Dataset Information

Phenotypic Variation in Mojave Rattlesnake (Crotalus scutulatus) Venom Is Driven by Four Toxin Families.

ABSTRACT: Phenotypic diversity generated through altered gene expression is a primary mechanism facilitating evolutionary response in natural systems. By linking the phenotype to genotype through transcriptomics, it is possible to determine what changes are occurring at the molecular level. High phenotypic diversity has been documented in rattlesnake venom, which is under strong selection due to its role in prey acquisition and defense. Rattlesnake venom can be characterized by the presence (Type A) or absence (Type B) of a type of neurotoxic phospholipase A 2 (PLA 2 ), such as Mojave toxin, that increases venom toxicity. Mojave rattlesnakes (Crotalus scutulatus), represent this diversity as both venom types are found within this species and within a single panmictic population in the Sonoran Desert. We used comparative venom gland transcriptomics of nine specimens of C. scutulatus from this region to test whether expression differences explain diversity within and between venom types. Type A individuals expressed significantly fewer toxins than Type B individuals owing to the diversity of C-type lectins (CTLs) and snake venom metalloproteinases (SVMPs) found in Type B animals. As expected, both subunits of Mojave toxin were exclusively found in Type A individuals but we found high diversity in four additional PLA 2 s that was not associated with a venom type. Myotoxin a expression and toxin number variation was not associated with venom type, and myotoxin a had the highest range of expression of any toxin class. Our study represents the most comprehensive transcriptomic profile of the venom type dichotomy in rattlesnakes and C. scutulatus. Even intra-specifically, Mojave rattlesnakes showcase the diversity of snake venoms and illustrate that variation within venom types blurs the distinction of the venom dichotomy.

SUBMITTER: Strickland JL

PROVIDER: S-EPMC5923301 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Phenotypic Variation in Mojave Rattlesnake (Crotalus scutulatus) Venom Is Driven by Four Toxin Families.

Strickland Jason L JL Mason Andrew J AJ Rokyta Darin R DR Parkinson Christopher L CL

Toxins 20180323 4

Phenotypic diversity generated through altered gene expression is a primary mechanism facilitating evolutionary response in natural systems. By linking the phenotype to genotype through transcriptomics, it is possible to determine what changes are occurring at the molecular level. High phenotypic diversity has been documented in rattlesnake venom, which is under strong selection due to its role in prey acquisition and defense. Rattlesnake venom can be characterized by the presence (Type A) or ab ...[more]

PMID: 29570631

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Project description:As trophic adaptations, rattlesnake venoms can vary in composition depending on several intrinsic and extrinsic factors. Ontogenetic changes in venom composition have been documented for numerous species, but little is known of the potential age-related changes in many rattlesnake species found in México. In the current study, venom samples collected from adult and neonate Crotalus polystictus from Estado de México were subjected to enzymatic and electrophoretic analyses, toxicity assays (LD50), and MALDI-TOF mass spectrometry, and a pooled sample of adult venom was analyzed by shotgun proteomics. Electrophoretic profiles of adult males and females were quite similar, and only minor sex-based variation was noted. However, distinct differences were observed between venoms from adult females and their neonate offspring. Several prominent bands, including P-I and P-III snake venom metalloproteinases (SVMPs) and disintegrins (confirmed by MS/MS) were present in adult venoms and absent/greatly reduced in neonate venoms. Age-dependent differences in SVMP, kallikrein-like, phospholipase A₂ (PLA₂), and L-amino acid oxidase (LAAO) activity levels were confirmed by enzymatic activity assays, and like many other rattlesnake species, venoms from adult snakes have higher SVMP activity than neonate venoms. Conversely, PLA₂ activity was approximately 2.5 × greater in venoms from neonates, likely contributing to the increased toxicity (neonate venom LD50 = 4.5 μg/g) towards non-Swiss albino mice when compared to adult venoms (LD50 = 5.5 μg/g). Thrombin-like (TLE) and phosphodiesterase activities did not vary significantly with age. A significant effect of sex (between adult male and adult female venoms) was also observed for SVMP, TLE, and LAAO activities. Analysis of pooled adult venom by LC-MS/MS identified 14 toxin protein families, dominated by bradykinin-inhibitory peptides, SVMPs (P-I, P-II and P-III), disintegrins, PLA₂s, C-type-lectins, CRiSPs, serine proteinases, and LAAOs (96% of total venom proteins). Neonate and adult C. polystictus in this population consume almost exclusively mammals, suggesting that age-based differences in composition are related to physical differences in prey (e.g., surface-to-volume ratio differences) rather than taxonomic differences between prey. Venoms from adult C. polystictus fit a Type I pattern (high SVMP activity, lower toxicity), which is characteristic of many larger-bodied rattlesnakes of North America.

