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The Auk 118(3):665-677, 2001 COMPARATIVE FUEL USE OF MIGRATING FAT STORES, MIGRATION LEONARD PASSERINES: EFFECTS OF DISTANCE, AND DIET Z. GANNES t Departmentof Ecologyand EvolutionaryBiology,PrincetonUniversity,Princeton,New Jersey08544, USA; and Mitrani Department of DesertEcology, Blaustein Institutefor DesertResearch, Ben-GurionUniversityof theNegev, Midreshet Ben-Gurion, 84990, Israel ABSTRACT.--Lipids are the dominant fuel sourceduring migratory flight, but the factors controllingthe relative importanceof lipid, protein, and carbohydrateto flight metabolism remain unclear.I tested the nonexclusivehypothesesthat diet, migrationdistance,or endogenouslipid reserves mediatevariation in the fuels birds catabolizeduring migration. Bloodplasmametaboliteconcentrations were significantlydifferent amongspecies,and indicated clear differencesin protein and lipid utilization among three turdid chat and five sylviid warbler speciescaughtduring spring migrationin the Negev Desert, Israel. Fruiteating species(omnivores)catabolizedless protein and morelipid during migrationthan insectivores.Metaboliteconcentrations of omnivorousBlackcaps(Sylviaatricapilla),Garden Warblers (S. borin), and Lesser Whitethroats (S. curruca) were consistent with low rates of proteolysis(low uric acid), and high ratesof lipolysis (high free-fatty acid and •3-hydroxybutyrate). On the other hand, metaboliteconcentrationsof insectivorousRedstarts(Phoenicurusphoenicurus), Nightingales (Lusciniamegarhynchos), Thrush Nightingales(L. luscinia), Barred Warblers(S. nisoria),and Orphean Warblers(S. hortensis)indicated increasedproteolysisand decreasedlipolysis.Blood metaboliteconcentrations,however,were not correlated with migrationdistance,and the resultsdo not support the hypothesisthat long-distance migrants use fuel differently than short-distance migrants. Triacylglycerol mobilization was positively correlated with the amount of visible subcutaneousfat, but blood metabolitecompositionwas more stronglyaffectedby diet. Omnivoresand insectivoresexhibit different fuel-usestrategiesto overcomethe physiologicalchallengesof migration.Received 29 March 2000, accepted 28 February2001. LIPIDSAREan energy-densefuel source for volant migrants, and provide the majority of the energy used during long-durationflights in birds (Rothe et al. 1987, Ramenofsky1990, off, burst flight, and fuel delivery to glucosedependent tissues (e.g. central nervous system; Rothe et al. 1987, Schwilch et al. 1996, Jenniand Jenni-Eiermann1998). Unlike lipid Jenni-Eiermann and Jenni 1991). Previous au- and carbohydratereserves,protein is stored thors have suggestedthat only lipids are ca- as functioning tissues.The roles of oxidized tabolized, and that protein massremains con- protein during migration are likely multifacstant during migration (e.g. Odum et al. 1964). eted and may includereplacingKrebscycleinHowever, body-masschangespreceding and termediaries,maintaining water balance,supduring migration (Marsh 1983, 1984; Piersma plying amino acids for gluconeogenesis,or and Jukema1990, Wingfield et al. 1990, Lind- balancing changesin mass-dependentpower str6m and Piersma 1993, Karasov and Pinrequirements (Biebach 1996). However, the show 1998, Klassen et al. 2000) and direct mechanisms controlling fuel composition evidence from blood metabolites (Jenni-Eier(contribution of lipid, protein, and carbohymann and Jenni 1991, Bairlein and Totzke drate) during migration remain enigmatic. 1992)indicatethat carbohydratesand proteins Previous studies are compatible with the also are oxidized during migration. Carbononexclusivehypothesesthat migration dishydrate storesare small relative to lipids, but tance or endogenouslipid reserves,or both, glycogenplays an important role during take- mediate variation in the fuels birds catabolize • Present address: Environmental Studies, Prescott College,220 Grove Avenue,Prescott,Arizona 86301, USA. E-mail: [email protected] 665 during migration.A long-distancemigrant,the GardenWarbler(Sylviaborin),usedlessprotein and more lipid during fall migration than a short-distancemigrant, the European Robin 666 LEONARD Z. GANNES [Auk, Vol. 118 Distribution (Erithacus rubecula;Jenni-Eiermann and Jenni 1991). Alternatively, the quantity of endogenous lipid reservescould explain the observed fuel-use differences.In the abovestudy, European Robins had smaller visible fat reserves than Garden Warblers (Jenni-Eiermann and Jenni 1991). Lean passerines,captured in the Sahara during fall migration, had elevated Lus.'iniamegarh Nightingale Turd Luscinia luscinia • • Thrushnightingale blood concentrations of urea, consistent with Phoenicuruspho