Montane Rattlesnakes in México: Venoms of Crotalus tancitarensis and Related Species within the Crotalus intermedius Group

Abstract

The Crotalus intermedius group is a clade of rattlesnakes consisting of several species adapted to a high elevation habitat, primarily in México. Crotalus tancitarensis was previously classified as C. intermedius, until individuals occurring on Cerro Tancítaro in Michoacán, México, were reevaluated and classified as a new species (C. tancitarensis) based on scale pattern and geographic location. This study aimed to characterize the venom of C. tancitarensis and compare the venom profile to those of other species within the Crotalus intermedius group using gel electrophoresis, biochemical assays, reverse-phase high performance liquid chromatography, mass spectrometry, and lethal toxicity (LD₅₀) assays. Results show that the venom profiles of species within the Crotalus intermedius group are similar, but with distinct differences in phospholipase A₂ (PLA₂), metalloproteinase PI (SVMP PI), and kallikrein-like serine proteinase (SVSP) activity and relative abundance. Proteomic analysis indicated that the highland forms produce venoms with 50-60 protein isoforms and a composition typical of type I rattlesnake venoms (abundant SVMPs, lack of presynaptic PLA₂-based neurotoxins), as well as a diversity of typical Crotalus venom components such as serine proteinases, PLA₂s, C-type lectins, and less abundant toxins (LAAOs, CRiSPs, etc.). The overall venom profile of C. tancitarensis appears most similar to C. transversus, which is consistent with a previous mitochondrial DNA analysis of the Crotalus intermedius group. These rattlesnakes of the Mexican highlands represent a radiation of high elevation specialists, and in spite of divergence of species in these Sky Island habitats, venom composition of species analyzed here has remained relatively conserved. The majority of protein family isoforms are conserved in all members of the clade, and as seen in other more broadly distributed rattlesnake species, differences in their venoms are largely due to relative concentrations of specific components.

Keywords: RP-HPLC; SDS-PAGE; enzymes; evolution; mass spectrometry; phenotype; snake venom metalloproteinase; toxin; venom.

Figure 1

(A) Distribution of C. tancitarensis (International Union for Conservation of Nature 2007. Crotalus tancitarensis. Attribution-Share Alike Creative Commons License). (B) Adult female C. tancitarensis; photo by SPM.

Figure 2

Condensed rattlesnake phylogeny inferred from 6727 bp of combined mitochondrial and nuclear DNA (fossil-calibrated); adapted from Blair and Sánchez-Ramírez [2]. 2 MY, 2 million years.

Figure 3

SDS-PAGE gel (12%) of various Crotalus species of central México compared to neonate and adult C. tancitarensis venoms, and C. tancitarensis adult venoms compared to other adult venoms in the Crotalus intermedius group. Crotalus p. pricei were from various geographic locations: Chiricahua Mountains (Chir.), Pinaleño Mountains (Pina.), Santa Rita Mountains (Santa.), and Durango, México. Approximate molecular mass is displayed to the right and shown in kDa. All proteins were reduced with DTT and visualized using Coomassie Brilliant Blue dye. Typical protein families, as determined by mass and previous experiments with purified toxins, are shown on the left.

Figure 4

Average enzyme activities of mountain rattlesnake venoms.

Figure 5

Reverse-phase HPLC chromatograms of C. intermedius clade species (2.0 mg venom each). (A) Crotalus tancitarensis (adult) venom. (B) Crotalus transversus venom. (C) Crotalus price pricei venom. (D) Crotalus intermedius venom. Elution gradient is indicated by the blue line.

Figure 6

Mass spectrometric analysis of individual venoms. (A) Venom protein families and subfamilies identified in each venom by shotgun proteomic analysis. Bars represent total proteins identified, and colors indicate number of proteins in each protein family. (B) Summed intensities of peptides per protein family and subfamily for venom proteins. Intensities of each shared peptide in an identified protein were summed based on protein family and subfamily and used for relative quantitative comparison of protein abundances between the different venoms. Abbreviations: bradykinin-potentiating peptides (BPP); cysteine-rich secretory protein (CRISP); C-type lectin (CTL); glutaminyl-peptide cyclotransferase (GPC); hyaluronidase (Hyal); L-amino acid oxidase (LAAO); nerve growth factor (NGF); 5′ nucleotidase (NTD); phosphodiesterase (PDE); PI, PII, PIII snake venom metalloprotease (PI, PII, PIII SVMP); phospholipase A₂ (PLA₂); phospholipase B (PLB); (SVSP); thrombin-like serine proteinase (TLE); vascular endothelial growth factor (VEGF).

Figure 7

Venn diagrams showing overlapping proteins between the (A) C. pricei group (the four geographical locations plus C. p. miquihaunus) and (B) each species. For (B), we merged protein identifications for the two individual C. tancitarensis, for the two individual C. intermedius venoms, and for the five venoms in the C. pricei group to permit cross-species comparisons. Toxins unique to each venom are indicated in bold print. Note that in both Venn diagrams, most proteins (29, 32) are shared among all members. Abbreviations: C-type lectin (CTL); glutaminyl-peptide cyclotransferase (GPC); phosphodiesterase (PDE); PI, PIII snake venom metalloprotease (PI, PIII SVMP); phospholipase A₂ (PLA₂); serine proteinase (SVSP).

Figure 8

Lethal toxicity (μg/g) of adult C. tancitarensis venom and adult C. p. pricei venom toward Hemidactylus frenatus.