Does Behavior Evolve First? Correlated Responses to Selection for Voluntary Wheel-Running Behavior in House Mice.

AbstractHow traits at multiple levels of biological organization evolve in a correlated fashion in response to directional selection is poorly understood, but two popular models are the very general "behavior evolves first" (BEF) hypothesis and the more specific "morphology-performance-behavior-fitness" (MPBF) paradigm. Both acknowledge that selection often acts relatively directly on behavior and that when behavior evolves, other traits will as well but most with some lag. However, this proposition is exceedingly difficult to test in nature. Therefore, we studied correlated responses in the high-runner (HR) mouse selection experiment, in which four replicate lines have been bred for voluntary wheel-running behavior and compared with four nonselected control (C) lines. We analyzed a wide range of traits measured at generations 20-24 (with a focus on new data from generation 22), coinciding with the point at which all HR lines were reaching selection limits (plateaus). Significance levels (226 P values) were compared across trait types by ANOVA, and we used the positive false discovery rate to control for multiple comparisons. This meta-analysis showed that, surprisingly, the measures of performance (including maximal oxygen consumption during forced exercise) showed no evidence of having diverged between the HR and C lines, nor did any of the life history traits (e.g., litter size), whereas body mass had responded (decreased) at least as strongly as wheel running. Overall, results suggest that the HR lines of mice had evolved primarily by changes in motivation rather than performance ability at the time they were reaching selection limits. In addition, neither the BEF model nor the MPBF model of hierarchical evolution provides a particularly good fit to the HR mouse selection experiment.

Effects of long-term voluntary wheel running and selective breeding for wheel running on femoral nutrient canals.

The nutrient artery provides ~50%-70% of the total blood volume to long bones in mammals. Studying the functional characteristics of this artery in vivo can be difficult and expensive, so most researchers have measured the nutrient foramen, an opening on the outer surface of the bone that served as the entry point for the nutrient artery during development and bone ossification. Others have measured the nutrient canal (i.e., the passage which the nutrient artery once occupied), given that the external dimensions of the foramen do not necessarily remain uniform from the periosteal surface to the medullary cavity. The nutrient canal, as an indicator of blood flow to long bones, has been proposed to provide a link to studying organismal activity (e.g., locomotor behavior) from skeletal morphology. However, although external loading from movement and activity causes skeletal remodeling, it is unclear whether it affects the size or configuration of nutrient canals. To investigate whether nutrient canals can exhibit phenotypic plasticity in response to physical activity, we studied a mouse model in which four replicate high runner (HR) lines have been selectively bred for high voluntary wheel-running behavior. The selection criterion is the average number of wheel revolutions on days 5 and 6 of a 6-day period of wheel access as young adults (~6-8 weeks old). An additional four lines are bred without selection to serve as controls (C). For this study, 100 female mice (half HR, half C) from generation 57 were split into an active group housed with wheels and a sedentary group housed without wheels for 12 weeks starting at ~24 days of age. Femurs were collected, soft tissues were removed, and femora were micro-computed tomography scanned at a resolution of 12 µm. We then imported these scans into AMIRA and created 3D models of femoral nutrient canals. We tested for evolved differences in various nutrient canal traits between HR and C mice, plastic changes resulting from chronic exercise, and the selection history-by-exercise interaction. We found few differences between the nutrient canals of HR versus C mice, or between the active and sedentary groups. We did find an interaction between selection history and voluntary exercise for the total number of nutrient canals per femur, in which wheel access increased the number of canals in C mice but decreased it in HR mice. Our results do not match those from an earlier study, conducted at generation 11, which was prior to the HR lines reaching selection limits for wheel running. The previous study found that mice from the HR lines had significantly larger total canal cross-sectional areas compared to those from C lines. However, this discrepancy is consistent with studies of other skeletal traits, which have found differences between HR and C mice to be somewhat inconsistent across generations, including the loss of some apparent adaptations with continued selective breeding after reaching a selection limit for wheel-running behavior.

