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Metabolism and performance : a study of provisioning in the tree swallow, Tachycineta bicolor Burness, Gary P. 2000

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METABOLISM AND PERFORMANCE: A STUDY OF PROVISIONING IN T H E T R E E SWALLOW, Tachycineta bicolor by G A R Y P. B U R N E S S  B.Sc. (Hons), Memorial University o f Newfoundland, 1989 M . S c , B r o c k University, 1992  A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF D O C T O R OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES. (Department o f Zoology).  W e accept this thesis as conforming to the required standard  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A September 2000  © Gary P. Burness, 2000  In  presenting this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  scholarly purposes may be her  representatives.  permission.  Z^ffi  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  8  StjJ.  ZOOO  for  an advanced  Library shall make it  agree that permission for extensive  It  publication of this thesis for financial gain shall not  Department of  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  is  granted  by the  understood  that  head of copying  my or  be allowed without my written  ABSTRACT  One goal of evolutionary physiology is to relate phenotypic variation to Darwinian fitness via organismal performance. Within this framework, I used breeding tree swallows, (Tachycineta bicolor) to identify physiological correlates and potential fitness consequences of inter-individual variation in parental energy expenditure (sustained metabolic rate, SusMR). I measured parental S u s M R using the doubly labelled water technique and correlated it with variation in natural brood size and nestling growth rate and mass. S u s M R was independent o f natural brood size, although large broods had greater mass gain than small broods. I hypothesized that parental efficiency increases with brood size. A m o n g adults rearing the same sized broods, S u s M R increased with brood mass, and i n one year, female S u s M R and nestling growth rate were positively correlated. Natural selection is defined as correlation between variation in a phenotypic trait and variation in fitness. If nestling mass or growth rate are accurate indices of fitness, S u s M R was under selection in this population. Individuals with high S u s M R had relatively large intestines; presumably increasing digestive capacity. This may result i n an increased resting metabolic rate and identify a potential energetic trade-off. I determined the influence o f body composition on resting oxygen consumption rate (VO2). The mass o f most organs differed between breeding seasons, possibly due to environmental conditions. Individuals with high resting VO2 had large kidneys but relatively small intestines. The basis o f a negative relationship is unclear because the intestine contributes positively to VO2 i n other species. A major determinant of parental life-time reproductive success is the survival of offspring to breeding. This is influenced by the quality o f the rearing environment and its affect on offspring condition. Few studies have investigated what physiological and biochemical characters underlie variation in condition. I manipulated the number of nestlings in a brood and followed growth and resting VO2 until near fledging. Surprisingly, many  characters were insensitive to environmental variation. Nonetheless, nestlings in reduced broods had a greater mass o f lipid, increased cardiac enzyme activity, and higher size-specific resting VO2 than individuals raised in enlarged broods. H o w these characters affect survival or the future adult phenotype remains unknown.  TABLE OF CONTENTS  Abstract  u  Table o f Contents  ...iv  List o f Tables  vi  List o f Figures  vii  Acknowledgments  '*  Preface  ".  CHAPTER ONE:  General introduction  CHAPTER TWO:  Physiological correlates o f parental quality i n breeding tree swallows, Tachycineta  CHAPTER THREE  1  bicolor.  Introduction  22  Materials and Methods  24  Results  33  Discussion  48  Conclusion  54  Inter-individual variability i n body composition and resting oxygen consumption rate i n breeding tree swallows Preface  55  Introduction  55  Materials and Methods  57  Results  62  Discussion  72  Conclusion  77  CHAPTER FOUR  Effect o f brood size manipulation on offspring physiology: an experiment with passerine birds.  CHAPTER FIVE  Preface  78  Introduction  78  Materials and Methods  79  Results  85  Discussion  94  Conclusion  99  General conclusions  L I T E R A T U R E CITED  APPENDIX 1  100  107  Rates o f C O 2 production and estimated sustained metabolic rates o f male and female tree swallows rearing natural sized broods  121  vi  LIST O F T A B L E S  2.1  Variation in haematocrit of adult tree swallows rearing different sized broods  41  2.2  Physiological correlates o f brood size in female tree swallows  42  2.3  Physiological correlates o f brood size in male tree swallows  43  2.4  Correlates o f total lipid mass i n female tree swallows  44  2.5  Correlates of total lipid mass in male tree swallows  45  2.6  Enzyme activity in the pectoralis of adult tree swallows  47  3.1  Allometric relationships and descriptive statistics for variables measured in tree swallows  3.2  64  External morphometries between years for male and female tree swallows  68  3.3  Inter-individual Pearson correlations among tissue and organ masses  71  4.1  Rates of resting oxygen consumption and carbon dioxide production of tree swallow nestlings  4.2  Effect of brood manipulation on organ masses o f 16 day old tree swallows  4.3  4.4  88  91  Effect of brood manipulation on muscle mass and water fraction of 16 day old tree swallows  92  M a x i m u m enzyme activities from tissues o f 16 day old tree swallows  93  vii  LIST O F FIGURES  1.1  Organismal performance paradigm  2.1  Box plots of growth rates between days 4 and 8 of (A) entire tree swallow broods and (B) individual nestlings  2.2  35  Sustained metabolic rate of adult tree swallows rearing natural sized broods  2.4  34  Allometric scaling of parental sustained metabolic rate and body mass in breeding tree swallows  2.3  3  37  Sustained metabolic rate of adult tree swallows and (A) the residual growth rate of their broods between days 4 and 8 and (B) the residual mass of their broods on day 8  39  2.5  Parental sustained metabolic rate and the wet mass of the small intestine........ 46  3.1  Allometric scaling of body mass and (A) resting oxygen consumption rate, (B) heart mass, (C) kidney mass, (D) small intestine mass, (E) liver mass, and (F) pectoralis mass  66  3.2  Correlation between resting oxygen consumption rate and kidney mass  70  4.1  (A) Body mass, and (B) body mass adjusted-resting oxygen consumption rate of tree swallow nestlings, as a function of age and treatment  4.2  86  Lipid mass of 16 day old tree swallow nestlings, adjusted for body size (PCI), as a function of brood manipulation  89  viii  PREFACE Portions o f this thesis have been previously published as the following: Burness, G.P., R. C. Ydenberg and P. W . Hochachka. 1998. Interindividual variability in body composition and resting oxygen consumption rate in breeding tree swallows, Tachycineta bicolor. Physiol. Zool. 71: 247-256. Burness, G.P., G . B . McClelland, S. L . Wardrop, and P. W . Hochachka. (2000). Effect o f brood size manipulation on offspring physiology: an experiment with passerine birds. J. exp. B i o l . (In press). This work was performed in Dr. Peter Hochachka's lab at the Dept. o f Zoology, the University o f British Columbia. Experimental design and execution of the research, as well as all data analyses and the writing o f the manuscripts were the responsibility o f Gary Burness, a Ph.D. candidate under my supervision.  Peter Hochachka, Ph.D. Dept. o f Zoology U . B . C . 07/09/00  ACKNOWLEDGMENTS  I would like to thank my supervisor and mentor, D r . Peter Hochachka, for his inexhaustible enthusiasm for science and for providing an environment in which I was free to develop a thesis topic outside of his general research area. B y combining a group o f graduate students with diverse research interests he created an academic environment that was second to none. I also wish to thank Dr. Ron Ydenberg for first identifying the research question that I explore in this thesis, for providing facilities i n the field and for always being willing to discuss interpretations o f the data. Completion o f this thesis would not have been possible without Peter's and Ron's combined input. This study would also not have been possible without the capable field and lab assistance of: Shawn Taylor, Julie Hunter, M i c k Legare, Fintan Maguire, Lara Webster, Chris Tucker, Sharilynn Wardrop, James Burns, Arlen Cosh, Margie Cooper, Melanie Grant, Dianna Dyke, Debbie Higgins, Jackson Lau, W i l l Park, Marc-Andre Beaucher and Scott Dicken. T o each o f them I am grateful. The staff o f the Creston Wildlife Management Area allowed me to work i n the Area and provided me with logistic support and friendships in the field. Brian Stushnoff, Gillian Cooper, Anne de Jager, Brenda Bruns, D o n Grayson, D o n Bjarnason, and Rosamond Eben - thanks to each o f you. The Hochachka lab was an amazing place in which to work. This was due in no small part to friendships and discussions with: Les Buck, Steve Land, J i m Staples, T i m West, Raul Suarez, Carole Stanley, Mark Mossey, Mark Trump, Petra Mottishaw, Jim Rupert, Charles-Antoine Darveau, K e v i n Campbell, Cheryl Beatty, Susan Shinn, Chris Guglielmo and Y a n Burelle. Special thanks to Sheila Thornton and Grant M c C l e l l a n d for delaying their theses write-ups so as not to finish too far ahead o f me. Thanks to Drs Lee Gass, B i l l Milsom, and Raul Suarez who made numerous useful suggestions throughout the development o f this project, and improved the text o f the thesis. Finally, I would like to thank my M o m for her unending support, both emotional and financial, and Antje for her friendship and love during the seemingly never-ending writing stage o f this thesis.  1  CHAPTER 1 GENERAL INTRODUCTION  Over 140 years ago, Darwin proposed that variation among species was the evolutionary result o f natural selection acting on phenotypic variation among individuals. Despite the fact that variation is the cornerstone upon which the theory o f evolution was built, it is only over the past 20 years or so that studies have investigated the extent o f physiological variation that exists among members of the same population. Strictly speaking, inter-individual variation in physiological characters has been recognized and studied for over 75 years (Prosser 1955). Such studies begin with the observation that populations o f the same species experience different environmental conditions due to geographic location. The average value for a character o f interest is reported for each population, with an associated standard deviation or error to indicate the degree o f confidence in the values. Although these types o f studies investigate members o f a single species, biological differences they identify are really among populations rather than among individuals per se (Garland and A d o l p h 1991). A description o f central tendency is useful for addressing numerous questions concerning function. Such a design, however, ignores and indeed attempts to minimize an additional source o f variation: that detectable among individuals within each population (Bennett 1987). The realization that repeatable and heritable variation is necessary for evolution via natural selection has resulted in increased interest in the causes and consequences o f what has traditionally been viewed in many physiological studies as "noise" (Bennett 1987). In this thesis, I focused on the variation that exists among individuals within a single population o f birds. I looked for physiological correlates o f sustained performance and attempted to link these via behavioural traits with Darwinian fitness. Accordingly, when  2  referring to intra-specific or inter-individual variation, I follow Darwin's definition, that o f "...differences...observed in the individuals o f the same species inhabiting the same confined locality" (Darwin 1888, p34). In this introduction, I first review the organismal performance paradigm o f Arnold (1983) and introduce approaches to studying inter-individual variation. A s most work on performance to date has focused on non-sustainable measures o f energy expenditure (e.g., sprinting or endurance), I review some o f these studies before introducing the concept o f sustained energy expenditure. I introduce breeding birds as a model system with which to study the evolution o f sustained energy expenditure, and discuss the potential importance o f the rearing environment in determining phenotypic variation. Finally, I introduce my study system and identify the aims of each research chapter.  Organismal performance  paradigm  Over the past 20 years, the study o f inter-individual variation has increased, particularly at the physiological level. M u c h o f this is due to the influential papers o f Lande and Arnold (1983), Bennett (1987) and the formulation o f the "organismal performance paradigm" (Arnold 1983). This paradigm provided a method by which selection on morphological traits could be measured i n natural populations. In its original formulation the paradigm focused on morphology, however, it can equally be applied to structure, physiology and behaviour. A s outlined by A r n o l d (1983), the task o f measuring selection on physiological traits can be broken into two parts: (1) the measurement o f the effects of physiological variation on performance and (2) identifying relationships between performance on fitness. This paradigm was later modified by Garland and Carter (1994) to include additional terms within the causal pathway (e.g., behaviour). Figure 1.1 outlines the organismal performance paradigm. In this version, an individual's genotype and its environment during development and ontogeny interact to  3  Genotype  Morphology Physiology Biochemistry  Environment  PERFORMANCE (Sustained energy expenditure)  BEHAVIOUR (Provisioning capacity)  NATURAL SELECTION  Figure 1.1 Organismal performance paradigm. The bracketed terms are examples o f a performance and a behaviour found i n my study (after Garland and Carter 1994).  4  determine its primary phenotypic characters. These characters are defined simply by the broad terms "morphology, physiology and biochemistry." These characters are assumed to act individually or together to set the upper limits on performance capacity. A n example o f a biochemical character would be the activity of hexokinase, an enzyme exerting considerable control over capacity for flux through glycolysis (e.g., Kashiwaya et al. 1994). Capacity for flux though a metabolic pathways likely limits an organism's performance abilities under various circumstances (e.g., Suarez 1996). Organismal performance is an extremely broad concept representing numerous different activities. For example, in his review Pough (1989) identified over 120 studies falling into five categories o f performance: (1) forced activity, (2) social behaviour and reproduction, (3) natural activity and foraging, (4) predatory activity and feeding, and (5) defensive behaviour.  Within the evolutionary physiology literature, organismal performance  has been most frequently used to describe the first two. However, regardless o f the activity that it describes, organismal performance defines the limits of what an individual is capable of, while 'behaviour' defines what an individual actually does (Garland and Carter 1994). For example, although a female mouse may be physiologically capable o f feeding 14 pups (Hammond and Diamond 1992), this may never occur in nature due to environmental constraints on foraging behaviour, such as the risk of predation (e.g., L i m a and D i l l 1990). Similarly, day length may place limits on parental energy expenditure through limiting available foraging time (Tinbergen and Verhulst 2000). In F i g 1.1, such constraints are indicated by the dashed lines which link the term Environment with Performance and Behaviour. Natural selection acts on behaviours (Fig 1.1). I f variation at the genotypic level affects behaviour v i a performance, there is potential for selection and evolutionary change. Studies o f the physiological correlates o f whole animal performance are useful as they can identify traits that may evolve under selection for increased whole organism performance.  It  5  should be noted that selection is defined operationally as the correlation between Darwinian fitness and variation in a phenotypic trait (Garland and Carter 1994). This definition emphasizes that selection acts on phenotypes, irrespective o f their genetic basis. Demonstration of selection, therefore, cannot be equated with evolutionary change.  