NEST PREDATION, CLUTCH SIZE, AND PHYSIOLOGICAL� COSTS OF EGG PRODUCTION IN THE SONG SPARROW�

(MELOSPIZA MELODIA).

by

Marc Travers� B.Sc. Bishop's University�

THESIS SUBMITTED IN PARTIAL FULFILLMENT OF� THE REQUIREMENTS FOR THE DEGREE OF�

MASTER OF SCIENCE�

In the� Department�

of� Biological Sciences�

© Marc Travers 2009

SIMON FRASER UNIVERSITY

Spring 2009

All rights reserved. This work may not be� reproduced in whole or in part, by photocopy�

or other means, without pennission of the author.�

APPROVAL

Name: Marc Travers

Degree: Master of Science

Title of Thesis:

Nest predation, clutch size, and physiological costs of egg production in the song sparrow (Melospiza melodia)

Examining Committee:

Chair: Dr. J. Guttman, Assistant Professor

Dr. T. Williams, Professor, Co-Senior Supervisor� Department of Biological Sciences, S.F.U.�

Dr. L. Zanette, Associate Professor, Co-Senior Supervisor Department of Biology, University of Western Ontario

Dr. D. Green, Assistant Professor� Department of Biological Sciences, S.F.U.�

Dr. B. Roitberg, Professor� Department of Biological Sciences, S.F. U.� Public Examiner�

22 January 20_0.,-9 _ Date Approved

11

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l.Ast: "'''.SIOI''I. Summer 2007

ABSTRACT

We examined the effects of nest predation on both clutch Size and the

physiological cost of egg production using a clutch removal experiment in free-living

song sparrows (Melospiza melodia), inducing "high nest predation" (HNP) females to

produce many replacement clutches compared to "low nest predation" (LNP) females. In

a preliminary analysis we investigated the utility of multiple measures to assess

"physiological condition", including inter-correlations between physiological traits, sex

differences, and the relationship between physiological traits and reproductive

perfonnance (laying date). In our main study, experimental nest predation resulted in

HNP females laying 11 % fewer eggs per replacement clutch. As a result of frequent re-

nesting, HNP birds produced 57% more clutches (3.0 vs 4.7) and laid 41 % more eggs in

total. Physiological condition of HNP females' declined during the experiment associated

with the increase in egg production and we suggest these results are consistent with

physiological costs of egg production.

Keywords: clutch size, nest predation, cost of reproduction, condition, hematology, oxidative stress, song sparrows (Melospiza melodia).

Subject Terms: Predation, clutch size, physiology, song sparrows (Melospiza melodia)

III

ACKNOWLEDGEMENTS

1 would like to thank my supervisors, Dr. Tony Williams, Dr. Liana Zanette, and

Dr. Mike Clinchy for their support and guidance throughout this project. I have learned a

great deal from all of your advice. 1 would also like to thank Dr. David Green for sitting

on my supervisory committee, and providing helpful comments and statistics advice

during this thesis. Sophie Bourgeon, Oliver Love, and Emily Wagner deserve a big thank

you for putting in many hours helping me with lab work. I would also like to thank the

entire Williams lab for helpful comments, advice and coffee breaks. Robert DeCaire,

Katie Pagnucco, and Renaud Rincent also deserve a big thank you for putting in long

days and conducting meticulous work in the field. I would like to thank Beryl Clinchy for

providing wonderful meals and a warm place to stay at the start of the field season. I

would like to thank my family for having provided me with unconditional support in any

interest or endeavour in my life. I could not have made it to SFU without your help. It is

impossible to express how much help Marlena has provided me over the last few years.

From providing stats advice to mental support, she was always there for me and I am

deeply indebted to her.

jV

TABLE OF CONTENTS

Approval ii�

Abstract iii�

Acknowledgements iv�

Table of Contents v�

List of Figures vii�

List of Tables viii�

Chapter 1 General Introduction 1�

I. I Introduction: 2� 1.2 Nest Predation and Clutch Size 3� 1.3 Cost of Egg Production 4� 1.4 Study species- The Song Sparrow (Melospiza melodia) 8� 1.5 Summary of Thesis Chapters 9� 1.6 References II�

Chapter 2 Multivariate analysis of physiological condition in relation to� reproductive quaHty and sex 16�

2.1 Introduction 17� 2.2 Materials & Methods 19�

2.2.1 Study Species and Field Methods 19� 2.2.2 Measurement of Physiological Traits 21� 2.2.3 Statistical Analysis 24�

2.3 Results 25� 2.3.1 Relationship Between Physiological Traits 25� 2.3.2 Differences Between Sexes 27� 2.3.3 Physiology and Proximity to Egg-Laying 27� 2.3.4 Physiology and Initiation Date 28�

2.4 Discussion 28� 2.4.1 Overall Relationship Between Physiological Traits 28� 2.4.2 Individual Relationships Between Physiological Traits 30� 2.4.3 Differences Between Sexes 31� 2.4.4 Physiology and Proximity to Egg-Laying " 32� 2.4.5 Physiology and Initiation Date 33�

2.5 References 34�

v

44 Chapter 3 Experimental evidence that nest predation affects clutch size and the cost of reproduction in a free living song bird

3.1� Introduction 45 3.2� Methods 49

3.2.1� Study Species 49 3.2.2� Food Supplementation 50 3.2.3� Experimental Manipulation of Nest Predation and Clutch Number. 51 3.2.4� Measurement of Physiological Traits 53 3.2.5� Hatching Success and Nestling Growth 56 3.2.6� Statistical Analyses 56

3.3 3.3.1� Clutch Size Effects 59 3.3.2� Clutch Mass & Egg Mass Effects 60 3.3.3� Cumulative Effects of Nest Predation on Total Egg Production 61 3.3.4� Physiological Cost of Egg Production 61 3.3.5� Hatching Success and Nestling Growth 62

3.4� Discussion 62

3.5� References 72

3.4.1

Results 59

Total Cumulative Egg Production & the Physiological Cost of Egg Production 66

Chapter 4 General sythesis and future directions 83�

4.1 Synthesis 83� 4.2 Future Directions 85� 4.3 References 87�

VI

LIST OF FIGURES

Figure 2.1 Male and female mean ± SE of all distinct correlations presented in Table 2.1 40

Figure 3.1 Treatment effects on the observed mean ± SE of a) clutch size b) clutch mass C) and total seasonal egg number laid by HNP (black) and LNP (grey) females in replacement clutches 78

Figure 3.2 Relationship between treatment effects and date on a) clutch size and b) clutch mass predicted by the mixed model (REML) for HNP (solid line) and LNP (dashed line) females. Dotted lines indicate the SE around the predicted line for HNP females and dashed/dotted lines indicate SE for LNP females. Numbers 1-4.7 represent clutch number and horizontal bars indicate the SE for the average date clutches were produce. For HNP females clutch number 4 indicates the date of the 4th

clutch for females that produced a total of 5 clutches. Clutch 4.7 is the final clutch for all HNP females (i.e. either 4 or 5) 79

Figure 3.3 Proportion of3, 4, and 5 egg clutches produced by HNP (black) and LNP (grey) females across the breeding season 80

Figure 3.4 Standardized effects sizes for treatment effects on HNP females relative to LNP females. Effect size; small := 0.2, medium := 0.5, larger := 0.8 (Cohen 1992). TAC = Total antioxidant capacity, TOS := Total oxidative status 82

VlI

LIST OF TABLES�

Table 2.1 Spearman rank correlations of condition indices. Female correlations are presented below diagonal 1s with male correlation above. * Significant correlation after controlling for multiple comparisons. t Moderate to large correlations (i.e. r> 0.4 Cohen 1988) 41

Table 2.2 Mean ± SE of female and male physiological traits. Paired t-test were conducted to determine statistical differences between social pairs. * Indicates significant values after controlling for multiple comparisons. Fat score was tested using a Wilcox Sign-rank test. TAC == total antioxidant capacity, TAC-res == residuals ofTAC after controlling for uric acid. Cart == corticosterone, NEFA == non-esterified free fatty acid 42

Table 2.3 Summary of regression analysis for female and male physiological traits correlated with the days until initiation of laying. NEFA == non­esterified free fatty acids. TAC-residuals == antioxidant capacity of plasma controlling for uric acid .. "'''''''''''' 43

Table 3.1 The effect of nest predation on physiological condition. Means ± SE for physiological traits measured in females at pre-breeding and post manipulation (incubation). See chapter 2 for units. Descriptive statistics from the OFA along with standardized effect sizes for each index. Effect size; small == 0.2, medium == 0.5, larger == 0.8 (Cohen 1992) 8.l

V!l[

CHAPTER 1 GENERAL INTRODUCTION

1.1 Introduction:

Predators can affect prey demography through consumption of individuals or their

offspring and through non-consumptive costs resulting from the response of prey to

predators (Creel and Christianson 2008). Non-consumptive costs originate from

interactions between predators and their prey (i.e. failed attack, consumption of

conspecifics/young) that do not result in death to the individual (Peckarsky et a1. 1993),

but that alter prey behaviour (e.g. altered reproductive strategies, increased parental

effort) or physiology (Boonstra et a1. 1998, Carter et al. 2008, Morat et a1. 2008).

Although these responses may be the best strategy to cope with predation, they ultimately

lead to reductions in reproductive success (Nelson et a1. 2004, Creel et al. 2007, Pangle et

a1. 2007, Carter et al. 2008), physiological condition (Boonstra et a1. 1998, Clinchy et al.

2004) and survival (Creel and Christianson 2008). Therefore, in addition to the

consumptive cost of predation, a full understanding of predator-prey interactions requires

an examination of non-consumpti ve predator effects (Peckarsky et a!. 1993, Lima 1998).

In a wide range of avian species, nest predation (i.e. a consumptive cost) accounts

for the loss of approximately half of all nesting attempts and more than 80% of all nest

failures (Ricklefs 1969, Martin 1992, Martin 1995, Newton 1998). Although much is

known about the frequency and universality of nest predation, little experimental work

has examined the non-consumptive cost that may occur as a result of nest predation.

Therefore, the main objective of this thesis was to examine two potential non­

consumptive costs that may result from the predator-prey interactions of nest predation;

reductions in clutch size and increased costs of egg production. What follows is a brief

2

overview of the evidence and proposed mechanistic explanations for both, the

relationship between nest predation and clutch size, and the cost of egg production.

1.2 Nest Predation and Clutch Size

In a 1947 article, Alexander Skutch first proposed that nest predators may limit

avian clutch size. Since that time, numerous descriptive studies have reported negative

correlations between clutch size and nest predation rates across species (Martin 1995,

Martin et al. 2006), habitats (Ferretti and Martin 2005, Zanette et al. 2006b, Olsen et al.

2008), and years (Julliard and Pemns 1997, Zanette et al. 2006b). Descriptive studies like

these provide valuable insight into factors associated with clutch size variation, but do not

allow us to infer a causal relationship. To date, two experimental studies have reported a

relationship between nest predation and clutch size. Eggers et al. (2006) found that

Siberian jays (Perisoreus in/austus) reduced their clutch size in response to recordings of

nest predator calls presented on their territories. Doligez and Clobert (2003)

experimentally elevated nest predation rates and displayed predator models to collared

flycatchers (Ficedula albicollis) and reported a reduced clutch size for the population in

the following year.

Slagsvold (1982) summarized the many proposed mechanisms by which nest

predation may reduce clutch size, classifying them into mechanisms that assume clutch

size affects the probability of nest predation occurring and those that assume nest

predation is independent of clutch size. With regards to the former, smaller clutches, and

the subsequent smaller broods, may incur less nest predation because of both a shorter

nest duration (i.e. fewer exposure days) and reduced nest conspicuousness. For example,

3

smaller clutches take fewer days to lay and smaller broods may fledge early if parents can

provide each nestling with more food (Perrins 1977). Smaller broods may also be less

conspicuous because better fed nestlings will beg less or because parents can make fewer

feeding trips to and from the nest (Eggers et at. 2006).

If nest predation is independent of clutch size, females may also reduce clutch

size to limit any costs (i.e. date effects or physiological) they may incur if nest predation

does take place (bet hedging strategy, Slagsvold 1984). For example, smaller clutches

require less time to produce allowing females to re-nest earlier in the season when

nestlings have a better chance of survival to adulthood (Hochacka 1990). Smaller

clutches should also reflect lower energetic or physiological investment, thus increasing a

female's ability to re-nest or her probability of survival should nest predation occur

(Slagsvold 1984).

1.3 Cost of Egg Production

Nest predation frequently results in re-nesting (Newton 1998, Grzybowski and

Pease 2005), which necessarily leads to an increase in egg production. As egg production

can incur costs, it follows that re-nesting in response to nest predation may increase the

costs that result from egg production. Experiments that have increased egg production

can provide examples for costs that may result from nest predation induced re-nesting.

