ASSESSING TARGET SPECIFICITY OF THE SMALL MOLECULE INHIBITOR MARIMASTAT TO SNAKE VENOM TOXINS: A NOVEL APPLICATION OF THERMAL PROTEOME PROFILING.

New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin inhibitors have shown promising cross-species neutralization of medically significant venom toxins in vivo and in vitro. The development of high-throughput approaches for the screening of such inhibitors could accelerate their identification, testing, and implementation, and thus holds exciting potential for improving the treatments and outcomes of snakebite envenomation worldwide. Energetics-based proteomic approaches, including Thermal Proteome Profiling (TPP) and Proteome Integral Solubility Alteration (PISA) assays, represent "deep proteomics" methods for high throughput, proteome-wide identification of drug targets and ligands. In the following study, we apply TPP and PISA methods to characterize the interactions between venom toxin proteoforms in Crotalus atrox (Western Diamondback Rattlesnake) and the snake venom metalloprotease (SVMP) inhibitor marimastat. We investigate its venom proteome-wide effects and characterize its interactions with specific SVMP proteoforms, as well as its potential targeting of non-SVMP venom toxin families. We also compare the performance of PISA thermal window and soluble supernatant with insoluble precipitate using two inhibitor concentrations, providing the first demonstration of the utility of a sensitive high-throughput PISA-based approach to assess the direct targets of small molecule inhibitors for snake venom.

Hunting behavior and feeding ecology of Mojave rattlesnakes (Crotalus scutulatus), prairie rattlesnakes (Crotalus viridis), and their hybrids in southwestern New Mexico.

Predators must contend with numerous challenges to successfully find and subjugate prey. Complex traits related to hunting are partially controlled by a large number of co-evolved genes, which may be disrupted in hybrids. Accordingly, research on the feeding ecology of animals in hybrid zones has shown that hybrids sometimes exhibit transgressive or novel behaviors, yet for many taxa, empirical studies of predation and diet across hybrid zones are lacking. We undertook the first such field study for a hybrid zone between two snake species, the Mojave rattlesnake (Crotalus scutulatus) and the prairie rattlesnake (Crotalus viridis). Specifically, we leveraged established field methods to quantify the hunting behaviors of animals, their prey communities, and the diet of individuals across the hybrid zone in southwestern New Mexico, USA. We found that, even though hybrids had significantly lower body condition indices than snakes from either parental group, hybrids were generally similar to non-hybrids in hunting behavior, prey encounter rates, and predatory attack and success. We also found that, compared to C. scutulatus, C. viridis was significantly more active while hunting at night and abandoned ambush sites earlier in the morning, and hybrids tended to be more viridis-like in this respect. Prey availability was similar across the study sites, including within the hybrid zone, with kangaroo rats (Dipodomys spp.) as the most common small mammal, both in habitat surveys and the frequency of encounters with hunting rattlesnakes. Analysis of prey remains in stomachs and feces also showed broad similarity in diets, with all snakes preying primarily on small mammals and secondarily on lizards. Taken together, our results suggest that the significantly lower body condition of hybrids does not appear to be driven by differences in their hunting behavior or diet and may instead relate to metabolic efficiency or other physiological traits we have not yet identified.

Specimen collection is essential for modern science.

Natural history museums are vital repositories of specimens, samples and data that inform about the natural world; this Formal Comment revisits a Perspective that advocated for the adoption of compassionate collection practices, querying whether it will ever be possible to completely do away with whole animal specimen collection.

Signals of differential introgression in the genome of natural hybrids of Caribbean anoles.

Hybridization facilitates recombination between divergent genetic lineages and can be shaped by both neutral and selective processes. Upon hybridization, loci with no net fitness effects introgress randomly from parental species into the genomes of hybrid individuals. Conversely, alleles from one parental species at some loci may provide a selective advantage to hybrids, resulting in patterns of introgression that do not conform to random expectations. We investigated genomic patterns of differential introgression in natural hybrids of two species of Caribbean anoles, Anolis pulchellus and A. krugi in Puerto Rico. Hybrids exhibit A. pulchellus phenotypes but possess A. krugi mitochondrial DNA, originated from multiple, independent hybridization events, and appear to have replaced pure A. pulchellus across a large area in western Puerto Rico. Combining genome-wide SNP datasets with bioinformatic methods to identify signals of differential introgression in hybrids, we demonstrate that the genomes of hybrids are dominated by pulchellus-derived alleles and show only 10%-20% A. krugi ancestry. The majority of A. krugi loci in hybrids exhibit a signal of non-random differential introgression and include loci linked to genes involved in development and immune function. Three of these genes (delta like canonical notch ligand 1, jagged1 and notch receptor 1) affect cell differentiation and growth and interact with mitochondrial function. Our results suggest that differential non-random introgression for a subset of loci may be driven by selection favouring the inheritance of compatible mitochondrial and nuclear-encoded genes in hybrids.

