Venom phenotypes of the Rock Rattlesnake (Crotalus lepidus) and the Ridge-nosed Rattlesnake (Crotalus willardi) from México and the United States.

Although the Mexican Highlands has the highest diversity of small-bodied rattlesnakes in the world, studies on the species found throughout this region have been relatively scarce. This has led to challenges with examining venom phenotypic characteristics, as well as species misidentifications and misclassifications. In the current study we investigated venom variation among four subspecies of Crotalus lepidus (C. l. klaluberi, C. l. lepidus, C. l. maculosus, C. l. morulus) and four subspecies of C. willardi (C. w. amabilis, C. w. obscurus, C. w. silus, and C. w. willardi) that inhabit regions of southwestern United States and central México. SDS-PAGE patterns show the presence of many of the major compounds found in other rattlesnake venoms, although minor variations in protein banding patterns and intensity are recognizable. Most notably, PI-metalloproteinase (SVMP) bands appear to be very faint to absent in northern C. l. lepidus and C. l. klauberi subspecies, but are fairly prominent in all other C. lepidus and C. willardi subspecies. Enzyme activity assays revealed that C. lepidus subspecies exhibit higher SVMP and thrombin-like activities when compared to C. willardi subspecies. Significant differences between subspecies were also observed for kallikrein-like serine protease, L-amino acid oxidase, and phosphodiesterase activities, although these differences appear to be random and fail to follow a geographical or phylogenetic trend. The same relationship was also observed for fibrinogenolytic and coagulation assays. Toxicity assays conducted on lab mice (Mus musculus), house geckos (Hemidactylus frenatus), and house crickets (Acheta domestica) revealed varying toxicities between subspecies, with C. l klauberi being the most toxic towards mice (LD50 = 1.36 μg/g) and house geckos (LD50 = 0.17 μg/g), and C. w. silus being most toxic to house crickets (LD50 = 1.94 μg/g). These results provide additional evidence that geographical isolation, natural selection, and adaptive evolution in response to diets may be driving forces contributing to population-level variation in venom composition.