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

Transcription

1 662 Running Behvior nd Its Energy Cost in Mice Selectively Bred for High Voluntry Locomotor Activity Enrico L. Rezende* Fernndo R. Gomes Mrk A. Chppell Theodore Grlnd Jr. Deprtment of Biology, University of Cliforni, Riverside, Cliforni Accepted 1/14/2009; Electroniclly Pulished 10/2/2009 necessrily effect overll energy udgets. Running ehvior lso differed etween sexes: within S lines, mles rn with more ut shorter outs thn femles. Our results indicte tht selection effects on energy udgets cn differ drmticlly etween sexes nd tht energetic constrints in S mles might prtly explin the pprent selection limit for wheel running oserved for over 15 genertions. ABSTRACT Locomotion is centrl to ehvior nd intrinsic to mny fitnesscriticl ctivities (e.g., migrtion, forging), nd it competes with other life-history components for energy. However, detiled nlyses of how chnges in locomotor ctivity nd running ehvior ffect energy udgets re scrce. We quntified these effects in four replicte lines of house mice tht hve een selectively red for high voluntry wheel running (S lines) nd in their four nonselected control lines (C lines). We monitored wheel speeds nd oxygen consumption for h to determine dily energy expenditure (DEE), resting metolic rte (RMR), locomotor costs, nd running ehvior (out chrcteristics). Dily running distnces incresed roughly 50% 90% in S lines in response to selection. After we controlled for ody mss effects, selection resulted in 23% increse in DEE in mles nd 6% increse in femles. Totl ctivity costs (DEE RMR) ccounted for 50% 60% of DEE in oth S nd C lines nd were 29% higher in S mles nd 5% higher in S femles compred with their C counterprts. Energetic costs of incresed dily running distnces differed etween sexes ecuse S femles evolved higher running distnces y running fster with little chnge in time spent running, while S mles lso spent 40% more time running thn C mles. This increse in time spent running impinged on high energy costs ecuse the mjority of running costs stemmed from posturl costs (the difference etween RMR nd the zero-speed intercept of the speed vs. metolic rte reltionship). No sttisticl differences in these trits were detected etween S nd C femles, suggesting tht lrge chnges in locomotor ehvior do not * Corresponding uthor. Present ddress: Deprtment de Genètic i de Microiologi, Fcultt de Ciències, Edifici Cn, Universitt Autònom de Brcelon, Bellterr, Brcelon, Spin; e-mil: enrico.rezende@u.ct. Present ddress: Deprtmento de Fisiologi, Instituto de Biociêncis, Universidde Estdul Pulist, Botuctu, CEP , Brzil. Physiologicl nd Biochemicl Zoology 82(6): y The University of Chicgo. All rights reserved /2009/ $15.00 DOI: / Introduction Locomotion is fundmentl to most ehviors. It is intrinsic in movements relted to territoril defense, forging, predtor escpe, mting interctions, migrtion, nd others, nd it is the presumed ncestrl source of mny evolved displys, such s the ritulized locomotor ptterns used s signls in courtship, ggression, nd other socil interctions (e.g., Irschick nd Grlnd 2001; Alexnder 2003; Perry et l. 2004; Husk et l. 2006; Oufiero nd Grlnd 2007). In these contexts, oth the limits to performnce nd the energy costs of locomotion re of considerle interest for ehviorl nd ecologicl physiology. With respect to performnce, interspecific comprisons hve shown tht locomotor ilities cn e correlted with spects of ehviorl ecology (Grlnd et l. 1988; Grlnd 1999). With respect to energetics, energy gthering, ssimiltion, nd/or prtitioning re ssumed to e centrl to Drwinin fitness in rod rnge of studies, nd in mny nlyticl pproches (e.g., optiml forging models), energy is used s proxy for fitness when testing hypotheses. Numerous comprtive studies show tht locomotion cn e energeticlly demnding, nd in mny mmmls, the highest ttinle whole-niml metolic rtes re chieved during running or flying (e.g., Tylor et l. 1982; Weiel et l. 2004). Thus, the energy costs of locomotion could potentilly constitute sustntil portion of n niml s dily energy expenditure (DEE). Becuse nimls re generlly presumed to e limited in their ilities to cquire nd expend energy (e.g., see Kotej et l. 2001; Vnholt et l. 2007), iologists routinely ssume tht trde-offs exist etween expenditures on locomotion, other mintennce requirements, nd such fitnesscriticl life-history trits s growth nd reproduction (Krsov nd Mrtinez del Rio 2007). However, the importnce of locomotor costs to the overll energy udget nd hence their potentil importnce to ehviorl evolution remins mtter of dete, especilly for smll mmmls. Some studies hve suggested tht these costs cn hve significnt effects on DEE under nturl conditions (Krsov 1992; Gormn et l. 1998; Corp et l. 1999; Girrd 2001), ut others indicte tht loco-

2 Locomotor Behvior nd Energy Expenditure in Mice 663 motor costs often comprise minor frction of DEE (Grlnd 1983; Altmnn 1987; Budinette 1991). Whether energy use in locomotion is high or low hs importnt implictions, oth for the fitness consequences of ehviors tht require movement nd for the mechnistic, suorgnisml trits tht underlie locomotor ehvior. Along with potentil trde-offs within the energy udget, high costs of locomotion nd ctivity, when coupled with ecologicl or socil requirements for sustntil movement, re expected to generte selection fvoring incresed locomotor efficiency. For exmple, we would expect morphologicl, physiologicl, nd ehviorl trits tht reduce locomotor costs to evolve concomitntly with incresed home rnge re or dily movement distnces (DMDs), which show enormous vriility mong species of mmmls (Hrestd nd Bunnell 1979; Grlnd 1983; Grlnd et l. 1993; Corp et l. 1999; McLoughlin nd Ferguson 2000; Kelt nd Vn Vuren 2001; Crone et l. 2005). Indeed, some ntomicl correltes of interspecific vrition in locomotor movements nd costs involving lim structure nd dimensions hve een identified (e.g., Kelly et l. 2006; Sockol et l. 2007). Also, ehvior cn hve sustntil effect on locomotor costs through n niml s choice of speeds, git, or