sheet structures. Both species, although at significantly different concentrations, where found to contain proline. This amino acid is uncommon in insect silks, and likely involved in increasing fiber elasticity.

Phylogeny, envenomation syndrome, and membrane permeabilising venom produced by Australia's electric caterpillar Comana monomorpha.

Zygaenoidea is a superfamily of lepidopterans containing many venomous species, including the Limacodidae (nettle caterpillars) and Megalopygidae (asp caterpillars). Venom proteomes have been recently documented for several species from each of these families, but further data are required to understand the evolution of venom in Zygaenoidea. In this study, we examined the 'electric' caterpillar from North-Eastern Australia, a limacodid caterpillar densely covered in venomous spines. We used DNA barcoding to identify this caterpillar as the larva of the moth Comana monomorpha (Turner, 1904). We report the clinical symptoms of C. monomorpha envenomation, which include acute pain, and erythema and oedema lasting for more than a week. Combining transcriptomics of venom spines with proteomics of venom harvested from the spine tips revealed a venom markedly different in composition from previously examined limacodid venoms that are rich in peptides. In contrast, the venom of C. monomorpha is rich in aerolysin-like proteins similar to those found in venoms of asp caterpillars (Megalopygidae). Consistent with this composition, the venom potently permeabilises sensory neurons and human neuroblastoma cells. This study highlights the diversity of venom composition in Limacodidae.

Structure and bioactivity of an insecticidal trans-defensin from assassin bug venom.

Disulfide-rich peptides such as defensins play diverse roles in immunity and ion channel modulation, as well as constituting the bioactive components of many animal venoms. We investigated the structure and bioactivity of U-RDTX-Pp19, a peptide previously discovered in venom of the assassin bug Pristhesancus plagipennis. Recombinant Pp19 (rPp19) was found to possess insecticidal activity when injected into Drosophila melanogaster. A bioinformatic search revealed that domains homologous to Pp19 are produced by assassin bugs and diverse other arthropods. rPp19 co-eluted with native Pp19 isolated from P. plagipennis, which we found is more abundant in hemolymph than venom. We solved the three-dimensional structure of rPp19 using 2D 1H NMR spectroscopy, finding that it adopts a disulfide-stabilized structure highly similar to known trans-defensins, with the same cystine connectivity as human α-defensin (I-VI, II-IV, and III-V). The structure of Pp19 is unique among reported structures of arthropod peptides.

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.

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.

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.

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.

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.

Natural templates for coiled-coil biomaterials from praying mantis egg cases.

Whereas there is growing interest in producing biomaterials containing coiled-coils, relatively few studies have made use of naturally occurring fibrous proteins. In this study, we have characterized fibrous proteins used by mother praying mantises to produce an extensive covering for their eggs called an ootheca and demonstrate the production of artificial ootheca using recombinantly produced proteins. Examination of natural oothecae by infrared spectroscopy and solid-state nuclear magnetic resonance revealed the material to consist of proteins organized predominately as coiled-coils. Two structural proteins, Mantis Fibroin 1 and Mantis Fibroin 2, were identified in ootheca from each of three species. Between species, the primary sequences of both proteins had diverged considerably, but other features were tightly conserved, including low molecular weight, high abundance of Ala, Glu, Lys, and Ser, and a triblock-like architecture with extensive central coiled-coil domain. Mantis fibroin hydrophobic cores had an unusual composition containing high levels of alanine and aromatic residues. Recombinantly produced mantis fibroins folded into coiled-coils in solution and could be fabricated into solid materials with high coiled-coil content. The structural features of mantis fibroins and their straightforward recombinant production make them promising templates for the production of coiled-coil biomimetics materials.