Diet Selection in the Yellow-Bellied Slider Turtle, Trachemys Scripta: Ontogenetic Diet Shifts and Associative Effects Between Animal and Plant Diet Items

DIET SELECTION IN THE YELLOW-BELLIED SLIDER TURTLE, TRACHEMYS SCRIPTA: ONTOGENETIC DIET SHIFTS AND ASSOCIATIVE EFFECTS BETWEEN ANIMAL AND PLANT DIET ITEMS

SARAH S. BOUCHARD

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2004

Sarah Bouchard

This dissertation is dedicated to my father, Thomas Bouchard.

ACKNOWLEDGMENTS

I thank my advisor, Karen Bjorndal, as well as my committee members, Lauren

Chapman, Nat Frazer, Carmine Lanciani, and Doug Levey, for their invaluable support and guidance throughout this study. I also thank Alan Bolten who provided valuable assistance in the beginning stages of this work. I am extremely indebted to the following undergraduate research assistants whose hard work and willingness to do almost anything made this project possible: Craig Baker, Lindy Barrow, Ann Frial, Jennifer Hill, Rachel

Marcus, Natalie Marshall, and Carrie Newsom. Additionally, I am grateful to Paul

Coehler, Dan Connelly, Justin Congdon, and Bill Hopkins who assisted with the collection of turtles by providing equipment and access to ponds. Jim Watt also assisted by providing juvenile turtles from his turtle farm in Pt. Mayaka, Florida. David

Chynoweth, Kaoru Kitajima, Adegbola Adesogan, and David Hodell graciously allowed me to conduct analyses in their laboratories at the University of Florida, and Colin

Chapman, Jason Curtis, Patrick Haley, Nathan Kreuger, Frank Robbins, and Karyn Rode provided invaluable assistance with these analyses. Kavita Isvaran, Suhel Quader, and

Nat Seavy offered much appreciated statistical advice, and Joe Carlin provided constructive comments on earlier versions of these papers.

I also thank the graduate students, post docs, and faculty in the Department of

Zoology for providing intellectual and emotional support throughout my graduate career.

In particular, I would like to acknowledge Sophia Balcomb, Rico Holdo, Carlos Iudica,

Yolanda Leon, Yoshi Matsuzawa, Shannon McCauley, Alison McCombe, Ben Miner,

iv Kate Moran, Daphne Onderdonk, John Paul, Peter Piermarini, Greg Pryor, April Randle,

Kim Reich, Brian Riewald, Sarah Schaack, Jeff Seminoff, Laura Sirot, Mark Spritzer,

Manjula Tiwari, James Vonesh, and Amy Zanne. Finally, I am extremely grateful to my

husband, Cotton Randall, and my parents, Tom and Muriel Bouchard. Their constant

support and encouragement allowed me to persevere through the ups and downs

associated with this dissertation.

Funding for this work came from the Archie Carr Center for Sea Turtle Research,

Brian Riewald Memorial Fund, Chelonian Research Foundation, McLaughlin

Dissertation Fellowship, Sigma Xi, Society for the Study of Reptiles and Amphibians, and the University of Florida Women’s Club. The Institutional Animal Care and Use

Committee at the University of Florida approved this research.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... iv

LIST OF TABLES...... viii

LIST OF FIGURES ...... x

ABSTRACT...... xi

CHAPTER

1 INTRODUCTION ...... 1

2 ONTOGENETIC DIET SHIFTS AND DIGESTIVE CONSTRAINTS IN THE YELLOW-BELLIED SLIDER TURTLE, TRACHEMYS SCRIPTA ...... 5

Introduction...... 5 Methods ...... 9 Experimental Animals and Diets...... 9 Experimental Protocol...... 10 Nutrient Analyses ...... 12 Digestive Processing Calculations ...... 14 Juvenile Growth and Composition ...... 15 Results...... 16 Discussion...... 17 Juvenile Growth and Composition ...... 17 Digestive Processing ...... 18 Conclusions...... 21

3 ASSOCIATIVE EFFECTS BETWEEN ANIMAL AND PLANT DIET ITEMS IN THE YELLOW-BELLIED SLIDER TURTLE, TRACHEMYS SCRIPTA ...... 26

Introduction...... 26 Methods ...... 30 Experimental Animals and Diets...... 30 Experimental Protocol...... 32 Nutrient Analyses ...... 33 Digestive Processing Calculations ...... 35

vi Short-Chain Fatty Acid Concentrations ...... 37 Results...... 38 Feeding Trials with Adult Turtles ...... 38 Feeding Trials with Juvenile Turtles ...... 40 Discussion...... 41 Conclusions...... 45

4 MICROBIAL FERMENTATION IN JUVENILE AND ADULT YELLOW- BELLIED SLIDER TURTLES, TRACHEMYS SCRIPTA...... 58

Introduction...... 58 Methods ...... 60 Results...... 62 Discussion...... 63

5 EFFECT OF DIET ON GASTROINTESTINAL TRACT MORPHOLOGY IN THE YELLOW-BELLIED SLIDER TURTLE, TRACHEMYS SCRIPTA...... 71

Introduction...... 71 Methods ...... 73 Results and Discussion ...... 74

6 CONCLUSIONS ...... 79

APPENDIX

DIGESTIVE PROCESSING OF DRY MATTER BY ADULT TURTLES...... 82

LIST OF REFERENCES...... 86

BIOGRAPHICAL SKETCH ...... 95

vii

LIST OF TABLES

Table page

2-1 Nutrient composition of duckweed and shrimp diets fed to juvenile and adult turtles as well as nutrient composition of orts (remaining food) for each treatment...... 23

2-2 Digestive processing of duckweed and shrimp diets fed to juvenile and adult turtles...... 24

2-3 Growth and nutrient composition of juvenile turtles fed duckweed or shrimp...... 25

3-1 Summary of studies investigating associative effects in wildlife...... 47

3-2 Nutrient composition of duckweed and shrimp fed to adult turtles ...... 48

3-3 Nutrient composition of duckweed and shrimp fed to juvenile turtles ...... 49

3-4 Nutrient composition of duckweed and shrimp orts (remaining food) from feeding trials with adults ...... 50

3-5 Digestive processing of duckweed, shrimp, and mixed diets by adult T. scripta ....51

3-6 Differences between actual and predicted digestibilities, digestible intakes, and daily gains of adult turtles fed two mixed diets of duckweed and shrimp ...... 52

3-7 Short-chain fatty acid composition in the digestive tracts of adult turtles fed duckweed, shrimp, and mixed diets ...... 53

3-8 Digestive processing of duckweed, shrimp, and mixed diets by juvenile T. scripta...... 54

3-9 Differences between actual and predicted digestibilities, digestible intakes, and growth rates of juvenile turtles fed a mixed diet containing duckweed and shrimp...... 55

3-10 Growth and nutrient composition of juvenile turtles fed duckweed, shrimp, and mixed diets ...... 56

4-1 Nutrient composition of duckweed fed to juvenile and adult turtles. All values except energy are presented on a percent dry matter basis...... 66

viii 4-2 Wet mass of T. scripta fermentation contents compared with that predicted for herbivorous reptiles of this size based on equation in Bjorndal (1997a) ...... 67

4-3 Molar percentages of individual short-chain fatty acids in digestive tracts of juvenile and adult T. scripta...... 68

5-1 Nutrient composition of duckweed and shrimp ...... 76

5-2 Gut morphology, gut mass, and large intestine contents of turtles fed duckweed and shrimp diets ...... 77

A-1 Digestive processing of dry matter from duckweed and shrimp diets fed to juvenile and adult turtles in Chapter 2 ...... 83

A-2 Digestive processing of dry matter from duckweed, shrimp, and mixed diets fed to adult turtles in Chapter 3...... 84

A-3 Differences between actual and predicted dry matter digestibilities and digestible intakes of adult turtles fed two mixed diets of duckweed and shrimp in Chapter 3 ...... 85

LIST OF FIGURES

Figure page

3-1 Concentrations of SCFAs in the digestive tracts of adult T. scripta consuming duckweed, shrimp, and mixed diets ...... 57

4-1 Digestive tract of juvenile T. scripta fed duckweed for five weeks...... 69

4-2 Concentrations of total short-chain fatty acids in the digestive tracts of juvenile and adult T. scripta...... 70

5-1 Gastrointestinal tracts from T. scripta fed (A) shrimp and (B) duckweed for five weeks...... 78

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

DIET SELECTION IN THE YELLOW-BELLIED SLIDER TURTLE, TRACHEMYS SCRIPTA: ONTOGENETIC DIET SHIFTS AND ASSOCIATIVE EFFECTS BETWEEN ANIMAL AND PLANT DIET ITEMS

Sarah S. Bouchard

August 2004

Chair: Karen A. Bjorndal Major Department: Zoology

The diet choices of animals can directly affect growth, survivorship, and reproduction. Understanding diet selection and nutrition in an animal can therefore provide valuable insight into the evolution of its life history characteristics and behavior.

Various factors influence diet selection, including diet chemistry, digestive physiology and anatomy of the consumer, and associative effects (interactions) between simultaneously ingested diet items. The purpose of this dissertation was to examine how these factors influence diet selection in yellow-bellied slider turtles, Trachemys scripta.

As juveniles, these turtles are carnivores that feed on aquatic invertebrates, but as they mature, they become opportunistic omnivores that feed primarily on aquatic plants.

Although adult turtles are primarily herbivorous, they will supplement their diet with animal material when it is readily available. Therefore, associative effects between plant and animal diet items could be important.

xi In feeding trials, I fed juvenile and adult turtles plant, animal, and mixed diets.

During these trials, I measured various digestive parameters including intake, digestibility, and daily gain of nutrients and energy. I also measured short-chain fatty acid concentrations in the gastrointestinal tract. The results of this work allowed me to address a number of questions related to T. scripta diet selection. First, I examined the role digestive physiology plays in the ontogenetic diet shift and found that juveniles were unable to meet their nutritional demands on the plant diet because of limits imposed on intake. Second, I observed a negative associative effect between plant and animal diet items in both juveniles and adults; this effect varied with different ratios of plant to animal material. Third, I described the gastrointestinal tract morphology of juveniles and adults and found that both age classes harbor microbial fermentations in their hindguts and that gastrointestinal tract morphology does not vary with age. Finally, I found that gross gastrointestinal tract morphology is not flexible in response to plant and animal diets.

xii CHAPTER 1 INTRODUCTION

The food an animal consumes provides the energy and nutrients for much of its biological activities. The diet choices of animals can therefore have profound effects on critical life history characteristics, such as growth, survivorship, and fecundity. For this reason, biologists have had a strong interest in understanding the factors influencing diet selection in animals. For my dissertation research, I explored this topic in the yellow- bellied slider turtle.

Traditionally, biologists have attempted to understand animal diet choices by examining the nutritional content of food items and costs associated with their acquisition

(Emlen 1966; MacArthur & Pianka 1966). However, optimal foraging models based on this approach often do not accurately predict animal diets because, in addition to nutritional content, the value of a diet item depends on the ability of the animal to extract nutrients from it (Karasov & Diamond 1988; Bozinovic & Martínez del Rio 1996; Levey

& Martínez del Rio 2001). This ability can depend on various factors, including chemical composition of the item, digestive physiology and anatomy of the animal, and associative effects, or interactions, between simultaneously ingested items (Bjorndal

1991; Bozinovic & Martínez del Rio 1996). Studies that consider these factors can provide more insight into animal foraging decisions than can traditional optimal foraging approaches.

Plant and animal diet items represent two types of food that differ considerably in their chemical composition (Robbins 1993), and, consequently, in how they are processed

1 2 in the vertebrate gastrointestinal tract. Animal tissues tend to be high in energy and nutrients and are easily digested by endogenous enzymes in the vertebrate stomach and small intestine. Plant tissues, on the other hand, are much more dilute in energy and nutrients. Furthermore, vertebrates do not possess enzymes capable of breaking down cellulose, the primary component of plant cell walls. To digest plant material, many vertebrates rely on microbial symbionts in their gastrointestinal tracts. These symbionts ferment cell wall constituents and produce waste products in the form of short-chain fatty acids, which the host absorbs and uses as an energy source.

Differences in how herbivores and carnivores digest plant and animal diets, respectively, are associated with considerable differences in their gastrointestinal tracts.

For herbivorous vertebrates that rely on fermentation to meet their energy requirements, the fermentation chamber must be sufficiently large to delay passage of digesta so that cell wall constituents are fermented and microbes can reproduce without being flushed from the system. In most species, fermentation takes place in either the foregut

(stomach) or hindgut (large intestine); therefore, these regions are often greatly enlarged and compartmentalized (Stevens & Hume 1995). Because animal material is more easily digested than is plant material, and it does not require fermentation, the gastrointestinal tracts of carnivores are generally much simpler and have a smaller capacity than do those of herbivores. For those vertebrates that switch between carnivorous and herbivorous diets, gastrointestinal anatomy is sometimes flexible, changing capacity to match what is required for efficient digestion of the diet (Levey et al. 1999; McClelland et al. 1999;

Starck 1999b).

Because plant and animal diet items differ greatly in nutritional composition and because they are processed so differently within the gastrointestinal tract, associative effects are likely to occur when plant and animal diet items are simultaneously ingested

(Bjorndal 1991). Associative effects occur when diet items interact with one another such that the net energy and nutrient gain from the mixed diet differs from the summation of gains from individual items. Both positive and negative effects have been documented between a various food types in a diverse array of animals including insects, turtles, birds, ungulates and rodents (Baker & Hobbs 1987; Kukor et al. 1988; Rickson et al.

1990; Bjorndal 1991; Bozinovic & Muñoz-Pedreros 1995; Hailey et al. 1998; Matter et al. 1999; Hilton et al. 2000). The mechanisms underlying these effects vary, but they often involve alterations in microbial fermentation. For example, in domestic livestock, the addition of urea and a small quantity of easily fermented carbohydrate increases digestibility of the forage. This increase is due to extra nitrogen from the urea and readily available energy from the carbohydrate stimulating growth of the microbial population, which can then ferment the forage more efficiently (Pond et al. 1995).

The yellow-bellied slider turtle, Trachemys scripta, is an excellent model for exploring the influence of digestive physiology on the selection of plant and animal diets.

Depending on life stage, T. scripta vary significantly in their degree of carnivory and herbivory. As juveniles, they are carnivores that feed on aquatic invertebrates, but as they mature, they become opportunistic omnivores that feed primarily on aquatic plants

(Clark & Gibbons 1969; Hart 1983; Parmenter & Avery 1990). Although adult turtles are primarily herbivorous, they will supplement their diet with animal material whenever it is readily available. Therefore, associative effects could play an important role in T.

4 scripta diet choices. Bjorndal (1991) found that adult turtles experienced a positive associative effect when fed a mixed diet containing duckweed, Spirodela polyrhiza, and mealworms, Tenebrio sp. She attributed this effect to extra nitrogen from mealworms stimulating growth of microbial populations, which could then ferment the duckweed more efficiently.

The purpose of this dissertation was to explore the role of food chemistry, digestive physiology and anatomy, and associative effects in the nutritional ecology of T. scripta.

In feeding trials, I fed juvenile and adult turtles plant, animal, and mixed diets. The results of this work are presented in four papers, which comprise Chapters 2-5 of the dissertation. In Chapter 2, I describe the role digestive physiology plays in the ontogenetic diet shift from carnivory to herbivory. In Chapter 3, I investigate associative effects, using previously untested plant and animal diet items, and I discuss whether different ratios of these diet items influence the magnitude and direction of the effect and whether the effect varies with turtle age class. In Chapter 4, I describe the digestive tract morphology of T. scripta and determine if the ontogenetic diet shift is associated with ontogenetic changes in gastrointestinal tract capacity. Finally, in Chapter 5, I evaluate the extent to which plant and animal diets induce change in gross gastrointestinal tract morphology.

CHAPTER 2 ONTOGENETIC DIET SHIFTS AND DIGESTIVE CONSTRAINTS IN THE YELLOW- BELLIED SLIDER TURTLE, TRACHEMYS SCRIPTA

Introduction

The foods that animals consume provide the energy and nutrients for much of their bodily functions. Diets of animals can therefore directly affect growth, survivorship, and reproduction. Diets sometimes change ontogenetically as animals grow and mature. Such ontogenetic shifts can occur between foods that are nutritionally similar or disimilar. For example, some fish are carnivores throughout life, but sequentially shift from zooplankton to invertebrates to fish prey (Gilliam 1982). Most frogs, on the other hand, experience more extreme shifts from herbivorous to carnivorous diets when they metamorphose from tadpoles to adults (Duellman & Trueb 1986). Similarly dramatic shifts, but in the opposite direction also occur in lizards (Rocha 1998; Durtsche 2000; Fialho et al. 2000, Cooper & Vitt 2002) and fishes (Horn 1989; Benavides et al. 1994).

