267DIETARY FLEXIBILITY AND ENZYMATIC DIGESTIONRevista Chilena de Historia Natural78: 267-279, 2005

Feral pigeons in urban environments:dietary flexibility and enzymatic digestion?

Palomas domésticas en ambientes urbanos: ¿flexibilidad dietaria y digestión enzimática?

MARÍA EUGENIA CIMINARI1, 2, GRACIELA DEL VALLE MOYANO1, 2,JUAN GABRIEL CHEDIACK1, 2 & ENRIQUE CAVIEDES-VIDAL1, 2 *

1Área de Biología, Departamento de Bioquímica y Ciencias Biológicas, Facultad de Química, Bioquímica y Farmacia,Universidad Nacional de San Luis, CC 226, 5700, San Luis, Argentina

2Laboratorio de Biología “Prof. Enrique Caviedes Codelia”, Facultad de Ciencias Humanas,Universidad Nacional de San Luis, CONICET CC 226, 5700, San Luis, Argentina;

*e-mail for correspondence: enrique.caviedes@gmail.com

ABSTRACT

Columba livia, original from Europe, is at present widely distributed all over the world. These granivorescolonized urban environments where the availability of crops and seeds is not always permanent and, for thatare forced to exploit other resources with different composition, e.g. high protein foodstuff. Thus, feralpigeons should have the ability to survive on a diet rich in protein as they do with starchy items by having anadequate digestive biochemical machinery to process it. Phylogenetical and functional hypothesis has beenproposed linking dietary flexibility and enzyme lability. All Passeriformes studied to date show the expectedpositive correlation between aminopeptidase-N and dietary protein but not for intestinal carbohydrases.Conversely, all the non-passerine species modulate intestinal carbohydrases, but not peptidases. Moreover,different scenarios may be posed as the output of a phylogenetical effect, e.g., adding constraints to a labilityscheme in certain groups or just determining it (e.g., intestinal disaccharidases modulated in Galloanserae andpeptidases modulated in Passeriformes). Consequently, we tested the prediction that feral pigeons adjustdigestive enzyme activities according to the level of the respective substrate (e.g., carbohydrates, protein) inthe diet. Birds were fed for 15 days with two different diets, one with high protein content (low in starch)(HP) and the other rich in starch and low in proteins (HS). Pigeons fed on the HP were able to survive with noother dietary supplement, as predicted. Pancreatic enzymes did not change between diet treatments. Birds fedon HP exhibited the predicted upward modulation of aminopeptidase-N activity, when compared to birds onHS, while intestinal carbohydrases did not show differences between diets. These results give an apparentsupport to the functional hypothesis, but are not enough to reject that the observed intestinal protease labilityhas a phylogenetical component, because feral pigeons are closely related to Passeriformes. Finally, feralpigeons have the ability to take full advantage of the energy content from proteins, having enough pancreaticproteases activity and modulating the intestinal aminopeptidase-N. These abilities may allow them to useeffectively the nutrients found in the cities surviving even when grains are not available along the year.

Key words: feral pigeons, dietary flexibility, digestive plasticity, intestinal enzymes, pancreatic enzymes.

RESUMEN

La paloma doméstica, originaria de Europa, está distribuida alrededor del mundo. Estas aves granívorascolonizan zonas urbanas donde granos y semillas no están disponibles todo el año, por lo que deben explotarrecursos con una composición química diferente, (e.g., ricos en proteínas). Entonces, estas aves debieranposeer la capacidad de sobrevivir con una dieta proteica, utilizando una maquinaria bioquímica digestivaadecuada. Se han propuesto dos hipótesis, una funcional y otra filogenética, para explicar la flexibilidaddietaria y la labilidad de las enzimas intestinales en aves. Los Paseriformes estudiados al momento muestranuna correlación positiva entre N-aminopeptidasa y las proteínas de la dieta, aunque esto no es aparente paralas carbohidrasas intestinales. Por el contrario, todas las especies no paseriformes estudiadas modulan lascarbohidrasas intestinales, pero no las peptidasas. A su vez se pueden representar diferentes escenarios comoproducto de la filogenia, por ejemplo, agregando limitaciones a patrones de labilidad o directamentedeterminándolos (e.g., la modulación de las disacaridasas en Galloanserae y la modulación de las peptidasasen Paseriformes. Por esto, sometimos a prueba que las palomas ajustan la actividad de las enzimas digestivasde acuerdo al nivel de su respectivo sustrato (carbohidratos o proteínas) en la dieta. Dos grupos de palomasfueron alimentadas 15 días con dietas diferentes, una rica en proteínas y pobre en almidón (HP) y otra rica enalmidón y pobre en proteínas (HS). Las palomas alimentadas con HP sobrevivieron sin suplementos dietarios

268 CIMINARI ET AL.

adicionales. Las enzimas pancreáticas fueron iguales entre tratamientos. Las palomas alimentadas con HPexhibieron una actividad de N-aminopeptidasa mayor que la de HS, mientras que las carbohidrasas fueroniguales entre dietas. Estos resultados dan soporte a la hipótesis funcional, pero también son insuficientes pararechazar que la labilidad de la proteasa intestinal constituye un efecto filogenético, ya que estas avesconstituyen un grupo cercano a los Passeriformes. En síntesis, las palomas poseen la capacidad de utilizar lasproteínas debido a que poseen una actividad proteásica pancreática adecuada y modulan la N-aminopeptidasa.Estas características permiten que las palomas domésticas aprovechen efectivamente alimentos que ofrecenlas ciudades y sobrevivan aun cuando la disponibilidad de granos sea escasa.

Palabras clave: palomas domésticas, flexibilidad dietaria, plasticidad digestiva, enzimas intestinales, enzimaspancreáticas.

ecologically important for what it permits themdo and for how it constrains their feedingecology or other features of their biology(Caviedes-Vidal et al. 2000).

