This is the published version:   Buttemer,W.A.,Bech,C.andChappell,M.A.2010,Citratesynthaseactivitydoesnotaccountforage‐relateddifferencesinmaximumaerobicperformanceinHouseSparrows(Passerdomesticus).,Australianzoologist,vol.35,no.2,pp.378‐382.

Available from Deakin Research Online:  http://hdl.handle.net/10536/DRO/DU:30020868ReproducedwiththekindpermissionofthecopyrightownerCopyright:2010,RoyalZoologicalSocietyofNewSouthWales

Citrate synthase activity does not accountfor age-related differences in maximumaerobic performance in House Sparrows{Passer domesticus),

William A. Buttemer', Claus Bech^ and Mark A. ChappelP' School of Biological Sciences, University of Wollongong,Wollongong, NSW, 2522, Departmentof Biology, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway, and ^Department of Biology, University of California, Riverside, Riverside, California 92521, USA.

We measured basal (BMR) and peak metabolic rates (PMR) in juvenile and adult HouseSparrows. Juvenile birds had significantly higher BMR, but lower PMR than adult birds, despitehaving statistically indistinguishable body masses. We then evaluated the relation betweenPMR and masses of central and peripheral organs and found that pectoral muscle mass bestcorrelated with PMR in both groups, accounting for about 35% of the variation in PMR. Becausecitrate synthase (CS) has such major importance in affecting the first committed step in thetricarboxylic acid cycle, we characterized CS activity levels in extracted muscles to see if thisbetter explained age-related differences in peak aerobic performance. Surprisingly, juvenilesparrows had significantly higher CS activity levels than adults (197.8 vs. 179.0 JM g ' min ' ,respectively).This higher enzyme activity in juveniles was completely offset by their significantlysmaller proportion of flight musculature relative to body mass (17.7 % in adults vs. 15.3% injuveniles). Consequently, ontogenetic changes in relative sizes of organs best accounts for age-related differences in peak metabolic rate.

Key words: peak metabolic rate, citrate synthase, aerobic capacity, ontogeny, house sparrows

IntroductionFrom the time of the earliest hunters, humans have heenfascinated by the large amount of variation in animallocomotor endurance. This interest is still prevalentand has provoked research focused on understandingthe physiological and morphological factors affectingaerohic performance. These studies have led to diverginghypotheses as to which physiological/morphologicalcomponents set the limits to a given species aerohiccapability. Some have proposed that these limits are set bythe capacity of peripheral effector organs (Peterson et al.1990), by the capacity of central "supply" organs (Kirkwood1983; Weiner 1992; Koteja 1996), or are associated withequal capacity of all components (symmorphosis; Weihel etal. 1991). One way to test these hypotheses is to compareproportionate sizes of peripheral and central organs amongspecies that differ significantly in aerohic performance(e.g., Daan et al. 1991), or to do this among memhers ofthe same species showing a range of locomotor abilities.We previously used the latter approach to evaluate themorphological traits of juvenile and adult house sparrows(Passer dimxesticus) in relation to their hasal and peakmetabolic rates (BMR and PMR, respectively; Chappell etal. 1999). Because the sizes of hoth a central organ (heart)and a peripheral tissue (pectoral muscle) accountedsignificantly for variation in PMR, our results supportedthe symmorphosis model of aerobic capacity. Importantly,however, pectoral muscle mass best correlated with PMRand BMR among all tissues considered.

A tacit assumption of inferring organ capacity frommass alone is that the mass-specific capahilitiesof organs among species or of individuals withinspecies are the same. Studies comparing the structureof mammalian locomotor muscles among 'athletic'versus 'non-athletic' species reveal the weakness ofthis assumption (Weihel et al. 2004). Their studyshowed that highly aerobic species had muscleswith significantly greater mitochondrial and capillarydensities than their less athletic counterparts.Although we did not characterise mitochondrial orcapillary properties of sparrow locomotor muscles, wedid save leg and flight muscle samples to suhsequentlyexamine citrate synthase (CS) activities. This enzymecatalyses the first step of the tricarhoxylic acid cycleof mitochondrial respiration and is believed to bea surrogate of tissue aerobic capacity (Hochachkaet al. 1988). If sparrows show substantial variationin pectoral muscle CS activity, then mass-specificaerohic capacity of these muscles should vary tothe same extent. If this were so, we would expectmeasurements of total pectoral CS activity to correlatebetter with PMR than pectoral mass alone. We reporthere the effect of including CS activity in evaluatingthe relation hetween PMR, BMR and pectoral musclemass in a subset of sparrows we studied previously(Chappell et al. 1999).

Theme edition of Australian Zoologist "£co/ogy meets Physiology", ?i Gordon Grigg festschrift, edited by Lyn Beard,Daniel Lunney, Hamish McCallum and Craig Franklin.Australian Zoologist Vol 35 (2) 2010.

