Effects of physiological state, mass change and diet on plasma metabolite profiles in the western sandpiper Calidris mauri

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1 The Journl of Experimentl Biology 28, Pulished y The Compny of Biologists 25 doi:1.1242/je Effects of physiologicl stte, mss chnge nd diet on plsm metolite profiles in the western sndpiper Clidris muri D. A. Semn 1, *, C. G. Guglielmo 2 nd T. D. Willims 1, 1 Deprtment of Biologicl Sciences, Simon Frser University, 8888 University Drive, Burny, V5A 1S6, Cnd nd 2 Division of Biologicl Sciences, University of Montn, Missoul MT 59812, USA *Present ddress: Ecology nd Evolutionry Biology, University of Arizon, Tucson, AZ 85721, USA Author for correspondence (e-mil: tdwilli@sfu.c) Accepted 14 Decemer 24 We used food restriction/refeeding protocol to put irds through controlled cycle of mss loss nd mss gin to investigte the effects of rte nd phse of mss chnge on plsm metolite levels in reltion to diet. Despite mrked differences in ft content of the two diets (18% vs 4%) men rte of mss loss or mss gin ws independent of diet. There ws lso no effect of diet on plsm levels of ny of the four mesured metolite (triglyceride, glycerol, uric cid nd β-oh-utyrte) during mss loss. However, during mss gin irds on the low ft diet hd higher plsm levels of triglyceride nd uric cid nd lower β-oh-utyrte thn irds gining mss on the high-ft diet. Thus, diet composition cn ffect plsm metolite profiles independently of differences in rtes of mss chnge. Nevertheless, certin plsm metolites were relted to vrition in rtes of mss chnge cross physiologicl sttes. Glycerol levels Summry were negtively relted to the rte of mss chnge (independent of diet), nd utyrte ws negtively relted to the rte of mss chnge on oth diets (though the slope of this reltionship ws diet dependent). Uric cid ws positively relted to the rte of mss chnge ut only for irds on the low-ft diet. Our study therefore confirms tht mesurement of plsm metolites cn provide roust informtion on physiologicl stte (gin, loss) nd the rte of mss chnge (e.g. in free-living irds cught only once) lthough reserchers should e cogniscent of potentil confounding effects of diet composition for certin metolites, oth for field studies nd for future experimentl vlidtions of this technique. Key words: metolites, mss chnge, migrtion physiology, fttening, cptive shoreirds. Introduction Refueling or fttening rtes re thought to e importnt determintes of the timing nd success of long-distnce migrtion in irds (e.g. Alerstm nd Lindstrom, 199), nd recently decreses in refueling rtes hve een implicted in rpid popultion declines in some Arctic-reeding shoreirds (Brown et l., 21; Bker et l., 23). However, empiricl dt on rtes of refueling, i.e. mss or ft gin, re difficult to otin directly in free-living irds through mesurement of ody mss ecuse of prolems ssocited with recpturing individuls, especilly in very lrge, synchronously migrting popultions, nd potentil effects of cpture stress (Guglielmo et l., 25; ut see Dunn, 2). Furthermore, single mesurements of ody mss or condition cn provide unrelile informtion on dynmic rtes of mss chnge (Willims et l., 1999). Severl recent studies hve suggested tht mesurement of plsm metolite concentrtions might provide useful informtion on rtes of mss chnge or physiologicl stte (e.g. feeding vs fsting; Jenni-Eiermnn nd Jenni, 1994; Willims et l., 1999; Jenni nd Schwilch, 21), ptterns of fuel utiliztion (Gnnes, 21; Jenni-Eiermnn et l., 22) nd inter-popultion or inter-site vrition in fttening rtes (Schu nd Jenni, 21; Guglielmo et l., 22). Direct vlidtion of the use of plsm metolite profiles to predict mss chnge in free-living irds will require independent ssessment of fttening rtes, which is difficult ecuse of the prolems ssocited with multiple recptures of individuls, s outlined ove (ut see Guglielmo et l., 25). Thus, the iologicl interprettion of chnges in plsm metolite concentrtions in free-living individuls is currently sed on reltionships etween metolite concentrtions nd rte of mss chnge estlished in cptive individuls (e.g. Jenni- Eiermnn nd Jenni, 1994; Willims et l., 1999). However, there hve een reltively few thorough vlidtions of this technique, nd it is uncler how the reltionship etween metolites nd mss chnge is ffected y cptivity itself (see Lmrechts et l., 1999), or fctors such s vrition in diet qulity, or the rte of mss chnge itself. For exmple, DeGrw et l. (1979) found tht metolite concentrtions in cptive white-crowned sprrows (Zonotrichi leucophrys) differed

2 762 D. A. Semn, C. G. Guglielmo nd T. D. Willims from those in free-living individuls. Diet qulity hs een shown to influence fuel utiliztion nd fttening rtes in some psserines (e.g. Birlein, 1998; Gnnes, 21), ut, to our knowledge, the effect of diet composition on plsm metolite concentrtions hs not yet een tested. This informtion is importnt since it cn indicte how roust msschnge-dependent vrition in metolite levels will e in the context of even greter ecologicl vriility experienced y free-living irds. In this pper, we exmine the effects of rte nd trjectory of mss chnge nd diet qulity on plsm metolite levels (triglyceride, glycerol, uric cid nd β-oh-utyrte) in western sndpipers (Clidris muri, Cnis), long-distnce migrting, Arctic-nesting shoreird. The specific ojectives of the study were () to experimentlly mnipulte rtes of mss chnge nd mss trjectory, using high- nd low-ft diets, to investigte effects of