Therefore, our first goal in this study was tolearn if feral pigeons have the capacity tosurvive on a diet rich in protein as they do withstarchy items. To test this, we fed two groupsof birds with different diets: one with highcontent in starch and low in proteins, as itwould be when they feed on grains and seeds,and the other with high protein content and lowin starch, mimicking a diet rich on arthropodsand insects (see Bell 1990).

Chemical breakdown of the food by enzymesof the gastrointestinal tract is an essential traitthat relates to the capacity of vertebrates toextract nutrients from its food (Karasov & Hume1997). Enzymatic digestion capacity needs tomatch the diet switch by: (a) having a fixed highconstitutive level of the pancreatic and intestinalenzyme pool needed for to breakdown allfoodstuffs or, (b) changing the level of enzymesactivity in a positive relationship with thesubstrate that is being digested. Consequently,our second goal was to assess how pancreaticand intestinal enzymes of feral pigeons adequateto digest carbohydrates and proteins fed eitheron a high starch or a high protein diet. Toachieve this goal, we measured the activity ofsome pancreatic and intestinal enzymes. Weassayed amylase (EC 3.2.1.1), the mostimportant carbohydrase secreted by the pancreas(hydrolyses α-1,4 glucosidic bonds in largeoligosaccharides producing maltose and smalloligosaccharides) (Gray 1992). Thedisaccharides and oligosaccharides formed bythe amylase or directly ingested are hydrolysedto monosaccharides by enzymes located in thebrush border of intestinal cells. We determinedthe activity of two disaccharidases, maltase (EC3.2.1.20) and sucrase (EC 3.2.1.48). Maltase

INTRODUCTION

Columba livia, originally from Europe, is atpresent widely distributed all over the world.During the eighteenth and nineteenth centuries,feral pigeons were spread intentionally and/oraccidentally by travelers (Johnston & Janiga1995). These birds colonized urban environments(e.g., cities, towns, docks and others humandevelopments), although some of them settled inthe field. In certain city areas, they achieve highdensities, sometimes reaching levels that createdifficulties for urban life. Their successcolonizing metropolitan areas have beenexplained by lack or low predation and/orcompetence, availability of nesting places andfood in winter (Jokimaki & Suhonen 1998, Sol etal. 1998).

Feral pigeons are primary granivores, thuswhen grains or seeds (e.g., crop fields) areavailable around cities, they use themintensively by flying out of the cities during theday for foraging and coming back for sleeping(Johnston & Janiga 1995). In these areas, birdsmay reach very high densities. An interestingissue is that the quality of food and itsavailability in the field is not constant all yearround, so even having cultivated fields in thevicinities, seeds and grains availability mayvary seasonally. Consequently, feral pigeonsneed to ingest other food items. There areauthors that consider feral pigeons not as strictgranivores but as granivores/omnivores sincethey include in their diets small invertebrates,insects and high protein substances present inthe garbage (Jokimaki & Suhonen 1998,Lefebvre & Giraldeau 1984). Thus, these birdsshould have the ability to digest such foodstuffto use its built–in energy for maintenanceduring periods where most preferable fooditems (i.e., seeds, grains) are unavailable. Theextent of digestive flexibility in birds is

269DIETARY FLEXIBILITY AND ENZYMATIC DIGESTION

activity, resulting from the activity of twoenzymes, maltase-glucoamylase and sucrase-isomaltase is probably the single best estimatorof the ability to assimilate complex solublecarbohydrates (Noren et al. 1986). Sucrase-isomaltase is a relatively unspecific enzyme thathydrolyses sucrose, isomaltose and maltose(Hunziker et al. 1986). We also measuredtrypsin (EC 3.4.4.4) and chymotrypsin (EC3.4.4.5), since they are important pancreaticproteases. Trypsin hydrolyses peptide bonds thatare adjacent to a basic amino acid, whereaschymotrypsin hydrolyses bonds that are adjacentto an aromatic amino acid (Brannon 1990).Oligopeptides are then hydrolysed by a largevariety of peptidases in the brush-bordermembrane. We chose to measure the activity ofaminopeptidase-N (EC 3.4.11.2), also known asleucine-aminopeptidase and amino-oligopeptidase (Vonk & Western 1984), adipeptidase with broad specificity that appears toaccount for almost all peptidase activity in thebrush border membrane (Maroux et al. 1973).

Dietary modulation of digestive enzymes hasbeen studied in several vertebrates, includingbirds. Several patterns had been apparent frommodulation studies. Passerine species do notchange their disaccharidase activities in relationto dietary switch (Afik et al. 1995, Martínez delRío 1995, Sabat et al. 1998, Caviedes-Vidal etal. 2000) with the only exception of pinewarblers (Levey et al.1999). In contrast, allpasserine birds modulate intestinal peptidases(Afik et al. 1995, Martínez del Río 1995, Sabatet al. 1998, Levey et al. 1999, Caviedes-Vidal etal. 2000). Galliforms (i.e., chicks: Siddons 1972,Biviano 1993, Ciminari et al. unpublishedresults; turkeys: Sell et al. 1988) andAnseriforms (i.e., Canada and snow geese:Ciminari et al. 19991, Ciminari et al. 1998)2

adjust their disaccharidase activities matchingsubstrate load. Conversely, aminopeptidase-Nactivity did not show variation in chicken andgeese (Ciminari 1997, Ciminari et al.unpublished results) and we are not aware of any

study on turkey intestinal peptidases. In relationto the pancreatic enzymes, there are only a fewstudies on modulation of amylase and peptidasesin birds. Chicken modify amylase and peptidaseactivities following dietary changes (Imondi &Bird 1967, Hulan & Bird 1972). In passerines,no changes have been apparent (Caviedes-Vidal& Karasov. 19953, Ciminari et al. 2001) with theexception of pine warbler (Levey et al. 1999).

