Sexually mediated phenotypic variation within and between sexes as a continuum structured by ecology: The mosaic nature of skeletal variation across body regions in Threespine stickleback (Gasterosteus aculeatus L.).

Ecological character displacement between the sexes, and sexual selection, integrate into a convergent set of factors that produce sexual variation. Ecologically modulated, sexually mediated variation within and between sexes may be a major contributor to the amount of total variation that selection can act on in species. Threespine stickleback (Gasterosteus aculeatus) display rapid adaptive responses and sexual variation in many phenotypic traits. We examined phenotypic variation in the skull, pectoral and pelvic girdles of threespine stickleback from two freshwater and two coastal marine sites on the Sunshine Coast of British Columbia, Canada, using an approach that avoids a priori assumptions about bimodal patterns of variation. We quantified shape and size of the cranial, pectoral and pelvic regions of sticklebacks in marine and freshwater habitats using 3D geometric morphometrics and an index of sexually mediated variation. We show that the expression of phenotypic variation is structured in part by the effects of both habitat marine vs freshwater and the effects of individual sites within each habitat. Relative size exerts variable influence, and patterns of phenotypic variation associated with sex vary among body regions. This fine-grained quantification of sexually mediated variation in the context of habitat difference and different anatomical structures indicates a complex relationship between genetically inferred sex and environmental factors, demonstrating that the interplay between shared genetic background and sexually mediated, ecologically based selective pressures structures the phenotypic expression of complex traits.

From panic to pedagogy: Using online active learning to promote inclusive instruction in ecology and evolutionary biology courses and beyond.

The rapid shift to online teaching in spring 2020 meant most of us were teaching in panic mode. As we move forward with course planning for fall and beyond, we can invest more time and energy into improving the online experience for our students. We advocate that instructors use inclusive teaching practices, specifically through active learning, in their online classes. Incorporating pedagogical practices that work to maximize active and inclusive teaching concepts will be beneficial for all students, and especially those from minoritized or underserved groups. Like many STEM fields, Ecology and Evolution shows achievement gaps and faces a leaky pipeline issue for students from groups traditionally underserved in science. Making online classes both active and inclusive will aid student learning and will also help students feel more connected to their learning, their peers, and their campus. This approach will likely help with performance, retention, and persistence of students. In this paper, we offer broadly applicable strategies and techniques that weave together active and inclusive teaching practices. We challenge instructors to commit to making small changes as a first step to more inclusive teaching in ecology and evolutionary biology courses.

Mitochondrial haplotypes are not associated with mice selectively bred for high voluntary wheel running.

Mitochondrial haplotypes have been associated with human and rodent phenotypes, including nonshivering thermogenesis capacity, learning capability, and disease risk. Although the mammalian mitochondrial D-loop is highly polymorphic, D-loops in laboratory mice are identical, and variation occurs elsewhere mainly between nucleotides 9820 and 9830. Part of this region codes for the tRNAArg gene and is associated with mitochondrial densities and number of mtDNA copies. We hypothesized that the capacity for high levels of voluntary wheel-running behavior would be associated with mitochondrial haplotype. Here, we analyzed the mtDNA polymorphic region in mice from each of four replicate lines selectively bred for 54 generations for high voluntary wheel running (HR) and from four control lines (Control) randomly bred for 54 generations. Sequencing the polymorphic region revealed a variable number of adenine repeats. Single nucleotide polymorphisms (SNPs) varied from 2 to 3 adenine insertions, resulting in three haplotypes. We found significant genetic differentiations between the HR and Control groups (Fst = 0.779, p ≤ 0.0001), as well as among the replicate lines of mice within groups (Fsc = 0.757, p ≤ 0.0001). Haplotypes, however, were not strongly associated with voluntary wheel running (revolutions run per day), nor with either body mass or litter size. This system provides a useful experimental model to dissect the physiological processes linking mitochondrial, genomic SNPs, epigenetics, or nuclear-mitochondrial cross-talk to exercise activity.