Most egg production experiments are conducted on "indeterminate layers", birds

that continue to lay when eggs are removed from their nest. In these experiments, females

forced to lay additional eggs were found to have reduced physiological condition

(Kalmbach et al. 2004), reduced brood rearing capabilities (Monaghan et at. 1998), and

4

reduced local survival (Nager et al. 2001, Visser and LesseHs 2001), as well as delays in

the onset of subsequent breeding seasons (Kalmbach et al. 2004). Furthennore, increased

laying resulted in females laying poorer quality eggs (Nager and Houston 2000) that

hatch less often (Kalmbach et al. 2004), and their chicks had slower growth and increased

early mortality (Monaghan et al. 1995). Although, the continuous laying of indeterminate

layers may differ from the way predation increases egg production (i.e. through

mcreasmg complete laying bouts or number of clutches), the type of costs (those

mentioned above) and the underlying mechanisms are likely similar. Below are

descriptions of the two dominant perspectives thought to explain the mechanism(s)

underlying the cost of egg production; resource and non-resource based costs (Williams

2005).

The costs of egg laying are traditionally assumed to result from resource based

trade-offs, wherein females allocate a limited amount of resources between egg

production and their own physiological requirements (Williams 1966). During laying,

females must obtain the energy and nutrients required for self-maintenance (e.g. energy

balance, muscle repair, immune function) as well as the energy and nutrients required by

the organs involved in egg production (e.g. ovary, liver) and the egg (e.g. fat, protein,

calcium, carotenoids). If a female is un-able to obtain sufficient resources, one or all of

these functions must suffer (Partridge 2005).

In many avian systems, resource availability appears to limit egg production.

Numerous studies have found that providing supplemental food to egg laying females

will increase their clutch size (Carlson 1989, Nager et al. 1997, Clifford and Anderson

200], Zanette et al. 2006b). Resource availability has also been found to limit other

5�

aspects of egg production, such as clutch mass and clutch number. Nager (2006) reported

that females with lower endogenous protein stores prior to laying produced significantly

lighter clutches. When a subset of these females were given supplemental protein they

produced heavier clutches than control females with similar initial protein stores (Nager

2006). Blount et al. (2004) found that females supplemented with carotenoids produce

similar clutch sizes compared with control birds, but were one third more likely to

produce an additional clutch. These studies clearly show that resource availability

frequently limits egg production (i.e. reduction in current reproductive success).

However, resource limitation does not necessarily result in long tenn trade-offs as a result

of reallocation of nutrients between egg production and maternal health, as females may

simply match their investment in eggs with food availability.

More recently, it has been proposed that reproduction itself, or the regulatory

(physiological) processes controlling reproduction, may generate costs (Partridge et a!.

2005, Harshman and Zera 2007) which might be independent of resource allocation per

se (i.e. non-resource based costs, Williams (2005». Specifically, egg production costs

would be expected to occur even when a female has access to sufficient resources

(Wagner et a!. 2008b). Kalmbach et al (2004) forced two groups of great skuas

(Slercorarius skua) to lay additional eggs, one with and one without food

supplementation, and compared their timing of laying one year later with un-manipulated

controls (fewer eggs and no food supplementation). Increased egg production was found

to delay the onset of breeding similarly in both fed and un-fed birds compared to un-fed

controls (Kalmbach et a!. 2004), suggesting that additional resources during laying did

not eliminate the cost of egg production. Next, I will discuss the two most commonly

6

cited explanation for non-resource based costs: oxidative stress and negative honnone

pleiotropy (Williams 2005, Harshman and Zera 2007).

Oxidative stress is the imbalance between reactive oxygen specles (ROS) and

antioxidant defenses (Finkel and Holbrook 2000). ROS can damage bio-molecules such

as DNA, lipids and proteins (Beckman and Ames 1998), and it is the cumulative effect of

oxidative damage that is believed to result in reproductive senescence and reduced

longevity, i.e. the free radical theory of aging (Hannan 1956, Beckman and Ames 1998,

Finkel and Holbrook 2000). Oxidative stress is considered a compelling candidate for

mediating the cost of egg production in part because both the free radical theory of aging

and the cost of egg production (i.e. life history theory) similarly predict senescence and

reduced survival (Harshman and Zera 2007). In addition, egg production likely increases

a number of metabolic processes that result in the production of oxidants, including

general energy production, organ function (e.g. liver), and lipid peroxidation (Beckman

and Ames 1998). Furthermore, several studies have found that increased egg production

is associated with reduced resistance to oxidative stress. For example, fruit fly's

stimulated to lay more eggs showed increased susceptibility to experimentally elevated

oxidative stress (subjects died more rapidly), suggesting that diminished resistance to

oxidative stress is a direct physiological cost of egg production (Salmon et al. 2001,

Wang et al. 2001). In addition, Bertrand et a1. (2006) report that whole blood from zebra

finches that laid more eggs had lower resistance to oxidants (i.e. faster lysis of red blood

cells). Furthermore, Alonzo-Alverez et al (2006) found that zebra finches that laid more

clutches had lower resistance to oxidants and that resistance to oxidants was a significant

predictor of mortality up to three months after sampling.

7

Hormones are also considered likely mediators of the cost of reproduction

because of their plieotropic (multiple) effects on physiology (Ketterson and Nolan 1992,

Ketterson and Nolan 1999, Williams 2005). Honnone pleiotropy may result in costs

specific to egg production because hormones are essential regulators of egg production

(i.e. present during egg production, likely at elevated levels), but elevated plasma

hormone levels are also known to have negative effects on some aspects of physiological

condition. In birds, the erythropoietic (red blood cell production) suppressive effect of the

hormone estradiol has been proposed as one mechanistic explanation for the cost of egg

production (Kalmbach et al. 2004, Williams et a1. 2004, Williams 2005, Wagner et al.

2008a, b). Marked increases in estradiol observed during egg production (Williams et al.

2004) are known to regulate the production of yolk precursors (Christians and Williams

1999, Walzem et a1. 1999), oviduct development (Yu et a1. 1971) and reproductive

behaviour (Balthazart 1983). Yet high levels of estradiol can suppress red blood cell

production (Clermont and Schraer 1979, Blobel and Orkin 1996), which may explain the

anaemia reported in a wide range of egg laying birds (Williams 2005, Wagner et al.

2008b). Because the level of circulating red blood cells is related to flight perfonnance

and aerobic capacity (Viscor et a1. 1985, Hammond et al. 2000), egg laying induced

anaemia may ultimately lead to reduced reproductive success or survival it~ for example,

females with lower hematocrit are less able to rear broods or escape predation.

1.4 Study species- The Song Sparrow (Melospiza melodia)

Unless otherwise stated, the following was obtained from the Arcese et a1. (2002)

review in the Birds of North America. Song sparrows are one of the most widespread

8

songbirds in North America, found in habitats ranging from arctic-alpine, rain forest, to

desert scrub. Peak breeding densities are found in riparian areas, on islands, and

bordering tidal marshes. The diet of Song sparrows consists of seeds, fruits, and

invertebrates. Stomach content analysis suggests that plant material accounts for 86% of

the diet in winter, 54% in spring, 60% in summer, and 92% in fall. Song sparrows are

socially monogamous (high territory fidelity), although extra pair patemity rates of 15%

have been documented. Clutch sizes range from 2-5 eggs, egg mass ranges from 2.43­

3.28g, and females can produce 3-4 successful clutches and up to 8 unsuccessful clutches

- totalling 28 eggs laid in a season (Zanette et a1. 2006b, Travers unpublished data).

Females solely build nests (open cup), incubate eggs and brood nestlings. Males and

females share the feeding of nestlings and fledglings. Nests are not re-used after

successful or unsuccessful breeding attempts. The rate of nest predation varies

considerably by habitat. At the study site used for this thesis, 48-69% of nests were

consumed between 2000-2002 (Zanette et a1. 2006a). The maximum recorded life span of

a song sparrow is 8-9 years. On Mandarte Island, British Columbia, 56% of the variation

in life time reproductive success (number of recruits to the island) was partitioned to

offspring survival to independence, 10% to life span, and 5% to the number of eggs

produced (all variables were positively correlated with life time reproductive success,

Smith 1988).

1.5 Summary of Thesis Chapters

In chapter 2, we investigated the utility of using multiple physiological traits to

assess "physiological condition". We examine (1) whether a suite of physiological traits

9

are likely to provide redundant infonnation on physiological condition (inter-conelations

between traits), (2) sources of variation in physiological traits (sex differences), and (3)

the relationship between physiological traits and reproductive perfonnance (laying date).

In chapter 3, we experimentally test if nest predation affects clutch size and the

physiological condition of females. Using a clutch removal manipulation, we induced

"high nest predation" (HNP) females to produce many replacement clutches compared to

"low nest predation" (LNP) females. We predicted that high nest predation would

decrease clutch size, however, we also predicted that by inducing re-nesting predation

would increase total egg production, resulting in elevated physiological costs of egg

production in HNP females. We test for nest predation affects on average clutch size and

on the seasonal pattern of clutch size, total seasonal egg production, and female

physiological state, and we evaluate if physiological effects are consistent with the

various predictions for the cost of egg production. Because females with insufficient

resources may trade-off their offspring quality to maintain their own condition or because

costs incuned by mothers may be passed on to offspring, we test for predation affects on

hatching success of eggs and on nestling size. Throughout the experiment we provided

all birds with unlimited access to high quality feed For this reason we expected that

treatment effects would not be attributed to resource limitation.

In chapter 4, 1 briefly review implications of the main results from the preceding

chapters, and discuss possibilities for future research.

10

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Arcese, P., M. K. Sogge, A. B. MalT, and M. A. Patten. 2002. Song SpalTow (Melospiza melodia).in A. G. Poole, F, editor. The Birds of North America. The Birds of NOlih America, Inc., Philadelphia.

Balthazart, J. 1983. Hormonal cOlTelates of behaviour. Pages 221-365 in D. S. Farner, J. R. King, and K. C. Parkes, editors. Avian Biology. Academic Press, New York,.

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15

CHAPTER 2 Multivariate analysis of physiological condition in relation to reproductive quality and sex

Marc Travers l ,2, Liana Zanette2 Michael Clinch/, Tony D. Williams I

'Simon Fraser University, Department of Biological Sciences, Burnaby, B.C., Canada 2University of Westem Ontario, Department of Biological Sciences, London, Ontario, Canada 3University of Victoria, Department of Biological Sciences, Victoria, B.c. Canada

16

2.1 Introduction

Body condition is of particular interest in studies of free-living animals because it

is thought to indicate aspects of individual quality that are related to performance in many

quality-dependent traits such as lay date (Bety et a!. 2003, Ninni et a1. 2004), clutch size

(Andersson and Gustafsson 1995), egg size (Smith and Moore 2003) offspring size

(Atkinson and Ramsay 1995), and survival (Romero and Wekelski 200 I, Blums et al.

2005). Body condition is frequently measured with morphometric based indices like the

body condition index (BCl), which is a measure of mass controlling for structural body

size. The BCl is widely used because it is non-invasive, easy to obtain in field studies,

and may reflect aspects of physiological state widely considered important to an animal's

condition such as nutrient status and fat storage (Brown 1996, Glazier 2000, Smith and

Moore 2003). However, some authors have questioned the validity of using the BCl

because the basic statistical and allometric principles of the method are unlikely to be met

in most field studies (Green 2001, Hayes and Shonkwiler 2001). While some empirical

studies have found that Bel is significantly correlated with actual body fat (Ardia 2005,

Schulte-Hostedde et a1. 2005), others have concluded that BCl may not be a broadly

applicable index of condition because it accurately reflects fat content in some species

but not others (Spengler et a!. 1995).

More recently, physiological indices have been increasingly used to measure

condition and assess individual quality. Physiological indices are promising condition

indicators because these assays can be used on a wide range of species to measure diverse

aspects of physiological state. For example, researchers have used physiological indices

to measure energetic state (Jenni-Eiermann and lenni 1997, Williams et a1. 1999).

17

immune function (Bourgeon et a1. 2007), hormone profiles (Love et al. 2005), blood

constituents (Totzke et al. 1999) and oxidative stress (Alonso-Alvarez et a1. 2004).

Importantly, physiological indices have been related to fitness related traits like arrival

date following migration (Piersma 1996), timing of breeding and clutch size (Andersson

and Gustafsson 1995) and survival (Nadolski et aJ. 2006).

Most studies that have used physiological indices have attempted to measure

condition with a single physiological trait or index. However, the complexity of

organismal physiology would suggest that measuring a single aspect (e.g. oxygen

carrying capacity or fat content) of physiological state may not provide a complete

understanding of condition. Yet to date, there has been little work examining how much

individual measures tell us about other aspects of overall physiological state. If multiple

physiological indices are highly correlated with one another and are indicative of many

aspects of physiological state, then individual indices may be expected to provide

considerable infonnation about overall condition. However, if physiological traits are not

highly related to one another measurement of multiple physiological traits might be

required for a more accurate assessment of true "condition" (Seeman et a1. 2001).