One interesting question is whether thedifferences in enzyme modulation observedreflect a dietary pattern, a phylogenetic pattern, orchance. Under the optimality theory and, usingeconomical arguments it has been predicted thatphenotypic plasticity would be favored by naturalselection (Sibly 1981). Thus, birds feeding ondifferent substrate on a temporal base wouldexhibit a link between enzyme activity and itsspecific substrate (Caviedes-Vidal et al 2000,Martínez del Río et al. 1995), i.e., omnivore birdsshould exhibit more plasticity than specialists.Though, none of the passerines show the expectedpositive correlation between intestinalcarbohydrases and dietary carbohydrate, they allshow the expected positive correlation betweenaminopeptidase-N and dietary protein. In contrastall the nonpasserine species studied to date showup the expected correlation for the intestinalcarbohydrases but not for peptidase (Caviedes-Vidal et al. 2000). Another determinant of thedigestive plasticity may be phylogeny. Differentscenarios may be posed as the output of aphylogenetical effect, e.g. adding constraints to alability scheme in certain groups or justdetermining it completely, e.g., intestinaldisaccharidases modulated in Galliformes andAnseriformes and peptidases modulated inPasseriformes. In this context, it has beenproposed an additional hypothesis linkingfunctionally digestive lability of the intestinalenzyme activity to phylogenetical features such asthe presence or absence of a functional cecum(Caviedes-Vidal et al. 2000). To date, all birdslacking cecum (e.g., Passeriformes) modulateintestinal peptidase and in contrast, cecaldigesters have a fix level of this enzyme, e.g.,Canada geese (Ciminari 1997), chicken (Ciminariet al. unpublished results), common quail(Ciminari et al. unpublished results). Within this

1 CIMINARI ME, C CODORNIÚ, E CAVIEDES-VIDAL,S MC WILLIAMS & WH KARASOV (1999) Dietarymodulation of intestinal carbohydrases in the Arctic geese(Chen caerulescens) of North America. Biocell 23: 11.2 CIMINARI ME, E CAVIEDES-VIDAL, S MCWILLIAMS & WH KARASOV (1998) Dietary modulationof the intestinal disaccharidases maltase and sucrase in theCanada geese (Branta canadensis). Biocell 22: 16.

3 CAVIEDES-VIDAL E & WH KARASOV (1995)Influences of diet composition on pancreatic enzymeactivities in house sparrows. American Zoologist 35: 78A.

270 CIMINARI ET AL.

framework, feral pigeons may be considered animportant inclusion and a good study subject forseveral reasons: their food habits (i.e., pigeonsfrom the urban sites are omnivores), morphologyof the digestive system (i.e., they have a vestigialcecum, Mc Lelland 1989) and phylogeny (i.e.,pigeons belong to the Order Columbiformes,Superorder Passerimorphae, closely related topasserines, Sibley & Ahlquist 1990).

Therefore, our predictions to test in thisstudy are that feral pigeons: (1) may live on ahigh protein diet as they do with a highcarbohydrate diet, (2) modulate peptidaseactivity in a positive correlation with dietaryprotein level, (3) do not modulate neitherintestinal carbohydrases nor pancreaticenzymes. Lastly, we consider that this study ofdigestive enzymes in feral pigeons is relevantsince (a) it may help us to understand theirsuccess in urban colonizing and, (b) it relates tothe problem of digestive lability in birds. Weare aware that by itself is not going to give adefinitive answer, especially in relation to thephylogenetical aspect, because this will requirea large multi-species comparison using thecomparative method (Harvey & Pagel 1991,Garland 1992) but it will contribute to broadenthe range of species explored.

MATERIAL AND METHODS

Animal care and housing

Sixteen adult feral Pigeons (Columba livia)were captured with a live trap near theUniversidad Nacional de San Luis Campus(San Luis, Argentina). The birds were housedindividually in cages (0.50 x 0.30 x 0.35 m)indoors under relatively constant environmentalconditions (25.2 ± 0.3 °C, relative humidity of50 ± 9 %) on a photoperiod of 14:10 h (light:dark) with ad libitum water and food (ParrilleroIniciador, Ganave, Alimentos Pilar S.A.).Animals were acclimated to laboratoryconditions for two weeks prior to use inexperiments. Animal care and trial protocolsfollowed Universidad Nacional de San Luis.

Diet acclimation

After the adjustment period, pigeons weredivided randomly in two groups and fed withdifferent semi-synthetic diets: a high protein

diet (HP) and a high starch diet (HS)(Caviedes-Vidal et al 2000). Food and waterwere offered ad libitum for 15 days. To test ourfirst goal we monitored the body mass andobserved carefully the behaviour and externalcondition of a subset of both dietary groups(four individuals of each group, HP and HS).

Sample collection

The birds were anesthetized using ethyl ether,the abdominal cavity was opened and the entiregastrointestinal tract was removed and chilledin ice-cold avian saline (Caviedes-Vidal &Karasov 1996). Stomach was removed, cleanedof extraneous tissue and weighed. Pancreas wascarefully excised, cleaned of extraneous tissue,divided longitudinally into two parts, placed intared cryovials, weighed and immediatelyfrozen in a -140 °C freezer. The content of thesmall intestine (SI) was removed, and the SIwas measured for length and weighed. Ten-cmlength segments of the proximal, medial andmost distal intestine (proximal = near to pyloricsphincter) were sectioned for the enzymeassays. Pieces were placed in tared cryovials,weighed and rapidly stored in a -140 °Cfreezer.

