Gradual and Discrete Ontogenetic Shifts in Rattlesnake Venom Composition and Assessment of Hormonal and Ecological Correlates.

Ontogenetic shifts in venom occur in many snakes but establishing their nature as gradual or discrete processes required additional study. We profiled shifts in venom expression from the neonate to adult sizes of two rattlesnake species, the eastern diamondback and the timber rattlesnake. We used serial sampling and venom chromatographic profiling to test if ontogenetic change occurs gradually or discretely. We found evidence for gradual shifts in overall venom composition in six of eight snakes, which sometimes spanned more than two years. Most chromatographic peaks shift gradually, but one quarter shift in a discrete fashion. Analysis of published diet data showed gradual shifts in overall diet composition across the range of body sizes attained by our eight study animals, while the shifts in abundance of different prey classes varied in form from gradual to discrete. Testosterone concentrations were correlated with the change in venom protein composition, but the relationship is not strong enough to suggest causation. Venom research employing simple juvenile versus adult size thresholds may be failing to account for continuous variation in venom composition lifespan. Our results imply that venom shifts represent adaptive matches to dietary shifts and highlight venom for studies of alternative gene regulatory mechanisms.

Cryptic diversity in the Mexican highlands: Thousands of UCE loci help illuminate phylogenetic relationships, species limits and divergence times of montane rattlesnakes (Viperidae: Crotalus).

With the continued adoption of genome-scale data in evolutionary biology comes the challenge of adequately harnessing the information to make accurate phylogenetic inferences. Coalescent-based methods of species tree inference have become common, and concatenation has been shown in simulation to perform well, particularly when levels of incomplete lineage sorting are low. However, simulation conditions are often overly simplistic, leaving empiricists with uncertainty regarding analytical tools. We use a large ultraconserved element data set (>3,000 loci) from rattlesnakes of the Crotalus triseriatus group to delimit lineages and estimate species trees using concatenation and several coalescent-based methods. Unpartitioned and partitioned maximum likelihood and Bayesian analysis of the concatenated matrix yield a topology identical to coalescent analysis of a subset of the data in bpp. ASTRAL analysis on a subset of the more variable loci also results in a tree consistent with concatenation and bpp, whereas the SVDquartets phylogeny differs at additional nodes. The size of the concatenated matrix has a strong effect on species tree inference using SVDquartets, warranting additional investigation on optimal data characteristics for this method. Species delimitation analyses suggest up to 16 unique lineages may be present within the C. triseriatus group, with divergences occurring during the Neogene and Quaternary. Network analyses suggest hybridization within the group is relatively rare. Altogether, our results reaffirm the Mexican highlands as a biodiversity hotspot and suggest that coalescent-based species tree inference on data subsets can provide a strongly supported species tree consistent with concatenation of all loci with a large amount of missing data.

In vitro effects of Crotalus atrox snake venom on chick and mouse neuromuscular preparations.

The neuromuscular effect of venoms is not a major clinical manifestation shared between rattlesnakes native to the Americas, which showed two different venom phenotypes. Taking into account this dichotomy, nerve muscle preparations from mice and chicks were used to investigate the ability of Crotalus atrox venom to induce in vitro neurotoxicity and myotoxicity. Unlike crotalic venoms of South America, low concentrations of C. atrox venom did not result in significant effects on mouse neuromuscular preparations. The venom was more active on avian nerve-muscle, showing reduction of twitch heights after 120 min of incubation with 10, 30 and 100 µg/mL of venom with diminished responses to agonists and KCl. Histological analysis highlighted that C. atrox was myotoxic in both species of experimental animals; as evidenced by degenerative events, including edematous cells, delta lesions, hypercontracted fibers and muscle necrosis, which can lead to neurotoxic action. These results provide key insights into the myotoxicity and low neurotoxicity of C. atrox in two animal models, corroborating with previous genomic and proteomic findings and would be useful for a deeper understanding of venom evolution in snakes belonging to the genus Crotalus.

Rattling the border wall: Pathophysiological implications of functional and proteomic venom variation between Mexican and US subspecies of the desert rattlesnake Crotalus scutulatus.