Ontogenetic diet shifts from carnivory to herbivory are widespread in several turtle families including Emydidae (Sexton 1959; Moll 1976; Clark & Gibbons 1969; Bancroft et al.

1983; Parmenter & Avery 1990), Chelidae (Georges 1982; Chessman 1984; Chessman 1986;

Kennett & Tory 1996; Spencer et al. 1998), and Cheloniidae (Bjorndal 1997b). These diet shifts are often accompanied by habitat shifts from shallow to deeper water (Hart 1983;

Congdon et al. 1992). Therefore, ecological hypotheses, such as differences in prey availability between these zones, have often been proposed to explain chelonian diet shifts

(Hart 1983; Parmenter & Avery 1990). Although ecological factors are probably important,

5 6

digestive physiology may also play a critical role (Karasov & Diamond 1988; Whelen et al.

2000).

Juvenile turtles may be carnivorous because they cannot sufficiently process plant material to meet their nutritional needs. In most herbivorous reptiles, microbial symbionts in the hindgut play an important role in the digestion of plant material (Bjorndal 1997a). These symbionts ferment plant cell wall constituents, producing short-chain fatty acids as a waste product, which the host absorbs and uses as an energy source. The capacity of the fermentation chamber must be sufficiently large to delay passage of digesta so that cell wall components can be digested and microbes can reproduce. If passage is too rapid, microbial populations will be flushed from the digestive tract. In both mammals and reptiles, fermentation chamber capacity is directly proportional to body size (Parra 1978; Bjorndal

1997a); however, metabolic demands scale allometrically with body size to a power less than one (Bennett and Dawson 1976; Nagy et al. 1999). Consequently, the ratio of fermentation chamber capacity to metabolic rate decreases in smaller animals (Justice & Smith 1992).

Pough (1973, 1983) hypothesized that ontogenetic diet shifts occur because high mass- specific metabolic rates and small gut capacities that limit intake preclude small reptiles from herbivory. However, since Pough (1973, 1983) published his studies, many examples of small herbivorous lizards have been documented, and some species expected to experience an ontogenetic diet shift actually have herbivorous young (Troyer 1984b; Mautz & Nagy 1987;

Wikelski et al. 1993; Cooper & Vitt 2002). Results from these studies indicate that small reptiles can offset unfavorable ratios of gut capacity to metabolic rate. For example, to meet high mass-specific metabolic demands, juvenile reptiles can increase mass-specific intake and shorten gut transit times relative to adults (Troyer 1984b; Mautz & Nagy 1987; Bjorndal &

Bolten 1992; Wikelski et al. 1993). Because time for fermentation is reduced, this strategy

could decrease digestibility of the diet. However, small reptiles can compensate by

maintaining higher body temperatures that facilitate digestion (Troyer 1987; Avery et al.1993;

Wikelski et al. 1993). Additionally, small bite size has advantages that allow juveniles to maintain high intake without sacrificing digestibility (Bjorndal & Bolten 1992). Compared with adults, juveniles have a smaller bite size which allows them to ingest smaller food particles that are more rapidly fermented (Bjorndal et al. 1990) and to feed more selectively on plant parts that are higher in nitrogen and energy (Troyer 1984b; Mautz & Nagy 1987;

Bjorndal & Bolten 1992). Additionally, small bite size can allow juveniles to better break down structural barriers to digestion (Bjorndal & Bolten 1992). For example, in red-bellied

turtles, Pseudemys nelsoni, juvenile bites penetrated the waxy cuticle of duckweed fronds more

frequently than did adult bites, giving microbial symbionts greater access to cell wall

components.

Although studies indicate that juvenile reptiles can process and subsist on a plant diet,

the question remains why juveniles turtles are carnivorous. Studies addressing this question to

date have focused on the slider turtle, Trachemys scripta, a species in which the shift has been

particularly well studied (Clark & Gibbons 1969; Hart 1983; Parmenter & Avery 1990). As

juveniles, these turtles are carnivores that feed on aquatic invertebrates, but, as they mature,

they become opportunistic omnivores that feed primarily on aquatic plants. Although adult T.

scripta can process and subsist on both carnivorous and herbivorous diets (Bjorndal 1991), it is

unclear if juveniles can do so, because previous studies of juveniles using artificial diets have

produced conflicting conclusions. McCauley and Bjorndal (1999) found that juvenile T.

scripta fed a gelatin-based diet had higher mass-specific intake relative to adults. This study

8 suggested that juveniles can subsist on herbivorous diets because intake increased in response to nutrient-dilute diets with energy levels similar to plants. However, the digestibility of this diet was so high that potential limits on intake imposed by fermentation and gut capacity were not relevant. In another study, Avery et al. (1993) found that juvenile T. scripta fed a pelleted diet of 10% crude protein did not grow and concluded that protein levels in plants were not sufficient to maintain juvenile growth. Therefore, even if juvenile T. scripta can process plant material, individuals feeding on plants may be in poorer condition than are those feeding on animal material.

In this study, I conducted feeding trials in which I fed juvenile and adult T. scripta plant and animal diets. The goal of these feeding trials was to answer two questions: (1) does juvenile growth and composition vary with plant and animal diets and (2) to what extent are juveniles and adults able to process plant and animal material. If juveniles are carnivorous because they are unable to subsist on plant material, then juveniles fed plants may not grow or their tissues may be lower in water, organic matter, nitrogen, lipids, or energy compared with juveniles fed animal material. Additionally, Clark & Gibbons (1969) proposed that juvenile T. scripta are carnivorous to obtain sufficient calcium for shell mineralization; juveniles fed plant material may therefore also be lower in calcium relative to those fed animal material.

If differences exist between growth and composition of juveniles fed plant and animal material, then comparisons of digestive processing, as measured by intake, digestibility, and transit time of the diet, may provide insight into why. For example, juveniles may have reduced ability to digest plant material relative to adults. Alternatively, if juveniles can digest plant material, their small gut capacities may limit their ability to consume sufficient quantities to meet their nutritional needs. This would be suggested if juveniles fed plant material do not

9 have higher mass-specific intakes relative to adults. Because juveniles are carnivorous in the wild, they should efficiently process animal material. Juveniles should digest animal material at least as well as adults do, if not better. Also, juveniles should have higher mass-specific intakes of animal material relative to adults.

Methods

Experimental Animals and Diets

Juvenile yellow-bellied slider turtles were obtained as hatchlings from a commercial turtle farm in Pt. Mayaka, Florida, in mid June 2000. These turtles were the offspring of breeding adults collected from northern Florida, Georgia, and South Carolina. Sex of these turtles was unknown. Adult turtles were collected in May 2001 from Kathwood Ponds located in the Audubon Society's Silver Bluff Sanctuary in Aiken County, South Carolina. All adult turtles used in this trial were males.

The plant diet was duckweed, Lemna valdiviana, collected from a pond in Gainesville,

Florida. Duckweed is a small, floating aquatic plant consumed by T. scripta throughout much of its range (Parmenter & Avery 1990). The animal diet was freshwater grass shrimp,

Palaemontes paludosus, purchased from a bait shop that obtained the shrimp from Gainesville area lakes. Pretrial observations indicated juveniles tended not to eat the most anterior portion of the shrimp containing the eyes and antennae or the posterior portion containing the caudal fin. To ensure that all animals consumed the same diet, these parts were removed before shrimp were fed to juveniles and adults. Nutrient composition of each diet is described in

Table 2-1. Because adult and juvenile trials were not conducted simultaneously, composition of duckweed varied between the trials. In every case, the difference means a higher quality diet for juveniles compared to adults, 9% lower in cell wall content (NDF), 7% higher in

energy, and 22% higher in protein. If juveniles had difficulty processing the duckweed diet, it

was not because the diet was lower in nutrients and energy, relative to the adult diet.

Experimental Protocol

Juveniles were housed individually in square Rubbermaid containers (18 x 18 cm),

with four containers placed within a larger Nalgene tank (45 x 60 cm). Each day, juveniles were rotated between Nalgene tanks to avoid a tank effect. Each Nalgene tank was equipped with a 75-W floodlight and a 20-W full-spectrum natural light fluorescent bulb. Adults were housed individually in the same Nalgene tanks. All turtles experienced a twelve-hour photoperiod and temperatures between 25-26 °C.

Turtles were fed either duckweed (juveniles n = 7; adults n = 7) or shrimp (juvenile n =

7; adults n = 5) diets. Both juvenile and adult feeding trials lasted five weeks and consisted of a two-week acclimation period followed by a three-week experimental period during which daily food intake and feces production were quantified. The juvenile trial was conducted from

29 August to 2 October 2000, and the adult trial from 30 May to 2 July 2001. Before the juvenile trial began, adult feces were introduced into tanks so juveniles could acquire microbial gut symbionts (Troyer 1984a). At the onset of the juvenile trial, turtles had an average mass of

11.6 g (range: 9.4 – 16.6 g). At the onset of the adult trial, turtles had an average mass of

995.4 g (range: 375.2 – 1451.1 g), with average masses in the duckweed and shrimp treatments of 956.2 g (range: 375.3 – 1280.2 g) and 1081.1 g (range: 912.0 – 1251.1 g), respectively. Originally ten adult turtles were divided between duckweed and shrimp diets to evenly distribute size in both treatment. Two remaining turtles, which were smaller, were added to the duckweed diet to increase sample size for other studies involving these turtles

Chapters 4 & 5). Data were analyzed with and without these additional individuals and

because results were the same, they were included in this study.

To determine digestibility, I collected all feces during the experimental periods. Feces

were collected in water balloons (juveniles) and in condoms (adults) using techniques modified from Avery et al. (1993) and Bjorndal (1991). For juveniles, the fecal collection device

consisted of a small Nalgene tubing connector with a water balloon attached to one end. The

tail of the turtle was placed inside the open end of the connector, and two wires held the tubing

connector against the skin of the animal. The first wire wrapped around the tubing connector

with each of the two ends threaded through one of two holes placed in the posterior marginal

scutes of the carapace. The ends were twisted around each other on the dorsal side of the

carapace to secure the wire in place. The second wire looped from the connector to a small

brass ring super-glued to the posterior end of the plastron. This prevented the connector from

moving upwards and away from the skin of the animal. Silicone was inserted into any space

remaining between the connector and the skin of the turtle. Feces flowed through the

connector into the balloon, which could be easily removed.

The fecal collection device for adults was similar to that of juveniles except that vinyl

tubing and non lubricated Trojan brand condoms were used in place of tube connectors and

water balloons. The tubing was fitted to the adult in the same manner as was the juvenile

design, except the small brass ring was not necessary because adult plastrons were sufficiently

rigid to secure the tubing in place.

During both trials, water was drained from tanks every morning at 0800h so all turtles

could bask for the same amount of time each day and differential thermoregulation among

turtles could be controlled. At 1000h, feces were collected from each balloon and condom, and

tanks were refilled with water. At 1100h, turtles were fed a known mass of either duckweed or

shrimp. Enough food was provided to ensure turtles could feed ad libitum. Turtles fed ad

libitum for six hours until 1700h when orts (remaining food) were collected and weighed.

Previous trials with freshwater turtles indicate that intake of turtles fed for six hours does not

differ from that of turtles fed throughout the day (Karen Bjorndal, pers. comm.). At the

conclusion of each day, feces were collected from adults for a second time. It was not

necessary to collect feces from juveniles because they did not produce as much feces as adults

and water balloons were not full of feces.

Nutrient Analyses

During the experimental periods, samples of duckweed and shrimp diets were collected

daily. Daily diet, ort and fecal samples were dried overnight at 60 °C. Daily diet samples, as

well as daily ort and fecal samples for each turtle, were combined to obtain a composite sample

of each across the three week period. All samples, except juvenile fecal samples, were ground

to pass through a 1 mm screen in either a Wiley Mill or coffee grinder (Mr. Coffee, Model IDS

57). Juvenile fecal samples were not ground because (1) sample quantities were so small that

grinding would have resulted in losing a significant percentage of sample and (2) the entire

fecal sample for each turtle was used for analysis.

Diet samples were analyzed for dry matter, organic matter, neutral detergent fiber (NDF), acid detergent fiber (ADF), nitrogen, and energy content. Fecal samples were analyzed for dry and organic matter because juveniles produced insufficient quantities for further analyses.

Duckweed orts and shrimp orts were analyzed for nitrogen and energy content to test if turtles fed selectively.

Dry matter and organic matter content were determined by drying subsamples overnight

at 105 °C and then combusting them at 500 °C for three hours. The difference between these

two measures represents the ash, or mineral, component of the sample. NDF and ADF were

determined by sequentially refluxing samples with neutral detergent and acid detergent

solutions (Goering & Van Soest 1970) in an Ankom200 Fiber Analyzer according to the

guidelines supplied with the equipment (Ankom Technology 1998, 1999). NDF represents the

cell wall component of the plant diet (cellulose, hemicellulose, lignin and cutin), and ADF

represents the ligno-cellulose and cutin component. The ADF component of the shrimp diet

represents the exoskeleton (primarily chitin) fraction of the diet (Stelmock et al. 1985).

Nitrogen content of the samples was determined using a Carlo Erba elemental analyzer.

Energy content was determined with a Parr bomb calorimeter (Parr Instrument Company

1960).

All diet samples, fecal samples, and ort samples from adults were analyzed in duplicate.

Dry matter, organic matter, and energy duplicates were accepted within 2% relative error.

Nitrogen duplicates were accepted within 1% absolute error, and duplicates for NDF and ADF

were accepted within 3% absolute error. Because the entire juvenile fecal sample was used for

dry and organic matter determinations, no duplicates were run. Orts from juveniles were also

not analyzed in duplicate because of insufficient sample quantity.

To determine if juvenile turtles experienced an advantage of small bite size, I examined subsamples of feces from juvenile and adult turtles with a dissecting scope. The number of duckweed fronds that passed through the gut intact was counted. If juveniles experienced advantage of small bite size, they will have significantly lower percentage of intact duckweed fronds in feces.

Digestive Processing Calculations

Intake of dry and organic matter was calculated as the difference between the quantities of food offered and orts remaining each day multiplied by the fraction of dry matter and organic matter in the diet. Because nutrient composition of the orts was similar to that of the diet, no adjustments were necessary to account for selective feeding. Digestibility was determined using the equation:

(intake - feces)/ intake where intake is total g of dry matter or organic matter consumed during the trial, and feces is g dry matter or organic matter in the feces produced during the trial. Note that this equation calculates apparent digestibility because it does not correct for the introduction of nutrients into digesta from endogenous sources of the turtle or from the microbial symbionts. Daily digestible intake was calculated by multiplying intake and digestibility. For adults eating shrimp, digestible dry matter intakes were less than digestible organic matter intakes, suggesting that either the quantity of ash in the diet was underestimated or the quantity in the feces was overestimated. Because of this discrepancy, only organic matter digestibilities and digestible intakes were presented (dry matter values are shown in Appendix). Because of unequal variances, differences in all digestive parameters between treatments were evaluated with Kruskal-Wallis tests and post-hoc analyses according to Conover (1980)

Transit time of the diet was time elapsed from when a 3 mm round piece of plastic flagging was fed to turtles to when it appeared in feces. This flagging approximated the size of duckweed fronds, which were oblong and ranged 2-4 mm in length and 1-1.5 mm in width.

Differences in transit time between treatments were also evaluated with Kruskal-Wallis tests and post-hoc analyses according to Conover (1980).

Using the Statistical Package for Social Sciences (SPSS), I ran linear regressions on log- transformed data to determine the allometric slope between dry matter intake of shrimp and duckweed and turtle body mass. Ninety-five percent confidence intervals around these estimates were also obtained.

Juvenile Growth and Composition

Juvenile turtles were weighed once a week during the five-week trial to determine growth rate. At the conclusion of five weeks, turtles were euthanized with sodium pentobarbital. The digestive tracts were dissected out and gut contents removed. Digestive tracts were recombined with carcasses and then dried to constant mass at 60 °C. To determine nutrient composition of turtle tissue, I broke up dried juveniles with a mortar and pestle and then ground them in a Wiley mill to pass through a 1 mm screen. Juveniles were analyzed for dry matter, organic matter, nitrogen, lipid, energy, and calcium content. Methodologies were the same as for diet samples, except a Gentry-Wiegert Phillipson microbomb calorimeter

(Gentry Instruments) controlled by a data logger (21X, Cambell Scientific) was used for

energy analysis. Percent calcium of the tissue was determined by solubilizing samples in a

hydrochloric acid solution and analyzing filtrate with a Perkin-Elmer Model 5000 Atomic

Absorption Spectrophotometer (Hesse 1972). Lipid content was determined by extraction for

eight hours in a Soxhlet extractor, with diethyl ether and petroleum ether as the solvent.

Dry and organic matter, energy, and nitrogen were analyzed in duplicate. Dry matter,

organic matter, and energy duplicates were accepted within 2% relative error, and nitrogen

within 1.5% absolute error. Duplicates were not run for percent lipid and calcium because of

insufficient sample. Differences in nutrient contents between juveniles were compared using t-

tests. All percentage data were arcsin transformed before analysis.