The best of both worlds? Rattlesnake hybrid zones generate complex combinations of divergent venom phenotypes that retain high toxicity.

Studying the consequences of hybridization between closely related species with divergent traits can reveal patterns of evolution that shape and maintain extreme trophic adaptations. Snake venoms are an excellent model system for examining the evolutionary and ecological patterns that underlie highly selected polymorphic traits. Here we investigate hybrid venom phenotypes that result from natural introgression between two rattlesnake species that express highly divergent venom phenotypes: Crotalus o. concolor and C. v. viridis. Though not yet documented, interbreeding between these species may lead to novel venom phenotypes with unique activities that break the typical trends of venom composition in rattlesnakes. The characteristics of these unusual phenotypes could unveil the roles of introgression in maintaining patterns of venom composition and variation, including the near ubiquitous dichotomy between neurotoxic or degradative venoms observed across rattlesnakes. We use RADseq data to infer patterns of gene flow and hybrid ancestry between these diverged lineages and link these genetic data with analyses of venom composition, biological activity, and whole animal model toxicity tests to understand the impacts of introgression on venom composition. We find that introgressed populations express admixed venom phenotypes that do not sacrifice biological activity (lethal toxicity) or overall abundance of dominant toxins compared to parental venoms. These hybridized venoms therefore do not represent a trade-off in functionality between the typical phenotypic extremes but instead represent a unique combination of characters whose expression appears limited to the hybrid zone.

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.

Single-Cell Heterogeneity in Snake Venom Expression Is Hardwired by Co-Option of Regulators from Progressively Activated Pathways.

The ubiquitous cellular heterogeneity underlying many organism-level phenotypes raises questions about what factors drive this heterogeneity and how these complex heterogeneous systems evolve. Here, we use single-cell expression data from a Prairie rattlesnake (Crotalus viridis) venom gland to evaluate hypotheses for signaling networks underlying snake venom regulation and the degree to which different venom gene families have evolutionarily recruited distinct regulatory architectures. Our findings suggest that snake venom regulatory systems have evolutionarily co-opted trans-regulatory factors from extracellular signal-regulated kinase and unfolded protein response pathways that specifically coordinate expression of distinct venom toxins in a phased sequence across a single population of secretory cells. This pattern of co-option results in extensive cell-to-cell variation in venom gene expression, even between tandemly duplicated paralogs, suggesting this regulatory architecture has evolved to circumvent cellular constraints. While the exact nature of such constraints remains an open question, we propose that such regulatory heterogeneity may circumvent steric constraints on chromatin, cellular physiological constraints (e.g., endoplasmic reticulum stress or negative protein-protein interactions), or a combination of these. Regardless of the precise nature of these constraints, this example suggests that, in some cases, dynamic cellular constraints may impose previously unappreciated secondary constraints on the evolution of gene regulatory networks that favors heterogeneous expression.

Feasibility of detecting snake envenomation biomarkers from dried blood spots.

Biofluid proteomics is a sensitive and high throughput technique that provides vast amounts of molecular data for biomarker discovery. More recently, dried blood spots (DBS) have gained traction as a stable, noninvasive, and relatively cheap source of proteomic data for biomarker identification in disease and injury. Snake envenomation is responsible for significant morbidity and mortality worldwide; however, much remains unknown about the systemic molecular response to envenomation and acquiring biological samples for analysis is a major hurdle. In this study, we utilized DBS acquired from a case of lethal rattlesnake envenomation to determine the feasibility of discovering biomarkers associated with human envenomation. We identified proteins that were either unique or upregulated in envenomated blood compared to non-envenomated blood and evaluated if physiological response pathways and protein markers that correspond to the observed syndromes triggered by envenomation could be detected. We demonstrate that DBS provide useful proteomic information on the systemic processes that resulted from envenomation in this case and find evidence for a massive and systemic inflammatory cascade, combined with coagulation dysregulation, complement system activation, hypoxia response activation, and apoptosis. We also detected potential markers indicative of lethal anaphylaxis, cardiac arrest, and brain death. Ultimately, DBS proteomics has the potential to provide stable and sensitive molecular data on envenomation syndromes and response pathways, which is particularly relevant in low-resource areas which may lack the materials for biofluid processing and storage.

