Tracing dietary protein in red-backed voles (Clethrionomys gapperi) using stable isotopes of nitrogen and carbon

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1 717 Tring dietry protein in red-ked voles (Clethrionomys gpperi) using stle isotopes of nitrogen nd ron Dvid T.J. Sre, John S. Millr, nd Frederik J. Longstffe Astrt: We exmined the stle isotopes of nitrogen nd ron in smll mmml, the red-ked vole (Clethroinomys gpperi (Vigors, 1830)), to determine if isotope signtures reflet diet omposition. Nitrogen- nd ron-isotope rtios in tissues from voles mintined on different protein levels in the lortory were ompred with wild-trpped voles. The isotopi frtiontion of dietry nitrogen nd ron ws lso exmined s food ws digested in the stomh, inorported into one ollgen, ioptite, nd hir, nd exreted s fees. Nitrogen nd ron isotopes were frtionted differently depending on the isotopi omposition nd protein ontent of the diet. δ 15 N nd δ 13 C vlues pper to e influened y ftors in ddition to diet, suh s mronutrients metolized for respirtion, metoli rte, nd periods of protein shortge. Résumé : L exmen des isotopes stles d zote et de rone hez un petit mmmifère, le mpgnol à dos roux de Gpper (Clethrionomys gpperi (Vigors, 1830)), permis de vérifier si les signtures isotopiques reflètent ien le régime limentire. Nous vons ompré les rpports isotopiques d zote et de rone dns les tissus de mpgnols nourris de onentrtions diverses de protéines en lortoire à eux de mpgnols piégés en nture. Nous vons ussi suivi l frtiontion isotopique de l zote et du rone limentires à mesure que l nourriture est digérée dns l estom, inorporée u ollgène des os, à l ioptite et ux heveux et exrétée dns les fèes. L frtiontion de l zote et du rone se fit différemment en fontion de l omposition isotopique et du ontenu protéinique du régime. Les vleurs de δ 15 Netdeδ 13 C semlent être ffetées pr des fteurs utres que le régime limentire, tels que les mronutriments métolisés pour l respirtion, le tux métolique et les périodes de pénurie protéinique. [Trduit pr l Rédtion] Sre et l. II 75 Introdution Protein is the prinipl onstituent of the orgns nd soft strutures of nimls, nd ontinuous supply from food is needed for their growth nd repir. The trnsformtion of food protein into ody protein is n importnt prt of the nutrition proess (Mynrd et l. 1979), ut it is diffiult to ssess. Although protein ssimiltion hs trditionlly een estimted using dietry oservtion, otining unised nd omplete oservtions of forging ehviour is prolemti for mny popultions (Morrison et l. 1990; Kelly 000). Furthermore, nutrients ingested my not reflet nutrient ssimiltion urtely. The omposition of protein ssimilted in foods n e mesured using stle-nitrogen nd stle-ron isotopes euse the δ 15 N nd δ 13 C vlues of niml tissues re relted to diet (DeNiro nd Epstein 1978; Bender et l. 1981; Reeived 13 August 00. Aepted 3 My 005. Pulished on the NRC Reserh Press We site t on 8 July 005. D.T.J. Sre 1 nd J.S. Millr. Deprtment of Biology, The University of Western Ontrio, London, ON NA 5B7, Cnd. F.J. Longstffe. Deprtment of Biology nd Deprtment of Erth Sienes, The University of Western Ontrio, London, ON NA 5B7, Cnd. 1 Corresponding uthor (e-mil: dvidsre@hotmil.om). Mko et l. 198; Tieszen et l. 1983; Amrose nd DeNiro 198) in wys relted to the retention of isotopes in the ody. Hene, δ 15 N nd δ 13 C vlues n e used to reonstrut diets nd determine the fte of ssimilted nutrients (Gnnes et l. 1997). Nitrogen-isotope ompositions re trditionlly used to mesure ssimilted protein. On verge, niml δ 15 N vlues re 3 5 higher thn dietry nitrogen, minly euse of exretion of 1 N in urine (Peterson nd Fry 1987). Ctolism of ody proteins during periods of stress my lso elevte δ 15 N vlues (Hoson nd Clrk 199), s result of the reyling of ody proteins into the metoli mino-id pool (Hoson et l. 1993). Cron-isotope omposition of plnts is determined primrily y the frtiontion ssoited with ron