Enlightening the toxinological dark matter of spider venom enzymes.

Spiders produce highly adapted venoms featuring a complex mixture of biomolecules used mainly for hunting and defense. The most prominent components are peptidic neurotoxins, a major focus of research and drug development, whereas venom enzymes have been largely neglected. Nevertheless, investigation of venom enzymes not only reveals insights into their biological functions, but also provides templates for future industrial applications. Here we compared spider venom enzymes validated at protein level contained in the VenomZone database and from all publicly available proteo-transcriptomic spider venom datasets. We assigned reported enzymes to cellular processes and known venom functions, including toxicity, prey pre-digestion, venom preservation, venom component activation, and spreading factors. Our study unveiled extensive discrepancy between public databases and publications with regard to enzyme coverage, which impedes the development of novel spider venom enzyme-based applications. Uncovering the previously unrecognized abundance and diversity of venom enzymes will open new avenues for spider venom biodiscovery.

Comparative venom analysis between melanistic and normally coloured phenotypes of the common adder (Vipera berus).

Snake venom is an ecologically relevant functional trait directly linked with a snake's fitness and survival, facilitating predation and defence. Snake venom variation occurs at all taxonomic levels, but the study at the intraspecific level is still in its early stages. The common adder (Vipera berus) exhibits considerable variation in colour phenotypes across its distribution range. Melanistic (fully black) individuals are the subject of myths and fairytales, and in German folklore such 'hell adders' are considered more toxic than their normally coloured conspecifics despite any formal investigation. Here, we provide the first comparative analysis of venoms from melanistic and normally coloured common adders. Specifically, we compared the venom profiles by sodium dodecylsulfate polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography and tested the venoms' protease, phospholipase A2 and cytotoxic activities. Phospholipase A2 activity was similar in both phenotypes, whereas general protease activity was higher in the melanistic venom, which was also more cytotoxic at two concentrations (6.25 and 12.5 µg ml-1). These minor differences between the venoms of melanistic and normally coloured adders are unlikely to be of clinical relevance in the context of human envenomation. In light of our results, the claim that melanistic adders produce more toxic venom than their normally coloured conspecifics appears rooted entirely in folklore.

Proteogenomics-guided functional venomics resolves the toxin arsenal and activity of Deinagkistrodon acutus venom.

Snakebite primarily impacts rural communities of Africa, Asia, and Latin America. The sharp-nosed viper (Deinagkistrodon acutus) is among the snakes of highest medical importance in Asia. Despite various studies on its venom using modern venomics techniques, a comprehensive understanding of composition and function of this species' venom remains lacking. We combined proteogenomics with extensive bioactivity profiling to present the first genome-level catalogue of D. acutus venom proteins and their exochemistry. Our analysis identified an unusually simple venom containing 45 components from 20 distinct protein families. Relative toxin abundances indicate that C-type lectin and C-type lectin-related protein (CTL), snake venom metalloproteinase (svMP), snake venom serine protease (svSP), and phospholipase A2 (PLA2) constitute 90 % of the venom. Bioassays targeting key aspects of viperid envenomation showed considerable concentration-dependent cytotoxicity, particularly in kidney and lung cells, and potent protease and PLA2 activity. Factor Xa and thrombin activities were minor, and no plasmin activity was observed. Effects on haemolysis, intracellular calcium (Ca2+) release, and nitric oxide (NO) synthesis were negligible. Our analysis provides the first holistic genome-based overview of the toxin arsenal of D. acutus, predicting the molecular and functional basis of its life-threatening effects, and opens novel avenues for treating envenomation by this highly dangerous snake.

Divergent venom effectors correlate with ecological niche in neuropteran predators.

Neuropteran larvae are fierce predators that use venom to attack and feed on arthropod prey. Neuropterans have adapted to diverse and sometimes extreme habitats, suggesting their venom may have evolved accordingly, but the ecology and evolution of venom deployment in different families is poorly understood. We applied spatial transcriptomics, proteomics, morphological analysis, and bioassays to investigate the venom systems in the antlion Euroleon nostras and the lacewing Chrysoperla carnea, which occupy distinct niches. Although the venom system morphology was similar in both species, we observed remarkable differences at the molecular level. E. nostras produces particularly complex venom secreted from three different glands, indicating functional compartmentalization. Furthermore, E. nostras venom and digestive tissues were devoid of bacteria, strongly suggesting that all venom proteins are of insect origin rather than the products of bacterial symbionts. We identified several toxins exclusive to E. nostras venom, including phospholipase A2 and several undescribed proteins with no homologs in the C. carnea genome. The compositional differences have significant ecological implications because only antlion venom conferred insecticidal activity, indicating its use for the immobilization of large prey. Our results indicate that molecular venom evolution plays a role in the adaptation of antlions to their unique ecological niche.

