Nutritional ecology of Old-World fruit bats

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Nutritional ecology of Old-World fruit bats
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Table of Contents
    Title Page
        Page i
        Page ii
    Dedication
        Page iii
    Acknowledgement
        Page iv
        Page v
        Page vi
    Table of Contents
        Page vii
        Page viii
        Page ix
    List of Tables
        Page x
    List of Figures
        Page xi
    Abstract
        Page xii
        Page xiii
    Chapter 1. Introduction
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
    Chapter 2. Research study sites and species
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
    Chapter 3. Fruit choice and calcium block use by tongan fruit bats: Do fruit bats seek out calcium in their diet?
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
    Chapter 4. Folivory in fruit bats: Are leaves a natural calcium supplement?
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
    Chapter 5. Bioavailability and apparent absorption of minerals
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
    Chapter 6. Nutritional landscape ecology and habitat use by tongan flying foxes in American Samoa
        Page 66
        Page 67
        Page 68
        Page 69
        Page 70
        Page 71
        Page 72
        Page 73
        Page 74
        Page 75
        Page 76
        Page 77
        Page 78
        Page 79
        Page 80
        Page 81
        Page 82
        Page 83
        Page 84
        Page 85
    Chapter 7. Absorption and utilization of minerals consumed by captive lactating female malayan flying foxes (Pteropus vampyrus) and their pups
        Page 86
        Page 87
        Page 88
        Page 89
        Page 90
        Page 91
        Page 92
        Page 93
        Page 94
        Page 95
        Page 96
        Page 97
        Page 98
        Page 99
    Chapter 8. Sugar concentration preferences of two species of blossom-bats (Syconycteris australis and Macroglossus minimus) in Papua New Guinea
        Page 100
        Page 101
        Page 102
        Page 103
        Page 104
        Page 105
        Page 106
        Page 107
        Page 108
        Page 109
        Page 110
        Page 111
        Page 112
        Page 113
    Chapter 9. Conclusion and conservation recommendations
        Page 114
        Page 115
        Page 116
        Page 117
        Page 118
        Page 119
        Page 120
    List of references
        Page 121
        Page 122
        Page 123
        Page 124
        Page 125
        Page 126
        Page 127
        Page 128
        Page 129
        Page 130
        Page 131
        Page 132
        Page 133
        Page 134
        Page 135
        Page 136
        Page 137
        Page 138
        Page 139
    Biographical sketch
        Page 140
        Page 141
        Page 142
        Page 143
Full Text










NUTRITIONAL ECOLOGY OF OLD-WORLD FRUIT BATS:
A TEST OF THE CALCIUM-CONSTRAINT HYPOTHESIS















By

SUZANNE LINN NELSON


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


2003































COPYRIGHT 2003

by

Suzanne Linn Nelson


































To Darrin














ACKNOWLEDGMENTS

I am heavily indebted and very appreciative to my funding sources, without which

this work could not have been completed. I appreciate the generous support of the Luis F.

Bacardi Graduate Fellowship, the National Park of American Samoa, the Disney Wildlife

Conservation Fund, and Bat Conservation International. I would also like to thank the

members of my committee, Drs. Steve Humphrey (chair), Tom Kunz (co-chair), Mel

Sunquist, George Tanner, and Lee McDowell for their support and advice during my

studies and research. I appreciate their time, advice, and technical assistance on many

aspects of this project. Their help was very appreciated.

I thank Roger Haagenson and John Seyjagat (Lubee Foundation, Inc.) for

offering and administering the Bacardi Graduate Fellowship. Brian Pope and Kerri Van

Wormer were helpful in my understanding of the issues and care of captive flying foxes.

Dana LeBlanc was always helpful answering questions concerning captive bat nutrition

and management. Dr. Ellen Dierenfeld of the New York Zoological Society provided

helpful nutrition discussions and important wildlife nutrition literature.

There are many people to thank in American Samoa. I am grateful to the National

Park of American Samoa for their funding of my work and for their generosity with

trucks and field equipment. I extend special thanks to Charles Cranfield and Dr. Peter

Craig for their advice, generosity, kindness, and support, which truly influenced the

outcome of the research. I thank Epi Suafua and the staff of the National Park of

American Samoa for their time, advice, and help with my project.








I am very grateful to the U.S.D.A. Land Grant in American Samoa for building

the bat house and providing equipment and supplies. I extend grateful thanks to the

Forestry Crew, which includes Aitasi Sameli, Kitiona Fa'atamala, Ritofu Lotovale,

Falaniko Mika, Tony Magalei, Logona Misa, and Eric Pese. I thank Dr. Carol Whitacker,

Sheri Mann, and Orlo Collin Steele for their support of the bat house project and the land

donation for its placement. I give special thanks to Dr. Don Vargo for his advice and help

on many topics and for the use of his lab and drying ovens.

I thank my field assistants, Raymond Pasay, Siniva Satele, and Gasetoto Gasetoto

for their hard work. I especially thank Gasetoto Gasetoto for his exceptional efforts,

including climbing and setting netting lines high in coconut trees. Joe Satele generously

allowed us to use his land and house while we netted bats. I thank the Fuimono family at

Fagatele Bay for their patience as we walked on their property at all hours. I thank Ian

Gurr for showing me plots and bat roosts on the north side of the island. I also thank my

friends in American Samoa whose company and help with netting I enjoyed very much,

including Kelby Black, Alison Graves, Malcolm Gaylord, Catherine Buchanon, Josh

Craig, Martin McCarthy, Graham Dawson, and Shintaro and Sophia Okamoto. I also

thank Dan Smith and Jeff Marsh for the generous use of the hyperbaric chamber and their

diving accident expertise.

I wish to thank my friends and fellow Wildlife Ecology and Conservation

graduate students for their help, support, and encouragement while at the University of

Florida. I learned much from them and very much enjoyed talking with them about

Biology. I wish to thank Caprice McRae, Monica Lindberg, Polly Falcon, Patty Connolly

and Nat Frazer for always working on my behalf in financial matters and for providing








support and encouragement. I thank Nancy Wilkinson and Jan Kivipelto of Animal

Sciences for their help and advice on mineral analysis and laboratory techniques.

Statistical help was generously provided by Dr. Ray Littell, Dr. Ken Portier, and

Yongsung Joo. Dr. Clarence Ammerman provided insightful discussions on mineral

bioavailability and apparent absorption. I thank Cindy Whitehurst of Park Avenue

Women's Center for sharing information on bone densiometry and osteoporosis. Dr.

Robert Barclay's ideas were the inspiration behind this dissertation. I thank him for his

insight and support of my ideas and research. His essays and ideas on calcium were

intriguing and inspiring.

I thank my family and friends for their support and encouragement during my

many years in school. Dr. Ed Heske and Dr. Lowell Getz always supported me and

believed in an undergrad with lots of energy and bad grades. I am very grateful to have

been visited in American Samoa by Doug Nelson and Laurie and Scott Beavers. I also

thank Doug for providing pictures for my seminar. Both my immediate and extended

family is and always has been the backbone of my strength, and I am very grateful to

have them in my life. Lastly, I am greatly indebted to Darrin Masters for his patience,

assistance, love, and support. It made all the difference.















TABLE OF CONTENTS
page

ACKN OW LED GM EN TS ....................................................................................................... iv

LIST OF TABLES.............................................................................................................. x

LIST OF FIGURES ....................................................................................................... xi

ABSTRA CT...................................................................................................................... xii

CHAPTER:

1. INTRODU CTION .......................................................................................................... 1

N nutritional Ecology......................................................................................................... 2
Calcium ........................................................................................................................... 3
Previous W ork in Bat N utrition...................................................................................... 4
Bats and Flight................................................................................................................ 4
Reproductive Costs......................................................................................................... 5
Relieving M ineral D eficiencies ...................................................................................... 7
D issertation Focus........................................................................................................... 7

2. RESEARCH STUDY SITES AND SPECIES ............................................................. 10

Flying Foxes.................................................................................................................. 10
Am erican Sam oa........................................................................................................... 12
Pteropus tonganus ........................................................................................................ 15
Papua N ew Guinea and K au W wildlife Area.................................................................. 17
Blossom bats of Papua N ew Guinea............................................................................. 18
Syconycteris australis............................................................................................. 19
M acroglossus m inimus........................................................................................... 20
The Lubee Foundation, Inc ........................................................................................... 21
Pteropus vampyrus........................................................................................................ 21

3. FRUIT CHOICE AND CALCIUM BLOCK USE BY TONGAN FRUIT BATS: DO
FRUIT BATS SEEK OUT CALCIUM IN THEIR DIET? ............................................... 24

Introduction................................................................................................................... 24
M ethods......................................................................................................................... 25
Results ........................................................................................................................... 27
Choice of H igh-Calcium or Low -Calcium Fruits.................................................. 27








U se of the Calcium Blocks...................................................... ........................... 28
Sugar ............................................................................................................................. 29
Calcium ......................... ........................... ..................................................................... 29
D discussion ..................................................................................................................... 31

4. FOLIVORY IN FRUIT BATS: ARE LEAVES A NATURAL CALCIUM
SUPPLEM EN T? .................................. .................................................................... .......... 34

Introduction ................................................................................................................... 34
M methods ............................................................................................................... ....... ... 36
Results ........................................................................................................................... 39
D discussion ............. ........................................................................................................ 41

5. BIOAVAILABILITY AND APPARENT ABSORPTION OF MINERALS
CONSUMED BY WILD TONGAN FLYING FOXES IN AMERICAN SAMOA ......... 46

Introduction.......................... ............................................... ..........................................46
M methods ......................................................................................................................... 49
N getting and H housing of Bats .................................................................................. 49
M ineral M etabolism Experim ents .......................................................................... 50
Analysis of Sam ples ................................................. .............. .......... ........... 52
Statistical Analysis................................................ ...................... .......................... 53
Results.................................................................................................. .........................53
M ineral Consum ption ................................................................................... ...... 53
M ineral Absorption........................................................... ......... ........................ 56
D iscussion..................................................................................................................... 57
Bioavailability and Absorption ....................................................................... ...... 59
M ineral Stress ........................................................................................................ 61
Future research.............................................................. .......... ........................... 63

6. NUTRITIONAL LANDSCAPE ECOLOGY AND HABITAT USE BY TONGAN
FLYING FOXES IN AMERICAN SAMOA ............................................................... 66

Introduction.............................. .................................................................. ........ .......... 66
M methods ....................................................................................................................... 68
M ajor V egetation Types ............. ......................................................................... 68
Nutritional Classification of the Major Vegetation Types ................................. 69
N getting of Bats ................................... ................................................................. 70
Radiotelem etry....................................................................................................... 70
Radiotelem etry Error ................................... .................................. ...................... 72
Results...... ............................................................... ............................................73
Radiotelem etry Error ...........................................................................................73
H habitat Selection .................................................................................................... 73
D instance Traveled from Roost to Foraging Site .......................... ......... .................. 76
D iscussion.................................. ................................................................................... 80
H habitat Preference ...............................................................................................80
D instance Flown from Roost to Foraging Site ................ ......................................... 81








Foraging Distance and Roost Affi liation............................................................... 82
Foraging Patterns of Reproductive Female Bats.................................................... 83
Nutritional Landscape Ecology.............................................................................. 83

7. ABSORPTION AND UTILIZATION OF MINERALS CONSUMED BY CAPTIVE
LACTATING FEMALE MALAYAN FLYING FOXES (Pteropus vampyrus) AND
THEIR PUPS .....................................................................................................................86

Introduction........................................................................................................... ........ 86
M methods ...................................... .......................................................................... ......... 88
Results .......................................... ............................................................................... 91
Discussion ............................. ................................................. ............... ......... 95

8. SUGAR CONCENTRATION PREFERENCES OF TWO SPECIES OF BLOSSOM-
BATS (Syconycteris australis AND Macroglossus minimus) IN PAPUA NEW GUINEA100

Introduction ................................................................................................................. 100
M ethods............................................... ........................................................................ 103
Nectar Concentrations .......................................................................................... 103
Preference Tests................................................................................................... 104
Results......................................................................................................................... 106
Discussion ................................................................................................................... 108

9. CONCLUSION AND CONSERVATION RECOMMENDATIONS ....................... 114

Testing the Calcium-Constraint Hypothesis ....................................... ........................ 114
M ineral Compensation ......................................................................................... 115
Tongan and M alayan Fruit Bat Dietary Choice ................................................... 116
Summ ary..................................................................................................................... 117
Conservation Recommendations...................................................... .......................... 118
M ajor Threats to Fruit Bats.................................................................................. 118
The National Park of Am erican Samoa ........................................... .................... 118
Reducing Hunting Pressure and Bat Education Programs................................... 119
LIST OF REFERENCES................................................................................ ................. 121

BIOGRAPHICAL SKETCH ........................................................................................... 140














LIST OF TABLES


Table page

3-1. Conjoint analysis results for all bats........................................................................... 29

3-2. Conjoint analysis results for all females .................................................................... 29

3-3. Frequency of calcium block use by Tongan fruit bats................................................ 30

4-1. Age and gender of bats used in the experiment......................................................... 38

5-1. Results of the principal components analysis............................................................. 54

5-2. A comparison of nutrient levels offered and consumed............................................. 55

5-3. A comparison of mineral apparent absorption values .............................................. 56

5-4. Mineral apparent absorption values for selected monogastrics species ..................... 58

5-5. Apparent absorption values for minerals in animals under nutritional stress ............ 58

6-1 Nutrient classification of habitat types in American Samoa ....................................... 70

6-2. Summary of goodness-of-fit tests for habitat selection.............................................. 75

6-3. Data summary for the 20 Tongan radio collared fruit bats in American Samoa........77

7-1. Composition of food fed to P. vampyrus at the Lubee Foundation, Inc ..................... 89

7-2. A comparison of lactating female P.vampyrus with pups to nonbreeding................. 92

7-3. A comparison of the diet offered and consumed by P. vampyrus.............................. 93

7-4. A comparison of the mineral amounts for diet, orts, ejecta, mineral intake,..............95

8-1. Bats used in the nectar preference tests.................................................................... 106















LIST OF FIGURES


Figure page

3-1. Calcium block use by male, female and reproductive female Tongan fruit bats.......30

4-1. Supplemental calcium ingested (mg/g) by folivory for adult and juvenile males and
fem ales ................................................................................................................... 39

4-2. A comparison of total calcium ingested among habitual, occasional, and non-leaf-
eaters ...................................................................................................................... 4 1

4-3. Typical pattern of leaf consumption by Pteropus tonganus in American Samoa......41

6-1. Map of Tutuila, American Samoa showing the three island habitat types................. 71

6-2. Frequency of P. tonganus location estimates............................................................. 74

6-3. Percent of locations by habitat type on Tutuila, American Samoa ............................ 75

6-4. A map of Tutuila, American Samoa showing the mean distance flown .................... 79

8-1. Distribution of blossom bats in Meganesia ............................................................. 101

8-2. Banana flower nectar concentrations........................................................................ 104

8-3. Results of 15% or 30% nectar-preference test.......................................................... 107

8-4. Results of 15% or 7% nectar-preference test............................................................ 107

8-5. Average amount of nectar consumed over four nights for each age class ............... 108















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

NUTRITIONAL ECOLOGY OF OLD-WORLD FRUIT BATS:
A TEST OF THE CALCIUM-CONSTRAINT HYPOTHESIS

By

Suzanne Linn Nelson

May 2003


Chair: Dr. Stephen R. Humphrey
Co-Chair: Dr. Tom Kunz
Department: Wildlife Ecology and Conservation

Although most studies argue that energy or protein is the most limiting

component of the fruit bat diet, the calcium-constraint hypothesis proposes that

reproduction in bats may be constrained by calcium rather than energy.

To test this hypothesis, I experimentally tested if bats attempted to increase their

calcium ingestion through preferential selection and consumption of calcium-rich foods.

This research used four different species of fruit bats in three different geographic

locations to test this theory. The various methods used included mineral metabolism

trials, fruit choice and mineral block experiments, nectar concentration trials, and studies

of habitat use by radiotelemetry.

Results indicated that fruit bats seemed to base their food choices on the sugar

content of fruits rather than the calcium content. Fruit bats preferred high-sugar

agricultural fruits in all experiments, but bats did not meet their mineral requirements by








consuming them. To compensate for mineral deficiencies resulting from reproduction or

rapid growth, fruit bats may demonstrate an additional preference for concentrated

mineral sources. Reproductive females and subadult bats appeared to select for additional

calcium by consuming leaves and by using the calcium blocks. Future work should

examine larger numbers of reproductive females and should observe bat foraging

throughout the entire night to look for temporal deviations in resource use. Population

persistence of Tongan fruit bats through time will reveal if these dietary choices are

adaptive or maladaptive.

Fruit bats act as seed dispersers and pollinators and are considered keystone

species on isolated oceanic islands. Hunting, hurricanes, and habitat loss threaten bat

populations. Native forest roosts are an essential resource component of the landscape for

bat populations. This study suggests envisioning a nutrient landscape and then evaluating

its components as they contribute to the needs of the bat population. A full explication

will provide for conservation planning.













CHAPTER 1
INTRODUCTION

Old-World fruit bats, or flying foxes, opportunistically feed on a wide variety of

plants. Their diet includes fruits, flowers, leaves, shoots, buds, nectar, and pollen of

tropical forest trees and shrubs (Start and Marshall 1974, Marshall 1985, Pierson and

Rainey 1992, Wiles and Fujita 1992, Kunz and Diaz 1995, Banack 1996, Bonaccorso

1998, Tan et al. 1998). Current estimates are that flying foxes consume the fruit of 136

genera, flowers of 97 genera, and leaves of 10 genera (Courts 1998). Food choice may be

influenced by a myriad of factors, including energy needs, requirements for specific

nutrients, reproductive status, constraints of the digestive system, abundance, diversity,

and seasonality of different food items, and competition and predation (Fleming 1988,

Oftedal 1991).

Fruit bats in the wild appear to meet their nutrient needs by consuming large

quantities of a wide variety of native fruits (Dempsey 1999). Their food tends to be

conspicuous, abundant, and easily harvested within clumps (Mickleburgh et al. 1992).

Hundreds of bats may descend on a locally and temporarily abundant food source until it

is depleted (Pierson and Rainey 1992, Wilson and Engbring 1992). Fruit bats are often

regarded as "sequential specialists," favoring preferred resources among a group of foods

as they become seasonally available (Marshall 1983, 1985, Banack 1998).