The mitochondrial UPR regulator ATF5 promotes intestinal barrier function via control of the satiety response.

Organisms use several strategies to mitigate mitochondrial stress, including the activation of the mitochondrial unfolded protein response (UPRmt). The UPRmt in Caenorhabditis elegans, regulated by the transcription factor ATFS-1, expands on this recovery program by inducing an antimicrobial response against pathogens that target mitochondrial function. Here, we show that the mammalian ortholog of ATFS-1, ATF5, protects the host during infection with enteric pathogens but, unexpectedly, by maintaining the integrity of the intestinal barrier. Intriguingly, ATF5 supports intestinal barrier function by promoting a satiety response that prevents obesity and associated hyperglycemia. This consequently averts dysregulated glucose metabolism that is detrimental to barrier function. Mechanistically, we show that intestinal ATF5 stimulates the satiety response by transcriptionally regulating the gastrointestinal peptide hormone cholecystokinin, which promotes the secretion of the hormone leptin. We propose that ATF5 protects the host from enteric pathogens by promoting intestinal barrier function through a satiety-response-mediated metabolic control mechanism.

Evidence that genomic incompatibilities and other multilocus processes impact hybrid fitness in a rattlesnake hybrid zone.

Hybrid zones provide valuable opportunities to understand the genomic mechanisms that promote speciation by providing insight into factors involved in intermediate stages of speciation. Here, we investigate introgression in a hybrid zone between two rattlesnake species (Crotalus viridis and Crotalus oreganus concolor) that have undergone historical allopatric divergence and recent range expansion and secondary contact. We use Bayesian genomic cline models to characterize genomic patterns of introgression between these lineages and identify loci potentially subject to selection in hybrids. We find evidence for a large number of genomic regions with biased ancestry that deviate from the genomic background in hybrids (i.e., excess ancestry loci), which tend to be associated with genomic regions with higher recombination rates. We also identify suites of excess ancestry loci that show highly correlated allele frequencies (including conspecific and heterospecific combinations) across physically unlinked genomic regions in hybrids. Our findings provide evidence for multiple multilocus evolutionary processes impacting hybrid fitness in this system.

Integrating genomic and epidemiologic data to accelerate progress toward schistosomiasis elimination.

The global community has adopted ambitious goals to eliminate schistosomiasis as a public health problem, and new tools are needed to achieve them. Mass drug administration programs, for example, have reduced the burden of schistosomiasis, but the identification of hotspots of persistent and reemergent transmission threaten progress toward elimination and underscore the need to couple treatment with interventions that reduce transmission. Recent advances in DNA sequencing technologies make whole-genome sequencing a valuable and increasingly feasible option for population-based studies of complex parasites such as schistosomes. Here, we focus on leveraging genomic data to tailor interventions to distinct social and ecological circumstances. We consider two priority questions that can be addressed by integrating epidemiological, ecological, and genomic information: (1) how often do non-human host species contribute to human schistosome infection? and (2) what is the importance of locally acquired versus imported infections in driving transmission at different stages of elimination? These questions address processes that can undermine control programs, especially those that rely heavily on treatment with praziquantel. Until recently, these questions were difficult to answer with sufficient precision to inform public health decision-making. We review the literature related to these questions and discuss how whole-genome approaches can identify the geographic and taxonomic sources of infection, and how such information can inform context-specific efforts that advance schistosomiasis control efforts and minimize the risk of reemergence.

Origins, genomic structure and copy number variation of snake venom myotoxins.

Crotamine, myotoxin a and homologs are short peptides that often comprise major fractions of rattlesnake venoms and have been extensively studied for their bioactive properties. These toxins are thought to be important for rapidly immobilizing mammalian prey and are implicated in serious, and sometimes fatal, responses to envenomation in humans. While high quality reference genomes for multiple venomous snakes are available, the loci that encode myotoxins have not been successfully assembled in any existing genome assembly. Here, we integrate new and existing genomic and transcriptomic data from the Prairie Rattlesnake (Crotalus viridis viridis) to reconstruct, characterize, and infer the chromosomal locations of myotoxin-encoding loci. We integrate long-read transcriptomics (Pacific Bioscience's Iso-Seq) and short-read RNA-seq to infer gene sequence diversity and characterize patterns of myotoxin and paralogous ß-defensin expression across multiple tissues. We also identify two long non-coding RNA sequences which both encode functional myotoxins, demonstrating a newly discovered source of venom coding sequence diversity. We also integrate long-range mate-pair chromatin contact data and linked-read sequencing to infer the structure and chromosomal locations of the three myotoxin-like loci. Further, we conclude that the venom-associated myotoxin is located on chromosome 1 and is adjacent to non-venom paralogs. Consistent with this locus contributing to venom composition, we find evidence that the promoter of this gene is selectively open in venom gland tissue and contains transcription factor binding sites implicated in broad trans-regulatory pathways that regulate snake venoms. This study provides the best genomic reconstruction of myotoxin loci to date and raises questions about the physiological roles and interplay between myotoxin and related genes, as well as the genomic origins of snake venom variation.