Preference for Western diet coadapts in High Runner mice and affects voluntary exercise and spontaneous physical activity in a genotype-dependent manner.

Do animals evolve (coadapt) to choose diets that positively affect their performance abilities? We addressed this question from a microevolutionary perspective by examining preference for Western diet (WD: high in fat and sugar, but lower in protein) versus standard rodent chow in adults of both sexes from 4 lines of mice selectively bred for high levels of voluntary wheel running (High Runner or HR lines) and 4 non-selected control (C) lines. We also assessed whether food preference or substitution affects physical activity (wheel running and/or spontaneous physical activity [SPA] in the attached home cages). In experiment 1 (generation 56), mice were given 6days of wheel acclimation (as is used routinely to pick breeders in the selection experiment) prior to a 2-day food choice trial. In experiment 2 (generation 56), 17days of wheel acclimation allowed mice to reach a stable level of daily running, followed by a 7-day food-choice trial. In experiment 3 (generation 58), mice had 6days of wheel acclimation with standard chow, after which half were switched to WD for two days. In experiment 1, WD was highly preferred by all mice, with somewhat greater preference in male C mice. In experiment 2, wheel running increased and SPA decreased continuously for the first 14days of adult wheel testing, followed by 3-day plateaus in both. During the subsequent 7-day food choice trial, HR mice of both sexes preferred WD significantly more than did C mice; moreover, wheel running increased in all groups except males from C lines, with the increase being significantly greater in HR than C, while SPA declined further in all groups. In experiment 3, the effect of being switched to WD depended on both linetype and sex. On standard chow, only HR females showed a significant change in wheel running during nights 7+8, increasing by 10%. In contrast, when switched to WD, C females (+28%), HR females (+33%), and HR males (+10%) all significantly increased their daily wheel-running distances. Our results show for the first time that dietary preferences can coadapt in response to selection on activity levels.

Effects of early-onset voluntary exercise on adult physical activity and associated phenotypes in mice.

The purpose of this study was to evaluate the effects of early-life exercise on adult physical activity (wheel running, home-cage activity), body mass, food consumption, and circulating leptin levels in males from four replicate lines of mice selectively bred for high voluntary wheel running (High Runner or HR) and their four non-selected control (C) lines. Half of the mice were given wheel access shortly after weaning for three consecutive weeks. Wheel access was then removed for 52 days, followed by two weeks of adult wheel access for all mice. A blood sample taken prior to adult wheel testing was analyzed for circulating leptin concentration. Early-life wheel access significantly increased adult voluntary exercise on wheels during the first week of the second period of wheel access, for both HR and C mice, and HR ran more than C mice. During this same time period, activity in the home cages was not affected by early-age wheel access, and did not differ statistically between HR and C mice. Throughout the study, all mice with early wheel access had lower body masses than their sedentary counterparts, and HR mice had lower body masses than C mice. With wheel access, HR mice also ate significantly more than C mice. Early-life wheel access increased plasma leptin levels (adjusted statistically for fat-pad mass as a covariate) in C mice, but decreased them in HR mice. At sacrifice, early-life exercise had no statistically significant effects on visceral fat pad, heart (ventricle), liver or spleen masses (all adjusted statistically for variation in body mass). Results support the hypothesis that early-age exercise in mice can have at least transitory positive effects on adult levels of voluntary exercise, in addition to reducing body mass, and may be relevant for the public policy debates concerning the importance of physical education for children.

Effects of voluntary exercise on spontaneous physical activity and food consumption in mice: Results from an artificial selection experiment.

