The Auk 109(2):334-345, 1992

LIVING OFF THE WAX OF THE LAND: BAYBERRIES AND YELLOW-RUMPED WARBLERS

ALLEN R. PLACE • AND EDMUND W. STILES 2 •Centerof MarineBiotechnology, University of Maryland,600 EastLombard Street, Baltimore, Maryland 21202, USA; and 2Departmentof BiologicalSciences, Rutgers-The State University of New Jersey, Piscataway,New Jersey08855, USA

ABSTRACT.--Yellow-rumpedWarblers (Dendroica coronata) and Tree Swallows(Tachycineta bicolor)are among a small group of in temperateNorth America that regularly eat waxy fruits. During the autumn,winter, and spring,these species feed extensivelyon fruits of the bayberry ( spp.). Covering the pulp of these fruits is a solid, waxy material consisting primarily of saturatedlong-chain fatty acids.For mostanimals, saturated fatty acidsare poorly assimilated(< 50%).Using 3H-glyceroltriether as a nonabsorbablefat marker, we determined that Yellow-rumpedWarblers are capableof high assimilationefficiences (> 80%)of bayberry wax when fed berries recoatedwith radioactivewax tracers.Efficient fatty-acidassimilation extendsto berriescoated with cetyl palmitate,a commonmarine, saturatedwax ester(> 90%). The fatty-alcoholmoiety of the marine wax was assimilatedwith a much lower efficiency (<50%). A beeswaxcoating of the berries is assimilatedwith an efficiencyof approximately 50%.Similar assimilationefficiencies of each wax are recordedfor Tree Swallows feeding on recoatedbayberries. Yellow Warblers(D. petechia)rejected recoated bayberries and exhibited little (< 5%) lipid assimilationof radiolabeledlipids. Yellow-rumped Warblerspossess several gastrointestinaltraits that permit efficient saturated-fatassimilation. Among these are an apparent retrograde reflux of intestinal contentsto the gizzard, elevated gall-bladder and intestinalbile-salt concentration, and a slow gastrointestinaltransit of dietary lipids. These gastrointestinaltraits permit efficient assimilationof saturatedfatty acidson bayberry fruits and may allow these small passerinesto maintain more northerly wintering rangesthan closelyrelated species.Received 24 May 1991,accepted 5 November1991.

THE ASSOCIATIONbetween Yellow-rumped monly observed over much of its wintering Warblers (Dendroicacoronata) and Myrica (bay- range.The TreeSwallow winters along the coast, berry and wax myrtle) is one of the mostwidely occasionallyas far north as Massachusettsand recognized -plant associationsin North south into . In autumn, large America (Brewer 1840, Hausman 1927, Martin flocks (some over 50,000; Stewart and Robbins et al. 1951). In fact, until 1983 one form of the 1958),move southalong the coastand may strip Yellow-rumped Warbler was referred to as the Myricashrubs of all fruits in a matterof minutes Myrtle Warbler(AOU 1983).A similarbird-fruit (E. Stiles, pers. observ.). relationship is found between Tree Swallows Generally, bayberry is found on dunes, old- (Tachycinetabicolor) and Myrica (Hausman1927). fields, and dry hills from Quebecto Louisiana; During the breeding season Yellow-rumped wax-myrtle typically is found on damp, sandy Warblers and Tree Swallows feed primarily on soils from New Jersey to Florida and Texas insects,but during the autumn, winter, and (Gleason and Cronquist 1963). Fruits ripen in spring their diets include large proportions of August through October and persistwell into fruit, especiallybayberry (Myricapennsylvanica the winter, providing an energy-rich resource Loisel.),wax-myrtle (M. ceriferaL.) and M. pus- for birds residing or wintering in northern and silla Raf. coastalregions of the United States. In easternNorth America, the Yellow-rumped Bayberry pulp includes a waxy coating of Warbler winters from central Maine and south- mono-and diglyceridesof myristic,palmitic and ern Nova Scotia, west to Kansas and Missouri, stearic fatty acids. Most exhibit low as- and south to Panama, which coincides with the similation (<50%) of these high-melting-point entire range of the above Myrica species.The lipids. For example, with chickens,absorption Yellow-rumped Warbler is the most northerly of thesefatty acidsdecreases monotonically with wintering wood warbler, and it is very com- increasingmelting point (Rennerand Hill 1961; 334 April 1992] BayberryWax and Yellow-rumped Warblers 335

