Web of venom: exploration of big data resources in animal toxin research.

Research on animal venoms and their components spans multiple disciplines, including biology, biochemistry, bioinformatics, pharmacology, medicine, and more. Manipulating and analyzing the diverse array of data required for venom research can be challenging, and relevant tools and resources are often dispersed across different online platforms, making them less accessible to nonexperts. In this article, we address the multifaceted needs of the scientific community involved in venom and toxin-related research by identifying and discussing web resources, databases, and tools commonly used in this field. We have compiled these resources into a comprehensive table available on the VenomZone website (https://venomzone.expasy.org/10897). Furthermore, we highlight the challenges currently faced by researchers in accessing and using these resources and emphasize the importance of community-driven interdisciplinary approaches. We conclude by underscoring the significance of enhancing standards, promoting interoperability, and encouraging data and method sharing within the venom research community.

Venom gland organogenesis in the common house spider.

Venom is a remarkable innovation found across the animal kingdom, yet the evolutionary origins of venom systems in various groups, including spiders, remain enigmatic. Here, we investigated the organogenesis of the venom apparatus in the common house spider, Parasteatoda tepidariorum. The venom apparatus consists of a pair of secretory glands, each connected to an opening at the fang tip by a duct that runs through the chelicerae. We performed bulk RNA-seq to identify venom gland-specific markers and assayed their expression using RNA in situ hybridisation experiments on whole-mount time-series. These revealed that the gland primordium emerges during embryonic stage 13 at the chelicera tip, progresses proximally by the end of embryonic development and extends into the prosoma post-eclosion. The initiation of expression of an important toxin component in late postembryos marks the activation of venom-secreting cells. Our selected markers also exhibited distinct expression patterns in adult venom glands: sage and the toxin marker were expressed in the secretory epithelium, forkhead and sum-1 in the surrounding muscle layer, while Distal-less was predominantly expressed at the gland extremities. Our study provides the first comprehensive analysis of venom gland morphogenesis in spiders, offering key insights into their evolution and development.

Modern venomics-Current insights, novel methods, and future perspectives in biological and applied animal venom research.

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.

Convergent evolution of venom gland transcriptomes across Metazoa.

Animals have repeatedly evolved specialized organs and anatomical structures to produce and deliver a mixture of potent bioactive molecules to subdue prey or predators-venom. This makes it one of the most widespread, convergent functions in the animal kingdom. Whether animals have adopted the same genetic toolkit to evolved venom systems is a fascinating question that still eludes us. Here, we performed a comparative analysis of venom gland transcriptomes from 20 venomous species spanning the main Metazoan lineages to test whether different animals have independently adopted similar molecular mechanisms to perform the same function. We found a strong convergence in gene expression profiles, with venom glands being more similar to each other than to any other tissue from the same species, and their differences closely mirroring the species phylogeny. Although venom glands secrete some of the fastest evolving molecules (toxins), their gene expression does not evolve faster than evolutionarily older tissues. We found 15 venom gland-specific gene modules enriched in endoplasmic reticulum stress and unfolded protein response pathways, indicating that animals have independently adopted stress response mechanisms to cope with mass production of toxins. This, in turn, activates regulatory networks for epithelial development, cell turnover, and maintenance, which seem composed of both convergent and lineage-specific factors, possibly reflecting the different developmental origins of venom glands. This study represents a first step toward an understanding of the molecular mechanisms underlying the repeated evolution of one of the most successful adaptive traits in the animal kingdom.

The new COST Action European Venom Network (EUVEN)-synergy and future perspectives of modern venomics.

Venom research is a highly multidisciplinary field that involves multiple subfields of biology, informatics, pharmacology, medicine, and other areas. These different research facets are often technologically challenging and pursued by different teams lacking connection with each other. This lack of coordination hampers the full development of venom investigation and applications. The COST Action CA19144-European Venom Network was recently launched to promote synergistic interactions among different stakeholders and foster venom research at the European level.

Venom Systems as Models for Studying the Origin and Regulation of Evolutionary Novelties.

