Mackessy, Stephen P.
University of Northern Colorado
Type of Resources
Place of Publication
University of Northern Colorado
Venoms are complex mixtures of toxic constituents used by venomous snakes to incapacitate prey, to defend against threats, and to aid in pre-digestion of prey items. Snake venoms vary based on a number of characteristics, including but not limited to individual identity, species, geographic range, and ontogeny. While much is understood about snake venom variability, relatively little is known as to how venom changes or is stable through time. This doctoral dissertation focuses on elucidating the nature and mechanism of compositional change in the venoms of two rattlesnake species, the Northern Pacific Rattlesnake (Crotalus oreganus oreganus) and the Desert Massasauga (Sistrurus tergeminus edwardsii). Investigating compositional change in snake venom, this dissertation has three major objectives: O1 To verify the quality of long-term stored samples for use in comparative analyses. O2 To compare the structure and function of snake venoms collected from the same geographic location at two, distant time points. O3 To detect and describe venom resistance in a rodent prey species of the rattlesnake S. t. edwardsii from Lincoln County, Colorado. Tracking changes in populations over time requires the use of long-term stored samples, so the second chapter discusses the viability of using stored samples, evaluating whether degradation is a limiting factor. Venom samples collected ~35 years ago were examined to detect changes in protein content, identity and enzyme activity. Enzyme iv assay data collected at the time of venom sample extraction were compared to data collected using the same samples and assay procedure in the present. SDS polyacrylamide gel electrophoresis (SDS-PAGE) and reverse phase high performance liquid chromatography (RP-HPLC) were used to detect features indicating degradation, such as lateral shifts in the position of stereotypical RP-HPLC peaks or the appearance of extra, unexpected SDS-PAGE bands that may result from proteolysis. Qualitative analyses did not reveal significant effects of degradation, but two enzyme activities were significantly lower for stored samples. While proteomic information was retained, some enzyme activity was lost after long-term storage, indicating that stored snake venom samples are viable sources of biological information, but require careful scrutiny of which feature are reliably retained over time. Special attention should be paid to the effects of degradation from storage in venom toxin quantity and quality. The third chapter explores whether venom composition change is detectable after ~35 years in three populations of C. o. oreganus. Comparing samples from three populations in California collected ~35 years apart, changes in presence/absence of toxins by SDS-PAGE band analysis, in relative abundance of toxins by RP-HPLC peak area analysis, and in specific activities of six enzymes were evaluated. Evidence in the literature suggested that changes are possible, but it was hypothesized that there would be no major differences in venom composition between the two time points. Multivariate analyses revealed no significant changes in SDS-PAGE band presence/absence over ~35 years for any population. There was some evidence that enzyme activities differed significantly in one of three populations; however, activity values varied similarly between age group and geographic location, producing low support for ecologically v relevant differences between groups. There was a strong signal for separation of samples by their collection date in the HPLC peak area analysis; more recently collected samples all contained significantly more of a specific L-amino acid oxidase and snake venom metalloprotease compared to long-term stored samples. Multivariate approaches documented variation within and between samples; however, this variation appeared to be shared among samples, regardless of the date they were collected or their collection location. While rapid compositional change is hypothetically possible within such a short time period, the venoms of these populations have not diverged dramatically over the past four decades. The fourth chapter addresses the hypothesis that diet plays a major role in driving snake venom evolution. Previous work in Lincoln County, Colorado, with S. t. edwardsii indicated that the venom of this rattlesnake species might differentially affect possible mammalian prey species. Initial assays evaluated whether some prey species are more or less resistant to the venom of S. t. edwardsii from Lincoln County, Colorado. It was determined that Peromyscus maniculatus resistance was restricted to S. t. edwardsii venom and did not confer resistance to a second sympatric rattlesnake, the Prairie Rattlesnake (Crotalus viridis viridis). Additionally, resistance was only found in P. maniculatus captured in Lincoln Co., indicating that this is a local adaptation. Finally, standard protein chemistry methods were used to attempt to isolate and describe a resistance molecule. Previous studies have described serum inhibitor molecules in other systems; however, mouse serum was not able to neutralize venom enzyme activity, and mass spectrometry identified a candidate resistance molecule as mouse serum albumin. vi The final chapter summarizes the findings of the preceding chapters, and this dissertation presents a new trajectory in the study of natural toxins. The use of long-term stored snake venom samples is validated and comparing these samples against those collected from extant members of the same populations confirms that the continuum of a population’s venom variation remains stable over several generations. The mechanism of this stability is not yet known, but there is increasing evidence that diet plays a role in genetic fixation of the types of toxins and combinations of toxins found in a given venom.
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