increasedprotein oxidation,comparedwith fat birds (Bairlein and Totzke 1992). A controlled study of birds varying in both migration distance and fat reservesis required to disentangle thesealternativeexplanationsfor fuel-use differencesamongspecies. An alternative factor that has not previously been explored in migrating birds is that diet during and precedingmigration might affect the compositionof fuel oxidized during migration. Daysto weeksof eatinga diet type changes both substrateutilization and storagerates in exercisinghumansand rats(reviewedin Volek 1997, Hawley et al. 1998, Spriet and Peters 1998). Similarly, the daily nitrogen requirementsof strictly frugivorousand nectarivorous birds are lower than omnivores Diet Redstart Sylvia nisoria Barred warbler Sylvia hortensis Orpheanwarbler Sylvia curruca Lesser whitethroat Sylviidae Garden warbler •.-•'I• or carnivores (reviewed in Bairlein and Gwinner 1994; Mur- Sylvia atricapilla Blackcap phy 1996). Although the effect of diet on the fuel compositionof migrating birds is completely unknown, diet affectsthe rate of mass F•G. 1. Closely related sylviid and turdid species gain during migratory stopover(Bairlein and migrate both short and long distances,and eat both Gwinner 1994, Parrish 1997). insectivorous and fruit-based omnivorous diets. The In the present study, I used a comparative cladogramwas adapted from phylogeniesby Sibley approachto test the nonexclusivehypotheses and Ahlquist (1995) and Blondelet al. (1996). Distrithat migrationdistance,fat reserves,or diet ex- bution maps indicate northern and southern explain the differencesin fuel use during migra- tremesof winter and breeding ranges(from Cramp 1988, 1992). Specieswere categorizedas mostly intion. Old World warblers (Sylviidae)and chats sectivorous(fly) or omnivorous(berry) accordingto (Turdidae) are an excellentrepresentativesys- Cramp (1988, 1992), and dietary assignmentsagree tem to test these hypotheses.Closely related sylviid and turdid speciesmigrate both short and long distancesand eat omnivorousand insectivorousdiets (Fig. 1). If migrationdistance were an important determinant of migratory fuel use, then a species' migration distance shouldbe positively correlatedwith lipid oxidation and negatively correlatedwith protein oxidationduring migratory flight. Alternatively, if the fuels catabolized during migration were mediated by endogenouslipid reserves, then lean birds should have increased levels of proteolysisand decreasedlevels of lipolysis comparedwith fat birds. Finally, if diet mediates the fuels oxidized during migration, fuel with observations in the field (see methods). compositionshould group according to diet type rather than lipid reservesor migration distance. METHODS This study focused on three turdid chats:Redstarts (Phoenicurusphoenicurus), Nightingales (Lusciniamegarhynchos), and Thrush-Nightingales(L. luscinia); and five sylviid warblers: Blackcaps(Sylvia atricapilla),Garden Warblers,LesserWhitethroats(S. curruca),Orphean Warblers(S.hortensis), and Barred Warblers(S. nisoria).Birdswere captured in mist nets July2001] FuelUsein Passerine Migrants in the gardens at Midreshet Ben-Gurion, Israel (30ø52'N, 34ø46'E, 476 m above sea level). Located in the northern Negev Desert, Midreshet Ben-Gurion receives<100 mm rainfall annually. Migrants were capturedin mist nets daily 4 March to 18 May 1996 and 10 March to 21 May 1997. The nets were checked at 15-20 min intervals from 1 h 667 In this study,I used free-fatty acidsand •3-hydroxybutyrate concentrationsas indicatorsof lipid turnover, and uric acid as an indicator of protein turnover. However, I do not assume that glycerol, triacylglycerol, or glucoseconcentrationswere correlated with turnover. Radar data from the Negev Desert indicated that beforesunriseand for approximately4 h thereafter. migrating passerinesland at dawn (Bruderer and High midday temperaturesand frequent afternoon Liechti 1995). Becausethe goal of this study was to windstormsprecluded capturing birds all day long. comparefuel use during flight, new-arrival and reFlattenedwing cord and mass(Ohauselectronicbal- capturebirds were sampledonly during the first 2 h ance +0.1 g) were measured,and birds were banded after twilight. Twilight times were calculatedusing with aluminum bands obtained from the Israel the program availablefrom the Australian DepartBanding Center.In addition, visual subcutaneousfat ment of Industry, Scienceand Tourism. Time after was scored in half-units from 0 for no visible fat to 4 twilight was included asa factorin statisticalmodels for lipid storageregionsoverflowingwith fat (mod- (see below). ified from HelmsandDrury 1960).Fatscoreexplains To obtainblood samples,I