Lacewing Venom: Linking molecular and phenotypic evolution

• Mendel, Helen C; Hopping, Gene; Undheim, Eivind Andreas Baste; Zuegg, Johannes; Lewis, Richard J & Forbes, Briony E [Show all 8 contributors for this article] (2025). PeptideMiner—neuropeptide discovery across the animal kingdom. GigaScience. 14. doi: 10.1093/gigascience/giaf078. Full text in Research Archive Show summary

  Neuropeptides represent the largest and most diverse class of cell-to-cell signaling molecules, holding important roles in animal physiology and behavior. They are evolutionarily ancient and widely distributed across the animal kingdom. Although over 200 neuropeptides have been identified, only a small fraction has been functionally characterized. A recognized bottleneck is the lack of effective tools to study their biological roles and therapeutic potential. Interestingly, neuropeptide-like peptides are also found in animal venoms, where they contribute to prey capture or defensive strategies. Mapping neuropeptide families across the animal kingdom is challenging due to their high sequence divergence and short mature peptide sequences. To address this, we developed PeptideMiner, a search tool that employs profile-hidden Markov models (profile-HMMs) for family-specific peptide discovery. PeptideMiner was systematically validated and benchmarked against existing methods, demonstrating its superior performance. By applying PeptideMiner to several venom transcriptomes—including 24 previously unpublished datasets—we identified 10 novel natriuretic peptides from distantly related species and 57 novel insulin-like sequences from marine predatory cone snails. Chemical synthesis and structure–activity relationship studies of newly identified conoinsulins at human insulin receptors emphasized the value of our approach in elucidating ligand–receptor interactions and discovering new pharmacological probes and therapeutic leads. PeptideMiner offers a powerful platform for discovering new bioactive peptides and family-specific analogues, accelerating both natural product discovery and evolutionary research.

• Isaksen, Anders; Nachtigall, Pedro Gabriel; Araya, Robin Aasegg; Beh, Jia Hao; Robinson, Samuel D. & Hansen, Thomas Fredrik [Show all 7 contributors for this article] (2025). Genome of the green-head ant, Rhytidoponera metallica, reveals mechanisms of toxin evolution in a genetically hyper-diverse eusocial species. Genome Biology. ISSN 1474-7596. 26(1). doi: 10.1186/s13059-025-03777-2. Full text in Research Archive Show summary

  Background While ants are textbook examples of eusocial animals in which altruistic behavior is maintained through kin selection, several ants form genetically diverse colonies that challenge this concept. One example is the Australian green-head ant (Rhytidoponera metallica) whose colonies harbor such extreme genetic variation that they have been speculated to represent an unstable form of eusociality. Yet, R. metallica is among the most successful ants on the Australian subcontinent. This success has been hypothesized to be partly due to the diverse venoms harbored within each colony. However, the genomic basis and evolutionary scenarios that maintain this toxin diversity remain unknown. Results To examine toxin genomic architecture, quantify individual-level genetic variation, and identify both proximate and ultimate mechanisms that have facilitated the toxin diversity in R. metallica, we generate a high-quality draft genome from a single worker. Most ectatotoxin genes are in clusters that contain evidence of multiple, complex gene-family expansions, some of which are likely explained by the presence of transposable elements. We also show that toxin regions of the genome exhibit elevated genetic variation despite being under strong selection and that this variation can translate to phenotypic diversity through toxin alleles with different functional properties. Conclusions Taken together, our results point to classical gene duplication and diversification as the main evolutionary mechanism by which the main toxin family in ant venoms evolves, suggest toxin-gene functional diversification under frequency-dependent selection maintains colony-level venom hypervariability in R. metallica, and provide new insight into the role of multi-level selection in eusocial animals.

• Maurstad, Marius Filomeno; Ramiro, Iris Bea; Øyen, Jan Philip; Sombke, Andy; Büsse, Sebastian & Nachtigall, Pedro Gabriel [Show all 8 contributors for this article] (2025). The green lacewing venom system and the complex mechanisms underlying its evolution. Molecular Biology and Evolution (MBE). ISSN 0737-4038. 43. doi: 10.1093/molbev/msaf326. Full text in Research Archive Show summary

