Variation In Venom Composition Research Articles

Prey Shifts Drive Venom Evolution in Cone Snails.

Venom systems are complex traits that have independently emerged multiple times in diverse plant and animal phyla. Within each venomous lineage there typically exists interspecific variation in venom composition where several factors have been proposed as drivers of variation, including phylogeny and diet. Understanding these factors is of broad biological interest and has implications for the development of antivenom therapies and venom-based drug discovery. Because of their high species richness and the presence of several major evolutionary prey shifts, venomous marine cone snails (genus Conus) provide an ideal system to investigate drivers of interspecific venom variation. Here, by analyzing the venom gland expression profiles of ∼3,000 toxin genes from 42 species of cone snail, we elucidate the role of prey-specific selection pressures in shaping venom variation. By analyzing overall venom composition and individual toxin structures, we demonstrate that the shifts from vermivory to piscivory in Conus are complemented by distinct changes in venom composition independent of phylogeny. In vivo injections of venom from piscivorous cone snails in fish further showed a higher potency compared with venom of nonpiscivores demonstrating a selective advantage. Together, our findings provide compelling evidence for the role of prey shifts in directing the venom composition of cone snails and expand our understanding of the mechanisms of venom variation and diversification.

Transcriptome profiling of the posterior salivary glands of the cuttlefish Sepia officinalis from the Portuguese West coast

Cephalopods like octopuses and cuttlefishes are known to secrete a ‘toxic saliva’ to inject into their prey, especially crustaceans since the XIX century. However, only in the mid-XX century were the first coleoid-specific toxins successfully isolated. Motivated by the growing interest on the global ocean as an almost inexhaustible source of novel bioactive compounds, we used RNA-Seq – based transcriptomics and de novo assembly of transcriptomes to screen the posterior salivary glands of Sepia officinalis (the common cuttlefish) from the Portuguese West coast for toxins and other bioactive proteins and peptides. Supported by microanatomical analyses, the posterior salivary glands constitute indeed the ‘venom gland’ whereas the more elusive anterior salivary glands (embedded in the buccal mass) are responsible for the production of mucin-rich saliva that is effectively the vehicle that transports the toxins as the venom is injected into the prey. Indeed, the transcriptomic profiling suggests that the cuttlefish venom is complex mixture of bioactive proteins, among which neurotoxins are major players, together with enzymes whose function is to digest the extracellular matrix to facilitate diffusion of the toxins. Nonetheless, by comparing with previous RNA-Seq data obtained from S. officinalis collected from other biogeographical areas, it may be suggested that significant inter-populational variation in venom composition can occur, which may potentially increase the span of bioactives secreted by these animals. We isolated and validated the full coding sequences for three important toxins, a cysteine-rich venom protein (CRVP), a venom insulin (VIns) and a cephalotoxin (CTX). The toxins seem to be relatively conserved among coleoids but diverging from other venomous mollusks such as cone snails. Their properties as potent modulators of glucose (in the case of VIns) and as potential neurotoxins (like CRVP and CTX) can render them primer targets for drug development.

Snakes on a plain: biotic and abiotic factors determine venom compositional variation in a wide-ranging generalist rattlesnake

BackgroundSnake venoms are trophic adaptations that represent an ideal model to examine the evolutionary factors that shape polymorphic traits under strong natural selection. Venom compositional variation is substantial within and among venomous snake species. However, the forces shaping this phenotypic complexity, as well as the potential integrated roles of biotic and abiotic factors, have received little attention. Here, we investigate geographic variation in venom composition in a wide-ranging rattlesnake (Crotalus viridis viridis) and contextualize this variation by investigating dietary, phylogenetic, and environmental variables that covary with venom.ResultsUsing shotgun proteomics, venom biochemical profiling, and lethality assays, we identify 2 distinct divergent phenotypes that characterize major axes of venom variation in this species: a myotoxin-rich phenotype and a snake venom metalloprotease (SVMP)-rich phenotype. We find that dietary availability and temperature-related abiotic factors are correlated with geographic trends in venom composition.ConclusionsOur findings highlight the potential for snake venoms to vary extensively within species, for this variation to be driven by biotic and abiotic factors, and for the importance of integrating biotic and abiotic variation for understanding complex trait evolution. Links between venom variation and variation in biotic and abiotic factors indicate that venom variation likely results from substantial geographic variation in selection regimes that determine the efficacy of venom phenotypes across populations and snake species. Our results highlight the cascading influence of abiotic factors on biotic factors that ultimately shape venom phenotype, providing evidence for a central role of local selection as a key driver of venom variation.