Citrate synthase activity

Material and Methods ^Study AnimalsWe captured free-living house sparrows {Passer domesticus)residing in the IUawarra region of NSW hetweenNovember and December. Birds at this time of yearcomprise adults and juveniles that are mostly 2-4 monthsold. We obtained 36 adults and 30 juvenile birds withnear-equal gender distribution in each group for a studyexamining the relation between organ morphology andmetabolic performance (Chappell et al. 1999). We havesubsequently characterised citrate synthase activity inmtiscle samples ftom 32 of these adults (15 females, 17males) and 28 of these juveniles (13 females, 15 males)and report these results in relation to their metabolic andmorphological measurements. Because most methodsinvolved in these comparisons are detailed in Chappell etal. (1999), only an overview is provided here. -

Peak Metabolic RateOn a given day, 4 birds were caught early morningand placed in a cage with ad libitum access to seedand water. About midday, each hird was placed ina motorised, revolving 5-litre drum that had clearsides and carpet lining the inner rim. A mass-flowcontroller supplied air at 5 1 min' and the gas contentof inlet and outlet ports was measured with an oxygenanalyser (Sable Systems FC-1). Birds were introducedinto the chamber, allowed 2 min to settle, andthen the motor was activated. The flight drum alsocontained ping-pong balls, which encouraged birdsto maintain a series of rapid takeoffs and short-termflights. Measurements were terminated at the firstindication of exercise fatigue by a bird. The metabolicdata were transformed to 'instantaneous' values usingmethods described by Bartholomew et al. (2001). Thehighest 60-sec instantaneous oxygen consumptionrate averaged over a continuous 60-sec interval wastermed Peak Metabolic Rate (PMR). Birds were thenreturned to cages and allowed to feed and drink beforemeasurement of basal metabolic rates (BMR).

Basal Metabolic RateFood was removed from cages at 1500 and each bird wasremoved at 1800 and then transferred to a respirometerfashioned from a 2-1 paint can fitted with inlet andoutlet ports, a perch, and a thermocouple to measurechamher temperature. The respirometers were housedin a temperature-controlled cahinet set to 32.5 °C, athetmoneutral temperature for this species (Hudsonand Kimsey 1966), and were each supplied an airflowof 500 ml min'. Inlet and outlet oxygen concentrationswere measured on a 2-channel oxygen analyser (AppliedElectrochemistry S-3A) and recorded using an A-to-Dconverter (Data Electronics) connected to a Macintoshcomputer. Oxygen consumption measurements continueduntil the foUowing morning, with the lowest continuous10-min interval recorded over this petiod designated asBMR. Birds were then returned to cages and allowedto feed and drink for 2 hours before being killed formorphological characterization.

Morphological MeasurementsBirds were weighed to the nearest 0.05 g prior todissection. Organs were removed, trimmed of excess fatand connective tissue, rinsed with physiological saline,and blotted dry before being weighed to the nearest 0.1mg. The contents of the heart and intestine were emptiedbefore being rinsed, blotted, and weighed. Sampled organsincluded liver, lungs, gut (small and large intestines),gizzard, lungs, heart, kidneys, and reproductive organs.In addition, the entire right pectoral musculature and allleg muscles proximal to the tibiotarsus-tarsometatarsusjoint were removed and weighed. All organs and musclesamples were then dried to constant mass at 60 °C andthen reweighed. Samples of left pectoral muscles and leftleg were also excised, weighed, and snap frozen in liquidnitrogen for subsequent enzyme analyses.

Citrate synthase measurementsMuscle samples were minced with fine scissors and aportion of these then weighed to the nearest 0.1 mg beforebeing homogenised in 20 volumes of 100 mM potassiumphosphate and 2 mM EDTA. Homogenisation was firstperformed using 3 10-sec pulses of an Ultra-Turrax T-25tissue homogeniser while the sample was surroundedby ice, followed by 1-min of final homogenisation usingground-glass tissue grinders. Citrate synthase activity wasmeasured using Dawson and Olson's (2003) modificationof the method described by Srere (1969). This involvedadding a 0.1 ml volume of the muscle homogenateto a final 1 ml volume comprising 100 mM Tris-HCl,0.2 mM s-aceryl CoA, 0.1 mM DTNB (5,5'-dithiobis(2-nitrobenzoic acid)), 2.5 mM EDTA, 0.5 mM cis-oxaloacetate maintained at 25 °C. The reaction is initiatedhy addition of the oxaloacetate, with the rate of CoA-SHproduced measured by rate of change in absorbance at412 nm on a recording spectrophotometer (Beckman).All reagents and enzymes were purchased from the Sigma-Aldrich Chemical Co.