While some US populations of the Mohave rattlesnake (Crotalus scutulatus scutulatus) are infamous for being potently neurotoxic, the Mexican subspecies C. s. salvini (Huamantlan rattlesnake) has been largely unstudied beyond crude lethality testing upon mice. In this study we show that at least some populations of this snake are as potently neurotoxic as its northern cousin. Testing of the Mexican antivenom Antivipmyn showed a complete lack of neutralisation for the neurotoxic effects of C. s. salvini venom, while the neurotoxic effects of the US subspecies C. s. scutulatus were time-delayed but ultimately not eliminated. These results document unrecognised potent neurological effects of a Mexican snake and highlight the medical importance of this subspecies, a finding augmented by the ineffectiveness of the Antivipmyn antivenom. These results also influence our understanding of the venom evolution of Crotalus scutulatus, suggesting that neurotoxicity is the ancestral feature of this species, with the US populations which lack neurotoxicity being derived states.

Integrated Venomics and Venom Gland Transcriptome Analysis of Juvenile and Adult Mexican Rattlesnakes Crotalus simus, C. tzabcan, and C. culminatus Revealed miRNA-modulated Ontogenetic Shifts.

Adult rattlesnakes within genus Crotalus express one of two distinct venom phenotypes, type I (hemorrhagic) and type II (neurotoxic). In Costa Rican Central American rattlesnake, ontogenetic changes in the concentration of miRNAs modulate venom type II to type I transition. Venomics and venom gland transcriptome analyses showed that adult C. simus and C. tzabcan expressed intermediate patterns between type II and type I venoms, whereas C. culminatus had a canonical type I venom. Neonate/juvenile and adult Mexican rattlesnakes showed notable inter- and intraspecific variability in the number, type, abundance and ontogenetic shifts of the transcriptional and translational venom gland activities. These results support a role for miRNAs in the ontogenetic venom compositional changes in the three congeneric Mexican rattlesnakes. It is worth noting the finding of dual-action miRNAs, which silence the translation of neurotoxic heterodimeric PLA2 crotoxin and acidic PLA2 mRNAs while simultaneously up-regulating SVMP-targeting mRNAs. Dual transcriptional regulation potentially explains the existence of mutually exclusive crotoxin-rich (type-II) and SVMP-rich (type-I) venom phenotypic dichotomy among rattlesnakes. Our results support the hypothesis that alterations of the distribution of miRNAs, modulating the translational activity of venom gland toxin-encoding mRNAs in response to an external cue, may contribute to the mechanism generating adaptive venom variability.

No safety in the trees: Local and species-level adaptation of an arboreal squirrel to the venom of sympatric rattlesnakes.

Within some species, squirrels respond to variable selection from venomous snake predators by showing population-level variation in resistance, while between species, some rattlesnakes possess venom that is more effective at overcoming venom resistance in different species of squirrels. A functional evaluation of resistance variation to venom within and between species of squirrels and snakes can link resistance variation to its evolutionary causes across these different evolutionary scales. To do this, we compared the effectiveness of squirrel sera in inhibiting rattlesnake (Crotalus spp.) venom metalloproteinase activity between populations and between species to test for a response to local variation in selection from a single rattlesnake predator and for specialization of two resistant squirrel species to each of their distinct sympatric snake predators. We found that Timber Rattlesnake (Crotalus horridus) venom inhibition by Eastern gray squirrels (Sciurus carolinensis) is higher at a site where the rattlesnakes are present, which suggests selection may maintain venom resistance in populations separated by short distances. Next, we performed a reciprocal cross of venoms and sera from two rattlesnake and two squirrel species. This showed that squirrel resistance is lower when tested against venom from allopatric compared to sympatric rattlesnake species, demonstrating that squirrel inhibitors are specialized to sympatric venom and suggesting a tradeoff in terms of specialization to the venom of a specific species of rattlesnake predator. This pattern can be explained if inhibitors must recognize venom proteins and resistance evolution tracks venom evolution.

Early significant ontogenetic changes in snake venoms.

Snake venom plays a critical role in food acquisition, digestion, and defense. Venoms are known to change throughout the life of some snake species, but nothing is known about the venom composition of hatchling/neonate snakes prior to and just after their first shedding cycle, despite this being a critical time in the life of the snake. Using a cohort of Crotalus horridus and two cohorts of Crotalus adamanteus, we showed for the first time that snakes undergo significant changes in venom composition after the postnatal shedding event. The number of changes among cohorts ranged widely and there was wide variation in the direction of protein regulation, which appeared to be on a locus-specific level rather than protein-family level. These significant venom composition changes that take place in the first few weeks of life most likely play critical roles in venom economy and resource conservation and may partially explain the rare, post-birth maternal care found in some venomous species.

