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.

Crotalus Durissus Ruruima: Current Knowledge on Natural History, Medical Importance, and Clinical Toxinology.

Crotalus durissus ruruima is a rattlesnake subspecies mainly found in Roraima, the northernmost state of Brazil. Envenomings caused by this subspecies lead to severe clinical manifestations (e.g. respiratory muscle paralysis, rhabdomyolysis, and acute renal failure) that can lead to the victim's death. In this review, we comprehensively describe C. d. ruruima biology and the challenges this subspecies poses for human health, including morphology, distribution, epidemiology, venom cocktail, clinical envenoming, and the current and future specific treatment of envenomings by this snake. Moreover, this review presents maps of the distribution of the snake subspecies and evidence that this species is responsible for some of the most severe envenomings in the country and causes the highest lethality rates. Finally, we also discuss the efficacy of the Brazilian horse-derived antivenoms to treat C. d. ruruima envenomings in Roraima state.

Side-impact collision: mechanics of obstacle negotiation in sidewinding snakes.

Snakes excel at moving through cluttered environments, and heterogeneities can be used as propulsive contacts for snakes performing lateral undulation. However, sidewinding, which is often associated with sandy deserts, cuts a broad path through its environment that may increase its vulnerability to obstacles. Our prior work demonstrated that sidewinding can be represented as a pair of orthogonal body waves (vertical and horizontal) that can be independently modulated to achieve high maneuverability and incline ascent, suggesting that sidewinders may also use template modulations to negotiate obstacles. To test this hypothesis, we recorded overhead video of four sidewinder rattlesnakes (Crotalus cerastes) crossing a line of vertical pegs placed in the substrate. Snakes used three methods to traverse the obstacles: a Propagate Through behavior in which the lifted moving portion of the snake was deformed around the peg and dragged through as the snake continued sidewinding (115/160 runs), Reversal turns that reorient the snake entirely (35/160), or switching to Concertina locomotion (10/160). The Propagate Through response was only used if the anterior-most region of static contact would propagate along a path anterior to the peg, or if a new region of static contact could be formed near the head to satisfy this condition; otherwise, snakes could only use Reversal turns or switch to Concertina locomotion. Reversal turns allowed the snake to re-orient and either escape without further peg contact or re-orient into a posture amenable to using the Propagate Through response. We developed an algorithm to reproduce the Propagate Through behavior in a robophysical model using a modulation of the two-wave template. This range of behavioral strategies provides sidewinders with a versatile range of options for effectively negotiating obstacles in their natural habitat, as well as provide insights into the design and control of robotic systems dealing with heterogeneous habitats.

Intraspecific sequence and gene expression variation contribute little to venom diversity in sidewinder rattlesnakes ( Crotalus cerastes).

Traits can evolve rapidly through changes in gene expression or protein-coding sequences. However, these forms of genetic variation can be correlated and changes to one can influence the other. As a result, we might expect traits lacking differential expression to preferentially evolve through changes in protein sequences or morphological adaptation. Given the lack of differential expression across the distribution of sidewinder rattlesnakes ( Crotalus cerastes), we tested this hypothesis by comparing the coding regions of genes expressed in the venom gland transcriptomes and fang morphology. We calculated Tajima's D and FST across four populations comparing toxin and nontoxin loci. Overall, we found little evidence of directional selection or differentiation between populations, suggesting that changes to protein sequences do not underlie the evolution of sidewinder venom or that toxins are under extremely variant selection pressures. Although low-expression toxins do not have higher sequence divergence between populations, they do have more standing variation on which selection can act. Additionally, we found significant differences in fang length among populations. The lack of differential expression and sequence divergence suggests sidewinders-given their generalist diet, moderate gene flow and environmental variation-are under stabilizing selection which functions to maintain a generalist phenotype. Overall, we demonstrate the importance of examining the relationship between gene expression and protein-coding changes to understand the evolution of complex traits.

Evidence for divergent patterns of local selection driving venom variation in Mojave Rattlesnakes (Crotalus scutulatus).