Fig. 1). Similar results have been documented 100' in rats feeding on high-melting-point triglyc- erides (Clifford et al. 1986). 80- It may be that Yellow-rumped Warblers and Tree Swallows are able to successfullyoccupy 6O northern regions in winter becausethey can assimilateefficiently the high-melting-point 4O fatty acidsin bayberrywax that few fruit-eating animalscan digest.To investigatethe capacity 2O of Yellow-rumped Warblersand Tree Swallows 0 to assimilatewaxy coatingson bayberries,we removed the natural wax from bayberriesand -20 recoatedthem with radioactivelylabeled lipids. 2'0 4'0 6'0 80 We includedbayberry wax in our coatings,as well astwo othernaturally-occurring solid wax- Melting Point (øC) es known to be eatenby birds--cetyl palmitate Fig. 1. Absorbabilityof fatty acidsvs. melting and myrcin--the alcohol-insolublefraction of points in domestichens (solid squares)and chicks beeswax.Wax esters,like cetyl palmitate, are (opensquares; Renner and Hill 1961).Melting points majorfood sources for high-latitudemarine an- of saturatedfatty acids increase with chainlength and decreasewith degreeof unsaturation.Feeding studies imals, especiallypelagic seabirds(Roby et al. performedwith unesterifiedfatty acids.Saturated fat- 1986, Place and Roby 1986, Jacksonand Place ty acidsused were myristic,palmitic and stearicacid. 1990),and myrcin is consumedand digestedby Unsaturatedhomolog of stearicacid (i.e. oleic acid) Black-throatedHoneyguides (Indicator indicator; wasassimilated at greaterthan 85%efficiency. Diamond and Place 1988). We also measured the rate of bayberry-fruit consumptionof cap- tive Yellow-rumped Warblerswith fruits avail- able ad libitumto assesswhether fruit handling cation, an aliquot of extractedwax was hydrolyzed might limit ingestion rates. Finally, we char- with methanolic HCL, the methyl esters extracted acterized in Yellow-rumped Warblers the bili- into hexane,and an aliquot of the hexaneextract sub- jected to gas chromatographydirectly on a Hewlett- ary and pancreatic components known to be Packard model 5890A instrument fitted with a DB-% essentialfor efficient lipid assimilationin other column (30 x 0.25 mm i.d., 0.25-/•m-thick film, J. & species(Carey et al. 1983). W. Scientific Inc., Rancho Cordova, California) and a flame ionization detectorat 280øC.The oven temper- IV[ATERIALSAND METHODS ature was programmedfrom 50 to 255øCat a heating rate of 18øCmin • up to 125'C (thereafter4'C min-'). Studyspecies.--Yellow-rumped Warblers and Yel- Helium was usedas carrier gaswith a flow rate of 3.6 low Warblers(Dendroica petechia) were capturedwith ml min -1 . mist nets on 10 March and 7 November 1986 at the De-waxed bayberrieswere recoatedwith radioac- RutgersEcological Preserve in Piscataway,New Jer- tively-labeledwaxes by placing them in a melt of one sey. The Yellow-rumpedWarblers had been eating of the following materials:(1) bayberrywax contain- bayberry at the time of capture.Captured birds were ing 9.1 /•Ci of 3H-GTE/berry and 6.99/•Ci of [1-a4C] held at roomtemperature in darkenedholding cages palmiticacid/berry; (2) cetylpalmitate containing 7.33 until initiation of experimentson the sameday or the /•Ci of •H-GTE/berry and 5.5/•Ci of [1-a4C]cetyl pal- following morning.During the two- to three-daycap- mitate/berry or 4,7/•Ci of cetyl [1-'4C]palmitate/ber- tivity, birds were given water and food regularly ry; (3) myrcin (alcohol-insolublefraction of beeswax) (strainedpeaches and pears baby food mixed with containing 9.1 /•Ci of •H-GTE/berry and 8.2 /•Ci of hard-boiledegg). The TreeSwallows used in our study triacontanol[1-a4C] palmitate/berry. Each berry was were part of an experimentalcolony at the Monell recoatedwith 6 to 10 mg of lipid. Institute. Bayberry fruit structure was examined with and Bayberrywax removal and recoating.--Bayberries were without the wax coatingwith an Hitachi S-450Ascan- de-waxedby placingthem in a 2:1mixture of hexane ning electron microscopeusing an acceleratingvolt- and chloroformat roomtemperature for 15 min. The age of 10 KV. berries then were removed and air dried for 24 h. Feedingstudies.--Twelve Yellow-rumped Warblers The bayberry wax (24.3% of fruit mass or 54.1% of were included in the feeding trials with recoatedra- pulp mass)was recoveredby removing the solvent dioactively-labeledbayberries. Five were spring-cap- under nitrogen. For fatty-acidmethyl esterquantifi- tured birds (three males and two females), and seven 336 PLACEA!qD STILES [Auk,Vol. 109 were fall-captured birds (four males and three fe- used in the radiometric scanning was P-10 (90% ar- males).Sets of three birds were fed each type of re- gon, 10% methane) at a flow rate 0.5 to 1.0 L min-'. coatedbayberry. Only two Tree Swallowswere tested The spatial resolution for each scanwas set at 4 mm. (each three times). We also attempted feeding trials The distribution of label among the lipid classeswas with six Yellow Warblers.Two to three recoatedbay- estimated by integration of the counts under each berries were force fed to each bird. All birds took the peak after subtractionfor background.The overall feeding without regurgitationexcept for Yellow War- counting efficiencyfor •4Caveraged 10.5%, while that biers. for 3H averaged0.5% acrossthe plate. Labeled cetyl After ingestion,each bird wasplaced on a polyeth- oleate,cholesterol oleate, triolein, oleicacid, and cetyl ylene-mesh(•A") platform suspendedin an air-tight alcoholwere usedas standards to determinethe Rfof 7.6-L (2-gallon) Bain Marie polyethylene container these major lipid classes. (Cole-Palmer).We pumped CO2-freeair through the Biliaryand pancreatic components.--Bile was removed containers with aquarium pumps attached to soda- from the gall bladder with a sterile l-cc tuberculin lime (indicator grade) scrubbers.The flow rate of air syringe and placed in a sterile 0.5-ml polyethylene through the containers was adjusted to 1 L min •. centrifugetube. Total biliary protein was determined Containerswere kept in the dark and maintained at on aliquots using the dye-binding assayof Bradford 21 _+3øC. Respired carbon dioxide was collectedby (1976).Total bile saltswere assayedwith 3 a-hydroxy- passingthe air effluentfrom eachcontainer through steroid dehydrogenase(EC 1.1.150; Coleman et al. NCS (AmershamCorp.) solubilizer (CO2-trappingef- 1979). Whole-bile aliquots were analyzed by HPLC ficiencywas 88%;Place and Roby 1986).Respired CO2 on Waters Nova 5/• Radial packsusing a linear gra- was collectedin 15-min intervals for up to 4 h post- dient (1.2 to 34.0 min, flow rate 2.8 ml min •) from ingestion. It is especially important with lipid-ab- initial conditions (3.21 mM phosphoricacid, 3.75 mM sorption studiesthat somemeasure of metabolismbe KOH, and 4.00 mM KH2PO4, pH 4.32, 68:32 Iv/v] taken. Lipids can be stored and not utilized. After 3 methanol: water) to a final condition (3.21 mM phos- to 4 h, Yellow-rumped Warblerswere killed with an phoric acid, 3.75 mM KOH, and 11.00 mM KH2PO4, overdoseof sodiumpentobarbital, or were killed by pH 4.32, 68:32[v/v] methanol: water). Bile saltswere CO2asphyxiation. Accumulated excreta in eachcon- detected and quantitated by their absorbance at tainer were extractedfor lipids by the Bligh and Dyer 204 nm in comparisonwith standards. (1959) technique. We used a nonabsorbable lipid To assaybiliary lipids, an aliquot of the bile was marker, glycerol triether [*H-GTE] to estimate ab- spotted on pre-extracted (three times with chloro- sorption efficiencyand transit rate (Morgan and Hof- form:methanol 1:1) and preweighed 3-MM chro- mann 1970,Carlson and Bayley 1972a).An important matographyfilter paper. After drying at room tem- advantageof this method is that endogenousfecal fat perature for 4 h, the paper was extractedwith 5 ml is not measured(Carlson and Bayley 1972b).The per- of chloroform: methanol (1:1, v/v). Aliquots of the cent [•4C]-lipidabsorbed (L•) was calculatedas: extract were taken, dried, redissolved in chloroform: methanol (1:I, v/v), and assayedfor biliary phos- La= 10011- (t/f)], (I) pholipid phosphorous(Petitou et al. 1978).Addition- al sampleswere spotted on chromarodsand devel- where t is the •H/•4C in the testmeal and f is the 3H/ oped chloroform:methanol: acetic acid: water (75:25: '4C in the fecal collection. 3:1).Determination of neutral-lipid,phospholipid, and To assessthe distribution of lipid marker and la- bile-saltmass was by comparisonwith a standardcurve beled wax, each bird was dissected into liver, heart, generatedfor concentrationsof cholesterol,oleic acid, pectoral muscle,gizzard, 1-cm-length intestinal seg- triolein, phosphatidylcholine, and taurochenodeox- ments, pancreas,gall bladder, and remaining carcass. ycholate between 1 and 20/•g. Operating conditions The contentsof each gastrointestinalsegment were for the IatroscanTH-10 analyzer(Iatron Laboratories, extruded, and all fractions were stored frozen at -70øC Tokyo, Japan) were the same as those describedby until analyzed.Each body part and the intestinalcon- Rigler et al. (1983).Samples were spottedon type S-II tentswere extractedfor lipids by the Bligh and Dyer chromarods(RSS Incorporated,Costa Mesa, Califor- (1959) technique. nia), which were activatedby scanningthem twice The distribution of label among lipid classeswas through a hydrogen flame. Integration was per- determinedby TLC of the lipid extracts.Aliquots con- formed by a Hewlett-Packard 3390-A integrator taining equivalentcounts were spottedon the pre- (Avondale, Pennsylvania).Chromarods were devel- absorbentarea of channeledsilica G plates(Uniplates, oped in equilibrated filter paper-lined glass tanks Analtech). After developmentwith hexane:diethyl containing75 ml of solvent.When multiple systems ether: aceticacid (80:20:1), the platewas scanned with were used, chromarods were dried according to the a BioScan100 radiometricscanner. This solventsys- method of Harvey and Patton (1981). tem resolveswax esters,triacylglycerols, fatty acids, Titrametric assaysof lipolytic activity were per- fatty alcohols, 1,3-diacylglycerols,1,2-diacylglycer- formed usinga gum arabicstabilized emulsion of 0.2 ols, monoacylglycerols,and complex lipids, in order M cetyl oleate and 0.1 M triolein. Temperature was of decreasingrelative mobility (Rf). The carriergas maintained at 25øC,and pH maintained at 9.00 using April1992] BayberryWax and Yellow-rumped Warblers 337 a pH-stat (TTT80 Radiometer).Purified chickencol- U-tests for all simple comparisons.All comparisons ipasewas addedin a five-fold excessand sodiumcho- within each ANOVA were a priori.When more than late (8 mM) was added to the lipid emulsions. two contrastswere made,probability levels were ad- Materials.--Cholic acid, deoxycholic acid, cheno- justedby Duncan'snew multiple-rangetest (Steel and deoxycholicacid, sodium taurocholate,sodium tau- Torrie 1960).Differences were consideredsignificant rodeoxycholate,sodium taurochenodeoxycholate, when P -< 0.05. cholesterol,tripalmitin, palmitic acid, egg phospha- tidylcholine, triglyceride (procedureNo. 388), cho- RESULTS lesterol(procedure No. 351), and glucose(procedure No. 510)assay kits were purchasedfrom SigmaChem- Bayberryfruit characterization.--Themean di- ical (St. Louis,Missouri). Cetyl alcoholand 1-triacon- tanol were purchasedfrom Aldrich Chemical (Mil- ameter of M. pennsylvanicafruits used in our waukee,Wisconsin). The A-grade sodiumsalts of studywas 3.2 + 0.05mm (n = 40). Eachbayberry glycochenodeoxycholate,taurodeoxycholate, tauro- averaged20 + 0.8 mg (n = 40) and contained chenodeoxycholate,and taurocholate were purchased 4.5 + 0.2 mg of wax (n = 40). The wax coatsthe from Calbiochem (La Jolla, California). All other outsidelayer of the fruit (exocarp)and can be chemicalswere reagent grade unless specifiedoth- removedby organicsolvents without disrupt- erwise. All solvents were either pesticide or HPLC ing the fruit structure(Fig. 2). The baseof each grade.Whatman 3-MM chromatographicfilter paper bracteoleis looselyattached to the seedand is was obtained from VWR Scientific (Philadelphia, easilyremoved by rubbing the fruit. Pennsylvania). The waxy coaton a bayberryhas an energy We found [1-•4C]palmitic acid (5.7 mCi/retool) from densityof 39.3 KJ/g as determinedby micro- New England Nuclear (Boston,Massachusetts) and tri[1-'4C]palmitate (60 mCi/mmol), and [1-•4C]cetyl bomb calorimetry.The wax from the bayberry alcohol (24 mCi/mmol) from Amersham (Arlington consistedof mixed mono- and diglycerides of Heights, Illinois) to have radiopuritiesgreater than three saturatedfatty acids--myristic(14%), pal- 98% by thin-layer chromatography. mitic (85%), and stearic (1%) acids--and had a Fluors were OCS (Amersham,Arlington Heights, melting point of 42-48øC.The melting points Illinois) and Biosafe II (Research Products Interna- of the other syntheticwaxes used in recoating tional, Mount Prospect,Illinois). All sampleswere the bayberrieswere 51.4øCfor cetyl palmitate counted on a Beckman LS 3801 scintillation counter and 62 to 65øC for beeswax. with quenchcorrections for different extractsand tis- Feedingtrials.--The averagemass of the Yel- sue types. Labeledwax esters([1-•4C] cetyl palmitate, cetyl [1- low-rumped Warblers used in our study was •4C]palmitate, and triacontanol[1-•4C] palmitate), as 10.9 + 0.2 g (n = 12), the mean hematocritwas well as carrier cetyl and triacontanolpalmitate, were 53.3 + 3.7 (n = 7), and the average body lipid synthesizedand purified as describedby Place and was 11.2 + 1.2%of wet weight (n = 6). No bird Roby (1986). The cetyl wax esterswere dissolvedin died during the feeding trials. After 3 to 4 h, 250 •1 toluene and stored under nitrogen at -20øC. each bird had defecated 1.4 + 0.5 (n = 12) seeds The triacontanolpalmitate was stored dry at -20øC. of the 2.4 + 0.5 (n = 12) bayberriesfed. Yellow- Fromradiometric scanning, the radiopurity(2.2 mCi/ rumped Warblers separatethe seed and brac- mmol) of each wax ester was 98.7%,with an overall teoles (exocarp)in the gizzard' when fed bay- yield of 45 to 75%. Basedon TLC/FID using hex- berries. Partially-denuded bayberries were ane: diethyl ether: formic acid (85:15:0.1),the chem- found in the gizzard. The seed appears to pre- ical purity of each wax ester was greater than 98%. The 1-(9 cis-Octadecenyl) 2,3-didodecyl glycerol cedethe bracteolesduring gastrointestinaltran- triether was synthesizedas describedin Morgan and sit in that bracteoleswere frequentlyfound orad Hofmann (1970). The tritiated triether (3H-GTE) was to the seed. The bracteoles and seed accumulate prepared by reduction with platinum as a catalyst in the rectum and are defecatedtogether with (New England Nuclear, Boston,Massachusetts). Pu- urates.Although neither Yellow-rumped War- rified 3H-GTE (>98% radiopurity) was obtained by biers nor Tree Swallows regurgitated forced- chromatographyon a silicic-acidcolumn eluted with fed fruits, all Yellow Warblers regurgitated the hexane/diethyl ether 85:15 (v/v; Roby et al. 1989). fruits immediately upon being placed in the The solvent was removed with nitrogen evaporation metabolismchambers. We found no statistically andthe purifiedtriether dissolved in absolutealcohol significant(<5%) lipid absorptionor respired to a specificactivity of lmCi/ml. Statisticalanalysis.--Results are expressedas œ +- SE, radioactive CO2 of any wax with Yellow War- biers. with n being the number of birds. Comparisonsthat involve percentageswere performed on arcsin-trans- In a separate feeding trial, three Yellow- formed data.We usedpaired t-testsor Mann-Whitney rumped Warblers were kept in captivity and 338 PLACEAND ST•LES [Auk,VoL 109

