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.

Corticosteroid-binding globulins: Lessons from biomedical research.

Glucocorticoids (GCs) circulate in the plasma bound to corticosteroid-binding globulin (CBG). Plasma CBG may limit access of glucocorticoids to tissues (acting as a sponge: the free hormone hypothesis), or may solely serve as a transport molecule, releasing GCs to tissues as the plasma moves through capillaries (the total hormone hypothesis). Both biomedical (focused on human health) and comparative (focused on ecological and evolutionary relevance) studies have worked to incorporate CBG in glucocorticoid physiology, and to understand whether free or total hormone is the biologically active plasma fraction. The biomedical field, however, has been well ahead of the comparative physiologists, and have produced results that can inform comparative research when considering the import of total vs. free plasma hormone. In fact, biomedical studies have made impressive strides regarding the function of CBG in tissues as well as plasma; we, however, focus solely on the plasma functions in this review as this is the primary area of disagreement amongst comparative physiologists. Here we present 5 sets of biomedical studies across genomics, pharmacology, cell culture, whole animal research, and human medicine that strongly support a role for CBG limiting hormone access to tissue. We also discuss three areas of concern across comparative researchers. In contrast to former publications, we are not suggesting that all comparative studies in glucocorticoid physiology must measure CBG, or that only free corticosterone levels are valid. However, we propose that comparative physiologists be aware of biomedical results as they investigate glucocorticoids and interpret how total hormone may or may not impact behavior and physiology of free-living vertebrates.

Living on the edge: Glucocorticoid physiology in desert iguanas (Dipsosaurus dorsalis) is predicted by distance from an anthropogenic disturbance, body condition, and population density.

Ecological factors, such as habitat quality, influence the survival and reproductive success of free-living organisms. Urbanization, including roads, alters native habitat and likely influences physiology, behavior, and ultimately Darwinian fitness. Some effects of roads are clearly negative, such as increased habitat fragmentation and mortality from vehicle collision. However, roads can also have positive effects, such as decreasing predator density and increased vegetation cover, particularly in xeric habitats due to increased water run-off. Glucocorticoids are metabolic hormones that reflect baseline metabolic needs, increase in response to acute challenges, and may mediate endogenous resource trade-offs between survival and reproduction. Here we examined circulating concentrations of corticosterone (baseline and stress-induced) in desert iguanas (Dipsosaurus dorsalis) in relation to the distance from a major anthropogenic disturbance, a high-traffic road in Palm Springs, CA. Additionally, we analyzed body condition and population density as additional predictors of glucocorticoid physiology. Surprisingly, we found lower baseline CORT levels closer to the road, but no effect of distance from road on stress-induced CORT or stress responsiveness (difference between baseline and stress-induced concentrations). Both population density and body condition were negative predictors of baseline CORT, stress-induced CORT, and stress responsiveness. Given the known effect of roads to increase run-off and vegetation density, increased water availability may improve available forage and shade, which may then increase the carrying capacity of the habitat and minimize metabolic challenges for this herbivorous lizard. However, it is important to recognize that surfaces covered by asphalt are not usable habitat for iguanas, likely resulting in a net habitat loss.

Across time and space: Hormonal variation across temporal and spatial scales in relation to nesting success.