Snake venoms represent an enriched system for investigating the evolutionary processes that lead to complex and dynamic trophic adaptations. It has long been hypothesized that natural selection may drive geographic variation in venom composition, yet previous studies have lacked the population genetic context to examine these patterns. We leverage range-wide sampling of Mojave Rattlesnakes (Crotalus scutulatus) and use a combination of venom, morphological, phylogenetic, population genetic, and environmental data to characterize the striking dichotomy of neurotoxic (Type A) and hemorrhagic (Type B) venoms throughout the range of this species. We find that three of the four previously identified major lineages within C. scutulatus possess a combination of Type A, Type B, and a 'mixed' Type A + B venom phenotypes, and that fixation of the two main venom phenotypes occurs on a more fine geographic scale than previously appreciated. We also find that Type A + B individuals occur in regions of inferred introgression, and that this mixed phenotype is comparatively rare. Our results support strong directional local selection leading to fixation of alternative venom phenotypes on a fine geographic scale, and are inconsistent with balancing selection to maintain both phenotypes within a single population. Our comparisons to biotic and abiotic factors further indicate that venom phenotype correlates with fang morphology and climatic variables. We hypothesize that links to fang morphology may be indicative of co-evolution of venom and other trophic adaptations, and that climatic variables may be linked to prey distributions and/or physiology, which in turn impose selection pressures on snake venoms.

Interfang Distances of Rattlesnakes: Sexual, Interspecific, and Body Size-related Variation, and Implications for Snakebite Research and Management.

OBJECTIVES: Snakebite severity corresponds to size of snake because the amount of venom a snake injects is positively associated with snake size. Because fang marks are often present on snakebite patients, we tested whether the relationship between snake length and distance between fang puncture wounds can be generalized for rattlesnakes of genus Crotalus. METHODS: We measured 2 interfang distances from 79 rattlesnakes of both sexes, 5 species, and varying body length: 1) distance between fang bases in anesthetized snakes, and 2) distance between fang punctures in a membrane-covered beaker bitten defensively. RESULTS: Statistical analyses supported our 2 hypotheses, that 1) body size-related fang divergence during fang protraction (ie, anterolateral movement during fang erection), and 2) the relationship between snake length and interfang distance are similar between the sexes and among different rattlesnake species. We therefore derived a general equation to estimate snake length based on distance between fang marks, and recommended 5 snake size categories: very small (<10 mm), small (10-15 mm), medium (15-20 mm), large (20-25 mm), and very large (>25 mm). CONCLUSIONS: The distance between fang marks on a snakebite patient may be used to estimate the size or size category of the offending snake, which in some cases may have predictive value for overall clinical severity of a given envenomation. Assessing interfang distance from puncture wounds can improve snakebite research and anticipation of snakebite severity.

Histology of the gastrointestinal tract from Bothrops jararaca and Crotalus durissus / Aspectos histológicos do trato gastrointestinal de Bothrops jararaca e Crotalus durissus

Recent studies have discussed the importance of snakes, going beyond the context of Public Health (snakebites). Promising research demonstrates the utility of snake venom in several fields, including oncology, diagnosis, anticoagulant therapy, and pain treatment. Nevertheless, there are still gaps in the basic knowledge on these animals, specifically regarding the histological characterization of the gastrointestinal tract, which ultimately hinder comparative histology and pathology studies. In this context the present study intends to contribute with the advance of current knowledge on snake histology by describing and analyzing histological samples of the gastrointestinal tracts of Bothrops jararaca and Crotalus durissus. Samples were collected from 12 individuals (six from each species), up to 6 months of age and equally distributed between males and females. Histological slides were prepared from the organs collected and were stained with hematoxylin and eosin, periodic acid-Schiff and alcian blue. The slides were photographed with a high-resolution camera to create a portfolio representative of the histology of the gastrointestinal systems of these animals.(AU)

