The Auk 109(2):334-345, 1992
LIVING
OFF THE WAX
OF THE LAND:
YELLOW-RUMPED
BAYBERRIES
AND
WARBLERS
ALLEN R. PLACE • AND EDMUND
W. STILES 2
•Centerof MarineBiotechnology,
Universityof Maryland,600EastLombard
Street,Baltimore,
Maryland21202,USA;
and
2Department
of Biological
Sciences,
Rutgers-The
StateUniversityof New Jersey,
Piscataway,
New Jersey08855,USA
ABSTRACT.--Yellow-rumped
Warblers(Dendroica
coronata)
and Tree Swallows(Tachycineta
bicolor)are among a small group of birds in temperateNorth America that regularly eat waxy
fruits.During the autumn,winter, and spring,thesespeciesfeed extensivelyon fruits of the
bayberry(Myrica spp.). Covering the pulp of these fruits is a solid, waxy material consisting
primarily of saturatedlong-chainfatty acids.For mostanimals,saturatedfatty acidsare poorly
assimilated(< 50%).Using 3H-glyceroltriether asa nonabsorbablefat marker,we determined
that Yellow-rumpedWarblersare capableof high assimilationefficiences(> 80%)of bayberry
wax when fed berries recoatedwith radioactivewax tracers.Efficientfatty-acidassimilation
extendsto berriescoatedwith 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 assimilationefficienciesof eachwax are recordedfor Tree Swallowsfeeding on
recoatedbayberries.Yellow Warblers(D. petechia)rejectedrecoatedbayberriesand 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-saltconcentration,and a slow gastrointestinaltransitof dietary lipids. These
gastrointestinaltraits permit efficientassimilationof 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 ASSOCIATION
between Yellow-rumped
Warblers (Dendroicacoronata)and Myrica (bayberry and wax myrtle) is one of the mostwidely
recognized bird-plant associationsin North
monly observed over much of its wintering
range.The TreeSwallowwintersalongthe coast,
occasionallyas far north as Massachusetts
and
south into Central America. In autumn, large
America (Brewer 1840, Hausman 1927, Martin
et al. 1951). In fact, until 1983 one form of the
flocks (some over 50,000; Stewart and Robbins
relationship is found between Tree Swallows
(Tachycineta
bicolor)and Myrica(Hausman1927).
During the breeding season Yellow-rumped
Warblersand Tree Swallows feed primarily on
1958),move southalong the coastand may strip
Myricashrubsof all fruitsin a matterof minutes
(E. Stiles, pers. observ.).
Generally, bayberry is found on dunes, oldfields, and dry hills from Quebecto Louisiana;
wax-myrtle typically is found on damp, sandy
soils from New Jersey to Florida and Texas
insects,but during the autumn, winter, and
(Gleason and Cronquist 1963). Fruits ripen in
Yellow-rumped Warbler was referred to as the
Myrtle Warbler(AOU 1983).A similarbird-fruit
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.
coastalregionsof the United States.
In easternNorth America,the Yellow-rumped
Bayberry pulp includes a waxy coating of
Warbler winters from central Maine and southmono-and diglyceridesof myristic,palmiticand
ern Nova Scotia, west to Kansas and Missouri,
stearicfatty acids.Most animals exhibit low asand 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
monotonicallywith
wintering wood warbler, and it is very com- increasingmelting point (Rennerand Hill 1961;
334
April 1992]
Bayberry
WaxandYellow-rumped
Warblers
335
100'
Fig. 1). Similar results have been documented
in rats feeding on high-melting-point triglyc-
80-
erides (Clifford et al. 1986).
It may be that Yellow-rumped Warblers and
Tree Swallows are able to successfullyoccupy
northern regions in winter becausethey can
6O
4O
assimilateefficiently the high-melting-point
fattyacidsin bayberrywaxthat few fruit-eating
animalscan digest.To investigatethe capacity
2O
of Yellow-rumped Warblersand Tree Swallows
to assimilatewaxy coatingson bayberries,we
removed the natural wax from bayberriesand
recoatedthem with radioactivelylabeledlipids.
We includedbayberrywax in our coatings,as
well astwo othernaturally-occurring
solidwaxesknown to be eatenby birds--cetyl palmitate
and myrcin--the alcohol-insolublefraction of
beeswax.Wax esters,like cetyl palmitate, are
majorfoodsources
for high-latitudemarineanimals, especiallypelagic seabirds(Roby et al.
1986, Place and Roby 1986, Jacksonand Place
1990),and myrcin is consumedand digestedby
Black-throatedHoneyguides(Indicatorindicator;
0
-20
2'0
4'0
6'0
80
Melting Point (øC)
Fig. 1. Absorbabilityof fatty acidsvs. melting
points in domestichens (solid squares)and chicks
(opensquares;
RennerandHill 1961).Meltingpoints
of saturatedfattyacidsincreasewith chainlengthand
decrease
with degreeof unsaturation.
Feedingstudies
performedwith unesterifiedfatty acids.Saturatedfatty acidsusedwere myristic,palmitic and stearicacid.
Unsaturatedhomologof stearicacid (i.e. oleic acid)
wasassimilated
at greaterthan 85%efficiency.