A central goal in biology is to determine the ways in which evolution repeats itself. One of the most remarkable examples in nature of convergent evolutionary novelty is animal venom. Across diverse animal phyla, various specialized organs and anatomical structures have evolved from disparate developmental tissues to perform the same function, that is, produce and deliver a cocktail of potent molecules to subdue prey or predators. Venomous organisms therefore offer unique opportunities to investigate the evolutionary processes of convergence of key adaptive traits, and the molecular mechanisms underlying the emergence of novel genes, cells, and tissues. Indeed, some venomous species have already proven to be highly amenable as models for developmental studies, and recent work with venom gland organoids provides manipulatable systems for directly testing important evolutionary questions. Here, we provide a synthesis of the current knowledge that could serve as a starting point for the establishment of venom systems as new models for evolutionary and molecular biology. In particular, we highlight the potential of various venomous species for the study of cell differentiation and cell identity, and the regulatory dynamics of rapidly evolving, highly expressed, tissue-specific, gene paralogs. We hope that this review will encourage researchers to look beyond traditional study organisms and consider venom systems as useful tools to explore evolutionary novelties.

When one phenotype is not enough: divergent evolutionary trajectories govern venom variation in a widespread rattlesnake species.

Understanding the origin and maintenance of phenotypic variation, particularly across a continuous spatial distribution, represents a key challenge in evolutionary biology. For this, animal venoms represent ideal study systems: they are complex, variable, yet easily quantifiable molecular phenotypes with a clear function. Rattlesnakes display tremendous variation in their venom composition, mostly through strongly dichotomous venom strategies, which may even coexist within a single species. Here, through dense, widespread population-level sampling of the Mojave rattlesnake, Crotalus scutulatus, we show that genomic structural variation at multiple loci underlies extreme geographical variation in venom composition, which is maintained despite extensive gene flow. Unexpectedly, neither diet composition nor neutral population structure explain venom variation. Instead, venom divergence is strongly correlated with environmental conditions. Individual toxin genes correlate with distinct environmental factors, suggesting that different selective pressures can act on individual loci independently of their co-expression patterns or genomic proximity. Our results challenge common assumptions about diet composition as the key selective driver of snake venom evolution and emphasize how the interplay between genomic architecture and local-scale spatial heterogeneity in selective pressures may facilitate the retention of adaptive functional polymorphisms across a continuous space.

Widespread vulnerability of Malagasy predators to the toxins of an introduced toad.

Invasive species are a key factor contributing to the global decline of biodiversity, and understanding the underlying mechanisms is crucial to mitigate detrimental effects [1]. One such mechanism is the introduction of invasive species with defensive strategies, such as novel toxins, that can disrupt native predator communities [2]. Disruption of such communities can produce trophic cascades, impacting a diverse array of taxa [2]. Madagascar, a globally significant biodiversity hotspot, has recently experienced the introduction of a toxic bufonid amphibian, the Asian common toad (Duttaphrynus melanostictus) [3]. Since its invasion, the toad population has expanded rapidly, making control efforts problematic and eradication extremely difficult [4]. Previous cases of bufonid introductions, such as the ongoing spread of the cane toad (Rhinella marina) in Australia, have resulted in the decimation of many indigenous species [2], prompting fears that Madagascar may be similarly impacted [4]. Here we show that these fears are warranted: we demonstrate that many Malagasy vertebrates are likely to be susceptible to the toxins of this invasive toad.

Venom On-a-Chip: A Fast and Efficient Method for Comparative Venomics.

Venom research has attracted an increasing interest in disparate fields, from drug development and pharmacology, to evolutionary biology and ecology, and rational antivenom production. Advances in "-omics" technologies have allowed the characterization of an increasing number of animal venoms, but the methodology currently available is suboptimal for large-scale comparisons of venom profiles. Here, we describe a fast, reproducible and semi-automated protocol for investigating snake venom variability, especially at the intraspecific level, using the Agilent Bioanalyzer on-chip technology. Our protocol generated a phenotype matrix which can be used for robust statistical analysis and correlations of venom variation with ecological correlates, or other extrinsic factors. We also demonstrate the ease and utility of combining on-chip technology with previously fractionated venoms for detection of specific individual toxin proteins. Our study describes a novel strategy for rapid venom discrimination and analysis of compositional variation at multiple taxonomic levels, allowing researchers to tackle evolutionary questions and unveiling the drivers of the incredible biodiversity of venoms.

Predictors of elevational biodiversity gradients change from single taxa to the multi-taxa community level.