The largest genus of the Old World fruit bats, Pteropus, is primarily an island-

dwelling taxon, with 97% having some or all of their distribution on islands

(Mickleburgh et al. 1992, Pierson and Rainey 1992). Fruit bats are crucial to establishing








and maintaining forest composition on isolated oceanic islands that have a

characteristically limited suite of animal pollinators and dispersers (Thornton et al. 1990,

Cox et al. 1991, Elmqvist et al. 1992, Cox and Elmqvist 2000, Cook et al. 2001). Flying

foxes are highly mobile and can travel 40-60 km to reach a feeding area (Marshall 1983,

Rainey et al. 1995, Banack 1996). Thus, they are able to transport seeds great distances as

they drop or defecate them while in flight (Rainey et al. 1995, Shilton et al. 1999). Fruit

bats are particularly important to tropical forest regeneration following natural

catastrophes or forest destruction by humans (Bonaccorso and Humphrey 1978,

Whittacker and Jones 1994, Thornton et al. 1996,), and they can influence the

composition and distribution of food resources within the landscape (Kunz 1996).

Although contributions of fruit bats to tropical forests are well documented, the factors

that influence food choice are still highly debated and largely unknown. Factors that

influence food choice can be evaluated within the discipline of nutritional ecology.

Nutritional Ecology

Nutritional ecology includes the study of organic and mineral nutrients. Organic

nutrients consist of fiber, carbohydrates (fiber and soluble carbohydrates), protein

(nitrogen), fat and/or energy. Mineral nutrients are inorganic elements and include

calcium, phosphorus, and iron. Minerals required in gram quantities in the body are

referred to as macrominerals and consist of calcium, phosphorus, sodium, chlorine,

potassium, magnesium, and sulfur. Macrominerals are important structural components

of bone and other tissues and play vital roles in the maintenance of acid-base balance,

osmotic pressure, and membrane electrical potential (McDowell 1992, NRC 2001).

Minerals required in milligram or microgram amounts are referred to as trace minerals.

This group includes copper, iodine, iron, manganese, molybdenum, selenium, zinc, and








perhaps chromium and fluorine. Trace minerals are present in very low concentrations

and often serve as components ofmetalloenzymes, enzyme cofactors, or hormones in the

endocrine system (NRC 2001). Studies of dietary minerals quantify and compare mineral

concentrations in foods consumed by animals. The study of minerals, particularly

calcium, represents the primary focus of my research.

Calcium

About 98% of the calcium in the body is located within the skeleton (McDowell

1992). Calcium is essential for the formation of skeletal tissues, transmission of nervous

tissue impulses, excitation of skeletal and cardiac muscle contraction, blood clotting, and

as an important component of milk (NRC 2001). The calcium concentration of plasma

must be maintained at a relatively constant value of 1 to 1.25 mM to ensure normal nerve

membrane and muscle end plate electric potential and conductivity (NRC 2001).

Vertebrates have evolved an elaborate system to maintain calcium homeostasis whenever

there is a loss of calcium (NRC 2001). The parathyroid glands monitor the concentration

of calcium in carotid arterial blood. When levels drop, calcium can be replaced by

resorption of calcium stored in bone, by increased calcium absorption, or by reducing

urinary calcium loss (NRC 2001). Active transport of calcium, an energy-requiring

process, appears to be the major route of calcium absorption and is controlled by the

hormone form of vitamin D (Wasserman 1981, Bronner 1987, Holick 2002a, 2002b). If

dietary calcium is severely deficient for a prolonged period, an animal can develop severe

osteoporosis to the point of developing fractures (Radostits et al. 1994). Despite the

skeletal changes that result from prolonged calcium deficiency, plasma calcium levels are

homeostatically maintained and will only be slightly lower than normal (NRC 2001).








Previous Work in Bat Nutrition

Previous nutritional studies of fruit bats identified the organic nutrient protein

(nitrogen) as the major limiting nutrient in the diet (Thomas 1984, Herbst 1986, Steller

1986). It was previously thought that fruit bats consumed only fruits and did not

supplement their diet with other foods (Thomas 1984, Herbst 1986).Yet fruits were

considered nutrient-poor because of their low fat and protein content (Mattson 1980,

Herrera 1987, Witmner 1998). Daily fruit intake and preference were thought to be

dictated by protein rather than energy content of the diet. It was thought that bats over-

consumed energy to obtain adequate protein (Thomas 1984)

Later research showed that the diet of fruit bats includes much more than just fruit

(Banack 1996, Courts 1998). Fruit bats have been reported as deliberately ingesting

insects, pollen, and leaves, possibly to provide extra protein in the diet (Drew 1988,

Mickleburgh et al. 1992, Kunz and Diaz 1994, Kunz and Ingalls 1995, Kunz 1996,

Courts 1998). Thus, protein may not be a limiting nutrient in the bat diet. Instead,

minerals such as calcium may be deficient in the diet of bats (Barclay 1995). Barclay

(1994, 1995) proposed that because of bat's adaptation to flight, reproduction of females

bats is most constrained by their intake of calcium rather than their intake of protein or

energy.

Bats and Flight

The unique adaptation of flight in bats imposes certain restrictions on their

reproduction and development not experienced by other mammals. The wing skeleton of

a growing bat must acquire structural and material characteristics that will enable it to

withstand the mechanical pressures of flapping flight (Bernard and Davison 1996). This

includes adequate mineralization to confer strength and stiffness and to resist torsional






5

and/or bending stresses (Papadimitriou et al. 1996). Bats and similarly sized terrestrial

mammals produce litters with a mass averaging 25% that of the female (Kurta and Kunz

1987, Hayssen and Kunz 1996). Compared to other mammals, bats raise their young to a

significantly larger size because young cannot fly and gain independence until they are

almost fully grown (Barclay 1995). Juvenile bats are unable to fly or forage

independently until they have achieved approximately 70% of adult mass and more than

95% of adult skeletal size (Kurta and Kunz 1987, Barclay 1995,).

Rodents typically produce small litters that are weaned quickly at 30-44% of adult

size and obtain some of their nutrition by foraging for themselves (Millar 1977, Barclay

1995). In contrast, maternal milk is the only energy and nutrient source for dependent

young bats. Juvenile flying foxes associate with their mothers for up to a year (Kurta and

Kunz 1987, Mickleburgh et al. 1992, Pierson and Rainey 1992) and will opportunistically

nurse if in close proximity, especially in captivity (D. LeBlanc, pers. comm.). Thus, near

the end of lactation, females provide total nutrition to bat offspring that are nearly of

adult size (Kunz et al.1995, Kunz and Stern 1995, Hood et al. 2001). Overall, bat pups

are more expensive, in terms of energy and nutrients, to a female bat than is each young

to an equivalently sized terrestrial mammal (Barclay 1995). Each young requires a large

parental investment which may restrict the total number of young that can be raised

(Barclay 1994, Kunz and Hood 2000).

Reproductive Costs

Several studies indirectly addressed the question of calcium demand during

pregnancy and lactation, and showed that these are periods of calcium stress for bats

(Kwiecinski et al. 1987a, 1987b, Studier et al.1991, Sevick and Studier 1992, Studier et

al. 1994a, 1994b, Bernard and Davison 1996). Nutritional requirements for females








increase dramatically during reproduction, and females may be in negative calcium

balance from the onset of pregnancy to the end of lactation (Bernard and Davison 1996).

Females bats bear almost the entire mineral cost of raising their offspring by

allocating their own skeletal calcium reserves to build the skeletons of their young

(Bernard and Davison 1996, Papadimitriou et al. 1996). The bones of females become

more porous as stored calcium is depleted during prolonged lactation (Sevick and Studier

1992, Radostits et al. 1994). The excessive calcium demands of raising several young in

sequential years can result in osteoporosis in female bats, particularly in the mandible and

the long bones of the wings (Kwiecinski et al. 1987a). Through time, this has the

potential to decrease a female's fitness. The increased risk of wing-bone fractures can

impede her ability to fly and forage. Tooth loss and the subsequent inability to chew

fruits and leaves could affect longevity, fitness, and overall health (Barclay 1995).

Keeler and Studier (1992) found that among reproductive female bats, all caloric

requirements were met, but calcium intake was one-tenth the estimated requirement. For

a lactating female, inadequate calcium can result in low milk production (McDowell

1992). Several factors can influence the postnatal growth of mammals, including age,

nutritional and hormonal condition of the mother, and milk quality and quantity (Hoying

and Kunz 1998, Kunz and Hood 2000). For nursing bat pups, inadequate milk results in

inhibited growth and reduced mineralization of bone, which can result in lameness and

bone fractures (Radostits et al. 1994, NRC 2001). Studies of other mammals showed that

inadequate quantities of calcium in the diet can affect fecundity, number of litters, and

survival of offspring (Batzli 1986, Delgiudice et al. 1990). Calcium may be a limiting

nutrient for reproductive bats that could influence population density through time.








Relieving Mineral Deficiencies

Bats may be able to delay or reduce the effects of calcium deficiency by feeding

on calcium-rich foods or on concentrated calcium sources. For example, O'Brien et al.

(1998) proposed that frugivores eat figs because they have higher calcium concentrations

than many other native and agricultural fruits. Calcium concentration of figs in American

Samoa were three times that of other native fruits, and over ten times more concentrated

in calcium than agricultural fruits on the island (Nelson et al. 2000a). Leaves are also

especially rich in calcium and are consumed by bats (Lowry 1989, Kunz and Ingalls

1994, Kunz and Diaz 1995,Tan et al 1998, Ruby et al. 2000). Concentrated sources of

calcium can supplement the diet of fruit bats, and may be important in times of greater

physiological need such as pregnancy and lactation.

Dissertation Focus

Studies of female food choices that result from the energetic and mineral demands

of pregnancy and lactation are limited. Robert Barclay proposed bold new ideas on what

may motivate female food choice during reproduction (Barclay 1994, 1995). My research

tests Barclay's "calcium-constraint hypothesis" which proposes that reproductive females

are more constrained by calcium than energy in their diet (Barclay 1994, 1995). I

examined whether bats attempted to increase their consumption of deficient nutrients

through dietary choice.

It is a challenge to conduct nutrition work on Old World fruit bats because the

requirements for calcium and all other minerals needed to maintain bat health are

currently unknown. Previous dietary work most often used mammalian standards or

standards for rats to determine bat requirements, despite different diets, digestive systems

and metabolic rates of bats (Oftedal and Allen 1996). Thus, one of the goals of my








research was to establish mineral requirements for flying foxes that could be used for

future wild and captive nutrition studies. By offering free-choice diets to fruit bats and

quantifying mineral consumption, I hoped to create a mineral profile of bat feeding.

This research focused on three different geographic regions and four different fruit

bat species that represent three different size classes of bats. Each species was appropriate

to answer a specific question on fruit bat feeding and nutrition. Research was performed in

Papua New Guinea on the tiny fruit bats (18-20 g) Macroglossus minimus and

Syconycteris australis, in American Samoa on the mid-sized fruit bat (300-600 g),

Pteropus tonganus, and on one of the world's largest fruit bats (1000-1500 g), Pteropus

vampyrus, at the Lubee Foundation, Inc., in Gainesville, Florida.

Most of the nutritional work in my research involved the Tongan fruit bat in

American Samoa. This species was chosen because it is mid-sized (300-600 g), is one of

the most widely distributed of all Pteropus species (Koopman and Steadman 1995, Miller

and Wilson 1997), and it feeds in both native and agricultural habitats (Pierson and

Rainey 1992, Banack 1996). Together, these features suggest that this species is highly

adaptable and is an excellent general model. Therefore, results of nutritional studies in

this species should be widely applied to many other flying fox species. The Tongan

flying fox was used to generate new information on wild fruit bat mineral levels, leaf

consumption, use of concentrated mineral sources, and habitat use while foraging.

One of the world's largest flying foxes, Pteropus vampyrus, was used to study

mineral consumption in a population of captive lactating females and their dependent

pups. The large size of P. vampyrus may result in excessive mineral requirements as

females attempt to meet the nutrient demands of a large pup for up to a year. Thus,








quantifying the mineral content of the diet that can meet the requirements of this large

flying fox can be helpful in formulating diets for reproductive females of other captive

flying fox species. Mineral absorption values can also be compared to determine if wild

Tongan fruit bats are mineral deficient.

Because Barclay (1995) argued that reproductive females were more constrained

by calcium than energy, I examined energy consumption in two species of blossom bats in

Papua New Guinea. These species are among the smallest members of the Old World fruit

bats. They have a high metabolic rate and little or no means of storing large energy

reserves (Lemke 1984), so they may be energy-sensitive foragers limited by food

availability (Law 1992, 1993b, 1994a). Different nectar solutions representing different

energy concentrations were given to the bats in choice trials to determine whether energy

was a limiting nutrient for these species.

I used two methods new to the study of bats. To quantify bat mineral

consumption, I used apparent absorption, a method commonly used in animal science but

rarely in wildlife ecology because of the need to use captive animals. This method

accounts for minerals in the both the food and feces, and for minerals lost in the fibrous

pellet expelled while fruit bats feed (Lowry 1989, Kunz and Ingalls 1994). Also new to

the study of bats is the idea of nutritional landscape ecology, which examines whether

bats use mineral-rich habitats while foraging throughout the landscape.

For all these studies, I predicted that bats will seek out calcium-rich sources to

supplement their diet. The additive effect of the constraints of flight, reproductive costs,

and calcium-poor diets should make calcium consumption a priority.













CHAPTER 2
RESEARCH STUDY SITES AND SPECIES

Flying Foxes

The Order Chiroptera consists of two suborders: Microchiroptera and

Megachiroptera. Current estimates suggest that Megachiroptera separated from

Microchiroptera 50.2 million years ago (Bastian et al. 2001). Unlike Microchiroptera,

which has a global distribution, members of Megachiroptera, also called Old World fruit

bats, are limited to the Paleotropics. Megachiroptera comprise the single family

Pteropodidae, which contains 42 genera and 191 species (N. Simmons, pers.comm.).

Flying foxes are members of the genus Pteropus, which contains 58 species (Koopman

1993). Almost 97% of flying fox species are island dwelling, and 35 species are confined

to a single island or island group (Mickleburgh et al. 1992). Members of the genus

Pteropus range from Madagascar to India, and from Southeast Asia to Australia, and

reach as far east as the Cook Islands (Pierson and Rainey 1992).

The genus Pteropus feeds on fruit, leaves, nectar and pollen of trees found within

both native forests and agricultural areas. Across their range, Pteropus species are known

to visit over 92 genera of plants in 50 different plant families (Marshall 1985, Wiles and

Fujita 1992). Fruit bats are important pollinators and seed dispersers in tropical forest

ecosystems (Fleming 1988, Rainey et al. 1995, Banack 1996). On many isolated oceanic

islands with depauperate pollinator and seed disperser faunas, flying foxes are the only

animals capable of carrying large-seeded fruits. In island ecosystems of the south Pacific,

flying foxes are considered keystone species because their extinction could result in a








significant decline in both native forest diversity and regeneration (Cox et al. 1992,

Rainey et al. 1995). Their role as long distance seed dispersers (Shilton et al. 1999)

further demonstrates their critical role in maintaining forest structure and integrity. Seed

dispersal by bats may increase seed survival by decreasing seed predation and increasing

the chances of landing in favorable microhabitats (Jantzen et al. 1976, Augspurger 1983).

Flying foxes are phytophagus and consume fruits, pollen, flowers, nectar and

leaves of plants (Marshall 1985). They process fruit by pressing the tongue against the

palate to break up the fruit and ingest the pulp and juices. Flying foxes also consume

leaves as a regular part of their diet (Kunz and Ingalls 1994, Kunz and Diaz 1995). They

chew the leaves and swallow the juice, ejecting the fiber portion as a small compressed

pellet (Lowry 1989). The ingestion of mostly the liquid portion of fruits and leaves

results in a food transit time as low as 20 minutes (Tedman and Hall 1985). The

advantage of this foraging strategy to a flying mammal is reduced bulk and wing loading,

and reduced energy expenditure traveling to foraging areas (Kunz and Ingalls 1994).

Most flying fox species give birth to one offspring per year starting when they are

two years old (Pierson and Rainey 1992). Gestation periods range from 4-6 months

followed by a rearing interval of equal length, although young may stay associated with

their mothers for up to a year (Banack 1996, Hall and Richards 2000). A few species (P.

mariannus yapensis in Yap, P. molossinus in Pohnpei, and P. tonganus in Samoa) may

have more than one birth peak per year (Pierson and Rainey 1992). Birth peaks are often

correlated with seasonality and resource availability (Heideman 1995, Racey and

Entwistle 2000). Bats of the genus Pteropus typically give birth to a single young, but

twinning can occur.








Habitat alteration and habitat loss are the primary reasons for declining

populations of flying foxes (Cheke and Dahl 1981, Fujita and Tuttle 1991, Mickleburgh

et al. 1992, Pierson and Rainey 1992). Many species, particularly those inhabiting

mangrove swamps and lowland forest have lost critical roosting areas (Mickleburgh et al.

1992). Fruit bat populations are also heavily influenced by human depredation and

tropical storms (Wodzicki and Felton 1980, Cheke and Dahl 1981, Heaney and Heideman

1987, Craig et al. 1994a, 1994b, Loebel and Sanewski 1987).

American Samoa

The Samoan islands are a biogeographical unit politically divided into American

Samoa, an unincorporated territory of the United States, and Samoa, a sovereign country.

They lie in the South Pacific Ocean (14 S, 170 W), approximately 4000 km southwest

of Hawaii and 3000 km northwest of New Zealand. American Samoa comprises Tutuila,

Aunu'u, the Manu'a Islands (Ta'u, Olosega, and Ofu), Rose Atoll, and Swains Island.

The largest island in American Samoa is Tutuila, which is 142 km2 in area, supports 90%

of the human population, and contains the capital village of Pago Pago (Craig and Syron

1992).

The islands are volcanic in origin, having risen from hot spots on the ocean floor

in the late Pliocene or early Pleistocene (Kear and Wood 1959). The islands are now

highly eroded, resulting in extremely steep topography and deeply cut valleys. The

climate is warm and humid year-round and considered moist tropical, with an average

annual temperature of 25C. There are two distinct seasons, the wet and dry season,

although rain falls 300 days/year (Amerson et al. 1982). The wettest months are October

through March and rainfall averages 3200 mm annually (NOAA 1996).








Samoa lies within the South Pacific hurricane belt and is subject to hurricanes and

tropical storms. The Samoan archipelago was battered by three intense storms in 1986,

1990, and 1991. These were the most severe storms to occur in Samoa in over 160 years,

with each sustaining winds between 200 km/h and 240 km/h (Elmqvist et al. 1994).