fixtion during photosynthesis (DeNiro 1977). However, the δ 13 C vlues of plnts utilizing similr photosyntheti pthwys my vry depending upon iohemil omposition, sine lipids re depleted of 13 C reltive to rohydrtes nd proteins (Prk nd Epstein 191; O Lery 1981). Similrly, plnt orgns my e enrihed or depleted of 13 C reltive to whole-plnt vlues (Prk nd Epstein 190; Lowdon 199; O Lery 1981; Vogel 198; Tieszen nd Boutton 1989). Upon digestion of plnt mtter, ron n e routed to different tissues (Gnnes et l. 1998) nd given tissue my not lwys follow ulk-diet vlues. Most tissues of lrge herivores re enrihed in 13 C reltive to the diet, wheres fees nd respirtory CO re generlly depleted of 13 C (DeNiro nd Epstein 1978; Tieszen nd Boutton 1989). Cn. J. Zool. 83: (005) doi: /Z NRC Cnd

2 718 Cn. J. Zool. Vol. 83, 005 Red-ked voles (Clethrionomys gpperi (Vigors, 1830)) re n undnt herivore in the Knnskis Vlley, nd lthough they hve een shown to prefer low protein (1%) diets in the lortory (Ksprin nd Millr 00), the protein ontent of their nturl diet remins unknown. The purpose of this study ws to determine whether the stle isotopes of nitrogen nd ron ould e used to exmine protein in the diet of red-ked voles. To do this we first ssessed whether the digest (stomh nd fees) nd tissues (hir nd one ollgen) of voles fed diets vrying in protein (1%, 17%, %) differed in their nitrogen- nd ronisotope ompositions. We lso determined how voles rised on diets with different protein levels frtionted nitrogen nd ron isotopes in their digest nd tissues. Finlly, we ompred nitrogen- nd ron-isotope rtios of hir, one ollgen, nd ioptite (ron only) from lortory nd wild-trpped voles. Among diets, tissue δ 15 N vlues were expeted to vry inversely with dietry protein levels, sine nimls on proteinlimited diets should tolize ody tissues s mino id nd energy soures (Hoson et l. 1993). Tissues were lso expeted to hve higher δ 13 C vlues in nimls on high protein diets reltive to medium nd low protein diets, euse protein is enrihed in 13 C reltive to other mronutrients (Tieszen nd Boutton 1989). Tissues from red-ked voles on low protein diets were expeted to resemle ulk dietry δ 13 C vlues euse they my e synthesized prtly from nonprotein omponents of the ulk diet (Gnnes et l. 1998). The δ 13 C vlues of metolilly intive tissues suh s ollgen nd hir were predited to reflet diet during limited period of growth (Tieszen et l. 1983). Bioptite is metolilly tive ut is generlly onsidered to turn over t slower rte thn other metolilly tive tissues. Bioptite ws therefore expeted to reflet lifetime verge dietry vlues (Gnnes et l. 1998). To exmine the rte of turnover, the δ 18 O nd δ 13 C vlues of struturl ronte in ioptite were ompred etween lortory nd wild voles. Methods Study re nd nimls Red-ked voles were livetrpped in res of open nopy sprue fir forest (Millr et l. 1985) throughout the Knnskis Vlley, Alert (51 N, 115 W), from My through August 003, using Longworth trps insulted with nesting mteril nd ited with sunflower seeds nd ots. Cptured voles were sexed, weighed, uniquely tgged for identifition, nd their reeding ondition reorded. Voles were then housed t the Knnskis Field Sttion (University of Clgry) in 9 m 18.5 m 13 m plsti ges ontining wood shvings nd otton tting t 0 C nd 1 h light :8hdrk photoperiod. Wter ws provided d liitum. Body mss ws mesured weekly throughout the study. Adult voles olleted s wild smples (N = 13) were killed within dy of pture using CO nd frozen for future nlysis. Animl re nd tretment protools were pproved y the University Counil on Animl Cre (The University of Western Ontrio) nd were in ordne with the priniples outlined y the Cndin Counil on Animl Cre. Animl diets Livetrpped dult red-ked voles were mintined on three diets tegorized s high, medium, nd low proteins. Eh smple group (wild, high, medium, nd low) ontined minimum of three nonreeding femles. Sex ws not onsidered in this study euse metoli rtes do not vry