There is a renewed interest in investigating individual variation in hormone levels in relation to fitness metrics, as hormones act as mediators of life-history trade-offs. Hormone concentrations, however, are labile, responding to both internal and external stimuli, so the relationship between hormones and fitness can be non-consistent. One explanation of this inconsistent relationship is that a single hormone sample may not be representative of individual phenotypes in a free-living species. We addressed this issue by repeatedly sampling a free-living population of mountain white-crowned sparrows, Zonotrichia leucophrys oriantha, for baseline and stress-induced corticosterone (cort) and testosterone (T) across different stages of the breeding season. We measured (co)variation using three different methods, taking into account inter- and intra-individual variances, to determine whether hormone levels and the stress response are repeatable. We documented the temporal (over 3 months) and spatial (home-range) variation of individual hormone phenotypes and investigated how these components related to nesting success. At the population level, we found significant repeatability in male stress-induced cort concentrations but no repeatability in male or female baseline cort or male T concentrations. Using a new metric of intra-individual variance focusing on the stress response (profile repeatability), we found a wide range of variance scores, with most individuals showing high variation in their stress response. Similarly, we found a low level of repeatability of the reaction norm intercept and slope for the stress response across different life-history stages. Males with higher concentrations of stress-induced cort had more central home-ranges. Males with higher body condition had larger home-ranges; however, home-range size did not relate to male hormone concentrations or nesting success. We also did not find any significant relationship between variation in hormone levels and nesting success. We recommend that future studies combine both physiological and environmental components to better understand the relationship between hormones and fitness.

Stress-Induced Hyperglycemia in White-Throated and White-Crowned Sparrows: A New Technique for Rapid Glucose Measurement in the Field.

Organisms experience stressors, and the physiological response to these stressors is highly conserved. Acute stress activates both the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis, increasing epinephrine, norepinephrine, and glucocorticoids, collectively promoting glucose mobilization. While this is well characterized in mammals, the hyperglycemic response to stress in avian and nonavian reptiles has received less attention. A number of factors, ranging from time of day to blood loss, are reported to influence the extent to which acute stress leads to hyperglycemia in birds. Here we characterized the glycemic response to acute handling stress in two species of free-living sparrows: white-throated sparrows (WTSPs: Zonotrichia albicollis) in St. Mary's County, Maryland, and white-crowned sparrows (WCSPs: Zonotrichia leucophrys) in Tioga Pass Meadow, California. We validated a novel technique for rapid field measurement of glucose using a human blood glucose meter, FreeStyle Lite. As expected, acute handling stress elevated blood glucose at both 15 and 30 min postcapture as compared to baseline for both WTSPs and WCSPs. In addition, handling for 30 min without bleeding had the same hyperglycemic effect as handling with serial bleeds in WCSPs. Finally, body condition that was measured as abdominal fat score predicted stress-induced blood glucose in WTSPs but not in WCSPs. Our results are consistent with the mammalian literature on acute stress and energy mobilization, and we introduce a new field technique for avian field biologists.

Effects of activity, genetic selection and their interaction on muscle metabolic capacities and organ masses in mice.

Chronic voluntary exercise elevates total daily energy expenditure and food consumption, potentially resulting in organ compensation supporting nutrient extraction/utilization. Additionally, species with naturally higher daily energy expenditure often have larger processing organs, which may represent genetic differences and/or phenotypic plasticity. We tested for possible adaptive changes in organ masses of four replicate lines of house mice selected (37 generations) for high running (HR) compared with four non-selected control (C) lines. Females were housed with or without wheel access for 13-14 weeks beginning at 53-60 days of age. In addition to organ compensation, chronic activity may also require an elevated aerobic capacity. Therefore, we also measured hematocrit and both citrate synthase activity and myoglobin concentration in heart and gastrocnemius. Both selection (HR versus C) and activity (wheels versus no wheels) significantly affected morphological and biochemical traits. For example, with body mass as a covariate, mice from HR lines had significantly higher hematocrit and larger ventricles, with more myoglobin. Wheel access lengthened the small intestine, increased relative ventricle and kidney size, and increased skeletal muscle citrate synthase activity and myoglobin concentration. As compared with C lines, HR mice had greater training effects for ventricle mass, hematocrit, large intestine length and gastrocnemius citrate synthase activity. For ventricle and gastrocnemius citrate synthase activity, the greater training was quantitatively explainable as a result of greater wheel running (i.e. 'more pain, more gain'). For hematocrit and large intestine length, differences were not related to amount of wheel running and instead indicate inherently greater adaptive plasticity in HR lines.

Acute Restraint Stress Alters Wheel-Running Behavior Immediately Following Stress and up to 20 Hours Later in House Mice.