Because the storms occurred so close together in time, they resulted in extensive damage

to native forest trees. Fruit bat populations were severely decimated due to loss of food,

roosts, and increased hunting immediately after the storms (Craig et al. 1994b, Elmqvist

et al. 1994, Pierson et al. 1996, Hjerpe et al. 2001). A sharp rise in air temperature over

the past decade (NOAA 1999) suggests further climatic uncertainty and a probable

increase in the frequency of hurricanes in the area (Craig et al. 2000).

Pacific island archipelagos display very high levels of endemism; typically 30%-

50% of the plants occur nowhere else (Brautigam and Elmqvist 1990, Cox et al. 1992).

More than 326 genera of vascular plants can be found in the Samoan archipelago

(Christophersen 1935) and at least 68 are endemic plant species (Amerson et al. 1982).

The affinities of most plants are Australian or Malesian (Whistler 1992). Paleotropical

rainforest is the natural vegetation of Samoa.

Rainforest originally covered nearly the entire surface of Samoa. Mature rain

forest is now restricted to the least accessible areas such as steep interior slopes and the

wet, cool montane regions away from villages. Rainforest is typically a tall forest with a

canopy up to 30 m in height (Whistler 1992). Rainforest trees in American Samoa

produce fleshy fruits adapted for dispersal by frugivorous birds and bats (Freifeld 1998).

The four types of rainforest are coastal, lowland, montane, and cloud forest, and each is

most easily distinguished by the plant species found within them (Whistler 1992).








Anthropogenic disturbances such as shifting cultivation and the development of

agroforestry have replaced much rainforest with secondary forest and agricultural lands.

Secondary forest is less diverse than mature rainforest, and is dominated by shade-

intolerant trees that quickly establish in disturbed areas (Whistler 1992 Freifeld 1998).

Cultivated lands consist of local slash-and-bum plots growing together in a mix of forest

and agricultural plants and are found in valleys and near villages (Cole et al. 1988).

The flora of American Samoa includes over 800 species of angiosperms that are

pollinated by 11 species of wasps, 9 species of frugivorous/nectarivorous birds, and 2

species ofphytophagus bats (Cox et al. 1992). Other animals on the islands include

snakes, skinks, toads, and large land snails. Rats, pigs, cows, dogs, and cats have been

introduced by humans. Feral pigs threaten native forest trees by destroying tree bark and

disturbing seed banks of native species. Feral cats penetrate secondary and agroforest

areas and prey on resident and migratory birds (S. Nelson, pers.obs.).

The Samoan islands have been inhabited by humans for approximately 3,000

years (Kirch and Hunt 1993, Petchey 2001). Today, the most serious environmental and

social problem facing American Samoa is its rapid human population growth (Craig et al.

2000). The population estimate of 63,000 for the year 2000 is increasing at a rate of

approximately 2.5% annually, which will result in a doubling time of only 28 years. A

continued increase is expected given the high birth rate (4.5 children per female) and high

proportion ofpre-reproductives in the population; nearly 50% of the population is less

than 20 years old (Craig et al. 2000). This exponential increase in the human population

is coupled with a substantial degree of habitat loss due to the confines of a small oceanic

island. Annually, 1-2% of rainforest is lost to agroforest in Samoa (Cole et al. 1988),








although current rates of forest loss may be higher. Land-use practices on Tutuila are

largely influenced by the steep topography of the island. With 50% of the land area

having a slope greater than 70%, there is relatively little land to use for agriculture and

housing (Craig et al. 2000). As the human population expands, hillsides are being

developed as are areas that were not formerly considered.

One area of refuge from development and human encroachment is the National

Park of American Samoa. The National Park of American Samoa was officially

established in September 1993 when a 50-year lease was signed between the National

Park Service and the American Samoa Government representing the villages in the Park.

The 9,000 acre park is spread out over three islands (Tutuila, Ta'u, Ofu), and includes

1000 acres that are underwater. The National Park of American Samoa contains many

critical roosting sites for both species of fruit bats (Brooke 1998, Brooke 2001) and

provides valuable native habitat for other species on the island (Freifeld 1999).

Pteropus tonganus

Three species of bats occur in the Samoan archipelago, Pteropus samoensis,

Pteropus tonganus, and Emballanura semicaudata (Peale 1848, Andersen 1912). E.

semicaudata is currently on the verge of local extinction in Samoa (Grant et al. 1994) or

may already be extinct. The two pteropodid bat species found on the Samoan islands are

the Samoan flying fox (Pteropus samoensis Peale), and the Tongan flying fox (Pteropus

tonganus Quoy and Gaimard). The Samoan flying fox is solitary, diurnal, and prefers to

forage on native forest fruits (Wilson and Engbring 1992, Thomson et al. 1998, Brooke et

al. 2000, Brooke 2001). In contrast, the Tongan flying fox roosts in noisy colonies of

thousands of bats, is primarily nocturnal, and forages both in native forest and

agricultural areas (Quoy and Gaimard 1830, Banack 1996). Also called the white-








collared or insular flying fox, P. tonganus is a medium-sized fruit bat that weighs

between ca. 300-600g, with a forearm length of 120-160 mm (Miller and Wilson 1997).

Males are generally larger than females (Flannery 1995b). The fur is black or seal brown

with a contrasting creamy yellow mantle (Miller and Wilson 1997).

The Tongan flying fox is common throughout the South Pacific and has the

largest geographic range of any pteropodid species in Oceania. It is one of the most

widely distributed of all Pteropus species (Koopman and Steadman 1995) and is found

south of the equator from the Schouten Islands offNE New Guinea, south to New

Caledonia, and east to the Cook Islands (Koopman 1993). The distribution of P. tonganus

includes the easternmost limit for the Pteropodidae. The Tongan flying fox is described

by Koopman (1979) as a "supertramp" species, referring to its absence from the largest

and most species-rich islands and its prevalence on small, species-poor ones (Pierson and

Rainey 1992, Mickleburgh et. al. 1992). There are few morphological differences among

populations of P. t. tonganus separated by several hundred kilometers, although animals

from Niue and the Cook Islands tend to be slightly smaller (Wodzicki and Felton 1980).

Tongan fruit bats roost colonially. Colonies can range in size from several

individuals to several thousand. Roosts in coastal forest are in undisturbed areas on steep

slopes immediately above the ocean or in upland forest (Brooke 1998). Within the roost,

bats hang together in harem groups consisting of a single dominant adult male with two

to sixteen adult females and their young, but group composition is highly labile (Banack

1996). Tongan fruit bat births have been observed year round in Samoa. The high number

of pregnant and lactating females throughout the year suggests the ability of this species

to rapidly increase its population size under optimal conditions (Banack 1996). The








mother carries her young until they are able to fly at 2-3 months of age (Brooke 1999).

About 50% of copulations observed involved females that were nursing young,

suggesting postpartum estrus in this species (Banack 1996).

Tongan fruit bats disperse from colonies to forage in native forests, agricultural

areas, and residential areas (Banackl996). They are described as favoring agricultural

areas, but the extent of use for each habitat type is currently unknown (Brooke 1998).

They are phytophagus and visit numerous plants to consume fruit, nectar, pollen and

leaves. A highly plastic forager, Pteropus tonganus uses 42 species of plants in American

Samoa and has the ability to find food despite seasonal and distributional changes in food

availability (Elmqvist et al. 1992, Banack 1998).

Mortality among Tongan flying foxes includes predation from raptors and snakes,

disease epidemics, hurricanes, hunting by local people, and habitat loss (Brooke 1998). In

many Pacific islands, fruit bats are considered a delicacy and consumed by local people,

and were previously hunted to supply a luxury food trade (Craig et al. 1994a, Wiles et al.

1997). Archeological records indicate that P. tonganus has been hunted and eaten for at

least the last 1,000 years (Steadman and Kirch 1990). In American Samoa, a ten year

hunting ban was enacted to limit depredation (Craig and Syron 1992, Craig et al. 1994b,

Brooke 2001). A current population estimate is 6300 or more Tongan fruit bats in

American Samoa (A.P. Brooke, pers. comm.). Pierson et al. (1992) listed this species as

priority grade H (not threatened) with priority status unknown.

Papua New Guinea and Kau Wildlife Area

Papua New Guinea comprises over 600 islands and includes the eastern half of

the island of New Guinea, the two northernmost islands of the Solomon chain

(Bougainville and Buka), and the Bismarck and Admiralty archipelagos. Papua New








Guinea lies between the Coral Sea and the South Pacific Ocean and is north of Australia.

The country is a member of the British Commonwealth with Port Moresby as its capital.

It is home to approximately five million people that speak over 800 languages.

Of the estimated 15,000 to 21,000 vascular plants found in Papua New Guinea,

more than half are believed to be endemic (Mittermeier et al. 1997). Similarly, mammals

are very diverse, with 242 species represented, 57 of which are endemic (Bonaccorso

1998). Bats, rodents and marsupials account for the bulk of the mammalian diversity, but

bat species are the most numerous and highly diverse. Thirty-four species of Old World

fruit bats are found in Papua New Guinea.

Fieldwork was done in the northern province of Madang in the Kau Wildlife

Area. The forest at Kau Wildlife Area is an old and relatively undisturbed lowland

rainforest tract of 300 ha that is an owned by the Dipida Clan. This area has been

untouched by logging, shifting cultivation, burning, or hunting with firearms since 1963

when it was put aside by the clan for conservation. Laboratory work was conducted at the

former Christensen Research Institute near Madang.

Blossom bats of Papua New Guinea

Both Syconycteris australis and Macroglossus minimus belong to the subfamily

Pteropodinae (formerly Macroglossinae) (Kirsch et al. 1995) which reaches its maximum

diversity in New Guinea (Flannery 1995a). Both S. australis and M minimus have large

geographic distributions and exhibit energetic plasticity. They are able to live in a variety

of environments, including small islands, disturbed successional forests, lowlands, and

montane rain forests (Bonaccorso and McNab 1997). S. australis is often described as a

feeding and habitat generalist in Papua New Guinea, while M minimus is a nectar

specialist (Bonaccorso 1998) This is opposite of Australia, where S. australis is a nectar








specialist (Law 1992). Morphologically, the two species of bats are almost identical in

linear size and body mass (approx. 15-20 g). They both have an elongated rostrum and a

slender, protrusible tongue with brushlike projections to gather nectar and pollen. Their

broad, short wings permit hovering and maneuverability (Gould 1978, Nowak 1999).

Syconycteris australis

The northern blossom bat, S. australis, resides in a variety of habitats, including

lowland rain forest, dry sclerophyll woodland, montane/hill forest, and swamp forest

(McKean 1972, Bonaccorso 1998). Its geographic distribution includes New Guinea and

the east coast of Australia into New South Wales (Richards 1983, Bonaccorso and

McNab 1997). This species weighs approximately 18 g and has an average forearm

length of 39 mm (Law 1992, Bonaccoso 1998, Nowak 1999).

During the day, S. australis roosts singly in the foliage of trees and shows fidelity

to day roost areas (Law 1996, Winkelmann et al. 2000). Northern blossom bats are

excellent thermoregulators and are able to inhabit almost the entire range of elevations in

New Guinea (McNab and Bonaccorso 1995). S. australis has a field metabolic rate that is

double that predicted for an animal its size but a basal metabolic rate lower than predicted

for its size (Geiser and Cobumrn 1999). An ability to undergo short periods of torpor may

explain its extended distribution range and counteract unpredictable nectar availability

and extended day roosting (Geiser et al. 1996, Cobumrn and Geiser 1998, McNab and

Bonaccorso 2001).

S. australis is distinguished from M minimus by a more robust dentition, which is

better suited for feeding on fruits, but it also consumes nectar, pollen, and occasional

insects (Bonaccorso 1998, Flannery 1995a). S. australis displays geographic variation in

its diet within Australia. Law (1992, 1994) found S. australis to be a nectar specialist in








its range in southern Australia, but is a frugivore and partial folivore in the more northern

part of its range. S. australis actively discriminated between sugar concentrations in

concentration preference tests (Law 1993). Flower morphology may influence S.

australis foraging, and result in resource partitioning within habitat areas (Nicolay and

Dumont 2000). The northern blossom bat is abundant and ubiquitous in distribution

throughout a wide range of habitat types and is listed as lower risk: least concern in the

1996 IUCN Red List of Threatened Animals (Baillie and Groombridge 1996).

Macroglossus minimus

Macroglossus minimus, also called the southern blossom bat, is the smallest of the

blossom bats found in New Guinea. This bat weighs only 12-18g and has a forearm

averaging 37.5 mm (Flannery 1995a, Nowak 1999). It is similar morphologically to S.

australis, but is unique in its feeding habits and more limited in its distribution. M.

minimus is a nectar specialist that occurs from sea level to 1000 m (Flannery 1995a,

Bonaccorso 1998). Specimens from New Guinea lacked fruit in the stomach (McKean

1983), and captive individuals refused to eat fruit (Mickleburgh et al. 1992). Instead, M

minimus seems to prefer the nectar and pollen (Nowak 1999).

M. minimus is widespread in lowland New Guinea, where it is often found in

secondary rain forest, hill forests, or coastal areas near mangroves (Mickleburgh et al.

1992, Bonaccorso 1998). It feeds in disturbed habitats and orchards due to its preference

for domesticated bananas (Gould 1978, Bonaccorso 1998). This species roosts singly, in

mother-infant pairs, or in small groups on the underside of large leaves, tree branches, or

roofs of abandoned buildings (Bonaccorso 1998). Births can occur in any month, perhaps

due to sperm storage (Hood and Smith 1989).








The southern blossom bat has a metabolic rate that is much lower than expected

for a mammal of its body mass (Bonaccorso and McNab 1997). This species may be

restricted to tropical areas because it can enter shallow daily torpor (Bartels et al. 1998).

M minimus is abundant and widespread throughout its range and is not currently

threatened (Mickleburgh et al 1992); the small size and cryptic roosting may contribute to

its listing as low risk: least concern in the 1996 IUCN Red List (Baillie and Groombridge

1996).

The Lubee Foundation, Inc.

The Lubee Foundation, Inc. was founded by the late Luis F. Bacardi in 1990 as a

non-profit organization involved in the conservation of threatened and endangered

species of Old World fruit bats. It maintains captive breeding populations and supports

research of both captive and wild populations of bats at its facility near Gainesville,

Florida. Bats are housed in outdoor circular flight exclosures that surround temperature-

controlled roosting quarters. Eleven species of bats are currently housed at this facility,

representing both fruit and nectar feeding bats. These species include Cynopterus

brachyotis, Eidolon helvum, Epomophorous wahlbergi, Glossophaga soricina, Pteropus

giganteus, Pteropus hypomelanus, Pteropus poliocephalus, Pteropus pumilus, Pteropus

rodricensis, Pteropus vampyrus, and Rousettus aegypticus. In total there are over 600

fruit bats currently housed at the facility. My research at The Lubee Foundation, Inc.

focused on P. vampyrus.

Pteropus vampyrus

P. vampyrus, also called the Malayan flying fox, is one of the world's largest

flying foxes, and can attain a wingspan of 2 m. Members of this species weigh between

645-1092 g and have a forearm of 180-220 mm. P. vampyrus has a distinctively dog or








foxlike face, and pelage color ranges from mahogany or orange to black with a black,

brown or grey/silver underbelly (Kunz and Jones 2000). The Malayan flying fox inhabits

Burma, Thailand, the Phillipines, Sumatra, Java, Borneo, the Lesser Sundas, and adjacent

islands (Andersen 1912, Medway 1969, Corbet and Hill 1992). There are currently seven

recognized subspecies of P. vampyrus (Bastian et al. 2001).

The Malayan flying fox is found in a variety of habitats including primary forest,

mangrove forests, mixed fruit orchards, and coconut groves (Medway 1969, Payne et al.

1985, Heideman and Heaney 1989). In Malaysia, P.vampyrus is most often found

roosting in isolated and inaccessible areas such as mangrove forests and freshwater

swamps (Payne et al.1985, Mohd-Azlan et al. 2001). Roosts are in the canopies of

emergent trees and are often shared with A. jubatus. Mixed groups of these two species

may range from 500 to 150,000 individuals (Mudar and Allen 1986, Heideman and

Heaney 1989). Roosts abandonment is most often due to disturbance, habitat loss, or

hunting (Mohd-Azlan et al. 2001).

Malayan flying foxes fly up to 50 km to reach their feeding grounds and shift

feeding sites in response to changes in food availability (Medway 1969). They feed on

flowers, nectar, and fruit, but most often on flowers and nectar (Gould 1977, Goodwin

1979, Payne et al. 1985). Pollen, nectar, and flowers of coconut and durian trees (Durio

zizebethinus), fruits oframbutan (Nephelium lappaceum), figs (Ficus spp.) and langsat

(Lansium domesticum) trees, in addition to fruits such as mangos and bananas are all

preferred foods (Heideman and Heaney 1989, Kunz and Jones 2000) and are actively

defended (Gould 1977). Figs are a dietary staple of P. vampyrus, while other foods are

utilized on a more sequential basis throughout the year (Stier and Mildenstein 2001).








Female P. vampyrus give birth synchronously during a single annual peak,

although the peak varies geographically and seasonally. Generally, females give birth

between March and May to a single offspring, but twinning does occur (Medway 1969,

Mickelburgh 1992). The gestation period is approximately 180 days (Kunz and Jones

2000). Young bats are carried by their mothers for the first few days, but later are left at

the roosts while their mothers forage. Young suckle from their mothers for 2-3 months

(Lekagul and McNeely 1977).

Camps of P. vampyrus in the Philippines once contained up to 100,000 bats

(Mickleburgh et al. 1992, Nowak 1999). Unregulated hunting and habitat loss are the

primary reasons for the decline in abundance for P. vampyrus (Mohd-Azlan et al. 2001).

In many areas, this species is considered a nuisance because it feeds in fruit orchards

(Medway 1969) and/or because of their noisy and conspicuous roosts (Kunz and Jones

2000). P. vampyrus is also hunted for local consumption and controls on hunting are

considered unenforceable. As a result, their numbers have declined severely (Heideman

and Heaney 1989). P. vampyrus is also threatened by the rapid loss and degradation of

mangroves for coastal reclamation and aquaculture, and by commercial logging and land

clearing for palm/rubber estates (Heideman and Heaney 1989). The Malayan flying fox is

listed as a species that that may become threatened with extinction if trade is not

regulated (Brautigam 1992)














CHAPTER 3
FRUIT CHOICE AND CALCIUM BLOCK USE BY TONGAN FRUIT BATS: DO
FRUIT BATS SEEK OUT CALCIUM IN THEIR DIET?