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.

Integrated "omics" profiling indicates that miRNAs are modulators of the ontogenetic venom composition shift in the Central American rattlesnake, Crotalus simus simus.

BACKGROUND: Understanding the processes that drive the evolution of snake venom is a topic of great research interest in molecular and evolutionary toxinology. Recent studies suggest that ontogenetic changes in venom composition are genetically controlled rather than environmentally induced. However, the molecular mechanisms underlying these changes remain elusive. Here we have explored the basis and level of regulation of the ontogenetic shift in the venom composition of the Central American rattlesnake, Crotalus s. simus using a combined proteomics and transcriptomics approach. RESULTS: Proteomic analysis showed that the ontogenetic shift in the venom composition of C. s. simus is essentially characterized by a gradual reduction in the expression of serine proteinases and PLA2 molecules, particularly crotoxin, a ß-neurotoxic heterodimeric PLA2, concominantly with an increment of PI and PIII metalloproteinases at age 9-18 months. Comparison of the transcriptional activity of the venom glands of neonate and adult C. s. simus specimens indicated that their transcriptomes exhibit indistinguisable toxin family profiles, suggesting that the elusive mechanism by which shared transcriptomes generate divergent venom phenotypes may operate post-transcriptionally. Specifically, miRNAs with frequency count of 1000 or greater exhibited an uneven distribution between the newborn and adult datasets. Of note, 590 copies of a miRNA targeting crotoxin B-subunit was exclusively found in the transcriptome of the adult snake, whereas 1185 copies of a miRNA complementary to a PIII-SVMP mRNA was uniquely present in the newborn dataset. These results support the view that age-dependent changes in the concentration of miRNA modulating the transition from a crotoxin-rich to a SVMP-rich venom from birth through adulthood can potentially explain what is observed in the proteomic analysis of the ontogenetic changes in the venom composition of C. s. simus. CONCLUSIONS: Existing snake venom toxins are the result of early recruitment events in the Toxicofera clade of reptiles by which ordinary genes were duplicated, and the new genes selectively expressed in the venom gland and amplified to multigene families with extensive neofunctionalization throughout the approximately 112-125 million years of ophidian evolution. Our findings support the view that understanding the phenotypic diversity of snake venoms requires a deep knowledge of the mechanisms regulating the transcriptional and translational activity of the venom gland. Our results suggest a functional role for miRNAs. The impact of specific miRNAs in the modulation of venom composition, and the integration of the mechanisms responsible for the generation of these miRNAs in the evolutionary landscape of the snake's venom gland, are further challenges for future research.

Ablation of the ability to control the right-to-left cardiac shunt does not affect oxygen uptake, specific dynamic action or growth in the rattlesnake Crotalus durissus.

The morphologically undivided ventricle of the heart in non-crocodilian reptiles permits the mixing of oxygen-rich blood returning from the lungs and oxygen-poor blood from the systemic circulation. A possible functional significance for this intra-cardiac shunt has been debated for almost a century. Unilateral left vagotomy rendered the single effective pulmonary artery of the South American rattlesnake, Crotalus durissus, unable to adjust the magnitude of blood flow to the lung. The higher constant perfusion of the lung circulation and the incapability of adjusting the right-left shunt in left-denervated snakes persisted over time, providing a unique model for investigation of the long-term consequences of cardiac shunting in a squamate. Oxygen uptake recorded at rest and during spontaneous and forced activity was not affected by removing control of the cardiac shunt. Furthermore, metabolic rate and energetic balance during the post-prandial metabolic increment, plus the food conversion efficiency and growth rate, were all similarly unaffected. These results show that control of cardiac shunting is not associated with a clear functional advantage in adjusting metabolic rate, effectiveness of digestion or growth rates.

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.

Seasonal variation in hormonal responses of timber rattlesnakes (Crotalus horridus) to reproductive and environmental stressors.

