Evolutionary convergence of muscle architecture in relation to locomotor ecology in snakes.

The epaxial muscles in snakes are responsible for locomotion and as such can be expected to show adaptations in species living in different environments. Here, we tested whether the structural units that comprise the superficial epaxial muscles (semispinalis-spinalis, SSP; longissimus dorsi, LD; iliocostalis, IC) were different in animals occupying similar habitats. To do so, we analyzed and compared the muscle architecture (mass, fiber length, and physiological cross-sectional area) of the superficial epaxial muscle segments in snakes that differ in their habitat use (e.g., arboreal, terrestrial, and aquatic). Our results showed that arboreal species have on average longer muscles and tendons spanning more segments likely important during gap bridging. Moreover, aquatic snakes show relatively heavier semispinalis-spinalis muscles with a greater cross-sectional area. The longissimus dorsi muscles also showed a greater cross-sectional area compared with terrestrial and especially arboreal snakes. Whereas the more strongly developed muscles in aquatic snakes are likely associated with the dense and viscous environment through which they move, the lighter muscles in arboreal snakes may provide an advantage when climbing. Future studies comparing other ecologies (e.g., burrowing snakes) and additional muscle units (e.g., multifidus; hypaxial muscles) are needed to better understand the structural features driving variation in locomotor performance and efficiency in snakes.

Measuring the 3D wake of swimming snakes (Natrix tessellata) using volumetric particle image velocimetry.

We describe a method for measuring the 3D vortical structures produced by an anguilliform swimmer using volumetric velocimetry. The wake of freely swimming dice snakes (Natrix tessellata) was quantified, revealing the creation of multiple vortices along the body of the snake due to its undulation. The 3D structure of the vortices generally consisted of paired vortex tubes, some of which were linked together to form a hairpin structure. The observations match predictions from computational fluid dynamic studies of other anguilliform swimmers. Quantitative measurements allowed us to study vortex circulation and size, and global kinetic energy of the flow, which varied with swimming speed, vortex topology and individual characteristics. Our findings provide a baseline for comparing wake structures of snakes with different morphologies and ecologies and investigating the energetic efficiency of anguilliform swimming.

Spot the odd one out: do snake pictures capture macaques' attention more than other predators?

Detecting and identifying predators quickly is key to survival. According to the Snake Detection Theory (SDT), snakes have been a substantive threat to primates for millions of years, so that dedicated visual skills were tuned to detect snakes in early primates. Past experiments confronted the SDT by measuring how fast primate subjects detected snake pictures among non-dangerous distractors (e.g., flowers), but did not include pictures of primates' other predators, such as carnivorans, raptors, and crocodilians. Here, we examined the detection abilities of N = 19 Tonkean macaques (Macaca tonkeana) and N = 6 rhesus macaques (Macaca mulatta) to spot different predators. By implementing an oddity task protocol, we recorded success rates and reaction times to locate a deviant picture among four pictures over more than 400,000 test trials. Pictures depicted a predator, a non-predator animal, or a simple geometric shape. The first task consisted of detecting a deviant picture among identical distractor pictures (discrimination) and the second task was designed to evaluate detection abilities of a deviant picture among different distractor pictures (categorization). The macaques detected pictures of geometric shapes better and faster than pictures of animals, and were better and faster at discriminating than categorizing. The macaques did not detect snakes better or faster than other animal categories. Overall, these results suggest that pictures of snakes do not capture visual attention more than other predators, questioning previous findings in favor of the SDT.

Plasticity matches phenotype to local conditions despite genetic homogeneity across 13 snake populations.