Project description:BACKGROUND: Snake venoms have significant impacts on human populations through the morbidity and mortality associated with snakebites and as sources of drugs, drug leads, and physiological research tools. Genes expressed by venom-gland tissue, including those encoding toxic proteins, have therefore been sequenced but only with relatively sparse coverage resulting from the low-throughput sequencing approaches available. High-throughput approaches based on 454 pyrosequencing have recently been applied to the study of snake venoms to give the most complete characterizations to date of the genes expressed in active venom glands, but such approaches are costly and still provide a far-from-complete characterization of the genes expressed during venom production. RESULTS: We describe the de novo assembly and analysis of the venom-gland transcriptome of an eastern diamondback rattlesnake (Crotalus adamanteus) based on 95,643,958 pairs of quality-filtered, 100-base-pair Illumina reads. We identified 123 unique, full-length toxin-coding sequences, which cluster into 78 groups with less than 1% nucleotide divergence, and 2,879 unique, full-length nontoxin coding sequences. The toxin sequences accounted for 35.4% of the total reads, and the nontoxin sequences for an additional 27.5%. The most highly expressed toxin was a small myotoxin related to crotamine, which accounted for 5.9% of the total reads. Snake-venom metalloproteinases accounted for the highest percentage of reads mapping to a toxin class (24.4%), followed by C-type lectins (22.2%) and serine proteinases (20.0%). The most diverse toxin classes were the C-type lectins (21 clusters), the snake-venom metalloproteinases (16 clusters), and the serine proteinases (14 clusters). The high-abundance nontoxin transcripts were predominantly those involved in protein folding and translation, consistent with the protein-secretory function of the tissue. CONCLUSIONS: We have provided the most complete characterization of the genes expressed in an active snake venom gland to date, producing insights into snakebite pathology and guidance for snakebite treatment for the largest rattlesnake species and arguably the most dangerous snake native to the United States of America, C. adamanteus. We have more than doubled the number of sequenced toxins for this species and created extensive genomic resources for snakes based entirely on de novo assembly of Illumina sequence data.

Project description:The same selective forces that give rise to rapid inter- and intraspecific divergence in snake venoms can also favor differences in venoms across life-history stages. Ontogenetic changes in venom composition are well known and widespread in snakes but have not been investigated to the level of unambiguously identifying the specific loci involved. The eastern diamondback rattlesnake was previously shown to undergo an ontogenetic shift in venom composition at sexual maturity, and this shift accounted for more venom variation than geography. To characterize the genetics underlying the ontogenetic venom compositional change in C. adamanteus, we sequenced adult/juvenile pairs of venom-gland transcriptomes from five populations previously shown to have different adult venom compositions. We identified a total of 59 putative toxin transcripts for C. adamanteus, and 12 of these were involved in the ontogenetic change. Three toxins were downregulated, and nine were upregulated in adults relative to juveniles. Adults and juveniles expressed similar total levels of snake-venom metalloproteinases but differed substantially in their featured paralogs, and adults expressed higher levels of Bradykinin-potentiating and C-type natriuretic peptides, nerve growth factor, and specific paralogs of phospholipases A2 and snake venom serine proteinases. Juvenile venom was more toxic to mice, indicating that the expression differences resulted in a phenotypically, and therefore potentially ecologically, significant difference in venom function. We also showed that adult and juvenile venom-gland transcriptomes for a species with known ontogenetic venom variation were equally effective at individually providing a full characterization of the venom genes of a species but that any particular individual was likely to lack several toxins in their transcriptome. A full characterization of a species' venom-gene complement therefore requires sequencing more than one individual, although the ages of the individuals are unimportant.

Dataset Information

Phenotypic Variation in Mojave Rattlesnake (Crotalus scutulatus) Venom Is Driven by Four Toxin Families.

Publications

Phenotypic Variation in Mojave Rattlesnake (Crotalus scutulatus) Venom Is Driven by Four Toxin Families.

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