diet qulity on metolite levels during different mss cycle phses or physiologicl sttes (i.e. mss loss, mss gin, nd stle mss), nd () to determine the effect of diet qulity on the reltionship etween plsm metolite levels nd rte of mss chnge. Finlly, since there is often mrked individul vriility in plsm metolite levels, even for given mss or physiologicl stte (e.g. Guglielmo et l., 22; Ydenerg et l., 22) we lso investigted whether mesures of ody condition or plsm metolite levels t time of cpture (i.e. in free-living individuls) predicted vrition in these prmeters in the sme irds in cptivity. Mterils nd methods Animls nd diets Femle juvenile western sndpipers were cptured t two migrtory stopover sites in the Frser River Delt, Boundry By (49 4, ) nd Roert s Bnks (49 3, ), during the southwrd migrtion in August 21 (N=1) nd 22 (N=36), using mist nets (Avinet, Dryden, New York, USA). When possile, lood smples were tken from individuls upon cpture ( wild phse) to otin free-living plsm metolite levels. Cpture nd nding ws crried out under United Sttes Fish nd Wildlife Service, Wshington Deprtment of Fish nd Wildlife, Environment Cnd, nd Simon Frser University Animl Cre permits. Birds were mintined in cptivity t the Animl Cre Fcility t Simon Frser University, in outdoor viries (3 m 6 m 2 m high) on nturl light cycle, with n infrred het lmp nd d liitum ccess to wter. Birds were provided with diet of oiled egg (with shell) nd melworms for the first few dys of cptivity nd were trnsitioned slowly to diet of Clrke s Fry trout chow (see elow). After irds were successfully trnsitioned to the stndrd diet, sed on stiliztion of ody mss, ll irds were lood smpled to otin pre-tretment metolite vlues with d liitum food ( pre-tretment phse) prior to further diet mnipultion. Two different diets were used in this experiment. Our stndrd diet ws 1.5 mm (width) pelleted Clrk s Fry trout chow (Moore-Clrk, Vncouver, Cnd); the sme diet ws used in our previous studies (Willims et l., 1999; Egeler et l., 23) nd is similr to tht used in other studies (Jenni- Eiermnn et l., 22). This high-ft, high-protein diet, referred to herefter s the high-ft diet, is primrily fish melsed nd consists of 47% protein, 18% ft, 2% fier, 9% sh nd 16.5% nitrogen-free extrct (NFE), with totl energy content of 18.2 kj g 1. The second diet used ws Hikri Cichlid By 2. mm (dimeter) pellet (Hikri USA, Hywrd, Cliforni, USA), low-ft, high-protein diet, referred to herefter s the low-ft diet. This is primrily fish mel-sed nd consists of 35% protein, 4% ft, 5% fier, 9% sh nd 37% NFE, with totl energy content of 16.2 kj g 1 (this second, low-ft diet more closely pproximtes to the ft content of nturl prey items; see Egeler nd Willims, 2). Generl experimentl protocol Dily food consumption (g ird 1 dy 1 ) ws mesured s the comined mount for ll irds in ech experimentl group throughout the experiment (see elow for finl smple sizes). All irds were weighed weekly (±.1 g), nd irds were weighed every other dy during periods of mss loss nd gin, to closely monitor mss chnge. Any individuls with ody mss <22 g (len mss in this species; Guglielmo nd Willims, 23) were seprted out nd excluded from the experiment. These individuls were held in seprte pen on the high-ft diet nd if they recuperted to mss >22 g they were used in susequent experiments. Birds were cught few t time (4 5 irds per experimentl group) nd lood smpled indoors to llow the remining individuls to return to norml forging ctivity etween disturnces; irds returned to forging within 1 min of the resercher vcting the viry. Totl disturnce time, the numer of minutes etween the first disturnce of irds in cge nd the finl ird eing cught nd smpled in tht cge, verged 23.7±14.9 min. Bleed time, etween cpture nd lood smpling of ech individul, verged 7.6±3.7 min, nd 9% of the leed times were less thn 12 min. All irds were lood smpled nd mss recorded etween 1. h nd 12. h PST (Pcific stndrd time; so time of dy, or time since lst mel ws not confounding fctor in our study, see Discussion). Birds were lood smpled vi rchil venipuncture with 26.5 guge needle nd lood (up to 3 µl) ws collected with heprinized cpillry tues nd centrifuged t 18 g for 1 min. Plsm ws drwn off using heprnized cpillry tues nd ws stored t 2 C until ssyed. Diet experiments Experimentl work ws conducted over severl months eginning in Octoer 22. Ad liitum food ws defined s 12.5 g ird 1 dy 1, sed on preliminry experiments where verge food consumption ws 6.6±1.7 g ird 1 dy 1. Birds were divided into two experimentl groups (low-ft nd highft diets), eqully divided in terms of ge (juveniles, N=36, cught in August 22, nd yerlings, N=4, cught in 21) nd cpture site (Boundry By nd Roert s Bnk). Birds