Here we take a comprehensive approach to measuring physiological condition in

male and female song sparrows (Melospiza melodia) during the pre-breeding and brood

rearing period. Specifically, we measured multiple physiological traits as putative indices

of condition (15 indices in total) relating to energetic state, hematology, oxidative stress,

and immune function. We first test for correlations between the individual physiological

traits we measured to examine the overall strength of relationships between common

indices of condition. We then compare physiological traits in female and male social

18

pminers to test whether sex is an important source of variation in the indices measured

here. Male and female physiology may differ as a result of differing roles (demands)

during some stages of breeding. Alternatively, sexes may show similar physiological

scores due to assortative mating or territory quality. Basic knowledge of sources of

variation should aid future studies in differentiating between environmental noise and

experimental effects. Final1y, we use a multivariate approach to test if an integrated

assessment of condition explains variation in the date females initiate breeding, and we

conduct these correlations in both females and their male social partners. Male condition

may be related to lay date if females paired to high quality males lay earlier or if territory

quality is also an important factor influencing lay date. Lay date is a good trait to

examine if the physiological traits measured here could be used as an indicator of

individual quality because 1) there is considerable evidence linking lay date with

reproductive success in birds (Daan et al. 1990, Verhulst and Tinbergen 1991, Sanz 1999,

Brinkhof et a1. 2002), indicating that lay date is a measure of reproductive quality and

because 2) lay date is considered a condition dependent trait (Bearhop et a1. 1999,

Kalmbach et a1. 2004).

2.2 Materials & Methods

2.2.1 Study Species and Field Methods

We studied song sparrows (Mefospiza mefodia) on the Southern Gulf Islands of

British Columbia, Canada. Song sparrows in this area are resident year round and multi­

brooded. Breeding typically begins mid April and ends in early July. Pre-breeding song

19

sparrows were captured between March 6 and March 28 (Julian capture date mean ± SD

= 76.8 ± 7.4) using baited potter traps. We pre-baited potter traps with 2.5ml of white

millet per day, for an average of 5 days prior to capture. Once captured, all birds were

colour-banded for individual identification and a blood sample (up to 300j..d) was

collected from the brachial vein using heparanized capillary tubes. We measured blood

glucose concentration (see below) and prepared blood smears immediately after releasing

the bird. Blood smears were air dried and stored in slide boxes containing desiccation

packs. We refrigerated blood in cooler packs for a maximum of 10 hours before

separating the plasma from the red blood cells. Plasma was stored on dry ice until

pennanent laboratory storage at -20°e. For all females sampled during the pre-breeding

period we attempted to locate their first nest. If a nest was found after incubation had

begun, we back calculated the lay date of the first egg by aging the eggs with the aid of

an egg candler. Laying date or initiation of breeding was then defined as the date when a

female laid her first egg in her first nest of the season (Julian lay date mean ± SD = I 07.8

± 8.8). We also captured and collected blood from females and males during the breeding

season. Breeding birds were sampled on the 61h day of the nestling phase using mist nets

placed across the flight path from the nest to their foraging areas. Blood used for

corticosterone analysis was collected in under 3 min from the time the bird hit the net.

Other than capture methods, blood collection procedures and storage were identical to

pre-breeding methods.

20�

2.2.2 Measurement of Physiological Traits

We examined fifteen physiological traits measuring components of hematological

state and oxygen carrying capacity (hematocrit, hemoglobin, and polychromatic, i.e.

proportion of reticulocytes or immature red blood cells), oxidative stress (total oxidative

status (TOS), total antioxidant capacity (TAC)), uric acid levels, plasma carotenoids, and

TAC-residuals controlling for uric acid, (i.e. antioxidant capacity not attributed to uric

acid), immunological status (plasma immunoglobulin levels), honnonal (total

corticosterone), and energetic state (plasma glucose, NEFA, triglyceride, glycerol). In

addition, we measured more widely used indices of body condition: body mass (g), body

condition index and fat score. Body condition index scores are mass divided by tarsus

length. We quantified visible subcutaneous fat stores under the wing, and in the furcular

and abdominal region using a 7 point scale (DeSante et a1. 2008). Physiological traits

were measured as follows:

We used colorimetric assays to measure plasma levels of total antioxidant

capacity (TAC), uric acid, total oxidant status (TOS), carotenoids, immunoglobulin (lg),

triglycerides, free glycerol, NEFA, and whole blood levels of hemoglobin. We

detennined assay variation using a hen plasma standard. Intra-assay variation was 4.6%,

5.4%, 2.7%, 2.6% 1.7%, 3%, 5.7% and 2.2% for triglyceride, uric acid, NEFA, 19,

carotenoids, TAC, TOS, and hemoglobin respectively. Inter-assay variation was 2.4%,

4.4%, 1.1 %, 19.4%, 5.9%, 2.3%, 9.2% for triglyceride, uric acid, NEFA, Ig, carotenoids,

TAC, and TOS respectively (we did not obtain an inter-assay CV for hemoglobin).

21

Hematological status: Hematocrit was measured using standard techniques (Campbell

1995) after centrifugation for 10mln using a Zipocrit (Laboratory Essentials, USA)

portable centrifuge. Hemoglobin concentration in whole blood was determined using the

cyanomethemoglobin method (Drabkin and Austin 1932) modified for a colorimetric

assay, using 5~1 of whole blood diluted in 1.25ml Drabkin's reagent (05941, Sigma­

Aldrich, Canada) with absorbance measured at 540nm. Polychromasia was counted on

smears stained with Wright-Giemsa (Sigma-Aldrich Canada, Ltd) and was calculated as

the proportion of red blood cells (RBC) that are immature (Campbell 1995). A single

observer, who was blind to experimental 10, counted (>500 RBC per count) all smears in

triplicate and had an intra-count variation of <4%. Polychromasia scores and intra-count

variation were calculated from the two most similar scores of 3 repeated counts of the

same smear.

Oxidative stress: Total antioxidant capacity (TAC) was determined using a modified

Randox-TEAC assay described by Ere! (2004). TAC results are reported in mmol Trolox

equivalent.L-1 (238813. Sigma-Aldrich). Plasma uric acid concentrations were measured

in duplicate using the QuantiChrom™ uric acid kit (DIUA-250; BioAssay Systems,

USA). Total oxidative status (TaS) was determined as described by Erel (2005). TOS

results are reported in H202 equivalent.L- I. We prepared the carotenoid assay (see

Alonso-Alvarez et a1. 2004) by vortexing 20~1 of plasma diluted in 180~1 of absolute

ethanol, centrifuging the plasma/ethanol at 1500g for 10min, and recovering the

supernatant. Plasma carotenoid concentrations were determined in triplicate using 50~1 of

supernatant per well, with absorbance measured at 450nm and concentrations calculated

22�

usmg a standard curve of lutein (minimum 70% xanthophyll from alfalfa; Sigma­

Aldrich).

Immune status: Plasma immunoglobulins (Ig) were determined using the ELISA method

with commercial anti-chicken antibodies as reported by Martinez et al. (2003). We

adapted the method for use in song sparrows by determining the sparrow appropriate

plasma dilution (1/8000). Ig results are presented in units of absorbance.

Hormonal. The concentration of total corticosterone (see Breuner and Orchinik 2002,

Love et al. 2004, 2005) in non-extracted plasma was determined using a corticosterone

Enzyme-linked-immunoabsorbent Assay (EIA - Assay Designs Inc., Michigan USA,

catalog # 901-097) with a 4-parameter logistic fit.

Energetic status: The plasma concentration of free glycerol and total triglycerides were

determined in duplicate samples with absorbance measured at 540nm after 10min of

incubation at 37°C (see Seaman et al. 2006), using Sigma-Aldrich reagents (Triglyceride

reagent & Free glycerol reagent, Sigma-Aldrich Canada). Triglyceride concentrations

were calculated by subtracting free glycerol from total triglyceride. Non-esterified free

fatty acids concentrations were determined in triplicate using a NEFA kit (NEFA-HR(2);

Wako Diagnostics, USA). Blood glucose scores were measured at the time of capture

using Acensia® glucose meter (Bayer Inc).

23�

2.2.3 Statistical Analysis

We used Speannan rank cOlTelations to examine the relationships between all

physiological traits to limit the influence of outliers on our statistical tests (Myers and

Well 2003). The r-values from the speannan rank correlations are presented in Table 2.1

as a correlation matrix. We first test whether all correlations (r-values) within the matrix

(separately for males and females) were significantly larger than would be expected by

chance when conducting this many correlations (i .e. were the matrix wide correlations

significantly different from zero given our sample size and number of correlations). We

used the program Multicorr to conduct the global test of the correlation matrix (Steiger

1979, 1980,2005). We then tested whether individual correlations within the matrix were

significant using standard t-tests, setting alpha at 0.05 and adjusting P-values for the

number of statistical comparisons conducted using the false discovery method (FOR)

(Benjamini and Hochberg 1995). For these correlations, we did not control for any

potential environmental effects like date, time of day or temperature because we wanted

to see how strongly traits are actually related to each other. Fore example, we know that

time of day affects some traits and not others. This indicates that traits are not necessarily

or always functionally linked, and the extent to which traits are, or are not, linked is

exactly what we are interested in testing. We used paired t-tests to examine sex

differences between social pairs, allowing us to test sex differences controlling for

territory quality. We again adjusted P-values using FOR. We used forward stepwise

regressions to examine if physiological traits of females are 1) related to the proximity

(days) from sampling to the beginning of egg-laying so that we could examine whether

physiology changes as females approach egg-laying and; 2) related to initiation date (date

on which the first egg of the season is laid) to assess whether early breeders are

24

physiologically distinct from those that begin their breeding season later. For males, we

are testing whether male physiology is related to 1) the proximity of sampling to their

partner's initiation of breeding and 2) the date their pattner started breeding. For the

stepwise regression analysis we set the F to enter at I, which allowed non-signiflcant

contributors to enter the model in some cases. Prior to the regression analyses we found

that lay date and the number of days from blood sampling to laying were highly

correlated requiring us to statistically partition out their shared affects on physiology in

the two models described above. To do this we entered lay date as an independent

variable in the model with days to laying as the dependent variable (model 1) and vice

versa for the model with lay date as the dependent variable (model 2). For the

physiological variables entered into the regression analyses we first examined lf capture

date, time, and temperature were significantly related to physiological traits. For traits

correlated with one or more of these confounding variables, we Obtained the residuals of

the regression, which were then used in the regression analyses in which proximity to

egg-laying and lay date were dependent variables. It should be noted that polychromasia

was not measured in males and corticosterone was not measured in pre-breeding samples.

Prior to any analyses all variables were Box-cox transformed (Krebs 1999) and tested for i

nonnality using the Shapiro-Wilks test.

2.3 Results

2.3.1 Relationship Between Physiological Traits

Overall, the inter-correlations between physiological traits (Table 2.1) were

significantly different from zero when simultaneously considering all possible

25

correlations (females: X2 120 = 256.2, P< 0.0001; Males: X2

105 = 213.1, P< 0.0001).

However, the magnitude of the correlations between all the physiological traits were

generally weak to moderate (Table 2.1), considering Cohens (1988) guidelines for the

strength of correlation effects (i.e. small= 0.1, medium= 0.3, large= 0.5). The average

correlation after removing the direction of etIect (- or +), was 0.22 (SO = 0.16, range =0­

91) for females, and 0.25 (SD = 0.17, range = 0-89) for males.

After controlling for the number of comparisons conducted, significant positive

correlations were found between total antioxidant capacity (TAC) and uric acid (Females:

t20= 9.66, P< 0.001, spearman r = 0.91; Males: tl7= 4.60, P< 0.001, spearman r = 0.74 )

and total oxidative status (TOS) and triglycerides (Females: t20= 3.97, P< 0.001,

spearman r = 0.66; Males: tl7= 3.58, P< 0.001, spearman r = 0.66) in both females and

males (Table 2.1). In Table 2.1, we also highlight moderate to large (r > OA, Cohen 1988)

correlations in female and male physiological traits.

2.3.1.1� Mass, Body Condition Index, and Fat Score

These more commonly used indices of condition also generally had a weak to

moderate average correlation with other traits (Cohen 1988) and, mass and condition

generally had similar relationships with other condition indices (Table 2.1). Female mass

and BCI had mainly negative relationships with other indices, with the notable exception

that mass and BCI tended (after correction for multiple comparisons) to be positively

related with NEFA (Table 2.1). In males, mass was significantly negatively correlated

with TAC (tI7= 3.92, P< 0.001, spearman r = -0.69), with BCI following a similar trend.

In males only, fat scores tended non-significantly to be positively related to uric acid and

glucose, while female fat scores were weakly correlated with all other variables.

26

2.3.2� Differences Between Sexes

Females were significantly lighter than their social mate at pre-breeding capture

(Table 2.2). Females tended to have higher levels of antioxidants other than uric acid (i.e.

TAC-residuals), and higher levels of oxidants (TOS), although both were not significant

after controlling for multiple comparisons. During the nestling phase, females were again

significantly lighter than their social mate, but also had a significantly lower body

condition index (Table 2.2). Females also tended to have lower hemoglobin during the

nestling phase (P= 0.051).

2.3.3� Physiology and Proximity to Egg-Laying

Several physiological traits in non-breeding females and males were significantly

related to the number of days until females laid their first clutch, i.e. days from sampling

to initiation of breeding. In females, hematocrit, triglycerides, and immunoglobulin (Ig),

loaded into the stepwise procedure as significantly positively correlated with the days

until laying (i.e. females with lower levels of these variables were closer to laying, Table

2.3). TAC-residual, which is a measure of antioxidant capacity not attributed to uric acid,

tended to be positively correlated with days until laying (Table 2.3). In contrast, glucose,

fat score, and uric acid were significantly negatively correlated with days to egg-laying.