Fig. 2. Scanning-electronmicrographs of fruits from bayberries(Myrica pennsylvanica)(A) with and (B) without the waxycoat. Fruit of bayberryis a smalldrupe envelopedby persistingoutgrowths of the exocarp (bracteoles)covered with a whitish waxy coating(Lawrence 1951). White bar represents0.34 nun. given bayberry fruits with water ad libitumto tabolizedC-14 label was found in adiposetissue assessthe quantity of bayberriesthey could in- in the carcassas triglyceride (>80%), with the gestdaily. On average,each bird ate 163 + 24 pectoral muscle the next highest in label ac- bayberriesper day.However, each bird lost 1.58 cumulation(5-8%). Only 2 to 4% of the label + 0.28 g of body weight per day during the was found in the liver. bayberry feeding trials. The fatty-alcohol moiety of the marine wax Tracer and marker recoveryand distribution.- ester was poorly assimilated and metabolized Within 15 rain of ingestion,radiolabeled CO2 by Yellow-rumped Warblers, despite nearly was collected when Yellow-rumped Warblers (> 95%)complete hydrolysis of the wax esterin were fed recoatedbayberries (Fig. 3). The high- the intestinal lumen. Palmitate, whether as a est rates and amount of respired label (40-60% free fatty acid or as the fatty-acid moiety in a of ingestedtracer) as well as the highestassim- cetyl palmitate,was absorbed with an efficiency ilation efficiencies (F = 10.44, df = 3 and 11, P of greaterthan 88%.When esterifiedto the 30- = 0.004) were observedwith [1-'4C]palmitate carbon-long fatty alcohol, triacontanol, the in bayberrywax (the natural food) and cetyl [1- efficiencywas reducedto lessthan 60%and la- •4C]palmitate in cetyl palmitate (a marine wax beled palmitate appeared to reside in the in- ester). Lower rates of lipid metabolismand as- testinal lumen longer (Fig. 4). However, assim- similation efficiencywere observedwhen birds ilation of the 16-carbon-lengthfatty alcohol, were fed beeswaxor cetyl palmitatelabeled in hexadecanol(cetyl alcohol),was lessthan 40%. the fatty-alcoholmoiety (Fig. 3 and Table 1). Overall, we accounted for 96 + 8.2% of the in- The rate of label respirationreflects the over- gestedC-14 tracerin our feedingtrials. all ratesof assimilation(i.e. hydrolysis,luminal No statisticallysignificant differencesin ex- absorption,repacking, transport from the en- creta3H-GTE recovery (F = 1.19,df = 3 and 11, terocytesand oxidation)for eachwax. With bay- P-- 0.372)or in total3H-GTE recovery (F = 0.623, berry wax and cetyl palmitate,only 15-17% of df = 3 and 11, P = 0.62) were observedamong the radiocarbon(as comparedto 33-35%of the feeding trials. The overall recoveryof the •H- nonabsorbablelipid marker, •H-GTE) was pres- GTE was 104 + 6.3%(n = 12), and greater than ent in the intestinal lumen after 180 rain (Fig. 98% of the recovered tritium label co-chromato- 4). The major portion (20-60%) of the nonme- graphed with pure glycerol triether. Becauseit April1992] BayberryWax and Yellow-rumped Warblers 339

