Physiological and Ecological Adaptations to Feeding in Vertebrates

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Physiological and Ecological Adaptations to Feeding in Vertebrates

Editors

J. Matthias Starck Department of Biology 11, University of Munich (LMU), Germany

Tobias Wang Department of Zoophysiology University of Aarhus, Denmark

Science Publishers, Inc. Enfield (NH),USA

Plymouth, UK

SCIENCEPUBLISHERS, INC. Post Office Box 699 Enfield, New Hampshire 03748 United States of America Internet site :http://www.scipub.net

[email protected](marketing department) [email protected](editorial department) [email protected](for all other enquiries) Library of Congress-in-Publication Data Physiological and ecological adaptations to feeding in vertebrates / editors, J. Matthias Starck, Tobias Wang p. ; cm. Includes bibliographical references and index. ISBN 1-57808-246-3 1.Digestion. 2. Physiology, Comparative. 3. Adaptation (Biology). 4. Animal Feeding. I. Starck, J. Matthias, 1958-11 Wang, Tobias. [DNLM: 1. Adaptation, Physiological. 2. Vertebratesphysiology. 3. Digestive Physiology. 4. Digestive Systemanatomy & histology. 5. Feeding Behavior--physiology. QP 82P5783 20041 QP145.P46 2004 573.3'16--dc22

ISBN 1-57808-246-3

O 2005, Copyright Reserved All right reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying or otherwise, without the prior permission of the publisher. The request to produce certain material should include a statement of the purpose and extent of the reproduction. Published by Science Publishers Inc., Enfield, NH, USA Printed in India

All animals must eat to obtain energy for resting metabolism and to cover the energetic expenses associated with reproduction and behavioral activities. Utilization of the ingested meals requires digestion and subsequent absorption of nutrients. Given the extreme diversity in foods selection and food abundance between different environments and the large differences in metabolic needs among different animals, it is not surprising that feeding habits and strategies differ enormously among vertebrates. Some species which inhabit environments where food abundance is either scarce or fluctuates on a seasonal basis exhibit extraordinary adaptations to long-term fasting. Other species show extraordinary specialization for procuring, subduing and digesting particular foods items. Such morphological and physiological specializations have evolved in response to environmental conditions, but may be physiologically or functionally constrained. Apart from the need to acquire energy and absorb nutrients, the form and functionsof the gastrointestinal organs have been shaped to reject a plethora of antigens, bacteria and viruses that attempt to invade the body through this open barrier. In recent years interest in the gastrointestinal tract has come in to focus for studies on physiological and evolutionary adaptations to the environment. Physiological ecology and functional ecological morphology have increasingly recognized the model character of the gastrointestinaltract for studies in physiological and ecological adaptation to fluctuating environmental conditions. Apart from studies seeking to understand the basic biological questions of how animals interact with their environment, a number of comparative studies have attracted model organisms to investigate particular physiological mechanisms shared among all animals. Given the recent surge in interest pertaining to the physiology, morphology and function of the gastrointestinal organs and the process of digestion, it seemed timely to us to summarize the current state of the knowledge taking an integrative view. We intended to present a perspective that focuses on the gastrointestinaltract from an integrative ecological and evolutionary perspective, which is based on a physiological foundation where basic mechanisms are understood and quantified. We have brought together experts from a variety of specialized fields from comparative morphology through ecological and molecular physiology, immunology and ecology.All are involved in studies of different aspects of the gastrointestinal tract, but

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Physiological and ecological adaptations t o feeding in vertebrates

each applies different techniques and takes a different intellectual and experimental approach to understanding the gastrointestinalfunction. We are fully aware that this book cannot cover all aspects of physiological and ecological adaptations to feeding in vertebrates, but we hope it stimulates discussion and future interest in the question of how organisms adjust their form and function to fluctuating external conditions. The book opens with a comparative morphological chapter that outlines the evolution of the feeding apparatus in vertebrates. The following three chapters provide reviews of concepts of digestive efficiency, absorption models, the role of adaptation and constraints in the evolution of the gastrointestinal tract. The remainder of the book comprise a selection of chapters by experts delving into ecological questions to nutrient absorption in different taxa of vertebrates. Although the book could not cover all topics relevant for the adaptation of the gastrointestinaltract, we think it provides an overview of our present state of knowledge. Also, each chapter provides a perspective paragraph that will hopefully stimulate future research. The book was compiled as a state of the art document and is addressed to all those seriously interested in physiological and ecological adaptations of the gastrointestinal system of vertebrates. This includes graduate student as well as professionals from such fields as animal science, vertebrate biology, veterinary science, animal nutrition and medical gastroenterology. On behalf of all authors we would like to thank Paul Andrews, Victor Apanius, Michael V. Bell, Walter Bock, Colin Brauner, Dominique Burrau, Ian Gibbins, Kirrtberly Hammond, M.M. Hemphiers, Susanne Holrngren, Ian Hume, William Karasov, Marek Konarzewski, Mads Lomholt, Carlos Martinez del Rio, Adam Summers, Scott McWilliams, Mike Rust for reviewing individual chapters of the book. The reviewers provided valuable comments and advise for the individual chapters. Munich, J. Matthias Starck June 2004

Aarhus, Tobias Wang

LIST OF CONTRIBUTORS

Augusto S. Abe Departamento de Zoologia, c.p. 199, Universidade Estadual Paulista, 13506-900, Rio Claro, SP, Brasil. JohnnieB. Andersen Department of Zoophysiology, University of Aarhus, Universitetsparken, Aarhus, Denmark. Denis V. Andrade Departamento de Zoologia, c. p. 199, Universidade Estadual Paulista, 13506900, Rio Claro, SP, Brasil. Phil F. Battley Department of Mathematics and Statistics, University of Otayo, Dunedin, Nezu Zealand Hannah V. Carey Department of Comparative Biosciences, University of Winconsin, School of Veterinary Medicine, 2015, Linden Dr. Madison, W I 53076, U S A . Luis E.C. Conceiqiio CCMAR- Centro de Clencias do Mar, Universidade do Algarve, Campus de Gambela, P-8000-117 Faro, Portugal. Ariovaldo P. Cruz-Neto Departamento de Zoologia, c. p. 199, Universidade Estadual Paulista, 13506900, Rio Claro, SP, Brasil. JamesW. Hicks Department of Ecology and Evolutionary Biology, University of California, Irvine, Irvine, C A . Anna Holmberg Department of Zoophysiology, University of Goteborg, Box 463, SE 405 30 Goteborg, Sweden. Susanne Holmgren Department of Zoophysiology, University of Goteborg, Box 463, SE 405 30 Goteborg, Sweden. Ian Hume University of Sydney, Biological Sciences A08, NS W 2006, Australia.

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Physiological and ecological adaptations t o feeding in vertebrates

William H. Karasov Department of Wildlife Ecology, 226 Russell Labs, 1630 Linden Drive, University of Wisconsin, Madison, W I 53706, U S A . Kirk Klasing Department of Animal Science, University of Calijornia, Davis, C A 9561 6 , U S A . Carlos Martinez del Rio Department of Zoology and Physiology, University of Wyoming, Laramie, W Y 82071 -31 66, U S A . David J. McKenzie Centre de Recherche sur les ~ c o s ~ s t 2 mMarins es et Aquacoles de L'Houmeau, UMR 10 CNXS-lfvemer, Place du Stminaire, B.P 5,171 37 L'Houmeau, France. Todd J. McWhorter Department of Wildlife Ecology, 226 Russell Labs, 1630 Linden Drive, University of Wisconsin, Madison, W I 53706, U S A . Scott R. McWilliams Department of Natural Resourses, 116 Coastal Institute in Kingston, University of Rhode Island, Kingston, RI 02881, U S A . Theunis Piersma Department of Animal Ecology, Centrefor Ecological and Evolutionary Studies, University of Groningen, PO Box 14,9750 AA Haren, The Netherlands, and Department of Marine Ecology, Royal Netherlands Institute for Sea Research (NIOZ),PO Box 59,1790 A B Den Burg, Texel, The Netherlands Ivar Rsnnestad Department of Zoology, University of Bergen, Allegatan 41, N-5007 Bergen, Norway. Margaret Rubega Department of Ecology and Evolutionary Biology, University of Connecticut, S ~ O W CT S , 06269-3043, U S A . Kurt Schwenk Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT 06269-3043, U S A . J. Matthias Starck Department of Biology 11, University of Munich (LMU),Grophaderner Str. 2, D-82152, Planegg-Martinsried, Germany. Tobias Wang Department of Zoophysiology, Building 131, University of Aarhus, Universitetsparken,Aarhus, Denmark. Blair 0.Wolf Biology Department, 167 Castetter Hall, The University of New Mexico, Albuquerque, N M 87131-1 091, U S A .

CONTENTS

Preface List of Contributors 1

The Diversity of Vertebrate Feeding Systems Kurt Schwenk and Margaret Rubega

2

Concepts of Digestive Efficiency Ian D. Hume

3

Carbohydrate Hydrolysis and Absorption: Lessons from Modeling Digestive Function Todd J. Mc Whortev

4

Digestive Constraints in Mammalian and Avian Ecology William H. Karasov and Scott R. Mc Williams

5

Paracellular Intestinal Absorption of Carbohydrates in Mammals and Birds Todd J.Mc Whovtev

6

Mass Balance Models for Animal Isotopic Ecology Carlos Martinez del Rio and Blair 0. Wolf

7

Structural Flexibility of the Digestive System of Tetrapods -Patterns and Processes at the Cellular and Tissue Level J. Matthias Starck

8

Adaptive Interplay between Feeding Ecology and Features of the Digestive Tract in Birds Phil F. Butt ley and Theunis Piersma

9

Gastrointestinal Responses to Fasting in Mammals Lessons from Hibernators Hannah V Carey

229

10

Interplay between Diet, Microbes, and Immune Defenses of the Gastrointestinal Tract Kirk Klasing

255

11

Effects of Digestion on the Respiratory and Cardiovascular Physiology of Amphibians and Reptiles Tobias Wang,Johnnie B. Andersen and James W . Hicks

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Physiological and ecological adaptations t o feeding i n vertebrates

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12

Specific Dynamic Action in Ectothermic Vertebrates: A General Review on the Determinants of Post-Prandial Metabolic Response in Fishes, Amphibians, and Reptiles Denis I? Andrade, Ariovaldo I? Cruz-Neto,Augusto S. Abe, and Tobias Wang

13

Control of Gut Motility and Secretion in Fasting and Fed Non-Mammalian Vertebrates Susanne Holmgren and Anna Holmberg

14

Effects of Dietary Fatty Acids on the Physiology of Environmental Adaptation in Fish. David J. McKenzie

15

Aspects of Protein and Amino Acids Digestion and Utilization by Marine Fish Larvae Ivar Rsnnestad and Luis E.C. Concei@io

Index

Diversity of Vertebrate Feeding Systems Kurt Schwenk* and Margaret Rubega Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT, USA

SYNOPSIS The vertebrate gut tube can be divided into "front-end and "back-end" components according to topology, function, and research tradition. The purpose of front-end feeding systems is to acquire food to be delivered to the back-end for chemical digestion and assimilation. In accomplishing this, the feeding system faces as many as five separate mechanical tasks recognized as "feeding stages:" capturelsubjugation, ingestion, transport, processing, and swallowing. In general, aquatic species exploit the high density of water to manipulate food items by modulating water flow through the mouth and pharynx. In contrast, terrestrial vertebrates typically employ some form of hyolingual feeding in which movements of the tongue and hyobranchial apparatus take the place of water flow in capturirrg, supporting and manipulatingfood. The condition of the bolus, when swallowed, varies markedly among taxa. Most ectotherms process their food little, if at all, whereas mammals and birds typically reduce their food to small particles (in mammals, by oral mastication; in birds, within the "gastric mill" of the gizzard). This fundamental difference probably relates to the need of endotherms to increase gut passage rates.

The study of vertebrate feeding proceeded historically along two lines. On the one hand, functional morphologists examined the myriad mechanisms by which vertebrates procure, process and swallow food. On the other, physiologists studied the structure and function of the gut during digestion and absorption of the food swallowed. These distinct fields of endeavor reflect not only different topological foci ("front end" vs "back end" of the gut tube),

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Physiological and ecological adaptations t o feeding in vertebrates

but a long-standing division of the life sciences into morphology and physiology (Owen, 1866; Gegenbauer, 1878; Woodger, 1929; Russell, 1916; see Schwenk, 2000d for discussion). Although the modern separation of physiological and morphological approaches in the study of vertebrate feeding is justified to some extent by differences in research methodology, their continued isolation seems to be more a manifestation of historical inertia than of biological reality. It is clear that form and function of the gut's front end and back end are inseparately related and that evolutionary changes in one are likely to have consequences for the other. This statement is so obvious as to be nearly superfluous. Nonetheless, it is worth emphasizing that the traditional partitioning of the field obscures the reality of feeding system evolution. Feeding function Sensu Lato requires the functional and anatomical integration of many separate components. The evolution of such integrated systems poses a major challenge in current evolutionary theory and is poorly understood in the context of traditional atomistic or reductionist approaches (Wagner and Schwenk, 2000; Schwenk, 2001b; Schwenk and Wagner, 2001).While some kind of rapprochement is necessary in the study of vertebrate feeding systems is widely acknowledged-indeed, it is a theme central to this volume-a truly synthetic understanding of the evolution of the feeding system still eludes us. Given the functional and evolutionary integration that necessarily exists between front and back ends of the gut, it is not surprising that many common factors can be found to have influenced their phenotypes-ectothermy vs endothermy, and diet. Nonetheless, it is apparent that common environmental "problems" (e.g.herbivory) have often resulted in clade-specificsolutions and that similarities among taxa in one end of the system (e.g. longer relative gut length)are not always paralleled in the other (e.g.different mechanisms of reduction). Thus, integration does not imply that front and back ends must always evolve in lock step-only that evolutionary changes in one are likely to have consequencesfor the other. Indeed, the extent to which feeding mechanisms and gut physiology are deterministically coupled remains an open and important question. In this chapter we provide an overview of the mechanical tasks faced by vertebrates when they feed and consider some of the diverse ways these tasks are executed in different taxa. The feeding systems of larvae are not considered in this review due to limited space and the fact that many larval forms are exceptionally small, particularly in fishes, and operate within the different physical paradigm of low Reynolds numbers where viscous forces predominate. Clearly the question of how such organisms feed and metamorphose into a high Reynolds number world is of great interest, but one we cannot treat here. Readers are referred to reviews provided by Sanderson and Kupferberg (1999)and Wassersug and Yamashita (2001).

Vertebrate feeding systems

3

MORPHOLOGY OF THE FEEDING APPARATUS A complete account of feeding system morphology is beyond the scope of this chapter but an introduction to relevant structures and terminology will help to clarify the following sections. Overviews of trophic morphology in fishes can be found in Lauder (1985a), Vandewalle et al. (1994), and Motta and Wilga (2001), and for tetrapods in Bramble and Wake (1985), H"iiemae and Crompton (1985),Schwenk (2000a), and in the taxon-specific chapters within Schwenk (2000b).General references that include excellent sections on feeding structure include Liem et al. (2001)and Hildebrand and Goslow (2001).As discussed, a traditional consideration of feeding form and function begins at the head and ends at the esophagus, so we confine our discussion to front-end components of the feeding system. The anterior end of the gut tube in deuterostome embryos opens through the stomadeurn to form the mouth. Between the opening of the mouth and the entrance to the esophagus lies a cavity somewhat arbitrarily divided into an anterior buccal cavity and a posterior pharynx. A hallmark of vertebrate evolution was the origin of a series of U-shaped skeletal arches supporting the pharynx. These so-called visceral arches form from a novel embryonic tissue, the neural crest (e.g. Thorogood, 1993).Ancestrally the pharyngeal skeleton formed a kind of "basket" that functioned as a filter to trap suspended food particles brought into the mouth by ciliary currents. The evolution of joints and associated branchiomeric musculature led to flexion of the skeletal elements and active pumping of water for feeding and respiratory function (Mallatt, 1996).This system is retained more or less unchanged in larval lampreys. The origin of jaws from an anterior visceral arch was probably associated with increasingly active and predaceous behavior (Northcutt and Gans, 1983)and a transition from suspension feeding to prehension of individual food particles (Mallatt, 1984).Arguably, jaws were a key innovation in the vertebrate lineage, leading to an explosion of gnathostome (jawed vertebrate) diversity and ultimately the demise of most jawless clades. We can reasonably infer that a great deal of this diversity was engendered by the trophic flexibility of jaw-based feeding systems, which permitted the invasion of new adaptive zones. Thus the jaws and their associated teeth and musculature, were established early in vertebrate evolution as the central elements of vertebrate feeding system evolution. They are the focus of most feeding studies. Vertebrate jaws are complex structures with multiple evolutionary and developmental sources. They develop from cartilages of the first visceral arch, i.e. the palatoquadrate in the upper jaw and the mandibular (or Meckel's) cartilage in the lower jaw. In most adult vertebrates the jaws are primarily composed of dermal (membrane)bones that invest the cartilages during later development.A few parts ossify directly as endochondral bones, notably at

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Physiological and ecological adaptations t o feeding i n vertebrates

the jaw joint, to form an upper quadrate bone and a lower articular, but the cartilages usually atrophy and in many species disappear. In basal bony fishes and tetrapods, the upper jaw fuses to other dermal bones of the facial skeleton,but retains independence, or at least limited mobility, in many taxa, especially fishes (e.g. Lauder, 1985a; Motta and Wilga, 2001). Indeed, the ability to protrude the jaws is an essential component of feeding in many fishes, particularly in suction feeders (Fig. 1.1) (Lauder, 1985a; Westneat, 1990; Motta and Wilga, 2001; Wilga et al., 2001). In tetrapods, the jaws are never protrusible; however, several lineages, notably birds and squamate reptiles, have evolved kinetic joints in the dermal skull so that the upper jaws and other skull elements can flex relative to the braincase (Beecher, 1962; Frazzetta, 1962;Bock, 1964; Zusi, 1993;Herrel et al., 1999;Hoese and Westneat, 1996; Arnold, 1998; Bout and Zweers, 2001; Metzger, 2002). Such "cranial kinesis" is most highly developed in advanced (macrostomatan)snakes, in which upper and lower jaw and palatal bones are independently and unilaterally mobile (Gans, 1961; Cundall and Greene, 2000). This increases gape and generates ratchet movements of the toothed elements, one side at a time, to pull the snake's head and body over a prey item. Mammals are distinguished by an akinetic and generally robust skull (Davis, 1961).The endochondral jaw joint bones of other vertebrates have been miniaturized and displaced to the middle ear in mammals, where they contribute to the auditory apparatus (Allin, 1975; Novacek, 1993; Rowe, 1996).A new jaw joint has evolved between two dermal bones, the dentary of the lower jaw and squamosal of the upper (thelatter element is usually fused with others to form the temporal bone).A diagnostic feature of living mammals and closely related fossil taxa is the presence of only a single bone, the dentary in the lower jaw. The dentary is, itself, a developmental composite, comprising the fusion of six separate Anlage (Atchley, 1993). The dentary bones of each side are joined anteriorly by a fibrous symphysis to form the mandible.The strength of the symphysis and the extent to which it transmits forces from one half of the mandible to the other varies among species (e.g. Beecher, 1979;Lieberman and Crompton, 2000). Teeth evolved in association with jaws. They are composed primarily of dentine and enamel, ancient hard tissues that invested the armor plates of ancestral jawless fishes (Butler and Joysey, 1978; Reif, 1982). Primitively, teeth were found throughout the buccal cavity and pharynx on various elements of the palate and pharyngeal skeleton, but palatal and pharyngeal teeth are often lost, especially in tetrapods.The marginal teeth of the jaws are restricted to the dentary of the lower jaw, and the maxilla and premaxilla of the upper jaw in bony vertebrates. In mammals and crocodilians, the teeth are rooted in deep sockets within the bone, but in most vertebrates they are cemented to the apical or medial jaw surfaces. The exposed, or crown portion of the tooth varies extensively in form, even among closely related species in some cases, variation that may be functionally related to

Vertebrate feeding systems

s IM

IHG

IHG

Fig. 1.1. Feeding in aquatic vertebrates usually involves manipulation of food particles indirectly through the modulation of water flow (left), whereas in tetrapods, the tongue and hyobranchial apparatus take over this role (right). Suction feeding in bony fishes (left) results from an explosive expansion of the mouth and pharynx caused by protrusion of the jaws, hyoid retraction, and opercular abduction. The negative pressure generated within the mouth causes an inrush of water that drags prey in. Tetrapods often capture food with the tongue which also supports and manipulates it within the mouth (right). Cyclical movements of the hyolingual apparatus transport the food item back to the pharynx for swallowing. ASHG: anterior suprahyoid muscle group; Bh: basihyal element of hyobranchium; Cb: ceratobranchial element of hyobranchium; DM: depressor mandibulae muscle; EAC: external adductor musles; ECC: epaxial cervical muscles; IAC: internal adductor muscles; IHG: infrahyoid muscle group; IM: intermandibularis muscle. Left side figures from Karel F. Liem (1979). Reprinted by permission of WileyLiss, Inc., a subsidiary of John Wiley and Sons, Inc. Right side figures from Bramble and Wake (1985), reprinted by permission of the publisher and President and Fellows of Harvard College.

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Physiological and ecological adaptations to feeding in vertebrates

the types of food eaten and the manner in which it is procured and processed in the mouth (Fig. 1.2).This is especially true for mammals because they masticate their food (see later).The pharyngeal teeth of some derived teleost fishes show similar, diet-based adaptations in crown form (e.g. Liem, 1973; Sibbing, 1991).Tooth reduction or loss is relatively commonplace among many clades. Turtles and birds have lost their teeth altogether and replaced them with a horny (keratinous)investment of the jaws called a beak or bill.

MORTAR-PESTLE

SHEARING BLADES

w 0

.

0 .

,

m SERIAL ARRAYS -LOW PROFILE BLADES

Fig. 1.2. Crown form in mammalian teeth is closely tied to the nature of the food eaten. Functional specialization of teeth is part of a suite of derived mammalian traits associated with the evolution of mastication. Mastication results in comminution of food, i.e. its reduction to a slurry of fine particles mixed with saliva. From Hiiemae (2000), reproduced with permission of Elsevier Science.

Vertebrate feeding systems

7

In most vertebrates, lower jaw motion is mostly limited to dorsoventral movements. Jaw closing is effected by adductor mandibulae musculature that is variously subdivided in different taxa. Adductor muscles run from the cranium to the lower jaw and are innervated by the trigerninal nerve (cranial nerve V). In most fishes, jaw opening is caused by the action of ventromedial hypobranchial muscles that run anteriorly from the pectoral girdle to the lower jaw (Wilga et al., 2000). These are innervated by spinooccipital nerves and/or the hypoglossal (c.n. XII). In nonmammalian tetrapods, a depressor mandibulae takes over jaw opening. It runs from the back of the cranium and neck to the retroarticular process of the mandible, depressing it by pulling up behind the jaw joint. Since this muscle is developmentally and evolutionarily derived from the superficial constrictor musculature of the throat, it is innervated by the facial nerve (c. n. VII). Jaw opening is often accompanied by elevation of the cranium caused by contraction of the epaxial neck musculature. Mammals use a novel muscle, the digastric, to depress the lower jaw. Its name derives from the fact that in many species it comprises two distinct bellies separated by a short tendon, each innervated by a different nerve (c.n.V and VII).It runs from the paroccipital process of the skull base to the anterior end of the mandible. During the evolution of mastication (see below) mandibular movements in most mammals became complex. They are mostly dorsoventral in carnivorous species but in herbivores include dramatic mediolateral and/or anteroposterior movements. The jaw joint is variously modified to accommodate such mobility. Mastication in mammals is associated with the evolution of a novel adductor muscle, the masseter, running from the zygomatic arch (cheek bone) to the lateral surface of the mandible. The masseter adds lateral and anterior components to jaw movement that are balanced by the dorsal and medial components of pterygoideus and temporalis adductors. The pharyngeal skeleton is an essential part of the feeding apparatus in both fishes and tetrapods. Ancestrally, there were seven visceral arches constituting the pharyngeal skeleton (splanchnocranium) of gnathostomes. Each arch is composed of several jointed elements joined in the ventral midline. The first, most anterior arch is the mandibular, comprising upper and lower jaws. The second is the hyoid arch and the remaining five the branchial (or gill) arches. The upper part of the hyoid arch in fishes (hyomandibula)runs from the jaw joint to the neurocranium and variously supports, braces or suspends the jaws. It is homologous to the columella (stapes),a middle ear ossicle, in tetrapods. The lower part (ceratohyal)is usually highly mobile. When at rest, the paired ceratohyals lie within the arc of the lower jaw, but when pulled back by hypobranchial muscles, the hyoid arch swings posteroventrally, depressing the floor of the pharynx and increasing its volume. Rapid hyoid retraction, along with elevation of the neurocranium and lateral movement of the opercular bones, are used by many fishes to generate suction within the mouth and pharynx to create currents for suspension

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Physiological and ecological adaptations t o feeding i n vertebrates

feeding,prey capture, and/or prey manipulation (seebelow).Many elements of the pharyngeal skeleton in bony fishes bear teeth (as do palatal and neurocranial bones) which are used to hold, grasp, manipulate, transport, and process food. In some teleosts, upper and lower tooth-bearing elements of the pharyngeal arches interact to form an internal set of "pharyngeal jaws" used in elaborate manipulatory and processing behavior (Liem, 1973; Liem and Osse, 1975; Liem and Greenwood, 1981; Lauder, 198313, 1985a; Sanford and Lauder, 1989;Sibbing, 1991; Vandewalle et al., 1994; Galis and Drucker, 1996),a condition known as "pharyngognathy" (Liem and Greenwood, 1981;Fig. 1.3). Fish use the pharyngeal skeleton, in particular the hyoid arch, to create feeding currents through volumetric changes of the pharynx. This is only possible in water because the density of prey is closely matched by the surrounding density of the medium. Rapid water flow is thus able to overcome

Fig. 1.3. Schematic representation of the pharyngeal jaw apparatus in four families of teleostean fish. The pharyngeal jaws are lightly stippled. Letters refer to muscle groups that act on the jaws to produce complex crushing, grinding, and transport movements. From Liem and Greenwood (1981), reproduced with permission of the Society for Integrative and Comparative Biology.

Vertebrate feeding systems

9

the prey's inertia. Tetrapods could not employ such inertial suction when they first began to feed in the terrestrial environment, but they nonetheless exploited the hyoid mobility inherited from their piscine ancestors when feeding on land (Shaffer and Lauder, 1988; Gillis and Lauder, 1994, 1995; Reilly, 1996).They accomplished this primarily through the evolution of a novel structure, the mobile, muscular tongue. The tongue evolved by elaboration of hypoglossal muscles associated with the hyoid and the first two or three branchial arches (Kallius, 1901). The reduced (compared to fishes) pharyngeal skeleton of tetrapods is called the hyobranchial apparatus or hyobranchium, and it supports the tongue and throat musculature (e.g. Fiirbringer, 1922; Weissengruber et al., 2003). The hyobranchium of nonmammalian tetrapods is often inaccurately referred to as the "hyoid apparatus," a term appropriately applied only to mammals in which the branchial arch contribution is lost or greatly reduced (Schwenk, 2000a). In combination,the tongue and hyobranchium are called the hyolingual apparatus. Instead of modulating water flow, movements of the tetrapod hyobranchium move the tongue which, in effect, takes the place of water in capturing, supporting, and manipulating food particles (Fig. 1.1).Tongue movement that is extrinsically generated by hyobranchial movment is enhanced in many tetrapods by intrinsically generated shape changes of the tongue's soft tissues (seebelow).In secondarily aquatic tetrapods that revert to suction feeding, the hyobranchial apparatus is once again used to modulate the flow of water by changing pharyngeal volume (Lauder, 1985a;Van Damme and Aerts, 1997; Deban and Wake, 2000; Aerts et al., 2001; Lemell et al., 2002). It is noteworthy that in these species the tongue is almost always reduced or even lost -an indication of its uniquely terrestrial role in feeding (Brambleand Wake, 1985). The tongue is a critical element of the tetrapod feeding system, largely overlooked in earlier studies of feeding. In many taxa, it participates in all stages of feeding, from prey capture to swallowing. Its morphology is diverse, ranging from little more than an epithelium covered part of the hyobranchium, to an astoundingly complex muscular organ capable of extreme changes in length and shape. The protean nature of tongue form in some taxa (notably mammals and some squamate reptiles) arises from its unusual biomechanical properties. The tongue is one of the very few vertebrate organs capable of hydrostatic deformation (Owen, 1868;Kier and Smith, 1985; Smith and Kier, 1989).Such so-called "muscular hydrostats" comprise solid muscle masses with a complex histology in which fiber systems are arrayed orthogonally, sometimes includinghelical systems as well (Kier and Smith, 1985; Smith and Kier, 1989; Schwenk, 2001a). Because the organ retains a constant volume and the intracellular fluid within it is incompressible, local or global reductions in diameter cause elongation and/or shape change. For example, myrmecophagous mammals use extreme length changes in their serpentine tongues to probe ant and termite nests (Reiss,

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Physiological and ecological adaptations t o feeding in vertebrates

2000); generation of intraoral suction within the buccal cavity during suckling in infant mammals is made possible by an oropharyngeal seal at the back of the mouth created by shape changes in the tongue (German and Crompton, 2000);lizards, terrestrial turtles and mammals form humps and cup-shaped depressions in the tongue to hold and push the bolus during hyolingual transport through the mouth (Brambleand Wake, 1985;Hiiemae, and hydrostatic elongation of the tongue in combi2000; Schwenk, 2000~); nation with a uniquely shaped hyobranchium, provides the explosive trigger that projects the chameleon's tongue out of its mouth (Wainwright and Bennett, 1992).

OVERVIEW OF VERTEBRATE FEEDING Feeding Mode, Sensory Biology and Foraging Strategy Before a feeding bout can begin, it is necessary for an animal to find a potential food item and to identify it as appropriate. This requires the use of various sensory systems and some kind of foraging strategy. The evolutionary interplay of sensory function, foraging, and feeding mode has rarely been explored in detail, but the need for their coordinated function is obvious. Vitt et al. (2003), for example, provide a case study of squamate reptiles showing that historical changes in the feeding system have had cascading effects on the evolution of sensory systems and ultimately, community ecology and geographic distribution. The study illustrates that an understanding of the integration of feeding mode, sensory biology, and foraging mode can potentially lead to compelling explanations for higher level patterns, in this case, the structuring of squamate communities on a global scale (Vitt et al., 2003). These patterns are necessarily taxon specific but the important role other systems and behavior play in feeding biology is noteworthy. The remainder of this chapter primarily concerns feeding function from the moment an appropriate prey item is within striking distance until it passes into the esophagus. In studying this behavior, several stages of feeding are formallyrecognized. Vertebrate Feeding Stages In order to acquire and digest food successfully, it is necessary for an animal to proceed through a series of different mechanical tasks, concluding with passage of the food bolus into the esophagus. These tasks are identified technically as "feeding stages" to highlight their different functions and to facilitate description and comparisons of feeding across taxa (Bramble and Wake, 1985; Hiiemae and Crompton, 1985; Schwenk and Throckmorton, 1989;Schwenk,2000a).However, it is necessary to state several caveats about

Vertebrate feeding systems

11

feeding stages before describing them. First, as noted, their recognition is based on the differing functional roles they play during a feeding bout. As such, feeding stages can be thought of as phenotypic "solutions" to a series of mechanical "problems" encountered during the course of getting food from the environment into the esophagus.There is potentially more than one solution to any given mechanical problem, so different taxa sometimes carry out the same feeding stage in mechanistically different ways. This leads to the second point, namely that use of a common name for the "same" feeding stage in different species should not be taken as an assertion of evolutionary homology. The homology of feeding stages among taxa is an open and critical research question, as is, for that matter, the extent to which the stages can be considered evolutionary "characters" at all (Reilly and Lauder, 1990; Smith, 1994; Schwenk, 2000a; Wainwright and Friel, 2001; McBrayer and Reilly 2002b). Third, any given species or individual might exhibit only a subset of all possible feeding stages (described below). Individuals might also vary in the particular feeding stages manifested during one feeding bout compared to another, or might vary the mechanism employed during a particular feeding stage depending on the nature of the fqod eaten or other local circumstances.Finally although some stages must necessarily precede other stages (e.g. capture must precede swallowing), some do not always occur sequentially. For example, capture/subjugation and ingestion are often combined into a single stage (ingestion), whereas processing and transport cycles are occasionally alternated or interspersed (e.g. Hiiemae and Crompton, 1985; Schwenk and Throckmorton, 1989; Hiiemae, 2000; Schwenk, 2000c; McBrayer and Reilly, 2002a). In all vertebrates, feeding emerges from the complex coordinationof skull, jaw, tongue, and hyobranchial movements. In tetrapods especially these movements are rhythmic and cyclic, leading to the suggestion that they are controlled by pattern generators in the central nervous system (Dellow and Lund, 1971; Thexton, 1973; Hiiemae, 2000). Bramble and Wake (1985)proposed that there is a basic or "model" feeding cycle that we might infer represents the ancestral or primitive pattern of coordinated movements and motor patterns in tetrapod feeding. There has been a great deal of discussion regarding the utility of the Bramble-Wake model in predicting the feeding kinematics of different taxa and during different feeding stages (e.g.Schwenk and Throckmorton, 1989;Reilly and Lauder, 1990; Delheusy and Bels, 1992; Bels et al., 1994;Lauder and Gillis, 1997;Schwenk, 2000c; Herrel et al., 2001), with no consensus emerging. Regardless, the critical point is that tetrapod feeding, at least, results from rhythmic, cyclical, and coordinated movements of the aforementioned parts. The fundamental unit of this behavior is the "gape cycle," representing a single excursion of the jaws from closed to open and back to closed (Bramble and Wake, 1985;Hiiemae and Crompton, 1985; Schwenk, 2000a).Movements of all other components of the feeding system are usually described relative to the gape cycle to facilitate comparisons,

12

Physiological and ecological adaptations t o feeding in vertebrates

although in some cases jaw movements are relatively trivial compared to movements of other parts, such as the hyobranchial apparatus. A single feeding bout represents a variable number of sequential gape cycles, with modulation of kinematic patterns occurring throughout, depending on the mechanical task at hand (the feeding stage) and the position and condition of the bolus. Although the different feeding stages outlined below can sometimes be differentiated qualitatively as well as quantitatively, only rarely are the kinematic transitions between them sharply defined. Active prey must first be captured and subjugated before it can be eaten. Once subdued, it can be brought into the mouth (ingestion).These actions represent nominally the first and second stages of feeding. However, in most vertebrates food is captured with the mouth so that capture and subjugation of prey occurs at the same time that it is moved into the oral cavity. Thus, in most vertebrates the single stage of ingestion accomplishes all three functions. However, in many species, particularly mammalian predators that feed on relatively large prey, a separate capture/subjugation stage is necessary before the food can be ingested. Such mammals typically run down and attack prey with the forelimbs and jaws to subdue and kill them (Ewer, 1973; Van Valkenburgh, 1996). Once quiescent, the prey can be consumed and ingestion initiated. Primates also typically use the forelimbs to grab, manipulate and sometimes kill a food item before placing it in the mouth. Although less common, a separate capture/subjugation stage occasionally occurs in nonmammalian taxa, such as fish-spearing with the bill in some wading birds, or raptorial capture and killing with the feet in other birds. The venomous crotalid snakes (rattlesnakes and kin) are particularly illustrative because after an envenomating bite they release their prey and allow it to die before ingesting it (Cundalland Greene, 2000). Rarely, capture and subjugation are performed as separate tasks, as in constricting snakes that capture a prey item with the jaws, but hold and subjugate it in coils of the body until it can be transported (Cundall and Greene, 2000).

Ingestion Ingestion refers to the transfer of a food item from the environment into the oral cavity.As noted, in most vertebrates this is accomplished with the mouth so that capture/subjugation and ingestion are combined into a single ingestion stage. In aquatic vertebrates, ingestion occurs in one of three ways: suspension feeding (sometimesinaccurately called "filter feeding"), suction feeding or jaw prehension. In suspension feeding, small food particles are collected from the water surface, water column or the benthos when water is passed through a porous structure, such as the gill rakers of the pharyngeal skeleton in many fishes or the baleen plates of mysticete whales (e.g. Northcott and

Vertebrate feeding systems

13

Beveridge, 1988;Sanderson and Wassersug, 1993;Goodrich et al., 2000; Werth, 2000b).Many aquatic birds use elaborations at the margins of the bill to filter algae and other suspended food particles from the water (Zweerset al., 1977, 1995;Kooloos et al., 1989).Some freshwater turtles draw small, floating food particles into the mouth (Belkin and Gans, 1968), trapping them in esophageal papillae when the water is expelled (Vogt et al., 1998). Suspension feeders either actively pump water into the mouth and pharynx, or engulf suspended food particles as they move their bodies forward with mouth agape. Many fishes can shift between these two modes of suspension feeding. However, drawing larger food items into the mouth requires the rapid generation of substantial negative pressure within the mouth and pharynx. This is referred to as suction feeding. As water is accelerated into the mouth, its rapid flow draws the prey along with it. There is a continuum among taxa in the amount of suction used and the extent to which the prey moves into the mouth versus the mouth over the prey (Norton and Brainerd, 1993;Liem, 1993; Nemeth, 1997;Van Damme and Aerts, 1997; Summers et al., 1998;Wainwright et al., 2001). In "inertial suction" the prey item is accelerated relative to a fixed point and moved into the mouth along with the water, whereas in "compensatory suction" the negative pressure generated by the predator is only enough to overcome the effects of its own bow wave as it moves forward, engulfing a stationary prey item. Such "ram feeders" attempt to mitigate the pressure wave by means of a large gape, capacious pharynx and unilateral flow of water. In bony fishes, suction is generated by an explosive expansion of the mouth and pharynx caused by expansive movements of the jaws, neurocranium, opercular bones and pharyngeal skeleton, especially retraction and depression of the hyoid arch (e.g. Lauder, 1983a, b, 1985a, b; Miiller and Osse, 1984; Bemis and Lauder, 1986;Bemis, 1987; Lauder and Shaffer, 1993; Liem, 1993; Gillis and Lauder, 1995; De Visser and Barel, 1998; Ferry-Graham and Lauder, 2001; Grubich, 2001; Westneat, 2001; Sanford and Wainwright, 2002). Upper jaw protrusion and kinesis create a small, round, anteriorly directed gape that increases water velocity and targets prey directly in front of the fish (Fig. 1.1). The cartilaginous elasmobranchs obviously lack the opercular apparatus and other elements of the bony fish skull and rely primarily on retraction of the hyoid arch to generate suction (Wu, 1994; Wilga and Motta, 1998; Edmonds et al., 2001; Motta and Wilga, 2001). A labial cartilage functions to restrict gape and accelerate flow. Many aquatic tetrapods, especially salamanders and turtles, have secondarily reverted to suction feeding (Erdman and Cundall, 1984;Lauder, 1985; Lauder and Shaffer, 1985; Elwood and Cundall, 1994; Lauder and Reilly, 1994; Van Damme and Aerts, 1997; Deban and Wake, 2000; Aerts et al., 2001; Deban and Marks, 2002; Lemell et al., 2002). As in fish, suction is generated by rapid expansion of the pharynx (Fig.l.4).These taxa typically have an elaborate and robust hyobranchial apparatus that is articulated in

14

Physiological and ecological adaptations t o feeding in vertebrates

Fig. 1.4. Skull (white) and hyobranchial apparatus (gray) of a larval aquatic salamander in lateral view. At left, the hyobranchium is at rest, lying flat within the throat. When retracted, at right, the downward pivoting of the ceratohyal and the rest of the hyobranchial apparatus causes a massive expansion of buccal and pharyngeal cavities to generate suction for the modulation of water flow. BP: branchial plate; CH: ceratohyal; EB1: first epibranchial. From Deban and Wake (2000), reproduced with permission of Elsevier Science.

such a way that at rest it lies flat in the floor of the mouth and throat, but when retracted it unfolds, dropping dramatically to vastly increase pharyngeal volume (e.g. Van Damme and Aerts, 1997; Deban and Wake, 2000). Esophageal expansion during the strike in some turtles helps prevent back pressure, suggesting that suction may be compensatory rather than inertial in these species (Lauder and Prendergast, 1992; Lemell et al., 2000). Longnecked turtles use a snake-like strike to propel the head toward the prey (Weisgram and Splechtna, 1992; Van Damme and Aerts, 1997,2002; Aerts et al., 2001). Patent gill slits allow fish and larval salamanders to draw water through the pharynx in one direction during suction feeding (unidirectional flow), whereas in metamorphosed salamanders, turtles, and mammals the water must exit the mouth during the compressive stage immediately following capture (bidirectionalflow), leading to a putative mechanical inefficiency (Lauderand Shaffer, 1986).Nonetheless, feeding performance in some highly aquatic adult salamanders is superior to larval forms with unidirectional flow, suggesting that morphological and behavioral adaptations can overcome this potential handicap (Miller and Larsen, 1989). A very few salamanders feed underwater using the terrestrial mechanism of tongue prehension (Deban and Wake, 2000). All neonate mammals (with the possible exception of some monotremes) use suction for ingestion of milk (e.g.German and Crompton, 2000) but suction feeding is uncommon in adult mammals. Walruses retract the tongue in the mouth like a piston to suck molluscs from the substrate (Kasteleinet al., 1994) and several other pimipeds may also use suction (Werth, 2000b). Some blunt-headed cetaceans, such as pilot whales, probably use suction for ingestion of food items such as squid (Werth,2000a, b). Jaw prehension of prey during ingestion is less common in aquatic vertebrates than suction and suspension feeding. Large, predaceous sharks, such as larnniforms and carchariniforms tend to overcome a prey item by

Vertebrate feeding systems

15

rapidly overtaking it, either engulfing it within the mouth or biting it. Some specialized biters, however, remove mouth-sized chunks of flesh from their prey using protrusible jaws and rows of sharp, serrated teeth (Frazzetta and Prange, 1987; Frazzetta, 1988, 1994; Motta and Wilga, 2001; Wilga et al., 2001).Some bony fish use the jaws and marginal teeth to grasp prey directly or to scrape food, such as algae, off the substrate (e.g.Liem, 1980; Turingan and Wainwright, 1993; Wainwright and Turingan, 1993; Alfaro and Westneat, 1999; Wainwright et al., 2000; Alfaro et al., 2001). Many species bite pieces from large prey in much the way some sharks do (Liem, 1980; Alfaro et al., 2001). Jaw feeding fish typically retain the use of suction to manipulate and transport prey within the pharynx. Crocodilians use the jaws to capture prey in water, typically with a rapid, sideways jerk of the head (Busbey, 1989; Davenport et al., 1990; Cleuren and De Vree, 2000). Some species, such as gavials, have long, narrow snouts lined with needlelike teeth specialized for the prehension of fish in water (Cleuren and De Vree, 2000). Either the tongue or the jaws are used as prehensile organs during ingestion in the vast majority of terrestrial vertebrates (Schwenk,2000b). During metamorphosis, salamanders with aquatic larvae shift from suction feeding to lingual prehension (Lauder and Shaffer, 1988; Shaffer and Lauder, 1988; Reilly, 1996; Deban and Marks, 2002), a transition associated with remodeling of the hyobranchial apparatus, closure of the gill slits and development of the tongue (Wake, 1982;Wake and Deban, 2000).Some aquatic sala-manders remain specialized suction feeders as adults, but postmetamorphic terrestrial species feed in water only infrequently and inefficiently. Tongue protrusion during lingual prehension is coupled to hyobranchial protraction, which varies from modest to extreme (e.g. Larsen et al., 1989; Findeis and Bemis, 1990;Lauder and Reilly, 1994;Wake and Deban, 2000; Deban et al., 2001).In plethodontids the hyobranchial apparatus is folded into a cylinder and protruded or projected out of the mouth along with the tongue (Fig. 1.5;Lombard and Wake, 1976; Deban et al., 1997; Wake and Deban, 2000). Prey items adhere to the sticky tongue pad. Virtually all terrestrial salamanders ingest prey with lingual prehension, but some, in particular larger species, resort to jaw prehension for large prey (Wake and Deban, 2000). Most frogs are also obligate lingual feeders, although some species occasionally approach large or difficult to capture prey closely enough to use the jaws (Anderson, 1993; Nishikawa, 2000). Frogs use three different mechanisms of tongue protrusion, i.e. mechanical pulling, inertial elongation,and hydrostatic elongation, each manifesting a characteristic suite of functional traits. Although hydrostatic elongation of the tongue is commonplace in mammals and some lizards, it is exceptional in frogs and restricted tospecies in two families with unusual tongue morphology (Ritter and Nishikawa, 1995; Nishikawa et al., 1999; Nishikawa, 2000). Unlike

16

Physiological and ecological adaptations t o feeding in vertebrates

Ton~wepad

h

Fig. 1.5. Schematic representation of tongue projection during lingual prey capture (ingestion) in a plethodontid salamander. Note that the hyobranchial apparatus (tongue skeleton) is folded and projected from the mouth along with the tongue. DNIP: depressor mandibulae muscle; RCP: rectus cervicis profundus muscle; SAR: subarcualis rectus muscle. From Deban and Dicke (1999), reproduced with permission of The Company of Biologists.

salamanders and lizards, the hyobranchial apparatus in these frogs participates only indirectly in tongue protrusion. Among lepidosaurian reptiles, tuatara and iguanian lizards rely on lingual prehension (Schwenk and Throckmorton, 1989; Bels et al., 1994; Schwenk, 2000~).A few species are obligate tongue feeders but most decrease tongue protrusion distance as prey become larger, eventually shifting to jaw prehension (Gorniaket al., 1982;Schwenk and Throckrnorton, 1989; Schwenk, 2000c; Kardong and Bels, 2001). Tongue protrusion during feeding is coupled to hyobranchial protraction, as in salamanders, but movement is much more limited and the hyobranchium never leaves the mouth. The tongue curls around the margin of the lower jaw and prey adhere to its sticky, papillose surface. Chameleons have modified this basic system by inserting a ballistic, projection phase in which the tongue is launched off a supporting process of the hyobranchium out of the mouth (Schwenk and Bell, 1988; Wainwright and Bennett, 1992; Schwenk, 2000~). The tongue surface is actively dimpled during prey prehension, generating suction to provide extra adhesion for relatively large prey (Herrelet al., 2000). With very few exceptions, the remaining lizards are obligate jaw feeders. Many of these have kinetic skulls that improve the speed and precision of jaw capture by allowing simultaneous movement of upper and lower jaws in a pincer-like action (Frazzettta, 1983; Schwenk, 2000c; Metzger, 2002). Snakes have taken jaw prehension and cranial kinesis to its most extreme form (Cundall and Greene, 2000).