In mates, the traits fat score and TAC-residuals were significantly positively correlated,

while NEFA (non-esterified free fatty acids) were significantly negatively correlated with

27�

days until their partner begins egg-laying (Table 2.3). Triglycerides and hemoglobin were

non-significant contributors in the stepwise procedure.

2.3.4 Physiology and Initiation Date

Several physiological traits were related to initiation date after controlling for the

proximity of blood sampling to egg-laying date. Females that staried breeding earlier in

the season had lower immunoglobulin scores (~= 0.38, t16= 2.38, P= 0.03) and tended to

have higher levels of free glycerol (~= -0.35, t16= -2.04, P= 0.058) than females that

initiated later, while hematocrit and NEFA were non-significant contributors in the

stepwise procedure (hematocrit: p= -0.08, t16= -0.59, P= 0.56; NEFA: p= 0.18, t16= 1.33,

P= 0.19). Males paired to females that initiated breeding earlier in the season had higher

TAC-residual (p= -0.24, tl3= -2.19, P= 0.047), and fat (P= -0.32, t13= -2.35, P= 0.03) and

tended to have higher hemoglobin (P= -0.25, tl3= -2.12, P= 0.054) and lower free glycerol

(P= 0.21, tl3= 1.72, P= 0.11).

2.4 Discussion

2.4.1 Overall Relationship Between Physiological Traits

We found significant overall relationships (global assessment) between all the

physiological traits measured, as would be expected if each trait measures some aspect of

overall condition. That is, we would predict that animals in good condition would have

better scores on many or all traits with the opposite occurring in poor condition

28�

individuals. However, if these traits do measure aspects of overall condition or quality it

is somewhat surprising that the relationships between traits were generally weak to

moderate. Particularly interesting is body condition index (BCI), which is frequently used

as an indicator of general nutritional state, but as are results show had only a small to

moderate correlation with our other variables including those that measure energetic and

nutritional status. Furthermore, our results indicate that several traits were related to each

other in a manner inconsistent with individuals being in either good or poor condition

(i.e. traits were inversely related with respect to interpretation of condition). For example,

mass and BCI were negatively related to many traits in both sexes, indicating that lighter

birds were in better condition. 'Unexpected inverse correlations' could arise if birds in

better condition can better afford to lose mass while gaining in some other aspect of

reproduction, such as greater territorial defence or increased foraging effort for specific

nutrients. Alternatively, birds may facultatively reduce mass (and BCI) to increase flight

performance when they are highly active (Norberg 1981, Gaston and Jones 1989).

Regardless of the exact reason for 'unexpected inverse correlations', such results

suggest that jf physiological traits are measured i.n isolation researchers may come to

incorrect conclusions about an animal's condition. Furthermore, the small to moderate

cOlTelations found between all the traits suggest that no single trait is likely to tell us

much about the variation in an animal's overall physiological state; implying that

individual physiological traits may also not be strongly related to quality dependent traits

like survival or fecundity, etc. In support ofthis idea, Seeman et al. (2001), in a study on

human aging, found that individually none of the 10 physiological traits they measured

predicted the timing of death. However, the cumulative measure of condition produced

29�

after combining the 10 traits did in fact significantly predict mortality. Seeman et a1.

(2001) suggested that to better understand condition we need to measure the combined

'physiological burden' incurred by individuals using measures that incorporate multiple

aspect of physiological state (see also chapter 3).

2.4.2 Individual Relationships Between Physiological Traits

Our results indicate that there were several moderate to strong correlations

between individual traits, however only a few correlations related to oxidative stress

remained significant after controlling for multiple comparisons. Here TAC (total plasma

antioxidant capacity) and uric acid were significantly positively correlated in both males

and females, which is consistent with uric acid acting as a strong antioxidant (Erel 2004).

In a comparison across 200 avian species, Cohen (2007) found an average correlation of

0.79 between uric acid and TAC, which is similar to the correlations (0.74 males, 0.91

females) found in this study. We also found strong positive correlations between TOS

(total plasma oxidative status) and triglycerides in both males and females. Positive

correlations between TOS and triglycerides may be explained by findings that

triglycerides are correlated with the release of ROS (reactive oxygen species) from white

blood cells in humans (Araujo et a1. 1995, Katsuki et al. 2004, Mazor et a1. 2008). In

males, mass and TAC were negatively correlated. Similar inverse relationships were

evident between mass and both uric acid and TAC residuals (antioxidant capacity not

attributed to uric acid), indicating that the higher TAC of lighter males was due to higher

levels of both uric acid and TAC residuals.

30

2.4.3 Differences Between Sexes

Most physiological traits were similar between male and female social mates at

both pre-breeding and brood rearing, with the exception that brood rearing females had

lower body condition index scores. The results of previous sex comparisons of

physiological traits vary considerably across species. Several studies have found that

measures of energetics, hematology and immune function differed between the sexes at

brood rearing (Kern et al. 2005, Owen et al. 2005, Kilgas et al. 2006) and pre-breeding

(Horak et al. 1998) while others have found no sex differences at various times

throughout the annual cycle (Acquarone et a1. 2002, Hauptmanova et al. 2002, Masello

and Quillfeldt 2004, Sanchez-Guzman et al. 2004). Owen et al. (2005) attributed sex

differences in physiological traits during brood rearing to the greater work load of males

who not only provision nestlings but defend territories as well. In a similar vein,

behavioural similarities between the sexes at brood rearing may explain why song

sparrows in the present study had comparable scores for most physiological traits. Here,

males and females were captured on the 6lh day of brood rearing; a time when the sexes

appear behaviourally the most similar during the breeding season. By this stage both

sexes appear to be equally focused on nestling provisioning as females greatly reduce

brooding during day light hours at this time and males appear less territorial as indicated

by less singing and a lack of response to song play backs (per. observation). At pre­

breeding, similarities in the physiology of male and female partners also may indicate

similar behaviour and energetic demands, or potentially different behaviour with similar

net energetic demands or effects on physiology. Although not tested here, it may also be

possible that small sex differences when added across the suite of physiological traits

31

may in fact indicate a cumulative physiological difference between the sexes at both

stages examined here (Seeman et a1. 2001).

2.4.4 Physiology and Proximity to Egg-Laying

Several physiological traits in both males and females appear to be related to the

proximity of egg-laying. Males had lower fat scores, less circulating antioxidants and

higher levels of free fatty acids (NEFA). These data suggest that males were more active

as the date their partner initiated egg-laying approached, but that this activity was not

directed towards increasing fat reserves or obtaining antioxidant rich foods. The lower fat

and higher free fatty acid scores of males suggest that fat is being converted to fatty acids

to fuel flight muscles (Butler and Bishop 2000) and high activity levels are potentially

metabolising antioxidants or reducing the foraging opportunities to replace them. Higher

activity levels of male song sparrows may be a result of increased territorial behaviour

(prior to their social partners entering oestrous), which has been shown to result in

reduced male energetic state (Komdeur 2001). Females appeared to differ from males in

several ways, though these differences were not due to egg laying per say, as birds in this

study were sampled an average of 31 days (range 56-12) before laying their first egg and

would not have started to produce eggs or have started to nest build. In contrast to males,

females sampled closer to their date of egg-laying had higher fat scores, yet their lower

triglyceride levels suggest they were not putting on fat at the time of sampling. Closer to

egg-laying females also had higher uric acid, indicating potentially higher dietary

consumption of protein (or catabolism), as well as higher glucose scores. Glucose is

known to power leg muscles in birds (Butler and Bishop 2000), and in contrast to males,

32�

high glucose but not high fatty acids, may indicate that females are more actively

foraging (song sparrows predominantly forage on the ground through hopping and

scratching) for protein (high uric acid) or other nutrients like calcium that are known to

be important in egg-laying (Graveland et al. 1994, Selman and Houston 1996). The lower

levels of Ig of females closer to egg-laying may result from increased locomotor activity

(Pedersen and Hoffman-Goetz 2000, Merino et al. 2006) associated with increased

foraging (see below for further discussion of Ig). The reduced hematocrit observed in

female song sparrows as lay date approached may also result from an increase in

locomotor activity (Birkhead et al. 1998), however inCrease oxygen demands (generally

associated with increased activity) are also reported to increase hematocrit (Viscor et a1.

1985).

2.4.5 Physiology and Initiation Date

Out of the suite of 15 physiology traits only Ig was significantly related to

initiation date. Here, females that laid earlier in the season had lower 19 scores. This may

indicate that early laying birds had lower parasite loads as blue tits Cyanistes caeruleus

infected with the blood parasite Haemoproteus were reported to have elevated Ig scores

(Ots and Horak 1998) and antibiotic-treated blue tits were found to have fewer parasites

and lower 19 relative to controls (Tomas et al. 2007). However, interpreting Ig scores can

be difficult as low scores have been reported for animals considered in both good (see

above) and poor health (Apanius and Nisbet 2006, Merino et al. 2006). In our case, no

traits other than 19 were strongly related to initiation date, giving us no reference point to

further interpret why early layers had lower Ig. It is surprising that other physiological

33�

traits were not correlated with initiation date, particularly energetic measures, as lay date

is thought to be condition dependent (Kalmbach et al. 2004). Potentially other factors like

age/experience, which are known to affect initiation date (Vergara et al. 2007), may not

be directly related to physiological state and therefore obscure potential relationships

between physiology and lay date. However, males paired to females that initiated

breeding earlier did appear to be in better condition based on higher levels of antioxidants

and fat and tended to have higher hemoglobin. This may indicate that male quality

influences female lay date (Bearhop et al. 1999) or potentially that pairs that started

breeding earlier had higher quality territories, and the physiological signature of territory

quality was, for some reason, only evident in males at the time of sampling.

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39

Figure 2.1 Male and female mean ± SE of all distinct correlations presented in Table 2.1.

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30

085

Table 2.3 Summary of regression analysis for female and male physiological traits correlated with the days until initia tion of laying. NEFA =non-esterified free fa tty acids. TAC-resid uals = antioxidant capacity of plasma controlling for uric acid.

Variables Beta SE of Beta t P

Female Measures Hematocrit 0.40 0.09 4.27 0.001 Tliglycerides 0.34 0.09 3.79 0.002 Glucose -0.35 0.09 -3.67 0.003 Fat Score -0.25 0.09 -2.81 0.02 Uric Acid -0.49 0.13 -3.72 0.003 Immunoglobulin 0.43 0.14 3.04 0,009 TAC-residual 0,18 0.08 2,11 0.055

Male Measures Fat Score 0.29 0.10 3.03 0.01 NEFA -0.28 0.11 -2.59 0.02 TAC-residuals 0.22 0,10 2.24 0.045 Triglycerides -0.13 0.10 -1,23 0.24 Hemoglobin 0.12 0,10 1,13 0.28

43

CHAPTER 3 EXPERIMENTAL EVIDENCE THAT NEST PREDATION AFFECTS CLUTCH SIZE AND THE COST OF REPRODUCTION IN A FREE LIVING SONG BIRD

Marc Travers l,2, Michael Clinchy3, Tony D. Williams l

, Liana Zanette2

lSimon Fraser University, Department of Biological Science, Burnaby, B.C., Canada 2University of Western Ontario, Department of Biological Science, London, Ontario, Canada 3University of Victoria, Department of Biological Science, Victoria, B.c. Canada

44

3.1 Introduction

For decades ecologists have been interested in whether clutch size in birds is

affected more by food availability or nest predation (Lack 1947, Skutch 1949, Pemns

1977, Martin 1987, Newton 1998). Whereas numerous food supplementation experiments

have demonstrated that food availability has proximate effects on clutch size (reviewed in

Nager 2006), only two experimental studies to date have shown that nest predation can

have proximate effects on clutch size. Eggers et al. (2006) showed that Siberian jays

(Perisoreus in/austus) reduced their clutch size in response to recordings of nest predator

calls presented on their territories. Doligez and Colbert (2003) experimentally elevated

nest predation rates and displayed predator models to collared flycatchers (Ficedula

albicollis) and reported a reduced clutch size for the population in the subsequent year.

In both studies clutch size effects were reported to be in response to predator cues

indicating the presence of predators rather than an individual's direct experience of nest

predation (i.e. offspring loss). To the best of our knowledge, no experimental study to

date has demonstrated that an individual's experience of nest predation affects the size of

subsequent clutches. Because direct nest predation is a common experience across many

avian species (Ricklefs 1969, Martin and Li 1992, Martin 1995), such data are critical for

furthering our understanding of predation effects on clutch size.

Martin (1995) conducted a comparative study examining the relationship between

direct nest predation and clutch size in 123 species of Passeriformes and Piciformes, and

reported an inverse correlation between direct nest predation and clutch size. Marti n

(1995) also found that nest predation was more strongly correlated with clutch number

(i.e. the number of clutches laid in a season) than with clutch size and that clutch number

45�

and clutch size were inversely correlated. Zanette et a!. (2006b) reported results from a

bi-factorial food supplementation and natural predator reduction experiment on song

sparrows (Melospiza melodia) that suggested direct nest predation may have proximate

effects on clutch size similar to those described by Martin (1995) at the inter-specific

level. Zanette et a1. (2006b) found that nest predation increased clutch number and that

clutch number and average clutch size were inversely correlated, which suggested that

birds under high nest predation were forced to re-nest more frequently and consequently

laid smaller clutches in each nest (Zanette et a1. 2006b). These studies suggest that direct

nest predation may in fact reduce clutch size, but that clutch size effects may be the result

of predation effects on clutch number.

