Comparative venomics of the Prairie Rattlesnake (Crotalus viridis viridis) from Colorado: Identification of a novel pattern of ontogenetic changes in venom composition and assessment of the immunoreactivity of the commercial antivenom CroFab®.

Here we describe and compare the venomic and antivenomic characteristics of both neonate and adult Prairie Rattlesnake (Crotalus viridis viridis) venoms. Although both neonate and adult venoms contain unique components, similarities among protein family content were seen. Both neonate and adult venoms consisted of myotoxin, bradykinin-potentiating peptide (BPP), phospholipase A2 (PLA2), Zn(2+)-dependent metalloproteinase (SVMP), serine proteinase, L-amino acid oxidase (LAAO), cysteine-rich secretory protein (CRISP) and disintegrin families. Quantitative differences, however, were observed, with venoms of adults containing significantly higher concentrations of the non-enzymatic toxic compounds and venoms of neonates containing higher concentrations of pre-digestive enzymatic proteins such as SVMPs. To assess the relevance of this venom variation in the context of snakebite and snakebite treatment, we tested the efficacy of the common antivenom CroFab® for recognition of both adult and neonate venoms in vitro. This comparison revealed that many of the major protein families (SVMPs, CRISP, PLA2, serine proteases, and LAAO) in both neonate and adult venoms were immunodepleted by the antivenom, whereas myotoxins, one of the major toxic components of C. v. viridis venom, in addition to many of the small peptides, were not efficiently depleted by CroFab®. These results therefore provide a comprehensive catalog of the venom compounds present in C. v. viridis venom and new molecular insight into the potential efficacy of CroFab® against human envenomations by one of the most widely distributed rattlesnake species in North America. BIOLOGICAL SIGNIFICANCE: Comparative proteomic analysis of venoms of neonate and adult Prairie Rattlesnake (Crotalus viridis viridis) from a discrete population in Colorado revealed a novel pattern of ontogenetic shifts in toxin composition for viperid snakes. The observed stage-dependent decrease of the relative content of disintegrins, catalytically active D49-PLA2s, L-amino acid oxidase, and SVMPs, and the concomitant increase of the relative abundance of paralytic small basic myotoxins and ohanin-like toxin, and hemostasis-disrupting serine proteinases, may represent an age-dependent strategy for securing prey and avoiding injury as the snake switches from small ectothermic prey and newborn rodents to larger endothermic prey. Such age-dependent shifts in venom composition may be relevant for antivenom efficacy and treatment of snakebite. However, applying a second-generation antivenomics approach, we show that CroFab®, developed against venom of three Crotalus and one Agkistrodon species, efficiently immunodepleted many, but not all, of the major compounds present in neonate and adult C. v. viridis venoms.

Modeling energetic and theoretical costs of thermoregulatory strategy.

Poikilothermic ectotherms have evolved behaviours that help them maintain or regulate their body temperature (T (b)) around a preferred or 'set point' temperature (T (set)). Thermoregulatory behaviors may range from body positioning to optimize heat gain to shuttling among preferred microhabitats to find appropriate environmental temperatures. We have modelled movement patterns between an active and non-active shuttling behaviour within a habitat (as a biased random walk) to investigate the potential cost of two thermoregulatory strategies. Generally, small-bodied ectotherms actively thermoregulate while large-bodied ectotherms may passively thermoconform to their environment. We were interested in the potential energetic cost for a large-bodied ectotherm if it were forced to actively thermoregulate rather than thermoconform. We therefore modelled movements and the resulting and comparative energetic costs in precisely maintaining a T (set) for a small-bodied versus large-bodied ectotherm to study and evaluate the thermoregulatory strategy.

Snakes survive starvation by employing supply- and demand-side economic strategies.