In a widespread species, a matching of phenotypic traits to local environmental optima is generally attributed to site-specific adaptation. However, the same matching can occur via adaptive plasticity, without requiring genetic differences among populations. Adult sea kraits (Laticauda saintgironsi) are highly philopatric to small islands, but the entire population within the Neo-Caledonian Lagoon is genetically homogeneous because females migrate to the mainland to lay their eggs at communal sites; recruits disperse before settling, mixing up alleles. Consequently, any matching between local environments (e.g. prey sizes) and snake phenotypes (e.g. body sizes and relative jaw sizes (RJSs)) must be achieved via phenotypic plasticity rather than spatial heterogeneity in gene frequencies. We sampled 13 snake colonies spread along an approximately 200 km northwest-southeast gradient (n > 4500 individuals) to measure two morphological features that affect maximum ingestible prey size in gape-limited predators: body size and RJS. As proxies of habitat quality (HQ), we used protection status, fishing pressure and lagoon characteristics (lagoon width and distance of islands to the barrier reef). In both sexes, spatial variation in body sizes and RJSs was linked to HQ; albeit in different ways, consistent with sex-based divergences in foraging ecology. Strong spatial divergence in morphology among snake colonies, despite genetic homogeneity, supports the idea that phenotypic plasticity can facilitate speciation by creating multiple phenotypically distinct subpopulations shaped by their environment.

Combining Freehand Ultrasound-Based Indentation and Inverse Finite Element Modeling for the Identification of Hyperelastic Material Properties of Thigh Soft Tissues.

Finite element analysis (FEA) is a numerical modeling tool vastly employed in research facilities to analyze and predict load transmission between the human body and a medical device, such as a prosthesis or an exoskeleton. Yet, the use of finite element modeling (FEM) in a framework compatible with clinical constraints is hindered by, among others, heavy and time-consuming assessments of material properties. Ultrasound (U.S.) imaging opens new and unique opportunities for the assessment of in vivo material properties of soft tissues. Confident of these advances, a method combining a freehand U.S. probe and a force sensor was developed in order to compute the hyperelastic constitutive parameters of the soft tissues of the thigh in both relaxed (R) and contracted (C) muscles' configurations. Seven asymptomatic subjects were included for the experiment. Two operators in each configuration performed the acquisitions. Inverse FEM allowed for the optimization of an Ogden's hyperelastic constitutive model of soft tissues of the thigh in large displacement. The mean shear modulus identified for configurations R and C was, respectively, 3.2 ± 1.3 kPa and 13.7 ± 6.5 kPa. The mean alpha parameter identified for configurations R and C was, respectively, 10 ± 1 and 9 ± 4. An analysis of variance showed that the configuration had an effect on constitutive parameters but not on the operator.

Seasonal Variations in Femoral Gland Secretions Reveals some Unexpected Correlations Between Protein and Lipid Components in a Lacertid Lizard.

Animals modulate intraspecific signal shape and intensity, notably during reproductive periods. Signal variability typically follows a seasonal scheme, traceable through the expression of visual, acoustic, chemical and behavioral patterns. The chemical channel is particularly important in lizards, as demonstrated by well-developed epidermal glands in the cloacal region that secrete lipids and proteins recognized by conspecifics. In males, the seasonal pattern of gland activity is underpinned by variation of circulating androgens. Changes in the composition of lipid secretions convey information about the signaler's quality (e.g., size, immunity). Presumably, individual identity is associated with a protein signature present in the femoral secretions, but this has been poorly investigated. For the first time, we assessed the seasonal variability of the protein signal in relation to plasma testosterone level (T), glandular activity and the concentration of provitamin D3 in the lipid fraction. We sampled 174 male common wall lizards (Podarcis muralis) over the entire activity season. An elevation of T was observed one to two months before the secretion peak of lipids during the mating season; such expected delay between hormonal fluctuation and maximal physiological response fits well with the assumption that provitamin D3 indicates individual quality. One-dimensional electrophoretic analysis of proteins showed that gel bands were preserved over the season with an invariant region; a result in agreement with the hypothesis that proteins are stable identity signals. However, the relative intensity of bands varied markedly, synchronously with that of lipid secretion pattern. These variations of protein secretion suggest additional roles of proteins, an issue that requires further studies.

Spinopelvic sagittal alignment of patients with transfemoral amputation.