puncturedthe vena ul-50% of the variation in total body fat of other mi- naris with a 26 gauge hypodermic needle and colgrant passerines(Rogers 1987, Krementz and Pen- lectedthe blood in heparinizedcapillary tubes(60dleton 1990,Sprengleret al. 1995),particularlywhen 120 [tL). Blood samples were kept on ice for a interobserver variation is reduced (Krementz and maximum of 3 h until they were spun in a microhePendleton1990).Throughoutboth seasons,threeob- matocrit centrifuge (5 min, 12,000 g). Blood metabservers were frequently crosscheckedfor internal olite concentrations did not changesignificantlyover consistency in scoringfat. Birdswere generallyheld this time period (L. Gannes unpubl. data). I mea<30 min, including time spent in the net and sured hematocrit and discarded the red blood cells. processing. Blood plasma was diluted gravimetricallyby oneBecausethe samesite was intensivelytrapped dai- half with isotonic saline solution, and diluted samly, individuals captured without a band (after the pleswere storedat -80øC until analyzed. first 10 days)were callednewarrivals.Birdscaptured Triacylglycerol,glycerol,uric acid, •3-hydroxybu->1 day after their first capture were termed tyrate, glucose(SigmaDiagnostic,St. Louis,Missourecaptures. ri) and free-fatty acid (Boehringer-Mannheim,IndiBloodmetabolite analyses.--Evidence for lipid, pro- anapolis, Indiana) concentrationswere analyzed tein, and carbohydrate catabolism can be found in using spectrophotometric assaysmodified for small the blood,becausethe majority of an animal'senergy volumes.The triacylglycerolvaluesreported in this reservesare stored externally to metabolizing cells. study were calculatedby subtractingfree glycerol Lipolysis is associatedwith elevatedconcentrations from the total bound and unbound glycerol ("true of free-fatty acid, glycerol,and [3-hydroxybutyrate, triacylglycerol").Absorbancevaluesof samples(in and elevated triacylglycerol levels are associated duplicate, 3-10 [tL) and standards were measured with net lipogenesisin the liver (Hurley et al. 1986, with a spectrophotometer. Jenni-Eiermannand Jenni1992,1994).Bloodplasma Migration distance.--Minimumand maximum mifree-fatty acid concentrations were significantlycor- gration distanceswere compared among species. related (r = 0.9446, Pearson correlation P = 0.0045) The minimum migration length was calculatedby with lipid turnover in chickens(Gallusgallusdomes- subtracting the northernmost wintering from the ticus)run on a treadmill (reanalysisof data from Vin- southernmostbreeding ranges, in degreeslatitude cent and Brackenbury1988). In addition, free-fatty (Cramp 1988, 1992).Similarly,the maximum migraacid and [3-hydroxybutyrateconcentrationswere tion distance was calculated by subtracting the positively correlatedin flying and resting pigeons southernmost wintering from the northernmost (mean r = 0.783 _+0.042; Gannes et al. 2001). Uric breeding ranges. Becausethe two measuresof miacid is the major end product of protein catabolism gration distancewere significantlycorrelated(pairin birds. In a variety of birds,bloodplasmauric acid wise correlation,R; 0.884,P = 0.0036),only the reconcentrationswere correlated with protein oxida- sults from the minimum migration distance were tion measured by excreted nitrogen (Mori and reported below. George 1978, Robin et al. 1987, Cherel et al. 1988, Diet.--I usedCramp (1988,1992)for assigningdiet Lindggrd et al. 1992).In contrast,blood glucosecon- types to species.During the nonbreeding season, centrationsare not necessarilycorrelated with glu- Blackcaps, Garden Warblers, and Lesser Whitecoseoxidation. Many birds regulate blood glucose throats depend to a large degree on noninsectfood concentrationswithin narrow limits (e.g.Hazelwood sources (berries and fruits), and will be referred to 1986, Swain 1992a),but somespeciesapparently do as omnivores.On the other hand, Nightingales, not (e.g. Swain 1987,Jenni-Eiermannand Jenni1997). Thrush-Nightingales, Redstarts, Barred Warblers, 668 LEONARDZ. GANNES [Auk, Vol. 118 TABLE1. Resultsfrom separateANCOVA modelsfor glycerol,triacylglycerol,free-fattyacid,uric acid, •hydroxybutyrate,and glucoseconcentrationin new-arrival passerinemigrants.Fat (visiblesubcutaneous fat score)and Timeof day(hoursafter twilight) were includedas covariatesand Species and Yearwere includedas factors.Significanteffectsare in bold. Fat Metabolite Slopea Glycerol Triacylglycerol Free-fattyacid 0.046 0.240 -0.014 Uric acid -0.036 •-hydroxybutyrate Glucose PC1 PC2 0.027 0.145 -0.058 0.195 F Species P F 2.2 0.1432 13.9 0.0003 0.0 0.8920 1.3 2.8 2.7 0.8 0.3613 5.0 0.1 0.7425 4.0 0.3 0.6 5.1 0.5873 0.4491 0.0263 4.7 10.3 2.4 Year P F 0.2418 0.0095 0.0121 <0.0001 0.0004 