Gape drives regional variation in temporalis architectural dynamics in tufted capuchins.

Dynamic changes in jaw movements and bite forces depend on muscle architectural and neural factors that have rarely been compared within the same muscle. Here we investigate how regional muscle architecture dynamics-fascicle rotation, shortening, lengthening and architectural gear ratio (AGR)-vary during chewing across a functionally heterogeneous muscle. We evaluate whether timing in architecture dynamics relates to gape, food material properties and/or muscle activation. We also examine whether static estimates of temporalis fibre architecture track variation in dynamic architecture. Fascicle-level architecture dynamics were measured in three regions of the superficial temporalis of three adult tufted capuchins (Sapajus apella) using biplanar videoradiography and the XROMM workflow. Architecture dynamics data were paired with regional fine-wire electromyography data from four adult tufted capuchins. Gape accounted for most architectural change across the temporalis, but architectural dynamics varied between regions. Mechanically challenging foods were associated with lower AGRs in the anterior region. The timing of most dynamic architectural changes did not vary between regions and differed from regional variation in static architecture. Collectively these findings suggest that, when modelling temporalis muscle force production in extant and fossil primates, it is important to account for the effects of gape, regionalization and food material properties. This article is part of the theme issue 'Food processing and nutritional assimilation in animals'.

Measuring Effects of Dietary Fiber on the Murine Oral Microbiome with Enrichment of 16S rDNA Prior to Amplicon Synthesis.

The oral cavity houses a diverse consortium of microorganisms corresponding to specific microbial niches within the oral cavity. The complicated nature of sample collection limits the accuracy, reproducibility, and completeness of sample collection of the dentogingival microbiome. Moreover, large variability among human oral samples introduces inexorable confounds. Here, we introduce a method to study the dentogingival microbiome using a murine model that allows for greater control over experimental variability and permits collection of the dentogingival microbiome in an intact state and in its entirety.As an example of this approach, this chapter provides a workflow to explore the effect of dietary fiber consumption on the murine dentogingival microbiome . Mice are fed diets corresponding to Fiber, Sugar, Fiber + Sugar, and Control groups for 7 weeks. A whole-mandible extraction technique is described to isolate the mandibular dentogingival surfaces. 16S rRNA gene analysis is coupled with removal of unwanted host DNA amplification products to allow an investigation of the dental microbiome in the presence of increased fiber in terms of microbial taxonomic abundance and diversity.

Odd-nosed monkey scapular morphology converges on that of arm-swinging apes.

Odd-nosed monkeys 'arm-swing' more frequently than other colobines. They are therefore somewhat behaviorally analogous to atelines and apes. Scapular morphology regularly reflects locomotor mode, with both arm-swinging and climbing anthropoids showing similar characteristics, especially a mediolaterally narrow blade and cranially angled spine and glenoid. However, these traits are not expressed uniformly among anthropoids. Therefore, behavioral convergences in the odd-nosed taxa of Nasalis, Pygathrix, and Rhinopithecus with hominoids may not have resulted in similar structural convergences. We therefore used a broad sample of anthropoids to test how closely odd-nosed monkey scapulae resemble those of other arm-swinging primates. We used principal component analyses on size-corrected linear metrics and angles that reflect scapular size and shape in a broad sample of anthropoids. As in previous studies, our first component separated terrestrial and above-branch quadrupeds from clambering and arm-swinging taxa. On this axis, odd-nosed monkeys were closer than other colobines to modern apes and Ateles. All three odd-nosed genera retain glenoid orientations that are more typical of other colobines, but Pygathrix and Rhinopithecus are closer to hominoids than to other Asian colobines in mediolateral blade breadth, spine angle, and glenoid position. This suggests that scapular morphology of Pygathrix may reflect a significant reliance on arm-swinging and that the morphology of Rhinopithecus may reflect more reliance on general climbing. As 'arm-swinging' features are also found in taxa that only rarely arm-swing, we hypothesize that these features are also adaptive for scrambling and bridging in larger bodied anthropoids that use the fine-branch component of their arboreal niches.

Mouse hallucal metatarsal cross-sectional geometry in a simulated fine branch niche.