In vertebrates, acute stressors-although short in duration-can influence physiology and behavior over a longer time course, which might have important ramifications under natural conditions. In laboratory rats, for example, acute stress has been shown to increase anxiogenic behaviors for days after a stressor. In this study, we quantified voluntary wheel-running behavior for 22 h following a restraint stress and glucocorticoid levels 24 h postrestraint. We utilized mice from four replicate lines that have been selectively bred for high voluntary wheel-running activity (HR mice) for 60 generations and their nonselected control (C) lines to examine potential interactions between exercise propensity and sensitivity to stress. Following 6 d of wheel access on a 12L∶12D photo cycle (0700-1900 hours, as during the routine selective breeding protocol), 80 mice were physically restrained for 40 min, beginning at 1400 hours, while another 80 were left undisturbed. Relative to unrestrained mice, wheel running increased for both HR and C mice during the first hour postrestraint (P < 0.0001) but did not differ 2 or 3 h postrestraint. Wheel running was also examined at four distinct phases of the photoperiod. Running in the period of 1600-1840 hours was unaffected by restraint stress and did not differ statistically between HR and C mice. During the period of peak wheel running (1920-0140 hours), restrained mice tended to run fewer revolutions (-11%; two-tailed P = 0.0733), while HR mice ran 473% more than C (P = 0.0008), with no restraint × line type interaction. Wheel running declined for all mice in the latter part of the scotophase (0140-0600 hours), restraint had no statistical effect on wheel running, but HR again ran more than C (+467%; P = 0.0122). Finally, during the start of the photophase (0720-1200 hours), restraint increased running by an average of 53% (P = 0.0443) in both line types, but HR and C mice did not differ statistically. Mice from HR lines had statistically higher plasma corticosterone concentrations than C mice, with no statistical effect of restraint and no interaction between line type and restraint. Overall, these results indicate that acute stress can affect locomotor activity (or activity patterns) for many hours, with the most prominent effect being an increase in activity during a period of typical inactivity at the start of the photophase, 15-20 h poststressor.

Exercise training effects on hypoxic and hypercapnic ventilatory responses in mice selected for increased voluntary wheel running.

NEW FINDINGS: What is the central question of this study? We used experimental evolution to determine how selective breeding for high voluntary wheel running and exercise training (7-11 weeks) affect ventilatory chemoreflexes of laboratory mice at rest. What is the main finding and its importance? Selective breeding, although significantly affecting some traits, did not systematically alter ventilation across gas concentrations. As with most human studies, our findings support the idea that endurance training attenuates resting ventilation. However, little evidence was found for a correlation between ventilatory chemoreflexes and the amount of individual voluntary wheel running. We conclude that exercise 'training' alters respiratory behaviours, but these changes may not be necessary to achieve high levels of wheel running. Ventilatory control is affected by genetics, the environment and gene-environment and gene-gene interactions. Here, we used an experimental evolution approach to test whether 37 generations of selective breeding for high voluntary wheel running (genetic effects) and/or long-term (7-11 weeks) wheel access (training effects) alter acute respiratory behaviour of mice resting in normoxic, hypoxic and hypercapnic conditions. As the four replicate high-runner (HR) lines run much more than the four non-selected control (C) lines, we also examined whether the amount of exercise among individual mice was a quantitative predictor of ventilatory chemoreflexes at rest. Selective breeding and/or wheel access significantly affected several traits. In normoxia, HR mice tended to have lower mass-adjusted rates of oxygen consumption and carbon dioxide production. Chronic wheel access increased oxygen consumption and carbon dioxide production in both HR and C mice during hypercapnia. Breathing frequency and minute ventilation were significantly reduced by chronic wheel access in both HR and C mice during hypoxia. Selection history, while significantly affecting some traits, did not systematically alter ventilation across all gas concentrations. As with most human studies, our findings support the idea that endurance training (access to wheel running) attenuates resting ventilation. However, little evidence was found for a correlation at the level of the individual variation between ventilatory chemoreflexes and performance (amount of individual voluntary wheel running). We tentatively conclude that exercise 'training' alters respiratory behaviours, but these changes may not be necessary to achieve high levels of wheel running.