Venom Ex Machina? Exploring the Potential of Cell-Free Protein Production for Venom Biodiscovery.

Venoms are a complex cocktail of potent biomolecules and are present in many animal lineages. Owed to their translational potential in biomedicine, agriculture and industrial applications, they have been targeted by several biodiscovery programs in the past. That said, many venomous animals are relatively small and deliver minuscule venom yields. Thus, the most commonly employed activity-guided biodiscovery pipeline cannot be applied effectively. Cell-free protein production may represent an attractive tool to produce selected venom components at high speed and without the creation of genetically modified organisms, promising rapid and highly efficient access to biomolecules for bioactivity studies. However, these methods have only sporadically been used in venom research and their potential remains to be established. Here, we explore the ability of a prokaryote-based cell-free system to produce a range of venom toxins of different types and from various source organisms. We show that only a very limited number of toxins could be expressed in small amounts. Paired with known problems to facilitate correct folding, our preliminary investigation underpins that venom-tailored cell-free systems probably need to be developed before this technology can be employed effectively in venom biodiscovery.

Diversely evolved xibalbin variants from remipede venom inhibit potassium channels and activate PKA-II and Erk1/2 signaling.

BACKGROUND: The identification of novel toxins from overlooked and taxonomically exceptional species bears potential for various pharmacological applications. The remipede Xibalbanus tulumensis, an underwater cave-dwelling crustacean, is the only crustacean for which a venom system has been described. Its venom contains several xibalbin peptides that have an inhibitor cysteine knot (ICK) scaffold. RESULTS: Our screenings revealed that all tested xibalbin variants particularly inhibit potassium channels. Xib1 and xib13 with their eight-cysteine domain similar to spider knottins also inhibit voltage-gated sodium channels. No activity was noted on calcium channels. Expanding the functional testing, we demonstrate that xib1 and xib13 increase PKA-II and Erk1/2 sensitization signaling in nociceptive neurons, which may initiate pain sensitization. Our phylogenetic analysis suggests that xib13 either originates from the common ancestor of pancrustaceans or earlier while xib1 is more restricted to remipedes. The ten-cysteine scaffolded xib2 emerged from xib1, a result that is supported by our phylogenetic and machine learning-based analyses. CONCLUSIONS: Our functional characterization of synthesized variants of xib1, xib2, and xib13 elucidates their potential as inhibitors of potassium channels in mammalian systems. The specific interaction of xib2 with Kv1.6 channels, which are relevant to treating variants of epilepsy, shows potential for further studies. At higher concentrations, xib1 and xib13 activate the kinases PKA-II and ERK1/2 in mammalian sensory neurons, suggesting pain sensitization and potential applications related to pain research and therapy. While tested insect channels suggest that all probably act as neurotoxins, the biological function of xib1, xib2, and xib13 requires further elucidation. A novel finding on their evolutionary origin is the apparent emergence of X. tulumensis-specific xib2 from xib1. Our study is an important cornerstone for future studies to untangle the origin and function of these enigmatic proteins as important components of remipede but also other pancrustacean and arthropod venoms.

Determining the pharmacological potential and biological role of linear pseudoscorpion toxins via functional profiling.

Arthropod venoms contain bioactive molecules attractive for biomedical applications. However, few of these have been isolated, and only a tiny number has been characterized. Pseudoscorpions are small arachnids whose venom has been largely overlooked. Here, we present the first structural and functional assessment of the checacin toxin family, discovered in the venom of the house pseudoscorpion (Chelifer cancroides). We combined in silico and in vitro analyses to establish their bioactivity profile against microbes and various cell lines. This revealed inhibitory effects against bacteria and fungi. We observed cytotoxicity against specific cell types and effects involving second messengers. Our work provides insight into the biomedical potential and evolution of pseudoscorpion venoms. We propose that plesiotypic checacins evolved to defend the venom gland against infection, whereas apotypic descendants evolved additional functions. Our work highlights the importance of considering small and neglected species in biodiscovery programs.

