Intraspecific venom variation in the medically significant Southern Pacific Rattlesnake (Crotalus oreganus helleri): biodiscovery, clinical and evolutionary implications.

Due to the extreme variation of venom, which consequently results in drastically variable degrees of neutralization by CroFab antivenom, the management and treatment of envenoming by Crotalus oreganus helleri (the Southern Pacific Rattlesnake), one of the most medically significant snake species in all of North America, has been a clinician's nightmare. This snake has also been the subject of sensational news stories regarding supposed rapid (within the last few decades) evolution of its venom. This research demonstrates for the first time that variable evolutionary selection pressures sculpt the intraspecific molecular diversity of venom components in C. o. helleri. We show that myotoxic β-defensin peptides (aka: crotamines/small basic myotoxic peptides) are secreted in large amounts by all populations. However, the mature toxin-encoding nucleotide regions evolve under the constraints of negative selection, likely as a result of their non-specific mode of action which doesn't enforce them to follow the regime of the classic predator-prey chemical arms race. The hemorrhagic and tissue destroying snake venom metalloproteinases (SVMPs) were secreted in larger amounts by the Catalina Island and Phelan rattlesnake populations, in moderate amounts in the Loma Linda population and in only trace levels by the Idyllwild population. Only the Idyllwild population in the San Jacinto Mountains contained potent presynaptic neurotoxic phospholipase A2 complex characteristic of Mohave Rattlesnake (Crotalus scutulatus) and Neotropical Rattlesnake (Crotalus durissus terrificus). The derived heterodimeric lectin toxins characteristic of viper venoms, which exhibit a diversity of biological activities, including anticoagulation, agonism/antagonism of platelet activation, or procoagulation, appear to have evolved under extremely variable selection pressures. While most lectin α- and β-chains evolved rapidly under the influence of positive Darwinian selection, the β-chain lectin of the Catalina Island population appears to have evolved under the constraint of negative selection. Both lectin chains were conspicuously absent in both the proteomics and transcriptomics of the Idyllwild population. Thus, we not only highlight the tremendous biochemical diversity in C. o. helleri's venom-arsenal, but we also show that they experience remarkably variable strengths of evolutionary selection pressures, within each toxin class among populations and among toxin classes within each population. The mapping of geographical venom variation not only provides additional information regarding venom evolution, but also has direct medical implications by allowing prediction of the clinical effects of rattlesnake bites from different regions. Such information, however, also points to these highly variable venoms as being a rich source of novel toxins which may ultimately prove to be useful in drug design and development.
Biological Significance: These results have direct implications for the treatment of envenomed patients. The variable venom profile of Crotalus oreganus helleri underscores the biodiscovery potential of novel snake venoms.
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