Environmentally cued parturition in a desert rattlesnake,Crotalus atrox

Embryonic development in animals is dynamically regulated by physiological, behavioural, and environmental factors (temperature, precipitation, humidity), which in turn influence the timing of birth or hatching. In the present study, we provide evidence that parturition in a large‐bodied North American pitviper, the western diamond‐backed rattlesnake (Crotalus atrox), is environmentally cued. Specifically, we tested the hypothesis of births coinciding with rainfall events during the second‐half of the monsoon season (late July to mid September) using randomization modelling. Twenty‐one adult females surgically implanted with radio‐transmitters were tracked for extended periods from 2001–2010. From 2003 to 2007, the 21 females gave birth to 38 litters, generating sufficient data to test our hypothesis. In all years, births were restricted to a 4‐week period from 5 August to 7 September, which spanned between 6 and 19 days (mean ± SD, 15 ± 5.2 days). Most births (92.1%) occurred in August. Births were significantly associated with rainfall events in 2007, although births in 2003 and 2005 occurred closer to rain events than randomly generated births for respective years. However, when birth events across all 5 years were pooled, the model indicated a significance difference in mean rain‐days versus random rain‐days. Hence, births occurred more closely to rain events than random days. Other variables associated with monsoon events (increases in cloud cover and humidity; changes in barometric pressure) were not measured but constitute potential cues. The present research is the first long‐term, individual‐based radio‐telemetric study of a snake species to investigate environmental cues related to parturition using procedures of randomization modelling. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 866–877.     

Mechanisms controlling venom expulsion in the western diamondback rattlesnake, Crotalus atrox

Although many studies have documented variation in the amount of venom expended during bites of venomous snakes, the mechanistic source of this variation remains uncertain. This study used experimental techniques to examine how two different features of the venom delivery system, the muscle surrounding the venom gland (the Compressor Glandulae in the rattlesnake) and the fang sheath, could influence venom flow in the western diamondback rattlesnake, Crotalus atrox. Differential contraction of the Compressor Glandulae explained only approximately 30% of the variation in venom flow. Lifting (compression) of the fang sheath as occurs during a normal strike produced marked increases in venom flow; these changes were closely correlated and exceed in magnitude by almost 10 x those recorded from the Compressor Glandulae alone. These results suggest that variation in these two aspects of the venom delivery system--both in terms of magnitude and temporal patterning--explain most of the observed variation in venom injection. The lack of functional or mechanical links between the Compressor Glandulae and the fang sheath, and the lack of skeletal or smooth muscle within the fang sheath, make it unlikely that variation in venom flow is under direct neural control. Instead, differential venom injection results from differences in the pressurization by the Compressor Glandulae, the gate keeping effects of the fang sheath and enclosed soft-tissue chambers, and by differences in the pressure returned by peripheral resistance of the target tissue.     

Characterization of a protease with alpha- and beta-fibrinogenase activity from the Western diamondback rattlesnake, Crotalus atrox.

A metalloprotease from the rattlesnake Crotalus atrox venom was isolated and purified from multiple-step chromatographies including anion-exchange chromatography, gel permeation and reversed-phase HPLC. The fraction was shown to be homogeneous as judged by SDS-gel electrophoresis. It also showed a high proteolytic activity against alpha- and beta-chains of fibrinogen molecules. Further characterization of the purified fraction with fibrinogenase activity indicated that it is a single-chain protease with a molecular mass of about 24 kDa and an acidic isoelectric point. It is relatively heat stable up to about 65 degrees C, inhibited by EDTA, beta-mercaptoethanol, but not by phenylmethanesulfonyl fluoride, N alpha-p-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone, soybean trypsin inhibitor and aprotinin. Amino acid analysis showed that the enzyme possesses an amino acid composition very similar to some metalloproteases characterized before from the closely related rattlesnake venoms. N-Terminal sequence analysis of the enzyme corroborated some similarity between this enzyme and the reported sequences of these enzymes characterized from the Crotalidae snake family. This study indicated the presence of a novel fibrinogenase (termed Catroxase) with N-terminal sequence different from the metalloprotease with hemorrhagic activity isolated from the same Western diamondback rattlesnake.     

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. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

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.     