PURPOSE: This study aims to describe the spinopelvic sagittal alignment in transfemoral amputees (TFAs) from a radiologic study of the spine with a postural approach to better understand the high prevalence of low back pain (LBP) in this population. METHODS: TFAs underwent X-rays with 3-D reconstructions of the full spine and pelvis. Sagittal parameters were analyzed and compared to the literature. Differences between TFAs with and without LBP were also observed. RESULTS: Twelve subjects have been prospectively included (TFA-LBP group (n = 5) and TFA-NoP group (n = 7)). Four of the five subjects of the TFA-LBP group and two of the seven in TFAs-NoP group had an imbalanced sagittal posture, especially regarding the T9-tilt, significantly higher in the TFA-LBP group than in the TFA-NoP (p = 0.046). Eight subjects (6 TFA-NoP and 2 TFA-LBP) had abnormal low value of thoracic kyphosis (TK). Moreover, the mean angle of TK in the TFA-NoP group was lower than in the TFA-LBP group (p = 0.0511). CONCLUSION: In the considered sample, TFAs often present a sagittal imbalance. A low TK angle seems to be associated with the absence of LBP. It can be hypothesized that this compensatory mechanism of the sagittal imbalance is the most accessible in this population. This study emphasizes the importance of considering the sagittal balance of the pelvis and the spine in patients with a TFA to better understand the high prevalence of LBP in this population. It should be completed by the analysis of the spinopelvic balance and the lower limbs in 3D. These slides can be retrieved under Electronic Supplementary Material.

Real Time Estimation of the Pose of a Lower Limb Prosthesis from a Single Shank Mounted IMU.

The command of a microprocessor-controlled lower limb prosthesis classically relies on the gait mode recognition. Real time computation of the pose of the prosthesis (i.e., attitude and trajectory) is useful for the correct identification of these modes. In this paper, we present and evaluate an algorithm for the computation of the pose of a lower limb prosthesis, under the constraints of real time applications and limited computing resources. This algorithm uses a nonlinear complementary filter with a variable gain to estimate the attitude of the shank. The trajectory is then computed from the double integration of the accelerometer data corrected from the kinematics of a model of inverted pendulum rolling on a curved arc foot. The results of the proposed algorithm are evaluated against the optoelectronic measurements of walking trials of three people with transfemoral amputation. The root mean square error (RMSE) of the estimated attitude is around 3°, close to the Kalman-based algorithm results reported in similar conditions. The real time correction of the integration of the inertial measurement unit (IMU) acceleration decreases the trajectory error by a factor of 2.5 compared to its direct integration which will result in an improvement of the gait mode recognition.

When carapace governs size: variation among age classes and individuals in a free-ranging ectotherm with delayed maturity.

Juvenile growth strongly impacts life-history traits during adulthood. Yet, in juveniles with delayed maturity, elusiveness has hindered age-specific studies of growth, precluding any detailed research on its consequences later in life. Different complex growth patterns have been extracted from captive animals, suggesting species-specific trajectories occur in free-ranging animals. How pronounced are growth and body size variation (VBS) throughout a long-lived ectotherm's life? Is VBS constant among age classes prior to maturity, or do compensatory and/or cumulative effects driven by long-lived-animal-specific strategies create distinct VBS cohorts, to ensure survival? To tackle the issue, we modelled growth data from continuous and dense annual capture-mark-recapture sampling (5096 body measurements of 1134 free-ranging individuals) of both immature and mature, long-lived Hermann's tortoises. We analysed population, cohort, and individual-based growth and VBS. Growth ring inferred ages were cross validated with annual recaptures in 289 juveniles. Analyses unravelled an S-shaped growth curve and identified three age cohorts across which VBS increases in a step-wise manner. Neonate-specific constraints and compensatory effects seem to control VBS until 4 years of age, possibly promoting survival with size. Subsequently, a hardened carapace takes over and cumulative effects fuelled by faster growth progressively increase VBS. Whereas ungulates are in a hurry to attain adult size before growth ceases (minimizing VBS), indeterminately growing tortoises can shape individual asymptotic sizes even after growth decelerates. Tortoise size is clearly shaped by age-specific ecological constraints; interestingly, it is likely the carapace that conducts the strategy, rather than maturity per se.

Trace Element Concentrations in European Pond Turtles (Emys orbicularis) from Brenne Natural Park, France.

We assessed trace elements concentration in European pond turtle (Emys orbicularis) from Brenne Natural Park (France). We sampled road-killed turtles (N = 46) to measure the concentrations of 4 non-essential (Ag, Cd, Hg, and Pb) and 10 essential (As, Co, Cr, Cu, Fe, Mn, Ni, Se, V, and Zn) elements in muscle, skin, liver and claws. Body size or sex did not influence the concentrations of most elements; except for Hg (liver, skin and claws) and Zn (muscle) which increased with body size. We found relatively high concentrations of Hg and Zn, possibly linked to fish farming. This result deserves future investigations to evaluate possible ecotoxicological effects on E. orbicularis.