Introduction

Diets of wild animals can be low in essential nutrients. When minerals are

deficient in the diet, animals often seek out concentrated sources of these nutrients. These

sources may include natural mineral licks and foods that are rich in the deficient mineral

(Klaus et al. 1998). High sodium concentrations often attract wild animals to mineral

licks (Belovsky and Jordan 1981, Moe 1993, Tracy and McNaughton 1995), although

minerals such as calcium and magnesium may be equally important (Jones and Hanson

1985, Holl and Bleich 1987). When consumed by animals, these concentrated sources of

minerals may help compensate for mineral deficiencies in the diet (Klaus et al. 1998).

The idea that animals preferentially select nutrient-rich foods or a nutritionally

balanced diet from among a broad array or foods is coined "nutritional wisdom" and is

highly controversial. When given a choice of diets, animals often chose foods with the

highest nutrient content or minerals in which they were deficient (Ozanne and Howes

1971, Batzli and Pitelka 1983, Bromage and DeLuca 1984, Barclay 2002). However,

other studies have found that animals failed to select foods that either met their dietary

requirements or corrected for their nutritional deficiencies, but instead preferred palatable

but nutritionally poor diets (Arnold 1964, Coppock et al. 1972, Muller et al. 1977,

Oftedal and Allen 1996, Dierenfeld and McCann 1999, Zervas et al. 2001).








Selection for concentrated sources of minerals may be associated with the sex of

the consuming animal. For example, adult females of many species were more likely to

use mineral licks, especially during pregnancy and lactation (Faber et al. 1993,

Montenegro 1998). It has been postulated that reproduction in bats may be limited by

calcium deficiency (Barclay 1994,1995). To provide supplemental calcium to their diet,

fruit bats on occasion seek out calcium-rich fruits and leaves (Barclay 2002).

This study was designed to explore whether wild Tongan fruit bats (Pteropus

tonganus) sought out and preferentially consumed calcium when it was made available.

Captive bats were tested as follows: (1) by documenting preference or avoidance of

calcium-rich fruits and (2) by documenting use of commercial calcium blocks. It is

hypothesized that bats would seek out calcium in their diet by consuming calcium-rich

fruits and using the calcium blocks.

Methods

This study was conducted from December 2000 to August 2001 on the island of

Tutuila, American Samoa (14 S, 170 W) in the South Pacific Ocean. The Tongan flying

fox, P. tonganus, a medium-sized fruit bat (300-600 g), was used for this study. P.

tonganus is common throughout the South Pacific (Miller and Wilson 1997) and is a

feeding generalist that forages in both native forest and agricultural areas (Banack 1996).

Tongan fruit bats (13 males, 8 non-reproductive females, 2 (lactating)

reproductive females) were captured in mist nets and transported to a 4 x 3 meter

screened outdoor structure (the "bat house") to allow for movement and limited flight by

the captive bats. Following a two-day acclimation period, bats were individually tested

for five days, for a total of seven days in captivity. Bats were given twice their body mass

in food nightly (wet weight) of a high and low calcium fruit suspended from plastic cable








ties on a large wooden dowel rod (the food bar). The types of fruit given to the bats

varied every day depending on fruit availability on the island. The high-calcium fruits

used in the experiments were 2-3 times higher in calcium than the low-calcium fruits

(1.06-12.28 mg/g, 5.75 + 1.39 mg/g for high-calcium fruits, 0.55-2.46 mg/g, 1.30 + 0.59

mg/g for low-calcium fruits). Mineral concentrations for all native and agricultural fruits

used in this study were based on previous mineral analyses (Nelson et al. 2000a). All fruit

given to the bats were preferred foods of P. tonganus in American Samoa (Banack 1996).

The most commonly used combination of fruits was local bananas and papaya, because

they were readily available at local markets when native fruit species were unavailable. In

addition to the high-calcium and low-calcium food types, a calcium block was suspended

nightly from the food bar. The position of the calcium block was randomly assigned each

night. The calcium block consisted of calcium sulfate and ground limestone, and

contained 21-26% calcium (8 inl Pet Products, Inc., Fairport Harbor, OH). Water and a

salt lick were available ad libitum.

An infrared video camera (Sony Digital Handycam DCR-TRV 120) was placed in

the bat house in front of the food bar each night to record fruit choices by the bat. The

videotape recorded the first 1.5 hours of each nightly feeding session. Use of the calcium

block was documented for each bat, as was the sequence of fruit choices. The first five

fruit choices made by the bat were considered an indicator of fruit preference. The

number of high-calcium fruits and low-calcium fruits chosen from the first five choices

were analyzed statistically to document if bats sought out calcium in their diet. A

binomial test was used to analyze preference for high or low calcium fruits (Hollander

and Wolfe 1999).








To better understand preferences for fruit choice, I incorporated the effect of

sugar and analyzed the relationship between calcium and sugar content using the

multivariate technique Conjoint Analysis (Hair et al.1998). Conjoint analysis predicted

bat choice for fruits when bats were given a subset of all fruits found on the island (see

Green and Srinivasan 1978). Differences in sugar concentrations between samples of

native and agricultural fruit were tested using a Mann-Whitney rank sum test (Sokal and

Rohlf 1995). Sugar concentration of fruits was considered high if it was over 10%,

intermediate if it was between 5-10% and low if it was less than 5% sugar. A sugar

refractometer (Model # 300010, Sper Scientific, Scottsdale, AZ) was used to determine

sugar values of different fruit types. High, medium and low calcium values were

determined from previous mineral analysis of fruits (Nelson et al. 2000a). Both the sugar

concentration and the calcium content of fruits were evaluated in Conjoint analysis to

form part-worth estimates that were summed to totals. The largest part-worth totals

resulted in high rankings for fruit preference.

Results

Choice of High-Calcium or Low-Calcium Fruits

Results of 63 trials and 146 h of video were analyzed to test whether bats

preferred high or low calcium fruits when given a choice among them. The first five

choices made by each bat in a trial were documented, resulting in a total of 262 choices.

Low calcium fruits were chosen 200/262 times, and preferred 76% of the time. Low-

calcium fruits were highly preferred over high-calcium fruits (p < 0.001). Fifty-five of the

62 times that high-calcium fruits were chosen, bats chose papaya. When papaya was

removed from the high calcium fruit choice set, then low-calcium fruits were preferred








97% of the time (p < 0.001). Native fruit was chosen in the first five choices only once in

262 trials, by a bat that took a single small bite and then did not choose it again.

A native fruit (a fig, Ficus tinctoria) and an agricultural fruit (papaya, Carica

papaya) were compared for sugar concentration using a Brix sugar refractometer. Papaya

samples (n=12) averaged 12.6% sugar and were significantly higher in sugar (p = 0.001)

than fig samples (n=1 5), which averaged 2.4% sugar. Although they provide an excellent

source of calcium (O'Brien et al. 1998), figs were never consumed by bats and were

highly avoided in this study. Despite using fresh, frozen, and three different species of

figs (Ficus tinctoria, F. unirauniculata, F. scabra), figs were never eaten by the bats.

Results of a Conjoint analysis indicated the sugar content of fruit was the basis for

fruit preference and selection by all of the bats that were tested (Table 3-1). Fruits that

were the most preferred were high in sugar and low in calcium, and the least preferred

fruits were low in sugar and high in calcium. Even if the sugar was high, high-calcium

fruits were still avoided by bats. I then reanalyzed the data to evaluate female choice

among fruits (Table 3-2). The results were very similar to those for the entire data set,

indicating that individual females did not forage differently from the group, they avoided

high-calcium fruits and preferred high sugar fruit. Sample sizes were too small to perform

a Conjoint analysis for reproductive females.

Use of the Calcium Blocks

Twice as many females used the calcium blocks as did males (4 females, 2

males), and almost half (40%) of the females used the calcium blocks, including both

reproductive females (Figure 3-1). Use of the calcium blocks by males was limited; only

15% of the males (2 subadults) used the blocks.









Table 3-1. Conjoint analysis results for all bats showing part-worth estimate totals and the
resultant rankings of fruit characteristics to determine fruit preference by P. tonganus.
Sugar Calcium
Part worth Part worth
Level Level evelPart worth Total Ranking
estimate estimate___________
High 0.27 Low 0.38 0.65 1
Medium -0.1 Low 0.38 0.28 2
High 0.27 Medium -0.05 0.22 3
Medium -0.1 Medium -0.05 -0.15 4
High 0.27 High -1.57 -1.30 5
Low -1.87 Low 0.38 -1.49 6
Medium -0.1 High -1.57 -1.67 7
Low -1.87 Medium -0.05 -1.92 8
Low -1.87 High -1.57 -3.44 9


Table 3-2. Conjoint analysis results for all females showing part-worth estimate totals
and the resultant rankings of fruit characteristics to determine fruit preference by P.
tonganus..__________________


Sugar


Calcium


S, Part worth Part worth ,, ,
Level estimate Level art worth Total Ranking
estimate estimate

High 0.27 Low 0.56 0.83 1
Medium -0.09 Low 0.56 0.47 2
High 0.27 Medium -0.27 0.00 3
Medium -0.09 Medium -0.27 -0.36 4
High 0.27 High -1.41 -1.14 5
Low -1.88 Low 0.56 -1.32 6
Medium -0.09 High -1.41 -1.50 7
Low -1.88 Medium -0.27 -2.15 8
Low -1.88 High -1.41 -3.29 9









Frequency of use was calculated as the number of times bats used the calcium lick

divided by how many nights it was available to them. Frequency of use by males and

females for the calcium blocks was very similar and did not exceed 10% (Table 3-3).

Reproductive females used the calcium blocks with approximately four times the

frequency (25%) of males or non-reproductive females. Most of the bats that used the

calcium block were either reproductive females or subadults. In some cases, they used the

calcium block before they ever chose fruit, and returned to use the calcium block

intermittently while consuming fruit.


fa 100
90
80
70
60
50 0
,,. 'm 40
400
-, 30
20
10
0]
Males (n=13) Non-reproductive Reproductive females
females (n=l 0) (n=2)
Figure 3-1. Calcium block use by male, female and reproductive female Tongan fruit
bats. Thirteen males, ten females and two reproductive females were tested.


Table 3-3. Frequency of calcium block use by Tongan fruit bats.
Group Number of Total number of Total Frequency of use
bats in group trials mineral days of in the trials
used trial
Males 13 3 47 6%
Non-reproductive 8 3 42 7%
females 3 42 7
Reproductive 2 2 8 25%
females2225








Discussion

Tongan fruit bats did not consistently seek out concentrated sources of calcium by

preferring calcium-rich fruits or by using commercial calcium blocks with high

frequency. Instead, the bats in this study preferred fruits that were high in sugar and low

in calcium. If high-calcium fruits were chosen, the fruit was usually papaya, a preferred

fruit that is high in sugar. Females used the calcium blocks more often than males.

Reproductive females used the calcium blocks at more than four times the frequency of

non-reproductive females and males. Subadult males were the only males to use the

calcium blocks.

Mammals often select foods to maximize their intake of carbohydrates like sugar

(Provenza et al. 1996, Kimball et al. 1998), and fruit bats are no exception. Fruits high in

sugars are highly preferred because they represent an important energy source for

frugivorous bats (Dierenfeld and Seyjagat 2000b). Fruit bats prefer fruits such as papaya

that are soft, succulent, and high in sugar, and will choose them over fruits low in sugar

(Courts and Feistner 2000). Sugar, rather than calcium, appears to motivate dietary

selection for fruits despite the importance of minerals to bat reproduction (Barclay 1995).

There may be a temporal component to resource use by bats. In other studies,

high-energy fruits were consumed first by hungry and dehydrated bats emerging from the

day roost, followed by consumption of mineral-rich leaves later in the night (Kurta et al.

1989, Elangovan et al. 2001). Tongan bats in this study had food available to them for ten

hours a night, but only the first 1.5 h of feeding were recorded due to limited battery

power and a lack of electricity in the bat house. This may have biased data collection

toward documenting foraging for high-energy foods rather than calcium-rich foods. Thus,

these results may not reflect feeding to consume deficient minerals that occurred later in








the night. Subsequent studies should record bat feeding at all times of the night to see if

there is a temporal component to resource selection.

It was hypothesized that bats would use concentrated calcium sources such as

calcium blocks to relieve mineral deficiencies. Overall, frequency of use was low for the

calcium blocks, but noteworthy gender differences emerged. The only male bats to use

the calcium blocks were two subadults. They licked the calcium blocks in almost half of

their trials, and sampled high-calcium native fruits that were generally ignored by other

bats. Subadult males may experience calcium deficiency due to rapid growth, and may

ingest supplemental calcium to relieve temporary deficiencies.

Females, particularly reproductive females, used the calcium blocks in greater

numbers and with greater frequency than did males. Twice as many females as males

used the blocks, including use among all reproductive females tested. The two

reproductive females used the calcium blocks at four times the frequency of either the

males or non-reproductive females, and often used the calcium blocks before consuming

any fruit. One of the lactating females removed high-calcium leaves from the

Callophyllum neo-ebuticum (Clusaceae) tree in the cage and was seen consuming them at

the food bar within 20 minutes of receiving sugar-rich agricultural fruits. Leaf

consumption was also very high among reproductive females, resulting in supplemental

calcium ingestion (Chapter 4). These results seem to indicate that in some cases,

reproductive females may prioritize calcium ingestion over the ingestion of high-energy

foods. This may indicate calcium deficiency among reproductive females. Unfortunately,

very few reproductive females were caught, and their small sample size limits potential









interpretation of these data. Future work should examine food choice using larger

numbers of reproductive females.

It is difficult to assess if animals in this study foraged with "nutritional wisdom."

The assumption of this work was that calcium was the most deficient component of the

diet, and would be pursued first by bats when foraging. Instead, the majority of P.

tonganus fed on high-sugar, high-energy, agricultural fruits soon after their presentation,

potentially to maximize energy consumption. Although female Tongan bats were

potentially deficient in calcium (Barclay 1995), they did not choose high-calcium fruits

from among those offered. A limited number of bats did seek out minerals by using the

calcium blocks. Pregnant females and rapidly-growing subadult bats used the calcium

blocks the most, and were the most likely candidates for calcium deficiency.

Factors that motivate fruit selection among Tongan fruit bats warrant further

research, with experiments that include a larger number of reproductive females, and

observations of feeding done at different times throughout the night. This may create a

more complete picture of nutritional priorities and how they affect temporal patterns of

resource use.













CHAPTER 4
FOLIVORY IN FRUIT BATS: ARE LEAVES A NATURAL CALCIUM
SUPPLEMENT?

Introduction

Folivory, or leaf-eating by bats, is a well documented phenomenon (Marshall

1985, Lowry 1989, Funakoshi et al. 1993, Kunz and Ingalls 1994, Kunz and Diaz 1995,

Banack 1996, Tan et al. 1998, Ruby et al. 2000). Leaves are an important dietary source

of minerals, carbohydrates, and protein, and are especially rich in calcium (Tan et al

1998, Nelson et al. 2000b, Ruby et al. 2000,). Leaves are a consistent food source for

bats; they are available year-round and are predictable in time and space (Kunz and

Ingalls 1994, Rajan et al. 1999). Thus, leaves may provide a greater net return per

foraging bout than ingestion of large amounts of low-protein fruit or the active pursuit of

insects (Thomas 1984, Kunz and Ingalls 1994, Tan et al. 1998). In addition, steroid

hormones found in leaves may influence bat reproductive activity (Wickler and Seibt

1964, Kunz and Diaz 1995).

Bats consume leaves by leaf-fractionation. This process includes masticating the

leaves into a bolus, swallowing the liquid portion, and ejecting the flattened fibrous pellet

(Lowry 1989, Funakoshi et al. 1993, Kunz and Ingalls 1994, Kunz and Diaz 1995). By

rejecting the fibrous portion, bats are able to consume leaf nutrients without altering their

digestive tract or increasing wing loading (Kunz and Ingalls 1994). Frugivorous bats

appear to be pre-adapted for folivory by leaf fractionation; their dentition and gut

morphology are specialized for extracting and digesting a largely liquid diet (Tedman and








Hall 1985, Kunz and Ingalls 1994). To shift their diet alternately between one of fruits to

leaves would involve little, if any, change in form of function of the gut or dentition

(Kunz and Diaz 1994).

Folivory was once thought to be rare among fruit bats, with leaves taken only

when other food sources were scarce (Marshall 1985, Funakoshi et al. 1993, Pierson et al.

1996). However, recent studies have shown that leaf-eating is both common and

widespread among Old World flying foxes (Banack 1996, Tan et al. 1998, Ruby et al.

2000). Folivory has been reported for at least 17 species of Old World Megachiroptera,

and leaves eaten by bats include 44 species of plants represented by 23 families (Kunz

and Diaz 1995). Bats locally consume a large variety of leaves. For example, Cynopterus

brachyotis fed regularly on the leaves of 14 plant species in southern India (Tan et al.

1998), and Pteropus dasymallus on nine species in Taiwan (Funakoshi et al 1993). The

incidence of leaf pellets under feeding roosts in Taiwan was 37-50%, and occurred

almost throughout the year (Funakoshi et al 1993). However, this may be an

underestimate. Leaf pellets are often not noticed because they are inconspicuous among

other plant material on the forest floor (Kunz and Ingalls 1994).

Calcium is of particular interest in bat biology (Barclay 1994,1995, Kunz et al.

1995, Bernard and Davison 1996). It has been proposed that females may be stressed for

calcium due to the mineral demands of both pregnancy and lactation (Barclay

1994,1995). To compensate for the large size of their offspring, bats donate their own

skeletal calcium to build the bones of their young (Barclay 1995). Leaves represent a rich

and consistent source of calcium to bats that are mineral stressed. Calcium concentrations

are often much higher in leaves than in fruit (Nelson et al. 2000b, Ruby et al. 2000).








While some fruits may be high in calcium, it is not readily available if the Ca:P ratio is

less than the optimum of 2 tol (McDowell 1992, Robbins 1993). The Ca:P ratio is three

times higher in leaves than in fruits, which further suggests that leaves may be valuable

for their high calcium content (Kunz and Diaz 1995, Ruby et al. 2000).

This study examined if folivory among a sample of captive, wild-caught Tongan

fruit bats (Pteropus tonganus). This study is the first to examine the amount of leaves

that are consumed by individual fruit bats in a single night, and to calculate how much

calcium folivory contributes to total daily calcium intake. I also describe gender and age

differences in leaf consumption and explain an observed pattern of leaf eating.

Methods

Research was conducted from December 2000 to August 2001 on the island of

Tutuila, American Samoa (14 S, 170 W) in the South Pacific Ocean. All 23 (13 male,

10 female) Tongan fruit bats (Pteropus tonganus) were caught using large mist nets and

transported to the "bat house." The bats consisted of four adult male, nine juvenile male,

four adult female, and six juvenile female bats. Two of the adult females were lactating.