movement intermittency (Hoyt nd Tylor 1981; Kengy nd Hoyt 1989; references in Girrd et l. 2001). Given the presumed importnce of locomotion nd energetics in ecologicl nd evolutionry physiology, direct, controlled tests of how vrition in locomotor ehvior ffects energy udgets or how selection on locomotor ehvior ffects running economy re surprisingly rre. Numerous comprtive studies provide useful insights into metolic rtes during locomotion nd the correlted evolution of ehviorl nd physiologicl trits (Budinette 1991; Grlnd 1999; Irschick nd Grlnd 2001; Rezende et l. 2004; Hely et l. 2005; Kelly et l. 2006), ut without informtion on running time or distnce or ehviorl or physiologicl trde-offs etween locomotion nd other energetic demnds, they provide limited insight s to the significnce of locomotion in energy udgets. Moreover, interspecific comprtive nlyses re complicted y sttisticl issues relted to phylogenetic signl nd often y confounding of independent vriles (e.g., Cloert et l. 1998; review in Grlnd et l. 2005). For exmple, mong mmmls, the species with the lrgest home rnges nd DMDs tend to e crnivores in the order Crnivor (Hrestd nd Bunnell 1979; Grlnd 1983; Grlnd et l. 1993; Corp et l. 1999; Kelt nd Vn Vuren 2001; Crone et l. 2005), so it is difficult to determine whether locomotor ehvior nd energetics hve evolved in concert with diet, ctivity levels, or other trits tht differ, on verge, etween Crnivor nd other mmmls. Another potentil prolem with most estimtes of locomotor energetics is tht costs re typiclly mesured during forced exercise t constnt speeds over long durtions (e.g., Tylor et l. 1982), sitution tht contrsts with the often highly intermittent running nd wlking ehvior of freely moving nimls (Kengy nd Hoyt 1989; Girrd et l. 2001; Vásquez et l. 2002). Here, we use n experimentl evolution pproch (Grlnd 2003; Swllow nd Grlnd 2005; Grlnd nd Kelly 2006; Grlnd nd Rose 2009; Rhodes nd Kwecki 2009) to exmine how locomotor ctivity nd energy udgets evolve in smll mmml. This study is unique for two resons. First, it employs respirometry system tht llows mesurements of ehvior nd energy costs of voluntry exercise with high temporl resolution over long periods (up to severl dys). We developed n enclosed running wheel coupled with stndrd housing cge tht permitted essentilly continuous mesurements of oxygen consumption ( Vo 2, stndrd mesure of metolic rte) nd running performnce. A study of deer mice (Peromyscus mnicultus; Chppell et l. 2004) vlidted the method nd showed tht individul vrition in running ehvior ws sustntil, tht mice rrely used speeds tht pproched mximum sustinle speeds, nd tht mice rn t wide rnge of speeds (contrry to hypotheses tht high speeds should e preferred to mximize trnsport efficiency; see lso Chppell et l on gerils). Second, this study involves mice from long-term selective reeding experiment (Swllow et l. 1998; Grlnd 2003; Rezende et l. 2006). This project hs produced four replicte lines of mice tht hve undergone multigenertionl selection for high voluntry wheel running (S lines) nd four nonselected control lines (C lines). After more thn 40 genertions, the S lines, compred with the C lines, run more thn twice s fr per dy on wheels during dys 5 nd 6 of 6-d period of wheel ccess nd show similr elevtion of home cge ctivity when housed without wheels (Rhodes et l. 2001; Mlisch et l. 2008, 2009; Vnholt et l. 2008). The motivtion nd/or rewrd systems of the S lines hve diverged in terms of wheel-running ehvior (Belke nd Grlnd 2007), gene expression in the hippocmpus (Bronikowski et l. 2004), nd rin neurochemistry nd phrmcologicl responses relted to wheel running (review in Rhodes et l. 2005; Keeney et l. 2008). Beyond locomotor ctivity, the S lines show differences from the C lines in predtory ggression (Gmmie et l. 2003), thermoregultory nest uilding (Crter et l. 2000), open-field tests (Bronikowski et l. 2001), nd other ehviors when oserved in regulr housing cges (Kotej et l. 1999). Compred with mice from the C lines, mice from the S lines re smller in ody size, hve less ody ft, nd show differences in reltive orgn sizes nd hind lim one dimensions (Swllow et l. 1999, 2005; Houle- Leroy et l. 2003; Grlnd nd Freemn 2005; Kelly et l. 2006; Middleton et l. 2008), nd t lest some of these ltertions seem clerly relted to running ilities (e.g., Grlnd nd Freemn 2005). The S lines show elevted mximl oxygen consumption during forced tredmill exercise (Rezende et l. 2006, 2006), higher tredmill endurnce (Meek et l. 2009), nd incresed insulin-stimulted glucose uptke y isolted extensor digitorum longus muscles (Dumke et l. 2001) ut few differences in resting glycogen levels or in depletion of glycogen stores during nightly running (Gomes et l. 2009), side from those relted to individuls expressing the mighty minimuscle phenotype (Houle-Leroy et l. 2003; Hrtmnn et l. 2008). Mice from the S lines show differences in circulting corticosterone, leptin, nd diponectin levels (Girrd nd Grlnd 2002; Girrd et l. 2007; Mlisch et l. 2007, 2008; Vnholt et

3 664 E. L. Rezende, F. R. Gomes, M. A. Chppell, nd T. Grlnd Jr. l. 2007, 2008) compred with those of the C line mice, ut whether these ffect locomotor ilities or energetics is uncler. As of genertion 21 22, the S mice showed no evidence of reduced reproductive performnce (Girrd et l. 2002; see lso Pontzer nd Kmilr 2009). Mice from S lines, compred with those from C lines, show higher ody tempertures t night when they re ctive (Rhodes et l. 2000) nd mintin ody mss etter when cold chllenged (Kotej et l. 2001) ut pprently do not differ in mximum cold-induced food consumption (Kotej et l. 2001) or in mximum food consumption when forced to work (run on wheel) to otin food (Vnholt et l. 2007). Overll, the S nd C lines cn offer roust insights into how locomotor propensities codpt with other trits, including energy udgets, nd the wys in which physicl ctivity cn ffect energy expenditure in freely locomoting nimls. This study hd three primry gols. The first ws to mesure with unprecedented ccurcy the frction of the energy