with sex nd reprodutive sttus in smll mmmls (Hvelk 00). Protein ontent ws lulted y multiplying the nitrogen onentrtion of eh diet (mesured using n elementl nlyzer) y ftor of.5 (Jones 191). Eleven dult voles were mintined on high protein diet of mixed greens nd sunflower seeds (% protein), 10 dult voles were mintined on medium protein diet onsisting of mixed greens nd ots (17% protein), nd 10 dult voles were mintined on low protein diet onsisting of mixed greens (1% protein). The mixed greens, omposed of the stems nd leves of C 3 plnts (Teeri nd Stowe 197; Tieszen et l. 1979), were seleted sed upon sesonl vilility nd vole preferene (see Norrie nd Millr 1990) nd inluded speies of Slix L., Trxum offiinle G.H. Weer ex Wiggers, Shepherdi ndensis (L.) Nutt., nd Populus tremuloides Mihx. Diets were seleted sed on pltility (Norrie nd Millr 1990; Ksprin nd Millr 00). For the low protein diet, protein ontent ws lulted using the verge protein ontent of ll types of forge, sine they hve een shown to hve pproximtely eqully high pltility (Norrie nd Millr 1990). For the medium nd high protein diets, protein ontent ws lulted using ots nd sunflower seeds, respetively, euse these hve een shown to e more pltle thn wild forge (Ksprin nd Millr 00). Wild forge ws provided in the se of medium nd high protein diets s supplement to ensure tht ll nutritionl requirements were met. To ount for sesonl vrition in isotopi omposition, vegettion ws olleted nd nlyzed in eh of July nd August. Smples of sunflower seeds nd ots used s feed were lso nlyzed. Red-ked voles were mintined on high, medium, nd low protein diets for ± 1 (men ± SD) dys. At the end of the study period, fresh fees were olleted from the ge, nd voles were killed using CO nd frozen for future nlysis. Hir smples were tken from the dorsl posterior of the vole for isotopi nlysis. Beuse the isotopi omposition of pelge reflets forging onditions t the time of pelge growth rther thn onditions t the time of smpling, voles were killed post moult (Sre et l. 005). Stomh ontents were removed nd voles were skeletonized using dermestid eetles to otin one smples for isotopi nlysis of ollgen nd ioptite. Stomh ontents nd fees of wild voles were not nlyzed euse the it used in the trps ws expeted to influene nitrogen- nd ron-isotope ompositions. Isotopi nlyses Hir smples were rinsed repetedly in :1 hloroform: methnol solution nd exmined under mirosope to ensure tht impurities nd surfe oils hd een removed. Fees nd plnt smples were dried for 9 h t 70 C nd stomh ontents were freeze-dried. All smples were ground to <1 mm using Wig-L-Bug. 005 NRC Cnd

3 Sre et l. II 719 Collgen ws extrted using modified Longin (1970) method. Bone smples were rinsed repetedly in 0:10:8 methnol:hloroform:wter solution nd lened using methnol to remove lipids (Bligh nd Dyer 1959), then reted with 0 80 ml of 0.5 mol/l of HCl for 1htodegrde the ollgen. The mixture ws then entrifuged to remove the ollgen nd ground to <1 mm using mortr nd pestle. Bone smples used for ioptite nlysis were rinsed repetedly with 0:10:8 methnol:hloroform:wter solution nd lened using methnol to remove lipids. Bone ioptite smples were lso ground to <1 mm using Wig-L-Bug. Nitrogen-, ron-, nd oxygen-isotope results re reported using the stndrd delt (δ) nottion in prts per thousnd ( ) reltive to tmospheri nitrogen, Vienn PDB (VPDB), nd Vienn Stndrd Men Oen Wter (VSMOW), respetively, (Coplen et l. 199; Coplen 199). Differenes in δ vlues etween two sustnes, nd, re defined s δ δ = (e.g., δ 15 N hir δ 15 N diet = hir diet ). Nitrogen- nd ron-isotope ompositions for hir, ollgen, stomh ontents, fees, nd plnt smples were otined using Thermo Finnign DeltPlusXL stle isotope rtio mss spetrometer oupled to Costeh elementl nlyzer in ontinuous flow mode (Lortory for Stle Isotope Siene t The University of Western Ontrio). The δ 15 N vlues were lirted using IAEA-N1 (epted vlue, 0.0 ) nd IAEA- N (epted vlue, 0.30 ). Vlues for mesurements