Results

Mass-specific intake varied significantly between treatments for dry matter (p = 0.002),

organic matter (p = 0.002), energy (p < 0.001), and nitrogen (p < 0.002) (Table 2-2). Juveniles

fed shrimp consumed 265% more mass-specific dry matter than those fed duckweed, whereas

adults fed shrimp consumed 60% more those fed duckweed. Organic matter digestibility also

varied significantly between diet (p < 0.001). In juveniles, shrimp digestibility (97.2%) was

48% higher than duckweed digestibility, whereas in adults, it (89.4%) was 30% higher than

duckweed digestibility. Mass-specific digestible organic matter intake also varied significantly

with diet (p < 0.001), with turtles fed shrimp consuming greater quantities of mass-specific

digestible organic matter than those fed duckweed (433% more for juveniles, 53% more for

adults).

On the shrimp diet, juveniles consumed nearly 200% more mass-specific dry matter,

organic matter, energy, and nitrogen than did adults (Table 2-2). Dry matter intake of shrimp

scaled to body mass with an allometric slope of 0.815 (F1, 10 = 119.21, p < 0.001) with an upper

95% confidence interval of 0.649 and a lower 95% confidence interval of 0.981. Overall, juvenile shrimp digestibility was 9% higher than that of adults. Although mass-specific digestible organic matter intake was not significantly different, it tended it be higher in juveniles (71%), and this trend approached significance (Tcritical of Conover (1980) post hoc test

= 5.0, Tcalculated = 4.7).

On the duckweed diet, mass-specific intakes of dry and organic matter as well as energy

were not significantly different between juveniles and adults (Table 2-2). Dry matter intake

scaled to body mass with an allometric slope of 0.948 (F1, 12 = 836.54, p < 0.001) with an upper

95% confidence interval of 0.877 and a lower 95% confidence interval of 1.018. Both age classes achieved equivalent digestibilities and consumed similar quantities of digestible

organic matter on this diet. Additionally, juveniles had significantly fewer intact duckweed

fronds in their feces (median = 38% range = 33 – 47%) than did adults (median = 49%, range =

37 – 61%) (one tailed Mann-Whitney U test, U = 7.00, p = 0.039).

There were no significant differences in transit time between treatments (p = 0.132,

Table 2-2). However, in adults, median duckweed transit time was over twice as long as that

of shrimp (7 days vs. 3 days). Additionally, duckweed tended to have a longer transit time in

adults than in juveniles. Transit times per cm of digestive tract were significantly slower in

juveniles than in adults, but did not vary with diet.

Juveniles fed shrimp grew 216% faster than did juveniles fed duckweed (p = 0.015) and were 10% lower in body water (p < 0.001), 9% lower in nitrogen (p < 0.001), and 31% lower in sodium (p = 0.002) (Table 2-3). There were no differences in any other body tissue parameters measured (p > 0.1) (Table 2-3).

Discussion

Juvenile Growth and Composition

Ultimately, the value of a diet to juvenile turtles is best reflected in the growth and condition of individuals feeding on that diet. If juvenile T. scripta are carnivorous because they are not able to subsist on a plant diet, then juveniles fed plants may not grow or their body tissues may be significantly lower in nutrients than those of juveniles fed an animal diet.

Juveniles fed shrimp grew 3.2 times faster than did juveniles fed duckweed. Such dramatic variation in growth probably resulted from differences in energy and nitrogen gains from each diet. Juveniles fed shrimp consumed 4.2 times more energy and 9.1 times more nitrogen than did juveniles fed duckweed. Although diet composition does not equal availability, juveniles fed shrimp probably assimilated substantially more energy and nitrogen than did those fed duckweed. Although juveniles fed duckweed did increase in mass over the course of the trial,

18 their tissues contained 11% more water than did tissues of turtles fed shrimp. Therefore, the mass gained may indicate more a gain in water than in new tissue. Juveniles fed duckweed gained an average of 994.3 mg wet mass during the trial, whereas those fed shrimp gained

3,142.9 mg. Assuming the mass gained during the trial had the same water content as the tissue at the end of the trial, juveniles fed duckweed gained only 161.1 mg of dry matter compared to 770.0 mg for those fed shrimp.

Given differences in intake and composition between diets, the nutrient composition of turtle tissue varied surprisingly little. Clark & Gibbons (1969) hypothesized that juvenile turtles may be carnivorous to obtain sufficient calcium for shell hardening after hatching.

However, no difference was found in percent calcium of juvenile tissue between treatments.

Turtles fed duckweed were 10% higher in percent than those fed shrimp. This result is surprising and difficult to explain, given that on the duckweed diet, juvenile nitrogen intake was 89% less than on the shrimp diet.

Digestive Processing

Comparisons of juvenile and adult digestive processing of duckweed and shrimp provide additional insight into why juvenile T. scripta are carnivorous. Juvenile digestive performance was exceptional on the shrimp diet. Not only did juveniles digest shrimp to a greater extent that did adults, they had higher mass-specific intakes as would be predicted based on metabolic demands. Although, juvenile mass-specific digestible organic matter intake was not significantly higher that that of adults, there was a strong trend. The lack of statistical significance probably stemmed for low statistical power due to small sample size.

In carnivorous reptiles, dry-matter intake scales to body mass with an allometric slope of

0.963 (Nagy 2001). Dry-matter intake of shrimp in T. scripta scaled to body mass with an allometric slope of 0.815. Ninety-five percent confidence intervals around the T. scripta

estimate included the allometric slope for carnivorous reptiles as well as the allometric slope

for the relationship between chelonian metabolic rate and body mass (0.86; Bennett & Dawson

1976). The fact that T. scripta dry matter intake scaled with body mass similarly to these other

scaling relationships suggests that juvenile intake of shrimp was not limited and that juveniles

could probably meet their metabolic demands on this diet.

Juveniles shrimp digestibility (97%) was remarkably high significantly greater than that

of adults. Similar results were found with spiny-tailed iguanas, Ctenosaura pectinata, a

species that also shifts diet ontogenetically from carnivory to herbivory (Durtsche 1999).

Although digestibilities were not as high as were those in juvenile T. scripta (organic matter

digestibility: 82.9% vs. 97.2%), juvenile C. pectinata assimilated 20-25% more energy and

nutrients from insect larvae than did adults. Such differences in the abilities of juveniles and

adults to digest animal material may be attributed to ontogenetic shifts in enzyme production or

in the densities and types of nutrient transporters (Buddington 1992).

Juvenile digestive processing of duckweed differed dramatically from that of shrimp.

Although juveniles were able to digest duckweed to the same extent as adults, they consumed equivalent quantities of mass-specific digestible organic matter as adults. Dry matter intake of duckweed scaled to body mass with an allometric slope of 0.948. The ninety five percent confidence intervals around this estimate do not include the allometric slope for the relationship between dry matter intake and body mass in herbivorous reptiles (0.717; Nagy

2001) nor the allometric slope for the relationship between chelonian metabolic rate and body mass. Assuming that adult turtle intake was not limited, these results suggest that juvenile intake was constrained on the duckweed diet and that juveniles probably had difficulty meeting metabolic demands.

Juvenile and adult duckweed intake patterns contrast with those of a previous study in which juvenile T. scripta fed a gelatin-based diet had higher mass-specific intake than did adults (McCauley & Bjorndal 1999). The results of these studies may differ because microbial fermentation was probably not required to digest the gelatin-based diet, whereas it may have used been to digest the duckweed. Although fiber digestion was not measured in this study, juvenile T. scripta fed duckweed in another study had short-chain fatty acids concentrations in

their large intestines indicative of active fermentation (Chapter 4). If juveniles rely on this

fermentation to meet a significant percentage of their energy requirements, then juvenile intake

of plant material may have been limited by a minimum gut residence time required for

adequate fermentation of the diet.

Juvenile and adult intake patterns also contrast with those of other reptiles, including the green iguana, Iguana iguana (Troyer 1984b), desert iguana, Dipsosaurus dorsalis (Mautz &

Nagy 1987), marine iguana, Amblyrhynchus cristatus (Wikelski et al. 1993), and red-bellied

turtle, Pseudemys nelsoni (Bjorndal & Bolten 1992). Unlike T. scripta, juveniles of these

species can maintain higher mass-specific intakes compared with adults. The difference

between T. scripta and these species may be related to differences in their gastrointestinal

tracts. Iguanas possess either spiral valves or transverse folds in the large intestine, which slow

the passage of digesta and increase the surface area for absorption (Iverson 1980). Red-bellied

turtles lack such valves, but the fermentation chamber in this turtle has expanded to include the

small as well as large intestine (Bjorndal & Bolten 1990). The fact that T. scripta

gastrointestinal tracts do not have these or any other obvious modifications for herbivory

(Chapter 4) may explain why juvenile T. scripta are less efficient herbivores than juveniles of

these other species.

Conclusions

Digestive physiology plays an important role in the ontogenetic diet shift of T. scripta.

Juveniles, which consume a carnivorous diet in the wild, were extremely efficient on the shrimp diet with high mass-specific intakes relative to adults and remarkably high digestibilities. Juveniles did not fare nearly as well on the duckweed diet, despite that fact that duckweed is a preferred food item of adult T. scripta (Parmenter & Avery 1990). Although juveniles were able to digest duckweed as well as adults, the allometric slope for the relationship between dry matter intake and body mass was significantly greater than was the slope for herbivorous reptiles. Therefore, juveniles fed duckweed did not have higher mass- specific intakes relative to adults as would be expected based on metabolic demands. Gut capacity and the time required for fermentation may critically limit juvenile intake of this diet.

Juveniles on the duckweed diet grew significantly slower than did those on the shrimp diet. Such differences in juvenile growth have important implications for T. scripta survival and reproduction. Because juvenile turtles have higher mortality than adults (Frazer et al.

1990; Bodie & Semlitsch 2000), rapid growth allows juveniles to pass through a vulnerable life stage more quickly. Additionally, male T. scripta mature upon reaching a certain size, whereas females tend to mature at a certain age regardless of size (Gibbons et al. 1981). Faster juvenile growth therefore decreases age at maturity for males and increases size at maturity for females.

Early maturation is advantageous for males because it allows males to obtain more matings over the course of their life span. Large size at maturity is advantageous for females because growth slows significantly at maturity and both reproductive output and survivorship of nesting

females are positively correlated with size (Congdon & Gibbons 1983; Tucker et al.1999).

Overall, this study provides a physiological explanation for the carnivorous diet of juvenile T. scripta. The question remains, however, why turtles switch to an herbivorous diet as they mature, despite the fact that they can easily process animal material. Possible explanations may relate to costs associated with the pursuit and capture of animal prey by adults. Because they are larger, adults have greater absolute metabolic demands and greater locomotory costs than do juveniles. Consequently, even without the digestive differences measured in this study, the net gain from a given animal prey item is less for adults than for juveniles (Parmenter & Avery 1990). Additionally, larger adults may have more difficulty foraging for animal prey than do juveniles because of maneuverability constraints in the littoral zone where animal prey is presumably most abundant (Hart 1983). Our understanding of the ontogenetic diet shift of T. scripta will not be complete until studies exploring these potential costs associated with prey acquisition by adults are done.

Table 2-1. Nutrient composition of duckweed and shrimp diets fed to juvenile and adult turtles as well as nutrient composition of orts (remaining food) for each treatment. Ort values are means ± standard errors for turtles in each treatment. All values except energy are presented on a percent dry matter basis. Note that shrimp values are for shrimp with anterior and posterior portions removed.

Duckweed Shrimp

Juvenile Trial Adult Trial Juvenile Trial Adult Trial Diet Organic Matter (%) 86.4 85.5 88.0 87.1

Fiber (%)* NDF 41.2 45.2 ADF 19.7 21.4 6.4 4.8

Nitrogen (%) 5.0 4.1 12.6 12.6

Energy (kJ g-1 DM) 18.49 17.35 21.75 20.91

Calcium (%) 0.8 -- 2.2 --

Orts Nitrogen (%) 4.8 ± 0.1 4.3 ± 0.4 12.6 ± 0.2 12.0 ± 0.0

Energy (kJ·g-1 DM) 17.9 ± 0.04 18.1 ± 3.2 21.6 ± 0.2 20.9 ± 0.1

*Neutral detergent fiber (NDF) represents cellulose, hemicellulose, lignin and cutin, whereas acid detergent fiber (ADF) represents cellulose, lignin, and cutin of duckweed. ADF represents the chitin component of shrimp.

Table 2-2. Digestive processing of duckweed and shrimp diets fed to juvenile and adult turtles. Comparisons between groups were made with Kruskal-Wallis tests and post hoc tests according to Conover (1980). Values are medians (ranges), and different superscripts across rows indicate significant differences between treatments.

Duckweed Diet Shrimp Diet Juveniles Adults Juveniles Adults (n = 7) (n = 7) (n = 7) (n = 5) H p Intake (mg⋅g turtle-1⋅day-1) Dry matter 2.6a,c (1.6 – 2.9) 2.0a (1.3 – 3.2) 9.5b (3.1 – 16.9) 3.2c (2.0 – 4.7) 14.676 0.002 Organic matter 2.3a,c (1.4 – 2.5) 1.8a (1.1 – 2.8) 8.3b (2.7 – 14.9) 2.8c (1.8 – 4.1) 15.338 0.002 a a b c Energy*(kJ⋅g turtle-1⋅day-1) 48.4 (29.3 – 53.1) 34.3 (23.4 – 54.0) 206.1 (66.7 – 367.7) 66.0 (42.6 – 97.7) 18.359 < 0.001 Nitrogen 0.13a (0.08 – 0.14) 0.07b (0.06 – 0.11) 1.19c (0.39 – 2.13) 0.40 d (0.26 – 0.59) 21.570 < 0.001

Organic matter digestibility (%) 65.7a (61.3 – 71.0) 68.6a (63.2 – 77.6) 97.2b (96.1 – 99.0) 89.4c (84.9 – 93.4) 21.000 < 0.001

Organic matter digestible intake (mg⋅g turtle-1⋅day-1) 1.5a (0.8 – 1.7) 1.3a (0.8 – 1.8) 8.0b (2.7 – 14.4) 2.3b (1.6 – 3.6) 18.406 < 0.001 24

Transit time Total hours 90.0a (64.0 – 97.5) 170.5a (94.8 – 199.5) 95.5a (77.5 – 120.0) 72.5a (37.5 – 143.8) 5.609 0.132 Hours⋅cm GI tract-1 4.6a (1.1 – 5.1) 1.5b (0.7 – 2.0) 7.1a (3.9 – 8.2) 0.8b (0.3 – 1.4) 10.046 0.018 Sample size for transit time (n = 5) (n = 5) (n = 3) (n = 5)

Table 2-3. Growth and nutrient composition of juvenile turtles fed duckweed or shrimp. Note that shrimp values are for shrimp with anterior and posterior portions removed. Values are means ± standard errors, and bold values indicate significant differences between treatments.

Diet

Duckweed Shrimp t p

Growth rate (mg⋅week-1) 195.0 ± 54.6 616.2 ± 126.6 3.056 0.015

Composition Body water (%) 83.8 ± 0.3 75.5 ± 0.7 10.987 < 0.001 * Organic matter (%) 84.6 ± 0.9 84.1 ± 0.4 0.685 0.507 * Nitrogen (%) 12.5 ± 0.1 11.4 ± 0.1 6.220 < 0.001 Lipid free basis 2.289 0.043 * 17.4 ± 0.4 16.4 ± 0.3 Lipid (%) 1.401 0.189 -1 * 28.2 ± 1.5 30.6 ± 0.9 Energy (kJ⋅g ) 21.23 ± 0.39 21.70 ± 0.22 1.087 0.300

* Minerals (%) 15.4 ± 0.9 15.9 ± 0.4 0.203 0.843 Calcium (%)* 0.885 0.410 * 3.26 ± 0.27 3.67 ± 0.37 Sodium (%) 1.08 ± 0.03 0.74 ± 0.07 4.158 0.002 * 1.016 0.331 Potassium (%) 0.63 ± 0.14 0.49 ± 0.06 Magnesium(%)* 1.473 0.169 0.11 ± 0.01 0.09 ± 0.01

*Dry matter basis

CHAPTER 3 ASSOCIATIVE EFFECTS BETWEEN ANIMAL AND PLANT DIET ITEMS IN THE YELLOW-BELLIED SLIDER TURTLE, TRACHEMYS SCRIPTA

Introduction

Dietary mixing is widespread among animal, commonly occurring in many

vertebrate and invertebrate species (Robbins 1993; Steven & Hume 1995; Coll &

Guershon 2002). Associative effects between diet items may play an important role in

the selection of mixed diets, particularly for diet items that differ radically from each

other in nutritional composition or in how they are processed in the digestive tract

(Bjorndal 1991; Robbins 1993; Bozinovic & Martínez del Rio 1996). These effects occur

when diet items interact with one another such that the net energy and nutrient gain from

the mixed diet differs from the net gain predicted by summing the gains from individual diet components. Although many studies have acknowledged the potential importance of associative effects in their study species (Meienberger et al. 1993; Pennings et al. 1993;

Barboza 1995; Campbell & MacArthur 1996; Nagy et al. 1998; Spencer et al. 1998;

Chen & Lue 1999; Durtsche 2000; Sales & Britz 2002), few studies have tested for and

quantified these effects (Table 3-1).

