Abstract
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.
Highlights
The rattlesnakes Crotalus and Sistrurus comprise a monophyletic lineage of vipers unique to the Americas, and they are recognized by the presence of the caudal rattle
Exceptions include species that represent the greatest risks to humans, such as C. simus [5,6] and C. scutulatus scutulatus [7]
We report here results of a detailed analysis changes in venomchanges composition, sex-related differences in toxin activities, andinLC-MS-MS
Summary
The rattlesnakes Crotalus and Sistrurus comprise a monophyletic lineage of vipers unique to the Americas, and they are recognized by the presence of the caudal rattle. It has been hypothesized that rattlesnakes originated in México [2,3], diversifying and dispersing to the north and south, and they have a current range extending over 70 degrees of latitude, from southern Canada to central Argentina and southern Uruguay [4]. Even with this wide geographical distribution, the highest diversity of rattlesnakes occurs on the Central. Mexican Plateau and surrounding highlands [4] Despite this diversity, venoms of most Mexican rattlesnakes, the montane species, are poorly known. Several recent studies have examined the venom compositional patterns of the diminutive C. lepidus [8,9,10]
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