Data addressing adrenocortical modulation across taxonomic groups are limited, especially with regard to how female reproductive condition influences the sensitivity of the hypothalamus-pituitary-adrenal axis. We investigated seasonal and reproductive variation in basal and stress-induced hormone profiles in a population of free-ranging timber rattlesnakes (Crotalus horridus) in north-central Pennsylvania during spring (i.e., May), summer (i.e., July), and early fall (i.e., September). Baseline corticosterone concentrations varied seasonally and were significantly lower during the summer sampling period in July. We observed a significant negative relationship between baseline corticosterone and testosterone in male snakes, while baseline corticosterone and estradiol tended to be positively correlated in females. Treatment of snakes with 1 h of capture stress significantly increased corticosterone across all seasons. However, there was a significant interaction between corticosterone responses to capture stress and season, suggesting that adrenocortical function is modulated seasonally. Because elevated corticosterone may be associated with reproduction, we asked whether hormonal stress responses vary with female reproductive condition. Although sample sizes are low, reproductive snakes had significantly higher baseline and stress-induced corticosterone concentrations than non-reproductive or post-parturient females. Further, despite similar baseline corticosterone concentrations between non-reproductive and post-parturient rattlesnakes, post-parturient females responded to capture stress with a significantly higher increase in corticosterone. Collectively, these data suggest that the sensitivity of the hypothalamus-pituitary-adrenal axis varies both seasonally and with changing reproductive states.

Structural and biological characterization of two crotamine isoforms IV-2 and IV-3 isolated from the Crotalus durissus cumanensis venom.

In this work, we isolated the two new crotamine isoforms from the Crotalus durissus cumanensis rattlesnake venom and its "in vitro" neurotoxic, myotoxic and lethality (DL(50)) intracerebroventricular (i.c.v.) effects were characterized. These proteins were named IV-2 and IV-3 and were purified by combination of two chromatographic steps on molecular exclusion chromatography on Superdex 75 and reverse phase HPLC (mu-Bondapack C18). The molecular mass of the crotamine isoforms was 4905.96 Da for isoform IV-2 and 4956.97 Da for IV-3 and, as determined by mass spectrometry, and both contained six Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of both isoforms. The positions of five sequenced tryptic peptides, including the N-terminal of the isoform IV-2 and four from isoform IV-3 were deduced by comparison with a homologous protein from the crotamine family. The isoforms IV-2 and IV-3 had a sequence of amino acids of 42 amino acid residues IV-2: YKRCHIKGGH CFPKEKLICI PPSSDIGKMD CPWKRKCCKK RS and pI value 9.54 and IV-3: YKQCHKKGGH CFPKEVLICI PPSSDFGKMD CRWKRKCCKK RS with a pI value of 9.54. This protein showed high molecular amino acid sequence identity with other crotamine-like proteins from Crotalus durissus terrificus. These new crotamine isoforms induced potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and potent myotoxic effect. In mice, both isoforms induced myonecrosis, upon intramuscular or subcutaneous injections. These activities were modulated by the presence of positively charged amino acid residues. The LD(50) of isoform IV-2 was 0.07 mg/kg and isoform IV-3 was 0.06 mg/kg the animal weight, by i.c.v. route.

Expression of maternal isotopes in offspring: implications for interpreting ontogenetic shifts in isotopic composition of consumer tissues.

When evaluating ontogenetic shifts in isotopic composition of consumer tissues within the context of a dietary analysis, the isotopic starting point of consumers in the population should not be ignored. Neonate isotopic composition may be different from that of juveniles and adults; in general, neonate tissues are built from maternal resources rather than food resources. Thus, the range of isotope values observed within a population of consumers may be significantly impacted by consumer isotope ratios at birth. Long-term goals of my research involve the use of stable isotopes to assess the role diet plays in driving population level differences in life history and demography observed among three pigmy rattlesnake (Sistrurus miliarius; family Viperidae) populations. For meaningful interpretation of field-collected data, it was important to determine starting (i.e., at birth) isotopic compositions of rattlesnakes from the study populations. We quantified isotopic composition of pregnant pigmy rattlesnake scale tissue, isotopic composition of neonate scale tissue and the degree that neonate scale tissue isotopic composition reflected the isotopic composition of maternal scale tissue. Collectively, neonate isotopic composition was highly variable among-litters; average litter delta(13)C values spanned 7 per thousand and average litter delta(15)N values spanned 2.8 per thousand. Over 95 % of the variation in offspring isotopic composition was expressed among litters. Thus, high levels of among-litter isotopic variation were largely due to the retention of a maternal signal. Results of the enclosure study suggest that highly variable isotopic signatures in young animals within field populations could easily reflect the retention of a maternal signal rather than differences in resource utilisation among younger snakes.