Vertebrate feeding systems

17

Lingual ingestion in turtles is restricted to terrestrial species (Summerset al., 1998; Wocheslander et al., 1999).Tongue protrusion is limited, with the tongue usually making contact with the food item at or near the jaw margins, almost within the mouth. Tongue retraction is typically accompanied by a bite. Semiaquatic species feeding on land use the jaws for prehension. The highly reduced tongue of most birds makes lingual ingestion impracticable for many species. Nectivores, however, are specialized lingual feeders. Hummingbirds, for example, probe flower nectaries with their very long tongues, using narrow channels in the tongue to acquire nectar by capillarity. Other nectivorous species, such as lories, have keratinous, brush-like tongue tips to increase surface area for nectar retrieval (McLelland, 1979).Woodpeckers use exceptionally long tongues to probe holes and crevices, using them to ingest larval insects (McLelland, 1979; Zweers and Berkhoudt, 2001) and crossbills pull seeds from cones using the tongue (Benkrnan, 1987). The extent to which mammals use lingual ingestion is relatively unstudied, but we suspect it is more common than supposed. Certainly specialized myrmecophages are well known to use their long, extensible tongues to probe ant and termite nests for prey (Reiss, 2000). Many nectar- and fruit-eating bats ingest liquid or soft food by lapping, often evincing brush-like tongue tips (like some nectar-feedingbirds) to maximize adherent food (Griffiths, 1982).Giraffes and related okapi use exceptionally protrusible tongues to strip leaves off trees (Owen, 1868; Kingdon, 1979)and some grazing bovids pull grass into the mouth for cropping using a prehensile tongue (KS, pers. obs.). Jaw prehension of food is common and general among tetrapods. Crocodilians lack a protrusible tongue and are obligate jaw feeders (see above). Similarly, gymnophione amphibians (caecilians) lack protrusible tongues and use the jaws to capture prey (Bemis et al., 1983; O'Reilly, 2000). The majority of birds are specialized for jaw prehension. They have modified the bill and rhamphotheca (the keratinous part) in myriad ways for this purpose (Fig. 1.6; Zweers, 1985;Zweers et al., 1994,1997;Rubega, 2000; Zweers and Berkhoudt, 2001).Scleroglossanlizards and snakes have modified the tongue for chemoreception and, with very few exceptions, rely on the jaws for ingestion (Cundall and Greene, 2000; Schwenk, 2000c; Kley, 2001). Most mammals are jaw feeders as well. Except for mammals, most jaw-feeding vertebrates have reduced, simplifiedor immobile tongues. Infrequently, structures other than the jaws and tongue are used for ingestion in tetrapods. The forelimbs are used in some mammals, such as primates and rodents, and in some frogs (Gray et al., 1997).Rarely, the hind limbs are used (as in some birds such as raptors), or other structures, such as an elephant's mobile trunk or the prehensile lips of black rhinoceroses and some other mammals (Kingdon, 1979).Whatever specific mechanism is used, ingestion results in the placement of the food item in the mouth where it is

18

Physiological and ecological adaptations t o feeding i n vertebrates

Fig. 1.6. Diversity of the jaws and beak in birds. Birds lack teeth and do little oral food processing, but the beak is specialized in various ways for food acquisition. (A) hyacinth macaw; (B) southern giant petrel; (C) parakeet auklet; (D) wrybill; (E) Andean avocet; (F) whippoorwill; (G) African spoonbill. From Rubega (2000), reproduced with permission of Elsevier Science.

Vertebrate feeding systems

19

positioned for processing or for immediate transport to the pharynx for swallowing. In some cases, a killing bite and/or head shake is interposed here (see processing,below).

Intraoral Transport Intraoral transport (or simply transport) usually refers to posterior movement of the food item through the oral cavity to the pharynx where it can be swallowed, but more generally it can be taken to mean any intraoral movement and manipulation of the food item once held in the mouth. For example, food that is chewed is often laterally repositioned so that it lies between upper and lower tooth rows. Side-switching during chewing is common in mammals and in many lizards (Hiiemae and Crompton, 1985; Hiiemae, 2000; Schwenk, 2000c; Reilly et al., 2001; McBrayer and Reilly, 2002a).A food item is sometimes transported anteriorly if, after ingestion, it comes to lie too far back in the mouth for processing (Schwenk and Wake, 1993; Schwenk, 2000a, c). In mammalian studies, two discrete types of transport are often distinguished (Hiiemae et al., 1978; Hiiemae and Crompton, 1985;Hiiemae, 2000).In stage I transport, food is moved from the incisive area at the front of the mouth to the postcanine region for processing. In stage 11transport, liquids and reduced food are moved posteriorly through the fauces (the posterior border of the oral cavity demarcated by the vertical columns of the palatoglossal muscles), either for bolus formation or for immediate swallowing. In aquatic vertebrates, transport is usually accomplished hydrodynamically (hydraulically)by creating pressure gradients within the oropharyngeal (and opercular) cavities (e.g. Lauder, 1985a; Bemis, 1987; Liem, 1990; Gillis and Lauder, 1994,1995; Lauder and Gillis, 1997).In some teleost fishes, tooth-bearing pharyngeal jaws are used to manipulate prey, moving it toward the esophagus (e.g. Liem and Greenwood, 1981; Sibbing, 1982; Lauder, 1983, 1985a; Sibbing et al., 1986; Vandewalle et al., 1994). Many cetaceans and pinnipeds also use hydrodynamic transport of captured food (Werth, 2000a, b). In terrestrial vertebrates, most transport and manipulation of food is hyolingual (Bramble and Wake, 1985; Hiiemae and Crompton, 1985; Schwenk, 2000a),meaning that it is mediated by coordinated, cyclical movements of the tongue and the hyobranchial skeleton (Fig. 1.1).During transport the food item sits on the tongue while cyclical motions of the tongue and hyobranchium move it toward the pharynx for swallowing,or reposition it in the mouth for processing (see next section). In taxa with muscular or fleshy tongues (mammals, many lizards, some turtles, parrots, possibly waterfowl), the tongue forms itself around the food item during transport, cupping it, or humps up in front of it, pushing it. In one bird lacking a fleshy tongue (and probably others), the tongue is bent sharply downward at an intrinsic hyobranchial joint and the food item is transported on the tongue

20

Physiological and ecological adaptations t o feeding in vertebrates

behind the peak (Rubega et al., submitted manuscript ). Among various tetrapods, palatal teeth, palatal rugae, or other keratinous projections on the palate prevent the bolus from moving forward while the hyolingual apparatus protracts beneath it in preparation for the next transport cycle (Bramble and Wake, 1985;Hiiemae and Crompton, 1985; Zweers, 1985;Hiiemae, 2000). In frogs, hyolingual transport may be unnecessary due to the brevity of the pharynx in this virtually neckless group. Except in a few species that use hydrostatic tongue elongation, the frog tongue is attached at the front of the mandible so that when it flips back into the mouth during ingestion, the adherent prey item is placed at the rear of the throat in position for immediate swallowing. Many tetrapods sometimes replace hyolingual transport with inertial transport, in which the food item is released or tossed by the jaws (and the head repositioned over it) so that it comes to lie farther back in the mouth (Gans, 1969).Inertial transport is especially typical of many reptiles, including some lizards, crocodilians, and birds (Gans, 1969; Smith, 1986; Zweers et al., 1994; Cleuren and de Vree, 2000; Schwenk, 2000c; Tomlinson, 2000), but it is also exhibited by ancestral mammals such as opossums, tenrecs, and tree shrews (Tupaia),as well as carnivoran species that bolt chunks of flesh (Hiiemae and Crompton, 1985;Van Valkenburgh, 1996). In snakes, the hyolingual apparatus is so specialized for chemosensory function that its role in feeding is entirely lost (Schwenk, 2000~).Intraoral transport is accomplished with movements of the highly kinetic skull, including unilateral movements of toothed jaw and palatal bones that alternately "grasp" a prey item on one side and then the other. Although small prey items are potentially pulled through the mouth and into the pharynx, most snakes feed on relatively large prey. In these species the kinetic skull mechanism is more accurately said to pull the snake's head and body over a stationary food item stabilized by its own mass. Thus, the snake transport mechanism is considered to be a type of inertial feeding (Cundall and Greene, 2000).Scolecophidiansnakes employ unique mechanisms of upper and lower jaw kinesis to "rake" prey into the mouth and push it into the pharynx (Kley 2001). Axial bending of the anterior trunk supplements cranial transport once the bolus is far enough back (Moon, 2000; Kley and Brainerd, 2002). Surface tension transport is a specialized mechanism of intraoral transport characteristic of shorebirds. Tiny prey items are suspended within a drop of water between the jaws while surface tension drives the drop along the bill as upper and lower jaws are spread apart (Rubega and Obst, 1993; Rubega, 1996,1997). In all cases, the outcome of transport is placement of food in the pharynx, ready to be swallowed. In many taxa, however, the food is mechanically reduced or otherwise processed before transport is completed.

Vertebrate feeding systems

Processing Processing refers to any mechanical reduction or preparation of the food before it is swallowed. However, many taxa, including most fish, most amphibians, many birds and snakes, do not process their food at all (other than lubrication with saliva) -they simply swallow it whole directly after ingestion and transport. And although most processing occurs within the oral cavity, some vertebrates do a significant amount of food preparation before or during the act of ingestion. Carnivoran mammals, for example, often rend pieces from their prey with the jaws, sometimes aided by the forelimbs (Ewer, 1973). Many terrestrial turtles do something similar by pinning a food item against the substrate with the forelimbs while tearing pieces off with the beak (KS, pers. obs.), as do many raptorial birds. Mammals, especially rodents and primates, hold a food item in the forelimbs and bite off small pieces for further processing within the mouth. Galapagos land iguanas often use their forelimbs to scrape the spines off the prickly pear cactus fruit they favor (H. K. Snell, pers. comm.). Crocodilians rend chunks from large prey by grasping the prey in the jaws and spinning violently on their axes (Cott, 1961; Pooley and Gans, 1976; Taylor, 1987).They also sometimes cache dispatched prey underwater to store it for later consumption and possibly to soften it before ingestion.Similarly,shrikes (passerine birds) impale prey on thorns or barbed wire, returning to feed on it later. Many sharks and other predatory fish tear or bite pieces from larger prey (Frazzetta and Prange, 1987; Motta and Wilga, 2001), as do some bony fish. Although processing of any kind is otherwise unknown in snakes, two species tear apart freshly molted crabs by pulling them through a loop of the body (Jayne et al., 2002). Granivorousbirds sometimes hold hard seeds with the feet while cracking them with the beak, or husk them directly in the bill, ingesting only the inner kernel (Ziswiler and Farner, 1972; Zweers et al., 1994; Nuijens and Zweers, 1997). Parrots employ an elaborate shelling behavior involving the beak and tongue (Homberger, 1980,1986).Kingfishers beat a captured fish against a perch, both to subdue and to soften it by breaking bones, then swallow it whole. Uniquely, many birds process food after it is swallowed, in specialized partitions of the esophagus and stomach (seelater). In tetrapods, most processing occurs within the mouth by crushing or biting with the teeth. Sometimes this is restricted to cropping during ingestion, or killing bites and head shakes immediately upon capture, but usually prey is further chewed with the teeth. Chewing involves repeated, cyclical biting movements that crush, puncture, shear and/or grind the food item, mechanically reducing it in preparation for swallowing. In nonmammalian taxa that chew, this behavior is referred to descriptively as puncture-crushing, as typified by lizards (Schwenk, 2000c; McBrayer and Reilly, 2002). Food is pierced by sharp, pointed teeth or crushed between blunt, molariform teeth, but there is little, if any, fragmentation of the bolus

22

Physiological and ecological adaptations t o feeding in vertebrates

and certainly no true comminution (see below).Puncture-crushingserves to soften the food item, to lubricate it with copious saliva, and potentially to introduce salivary enzymes into the bolus, initiating chemical digestion. Durophagous species feeding on snails and large arthropods may use temporal summation of pulsatile adductor contractions to increase bite force (Gans and De Vree, 1986). Chewing in nonmammalian taxa is often erroneously referred to as "mastication", but this term is accurately applied only to mammals (Davis, 1961; Schwenk, 2000a). Mastication is a derived and specialized form of chewing associated with a suite of mammalian novelties, including functional specialization of teeth along the tooth row (heterodonty),precise, unilateral occlusion of upper and lower teeth, a masticatory cycle including lateral and/or anteroposterior movements of the lower jaw, a derived tongue morphology, and the evolution of a muscular pharynx associated with a unique form of swallowing (Hiiemae and Crompton, 1985;Crompton, 1989, 1995;Smith, 1992; Herring, 1993; Weijs, 1994; Thexton and Crompton, 1998; Hiiemae, 2000; Schwenk, 2000a, 2001a). The important feature of mastication, in contrast to puncture-crushing, is that it reduces ingested food to a fine slurry of tiny particles mixed with saliva, a process referred to as comminution (Fig. 1.2). Food in this semiliquid state is moved during stage 2 transport into the pharynx where it is temporarily held or swallowed immediately. Uniquely, mammals often interpose swallow cycles amidst a series of masticatory cycles (Thexton and Crompton, 1998;Hiiemae, 2000), whereas other tetrapods only swallow a bolus once chewing is completed. Some mammals have secondarily reduced or lost their ability to masticate. This is usually correlated with modification, reduction or loss of the teeth associated with specialized diets such as insectivory and piscivory (e.g. odontocete cetaceans) or secondary reversion to suspension feeding (e.g. mysticete cetaceans).Modern monotremes have lost their teeth altogether, substituting keratinized structures on the tongue and palate to rasp their food (Owen, 1868; Doran and Baggett, 1972; Griffiths, 1978). Turtles and modern birds entirely lack teeth and rely on the keratinous rhamphotheca for whatever oral processing they do (Fig. 1.6).In turtles the apical edges of the beak are sharp and the lower jaw fits snugly within the upper forming an effective shearing mechanism. Some turtles also crush food between upper and lower plates (trituratingsurfaces) at the beak's front end (Gaffney, 1979).Birds, in general, do very little oral food processing, for reasons discussed in a subsequent section; however some birds shear food with the sharp edges of the beak. Owls and raptors, for example, often hold prey with the feet and use the beak to tear it into bits and some frugivorous parrots similarly shear off pieces of fruit (Zweersand Berkhoudt, 2001). Most sharks do no processing with the marginal teeth other than killing bites and/or excision of pieces from larger prey (Frazzetta and Prange, 1987; Frazzetta, 1994;Motta and Wilga, 2001).Some elasmobranchs,however, crush

Vertebrate feeding systems

23

hard-bodied prey, such as mollusks, with plates of flattened teeth (Moss, 1977; Summers, 2000; Wilga and Motta, 2000). In these taxa the cartilaginous jaws may be strengthened by unusual "trabecular cartilage" (Summers, 2000).Relatively few bony fish use the jaws and marginal teeth for processing, probably because without cheeks and lips, reduced food particles would be lost in the water (Vandewalleet al., 1994).However, some taxa do manage to reduce food in the marginaljaws (e.g.Hernandez and Motta, 1997),but in most bony fish that process their food, it is crushed, ground or pierced by intraoral and/or intrapharyngeal teeth on the palate and hyobranchial skeleton. Indeed, in derived taxa, such as the Cichlidae and Labridae, teeth are restricted solely to tooth plates on the pharyngeal jaws which are used to process the food before it is swallowed (e.g.Liem, 1973; Sibbing, 1982,1991; Liem and Sanderson, 1986;Vandewalle et al., 1994; Galis and Drucker, 1996; Grubich, 2000). Some aquatic taxa pump water in and out of the mouth, lacerating prey as it is raked across the marginal teeth (Bemis, 1987;Elwood and Cundall, 1994). Although salamanders virtually never process their food, one group (thedesmognathine plethodontids) routinely delivers crushing bites using "head tucking"behavior in which force is transmitted to the lower jaw via a ligamentous connection to the cervical vertebrae (Schwenk and Wake, 1993).

Swallowing During swallowing (also called pharyngeal emptying; Smith, 1992;Schwenk, 2000a), the bolus is moved from the pharynx into the esophagus where peristalsis takes over the task of its transport through the remainder of the gut. Depending on the particular mechanism of swallowing used and/or the relative length of the prey item compared to the pharynx, the transition between intraoral transport and swallowing is often blurred. An extreme example is evident in macrostomatan snakes, which typically eat relatively large or elongate prey (Cundall and Greene, 2000). One end of a prey item often extends from the mouth even as the other end enters the esophagus! Initially, unilateral, alternating movements of the kinetic skull and jaws are used to move the prey toward the esophagus. These are supplemented and then replaced by axial bending movements of the trunk as the food item moves farther into the esophagus (Moon, 2000; Kley and Brainderd, 2002), so that one feeding stage blends imperceptibly into the next. A similar situation occurs in some seabird chicks feeding on relatively long fish. In most taxa there is a somewhat gradual transition between transport and swallowing cycles rather than a sharp demarcation. In mammals, as noted, swallowing cycles are often interposed among a series of masticatory cycles so there may not be a terminal swallowing stage, per se. Swallowingin fishes is poorly understood, but in many teleosts, at least, it is accomplished with manipulatory movements of the pharyngeal jaws that push the bolus into the esophagus (Lauder, 1983a, b, 1985a).How it

24

Physiological and ecological adaptations t o feeding in vertebrates

occurs in taxa lacking pharyngeal jaws is not clear (see Lauder, 1983a).Some suspension feeding fish trap small food particles in mucous strands that move into the esophagus with water flow (Sandersonet al., 1996).Recently, Sanderson et al. (2001)showed that suspension-feedingteleosts do not trap particles directly in the gill rakers as previously thought ("sieving"), but rather capture them using "crossflow filtration" in which food particles are concentrated in the oral cavity and then swept across the rakers toward the throat, thus solving "the mystery of particle transport to the oesophagus" (Sanderson et al., 2001). Teleosts have an esophageal sphincter that putatively prevents them from swallowing too much water (Stevens and Hume, 1995). Most tetrapods use cyclical movements of the tongue and hyobranchial skeleton, as well as compression of the pharynx with the superficial constrictor musculature, to push or squeeze food into the esophagus. In lizards and terrestrial turtles, for example, the posterior end of the tongue is used to tamp food into the throat ("pharyngeal packing") and this is followed or in some cases, replaced with pharyngeal compression (Fig. 1.7; Smith, 1984, 1986; Bels et al., 1994; Schwenk, 2000~).In amphibians, swallowing is accomplished primarily with pharyngeal compression, often accompanied by retraction of the eyeballs, which may help to force the bolus into the esophagus (Duellman and Trueb, 1986; Deban and Wake, 2000). In taxa with reduced tongues, such as crocodilians and birds, pharyngeal compression is supplemented with inertial movements of the head, as well as gravity (Cleuren and De Vree, 2000; Tomlinson, 2000; Zweers and Berkhoudt, 2001). Some birds also use sinuous contractions of the floor of the pharynx ("properistalsis") to move food into the esophagus (Zweers, 1985; Zweers and Berkhoudt, 2001). Mammals employ a uniquely derived form of swallowing called deglutition (Thexton and Crompton, 1998; Hiiemae, 2000). Like the term "mastication", deglutition is often incorrectly applied to nonrnammalian taxa. Mammalian deglutition is associated with a derived tongue morphology and especially the presence of a soft palate and pharyngeal musculature (Smith, 1992;Hiiemae, 2000).Mammals can form a sphincter-like seal at the base of the tongue (at the fauces), functionally subdividing the buccal and pharyngeal space (Hiiemae,2000). Masticated food is passed through this seal during stage 2 transport to accumulate in the oropharynx.When a bolus is sufficientlylarge, an explosive contraction of the tongue base and soft palate, in conjunction with peristaltic waves of contraction in the pharyngeal musculature, propel food into the esophagus. CONSEQUENCES OF FEEDING-TAXONOMIC HIGHLIGHTS Vertebrate feeding systems were surveyed above according to the mechanical task associated with each feeding stage. The review revealed a great deal

Vertebrate feeding systems

Fig. 1.7. Swallowing in a terrestrial turtle based on individual frames from cineradiographic film. Numbers indicate frame number. Note shape changes in the tongue as it moves in front of the bolus and forms a seal with the palate. It then pushes the bolus posteriorly, into the esophagus, where peristalsis takes over. Tongue movement is accompanied by hyobranchial movement. ASHG: anterior suprahyoid muscles. From Bramble and Wake (1985), reprinted by permission of the publisher and President and Fellows of Harvard College.

of diversity in the manner that vertebrates procure, process, transport, and swallow food. Despite this diversity, all systems share the common outcome of moving the bolus into the esophagus for further digestion and nutrient assimilation. Therefore, the mechanical and chemical condition of the bolus at the point of swallowing, as determined by the front-end feeding mechanism, has obvious significance for physiological functioning in the remainder of the gut and it is this inescapable connection we turn to here.

Fishes Obviously, fishes are phylogenetically and taxonomically disparate, but within any clade the nature of the food swallowed devolves to one of five

26

Physiological and ecological adaptations t o feeding in vertebrates

types: particulate food collected by suspension feeding; tiny food items taken individually; relatively larger, whole, unprocessed food items; minimally processed food consisting of large pieces or chunks excised by the marginal teeth; and food that is processed, sometimes sigruficantly,by the marginal or pharyngeal jaws. Whether they trap suspended food from the water column or substrate, or ingest it one bit at a time, microphagous fishes swallow food that does not require further reduction and is ready for passage into the gut and assimilation. Whole food particles, however small, may nonetheless pose a different digestive challenge compared to food that has been milled, and therefore ruptured, by the pharyngealjaw apparatus. Pharyngognathy opens the door to significant food processing potentially rivaling that of mammals (e.g. Sibbing, 1991),but the nature of the processed food is rarely studied. Stomach content analyses of labrids by Wainwright (1987, 1988) showed that snail and crab shells were crushed and fragmented by the pharyngeal.jaws, with most of the shell fragmentswinnowed out before swallowing.Parrotfish process algae (and the coral with which it is associated) into a fine paste (Bellwoodand Choat, 1990;Bellwood, 1996,Choat et al., 2002) and presumably this is why they do not require a stomach (Horn, 1989).There seems to be a strong correlation between the degree of pharyngeal processing and gut form, at least in herbivorous species (Horn, 1989, 1992). Some seedeating species may use pulsatile contractions of the pharyngeal jaw musculature to increase crushing forces by means of temporal summation (Irish, 1983).Pharyngognathy may be particularly important to herbivorous and durophagous species, as might be expected, but this is an untested assumption. Contrastingly, in fishes that swallow entire prey or large pieces thereof, the gut must complete digestion chemically. Such species possess an extensible stomach for storage and face the potentially daunting digestive problem of the food item's high volume-to-surface area ratio. These issues are less problematic for carnivorous species because flesh is more easily digested. Herbivorous fishes are either inefficient in digestion, highly selective in diet, restricted to microphagy, or employ significant pharyngeal processing.

Amphibians Amphibians are nearly universal in their lack of prey processing and thus, with few exceptions, swallow whole, unreduced prey items. Notable exceptions are aquatic salamanders that shred prey items against the teeth as they are sucked in and out the mouth (Elwood and Cundall, 1994),desmognathine salamanders that routinely crush prey between the jaws (Schwenk and Wake, 1993),and caecilians that shear off pieces of prey with specialized dentition and/or axial rotation of the body while withdrawing into a burrow (Bemiset al., 1983; O'Reilly, 2000). Virtually no adult amphibians are herbivorous,

Vertebrate feeding systems

27

although two frog species are reported to consume more than incidental quantities of fruit or leaves (da Silva et al., 1989; Das, 1995). Despite the lack of herbivory, amphibian diets are diverse, particularly in prey size, which ranges from mites to vertebrates.Disparity in prey size and type, coupled to constraints on gut length imposed by the anuran body plan, make frogs an ideal group in which to examine diet-gut relationships. The only known folivorous frog, for example, was found to have an unusually long gut that may contain fermentative microorganisms (Das, 1995).A number of frog groups share a suite of characters related to microphagy, including cutaneous sequestering of dietary alkaloids (Vences et al., 1997/98). Microphagy increases the surface-area-to-volume ratio of the food particles swallowed (circumventing the problem of no chewing), but the arthropod prey eaten (mostlyants) tend to be noxious and/or low in nutritive value. A termite specialist, for example, was found to have a relatively longer gut than other species, putatively because termites are relatively hard to digest (Das, 1995).

Nonavian Reptiles Most reptiles do little or no processing and swallow food largely intact, either whole or in large pieces. Crocodilians exemplify this mode of feeding: small prey are simply tossed into the back of the mouth and swallowed (sometimes after a crushing or killing bite). Larger prey are dismembered or tom apart and the pieces bolted. Both feeding modes are enhanced by an exceptionally powerful bite (Ericson et al., 2003). The stomach is divided into a cranial glandular part and a caudal muscular part (the pylorus) that appears as a separate chamber when the stomach is empty (Richardson et al., 2002). The pylorus is often referred to as a "gizzard". Its homology to the bird gizzard is assumed and not certain. In any case, crocodilians, like birds, often swallow grit or small stones called gastroliths that are held within the gizzard and presumed to aid in the mechanical reduction of food. However, the evidence for this in crocodiliansis weak, gastroliths may also function as ballast (Taylor, 1993).Mechanical processing in the muscular gizzard, possibly aided by gastroliths, may help to mitigate the problem of digesting large, mostly intact pieces of food. Crocodiliansare also reputed to have exceptionally acidic gastric secretions that promote rapid digestion (Richardsonet al., 2002). Turtles do little or no intraoral processing, but do crop bite-sized pieces of food with their sharp beaks. Small, cropping bites may be particularly important in herbivorous species (Bjomdal and Bolten, 1992). Carnivorous and durophagous species generate more powerful bites than species with other diets (Herrel et al., 2002) but even extreme dietary specialization is possible without attendant specializationof the feeding system and gut (e.g. Meylan, 1988). Suction feeding aquatic species potentially swallow relatively large, whole prey.

28

Physiological and ecological adaptations t o feeding in vertebrates

Snakes, except for the two crab-eatingspecies mentioned above, virtually always swallow whole prey with no intraoral processing. However, the venom that crotalid snakes (rattlesnakes and their relatives) inject into their prey contains proteolytic digestive enzymes that initiate digestion from inside-out (Thomas and Pough, 1979; Cundall and Greene, 2000). Relative prey size is exceptionally large in this group, sometimes exceedingthe snake's own mass, and the prey's relatively small surface area could challenge the gut's ability to digest it extrinsically before it putrefies, This problem is exacerbated by the fact that the gut takes time to be upregulated after a period of quiescence (e.g. Secor and Diamond, 1998; Starck and Beese, 2001). Some snakes are able to crush bird eggs within the gut, retaining the liquid contents while regurgitating the indigestible shell (Gans, 1952). Although many lizards swallow small prey whole, lepidosaurs are exceptional among reptiles in the extent of their intraoral processing. Tuatara and many lizards initially crop larger food items into mouth-sized pieces. This is particularly true for herbivores whose teeth are often specialized for this purpose (Schwenk, 2000~).Sometimes inertial shaking is used to dismember prey. Most food is then chewed between marginal tooth rows, sometimes aided by palatal teeth. Chewing typically crushes, pierces, and softens the food item, but rarely results in significant trituration, even in herbivores. However, it is very likely that chewing introduces salivary enzymes and presumably the soft, well-lubricated bolus is more easily swallowed and digested.

Birds Although many birds reduce prey to some extent before ingestion, loss of teeth precludes significant intraoral processing. Consequently, birds often swallow whole, large, hard, or refractory food particles such as seeds without the benefit of front-end processing. Instead, they have adopted a novel strategy for processing that relies on specialization of the anterior gut. The esophagus is extremely extensible in species that swallow large prey and in many taxa it includes an expanded region where food can be temporarily stored. When evident as a distinct diverticulum, this structure is called a crop (Ziswiler and Farner, 1972;McLelland, 1979).The principal function of the crop is to store excess food and to regulate its rate of delivery to the stomach. In pigeons the crop produces and stores a liquid slurry of shed cells that is regurgitated to feed chicks. In many other species, the crop stores food and water for later regurgitation feeding of chicks. Although no significant chemical digestion occurs here, grain and other hard foods are moistened and softened. The single exception is the folivorous hoatzin in which the muscular crop serves as the site of fermentative digestion (Grajal, 1995). The stomach comprises two principal chambers, a cranial proventriculus and a caudal gizzard or ventriculus (Fig. 1.8, Ziswiler and Farner, 1972; McLelland, 1979). The proventriculus is glandular and the site of most

29

Vertebrate feeding systems

chemical secretion and digestion while the gizzard is extremely muscular in most species and responsible for mechanical processing. The gizzard is especially well developed in taxa whose food requires mechanical reduction, i.e. omnivores, insectivores,herbivores, and granivores (in some carnivores it is reduced or virtually absent). In these species, the gizzard is lined with a thick, hard cuticle forming dorsal and ventral "grinding plates" covered with ridges. Food is ground or crushed between the plates, with reduction facilitated by grit intentionally swallowed. The amount of grit in the gizzard has been correlated with diet and found to be more prevalent in granivorous birds, while grit size correlates with body size (Gionfriddo and Best, 1996).The crushing action of the gizzard in some species is considerable, i.e. whole, hard-shelled nuts are fragmented by turkeys within hours and there are even reports of metal objects being folded and ground into fragments (Welty, 1975).The gizzard apparatus is often referred to as a "gastric mill" and has been compared to the mammalian masticatory apparatus (e.g. King and King, 1979).

Papilla proventricularis

Isthmus gastris -

r, Lateral muscle

G* N Y

b,

Saccus cauda Fig. 1.8. The stomach of a granivorous bird (rock dove), showing its division into a cranial proventriculus and a caudal gizzard. The gizzard is highly muscular and lined with a thick, abrasive cuticle which, in conjunction with swallowed grit, mechanically processes food, reducing it to small particles. From N. S. Proctor and P. J. Lynch (1993), reproduced with permission of Yale University Press.

30

Physiological and ecological adaptations t o feeding in vertebrates

Mammals Evolution of mastication is a key theme in the history of mammals. It is at the heart of a sweeping reorganization of the skull, palate, dentition, tongue, pharynx, jaw muscles, and ear, and is intimately associated with the evolution of endothermy. It uniquely distinguishesmammals from all other vertebrates. The masticatory apparatus evolved incrementally within the synapsid stem lineage and was present essentially in the modern form once a fully functional dentary-squamosaljaw joint replaced the increasingly weak, ancestral quadrate-articular joint (Crompton, 1989,1995). The critical feature of the masticatory system is its ability to reduce food quickly to a mash of tiny particles mixed with copious enzyme-containing saliva (Fig. 1.2). Compared to other vertebrates, the bolus is virtually "predigested when swallowed. This has obvious relevance for passage rates in the gut. Although there are many secondary departures from the fundamental pattern of mastication, these usually occur in taxa that consume flesh or other types of food relatively more easily digested.Conversely the masticatory mills of herbivores and taxa that consume other types of refractory foods are elaborate. General Patterns One noteworthy pattern that emerges from the preceding consideration of the bolus condition is that in ectothermic vertebrates, food is processed relatively little, if at all, whereas in birds and mammals, food entering the intestine is usually extensively triturated. This distinction probably relates to the greater energetic demands of endotherms and their need to increase gut passage rates. Increased passage rates, as well as greatly increased gut surface area, are necessary in endotherms because they are no more efficient in extracting energy from their food than are ectotherms(Karasov, 1987;Karasov and Diamond, 1985,1988).By essentially "predigesting" their food, birds and mammals decrease the time necessary to hold it in the gut for chemical breakdown. In contrast, ectotherms can afford the low gut passage rates required for adequate digestion by virtue of their modest energetic demands. Mammals employ a complex oral masticatory system to process their food, whereas birds rely on a "gastric mill". In birds, the anterior gut effectively functions as part of the front-end feeding system. The evolutionary transfer of food processing from the oral apparatus to the gizzard, along with the loss of teeth, shifted the mass of the prey-reduction apparatus toward the center of gravity, an obvious advantage for a volant animal. These general patterns potentially have had important consequences for patterns of evolution in front-end phenotypes, a topic we explore elsewhere (Schwenk and Rubega, in litt.).

Vertebrate feeding systems

Acknowledgments

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Weijs W. A. 1994. Evolutionary approach of masticatory motor patterns in mammals. In: Biomechanics of Feeding in Vertebrates (Advances in Comparative & Environmental Physiology 18). V. L. Bels, M. Chardon, and P. Vandewalle (eds.). Springer-Verlag, Berlin, pp. 281-320. Weisgram J. and Splechtna H. 1992. Cervical movement during feeding in Chelodina novaeguinaeae (Chelonia, Pleurodira). Zool. Jb. Anat. 122: 331-337. Weissengruber G. E., Forstenpointner G., Peters G., Kiibber-Heis A. and Fitch W. T. 2003. Hyoid apparatus and pharynx in the lion (Panthera leo), jaguar (Panthera onca), tiger (Panthera tigris), cheetah (Acinonyx jubatus) and domestic cat (Felis selvestris f. cafus). J. Anat. 201: 195-210. Welty J. C. 1975. The Life of Birds. W. B. Saunders, Philadelphia, PA, USA, (2nded.). Werth A. 2000a. A kinematic study of suction feeding and associated behavior in the long-finned pilot whale, Globicephala melas (Traill). Mar. Mam. Sci. 16: 299-314. Werth A. 2000b. Feeding in marine mammals. In Feeding. Form, Function and Evolution in Tetrapod Vertebrates. K. Schwenk (ed.). Acad. Press, San Diego, CA, USA, pp. 487-526. Westneat M. W. 1990. Feeding mechanics of teleost fishes (Labridae): a test of four-bar linkage models. J Morph. 205: 269-295. Westneat M. W. 2001. Ingestion in fishes. In Encyclopedia of Life Sciences. Macmillan Publ., London, UK. Wilga C. D. and Motta P. J. 1998. Conservation and variation in the feeding mechanism of the spiny dogfish Squalus acanthias. J. Exp. Biol. 201: 1345-1358. Wilga C. D. and Motta P. J. 2000. Durophagy in sharks: feeding mechanics of the hammerhead Sphyrna tiburo. J. Exp. Biol. 203: 2781-2796. Wilga C. D., Wainwright P. C. and Motta P. J. 2000. Evolution of jaw depression mechanics in aquatic vertebrates: insights from Chondrichthyes. Biol. J. Linn. Soc. 71: 165-185. Wilga C. D., Hueter R. E., Wainwright P. C . and Motta P. J. 2001. Evolution of upper jaw protrusion mechanisms in elasmobranchs. Amer. Zool. 41: 1248-1257. Wocheslander R., Hilgers H. and Weisgram J. 1999. Feeding mechanism of Tesfudo hermanni boettgeri (Chelonia, Cryptodira). Nefh. J. Zool. 49: 1-13. Woodger J. H. 1929. Biological Principles. A Critical Study. Kegan Paul, Trench, Trubner, New York, NY. Wu E. H. 1994. Kinematic analysis of jaw protrusion in orectolobiform sharks: a new mechanism for jaw protrusion in elasmobranchs. J. Morph. 222: 175-190. Ziswiler V. a n d Farner D. S. 1972. Digestion a n d the digestive system. In: Avian Biology, vol. 11. D. S. Farner and J. R. King (eds.). Acad. Press, New York, NY, pp. 343-430. Zusi R. L. 1993. Patterns of diversity in the avian skull. In: The Skull, Vol. 2. Patterns of Structural and Systematic Diversify. J. Hanken and B. K. Hall (eds.). Univ. Chicago Press, Chicago, IL, pp. 391-437. Zweers G. A. 1985. Generalism and specialism in avian mouth and pharynx. In: Functional Morphology in Vertebrates (Fortschr. Zool. 30). H.-R. Duncker and G. Fleischer (eds.). Gustav Fischer Verlag, Stuttgart, pp. 189-201. Zweers G. A. and Berkhoudt H. 2001. Ingestion in birds. In: Encyclopedia of Life Sciences. Macmillan Publ., London, LTK. Zweers G. A., Gerritsen A. F. C. and van Kranenburg-Vood P. J. 1977. Mechanics of feeding of the mallard (Anas platyrhynchos L.; Aves, Anseriformes). Contrib. Vert. Evol. Karger, Basel. Zweers G. A., Berkhoudt H. and Vanden Berge J. C . 1994. Behavioral mechanisms of avian feeding. In: Biomechanics of Feeding in Vertebrates (Advances in Comparative & Environmental Physiology 18). V. L. Bels, M. Chardon, and P. Vandewalle (eds.). SpringerVerlag, Berlin, pp. 241-279. Zweers G. A., Vanden Berge J. C. and Berkhoudt H. 1997. Evolutionary patterns of avian trophic diversification. Zoology 100: 25-57. Zweers G . A., d e Jong F., Berkhoudt H. and Vanden Berge J. C. 1995. Suspension feeding in flamingos (Phoenicopferus ruber). Condor 97: 297-324.

Concepts of Digestive Efficiency Ian D. Hume University of Sydney, Biological Sciences, NSW, Australia

SYNOPSIS This chapter defines digestibility (digestive efficiency) and addresses three issues associated with the concept of digestive efficiency: the problem of terminology and confusion by some workers of digestibility with metabolizability;the trade-off between maximizing digestive efficiency and rate of net energy gain; and the various factors that affect digestive efficiency.The main animal factors include mean retention time of food in the digestive tract, food particle size and effectiveness of mastication, level of food intake, digestive tract capacity and morphology, hydrolytic enzyme activities, and absorptive capacities.

The potential value of a food for supplying energy or a particular nutrient can be determined by chemical analysisbut the actual value of the food to the animal can be arrived at only after allowing for the inevitable losses that occur during digestion, absorption, and metabolism. Figure 2.1 shows the various avenues of loss for food energy.Of the three main avenues of nutrient loss, that in the feces is usually the greatest by far and also the easiest to measure. Hence, digestibility, the proportion of food not appearing in the feces (Fig.2.1),is used as an attribute of a food almost as much as its chemical composition. Stated another way, digestibilityis the ratio of food absorbed to food ingested. Ratios such as this may be the only meaningful way of interpreting some biological processes (Sokal and Rohlf, 1995),but there can be statistical problems with their use, especially in correlation analysis (Raubenheimer, 1995). Transformation of the data to render them approximately normally distributed can be used to overcome some of the

44

Physiological and ecological adaptations t o feeding in vertebrates

statistical drawbacks (Sokal and Rohlf, 1995).Digestibility (or digestive efficiency) is an example of a biologically meaningful ratio. It has important implications in studies of nutritional ecology, resource exploitation, and energy flow through ecosystems. Its measurement is also critical in the design of feeding systems to maximize production efficiency in commercial livestock feeding programs. Thus there are two distinct contexts in which digestive efficiency can be discussed: the nutritional ecology of wild animals and livestock production systems. The term "digestibility" is more commonly used in livestock production circles, and "digestive efficiency" in the nutritional ecology of wild animals. They are equivalent terms.

U+G=U,+G, +Ue+Ge

- Heat increment of feeding (HIF) - Foraging - Tissue synthesis

Fig. 2.1. Relationships among energy terms used in this chapter, modified from Kleiber (1961). Fe, Ue, and Ge denote energy of endogenous origin in feces, urine, and gases respectively. F,, U,, and G, denote energy of food origin in feces, urine, and gases respectively. The total heat production is divided into basal metabolic rate (BMR) which has a fixed and a variable (flexible) component, and other processes and activities. The efficiencies depicted are apparent efficiencies; for true efficiencies F, U, and G in these equations should be replaced by F,, U,and G, respectively.

Concepts of digestive efficiency

45

In the ecological context two points should be considered before exploring factors that affect digestive efficiency. The first is the choice of the most appropriate unit of measurement. In most cases it is probably energy rather than protein or some other specific nutrient because animals tend to eat to meet their requirements for energy under most circumstances.This is particularly so for birds and mammals because of the high energetic costs of endothermy. However, it is not always convenient or possible to measure the energy content of food and feces and dry matter may be more appropriate. The second point is the question of whether animals attempt to maximize digestive efficiency, or try to maximize the rate of digestible energy intake or some more specific nutrient. Karasov and Hume (1997)concluded that the structure and function of the gastrointestinaltract did not necessarily evolve toward maximal digestive efficiency. They proposed that a more appropriate evolutionary goal might be maximization of the rate of extraction of nutrients, be it in terms of energy or some other limiting resource. Penry and Jumars (1987) in their application of chemical reactor theory to animal digestive systems, defined their design objective as the maximum conversion of ingested food to assimilableproducts in the minimum of time and gut-reactor volume. This chapter accepts the premise that for most foraging animals the objective is to maximize the rate of net energy gain. This premise is the basis for the simple model of digestion introduced by Sibly (1981;Fig. 2.2). In this model the rate of release of net energy is initially negative as energy is expended by the animal in overcoming the physical defenses of the ingested food, such as the chitinous exoskeletonof arthropods or tough seed coats. This is followed by a period of rapid digestion (e.g. of hemolymph and soft tissues of arthropods, and the contents of plant cells), but thereafter digestion rate falls as digestion is progressively confined to less tractable food components such as the structural proteins of animal tissues and the structural carbohydrates (cellulose and hemicelluloses) of plant cell walls. Optimal digestion time is given by the straight line from the origin tangential to the curve. A

?

Maximal rate of digestion Energy

food

B

/

t

high

Optimal digestion ;/time short

+ Mean retention time of food

Fig. 2.2. Model of digestion in a continuous-flow system for a high-quality (A) and a lowquality (B) food. Modified from Sibly (1981) by Hume (1989).

46

Physiological and ecological adaptations t o feeding in vertebrates

Several predictions arise from the Sibly model. First, optimal digestion time will vary among foods, being longer for poor-quality foods (e.g. hardbodied adult insects) than those of higher quality (soft-bodied insect larvae) (Fig.2.2). Second, because of longer digestion times, animals routinely eating poorer quality foods should have larger digestive tracts (e.g.herbivores versus carnivores, larger versus smaller animals of the same species, and larger-size species of similar gastrointestinal tract morphology such as colon fermenters; Hume, 1989). Third, if gastrointestinal tract capacity is limiting (as in young animals), the optimal strategy is to maximize digestion rate by selecting only high-quality foods. Finally, at any given level of intake, an animal should maximize retention of food in order to maximize the amount of energy absorbed (Sibly, 1981). This is particularly so when food availability is limited, although clearly there is a holding time beyond which net loss occurs. These predictions are explored in the rest of this chapter.

DEFINITIONS AND TERMINOLOGY There is some confusion in the literature as to what is actually being measured by digestive efficiency, partly because synonyms for digestive efficiency are loosely employed. Efficiency implies a ratio, usually between output and input. Digestive efficiency is the ratio (often expressed as a percent) between output in terms of dry matter, energy, or a nutrient absorbed (i.e.not eliminated in the feces) and its intake. Feces consist only of undigested food residues and metabolic products (including bacteria) of the gut and not renal excretory products. Absorption is usually taken to be synonymous with digestion, that is, an item digested is assumed to be absorbed. Thus equivalent terms for digestive efficiency are absorbability (Kleiber, 1961) and absorption efficiency (Speakman, 1987). The assumption of synonymy between digestion and absorption is probably valid in a goodly majority of cases, but instances in which the absorptive capacity of the gut can be exceeded are known (see below). Other alternative terms for digestive efficiency are extraction efficiency (Karasov, 1996;Karasov and Hume, 1997), assimilable mass coefficient, and utilization efficiency (Guglielmo and Karasov, 1993). "Digestibility", "digestion coefficient" or "digestive efficiency" are the preferred terms because they clearly exclude considerations of the efficiency with which nutrients are utilized once they have been absorbed. Also eliminated in the feces are materials not directly of dietary origin but from the animal itself. Of metabolic origin, they are referred to as metabolic fecal dry matter or metabolic fecal nitrogen (MFN) for example. An alternative to and perhaps more precise term than MFN is nondietary fecal nitrogen (NDFN)(Mason,1969)because the microbial component of MFN is not strictly of endogenous (animal) origin. If metabolic fecal material is not accounted

Concepts o f digestive efficiency

47

for, digestibility is "apparent digestibility", as distinct from "true digestibility" if allowance is made for metabolic fecal material. Unless reported digestibilities are defined as true digestibilities they can be assumed to be apparent digestibilities.True digestibility is the ratio of quantity absorbed to quantity ingested (Sibly, 1981) and is always greater than apparent digestibility by the amount of metabolic fecal material (Van Soest, 1994). Measurement of metabolic fecal material is fraught with difficulties. For dry matter and energy it means measurement of fecal output at zero intake of dry matter. Long collection periods from starving animals would be necessary but not ethically acceptable. Also, loss of metabolic fecal material at zero food intake may not reflect losses at higher intakes. Determination of metabolic fecal nitrogen is based on measurement of fecal output on a nitrogen-free diet, but animals usually refuse to eat such unbalanced food. Alternatively, fecal output at zero intake can be estimated by extrapolation from a range of diets of decreasing nitrogen concentration, but rarely can this be done with any degree of confidence. Whenever true digestibilities of energy or dry matter have been estimated in animals at or above maintenance (bear in mind the difficulties in doing this) the differencebetween true and apparent digestibility has usually been small, less than two percentage units (Miller and Reinecke, 1984). However, at low levels of food intake it can be greater, which leads to erroneous ctsnclusions about the efficiency with which energy is extracted from a particular food (Guglielmo and Karasov, 1993).The problem is most acute for protein because fecal metabolic losses of nitrogen are so large, equivalent to about 4 percentage units of crude protein (nitrogen times 6.25). Thus when dietary crude protein falls to 4% of dry matter, as it does in some cereal straws, apparent digestibilityof nitrogen falls to zero, even though its true digestibility may be 85-90% (Van Soest, 1994).For some minerals it is essential to distinguish in the feces that portion which is unabsorbed material from that portion excreted from the body, perhaps by labeling the body pool of that mineral with a radioactive isotope. This is especially so for calcium. Feces are the principal means of elimination of calcium from the body. Apparent digestion coefficients for this mineral are quite meaningless (McDonald et al., 1995). In amphibians, reptiles and birds, feces cannot readily be separated from urine. Inclusion of urine in the excreta yields "apparent metabolizability" rather than apparent digestibility, a distinction often not made in the literature. The problem is greatest with the total collection method of determining digestibility. To minimize the problem, Speakman (1987) used the ratio of natural ash content (ash equivalent) of excreta and food of birds to calculate apparent digestive efficiency, with the assumption that negligible dietary ash was excreted in the urine. However, the result is a compromise at best and does not negate the need for using "metabolizability" whenever urine is included in the excreta.

