The genetic regulatory architecture and epigenomic basis for age-related changes in rattlesnake venom.

Developmental phenotypic changes can evolve under selection imposed by age- and size-related ecological differences. Many of these changes occur through programmed alterations to gene expression patterns, but the molecular mechanisms and gene-regulatory networks underlying these adaptive changes remain poorly understood. Many venomous snakes, including the eastern diamondback rattlesnake (Crotalus adamanteus), undergo correlated changes in diet and venom expression as snakes grow larger with age, providing models for identifying mechanisms of timed expression changes that underlie adaptive life history traits. By combining a highly contiguous, chromosome-level genome assembly with measures of expression, chromatin accessibility, and histone modifications, we identified cis-regulatory elements and trans-regulatory factors controlling venom ontogeny in the venom glands of C. adamanteus. Ontogenetic expression changes were significantly correlated with epigenomic changes within genes, immediately adjacent to genes (e.g., promoters), and more distant from genes (e.g., enhancers). We identified 37 candidate transcription factors (TFs), with the vast majority being up-regulated in adults. The ontogenetic change is largely driven by an increase in the expression of TFs associated with growth signaling, transcriptional activation, and circadian rhythm/biological timing systems in adults with corresponding epigenomic changes near the differentially expressed venom genes. However, both expression activation and repression contributed to the composition of both adult and juvenile venoms, demonstrating the complexity and potential evolvability of gene regulation for this trait. Overall, given that age-based trait variation is common across the tree of life, we provide a framework for understanding gene-regulatory-network-driven life-history evolution more broadly.

Functional genomic diversity is correlated with neutral genomic diversity in populations of an endangered rattlesnake.

Theory predicts that genetic erosion in small, isolated populations of endangered species can be assessed using estimates of neutral genetic variation, yet this widely used approach has recently been questioned in the genomics era. Here, we leverage a chromosome-level genome assembly of an endangered rattlesnake (Sistrurus catenatus) combined with whole genome resequencing data (N = 110 individuals) to evaluate the relationship between levels of genome-wide neutral and functional diversity over historical and future timescales. As predicted, we found positive correlations between genome-wide estimates of neutral genetic diversity (π) and inferred levels of adaptive variation and an estimate of inbreeding mutation load, and a negative relationship between neutral diversity and an estimate of drift mutation load. However, these correlations were half as strong for projected future levels of neutral diversity based on contemporary effective population sizes. Broadly, our results confirm that estimates of neutral genetic diversity provide an accurate measure of genetic erosion in populations of a threatened vertebrate. They also provide nuance to the neutral-functional diversity controversy by suggesting that while these correlations exist, anthropogenetic impacts may have weakened these associations in the recent past and into the future.

Snake venom gene expression is coordinated by novel regulatory architecture and the integration of multiple co-opted vertebrate pathways.

Understanding how regulatory mechanisms evolve is critical for understanding the processes that give rise to novel phenotypes. Snake venom systems represent a valuable and tractable model for testing hypotheses related to the evolution of novel regulatory networks, yet the regulatory mechanisms underlying venom production remain poorly understood. Here, we use functional genomics approaches to investigate venom regulatory architecture in the prairie rattlesnake and identify cis-regulatory sequences (enhancers and promoters), trans-regulatory transcription factors, and integrated signaling cascades involved in the regulation of snake venom genes. We find evidence that two conserved vertebrate pathways, the extracellular signal-regulated kinase and unfolded protein response pathways, were co-opted to regulate snake venom. In one large venom gene family (snake venom serine proteases), this co-option was likely facilitated by the activity of transposable elements. Patterns of snake venom gene enhancer conservation, in some cases spanning 50 million yr of lineage divergence, highlight early origins and subsequent lineage-specific adaptations that have accompanied the evolution of venom regulatory architecture. We also identify features of chromatin structure involved in venom regulation, including topologically associated domains and CTCF loops that underscore the potential importance of novel chromatin structure to coevolve when duplicated genes evolve new regulatory control. Our findings provide a model for understanding how novel regulatory systems may evolve through a combination of genomic processes, including tandem duplication of genes and regulatory sequences, cis-regulatory sequence seeding by transposable elements, and diverse transcriptional regulatory proteins controlled by a co-opted regulatory cascade.

A novel broad spectrum venom metalloproteinase autoinhibitor in the rattlesnake Crotalus atrox evolved via a shift in paralog function.

