RESEARCH ARTICLE Carbon isotopic fractionation in eider adipose tissue varies with fatty acid structure: implications for trophic studies

Transcription

1 379 The Journl of Experimentl Biology 214, Published by The Compny of Biologists Ltd doi:1.1242/jeb RESEARCH ARTICLE Crbon isotopic frctiontion in eider dipose tissue vries with ftty cid structure: implictions for trophic studies Suznne M. Budge 1, *, Shiwy W. Wng 2,3, Tuul E. Hollmén 4,5 nd Mtthew J. Wooller 2,4 1 Deprtment of Process Engineering nd Applied Science, Dlhousie University, Hlifx, NS, Cnd, B3J 2X4, 2 Alsk Stble Isotope Fcility, Wter nd Environmentl Reserch Center, University of Alsk Firbnks, Firbnks, AK 99775, USA, 3 Sedn Ecologicl, Inc., Firbnks, AK 9977, USA, 4 School of Fisheries nd Ocen Sciences, University of Alsk Firbnks, Sewrd, AK 99664, USA nd 5 Alsk SeLife Center, Sewrd, AK 99664, USA *Author for correspondence (suznne.budge@dl.c) Accepted 31 August 211 SUMMARY Crbon isotopic frctiontion ws investigted in ftty cids (FA) of dipose tissue nd blood serum of thretened Stellerʼs eiders (Polystict stelleri) nd spectcled eiders (Somteri fischeri) reltive to the FA in their diets. Cptive eiders were fed known diet for 18dys with serum smpled t 6, 12 nd 18dys immeditely fter 12h fst; dipose ws collected t 18dys. Essentil FA (EFA) in the dipose showed vrying degrees of isotope frctiontion ( 4 ), depending on FA structure. The d 13 C vlues of long-chin FA 2:5n-3 nd 22:6n-3 did not differ from those in the diet, while those of 18:2n-6 nd 18:3n-3 were ~2 greter thn in the diet. The d 13 C vlues of free FA (FFA) in serum were not consistent within individuls or smpling dtes; frctiontion vried rndomly, suggesting tht FFA were rising from diet, rther thn mobiliztion from dipose tissue. Discrimintion fctors were used in combintion with mixing model incorporting FA nd lipid concentrtions to estimte the diet of eiders fed binry mixture with contrsting isotopic signtures. Diet estimtes vried with FA but men vlues closely pproximted the ctul proportions consumed. By trcking EFA, this study voided the complictions in interprettion rising from isotopic routing of crbon in bulk isotope nlyses nd serves s bsis for the development of compound-specific isotopic methods to trce dietry input in wild eiders. However, our understnding of the processes contributing to the vrition in isotopic signtures of FA in nture is currently limited, nd we recommend tht future reserch directions focus on elucidting these mechnisms. Key words: diet estimte, discrimintion, lipid, compound-specific isotope nlysis, stble isotope, Steller s eider, spectcled eider. INTRODUCTION The spectcled eider, Somteri fischeri (Brndt 1847), nd the Alskn breeding popultion of the Steller s eider, Polystict stelleri (Plls 1769), hve experienced precipitous popultion declines of breeding birds in Alsk nd re currently listed s thretened under the provisions of the US Endngered Species Act (Federl Register, 1993; Federl Register, 1997). Resons for the declines remin lrgely unknown, but chnges in the mrine ecosystem leding to vrition in the vilbility of food resources hve been listed s potentil cuses nd threts to the recovery of both species (US Fish nd Wildlife Service, 1996; US Fish nd Wildlife Service, 22). Both Steller s nd spectcled eiders use rnge of mrine hbitts in the Bering Se for stging, molting nd wintering, nd re thought to forge on vriety of prey items, including bivlves, crustcens nd snils (Petersen, 198; Petersen, 1981; Petersen et l., 1998). Both eider species nest in tundr hbitts long the costl Bering nd Beufort Ses, nd feed on insects, insect lrve, seeds nd plnt mterils in tundr ponds (Petersen et l., 2; Fredrickson, 21). Reproductive problems nd low productivity hve been documented for both species (Grnd et l., 22; Rojek, 28). In Steller s nd spectcled eiders, the reltive importnce of mrine nd terrestril nutrients for reproduction is unknown, but there is evidence to suggest tht some se duck species cquire nutrients for successful breeding from both the terrestril nesting nd mrine stging grounds (Bond et l., 27; Sénéchl et l., 211). A better understnding of diet sources nd timing of nutrient cquisition for these eiders will provide informtion to help identify importnt pre-breeding hbitts of thretened eider popultions in Alsk. There re severl well-known chllenges nd bises ssocited with trditionl methods of estimting mrine bird diets (reviewed in Brrett et l., 27). New techniques using minimlly invsive smpling methods nd biochemicl signtures in stored ft (Iverson et l., 27; Wng et l., 21), blood (Käkelä et l., 29; Federer et l., 21), stomch oil (Connn et l., 25; Wng et l., 27), fethers (Hobson nd Clrk, 1992) or eggs (Hobson, 1995; DeVink et l., 211) provide promising lterntives for diet ssessments in vin popultions. These biomrker techniques typiclly involve either stble isotope or ftty cid (FA) nlysis. For instnce, stble isotopic nlyses of crbon hve been used to determine the reltive contributions of sources of diet items (i.e. mrine versus terrestril, benthic versus pelgic), while stble nitrogen isotope nlyses cn indicte n niml s trophic level (Hobson nd Welch, 1992; Hobson et l., 22). However, there re limittions to the bulk isotope (i.e. mesurement of the isotopic composition of totl orgnic crbon or nitrogen in smple) pproches, including isotopic routing nd frctiontions from diet to tissue tht cn complicte interprettion (Federer et l., 21). As n lterntive, FA signture nlysis hs been used to investigte diets