Diamond and Place 1988). We also measured
the rate of bayberry-fruit consumptionof captive Yellow-rumped Warblerswith fruits avail-
ablead libitumto assess
whether 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 biliary and pancreatic components known to be
essentialfor efficientlipid assimilationin other
species(Carey et al. 1983).
IV[ATERIALSAND METHODS
Studyspecies.--Yellow-rumpedWarblers and Yellow Warblers(Dendroica
petechia)
were capturedwith
mist nets on 10 March
and 7 November
1986 at the
into hexane,and an aliquot of the hexaneextractsubjectedto gas chromatographydirectly on a HewlettPackard
model
5890A
instrument
fitted with
a DB-%
column (30 x 0.25 mm i.d., 0.25-/•m-thick film, J. &
W. Scientific Inc., Rancho Cordova, California) and a
flame ionization detectorat 280øC.The oven temperature wasprogrammedfrom 50 to 255øCat a heating
rate of 18øCmin • up to 125'C (thereafter4'C min-').
Helium was usedas carriergaswith a flow rate of 3.6
ml min -1 .
De-waxed bayberrieswere recoatedwith radioactively-labeledwaxesby placingthem in a melt of one
sey.The Yellow-rumpedWarblershad been eating of the following materials:(1) bayberrywax containbayberryat the time of capture.Capturedbirds were ing 9.1 /•Ci of 3H-GTE/berry and 6.99/•Ci of [1-a4C]
held at roomtemperaturein darkenedholdingcages palmiticacid/berry;(2) cetylpalmitatecontaining7.33
until initiation of experimentson the sameday or the /•Ci of •H-GTE/berry and 5.5/•Ci of [1-a4C]cetyl palfollowing morning.During the two- to three-daycap- mitate/berry or 4,7/•Ci of cetyl [1-'4C]palmitate/bertivity, birds were given water and food regularly ry; (3) myrcin (alcohol-insolublefraction of beeswax)
(strainedpeachesand pears baby food mixed with containing 9.1 /•Ci of •H-GTE/berry and 8.2 /•Ci of
hard-boiledegg).The TreeSwallowsusedin 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
Bayberry
waxremoval
andrecoating.--Bayberries
were without the wax coatingwith an Hitachi S-450Ascande-waxedby placingthemin a 2:1mixtureof hexane ning electronmicroscopeusing an acceleratingvoltand chloroformat roomtemperaturefor 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 massor 54.1% of were included in the feeding trials with recoatedrapulp mass)was recoveredby removing the solvent dioactively-labeledbayberries.Five were spring-capunder nitrogen.For fatty-acidmethyl esterquantifi- tured birds (three males and two females), and seven
RutgersEcologicalPreservein Piscataway,New Jer-
336
PLACE
A!qDSTILES
were fall-captured birds (four males and three females).Setsof three birds were fed each type of recoatedbayberry.Only two Tree Swallowswere tested
(each three times). We also attempted feeding trials
with six Yellow Warblers.Two to three recoatedbayberries were force fed to each bird. All birds took the
feeding without regurgitationexceptfor Yellow Warbiers.
After ingestion,eachbird wasplacedon a polyethylene-mesh(•A")platform suspendedin an air-tight
7.6-L (2-gallon) Bain Marie polyethylene container
(Cole-Palmer).We pumped CO2-freeair through the
containerswith aquarium pumps attached to sodalime (indicator grade) scrubbers.The flow rate of air
through the containerswas adjustedto 1 L min •.
Containerswere kept in the dark and maintained at
21 _+3øC.Respiredcarbon dioxide was collectedby
passingthe air effluentfrom eachcontainerthrough
NCS (AmershamCorp.) solubilizer (CO2-trappingefficiencywas88%;Placeand Roby 1986).RespiredCO2
was collectedin 15-min intervals for up to 4 h postingestion. It is especially important with lipid-absorptionstudiesthat somemeasureof metabolismbe
taken. Lipids can be storedand not utilized. After 3
to 4 h, Yellow-rumped Warblerswere killed with an
overdoseof sodiumpentobarbital,or were killed by
CO2asphyxiation.Accumulatedexcretain eachcontainer were extractedfor lipids by the Bligh and Dyer
(1959) technique. We used a nonabsorbable lipid
marker, glycerol triether [*H-GTE] to estimate absorption efficiencyand transit rate (Morgan and Hofmann 1970,Carlsonand Bayley1972a).An important
advantageof this methodis that endogenousfecalfat
is not measured(Carlsonand Bayley1972b).The percent [•4C]-lipidabsorbed(L•) was calculatedas:
[Auk,Vol. 109
used in the radiometric scanningwas P-10 (90% argon, 10% methane) at a flow rate 0.5 to 1.0 L min-'.
The spatial resolution for each scanwas set at 4 mm.
The distribution of label among the lipid classeswas
estimatedby integration of the counts under each
peak after subtractionfor background.The overall
countingefficiencyfor •4Caveraged10.5%,while that
for 3H averaged0.5% acrossthe plate. Labeled cetyl
oleate,cholesterololeate,triolein, oleicacid,and cetyl
alcoholwere usedasstandards
to determinethe Rfof
these major lipid classes.
