Fernando R. Gomes

Circadian pattern of total and free corticosterone concentrations, corticosteroid-binding globulin, and physical activity in mice selectively bred for high voluntary wheel-running behavior

In vertebrates, baseline glucocorticoid concentrations vary predictably on a diel basis, usually peaking shortly before the onset of activity. Presumably, circadian patterns in glucocorticoid secretion have evolved to match predictable rises in energetic need. In mice from lines selectively bred for high voluntary wheel-running, previous studies have reported that baseline plasma corticosterone concentrations at two different times during the photophase are elevated twofold above those of non-selected control lines. Here, we tested the hypothesis that the elevated daytime corticosterone levels could be explained by a shift in the circadian pattern of corticosterone levels. We measured baseline total plasma corticosterone levels, corticosteroid-binding globulin (CBG) capacity, and calculated free corticosterone levels (corticosterone not bound to corticosteroid-binding globulin and potentially biologically active) at six points during the 24-hour cycle in males on a 12:12 photoperiod. We also examined the daily pattern of both wheel-running and home-cage activity. Based on combined analysis of all six points, the circadian pattern of total corticosterone, corticosteroid-binding globulin, and free corticosterone levels did not significantly differ between high-runner and control mice (linetype * time interaction P=0.56, 0.45, and 0.55, respectively); however, all varied with time (all P<0.0001) and mice from the selected lines had significantly elevated total (P=0.0125) and free (P=0.0140) corticosterone, with no difference in CBG binding capacity (P=0.77). All mice were active primarily during the dark phase, and the factorial increase in activity of selected relative to controls lines was 2.33 for total daily wheel revolutions and 2.76 for total daily home-cage activity. The onset of the active period for both measures of locomotor activity coincided with peak total and free corticosterone levels in both selected and control lines. These findings lend support to our hypothesis that elevated circulating corticosterone levels have evolved as an adaptation to support increased locomotor activity in the selected lines.

Baseline and stress-induced plasma corticosterone concentrations of mice selectively bred for high voluntary wheel running.

The hypothalamic‐pituitary‐adrenal (HPA) axis is important in regulating energy metabolism and in mediating responses to stressors, including increasing energy availability during physical exercise. In addition, glucocorticoids act directly on the central nervous system and influence behavior, including locomotor activity. To explore potential changes in the HPA axis as animals evolve higher voluntary activity levels, we characterized plasma corticosterone (CORT) concentrations and adrenal mass in four replicate lines of house mice that had been selectively bred for high voluntary wheel running (HR lines) for 34 generations and in four nonselected control (C) lines. We determined CORT concentrations under baseline conditions and immediately after exposure to a novel stressor (40 min of physical restraint) in mice that were housed without access to wheels. Resting daytime CORT concentrations were approximately twice as high in HR as in C mice for both sexes. Physical restraint increased CORT to similar concentrations in HR and C mice; consequently, the proportional response to restraint was smaller in HR than in C animals. Adrenal mass did not significantly differ between HR and C mice. Females had significantly higher baseline and postrestraint CORT concentrations and significantly larger adrenal glands than males in both HR and C lines. Replicate lines showed significant variation in body mass, length, baseline CORT concentrations, and postrestraint CORT concentrations in one or both sexes. Among lines, both body mass and length were significantly negatively correlated with baseline CORT concentrations, suggesting that CORT suppresses growth. Our results suggest that selection for increased locomotor activity has caused correlated changes in the HPA axis, resulting in higher baseline CORT concentrations and, possibly, reduced stress responsiveness and a lower growth rate.

Maximal metabolic rates during voluntary exercise, forced exercise, and cold exposure in house mice selectively bred for high wheel-running

SUMMARY Selective breeding for high wheel-running activity has generated four lines of laboratory house mice (S lines) that run about 170% more than their control counterparts (C lines) on a daily basis, mostly because they run faster. We tested whether maximum aerobic metabolic rates (V̇O2max) have evolved in concert with wheel-running, using 48 females from generation 35. Voluntary activity and metabolic rates were measured on days 5+6 of wheel access (mimicking conditions during selection), using wheels enclosed in metabolic chambers. Following this, V̇O2max was measured twice on a motorized treadmill and twice during cold-exposure in a heliox atmosphere (HeO2). Almost all measurements, except heliox V̇O2max, were significantly repeatable. After accounting for differences in body mass (S<C) and variation in age at testing, S and C did not differ in V̇O2max during forced exercise or in heliox, nor in maximal running speeds on the treadmill. However, running speeds and V̇O2max during voluntary exercise were significantly higher in S lines. Nevertheless, S mice never voluntarily achieved the V̇O2max elicited during their forced treadmill trials, suggesting that aerobic capacity per se is not limiting the evolution of even higher wheel-running speeds in these lines. Our results support the hypothesis that S mice have genetically higher motivation for wheel-running and they demonstrate that behavior can sometimes evolve independently of performance capacities. We also discuss the possible importance of domestication as a confounding factor to extrapolate results from this animal model to natural populations.