2 Crbon isotopes of ftty cids in eiders 3791 of higher predtors (e.g. Thiemnn et l., 27; Willims et l., 28; Cooper et l., 29), including cptive eiders (Wng et l., 21). FA re the primry components of most lipids nd hve vriety of structures. Becuse of biochemicl restrictions, mny dietry polyunsturted FA (PUFA) re incorported into mrine nimls with little modifiction to structure, mking these FA vluble s indictors, or biomrkers, of their source. However, these biomrker techniques cn only offer generl informtion on temporl nd sptil vrition in consumer diets; much more complex models (i.e. quntittive ftty cid signture nlysis, QFASA) (Iverson et l., 24) re necessry to estimte proportions nd types of prey species in predtor diets using FA. Recently, we demonstrted the use of stble crbon isotopes of individul FA to trce crbon flow from prey to predtor cross multiple trophic levels in the Arctic (Budge et l., 28). This pproch trcks the stble crbon isotopes of specific FA structures tht cn only rise from diet nd hs two cler dvntges over the bulk isotope technique. First, this technique voids the problem of isotopic routing encountered with bulk isotope nlyses. Isotopic routing occurs when the crbon ssocited with dietry component (protein, crbohydrte or lipid) is routed to specific tissue component. For instnce, dietry protein crbon is likely to be routed to body muscle tissue. Difficulties rise, however, when dietry component, such s crbohydrte or lipid, is ctbolized nd used s crbon source to synthesize nother component, such s protein. The extent of routing cn be influenced by nutrient content nd digestibility of diets (Focken, 24; Codron et l., 211) nd environmentl fctors (Bloomfield et l., 211), nd techniques such s lipid removl before isotopic nlysis ttempt to minimize its impct; however, it remins compliction in the interprettion of bulk crbon isotope nlysis (Gnnes et l., 1997; Wolf et l., 29). Anlysis of essentil dietry FA tht cnnot be synthesized by nimls thus voids some of the difficulties ssocited with isotopic routing. Second, this technique cquires d 13 C dt of severl FA within ech smple ner-simultneously; when combined with mixing models to estimte source contributions, this llows severl independent ssessments of diet to be mde, ech bsed on n individul FA, thereby incresing the ccurcy of the results. Thus, the combintion of these fetures in the nlysis of stble crbon isotopes of essentil FA (EFA) offers cler benefits compred with the nlysis of isotopes of bulk crbon. While severl studies hve reported the stble crbon isotope composition (expressed s d 13 C vlues) of FA in members of vrious food webs (Hmmer et l., 1998; Ruess et l., 25; Budge et l., 28) nd provided evidence to suggest tht essentil dietry FA d 13 C vlues re preserved in the predtor s tissues (Stott et l., 1997; Howlnd et l., 23), none of these hve dequtely ddressed the issue of moleculr frctiontion, or chnge in d 13 C vlues during metbolism of dietry FA. The bsic processes involved in vin lipid digestion, trnsport nd deposition re firly well understood (Klsing, 1998). In the simplest cse, dietry lipids re predominntly tricylglycerols (TAG) consisting of three FA esterified to glycerol bckbone. FA in TAG molecules undergo number of enzymtic processes (e.g. lipolysis nd re-esterifiction) before being deposited in the dipose for storge. During periods of fsting, FA in the dipose re mobilized for energy nd trnsported in the blood, bound to proteins, s free FA (FFA). FA my lso be ctbolized for energy in the liver or, in some situtions, elongted or desturted. These processes ll involve enzyme-ctlyzed rections nd introduce the potentil for isotopic frctiontion of the FA. Such frctiontion my hve substntil impct on the interprettion of the FA d 13 C vlues; thus, the extent of frctiontion must be known before ttempting to relte consumers stble crbon isotope compositions to their diets. Our objective ws to determine the extent of moleculr crbon isotopic frctiontion tht dietry FA experience during ft deposition nd mobiliztion through controlled feeding studies with cptive Steller s nd spectcled eiders. Specificlly, we mesured the d 13 C vlues of severl essentil nd non-essentil FA in diet, dipose nd serum to crete mixing model incorporting informtion from moleculr frctiontion in the eider tissues, nd proportions nd d 13 C vlues of specific FA in dietry end-members. We then tested this model on smples preserved from previous cptive feeding experiment (Wng et l., 21) in which eiders were switched from diet bsed on terrestril mteril to experimentl diets spiked with different mrine food item. MATERIALS AND METHODS Controlled feeding studies Feeding trils were conducted during the non-breeding seson with eight mle Steller s eiders nd eight mle spectcled eiders housed t the Alsk SeLife Center in Sewrd, Alsk. Reserch ws conducted under Institutionl Animl Cre nd Use protocol number 9-8. For 2 yers, these birds hd been consistently fed diet bsed on Mzuri se duck formul (Purin Mills, St Louis, MO, USA), which consisted of pproximtely 6.5% lipid, 21.6% protein, 8.4% fiber, 1.9% sh nd 46.6% nitrogen-free extrct long with vitmins ( ccessed 15 Mrch 26). This ws supplemented with 5% of other diet items, including Antrctic krill (Euphusi superb), Atlntic silverside (Menidi menidi), Atlntic surf clm (Spisul solidissim) nd blue mussel (Mytilus edulis) (diet 1). This diet ws lso supplied throughout the 18 dys of this experiment, with the intke of diet items recorded dily by species so tht the ctul diet could be clculted. Blood serum ws smpled between 9: h nd 12: h t 6, 12 nd 18 dys immeditely fter 12 h fst. Up to 3 ml of whole blood ws collected from the jugulr vein using 23 guge needle nd 5 ml syringe, trnsferred to collection tubes (BD Vcutiner SST Plus Blood Collection Tubes, Frnklin Lkes, NJ, USA) nd llowed to clot t 4 C. Adipose tissues were collected from ll birds t the end of the 18 dys. A biopsy technique ws used to obtin pproximtely.1 g of synscrl dipose tissue smples from individuls vi 1 cm skin incision (Iverson et l., 27; Wng et l., 21). The re ws disinfected prior to biopsy with betdine swb nd lidocine spry. Lidocine ws lso injected subcutneously. After surgery, the biopsy site ws seled with Vet-bond (3M, St Pul, MN, USA). The dipose tissue smple ws plced in vil of chloroform with.1% butylted hydroxytoluene dded s n ntioxidnt. For comprison, smples from previous controlled feeding experiment (Wng et l., 21) were lso nlyzed. In tht study, eiders were fed consistent diet of predominntly Mzuri (88%), supplemented with the sme diet items s in diet 1 described bove. After 69 dys, the diets of the Steller s nd spectcled eiders were switched to diet 2 which consisted of only Mzuri nd krill, t proportions of 66% nd 34%, nd 56% nd 44%, respectively. These diets were mintined for 21 dys nd synscrl dipose tissue ws collected s described bove. For both experiments, smples of the Mzuri nd diet supplements were collected ech month to monitor the consistency of FA level nd isotopic composition (N 25; 5 smplings 5 diet types consisting of Mzuri nd supplements of krill, silverside, clm nd mussel). Smples were frozen intct nd stored t 2 C until extrcted.