Biliaryandpancreatic
components.--Bile
wasremoved
from the gall bladder with a sterile l-cc tuberculin
syringe and placed in a sterile 0.5-ml polyethylene
centrifugetube. Total biliary protein was determined
on aliquots using the dye-binding assayof Bradford
(1976).Totalbile saltswere assayedwith 3 a-hydroxysteroid dehydrogenase(EC 1.1.150; Coleman et al.
1979). Whole-bile aliquots were analyzed by HPLC
on Waters Nova 5/• Radial packsusing a linear gradient (1.2 to 34.0 min, flow rate 2.8 ml min •) from
initial conditions (3.21 mM phosphoricacid, 3.75 mM
KOH, and 4.00 mM KH2PO4, pH 4.32, 68:32 Iv/v]
methanol: water) to a final condition (3.21 mM phosphoric acid, 3.75 mM KOH, and 11.00 mM KH2PO4,
pH 4.32, 68:32[v/v] methanol: water). Bile saltswere
detected and quantitated by their absorbanceat
204 nm in comparisonwith standards.
To assaybiliary lipids, an aliquot of the bile was
spotted on pre-extracted (three times with chloroform:methanol 1:1) and preweighed 3-MM chromatographyfilter paper. After drying at room temperature for 4 h, the paper was extractedwith 5 ml
of chloroform: methanol (1:1, v/v). Aliquots of the
extract were taken, dried, redissolved in chloroform:
methanol (1:I, v/v), and assayedfor biliary phospholipid phosphorous(Petitou et al. 1978).Additional sampleswere spotted on chromarodsand develwhere t is the •H/•4C in the testmealand f is the 3H/ oped chloroform:methanol:aceticacid: water (75:25:
'4C in the fecal collection.
3:1).Determinationof neutral-lipid,phospholipid,and
To assessthe distribution of lipid marker and la- bile-saltmasswasby comparisonwith a standardcurve
beled wax, each bird was dissected into liver, heart,
generatedfor concentrationsof cholesterol,oleicacid,
pectoral muscle,gizzard, 1-cm-lengthintestinal seg- triolein, phosphatidylcholine, and taurochenodeoxments, pancreas,gall bladder, and remaining carcass. ycholate between 1 and 20/•g. Operating conditions
The contentsof each gastrointestinalsegmentwere 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.Eachbody part and the intestinalcon- Rigler et al. (1983).Sampleswere spottedon type S-II
tentswere extractedfor lipids by the Bligh and Dyer chromarods(RSSIncorporated,CostaMesa, Califor(1959) technique.
nia), which were activatedby scanningthem twice
The distribution of label among lipid classeswas through a hydrogen flame. Integration was perdeterminedby TLC of the lipid extracts.Aliquotscon- formed by a Hewlett-Packard 3390-A integrator
taining equivalentcountswere spottedon the pre- (Avondale, Pennsylvania).Chromarodswere develabsorbentareaof channeledsilicaG plates(Uniplates, oped in equilibrated filter paper-lined glass tanks
Analtech). After developmentwith hexane:diethyl containing75 ml of solvent.When multiple systems
were used, chromarodswere dried according to the
ether: aceticacid(80:20:1),the platewasscannedwith
a BioScan100 radiometricscanner.This solventsys- method of Harvey and Patton (1981).
tem resolveswax esters,triacylglycerols,fatty acids,
Titrametric assaysof lipolytic activity were perfatty alcohols, 1,3-diacylglycerols,1,2-diacylglycer- formed usinga gum arabicstabilizedemulsionof 0.2
ols, monoacylglycerols,and complexlipids, in order M cetyl oleate and 0.1 M triolein. Temperature was
of decreasing
relativemobility(Rf).The carriergas maintained at 25øC,and pH maintained at 9.00 using
La= 10011- (t/f)],
(I)
April1992]
Bayberry
WaxandYellow-rumped
Warblers
a pH-stat(TTT80Radiometer).Purifiedchickencolipasewasaddedin a five-fold excessand sodiumcholate (8 mM) was added to the lipid emulsions.
Materials.--Cholic acid, deoxycholicacid, chenodeoxycholicacid, sodiumtaurocholate,sodiumtaurodeoxycholate,sodium taurochenodeoxycholate,
cholesterol,tripalmitin, palmitic acid, egg phosphatidylcholine, triglyceride (procedureNo. 388), cholesterol(procedureNo. 351), and glucose(procedure
No. 510)assaykitswere purchasedfrom SigmaChemical (St. Louis,Missouri).Cetyl alcoholand 1-triacontanol were purchasedfrom Aldrich Chemical (Mil-
337
U-tests for all simple comparisons.All comparisons
within each ANOVA were a priori.When more than
two contrastswere made,probabilitylevelswere adjustedby Duncan'snew multiple-rangetest(Steeland
Torrie 1960).Differenceswere consideredsignificant
when
P -< 0.05.