of IAEA-N1 nd 3 mesurements of IAEA-N vried y no more thn ±0.18 during the ourse of the investigtion. For 8 mesurements of n internl niotinmide stndrd, δ 15 N vlue of 1.59 ± 0.1 ws otined, whih ompres well with the epted vlue of The δ 13 C vlues were lirted using NBS- (epted vlue, ) nd ANU-Surose (epted vlue, ). Vlues for mesurements of NBS- nd 5 mesurements of ANU-Surose vried y no more thn ±0.11 during the ourse of the investigtion. For 8 mesurements of niotinmide, δ 13 C vlue of 3.5 ± 0.10 ws otined, whih ompres well with the epted vlue of 35.. The δ 13 C nd δ 18 O vlues of ioptite ronte were otined using Miromss Multi-Prep online system tthed to Miromss Optim dul inlet stle isotope rtio mss spetrometer. The δ 13 C vlues were lirted using NBS-19 (epted vlue, 1.95 ) nd n internl lite stndrd (Suprpur; epted vlue, 35.8 ). Vlues for five mesurements of NBS-19 nd three mesurements of Suprpur vried y no more thn ±0. during the ourse of the investigtion. For three mesurements of NBS-18, δ 13 C vlue of.87 ± 0.17 ws otined, whih ompres well with the epted vlue of 5.0. For three mesurements of n internl stndrd (WS lite), δ 13 C vlue of 0.7 ± 0.10 ws otined, whih ompres well with the epted vlue of The δ 18 O vlues were lirted using NBS-18 (epted vlue, 7.0 ) nd NBS-19 (epted vlue, 8. ). Vlues for three mesurements of NBS-18 vried y no more thn ±0.08 nd four mesurements of NBS-19 vried y no more thn ±0.03 over the ourse of the investigtion. A δ 18 O vlue of 13.1 ± 0. ws otined for three mesurements of Suprpur, whih ompres well with the epted vlue of A δ 18 O vlue of. ± 0.1 ws otined for three mesurements of seond internl lite stndrd (WS), whih ompres well with the epted vlue of.9. Nitrogennd ron-isotope δ vlues were ompred ross the three diets, s well s mong tissues. All omprisons were performed using one-wy ANOVAs (α = 0.05). When ANOVAs indited signifint vrition (P < 0.05), Tukey s honestly signifint differene (HSD) tests were used to test whih tegories differed. Results re presented s mens ± SE. Results Nitrogen isotopes Whole-diet δ 15 N vlues inresed with protein ontent (F [,0] = 1.13, P < 0.001; Fig. 1). The δ 15 N vlues of stomh ontents (F [,] = 7., P < 0.001; Fig. 1) nd fees (F [,] = 0.0, P < 0.001; Fig. 1) lso inresed with protein ontent. The δ 15 N vlues of one ollgen were similr etween low nd medium protein diets ut higher in wild nd high protein diets (Fig. 1d). A similr trend ws seen in hir (Fig. 1e). Red-ked voles on the high protein diets were enrihed in 15 N y pproximtely.5 (ollgen) nd 1.5 (hir) reltive to voles on the low nd medium protein diets, though the differenes etween the low, medium nd high protein diets for hir were not signifint (Figs. 1d, 1e). Collgen nd hir δ 15 N vlues did not differ signifintly from eh other within eh diet (low protein diet: Tukey s HSD, P = 0.9; medium protein diet: Tukey s HSD, P = 0.71; high protein diet: Tukey s HSD, P = 0.8), lthough ollgen tended to e more enrihed in 15 N reltive to diet thn hir (Tle 1). Nitrogen isotopes were frtionted upon ingestion y voles rised on low, medium, nd high protein diets (F [,1] = 5.35, P < 0.001; F [,38] = 13.80, P < 0.001; F [,37] = 3.8, P = 0.011, respetively). The 15 N/ 1 N rtio ws generlly not hnged in the digest, exept tht stomh ontents nd fees were enrihed in 15 N in the low protein diet (Tle 1). 15 N ws lwys preferentilly inorported into one ollgen (Tle 1). Hir lso ws signifintly enrihed in 15 N reltive to the low nd medium protein diets, ut not the high protein diet (Tle 1). Voles on the low protein diet showed the lrgest nitrogen-isotope frtiontion etween tissue nd diet (Tle 1). Collgen (F [3,3] = 9.8, P < 0.001; Fig. 1d) nd hir (F [3,39] = 7.79, P < 0.001; Fig. 1e) were signifintly enrihed in 15 Nin wild voles reltive to lortory voles rised on the low nd medium protein diets, ut not reltive to voles rised on high protein diets. Cron nd oxygen isotopes Whole-diet δ 13 C vlues differed