After a predation event, many avian species can re-nest (Grzybowski and Pease

2005). While re-nesting increases the chances that parents will successfully rear at least

one offspring in a breeding season, re-nesting necessarily leads to an increase in clutch

number and therefore egg production. Martin (1995) proposed that increases in egg

production as a consequence of predation may increase the energetic demands on

female's, which may lead to energy trade-offs resulting in the inverse relationship

between clutch number and clutch size; i.e. females not able to obtain sufficient resources

to meet the demands of increases in clutch number lay smaller clutches. Clearly increases

in egg production require females to obtain some amount of additional resources, and

supplemental feed high in energy and nutrients has been shown to increase clutch size in

song sparrows (Zanette et a1. 2006b) and other species (Nager 2006). However, Zanette et

al. (2006b) reported a similar inverse relationship between clutch number and clutch size

in both food supplemented and non food supplemented song sparrows; suggesting that, at

46�

least at the intra-specific level, the inverse relationship between clutch number and clutch

size is not the result of a resource-based mechanism. Therefore, experimentally

examining the effects of nest predation on clutch number and clutch size when food is

super abundant, is necessary to determine if direct nest predation does in fact affect

clutch size, and whether, as suggested by Zanette et a1. (2006b), the effects can occur

when food is abundant.

The demands of increased egg production have been shown experimentally to

negatively affect a female's physiological condition (Kalmbach et a1. 2004) as well as the

quality of the offspring she can produce; indicated by reduced hatching success

(Kalmbach et a1. 2004) and lower mass of nestlings that do hatch (Monaghan et a1. 1995).

The costs of egg laying are traditionally assumed to result from resource based trade-offs

where females allocate a limited amount of resources between egg production and their

own physiological systems (Williams 1966); thus, resource allocation trade-offs should

be evident only when resources are scarce. More recently, it has been proposed that

reproduction itself, or the regulatory (physiological) processes controlling reproduction,

may generate physiological costs to the female (Partridge et al. 2005, Harshman and Zera

2007) which might be independent of allocation of resources per se (i.e. non-resource

based costs, Williams (2005)). As one example, the hormone estradiol (E2) is necessary

to produce eggs but increased levels during egg production can cause a transient

inhibition of red blood cell production resulting in reproduction-induced anaemIa

(Williams et a1. 2004, Wagner et a1. 2008a). Despite some progress, the physiological

mechanisms underlying the costs of egg production remain poorly understood (Williams

2005, Harshman and Zera 2007). Therefore, incorporating measurements of physiological

47

condition into experimental studies which manipulate egg production and resource (food)

availability might identify specific mechanisms underlying costs of egg production (e.g.

resource vs non-resource) and if this is done in the context of nest predation may further

our understanding of the ways in which predation can impact prey. Furthennore, if the

inverse relationship between clutch number and clutch size was the result of an energetic

trade-off (sensu Martin 1995) we would expect that nest predation induced increases in

clutch number/egg production would result in either females ending up in a poor

energetic state, or that their offspring were be of poorer quality, or both. Yet, if costs of

egg production exist even when females are not limited by food availability, the

possibility would remain that the inverse relationship between clutch number and clutch

size is the result of egg production costs (Martin 1995), but of a non-resource based type.

We conducted a clutch removal manipulation to examine the effects of

experimental nest predation on clutch number and clutch size in a multi-brooded species,

the song sparrow (Melospiza melodia). Following Zanette et aI's (2006b) study we were

specificall y interested in testing the effects of nest predation independent of food

availability and so we provided all birds with un-limited access to high quality food

throughout the entire breeding season. In addition to testing the affects of nest predation

on clutch size, we examined the cumulative affects of nest predation on total seasonal egg

production to investigate whether predation resulted in increased physiological 'costs of

egg production' for mothers as well as reduced hatching success and growth of offspring.

We assessed physiological costs of egg production using a range of physiological traits

which should reflect the specific mechanisms introduced above (resource-based

mechanisms affecting energetic balance, and non-resource based mechanisms affecting

48�

oxidative stress and hematology), along with general indices of condition. We predicted

that in food-supplemented females with access to ad libitum high quality food; 1)

experimentally-increased nest predation will increase clutch number and decrease clutch

size, 2) that high-predation females with a higher frequency of re-nesting (i.e. elevated

total seasonal egg production) will be in poorer physiological condition reflecting

increased costs of egg production, and 3) because of food supplementation, we expect

costs will be consistent with non-resource rather than resource based hypotheses for the

costs of egg production. Finally, Zanette et al. (2006a,b) showed that food availability

reduces the occurrence of nest predation. If nest predation affects clutch size even when

food is not limiting, then because food affects nest predation, food and predators would

have combined effects on clutch size. Whereas speaking of either food or predator effects

may help us disentangle specific mechanisms, in many real-world situations this either/or

likely represents a false dichotomy (Zanette et al. 2006b).

3.2 Methods

3.2.1 Study Species

We worked on a colour banded population of song sparrows (Melospiza melodia)

on the Southern Gulf Islands, British Columbia, Canada. Song sparrows in this area are

resident year round and multi-brooded. Song sparrows in the region can successfully rear

3-4 broods, and when nest predation is high females can produce up to 8 clutches and 28

eggs in a season (Zanette et aJ. 2006b). Clutch size can range from 2-5 eggs but IS

typically 3-4 eggs and egg-laying typically begins in April and ends in early July.

49�

3.2.2 Food Supplementation

As described in the introduction, we were interested in investigating the effects of

nest predation on clutch size and physiological costs of egg production independent of

food availability, i.e. at high-resource levels. To remove the potential effects of limiting

resources on egg production and female condition we provided ad libitum supplemental

food (from March 31 to the end of the breeding season) to all females via feeders placed

on each territory (following Zanette et a!. 2003, 2006a,b). Previous work has shown that

song sparrows supplemented with food high in carbohydrates, fat, protein, and calcium

had greater foraging efficiency (Duncan-Rastogi et a1. 2006), nested earlier and for

longer, produced larger clutches, more clutches, and increased total seasonal egg

production (Zanette et al. 2006b), were in better physiological condition (Clinchy et a!.

2004), and had higher annul reproductive success (Zanette et a1. 2003, Zanette et a!.

2006a). Here, we further improved on the supplemental food provided by adding whole

egg powder and carotenoids. Whole egg powder was used to provide the sulphur rich

amino acids (Houston et aL 1995) in the appropriate ratios (Ramsay and Houston 1998)

thought to limit clutch size and egg mass (Selman and Houston 1996). In general, whole

egg powder would also presumably provide all nutrients or ratios of nutrients impOltant

for egg production. Carotenoids have been shown to limit re-nesting in gulls (Blount et

a!. 2004) and may also have beneficial effects on physiological condition (McGraw et al.

2006) and nestling development (De Neve et a1. 2008). The feed used in this study

consisted per kilogram of: 600.90g white millet, 315.47g of high-fat high-protein pellets

(Aquamax Grower 400), 2I.03g of crushed oyster shell, 60g of dried whole egg powder,

and 2.6g of Oro Glo pigrnenter, Kemin industries (equivalent to O.04g xanthophyll

carotenoids/kg of feed). We estimated that 60g of dried whole egg is equivalent to 240g

50

of wet egg based on the 75% water content of eggs reported by Carey et al. (1980). To

continn that specitic components of the supplemental feed were consumed and absorbed

we conducted a feeding validation using plasma carotenoid levels from fed and un-fed

birds as a marker of dietary intake.

3.2.3 Experimental Manipulation of Nest Predation and Clutch Number

To experimentally manipUlate nest predation we needed to control the level of

natural nest predation. Consequently, we conducted our nest predation manipulation on

islands with comparatively low levels of natural nest predation (Zanette et al. 2006a)

where, as part of another experiment, natural nest predation was further reduced by the

removal of a principal nest predator (the brown-headed cowbird).

We experimentally manipulated nest predation to generate a High Nest Predation

(HNP; n = 14 females) and a Low Nest Predation (LNP; n = 11 females) group. To

generate high nest predation we removed all eggs from a female's nest on the 6th day of

incubation which caused females to re-nest. We repeated this process until females in the

HNP group had produced 4 or 5 clutches. We generated low nest predation by removing

all eggs from a female's nest on the 6th day of incubation and replacing them with clay

eggs fashioned to resemble those of song sparrows. Females in the LNP group incubated

the day eggs and because the eggs did not hatch, the duration of the incubation period

was extended (normal duration of incubation = 13 days; mean extension ± S. D.= 3.65 ±

1.93 days). Females in the LNP group eventually abandoned these nests, giving them the

opportunity to re-nest. We repeated the clay egg manipulation until LNP females had

produced 3 clutches over the breeding season. Incubating a clutch that does not hatch is

51

an experience females song sparrows sometimes undergo because the clutch is infertile,

but more frequently in this region this is due to a female cowbird having visited the nest

and punctured the eggs unbeknownst to the mother sparrow. Mother sparrows in this

population have consequently been recorded incubating up to an additional II days (i.e.

nearly double the normal incubation peliod; CJinchy & Zanette unpublished data). Here

our aim was to address factors that result from increased egg production, as opposed to

brood rearing. For this reason, we followed the clay egg procedure for the LNP group to

ensure that these females, like their HNP counterparts, only experienced egg-stage­

related phenomenon, and did not raise young during the course of the experiment. The

term low nest predation treatment refers to how LNP nests lasted longer (rather than LNP

females actually experiencing predation), thus simulating longer nest durations that

would occur in low predation areas relative to high predation areas. That is, when the

predation rate is high nests are likely to be consumed early in the cycle. Conversely,

when nest predation is lower nest are likely to be consumed later in the cycle, thus lasting

longer.

To experimentally control for initiation date (date of the first egg laid in the

season) we assigned females to treatments by pairing birds with similar initiation dates

and then randomly assigning each female to one of the treatments. For HNP females'

penultimate clutch (3 or 4) only, we timed the removal of the clutch to ensure that HNP

females produced their final experimental clutch (clutch # 4 or 5) at the same time as

LNP females produced their third clutch.

52

3.2.4 Measurement of Physiological Traits

We captured pre-breeding song sparrows between March 6-282007 using baited

potter traps in order to obtain a baseline measure of physiological state. Once captured,

we colour banded all birds for individual identification and collected up to 300111 of blood

from the brachial vein using heparanized capillary tubes. We measured blood glucose

concentration (see below) and prepared blood smears immediately after releasing the

bird. Blood smears were air dried and stored in slide boxes containing desiccation packs.

We refiigerated blood in cooler packs for a maximum of 10 hours before separating the

plasma from the red blood cells. Plasma was stored on dry ice until pennanent laboratory

storage at -20°C. Females were captured and blood sampled a second time on the 8-1 Olh

day of incubation of their final experimental clutch using mist nets. Blood used for

corticosterone analysis was collected in under 3 min from the time the bird hit the net.

Other than capture methods, blood collection procedures and storage were identical to

pre-breeding methods. Treatments did not differ in the date of blood sampling at the

beginning (pre-breeding tlll= 0.33, P= 0.75) or end of the experiment (final clutch: tl8=

0.03, p= 0.97)

We used colorimetric assays to measure plasma levels of triglycerides, NEFA,

immunoglobulin (Ig), carotenoids, total antioxidants capacity (TAC), total oxidant status

(TOS) and whole blood levels of hemoglobin. We detennined assay variation using a hen

plasma standard. Intra-assay variation range was 4.6%, 2.7%, 2.6% 1.7%, 3%, 5.7% and

2.2% for triglyceride, NEFA, Ig, carotenoids, TAC, TOS, and hemoglobin respectively.

Inter-assay variation was 2.4%, 1.1 %, 19.4%, 5.9%, 2.3%, 9.2% for triglyceride, NEFA,

19, carotenoids, TAC, and TOS respectively (we did not obtain an inter-assay CY for

hemoglobin).

53

3.2.4.1� Energetic measures

Plasma concentrations of total triglycerides were determined in duplicate samples

with absorbance measured at 540nrn after 10min of incubation at 37°C (see Seaman et al.

2006), using Sigma-Aldrich reagents (Triglyceride reagent & free glycerol reagent,

Sigma-Aldlich Canada). Triglyceride concentrations were calculated by subtracting free

glycerol from total triglyceride. Non-esterified free fatty acids concentrations were

determined in triplicate using a NEFA kit (NEFA-HR(2); Wako Diagnostics, USA).