Variation in oxygen consumption of the western diamondback rattlesnake (Crotalus atrox): implications for sexual size dimorphism

Variation in metabolism affects energy budgets of individuals and may serve as a mechanism that influences variation at whole organism or population levels. For example, sex differences in metabolic expenditure may contribute to bioenergetic sources of sexual size dimorphism. We measured oxygen consumption rates of 48 western diamondback rattlesnakes (Crotalus atrox) from a sexually dimorphic population and tested the effects of body mass, body temperature and time of day, in three groups of snakes: males, non-reproductive females, and vitellogenic females. Metabolic rates of male and non-reproductive female C. atrox were similar to rates reported for other rattlesnakes (mass exponents ranging from 0.645–0.670). Oxygen consumption was affected by body mass, body temperature and time of day, and was approximately 1.4 times greater in vitellogenic females than in non-reproductive females. No differences were found between males and non-reproductive females. Accordingly, differences in metabolic rate apparently do not contribute directly to sexual dimorphism in this population. Nevertheless, estimates of size-dependent maintenance expenditure lead us to hypothesize that adult female body size may represent a compromise between selection for increased litter size (accomplished by increasing body size), and selection for increased reproductive frequency (accomplished by decreasing body size, and, therefore inactive maintenance expenditure); this is a mechanistic scenario suggested previously for some endotherms. Accepted: 20 May 1998     

A new dwarf boa (Serpentes, Booidea, 'Tropidophiidae') from the Early Oligocene of Belgium: a case of the isolation of Western European snake faunas

Falseryx neervelpensis sp. nov. (Booid-grade, 'Tropidophiidae') from the earliest Oligocene (MP 21) of Belgium is described on the basis of vertebrae coming from all major portions of the vertebral column. In its peculiar caudal osteology, the snake approaches the unique morphological pattern characteristic of the living Neotropical Tropidophiinae. This is the first time such a complete and informative vertebral column of a dwarf boa has been described. The genus Falseryx was absent from Western Europe in younger parts of the Oligocene, but reappeared at the end of the Early Miocene. This dispersal pattern provides additional evidence that in most phases of the Oligocene and Early Miocene Western European snake faunas were effectively isolated from possible influences from the East.  © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society , 2008, 152 , 393–406.     

Aggressive mimicry in neonates of the sidewinder rattlesnake,Crotalus cerastes (Serpentes: Viperidae): stimulus control and visual perception of prey luring

Aggressive mimicry in vertebrates remains understudied relative to other categories of mimetic systems, such as Batesian mimicry. Prey attraction through caudal luring (CL) is a type of aggressive mimicry that constitutes a tripartite relationship in which a predator (mimic, S2), typically a snake, produces a highly specific tail display in the presence of a prey species (receiver or operator, R) to produce a resemblance to a prey animal (model, S1), such as a worm or insect, that the receiver mistakes for food and attempts to capture. Most reports of CL in snakes, however, are not hypothesis‐based and provide limited information on the cognitive interplay between predator and prey. In two experiments, CL was studied using a large sample (N = 40) of neonatal sidewinder rattlesnakes (Crotalus cerastes) and lizards (N = 12 species) to investigate stimulus control and visual perception. In experiment 1, CL was elicited in 110 trials using lizards that were either syntopic (N = 6 species) or nonsympatric (N = 6 species) to C. cerastes, and CL occurred at a significantly greater frequency when using syntopic taxa. Similarly, syntopic lizards were attracted to luring snakes significantly more than their nonsympatric counterparts. The presence of CL in C. cerastes was not ubiquitous and we provide preliminary evidence that this behaviour varies geographically and thus has a genetic basis. In experiment 2, a potential predator (live toad) was introduced to subjects that had been stimulated to lure by means of a prey‐dummy and, in all (N = 8) trials, there was an immediate shift in the behaviour of the snakes. The most notable changes were termination of CL and a transition to species‐typical defensive displays, which included rapid tail vibration and audible rattling in individuals with two (or more) rattle segments. We discuss future directions of CL research in snakes, especially with regard to expanding the types of cognitive tests. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 81–91.     

Hemorrhagic toxins from Western diamondback rattlesnake (Crotalus atrox) venom: isolation and characterization of five toxins and the role of zinc in hemorrhagic toxin e.