udget composed of locomotor ctivity to determine whether it is potentilly significnt, tht is, more thn the roughly 1% of DEE predicted y severl uthors (Grlnd 1983; Altmnn 1987; Budinette 1991). This is essentil for guging the likelihood tht locomotor costs hve sustntil effect on DEE nd components of Drwinin fitness (e.g., litter size) nd hence led to dpttions tht reduce locomotor costs. The second ws to nlyze how the evolution of locomotor ctivity cn ffect energy expenditure nd vice vers. Specificlly, we tested whether the evolutionry increse in dily movement distnce in S lines resulted in proportionlly incresed DEE nd ddressed whether energetic constrints might e involved in the selection limit in running distnces oserved fter 16 genertions of selection (Grlnd 2003). The third ws to test whether differences in running ehvior etween S nd C lines (running speeds, numer nd durtion of running outs, etc.) ffect locomotor efficiency nd energy expenditure. Mteril nd Methods Animls nd Selection Procedure As descried y Rezende et l. (2006), we studied mice from genertions 32 (mles) nd 34 (femles) of the ongoing selection experiment. As detiled y Swllow et l. (1998), the originl progenitors were outred, geneticlly vrile lortory house mice (Mus domesticus) of the Hsd:ICR strin (Hrln Sprgue Dwley). After two genertions of rndom mting, mice were rndomly pired nd ssigned to eight closed lines (10 15 pirs in ech; out 10 fmilies per line were selected). In ech susequent genertion, 6 8-wk-old offspring were housed individully, with ccess to running wheel for 6 d. Wheel running ws monitored y n utomted system nd quntified s the totl numer of revolutions on dys 5 nd 6 of the 6-d test. In four selected lines (S), the highest-running mle nd femle from ech fmily were selected s reeders for the next genertion. In four control lines (C), mle nd femle were rndomly chosen from ech fmily. Within ll lines, siling mtings were not llowed. All spects of niml housing nd experimentl procedures were pproved y the University of Cliforni, Riverside, Institutionl Animl Cre nd Use Committee nd re in complince with U.S. lws. Metolic Rte Mesurements To determine energy costs of voluntry running, we used the protocol reported y Chppell et l. (2004) nd Rezende et l. (2006). Briefly, we enclosed commercilly ville rodent wheel (circumference 1.12 m; the sme model used in the selection procedure) nd stndrd plstic mouse cge within n irtight Lucite chmer (Fig. 1 in Chppell et l. 2004). The mouse cge contined edding (wood shvings) s well s food hopper nd wter tue tht were ville d li. Mice entered nd exited the wheel t will through n ccess port cut into the side of the mouse cge nd could run t speeds, directions, nd durtions of their choice. The enclosures hd pired incurrent nd excurrent ports for irflow nd n internl fn tht rpidly recirculted ir to fcilitte mixing. Two wheel enclosures were housed in lrge environmentl cinet tht controlled mient temperture (T ;20 27 C) nd photoperiod (12L : 12D; drk period hours). Temperture nd light cycles during mesurements were similr to conditions in our niml room. We regulted irflow to the enclosures (2,500 ml/min 1%) with upstrem mss flow controllers (Applied Mterils, Sunnyvle, CA; Tyln, Billeric; or Porter Instruments, Htfield, PA). Aout 100 ml/min of excurrent ir ws susmpled nd dried (mgnesium perchlorte) for gs nlysis y Sle Systems (Ls Vegs, NV) Oxzill dul-chnnel O 2 nlyzer nd Sle Systems CA-2A CO 2 nlyzers. Oxygen nd CO 2 concentrtion, flow rtes, wheel speed, nd T were recorded every 1.5 s on Mcintosh computers equipped with Ntionl Instruments A-D converters nd LHelper softwre (Wrthog Systems, Wheel speed ws mesured with tchometers (not y counting wheel revolutions; Eikeloom 2001; Kotej nd Grlnd 2001). Computer-controlled vlves took 2.5-min gs reference redings every 45 min. Procedures for seline djustment, smoothing, respirometry clcultions, nd instntneous conversions to estimte oxygen consumption rtes ( Vo ) nd CO 2 production rtes ( 2 Vco2) concomitntly with wheel running re detiled elsewhere (Chppell et l. 2004; Rezende et l. 2006). Experimentl Protocol To mimic conditions during selection trils nd llow cclimtion to the wheel system, mice tht were used for exercise cost trils hd ccess to stndrd running wheel nd mouse cge setup for 4 5 d efore mesurements. Mice were weighed to the nerest 0.1 g efore entering the cclimtion wheel (dy

4 Locomotor Behvior nd Energy Expenditure in Mice 665 0). Following the cclimtion period, they were plced in wheel respirometry chmer etween 1130 nd 1200 hours (i.e., the middle of the inctive phse of the dily cycle) for recording of ehvior nd metolism. The home cge ws trnsferred to the wheel chmer with ech mouse, so nimls were exposed to fmilir edding nd odors during mesurements. However, it ws not fesile to completely clen the wheel chmers etween trils. We first tested set of mle retired reeders from genertion 32 postselection. These mice were wheel cclimted for 5 d nd mesured on dy 6; we report dt for 49 individuls tht provided relile mesurements ccording to visul inspection of the originl records. Susequently, we tested 48 femles from genertion 34 postselection (six individuls from ech of four S nd four C lines, ech individul from different fmily). After wening t 21 d of ge, femles were rndomly mixed four per cge (except to meet the condition of two individuls from the C line nd two from the S line per cge). Mesurements egn t out 8 wk of ge. In slight chnge from the procedure for mles, the femles hd ccess to cclimtion wheels for 4 d. At dy 5, they were plced inside metolic chmer, nd metolism nd ehvior were recorded during dys 5 nd 6; we used the dt from dy 6 in nlyses compring mles nd femles (see Rezende et l. 2006). Becuse only two nimls could e mesured simultneously, mesurements were rndomly scheduled cross lines, except tht we roughly ttempted to control for ge effects (e.g., mice tht were orn first were lso mesured first), nd we lwys ttempted to mesure one S nd one C mouse concomitntly. The ltter criterion ws not lwys met