  Abstract Venom has independently evolved across many lineages, yet relatively few have been studied in detail, particularly among insects. Of these, Neuroptera (lacewings, antlions and relatives) remain largely unexplored, despite being widespread with agriculturally important groups such as green lacewings. While adults are non-venomous, neuropteran larvae are ferocious predators that use pincer-like mouthparts to inject paralysing and liquefying venom to subdue and consume their prey. Here, we provide a comprehensive investigation of the venom system in Neuroptera by integrating a high-quality genome, long-read transcriptomes spanning all life stages, microCT-reconstruction of venom glands, tissue-specific expression analyses, venom proteomics, and functional assays of the common green lacewing Chrysoperla carnea. We provide a re-description of the neuropteran venom system, demonstrate the venom’s insecticidal and cytotoxic activity, and show the venom comprises diverse toxin gene families and is richer and more similar to the venom of antlions than previously proposed. We show that this toxin arsenal is the result of a multitude of evolutionary events that include co-option, recruitment following gene duplication, diversification of toxin-paralogs by gene duplication, and functional innovation of new paralogs through both small structural and large architectural changes. In addition, we find that alternative splicing of toxin genes is an important contributor to the biochemical arsenal, which is a mechanism rarely documented among venomous animals. Our results demonstrate how multiple genomic and evolutionary mechanisms together contribute to the emergence and evolution of a complex molecular trait, and provide new insights into the evolution of venom in insects.

• Schendel, Vanessa; Hamilton, Brett R.; Robinson, Samuel D.; Green, Kathryn; Sayre, Marcel E. & Brown, Darren [Show all 13 contributors for this article] (2025). Exaptation of an evolutionary constraint enables behavioural control over the composition of secreted venom in a giant centipede. Nature Ecology and Evolution. 383. doi: 10.1038/s41559-024-02556-9. Full text in Research Archive Show summary

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• Seldeslachts, Andrea; Maurstad, Marius Filomeno; Øyen, Jan Philip; Undheim, Eivind Andreas Baste; Peigneur, Steve & Tytgat, Jan (2024). Exploring oak processionary caterpillar induced lepidopterism (Part 1): unveiling molecular insights through transcriptomics and proteomics. Cellular and Molecular Life Sciences (CMLS). ISSN 1420-682X. 81. doi: 10.1007/s00018-024-05330-z. Full text in Research Archive Show summary

  Lepidopterism, a skin inflammation condition caused by direct or airborne exposure to irritating hairs (setae) from processionary caterpillars, is becoming a significant public health concern. Recent outbreaks of the oak processionary caterpillar (Thaumetopoea processionea) have caused noteworthy health and economic consequences, with a rising frequency expected in the future, exacerbated by global warming promoting the survival of the caterpillar. Current medical treatments focus on symptom relief due to the lack of an effective therapy. While the source is known, understanding the precise causes of symptoms remain incomplete understood. In this study, we employed an advanced method to extract venom from the setae and identify the venom components through high-quality de novo transcriptomics, venom proteomics, and bioinformatic analysis. A total of 171 venom components were identified, including allergens, odorant binding proteins, small peptides, enzymes, enzyme inhibitors, and chitin biosynthesis products, potentially responsible for inflammatory and allergic reactions. This work presents the first comprehensive proteotranscriptomic database of T. processionea, contributing to understanding the complexity of lepidopterism. Furthermore, these findings hold promise for advancing therapeutic approaches to mitigate the global health impact of T. processionea and related caterpillars.

• Schendel, Vanessa; Müller, Carsten H. G.; Kenning, Matthes; Maxwell, Michael; Jenner, Ronald A. & Undheim, Eivind Andreas Baste [Show all 7 contributors for this article] (2024). The venom and telopodal defence systems of the centipede Lithobius forficatus are functionally convergent serial homologues. BMC Biology. 22(1). doi: 10.1186/s12915-024-01925-x. Full text in Research Archive Show summary

  Background Evolution of novelty is a central theme in evolutionary biology, yet studying the origins of traits with an apparently discontinuous origin remains a major challenge. Venom systems are a well-suited model for the study of this phenomenon because they capture several aspects of novelty across multiple levels of biological complexity. However, while there is some knowledge on the evolution of individual toxins, not much is known about the evolution of venom systems as a whole. One way of shedding light on the evolution of new traits is to investigate less specialised serial homologues, i.e. repeated traits in an organism that share a developmental origin. This approach can be particularly informative in animals with repetitive body segments, such as centipedes. Results Here, we investigate morphological and biochemical aspects of the defensive telopodal glandular organs borne on the posterior legs of venomous stone centipedes (Lithobiomorpha), using a multimethod approach, including behavioural observations, comparative morphology, proteomics, comparative transcriptomics and molecular phylogenetics. We show that the anterior venom system and posterior telopodal defence system are functionally convergent serial homologues, where one (telopodal defence) represents a model for the putative early evolutionary state of the other (venom). Venom glands and telopodal glandular organs appear to have evolved from the same type of epidermal gland (four-cell recto-canal type) and while the telopodal defensive secretion shares a great degree of compositional overlap with centipede venoms in general, these similarities arose predominantly through convergent recruitment of distantly related toxin-like components. Both systems are composed of elements predisposed to functional innovation across levels of biological complexity that range from proteins to glands, demonstrating clear parallels between molecular and morphological traits in the properties that facilitate the evolution of novelty. Conclusions The evolution of the lithobiomorph telopodal defence system provides indirect empirical support for the plausibility of the hypothesised evolutionary origin of the centipede venom system, which occurred through functional innovation and gradual specialisation of existing epidermal glands. Our results thus exemplify how continuous transformation and functional innovation can drive the apparent discontinuous emergence of novelties on higher levels of biological complexity.