Structural characterization and bioactivity profiling of the fungal peptaibiotic tolypin reveal protective effects against influenza viruses.

In a bioprospection for new antivirals, we tested nonribosomally biosynthesized polypeptide antibiotics in MDCK II cells for their actions on influenza A and B viruses (IAV/IBV). Only tolypin, a mixture of closely related 16-residue peptaibiotics from the fungus Tolypocladium inflatum IE 1897, showed promising activity. It was selected for further investigation and structural characterization by ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HR-MS/MS) and ultrahigh performance liquid chromatography coupled to in-source collision-induced dissociation tandem mass spectrometry (UHPLC-isCID-HR-MS/MS), revealing 12 partially co-eluting individual peptides that were fully sequenced. Since tolypin-related efrapeptins are potent inhibitors of F1/Fo-ATPase, we screened tolypin for its toxicity against MDCK II cells and larvae of the greater wax moth Galleria mellonella. We found that a nontoxic concentration of tolypin (1 µg/mL) reduced the titer of two IBV strains by 4-5 log values, and that of an H3N2 strain by 1-2 log values, but the H1N1pdm strain was not affected. The higher concentrations of tolypin were cytostatic to MDCK II cells, shifted their metabolism from oxidative phosphorylation to glycolysis, and induced paralysis in G. mellonella, supporting the inhibition of F1/Fo-ATPase as the mode of action. Our results lay the foundations for future work to investigate the interplay between viral replication and cellular energy metabolism, as well as the development of drugs that target host factors.

Current Insights into Sublethal Effects of Pesticides on Insects.

The effect of pesticides on insects is often discussed in terms of acute and chronic toxicity, but an important and often overlooked aspect is the impact of sublethal doses on insect physiology and behavior. Pesticides can influence various physiological parameters of insects, including the innate immune system, development, and reproduction, through a combination of direct effects on specific exposed tissues and the modification of behaviors that contribute to health and reproductive success. Such behaviors include mobility, feeding, oviposition, navigation, and the ability to detect pheromones. Pesticides also have a profound effect on insect learning and memory. The precise effects depend on many different factors, including the insect species, age, sex, caste, physiological condition, as well as the type and concentration of the active ingredients and the exposure route. More studies are needed to assess the effects of different active ingredients (and combinations thereof) on a wider range of species to understand how sublethal doses of pesticides can contribute to insect decline. This review reflects our current knowledge about sublethal effects of pesticides on insects and advancements in the development of innovative methods to detect them.

Engineering a wolf spider A-family toxin towards increased antimicrobial activity but low toxicity.

Spider-derived peptides with insecticidal, antimicrobial and/or cytolytic activities, also known as spider venom antimicrobial peptides (AMPs), can be found in the venoms of RTA-clade spiders. They show translational potential as therapeutic leads. A set of 52 AMPs has been described in the Chinese wolf spider (Lycosa shansia), and many have been shown to exhibit antibacterial effects. Here we explored the potential to enhance their antimicrobial activity using bioengineering. We generated a panel of artificial derivatives of an A-family peptide and screened their activity against selected microbial pathogens, vertebrate cells and insects. In several cases, we increased the antimicrobial activity of the derivatives while retaining the low cytotoxicity of the parental molecule. Furthermore, we injected the peptides into adult Drosophila suzukii and found no evidence of insecticidal effects, confirming the low levels of toxicity. Our data therefore suggest that spider venom linear peptides naturally defend the venom gland against microbial colonization and can be modified into more potent antimicrobial agents that could help to battle infectious diseases in the future.

Gene silencing in the aedine cell lines C6/36 and U4.4 using long double-stranded RNA.