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.

Effects of reproductive status on behavioral and endocrine responses to acute stress in a biparental rodent, the California mouse (Peromyscus californicus).

In several mammalian species, lactating females show blunted neural, hormonal, and behavioral responses to stressors. It is not known whether new fathers also show stress hyporesponsiveness in species in which males provide infant care. To test this possibility, we determined the effects of male and female reproductive status on stress responsiveness in the biparental, monogamous California mouse (Peromyscus californicus). Breeding (N=8 females, 8 males), nonbreeding (N=10 females, 10 males) and virgin mice (N=12 females, 9 males) were exposed to a 5-min predator-urine stressor at two time points, corresponding to the early postpartum (5-7 days postpartum) and mid/late postpartum (19-21 days postpartum) phases, and blood samples were collected immediately afterwards. Baseline blood samples were obtained 2 days prior to each stress test. Baseline plasma corticosterone (CORT) concentrations did not differ among male or female groups. CORT responses to the stressor did not differ among female reproductive groups, and all three groups showed distinct behavioral responses to predator urine. Virgin males tended to increase their CORT response from the first to the second stress test, while breeding and nonbreeding males did not. Moreover, virgin and nonbreeding males showed significant behavioral changes in response to predator urine, whereas breeding males did not. These results suggest that adrenocortical responses to a repeated stressor in male California mice may be modulated by cohabitation with a female, whereas behavioral responses to stress may be blunted by parental status.

How to run far: multiple solutions and sex-specific responses to selective breeding for high voluntary activity levels.

The response to uniform selection may occur in alternate ways that result in similar performance. We tested for multiple adaptive solutions during artificial selection for high voluntary wheel running in laboratory mice. At generation 43, the four replicate high runner (HR) lines averaged 2.85-fold more revolutions per day as compared with four non-selected control (C) lines, and females ran 1.11-fold more than males, with no sex-by-linetype interaction. Analysis of variance indicated significant differences among C lines but not among HR for revolutions per day. By contrast, average speed varied significantly among HR lines, but not among C, and showed a sex-by-linetype interaction, with the HR/C ratio being 2.02 for males and 2.45 for females. Time spent running varied among both HR and C lines, and showed a sex-by-linetype interaction, with the HR/C ratio being 1.52 for males but only 1.17 for females. Thus, females (speed) and males (speed, but also time) evolved differently, as did the replicate selected lines. Speed and time showed a trade-off among HR but not among C lines. These results demonstrate that uniform selection on a complex trait can cause consistent responses in the trait under direct selection while promoting divergence in the lower-level components of that trait.

Corticosterone and cortisol binding sites in plasma, immune organs and brain of developing zebra finches: intracellular and membrane-associated receptors.

Glucocorticoids (GCs) affect the development of both the immune and nervous systems. To do so, GCs bind to intracellular receptors, mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). In addition, GCs bind to membrane-associated corticosteroid receptors (mCR). Two well-known GCs are corticosterone and cortisol. Whereas corticosterone is the primary GC in zebra finch plasma, cortisol is the primary GC in zebra finch lymphoid organs and is also present in the brain and plasma during development. Here, we characterized binding sites for corticosterone and cortisol in plasma, liver, lymphoid organs, and brain of developing zebra finches. In tissues, we examined both intracellular and membrane-associated binding sites. For intracellular receptors, there were MR-like sites and GR-like sites, which differentially bound corticosterone and cortisol in a tissue-specific manner. For mCR, we found little evidence for membrane-associated receptors in immune organs, but this could be due to the small size of immune organs. Interestingly, cortisol, but not corticosterone, showed a low amount of specific binding to bursa of Fabricius membranes. For neural membranes, corticosterone bound to one site with low affinity but a relatively high B(max), and in contrast, cortisol bound to one site with high affinity but a lower B(max). Our results indicate that intracellular and membrane-associated receptors differentially bind corticosterone and cortisol suggesting that corticosterone and cortisol might have different roles in immune and nervous system development.