RESULTS
Bayberry
fruit characterization.--The
mean diameter of M. pennsylvanica
fruits used in our
waukee,Wisconsin).The A-gradesodiumsaltsof studywas3.2 + 0.05mm (n = 40). Eachbayberry
glycochenodeoxycholate,taurodeoxycholate,tauro- averaged20 + 0.8 mg (n = 40) and contained
chenodeoxycholate,
andtaurocholate
werepurchased 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 unlessspecifiedoth- removedby organicsolventswithout disrupterwise. All solvents were either pesticide or HPLC
ing the fruit structure(Fig. 2). The baseof each
grade.Whatman3-MM chromatographic
filter paper bracteoleis looselyattachedto the seedand is
was obtained from VWR Scientific (Philadelphia, easilyremovedby rubbingthe fruit.
Pennsylvania).
The waxy coaton a bayberryhasan energy
We found [1-•4C]palmiticacid(5.7 mCi/retool) from
density
of 39.3 KJ/g as determinedby microNew 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%),pal98% by thin-layer chromatography.
Fluorswere OCS (Amersham,Arlington Heights,
Illinois) and Biosafe II (ResearchProducts International, Mount Prospect,Illinois). All sampleswere
counted
on a Beckman
LS 3801 scintillation
counter
with quenchcorrectionsfor different extractsand tissue types.
Labeledwax esters([1-•4C]cetyl palmitate, cetyl [1•4C]palmitate,and triacontanol[1-•4C]palmitate),as
well as carrier cetyl and triacontanolpalmitate, were
synthesizedand purified as describedby Place and
Roby (1986). The cetyl wax esterswere dissolvedin
250 •1 toluene and stored under nitrogen at -20øC.
The triacontanolpalmitate was stored dry at -20øC.
Fromradiometricscanning,the radiopurity(2.2mCi/
mmol) of each wax ester was 98.7%,with an overall
yield of 45 to 75%. Basedon TLC/FID using hexane: diethyl ether: formicacid (85:15:0.1),the chemical purity of each wax ester was greater than 98%.
The 1-(9 cis-Octadecenyl)2,3-didodecyl glycerol
triether was synthesizedas describedin Morgan and
Hofmann (1970). The tritiated triether (3H-GTE) was
mitic (85%), and stearic (1%) acids--and had a
melting point of 42-48øC.The melting points
of the other syntheticwaxesusedin recoating
the bayberrieswere 51.4øCfor cetyl palmitate
and 62 to 65øC for beeswax.
Feedingtrials.--The averagemassof the Yellow-rumped Warblers used in our study was
10.9 + 0.2 g (n = 12), the mean hematocritwas
53.3 + 3.7 (n = 7), and the averagebody lipid
was 11.2 + 1.2%of wet weight (n = 6). No bird
died during the feeding trials. After 3 to 4 h,
each bird had defecated 1.4 + 0.5 (n = 12) seeds
of the 2.4 + 0.5 (n = 12) bayberriesfed. Yellowrumped Warblers separatethe seed and bracteoles (exocarp)in the gizzard'when fed bayberries. Partially-denuded bayberries were
found in the gizzard. The seed appearsto pre-
cedethe bracteolesduring gastrointestinal
transit in that bracteoles
were frequentlyfoundorad
to the seed. The bracteoles and seed accumulate
in the rectum and are defecatedtogether with
urates.Although neither Yellow-rumped Warbiers nor Tree Swallows regurgitated forcedfed fruits, all Yellow Warblers regurgitated the
fruits immediately upon being placed in the
The solvent was removed with nitrogen evaporation
metabolismchambers.We found no statistically
andthepurifiedtrietherdissolvedin absolutealcohol
significant(<5%) lipid absorptionor respired
to a specificactivity of lmCi/ml.
radioactive
CO2 of any wax with Yellow WarStatistical
analysis.--Results
are expressedasœ+- SE,
prepared by reduction with platinum as a catalyst
(New England Nuclear, Boston,Massachusetts).
Purified 3H-GTE (>98% radiopurity) was obtained by
chromatographyon a silicic-acidcolumnelutedwith
hexane/diethyl ether 85:15 (v/v; Roby et al. 1989).
biers.
with n being the number of birds. Comparisonsthat
In a separate feeding trial, three Yellowinvolve percentageswere performedon arcsin-transformeddata.We usedpaired t-testsor Mann-Whitney rumped Warblers were kept in captivity and
338
PLACE
ANDST•LES
[Auk,VoL 109
Fig. 2. Scanning-electronmicrographsof fruits from bayberries(Myrica pennsylvanica)
(A) with and (B)
withoutthe waxycoat.Fruit of bayberryis a smalldrupeenvelopedby persistingoutgrowthsof the exocarp
(bracteoles)
coveredwith a whitish waxy coating(Lawrence1951).White bar represents0.34nun.
given bayberry fruits with water ad libitumto
assess
the quantity of bayberriesthey couldin-
gestdaily. On average,eachbird ate 163 + 24
bayberriesper day.However,eachbird lost1.58
+ 0.28 g of body weight per day during the
bayberry feeding trials.
Tracer and marker recoveryand distribution.-
tabolizedC-14 label wasfound in adiposetissue
in the carcassas triglyceride (>80%), with the
pectoral muscle the next highest in label accumulation(5-8%). Only 2 to 4% of the label
was found
in the liver.
