Crotalus Viridis Research Articles

Foraging and scavenging behaviour of the prairie rattlesnake (Crotalus viridis): no evidence that envenomation cues facilitate kleptoparasitism of struck prey

Abstract Most viperids are ambush predators that primarily use venom to subdue prey, employing a strike-release-trail hunting strategy whereby snakes follow the unique scent of envenomated prey to locate carcasses they have bitten and released. In addition to killing prey, rattlesnakes (like most carnivores) will also opportunistically scavenge carrion. This scavenging strategy likely includes the occasional consumption of carcasses killed by other snakes (i.e., kleptoparasitism). In areas with high densities of other pitvipers, utilizing the unique scent of animals envenomated by other snakes might be a viable alternative foraging strategy. We evaluated this possibility experimentally using a series of captive behavioural trials on prairie rattlesnakes (Crotalus viridis) to determine whether conspecific or heterospecific (C. scutulatus, C. ornatus) envenomation cues might increase the likelihood of kleptoparasitism. Rattlesnakes did not prefer envenomated prey over nonenvenomated prey, nor did they prefer venom cues of one species over another. Although they did frequently scavenge carcasses, in the absence of striking, snakes generally located carcasses using random searching movements instead of scent trails. Additionally, the amount of time rattlesnakes spent investigating carcass trails did not differ significantly among treatments, suggesting that striking, and the resultant formation of a chemical search image of prey, is more crucial to trailing behaviour than venom cues. Moreover, a high degree of behavioural variation among individuals was observed, suggesting that scavenging and kleptoparasitism in rattlesnakes is more complex than previously realized, and making generalizations about these behaviours is challenging.

EFFECT OF BODY POSITION ON ECHOCARDIOGRAPHIC PARAMETERS IN PRAIRIE RATTLESNAKES (CROTALUS VIRIDIS).

Echocardiography is a noninvasive diagnostic tool that can provide instantaneous information about cardiac function, but it is uncommonly used by veterinarians to assess reptilian patients. Echocardiograms were performed on 14 clinically healthy, adult prairie rattlesnakes (Crotalus viridis), and cardiac measurements were taken in a horizontal and vertical position. Cardiac parameters including ventricular volume in systole and diastole, as well as the diameter of the left atrium, pulmonary artery, and paired left and right aortic arches were obtained. No evidence of cardiac disease was noted in any of the study animals. Males had a greater percentage of ventricular volume change (VVC) than females in the vertical position (P = 0.043). The percentage of ventricular volume change was significantly lower in the horizontal compared with the vertical position (P = 0.032) and was not different by sex. For the short-axis views, the right atrial diameter and short-axis ventricular area in diastole and systole were significantly smaller in snakes in the vertical compared with the horizontal position. This study is the first to obtain echocardiographic measurements in North American vipers and adds to the understanding of techniques used to evaluate the cardiac function of these species.

Patterns of Survival of a Communally Overwintering Rattlesnake Using an Artificial Hibernaculum

Avoiding thermal stress by using hibernacula is fundamental to survival of snakes in temperate environments. Snakes may overwinter alone or aggregate in communal hibernacula to avoid temperature extremes. Limited information is available regarding the overwintering ecology of rattlesnakes, and basic demographic information is necessary for understanding population dynamics, habitat requirements, and management of rattlesnakes. Even less is known about the demographics of species and populations utilizing artificial hibernacula, which may become increasingly important as habitat is lost and/or becomes more fragmented. We used seven years of mark–recapture data of the Western Rattlesnake (Crotalus viridis) at an artificial hibernaculum to estimate annual apparent survival, encounter rates, and transition rates between life stages (mature vs. immature) to investigate how these rates vary over time and in response to drought conditions. Further, we described trends in body condition and age structure, as well as trends in the proportion of reproductive females. Apparent survival rates were consistent and high across the study period for all snakes (including immature and mature females and all males; 0.66). The probability of an immature female surviving and transitioning to a mature adult was high and consistent across the study period (0.73). The stable survival and transition rates observed in this study may indicate other demographic rates, such as reproductive output, may be important drivers of population dynamics. This study contributes basic knowledge about population dynamics of communally overwintering rattlesnakes and suggests survival rates were relatively stable for snakes regularly utilizing this artificial hibernaculum.