BACKGROUND: RNA interference (RNAi) is a target-specific gene silencing method that can be used to determine gene functions and investigate host-pathogen interactions, as well as facilitating the development of ecofriendly pesticides. Commercially available transfection reagents (TRs) can improve the efficacy of RNAi. However, we currently lack a product and protocol for the transfection of insect cell lines with long double-stranded RNA (dsRNA). METHODS: We used agarose gel electrophoresis to determine the capacity of eight TRs to form complexes with long dsRNA. A CellTiter-Glo assay was then used to assess the cytotoxicity of the resulting lipoplexes. We also measured the cellular uptake of dsRNA by fluorescence microscopy using the fluorophore Cy3 as a label. Finally, we analyzed the TRs based on their transfection efficacy and compared the RNAi responses of Aedes albopictus C6/36 and U4.4 cells by knocking down an mCherry reporter Semliki Forest virus in both cell lines. RESULTS: The TRs from Biontex (K4, Metafectene Pro, and Metafectene SI+) showed the best complexing capacity and the lowest dsRNA:TR ratio needed for complete complex formation. Only HiPerFect was unable to complex the dsRNA completely, even at a ratio of 1:9. Most of the complexes containing mCherry-dsRNA were nontoxic at 2 ng/µL, but Lipofectamine 2000 was toxic at 1 ng/µL in U4.4 cells and at 2 ng/µL in C6/36 cells. The transfection of U4.4 cells with mCherry-dsRNA/TR complexes achieved significant knockdown of the virus reporter. Comparison of the RNAi response in C6/36 and U4.4 cells suggested that C6/36 cells lack the antiviral RNAi response because there was no significant knockdown of the virus reporter in any of the treatments. CONCLUSIONS: C6/36 cells have an impaired RNAi response as previously reported. This investigation provides valuable information for future RNAi experiments by showing how to mitigate the adverse effects attributed to TRs. This will facilitate the judicious selection of TRs and transfection conditions conducive to RNAi research in mosquitoes.

Radioactivity and GMO-Free Sterile Insect Technology for the Sustainable Control of the Invasive Pest Drosophila suzukii.

Drosophila suzukii (D. suzukii), commonly known as the spotted wing drosophila, is a highly invasive crop pest that is difficult to control using chemical insecticides. To address the urgent need for alternative and more sustainable control strategies, the sterile insect technique (SIT) is improved, which involves the release of sterilized male insects to mate with fertile conspecifics, thereby reducing the size of the pest population in the subsequent generation. The three critical aspects that influence the success of SIT programs in D. suzukii are addressed. First, an accurate and nondestructive method is established to determine the sex of individual insects based on the differential weight of male and female pupae. Second, conditions for X-ray sterilization are systematically tested and an optimal dose (90 kV/40 Gy) is identified that ensures the efficient production of sterile D. suzukii for release. Finally, the inherent thermosensitivity of D. suzukii males is exploited to develop a temperature-based sterilization technique, offering an alternative or additional SIT method for this pest. These advances will contribute to the development of a comprehensive and effective strategy for the management of D. suzukii populations, reducing their impact on agriculture and helping to safeguard crop yields.

Prevalence in Potato of 'Candidatus Arsenophonus Phytopathogenicus' and 'Candidatus Phytoplasma Solani' and Their Transmission via Adult Pentastiridius leporinus.

The planthopper Pentastiridius leporinus (Hempiptera: Cixiidae) is the main vector of two bacterial pathogens: the γ-proteobacterium 'Candidatus Arsenophonus phytopathogenicus' and the stolbur phytoplasma 'Candidatus Phytoplasma solani'. These pathogens cause the disease syndrome basses richesses (SBR) in sugar beet (Beta vulgaris), which reduces the yields and sugar content. In 2022, potato (Solanum tuberosum) fields were found to be colonized by P. leporinus, and the transmission of Arsenophonus was confirmed, resulting in symptoms like wilting, yellow leaves, and rubbery tubers. We monitored both pathogens in Southwest Germany in 2022 and 2023. This revealed their widespread presence in potato tubers, although there were differences in regional prevalence. The broad prevalence of Arsenophonus was maintained in 2023, whereas the prevalence of stolbur increased in most locations. We confirmed that P. leporinus adults can transmit both pathogens to potatoes, but neither pathogen reduced the germination rate of tubers, and no plants showed abnormal growth after germination. Arsenophonus was not detected in germinated shoots, but 5.4% contained stolbur, emphasizing the need for plant material testing to maintain phytosanitary conditions.

An enteric ultrastructural surface atlas of the model insect Manducasexta.

The tobacco hornworm is a laboratory model that is particularly suitable for analyzing gut inflammation, but a physiological reference standard is currently unavailable. Here, we present a surface atlas of the healthy hornworm gut generated by scanning electron microscopy and nano-computed tomography. This comprehensive overview of the gut surface reveals morphological differences between the anterior, middle, and posterior midgut, allowing the screening of aberrant gut phenotypes while accommodating normal physiological variations. We estimated a total resorptive midgut surface of 0.42 m2 for L5d6 larvae, revealing its remarkable size. Our data will support allometric scaling and dose conversion from Manduca sexta to mammals in preclinical research, embracing the 3R principles. We also observed non-uniform gut colonization by enterococci, characterized by dense biofilms in the pyloric cone and downstream of the pylorus associated with pore and spine structures in the hindgut intima, indicating a putative immunosurveillance function in the lepidopteran hindgut.