• Seldeslachts, Andrea; Undheim, Eivind Andreas Baste; Vriens, Joris; Tytgat, Jan & Peigneur, Steve (2024). Exploring oak processionary caterpillar induced lepidopterism (part 2): ex vivo bio-assays unmask the role of TRPV1. Cellular and Molecular Life Sciences (CMLS). ISSN 1420-682X. 81. doi: 10.1007/s00018-024-05318-9. Full text in Research Archive Show summary

  As human skin comes into contact with the tiny hairs or setae of the oak processionary caterpillar, Thaumetopoea processionea, a silent yet intense chemical confrontation occurs. The result is a mix of issues: skin rashes and an intense itching that typically lasts days and weeks after the contact. This discomfort poses a significant health threat not only to humans but also to animals. In Western Europe, the alarming increase in outbreaks extends beyond areas near infested trees due to the dispersion of the setae. Predictions indicate a sustained rise in outbreaks, fueled by global changes favoring the caterpillar’s survival and distribution. Currently, the absence of an efficient treatment persists due to significant gaps in our comprehension of the pathophysiology associated with this envenomation. Here, we explored the interaction between the venom extract derived from the setae of T. processionea and voltage- and ligand-gated ion channels and receptors. By conducting electrophysiological analyses, we discovered ex vivo evidence highlighting the significant role of TPTX1-Tp1, a peptide toxin from T. processionea, in modulating TRPV1. TPTX1-Tp1 is a secapin-like peptide and demonstrates a unique ability to modulate TRPV1 channels in the presence of capsaicin, leading to cell depolarization, itch and inflammatory responses. This discovery opens new avenues for developing a topical medication, suggesting the incorporation of a TRPV1 blocker as a potential solution for the local effects caused by T. processionea.

• Ashwood, Lauren M.; Elnahriry, Khaled A.; Stewart, Zachary K.; Shafee, Thomas; Naseem, Muhammad Umair & Szanto, Tibor G. [Show all 23 contributors for this article] (2023). Genomic, functional and structural analyses elucidate evolutionary innovation within the sea anemone 8 toxin family. BMC Biology. 21, p. 1–25. doi: 10.1186/s12915-023-01617-y. Full text in Research Archive Show summary

  Background The ShK toxin from Stichodactyla helianthus has established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in Actiniarians remain uncharacterised. One such peptide family, sea anemone 8 (SA8), is present in all five sea anemone superfamilies. We explored the genomic arrangement and evolution of the SA8 gene family in Actinia tenebrosa and Telmatactis stephensoni, characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom of T. stephensoni. Results We identified ten SA8-family genes in two clusters and six SA8-family genes in five clusters for T. stephensoni and A. tenebrosa, respectively. Nine SA8 T. stephensoni genes were found in a single cluster, and an SA8 peptide encoded by an inverted SA8 gene from this cluster was recruited to venom. We show that SA8 genes in both species are expressed in a tissue-specific manner and the inverted SA8 gene has a unique tissue distribution. While the functional activity of the SA8 putative toxin encoded by the inverted gene was inconclusive, its tissue localisation is similar to toxins used for predator deterrence. We demonstrate that, although mature SA8 putative toxins have similar cysteine spacing to ShK, SA8 peptides are distinct from ShK peptides based on structure and disulfide connectivity. Conclusions Our results provide the first demonstration that SA8 is a unique gene family in Actiniarians, evolving through a variety of structural changes including tandem and proximal gene duplication and an inversion event that together allowed SA8 to be recruited into the venom of T. stephensoni.

• Robinson, Samuel D.; Deuis, Jennifer R.; Touchard, Axel; Keramidas, Angelo; Mueller, Alexander & Schroeder, Christina I. [Show all 16 contributors for this article] (2023). Ant venoms contain vertebrate-selective pain-causing sodium channel toxins. Nature Communications. 14, p. 1–11. doi: 10.1038/s41467-023-38839-1. Full text in Research Archive Show summary

  Abstract 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 (Na V ) 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.

• Robinson, Samuel D.; Schendel, Vanessa; Schroeder, Christina I.; Moen, Sarah; Mueller, Alexander & Walker, Andrew A. [Show all 11 contributors for this article] (2023). Intra-colony venom diversity contributes to maintaining eusociality in a cooperatively breeding ant. BMC Biology. 21(5), p. 1–19. doi: 10.1186/s12915-022-01507-9. Full text in Research Archive Show summary

  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.