The factors determining gradients of biodiversity are a fundamental yet unresolved topic in ecology. While diversity gradients have been analysed for numerous single taxa, progress towards general explanatory models has been hampered by limitations in the phylogenetic coverage of past studies. By parallel sampling of 25 major plant and animal taxa along a 3.7 km elevational gradient on Mt. Kilimanjaro, we quantify cross-taxon consensus in diversity gradients and evaluate predictors of diversity from single taxa to a multi-taxa community level. While single taxa show complex distribution patterns and respond to different environmental factors, scaling up diversity to the community level leads to an unambiguous support for temperature as the main predictor of species richness in both plants and animals. Our findings illuminate the influence of taxonomic coverage for models of diversity gradients and point to the importance of temperature for diversification and species coexistence in plant and animal communities.

Is Hybridization a Source of Adaptive Venom Variation in Rattlesnakes? A Test, Using a Crotalus scutulatus × viridis Hybrid Zone in Southwestern New Mexico.

Venomous snakes often display extensive variation in venom composition both between and within species. However, the mechanisms underlying the distribution of different toxins and venom types among populations and taxa remain insufficiently known. Rattlesnakes (Crotalus, Sistrurus) display extreme inter- and intraspecific variation in venom composition, centered particularly on the presence or absence of presynaptically neurotoxic phospholipases A2 such as Mojave toxin (MTX). Interspecific hybridization has been invoked as a mechanism to explain the distribution of these toxins across rattlesnakes, with the implicit assumption that they are adaptively advantageous. Here, we test the potential of adaptive hybridization as a mechanism for venom evolution by assessing the distribution of genes encoding the acidic and basic subunits of Mojave toxin across a hybrid zone between MTX-positive Crotalus scutulatus and MTX-negative C. viridis in southwestern New Mexico, USA. Analyses of morphology, mitochondrial and single copy-nuclear genes document extensive admixture within a narrow hybrid zone. The genes encoding the two MTX subunits are strictly linked, and found in most hybrids and backcrossed individuals, but not in C. viridis away from the hybrid zone. Presence of the genes is invariably associated with presence of the corresponding toxin in the venom. We conclude that introgression of highly lethal neurotoxins through hybridization is not necessarily favored by natural selection in rattlesnakes, and that even extensive hybridization may not lead to introgression of these genes into another species.

Reptiles as Reservoirs of Bacterial Infections: Real Threat or Methodological Bias?

Bacterial infections secondary to snakebites and human pathogens (e.g., Salmonella) have been linked to the oral microbiota of snakes and pet reptiles. Based on culture-dependent studies, it is speculated that snakes' oral microbiota reflects the fecal flora of their ingested preys. However, cultured-based techniques have been shown to be limited as they fail to identify unculturable microorganisms which represent the vast majority of the microbial diversity. Here, we used culture-independent high-throughput sequencing to identify reptile-associated pathogens and to characterize the oral microbial community of five snakes, one gecko, and two terrapins. Few potential human pathogens were detected at extremely low frequencies. Moreover, bacterial taxa represented in the snake's oral cavity bore little resemblance to their preys' fecal microbiota. Overall, we found distinct, highly diverse microbial communities with consistent, species-specific patterns contrary to previous culture-based studies. Our study does not support the widely held assumption that reptiles' oral cavity acts as pathogen reservoir and provides important insights for future research.

Comparative landscape genetics of two river frog species occurring at different elevations on Mount Kilimanjaro.

Estimating population connectivity and species' abilities to disperse across the landscape is crucial for understanding the long-term persistence of species in changing environments. Surprisingly, few landscape genetic studies focused on tropical regions despite the alarming extinction rates within these ecosystems. Here, we compared the influence of landscape features on the distribution of genetic variation of an Afromontane frog, Amietia wittei, with that of its more broadly distributed lowland congener, Amietia angolensis, on Mt. Kilimanjaro, Tanzania. We predicted high gene flow in the montane species with movements enhanced through terrestrial habitats of the continuous rainforest. In contrast, dispersal might be restricted to aquatic corridors and reduced by anthropogenic disturbance in the lowland species. We found high gene flow in A. wittei relative to other montane amphibians. Nonetheless, gene flow was lower than in the lowland species which showed little population structure. Least-cost path analysis suggested that dispersal is facilitated by stream networks in both species, but different landscape features were identified to influence connectivity among populations. Contrary to a previous study, gene flow in the lowland species was negatively correlated with the presence of human settlements. Also, genetic subdivision in A. wittei did not coincide with specific physical barriers as in other landscape genetic studies, suggesting that factors other than topography may contribute to population divergence. Overall, these results highlight the importance of a comparative landscape genetic approach for assessing the influence of the landscape matrix on population connectivity, particularly because nonintuitive results can alter the course of conservation and management.