Animals vary widely in their abilities to tolerate extended periods of food limitation. Although some snakes are known for their unique ability to survive periods of inanition that last up to 2 years, very little is known about the biological mechanisms that allow them to do this. Consequently, the present study examined physiological, compositional, and morphological responses to 168 days of starvation among three distantly related snake species (i.e., ball python, Python regius; ratsnake, Elaphe obsoleta; and western diamondback rattlesnake, Crotalus atrox). Results revealed that each of these species was able to successfully tolerate starvation by adaptively utilizing supply- and demand-side regulatory strategies. Effective demand-side strategies included the ability of snakes to depress their resting metabolic demands by up to 72%. Moreover, supply-side regulation of resources was evidenced by the ability of snakes to spare their structurally critical protein stores at the expense of lipid catabolism. Such physiological strategies for minimizing endogenous mass and energy flux during periods of resource limitation might help explain the evolutionary persistence of snakes over the past 100 million years, as well as the repeated radiation of snake lineages into relatively low-energy environments. The final section of this study outlines a novel modeling approach developed to characterize material and chemical flux through animals during complete inanition. This approach was used to make comparisons about the efficacy of various supply- and demand-side starvation strategies among the three species examined, but could also be used to make similar comparisons among other types of animals.

From physiology to fitness: the costs of a defensive adaptation in rattlesnakes.

The costs of using and maintaining presumed adaptations are unknown for most animals. Energetically expensive traits, such as some agonistic and antipredator behaviors in animals, may incur trade-offs with other aspects of an animal's life history, such as feeding and reproduction. However, infrequent and brief use may reduce the costs of vigorous behaviors. The shaker muscles in the tails of rattlesnakes are an excellent system for studying the potential costs of a specialized defensive system. The high energetic cost of rattling may increase feeding requirements or use energy that could otherwise be available for reproduction. I used energetic modeling to test whether the cost of rattling in western diamond-backed rattlesnakes (Crotalus atrox) can be high enough to increase feeding demands or reduce fecundity and fitness. Only very frequent and prolonged rattling would increase feeding needs and perhaps reduce fecundity to some degree. Typically, rattling probably incurs very low costs to feeding, reproduction, and hence fitness. These and other results suggest that many seemingly expensive adaptations may have minimal costs to energy budgets, reproduction, and fitness.

Mechanical trade-offs explain how performance increases without increasing cost in rattlesnake tailshaker muscle.

Rattling by rattlesnakes is one of the fastest vertebrate movements and involves some of the highest contraction frequencies sustained by vertebrate muscle. Rattling requires higher accelerations at higher twitch frequencies, yet a previous study showed that the cost per twitch of rattling is independent of twitch frequency. We used force and video recordings over a range of temperatures to examine how western diamondback rattlesnakes (Crotalus atrox) achieve faster movements without increases in metabolic cost. The key findings are (i) that increasing muscle twitch tension trades off with decreasing twitch duration to keep the tension-time integral per twitch nearly constant over a wide range of temperatures and twitch frequencies and (ii) that decreasing lateral displacement of the rattle joint moderates the mechanical work and power required to shake the rattle at higher frequencies. These mechanical trade-offs between twitch tension and duration and between joint force and displacement explain how force, work and power increase without an increase in metabolic cost.

Scaling of CO2 production in the timber rattlesnake (Crotalus horridus), with comments on cost of growth in neonates and comparative patterns.

To understand the bioenergetic fluxes of free-ranging timber rattlesnakes (Crotalus horridus) better, we measured CO(2) production rate of 83 snakes in response to body mass, body temperature, time of day, sex, and geographic locality (northwest Arkansas and coastal Virginia). Effects of body mass, temperature, time of day, and the temperature-by-time interaction were remarkably similar to effects reported for other rattlesnakes. We noted that C. horridus has relatively high, but precedented, Q(10) (3.71-4.78); however, the adaptive significance of this observation, if any, remains obscure. Once the confounding effect of body mass was statistically adjusted, C. horridus exhibited no sex-specific effects; however, there was a significant locality-by-time effect, which is of equivocal biological significance. In contrast to the findings of a recent review on cost of growth in neonatal reptiles, C. horridus neonates exhibited metabolic rates that were from 200% to 400% greater than expectations from the mass scaling of yearlings and older animals. We interpreted this as evidence for a cost of synthesis in growing neonates. We report regression equations for the estimation of resting CO(2) production rate in C. horridus as a function of body mass, body temperature, and time of day. Our data contribute to a growing, comparative database documenting rattlesnakes as low-energy specialists.