0.0001 <0.0001 0.0249 Time of day P F n 2.0 0.2 2.7 0.1599 0.6240 0.1000 2.1 0.1498 3.4 0.0684 180 0.7 0.4199 4.1 0.0437 159 3.9 6.0 0.0 0.0514 0.0159 0.9996 131 122 122 49.0 2.0 10.0 <0.0001 0.1646 0.0020 2.3 0.0 0.0 P 0.1305 180 0.8916 180 0.9236 171 Slopeof ANCOVA effectof fat on metaboliteconcentrations, significantslopesare in bold. and Orphean Warblers take almost exclusivelyinsects,and will be referred to asinsectivores. Although many avian migrantschangedietsduring migration (reviewed in Bairlein and Gwinner 1994), observations from Midreshet Ben-Gurion agreed with Cramp'sdietary generalizations.Approximately2030% of the Blackcaps,Garden Warblers,and Lesser Whitethroats captured in 1997 had plant parts in their feces, or their bill, forehead, and crown were coveredwith pollen and nectar (L. Gannesunpubl. data). The defecatedseedswere mostly from acacia pods (Acaciasp.)and the acaciatreeswere oftencovered with feeding Blackcapsand Lesser Whitethroats.In addition,I frequentlysawLesserWhitethroatsprobingflowers,presumablyfor nectar.Less than 3% of the Redstarts,Orphean Warblers,Barred Warblers, Nightingales, and Thrush-Nightingales had plant parts in their fecesand nonewere covered with pollen (L. Gannes unpubl. data). In addition, Redstartsand OrpheanWarblersfed adlibitumin the laboratoryrefusedto eat a fruit-baseddiet ("banana mash"; Denslow et al. 1987), but readily consumed mealworms (Tenebriolarvae; L. Gannes unpubl. data). On the other hand, Blackcapsreadily ate and gained masson either the fruit-basedor mealworm diets (L. Gannesunpubl. data). Statisticalanalyses.--Separate ANCOVA modelsfor eachmetabolitewere used to interpret interspecific and recapturedbirds, and betweenomnivoresand insectivores. Both models included time after twi- light as a covariate,and year and speciesas factors. Principal componentanalysis(PCA) was used to clarify interspecificvariation in blood-metabolite concentrations. PCA combines correlated variables into independentand interpretableaxes.The new PCA values can then be used to comparecorrelated variables among species.All six blood metabolites were usedin the PCA,but birds missingoneor more metabolite measures were excluded from the analysis. The data in the ANCOVA models did not meet the assumptionsof normality. Natural-log transformed variablesdid meetthe assumptionsof normality,but the transformationsdid not changethe resultsqualitatively. Therefore,data were not transformedbefore analysis.Discrepanciesin samplesize are a result of incompletesamples,and all meansare given _+SE.Statisticalmodelsand PCA were analyzed using JMP 3.1 for the Macintosh(SASInstitute 1994). RESULTS Interspecific bloodmetabolites.--Blood-metabolite concentrationsof newly arrived birds were significantlydifferent amongthe species differences in blood metabolites, with fat score as a independentof fat score(Table1). In newly arcovariate.We included time after twilight as a cov- rived birds, free-fattyacid valueswere highest ariate in all ANCOVA models, because previous in Blackcaps and GardenWarblers,and lowest studies have found that metabolite concentrations in Redstarts,Nightingales,and OrpheanWarchangeover the courseof the day (Jenni-Eiermann blers(Fig. 2). Blackcaps, GardenWarblers,and and Jenni 1997, Jenni and Jenni-Eiermann 1996). Year Lesser Whitethroats had significantly lower was also included to control for differences between 1996 and 1997.The least squaremeans(LSM) from the ANCOVA models were used to make ad hoc com- parisons of metabolite values independent of fat score,time after twilight, and year. Similarly, ANCOVA models were used to interpret blood-metabolite concentration differences between new-arrival uric acid concentrations than redstarts or Or- phean Warblers (Fig. 2). Blackcaps,Garden Warblers,and LesserWhitethroatshad higher plasma •3-hydroxybutyrate levelsthan Nightingales, Redstarts,and Thrush-Nightingales (Fig. 2). Glycerol concentrationswere not sig- July2001] 3.0 FuelUsein Passerine Migrants ab a a b b ab a ab 669 1.0- il; t 0.8 20 23 54 34 8 0,2 6 8 26 55 34 21 ac ad a b bc cd a 3.0 ac e• ;i[i 8 20 25 58 20 9 ac a ab b a b 8 21 26 55 34 21 6 0.0-- a a bc bc c ab ac ab ab 9 6 P '-- 0.0095 0.0 a 6 1.0 P < 0.0001 34 9 P = 0.1432 0.0 .•1 3.0 21 P = 0.0121 0.0 1.5- 18 30.0 ac ac b c bc ac bc 6 11 18 48 25 9 8 a 2.0 •'• 20.0 1.0 8 0.0 15 22 56 29 14 9 6 P= 0.0004 ........ L1 Lm Pp Sa Sb Sc Sh Sn 15.0 5 P= 0.0001 L•ILm ' Pp ' S•tS• S• S[•Sn ' FIG.2. Blood-plasma concentrations (retool/L)of free-fatty acids,uricacid,•-hydroxybutyrate, glycerol, triacylglycerol, and glucosesignificantly vary amongmigratoryspecies. Least-square means(LSM ___ SE) givenfor furdidchats(squares): Thrush-Nightingales (L1),Nightingales (Lm),Redstarts (Pp);andsylviid warblers(circles): Blackcaps (Sa),GardenWarblers(Sb),LesserWhitethroats (Sc),OrpheanWarblers(Sh), BarredWarblers(Sn).P-valuesindicateresultsof speciesdifferencesin ANCOVAmodels(Table1), and differentlettersindicatesignificantly differentmeans,by linearcontrasts. Samplesizesgivenbelowtheerror bars.Omnivores andinsectivores areindicated by filledandemptysymbols, respectively. nificantlydifferentamongthe species(Fig. 2). explained48% of the variation (Table2). The Plasma triacylglycerolconcentrationsin new- first principalcomponentaxis(PC1)wasposiarrival Blackcapsand Lesser Whitethroats tively correlated with concentrationsof free- were lower than those of Garden Warblers, fatty acidsand •-hydroxybutyrateand negaNightingales,and Thrush-Nightingales(Fig. tively correlated with concentrationsof uric 2). Redstartsand GardenWarblershad higher acid and triacylglycerol(Table 2). PC1 exlevelsof plasmaglucosethan BarredWarblers, plained 26% of the total variance.BecausePC1 Nightingales,or Thrush-Nightingales (Fig.2). was positively correlatedwith lipid metaboPrincipalcomponentanalysis(PCA)clarified lites and negativelycorrelatedwith uric acid,I the complexpatternsof interspecific bloodme- interpretedPC1 as the "lipolysis-proteolysis tabolites.The first two principalcomponents axis."PC1valuesfromBlackcaps, GardenWar- 670 LEONARD Z. GANNES [Auk,Vol. 118 TABLE 2. Results from PCA of blood metabolites for TABLE3. Least square means (LSM + SE), minimum and maximum visible subcutaneous fat new-arrival migrants. Valuesindicate correlation coefficientsof first (PC1) and second principal scoresfor studyspecies.LSM from ANOVAmodel componentaxes(PC2). PC1 was positivelycorreof species(F = 5.92, df = 7 and 183,P < 0.0001) lated with lipid metabolites(B-hydroxybutyrate, controllingfor year (F = 25.9, df = 1 and 183,P < free fatty acids),negativelycorrelatedwith protein 0.0001).Differentlettersindicatesignificantlydifmetabolite(uric acid), and interpreted as the "liferentmeansby linear constrasts. polysis-proteolysis axis."PC2waspositivelycorrelatedwith lipid metabolites(glycerol,triacylgly- Species Fat score Min Max n cerol, free-fatty acid), negatively correlatedwith 2.19 + 0.3b 0.5 3.5 8 glucoseconcentration,and interpretedas the "li- ThrushNightingale (Luscinialuscinia) polysis-glycolysis axis." Nightingale 1.96 +_0.19b 0.5 3.0 21 (L. megarhynchos) Metabolite PC1 PC2 % Explained Glycerol Triacylglycerol Free-fattyacid 26 0.12 - 0.42 0.45 Uric acid -0.75 •-hydroxybutyrate 22 0.52 0.64 0.50 0.72 Glucose -0.03 Redstart 0.81 _+ 0.17 a (P.phoenicurus) Blackcap (Sylviaatricapilla) 1.68 _+0.11b 0 4.0 58 Garden 1.25 _+ 0.15 a 0 2.5 34 1.81 _+ 0.19 b 0 3.5 21 Warbler 0.13 (S. borin) 0.22 Lesser Whitethroat -0.65 0 2.5 26 (S. curruca) Orphean Warbler (S. hortensis) 2.24 _+0.30b 0 3.0 9 Barred Warbler 2.21 _+ 0.37 b 3.0 6 2.0 (S. nisoria) biers, and LesserWhitethroatsindicatedhigh levelsof lipolysisand low levelsof proteolysis, but metabolitesfrom Orphean Warblers,Redstarts, Nightingales, Barred Warblers, and Thrush-Nightingalesindicatedlow levelsof lipolysisand high levelsof proteolysis(Fig. 3). The second principal component axis (PC2) wasnegativelycorrelatedwith glucoseconcentration and positivelycorrelatedwith glycerol, triacylglycerol,and free-fatty acid concentrations.This axis explainedan additional22% of LLm• SnlSa r• b-•b •Sc - 0.5 1.5 i i -0.5 0 UA, TAG PC1 Proteolysis i 0.5 i 1 FF^, •-HB^ Lipolysis F•G.3. Protein and lipid oxidationduring migration significantly differ among species.The first (PC1) and second(PC2) principalcomponents were interpreted as the "lipolysis-proteolysis"and the "lipolysis-glycolysis"axes,respectively(SeeTable 2). PC1valuesfrom Blackcaps, GardenWarblers,and LesserWhitethroatsindicatedhigh levelsof lipolysis and low levelsof proteolysis,but metabolitesfrom OrpheanWarblers,Redstarts,Nightingales,Barred Warblers, and Thrush-Nightingalesindicated low levelsof lipolysisand high levelsof proteolysis.Redstartsand OrpheanWarblershad significantlylower PC2 valuesthan Nightingales,Thrush-Nightingales, Blackcaps,and Barred Warblers.Valuesare LSM _+ SE.SeeFigure2 for explanationof symbolsandTable i for full ANCOVA models. the overall variance (Table 2). PC2 can be inter- preted as the lipolysis-glycolysisaxis. Redstartsand OrpheanWarblershad significantly lower PC2 valuesthan Nightingales,ThrushNightingales, Blackcaps,and Barred Warblers (Fig. 3). Fat stores.