The Evolutionary Economics of Embryonic-Sac Fluids in Squamate Reptiles.

The parchment-shelled eggs of squamate reptiles take up substantial water from the nest environment, enabling the conversion of yolk into neonatal tissue and buffering the embryo against the possibility of subsequent dry weather. During development, increasing amounts of water are stored in the embryonic sacs (i.e., membranes around the embryo: amnion, allantois, and chorion). The evolution of viviparity (prolonged uterine retention of developing embryos) means that embryonic-sac fluid storage now imposes a cost (increased maternal burdening), confers less benefit (because the mother buffers fetal water balance), and introduces a potential conflict among uterine siblings (for access to finite water supplies). Our data on nine species of squamate reptiles and published information on three species show that the embryonic-sac fluids comprise around 33% of neonatal mass in viviparous species versus 94% in full-term eggs of oviparous squamates. Data on parturition in 149 vipers (Vipera aspis, a viviparous species) show that larger offspring store more fluids in their fetal sacs and that an increase in litter size is associated with a decrease in fluid-sac mass per offspring. Overall, the evolutionary transition from oviparity to viviparity may have substantially altered selective forces on offspring packaging and created competition among offspring for access to water reserves during embryonic development.

Seasonal variations of plasma testosterone among colour-morph common wall lizards (Podarcis muralis).

Sexual steroids influence reproductive behaviours and promote secondary sexual traits. In male lizards, increasing levels of testosterone (T) bolster conspicuous colouration, stimulate territoriality, and trigger antagonistic interactions among rivals. Moreover, in colour polymorphic species, reproductive strategy, aggressiveness and T levels can differ between morphs. Therefore, T level is considered as an important mechanism that regulates the expression of colour polymorphism and sexual behaviours of males. But in the polymorphic territorial wall lizard (Podarcis muralis), a lack of relationship between morphs and aggressiveness challenges the notion that T plays such a role. To examine this issue, we compared adult T levels among three colour morphs (white, yellow and red) through repeated sampling during the mating season. High T levels were observed at the onset of the mating season followed by a significant decrease, a pattern documented in other lizard species. Mean T levels did not differ among morphs. However, yellow males maintained significantly higher T levels over time and displayed a stronger subsequent decline. Overall, in this species, seasonal T patterns differ among morphs, not mean values. Previous studies revealed that T suppresses the immune response; suggesting that a strong initial investment promoted by high T levels may trade-off against immunity (maintenance). Further experimental investigations are required to clarify the relationship between T and reproductive effort in polymorphic species that exhibit complex temporal pattern of T levels.

Cross-Slope and Level Walking Strategies During Swing in Individuals With Lower Limb Amputation.

OBJECTIVE: To quantitatively analyze prosthetic limb swing phase gait strategies used to adapt to cross slopes compared with flat surfaces. DESIGN: Cross-sectional study. SETTING: Gait laboratory. PARTICIPANTS: A volunteer sample (N=49) of individuals with transfemoral amputation (n=17), individuals with transtibial amputation (n=15), and able-bodied individuals (n=17). INTERVENTIONS: Participants walked on flat and 6° (10%) inclined cross-slope surfaces at a self-selected walking speed. MAIN OUTCOME MEASURES: Gait speed, step width, sagittal plane kinematics (ankle, knee, hip) on the prosthetic side during swing (uphill limb) and on the contralateral side during stance (downhill limb), frontal plane pelvic kinematics on the prosthetic side during swing, contralateral side ankle power during stance, and timing of gait events. RESULTS: All groups reduced gait speed and downhill limb knee flexion during the stance phase. Able-bodied participants adjusted their uphill limb ankle flexion during the swing phase. Participants with lower limb amputation used additional adjustments during the swing phase of the prosthetic limb when positioned uphill on cross slopes. Transtibial amputee participants mainly adapted with increased flexion of the residual hip and knee joints. Transfemoral amputee participants primarily compensated using increased pelvic hiking and vaulting gait strategies. CONCLUSIONS: The swing phase of the uphill limb during cross-slope walking results in compensatory mechanisms that should be addressed during rehabilitation to gain confidence and reduce avoidance when encountering cross slopes in daily life.