80 timate of 232 ,in for the mean retention time 70 of the 3H-GTE marker. This estimate is based on ß 90.1% force-fed single meals and may overestimate mean retention time in freely-feedingbirds. mmmmmmm The assimilation efficiencies recorded for the 40 two Tree Swallows (Table 2) when feeding on ß 0 0 0 0 84'4ø'• 30 0 0ø recoatedbayberries parallels those obtained for Yellow-rumpedWarblers. No statisticalanaly- 20 mmoø []43.7øA 0 0 • 23.1ø• siswas performed on thesedata because of the 10 small samplesize. Bileof Yellow-rumpedWarblers.--The bile-salt componentsin Yellow-rumped Warbler bile -10 0 5•) 160 1•0 260 25O were predominantlytaurine conjugatesof the Time (.in) primary bile saltscholate and chenodeoxycho- Fig. 3. Representativetime coursesof respiredcar- late (Table 3). Relatively large amountsof tau- bon-14 labeled CO2in four Yellow-rumped Warblers rine and glycineconjugates of ursocholatealso fed bayberriescoated with four different radiolabeled were found. Gall-bladder bile-salt concentra- waxes.Symbols represent percent of ingestedlabel tion in individuals 4 h after feeding was 607 + recoveredat eachtime point for the following waxes: 16.2 raM, as determined by HPLC and 598 + cetyl[1-•4C] palmitate (solid squares), [1-14C] cetyl pal- 75 raM, as determined by enzymatic analysis. reitate (open squares),[1-14C] palmitate in bayberry The phospholipidcontent (~ 1.0 raM) was rel- wax (open circles),and triacontanol[1-•4C] palmitate in beeswax(open triangles).Assimilation efficiency atively low, while the neutral lipid content(~ 2 of each bird for the wax coating presented to right raM) was relatively high comparedto mam- of each curve. malian bile (phospholipid8.1 mM and a trace of neutrallipid in sevenmammalian biles; Cole- man et al. 1979). Luminal bile-salt concentrations and distributions was impossible to recoat each berry with an ofintestinal markers and labels.--The elevated level identical quantity of the 3H-GTE marker, the of bile salts in the gall bladder was reflected percent recovery is based on the average in high levels of bile saltsin the proximal half GTE found with 10 randomly selectedrecoated of the intestinallumen (Fig. 5A). After segment bayberries. 8, the luminal levels droppedbelow our detec- Although we did not set out to measuremean tion limit. Levels of bile salts approaching50 retention time directly in our feeding trials, an mM were found in the gizzard, nearly two seg- estimate can be made from the 3H-GTE marker ments orad to the biliary and pancreaticducts. recovery (Table 1). Excreta in different birds After 4 h postingestion,less than 2% of either were collectedfrom 180 to 281 ,in postinges- label was found in each segmentof the intes- tion. If we use each trial as a separateobser- tinal lumen (Fig. 5B). Lessthan 0.1% of the 3H- vation in describing the cumulative excretion GTE marker was recoveredfrom eachsegment curve and calculate the mean retention time as of the intestinal tissue(Fig. 5C), indicative of described by Warner (1981), we obtain an es- the marker'slow absorptionefficiency. The [1-