48

Physiological and ecological adaptations to feeding in vertebrates

True metabolizability takes into account endogenous urinary losses as well as metabolic fecal losses. Methods for estimating endogenous urinary losses are analogous to methods for estimating metabolic fecal losses and suffer from similar shortcomings. Endogenous urinary nitrogen is particularly sensitive to the energy and protein status of the animal. For instance, catabolism of protein to meet essential energy needs elevates endogenous urinary nitrogen (Kleiber,1961).

-

Iguana SNAIL

rusta ace ins

Horse

-

Fish

(LIVEI

-

Orthoptera

. I

Lepidoptera

Change

DRIED

~n

FRESH

61rd(Chicken) Oiptera

Carcass

Hornoptera

Hymenoptera Beef

I

..** oleop pi era

-

Henegg * * * * * ' mollusc^

~soptera

**,

-

~=I!JNG~L

MYCELLA Soyabean

Legumes Pea Peanuts

Otl seed

Cereals

RE PLANTS

Cheslrmts

Acorns

Leaves

I

I

I

I

20

40

60

80

FM FLUID PHLOEM FI UID

Water content (% wet mass)

Fig. 2.3. Materials used by animals as food arranged according to their water (abscissa) and total nitrogen (ordinate) contents, two measures of quality. The nitrogen contents for phloem and xylem are percent of fresh mass; all other nitrogen contents are percent of dry mass from Chivers and Langer (1994) and Slansky and Scriber (1985).

Concepts o f digestive efficiency

FACTORS AFFECTING DIGESTIVE EFFICIENCY The wide variation in digestive efficienciesobserved in nature can be attributed to both dietary and animal effects.

Dietery Effects The diets of vertebrates range from microbial through animal to plant and fungal tissues (Fig. 2.3).In general, animal tissues are more digestible than plant tissues but exceptions include the low digestibility of the exoskeleton of invertebrates and the endoskeleton of vertebrate prey. Plant material can be divided into four major components: cell contents, cell walls, exudates, and secondary metabolites. The digestibility of plant material is often determined by the ratio of cell walls to cell contents (Hume, 1989). Cell contents include cytoplasmic elements that are largely soluble (e.g. enzymes, organic acids, simple sugars) and storage forms of energy (e.g.starch, triacylglycerols). Fruits, seeds and young leaves are high in cell contents and are therefore generally of high digestibility. Cell walls contain (in order of increasing difficulty to digest)pectin, hemicelluloses, cellulose, and lignin. Ligrun is virtually indigestible in the anaerobic conditions of the vertebrate digestive system and reduces the digestibility of hemicelluloses and cellulose by forming a physical barrier between them and hydrolytic enzymes secreted mainly by symbiotic microbes in the digestive tract. Mature leaves and other structural parts of plants (stems,petioles) are high in cell wall content and are therefore highly variable but relatively low in digestibility. Roots may be highly fibrous (and hence of low digestibility)or may contain storage forms of carbohydrate (starch, oligosaccharides)and hence be of higher digestibility. Plant exudates include (in increasing difficulty to digest) nectar, saps, gums and resins. Nectar and saps consist mainly of water and sugars, gums are mainly polymers of sugars and approach hemicellulose in degree of difficulty of digestion, and resins are virtually indigestible. Plant secondary metabolites interfere with digestion (e.g.tannins) or with metabolism of the animal and inhibit food ingestion (e.g.terpenes). Dietary characteristics thus play an important role in determining digestive efficiency. The effects of diet are often clear but in other cases differences in digestive efficiency among animals can be subtle, but nevertheless significant. It is therefore essential that comparisons in digestive efficiency across animal taxa are made on a common diet. This is often not done, leading to misleading statements about taxonomic differences that are confounded by dietary effects. Animal Effects Once potential differences in digestibility of foods are controlled, the greatest differences in digestive efficiency among vertebrates are between taxa. The

50

Physiological and ecological adaptations t o feeding in vertebrates

most potent factor affecting digestive efficiency is likely to be mean retention time (MRT),which is the average time that food stays within the gastrointestinal tract. MRT determines the time that dietary substrates are subjected to attack by digestive enzymes (Fig.2.2). These enzymes come principally from the animal itself in the case of carnivores and some exudivores (animalsthat feed on some form of plant exudate), but in omnivores and herbivores digestive enzymes come from both the animal and mutualistic microorganisms (bacteria, protozoa and sometimes anaerobic fungi) in the gastrointestinal tract. Stevens and Hume (1998) emphasized the ubiquitous role that gut microbes play in the conversion and conservation of nutrients in all vertebrates. Mean retention times are determined by factors such as particle sizes of solid digesta and thus the effectiveness of mastication, level of food intake, and gastrointestinal tract capacity and morphology (Stevens and Hume, 1995), as well as the suite of hydrolytic enzymes in the small intestine, nutrient absorption rates, and the location and size of the microbial population in the hind gut (cecum and proximal colon) and/or foregut (forestomach) of herbivores. The relationship between digestive efficiency and MRT depicted in the Sibly model (Fig.2.1) is well illustrated by the study of Hilton et al., (2000a)in which eight North Atlantic seabird species were offered two fish species commonly found in the diet of wild seabirds. There were small but statistically significant differences in digestive efficiency among the seabirds. True metabolizable energy coefficients (Miller and Reinecke, 1984)varied between 75% in black-legged kittiwakes and 83%in northern fulmars on a common diet of sand eel, and between 77% and 84% in the same two species on a common diet of whiting. Correlation was positive between digestive efficiency and mean retention time, which Hilton et al. (2000a)suggested represented a trade-off between the conflicting benefits of maximizing digestive efficiency and minimizing time spent feeding and digesting food. Retention time of digesta in the stomach was the most important component of total tract MRT and was greater in species with larger stomachs. Mean retention time of digesta depends not only on gut capacity but also on the rate of digesta flow, so that MRT (h)is proportional to digesta volume (mL) / digesta flow (mL/h-') (Karasov, 1996). The factor most strongly influencing digesta flow rate is food ingestion rate. Thus, in the absence of any change in gut capacity (digesta volume), an increase in food intake will reduce MRT and, in turn, digestive efficiency. However, intake of metabolizable energy may be maximized in the process. Thus there is an apparent trade-off between digesta MRT and digestive efficiency, and animals do not always attempt to maximize digestive efficiency. Hilton et al. (2000b)concluded that rapid (and thus less complete) digestion is likely to be favored when the energy costs of commuting between feeding and nesting sites are large and there is selective advantage in maximizing metabolizable energy intake. In contrast, slow (but more complete) digestion is preferable when

Concepts of digestive efficiency

51

commuting costs are small. In raptors, rapid digestion appears to be associated with a pursuit foraging mode, where the weight savings that can be achieved through rapid digestion (smaller gut capacity, smaller digesta load) exceed the costs in reduced digestive efficiency (Hilton et al., 1999). In contrast, slow digestion tends to be found in species with a searching foraging mode. These species may be able to exploit a wider range of food types, including lower quality prey, than raptors that actively pursue aerial prey. If juveniles are poorer foragers than adults and thus not able to always select high-quality foods (as predicted above from the Sibly model), they may be expected to maximize efficiencyby having slower passage rates and larger guts (Castro et al., 1989; Jackson, 1992; Barton and Houston, 1993). Particle size and mastication Oral processing is the first stage of digestion in most vertebrates. In most fishes, amphibians, reptiles, and birds, oral processing may result in only limited comminution of food, but in mammals the teeth act together with soft tissues to fracture food particles inside the mouth before swallowing (Lucas, 1994). The more finely the food is ground, the greater the surface area available for attack by digestive enzymes and bacteria. The efficiencywith which food is comminuted in the mouth of mammals depends malnly on the degree of tooth wear, and thus with age of the animal. For instance, Gipps and Sanson (1984) showed that as common ringtail possums (Pseudocheirus peregrinus) aged their teeth became more worn. As a result, the ratio of small particles (< 280 pm) to large particles (> 560 pm) of Eucalyptus leaf in their stomach decreased from 0.85 to 0.55, and the digestibility of dry matter decreased by 7% and that of cell walls by 46%. In koalas (Phascolarctos cinereus), both very young and very old animals were found to be less efficient in comminuting eucalypt leaves, indicating that some tooth wear is necessary in order to maintain maximal total length of cutting edges of the molars (Lanyon and Sanson, 1986). The implication is that digestive efficiency is likely to be greatest in young adult koalas (Hume, 1999). Level of food intake If digestive tract capacity remains constant, increased rates of food intake lead to shorter MRTs and, in turn, lowered digestive efficiency. The effect is likely to be greatest on low-quality (low-digestibility)foods. Thus, on a highfiber synthetic diet of 41% neutral-detergent fiber (NDF),common brushtail possums (Trichosurus vulpecula) eating 10 g dry matter.kg-o-75. dayldigested 88% of dry matter and 77% of NDF consumed, but those eating 50 gkg-0.75dayldigested only 71% dry matter and 42% NDF (Wellard and Hume, 1981). However, digestible energy intake remained constant, supporting the contention that animals tend to maximize the rate of extracting energy rather than digestive efficiency. On a low-fiber (17% NDF) diet there was no effect of feeding level on digestive efficiency. Similar responses were reported in ruminants fed early- and late-cut hays (Blaxter, 1962).

52

Physiological and ecological adaptations t o feeding in vertebrates

Digestive tract capacity Animals are often able to accommodate increased food loads if given time to acclimatize to the new condition. There appear to be two ways that animals meet increases in energy requirements and thus food intake: 1)more rapid digesta flow through a digestive tract of unchanged dimensions, which results in lower digestive efficiencies because ingested food is exposed to digestive processes for shorter periods; or 2) more rapid digesta flow through an enlarged digestive tract, resulting in similar turnover times and MRT, with no change in digestive efficiency. Karasov and Hurne (1997)concluded that for modest increases in energy requirements, the first alternative probably operates, with a small reduction in digestive efficiency but an increase in metabolizableenergy intake. The response of meadow voles with thyroxine implants eating 40% more than maintenance followed this strategy (Derting and Bouge, 1993). For larger increases in energy requirements, the second alternative appears to hold; more rapid digesta flow through a larger digestive tract so that MRT does not change and digestive efficiency remains the same, with a significant increase in the rate of metabolizable energy intake. The response patterns of mice (Toloza et al., 1991)and house wrens (Dykstraand Karasov, 1992)exposed to cold most closely fitted this strategy; digestive tract capacity increased, MRT did not change, and feeding rate more than doubled with no decrease in digestive efficiency. A special case of this latter response was reported in the garden warbler (Sylvia borin), a long-distancemigratory species. Bairlein (1985) and Hume and Biebach (1996) found that the 33% increase in daily food intake during the fattening period just prior to the autumnal migratory flight from Europe to Africa was associated not just with maintenance of energy metabolizability but with increased energy metabolizability. Hume and Biebach (1996) showed that MRT did not change, indicating that digestive tract capacity must have increased substantially to accommodate greater gut loading. Why the birds do not retain this peak digestive efficiency outside the premigration fattening period is a question that requires an answer (Biebach, 1996).It may be related to the costs of maintaining such a large mass of gut tissue; the gastrointestinal tract is responsible for a disproportionately high fraction of whole-body protein turnover and energy utilization (McBride and Kelly 1990).During the migratory fight itself there is atrophy of the digestive tract, which saves weight, reduces the maintenance cost of a tissue not being used, and partially fuels the flight. After a 48-hour period of starvation of fattened birds to partially simulate a migratory flight over desert, digestive tract tissue mass fell by 50% and that of the small intestine by 63% (Hume and Biebach, 1996). Note that the small intestine is by far the most active tissue of the whole tract; in alpine marmots (Marmota marmots), mitotic indices in the small intestine ranged from 40% to 60% during the active (feeding)season, but in the stomach, cecum, and colon mitotic indices never rose above 4%

Concepts of digestive efficiency

53

(Hume et al., 2002). Thus the disadvantage of maintaining peak digestive efficiency during long migratory flights is clear. It may be that at other times of the year when energy demands are lower, the costs of maintaining a large gut tissue mass outweigh the energetic benefits of maintaining digestive efficiency at a peak level. Another way an increased food load may come about is through a decrease in food quality. Increases in digestive tract capacity have been reported in a range of types and sizes of mammalian herbivores in response to seasonal or experimental increases in the plant cell wall (fiber) content of the diet (e.g. Gross et al., 1985; Green and Millar, 1987; Yahav and Choshniak, 1990; Nagy and Negus, 1993; Hammond and Wunder, 1995; Loeb et al., 1991; Bozinovic, 1995; Castle and Wunder, 1995). In each case, although digestive efficiency was depressed by the increased fiber content of the diet, higher food intakes meant that intakes of metabolizable energy either remained constant or declined only slightly. The greatest increases in volumetric capacity were in parts of the tract associated with microbial fermentation of plant cell walls, namely the cecum in the small hind-gut fermenters studied. Morphological features that retard digesta flow In addition to increased capacity of the digestive tract, numerous morphological features of the tract also serve to retard digesta flow. Often this affects one phase of the digesta more than another. For instance, the stomach of all vertebrates, as far as we know, selectively retards flow into the duodenum of large food particles relative to fluid, solutes, and small particles (Stevens and Hume, 1995). Selective retention of large food particles is enhanced in the stomach of foregut fermenting herbivores by compartmentalization of the forestomach into several chambers, and morphological specializations such as the reticulo-omasal orifice of ruminants and the haustrated "colon-like" forestomach of kangaroos (Hume, 1989).Large hindgut fermenting herbivores (colon fermenters) also selectively retain large particles, enhancing microbial breakdown in a large haustrated proximal colon. In contrast, many small hindgut fermenters, the cecum fermenters, feature selective retention not of the large particles but the small particles (including bacteria), along with fluid and solutes, in an enlarged cecum. This strategy enhances microbial breakdown of food in the cecum while concomitantly facilitating passage through the colon of large, less digestible food particles. This clears the digestive tract of indigestible bulk and allows much higher intakes of plant material than would otherwise be the case. As a result, cecum fermenters, most of which are below 10kg in body mass, are able to process plant material of much higher fiber content than would be predicted on the basis of body size and thus digestive tract size alone. The mechanisms responsible for selective retention of fluid, solutes and small particles in the cecum have been termed "colonic separation mechanisms" and are reviewed by Bjornhag (1987, 1994).

54

Physiological and ecological adaptations t o feeding i n vertebrates

Table 2.1. Digestive efficiency in four rodents on a common diet of commercial rabbit pellets containing 35% neutral-detergent fiber (Hume et al., 1993) Species

Colonic separation mechanism

Body mass (8)

Digestibility of dry matter ( "10

Yellow pine chipmunk Columbian ground squirrel

Yes No No

61 55 629

Hoary marmot

No

2,522

+ 1.0" + 3.0b + l.lb 50.3 + 1.2"

Townsend's vole

>

50.9 40.6 40.9

The interplay between digestive tract capacity and morphology on digestive efficiency is illustrated by the example in Table 2.1. The 25% greater digestive efficiency in the vole (Microtustownsendii)than a sciurid rodent (the yellow pine chipmunk, Eutarnias arnoenus ) of similar body size is due to the presence in the hindgut of all microtine rodents (voles and lemmings) of a colonic separation mechanism (CSM). In contrast, no sciurid rodent (the squirrels)has been found to have a CSM. A 10-fold increase in body size, and thus of absolute gut capacity in the Columbian ground squirrel (Sperrnophilus colurnbianus)did not compensate for the lack of a CSM in the sciurid digestive tract (digestive efficiency was still 25% greater in the vole), but a 40-fold increase (in the hoary marmot, Marrnota caligata) did. Thus greater gut capacity can compensate for the lack of a CSM-but only at the body sizes reached by marmots.

Hydrolytic enzymatic activity Most vertebrates can digest disaccharides in the small intestine, but with one notable, well-researched exception:American robins ( Turdus rnigratorius) and all other birds in the sturnid-muscicapid lineage (starlings, thrushes and Old World flycatchers) examined to date lack expression of the intestinal enzyme sucrase (Levey and Martinez del Rio, 2001).Consequently, those birds are unable to hydrolyze sucrose. This extreme example serves to illustrate how the digestibility of certain foods can be zero in some species but close to 100%in others. Within these extremes there are numerous examples of differences among vertebrate species in their capacity to digest fruit diets. However, the bases for the differences invariably seem not to lie in levels of hydrolytic enzyme activity or rates of nutrient uptake per unit of small intestine, but in increases in small intestinal length, mass, and surface area (McWilliams et al., 1999). Absorptive capacities Comparative aspects of the mechanisms of nutrient uptake in vertebrates were reviewed by Stevens and Hume (1995)and Karasov and Hume (1997). Absorptive capacity is the product of absorption rate per unit surface area of intestine (which depends on the transport properties of the intestinal tissue) and total surface area available for absorption. From data available at that

Concepts o f digestive efficiency

55

time, Karasov and Hume (1997)concluded that absorptive capacity among vertebrates was most strongly determined by differences in absorptive surface area rather than area-specific uptake rates. Within fish, reptiles, mammals, and birds there does not appear to be any dependence of nutrient uptake per unit surface area of intestine on body size or taxon. However, small intestinal nominal surface areas scale to body mass in all vertebrate groups to a common slope of 0.71, suggesting a match between absorptive area and food intake that scales in a similar fashion. Proportionality coefficients of nominal surface areas calculated by Karasov and Hume (1997) were 1.06,0.63,1.08,1.43,and 2.47 for fish, amphibians, reptiles, birds, and mammals respectively. Thus the higher absorptive capacities of mammals and birds than of fish, amphibians and reptiles can be explained largely by their longer small intestines (Stevens and Hume, 1995) and thus greater nominal surface areas. Total surface area for absorption includes the contribution of villi and microvilli as well, which increase the mucosal area of the intestine several orders of magnitude above the nominal area. So far, no pattern has emerged between villus or microvillus multiplication factors and taxon. On average, the villi increase the potential absorptive area of the mammalian small intestineby 6.7 times, and the microvilli by 53 times. However, nutrient uptake takes place mostly by epithelial cells closer to the villus tip, so actual absorptive surface areas are likely to be much less than potential uptake surface areas. The matches among the capacity for nutrient absorption and the capacity for chemicalbreakdown, digesta MRT, and overall digestive efficiency are of great interest. The principle of symmorphosis (Weibel et al., 1998)states that there should be close matches among these component parts of the system for delivery of nutrients to animal tissues. That is, there should not be gross overenpeering of any one component of the system, otherwise energy would be wasted in the maintenance of unused capacity and there would be a waste of physical space within the organism. However, with increased environmental stochasticitythe need for a higher than average capacity in one or more components of the delivery system might be expected in order to cope with peaks in nutrient supply. The existence of such "safety margins" (Diamond, 1998)will inevitably obscure symmorphosis. The size of any safety margin is likely to be related to a) the expected frequencyof peaks in nutrient supply, and b) the consequences of overload that is unprocessed. Digestive and/or absorptive capacity of the intestine can be exceeded, as seen in grain overload caused by fermentationof undigested grain in the hindgut of horses.

CONCLUSION Digestive efficiencyis a central concept in the nutritional ecology of animals, and digestibility (or digestion coefficient), the proportion of food not

56

Physiological and ecological adaptations t o feeding in vertebrates

appearing in the feces, is an important measurement of food quality and animal digestive performance.However, from theoretical models of the digestive tract and from accumulating evidence in the literature, it appears that animals only rarely seek to maximize digestibility. Instead, for most foragmg animals, the objective seems to be maximizing the rate of net energy gain. That is, it can be costly in terms of net energy gain to maximize digestibility. Also, because of the high maintenance costs of the gastrointestinal tract, particularly the small intestine, it may be an optimal strategy to maintain digestive efficiency at somethingbelow peak performance except at those times of the year when energy demands are especially high. Examples include the premigratory fattening period of some long-distance migratory birds, the short summer period for reproduction, growth and fattening of some rodents that hibernate at high altitudes and/or high latitudes, and the periods of intense cold faced by some high latitude nonmigratory avian species. That digestive efficiency on a given diet is not a fixed parameter attests to the highly flexible nature of the vertebrate digestive tract. Acknowledgment I thank Marcel Klaassen for his valuable insights into concepts of digestive efficiency and for discussions on how we might include the costs of maintaining the tissues of the gastrointestinaltract in future models of digestion.

REFERENCES Bairlein F. 1985. Efficiency of food utilization during fat deposition in the long-distance migrating garden warbler, Sylvia borin. Oecologia 68: 118-125. Barton N.W.H. and Houston D.C. 1993. The influence of gut morphology on digestion time in raptors. Comp. Biochem. Physiol. 105A: 571-578. Biebach H. 1996. Energetics of winter and migratory fattening. In: Avian Energetics and Nutritional Ecology. C. Carey (ed.). Chapman and Hall, New York, NY, pp. 280-323. Bjornhag G. 1987. Comparative aspects of digestion in the hindgut of mammals. The colonic separation mechanism (CSM) (a review). Dtsch. Tierarztl. Wschr. 94: 33-36. Bjornhag G. 1994. Adaptations of the large intestine allowing small animals to eat fibrous foods. In: The Digestive System in Mammals: Food, Form and Function. D.J. Chivers and P. Langer (eds.). Cambridge Univ. Press, Cambridge, UK, pp. 287-309. Blaxter K.L. 1962. The Energy Metabolism of Ruminants. Hutchinson, London, U.K. Bozinovic F. 1995. Nutritional energetics and digestive responses of an herbivorous rodent (Octodon degus) to different levels of dietary fiber. J. Mammal. 76: 627-637. Castle K.T. and Wunder B.A. 1995. Limits to food intake and fiber utilization in the prairie vole, Microtus ochrogaster: Effects of food quality and energy need. J. Comp. Physiol. B164: 609-617. Castro G., Stoyan N. and Myers J.P. 1989. Assimilation efficiency in birds: a function of taxon or food type? Comp. Biochem. Physiol. 92A: 271-278. Chivers D. J. and Langer, P. (eds.) 1994. The Digestive System in Mammals: Food, Form and Function. Cambridge Univ. Press, Cambridge, UK. Derting T.L. and Bouge B.A. 1993. Responses of the gut to moderate energy demands in a small herbivore (Microtus pennsylvanicus). J. Mammal. 74: 58-68.

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Diamond J. M. 1998. Evolution of biological safety factors: a cost/benefit analysis. In: Principles of Animal Design. E.R. Weibel, C.R. Taylor, and L. Bolis (eds.). Cambridge Univ. Press, Cambridge, UK, pp. 21-27. Dykstra C. R. and Karasov W.H. 1992. Changes in gut structure and function of house wrens (Troglodytes aedon) in response to increased energy demands. Physiol. Zool. 65: 422-442. Gipps J.M. and Sanson G.D. 1984. Mastication and digestion in Pseudocheirus. In: : Possums and Gliders. A.P. Smith and I.D. Hume (eds.). Australian Mammal Soc., Sydney, NSW, Australia, pp. 237-246. Green D.A. and Millar J.S. 1987. Changes in gut dimensions and capacity of Peromyscus maniculatus relative to diet quality and energy needs. Can. J. Zool. 65: 2159-2162. Gross J.E., Wang Z. and Wunder B.A. 1985. Effects of food quality and energy needs: changes i n gut morphology and capacity of Microtus ochrogaster. J. Mammal. 66: 661-667. Guglielmo C.G. and Karasov W.H. 1993. Endogenous mass and energy losses in ruffed grouse. Auk 110: 386-390. Hammond K.A. and Wunder B.A. 1995. Effect of cold temperatures on the morphology of gastrointestinal tracts of two microtine rodents. J. Mammal. 76: 232-239. Hilton G.M., Furness R.W. and Houston D.C. 2000a. A comparative study of digestion in North Atlantic seabirds. J. Avian Biol. 31: 3646. Hilton G.M., Furness R.W. and Houston D.C. 2000b. The effects of switching and mixing on digestion in seabirds. Funct. Ecol. 14: 145-154. Hilton G.M., Houston D.C., Barton N.W.H., Furness R.W. and Ruxton G . D . 1999. Ecological~constraintson digestive physiology in carnivorous and piscivorous birds. J. Exp. ZOO/.283: 365-376. Hume I.D. 1989. Optimal digestive strategies in mammalian herbivores. Physiol. Zool. 62: 1145-1163. Hume I.D. 1999. Marsupial Nutrition. Cambridge Univ. Press: Cambridge, U.K. Hume I.D. and Biebach H. 1996. Digestive tract function in the long-distance migratory garden warbler, Sylvia borin. J. Comp. Physiol. B166: 388-395. Hume I.D., Morgan K.R. and Kenagy G.J. 1993. Digesta retention and digestive performance in sciurid and microtine rodents: Effects of hindgut morphology and body size. Physiol. Zool. 66: 396411. Hume I.D., Beiglboeck C., Ruf T., Frey-Roos F., Bruns U. and Arnold W. 2002. Seasonal changes in morphology and function of the gastrointestinal tract of free-living alpine marmots (Marmota rnarmota). J. Comp. Physiol. B172: 197-207. Jackson S. 1992. Do seabird gut sizes and mean retention times reflect adaptation to diet and foraging method? Physiol. Zool. 65: 674-697. Karasov W.H. 1996. Digestive plasticity in avian energetics and feeding ecology. In: Avian Energetics and Nutritional Ecology. C. Carey (ed.). Chapman Hall, New York, NY, pp. 61-84. Karasov W.H. and Hume I.D. 1997. Vertebrate gastrointestinal system. In: Handbook of Physiology. Sec. 13: Comparative Physiology. W. H. Dantzler (ed.). Oxford Univ. Press, New York, NY, pp. 409-480. Kleiber M. 1961. The Fire of Life. Wiley, New York, NY. Lanyon J.M. and Sanson G.D. 1986. Koala (Phascolarctos cinereus) dentition and nutrition. 11. Implications of toothwear in nutrition. J. Zool. Lond. (A) 209: 169-181. Levey D.J. and Martinez del Rio C. 2001. It takes guts (and more) to eat fruit: lessons from avian nutritional ecology. Auk 118: 819-831. Loeb S.C., Schwab R.G. and Demment M.W. 1991. Responses of pocket gophers (Thomomys bottae) to changes in diet quality. Oecologia 86: 542-551. Lucas P.W. 1994. Categorisation of food items relevant to oral processing. In: The Digestive System in Mammals: Food, Form and Function. D.J. Chivers and P. Langer (eds.). Cambridge Univ. Press, Cambridge, LTK, pp.197-218.

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Mason V.C. 1969. Some observations on the distribution and origin of nitrogen in sheep feces. J. Agric. Sci. 73: 99-111. McBride B.W. and Kelly J.M. 1990. Energy cost of absorption and metabolism in the ruminant gastrointestinal tract and liver: a review. J. Anim. Sci. 68: 2997-3010. McDonald P., Edwards R.A., Greenhalgh J.F.D. and Morgan C.A. 1995. Animal Nutrition, Longman: Harlow, Essex, UK, (5thed.). McWilliams S.R., Caviedes-Vidal E. and Karasov W.H. 1999. Digestive adjustments in cedar waxwings to high feeding rate. J. Exp. Zool. 283: 394407. Miller M.R. and Reinecke K.J. 1984. Proper expression of metabolizable energy in avian energetics. Condor 86: 396-400. Nagy T.R. and Negus N.C. 1993. Energy acquisition and allocation in male collared lemmings (Dicrostonyx groenlandicus): Effects of photoperiod, temperature and diet quality. Physiol. Zool. 66: 537-560. Penry D.L. and Jumars P.A. 1987. Modeling animal guts as chemical reactors. Amer. Nat. 129: 69-96. Raubenheimer D. 1995. Problems with ratio analysis in nutritional studies. Funct. Ecol. 9: 21-29. Sibly R.M. 1981. Strategies of digestion and defecation. In: Physiological Ecology: An Evolutionary Approach to Resource Use. C.R. Townsend and P. Calow (eds.). Sinauer, Sunderland, MA (USA), pp. 109-139. Slansky F. and Scriber J.H. 1985. Food consumption and utilization. In: Comprehensive lnsect Physiology, Biochemistry and Pharmacology, Vol. 4: Regulation: Digestion, Nutrition, Excretion. G.A. Kerkut and L.I. Gilbert (eds.). Pergamon: Oxford, U.K. pp.87-163. Sokal R.R. and Rohlf F.J. 1995. Biometry: T? Principles and Practice of Statistics in Biological Research, Freemans, New York, NY, (3 ed.). Speakman J.R. 1987. Apparent absorption efficiencies for redshank (Tringa totanus L.) and oystercatcher (Haematopus ostralegus L.): implications of optimal foraging models. Amer. Nat. 130: 677-691. Stevens C.E. and Hume I.D. 1995. Comparative Physiology of the Vertebrate Digestive System. Cambridge Univ. Press, Cambridge, UK, (2nded.). Stevens C.E. and Hume I.D. 1998. Contributions of microbes in vertebrate gastrointestinal tract to production and conservation of nutrients. Physiol. Rev. 78: 393427. Toloza E.M., Lam M. and Diamond J. 1991. Nutrient extraction by cold-exposed mice: a test of digestive safety margins. Amer. J. Physiol. 261: G6084620. Van Soes5P.J. 1994. Nutritional Ecology of the Ruminant. Cornell Univ. Press, Ithaca, IVY, (2 ed.). Weibel E.R., Taylor C.R. and Bolis L., (eds.) 1998. Principles of Animal Design. Cambridge Univ. Press, Cambridge, UK. Wellard G.A. and Hume I.D. 1981. Digestion and digesta passage in the brushtail possum, Trichosurus vulpecula (Kerr). Ausr. J. Zool. 29: 147-156. Yahav S. and Choshniak I. 1990. Response of the digestive tra to low quality dry food in the fat jird (Microtus crassus) and the levant vole (Microtus guentheri). J. Arid Environ. 19: 209-215.

Carbohydrate Hydrolysis and Absorption: Lessons from Modeling Digestive Function Todd J. McWhorter

Department of Wildlife Ecology University of Wisconsin, Madison, WI, USA

SYNOPSIS Nutrient assimilation is a complex phenomenon involving numerous enzymatic and transport pathways taking place in a variety of organs within the gastrointestinal tract (GIT). The structure and surface area of the GIT, factors affecting motility and thus the throughput of digesta, and the GIT's ability to chemically break down or ferment macromolecules and then absorb the resultant products affect an animal's ability to extract nutrients from its food. Digestive physiologists tend either to focus narrowly (working at the cellular, biochemical and molecular levels), or to analyze digestive performance at the whole-animal level (measuring digestive efficiency or retention time of food in the gut). As a consequence of this dichotomy, we have understood very little about how the fine details of nutrient digestion and uptake affect whole organism digestive efficiency and food intake rates until relatively recently. Since the pioneering work of Penry and Jumars and Martinez del Rio and Karasov, digestive physiologists have borrowed concepts from chemical reactor theory to model gut function. These models have provided the theoretical framework needed to integrate digestive processes with gut morphology and the chemical properties of food. This chapter reviews the application of chemical reactor theory to the study of digestion and digestive capacities in vertebrates, using recent studies of sucrose hydrolysis and hexose absorption in nectar- and fruit-eating birds as examples.

MODELING GUT FUNCTION: LESSONS FROM NECTAR- AND FRUIT-EATING BIRDS*

In the past two decades, models adopted from optimality theory (Sibly,1981) and chemical reactor theory (see for example Froment and Bischoff,1990; * See Table 3.1 for explanation of symbols and units

60

Physiological and ecological adaptations t o feeding in vertebrates

Levenspiel, 1998;Carberry, 2001) have provided the theoretical framework needed to integrate digestive processes with gut morphology and the chemical properties of food. Such models are useful because they: (1)clarify the relationships of gastrointestinal tract (GIT)attributes to one another, (2)identify those aspects of the GIT that determine the rate and efficiency of nutrient extraction from food, and (3)reduce the complexity of GIT systems within and between phyletic lines (Karasov and Hume, 1997). Models are important tools in mechanistic and comparativestudies of complex systems. They also provide a means for making inferences about whole-animal function from digestive processes, and are thus of ecological importance. Penry and Jumars (1986,1987)first recognized that gut function could be modeled using analogies to man-made chemical reactors. The task of the digestive physiologist parallels that of the chemical engineer evaluating the performance of reactor designs (gut functional morphologies)with the goal of maximizing yield (energy or nutrient assimilation, the optimizationcriterion or design objective),given a series of chemical reactions (digestivehydrolysis and nutrient uptake) taking place within these reactors (Martinez del Rio et al., 1994). Dade et al. (1990) and Martinez del Rio and Karasov (1990) extended the approach to include both hydrolysis and absorption and to predict the ingestion rate that maximizes net rate of absorptive gain to the animal. The first generation of chemical reactor models were "strategical" (sensu Levins, 1966),providing primarily qualitativepredictions, sacrificing precision to realism and generality (Martinez del Rio et al., 1994).This approach differed from previous "tactical" attempts (Levins, 1966)to model gut function, which emphasized precision, were complex and rich in detail but had limited generality. Newer generations of reactor models include significantly more physiological and ecological detail but the accompanying gain in precision comes at a loss of generality. The application of reactor theory to digestive processes has significantly broadened our perspective and allowed us to recognize two important things. First, that the tubular portions of the gut are not merely connectors, but reaction vessels in their own right. Second, that considering animal guts as cornbinationsof different kinds of reaction vessels (e.g.tubular, mixing, batch) raises important questions about the relative function of these vessels, how they interact, and what combination of vessels is optimal under different physiological and ecological conditions (see for example, Alexander, 1994). Reactor theory has allowed us to appreciate the significance of guts with widely ranging proportions of mixing to nonmixing compartments. The approach can be used to model alternate ways in which foods can be processed (Sibly, 1981; Penry and Jumars, 1987)and thus aid in our understanding of GIT evolution (Prop and Vulink, 1992; Karasov and Cork, 1996;Karasov and Hume, 1997). Chemical reactor theory is general and flexible: it permits the study of digestion in animals with diverse diets and digestive modes under a common paradigm (Martinez del Rio et al., 1994). It has been applied to

Modeling digestive function Table 3.1. Symbols and units Symbol Definition

Units

pmol-pL-' substrate or dietary sugar concentration pmol.pL-I glucose concentration per unit volume of digesta sucrose concentration per unit volume of digesta pmol.pL-I pmol-pL-I initial sucrose concentration per unit volume of digesta pmol.pL-' final sucrose concentration per unit volume of digesta min-' coefficient of intestinal passive permeability of hexoses pmol-pL-' Michaelis constant (K,) of intestinal uptake of glucose Michaelis constant (K,) of intestinal hydrolysis of sucrase pmol-pL-' reaction or uptake rate pmol.(pL.min)-' pmol.(pL.min)-' rate of glucose uptake in the intestine rate of hexose (glucose and fructose) uptake in the intestine pmol.(pL.min)-' rate of sucrose hydrolysis in the intestine pmol.(pL.min)-' intestinal throughput or retention time min optimal intestinal throughput or retention time min min intestinal throughput time that maximizes extraction efficiency pL-mini digesta flow rate in the intestine or food intake rate gut volume PL maximal rate (Vmax) of glucose uptake in the intestine pmol.(pL.min)-' pmol.(pL.min)-' maximal rate (Vmax) of hexose uptake in the intestine maximal rate (Vmax) of sucrose hydrolysis in the intestine pmol.(pL.min)-' reaction efficiencv

understanding the relationship between ingestion and downstream digestive processes in a wide variety of taxa, including marine invertebrates (e.g. Penry and Jumars, 1990;Plante et al., 1990),herbivorous mammals (e.g.Hwne, 1989),carnivores (Cochran, 1987),nectar- and fruit-eating birds (e.g.Martinez del Rio and Karasov, 1990; Lopez-Calleja et al., 1997; Levey and Martinez del Rio, 1999; McWhorter and Martinez del Rio, 2000), insectivorous birds (Dykstra and Karasov, 1992), and marine herbivorous fishes (Horn and Messer, 1992).Indeed, chemical reactor theory is so general and flexible that it is also widely applied by biologists, hydrologists, and geochemists in examining transport time scales (e.g. residence or retention time of water or solutes in bodies of water, see Monsen et al., 2002). The purpose of this chapter is not to provide a comprehensive review of the application of reactor engineering concepts to the study of digestion, nor to provide detailed theoretical derivations for reactor models. Interested readers are referred to the fine reviews by Martinez del Rio et al. (1994) and Karasov and Hume (1997) for the former, and to Penry and Jumars (1987), Dade et al. (1990), Martinez del Rio and Karasov (1990),Horn and Messer (1992),Jumars and Martinez del Rio (1999),and Jumars (2000a, 2000b) for the latter. In addition,

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Physiological and ecological adaptations t o feeding in vertebrates

any chemical engineering textbook (e.g. Levenspiel, 1998) will provide a formal exposition of reactor performance analysis. Rather, the purposes of this chapter are to: I ) provide readers with a brief introduction to chemical reactor models and 2) review the recent and rapidly accelerating use of nectar- and fruit-eating birds as valuable test cases for the description of guts as chemical reactors and of optimization premises.

Guts as Chemical Reactors Building mathematical models of the digestive process using the reactor approach relies on three steps. (1)An optimization criterion (or design objective, Penry and Jumars, 1987)must be defined. Researchers have proposed that maximization of the rate of nutrient and/or energy extraction is an appropriate criterion, although many others are possible (e.g.extraction efficiency may be useful for examiningboth mechanistic and evolutionary questions; see Prop and Vulink, 1992;Karasov and Cork, 1996). The former will be a primary focus of my discussion, as it was the criterion adopted in most early models of gut function in nectar- and fruit-eating birds (e.g.Martinez del Rio and Karasov, 1990; Martinez del Rio et al., 1994).(2) Explicit analogies between gut morphologies and reactor designs must be established. Penry and Jumars (1987)identified three basic types of reactors analogous to GIT organs: batch reactors, continuous-flow stirred tank reactors (CSTR), and plug-flow reactors (PFR).Complex gut functional morphologies can be modeled by setting two or more reactors in series or parallel (Levenspiel, 1998). (3)Physiological measurements such as affinities and maximal rates of enzymes and transporters must be employed as parameters in performance equations to predict reactor configurations and/ or digestive behaviors that maximize the optimization criterion (Hume, 1989; Dade et al., 1990; Martinez del Rio et al., 1994; Karasov and Hume, 1997). Modeling guts as chemical reactors requires one to make, and hence eventually validate, explicit assumptions about the digestive process (see Martinez del Rio et al., 1994),but it also allows one to make crisp falsifiablepredictions about wholeorganism outcomes from measurements done in vitro at the organ and tissue level (Martinez del Rio and Karasov, 1990). The important interrelationships in digestive function that facilitate use of reactor theory are outlined below, followed by a description of the morphological analogies that have been established between guts and reactors. The physiological measurements employed as parameters in performance equations are discussed in the section Reactor Models Applied to Nectar- and Fruit-eating Birds. General features of digestion models Three distinct phases of digestion can be distinguished from a simple graphical model conceived by Sibly (1981)and refined by Karasov and Hume (1997) (Fig. 3.1). First, time and energy are invested in extraction (mechanicaland chemical breakdown and fermentation);next, there is a phase of rapid absorption, and third the rate of absorption declines as digestion is completed.

Modeling digestive function

63

Net energy or nutrient gain (defined as the amount absorbed minus the amount invested in digestion, Sibly, 1981)is a positive function of the time that digesta is retained in the GIT (the reaction chamber).For a given retention time, z, net gain is a positive function of reaction rate, r. For the GIT, r is equal to the rate of extraction and absorption (essentially the instantaneous slope of the curve f(z) in Fig. 3.1).The efficiency of any given reaction, X, is defined as the amount of product formed or nutrient absorbed relative to the initial amount of substrate present, C. Gut functional morphology affects digesta retention time, or the time required to process one reactor volume of digesta input (Penry and Jumars, 1987). Given that digesta flow, v,,, is constant, as the volume, V, of any region of the gut increases the retention time in that region also increases. As gut motility, and hence flow of digesta, increases, the retention time decreases given that V is constant.

I

Digestion corr~plete Slope = f(~)/a,or net rate of extraction

I

Ingestion Fig. 3.1. A simple graphical model of digestion. The net amount of energy or nutrient obtained (f(z), on the y-axis) is a positive function of retention time (7, on the x-axis). f(z) may decline with time during the initial phases of extraction (between the time of ingestion and absorption of breakdown products) but then increases rapidly during the phase of maximal absorption. As digestion nears completion, the rate of absorption declines. f(z) has the same form as digestibility (extraction efficiency) plotted as a function of retention time. The maximum net rate of extraction, f(z)/z, occurs at the retention time z* where a line through the origin is tangent to f(z)),while the efficiency of extraction is maximized at z" (adapted from Sibly, 1981; Karasov and Hume, 1997).

64

Physiological and ecological adaptations t o feeding i n vertebrates

These relationships can be integrated by a general model presented by Karasov and Hume (1997): ( X . C ) / r rn z rn V/v, (1) The relationships in Figure 3.1 and eqn. (1)apply to the digestion of whole meals, as well as to specific reactions (e.g. breakdown of disaccharides or uptake of hexoses) in the GIT or portions there of, and to models of different kinds and combinations of reactor types (below).

Morphologicalanalogies Modeling gut function is facilitated by establishing analogies between digestive organs and one or a combination of several reactor types. Chemical engineers recognize three basic types of reactors: batch reactors, continuous-flow stirred tank reactors (CSTR),and plug-flow reactors (PFR). The ideal forms of these reactors and their performance characteristics are briefly described here and examples of gut structures analogous to each reactor type are provided. Batch reactors process substrates (reactants) in discrete batches. Reaction periods alternate with idle periods during which the reactor is emptied of reaction products and unreacted components and reloaded. This interruption of material flow may result in a low overall extraction rate capacity unless the volume of the reactor is very large. The contents of an ideal batch reactor are perfectly mixed (i.e. spatially homogeneous at any given time); changes in reactant concentration occur only with respect to time. Figure 3.2a shows the change in concentration of products and reactants over time in an ideal batch reactor. Examples of batch reactors are found in coelenterates (Yonge, 1937), and in the blind compartments of vertebrate guts (e.g. ceca of herbivorous birds or the stomachs of carnivores, Cochran, 1987;Duke, 1989),assuming that meals are processed as separate batches. Continuous-flowstirred tank reactors are characterized by a continuous flow of reactants into and products out of a perfectly mixed reaction chamber. The composition of reactants within a CSTR operating at steady state are spatially homogeneous and constant with time. Incoming substrates are immediately diluted by recirculating materials upon entry, which reduces reaction rate, but efficiency can be high if the flow rate is low enough (i.e. retention time is long enough). Figure 3.2b shows that efficiency and rate of product formation are a function of flow rate through a CSTR. These conditions are thought to occur in the reticulorumen of the ruminant stomach, the camelid forestomach,and the sacciform region of the rat-kangaroo forestomach (Hume, 1989). These examples deviate from ideal CSTR conditions in that input is not continuous but outflow is modulated and probably more continuous. Plug-flow reactors consist of (usually) tubular reaction vessels through which there is a continuous orderly flow of material. Perfect radial mixing and negligible axial mixing (along the length of the reactor) are assumed;

Modeling digestive function

- Product

Contents mixed

Reactant md OUQU?3!

Holding time (b)

8

continuous

.H

I8'

Continuous

d 0

Low flaw rate

1P

2

output

High flow rate

Interior of reactor

&

\ I

0

I

Exit

_ - - _Low _ flow rate

."Y

D

High flow rate

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_I.#--

#

','

/;

*I

t,

High flow rate Low flow rate

4IxIIlcornposition'uniform in cross-sectionat steady state, unchanging with time.

Exit Position along the reactor

0

Fig. 3.2. Ideal chemical reactors analogous to digestive organs. (a) In batch reactors the concentration of reactants and products changes over time. (b) In continuous-flow stirred tank reactors (CSTR), flow of reactants into and products out of the reactor maintains constant concentrations and reaction rates. (c) In plug-flow reactors (PFR) there is continuous flow and a steady state gradient in the concentration of reactants, products, and reaction rates along the length of the reactor (adapted from Penry and Jumars, 1987; Martinez del Rio et al., 1994; Karasov and Hume, 1997).

reactants are thus uniform in any given cross section and form a constant gradient along the length of the reactor during steady-state function. Figure 3 . 2 illustrates ~ efficiency and rate of product formation as a function of flow rate through an ideal PFR. Reactant concentration, and thus reaction rate, decline along the length of the vessel, although PFRs provide the highest rate of reaction in the minimum time and volume under most conditions (see Penry and Jumars, 1987). The small intestine of many vertebrates, assuming that they exhibit little axial mixing, and the simple tubular guts of deposit feeders probably function as PFRs. With few exceptions, the GI% of vertebrates are complex and contain elements of many reactor types whose function may deviate significantly

66

Physiological and ecological adaptations t o feeding in vertebrates

from the ideal conditions described above. Penry and Jumars (1986,1987), Martinez del Rio et al. (1994), Karasov and Hurne (1997) and Jumars (2000a, 2000b) extensively discuss reactor performance and the application of chemical reactor theory to the study of digestion in a wide variety of vertebrate and nonvertebrate taxa. Penry and Jumars (1987) and Horn and Messer (1992)provide analytical derivations for mass-balance equations for the ideal reactor types. The discussion herein is focused on the recent use of chemical reactor theory to model gut function in nectar- and fruit-eating birds.