Approaches to studying intra-specific variation Within the organismal performance paradigm, there are currently two complimentary approaches in the study of intra-specific variation: (i) the gene-to-performance approach and (ii) the performance-to-gene approach (Pough 1989). In the first approach, a site of genetic variation (e.g., allelic isozymes) is identified and an attempt is made to trace the variation to higher levels of biological organization. This first approach is exemplified by the studies o f Powers and colleagues o f latitudinal variation i n the lactate dehydrogenase protein (Ldh-B) o f killifish, Fundulus heteroclitus along the eastern seaboard of North America (reviewed i n Powers and Schulte 1998). These studies have shown that variation i n Ldh-B results i n differences in physiological function, which in turn correlate with survival at high temperatures. Differences among populations in Ldh-B are found in terms o f enzyme activity, m R N A levels and transcription rate. Recent work has shown that variation in transcription rates among populations is due to variation in regulatory sequences, suggesting the action o f natural selection. Gene-to-performance studies are o f inherent interest from the perspective of the evolution and adaptation o f the products o f a single locus. Unfortunately, they tell little about the evolution o f the complex physiology in which the allozymes function (Mangum and Hochachka 1998). The second approach to studying performance (performance-to-gene) begins by recognizing the existence o f variation in performance at the whole animal level. Rather than being under the control o f a single gene, whole animal performance is likely a quantitative trait under the control o f numerous genes (e.g., D o h m et al. 1996, Swallow et al. 1998).  6  Investigation proceeds outward in two directions; one direction investigates how variation in performance affects fitness, while the other direction seeks to identify physiological or biochemical correlates of the observed differences in performance. To date, performance-togene studies have most frequently looked at forced activity, including burst sprinting, endurance and thermogenic capacity (e.g., Garland 1984, Garland and Else 1987, Bennett et al. 1989, Konarzewski and Diamond 1994, Hayes and O'Connor 1999). Other areas o f study have included dominance (e.g., Roskaft et al. 1986), vocalization during courtship (e.g., Zimmitti 1999), lactational capacity (Hammond and Diamond 1992), and a combination o f lactation and cold exposure (Hammond et al. 1994). Regardless what the performance trait is, the first step i n such studies is to assess the magnitude o f inter-individual variation. With the exception o f humans (e.g. Bouchard et al. 1992), by far the greatest amount o f work on inter-individual variation i n performance has been in ectotherms: fish (e.g., Micropterus salmoides K o l o k 1992); lizards, (e.g., Ctenosaura similis Garland 1984; Amphibolurus nuchalis Garland and Else 1987; Sceloporus  merriami  Huey et al. 1990); salamanders (e.g., Ambystoma tigrinum nebulosum Bennett et al. 1989); snakes (e.g., Thamnophis sirtalis Jayne and Bennett 1990, Peterson et al. 1998); and anurans (e.g., Hyla versicolor Taigen et al. 1985., Bufo woodhousei fowleri Walton 1988, Pseudacris crucifer Zimmitti 1999). The study o f inter-individual variation i n small mammals and birds within an organismal performance paradigm is relatively recent, and increasing in frequency (e.g., Hayes et al. 1992, D o h m et al. 1996, Chappell et al. 1995, 1996,1999). Numerous studies have demonstrated that inter-individual variation in organismal performance is detectable, and often large. For example, the size corrected coefficient o f variation ( C V ) for endurance was 63% in adult lizards (C. similis, Garland 1984). A m o n g neonatal garter snakes, endurance has been shown to vary by up to 100-fold (Jayne and Bennett 1990). Variation among individuals can be even more extreme, and within a single population o f canyon lizards (S. merriami), two females had endurance times in excess o f six standard deviations o f the mean (Huey et al. 1990). Although such aberrant values would  7  have traditionally been assumed to be 'outliers' due to measurement error, endurance shows high repeatability among trials (Van Berkum et al. 1989). Performance in mammals (other than humans), has been studied less frequently, however there is evidence of considerable phenotypic variation. In studies of individual house mice (Mus domesticus), some individuals run for many k m per day, while others are virtually sedentary (Friedman et al. 1992, D o h m et al. 1994). In birds, inter-individual variation appears modest, but is detectable. In house sparrows, Passer domesticus, for example, the C V for V 0 2 m a x  w  a  s  approximately 16%  (Chappell et al. 1999). This is comparable to that reported i n reptiles (17%, Garland 1984).  Performance-to-jitness Central to the study of organismal performance is the notion that those individuals with higher performance capacities w i l l make greater contributions to the gene pool. Quantifying selection, however, requires measurement of individual differences i n Darwinian fitness.  A s fitness is difficult to measure, studies frequently use correlates, for example,  clutch size, seasonal reproductive success or survivorship.  In the first study to demonstrate  selection on performance traits in a natural population, Jayne and Bennett (1990) measured the locomotor capacity o f newborn garter snakes i n the laboratory and released them into their original environment. Variation in locomotor capacity as measured in the lab significantly predicted subsequent survivorship. A s differential survivorship occurred prior to reproductive age, locomotor capacity was under natural selection in this population (Jayne and Bennett 1990). Hayes and O'Connor (1999) recently demonstrated that thermogenic capacity was under significant directional selection in their study population o f high altitude deer mice. Individual mice were captured in the field, had their thermogenic capacity measured ( V 0 2 m a x ) v i a cold exposure, and were released into their original population. Attempts were made to capture individuals approximately 2 months later. In one of two years, individual  8  mice with relatively high thermogenic capacity had a greater probability o f being recaptured, suggesting increased survivorship. I am aware o f only a single study o f birds that has attempted to relate variation in lifetime reproductive success (LRS) with organismal performance. Bryant (1991) showed that i n house martins, Delichon urbica, the relationship between daily energy expenditure and L R S followed a quadratic relationship; individuals with the highest L R S had intermediate expenditures. This suggests potential selection against individuals with high energy expenditures. The data set, however, included artificially enlarged broods, which in other studies has been shown to result i n increased parental mortality rates (e.g., Daan et al. 1996). In contrast to house martins (Bryant 1991), other species show a positive relationship between energy expenditure and correlates o f fitness (fledgling mass or condition; Merino et al. 1996, Moreno et al. 1997).  Physiology-to-performance Studies such as those o f Jayne and Bennett (1990) and Hayes and O'Connor (1999) are rare, and have successfully tackled one-half o f the organismal performance paradigm: the relationship between performance and fitness. But what about the relationship between performance and its physiological or biochemical correlates? Inter-individual differences in performance have been studied primarily in ectotherms, and have been shown to correlate with various morphological and physiological variables. In one o f the earliest intra-specific studies, Garland (1984) demonstrated that i n the lizard, C. similis, ninety percent o f the sizecorrected inter-individual variation i n endurance could be explained by four variables: V 0 2 m a x , heart and thigh-muscle mass, and hepatic aerobic enzyme activity.  Physiological  predictors o f performance are not universal across species. In A nuchalis, another lizard, endurance was best predicted by heart lactate dehydrogenase and thigh pyruvate kinase and/or citrate synthase (Garland and Else 1987); none o f which was a predictor i n C. similus.  9 Recent work on vocalizations in male spring peepers, P. crucifer, has looked for physiological and biochemical correlates of calling rate (Zimmitti 1999). In this species, males with higher calling rates have increased mating success, while in a related species, H. versicolor, the offspring o f males with long calls have higher growth rates and fitness (Welch et al. 1998). These studies provide a plausible link between calling performance and Darwinian fitness. Within a population of spring peepers, males with relatively high calling rates had significantly heavier ventricles, higher blood haemoglobin concentrations, and higher enzyme activities in the trunk muscles, than individuals that called less frequently (Zimmitti 1999). A s some o f these physiological and biochemical characters likely have a heritable basis (Garland et al. 1990), selection for increased calling frequency, may result in evolution of these characters. While considerable work has been undertaken on inter-individual performance in ectotherms, relatively few studies have considered performance i n homeotherms, particularly birds (but see Chappell et al. 1999). The evolution o f flight likely places quite different selective pressures on locomotory behaviours. Although burst sprinting (e.g., take-off speed) has ecological relevance in terms o f avoiding predators, in species with parental care an additional trait that is o f likely selective importance is the capacity for long-term sustained energy expenditure (Peterson et al. 1990, Hammond and Diamond 1997).  Sustained energy expenditure In studies of exercise performance in terrestrial vertebrates, three types o f activity have been commonly examined: capacity for burst sprinting, maximal exertion and endurance (e.g., Bennett 1991). These three measures represent a broad spectrum o f maximal activities and presumably influence how well an individual avoids predators, obtains mates or captures prey (Jayne and Bennett 1990). Although these measures o f performance differ in terms o f fuel utilization and time to fatigue (e.g., Roberts et al. 1996), one thing they have in common is that they rely on stored energy reserves.  10 There is a well recognized negative relationship between power output and duration o f activity. Using an Olympic runner as an example (e.g., Peterson et al. 1990), the power output o f an individual sprinting 100 m is considerably greater than that o f a 1000 m runner, which is in turn greater than a 10,000 m runner. To supply adequate levels of A T P for muscle contraction, short, high intensity bouts o f activity rely on phosphocreatine (PCr) hydrolysis and anaerobic glycolysis. In contrast, lower intensity exercise o f higher duration (e.g., runs o f 10,000 m) relies primarily on lipid oxidation or a mix o f substrates (e.g., Roberts et al. 1996). In each o f these cases individuals are relying on finite energy stores, and therefore performance cannot be sustained indefinitely. It has been hypothesized that with increasing duration o f activity, exercise intensity declines and approaches an asymptote (Peterson et al. 1990). This asymptotic level o f energy expenditure has been called the sustained metabolic rate (SusMR). A n individual's (or species') S u s M R is its metabolic rate time-averaged over periods long enough that metabolism is fueled by food intake rather than depletion o f energy reserves (Peterson et al. 1990). A s it is a time-averaged measure, it includes the costs of resting metabolism, thermoregulation, feeding, and a variety o f other expenses.  Time averaged measures o f energy expenditure have  various names in the literature. A s they are frequently measured i n free living animals in the field, they are often referred to as field metabolic rate ( F M R ) . Alternate names are daily energy expenditure ( D E E ) and average daily metabolic rate ( A D M R , Speakman 1997). Techniques for measuring sustained energy expenditure vary depending on the question asked and species studied (reviewed by Speakman 1997). I f long-term energy turnover is to be measured i n the field the most powerful technique to date involves the use of doubly labelled water ( D L W , Lifson and McClintock 1966). This technique involves introduction o f oxygen and hydrogen isotopes ( 0 , and either H or H ) in the form of 1 8  3  2  enriched water. After an animal has been injected (or fed) with enriched water, the washoutrates o f the isotopes can be measured by taking blood samples before and after an observation period. These washout-rates are then used to calculate respiratory C O 2 production, and  11 hence energy expenditure. Apart from the use o f telemetry (e.g., Bevan et al. 1995), the D L W technique is the only method that allows for measurement o f total rates o f metabolism in free-ranging animals in their natural environment.  Physiological  correlates of sustained energy expenditure  There is considerable inter- and intra-specific variation in levels o f sustained energy expenditure (e.g., Konarzewski and Diamond 1994, Peterson et al. 1998, Nagy et al. 1999). But what are the proximate factors underlying differences among individuals and species? In the presence o f excess food, a ceiling on S u s M R is likely imposed either centrally or peripherally. A central limitation occurs i f activity is limited by machinery shared by various energy consuming pathways. The most likely site o f a central limitation would be in the capacity o f the gut to digest and absorb food.  Alternatively, a limitation to S u s M R may  reside in the energy consuming tissues themselves, for example, in the properties o f skeletal muscle (reviewed by Hammond and Diamond 1997). T o distinguish between these two hypotheses, experiments have pushed laboratory mice to their maximal S u s M R using different modes o f energy expenditure (e.g., cold exposure, lactation, running on treadmills). The level o f metabolic ceilings differed between modes o f energy expenditure, indicating that limitations were unlikely to reside i n a shared pathway, but rather i n peripheral tissues (Hammond and Diamond 1992, 1994, Hammond et al. 1994, Konarzewski and Diamond 1994). In nature it is unlikely that individuals w i l l ever be capable o f obtaining unlimited food.  However, despite this an important observation from laboratory studies was that mice  forced to increase their energy expenditures displayed hypertrophy o f the small intestine, kidneys, liver, and heart (Hammond and Diamond 1994, Hammond et al. 1994, Konarzewski and Diamond 1994). This supports previous suggestions from studies o f shorebirds that a high S u s M R requires a high level o f support from the organs o f the abdominal cavity (Kersten and Piersma 1987). A s organs of the abdominal cavity have exceptionally high  12  mass-specific metabolic rates (Krebs 1950, Scott and Evans 1992), although they contribute only a fraction o f an individual's total body mass, they contribute disproportionately to resting metabolic rate (e.g., Daan et al. 1989, 1990, Konarzewski and Diamond 1994, Meerlo etal. 1997). If large internal organs are required to attain a high SusMR, and these organs contribute disproportionately to an individual's R M R , is there a correlation between S u s M R and R M R ?  Evidence for this relationship is mixed (e.g., Hayes and Garland 1995). In  laboratory mice there was a relationship between maximum daily energy intake (a surrogate o f S u s M R ) and the masses of the small intestine and kidney, and between R M R and both heart and kidney mass. There was, however, no relationship between S u s M R and R M R (Konarzewski and Diamond 1994). There has also been a failure to detect such a relationship in field caught voles (Microtus agrestis, Meerlo et al. 1997). A n inability to detect a relationship between S u s M R and R M R may be due to the relatively narrow range of energy expenditures found intra-specifically; the noise simply exceeds the signal. I f the range o f energy expenditures is enlarged through comparisons among species, mammals with a relatively high S u s M R do have a relatively high R M R before and after correcting for the influence o f phylogeny (Daan et al. 1991, Ricklefs et al. 1996). Although an inter-specific relationship between S u s M R and R M R exists i n birds (Daan et al. 1990), this relationship is reduced considerably after controlling for phylogeny (Ricklefs et al. 1996). A failure to detect a relationship between S u s M R and R M R i n birds may be due to differences among species in the intensity o f energy expenditures during parental care, and the existence of'safety margins' (Toloza et al. 1990). If species with chronically high or low energy budgets are compared, there are apparent couplings among organ sizes, R M R and SusMR. Tropical birds and mammals tend to have smaller organs o f the abdominal cavity and R M R than temperate species, and typically have a lower S u s M R due to their decreased thermal requirements (Rensch and Rensch 1956, in Daan et al. 1990). Similarly, endotherms have SusMRs considerably higher than those o f ectotherms (Nagy et al. 1999). The organs o f  1 3  the abdominal cavity o f endotherms are also considerably larger than those of ectotherms (Else and Hulbert 1981).  