The complexity of snake venom composition reflects adaptation to the diversity of prey and may be driven at times by a coevolutionary arms race between snakes and venom-resistant prey. However, many snakes are also resistant to their own venom due to serum-borne inhibitors of venom toxins, which raises the question of how snake autoinhibitors maintain their efficacy as venom proteins evolve. To investigate this potential three-way arms race among venom, prey, and autoinhibitors, we have identified and traced the evolutionary origin of serum inhibitors of snake venom metalloproteinases (SVMPs) in the Western Diamondback rattlesnake Crotalus atrox which possesses the largest known battery of SVMP genes among crotalids examined. We found that C. atrox expresses five members of a Fetuin A-related metalloproteinase inhibitor family but that one family member, FETUA-3, is the major SVMP inhibitor that binds to approximately 20 different C. atrox SVMPs and inhibits activities of all three SVMP classes. We show that the fetua-3 gene arose deep within crotalid evolution before the origin of New World species but, surprisingly, fetua-3 belongs to a different paralog group than previously identified SVMP inhibitors in Asian and South American crotalids. Conversely, the C. atrox FETUA-2 ortholog of previously characterized crotalid SVMP inhibitors shows limited activity against C. atrox SVMPs. These results reveal that there has been a functional evolutionary shift in the major SVMP inhibitor in the C. atrox lineage as the SVMP family expanded and diversified in the Crotalus lineage. This broad-spectrum inhibitor may be of potential therapeutic interest.

SEQUENCE SLIDER: integration of structural and genetic data to characterize isoforms from natural sources.

Proteins isolated from natural sources can be composed of a mixture of isoforms with similar physicochemical properties that coexist in the final steps of purification. Yet, even where unverified, the assumed sequence is enforced throughout the structural studies. Herein, we propose a novel perspective to address the usually neglected sequence heterogeneity of natural products by integrating biophysical, genetic and structural data in our program SEQUENCE SLIDER. The aim is to assess the evidence supporting chemical composition in structure determination. Locally, we interrogate the experimental map to establish which side chains are supported by the structural data, and the genetic information relating sequence conservation is integrated into this statistic. Hence, we build a constrained peptide database, containing most probable sequences to interpret mass spectrometry data (MS). In parallel, we perform MS de novo sequencing with genomic-based algorithms to detect point mutations. We calibrated SLIDER with Gallus gallus lysozyme, whose sequence is unequivocally established and numerous natural isoforms are reported. We used SLIDER to characterize a metalloproteinase and a phospholipase A2-like protein from the venom of Bothrops moojeni and a crotoxin from Crotalus durissus collilineatus. This integrated approach offers a more realistic structural descriptor to characterize macromolecules isolated from natural sources.

The Tiger Rattlesnake genome reveals a complex genotype underlying a simple venom phenotype.

Variation in gene regulation is ubiquitous, yet identifying the mechanisms producing such variation, especially for complex traits, is challenging. Snake venoms provide a model system for studying the phenotypic impacts of regulatory variation in complex traits because of their genetic tractability. Here, we sequence the genome of the Tiger Rattlesnake, which possesses the simplest and most toxic venom of any rattlesnake species, to determine whether the simple venom phenotype is the result of a simple genotype through gene loss or a complex genotype mediated through regulatory mechanisms. We generate the most contiguous snake-genome assembly to date and use this genome to show that gene loss, chromatin accessibility, and methylation levels all contribute to the production of the simplest, most toxic rattlesnake venom. We provide the most complete characterization of the venom gene-regulatory network to date and identify key mechanisms mediating phenotypic variation across a polygenic regulatory network.

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

BACKGROUND: Snake 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. RESULTS: Using 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. CONCLUSIONS: Our 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.

Inferring population connectivity in eastern massasauga rattlesnakes (Sistrurus catenatus) using landscape genetics.

Assessing the environmental factors that influence the ability of a threatened species to move through a landscape can be used to identify conservation actions that connect isolated populations. However, direct observations of species' movement are often limited, making the development of alternate approaches necessary. Here we use landscape genetic analyses to assess the impact of landscape features on the movement of individuals between local populations of a threatened snake, the eastern massasauga rattlesnake (Sistrurus catenatus). We linked connectivity data with habitat information from two landscapes of similar size: a large region of unfragmented habitat and a previously studied fragmented landscape consisting of isolated patches of habitat. We used this analysis to identify features of the landscape where modification or acquisition would enhance population connectivity in the fragmented region. We found evidence that current connectivity was impacted by both contemporary land-cover features, especially roads, and inherent landscape features such as elevation. Next, we derived estimates of expected movement ability using a recently developed pedigree-based approach and least-cost paths through the unfragmented landscape. We then used our pedigree and resistance map to estimate resistance polygons of the potential extent for S. catenatus movement in the fragmented landscape. These polygons identify possible sites for future corridors connecting currently isolated populations in this landscape by linking the impact of future habitat modification or land acquisition to dispersal ability in this species. Overall, our study shows how modeling landscape resistance across differently fragmented landscapes can identify habitat features that affect contemporary movement in threatened species in fragmented landscapes and how this information can be used to guide mitigation actions whose goal is to connect isolated populations.