3 3792 S. M. Budge nd others Isoltion of blood lipids, lipid extrction nd FA nlyses Whole-blood smples were centrifuged to isolte serum t 15 g for 1 min in Cly Adms TRIAC centrifuge (Becton Dickinson Compny, Frnklin Lkes, NJ, USA) within 1 2 h of the blood drw. Lipids were extrcted from ll smple types (serum, dipose, diet smples) using modified Folch extrction (Folch et l., 1957; Budge et l., 26) with 2:1 chloroform:methnol. Thin-lyer chromtogrphy using developing solvent of 85:15:1 hexne:diethyl ether:cetic cid ws used to isolte FFA in fsting blood smples. FA methyl esters (FAME) were prepred directly from 1 mg of the extrcted nd isolted lipid using H 2 SO 4 in methnol. FAME were then extrcted into hexne nd concentrted to 5 mg ml 1. FAME were quntified using temperture-progrmmed gs chromtogrphy (GC) s described previously (Budge et l., 26), using Perkin Elmer Autosystem II (Perkin Elmer, Boston, MA, USA) flme ioniztion detector (FID) gs chromtogrph fitted with 3 m.25 mm i.d. column coted with 5% cynopropyl polysiloxne (.25 m film thickness; J&W DB-23; Folsom, CA, USA) nd operting in split injection mode. For ll smple types, 1. l of 5 mg ml 1 solution of FAME in hexne ws injected. FA were identified using known stndrd mixtures (Nu Check Prep., Elysin, MN, USA) nd GC-mss spectrometry (Thermo Finnign Polris Q; Thermo Finnign, Austin, TX, USA). All smple chromtogrms nd FA identifictions were individully checked, corrected, nd reintegrted s necessry. Ech FA ws described using the shorthnd nomenclture of A:Bn-X, where A represents the number of crbon toms, B the number of double bonds nd X the position of the double bond closest to the terminl methyl group. The d 13 C vlues of FAME sub-smples were determined by routing the effluent from GC through combustion interfce (IsoLink; to n isotope rtio mss spectrometer (IRMS) (Thermo Finnign Delt V). FAME were seprted using the sme GC column nd temperture progrm s described bove. The GC ws operted in splitless injection mode nd for dipose nd serum smples, 1. l of 1. mg ml 1 FAME solution in hexne ws injected. For diet smples, the concentrtion ws reduced to.5 mg ml 1. Monounsturted FAME isomers were dequtely resolved by GC-FID but co-eluted with GC-IRMS so 16:1 nd 18:1 represent the sum of those monounsturted FAME species. FAME nlyzed here differed from the FA present in the source mteril becuse of the ddition of methyl group derived from the methnol used in trnsesterifiction. To correct for the contribution from this extr crbon, we trnsesterified individul FFA stndrds 16: nd 18: with the sme regents described bove. The d 13 C vlues for FFA nd FAME derived from those were determined using n elementl nlyzer (Costech ECS41; Vlenci, CA, USA) routed to the IRMS. The difference between the d 13 C vlues for FFA nd FAME with the sme chin length ws due to the dded methyl group. The proportionl contribution of this methyl group to given FA depends on the chin length; n verge d 13 C vlue for this dded methyl group ws therefore clculted using the following eqution: d 13 C (n + 1) [d 13 C FAME ] n[d 13 C FFA ], (1) where n is the number of C toms in the FFA (Abrjno et l., 1994). The methyl C hd n verge vlue of 49.±.3. All FAME dt were corrected for the contribution of this methyl group by rerrnging the bove eqution. Anlyticl precision throughout the runs ws trcked using n 18: stndrd nd ws.3. All d 13 C vlues re reported reltive to Vienn Pee Dee Belemnite using delt nottion. These vlues were clibrted using stndrd mixture consisting of ethyl nd methyl esters of 14:, 16:, 18: nd 2: supplied by Indin University Stble Isotope Reference Mterils, where r 2 of the known versus expected reltionship ws >.98. Dt nlyses A three-level one-wy nlysis of vrince (ANOVA) ws used to test for differences in d 13 C vlues of ech FA between species nd diet 1. The d 13 C vlues of FA of diet 1 were identicl for the two species so were not compred. Post hoc testing ws crried out using Tukey s test with n djusted -level of.17 (.5/3). The d 13 C vlues of FA of diet 2 were not identicl so four-level one-wy ANOVA ws conducted using n djusted -level of.13 (.5/4) for post hoc testing. A two-level ANOVA ws used to compre d 13 C of FA of diet nd the third serum smple, while repetedmesures ANOVA ws used to test for differences between dipose nd serum. All nlyses were performed using SPSS 11. Stble isotopic discrimintion fctors ( ) between FA in diet (D) nd dipose (A) were clculted by subtrcting the isotopic vlue of diet for ech FA nd individul eider from the corresponding vlue in the dipose tissue, so tht A D A D. Similrly, discrimintion fctors were clculted between dipose (A) nd serum (S) following the sme eqution ( S A S A). To test the efficcy of FA-specific isotope nlysis in estimting dietry contributions, two-end member mixing model ws used to determine the reltive contributions of Mzuri nd krill (diet 2) (Wng et l., 21) to crbon in the FA exmined: d 13 C eider,j j x j d 13 C M + (1 x j ) d 13 C k, (2) where d 13 C eider,j is the d 13 C of the eider dipose for j, the FA of interest, j is the discrimintion fctor for diet to dipose trnsfer for j, x j is the proportion of Mzuri crbon contribution to j, d 13 C M is the d 13 C of the Mzuri FA nd d 13 C k is the d 13 C of the krill FA. To determine the contribution of Mzuri crbon to ll FA in generl in the eider dipose tissue, the reltive mounts of lipid nd FA in the diet items must be tken into ccount: RESULTS The mjor FA in ll diet items were similr, with 16:, 18: nd 18:1 generlly dominting the profiles (Tble 1). The Mzuri contined 18:2n-6 t 38% by mss of totl FA nd ws distinct from the other diet items, ll of which hd 18:2n-6 levels <2%. Except for the mussel, levels of 18:3n-3 were lso much greter in Mzuri (4% versus <1%) thn in the diet supplements. Conversely, clm, krill, mussel nd silverside contined the mrine FA 2:5n-3 nd 22:6n-3 t levels 3 1 times greter thn Mzuri. The FA compositions in dipose nd serum were similr for both Steller s nd spectcled eiders. The dipose FA of both eider species were brodly similr to tht of Mzuri, the mjor component in diet 1, with 16:, 18:1 nd 18:2n- 6 dominting both profiles; however, reltive proportions in dipose nd Mzuri differed (Tbles 1 nd 2). For exmple, the rtio of 18:2n- x j FA j,m L M x FA = x j FA j,m L M + 1 x j FA j,k L k, (3) where x FA is the proportionl contribution of Mzuri to ll FA in the eider dipose, FA j,m is the verge proportion of FA j in the Mzuri, FA j,k is the verge proportion of FA j in the krill, L M is the verge lipid content of the Mzuri nd L k is the verge lipid content of the krill.