Spatial variation in age structure among colonies of a marine snake: the influence of ectothermy.

Several tetrapod lineages that have evolved to exploit marine environments (e.g. seals, seabirds, sea kraits) continue to rely upon land for reproduction and, thus, form dense colonies on suitable islands. In birds and mammals (endotherms), the offspring cannot survive without their parents. Terrestrial colonies contain all age classes. In reptiles (ectotherms), this constraint is relaxed, because offspring are independent from birth. Hence, each age class has the potential to select sites with characteristics that favour them. Our studies of sea snakes (sea kraits) in the lagoon of New Caledonia reveal marked spatial heterogeneity in age structure among colonies. Sea krait colonies exhibit the endothermic 'seal-seabird' pattern (mixed-age classes within populations) only where the lagoon is narrow. Where the lagoon is wide, most snake colonies are comprised primarily of a single age cohort. Nurseries are located near the coast, adult colonies offshore and mixed colonies in-between. We suggest that ectothermy allows individuals to utilize habitats that are best suited to their own ecological requirements, a flexibility not available to endothermic marine taxa with obligate parental care.

Effect of water deprivation on baseline and stress-induced corticosterone levels in the Children's python (Antaresia childreni).

Corticosterone (CORT) secretion is influenced by endogenous factors (e.g., physiological status) and environmental stressors (e.g., ambient temperature). Heretofore, the impact of water deprivation on CORT plasma levels has not been thoroughly investigated. However, both baseline CORT and stress-induced CORT are expected to respond to water deprivation not only because of hydric stress per se, but also because CORT is an important mineralocorticoid in vertebrates. We assessed the effects of water deprivation on baseline CORT and stress-induced CORT, in Children's pythons (Antaresia childreni), a species that experiences seasonal droughts in natural conditions. We imposed a 52-day water deprivation on a group of unfed Children's pythons (i.e., water-deprived treatment) and provided water ad libitum to another group (i.e., control treatment). We examined body mass variations throughout the experiment, and baseline CORT and stress-induced CORT at the end of the treatments. Relative body mass loss averaged ~10% in pythons without water, a value 2 to 4 times higher compared to control snakes. Following re-exposition to water, pythons from the water-deprived treatment drank readily and abundantly and attained a body mass similar to pythons from the control treatment. Together, these results suggest a substantial dehydration as a consequence of water deprivation. Interestingly, stress-induced but not baseline CORT level was significantly higher in water-deprived snakes, suggesting that baseline CORT might not respond to this degree of dehydration. Therefore, possible mineralocorticoid role of CORT needs to be clarified in snakes. Because dehydration usually induces adjustments (reduced movements, lowered body temperature) to limit water loss, and decreases locomotor performances, elevated stress-induced CORT in water-deprived snakes might therefore compensate for altered locomotor performances. Future studies should test this hypothesis.

Reduce, Replace, Refine: Determining A Posteriori Reference Intervals for Biochemistry in Hermann's Tortoise (Testudo hermanni).

Biochemical and hematological analyses are important for the assessment of animal health. However, for most wild species their use is hindered by the scarcity of reliable reference intervals. Indeed, collecting body fluids (e.g., blood, urine) in free-ranging animals is often technically challenging. Further, sampling many individuals would be essential to consider major sources of variations, such as species, populations, sex, age, and seasons. One alternative, according to the reduction, refinement, and replacement framework, is to establish reference intervals a posteriori using literature survey and unpublished data. We produced reference intervals for free-ranging Hermann's tortoises (Testudo hermanni), using analyses performed on blood samples collected in previous studies and conservation programs conducted in the field between 2010 and 2016 in southern France (n=195 individuals). Thirteen parameters were analyzed: packed-cell volume, blood concentrations of corticosterone, testosterone, glycemia, cholesterol, triglyceride, urea, uric acid, calcium, sodium, potassium, asparagine aminotransferase (AST), and alanine aminotransferases (ALT). Reference intervals for subgroups defined by sex and season were relevant for corticosterone, triglyceride, and calcium (sex) and cholesterol (season). Comparing our results with those obtained in captive individuals in Germany, except for urea and AST levels the intervals from both free-ranging versus captive tortoises were similar, suggesting that reference intervals established from captive individuals may be suitable for free-ranging populations in this species.