signifintly mong the diets (F [,0] = 17.9, P < 0.001) ut did not vry onsistently with protein ontent (Fig. ). For eh diet, oth stomh (F [,3] = 78.75, P 0.001, Fig. ) nd fees (F [,] = 7., P < 0.001; Fig. ) refleted dietry δ 13 C vlues nd differed signifintly mong low, medium, nd high protein diets. Collgen smples from red-ked voles on low protein diets were slightly ut signifintly depleted of 13 C reltive to other diets (Fig. d), while differenes in δ 13 C vlues mong diets were negligile upon inorportion into hir (Fig. e). Bioptite from voles rised on high protein diets ws signifi- 005 NRC Cnd

4 70 Cn. J. Zool. Vol. 83, 005 Fig. 1. Men (±SE) δ 15 N vlues of () ulk diet, () stomh, () fees, (d) ollgen, nd (e) hir tissues of red-ked voles, Clethrionomys gpperi, fed on low, medium, or high diets. The δ 15 N vlues otined for wild red-k voles re lso illustrted for (d) ollgen nd (e) hir tissues. Brs with different letters indite results tht re signifintly different (Tukey s HSD, P < 0.05). () () δ N( ) δ N( ) 0 () Dietry protein ( ) 15 δ N δ 15 N( ) (d) Dietry protein 0 Dietry protein Dietry protein 8 (e) ( ) 15 δ N Dietry protein Tle 1. Men (±SE) 15 N tissue diet for red-ked voles, Clethrionomys gpperi, fed three different levels of dietry protein (low, medium, high). 15 N tissue diet ( ) Smple Low (1%) Medium (17%) High (%) Stomh ontents 1.85±0.37* 1.7± ±0. Fees 1.7±0.38* 1.17± ±0.5 Collgen 5.09±0.38* 3.37±0.51*.0±0.* Hir.59±0.37*.77±0.53* 1.±0.58 *Smple ws signifintly different from diet (Tukey s HSD, P < 0.05). 005 NRC Cnd

5 Sre et l. II 71 Fig.. Men (±SE) δ 13 C vlues of () ulkdiet,() stomh, () fees, (d) ollgen, (e) hir, nd (f) ioptite tissues for red-ked voles fed on low, medium, or high protein diets. The δ 13 C vlues otined for wild red-ked voles re lso illustrted for (d) ollgen, (e) hir, nd (f) ioptite tissues. Brs with different letters indite results tht re signifintly different (Tukey s HSD, P < 0.001). () () Dietry Protein Dietry Protein () (d) Dietry Protein Dietry Protein (e) (f) Dietry Protein -0 Dietry Protein ntly depleted of 13 C reltive to ioptite from voles rised on low nd medium protein diets (Fig. f). Cron isotopes were vrily frtionted upon ingestion y red-ked voles rised on low, medium, nd high protein diets (F [5,51] = 553.8, P < 0.001, F [5,5] = 38., P < 0.001, F [5,] = , P < 0.001, respetively). The 13 C/ 1 C rtio did not hnge s the low nd high protein diets pssed into the stomh nd were exreted s fees (Tle ). Cron isotopes lso were not frtionted s the medium protein diet pssed into the stomh, ut fees were signifintly depleted of 13 C reltive to diet (Tle ). Collgen, hir, nd ioptite were signifintly enrihed in 13 C reltive to ll diets, with ioptite hving the highest δ 13 C vlue (Tle ). Voles rised on the low protein diet showed the gretest frtiontion of ron isotopes etween tissue nd diet (Tle ). δ 13 C vlues of ollgen (F [3,3] = 11.19, P < 0.001; Fig. d), hir (F [3,3] = 5.0, P < 0.001; Fig. e), nd ioptite (F [3,3] = 10.19, P < 0.001; Fig. f) differed signifintly etween lortory nd wild red-ked voles. Collgen ws signifintly enrihed in 13 C in wild voles reltive to lortory voles on the low protein diet, tended to e enrihed in 005 NRC Cnd

6 7 Cn. J. Zool. Vol. 83, 005 Tle. Men (±SE) 13 C tissue diet for red-ked voles fed three different levels of dietry protein (low, medium, high). 13 C tissue diet ( ) Smple Low (1%) Medium (17%) High (%) Stomh ontents 0.5±0.3 0.± ±0.37 Fees 0.51± ±0.33* 0.±0.38 Collgen 7.