4 Crbon isotopes of ftty cids in eiders 3793 Tble 1. Mss percent nd d 13 C vlues of FA in diet items for diet 1 (this study) nd diet 2 (Wng et l., 21) Diet 1 Diet 2 Mzuri Krill Clm Mussel Silverside Mzuri Krill (N 5) (N 5) (N 5) (N 4) (N 5) (N 3) (N 3) FA composition (mss%) 16: 15.87± ± ± ± ± ± ±.19 16:1 2.59± ± ± ± ± ± ±.37 18: 6.3± ± ± ± ± ± ±.4 18:1 2.68± ± ± ± ± ± ±.34 18:2n ± ±.8.41±.2 2.3±.21.84± ± ±.8 18:3n ±.6.95±.14.3± ±.14.7± ±.4.65±.6 2:5n ± ± ± ± ± ± ±.81 22:6n ± ± ± ± ± ± ±.12 FA d 13 C ( ) 16: 26.± ± ± ± ± ± ±1.9 16:1 24.9± ± ± ± ± ± ±1.1 18: 27.1± ± ± ± ± ± ±.8 18:1 25.8± ± ± ± ± ± ±.9 18:2n ± ±.2 NA 29.2± ± ± ±2.7 18:3n-3 32.±. 38.5±.2 NA 3.9±.3 32.± ±.5 4.6±2.8 2:5n ± ± ± ± ± ± ±.8 22:6n ±.3 34.± ± ± ± ± ±.3 Actul proportion consumed (%) Steller s eider Spectcled eider Mss% nd d 13 C dt re mens ± s.e. FA, ftty cids. Tble 2. FA composition in dipose tissue nd blood serum of Steller s nd spectcled eiders fed diet 1 Adipose Serum Serum Serum 18 dys 6 dys 12 dys 18 dys Steller s eider (N 8) (N 8) (N 7) (N 8) 16: 18.92± ± ± ±.9 16:1 2.52± ± ± ±.1 18: 8.7± ± ± ±.91 18: ± ± ± ±1.2 18:2n ± ± ± ±.74 18:3n ±.3.88± ±.6.79±.7 2:5n-3.24±.2.25±.5.38±.7.27±.6 22:6n-3.46±.5.35±.3.46±.5.38±.4 Spectcled eider (N 8) (N 8) (N 8) (N 8) 16: 17.18± ± ± ±1. 16:1 1.98± ± ± ±.2 18: 8.35± ± ± ±.87 18: ± ± ± ± :2n ± ± ± ±1.8 18:3n ±.5.83±.8 1.2±.7.8±.1 2:5n-3.17±.2.24±.4.35±.4.17±.2 22:6n-3.36±.6.47±.7.51±.5.29±.5 FA (ftty cid) composition is given s mss% totl FA (mens ± s.e.). 6 to 18:1 in Mzuri ws pproximtely 2:1 but only 1:2 in the dipose. Serum FFA lso showed vrition in proportions reltive to the dipose from which they were mobilized. For instnce, serum consistently contined greter mounts of 16: nd 18:, nd lower levels of 18:1 nd 18:2n-6 thn dipose. The d 13 C vlues of individul FA were generlly similr in the Mzuri, clm, mussel nd silverside in diet 1 (Tble 1). For instnce, the d 13 C vlues of 18:2n-6 rnged from 29.2 to 28.6 in the four diet items, while 22:6n-3 rnged from 26.5 to Krill FA were ll much more isotopiclly depleted, with d 13 C vlues in the six FA nlyzed vrying from 39.2 to Similr ptterns were evident in the Mzuri nd krill of diet 2. Bsed on the known lipid contents (dt not shown), concentrtions nd d 13 C vlues of FA in individul diet items (Tble 1), nd proportions of ech diet item consumed (Tble 1), d 13 C vlue of ech FA in the totl diet ws clculted (Fig. 1). For ll FA except 2:5n-3 nd 22:6n-3, consistent nd significnt (P<.17) isotopic enrichment in crbon of 1 4 ws evident in the dipose of both eider species reltive to diet 1 (Fig. 1). In generl, this enrichment ws less for EFA (18:2n-6, 18:3n-3, 2:5n-3 nd 22:6n-3) thn for non-efa,

5 3794 S. M. Budge nd others A 16: 16:1 18: 18:1 18:2 18:3 2:5 22:6 Fig. 1. d 13 C vlues (mens nd s.e.) of diet, dipose nd serum ftty cids (FA) for (A) Steller s eider nd (B) spectcled eider fed diet 1 for 18 dys. The sme letters indicte sttisticlly similr vlues. All other comprisons re significntly different (P<.17). FA δ 13 C ( ) B Diet Adipose Serum b,b with the exception of 16:1 (Fig. 1); this FA hd A D of ~1, similr to tht of the EFA. Vlues for d 13 C of 2:5n-3 nd 22:6n-3 in the dipose of both species were not significntly different from diet 1. There ws lso very limited species effect for eiders fed diet 1; only d 13 C of 16: showed subtle but significnt difference (P<.17) between the two species ( 23.7±.2 nd 24.4±.2, for Steller s nd spectcled eiders, respectively). Isotopic enrichment of the FA in eider dipose ws less consistent between species for eiders fed diet 2 (Fig. 2); however, no significnt differences in d 13 C vlues of ech FA in dipose between species were found, due to the lrge vrince (up to ~2 ) ssocited with the mesurements. Isotopic vlues were more similr between diet 2 nd dipose, with 16:, 18: nd 18:3n-3, in ddition to 2:5n-3 nd 22:6n-3, showing similrity (P>.13) in d 13 C vlues in both species. Men stble isotopic discrimintion fctors of FA in dipose nd diet vried from.5 to +4 within species (Tble 3); within FA, there ws not significnt difference between species (Tble 3). The estimted proportions of Mzuri consumed in diet 2 were clculted bsed on the discrimintion fctors of strictly dietry FA (18:2n-6, 18:3n-3, 2:5n-3 nd 22:6n-3; Tble 3), the mesured FA d 13 C vlue in the dipose (Fig. 2), nd the lipid content (not shown), FA proportion nd FA d 13 C vlue of ech of the two diet items (Tble 1), following Eqns 2 nd 3. Estimted proportions vried widely mong the FA but estimtes bsed on 2:5n-3 were closest to ctul Tble 3. Discrimintion fctors clculted from comprison of d 13 C vlues in dipose nd diet 1 Discrimintion fctors ( A D ) Estimted proportion Mzuri consumed Clculted Steller s eider Spectcled eider Steller s eider Spectcled eider 16: 4.4± ±.24 NA NA 16:1 1.28±.3 1.6±.46 NA NA 18: 3.69± ±.23 NA NA 18:1 3.38± ±.5 NA NA 18:2n-6 1.9± ±.29 69±5 55±4 18:3n ± ±.26 14±8 27±9 2:5n-3.53±.82.52±.98 3±79 46±116 22:6n-3.84±.93.77±.8 2±51 36±33 Men NA NA 29±95 41±121 Men 18:2 nd 18:3 only NA NA 42±9 41±1 Assumed 2:5n-3 33±21 11±22 22:6n-3 34±362 54±21 Discrimintion fctor dt re mens ± s.e. Proportions of Mzuri consumed in diet 2 (mens ± s.e.) were estimted bsed on derived discrimintion fctors, FA (ftty cid) proportions, lipid levels nd d 13 C of FA in Mzuri nd krill. Actul proportions of Mzuri fed were 34% nd 44% for Steller s nd spectcled eiders, respectively.