T^i•i,E1. Assimilationefficiencies and marker recoveries(œ + SD) of fed radiolabeledwaxes to Yellow-rumped Warblers(n = 3). Matching letters that follow values representsignificant differences (P < 0.05, multiple range test).

Percent marker Total percent Assimilation recovery in marker Carrierlipid Label efficiency excreta recovery Bayberrywax [1-•4C]palmitate 88.3 + 3.1" 63.2 + 26.7 93.8 + 9.1 Cetyl palmitate Cetyl [1-•4C]palmitate 88.9 + 2.5b 44.8 + 12.3 98.2 + 9.1 Cetyl palmitate [1-•4C]Cetyl palmitate 39.7 + 16.6a,b 47.3 + 11.8 98.6 + 9.1 Beeswax Triacontanol[1-•C] palmitate 56.3 + 21.9',• 32.3 + 27.5 116 + 10.8 340 PLACEAND STILES [Auk, Vol. 109

TABLE3. Yellow-rumped Warbler gall-bladder bile [] Beeswax composition(• + SD, n = 6). [] Cetyl palmirate ß Bayberry Concentration Constituent (mM) Tauroursocholate 20.3 + 6.66 Taurocholate 181.5 + 19.3 Taurochenodeoxycholate 341.8 + 42.2 Taurolithocholate 5.1 + 0.87 Glycoursocholate 58.5 + 0.7 Phospholipid 1.18 + 0.65 Cholesterol+ triglyceride + fatty acid 2.6 _+0.59

+ 0.3 •moles min -• mg-• while that for wax esterlipolysis was 2.5 + 0.5 •moles min -1 mg-•. Fig.4. Distributionof carbon-14 radioactivetracer Hence,under thesein vitroassay conditions wax in respiredCO•, excreta,and tissues.Data presented esterswere hydrolyzed 8.3 + 2.3 times slower for three Yellow-rumped Warblers fed different re- than triglycerides. coatedbayberries 180 min postingestion.