±0.37*.7±0.3* 7.3±0.39* Hir.15±0.3*.38±0.3* 5.08±0.3* Bioptite 13.0±0.3* 10.01±0.3* 11.3±0.38* *Smple ws signifintly different from diet (Tukey s HSD, P < 0.05). Fig. 3. Men (±SE) δ 18 O vlues of struturl ronte in ioptite for red-ked voles fed on low, medium, nd high protein or wild diets. Brs with different letters indite results tht re signifintly different (Tukey s HSD, P < 0.001). δ 18 O(%) C reltive to voles fed the medium protein diet, ut did not differ from those fed the high protein diet (Fig. d). Hir ws signifintly enrihed in 13 C in wild voles reltive to lortory voles fed the low, medium, nd high protein diets (Fig. e). Bioptite from wild voles ws signifintly enrihed in 13 C reltive to voles rised on the high protein diet (Fig. f). Bioptite δ 18 O vlues did not vry etween groups rised in the lortory (F [,] = 0.57, P = 0.57; Fig. 3), ut wild voles were signifintly enrihed in 18 O reltive to lortory voles (F [1,3] =.19, P < 0.01; Fig. 3). Disussion Dietry Protein Nitrogen isotopes Whole-diet δ 15 N vlues inresed with protein ontent, ontrry to wht ws expeted euse plnts do not generlly eome enrihed in 15 N during nitrte sorption; plnt δ 15 N vlues generlly vry with soil δ 15 N vlues rther thn protein ontent (Mriotti et l. 1980; Hndley nd Rven 199). There is no evidene to suggest tht soil δ 15 N vlues should vry onsistently with protein ontent. However, the δ 15 N vlues of the diet my vry depending on the hemil onstituents of plnt s orgns (Hndley nd Rven 199); plnt protein is generlly enrihed y 3 reltive to totl ell N (Mko et l. 198, 1987). Therefore, protein ontent my e importnt for understnding vrition in plnt δ 15 N vlues (Amrose 1991). The δ 15 N vlues of red-ked vole stomh ontents nd fees differed signifintly mong ll groups nd inresed with dietry δ 15 N vlues, nd pper to therefore illustrte the diretion of hnge in the nitrogen-isotope omposition of diet (Kelly 000). Nitrogen isotopes were not signifintly frtionted s the medium nd high protein diets pssed through the digestive trt, inditing no differentil ssimiltion of isotopes during digestion. Voles on the low protein diet hd stomh ontents signifintly enrihed in 15 N over diet, ut no further enrihment ws oserved in fees, gin inditing no differentil ssimiltion of isotopes during digestion. The enrihment in 15 N of vole fees reltive to diet my result from the preferentil removl of 1 N in the digestive trt (Peterson nd Fry 1987; Steele nd Dniel 1978; Hre et l. 1991; Sutoh et l. 1987). However, this differene is lso evident in stomh ontents, prior to ssimiltion in the intestines, nd my reflet the seretion of digestive enzymes into the digest. Contrry to our predition, the δ 15 N vlues of tissues inresed rther thn deresed with inresing protein levels. The vrition oserved in tissue δ 15 N vlues refleted vrition in whole-diet δ 15 N vlues, whih in turn refleted protein ontent. Red-ked voles onsuming exess quntities of protein my hve higher metoli osts thn those on low protein diets (Mynrd et l. 1979), nd higher metoli rte in voles on high versus low protein diets my hve ontriuted to the higher δ 15 N vlues. Collgen nd hir re typilly enrihed in 15 N y over diet in mmmls (DeNiro nd Epstein 1981; Hre et l. 1991; Sutoh et l. 1987) euse of the preferentil exretion of 1 N in urine (Peterson nd Fry 1987). This ws oserved in tissues from red-ked voles rised on medium nd high protein diets, ut voles rised on low protein diets were even more enrihed in 15 N. As result, differenes mong δ 15 N vlues in tissues from voles fed low, medium, nd high protein diets were not s gret s differenes mong whole-diet δ 15 N vlues. Voles rised on low protein diets my hve high δ 15 N vlues euse of toliztion of energy stores (Hoson nd Clrk 199). Collgen nd hir δ vlues did not differ from eh other within eh diet, lthough ollgen tended to e more enrihed in 15 N reltive to diet thn hir. Hilderrnd et l. (199) lso found tht mmmls on onstnt diet show similr degrees of 15 N enrihment in ollgen nd hir. The tendeny in red-ked voles for ollgen to e more enrihed in 15 N thn hir proly reflets differene in the mino-id omposition etween the two tissues (Hre et l. 1991). Collgen nd hir from wild red-ked voles were signifintly enrihed in 15 N reltive to voles rised on the low 005 NRC Cnd