Statistical AnalysesBecause most morphological and physiological featuresof animals are influenced hy body mass, examination ofgender and age effects on these variables were analysedusing an ANCOVA general linear model, with genderand age as fixed factors and body mass as a covariate(SPSS). Correlation analyses were used to compare theamount of variance accounted by different regressionmodels. Some post-hoc analyses employed T-tests, withmultiple comparisons using Bonferroni correction factorsto correct for accrual of Type I errors. The level ofsignificance was p<0.05 for all statistical comparisons.Unless stated otherwise, all values are reported as mean± one standard error

Results and DiscussionAlthough house sparrows are known to be size dimorphic,these tendencies are greatly reduced in populationsliving in regions with limited annual variation intemperature (Murphy 1985). This may explain thenear absence of sexual dimorphism in the traits we

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measured in Illawarra birds. The only organs to differbetween sexes were the slightly larger liver and gizzardin adult females compared to adult males (Ghappellet al. 1999). By contrast, juvenile and adult sparrowsdiffered significantly in a number of morphologicalcharacteristics. Gompared to adults, juveniles displayedhypertrophied digestive organs (gut, gizzard, liver,and kidneys; Table 1). This enlargement most likelyrepresents the retention of traits permitting very rapidgrowth as chicks by recently fledged juveniles (Klaassenand Drent 1991). In contrast to diffetences in digestiveorgan sizes, leg and pectoral muscles were significantlysmaller in juveniles than in adults (Table 1; Fig. 1).Because these groups had statistically indistinguishablebody masses, this implies that juveniles have potentiallyless locomotor capacity than adults. Apart from thesemorphological differences, there were significant age-related differences in both basal and peak metabolicrates (Table 1), witb juveniles having higher BMR,yet lower PMR than their adult counterparts. It isworthwhfle to see how the age-related differences inmorphological features are associated with variation inaerobic metabolism.

Qne of the first studies to identify morphologicalinfluences on BMR concluded that interspecificvariation was largely due to proportionate differencesin the relative size of hearts and kidneys (Daan et al.1990). This approach has been extended to evaluateintraspecific variation in BMR, but typically with theorgans appearing to be responsible for this variationdiffering between species (e.g., Daan et al. 1990versus Ghappell et al. 1999) or at different times ofyear within species (Vezina and Williams 2003). Ithas been suggested that the lack of consistency inexplaining variation in BMR is due to its reliance onthe assumption that the organs being compared havethe same metabolic intensity (Vezina and Williams2005). Although we did not measure tissue-specificmetabolic rate, the activity of citrate synthase (GS)is believed to be a surrogate of tissue aerobic capacity(Hochachka et al. 1988; Vezina and Williams 2005).

Pectoralmuscles

Legmuscles

LocomotorTotal

Figure I. Pectoral, leg, and total (leg + pectoral) musclemasses as proportions of total body mass in adult andjuvenile house sparrows, juvenile proportions are allsignificantly smaller than tbose of adults (P<O.OI5 for allcomparisons). Columns represent mean proportions fortbe 32 adults and 28 juveniles sampled and the error barsdesignate I standard error

Qur measurements of GS activity show that thepectoral muscles of juvenfles have significantly highermass-specific activity, resulting in there being nodifference in total muscle GS activity between the agegroups (Table 2). The GS levels measured in juvenilepectoral muscles agree closely with values reported byQ'Gonnor and Root (1993) for house sparrows capturedin Michigan in late autumn. In contrast to pectoralmuscle, leg muscle GS mass-specific activity did notdiffer between groups, but because leg muscle mass wassignificantly smaller in juveniles, their total GS activitywas significantly lower than in adults (Table 2).

Table I. Age effectscomparisons for alldifferences betweenbetween adults and

on morphological and physiological variables of 32 adult and 28 juvenile house sparrows. Statisticalvariables except body mass are based on ANCOVA with body mass as a covariate. Body massage classes are based on ANOVA. P values of less than 0.05 identify statistically significant differencesjuveniles.

Variable, units

Body Mass, g

Lungs, g

Heart, g

Gizzard, g

Liver; g

Gut, g

Kidneys

Pectoral muscles, g

Leg Muscles, g

BMR, ml O j min-'

PMR, ml Oj min-'

Adults

23.10 + 0.35

0.246 ± 0.009

0.262 + 0.007

0.872 + 0.028

0.619 + 0.018

0.430 + 0.017

0.240 ± 0.006

2.051 + 0.046

0.977 ± 0.022

0.97 + 0.03

9.99 ± 0.32

Juveniles

22.34 ± 0.40

0.251 + 0.005

0.241 ±0.004

1.054 + 0.037

0.786 + 0.020

0.659 ± 0.023

0.284 ± 0.005

1.886 ±0.038

0.899 ± 0.020

1.06 ± 0.02

8.81 ±0.26

P adults vs.Juv.