DISCUSSION

CTM] palmitate isolatedfrom the intestinal tissue Frugivoryof waxy fruits.--The associationof waslargely incorporated in triglyceride(> 80%), Yellow-rumped Warblerswith waxy fruits rep- although substantialfree fatty acid (>8%) was resentsone of the most specialized fruit/fru- also recovered. givore relationshipsso far reported,similar to Similar results were obtained for birds fed thoseof the Phainopepla(Phainopepla nitens) and cetyl [1-C•4] palmitate and triacontanol[I-C •4] mistletoe (Phoradendroncalifornicum) in the palmitate.Although in the latter caseconsid- southwestern United States (Walsberg 1975), erably more unhydrolyzed wax ester could be CedarWaxwings (Bombycilla cedrorum) and mis- extracted from the lumen. With birds fed [I-C •4] tletoe (P. serotinum)in the southeasternUnited cetyl palmitate,significant levels of free [I-C 14] States(Skeate 1985), and Asian flowerpeckers cetyl alcohol could be found in the intestinal and mistletoes (Docters van Leeuwen 1954). lumen. In intestinal tissue, only labeled fatty Waxy fruits have been identified as common acid and triglyceride were detected. food items in the diets of Northern Flickers Lipolysisby Yellow-rumpedWarblers.--Extracts (Colaptes auratus ), Downy Woodpeckers(Picoides of pancreatictissue from Yellow-rumped War- pubescens),and other woodpeckers,as well as blers were capableof hydrolysis of both tri- Tree Swallows and Yellow-rumped Warblers glycerideand wax esteremulsions in vitro.The (Martin et al. 1951), yet consumptionof large specificactivity of triglyceridelipolysis was 21 numbersof thesefruits by pen-raisedNorthern Bobwhites(Colinus virginianus) has been found to interfere with digestionand may even prove TABLE2. Assimilationefficiency (• + SD) of radio- fatal (Martin et al. 1951). labeled waxes for two Tree Swallows. The frugivorous diet of the Yellow-yumped Warblers is not restrictedto Myrica. In the in- Assimila- terior of the United States, where species of tion Myrica are lesscommon, Yellow-rumped War- Carrier lipid Label efficiency biers commonly eat poison-ivy fruits (Toxico- Bayberry wax [1-•C] palmitate 66.4 + 0.6 dendronradicans; Ridgeway 1889, Graber and Tri [I~I•C] palmitin 77.6 + 0.8 Tripalmitin Graber 1979, Graber et al. 1983), another solid Cetyl palmitate Cetyl [1-•C] palmirate 79.6 + 0.8 Cetyl palmitate [1-•C] Cetyl palmirate 47.8 + 0.8 and waxy fruit. In fact, the waxy mesocarpof Cetyl alcohol [1-a•C]Cetyl alcohol 55.4 + 1.0 Toxicodendronconsists primarily of glyceridesof Beeswax Triacontanol [1-•C] palmiticand oleicacids (Brizicky 1963), similar palmitate 18.8 + 5.3 in compositionto the waxy pulp of Myricafruits. April 1992] BayberryWax and Yellow-rumped Warblers 341

6O A (Vitis), Virginia creeper (Parthenocissus),and 5o sumac (Rhus;Bent 1953, Griscom and Sprunt 1957).This warbler speciesis secondonly to the 4O Yellow-rumped Warbler in the northern extent of its wintering range (AOU 1983). The Bay- 30 breastedWarbler rarely is frugivorouswithin

20 the United States(Martin et al. 1951, Bent 1953), but consumesfruits from 21 plant speciesin 10 Panama(Greenberg 1981). The Chestnut-sided