The roles of balancing selection and recombination in the evolution of rattlesnake venom.

The origin of snake venom involved duplication and recruitment of non-venom genes into venom systems. Several studies have predicted that directional positive selection has governed this process. Venom composition varies substantially across snake species and venom phenotypes are locally adapted to prey, leading to coevolutionary interactions between predator and prey. Venom origins and contemporary snake venom evolution may therefore be driven by fundamentally different selection regimes, yet investigations of population-level patterns of selection have been limited. Here, we use whole-genome data from 68 rattlesnakes to test hypotheses about the factors that drive genomic diversity and differentiation in major venom gene regions. We show that selection has resulted in long-term maintenance of genetic diversity within and between species in multiple venom gene families. Our findings are inconsistent with a dominant role of directional positive selection and instead support a role of long-term balancing selection in shaping venom evolution. We also detect rapid decay of linkage disequilibrium due to high recombination rates in venom regions, suggesting that venom genes have reduced selective interference with nearby loci, including other venom paralogues. Our results provide an example of long-term balancing selection that drives trans-species polymorphism and help to explain how snake venom keeps pace with prey resistance.

The Rattlesnake W Chromosome: A GC-Rich Retroelement Refugium with Retained Gene Function Across Ancient Evolutionary Strata.

Sex chromosomes diverge after the establishment of recombination suppression, resulting in differential sex-linkage of genes involved in genetic sex determination and dimorphic traits. This process produces systems of male or female heterogamety wherein the Y and W chromosomes are only present in one sex and are often highly degenerated. Sex-limited Y and W chromosomes contain valuable information about the evolutionary transition from autosomes to sex chromosomes, yet detailed characterizations of the structure, composition, and gene content of sex-limited chromosomes are lacking for many species. In this study, we characterize the female-specific W chromosome of the prairie rattlesnake (Crotalus viridis) and evaluate how recombination suppression and other processes have shaped sex chromosome evolution in ZW snakes. Our analyses indicate that the rattlesnake W chromosome is over 80% repetitive and that an abundance of GC-rich mdg4 elements has driven an overall high degree of GC-richness despite a lack of recombination. The W chromosome is also highly enriched for repeat sequences derived from endogenous retroviruses and likely acts as a "refugium" for these and other retroelements. We annotated 219 putatively functional W-linked genes across at least two evolutionary strata identified based on estimates of sequence divergence between Z and W gametologs. The youngest of these strata is relatively gene-rich, however gene expression across strata suggests retained gene function amidst a greater degree of degeneration following ancient recombination suppression. Functional annotation of W-linked genes indicates a specialization of the W chromosome for reproductive and developmental function since recombination suppression from the Z chromosome.

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 genomic can of worms for schistosome host-specificity.

Understanding the genetic underpinnings of schistosome host preferences is critical. Luo et al. recently identified genes associated with intermediate and definitive host-switching based on a new chromosome-level genome for Schistosoma japonicum, population genetic comparisons, and follow-up experiments. This represents a guide to fully map-selected schistosome genes using population genetics.

A chromosome-level genome assembly and annotation of the desert horned lizard, Phrynosoma platyrhinos, provides insight into chromosomal rearrangements among reptiles.