We evaluated the effect of voluntary exercise on spontaneous physical activity (SPA) and food consumption in mice from 4 replicate lines bred for 57 generations for high voluntary wheel running (HR) and from 4 non-selected control (C) lines. Beginning at ~24 days of age, mice were housed in standard cages or in cages with attached wheels. Wheel activity and SPA were monitored in 1-min intervals. Data from the 8th week of the experiment were analyzed because mice were sexually mature and had plateaued in body mass, weekly wheel running distance, SPA, and food consumption. Body mass, length, and masses of the retroperitoneal fat pad, liver, and heart were recorded after the 13th week. SPA of both HR and C mice decreased with wheel access, due to reductions in both duration and average intensity of SPA. However, total activity duration (SPA+wheel running; min/day) was ~1/3 greater when mice were housed with wheels, and food consumption was significantly increased. Overall, food consumption in both HR and C mice was more strongly affected by wheel running than by SPA. Duration of wheel running had a stronger effect than average speed, but the opposite was true for SPA. With body mass as a covariate, chronic wheel access significantly reduced fat pad mass and increased heart mass in both HR and C mice. Given that both HR and C mice housed with wheels had increased food consumption, the energetic cost of wheel running was not fully compensated by concomitant reductions in SPA. The experiment demonstrates that both duration and intensity of both wheel running and SPA were significant predictors of food consumption. This sort of detailed analysis of the effects of different aspects of physical activity on food consumption has not previously been reported for a non-human animal, and it sets the stage for longitudinal examination of energy balance and its components in rodent models.

Shape-shift: semicircular canal morphology responds to selective breeding for increased locomotor activity.

Variation in semicircular canal morphology correlates with locomotor agility among species of mammals. An experimental evolutionary mouse model was used to test the hypotheses that semicircular canal morphology (1) evolves in response to selective breeding for increased locomotor activity, (2) exhibits phenotypic plasticity in response to early-onset chronic exercise, and (3) is unique in individuals possessing the minimuscle phenotype. We examined responses in canal morphology to prolonged wheel access and selection in laboratory mice from four replicate lines bred for high voluntary wheel-running (HR) and four nonselected control (C) lines. Linear measurements and a suite of 3D landmarks were obtained from 3D reconstructions of µCT-scanned mouse crania (µCT is microcomputed tomography). Body mass was smaller in HR than C mice and was a significant predictor of both radius of curvature and 3D canal shape. Controlling for body mass, radius of curvature did not differ statistically between HR and C mice, but semicircular canal shape did. Neither chronic wheel access nor minimuscle affected radius of curvature or canal shape These findings suggest that semicircular canal morphology is responsive to evolutionary changes in locomotor behavior, but the pattern of response is potentially different in small- versus large-bodied species.

Immune response to a Trichinella spiralis infection in house mice from lines selectively bred for high voluntary wheel running.

Four lines of mice bred for high voluntary wheel running (HR lines) have high baseline circulating corticosterone levels and increased daily energy expenditure as compared with four non-selected control (C) lines. High corticosterone may suppress immune function and competing energy demands may limit ability to mount an immune response. We hypothesized that HR mice have a reduced immune response and therefore a decreased ability to fight an infection by Trichinella spiralis, an ecologically relevant nematode common in mammals. Infections have an acute, intestinal phase while the nematode is migrating, reproducing and traveling throughout the bloodstream, followed by a chronic phase with larvae encysted in muscles. Adult males (generation 55 of the selection experiment) were sham-infected or infected by oral gavage with ~300 J1 T. spiralis larvae. During the chronic phase of infection, mice were given wheel access for 6 days, followed by 2 days of maximum aerobic performance trials. Two weeks post-infection, infected HR had significantly lower circulating immunoglobulin E levels compared with infected C mice. However, we found no statistical difference between infected HR and C mice in numbers of encysted larvae within the diaphragm. As expected, both voluntary running and maximum aerobic performance were significantly higher in HR mice and lower in infected mice, with no line type-by-infection interactions. Results complement those of previous studies suggesting decreased locomotor abilities during the chronic phase of T. spiralis infection. However, despite reduced antibody production, breeding for high voluntary wheel exercise does not appear to have a substantial negative impact on general humoral function.