0' Warbler eats some fruits on the wintering Gizzard I 2 3 4 5 6 7 8 9 10 grounds in Central America (Howe and De- Steven 1979,Greenberg 1979, 1981).Greenberg (1981) found that a major fruit usedselectively 0.4 by Bay-breastedand Chestnut-sidedwarblers in Panama was from Lindackeffa laurina (Flacour- 0.3 tiaceae),and he identified the aril as having "a distinctly waxy texture and odor." Skeate(1985) 0.2 found that 91% of fruit removed from Myrica ceriferain hammock communities in central 0.1 Florida was taken by Yellow-rumpedWarblers and that poisonivy was eaten only by Yellow- 0.0 Gizzard I 2 3 4 5 6 7 8 9 I 0 rumped Warblers and woodpeckers.The Tree Swallow is the only member of North American swallowsto eat fruit asa regularpart of the diet (Bent 1942).Birds eating waxy fruits represent an unusualassociation of frugivoresas they be- long to families or subfamiliesof birds that are generally consideredhighly insectivorous. 0.1ø'21 C Yellow-rumpedWarblers feed on bayberries to eat the wax.--The rapid and extensive release of radiolabeledCO2 for Yellow-rumped Warblers fed bayberriesrecoated with [1-C•4] palmitate is 06 strongevidence supporting a metabolicfuel role I 2 3 4 5 6 7 8 9 10 for the natural waxy coatingon bayberry.Me- Gastrointestinal Segment tabolism of dietary fat occurred in birds with Fiõ. 5. (A) Concentration and localization of bile greater than 10% body fat, a fat content that salts.3H-GTE (open bars) and [1-C•4] palmitic acid (hatchedbars) in (B)luminal contents and (C) tissues agreesclosely with prior studieson autumn and in a Yellow-rumpedWarbler's gastrointestinal tract 4 spring Yellow-rumped Warblers (Yarborough h after feedinga recoatedbayberry. Each segment and Johnson,1965). With an averagecoating of numberrepresents 1 cm of intestine,progressing to- 4.5 to 7.3 mg of wax per fruit, we estimatethat ward the cloaca.Average intestinal length in exper- a Yellow-rumped Warbler eating between 170 imentalbirds was 10.9 + 0.27 cm (n = 9). Intactbay- and 280bayberries would meeta 50 Kj/day dai- berry seedsfound in segments3 and 5 for this ly energy expenditure. Our feeding trials in- individual. Means, with SE indicated. dicatethat they are capableof eating over 160 fruits per day. However,they lost nearly 1.5 g of body weight per day on a bayberry diet, in- Of the 21 membersof the genus Dendroica dicatingthat for ourcaptive birds the waxycoat- that breed in the United States and Canada, ing was not sufficientfor weight maintenance. only the Yellow-rumped,Pine (D. pinus),Chest- The lossin bodyweight alsomay be attributable nut-sided(D. pennsylvanica),and Bay-breasted to low nitrogen contentin the coatingor to fat- (D. castanea)warblers have been identified as soluble toxins present in the wax (Levey and including fruit asa largepart of their diets.Pine Karasov 1989). Warblersfeed on many fruits including poison Determinantsof highlipid digestiveefficiency in ivy, bayberry,dogwood, (Cornus), wild grape Yellow-rumpedWarblers.--Ingested fat, especial- 342 PLACEAi•IIV SI'II. ES [Auk,Vol. 109