3 Plsm metolites nd mss chnge 763 were trnsitioned from the high- to low-ft diet over 2 week period. In Tril 1, oth the high-ft nd low-ft groups were cycled through mss loss (food restriction) nd mss gin (refeeding). Blood smples were tken in the middle of the Loss phse (dy 7 reltive to the end of the experiment, see Fig. 1) nd 2 dys fter refeeding during the Gin phse (dy 2, Fig. 1). Mss loss ws chieved y food restriction, during which time irds received 85% of the verge food consumption (g ird 1 dy 1 ) for tht group. To ensure continued mss loss, food ws further restricted to 8% of verge consumption during the finl 2 dys of the Loss phse. At the end of the mss Gin phse, once ody mss stilized, irds were lood smpled gin during d liitum food consumption ( Adli phse), nd the irds were left on tht diet for t lest 2 weeks fter the lst lood smpling efore eing switched to the lternte diet nd used in Tril 2. The sme cycle of mss loss/gin ws then repeted on the lternte diet (Tril 2) so tht ech individul served s its own control. This rndom order design ccounts for possile time of yer effects, given tht minimum of 1 month seprted the two cycles ecuse of recovery nd trnsition periods. This experimentl design lso controls for possile effects of experience (i.e. irds hving previously een through loss gin cycle), ecuse hlf of the irds experienced diet mnipultion for the first time on the high-ft diet nd the other hlf experienced the mnipultion for the first time on the lowft diet. Some irds (N=7) in the first group trnsitioned to the low-ft diet were unle to stilize ody mss on tht diet nd were isolted from the rest of the irds. As soon s their ody mss did stilize they were run through the mss cycle on the low-ft diet s seprte group. There ws no significnt difference etween these two groups of irds in the rte of mss chnge during either the Adli or Loss phses (P>.7, oth phses). The lighter irds tht were seprted chieved mrginlly lower rte of mss gin thn the originl group (F 1,13 =5.4, P=.45); however, there ws no significnt difference in the percentge of mss gin or loss chieved etween these two groups (P>.1, oth prmeters). Therefore, the results of these two groups were pooled together nd nlyzed s single set of irds on the low-ft diet in Tril 1. Dt were pooled etween trils nd nlyzed for ech diet tretment (low- or high-ft). Finl smple sizes were N=19 for irds cycled through loss gin cycle on the low-ft diet, N=28 for on the high-ft diet nd N=15 for the mss cycle on oth diets. Plsm metolite ssys Free plsm glycerol nd triglyceride were ssyed using sequentil color endpoint ssy (Trinder regent A nd B, respectively, Sigm-Aldrich Cnd, Okville, Ontrio, Cnd), using 5 µl of smple with 24 µl nd 6 µl of regents A nd B respectively, with reding tken t 54 nm fter 1 min of incution t 37 C fter the ddition of ech regent. Triglyceride concentrtion (mmol l 1 ) ws clculted y sutrcting free glycerol from totl glycerol. Uric cid ws ssyed vi color endpoint ssy (WAKO USA, Richmond, Virgini, USA), using 5 µl of smple with 3 µl of regent, with reding tken t 55 nm fter 1 min of incution t 37 C. Assys were run in 4 µl flt-ottom 96-well micropltes (NUNC, Denmrk) nd red with microplte spectrophotometer (Biotek 34EL). Ech plte ws run with stndrd curve sed on seril dilution of 2.54 mmol glycerol (Sigm-Aldrich Cnd, Okville, Ontrio, Cnd) for the triglyceride-glycerol ssy nd 2.97 mmol uric cid (prepred in our lortory) for the uric cid ssys. Ech plte lso included 19-dy-old hen plsm pool used to clculte interssy coefficient of vrition. Inter-ssy coefficients of vrition were 3.1% (N=11), 7.% (N=11), 6.1% (N=4) nd 9.4% (N=5), nd intr-ssy coefficients of vrition were 3.2% (N=6), 3.9% (N=6) nd 3.1% (N=17) for glycerol, triglyceride nd uric cid respectively. β-oh-utyrte ws nlyzed y one of us (C.G.G.) vi kinetic endpoint ssy (Guglielmo et l., 25). Sttisticl nlysis All four metolite levels were non-normlly distriuted, so we trnsformed the dt using ln(metolite) to pproximte normlity nd trnsformed dt were used in ll nlyses. Owing to the very short period of mss gin, we clculted Mss over the first two dys of mss gin (etween dy 4 nd 2). However, the period of mss loss ws more prolonged. We therefore clculted Mss over the 3-dy period prior to the Loss lood smple (i.e. etween dy 1 nd dy 7, Fig. 1). We considered the shorter intervls of mss gin nd loss (2 3 dys) s more iologiclly relevnt since they pproximte verge flight nd stop-over times for migrting western sndpipers (Wrnock nd Bishop, 1998). The reltionship etween the rte of mss chnge ( Mss; g dy 1 ) nd residul metolite vlues (controlling for covrites s necessry, see elow) ws tested for ech metolite independently on pooled dt set including individuls nd in ll three mss cycle phses, using liner mixed model with repeted mesures to ccount for repetition of individuls with diet s min effect. Testing the mss chnge-metolite reltionship for ll phses of the loss-gin cycle with single model llowed for mximiztion of rnge of mss chnge. In ech cse if the Mss*diet interction term ws significnt the reltionship etween Mss nd metolite concentrtion ws tested seprtely y diet; otherwise the interction term ws dropped from the model. All sttisticl nlyses were crried out using SAS (SAS Institute, 199). Results Vrition in rtes of mss chnge in reltion to diet mnipultion On oth diets the trjectories of mss chnge were similr for Trils 1 nd 2 (Fig. 1). On the low-ft diet, there ws no difference in initil, lowest or finl mss etween trils (P>.5 in ll cses; Tle 1). In contrst, on the high ft diet initil nd lowest msses were lower in Tril 2 thn Tril 1 (P<.5), ut there ws no difference in finl mss (Tle 1). For rte of