Effects of size, sex, and voluntary running speeds on costs of locomotion in lines of laboratory mice selectively bred for high wheel-running activity.

Selective breeding for over 35 generations has led to four replicate (S) lines of laboratory house mice (Mus domesticus) that run voluntarily on wheels about 170% more than four random‐bred control (C) lines. We tested whether S lines have evolved higher running performance by increasing running economy (i.e., decreasing energy spent per unit of distance) as a correlated response to selection, using a recently developed method that allows for nearly continuous measurements of oxygen consumption (V̇o2) and running speed in freely behaving animals. We estimated slope (incremental cost of transport [COT]) and intercept for regressions of power (the dependent variable, V̇o2/min) on speed for 49 males and 47 females, as well as their maximum V̇o2 and speeds during wheel running, under conditions mimicking those that these lines face during the selection protocol. For comparison, we also measured COT and maximum aerobic capacity (V̇o2max) during forced exercise on a motorized treadmill. As in previous studies, the increased wheel running of S lines was mainly attributable to increased average speed, with males also showing a tendency for increased time spent running. On a whole‐animal basis, combined analysis of males and females indicated that COT during voluntary wheel running was significantly lower in the S lines (one‐tailed P = 0.015). However, mice from S lines are significantly smaller and attain higher maximum speeds on the wheels; with either body mass or maximum speed (or both) entered as a covariate, the statistical significance of the difference in COT is lost (one‐tailed P ≥ 0.2). Thus, both body size and behavior are key components of the reduction in COT. Several statistically significant sex differences were observed, including lower COT and higher resting metabolic rate in females. In addition, maximum voluntary running speeds were negatively correlated with COT in females but not in males. Moreover, males (but not females) from the S lines exhibited significantly higher treadmill V̇o2max as compared to those from C lines. The sex‐specific responses to selection may in part be consequences of sex differences in body mass and running style. Our results highlight how differences in size and running speed can account for lower COT in S lines and suggest that lower COT may have coadapted in response to selection for higher running distances in these lines.

Running Behavior and Its Energy Cost in Mice Selectively Bred for High Voluntary Locomotor Activity

Locomotion is central to behavior and intrinsic to many fitness‐critical activities (e.g., migration, foraging), and it competes with other life‐history components for energy. However, detailed analyses of how changes in locomotor activity and running behavior affect energy budgets are scarce. We quantified these effects in four replicate lines of house mice that have been selectively bred for high voluntary wheel running (S lines) and in their four nonselected control lines (C lines). We monitored wheel speeds and oxygen consumption for 24–48 h to determine daily energy expenditure (DEE), resting metabolic rate (RMR), locomotor costs, and running behavior (bout characteristics). Daily running distances increased roughly 50%–90% in S lines in response to selection. After we controlled for body mass effects, selection resulted in a 23% increase in DEE in males and a 6% increase in females. Total activity costs (DEE − RMR) accounted for 50%–60% of DEE in both S and C lines and were 29% higher in S males and 5% higher in S females compared with their C counterparts. Energetic costs of increased daily running distances differed between sexes because S females evolved higher running distances by running faster with little change in time spent running, while S males also spent 40% more time running than C males. This increase in time spent running impinged on high energy costs because the majority of running costs stemmed from “postural costs” (the difference between RMR and the zero‐speed intercept of the speed vs. metabolic rate relationship). No statistical differences in these traits were detected between S and C females, suggesting that large changes in locomotor behavior do not necessarily effect overall energy budgets. Running behavior also differed between sexes: within S lines, males ran with more but shorter bouts than females. Our results indicate that selection effects on energy budgets can differ dramatically between sexes and that energetic constraints in S males might partly explain the apparent selection limit for wheel running observed for over 15 generations.

Environmental and Physiological Factors Influence Antipredator Behavior in Scinax hiemalis (Anura: Hylidae)

Abstract Individual variation in antipredator behavior may be influenced by ecological factors, such as ambient temperature and predation pressure, and by intrinsic factors, such as physiological condition. We tested the hypothesis that both types of factors interact to induce a specific antipredator response in the treefrog, Scinax hiemalis. When disturbed by the threat of predation, individuals of this species exhibit either a passive response by feigning death or immobility, or an active escape response by jumping away. We examined the responses of 24 adult male S. hiemalis to simulated predation in the laboratory at 10, 15, and 20 C. In addition, responses from a single stimulus were compared with those from a series of stimuli. We determined physiological condition from measures of body length and mass, jumping performance, aerobic metabolism, and estimated energy reserves. Temperature had the most influence on antipredator behavior, with more frogs exhibiting passive responses at 10 C than at higher temperatures. If stimulated more than once, the proportion of active responses increased at all three temperatures. Larger individuals were more likely to exhibit an active response, but no mass-independent physiological variables were related to response type. These results suggest that frogs respond to both extrinsic and intrinsic factors that may affect their behavioral performance.