The bat house was a 4 x 3 m outdoor wooden structure with an adjoining 4 x 3 m

screened outdoor pen specifically built to house bats for these experiments. The outdoor

pen contained a single Callophyllum neo-ebudicum (Clusiaceae) tree for roosting and leaf

consumption (Trail 1994, Whistler 1994). This tree was the only leaf source for the bats

in the present study. The bats could fly and move easily within the outdoor enclosure.

Each night, bats were offered twice their body mass in confirmed bat foods from the

island (Banack 1996). The fruit type varied each day depending on fruit availability on

the island. See Chapter 3 for further details. Salt rings comprised of salt and mineral oil

and contained 96-99 % salt (Pet Products, Inc., Hauppauge, N.Y.) as well as collected








rainwater were available to the bats ad libitum. Feeding trials were conducted on

individual bats and lasted three to five days following a two day acclimation period, for a

total of seven days in captivity. Only one bat was present and tested at a time in the

outdoor enclosure. Fruit traps, or raised screen platforms, covered the ground of the

enclosure to catch food and leaves dropped by the bats while they were feeding.

The number of leaves and percentage of leaf eaten were recorded for each bat

daily. Limited samples of representative leaves were collected and dried at 105C for 24

h. Leaf samples included both whole leaves and leaves partially eaten by bats. Samples

were analyzed at the University of Florida in Gainesville, Florida USA. Dried samples

were prepared and digested according to Miles et al. (2000). Calcium concentrations

(ppm) were assessed by atomic absorption spectrophotometry (Perkin-Elmer AAS 5000

Norwalk, CT.). All values were calculated on a dry matter basis.

Males and females, and juveniles and adults, were compared to identify

differences in leaf-eating behavior. The bats seemed to fall into three general categories

from visual observation of Table 4-1. Bats that habitually consumed leaves more than

50% of the days they were in the pen were classified as habitual leaf-eaters. Some of the

bats consumed leaves occasionally, less than half of the days that they were in the

experiments, and were classified at occasional leaf-eaters. Some bats never consumed

leaves and were therefore classified as non-leaf eaters. These three groups were later

compared for total calcium consumption.

Supplementary calcium values for each group were calculated as the total amount

of leaf matter eaten by a bat multiplied by the calcium (Ca) concentration of the leaves

(8861.47 mg/g Ca). To determine how much calcium folivory contributes to total daily









Table 4-1. Age and gender of bats used in the experiment, and number of leaves, amount
of total leaf matter eaten (g), calculated calcium supplement (mg) gained by leaf-eating.


Gender Age


Male Adult
Female Juvenile
Female Adult
Female Adult
Male Juvenile
Male Juvenile
Male Juvenile
Female Juvenile
Female Juvenile
Male Juvenile
Male Juvenile
Male Juvenile
Female Adult
Male Adult
Female Adult
Male Juvenile
Male Adult
Male Juvenile
Female Juvenile
Male Adult
Female Juvenile
Male Juvenile
Female Juvenile


Leaf
eater?
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
no
no
no
no


calcium intake, I calculated the average amount of supplemental calcium consumed for

each of the three groups, and compared that to the average total calcium ingested by that

group. Differences in consumption of leaves between gender and age were compared

using two-tailed t tests. The Kolmogorov-Smimrnov test was used to evaluate assumptions

of normality for each variable, and Levene's test was used to evaluate the assumption of


Days
leaves
eaten (%)
50
20
100
100
100
100
33
25
40
100
60
60
67
100
67
80
50
50
25
0
0
0
0


Total
leaves
eaten
5
0.75
4
7
26
12
1
2
3
8
12
10
2
14
4
11
2
4
1
0
0
0
0


Total leaf Calcium
matter eaten (g) supplement (mg)
1.45 12.85
0.3 2.66
1.07 9.47
1.82 16.13
6.76 59.91
3.12 27.65
0.26 2.31
0.52 4.61
0.81 7.18
2.08 18.43
3.12 27.65
2.6 23.04
0.55 4.87
3.64 32.26
1.04 9.22
2.86 25.34
0.52 4.61
1.07 9.48
0.26 2.31
0 0
0 0
0 0
0 0








normality between groups (Sokal and Rohlf 1995). Total calcium consumption for the

three leaf-consumption groups were compared using a one way ANOVA.

Results

Ninety-four feeding trials were performed on 23 Tongan fruit bats. Leaves were

consumed by 82.7% of the bats in this study. More males (92%) consumed leaves than

females (70%). The total leaf mass eaten (g) differed (p = 0.02) between the sexes; males

consumed an average of 9.55 + 6.99 SD leaves, and females consumed an average of

2.97 + 2.04 leaves over the period of the feeding trial. This resulted in 22.14 + 16.00 SD

(g) of additional calcium for males and 7.06 + 4.56 SD (g) for females. The amount of

additional ingested calcium was different (p = 0.02) between males and females. Juvenile

male and juvenile female bats differed (p = 0.04) in their consumption of leaves (24.22 +

17.03 and 4.19 + 2.24, respectively); but male and female adult bats did not (p = 0.40).

Twice as many juvenile males ate leaves than juvenile females (8 males: 4 females).The

maximum number of leaves eaten in a single night (26) was by a young male.


45
40
g 40-
S 35
30 -
I:o 25-
20
15 1
S 10 -
5 5
0 -1
Adult males Adult females Juvenile males Juvenile females

Figure 4-1. Supplemental calcium ingested (mg/g) by folivory for adult and juvenile
males and females.








The number of leaves eaten by individuals over a five-day period ranged from

0.75 to 26 leaves. Overall, folivory provided 2.3 to 32.26 mg of additional calcium to the

diet of fruit bats in the present study. There were similar numbers of habitual and

occasional leaf eaters (11 and 8, respectively); however, habitual leaf eaters consumed

significantly more calcium through leaf-eating than occasional leaf eaters (p = 0.04).

Habitual leaf-eaters consumed an average of 10 + 6.56 SD leaves, which contributed an

additional 23.89 + 15.05 SD mg/g of dietary calcium (Figure 4-2). This represented an

average dietary increase in calcium of 11-46% when compared to the daily calcium

consumption for each bat in that group. Occasional leaf-eaters consumed an average of

2.34 + 1.54 SD leaves, which added an additional 5.75 + 3.82 g of calcium to their diet.

Occasional leaf-eating represented an average dietary increase in calcium of 3-22% when

compared to the daily calcium consumption for each bat. Non-leaf-eaters (n = 4) added

no additional calcium to their diet.

When eating leaves, P. tonganus often avoided the fibrous midrib, and instead ate

around it (Figure 4-3). I chemically analyzed portions of a leaf cut along the midrib that

did not contain the midrib to imitate leaf consumption by bats. These samples were

compared to whole leaves that contained the fibrous midrib. The portions with the midrib

contained 15.08 mg/g of calcium, and leaves without the veins contained 8.86 mg/g of

calcium. Unfortunately, the work was largely exploratory and several leaves were used to

produce only one analyzed sample for both the leaves containing the midrib and those

that did not contain the midrib. Thus, a statistical analysis of this sample was not

possible.










* Average daily calcium
consumption
* Supplemental calcium
from leaf-eating


0 -
Habitual leaf-eaters


Occasional leaf-eaters


Non-leaf-eaters


Figure 4-2. A comparison of total calcium ingested among habitual, occasional, and non-
leaf-eaters. Total calcium is average daily calcium and supplemental calcium from leaf-
eating combined. Total average daily calcium values for each group are calculated from
values found in Chapter 5.


Figure 4-3. Typical pattern of leaf consumption by Pteropus tonganus in American
Samoa.

Discussion

Previous studies of folivory (Marshall 1985, Lowry 1989, Funakoshi et al. 1993,

Kunz and Ingalls 1994, Kunz and Diaz 1995, Banack 1996, Tan et al. 1998, Ruby et al.








2000) were based on indirect means of quantifying leaf-eating by bats and thus were

unable to describe either the amount of leaves eaten per bat, or the sex and age of the leaf

consumer. This study quantified both the amount of leaves consumed by individual bats

and how much folivory contributed to total dietary calcium intake. The majority (83%) of

wild-caught bats engaged in leaf-eating while in captivity. Both sexes consumed leaves,

but male bats consumed more leaves than females, and juvenile males consumed both the

greatest number and volume of leaves of all groups. Habitual leaf-eating bats could

potentially increase their dietary calcium consumption by 46%. Clearly, folivory is both

widely and frequently practiced by P. tonganus, and it has the potential to contribute

significantly to the total amount of ingested calcium.

The assumption in conducting this study was that the brevity of the time that bats

spent in captivity would neither alter nor adversely affect the current mineral status of the

bats used in the experiments. That is, the deficiencies or excesses of their native diet

would influence their consumption patterns while in captivity. If this is true, then both

male and female bats consume leaves as a regular part of their diet in the wild, but

consumption patterns and volume of leaves eaten differs among sexes and groups.

Some bats consistently consumed leaves each day of the experiment, while others

only sampled the leaves intermittently. An interesting outcome of this analysis is that

although the average daily (dietary) calcium ingestion was lower for habitual leaf-eaters

than for occasional leaf-eaters, both groups were appeared similar in the total amount of

calcium ingested. Habitual leaf-eaters exhibited the greatest variation in daily dietary

calcium ingestion. Non-leaf eaters ingested the least calcium, but calcium ingestion by








non-leaf-eaters was very similar to the average daily calcium ingested by the habitual

leaf-eaters.

Kunz and Diaz (1995) observed only mature males carrying leaves, and

hypothesized that folivory may be limited to adult male bats. Overall, adult males

consumed significantly more leaves and ingested more leaf matter than adult females in

this study. Interestingly, leaf-eating was practiced most often by juvenile males. Other

work has suggested that compounds extracted by leaf fractionation could be possible

regulators of reproductive activity for bats (Kunz and Ingalls 1994, Kunz and Diaz 1995).

Erythrina leaves may contain one or more metabolites (alkaloids) important for

reproduction, and they are consumed by P. tonganus on the island of Tonga (Harris and

Baker 1959). Perhaps, in addition to being a rich calcium source that supports rapid

growth, leaves may also influence reproductive activity in young male bats.

Female bats consume leaves to access nutrients and minerals not available in

fruits (Kunz and Diaz 1995). Leaves analyzed from American Samoa were very rich

sources of calcium and other macrominerals. Leaves contained concentrations of many

nutrients comparable or higher than those of ripe fruit preferred by fruit bats in Samoa

(Nelson et al. 2000b). Leaves tended to have higher levels of calcium, sodium,

manganese, and magnesium than ripe native or agricultural fruits (Nelson et al. 2000b).

Also, leaves are widely available in both the wet and dry season (Whistler 1994). Thus,

leaves may represent a rich, year-round, readily accessible source of concentrated

minerals for female bats.








P. tonganus females in Samoa may also consume leaves for their high calcium

content. Banack (1996) found that female Tongan fruit bats in American Samoa gave

birth year-round, and young were seen on mothers all months of the year. She also

observed copulations with pregnant females, suggesting that the female was nursing

while allocating her own calcium for the skeletal formation of a new offspring (Barclay

1995, Banack 1996). Both gestation and lactation are nutritionally demanding, and their

combined effect may promote leaf-eating in females. In addition, the population of

P. tonganus in American Samoa has increased 3-fold over the last decade, following a

series of three destructive hurricanes that decimated the bat population (Craig et al.

1994b, Elmqvist et al. 1994, Brooke 1998). This population's rapid expansion may have

resulted in additional calcium stress to reproductive females. However, despite this

potential calcium stress, P. tonganus in American Samoa consistently chose low-calcium,

high-sugar agricultural foods that resulted in inadequate calcium consumption, and high

levels of retention that suggest calcium stress (Chapter 5). All together, the cumulative

demands of gestation and lactation, overlapping generations, a rapid population increase,

and a diet low in calcium, may promote in leaf-eating by female bats.

Recent evidence indicates that leaves are consumed by bats throughout the year.

There are reports of year-around leaf consumption based on either analysis of fecal

remains, leaf parts discarded beneath roosts (Lowry 1989, Parry-Jones and Augee 1991a,

Bhat 1994), or direct observation (Zortea and Mendes 1993). Bhat (1994) noted that leaf-

eating was common in each month of the year in C sphinx. Banack (1996) described

year-round leaf use by both Pteropus samoensis and P. tonganus in American Samoa.

This study is consistent with her findings; leaves were consumed in each of the eight








months of this study, with no apparent differences between months or seasons. However,

the manner in which Tongan fruit bats consumed leaves suggests that they are actively

avoiding the fibrous midrib, despite it being a rich source of calcium. Bats may avoid the

midrib because it has high levels of tannins or secondary compounds (Dasilva 1994).

This study has shown that males consumed significantly more leaves than

females, and that folivory can contribute significantly to the total dietary calcium of leaf-

eating bats. However, the motivation for folivory still cannot be ascribed to a single

factor; both the high calcium content and presence of hormonal compounds in leaves may

play integral roles. The propensity of males to consume large amounts of leaves suggests

a hormonal motivation, but the calcium contribution to the diet due to folivory is

significant and noteworthy. Future research should test whether hormonal compounds are

present in the Callophyllum neo-ebudicum leaves and if these compounds influence

reproductive cycles in this bat. Meanwhile, leaf-eating is a common practice among

Tongan fruit bats, and the leaves provide a rich and concentrated calcium supplement to

the often calcium-poor diet of fruit bats.













CHAPTER 5
BIOAVAILABILITY AND APPARENT ABSORPTION OF MINERALS CONSUMED
BY WILD TONGAN FLYING FOXES IN AMERICAN SAMOA

Introduction

Previous research on nutrition in bats has concluded that energy and protein are

the most important dietary nutrients (Thomas 1984, Herbst 1986, Fleming 1988).

Although dietary mineral composition and concentration are not often measures of

dietary quality (Cole and Batzli 1979, Batzli 1986), recent studies have illustrated the

importance of minerals in bat nutrition (Uhland et al. 1992, Barclay 1994,1995,).

Nutrients that are consumed at marginal or inadequate levels with respect to requirements

may limit animal performance (Oftedal 1991). Mineral nutrition can affect fecundity,

number of litters, and survival of offspring (Batzli 1986, Delgiudice 1990) and are critical

to the basic physiological functions of animals (McDowell 1992). Despite their

importance to survival and fecundity, mineral requirements remain largely unknown for

most species.

Calcium is the most abundant mineral in the body, and one of interest in bat

biology. For a pregnant or lactating female, inadequate calcium intake causes weakened

bones and low milk production (McDowell 1992). For her dependent young, inadequate

calcium from the mother results in inhibited growth, loss of body mass, and reduced

mineralization of bone that can result in lameness and bone fractures to young bones

(Radostits et al. 1994). Low dietary intakes also affect successive generations; offspring

of rats fed a poor calcium diet survived but could not reproduce and had only 75-80% of








the normal skeletal calcium content (Brommage and DeLuca 1984). The excessive

mineral demands of pregnancy and lactation result in a negative calcium balance as

females donate their own skeletal calcium reserves to build the skeletons of their young

(Radostits et al. 1994, Bernard and Davison 1996). Raising several young in sequential

years may result in osteoporosis in females, resulting in bones that fracture and easily

break (Keeler and Studier 1992, Studier et al. 1994a).

Requirements for calcium and all other minerals needed to maintain the health of

bats are currently unknown. To examine mineral levels in bats, previous studies have

used indirect methods to quantify minerals in the diet. Methods of calcium status

evaluation have included analysis of blood plasma (Kunz and Stem 1995, Heard and

Whittier 1997, Dierenfeld and Seyjagat 2000a, Kwiecinski et al. 2001), whole body

mineral composition (Studier 1994, Studier and Kunz 1995), and fecal analysis without a

knowledge of the types or quantities of foods consumed in the diet (Studier et al. 1991,

Keeler and Studier 1992, Studier et al. 1994b). However, none of these methods

quantified the amount of minerals consumed, absorbed, or the absorption efficiency of

minerals. Together, these factors can create a portrait of bat feeding and demonstrate the

degree of mineral inadequacy in an individual.

The present study was designed to determine mineral absorption efficiencies of

flying foxes using apparent absorption. Apparent absorption has been used in a limited

number of nutritional studies (Belovsky and Jordan 1981, Dierenfeld and Seyjagat

2000b), but not on wild bats. This method measures both mineral intake and fecal

excretion, and it can account for the unique manner of feeding by fruit bats. To feed on

fruit and leaves, fruit bats chew the plant matter into a fibrous pellet, swallow the juice,








and expel the flattened pellet (Lowry 1989, Kunz and Ingalls 1994, Kunz and Diaz 1995).

Previous studies were not able to account for minerals found in the expelled pellet.

Due to a lack of mineral requirement standards for fruit bats, target levels for

nutrients are based on estimated requirements reported for other mammals (NRC 1995),

or what is fed in captivity to maintain breeding populations (Courts 1998). Previous

research has compared values either to those of domestic laboratory mammals or to a

generalized mammalian standard (Oftedal and Allen 1996, Dierenfeld and Seyjagat

2000a). Mineral requirements established for rats and primates seem the most appropriate

models to compare to fruit bats in the absence of true bat values. Rats are similar in size

to bats and are monogastric, whereas primates are larger but share a monogastric gut,

similar feeding habits, and perhaps an evolutionary past with bats (Pettigrew 1991).

No studies have confirmed that bats are significantly different than other

mammals in their general nutrition needs, but differences in absorption of minerals can

occur among similar species, and dietary diversity is high within the family Pteropodidae

(Walinski and Guggenheim 1974, Marshall 1985, Courts and Feistner 2000). Nutrient

requirements are affected by such factors as growth rate, reproductive output, and

metabolic needs (Oftedal and Allen 1996). These factors can be markedly different for

rats and primates when compared to bats. Moreover, published requirements for

laboratory animals are often in excess of true requirements to allow for ingredient

variation and other margins of safety (Oftedal and Allen 1996). Thus, mineral

requirements of domestic animals may be an inappropriate standard for fruit bats.

The Tongan flying fox, Pteropus tonganus (Quoy and Gaimard, 1830) was used

in this study. P. tonganus is one of the most widely distributed of all Pteropus species








(Koopman and Steadman 1995) and has adapted to many habitats and food types. It is a

highly plastic forager and feeds on both native and agricultural fruits (Banack 1996,

Pierson and Rainey 1992). Its wide geographic range and use of both native and

agricultural fruits suggests a highly generalized digestive system. Additionally, the

Tongan fruit bat is a mid-size fruit bat (300-600 g; Miller and Wilson 1997) so mineral

absorption values are applicable to a wide range of body sizes. Overall, the Tongan fruit

bat is an excellent study species because results should be widely applicable to many

other flying fox species.