• Maxwell, Michael J.; Thekkedam, Chris; Lamboley, Cedric; Chin, Yanni K.-Y.; Crawford, Theo & Smith, Jennifer J. [Show all 14 contributors for this article] (2023). A bivalent remipede toxin promotes calcium release via ryanodine receptor activation. Nature Communications. 14(1), p. 1–13. doi: 10.1038/s41467-023-36579-w. Full text in Research Archive Show summary

  Abstract Multivalent ligands of ion channels have proven to be both very rare and highly valuable in yielding unique insights into channel structure and pharmacology. Here, we describe a bivalent peptide from the venom of Xibalbanus tulumensis , a troglobitic arthropod from the enigmatic class Remipedia, that causes persistent calcium release by activation of ion channels involved in muscle contraction. The high-resolution solution structure of φ-Xibalbin3-Xt3a reveals a tandem repeat arrangement of inhibitor-cysteine knot (ICK) domains previously only found in spider venoms. The individual repeats of Xt3a share sequence similarity with a family of scorpion toxins that target ryanodine receptors (RyR). Single-channel electrophysiology and quantification of released Ca 2+ stores within skinned muscle fibers confirm Xt3a as a bivalent RyR modulator. Our results reveal convergent evolution of RyR targeting toxins in remipede and scorpion venoms, while the tandem-ICK repeat architecture is an evolutionary innovation that is convergent with toxins from spider venoms.

• Liu, Junyu; Maxwell, Michael; Cuddihy, Thom; Crawford, Theo; Bassetti, Madeline & Hyde, Cameron [Show all 10 contributors for this article] (2023). ScrepYard: An online resource for disulfide-stabilized tandem repeat peptides. Protein Science. ISSN 0961-8368. 32(2), p. 1–12. doi: 10.1002/pro.4566. Full text in Research Archive Show summary

  Abstract Receptor avidity through multivalency is a highly sought‐after property of ligands. While readily available in nature in the form of bivalent antibodies, this property remains challenging to engineer in synthetic molecules. The discovery of several bivalent venom peptides containing two homologous and independently folded domains (in a tandem repeat arrangement) has provided a unique opportunity to better understand the underpinning design of multivalency in multimeric biomolecules, as well as how naturally occurring multivalent ligands can be identified. In previous work, we classified these molecules as a larger class termed secreted cysteine‐rich repeat‐proteins (SCREPs). Here, we present an online resource; ScrepYard, designed to assist researchers in identification of SCREP sequences of interest and to aid in characterizing this emerging class of biomolecules. Analysis of sequences within the ScrepYard reveals that two‐domain tandem repeats constitute the most abundant SCREP domain architecture, while the interdomain “linker” regions connecting the functional domains are found to be abundant in amino acids with short or polar sidechains and contain an unusually high abundance of proline residues. Finally, we demonstrate the utility of ScrepYard as a virtual screening tool for discovery of putatively multivalent peptides, by using it as a resource to identify a previously uncharacterized serine protease inhibitor and confirm its predicted activity using an enzyme assay.

• Dashevsky, Daniel; Baumann, Kate; Undheim, Eivind Andreas Baste; Nouwens, Amanda; Ikonomopoulou, Maria P. & Schmidt, Justin O. [Show all 10 contributors for this article] (2023). Functional and Proteomic Insights into Aculeata Venoms. Toxins. 15(3), p. 1–25. doi: 10.3390/toxins15030224. Full text in Research Archive Show summary

  Aculeate hymenopterans use their venom for a variety of different purposes. The venom of solitary aculeates paralyze and preserve prey without killing it, whereas social aculeates utilize their venom in defence of their colony. These distinct applications of venom suggest that its components and their functions are also likely to differ. This study investigates a range of solitary and social species across Aculeata. We combined electrophoretic, mass spectrometric, and transcriptomic techniques to characterize the compositions of venoms from an incredibly diverse taxon. In addition, in vitro assays shed light on their biological activities. Although there were many common components identified in the venoms of species with different social behavior, there were also significant variations in the presence and activity of enzymes such as phospholipase A2s and serine proteases and the cytotoxicity of the venoms. Social aculeate venom showed higher presence of peptides that cause damage and pain in victims. The venom-gland transcriptome from the European honeybee (Apis mellifera) contained highly conserved toxins which match those identified by previous investigations. In contrast, venoms from less-studied taxa returned limited results from our proteomic databases, suggesting that they contain unique toxins.