The concept of associative effects was first demonstrated in studies of domestic

livestock nutrition. Like many herbivorous wildlife species, livestock, such as cattle, use

microbial gut symbionts to digest plant material. These symbionts ferment plant cell wall

components and produce waste products in the form of short-chain fatty acids (SCFA),

which the host absorbs and uses as an energy source. Associative effects found in

26 27 livestock often result from alterations in microbial fermentation. For example, adding grain to a forage diet depresses digestibility because gut symbionts preferentially attack the easily fermentable grain carbohydrates rather than the structural carbohydrates of the forage. This rapid fermentation produces high concentrations of SCFAs that lower pH of the fermentation chamber and create an unfavorable environment for symbionts

(Schneider & Flatt 1975). However, if urea and a small quantity of easily fermented carbohydrate are added to forage, digestibility increases. This increase is due to extra nitrogen from the urea and readily available energy from the carbohydrate, stimulating growth of the microbial population, which can then ferment the forage more efficiently

(Pond et al. 1995).

Associative effects have been demonstrated in a diverse array of wild species including insects, turtles, birds, ungulates, and rodents (see Table 3-1 for summary and references). In some cases, the possible mechanisms underlying these effects mirror those found in domestic livestock. For example, Bjorndal (1991) found a positive associative effect in yellow-bellied slider turtles, Trachemys scripta, fed a diet comprised of 77% duckweed, Spirodela polyrhiza, and 23% Tenebrio larvae (dry matter basis).

Adult yellow-bellied slider turtles are opportunistic omnivores that feed primarily on aquatic plants (Parmenter & Avery 1990), and the ratio of plant to animal material in that study approximated that consumed by a wild population of adult T. scripta (Bjorndal

1991). Bjorndal (1991) hypothesized that the positive associative effect between duckweed and insect larvae was due to nitrogen in the larvae stimulating growth of the microbial symbiont population. She proposed this hypothesis because T. scripta use microbial gut symbionts to digest plant material (Chapter 4; Bjorndal & Bolten 1993) and

because the cell wall, or fiber, component of the diet was most affected by the associative

effect.

The inclusion of animal material in a plant diet, however, does not consistently

produce a positive associative effect. For example, an omnivorous tortoise, Kinixys

spekii, experienced a negative associative effect when fed a diet comprised of 74.2%

kale, Brassica oleracea, and 25.8% millipedes, Alloporus sp. (dry matter basis) (Hailey et al. 1998). This negative effect was attributed to kale, with its relatively short gut transit

time, decreasing millipede transit time, thus depressing digestibility. Studies of K. spekii

and T. scripta (Bjorndal 1991) demonstrate that plant and animal diet items do not always

interact in predictable ways. Because associative effects can significantly alter diet value,

better knowledge of these effects is required to understand more completely the

nutritional ecology of animals consuming mixed diets.

The purpose of this study was to quantify associative effects in T. scripta, using previously untested diet items, duckweed, Lemna valdiviana, and freshwater grass shrimp, Palaemontes paludosus. I performed a series of feeding trials in which I fed adult turtles 100% duckweed, 100% shrimp, and two mixed diets containing 67% duckweed, 33% shrimp and 86% duckweed, 14% shrimp (dry matter basis). During the feeding trials, I measured intake, digestibility, and transit time of the diets. At the conclusion of each trial, I measured SCFA concentrations in the digestive tracts of turtles on each diet. The results of these feeding trials allowed me to (1) determine if associative effects exist between duckweed and shrimp and (2) begin to understand possible mechanisms underlying these effects. By examining two mixed diets, I was able to assess if any existing associative effect varied with the ratio of plant to animal material in

29 the diet. This question is important because the relative proportions of plant and animal material in the natural diets of T. scripta vary widely depending on food availability (Hart

1983; Parmenter 1980; Parmenter & Avery 1990). Studies of domestic livestock nutrition have demonstrated that the magnitude of an associative effect can vary with different ratios of diet components (Van Soest 1994). Additionally, it is theoretically possible that the direction of the effect varies with different ratios, although it has yet to be demonstrated.

By measuring digestibility of individual diet components (organic matter, fiber, lipid, energy, and nitrogen), transit time, and concentrations of SCFA insight can be gained in possible mechanisms underlying associative effects. For example, if fiber digestibility is most altered by the effect, alterations in microbial fermentation may be involved. This could result from differential transit times of the diet components (Baker

& Hobbs 1987; Hailey et al. 1998) or preferential fermentation of one component over the other (Schneider & Flatt 1975). Preferential fermentation would be revealed by examining relative concentrations of individual SCFAs (acetate, propionate, and butyrate) produced on each diet. Different ratios can indicate different substrates for fermentation.

In addition to assessing associative effects in adult T. scripta, I also determined if an associative effect exists in juveniles fed a mixed diet containing 81% duckweed, 19% shrimp. I looked for an associative effect in terms of diet digestibility, digestible organic matter intake, and growth rate. I also compared nutrient composition (water, organic matter, nitrogen, lipid, energy, and calcium content) of juvenile turtles maintained on the mixed diet with that of juveniles maintained on a pure diet (Chapter 2). These turtles

30 experience an ontogenetic diet shift from carnivory to herbivory (Parmenter & Avery

1990), and depending on the population, this shift can occur gradually (Hart 1983) or suddenly (Clark & Gibbons 1969). For those populations where the shift occurs gradually, juvenile turtles consume a broad range of plant to animal ratios as they mature.

Associative effects could play an important role in the nutrition of juveniles during that time.

Methods

Experimental Animals and Diets

Two feeding trials with adults and one with juveniles were conducted to compare how yellow-bellied slider turtles process plant, animal, and mixed diets. In the first trial with adults, turtles were fed pure diets of either duckweed, Lemna valdiviana (n = 7), or a freshwater grass shrimp, Palaemontes paludosus (n = 8). In the second trial with adults, turtles were fed a mixed diet by dry mass of either 67% duckweed, 33% shrimp (n = 4) or

14% duckweed, 86% shrimp (n = 3). In the trial with juveniles, turtles were fed a mixed diet by dry mass of 81% duckweed, 19% shrimp (n = 8). These ratios of plant to animal material are within ranges measured for natural populations of T. scripta (Parmenter

1980; Parmenter & Avery 1990). During the first trial with adults and the trial with juveniles, duckweed was collected from a local pond in Gainesville, Florida. Because this pond dried up before the onset of the second trial with adults, duckweed for that trial was purchased from an aquarium store. Grass shrimp for all trials were purchased from a bait shop that obtained the shrimp from Gainesville area lakes. Because some turtles did not eat the anterior most portion of the shrimp containing the eyes and antennae or the posterior portion containing the caudal fin, these parts were removed before shrimp were

31 fed to turtles. This ensured all animals consumed the same diet. The nutrient composition of all diets is described in Tables 3-2 and 3-3.

Adult turtles were collected from ponds located at Savannah River Ecology

Laboratory and Audubon Society's Silver Bluff Sanctuary in Aiken County, South

Carolina. Turtles for the first adult trial were collected in May 2001, and those for the second in September 2001. Juvenile turtles were obtained from a commercial turtle farm in Pt. Mayaka, Florida, in mid June 2000. These turtles were the offspring of adults collected from northern Florida, Georgia, and South Carolina. The juvenile trial was conducted from 29 August to 2 October 2000, the first adult trial from 30 May to 2 July

2001, and the second adult trial from 4 January to 8 February 2002. Before the trials, turtles were maintained on a mixture of aquatic plants (primarily duckweeds) and invertebrates collected from a local pond. At the onset of the trial, turtles were abruptly switched to the experimental diet; no turtle demonstrated any obvious difficulty with this switch. All trials lasted five weeks and consisted of a two-week acclimation period followed by a three-week experimental period during which daily food intake and feces production were quantified. Mean turtle mass at the beginning the juvenile trial was

11.24 g (range: 8.94 – 14.05 g); at the beginning of the first and second adult trials, it was

995.4 g (range: 375.2 – 1451.1 g) and 1340.2 g (range: 812.2 – 1810.9 g), respectively.

Originally ten adult turtles were divided between duckweed and shrimp diets to evenly distribute size in both treatment. Two remaining turtles, which were smaller, were added to the duckweed diet to increase sample size for other studies involving these turtles

(Chapters 4 & 5). Data were analyzed with and without these additional individuals and because results were the same, they were included in this study.

Experimental Protocol

Adult turtles were housed individually in square Nalgene tanks (45 x 60 cm) equipped with a 75-W floodlight and a 20-W full spectrum natural light fluorescent bulb.

Juveniles were housed individually in square Rubbermaid containers (18 x 18 cm) placed within the larger Nalgene tanks (45 x 60 cm). During the trials, turtles experienced a 12 hour photoperiod and temperatures between 25-26 °C. To determine digestibility, I collected and quantified all feces produced during the experimental periods. Turtles were therefore fitted with fecal collection devices after Avery et al.

(1993) and Bjorndal (1991) (see Chapter 2 for a detailed description).

During the trials, water was drained from tanks every morning at 0800h so all turtles could bask for the same amount of time each day and differential thermoregulation among turtles could be controlled. At 1000h, feces were collected, and tanks were refilled with water. At 1100h, turtles were fed a known mass of either duckweed or shrimp, with turtles on the mixed diet receiving only the duckweed fraction of their diet.

Turtles fed ad libitum for six hours until 1700h when orts (remaining food) were collected and weighed. Turtles on the mixed diets were then fed a quantity of shrimp that resulted in the appropriate ratio of duckweed to shrimp depending on the amount of duckweed consumed that day. This ensured they consumed a constant ratio of plant to animal matter despite daily fluctuations in duckweed intake. Turtles on the mixed diet immediately consumed all shrimp offered to them at the end of the day. Analysis of feces and digestive tract contents indicated that shrimp and duckweed components of the diet were well mixed after consumption. During the adult trials, feces were collected again at 1700h hours.

During the juvenile trial, turtles were weighed once a week to determine growth rate. At the conclusion of five weeks, turtles were euthanized with sodium pentobarbital.

The digestive tracts were dissected out and gut contents were removed. Carcasses were dried to constant mass at 60 °C with their respective digestive tracts and then analyzed for nutrient composition. Gut contents were removed so that nutrient composition of digesta did not influence nutrient composition of turtle carcasses.

Nutrient Analyses

During the experimental periods, duckweed and shrimp diet samples were collected daily in addition to feces and orts from each turtle. All samples were dried overnight at

60 °C. Daily diet samples, as well as daily ort and fecal samples from each turtle, were combined to obtain a composite sample of each. All samples were ground to pass through a 1 mm screen in either a Wiley Mill or coffee grinder (Mr. Coffee, Model IDS

57). All diet samples and adult fecal samples were analyzed for dry matter, organic matter, neutral detergent fiber (NDF), acid detergent fiber (ADF), nitrogen, lipid, and energy content. Adult duckweed and shrimp orts were also analyzed for these dietary components to test if turtles fed selectively. Because juveniles produced such small quantities of feces, juvenile feces were analyzed only for dry and organic matter.

Dry matter content was determined by drying subsamples overnight at 105 °C.

Organic matter content was then determined by combustion of subsamples at 500 °C for three hours. The difference between these two measures represents the ash, or mineral, component of the sample. NDF and ADF were determined by sequentially refluxing subsamples in neutral detergent and acid detergent solutions (Goering & Van Soest 1970) in an Ankom200 Fiber Analyzer according to the guidelines supplied with the equipment

(Ankom Technology 1998, 1999). NDF represents the cell wall component of duckweed

(cellulose, hemicellulose, lignin and cutin), and ADF represents the ligno-cellulose and

cutin component. The ADF component of shrimp represents the exoskeleton (primarily

chitin) fraction of the diet (Stelmock et al. 1985). Lipid content was determined with a

Soxhlet extractor, using diethyl ether and petroleum ether as the solvent. Nitrogen content of the samples was determined using a Carlo Erba elemental analyzer. Energy content was determined with a Parr bomb calorimeter (Parr Instrument 1960).

All diet, ort, and adult fecal samples were analyzed in duplicate. Dry and organic matter and energy duplicates were accepted within 2% relative error. Nitrogen duplicates were accepted within 1% absolute error, and duplicates for lipid, NDF and ADF were accepted within 3% absolute error. Juvenile feces were not analyzed in duplicate because of insufficient sample quantities.

Juvenile tissues were analyzed for dry matter, organic matter, nitrogen, lipid, energy, and mineral content. Initially, dried juvenile carcasses were broken up with a mortar and pestle and then ground in a Wiley mill to pass through a 1mm screen.

Methodologies for nutrient analyses were then the same as for diet samples except a

Gentry-Wiegert Phillipson microbomb calorimeter (Gentry Instruments) controlled by a data logger (21X, Cambell Scientific) was used for energy analysis. Mineral content of the tissue was determined by solubilizing samples in a hydrochloric acid solution and analyzing filtrate with a Perkin-Elmer Model 5000 Atomic Absorption

Spectrophotometer (Hesse 1972). Dry and organic matter, energy, and nitrogen were analyzed in duplicate. Dry and organic matter and energy duplicates were accepted

within 2% relative error, and nitrogen within 1.5% absolute error. Duplicates were not run for percent lipid and calcium because of insufficient sample.

Digestive Processing Calculations

Dry and organic matter intakes were calculated as the difference between the amount of food offered and orts remaining each day multiplied by the fraction of dry and organic matter in the diet. Because nutrient composition of the orts was similar to that of the diet, no adjustments were necessary to account for selective feeding. Digestibility of dry and organic matter, NDF, ADF, lipid, energy and nitrogen was determined using the following equation:

digestibility = (intake - feces)/ intake where intake was total grams of the dietary component consumed during the experimental period, and feces was total grams of that component in the feces produced.

Digestible intakes (dry and organic matter) and daily gains (energy and nitrogen) were calculated by multiplying daily intake of the component by its digestibility. For adults eating shrimp, digestible dry matter intakes were less than digestible organic matter intakes, suggesting that either the quantity of ash in the diet was underestimated or the quantity in the feces was overestimated. Because of this discrepancy, only organic matter digestibilities and digestible intakes were presented for adults (dry matter values are shown in the Appendix). Transit time of the diet was time elapsed from when a 3 mm round piece of plastic flagging was fed to turtles to when it appeared in feces. This flagging approximated the size of duckweed fronds which were oblong and ranged 2-4 mm in length and 1.0-1.5 mm in width. Because of unequal variances, differences in digestive parameters between treatments were evaluated with Kruskal-Wallis tests with post hoc analyses according to Conover (1980).

To test for associative effects between diet items, I compared digestibilities, digestible intakes, and daily gains (adults only) of turtles fed mixed diets with predicted values based on results from turtles eating 100% duckweed and 100% shrimp diets.

Juvenile data were compared with data from juveniles fed pure diets in a previous study

(Chapter 2). Predicted digestibilities for each component were calculated with the equation used by Bjorndal (1991):

VP = (VD x FD) + (VS x FS)

VP = predicted digestibility

VD = actual digestibility of component 100% duckweed diet

FD = fraction of that component contributed by duckweed to the mixed diet

VS = actual digestibility of component in 100% shrimp diet

FS = fraction of that component contributed by shrimp to the mixed diet

Predicted digestible organic matter intakes were calculated by multiplying Vp for organic matter by mass-specific intake of the mixed diet and organic matter content of the mixed diet (Bjorndal 1991). Predicted energy and nitrogen daily gains were calculated the same way using the appropriate values for energy and nitrogen.

For each digestibility VD and VS were random numbers generated from distributions with means and standard deviations as determined from turtles fed pure duckweed and shrimp diets. Random numbers from those distributions were used rather than means to maintain variance in predicted values. Reduced variance in predicted values could increase the likelihood of finding a significant difference between actual and predicted values.

To compare actual and predicted values, I used the following procedure in the

statistical and programming language R (Ihaka and Gentleman 1996). For each

digestibility random numbers (n = 4 for adult diet with 67% duckweed; n = 3 for adult

diet with 14% duckweed; n = 8 for juvenile diet with 81% duckweed) were generated

from the corresponding duckweed and shrimp distributions. Using the preceding

equation, random numbers were used to calculate a predicted digestibility for each turtle in the treatment. These predicted digestibilities were then used to calculate the corresponding predicted digestible intake or daily gain. Differences between predicted and observed values for each turtle were calculated, and the mean difference for all turtles in the treatment was determined. This entire procedure was repeated 1000 times with new random numbers generated each time. The mean difference for each iteration was then plotted in a histogram, and 95% confidence intervals were determined. Actual values were considered different from predicted values if confidence intervals did not overlap with zero.