for mles ecuse only one metolic chmer ws ville during some trils. For femles, we tested rottionl resistnce efore nd fter ech mesurement y spinning wheels to high speed ( 80 rpm) with n electric drill fitted with ruer friction disk nd then monitoring the time needed for speed to decy to 0. No chnges in resistnce occurred during the trils. The following vriles were extrcted for ech tril (totl recording period of h, etween 1200 nd roughly 1130 hours on the following dy): Vo 2 vm, mximum 1-min verge Vo 2 during tril (ml/min); RMR, resting metolic rte (mesured s the lowest continuous 5-min running verge of Vo 2 during periods of inctivity; ml/min); DEE, verge dily energy expenditure (totl Vo 2 /totl tril durtion; ml/ min); D run nd T run, totl distnce run (m) nd time spent running (clculted using speeds ove 0.5 rpm [0.56 m/min] to remove effects of electricl noise; min); V men, men running speed (D run /T run ; m/min, log trnsformed to improve normlity); V mx 1, mximum 1-min verge wheel speed (m/min); V pek, mximum instntneous wheel speed over 1.5 s (m/min, log trnsformed to improve normlity); N outs, numers of running outs (log trnsformed), defined s period of wheel rottion lsting 3 s or more t speeds ove 0.5 rpm in either direction of rottion (Chppell et l. 2004); nd BD men nd BD mx, men nd mximum durtion of running outs, respectively (expressed in s, log trnsformed in oth cses). Comprisons etween mesurements in dy 5 versus dy 6 in femles show tht these vriles re highly repetle (onetiled P in ll cses), so the oserved vrition should ccurtely reflect interindividul differences in physiology nd ehvior (detiled results re listed in Rezende 2005, p. 215). We lso clculted the cost of trnsport (COT) from the reltionship etween wheel speed nd Vo 2 for ech mouse. Asolute COT t given speed is Vo 2 /speed. The slope of the speed versus Vo 2 regression is the incrementl COT (the energy costs strictly ssocited with displcement, in units of energy distnce 1 ; Tylor et l. 1982; Grlnd 1983). The zero-speed intercept of this regression is typiclly elevted ove RMR, nd the difference (intercept RMR) is ssumed to e posturl cost of locomotion (Tylor et l. 1982; Fig. 1; see lso Dlugosz et l. 2009). Anlyses of incrementl COT for these mice re shown y Rezende et l. (2006). Figure 1. Interctions etween running speed nd metolic rte for typicl smll mmml, including the mice in this study (top). Resting metolic rte (RMR) is the miniml rte of energy expenditure of n inctive niml nd is ssumed not to chnge during running. Asolute cost of trnsport t given running speed is metolic rte/speed. The slope of the regression of metolic rte on speed is the incrementl costs of trnsport (icot, in units of energy per unit distnce). The intercept of the regression is typiclly elevted ove RMR, nd this difference is clled the posturl cost of locomotion. Continuous records of oxygen consumption (middle) nd wheel speeds (ottom) llowed us to prtition energy expenditure into different components, s shown digrmmticlly for dily energy expenditure (DEE), where DEE p RMR ctivity costs (gry, middle). Activity costs cn e susequently prtitioned into posturl costs, dily locomotor costs (DCOT), nd extr costs, s detiled in Mteril nd Methods.

5 666 E. L. Rezende, F. R. Gomes, M. A. Chppell, nd T. Grlnd Jr. Energy Budgets To clculte the components of energy udgets for ech individul, DEE ws roken down into RMR nd ctivity costs (net ctivity costs in ml O 2 /min p DEE RMR in ml/min, nd reltive ctivity costs p net costs/dee, expressed s %). Note tht costs of ctivity, s defined here, refer to the totl oxygen consumption ove RMR verged throughout the entire tril nd not costs ssocited only with wheel running (Fig. 1). Costs of locomotion (totl dily costs of trnsport [DCOT] posturl costs) re suset of totl ctivity costs (Fig. 1). We clculted DCOT s incrementl COT # D run. Similrly, we estimted the dily contriution of posturl costs s (intercept RMR)# T run. For comprisons with rtes of energy use (e.g., DEE, RMR), we divided DCOT nd posturl costs y tril durtion (c h). Activity costs include dditionl energy expenditures not directly relted to wheel locomotion (e.g., thermoregultion, digestion, ctivity outside of the running wheel). We estimted the contriutions of these extr fctors y sutrcting locomotion costs from ctivity costs (i.e., extr p net ctivity costs [DCOT posturl costs]). Sttisticl Anlyses Becuse mles nd femles were mesured under similr ut not identicl conditions (Rezende 2005, pp ) nd the sexes hve incresed totl running distnce in somewht different mnners in response to selection (Swllow et l. 1998; Grlnd 2003), nlyses were initilly performed seprtely for ech sex. However, results were consistent etween sexes, nd so we lso report nlyses of pooled dt (oth sexes) with sex in the model s n dditionl fctor. Age ws removed from these nlyses ecuse it ws highly correlted with mss when sexes were pooled tht is, mles were considerly older nd lrger thn femles (Tle 2). Effects of selection (line type; S vs. C) were estimted using one-wy (mles nd femles seprtely) or two-wy (pooled sexes) nested ANCOVA with Type III tests of fixed effects using mixed models in SPSS for Windows nd SAS PROC MIXED (ver. 8; SAS Institute, Cry, NC). Line-type effects with 1 nd 6 df were tested over the replicte lines rndom effect ( N p 8) nested within line type. Testing the effect of line type reltive to the vrition mong lines is necessry in principle ecuse it mtches the experimentl design (Grlnd nd Rose 2009) nd in prctice ecuse vrious trits show significnt differences mong the replicte lines (e.g., Kotej et l. 1999; Swllow et l. 2001; Gmmie et l. 2003; Bronikowski et l. 2006; Rezende et l. 2006; Mlisch et l. 2007; Kne et l. 2008; results of this study). Body mss nd ge were initilly included in the model s covrites. In some cses where solute rther thn mss-corrected vlues re of interest (e.g., running speeds nd distnces), nlyses were lso performed without ody mss in the model. Some trits were log trnsformed to improve normlity of residuls or to llow estimtion