Reactor Models Applied to Nectar- and Fruit-eating Birds The energy in nectar and the pulp of many fruits is primarily in the form of simple sugars (disaccharides and hexoses, Baker and Baker, 1983; Martinez del Rio et al., 1992), and nectarivorous and frugivorous animals tend to possess relatively simple guts. The digestive processes of animals feeding on nectars and sugary fruits (vs. waxy fruits, see Snow, 1981)may therefore be simpler to study than those of animals feeding on more complicated foods. Martinez del Rio and Karasov (1990) recognized the value of nectar- and fruit-eatingbirds as test cases for the description of guts as chemical reactors and of optimization premises. They extended the approach advocated by Penry and Jumars (1987)to include both hydrolysis and absorption and to predict the ingestion rate that maximizes net rate of absorptive gain to the animal. The two areas they addressed are: (1)how digestive constraints influence sugar and hence resource preferences and (2)how digestive traits and strategies are modified by the sugar composition and concentration of food. It has often been suggested that the chemical characteristics of the "rewards" (e.g. sugars) offered by plants reflect the dietary preferences of pollinators (Baker and Hurd, 1968;Howell, 1974; Calder, 1979),and ingestion rate is a functional response that connects individuals with populations and ecosystem processes (Karasov,1990;Jurnars and Martinez del Rio, 1999). Thus, studying the digestive strategiesof birds feeding on sugars can provide ecological and evolutionary insight. The theoretical framework provided by Martinez del Rio and Karasov (1990)has been employed by researchers at an accelerating pace during the past decade (Karasov and Cork, 1996; Downs, 1997; Lopez-Calleja et al., 1997; McWilliams and Karasov, 1998b; Levey and Martinez del Rio, 1999). The general conclusion from these empirical tests of the first generation of reactor models is that the results do not match predictions under the optimization premise of maximizing net rate of energy gain (Jumars and Martinez del Rio, 1999).The second generation of chemical reactor models, applied outside the context of maximization of net rate of energy or nutrient gain to estimate digestive capacities in nectar-eating birds, have yielded some remarkably accurate predictions. The digestive limitations identified by these models are informing recent tests of the sugar composition and

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concentrationpreferences of nectar-eatingbirds (e.g.Schondube and Martinez del Rio, 2003; Fleming et al., 2004.)and community level studies of resource use (Schondube,2003). In this section Martinez del Rio and Karasov's (1990) models are described:the morphological analogies drawn, the physiological parameters and performance equations used, and the resultant predictions. Mismatches between the predictions of these models and empirical results are then briefly described, focusing on how digestive traits and strategies are modified by the sugar composition and concentration of food and foraging costs. This is followed by a discussion of the second generation of chemical reactor models, their assumptions, and how the accuracy of their predictions differsbetween hummingbirds and nectar-eating passerine birds. The section concludes with suggestions for future directions and a discussion of the potential for chemical reactor models to test the ecological and evolutionary consequences of digestive constraints under natural conditions. Although Martinez del Rio and Karasov (1990) make explicit predictions about the diet preferences of nectar- and fruit-eating birds, the matches between these predictions and empirical results are not discussed. The sugar composition and concentrationpreferences of nectar- and fruit-eatingbirds seem to be the result of a complex interaction of digestive and osmoregulatory constraints on the part of the animal, and plant nectar characteristics;interested readers are referred to Martinez del Rio and Karasov (1990),Martinez del Rio (1990b), Martinez del Rio et al. (2001),Nicolson (2002),and Schondube and Martinez del Rio (2003). Modeling from the bottom up: tractability from digestive simplicity Martinez del Rio and Karasov (1990)modeled the guts of nectar- and fruiteating birds as PFRs because the digestion and absorption of sugars takes place entirely within a relatively simple tubular intestine. They included parameters describing both carrier-mediated and diffusive absorption of hexose sugars in their performance equations. Carrier-mediated absorption (active transport for glucose, facilitated diffusion for fructose, Karasov and Diamond, 1983; Alpers, 1987) is described by Michaelis-Menten kinetics with parameters of maximal absorption rate (Vp)and Michaelis constant (k 8' the sugar concentration at which absorption is equal to Vg/2, which is reciprocally related to the affinity of the carrier system for the sugar). Simple diffusive absorption is described as the product of luminal glucose concentration (G) and a permeability coefficient (k,). The rate of intestinal absorption of a single hexose such as glucose can be modeled as: rg= (G.Vg)/(kg + G) + (k,.G) (2) with units of p o l (pL&)-I, p o l pL-Iand min-lfor Vg,kg and k, respectively. In this case, digestion can be modeled simply as the rate of absorption: d G / d ~= -up (3)

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where z is a time constant (in units of min) equal to the ratio of the volume of the intestine to the rate of entrance of food into the intestine (v,).In an ideal PFR, equals the mean retention time of the reactor (also called throughput time, see above and Martinez del Rio and Karasov, 1990; Levenspiel, 1998). When Gis initially high, absorption rate is maximal (equalto Vg+ k,.G if G >> $). As glucose is absorbed, the lumenal concentration, and hence absorption rate drops, at an accelerating rate once concentration is in the range of kg. These kinetic features determine the form of the relationship between glucose (or energy) absorbed as a function of z (Fig. 3.1) and can be seen as boundary conditi&s in an optimization model wherein the control variable is z. ~ h e i that maximizes the net rate of energy intake (z*)is that at which a straight

a, V)

2 0

3 V)

u

s (CI

h

-s (b) a,

x

(CI

w

Q 3

Concentration of hexoses Fig. 3.3. Predicted absorption kinetics as a function of hexose concentration for Martinez del Rio and Karasov's (1990) (a) "hummingbird" and (b) "frugivore" models. Curve C represents the absorption of hexoses resulting exclusively from carrier-mediated processes. Curve C + P shows the sum of carrier-mediated uptake and passive diffusion of hexoses. Rates of sucrose hydrolysis (rs) are shown as lines parallel to the concentration axes for scenarios in which sucrase activity is limiting (2Vs < V,) and hexose uptake is limiting (2Vs> V,). Predictions were based on kinetic parameters resembling those of a hummingbird and a representative frugivore (adapted from Martinez del Rio and Karasov, 1990).

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line passing through the origin is tangent to the absorption curve. Modeling the digestion and absorption of the disaccharide sucrose, which must be hydrolyzed prior to the absorption of its hexose components glucose and fructose, requires an additional equation for the hydrolysis step (seeequations 3 and 4 in Martinez del Rio and Karasov, 1990).The overall hexose absorption curve in the case of a PFR with sucrose as the feed therefore depends on the relative rates of sucrose hydrolysis and hexose absorption (see Fig. 3.3b and Martinez del Rio and Karasov, 1990). Martinez del Rio and Karasov (1990) analyzed the behavior of two hypothetical systems: one in which all intestinaltransport is carrier-mediated, and a second which includes a passive component to absorption. Hummingbirds were thought to show no passive absorption of glucose (Diamondet al., 1986),while the frugivorous birds studied to date did absorb glucose passively (Karasovand Levey 1990);hence the systems were dubbed the "hummingbird model" and the "frugivore model", respectively. The authors assumed that sucrose was the primary dietary substrate for hummingbirds and considered both sucrose and hexose meals for frugivores. Figure 3.3 summarizes the main difference between these models. In the hummingbird model, the rate of hexose absorption (r,, includingboth glucose and fructose absorption)tends asymptotically to twice the maximum rate of sucrose hydrolysis (2VJ if sucrose hydrolysis is the rate-limitingstep (i.e.2Vs < V,, the maximal rate of hexose absorption)and to Vhif absorption of hexoses is the rate-limiting step (2Vs> V,), provided that the initial concentration of sucrose is higher than the Michaelis constant of sucrase (probably valid for hummingbirds, see Martinez del Rio and Karasov, 1990; Fig. 3.3a). The rate of hexose absorption from sucrose is therefore constant and approximately equal to the value of the rate-limiting step (see Fig. l b and Appendix A in Martinez del Rio and Karasov, 1990). In the frugivore model with hexose meals, the rate of hexose absorption depends on the lumenal concentration of hexoses (see example for glucose above). The maximal rate of absorption for a frugivore feeding on sucrose is equal to 2Vs (Fig. 3.3b). In this case sucrose hydrolysis is the rate-limiting step unless the Vq/V, ratio is extremely high. Because of the high sugar concentrations found in most fruit pulps and nectars (Lee et al., 1970; Levey, 1987), it is more likely for a bird with substantial passive absorption of hexoses to be limited by sucrose hydrolysis than a bird that relies only on carrier-mediated uptake (Martinezdel Rio and Karasov, 1990). Martinez del Rio and Karasov (1990)explored optimal digestion strategies for animals feeding on sucrose or hexoses and possessing intestines with the characteristics described above for the hummingbird and frugivore models. Their optimization criterion was maximization of the rate of net energy gain. These authors and Martinez del Rio et al. (1994) discuss the mechanics of including foraging, food processing, and maintenance costs in optimal digestion cost-benefit analyses, so these subjects are not discussed

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here. In the hummingbird model absorption curves are approximately linear except at very low sugar concentrations (see Fig. 2 in Martinez del Rio and Karasov, 1990).The optimal value of z for the hummingbird model is that at which almost all sugar is absorbed; thus hummingbirds should exhibit nearly 100%efficiency in digestion of sugars. The value of z* is always a linearly increasing function of the initial sugar concentration, which implies that throughput time should increase with increasing nectar sugar concentration and animals should prefer energy concentrated foods. Given a choice between foods with equicaloric concentrations of sucrose and hexoses (the energy per mole of the former being twice that of the latter),energy-maximizing birds should prefer hexoses when sucrose hydrolysis is limiting (2Vs< Vh) and be indifferent if hexose absorptionis limiting (2Vs> V,). As a consequence of the linearity of the cost-benefit curve in the hummingbird model, T*, and thus the time invested in digesting a meal, is independent of the cost of obtaining it. In the frugivore model when sucrose is the main food and 2Vs < V,, the absorption curves are approximately linear, T* is a linearly increasing function of sucrose concentration in food, and optimal sucrose digestibility is near 100%. As in the hummingbird model, time invested in digestion is independent of cost of acquisition. When hexoses are the main sugar in food, the maximal rate of absorption occurs at the beginrung of the absorptive process and decreases thereafter (see above and Fig. 3.1). In this case, both the concentration of hexose in food and the cost of feeding influence T*. Increasing the cost of feeding increases T*:food that costs more to acquire should be retained longer and digested more thoroughly. Increasing the concentration of hexoses in food decreases z*:energy-rich foods should be retained for a shorter time and digested less thoroughly. Optimal energy intake rate increaseswith sugar concentration as in the hummingbird model and animals should thus prefer concentrated over dilute nectars. Martinez del Rio and Karasov (1990)predicted that birds with an important passive component to nutrient absorption and feeding on sucrose are more likely to be limited by sucrose hydrolysis, and thus should prefer hexoses to equicaloric sucrose. They predicted that this preference should increase with increasing energy density in food. Mismatch between predictions about digestive strategies and empirical data Martinez del Rio and Karasov (1990) used explicit assumptions about digestive processes and invitro measurements done at the organ and tissue level to generate predictions about whole-organism digestive (and thus behavioral) outcomes. How well did their first generation models and predictions about digestive strategies conform to reality? The general conclusion drawn from early empirical tests of reactor-based digestion models in nectar- and fruit-eating birds is that the results do not match predictions

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under the optimization premise of maximizing net rate of energy gain (Jumars and Martinez del Rio, 1999; Levey and Martinez del Rio, 2001). The mismatches between predictions and empirical results and the lessons learned from early applications of guts-as-reactor models to hexose- and sucrose-eatingbirds are briefly described in this section. Karasov (1999)also summarized some of these tests and provides a useful discussion of methodological details. Tests of optimal digestion models in sucrose- and hexose-eating birds have involved manipulation of food quality (sugar concentration,Karasov and Cork, 1996;Downs, 1997; Lopez-Calleja et al., 1997;Witmer, 1998;Levey and Martinez del Rio, 1999)and energetic demands (ambient temperature and foraging costs, McWilliams and Karasov, 1998b). Three observations seem to apply broadly to the results of food-quality manipulation studies. First, increasing the sugar concentration in food leads to decreased food intake rates. Second, assimilationefficiency is very high (>90%)and appears to be independent of sugar concentration in food. Third, net assimilation rate is relatively constant in spite of variation in sugar concentration in food (Karasov and Cork, 1996; Downs, 1997; Witmer, 1998). In other words, gut retention time either remains constant or increases with increment in sugar concentration (Levey and Martinez del Rio, 1999). In addition, tests of the predictions that extraction efficiency and retention time should increase with foraging costs (e.g.longer intermeal intervals)have also failed to support the model (McWilliamsand Karasov, 1998a, 1998b). Taken together, these results call into question the optimization premise and/or the physiological assumptions of the model (Jumarsand Martinez del Rio, 1999; Levey and Martinez del Rio, 2001). One possibility is that the optimizationpremise is incorrect. Nectar- and fruit-eating birds may not maximize their net rate of energy assimilationbut rather maintain constant rates of energy intake or minimize feeding time by maximizing digestive efficiency. Lopez-Calleja et al. (1997)investigated the effect of varying sucrose concentration on feeding patterns, gut function, and energy management in hummingbirds. They found that hummingbirds exhibited almost complete assimilation of sugars and increased meal retention times and intermeal intervals with increased sugar concentration, in agreement with the predictions of Martinez del Rio and Karasov's (1990) model. However, hummingbirds showed no significant differences in daily energy intake when fed different sugar concentrations. This constant daily rate of energy intake can seemingly be interpreted as two alternatives:(1)at the scale of a day, hummingbirds were not acting as "energy maximizers" (sensu Schoener, 1971),or (2)energy intake was constrained by maximal gut processing rates (Karasov et al., 1986; Levey and Martinez del Rio, 1999). The feeding patterns and energy intake rates observed by Lopez-Calleja et al. (1997) in hummingbirds indicated that they were not using their guts to maximal capacity. The authors concluded that energy maximization is

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probably an inappropriate assumption for birds that are not growing, storing fat, or reproducing (see also Karasov and Cork, 1996; Karasov, 1999),and proposed an alternativemodel (or rather an alternative optimizationpremise) which assumes that birds which vary their food intake maintain constant energy intake. This model also predicts complete sugar assimilation and increased retention time with increasing sugar concentration,but in contrast to Martinez del Rio and Karasov's (1990) model, it predicts that as sugar concentration increases the gut spends increasing amounts of time idle. McWhorter and Martinez del Rio (2000)and Martinez del Rio et al. (2001) developed and tested models which differentiatebetween constant energy intake and digestive constraints to energy assimilation (see below: Second Generation Reactor Models: Gut Function in Nectar-eating Birds). The hypothesis that birds minimize feeding time by maximizing digestive efficiency is not mutually exclusive to these alternatives. They may reduce their need to forage (andthus expend energy and risk predation)by thoroughly digesting everything they consume (Levey and Martinez del Rio, 2001). McWilliams and Karasov (1998b) found that cold-acclimated frugivorous cedar waxwings (Bombycilla cedrorum) behaved as time minimizers while maintaining maximal glucose extraction efficiency. A second, and by no means mutually exclusive possibility, is that one or more of the physiological assumptions of the model are incorrect. These assumptions include descriptions of food acquisition and processing costs, constancy of reactant volumes, and reaction kinetics or mixing (Jumarsand Martinez del Rio, 1999). The reaction kinetics assumed by Martinez del Rio and Karasov's (1990)model for frugivorous birds feeding on hexoses appears to be correct: a substantial body of research supports the notion that there is significant passive, presumably paracellular, absorption of glucose in passerine birds (reviewedby Afik et al., 1997). It remains to be seen whether absorption of hexoses by hummingbirds is entirely carrier mediated as previously thought (Diamond et al., 1986);recent evidence suggests that this may not be the case (see Paracellular Intestinal Absorption of Carbohydrates in Mannals and Birds in chapter 5by McWhorter, present volume). Preingestional food processing costs that rise with sugar concentration (e.g.increasing nectar viscosity; see Kingsolver and Daniel, 1983)may overturn predictions about feeding rates but have not been included in models to date, which instead assume fixed costs per time that are proportional to volumetric ingestion rates (Jumarsand Martinez del Rio, 1999). Functional response models (see Jeschke et al., 2002) may provide a useful framework for linking digestive and preingestional limitations on intake rate. The assumption of constant gut volume may be violated by water-balance constraints: birds feeding on solutions of hexoses at concentrations that exceed the osmolality of plasma dilute incoming food with intestinal secretions (Chang and Rao, 1994), and some nectar-eating passerines may modulate water absorption across the gut more or less depending on food sugar concentration

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(McWhorter et al., 2003). A crucial assumption of the optimal digestion model in frugivores is that animals can modulate assimilation efficiency. However, the consistently high sugar assimilation efficiencies exhibited by these birds casts doubt on this assumption. Indeed, placing a high concentration of undigested, osmotically active substrates (i.e. sugars) in the lower gut may cause osmotic diarrhea and impair ability to reabsorb water and electrolytes (Malcarneyet al., 1994;Levey and Martinez del Rio, 1999).Lastly, Levey and Martinez del Rio (1999) found evidence of significant axial (i.e. longitudinal) mixing in the guts of cedar waxwings, invalidating the simplifying assumption of Martinez del Rio and Karasov (1990) that no axial mixing takes place. A theoretical framework is useful to the extent that it spawns empirical tests of its predictions and facilitates interpretation of experimental results. The first generation of chemical reactor models applied to nectar- and fruiteatingbirds were fertile at generating empirical work, but this work led to the conclusion that the optimization premise and/or physiological assumptions of the models were incorrect. Optimization studies often cycle between the process of model building and empirical performance tests (Seger and Stubblefield, 1996);modeling gut function in nectar-and fruit-eating birds is certainly an example of the cyclical nature of this process. Levey and Martinez del Rio (1999) proposed alternative models of gut function in frugivores, modifying assumptions from Martinez del Rio and Karasov (1990) for a better match between predictions and empirical data. McWhorter and Martinez del Rio (2000),Martinez del Rio et al. (2001),and McWhorter (2002) tested several of those alternatives in nectar-eating birds. This second generation of chemical reactor models is discussed in the following section.

Second Generation Reactor Models: Gut Function in Nectareating Birds Nectar-eating birds generally respond to experimentally increased sugar concentration in food by decreasing their food intake rates (Collins, 1981; Downs, 1997; L6pez-Calleja et al., 1997;Lotz and Nicolson, 1999; McWhorter and Lopez-Calleja, 2000; McWhorter and Martinez del Rio, 2000; Martinez del Rio et al., 2001; McWhorter et al., 2003).Similar reciprocal relationships have been described for a wide variety of species (Montgomery and Baumgardt, 1965; Batzli and Cole, 1979; Simpson et al., 1989; Nagy and Negus, 1993; Castle and Wunder, 1995) and have often been attributed to compensatory feeding (Simpsonet al., 1989).According to this explanation, animals regulate food intake to maintain a constant flux of assimilated energy or nutrients (Montgomeryand Baumgardt, 1965;Slansky and Wheeler, 1992). If the energy of nutrient density of food is decreased, animals compensate by increasing intake. Indeed, this inverse relationship often leads to relatively constant energy intake by nectar-eating birds (e.g. Beuchat et al., 1979; Lopez-Calleja et al., 1997; Lotz and Nicolson, 1999; McWhorter and

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Martinez del Rio, 2000). An alternative explanation often proposed is that intake may be constrained by the ability of animals to process the nutrients contained in food (see above and Karasov et al., 1986;Levey and Martinez del Rio, 1999).Two complementary approaches may be used to differentiate compensatory feeding from physiological constraint. The first is descriptive and relies on examining the functional structure of the intake response, in nectar-eating birds usually a power function of the form v, = (4) where v, is food intake rate, C equals sugar concentration, and a and b are empirically derived constants (McWhorterand Martinez del Rio, 1999,2000). Because v, decreasesas a power function of C, the amount of energy ingested is also a power function of C. Animals exhibiting values of b equal to 1show "perfect" compensation for the energy density of food, and thus sugar intake that is independent of concentration. This approach provides only inferential evidence of constraintson intake, that is, a slope (b) less than one implies but does not demonstrate constraint. Martinez del Rio et al. (2001) discuss this approach in detail and provide a general review of the intake responses of nectar-eating birds. The second approach, described below, is experimental and relies on determining the effect of changing energetic demands on the intake response. The chemical reactor models described in this section predict how capacity for sucrose digestion, and thus food intake, changes with food sucrose concentration.These models provide a mechanistic bridge between gut function and feeding behavior. They allow one to determine whether the intake response observed in sucrose-eating nectarivorous birds is the result of constraints imposed by digestive processes or the result of compensatory feeding. In this section the experimental approach to differentiating between compensatory feeding and constraint is outlined, followed by a brief description of the digestive capacity model developed by McWhorter and Martinez del Rio (2000).Explicit predictions made by Martinez del Rio et al. (2001), based on this model, about how compensatory feeding and physiological constraint shape the intake responses of nectar-eating birds are then discussed. Lastly, tests of the model made by McWhorter (2002)in nectar-eatingpasserine sunbirds and Schondube (2003)in a community of nectar-eating birds are described. Apparent mismatches in the accuracy of the predictions of this model between hummingbirds and passerine birds are pointed out. McWhorter and Martinez del Rio (2000)addressed the question of whether the intake-responserelationship observed in hummingbirds is the result of compensatory feeding or a digestive constraint to energy assimilation.They exposed broad-tailedhummingbirds (Selasphorus platycercus) to ambient temperatures of 22°C and 10°Cand fed them diets ranging in sucrose concentration from 292 to 1,168mM. Because chronic cold exposure in endotherms is

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often accompanied by increased digestive and metabolic capacities (Konarzewski and Diamond, 1994 and references therein), hummingbirds were acutely exposed to the lower temperature. The resting metabolic rate of broad-tailed hummingbirds is considerably higher at 10°C than at 20°C (Bucher and Chappell, 1988).Based on this observation, McWhorter and Martinez del Rio (2000)hypothesized that for a given food energy density birds exposed to lower temperatures would show increased food intake. An increase in sugar intake under energetically demanding conditions would support the compensatory feeding explanation. Conversely, the opposite effect would provide evidence that a physiological process limits sugar assimilation. Although the birds exhibited the expected intake-responserelationship, they did not significantly increase food consumption when exposed to the lower ambient temperature (McWhorter and Martinez del Rio, 2000). In addition, at 10°C birds lost significantly more body mass, were often observed emerging from torpor in the morning, and exhibited other behavior commonly associated with energy conservation (ptiloerection,decreased flying time, feet held close to the body in flight, Gass and Montgomerie, 1981; Udvardy, 1983). Regardless of any energy conserving mechanisms employed, it appeared that acutely cold-exposed hummingbirds could not assimilate energy fast enough to compensate for their higher energy demands. These observationswere interpreted as evidence of a physiologcal constraint to energy assimilation. The apparent inability of hummingbirds to increase energy assimilation when acutely subjected to higher energetic demands led McWhorter and Martinez del Rio (2000) to speculate about the factors imposing an upper limit to food intake. Because hummingbirds obtain the vast majority of their energy from the sugars contained in sucrose-rich floral nectars, physiological processes that determine rates of sucrose assimilation were identified as potential limiting factors. Sucrose ingestion can be limited by rates of sucrose hydrolysis or transport of the resulting monosaccharides (Karasov et al., 1986;Martinez del Rio, 1990a),and by rates of sugar catabolism or biosynthetic processes (Suarez et al., 1988; Suarez et al., 1990). McWhorter and Martinez del Rio (2000)focused on the potential role of digestive processes in limiting energy assimilation and assumed that sucrose hydrolysis in the small intestine is the limiting step in sucrose assimilation (or that digestive and metabolic processes such as hydrolysis and uptake are matched to one another, Hamrnond and Diamond, 1997). They used in vitro data on enzyme (sucrase-isomaltase)activity and gut volume to predict maximum sucrose digestive capacity as a function of sugar concentration in food. They assumed that the guts of hummingbirds function as PFRs (see above and Penry and Jumars, 1987)and included significantlymore physiological detail than previous attempts to estimate hydrolytic capacity in animal guts (e.g. Diamond and Hammond, 1992). Most significantly their model takes into account the decline in sucrose concentration along the length of the gut that

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accompanies hydrolysis. Previous analyses of guts as chemical reactors used models to predict throughput times that maximize the net rate of nutrient or energy assimilation (Dade et al., 1990;Martinez del Rio and Karasov, 1990; Jumars and Martinez del Rio, 1999). McWhorter and Martinez del Rio's (2000) model was designed outside this context of optimality. Following Levey and Martinez del Rio (1999),they assumed that hummingbirds must show high assimilation efficiencies to prevent osmotic imbalances in the lower gut, and used this physiological detail as a constraint on the model to estimate intake rates. McWhorter and Martinez del Rio (2000)made two critical assumptions in their model: (1)that digesta flows unidirectionally (Jumars and Martinez del Rio, 1999) and (2) that the rate at which sucrose is hydrolyzed in the intestine (rs)follows simple Michaelis-Menten kinetics: r5= Vs-S.(ks + S)-' (5) where Vsequals the maximum rate of hydrolysis along the intestine (in pmol min-I pLal),ks the Michaelis constant of sucrase (in pmol pL-I), and S the concentration of sucrose (inpnol pL-l)down the intestine or over time (Jumars and Martinez del Rio, 1999). Equation (5) can be integrated to yield the throughput time (7) required to reduce the initial sucrose concentration (So) to a given final value (Sf,based on assimilation efficiency): In plug flow reactors if one knows z and the volume of gut contents (Vin pL), intake rate (voin pL min-I)can be estimated as: v = V.z-'

The model predicted that the relationship between maximal food intake rate and sugar concentration should follow a power function with a slope (b) lower than one. The intake rates predicted for broad-tailed hummingbirds slightly overestimated observed intake rates (by a m a r p that increased from 15%at the lowest concentration to 35% at the highest) but there was a remarkable qualitative match between the model's output and bird behavior (Fig. 3.4a). The model also predicted that sugar assimilation rate should increase with increased concentration in food. This prediction was upheld in broad-tailed hummingbirds. Although there was no significant increase in sucrose intake between the two ambient temperatures,there was a signlficant effect of concentration on sucrose intake, a consequence of lower average hydrolysis rates at lower food concentrations (McWhorter and Martinez del Rio, 2000). Sucrose apparent assimilation efficiency was high (>95%)and independent of sugar concentration. Taken together, these observations provide compelling evidence for a physiological constraint to energy assimilation. Martinez del Rio et al. (2001)extended the approach of McWhorter and Martinez del Rio (2000)to make explicit predictions as to the conditions that

Modeling digestive function

Sucrose concentration (mM)

Fig. 3.4. (a) Food intake rate (v,) of broad-tailed hummingbirds at 22 "C (solid circles) and 10°C (open circles) decreased as a common power function of diet sugar concentration (v, = 1000.C-0.77, rZ = 0.87, dashed line). Hummingbirds were unable to increase food

consumption when abruptly exposed to low ambient temperature (and hence increased metabolic demands). Solid line represents predicted maximal food intake rate (vo= 653.CRedrawn from McWhorter and Martinez del Rio (2000). (b) Hypothetical relationship between daily food intake rate, sucrose concentration in food, and energy expenditures for magnificent hummingbirds. Thick line represents predicted maximal food intake rate Observed food based on McWhorter and Martinez del Rio's (2000) model (v, = 4030.C-0.82). intake rates for birds feeding under mild ambient conditions were lower than predicted maximal intake rates, but the exponent of this relationship was higher than that of the r2 = 0.98; data not shown). Other lines represent predicted relationship (v, = 7229~C-O.~~, hypothetical intake responses for three levels of daily energy expenditure (DEE). Note that at higher levels of energy expenditures, this hypothesis predicts "broken" intake responses described by two power functions with different slopes. Redrawn from Martinez del Rio et al. (2001). (c) Food intake rate of Palestine sunbirds at 30°C (open crosses), 15°C (solid circles), and 5°C (open circles) decreased as power functions of diet sugar concentration (v, = 966.C-076, r2 = 0.91 for 30°C, lower dashed line, and v, = 3296.C-0.92, 12 = 0.97 for 5 and 15"C, upper dashed line). Solid line represents predicted maximal food intake rate (v, = 1472.C-075). Redrawn from McWhorter (2002). Note that all axes are logarithmic and vary in scale among panels.

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determine whether compensatory feeding or physiological constraint shape the intake response of nectar-eating birds. They applied McWhorter and Martinez del Rio's (2000) model to magnificent hummingbirds (Eugenes fulgens)and similarly found that the model's output and the actual intake response of birds were both well described by power functions with negative exponents. In this case, however, the slope of the predicted intake response (0.822)was significantly lower than that of the observed relationship (0.942 + 0.047 SD), suggesting that under the mild conditions of the experiment birds were exhibiting compensatory feeding (Martinez del Rio et al., 2001). The birds appeared to possess digestive "spare capacity" (Diamond, 1991). The digestive safety factors (defined as the ratio of capacity to load, Diamond and Hammond, 1992) for magnificent hummingbirds were modest and ranged from 1.09 to 1.26 from the lowest to the highest food sugar concentration. McWhorter and Martinez del Rio (2000)reported similarly modest safety factors for broad-tailed hummingbirds (averaging 1.2 0.2 SD). Although Martinez del Rio et al. (2001) did not expose magnificent hummingbirds to low ambient temperatures, they predicted that acutely increasing energy demands would reduce digestive safety margins and force birds to shift from an intake response attributable to compensatory feeding to one shaped by digestive constraints. Figure 3.4b shows the hypothetical effect of increasing energy demands on the intake response of magnificent hummingbirds.Note that at higher levels of energy expenditures this model predicts a "broken" intake response described by two power functionswith different slopes (Martinez del Rio et al., 2001). McWhorter (2002) examined the interplay between compensatory and constrained feeding in a nectar-eating passerine bird, the Palestine sunbird (Nectarinia osea). Sunbirds were fed on sucrose solutions of varying concentration and exposed to two ambient temperatures within their acclimatized range (15and 30°C),and acutely to one temperature well below this range (5°C).The intake responses of sunbirds were compared with maximal intake rates predicted using McWhorter and Martinez del Rio's (2000)model. As expected, surlbirds decreased their food intake rates in response to sugar concentration in relationships described by power functions with negative exponents (Fig. 3.4~). At 15°C and 30°C, they were able to compensate for differences in food energy density and increased metabolic demands. When exposed to a relatively sudden drop in ambient temperature (to 5°C) and hence to an acute increase in thermoregulatory and food-warming energy expenditures, surlbirds were unable to increase their rates of food and energy intake. Predicted maximal sucrose and food intake rates matched actual intake rates at low food sucrose concentrations (292 mM) and low )~ that under temperatures (5 and 15°C) very closely (Fig. 3 . 4 ~suggesting these conditions intestinal sucrose hydrolysis rates were near maximal. Although the intake response at 5°C was not statistically distinguishable from that at 15"C, data for 5°C considered separately suggest a broken intake

*

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response as predicted by Martinez del Rio et al. (2001). Unlike the broadtailed hummingbirds studied by McWhorter and Martinez del Rio (2000), Palestine sunbirds exposed to ambient temperatures below their acclimatized range and subjected to digestive limitations to energy assimilation gained body mass. They appeared to balance their energy budgets using behavioral reductions in energy expenditures (McWhorter,2002). Taken together the patterns of food and energy intake exhibited by Palestine sunbirds and modeling of their digestive capacities provide compelling evidence for a physiological constraint to food intake when they feed on dilute diets under energetically demanding conditions. Like the hummingbirds studied by McWhorter and Martinez del Rio (2000) and Martinez del Rio et al. (2001), sunbirds appear to operate with small digestive spare capacities.Safety factors for sunbirds feeding at 15OCwere modest and ranged from 1.05to 1.5from the lowest to the highest sucrose concentration.At 30°C, safety factors were slightly larger, ranging from 1.62 to 1.66. McWhorter and Martinez del Rio's (2000)model overestimated maximal intake rates for broad-tailed hummingbirds (between 10% and 35'10, depending on sugar concentration),but appeared to very accurately predict maximal intake rates for Palestine sunbirds. What might explain this difference? One possibility is differences in the mechanism of hexose absorption between hummingbirds and sunbirds. Hummingbirds are thought to absorb glucose entirelyby mediated uptake (Diamond et al., 1986)whereas passive, presumably paracellular absorption of glucose may be very significant in fruit- and nectar-eating passerine birds (see Afik et al., 1997 and Chapter 5 by McWhorter, this volume). McWhorter and Martinez del Rio (2000)did not include the kinetics of glucose and fructose absorption in their model and assumed that sucrose hydrolysis was the limiting step in digestion. However, they found more glucose and fructose than sucrose in the excreta of broad-tailed hummingbirds, suggesting that hexose uptake capacities in hummingbirds may be more limiting than sucrose hydrolysis. It is possible that the inclusion of hexose uptake in the model would yield lower predicted intake rates for hummingbirds. Unfortunately, the method most widely used to estimate intestinal uptake of hexoses in vitro, the intestinal everted sleeve (Karasov and Diamond, 1983),may cause serious damage to intestinal tissues and lead to large underestimates of in vivo uptake rates (Starck et al., 2000). Indeed, glucose uptake rates measured in vitro using everted sleeves in rufous hummingbirds were approximately four times lower than glucose absorption rates observed in vivo (see Karasov et al., 1986).Note that this also presents a technical challenge for modeling digestive capacities in frugivores feeding on hexose diets. The passive absorption of glucose has not been measured in any species of sunbird, but it is likely to be similar to that in other nectar-eating passerines. Martinez del Rio and Karasov (1990)predicted that sucrose hydrolysis should be more limiting for birds with significantpassive components to hexose absorption.

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McWhorter and Martinez del Rio's (2000)assumption that sucrose hydrolysis is the limiting step may therefore be correct for nectarivorous passerines feeding on sucrose, but incorrect for hummingbirds.Indeed, Schondubeand Martinez del Rio (2003)recently found that nectar-eating passerine flowerpiercers (Diglossa baritula) were able to consume and assimilate 10%more of a hexose (glucoseand fructose) solution than an equicaloric sucrose solution. A second possibility is that the discrepancy in accuracy of the model's predictions is due to differences in the roles of the guts of hummingbirds and sunbirds in osmoregulation.McWhorter and Martinez del Rio (2000)assumed that the concentration of sucrose in the intestinal lumen changed simply as a result of hydrolysis. In reality, sucrose concentration changes with hydrolysis and the addition or removal of water by secretion and absorption into and from the intestinal lumen (Chang and Rao, 1994). McWhorter and Martinez del Rio (1999) showed that hummingbirds absorb essentially all ingested water across the intestine, but McWhorter et al. (2003)found that Palestine sunbirds shunt up to 60%of ingested water through their intestines. The model's simplifying assumptions about the decline in sucrose concentration and the constancy of digesta volume may therefore be more correct for sunbirds than for hummingbirds. Data on the concentration of solutes in the intestines of nectar-eating birds can help evaluate the validity of these assumptions (see Ferraris et al., 1990). Chemical reactor models of digestive capacity have the useful feature of generating precise predictions about the form of the intake response based on energy expendituresand the magnitude of physiological traits. They allow probing the relative importance of behavioral changes and physiological mechanisms for balancing the energy budgets of small endotherms over short timescales. The picture emerging from recent tests of these models is that physiologicalconstraints and compensatory feeding are complementary mechanisms shaping the behavioral responses of nectar-eating birds to varying food energy density (Martinez del Rio et al., 2001). Rapid-exposure experiments such as those employed by McWhorter and Martinez del Rio (2000)and McWhorter (2002))used within the framework of modeling, are informative because they reveal the immediate short-term digestive spare capacity of an animal. Small digestive safety factors may be a general trait of nectar-eating birds, which appear to have considerable flexibility in modulating their energy expenditureswhen faced with digestive constraints and/or increased thermoregulatory energy demands (Martinez del Rio et al., 2001; McWhorter, 2002).The approach described here may also be useful in determining how digestive safety factors change during growth, reproduction, and migration (McWhorterand Lopez-Calleja, 2000).Chapter 4 in this volume (Karasov and McWilliams) presents a general discussion of the adaptationof digestivecapacities to the amounts and types of food eaten, and the use of cold-exposuretests to measure digestive capacities. McWhorter and Lopez-Calleja (2000)review the factors that may impose ceilings on the energy budgets of hummingbirds during chronic cold exposure.

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Models of digestive capacity are also useful tools in community-level studies of resource use. Schondube (2003) used McWhorter and Martinez del Rio's (2000)approach to predict the maximal sucrose digestive capacities of a community of 10nectar-feedingbirds (7hummingbirds, 2 warblers, and 1 flowerpiercer). He compared the birds' predicted maximal rate of energy intake with their field metabolic rates (FNIRs) estimated from published allometric equations, and with their food intake rates. Predicted maximal energy intake rates for hummingbirds were either equal to or higher than their expected FMRs and exceeded observed food intake by 10 to 70%. Predicted maximal energy intake values for passerines were either equal to or lower than their predicted FMRs and digestion of sucrose seemed to limit ingestion rate. Knowledge of interspecific variation in digestive capacities can clearly lead to testable predictions about resource use on a community scale (but see caveats regarding the differences in digestive performance among hummingbirds and passerines above).

Chemical reactor models have provided the theoretical framework needed to integrate digestive processes with gut morphology and the chemical properties of food. Models force us to make o w assumptions explicit, provide falsifiable predictions, and locate areas where research is needed. They provide a means to make inferences about whole-animal function from digestive processes, and are thus of ecological importance. The approach can also be used to model alternate ways in which foods can be processed and thus augment our understanding of GIT evolution. The cyclical, iterative process of model building, empirical testing, and reformulation has been fruitful (see Levey and Martinez del Rio, 1999;Levey and Martinez del Rio, 2001). Although many of the predictions of early guts-as-reactor models have been proven false, much has been learned in the process. Secondgeneration models in nectar-eating birds, developed outside the context of optimization, have generated some remarkable predictions. Digestive limitations identified by these approaches are already informing recent tests of the sugar composition and concentration preferences of nectar-eating birds (e.g. Nicolson, 2002; Schondube and Martinez del Rio, 2003; Fleming et al., 2004) and community level studies of resource use (Schondube,2003). Future gut function models in frugivores will likely not emphasize energy intake maximization, but instead explore efficient gut designs that permit digestive processes to take place at the rate dictated by metabolic demands (Jumars, 2000b). These models may include design objectives such as optimizing levels of expression and distributions of enzymes and transporters along the gut (Levey and Martinez del Rio, 2001). Modeling digestive capacities in frugvores feeding on hexoses using McWhorter and Martinez del Rio's (2000)

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approach described above will require overcoming technical problems in estimating the rate of hexose transport in the intestine. Functional response models (see Jeschke et al., 2002) are consistent with chemical reactor theory and may provide a useful framework for linking digestive and preingestional limitations on intake rate. Integrating pre- and postingestional constraints with foraging costs and predation risks may increase the value of digestive models for interpreting ecological and behavioral patterns (see Bednekoff and Houston, 1994). Nectar- and fruit-eating birds present an unparalleled opportunity to explore the interaction between gut and metabolic function, and to test the notion that digestive constraints have ecological and evolutionary consequences for animals (and plants) under natural conditions. The techniques necessary to test the predictions of the model presented by McWhorter and Martinez del Rio (2000)and refined by Martinez del Rio et al. (2001)in the field are readily available: daily energy expenditures can be measured using standard methods (Powers and Nagy, 1988; Tiebout and Nagy, 1991) and digestive capacities can be estimated from physiological measurements and estimates of sugar concentration and composition of floral nectars.

Acknowledgements Supported by NSF (IBN - 02 16709)to William H. Karasov. REFERENCES Afik D., McWilliams S. R. and Karasov W. H. 1997. A test for passive absorption of glucose in yellow-rumped warblers and its ecological implications. Physiol. Zool. 70: 370-377. Alexander Mc. R. 1994. Optimum gut structure for specified diets. In: The Digestive System in Mammals: Food, Form and Function. D. J . Chivers and P. Langer (eds.). Cambridge Univ. Press, Cambridge, pp. 54-62. Alpers D. H. 1987. Digestion and absorption of carbohydrates and proteins. In: Physiology of the Gastrointestinal Tract, vol. 2. L. R. Johnson (ed.) Raven Press, New York, NY, pp. 1469-1487. Baker H. G. and Baker I. 1983. Chemical constituents of nectar in relation to pollination mechanisms and phylogeny. In: Handbook of Experimental Pollination Biology, pp. 131-171. Baker H. G. and Hurd P. H. 1968. Intrafloral ecology. Annu. Rev. Enfomol. 13: 385414. Batzli G . 0. and Cole F. R. 1979. Nutritional ecology of microtine rodents: digestibility of forage. Mammal 60: 740-750. Bednekoff P. A. and Houston A. I. 1994. Avian daily foraging patterns: effects of digestive constraints and variability. Evol. Ecol. 8: 36-52. Beuchat C. A., Chaplin S. B. and Morton M. L. 1979. Ambient temperature and the daily energetics of two species of hummingbirds, Calypte anna and Selasphorus rufus. Physiol. Zool. 52: 280-295. Bucher T. L. and Chappell M. A. 1988. Energy metabolism and patterns of ventilation in euthermic and torpid hummingbirds. In: Physiology of Cold Adaptation in Birds C. Bech and R. E. Reinersten (eds.). Plenum Press, New York, NY, pp. 187-195.

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Calder W. A., 111. 1979. On the temperature-dependency of optimal nectar concentrations for birds. J. Theor. Biol. 78: 185-196. Carberry J. J. 2001. Chemical and Catalytic Reaction Engineering. Dover Publ. New York, NY. Castle K. T. and Wunder B. A. 1995. Limits to food intake and fiber utilization in the prairie vole, Microtus orchrogaster: effects of food quality and energy need. J. Comp. Physiol. B 164: 609-617. Chang E. B. and Rao M. C. 1994. Intestinal water and electrolyte transport. In: Physiology of the Gastrointestinal Tract L. R. Johnson (ed.). Raven Press, New York, NY, pp. 2027-2081. Cochran P. A. 1987. Optimal digestion in a batch-reactor gut: the analogy to partial prey consumption. Oikos 50: 268-270. Collins B. G. 1981.Nectar intake and water balance for two species of Australian honeyeater, Lichmera indistincta and Acanthorynchus superciliosis. Physiol. Zool. 54: 1-13. Dade W. B., Jumars P. A. and Penry D. L. 1990. Supply-side optimization: maximizing absorptive rates. In: Behavioral Mechanisms of Food Selection, R. N. Hughes (ed.). Springer, Berlin, pp. 531-556. Diamond J. 1991. Evolutionary design of intestinal nutrient absorption: enough but not too much. News Physiol. Sci. 6: 92-96. Diamond J. M. and Hammond K. A. 1992. The matches, achieved by natural selection, between biological capacities and their natural loads. Experientia 48: 551-557. Diamond J. M., Karasov W. H., Phan D. and Carpenter F. L. 1986. Digestive physiology is a determinant of foraging bout frequency in hummingbirds. Nature 320: 62-63. Downs C. T. 1997. Sugar digestion efficiencies of Gurney's sugarbirds, malachite sunbirds, and black sunbirds. Physiol. Zool. 70: 93-99. Duke G. E. 1989. Relationship of cecal and colonic motility to diet, habitat, and cecal anatomy in several avian species. J. Exp. Biol. Suppl. 3: 3847. Dykstra C. R. and Karasov W. H. 1992. Changes in gut structure and function of house wrens (Troglodytes aedon) in response to increased energy demands. Physiol. Zool. 65: 422-442. Ferraris R. P., Yasharpour S., Lloyd K. C. K., Mirzayan R. and Diamond J. M. 1990. Luminal glucose concentrations in the gut under normal conditions. Amer. J. Physiol. 259: G822-G837. Fleming P. A., Hartman Bakken B., Lotz C. N. and Nicolson S. W. 2004. Concentration and temperature effects on sugar intake and preferences in a sunbird and a hummingbird. Funcf. Ecol. 18: 223-232. Froment G. E. and Bischoff K. B. 1990. Chemical Reactor Analysis and Design. John Wiley & Sons, New York, NY, (2nded.). Gass C. L. and Montgomerie R. D . 1981. Hummingbird foraging behavior: decisionmaking and energy regulation. In: Foraging Behavior: Ecological, Ethological and Physiological Approaches. A. C. Kamil and T. D. Sargent (eds.). Garland STPM, New York, NY, pp. 159-194. Hammond K. A. and Diamond J. 1997. Maximal sustained energy budgets in humans and animals. Nature 386: 457-462. Horn M. H. and Messer K. S. 1992. Fish guts as chemical reactors: a model of the alimentary canals of marine herbivorous fishes. Marine Biol. 113: 527-535. Howell D . J . 1974. Bats and pollen: physiological aspects of the syndrome of chiropterophily. Comp. Biochem. Physiol. 48: 263-276. Hume I. D. 1989. Optimal digestive strategies in mammalian herbivores. Physiol. Zool. 62: 1145-1163. Jeschke J. M., Kopp M. and Tollrian R. 2002. Predator functional responses: discriminating between handling and digesting prey. Ecol. Monogr. 72: 95-112. Jumars P. A. 2000a. Animal guts as ideal chemical reactors: maximizing absorption rates. Amer. Nat. 155: 527-543.