0.15

0.28

0.051

<O.OOOI

<O.OOOI

<O.OOOI

<O.OOOI

0.025

0.037

0.001

0.017

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volume 35 (2) 2010

Citrate synthase activity

Table 2. Age effects on citrate synthase activity in leg and pectoral muscles of 32 adult and 28 juvenile house sparrows.Statistical comparisons are based on ANCOVA with body mass as a covariate. P values of less than 0.05 identifystatistically significant differences between adults and juveniles. Specific activity (SA) is in relation to a gram of muscletissue, whereas total activity (TA) is the product of specific activity and total mass of a given muscle.

Variable, units

Leg SA, |j.mol min' g"'

Leg TA, |imol min '

Pectoral SA, |amol min'' g"

Pectoral TA, i^mol min

Adults

67.8 ± 2.3

132.7 ± 5.6

173.8 + 4.8

707.8 ± 24.7

Juveniles

61.7 + 2.2

1 1 1.4 ±4.8

197.8 + 5.7

751.3 ± 30.7

P Adults vs. Juv.

0.088

0.008

0.028

0.091

If characterisation of CS activity in muscles betteraccounts for their resting metabolic needs as suggestedby Vezina and Williams (2005), we would expect tbetotal CS activity, the product of CS mass-specific activityand total muscle mass, to better correlate with BMRthan muscle mass alone. After controlling for body massinfluences, the correlation between pectoral total CSactivity and BMR in botb groups combined is, in fact,lower (r = 0. 495) than for pectoral muscle mass alone(r = 0.506; 47 dO. Furthermore, total pectoral CS ofjuvetiile sparrows showed much weaker correlation withBMR (r = 0.39; P=0.043) than did wet pectoral mass(r = 0.56; p=0.003). This trend is also evident for legmuscles in that the correlation between leg mtiscle totalCS activity and BMR was weaker (P=0.51) than for legmuscle mass alone (P=0.18). On the other hand, if CSactivity is a valid indicator of tissue oxidative capacity(Emmett &. Hochachka 1981; Hochachka et al. 1988), wewould expect total pectoral CS activity to have a strongercorrelation with PMR than pectoral mass alone. Somewhatsurprisingly after controlling for body mass, variation inpectoral muscle mass better explains PMR differencesamong all sparrows measured (r = 0.58, P <0.001; Fig.

3 4 5 6

Pectoral Muscle Mass, gFigure 2. Peak metabolic rate during flight exercise inrelation to pectoral mass of adult (open circles) andjuvenile (filled circles).

2) than does variation in pectoral total CS activity (r =0.29, P = 0.03; Fig. 3). Among adult sparrows, PMR wassignificantly correlated with pectoral mass (r = 0.63, p =0.001), but not with pectoral total CS activity (r = 0.33,P = 0.07). In contrast, both pectoral mass and pectoraltotal CS activity were significantly correlated with PMRin juveniles (P < 0.035 for both comparisons).

Assuming pectoral total CS activity to be a valid descriptorof that muscle's aerobic capacity, we would have toconclude that the significantly lower PMR of juvenilesis not due to age-related differences in pectoral muscleoxidative potential. This assumption depends, of course,on how well muscle CS activity describes its aerobiccapability. It is revealing that pectoral muscle mass-specificCS activities tend to be slightly higher in chicks aboutto fledge than in adults in both common terns (Sternahmmda; Burness et al. 2005) and barnacle geese i^rantaleucopsis; Bishop et al. 1995). Given the absence of flightexperience in these chicks, it would be very surprising iftheir flight PMR could match that of experienced adults.On the other hand, Hammond et al. (2000) found thatmale red junglefowl (Callus galhts) had significantly higherPMR and muscle citrate synthase activities than females,

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Figure 3. Peak metabolic rate during flight exercise inrelation to total citrate synthase activity of adult (opencircles) and juvenile (filled circles) pectoral muscles.

2010 z!oologist volume 35 (2) 381

Buttemer et al.

but the highly dimorphic nature of this species makes itdifflcult to gauge the contribution of CS activity to PMRdifferences. While they did report dominant males tohave higher mass-specific CS activities than subordinates,these social groups did not differ in PMR (Hammond etal. 2000). It is also important to recognise that aerobicmetabolic pathways involve many enzymes, which rarely

show concordant scaling with overall aerobic performance(Darveau et al. 2005). What we can conclude is thatmuscle CS activity does not constrain aerobic capacity injuvenile house sparrciws. Instead, attainment cif adult-levelPMR may require ontogenetic changes in morphologicalstructures that are associated with adult peak aerobicperformance.

AcknowledgmentsWe thank the Australian Research Council and theInstitute of Conservation Biology at the University ofWoUongong for supporting this research. All experiments

were approved by the University of WoUongong AnimalEthics Committee in accordance with National Healthand Medical Research Council guidelines.

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