Sample preparation

Intestinal segments were thawed at 4 °C andhomogenized for 30 s using a Fisher Scientifichomogenizer (setting high) in 350 mM mannitolin 1 mM Hepes/KOH buffer (pH 7), using 10mL per g tissue. We measured activity ofmembrane-bound enzymes in whole tissuehomogenates rather than in mucosal samples orisolated brush border membrane preparations toavoid underestimation of activity as previouslyreported (Martínez del Río 1990). One part ofthe pancreas tissue was used for amylase assays.The tissue was thawed at 4 °C and homogenizedfor 30 s using a Fisher Scientific homogenizer(setting: high) in homogenizing buffer Trisbuffer (pH 8.2) containing 3 mM sodiumtaurocholate acid, 0.2 % (w/v) Triton X-100, 1mM benzamidine and 2 mM hydrocinnaminicacid) using 10 mL g-1 tissue. The other pancreastissue portion was treated identically than foramylase except that both protease inhibitors(hydrocinnaminic acid and benzamidine) wereomitted in the homogenizing buffer.

271DIETARY FLEXIBILITY AND ENZYMATIC DIGESTION

Pancreatic enzymes

Analysis of trypsin and chymotrypsin requiresprior activation of the zymogens. Trypsinogenwas activated to trypsin by using bovine intestineenterokinase (≥ 0.5 units per mg solid). Inpreliminary studies we observed that activationfor 30 min at 25 °C with 5 U mL-1 of enterokinasein 40 mM succinate buffer (pH 5.6) was sufficientto completely activate avian trypsinogen. After 30min, the activation was stopped by the addition of1.0 volume of 40 mM HCl containing 5 mMCaCl2 and placement on ice. Chymotrypsinogenwas converted to chymotrypsin in the presence ofbovine pancreatic TPCK trypsin (11,700 U mg-1).We observed that activation for 40 min at 25 °Cwith 15 µg mL-1 in 40 mM succinate buffer (pH5.6) was sufficient to completely activate avianchymotrypsinogen. Samples were centrifuged at20,000X g for 15 min at 4 °C. After 40 minactivation was stopped by addition of 1.0 volumeof 40 mM HCl containing 5 mM CaCl2 andplacement on ice.

Trypsin assay

We determined the activity of trypsin based onthe method of Erlanger et al. (1961). Aliquots of16 µL, from the homogenate appropriatelydiluted, were incubated with 800 µL of 2 mMDL-BAPNA (benzoyl-arginine-p-nitroanilide) atpH 9 during 10 min at 40 °C. The reaction wasterminated by adding 160 µL of 30 % aceticacid. Absorbance at 410 nm was read and usinga p-nitroanilide standard curve it was estimatedthe amount of p-nitroanilide liberated. Allabsorbances were read using a Spectronic 21D(Milton Roy, USA) spectrophotometer

Chymotrypsin assay

The activity of chymotrypsin was also measuredby the amount of p-nitroanilide released by thehydrolysis of GPNA (N-glutaryl-L-phenylalanine-p-nitroaniline) 1 mM solution at pH 7.4 and 40 °C.The reaction was terminated by adding 160 µL of30 % acetic acid and the absorbance was read at410 nm (Erlanger et al. 1966).

Amylase assay

The activity of amylase was measured by amodification of the 3,5-dinitrosalicylate

method (Dahlqvist 1962). Aliquots of 100 µLfrom the homogenate appropriately dilutedwere incubated with 2 % potato soluble starchACS reagent at 40 °C for 3 min. The reactionwas terminated by the addition of 1 mLdinitrosalicylate reagent. The tubes wereimmersed in boiling water during 10 min. Theabsorbance was read at 530 nm.

Disaccharidase assays

We determined the activity of thedisaccharidases maltase and sucrase in theintestinal homogenate. We used thecolorimetric method developed by Dahlqvist(1984) and modified by Martínez del Río(1990). Aliquots of 40 µL of t issuehomogenate, appropriately diluted, wereincubated with 40 µL of 56 mM sugar (maltoseand sucrose) solutions in 0.1 M maleate/NaOHpH 6.5. After 10 min incubation at 40 °C wearrested the reaction adding 1 mL of GlicemiaEnzimática reagent (Wiener LaboratoriosSAIC, Rosario, Argentina). Sample solutionswere allowed to stand for 20 min and then theabsorbances were measured at 505 nm al roomtemperature. Enzyme activity was determinedusing a glucose standard curve.

Aminopeptidase-N assay

We assayed aminopeptidase-N using L-alanine-p-nitroanilide as a substrate (Maroux et al1973). We started the reaction adding aliquotsof 10 µl of the tissue homogenate to 1 ml assaysolution, made of 2.0 mM L-alanine-p-nitroanilide in 0.2 M phosphate buffer(NaH2PO4/Na2HPO4, pH 7). The reaction wasincubated during 10 min at 40 °C and thenarrested with 3 ml of chilled 2 M acetic acid.The absorbance was measured at 384 nm, andactivity was determined using a p-nitroanilidestandard curve.

Protein measurement

The protein concentration of the samples wasestimated using the commercial Wiener LabProti 2 Assay Kit (EDTA/Cu Reagent, fromWiener Laboratorios SAIC, Rosario).Absorbances were read at 540 nm and theserum standard provided with the kit was usedas standard.

272 CIMINARI ET AL.

Standardization of enzyme activities and calcu-lation of summed hydrolysis activity

Total (summed) and standardized intestinalactivities were calculated for all enzymes.Activities are presented as total hydrolyticactivity (µmol min-1), activity per unitintestinal (or pancreas) wet mass (µmol min-1 gwet tissue-1), and activity per g of protein(µmol min-1 g protein-1). The advantages of ournormalization procedures are discussed byMartinez del Río (1990). We calculated thesummed hydrolysis activity of the pancreas, bymultiplying the activity per gram tissue by thepancreas mass, and the summed hydrolysisactivity of the entire small intestine bymultiplying activity per gram tissue in eachregion by 1/3 of the small intestine total mass,and summed over the three regions.