A genome assembly for the southern Pacific rattlesnake, Crotalus oreganus helleri, in the western rattlesnake species complex.

Rattlesnakes play important roles in their ecosystems by regulating prey populations, are involved in complex coevolutionary dynamics with their prey, and exhibit a variety of unusual adaptations, including maternal care, heat-sensing pit organs, hinged fangs, and medically-significant venoms. The western rattlesnake (Crotalus oreganus) is one of the widest ranging rattlesnake species, with a distribution from British Columbia, where it is listed as threatened, to Baja California and east across the Great Basin to western Wyoming, Colorado and New Mexico. Here, we report a new reference genome assembly for one of six currently recognized subspecies, C. oreganus helleri, as part of the California Conservation Genomics Project (CCGP). Consistent with the reference genomic sequencing strategy of the CCGP, we used Pacific Biosciences HiFi long reads and Hi-C chromatin-proximity sequencing technology to produce a de novo assembled genome. The assembly comprises a total of 698 scaffolds spanning 1,564,812,557 base pairs, has a contig N50 of 64.7 Mb, a scaffold N50 of 110.8 Mb, and BUSCO complete score of 90.5%. This reference genome will be valuable for studies on the genomic basis of venom evolution and variation within Crotalus, in resolving the taxonomy of C. oreganus and its relatives, and for the conservation and management of rattlesnakes in general.

The origins and evolution of chromosomes, dosage compensation, and mechanisms underlying venom regulation in snakes.

Here we use a chromosome-level genome assembly of a prairie rattlesnake (Crotalus viridis), together with Hi-C, RNA-seq, and whole-genome resequencing data, to study key features of genome biology and evolution in reptiles. We identify the rattlesnake Z Chromosome, including the recombining pseudoautosomal region, and find evidence for partial dosage compensation driven by an evolutionary accumulation of a female-biased up-regulation mechanism. Comparative analyses with other amniotes provide new insight into the origins, structure, and function of reptile microchromosomes, which we demonstrate have markedly different structure and function compared to macrochromosomes. Snake microchromosomes are also enriched for venom genes, which we show have evolved through multiple tandem duplication events in multiple gene families. By overlaying chromatin structure information and gene expression data, we find evidence for venom gene-specific chromatin contact domains and identify how chromatin structure guides precise expression of multiple venom gene families. Further, we find evidence for venom gland-specific transcription factor activity and characterize a complement of mechanisms underlying venom production and regulation. Our findings reveal novel and fundamental features of reptile genome biology, provide insight into the regulation of snake venom, and broadly highlight the biological insight enabled by chromosome-level genome assemblies.

Insights on the inhibition properties of Jatromollistatin (a cyclic heptapeptide) against Crotalus adamanteus metalloendopeptidase using molecular docking analysis.

Jatropha mollissima is endemic to Brazil and is used for traditional medicinal purposes, including the treatment of snakebite. In this study, latex obtained from this plant was fractioned using reversed-phase chromatography, and the fractions were then screened for peptides. A 755 g/mol peptide was obtained, and MS/MS analyses indicated it had a cyclic sequence (Pro-Leu-Gly-Val-Leu-Leu-Tyr). This peptide sequence was present in the Jatropha genome database, and an identity value of 90.71%, an E-value of 0.0, and a score of 883 with NO-associated protein 1/chloroplastic/mitochondria of Jatropha curcas were obtained from the NCBI nonredundant protein sequence (nr) database. Molecular docking analyses performed with the peptide against a metalloendopeptidase belonging to Crotalus adamanteus snake venom suggested the cyclic peptide establishes favorable interactions with the catalytic site of the enzyme. Therefore, it could inhibit enzyme catalysis. This belief was corroborated by the formation of 6 hydrogen bonds with the linear form of the peptide. Tighter complexation of the cyclic form (41 kcal/mol more energetic) revealed better spatial blocking. The linear form outperformed the cyclic form in complexing the required energy, recruiting more catalytic residues (6/2), and in establishing more hydrogen bonds (6/3). However, cyclic folding provided a more significant spatial block within the catalytic site. The set of results suggests that the cycle peptide, here called Jatromollistatin, which was previously described as jatrophidin and pohlianin A in two other species of Jatropha, is a promising candidate to inhibit venom proteases. This belief is corroborated by the topical use of the latex for initial treatment of snakebites.