--Althoughthere was a large variation in fat score,only triacylglycerolconcentrations were significantlycorrelatedwith fat score.Among species,birds coveredthe entire fat-scorerange(0-4), and mostspeciesalsohad individuals from all categories(Table3). Garden Warblersand Redstartshad significantly lower fat scoresthan any of the other species (Table3). ANCOVA analysesindicatedthat fat scorecovariedwith triacylglycerolvaluesregardlessof speciesdifferencesand controlling for year and time after twilight (Table1). Not surprisingly,PC2 was also positively correlat- July2001] FuelUsein Passerine Migrants 671 Sa •.• 0.5 0 Sh • tSn • S -1.0 R = 0.168, P > 0.6 i i 10 15 i 20 i 25 i 30 i 35 40 v v Minimum migrationdistance(degreeslatitude) FIG. 4. Minimum migration distance did not explain the variation of lipid and protein use in migrating songbirds.Least-squaremeans(LSM _+SE) of the first principalcomponent(PC1) were not significantly correlatedwith a species'minimum migration distance(R and P for pairwise correlations). Migration distancetaken from Cramp (1988, 1992). SeeFigure 2 for explanationof symbolsand Table1 for completeANCOVA model. v ed with fat score, because PC2 is associated with triacylglycerolconcentrations(Table 2). However,the covarianceof fat scorewith glycerol, glucose,free-fatty acids,uric acid, fi-hydroxybutyrate and PC1 were not significant (Table 1). Migrationdistance.--Thewintering rangesof the sylviids and turdids in this study span from north of the equatorto the southerntip of Africa, and the breeding rangesbegin in the northern Mediterranean and most extend above the Arctic Circle (Fig. 1; Cramp 1988, 1992). The species'meansof the first principal componentaxis (PC1) were not correlatedwith minimum migrationdistanceof a species(pairwise correlations; Fig. 4). Similarly,noneof the individual metabolitesor PC2 correlatedsignificantlywith minimum migrationdistance(P > 0.2). Diet.--Diet explained much of the variation in bloodmetaboliteconcentrations of newly arrived migrants (Table 4). Fruit-eating omnivores(Blackcaps,Garden Warblers,and Lesser Whitethroats)had significantlyhigherfree-fatty acid and fi-hydroxybutyrate,and lower uric acid and triacylglycerolplasma concentrations than insectivores (Orphean Warblers, Redstarts, Barred Warblers, Nightingales, and +1 +1 +1 +1 +1 +1 +1 +1 c.l I I 672 LEONARD Z. GANNES [Auk, Vol. 118 Thrush-Nightingales). As a result, omnivores had significantly higher means on the lipolysis-proteolysisaxis (PC1) than did insectivores (Fig. 2). Glycerol, glucose,and PC2 were not significantlydifferentbetweenthe diet groups (Table 4). Metabolitesof newly arrived and recaptured birds.--A five-speciessubset (Lesser Whitethroat, Nightingale, Redstart, Blackcap,Garden Warbler) of the eight study specieswere recapturedin large enoughnumbersto compare blood metabolite concentrationsof newly arrived and recaptured birds. Repeated measures on the same bird were not possible,because few birds were sampled both as new arrivals and recaptures.Newly arrived birds had significantlyhigher glyceroland free-fatty acid, and lower uric acid concentrations than recapturedbirds (Table5). Bloodglucose,•3-hydroxybutyrate and triacylglycerollevelswere not significantlydifferent betweennew arrival and recaptured birds (Table 5). In addition, blood hematocritvalueswere significantlylower in recaptured (0.443 ___ 0.007) than in new arrival birds (0.492 +__ 0.004; F = 46.1, df = i and 190, P < 0.0001), but neither species(F = 1.9, df = 4 and 190, P > 0.1) nor year (F = 0.2, df = i and 190, P > 0.6) were statistically significant. DISCUSSION Blood plasma metabolite concentrations were significantlydifferent amongspeciesand indicatedclear differencesin protein and lipid utilization. Metabolite concentrations in Black- caps, Garden Warblers, and Lesser Whitethroatswere consistentwith low levelsof proteolysis (low uric acid), and a high degree of lipid oxidation(indicatedby high free-fattyacids and 13-hydroxybutyrate; Fig. 2). On the other hand, metabolite concentrations of Red- starts, Nightingales, Thrush-Nightingales, Barred Warblers, and Orphean Warblers suggesteda higher level of proteolysisand lower lipolysis (Fig. 2). PCA separatedspeciesalong the lipolysis-proteolysisaxis (PC1; Fig. 3). The resultswere consistentwith Blackcaps,Garden Warblers, and Lesser Whitethroats using less protein and more lipids during migrationthan Redstarts,Orphean Warblers,BarredWarblers, Nightingales,and Thrush-Nightingales. vvv July2001] FuelUsein Passerine Migrants Levels of proteolysis and lipolysis clearly groupedaccordingto diet. Fruit-eatingomnivores(Blackcaps,Garden Warblers,and Lesser Whitethroats;Fig. 1) had lower uric acid, and higher free-fatty acid and [•-hydroxybutyrate concentrations than insectivores (Orphean Warblers, Redstarts, Barred Warblers, Nightingales, and Thrush-Nightingales;Table 4). Speciesshare a commonevolutionaryhistory, and variousauthorshave pointed to the