The ontogeny of contractile performance and metabolic capacity in a high-frequency muscle.

High-performance muscles such as the shaker muscles in the tails of western diamond-backed rattlesnakes (Crotalus atrox) are excellent systems for studying the relationship between contractile performance and metabolic capacity. We observed that shaker muscle contraction frequency increases dramatically with growth in small individuals but then declines gradually in large individuals. We tested whether metabolic capacity changed with performance, using shaker muscle contraction frequency as an indicator of performance and maximal activities of citrate synthase and lactate dehydrogenase as indicators of aerobic and anaerobic capacities, respectively. Contraction frequency increased 20-fold in 20-100-g individuals but then declined by approximately 30% in individuals approaching 1,000 g. Mass-independent aerobic capacity was positively correlated with contractile performance, whereas mass-independent anaerobic capacity was slightly but negatively correlated with performance; body mass was not correlated with performance. Rattle mass increased faster than the ability to generate force. Early in ontogeny, shaker muscle performance appears to be limited by aerobic capacity, but later performance becomes limited equally by aerobic capacity and the mechanical constraint of moving a larger mass without proportionally thicker muscles. This high-performance muscle appears to shift during ontogeny from a metabolic constraint to combined metabolic and mechanical constraints.

Sexual size dimorphism and growth plasticity in snakes: an experiment on the Western Diamond-backed Rattlesnake (Crotalus atrox).

We conducted an experiment to examine the effects of sex and food intake on growth, mass gain, and attainment of sexual maturity in Western Diamond-backed Rattlesnakes (Crotalus atrox). We also measured testosterone levels to determine whether testosterone might be involved in the male-biased sexual size dimorphism observed in this species. We collected neonate rattlesnakes and raised them in the laboratory for 2 years on either a high-intake diet (fed one mouse per week) or a low-intake diet (fed one mouse every 3 weeks). High-intake snakes grew and gained mass more rapidly than low-intake snakes, but males did not grow or gain mass more rapidly than females in either treatment group. High-intake snakes attained reproductive maturity earlier than low-intake snakes, indicating that size, not age, is the critical determinant of reproductive maturity. Males had higher levels of testosterone than females but did not grow more quickly, suggesting that testosterone may not affect growth in this species and may therefore not be the proximate determinant of sexual size dimorphism.

Effects of food supplementation on the physiological ecology of female Western diamond-backed rattlesnakes (Crotalus atrox).

Food availability is an important factor in the life histories of organisms because it is often limiting and thus can affect growth, mass change, reproduction, and behaviors such as thermoregulation, locomotion, and mating. Experimental studies in natural settings allow researchers to examine the effects of food on these parameters while animals are free to behave naturally. The wide variation among organisms in energy demands and among environmental food resources suggest that responses to changes in food availability may vary among organisms. Since most supplemental feeding field experiments have been conducted on species with high energy demands, we conducted a supplemental feeding study on free-ranging, female Western diamond-backed rattlesnakes (Crotalus atrox), a species with low energy demands and infrequent reproductive investment. Snakes were offered thawed rodents 1-4 times per week. Over two active seasons, we collected data on surface activity, home range size, growth, mass change, and reproduction of supplementally fed and control snakes. Fed and control snakes did not differ in surface activity levels (proportion of time encountered above versus below ground) or home range size. Fed snakes grew and gained mass faster, and had a dramatically higher occurrence of reproduction than control snakes. Also, fed snakes were in better body condition following reproduction than snakes that were not fed. However, litter characteristics such as offspring number and size were not increased by feeding, suggesting that these characteristics may be fixed. These data experimentally demonstrate that food availability can directly impact some life history traits (i.e., growth and reproduction for C. atrox), but not others (i.e., surface activity and home range size for C. atrox). The relationship between food availability and life history traits is affected in a complex way by ecological traits and physiological constraints, and thus interspecific variation in this relationship is likely to be high.

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.