4 764 D. A. Semn, C. G. Guglielmo nd T. D. Willims Body mss (g) A B Loss Gin Dys from end of experiment Fig. 1. Men ody mss of cptive western sndpipers during mss loss nd gin cycles (A) during Tril 1 (open symols) nd Tril 2 (closed symols) on low-ft (squres) nd high-ft (circles) diets, nd (B) pooled dt for the two trils on different diets. Dys of experiment hve een stndrdized from the end of the experiment. Arrows indicte time of lood smpling for the loss nd gin phses. mss chnge, there ws no difference etween trils for either mss gin or loss on the low ft diet (P>.5). For the high-ft diet there ws similrly no tril difference for mss loss (P>.3), ut rte of mss gin ws higher in tril 2 compred with tril 1 (P<.1). However, since we were primrily interested in the reltionship etween vrition in mss chnge nd plsm metolites, nd since there ws control for ody mss, we pooled dt from Trils 1 nd 2 for susequent nlysis (see Fig. 1). For the pooled dt, the lowest mss reched during Loss nd the finl mss reched during Gin were oth lower for irds on the low-ft diet thn for irds on the high-ft diet (lowest mss, dy 4, t 43 = 3.4, P<.1; finl mss, dy, t 41 = 2.26, P<.5; Fig. 1); however, initil mss ws independent of diet (t 42 = 1.11, P>.2; Fig. 1B). As we hd nticipted, mss cycle phse (gin, loss, d li) hd highly significnt effect on rte nd trjectory of mss chnge (lowft diet, F 2,16 =75.2, P<.1; high-ft diet, F 2,21 =32.3, P<.1; Tle 2). For oth diets, ll pir-wise contrsts of mss chnge etween mss cycle phses were highly significnt (P<.1 in ll cses). However, there ws no effect of diet on rte of mss chnge for either the Loss, Gin or Adli phses of mss chnge (P>.6 in ll cses; Tle 2). Men Tle 1. Initil, lowest nd finl ody mss (g) of cptive western sndpipers during mss loss nd gin cycles y tril (Trils 1 nd 2) nd diet (high ft nd low ft) Diet Mss (g) Tril 1 Tril 2 P High ft (N=15,13) Initil 29.1± ±5.3.2 Lowest 25.6± ± Finl 3.8± ± Low ft (N=14,5) Initil 27.7± ± Lowest 23.2± ±.9.81 Finl 28.5± ±3..79 Vlues re mens ± S.D.; N vlues in prentheses re for Trils 1 nd 2, respectively. mss chnge ws significntly different from zero for the Loss nd Gin phses, ut not when irds were on Adli food (Tle 2). For ech diet, size-corrected ody mss (controlling for trsus length) ws positively correlted etween ll three mss cycle phses (low-ft: r>.5, P<.2; high-ft: r>, P<.4 ll cses), i.e. the heviest individul during Loss would lso e the heviest individul during Gin. In contrst, none of the four metolite vlues were correlted within individuls etween different phses of mss chnge on either diet (P>.7, ll cses). Effects of leed time, hndling time nd ody mss on metolite levels Plsm glycerol levels were independent of plsm levels of the other three mesured metolites (P>.1 for ech comprison with the three other metolites), so this metolite ws nlyzed seprtely. There ws positive correltion etween triglyceride nd uric cid levels (r=7, P<.1), nd oth triglyceride nd uric cid were negtively correlted with β-oh-utyrte (triglyceride, r= 7, P<.1; uric cid, r=.56, P<.1). Plsm triglyceride levels were positively relted to leed time (triglyceride: F 1,25 =6.64, P<.2, =.16), wheres glycerol, uric cid, nd Tle 2. Rtes of mss chnge for cptive western sndpipers during different phses of n experimentlly induced mss loss nd gin cycle on low-ft nd high-ft diet Phse Diet Mss chnge (g dy 1 ) Rnge Loss Low-ft.57±.12** 1.14 to.17 High-ft.61±.38* 2.89 to 2.11 Gin Low-ft 2.3±.87**.75 to 4.43 High-ft 2.16±1.31** to 5.56 Adli Low-ft.7± to.98 High-ft.± to 2.64 Vlues re mens ± 95% CI, with rnges; N=18 (low-ft) nd N=23 (high-ft) diet. Asterisks indicte mss chnge is significntly different from zero, *P=.1, **P<.1.

5 Plsm metolites nd mss chnge 765 β-oh-utyrte levels were independent of leed time (P>.1, in ll cses). In ddition, plsm triglyceride nd uric cid were negtively relted to totl disturnce time (triglyceride, F 1,172 =5.7, P<.2, =.7; uric cid: F 1,171 =16.9, P<.1, =.11), while β-oh-utyrte levels were positively relted to totl disturnce time (F 1,158 =7.75, P<.1, =.9). Plsm triglyceride nd uric cid were positively relted to ody mss (triglyceride, F 1,25 =56.68, P<.1, =.69; uric cid, F 1,23 =3.26, P=.7, m=.18) wheres β-oh-utyrte ws negtively relted to ody mss (F 1,178 =12.97, P<.1, =.41). For susequent nlyses, of triglyceride, uric cid nd β-oh-utyrte residul metolite vlues were clculted from regression nlysis including mss, disturnce time or leed time s fctors where pproprite. Glycerol ws independent of ll three fctors (ody mss, leed time, nd totl disturnce time, P>.3, in ll cses) nd log trnsformed plsm glycerol concentrtions will e referred to s glycerol levels herefter. Effect of mss cycle phse nd diet on plsm metolite levels On the low-ft diet, there ws n overll phse effect for ll four metolite vlues, i.e. metolite levels vried significntly in reltion to phse of mss chnge (repeted mesures ANOVA, P<.1 in ll cses; Fig. 2). In contrst, on the high-ft diet, there ws significnt overll phse effect for glycerol nd uric cid (P<.2 in oth cses), ut triglyceride nd β-oh-utyrte levels were independent of phse of mss chnge (P>.1; Fig. 2). Birds losing mss hd higher glycerol levels thn irds in oth Gin nd Ad Li phses of mss chnge, on oth diets (P<.5), with no difference in glycerol levels etween Gin nd Adli phses (Fig. 2B). Similrly, on the low ft diet, irds losing mss hd lower levels of uric cid nd higher levels of β-oh-utyrte thn irds in oth Gin nd Adli phses (P<.5; Fig. 2C,D). On the high ft diet, irds losing mss lso hd lower uric cid levels thn in the Adli phse (P<.1), ut uric cid levels were only mrginlly different etween Loss nd Gin phses (P=.8; Fig. 2C). There were few significnt differences in metolite levels etween Gin nd Adli phses of mss chnge, except tht triglyceride levels were higher during the Adli phse on the high-ft diet (P<.5; Fig. 2A). Compring metolite levels etween diets, within ech phse of mss chnge there were no significnt differences in metolite levels etween the low- nd high-ft diets for the Loss phse (P>.1; ll four