Five previously unknown hemorrhagic proteins, designated hemorrhagic toxins a,b,c,d, and e, were isolated from the venom of the western diamondback rattlesnake (Crotalus atrox). Molecular weights of hemorrhagic toxins a-e were determined to be 68 000, 24 000, 24 000, 24 000, and 25 700, respectively, by sodium dodecyl sulfate-phosphate gel electrophoresis using various polyacrylamide gel concentrations. Amino acid composition showed a total of 636, 200, 213, 214, and 219 amino acids for hemorrhagic toxins a-e, respectively. All the hemorrhagic toxins were found to lose their hemorrhagic activities with the metal chelators ethylenediaminetetraacetic acid and 1, 10-phenanthroline. All the hemorrhagic toxins were found to contain approximately 1 mol of zinc/mol of toxin, and they were all demonstrated to be proteolytic when dimethylcasein and dimethylhemoglobin were used as substrates. When zinc was removed from hemorrhagic toxin e with 1,10-phenanthroline, both both the proteolytic and hemorrhagic activities were equally inhibited. When the apohemorrhagic toxin e thus produced was incubated with zinc, the hemorrhagic and proteolytic activities were regenerated to the same extent. CD, UV, and Raman spectroscopy were used to study the structure of native hemorrhagin toxin e as well as the structural changes caused by zinc removal. From CD spectroscopy the native toxin was estimated to consist of 23% alpha helix, 6% beta structure, and 71% random-coil conformation. When over 90% of the zinc was removed, the alpha-helix content dropped from 23 to 7%.     

Post‐fire habitat use of the golden‐backed tree‐rat (Mesembriomys macrurus) in the northwest Kimberley,Western Australia

Fire regimes are changing throughout the world. Changed fire patterns across northern Australian savannas have been proposed as a factor contributing to recent declines of small‐ and medium‐sized mammals. Despite this, few studies have examined the mechanisms that underpin how species use habitat in fire‐affected landscapes. We determined the habitats and resources important to the declining golden‐backed tree‐rat (Mesembriomys macrurus) in landscapes partially burnt by recent intense fire. We aimed to (i) compare the relative use of rainforest and savanna habitats; (ii) examine the effect of fire history on use of savanna habitats; and (iii) identify key foraging and denning resources. Habitat selection was examined by comparing the availability of eight habitat types around real (used) and generated (available) location points. Individuals used a range of habitats, but consistently selected long unburnt rainforest in preference to recently burnt savanna (1–12 months post‐fire); however, recently burnt savanna was used in preference to long unburnt savanna. Tree‐rats foraged in Terminalia hadleyana, Planchonia rupestris, Celtis philippensis and Owenia vernicosa, tree species that are found in a variety of habitat types. Individuals used a range of den sites, including cliffs, trees, logs, scree and stags found throughout the study area. Although multiple factors may have led to the decline of Mes. macrurus across its range, these results are consistent with the idea that changes in the savanna structure as a consequence of contemporary fire patterns could also have a role. The continued persistence of Mes. macrurus in the northwest Kimberley may be supported by land management strategies that conserve fruiting and hollow‐bearing trees, and maintain the availability of fire‐sensitive vegetation types.     

PCR primers for microsatellite loci in the tiger rattlesnake (Crotalus tigris,Viperidae)

The tiger rattlesnake (Crotalus tigris) is found only in the Sonoran Desert of North America, where it inhabits rocky foothills that are increasingly being affected by urban encroachment. In order to continue a long‐term study of the demography and movements of tiger rattlesnakes around Tucson, Arizona, we identified six novel polymorphic microsatellite loci for this species. All loci exhibited high variability (five to 41 alleles). These markers are useful for studies of paternity, population structure, and assessing appropriate destinations for translocated animals.     

From lizard body form to serpentiform morphology: The atlas–axis complex in African cordyliformes and their relatives

The comparative vertebral morphology of the atlas–axis complex in cordyliforms, xantusiid and several skinks is studied here. These lizards are particularly interesting because of their different ecological adaptations and anti‐predation strategies, where conformation ranges from the lizard‐like body to a snake‐like body. This transition to serpentiform morphology shows several evolutionary patterns in the atlas–axis complex: 1) the zygapophyseal articulations are lost in the early stage of the transition. In contrast to mammals, the atlas is more or less locked to the axis in lepidosaurs, but the absence of zygapophyseal articulation releases this locking for rotation. However despite its serpentiform morphology, Chamaesaura is different, in possessing this articulation; 2) the first intercentrum of Chamaesaura and Tetradactylus africanus (serpentiform grass‐swimmers) is fully curved anteriorly, underlying the occipital condyle. While this limits ventral skull rotation beyond a certain angle, it locks the skull, which is a crucial adaptation for a sit‐and‐wait position in grassland habitats that needs to keep the head stabilized; and 3) in Acontias, most of the atlas articular surface with the occipital condyle is formed by the lateral aspect of the articulation area relative to the area located in the dorsal region of the slightly reduced intercentrum. A similar state occurs in amphisbaenians, most likely reflecting a fossorial lifestyle of the limbless lizards. Although Chamaesaura and Tetradactylus live sympatrically in grasslands, Chamaesaura differs in several ways in atlas–axis complex: for example, aforementioned presence of the atlas–axis zygapophyseal articulation, and long posterodorsal processes. Its occipital condyle protrudes further posteriorly, placing the atlas–axis complex further from the endocranium than in Tetradactylus. Hence, adaptation in the same niche, even among sister clades, can lead to different atlas–axis morphology due to different lifestyle strategies, for example, different foraging mode, while similar atlas–axis morphology can evolve in two lineages occupying different niches, as in Ablepharus and Scelotes. J. Morphol. 277:512–536, 2016. © 2016 Wiley Periodicals, Inc.     