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Jumars P. A. 2000b. Animal guts as nonideal chemical reactors: partial mixing and axial variation in absorption kinetics. Amer. Nat. 155: 544-555. Jumars P. A. and Martinez del Rio C. 1999. The tau of continuous feeding on simple foods. Physiol. Biochem. Zool. 72: 633-641. Karasov W. H. 1990. Digestion in birds: chemical and physiological determinants and ecological implications. S fudies Avian Biol. 13: 391415. Karasov W. H. 1999. Optimal digestive responses to changing diet and foraging costs. In: Proc. 22nd Inf. Ornithological Congress, Durban. N . J. Adams and R. H. Slotow (eds.). BirdLife South Africa, Johannesburg, pp. 2247-2258. Karasov W. H. and Diamond J. M. 1983. A simple method for measuring intestinal solute uptake in vitro. J. Comp. Physiol. 152: 105-116. Karasov W. H. and Levey D. J. 1990. Digestive system trade-offs and adaptations of frugivorous passerine birds. Physiol. Zool. 63: 1248-1270. Karasov W. H. and Cork S. J. 1996. Test of a reactor-based digestion optimization model for nectar-eating rainbow lorikeets. Physiol. Zool. 69: 117-138. Karasov W. H. and Hume I. D. 1997. Vertebrate gastrointestinal system. In: Handbook of Comparative Physiology. W. Dantzler (ed.). Amer. Physio. Soc., Bethesda, MD, pp. 409-480. Karasov W. H., Phan D., Diamond J. M., and Carpenter F. L. 1986. Food passage and intestinal nutrient absorption in hummingbirds. Auk 103: 453464. Kingsolver J. and Daniel T. 1983. Mechanical determinants of feeding strategy in hummingbirds: energetics, tongue morphology, and licking behavior. Oecologia 60: 214-226. Konarzewski M. and Diamond J. 1994. Peak sustained metabolic rate and its individual variation in cold-stressed mice. Physiol. Zool. 67: 1186-1212. Lee C. Y., Shallenberg R. S. and Vittum M. T. 1970. Free sugars in fruits and vegetables. Food Life Sci. Bull. 1: 1-12. Levenspiel 0. 1998. Chemical Reaction Engineering, John Wiley & Sons, New York, NY (3rded.). Levey D. J. 1987. Sugar-tasting ability and fruit selection in tropical fruit-eating birds. A u k 104: 173-179. Levey D. J. and Martinez del Rio C. 1999. Test, rejection, and reformulation of a chemical reactor-based model of gut function in a fruit-eating bird. Physiol. Biochem. Zool. 72: 369-383. Levey D. J. and Martinez del Rio C. 2001. It takes guts (and more) to eat fruit: lessons from avian nutritional ecology. Auk 118: 819-831. Levins R. 1966. The strategy of model building in population biology. Amer. Sci. 54: 421-431. Lopez-Calleja M. V., Bozinovic F. and Martinez del Rio C. 1997. Effects of sugar concentration on hummingbird feeding and energy use. Comp. Biochem. Physiol. 118A: 1291-1299. Lotz C. N. and Nicolson S. W. 1999. Energy and water balance in the lesser doublecollared sunbird (Nectarinia chalybea) feeding on different nectar concentrations. J. Comp. Physiol. B 169: 200-206. Malcarney H. L., Martinez del Rio C. and Apanius V. 1994. Sucrose intolerance in birds: simple nonlethal diagnostic methods and consequences for assimilation of complex carbohydrates. Auk 111: 170-177. Martinez del Rio C. 1990a. Dietary, phylogenetic, and ecological correlates of intestinal sucrase and maltase activity in birds. Physiol. Zool. 63: 987-1011. Martinez del Rio C. 1990b. Sugar preferences in hummingbirds: the influence of subtle chemical differences on food choice. Condor 92: 1022-1030. Martinez del Rio C. and Karasov W. H. 1990. Digestion strategies in nectar- and fruiteating birds and the sugar composition of plant rewards. Amer. Nat. 136: 618-637. Martinez del Rio C., Baker H. G . and Baker I. 1992. Ecological and evolutionary implications of digestive processes: bird preferences and the sugar constituents of floral nectar and fruit pulp. Experientia 48: 544-551.

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Martinez del Rio C., Cork S. J. and Karasov W. H. 1994. Modelling gut function: an introduction. In: The Digestive System in Mammals: Food, Form and Function. D. J. Chivers and P. Langer (eds.). Cambridge Univ. Press, Cambridge, pp. 25-53. Martinez del Rio C., Schondube J. E., McWhorter T. J. and Herrera L. G . 2001. Intake responses in nectar-feeding birds: digestive and metabolic causes, osmoregulatory consequences, and coevolutionary effects. Amer. Zool. 41: 902-915. McWhorter T. J. 2002. The integration of digestive, metabolic and osmoregulatory processes in nectar-eating birds. PhD Diss; Dept. Ecol. Evol. Biol., University Arizona, Tucson, AZ. McWhorter T. J. and Martinez del Rio C. 1999. Food ingestion and water turnover in hummingbirds: how much dietary water is absorbed? J. Exp. Biol. 202: 2851-2858. McWhorter T. J. and L6pez-Calleja M. V. 2000. The integration of diet, physiology, and ecology of nectar-feeding birds. Rev. Chil. Hist. Nut. 73: 451460. McWhorter T. J. and Martinez del Rio C. 2000. Does gut function limit hummingbird food intake? Physiol. Biochem. Zool. 73: 313-324. McWhorter T. J., Martinez del Rio C. and Pinshow B. 2003. Modulation of ingested water absorption by Palestine sunbirds: evidence for adaptive regulation. J. Exp. Biol. 206: 659-666. McWilliams S. R. and Karasov W. H. 1998a. Test of a digestion optimization model: effect of variable-reward feeding schedules on digestive performance of a migratory bird. Oecologia 114: 160-169. McWilliams S. R. and Karasov W. H. 1998b. Test of a digestion optimization model: effects of costs of feeding on digestive parameters. Physiol. Zool. 71: 168-178. Monsen N. E., Cloern J. E., Lucas L. V. and Monismith S. G . 2002. A comment on the use of flushing time, residence time, and age as transport scales. Limnol. Oceanog. 47: 1545-1553. Montgomery M. J. and Baumgardt B. R. 1965. Regulation of food intake in ruminants. 1. Pelleted rations varying in energy concentration. J. Dairy Sci. 48: 569-574. Nagy K. A. and Negus N. C. 1993. Energy acquisition and allocation in male collared lemmings Dicrostonyx groenlandicus: effects of photoperiod, temperature, and diet quality. Physiol. Zool. 66: 537-560. Nicolson S. W. 2002. Pollination by passerine birds: why are the nectars so dilute? Comp. Biochem. Physiol. B 131: 645 -652. Penry D. L. and Jumars P. A. 1986. Chemical reactor analysis and optimal digestion. Biosci. 36: 310-315. Penry D. L. and Jumars P. A. 1987. Modeling animal guts as chemical reactors. Amer. Nut. 129: 69-96. Penry D. L. and Jumars P. A. 1990. Gut architecture, digestive constraints and feeding ecology of deposit-feeding and carnivorous polychaetes. Oecologia 82: 1-11. Plante C. J., Jumars P. A. and Baross J. A. 1990. Digestive association between marine detritivores and bacteria. Ann. Rev. Ecol. and System. 21: 93-127. Powers D. R. and Nagy K. A. 1988. Field metabolic rate and food consumption by freeliving Anna's hummingbirds (Calypte anna). Physiol. Zool. 61: 500-506. Prop J. and Vulink T. 1992. Digestion by barnacle geese in the annual cycle: the interplay between retention time and food quality. Funct. Ecol. 6: 180-189. Schoener T. W. 1971. Theory of feeding strategies. Ann. Rev. Ecol. System. 2: 369-404. Schondube J. E. 2003. Flowerpiercers and hummingbirds: a comparative study of nectar feeding strategies in birds. PhD Diss. Dept. Ecol. Evol. Biol. Univ. Arizona, Tucson, AZ. Schondube J. E. a n d Martinez del Rio C. 2003. Concentration-dependent sugar preferences in nectar-feeding birds: mechanisms and consequences. Funct. Ecol. 17: 445-453. Seger J. and Stubblefield J. W. 1996. Optimization and adaptation. In: Adaptation, M. R. Rose and G. V. Lauder (eds.). Academic Press, New York, NY, pp. 93-124.

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Digestive Constraints in Mammalian and Avian Ecology William H. Karasovl and Scott R. McWilliams2

'University Wisconsin, Department of Wildlife Ecology, Madison, WI, USA University Rhode Island, Department of Natural Resources, Kingston, RI, USA

SYNOPSIS The difference between the rate of nutrient intake for maintenance and the maximum rate of digestion, termed spare digestive capacity, potentially limits energy allocation. Because the maximum digestion rate can be adjusted upward in relation to factors such as diet quality and quantity there are both immediate and long-term spare capacities. We review their quantitation and time course for change, which are both ecologically important. A critical design feature of most studies measuring immediate spare capacity is that they quickly challenge animals to increase rate of digestion through cold stimuli, forced activity, or reduction in feeding time. A rapid time course is important because within a few days adjustments occur in the digestive tract that increase digestive capacity, in which case immediate spare capacity is no longer measured. Technical reasons why biochemical measures of spare capacity may not necessarily establish limitation at the whole-animal level are discussed herein. The majority of species studied had quite modest immediate spare capacities (range 950%). But in the same species the long-term spare capacity was about 100-125% above routine rates of nutrient intake or digestion. In laboratory mice digestive capacity increased to match any demand put on it, but whether the gut sometimes ultimately limits the energy budget is unknown for most animals. We review examples in which digestive limits are apparently dictated by the volumetric capacity of the gut or the rates at which food is either mechanically or biochemically broken down, but we know of no examples of limiting absorption.

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DIGESTIVE CONSTRAINTS: SIGNIFICANCE AND GENERAL PRINCIPLES Wild mammals and birds undergo a range of food intake from hyperphagia during low temperature acclimation or periods of production (e.g.lactation, migratory fattening), which may require an enlarged gut, to restricted feeding or fasting which may cause disuse atrophy (see examples below and in Chapters 8,9, and 13 of this volume). Omnivorous species encounter different types of food on a daily and/or seasonal basis, and this may require biochemical adjustments for breaking down and absorbing different substrates (Karasov,1996;Karasov and Hume, 1997). Our interpretation that the attendant changes in gastrointestinal (GI) tract structure and biochemistry are "adaptive" rests on our assumption that the GI tract digestive characteristics (e.g.size, enzymes, etc.) are matched to the prevailing diet composition and feeding rate, and that these characteristics do not provide a digestive capacity in great excess of what is necessary for the prevailing diet and feeding rate. The first part of this idea is very well supported by many studies that show a positive correlation between size and enzyme content of the GI tract and daily feeding rate, a positive correlation between enzyme levels and the diet concentration of the enzyme primary substrates, and a positive correlationbetween diet nutrient density and retention time of digesta in the gut (Karasov and Hume, 1997).The second part of the idea is more often asserted than actually demonstrated. We assume that when load is increased, the animal's feeding may be constrained until digestive capacity is increased via the aforementioned adjustments in digestive characteristics. The idea of digestive limitation, besides operating as an important interpretive paradigm, could also be important if digestive processing limited energy flow or other ecological processes (e.g. diet selection). Wild animals do appear to have maximal sustained metabolic rates and if the limit is not imposed by food availability, three physiological hypotheses about the proximate factor(s)have been proposed (Karasov, 1986; Weiner, 1992;Hammond and Diamond, 1997). The central limitation hypothesis suggests that the bottleneck resides in physiological processes and systems, including the digestive system, that are involved in acquiring, processing, and distributing energy to energy-consumingorgans such as muscle or mammary glands. The peripheral limitation hypothesis suggests that processes (such as thermoregulation, lactation, activity)within the energy-consuming organs each have their own metabolic ceilings and this determines the maximum sustained metabolic rate. Finally, the idea of "symmorphosis" proposes that capacities of several of these potentially Limiting factors might be matched to each other and to natural loads (Taylor and Weibel, 1981;Weibel, 2000). One theme of interest here is that maximum sustained metabolic rate in many wild vertebratesmay be determined by the capacity of their digestive system.

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The concept of a digestive limitation can be traced back at least to the work of Max Kleiber (Kleiber, 1933, cited in Kleiber, 1961) on the maximal food capac:ty of domestic animals, and more recently updated by several authors (Kendeigh, 1949;West, 1960;Kirkwood, 1983;Karasov, 1986;Weiner, 1992).Most recently we have come to appreciate that digestion may represent a flexible limit because there is considerable evidence in birds (Karasov, 1996)and mammals (Karasov and Hume, 1997)that digestive features that may limit food processing are adjusted in relation to factors such as diet quality and quantity.Digestion rate for a particular food or substrate can be greatly increased through changes in digestive organ size, changes in the complement of enzymes and transport mechanisms for breaking down and absorbing food and substrate, and changes in alimentary tract muscular activity that affect the contact time between food or substrates and the gastrointestinal (GI)processes. The relative differences (orratios) between either the current or the absolute maximal digestion rate and the current food intake rate are measures of an animal's "safety margin" (Diamond, 1991) or "reserve capacity" (Diamondand Hamrnond, 1992)for responding to changes in environmentalconditions over different timescales.These concepts of GI flexibility and spare capacity are illustrated in Fig. 4.1. Two points deserve highlighting in Fig. 4.1: (1)at any given time an animal has some limited spare capacity (called "immediate spare capacity") but this decreases in extent as the GI system reaches its long-term capacity (Hammond et al., 1994);and (2)phenotypic flexibility of the GI organs is primarily responsible for an animal's ability to change food intake and diet (i.e.it represents most of the "long-term capacity"); however, such phenotypic flexibility requires acclimation time. Explicit references in ecology to possible digestive constraints actually predate most of the works on digestive constraint. For example, C.S. Holling criticized early predator-prey models because they assumed a linear relationship between an individual predator's consumption rate of prey and the prey's density. He proposed instead a "functional response" whereby the consumption rate increased with prey density but reached a plateau value beyond which consumption would not increase (Holling, 1959). Though many ecologists today associate the plateau value with a handling time that defines maximal intake in Holling's "disc equation" or with an herbivore's maximum rate of cropping and chewing (Gross et al., 1993),some ecologists, even in Holling's time, have recognized that maximal digestion rate could also dictate the plateau value (Jeschkeet al., 2002).For example, Mook (1963), observing clear satiation of wild bay-breasted warblers feeding on spruce budworms, modeled predation by including a functional response that included a digestive pause of two hours. Besides potentially limiting energy intake and thus growth, storage and reproductive rates, digestive limitations can be important in behavioral models of optimal diet, models of

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.

d

Acclimation time

Fig. 4.1. Immediate spare capacity and long-term capacity (phenotypic flexibility plus immediate spare capacity) for a hypothetical animal exposed to increasing energy demands (e.g. during migration, during cold weather). The solid lower line represents the nutrient load from feeding. Its baseline corresponds to the animal's routine energy demands (e.g. not during migration or at thermoneutral temperatures). The solid upper line represents the capacity of the gut for processing that nutrient load. Capacity on the y-axis could be total digestion rate, volumetric intake, nutrient uptake capacity, rate of digestive enzyme activity or some other performance measure of the animal. The x-axis is time since the start of an increase in energy demand or change in diet quality. At any given time, an animal can increase its food intake only within the limits set by the level of immediate spare capacity, which decreases as the animal approaches its longterm capacity. When energy and nutrient demands increase, and if the animal has been given time to fully acclimate to these elevated energy demands, then phenotypic flexibility in the digestive system of the animal enables increased energy intake (shown as the increase in solid lower line above the baseline nutrient load). These changes in digestive capacity are critically important in allowing animals to overcome the challenges associated with changing diet quality or quantity (adapted from Diamond, 1991; Diamond and Hammond, 1992).

territorial defense, daily foraging patterns, and optimal migration strategy (examples in Bednekoff and Houston, 1994; Karasov, 1996).

Digestive constraints in mammalian and avian ecology

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The easiest way to detect a digestion-limited animal is to directly measure food collection rate (or handling time) and digestion rate (or digestion time) and compare them, although there are some other methods as well (Jeschke et al., 2002). Thus, insectivorous house wrens (Troglodytes aedon) were found capable of collecting at least 21 grams dry mass arthropods/day (Dykstra and Karasov, 1993),three times as much as their maximal digestion rate (Dykstra and Karasov, 1992). Voles can consume herbage at a rate of 0.15 g min-', at least ten times faster than they can process it (Zynel and Wunder, 2002).Classic examples of digestive limitation are provided by some avian herbivores (Kenward and Sibly 1977;McWilliams and Raveling, 2004) and by ruminant herbivores, for whom rate of intake may be limited by biting, chewing, and rumination, and not plant abundance (e.g. Spalinger et al., 1986;Spalinger et al., 1988).Similar kinds of arguments have been made for nectar-feeding hummingbirds (Diamond et al., 1986),and for bivalve- or crab-consuming birds (Zwarts and Dirksen, 1990; Kersten and Visser, 1996; Guillemette, 1994;Guillemette, 1998).Granivores ought to provide other examples of digestive limitation because they forage on a sometimes highly available food resource yet have a relatively long digestive processing time (Karasov, 1990),but to our knowledge no one has adduced an example to date. According to Jeschke et al. (2002), all animals for which measures of both digestion and handling time are available are digestion limited. One goal here is to review the quantitation of digestive capacity. There have been far more estimates of long-term than immediate spare capacity and the two are sometimes not distinguished. Further, we think some current estimates of spare capacity are inaccurate and have illustrated how it can be more accurately estimated. While digestive Limitation can have clear ecological significance, its mechanistic basis is rarely defined. The magnitude of the limit might be dictated by the volumetric capacity of the gut or the rates at which food are either mechanically or biochemically broken down or absorbed. Whichever feature(s)dictates the digestion limit, its time course for change is also rarely defined, though that too has important ecological implications. Without knowing such details, the quantitative integration of digestion with postabsorptive metabolism in the overall scheme of nutrient processing cannot be completely achieved in a fashion analogous to that achieved for respiratory and metabolic physiology (Weibel,2000). To further this endeavor, and in light of their ecological significance, we therefore focus on these mechanistic details. We think that the magnitude of immediate and long-term spare capacity, and the time course over which digestive capacity can be increased, are the two keys to understanding the digestive challenges that animals face under a variety of interesting ecological situations.

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DEFINING THE LIMITS: MAGNITUDE OF OVERALL DIGESTIVE CAPACITY AND I T S MEASUREMENT I N FEEDING TRIALS Immediate and long-term spare capacity can be estimated in balance trials in which maximum feeding and digestion rates are measured in animals highly motivated to feed, presumably at maximal levels. The spare capacity is the relative difference (or ratio) between the rates measured under those conditions and the rates measured under more routine or baseline conditions. The method is exemplified in Weiner's (1987)study of energetics of Djungarian hamsters. He took hamsters acclimated to room temperatures (22°C) and switched them to cold conditions (-2°C) either quickly or gradually over many days. In the cold, hamsters must eat more to balance higher heat loss or they will catabolize their body tissues to supply the extra energy. Hamsters switched quickly increased their feeding and digestion rate only 15% and lost body mass, whereas hamsters acclimated slowly increased their rates 92% and maintained body mass. Thus, hamsters switched quickly experienced an energy deficit and should have been motivated to eat more, but did not. Presumably, they did not have the spare digestive capacity to do so; their "immediate spare capacity" was only 15% above what they needed routinely for energy balance at 22°C. Hamsters switched more gradually were able to increase their digestive capacity, but their 92% increase in response to cold acclimation was still less than their long-term spare capacity. Weiner (1987) found that hamsters at peak lactation could increase their digestion rate 116 O/O compared with nonreproductives, so this would be a closer estimate of their long-term spare capacity. There have been many measurements of feeding and digestion rates in mammals and birds highly motivated to feed, presumably at near-maximal levels. Typically they involve animals acclimated to very low temperatures, ideally at their limit of thermal tolerance, high levels of forced activity or hyperphagic animals during lactation, storing energy for migration and hibernation, or engaged in rapid growth. Some of these data have been Table 4.1. Relationships between near maximum metabolizable energy intake (MEmax, kJ/d) and body mass (m in g). Group

Energy demanding situation1 Mammals and birds C, L, G Mammals L Birds C Passerine birds M Shorebirds M

Allometric equation

Reference

MEmax= 11.84 m0.72 (Kirkwood, 1983) MEmax= 18.49 nzO66 (Weiner, 1992) (Karasov, 1990) MEmax= 16.42 MEmax= 16.09 m0.70(Lindstrom and Kvist, 1995) MEmax= 11.7 moR2 (Kvist, 2001)2

Energy demanding situations: C = cold acclimation; L = lactation; G = growth; M migratory fattening. Equation calculated by us.

=

Digestive constraints in mammalian and avian ecology

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summarized in allometric equations that express maximum metabolizable as a function of body mass (Table 4.1). As a general energy intake (MEmax) rule, MEmxscales with body mass in a fashion similar to other metabolic rates (i.e.with mass2/3-3/4; note though that none of the estimates control for phylogenetic association among the data) and is 4 - 7 times basal metabolic rate (Hammond and Diamond, 1997).The highest value we know of, measured in lactating mice exposed to low temperature, is 7.7X resting metabolism (Johnsonand Speakman, 2001). MEmxcan depend on the nature of the food. For example, shorebirds that must crush hard shellfish in their gizzards cannot sustain the very high rates they achieve when eating commercially prepared, soft trout food or mealworms (below). Also, Kvist and Lindstrom (2000)made an important point that the absolute amount of food digested per day is influenced not just by the hourly rate, but also the total hours available for feeding (also see McWilliams and Raveling, 2004). The digestive adjustments of mammals and birds acclimated to high feeding rate almost always include increased gut size (though see Johnson and Speakman,2001) and consequently increased amounts of digestive enzymes and nutrient transporters (Karasov and Hume, 1997; McWilliams and Karasov, 2001). Unfortunately, there is no published study for any vertebrate of both rapid and gradual adjustment of feeding and digestion to high energy demand that includes corresponding changes in gut size and biochemistry. The rapid-adjustment experiments, which have been least often performed, are perhaps most interestingbecause they reveal the immediate spare digestive capacity of the animal. We designed a comprehensive study with white-throated sparrows (Zonotrichia albicollis) to determine their response to both rapid and gradual increase in energy demand in order to estimate the level of spare capacity and phenotypic flexibility in their digestive system in response to changes in feeding rate. The experiment involved manipulating ambient temperature, which caused changes in the metabolic rate of sparrows (i.e.increased metabolic rate with lower ambient temperature) and thus induced changes in their food intake to maintain their body temperature constant. By random assignment, sparrows were either held continuously at +21°C,switched rapidly from +21°Cto -20°C, or gradually acclimated to -20°C over 50 days. We measured daily food intake and digestive efficiency of starch (the primary nutrient in their semisynthetic diet) in the three groups of sparrows. The prediction was that sparrows switched rapidly from warm to cold temperatures would maintain digestive efficiency constant only if some safety margin of nutrient absorption capacity over nutrient intake existed before the temperature switch. White-throated sparrows at -20°C required 83% more food than birds at +21°C, as indicated by the comparison of feeding rates of acclimated sparrows in steady state at -20°C and +21°C (Fig.4.2).When birds were switched rapidly from +21°Cto -20°C they increased feeding rate only 45%, a level of

ysiological and ecological adaptations t o feeding

rtebr

h

u

A

Fig. 4.2. Food intake, body mass change, retention time of digesta, and digestive efficiency of starch in white-throated sparrows that were either acclimated to +21°c or - 2 0 ' ~ or switched immediately from +21°c to -20'~. Sparrows acclimated to -20°c ate more than sparrows acclimated at +21°c although both groups of sparrows maintained similar body mass. Sparrows in all three treatment groups had similar digestive efficiency and retention times. Thus, sparrows acclimated to +21°c have a limited spare capacity of about 45% as indicated by an increase in food intake of this magnitude for birds switched rapidly to colder temperatures. However, this limited increment in food intake did not suffice to satisfy the energy demands imposed by a rapid switch from +21°c to - 2 0 ' ~ given these birds lost body mass. This indicates that phenotypic flexibility in digestive features is necessary for sparrows to achieve their long-term capacity.

food intake which was not sufficient to satisfy the extra energy demands, as evidenced by body mass loss (Fig.4.2). Interestingly, birds in all three treatment groups had similar digestive efficiency and retention times (Fig.4.2). Thus, sparrows have some spare capacity (of about 45%) but it did not suffice to satisfy the energy demands imposed by a rapid switch from +21°Cto -20°C. If given enough time for acclimation to the cold, however, sparrows can satisfy the elevated energy demands associated with living in the cold, as evidenced by their ability to maintain body mass after 50 days of acclimation at -20°C. The digestive adjustments to increased feeding rate that occurred during acclimation to the cold included an increase in size of small intestine (Fig. 4.3),large intestine, and liver but not gizzard and pancreas. We are currently completing analysis of digestive enzyme activity and nutrient uptake rates to determine whether adjustments in these digestive features are involved along with changes in gut size. Notice that the 57% increase in

Digestive constraints in mammalian and avian ecology

Fig. 4.i. Small intestine mass (g) of white-throated sparrows that were either acclimated to +21 C or - 2 0 ' ~ or switched immediately from +21 C to -20'~. Sgarrows acclimated to - 2 0 ' ~ had larger small intestines than sparrows acclimated at +21 C, whereas sparrows switched immediately from +21°C to - 2 0 ' ~ had similar small intestine mass as sparrows acclimated to +21°C. See text for a discussion of how these increases in gut size along with the results shown in Fig. 4.2 can be used to estimate the immediate spare capacity and long-term capacity of white-throated sparrows (depicted hypothetically in Fig. 4.1).

small intestine sufficed to accommodate the 83'10 higher feeding rate in birds acclimated at -20°C. This is apparent because mean retention time, efficiency digesting starch, and body mass did not decline significantly with cold acclimation (Fig.4.2). If one considers that sparrows acclimated to +21°Chad a spare capacity of 45% to start with, adding an increase in gut size of 57% to that can more than account for the 87% increased ability to process food. The two measures together imply that sparrows acclimated to -200C probably still had some immediate spare capacity and therefore their long-term digestive capacity was higher than 87% above their digestion rate when held at +21°C. This makes sense because it is known that captive white-throated sparrows can tolerate temperatures down to -29°C when feeding rates are 2.26 (126%)times higher than at +21°C (Kontogiannis,1968). Interestingly, white-throated sparrows engaged in forced activity could tolerate temperatures down to only -5°C but their digestion rates were similar to those of birds acclimated to -29"C, which is consistent with a central limitation set by nutrient processing rather than a peripheral limitation set by heat generation. Thus, the results from the experiment with white-throated sparrows, along with those of Kontogiannis (1968), conform nicely to the model presented in Fig. 4.1 and imply that immediate spare capacity is around 45%

96

Physiological and ecological adaptations t o feeding i n vertebrates

but that after long term acclimation the long-term capacity is around 126% above "baseline". Another method to estimate the immediate spare capacity is to measure intake/digestion rate during periods of restricted feeding. The approach is exemplified in the study by Winter (1998)on a single nocturnal nectarivorous bat (Glossophaga longirostris, 16.4 g), and other examples are provided below. Winter (1998)manipulated the ratio of day-to-night length and forced the bat to eat and digest relatively large amounts of sugar in relatively short amounts of time. He pointed out that after the first hour of feeding during which the gut becomes essentially filled, there is a steady-state period during which food intake can be no faster than the rate of processing, which includes both sugar (sucrose + glucose + fructose) digestion/absorption, postabsorptive processing, and excess water excretion. When feeding time was decreased after 7 d at 12 h / d to 2 h/d, the rate of glucose assimilation during the steady-state period of the night increased to a level 73% higher than during 12-hnights. But the bat feeding for just 2 h/d lost mass and so after 2 d it was switched to 4 h / d and then to 6 h / d feeding throughout which it maintained mass and continued to have an elevated hourly feeding rate as when given 2 h / d to feed. As the bat's energy budget was more and more stressed, it reduced its energy expenditures by reducing flight time. The results thus implied that when motivated to feed, the bat utilized its immediate spare digestive capacity to the maximum, which was apparently at least 73%. With the data it cannot be decided whether this is characteristic of the species (only one individual was studied), nor whether food intake was limited by sugar absorption capacity or water clearance capacity. In a third method to infer immediate spare digestive capacity, which we term a hybrid method, results of feeding trials and in vitro assays are combined to yield estimates of spare capacity. A study on migratory yellowrumped warblers (Dendroica coronata) can be used to illustrate the method (Leeet al., 2002). The birds were captured during migration and habituated to a diet of fruit mash and mealworms. Controlbirds were fed ad libitum but experimentalbirds were food restricted for 3 days by providing 44% of the ad libitum level of food. One purpose of the food restriction was to increase their motivation to feed maximally, and once they were again provided food ad libitum they increased their feeding and digestion rate by 18% compared with controls and the birds gained body mass. This suggests a spare digestive capacity of at least 18%,but other measures of digestive enzymes indicated that it was actually greater than this. The authors showed that the food restriction caused a 20% decline in intestine mass, declines of about 40% in intestinal enzymatic capacities (sucrase, maltase, and aminopeptidase activities were measured along the intestine), and pancreatic enyzme levels were not significantlyaffected (trypsin and chymotripsin) except for a 36% reduction in amylase. If the previously food-restricted birds could increase their digestion rate by 18% compared with controls while concomitantly

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possessing around 40% less enzyme activity than controls, apparently the immediate spare capacity of the control birds must have been approximately 58% (= 18 + 40). There is some support for this estimate. McWilliams and Karasov (1998b)employed a time-limited intermittent feeding protocol and also found evidence of immediate spare digestivecapacity in yellow-rumped warblers. In that study, yellow-rumped warblers were capable of increasing their food intake by 50% in a matter of hours with no change in digestive efficiency or mean retention time. With longer acclimation time warblers increase their feeding and digestion rates more than this during the migratory phase which can be induced by changes in light cycle. For example, warblers on long daylength (16L:8D)had hourly feeding/digestion rates 130%higher than warblers on short daylength (10L:14D;McWilliams and Karasov, 1998a). We are aware of only seven balance studies that permit estimation of immediate spare digestive capacity in mammals and birds (Table 4.2). For now we have excluded a number of studies that estimated immediate spare capacity solely on the basis of biochemical measures (e.g. Buddington and Diamond, 1990, 1992; Toloza et al., 1991; Toloza and Diamond, 1992;Jackson and Diamond, 1995;Weiss et al., 1998)because they do not necessarily establish limitation at the whole-animal level (i.e. the chosen biochemical measure may have higher spare capacity compared with some other step of digestion) and because we have concerns (discussed below) about their accuracy. Of the balance trials, four were discussed above and the others will be considered shortly. The critical design feature of most of these studies is that they quickly challenged animals to increase rate of feeding and digestion, either through cold stimulus, forced activity, or reduction in feeding time. The last, hybrid method essentially inferred the immediate spare capacity by comparing digestive responses of control animals feeding at routine levels with experimental animals with reduced digestive tracts. Another method, never tried but which might be considered, is experimental ablation of the brain's food intake control center (ventromedial hypothalamus)which rapidly brings about hyperphagia.Whatever the method, a rapid time course is important because within a few days adjustmentsoccur in the GI tract that increase the digestive capacity (discussedbelow), in which case immediate spare capacity is no longer measured. All the species studied had quite modest immediate spare capacities (range 9-5O0/0), excluding the measurement on a single bat. This implies that in the wild sudden larger increases in energy needs due to increased activity or thermoregulatory costs cannot be immediatelycompensated by increased food intake even if food is abundant; instead, behavior patterns must be altered to save energy or energy stores must be recruited. But in the same species the long-term spare capacity, achieved partly through adjustments in the GI tract over the course of several days (below) is about 100-125% above routine rates of feeding/digestion (much higher in mice; Table 4.2).

26.4

24

16.4

51

11

3.3

Mus rnusculus

White-throated sparrow

Glossophaga Iongiros tris

Prairie vole

Yellow-rumped warbler

Broad-tailed hummingbird

Daily digestion rate at 22OC

Daily digestion rate at 23°C Daily digestion rate at 21°C

Daily digestion of lactating females at 21°C Daily digestion rate at 21°C Hourly digestion rate for 12 h/d

Daily digestion rate at 22°C

"Baseline" conditions

50

58%

20%

-

9%

73?hd

45%

10%

15%

126%

300hb 488%"

116°/0

1

Cold acclimation, 4 lactation Migratory mode 5 induced by increasing daylength 6

Lactating females acclimated to 8 "C 7 Acclimated to -29 "C 2

Peak lactation

Long-term spare capacity Ref ." Increase Method of over baseline determination

Reduced feeding Not determined time to 2 - 4 h/d Reduced feeding time 95% and feeding bout duration Hybrid estimate 130% and reduced feeding time Measurement Not of sucrase and determined switched to 10°C

Lactating females switched to 8OC Switched to -20°C

Switched to -2OC

Immediate spare capacity Increase Method of over baseline determination

"eferences: 1 Weiner (1987); 2 McWilliams and Karasov (2002); Kontogiamis (1968); 3 Winter (1998); 4 Zynel and Wunder (2002); 5 McWilliams and Karasov (1998a); Lee et al. (2002); 6 McWhorter and Martinez del Rio (2000); 7 Johnson and Speakman (2001) bincrease compared with lactating females at 21°C 'increase compared with nonreproductive females at 21°C donly a single bat studied

35

Mass (g)

Djungarian hamster

Species

Table 4.2. Immediate and long-term spare capacity estimated in balance trials in which maximum feeding and digestion rates were measured in animals highly motivated to feed, presumably at maximal levels. The critical design feature of most of these studies is that they quickly challenged animals to increase rate of feeding and digestion, either through cold challenge, forced activity, or reduction in feeding time.

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DEFINING MECHANISMS: DIGESTIVE FEATURESTHAT MIGHT LIMIT OVERALL DIGESTIVE CAPACITY The magnitude of the digestive limit might be dictated by the volumetric capacity of the gut or the rates at which food are either mechanically or biochemically broken down or absorbed. There are plausible examples of most of these.

Limited Gastrointestinal Tract Volume as a Digestive Constraint Zynel and Wunder (2002),employing a protocol of reduced feeding time (see above), described an apparent gut volume limitation in captive, nonreproductive herbivorous prairie voles (Microtus ochrogaster). They held the animals at 23°C and fed the controls ad libitum and the experimentalseither in a single 3-hour time block per day or in six half-hour time blocks spread every 4 h through the day (still 3 h total feeding time). Three hours of feeding was chosen for the experimental voles because this was more than enough time for them to ingest and chew their daily food requirement of 7.7 g d-'. Voles in both the experimental groups rapidly filled their stomachs with up to 1.4g dry food, the maximum stomach capacity determined in earlier studies. Voles fed in a single time block could not maintain body mass constant whereas voles fed in multiple time blocks could. In a single 3-h time block voles could apparently process at most 2 g if they continually "topped-off" as digesta moved from the stomach through the distal GI tract. In contrast, if voles filled their stomach with 1.4g once every 4 h, which is apparently time enough to clear the stomach, they potentially could digest 8.4 g d-' (= 1.4 g x 24 h/4 h), which suggests an immediate spare capacity of 9% (8.4/7.7 = 1.09). Voles can increase their feeding and digestion rate much more than this when chronically acclimated to low temperature or during lactation. Their long-term spare capacity is about double the routine digestion rate of the controls in this study (Zynel and Wunder, 2002). Though we have described this as an example of a volumetric constraint, perhaps it would be more accurate to say that the bottleneck might lie in the volumetric turnover in g/h, or rate of emptying in g/h of the stomach. This expression puts the bottleneck in the same units as the rate being limited (feeding rate in g/h). On the one hand the distinction seems moot if voles exhibit near instantaneous stomach filling time in relation to stomach emptying time, but on the other hand it begs the question of whether subsequent digestive processes (breakdown,absorption, etc.) are too slow to permit more rapid emptying of the stomach into the small intestine and thus signal the stomach by negative feedback. In any event, an important ecological interpretation of this putative bottleneck is framed in terms of time, i.e. that this bottleneck causes optimally spaced rest bouts between feeding and thus is the primary cause of the observed ultradian rhythm in voles (see discussion in Zynel and Wunder, 2002).

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Physiological and ecological adaptations t o feeding in vertebrates

Limited Rate of Mechanical Breakdown as a Digestive Constraint Birds that consume and crush shellfish provide a compelling example of this kind of limitation. The limitation is suggested for red knots (Calidris canutus) by the fact that they achieve lower rates of MErnx(by less than half) when consuming whole bivalves, whose shells they must crush and excrete via the cloaca, than when consuming the flesh alone which has been removed from shells (T. Piersma, pers. comm.).The increase in gizzard muscularity when knots are transited from soft food to whole bivalves (Piersma et al., 1993; Dekinga et al., 2001) is consistent with the idea that mechanical breakdown is an important limitation to overall digestion rate. The higher maximum rate on flesh perhaps reflects limits in digestive or postabsorptive biochemical processing of the primarily proteinaceous material. Another example of an apparent bottleneck caused by limiting rate of mechanical breakdown might be rumen clearance in ruminants (Van Soest, 1994). The orifice between the rumenoreticulum and omasum functions like a particle size or density sieve so that particles do not escape the rumen until they are sufficiently reduced in size. Reduction is achieved partly through mechanical means (muscular activity of the rumen in conjunction with rumination and chewing) and partly through biochemical means (fermentation rate). Intake of additional food must be matched to the rate of clearance from the rumen. There are other interesting cases of possible limitation by mechanical breakdown that beg to be studied. As mentioned above, among birds granivores have relatively long digesta processing times but the possibility of this being a mechanical digestion limitation has not been systematically explored. Insectivores have been little studied, but Hanski (1984)reported that apparent digestive pauses became more evident when shrews were fed heavily chitinized beetles than when fed lightly chitinized insect pupae. It seems reasonable to apply the same research approach to these situations as described above for red knots: present the same food either intact or mechanically preprocessed under conditions that motivate the animals to feed maximally. Limited Rate of Biochemical Breakdown as a Digestive Constraint McWhorter and Martinez del Rio (2000)proposed that food intake by migratory broad-tailed hummingbirds (Selasphorus platycercus) is limited by rates of hydrolysis. These birds digest mainly sucrose and so sucrase activity in the intestine's brush border was measured in vitro. The in vitro measurement was made with homogenates of tissues collected along the length of the intestine under conditions that saturate the enzyme(s) so that the maximal reaction velocity (V_J could be integrated along the length to yield a total hydrolytic capacity. This capacity was about 120% higher than the

Digestive constraints in mammalian and avian ecology

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observed rates of sucrose intake and digestion, implying that the immediate spare capacity was quite high. But, as the authors pointed out, the common procedure of using the VmaX over the entire intestinal length is physiologically unrealistic because the sucrose concentration progressively lowers as the digesta flows distally along the gut during digestion. Using a more sophisticated model of the gut as a plug-flow chemical reactor, Jumars and Martinez del Rio (1999)calculated a lower digestive capacity that was only 15-35% higher than observed rates of sucrose intake/digestion. They considered this to be the more accurate estimate of the immediate spare digestive capacity of the broad-tailed hummingbird. Support for their argument came in trials in which they rapidly exposed the hummingbirds to low temperature. The birds did not (could not?) increase their intake but instead reduced their expenditure by utilizing torpor. In a similar kind of experiment rufous hummingbirds (Selasphorus rufus, 3.2 g) switched suddenly to low temperature did not (could not?) sufficiently increase their intake and lost body mass (Gass et al., 1999). The study by McWhorter and Martinez del Rio (2000)underscored some important considerationsin estimating digestive capacity by extrapolation from measures of maximum enzymatic breakdown rate in vitro. First, the method assumes that hydrolysis rates measured in vitro correspond to actual rates in vivo. This may apply best for digestion of sucrose for which hydrolysis depends only on an enzyme bound to the intestine's brush border that is easily measured. Second, for most foods, besides sucrose-rich nectars, there are multiple substrates (e.g. starch, protein, fat) whose digestion is much more complex involving gastric and/or pancreatic enzymes that act in addition to multiple intestinal brush border enzymes. This complexity far exceeds our current abilities to model the overall process. Third, the hydrolysis rate is concentration dependent over some substrate range. Though most other studies estimating hydrolytic capacity (e.g.Hammond et al., 1994; Weiss et al., 1998; Martinez del Rio et al., 2001) have assumed constant saturating substrate concentrations, the newer, more physiologically realistic approach by McWhorter and Martinez del Rio (2000)showed that the aforesaid studies surely overestimated the hydrolytic capacity.

Limited Rate of Nutrient Absorption as a Digestive Constraint We know of no published study that provides strong evidence of nutrient absorption acting as a digestive bottleneck. Earlier studies that stimulated much interest in digestive bottlenecks (Karasov et al., 1986;Diamond et al., 1986)may be used to illustrate the problem. The intestinal glucose uptake capacity of rufous hummingbirds (SeIasphorusrufous, 3.2 g) was estimated to be 87.7 pmol h-' based on in-vitro measurement. How does this compare with actual intake? A 3-g hummingbird held at room temperature digested 1.5 - 2 g sucrose d-' (McWhorter and Martinez del Rio, 2000) or 4.4

102

Physiological and ecological adaptations t o feeding in vertebrates

- 5.8 mmol/d-'. Assuming that all this was digested in 16 h and that half was glucose, the bird's actual glucose absorption rate was thus at least 138 pmol h-I, 56% higher than the maximal uptake capacity in vitro! In some other studies when the in vitro measurement of D-glucose absorption was less than what animals actually achieved, the authors argued that glucose that was not absorbed by the intestine was later fermented in the hind gut. But this kind of explanation cannot apply to a hummingbird (no hind gut). The simplest explanation is that the in vitro measurement underestimated actual glucose absorption rate. There is valid concern that in other studies a similar underestimation occurred but was overlooked by invoking cecal fermentation. Consider some of the problems that plague estimates of nutrient absorption capacity, which can easily lead to either over- or underestimation. Overestimation of absorption rate, as for hydrolysis rate, is possibly caused by improper assumptions about lumenal nutrient concentrations.For example, when Toloza and Diamond (1992)estimated the immediate spare absorptive capacity of adult laboratory rats they found that mediated glucose absorption was 130% higher than daily glucose intake rate when they assumed lumenal concentrationwas 50 mM, but only 20% higher when they assumed the lower actual determinations of lumenal glucose concentration because absorption rate is much lower at low concentration (Fig.4.4).Several factors can lead to underestimation of absorption rate. It is possible that absorption rates measured in vitro are less than the rates in vivo because isolated tissue may become damaged and lead to underestimation of active transport rates (Starck et al., 2000). Also, measures of absorption with isolated intestinal tissue apparently fail to incorporate processes that may function in the intact animal such as trafficking of additional glucose transporters (GLUT 2) to the brush border stimulated by the presence of lumenal sugar (Kellett and Helliwell, 2000), and an important passive absorption pathway that seems very important, at least in birds (Karasov and Cork, 1994; Caviedes-Vidal and Karasov, 1996; Chediack et al., 2001) and probably in mammals (Pappenheimer, 1998; Fig. 4.4). Other kinds of absorption measures in vivo in anesthetized animals may be suspect because the anesthesia can influence the rates of absorption (Uhing and Kimura, 1995).Weber and Ehrlein (1998) arguably misestimated spare capacity by overlooking the very real physiological constraint that animals do not excrete a large amount of unabsorbed solute (see Mc Whorter and Martinez del Rio 2000),and by assuming that the apparent maximum absorption rate at their test concentration would represent the maximum absorption rate at higher test concentrations. Estimation of whole-animal glucose absorptive capacity by in vitro methodology has rarely been validated and in one of the earlier studies applying it, Toloza and Diamond (1992) pointed out that the calculation, which has numerous approximations, should be considered meaningful to an order of magnitude. Setting aside the issue of quantitative accuracy, we

Digestive constraints in mammalian and avian ecology

1 0

de+;*

METHOD in vitro

A

10 20 30 40 50 60 70 80 90 100 Glucose concentration (mM)

Fig. 4.4. Estimation of nutrient absorption capacity depends on method used and concentration assumed. This is illustrated in the comparison of measures in jejunum of adult laboratory rats. Many studies have applied the everted sleeve method (Karasov and Diamond, 1983) which was used by Debnam et al., (1988) to measure mediated Dglucose uptake ("in-vitro", open triangles, solid line). These researchers also measured mediated D-glucose absorption in perfused jejunum of anesthetized rats ("in-vivo A", open circles, dashed line). At low concentrations the rate is lower than in the in-vitro preparation because of unstirred layer effects, but maximal mediated uptake (plateau values) is fairly similar. Both measures, as well as the single highest reported maximal mediated in-vitro uptake in rats (431 nmol min-' ~ m -Toloza ~; and Diamond, 1992) are lower than absorption rates measured in chronically perfused, unanesthetized adult rats (Ugolev et al., 1986)) interpreted by Pappenheimer, 1998) ("in vivo C", open squares, dotted line). This latter measurement may include the effect of recruitment of additional glucose transporters (GLUT 2) that have lower affinity than the brush border glucose transporter (SGLT 1)(Kellett and Helliwell, 2000) and includes passive absorption, typically neglected in calculations of absorption capacity but which becomes especially important as concentration increases.

do think that in vitro measures are very useful for indicating qualitative changes in digestive capacity. Furthermore, when in vitro biochemical measures are made in conjunction with other whole-animal measures, perhap they can lead to useful hybrid estimates of immediate spare capacity, as described above.

HOW QUICKLY DOES DIGESTIVE CAPACITY INCREASE?