This study used apparent absorption to quantify mineral ingestion and absorption

in P. tonganus. Mineral absorption results were compared to both mammalian standards

and other fruit bat mineral retention values to quantify if Tongan fruit bats met

recommended mineral standards or were mineral stressed as determined by elevated

absorption levels. This was the first attempt to quantify ingestion, absorption, and

retention for minerals in wild populations of flying foxes.

Methods

Netting and Housing of Bats

Research was conducted from September 2000 to August 2001 on the island of

Tutuila, American Samoa (14 S, 170 W) in the South Pacific Ocean. Tongan fruit bats

(13 males, 10 females) were captured in large mist nets (6-18 m, 4 inch mesh, Avinet,

Inc.) attached to pulleys set high in coconut trees or on tall poles. Nets were raised at

sunset and were checked every 30 minutes until midnight when they were taken down.

Netting was conducted all over the island so that the bats used in the mineral retention

trials would represent the bat population of the entire island.








Following capture, a single bat was transported to the "bat house," a 4 x 3 m

wooden building with an adjoining 4 x 3 m screened outdoor structure. Both structures

were built to temporarily house bats for these experiments. The outdoor structure was a

wooden frame enclosed with rat wire and screen, and was lined with fishing nets to

facilitate roosting and movement of bats. It contained a single Callophyllum neo-

ebudicum (Clusiaceae) tree that could be used by bats for roosting and leaf consumption.

The bats could fly and move easily within the outdoor structure. Water and salt licks were

available to the bats ad libitum inside the outdoor structure at all times. Calcium blocks

were occasionally available to bats as part of another experiment (Chapter 3). Salt licks

consisted of salt and mineral oil, and calcium blocks consisted of calcium sulfate and

ground limestone (8 inl Pet Products, Inc., Fairport Harbor, OH).

Mineral Metabolism Experiments

Bats were sexed, weighed, measured, and examined for injuries upon capture.

Following a two day acclimation period, the bats were tested for five days in the bat

house. Each bat spent a total of seven days in captivity and only one bat was present and

tested at a time in the outdoor structure. Bats were given twice their body mass in food

nightly (wet weight) so that hunger would not result in atypical food choices. To test if

Tongan fruit bats would choose high calcium fruits if they were available (Chapter 3),

each bat was presented nightly with equal masses of one high-calcium fruit and one low-

calcium fruit. The fruit type varied each day depending on fruit availability on the island.

Native fruits were typically high in calcium and agricultural fruits were low in calcium

(Nelson et al. 2000a). The high-calcium fruits used in the experiments were 2-3 times

higher in calcium than the low-calcium fruits (1.06-12.28 mg/g, 5.75 1.39 mg/g for

high-calcium fruits, 0.55-2.46 mg/g, 1.30 0.59 mg/g for low-calcium fruits). Mineral








concentrations for all native and agricultural fruits were based on previous work

analyzing the mineral concentrations of bat fruit in Samoa (Nelson et al. 2000a, 2000b).

According to Banack, all fruits given to the bats were preferred P. tonganus foods in

American Samoa (Banack 1996,1998).

During fruit preparation and handling, disposable plastic gloves were worn, and

stainless steel or plastic utensils were utilized to avoid mineral contamination. Utensils

were washed using 1:1 vinegar and distilled water solution. Fruits were cut into equal-

sized cubes, weighed, and suspended on plastic cable ties from a large wooden dowel rod

(the food bar). Desiccation factors were determined from sub-samples of both high and

low-calcium fruit, handled and prepared in the exact same way as the fruits given to the

bats, but placed in a separate small cage within the outdoor structure each night. The

representative fruits were weighed the next morning to establish a desiccation factor for

each fruit type that was later subtracted from the samples to yield an accurate estimated

wet weight value. Fruit traps, or raised screen platforms, covered the ground of the

outdoor structure to catch food and leaves dropped by the bats while they were feeding.

The fruit traps were washed daily with collected rainwater.

All samples were collected the following morning. Food remains were separated

into two categories: uneaten food still hanging from the food bar or dropped to the fruit

traps below (hereafter called orts), and food that had been chewed on and sucked of all its

juice and then spit out as small flattened disks (called ejecta). Fecal matter, representative

samples for both fruit types, and partially eaten leaves were also collected, separated, and

weighed. A wet mass was recorded for each type of sample (orts, ejecta, fecal,

representative fruit, and leaves) for each day. Seeds were removed from all fruits before








weighing. Samples were placed in a drying oven for 24 hours at 1050 C and dried to a

constant mass and reweighed. Urine was not collected separately because it is not used

for apparent absorption (McDowell 1992). All samples were stored in plastic bags in

airtight plastic containers containing desiccant. The containers were stored in an air-

conditioned laboratory to prevent mold growth until laboratory analyses were done.

Analysis of Samples

Samples were analyzed at the Animal Nutrition Laboratory at the University of

Florida in Gainesville, Florida. Dried samples were weighed and dry ashed at 550C for

12 hours. Samples were prepared and digested according to the procedures of Miles et al.

(2001). Mineral concentrations of Ca, Cu, Fe, Mg, Fe, Mn and Zn were assessed using

flame atomic absorption spectrophotometry using a Perkin-Elmer AAS 5000 (Perkin-

Elmer 1980) after wet digestion in HCL and dilution in 1% lanthanum solution.

Phosphorus was measured separately using a colorimetric assay (Harris and Popat 1954).

Samples were analyzed in duplicate if sample size allowed. Standard reference material

(citrus leaves 1572, National Institute of Standards and Technology, Gaithersburg, MD.)

was run with each sample set.

Data analyses included calculating the total mineral consumption by each bat for

each day. The mineral concentration numbers of fruit were multiplied by the amount of

fruit that was consumed each day. This resulted in a value for total mineral intake.

Apparent absorption was calculated using the following equations. Mineral

calculations were calculated on a dry matter basis. Each calculation was performed for

each mineral, for each bat, for each day they were housed in the outdoor structure,

resulting in 805 apparent absorption values. The average absorption value for each bat,

for each mineral, for all days in captivity was averaged and compared.








Total mineral intake (g) =
(total amount of fruit offered (g)) (total amount of orts (g)) (total amount of
ejecta (g))

Apparent mineral absorption (%) =
(total mineral intake) (fecal mineral)
(total mineral intake) x 100

Statistical Analysis

To statistically analyze the data set, mineral apparent absorption values among all

bats were tested for normality using the univariate Shapiro-Wilkes test. The values were

not normally distributed, so the data were rank-transformed (Sokal and Rohlf 1995). The

transformed data were analyzed using Principal Components Analysis (PCA) to evaluate

patterns of variation between different bat sexes, ages, and reproductive states of the bats.

PCA assesses relationships of independent variables within a single data set and places

factors that are ecologically similar in close proximity in ordination space (Garigal et al.

2000). Comparisons of apparent absorption values for each mineral between P. tonganus

and the giant flying fox Pteropus vampyrus (Chapter 7) were analyzed using a two

sample t-test assuming equal variance (Sokal and Rohlf 1995).

Results

Mineral Consumption

A total of 115 feeding trials were performed on 23 wild Tongan fruit bats. The

bats consisted of four adult male, nine juvenile male, four adult female, and six juvenile

female bats. Two of the adult females were lactating. When all values for average mineral

absorption values were analyzed, PCA resulted in no clear patterns or trends for either the

macrominerals or trace elements (Table 5-1). Subsequent t-tests failed to distinguish clear

patterns among the data for different sexes, ages, and reproductive classes for all








minerals. Thus, data were grouped and evaluated as a single data set for all future

analyses.



Table 5-1. Results of the principal components analysis comparing average mineral
absorption values for all bats.
Factor 1 Factor 2
Eigenvalue 3.33 1.26
Percent variation explained 47.50 17.98
Contributions of each individual variable
Calcium 0.45 0.26
Phosphorus 0.32 0.52
Magnesium 0.34 0.14
Zinc 0.29 -0.69
Iron 0.28 0.24
Manganese 0.48 -0.12
Copper 0.42 -0.32

The results of the PCA for mineral components identified two factors with

eigenvalues > 1 that together explained 65.48% of the variation. Factor 1 loaded

positively for all minerals, but the factor scores were highest for calcium, manganese and

copper. Factor 2 loaded positively for all minerals but zinc, manganese, and copper.

Phosphorus loaded most heavily and positively of all the minerals in Factor 2 (0.52), and

calcium was the second highest with the factor score 0.26.

Bats were offered an average of 566.7 + 109.30 g (wet weight) of food per day of

which an average of 332.2 + 113.00 g was consumed. Tongan bats consumed 85% of

their body mass daily on a wet matter basis and 17% on a dry matter basis. Despite the

large amount of food rejected daily, both the amount of food offered and the amount

consumed consistently failed to meet the required mineral standards established for

laboratory animals (Table 5-2). Calcium (Ca), phosphorus (P), manganese (Mn), and

copper (Cu) were below the required levels for both the diet offered and consumed. Zinc








(Zn) greatly exceeded the rat and primate requirements in both what was offered and

consumed. The amount of magnesium (Mg) offered was sufficient, but it was not

consumed in adequate amounts to meet requirements. Both the amount of iron (Fe)

offered and consumed met the requirements for the rat but not for the primate.



Table 5-2. A comparison of nutrient levels offered and consumed by wild P. tonganus to
values for standard diets of rats and primates..

Ca P Mg Zn Fe Mn Cu
Rat' 0.56 0.33 0.06 11 39 11 6
Primate2 0.54 0.43 0.16 11 196 30 10
Diet offered to bats 0.23 0.23 0.21 54 100 9 5
Diet consumed by bats 0.07 0.11 0.07 50 69 4 2
Diet consumed by bats 0.08 0.11 0.08 50 70 5 2
eating leaves*
1= NRC 1995, 2= Ofiedal and Allen 1996.
*Folivory data are from Chapter 5
Zn, Fe, Mn, and Cu are reported as ppm, and values for Ca, P, and Mg are reported as
percent

The addition of minerals due to leaf-eating (Chapter 4) slightly raised ingested

mineral levels but overall had little impact on mineral consumption for Tongan fruit bats.

Folivory slightly raised Ca and Mg levels, but only to one-seventh of the calcium

requirement for rats and one half the magnesium requirement of primates.

Calcium and phosphorus interact and influence the absorption of each other.

Calcium is best absorbed when it occurs at a Ca:P ratio of 1:1 to 2:1 (McDowell 1992). In

this study, the expected quantity of calcium as well as the Ca:P ratio of 1:1 were not

achieved in the food consumed by P. tonganus. The diet offered contained a 1:1 ratio,

but the Ca:P ratio for ingested food was 0.6:1; approximately half the expected

requirement. A low ratio can inhibit calcium absorption making less available to the

animal for physiological functions (McDowell 1992).








Additional minerals were available to bats from drinking water. Collected rain

water was given daily to bats. The values for 10 ml of Samoan rainwater (n=2 samples)

are reported in ppm: Ca = 0.11, P = 0.06, Mg = 0.09, Zn = 0.05, Fe = 0.0, Mn = 0.02,

Cu = 0.02. The amount of water consumed by bats remains unknown, so the mineral

contribution of water to the diet could not be calculated. Salt licks and calcium blocks

may have contributed to mineral ingestion, but use and amount of minerals ingested

could not be quantified.

Mineral Absorption

Results of the present study were compared to a parallel investigation on the

captive giant flying foxes P. vampyrus (Table 5-3). The P. vampyrus study was

performed on groups of captive bats (Chapter 7) while P. tonganus were tested

individually, but all other techniques used were identical for both studies. The captive

colony of P. vampyrus includes lactating females and their offspring as well as non-

reproductive females. Mineral apparent absorption values for the 23 P. tonganus used in

the present study and those of the three pens of P.vampyrus have been averaged and

presented in Table 5-3 as a single value for each species and mineral.

The apparent absorption values reported for both P. vampyrus and P. tonganus are

similar. P. vampyrus met their mineral needs for both amount of minerals offered and

consumed for all minerals but copper (Chapter 7). In contrast, P. tonganus was deficient

in amounts consumed for Ca, P, Mn, and Cu (Table 5-2). There was a difference (p <

0.05) in apparent absorption values between P. tonganus and P. vampyrus for calcium (p

= 0.04), phosphorus (p = 0.001), zinc (p = 0.001) and iron (p = 0.001), where P. tonganus

had higher absorption values for those minerals.








Table 5-3. A comparison of mineral apparent absorption values for two species of flying
foxes, Pteropus tonganus and Pteropus vampyrus*.

Pteropus vampyrus Pteropus tonganus
Calcium** 65% 79%
Phosphorus** 63% 84%
Magnesium 68% 73%
Zinc** 39% 85%
Iron** 63% 76%
Manganese 69% 68%
Copper 72% 75%
*Data sets used identical analytical procedures. P. vampyrus information is found in
Chapter 7.
** significantly different (p < 0.05) absorption values between species

Discussion

This study showed that wild Tongan fruit bats have high mineral absorption

values. The absorption values for P. tonganus are more similar to those found for P.

vampyrus, another flying fox species, than to either rats or primates (see Table 5.4).

However, when compared to P. vampyrus, the Tongan fruit bat had significantly higher

absorption rates for several important minerals. This suggests that the Tongan fruit bat

population may be highly mineral stressed for several macrominerals that are only

available in minimal quantities in their diet.

Because the apparent absorption technique has never been attempted before on

wild Tongan fruit bats, it is a challenge to find values to compare to those found in the

present study. Apparent absorption values for monogastric animals and another species of

bat, Desmodus rotundus, are presented in Table 5-4 to serve as a basis of comparison to

values found for P. tonganus in the present study. The values presented are for typical

animals that are not under conditions of mineral stress. For the minerals being studied,








values for monogastric animals are typically low and do not exceed 50% absorption

under normal conditions.



Table 5-4. Mineral apparent absorption values for selected monogastrics species.
Absorption values found in this table are from non-mineral stressed individuals.
Mineral Animal Absorption (%) Reference
Calcium Human 21% Coudray et al. 1997
Fox squirrel 30-39% Havera 1978
Vampire bat 16-24% Coen 2002
Phosphorus Human 24-31% Hevesy 1948
Pig 17-47% Jungbloed and Kemme 1990
Vampire bat 18-44% Coen 2002
Magnesium Human 46% Coudray et al. 1997
Pig 50-60% Miller 1980
Vampire bat 25-29% Coen 2002
Zinc Human 14% Coudray et al. 1997
Rat 17-20% Tidehagetal. 1988
Manganese Human 3-4% Hurley and Keen 1987
Rat 3-4% Greenberg et al. 1943
Iron Human 22% Coudray et al. 1997
Rat 9-60% Fairweather-Tait and Wright 1991
Vampire bat 5-14% Coen 2002
Copper Human 25-70% Strickland et al. 1972
Sheep 3-13% Suttle 1991

However, when dietary minerals are deficient in the diet, mineral absorption often

increases due to homeostatic mechanisms that compensate for inadequate intake of that

nutrient (Ammerman 1995). Values for normal, non-stressed animals are given as a

reference for each mineral. Absorption values for mineral deficient animals are typically

3-4 times higher than are found for animals with adequate mineral intake.

Together, these two tables illustrate how elevated the apparent absorption values

are for P. tonganus in this study. These values for P. tonganus are much higher than are

typical of other monogastric animals under normal conditions, and are instead similar

tothose found for animals that are nutritionally stressed for minerals. Mineral apparent








Table 5-5. Apparent absorption values for minerals in animals under nutritional stress
compared to animals at normal mineral intake levels.
Mineral Animal Absorption (%) Mineral Status Reference
Calcium Human 28% normal intake Brine and Johnson 1955
43% deficient intake Brine and Johnson 1955
58% preterm infant Bronner et al. 1992
75% growing child RDA 1989
Phosphorous Human 24-31% adult Hevesy 1948
71% preterm infant Koo and Tsang 1991
Magnesium Rat 26% normal intake Brink et al.1992
57% deficient intake Brink et al. 1992
Iron Human 2-15% normal adult Josephs 1958
20-60% anemic adult Josephs 1958
Manganese Rat 3-4% adult Greenberg et al. 1943
20% young Keen et al. 1986

absorption values for P. tonganus are most like those of P. vampyrus, but P. tonganus

values are still significantly higher for several minerals. However, the majority of

absorption values for P. vampyrus are those of lactating females and their pups. The

highly elevated apparent absorption values for P. tonganus suggest that this population

may be highly stressed for mineral nutrients while consuming its current diet in the wild.

The diet of P. tonganus typically includes a large volume of low-nutrient agricultural

fruits (Banack 1996), which potentially contributes to nutrient deficiency in this species.

Bioavailability and Absorption

Bioavailability is defined as the degree to which an ingested nutrient is absorbed

in a form that can be used for metabolic functions by an animal (Ammerman 1995). Total

intake of a nutrient depends on both the intake and bioavailability of the nutrient (Oftedal

1991). The absorption level of a mineral provides an estimate of its bioavailability. For

example, high absorption indicates that the mineral's bioavailability is high and low

absorption indicates that the mineral's bioavailability is low. Minerals found in plants are

often less bioavailable than in animal sources because fiber in plants binds to minerals








and makes them unavailable for absorption (Soares 1995). Because of this, only 30-50%

of ingested calcium in humans is absorbed by the body (Arnaud and Sanchez 1996,

Bronner 1998).

Fruit bats consume foods by chewing them into a bolus, swallowing the liquid

portion, and ejecting the flattened fibrous pellet (Lowry 1989, Funakoshi et al. 1993,

Kunz and Ingalls 1994, Kunz and Diaz 1995). By rejecting the fibrous portion and

swallowing the juice, fruit bats may be increasing the bioavailability of the minerals in

fruits. Levels of minerals were usually more concentrated in the ejecta pellet than in the

diet samples in this study. This suggests that the minerals are in a less soluble form than

in fruit and remain in the fiber portion after it has been ejected. To test this hypothesis,

apparent absorption was recalculated by including the fibrous pellet in the feces

calculation, assuming the portion of calcium in the pellet would not have been absorbed

in the gut. The calculation resulted in a 5-20% decrease in mineral apparent absorption

values. Thus, the unique pattern of bat consumption may remove the highly absorbable

minerals from the pellet by placing them in solution. Because calcium must be in solution

to be absorbed (Bronner and Pansu 1999), apparent absorption will be high after

ingesting the fruit juices because the ingested minerals are highly bioavailable (Pansu et

al. 1993, Duflos et al. 1995,). This manner of feeding results in minerals that are readily

absorbed, resulting in high absorption values. Bioavailability and solubility of minerals

are crucial under conditions of low mineral intake (Bronner and Pansu 1999).