Interindividual Differences in Leg Muscle Mass and Pyruvate Kinase Activity Correlate with Interindividual Differences in Jumping Performance of Hyla multilineata

Frog jumping is an excellent model system for examining the structural basis of interindividual variation in burst locomotor performance. Some possible factors that affect jump performance, such as total body size, hindlimb length, muscle mass, and muscle mechanical and biochemical properties, were analysed at the interindividual (intraspecies) level in the tree frog Hyla multilineata. The aim of this study was to determine which of these physiological and anatomical variables both vary between individuals and are correlated with interindividual variation in jump performance. The model produced via stepwise linear regression analysis of absolute data suggested that 62% of the interindividual variation in maximum jump distance could be explained by a combination of interindividual variation in absolute plantaris muscle mass, total hindlimb muscle mass (excluding plantaris muscle), and pyruvate kinase activity. When body length effects were removed, multiple regression indicated that the same independent variables explained 43% of the residual interindividual variation in jump distance. This suggests that individuals with relatively large jumping muscles and high pyruvate kinase activity for their body size achieved comparatively large maximal jump distances for their body size.

Variation in Call Structure and Calling Behavior in Treefrogs of the Genus Scinax

Abstract We investigated calling behavior and call structure for eight species of treefrogs in the genus Scinax, and document noteworthy patterns of interspecific variation that largely reflect habitat and social environment in this report. The five species in the S. ruber clade call in open areas or forest edges near standing water and produce simple calls at relatively high rates and low to moderate frequencies. Species from the S. catharinae clade call from more forested habitats and produce complex, higher frequency calls at relatively low rates. Differences in the range of activity temperatures and habitats among species reflect differences in geographical distribution and spatio-temporal selection of microhabitat during calling. In general, species with high calling rates also had high rates of locomotion during calling activity and were active at warmer temperatures. One exception was S. crospedospilus, which has an extremely high calling rate but exhibits among the lowest rates of locomotion and activity temperatures. Complex calls delivered at lower rates and simple calls produced at high rates represent extremes of the range of calling behavior among these species of Scinax.

An evolutionary frame of work to study physiological adaptation to high altitudes

How complex physiological systems evolve is one of the major questions in evolutionary physiology. For example, how traits interact at the physiological and genetic level, what are the roles of development and plasticity in Darwinian evolution, and eventually how physiological traits will evolve, remains poorly understood. In this article we summarize the current frame of work evolutionary physiologists are employing to study the evolution of physiological adaptations, as well as the role of developmental and reversible phenotypic plasticity in this context. We also highlight representative examples of how the integration of evolutionary and developmental physiology, concomitantly with the mechanistic understanding of physiological systems, can provide a deeper insight on how endothermic vertebrates could cope with reduced ambient temperatures and oxygen availability characteristic of high altitude environments. In this context, high altitude offers a unique system to study the evolution of physiological traits, and we believe much can be gained by integrating theoretical and empirical knowledge from evolutionary biology, such as life-history theory or the comparative method, with the mechanistic understanding of physiological processes.

Calling Behavior and Parasite Intensity in Treefrogs, Hypsiboas prasinus

Abstract A negative relationship between parasite intensity and male ornament condition or sexual display rate is one of the conditions of the parasite-mediated sexual selection model. In anurans, temporal properties of calling behavior, and particularly calling rates, are the best candidates to express a negative relationship with parasite intensity, given the high energy costs of calling and the fact that calls are potentially under strong intersexual selection. We studied the relationship between call parameters and helminth parasite intensity in males of a Brazilian subtropical treefrog, Hypsiboas prasinus. We tested the hypotheses that: 1) calling characteristics are correlated negatively to parasite intensity; and 2) the relationship between calling performance and parasite intensity is more pronounced when dynamic properties are considered. According to our predictions, only rate, the most important dynamic property of calling behavior, is associated with individual variation in parasite intensity. Males that call at higher rates show lower total parasite intensity. The negative relationship between parasite intensity and calling rate in H. prasinus could be because of the higher energy cycles associated with the maintenance of high calling performance. Also, theoretically, calling rate could work as an honest signal of anuran male quality, although the causal relationship between calling variation and parasite intensity, as well as the relevance of this relationship for female choice and male reproductive success, remain to be investigated.