Blood glucose scores were measured at the time of capture using Acensia® glucose meter

(Bayer Inc). We quantified visible subcutaneous fat stores under the wing, and in the

furcular and abdominal region using a 7 point scale (DeSante et al. 2008)

3.2.4.2� Non-energetic measures

This included measures of hematological (hematocrit, hemoglobin,

polychromasia), immunological (lg), corticosterone and oxidative stress (TAC, TOS,

carotenoids) status. Hematocrit was measured using standard techniques (Campbell and

Ellis 2007) after centrifugation for lOmin using a Zipocrit (Laboratory Essentials, USA)

portable centrifuge. Hemoglobin concentration in whole blood was determined using the

cyanomethemoglobin method (Drabkin and Austin 1932) modified for a colorimetric

assay, using 5Jll of whole blood diluted in 1.25ml Drabkin's reagent (D5941, Sigma­

Aldrich, Canada) with absorbance measured at 540nm. Polychromasia was counted on

smears stained with Wright-Giemsa (Sigma-Aldrich Canada, Ltd) and was calculated as

54

the proportion of red blood cells (RBC) that are immature (Campbell and Ellis 2007). A

single observer, who was blind to experimental ID, counted (>500 RBC per count) all

smears in triplicate and had an intra-count variation of <4%. Polychromasia scores and

intra-count variation were calculated from the two most similar scores of 3 repeated

counts of the same smear. Plasma immunoglobulins (lg) were detennined using the

ELISA method with commercial anti-chicken antibodies as reported by Martinez et al.

(2003). We adapted the method for use in song sparrows by determining the sparrow

appropriate plasma dilution (1/8000). 19 results are presented in units of absorbance. The

concentration of total corticosterone (see Breuner and Orchinik 2002, Love et al. 2004,

2005) in non-extracted plasma was detennined using a corticosterone Enzyrne-linked­

immunoabsorbent Assay (EIA - Assay Designs Inc., Michigan USA, catalog # 901-097)

with a 4-parameter logistic fit. Total antioxidant capacity (TAC) was detennined using a

modified Randox-TEAC assay described by Ere1 (2004). TAC results are reported in

mmol Trolox equivalent.L- 1 (238813, Sigma-Aldrich). Total oxidative status (TOS) was

determined as described by Erel (2005). TOS results are reported in Ilmo1 H202

equivalent.C'. We prepared the carotenoid assay (see Alonso-Alvarez et al. 2004) by

vortexing 20111 of plasma diluted in 180111 of absolute ethanol, centrifuged the

plasma/ethanol at 1500g for 10min, and recovered the supernatant. Plasma carotenoid

concentrations were detennined in triplicate using SOIlI of supernatant per well, with

absorbance measured at 450nm and compared with a standard curve of lutein (minimum

70% xanthophyll from alfalfa; Sigma-Aldrich).

55

3.2.5 Hatching Success and Nestling Growth

On the final experimental clutch (clutch 3 for LNP and clutch 4 or 5 for HNP) we

removed one egg from each nest for egg composition analysis (results not discussed in

this paper) and the remaining eggs were left un-manipulated in the nest. For those nests

that survived natural predation, we examined treatment affects on post-manipulation

hatching success and nestling growth.

3.2.6 Statistical Analyses

We obtained complete information on clutch size for 85 nests and egg/clutch mass

for 68 nests and used these in our analyses. We conducted 3 sets of analyses. In the first

set, we evaluated treatment effects on clutch size, clutch mass, and egg mass using mixed

models. We first tested for a main effect oftreatment on each egg variable in replacement

clutches (i.e. those laid after the first clutch of the season) by conducting a one-way

ANOVA with female identification as a random effect. We then examined how our 3

egg variables changed over the season in the two groups by conducting a one-way

ANCOVA with female entered as a random effect and treatment and date as fixed effects.

In the latter analysis we include all clutches including the first clutch of the season (i.e.

clutch sizes produced before the beginning of the experiment) and enter both date and the

quadratic of date (date2) into the model to test for trt*date and trt*date2 interactions. Both

date tenns are important because song sparrows often follow a curvilinear clutch size

pattern across the season. Clutch size often increases from first to latter clutches because

sparrows at our site do not produce first clutches of 5 eggs, but do produce 5 egg clutches

later. Though, clutch size often decreases after mid season, potentially as a result of a

56�

negative relationship between lay date and recruitment reported in song sparrows <1 Okm

away on Mandarte island (Hochacka 1990). We also examine treatment affects on the

distribution of clutch sizes laid using a chi squared test.

In the second set of analyses we evaluate treatment effects on total cumulative

egg production (clutch number and total egg number) and we compare HNP and LNP

females' experimental season length, nest duration, and re-nesting period (i.e. days from

clutch removal or abandonment to the laying of the first egg in replacement clutch) to

detennine a) how these are driving the clutch number and egg number difference, and 2)

potentially influencing any treatment effects on clutch size and other egg production

variables. We use a non-parametric Mann-Whitney U test to evaluate treatment effects

on the number of clutches produced by HNP and LNP females and we use a t-test to

compare treatment effects on total egg production (total = clutch number * dutch size).

We restrict the latter analysis to females (n= 9 HNP, n= 8 LNP in which we obtained

complete clutch size information on every clutch produced throughout the season. (i.e.

actual total egg production), rather than multiplying average clutch number by average

clutch size for each individual; although these two methods result in virtually identical

outcomes. We use a t-test to evaluate treatment differences in season length, nest

duration, and average inter-nest interval. Nest duration and inter-nest interval were

calculated as an average for each female across all the nests they produced.

In the third set of analyses, we evaluate the cumulative effects of nest predation

on the physiological profile of females in our two groups using a discriminant function

analysis (DFA). Model significance was determined using a pennutation test iterated

10,000 times in R statistical software (see Mundry and Sommer 2007 for a description of

57

OFA with pennutation test (pDFA)). Our test statistic for the pennutation test was the

eigen-value from the DFA, rather than the classification accuracy used in Mundry &

Sommer (2007). Much like the F statistic, the eigen-value is the between-group variation

divided by the within group variation (in canonical scores) (McGarigal et al. 2000), and

therefore does not have an upper bound (maximum value) like classification accuracy

(i.e. 100%), thus removing the problem of over-fitting when using many discriminating

variables. We report descriptive statistics from the discriminant function analysis (Beta

values) along with standardized effect sizes (Cohen 1992) for each variable in the model

(Table 3.1). We first conducted a DFA comparing the pre-breeding (base line) physiology

of HNP and LNP females testing for initial treatment biases in condition. To test for

predation affects on physiology we use a repeated measures design in the fonn of

difference scores (second capture score - first capture score) in the pDFA. We include

only second capture scores of corticosterone because corticosterone was not assayed in

pre-breeding samples. We used a chi squared test to examine treatment effects on

hatching success of the final experimental clutches (i.e. clutch 3 LNP & clutch 4 or 5

HNP). Finally, we used a t-test to examine treatment affects on nestling mass and

structural size. We used residuals for mass after controlling for the time of day sampled.

Nestling structural size was a combined measure of wing cord and tarsus length produced

using a principle components analysis.

All mixed models were fitted using the restricted maximum likelihood procedure

in Genstat. In the mixed models we test for un-equal variances and deviations from

nonnality using residual and nonnaI probability plots. For all t-tests, we conduct similar

tests using Levene's test of un-equal variance and the Shapiro-Wilk's nonnality test.

58

3.3� Results

Our stratified random design resulted in females in both treatments having similar

initial lay dates (Julian date: HNP females = 94.1 ± 9.1, LNP females = 93.3 ± 8.3; t23 =

0.23, P = 0.83). Also, HNP and LNP females laid their final experimental clutch on a

similar date (HNP females = 149 ± 8.8, LNP females =147 ± 7.8; t22 = -0.57, P = 0.57).

We found no significant differences in clutch size (Mann-Whitney U= 31.5, N ,= 9, N2=

9, p= 0.42), clutch mass (t8= 0.63, P= 0.55), or egg mass (t8= 0.25, P= 0.80) for the first

clutches of females subsequently assigned to either the high predation or low predation

treatments. We confirmed that the experimental diet was consumed and specific

components absorbed based on analysis of plasma carotenoid levels: fed and un-fed birds

were captured on day 6 of brood rearing, after the clutch number manipulation, at which

time fed birds had significantly higher plasma levels of carotenoids than un-fed birds (t41

= -2.93, P < 0.01).

3.3.1� Clutch Size Effects

Experimental nest predation significantly increase clutch number and significantly

decreased clutch size: HNP females laid 57% more clutches in total and in replacement

clutches laid 11 % fewer eggs per clutch or nearly half an egg less, compared to LNP

females (Fig 3.1a). Mean clutch number was 4.71 ± 0.47 for HNP females and 3.00 ±

0.00 for LNP females (Mann-Whitney U= 0.00, P< 0.001). Mean clutch size for

replacement clutches was 3.75 ± 0.07 for HNP females and 4.21 ± 0.14 for LNP females

59

(treatment effect, F L 64 = 10.70, P = 0.002). When considering clutch size across the

entire season, the effect of date on clutch size significantly varied between the two

treatments (treatment*Date, Fl,78 = 8.01, P < 0.01; treatment*date-) = FUBI = 7.21, P <

0.01; Fig 3.2a). The nest predation treatment had significant effects on the distribution of

clutch sizes laid by HNP and LNP females (X22= 14.23, P < 0.001), with HNP females

laying proportionally more small and medium sized clutches, but fewer large clutches.

HNP females laid 8.5% and 11.2% more 3 and 4 egg clutches respectively, but 19.7%

fewer 5 egg clutches (Fig 3.3).

3.3.2� Clutch Mass & Egg Mass Effects

The effect of experimentally-increased nest predation on total clutch mass

followed a similar trend as that seen for clutch size: HNP females laid replacement

clutches which were on average 6.5% lighter (Fig 3.1 b) and the effect of date on clutch

mass tended to vary between the treatments mirroring the clutch size results (Fig 3.2b).

However, the effect of experimental nest predation on the average clutch mass of

replacement clutches was marginally non-significant (mean ± SE (g), HNP == 11.24 ±

0.28, LNP = 12.04 ± 0.42; treahnent effect, F I, 55. = 2.46, P = 0.12), as was the interaction

between date and treatment on the seasonal pattern of clutch mass (treatment*date = F 1,61

== 2.77, P =0.10; treatment*date2, F 1.61 = 3.01, P = 0.09). Egg mass was not affected by

the treatment (F 1.56 = 0.24, P == 0.63) or by treatment by date interactions (treatment*date

)

= FI.62 = 0.05, P = 0.81; treatment*date-, FI.62 = 0.01, P = 0.91).

60�

3.3.3� Cumulative Effects of Nest Predation on Total Egg Production

As a result of producing 57% more clutches HNP females laid 41.5% more eggs

(Fig 3.1b) over the course of the experiment (HNP = 17.33 ± 0.5, LNP = 12.25 ± 0.37;

t15= 8.02, P < 0.001), again revealing that HNP females laid fewer eggs per replacement

clutch since the increase in total egg production (41 %) was less than expected from the

increase in clutch number (57 %). The clutch number difference (and the clutch number

affect on egg number) occurred within the same number of days, i.e. the length of the

experimental breeding season was similar when comparing HNP and LNP females

(season length, t22 = -0.31, P == 0.75). Constrained within the same season length, the

clutch number difference must be due to treatment effects on either the duration of each

nesting attempt (i.e. days from laying the first egg to clutch removal or abandonment)

and/or the duration of the re-nesting period between nesting attempts. HNP females nests

lasted 64% fewer days than LNP females' nests (HNP = 6.52 ± 0.45, LNP = 17.95 ±

0.79; t23= -13.26, P< 0.001), but HNP and LNP females did not differ in the length of the

re-nesting period (t23= -0.74, P= 0.47). This indicates that the clutch number difference

was solely due to predation effects on nest duration.

3.3.4� Physiological Cost of Egg Production

Prior to the start of breeding HNP and LNP females were in similar physiological

state when compared using our integrated measure of condition based on a suite of

physiological traits (DFA Eigen-value = 2.32, Wilks' Lambda = 0.30, pDFA P = 0.51).

However, at the tennination of the experimental nest predation manipulation HNP and

LNP females had significantly different physiological profiles (DFA Eigen-value = 32.7,

61

Wilks' Lambda = 0.03, pDFA P = 0.0012; Table 3.1). Looking at effect sizes> 0.2

(Table 3.1 & Fig 3.4), following Cohen's (1992) guidelines for the magnitude of effect

sizes (i.e. small= 0.2, medium== 0.5, large== 0.8), females in the high nest predation

treatment had higher polychromasia (immature red blood cells), a larger imbalance

between total plasma antioxidants (TAC) and oxidative status (TOS), lower plasma

carotenoids and plasma imunoglobulin levels, higher plasma corticosterone levels, and

lower body fat.

3.3.5 Hatching Success and Nestling Growth

Hatching success of eggs within the final experimental clutches did not differ

between HNP and LNP females (X2, == 0.13, P == 0.72). There was a trend for HNP

nestlings to be lighter 6 days after hatching, however the difference was non-significant

(ts = -1. 74, P = 0.12), and nestlings of HNP and LNP females were found to be of similar

structural size (ts = -0.44, P = 0.73).