The fatty-alcohol moiety of the marine wax
ester was poorly assimilated and metabolized
Within 15 rain of ingestion,radiolabeledCO2 by Yellow-rumped Warblers, despite nearly
was collected when Yellow-rumped Warblers (> 95%)completehydrolysisof the wax esterin
werefed 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-acidmoiety in a
of ingestedtracer)aswell asthe highestassim- cetylpalmitate,wasabsorbedwith 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 inester).Lower ratesof lipid metabolismand as- testinallumen longer (Fig. 4). However, assimsimilation efficiencywere observedwhen birds ilation of the 16-carbon-lengthfatty alcohol,
were fed beeswaxor cetyl palmitatelabeledin hexadecanol(cetyl alcohol),was lessthan 40%.
Overall, we accounted for 96 + 8.2% of the inthe fatty-alcoholmoiety (Fig. 3 and Table 1).
The rate of label respirationreflectsthe over- gestedC-14 tracerin our feedingtrials.
all ratesof assimilation(i.e. hydrolysis,luminal
No statisticallysignificant differencesin exabsorption,repacking,transportfrom the en- creta3H-GTErecovery(F = 1.19,df = 3 and 11,
terocytesand oxidation)for eachwax.With bay- P-- 0.372)or in total3H-GTErecovery(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 •Hnonabsorbablelipid marker, •H-GTE) was pres- GTE was 104 + 6.3%(n = 12), and greaterthan
ent in the intestinal lumen after 180 rain (Fig. 98% of the recovered tritium label co-chromato4). The major portion (20-60%) of the nonme- graphedwith pure glycerol triether. Becauseit
April1992]
Bayberry
WaxandYellow-rumped
Warblers
timate
of 232 ,in
for the mean retention time
of the 3H-GTE marker. This estimate is based on
80
70
ß
90.1%
mmm
mmmm
40
ß
30
0 0ø
000
mmo
ø
20
00
10
-10
339
0
5•)
160
0
•
1•0
force-fed single meals and may overestimate
mean retention time in freely-feedingbirds.
The assimilation
efficiencies recorded for the
two Tree Swallows (Table 2) when feeding on
84'4ø'• recoatedbayberriesparallelsthoseobtainedfor
Yellow-rumpedWarblers.No statisticalanaly[]43.7øA
siswasperformedon thesedatabecauseof the
23.1ø•
260
25O
Time (.in)
Fig. 3. Representativetime coursesof respiredcarbon-14 labeled CO2in four Yellow-rumped Warblers
fed bayberriescoatedwith four different radiolabeled
waxes.Symbolsrepresentpercentof ingestedlabel
recoveredat eachtime point for the following waxes:
cetyl[1-•4C]palmitate(solidsquares),[1-14C]
cetylpalreitate (open squares),[1-14C]palmitate in bayberry
wax (open circles),and triacontanol[1-•4C]palmitate
in beeswax(open triangles).Assimilationefficiency
of each bird for the wax coating presentedto right
of each curve.
small samplesize.
Bileof Yellow-rumped
Warblers.--Thebile-salt
componentsin Yellow-rumpedWarbler bile
were predominantlytaurine conjugatesof the
primary bile saltscholateand chenodeoxycholate (Table 3). Relativelylarge amountsof taurine and glycineconjugates
of ursocholate
also
were
found.
Gall-bladder
bile-salt
concentra-
tion in individuals 4 h after feeding was 607 +
16.2 raM, as determined by HPLC and 598 +
75 raM, as determined by enzymaticanalysis.
The phospholipidcontent(~ 1.0 raM) was relatively low, while the neutrallipid content(~ 2
raM) was relatively high comparedto mammalian bile (phospholipid8.1 mM and a trace
of neutrallipid in sevenmammalianbiles;Coleman et al. 1979).
Luminal bile-salt concentrations and distributions
ofintestinal
markers
andlabels.--Theelevatedlevel
of bile salts in the gall bladder was reflected
in high levels of bile saltsin the proximal half
of the intestinallumen (Fig. 5A). After segment
8, the luminal levelsdroppedbelow our detection limit. Levels of bile saltsapproaching50
mM were found in the gizzard,nearly two segestimate 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,lessthan 2% of either
were collectedfrom 180 to 281 ,in postinges- label was found in eachsegmentof the intestion. If we use each trial as a separateobser- tinal lumen (Fig. 5B). Lessthan 0.1%of the 3Hvation in describingthe 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-
was impossible to recoat each berry with an
identical quantity of the 3H-GTE marker, the
percent recovery is based on the average
GTE found with 10 randomly selectedrecoated
bayberries.
Although we did not setout to measuremean
retention time directly in our feeding trials, an
T^i•i,E1. Assimilationefficienciesand marker recoveries(œ+ SD) of fed radiolabeledwaxesto Yellow-rumped
Warblers(n = 3). Matching lettersthat follow valuesrepresentsignificantdifferences(P < 0.05, multiple
range test).