Locomotor play behavior evolves by random genetic drift but not as a correlated response to selective breeding for high voluntary wheel-running behavior.

Locomotor play is vigorous and seemingly purposeless behavior, commonly observed in young mammals. It can be costly in terms of energy expenditure, increased injury risk, and predator exposure. The main hypothesized benefit of locomotor play is enhancement of neuromuscular development, with effects persisting into adulthood. We hypothesized that levels of locomotor play would have evolved as a correlated response to artificial selection for increased voluntary exercise behavior. We studied mice from 4 replicate lines bred for voluntary wheel running (High Runner or HR) at 6-8 weeks of age and four non-selected Control (C) lines. Mice were weaned at 21 days of age and play behavior was observed for generations 20 (22-24 days old), 68 (22-23 days old), and 93 (15 days old). We quantified locomotor play as (1) rapid, horizontally directed jerk-run sequences and (2) vertical "bouncing." We used focal sampling to continuously record behavior in cages containing 4-6 individuals during the first 2-3 h of the dark cycle. Observations were significantly repeatable between observers and days. A two-way, mixed-model simultaneously tested effects of linetype (HR vs. C), sex, and their interaction. Contrary to our hypothesis, HR and C lines did not differ in any generation, nor did we find sex differences. However, differences among the replicate HR lines and among the replicate C lines were detected, and may be attributed to the effects of random genetic drift (and possibly founder effects). Thus, play behavior did evolve in this selection experiment, but not as a correlated response to selection for voluntary exercise.

Selectively breeding for high voluntary physical activity in female mice does not bestow inherent characteristics that resemble eccentric remodeling of the heart, but the mini-muscle phenotype does.

Physical activity engagement results in a variety of positive health outcomes, including a reduction in cardiovascular disease risk partially due to eccentric remodeling of the heart. The purpose of this investigation was to determine if four replicate lines of High Runner mice that have been selectively bred for voluntary exercise on wheels have a cardiac phenotype that resembles the outcome of eccentric remodeling. Adult females (average age 55 days) from the 4 High Runner and 4 non-selected control lines were anaesthetized via vaporized isoflurane, then echocardiographic images were collected and analyzed for structural and functional differences. High Runner mice in general had lower ejection fractions compared to control mice lines (2-tailed p â€‹= â€‹0.023 6) and tended to have thicker walls of the anterior portion of the left ventricle (p â€‹= â€‹0.065). However, a subset of the High Runner individuals, termed mini-muscle mice, had greater ejection fraction (p â€‹= â€‹0.000 6), fractional shortening percentage (p â€‹< â€‹0.000 1), and ventricular mass at dissection (p â€‹< â€‹0.002 7 with body mass as a covariate) compared to non-mini muscle mice. Mice from replicate lines bred for high voluntary exercise did not all have inherent positive cardiac functional or structural characteristics, although a genetically unique subset of mini-muscle individuals did have greater functional cardiac characteristics, which in conjunction with their previously described peripheral aerobic enhancements (e.g., increased capillarity) would partially account for their increased V˙ O2max.

Selective breeding for high voluntary exercise in mice increases maximal (V̇O2,max) but not basal metabolic rate.