6 Crbon isotopes of ftty cids in eiders 3795 FA δ 13 C ( ) A B 16: 16:1 18: 18:1 18:2 18:3 2:5 22:6 Diet Adipose in both species (Tble 3). However, the errors ssocited with estimtes tht included contributions from 2:5n-3 nd 22:6n-3 were lrge becuse of the lrge uncertinty ssocited with the discrimintion fctors for those two FA. Accurte estimtes with much lower errors were obtined when mens were clculted using only 18:2n-6 nd 18:3n-3 (estimtes of 42±9% nd 41±1% versus ctul of 34% nd 44% in Steller s nd spectcled eiders, respectively). With the exception of 16:1 in the Steller s eiders nd 18:3n-3 in both species, d 13 C vlues of serum FA were ll significntly depleted (P<.5) reltive to dipose, nd were much more similr to dietry isotopic vlues (Fig. 1). Discrimintion fctors for the mobiliztion of FA from dipose to serum vried widely nd showed little consistency within individuls nd FA (Fig. 3). The most extreme exmple of vribility mong serum collections in one individul occurred for 18:3n-3 in spectcled eider 16, where discrimintion fctors rnged from low of 7.1 to high of In mny individuls, the isotopic vlues of two of the three serum smples greed quite well (Fig. 3); however, there ws no regulrity in the pttern nd the dissimilr smple ws not ssocited with prticulr blood collection. DISCUSSION Diet to dipose isotopic discrimintion fctors Potentil explntions for the vrition in discrimintion fctors with FA structure require considertion of the metbolic pthwys tht FA cn follow fter consumption (see Stevens, 1996; Klsing, 1998; Price, 21). Dietry lipids, fter crossing through the epithelil cells in the intestine, re first trnsported in the blood s portomicrons, the vin equivlent of mmmlin chylomicrons, nd routed directly to the liver. There they cn be ressembled into very lowdensity lipoproteins (VLDL) for trnsport to other tissues (e.g. muscle nd dipose), elongted/desturted, stored or ctbolized for energy (Stevens, 1996). If dietry lipids re in excess, some Fig. 2. d 13 C vlues (mens nd s.e.) of diet nd dipose FA for (A) Steller s eider nd (B) spectcled eider fed diet 2 for 21 dys. The sme letters indicte sttisticlly similr vlues. All other comprisons re significntly different (P<.13). Discrimintion fctors (Δ A S ) : : : 18:1 18:2n-6 18:3n Steller's eider Spectcled eider Fig. 3. Discrimintion fctors for the mobiliztion of free FA (FFA) from dipose to serum. Dt re displyed for ech individul t ech of three collection periods (circle, 6 dys; squre, 12 dys; tringle, 18 dys). proportion is trnsported s VLDL to dipose tissue for storge. In fsting stte, FFA (or non-esterified FA) re mobilized from dipose for trnsport to tissues where they re ctbolized s fuel. Within this context, number of mechnisms could explin our results. De novo synthesis of the sturted nd monounsturted FA could be occurring nd would explin some of the observed frctiontion. Similrly, elongtion nd desturtion of precursors could lso occur to form sturtes nd monounsturtes, likely resulting in frctiontion (Monson nd Hyes, 1982). However, the birds were fed nutritionlly sound diets with n excess of lipids nd in tht sitution, enzymes ssocited with these processes re commonly down-regulted so it is unlikely tht either biosynthesis or modifiction of FA ws occurring (Stevens, 1996; Gurr, 1997). Synthesis of the n-3 nd n-6 PUFA is impossible, s vertebrtes do not possess the necessry enzymes to do so.