Taxon-selective venom variation in adult and neonate Daboia russelii (Russell's Viper), and antivenom efficacy

Major variations in venom composition can occur between juvenile and adult venomous snakes. However, due to logistical constraints, antivenoms are produced using adult venoms in immunising mixtures, possibly resulting in limited neutralisation of juvenile snake venoms. Daboia russelii is one of the leading causes of snakebite death across South Asia. Its venom is potently procoagulant, causing stroke in prey animals but causing in humans consumptive coagulopathy—a net anticoagulant state—and sometimes death resulting from hemorrhage. In this in vitro study, we compared the venom activity of—and antivenom efficacy against—six 2-week-old D. russelii relative to that of their parents. Using a coagulation analyser, we quantified the relative coagulotoxicity of these venoms in human, avian, and amphibian plasma. The overall potency on human plasma was similar across all adult and neonate venoms, and SII (Serum Institute of India) antivenom was equipotent in neutralising these coagulotoxic effects. In addition, all venoms were also similar in their action upon avian plasma. In contrast, the neonate venoms were more potent on amphibian plasma, suggesting amphibians make up a larger proportion of neonate diet than adult diet. A similar venom potency in human and avian plasmas but varying selectivity for amphibian plasma suggests ontogenetic differences in toxin isoforms within the factor X or factor V activating classes, thereby providing a testable hypothesis for future transcriptomics work. By providing insights into the functional venom differences between adult and neonate D. russelii venoms, we hope to inform clinical treatment of patients envenomated by this deadly species and to shed new light on the natural history of these extremely medically important snakes.

Assessment of quality and pre-clinical efficacy of a newly developed polyvalent antivenom against the medically important snakes of Sri Lanka

Snake envenomation is a severe problem in Sri Lanka (SL) and Indian polyvalent antivenom (PAV) is mostly used for treating snakebite albeit due to geographical variation in venom composition, Indian PAV shows poor efficacy in neutralizing the lethality and toxicity of venom from the same species of snakes in SL. Therefore, the quality and in vivo venom neutralization potency of a country-specific PAV produced against the venom of the five most medically important snakes of SL (Daboia russelii, Echis carinatus, Hypnale hypnale, Naja naja, Bungarus caeruleus) was assessed. LC-MS/MS analysis of two batches of PAV showed the presence of 88.7–97.2% IgG and traces of other plasma proteins. The tested PAVs contained minor amounts of undigested IgG and F(ab′)2 aggregates, showed complement activation, were devoid of IgE, endotoxin, and content of preservative was below the threshold level. Immunological cross-reactivity and in vitro neutralization of enzymatic activities, pharmacological properties demonstrated superior efficacy of SL PAV compared to Indian PAV against SL snake venoms. The in vivo neutralization study showed that the tested PAVs are potent to neutralize the lethality and venom-induced toxicity of SL snake venoms. Therefore, our study suggests that introduction of SL-specific PAV will improve snakebite management in SL.

King Cobra and snakebite envenomation: on the natural history, human-snake relationship and medical importance of Ophiophagus hannah.