Assimilation
Carrierlipid
Bayberrywax
Cetyl palmitate
Cetyl palmitate
Beeswax
Label
[1-•4C]palmitate
Cetyl [1-•4C]palmitate
[1-•4C]Cetyl palmitate
Triacontanol[1-•C] palmitate
efficiency
88.3 +
88.9 +
39.7 +
56.3 +
3.1"
2.5b
16.6a,b
21.9',•
Percentmarker
recovery in
excreta
63.2 +
44.8 +
47.3 +
32.3 +
26.7
12.3
11.8
27.5
Total percent
marker
recovery
93.8 +
98.2 +
98.6 +
116 +
9.1
9.1
9.1
10.8
340
PLACEANDSTILES
[]
[Auk, Vol. 109
TABLE3. Yellow-rumped Warbler gall-bladder bile
composition(• + SD, n = 6).
Beeswax
[] Cetyl palmirate
ß Bayberry
Concentration
Constituent
(mM)
Tauroursocholate
Taurocholate
20.3 + 6.66
181.5 + 19.3
Taurochenodeoxycholate
341.8 + 42.2
Taurolithocholate
5.1 + 0.87
Glycoursocholate
Phospholipid
58.5 + 0.7
1.18 + 0.65
Cholesterol+ triglyceride +
fatty acid
2.6 _+0.59
+ 0.3 •moles min -• mg-• while that for wax
esterlipolysiswas2.5 + 0.5 •moles min -1 mg-•.
Fig.4. Distributionof carbon-14radioactivetracer Hence,under thesein vitroassayconditionswax
in respiredCO•,excreta,and tissues.Datapresented esterswere hydrolyzed 8.3 + 2.3 times slower
for three Yellow-rumpedWarblersfed different re- than triglycerides.
coatedbayberries180 min postingestion.
DISCUSSION
Frugivoryof waxyfruits.--The associationof
CTM]
palmitateisolatedfrom the intestinal tissue
waslargelyincorporatedin triglyceride(> 80%), Yellow-rumped Warblerswith waxy fruits repalthough substantialfree fatty acid (>8%) was resentsone of the most specialized fruit/frualso 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),
cedrorum)
and miserably more unhydrolyzed wax estercould be CedarWaxwings(Bombycilla
extracted from the lumen. With birds fed [I-C •4] tletoe (P. serotinum)in the southeasternUnited
cetylpalmitate,significantlevelsof 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
food items in the diets of Northern
Flickers
acid and triglyceride were detected.
auratus
), Downy Woodpeckers(Picoides
Lipolysis
by Yellow-rumped
Warblers.--Extracts (Colaptes
and other woodpeckers,as well as
of pancreatictissuefrom Yellow-rumpedWar- pubescens),
blers were capableof hydrolysisof both tri- Tree Swallows and Yellow-rumped Warblers
glycerideand wax esteremulsionsin vitro.The (Martin et al. 1951), yet consumptionof large
specificactivity of triglyceridelipolysiswas 21 numbersof thesefruits by pen-raisedNorthern
Bobwhites(Colinusvirginianus)has been found
to interfere with digestionand may even prove
TABLE2. Assimilationefficiency(• + SD) of radiolabeled
waxes for two Tree Swallows.
Assimilation
Carrier lipid
Label
Cetyl alcohol
[1-•C] palmitate
Tri [I~I•C] palmitin
Cetyl [1-•C] palmirate
[1-•C] Cetyl palmirate
[1-a•C]Cetyl alcohol
Beeswax
Triacontanol [1-•C]
Bayberry wax
Tripalmitin
Cetyl palmitate
Cetyl palmitate
palmitate
efficiency
66.4 +
77.6 +
79.6 +
47.8 +
55.4 +
0.6
0.8
0.8
0.8
1.0
18.8 + 5.3
fatal (Martin et al. 1951).
The frugivorous diet of the Yellow-yumped
Warblers is not restrictedto Myrica. In the interior of the United States, where species of
Myrica are lesscommon,Yellow-rumpedWarbiers commonly eat poison-ivy fruits (Toxicodendronradicans;Ridgeway 1889, Graber and
Graber 1979, Graber et al. 1983), another solid
and waxy fruit. In fact, the waxy mesocarpof
Toxicodendron
consistsprimarily of glyceridesof
palmiticand oleicacids(Brizicky1963),similar
in compositionto the waxy pulp of Myricafruits.