Do mice bred selectively for high locomotor activity have a greater reliance on lipids to power submaximal aerobic exercise?

Patterns of fuel use during locomotion are determined by exercise intensity and duration, and are remarkably similar across many mammalian taxa. However, as lipids have a high yield of ATP per mole and are stored in large quantities, their use should be favored in endurance-adapted animals. To examine the capacity for alteration or differential regulation of fuel-use patterns, we studied two lines of mice that had been selectively bred for high voluntary wheel running (HR), including one characterized by small hindlimb muscles (HR(mini)) and one without this phenotype (HR(normal)), as well as a nonselected control line. We evaluated: 1) maximal aerobic capacity (Vo(2 max)); 2) whole body fuel use during exercise by indirect calorimetry; 3) cardiac properties; and 4) many factors involved in regulating lipid use. HR mice achieved an increased Vo(2 max) compared with control mice, potentially in part due to HR cardiac capacities for metabolic fuel oxidation and the larger relative heart size of HR(mini) mice. HR mice also exhibited enhanced whole body lipid oxidation rates at 66% Vo(2 max), but HR(mini), HR(normal), and control mice did not differ in the proportional mix of fuels sustaining exercise (% total Vo(2)). However, HR(mini) gastrocnemius muscle had elevated fatty acid translocase (FAT/CD36) sarcolemmal protein and cellular mRNA, fatty acid binding protein (H-FABP) cytosolic protein, peroxisome proliferator-activated receptor (PPAR) α mRNA, and mass-specific activities of citrate synthase, ß-hydroxyacyl-CoA dehydrogenase, and hexokinase. Therefore, high-running mouse lines had whole body fuel oxidation rates commensurate with maximal aerobic capacity, despite notable differences in skeletal muscle metabolic phenotypes.

Within-lifetime trade-offs but evolutionary freedom for hormonal and immunological traits: evidence from mice bred for high voluntary exercise.

Chronic increases in circulating corticosterone (CORT) generally suppress immune function, but it is not known whether evolved increases necessarily have similar adverse effects. Moreover, the evolution of immune function might be constrained by the sharing of signaling molecules, such as CORT, across numerous physiological systems. Laboratory house mice (Mus domesticus Linnaeus) from four replicate lines selectively bred for high voluntary wheel running (HR lines) generally had baseline circulating CORT approximately twofold higher than in four non-selected control (C) lines. To test whether elevated baseline CORT suppresses the inflammatory response in HR mice, we injected females with lipopolysaccharide (LPS). All mice injected with LPS exhibited classic signs of an inflammatory response, including sickness behavior, loss of body mass, reduced locomotor activity (i.e. voluntary wheel running), enlarged spleens and livers, elevated hematocrit and elevated inflammatory cytokines. However, as compared with C mice, the inflammatory response was not suppressed in HR mice. Our results, and those of a previous study, suggest that selective breeding for high voluntary exercise has not altered immune function. They also suggest that the effects of evolved differences in baseline CORT levels may differ greatly from effects of environmental factors (often viewed as 'stressors') that alter baseline CORT during an individual's lifetime. In particular, evolved increases in circulating levels of 'stress hormones' are not necessarily associated with detrimental suppression of the inflammatory response, presumably as a result of correlated evolution of other physiological systems (counter-measures). Our results have important implications for the interpretation of elevated stress hormones and of immune indicators in natural populations.

The biological control of voluntary exercise, spontaneous physical activity and daily energy expenditure in relation to obesity: human and rodent perspectives.