Mice raised in experimental habitats containing an artificial network of narrow "arboreal" supports frequently use hallucal grasps during locomotion. Therefore, mice in these experiments can be used to model a rudimentary form of arboreal locomotion in an animal without other morphological specializations for using a fine branch niche. This model would prove useful to better understand the origins of arboreal behaviors in mammals like primates. In this study, we examined if locomotion on these substrates influences the mid-diaphyseal cross-sectional geometry of mouse metatarsals. Thirty CD-1/ICR mice were raised in either arboreal (composed of elevated narrow branches of varying orientation) or terrestrial (flat ramps and walkways that are stratified) habitats from weaning (21 days) to adulthood (≥4 months). After experiments, the hallucal metatarsal (Mt1) and third metatarsal (Mt3) for each individual were isolated and micro-computed tomography (micro-CT) scans were obtained to calculate mid-shaft cross-sectional area and polar section modulus. Arboreal mice had Mt1s that were significantly more robust. Mt3 cross sections were not significantly different between groups. The arboreal group also exhibited a significantly greater Mt1/Mt3 ratio for both robusticity measures. We conclude that the hallucal metatarsal exhibits significant phenotypic plasticity in response to arboreal treatment due to habitual locomotion that uses a rudimentary hallucal grasp. Our results support the hypothesis that early adaptive stages of fine branch arboreality should be accompanied by a slightly more robust hallux associated with the biomechanical demands of this niche.

Plasticity in the cerebellum and primary somatosensory cortex relating to habitual and continuous slender branch climbing in laboratory mice (Mus musculus).

The origin of the mammalian order Primates is nested within a Euarchontan ancestry that was probably exploiting the fine branch arboreal niche in a facultative way. A putative transition into this habitat may have begun with a more generalized small-bodied mammal that lacked climbing specializations for grasping hands and feet. Here, we investigate whether mice exhibit central nervous system (CNS) plasticity associated with learning to grasp/climb proficiently. House mice were used to study phenotypic plasticity within the cerebellum and primary somatosensory cortex associated with the fine branch niche. This experimental treatment has previously been shown to influence skeletal plasticity in part because climb-training encourages tail use and facultative pedal grasping. The CNS necessary to coordinate and control these locomotor behaviors was investigated in a standard mouse model (N = 10 male CD-1/ICR mice), and plasticity was detected by histomorphometric and immunohistologic changes within the cerebellum and cerebrum. The climbing group had a significantly smaller relative granule cell layer in cerebellar lobule 1-3 than the control group (P < 0.10), but increased nerve growth factor immunoreactivity in white matter tracts of these lobules (P < 0.05). Qualitative observations in the primary somatosensory cortex revealed greater pyramidal/stellate cell counts in climbers. We suggest that coordinated tail and hindlimb learning within the arboreal milieu is facilitated by increased growth factor expression and neuronal alterations in the CNS. These findings suggest that mammals with a generalized Euarchontogliran body plan were capable of facultative pedal grasping and tail use so as to exploit the terminal branch niche.

The role of the sutures in biomechanical dynamic simulation of a macaque cranial finite element model: implications for the evolution of craniofacial form.

The global biomechanical impact of cranial sutures on the face and cranium during dynamic conditions is not well understood. It is hypothesized that sutures act as energy absorbers protecting skulls subjected to dynamic loads. This hypothesis predicts that sutures have a significant impact on global patterns of strain and cranial structural stiffness when analyzed using dynamic simulations; and that this global impact is influenced by suture material properties. In a finite element model developed from a juvenile Rhesus macaque cranium, five different sets of suture material properties for the zygomaticotemporal sutures were tested. The static and dynamic analyses produced similar results in terms of strain patterns and reaction forces, indicating that the zygomaticotemporal sutures have limited impact on global skull mechanics regardless of loading design. Contrary to the functional hypothesis tested in this study, the zygomaticotemporal sutures did not absorb significant amounts of energy during dynamic simulations regardless of loading speed. It is alternatively hypothesized that sutures are mechanically significant only insofar as they are weak points on the cranium that must be shielded from unduly high stresses so as not to disrupt vitally important growth processes. Thus, sutural and overall cranial form in some vertebrates may be optimized to minimize or otherwise modulate sutural stress and strain.

The global impact of sutures assessed in a finite element model of a macaque cranium.

The biomechanical significance of cranial sutures in primates is an open question because their global impact is unclear, and their material properties are difficult to measure. In this study, eight suture-bone functional units representing eight facial sutures were created in a finite element model of a monkey cranium. All the sutures were assumed to have identical isotropic linear elastic material behavior that varied in different modeling experiments, representing either fused or unfused sutures. The values of elastic moduli employed in these trials ranged over several orders of magnitude. Each model was evaluated under incisor, premolar, and molar biting conditions. Results demonstrate that skulls with unfused sutures permitted more deformations and experienced higher total strain energy. However, strain patterns remained relatively unaffected away from the suture sites, and bite reaction force was likewise barely affected. These findings suggest that suture elasticity does not substantially alter load paths through the macaque skull or its underlying rigid body kinematics. An implication is that, for the purposes of finite element analysis, omitting or fusing sutures is a reasonable modeling approximation for skulls with small suture volume fraction if the research objective is to observe general patterns of craniofacial biomechanics under static loading conditions. The manner in which suture morphology and ossification affect the mechanical integrity of skulls and their ontogeny and evolution awaits further investigation, and their viscoelastic properties call for dynamic simulations.