7 3796 S. M. Budge nd others When FA re pckged into portomicrons for trnsport in the blood or deposited in the liver for storge, they must first be hydrolyzed, or cleved, from glycerol bckbone to cross the cell membrne of epithelil nd endothelil cells nd then re-esterified into n intct TAG. The hydrolysis nd re-esterifiction rections re medited by enzymes nd therefore re subject to kinetic frctiontion. However, both hydrolysis nd re-esterifiction involve breking or forming bond between crbon tom in the glycerol bckbone nd n oxygen tom in the ftty cyl chin; there is no resonble mechnism for FA with n isotopiclly lighter crbon tom to be selected, s only the oxygen tom on the FA chin tkes prt in the rection. Thus, portomicron formtion nd FA deposition in the liver cn be eliminted s cuses of the observed frctiontion. In fct, ll lipid-trnsport processes supplying TAG to tissues involve these processes of hydrolysis nd re-esterifiction nd cn be eliminted s sources of frctiontion. Similrly, selective mobiliztion of isotopiclly lighter FA from the dipose tissue during fsting proceeds through hydrolysis of TAG nd is unlikely to result in frctiontion for the sme reson. The only probble explntion tht remins for frctiontion is ctbolism of FA for energy. Ctbolism of FA in the liver before deposition in dipose could led to these results if enzymes ssocited with -oxidtion discriminte FA substrtes ccording to their msses nd fvor those with lighter msses. This selection would result in more rpid oxidtion of the isotopiclly lighter FA, leving greter proportion of isotopiclly hevier FA to be routed to the dipose for deposition, nd offers resonble explntion for the enrichment in 13 C found in most FA in the dipose reltive to the diet. Such process lso grees with the originl dt of Deniro nd Epstein where respired CO 2 ws found to be isotopiclly lighter thn tht of either diet or tissue, likely the result of ctbolism (Deniro nd Epstein, 1978). McMhon nd collegues found vrible crbon isotopic frctiontion in non-essentil mino cids between diet nd muscle tissue in fish species, but prcticlly no frctiontion of essentil mino cids (McMhon et l., 21); similr results hve lso been reported for mino cids in insects (O Brien et l., 25). Here, we present dt for four FA tht would normlly be considered essentil, bsed on n inbility of the eiders s vertebrtes to synthesize these FA de novo: 18:2n-6, 18:3n-3, 2:5n-3 nd 22:6n- 3 (Stevens, 1996; Klsing, 1998; Cunnne, 23). We found no discrimintion of 2:5n-3 nd 22:6n-3 between diet nd dipose (Figs 1 nd 2); however, we found substntil vrition in the discrimintion fctors of 18:2n-6 nd 18:3n-3 (~2 nd 1.5, respectively). This seemingly differentil frctiontion of EFA my be tied to the criteri used to define essentility. Vertebrtes lck the enzymes necessry to form 18:2n-6 nd 18:3n-3 from precursors so these FA re normlly considered essentil becuse they cnnot be synthesized de novo. However, it is the longer chin PUFA, 2:4n-6, 2:5n-3 nd 22:6n-3, rther thn 18:2n-6 nd 18:3n-3, tht hve importnt physiologicl functions, such s nervous system development nd hormone signling (Gurr, 1997; Ackmn nd Cunnne, 1992). Although 18:2n-6 nd 18:3n-3 themselves hve no known essentil functions, they cn serve s precursors for the synthesis of the longer chin PUFA through elongtion nd desturtion, nd eliminte symptoms of EFA deficiency (Ackmn nd Cunnne, 1992; Cunnne, 23), so they re therefore lso considered EFA [see Cunnne (Cunnne, 23) for full discussion of criteri for essentility]. There is evidence from fish (Tocher, 23) tht the enzymes responsible for elongtion nd desturtion of precursors re much less ctive in mrine environments where long chin essentil PUFA re plentiful. Similr rguments hve been proposed to explin the inbility of most humns in Western society to form long chin PUFA from precursors when consuming diets high in PUFA (Siguel nd Mclure, 1987; Ackmn nd Cunnne, 1992). If the essentil function of 18:2n-6 nd 18:3n-3 is to serve s precursors for synthesis of 2:5n-3, 2:4n-6 nd 22:6n- 3 but those long chin PUFA re not being formed by elongtion nd desturtion, then 18:2n-6 nd 18:3n-3 would not hve direct essentil function. In tht sitution, there would be no need to spre them from oxidtion nd we might then expect discrimintion of these FA. However, this line of resoning fils for 18:3n-3 when diet 2 is considered. For diet 2, 18:3n-3 showed no net frctiontion from diet to dipose, despite the presence of even lrger mounts of 2:5n-3 nd 22:6n-3 in diet 2 thn in diet 1 (Tble 4). If 18:3n- 3 were being ctbolized becuse it ws not essentil s result of sufficient levels of long chin PUFA in the diet, one would expect ctbolism to continue in eiders fed diet 2, which contins even higher levels of those PUFA. Ctbolism of specific FA my lso be driven by dietry excess of those FA. Essentility implies tht there is some bsolute dietry intke of FA tht must be met. Amounts consumed in excess of tht minimum mount could simply be used s substrte for oxidtion, like non-essentil FA. The mjor FA in diet 1 ws 18:2n-6 (Tble 4), t pproximtely 39% of totl FA. Free-rnging eiders would not normlly consume such high levels of 18:2n-6, with tht FA typiclly present in proportions <12% in most mrine bivlve species (Tble 1) (Joseph, 1982), s well s qutic insects (Goedkoop et l., 2; Sushchik et l., 23) nd plnts (e.g. Copemn et l., 29). Known diet items for free-rnging eiders would lso be expected to hve low lipid contents, so tht 18:2n-6 would mke very little contribution to totl FA consumed. In cptive eiders fed high qulity diet d libitum, it is possible tht the 18:2n- 6 in the diets is in excess of their dietry needs. Such n excess is likely to be ctbolized for energy, s with ny other FA. In fct, it might be tht 18:2n-6 is being preferentilly ctbolized simply becuse of this potentil excess in the diet. Ackmn nd Cunnne proposed the sme explntion for the preferentil oxidtion of 18:2n-6 in Western humn diets (Ackmn nd Cunnne, 1992); the effect is lso well known in quculture fish species (Turchini et l., 29). A similr scenrio could lso be true with 18:3n-3. The Tble 4. Approximte concentrtions of FA consumed with ech diet FA composition Diet 1 Diet 2 Steller s eider 16: 814±4 1627±29 16:1 146±7 65±17 18: 35±16 158±2 18:1 117± ±26 18:2n ±94 733±15 18:3n-3 195±1 111±3 2:5n-3 142±6 834±33 22:6n-3 166±7 49±9 Spectcled eider 16: 812±4 15±25 16:1 143±7 531±14 18: 37±16 175±3 18:1 118± ±24 18:2n ±95 912±18 18:3n-3 197±1 125±2 2:5n-3 138±6 723±28 22:6n-3 16±7 368±7 Dt re mg 1 g 1 (totl), mens ± s.e. FA, ftty cids.