Short-Chain Fatty Acid Concentrations

At the conclusion of adult trials, turtles were euthanized with sodium pentobarbital.

Three additional adults (one fed duckweed and two fed shrimp) were also included in this portion of the study. Although intake and digestibility were not measured for these animals, they were maintained under feeding trial conditions for five weeks before being euthanized.

Digesta was analyzed for short-chain fatty acid concentrations (SCFA). Whenever sufficient digesta was present, samples were collected from five gut sections (stomach, anterior and posterior small intestine, and anterior and posterior large intestine) and preserved in 20% phosphoric acid. Samples were centrifuged and the SCFA

concentrations of the supernatants were measured using a Shimadzu gas chromatograph

(Model GC-9AM) with a Perkin Elmer Computing Integrator (LCI-100). The remaining

contents of these gut sections were removed, weighed, and dried at 60 °C to determine total content mass for each section. After SCFA concentrations were determined, the phosphoric acid was evaporated from vials to determine dry mass of samples used for

SCFA analyses.

SCFA concentrations were compared between turtles fed duckweed (n = 5) and shrimp (n = 6) using a repeated-measures analysis of variance. This analysis was used to partition inter and intra-individual variation in SCFA concentrations. Measurements from one turtle fed duckweed were not included in the analysis because SCFA concentrations were over four standard deviations lower than the average. There was most likely a problem with the handling of the sample because this turtle was able to digest ADF (cell wall) component of diet as well as other turtles (65.0%). An additional turtle fed duckweed and one fed shrimp were also not included in the analysis because they did not have sufficient digesta in every gut region for SCFA analysis. For those regions where there was sufficient digesta, these turtles had SCFA concentrations within

the range found in other turtles in the same treatment. Turtles fed mixed diets were not

included in this analysis because only two turtles in each treatment had sufficient digesta

in every gut region for SCFA analysis. However, SCFA concentrations for turtles on

mixed diets are presented for comparison.

Results

Feeding Trials with Adult Turtles

Mass-specific intake did not vary significantly among adult turtles fed the four diets (dry matter: p = 0.089; organic matter: p = 0.062; Table 3-5). However,

39 digestibilities of every dietary component did vary significantly with diet (organic matter: p = 0.004; NDF: p = 0.002; ADF: p 0.003; lipid: p = 0.003; energy: p = 0.004; nitrogen: p

= 0.001) as did digestible intakes of organic matter (p = 0.009) and daily gains of energy

(p = 0.004) and nitrogen (p = 0.002). Transit time did not vary significantly among diets

(p = 0.221), although median transit time of duckweed was over twice as long as that of shrimp.

On the 67% duckweed diet, digestibility of all dietary components except nitrogen was significantly less than predicted (organic matter: lower 95% Confidence Interval

(C.I.). = -14.5, upper C.I. = -6.5; NDF: lower C.I. = -22.8, upper C.I. = -10.2; ADF: lower C.I. = -45.4, upper C.I. = -26.9; lipid: lower C.I. = -13.6, upper C.I. = -2.9; energy: lower C.I. = -12.4, upper C.I. = -4.6; Table 3-6). This effect was most dramatic for the

ADF portion of the diet, which was only half as digestible as predicted. Digestible organic matter intakes were 15% less than predicted (lower C.I. = -0.19, upper C.I. = -

0.07) and daily energy gains were 8% less than predicted (lower C.I. = -3.2, upper C.I. =

-0.8). There was no significant differences for daily nitrogen gain lower C.I. = 0.00, upper C.I. = 0.01).

On the 14% duckweed diet, NDF and ADF digestibilities were depressed relative to expected values by 9% and 8%, respectively (NDF: lower C.I. = -10.5, upper C.I = -5.7;

ADF: lower C.I. = -11.8, upper C.I. = -0.8). All other digestibilities were significantly higher than predicted, including organic matter (lower C.I. = 0.5, upper C.I. = 6.4), lipid

(lower C.I. = 4.8, upper C.I. = 13.9), energy (lower C.I. = 0.1, upper C.I. = 6.6), and nitrogen (lower C.I. = 1.1, upper C.I. = 5.2). This effect was most dramatic for the lipid component of the diet, which was 12% higher than predicted. Digestible organic matter

intake and daily energy gain were both 4% higher than expected (digestible organic

matter intake: lower C.I. = 0.02, upper C.I. 0.13; energy daily gain: lower C.I. = 0.20,

upper C.I. = 3.23), whereas there was no difference for nitrogen daily gain (lower C.I. =

0.00, upper C.I = 0.01)

The digestive tracts of turtles on each of the four diets contained short-chain fatty

acids (Figure 3-1). These SCFAs consisted of acetate, propionate, butyrate, isobutryate,

valerate, and isovalerate (Table 3-7). In the anterior large intestine, where SCFAs

peaked, acetate was the primary acid produced followed by propionate and butyrate. For

those turtles fed pure shrimp, acetate concentrations were 19-23% lower and proprionate

and butyrate concentrations approximately 55-60% higher relative to concentrations

measured in turtles fed the other diets.

SCFA concentrations varied significantly between gut regions (Figure 3-1; molar

basis: F4, 36 = 12.64, p < 0.001; dry mass basis: F4, 36 = 11.33, p < 0.001). However,

there was no difference between duckweed and shrimp diets (molar basis: F1, 9 = 2.86, p

= 0.125; dry mass basis: F1, 9 = 0.74, p = 0.411), and there was no interaction between

gut region and diet (molar basis: F4, 36 = 1.08, p = 0.361; dry mass basis: F4, 36 = 1.60, p

= 0.196). The SCFA concentrations in the digestive tracts of turtles fed the mixed diets were comparable to those of turtles fed pure diets (Figure 3-1).

Feeding Trials with Juvenile Turtles

Mass-specific intake, digestibility, and mass-specific digestible intake of the mixed diet were 74%, 28%, and 83% less than that of the pure shrimp diet, respectively (Table

3-8). None of these variables were significantly different from the pure duckweed diet.

(Table 3-9). Digestibility of the mixed diet was 9% less than predicted (dry matter: lower

C.I. = -8.8, upper C.I. = -5.2; organic matter: lower C.I. = -9.2, upper C.I. = -5.5), and

41 digestible intake was 5% less than predicted (dry matter: lower C.I. = -0.12, upper C.I. =

-0.03; organic matter: lower C.I. = -0.23, upper C.I. = -0.13). Additionally, juvenile growth on the mixed diet was half predicted growth (lower C.I. = -227.9, upper C.I. = -

46.5). Juveniles fed the mixed diet were 8% higher in water content, 20% lower in lipid, and 25% lower in energy content than juveniles fed shrimp; they did not differ in nutrient composition from those fed the duckweed diet (Table 3-10).

Discussion

Daily energy and nitrogen gain are the best measures of diet value for adults because these measures integrate both the quantity of food consumed and the ability of the animal to digest it (Bjorndal & Bolten 1993). In terms of energy gain, adult turtles on both mixed diets experienced an associative effect, and the direction of this effect switched at different diets ratios. Turtles fed the 67% duckweed diet experienced a significant negative associative effect, whereas those fed the 14% mixed diet experienced a significant positive effect. To the best of my knowledge, this is the first report of two diet items producing opposite effects when fed in different ratios. In terms of nitrogen gain, adult turtles did not experience an associative effect on either mixed diet.

The negative associative effect experienced by adult turtles on the 67% duckweed diet was most dramatic in terms of ADF digestibility. This component of the diet was primarily derived from duckweed fiber (86%), which is usually fermented by symbionts in the hindgut. These results therefore suggest that the negative associative effect arose from an alteration in microbial fermentation. Concentrations of SCFAs in turtle large intestines indicate that fermentation played at least some role in the digestion of all four diets, including shrimp. Possible substrates for shrimp fermentation included proteins, lipids, and chitin. Chitin, as measured by ADF, was 83% digestible in the pure shrimp

42 diet and was the most likely substrate for fermentation because most proteins and lipids are typically digested and absorbed before digesta reaches the hindgut. This would be especially true in this study because shrimp exoskeletons were opened at the anterior and posterior ends exposing the underlying soft tissue. Chitin fermentation has also been observed in minke whales, Balaenoptera acutorostrata (Olsen et al. 2000), bowhead whales, Balaena mysticetus (Leedle et al. 1995), and Adélie penguins, Pygoscelis adeliae

(Stemmler et al. 1984) feeding on krill and other invertebrates high in chitin.

The negative associative effect on the 67% duckweed diet could have arisen if shrimp was more easily fermented than duckweed, and gut symbionts preferentially fermented shrimp over duckweed. However, duckweed and shrimp fermentation produced individual SCFAs in different proportions, with shrimp fermentation producing less acetate and more propionate and butyrate than duckweed fermentation. The relative proportions of SCFAs produced in the large intestine during fermentation of the mixed diets more closely resembled those of duckweed fermentation (Table 3-7), suggesting shrimp was not preferentially fermented over duckweed.

Associative effects in some animals have been attributed to differences in transit time between diet items (Table 3-1). Such an explanation may apply to the negative effect experienced by turtles in this study. Although there was no statistically significant difference in transit time between duckweed and shrimp, the median transit time of duckweed in adults was more than twice as long as shrimp (7.1 vs. 3.0 days). The lack of statistical significance may stem from the crude method of measuring transit time and low statistical power associated with small sample size. If including shrimp in the duckweed diet stimulated an increased passage rate, digesta would be exposed to gut

symbionts for less time and digestibility could be reduced, particularly in terms of fiber.

The median transit times of the 67% duckweed diet and the juvenile mixed diet suggest

that this may be possible (Tables 3-5 and 3-8). However, before any conclusions can be

drawn, this hypothesis must be explored in more individuals using more accurate

methodologies.

This proposed explanation for the negative associative effect does not explain why turtles fed the mixed diet containing 67% duckweed experienced a positive associative effect in terms of nitrogen digestibility. This result is difficult to understand given that digestibility of all other dietary components was depressed. However, digestibilities

measured in this study were apparent rather than true digestibilities. Therefore, the

elevated nitrogen digestibility could be attributed to a decrease in nitrogen loss from

intestinal sloughing, mucus production, or bacterial protein, rather than a change in

digestibility of the diet. Regardless of the explanation, the most meaningful measure

from the perspective of the animal remains the overall daily nitrogen gain which

demonstrated a negative associative effect.

The negative associative effects measured in this study contrast with the positive

effect previously measured in adult T. scripta fed a diet containing 77% duckweed, S.

polyrhiza, and 23% mealworm larvae, Tenebrio sp. (Bjorndal 1991). These conflicting

results may be related to differences in how turtles processed the different diet items.

Trachemys scripta did process shrimp differently than mealworms (Bjorndal 1991,

mealworm intake = 6.3 mg·g turtle-1·day-1, organic matter digestibility = 88%, energy

gain = 157 kJ·g turtle-1·day-1); however, differences in digestive processing were much more pronounced between the two duckweed species. Although the nutrient composition

of S. polyrhiza (88.6% organic matter, 38.5% NDF, 20.2% ADF, 5.1% nitrogen, 5.1%

lipids and 18.1 kJ·g dry matter-1) was similar to that of L. valdiviana, daily energy gain from S. polyrhiza was 33% less than that from L. valdiviana. Spirodela polyrhiza also had a higher intake (3.0 mg dry matter · g turtle-1 · day-1), more rapid transit time (3.0

days) and lower digestibility, particularly with respect to fiber (NDF digestibility = 25%;

ADF = 9%). Such differences suggest that microbial fermentation played a less

important role in the digestion of S. polyrhiza than in the digestion of L. valdiviana, as

was found in a similar comparison between T. scripta fed S. polyrhiza and another

aquatic plant, Hydrilla verticillata (Bjorndal & Bolten 1993). The positive associative

effect experienced on the S. polyrhiza mixed diet could have resulted from larval

nutrients stimulating microbial population growth to a size that allowed fermentation to

play a more substantive role in duckweed digestion. Such an input of nutrients was not

required for effective fermentation of L. valdiviana.

Several differences in the physical structure and chemical composition of these

duckweeds may explain why T. scripta relied on fermentation to varying degrees for their digestion. First, duckweed fronds are surrounded by a waxy cuticle that acts as a physical barrier to fermentation by gut symbionts (Bjorndal & Bolten 1992); this cuticle is significantly thicker in S. polyrhiza than in L. valdiviana (Elias Landolt, pers. comm.).

Second, S. polyrhiza and L. valdiviana differ in the quantity and composition of lignins and tannins, compounds that are known to influence herbivore digestive processing

(Robbins 1993). Spirodela polyrhiza is 165% higher in lignin content than L. valdiviana and contains two benzaldehydes, vanillin and syringaldehyde, that are not found in L. valdiviana (Blazey & McClure 1968). Additionally, S. polyrhiza contains tannins,

whereas L. valdiviana does not (Elias Landolt, pers. comm.). Differences in these

compounds may influence palatability, transit time, and digestibility of these duckweeds.

Contrary to the negative effect observed on the 67% duckweed diet, turtles fed the

14% duckweed diet experienced a positive associative effect in terms of energy gain.

Although ADF digestibility was depressed, the negative effect was less pronounced than in the 67% duckweed diet. Because ADF comprised a much smaller percentage of the total diet (6%), a reduction in its digestibility did not alter daily energy gain relative to expected values. The digestibilities of all other dietary components were significantly elevated compared to expected values. This effect was most dramatic for lipid digestibility which was 12% higher than expected. Because 89% of the lipids in this mixed diet were derived from the shrimp, one might speculate that digestibility of shrimp lipids was somehow enhanced with the addition of a small quantity of duckweed. One possibility is that the addition of duckweed to the shrimp diet slowed passage rate allowing diet to be digested to a greater extent. The assimilation of lipids requires emulsification with bile followed by micelles formation (Maynard et al. 1979). Because this process requires time, increased transit time caused by the addition of duckweed could facilitate digestibility of lipid shrimp. However, as previously stated, such a hypothesis must be explored with more accurate methods of assessing transit time.

Conclusions

This study demonstrates that both positive and negative associative effects can occur when animals consume the same diet items in different ratios. In natural populations of T. scripta, the ratio of plant to animal material in the diet can vary widely depending on the availability of resources (Hart 1983; Parmenter 1980; Parementer &

Avery 1990). Because associative effects can vary with different ratios of plant to animal

46 material, turtles may benefit from positive associative effects under some conditions, whereas they may incur costs from negative effects under other conditions. These results highlight that in studies designed to examine the role of associative effects in wildlife nutrition, care should be taken to accurately define the most biologically relevant diet ratios because different ratios can produce opposite results.

Different plant and animal diet items can produce different associate effects even when fed in similar ratios. Both positive and negative effects have been demonstrated in

T. scripta when animal material is added to a predominately plant diet (this study and

Bjorndal 1991). Additional research is needed with more diet items to determine how prevalent each type of effect is in wild T. scripta consuming this kind of diet. If negative effects prevail, then there may be no energetic advantage to including animals in a plant diet. However, turtles may include animal material for other dietary constituents, such as nitrogen. In this study, there was no negative associative effect in terms of nitrogen gain.

Therefore, the benefits of acquiring nitrogen from animal material could outweigh the energetic costs.

Table 3-1. Summary of studies investigating associative effects in wildlife.

Type of Study species Diet items interaction Proposed mechanism Source

Yellow-bellied slider turtle Duckweed plant + Nitrogen in larvae stimulated growth of Bjorndal 1991 (Trachemys scripta) (Spirodela polyrhiza) gut microbial population which Beetle larvae digested plant fiber more efficiently (Tenebrio sp.)