7 Sre et l. II 73 nd medium protein diets, lthough not reltive to those on high protein diets. High tissue δ 15 N vlues of wild voles proly do not reflet high protein diet euse voles re herivorous nd prefer reltively low protein diets (Norrie nd Millr 1990; Ksprin nd Millr 00). The enrihment of 15 N in wild voles my result from higher metoli stress inurred y voles exposed to older tempertures thn lortory voles. Smll mmmls exposed to old stress hve higher metolism thn those in thermoneutrl environment (Rndolph 1980; Hvelk 00), nd δ 15 N vlues my inrese with higher metoli rte. Cron nd oxygen isotopes Men δ 13 C vlues of diets were in the rnge oserved for terrestril C 3 plnts (O Lery 1981). However, the δ 13 Cvlues did not vry onsistently with protein ontent. The medium protein diet ws enrihed in 13 C reltive to the low nd high protein diets, perhps euse different mino ids hve hrteristi isotopi vlues (Gnnes et l. 1998). The reltively high ft ontent of the high protein diet (9% vs. 7% in the medium protein diet; Sherz nd Senser 199) proly ontriuted to lower δ 13 C vlues, given tht lipids n e depleted of 13 C y up to 8 reltive to whole plnt vlues (Prk nd Epstein 191). The δ 13 C vlues of stomh ontents nd fees differed mong ll diets in the sme mnner s dietry δ 13 C vlues. Although the digest omposition ws funtion of diet, suggesting tht red-ked voles were not forging seletively, diets did not vry solely with protein ontent nd therefore did not predit food qulity. Cron-isotope frtiontion did not hnge s food pssed through the digestive trt, lthough fees from nimls rised on the medium protein diet were depleted of 13 C y pproximtely reltive to oth diet nd stomh ontents. Fees in lrge herivores re ommonly depleted of 13 C reltive to diet y slightly more thn 1 (Tieszen nd Boutton 1989; DeNiro nd Epstein 1978; Jones et l. 1979), possily euse of miroil 13 C disrimintion or ontmintion in the gut y tissues or fluids with lower δ 13 C vlues. Collgen nd hir δ 13 C vlues were not losely relted to the isotopi omposition of the ulk diet, nd ollgen smples from red-ked voles rised on low protein diets hd signifintly lower δ 13 C vlues thn medium nd high diet ollgen smples. These tissues re derived minly from the protein portion of the diet, whih ounts for 91% of the isotopi vlue of one ollgen nd 8% of the isotopi vlue of hir (Tieszen nd Fgre 1993). Collgen nd hir δ 13 C vlues re lso modulted y the proportion of protein in the diet nd differenes in δ 13 C vlues etween proteins nd lipids (Amrose nd Norr 1993). Animls on high protein diets n synthesize ollgen diretly from dietry protein, wheres nimls rised on low protein diets must synthesize some ollgen from fts nd rohydrtes, whih generlly hve low δ 13 C vlues (Gnnes et l. 1998). As result, low δ 13 C vlues in ollgen my indite prolonged periods of protein shortge, irrespetive of dietry δ 13 C vlues. The δ 13 C vlue of vole hir ws similr mong ll diets, suggesting tht the protein omponent of ll diets ontined the mino ids required for hir prodution, nd tht these mino ids hd similr δ 13 C vlues. The differene in δ 13 C vlues etween ollgen nd hir my reflet vritions in the mino ids utilized in their formtion (Tieszen nd Fgre 1993), euse pools of mino ids re thought to e in isotopi equilirium throughout the ody (Hre et l. 1991). Although ollgen nd hir were enrihed in 13 C reltive to ll diets, red-ked voles rised on low nd high protein diets showed the lrgest tissue diet frtiontion. In previously studied smll mmmls, one ollgen is typilly enrihed in 13 C y 3% 5, wheres hir is typilly enrihed in 13 C y pproximtely 1 reltive to diet (DeNiro 1977; DeNiro nd Epstein 1978; Bender et l. 1981; Krueger nd Sullivn 198; Tieszen nd Fgre 1993). The ollgen nd hir δ 13 C vlues of voles losely pproximted those of mie (Mus musulus L., 1758), suggesting tht the mino ids used in tissue synthesis hd similr δ 13 C vlues. These tissues in voles were onsiderly more enrihed in 13 C reltive to their diet thn were mie (Tieszen nd Fgre 1993). We suggest tht voles otin suffiient dietry protein to synthesize tissues