8 Crbon isotopes of ftty cids in eiders 3797 mount of tht FA consumed by the eiders is fr less thn tht of 18:2n-6 but it might remin in excess of some unknown minimum, thus resulting in selective ctbolism nd the observed frctiontion when diet 1 is consumed. For diet 2, the levels of 18:3n-3 consumed dropped substntilly so tht mounts of 18:3n-3 were much less thn those of 2:5n-3 nd 22:6n-3 nd there ws no net frctiontion between diet nd dipose. It my be tht mounts of 18:3n-3 hd fllen below some minimum dietry threshold so there ws no excess to oxidize, while 2:5n-3 nd 22:6n-3 remined below their minimum necessry levels, suppressing ctbolism of those FA. A similr explntion my pply to the discrimintion fctor observed for 16:1. This FA ws present t mounts <15 mg 1 g 1 in diet 1, levels similr to 18:3n-3, 2:5n-3 nd 22:6n-3; these reltively low levels my mke it poor substrte for oxidtion. In diet 2, the concentrtion of 16:1 incresed lmost 4-fold, nd showed frctiontion more similr to the other bundnt FA. Finlly, our results my lso be influenced by turnover times of FA in the dipose tissue. If one ccepts the ide tht frctiontion is due to oxidtion of excess FA, one might expect tht less frctiontion would be observed with drop in levels of dietry 18:2n-6 nd 18:3n-3. Certinly, the result for 18:3n-3 in diet 2 grees with this; however, very similr isotopic enrichment ws observed for 18:2n-6 in the two diets, despite the drop in intke. Eiders were only fed diet 2 for 21 dys; tht period of time my not hve been sufficient to fully replce the lrge mount of 18:2n-6 originlly deposited in the dipose tissue. Indeed, the proportions of 18:2n-6 did not fll to mtch dietry levels, unlike most other FA including 18:3n-3, 2:5n-3 nd 22:6n-3 [dt in Wng et l. (Wng et l., 21)]. Thus, it is likely tht the d 13 C vlue for dipose fter 21 dys on diet 2 ws still hevily influenced by the d 13 C of the previously fed diet, rther thn the new diet. All of this discussion rises importnt questions bout frctiontion of FA tht should be ddressed before FA-specific isotope nlysis is used to estimte diets of consumers. Bsed on the bove rguments, only PUFA tht cnnot be synthesized de novo would be useful in trcking diet nd it seems resonble to conclude tht eiders forging in their nturl environment would experience little frctiontion in 18:3n-3, 2:5n-3 nd 22:6n-3. However, from the present dt, it is difficult to ssign relible discrimintion fctor to 18:2n-6. More experimenttion, using diets tht re more similr to those consumed by free-rnging eiders, is needed to ddress these questions. Adipose to serum isotopic discrimintion fctors During fsting, FA re mobilized from dipose nd trnsported in the blood to other tissues where they cn be ctbolized for energy (Stevens, 1996; Price, 21). Thus, it is resonble to expect tht the d 13 C vlues of serum FFA would resemble the dipose lipids tht they were derived from. Anlysis of serum FFA therefore represented much less invsive mnner to indirectly mesure dipose FA d 13 C vlues. The process of mobiliztion involves hydrolysis of FA from TAG molecules in the dipose nd, s described bove, does not involve the clevge of bond involving crbon tom in the FA chin. There is lso no opportunity during this mobiliztion for synthesis or modifiction of FA so ll six FA mesured in serum, including sturted nd monounsturted FA, could be compred between the tissues without concern for biosynthetic modifiction of d 13 C of FA. Becuse of these restrictions, we did not expect to observe frctiontion rising from the mobiliztion of FA from dipose to serum. Insted, we found frctiontion for tht trnsfer nd it ppered to be rndom (Fig. 3), which suggests some chllenges ssocited with the methodology. Fsting, s well s strenuous exercise, results in incresed mobiliztion of FFA from dipose stores into the bloodstrem in both humns (Cortright et l., 1997) nd birds (Lien et l., 1999; Price et l., 28) but there re few dt vilble to indicte the minimum time period necessry to promote this mobiliztion. Lien nd collegues (Lien et l., 1999; Lien nd Jn, 23) used fsting period of 3 dys with Tsiy ducks (Ans pltyrhynchos vr. domestic), lrger species, while Käkelä nd collegues (Käkelä et l., 26; Käkelä et l., 27; Käkelä et l., 29) used period of 24 h to ensure tht portomicrons derived directly from diet were eliminted from circultion in severl sebird species. Eiders in this study were fsted for shorter period of 12 h prior to blood smpling. We used thin-lyer chromtogrphy to isolte FFA from other lipid clsses in the serum smples, so tht the d 13 C dt would only reflect those of FFA mobilized from dipose. However, FFA cn lso rise through nturl hydrolysis of lipids, such s TAG nd phospholipids, during smple collection nd nlysis (Christie, 23), so the FFA tht were isolted in eider serum my hve represented degrdtion products of either dietry lipids in the form of portomicrons or lipids present in blood lipoproteins. Typicl fsting levels of FFA seem to vry by species nd likely by body mss but re generlly of the order of 2 5% tht of TAG. For instnce, Lien nd collegues found FFA t ~25% of TAG fter 3 dy fst in ducks (Lien et l., 1999). With much smller body msses of <3 g, white-throted sprrows (Zonotrichi lbicollis) lso hd FFA levels rnging from 25% to 5% tht of TAG fter n overnight fst of unspecified durtion (Smith et l., 27). In contrst, nlysis of lipid clss dt showed FFA t trce levels (<5% of TAG) in ll eider serum smples, suggesting tht the FFA were derived from the brekdown of circulting lipids, rther thn mobilized from TAG in dipose. The d 13 C vlues of serum FFA were intermedite between those of diet nd dipose (Fig. 1) nd, for 16:1 nd 18:2n-6, were not significntly different from diet in both eider species. This suggests strong dietry influence; if portomicrons were still circulting fter 12 h fst, hydrolysis of the dietry TAG crried within them would likely result in the similrity in d 13 C vlues of the two FA. It is likely tht longer fsting period is necessry to reduce levels of circulting TAG nd to promote mobiliztion of FFA from dipose. Becuse it ws not possible to smple dipose t ll three time points when blood ws collected, it ws necessry to mke the ssumption tht the d 13 C of FA in dipose did not vry from dy 6 to dy 18 so tht discrimintion fctors clculted t 6 nd 12 dys incorported d 13 C from dipose t 18 dys. The diets fed for the durtion of the experiment were very similr to the mintennce diets fed prior to nd fter the study, consisting of ~95% Mzuri with supplementtion with other diet items. Thus, it is likely tht the d 13 C vlues of FA in dipose were quite similr t ll time points. Regrdless of the cuse of the vrince in discrimintion fctors for mobiliztion of FFA from dipose to blood, there is clerly need for further reserch. A first essentil step is to determine the minimum time necessry to promote mobiliztion of FFA from dipose. This could be ccomplished by simple trcer experiments where dietry pulse of FA tht is not present in the regulr diet is provided. For instnce, 23: could be fed in lrge dose to the eider nd the time required for its clernce from the blood determined by periodic nlysis of serum FFA. With tht time frme estblished, it would be reltively simple to repet the serum nd dipose collections to initilly estblish consistency of discrimintion fctors for FA within individuls nd likely within species, ssuming constnt diet ws mintined.

