Elsevier

Toxicon

Volume 57, Issue 5, April 2011, Pages 657-671
Toxicon

A high-throughput venom-gland transcriptome for the Eastern Diamondback Rattlesnake (Crotalus adamanteus) and evidence for pervasive positive selection across toxin classes

https://doi.org/10.1016/j.toxicon.2011.01.008Get rights and content

Abstract

Despite causing considerable human mortality and morbidity, animal toxins represent a valuable source of pharmacologically active macromolecules, a unique system for studying molecular adaptation, and a powerful framework for examining structure-function relationships in proteins. Snake venoms are particularly useful in the latter regard as they consist primarily of a moderate number of proteins and peptides that have been found to belong to just a handful of protein families. As these proteins and peptides are produced in dedicated glands, transcriptome sequencing has proven to be an effective approach to identifying the expressed toxin genes. We generated a venom-gland transcriptome for the Eastern Diamondback Rattlesnake (Crotalus adamanteus) using Roche 454 sequencing technology. In the current work, we focus on transcripts encoding toxins. We identified 40 unique toxin transcripts, 30 of which have full-length coding sequences, and 10 have only partial coding sequences. These toxins account for 24% of the total sequencing reads. We found toxins from 11 previously described families of snake-venom toxins and have discovered two putative, previously undescribed toxin classes. The most diverse and highly expressed toxin classes in the C. adamanteus venom-gland transcriptome are the serine proteinases, metalloproteinases, and C-type lectins. The serine proteinases are the most abundant class, accounting for 35% of the toxin sequencing reads. Metalloproteinases are the most diverse; 11 different forms have been identified. Using our sequences and those available in public databases, we detected positive selection in seven of the eight toxin families for which sufficient sequences were available for the analysis. We find that the vast majority of the genes that contribute directly to this vertebrate trait show evidence for a role for positive selection in their evolutionary history.

Introduction

Venoms can be viewed as inverse biochemical fingerprints of the most fundamental physiological processes sustaining a prey animal’s life. In their capacity to interfere with multifarious physiological, neurological, and hemostatic processes, animal toxins contain a wealth of information about their targets and have numerous applications in medical treatments or their design (Harvey et al., 1998, Ménez 1998, Escoubas and King, 2009). From the perspective of evolutionary biology, snake venoms represent a tremendous opportunity to study a large, integrated system of proteins that contribute to a single, well-defined, ecologically critical phenotypic trait. Venoms function in procuring and digesting prey and in defense, and therefore they directly contribute to fitness. The availability of representative structures for the major toxin classes (see, e.g., Holland et al., 1990, Gomis-Rüth et al., 1993, Zhang et al., 1994, Kumasaka et al., 1996, Gong et al., 1998, Pawelek et al., 2000, Watanabe et al., 2003, Lou et al., 2005) could eventually enable molecular evolutionary study to rival the level of detail found in current research on viral evolution (e.g., Bull et al., 2000, Rokyta and Wichman, 2009), but for a complex trait in vertebrates. Toxin genes show accelerated evolution, exon shuffling, transcriptional splicing, and gene fusion (Pahari et al., 2007). Work on a handful of toxin gene families has demonstrated an important role for positive selection in their evolution (Kordiš and Gubenšek, 2000, Lynch, 2007, Gibbs and Rossiter, 2008), but determining the prevalence of this type of selection in general awaits a more complete study of variation across the large number of genes that contribute to venom, which will first require a complete characterization of all of the venom genes.