• Kazandjian, Taline D.; Hamilton, Brett R.; Robinson, Samuel D.; Hall, Steven R.; Bartlett, Keirah E. & Rowley, Paul [Show all 9 contributors for this article] (2022). Physiological constraints dictate toxin spatial heterogeneity in snake venom glands. BMC Biology. 20(1). doi: 10.1186/s12915-022-01350-y. Full text in Research Archive Show summary

  Background Venoms are ecological innovations that have evolved numerous times, on each occasion accompanied by the co-evolution of specialised morphological and behavioural characters for venom production and delivery. The close evolutionary interdependence between these characters is exemplified by animals that control the composition of their secreted venom. This ability depends in part on the production of different toxins in different locations of the venom gland, which was recently documented in venomous snakes. Here, we test the hypothesis that the distinct spatial distributions of toxins in snake venom glands are an adaptation that enables the secretion of venoms with distinct ecological functions. Results We show that the main defensive and predatory peptide toxins are produced in distinct regions of the venom glands of the black-necked spitting cobra (Naja nigricollis), but these distributions likely reflect developmental effects. Indeed, we detected no significant differences in venom collected via defensive ‘spitting’ or predatory ‘biting’ events from the same specimens representing multiple lineages of spitting cobra. We also found the same spatial distribution of toxins in a non-spitting cobra and show that heterogeneous toxin distribution is a feature shared with a viper with primarily predatory venom. Conclusions Our findings suggest that heterogeneous distributions of toxins are not an adaptation to controlling venom composition in snakes. Instead, it likely reflects physiological constraints on toxin production by the venom glands, opening avenues for future research on the mechanisms of functional differentiation of populations of protein-secreting cells within adaptive contexts.

• Reumont, Bjoern M. von; Anderluh, Gregor; Antunes, Agostinho; Ayvazyan, Naira; Beis, Dimitris & Caliskan, Figen [Show all 35 contributors for this article] (2022). Modern venomics-Current insights, novel methods, and future perspectives in biological and applied animal venom research. GigaScience. 11. doi: 10.1093/gigascience/giac048. Full text in Research Archive Show summary

  Venoms have evolved >100 times in all major animal groups, and their components, known as toxins, have been fine-tuned over millions of years into highly effective biochemical weapons. There are many outstanding questions on the evolution of toxin arsenals, such as how venom genes originate, how venom contributes to the fitness of venomous species, and which modifications at the genomic, transcriptomic, and protein level drive their evolution. These questions have received particularly little attention outside of snakes, cone snails, spiders, and scorpions. Venom compounds have further become a source of inspiration for translational research using their diverse bioactivities for various applications. We highlight here recent advances and new strategies in modern venomics and discuss how recent technological innovations and multi-omic methods dramatically improve research on venomous animals. The study of genomes and their modifications through CRISPR and knockdown technologies will increase our understanding of how toxins evolve and which functions they have in the different ontogenetic stages during the development of venomous animals. Mass spectrometry imaging combined with spatial transcriptomics, in situ hybridization techniques, and modern computer tomography gives us further insights into the spatial distribution of toxins in the venom system and the function of the venom apparatus. All these evolutionary and biological insights contribute to more efficiently identify venom compounds, which can then be synthesized or produced in adapted expression systems to test their bioactivity. Finally, we critically discuss recent agrochemical, pharmaceutical, therapeutic, and diagnostic (so-called translational) aspects of venoms from which humans benefit.

• Finol-Urdaneta, Rocio K.; Ziegman, Rebekah; Dekan, Zoltan; McArthur, Jeffrey R.; Heitmann, Stewart & Luna-Ramirez, Karen [Show all 20 contributors for this article] (2022). Multitarget nociceptor sensitization by a promiscuous peptide from the venom of the King Baboon spider. Proceedings of the National Academy of Sciences of the United States of America (PNAS). ISSN 0027-8424. 119(5). doi: 10.1073/pnas.2110932119. Full text in Research Archive Show summary

  Significance Pain development and discomfort are universal features of spider envenomation, yet severe pain arising from bites by Old World spiders is poorly understood. Molecular analyses of the venom of the King Baboon spider revealed abundant expression of the inhibitory cystine knot peptide Pm1a. Synthetic Pm1a induces pain in mice while simultaneously enhancing proexcitatory sodium currents and decreasing inhibitory potassium currents. These concomitant effects promote hyperexcitability in pain-sensing neurons that can be reversed by pharmacological inhibition of voltage-gated sodium channels. The coordinated modulation of excitatory and inhibitory ion channels involved in pain propagation may represent an economical and effective defense strategy in pain-inducing defensive venoms.