Organismal performance and breeding birds A s variation exists among individuals in burst sprinting or endurance exercise (Garland 1984, Garland and Else 1987, Bennett et al. 1989, Huey et al. 1990, Friedman et al. 1992, D o h m et al. 1994), it is not surprising that such variation also exists in sustained energy expenditures (e.g., Bryant and Westerterp 1982, Williams 1987, Moreno 1989, Konarzewski and Diamond 1994, Merino et al. 1996, Peterson et al. 1998, Potti et al. 1999). Birds are excellent models for studies o f the evolution o f performance, i n part because individuals within the same environment often display extensive variation in fitness related traits (e.g., Masman et al. 1989, Hochachka 1993, Pettifor 1993b, Blomqvist et al. 1996, Wendeln and Becker 1999). These traits include egg size (Blomqvist et al. 1996), clutch size (Pettifor 1993a), parental body condition (Wendeln and Becker 1999), and provisioning capacity (Wardrop 2000). Repeated measurements o f the same adults have shown that i n consecutive years individuals often lay similar sized clutches and have eggs o f similar mass (Wiggins 1990, Pettifor 1993b). Two recent avian studies have raised the additional exciting possibility that measurements of resting and sustained energy expenditure are repeatable between breeding seasons (Bech et al. 1999, Potti et al. 1999). This suggests that individuals may differ consistently in their energy expenditure, and that traits associated with them may retain some genetic variance (Potti et al. 1999). A s energy requirements o f offspring increase with increasing brood size, in theory so should parental energy expenditure. In fact, it has been hypothesized that the number o f offspring that parents can raise may be limited by their physiological capacity to feed their young (Drent and Daan 1980). Because breeding adults remain in approximate energy balance, the most likely site of limitation on energy expenditure is in the rates o f nutrient intake, digestion or assimilation (Kirkwood 1983, Masman et al. 1989, Peterson et al. 1990,  1 4  Weiner 1992). This hypothesis was tested by Dykstra and Karasov (1992, 1993). They compared near maximal rates of energy flow in house wrens, Troglodytes aedon, exposed to a combination o f cold and exercise in the laboratory, with that o f individuals o f the same species rearing manipulated broods in the field. A s the rate of parental energy expenditure in the field was considerably below that measured in the lab, they rejected the hypothesis that maximum rates o f energy flow limit brood size (Dykstra and Karasov 1992, 1993). Although an individual's brood size is probably not limited by the maximal rate of energy flow, this does not necessarily mean that brood size and parental daily energy expenditure are uncoupled. Support for such a relationship between S u s M R and behavioural traits such as brood or clutch size is, however, equivocal. In their recent review, Williams and Vezina (2000) noted that only 6 o f 20 avian studies (30%) could detect a relationship between the number o f nestlings i n a brood and parental S u s M R . However, some o f the studies in their review were o f species that hold feeding territories (e.g., European kestrels, Falco tinnunculus, and wheatears, Oenanthe oenanthe). In these species clutch size may be adjusted to the quality o f an individual's territory (Hogstadt 1980). Individuals on l o w quality territories may have small clutches and expend the same amount o f energy as individuals on high quality territories with larger clutches. A similar interpretation could explain (in part) why only 5 o f 15 studies reviewed by Williams and V e z i n a (2000) could detect a relationship between S u s M R and parental provisioning rate. Although some studies fail to detect relationships between S u s M R and surrogates of fitness, very convincing relationships are found in others. For example, when the brood size of pied flycatchers, Ficedula hypoleuca, was manipulated, the S u s M R o f males and females was positively correlated with nestling mass and tarsus length (Moreno et al. 1997). In addition, female S u s M R was positively correlated with nestling quality, as indicated by Trypanosoma infection (Merino et al. 1996). These studies led Moreno et al. (1997) to suggest that parental energy expenditure ( S u s M R ) was analogous to a performance trait,  15  constrained by "parental time-activity budgets or by condition-dependent physiological limits." Adults from a number o f species appear unable to respond to the energetic challenge of an increased brood size. For example, the recent work o f Tinbergen and Verhulst (2000) showed that although female great tits, Parus major, reduced their energy expenditures when the number o f nestlings was artificially reduced, under conditions o f increased brood requirements they failed to elevate their SusMR. This supports the existence o f an energetic ceiling set at their natural brood size. Tinbergen and Verhulst (2000) suggested that female energy expenditure is constrained by day length, which in turn limits available foraging time. Additional support for an energetic ceiling comes from studies that manipulated female energy expenditure independent o f brood size. Moreno et al. (1999) clipped the primary feathers o f female pied flycatchers (to increase wing loading). Females with clipped wings did not spend any more energy than controls (undipped), and as a consequence their nestlings suffered a reduced weight gain. This again suggests that females were unable (or unwilling) to increase energy expenditure. Despite these studies, numerous others have shown that parents can increase the energy expenditures (Masman et al. 1989), but may suffer reduced condition or increased mortality as a consequence (Winkler and A l a n 1995, Daan e t a l . 1996). Dykstra and Karasov (1992, 1993) showed that maximum rates o f energy flow likely do not limit brood size. However, i f i n order to attain a high S u s M R hypertrophy o f the organs o f the abdominal cavity is necessary, individuals with relatively high S u s M R may have to 'pay' in terms o f increased energy expenditures while resting (Hammond and Diamond 1997). Consequently, the number and quality o f nestlings that an adult produces may represent a balance between the fitness benefits derived from increased rates o f recruitment, and the costs o f maintaining the organs necessary to attain a high S u s M R . T o date, no study has investigated the physiological and fitness correlates o f variation in S u s M R ; this is a major component o f my thesis, and is presented in Chapters 2 and 3.  1 6  Environmental components of variation Most o f this thesis focuses on adults. However, as the fitness o f adults depends on the number of their offspring recruited into the next generation, it is necessary to consider how variation in the quality of parental care may influence variation in the quality of nestlings. Next to successful mating, the most important determinant o f lifetime reproductive success in many species is the survival and recruitment of offspring (Clutton-Brock 1988). There is increasing evidence that variation in the quality o f the rearing environment affects both nestling survival and subsequent future reproductive performance (e.g., Lindstrom 1997). Individuals that experience adverse conditions during early development are often smaller and lighter in mass at independence, and have decreased survival probabilities (Boag 1987, Richner 1989, Dijkstra et al. 1990, Korpimaki and Rita 1996, de Kogel 1997, Koskela 1998). Differences i n body size that are established during the nestling phase and that are often maintained in adults, contribute to the non-heritable environmental component of variation (James 1983, Boag 1987, Richner 1989, Alatalo et al. 1990, de Kogel 1997). A n elegant experiment demonstrated the importance o f the rearing environment in determining adult structural size. James (1983) transplanted red-winged blackbirds from either end o f a geographic cline in body size. Nestlings grew to resemble their foster parents rather than biological parents, demonstrating that much o f the clinal variation in body size was due to environmental effects. A more common way to experimentally manipulate the environment during development is through increasing or decreasing the number of nestlings in a brood. Parents often fail to meet the energetic challenge o f the increased brood size resulting in poor nestling growth. For example, in the laboratory, de K o g e l (1997) produced morphologically stunted adult zebra finches by increasing the number o f nestlings in the brood i n which they were reared. In addition to influencing skeletal morphology, conditions during early development can also affect future reproductive potential. Through presumed condition-mediated  17  mechanisms, female collard flycatchers reared in artificially enlarged broods laid fewer eggs in their first breeding attempt than individuals that were reared in control or reduced broods (Schluter and Gustafsson 1993). Increasing or decreasing brood size also affected subsequent sexual attractiveness o f males (Gustafsson et al. 1995). During their first breeding season, males that were raised in artificially enlarged broods had a smaller forehead patch (a secondary sexual character) than individuals reared in reduced or control broods. This has reproductive consequences as males with large patches mate with more females (Gustafsson et al. 1995). Similarly, male zebra finches reared in small broods developed redder bills and were more attractive to females than their siblings reared i n larger broods (de Kogel and Prijs 1996). Implicit in these studies is that variation in the environment experienced during early development affected an individual's 'condition' when an adult. Apart from recent work on immunosuppression (e.g. Merino et al. 1996), the physiological and biochemical traits defining condition remain relatively unexplored. Recent studies have shown that estimates o f condition (residuals o f mass on tarsus length) of nestlings are good predictors o f their fat reserves up to 4 months later during migration (Merila and Svensson 1997). This suggests that the window for selective events leading to a relationship between condition and survival maybe quite wide (Merila and Svensson 1997). There exists considerable variation among adult birds i n body composition and resting metabolism (Chapter 3). Some o f this variation likely correlates with inter-individual variation in aerobic capacity and perhaps dominance, as has been reported in other species (Raskaft et al. 1986, Bryant and Newton 1994, Chappell et al. 1999). Although there is a heritable basis to variation in physiological traits such as resting metabolism and the size of many internal organs (Schlager 1968, M c K i t r i c k 1990, Mahaney et al. 1993), the importance of early development i n modifying this variation remains relatively unexplored (but see Dohm et al. 1996). Partitioning the relative influence o f different sources o f variation in these traits would be o f interest, but would be a difficult task given the complexity o f the experimental design (cross-fostering) and large sample sizes required (e.g., Garland et al. 1990). A useful  18 first step is to determine to what extent physiological and biochemical traits (e.g., heart mass or enzyme activity) are shaped by environment variation; this was performed in Chapter 4.  Study species and location One o f the best species in which to address questions o f organismal performance is the tree swallow, Tachycineta bicolor.  This species is an aerial insectivore that forages for  up to 15 hours per day when feeding dependent young (Wiggins 1990). This results in adults having one o f the highest S u s M R ever measured, sometimes i n excess o f 6.0 X R M R (Williams 1988). A s this value approaches the hypothesized ceiling o f 7.0 X R M R for all species (Peterson et al. 1990, Hammond and Diamond 1997), it suggests a relatively small margin o f safety (e.g., Toloza et al. 1990). First time breeding female swallows are identifiable on the basis o f plumage (1-year old, Hussell 1983), and were excluded from the present study. In females older than 2 years the confounding affects of age and breeding experience on fitness correlates such as clutch size are minimal (Strutchbury and Robertson 1988, Robertson et al. 1992). Incubation is performed exclusively by the female and lasts 14-15 days (Robertson et al. 1992). After the young hatch, both parents feed the nestlings at similar rates (Quinney 1986). Nestlings follow a sigmoidal growth curve, attain peak mass by approximately day 12 post hatch, and fledge at 18-22 days (Robertson et al. 1992). During brood rearing, parents that feed their nestlings more frequently have higher energy expenditures (Williams 1988). Assuming that larger broods require more food (have increased energy requirements, Drent and Daan 1980), this suggests that parents feeding a larger number o f nestlings w i l l have a higher S u s M R . A s tree swallows do not hold feeding territories, I assumed that i n the present study all individuals had similar access to food resources. Data i n this thesis were collected over 5 field seasons, 1994-1998, at the Creston Valley Wildlife Management Area, a 7000 hectare wetland area i n southeastern British  1 9  Columbia. Throughout the Management Area there are a series of man-made dikes, upon which I placed between 140 and 200 nest boxes (depending on the year). Although tree swallows have been studied at this location since the early 1980's, the number of banded individuals represents <50% o f the population.  Aims of thesis To date, no study has looked for physiological correlates o f sustained performance and attempted to link them via behavioural traits with fitness.  In Chapters 2 and 3,1 sought  answers to three primary questions: (i) what are the physiological and biochemical correlates of one indicator o f whole animal performance (SusMR); (ii) what is the relationship between performance (SusMR) and Darwinian fitness; (iii) what is the relationship between variation in organ size and resting rates o f metabolism? In Chapter 4,1 asked how variation i n the environment during early development affects the physiology and biochemistry o f individual nestlings near fledging. CHAPTER  2  I measured the S u s M R o f adult tree swallows rearing different natural sized broods using the D L W technique. Correlations were sought among variation in performance (SusMR), natural brood size and both nestling mass and growth rates (as indices o f fitness). Following measurement o f their performance, I sacrificed a sample o f adults to determine i f variation in parental energy expenditure was associated with variation in physiological and biochemical characters.  The following hypotheses were tested: 1 ) There are positive relationships among parental S u s M R , brood size, nestling mass and growth. That is, parents that can attain a high S u s M R , w i l l have larger broods and faster growing nestlings.  20 2) Individuals with a relatively high S u s M R w i l l have relatively large internal organs, and relatively high metabolic capacities in the pectoral muscle (as indicated by the activities o f key enzyme activities from various metabolic pathways).  CHAPTER  3  There is intra-specific evidence that maintenance o f relatively large organs in the abdominal cavity is metabolically costly for mammals, resulting in elevation o f resting metabolic rate (Daan et al. 1991, Konarzewski and Diamond. 1994, 1995, Meerlo et al. 1997). Although this would be expected intra-specifically in birds, prior to the publication o f Chapter 3 (Burness et al. 1998) no such data existed; nor had anyone reported inter-annual differences in the sizes o f internal organs.  The following hypotheses were tested: 1) There w i l l be a positive relationship between adult resting metabolic rate and the size o f metabolically active organs of the abdominal cavity.  2) Resting metabolic rate and the size o f internal organs w i l l show inter-annual variation.  CHAPTER  4  I performed brood manipulations i n the field to mimic environmental variation. I considered nestlings reared in enlarged broods to be in a "poor quality" environment, resulting from a dilution i n parental care. For nestlings i n reduced broods, the reverse was true. A t 16 days o f age, I measured the resting metabolic rate of nestlings representing the average individual for each brood. I then sacrificed these individuals to determine body composition and tissue biochemistry.  2 1 The following hypotheses were tested: 1) For a given structural size, nestlings in "good quality" environments w i l l have larger hearts, liver and kidneys, greater mass of lipid and pectoral muscle, higher activities of oxidative and glycolytic enzymes, and higher resting metabolic rates, than individuals in "poor quality" environments.  