In general, sustained high rates of physical activity require a high maximal aerobic capacity (V̇O2,max), which may also necessitate a high basal aerobic metabolism (BMR), given that the two metabolic states are linked via shared organ systems, cellular properties and metabolic pathways. We tested the hypotheses that (a) selective breeding for high voluntary exercise in mice would elevate both V̇O2,max and BMR, and (b) these increases are accompanied by increases in the size of some internal organs (ventricle, triceps surae muscle, liver, kidney, spleen, lung, brain). We measured 72 females from generations 88 and 96 of an ongoing artificial selection experiment comprising four replicate High Runner (HR) lines bred for voluntary daily wheel-running distance and four non-selected control lines. With body mass as a covariate, HR lines as a group had significantly higher V̇O2,max (+13.6%, P<0.0001), consistent with previous studies, but BMR did not significantly differ between HR and control lines (+6.5%, P=0.181). Additionally, HR mice did not statistically differ from control mice for whole-body lean or fat mass, or for the mass of any organ collected (with body mass as a covariate). Finally, mass-independent V̇O2,max and BMR were uncorrelated (r=0.073, P=0.552) and the only statistically significant correlation with an organ mass was for V̇O2,max and ventricle mass (r=0.285, P=0.015). Overall, our results indicate that selection for a behavioral trait can yield large changes in behavior without proportional modifications to underlying morphological or physiological traits.

Effects of early-life voluntary exercise and fructose on adult activity levels, body composition, aerobic capacity, and organ masses in mice bred for high voluntary wheel-running behavior.

Fructose (C6H12O6) is acutely obesogenic and is a risk factor for hypertension, cardiovascular disease, and nonalcoholic fatty liver disease. However, the possible long-lasting effects of early-life fructose consumption have not been studied. We tested for effects of early-life fructose and/or wheel access (voluntary exercise) in a line of selectively bred High Runner (HR) mice and a non-selected Control (C) line. Exposures began at weaning and continued for 3 weeks to sexual maturity, followed by a 23-week "washout" period (equivalent to ∼17 human years). Fructose increased total caloric intake, body mass, and body fat during juvenile exposure, but had no effect on juvenile wheel running and no important lasting effects on adult physical activity or body weight/composition. Interestingly, adult maximal aerobic capacity (VO2max) was reduced in mice that had early-life fructose and wheel access. Consistent with previous studies, early-life exercise promoted adult wheel running. In a 3-way interaction, C mice that had early-life fructose and no wheel access gained body mass in response to 2 weeks of adult wheel access, while all other groups lost mass. Overall, we found some long-lasting positive effects of early-life exercise, but minimal effects of early-life fructose, regardless of the mouse line.

Oral antibiotics reduce voluntary exercise behavior in athletic mice.

The gut microbiome can affect various aspects of both behavior and physiology, including exercise ability, but effects on voluntary exercise have rarely been studied. We studied females from a selection experiment in which 4 replicate High Runner (HR) lines of mice are bred for voluntary exercise and compared with 4 non-selected control (C) lines. HR and C mice differ in several traits that likely interact with the gut microbiome, including higher daily running distance, body temperatures when running, spontaneous physical activity when housed without wheels, and food consumption. After two weeks of wheel access to reach a stable plateau in daily running, mice were administered broad-spectrum antibiotics for 10 days. Antibiotic treatment caused a significant reduction in daily wheel-running distance in the HR mice (-21%) but not in the C mice. Antibiotics did not affect body mass or food consumption in either HR or C mice, and we did not observe sickness behavior. Wheel running by HR mice did not recover during the 12 days following cessation of antibiotics. The decreased wheel-running in HR but not C mice, with no apparent negative side effects of antibiotics, suggests that the HR microbiome is an important component of their high-running phenotype.

Effects of selective breeding for voluntary exercise, chronic exercise, and their interaction on muscle attachment site morphology in house mice.