Na atualidade, a importância das serpentes extravasa a conotação em saúde pública (acidentes ofídicos), e pesquisas promissoras têm demonstrado a utilidade dos venenos das serpentes em diversas áreas como oncologia, diagnóstico, terapia anticoagulante e tratamento da dor. Ainda assim, existem lacunas no conhecimento básico sobre esses animais, tais como a caracterização histológica do seu sistema gastrointestinal, que se tornam obstáculos para estudos nos campos da histologia e patologia comparadas. Nesse contexto, o presente trabalho pretende contribuir com o avanço do conhecimento sobre histologia de serpentes a partir da descrição e análise histológica de amostras do trato gastrointestinal de exemplares das espécies Bothrops jararaca e Crotalus durissus. Para tanto, foram coletadas amostras de 12 indivíduos com até seis meses de idade, seis por espécie (distribuídos igualmente entre machos e fêmeas). A partir dos órgãos colhidos, lâminas histológicas foram preparadas e coradas pela técnica de hematoxilina e eosina, bem como PAS com Alcian Blue. As lâminas foram fotografadas por câmera de alta resolução, resultando em um portfólio representativo da histologia do sistema gastrointestinal desses animais.(AU)

Deconstructing a Species-Complex: Geometric Morphometric and Molecular Analyses Define Species in the Western Rattlesnake (Crotalus viridis).

Morphological data are a conduit for the recognition and description of species, and their acquisition has recently been broadened by geometric morphometric (GM) approaches that co-join the collection of digital data with exploratory 'big data' analytics. We employed this approach to dissect the Western Rattlesnake (Crotalus viridis) species-complex in North America, currently partitioned by mitochondrial (mt)DNA analyses into eastern and western lineages (two and seven subspecies, respectively). The GM data (i.e., 33 dorsal and 50 lateral head landmarks) were gleaned from 2,824 individuals located in 10 museum collections. We also downloaded and concatenated sequences for six mtDNA genes from the NCBI GenBank database. GM analyses revealed significant head shape differences attributable to size and subspecies-designation (but not their interactions). Pairwise shape distances among subspecies were significantly greater than those derived from ancestral character states via squared-change parsimony, with the greatest differences separating those most closely related. This, in turn, suggests the potential for historic character displacement as a diversifying force in the complex. All subspecies, save one, were significantly differentiated in a Bayesian discriminant function analysis (DFA), regardless of whether our priors were uniform or informative (i.e., mtDNA data). Finally, shape differences among sister-clades were significantly greater than expected by chance alone under a Brownian model of evolution, promoting the hypothesis that selection rather than drift was the driving force in the evolution of the complex. Lastly, we combine head shape and mtDNA data so as to derived an integrative taxonomy that produced robust boundaries for six OTUs (operational taxonomic units) of the C. viridis complex. We suggest these boundaries are concomitant with species-status and subsequently provide a relevant nomenclature for its recognition and representation.

Phenotypic integration in the feeding system of the eastern diamondback rattlesnake (Crotalus adamanteus).

Selection can vary geographically across environments and temporally over the lifetime of an individual. Unlike geographic contexts, where different selective regimes can act on different alleles, age-specific selection is constrained to act on the same genome by altering age-specific expression. Snake venoms are exceptional traits for studying ontogeny because toxin expression variation directly changes the phenotype; relative amounts of venom components determine, in part, venom efficacy. Phenotypic integration is the dependent relationship between different traits that collectively produce a complex phenotype and, in venomous snakes, may include traits as diverse as venom, head shape and fang length. We examined the feeding system of the eastern diamondback rattlesnake (Crotalus adamanteus) across environments and over the lifetime of individuals and used a genotype-phenotype map approach, protein expression data and morphological data to demonstrate that: (i) ontogenetic effects explained more of the variation in toxin expression variation than geographic effects, (ii) both juveniles and adults varied geographically, (iii) toxin expression variation was a result of directional selection and (iv) different venom phenotypes covaried with morphological traits also associated with feeding in temporal (ontogenetic) and geographic (functional) contexts. These data are the first to demonstrate, to our knowledge, phenotypic integration between multiple morphological characters and a biochemical phenotype across populations and age classes. We identified copy number variation as the mechanism driving the difference in the venom phenotype associated with these morphological differences, and the parallel mitochondrial, venom and morphological divergence between northern and southern clades suggests that each clade may warrant classification as a separate evolutionarily significant unit.