Effect of pH and kinetics: pancreatic enzymes

In order to compare maximal activities, weassessed the effect of pH on the activity ofamylase, trypsin and chymotrypsin in thepancreas. Thus, the selected pHs for measuringthe activities were the optimum for each enzyme.(chymotrypsin 7.4, trypsin 9 and amylase 5.5). Inaddition, we measured the substrate concentrationeffect on enzyme activities. Trypsin andchymotrypsin hydrolysis rates exhibited saturablekinetics that was adequately described by theMichaelis-Menten function. Consequently, all theenzyme activities were assayed at a substrateconcentration that provides Vmax.

Effect of pH and kinetics: intestinal enzymes

The chosen pHs for assaying the activities werethe optimum we measured for each enzyme (7for aminopeptidase-N, 6 for sucrase and 6.5 formaltase. These pH*optima are the same or almostfor most of other bird studies (Afik et al. 1995,Sabat et al. 1998, Caviedes-Vidal et al. 2000).

The apparent binding constants K*m and Vmax

were determined at a substrate concentrationvarying from 1 to 100 mM for disaccharidasesand from 1 to 20 mM for aminopeptidase-N.Aminopeptidase-N, sucrase and maltase exhibitedsaturable kinetics that was adequately describedby Michaelis-Menten functions. The coefficientsof correlation (r2) for the individual birds testedranged from 0.967 to 0.999. Diets did not affectthe values of K*m in aminopeptidase-N (F1,6 =

2.896; P = 0.140), sucrase (F1,6 = 1.078; P =0.338) and maltase (F1,6 = 0.0085; P = 0.929).Aminopeptidase-N K*m averaged 1.648 ± 0.219,sucrase K*m averaged 4.456 ± 0.297 and maltaseK*m averaged 6.128 ± 0.772.

Data analysis

Results are given as means ± 1 SE, n is number ofindividuals. Standard least-squares methods wereused to estimate parameters of linear regression.One-way analysis of variance (ANOVA) wasused to compare the pancreatic enzymes activitiesamong diets. Repeated measures analysis ofvariance (RM-ANOVA) was used to examine theeffect of diet and intestinal region on enzymeactivities. The significance level was set at P <0.05. Kinetic parameters were determined byfitting the kinetic data by non-linear curve fitting(gauss Newton routine, SYSTAT, Wilkinson1992) to the equation relative activity = (Vmax xconcentration)/(K*m + concentration).

RESULTS

Acclimation to the treatment diets

Birds averaged a 3.2 to 13.6 % decrease of theirinitial body mass after 15 days of trial under bothtreatment diets (high protein and high starch)(Table 1), although this change was not significantamong diets (F1,14 = 2.51, P = 0.135). We also didnot observe any variation of the behavior or theexternal appearance of the birds. This observationsuggests that, as we predicted, pigeons feed onfoodstuff made of high protein content and low incarbohydrates with no apparent harm.

Gut morphometrics

The mass and length of the small intestine andthe pancreas mass did not vary between the twodietary groups (Table 1) suggesting that noadequacy of the gut morphology undergoeswith the different diets.

Specific and summed activity

The three pancreatic enzymes assayed were nosignificantly affected by the treatment diets(amylase: F1,14 = 0.804, P = 0.385; chymotrypsin:F1,13 = 3.22, P = 0.096; trypsin: F1,12 = 1.115, P =

273DIETARY FLEXIBILITY AND ENZYMATIC DIGESTION

0.312; Fig. 1A, 1B, and 1C) on a tissue specificactivity base (per gram wet tissue), neither ifexpressed on a protein specific base (data notshown). As for the specific activity, summedhydrolysis activity of the whole pancreas did alsonot differ significantly between diets for amylase,(F1,14 = 0.923, P = 0.353) chymotrypsin (F1,13 =

0.135, P = 0.719) and trypsin (F1,13 = 0.562, P =0.467) (Fig. 1D, 1E, and 1F). Pancreas proteincontent did not vary between diets in the birds(F1,14 = 4.331, P = 0.056) and averaged 0.161 ±0.008 g protein per gram pancreas. Pancreaticprotein content was significantly correlated withpancreatic mass (F1,14 = 49.68, P < 0.001).

TABLE 1

Final body mass and SI and pancreas measures of feral pigeons fed on either a diet rich inprotein (HP) or rich in starch (HS)

Masa corporal final y medidas del intestino delgado y páncreas de las palomas domésticas alimentadas con una dieta ricaen proteínas (HP) y una rica en almidón (HS)

Diet n Final Mb (g) Change in Mb (g) SI mass (g) SI length (cm) Pancreas mass (g)

HP 8 311.44 ± 15.04 -10.1 ± 4.96 6.90 ± 0.48 64.29 ± 6.25 0.759 ± 0.055

HS 8 293.71 ± 16.23 -40.07 ± 18.2 6.81 ± 0.67 62.72 ± 6.60 0.634 ± 0.032

P-value 0.59 0.135 0.99 0.99 0.071

*Values are means ± SE (n = number of pigeons)

Fig. 1: Activity of specific and summed pancreatic enzymes in feral pigeons fed on two differentdiets. Bars are means ± 1 SE. (HP: High protein diet; HS: High starch diet).Actividad específica y total de las enzimas pancreáticas en palomas domésticas. Las barras representan las medias ± 1 ES.(HP: Dieta rica en proteínas; HS: Dieta rica en almidón).