Mechanical impact of incisor loading on the primate midfacial skeleton and its relevance to human evolution.

The midfacial skeleton in the human lineage demonstrates a wide spectrum of variation that may be the consequence of different environmental and mechanical selective pressures. However, different facial configurations may develop under comparable selective regimes. For example, the Neanderthal high and projected face and the Inuit broad and flat face are hypothesized to be the consequence of (1) life in a cold climate, and (2) excessive paramasticatory stresses focused on the anterior dentition. In this study, the second of these two hypotheses is tested using finite element analyses of a monkey skull. Results indicate that incisor loading induces heavy stress in the anterior midface of macaques. Additional analyses using incremental increases in the anteroinferior tilt of the skull to simulate different magnitudes of facial projection revealed that comparable muscular force generates less stress in a less-projected face. However, the findings of our final analyses, which attempted to combine biting with the incisors and pulling with the hands, differed from the analyses that mimicked only incisor loading (without any sort of anterior pulling component). These findings suggest that shortening the face may be the most effective way to compensate for anterior dental loading but not necessarily offset the forces incurred when using the anterior dentition as a vice for various paramasticatory behaviors. Although Neanderthals may have frequently loaded their anterior dentition, countervailing selection pressures, such as the inclusion of tough foods in the diet that demanded molar grinding, may have selected for a longer face with a lower load- to lever-arm ratio.

Simulating the fine-branch arboreal niche and exercising mice to elicit above-branch quadrupedal grasping and climbing.

The fine-branch niche is a natural setting found among the slender vines and terminal branches of shrubs and tree canopies. In this study, the authors designed two simulations of this setting for laboratory mice. Their main goal was to model phenotypic plasticity in a small, clawed mammal, in order to better understand the effect of a thin-branch arboreal setting on musculoskeletal growth and behavior of these animals. The authors exposed mice to the smaller climbing setting for limited amounts of time (e.g., 30 min) and used the larger setting to permanently house another group of mice. Mice in both the limited and continuous climbing groups succeeded at quadrupedal climbing among a complex of thin branch segments. This led the authors to postulate that similarly sized pre-primates that lacked the unique features of today's primates could potentially have exploited this niche. The mice housed in the continuous model remained healthy and showed no signs of aggression, leading the authors to suggest that animal care personnel could use similar models as forms of enrichment for laboratory mice.

Cranial suture morphology and its relationship to diet in Cebus.

Cranial sutures are complex morphological structures. Four Cebus species (C. albifrons, C. apella, C. capucinus, C. olivaceus) are used here to test the hypothesis that sagittal suture complexity is enhanced in animals that eat materially challenging foods. These primates are ideal for such comparative studies because they are closely related and some are known to exhibit differences in the material properties of the foods they ingest and masticate. Specifically, Cebus apella is notable among members of this genus for ingesting food items of high toughness as well as consistently demonstrating a relatively robust cranial morphology. Consistent with previous studies, C. apella demonstrates significantly more robust mandibular and temporal fossa morphology. Also, C. apella possesses sagittal sutures that are more complex than congenerics. These data are used to support the hypothesis that cranial suture complexity is increased in response to consuming diets with more obdurate material properties. One interpretation of this hypothesis is that, compared to non-apelloids, total strain in the apelloid cranial suture connective tissue environment is elevated due to increased jaw muscle activity by increases in either force magnitudes or the number of chewing events. It is argued that greater masticatory function enhances the growth and modeling of cranial suture interdigitation. These data show that cranial suture complexity is one more hard tissue feature from the skull that might be used to inform hypotheses of dietary functional morphology.

Enlargement of the temporalis muscle and alterations in the lateral cranial vault.

The purpose of this study was to test the hypothesis that increased masticatory muscle accompanied morphologic changes in the temporal bone and squamosal suture. Ten mice deficient for the protein myostatin (Mstn -/-) had significantly increased skeletal muscle mass and were compared with nine controls (Mstn +/+). Variables measured include linear and areal metrics describing temporal size and temporal bone shape as well as the extent of the area of the squamosal suture that overlaps, or bevels, with parietal bones. Mstn-/- mice showed significantly larger temporalis muscles. Their temporal bones showed significantly decreased size as well as decreased beveling of the squamosal suture. These decreases were absolute as well as relative and were not restricted to either vertical or horizontal axes. The increased masticatory musculature of Myostatin-null mice had a shrinking effect on the temporal aspect of the cranium. These results are inconsistent with the interpretation that increased temporalis mass induces morphologic changes in temporal bone that compensate for putative increases in compressive forces transduced at this region. Rather than increase in the area of overlap between two calvarial bones, potential increase in biomechanical loading along the temporal squama led to a smaller bevel which would presumably weaken this joint. It is unclear why this is so. Either compressive forces are not anabolic to suture beveling or they do upregulate growth of the suture bevel, with compression not being the primary loading regime at this suture.