King Cobra (Ophiophagus hannah) has a significant place in many cultures, and is a medically important venomous snake in the world. Envenomation by this snake is highly lethal, manifested mainly by neurotoxicity and local tissue damage. King Cobra may be part of a larger species complex, and is widely distributed across Southeast Asia, southern China, northern and eastern regions as well as the Western Ghats of India, indicating potential geographical variation in venom composition. There is, however, only one species-specific King Cobra antivenom available worldwide that is produced in Thailand, using venom from the snake of Thai origin. Issues relating to the management of King Cobra envenomation (e.g., variation in the composition and toxicity of the venom, limited availability and efficacy of antivenom), and challenges faced in the research of venom (in particular proteomics), are rarely addressed. This article reviews the natural history and sociocultural importance of King Cobra, cases of snakebite envenomation caused by this species, current practice of management (preclinical and clinical), and major toxinological studies of the venom with a focus on venom proteomics, toxicity and neutralization. Unfortunately, epidemiological data of King Cobra bite is scarce, and venom proteomes reported in various studies revealed marked discrepancies in details. Challenges, such as inconsistency in snake venom sampling, varying methodology of proteomic analysis, lack of mechanistic and antivenomic studies, and controversy surrounding antivenom use in treating King Cobra envenomation are herein discussed. Future directions are proposed, including the effort to establish a standard, comprehensive Pan-Asian proteomic database of King Cobra venom, from which the venom variation can be determined. Research should be undertaken to characterize the toxin antigenicity, and to develop an antivenom with improved efficacy and wider geographical utility. The endeavors are aligned with the WHO´s roadmap that aims to reduce the disease burden of snakebite by 50% before 2030.

A therapeutic combination of two small molecule toxin inhibitors provides broad preclinical efficacy against viper snakebite

Snakebite is a medical emergency causing high mortality and morbidity in rural tropical communities that typically experience delayed access to unaffordable therapeutics. Viperid snakes are responsible for the majority of envenomings, but extensive interspecific variation in venom composition dictates that different antivenom treatments are used in different parts of the world, resulting in clinical and financial snakebite management challenges. Here, we show that a number of repurposed Phase 2-approved small molecules are capable of broadly neutralizing distinct viper venom bioactivities in vitro by inhibiting different enzymatic toxin families. Furthermore, using murine in vivo models of envenoming, we demonstrate that a single dose of a rationally-selected dual inhibitor combination consisting of marimastat and varespladib prevents murine lethality caused by venom from the most medically-important vipers of Africa, South Asia and Central America. Our findings support the translation of combinations of repurposed small molecule-based toxin inhibitors as broad-spectrum therapeutics for snakebite.

Variation in venom composition in the Australian funnel-web spiders Hadronyche valida.

Mygalomorph venom properties and active components, which have importance in medicine, agronomy, venomics, ecology and evolution, have been widely studied, but only a small fraction have been characterised. Several studies have shown inter-individual variation in the composition of venom peptides based on ontogeny, sexual dimorphism, season and diet. However, intra-individual variation in venom composition, which could play a key role in the evolution, diversification and function of toxins, is poorly understood. In this study, we demonstrate significant intra- and inter-individual variation in venom composition in the Australian funnel-web spider Hadronyche valida, highlighting that individuals show different venom profiles over time. Fourteen (four juvenile and ten adult females) funnel-web spiders, maintained under the same environmental conditions and diet, were milked a total of four times, one month apart. We then used reversed-phase high performance liquid chromatography/electrospray ionisation mass spectrometry to generate venom fingerprints containing the retention time and molecular weights of the different toxin components in the venom. Across all individuals, we documented a combined total of 83 individual venom components. Only 20% of these components were shared between individuals. Individuals showed variation in the composition of venom peptides, with some components consistently present over time, while others were only present at specific times. When individuals were grouped using the Jaccard clustering index and Kernel Principal Component Analysis, spiders formed two distinct clusters, most likely due to their origin or time of collection. This study contributes to the understanding of variation in venom composition at different levels (intra-individual, and intra- and inter-specific) and considers some of the mechanisms of selection that may contribute to venom diversification within arachnids. In addition, inter-specific variation in venom composition can be highly useful as a chemotaxonomic marker to identify funnel-web species.