Prioritizing human safety and multispecies connectivity across a regional road network

AbstractThe intersection of wildlife and people on roads raises two critical issues: the barrier and mortality effects of roads on wildlife and risks to people from animal‐vehicle collisions (AVCs). Road mitigation decisions are typically made at the discretion of transportation departments that are mandated to primarily address motorist safety. Therefore, prioritization of road sections for mitigation currently focuses on identification of spatial clusters of AVCs. We sought to understand if AVC clusters align with multispecies connectivity across roads to accurately identify multipurpose mitigation hotspots. We developed a decision‐support tool based on weighted priorities for mitigation planning across 7,900 km of roads over an 84,000‐km2 area of southern Alberta, Canada. To assess alignment, we built functional connectivity models for four focal species (prairie rattlesnake, grizzly bear, mule deer, and pronghorn) and a species‐neutral structural connectivity model. We integrated AVC risk and wildlife connectivity indices into Mitigation Priority Indices that varied the weighting of individual indices. Our results demonstrated poor spatial alignment between road sections of high motorist safety risk and those of high value for wildlife connectivity. Transportation planning would benefit from integrating motorist safety risk and wildlife management needs to prioritize mitigation neighborhoods along roadways.

Comparison of isoflurane, sevoflurane, and desflurane as inhalant anesthetics in prairie rattlesnakes (Crotalus viridis).

To characterize induction and recovery characteristics of 3 commonly used inhalant anesthetics in prairie rattlesnakes (Crotalus viridis): isoflurane, sevoflurane, and desflurane. 12 healthy adult prairie rattlesnakes. In a randomized crossover design, snakes underwent anesthetic induction with 5% isoflurane, 8% sevoflurane, or 18% desflurane, with a washout period of ≥ 7 days between anesthetic events. Anesthetic depth parameters were recorded throughout induction and recovery, including time to loss and return of righting reflex, muscle tone, ability to intubate, response to pressure, and time to return to spontaneous respiration. Every 5 minutes throughout the anesthetic procedures, heart rate, respiratory rate, and percentage expired anesthetic gas were recorded. No snakes died during the study. Sevoflurane anesthesia resulted in anesthetic gas avoidance behavior in snakes during induction and had the significantly longest recovery time to extubation and time to return of pressure response, compared with the other inhalant anesthetics. Anesthesia with isoflurane resulted in a significantly longer time to return of righting reflex, compared with sevoflurane or desflurane. No significant difference was noted in time to loss of pressure response among the 3 anesthetic gases. Desflurane anesthesia resulted in the significantly quickest loss of righting reflex among the anesthetic protocols; despite this, 4 of 12 desflurane anesthetized snakes did not achieve an anesthetic plane deep enough for intubation. Isoflurane and sevoflurane, but not desflurane, inhalation anesthesia resulted in consistent and predictable loss of righting reflex and induction of anesthesia deep enough to allow intubation in snakes.

Asymmetrical expression of toxins between the left and right venom glands of an individual prairie rattlesnake (Crotalus viridis viridis)

It is assumed that toxin expression is equivalent between left and right glands of a single snake. In the current study, we report venoms that differ in enzyme functionality and overall composition between the left and right gland of a single snake. The right gland produced venom of comparable composition to venom previously extracted from the same individual; however, the left gland produced venom with overall lower protein content and considerably less enzyme activity.

Characterization of plasma secretory phospholipase A2 activity in the prairie rattlesnake (Crotalus viridis)

BackgroundPlasma phospholipase A2 (PLA2) enzyme activity is a key component of innate immunity in most vertebrates. We evaluated circulating secreted PLA2 activity of prairie rattlesnakes (Crotalus viridis) by incubation of plasma with bacteria labeled with fluorescent membrane lipids.ResultsIncubation of bacteria with increasing volumes of plasma resulted in volume-dependent lysis of fatty acids from bacterial membranes. The activity was rapid, with substantial activity recorded after only 5 min of incubation with labeled bacteria, and a linear response for 20 min. In addition, the lysis activity was temperature-dependent, increasing activities from 5 to 20 °C, peak activities at 25–30 °C, and then decreasing activities from 35 to 40 °C. Furthermore, the activity was inhibited in a concentration-dependent manner by p-bromophenacyl bromide, a specific inhibitor of PLA2 activity, which indicated that the observed activities were due to the presence of PLA2 in the plasma of C. viridis.ConclusionsThis study represents the first description of secretory PLA2 activity in the plasma of a snake. Our study shows that in addition to being an important component of snake venom, PLA2 enzymes play an important role in the snake’s immune response.

