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1. Duvernoy’s Gland Transcriptomics of the Plains Black-Headed Snake, Tantilla nigriceps (Squamata, Colubridae): Unearthing the Venom of Small Rear-Fanged Snakes

   https://doi.org/10.3390/toxins13050336  

   Hofmann, Erich P.; Rautsaw, Rhett M.; Mason, Andrew J.; Strickland, Jason L.; Parkinson, Christopher L. (May 2021, Toxins)

   null (Ed.)

   The venoms of small rear-fanged snakes (RFS) remain largely unexplored, despite increased recognition of their importance in understanding venom evolution more broadly. Sequencing the transcriptome of venom-producing glands has greatly increased the ability of researchers to examine and characterize the toxin repertoire of small taxa with low venom yields. Here, we use RNA-seq to characterize the Duvernoy’s gland transcriptome of the Plains Black-headed Snake, Tantilla nigriceps, a small, semi-fossorial colubrid that feeds on a variety of potentially dangerous arthropods including centipedes and spiders. We generated transcriptomes of six individuals from three localities in order to both characterize the toxin expression of this species for the first time, and to look for initial evidence of venom variation in the species. Three toxin families—three-finger neurotoxins (3FTxs), cysteine-rich secretory proteins (CRISPs), and snake venom metalloproteinases (SVMPIIIs)—dominated the transcriptome of T. nigriceps; 3FTx themselves were the dominant toxin family in most individuals, accounting for as much as 86.4% of an individual’s toxin expression. Variation in toxin expression between individuals was also noted, with two specimens exhibiting higher relative expression of c-type lectins than any other sample (8.7–11.9% compared to <1%), and another expressed CRISPs higher than any other toxin. This study provides the first Duvernoy’s gland transcriptomes of any species of Tantilla, and one of the few transcriptomic studies of RFS not predicated on a single individual. This initial characterization demonstrates the need for further study of toxin expression variation in this species, as well as the need for further exploration of small RFS venoms. 

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2. Venom production and secretion in reptiles

   https://doi.org/10.1242/jeb.227348  

   Mackessy, Stephen P. (April 2022, Journal of Experimental Biology)

   ABSTRACT The venom glands of reptiles, particularly those of front-fanged advanced snakes, must satisfy conflicting biological demands: rapid synthesis of potentially labile and highly toxic proteins, storage in the gland lumen for long periods, stabilization of the stored secretions, immediate activation of toxins upon deployment and protection of the animal from the toxic effects of its own venom. This dynamic system could serve as a model for the study of a variety of different phenomena involving exocrine gland activation, protein synthesis, stabilization of protein products and secretory mechanisms. However, these studies have been hampered by a lack of a long-term model that can be propagated in the lab (as opposed to whole-animal studies). Numerous attempts have been made to extend the lifetime of venom gland secretory cells, but only recently has an organoid model been shown to have the requisite qualities of recapitulation of the native system, self-propagation and long-term viability (>1 year). A tractable model is now available for myriad cell- and molecular-level studies of venom glands, protein synthesis and secretion. However, venom glands of reptiles are not identical, and many differ very extensively in overall architecture, microanatomy and protein products produced. This Review summarizes the similarities among and differences between venom glands of helodermatid lizards and of rear-fanged and front-fanged snakes, highlighting those areas that are well understood and identifying areas where future studies can fill in significant gaps in knowledge of these ancient, yet fascinating systems. 

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3. Comparative venom-gland transcriptomics and venom proteomics of four Sidewinder Rattlesnake (Crotalus cerastes) lineages reveal little differential expression despite individual variation

   https://doi.org/10.1038/s41598-018-33943-5  

   Hofmann, Erich P.; Rautsaw, Rhett M.; Strickland, Jason L.; Holding, Matthew L.; Hogan, Michael P.; Mason, Andrew J.; Rokyta, Darin R.; Parkinson, Christopher L. (October 2018, Scientific Reports)

   Abstract Changes in gene expression can rapidly influence adaptive traits in the early stages of lineage diversification. Venom is an adaptive trait comprised of numerous toxins used for prey capture and defense. Snake venoms can vary widely between conspecific populations, but the influence of lineage diversification on such compositional differences are unknown. To explore venom differentiation in the early stages of lineage diversification, we used RNA-seq and mass spectrometry to characterize Sidewinder Rattlesnake (Crotalus cerastes) venom. We generated the first venom-gland transcriptomes and complementary venom proteomes for eight individuals collected across the United States and tested for expression differences across life history traits and between subspecific, mitochondrial, and phylotranscriptomic hypotheses. Sidewinder venom was comprised primarily of hemorrhagic toxins, with few cases of differential expression attributable to life history or lineage hypotheses. However, phylotranscriptomic lineage comparisons more than doubled instances of significant expression differences compared to all other factors. Nevertheless, only 6.4% of toxins were differentially expressed overall, suggesting that shallow divergence has not led to major changes in Sidewinder venom composition. Our results demonstrate the need for consensus venom-gland transcriptomes based on multiple individuals and highlight the potential for discrepancies in differential expression between different phylogenetic hypotheses. 

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4. Venom Gene Sequence Diversity and Expression Jointly Shape Diet Adaptation in Pitvipers

   https://doi.org/10.1093/molbev/msac082  

   Mason, Andrew J.; Holding, Matthew L.; Rautsaw, Rhett M.; Rokyta, Darin R.; Parkinson, Christopher L.; Gibbs, H. Lisle (April 2022, Molecular Biology and Evolution)

   Yoder, Anne (Ed.)

   Abstract Understanding the joint roles of protein sequence variation and differential expression during adaptive evolution is a fundamental, yet largely unrealized goal of evolutionary biology. Here, we use phylogenetic path analysis to analyze a comprehensive venom-gland transcriptome dataset spanning three genera of pitvipers to identify the functional genetic basis of a key adaptation (venom complexity) linked to diet breadth (DB). The analysis of gene-family-specific patterns reveals that, for genes encoding two of the most important venom proteins (snake venom metalloproteases and snake venom serine proteases), there are direct, positive relationships between sequence diversity (SD), expression diversity (ED), and increased DB. Further analysis of gene-family diversification for these proteins showed no constraint on how individual lineages achieved toxin gene SD in terms of the patterns of paralog diversification. In contrast, another major venom protein family (PLA2s) showed no relationship between venom molecular diversity and DB. Additional analyses suggest that other molecular mechanisms—such as higher absolute levels of expression—are responsible for diet adaptation involving these venom proteins. Broadly, our findings argue that functional diversity generated through sequence and expression variations jointly determine adaptation in the key components of pitviper venoms, which mediate complex molecular interactions between the snakes and their prey. 

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

   https://doi.org/10.1101/gr.276251.121  

   Perry, Blair W.; Gopalan, Siddharth S.; Pasquesi, Giulia I.M.; Schield, Drew R.; Westfall, Aundrea K.; Smith, Cara F.; Koludarov, Ivan; Chippindale, Paul T.; Pellegrino, Mark W.; Chuong, Edward B.; et al (June 2022, Genome Research)

   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. 

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