Species-specific and geographical variation in venom composition of two major cobras in Indian subcontinent: Impact on polyvalent antivenom therapy

While snakebite is a severe problem for several countries throughout the world the Indian subcontinent has witnessed the highest global incidence of snakebite which represents an occupational health hazard. Belonging to the family Elapidae, the Indian spectacled cobra (Naja naja), is one of the members of the “Big Four” venomous snakes found throughout the Indian subcontinent. Indian monocled cobra Naja kaouthia is prevalent in eastern and north-eastern India as well as in Bangladesh, Nepal, and Bhutan. Species-specific and geographical differences in venom composition of two major Indian cobras (N. naja vs. N. kaouthia) may limit the effectiveness of antivenom treatment. Therefore, exploring the venom proteomes of N. naja and N. kaouthia from different geographical locales of the Indian subcontinent, and the effect of this variation on antivenom efficacy is warranted. Proteomic analyses have demonstrated that although eastern Indian N. naja and N. kaouthia venoms have similarities, species-specific and geographical variation is evident by differences in their venom toxicity values and pathobiological effects in experimental animals. This is the major reason as to why a polyvalent antivenom against N. naja venom exhibited only partial neutralization of enzymatic activity and immunological cross-reactivity against cobra venoms from other regions of India. This suggests that addition of polyclonal antibodies against N. kaouthia venom to polyantivenom, or preparation/administration of region-specific polyantivenom will provide better treatment of cobra bites in the Indian subcontinent.

Interventions for the management of snakebite envenoming: An overview of systematic reviews.

IntroductionSnakebite is a neglected tropical disease that leads to more than 120,000 deaths every year. In 2019, World Health Organization (WHO) launched a strategy to decrease its global burden by 2030. There is a range of issues around different interventions for the management of snakebite. Decisions around these interventions should be informed by evidence from systematic reviews (SR).MethodsAn overview of SRs was conducted by searching 12 electronic databases, PROSPERO, contacting experts and screening the bibliography of included reviews. Screening, data extraction, and quality assessment (through AMSTAR-2) was done by at least two overview authors independently with discrepancies sorted by consensus. A narrative synthesis was conducted.Principle findingsThe overview found 13 completed SRs that has looked at various aspects of management of snakebite envenomation. There was one SR on first aid, nine on effectiveness and safety of snake anti-venom (SAV), two on drugs to prevent adverse reactions due to SAV therapy, and one on surgical interventions for management of snakebite envenomation. All, except one, SR was appraised to have critically low confidence as per AMSTAR-2 Criteria. Evidence base was restricted to few studies for most interventions.DiscussionHigh quality evidence from SRs is required to inform guidelines and health system decisions which can bring down the burden of snakebite. The review indicates the need to fund high-quality SRs, evidence gaps and core outcome sets which can inform guideline recommendations, funding priorities for conduct of future trials. Variation in species distribution as well as intra-species variation in venom composition implies the need for conduct of region or, nation or state (sub-national) specific randomised controlled trials and SRs on different SAVs and their dosing regimens.

Trimeresurus albolabris snakebite treatment implications arising from ontogenetic venom comparisons of anticoagulant function, and antivenom efficacy

Does the venom of Trimeresurus albolabris (white-lipped pit viper) differ between neonate and adults? This species is responsible for most snakebites within south and southeast Asia, yet it is unknown whether ontogenetic variation in venom composition occurs in this species, or how this might affect antivenom efficacy. Using a coagulation analyser robot, we examined the anticoagulant activity of T. albolabris venom from eight individuals across multiple age classes. We then compared the efficacy of Thai Red Cross Green Pit Viper Antivenom across these age classes. Venoms from all age classes were equally potent in their pseudo-procoagulant, fibrinogenolytic activity, in that fibrinogen was cleaved to form weak, unstable fibrin clots that rapidly broke down, thus resulting in a net anticoagulant state. Similarly, this coagulotoxic activity was well neutralised by antivenom across all venoms. Given that coagulotoxicity is the primary serious pathology in T. albolabris envenomations, we conclude that Thai Red Cross Green Tree Pit Viper Antivenom is a valid treatment for envenomations by this species, regardless of age or sex of the offending snake. These results are relevant for clinical treatment of envenomations by T. albolabris.