The insect microbiome is a vast source of bioactive small molecules.

Covering: September 1964 to June 2023Bacteria and fungi living in symbiosis with insects have been studied over the last sixty years and found to be important sources of bioactive natural products. Not only classic producers of secondary metabolites such as Streptomyces and other members of the phylum Actinobacteria but also numerous bacteria from the phyla Proteobacteria and Firmicutes and an impressive array of fungi (usually pathogenic) serve as the source of a structurally diverse number of small molecules with important biological activities including antimicrobial, cytotoxic, antiparasitic and specific enzyme inhibitors. The insect niche is often the exclusive provider of microbes producing unique types of biologically active compounds such as gerumycins, pederin, dinactin, and formicamycins. However, numerous insects still have not been described taxonomically, and in most cases, the study of their microbiota is completely unexplored. In this review, we present a comprehensive survey of 553 natural products produced by microorganisms isolated from insects by collating and classifying all the data according to the type of compound (rather than the insect or microbial source). The analysis of the correlations among the metadata related to insects, microbial partners, and their produced compounds provides valuable insights into the intricate dynamics between insects and their symbionts as well as the impact of their metabolites on these relationships. Herein, we focus on the chemical structure, biosynthesis, and biological activities of the most relevant compounds.

The Crayfish Plague Pathogen Aphanomyces astaci in Ireland.

Crayfish plague is a devastating disease of European freshwater crayfish and is caused by the oomycete Aphanomyces astaci (Ap. astaci), believed to have been introduced to Europe around 1860. All European species of freshwater crayfish are susceptible to the disease, including the white-clawed crayfish Austropotamobius pallipes. Ap. astaci is primarily spread by North American crayfish species and can also disperse rapidly through contaminated wet gear moved between water bodies. This spread, coupled with competition from non-indigenous crayfish, has drastically reduced and fragmented native crayfish populations across Europe. Remarkably, the island of Ireland remained free from the crayfish plague pathogen for over 100 years, providing a refuge for A. pallipes. However, this changed in 1987 when a mass mortality event was linked to the pathogen, marking its introduction to the region. Fortunately, crayfish plague was not detected again in Ireland until 2015 when a molecular analysis linked a mass mortality event in the Erne catchment to Ap. astaci. Since then, the pathogen has appeared across the island. Between 2015 and 2023, Ap. astaci was detected in 18 water catchments, revealing multiple genotypes. Intriguingly, the pathogen in Ireland is present without its natural host species. The uneven distribution of various genetic lineages strongly suggests the human-mediated transport of zoospores via contaminated water equipment as a primary cause of spread. This review details the timeline of these events, Ap. astaci's introduction into Ireland, and its rapid spread. As well, this review references the genotypes that have been determined, and discusses the issue of non-indigenous crayfish species in Ireland and management efforts.

Design of Polymer Carriers for Optimized Pheromone Release in Sustainable Insect Control Strategies.

Semiochemicals such as pheromones play a major role in communication between insects, influencing their spatial orientation, aggregation, defense, and mating. The rational chemical design of precision pheromone-releasing materials are increased the efficiency of pheromone-based plant protection agents. Decades of research is begun to unravel the complex communication structures regulated by semiochemicals, from the neuronal perception of specific chemical substances to the behavioral responses in hundreds of species, including many devastating pest insects. This article summarizes the most effective uses of semiochemicals in agriculture, the behavioral responses of selected target species, and controlled-release strategies based on formulations such as novel fibrous polymer carriers. This study helps scientists, decision-makers, farmers, and the public understand the importance of appropriate mating disruption techniques that reduce the need for broad-spectrum insecticides and limit their impact on non-target and beneficial insects.

Prevalent bee venom genes evolved before the aculeate stinger and eusociality.