Skeletal muscles attach to bone at their origins and insertions, and the interface where tendon meets bone is termed the attachment site or enthesis. Mechanical stresses at the muscle/tendon-bone interface are proportional to the surface area of the bony attachment sites, such that a larger attachment site will distribute loads over a wider area. Muscles that are frequently active and/or are of larger size should cause attachment sites to hypertrophy (training effect); however, experimental studies of animals subjected to exercise have provided mixed results. To enhance our ability to detect training effects (a type of phenotypic plasticity), we studied a mouse model in which 4 replicate lines of High Runner (HR) mice have been selectively bred for 57 generations. Selection is based on the average number of wheel revolutions on days 5 & 6 of a 6-day period of wheel access as young adults (6-8 weeks old). Four additional lines are bred without regard to running and serve as non-selected controls (C). On average, mice from HR lines voluntarily run ~3 times more than C mice on a daily basis. For this study, we housed 50 females (half HR, half C) with wheels (Active group) and 50 (half HR, half C) without wheels (Sedentary group) for 12 weeks starting at weaning (~3 weeks old). We tested for evolved differences in muscle attachment site surface area between HR and C mice, plastic changes resulting from chronic exercise, and their interaction. We used a precise, highly repeatable method for quantifying the three-dimensional (3D) surface area of four muscle attachment sites: the humerus deltoid tuberosity (the insertion point for the spinodeltoideus, superficial pectoralis, and acromiodeltoideus), the femoral third trochanter (the insertion point for the quadratus femoris), the femoral lesser trochanter (the insertion point for the iliacus muscle), and the femoral greater trochanter (insertion point for the middle gluteal muscles). In univariate analyses, with body mass as a covariate, mice in the Active group had significantly larger humerus deltoid tuberosities than Sedentary mice, with no significant difference between HR and C mice and no interaction between exercise treatment and linetype. These differences between Active and Sedentary mice were also apparent in the multivariate analyses. Surface areas of the femoral third trochanter, femoral lesser trochanter, and femoral greater trochanter were unaffected by either chronic wheel access or selective breeding. Our results, which used robust measurement protocols and relatively large sample sizes, demonstrate that muscle attachment site morphology can be (but is not always) affected by chronic exercise experienced during ontogeny. However, contrary to previous results for other aspects of long bone morphology, we did not find evidence for evolutionary coadaptation of muscle attachments with voluntary exercise behavior in the HR mice.

Cross-fostering selectively bred High Runner mice affects adult body mass but not voluntary exercise.

While nursing, mammals progress through critical developmental periods for the cardiovascular, musculoskeletal, and central nervous systems. The suckling period in mammals is therefore especially vulnerable to environmental factors that may affect the "developmental programming" of many complex traits. As a result, various aspects of maternal behavior and physiology can influence offspring in ways that have lasting effects into adulthood. Several recent studies of animal models have shown that maternal effects can partially program adult activity behaviors, which has important implications for health and locomotor performance. Here, we used cross-fostering to test for possible maternal effects on adult wheel-running behavior (voluntary exercise), maximal aerobic capacity during forced exercise (VO2max), body mass and composition, and organ masses. Subjects were from a line of mice that has been selectively bred for ∼90 generations for high voluntary wheel-running behavior (High Runner; HR) and a non-selected Control (C) line. Adult HR mice run ∼3-fold the daily distances of C mice and have evolved other differences associated with exercise capacity, including elevated VO2max, reduced body mass and fat mass, and larger hearts. At birth, we fostered offspring to create 4 experimental groups: C pups to other C dams (in-foster), HR pups to other HR dams (in-foster), C pups to HR dams (cross-foster), HR pups to C dams (cross-foster). Thus, all pups were fostered to a different mother. Mice were weaned 3 weeks later, and adult testing began at ∼6 weeks of age. At weaning, pups raised by HR dams were smaller than those raised by C dams for both sexes and as expected, HR pups raised by HR dams weighed less than C pups raised by C dams. As adults, mice raised by HR dams continued to have reduced body masses. As expected, adult HR mice ran approximately 3-fold more than their C counterparts and females ran more than males. However, cross-fostering did not statistically affect any aspect of wheel-running behavior (distance, duration, speed). Similarly, with body mass as a covariate, HR mice had higher VO2max than C mice, and males had higher VO2max than females, but cross-fostering had no effect. With body mass as a covariate, cross-fostering had variable effects on adult organ masses in a sex-specific manner. Overall, our results indicate that development of the adult High Runner phenotype does not require rearing by an HR dam, suggesting that high adult activity in humans may be independent of high maternal activity.