of llometric scling exponents. Adjusted lest squres mens nd ssocited stndrd errors were clculted to estimte the mgnitude of differences etween S nd C lines. Although we report two-tiled P vlues in the tles for simplicity (unless otherwise noted), we tested directionl hypotheses whenever pproprite to increse sttisticl power. For exmple, we expect tht selection hs led to higher DEE nd totl running distnces in S mice ecuse these lines voluntrily run more thn C lines. To nlyze how ehvior, running performnce, nd metolic rtes correlte t the level of individul vrition, we employed Person product-moment correltions etween residuls of ech vrile s computed from the nested ANCOVAs descried ove. We controlled for Type I errors in these multiple simultneous tests y use of the flse discovery rte method (Storey nd Tishirni 2003), testing P vlues with the QVALUE lirry ( in the R sttisticl pckge (R Foundtion for Sttisticl Computing). Results (see Tle 3) were considered sttisticlly significnt if the flse discovery rte ssocited with ech test tht resulted in P! 0.05 ws smller thn 5% (i.e., Q! 0.05). Results We se our clcultions of metolism nd energy use on rtes of oxygen consumption nd used the Vco 2 dt to test for selection-induced chnges in respirtory exchnge rtio ( RER p Vco 2/Vo2). We found no sttisticl differences etween S nd C mice in verge dily RER ( F1, 6 p 3.49, P p 0.11), RER t rest (i.e., estimted during the 5-min period of RMR; F p 0.22, P p 0.65), or RER during mximum vol- Tle 1: Effects of selective reeding for high voluntry wheel running (S vs. C lines), vrition mong replicte lines, nd sex differences while controlling for effects of ody mss P selection P lines P sex P ody mss P sex # selection Body mss ! Vo 2 vm ! DEE ! RMR ! DEE RMR V mx V pek V men D run T run.3916! N outs BD men BD mx 1, 6 Note. Comprisons etween S nd C lines in the nested model included sex s fixed fctor (SAS PROC MIXED). Age ws not included in the model ecuse it ws highly correlted with mss when sexes were pooled (i.e., mles were considerly older nd hevier thn femles). All P vlues re for twotiled tests, nd vlues significnt t P! 0.05 re in old. See text for descriptions of vriles nd revitions. Tested over the sex # line interction ( df p 1, 6). Anlyses performed with log-trnsformed dt (see text).

6 Locomotor Behvior nd Energy Expenditure in Mice 667 Tle 2: Adjusted lest squres mens nd stndrd errors for metolic nd locomotor trits sed on seprte nlyses of mles nd femles Selected Control S/C P selection P lines P ody mss Mles (N p 49): Vo 2 vm (ml O 2 /min) DEE (ml O 2 /min) RMR (ml O 2 /min) DEE RMR (ml O 2 /min) V mx 1 (m/min) V mx 1 (m/min) V pek (m/min), [33.46, 38.33] [28.24, 32.37] V pek (m/min) [33.78, 38.35] [28.23, 32.04] V men (m/min), [16.75, 19.88] [11.95,14.40] 1.39! V men (m/min) [17.24, 19.87] [11.97, 13.93] D run (m) 7, , !.0001 D run (m) 7, , T run (min) !.0001 T run (min) ! N outs 463 [326,655] 565 [399,802] BD men (s) 45.3 [38.5, 53.3] 27.7 [23.2, 33.1] BD mx (s) [314.0, 581.3] [190.5, 353.7] Femles (N p 47): Vo 2 vm (ml O 2 /min) DEE (ml O 2 /min) RMR (ml O 2 /min) !.001 DEE RMR (ml O 2 /min) V mx 1 (m/min) V mx 1 (m/min) V pek (m/min), [36.15, 43.08] [25.27, 30.00] V pek (m/min) [35.17, 43.59] [24.99, 30.79] V men (m/min), [18.22, 24.11] [11.46, 15.07] V men (m/min) [17.67, 24.89] [11.12, 15.51] D run (m) 9, , D run (m) 9, , T run (min) T run (min) N outs 228 [170,307] 471 [353,629] BD men (s) 95.3 [72.9, 124.4] 48.8 [37.6, 63.26] BD mx (s) [563.7,925.5] [330.6,535.4] Note. Significnce of the effects of selection history (S vs. C), line, nd ody mss re from nested ANCOVA, including ge s covrite. Adjusted mens SEs were clculted for mle of 37.4 g (125 d of ge) nd femle of 25.3 g of 71 d of ge. P vlues re for two-tiled tests, nd significnt vlues re in old. See text for descriptions of vriles nd revitions. Body mss not included in the model. Vlues re ck trnsformed from log mens otined in the mixed model; ck-trnsformed 95% confidence intervls re shown in rckets. untry Vo 2 ( F1,6p 3.35, P p 0.12). However, in ll cses femles hd significntly lower RER thn mles, nd the sex # line type interction ws never significnt. Although sttisticl comprisons were not presented, Figure 1 of Kne et l. (2008) lso shows tht femles hd lower RERs thn mles under sl conditions (fsted 3 h) t 23 mo of ge. However, study of one S nd one C line y Vnholt et l. (2008) did not show sex differences for RER over 24 h on either highcrohydrte or high-ft diet (see their Tles 2 nd 3; gin, sttisticl comprisons were not presented). Body Size, Resting Metolic Rte, nd Dily Energy Expenditure Body mss ws significntly smller in S lines regrdless of sex, nd mles were significntly lrger thn femles (Tle 1). Men mss ws g for S femles versus g for C femles; S mles verged g, wheres C mles weighed g. Accordingly, we controlled for mss nd sex when compring mong ehviorl nd metolic trits. Both ody mss nd sex (femles higher) strongly ffected

7 668 E. L. Rezende, F. R. Gomes, M. A. Chppell, nd T. Grlnd Jr. femles ws out 49% higher thn tht of mles, nd the RMR of S mice ws roughly 6% higher thn tht of C mice (not significnt). A previous study of sl (i.e., fsted) metolic rte in ICR mice from the sme source tht ws used to estlish the selection experiment discovered no sttisticl effect of sex (Dohm et l. 2001), so the difference found here for RMR my reflect the fct tht mles were much older thn femles (see lso Kne et l. 2008). Body mss lso significntly ffected DEE, nd fter correcting for running distnce, line type did not. However, in contrst to RMR, DEE ws not ffected y sex (Tle 1; Fig. 2). For DEE in n S line mle, generlized eqution tht corrects for running distnce is log DEE p ( ) ( ) log mss ( )D, run Figure 2. Reltionship etween ody mss, resting metolic rte (RMR), nd dily energy expenditure (DEE) for 49 mles (squres) nd 47 femles (circles). Symols re rw vlues for DEE (top; the verge energy expenditure for the entire 23.5-h period) nd RMR (middle; the lowest 5-min verge in the entire tril). After log trnsformtion, slopes ( SE) nd