Variations in neurotoxicity and proteome profile of Malayan krait (Bungarus candidus) venoms.

Malayan krait (Bungarus candidus) is a medically important snake species found in Southeast Asia. The neurotoxic effects of envenoming present as flaccid paralysis of skeletal muscles. It is unclear whether geographical variation in venom composition plays a significant role in the degree of clinical neurotoxicity. In this study, the effects of geographical variation on neurotoxicity and venom composition of B. candidus venoms from Indonesia, Malaysia and Thailand were examined. In the chick biventer cervicis nerve-muscle preparation, all venoms abolished indirect twitches and attenuated contractile responses to nicotinic receptor agonists, with venom from Indonesia displaying the most rapid neurotoxicity. A proteomic analysis indicated that three finger toxins (3FTx), phospholipase A2 (PLA2) and Kunitz-type serine protease inhibitors were common toxin groups in the venoms. In addition, venom from Thailand contained L-amino acid oxidase (LAAO), cysteine rich secretory protein (CRISP), thrombin-like enzyme (TLE) and snake venom metalloproteinase (SVMP). Short-chain post-synaptic neurotoxins were not detected in any of the venoms. The largest quantity of long-chain post-synaptic neurotoxins and non-conventional toxins was found in the venom from Thailand. Analysis of PLA2 activity did not show any correlation between the amount of PLA2 and the degree of neurotoxicity of the venoms. Our study shows that variation in venom composition is not limited to the degree of neurotoxicity. This investigation provides additional insights into the geographical differences in venom composition and provides information that could be used to improve the management of Malayan krait envenoming in Southeast Asia.

Geographic variation in morphology in the Mohave Rattlesnake (Crotalus scutulatus Kennicott 1861) (Serpentes: Viperidae): implications for species boundaries.

The Mohave Rattlesnake (Crotalus scutulatus) is a highly venomous pitviper inhabiting the arid interior deserts, grasslands, and savannas of western North America. Currently two subspecies are recognized: the Northern Mohave Rattlesnake (C. s. scutulatus) ranging from southern California to the southern Central Mexican Plateau, and the Huamantla Rattlesnake (C. s. salvini) from the region of Tlaxcala, Veracruz, and Puebla in south-central Mexico. Although recent studies have demonstrated extensive geographic variation in venom composition and cryptic genetic diversity in this species, no modern studies have focused on geographic variation in morphology. Here we analyzed a series of qualitative, meristic, and morphometric traits from 347 specimens of C. scutulatus and show that this species is phenotypically cohesive without discrete subgroups, and that morphology follows a continuous cline in primarily color pattern and meristic traits across the major axis of its expansive distribution. Interpreted in the context of previously published molecular evidence, our morphological analyses suggest that multiple episodes of isolation and secondary contact among metapopulations during the Pleistocene were sufficient to produce distinctive genetic populations, which have since experienced gene flow to produce clinal variation in phenotypes without discrete or diagnosable distinctions among these original populations. For taxonomic purposes, we recommend that C. scutulatus be retained as a single species, although it is possible that C. s. salvini, which is morphologically the most distinctive population, could represent a peripheral isolate in the initial stages of speciation.

Case reports of envenomation and venom composition differences between two Arizona populations of the Southwestern Speckled Rattlesnake, Crotalus pyrrhus (Cope, 1867)

Envenomations by the Southwestern Speckled Rattlesnake (Crotalus pyrrhus) are fairly rare. Previous descriptions in the literature do not include locality, an important factor in the clinical symptoms or syndromes of snakebites resulting from geographic variation in venom composition. Here, we describe two cases of envenoming by C. pyrrhus from two Arizona localities (Tinajas Altas Mountains, Yuma County, and Phoenix Mountains, Maricopa County). Both patients experienced swelling, but neither demonstrated coagulopathy, thrombocytopenia, or hypofibrinogenemia. The Phoenix Mountains patient developed hemorrhagic bullae and tissue damage in his bitten extremity, necessitating the amputation of the distal portion of his middle finger. Treatment for both consisted of medication for pain, isotonic crystalloid, and antivenom therapy with recovery in each case. Based on visual inspection of 1D-gels and RP-HPLC chromatograms, venom samples were largely similar but appeared to differ quantitatively for several toxin families between and within populations.