2 ) Nestlings o f a given structural size would differ between treatments in the size o f their intestines and gizzards. I could not predict a priori whether there would be an increase or decrease in the size o f these organs with increasing brood size (under food restriction either response is plausible, Galuso and Hayes 1998).  22  CHAPTER 2 PHYSIOLOGICAL CORRELATES OF PARENTAL QUALITY IN BREEDING TREE S W A L L O W S , TACHYCINETA  BICOLOR  INTRODUCTION Many populations o f passerine birds exhibit considerable variation i n clutch size, even though individuals laying the largest numbers o f eggs often raise the most recruits (e.g., Boyce and Perrins 1987). One hypothesis to explain this variation proposes that females adjust their clutch size to their own individual circumstances (individual optimization hypothesis, Perrins and M o s s 1975, Pettifor et al 1988); for example, to the quality o f their territory (Hogstedt 1980) or to their own individual abilities. Although territory quality can be defined in terms of ecological variables such as predation risk or food resources, variables defining individual quality are less clear. The provisioning o f dependent nestlings requires an elevation o f parental activity and consequently an elevation o f metabolic rate (Drent and Daan 1980). One character that may differ among individuals within the same population is the level to which each individual can elevate its energy expenditure. This is an individual's sustained metabolic rate (SusMR), defined as the metabolic rate time-averaged over periods long enough that metabolism is fueled by food intake rather than depletion o f energy reserves (Peterson et al. 1990). Because metabolism is fueled by food intake, individuals remain i n energy balance. Provisioning of dependent young often lasts many weeks, and as there is a negative relationship between the intensity of activity and its duration, S u s M R s during parental care are typically only a few times the basal or resting metabolic rate (Drent and Daan 1980, Peterson et al. 1990, Hammond and Diamond 1997). In the same way that inter-individual variation exists i n capacity for short term energy expenditure (V02max> Chappell et. al 1999), variation i n S u s M R has also been reported (e.g., Konarzewski and Diamond 1994, Bryant 1991). A recent study o f birds indicated that in  23  females (although not males) estimates of SusMR were repeatable between breeding seasons (Potti et al. 1999). The stability o f such a trait over a period o f a year suggests that it may have a genetic component. In this context, sustained energy expenditure may be viewed as an organismal performance trait with potential links to fitness (Garland and Carter 1994). Presumably, individuals with high S u s M R w i l l display various physiological adaptations, including relatively large intestines (Konarzewski and Diamond 1994), hearts and kidneys (Daan et al. 1990), and the capacity for high flux rates through various metabolic pathways.  Laboratory studies of small mammals have shown that inter-individual variation  in maximum sustained energy expenditure does correlate with differences in body composition (Konarzewski and Diamond 1994, Koteja 1996). For example, individual mice with relatively high energy intake rates had relatively heavy kidneys and small intestines (Konarzewski and Diamond 1994). A s the kidney accounts for a large fraction o f resting metabolic rate, these individuals had an increased resting energy expenditure (Konarzewski and Diamond 1994). These data suggest the existence o f a trade-off between the potential benefits o f attaining an high S u s M R and the costs o f maintaining the organs necessary to do it (Kersten and Piersma 1987, Hammond and Diamond 1997). Even though many physiological and biochemical characters display considerable plasticity, they likely still retain some genetic variance (Schlager 1968, Garland et al. 1990, M c K i t r i c k 1990, Mahaney et al 1993, Konarzewski and Diamond 1995). Identification o f the physiological and biochemical correlates of S u s M R may give insight into which characters would be subject to potential evolutionary change under selection for whole animal performance. In an attempt to better understand the physiological causes and ecological consequences o f variation in whole animal performance, I studied breeding tree swallows, Tachycineta bicolor. This species is an aerial insectivore and does not hold feeding territories (Robertson et al. 1992). Consequently, variance in nestling growth, a surrogate o f fitness, is presumably due i n large part to differences in individual parental quality (e.g., DeSteven  24  1980). I asked three primary questions: (1) Do parents rearing large natural broods trade-off nestling quality for quantity? (2) Does parental S u s M R correlate with indices o f fitness (brood size and nestling mass)? (3) What are the physiological and biochemical correlates o f parental S u s M R , and do these differ between adults rearing different sized broods?  MATERIALS AND METHODS: Study site and choice of study nests The field component o f this study was performed in May-June 1996 and 1997 at the Creston Valley Wildlife Area, near Creston, British Columbia, Canada. Approximately 180 nest boxes were erected approximately 15-20 m apart, along man-made dikes within the Wildlife Area. Beginning in the first week of May, boxes were checked daily for signs of breeding and the presence o f eggs. Females lay a single egg per day, typically on consecutive days, until clutch completion. Clutch completion is followed by 14-15 days o f incubation (Robertson et al. 1992). To minimize disturbance, no nest checks were conducted during incubation. Within 1 -2 days o f predicted hatch dates, nest checks were resumed to record dates o f hatching (hatch = day 1). Within a clutch, hatching was relatively synchronous and was typically complete within 1-2 days. In both 1996 and 1997, egg laying began during the first week o f M a y and continued into early June. To minimize the possibility o f including females laying replacement clutches, I only considered nests with clutches initiated i n M a y . Study nests were chosen based on their original clutch sizes (5, 6 or 7 eggs). To minimize date as a correlate o f clutch/brood size (Winkler and A l l e n 1996), I randomized the choice o f study nests across each breeding season (i.e., not all 7 egg nests were selected early in the season).  25  Nestling mass and growth rate O n day 4,1 weighed nestlings from each study nest using a spring-loaded scale (± 0.5 g) and banded them loosely. If a nestling was too small to be banded it was marked with indelible marker and banded within a few days. O n day 8, nestlings were re-weighed and the bands tightened.  If an egg failed to hatch by day 4, it was replaced by a 4 day old nestling  (± 1 day) from another nest. Similarly, i f a nestling died between days 4 and 8, to maintain the original brood size it was replaced by a nestling of similar age. However, no measure of growth for that brood was recorded. Nestlings whose parents were involved in a study of energetics (below) were weighed a third time on day 9. This third weighing was used as an indirect measure of whether parents were behaving normally following injection and release on day 8 (i.e., did nestlings lose weight over the energetic study).  Doubly labeled water I measured the sustained metabolic rate (SusMR) o f adult tree swallows rearing natural broods of 5, 6 or 7 nestlings using the doubly labeled water technique ( D L W ; Lifson and M c C l i n t o c k 1966). To standardize for brood age (e.g., Sanz and Tinbergen 1999), all adults were captured at the nest box on day 8 o f chick rearing. In broods o f 5 and 7, attempts were made to capture both members o f the pair, while in broods o f 6, a single parent was captured. I prepared the D L W injection solution by mixing 0.120 m L o f 2.99 m C i H 2 0 with 3  8.97 m L o f 97 atom % H 2 0 . Using a calibrated glass syringe, I injected 0.10 m L o f solution 1 8  (ca. 3 3 u C i tritium per individual) into the pectoral muscle o f each adult. Each adult was then weighed using a spring-loaded scale (± 0.5 g), banded and held for 1 hr in an individual brown paper bag.  This was sufficient time to allow for equilibration o f the isotopes with the body  water (e.g., Williams and Nagy 1984). Following equilibration, I collected approximately 0.150 m L o f blood from the brachial vein into heparinized microcapillary tubes and then  26  released the bird. After approximately 24 hours, the bird was recaptured and a second set of blood samples was taken from the other wing.  In each year, 2 non-experimental females  were captured at the study site and a blood sample was taken to determine background levels of  1 8  Oand H . 3  In 1996, microcapillary tubes containing blood samples were immediately flamesealed in the field using a butane torch. In 1997, tubes were first sealed with Critocaps, and then flame-sealed upon return to the lab at the end o f the day. A l l blood samples were stored at 4 ° C until distillation and analysis by Dr. K . A . Nagy's Laboratory o f Biomedical and Environmental Sciences, U C L A .  1 8  0 concentration was measured in triplicate using  cyclotron-generated proton activation analysis.  3  H activity was measured i n duplicate using  a liquid scintilation counter. Adults rearing 6 nestlings were released following the second blood sample. To address questions o f physiological and biochemical correlates of clutch size and energy expenditure, I sacrificed adults rearing either 5 or 7 nestlings (see below). Their nestlings were distributed among non-study nests i n the population.  Environmental  temperature  The daily maximum and minimum air temperatures during the study period were obtained from an Atmospheric Environment Service weather station, approximately 5 k m from the study site. Most adults were captured and injected between 10:00 and 13:00. Consequently, the maximum temperature experienced during the D L W trial was assumed to occur on the day o f capture (nestling day 8). The minimum temperature most likely occurred between approximately 00:00 and 0500, and was considered to be the lowest temperature recorded for the day o f re-capture (nestling day 9).  27 Haematocrit and haemoglobin I collected an additional 100-200 ul blood sample from adults rearing either 5 or 7 nestlings. T o determine haematocrit (Het, %), microcapillary tubes containing the samples were spun at maximum speed for 10 min. using an Adams micro-haematocrit bench top centrifuge. The percentage of the tube occupied by packed cells was then measured. Concentration of haemoglobin [Hb, g d L ] was determined using a portable H e m o C u e ® B _ 1  Hemoglobin photometer (Angelholm, Sweden). The number of replicates for each character was determined by the size o f the blood sample and ranged from one to three (which were averaged). For 5 individuals in which H b but not Het was measured, I estimated Het from regressions o f Het on H b for the 29 individuals in which both characters were measured. Separate predictive equations were necessary for each year because the slopes o f the regression lines differed ( A N C O V A , P<0.15). 1996: Het = 13.83 + 1.80[Hb], r =0.77, 2  N=12, P O . 0 0 1 ; 1997: Het = 3.59 + 2.69[Hb], r =0.83, N=19, P O . 0 0 1 ) . A s Het and H b 2  correlate strongly (r=0.83, N=29, P<0.001), only results for Het are presented.  Body composition During two breeding seasons, I sacrificed 49 adults (29 females and 20 males) rearing either 5 or 7 nestlings immediately following blood sampling (following the guidelines of the Canadian Committee on A n i m a l Care). Within 1-2 m i n . o f death, a sample (ca. 300 mg) o f the right pectoralis major was removed from each individual and immediately frozen in a liquid N2-charged dry shipper for enzyme assays. The remainder o f the pectoralis and supracoracoideous (hereafter, "pectoralis") was then removed, followed by the heart, liver, small intestine, gizzard, and kidney. A l l tissues except the gizzard were stored i n air-tight cryovials and frozen in the dry shipper. Each carcass (including the gizzard) was double bagged and stored at -20°C. U p o n return from the field, I transferred the samples o f  28  pectoralis to liquid N 2 , and stored the remainder of the tissues and carcass at either -20°C or 80°C. Wet weights were determined for all organs and tissues (± 0.0001 g). The small intestine and gizzard were initially weighed full. The small intestine was cut into three sections of equal length. The gizzard, and each section of the small intestine were then cut longitudinally and the contents rinsed out with 0.9% N a C l .  Each tissue was then blotted dry  and re-weighed to determine empty mass. Determination o f lipid levels was restricted to the carcass and pectoralis. In preparation for fat extraction, carcasses were partially thawed, plucked o f all feathers, and weighed (± 0.000 lg). The carcass and pectoralis were dried to constant mass in a 70°C oven and freeze dryer respectively. These dried samples were then fat extracted for 7 hours in a Soxhlet apparatus containing petroleum ether as the solvent (Dobush et al. 1985). Following extraction, the carcass and pectoralis were placed in a fume hood to evaporate any remaining solvent, oven-dried overnight, and then re-weighed. The difference between the preextraction and post-extraction mass represented the mass o f lipid.  Biochemical analyses The subsamples (ca. 300 mg) o f the pectoralis major were removed from liquid N 2 , weighed frozen (± 0.000lg) and added to 9 volumes o f 0°C homogenization buffer (20mM N a 2 H P 0 4 , 0.2% B S A (defatted), 5 m M 6-mercaptoethanol, 0 . 5 m M E D T A , lOOug/mL aprotinin, glycerol 50% v/v, p H 7.4 at 21°C; Mommsen and Hochachka 1994). Each sample was minced on ice for 1 minute using scissors, followed by homogenization using a hand held Tissue Tearor ( 3 X 1 0 sec. bursts separated by 30 sec. breaks). Samples were further homogenized for 3 min. using a Lurex ground glass-on-glass homogenizor, and then sonicated for 3 X 10 sec bursts, separated by 30 sec breaks, using a Kontes Micro-ultrasonic cell disrupter. Homogenates were stored at -80°C until assaying (maximum 4 months). This  29  homogenization buffer allows samples to be frozen for extended periods with no loss of enzyme activity (Mommsen and Hochachka 1994). A s an index of maximum capacity for flux at specific steps through various metabolic pathways, I measured the maximum catalytic activity ( V  m a x  ) o f key metabolic enzymes  under optimal conditions. A l l assays were performed on a temperature controlled 6 cuvette spectrophotometer (Perkin Elmer Lamda 2). Assay temperature was maintained at 42°C using a Lauda R M 6 circulating water bath and water jacketed cuvette holders. In all assays, uncentrifuged homogenates were used to avoid potential loss o f activity in the pellet. Each reaction was replicated in 3 cuvettes. The two cuvettes with the most similar activity were averaged; in cases where values from the three cuvettes were equidistant, all three were averaged. Preliminary experiments confirmed that all substrates and cofactors were saturating but not inhibitory. W i t h the exception o f citrate synthase, all assays were at p H 7.0 and 340 nm. Citrate synthase was assayed at p H 8.0, 412 nm. Enzyme activities are expressed as international units (umoles substrate converted to product per minute) per gram wet weight o f tissue. Assays were performed as follows. Lactate dehydrogenase ( E C 1.1.1.27; L D H ) : 5 0 m M Imidazole, 0 . 1 5 m M N A D H , l O m M B-mercaptoethanol, 1.0 m M N a C N , 1.0 m M Pyruvate. 3-hydroxyacyl-CoA dehydrogenase ( E C 1.1.1.35; H O A D ) : 5 0 m M Imidazole, 0.15mM N A D H , l O m M B-mercaptoethanol, 1.0 m M N a C N , 0.05 m M acetoacetyl C o A . Citrate synthase ( E C 4.1.3.7; CS): 5 0 m M Tris buffer, 0.05% Triton X - 1 0 0 , 0 . 2 m M D T N B , 0 . 2 m M acetyl C o A , 0 . 5 m M oxaloacetate (omitted from the control cuvette).  Pyruvate  kinase ( E C 2.7.1.40; P K ) : 5 0 m M Imidazole, 0.15mM N A D H , l O m M B-mercaptoethanol, 1.0 m M N a C N , l O O m M K C I , l O m M M g C l , l O u M fructose 1-6 bisphosphate, 5.0 m M A D P , 2  5 m M phospho (enol) pyruvate, excess L D H (ca 5 U / m L ) . To account for P K contamination in the coupling enzyme ( L D H ) , I ran 6 additional control reactions containing no homogenate. The rate of change in absorbance over time was calculated for each o f these 6 control reactions and averaged. The average control rate was subtracted from all P K reaction rates before  30 calculating enzyme activity. Additional control reactions (containing no substrate) were initially run for each enzyme. The rates o f control reactions were l o w for all enzymes except CS and were subsequently omitted.  Calculations of sustained metabolic rate A n individual's initial total body water pool ( T B W i , in m L ) was estimated using the 1 8  0 dilution space, calculated according to Appendix I o f Nagy (1983). O n occasion a small  amount o f water remained on the skin following an injection. Due to the occasional uncertainty concerning the exact amount of water that was injected, instead of individual values, a mean percentage body water of 66.2% was assumed i n all cases. This percentage estimate was based on the mean dilution space calculated for 43 individuals with injections o f known volume. The final body water pool (TBWf, in m L ) , was estimated from T B W i , assuming both a linear change in pool size and that T B W f occupies the same percentage o f the body mass as T B W i .  