How acute is the acute stress response? Baseline corticosterone and corticosteroid-binding globulin levels change 24h after an acute stressor in Japanese quail.

Changes in plasma corticosteroid-binding globulin (CBG) capacity can alter free plasma concentration and tissue availability of glucocorticoids (GC) and hence alter the organismal response to stress. However, CBG change in response to stress has not been extensively studied. While it is clear that chronic stress can causes CBG decline and in some species acute stressors can reduce CBG during the 30-60 min of the stressor, more long-term changes in CBG following an acute stressor has received less attention. Here we investigated corticosterone (CORT: the primary GC in birds) and CBG levels 24h after an acute stressor in a unique study system: Japanese quail divergently selected for CORT reactivity to acute stress. Using this model, we examined the interaction of selected CORT reactivity with CBG response to determine if CBG shows a delayed decline in response to an acute stressor and if that decline varies by selected genetic background. We found lowered CBG capacity, elevated total CORT and free CORT 24h after acute stress in all three quail groups. These results demonstrate for the first time in an avian species that exposure to an acute stressor can affect CBG and CORT 24h later.

Steroid-binding proteins and free steroids in birds.

Within the comparative literature, corticosteroid-binding globulin (CBG) has recently emerged as a potential modulator of the glucocorticoids-driven stress response. Many avian field studies include the measurement of CBG with the goal of making behavioral and ecological inferences. However, the field of stress physiology is divided on how to interpret the biological importance of the different states of circulating hormones. Here we review evidence for the biological relevance of each fraction of glucocorticoid hormone; the CBG-glucocorticoid complex (the bound fraction) and the remainder which is either unbound or loosely attached to albumin (the free fraction). We suggest that the biological importance of free vs. bound hormone depends on the location of interest (plasma or tissues), and the time frame of interest (current or future need). While a large body of evidence suggests that free hormones are the biologically active fraction, evidence also suggests that the bound fraction is a biologically relevant reservoir of glucocorticoids. We review two salient topics from the avian stress literature; stress-induced decreases in CBG capacity and glucocorticoid influences in life history strategies. These topics are discussed with an emphasis on free vs. bound hormone concentration and how that compares to current vs. future glucocorticoid needs.

Lines of mice with chronically elevated baseline corticosterone levels are more susceptible to a parasitic nematode infection.

Chronically elevated circulating plasma glucocorticoid concentrations can have suppressive effects on immune function in mammals. House mice (Mus domesticus) that have been selectively bred for high voluntary wheel running exhibit chronically elevated (two-fold, on average) plasma corticosterone (CORT) levels and hence are an interesting model to study possible glucocorticoid-induced immune suppression. As an initial test of their immunocompetence, we compared the four replicate high runner (HR) lines with their four non-selected control (C) lines by subjecting them to infection by a parasitic nematode, Nippostrongylus brasiliensis. At generation 36 of the selection experiment, 10 adult males from each of the eight lines were inoculated subcutaneously with approximately 600 third-stage larval N. brasiliensis, and then sacrificed 12 days after injection. Neither spleen mass nor number of adult nematodes in the small intestine differed significantly between HR and C lines. However, the eight lines differed significantly in nematode counts, and the line means for nematode infestation were significantly positively related to baseline circulating CORT concentration measured in males from generations 34 and 39. Therefore, although selective breeding for high locomotor activity may not have resulted in a generally compromised immune response, results of this study are consistent with the hypothesis that glucocorticoids can have immunosuppressive effects.

Glycogen storage and muscle glucose transporters (GLUT-4) of mice selectively bred for high voluntary wheel running.