• Verdes, Aida; Taboada, Sergi; Hamilton, Brett R.; Undheim, Eivind Andreas Baste; Sonoda, Gabriel G. & Andrade, Sonia C.S. [Show all 10 contributors for this article] (2022). Evolution, Expression Patterns, and Distribution of Novel Ribbon Worm Predatory and Defensive Toxins. Molecular Biology and Evolution (MBE). ISSN 0737-4038. 39(5). doi: 10.1093/molbev/msac096. Full text in Research Archive Show summary

  Ribbon worms are active predators that use an eversible proboscis to inject venom into their prey and defend themselves with toxic epidermal secretions. Previous work on nemertean venom has largely focused on just a few species and has not investigated the different predatory and defensive secretions in detail. Consequently, our understanding of the composition and evolution of ribbon worm venoms is still very limited. Here, we present a comparative study of nemertean venom combining RNA-seq differential gene expression analyses of venom-producing tissues, tandem mass spectrometry-based proteomics of toxic secretions, and mass spectrometry imaging of proboscis sections, to shed light onto the composition and evolution of predatory and defensive toxic secretions in Antarctonemertes valida. Our analyses reveal a wide diversity of putative defensive and predatory toxins with tissue-specific gene expression patterns and restricted distributions to the mucus and proboscis proteomes respectively, suggesting that ribbon worms produce distinct toxin cocktails for predation and defense. Our results also highlight the presence of numerous lineage-specific toxins, indicating that venom evolution is highly divergent across nemerteans, producing toxin cocktails that might be finely tuned to subdue different prey. Our data also suggest that the hoplonemertean proboscis is a highly specialized predatory organ that seems to be involved in a variety of biological functions besides predation, including secretion and sensory perception. Overall, our results advance our knowledge into the diversity and evolution of nemertean venoms and highlight the importance of combining different types of data to characterize toxin composition in understudied venomous organisms.

• Eagles, David A.; Saez, Natalie J.; Krishnarjuna, Bankala; Bradford, Julia J.; Chin, Yanni K.-Y. & Starobova, Hana [Show all 14 contributors for this article] (2022). A peptide toxin in ant venom mimics vertebrate EGF-like hormones to cause long-lasting hypersensitivity in mammals. Proceedings of the National Academy of Sciences of the United States of America (PNAS). ISSN 0027-8424. 119(7). doi: 10.1073/pnas.2112630119. Full text in Research Archive Show summary

  Venoms are excellent model systems for studying evolutionary processes associated with predator–prey interactions. Here, we present the discovery of a peptide toxin, MIITX2-Mg1a, which is a major component of the venom of the Australian giant red bull ant Myrmecia gulosa and has evolved to mimic, both structurally and functionally, vertebrate epidermal growth factor (EGF) peptide hormones. We show that Mg1a is a potent agonist of the mammalian EGF receptor ErbB1, and that intraplantar injection in mice causes long-lasting hypersensitivity of the injected paw. These data reveal a previously undescribed venom mode of action, highlight a role for ErbB receptors in mammalian pain signaling, and provide an example of molecular mimicry driven by defensive selection pressure.

• Nixon, Samantha A.; Robinson, Samuel D.; Agwa, Akello J.; Walker, Andrew A.; Choudhary, Shivani & Touchard, Axel [Show all 13 contributors for this article] (2021). Multipurpose peptides: The venoms of Amazonian stinging ants contain anthelmintic ponericins with diverse predatory and defensive activities. Biochemical Pharmacology. ISSN 0006-2952. 192, p. 1–16. doi: 10.1016/j.bcp.2021.114693. Full text in Research Archive Show summary

  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.

• Robinson, Samuel D.; Kambanis, Lucas; Clayton, Daniel; Hinneburg, Hannes; Corcilius, Leo & Mueller, Alexander [Show all 15 contributors for this article] (2021). A pain-causing and paralytic ant venom glycopeptide. iScience. 24(10), p. 1–21. doi: 10.1016/j.isci.2021.103175. Full text in Research Archive Show summary

  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.

• Ashwood, Lauren M.; Mitchell, Michaela; Madio, Bruno; Hurwood, David A.; King, Glenn F. & Undheim, Eivind Andreas Baste [Show all 8 contributors for this article] (2021). Tentacle Morphological Variation Coincides with Differential Expression of Toxins in Sea Anemones. Toxins. 13(452). doi: 10.3390/toxins13070452. Full text in Research Archive Show summary

  Phylum Cnidaria is an ancient venomous group defined by the presence of cnidae, specialised organelles that serve as venom delivery systems. The distribution of cnidae across the body plan is linked to regionalisation of venom production, with tissue-specific venom composition observed in multiple actiniarian species. In this study, we assess whether morphological variants of tentacles are associated with distinct toxin expression profiles and investigate the functional significance of specialised tentacular structures. Using five sea anemone species, we analysed differential expression of toxin-like transcripts and found that expression levels differ significantly across tentacular structures when substantial morphological variation is present. Therefore, the differential expression of toxin genes is associated with morphological variation of tentacular structures in a tissue-specific manner. Furthermore, the unique toxin profile of spherical tentacular structures in families Aliciidae and Thalassianthidae indicate that vesicles and nematospheres may function to protect branched structures that host a large number of photosynthetic symbionts. Thus, hosting zooxanthellae may account for the tentacle-specific toxin expression profiles observed in the current study. Overall, specialised tentacular structures serve unique ecological roles and, in order to fulfil their functions, they possess distinct venom cocktails.