intercepts were clculted from one- or two-wy nested ANCOVAs controlling for ge nd running ctivity y including D run s covrite in DEE nlyses. Regressions for DEE nd RMR with oth sexes nd line types included (dotted lines) were clculted for 96-d-old individul running 5,975.4 m (equtions in top, middle). For sexes nlyzed seprtely (ut pooling line types), regressions (solid lines) were clculted for men ge of 125 d nd D run of 5,116.5 m for mles nd 72 d nd 6,870.8 m for femles (ottom). Allometries pper to e stright lines, despite the liner xes, ecuse the curvture is nerly imperceptile in this rnge of ody mss. Equtions for the complete models re shown in Results. RMR, ut line type (i.e., selection on voluntry running) hd no effect in either sex (Tles 1, 2; Fig. 2). If we reclculte the prtil regression coefficients from model similr to the one in Tle 1, generl eqution tht descries RMR in n S line mle is log RMR p ( ) ( ) log mss, where coefficients re shown SE, ody mss is in grms, nd RMR is in milliliters of oxygen per minute. For femles, ( 0.038) would e dded to the constnt, nd for C line mouse, the constnt would e decresed y ( 0.021). Accordingly, fter correcting for mss differences, the RMR of where DEE is in milliliters of oxygen per minute nd D run is in kilometers. For femle, the vlue ( ) would e dded to the constnt, nd for C line mouse, the term ( ) would e dded. Hence, t given mss nd running distnce, femle DEE is roughly 10% higher thn tht of mles, nd the DEE of C line mice is out 2% higher thn tht of S line mice, lthough neither difference is sttisticlly significnt. These equtions do not correspond exctly to the models in Tle 1: to estimte prtil regression coefficients, we log trnsformed dt to compute llometric reltionships, the sex # line type interction term ws not included (it ws never significnt when included; P ), nd D run ws dded s covrite for DEE. Comprisons of DEE nd RMR mong S nd C in Tle 1 were performed with untrnsformed dt, testing for sex # line type interction, nd testing for DEE did not control for differences in D run (which differed significntly etween S nd C lines; Tle 1). Activity nd Locomotor Costs As previously reported for this smple of mice (Rezende et l. 2006), individuls from S lines rn much more thn C mice (Tle 2): D run in S nd C lines ws out 7.51 km versus 3.92 km in mles (S 92% higher thn C) nd 9.64 km versus 5.83 km in femles (S 65% higher thn C). The chnge in running ehvior ws ssocited with chnges in energy metolism. Differences etween S nd C lines in DEE nd overll ctivity costs (DEE RMR) were sttisticlly significnt (one-tiled P! 0.05; Tle 1) ut proportionlly considerly smller thn for D run. In mles, mss-djusted DEE in S lines ws 22.8% higher thn in C lines (2.56 vs ml O 2 /min), ut in femles it ws only 5.8% higher (2.17 vs ml O 2 /min). After controlling for the effects of ody mss nd selection history, ll three metolic indexes (minimum, men, nd mximum power output; RMR, DEE, Vo 2 vm, respectively) were significntly positively correlted with ech other in oth mles nd femles (Tle 3). S mice of oth sexes hd higher Vo 2 vm thn

8 Locomotor Behvior nd Energy Expenditure in Mice 669 Tle 3: Person product-moment correltions etween residul metolic nd locomotor trits DEE RMR V mx 1 V pek V men D run T run N outs, BD men, BD mx, Mles (N p 49): Vo 2 vm DEE RMR V mx V pek V men D run T run N outs BD men.837 Femles ( N p 47): Vo 2 vm DEE RMR V mx V pek V men D run T run N outs ( N p 46), BD men ( N p 46),.735 Note. Correltions were performed etween residuls from one-wy nested ANCOVA with line type s the grouping fctor nd lines nested within line types (see Sttisticl Anlyses ). Age nd ody mss were included s covrites. Sttisticlly significnt correltions fter correcting for multiple comprisons (see Sttisticl Anlyses ) re shown in old. See text for descriptions of vriles nd revitions. Anlyses performed with log-trnsformed dt. Influentil point ws removed (femle 37255). C mice (Tle 2), consistent with the higher running speeds in S lines (Tles 1, 2). Additionlly, Vo 2 vm ws positively correlted to D run nd (in mles only) to T run. In summry, selection hs resulted in sustntil increse in dily wheel-running distnce in oth sexes, ut the effect on totl DEE is sustntil only in mles. After ccounting for differences in ody mss, ctivity costs (DEE RMR) in femles were not significntly different in S nd C lines, ut in mles, ctivity costs were 29.2% greter in S lines (one-tiled P p 0.055; Tle 2). Expressed s percentge of DEE, ctivity costs were positively correlted with ody mss ( F1, 79 p 4.05, two-tiled P p ) ut were significntly lower in femles (54.9 vs. 64.3%; F1, 6 p 13.21, P p ; Fig. 3). No significnt sex # line type interctions were detected. As previously reported (Rezende et l. 2006), S nd C mice do not differ in incrementl COT (i.e., the slope of the reltionship etween speed nd Vo 2 ) fter ccounting for differences in ody mss. In our smple, the net DCOT ws independent of ody size, regrdless of whether D run ws included in the model (Tle 4). As for D run nd (in mles) T run,oth selection history nd line significntly ffected DCOT, with S mice hving considerly higher DCOT thn C mice (mssdjusted DCOT out 40.5% higher in S thn in C in femles nd 77% higher in S thn in C in mles; Tle 4). As frction of DEE, DCOT ws lso higher in S mice thn in C mice, lthough the difference ws smll (out 6.3% 8.7% of DEE in C lines vs. 8.7% 12.7% in S lines; Tle 4; Fig. 3). There were lso significnt differences in DCOT mong lines, oth s mss-djusted vlue nd s percent of DEE (Tle 4). In ddition to DCOT, the other component of energy use in locomotion is posturl costs, which re dependent on T run ut independent of the speed of running. Posturl costs in our mice were independent of selection history nd sex ut were positively correlted to ody mss nd vried mong lines (Tle 4); T run did not differ significntly mong S nd C femles (Tle 2), nd T run in S mles ws out the sme s for S nd C femles (Tles 2, 4). Hence, C mles, which rn for considerly less time thn S mles or femles of either line type, tended to hve lower posturl costs thn other groups for oth mss-djusted vlues or percent of DEE (Tle 4). Differences in mss-djusted posturl costs pproched significnce in pirwise comprisons etween C mles nd oth S mles nd S femles (two-tiled P p nd P p 0.070, respectively). Posturl costs exceeded DCOT (oth mss djusted nd percent of DEE) y roughly fctor of three in oth sexes nd line types, with the smllest difference in C mles (Tle 4; Fig. 3). These results show tht posturl costs encompss the lrgest