In the wild when energy needs suddenly increase, the digestive system could act as a bottleneck over some short term even if it eventually adjusts to permit

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a higher rate of energy flow. The period of time over which this digestive constraint operates is dictated by the time it takes to increase digestive organ size or tissue-specific levels of digestive enzymes and nutrient absorption mechanisms. Relying on a rather limited number of studies, we can assemble a picture of the time course of digestive adjustment starting with turnover time of intestinal enzymes and epithelial cells and proceeding through rates of change of entire tissues to whole-animal feeding responses. The picture that emerges is that biochemical changes may occur faster than structural changes and changes may occur faster in small than in large animals. Starting with the most basic level, birds and mammals switched from carbohydrate-free diet to high carbohydrate diet could at least double the enzymes and/or nutrient specific activity of their ~arboh~drate-d;~estin~ transporter within 1-2 days of the dietdswitch (Karasov and Hume, 1997). An important mechanism is the replacement of intestinal cells with new cells possessing more copies of particular digestive enzymes (Karasov and Hume, 1997). In birds the rate of cell proliferation, indexed by the length of the S-phase (phase of DNA replication, measured by labeling in vivo) was measured in two different-sized species during growth (Starck, 1996).This rate did not differ markedly by age or species and so given a rather invariant S-phase (average 6 hours), the intestinal turnover time of small birds (replacementtime of intestinal cells)was 2-3 days compared with 8-12 days in larger birds (Starck, 1996). Among the six mammal species studied by Smith et al. (1984), however, there was no marked body-size dependent variation in enterocyte life span and, as in birds, enterocyte turnover rate was independent of age in mice (Ferraris and Vinakota, 1995). In laboratory rats, which have a one-day enterocyte turnover (Karasovand Diamond, 1987), following a fast the villi returned to their normal length within a day after initiation of feeding (Butset al., 1990;Hodin et al., 1994).The first responses of the atrophied gut of starved rats to initiation of feeding occurred as early as two hours after the first meal, when genes such as c-fos and c-jun, which represent the mitogenic response in many types of tissues, were first expressed in intestinal crypt cells (Hodin et al., 1994). Several studies, especially in birds, have monitored progressive changes in organ sizes following diet switches using destructive or nondestructive sampling methods. Fasted blackcaps (Sylvia atricapilla) that had reduced intestinal mass grew back their small intestine in two days or less once they were provided with food ad libitum (Karasovet al., 2004).Red knots switched from soft food to hard shellfish increased gizzard mass 147'/0 within 6 days (Dekinga et al., 2001). Japanese quail switched to high fiber diet increased gizzard mass 110% within 6 days, but significant increases were already apparent 1 day after the diet switch (Starck, 1999a). Reversible changes in gut length in response to changes in diet composition have been reported to occur within 3 4 weeks in grouse and quail (Moss and Parkinson, 1972;

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Savory and Gentle, 1976a, b) and ducks (Miller, 1975),with significant responses within 5 days in ducks (Drobney, 1984; Kehoe et al., 1988). Whole-animal feeding trials gave a similar picture of the time course for adjustment. American robins (Turdus migratorius) and European starlings (Sturnisvulgaris) switched from fruit to insect diets progressively increased digestive efficiency within three days of the diet switch (Levey and Karasov, 1989).Fasted blackcaps and thrush nightingales that had been food restricted, progressively increased their digestion rates to a maximum over the course of 3 days after returning to ad libitum feeding (Karasov and Pinshow, 2000; Kvist and Lindstrom, 2000). Red knots delayed accepting a new shellfish diet for at least 2 days when switched from soft food (Piersma et al., 1993; Dekinga et al., 2001). In summary, the response of the digestive system to changes in diet composition and feeding rate seems rapid. Even for structural measures (e.g. gizzard or intestine mass) that may respond more slowly than biochemical measures, statistically significant changes of a magnitude of 2040% are apparent in most species within 1-2 days of a change in diet (Starck, 199913). But systematic studies within and across species of correlated rates of change in digestive biochemistry and structure in response to whole-animal dietary adjustment are generally lacking. Long-term Digestive Capacity-How High can it Go? If birds are given adequate time to acclimate, then increases of at least two times in food intake and digestion rate are possible (Karasov,1996). Doubling food intake occurs commonly in birds preparing for migration (Berthold, 1975;Blem, 1980; Karasov, 1996)and in birds at cold temperatures (Dawson et al., 1983; Karasov, 1990; Dykstra and Karasov, 1992; McWilliams et al., 1999). Many mammals exhibit increases of similar magnitude (e.g. Tables 4.1 and 4.2) but some truly extraordinary increases have been recorded in laboratory mice (Hamrnond et al., 1994).For example, nonreproductiveSwissWebster female mice doubled their intake/digestion rate when switched from 23 to 5°C and could still increase it 3.3times more at peak lactation with very large litters. The net long-term digestive capacity was thus about 6.7 times the rate under routine conditions. The relative increase was similarly high, 5.9 times, in the MF1 strain of Mus musculus (Johnsonand Speakman, 2001). Are mice exceptional in this regard because of selection for high reproductive rate? In domesticated birds an important digestive change obtained as a result of artificial selection for more rapid growth was an increase in the relative size of the digestive organs (Lilja et al., 1985; Jackson and Diamond, 1996) which presumably permits relatively high digestion rates. As mentioned above, the digestive adjustmentsof mammals and birds to long-term acclimation to high feeding rate almost always include increased gut size and consequently increased amounts of digestive enzymes and

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nutrient transporters (Karasov and Hume, 1997). Interestingly,the processing time of each meal, measured as mouth-to-cloaca total mean retention time (MRT; an index of turnover), and digestive efficiency do not change markedly (Dykstra and Karasov, 1992; McWilliams et al., 1999; Fig. 4.2). Probably, feedback mechanisms in the digestive tract ensure that the rate food enters .the intestine from the stomach and travels distally along the intestine does not exceed the rate at which it is broken down and absorbed. What permits higher food intake (inflow)even though turnover time is held fairly constant, is the larger volumetric capacity (Karasov, 1996). The primary instance in which NIRT is altered is when food richness is altered, in which case MRT changes in a corresponding fashion with the result that movement of digesta is matched to breakdown and absorption rates and digestive efficiency is maintained (Karasov, 1996). Thus, for these cases in which the intestine's rate of breakdown and absorption is limiting, if the feeding rate or food richness is to increase, then the biochemical features (enzyme levels, nutrient absorption rates) must be increased through an increase in activity per unit tissue or an increase in total amount of tissue. Both kinds of adjustments occur in mammals (Weiss et al., 1998) and birds (McWilliams and Karasov, 2001).This kind of integrated analysis of how the gut functions and adjusts has not been performed for the types of feeders whose intake is possibly limited by the rate of physical breakdown of the food (e.g.feeders on bivalves and crabs; see below). Whether the digestive capacity can be increased to match any demand put on it or whether the gut sometimes ultimately limits the energy budget is not known for most animals. The issue has been thoroughly studied in laboratory mice challenged during cold acclimation, lactation, and a combination of these factors (Hammond et al., 1994; Johnson and Speakman, 2001). With each increasing energetic challenge Swiss-Webstermice increased gastrointestinal mass and hydrolytic and absorptive capacity and, for the highest load of lactation in the cold, the energy budget limit was not set by the digestive system but more likely by lactational performance (Hammond et al., 1996).For the MF1 strain Johnson and Speakman (2001)doubted that even lactational performance was a limit, at least during a female's first lactation. They speculated that in that strain females may limit themselves during their first reproduction perhaps to maximize lifetime reproduction. How can we test whether the gut limits the energy budget for an animal in the field? The method used so far has been to measure the long-term limit in laboratory studies and compare it with the field energy budget. Studies on house wrens (Dykstra and Karasov, 1992, 1993) and yellow-eyed Juncos (Juncophaeonotus) (Weathers and Sullivan, 1989), for example, rejected the hypothesis that rate of digestion might limit brood size proximally because parental energy expenditure,measured with doubly labeled water, was below the longer term digestive capacity.

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Should we generalize from these results with laboratory mice and two passerine species and conclude that for all mammals and birds that digestive capacity can be increased to match any demand put on it and will not be linliting in the ecological setting? This would be premature we think. As described above, there are interesting plausible examples of digestive bottlenecks involving animals eating foods quite differentfrom the formulated laboratory chow fed to mice. There are other energy intensive points in the life cycle, such as growth (Karasov and Wright, 2002) and migration, during which digestion may prove to be the limiting factor in the energy budget. Also, there may be situations in which the immediate spare digestive capacity may be ecolo~callyimportant even if over the longer term digestive capacity increases and the long-term capacity is not limiting. For example, a gutlimitation hypothesis for many migratory birds suggests that the initially slow rate of mass gain at stopover sites occurs because birds lose digestive tract tissue and hence function during fasting, and rebuilding of the gut takes time and resources and itself restricts the supply of energy and nutrients from food (McWilliamsand Karasov, 2001). For birds that fly, the size of the digestive tract is likely ultimately limited by mass balance requirements for flight (i.e.big guts can't fly; Piersma and Gill, 1998).

FUTURE DIRECTIONS

(1) The idea of a digestive constraint is most plausible when food collection rate and digestion rate are both measured and the former is higher than the latter. Granivores are good candidates for such digestive limitation but to our knowledge no one has yet provided an example. (2) There is no published study for any vertebrate of both rapid and gradual adjustment of feeding and digestion to high energy demand that includes corresponding changes in gut size and biochemistry. (3) Rapid-adjustment experiments, rarely performed (only seven studies that we know of),are perhaps most interesting because they reveal the immediate spare digestive capacity of the animal.

(4) Granivores and insectivores eating heavily chitinized prey, provide interesting cases of possible digestivelimitation by mechanicalbreakdown and beg to be studied. (5) Estimation of whole-animal hydrolytic and absorptive capacity by invitro methodology has rarely been validated which undercuts their application for quantitative estimation of spare digestive capacity. (6) More integrative studies are needed that simultaneously measure adjustments in gut anatomy, retention time of digesta, enzyme hydrolysis

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rates, nutrient absorption rates, and digestive efficiency in response to changes in food quantity and quality. (7) Our understanding of the time course of digestive adjustment starting with turnover time of intestinal enzymes and epithelial cells and proceeding through rates of change of entire tissues to whole-animal feeding responses is based on very few studies. (8) Can wild species or much larger species achieve the increases in longterm digestive capacity achieved by small rodents such as laboratory mice (6.7times the digestion rate under routine conditions)? (9) Do laboratory mice reflect the norm or are they exceptional in their ability to match digestive capacity to any demand put on it? (10) More integrative studies are needed that compare immediate and longterm digestive capacity with rates of energy flow in free-living animals at energy intensive points in their life cycle. Are there other ways to test the hypothesis that digestion proximally limits energy budgets in the field? Acknowledgments

Supportedby N.S.F. (IBN-9723793,IBN-0216709 to W.H.K. and IBN-9984920 to S.R.M.).

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Paracellular Intestinal Absorption of Carbohydrates in Mammals and Birds Todd J. McWhorter Department of Wildlife Ecology, University of Wisconsin, Madison, WI, USA

SYNOPSIS In most animals and for most dietary substrates, digestion comprises two steps: hydrolysis into smaller molecules and absorption across the gut wall. This chapter deals with the latter of these inextricably interrelated processes. Intestinal absorption and paracellular permeability are subjects of considerable interest, both because they determine capacities for nutrient uptake and because they have important consequences for exposure to natural and man-made water-soluble compounds including drugs, toxins, and toxicants. These subjects are reviewed in the context of our current understanding of intestinal carbohydrate absorption and some important, and sometimes controversial, differences among mammals and birds are highlighted. A short description of mediated carbohydrate absorption, outlining the roles played by specific transporters, is given and the modulation of solute uptake capacities is briefly discussed. The heated and ongoing debate regarding the relative importance of paracellular uptake (via diffusion and solvent drag) as a pathway for carbohydrate absorption in mammals is taken up next, followed by a discussion of the current evidence regarding paracellular absorption of carbohydrates in birds, and a critical examination of the experimental approaches that have been used in resolving the relative importance of this process. Lastly recent studies correlating functional and mechanistic aspects of paracellular flux in birds are reviewed.

INTESTINAL ABSORPTION OF CARBOHYDRATES Epithelial uptake of carbohydrates in the intestine is known to occur through both carrier-mediated (active or facilitative passive) and nonmediated mechanisms (Hopfer, 1987). Carrier-mediated transport is effected by

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specific membrane-associated carriers, and its rate follows saturation kinetics. By contrast, the rate of nonmediated passive uptake in the intestine varies linearly with solute concentration and does not obey saturation kinetics. This chapter commences with a discussion of each of these processes in turn and a brief outline of the modulation of solute uptake capacities. Carrier-mediated Transport As early as the 1960s it was recognized that active, carrier-mediated processes were important in carbohydrate absorption (Crane, 1960). The transcellular route for active D-glucose absorption involves carrier-mediated transport at both the lumenal (brush-border) and basolateral membranes. The concept of Na+-gradient-driven, secondary active transport was contrived by Crane et al., (1961) to explain concentrative absorption of D-glucose in the intestine. This insight was particularly remarkable, considering the paucity of data on cytosolic composition and intracellular processes available at that time (Hopfer, 1987).Crane et al. (1961)correctly inferred that the concentrative step is located at the brush-border membrane and is a result of cotransport with sodium. The specific protein transporter responsible was cloned by Wright and colleagues in 1987 and is now known as SGLTl (Hediger et al., 1987).It is a high-affinity, low-capacity transporter in which D-glucose translocation is coupled to the cotransport of two Na' (Hediger, 1994).The absorption of D-glucose across intestinal epithelial cells occurs against a concentration gradient by SGLTl in the brush-border membrane. The Na+/K+-ATPase(Skou and Esmann, 1992) located in the basolateral membrane maintains the electrochemical gradient required to drive uphill glucose transport. The other well-characterized member of the SGLT family is a low-affinity, high capacity brush-border Na+-glucose cotransporter (SGLT2, stoichiometry 1 Na' : 1 glucose) that is principally involved in glucose reabsorption in the kidney (Kanai et al., 1994). SGLT2 has not been documented in the intestine (Halaihel et al., 1999). D-glucose moves out of intestinal epithelial cells via GLUT2 in the basolateral membrane (Hediger, 1994). GLUT2 is a low-affinity member of the GLUT family of facilitated glucose cotransporters that permit movement of glucose across plasma membranes down its concentration gradient (Thorens, 1993;Hediger, 1994). Recent studies in perfused rat jejunum have described a mechanism for gut glucose (and fructose) absorption that involves GLUT2-mediated facilitated diffusion in the apical membrane (Helliwell et al., 2000a; Kellett and Helliwell, 2000; Kellett, 2001). This mechanism is initiated by the active transport of glucose via SGLT1, which causes the translocation of GLUT2 to the lumenal surface of the gut. Regulation appears to be via protein kinase C-dependent and mitogen-activatedprotein kinase signaling pathways (see Kellett, 2001 for review). This novel finding has direct bearing on the debate regarding paracellular glucose absorption (see below).

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Some researchers have suggested that a heterogeneity of D-glucose transport systems exists in the intestinal epithelial brush-border (Halaihel et al., 1999 and references therein). The proposed second active transport system, dubbed system 2 (S2), is different from SGLTl and basolateral GLUT2. Halaihel et al. (1999) used pig jejunal brush-border membrane vesicles to confirm that S2 is a low-affinity, high-capacity, D-glucose and D-mannose transporter, distinct from any previously known. The existence of 52 is somewhat controversial, having been questioned by researchers who affirm that a single system (i.e. SGLTl plus some passive up take) completely explains glucose uptake in the intestine (Malo, 1993; Hediger and Rhoads, 1994). Halaihel et al. (1999) carefully reexamined the heterogeneity question to determine whether previous experimental procedures could have caused the apparent presence of nonexistent transport systems, and to determine the minimum range of substrate concentrations needed to correctly fit saturation curves. Their reanalysis indicated that D-glucose contaminated with D-sorbitol cannot cause the spurious existence of transport systems (a criticism of Malo, 1993) and that a large range of substrate concentrations (2 50 mM) is necessary to correctly distinguish the kinetics of D-glucose transport processes (Halaihel et al., 1999). They affirmed that their results distinguish two kinetically distinct systems: high-affinity, low-capacity SGLTl and low-affinity, high capacity S2, which is not believed to be a member of the SGLT family. The debate continues and it remains to be seen whether a new transporter will be cloned (see for example, Doege et al., 2000). It is possible that this proposed S2 relies on the recruitment of GLUT2 to the brush-border membrane (see above). Regardless, most of the active D-glucose uptake at low concentrations appears to mediated by SGLTl (Halaihel et al., 1999). Fructose transport by the brush-border epithelia of the mammalian intestine is accomplished by another member of the GLUT family, GLUT5 (Burant et al., 1992; Rand et al., 1993). GLLTT5 is a sodium-independent, facilitative transporter that has a very poor affinity for D-glucose (Rand et al., 1993and references therein). Fructose absorption appears to be much more concentration-dependent than D-glucose transport (Holdsworth and Dawson, 1964), although there has been some suggestion of active fructose transport associated with sucrose digestion and disaccharidase activity (Shi et al., 1997and references therein). Transport of fructose across the basolateral membrane (and possibly also across the brush-border membrane, see above and Helliwell et al., 2000b) is carried out by the facilitative glucose transporter GLUT2, which transports galactose as well (Burant et al., 1992). Functional studies have demonstrated significant differences in the biochemical properties of rat and human GLLTT5, so it is possible that there are interspecific differencesin the regulation of dietary fructose uptake (Rand et al., 1993).

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Modulation of Solute Uptake Capacities Regulation of solute uptake capacities has been a well-known phenomenon for nearly 70 years (Ferraris et al., 1990). It has been described under a wide range of conditions, including pregnancy, lactation, exercise, exposure to a cold environment, and varying dietary carbohydrate levels (reviewed by Karasov and Diamond, 1983, see also Chapter 4 by Karasov and McWilliams, this volume; Ferraris and Diamond, 1989).Several functional considerations predict the pattern of uptake capacity regulation (termed the "adaptive modulation hypothesis" Karasov and Diamond, 1983; Karasov, 1992). Two of these considerations are relevant to the discussion here of carbohydrate absorption. First, transporters should be repressed if the biosynthetic costs of producing and maintaining the transporter exceed the benefits that the transporter provides. Second, transporters for nutrients that yield calories should be up-regulated by their substrates since metabolizable nutrients yield calories in proportion to amount of nutrient. Indeed, it has repeatedly been shown in a wide variety of animals that dietary glucose levels modulate uptake capacity (Karasovand Diamond, 1983; Diamond and Karasov, 1984; Ferraris and Diamond, 1989; Karasov, 1992).Similar modulations are known to occur as the result of varying dietary fructose levels (Ferraris and Diamond, 1989; Corpe et al., 1996; Shu et al., 1997). Carbohydrate uptake appears to be regulated primarily in those species that encounter significant and varying carbohydrate levels in their diets (Ferraris and Diamond, 1989; Afik et al., 1995). Such modulation is physiologically important in overcoming potential digestive bottlenecks (Ferrarisand Diamond, 1989)while avoiding waste of biosynthetic activity on unutilizable capacity (Diamond, 1991). The dietary modulation of transport capacity has recently been extensively studied in birds (e.g. Afik et al., 1995; Martinez del Rio et al., 1995; Levey et al., 1999). Nonmediated Uptake of Carbohydrates The paracellular component of the epithelial barrier is the pathway between adjacent epithelial cells. Transport across this pathway is restricted by the junctional complex and the lateral intercellular spaces (Ballard et al., 1995). The most important component of the junctional complex for restricting passage of small solutes through the paracellular pathway appears to be the tight-junction (Anderson and van Itallie, 1995;Anderson, 2001).This barrier is created where transmembrane protein strands from adjacent cells converge in the paracellular space (Tsukita and Furuse, 2000).There is thought to be a positive correlation between the number of strands and paracellular electrical resistance (the inverse of permeability).Claudins have been identified as the major protein constituents of tight-junction strands and these strands appear to be reorganized very dynamically (Sasaki et al., 2003). The standing osmotic gradient theory for solute and liquid absorption proposes that active transport of glucose and sodium drives paracellular liquid

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uptake by establishing a solute osmotic gradient across the enterocytes (Diamond and Bossert, 1967).Glucose is actively transported across the brushborder (apical)membrane and then moves across the basolateral membrane into the intercellular space (see above and Hediger, 1994). Sodium is actively transported across the basolateral membrane by the Na+/K+-ATPase.This buildup of osmotically active solutes drives net absorption of water (and theoretically accompanying nutrients via solvent drag) across intestinal epithelia (Diamond and Bossert, 1967). It is presently unclear whether water moves predominantly across cell membranes or through tight-junctions (Ballard et al., 1995), but see discussion below. In contrast with carriermediated transport, the rate of passive transport varies linearly with solute concentration and does not obey saturation kinetics (see Chap. 3, Fig. 3.3b). RELATIVE IMPORTANCE OF PARACELLULAR CARBOHYDRATE UPTAKE I N MAMMALS

Until just recently it was widely accepted that carbohydrate transport in the mammalian intestine occurs principally through active, carrier-mediated mechanisms (Crane, 1960,1975;Hopfer, 1987).Pappenheimer and coworkers proposed in the late 1980s that most glucose uptake occurs passively via the paracellular pathway instead (Madara and Pappenheimer, 1987; Pappenheimer, 1987;Pappenheimer and Reiss, 1987; Pappenheimer, 1990). This hypothesis was based on the observation that the permeability of the small intestine epithelia was increased when exposed to high concentrations of glucose in the lumen. It was controversial because it challenged conventional theories of intestinal solute and water absorption. The subject has been one of considerable debate ever since. In this section the evidence presented by Pappenheimer and coworkers in support of the notion that paracellular flux is the major pathway for the absorption of hexoses and other hydrosoluble nutrients is outlined, followed by a discussion of several criticisms of their hypothesis and a review of recent evidence bearing on this controversial subject. In a series of three papers published in 1987, Pappenheimer and colleagues presented evidence in support of their novel hypothesis (Madara and Pappenheimer, 1987; Pappenheimer, 1987; Pappenheimer and Reiss, 1987). The main tenet of this hypothesis is that glucose and amino acids taken up by enterocytes serve as intracellular signals to decrease tight-junction integrity and thereby facilitate the absorption of hydrophilic solutes by paracellular bulk flow. Based on the clearances of inert solutes (steady-state transepithelial fluxes per unit concentration) from the small intestines of anesthetized animals, Pappenheimer and Reiss (1987) calculated the fraction of fluid absorption which passes paracellularly. The addition of 25 mM

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D-glucose to the perfusion solution of an in vivo rat preparation doubled the estimated rate of paracellular liquid absorption (to approximately 50% of total). They concluded that above a lumenal concentration of 250 mM D-glucose (which may or may not be exceedingly high, see below), paracellular solvent drag is the principal route for intestinal absorption of glucose. They further proposed that Na+-coupledtransport of organic solutes from the lumen to intercellular spaces provides the principal osmotic force for fluid absorption and modifies tight-junction permeability. Pappenheimer (1987)addressed the question of whether Na+-coupledtransport increases solvent drag solely by its effects on fluid absorption, or whether it also opens epithelial.junctions. He found that the addition of small concentrations of D-glucose or amino acids to isolated intestinal segments greatly decreased transepithelial impedances. Impedance was interpreted in terms of junctional and lateral space resistance and surface area of basolateral membranes (i.e.decreased impedance = decreased resistance = increased permeability). The addition of 25 mM D-glucose decreased lateral space resistance by more than 50%. These results provided support for the theory that the surface area of lateral membranes and dimensions of epithelial cell junctions are regulated by the concentrationof nutrients in the intestinal lumen (Pappenheimer, 1987). Pappenheimer (1987) further supposed that Na+coupled solute transport triggers contraction of circumferential actomyosin fibers in the terminal web of the microvillar cytoskeletal system, thereby pulling apart tight-junctions to allow the paracellular absorption of solutes by solvent drag. Madara and Pappenheimer (1987)explored the structural correlates of the impedance and permeability changes induced by glucose and amino acids. Profound changes in ultrastructure were observed using light and electron microscopy and freeze-fracture techniques. Specifically, the addition of D-glucose or amino acids induced junctional dilation, expansion of lateral spaces, and condensation of actomyosin in the perijunctional ring. The authors interpreted these observations as providing support for the hypothesis that Na+-coupledtransport triggers the physical opening of intercellular channels and thus facilitates paracellular flux (Madara and Pappenheimer, 1987). Pappenheimer (1990) emphasized the importance of paracellular glucose transport by comparing the daily carbohydrateintake of several species with maximum predicted rates of transcellular Na+-coupledtransport. He estimated that total ingestion of sugars exceeds the capacity for active transcellular transport threefold in rats and fivefold in rabbits. The discrepancy between average rates of ingestion and maximum rates of transport increased exponentially with body weight. In humans, the ingestion-absorptionrate was predicted to be 10-20times greater than active transport. The discrepancy between these estimates of maximal active transport and ingested carbohydrate loads, and the recovery of >8O0/0of [3H]mannitol (a marker supposedly not absorbed by any carrier-mediatedprocess) in urine

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or other body fluids, led Pappenheimer to reiterate the conclusion that paracellular transport plays the major role in intestinal nutrient absorption (Pappenheimer, 1990). In fact, according to the hypotheses of Pappenheimer and coworkers, the primary role of Na+-coupled transport of solutes is to provide the osmotic force for convective flow and to effect the decrease in epithelial permeability necessary for increased paracellular flux (Pappenheimer and Reiss, 1987; Pappenheimer, 1990). Recent experiments using phloridzin blocking in dogs (Pencek et al., 2002; Pencek et al., 2003) confirm that SGLT1-mediated glucose uptake is indeed required to activate paracellular absorption of glucose; however, these studies also suggest that paracellular absorption is a relatively minor component of total uptake (see below). As is the case with any hypothesis that challenges conventional thought, there has been intense scrutiny of Pappenheimer's work. Some of these criticisms are discussed below. The alternative view to paracellular uptake, advanced by Ferraris and Diamond (1989; 1997),is that adaptation of brush-border membrane carbohydrate transporters (i.e.SGLT1, GLUT5, and based on recent evidence possibly the recruitment of GLUT2) is matched to dietary intake. A significant body of literature reports measurements of intestinal glucose transport as a function of lumenal concentration (usually based on perfusions). Knowledge of the glucose concentrations normally present in the small intestine of animals is essential for assessing the physiological meaning of these measurements. Ferraris et al. (1990) criticized previous work reporting exceptionally high lumenal glucose concentrations and subsequent studies that have based their conclusions on such observations. They argued that knowing the true physiological glucose concentration in the lumen is critical for determining: (1)whether or not the intestine possesses reserve absorptive capacity, (2)whether regulation of glucose transport at the membrane level has physiological significance, (3) the predominant mechanism of glucose absorption (i.e. paracellular vs. transcellular uptake), and lastly (4) the significance of Michaelis-Menten constants (K,) reported for glucose transport (Ferraris et al., 1990). Lumenal glucose concentrations in the small intestine were widely assumed to be in the range of 50-500 mM (Crane, 1975; Alpers, 1987; Pappenheimer and Reiss, 1987). Ferraris et al. (1990) pointed out that although reports of lumenal glucose concentrations appear to be unanimous, these exceptionally high values have several surprising implications. First, they imply that lumenal contents are very hypertonic, because in addition to glucose the lumen contains many other osmotically active substances (ions, amino acids, etc.). Since the small intestine is extremely permeable to water (Fromter and Diamond, 1972; Chang and Rao, 1994),this high concentration would presumably be quickly diluted to isotonicity. Second, the reported prevailing high glucose concentrations are nearly two orders of magnitude higher than Kmvalues for glucose absorption measured in vivo or in vitro. The authors suggested that this would be unusual, as most

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enzyme and transporter Kmvalues are in the range of prevailing substrate concentrations, permitting the efficient regulation of transport rates through modulation of Km(Hochachkaand Somero, 1984; Ferraris et al., 1990). Third, the quoted values imply that the small intestine's capacity for glucose absorption is up to two orders of magnitude higher than daily intakes. Ferraris et al. (1990) argued that this is also unusual, based on the observation that of enzymes tend to be in the range of premaximal reaction velocities (VmaX) vailing fluxes. However, if pa&kellular absorption is indeed a significant route of absorption (Pappenheimer, 1990),lumenal concentrations and abof an enzyme syssorptive fluxes considerably higher than the Kmand Vmax tem respectively,would not be surprising. Fourth, the reported values would make upregulation of glucose absorption pointless, since absorptive capacity would already be present in enormous excess. Modulation of absorptive capacity in response to energetic demands or dietary substrate concentration has been observed in many animals (Karasov and McWilliams, Chapter 4 this volume). Finally the authors argued that cited values imply that the contribution of carrier-mediated transport to total glucose absorption is increasingly minor as lumenal concentrations increase. This is in fact exactly the point made by Pappenheimer (1990; also see above), based on his estimates of maximal uptake and review of lumenal glucose concentrations (mostly > 250 mM). These implications motivated Ferraris et al. (1990) to measure glucose concentrations in the gut under "physiological" conditions. They allowed animals to consume diets typical to their species, and also created artificial, supraphysiological diets. Lumenal glucose concentrations ranged from 0.2 mM to 48 mM under all physiological conditions and did not exceed 100 mM even in rats fed 65% (wt/wt) glucose diets. Because concentrations were found to be relatively low, they concluded that glucose contributes only a small percentage of total lumenal osmolality. Direct measurements of lumenal osmolality confirmed that even at their peak values were only moderately hypertonic. The lumenal glucose concentrations observed by Ferraris et al. (1990) were considerably lower than those assumed by earlier studies (Crane, 1975; Pappenheimer and Reiss, 1987). They resolve this discrepancyby pointing out that earlier studies often infused high concentrations of glucose directly into the gut, used nonspecific assay methods and omitted controls. Reported lumenal glucose concentrationshave declined dramatically as a function of year of publication (i.e.with the use of modern specific assays and controls, Ferraris et al., 1990). Studies using modern methods (Olsen and Ingelfinger, 1968;Murakami et al., 1977; Ilundain et al., 1979)show lumenal glucose concentrations that agree well with the measurements of Ferraris et al. (1990). These much lower levels eliminate the potential of unlikely osmotic scenarios in the small intestine and also answer the apparent paradoxes presented by high lumenal glucose concentrations. Apparent Kmvalues (uncorrectedfor the effects of unstirred layers, USL, see Meddings and

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Westergaard, 1989) measured by Ferraris et al. (1990) were between 6 mM and 23 mM in vivo, values which accord well with prevailing lumenal glucose concentrations reported in mammals. The conclusion that lumenal glucose concentrations in mammals were not as high under normal conditions as previously believed (> 250 mM) undermined Pappenheimer's arguments that paracellular transport is the primary route for intestinal absorption of hydrosoluble nutrients. Pappenheimer (1990)may have also underestimated maximal active transport capacities. His calculations of maximal active transport rates were based on assumptions regarding the uniformity of uptake capacity throughout the intestine. It is unlikely these assumptions hold across all species examined (see McWhorter and Lopez-Calleja, 2000). In addition, Uhing and Kimura (199513) showed that in studies using anesthesia and surgical manipulation in rats active glucose transport may be inhibited by up to 86%. Ferraris et al. (1990) estimated that carrier-mediated D-glucose uptake capacity exceeds glucose intake by only about twofold in rats fed normal diets. Since there appears to be only a modest excess of uptake capacity over intake, upregulation serves to match capacity to demand (see discussion of adaptive modulation above). The considerable adaptability for glucose uptake shown by vertebrate intestines would be superfluous if such a grossly enormous margin in uptake capacity existed. Pappenheimer (1993)argued that the adaptive modulation of glucose transport capacity does not refute a major role for paracellular transport of nutrients. He contended that these changes alter absorptive capacity by regulating the permeability of tightjunctions and the concentration gradients available to drive fluid and nutrient uptake. Until it is possible to distinguish between active regulation of tight-junctions via cytoskeletal contraction and secondary dilation due to transported fluid, or the alteration of tight-junction structure for other purposes (e.g. translocation of GLUT2 to the apical membrane or immune-related responses) this argument is not testable. The conclusions of Pappenheimer and Reiss (1987) regarding the importance of paracellular flux were based on physiological glucose concentrations they believed to be >250 mM. In a later paper Pappenheimer states explicitly that measurements of glucose flux in vivo show that passive flux begins to exceed active transport at concentrations in the range of 25 to 50 mM (Pappenheimer, 1990 and references therein). Because of their finding of much lower lumenal glucose concentrations, Ferraris et al. (1990) argued that the conclusion of a significant paracellular component to glucose absorption is unwarranted under normal conditions. They did concede, however, that in animals fed supraphysiological glucose loads paracellular transport may play a significant role. Pappenheimer (1993) claimed that the lumenal glucose concentrations measured by Ferraris et al. (1990)are rnisleadmg because most glucose present in the small intestine is liberated from a-limit dextrins and disaccharides

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cleaved by membrane-bound enzymes. His argument was that the glucose concentration in the pericellular USL may be far greater than measured in the intestinal chyme and may exceed 300 mM close to the enterocytes (Pappenheimer, 1993,1998). Ballard et al. (1995)estimated that if this concentration of glucose was present in the USL, the osmolality could be up to 600 mOsm(kg H20)-',a value about twice that of plasma or intestinal chyme (Ferraris et al., 1990).They reiterated that the maintenance of such a large osmotic gradient is unlikely given the high osmotic water permeability of the intestinal epithelial barrier (Fromter and Diamond, 1972; Chang and Rao, 1994). Ballard et al. (1995) conducted a mathematical assessment of Pappenheimer's (1990) argument and confirmed that intestinal glucose concentrations in vivo (by recent measurements) do not reach levels at which paracellular transport could account for more than about 30% of total glucose uptake. Pappenheimer and Reiss (1987) argued that the principal role of carriermediated transport of solutes from the lumen to intercellular spaces is to provide the osmotic driving force for paracellular fluid absorption. The proposed mechanics of this process were discussed earlier. The addition of D-glucose to lumenal contents has been shown to cause significant increases in net active Na+absorption and in liquid absorption across intestinal epithelia (Pappenheimer and Reiss, 1987; Atisook et al., 1990). Furthermore, the addition of phloridzin (Heaton and Code, 1969),a selective inhibitor of Na+glucose cotransport, or replacement of Na' with other cations (Csaky and Zollicoffer,1960)blocks this increased liquid absorption. Ballard et al. (1995) pointed out that although the observation of increased fluid movement is not in dispute, it is debatable whether the water moves predominantly across cell membranes or through the tight-junctions. A study with Necturus gall bladder found that about 30% of osmotically driven water absorption passes transjunctionally (implying that up to 70% passes transcellularly; Ballard et al., 1995and references therein). Further, Loo et al. (1996)estimated that the translocation of each glucose molecule by SGLTl is coupled with the transport of up to 260 water molecules. In hummingbirds, the uptake of water associated with the hydration sphere of actively transported D-glucose alone can account for complete absorption of their immense daily water fluxes (McWhorter and Martinez del Rio, 1999).If a significant portion of water absorption occurs via a transcellular route, the importance of paracellular water (and thus hydrosoluble nutrient) absorption hypothesized by Pappenheimer and Reiss (1987) is questionable. The observation of phloridzin blocking water absorption (Heaton and Code, 1969)is consistent with the idea that significant water absorption occurs via active cotransport, although phloridzin would also block the building of osmotic gradients in intercellular spaces by actively transported glucose. Indeed, Pencek et al. (2003) recently found that phloridzin blocked both passive and active uptake of glucose (measured as uptake of radiolabeled L-glucose and

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D-glucose respectively) in chronically catheterized dogs and concluded that mediated glucose transport by SGLTl was necessary to activate passive transport. Ballard et al. (1995) made several criticisms of the conclusions of Pappenheimer's group regarding solute-induced changes in tight-junction permeability. First, they pointed out that because the concentration of ferrocyanide (animpermeant osmolyte)used by Madara and Pappenheimer (1987) only reduced liquid absorption by 20°/0, their conclusion that the morphological changes observed in tight-junctions resulted from contraction of cellular cytoskeletal elements and not from liquid absorption was unjustified. It is interesting to note, however, that if a significant volume of water is absorbed by Na+-coupledactive transport as discussed in the previous paragraph, this criticism may not be valid. Second, Ballard et al. (1995)pointed out that although several lines of evidence indicate that solute-induced changes in cellular cytoskeleton may modulate tight-junction integrity (Madara et al., 1987;Madara and Pappenheimer, 1987; Pappenheimer, 1987; Madara and Carlson, 1991; Pappenheimer and Volpp, 1992),some authors have challenged the notion that glucose-stimulated liquid absorption increases tight-junction permeability to hydrophilic solutes. Fine et al. (1993) found no changes in tight-junction permeability associated with glucosestimulated liquid absorption. It is not clear whether these differences are due to experimental or interspecifc differences. The problems of separating the direct effects of D-glucose and Na' transport make it difficult to determine whether changes in tight-junctionmorphology occur prior to or as a consequence of liquid absorption. Several recent studies appear to confirm the notion that paracellular transport plays only a minor role in intestinal carbohydrate absorption in mammals. Fine at al. (1993)found that in human in vivo jejunal perfusions the fraction of total glucose absorption that could be attributed to a passive mechanism averaged 5%. No passive absorption was detected in the human ileum in vivo. As already mentioned, Fine et al. (1993) likewise did not observe an increase in tight-junction permeability associated with Nai-dependent nutrient transport. Several recent studies in rats (O'Rourke et al., 1995;Schwartz et al., 1995;Uhing and Kimura, 1995a;Shi and Gisolfi, 1996) and dogs (Lane et al., 1999; Pencek et al., 2002; Pencek et al., 2003) have also concluded that paracellular flux is not a major absorptive pathway for hexoses, despite observations of increased fluid absorption (consistent with Pappenheimer's ideas) in many cases. Lane et al. (1999) used Thiry-Vella perfusion loops to quantify paracellular (i.e. L-glucose) uptake in dog jejunum. This preparation is highly controllableand thought to mimic natural digestive conditions very closely. At physiological concentrations of D-glucose (1-50 mM), the fractional absorption of L-glucose was only 4 to 7% of total glucose absorption. Even under supraphysiological conditions (perfusion of 150mM D-glucose, D-maltose, or D-mannitol),fractional absorption

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of L-glucose was low (2 to 5'/0). There was significant fractional water absorption in all experiments, which is a prerequisite for solvent drag (but see discussion above). Despite experimental conditions designed to maximize paracellular transport, their results were interpreted as evidence that paracellular transport of glucose plays a minor role in the dog (Lane et al., 1999). Pencek et al. (2003)recently found that the contribution of passive absorption to total glucose uptake in dogs was slightly higher than previously reported and increased after exercise relative to rested controls (from -11% to 18%of total). Interestingly, Schwartz et al. (1995)found that a significant fraction (71%) of administered L-glucose was absorbed in rat intestine, but only after Dglucose concentrations were reduced to negligible levels. Based on these measurements they concluded that L-glucose has a weak affinity for the Dglucose carrier and is therefore not an adequate marker for paracellular uptake. These authors cite differences among the absorption of L-glucose and other nonmetabolized hexoses as additional evidence that L-glucose interacts with an active transport system (Schwartzet al., 1995). Several older studies seem to show active transport of L-glucose (Caspary and Crane, 1968;Neale and Wiseman, 1968;Bihler, 1969;Hopfer et al., 1975);however, it is possible that in these older studies the L-glucose used could have been contaminated with D-glucose or other carbohydrates. A considerable volume of recent work, including some basolateral membrane vesicle uptake studies, indicates that L-glucose does not interact with the SGLTl transporter (Wright et al., 1980; Ikeda et al., 1989; Fine et al., 1993; Uhing and Kimura, 1995a; Lane et al., 1999). An additional criticism of Schwartz et al. (1995)is that gut absorption of hexoses was determined by their subsequent collection in urine, an indirect method subject to the effects of differential renal handling of hexoses (Ullrich and Papavassiliou, 1985). The absorption of these carbohydrateprobes also varies greatly with structure and molecular weight (e.g.Chediack et al., 2003). A criticism of the mammalian studies described above is that in most cases hexoses were perfused directly into the intestinal lumen and were therefore subject to the diffusion dampening effects of USL (Barry and Diamond, 1984;Meddings and Westergaard, 1989). This potential restriction to the diffusion could reduce the absorption of markers and thus lead to underestimates of paracellular uptake. Indeed, Michaelis-Menten constants for glucose transport are higher in vivo than in vitro (attesting to the effects of thicker USL in vivo; Meddings and Westergaard, 1989; Ferraris et al., 1990). It does appear, however, that diffusion-limited probes can be utilized to measure USL resistance in the intestine of a live animal so that absolute transport parameters can be determined in vivo in experimental animals (Westergaard et al., 1986). Such corrections and derivations of corrected kinetic constants (Meddings and Westergaard, 1989) would allow direct comparison among species and experimental protocols, remove this

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potential criticism, and assist in elucidating the true contribution of paracellular solvent drag to the absorption of dietary carbohydrates. Kellet and colleagues recently provided new evidence that an apparent passive component of glucose uptake indeed exists, but that it is mediated by GLUT2 in the brush-border membrane (see above and Helliwell et al., 2000a; Kellett and Helliwell, 2000; Kellett, 2001). They argued that this finding explains the discrepancy between the saturation of SGLTl at lumenal concentrations of 30-50 mM D-glucose and the long-established pattern of a linear increase in glucose uptake up to concentrations of several hundred millimolar (Kellett, 2001 and references therein). They further argued that the very presence of a facilitative transporter in the brush-border membrane invalidates the theory of paracellular solvent drag proposed by Pappenheimer and colleagues because this would prevent the concentration of glucose in intercellular spaces. They explained the direct relationship between water transport and glucose absorption at higher glucose concentrations (e.g. Fullerton and Parsons, 1956; Pappenheimer and Reiss, 1987)by invoking the idea that glucose transporters can act as low conductance water channels (see above and Fischbarg et al., 1990; Loo et al., 1999). Their conclusions were based on the premise that GLUT2 is the primary glucose transporter at high lumenal glucose concentrations. Their results confirmed that glucose absorption via SGLTl is necessary to activate passive absorption, consistent with Pappenheimer 's predictions, and that SGLTl plays an important regulatory role in regulating tight-junction structure. They argued that alterations in tight-junction permeability mediated by glucose transport may be associated with the translocation of GLUT2. Given the debate surrounding in vivo lumenal glucose concentrations (see above), the technical difficulties in quantifying recruitable brush-border GLUT2 activity (see Helliwell et al., 2000b; Kellett, 2001), and the fact that recruitable GLUT2 activity has only been studied in rats (which apparently have very low paracellular intestinal permeability, Schwartz et al., 1995), the significance of their findings remains unclear. Recent evidence suggests, for example, that GLUT2 recruitment is unlikely to account for the significant passive absorption of glucose observed in birds (seebelow and Chang, 2002), although there may be differences in paracellular permeability and uptake mechanisms between birds and mammals.