Rates o f C O 2 production ( r C 0 2 ) were calculated using equation (1) o f Nagy (1983), reexpressed i n m L d" : 1  rCOo = 622.32 ("TBWf - T B W i ) In [(Oi - O b Y H f - Hb) / ( O f - Ob) ( H i - Hb)]  (1)  In [ ( T B W f / T W B i ) ] (t)  Where: O i , Of, H i and H f are an individual's initial and final concentrations of Ob and H b are the background levels o f  1 8  1 8  0 and ^ H ,  0 and H measured in 2 non-experimental animals, 3  t refers to the time between release and recapture (in days), and In is natural logarithm. During the experiment, adult swallows lost an average o f 0.91 ± 2.85% of their mass per 24 hours (Range: -7.27% to + 4.17%, N=43). To allow for conversion of C 0  2  production to energy expenditure, it was necessary to assume that the majority o f individuals  31 were in approximate energy balance. I converted the rate of CO2 production to J d"  1  assuming 26.2 J m L " CO2 for insectivorous food (Weathers and Sullivan 1989). For birds 1  that lost >4% o f their initial body mass (N=6), I followed Weathers and Sullivan (1989) and included the heat produced from the oxidation o f fat in estimates o f SusMR. This resulted i n a marginal increase in my estimates of S u s M R (0.84 ± 0.16%, N=6). One swallow increased in mass by 0.78 g, representing a gain of 4.2%. A s changes in body composition between release and recapture were unknown, I made no attempt to correct my estimates o f S u s M R for mass gain. One individual in 1996 could not be re-captured within 24 hr and was captured the following morning (total elapsed time = 39 hr). This individual spent more time at rest than the other birds, which would lead to an underestimate o f its S u s M R i f expressed simply per 24 hours. In order to estimate the volume o f CO2 that this individual would have produced in a 24 hour day (15 hours o f daylight, 9 hours o f darkness), I needed to calculate the daytime and nighttime rates o f CO2 production (VCO2). I calculated the nighttime VCO2 from the estimated nighttime rate o f oxygen consumption (VO2), assuming a respiratory exchange ratio o f 0.75. The average nighttime VO2 was estimated as 1.9 X basal VO2 (Tinbergen and Dietz 1994), and I assumed the basal VO2 to be 75% o f the daytime resting VO2 (Aschoff and Pohl, 1970). I calculated daytime resting VO2 from a species-specific allometric equation generated for adults (Burness et al. 1998, Chapter 3). Based on the time o f initial capture and subsequent recapture, I estimated that this bird spent 18 hours at a nighttime VCO2 and the remaining 21 hours at a daytime "active" VCO2. T o calculate the volume o f CO2 produced during the daytime (in 21 hours), I subtracted the volume produced during the 18 hours o f darkness from the total CO2 produced i n 39 hours (from D L W ) .  B y knowing the  daytime and nighttime VCO2,1 could calculate the average VCO2 over 24 hours. Sixty-two adult tree swallows were successfully recaptured, and o f these, 47 yielded reliable estimates o f S u s M R during provisioning. Estimates o f S u s M R were considered to be unreliable, and were excluded from analyses i f either the final H or 3  1 8  0 values had decayed  32  to background ( N - 4 ) , or the estimated S u s M R was less than the allometrically predicted B M R (N=7). Four additional adults were omitted because either their S u s M R was < 1.5 X B M R (considered unlikely in an aerial insectivore provisioning young, Williams 1988), and/or their nestlings displayed large weight loss during the trial, suggesting potential negative effects of the injection on the parents.  Statistical analyses Data were transformed as necessary to meet assumptions o f multivariate statistical tests (e.g., normality o f residuals). The influence of potential covariates (e.g., time, date, body mass), main effects (e.g., year, sex or brood size) and interaction terms on dependent variables were first explored using either a forward or backward stepwise regression. Probabilities for inclusion and exclusion were set at 0.05 and 0.10 respectively. Terms significant in the stepwise regression were then included in 1 or 2-way analysis o f variance ( A N O V A ) , analysis o f covariance ( A N C O V A ) , or multiple regressions. Interaction terms were excluded from models when P>0.15.  In order for some results to be viewed graphically,  on occasion I analyzed residuals. In these cases P-values were corrected to account for the degrees o f freedom lost in generation o f the residuals (Hayes and Shonkwiler 1996). Whenever possible year and sex o f parents were pooled (with either year or sex included as a main effect). However, as both the male and female were often captured from the same nest, in analyses involving brood size or nestling mass and growth, sexes were considered separately. To avoid the possibility o f spurious autocorrelation i n analyses o f body composition, the mass o f each organ was subtracted from total body mass before each computation (Christians 1999). Unless otherwise noted, data are reported as least squares means ± 1 S . E . M . (standard error o f the mean) and probabilities are 2-tailed. Statistical significance was claimed at P O . 0 5 . Analyses were performed using J M P statistical software. I performed power analyses using P A S S 6.0.  33  RESULTS: Brood size and nestling growth rate Growth was followed in 52 unmanipulated nests between days 4 and 8 (22 i n 1996; 30 in 1997). The total mass gain o f broods per day (brood growth rate) increased with increasing natural brood size ^2,49=41.479, P O . 0 0 1 ; all brood sizes significantly different, Tukey H S D P O . 0 5 ; F i g 2.1 A ) . The growth rate of individual nestlings was independent of brood size (P=0.054, F i g 2.IB). Growth rates did not differ between years (P>0.10).  Potential correlates ofSusMR Various factors can influence estimates o f S u s M R (Speakman 1997). These need to be identified before relationships among SusMR, brood size and nestling growth can be determined. Year, sex and body mass The S u s M R o f adult tree swallows ranged from 56.1 - 136.3 k J d" , with an average 1  value of 101.5 k J d ' (S.D.=18.9, N=47); males and females did not differ (P>0.15). The 1  average S u s M R in 1996 was less than i n 1997 (Wilcoxon test, Z = -2.198, P O . 0 5 ) . 1996: 89.4 k J d " (S.D =25.5, N=14), 1997: 106.6 k i d " 1  1  (S.D.=12.6, N=33). There was a weak  but significant increase i n S u s M R with increasing body mass (Fi 44=4.759, P O . 0 5 , F i g 2.2), >  after controlling for year effects. When each year was considered separately, mass explained at most 11% o f the variance in S u s M R (1996: r =0.11, N=14, P O . 2 4 2 ; 1997: r =0.09, 2  2  N=33, P O . 0 8 4 ) ; neither regression was significant.  Environmental temperature Daily minimum temperature was not correlated with S u s M R i n either 1996 or 1997 (P>0.15). In 1997 only, there was a marginally significant decrease in S u s M R with increasing daily maximum temperature (r=-0.31, N=33, P=0.079).  34  Figure 2.1 B o x plots o f growth rates between days 4 and 8 o f (A) entire tree swallow broods and (B) individual nestlings. The solid horizontal line i n the middle o f each box is the median, the dashed line is the mean. The bottom and top o f the boxes are the 25th and 75th percentiles. The vertical lines above and below the boxes are the 1 Oth and 90th percentiles; data points falling outside this range are indicated by solid circles. Sample sizes are i n brackets.  35  -O  150  3  aT 130  I  -I  110  o  -Q CD 0)  90  E 0 c  70  'co  % CO  CP  50  -i  16  17  •  r  18  -i  19  1  r  20  21  22  Body mass (g)  Figure 2.2 Allometric scaling of parental sustained metabolic rate and body mass in breeding tree swallows, (o) 1996; (•) 1997. When considered independently, neither year was significant.  36  Mass change and recapture interval Adults lost on average 0.17 g d  _ 1  (S.D.=0.529, N=43, Appendix 1). Change in body  mass was not a significant predictor of S u s M R (P>0.50; both body mass and year were significant in the model, P<0.05). The mean elapsed time between release and recapture was 24 hr 41 min (S.D.=2 hr 10 min, N=47; Range: 18 hr 45 min to 28 hr 49 min). After controlling for body mass and year o f study, there was no relationship between S u s M R and the deviation o f the recapture from 24 hrs (recapture interval - 24 hr; Fi 43=0.083, P>0.50). (  Parental SusMR and correlates of fitness Brood size Parental S u s M R was independent o f brood size (P>0.50, F i g 2.3). A lack o f statistical significance was probably not due to insufficient power. F r o m F i g 2.1 A , I estimated that broods o f 7 nestlings had ~ 3 0 % greater mass gain per day than broods o f 5 nestlings. Consequently, I predicted a priori that the S u s M R o f individuals rearing 7 nestlings would be - 3 0 % higher than those rearing 5 nestlings. I had a power o f 0.80 to detect a 25% difference in energy expenditure among females rearing each of the 3 brood sizes, and the ability to detect a 35% difference among males. Finally, i f the sexes were pooled, parental S u s M R remained independent o f brood size (P>0.90) despite the ability to detect a 2 1 % difference among means (at a power o f 0.80). When parental S u s M R was expressed per nestling rather than per brood, females rearing broods o f 5 expended more energy per nestling than females rearing broods o f 6 or 7 (F ,26 2  = 14.393, P O . 0 0 1 ; Tukey H S D P O . 0 5 ; F i g 2.3). In males, S u s M R per nestling was  independent o f brood size (P=0.066, F i g 2.3). If years were considered separately, i n 1997 males rearing broods o f 5 or 6 nestlings expended significantly more energy per nestling than males rearing broods o f 7 ( F , i i = 7.134, P O . 0 5 ; Tukey H S D P O . 0 5 ) . S u s M R in 1996 was 2  measured in only 3 males, precluding a separate analysis.  37  -o 0 +-» CD I—  O  o 3  0  E  TD CD C 'CD -•—• CO  ZJ  :  120 i 100  Males  (5)  (6)  ()  (6) J  O  25  CO  15  5  6  7  Brood size  Figure 2.3 Sustained metabolic rate of adult tree swallows rearing natural sized broods. Circles represent the total parental energy expenditure, triangles are the energy expenditure per nestling. Least squares means ± 1 S.E.M., *P<0.05. Sample sizes are in brackets.  38  Nestling mass and growth rate Parental S u s M R was analyzed with respect to brood mass on day 8, and the previous mass gain o f the brood between days 4 and 8. Years and sexes were analyzed separately due to significant interaction terms. A s I predicted positive relationships among variables a priori, P-values are 1-tailed. In 1997, females with an high S u s M R were rearing broods that had previously displayed a high growth rate between days 4 and 8 (Fi i3=10.832, P O . 0 1 ; F i g . 2.4A). ;  A  single nest with a high studentized residual (-2.891) was omitted from analysis; the nestlings from this nest had the highest body mass on day 4 (>2.5 S.D. from the mean), and had little mass gain between days 4 and 8. Male S u s M R was independent of the previous mass gain of his brood (P=0.122). There was a positive correlation between female and male S u s M R and the total mass o f their broods on day 8 (Females: Fi i4=21.400, P O . 0 0 1 ; Males: ;  Fi =4.571, P O . 0 5 ; Figs. 2.4B). >9  In 1996, there was no relationship between female S u s M R and the mass gain o f her nestlings between days 4 and 8 (P>0.20). The correlation between female S u s M R and brood mass on day 8 was more complex than in 1997, and varied between brood sizes (SusMR*brood size interaction, P O . 0 1 ) . Females rearing broods o f 5 or 6 nestlings increased their S u s M R with increasing brood mass on day 8 (Fi 3=7.771, P=0.034, N=6). In j  contrast, females rearing 7 nestlings showed a significant negative correlation (r = -0.98, P O . 0 5 , two-tailed test, N=4).  A s only 3 males were labeled i n 1996, analysis was not  possible. Relationships among growth, brood mass and parental S u s M R were not driven by covariation with temperature (sensu, Dykstra and Karasov 1993). There was no relationship between temperature on day 8 (maximum or minimum) and either mass gain o f the brood between days 4 and 8, or total brood mass on day 8 i n either year (P>0.05).  39  Figure 2.4 Sustained metabolic rate o f adult tree swallows and ( A ) the residual growth rate o f their broods between days 4 and 8 and (B) the residual mass o f their broods on day 8. Residuals controlled for the effect o f brood size on the dependent variable. Females: solid circles, solid line; Males: open circles, dashed line Solid triangle was an outlier (see text) and was omitted from analyses. A l l correlations significant (P<0.05), except for that o f males in panel A (P=0.12).  40  Physiological correlates of brood size The mean Het was 43.4 % (S.D =5.1, N=46), and ranged from 33.2 - 54.3%. Mean Het did not vary between brood size or year o f study for either sex (P>0.30, Table 2.1). Females captured later in the season had significantly lower Het than those captured early (F 1,23=6.068, P<0.05); this relationship was not seen for males (P>0.40). Adult organ and tissue masses were unrelated to brood size (P>0.10), but differed between years (Tables 2.2, 2.3). In females, the mean mass o f the pectoralis and kidney were respectively, 8% and 15% greater i n 1997 than in 1996 (Pectoralis: Fi 4=7.303, P O . 0 5 ; ;2  Kidney: Fi 23=8.776). In males, mean liver mass was 35% greater in 1997 than 1996 ;  (Fi,14=5.864, P O . 0 5 ) . The mass o f stored lipid did not differ between size o f brood or years (Tables 2.4, 2.5). In females, a significant interaction term was due to a single point with a relatively large studentized residual (-2.487). Excluding this point, the only significant predictor of lipid mass was lean body mass (F] 23 5.622, P O . 0 5 ) . In males, total lipid mass was correlated =  >  with lean body mass ( P O . 0 5 ) and both the time and date on which individuals were captured (PO.05).  Physiological correlates of parental SusMR Sexes were pooled but data from the two years were considered separately.  In 1997,  the only organ to correlate with S u s M R was the small intestine. After controlling for both recapture date and body mass, individuals with relatively heavy intestines had a relatively high S u s M R ( F i 2 0 8 . 0 8 8 . P=0.01; Fig. 2.5). This relationship was dependent on inclusion =  of recapture date as a significant covariate. In 1996, an individual's S u s M R could not be predicted by the mass o f any o f its organs (P>0.05). In neither year was there a relationship between an individual's Het and its S u s M R (P>0.30).  Table 2.1 Variation in the haematocrit o f adult tree swallows rearing different sized broods.  Brood size Sex  N  5  P-value 7  Brood size  Year  Males  (10,9)  43.19 ± 1.98  41.72 ± 1.84  0.525  0.594  Females  (15,12)  44.56 ± 1.32  42.78 ± 1.47  0.375  0.592  Values (%) are least squares means ± S . E . M . (standard error o f mean) from A N C O V A . Sample sizes are in brackets.  4 2  Table 2.2. Physiological correlates o f brood size in female tree swallows.  Brood size Character  N  P-value  5  7  Brood size  Year  1  Pectoralis  (14, 14)  2.62 ± 0.05  2.62 ± 0.05  0.979  0.012  Heart  (15, 14)  0.23 ± 0.01  0.23 ± 0.01  0.643  0.562  Kidney  (14,14)  0.25 ± 0.01  0.25 ± 0.01  0.811  0.007  Liver  (15,13)  0.60 ± 0.02  0.64 ± 0.02  0.153  0.057  Intestine  (15,13)  0.68 ± 0.02  0.69 ± 0.03  0.865  0.162  Gizzard  (14, 14)  0.46 ± 0.02  0.43 ± 0.02  0.302  0.224  b  Values are least squares means ± S . E . M . (standard error o f mean) from A N C O V A . Masses are in grams. Sample sizes (bracketed) varied across organs due to missing data. The pectoral muscle, kidney, and gizzard each had a single outlier with a large studentized residual (>3.0 ) omitted. W h e n significant difference occurred between years, 1997>1996. Significant year*time of capture interaction. Significant P-values are underlined. a  b  4 3  Table 2.3  Physiological correlates o f brood size in male tree swallows.  Brood size Character  N  P-value  5  7  Brood size  Year  3  Pectoralis  (11,9)  2.71 ± 0.12  2.77 ± 0.12  0.722  0.084  Heart  (11,9)  0.25 ± 0.01  0.24 ± 0.01  0.527  0.850  Kidney  (11,9)  0.25 ± 0.01  0.26 ± 0.01  0.671  0.203  Liver  (10, 9)  0.65 ± 0.04  0.58 ± 0.04  0.249  0.030  Intestine  (11,9)  0.75 ± 0.03  0.71 ± 0.03  0.350  0.118  Gizzard  (10, 8)  0.45 ± 0.03  0.42 ± 0.03  0.605  0.090  Values are least squares means ± S.E.M.(standard error o f mean) from A N C O V A . Masses are in grams. Sample sizes (bracketed) varied across organs due to missing data. W h e n significant difference occurred between years, 1997>1996. Significant P-values are underlined. a  44  Table 2.4  Correlates o f total lipid mass in female tree swallows.  