9 670 E. L. Rezende, F. R. Gomes, M. A. Chppell, nd T. Grlnd Jr. Figure 3. Contriution of resting metolic rte (RMR), posturl costs, nd dily costs of trnsport (DCOT) to dily energy expenditure (DEE). Vlues re shown s verge Vo 2 for the entire 23.5-h tril, djusted for vrition in ody mss (left) nd reltive to DEE (p100%; right). The totl energy cost of running is the sum of posturl costs nd DCOT. Extr is the mount of DEE not included in RMR, posturl costs, or DCOT, nd it presumly results from thermoregultory costs, energy spent on digestion, nd ctivity out of the running wheel (see Mteril nd Methods ). Brs represent djusted mens for 31.4-g individul clculted with SAS PROC MIXED from two-wy nested ANCOVA controlling for ge nd for sex # line type interction (ut not for D run ; i.e., model 2 in Tle 3). frction of energy expenditure involved in wheel running in S nd C lines wheres net costs of displcement re less importnt. Although S mice rn lmost twice s fr per dy s C nimls, we did not find consistent line-type effect on totl running costs (DCOT posturl costs) in either sex (Tle 5). Line type ws not significnt predictor of totl running costs in most models, lthough it pproched significnce in one nlysis for femles. Running Behvior Numerous spects of running ehvior differed etween S nd C line types, ut some of these differences were dissimilr in mles nd femles, nd there ws lso considerle vrince mong the replicte lines. In S lines, femles incresed D run minly y incresing running speed with little chnge in running time, while in S mles, oth speed nd T run tended to increse (Tle 2; Fig. 4; Rhodes et l. 2000; Rezende et l. 2006). As expected, energy use ws ffected y running ehvior: correltions etween DEE nd D run or T run were positive nd highly significnt in oth sexes. After ccounting for ody mss differences, RMR ws strongly positively correlted with T run nd D run in mles ( P! in oth cses) ut not in femles (Tle 3). Net ctivity costs were highly correlted with D run in oth sexes, nd T run (rther thn speed) ws the min fctor ccounting for this ssocition fter controlling for other covrites (Tle 5). Similrly, reltive ctivity costs (% of DEE) were significntly correlted with T run in oth sexes ut not with running speed (Tle 5). Selective reeding incresed the mximum (V pek, V mx 1) nd the men voluntry wheel-running (V men ) speeds of oth sexes (Fig. 5), with corresponding increses in power use during running (Fig. 6). All three speed indexes were positively correlted with mximum power output ( Vo 2 vm) ut not with DEE or RMR. Selection lso ffected the numer of running outs (N outs ) nd their durtion (oth BD men nd BD mx ; Tle 2); sex lso ffected N outs nd BD men. In oth sexes, S mice hd fewer running outs (significntly in femles; Tle 2) ut incresed out durtion compred with C mice; D run ws lso significntly correlted with N outs mong femles ut not mles. Interestingly, mles ut not femles showed strong positive ssocition etween T run nd BD men nd BD mx.bothbd men nd BD mx were negtively correlted with N outs in oth sexes, lthough this ssocition ws weker in mles (not significnt for BD mx ; Tle 3). DEE ws positively correlted with N outs in mles ut not in femles. The sme ws true for correltions etween DEE nd out durtion (oth BD men nd BD mx ), which were positive nd significnt only for mles. In summry, energy costs seem to e primrily determined y the mount of time spent running rther thn running speeds, nd differences etween mles nd femles my stem from the different strtegies dopted y ech sex to run longer distnces. Discussion The intent of this study ws to exmine ehviorl nd energetic consequences of selection-induced vrition in voluntry running ehvior. These issues re of rod importnce for understnding the effects of ehviorl evolution on energy udgets nd vice vers. Physiologiclly, running is one of the most demnding nd energeticlly expensive of ll ehviors; therefore, it is intuitive to expect tht ehviors involving extensive locomotion might comprise sustntil portions of totl DEE. Whether selection fvoring incresed running ctivity in ny ecologicl or socil context cn result in correlted responses to improve running economy will depend on the effect of locomotion on totl time nd energy udgets. For instnce, sep-

10 Locomotor Behvior nd Energy Expenditure in Mice 671 Tle 4: Contriution of resting metolic rte (RMR), posturl costs, nd dily costs of trnsport (DCOT) to dily energy expenditure (DEE), expressed in solute vlues nd reltive to the totl RMR (ml O 2 /min) Posturl (ml O 2 /min) DCOT (ml O 2 /min) Extr (ml O 2 /min) RMR (% DEE) Posturl (% DEE) DCOT (% DEE) Extr (% DEE) Model 1: Femles C Femles S Mles C Mles S P: Line type Line Body mss! Sex Sex # line type D run.1327!.0001! !.0001!.0001!.0001!.0001 Model 2: Femles C Femles S Mles C Mles S P: Line type Line.8674! Body mss! Sex Sex # line type Note. Extr refers to the frction of DEE not ccounted for y the previous estimtes (see Mteril nd Methods ). Vlues re djusted mens SE otined from SAS PROC MIXED independently for ech trit, controlling or not for differences in totl running distnce (Model 1 nd Model 2, respectively). Significnce of ech fctor is listed for ech trit, nd vlues sttisticlly significnt (two-tiled P! 0.05 ) re in old. Percentges were lwys pproximtely normlly distriuted, so no trnsformtion ws performed efore nlyses. See text for descriptions of vriles nd revitions. rte selection experiments in rts nd mice support the hypothesis of positive genetic correltion etween voluntry ctivity nd mximum eroic performnce during