ABSORPTION OF CARBOHYDRATES BY THE AVIAN INTESTINE The evidence to date indicates that paracellular flux plays a relatively minor role in total dietary carbohydrate uptake in mammals. Paracellular permeability may be slightly increased in mammals after exercise (e.g.Pencek et al., 2003). There is a general consensus, however, that at high lumenal glucose

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concentrations (>50mM) paracellular transport may become more important (Ferrariset al., 1990; Pappenheimer, 1993). Although the diets of most animals do not routinely include carbohydrate levels high enough to create lumenal glucose concentrations >50 rnM (but see discussion above), nectarand fruit-eating animals may be an exception. The average nectar concentration found in the floral nectars of hummingbird pollinated plants for example, is about 23% sucrose (wt/vol; approximately 670 mM; Pyke and Waser, 1981),yielding an equal glucose concentration upon hydrolysis. The relative contribution of passive, paracellular hexose uptake (Karasov et al., 1986; Karasov and Cork, 1994; Karasov et al., 1996; Levey and Cipollini, 1996;Afik et al., 1997)and the correlation between functional and mechanistic aspects of paracellular absorption (Chediack et al., 2001; Chang, 2002; Chediack et al., 2003) have been extensively investigated in birds over the past decade. The findings of these studies, the methods employed, and their implicit assumptions are critically examined below. The spare capacity hypothesis proposes that the ability of the intestine to absorb nutrients is slightly greater than required to meet loads determined by food intake (Diamond, 1991; Diamond and Hammond, 1992). Nectarand fruit-eating birds routinely consume several times their body mass in food per day to meet energy demands (e.g. Rooke et al., 1983; Powers and Nagy, 1988; Beuchat et al., 1990; Williams, 1993; Powers and Conley 1994; Goldstein and Bradshaw, 1998; McWhorter and Martinez del Rio, 1999; Fleming and Nicolson, 2003). Although nectar diets consist mainly of water, these birds ingest and assimilate enormous carbohydrate loads. Humrningbirds by and large assimilate all of the carbohydrates in their diets, regardless of concentration (Hainsworth, 1974; Karasov et al., 1986; McWhorter and Martinez del Rio, 1999). Capacity therefore appears to be well matched to load in these animals (but see Second Generation Reactor Models: Gut Function in Nectar-eating Birds in chapter 3, by McWhorter, this volume). Until recently, it was not known whether nectar- and fruit-eating birds have exceptionally high active transport capacity or rely extensively on paracellular routes of carbohydrate uptake. It might be expected that given potentially high lumenal glucose concentrations, these birds could save the energy required for active transport if the intestine's passive permeability were sufficiently high. Karasov et al. (1986) evaluated both carrier-mediated active transport and passive permeability of the intestinal epithelia in hummingbirds. Their results indicated that the passive permeability of hummingbird intestines was immeasurably low but that capacity for carrier-mediatedsugar absorption was the highest measured for any animal to date. They calculated that active transport alone was sufficient to account for all glucose absorption over a brief experimental period. Since passive absorption was apparently unnecessary to absorb sugars, they speculated that the intestine's low passive permeability might be an adaptation to high rates of fluid transit,

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protecting the animal from losing valuable solutes by dialysis (Karasov et al., 1986). It is important to point out, however, that the validity of the everted intestinal sleeve method used to measure these uptake parameters has recently come into question. The method employs an in vitro preparation where a section of intestine is removed, everted, and placed on a glass or metal rod. The tissue is then immersed in a rapidly stirred nutrient bath to measure ~ (but apparently not in mammals) uptake. It has been discovered that i r birds the shearing force of the stirred bath and tissue manipulation during preparation significantlycompromise the epithelia (Starck et al., 2000), and tissue bioactivity may also be reduced (Karasov and Debnam, 1987).In addition, the everted sleeve method has been criticized for ignoring physiological conditions relevant to accurately characterizing passive transport (Pappenheimer, 1990; Karasov et al., 1996). Powers and Nagy (1988) estimated the field metabolic rate (FMR)of Anna's hummingbirds (Calypteanna, one of the species studied by Karasov et al., 1986)to be 32 kJd-'. This energy requirement is far above the maximum estimates of transport based on active mechanisms alone (6.2 kJ d-l, calculated from Karasov et al., 1986). Two possibilities arise from this line of reasoning: (1)the active transport rates in hummingbird intestines were underestimated due to methodological problems and (2)paracellular absorption of nutrients plays a major role in hummingbirds. These possibilities are, of course, not mutually exclusive. A discussion of more recent studies of passive transport in birds and analysis of the methods employed provides additional insight. Karasov and Cork (1994)tested the hypothesis that most glucose absorption across the small intestine's brush border is by active cotransport with sodium in nectar-eating rainbow lorikeets (Trichoglossus haema todus). Maximal mediated D-glucose uptake summed along the entire intestine using the in vitro everted sleeve method was an order of magnitude too low to explain observed rates of glucose assimilation in vivo. This result implied a predominant role for passive glucose absorption in these birds. These authors applied a pharmacokinetic technique to measure passive absorption in vivo and found that 80% of ingested L-glucose was absorbed, providing evidence for significant nonrnediated transport (Karasov and Cork, 1994). Several additional studies using Karasov and Cork's (1994)method provided similar estimates of L-glucose absorption: 52% to 92% in Northern bobwhite quail (Levey and Cipollini, 1996);91% in omnivorous yellow-rumped warblers (Afik et al., 1997);75% in granivorous house sparrows (Caviedes-Vidal and Karasov, 1996); and 79% in frugivorous cedar waxwings (D.J. Levey pers. comm.). In addition, McWhorter (unpub. data), using this method, recently found that between 37% and 85% of ingested L-glucose was absorbed by nectarivorous broad-tailed hummingbirds. There seems to be considerable evidence that paracellular absorption is a significant route for carbohydrate absorption in the avian intestine. The consistency of this finding in birds with diverse diets and taxonomic associations suggests a general

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phenomenon in birds. Because all of the abovementioned studies rely on the same model, however, careful examination of the method and its assumptions is prudent. Karasov and Cork's (1994)method relies on determining the steady-state concentration of labeled L-glucose (P)in the plasma of freely feeding birds, the marker ingestion rate (I),the marker distributionspace (D),and the elimination constant for removal of L-glucose from plasma (k,). Fractional absorption (F)is then calculated as: (1) F = (P. k;D)/I The L-glucose pool size and elimination constant are measured simultaneous with the steady-state feeding experiment by injecting L-glucose with an alternate label and taking consecutive blood samples. Although the model is relatively simple, it relies on several important assumptions. A primary one is that absorbed L-glucose is not metabolized but entirely excreted and that its excretion can be approximated by single pool kinetics. Karasov and Cork (1994) cited two lines of evidence in support of the assumption that L-glucose is not metabolized. First, injected Lglucose was 94%recovered in quantitative collections of excreta, which implies little or no metabolism or deposition in tissues. Second, the ratio of activity of excreta counted before and after drying was one, implying that label had not been transferred to water during metabolism. Further, CaviedesVidal and Karasov (1996)and Chang et al. (2004) found that essentially all radioactivity (95-98%) remained associated with L-glucose in house sparrow (Passerdomesticus)plasma, using thin-layer chromatography to separate [3H]L-glucoseand high-performance liquid chromatography (HPLC)to separate [14C]L-glucose respectively. Examination of plasma label decay curves followinginjection of labeled L-glucose led Karasov and Cork (1994)to conclude that excretion of L-glucose was approximated by single pool kinetics. Specifically,semilog plots of specific activity per gram excreta vs time were approximately linear, which implies single pool kinetics. McWhorter (unpubl. data) also observed linear semilog plots of [14C]L-glucoseactivity in excreta in hummingbirds. Karasov and Cork (1994) argued that changing the assumption about the number of compartments causes counterbalancing changes in the slope (-ke,)and intercept (the inverse of pool size)of the slowest components of the curve that are plotted at longer time points. In other words, a second compartment with slower turnover would lead to a shallower slope (and thus lower elimination constant), but a larger pool size. The authors argued that these changes would cancel each other out, which seems reasonable based on a brief graphical analysis of the model (Karasov and Cork, 1994). A second major assumption of the model is that L-glucose absorption can be equated with passive D-glucose absorption. This involves both the assumption that the diffusion coefficients across the epithelia are the same and

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that L-glucose is not recognized by a transporter. Karasov and Cork (1994) admitted that the assumption of equal diffusion coefficients is not strictly correct in every circumstance.The difference is due to the mediated transport of D-glucose into the intercellular space, potentially leading to a small or even negative diffusive term for D-glucose (whilethat of L-glucose remained positive). They pointed out, however, that if this is the case then L-glucose absorption actually underestimates D-glucose passive absorption and the conclusion of significant paracellular transport holds. Although Schwartz et al. (1995)concluded that L-glucose seems to have a weak affinity for SGLT1, most experimental evidence suggests that it does not (Wright et al., 1980; Ikeda et al., 1989; Fine et al., 1993; Uhing and Kimura, 1995a; Lane et al., 1999;Chang, 2002). It appears that the assumptions of Karasov and Cork's (1994)model are satisfied or that deviations from such do not impact conclusions significantly. If anything, deviations from the assumptions lead to underestimates of paracellular glucose absorption. Fractional absorption measurements using alternative markers and calculation methods suggest that previous measurements of L-glucose absorption are not artifacts of isotope separation or affinity for active transport systems (Chediack et al., 2001; Chediack et al., 2003). Chediack et al. (2001) extended the observations of hydrophilic absorption in birds using a pharmacokinetic technique that relies on the appearance of probes in the blood after feeding and injection (Caviedes-Vidal and Karasov, 1996). The absorption of carbohydrate probes (D-mannitoland L-arabinose,both thought to be abso:rbedonly via nonmediated processes, Dawson et al., 1987;Krugliak et al., 1994)was calculated by assuming a single compartment and first-order kinetics (Welling,1986). Inspection of plasma decay curves following injection of probes and comparative fitting of the data to one-compartment and two-compartment models lead the authors to conclude that this assumption was appropriate. Following typical procedures in pharmacokinetics, fractional absorption (F)was calculated using the areas under the post-gavage and -injection curves (AUC) of marker concentration vs time: = (AUCgavage/Dosegavage)/(AUCiniection/Doseiniection) (2). The authors found substantial absorption of D-mannitol and L-arabinose (69 -+ 3%, no difference between probes or among administered concentrations) in house sparrows using this method. Absorption of both probes correlated directly with their orally administered doses, indicating that uptake was nonmediated. These results are consistent with an earlier report of substantial (75%)absorption of [3H]L-glucosein house sparrows (Caviedes-Vidaland Karasov, 1996),providing an important confirmation of a substantial pathway for passive absorption in birds.

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Mechanistic Examinations of Paracellular Uptake Recent studies of the correlation between functional and mechanistic aspects of paracellular uptake in birds are providing further confirmation of the importance of this pathway for nutrient absorption and will certainly invite and inform more mechanistic future studies. These studies are briefly reviewed below. Chediack et al. (2003)used the pharmacokineticmethod described above (see eqn 2) to examine two mechanistic aspects of paracellular absorption of carbohydrates. The first objective of their study was to examine the affect of probe molecular size on absorption. Previous studies had shown a decline in absorption with increase in molecular weight of probes that was more rapid than expected based on their free aqueous diffusion coefficients (Hamilton et al., 1987;Meehye, 1996;Ghandehari et al., 1997;He et al., 1998). Chediack et al. (2003) recognized that this pattern is consistent with movement through effective pores in epithelia ("sieving", Chang et al., 1975;Friedman, 1987);such size selectivity partly determines the size range of hydrophilic nutrients or toxins that might be absorbed passively. Absorption of water-soluble compounds in vivo seems more likely to occur via solvent drag through intercellular channels than by diffusion through the cytoplasm of enterocytes (Pappenheimer,2001). The second objective of the Chediack et al. (2003) study was to test for modulation of paracellular absorption. Paracellular permeability may be altered by both endogenous and exogenous agents (the latter including nutrients such as glucose, amino acids and medium chain fatty acids, as well as toxins, see Chediack et al., 2003 and references therein). The mechanisms by which such modulation occurs are not known, but may include increased solvent drag and/or cytoskeletal contractions (Madara et al., 1986; Madara and Pappenheimer, 1987; Pappenheimer, 1987;Pappenheimer and Reiss, 1987;Madara et al., 1988)or protein strand alterations that change tight-junction effective pore size (see Sasaki et al., 2003). Chediack et al. (2003) measured the fractional absorption of metabolically inert carbohydrate probes ranging in molecular weight from 150.13to 342.2 Da in the presence and absence of both lumenal food and glucose to meet these objectives. They found that (1)fractional absorption decreased significantlywith increase in molecular weight (61 9% for L-arabinose, 64 13% for L-rhamnose, 46 2 8% for perseitol, and 15 + 5% for lactulose, averaged over all treatments), and (2) fractional absorption was significantly greater in the presence of food or D-glucose than in the presence of D-mannitol (not absorbed by mediated processes). The latter result was confirmed by Chang et al. (2004)who found that the fractional absorption of L-glucose was significantlyincreased in the presence of 3-0-methylD-glucose (3-OMG, an analogue of D-glucose that is transported but not metabolized). A theoretical understanding of how hydrophilic molecules cross epithelia can be applied profitably to the interpretation of such data

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(see Chediack et al., 2003 and references therein). Such interpretation may clarify the relative effects of changes in effective pore size and solvent flux for determining the magnitude of paracellular absorption within and among species, and may be important for predicting the oral bioavailability of water-soluble nutrients and natural and man-made xenobiotics (toxins,plant secondary compounds, toxicants, drugs, etc.). Differences in effective pore size among species or due to dietary or other modulation (He et al., 1998; Chediack et al., 2003) discovered using the aforesaid methods may also invite further comparative studies on the mechanism(s)of paracellular uptake. Kellet and colleagues recently suggested an alternative mechanism by which D-glucose absorption might be enhanced by mediated nutrient uptake: recruitment of GLUT2 to the brush-border membrane (see above and Helliwell et al., 2000b; Kellett and Helliwell, 2000). The apparent Kmof GLUT2 is higher than that of SGLT1; thus the authors argued that the effect may falselygive the appearance of an increase in nonmediated uptake. However, Chang et al. (2004)argued that observed increases in the absorption of L-glucose, which is not transported by GLUT2 expressed in Xenopus oocytes (Burant and Bell, 1992),and observations of increased absorption of other metabolically inert carbohydrates (Chediack et al., 2003),in the presence of lumenal glucose refutes this assertion, at least in birds. Chang et al. (2004)tested the hypothesis that paracellular absorption is an important component of glucose absorption in birds by comparing the apparent rate and extent of absorption of D- and L-glucose in vivo in house sparrows. These authors further predicted that the absorption of labeled Lglucose would not be depressed (by competitive inhibition, Malo and Berteloot, 1991)when measured in the presence of a high sugar concentration (i.e. 100 mM unlabeled D- or L-glucose) in vitro. The in vivo predictions were tested simultaneouslyby measuring the fractional absorption and apparent absorption rate of radiolabeled 3-OMG (see above) and L-glucose from the intestine. In vitro uptake experiments employed the everted sleeve method; however, the gross morphological damage and reduced bioactivity found in other avian species (Starck et al., 2000) were apparently not observed. The authors found that (1) uptake of L-glucose in vitro was not inhibited by high concentrations of unlabeled sugars, indicating that it does not interact with a transporter, and (2) the apparent rate of absorption and extent of D- and L-glucose uptake were similar in vivo and indicated that >7O0/0of glucose absorption was passive. Schwartz et al. (1995) suggested that simply comparing the fractional absorptions of D- and L-glucose was inappropriate because these isomers appeared to be absorbed in different regions in rats. Chang et al. (2004)asserted that this explanation does not apply to house sparrows; D- and L-glucose had similar apparent absorption rates throughout all experimental sampling time points, and absorption of L-glucose was not prolonged compared to D-glucose (it in fact occurred faster in the presence of lumenal D-glucose). These experiments confirmed

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that the vast majority of glucose is absorbed by a passive route in house sparrows. Because the fractional absorptions of D- and L-glucose were similar and L-glucose is apparently not transported by GLUT2 (Burant and Bell, 1992),it seems implausible that GLUT2 recruitment to the brush-border is entirely responsible for the passive component of glucose uptake in birds (see also Kellett, 2001). Chang and Karasov (2004)attempted to further verify and visualize the paracellular pathway in intact house sparrows using sodium fluorescein, which distributes only to the extravascular interstitial space (Nugent and Jain, 1984; Hurni et al., 1993; Sakai et al., 1997) and is widely used as a hydrophilic marker for in vitro paracellular permeability studies (Chao et al., 1998; Lindmark et al., 1998; Stagni et al., 1999; Clausen and BernkopSchnurch, 2000; Gaillard and de Boer, 2000). These authors predicted that the absorption of fluorescein would be only via the paracellular pathway and would be increased in the presence of lumenal D-glucose. Pharmacokinetic analysis indicated that the fractional absorption of fluorescein was approximately 40% and that of L-glucose was approximately 80%. Fluorescein absorption peaked faster in the presence of lumenal D-glucose (at 6 rnin vs 8 min in the D-mannitol control group), but fractional absorption was not sigruficantly different among treatments. Visualization using confocal laser scanning microscopy showed that fluorescein was found primarily in the paracellular space and villous core. Some fluorescence at the apical membrane was attributed to binding (Braginskaja et al., 1994).Three-dimensional image reconstruction confirmed that fluorescein was distributed extravascularly. A positive correlation between fluorescein and L-glucose absorption was interpreted as suggesting that these molecules share a common uptake pathway. Chang and Karasov (2004) argued that alternate explanations (transport of fluorescein into enterocytesfollowed by metabolism or rapid expulsion into intercellular spaces) are unlikely. Mediated transporters of fluoresceinmay be expressed in rat small intestine (see Walters et al., 2000; Cattori et al., 2001; Sun et al., 2001), however there is no direct evidence that fluorescein is transported in intestinal enterocytes.

A primary observation that led to the hypothesis that paracellular flux may be important for nutrient absorption was that maximal carrier-mediated transport was insufficient to explain rates of glucose assimilation in vivo (Pappenheimer, 1990). Most recent evidence suggests that paracellular flux plays a relatively minor role in the intestinal absorption of carbohydrates in mammals, but may play a much more sigruficant role in birds. This conclusion is robust even if the studies discussed above seriously underestimated mediated uptake capacities (see caveats for the everted sleeve method).

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Glucose-dependentrecruitment of GLUT2 to the brush-border membrane may constitute a significant pathway for passive transcellular glucose and fructose absorption in mammals (Kellett,2001) but may not play as significant a role in birds. Evidence clearly falsifying Pappenheimer 's paracellular solvent drag hypothesis in mammals, however, has not yet come to light. It remains to be seen whether significant paracellular flux is a general phenomenon among birds, or if rigorous testing of the assumptionsof previously employed models and use of alternative methods (e.g.in vivo blocking of SGLTl and GLUT2 with phloridzin and phloretin respectively) will lead to different conclusions. It is possible that paracellular absorption is a general vertebrate trait that has been particularly enhanced to augment mediated nutrient absorption in flying birds that are known to possess relatively less small intestinal tissue (Karasov and Hume, 1997)and exhibit relatively shorter digesta retention times. A systematic study across birds, mammals, and reptiles using uniform methodology would be an important contribution toward resolving this issue. Recent studies of the correlation among functional and mechanistic aspects of paracellular flux have important bearing on the emerging fields of comparative evolutionary physiology and ecotoxicology. These studies establish important limits on studies quantifying physiological capacity in relation to load (Diamond, 1991,1993). Passive absorption clearly must be considered when attempting to match nutrient absorption capacity and nutrient intake. Pappenheimer and colleagues almost single-handedly advanced the hypothesis of water-soluble solute absorption in a field dominated by the molecular biology of carbohydrate transporters from the late 1980s onward (Pappenheimer,1990,1993,1998,2001). They proposed that nutrient-stimulated water, and thus accompanying solute absorption increases with body size, whereas the importance of transcellular mediated absorption declines and therefore the importance of paracellular absorption must increase with body size. This prediction has important implications for nutrient absorption, oral drug deliver$ and exposure to natural and man-made toxins. Phylogenetic and body-size differences in, and modulation of, paracellular flux are important for understanding and predicting the extent of water-soluble compound absorption (Anderbertet al., 1993;Chang and Rao, 1994; Bjork et al., 1995; Karlsson et al., 1999). Some studies that have compared water-soluble solute permeability among different size mammals seem to support Pappenheimer's predictions (seeChediack et al., 2003 for review). Other studies on humans (Fine et al., 1993),rats (Schwartzet al., 1995;Uhing and Kimura, 1995a), and dogs (Lane et al., 1999; Pencek et al., 2002; Pencek et al., 2003) have concluded that most glucose absorption in mammals is mediated. A theoretical framework based on the flux of solutes through porous epithelia (Kedem and Katchalsky, 1958;Chang, 2002; Chediack et al., 2003) can be used to describe the paracellular absorption of water-soluble

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molecules across the small intestine's mucosal epithelium and to separate the relative importance of diffusion and solvent drag. Such an understanding will invite and inform future mechanistic and comparative studies of nutrient absorption. The molecular events that result in tight-junctionregulation (Anderson, 2001; Kellett, 2001), phylogenetic differences in tight-junction structure (Sasakiet al., 2003),and the regulation and generality of GLUT2 recruitment to the brush-border membrane (Kellett, 2001), will certainly be important areas of future research. Acknowledgments

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Mass-Balance Models for Animal Isotopic Ecology Carlos Martinez del Riol and Blair 0.WolfZ University Wyoming, ~ e ~ a r t m eof n tZoology Physiology, Laramie, WY, USA, University New Mexico, Department of Biology, Albuquerque, NM, USA

SYNOPSIS Analysis of natural stable isotope ratios has created a methodological upheaval in animal ecology. Because the distribution of stable isotopes in organisms follows reliable patterns, their analyses have become established useful methods for animal ecologists. However, because animal ecologists have adopted a phenomenological approach to the use of stable isotopes, the mechanisms that create isotope variation patterns remain unexplored. The mass-balance models that can provide a mechanistic, and hence predictive foundation for animal isotopic ecology are presented here. We review and elaborate the current mixing models used to reconstruct animal diets and develop new mathematical models to explain one of the most widely used patterns in animal isotopic ecology: enrichment in 15N observed acrpss trophic levels. Construction of element and isotope budgets is central to testing the mass-balance models described herein. Because the concept of a budget is central to all animal physiological ecology, development of a mechanistic and predictive framework for isotopic animal ecology falls naturally on physiological ecologists. We argue that progress in isotopic animal ecology hinges on laboratory experiments that explore mechanism, documentation of pattern in the field, and theoretical integration of mechanism and pattern.

Democritus was right: living organisms are collections of interacting atoms. We now believe that atoms are made of electrons clouding around a

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nucleus made up of protons and neutrons. The numbers of charged particles (electrons and protons) within the atom are equal, so the whole atom is electrically neutral. The neutrons stop the nucleus from tearing itself apart. They work as a glue that bonds with the protons and provides cohesion within the nucleus. Elements with the same number of protons but a different number of neutrons are called isotopes and vary in mass. Most of these isotopes are stable (do not undergo radioactive decay) and can be distinguished by their mass. Many physicochemical processes are sensitive to differences in the dissociation energies of molecules, which often depend on the mass of the elements of which these molecules are made [Ball (2002) provides a particularly good introduction to atoms, elements, and isotopes]. The enzymatic pathways that organisms use to manufacture and transform organic molecules for example, can be isotopically discriminating. In general, it is easier to form, or break, bonds that contain lighter isotopes. The result is that molecules that contain the lighter isotope are preferentially incorporated into the products of incomplete reactions. As a result, the unreacted residues become enriched in the heavier isotope (Hoeffs, 1997). These isotopic effects are useful. The isotopic composition of many materials, including the tissues of organisms, often contains a label of the process that created it. Ecologists and physiologists can use these labels or isotopic signatures to detect the imprint of processes at a variety of scales. Plant physiologists, atmospheric scientists, and geochemists have relied on the measurement of natural stable isotope signatures for decades (Lajtha and Michener, 1994).Animal physiologists and ecologists, on the other hand, have been tardy in joining the isotopic research enterprise.Only one chapter in a recent review on the use of stable isotopes to integrate biological, ecological, and geochemical processes deals with animals (Griffith, 1998).Interestingly, the animals that this chapter deals with are extinct (Cerling et al., 1998 in Griffith, 1998).Indeed, paleontologists and archaeologists have been unusual among zoologists in their reliance on stable isotopes as tools in the reconstruction of the diets and habits of extinct animals and ancient humans (Koch et al., 1994 and references therein). Although zoologists have been latecomers, we have recently been active. The number of publications in animal ecology and physiological ecology that use stable isotopes has doubled every 3 years over the last 10 years (Fig. 6.1).This is a phenomenal rate of increase for the incorporation of any scientific methodology,As the following,almost certainly incompletelist attests, a large variety of phenomena in animal ecology can be informed by an isotopic approach. Stable isotopes have been used to reconstruct animal diets (Hobsonet al., 1994),determine patterns of resource allocation to reproduction (O'Brien et al., 2002), track animal migration (Hobson, 1999),assess the flux of materials from the sea into terrestrial food webs (Ben David et al., 1998), assign trophic levels (Post, 2002), and to determine the structure of food webs (France, 1995).

Mass-balance models

year Fig. 6.1. Number of publications on animal ecology and physiological ecology that rely on stable isotopes has increased exponentially (r2 = 0.81) in the last 10 years with a proportional rate of increase of 23%. Number of publications in this data set obtained by searching Biological Abstracts using "stable isotopes" and at least one of the following terms as key words: "animal", "diet", "food web", and "migration".

In geochemistry, plant physiology, physiological ecology, progress in the use of stable isotopes relies on vigorous interaction between theory, laboratory research, and field study [the chapters in Griffiths (1998)volume are superb examples].With few exceptions (someof which are reviewed below), animal ecologistshave adopted a different pathway. The vast majority of our field isotopic studies are phenomenological and a well-developed theoretical edifice does not inform our laboratory experiments. The objective of this chapter is to outline what we believe are some of the elements of a mechanistic theoretical framework for the isotopic ecology of animals. Like work in plant physiology and geochemistry,we too rely on mass-balance models to disentangle the relative importance of the factors that determine animal tissue stable isotopic composition. The two broad themes considered are (1) what is the timescale of incorporation of an isotopic signal into an animal's tissues and (2) why does the isotopic composition of animal tissues often differ from that of the resources they use. We review and elaborate on the current mixing models used to reconstruct animal diets and develop new models to explain one of the most widely used patterns in animal isotopic

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ecology: enrichment in 15Nobserved across trophic levels. Although we focus on the isotopes of carbon and nitrogen for clarity, mass-balance models described here can be easily applied to other elements. Geochemists developed an arcane, but precise isotopicjargon. Before dealing with mass-balance models, we must introduce the terminology that isotopic ecology shares with the atmospheric and geological sciences.

STABLE ISOTOPES: TERMINOLOGY PRIMER The isotopic composition of a sample is measured as the ratio of one isotope to another: In most cases the abundance of one isotope (generally the lightest) exceeds that of the other by a large margin. For example, 13Cmakes up only 1.l0/0of the total carbon on earth and 15Nconstitutes only 0.37O/0 of the nitrogen (Richardson and McSween, 1989).Consequently this ratio can be a very small number. To make measurements of the relative abundance of two isotopes graspable, geochemists express the isotopic composition of most materials as the normalized ratio of the sample to a standard in parts per thousand (per md, %o):

a=[

sample

-

standard

]xlOOO

(1)

R stan,, where X is an element, and Rsamp, and R,an,a, are the ratios of the heavy to the light isotopes for the sample and standard, respectively, In some cases, it is useful to transform from fractions or percentages to ratios and 6 values with the transformation f

where f, is the fraction of the heavy isotope. For values of fH < 0.1, Rsample can be approximated very closely by f, (RSample=fH). Although some of the standards chosen by geochemists seem capricious to biologists, at this point we have no say in the matter. Amarine belemnite for the Pee Dee Formation (VPDB) and ocean water (standard mean ocean water = SMOW) are used as standards for nitrogen and carbon respectively. Thus, isotope ratios are commonly expressed as %o SMOW or %o VPDB. The words "depleted" and "enriched" refer to the heavy, and often less abundant isotope of a pair: Depleted means a more negative 6 value whereas enriched means a more positive 6 value.

Fractionation As mentioned in the introduction, the natural variation in the relative abundance of stable isotopes in any substance is the consequence of tiny mass differences that cause the isotopes to behave differently in both physical

Mass-balance models

145

processes and chemical reactions. In general, the lighter isotope (I2C,or 14N) tends to form weaker bonds and to react faster than the heavier isotope (13C, or 15N).As a consequence, the abundance of stable isotopes of an element will vary among the reactants and products of a chemical reaction. The change in isotopic abundance between chemical species (i.e. reactants or products) resulting from physical and chemical processes is called fractionation. Fractionation (a,.,) between the chemical species A and B is described in terms of the ratio in delta (6) values between the species:

Values of a are usually very close to 1, so the difference between two delta values is often reported and denoted by the discrimination factor aA.,(aA-, = - 6,). Two types of fractionation have relevance for biologists. Equilibrium fractionation occurs among chemical molecules linked by equilibria as a result of bond strength differences between the isotopic species. For example, carbonate in bone is probably derived from blood bicarbonate. Carbon and oxygen isotopes are rapidly exchanged among blood bicarbonate, dissolved blood carbon dioxide, and body water by the following equilibria:

The isotope equilibrium of bone carbonate is controlled by the composition of dissolved CO,, which is produced by respiration, and fractionation associated with equilibrium exchanges of carbon. Suppose that one is attempting to estimate the isotopic composition of the diet of an extinct mammal from the carbon in the apatite of its teeth. At mammalian body temperatures, the fractionation (E) from C0, to HCOl is about 8%0 (Mook, 1986). Assuming that E between dissolved bicarbonate and carbonate in apatite is l % o 0r2%~ (theE value for calcium carbonate), then apatite carbonate should have a 613Cvalue approximately9%0to 10%ogreater than that of respired CO, which presumably reflects that of diet. Kinetic fractionation effects occur because of differences in the rate of transport or rate of reaction of isotope species. For reactions catalyzed by enzymes, the magnitude of fractionation can be used to approximate the relative affinity of an enzyme for a compound with one isotope or another. An example of a kinetic fractionation is the reaction catalyzed by the enzyme glutamic oxaloacetic transaminase. This enzyme catalyzes the symmetrical reaction that transfers an amino group from glutamic acid to oxalacetic acid to yield a-ketoglutaric acid and aspartic acid (Mackoet al., 1986).Glutamic oxaloacetic transaminase transfers 14NH2from glutamic acid to aspartic acid 1.0083times faster than 15NH2.In the reverse reaction l4NH2is incorporated

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into a-ketoglutarate 1.0017 times faster than 15NH. Transaminases catabolize nitrogen transfers for 12 other amino acids. Their kinetic discrimination against 15N may explain the observed 15Nenrichment between diet and nonessential amino acids, as well as the progressive enrichment in 15Nacross trophic levels (Gaebler et al., 1966; Post 2002; and this chapter).

Terminology : Caveat Many processes can lead to differencesin composition between an organism's tissues and its diet. For example, the enzymatic fractionation resulting from the action of transaminases described above, produces tissue proteins that most likely tend to be enriched in 15Nrelative to diet. Some of the differences between the isotopic composition of diet and a consumer's tissues are the result of fractionating processes. Others are the result of stoichiometric effects and what has been called isotopic routing (see subsequent sections). Because several processes can lead to differences in the isotopic composition of diet and animal tissues, it is inappropriate to call these differences "fractionation". Cerling and Harris (1999)proposed the term discrimination faca,,,) for the differencebetween the isotopic composition of tor (A,,, = 6t,ssu_diet and that of consumer tissues. Using the term "fractionation" to describe differences between the isotopic composition of a resource and the tissues of a consumer is inappropriate for two reasons: (1)it confuses pattern with process (fractionation is only one of the processes that produce discrimination) and (2) it is inconsistent with usage in other fields.

MIXING MODELS: GUIDE FORTHE PERPLEXED Stable isotopes are widely used to reconstruct animal diets. The basic idea has been summarized in the phrase "animals are what they eat". The isotopic composition of an animal tissue reflects the contribution of dietary components with different isotopic compositions (DeNiro and Epstein, 1978, 1981).Two types of approaches have been used to reconstruct animal diets from isotopic data: Euclidean distance methods and mixing mass-balance models (reviewed by Phillips, 2001). Phillips (2001)demonstrated that Euclidean distance methods do not estimate diet proportions correctly, Thus, we do not deal with these models here. Rather, we review in some detail mass-balancemixing models and their assumptions. Our description of rnixing models relies heavily on the papers by Phillips (2001)and Phillips and Koch (2001).

Linear Mixing Models The simplest of the mass-balance mixing models assumes that the isotopic composition of their tissues equals the weighed average of the isotopic composition of the diet constituents. For two diet constituents:

Mass-balance models

147

where p equals the fraction of diet A and 6XAand 6XBare the isotopic composition of diet components A and B. Provided that the isotopic composition of two elements is used, eqn (4) can be extended to estimate the fraction (pi)of the diet comprised by three types of items (Phillips,2001, Ben David and Schell, 2001). For carbon (C) and nitrogen (N) this requires solving the following system of linear equations in which A, B, and C are three different food types, and p, + p, + p, are the contributions of each food type to the animal diet:

= P A + P B+PC In general, one can use n-1 isotopes to discriminate the contribution of n food sources. Because eqns (4)and (5)depend linearly on p, the mixing relations they depict can be labeled "linear mixing models". Although eqns (4)and (5)look reasonable, they contain a variety of unrealistic assumptions.First, they assume that food types are stoichiometrically identical, i.e. that food type A and B contain exactly the same relative carbon and nitrogen contents. Second, they assume that all dietary items are assimilated with equal efficiency. Lastly, eqns (4) and (5) assume that isotopes are completely homogenized in the consumer's body prior to tissue synthesis. Phillips and Koch (2001)refined mixing models to incorporatedifferences in food stoichiometry and assimilation efficiency.We deal with the homogeneity assumption in a later section.

Concentration-dependent Mixing Models Phillips and Koch's (2001)concentration-dependentmixing models assume that the contribution of a given dietary item to an animal's carbon (or nitrogen) pool depends on how much carbon (or nitrogen)that item contains. The difference in the results of using linear mixing models and concentrationdependent mixing models is best illustrated with one isotope and two diet types. Let us call B the total assimilated biomass, p the fraction of total assimilation contributed by diet 1, (1-p)the fraction of total assimilation contributed by diet 2, and [C,] and [C,] the concentrations of element X in diets 1and 2, respectively.The relative contribution of diet 1to the pool of element X in the consumers tissues will be

and the isotopic composition of the pool of element X will be

148

Physiological and ecological adaptations to feeding in vertebrates

Figure. 6.2 illustrates the potentially large errors that can be committed by assuming a linear mixing model when the two diets differ significantly in elemental composition. The concentration-dependent mixing model can be easily modified for more than one isotope and more than one diet. Again, n-1 isotopes can be use to differentiate among n diets. For more than one isotope, pi is the fraction of total assimilated biomass (B) contributed by item i and Pxirepresents the fraction of assimilated element X Pxi

-

Bpix.

-

p.x.

~f. pix, 2 PjXj

'

j=1

(8)

j= 1

For three food sources, and two elements for example, carbon and nitrogen with concentrations [Ci]and [Nil (i = 1,2, and 3) and isotopic compositions 'Tiand 15Ni,we have that:

Of course, eqn (9)reduces to eqn (5)if all the diet components have the same elemental composition (i.e. [C,] = [C,] = [C,], and [N,]= [N, ] = [N,]). The error caused by neglecting concentration dependence increases as the differences in elemental composition among dietary components increase.With a bit of algebra, eqn (9) can be written in matrix form as a system of 3 linear equations in 3 unknowns: AP=B where

and

r P,I

P =I p,

lP3

roi

1,

1

and B =I 0 1

1

11

Phillips and Koch (2001) provide an algorithm to solve for vector P in eqn (10). The concentration-dependent mixing model proposed by Phillips and Koch (2001)assumes that all elements in a diet are assimilated with the same efficiency,which is not necessarily the case. Fortunately, element-dependent

Mass-balance models

149

variation in assimilation efficiency can be incorporated into the model. Let us call B' the total biomass ingested and eyithe efficiency with which element X is assimilated in diet i. Then eqn (8)must be modified as

j =l

j=1

Equation (9) has to be modified accordingly and the value of eximust be estimated experimentally. Although physiological ecologists estimate the assimilation efficiency for food types and even specific nutrients routinely, there are few accounts of the efficiency with which different elements are assimilated. We emphasize that the term exin eqn (11)represents "true" assimilation efficiency rather than the apparent assimilation efficiency so often reported. True assimilation efficiency is the fraction of the ingested element absorbed (i.e.ex= amount of element x not assimilated/amount of element x ingested), whereas apparent assimilation efficiency includes endogenous fecal losses (apparent assimilation efficiency = [amount of element x not assimilated + endogenous fecal losses]/amount of element x ingested). Readers can find a lucid explanation of the difference between true and apparent assimilation in Karasov (1990). Incorporating food stoichiometry in mixing models requires more empirical work. It requires analyzing (or at least estimating)the food's elemental composition and may require determining the efficiency with which different elements in each diet are assimilated. Field researchers may understandably complain that concentration-dependent models require more additional data and assumptions than simple linear mixing models (Robbins et al. 2002). However, simple models that make seriously wrong assumptions can yleld seriously erroneous results. The "collect-combust-and infer" approach that has characterized animal isotopic ecology so far has been fruitful. Although it will probably remain the approach of choice for some problems that can be solved by qualitativeapproaches, it has serious limitations. The simple linear mixing models that dominate the literature are misleading if the elemental composition of diet components differs substantially (Fig.6.2).Considering the potential effect of food's elemental composition and differential assimilation on isotopic incorporation adds realism to mixing models. In some cases, however, even the detail provided by concentration-dependentmodels may not suffice and additional assumptions may need to be incorporated.

Isotopic Routing Mass-balance mixing models make a crucial assumption which is almost certainly wrong in many animals. They assume that the isotopes of the elements contained in all dietary sources are completely homogenized ("mixed")

150

Physiological and ecological adaptations t o feeding in vertebrates

Fig. 6.2. Isotopic composition of a homogeneous mixture of two materials depends on two factors: 1) the fraction of each material in the mixture and 2) the elemental composition of the two materials. In the example depicted by the family of curves, p equals the fraction of material 1 in the mixture and 1-p is the fraction of material 2. The isotopic composition of materials 1 and 2 is 615N,=13.2and 615N, = -0.9 respectively. To construct the curves we maintained C, constant (C, = 0.12) and varied C, from 0.01 (punctate curve) to 0.12 (thick line). The values for C, in the remaining curves, from top to bottom are 0.02, 0.04, and 0.08. A linear mixing model [see eqn (4)]assumes that C, = C , and hence always predicts a straight line. Mixing models that account for the elemental composition of the mixture yield curves rather than straight lines if C, z C,. The isotopic and elemental compositions of this artificial example correspond to the values of salmon (material 1) and plants (material 2) ingested by brown bears (Ursus arctos, after Phillips and Koch, 2001).

in the animal body before tissues are synthesized. The animals that best fit this assumption are foregut fermenters in which nutrients are homogenized to the common denominator of volatile fatty acids and bacterial protein before being absorbed. However, even in ruminants many nutrients escape the fermentative chamber and are absorbed intact in the lower gut (Van Soest, 1994).Once absorbed, nutrients enter a variety of metabolic pathways and the elements in them can undergo varying degrees of mixing (Fig. 6.3).The mixing assumption is problematic whenever diet components differ in macronutrient content. Synthesis of one macronutrient from another can be difficult (e.g. glucose and glucogenic amino acids cannot be synthesized from fatty acids) and is always energetically expensive (Fig. 6.3). Thus,

Mass-balance models

sloughed cells, hair, etc.

sloughed

Acid Pools

Pool

uric acid

cr urea

t

'Yz

3-phosphoglyceraldehyde

carrier-Hz

IATplt(gbmgenica'a')

&Toketone

t pyruvic acid

acetyl CoA

4a-ketoglutaric acid

bodies

YT

cycle

~ a 2 r - H4~ Aw+p,=m

lactic acid

t ,

4

carrier

Fig. 6.3. Carbon and nitrogen in organisms are found as components of macronutrients. This scheme outlines the potential interconversions among the primary macronutrients in an animal body. Although there is potential for considerable mixing of elements among the different macronutrient pools, mixing may be energetically expensive. Recall that in general catabolism generates ATP. Synthesis, however, requires both ATP and reducing equivalent (such as NADHP). For example, lipids can be synthesized from both carbohydrates and proteins but lipid synthesis entails a high cost (the synthesis of a single palmitate molecule from 8 Acetyl-CoA requires 7 ATPs and 14 NADPH). In a similar fashion, although dispensable amino acids can be synthesized from the carbon skeletons resulting from both carbohydrate and lipid metabolism, this process is ATP dependent. Furthermore, the addition of amino acids to a peptide chain requires ATP (Mathews et al. 2000). A corollary of this observation is that organisms should route dietary macronutrients.

animals should route macronutrients and the isotopes in them from diet into the same macronutrient types in their tissues. This phenomenon has been called isotopic or nutrient routing. Paleontologists have recognized the problems posed by isotopic routing for quite some time. For example, anthropologists and paleontologistshave traditionally used bone collagen, largely composed of protein, to analyze isotopic composition for dietary reconstruction.Collagen has two problems: (1)it contains 33%glycine, which is a relatively 13C-enriched amino acid, so collagen tends to be 13C-enrichedrelative to other tissues; and (2) collagen is largely composed of protein and the composition of body protein in omnivores often reflects the isotopic composition of dietary protein (Ambroseand

Physiological and ecological adaptations t o feeding in vertebrates

152

Norr, 1993). Recognition of the principle that in omnivores the isotopic composition of tissue protein often reflects that of dietary protein, and not that o€bulk diet, has led researchers to analysis of the carbonates in bone apatite (Tieszen and Fagre, 1993).These are synthesized from circulating bicarbonate derived from CO, and hence probably reflect the components of the diet that are catabolized for energy (Ambrose and Norr, 1993).Omnivorous animals feeding on diets with low protein content should allocate dietary protein for tissue maintenance and repair, rather than catabolize it for energy. Consequently apatite carbonates may underestimate the contribution of dietary protein (see below).

Macronutrient Concentration-dependent Mixing Models The notion that protein should be routed to protein can be formalized in mixing models that depend on the content of macronutrients (protein, carbohydrate, and lipid) in each diet component. For element X (carbon or nitrogen) in protein:

a

tissue protein

1I

=(

P[P,I + (1-p)[P,

I

)(=Pl~[pll +=p,(l-p)[p,l)

(12)

where [PI]and [P,] are the protein contents of diet components 1 and 2 respectively and 6X,,, and 6X,,, are the isotopic compositions of the protein in these components. Equation (12) assumes that the concentration of element X in the protein of components 1 and 2 is the same, which is a reasonable assumption. It also assumes that protein in both components is assimilated with equal efficiency. Differences in the elemental concentration and in assimilation efficiency between the protein contained in diet components can be easily incorporated into eqn (12) [see eqns (8)and (lo)]. Figure 6.4 illustrates the difference between the results of a concentrationdependent model and one that incorporates differences in protein content between diet components. The differences between the results of the two models increase as the disparity in protein composition between diets increases. Note the large errors a concentration-dependentmodel can engender if there is routing. Suppose that [P,] = 0.07 and 613C= -24 for the example depicted in Fig. 6.4.If there is protein routing this value represents a p of 0.25. The concentration-dependentmodel would estimate p as 0.75! A protein concentration-dependentmixing model for n-1 isotopes and n diets can be constructed as:

xtissue

protein

-

i=l

2piepi[pi1 i =l

Mass-balance m o d e l s

-28

1 0

I

I

I

I

I

153

I

I

I

I

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 P

1

Fig. 6.4. Mixing models that assume isotopic routing of dietary protein into tissue protein yield results different from those of concentration-dependent models. In this example, we applied a protein routing model [eqn (11)] to two diets with contrasting isotopic composition (PIC, = -27 and 613C2= -12). Diet 1 had a fraction of assimilated biomass equal to p and a protein content ([P,] on a dry mass basis) of 0.75. The protein content of diet 2 ([I-',]) varied from 0.07 (dashed line) to 0.75 (thick line). The values for P, in the remaining curves, from top to bottom, are 0.14, 0.28, and 0.54. The concentration-dependent model [from eqn (7)] assumes that [C,] = 57.7 and [C,] = 46.8 (i.e. the values of lean deer meat and corn respectively. The thick line represents both the concentration-dependent model and a protein routing model that assumes identical protein contents in diet components 1 and 2. Because the carbon contents of the two diet components are similar, the concentration-dependent model yields a linear mixing relationship.

where pi is the fraction of total biomass ingested comprised by component i, eI,ithe efficiency with which the protein contained in diet component i is assimilated, [Pi] its protein content, and 6X, the isotopic composition for element X of the protein in component i. For N and C, the matrix form of this linear system is

A,P=B,

(14)

where

r (6l3CI- B3CtisSue )epl[p,I

I AP =

(613C2- G3CtisSue )ep2[p21 (613C3- 83Ctissue )eP3[P3 I1

( g 5 ~- ~, 5 ~ , . s u e ) e p l ([ pg l 5~ ~-,6 1 5 ~ t i)ep2 s s u[p21 e ( 6 1 5 ~-,615~tis,e)e,[p, II

1

1

1

1

154

Physiological and ecological adaptations to feeding in vertebrates

r pI1

and

~01

P =I p, 1 , and B =I 0 1

Ip3J

111

The mixing model described by eqn (12) assumes that protein is always routed into protein. This assumption is likely to be correct if, when eaten alone, all diet components satisfy the animal's protein requirements. However, many interesting situations involve one dietary component that does not provide sufficient protein but which is abundant and provides energy in the form of carbohydrates and lipids. Good examples of these situations are fruit- and nectar-eatingbirds that satisfy most of their protein requirements with insects (Martinezdel Rio, 1994)and carnivorous mammals that ingest fruit and plants in addition to meat (Pritchard and Robbins, 1990).When one of the diets is protein deficient, at low ingestion levels of the protein-rich alternative there is probably significant routing of carbon from carbohydrates and, to a lesser extent from lipids, to protein. A mixing model that incorporates this added level of realism requires many assumptions and cannot be solved analytically for p. It is useful, however, to ascertain how wrong the results of our mixing models can be. We explored the potential effect of isotopic routing among macronutrients with an artificial situation. We assumed a large animal (65 kg) that consumes two diet components with contrasting isotopic and macronutrient compositions(Fig.6.5).The protein content of these two diets is such that only one of the diet components (component 1) has sufficient protein to g day-') if it ingests satisfy the animal's minimal protein requirements (PRmir, enough of it to satisfy its energy needs. The other diet component (component 2) is protein deficient. We assumed this animal to ingest enough mass of each diet component to maintain neutral energy balance. However, we also assumed that the animal protein intake was insufficient to satisfy its minimum protein requirements when it ate only component 2. Assuming the total protein consumption as equal to or higher than PRmin, we calculated the animal tissue protein 613Cusing eqn (11).However, were protein ingestion less than PR-, we assumed that the carbon needed to synthesize the protein deficit was derived from amination of carbon skeletons derived from carbohydratesin component 2 (the carbohydrate-rich, but protein-deficient component):

'

'"'tissue

protein

=

{

I

((pRmn - B ( p [ y 1 + ( 1 - p ) [ P , 1 ) ) 6 q if PRmmn > B(p[ql+(l- p)[P21) P R ~ ~ ~ and

Mass-balance models

155

-

813Ccat(non-protein) concentrationdependent mixing model perfect routing (protein to protein)

.........0 ........ imperfect routing

Fig. 6.5. A more realistic model of isotopic routing for two diet components differing in protein content. The protein content and isotopic composition of diet components 1 and 2 are identical to those in Fig. 6.4. The energy content of diet component 1was 25.1 kJ g-' and that of diet component 2.17 kJ g-' and we assumed that the animal ingested a mixture of diet components that satisfied its daily maintenance energy requirements (11,241 kJ day-'). We also assumed that the minimal protein requirements (PRmi,)equaled 77 grams day-' (these data correspond to a 65 kg human exercising moderately; Reeds and Becket, 1996). Finally, we assumed that diet component 1 contained 25% lipid and no carbohydrates and component 2 contained 89% carbohydrate and 4% lipid. The thick curve assumes concentration-dependent mixing. Note that assuming that some carbohydrate is routed to protein (curve with open circles, "imperfect routing") at low protein intakes does not yield results very different from those of a mixing curve that assumes that only protein is routed into protein. The curve labeled with diamonds assumes that the isotopic composition of the nutrients catabolized for energy reflects a mixture of the components that remain after protein has been allocated to satisfy minimal protein requirements. If animals route protein to protein and allocate carbohydrates and lipids to energy production, a concentration-dependent mixing model underestimates the contribution of the protein-rich component. Using a concentration dependent-model a value of 613C= -14%0in exhaled breath (or bone apatite) corresponds with a p - 0.12. However, if there is protein routing p = 0.19 (dotted lines).