Snake Recombination Landscapes Are Concentrated in Functional Regions despite PRDM9.

Meiotic recombination in vertebrates is concentrated in hotspots throughout the genome. The location and stability of hotspots have been linked to the presence or absence of PRDM9, leading to two primary models for hotspot evolution derived from mammals and birds. Species with PRDM9-directed recombination have rapid turnover of hotspots concentrated in intergenic regions (i.e., mammals), whereas hotspots in species lacking PRDM9 are concentrated in functional regions and have greater stability over time (i.e., birds). Snakes possess PRDM9, yet virtually nothing is known about snake recombination. Here, we examine the recombination landscape and test hypotheses about the roles of PRDM9 in rattlesnakes. We find substantial variation in recombination rate within and among snake chromosomes, and positive correlations between recombination rate and gene density, GC content, and genetic diversity. Like mammals, snakes appear to have a functional and active PRDM9, but rather than being directed away from genes, snake hotspots are concentrated in promoters and functional regions-a pattern previously associated only with species that lack a functional PRDM9. Snakes therefore provide a unique example of recombination landscapes in which PRDM9 is functional, yet recombination hotspots are associated with functional genic regions-a combination of features that defy existing paradigms for recombination landscapes in vertebrates. Our findings also provide evidence that high recombination rates are a shared feature of vertebrate microchromosomes. Our results challenge previous assumptions about the adaptive role of PRDM9 and highlight the diversity of recombination landscape features among vertebrate lineages.

The Chemosensory Repertoire of the Eastern Diamondback Rattlesnake (Crotalus adamanteus) Reveals Complementary Genetics of Olfactory and Vomeronasal-Type Receptors.

Pitviper sensory perception incorporates diverse stimuli through the integration of trichromatic color vision, bifocal heat-sensing, and dual-system chemoperception. Chemoperception, or olfaction, is mediated by chemoreceptors in the olfactory bulb and the vomeronasal organ, but the true genomic complexity of the gene families and their relative contributions is unknown. A full genomic accounting of pitviper chemoperception directly complements our current understanding of their venoms by generating a more complete polyphenic representation of their predatory arsenal. To characterize the genetic repertoire of pitviper chemoperception, we analyzed a full-genome assembly for Crotalus adamanteus, the eastern diamondback rattlesnake. We identified hundreds of genes encoding both olfactory receptors (ORs; 362 full-length genes) and type-2 vomeronasal receptors (V2Rs; 430 full-length genes). Many chemoreceptor genes are organized into large tandem repeat arrays. Comparative analysis of V2R orthologs across squamates demonstrates how gene array expansion and contraction underlies the evolution of the chemoreceptor repertoire, which likely reflects shifts in life history traits. Chromosomal assignments of chemosensory genes identified sex chromosome specific chemoreceptor genes, providing gene candidates underlying observed sex-specific chemosensory-based behaviors. We detected widespread episodic evolution in the extracellular, ligand-binding domains of both ORs and V2Rs, suggesting the diversification of chemoreceptors is driven by transient periods of positive selection. We provide a robust genetic framework for studying pitviper chemosensory ecology and evolution.

Genomic signatures of inbreeding and mutation load in a threatened rattlesnake.

Theory predicts that threatened species living in small populations will experience high levels of inbreeding that will increase their genetic load, but recent work suggests that the impact of load may be minimized by purging resulting from long-term population bottlenecks. Empirical studies that examine this idea using genome-wide estimates of inbreeding and genetic load in threatened species are limited. Here we use individual genome resequencing data to compare levels of inbreeding, levels of genetic load (estimated as mutation load) and population history in threatened Eastern massasauga rattlesnakes (Sistrurus catenatus), which exist in small isolated populations, and closely related yet outbred Western massasauga rattlesnakes (Sistrurus tergeminus). In terms of inbreeding, S. catenatus genomes had a greater number of runs of homozygosity of varying sizes, indicating sustained inbreeding through repeated bottlenecks when compared to S. tergeminus. At the species level, outbred S. tergeminus had higher genome-wide levels of mutation load in the form of greater numbers of derived deleterious mutations compared to S. catenatus, presumably due to long-term purging of deleterious mutations in S. catenatus. In contrast, mutations that escaped species-level drift effects within S. catenatus populations were in general more frequent and more often found in homozygous genotypes than in S. tergeminus, suggesting a reduced efficiency of purifying selection in smaller S. catenatus populations for most mutations. Our results support an emerging idea that the historical demography of a threatened species has a significant impact on the type of genetic load present, which impacts implementation of conservation actions such as genetic rescue.