In addition to their unique feeding behavior, fruit bats have several anatomical

adaptations that further increase mineral absorption. The stomachs of fruit bats are large,

and the small intestine is long and convoluted, both potential adaptations to increase








absorptive surface area (Dempsey 1999). In addition to the solubility of the mineral, time

spent in the intestine is the differentiating factor as to how much mineral is absorbed

(Bronner 1998). The relatively long intestine in Old World fruit bats may be up to nine

times their body length (Okon 1977). The added surface area of the stomach and

intestines may be essential to counteract short gut retention times among fruit bats (30

minutes, Tedman and Hall 1985), and to increase nutrient absorption time of

nutritionally-poor foods.

Mineral Stress

Fruit bats may have higher mineral absorption values than other monogastric

animals due to their unique feeding patterns and the potentially high bioavailability of

minerals in their food. However, high absorption values also suggest mineral stress. An

animal will absorb more of a nutrient if the nutrient is deficient in the body or diet

(Ammerman 1995). P. tonganus had higher absorption levels for many critical nutrients

when compared to P. vampyrus, a flying fox twice its size (Kunz and Jones 2000). The

highly elevated absorption values of P. tonganus are of particular interest because P.

tonganus did not meet its expected mineral requirements for either offered or consumed

food, whereas P. vampyrus met all of its expected nutrient requirements.

Although agricultural fruits were nutrient poor, and Tongan fruit bats did not meet

their expected mineral requirements, bats consistently rejected 41% the offered food and

consumed only 85% of their body mass in food nightly. Unlike other bats that consume

agricultural fruits, P. tonganus did not consume 2.5 times their body mass to meet its

nutrient requirements (Dempsey 1999). Although low in nutrients, foods offered to P.

tonganus were documented as preferred bat foods in American Samoa (Banack 1996,

1998), and were highly preferred to native fruits by fruit bats in fruit preference








experiments (Chapter 3). In addition to being low in nutrients, the foods ingested resulted

in a low Ca:P ratio of less than 1:1, which further inhibited mineral absorption

(McDowell 1992). Tongan fruit bats consistently chose and consumed low-nutrient fruits

that did not meet the mineral requirements of the rat, primate or mammalian standard.

Tongan fruit bats in this study consumed only one-eighth of the expected

requirements for calcium and one-third of the phosphorus requirements required for a rat.

Yet despite not meeting these requirements, P. tonganus births have been observed year

round in Samoa, with high numbers of pregnant and lactating females seen throughout

the year (Banack 1996). The population has increased three-fold in the last decade

following a series of hurricanes that had severely reduced the population (Craig et al.

1994b, Pierson et al.1996, Brooke 1998). Thus, rapid population expansion coupled with

the high nutrient cost of bearing a single young (Barclay 1994, Kunz and Stem 1995),

and despite the consumption of nutrient-poor food, brings into question the source of

minerals, particularly calcium, to support rapid population growth.

The priority of all mammals is to maintain calcium concentrations in plasma close

to 2.5 mmol (100 mg) 1' despite fluctuations in the amount of calcium ingested (Amaud

and Sanchez 1996, Hurwitz 1996). This concentration is needed to maintain calcium

functions such as cellular metabolism, blood clotting, enzyme activation, and

neuromuscular action (McDowell 1992, Soares 1995). Plasma levels will not reflect

mineral deficiency for minerals such as calcium, because homeostatic mechanisms

maintain calcium levels despite dietary deficiencies (McDowell 1992). Bone acts as a

large storehouse for calcium: 99% of the calcium in the body is stored in bone. If plasma

calcium concentrations begin to decrease, calcium is quickly mobilized and resorbed








from bone to return plasma calcium levels back to normal (Bronner 1992, Garel 1987).

Bone calcium is in a constant state of flux, and it is resorption rather than accretion that is

highly responsive in restoring plasma calcium levels (Kwiecinski et al. 1987a).

Changes in bone calcium therefore reflect the extent to which dietary calcium

meets the calcium requirements of bats (Bernard and Davison 1996). Females readily

allocate their own skeletal calcium to build the skeletons of their offspring, and exhibit

marked bone-thinning and structural changes to bone as a result of the calcium demands

of pregnancy and lactation (Kwiecinski et al. 1987b, DeSantiago et al. 1999). Calcium

levels and bone density can be restored following lactation, with the consumption of an

adequate calcium diet (Kwiecinski et al. 1987b). However, Tongan fruit bat females in my

feeding trials were not consuming a nutritionally adequate diet based on expected values.

Their diet was marginal for most of the macronutrients examined in this study, and

extremely poor in calcium. Thus, if and how Tongan fruit bats are able to rebuild their

skeletons following pregnancy and lactation remains unknown.

Future research

The only true measure of female calcium levels is a bone density test. Porosity of

bone serves as an indicator of the calcium status and could determine if the skeletons of

P. tonganus females are porous and osteoporitic, or are healthy, having recovered from

the demands of raising young. To test these ideas further, I predict the following

concerning bone density.

1. Flying fox species that prefer agricultural fruits should have lower bone density
than those that prefer nutrient-dense native forest fruits. This prediction could be
tested using P. tonganus and its congener Pteropus samoensis in American Samoa
(Banack 1996, Pierson and Rainey 1992, Nelson et al. 2000a) or Pteropus
vampyrus lanensis and Acerodonjubatus in the Phillipines (Steir and Mildenstein
2001, Mildenstein 2002).








2. Tongan fruit bat's preference for nutrient-poor agricultural fruits is relatively
recent and corresponds with human settlement of the South Pacific islands. This
could be tested by conducting bone porosity tests on archaeological bat remains
that predate human settlement of the islands and comparing them to the bones of
modem Tongan fruit bats (see Steadman 1991, Kirch et. al. 1992).

3. Female bats that consume agricultural fruits and have had many pups should have
lower bone density and higher bone porosity than adult non-parous females or
adult males of the same species that consume an agricultural diet.

4. Supplementing the diet with calcium should lower mineral absorption rates and
increase bone density. The diet of P. vampyrus in captivity was supplemented
with additional Ca. Therefore, with Ca supplementation, P. tonganus should
increase bone density and decrease mineral absorption over time, resulting in
values similar to those of P. vampyrus.

Theoretically, animals should evolve feeding behaviors that enhance the intake of

limiting nutrients (Oftedal 1991). Fruit bats may have adapted behaviorally and

anatomically to increasing the bioavailability and absorption of minerals from their diet.

These adaptations probably arose in response to the evolutionary constraints on increased

wing loading from fiber in fruits (Dudley and Vermeij 1992,1994) and to flying fox's

preference for high-sugar, low-nutrient fruits (Parry-Jones and Augee 1991 a, Nelson et

al. 2000a). However, it is not yet known if the preference for high-sugar, low nutrient

foods predates the arrival of humans and agriculture on South Pacific islands.

In conclusion, by producing a rejected fiber pellet and swallowing only the juice

while eating fruits and leaves, bats may place highly bioavailable minerals in solution.

These minerals can then be readily absorbed in the abundant surface area of a large and

highly convoluted stomach and small intestine. The elevated apparent absorption values

found in P. tonganus suggest that ingested minerals are either highly bioavailable and

readily absorbed, or this population is in severe mineral stress due to its preference for

agricultural fruits. Future work may distinguish if elevated absorption values are unique

to Tongan fruit bats or are typical of other flying fox populations. Much work remains to





65


be done on the mineral nutrition of fruit bats so that base values can be established for

different species and animals in different physiological states.













CHAPTER 6
NUTRITIONAL LANDSCAPE ECOLOGY AND HABITAT USE BY TONGAN
FLYING FOXES IN AMERICAN SAMOA

Introduction

Flying foxes of the genus Pteropus are strong fliers capable of traveling long

distances (Nelson 1965, Eby 1991, Spencer et al. 1991, Banack 1996, Palmer and

Woinarski 1999, Shilton et al. 1999, Palmer et al. 2000). Pteropus species may commute

up to 50 km nightly, traveling at speeds of 40 km/h while searching and foraging among

food patches throughout the landscape (Richards 1990, Spencer et al. 1991, Palmer and

Woinarski 1999, Banack 1996). This allows flying foxes to access patchily distributed

fruit, nectar, and flowers, and to avoid local food shortages by seeking out distant,

scattered food resources (Bronstein 1995). Thus, an entire forest or all of a small oceanic

island may represent potential foraging habitat to a flying fox.

The selection of a foraging patch within a heterogeneous landscape has been

described as a hierarchical decision process that occurs at different levels: regional,

landscape, plant community, or at the level of the individual plant (Senft et al. 1987).

Potentially using significant powers of spatial memory and learning, flying foxes range

widely with information on both the location and quantity of resource patches within the

landscape (see Lima and Zollner 1996, Zollner and Lima 1999). For example, the black

flying fox (Pteropus alecto) exploited landscape patchiness at two scales, between broad

vegetation types and within vegetation types, and selected sites that were rich in

resources from among the homogeneous forest matrix (Palmer et al. 2000). Its selection








highlighted the patchiness of resources in the landscape (Palmer et al. 2000) and

demonstrated the bat's awareness of differences in habitat quality among the patches.

Animals should utilize resource patches within the landscape in a way that

maximizes fitness (Lima and Zollner 1996). In certain stages of a life cycle, nutritional

needs can be very specific, and filling those needs can be crucial for population survival.

Habitats in which such needs can be met are often considered key to population

persistence (Kozakiewicz 1995). For example, calcium is often limited in the diet of bats,

and females may be calcium stressed during pregnancy and lactation (Keeler and Studier

1992, Radostits et al. 1994, Studier et al. 1994a, Bernard and Davison 1996). Inadequate

levels of dietary calcium can result in low milk production in females and inhibit growth

in offspring (McDowell 1992, Radostits et al. 1994). Limited calcium availability in the

diet has the potential to limit fecundity and survivorship of animal young (Batzli 1986),

which may affect population levels in bats (Barclay 1995). Increased calcium

requirements during pregnancy and lactation may be relieved by the consumption of

concentrated sources of calcium such as figs (Ficus spp.) or other calcium-rich fruits

(Nelson et al. 2000a, Ruby et al. 2000). Foraging areas that contain concentrated sources

of calcium should potentially be important for reproductive bat populations and for

population persistence.

Before the arrival of Polynesians more than 3,000 years ago, most of Tutuila,

American Samoa, was covered in native rainforest (Cole et al. 1988, Whistler 1992, Hunt

and Kirch 1997). Since then, human activities such as land clearing and shifting

cultivation have altered much of the forest area and replaced rainforest with mixed crops

and residential areas (Cole et al. 1988). Previous nutritional analysis of native and








agricultural fruits indicate that native fruits are a much more concentrated source of

nutrients, particularly for the mineral calcium (Nelson et al. 2000a). Thus, patches that

contain native fruits may represent higher quality habitat than those that contain nutrient-

poor agricultural fruits. Habitat patches that contain calcium-rich fruits could potentially

be used to increase dietary calcium consumption.

The Tongan fruit bat, P. tonganus, is a habitat and feeding generalist, that forages

in both native and agricultural areas on 42 species of plants on Tutuila, American Samoa

(Wilson and Engbring 1992, Trail 1994, Banack 1996), but the extent of use of each

habitat type is unknown (Brooke 1998). Tongan flying foxes are able to transverse the

length of Tutuila island in a single night, and use different sides of the island for foraging

throughout the night (Banack 1996). Tutuila is isolated from other islands by an ocean

barrier of 100 km. Thus, the island represents a single, isolated, heterogeneous foraging

area. This results in a unique situation that allows one to study the entire foraging area

available to P. tonganus in American Samoa.

Nutritional landscape ecology combines the concepts of nutritional ecology and

landscape ecology to determine if animals select nutrient-rich areas as they forage within

the landscape. In the present study, I evaluated whether Tongan fruit bats foraged

preferentially in high calcium habitat types to increase consumption of calcium. It was

predicted that Tongan flying foxes, particularly pregnant and/or lactating females, would

forage within calcium-rich areas to obtain supplemental calcium in their diet.

Methods
Major Vegetation Types

This study was conducted between February 2000 and August 2001 on Tutuila,

the largest island in American Samoa (14 S, 170 W) in the South Pacific Ocean. The








three major vegetation types on present-day Tutuila are native forest, mixed agroforest,

and village agricultural areas. Native forest is the climax forest for the island and includes

upland, mangrove, moss, and coastal forest (Cole et al. 1988, Whistler 1994). Native

forest on Tutuila includes a rich diversity of plant and tree species with a 30% level of

endemism (Whistler 1992, 1994). Mixed agroforest results from disturbance, either

natural or anthropogenic, and is a transitional stage between plantation land and native

forest. In this forest type, fruit trees are planted among secondary growth forest trees

(Cole et al. 1988). Village agricultural lands include land cleared to grow fruit trees in

sparse density, and are adjacent to residential areas that include villages, plantations, and

roads (Cole et al. 1988). A vegetation habitat map of Tutuila was available for Tutuila

and coded for the three major vegetation types (Cole et al. 1988, Freifeld 1998).

Nutritional Classification of the Major Vegetation Types

The three vegetation types of the island were categorized as either high,

intermediate, or low in nutrient availability based on the calcium content of the fruits

found within each habitat (Table 6-1). Nutritional analysis indicated that minerals were

more concentrated in native fruits than in agricultural fruits (Nelson et al. 2000a). Figs

are an especially rich source of calcium and are found mostly in native forests (Whistler

1992, Nelson et al. 2000a). Because native forests contain calcium-rich fruits and figs,

these forests represented calcium-rich habitat. Mixed agroforest represents intermediate-

calcium habitat because it contained both native and agricultural fruits. Village

agricultural land was considered calcium-poor habitat because it contained only

agricultural fruits that were nutrient-poor and sparsely distributed in the landscape.








Table 6-1 Nutrient classification of habitat types in American Samoa.
Habitat type Sample of trees used by Calcium Habitat classification
fruit bats* content
(mg/g)**
Native forest Ficus scabra 10.30 Calcium-rich
Planchonella garberi 4.66
Mixed agroforest Carica papaya 2.46 Intermediate calcium
Myrsticafatua n/a
Village agriculture Musa spp. 0.55 Calcium-poor
Artocarpus altilus 0.91
* from Whistler 1992, 1994, Cole et al. 1988, Banack 1996.
** from Nelson et al. 2000a

Netting of Bats

Bats were captured using mist nets set at several foraging sites throughout the

island. Sites were chosen by watching animal movements at dusk, by finding fruit ejecta

pellet locations, and on the advice of local residents. Bats were captured in large mist nets

(6-18 m, 4 inch mesh, Avinet, Inc., Dryden NY) attached to pulleys placed high in

coconut trees (approx. 15 m) or on tall poles. Nets were raised at sunset and were

checked every 30 minutes until midnight when they were taken down. Netting was

terminated in the event of rain, a full moon, or excessive wind. Bats were caught from the

center, east, and west side of the island so that results represent the entire island

population. After capturing a bat, its sex, reproductive status, overall body condition,

forearm length, body mass, and time of capture were recorded (Racey 1988).

Radiotelemetry

Radiocollars (Model RI-2D, Holohil Systems, Ltd) were fitted around the necks

of bats using embroidery thread strung inside Tygon rubber tubing (2 mm). The

transmitters weighed 8.5 g and represented 3% of the bat's body weight. Collars often fell

off or were removed by bats but were retained for an average of 2.5 months. They were

recovered, refurbished, and used again. Nocturnal radiotracking involved tracking bats on









Vamnuu Point










Wsand Habitat Types
[jMixed Agroforest
Native Forest
-Village Agriticture



Fagate Bay
0 5 10 15 Kllometers

Figure 6-1. Map of Tutuila, American Samoa showing the three island habitat types.
Map follows Freifeld 1998.

foot or from a truck along roads and trails. The loudest signal method (Springer 1979,

Kenward 2001) was used to determine the direction to the signal. By sighting down the

mast of the antennae with a compass, an azimuth was taken. Three azimuths whose

intersecting angles were generally 30 but no less than 20 apart were used to estimate

the bat's location. The program Locate (version 2.82, Nams 2001) was used to generate

the location estimates.

Bats were tracked two to three nights a week and only once per night to maintain

independence of locations (Erickson et al. 2001, Kenward 2001). Locations were

collected in the first six hours of the night (18:00-24:00 h), beginning shortly after bats

left the roost to forage and continuing until midnight. Collection terminated at midnight

because bats displayed a sharp decrease in activity beginning at midnight and continuing

until early morning (Banack 1996). The sequence in which individual bats were located








was random and opportunistic. Locations taken early in the night were preferred to test if

calcium was sought first, although previous radio-tracking work indicated that P.

tonganus often used a single foraging area throughout the night (Banack 1996).

A digital USGS 7.5" map of Tutuila was proportioned and georeferenced by using

ESRI's Image Analyst and 18 GPS locations taken across the island. The Freifeld

vegetation types map (Freifeld 1998) was overlaid onto the USGS map and proportioned

using Image Analyst. The map image was then converted to an ArcView shape file for

use in habitat analysis. The map was ground-verified at 175 points to check for accuracy

of the resulting map. The three areas that received the most bat use were mapped with a

GPS, converted to GIS, and used for the habitat analysis. Habitat use was determined by

the number of times each bat was located within each habitat type. The Chi-square

goodness of fit test and simultaneous Bonferroni confidence intervals (Neu et al. 1974)

was used to determine if habitat types were used in proportion to their availability (see

Erickson et al. 2001). Tests of habitat selection were performed for three groups; males,

reproductive females, and non-reproductive females. To evaluate differences in foraging

distances flown by bats, SPSS (Norusis 1993) was used. A Kolmogorov-Smimov test

revealed that the data were not normally distributed, so the non-parametric Mann-

Whitney test was used to analyze the data (Sokal and Rohlf 1995).

Radiotelemetry Error

Data obtained by two independent observers were evaluated for telemetry error

using the Location Error method (Zimmerman and Powell 1995) and error ellipse results

from the Locate program. Fourteen test collars were placed in different habitat types and

at different distances around the study area to account for the different effects each

habitat type and distance may have on the radio signal. True collar locations were








determined using a handheld GPS (GeoExplorer 2, Trimble Navigation, Ltd., Sunnyvale,

CA). Error was assessed for both observers independently taking radiotelemetry

locations. A two-sample t-test (Sokal and Rohlf 1995) was performed on the results of 27

collar locations to evaluate the potential difference in performance between the

individuals radio-locating bats. The data were transformed by natural log to approximate

a normal distribution.