Predator-prey interactions and venom composition in a high elevation lizard specialist, Crotalus pricei (Twin-spotted Rattlesnake)

The Twin-spotted Rattlesnake (Crotalus pricei) is a small lizard specialist restricted to higher montane habitat in the Sky Islands of Arizona and México. Though this species is restricted to high elevations and dispersal between mountaintops is impossible, few studies have investigated venom composition or the predator-prey relationship between C. p. pricei and its primary prey source, Yarrow's Spiny Lizard (Sceloporus jarrovii). Because of current isolation of populations in disjunct mountain ranges, it is possible that populations show local adaptation to prey and/or environmental conditions, as reflected by distinct venom phenotypes. Here we characterize venom composition of C. pricei from several different Sky Island mountain ranges and its relationship with S. jarrovii by using various analytical techniques and comparative toxicity tests. Results of venom analyses indicate that there is limited geographic variation in venom composition, occurring primarily in venoms of C. pricei from Durango, México, which had unusual PLA2 isoforms that are lacking from venoms of U.S. populations. Toxicity assays reveal that S. jarrovii has not developed resistance specific to C. p. pricei venom but does display a general tolerance to venom of several snakes in the genus Crotalus. These results provide insight into the evolutionary relationship between a lizard specialist and its natural prey, in addition to novel information on the venom composition of a little-studied species with a narrow range in the United States.

Venom Costs and Optimization in Scorpions

Scorpions use venoms as weapons to improve prey capture and predator defense, and these benefits must be balanced against costs associated with its use. Venom costs involve direct energetic costs associated with the production and storage of toxins, and indirect fitness costs arising from reduced venom availability. In order to reduce these costs, scorpions optimize their venom use via evolutionary responses, phenotypic plasticity, and behavioral mechanisms. Over long timescales, evolutionary adaptation to environments with different selection pressures appears to have contributed to interspecific variation in venomcomposition and stingermorphology. Furthermore, plastic responses may allow scorpions to modify and optimize their venom composition as pressures change. Optimal venomuse can vary when facing each prey itemand potential predator encountered, and therefore scorpions display a range of behaviors to optimize their venom use to the particular situation. These behaviors include varying sting rates, employing dry stings, and further altering the volume and composition of venom injected. Whilst these cost-reducing mechanisms are recognized in scorpions, relatively little is understood about the factors that influence them. Here, we review evidence of the costs associated with venom use in scorpions and discuss the mechanisms that have evolved to minimize them.

Effects of Predator-Prey Interactions on Predator Traits: Differentiation of Diets and Venoms of a Marine Snail

Species interactions are fundamental ecological forces that can have significant impacts on the evolutionary trajectories of species. Nonetheless, the contribution of predator-prey interactions to genetic and phenotypic divergence remains largely unknown. Predatory marine snails of the family Conidae exhibit specializations for different prey items and intraspecific variation in prey utilization patterns at geographic scales. Because cone snails utilize venom to capture prey and venom peptides are direct gene products, it is feasible to examine the evolution of genes associated with changes in resource utilization. Here, we compared feeding ecologies and venom duct transcriptomes of individuals from three populations of Conus miliaris, a species that exhibits geographic variation in prey utilization and dietary breadth, in order to determine the extent to which dietary differences are correlated with differences in venom composition, and if expanded niche breadth is associated with increased variation in venom composition. While populations showed little to no overlap in resource utilization, taxonomic richness of prey was greatest at Easter Island. Changes in dietary breadth were associated with differences in expression patterns and increased genetic differentiation of toxin-related genes. The Easter Island population also exhibited greater diversity of toxin-related transcripts, but did not show increased variance in expression of these transcripts. These results imply that differences in dietary breadth contribute more to the structural and regulatory differentiation of venoms than differences in diet.