L i p i d mass F  P-value  0.010  0.490  0.491  1  0.032  1.533  0.229  1  0.106  5.117  0.034  Capture date  1  0.125  6.023  0.023  L B M * capture date  1  0.122  5.897  0.024  Source  df  SS  Brood size  1  Year LBM  b  22  Error a  a  0.455  Partial sum o f squares. L B M (Lean body mass). Significant P-values are underlined. N=28. D  Table 2.5  Correlates of total lipid mass in male tree swallows.  L i p i d mass F  P-value  0.006  0.473  0.504  1  O.001  0.001  0.977  Lean body mass  1  0.077  6.353  0.026  Capture time  1  0.104  8.620  0.012  Capture date  1  0.279  23.122  <0.001  13  0.157  Source  df  SS  Brood size  1  Year  Error a  a  Partial sum o f squares. Significant P-values are underlined. N=19.  46  •  r = 0.54  • P< 0.05  #  •  •  / • •  •• • •  »  •  I -0.30  -0.15  0.00  0.15  0.30  Residuals of intestine mass  Figure 2.5 Parental sustained metabolic rate and the wet mass o f the small intestine. Residuals controlled for the effects o f body mass and date.  4 7  Table 2.6  Enzyme activity in the pectoralis o f adult tree swallows.  Statistics  Brood size t  7  5  Character  P-value  Females PK  761.7  ± 69.84  736.4 ±  77.00  0.706  0.491  CS  277.6  ± 31.62  284.5 ±  39.59  0.391  0.701  HOAD  115.1  ±  117.8 ± 23.76  0.255  0.802  LDH  349.4  ± 41.44  326.2 ±  31.90  1.300  0.213  PK  696.7  ± 80.82  669.5 ± 23.59  0.859  0.405  CS  288.6  ± 39.84  276.1 ± 22.38  0.737  0.473  HOAD  117.2  ± 18.7  131.3 ± 27.39  1.220  0.243  LDH  350.3  ± 77.7  364.0 ± 49.79  0.407  0.690  18.80  Males  Enzyme activities are in U / g tissue, expressed as means ± 1SD. Sample sizes: Females, 5 chicks (N=9), 7 chicks (N=8); Males, 5 chicks (N=9), 7 chicks (N=7).  48  Biochemical correlates of brood size and parental  SusMR  The only enzyme that demonstrated a significant allometric scaling with mass was pyruvate kinase ( l o g i P K = 3.54 + -0.54 logioMass; r =0.15, N=33, P O . 0 5 ) . In contrast to 2  0  previous studies o f other taxa (e.g., mammals, Emmett and Hochachka 1981), the slope o f the allometric relationship was negative rather than positive. Despite considerable variance in pectoral muscle enzyme activity, activity and brood size were unrelated (P>0.25; Table 2.6). Enzyme activity was also unrelated to parental S u s M R (P>0.30).  DISCUSSION This study o f natural brood size variation in tree swallows demonstrated that: (i) adults did not trade-off nestling quality for quantity; (ii) S u s M R and brood size were unrelated; (iii) parental S u s M R and brood mass were related; (iv) adult body composition and muscle biochemistry were unrelated to brood size; and (v) parental S u s M R was correlated with the mass o f the small intestine.  Parental effort: brood size and SusMR The growth rate o f nestlings in large natural broods was the same as in small natural broods, indicating that parents did not trade-off nestling quality for their quantity. Although energetic savings due to decreased heat loss per nestling in large broods may play a role in the observed patterns (Royama 1966), variance in parental ability is probably more important. Indirect evidence for this comes from brood enlargements in this same population. Nestlings in artificially enlarged broods gained less mass than those in control broods ( G P B unpublished data, see also Chapter 4), which is inconsistent with energetic savings i n large broods. Despite differences in the total mass gain among broods o f different sizes, I could detect no relationship between either male or female S u s M R and the number o f nestlings  49  reared. Adults rearing broods o f 7 spent significantly less energy, per nestling, than parents rearing broods o f 5. One possible explanation is that parents rearing 7 nestlings were energetically more efficient than those rearing 5. The reason for this is unknown, but may be related to differences in experience or skill levels. For example, individual European kestrels (Falco tinnunculus) with differing natural brood sizes had the same estimated S u s M R (based on time-activity budgets), spent the same amount o f time i n flight and delivered the same amount o f food per nestling (Masman et al. 1989). In that study, males with larger natural broods had better territories and a greater rate o f prey capture per unit time. In an aerial insectivore such as the tree swallow, variance among individuals in foraging ability is likely important. In addition to increasing their energy expenditure, provisioning parents have many ways to deal with variation i n brood demand. For example, they may increase their load size or change the type o f prey delivered to nestlings (Wright et al. 1998). Whether adult swallows forage in different locations is unknown. A lack o f relationship between natural or manipulated brood size and S u s M R is not unusual. Williams and Vezina (2000) reviewed 20 studies that attempted to correlate S u s M R with brood size. O f these, 14 (70%) failed to detect a significant correlation. Even when a significant correlation was reported, it was not necessarily consistent between sexes, populations or nestling ages. These previous studies, and the present one, support the hypothesis that parents adjust their brood size to their own feeding capacity. This may allow all adults to work at similar levels o f energy expenditure irrespective o f the number o f nestlings (Drent and Daan 1980).  Parental effort: SusMR and brood mass Despite a lack of difference in S u s M R between brood sizes, among broods o f a given size parental S u s M R was correlated with nestling mass. Moreno et al. (1997) found a similar correlation in the pied flycatcher (Ficedula hypoleuca).  In that species, there was a strong  positive correlation between female S u s M R and both nestling mass and tarsus length. In  5 0  males, nestling tarsus length could be predicted from S u s M R , but only after controlling for the effect o f treatment (brood enlargement). The data in the present study and that o f Moreno et al. (1997) are correlational. Consequently, a relationship between parental S u s M R and brood mass can be interpreted in two ways: (1) parents were adjusting their S u s M R to the food requirements o f their nestlings, with heavier nestlings requiring more food, (2) parental effort, as reflected by S u s M R , determines nestling growth rates and mass.  Traditionally, it has been assumed that  nestling food requirements determine feeding rate, resulting in variation in parental S u s M R (e.g., Tinbergen and Dietz 1994). In contrast to this, Moreno et al. (1997) argued that an individual's S u s M R is relatively constrained, and should therefore be viewed as the independent rather than dependent variable. A s parental S u s M R rarely responds to changes in brood demand, they argued that levels o f activity are constrained by either a parental timeactivity budget or a physiological limit to energy expenditure (Drent and Daan 1980, Weiner 1992). Additional support for a constraint on energy expenditure comes from the recent studies o f Moreno et al. (1999). They increased flight costs o f female pied flycatchers directly by clipping two primary flight feathers from each wing. Females with clipped feathers maintained the same body mass and did not change their S u s M R relative to controls. The nestlings o f females with clipped feathers had lower body mass and increased mortality rates. These data suggested that females were unable or unwilling to increase their energy expenditure. Wardrop (2000) has shown that individual tree swallows vary i n their capacity to respond to an energetic challenge. She enlarged broods by 2 nestlings, and followed nestling mass gain (as an index o f parental provisioning capacity) over the next two days. The entire brood was then replaced with nestlings from elsewhere i n the population (to avoid autocorrelation), and the growth o f the replacement nestlings was followed until day 15. Parents who had a relatively high provisioning capacity early in the breeding season produced  5 1  relatively heavy replacement fledglings. These results demonstrated that parents differed in their abilities to respond to an energetic challenge, that these differences were maintained throughout the breeding season, and that they likely had fitness consequences. The recent finding that estimates o f S u s M R i n female pied flycatchers are repeatable between years (Potti et al. 1999) suggests that energy expenditure may retain a genetic component o f variation. In light o f this finding, and the work o f Wardrop (2000), the variation in nestling body mass and growth rates that I detected likely reflect differences among parents in their capacity to attain a high S u s M R .  Physiological  correlates of SusMR  In this study, I viewed parental S u s M R as a performance trait, much like V 0 2 m a x - In the same way that correlates o f V02max have been identified previously (e.g., Garland 1984, Taylor et al. 1987, Chappell et al. 1999), I sought physiological correlates o f an individual's SusMR. These would define a 'high quality' individual physiologically, and identify characters that may be subject to evolutionary change under selection for whole animal performance. In contrast to short term measures o f activity such as V02max> sustained energy expenditures require that an individual remain in approximate energy balance, i.e., energy intake must equal energy output. A t a mechanistic level, sustained energy expenditures are likely constrained by either: (1) an individual's investment i n nutrient acquisition (including foraging, digestion, absorption; Weiner 1992) or (2) at the site o f energy expenditure (e.g., contractile properties o f skeletal muscle). Alternatively, there may be no single site o f limitation, and the capacities o f various steps may be approximately equal (symmorphosis, Taylor and Weibel 1981). There is increasing laboratory evidence that i n the presence o f excess food, maximum S u s M R is more likely limited by peripheral tissues (e.g., working muscles) than centrally (e.g., by digestion; Hammond and Diamond 1997). Although individual tree swallows varied considerably in pectoral muscle mass and enzyme activity,  52  this variance was unrelated to SusMR. This suggests that the upper limit to an individual tree swallow's S u s M R in the field was unlikely to be limited peripherally. There was, however, a positive correlation between an individual's S u s M R and the mass of its small intestine. A similar correlation has been found i n laboratory studies o f small mammals (Konarzewski and Diamond 1994). T o my knowledge, this is the first demonstration o f such a relationship under field conditions. Although my study suggests that attainment of a relatively high S u s M R requires a relatively large gastro-intestinal system, a simple correlation is not sufficient to identify the intestine as a bottleneck.  Only through a  comparison o f energy expenditure during provisioning with that during other activities (e.g., shivering thermogenesis) could the proximate factors imposing a ceiling be identified (Peterson et al. 1990, Hammond and Diamond 1997). For example, i f the S u s M R during shivering was greater than during provisioning, a ceiling on provisioning could not be determined by shared machinery (e.g., intestine). It is well recognized that individuals w i l l undergo intestinal hypertrophy under conditions of chronically high energy expenditure (e.g., Dykstra and Karasov 1992, Hammond and Diamond 1997). It is unknown whether inter-individual variation in intestine mass allowed some swallows to attain a high S u s M R , or whether individuals underwent intestinal hypertrophy in response to a high S u s M R . Use o f non-invasive techniques that allow repeated sampling of the same individual (e.g., ultrasonography, Dietz et al 1999) may allow for these two possibilities to be disentangled. Dykstra and Karasov (1992, 1993) argued that provisioning effort in the field is unlikely to be constrained physiologically. They showed that the S u s M R o f house wrens feeding nestlings was considerably below that measured in the lab under conditions o f cold and exercise. In support o f these findings, Tinbergen and Verhulst (2000) recently showed that although an energetic ceiling is apparently set for female great tits (Parus major) at the level o f their unmanipulated brood size, the ceiling varied between years. Although this does not negate the need for an increased digestive capacity under conditions o f high energy  53  expenditure, it does suggest that a ceiling on S u s M R i n the field is more likely set by ecological factors (e.g., day length) rather than physiological ones (e.g., digestive capacity).  Inter-annual variation SusMR and body composition There was a significant inter-annual difference in SusMR. This difference was due in part to 5 o f 14 individuals, each with a particularly l o w S u s M R i n 1996 (Fig 2.2). A s I performed no behavioural observations I cannot tell whether these individuals were behaving normally during the 24 hour trial. If the low values were a consequence o f handling stress, low S u s M R s would have also been expected in 1997, yet these were not observed. In addition to low values for SusMR, individuals sacrificed in 1996 had on average smaller internal organs than in 1997. Whether interannual differences in body composition were related to differences i n S u s M R is unknown, although relatively large internal organs may be a prerequisite for an elevated S u s M R (e.g., Kersten and Piersma 1987, Daan et al. 1990). Inter-annual differences in both S u s M R and organ size may be related to variation in food availability. It is unclear, however, whether an increase or decrease in the size o f internal organs would be predicted in response to increased food abundance (e.g., Daan et al. 1989, Geluso and Hayes 1999). Regardless, inter-annual differences in organ mass have been found for this same population in other years (Chapter 3), suggesting that phenotypic flexibility is a general phenomenon (Piersma and Lindstrom 1997).  SusMR and fitness. There is increasing evidence that rates o f energy expenditure in birds are stable over relatively long time periods (Chappell et al. 1996, Potti et al. 1999, B e c h et al. 1999). But why do only some individuals attain a high S u s M R i f it is potentially linked with Darwinian fitness? One possibility is that in order to attain a high S u s M R it is necessary to maintain a relatively large gut (e.g., Konarzewski and Diamond 1994, Dykstra and Karasov 1992). This may result in an elevation o f an individual's energy expenditure while resting (Piersma et al.  54  1996, Chappell et al. 1999, but see Chapter 3), which likely carries associated costs. Correlations between S u s M R and intestinal mass would presumably result in covariation between resting and sustained metabolic rates (aerobic capacity model, Bennett and Ruben 1979). Support for such covariation remains equivocal (Hayes and Garland 1995, Ricklefs et al. 1996).  Conclusion Energy allocation by parents to their to offspring is predicted to increase with increasing brood size. Despite this, I could not detect a relationship between brood size and parental energy expenditure. One explanation is that there exists inter-individual variation in parental foraging efficiency. This supports a previous suggestion i n the literature that clutch size is adjusted to the amount o f food that can be delivered to nestlings for the same parental energy expenditure (Masman et al. 1989). In one o f two years, there was a positive relationship between parental S u s M R and brood mass, suggesting potential reproductive benefits.  Individuals with relatively high S u s M R had relatively large intestines, which  presumably allowed for an increased digestive capacity. This suggests the existence o f a trade-off between the reproductive benefits o f attaining a high sustained energy expenditure, and the costs associated with maintaining expensive metabolic machinery.  55  CHAPTER 3 INTER-INDIVIDUAL VARIABILITY IN BODY COMPOSITION AND RESTING OXYGEN CONSUMPTION RATE IN BREEDING TREE SWALLOWS  PREFACE This chapter is adapted from a paper published by G . P. Burness, R . C . Ydenberg and P. W . Hochachka (Physiol. Zool. 71: 247-256). I was responsible for all data collection, analysis and presentation. M y co-authors provided guidance and editorial advice.  INTRODUCTION Basal metabolic rate ( B M R ) is defined as the minimum rate o f energy expenditure in a non-growing, post-absorptive organism, at rest in its fhermoneutral zone (Brody 1945), during its period o f daily inactivity (Aschoff and Pohl 1970). A large comparative data set exists relating B M R to body mass across a wide variety o f taxa. Studies investigating B M R have often been concerned with accurate determination o f the slopes o f these allometric relationships (e.g., mammals , Elgar and Harvey 1987; birds, Bennett and Harvey 1987). These studies and others (e.g., Koteja and Weiner 1993) have also been interested i n species that deviate from the regression lines. For a given body mass, two species can vary considerably in their B M R s . A s an example, the Virginia opossum (Didelphis virginiand) has a B M R 30% lower than predicted for a similarly sized eutherian mammal (Fournier and Weber 1994). Such deviations also exist in birds. For example, island species have much lower B M R s than mainland species o f the same body size ( M c N a b 1994). The mechanistic basis underlying variability in B M R among similarly sized species is gradually being determined.  