Experimental modification of morphology reveals the effects of the zygosphene-zygantrum joint on the range of motion of snake vertebrae.

Variation in joint shape and soft tissue can alter range of motion (ROM) and create trade-offs between stability and flexibility. The shape of the distinctive zygosphene-zygantrum joint of snake vertebrae has been hypothesized to prevent axial torsion (twisting), but its function has never been tested experimentally. We used experimental manipulation of morphology to determine the role of the zygosphene-zygantrum articulation by micro-computed tomography (μCT) scanning and 3D printing two mid-body vertebrae with unaltered shape and with the zygosphene digitally removed for four species of phylogenetically diverse snakes. We recorded the angular ROM while manipulating the models in yaw (lateral bending), pitch (dorsoventral bending) and roll (axial torsion). Removing the zygosphene typically increased yaw and dorsal pitch ROM. In the normal vertebrae, roll was <2.5deg for all combinations of pitch and yaw. Roll increased in altered vertebrae but only for combinations of high yaw and ventral pitch that were near or beyond the limits of normal vertebra ROM. In the prairie rattlesnake and brown tree snake, roll in the altered vertebrae was always limited by bony processes other than the zygosphene, whereas in the altered vertebrae of the corn snake and boa constrictor, roll ROM was unconstrained when the pre- and post-zygapophyses no longer overlapped. The zygosphene acts as a bony limit for yaw and dorsal pitch, indirectly preventing roll by precluding most pitch and yaw combinations where roll could occur and potentially allowing greater forces to be applied across the vertebral column than would be possible with only soft-tissue constraints.

PREDATION ON NORTHERN BOBWHITE AND SCALED QUAIL BY SNAKES IN TEXAS, USA

Snakes are a known predator of quail; however, the influence of snake predation on quail populations is unknown. We used a long-term data set collected by eight radiotelemetry studies in Texas, encompassing 23 years (1994–2017) and 5,369 quail, to quantify snake predation on quail. We documented that northern bobwhite (Colinus virginianus) and scaled quail (Callipepla squamata) mortalities were caused by three species: western diamondback rattlesnakes (Crotalus atrox), prairie rattlesnakes (Crotalus viridis viridis), and Texas rat snakes (Pantherophis obsoletus). This study is the first to document predation of quail (Odontiphoridae) by the latter two species. We found 30 mortalities (0.6%) attributable to western diamondback rattlesnakes. We found 1 mortality (0.03%) attributable to a prairie rattlesnake and 2 mortalities (0.06%) attributable to Texas rat snakes of the 3,206 quail exposed to predation by those two species. We concluded that snakes are not major predators of adult quail, but that they will prey opportunistically upon them.

Allopatric divergence and secondary contact with gene flow: a recurring theme in rattlesnake speciation

AbstractThe study of recently diverged lineages whose geographical ranges come into contact can provide insight into the early stages of speciation and the potential roles of reproductive isolation in generating and maintaining species. Such insight can also be important for understanding the strategies and challenges for delimiting species within recently diverged species complexes. Here, we use mitochondrial and nuclear genetic data to study population structure, gene flow and demographic history across a geographically widespread rattlesnake clade, the western rattlesnake species complex (Crotalus cerberus, Crotalus viridis, Crotalus oreganus and relatives), which contains multiple lineages with ranges that overlap geographically or contact one another. We find evidence that the evolutionary history of this group does not conform to a bifurcating tree model and that pervasive gene flow has broadly influenced patterns of present-day genetic diversity. Our results suggest that lineage diversity has been shaped largely by drift and divergent selection in isolation, followed by secondary contact, in which reproductive isolating mechanisms appear weak and insufficient to prevent introgression, even between anciently diverged lineages. The complexity of divergence and secondary contact with gene flow among lineages also provides new context for why delimiting species within this complex has been difficult and contentious historically.