Effect of the ischial support on muscle force estimation during transfemoral walking.

BACKGROUND: Transmission of loads between the prosthetic socket and the residual limb is critical for the comfort and walking ability of people with transfemoral amputation. This transmission is mainly determined by the socket tightening, muscle forces, and socket ischial support. However, numerical investigations of the amputated gait, using modeling approaches such as MusculoSkeletal (MSK) modeling, ignore the weight-bearing role of the ischial support. This simplification may lead to errors in the muscle force estimation. OBJECTIVE: This study aims to propose a MSK model of the amputated gait that accounts for the interaction between the body and the ischial support for the estimation of the muscle forces of 13 subjects with unilateral transfemoral amputation. METHODS: Contrary to previous studies on the amputated gait which ignored the interaction with the ischial support, here, the contact on the ischial support was included in the external loads acting on the pelvis in a MSK model of the amputated gait. RESULTS: Including the ischial support induced an increase in the activity of the main abductor muscles, while adductor muscles' activity was reduced. These results suggest that neglecting the interaction with the ischial support leads to erroneous muscle force distribution considering the gait of people with transfemoral amputation. Although subjects with various bone geometries, particularly femur lengths, were included in the study, similar results were obtained for all subjects. CONCLUSIONS: Eventually, the estimation of muscle forces from MSK models could be used in combination with finite element models to provide quantitative data for the design of prosthetic sockets.

Shelter availability, stress level and digestive performance in the aspic viper.

The lack of shelter can perturb behaviors, increase stress level and thus alter physiological performance (e.g. digestive, immune or reproductive functions). Although intuitive, such potential impacts of lack of shelter remain poorly documented. We manipulated shelter availability and environmental and physiological variables (i.e. access to a heat source, predator attack, feeding status) in a viviparous snake, and assessed sun-basking behavior, digestive performance (i.e. digestive transit time, crude estimate of assimilation, regurgitation rate) and plasma corticosterone levels (a proxy of stress level). Shelter deprivation provoked a strong increase in sun-basking behavior and thus elevated body temperature, even in unfed individuals for which energy savings would have been otherwise beneficial. The lack of heat was detrimental to digestive performance; simulated predator attacks worsened the situation and entailed a further deterioration of digestion. The combination of the lack of shelter with cool ambient temperatures markedly elevated basal corticosterone level and was associated with low digestive performance. This hormonal effect was absent when only one negative factor was involved, suggesting a threshold response. Overall, our results revealed important non-linear cascading impacts of shelter availability on stress-hormone levels, behaviors and physiological performance. These results infer that shelter availability is important for laboratory studies, captive husbandry and possibly conservation plans.

Variations of natremia in sea kraits (Laticauda spp.) kept in seawater and fresh water.

Marine tetrapods evolved specific excretory structures (e.g. salt glands) that maintain salt concentrations within a narrow range of variation. However, recent investigations showed that in some lineages (sea snakes), individuals dehydrate in seawater and cannot equilibrate their hydromineral balance without access to fresh water. How these marine species cope with salt gain is therefore puzzling. We sampled two species of amphibious sea kraits (Laticauda saintgironsi and L. laticaudata) in the field. We also experimentally investigated patterns of salt regulation, specifically variations in natremia (plasma sodium) and body mass (net water flow), in individuals transferred first to fresh water and then to seawater. Our results show that free-ranging sea kraits display hypernatremia (up to 205mmol·l(-1)). Experimental data showed that natremia markedly decreased in snakes exposed to fresh water and increased when they were transferred to saltwater, thereby demonstrating a marked flexibility in their relation to environmental conditions. A literature survey indicated that all free-ranging marine snake species usually display hypernatremia despite having functional salt glands. Overall, sea snakes exhibit a marked tolerance to salt load compared to other marine tetrapods and apparently trigger substantial salt excretion only once natremia exceeds a high threshold. We hypothesise that this high tolerance significantly decreases energetic costs linked to salt gland functioning.