Alterations of temporalis muscle contractile force and histological content from the myostatin and Mdx deficient mouse.

Myostatin (GDF8) and dystrophin are critical molecules for muscle organisation. Myostatin is involved in regulating muscle growth and development, whereas dystrophin is part of the dystrophin-glycoprotein complex (DGC), which anchors the cytoskeleton to the sarcolemma. We examined temporalis muscle morphology and function in myostatin deficient and dystrophin deficient (Mdx) mice in order to determine how myostatin and dystrophin affect bite force and muscle fibre composition. Bite forces from 4-month-old myostatin-/-, dystrophin deficient (Mdx) and normal control mice were measured by load cell and field stimulation of the temporalis muscle. Tissue sections were stained with haemotoxylin and eosin (H&E) and the periodic acid-Schiff reaction (PAS) to assess morphology and fibre type differences. A positive relationship between bite force and muscle mass for both genetic models was observed. Both Mstn-/- and Mdx mice showed significant elevation in bite force and muscle mass. Histological examination revealed greater muscle fibre cross-sectional area variability in Mdx mice (ANOVA, F=5.6, P<0.01). Surprisingly, the Mstn-/- mice demonstrated a disproportionate increase in bite force at higher stimulation frequencies with comparison of regression lines for force-frequency data (ANOVA, F=3.46, P<0.07). Muscle fibre typing using a PAS staining technique revealed significantly more type IIx/b glycolytic muscle fibres in the Mstn-/- mice. Our results suggest that histopathologies associated with Mdx mice did not diminish gross temporalis structure or function, whilst the force-frequency changes associated with Mstn-/- mice were reflected in an elevation of type IIx/b fibres.

Effects of increased muscle mass on mouse sagittal suture morphology and mechanics.

The purpose of this study is to test predicted form-function relationships between cranial suture complexity and masticatory muscle mass and biomechanics in a mouse model. Specifically, to test the hypothesis that increased masticatory muscle mass increases sagittal suture complexity, we measured the fractal dimension (FD), temporalis mass, and temporalis bite force in myostatin-deficient (GDF8(-/-)) mice and wild-type CD-1 mice (all male, 6 months old). Myostatin is a negative regulator of muscle mass, and myostatin-deficient mice show a marked increase in muscle mass compared to normal mice. We predicted that increased sagittal suture complexity would decrease suture stiffness. The data presented here demonstrate that increased suture complexity (measured as FD) was observed in a hypermuscular mouse model (GDF8(-/-)) with significantly increased temporalis muscle mass and bite forces. Hypermuscular mice were also found to possess suture connective tissue that was less stiff (i.e., underwent more displacement before failure occurred) when loaded in tension. By decreasing stiffness, suture complexity apparently helps to dissipate mechanical loads within the cranium that are related to chewing. These results suggest that cranial suture connective tissue locally adapts to functional demands of the biomechanical suture environment. As such, cranial sutures provide a novel model for studies in connective tissue mechanotransduction.

Bone architecture and disc degeneration in the lumbar spine of mice lacking GDF-8 (myostatin).

GDF-8, also known as myostatin, is a member of the transforming growth factor-beta superfamily of secreted growth and differentiation factors that is expressed in vertebrate skeletal muscle. Myostatin functions as a negative regulator of skeletal muscle growth and myostatin null mice show a doubling of muscle mass compared to normal mice. We describe here morphology of the lumbar spine in myostatin knockout (Mstn(-/-)) mice using histological and densitometric techniques. The Mstn(-/-) mice examined in this study weigh approximately 10% more than controls (p<0.001) but the iliopsoas muscle is over 50% larger in the knockout mice than in wild-type mice (p<0.001). Peripheral quantitative computed tomography (pQCT) data from the fifth lumbar vertebra show that mice lacking myostatin have approximately 50% greater trabecular bone mineral density (p=0.001) and significantly greater cortical bone mineral content than normal mice. Toluidine blue staining of the intervertebral disc between L4-L5 reveals loss of proteoglycan staining in the hyaline end plates and inner annulus fibrosus of the knockout mice. Loss of cartilage staining in the caudal end plate of L4 is due to ossification of the end plate in the myostatin-deficient animals. Results from this study suggest that increased muscle mass in mice lacking myostatin is associated with increased bone mass as well as degenerative changes in the intervertebral disc.