M c N a b (1994) found a positive correlation between B M R and  pectoral muscle mass in many flightless birds.  Kersten and Piersma (1987) speculated that  inter-specific differences in B M R s among birds reflected differences in the size of a species'  56  "metabolic machinery". Daan et al. (1990) subsequently demonstrated that those species o f birds with relatively high B M R s for their body size have relatively large masses o f hearts and kidneys. In fact, in an analysis o f 22 avian species, these two organs, which contribute only 0.61% o f body mass, explained 50% o f the variation in B M R (Daan et al. 1990). Both o f these organs have exceptionally high oxygen consumption rates i n tissue slice preparations (Krebs 1950, Scott and Evans 1992). Within a species the relationships between B M R and organ masses are unclear.  As  the slopes from regressions o f B M R on mass vary depending on the taxonomic level studied (e.g., Bennett and Harvey 1987) different mechanisms may be acting within species from those acting between species. To date most research on this question has considered small mammals (Konarzewski and Diamond 1994, 1995, Koteja 1996, Speakman and McQueenie 1996, Meerlo et al. 1997) and lizards (Garland 1984, Garland and Else 1987). A s an estimate o f B M R many studies measure resting metabolic rate ( R M R ) , which does not assume that individuals are post-absorptive or in their period o f daily inactivity. In comparisons among and within strains of inbred mice, Konarzewski and Diamond (1994, 1995) demonstrated that individuals with high R M R s have relatively large kidneys, livers, hearts, and intestines. In contrast, relationships between organ mass and B M R are weak or absent in Peromyscus maniculatus (Koteja 1996). Meerlo et al. (1997) demonstrated a significant positive relationship between mass-independent residuals o f B M R and heart mass in the field vole (Microtus agrestis).  Finally, using principal component analysis, Speakman  and McQueenie (1996) suggested a relationship between B M R and organs o f the digestive system in mice. A s variability is necessary for the evolution o f a trait through natural selection, assessment o f trait variability is important.  This was the first field study o f any avian  species to relate inter-individual variability in R M R with body composition (but see Chappell et al. 1999, Bech et al. 1999). In addition, as food resources likely vary across years, there may be considerable variability i n R M R and the masses o f energetically  57  expensive organs between breeding seasons.  Although individual body composition changes  cannot be followed practically across more than one breeding season, annual averages are informative. I present data from tree swallows (Tachycineta bicolor) collected over two breeding seasons. This study had two aims: (1) to determine the extent o f variation i n R M R (as an approximation o f B M R ) and body composition in a w i l d population o f birds, and (2) to address the question o f relationships between R M R and organ and tissue masses.  MATERIALS AND METHODS This study took place during May-June, 1994 and 1995, i n Creston, British Columbia, Canada. Tree swallows in this population typically lay between one and eight eggs in nest boxes, with a modal clutch of six. Only those pairs that laid 6 eggs were chosen for study. First-time breeding females can be distinguished on the basis o f plumage (Hussell 1983) and were excluded from this study. In 1994, tree swallows were captured at the nest box 8-9 days after hatch o f chicks and transported to a field lab for resting oxygen consumption rate measurements (VO2).  In 1995, the experimental protocol was modified  slightly. First, to increase parental effort, when chicks were 4 d old one additional nestling was added to each brood. This increased brood sizes from six to seven.  Second, as part of  another study on foraging energetics, all experimental adults were captured and injected intramuscularly on day 8 with 100 o L o f doubly labeled water ( H 2 0 ; see Chapter 2 for 3  , 8  methodology). Birds were held for 1 h, a sample o f blood (150ul) was taken, and the bird was released. U p o n recapture after 24 h, a second blood sample was taken, and the birds were transported to the field lab as in 1994. Although there was a difference in field protocols between 1995 and 1994 , the gross morphological measurements I performed on adults were unlikely to have been affected by such protocol changes.  58  Resting oxygen consumption A s an approximation o f B M R , I measured daytime resting VO2. In both 1994 and 1995, VO2 was determined using closed system manometry (e.g., Williams and Prints 1986, O b s t e t a l . 1987). After capture, birds were immediately transported to a field lab for VO2 measurements. Adult swallows were weighed on an electronic pan balance (to the nearest 0.2 g).  Flattened wing chord (measured from the wrist to the distal tip o f the ninth primary  feather, ±0.5 mm) was measured using a ruler with a stop at one end. K e e l length was measured using dial calipers (±0.05 mm).  Birds were then placed in a 1,000-mL black  Plexiglas metabolic chamber. The floor o f the chamber contained soda lime and Drierite to absorb CO2 and H 0 , respectively. A steel mesh covered with a sheet o f tissue paper on 2  which the bird stood was placed over the chemicals. After an equilibration time o f 1.5-2.5 h, a V-shaped Plexiglas manometer filled with Krebs manometer fluid was attached to the chamber. Five milliliters o f oxygen was injected from a calibrated glass syringe, causing an increase in chamber pressure.  The time taken for the bird to consume the 5 m L o f 0  2  was  indicated by movement o f the manometer fluid. This procedure was repeated over the next 45-60 min, and the values were averaged. The minimum acceptable number o f replicates for a successful trial was 7 with a maximum o f 12. The mode was 10. If the bird was active in the chamber, the manometer fluid would 'jump'. Only measurements in which the level of the manometer fluid decreased linearly and smoothly were used, indicating quiescence.  Similar manometric systems to the one in this study have been  in good agreement when compared against flow through systems (Obst et al. 1986, Williams and Prints 1986). In addition, the coefficients o f variation for VO2 for birds measured in the present study fell well within the range calculated for similarly sized passerines (e.g., Dutenhoffer and Swanson 1996). The thermoneutral zone for tree swallows is between approximately 30° and 35°C (Williams 1988). Thermoneutrality was maintained by submerging the chambers in a Lauda  59  R M 6 temperature-controlled water bath.  Chamber temperature was monitored (±0.1 °C)  using a thermocouple inserted approximately 4 c m down into the sealed chamber. Daily changes in room temperature and consequently water bath settings resulted in slightly different chamber temperatures between individuals. Such temperatures were, however, always within the thermoneutral zone. The thermocouple also recorded rapid temperature fluctuations coincident with 'jumping' o f the manometer fluid. This indicated activity in the chamber, and recordings were not attempted during such periods. To minimize variability due to circadian rhythms (Aschoff and Pohl 1970), all measurements were performed between 1000 and 1800 hours. Forty-eight o f 51 birds captured had their V O 2 measured on the same day as capture. Three in 1995 were captured late in the day, and to avoid variability due to circadian rhythms were held overnight until measurement the following day. Birds were left in the chamber for between 1.5 and 2.5 h before beginning measurements, so it is unknown i f they were all post-absorptive. To assess this, small intestine contents were measured and correlated against mass independent V O 2 to determine i f there was an apparent heat increment o f feeding. During trials, room temperature and barometric pressure were recorded and all values o f V O 2 were corrected to S T P D .  Haematocrit Following measurement o f resting V O 2 , birds were removed from the chamber and reweighed. The before and after weights were averaged. To address variability in Haematocrit (Het) i n 1995, blood samples were collected into heparinized microcapillary tubes. These tubes were immediately spun at maximum speed for 10 min using an Adams microhaematocrit centrifuge. The percentage o f the tubes occupied packed cells was measured. The number o f replicates was determined by the size o f the blood sample and ranged from one to three replicates.  60 Tissue masses In both years birds were killed by cervical dislocation immediately following measurement o f V O 2 . Both the left and right pectoralis major and supracoracoideous were removed together (referred to as pectoralis), followed by the heart, liver, small intestine, and kidney. A l l tissues were stored i n air-tight cryovials, frozen in a portable dry shipper charged with liquid N 2 , and transported back to Vancouver, where they were transferred to a -80°C freezer until analysis. Wet weights were determined for all organs and tissues (± 0.0001 g). Two individuals from 1994 did not have their left pectoralis weighed. These were estimated from a regression of left pectoral muscle mass on right pectoral muscle mass generated from 18 other individuals (r =0.70, P O . 0 0 1 ) . 2  The small intestine was initially weighed full and empty  mass was determined as in Chapter 2. The difference between full and empty intestine mass was assumed to be gut contents.  The intestine was then dried to a constant mass for 36 h at  75°C. I report dry tissue mass only, as the correlation between wet and dry mass is high (r = 0.93, P < 0.001). N o other tissues were dried.  Statistical analyses A l l variables measured are likely to scale allometrically with body mass. A l l data with the exception o f small intestine contents were logio - transformed and regressed on mass using simple linear regression. To improve normality, intestine contents were square-root transformed. The effect o f year and sex on the dependent variables (masses o f heart, kidney, pectoralis, liver, small intestine, and intestine contents) were explored using an A N C O V A . I initially included the interaction terms o f year and mass, and sex and mass as additional covariates. A s no significant interactions were found, all further multiple regressions were performed including only the covariate (mass) and main effects (year or sex). Residuals were then generated from either A N C O V A s or, when the effect was not significant, from simple  61  linear regressions. Residual analysis removes the effect o f the covariate (mass) from the dependent variable. I report model and error degrees o f freedom following the F-Value. To test for outliers, the studentized residuals for the dependent variable were generated from the multiple regressions on mass. These residuals follow a t-distribution (Wilkinson et al. 1992). I felt that characters such as resting V 0 were more likely to be 2  influenced by measurement error (e.g., stress o f birds) than were tissue masses. Consequently, for resting V O 2 data, studentized residuals significant at P < 0.05 were excluded; for all other variables, P < 0.001. To examine the variability o f each dependent variable, I followed Garland (1984) and compared the standard deviation o f the residuals generated from the simple linear regressions on body mass. The standard deviation o f residuals from a l o g transformed data set is e  approximately equal to the coefficient o f variation o f the untransformed data set after removal of the effect o f body mass. A s residuals in the present study were generated from logio-logio regressions on mass, and not l o g , data were converted from logio to l o g . e  e  To convert from  logio to loge the standard deviation o f the residuals was multiplied by 2.3026.  When a  significant regression could not be generated, the standard deviation o f the residuals is simply equal to the coefficient o f variation o f the data set before log transformation. Conversion o f the standard deviation o f the residuals to coefficient of variation allows comparison with literature values. To determine the extent to which variation in resting V O 2 reflected variation in organ and tissue mass, residuals from multiple regressions were included i n correlations and a stepwise multiple regression. Residuals o f resting V O 2 were first correlated against residuals of resting organ and tissue masses using a Pearson-product moment correlation or, in the case of the non-normally distributed Het data, a Spearman-rank correlation.  A predictive  equation for residual resting V O 2 was generated using a forward stepping multiple regression. Residuals o f Het and organ masses were correlated. To control the probability o f a Type I error (rejecting the null hypothesis when it should not be rejected), a sequential  62  Bonferroni correction (Rice 1989) was applied to the data to correct for the number of tests. Correlations within this matrix were considered significant at P < 0.002 (alpha = 0.05). A l l other comparisons were considered significant at P < 0.05. Most analyses utilize mass corrected residuals. To account for the degrees of freedom lost in generating the residuals, N-3 degrees of freedom were used when testing significance of Pearson correlations (Hayes and Shonkwiler 1996), andN-1 for Spearman-rank correlations. As significant P-values were very small in both the correlation matrix (Table 3.3) and stepwise regressions, the degrees of freedom were not adjusted. Analyses were performed using Systat 5.2.1 (Wilkinson et al. 1991). 95% confidence intervals for slopes and intercepts of regressions were generated using SAS (SAS Institute Inc. 1988).  RESULTS Allometric  relationships  I measured V O 2 and body composition for a total of 51 individuals. When V O 2 was plotted against mass, three points had exceptionally high studentized residuals (3.033; 2.312; 2.036 ; all P < 0.05). Similarly, when heart, pectoralis, and intestine mass were when plotted against body mass each had a single point identified as an outlier (studentized residuals: 3.672; 3.606; 6.528 , P < 0.001). These points were included in the figures although not in the analyses. Males were significantly heavier than females (Males, 18.44 ± 0.97 g; Females, 16.95 ± 0.92 g: t = 5.610, df = 49, P < 0.001). Resting V O 2 , and most tissue and organ masses showed no significant sex differences when body mass was included as a covariate: V O 2 (P 0.886), heart (P = 0.057), kidney (P = 0.895), liver (P = 0.675), small intestine (P = 0.240). A significant difference was found between the sexes for pectoralis (Fj^s = 7.811, P = 0.007). This was probably attributable to the terms sex and mass sharing considerable variance. the pectoralis, residuals were generated from regressions with and without the term 'sex' included. In subsequent analyses of these residuals, inclusion of 'sex' did not change any  For  63  trends or conclusions. A s the pectoralis was the only tissue showing significant sex differences, I pooled the sexes for all variables and report results o f pooled analyses only. To generate allometric equations and assess the amount o f variance attributable to body mass alone, data were pooled across the two years. The mean body mass was 17.7± 1.2 g, and ranged from 15.3 g - 20.6 g. A l l variables except Het (P - 0.939) and gut contents (P = 0.156) scaled significantly with mass (Table 3.1). Using A N C O V A with body mass as a covariate, I found no difference between years in the slopes o f regressions for heart (P = 0.223; Fig. 3 . I B ) ; kidney (P = 0.377; F i g . 3.1C), small intestine (P = 0.699; Fig. I D ) and liver (P = 0.800; Fig. IE). For a given body mass these organs were, however, heavier in 1994 than in 1995: heart (Fi^j kidney ( F i (Fi,48  =  > 4 8  = 45.760, P < 0.001), small intestine ( F i  > 4 7  = 19.967, P < 0.001),  = 12.457, P = 0.001), and liver  17.780, P < 0.001). N o significant difference was found for resting V O 2 (Fig. 3.1A)  in either slope (P = 0.878) or intercept (P = 0.839). Similarly, no significant differences were found between years for pectoral muscle either in slope (P = 0.935) or intercept (P = 0.493, Fig. 3. IF).  In all cases where there were differences in intercept o f the regression lines over  the range o f values measured, the lines for 1994 fell above those o f 1995. To explore the inter-annual variation further, I generated an index o f structural size using principal component analyses (e.g., Cooch et al. 1991). I extracted the first principal component ( P C I ) from the correlation o f wing (mm) and keel length (mm) for the 45 individuals for which I had both measures. P C I explained 79% o f the variation in these characters. I then included P C I as a covariate and regressed mass against it and included year as the main effect. The interaction term was not significant (P = 0.511) and was excluded from the model. For a given body size, birds in 1994 were not significantly heavier than those i n 1995 (P = 0.285).  