forced exercise (Swllow et l. 1998; Rezende et l. 2005, 2006; Wters et l. 2008), ut it remins uncler to wht extent incresed wheel-running distnces dd dditionl costs to DEE. In this context, the results of our study re unique ecuse we were le to directly estimte the ssocitions etween running ehvior, locomotor performnce, nd energy udgets s well s how they evolve in response to selection for high ctivity levels. Costs of Locomotion nd Running Behvior Although locomotion costs re frequently ssumed to e sustntil portion of totl energy use, rodscle comprtive nlyses y Grlnd (1983), Altmnn (1987), nd Budinette (1991) predicted trnsport costs of roughly 1% of DEEs in mmmls smller thn 100 g rising to perhps 10% 15% of DEE in lrge, moile crnivores. These surprisingly low vlues derive from clcultions sed on incrementl costs of trnsport (icot; Fig. 1) nd estimtes of DMD in nturl hitts (i.e., locomotor cost p icot # DMD); Grlnd (1983) termed this the ecologicl cost of trnsport (ECT). The ECT is useful in n ecologicl context ecuse its units (energy mss 1 distnce 1 ) re independent of speed nd, ccordingly, its estimtion does not require detiled informtion on locomotor ehvior. An ECT mounting to 1% of DEE seems unlikely to hve much effect on resource lloction nd hence spects of Drwinin fitness, suggesting little potentil for selection to improve running economy nd little selection ginst incresed moility (t lest from the stndpoint of energy costs). However, our results indicte considerly higher locomotor costs. If running costs re clculted s DCOT (DCOT p icot # DMD; Tle 4), then our mice expended from 6.3% to 12.7% of their DEE on locomotor ehvior, with S mice hving slightly higher DCOT thn C mice (Fig. 3). These estimtes re similr to previous reports of DCOT in mice from erlier genertions of our selection experiment (4.4% 7.5% of DEE sed on food consumption nd wheel-running dt; Kotej et l. 1999) nd lso resemle results from deer mice (Peromyscus mnicultus) tested in the sme wheel respirometers (6.3% of DEE; Chppell et l. 2004) nd from free-living golden-mntled ground squirrels (Spermophilus sturtus; 13%; Kengy nd Hoyt 1989). Wht ccounts for the contrst etween the very low predicted ECT nd the much higher mesured DCOT? To some

11 672 E. L. Rezende, F. R. Gomes, M. A. Chppell, nd T. Grlnd Jr. Tle 5: Reltionship of ctivity nd running costs with line type (S vs. C lines), totl running distnces, totl running time, nd verge running speed in mles nd femles Model df Totl Activity Costs (DEE RMR) Running Costs (DCOT Posturl Costs) Net (ml O 2 /min) Reltive (% DEE) Net (ml O 2 /min) Reltive (% DEE) F P F P F P F P Mles: Line type 1, Line type 1, D run 1, ! ! !.0001 N outs 1, ! Line type 1, T run 1, ! ! !.0001 V men 1, N outs 1, Femles: Line type 1, Line type 1, D run 1, ! ! !.0001 N outs 1, ! !.0001 Line type 1, T run 1, ! ! !.0001 V men 1, N outs 1, Note. One-wy ANCOVAs with replicte lines nested within line types (SAS PROC MIXED) were performed seprtely for mles nd femles. Body mss nd ge were included s covrites in ll models. Numer of outs ws included in some models to control for vrition in running ehvior. All sttisticlly significnt effects hd positive sign. P vlues re from two-tiled tests. See text for descriptions of vriles nd revitions. Log-trnsformed to improve normlity. Significnt line differences ( P! 0.01). degree, methodologicl differences my e responsile. Estimtes of running costs derived from tredmill tests might e ised reltive to costs of running in the field for severl resons (see Altmnn 1987; Krsov 1992; Corp et l. 1999; Rezende et l. 2006). Similrly, running in wheels my hve somewht different costs from running over typicl terrin, lthough wheel-derived slopes (icot) re not sustntilly different from those otined vi tredmill testing (Chppell et l. 2004; Rezende et l. 2006). Another possile prolem is tht running time in wheels is often tulted in 1-min ins, which hs een criticized for not ccurtely reflecting running ehvior (Eikeloom 2001; Girrd et l. 2001; Kotej nd Grlnd 2001). However, the most likely cuse of the ECT-DCOT discrepncy is estimtes of movement distnces. Some studies hve rgued tht ECT clcultions were ised due to underestimtion of DMD in the field (Altmnn 1987; see lso Kengy nd Hoyt 1989; Budinette 1991; Krsov 1992; Corp et l. 1999). For exmple, dily wheel running in g muroid rodents rnges from 3 to 16 km (Dewsury 1980; Grlnd 2003; Tle 2), ut estimted DMD from field studies of smll (!100 g) rodents is less thn 1.0 km (Grlnd 1983; Crone et l. 2005). It is certinly possile tht l-rered nimls with d li. food, no risk of predtion, nd so on, run more thn would e the cse for free-living wild nimls (see Sherwin 1998), ut it seems unlikely tht this lone could ccount for the severlfold difference etween field nd lortory estimtes of DMD, ECT, nd DCOT. Clerly, ccurte informtion on dily movements in nturl hitts is prerequisite for roust estimtion of ECT for free-living nimls, s ws emphsized y Grlnd (1983). A criticl considertion for clculting the energetic consequences of running ehvior is the so-clled posturl costs of locomotion: the elevtion ove resting metolism of the intercept of the speed versus metolic rte regression (see lso Dlugosz et l. 2009). Posturl costs re independent of speed nd pper to e n unvoidle expense in studies of oth forced (e.g., Tylor et l. 1982) nd voluntry (Chppell et l. 2004; Rezende et l. 2006) running, lthough they my e lower in the ltter (Chppell et l. 2004, 2007). In our mice, posturl costs were severl times lrger thn DCOT in oth S nd C lines (Tle 4; Fig. 3). Consequently, the totl cost of running (DCOT posturl cost) comprised 26% 28% of the energy udget in C lines nd 32% 36% of the energy udget in S lines. The greter running distnce in S lines compred with C lines did increse running expenditures nd DEE. However, those increses were proportionlly quite smll compred with the lmost twofold difference in running distnce etween line types nd generlly did not ttin sttisticl significnce (Tle 5). Similrly, t genertion 10, Kotej et l. (1999) found