Genome-wide data reveal extensive gene flow during the diversification of the western rattlesnakes (Viperidae: Crotalinae: Crotalus).

Hybridization and introgression are important, but often overlooked processes when inferring phylogenies. When these processes are not accounted for and a strictly diverging phylogenetic model is applied to groups with a history of hybridization, phylogenetic inference and parameter estimation can be inaccurate. Recent developments in phylogenetic network approaches coupled with the increasing availability of genomic data allow inferences of reticulate evolutionary histories across the tree of life. The western rattlesnake species group (C. viridis species complex, C. mitchellii species complex, C. scutulutas, and C. tigris) is an iconic snake lineage that is widespread across western North America. This group is composed of several species complexes with unclear species limits, likely the result of ongoing gene flow among nascent lineages. Here I generate reduced representation genomic data and test for a history of reticulation within this group. I demonstrate that all species have undergone hybridization with at least one other lineage, suggesting introgression is widespread in this group. Topologies differ between phylogenies estimated under the multispecies coalescent and multispecies network coalescent methods, indicating that gene flow has obscured phylogenetic relationships within this group. These past introgression events are predominantly restricted to species that co-occur geographically. However, within species that have a history of introgression, this signature is detected regardless of specimen sampling across geography. Overall, my results suggest the accumulation of reproductive isolating barriers occurs slowly in rattlesnakes which likely leads to the difficulty in delimiting species, furthermore, the results of this study have implications for trait evolution in this group.

Population Genomic Analyses Confirm Male-Biased Mutation Rates in Snakes.

Male-biased mutation rates occur in a diverse array of organisms. The ratio of male-to-female mutation rate may have major ramifications for evolution across the genome, and for sex-linked genes in particular. In ZW species, the Z chromosome is carried by males two-thirds of the time, leading to the prediction that male-biased mutation rates will have a disproportionate effect on the evolution of Z-linked genes relative to autosomes and the W chromosome. Colubroid snakes (including colubrids, elapids, and viperids) have ZW sex determination, yet male-biased mutation rates have not been well studied in this group. Here we analyze a population genomic dataset from rattlesnakes to quantify genetic variation within and genetic divergence between species. We use a new method for unbiased estimation of population genetic summary statistics to compare variation between the Z chromosome and autosomes and to calculate net nucleotide differentiation between species. We find evidence for a 2.03-fold greater mutation rate in male rattlesnakes relative to females, corresponding to an average µZ/µA ratio of 1.1. Our results from snakes are quantitatively similar to birds, suggesting that male-biased mutation rates may be a common feature across vertebrate lineages with ZW sex determination.

Tipping the Scales: The Migration-Selection Balance Leans toward Selection in Snake Venoms.

The migration-selection interaction is the strongest determinant of whether a beneficial allele increases in frequency within a population. Results of empirical studies examining the role of gene flow in an adaptive context, however, have largely been equivocal, with examples of opposing outcomes being repeatedly documented (e.g., local adaptation with high levels of gene flow vs. gene swamping). We compared neutral genomic and venom expression divergence for three sympatric pit vipers with differing ecologies to determine if and how migration-selection disequilibria result in local adaptation. We specifically tested whether neutral differentiation predicted phenotypic differentiation within an isolation-by-distance framework. The decoupling of neutral and phenotypic differentiation would indicate selection led to adaptive divergence irrespective of migration, whereas a significant relationship between neutral and venom expression differentiation would provide evidence in favor of the constraining force of gene flow. Neutral differentiation and geographic distance predicted phenotypic differentiation only in the generalist species, indicating that selection was the predominant mechanism in the migration-selection balance underlying adaptive venom evolution in both specialists. Dispersal is thought to be a stronger influence on genetic differentiation than specialization, but our results suggest the opposite. Greater specialization may lead to greater diversification rates, and extreme spatial and temporal variation in selective pressures can favor generalist phenotypes evolving under strong stabilizing selection. Our results are consistent with these expectations, suggesting that the equivocal findings of studies examining the role of gene flow in an adaptive context may be explained by ecological specialization theory.