As Fig. 6.5 shows, making this more realistic assumption yields results very similar to those obtained by assuming a simple protein-to-protein routing model (the maximal difference between the two models is 1.4%O ). Equation (15) assumes that only carbohydrate carbon from diet component 2 is incorporated into protein. An alternative is to assume that the carbon used to compensate the protein deficit is derived from aminating carbon skeletons from a mix of all macronutrients. This assumption yields almost identical results as eqn (15).

156

Physiological and ecological adaptations t o feeding in vertebrates

The critical assumption of eqn (15) is that carbon skeletons of carbohydrates can be aminated. Why would an animal with limited protein transfer an amino group from protein into carbohydrate.The reason lies in the inefficiency of metabolism. Even animals in negative nitrogen balance continue to catabolize protein. O'Brien et al. (2000,2002) have shown that hawkmoths feeding on sucrose-rich but nitrogen-free nectar, use nitrogen stored as larvae to synthesize dispensable amino acids that are incorporated into egg proteins. These authors found the carbon isotopic composition of nectar sugars in dispensable ("nonessential") amino acids, but not in indispensable ("essential"). We speculate that when nitrogen is limited, the ammonia resulting from inevitable protein turnover will be incorporated into carbon skeletons derived from carbohydratesto synthesize dispensable (nonessential) amino acids. Of course, essential amino acids are always derived exclusively from dietary protein. This hypothesis can be tested by analyzing the composition of specific amino acids (see O'Brien, 2002). Equations (12)and (15)can be used to predict the carbon isotopic composition of tissue protein. Assuming that protein is routed to protein, the isotopic composition of the mixture of macronutrients catabolized for energy (Fl3CCat) should reflect the isotopic composition of carbon contributed by the macronutrients that remain after allocation to protein. Estimating Fl3CCat is a simple exercise in accounting but because the resultant equation is long and awkward, it is not presented here. The value of Fl3CCat reflects the increased importance of the protein-deficient diet as a source of energy. If there is protein routing and a concentration-dependent mixing model is used to estimate diet composition from the 613Cof nonprotein tissue (e.g.breath CO, and bone apatite; Hatch et al., 2002), the fraction of the protein-rich component (p) will be underestimated (Fig.6.5). Several authors have pointed out that the carbon isotope ratio of breath CO, is a reliable indicator of the carbon isotope composition of bulk diet (Hatch et al., 2002 and references therein). The results depicted in Fig. 6.5 cast doubt on this assertion. Indeed, if diet components differ in protein content, breath CO, will have a carbon isotope will composition closer to bulk diet than the 613Cof body protein. Yet 6l3CCat have a value biased toward that of the protein-deficient component of the diet. This bias should increase with (a) the protein requirements of the animal and (b)the disparity in protein content between dietary components. The message of our admittedly simplistic routing models may be disappointing for ecologists eager to use stable isotopes to find out what their study animals eat. Our models suggest that isotopic ecology and nutritional ecology are inextricably linked. To understand incorporationof the isotopic signal of different diet components into animal tissues it appears that we must know not only the macronutrient content of these components,but also the efficiency with which these macronutrients are assimilated. Using concentration-dependent or macronutrient content-dependent models to reconstruct animal diets using stable isotopes cannot be done with

Mass-balance models

157

confidence until we validate the relative performance of these models in the laboratory. The models described in this section outline a research agenda. Although we believe that isotopic ecology can benefit from the adoption of more realistic mixing models, there will always be a place for the simple mixing models outlined in eqns (4) and (5).These models can be used to accurately describe the proportion of diferent diets incorporated into a given tissue. As emphasized above, these proportions may/may not represent the proportion in which these diets are ingested. Using any of the models described above is "correct" provided that in each case the assumptions of the models are identified and that the limitations of the inferences that can be derived from them are recognized.

Compound Specific Isotopic Analysis: I s I t a Way Out? The ability to measure the isotopic composition in specificbiochemical compounds (e.g. fatty acids, cholesterol, and amino acids) suggests an alternative to avoid the problem of nutrient routing in diet reconstruction (Hammer et al., 1998).The isotopic composition of indispensable ("essential") nutrients that the animal cannot synthesize must reflect the composition of the mix of these nutrients in the diet (O'Brien et al., 2002). There is enormous potential for use of the isotopic composition of individual essential nutrients to sort out an animal's dietary components.However, this new level of technological sophistication does not liberate us from mixing models. It is an easy exercise to modify eqn (12)to determine diet components from the isotopic composition of an essential nutrient (just substitute Pi for E, the concentration of the essential nutrient E in diet, and FX, for FX,,). he isotopic composition of an essential nutrient in animal tissues is the result of the concentration of this nutrient in each diet component and thus its isotopic composition in the diet in toto.

DYNAMICS OF ISOTOPIC INCORPORATION Mixing models assume equilibrium. They assume that an animal has ingested the diet components in a fixed combination long enough for the isotopic composition of its tissues to have reached a steady state. Because animals shift diets such is rarely the case. Indeed, stable isotopes have proven useful tools in documenting diet shifts in animals (Wolf and Martinez del Rio, 2000).The isotopic composition of a tissue is the result of the integration of isotopic inputs over some time in the past. Thus, using tissues with different turnovers will give information of the past diet of an animal over different time intervals. The turnover rate of tissue constituents governs the time window of isotopic incorporation (Tieszen et al., 1983). The dynamics of incorporation of an isotopic "signature" into a tissue depends on the rate at which the materials in the tissue turn over. Consider

158

Physiological and ecological adaptations t o feeding in vertebrates

Fig. 6.6. In a pool of element X of size 4,the rate at which the amount of heavy isotope changes equals the difference between the rate at which this isotope enters the pool (Ap,f,) minus the rate at which it exits the pool (A?"f,). A, is the size of the pool (in mols), ro and ri are the fractional rates of input and output of element X into the pool (with units equal to time-') respectively, f, the fraction of heavy isotope in the pool, and f,, the fraction of heavy isotope in diet. 1-(

df, A , dt

a tissue that contains A, mols of the element in question. We can envision A, as the pool of element X in a tissue. Then consider the amount (A, = f,AT) of the heavy isotope (13Cor 15N)in this pool. Let us call ri and ro (with units equal to time-l)the fractionalrates at which the element enters and leaves the pool respectively, and f,, and f, the fractions of the heavy isotope in the incoming materials (d for diet) and in the pool (b for body) respectively (Fig. 6.6).Then

and

Combining eqns (16)and (17),and because, we have.

1 dA = (5 - ro) A, dt

(-)-

1

Mass-balance models

159

Assuming that at time 0, f, = f,(O), and that fHd(t) is constant (f,,(t) = fHd), we may then integrate eqn (18)to yield:

Several studies have investigated the change in tissue isotopic composition after an animal has been subjected to two diets that differ markedly in isotopic composition (O'Brien et al., 2000 and references therein). These authors used coexponential functions of the form

to describe their data. Because the heavy isotope is usually rare (i.e. fH

--

60

40

0

a

a

20

3

0

+

LIVER

u

CARCASS

2

4

6

8

Time after tubefeeding (h) Fig. 15.6. Postprandial handling of a tube-fed a mix of dissolved FAA (based on the composition of Bovine Serum Albumin) with added I4C-labeled lysine in Atlantic halibut (Hippoglossus hippoglossus) at 59-60 days post-first feeding (adapted from Rojas-Garcia and Rernnestad, 2003a).

The benefits of using a CO, trap when studying evacuation of intact FAA was demonstrated in the studies of Rlannestad et al. (2001a,b; Fig. 15.6). Taking alanine and glutamate as examples, these authors showed that 45% and 68% of the total labeled FAA tube-fed to Senegalese sole were recovered in the water and that virtually all (95%)of this was due to released CO,, i.e. catabolism (bnnestad et al., 2001b).This emphasizesthe need for separating evacuation and excretion in studies of digestive absorption efficiency. At the

408

Physiological and ecological adaptations t o feeding in vertebrates

same time, the high catabolism of AA demonstrated in this study, 41% and 65% of total fed AA (Rannestad et al., 2001b), supports the earlier estimates of AA catabolism based on apparent NQ. Further studies with increasing concentrations of FAA should be performed in order to determine the limit above which the FAA begins to be evacuated. The challenge in optimizing the intestinal performance of fish larvae in relation to dietary AA is probably to find the ideal balance between the different molecular forms of AA. Furthermore, improvement of dietary AA utilization will also depend on defining the ideal dietary IAA profile at different larval stages (Conceiqsoet al., 2003).Finally, despite the relatively immature digestive system of marine fish larvae, it should be stressed that they have one of the highest potential growth rates among vertebrates. Acknowledgments

Supported by Research Council of Norway projects 141990/120,138382/ 140,115876/122, FCT (Portugal) grant - SFRH/BPD/7149/2001, and project POCTI/ 1999/CVT/34608 (FCT,Portugal and FEDER, European Union).This is publication #I32 from the University of Bergen Fish Larval Locus. REFERENCES Alvarez M.d.C., Perez R., Seikai T., Takahashi Y. and Tanaka M. 1999. Ontogenetic development of the digestive enzyme activities under different feeding regimes during the early life stages in Japanese flounder, Paralichthys olivaceus. J. Fish Biol. in press. Applebaum S.L and Rsnnestad I. 2004. Absorption, assimilation and catabolism of individual free amino acids by late larval Atlantic halibut (Hippoglossus hippoglossus). Aquaculture 230: 313-322. Barr Y., Rojas-Garcia C.R. and Rsnnestad I. 2001. The digestion capacity of protein and amino acids in the Atlantic halibut (Hippoglossus hippoglossus). In: Larvi 2001. C.I. Hendry, G. van Stappen, W. Willie, and P. Sorgeloos (eds.). Eur. Aquacult. Soc., Oostende, Belgium, Sp. Publ. no. 30, pp. 50-53. Bender D.A. 1985. Amino Acid Metabolism. John Wiley & Sons, New York, NY. Bengtson D.A., Borms D.N., Leibovitz H.E. and Simpson K.L. 1993. Studies on structure and function of the digestive system of Medidia beryllina (Pisces, Atherinidae). In: Physiology and Biochemistry of Fish Larval Development. B.T. Walther and H.J. Fyhn (ed.). Univ. Bergen, Bergen Press, Bergen, Norway, pp. 199-208. Berge G.E., Lied E. and Espe M. 1994. Absorption and incorporation of dietary free and protein bound (U14C)-lysinein Atlantic cod (Gadus morhua ). Comp. Biochem. Physiol. 109A: 681-688. Bowen S.H. 1987. Dietary protein requirement of fishes - a reassessment. Can. J. Fish. Aquat. Sci. 44: 1995-2001. Brett J.R. and Zala C.A. 1975. Daily pattern of nitrogen excretion and oxygen consumption of sockeye salmon (Oncorhynchus nerka) under controlled conditions. J. Fish. Res. Bd Can. 32: 2479-2486. Buddington R.D. 1985. Digestive secretions of lake sturgeon, Acipenser fulvescens, during early development. J. Fish Biol. 26: 715-723. Cahu C.L. and Zambonino Infante J.L. 1995a. Effect of the molecular form of dietary nitrogen supply in sea bass larvae: Response of pancreatic enzymes and intestinal peptidases. Fish Physiol. Biochem. 14: 209-214.

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Terjesen B.F., Finn R.N., Norberg B. and Rennestad I. 2002. Kinetics and fates of ammonia, urea, and uric acid during oocyte maturation and ontogeny of Atlantic halibut (Hippoglossus hippoglossus). Comp. Biochem. Physiol. 131A: 443-455. Thorsen A. 1995. Oogenesis in marine bony fishes: physiological mechanisms of oocyte hydration and egg buoyancy. PhD, University of Bergen, Bergen, Norway. Tonheim S.K., Koven W.M. and Rennestad I. 2000. Enrichment of Arternia with free methionine. Aquaculture. 190: 223-235. Tonheim, S. K, M. Espe, A. J. Raae, M.J. Darias and I. Rennestad. 2004. In vivo incorporation of [U]-14C-amino acids : An alternative protein labelling procedure for use in examining larval digestive physiology. Aquaculture. 235: 553-567. Tytler P., Bell M.V. and Robinson J. 1993. The ontogeny of osmoregulation in marine fish, with particular reference to the effects of changes in salinity and temperature. In: Physiology and Biochemistry of Fish Larval Development. B.T. Walther and H.J. Fyhn (eds.). Univ. Bergen, Bergen Norway, pp. 249-258. Ueberschar B. 1988. Determination of the nutritional condition of individual marine fish larvae by analysing their proteolytic enzyme activities with a highly sensitive fluorescence technique. Meeresforsch. 32: 144-154. van der Meeren T. 1991. Algae as first food for cod larvae, Gadus morhua L.: filter feeding or ingestion by accident? J. Fish Biol. 39: 225-237. van der Meeren T., Fyhn H.J., Pickova J. and Hamre K. 2001. Biochemical composition of copepods: Seasonal variation in lagoon-reared zooplankton. In: Larvi 2001 C.I. Hendry, G. van Stappen, W. Willie, and P. Sorgeloos (eds.). Eur. Aquacult. Soc., Oostende, Belgium, Sp. Publ. no. 30, pp. 614-615. van Waarde A. 1983. Aerobic and anaerobic ammonia production by fish. Comp. Biochem. Physiol. 74B: 675-684. van Waarde A. 1988. Biochemistiy of non-protein nitrogenous compounds in fish including the use of amino acids for anaerobic energy production. Comp. Biochem. Physiol. 91B: 207-228. Verreth J., Polat A*, van Herwaarden H., ConceiqZo L. and Huisman E.A. 1995. A comparison of methods to study energy resource partitioning in early life stages of fish, with special reference to the African catfish, Clarias gariepinus Burchell. ICES Mar. Sci. Symp. 201: 57-63. Verreth J.A.J., Torreele E., Spazier E., van der Sluiszen A., Rombout J.H.W.M. and Booms R. 1992. The development of a functional digestive system in the African Catfish Clarias gariepinus (Burchell). J. World Aquacult. Soc. 23: 286-298. Walford J. and Lam T.J. 1993. Development of digestive tract and proteolytic enzyme activity in seabass (Lutes calcarfer). Aquaculture 109: 187-205. Walton M.J. 1985. Aspects of amino acid metabolism in teleost fish. In: Nutrition and Feeding in Fish. C.B. Cowey, A.M. Mackie, and J.G. Bell (eds.). Acad. Press, London, UK, pp. 47-67. Watanabe T. and Kiron V. 1994. Prospects in larval fish dietetics. Aquaculture 124: 223251. Watanabe Y. 1984. Morphological and functional changes in rectal epithelium cells of pond smelt during post embryonic development. Bull. Jpn. Soc. Sci. Fish. 50: 805-814. Werner R.G. and Blaxter J.H.S. 1980. Growth and survival of larval herring (Clupea haerengus) in relation to prey density. Can. J. Fish. Aqua. Sci. 37: 3545. Wilson R.P. 1989. Amino acids and proteins. In: Fish Nutrition. J.E. Halver (eds.). Acad. Press, New York, NY, pp. 111-151. Wright P.A. and Fyhn H.J. 2001. Nitrogen excretion and metabolism in early life stages. In: Fish Physiology, vol. XX. P.A. Wright and P.M. Anderson (eds.). Acad. Press, New York, NY, pp. 178-252. Wright P.A., Felskie A. and Anderson P.M. 1995. Induction of ornithine-urea cycle enzymes and nitrogen metabolism and excretion in rainbow trout (Oncorhynchusmykiss) during early life stages. J. Exp. Biol. 198: 127-135.

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INDEX Atrophy 4, 2, 88, 191, 193, 210, 211, 222, A-linolenic acid 365, 374 Acid-base status of arterial plasma 286 Active transport 67, 1, 02, 114, 115, 116, 118, 121, 123, 124, 126, 127, 129, 135, 140, 243, 387

Actomyosin 118 Adaptive modulation hypothesis 116, 121, 137

Adductor mandibulae 7 Adductor muscles 5, 33 Adriatic sturgeon (Acipenser naccarii) 367, 369, 373, 379, 386

Agkistrodon piscivorus 283, 306 Alkaline tide 279, 280, 284, 285, 286, 287, 288, 295, 297, 299, 300-303

Autonomic nervous system 302, 325, 326, 328, 355, 357, 358, 359, 360

B B-lymphocytes 263, 267, 270 Bar-tailed godwit 210, 218, 220, 226, 227 Bats 17, 34, 238 Beak 6, 18, 21, 22, 27, 411 Bears 150, 173, 177, 190, 198, 230, 233, 238, 239, 244, 249, 250, 251, 254, 318

Bidirectional flow 14, 35, 37 Biting 15, 21, 31, 33, 74, 78, 91, 357, 375 Bivalve 91, 100, 106, 215, 216, 217, 218, 220, 221

AlkaIoids 27, 257 Allies scolopacidae 218 Alligator mississippiensis 32, 33, 184, 286, 289, 291, 295, 299, 300, 311, 319

Allometry 81, 92, 93, 206, 218, 320 Alsophis portoricensis 312, 322 Ambystoma 33, 35, 36, 39, 341, 359 American robins (Turdus migratorius) 105 Ammotretis rostrata 330, 355 Amylase 96, 239, 250 Antinutrients 257 Antioxidants 245 Apoptosis 192, 193, 194, 197, 198, 238, 247, 252, 261

Apparent digestibility 47 Apfenodytes forsteri 176, 197 Arachidonic acid 365, 380, 385 Arctic ground squirrels (Spermophilus parryii) 240, 253 Assimilation efficiency 56, 71, 73, 76, 147, 149, 152, 309, 397, 406

Blackcaps 104, 105, 110, 179, 180, 181, 183, 197, 226

Bladder bile 239 Boa constrictor 290, 293, 299, 303, 322, 324 Bolus 1, 10, 12, 19, 20, 21, 22, 23, 24, 25, 28, 30, 344, 345, 346, 409

Bovids 17 Bramble-wake model 11 Branchiomeric musculature 3 Broad-tailed hummingbirds (Selasphorus platycercus) 74, 100 Brush-border membrane 110, 114, 115, 119, 125, 131, 133, 134, 137, 138, 232, 235, 254

Buccal cavity 3, 4, 10 Bufo 282, 287, 288, 289, 290, 295, 296, 297, 299, 300, 301, 303, 323, 324, 348, 355

Bufo marinus 290, 296, 299, 300, 303, 323, 324, 355

Burmese pythons 199, 302, 312, 322, 323 C

Atlantic salmon 354, 372, 374, 375, 376, 377f 3831 384f 386, 387, 400,

229, 231, 234, 235, 236, 238, 240, 242, 243, 249

409t

410

Caecilians 17, 26, 37 Caiman 183, 185, 199, 316, 320, 338, 356

418

Physiological and ecological adaptations t o feeding i n vertebrates

Capillarity 17 Capture 1, 5, 8, 9, 11, 12, 14, 15, 16, 17, 19,

Compensatory feeding 73, 74, 75, 76, 78,

21, 24, 32, 33, 34, 35, 36, 37, 38, 39, 40, 96, 179, 190, 294, 308, 326

Compensatory suction 13 Concentration-dependent mixing models

Capture/subjugation 1, 11, 12 Carbohydrate 45, 49, 59, 72, 82, 84, 104, 108, 121, 131, 140, 311,

113, 123, 132, 151, 345,

114, 124, 133, 152, 402,

115, 125, 134, 154, 404,

116, 126, 135, 155, 405,

117, 127, 136, 156, 413,

118, 129, 137, 196, 424,

119, 130, 139, 276,

Cardiac performance 363, 372, 383, 384 Cardiolivin 380 Carrier-mediated absorption 67 Carrier-mediated transport 113, 114, 117, 120, 122, 132, 380

Catfishes 311 Ceca 50, 52, 53, 64, 83, 102, 203, 204, 205, 211, 222, 235, 244, 255, 257, 265, 266, 267, 268, 334, 335, 346, 34% 353; 359, 414

Cell division 199, 261 Cell loss 191, 193, 231 Cell membranes 117, 122, 365 Cell proliferation 104, 182, 191, 192, 193, 194, 198,c 199, 231, 236, 237, 247, 250, 252, 273

80, 86

147, 152

Cost-benefit curve 70

Coturnix japonica 110, 111, 169, 199, 355 Cranial kinesis 4, 16, 31, 33, 34, 37 Crocodiles 4, 15, 17, 20, 21, 24, 27, 32, 38, 183, 184, 190, 286, 311, 321, 333, 348

Crocodylus 32, 292, 293, 299, 301, 311, 321, 338, 356

Crop 17, 21, 26, 27, 28, 40, 89, 60, 188, 203, 221, 226, 262, 263, 269, 310, 317, 319, 331, 341, 346, 412

Crossflow filtration 24, 38

Crotalus cerastes 306, 323 Crotalus durissus 189r 299, 300, 318, 319 Crotalus horridus 200, 303, 313, 324 Crotalus viridis 189, 322 Ctenosaura 39, 316, 322 Cuticle 29 Cyclooxygenase 381 Cytokines 247, 262, 270, 271, 272, 276

Cellular dystrophy 182 Cellular hypotrophy 182

Defenses 45, 250, 255, 259, 260, 262, 268,

Cephaloscyllum ventriosum 312 Ceratophrys cranwallii 189, 316, 322

Defensins 262, 277 Deglutition 24 Dendritic cells 263, 269 Desmognath 23, 26, 39 Diatoms 381 Diet hardness 209, 214, 218 Diet microflora 256 Diet preferences 67 Diet quality 57, 58, 85, 87, 89, 90, 111, 174,

Chameleons 16, 34, 40 Charadriiformes 202, 212, 218 Chemical digestion 1, 22, 28 Chemical reactors 45, 58, 59, 60-62, 6567, 73-75, 80-85

Chemoreception 17

Chen rossii 167 Chewing 19, 21, 22, 27, 28, 89, 91, 100 Claudins 116, 140 Cold exposure 74, 80 Colon 46, 50, 52, 53, 54, 56, 83, 136, 203, 235, 253, 258, 259, 265, 266, 272, 273, 275, 283, 330, 335, 341, 358, 360, 364, 366, 382

Comminution 6, 22, 51

271, 272, 424

209, 211, 226, 381

Diffusive absorption 67 Digestibility 43, 44, 46, 47, 49, 51, 54, 55, 56, 63, 70, 82, 210, 211, 248, 277, 312, 361, 391, 393, 396, 397, 401, 402, 414

Digestion coefficient 46, 47, 55 Digestive constraints 66, 67, 72, 78, 80, 82, 85, 87, 88, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 108, 109, 111, 424

Index Digestive efficiency 31, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 59, 71, 72, 93, 94, 97, 105, 106, 108, 182, 204, 209, 211, 225, 227, 256, 321, 391, 396, 421, 424 Digestive enzymes 28, 50, 51, 93, 96, 104, 105, 163, 203, 239, 248, 270, 312, 315, 316, 390, 391, 411, 412 Digestive function 59, 61, 62, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 229, 239, 248, 250, 314, 391, 409, 424 Digestive spare capacity 80 Dinoflagellates 381 Discrimination 136, 145, 146, 159, 164, 165, 249 Djungarian hamsters 92 Docosahexanoic acid 365 Dormice (Eliomys quercinus) 238 Dover sole (Solea solea) 378 Ducks 105, 109, 172, 198, 225, 226, 227, 228, 348

E Eicosanoids 262, 364, 365, 380, 381, 387 Elaphe obsolete 315 Elasmobranchs 13, 22, 33, 41, 330, 349, 350, 362 Electrolytes 73, 137, 243, 249 Energy assimilation 71, 72, 74, 75, 76, 79, 205 Energy substrates 365, 389, 404 Enteric nervous system 325, 326, 327, 328, 332, 335, 353, 355, 358, 359, 360 Enteric neurons 240, 327, 328, 329, 330, 341, 344, 354, .-61 Enterochromaffin cells 349 Enterocyte 104, 109, 111, 117, 122, 130, 132, 180, 184, 185, 186, 187, 188, 191, 192, 231, 232, 235, 236, 237, 238, 241, 242, 243, 245, 246, 247, 249, 252, 258, 260, 261, 262, 263, 269, 270, 271, 283 Enterocyte life span 104 Enterocyte turnover 104, 192 Environmental stressors 363, 378 Eosinophils 262, 263 Epithelial barrier 16, 122, 271

419 Epithelial configuration 184, 186 Epithelium 9, 134, 136, 138, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 192, 193, 194, 199, 231, 233, 236, 238, 243, 244, 252, 255, 256, 258, 259, 260, 261, 262, 263, 268, 269, 270, 271, 273, 275, 276, 328, 354, 415 Equilibrium fractionation 145 Esophageal papillae 13, 40 Esophagus 3, 10, 11, 19, 21, 23, 24, 25, 28, 203, 260, 261, 267, 326, 327, 330, 343, 344, 346, 348, 351, 361, 391 Eulamprus quoyii 314 Eurasian curlew 218, 220 European eel (Anguilla anguilla) 367, 386 European starlings (Sturnis vulgaris) 105 Eutamias amoenus 54 Everted intestinal sleeve method 127

F Fasting tolerance 176 Fat stores 176, 229, 233, 248 Fecal dry matter 46 Feeding bout 10, 11, 12 Feeding cycle 11 Feeding ecology 36, 57, 85, 110, 171, 189, 190, 195, 201, 217, 424, 310 Feeding mode 10, 27, 36 Feeding stages 1, 10, 11, 12, 402, 405 Filter feeding 12, 415 Fish larvae 363, 378, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 401, 403, 404, 405, 406, 408, 409, 410, 411, 412, 414, 415, 425 Fish-spearing 12 Fluctuations in food availability 176 Food webs 142, 172, 365, 366, 376, 381, 383, 386 Foraging strategy 10 Fractionation 144, 145, 146, 162, 164, 166, 168, 172, 173, 174 Frogs 15, 16, 17, 20, 27, 32, 37, 40, 189, 190, 286, 302, 354, 358 Fructose transport 115, 135, 137, 139, 140 Frugivory 22, 66, 69, 72, 84, 127, 228, 272

420

Physiological and ecological adaptations to feeding in vertebrates

Gadus morhua 299, 301, 309, 310, 323, 336, 338, 348, 353, 355, 356, 357, 358, 359, 360, 385, 387, 408, 410, 412, 413, 415 Galliformes 202, 204, 205, 206, 207, 209, 212, 213, 225 Gape cycle 11, 12 Gastric acid secretion 283, 284, 285, 286, 288, 299, 300, 302, 303, 306, 338, 347, 348, 349, 350, 353, 356, 358, 359, 361 Gastric mill 1, 29, 30 Gastrin 325, 334, 335, 336, 337, 345, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 361 Gastrointestinal blood flow 292, 293, 299, 301 Gastrointestinal defenses 259 Gastroliths 27, 39 Gavials 15 Ghrelin 325, 335, 337, 338, 339, 352, 355, 357, 358, 359, 362 Gilthead seabream (Sparus aurata) 378, 385, 4 12 Giraffes 17 Gizzard 1, 27, 28, 29, 30, 93, 94, 100, 104, 105, 109, 111, 177, 181, 182, 196, 199,

Glossophaga longirostris 96 GLUT2 102, 103, 110, 114, 115, 119, 121, 125, 131, 132, 133, 134, 135, 137, 138 GLUT5 115, 119, 135, 137, 139, 140 Glutathione 245, 250, 252 Golden hamsters (Mesocricetus auratus) 240 Granivores 29, 91, 100, 107, 207, 257, 269 Granulocytes 263 Great knots 203, 210, 224 Grinding plates 29 Grit 27, 29, 33, 117, 123, 229, 240, 250, 255, 270, 271, 339, 365 Ground squirrels 172, 230, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 250, 251, 252, 253, 254, 320 Grouse 57, 104, 111, 204, 206, 207, 211,

Gut motility 63, 262, 315, 325, 326, 328, 329, 331, 333, 334, 335, 337, 339, 341, 342, 343, 344, 345, 347, 351, 352, 353, 355, 357, 359, 360, 425 Gut volume 61, 72, 75, 99

327, 338, 349, 361,

Gut-associated lymphoid tissue (GALT) 255, 256 Gymnophione amphibians 17

Haematopodidae 218 Halibut (Hippoglossus hippoglossus) 378, 408, 411, 412, 413, 414, 415 Hamsters (Cricetus cricetus) 238 Handling stress 378, 385 Harpagifer antarcticus 310, 319 Heart rate 293, 294, 295, 296, 297, 300, 303, 324, 374 Helicobacter 261, 275 Herbivores 7, 28, 29, 30, 46, 50, 53, 57, 83, 91, 109, 164, 201, 226, 256, 257, 267, 271, 326 Herring (Clupea haringus) 378 Heterodonty 22 Hexose absorption 59, 69, 70, 79 Hibernation 92, 176, 192, 196, 229, 231, 233, 234, 235, 236, 237, 238, 240, 241, 242, 243, 244, 245, 246, 248, 249, 250, 251, 252, 253, 254, 362, 368 Hoatzin 28, 34 Homology 11, 27 Horseshoe crabs 216 Hummingbirds 17, 67, 69, 70, 71, 72, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 91, 100, 101, 110, 122, 127, 128, 129, 130, 136, 137, 139, 140, 141 Hydrostatic elongation of the tongue 10, 15 Hyobranchial apparatus 1, 5, 9, 12, 13, 14, 15, 16 Hyoid apparatus 7, 8, 9, 13, 34, 39, 41 Hyolingual apparatus 5, 9, 20 Hyolingual transport 10, 20 Hyperplasia 182, 192, 252

Index Hypertrophy 182, 192, 211, 225, 271, 280, 28 1 Hypobranchial muscles 7 Hypoxia 299, 301, 363, 367, 368, 369, 370, 371, 372, 374, 378, 379, 380, 382, 384, 386, 387

Iguana 16, 21, 368 Immune system 233, 244, 2 56, 257, 259, 260, 261, 262, 263, 265, 267, 268, 269, 270, 271, 272, 273, 275, 277 Inertial suction 9, 13 Inertial transport 20 Ingestion 1, 11, 12, 14, 15, 16, 17, 19, 20, 21, 28, 41, 49, 50, 60, 61, 63, 66, 72, 75, 81, 82, 85, 118, 128, 138, 154, 165, 233, 241, 245, 248, 249, 279, 280, 283, 284, 286, 293, 294, 300, 306, 308, 309, 310, 314, 318, 390, 396, 406, 415 Insectivory 22 Intake response 74, 77, 78, 80, 85, 111 Interstitial cells of Cajal 325, 328, 333, 354, 357, 359, 360, 361, 362 Intestinal absorption 67, 72, 113, 118, 121, 132, 135, 136, 139, 253, 272, 274, 315, 406, 412, 424 Intestinal blood flow 292, 293, 299, 301 Intestinal crypts 182, 184, 191, 192, 193 Intestinal epithelia 114, 115, 117, 122, 126, 134, 135, 137, 138, 196, 231, 233, 235, 236, 243, 244, 249, 250, 252, 253, 258, 260, 262, 269, 270, 276 Intraepithelial lymphocytes 244, 263, 267, 268, 269, 271, 272, 277 Intraoral transport 19, 20, 23 Isolated heart 373, 374, 380 Isotopic composition 142, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 156, 157, 159, 160, 161, 162, 163, 164, 166, 167, 168, 169, 171, 173, 174 Isotopic routing 146, 149, 151, 153, 154, 155 Isotopic signature 142, 160

Japanese flounder (Paralichfhys olivaceus) 378, 387

421 Japanese quail 104, 110, 111, 159, 169, 179, 181, 182, 198, 199, 225, 227, 228, 355 Jaw muscles 30 Jaw prehension 12, 14, 15, 16, 17

K Kidneys 189, 195, 285, 288 Kinetic fractionation 145, 173 Kinetic skulls 16 Kingfishers 21 Kinixys spekii 311, 321 Knots (Calidris canutus) 100, 210

L Lactation 88, 92, 98, 99, 105, 106, 109, 116, 191, 254 Large intestine 56, 94, 138, 203, 254, 255, 263, 265, 266, 267, 268, 275, 276, 332, 340, 354, 355, 357, 362 Lepidosaurian reptiles 16 Linear mixing models 146, 147, 149 Lingual ingestion 17 Lingual prehension 15, 16 Linoleic acid 364, 365, 375, 377, 379, 383 Lipid storage 214 Lipoxygenase 381 Lips 17, 20, 23, 146, 147, 148, 150, 173, 174, 296, 302, 311, 323 Long-distance migratory species 52

Lumbricus festivus 166, 174 Lymphocytes 244, 252, 255, 256, 257, 259, 263, 264, 265, 267, 268, 269, 270, 271, 272, 276, 277 Lysozyme 262

M M cells 192, 263, 269, 274, 275, 328, 415 Macrostomatan snakes 23 Marginal teeth 4, 15, 22, 23, 26 Marmofa marmofa 52, 54, 57, 192, 230, 233, 234, 235, 237, 244, 252, 321 Masseter 7 Mast cells 262, 263 Mastication 1, 6, 7, 22, 24, 30, 32, 34, 39, 43, 50, 51, 57, 308, 310

422

Physiological and ecological adaptations t o feeding i n vertebrates

Masticophis flagellum 306, 323 Maximum metabolizable energy 92, 93 Mean retention time 43, 50, 57, 68, 95, 97, 106 Menidia menidia 309, 310, 316 Metabolic fecal dry matter 46 Metabolic fecal losses 48 Metabolic fecal nitrogen 46, 47, 163 Metabolic rate 44, 75, 81, 84, 85, 88, 93, 109, 111, 127, 139, 191, 200, 224, 225, 226, 230, 246, 279, 280, 281, 288, 289, 291, 292, 293, 298, 300, 301, 306, 307, 308, 312, 313, 315, 317, 318, 319, 320, 321, 323, 324, 363, 368, 370, 371, 372, 374, 379, 380, 381, 382, 386 Metabolizability 43, 44, 47, 48, 52 Microbes 49, 50, 58, 198, 255, 256, 257, 261, 263, 268, 269, 276, 424 Microflora 244, 255, 256, 257, 258, 259, 260, 261, 263, 264, 268, 269, 271, 272, 273, 274, 275, 276, 277 Microphagous fishes 26 Micropterus salmoides 310, 317, 319 Microtus townsendii 54 Migrating motor complexes 339, 343 Mixing models 141, 143, 146, 147, 149, 150, 152, 153, 154, 157, 172, 173, 174 Mollusks 23, 201, 218, 220, 221 Molt 21, 209, 210, 214, 376, 377, 383, 385, 386, 387 Monotremes 14, 22, 34, 348 Mucosal defense system 262 , Mucosal epithelium 134, 179, 180, 181, 182, 183, 184, 185, 187, 189, 190, 192, 194, 328 Mus musculus 98, 105, 109 Muscardinus avellanarius 239 Muscular hydrostats 9 Myrmecophages 17 Mysticete whales 12

Net assimilation rate 71 Neuropeptides 293, 301, 334, 340, 353, 356, 358, 359 Neurotransmitters 325, 330, 334, 339, 341, 344, 346, 349, 357, 359 Nonadrenergic-noncholinergic (NANC) 293 Nonmediated passive uptake 114 Nutrient absorption 50, 55, 70, 83, 84, 93, 101, 102, 103, 104, 106, 108, 109, 110, 112, 119, 130, 132, 133, 134, 136, 137, 203, 232, 242, 243, 249, 258, 260, 262, 410, 421 Nutrient assimilation 25, 59, 60, 110, 225, 226, 396, 414 Nutrient uptake 54, 55, '60, 86, 90, 94, 109, 112, 113, 121, 131, 140

N Natrix maura 317, 321 Nectarivory 17, 66, 74, 80, 96, 110, 127, 137, 206, 272 Neonate mammals 14

Paracellular permeability 113, 125, 130, 132, 134, 138 Parasites 256, 257 Parasympathetic 293, 325, 328, 329, 330, 353

0 Occlusion 22 Okapi 17 Opossums 20 Optimal digestion 45, 46, 69, 71, 72, 83, 85 Oral cavity 12, 19, 21, 24 Osmoregulation 80, 134, 136, 363, 376, 377, 382, 394, 415 Owls 22, 361 Oxidative stress 245, 246, 250, 251, 252 Oxygen uptake 280, 281, 282, 293, 298, 303, 307, 312, 317, 368, 369, 370 Oystercatchers 218

P Palatal teeth 20, 28 Palate 4, 20, 22, 23, 24, 25, 30 Pancreas 94, 203, 222, 238, 239, 251, 253, 328, 347, 353, 354, 390, 405, 412 Pancreatic digestive enzymes 248 Paneth cells 262 Panurus biarmicus 177, 199 Paracellular flux 113, 117, 118, 119, 121, 123, 125, 132, 133

Index Parietal cell 284, 285, 347, 349, 358 Parrots 19, 21, 22, 206 Passage rates 1, 30, 51, 391 Passerines 72, 79, 80, 81, 204, 207, 208, 209, 210, 211, 213 Passive absorption 69, 79, 82, 102, 103, 108, 133, 135, 123, 124, 125, 126, 127, 129, 134, 135, 136, 139 Passive permeability 61, 126 Pattern generators 11 Pepsin / pepsinogen 351 Peristalsis 23, 24, 25, 256, 261, 308, 325, 334, 337, 344, 345, 346, 356, 414 Peyer patches 257, 262, 263, 267, 269, 270 Pharmacokinetics 129, 139, 140 Pharyngeal jaws 8, 19, 23, 24, 26, 36 Pharyngeal musculature 24 Pharyngeal skeleton 3, 4, 7, 8, 9, 12, 13 Pharyngognathy 8, 26 Pharynx 1, 3, 4, 5, 7, 8, 13, 14, 15, 19, 20, 22, 23, 24, 30, 39, 41, 267, 275 Phascolarctos cinereus 51, 57, 274 Phenotypic flexibility 89, 90, 93, 94, 111, 176, 177, 190, 194, 195, 199, 201, 223, 225, 227, 228, 231, 250, 314, 322, 324 Phloretin 133 Phloridzin 119, 122, 133, 243 Phospholipase 381, 383 Phytohemagglutinins 257, 271 Pinnipeds 14, 19 Piscivory 22 Pleuronectes platessa 316, 321 Plovers Charadriidae 218

Podarcis s. sicula 345, 358 Polyunsaturated Fatty acids (PUFA) 364 Postprandial response 185, 188, 199, 307, 312 Postprandial stimulation 308 Prairie voles (Microtus ochrogaster) 99 Prey capture 9, 16, 32, 33, 34, 35, 36, 37, 38, 39, 40 Prey manipulation 8, 34 Prey size 27, 28, 35, 317 Primary producers 161, 365, 381 Primates 12, 17, 21

423 Properistalsis 24 Protein metabolism 170, 214, 311, 414 Protein reallocation 222 Protein synthesis 165, 173, 191, 237, 252, 254, 279, 280, 283, 303, 306, 312, 314, 319, 398, 402, 406, 409, 411 Proton leak 380 Proventriculus 28, 29, 203, 215, 337, 339, 353, 355 Pseudocheirus peregrinus 51, 274 Pseudostratified epithelium 184, 185, 186, 190 Puncture-crushing 21, 22 Pylorus 27 Python molurus 189, 198, 199, 200, 294, 296, 297, 302, 320, 322, 323, 324, 337, 339, 340, 341 Python regius 189, 303

R

Raja sp. 336 Ram feeders 13 Rana catesbeiana 285, 286, 287, 300, 320, 336, 338, 351, 352, 358, 359, 361 Rana pipiens 31, 342 Raptors 17, 22, 51, 56, 204 Rattlesnakes 12, 28, 189, 300, 310, 319, 324 Regurgitation 28, 315 Reserve capacity 89 Retention times 50, 57, 71, 94, 133 Rhamphotheca 17, 22 Rhinoceroses 17 Rhombosolea tapirina 330, 355 Rhythmic oscillating complexes 343, 361 Rodents 17, 21, 54, 56, 57, 82, 108, 173, 190, 230, 245, 269, 270 57, 218, 221, 277 Rumenoreticulum 100 Ruminant herbivores 91 S Safety margin 55, 58, 78, 89, 93, 112 Salamanders 13, 14, 15, 16, 23, 26, 32, 35, 36, 39, 41 Salinity challenge 378

424

Physiological and ecological adaptations to feeding in vertebrates

Saliva 6, 21, 22, 28, 30, 323, 347 Salmo salar 336, 354, 372, 374, 377, 383, 384, 386, 387, 409, 410 Sandpipers 218 Sauropsids 176, 177 Scleroglossan lizards 17 Scolecophidian snakes 20 SDA 84, 110, 137, 174, 185, 186, 279, 281, 282, 283, 289, 292, 293, 295, 299, 300, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 321, 323, 409 Seabass (Dicentrarchus labrax) 378 SGLTl 114, 115, 119, 122, 123, 124, 125, 129, 131, 133, 242, 243 SGLT2 114, 137 Shorebirds 20, 38, 92, 93, 111, 202, 203, 204, 217, 218, 224, 225, 227, 228 Shrikes 21 Sieving 24, 130 Simple diffusive absorption 67 Sodium fluorescein 132, 13 Soft palate 24 Solvent drag 113, 117, 118, 124, 125, 130, 133, 134, 139 Spare capacity 78, 80, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 111, 126 Specific dynamic action 185, 200, 279, 281, 299, 301, 302, 303, 305, 306, 307, 308, 319, 320, 321, 322, 323, 324, 424 Spermophilus columbianus 54 Spiders 215, 220 Spinal autonomic system 330 Splanchnocranium 7 Squalus acanfhias 41, 336, 338, 348, 352, 356, 360 Stable isotopes 141, 142, 143, 144, 145, 146, 156, 157, 170, 172, 173 Standard metabolic rate (SMR) 368 Stomach mass 214, 215, 216, 218 Stratified epithelium 184, 185, 260 Stress tolerance 378, 386, 387 Sfurnus vulgaris 111, 138, 178, 195, 225, 228 Sucrase 54, 61, 68, 69, 75, 76, 84, 96, 98, 100, 240, 241

Sucrose 54, 59, 61, 68, 69, 70, 71, 74, 75, 76, 77, 78, 79, 80, 81, 84, 96, 100, 101, 115, 126, 156, 272 Sucrose hydrolysis 59, 61, 68, 69, 70, 75, 78, 79 Suction feeding 5, 9, 12, 13, 14, 15, 27, 31, 32, 34, 37, 41 Surface tension transport 20 Suspension feeding 3, 7, 12, 13, 14, 22, 24, 26, 35, 37, 38, 42, 190 Sustained aerobic exercise 374, 382 Swallowing 1, 5, 9, 11, 19, 20, 21, 22, 23, 24, 25, 26, 34, 37, 40, 51, 344 Swimming performance 299, 375, 383, 384, 386 Sylvia atricapilla 179, 180, 197, 226 Sylvia borin 52, 56, 57, 179, 195, 196, 197, 224, 225, 226 Sympathetic nervous system 293, 294, 325, 328, 329, 330, 332, 353, 354, 357 T T-lymphocytes 259, 263, 269, 270, 271 Teeth 3, 4, 6, 8, 15, 18, 20, 21, 22, 23, 26, 28, 30, 32, 33, 51, 145 Teleosts 8, 23, 24, 36, 336, 343, 349, 350, 4 12 Temporalis 7 Tenrecs 20 Thamnophis sirtalis 189, 320 Throughput time 61, 68, 70, 76 Tight-junction 116, 117, 118, 121, 122, 123, 125, 130, 134 Tilapia 37, 38, 341, 362 Tinca tinca 330 Tongue 1, 5, 9, 10, 11, 14, 15, 16, 17, 19, 20, 21, 22, 24, 25, 30, 32, 33, 35, 36, 37, 38, 39, 40, 84, 250, 324 Tooth reduction 6 Torpor 75, 101, 229, 230, 233, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 253, 368 Transepithelial impedances 118 Transitional epithelium 188, 189, 190, 193, 194 Tree shrews 20 True digestibility 47

Index Trunk 17, 20, 23, 35, 39, 294, 329, 357 Tuatara 16, 28, 34 Tupinambis merianae 317 Turdus migratorius 54, 105 Turnover time 52, 104, 106, 108, 191, 237 Turtles 6, 10, 13, 14, 17, 19, 21, 22, 24, 27, 31, 33, 34, 37, 39, 40, 313, 315, 323, 333

u Unidirectional flow 14 Urea 163, 19-69, 172, 177, 244, 251, 253, 254, 405, 415, 416 Urinary losses 48 Ursus americanus 238, 251, 253

v Vagus 329, 330, 335, 338, 343, 344, 348, 350, 353, 355, 360 Varanus 39, 282, 287, 293, 294, 296, 297, 298, 300, 301, 302, 311, 316, 317, 318, 322, 323, 324 Varanus albigularis 296, 302, 311, 323 Varanus exanthematicus 293, 294, 297, 298, 300, 301 Venom 12, 28, 40, 312, 322, 324 Ventilation 36, 82, 286, 287, 289, 300, 303, 317, 369, 370 Ventriculus 28, 29, 203, 215, 337, 339, 353, 355

425

Villus 55, 139, 180, 191, 192, 199, 229, 231, 235, 236, 237, 238, 249, 253 Visceral arches 3, 7 W Waders 201, 202, 204, 209, 210, 211, 213, 214, 215, 217, 218, 223 Walleye (Stizostedion vitreum) 378 Walruses 14 Water absorption 72, 85, 117, 122, 124 Water-balance 72 Waterfowl 19, 202, 204, 208, 209, 210, 211, 213, 214, 228 White-throated sparrows 93, 94, 95 Woodpeckers 17 X

Xenopus laevis 131, 190, 338, 341-343, 356, 357, 358, 359, 360

354,

Y

Yellow tail (Seriola quinqueradiata) 378 Yellow-eyed Juncos (Junco phaeonotus) 106 Yellow-rumped warblers 82, 96, 97, 110, 127, 134, 165

z Zonotrichia albicollis 34, 93