Cerambycid beetles Fungus and wood + Ingestion of fungus provided cellulytic Kukor et al. 1988 Siricid woodwasps enzymes which facilitated digestion of Kukor & Martin 1983 Termites plant parts Martin & Martin 1978

Beetle Peach palm trichomes and + Ingestion of highly lignified trichomes Rickson et al. 1990 (Cyclocephala amazona) pollen (Bactris gasipaes) crush pollen allowing it to be digested

Rodent Fungus (Boletus edulis) + Nitrogen in larvae stimulated growth of Bozinovic & Muñoz-Pedreros 1995 (Abrothrix longipilis) insect larvae gut microbial population which

(species not given) digested fungus carbohydrates more 47 efficiently

Hingeback Tortoise Millipedes (Alloporus sp.) - Transit time of kale reduced transit Hailey et al. 1998 (Kinixys spekii) Kale leaves time of millipedes (Brassica oleracea)

Beetle Milkweed flowers and - Secondary compounds in foliage Matter et al. 1999 (Tetraopes tetraophthalmus) foliage (Asclepias syriaca) depressed floral digestion

Lesser Black-Backed Gulls Whiting no effect (Larus fuscus) (Merlangius merlangus) None given Hilton et al. 2000 Sprat Common Guillemots (Sprattus sprattus) - (Uria aalge)

Elk (Cervis elaphus) Browse stems + Transit time of browse increased transit Baker & Hobbs 1987 (Vaccinium sp.) time of grass, facilitating fiber Mountain sheep Grass hay + digestion (Ovis canadensis) (Bromus inermis)

Table 3-2. Nutrient composition of duckweed and shrimp fed to adult turtles. All values except energy are presented on a percent dry matter basis. Note that shrimp values are for shrimp with anterior and posterior portions removed. Mixed Diets

Duckweed Duckweed (from pond) Shrimp (from store) Shrimp

Organic Matter (%) 85.5 87.1 75.9 87.0

Fiber (%)* NDF 45.2 22.5 42.9 22.5

ADF 21.4 4.8 15.0 4.7 48 Nitrogen (%) 4.1 12.6 4.1 12.2

Lipids (%) 13.2 15.1 12.0 15.8

Energy (kJ g-1 DM) 17.4 20.9 14.5 20.8

Table 3-3. Nutrient composition of duckweed and shrimp fed to juvenile turtles. All values except energy are presented on a percent dry matter basis. Note that shrimp values are for shrimp with anterior and posterior portions removed.

Duckweed (from pond) Shrimp Diet Organic Matter (%) 86.4 88.0

Fiber (%) NDF 41.2 20 ADF 19.7 6.4

Nitrogen (%) 5.0 12.6

Energy (kJ·g-1 DM) 18.49 21.75

Table 3-4. Nutrient composition of duckweed and shrimp orts (remaining food) from feeding trials with adults. All values except energy are presented on a percent dry matter basis. Ort values are means ± standard errors for all turtles in each treatment. No shrimp ort values are presented for mixed diets because turtles consumed all shrimp in these treatments.

Mixed Diets

Duckweed Duckweed (67% duckweed, (14% duckweed, Duckweed Shrimp 33% shrimp diet) 86% shrimp diet)

Organic Matter (%) 85.8 ± 0.4 87.6 ± 0.1 76.7 ± 0.2 78.0 ± 0.1

Fiber (%) 50 NDF 45.7 ± 1.5 -- 41.1 ± 0.9 41.1 ± 0.4

ADF 24.0 ± 1.7 6.3 ± 0.1 17.5 ± 0.9 16.7 ± 0.2

Nitrogen (%) 4.3 ± 0.4 12.0 ± 0.0 4.0 ± 0.1 4.3 ± 0.0

Lipids (%) 13.5 ± 1.2 13.3 ± 0.3 9.6 ± 0.2 10.7 ± 0.2

Energy (kJ g-1 DM) 18.1 ± 3.2 20.9 ± 0.1 15.4 ± 0.0 15.5 ± 0.2

Table 3-5. Digestive processing of duckweed, shrimp, and mixed diets by adult T. scripta. Differences between treatments were determined by Kruskal-Wallis tests and post hoc tests according to Conover (1980). Values are medians (range), and different superscripts across rows indicate significant differences between treatments.

67% Duckweed 14% Duckweed 100% Duckweed 33% Shrimp 86% Shrimp 100% Shrimp (n = 7) (n = 4) (n = 3) (n = 5) H p

Intake (mg⋅g turtle-1⋅day-1) Dry matter 2.0 (1.3 – 3.2) 1.4 (0.7 – 2.7) 2.1 (2.0 – 2.4) 3.2 (2.0 – 4.7) 6.518 0.089 Organic matter 1.8 (1.1 – 2.8) 1.1 (0.5 – 2.2) 1.8 (1.7 – 2.1) 2.8 (1.8 – 4.1) 7.341 0.062

Digestibility (%) Organic matter 68.6a (63.1 – 77.6) 67.0a (62.3 – 70.9) 91.4b (86.7 – 93.1) 89.2b (84.9 – 93.4) 13.548 0.004 NDF 74.5a (59.4 – 82.8) 60.7b (56.3 – 67.1) 81.3c (72.1 – 87.5) 94.1c (91.7 – 94.3) 14.472 0.002 ADF 65.0a (41.9 – 78.2) 32.2b (23.2 – 38.5) 74.5a (55.7 – 79.3) 83.3c (74.6 – 84.9) 13.687 0.003 Lipid 60.8a (49.4 – 72.4) 59.2a (52.8 – 68.5) 88.5b (84.5 – 88.5) 77.8b (76.6 – 87.6) 13.961 0.003 51 Energy 65.8a (59.1 – 75.8) 68.6a (64.6 – 73.6) 92.5b (88.6 – 93.4) 90.1b (85.5 – 94.5) 13.288 0.004 Nitrogen 74.6a (70.7 – 81.5) 87.1b (86.5 – 92.4) 96.5c (92.4 – 97.2) 94.0c (90.2 – 95.0) 15.517 0.001

Digestible organic matter intake (mg⋅g turtle-1⋅day-1) 1.3a (0.8 – 1.8) 0.7a (0.4 – 1.7) 1.7a,b (1.5 – 1.9) 2.3b (1.6 – 3.6) 11.561 0.009

Daily gain Energy (kJ⋅g turtle-1⋅day-1) 24.4a (15.4 – 34.0) 15.4a (8.6 – 36.1) 39.9b (35.7 – 44.9) 56.5b (38.4 – 86.9) 13.596 0.004 a a b b Nitrogen (mg⋅g turtle-1⋅day-1) 0.05 (0.04 – 0.08) 0.08 (0.04 – 0.18) 0.23 (0.21 – 0.26) 0.36 (0.24 – 0.55) 14.519 0.002

Transit time (hours) 170.5 (94.8 – 199.5) 91.0 (55.5 – 126.5) 135.0 (114.5 – 143.8) 72.5 (37.5 – 143.8) 4.407 0.221 Samples sizes for transit time (n = 5) (n = 2) (n = 3) (n = 5)

Table 3-6. Differences between actual and predicted digestibilities, digestible intakes, and daily gains of adult turtles fed two mixed diets of duckweed and shrimp. Bold values indicate those confidence intervals that do not include zero.

67% Duckweed 14% Duckweed 33% Shrimp 86% Shrimp 95% confidence 95% confidence Mean difference interval Mean difference interval from predicted lower upper from predicted lower upper

Digestibility (%) Organic matter -9.9 -14.2 -6.0 3.7 0.7 6.9 NDF -19.3 -24.1 -14.4 -10.5 -12.1 -8.9 ADF -39.3 -47.4 -32.1 -10.1 -14.5 -5.3 Lipid -7.2 -13.5 -1.7 10.0 5.9 14.2 52 Energy -6.8 -10.9 -2.1 4.3 1.2 7.6 Nitrogen 6.8 4.5 9.3 4.5 2.7 6.4

Digestible intake (mg⋅g turtle-1⋅day-1) Organic matter -0.12 -0.19 -0.07 0.07 0.02 0.13

Daily gain Energy (kJ⋅g turtle-1⋅day-1) -2.02 -3.20 -0.80 1.71 0.20 3.23 Nitrogen (mg⋅g turtle-1⋅day-1) 0.00 0.00 0.01 0.01 0.00 0.01

Table 3-7. Short-chain fatty acid composition in the digestive tracts of adult turtles fed duckweed, shrimp, and mixed diets. Values are mean percentages of total SCFAs ± standard errors.

Short-chain fatty acid

n Acetate Propionate Butyrate Isobutyrate Valerate Isovalerate Stomach 100% duckweed 5 67.1 ± 1.7 20.3 ± 5.8 0.0 ± 0.0 5.5 ± 3.4 0.0 ± 0.0 7.2 ± 4.7 67% duckweed, 33% shrimp 3 67.1 ± 2.4 25.6 ± 5.8 0.0 ± 0.0 7.3 ± 7.3 0.0 ± 0.0 0.0 ± 0.0 14% duckweed, 86% shrimp 2 59.0 ± 7.5 9.6 ± 4.0 0.8 ± 0.8 8.6 ± 1.4 0.0 ± 0.0 22.0 ± 1.3 100% shrimp 6 74.6 ± 22.1 20.0 ± 4.3 7.0 ± 3.6 0.0 ± 0.0 0.0 ± 0.0 10.4 ± 3.0

Anterior small intestine 100% duckweed 5 60.0 ± 3.6 20.7 ± 4.7 0.0 ± 0.0 3.7 ± 1.8 0.0 ± 0.0 10.5 ± 4.9 67% duckweed, 33% shrimp 3 67.4 ± 12.8 19.1 ± 1.0 0.0 ± 0.0 2.6 ± 2.6 0.0 ± 0.0 10.9 ± 10.9 53 14% duckweed, 86% shrimp 3 73.4 ± 7.7 10.1 ± 5.0 5.9 ± 5.9 4.4 ± 0.2 0.8 ± 0.8 5.4 ± 2.7 100% shrimp 7 53.1 ± 3.7 23.2 ± 5.1 21.3 ± 7.8 0.2 ± 0.2 0.0 ± 0.0 2.3 ± 1.6

Posterior small intestine 100% duckweed 5 75.9 ± 1.8 9.7 ± 2.0 9.0 ± 2.0 2.9 ± 0.4 0.0 ± 0.0 2.5 ± 0.7 67% duckweed, 33% shrimp 3 81.2 ± 1.4 1.8 ± 0.6 8.2 ± 1.1 4.5 ± 1.2 0.0 ± 0.0 4.4 ± 1.2 14% duckweed, 86% shrimp 3 65.8 ± 3.8 2.7 ± 0.8 25.6 ± 4.9 2.9 ± 1.3 0.0 ± 0.0 3.0 ± 0.7 100% shrimp 7 6.01 ± 5.2 12.9 ± 2.1 13.2 ± 2.5 2.0 ± 0.8 0.7 ± 0.7 6.2 ± 2.2

Anterior large intestine 100% duckweed 5 78.7 ± 2.9 10.3 ± 2.4 8.7 ± 0.4 1.1 ± 0.2 0.2 ± 0.2 1.0 ± 0.3 67% duckweed, 33% shrimp 4 74.0 ± 6.4 11.3 ± 3.9 8.9 ± 3.5 2.1 ± 0.4 0.6 ± 0.3 3.0 ± 0.9 14% duckweed, 86% shrimp 3 74.5 ± 1.5 8.3 ± 4.2 9.8 ± 0.9 3.4 ± 2.3 1.7 ± 0.5 2.3 ± 0.1 100% shrimp 6 59.8 ± 5.1 15.6 ± 3.3 14.0 ± 2.2 2.7 ± 0.7 2.6 ± 1.2 5.2 ± 1.7

Posterior large intestine 100% duckweed 5 66.4 ± 4.5 9.7 ± 2.0 14.8 ± 2.5 6.8 ± 1.8 0.2 ± 0.2 2.1 ± 0.5 67% duckweed, 33% shrimp 3 74.3 ± 1.6 4.5 ± 2.8 8.45 ± 4.6 7.5 ± 3.5 0.8 ± 0.8 4.5 ± 1.4 14% duckweed, 86% shrimp 3 69.5 ± 1.6 4.4 ± 4.4 8.5 ± 0.8 9.9 ± 2.7 1.0 ± 0.5 6.6 ± 0.4 100% shrimp 7 64.0 ± 18.9 21.8 ± 8.8 16.8 ± 7.1 8.4 ± 4.1 4.5 ± 2.4 15.2 ± 5.9

Table 3-8. Digestive processing of duckweed, shrimp, and mixed diets by juvenile T. scripta. Differences between treatments are determined by Kruskal-Wallis tests and post hoc tests according to Conover (1980). Values are medians (range), and different superscripts across rows indicate significant differences between treatments. Data from turtles fed pure diets are from Chapter 2.

Diet

81% Duckweed Duckweed 19% Shrimp Shrimp (n = 7) H p (n = 7) (n = 8)

Intake (mg⋅g turtle-1⋅day-1) Dry matter 2.6a (1.6 – 2.9) 2.5a (0.3 – 5.0) 9.5b (3.1 – 16.9) 10.794 0.005 Organic matter 2.3a (1.4 – 2.5) 2.3a (0.3 – 4.7) 8.3b (2.7 – 14.9) 10.536 0.005

54 Digestibility (%) Dry matter 65.2a (61.2 – 70.5) 69.1a (47.1 – 80.0) 96.9b (95.4 – 98.5) 13.753 0.001 Organic matter 65.7a (61.3 – 71.0) 69.6a (45.9 – 79.8) 97.2b (96.1 – 99.0) 13.753 0.001

Digestible intake (mg⋅g turtle-1⋅day-1) Dry matter 1.7a (1.0 – 1.9) 1.5a (0.1 – 3.0) 9.0b (3.0 – 16.3) 13.702 0.001 Organic matter 1.5a (0.8 – 1.7) 1.4a (0.1 – 2.8) 8.0b (2.7 – 14.4) 13.193 0.001

Transit time (hours) 90.0 (64.0 – 97.5) 61.3 (30.5 – 105.5) 95.5 (77.5 – 120.0) 3.675 0.159 Sample sizes for transit time (n = 5) (n = 7) (n = 3)

Table 3-9. Differences between actual and predicted digestibilities, digestible intakes, and growth rates of juvenile turtles fed a mixed diet containing duckweed and shrimp. Bold values indicate confidence intervals that do not include zero.

81% Duckweed 19% Shrimp

95% confidence Mean difference interval from predicted lower upper

Digestibility (%) Dry matter -7.0 -8.8 -5.2 Organic matter -7.2 -9.2 -5.5

Digestible intake (mg⋅g turtle-1⋅day-1) Dry matter -0.08 -0.12 -0.03 Organic matter -0.18 -0.23 -0.13

Growth (mg·week-1) -140.7 -227.9 -46.5

Table 3-10. Growth and nutrient composition of juvenile turtles fed duckweed, shrimp, and mixed diets. Comparisons were made with ANOVAs. Values are means ± standard errors, and different superscripts across rows indicate significant differences between treatments. Data for turtles fed the pure diets are from Chapter 2.

Diet

81% Duckweed Duckweed 19% Shrimp Shrimp (n = 7) F p (n = 6) (n = 8)

Growth rate (mg⋅week-1) 195.0a ± 54.6 135.5a ± 38.8 616.2b ± 126.6 10.683 0.001

Composition Body water (%) 83.8a ± 0.3 82.5a ± 0.9 75.5b ± 0.7 39.127 < 0.001 56 * Organic matter (%) 84.6 ± 0.9 83.1 ± 0.6 84.1 ± 0.4 1.455 0.260 * Nitrogen (%) 12.5a ± 0.1 12.6a ± 0.3 11.4b ± 0.1 11.188 0.001 Lipid free basis 2.736 0.092 * 17.4 ± 0.4 16.9 ± 0.3 16.4 ± 0.3 Lipid (%) a,b a b 4.513 0.026 -1 * 28.2 ± 1.5 25.5 ± 1.3 30.6 ± 0.9 Energy (kJ⋅g ) 21.23a,b ± 0.39 20.45a ± 0.26 21.70b ± 0.22 5.113 0.017

Minerals* Calcium (%) 3.26 ± 0.27 4.02 ± 0.64 3.67 ± 0.37 0.573 0.574 Sodium (%) 1.08a ± 0.03 1.00a ± 0.06 0.74b ± 0.07 8.334 0.003 Potassium (%) 0.63 ± 0.14 0.78 ± 0.10 0.49 ± 0.06 2.200 0.141 Magnesium (%) 0.11 ± 0.01 0.09 ± 0.01 0.09 ± 0.01 1.628 0.224

*Dry matter basis

140 A . 120

) juveniles

M adults m 100 n ( o i at

r 80 ent 60 conc

FA 40 SC 20

0 .

) 1200 r e t t

a B 1000 y m g dr /

s 800 e l o m u 600 on ( i at r 400 ent

conc 200 FA

SC 0 anterior posterior anterior posterior stomach small small large large intestine intestine intestine intestine

Figure 3-1. Concentrations of SCFAs in the digestive tracts of adult T. scripta consuming duckweed, shrimp, and mixed diets. Samples sizes are indicated in parentheses under each column. A) Concentrations are presented on molar basis. B) Concentrations are presented on dry matter basis.

CHAPTER 4 MICROBIAL FERMENTATION IN JUVENILE AND ADULT YELLOW-BELLIED SLIDER TURTLES, TRACHEMYS SCRIPTA

Introduction

Microbial gut symbionts play a critical role in the digestive physiology of herbivorous reptiles (Zimmerman & Tracy 1989; Stevens & Hume 1995; Bjorndal

1997a). These symbionts ferment the cell wall constituents of plants, producing short- chain fatty acids (SCFAs) that the host absorbs and uses as an energy source. For reptiles that rely on fermentation to help meet their energy requirements, the capacity of the fermentation chamber must be sufficiently large or negative peristalsis must be used to delay passage of digesta so that cell wall constituents can be digested and microbes can reproduce. In herbivorous reptiles, fermentation chamber capacity (as measured by mass of fermentation contents) scales with body size according to the following equation: capacity = 0.0926 body size0.9919 (Bjorndal 1997a).