April 1992]
Bayberry
WaxandYellow-rumped
Warblers
6O
A
5o
4O
341
(Vitis), Virginia creeper (Parthenocissus),
and
sumac (Rhus;Bent 1953, Griscom and Sprunt
1957).This warblerspeciesis secondonly to the
Yellow-rumped Warbler in the northern extent
30
of its wintering range (AOU 1983).The BaybreastedWarbler rarely is frugivorouswithin
20
the United States(Martin et al. 1951, Bent 1953),
but consumesfruits from 21 plant speciesin
Panama(Greenberg 1981). The Chestnut-sided
Warbler eats some fruits on the wintering
grounds in Central America (Howe and DeSteven 1979,Greenberg1979, 1981).Greenberg
(1981) found that a major fruit usedselectively
by Bay-breastedand Chestnut-sidedwarblers in
10
0'
Gizzard
I
2
3
4
5
6
7
8
9
10
0.4
Panama was from Lindackeffalaurina (Flacour0.3
0.2
0.1
0.0
Gizzard
I
2
3
4
5
6
7
8
9
ø'21
I 0
C
0.1
Yellow-rumped
Warblers
feedonbayberries
toeat
06
I
2
3
4
5
6
tiaceae),and he identified the aril as having "a
distinctlywaxy textureand odor." Skeate(1985)
found that 91% of fruit removed from Myrica
ceriferain hammock communities in central
Florida was taken by Yellow-rumpedWarblers
and that poisonivy was eaten only by Yellowrumped Warblers and woodpeckers.The Tree
Swallow is the only memberof North American
swallowsto eat fruit asa regularpart of the diet
(Bent 1942).Birdseating waxy fruits represent
an unusualassociation
of frugivoresasthey belong to families or subfamiliesof birds that are
generally consideredhighly insectivorous.
7
8
9
10
Gastrointestinal Segment
the wax.--The rapid and extensive release of
radiolabeledCO2for Yellow-rumpedWarblers
fed bayberriesrecoatedwith [1-C•4]palmitateis
strongevidencesupportinga metabolicfuel role
for the naturalwaxy coatingon bayberry.Me-
tabolism of dietary fat occurredin 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
agrees
closelywith prior studieson autumn and
(hatchedbars)in (B)luminalcontentsand (C) tissues
spring
Yellow-rumped Warblers (Yarborough
in a Yellow-rumped
Warbler'sgastrointestinal
tract4
and
Johnson,
1965).With an averagecoatingof
h after feedinga recoatedbayberry.Eachsegment
numberrepresents
1 cmof intestine,progressing
to- 4.5 to 7.3 mg of wax per fruit, we estimatethat
ward the cloaca.Averageintestinallengthin exper- a Yellow-rumped Warbler eating between 170
imentalbirdswas 10.9 + 0.27cm (n = 9). Intactbayberry seedsfound in segments3 and 5 for this
and 280bayberrieswould meeta 50 Kj/day daily energy expenditure.Our feeding trials inindividual. Means, with SE indicated.
dicatethat they are capableof eating over 160
fruitsper day.However,they lostnearly 1.5g
of body weight per day on a bayberrydiet, inOf the 21 membersof the genusDendroica dicatingthatfor ourcaptivebirdsthewaxycoatthat breed in the United States and Canada,
ing was not sufficientfor weight maintenance.
only the Yellow-rumped,Pine (D. pinus),Chest- The lossin bodyweight alsomaybe attributable
nut-sided(D. pennsylvanica),
and Bay-breasted to low nitrogencontentin the coatingor to fat(D. castanea)warblers have been identified as soluble toxins present in the wax (Levey and
includingfruit asa largepart of their diets.Pine Karasov 1989).
Warblersfeed on many fruits including poison
Determinants
of highlipiddigestive
efficiency
in
ivy, bayberry,dogwood,(Cornus),wild grape Yellow-rumped
Warblers.--Ingested
fat, especial-
342
PLACE
Ai•IIV
SI'II.ES
ly high-melting-pointfatty acids,mustbe emulsified or solubilized in the stomachprior to the
formationof miceliesand absorptionin the duOdehum. Potential
emulsifiers
that can function
[Auk,Vol. 109
We hypothesize that Yellow-rumped Warbiers alsomay exhibit a gastrointestinalreflux
basedon our measurements
of biliary products
found in the gizzard. Bile-salt concentrations
exceeding50 mM are recordedin the gizzard
alongwith lipolysisproductsof dietary lipids.
Direct radiographic observationswill be necessaryto substantiatethis hypothesis,sincethe
accumulationof biliary and intestinal contents
in the gizzard may have resulted during eu-
in the acidmilieu of the stomachincludepeptic
digestsof dietary protein, complexpolysaccharides and dietary phospholipids.In mammals,
someenzymatic hydrolysisof dietary triglyceridesoccursin the stomach,resultingin gastric
digestionof up to 30%of fats(Careyet al. 1983).
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(Robyet al. 1986,PlaceandRoby the solubilityof unconjugatedbile salts(Carey
1986, Place et al. 1989, Jackson and Place 1990, et al. 1983), it is important that these natural
Place1992).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,becauseof taurine conjugation,bile
fowl, as in mammals,gastricmotility is inhib- saltsdo not precipitatein the gizzard. Greater
ited by intraduodenalinjectionsof 0.1 N I-IC1, than 90% of the bile salts in Yellow-rumped
1,600 mOsM solutions of NaC1, corn oil, or amiWarbler bile are taurine conjugates(Table 3).
no acids, as well as by intraduodenal balloon Taurine bile-salt conjugatesremain solubleat a
inflation (Duke and Evanson 1972, Duke et al. pH below 1.0 (Carey et al. 1983).