BACKGROUND: Venoms, which have evolved numerous times in animals, are ideal models of convergent trait evolution. However, detailed genomic studies of toxin-encoding genes exist for only a few animal groups. The hyper-diverse hymenopteran insects are the most speciose venomous clade, but investigation of the origin of their venom genes has been largely neglected. RESULTS: Utilizing a combination of genomic and proteo-transcriptomic data, we investigated the origin of 11 toxin genes in 29 published and 3 new hymenopteran genomes and compiled an up-to-date list of prevalent bee venom proteins. Observed patterns indicate that bee venom genes predominantly originate through single gene co-option with gene duplication contributing to subsequent diversification. CONCLUSIONS: Most Hymenoptera venom genes are shared by all members of the clade and only melittin and the new venom protein family anthophilin1 appear unique to the bee lineage. Most venom proteins thus predate the mega-radiation of hymenopterans and the evolution of the aculeate stinger.

The resuscitation-promoting factor (Rpf) from Micrococcus luteus and its putative reaction product 1,6-anhydro-MurNAc increase culturability of environmental bacteria.

Dormant bacterial cells do not divide and are not immediately culturable, but they persist in a state of low metabolic activity, a physiological state having clinical relevance, for instance in latent tuberculosis. Resuscitation-promoting factors (Rpfs) are proteins that act as signalling molecules mediating growth and replication. In this study we aimed to test the effect of Rpfs from Micrococcus luteus on the number and diversity of cultured bacteria using insect and soil samples, and to examine if the increase in culturability could be reproduced with the putative reaction product of Rpf, 1,6-anhydro-N-acetylmuramic acid (1,6-anhydro-MurNAc). The rpf gene from Micrococcus luteus was amplified and cloned into a pET21b expression vector and the protein was expressed in Escherichia coli BL21(DE3) cells and purified by affinity chromatography using a hexa-histidine tag. 1,6-Anhydro-MurNAc was prepared using reported chemical synthesis methods. Recombinant Rpf protein or 1,6-anhydro-MurNAc were added to R2A cultivation media, and their effect on the culturability of bacteria from eight environmental samples including four cockroach guts and four soils was examined. Colony-forming units, 16S rRNA gene copies and Illumina amplicon sequencing of the 16S rRNA gene were measured for all eight samples subjected to three different treatments: Rpf, 1,6-anhydro-MurNAc or blank control. Both Rpf and 1,6-anhydro-MurNAc increased the number of colony-forming units and of 16S rRNA gene copies across the samples although the protein was more effective. The Rpf and 1,6-anhydro-MurNAc promoted the cultivation of a diverse set of bacteria and in particular certain clades of the phyla Actinomycetota and Bacillota . This study opens the path for improved cultivation strategies aiming to isolate and study yet undescribed living bacterial organisms.

Venomics of the milos viper (Macrovipera schweizeri) unveils patterns of venom composition and exochemistry across blunt-nosed viper venoms.

Introduction: Snakebite is a neglected tropical disease and a globally important driver of death and morbidity. Vipers of the genus Macrovipera (Viperidae: Viperinae) are among the snakes of higher medical importance in the Old World. Despite the medical relevance of Macrovipera venoms, the knowledge regarding them is heterogeneously distributed with virtually all works conducted so far focusing on subspecies of Macrovipera lebetinus, while other species within the genus are largely overlooked. Here we present the first proteomic evaluation of the venom from the Greek endemic Milos viper (Macrovipera schweizeri). In line with clinical symptoms typically elicited by Macrovipera envenomations, Milos viper venom primarily comprises coagulotoxic and cytotoxic protein families, such as metalloproteinases (svMP) and serine proteases (svSP). Methods: We conducted comparative bioactivity assays on venoms from M. schweizeri and the M. lebetinus subspecies M. lebetinus cernovi, M. lebetinus obtusa, and M. lebetinus turanica, and showed that they all exhibit similarities in levels of cytotoxicity proteolytic activity, and inhibition of prokaryotic growth. Lastly, we compared Macrovipera venom profiles by 1D-SDS-PAGE and RP-HPLC, as well as our proteomic data with previously published Macrovipera venom proteomes. Results and discussion: The analyzes performed to reveal that a general venom profile seems to be conserved across blunt-nosed vipers, and that, M. schweizeri envenomations, similarly to those caused by other blunt-nosed vipers, are able to cause significant tissue damage. The present work represents an important starting point for the development of comparative studies across the full taxonomic range of the genus Macrovipera and can potentially help optimize the treatment of envenomations caused by M. schweizeri.