3.4 Discussion

Consistent with our first prediction, our results clearly show that experimentalIy­

increased nest predation increased clutch number but decreased clutch size of

replacement clutches. In tenns of clutch size, females that experienced nest predation a)

laid smaller replacement clutches, and b) had a significantly altered pattern of seasonal

variation in clutch size. As a result, females that experienced high nest predation laid

proportionally more 3- and 4-egg clutches but fewer 5-egg clutches. Given the effects of

62�

nest predation on clutch size it was surprising that there were no significant predation

affects on clutch mass, although the affects of nest predation on clutch mass did closely

mirror those of predation on clutch size. 11 appears that indlvidual variation in egg size

and a slight non-significant increase in egg size by HNP females dampened nest

predation affects on clutch mass. Nest predation dld not significantly affect the mass of

eggs laid in replacement clutches or the seasonal pattern of variation in egg mass. In line

with our second prediction, our results show that high predation females (HNP) with

increased total egg production were in worse physiological condition, consistent with nest

predation inducing an increased cost of egg production. We suggest that treatment effects

on physiological condition were largely consistent with the hypothesis of non-resource

based mechanisms underlying costs of egg production, i.e. this involved changes in traits

related to hematology and oXldative stress.

Our study has demonstrated experimentally that the direct effect of nest predation

per se affects the clutch size of replacement clutches laid in subsequent nests, a finding

that contrasts with that of previous studies. For example, Doligez & Colbert (2003)

manipulated nest predation and also the visual cues of common nest predators (i.e. they

presented stuffed models at the nest) but found that clutch size did not differ between

birds that experienced nest predation and those that had not. Rather, Doligez & Colbert

(2003) found that clutch size was reduced in the experimental areas in the year following

experimental manipulation because females laid smaller clutches when paired to males

that had resided in the experimental area during the manipulation and laid larger clutches

when paired to newly immigrated males. Doligez & Colbert (2003) concluded that clutch

size adjustments were a result of infOlmation on predation risk, but that this information

63

was not based on direct nest predation. Eggers et al. (2006) examined the effects of

auditory cues to predator presence and found that Siberian jays reduced clutch size in

response to increased vocalizations of common nest predators. Fountain and Martin

(2006), using a predator removal experiment, show that in habitats with both more

predator cues and higher levels of nest predation, birds produced smaller eggs, but not

smaller clutches. In our study we manipulated nest predation only, and not the visual or

auditory cues of predators. In this case both treatments were equally exposed to the nest

predator (the researcher) and would presumably have similar background cues from

natural predators at our study site. The reduction in clutch size that we report thus

demonstrates that nest predation itself can affect clutch size through effects on clutch loss

and is, to the best of our knowledge, the first experimental evidence that direct nest

predation affects clutch size.

As we discussed in the introduction, Martin (1995) proposed that the inverse

correlation between clutch number and clutch size observed across species may have

been the result of an energetic trade-off. In the present study nest predation increased

clutch number and decreased clutch size when energy and nutrients were super-abundant,

suggesting that food availability can not eliminate the effects of direct nest predation on

clutch size in song sparrows. Yet, it is possible that food limitation may remain the

underlying mechanism for the clutch size reduction that we report. For example, the

amount of food consumed by prey may be affected by predator' intimidation', resulting

in predators affecting clutch size indirectly through effects on food availability. Martin

(1992) described one such means where nest predation affects "perching time" (i.e.,

vigilance), which affects "foraging time," which through reduced food consumption

64�

affects clutch size. However, our work and previous work on song sparrows suggests that

predator intimidation did not limit song sparrows access to food. In a bi-factorial study of

food and predators, Zanette et al. (2006b) found that food availability is an important

factor detennining the length of time required for re-nesting, with fed birds re-nesting in

significantly fewer days than un-fed birds. Therefore, if predator intimidation reduced

song sparrows access to food we would expect that predators would then also affect the

re-nesting duration. Zanette et al. (2006b) reported that predators did not affect the re­

nesting duration for both fed and un-fed birds. In the present study we also found the nest

predation did not affect the re-nesting duration suggesting that predator intimidation did

not affect song sparrows access to food. Overall, we suggest that our results demonstrate

that nest predation in song sparrows affects clutch size by a mechanism that is

independent of food availability.

In showing that nest predation can affect clutch size when food is abundant, our

results also confinn that food and predators can have combined effects on clutch size via

food acting on the rate of nest predation. Zantte et al. (2006a) found that food availability

reduced the occurrence of nest predation in song sparrows and Duncan-Rastogi et aJ.

(2006) showed this was likely because fed birds spent more time on the nest and made

fewer foraging trips of shorter duration then un-fed birds. Combined these results

suggested that if nest predation does affect clutch size then because food availability

reduced the occurrence of nest predation, food may reduce the opportunity for predation

to affect clutch size (i.e. a food and predator interaction on clutch size) (Zanette et al.

2006b). Therefore, our results demonstrating that predators can affect clutch size also

confinns that food and predators can have combined affects on clutch size (Zanette et al.

6S�

2006b). Taken together, we suggest that work on food and predators in song sparrows

sheds light on the decades long dichotomy over whether food or predators affect clutch

size (Lack 1947, Pemns 1977, Martin 1987, Newton 1998).

3.4.1� Total Cumulative Egg Production & the Physiological Cost of Egg Production

Our results suggest that there is a physiological cost of increased egg production

as a result of direct nest predation stimulating more frequent re-nesting and thus, total egg

production. Here experimentally-increased nest predation increased total egg production

with HNP females laying an average of 1.7 more clutches, which resulted in HNP

females producing an additional 5 eggs over the season. We believe that the effects of

nest predation on egg production that we found are not specific to our study. In birds,

direct nest predation 1s the primary source of nest failure (Ricklefs 1969, Martin 1995),

and has frequently been reported to stimulate re-nesting and thus increase clutch number

in many species (Grzybowski and Pease 2005, Zanette et a1. 2006b). For example, Catlin

& Rosenberg (2008) found that nest predation in burrowing owls (Athene mniculari)

resulted in both increased clutch number and total egg number. Other studies directly

manipulating egg production by egg removal, have shown that increased egg production

effort can have negative affects on female survival (Nager et a!. 200 I, Visser and Lessells

2001), a female's ability to rear chicks (Monaghan et a1. 1998), and can delay subsequent

breeding (Kalmbach et a1. 2004), negatively impact a female's physiological condition,

reduced hatching success of eggs (Kalmbach et a1. 2004), and reduced nestling size

(Monaghan et a!. 1995).

66

In our study, HNP females were in worse overall physiological condition at the

end of the season, consistent with costs incurred from greater total seasonal egg

production. We found that treatment effects on condition indices were largely consistent

with predictions for the non-resource based costs of egg production discussed in the

introduction (Williams 2005). However, traditionally the cost of egg production is

explained by a resource based trade-off where females are forced to allocate a limiting

amount of food resources between there own energetic needs or those of their offspring.

For example, Kalmbach et al. (2004) found that increased egg production in great skuas

(Stercorarius skua) resulted in both a decrease in female mass and reduced hatching

success of later laid eggs. In the present study, we supplemented all birds a high quality

diet, and therefore did not expect to find resource based trade-offs. Even if we assumed

resources were still limiting, but by an internal limitation (e.g. absorption rate) rather than

external quantity (Speakman and Krol 2005), we would expect treatment effects on

energetic traits and or a trade-off with offspring quality. However, we generally did not

detect evidence for resource based trade-off. Here, there were significant increases in our

two measures of egg production, yet there were generally weak and inconsistent

treatment affects on female energetic balance (with the exception of fat); in fact there was

a slight positive affect on HNP mass. Furthermore, female energetic condition does not

appear to have been maintained at the expense of offspring quality. HNP females actually

laid slightly heavier eggs (3.00g vs 2.89g, non-significant difference) in their final

clutches, and there were no treatment effects on hatching success, or nestling growth. We

suggest that the general lack of effects, or trade-off, predicted by the resource based

hypothesis, indicates that females were not limited by food availability; which is

67

consistent with the super-abundance of food and is similar to our clutch size results. That

said, there was a large negative treatment effect on fat scores, suggesting that despite an

abundance of food HNP females were not able to absorb or assimilate energy at a high

enough rate to maintain fat stores during increased egg production (Speakman and Krol

2005). Alternatively, temporal differences in access to food may have influenced fat

dynamics between treatments. HNP females spent less time incubating nests and more

time re-nesting as a result of predation, reducing the number of days spent in continuous

short term incubation fasts. Energy during short term fasts is fuelled by fat stores (Jenni­

Eiermann and Jenni 1997), however, because converting food into fat and then

metabolizing fat into usable energy is inefficient (Cope 2003), animals with continuous

access to food may facultatively reduce fat storage (i.e. similar to the programmed

anorexia hypothesis Norberg 1981, Gaston and Jones 1989).

In contrast to effects on "energetic" physiological traits, overall there were

consistent negative effects on non-energetic indices. In terms of the different non­

resource based mechanisms for costs of egg production described in the introduction,

HNP females had a greater imbalance between antioxidants and oxidative status (reactive

oxygen species) consistent with oxidative stress as an underlying mechanism for the cost

of egg production (Salmon et al. 2001, Wang et al. 2001, Alonso-Alvarez et al. 2006,

Bize et al. 2008). HNP females also had higher polychromasia (immature red blood cells)

as predicted by the idea of reproductive anaemia as a result of estrogen-dependent

transient (i.e. during egg laying) suppression of red blood cell (RBC) production

(Williams et al. 2004, Wagner et al. 2008a). This suggests that a greater number of bouts

of egg laying (i.e. clutch number) with high nest predation might involve more frequent

68�

exposure to reproductive hormones which would enhance the negative pleiotropic

(multiple) effects of gonadal estrogens. The slightly higher levels of hematocrit (which

appears inconsistent with estrogen suppression) of HNP females may be due to the

increased proportion of the larger (volume) immature red blood cells (Campbell and Ellis

2007) found in HNP females blood. We obtained blood samples approximately 9 days

after estrogens would have decreased (i.e. at clutch completion (Williams et al. 2004),

which is sufficient time for regenerative anaemia to recover hematocrit levels (Domm

and Taber 1946, Clark et al. 1988) through increasing cell numbers but also simply

because of the larger contribution of each immature cell to hematocrit volume (Fernandez

and Grindem 2006). Our general indices of condition, immunogloblins and

corticosterone, are also consistent with HNP females being in worse condition, and

potentially, at least in the case of imunoglobulins, as a result of increased egg production.

Merino et al. (2006) found that increased breeding effort reduced immunoglobulin levels

and Ochs et a1. (2008) found that females that produced more eggs (i.e. larger clutches)

had declines in general measures of immune function. Finally, numerous studies have

shown that plasma corticosterone levels are negatively correlated with condition

(Kitaysky et al. 1999, Romero and Wekelski 2001, Lobato et a1. 2008).

A further explanation for our results is that many of these physiological effects we

observed might also be associated with the elevated corticosterone levels in HNP

females, potentially because increased nest predation induced higher 'stress'. The chronic

stress hypothesis (conflict between obtaining food and avoiding being eating) proposed

by Boonstra et al. (1998) for mammals and followed up by Clinchy et a1. (2004) in birds,

also predicts a number of broad scale physiological changes observed here (excluding

69�

oxidative stress). To examIne this hypothesis we looked at the direction of the

relationship between corticosterone (stress honnone) and all other indices of condition at

post breeding (but not reported in this paper). We found that the direction of the

relationship between indices and corticosterone was not consistent with treatment effects

on indices (7 out 12 times), suggesting corticosterone did not cause many of the treatment

effects. Although, corticosterone can have wide spread influence on many variables we

examined, we suggest that it does not provide a better explanation then predictions made

by the cost of egg production but may have also contributed to our results.

Our results provide initial evidence consistent with the possibility that the effect

of nest predation on increased clutch number and decreased clutch size is the result of a

trade-off influenced by physiological costs of egg production. Gasparini et al. (2006)

found that experimentally inducing re-nesting (i.e. increasing clutch number) in

kittiwakes resulted in reduced clutch mass in both fed and un-fed birds replacement

clutches (i .e. simi lar to our findings food did not eliminate the inverse relationship),

which they suggest may be the result of a physiological constraint influencing egg

production decisions. If in the present study clutch size reductions are the result of

physiological cost of egg production, the influence of such costs would have to occur

early in the season because HNP females produced smaller clutches as early as the

second clutch, i.e. the first replacement clutch. However, in the present study we are

unable to detennine if increased physiological costs of egg production are due to a greater

total egg production (i.e. more bouts and more eggs), which would not fully explain the

early clutch size reduction or, due to the fact that high-predation females had an increased

frequency of egg laying with less "recovery" time between laying events; which would

70�

be consistent with an early reduction of clutch size. With regards to shorter recovery

explanation, incubation, although energetically demanding, can allow for physiological

recovery from egg production (Hario et al. 1991, Alonso-Alvarez et al. 2002, Williams et

al. 2004, Navarro et al. 2007, Wagner et aJ. 2008b). In our study, increased nest predation

shortened the incubation period reducing the potential for incubation recovery. As well,

shorter incubation periods were not compensated for by longer re-nesting duration. Such

behaviour may be due to the fact that annual reproductive success in song sparrows and

other species is strongly influence by clutch number (Nagy and Holmes 2004, Zanette et

al. 2006a), therefore birds may be 'driven' to re-nest without fully recovering

physiological state, with physiological state then influencing clutch size decisions.