BACKGROUND: The increasing number of chromosome-level genome assemblies has advanced our knowledge and understanding of macroevolutionary processes. Here, we introduce the genome of the desert horned lizard, Phrynosoma platyrhinos, an iguanid lizard occupying extreme desert conditions of the American southwest. We conduct analysis of the chromosomal structure and composition of this species and compare these features across genomes of 12 other reptiles (5 species of lizards, 3 snakes, 3 turtles, and 1 bird). FINDINGS: The desert horned lizard genome was sequenced using Illumina paired-end reads and assembled and scaffolded using Dovetail Genomics Hi-C and Chicago long-range contact data. The resulting genome assembly has a total length of 1,901.85 Mb, scaffold N50 length of 273.213 Mb, and includes 5,294 scaffolds. The chromosome-level assembly is composed of 6 macrochromosomes and 11 microchromosomes. A total of 20,764 genes were annotated in the assembly. GC content and gene density are higher for microchromosomes than macrochromosomes, while repeat element distributions show the opposite trend. Pathway analyses provide preliminary evidence that microchromosome and macrochromosome gene content are functionally distinct. Synteny analysis indicates that large microchromosome blocks are conserved among closely related species, whereas macrochromosomes show evidence of frequent fusion and fission events among reptiles, even between closely related species. CONCLUSIONS: Our results demonstrate dynamic karyotypic evolution across Reptilia, with frequent inferred splits, fusions, and rearrangements that have resulted in shuffling of chromosomal blocks between macrochromosomes and microchromosomes. Our analyses also provide new evidence for distinct gene content and chromosomal structure between microchromosomes and macrochromosomes within reptiles.

Identification of an integrated stress and growth response signaling switch that directs vertebrate intestinal regeneration.

BACKGROUND: Snakes exhibit extreme intestinal regeneration following months-long fasts that involves unparalleled increases in metabolism, function, and tissue growth, but the specific molecular control of this process is unknown. Understanding the mechanisms that coordinate these regenerative phenotypes provides valuable opportunities to understand critical pathways that may control vertebrate regeneration and novel perspectives on vertebrate regenerative capacities. RESULTS: Here, we integrate a comprehensive set of phenotypic, transcriptomic, proteomic, and phosphoproteomic data from boa constrictors to identify the mechanisms that orchestrate shifts in metabolism, nutrient uptake, and cellular stress to direct phases of the regenerative response. We identify specific temporal patterns of metabolic, stress response, and growth pathway activation that direct regeneration and provide evidence for multiple key central regulatory molecules kinases that integrate these signals, including major conserved pathways like mTOR signaling and the unfolded protein response. CONCLUSION: Collectively, our results identify a novel switch-like role of stress responses in intestinal regeneration that forms a primary regulatory hub facilitating organ regeneration and could point to potential pathways to understand regenerative capacity in vertebrates.

Prospects for genomic surveillance for selection in schistosome parasites.

Schistosomiasis is a neglected tropical disease caused by multiple parasitic Schistosoma species, and which impacts over 200 million people globally, mainly in low- and middle-income countries. Genomic surveillance to detect evidence for natural selection in schistosome populations represents an emerging and promising approach to identify and interpret schistosome responses to ongoing control efforts or other environmental factors. Here we review how genomic variation is used to detect selection, how these approaches have been applied to schistosomes, and how future studies to detect selection may be improved. We discuss the theory of genomic analyses to detect selection, identify experimental designs for such analyses, and review studies that have applied these approaches to schistosomes. We then consider the biological characteristics of schistosomes that are expected to respond to selection, particularly those that may be impacted by control programs. Examples include drug resistance, host specificity, and life history traits, and we review our current understanding of specific genes that underlie them in schistosomes. We also discuss how inherent features of schistosome reproduction and demography pose substantial challenges for effective identification of these traits and their genomic bases. We conclude by discussing how genomic surveillance for selection should be designed to improve understanding of schistosome biology, and how the parasite changes in response to selection.

The effects of climate and demographic history in shaping genomic variation across populations of the Desert Horned Lizard (Phrynosoma platyrhinos).

Species often experience spatial environmental heterogeneity across their range, and populations may exhibit signatures of adaptation to local environmental characteristics. Other population genetic processes, such as migration and genetic drift, can impede the effects of local adaptation. Genetic drift in particular can have a pronounced effect on population genetic structure during large-scale geographic expansions, where a series of founder effects leads to decreases in genetic variation in the direction of the expansion. Here, we explore the genetic diversity of a desert lizard that occupies a wide range of environmental conditions and that has experienced post-glacial expansion northwards along two colonization routes. Based on our analyses of a large SNP data set, we find evidence that both climate and demographic history have shaped the genetic structure of populations. Pronounced genetic differentiation was evident between populations occupying cold versus hot deserts, and we detected numerous loci with significant associations with climate. The genetic signal of founder effects, however, is still present in the genomes of the recently expanded populations, which comprise subsets of genetic variation found in the southern populations.