• Calvete, Juan J.; Lomonte, Bruno; Saviola, Anthony J.; Bonilla, Fabián; Sasa, Mahmood & Williams, David J. [Show all 9 contributors for this article] (2021). Mutual enlightenment: A toolbox of concepts and methods for integrating evolutionary and clinical toxinology via snake venomics and the contextual stance. Toxicon: X. 9-10, p. 1–16. doi: 10.1016/j.toxcx.2021.100070. Full text in Research Archive Show summary

  Snakebite envenoming is a neglected tropical disease that may claim over 100,000 human lives annually worldwide. Snakebite occurs as the result of an interaction between a human and a snake that elicits either a defensive response from the snake or, more rarely, a feeding response as the result of mistaken identity. Snakebite envenoming is therefore a biological and, more specifically, an ecological problem. Snake venom itself is often described as a “cocktail”, as it is a heterogenous mixture of molecules including the toxins (which are typically proteinaceous) responsible for the pathophysiological consequences of envenoming. The primary function of venom in snake ecology is pre-subjugation, with defensive deployment of the secretion typically considered a secondary function. The particular composition of any given venom cocktail is shaped by evolutionary forces that include phylogenetic constraints associated with the snake's lineage and adaptive responses to the snake's ecological context, including the taxa it preys upon and by which it is predated upon. In the present article, we describe how conceptual frameworks from ecology and evolutionary biology can enter into a mutually enlightening relationship with clinical toxinology by enabling the consideration of snakebite envenoming from an “ecological stance”. We detail the insights that may emerge from such a perspective and highlight the ways in which the high-fidelity descriptive knowledge emerging from applications of -omics era technologies – “venomics” and “antivenomics” – can combine with evolutionary explanations to deliver a detailed understanding of this multifactorial health crisis.

• Ashwood, Lauren M.; Undheim, Eivind Andreas Baste; Madio, Bruno; Hamilton, Brett R.; Daly, Marymegan & Hurwood, David A. [Show all 8 contributors for this article] (2021). Venoms for all occasions: The functional toxin profiles of different anatomical regions in sea anemones are related to their ecological function. Molecular Ecology. ISSN 0962-1083. p. 1–18. doi: 10.1111/mec.16286. Full text in Research Archive
• Undheim, Eivind Andreas Baste & Jenner, Ronald A. (2021). Phylogenetic analyses suggest centipede venom arsenals were repeatedly stocked by horizontal gene transfer. Nature Communications. 12(1), p. 1–14. doi: 10.1038/s41467-021-21093-8. Full text in Research Archive Show summary

  Venoms have evolved over a hundred times in animals. Venom toxins are thought to evolve mostly by recruitment of endogenous proteins with physiological functions. Here we report phylogenetic analyses of venom proteome-annotated venom gland transcriptome data, assisted by genomic analyses, to show that centipede venoms have recruited at least five gene families from bacterial and fungal donors, involving at least eight horizontal gene transfer events. These results establish centipedes as currently the only known animals with venoms used in predation and defence that contain multiple gene families derived from horizontal gene transfer. The results also provide the first evidence for the implication of horizontal gene transfer in the evolutionary origin of venom in an animal lineage. Three of the bacterial gene families encode virulence factors, suggesting that horizontal gene transfer can provide a fast track channel for the evolution of novelty by the exaptation of bacterial weapons into animal venoms.

• Kazandjian, Taline D.; Petras, Daniel; Robinson, Samuel D.; Thiel, Jory van; Greene, Harry W. & Arbuckle, Kevin [Show all 29 contributors for this article] (2021). Convergent evolution of pain-inducing defensive venom components in spitting cobras. Science. ISSN 0036-8075. 371(6527), p. 386–390. doi: 10.1126/science.abb9303. Full text in Research Archive Show summary

  Convergent evolution provides insights into the selective drivers underlying evolutionary change. Snake venoms, with a direct genetic basis and clearly defined functional phenotype, provide a model system for exploring the repeated evolution of adaptations. While snakes use venom primarily for predation, and venom composition often reflects diet specificity, three lineages of cobras have independently evolved the ability to spit venom at adversaries. Using gene, protein, and functional analyses, we show that the three spitting lineages possess venoms characterized by an up-regulation of phospholipase A2 (PLA2) toxins, which potentiate the action of preexisting venom cytotoxins to activate mammalian sensory neurons and cause enhanced pain. These repeated independent changes provide a fascinating example of convergent evolution across multiple phenotypic levels driven by selection for defense.