statistical pitfalls of treating speciesas statistically independentobservations(reviewed in Felsenstein 1985, Garland and Carter 1994). However, 673 Diet potentially can affect fuel use in flight by long-termbiochemicaladaptationto a diet, short-term replenishment of endogenousreserves,or both. The daily nitrogen requirements of frugivorous and nectarivorousbirds are lower than omnivores or carnivores (re- viewed in Bairlein and Gwinner 1994,Murphy 1996), and changingdiet type for a period of days to weeks affects substrateutilization in exercisinghumansand rats (Hawley et al. 1998, Spriet and Peters 1998). However, proximate mechanismsof dietary adaptationare not well understood, and it remains unclear how diet- induced fuel-use differences might translate sectivorous Orphean and Barred warblers into fuel-use differences during migratory closelyresembledthe concentrations of insec- flight. Alternatively,dietary intake and compositionhasa short-termeffect,takinghoursto tivorous turdid chats rather than their omnivcarbohydrate orous congeners(Fig. 1). Migratory fuel-use days,on an animal'sendogenous stores (Hazelwood 1986, Swain 1992a, Spriet splittingaccordingto dietarypreference,rather than phylogeneticrelationships,supportsthe and Peters1998). Becauseglycogenstorescan hypothesisthat diet wasa moreimportantfac- be depletedquicklyduring exerciseand are retor in migratory fuel-use than phylogenetic plenishedindependentof lipid reserves,evena highly obesemigrant may have very reduced history. glycogen reserves after a nocturnal flight. Some individuals labeled as new arrivals Fruit-eating omnivores eating carbohydratemay not have been captured when they first rich diets (i.e. fruit, nectar)may havelarger glyarrived at the study site, but severallines of cogenreservesat the beginningof a migratory reasoningsuggestthat blood-metaboliteconflight than insectivoreseating carbohydratecentrationsof new-arrival birds were represenpoor diets, similar to mammals (reviewed in tative of in-flight concentrationsof migrating Volek 1997, Hawley et al. 1998, Spriet and Pebirds. First, sampleswere only taken during ters1998).In postabsorptive animalswhen glythe period when radar indicatedmost passer- cogenstoresare reduced, protein becomesthe ines make landfall (Bruderer and Liechti 1995). dominant precursor for gluconeogenesis Second,only a small minority of the individu- (Maughan et al. 1997).As a result, a bird with als remainedat the sitefor longerthan oneday. large glycogenstoresmay catabolizelessproEightyto 90% of the migrantsin 1996and 1997 tein to maintain blood glucoselevels during were only captured once,and the majority of migration than a bird with small glycogenrethosebirds probablycontinuedmigrating the serves.In addition,the two effectsprobablyare sameeveningof the day they arrived (L. Gan- not mutually exclusiveand couldactin concert: nes unpubl. data). Third, blood metabolites enzymatic activity reducing body tissues'glu(glycerol,free-fatty acid,uric acid) and hemat- cose requirements,and therefore conserving ocrit were significantlydifferentbetweennew- glycogenreservesand thusbody protein.Howarrival and recaptured birds, suggestingthey ever,without knowing the stopoverand feedcamefrom two different "groups" (Table5). Fi- ing history of individual birds, it is impossible nally,otherwork hasshownthatbloodconcen- to separatelong-term dietary adaptationfrom trations of glycerol, free-fatty acid, and uric short-termreplenishmentof glycogenreserves. acid were not significantly different between The resultsfrom this studyarenot consistent EuropeanRobinscapturedin flight and those with the hypothesis that long-distancemicaptured soon after landing (Jenni-Eiermann grantsuse fuel differently than short-distance and Jenni1991). In the present study, we in- migrants (cf. Jenni-Eiermannand Jenni 1991, cluded time after twilight as a covariateto con- Jenni and Jenni-Eiermann 1998). Individual trol for potential changesin metaboliteconcen- blood-metaboliteconcentrationsand principal component values were not correlated with tration during the 2 h samplingperiod. the blood-metabolite concentrations of the in- 674 LEONARD Z. GANNES minimum migrationdistancesthat varied over 20ølatitude (-2,200 km; Fig. 4). All the species in the presentstudywinter southof the Sahara, and crossingthe Saharamay exert similar selectivepressurefor differentialfuel useregardless of total migration distance.However, the European Robin studied by Jenni-Eiermann and Jenni(1991) winters on the northern edge of the Sahara (Cramp 1988), and used more protein and lesslipid than the Garden Warbler that winters in southernAfrica (Cramp 1992). As would be predictedfrom the presentstudy, the