Variation in the Venom of Parasitic Wasps, Drift, or Selection? Insights From a Multivariate QST Analysis

Differentiation of traits among populations can evolve by drift when gene flow is low relative to drift or selection when there are different local optima in each population (heterogeneous selection), whereas homogeneous selection tends to prevent evolution of such a differentiation. Analyses of geographical variations in venom composition have been done in several taxa such as wasps, spiders, scorpions, cone snails and snakes, but surprisingly never in parasitoid wasps, although their venom should constrain their ability to succeed on locally available hosts. Such a study is now facilitated by the development of an accurate method (quantitative digital analysis) that allows analyzing the quantitative variation of large sets of proteins from several individuals. This method was used here to analyse the venom-based differentiation of four samples of Leptopilina boulardi and five samples of L. heterotoma from populations along a 300 km long south-north gradient in the Rhone-Saone valley (South-East of France). A major result is that the composition of the venom allows to differentiate the populations studied even when separated by few kilometers. We further analyzed these differentiations on the populations (reared under similar conditions to exclude environmental variance) with a QST analysis which compared the variance of a quantitative trait (Q) among the subpopulations (S) to the total variance (T). We also used random forest clustering analyses to detect the venom components the most likely to be adapted locally. The signature of the natural selection was strong for L. heterotoma and L. boulardi. For the latter, the comparison with the differentiation observed at some neutral markers revealed that differentiation was partly due to some local adaptation. The combination of methods used here appears to be a powerful framework for population proteomics and for the study of eco-evolutionary feedbacks between proteomic level and population and ecosystem levels. This is of interest not only for studying field evolution at an intermediate level between the genome and phenotypes, or for understanding the role of evolution in chemical ecology, but also for more applied issues in biological control.

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.

Intact protein mass spectrometry reveals intraspecies variations in venom composition of a local population of Vipera kaznakovi in Northeastern Turkey

We report on the variable venom composition of a population of the Caucasus viper (Vipera kaznakovi) in Northeastern Turkey. We applied a combination of venom gland transcriptomics, de-complexing bottom-up and top-down venomics. In contrast to sole bottom-up venomics approaches and gel or chromatography based venom comparison, our combined approach enables a faster and more detailed comparison of venom proteomes from multiple individuals. In total, we identified peptides and proteins from 15 toxin families, including snake venom metalloproteinases (svMP; 37.8%), phospholipases A2 (PLA2; 19.0%), snake venom serine proteinases (svSP; 11.5%), C-type lectins (CTL; 6.9%) and cysteine-rich secretory proteins (CRISP; 5.0%), in addition to several low abundant toxin families. Furthermore, we identified intraspecies variations of the venom composition of V. kaznakovi, and find these were mainly driven by the age of the animals, with lower svSP abundance detected in juveniles. On the proteoform level, several small molecular weight toxins between 5 and 8 kDa in size, as well as PLA2s, drove the differences observed between juvenile and adult individuals. This study provides novel insights into the venom variability of V. kaznakovi and highlights the utility of intact mass profiling for fast and detailed comparison of snake venom. Biological significancePopulation level and ontogenetic venom variation (e.g. diet, habitat, sex or age) can result in a loss of antivenom efficacy against snakebites from wide ranging snake populations. The current state of the art for the analysis of snake venoms are de-complexing bottom-up proteomics approaches. While useful, these have the significant drawback of being time-consuming and following costly protocols, and consequently are often applied to pooled venom samples. To overcome these shortcomings and to enable rapid and detailed profiling of large numbers of individual venom samples, we integrated an intact protein analysis workflow into a transcriptomics-guided bottom-up approach. The application of this workflow to snake individuals of a local population of V. kaznakovi revealed intraspecies variations in venom composition, which are primarily explained by the age of the animals, and highlighted svSP abundance to be one of the molecular drivers for the compositional differences observed.