Enz

yme

acti

vity

(µ

mol

g-1

min

-1)

100

80

60

40

20

0

80

60

40

20

0

Enz

yme

capa

city

(µ

mol

min

-1)

B Trypsin E Trypsin

Treatment diets

C Chymotrypsin

High Protein High Starch

Treatment diets

High Protein High Starch

80

60

40

20

0

0.6

0.4

0.2

0

A Amylase D Amylase

60

40

20

0

0.3

0.2

0.1

0.0

F Chymotrypsin

274 CIMINARI ET AL.

Intestinal activity: specific activity

Effect of diet. Our second prediction wassupported; aminopeptidase-N exhibited adietary effect in the predicted direction, birdsfed on high protein diet had significantly largeractivities of this enzyme when compared tobirds fed on high starch (Table 2, Fig. 2C).Multiple comparisons Tukey HSD test revealeda dietary effect on the proximal and medialaminopeptidase-N activities on birds fed onhigh protein diet (HP > HS, P < 0.05), while inthe distal portion of the small intestine nodifference was observed (HP = HS, P >0.05).Proximal and medial section activities of birdsfed on high protein diet were, respectively, 40and 88 % higher than those of birds fed on highstarch diet.

Our third prediction was sustained as well,no diet effect on the carbohydrases activitieswas apparent (Table 2, Fig. 2A and 2B).

Positional effect. All three enzymesexhibited a significant positional effect (Table2), where the proximal and medial activitieswere higher than the distal despite the diet(Tukey HSD, P< 0.05).

Protein content. The small intestine proteincontent did not differ significantly betweendiets in any of the intestinal regions (data notshown) and the averaged 0.233 ± 0.016 gprotein per g of small intestine. Intestinalprotein content was significantly correlatedwith intestinal mass (F1,9 = 19.97, P < 0.01).

Summed activity

Diet did not affect the summed hydrolysis ratesof maltase (F1,14 = 0.202, P = 0.659) andsucrase (F1,14 = 1.080, P = 0.316), nevertheless,pigeons fed on the high protein diet exhibited ahigher aminopeptidase-N summed activity(F1,14 = 4.585, P < 0.05) when compared tobirds fed on the HS diet (Fig. 2D, 2E, and 2F).

Relationship between maltase and sucrase

As a partial test for sucrase-independentmaltase-glucoamylase activity (Martínez delRío 1990), we regressed the summed activity ofmaltase against sucrase. Theoretically, theintercept of this relationship estimates theactivity of sucrase-independent maltase (i.e.,maltase-glucoamylases; Semenza & Auricchio

1989) and the slope estimates the contributionof sucrase-isomaltase to maltose hydrolysis. Inour birds, intestinal maltase activity wassignificantly correlated with intestinal sucraseactivity (F1,14 = 14.665, P = 0.002). Nodifference was apparent for the relationshipmaltase vs. sucrase activity relationshipsamong diets (F1,13 = 0.764, P = 0.398), thus weregressed all data together y = 243.52 (±110.52) + 10.11(± 2.64). The intercept waspositive and statistically different from zero (t= 2.202, P =0.045). The contribution of sucraseto maltase activity in the intestine wasestimated by multiplying the slope of themaltase vs. sucrase relationship by the summedsucrase activity (39.96 ± 3.24 µmol min-1).Sucrase, thus apparently accounted for 62.38 %of the maltase activity (647.49 ± 45.83 µmolmin-1). In other words there was evidence formaltase-glucoamylase activity independent ofsucrase that accounted for 37.62 % (100-62.38)of all maltase activity.

DISCUSSION

Acclimation to a diet rich in protein content

In this study wild captured feral pigeons werehabituated for 15 days to synthetic diets thatvaried in protein and carbohydrate contents. Intotal agreement with our first prediction, birdsfed on a high protein diet were able to survivewith no other dietary supplement, maintainingbody mass and exhibiting no apparentmalnutrition symptoms. Indeed, there was nodifference in body mass between both highprotein and high starch diet birds (see results).The inclusion by feral pigeons of other fooditems different from starchy substances (e.g.,seeds, grains), like high-protein foods (e.g.,arthropods, animal made up pieces), may playan important role in pigeons diet. Thus, pigeonsthat live in or around cities are opportunistic,exploiting available resources with differentchemical composition. This hypothesis maycontribute to explain the success of feralpigeons in urban sites. An interestingevolutionary question that emerges from thisstudy that begs for testing is to know ifcongener species of feral pigeons have thesame capacities to exploit food items rich inhigh protein and survive.

275DIETARY FLEXIBILITY AND ENZYMATIC DIGESTION

Fig. 2: Intestinal brush border enzyme activity in feral pigeons fed on two different diets. Valuesare coded according to the feral pigeon’s diet (black solid line and circle, HP: High protein diet;unfilled dashed line and triangles, HS: High starch diet). Statistical comparisons are in Table 2.Actividad de las enzimas intestinales en palomas domésticas. Los valores están codificados de acuerdo a la dieta de laspalomas (línea continua y círculo lleno, HP: Dieta rica en proteínas; línea cortada y triángulos, HS: Dieta rica en almidón).Las comparaciones estadísticas están en la Tabla 2.