European Robin is mainly insectivorous [Auk, Vol. 118 and may provideimportantnutrientsand fatty acids (reviewed in Bairlein and Gwinner 1994, Parrish 1997). In addition, the present work suggeststhat the switch from insectivoryto fruit-eating omnivory is also associatedwith decreased catabolism of endogenousbody protein. We might expect a strong selectivepressure on insectivores to reduce protein oxidation. Unlike lipid and carbohydratereserves,protein is stored as functioningtissue.However, birds may preferentially catabolizeassimilation organsto avoid catabolizingflight musand the Garden Warbler is omnivorous (Bert- cles that could impair flying ability (Swain hold 1976, Cramp 1988, 1992; L. Gannes un- 1992b,Kaspereket al. 1992, Karasovand Pinshow 1998, Biebach1998). As a result, protein publ. data). Previous authors have hypothesized that oxidation may lead to decreasedperformance speciesdifferencesin fuel use were mediated of absorptiveorgans (e.g. Biebach1998, Karby fat reserves:fat birds usemorelipid and less asov and Pinshow 1998, Piersma 1998), that protein than thin birds (Jenni-Eiermannand could reduce a migrant's ability to refuel enJenni 1991, Bairlein and Totzke 1992). The re- route. Compared to lipids, protein is a relasuitsfrom thepresentstudypartially agree.Fat tively heavy fuel (Schmidt-Nielsen1990).Probirds had higher triacylglycerolconcentrations tein oxidation actually could reduce the than lean birds, but none of the other metabo- amount of weight birds must carry during lites covaried with fat score (Table 1). It seems long-duration flights (e.g. Piersma and Lindthat fat migrantsincreasedtriacylglycerolmo- strom 1997, Piersma and Gill 1998), perhaps bilization without changingproteolysisor oth- increasingpotential flight range. Ultimately, er lipolysis metabolites.Migrating passerines the selectivepressureson migrantswill be the may supply metabolizing cells directly with result of how diet affectstotal migration dutriacylglycerol,circumventing the potentially ration throughproximatefactorssuchasstoprate-limiting albumin-mediatedtransport of over site suitability or availability, stopover free-fatty acids (Weber 1988, Jenni-Eiermann duration, and maximum migration distance and Jenni 1992). The present study suggests without refueling. It seems,however,that inthat fat birds might be able to use the triacyl- sectivoresand fruit-eating omnivoresexhibit glycerolpathway to a greaterdegreethan lean different migrationstrategies,eachwith an asbirds. However,species-level differencesin fuel sociatedsuite of morphologicaland behavioruseoccurafter controllingfor differencesin fat al adaptationsto overcomethe physicalchallengesof migration. score (Table 1). Conclusions.--Spring migrants minimize the ACKNOWLEDGMENTS time spenton migration,presumablyto acquire good breeding territories (Lindstr0m and A1This researchis dedicated to my grandparents erstam 1992,Klaassen1996).Over evolutionary (Jackand Molly Rayman, Miriam and Abe Gannes) time, migrantshave"solved" migrationalchal- who havebeena sourceof inspirationfor as long as lengeswith a suite of morphologicaland be- I can remember. The fieldwork would have been imhavioral adaptations(reviewedin Bertholdand possible without skilled assistancefrom Wendy Terrill 1991, Gwinner 1990, Berthold and Hel- Schelsky,Nathaniel Gerhart, Itai Shani, Nir Sapir, big 1991, Bairlein 1991). One such adaptation exhibited by many passerine migrants is switching from a mainly insectivorousdiet to a fruit-baseddiet precedingand during migra- Norm Budnitz, Itamar Giladi, Gilead Michaeli, Kevin tion (reviewed in Bairlein and Gwinner 1994). constructive Schwartz,and Garr. I thank Berry Pinshowfor making facilities availableto me at the Mitrani Department of Desert Ecology,Ben-GurionUniversity,Israel. This manuscript was greatly improved with comments on earlier drafts from Carlos Switching to a fruit-dominated diet increases Martfnez del Rio, Diane O'Brien, Scott McWilliams, premigrationaland en-routerate of massgain, Lila Fishman,William Karasov,ChrisGuglielmo,Ulf July2001] FuelUsein Passerine Migrants Bauchinger,and Herbert Biebach.Insightful commentsfrom two anonymousreviewersimprovedthe clarity of the manuscript.This work was funded by a National Science Foundation Doctoral Dissertation Improvement Grant (# 9623836),a Sigma Xi GrantIn-Aid of Research,and PrincetonUniversityfunds. The researchwas conductedwith researchpermits from the Israeli Nature ReservesAuthority. This is contributionnumber324 of the Mitrani Department for Desert Ecology. LITERATURE CITED 675 Birds of the Western Palearctic: Warblers. Ox- ford UniversityPress,Oxford. DENSLOW,J. 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