In conclusion, our results further highlight how predators can negatively affect

prey beyond simply killing them or their offspring (Lima 1998, Creel and Christianson

2008). Here, song sparrows that experienced nest predation laid smaller subsequent

clutches and ending up in worse physiological condition, consistent with predation effects

increasing the cost of egg production. Furthermore, our results suggest that direct nest

predation does affect clutch size and can affect physiological condition independent of

food availability, but importantly, we do not imply that this means that food does not also

affect the variables we measured. Our results confinn a previous hypothesis that food and

predators have combined effects on clutch size (Zanette et al. 200Gb), and highlights the

importance of simultaneously considering the affects of food and predators.

71

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Zanette, L., M. Clinchy, and J. N. M. Smith. 2006a. Combined food and predator effects on songbird nest survival and annual reproductive success: results from a bi­factorial experiment. Oecologia 147:632-640.

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77

Figure 3.1 Treatment effects on the observed mean ± SE of a) clutch size b) clutch mass C) and total seasonal egg number laid by HNP (black) and LNP (grey) females in replacement clutches.

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78

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18

Figure 3.4 Standardized effects sizes for treatment effects on HNP females relative to LNP females. Effect size; small = 0.2, medium == 0.5. larger == 0.8 (Cohen 1992). TAC == Total antioxidant capacity, TOS == Total oxidative status

Hematocrit

Hemoglobin

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TAC

TOS

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82�

CHAPTER 4 GENERAL SYTHESIS AND FUTURE DIRECTIONS

4.1 Synthesis

What follows is a discussion of our results from chapter 2 and 3 usmg a new

conceptual frame work (allostasis) to integrate the findings of both chapters. I have

chosen to discuss allostasis because I believe that ecophysiology studies could benefit

from working within the allostasis framework. In addition, although not discussed below,

I have found that the analytical tools used in studies of allostasis may also be beneficial to

ecologists that use multiple variables to measure physiological state.

A goal of many studies of free living animals is to identify physiological traits

that indicate the health and fitness potential of an individual (Bortolotti et al. 2002, Hill

and Fanner 2005, Ochs and Dawson 2008). Numerous studies report relationships

between hormonal, immunological or energetic measures and traits related to

reproductive success and survival (Andersson and Gustafsson 1995, Atkinson and

Ramsay 1995, Romero and Wekelski 2001, Bety et al. 2003, Blums et al. 2005).

However, no universal trait has yet immerged that ecologist, or doctors for that matter,

use to determine the health or fitness potential of an individual. Our results (chapter 2)

suggest that this may be due to the fact that no single trait explains much of the variation

in overall physiological state. From this, we concluded that measurements of condition

should integrate multiple traits to obtain a better understanding of global physiological

state. The results of chapter 3 provide further support for this earlier finding. Rather than

83

large effects on a few physiological traits, our analysis of condition revealed that it was

the cumulative effect of predation spread across a number of traits that resulted in

treatment differences.

The results from a number of human epidemiology studies appears to support our

conclusion that a more integrated measure will aid in more accurately assessing condition

(McEwen 1998, Seeman et al. 2001, Glei et al. 2007, Nelson et al. 2007), but these

studies also provide a framework to better understand physiological results. Many

medical studies are examining physiology and health within the conceptual framework of

allostasis (Stewart 2006). Allostasis is the maintenance of stability (in traits key to

immediate survival-pH, glucose, temperature, oxygen levels) through change in other

physiological systems or through behavioural changes (e.g. the laying of smaller clutches

found in chapter 3) (McEwen and Seeman 1999). When the body is challenged (e.g. as

mundane as standing up in the morning or as serious as fleeing a predator), stability in the

four key traits is maintained by honnone regulated changes in physiology (e.g. mobilise

fat stores when glucose is low)(McEwen and Wingfield 2003). When larger challenges

are frequent and long lasting, the bodies physiological response inevitably leads to wear

and tear on its own system (i .e. allostatic load) (Seeman et al. 200 I, McEwen 2008).

Allostasis and allostatic load appear to provide a good explanation for why we

found significant but weak correlation among traits (chapter 2) and why we might expect

that predation effects were only moderate but spread over many traits (chapter 3).

Allostasis is regulated by honnones which act (sometimes in detrimental fashion) on

many tissues throughout the body and can have suppressive and stimulatory effects on

other honnones; e.g. as one mediator increases there are compensatory changes else

84�

where (McEwen 2008). This highlights the interconnected nature of organismal

physiology and would likely result in correlations between traits like we found in chapter

2. However, for much of the same reasons, the allostatic response can be very complex;

with multiple pathways to the same end point (McEwen 2008). In this case, the

relationship between traits would be weakened. Furthennore, multiple physiological

pathways to solve one challenge (e.g. recovery from egg laying) may result in spreading

physiological 'costs' over multiple traits. For these reasons, researchers who use the

concept of allostasis propose that obtaining a more comprehensive measure of

physiological condition will provide a better health index (Seeman et al. 2001).

One mechanism that generates allostatic load is repeated events that result JO

elevations of the al10static response (Logan and Barksdale 2008). It seems that nest

predation induced laying of addition clutches could be such a repeated event. A second

type of allostatic load involves the failure to habituate to the same stressor (i.e. the failure

ofthe body to dampen the honnonal response) (McEwen and Seeman 1999). Again, with

respect to predation, females that are laying additional clutches can not 'habituate' to the

reproductive honnones necessary for egg production. In this sense, exposure to estradiol

and subsequent proposed suppression of erythropoieses during laying would represent the

second type of alJostatic load.

4.2 Future Directions

Future studies should test if reductions in condition like those reported here affect

reproduction and survival outside of the experimental breeding season, as these data

85

would be critical to quantify the long tenn demographic costs of predation and increased

egg production. Directly manipulating condition in a manner predicted by the cost of egg

production would seem like the most expedient way to examine the long tenn effects of

increased egg laying. Administration of caffeine (Olcina et al. 2008) or nicotine (Neogy

et al. 2008) has previously been used to increase oxidative stress. And injections of

phenylhydrazine hydrochloride can be used to induce anaemia (Clark et al. 1988, Wagner

2008). These manipulations could be conducted at the end of the breeding season to

examine the effects of egg production on over winter survival and subsequent breeding

perfonnance.

In the present study we were not able to determine whether clutch size reductions

we reported are the result of egg production costs influencing clutch size decisions (i.e.

bet hedging strategy, Slagsvold 1984) or whether females reduced clutch size as a

strategy to limit the likelihood of future predation (i.e. nest survival strategy, Skutch

1949). As we suggested in chapter 3, are data are consistent with costs of egg production

influencing clutch size. However, our results are not inconsistent with females having

reduced clutch size to limit predation occurring on subsequent nest (i.e. reduced nest

duration or reduced conspicuousness hypothesis, Slagsvold 1982). Indirect evidence

reported in song sparrows suggests that reduction in clutch size may in fact reduce the

likelihood of predation occurring. Zanette et al. (2006) found that fed birds were less

likely to have their nests predated, which may result from fed females having longer

bouts on and shorter bouts off the nest during incubation (Duncan-Rastogi et al. 2006).

These results suggest that activity at the nest may in fact be an important factor

influencing predation. A valuable first step in differentiating between these two

86�

hypothesis would be to first test the relationship between clutch size and condition. This

could be accomplished using the phenotypic manipulations of oxidative stress and

hematocrit mentioned above. Regardless, of the specific nest predation hypothesis, this

work may provide valuable insight into proximate mechanisms affecting clutch size,

particularly as previous studies have found correlations between clutch size and both

oxidative stress (Bize et a1. 2008) and hematocrit (Dufva 1996).

4.3 References

Andersson, M. and L. Gustafsson. 1995. Glycosylated hemoglobin - a new measure of condition in birds. Proceedings of The Royal Society of London Series B­Biological Sciences 260:299-303.

Atkinson, S. and M. A. Ramsay. 1995. The effects of prolonged fasting of the body­composition and reproductive success of female polar bears (Ursus-maritimus). Functional Ecology 9:559-567.

Bety, 1., G. Gauthier, and 1. F. Giroux. 2003. Body condition, migration, and timing of reproduction in snow geese: A test of the condition-dependent model of optimal clutch size. American Naturalist 162:110-121.

Bize, P., G. Devevey, P. Monaghan, B. Doligez, and P. Christe. 2008. Fecundity and survival in relation to resistance to oxidative stress in a free-living bird. Ecology 89:2584-2593.

Slums, P., J. D. Nichols, J. E. Hines, M. S. Lindberg, and A. Mednis. 2005. Individual quality, survival variation and patterns of phenotypic selection on body condition and timing of nesting in birds. Oecologia 143:365-376.

Bortolotti, G. R., R. D. Dawson, and G. L. Murza. 2002. Stress during feather development predicts fitness potential. Journal of Animal Ecology 71 :333-342.

Clark, M. W., R. P. Gildersleeve, J. P. Thaxton, C. R. Parkhurst, and D. 1. McRee. 1988, Hematological effects of ethyl methanesulfonate, paraquat and phenylhydrazine in japanese quail. Comparative Biochemistry and Physiology C-Phannacology Toxicology & Endocrinology 89: 15-30.

87

Dufva, R. 1996. Blood parasites, health, reproductive success, and egg volume in female Great Tits Panls major. Journal of Avian Biology 27:83-87.

Duncan-Rastogi, A, L. Zanette, and M. Clinchy. 2006. Food availability affects diurnal nest predation and adult antipredator behaviour in song sparrows, MeLospiza melodia. Animal Behaviour 72:933-940.

Glei, D. A., N. Goldman, Y. L. Chuang, and M. Weinstein. 2007. Do chronic stressors lead to physiological dysregulation? Testing the theory of allostatic load. Psychosomatic Medicine 69:769-776.

Hill, G. E. and K. L. Fanner. 2005. Carotenoid-based plumage coloration predicts resistance to a novel parasite in the house finch. Naturwissenschaften 92:30-34.

Logan, J. G. and D. J. Barksdale. 2008. Allostasis and allostatic load: expanding the discourse on stress and cardiovascular disease. Journal of Clinical Nursing 17:201-208.

McEwen, B. S. 1998. Protective and damaging effects of stress mediators. New England Joumal of Medicine 338: 171-179.

McEwen, B. S. 2008. Central effects of stress honnones in health and disease: Understanding the protective and damaging effects of stress and stress mediators. European Journal of Phannacology 583: 174-185.

McEwen, B. S. and T. Seeman. 1999. Protective and damaging effects of mediators of stress - Elaborating and testing the concepts of allostasis and allostatic load. Pages 30-47. New York Acad Sciences.

McEwen, B. S. and 1. C. Wingfield. 2003. The concept of allostasis in biology and biomedicine. Honnones and behavior 43:2-15.

Nelson, K. M., G. Reiber, T. Kohler, and E. J. Boyko. 2007. Peripheral arterial disease in a multiethnic national sample: The role of conventional risk factors and allostatic load. Ethnicity & Disease 17:669-675.

Neogy, S., S. Das, S. K. Mahapatra, N. Mandai, and S. Roy. 2008. Amelioratory effect of Andrographis paniculata Nees on liver, kidney, heari, lung and spleen during nicotine induced oxidative stress. Environmental Toxicology and Phannacology 25:321-328.

Ochs, C. L. and R. D. Dawson. 2008. Patterns of variation in leucocyte counts of female tree swallows, Taehycineta hieoLor: Repeatability over time and relationships with condition and costs of reproduction. Comparative Biochemistry and Physiology A-Molecular & Integrative Physiology 150:326-331.

88�

Olcina, G. J., R. Timon, D. Munoz, J. J. Maynar, M. J. Caballero, and M. Maynar. 2008. Caffeine ingestion effects on oxidative stress in a steady-state test at 75% V-02 (max). Science & Sports 23:87-90.

Romero, M. L. and M. Wekelski. 2001. Corticosterone levels predict survival probabilities of Galapagos marine iguanas during El Nino events. Proceedings of the National Academy of Sciences of the United States of America 98:7366-7370.

Seeman, T. E., B. S. McEwen, J. W. Rowe, and B. H. Singer. 2001. Allostatic load as a marker of cumulative biological risk: MacArthur studies of successful aging. Proceedings of the National Academy of Sciences of the United States of America 98:4770-4775.

Skutch, A. F. 1949. Do tropical birds raise as many young as they can nourish? Ibis 91:430-455.

Slagsvold, T. 1982. Clutch size vanatlOn In passenne birds - the nest predation hypothesis. Oecologia 54:159-169.

Slagsvold, T. 1984. Clutch size variation of birds in relation to nest predation - on the cost of reproduction. Journal of Animal Ecology 53:945-953.

Stewart, J. A. 2006. The Detrimental Effects of Allostasis:Allostatic Load as a Measure of Cumulative Stress. Journal of physiological Anthropology 25: 133-145.

Wagner, E. 2008. Anemia: A pysiological mechanism underlying the cost of egg production. Simon Fraser University, Burnaby.

Zanette, L., M. Clinchy, and J. N. M. Smith. 2006. Combined food and predator effects on songbird nest survival and annual reproductive success: results from a bi­factorial experiment. Oecologia 147:632-640.

89