Mammals expend energy in many ways, including basic cellular maintenance and repair, digestion, thermoregulation, locomotion, growth and reproduction. These processes can vary tremendously among species and individuals, potentially leading to large variation in daily energy expenditure (DEE). Locomotor energy costs can be substantial for large-bodied species and those with high-activity lifestyles. For humans in industrialized societies, locomotion necessary for daily activities is often relatively low, so it has been presumed that activity energy expenditure and DEE are lower than in our ancestors. Whether this is true and has contributed to a rise in obesity is controversial. In humans, much attention has centered on spontaneous physical activity (SPA) or non-exercise activity thermogenesis (NEAT), the latter sometimes defined so broadly as to include all energy expended due to activity, exclusive of volitional exercise. Given that most people in Western societies engage in little voluntary exercise, increasing NEAT may be an effective way to maintain DEE and combat overweight and obesity. One way to promote NEAT is to decrease the amount of time spent on sedentary behaviours (e.g. watching television). The effects of voluntary exercise on other components of physical activity are highly variable in humans, partly as a function of age, and have rarely been studied in rodents. However, most rodent studies indicate that food consumption increases in the presence of wheels; therefore, other aspects of physical activity are not reduced enough to compensate for the energetic cost of wheel running. Most rodent studies also show negative effects of wheel access on body fat, especially in males. Sedentary behaviours per se have not been studied in rodents in relation to obesity. Several lines of evidence demonstrate the important role of dopamine, in addition to other neural signaling networks (e.g. the endocannabinoid system), in the control of voluntary exercise. A largely separate literature points to a key role for orexins in SPA and NEAT. Brain reward centers are involved in both types of physical activities and eating behaviours, likely leading to complex interactions. Moreover, voluntary exercise and, possibly, eating can be addictive. A growing body of research considers the relationships between personality traits and physical activity, appetite, obesity and other aspects of physical and mental health. Future studies should explore the neurobiology, endocrinology and genetics of physical activity and sedentary behaviour by examining key brain areas, neurotransmitters and hormones involved in motivation, reward and/or the regulation of energy balance.

Sex-specific heterosis in line crosses of mice selectively bred for high locomotor activity.

When populations with similar histories of directional selection are crossed, their offspring may differ in mean phenotype as compared with the average for the parental populations, often exhibiting enhancement of the mean phenotype (termed heterosis or hybrid vigor). We tested for heterosis in a cross of two replicate lines of mice selectively bred for high voluntary wheel running for 53 generations. Mice were paired to produce four sets of F1 offspring: two purebred High Runner (HR) lines and the hybrid reciprocal crosses. The purebred HR showed statistically significant, sex-dependent differences in body mass, wheel revolutions, running duration, mean running speed, and (controlling for body mass) organ masses (heart ventricles, liver, spleen, triceps surae muscle). Hybrid males ran significantly more revolutions than the purebred males, mainly via increased running speeds, but hybrid females ran intermediate distances, durations, and speeds, as compared with the purebred females. In both sexes, ventricles were relatively smaller in hybrids as compared with purebred HR. Overall, our results demonstrate differential and sex-specific responses to selection in the two HR lines tested, implying divergent genetic architectures underlying high voluntary exercise.

Effects of parity on pelvic size and shape dimorphism in Mus.

The pelvis is a sexually dimorphic structure and although the causes of that dimorphism have long been studied, relatively little is known regarding the effects of partuitive events on the magnitude of that dimorphism. Here, we use a sample of Mus musculus domesticus to contrast dimorphism in body length and os coxae size and shape between males and parous and nulliparous females. We also test for correlations between relative litter size (L/M) and relative offspring size (O/M) with body length and os coxae size and shape in parous females. Males had greater body length than nulliparous females but were not different from parous females. Females as a whole had the largest os coxae, with parous females having the largest and males the smallest. Os coxae shape was also significantly different between groups and was most divergent between parous females and males than between nulliparous females and males. Os coxae shape differences between females are associated with differences in body length between females and O/M is correlated with os coxae shape in parous females such that females with the largest offspring have the most divergent shapes along the relative warp one axis. Pelvic shape differences between males and females were consistent with previous findings in other taxa which identify the pubo-ischial complex as the primary region of dimorphism.