Carbon monoxide releasing molecule enhances coagulation and decreases fibrinolysis in canine plasma exposed to Crotalus viridis venom in vitro and in vivo.

Carbon monoxide releasing molecule-2 (CORM-2), an emerging therapeutic in human medicine, enhances plasmatic coagulation and attenuates fibrinolysis in vitro in human, rabbit and horse plasma and ameliorates hypocoagulation and hyperfibrinolysis secondary to venom exposure in human plasma in vitro. Fibrinogenases in rattlesnake venom cause decreased clot strength, and in the presence of tissue plasminogen activator (tPA) in vitro, a markedly increased rate of clot lysis. CO interacts with a haem group on fibrinogen, changing its configuration so that the fibrin clot is strengthened and more resistant to fibrinolysis. We hypothesized that CORM-2 enhances coagulation and attenuates fibrinolysis in canine plasma exposed to Cviridis venom. We measured the effects of Cviridis venom on clot strength, rates of coagulation and fibrinolysis in both pooled canine plasma and plasma from individual naturally envenomed dogs, with and without CORM-2, using thromboelastography (TEG). We tested venom effects on coagulation using tissue factor (TF) activated TEG and on both coagulation and fibrinolysis using TF-activated TEG with added tPA. We found that 17.9µg/mL of venom causes a mean 26.4% decrease in clot strength, a 61.8% decrease in maximum rate of thrombus generation, 75% faster clot lysis, a 226% increase in maximum rate of lysis and a 92% decrease in total clot life span (CLS). CORM-2 ameliorated these effects, increasing CLS in the presence of venom by 603%. Additionally, we showed that CORM-2 has similar effects in vitro on plasma from naturally envenomed dogs, showing promise as an adjunct therapy for snake envenomation.

The origins and evolution of chromosomes, dosage compensation, and mechanisms underlying venom regulation in snakes.

Here we use a chromosome-level genome assembly of a prairie rattlesnake (Crotalus viridis), together with Hi-C, RNA-seq, and whole-genome resequencing data, to study key features of genome biology and evolution in reptiles. We identify the rattlesnake Z Chromosome, including the recombining pseudoautosomal region, and find evidence for partial dosage compensation driven by an evolutionary accumulation of a female-biased up-regulation mechanism. Comparative analyses with other amniotes provide new insight into the origins, structure, and function of reptile microchromosomes, which we demonstrate have markedly different structure and function compared to macrochromosomes. Snake microchromosomes are also enriched for venom genes, which we show have evolved through multiple tandem duplication events in multiple gene families. By overlaying chromatin structure information and gene expression data, we find evidence for venom gene-specific chromatin contact domains and identify how chromatin structure guides precise expression of multiple venom gene families. Further, we find evidence for venom gland-specific transcription factor activity and characterize a complement of mechanisms underlying venom production and regulation. Our findings reveal novel and fundamental features of reptile genome biology, provide insight into the regulation of snake venom, and broadly highlight the biological insight enabled by chromosome-level genome assemblies.

Incidentally gathered natural history information on Bullsnakes (&lt;i&gt;Pituophis catenifer sayi&lt;/i&gt;) in southeastern Alberta

We present observations on Bullsnakes (Pituophis catenifer sayi) gathered during a study of Prairie Rattlesnakes (Crotalus viridis) in a multiple-use, mixed grass landscape adjacent to the South Saskatchewan River, ~30 km northeast of Medicine Hat, Alberta, in May–October 1997. Hibernacula shared with rattlesnakes were located close to the river. We captured 31 Bullsnakes, either in a drift fence array around a hibernaculum or by hand; three were recaptured once. Emergence from the hibernaculum ended in mid-May, and return to it began in early September. A gap in capture events occurred between early July and late August, possibly attributable to fossorial activity during the height of the summer. The sex ratio of captured adult snakes was 0.64 in favour of males. Males attained the greatest maximum body sizes, but there was no significant size dimorphism by sex. Bullsnakes were assignable to juvenile, subadult, and adult classes by body size. Most captures were made on slopes in the immediate vicinity of the river, in areas classed as “thin breaks”, but four captures, about 7 km east of the river, provide evidence of long-distance movements from hibernacula. Captures were seldom made in the vicinity of anthropogenic features. Gas field development has increased greatly in the years since these data were collected. Our findings provide a baseline for Bullsnake population responses to such changes.