metolites). For oth the Gin nd Adli phses of mss chnge, irds on the low-ft diet hd higher triglyceride nd uric cid levels nd lower β-ohutyrte levels thn those on the high-ft diet (P<.1 in ll cses), ut glycerol levels were independent of diet (P> in oth cses; Fig. 2). Reltionship etween metolites nd rte of mss chnge For triglyceride nd glycerol levels, the Mss*diet interction ws not significnt (P>.9), so it ws dropped from the model. Triglyceride levels were independent of the rte of mss chnge (F 1,41 =2.2, P>.1; Fig. 3A). However, there ws significnt effect of diet for this reltionship with irds on the low-ft diet hving higher triglyceride levels thn irds on the high-ft diet (F 1,27 =83.63, P<.1), consistent with the nlysis of men metolite levels for ech phse. Glycerol levels were negtively relted to the rte of mss chnge (F 1,4 =14.59, P<.5, =.1) nd this reltionship ws independent of diet (F 1,24 =.19, P>.6; Fig. 3B). For uric cid nd β-oh-utyrte, there ws significnt Mss*diet interction term (uric cid: F 1,32 =6.2, P<.2; β-oh-utyrte: Residul ln(triglyceride) A C Residul ln(uric cid) ln(glycerol) B Loss Gin Adli Loss Gin Adli Phse Phse D Residul ln(β-oh-utyrte) Fig. 2. Vrition in plsm metolite levels in reltion to phse of mss chnge for (A) triglyceride, (B) glycerol, (C) uric cid nd (D) β-ohutyrte levels in cptive western sndpipers on low-ft (squres) nd high-ft (circles) diets. For ech metolite, nd within ech diet tretment, points with the sme letter re not significntly different (P>.5).

6 766 D. A. Semn, C. G. Guglielmo nd T. D. Willims A C Residul ln(triglyceride) Residul ln(uric cid) ln(glycerol) B Mss (g dy 1 ) D Mss (g dy 1 ) Residul ln(β OH utyrte) Fig. 3. Reltionship etween the rte of mss chnge ( Mss) nd (A) triglyceride, (B) glycerol, (C) uric cid nd (D) β-oh-utyrte levels for cptive western sndpipers on low-ft diet (closed squres, roken line) nd high-ft diet (open circles, solid line). F 1,28 =25.64, P<.1); therefore we nlyzed ech diet seprtely for these metolites. Uric cid ws positively relted to the rte of mss chnge for irds on the low-ft diet (F 1,23 =1.2, P<.5, =.12); however, uric cid levels were independent of the rte of mss chnge on the high-ft diet (F 1,32 =.83, P>; Fig. 3C). β-oh-utyrte ws negtively relted to the rte of mss chnge on oth diets (low-ft: F 1,15 =73.81, P<.1, =.3; high-ft: F 1,27 =5.13, P=.3, =.6; Fig. 3D). Comprison of mss nd metolite levels in free-living nd cptive irds There ws no correltion etween size-corrected ody mss nd ny metolite vlues etween irds t the time of originl cpture nd the Pre-tretment phse (P>.2, ll prmeters). Similrly, there ws no correltion for these prmeters etween time of cpture nd irds in the Wild nd Gin phses on either diet (P>.1, ll cses). Birds fter trnsition into cptivity, given n d liitum high-ft diet, prior to diet mnipultion (Pre-tretment) hd lower uric cid levels nd higher glycerol nd β-oh-utyrte levels thn they did t the time of originl cpture (glycerol: t 35 =2.54, P<.2; uric cid: t 31 = 4.4, P<.5; β-oh-utyrte: t 17 =4.48, P<.5). However, triglyceride levels did not differ etween pretretment nd time of cpture (t 33 = 1.17, P>.2). Birds ctively gining mss (Gin) on the sme high-ft diet hd higher vlues of β-oh-utyrte nd lower vlues of the other three metolites thn t the time of cpture (triglyceride: t 25 = 5.83, P<.1; glycerol: t 26 = 2.82, P<.1; uric cid: t 23 = 5.11, P<.1; β-oh-utyrte: t 15 =5.19, P<.1). In contrst, irds during the Gin phse on the low-ft diet did not differ from the originl time of cpture in ny of the metolite levels (glycerol: P>.5; ll others: P>), i.e. the plsm metolite profiles on the low-ft diet were more similr to those of free-living, migrtory irds. Discussion Food restriction/refeeding llowed us to successfully put irds through controlled cycle of mss loss nd mss gin, to rigorously investigte the effects of phse of mss chnge nd diet, on plsm metolite levels. Surprisingly, even though we used experimentl diets of very different ft content (18% nd 4%) there ws no difference in the men rte of mss loss or mss gin etween diets. Consistent with this lck of difference in rte of mss chnge, during mss loss there ws no effect of diet on plsm levels of ny of the four mesured metolites (triglyceride, glycerol, uric cid nd β-oh-utyrte). However, during mss gin irds on the low-ft diet hd higher plsm levels of triglyceride nd uric cid nd lower β-oh-utyrte thn irds gining mss on the high-ft diet, despite the fct tht rte of mss gin ws not different. This diet effect on plsm metolites ws lso pprent in irds on d liitum food when ody mss ws more or less stle, confirming tht diet composition cn ffect plsm metolite profiles independently of differences in rtes of mss chnge. Nevertheless, we confirmed tht certin plsm metolites cn provide informtion on vrition in rtes of mss chnge