Sidewinding with minimal slip: snake and robot ascent of sandy slopes.

Limbless organisms such as snakes can navigate nearly all terrain. In particular, desert-dwelling sidewinder rattlesnakes (Crotalus cerastes) operate effectively on inclined granular media (such as sand dunes) that induce failure in field-tested limbless robots through slipping and pitching. Our laboratory experiments reveal that as granular incline angle increases, sidewinder rattlesnakes increase the length of their body in contact with the sand. Implementing this strategy in a physical robot model of the snake enables the device to ascend sandy slopes close to the angle of maximum slope stability. Plate drag experiments demonstrate that granular yield stresses decrease with increasing incline angle. Together, these three approaches demonstrate how sidewinding with contact-length control mitigates failure on granular media.

Organotopic organization of the primary Infrared Sensitive Nucleus (LTTD) in the western diamondback rattlesnake (Crotalus atrox).

Pit vipers (Crotalinae) have a specific sensory system that detects infrared radiation with bilateral pit organs in the upper jaw. Each pit organ consists of a thin membrane, innervated by three trigeminal nerve branches that project to a specific nucleus in the dorsal hindbrain. The known topographic organization of infrared signals in the optic tectum prompted us to test the implementation of spatiotopically aligned sensory maps through hierarchical neuronal levels from the peripheral epithelium to the first central site in the hindbrain, the nucleus of the lateral descending trigeminal tract (LTTD). The spatial organization of the anatomical connections was revealed in a novel in vitro whole-brain preparation of the western diamondback rattlesnake (Crotalus atrox) that allowed specific application of multiple neuronal tracers to identified pit-organ-supplying trigeminal nerve branches. After adequate survival times, the entire peripheral and central projections of fibers within the pit membrane and the LTTD became visible. This approach revealed a morphological partition of the pit membrane into three well-defined sensory areas with largely separated innervations by the three main branches. The peripheral segregation of infrared afferents in the sensory epithelium was matched by a differential termination of the afferents within different areas of the LTTD, with little overlap. This result demonstrates a topographic organizational principle of the snake infrared system that is implemented by maintaining spatially aligned representations of environmental infrared cues on the sensory epithelium through specific neuronal projections at the level of the first central processing stage, comparable to the visual system.

Multilocus species delimitation in the Crotalus triseriatus species group (Serpentes: Viperidae: Crotalinae), with the description of two new species.

Members of the Crotalus triseriatus species group of montane rattlesnakes are widely distributed across the highlands of Mexico and southwestern USA. Although five species are currently recognized within the group, species limits remain to be tested. Genetic studies suggest that species may be paraphyletic and that at least one cryptic species may be present. We generate 3,346 base pairs of DNA sequence data from seven nuclear loci to test competing models of species delimitation in the C. triseriatus group using Bayes factor delimitation. We also examine museum specimens from the Trans-Mexican Volcanic Belt for evidence of cryptic species. We find strong support for a nine-species model and genetic and morphological evidence for recognizing two new species within the group, which we formally describe here. Our results suggest that the current taxonomy of the C. triseriatus species group does not reflect evolutionary history. We suggest several conservative taxonomic changes to the group, but future studies are needed to better clarify relationships among species and examine genetic patterns and structure within wide-ranging lineages.

Induction of phenotypic plasticity in rattlesnake trophic morphology by diet manipulation.

Gape-limited predators are restricted in the shape and size of prey items they can ingest by their trophic morphology. Evolutionary theory predicts that gape-limited predators, such as rattlesnakes, should possess plasticity in their trophic morphology to allow them to respond to environmental cues about their prey base. This study examined the effects of two possible influences over trophic morphology in the pit-viper Crotalus viridis viridis. Snakes from six litters were exposed to diet manipulations performed over 480 days. By day 480, snakes from two prey-size treatments exhibited significantly different head shapes. Snakes reared on whole rodents had broader heads, whereas snakes force-fed homogenized prey had narrower heads. Shape differences varied among litters, suggesting that not all litters responded the same to diet manipulations. Results suggest that trophic morphology of rattlesnakes is plastic, at least in some litters, and can be induced by prey items.