In most herbivorous reptiles studied to date, fermentation occurs primarily in the

large intestine (Troyer 1984c; Stevens & Hume 1995; Bjorndal 1997a). The only known

exception is the freshwater red-bellied turtle, Pseudemys nelsoni, which maintains

significant fermentation in the small, as well as large intestine (Bjorndal & Bolten 1990).

The small intestine is typically where carbohydrates and proteins are digested by

endogenous enzymes of the turtle. Fermentation in this region is therefore surprising

because endogenous enzymes must compete with microbial symbionts for these high

quality nutrients. However, Bjorndal and Bolten (1990) concluded that the capacity of

58 59

the large intestine alone did not provide an adequate fermentation chamber for an

herbivore the size of P. nelsoni. They hypothesized that, because of limited space within

the turtle shell, expansion of the large intestine would necessarily involve a decrease in

small intestine size. Although such a tradeoff would have potentially little consequence

for an herbivore relying on fermentation, it could compromise juvenile digestive

efficiency if young P. nelsoni are carnivorous like the young of many other freshwater

turtle species (Sexton 1959; Clark & Gibbons 1969; Moll 1976; Hart 1983). Reduced

juvenile digestive efficiency has fitness consequences because both survivorship and

future reproductive output are linked to rapid juvenile growth (Congdon & Gibbons

1983; Bodie & Semlitsch 2000).

Small intestine fermentation does not occur in other chelonians investigated to date including the green turtle, Chelonia mydas (Bjorndal 1979), red foot tortoise, Geochelone carbonaria (Guard 1980), and desert tortoise, Xerobates agassizii (Barboza 1995).

However, no freshwater species besides P. nelsoni have been examined. One freshwater turtle that could harbor small intestine fermentation is the yellow-bellied slider,

Trachemys scripta. As juveniles, these turtles are carnivores that feed on aquatic invertebrates, but, as they mature, they become opportunistic omnivores that feed primarily on aquatic plants (Clark & Gibbons 1969; Hart 1983). Adult T. scripta presumably maintain active microbial fermentation because they can digest plant cell wall constituents (Bjorndal 1991, 1997a). However, the presence of SCFAs and the location of the fermentation chamber have not been evaluated.

Additionally, it is unknown if significant microbial fermentation occurs in juvenile

T. scripta. Because gut tissue is energetically expensive (Cant et al. 1996) and because

digestion of animal matter requires a smaller gut capacity than digestion of plants,

juveniles may possess relatively smaller gut capacities than do adults. Such an

ontogenetic shift in gut capacity could allow juveniles to allocate more energy to growth,

maximizing survivorship and future reproduction (Congdon & Gibbons 1983; Bodie &

Semlitsch 2000). If relative gut capacity increases with age, juveniles may not possess a

sufficiently large fermentation chamber to maintain microbial fermentations when fed a

plant diet.

The purpose of this study was to measure SCFA concentrations in the digestive

tracts of juvenile and adult T. scripta to determine (1) if this species harbors small

intestine fermentation, (2) if juveniles possess significant concentrations of SCFAs compared with other reptiles known to rely on microbial fermentation, and (3) if the ontogenetic diet shift is accompanied by changes in fermentation chamber capacity.

Methods

Juvenile yellow-bellied slider turtles were obtained from a commercial turtle farm in Pt. Mayaka, Florida in mid June 2000. These turtles were the offspring of breeding adults collected from northern Florida, Georgia, and South Carolina. Adult turtles were collected in May 2001 from Kathwood Ponds located in the Audubon Society's Silver

Bluff Sanctuary in Aiken County, South Carolina.

Adults (n = 7) were housed individually in Nalgene tanks (45 x 60 cm) equipped with a 75-W floodlight and a 20-W full spectrum natural light fluorescent bulb. Juveniles

(n = 13) were divided in two groups and housed in one of two Nalgene tanks equipped with the same lighting as adult tanks. During both trials, turtles experienced a twelve- hour photoperiod and temperatures between 25-26 °C. For five weeks, all turtles were

maintained on duckweed, Lemna valdiviana, a small, floating aquatic plant consumed by

T. scripta throughout much of its range (Parmenter & Avery 1990). For the trial with

juveniles, duckweed was collected from a pond in Gainesville, Florida. Because this

pond dried up before the onset of the trial with adults, duckweed for that trial was

obtained from a local aquarium shop. Table 4-1 describes the nutrient composition of

duckweed from each source.

The trial with juveniles was conducted from 9 November to 14 December 2000,

and the trial with adults from 24 September to 1 November 2001. At the onset of the

study, juveniles and adults had an average mass of 12.3 g (range = 9.1 – 15.8 g) and

770.5 g (range = 375.2 – 1183.0 g), respectively. Before the onset of the juvenile trial,

adult feces were introduced into the tanks so turtles could acquire gut symbionts (Troyer

1984a).

After five weeks, turtles were euthanized with sodium pentobarbital. Turtles were

dissected, digestive tracts removed, and digesta samples collected. In adults, samples

were taken from five gut sections (stomach, anterior and posterior small intestine, and

anterior and posterior large intestine) and preserved in 20% phosphoric acid.

Preservation in the acid stopped fermentation and the production of SCFAs. The

remaining contents of these gut regions were removed, weighed and dried at 60 °C.

Because juvenile guts were so much smaller than those of adults, all digesta in each gut

region was used for analysis. Samples were collected and preserved from four gut

regions (stomach, small intestine, and anterior and posterior large intestine) because there

was not enough digesta in the small intestine for analyzing anterior and posterior sections separately. Samples from both age classes were centrifuged and SCFA concentrations of

the supernatants were measured using a Shimadzu gas chromatograph (Model GC-9AM)

with a Perkin Elmer Computing Integrator (LCI-100). After SCFA concentrations were

determined, the phosphoric acid was evaporated from vials to determine dry mass of

samples.

SCFA concentrations were compared between small and large intestines using a paired t-test. In juveniles, the composite small intestine sample was compared to the anterior large intestine sample, whereas in adults, the posterior small intestine sample was compared to the anterior large intestine sample. One adult turtle was not included in this analysis because SCFA concentrations in its digestive tract were over four standard

deviations below the mean. Because duckweed fed to adults and juveniles varied in fiber

content, no comparisons were made between SCFA concentrations in adults and

juveniles. However, in both age classes, SCFA concentrations indicated that the large

intestine was the main fermentation chamber; therefore, the mass of large intestine

contents was compared between juveniles and adults. This t-test was performed on a

mass-specific basis because fermentation chamber capacity scales isometrically with

body mass in reptiles (Bjorndal 1997a). A paired t-test was used to compare mass of

large intestine contents of juveniles and adults with values predicted for herbivorous

reptiles of those sizes based on the equation in Bjorndal (1997a).

Results

The gastrointestinal tracts of T. scripta were relatively simple, consisting of a

stomach, small intestine, and large intestine with a slight eccentric dilation at the

proximal end as described for other species by Guard (1980) (Figure 4-1). SCFAs were

found in the digestive tracts of both juveniles and adults with concentrations peaking in

the anterior large intestines of both age classes (Figure 4-2). In juveniles, concentrations

in the anterior large intestines were 148% higher on a molar basis and 200% higher on a

dry matter basis than in the composite small intestine of juveniles (molar basis: t =

3.096, p = 0.009; dry matter basis: t = 3.690, p = 0.003). For adults, concentrations in

the anterior large intestine were 68% higher on a molar basis and 144% higher on a dry

matter basis than in the posterior small intestine of adults (molar basis: t = 5.214, p =

0.006; dry matter basis: t = 3.564, p = 0.023). In the anterior large intestine, acetate was

produced in the highest proportions (juveniles = 81.9%; adults = 75.9%) followed by

propionate (juveniles = 12.0%; adults = 11.2) and butyrate (juveniles = 3.4%; adults =

10.2%) (Table 4-3).

Mass-specific wet mass of large intestine contents did not vary between age classes

(mean for juveniles = 49.49, mean for adults = 54.14; t = 0.867, p = 0.397). For both juveniles and adults, the mass of fermentation contents (i.e., large intestine contents) was approximately half that expected for herbivorous reptiles of those sizes (Table 4-2).

Discussion

Concentrations of total SCFAs in the large intestines of both juvenile and adult T.

scripta are indicative of an active microbial fermentation and are within the range found

in the hindguts of other herbivorous reptiles (range: 51 – 807 µmoles·ml-1; Bjorndal

1997a) and mammals (range: 18-236 µmoles·ml-1; Stevens & Hume 1995). The relative

proportions of these SCFAs were consistent with the pattern found in most reptiles:

acetate > propionate > butyrate > valerate (Bjorndal 1997a). Although SCFAs were also

found in the small intestine of T. scripta, they were at significantly lower levels than in

the anterior large intestine. This contrasts with the red-bellied turtle, P. nelsoni, which

had equally high concentrations in the small and large intestines. The main fermentation

chamber in T. scripta has therefore not expanded to include the small intestine as it has in

P. nelsoni (Bjorndal & Bolten 1990). This is somewhat surprising because large intestine contents in T. scripta are approximately half the mass of fermentation contents expected for an herbivorous reptile that size. However, adult T. scripta are much more omnivorous than P. nelsoni (Bjorndal & Bolten 1993) and probably rely less on fermentation as an energy source than do P. nelsoni. Consequently, there may be less selective pressure for T. scripta to expand their fermentation chamber capacity.

Differences in relative fermentation chamber size between T. scripta and P. nelsoni probably account for differences in digestive performance observed between these species. Bjorndal & Bolten (1993) found that both turtles had the same daily gain of energy and nitrogen on a diet that required limited microbial fermentation, Spirodela punctata. However, on a diet requiring more extensive fermentation, Hydrilla verticillata, P. nelsoni had greater daily gains than T. scripta. Similar results were found in a comparison between an omnivorous and herbivorous tortoise (Hailey 1997). The large intestine contents of the omnivorous tortoise, Kinixys spekii, were about half the mass of fermentation contents expected for an herbivorous reptile that size (52.8 g vs.

101.8 g), whereas the mass of large intestine contents of the herbivorous tortoise,

Geochelone pardalis, better approximated the expected value (285.6 g vs. 311.7 g). Both species digested a low fiber diet of kale, Brassica oleracea, to the same extent. However, the omnivore consumed only minimal quantities of a more fibrous, grass diet, Lolium sp., which the herbivore readily consumed and digested.

Comparisons between the large intestine contents of juveniles and adults indicated that the wet mass of T. scripta fermentation contents scales isometrically with body size, as has been found in mammals (Parra 1978) and other reptiles (Troyer 1984b; Bjorndal

1997a). Thus, the ontogenetic diet shift from carnivory to herbivory experienced by these turtles is not associated with a change in relative fermentation chamber capacity.

Juveniles might therefore be capable of digesting the fiber component of plant material, despite the fact that they are primarily carnivorous in the wild. However, since the mass of fermentation contents in juveniles was nearly half the predicted value, they may have difficulty with diets that require extensive fermentation.

Table 4-1. Nutrient composition of duckweed fed to juvenile and adult turtles. All values except energy are presented on a percent dry matter basis.

Duckweed

Juvenile Trial Adult Trial Diet Organic Matter (%) 86.4 86.4

Fiber (%)* NDF 41.2 46.7 ADF 19.7 29.9

Nitrogen (%) 5.0 2.7

Energy (kJ g-1 DM) 18.49 16.56

*Neutral detergent fiber (NDF) represents cellulose, hemicellulose, lignin and cutin, whereas acid detergent fiber (ADF) represents cellulose, lignin, and cutin.

Table 4-2. Wet mass of T. scripta fermentation contents compared with that predicted for herbivorous reptiles of this size based on equation in Bjorndal (1997a). Actual and predicted values were compared using a paired t-test. Values are means ± standard errors.

Actual mass of Predicted mass fermentation based on Bjorndal contents (g) (1997) (g) t p

Juveniles 0.64 ± 0.05 1.22 ± 0.05 21.909 < 0.001 (n = 13)

Adults 40.14 ± 5.21 71.47 ± 8.55 4.309 0.005 (n = 7)

Table 4-3. Molar percentages of individual short-chain fatty acids in digestive tracts of juvenile and adult T. scripta. Values are means ± standard errors.

Acetate Propionate Butyrate Isobutyrate Valerate Isovalerate

Stomach juveniles 55.2 ± 5.1 38.5 ± 5.0 2.2 ± 0.8 4.1 ± 2.3 0.0 ± 0.0 0.0 ± 0.0 adults 67.1 ± 1.7 20.3 ± 5.8 0.0 ± 0.0 5.5 ± 3.4 0.0 ± 0.0 7.2 ± 4.7

Small Intestine juveniles 73.6 ± 4.7 12.9 ± 1.6 1.7 ± 0.5 11.7 ± 4.7 0.0 ± 0.0 0.0 ± 0.0 adults anterior 65.1 ± 3.6 20.7 ± 4.7 0.0 ± 0.0 3.7 ± 1.8 0.0 ± 0.0 10.5 ± 4.9 posterior 75.9 ± 1.8 9.7 ± 2.0 9.0 ± 2.0 2.9 ± 0.4 0.0 ± 0.0 2.5 ± 0.7 68

Anterior Large Intestine juveniles 81.9 ± 2.1 12.0 ± 1.0 3.4 ± 0.4 2.7 ± 2.1 0.0 ± 0.0 0.0 ± 0.0 adults 75.9 ± 3.7 11.2 ± 2.9 10.2 ± 1.6 1.1 ± 0.1 0.4 ± 0.3 1.2 ± 0.3

Posterior Large Intestine juveniles 72.4 ± 3.9 23.0 ± 3.7 2.0 ± 0.6 2.5 ± 1.8 0.1 ± 0.1 0.0 ± 0.0 adults 64.3 ± 3.9 9.9 ± 1.5 13.2 ± 2.4 9.3 ± 2.7 0.2 ± 0.1 3.2 ± 1.1

Figure 4-1. Digestive tract of juvenile T. scripta fed duckweed for five weeks.

140 A . 120

) juveniles

M adults m 100 on ( i at

r 80 ent 60 conc

FA 40 SC 20

. 1200

) B M 1000 g D / s e l

o 800 m u on (

i 600 t a r t n 400 once c A

F 200 C S 0 anterior posterior anterior posterior stomach small small large large intestine intestine intestine intestine

Figure 4-2. Concentrations of total short-chain fatty acids in the digestive tracts of juvenile and adult T. scripta. Note that small intestine values for juveniles are from the entire small intestine and are not divided into anterior and posterior regions as in adults. A) Concentrations are presented on molar basis. B) Concentrations are presented on dry matter basis.

CHAPTER 5 EFFECT OF DIET ON GASTROINTESTINAL TRACT MORPHOLOGY IN THE YELLOW-BELLIED SLIDER TURTLE, TRACHEMYS SCRIPTA

Introduction

The gastrointestinal tract is an energetically expensive organ (Cant et al. 1996), which, in many species, morphologically changes in response to diet (Starck 1999b).

Such phenotypic flexibility may be an adaptation that conserves energy by matching gut size with that required for efficient digestion (Piersma & Lindstrom 1997; Secor 2001).

The most dramatic changes in gut morphology have been documented in snakes, which significantly up-regulate their gut upon consumption of a meal (Secor 2001). Changes in gut morphology also occur in migratory birds during hyperphagia and fasting

(McWilliams & Karasov 2001) and in mammals during hyperphagia associated with lactation and cold exposure (Hammond & Kristan 2000; Nespolo et al. 2002).

Gut morphology also responds to diet quality. For example, in birds and mammals, increases in gut size are associated with increases in dietary fiber (Owl & Batzli 1998;

Geluso & Hayes 1999; Starck 1999a; Pei et al. 2001) and with switches from insect to plant diets (Al-Jaborae 1980; Al-Dabbagh 1987; Levey et al. 1999; McClelland et al.

1999). The guts of freshwater turtles may also morphologically change in response to diet quality. Many freshwater turtles temporally vary the percentages of plant and animal material consumed. Such variation can occur seasonally (Mahmoud 1968; Schubauer &

Parmenter 1981; Parmenter & Avery 1990; Chen & Lue 1999) or ontogenetically, as

71 72

many species switch from carnivory to herbivory as they mature (Clark & Gibbons 1969;

Moll 1976; Hart 1983; Chessman 1986).