Elevated bile-salt levels were observed also
1973). In addition to this inhibition of gastric
emptying, the enterogastricreflex in birds in- in the proximal lumen of the intestine.The critcludes the occurrence of one or more intestinal
refluxesduring the period of gastricmotility
ical micelie
concentration
of bile salts in the
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 moveintestinallumen 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 (Meleagris
gallopavo)
indicatethat miceilarsolubilizationof lipolyticproductsand,
antiperistalsisincludesthe duodenumand pos- potentially,establishinga chemicaldriving force
sibly the upper jejunum (Duke 1986).Intestinal for passiveuptake of dietary fatty acids.
refluxesoccurapproximatelythree times more
The fact that wax estersare hydrolyzedeight
often in Leach'sStorm-Petrels (Oceanodroma
leu- times more slowly than triglyceridesand pancorhoa)than in fowl (Duke et al. 1989), and increaticextractsof Yellow-rumpedWarblersmay
volve the movement of intestinal contents back
explain the slower rates of metabolismof triato the proventriculus.
contanol palmitate. However, the fact that the
Thus, gastricemptying in thesebirds is close- fatty-acidmoiety in cetyl palmitateis metaboly 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-rumpedWarther processingin the gizzard. In the process, biers.
duodenalproductslike monoglyceridesand fatOne trait observedin seabirds,which appears
ty acids are refluxed to the gizzard along with
not presentin the two passetineswe analyzed,
biliary (bile salts,phospholipids,and triglyc- is efficient utilization of long-chain fatty alcoerides) and pancreatic products (lipases). Gas- hols. Whereasdietary fatty alcoholsare effitric productionof lipid emulsifiersis not found ciently assimilated(> 90%;Placeand Roby 1986,
in birds; instead, products of normal intestinal Jacksonand Place1990,Place1992)by seabirds,
lipolysisare refluxedto a highly efficientemul- both Yellow-rumped Warblersand Tree Swalsificationmill, the gizzard.
lows were relatively poor (<50%) at assimilat-
April 1992]
Bayberry
WaxandYellow-rumped
Warblers
343
ing long-chainfatty alcohols.This inefficiency an apparent retrograde intestinal reflux to the
in fatty-alcoholassimilationcausesfew prob- gizzard. Gastrointestinalmean retention time
lems for Yellow-rumped Warblers and Tree may alsobe longer in Yellow-rumped Warblers
Swallows, sincethey rarely ingest large quan- than expectedfor other frugivorousbirds. Detities of fatty alcohol in their natural diet (un- spite thesetraits, Yellow-rumped Warblersin
like seabirds).However, the findingsclearlyin- captivityare not ableto maintainconstantbody
dicatethat the capacityto oxidize fatty alcohols weight eatinga bayberrydiet. Leveyand Karacanbe modulatedindependently of the capacity sov (1989) concludedthat few temperatebirds
to hydrolyze wax esters.
eatingnonwaxyfruits couldmaintainlong-term
Many nonwaxy fruits are characterizedby a nutrient and energybalanceon a diet of solely
pulp with a dilute solution of sugars,low con- fruits. Our results with Yellow-rumped Warcentrations of amino acids, and a substantial
biers eating the waxy fruits with higher energy
portion of undigestibleseedmass.Accordingly, density are consistentwith their conclusion.
frugivorousbirds feeding on nonwaxy fruits
Both Yellow-rumped Warblers and Tree
haveshortdigestiveretentiontimes(Leveyand Swallowsare facultativein their frugivory,eatKarasov1989, Karasovand Levey 1990). Waxy ing insectswhen available.The gastrointestinal
fruits are characterizedby a pulp of increased traits we have documented in Yellow-rumped
energydensity,becauseof a high lipid content Warblersshouldenhancelipid absorptionoverin the wax. However, a paradoxseemsto exist, all, whether the lipid is derived from insectsor
sincefrugivorousbirds 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 correlatedwith reten- suspectthey are constitutive,sinceboth spring
tion time (e.g. Jacksonand Place 1990). More- and autumn birds displayedsimilar absorption
over,the level of dietaryfat stronglyinfluences capacities.We also suspectthat these two spegastrointestinaltransit (Mateos and Sell 1981, cies,especiallyYellow-rumpedWarblers,may
Mateos et al. 1982). We predict that, for birds have an enhanceddetoxificationmachineryfor
feeding on waxy fruits, gastrointestinalreten- plant secondarycompounds.The bile is a major
tion timeswould be longer than for birds feed- secretoryroute for detoxifiedcompounds.Yeling on nonwaxy fruits. Also,we predictthat the low-rumped Warblersand Tree Swallowscan
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,Placeand Roby1986,Diamond
waxy fruits than in birds feeding on nonwaxy and Place 1988, lackson and Place 1990) that
fruits. While we did not measure mean retenexhibit a distinctive ability to assimilateothtion directly in the current study, the estimate
we obtain from the 3H-GTElipid phasemarker
indicatesthat mean retention time of bayberry
wax in Yellow-rumped Warblers may be fivefold longer than Cedar Waxwings eating wild
erwiserefractorylipidsand"live off the wax of
the land."
LITEKATURE CITED
grapes(Karasovand Levey 1990).We believe a
detailed
examination
of mean retention
times
in birds feeding on waxy dietsmay be a useful
direction
for future
studies.
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