To examine the evolution of endurance-exercise behaviour, we have selectively bred four replicate lines of laboratory mice (Mus domesticus) for high voluntary wheel running (;high runner' or HR lines), while also maintaining four non-selected control (C) lines. By generation 16, HR mice ran approximately 2.7-fold more than C mice, mainly by running faster (especially in females), a differential maintained through subsequent generations, suggesting an evolutionary limit of unknown origin. We hypothesized that HR mice would have higher glycogen levels before nightly running, show greater depletion of those depots during their more intense wheel running, and have increased glycogen synthase activity and GLUT-4 protein in skeletal muscle. We sampled females from generation 35 at three times (photophase 07:00 h-19:00 h) during days 5-6 of wheel access, as in the routine selection protocol: Group 1, day 5, 16:00 h-17:30 h, wheels blocked from 13:00 h; Group 2, day 6, 02:00 h-03:30 h (immediately after peak running); and Group 3, day 6, 07:00 h-08:30 h. An additional Group 4, sampled 16:00 h-17:30 h, never had wheels. HR individuals with the mini-muscle phenotype (50% reduced hindlimb muscle mass) were distinguished for statistical analyses comparing C, HR normal, and HR mini. HR mini ran more than HR normal, and at higher speeds, which might explain why they have been favored by the selective-breeding protocol. Plasma glucose was higher in Group 1 than in Group 4, indicating a training effect (phenotypic plasticity). Without wheels, no differences in gastrocnemius GLUT-4 were observed. After 5 days with wheels, all mice showed elevated GLUT-4, but HR normal and mini were 2.5-fold higher than C. At all times and irrespective of wheel access, HR mini showed approximately three-fold higher [glycogen] in gastrocnemius and altered glycogen synthase activity. HR mini also showed elevated glycogen in soleus when sampled during peak running. All mice showed some glycogen depletion during nightly wheel running, in muscles and/or liver, but the magnitude of this depletion was not large and hence does not seem to be limiting to the evolution of even-higher wheel running.

Behavioral despair and home-cage activity in mice with chronically elevated baseline corticosterone concentrations.

Dysfunction of the hypothalamic-pituitary-adrenal axis resulting in elevated baseline glucocorticoid concentrations is a hallmark of stress-related human anxiety and affective disorders, including depression. Mice from four replicate lines bred for high voluntary wheel running (HR lines) run almost three times as much as four non-selected control (C) lines, and exhibit two fold elevated baseline circulating corticosterone levels throughout the 24 h cycle. Although elevated baseline CORT may be beneficial for high locomotor activity, chronic elevations can have deleterious effects on multiple systems, and may predispose for affective disorders. Because stressful events often precede a depressive bout, we quantified depressive-like behavior in the forced-swim (FST; generation 41) and tail-suspension tests (TST; generation 47) in HR and C mice that had wheel access for 6 days and then were deprived of wheels on day seven prior to the FST or TST. Male HR spent significantly more time immobile in the FST than C, suggesting that HR males have a predisposition for depression-like behavior. Both male and female HR (generation 43) were more active than same-sex controls in both wheel running and home-cage activity across 22 h (pooling the sexes, HR/C = 2.28 and 2.66, respectively).

Selective breeding for a behavioral trait changes digit ratio.

The ratio of the length of the second digit (index finger) divided by the fourth digit (ring finger) tends to be lower in men than in women. This 2D:4D digit ratio is often used as a proxy for prenatal androgen exposure in studies of human health and behavior. For example, 2D:4D ratio is lower (i.e. more "masculinized") in both men and women of greater physical fitness and/or sporting ability. Lab mice have also shown variation in 2D:4D as a function of uterine environment, and mouse digit ratios seem also to correlate with behavioral traits, including daily activity levels. Selective breeding for increased rates of voluntary exercise (wheel running) in four lines of mice has caused correlated increases in aerobic exercise capacity, circulating corticosterone level, and predatory aggression. Here, we show that this selection regime has also increased 2D:4D. This apparent "feminization" in mice is opposite to the relationship seen between 2D:4D and physical fitness in human beings. The present results are difficult to reconcile with the notion that 2D:4D is an effective proxy for prenatal androgen exposure; instead, it may more accurately reflect effects of glucocorticoids, or other factors that regulate any of many genes.