7 Plsm metolites nd mss chnge 767 cross physiologicl sttes. In prticulr, in this study, glycerol levels were negtively relted to the rte of mss chnge (independent of diet), nd β-oh-utyrte ws negtively relted to the rte of mss chnge on oth diets (though the slope of this reltionship ws diet dependent). Uric cid ws positively relted to the rte of mss chnge ut only for irds on the low-ft diet. Finlly, neither mss or metolite levels of free-living irds t cpture predicted their mss or metolite responses during cptivity when on the high-ft diet. It is importnt to note tht the low ft diet (4%) we used more closely pproximtes the verge lipid content of mrine inverterte prey trgeted y western sndpipers on mudflts (2%; Egeler nd Willims, 2). Consistent with this, the plsm metolite profiles of cptive irds gining mss on the low-ft diet were not significntly different from those of freeliving irds t the time of cpture (which we ssume were fttening t the stop-over site where they were cught). There re severl fctors tht could hve complicted our experimentl design, e.g. time of yer nd the effects of molt. Jenni-Eiermnn et l. (22) showed tht in congeneric shoreird, the red knot, there is nturlly occurring cycle in fttening cptivity, which is reflected in chnges in plsm metolite concentrtions. However, in our experiment irds went through the mss chnge cycle on the two different diets in rndom order. In ddition, our entire experiment took plce etween Octoer nd Jnury, which flls within stle mss phse in the nturl cycle reported in the study of Jenni- Eiermnn et l. (22). We re, therefore, confident tht time of yer effects do not confound our results. Plsm triglyceride levels hve een shown to vry with timing of molt (Jenni- Eiermnn nd Jenni, 1996; Totzke nd Birlein, 1998; Jenni- Eiermnn et l., 22). However, our study (Octoer to Jnury) occurred lmost exclusively outside of the periods of ody molt for the western sndpiper. No individuls in our study experienced wing molt. Body molt ws oserved in few of the individuls, however, only towrd the end of the second tril (Jnury), so we re confident tht molt does not confound our results. Plsm metolite concentrtions nd physiologicl stte Numerous studies in cptive psserines hve shown tht plsm metolite concentrtions reflect the physiologicl stte of ird in reltion to fttening or fsting (DeGrw et l., 1979; Jenni-Eiermnn nd Jenni, 1994; Totzke nd Birlein, 1998). Jenni-Eiermnn et l. (22) showed tht certin plsm metolites (triglyceride nd β-oh-utyrte) reflect sesonlly chnging metolic processes mong different life cycle stges (molt, migrtion, oversumering) in cptive red knot (with irds on similr diet to our high-ft diet). Our study confirms some of the results of Jenni-Eiermnn et l. (22; β-oh-utyrte), nd extends their results for other metolites (glycerol), ut there re lso differences etween their study nd ours for uric cid nd triglyceride. In oth the red knot nd the western sndpiper periods of mss gin re chrcterized y low β-ohutyrte levels, reltive to phses of mss loss. However, this reltionship etween mss gin nd β-oh-utyrte ws only evident on the low-ft diet in our study: on the high-ft diet β- OH-utyrte did not differ for irds losing or gining mss, even though solute rte of mss gin ws the sme s for the low-ft diet. In western sndpipers we found higher uric cid levels ssocited with mss gin, ut gin this ws only significnt on the low ft diet. Jenni-Eiermnn et l. (22) found no significnt chnge in uric cid levels cross different life stges, despite irds showing cler periods of ody mss increse. In their red knot study, Jenni-Eiermnn et l. (22) reported triglyceride including free glycerol (rther thn reporting free glycerol seprtely; see Willims et l., 1999) so their triglyceride results re not directly comprle to our study. However, Jenni-Eiermnn et l. (22) concluded tht life-stges with ody mss gin were lso ssocited with incresed plsm triglyceride levels, reflecting incresed turnover of lipids. In contrst, correcting for free glycerol, we found no difference in plsm triglyceride levels etween phses of mss gin nd loss on the low-ft diets, nd gin on the high-ft diet triglyceride levels did not vry with ny phse of mss chnge. Free glycerol ws higher during mss loss thn during mss gin, consistent with results from some psserine studies (Jenni-Eiermnn nd Jenni, 1994) nd this ws the only metolite in our study for which this reltionship ws independent of diet. Glycerol hs trditionlly een ssumed to increse during mss loss (i.e. when irds re in negtive energy lnce) ecuse it is relesed into the plsm during lipolysis of triglycerides in dipose tissue (Rmenofsky, 199). Recently Guglielmo et l. (25) suggested tht glycerol might demonstrte U-shped reltionship with ody mss, such tht it ws lso high in irds with very high rtes of mss gin, ut we found no evidence to support this in our study. The effect of diet composition on plsm metolite concentrtions of vin migrnts hs not, to our knowledge, een tested efore, lthough Birlein (1998) tested for effects of diet lipid nd protein content on rtes of migrtory fttening in the psserine grden wrler Sylvi orin. Birlein (1998) mesured fttening on more exhustive suite of 13 different diets nd the results offer insight into our study. In our study, the diets vried drmticlly in lipid content (18% vs 4%) ut protein content ws only slightly higher for the high-ft diet (47% vs 35%). Birlein (1998) demonstrted tht the fttening rte chieved y the wrlers depended on the percentge protein nd the reltive proportion of protein to ft, s well s the percentge lipid in the diet. The wrlers chieved the highest rte of fttening on diet consisting of 5% protein nd 1% ft (5:1), which is lower in protein ut similr in ft to their nturl diet (15:1). Furthermore, the wrlers chieved higher rte of fttening on the 5:1 diet thn on diet of identicl protein content ut higher ft content (5:2). Similrly, in our study, the irds on the diet lower in oth ft nd protein (~4:35 vs ~18:47) hd plsm metolite concentrtions indictive of higher fttening (lthough we cnnot rule out n effect of the different NFE content of our two diets).