Evaluation of vasoactivity after haemotoxic snake venom administration

The chick chorioallantoic membrane (CAM) is a widely used model in medical research and fulfils the requirements laid out in the 3 R's. The CAM, contains a dense network of blood vessels, essential for embryo development but also makes the chick embryo an invaluable resource for the study of angiogenesis and the haemotoxicity of substances. Non-neurotoxic snake venom is responsible worldwide for numerous deaths but also has been found to have a vast range of medicinal benefit. This study combines evaluating the time of onset and intensity of effects of three whole viper venoms (Bitis aritans, Crotalus viridis, Agkistrodon contortrix) at varying concentrations. They were applied onto the CAM, using the Luepke grading system as one method of determining their rapid irritation potential. Regarding the principles of 3 R's, this method helps to evaluate the haemotoxic effect of venom as an alternative method to the rodent tests. The information provided from these results can be used as a rapid tool for both medical management of snakebite wounds and the potential use of snake venom in medical treatments. Then, Luepke grading system can help to evaluate the haemotoxic effect of venom in combination with other appropriate methods.

Rarely Observed Behaviors and Occurrences of Black-footed Ferrets Preconditioning for Wild Release 2014–2017

Abstract Black-footed ferrets Mustela nigripes became extinct in the wild in 1987 when the last known wild animals were removed from a remnant population in an attempt to save the species by beginning a captive breeding program. Breeding efforts were successful, and since 1991 wildlife managers have annually released ferrets back into the wild at sites within the ferrets' historic range. Before their release into the wild, ferrets undergo a preconditioning stage in a quasi-natural habitat which better prepares them for life in the wild after a captive upbringing. Due to their nocturnal, fossorial, and largely solitary lifestyle, combined with their expansive habitat and endangered status, there are undoubtedly a multitude of behaviors ferrets exhibit that are not commonly observed. Here I detail multiple aspects of rarely described black-footed ferret ecology including black-tailed prairie dog Cynomys ludovicianus hunting stratagems, interactions with prairie rattlesnakes Crotalus viridis viridis, predation on birds, and sudden unexplained deaths of entire ferret family groups. The preconditioning setting that serves as an intermediate step between a captive and wild life for captive-reared ferrets allows for wildlife managers to witness ferret behaviors, though similar situations conceivably occur somewhat regularly in the wild.

Characterization of serum complement immune activity in the prairie rattlesnake (Crotalus viridis)

BackgroundReptile populations face a growing number of threats including global climate change and emerging pathogens. Unfortunately, research investigating the reptile immune system lags behind other taxa groups, hindering our ability to predict or mitigate species’ response to threats. Baseline studies are critical for our understanding of reptile immune response and overall health of wild populations.ResultsTreatment of unsensitized sheep red blood cells (SRBCs) with plasma collected from captive prairie rattlesnakes (Crotalus viridis) resulted in volume-dependent hemolysis, with a CH50 of 0.23 ± 0.01 mL. Kinetic analyses at different temperatures revealed that the hemolysis was relatively rapid, with 50% of hemolytic activity occurring within 15 min (25 °C), 18 min (35 °C), or 23 min (15 °C), and near maximum hemolysis within 60–90 min at all three temperatures. A comparative thermal profile revealed that complement activity was low at 5 °C, but increased sharply at 10 and 15 °C, and was maximal at 20–30 °C. A steep decline in activity was noted at temperatures > 30 °C. Mild heat treatment of the snake plasma (56 °C, 30 min) or treatment with proteases completely abolished the hemolytic activity. In addition, inclusion of 5 mM EDTA inhibited 90% of the hemolysis, but the activity could be reconstituted with the addition of 15 mM Ca2+ or Mg2+, but not Ba2+, Cu2+, or Fe2+. Furthermore, the hemolysis was unaffected in the presence of 20 mM methylamine, indicating that the alternative mechanism of complement activation is responsible for the observed activities.ConclusionsRattlesnakes show a relatively robust innate immune response as measured by hemolysis of SRBCs. However, hemolytic activity is reduced at high temperatures, indicating that rising global temperatures may have immune consequences for snake species, making them more vulnerable to known and emerging pathogens.