Results

Radiotelemetry Error

The results of the t-test showed no difference (p = 0.27) in the error distances

between the two observers, thus the results were pooled to produce an overall study error

distance for all radio-location estimates. The mean error for the straight-line distance

between a known location and a location estimate was 103 138 m. For comparison, the

mean error ellipse from the Locate location estimate was 1.72 2.63 ha with a median of

0.54 ha. The standard deviation of 2.63 ha is less than a 100 m straight-line distance.

Habitat Selection

Radiocollars were attached to twenty (11 males, 9 females) Tongan flying foxes.

Two of the nine females were lactating and thus classified as reproductive. All other

females were considered non-reproductive. Seven months of radiotelemetry resulted in

166 usable locations. Bats left the roost to begin foraging at approximately 18:00.

Radiotelemetry locations were taken between 18:00 and 24:00 h. (Figure 6-2)









40
35
30
S25







Time of night (h)
'20
o15
S10
o5
0 0
66 4i @\' 6; 0i 0 -' 4 en r6
~ eq N q eqi
Time of night (h)

Figure 6-2. Frequency of P. tonganus location estimates recorded from 18:00 to 0:00 on
Tutuila, American Samoa.

Mixed agroforest provided 70% of all radiotelemetry locations (116/166

locations) and was preferred (p < 0.001) over the native forest or village agricultural

habitat types (Table 6-2). Mixed agroforest was used by both sexes and all ages of bats

across a wide spectrum of time. Native forest was avoided by bats (p = 0.05) and was

used only 20% of the time. Village agriculture was used only by non-reproductive

females (n= 9 locations), and never by males or reproductive females. Reproductive

females (n=2) were located only within mixed agricultural areas. A broken collar on one

of the females prevented us from getting a greater number of locations for reproductive

females (n = 10). Eight dropped collars were found; five in mixed agricultural areas, two

in village agricultural areas, and one in native forest habitat over the seven months.










Table 6-2. Summary of goodness-of-fit tests for habitat selection for radio-collared
Tongan flying foxes on Tutuila, American Samoa.
Habitat typea
n X2 DF Village Mixed Native
agriculture agroforest forest
All bats 20 117.18* 19 NS Preferred Avoided
Males 11 69.43* 10 Avoided Preferred Avoided
Non-reproductive 7 41.69* 6 Preferred NS Avoided
females
Reproductive 2 87.70* 1 Avoided Preferred Avoided
females
* significant at p < 0.001, indicating that habitats were not used in proportion to
availability.
a Avoid = habitat used less than expected based on its availability
Prefer = habitat used more than expected based on its availability
NS= no selection, habitat used in proportion to its availability


120 0 Native forest
U Mixed agroforest
S100-
S100 0 Village agricultural

80

I2 60-
40

20

0 ,

Males Non-reproductive Reproductive
females females

Figure 6-3. Percent of locations by habitat type on Tutuila, American Samoa used by
Tongan flying fox males (n= 11), non-reproductive females (n = 7), and reproductive
females (n=2).









Distance Traveled from Roost to Foraging Site

All bats used in this study either roosted on the southwest side of the island at

Fagatele Bay on Mataautuloa Ridge, or on the northeast side of the island near Afono

Village at Vainuu Point on Ogetu Ridge (Figure 6-1). Each bat was tracked for an

average of 2.5 months before the collar fell off or we were unable to locate the bat on the

island (Table 6-3). The average flight distance for bats from roost to foraging site was 1.8

km. Males and females did not differ in the distance flown from roost to foraging site (1.5

km males, 2.3 km females). Both reproductive females were identical in their average

foraging distance (0.7 kmn). Although their average distance flown was much less than

that of other females (0.7 km vs 2.3 kmn, respectively), the results were not significantly

different (p = 0.55), most likely due to the small sample size of lactating females (n=2).

Bats from roosts on the west side of the island (at Fagatele Bay, n= 15) flew an

average of 0.87 km to their feeding locations, while bats on the east side (at Vainuu

Point, n=5) flew an average of 4.84 km (Figure 6-4). These foraging distances were

significantly different (p = 0.002). The single longest straight-line distance flown by a bat

from the east-side roost (Vainuu Point) was 16km by an adult female. The longest

straight-line distance flown from a bat from the west-side roost (Fagatele Bay) was 8.1

km by a young adult female.












Table 6-3. Data summary for the 20 Tongan radio collared fruit bats in American Samoa.

Individual Sex Age Number of Number of
Indviua Sx ge months tracked radiotelemetry fixes
1 M Subadult 2 10
2 F Subadult 2 3
3 M Subadult 2 11
4 F Adult 1 7
5 F Adult 4 10
6 M Subadult 2 6
7 F Subadult 2 4
8 M Subadult 2 8
9 M Adult 2 6
10 M Subadult 2 5
11 M Subadult 4 14
12 F Subadult 3 14
13 F Adult 2 5
14 F Adult 3 5
15 M Subadult 2 10
16 M Subadult 1 7
17 M Adult 4 21
18 F Adult 3 19
19 M Adult 2 14
20 F Adult 1 3


Ave. roost to foraging
distance(km)
0.9
0.6
0.4
0.7
2.4
0.9
0.6
0.7
0.7
0.7
7.2
3.2
2.8
9.0
2.8
0.9
0.7
0.7
0.6
0.7


Roost location
on Tutuila
West
West
West
West
East
West
West
West
West
West
East
West
East
East
East
West
West
West
West
West


































0 5 10 KMometers

Figure 6-4. A map of Tutuila, American Samoa showing the mean distance flown from each roost site for P. tonganus and the longest
distance moved by a bat from each roost.


HabitatTypqes
""] Miud g-'DforeI l
E N aiwa Forest
if v'tlage Aglicullural


Is BRIP0011
CD Marn Ditance Moved
U Long*St Disltnco Wsed








Discussion

Radio-tracking studies can highlight what habitat types are preferred by flying

foxes, and if reproductive females prefer nutrient rich areas for foraging. In this study,

Tongan flying foxes preferred the mixed agroforest habitat type to all other habitat types.

Mixed agroforest represented 70% of all radiotelemetry locations. Non-reproductive

females were the only group that preferred to forage in village agricultural areas, and

reproductive females foraged only within mixed agricultural areas. Bats roosting on the

east side of the island flew farther to forage than bats roosting on the west side of the

island. Reproductive females flew less distance than non-reproductive females to forage.

Habitat Preference

The observed preference that P. tonganus displayed for mixed agroforest habitat

in this study is consistent with other studies on this species (Pierson et al. 1992, Wilson

and Engbring 1992, Banack 1996, Brooke 1998). The results of this landscape-level

study are also in agreement with the results of fruit preference tests at the level of the

individual bat, where agricultural fruits were overwhelmingly preferred to native fruits in

feeding trials (Chapter 3). Dropped collar locations further support a preference for the

agroforest habitat type and may be a reflection of time spent foraging in that habitat type.

Tongan flying foxes exhibited variation in habitat use among males and females.

Non-reproductive females were the only individuals to use village agricultural areas for

foraging. Reproductive (lactating) females only fed in mixed agroforest areas. In contrast,

males were never located in village agricultural areas. In Australia, Spencer and Fleming

(1989) also found that females (Nyctimene robinsoni) were more likely to feed on fruit in

fruit orchards than males, whereas males preferred to feed on native figs. Because figs are

calcium rich (O'Brien et al. 1998, Nelson et al. 2000a), and reproductive or parous








females may be calcium deficient, it seems counterintuitive for males, but not females, to

prefer them. Elangovan et al (2001) found that carbohydrates and water were consumed

first following the extended day roosting period. Females may be more water or energy

stressed than males, particularly females while lactating. Agricultural fruits tend to be

both juicier and more sugar-rich than native fruits (Oftedal and Allen 1996). This may

contribute to a preference for agricultural fruits, particularly among reproductive females

that emerge following a period of fasting in their day roost and demands associated with

lactation.

Distance Flown from Roost to Foraging Site

Flying fox species typically fly 10-30 km between roost sites and feeding

locations (Mickleburgh et al. 1992, Palmer and Woinarsdki 1999). Previous research in

American Samoa indicates that Tongan flying foxes are capable of flying 45 km in a

single night (Banack 1996). While a single adult female in this study flew 16 km from a

roost to a foraging location, this behavior was not typical of the group. Instead, Tongan

flying foxes in this study averaged less than 2 km flying distance between their roost and

foraging locations. Bats flew approximately 1/10 the distance reported for P. tonganus in

1992-1994 on the same island (Banack 1996).

Differences in the distances flown by P. tonganus may be related to the years in

which the studies were done. Banack's study was conducted two years after a series of

destructive hurricanes battered the Samoan archipelago. With sustained winds in excess

of 200 km/hr, the hurricanes stripped trees of their fruit and leaves, and severely reduced

the food base of the island (Elmqvist et al. 1994, Pierson et al. 1996). For several years

following the hurricanes, food was scarce on Tutuila, and Tongan flying foxes had to fly








farther to find food (see Craig et al. 1994b, Nelson et al. 2000b). In the decade since the

hurricanes, food has become plentiful on the island. Monthly fruit surveys conducted in

2001 demonstrated that agricultural fruits were abundant year round on Tutuila (S.

Nelson, unpublished data, Trail 1994). The year-round abundance and availability of

agricultural fruits compared to native fruits may have resulted in both a preference for

them and shorter foraging distances from the roost to find them. Foraging distances have

been shown to decrease in other bat species when resources were plentiful and increase

when resources were scarce (Spencer and Fleming 1989, Palmer and Woinarski 1999).

Foraging Distance and Roost Affiliation

The greatest differences in foraging distances among P. tonganus were related to

roost affiliation. Bats that roosted on the east-side of the island traveled an average of 5.5

km farther to forage than did the bats that roosted on the west side. Both roosts were in

native habitat, as is typical of this species (Brooke 1998). However, the roost on the west

side of the island (at Fagatele Bay) was near a large agricultural area. In contrast, bats on

the east side of the island were near the National Park of American Samoa, which is

primarily native forest habitat (Cole et al. 1988, Whistler 1994). Although native forest

provided a nutritionally richer resource, bats from the east side flew across the island

nightly to feed in mixed agroforest areas. For example, an adult female flew an average

of 9 km each night from her east-side roost to feed within different mixed agroforest

areas on the west side. Other flying foxes that roost in native forests but feed in

agricultural areas also fly long distances to reach the agricultural areas (Mildenstein

2002).








Foraging Patterns of Reproductive Female Bats

Reproductive females exhibited atypical foraging behaviors when compared to

non-reproductive female bats. The two lactating females traveled less than any other

group of bats. Both lactating females roosted on the east side of the island (Fagatele Bay)

but flew less than the average foraging distance for other bats that also occupied that

roost. These two females also flew identical distances (0.7 km) and flew the same

distance each night they were tracked to foraging areas (n=l10). Foraging distances for

reproductive females may be restricted to areas near the maternity roost because they

have dependent young (Palmer and Woinarski 1999). This may explain the consistency in

their flight distances and the relatively short distances flown by both reproductive

females. Dominque (1991) found that pregnant and lactating Carollia perspicillata

females flew almost as many flying bouts as non-reproductive females, but their flights

were much shorter. In contrast, non-reproductive females performed longer exploratory

flights to survey for fruit abundance. Thus, the feeding behavior seen in reproductive

females may be a means of shifting energy from exploratory behavior to reproductive

effort (Dominique 1991).

Nutritional Landscape Ecology

Little is known about the kind of information available to animals at the scale of

ecological landscapes, and how this information is used with respect to habitat selection

(Lima and Zollner 1996). The central prediction of this study was that reproductive

females would forage in calcium-rich habitats to relieve calcium deficiencies that

potentially arise during pregnancy and lactation (Keeler and Studier 1992, Radostits et al.

1994, Studier et al. 1994a, Bernard and Davison 1996). However, a strong preference for

calcium-poor agricultural habitat areas and an avoidance of calcium-rich native forest








habitats suggests that Tongan flying foxes may forage to maximize the intake of nutrients

other than calcium. Agricultural fruits were selected despite the calcium deficiency that

resulted from consuming them, based on an assumption of the standard (Chapter 3,5).

While foraging, Tongan flying foxes appeared to seek out fruits that were high in energy-

rich carbohydrates (sugar), similar to what was reported for Cynopterus sphinx

(Elangovan et al. 2001, Chapter 3, 7).

A potential limitation of this study was the bias toward documenting initial

foraging flights. Elangovan et al. (2001) found that C. sphinx fed on predominantly

energy-rich fruits during the early hours of the night, and foraged for concentrated

mineral sources later in the night. Sugar-rich agricultural fruits provide the highest energy

return for an animal's foraging effort, and may relieve a carbohydrate and water debt

incurred while at the day roost (Kurta et al. 1989, Elangovan et al. 2001). Initial foraging

flights were prioritized under the assumption that bats would first forage to relieve

calcium deficiencies. Early night was also chosen to avoid the period of rest typical of P.

tonganus later in the night (Banack 1996). In addition, because all lands are private in

American Samoa, work was terminated at midnight to avoid disturbing residents sleeping

in open houses (fales) while tracking bats on private land. However, Banack (1996)

found that most P. tonganus foraged within a single area throughout the night, following

a period of foraging upon arrival in the area (Banack 1996). In another study, C. sphinx

left the roost to begin foraging at 18:00, and commenced leaf-eating at 19:30 h

(Elangovan et al. 2001). Almost 90% of the radiotelemetry locations in this study were

recorded after 19:30. Thus, these radiotelemetry locations may include leaf-eating, and

reflect the consumption of high-energy fruits earlier and leaves later in the night.








Due to the paucity of alternative vertebrate pollinators and seed dispersers, fruit

bats are considered keystone species on Tutuila (Cox et al. 1991, Banack 1998, Rainey et

al. 1995). A decline in bat population size could affect community structure and

biodiversity on the island. Mixed agroforests were highly preferred by Tongan fruit bats,

and their maintenance is important for successful foraging. P. tongnanus and its congener

on Tutuila, Pteropus samoensis, are both dependent on native forest for roosting (Brooke

1998, 2001). It is critical to preserve native forest on Tutuila to maintain roosting habitat

and to create a buffer from anthropomorphic disturbance. Disturbance of maternal roosts

can lead to abandonment of the roost and have population-level effects (Brooke 1998).

In summary, Tongan flying foxes showed a strong preference for agricultural

habitats. Bats commuted nightly over native forest habitat to feed in mixed agroforest

areas or fed in adjacent mixed agroforest areas near their roosts. Tongan flying foxes

appeared to forage in a manner that maximized their energy intake rather than their

calcium intake. Succulent and sugar-rich agricultural fruits were possibly preferred by

hungry and dehydrated bats emerging from the day roost as a source of quick, high-

energy food. Agricultural fruits were plentiful and available year-round on Tutuila, which

may have resulted in minimum foraging distances to find them. Reproductive females

may be constrained to forage near the maternity roost to support energy-demanding

lactation and, if so, reproductive females should prefer high-energy, locally abundant

agricultural fruits. Future habitat use studies of flying foxes should include radiotelemetry

locations taken throughout the night and an investigation of foraging within a larger

group of reproductive females. Together, these studies may prove decisive in determining

if P. tonganus attempts to increase their calcium intake by feeding in nutrient-rich areas.













CHAPTER 7
ABSORPTION AND UTILIZATION OF MINERALS CONSUMED BY CAPTIVE
LACTATING FEMALE MALAYAN FLYING FOXES (PTEROPUS VAMPYRUS)
AND THEIR PUPS

Introduction

Fruit bats of the suborder Megachiroptera and family Pteropodidae are

increasingly bred and maintained in captivity. However, little is known about either their

mineral requirements or the adequacy of the diets fed to them. Because nutritional

standards are unknown, dietary recommendations for fruit bats have been based on

standards for rats, averages for all mammals, or diets that maintain breeding colonies of

flying foxes in captivity (Fascione 1995, NRC 1995, Dierenfield and Seyjagat 2000b).

Two separate factors, the overfeeding of heterogeneous diets, and dominance

hierarchies in social species, are now recognized as important factors influencing the

nutritional intake of captive wildlife (Robbins 1993). The distribution and composition of

the daily diet given to fruit bats in captivity are quite different than that consumed by

free-ranging bats. In the wild, Pteropus vampyrus flys up to 50 km each night to reach its

feeding grounds, and the temporal and spatial distribution of food resources are complex,

so that food acquisition often requires a large proportion of an animal's time budget

(Medway 1969, Oftedal and Allen 1996, Kunz and Jones 2000). In addition, wild animals

do not eat more than they require because this effects their wing loading and ability to fly

(Dudley and Vermeij 1992). In captivity, energy requirements are less because bats do

not travel to their food source, and flight is restricted by cage size (Courts and Feistner

2000). Thus, the combination of reduced activity and plentiful food result in captive bats








that are heavier than their wild counterparts. This may lead to obesity in dominant

individuals (Allen and Oftedal 1996, LeBlanc 1999).

When captive animals are housed in groups, it is common practice to feed

amounts somewhat in excess of consumption to ensure that all individuals have access to

food, and that young and subordinate animals receive adequate quantities (Courts and

Feistner 2000). Animals often choose among the numerous food items offered, and may

ingest a diet that is much different from the diet that was offered. Therefore, assessments

of the nutritional adequacy of a diet should be based on what is actually eaten rather than

what is offered (Oftedal and Allen 1996). Intake levels also vary with origin, age,

dominance, and reproductive status of animals (Courts and Feistner 2000).

To accurately document mineral intake, this study evaluated mineral absorption

efficiencies using apparent absorption. This method measured both mineral intake and

excretion, and accounted for the unique manner of fruit bat feeding where bats chew the

plant matter into a fibrous pellet, swallow the juice, and eject the flattened pellet (Lowry

1989, Kunz and Ingalls 1994, Kunz and Diaz 1995). Previous studies failed to account

for minerals found in ejected pellets.

Growth, pregnancy, lactation, age, gender, nutrient interactions and illness can

influence nutrient requirements. Appropriate amounts of calcium and phosphorus are

especially critical for bats during early growth and peak lactation (Barclay 1995, Hood et

al. 2001). If dietary intake of calcium is inadequate, pregnant and/or lactating females

donate their own skeletal calcium to build the skeletons of their young (Bernard and

Davison 1996). Inadequate dietary calcium can result in weakened bones and low milk

production in females (Radostits et al. 1994). Patterns of post-natal growth in the pups are