Mating systems, reproductive success, and sexual selection in secretive species: a case study of the western diamond-backed rattlesnake, Crotalus atrox.

Long-term studies of individual animals in nature contribute disproportionately to our understanding of the principles of ecology and evolution. Such field studies can benefit greatly from integrating the methods of molecular genetics with traditional approaches. Even though molecular genetic tools are particularly valuable for species that are difficult to observe directly, they have not been widely adopted. Here, we used molecular genetic techniques in a 10-year radio-telemetric investigation of the western diamond-backed rattlesnake (Crotalus atrox) for an analysis of its mating system and to measure sexual selection. Specifically, we used microsatellite markers to genotype 299 individuals, including neonates from litters of focal females to ascertain parentage using full-pedigree likelihood methods. We detected high levels of multiple paternity within litters, yet found little concordance between paternity and observations of courtship and mating behavior. Larger males did not father significantly more offspring, but we found evidence for size-specific male-mating strategies, with larger males guarding females for longer periods in the mating seasons. Moreover, the spatial proximity of males to mothers was significantly associated with reproductive success. Overall, our field observations alone would have been insufficient to quantitatively measure the mating system of this population of C. atrox, and we thus urge more widespread adoption of molecular tools by field researchers studying the mating systems and sexual selection of snakes and other secretive taxa.

Does the rattle of Crotalus durissus terrificus reveal its dietary history?

Background Environmental devastation threatens the survival of many species, including venomous snakes such as the South American rattlesnake Crotalus durissus terrificus. This observation is based on the decrease of snakes collected and donated to Brazilian research institutes. Nevertheless, some individuals have managed to survive and procreate. The question is how these snakes are adapting in these new environmental conditions.Methods To answer it, the carbon-13 level of rattlesnakes and their feed (either laboratory or wild mice) was evaluated by isotope-ratio mass spectrometry. Thus, rattle segments from 16 adults and 15 offspring of captive snakes, and of three wild newborn C. d. terrificus were evaluated as well as 17 Mus musculus mice captured in traps, four live feeder mice and the ration offered to mice at animal houses.Results The isotopic exchange time of the captive adult snakes (n = 16) varied between 33 and 37 months and of captive-born animals (n = 15), until reaching a plateau of equilibrium, varied from 18 to 24 months. Regarding the captured Mus musculus (n = 17), 88.23% (n = 15) were from a C4 environment. Of the six rattle rings from offspring of captured C. d. terrificus, five were from a C4environment, whereas of the 170 rattle rings studied, 60% originated from a C3 environment and 40% from a C4. The same carbon-13 values were found in captive snakes.Conclusions Based on the present results, it can be inferred that most C. d. terrificus snakes (60%) fed animals from a C3environment; birds consist of an alimentary alternative for snakes, as well as rodents, small reptiles and amphibians; different venom compositions among snakes from the same region may be related to the food type; the primary rattle of offspring reflects the maternal diet during gestation; and, finally, the different rattle rings indicate the alimentary history of these animals.(AU)

Evaporative respiratory cooling augments pit organ thermal detection in rattlesnakes.

Rattlesnakes use their facial pit organs to sense external thermal fluctuations. A temperature decrease in the heat-sensing membrane of the pit organ has the potential to enhance heat flux between their endothermic prey and the thermal sensors, affect the optimal functioning of thermal sensors in the pit membrane and reduce the formation of thermal "afterimages", improving thermal detection. We examined the potential for respiratory cooling to improve strike behaviour, capture, and consumption of endothermic prey in the South American rattlesnake, as behavioural indicators of thermal detection. Snakes with a higher degree of rostral cooling were more accurate during the strike, attacking warmer regions of their prey, and relocated and consumed their prey faster. These findings reveal that by cooling their pit organs, rattlesnakes increase their ability to detect endothermic prey; disabling the pit organs caused these differences to disappear. Rattlesnakes also modify the degree of rostral cooling by altering their breathing pattern in response to biologically relevant stimuli, such as a mouse odour. Our findings reveal that low humidity increases their ability to detect endothermic prey, suggesting that habitat and ambush site selection in the wild may be influenced by external humidity levels as well as temperature.