TABLE 2

Statistics of a repeated measure ANOVA for intestinal enzyme activities under two dietarytreatments and intestinal position

Valores estadísticos de un ANOVA de mediciones repetidas de las actividades de enzimas intestinales de dos dietasdiferentes, y de distintos segmentos intestinales

Treatment Degrees of Enzyme activityfreedom

Maltase Sucrase Aminopeptidase-N

F-value P-value F-value P-value F-value P-value

Diet 1,11 1.168 0.303 1.219 0.293 9.482 0.010

Position 2,22 30.150 <0.001 33.274 <0.001 9.788 0.001

Diet x position 4,22 3.090 0.066 3.608 0.044 2.638 0.094

A Maltase

E Sucrase

A Maltase

F Aminopeptidase-N

Treatment dietsIntestinal section

HP HSP M D

800

600

400

200

0

180

150

120

90

60

30

0

60

40

20

0

15

12

9

6

3

0

10

8

6

4

2

0

6

4

2

0

Enz

yme

acti

vity

(µ

mol

g-1

min

-1)

Enz

yme

capa

city

(µ

mol

min

-1)

B Sucrase

C Aminopeptidase-N

276 CIMINARI ET AL.

Gut morphometrics

In our experiments, the small intestine massand lengths of the pigeons were not affected byhigh protein treatment when compared to birdsfed on the starch diet. Among birds, a non-homogeneous morphological response of thegut has been found when shifting to a highprotein diet. Afik et al. (1995) contrastedyellow-rumped warblers after a seven dayacclimation to either a fruit (high carbohydrate)or an insect (high protein) diet. These birdsexhibited small intestines with similar length,but different mass (fruit>insect); yet authorscaution about a possible methodologicalimprecision when measuring the mass. In a 20days-trial, European starlings (Martínez del Ríoet al. 1995) fed on rich in protein diet hadlonger small intestines and larger nominalintestinal surface areas but not heavierintestines compared to birds supplied with ahigh carbohydrate diet. In an oppositedirection, Levey et al. (1999) reported thatwarblers acclimated for 54 days on severalcomposite diets, had heavier but not longerintestines on a high carbohydrate diet (fruits)when compared to birds maintained on a highprotein diet (insects). In two different studieson house sparrows fed with the samesemisynthetic diets (high protein or highstarch) revealed a dissimilar output (Caviedes-Vidal & Karasov 1996, Caviedes-Vidal et al.2000). In the first study, birds were acclimatedto the diets for 3 weeks and had different smallintestine mass and length (high protein>highstarch), while in the second, birds acclimatedfor ten days did not exhibit any morphometrydifferences in their intestine. Zonotrichiacapensis and Diuca diuca exposed to either acarbohydrate diet or a carbohydrate free diet(protein replaced carbohydrate) for 30 daysshowed no differences of the intestine massamong diets (Sabat et al. 1998). Examining theavailable evidence there is not a clearrelationship between the size of the intestinesand the diet. Why a bird would change the sizeof the intestines? Adjustments of the sizes havebeen related to incremented feeding rates, dueeither to cold, low quality food (high fiber) orreproduction (reviewed by Karasov 1996).Mechanistically, an enlargement may bepredicted if reaction rates (hydrolysis orabsorption) and their plasticities are not enough

to match an increase of the whole digesta orfrom a specific substrate with no decrease ofthe digestive efficiency. A shortening of the gutsize may be related with the maintenance costof a structure with excessive unused sparecapacity. In our study case, feral pigeons didnot seem to need an increase of the gut sincethey showed adequate fixed carbohydraseenzyme activity and for protein processing theyexhibited enough plasticity of the tissue-specific aminopeptidase-N activity (see belowfor further discussion of the activities).

As for intestines, pancreas mass was notsignificantly correlated to diets (pancreas massdid not change, Table 1). The same results wereobtained in two passerine species (pinewarblers (Levey et al. 1999) acclimated during54 days and, yellow-rumped warblers (Ciminariet al. 2001) acclimated during 7 days, pancreasmass did not change between diets contrastingin protein content, despite the differences inacclimation periods.

Summarizing, the gut morphometry of feralpigeons is not affected when these birds exploithigh protein food items. In other words, thedigestive plasticity to process contrasting dietcomposition does not rely on morphologicaladjustments of the gut, which is apparent inseveral other avian species.

Pancreatic enzyme activities

Very few data of specific (normalized by tissuesize) or total pancreatic enzymes activity areyet available preventing us to performcomparisons at the interspecies orinterpopulation levels. In this view, it is importto emphasize that feral pigeons have detectablelevels of all three pancreatic enzymes. Specificamylase activity per gram pancreas is lowcompared with other species (Ciminari et al.2001). Specific chymotrypsin activity is aslightly lower than in yellow-rumped warblersand trypsin is about a twice higher than theactivity of yellow-rumped warblers (Ciminariet al. 2001). Summed chymotrypsin and trypsinhydrolysis activities of the whole pancreas inpigeons were also higher than in yellow-rumped warblers (Ciminari et al. 2001).Therefore, for one the first relevant steps ofenzyme digestion, feral pigeons have anadequate biochemical machinery to afford thedigestion of carbohydrates and proteins.

277DIETARY FLEXIBILITY AND ENZYMATIC DIGESTION

Intestinal enzymes activity

Our specific disaccharidase activity values arehigher than the values reported for housesparrows, yellow-rumped warblers and pinewarblers (Afik et al. 1995, Levey et al. 1999,Caviedes-Vidal et al. 2000) but not for chickens(156.24 ± 29.51 Ciminari et al. unpublishedresults), where disaccharidases are much higherthan in all species cited. Conversely, pigeon’saminopeptidase-N activity reached the levels ofhouse sparrows, yellow-rumped warblers, pinewarbler (Afik et al. 1995, Levey et al. 1999,Caviedes-Vidal et al. 2000), but not thehydrolysis rate of chickens which is more thandouble of the level of pigeons (10.31 ± 0.95Ciminari et al. unpublished results).