• Walker, Andrew A.; Robinson, Samuel D.; Hamilton, Brett F.; Undheim, Eivind Andreas Baste & King, Glenn F. (2020). Deadly Proteomes: A Practical Guide to Proteotranscriptomics of Animal Venoms. Proteomics. ISSN 1615-9853. 20(1900324). doi: 10.1002/pmic.201900324. Full text in Research Archive
• Chow, Chun Yuen; Chin, Yanni K. Y.; Ma, Linlin; Undheim, Eivind Andreas Baste & Herzig, Volker (2020). A selective NaV1.1 activator with potential for treatment of Dravet syndrome epilepsy. Biochemical Pharmacology. ISSN 0006-2952. 181, p. 1–15. doi: 10.1016/j.bcp.2020.113991. Full text in Research Archive
• Aili, Samira R.; Touchard, Axel; Hayward, Regan; Robinson, Samuel D.; Pineda, Sandy S. & Lalagüe, Hadrien [Show all 14 contributors for this article] (2020). An integrated proteomic and transcriptomic analysis reveals the venom complexity of the bullet ant Paraponera clavata. Toxins. 12(5), p. 1–30. doi: 10.3390/toxins12050324. Full text in Research Archive Show summary

  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.

• Ashwood, Lauren M.; Norton, Raymond S.; Undheim, Eivind Andreas Baste; Hurwood, David A. & Prentis, Peter J. (2020). Characterising functional venom profiles of anthozoans and medusozoans within their ecological context. Marine Drugs. 18(202), p. 1–19. doi: 10.3390/md18040202. Full text in Research Archive Show summary

  This review examines the current state of knowledge regarding toxins from anthozoans (sea anemones, coral, zoanthids, corallimorphs, sea pens and tube anemones). We provide an overview of venom from phylum Cnidaria and review the diversity of venom composition between the two major clades (Medusozoa and Anthozoa). We highlight that the functional and ecological context of venom has implications for the temporal and spatial expression of protein and peptide toxins within class Anthozoa. Understanding the nuances in the regulation of venom arsenals has been made possible by recent advances in analytical technologies that allow characterisation of the spatial distributions of toxins. Furthermore, anthozoans are unique in that ecological roles can be assigned using tissue expression data, thereby circumventing some of the challenges related to pharmacological screening.

• Schendel, Vanessa; Rash, Lachlan D.; Jenner, Ronald A. & Undheim, Eivind Andreas Baste (2019). The diversity of venom: The importance of behavior and venom system morphology in understanding its ecology and evolution. Toxins. 11(11), p. 1–22. doi: 10.3390/toxins11110666. Full text in Research Archive Show summary

  Venoms are one of the most convergent of animal traits known, and encompass a much greater taxonomic and functional diversity than is commonly appreciated. This knowledge gap limits the potential of venom as a model trait in evolutionary biology. Here, we summarize the taxonomic and functional diversity of animal venoms and relate this to what is known about venom system morphology, venom modulation, and venom pharmacology, with the aim of drawing attention to the importance of these largely neglected aspects of venom research. We find that animals have evolved venoms at least 101 independent times and that venoms play at least 11 distinct ecological roles in addition to predation, defense, and feeding. Comparisons of different venom systems suggest that morphology strongly influences how venoms achieve these functions, and hence is an important consideration for understanding the molecular evolution of venoms and their toxins. Our findings also highlight the need for more holistic studies of venom systems and the toxins they contain. Greater knowledge of behavior, morphology, and ecologically relevant toxin pharmacology will improve our understanding of the evolution of venoms and their toxins, and likely facilitate exploration of their potential as sources of molecular tools and therapeutic and agrochemical lead compounds.

• Hamilton, Brett R.; Marshall, David L.; Casewell, Nicholas R.; Harrison, Robert A.; Blanksby, Stephen J. & Undheim, Eivind Andreas Baste (2019). Mapping enzyme activity on tissue by functional mass spectrometry imaging. Angewandte Chemie International Edition. ISSN 1433-7851. 59, p. 3855–3858. doi: 10.1002/anie.201911390. Full text in Research Archive Show summary

  Enzymes are central components of most physiological processes, and are consequently implicated in various pathologies. High‐resolution maps of enzyme activity within tissues therefore represent powerful tools for elucidating enzymatic functions in health and disease. Here, we present a novel mass spectrometry imaging (MSI) method for assaying the spatial distribution of enzymatic activity directly from tissue. MSI analysis of tissue sections exposed to phospholipid substrates produced high‐resolution maps of phospholipase activity and specificity, which could subsequently be compared to histological images of the same section. Functional MSI thus represents a new and generalisable method for imaging biological activity in situ.

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