Isolation and characterization of microsatellite loci from a threatened rattlesnake (New Mexico Ridge‐nosed Rattlesnake,Crotalus willardi obscurus)

Six novel microsatellite loci are identified from genomic DNA of the threatened New Mexico Ridge‐nosed Rattlesnake (Crotalus willardi obscurus). Data from the Animas Mountains (New Mexico) population demonstrate these loci: (i) are highly variable with 5–24 alleles per locus, expected heterozygosities between 0.35 and 0.92, and observed heterozygosities between 0.32 and 0.91; (ii) are sufficiently variable for assigning parentage with total exclusionary power for the first parent of 0.96, and 0.99 for the second parent; and (iii) amplify similar size fragments in other rattlesnakes (C. atrox, C. lutosus, C. scutulatus, and C. tigris).     

The ventilatory response to environmental hypercarbia in the South American rattlesnake,<Emphasis Type="Italic"> Crotalus durissus</Emphasis>

To study the effects of environmental hypercarbia on ventilation in snakes, particularly the anomalous hyperpnea that is seen when CO₂ is removed from inspired gas mixtures (post-hypercapnic hyperpnea), gas mixtures of varying concentrations of CO₂ were administered to South American rattlesnakes, Crotalus durissus, breathing through an intact respiratory system or via a tracheal cannula by-passing the upper airways. Exposure to environmental hypercarbia at increasing levels, up to 7% CO₂, produced a progressive decrease in breathing frequency and increase in tidal volume. The net result was that total ventilation increased modestly, up to 5% CO₂ and then declined slightly on 7% CO₂. On return to breathing air there was an immediate but transient increase in breathing frequency and a further increase in tidal volume that produced a marked overshoot in ventilation. The magnitude of this post-hypercapnic hyperpnea was proportional to the level of previously inspired CO₂. Administration of CO₂ to the lungs alone produced effects that were identical to administration to both lungs and upper airways and this effect was removed by vagotomy. Administration of CO₂ to the upper airways alone was without effect. Systemic injection of boluses of CO₂-rich blood produced an immediate increase in both breathing frequency and tidal volume. These data indicate that the post-hypercapnic hyperpnea resulted from the removal of inhibitory inputs from pulmonary receptors and suggest that while the ventilatory response to environmental hypercarbia in this species is a result of conflicting inputs from different receptor groups, this does not include input from upper airway receptors.Communicated by G. Heldmaier     

Genotyping‐in‐Thousands by sequencing reveals marked population structure in Western Rattlesnakes to inform conservation status

Delineation of units below the species level is critical for prioritizing conservation actions for species at‐risk. Genetic studies play an important role in characterizing patterns of population connectivity and diversity to inform the designation of conservation units, especially for populations that are geographically isolated. The northernmost range margin of Western Rattlesnakes (Crotalus oreganus) occurs in British Columbia, Canada, where it is federally classified as threatened and restricted to five geographic regions. In these areas, Western Rattlesnakes hibernate (den) communally, raising questions about connectivity within and between den complexes. At present, Western Rattlesnake conservation efforts are hindered by a complete lack of information on genetic structure and degree of isolation at multiple scales, from the den to the regional level. To fill this knowledge gap, we used Genotyping‐in‐Thousands by sequencing (GT‐seq) to genotype an optimized panel of 362 single nucleotide polymorphisms (SNPs) from individual samples (n = 461) collected across the snake's distribution in western Canada and neighboring Washington (USA). Hierarchical STRUCTURE analyses found evidence for population structure within and among the five geographic regions in BC, as well as in Washington. Within these regions, 11 genetically distinct complexes of dens were identified, with some regions having multiple complexes. No significant pattern of isolation‐by‐distance and generally low levels of migration were detected among den complexes across regions. Additionally, snakes within dens generally were more related than those among den complexes within a region, indicating limited movement. Overall, our results suggest that the single, recognized designatable unit for Western Rattlesnakes in Canada should be re‐assessed to proactively focus conservation efforts on preserving total genetic variation detected range‐wide. More broadly, our study demonstrates a novel application of GT‐seq for investigating patterns of diversity in wild populations at multiple scales to better inform conservation management.