from low protein, herivorous diet, wheres omnivores suh s mie my require greter mounts of dietry protein to synthesize tissues. The δ 18 O vlues of struturl ronte in ioptite showed sustntil turnover in the dult red-ked voles during their time in ptivity. There ws signifint differene in the δ 18 O vlues etween wild nd lortory voles (Fig. 3). The wild voles hd the highest δ 18 O vlues, whih likely indites onsumption of wter tht eme enrihed in 18 O during evportion (Dnsgrd 19). Prtilly evported smll pools re hrteristi of the hitt of voles in the Knnskis Vlley. Lortory voles hd muh lower δ 18 O vlues thn wild voles, whih my reflet the isotopi omposition of mountin-strem-fed reservoir used to supply their drinking wter. The similrity in δ 18 O vlues for ioptite mong ll three lortory diets lso indites tht the extent of one ioptite turnover ws similr, regrdless of diet. The ioptite δ 13 C vlues lso suggest sustntil turnover in dult red-ked voles. Cron in ioptite is derived from lood ironte, whih is generted y energy metolism (Amrose nd Norr 1993). As result, ioptite ron-isotope ompositions re funtion of the plnt mronutrients used for respirtion (Tieszen nd Fgre 1993). The highest ioptite δ 13 C vlues (Fig. f) were otined for wild voles, whih suggests tht their diet ws more enrihed in 13 C thn the diet of lortory voles. The δ 13 C vlues of ioptite from lortory voles were ll low. The seprtion etween ioptite nd lortory diet δ 13 C vlues is different in eh se, with voles fed the low protein diet showing the lrgest pprent frtiontion (Tle ). Bioptite δ 13 C vlues re generlly onsidered to pproximte lifetime dietry δ 13 C vlues ut n e modulted y the proportion of mronutrients ville for metolism (Gnnes et l. 1998). For exmple, ioptite from redked voles fed the low protein diet were enrihed in 13 Cto greter extent thn voles fed the other protein diets. This my indite greter ontriution of protein nd rohydrtes nd smller ontriution of lipids towrds metolism on the low protein diet. It is of interest tht the wild red-ked voles hd the highest ioptite δ 13 C vlues. This offers the possiility tht wild voles derived more energy from protein soures thn 005 NRC Cnd

8 7 Cn. J. Zool. Vol. 83, 005 lortory voles. Furthermore, the hir of wild voles ws enrihed in 13 C reltive to lortory voles for ll diets, while ollgen vlues of wild voles were enrihed in 13 C reltive to lortory voles fed the low protein diet. This my e inditive of high protein, low lipid diet in the wild. In generl, tissue δ 15 N vlues of red-ked voles vried positively rther thn negtively with dietry protein levels, while δ 13 C vlues did not vry systemtilly with dietry protein levels. Both lortory nd wild voles in the Knnskis Vlley otined suffiient protein from their diets to synthesize tissues; therefore, they do not pper to e limited y protein. Although the use of stle-nitrogen nd stle-ron isotopes to determine trophi reltionships nd dietry soures is well estlished, interprettion of δ vlues within trophi level is diffiult euse ftors other thn diet ontriute to isotopi vrition. Additionl experiments performed under ontrolled onditions, s well s isotopi nlysis of mronutrients isolted from the diet, re needed to seprte the potentil effets of dietry nd metoli frtiontion on smll-mmml stle-isotopi ompositions. Aknowledgements We thnk the stff t the Knnskis Field Sttion for their support during the summers. K. Lw nd L. Hung were most helpful in the Lortory for Stle Isotope Siene t The University of Western Ontrio. We thnk K. Ksprin nd L. Znette for vlule disussion of erly drfts of the mnusript. This study ws funded y the Nturl Sienes nd Engineering Reserh Counil of Cnd Disovery grnts to J.S.M. nd F.J.L. Referenes Amrose, S.H Effets of diet, limte, nd physiology on nitrogen isotope undnes in terrestril foodwes. J. Arheol. Si. 18: Amrose, S.H., nd DeNiro, M.J The isotopi eology of Est Afrin mmmls. 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