A number of research groups have sequenced portions of snake venom-gland transcriptomes to identify the genes contributing to venoms. These previous studies have relied on cloning of cDNA libraries and Sanger sequencing, generating important, but ultimately limited, data. More than half of the expressed sequence tags (ESTs) from these studies have, in most cases, been found to code for toxin genes (see, e.g., Junqueira-de Azevedo and Ho, 2002), and a large proportion of the remaining ESTs to code for genes involved in transcription and translation, cell regulation, and metabolism (Pahari et al., 2007, Leão et al., 2009). Previous work has focused on species native to South America (Junqueira-de Azevedo and Ho, 2002, Ching et al., 2006, Cidade et al., 2006, de Azevedo et al., 2006, Leão et al., 2009), Africa (Francischetti et al., 2004, Wagstaff and Harrison, 2006, Casewell et al., 2009), and China (Qinghua et al., 2006, Zhang et al., 2006). Two North American species (Sistrurus catenatus and Agkistrodon piscivorus) have been studied in this manner (Pahari et al., 2007, Jia et al., 2008). None of these previous studies used modern high-throughput sequencing technologies, and the majority of mRNAs identified were represented in the data by a single EST each (see, e.g., Qinghua et al., 2006, Leão et al., 2009), indicating low coverage and a high likelihood that many transcripts remained undetected or only partially sequenced. A comparison by Wagstaff and Harrison (2006) between this standard approach to transcriptomics and a proteomic analysis of venom revealed that the standard level of sequence coverage provides a far from complete characterization of snake venoms. The application of next-generation sequencing, though not without its own issues, should alleviate issues of low coverage and provide a more complete characterization of the genes contributing to snake venoms.

The Eastern Diamondback Rattlesnake (Crotalus adamanteus) is the largest member of the genus Crotalus, a group of New World pit vipers typified by the hollow segments on the end of the tail that form a rattle. The largest individual reliably reported measured 2.44 m, but more commonly, adults average between 1.2 and 1.5 m in length (Klauber, 1997). This species is restricted to the southeastern United States, where it historically occurred in seven states along the southeastern Coastal Plain (Conant and Collins, 1998). Currently, it is listed as endangered in North Carolina and has been essentially extirpated from Louisiana (Palmer and Braswell, 1995, Dundee and Rossman, 1996). Because of its large size, C. adamanteus preys primarily on small mammals and birds, seldom taking the various ectothermic prey items that many other rattlesnake species are known to consume. Various mouse and rat species, squirrels, and rabbits form the bulk of the diet, though ground-nesting birds, such as quail, are also routinely consumed (Klauber, 1997).

We present the first, to our knowledge, high-throughput venom-gland transcriptome for a snake species and the first transcriptomic characterization for a species of the genus Crotalus. In the work reported here, we focused on characterizing the most abundant toxin-encoding transcripts in the venom-gland transcriptome of C. adamanteus. In addition, we provide analyses of the molecular evolutionary forces responsible for the evolution of the identified toxins by testing for evidence of positive selection driving the sequence divergence between C. adamanteus and other species with representative sequences in public databases.

Section snippets

Snake venom-gland preparation

We sequenced the venom-gland transcriptome of a single animal from Florida (Wakulla County). The specimen was an adult female weighing 392.9 g with a snout-to-vent length of 792 mm and a total length of 844 mm. To stimulate transcription in the venom glands, venom was extracted by electrostimulation under anesthesia (McCleary and Heard, 2010). The snake was anesthetized by propofol injection (10 mg/kg). After venom extraction, the animal was allowed to recover for four days so that

Sequencing and assembly results

Our half run on the Roche GS FLX generated 635,484 reads of average length 300 nucleotides after trimming of adapters and low-quality sequence. Of these, 82,621 were singletons, and the remaining 552,863 were assembled into 24,773 contigs of average length 513 nucleotides and comprising on average 22 sequencing reads. We performed an in-depth analysis of those contigs with the highest coverages and identified as putative toxins. These results are described in the next section. The approach

Discussion

C. adamanteus is generally considered to be the most dangerous snake in the United States because of its extremely large size and commensurately large venom glands, which can yield in excess of 1 ml of venom (DRR, personal observation). It and the Western Diamondback Rattlesnake (C. atrox) are responsible for most snakebite deaths in the United States (Gold et al., 2002). Our results for C. adamanteus are the first transcriptomic treatment of a member of the genus Crotalus and, to our

Acknowledgments

The authors would like to thank Darryl Heard for providing training to DRR and KPW in the electrostimulation technique for venom extraction and Kathleen Harper for advice on reptile anesthesia and euthanasia.

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