8 768 D. A. Semn, C. G. Guglielmo nd T. D. Willims Reltionship etween metolite levels nd rte of mss chnge The reltionship etween the rte of mss chnge nd plsm concentrtions of three of the four metolites tested (triglyceride, β-oh-utyrte nd uric cid) ws lso dependent on diet composition in our study. For β-oh-utyrte we found negtive reltionship etween metolite level nd rte of mss chnge on oth diets, even though the slope of this reltionship differed mong diets. The sme reltionship hs een reported previously for severl psserines (Jenni- Eiermnn nd Jenni, 1994; Jenni nd Schwilch, 21) nd the red knot (Jenni-Eiermnn et l., 22). Fewer studies hve reported on the reltionship etween mss chnge nd uric cid, ut Jenni-Eiermnn nd Jenni (1994) lso found positive reltionship, s we found on our low-ft diet. However, this result contrsts with tht of Jenni-Eiermnn et l. (22) in which the uric cid levels of red knots were negtively relted to the rte of mss chnge over period of 5 7 dys. The resons for this difference re not cler; lthough in our study nd tht of Jenni-Eiermnn et l. (22) irds were fed trout chow, it is possile these differed in protein content, which is known to ffect uric cid levels (Jenni nd Jenni-Eiermnn, 1998). Furthermore, elevted uric cid levels cn e ssocited with oth protein ctolism nd high protein turnover during hyperphgi nd fttening (Jenni- Eiermnn nd Jenni, 1994; Jenni-Eiermnn nd Jenni, 1998). Therefore, differentil representtion of rnge of mss chnge (i.e. loss or gin) in the two studies is possile explntion for the positive versus negtive reltionship etween uric cid nd rte of mss chnge detected y our study nd tht of Jenni-Eiermnn et l. (22). We lso detected significnt negtive reltionship etween the rte of mss chnge nd plsm glycerol in cptive western sndpipers, which is consistent with the reltionship previously demonstrted in this species y Willims et l. (1999). As with dt on phses of mss chnge, this reltionship ws independent of diet. We did not detect ny reltionship etween plsm triglyceride levels nd mss chnge in the present study, which contrsts with the similr study of the sme species y Willims et l. (1999). Nevertheless, Willims et l. (1999) filed to detect reltionship etween triglyceride nd mss chnge over 7 dys in western sndpipers, wheres significnt positive reltionship etween plsm metolite concentrtions nd the rte of mss chnge ws detected over 1 2 dys, i.e. the reltionship etween mss chnge nd triglyceride ws less roust thn tht for glycerol nd mss chnge. Willims et l. (1999) lso generted mss loss over 1 2 dys vi food removl, not food restriction, so metolic responses might hve reflected short-term strvtion, with much higher rtes of mss loss thn in the present study. Conversely, the smpling periods of 7 dys tht were tested y Willims et l. (1999) were under nturl mss chnge conditions nd reflected less extreme men rtes of mss chnge (± S.E.M.; gin,.13±.8 g dy 1 ; loss,.36±.8 g dy 1, cf. rtes in Tle 2) thn those experienced y the irds on the experimentlly induced mss cycle in the present study (see Tle 1). Nevertheless, Jenni- Eiermnn et l. (22) lso found no reltionship etween triglycerides nd ody mss chnge in their study of cptive red knot. These inconsistent results for triglyceride in cptive sndpipers contrst with generlly consistent positive reltionships etween triglycerides nd mss chnge in psserines (e.g. Jenni-Eiermnn nd Jenni, 1994; Jenni nd Schwilch, 21). This difference might e due, in prt, to the fct tht some studies report totl triglyceride including free glycerol wheres others clculte triglyceride s totl glycerol free glycerol; free glycerol cn represent sustntil, nd highly vrile, proportion of totl glycerol. It is interesting tht the reltionship etween plsm triglyceride nd mss chnge is the most inconsistent of ll the metolites in experimentl studies of cptive irds, wheres plsm triglyceride ppers to e the most roust indictor of mss chnge in studies of free-living irds (e.g. Schu nd Jenni, 21; Guglielmo et l., 22, 25; D. S., unpulished dt). Our study suggests tht one reson for these inconsistencies might e effects of different qulity diets used in experimentl studies: high-ft, trout chow diets typiclly used in shoreird studies generlly pper to result in very different plsm metolite profiles, nd different reltionships etween metolites nd physiologicl stte or mss chnge, thn do low-ft diets (which more closely pproximte nturl shoreird diets). Plsm metolite profiles of western sndpipers gining mss on the low-ft diet were more consistent with dt from field studies nd, in our study, were more similr to those of free-living irds t the time of cpture. In conclusion, t lest on the low-ft diet (4%), which more closely represents the verge lipid content of mrine inverterte prey trgeted y western sndpipers, our study confirms tht mesurement of plsm metolites cn provide roust informtion on physiologicl stte (gin, loss) nd the rte of mss chnge in free-living shoreirds cught only once, s hs een demonstrted for psserines (e.g. Jenni nd Schwilch, 21; lthough we should stress tht these results re only vlid for irds tht hve een feeding for severl hours prior to smpling). Nevertheless, in migrtory species where the lipid composition of the nturl diet cn e high, or highly vrile (e.g. psserines feeding on fruit, or sndpipers feeding on cr or fish spwn), reserchers should e cogniscent of effects of diet composition for certin metolites. Future studies should consider possile confounding effects of experimentl diets in vlidting, or compring, plsm metolite profiles in reltion to mss chnge for free-living versus cptive irds, prticulrly for non-psserines. This project ws supported in prt y the Mrine Ecosystem Helth Progrm (grnt to T.D.W., C.G.G. nd R. W. Elner) through the Wildlife Helth Center, School of Veterinry Medicine, University of Cliforni, Dvis. Other funding ws provided y C. D. Nelson entrnce scholrship (Simon Frser University; SFU), nd Chir of Wildlife Ecology. Crl Schwrz provided vlule sttisticl dvice.