Drift, selection and adaptive variation in small populations of a threatened rattlesnake.

An important goal of conservation genetics is to determine if the viability of small populations is reduced by a loss of adaptive variation due to genetic drift. Here, we assessed the impact of drift and selection on direct measures of adaptive variation (toxin loci encoding venom proteins) in the eastern massasauga rattlesnake (Sistrurus catenatus), a threatened reptile that exists in small isolated populations. We estimated levels of individual polymorphism in 46 toxin loci and 1,467 control loci across 12 populations of this species, and compared the results with patterns of selection on the same loci following speciation of S. catenatus and its closest relative, the western massasauga (S. tergeminus). Multiple lines of evidence suggest that both drift and selection have had observable impacts on standing adaptive variation. In support of drift effects, we found little evidence for selection on toxin variation within populations and a significant positive relationship between current levels of adaptive variation and long- and short-term estimates of effective population size. However, we also observed levels of directional selection on toxin loci among populations that are broadly similar to patterns predicted from interspecific selection analyses that pre-date the effects of recent drift, and that functional variation in these loci persists despite small short-term effective sizes. This suggests that much of the adaptive variation present in populations may represent an example of "drift debt," a nonequilibrium state where present-day levels of variation overestimate the amount of functional genetic diversity present in future populations.

The molecular basis of venom resistance in a rattlesnake-squirrel predator-prey system.

Understanding how interspecific interactions mould the molecular basis of adaptations in coevolving species is a long-sought goal of evolutionary biology. Venom in predators and venom resistance proteins in prey are coevolving molecular phenotypes, and while venoms are highly complex mixtures it is unclear if prey respond with equally complex resistance traits. Here, we use a novel molecular methodology based on protein affinity columns to capture and identify candidate blood serum resistance proteins ("venom interactive proteins" [VIPs]) in California Ground Squirrels (Otospermophilus beecheyi) that interact with venom proteins from their main predator, Northern Pacific Rattlesnakes (Crotalus o. oreganus). This assay showed that serum-based resistance is both population- and species-specific, with serum proteins from ground squirrels showing higher binding affinities for venom proteins of local snakes compared to allopatric individuals. Venom protein specificity assays identified numerous and diverse candidate prey resistance VIPs but also potential targets of venom in prey tissues. Many specific VIPs bind to multiple snake venom proteins and, conversely, single venom proteins bind multiple VIPs, demonstrating that a portion of the squirrel blood serum "resistome" involves broad-based inhibition of nonself proteins and suggests that resistance involves a toxin scavenging mechanism. Analyses of rates of evolution of VIP protein homologues in related mammals show that most of these proteins evolve under purifying selection possibly due to molecular constraints that limit the evolutionary responses of prey to rapidly evolving snake venom proteins. Our method represents a general approach to identify specific proteins involved in co-evolutionary interactions between species at the molecular level.

Bioaffinity Fishing Procedure Using Secretory Phospholipase A2 for Screening for Bioactive Components: Modulation of Pharmacological Effect Induced by sPLA2 from Crotalus durissus terrificus by Hispidulin from Moquiniastrum floribundum.

Bioaffinity capturing of molecules allows the discovery of bioactive compounds and decreases the need for various stages in the natural compound isolation process. Despite the high selectivity of this technique, the screening and identification methodology depends on the presence of a protein to capture potential ligands. However, some proteins, such as snake secretory phospholipase A2 (sPLA2), have never been investigated using this approach. The purpose of this study was to evaluate the use of a new method for screening natural compounds using a bioaffinity-guided ultrafiltration method on Crotalus durissus terrificus sPLA2 followed by HPLC-MS to identify the compounds, and this method could be used to discover new anti-inflammatory compounds from the various organisms originating from biodiversity. Different extracts were selected to evaluate their ability to inhibit sPLA2 activity. The extracts were incubated with sPLA2 and the resulting mixture was ultrafiltrated to elute unbound components. The resulting compounds were identified by HPLC-MS. We identified hispidulin as one of the components present in the Moquiniastrum floribundum leaf and evaluated the ability of this isolated compound to neutralize the inflammatory activity of sPLA2 from Crotalus durissus terrificus.