ly high-melting-pointfatty acids,must be emul- We hypothesize that Yellow-rumped War- sified or solubilized in the stomachprior to the biers also may exhibit a gastrointestinalreflux formationof miceliesand absorptionin the du- basedon our measurementsof biliary products Odehum. Potential emulsifiers that can function found in the gizzard. Bile-salt concentrations in the acidmilieu of the stomachinclude peptic exceeding50 mM are recordedin the gizzard digestsof dietary protein, complexpolysaccha- along with lipolysisproducts of dietary lipids. rides and dietary phospholipids.In mammals, Direct radiographic observationswill be nec- some enzymatic hydrolysisof dietary triglyc- essaryto substantiatethis hypothesis,since the eridesoccurs in the stomach,resulting in gastric accumulationof biliary and intestinal contents digestionof up to 30%of fats(Carey et al. 1983). in the gizzard may have resulted during eu- It is thought that the monoglyceridesformed thanasia. during this gastriclipolysis of dietary-neutral An important corollary with respect to the lipids further aid emulsification. effectivenessof any biliary reflux is the nature In seabirds,little gastric lipolysis is found; of the bile salts. Because of the acidic nature in yet, thesebirds are highly successfulat neutral the gizzard and the marked pH dependencein lipid digestion(Roby et al. 1986,Place and Roby the solubility of unconjugatedbile salts(Carey 1986, Place et al. 1989, Jackson and Place 1990, et al. 1983), it is important that these natural Place 1992).This ability to assimilatenonpolar fatty-acid solubilizers remain in solution and lipids efficiently may be due to a unique char- not precipitatein the gizzard. In Yellow-rumped acter of the "enterogastricreflex" of birds. In Warblers,because of taurine conjugation,bile fowl, as in mammals,gastric motility is inhib- saltsdo not precipitatein the gizzard. Greater ited by intraduodenalinjections of 0.1 N I-IC1, than 90% of the bile salts in Yellow-rumped 1,600 mOsM solutions of NaC1, corn oil, or ami- Warbler bile are taurine conjugates(Table 3). no acids, as well as by intraduodenal balloon Taurine bile-salt conjugatesremain soluble at a inflation (Duke and Evanson 1972, Duke et al. pH below 1.0 (Carey et al. 1983). 1973). In addition to this inhibition of gastric Elevated bile-salt levels were observed also emptying, the enterogastric reflex in birds in- in the proximal lumen of the intestine. The crit- cludes the occurrence of one or more intestinal ical micelie concentration of bile salts in the refluxes during the period of gastric motility presenceof lipolytic products is below 5 mM inhibition (Duke et al. 1973, 1989, Duke 1986). (Carey et al. 1983). Thus, the bile salts in the In the chick of domestic chickens, this move- intestinal lumen of the Yellow-rumped Warbler ment appears to be continuous and regular are nearly 5 to 10 times above their critical mi- (Sklan et al. 1978), and observations in the do- celiar concentration,hence ensuring effective mesticturkey (Meleagrisgallopavo) indicate that miceilarsolubilization of lipolyticproducts and, antiperistalsisincludes the duodenum and pos- potentially,establishing a chemicaldriving force sibly the upper jejunum (Duke 1986). Intestinal for passiveuptake of dietary fatty acids. refluxes occurapproximately three times more The fact that wax estersare hydrolyzedeight often in Leach'sStorm-Petrels (Oceanodromaleu- times more slowly than triglycerides and pan- corhoa)than in fowl (Duke et al. 1989), and in- creaticextracts of Yellow-rumpedWarblers may volve the movement of intestinal contents back explain the slower rates of metabolismof tria- to the proventriculus. contanol palmitate. However, the fact that the Thus, gastricemptying in thesebirds is close- fatty-acidmoiety in cetyl palmitate is metabo- ly tied to the receptivenessof the duodenum lized as rapidly as the free fatty acid (Fig. 2) for additional digesta, and the reflux returns indicatesthat hydrolysisis not a rate-limiting the digesta(both gastricand duodenal) for fur- stepin lipid absorptionof Yellow-rumpedWar- ther processingin the gizzard. In the process, biers. duodenalproducts like monoglyceridesand fat- One trait observedin seabirds,which appears ty acids are refluxed to the gizzard along with not present in the two passetineswe analyzed, biliary (bile salts, phospholipids,and triglyc- is efficient utilization of long-chain fatty alco- erides) and pancreatic products (lipases). Gas- hols. Whereasdietary fatty alcoholsare effi- tric production of lipid emulsifiersis not found ciently assimilated(> 90%;Place and Roby 1986, in birds; instead, products of normal intestinal Jacksonand Place 1990,Place 1992)by seabirds, lipolysisare refluxedto a highly efficientemul- both Yellow-rumped Warblersand Tree Swal- sificationmill, the gizzard. lows were relatively poor (<50%) at assimilat- April 1992] BayberryWax and Yellow-rumped Warblers 343 ing long-chainfatty alcohols.This inefficiency an apparent retrograde intestinal reflux to the in fatty-alcoholassimilation causes few prob- gizzard. Gastrointestinalmean retention time lems for Yellow-rumped Warblers and Tree may alsobe longer in Yellow-rumped Warblers Swallows, since they rarely ingest large quan- than expectedfor other frugivorousbirds. De- tities of fatty alcohol in their natural diet (un- spite these traits, Yellow-rumped Warblers in like seabirds).However, the findingsclearly in- captivityare not ableto maintainconstant body dicatethat the capacityto oxidize fatty alcohols weight eatinga bayberrydiet. Leveyand Kara- canbe modulatedindependently of the capacity sov (1989) concludedthat few temperatebirds to hydrolyze wax esters. eating nonwaxyfruits could maintain long-term Many nonwaxy fruits are characterizedby a nutrient and energy balanceon a diet of solely pulp with a dilute solution of sugars,low con- fruits. Our results with Yellow-rumped War- centrations of amino acids, and a substantial biers eating the waxy fruits with higher energy portion of undigestibleseed mass. Accordingly, density are consistentwith their conclusion. frugivorous birds feeding on nonwaxy fruits Both Yellow-rumped Warblers and Tree have shortdigestive retention times (Levey and Swallowsare facultativein their frugivory, eat- Karasov 1989, Karasovand Levey 1990). Waxy ing insectswhen available.The gastrointestinal fruits are characterizedby a pulp of increased traits we have documented in Yellow-rumped energy density,because of a high lipid content Warblersshould enhance lipid absorptionover- in the wax. However, a paradox seemsto exist, all, whether the lipid is derived from insectsor since frugivorousbirds typically exhibit short fruits. Whether the traits are constitutive or can food-retention times (Karasov 1990), yet lipid be modulated by diet is not known, but we absorptionis positively correlated with reten- suspectthey are constitutive,since both spring tion time (e.g. Jacksonand Place 1990). More- and autumn birds displayedsimilar absorption over,the level of dietary fat stronglyinfluences capacities.We also suspectthat these two spe- gastrointestinaltransit (Mateos and Sell 1981, cies,especially Yellow-rumped Warblers,may Mateos et al. 1982). We predict that, for birds have an enhanceddetoxification machinery for feeding on waxy fruits, gastrointestinalreten- plant secondarycompounds. The bile is a major tion times would be longer than for birds feed- secretoryroute for detoxifiedcompounds. Yel- ing on nonwaxy fruits. Also, we predict that the low-rumped Warblers and Tree Swallows can gastrointestinalreflux rate would be higher (i.e. be added to the list of other birds (Obst 1986, more frequent and greater rate) in birds eating Robyet al. 1986,Place and Roby 1986,Diamond waxy fruits than in birds feeding on nonwaxy and Place 1988, lackson and Place 1990) that fruits. While we did not measure mean reten- exhibit a distinctive ability to assimilate oth- tion directly in the current study, the estimate erwiserefractory lipids and "live off the wax of we obtain from the 3H-GTElipid phasemarker the land." indicatesthat mean retention time of bayberry wax in Yellow-rumped Warblers may be five- fold longer than Cedar Waxwings eating wild LITEKATURE CITED grapes(Karasov and Levey 1990).We believe a detailed examination of mean retention times AMERICANORNITHOLOGISTS' UNION. 1983. Check-list in birds feeding on waxy dietsmay be a useful of North American birds, 6th ed. American Or- direction for future studies. nithologists'Union, Washington,D.C. In summary, we find that Yellow-rumped BENT,A. C. 1942. Life histories of North American Warblers and Tree Swallows are capableof ef- flycatchers,larks, swallows, and their allies.U.S. ficient absorption and metabolism of bayberry Natl. Mus. Bull. 179. wax. Efficientutilization extendsto coatingswith BENT,A.C. 1953. Life histories of North American a marine wax ester, and to a lesser extent with wood warblers, parts 1 and 2. U.S. Natl. Mus. Bull. 203. the major wax ester (triacontanolpalmitate) in BLIGH,E.G., AND W. J. DYER. 1959. A rapid method beeswax.Fatty alcohols are less efficiently as- of total lipid extractionand purification.Can. J. similatedthan the equivalent-chain-lengthfat- Blochem.Physiol. 37:911-917. ty acid. This unique assimilatorycapacity for BRADFORD,M. M. 1976. A rapid and sensitivemeth- high-melting-pointwaxes in Yellow-rumped od for the quantitationof microgramquantities Warblers is associatedwith an elevated gall- of proteinutilizing the principleof protein-dye bladder and luminal bile-salt concentration, and building.Anal. Blochem.72:248-254. 344 PLACEAND STILES [Auk,Vol. 109

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