Effect of intestinal position on activity

Feral pigeons display an unusual spatial pattern ofaminopeptidase-N activity. The expression of theenzyme remains unchanged along the smallintestine in birds fed on the high starch diet, butfor those on high protein diet, the activity wassignificantly higher in the medial portion. This iscoincident with the finding that proline uptake ishigher in the medial portion, than in the proximaland distal portions, in pigeons (Obst andDiamond 1989). Other patterns found among birdspecies include a high aminopeptidase-N activityin the distal section compared to the proximal andmedial section of the intestine, such the case ofhouse sparrows (Caviedes-Vidal et al. 2000),coastal Cinclodes (Sabat & González 2003) andCanada geese (Ciminari 1997), while for pinewarblers (Levey et al. 1999), yellow-rumpedwarblers (Afik et al. 1995) and European starlings(Martinez del Río et al. 1995) the proteinbreakdown membrane-bound related activityremains unchanged or decreases along theproximal to the medial and distal portions.

Disaccharidase activities along the smallintestine of feral pigeons, followed a generalpattern demonstrated in most of the birds studiedup to now, a pronounced decline towards thedistal region (Afik et al. 1995, Ciminari et al.19984, Levey et al. 1999, Caviedes-Vidal et al.2000, Sabat & González 2003).

Pattern and magnitude of dietary modulation ofpancreatic and intestinal enzymes

In our study any pancreatic enzyme activitychanged following an increase in their respectivesubstrate. Among species, there is a considerablevariation of the modulation of pancreaticenzymes as a response for a dietary switch.Different results have been observed withinomnivorous wild birds. House sparrows(Caviedes-Vidal & Kasarov. 19955) and yellow-rumped warblers (Ciminari et al. 2001) did notmodulate any of the enzymes, while pinewarbler (Levey et al. 1999) modulated amylase,trypsin and chymotrypsin. On the other hand, inthe chicken, the levels of amylase and proteaseschange in proportion to dietary content of theirrespective substrates (Imondi & Bird 1967,Hulan & Bird 1972). The number of speciesreported is small, so is premature to suggest adietary modulation pattern of pancreaticenzymes in birds. However, is noticeable thatferal pigeons show the same pattern as mostpasserines species dietary modulation of theirpancreatic enzymes activity. Two of the threepancreatic enzymes, amylase and trypsin, haveelevated constitutive levels, thus we may statethat pigeons have the required (and more)biochemical machine to break down thecarbohydrates that reach the digestive tract.

Intestinal enzyme results supported thedirection of our predictions and conform apattern for all passerine species studied up todate, with the solely exception of pine warbler.Modulation of the hydrolytic and transportactivity of the intestine’s brush border mayinvolve changes in absorption and hydrolysisrates due to altered densities of transporters andhydrolases per unit intestinal mass/area(“specific modulation”) or, may involve changesin intestinal mass and/or area (“non-specificmodulation”, Karasov & Diamond 1983). Weonly found specific modulation in one of theintestinal enzyme activity studied,aminopeptidase-N; this enzyme reached higherlevels in birds fed on the high protein diet vs.those fed low protein diet (high starch diet),although the increment was restricted to themedial portion. Disaccharidases did not show

4 CIMINARI ME, E CAVIEDES-VIDAL, S MCWILLIAMS & WH KARASOV (1998) Dietary modulationof the intestinal disaccharidases maltase and sucrase in theCanada geese (Branta canadensis). Biocell 22: 16.

5 CAVIEDES-VIDAL E & WH KARASOV (1996)Influences of diet composition on pancreatic enzymeactivities in house sparrows. American Zoologist 35: 78A.

278 CIMINARI ET AL.

significant differential levels between birds fedon the two diets. As for non-specific modulation,we did not find it in our study, an obviousconsequence when no changes of the intestinalmass and/or length undergo along with the diettreatments.

Ecological and evolutionary implications ofdietary modulation

As for intestinal enzymes, feral pigeonsfollowed the pattern observed for Passeriformesand for birds having vestigial and non-functionalcaeca. Thus, on one hand, our results giveapparent support to the functional hypothesis(Caviedes-Vidal et al. 2000), which states that asmall intestinal escape of amino nitrogen aspeptides to the cecum can support microbialgrowth, contrary to the small intestine of thepasserines, which may have been selected toextract the maximum available amino nitrogen,rather than excreting it as waste. Additionally,these different ways to extract nitrogen from thediet may reveal differences in the ecologicalbehaviour of the animals, principally concerningto the food choices that they make, in aparticular habitat. Recent studies in chickens andquails (Ciminari et. al unpublished results) andin geese (Ciminari 1997), gives support to thisidea too. On the other hand, our findings are notenough to reject that the observed intestinalprotease dietary modulation has a phylogeneticcomponent because feral pigeons are closelyrelated to Passeriformes, in fact they belong tothe parvclass Passerae. Summarizing, the datapresented here increase our knowledge and helpclarifying the scenario for to have a definitiveanswer, which would require testing Passeraespecies having a functional caeca andGalloanserae species lacking cecum.

Pigeons living on a high protein diet

In short, this is the first study that establishes arelationship between the energy acquisitionfrom food in pigeons that live in urbanenvironments and thriving in them. Feralpigeons have the ability to take full advantageof the energy content from proteins, havingenough pancreatic proteases activity andmodulating the intestinal aminopeptidase-N.These abili t ies may allow them to useeffectively the nutrients found in the cities,

especially when environmental availability ofstarchy foods are scarce.

ACKNOWLEDGMENTS

This study is dedicated to the memory of ourbeloved Prof. Mario Rosenmann, as a modestrecognition of all the academic and humanknowledge we received from him. We thankCarlos Codorniú for his logistic support in thelaboratory. MEC and JGC are UNSL andCONICET fellowship holders respectively. Thework was supported by CyT–UNSL 22Q151and FONCYT PICT98 01-3101 grants to EC-V.

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Associate Editor: Francisco BozinovicReceived December 2, 2004; accepted March 1, 2005