The Evolutionary Implications of Hemipenial Morphology of Rattlesnake Crotalus durissus terrificus (Laurent, 1768) (Serpentes: Viperidae: Crotalinae).

Most amniotes vertebrates have an intromittent organ to deliver semen. The reptile Sphenodon and most birds lost the ancestral penis and developed a cloaca-cloaca mating. Known as hemipenises, the copulatory organ of Squamata shows unique features between the amniotes intromittent organ. They are the only paired intromittent organs across amniotes and are fully inverted and encapsulated in the tail when not in use. The histology and ultrastructure of the hemipenes of Crotalus durissus rattlesnake is described as the evolutionary implications of the main features discussed. The organization of hemipenis of Crotalus durissus terrificus in two concentric corpora cavernosa is similar to other Squamata but differ markedly from the organization of the penis found in crocodilians, testudinata, birds and mammals. Based on the available data, the penis of the ancestral amniotes was made of connective tissue and the incorporation of smooth muscle in the framework of the sinusoids occurred independently in mammals and Crotalus durissus. The propulsor action of the muscle retractor penis basalis was confirmed and therefore the named should be changed to musculus hemipenis propulsor.The retractor penis magnus found in Squamata has no homology to the retractor penis of mammals, although both are responsible for the retraction of the copulatory organ.

Viperid venom glands with defective venom production. Morphological study.

The venom of viperid snakes is collected monthly at Butantan Institute for research purposes and production of antivenoms. Here we describe histological and ultrastructural changes on Crotalus durissus terrificus and Bothrops sp. venom glands with defective venom production. Secretory tubules commonly showed partial or total obliteration of their lumina by masses of necrotic cells and cellular debris. Secretory cells showed varying degrees of degenerative and/or metaplastic alterations seriously affecting the structures responsible for the synthesis and secretion of venom. The intertubular connective tissue presented fibroblast hyperplasia, inflammatory cells infiltration, vacuolated cells and blood vessels alterations. In two venom glands out of nineteen snakes examined, virus-like particles were found. The alterations observed in most of the glands could have been caused by excessive manual pressure, during venom extraction routine, causing disruption of the secretory tubules and leakage of venom to the intertubular connective tissue.

Gape size, its morphological basis, and the validity of gape indices in western diamond-backed rattlesnakes (Crotalus atrox).

Maximum gape is important to the ecology and evolution of many vertebrates, particularly gape-limited predators, because it can restrict the sizes and shapes of prey that can be eaten. Although many cranial elements probably contribute to gape, it is typically estimated from jaw length or jaw width, or occasionally from a combination of these two measures. We measured maximum gape directly for 18 individuals of the western diamond-backed rattlesnake, Crotalus atrox. We measured each individual's body length, several external cranial dimensions, several cranial osteological dimensions from cleaned skeletons, and we calculated gape index values from two published gape indices (GI). Cranial bone lengths and gape circumference showed negative allometry with snout-vent length (SVL), indicating that small individuals have relatively larger heads and gapes than their larger conspecifics. We then used Akaike's Information Criterion to determine which external and osteological measurements were the best predictors of gape. Body size (SVL) was the best predictor of maximum gape overall; however, when SVL was excluded from the analysis, quadrate (QL) and mandible lengths (MdLs) were the best predictors of maximum gape using both external and osteological measurements. Quadrate length probably contributes directly to gape; however, the importance of MdL to gape is less clear and may be due largely to its allometric relationships with head length and SVL. The two published GI did not prove to be better indicators of actual gape than the jaw and QLs in this study, and the gape values they produced differed significantly from our empirically determined gapes. For these reasons, we urge caution with the use and interpretation of computed GI in future studies. The extensive variation in quadrate and mandible morphology among lineages suggest that these bones are more important to variation in gape among species and lineages than within a single species.