9 Plsm metolites nd mss chnge 769 Memers of the SFU Western Sndpiper Reserch Network provided collortion, support nd feedck. Thnks to Will Stein nd mny others for ssisting in the cpture of the irds, nd to memers of the Willims l, prticulrly Ktrin Slvnte, nd the stff of SFU Animl Cre Fcility for ssistnce with the cre of irds. References Alerstm, T. nd Lindstrom, A. (199). Optiml ird migrtion: the reltive importnce of time, energy, nd sfety. In Bird Migrtion: Physiology nd Ecophysiology (ed. E. Gwinner), pp Berlin: Springer-Verlg. Bker, A. J., Gonzlez, P. M., Piersm, T., Niles, L. J., de Lim Serrno do Nscimento, I., Atkinson, P. W., Clrk, N. A., Minton, C. D. T., Peck, M. K. nd Arts, G. (23). Rpid popultion decline in red knots: fitness consequences of decresed refuelling rtes nd lte rrivl in Delwre By. Proc. R. Soc. Lond. B 271, Birlein, F. (1998). The effect of diet composition on migrtory fuelling in Grden Wrlers Sylvi orin. J. Avin Biol. 29, Brown, S., Hickey, C., Hrrington, B. nd Gill, R. (21). The United Sttes Shoreird Conservtion Pln, 2nd edn. Mnomet: Mnomet Center for Conservtion Sciences. DeGrw, W. A., Kern, M. D. nd King, J. R. (1979). Sesonl chnges in the lood composition of cptive nd free-living white-crowned sprrows. J. Comp. Physiol. 129, Dunn, E. H. (2). Temporl nd sptil ptterns in dily mss gin of Mgnoli Wrlers during migrtory stopover. Auk 117, Egeler, O. nd Willims, T. D. (2). Sesonl, ge, nd sex-relted vrition in ftty-cid composition of depot ft in reltion to migrtion in Western Sndpipers. Auk 117, Egeler, O., Semn, D. nd Willims, T. D. (23). The influence of diet on ftty cid composition of depot ft in Western Sndpipers. Auk 12, Gnnes, L. Z. (21). Comprtive fuel use of migrting psserines: effects of ft stores, migrtion distnce nd diet. Auk 118, Guglielmo, C. G. nd Willims, T. D. (23). Phenotypic flexiility of ody composition in reltion to migrtory stte, ge nd sex in the Western Sndpiper (Clidris muri). Physiol. Biochem. Zool. 76, Guglielmo, C. G., O Hr, P. D. nd Willims, T. D. (22). Extrinsic nd intrinsic sources of vrition in plsm lipid metolites of free-living western sndpipers (Clidris muri). Auk 119, Guglielmo, C. G., Cersle, D. J. nd Eldermire, C. (25). A field vlidtion of plsm metolite profiling to ssess refueling performnce of migrtory irds. Physiol. Biochem. Zool. In press Jenni, L. nd Jenni-Eiermnn, S. (1998). Fuel supply nd metolic constrints in migrting irds. J. Avin. Biol. 29, Jenni, L. nd Schwilch, R. (21). Plsm metolite levels indicte chnge in ody mss in reed wrlers Acrocephlus scirpceus. Avin Sci. 1, Jenni-Eiermnn, S. nd Jenni, L. (1994). Plsm metolite levels predict individul ody-mss chnges in smll long-distnce migrnt, the grden wrler. Auk 112, Jenni-Eiermnn, S. nd Jenni, L. (1996). Metolic differences etween the postreeding, moulting nd migrtory periods in feeding nd fsting psserine irds. Funct. Ecol. 1, Jenni-Eiermnn S., Jenni L., Kvist A., Lindstrom A., Piersm T. nd Visser, G. H. (22). Fuel use nd metolic response to endurnce exercise: wind tunnel study of long-distnce migrnt shoreird. J. Exp. Biol. 25, Jenni-Eiermnn, S., Jenni, L. nd Piersm, T. (22). Plsm metolites reflect sesonlly chnging metolic processes in long-distnce migrnt shoreird (Clidris cnutus). Zoology 15, Lmrechts, M. M., Perret, P., Mistre, M. nd Blondel, J. (1999). Do experiments with cptive non-domesticted nimls mke sense without popultion field studies? A cse study with lue tits reeding time. Proc. R. Soc. Lond. B 266, Rmenofsky, M. (199). Ft storge nd ft metolism in reltion to migrtion. In Bird Migrtion: Physiology nd Ecophysiology (ed. E. Gwinner), pp Berlin: Springer-Verlg. SAS Institute (199). SAS/STAT User s Guide, Relese 6.3 Edition. Cry, NC, SAS Institute. Schu, M., nd Jenni, L. (21). Vrition in fuelling rtes mong sites, dys nd individuls in migrting psserine irds. Funct. Ecol. 15, Totzke, U. nd Birlein, F. (1998). The ody mss cycle of the migrtory grden wrler (Sylvi orin) is ssocited with chnges in sl plsm metolite levels. Comp. Biochem. Physiol. 121A, Wrnock, N. nd Bishop, M. A. (1998). Spring stopover ecology of migrnt Western Sndpipers. Condor 1, Willims, T. D., Guglielmo, C. G., Egeler, O. nd Mrtyniuk, C. J. (1999). Plsm lipid metolites provide informtion on mss chnge over severl dys in cptive Western Sndpipers. Auk 116, Ydenerg, R. C., Butler, R. W., Lnk, D. B., Guglielmo, C. G., Lemon, M. nd Wolf, N. (22). Trde-offs, condition dependence, nd stopover site selection y migrting sndpipers. J. Avin Biol. 33,