9 3798 S. M. Budge nd others Diet estimtion The development of n dditionl technique to estimte diet in eiders ws the ultimte gol of this work. A necessry criterion for this ppliction is the presence of distinct isotopic signtures of FA in different sources. Mrine FA would be expected to follow the sme trend s observed with bulk crbon nlysis nd be enriched in 13 C reltive to terrestril plnt mteril produced vi C 3 photosynthesis. However, Mzuri is bsed primrily on corn, C 4 plnt tht is typiclly enriched in 13 C reltive to C 3 plnts, nd such enrichment in C 4 plnts cn led to isotopic signtures tht re very similr to mrine vlues. We clerly sw this effect in our dt with little consistent vrition in the d 13 C vlues of FA mong Mzuri, clm, mussels nd silverside. Conversely, the d 13 C vlues of Antrctic krill nlyzed here were more depleted by t lest 6 compred with the Mzuri in the four PUFA used in the diet estimtes (Tble 1); such depletion is typicl of Antrctic krill (Schmidt et l., 23) nd provided convenient opportunity to test our mixing model. Estimtes using ll four PUFA llowed n ccurte men to be clculted. In the wild, our interest in eider diets is to differentite between terrestril/qutic nd mrine sources but it is unlikely in tht ppliction tht 2:5n-3 nd 22:6n-3 will provide useful estimtes. Terrestril plnts will contin very little, if ny, of those two FA (Gunstone et l., 1986), nd it will be necessry to rely on FA with limited synthesis pthwys common to both systems, such s 18:2n-6 nd 18:3n-3. In the present study, if only those two FA re used, estimtes for diet 2 for both species would be ~41±1% Mzuri (Tble 3), despite very different estimtes for individul birds, suggesting tht the results would remin ccurte with fewer FA nlyzed. No significnt difference ws found between the d 13 C vlues of diet 1 nd dipose for 2:5n-3 nd 22:6n-3 becuse of their high vrince, implying tht A D. However, when such vlue ws used in the diet clcultion s the discrimintion fctor for 2:5n- 3, estimtes of proportions of Mzuri consumed for Steller s eiders hd meningless, negtive vlue (Tble 3). Thus, non-zero discrimintion fctors were clculted nd pplied. Erlier discussion hs offered explntions to support the similrity in d 13 C vlues in dipose nd diet for those FA, nd highlighted the implictions; the need to use non-zero A D does not necessrily contrdict this nd rises becuse of the high vrince in the mesurements rther thn cler dissimilrity in vlues. While the men of the individul estimtes did provide n ccurte ssessment of proportions, the error ssocited with 2:5n- 3 nd 22:6n-3 ws lrge. Although only three individuls of ech species were vilble to test diet estimtes, the lrge errors were not consequence of vrition due to low smple number; rther, they were derived from the discrimintion fctors clculted in the initil experiment where eight individuls were nlyzed. Both 2:5n-3 nd 22:6n-3 were present t low concentrtions (<.5% of totl FA), resulting in lower smple precision. These errors were then propgted through the clcultion, generting the high vrition in the individul estimtes for those FA. Conversely, 18:2n-6 nd 18:3n-3 were present t higher concentrtions (~22% nd 1.5% for 18:2n-6 nd 18:3n-3, respectively) nd vritions in the resulting estimtes were smll. When only those two FA were used to estimte diet, the ssocited errors were much more resonble t 9% nd 1% for Steller s nd spectcled eiders, respectively (Tble 3), demonstrting tht it is possible to chieve diet estimtes with low vrition. However, none of the estimtes bsed solely on individul FA were ccurte. Clerly, in this ppliction, the ccurcy of n estimte is not necessrily linked to its reproducibility. Implictions nd recommendtions This study estblishes strting point for estimting predtor diets using stble isotopes of FA, nd highlights severl key considertions. Perhps the most importnt issue in the ppliction of this technique is the reserch question to be ddressed. Like nlyses of bulk isotopes, this technique cn yield informtion bout the origin of mteril in predtor diets but only sources with distinct isotopic vlues cn be differentited. Thus, we would expect this method to be useful in situtions similr to those in which bulk isotope vlues re commonly used. For instnce, we hve previously demonstrted the potentil utility of the compound-specific pproch in differentiting between ice lgl nd phytoplnkton crbon (Budge et l., 28), nd it is likely to lso be useful in ddressing questions tht differentite between benthic nd pelgic sources, nd mrine nd terrestril mteril. In more complicted situtions, with more thn two distinct sources, simple mixing models will not suffice nd much more complex sttistics will be required to ssign sources (e.g. Wrd et l., 21). It my lso be possible to combine informtion from proportionl FA composition (e.g. Iverson et l., 24; Wng et l., 21) with d 13 C vlues of FA so tht more relible diet estimtes cn be mde. Severl issues remin to be ddressed before this technique cn be used to relibly trce crbon sources in free-rnging birds. For instnce, nutritionl qulity nd digestibility of food is known to influence discrimintion fctors t the bulk level (Codron et l., 211) nd the subjects in our cptive study showed differentil ctbolism, depending upon the mounts of EFA in their diets. In free-rnging birds, it would be impossible to know the levels of dietry FA consumed nd therefore difficult to ssign pproprite discrimintion fctors. More controlled feeding studies, with diets of vrying FA concentrtions similr to wild diets, would help resolve this issue. Specificlly, we must understnd the vrition in discrimintion fctors with chnging dietry FA levels. This lso reltes to second issue concerning the period of feeding represented by dietry FA in dipose tissues, s well s their turnover times. Both re poorly understood in birds nd will lso influence the ccurcy of diet estimtes. In fct, we suspect tht the vrition in our diet estimtes ws linked to filure in equilibrtion of dipose tissue FA with the new diet. Turnover time will depend on the concentrtions of FA in the diet nd would lso best be estblished by feeding ecologiclly relevnt diets. Thus, we recommend tht crefully designed feeding studies be used to ddress both issues of differentil ctbolism nd turnover time. Another hurdle is simply demonstrting the utility of this compound-specific pproch compred with bulk stble crbon isotope nlyses. We believe the dvntges ssocited with isotopic routing of EFA nd the bility to obtin multiple diet estimtes from severl FA in single smple mke the FA-specific pproch superior to nlyses of bulk stble crbon isotopes. However, this technique requires more involved smple preprtion nd expensive instrumenttion; it lso relies on informtion from lipids, dietry component tht is usully removed before bulk nlysis is conducted (Post et l., 27). A cler dvntge, either in ccurcy or reproducibility of diet estimtes generted with FA stble crbon isotopes, is required. We therefore recommend tht future studies lso determine bulk stble crbon isotopes of the sme lipidextrcted mteril s nlyzed with the FA-specific technique, so tht diet estimtes derived from the two methods bsed on different dietry components cn be compred. While some questions do remin concerning the ppliction of this FA-specific stble crbon isotope technique, we believe the study of EFA will llow us to ultimtely derive ccurte