Venomous Snakebites in Canada: A National Review of Patient Epidemiology and Antivenom Usage

There are 3 pit viper species in Canada. Limited Canadian literature exists on the epidemiology of venomous snakebites and the treatment patterns with antivenom. This study described the epidemiology, the utilization of antivenom, and estimated expenditures due to forfeited antivenom for pit viper envenomations in Canada. A retrospective review of the Health Canada Special Access Program records to generate descriptive statistics. Data are presented as mean±SD (range), as appropriate. The geographic distribution of Canadian pit viper species is presented. There were 99 envenomations reported in Canada from January 2009 to December 2015. The number of envenomations per year was 14±6 (6-21). CroFab and Antivipmyn are used in Canada to treat envenomations. The number of vials for patient treatment was 17±12 (3-66) and 16±9 (6-42) for CroFab and Antivipmyn, respectively. Antivenom stock usage for patient treatment varied across the country with provincial means reported for British Columbia (33%), Alberta (37%), Saskatchewan (27%), and Ontario (71%). The costs incurred secondary to forfeited stock where estimated as: $1,280,000 USD in British Columbia, $255,000 in Alberta, $60,000 in Saskatchewan, and $0 in Ontario. The absolute number of annual envenomations is small and the 3 Crotalinae species are limited to relatively narrow geographic areas in British Columbia, Alberta, Saskatchewan, and Ontario. The utilization of antivenom in the treatment of patients revealed that regions where the western and prairie rattlesnake reside forfeited a substantial amount of antivenom from 2009 to 2015. Organizations responsible for maintaining antivenom supplies on a provincial or regional level could use these data to guide antidote stocking and reduce costs.

Snake Venom Extracellular vesicles (SVEVs) reveal wide molecular and functional proteome diversity

Proteins constitute almost 95% of snake venom’s dry weight and are produced and released by venom glands in a solubilized form during a snake bite. These proteins are responsible for inducing several pharmacological effects aiming to immobilize and initiate the pre-digestion of the prey. This study shows that proteins can be secreted and confined in snake venom extracellular vesicles (SVEVs) presenting a size distribution between 50 nm and 500 nm. SVEVs isolated from lyophilized venoms collected from four different species of snakes (Agkistrodon contortrix contortrix, Crotalus atrox, Crotalus viridis and Crotalus cerberus oreganus) were analyzed by mass spectrometry-based proteomic, which allowed the identification of proteins belonging to eight main functional protein classes such as SVMPs, serine proteinases, PLA2, LAAO, 5′nucleotidase, C-type lectin, CRISP and Disintegrin. Biochemical assays indicated that SVEVs are functionally active, showing high metalloproteinase and fibrinogenolytic activity besides being cytotoxic against HUVEC cells. Overall, this study comprehensively depicts the protein composition of SVEVs for the first time. In addition, the molecular function of some of the described proteins suggests a central role for SVEVs in the cytotoxicity of the snake venom and sheds new light in the envenomation process.

Morphological traits as indicators of sexual dimorphism in Prairie Rattlesnakes (Crotalus viridis)

Morphological traits as indicators of sexual dimorphism in Prairie Rattlesnakes (Crotalus viridis)

Antibacterial properties of plasma from the prairie rattlesnake (Crotalus viridis)

The innate immune system functions to quickly respond to pathogens and is likely the primary line of defense for ectothermic vertebrates. Snake populations appear to be in widespread decline globally, but despite the threats from emerging pathogens, very little work has been conducted to characterize their basic immune function. We used a wide-ranging snake species, the Prairie Rattlesnake (Crotalus viridis), to measure effects of snake plasma on the growth of eight bacterial species. Additionally, we quantified bacterial killing ability and kinetics of the immune response. Our results show that Prairie Rattlesnakes have robust innate immune systems, and concentrations of 10% snake plasma inhibit growth of 6 of 8 bacteria tested. Undiluted snake plasma inhibited nearly all bacterial growth. The immune response was fairly rapid, inhibiting 73% of bacterial growth within 20 min of exposure. These results are encouraging for conservation of wild populations, as snakes appear to exhibit a strong innate immune response. However, further work needs to be directed toward the evaluation of immune system capabilities in individual populations of conservation concern, and against pathogens known to cause mortality in wild snakes.