Differences in the percentages of plant and animal material in the diet may lead to changes in turtle gut morphology because these diet types are processed differently within the gut. Animal material is typically digested by endogenous enzymes in the stomach and small intestine, whereas plant digestion depends on microbial symbionts in the large intestine (Bjorndal 1997a; Chapter 4). These symbionts ferment cell wall components and produce waste products in the form of short-chain fatty acids, which the turtle absorbs and uses as an energy source. For turtles consuming plant material, the capacity of the large intestine must be sufficiently large to delay passage of digesta so that cell wall components can be digested and microbes are able to reproduce without being flushed from the system. Therefore, one might expect the large intestines of turtles consuming plants to be larger than those of turtles consuming animals.

The yellow-bellied slider turtle, Trachemys scripta, is a freshwater turtle species that may have a morphologically flexible digestive tract. This species relies on hindgut fermentation to digest plants (Chapter 4) and varies, both seasonal and ontogenetically, in the ratio of dietary plant to animal material (Parmenter & Avery 1990). Secor &

Diamond (1999) did not find that T. scripta changed gut morphology in response to feeding after a prolonged fasting period. However, turtles in that study were fed a meal of beef. Consequently, potential changes in morphology induced by an animal to plant diet shift were not examined. Also, unlike snakes, a long fasting period is probably not typical of freshwater pond turtles. The purpose of this study was to evaluate the effect of plant and animal diets on T. scripta gut morphology.

Methods

Turtles used in this study were part of several larger studies designed to examine T. scripta digestive physiology (Chapters 2, 3, and 4). They were collected in May 2001 from Kathwood Ponds located in the Audubon Society's Silver Bluff Sanctuary in Aiken

County, South Carolina (mean turtle mass = 995.4 g; range = 375.2 – 1451.1 g). They were housed individually in Nalgene tanks (45 x 60 cm) equipped with a 75-W floodlight and a 20-W full spectrum natural light fluorescent bulb, and experienced a twelve-hour photoperiod and temperatures between 25-26 °C.

Turtles were fed either duckweed, Lemna valdiviana (n = 7), or freshwater grass shrimp, Palaemontes paludosus (n = 7), for five weeks from 24 September to 1

Novemeber 2001. Duckweed, a small, floating aquatic plant consumed by T. scripta throughout much of its range (Parmenter & Avery 1990) was obtained from an aquarium shop. Shrimp was purchased from a bait shop that obtained shrimp from Gainesville area lakes. Because some turtles did not eat the anterior most portion of the shrimp containing the eyes and antennae or the posterior portion containing the caudal fin, these parts were removed before shrimp was fed to turtles. This ensured all turtles in this treatment consumed the same diet. Table 1 provides nutritional composition of duckweed and shrimp diets, which were determined as described in Chapter 2.

After five weeks on experimental diets, turtles were euthanized with sodium pentobarbital. Digestive tracts were removed, stripped of mesenteries and laid out straight. Stomach, small intestine and large intestine lengths and widths were measured with calipers ± 0.01 cm. Contents of each gut region were then removed, and gut contents and gut tissues for each section were weighed separately and dried at 60 °C. Not

all digestive tracts were dried and weighed, so sample sizes for this measure are smaller

than are those for other morphological measurements. Differences in the lengths, widths,

and masses of turtle digestive tracts, as well as mass of large intestine contents, were

compared using analysis of covariance with body mass as the covariate.

Results and Discussion

Gross gastrointestinal tract morphology of T. scripta did not vary in response to plant and animal diets. Although turtles fed duckweed had significantly wider and longer large intestines than did turtles fed shrimp (Table 5-2, Fig. 5-1), no significant difference existed between the two diets in large intestine mass. Because turtles fed duckweed had

4.5 times more wet contents and 3.0 times more dry contents in their large intestines than did turtles fed shrimp (Table 5-3), differences in large intestine size can be attributed to distention of this region by gut contents rather than to differences in the amount of intestinal tissue.

Trachemys scripta fed this duckweed species are known to have SCFAs in their large intestines (Chapter 4) and to digest the fiber component of the plant (Chapter 2).

The gastrointestinal tract size measured in this study was therefore sufficient to allow efficient plant digestion. Thus, the capacity of the large intestine may be great enough to delay passage of digesta and ensure maintenance of the microbial symbiont population or negative peristalsis may be used to delay plant material.

If gut volume plays an important role in delaying digesta, turtles consuming animal material may maintain more gastrointestinal tract than is required for efficient digestion of an animal diet. Such an absence in gastrointestinal flexibility may be favored if the rate of morphological response to diet is slower than is the rate at which the diet changes

(Sabat et al. 1998). Rates of change in gut morphology are dependent on intestinal tissue

75 turnover rates. In birds and mammals, the turnover rate of intestinal tissue ranges from a few days to two weeks, allowing for significant changes in gastrointestinal size in one to two days (Starck 1999b). Intestinal turnover rates of turtles are unknown; however, studies of stable isotope turnover rates in other tissues indicate that rates in turtles are dramatically slower than in birds and mammals (Seminoff & Bjorndal unpublished data).

In adult T. scripta, the rate of change between plant and animal material in the diet may be too fast to favor flexibility in gastrointestinal tract morphology. Adult T. scripta are primarily herbivorous, opportunistically consuming animal material when available

(Parmenter & Avery 1990). They are therefore likely to consume plants before or after an animal meal. Because the transit time of plant material in adult T. scripta ranges between three to seven days (Chapter 2; Bjorndal & Bolten 1993), adult turtles probably have at least some plants in their gastrointestinal tracts at any given time.

Although gross gut morphology did not appear to be flexible in T. scripta, other aspects of digestive physiology may vary in response to plant and animal diets. For example, the gut transit times of some plant and animal diets may vary in T. scripta

(Chapter 3), and these differences could be mediated by diet-induced changes in gut motility (Bjorndal 1989). Additionally, the production of enzymes and nutrient transport systems may vary with plant and animal diets, as documented in some birds (Diamond

1991; Levey et al. 1999). These changes in the gastrointestinal tract may occur over a shorter time scale than changes in gross gastrointestinal morphology and may therefore be favored in T. scripta.

Table 5-1. Nutrient composition of duckweed and shrimp. All values except energy are presented on a percent dry matter basis. Note that shrimp values are for shrimp with anterior and posterior portions removed.

Duckweed Shrimp Diet Organic Matter (%) 86.4 86.6

Fiber (%)* NDF 46.7 -- ADF 29.9 5.7

Nitrogen (%) 2.7 12.0

Energy (kJ·g-1 DM) 16.56 21.07

*Neutral detergent fiber (NDF) represents cellulose, hemicellulose, lignin and cutin, whereas acid detergent fiber (ADF) represents cellulose, lignin, and cutin in duckweed and chitin in shrimp.

Table 5-2. Gut morphology, gut mass, and large intestine contents of turtles fed duckweed and shrimp diets. Treatments were compared with analysis of covariance using body mass as the covariate. Values are adjusted means ± standard errors. Sample size duckweed, shrimp Duckweed Shrimp F d.f. p

Stomach Length (cm) 7, 7 7.7 ± 0.3 7.9 ± 0.3 0.390 1, 11 0.545 Width at widest point (cm) 7, 7 2.3 ± 0.1 2.7 ± 0.1 5.639 1, 11 0.037 Width at narrowest point (cm) 7, 7 1.2 ± 0.1 1.2 ± 0.1 0.101 1, 11 0.756 Dry mass of gut section (g) * 4, 6 ------

Small intestine

Length (cm) 7, 7 81.8 ± 6.0 78.4 ± 6.0 0.134 1, 11 0.721 77 Average width (cm) 7, 7 0.9 ± 0.04 0.8 ± 0.04 1.565 1, 11 0.237 Dry mass of gut section(g) 4, 6 4.30 ± 0.65 5.16 ± 0.52 0.941 1, 7 0.364

Large intestine Length (cm) 7, 7 20.3 ± 0.8 13.7 ± 0.9 25.864 1, 11 < 0.001 Width at widest point (cm) 7, 7 3.1 ± 0.1 1.0 ± 0.2 42.820 1, 11 < 0.001 Width at narrowest point (cm) 7, 7 1.7 ± 0.2 0.8 ± 0.04 5.114 1, 11 0.045 Dry mass of gut section (g) 4, 6 1.25 ± 0.18 1.21 ± 0.15 0.029 1, 7 0.870 Wet mass of contents (g) 7, 7 43.09 ± 4.12 6.14 ± 4.42 29.553 1, 11 < 0.001 Dry mass of contents (g) 7, 7 4.58 ± 0.68 1.51 ± 0.68 8.621 1, 11 0.014

* Could not be compared with ANCOVA because slopes were not homogenous.

Figure 5-1. Gastrointestinal tracts from T. scripta fed (A) shrimp and (B) duckweed for five weeks. Note esophagus at top of each figure. Horizontal lines indicate 5 cm for scale.

CHAPTER 6 CONCLUSIONS

The purpose of this dissertation was to explore the role of food chemistry, digestive physiology and anatomy, and associative effects in Trachemys scripta diet selection. In

Chapter 2, I examined the role digestive physiology plays in the ontogenetic diet shift from carnivory to herbivory that these turtles experience. The goals of this chapter were to determine to what extent juveniles and adults process plant and animal material and to measure how juvenile growth and condition vary with plant and animal diets. The main conclusions were as follows.

• Juveniles, which consume a carnivorous diet in the wild, were extremely efficient on the animal diet with high mass-specific intakes relative to adults and remarkably high digestibilities. This age class, however, did not fare nearly as well on the plant diet. Although juveniles digested plant material as well as adults, they did not have higher mass-specific intakes as expected based on metabolic demands. Overall, juvenile and adult intake patterns on the plant and animal diets indicated that juveniles fed animal material were able to meet their nutritional demands, whereas those fed plant material were not.

• Conclusions drawn from intake patterns were also supported by the faster growth experienced by juveniles fed animal material. Although differences in growth on the two diets were not surprising given differences in energy and nitrogen intake, this result has important evolutionary implications for T. scripta because rapid juvenile growth is linked to higher survivorship and greater future reproductive success (Bodie & Semlitsch 2000).

• Overall, this study provides a strong link between the carnivorous diet of juvenile T. scripta and digestive physiology. The question remains, however, why turtles switch to an herbivorous diet as they mature. To understand more completely the ontogenetic diet shift of T. scripta, we need studies that examine the costs and benefits of herbivorous and carnivorous diets to adults.

In Chapter 3 of this dissertation, I investigated the role of associative effects in the

diet selection of T. scripta. I used previously untested plant and animal diet items to

79 80 assess to what extent these effects vary with different ratios of plant to animal material and to what extent they vary with turtle age class. The main conclusions from this chapter were as follows.

• In terms of nitrogen gain, no associative effect was found for either diet.

• In terms of energy gain, a negative effect was found on the 67% duckweed diet, but a positive effect was found for the 86% duckweed diet. This is the first report of two diet items producing positive and negative effects in different ratios.

• Differential transit time of the duckweed and shrimp can explain both the negative and positive effect, but before any definitive conclusions are drawn, this hypothesis must be explored in more individuals with more accurate methods of measuring passage rate.

In Chapter 4, I described the digestive tract morphology of T. scripta and determined (1) if this species harbors small intestine fermentation, (2) if juveniles possess significant concentrations of SCFAs compared with other reptiles known to rely on microbial fermentation, and (3) if the ontogenetic diet shift is accompanied by changes in fermentation chamber capacity. The main conclusions were as follows.

• Both juveniles and adults have relatively simple gastrointestinal tracts consisting of a stomach, small intestine, and large intestine with a slight eccentric dilation at the proximal end.

• Concentrations of total SCFAs in the large intestines of juvenile and adult T. scripta fed plant material indicate an active microbial fermentation and were within the range found in the large intestines of other herbivorous reptiles (Bjorndal 1997).

• Trachemys scripta has not expanded the fermentation chamber to include both the small and large intestine, as has the red-bellied turtle, Pseudemys nelsoni (Bjorndal & Bolten 1990). Differences in fermentation chamber location between these two species may relate to differences in their degree of herbivory and may explain differences in their digestive efficiency on diets that require extensive fermentation (Bjorndal & Bolten 1993).

• The ontogenetic diet shift is not accompanied by a shift in relative fermentation chamber capacity. Therefore, juveniles may be capable of digesting the fiber component of plant material, despite the fact that they are primarily carnivorous in the wild.

Finally, in Chapter 5, I evaluated the extent to which plant and animal diets induce change in gross gastrointestinal tract morphology. The main conclusions were as follows.

• Gross gastrointestinal tract morphology of T. scripta did not vary in response to plant and animal diets. Although turtles fed plant material had wider and longer large intestines than turtles fed animal material, these differences were explained by variation in large intestine contents, rather than by changes in tissue mass.

• Because T. scripta can digest the fiber component of the plant diet, the large intestine size measured in this study was sufficient to maintain microbial symbionts and allow efficient plant digestion. Either the capacity of the large intestine was great enough to sufficiently delay passage of digesta or negative peristalsis was used.

APPENDIX DIGESTIVE PROCESSING OF DRY MATTER BY ADULT TURTLES

Table A-1. Digestive processing of dry matter from duckweed and shrimp diets fed to juvenile and adult turtles in Chapter 2. Comparisons between groups were made with Kruskal-Wallis tests and post hoc tests according to Conover (1980). Values are medians (ranges), and different superscripts across rows indicate significant differences between treatments.

Duckweed Diet Shrimp Diet Juveniles Adults Juveniles Adults (n = 7) (n = 7) (n = 7) (n = 5) H p

Dry matter intake 2.6a,c (1.6 – 2.9) 2.0a (1.3 – 3.2) 9.5b (3.1 – 16.9) 3.2c (2.0 – 4.7) 14.676 0.002 (mg⋅g turtle-1⋅day-1)

Dry matter digestibility (%) 65.2a (61.2 – 70.5) 64.9a (58.3 – 75.8) 96.9b (95.4 – 98.5) 77.0c (66.1 – 85.6) 18.691 < 0.001

Digestible dry matter intake 1.7a (1.0 – 1.9) 1.4a (0.8 – 2.1) 9.0b (3.0 – 16.3) 2.1c (1.6 – 3.6) 17.785 < 0.001 -1 -1 83 (mg⋅g turtle ⋅day )

Table A-2. Digestive processing of dry matter from duckweed, shrimp, and mixed diets fed to adult turtles in Chapter 3. Differences between treatments were determined by Kruskal-Wallis tests and post hoc tests according to Conover (1980). Values are medians (range), and different superscripts across rows indicate significant differences between treatments.

67% Duckweed 14% Duckweed 100% Duckweed 33% Shrimp 86% Shrimp 100% Shrimp (n = 7) (n = 4) (n = 3) (n = 5) H p

Dry matter intake (mg⋅g turtle-1⋅day-1) 2.0 (1.3 – 3.2) 1.4 (0.7 – 2.7) 2.1 (2.0 – 2.4) 3.2 (2.0 – 4.7) 6.518 0.089

Dry matter digestibility (%) 64.9a (58.3 – 75.8) 62.1a (56.1 – 67.2) 85.0b (79.2 – 88.6) 77.0b (66.2 – 85.6) 11.632 0.009

Dry matter digestible intake -1 -1 a a b b (mg⋅g turtle ⋅day ) 1.4 (0.8 – 2.1) 0.8 (0.5 – 2.0) 1.9 (1.6 – 2.1) 2.1 (1.6 -3.6) 11.561 0.003 84

Table A-3. Differences between actual and predicted dry matter digestibilities and digestible intakes of adult turtles fed two mixed diets of duckweed and shrimp in Chapter 3. Bold values indicate those confidence intervals that do not include zero.

67% Duckweed 14% Duckweed 33% Shrimp 86% Shrimp 95% confidence 95% confidence Mean difference interval Mean difference interval from predicted lower upper from predicted lower upper

Dry matter digestibility (%) -8.0 -13.2 -2.7 9.285 2.2 16.3

Dry matter digestible intake 85 (mg⋅g turtle-1⋅day-1) -0.11 -0.20 -0.02 0.20 0.04 0.37

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BIOGRAPHICAL SKETCH

Sarah Bouchard was born in Detroit, Michigan in 1973. She attended college at

Kalamazoo College in Kalamazoo, MI, where she majored in biology. While there, she spent one quarter studying abroad at the International Institute in Madrid, Spain.

Additionally, she spent time on the Pacific coast of Costa Rica studying leatherback sea turtles and in the Galapagos Islands studying lava lizards. After graduating magna cum laude with honors in biology, she moved to the University of Florida where she received a M.S. degree from the Department of Zoology. Her thesis examined the role of loggerhead sea turtles in transporting nutrients between marine and beach ecosystems.

While at the University of Florida, she taught a wide variety of laboratory classes including Introductory Biology, Functional Vertebrate Anatomy, Animal Physiology,

Vertebrate Zoology, and Ecology. She also visited local elementary schools to give talks about local wildlife through the Alachua County School Volunteer Program. In the fall of 2004, she joins the faculty of the Life Science Department at Otterbein College in

Westerville, Ohio.