64  CO  ca  ON  CN T t  O o  oi  ^—i NO ID  ID  CO  CO CD  t-  NO  C-H  O  ON NO  o  o o  CN  oo  o  oo  CO  o  NO  o ©  ID CO  o  CN  r~ p d  o d  ,23  3  •r  1  CD  rd  v>  oo  T f  de\ for di  > u  OH  r ^  NO  ©  d  d  O  ©  r-»  cn  oo  oo  r--  >D  ON NO  o  O  -H  -H  CO  ©  -H  CN  ©  -H  CD  ^H  u c  x  ID  ON -H CD  OH  _o  -=t  3 0.5  S3  co T t  d  oo  1  i—i  o  r-  oo  d  -H  ——I  o  -H  co  p  c--  CN  CN  i  ON ID  l  ON IT) ID  00 NO NO  d-H  ©  CO  cn  ON O  00 00  ©  T t  r-  NO  ©  o  oo  •  o Tfr  o  ©  00 ID  d-H  NO CN  r-  i  •  i  -H  oo i  ID CO  d  -H ID CN  oo ON NO  d  NO CN  CN CN  d  CO  d  CO  —H  o  d  CN  d  p  o  CO CO ID  i  1  o  o  CN  o NO  ID CN  NO NO  NO NO .  O  ©  d  CN  OO  o ID  i—•l  O ID  T t  1—1  ID  m  fe  u  O  >  w  •e cd CD  ney (g  o h-1  iver (g)  CN  O Cd  O +-»  ^  CH  CJ  1  d  o ID  t~~ .—1  T f  o CO •  « B E d  s o -2 cd -=3  00  si CD  r—1 <—  2  w  .23 /  rH  §  W * on «  M  Inte stine (  CD  r-  Pec toralis  cd  CD  cC 3  ID CN  a  5  1  ID CO  T t  ca  CT N O CD C N  O  oo  TW  Cd  T t  i—i  i  C  " s  o  CN  i  -H  V i  ro  ON NO  •3 «  t3  CO  o  Cu) •*-»  '—  °glO  ii  NO  CN «D  1—1  b  ID ON  NO  Ion. cv% convert fro  u  1  1  :he stai e (see  o  co  culat  co  om regres sion Itipllied by 2.3(  (H  ard err or of esti: netric regressio  ID  CN  ON  +->  o X  * Id aJ «  o « CD  65  Figure 3.1 Allometric scaling o f body mass and (A) resting oxygen consumption rate, (B) heart mass, (C) kidney mass, (D) small intestine mass, (E) liver mass, and (F) pectoralis mass. Open circles are birds measured i n 1994; filled circles are for 1995. Triangles are outliers not included in analyses (see text). Dashed line is regression for 1994; solid line for 1995. If only one line present, there was no difference between years. Significance o f regressions is given in text. Axes are logio transformed.  66  0.36  15  16  17  18  17  21  19  Body mass (g)  18  10  21  20  Body mass (g) 0.25  0 36  0.32  i  D)  0.28  E  0.24 H  to to co >v  c  T3  0.20  15  16  17  18  19  20  16  15  21  17  18  19  Body mass (g)  Body mass (g) 4.0  A  LL  3.5  °c»  3  3.0  to to  •V.  CO £ 2.5  o> i  .52  s. o  •  t3  0) 2.0  1°  o  • • •  •  CL  1.5  15  Body mass (g)  16  17  18  19  Body mass (g)  20  21  67  In addition, apart from male wing length, there were no other differences between years for the other external characters that I measured (body mass and keel length; Table 3.2).  Variability among individuals The coefficient o f variation for each character was calculated as the standard deviation of the residuals generated from the regressions of Table 3.1; this demonstrates variation after the effects o f body mass have been removed. The coefficients o f variation ranged from a low of 7.8 for pectoral muscle mass to a high o f 18.4 for Het. Resting V O 2 showed moderate variability with a coefficient o f variation o f 9.4. The small intestine, a relatively plastic organ (e.g., Secor and Diamond 1995), not surprisingly had a high coefficient o f variation (16.3%).  Correlations among characters Residuals generated from either A N C O V A s or linear regressions were correlated against each other to address two questions: (1) D o individuals with a relatively high resting V O 2 for their body mass have relatively large organs and tissues? and (2) D o individuals with a relatively large mass o f one organ or tissue for their body mass have other relatively large organs and tissues? To assess i f all birds were post-absorptive, I first correlated residual intestine contents against residual V O 2 . Intestine contents were obtained for 43 birds. A s there was no significant relationship between the residuals (r = 0.114, P > 0.40), I concluded that any differences in V O 2 among individuals were unlikely to be due to a heat increment o f feeding. I correlated residual V O 2 against the residuals o f different tissues and organs. A s hypothesized, those individuals with a relatively high V O 2 had relatively large kidneys (r = 0.29, N = 48, P < 0.05. F i g 3.2). N o other morphological variable correlated with residual V 0 : (heart, r = -0.15, N = 47, P > 0.30; intestine, r = -0.19, N = 47, P > 0.20; liver, r = 2  0.056, N = 48, P > 0.70; pectoral muscle, r = -0.23, N = 47, P > 0.10).  68  Table 3.2 External morphometries between years for male and female tree swallows.  Year 1994  Statistic 1995  t  P  Body mass (g) Male  18.4 ±  0.70  (9)  18.4 ±  1.11 (16)  0.049  0.961  Female  16.9 ±  0.90  (10)  17.0 ±  0.96 (16)  0.427  0.673  Male  119.1 ±  2.80  (9)  122.7 ±  2.61 (15)  3.190  0.004  Female  114.6 ±  3.21  (9)  116.9 ±  2.38 (15)  2.049  0.053  Wing length (mm)  Keel length (mm) Male  21.26 ±  0.74  (8)  21.41 ±  0.72 (14)  0.449  0.658  Female  19.74 ±  0.33  (9)  20.19 ±  0.63 (16)  1.973  0.061  Note. Values are means ± 1 S.D. Significance assessed using t-tests on log-transformed data. Sample size is in parentheses.  69  Values for Het were non-normally distributed (Lillifore's test, P = 0.011) and were compared to residual V O 2 using a Spearman-rank correlation (the degrees o f freedom were reduced by 1). Residual Het correlated positively with residual V O 2 (r = 0.38, N = 29, P < 0.05). I performed a stepwise multiple regression with residual V O 2 as the dependent variable and the residual o f each organ or tissue as a potential predictor variable. The residuals o f kidney (t = 3.112, P = 0.003), and intestine mass (t = -2.616, P = 0.012) were the only significant predictors o f residual V O 2 . Although residual kidney mass loaded positively, residual intestine mass loaded negatively. Together, kidney and intestine explained 2 1 % o f the variation i n residual V O 2 ( N = 47, F2 44 = 5.855, P = 0.006). A second stepwise ;  regression was performed, including residual Het as an additional predictor. Residual kidney mass (t = 3.628, P = 0.001), and residual intestine mass (t = -3.267, P=0.003), were still significant. Residual Het approached significance (P = 0.053). Table 3.3 is a correlation matrix showing all possible correlations among characters. A l l significant correlations were positive. Without correcting for body mass, numerous correlations existed among organs and tissues. The number was reduced when body mass independent residuals were correlated. Individual swallows with relatively large livers for their body mass also had relatively large kidneys (r = 0.52, P < 0.001), and intestines (r = 0.50, P < 0.001). Individuals with relatively large hearts also had relatively large pectoral muscles (r = 0.49, P < 0.001). N o other correlations were significant.  70  CD CO *-  •*->  0.150 r = 0.29  •  P < 0.05  •  0.075 -  0.000  .•V•1 <•« • •  -0.075 H  •  ••  •  •  ••  -0.150 -0.150  -0.075  0.000  0.075  0.150  Residuals of kidney mass  Fig. 3.2  Correlation between resting oxygen consumption rate and kidney mass. Residuals controlled for the effect o f body mass on both variables.  71  oo (N  O i  ii  fl  to  cu  CO CU CO CO  cd  00  VH  O  rt o  cu  ©  ON OO T t  s  CN ©  in © oo  o ON o  0  ©  ©  CN © in  © © ©I  V  © ©  cn  CN  O V  ©  PH  s  OO ON ©  ©  CN © ©  ©* in  o  A^  in  cn ©  •—i  o  CN  cn r-CN  ©^  o  ©  .—i©  CN  ©  rtH  ©'  ©  in © © ©  i  m  o" A^  VO ©  ON T t  © ©  ©  A  oo 1 —1  oo m ©  Tt  ©  OO  VO  © © ©  cn cn © •  o  1—(  o>)  a  cd  vo cn ©  0  T3  fl  cd cu CO CO  00 fl O  s  >  CN  VO  Tt  in  cn  cu  fl -o  1 —1 o  i—i  ON VO  o  cn ©  © ©  2  oo © © ©  v  —'  in  i—i  m ©  ©  ON  VO  i—i  o  © ©  o  i  © © A^  cd  cd CO  A  o n  fc! o o  cu  >  CN CN  cn  CN CN  m  © ©  ©  V  VO  vo T t  © ©  ,—i » —i ©  r-H  t-VO  ©  © O  o  ©  in © © ©  in CN CN ©  1  © in © A^  Cl  o  to  cu PH  "cd T3 %  t. cd  cn  K  ©  oo in  cu  o o o V  ©  oo in  © o o V  CN  VO  O ©  ©  V  ON ON  cn ©  '  1  •Cl-HtH u  cn cn  cu  cd cu  cu >  >v cu  fl -a  2  cd  CU  fl  l-H  cu  o rt o  rt fl  cu  PH  a  o~ CN © A^  72  Analyses to avoid potential part-whole  correlation.  A potential spurious correlation can result i f the dependent variable is included in the independent variable (part-whole correlation; Sokal and R o h l f 1995, Christians 1999). The degree of importance varies with the magnitude of the dependent variable relative to the independent. To assess the potential for spurious correlations, I performed a second set o f analyses i n which the mass o f each organ or tissue was first subtracted from the body mass o f the animal before an allometric equation was generated. For no tissue or organ other than pectoralis muscle was there any change in significance. The pectoralis contributes approximately 10% o f the mass o f the bird. Removal o f the mass o f the pectoralis from body mass reduced the coefficient o f determination from 0.52 (Table 3.1) to 0.19. The regression was still significant (¥\^g = 11.41, P < 0.001). A significant negative relationship was now found between residual pectoral muscle mass and residual V O 2 (r = -0.29, N = 48, P < 0.05). The results from the stepwise regression did not change.  DISCUSSION Allometric  relationships.  Resting V O 2 scaled intra-specifically as m a s s  1 0 3  i n tree swallows. The 95%  confidence intervals are fairly large (Table 3.1); consequently this intra-specific slope is not significantly different from the phylogenetically corrected inter-specific slope o f m a s s 0  63  found for birds (Reynolds and Lee 1996). Similarly, the slopes o f all regressions for body components on mass were close to m a s s  1 0  (Table 3.1). Again, considering confidence  intervals, these are close to the avian inter-specific values, which range from m a s s 0  breast muscle to m a s s  1 0 4  95  for  for liver (Daan et al. 1990).  Relative to inter-specific studies, the fraction of the variance i n resting V O 2 explained by body mass in this study was low. For example, body mass explained between 95%-97% of the inter-specific variance in B M R (measured as V O 2 ) in birds (Daan et al. 1990). In  73  contrast, body mass explained only 35% o f the variance in this character within tree swallows (Table 3.1). This is likely due to the narrow range o f body masses occurring within a single population when compared to that occurring between species. In inter-specific studies to date, the heaviest species is 200 times the lightest (Daan et al. 1990). In contrast, in this study, the heaviest tree swallow (20.6 g) was only 1.3 times the lightest (15.3 g). The correlation coefficients for resting V O 2 in this study are, however, comparable to those from studies o f random-bred domestic mice (Mus domesticus) which cover a similarly narrow range of body mass (Hayes et al. 1992).  Inter-annual variability in tissue mass. For most organs and tissues there were significant inter-year differences. Animals captured in 1994 had larger tissues for a given body mass than those from 1995. I feel this was unlikely due to a systematic error between years; for example, the same pan balances were used i n both years. The capacity for individuals to demonstrate inter-annual up- and down-regulation o f organ sizes is not surprising. In birds, large scale changes in pectoral muscle mass occur during pre-migration (e.g., Marsh 1984) and moulting (Gaunt et al. 1990), while in snakes there are rapid changes i n gut mass during feeding (Secor and Diamond 1995). To my knowledge, however, such changes have never been demonstrated between consecutive breeding seasons. A s outlined i n Chapter 2,1 feel differences between years may be driven at least partly by the level o f food resources available to the breeding birds. Daan et al. (1989) demonstrated that adult kestrels kept on restricted diets have decreased masses o f hearts and kidneys. In the present study, both o f these tissues showed significant inter-annual differences.  Differences between years may also be analogous to a training effect. In 1995,  in order to increase the provisioning parents' work load, an additional chick was added to their nests; which may result in an increased parental visitation rate. Although small scale physiological changes in response to training can be seen over a period o f a week (e.g.,  74  increases in N a / K +  +  ATPase; Green et al. 1993), it is unknown i f the large scale adjustments  in organ sizes I saw can be made over a period o f 5 d (but see Stark 1999, Battley et al. 2000). Individuals with smaller organs for a given body mass were apparently not in poorer condition than those individuals measured the previous year.  They were not lighter in body  mass for a given body size ( P C I ) nor in mass, independent o f size (Table 3.2). A change in organ mass with little change in body mass has been found previously in house wrens challenged with increased exercise and cold (Dykstra and Karasov 1992). The study on wrens and this study suggest that use o f relationships between size and body mass as a condition index should be used with caution.  Variability among individuals For there to be evolution o f a trait, there must be variability in the population, and the trait must be heritable. One major aim o f this study was to assess this first point by determining the extent o f physiological variability among individuals within a population. After effects o f body mass had been removed (except for Het, which did not scale allometrically), the coefficients o f variation (Table 3.1) ranged from a low value o f 7.8 for pectoral muscle mass to a high o f 18.4 for Het. Resting V O 2 showed moderate variability ( C V = 9.4%) when compared with other characters measured. A s resting V O 2 is probably the most widely assessed trait in avian energetics (Daan et al. 1990), there is an enormous comparative data base on which to compare variability in V O 2 . A s an example, I calculated approximate coefficients o f variation for 10 species o f passerines whose B M R s were measured by Dutenhoffer and Swanson (1996). Using their reported mean masses, B M R s , and standard deviations, I calculated the range o f coefficients o f variation assuming a mass exponent o f 0.76 (their inter-specific exponent). The coefficients o f variation o f B M R ranged from a low o f 4.5% for the house sparrows (Passer domesticus; N = 6) to 2 1 % for the Eastern wood-pewee (Contopus  virens;  N = 5), with the average coefficient o f variation being 11.4%. The coefficient of variation for  75  tree swallows measured in the present study fell well within the range calculated for these other species. Few studies have considered the inter-individual variability in organ and tissue mass. The coefficients o f variation for organ and tissue masses i n this study (Table 3.1) are comparable to those o f one lizard (C. similus; Garland 1984), but lower than those o f a second species (A. nuchalis; Garland and Else 1987).  Correlations among characters. The second principal aim o f this study was to establish potential morphological and physiological correlates for the reported variability in resting V O 2 i n a w i l d population o f birds. I addressed the question: do individuals with relatively high resting VO2S for their body mass have relatively large metabolically active internal organs? Such a relationship was demonstrated only for the kidney, where a positive relationship was found between kidney mass and V O 2 (Fig 3.2). This has been shown previously, inter-specifically in birds (Daan et al. 1990) and intra-specifically in mice (e.g., Konarzewski and Diamond 1994, 1995). The kidney has one o f the highest VO2S o f any organ (Krebs 1950). Surprisingly, the heart, which is another metabolically active organ (Krebs 1950) and a significant predictor o f resting V O 2 in other inter- and intra-specific studies (e.g., Daan et al. 1990; Konarzewski and Diamond 1994, 1995; Meerlo et al. 1997, Chappell et. al 1999), showed no relationship with resting V O 2 in tree swallows. Those individuals with relatively high VO2S had relatively small pectoral muscles (when pectoral muscle mass was first subtracted from the dependent variable). The basis behind this relationship is unclear, but one possibility is that individuals with the smallest pectoral muscles had the greatest mitochondrial volume densities.  This would result in an  increased V O 2 per gram o f tissue. The activity o f citrate synthase, a mitochondrial marker enzyme, does not, however, support this contention ( G P B unpublished data).  76  N o other tissues correlated with residual V O 2 . In a stepwise regression, kidney mass was a significant positive predictor, and surprisingly, small intestine mass was a significant negative predictor. In tree swallows, after controlling for body mass and kidney mass, birds with higher resting VO2S have smaller intestines.  This is the reverse o f previous studies that  have found relationships between resting V O 2 and small intestine mass (Konarzewski and Diamond 1994, 1995, Koteja 1996). I correlated the masses o f internal organs and tissues (Table 3.3). Individuals with relatively large hearts also had relatively large pectoral muscles, suggesting a functional matching o f the two organs. This same correlation was recently reported by Chappell et al. (1999) in house sparrows.  In tree swallows, individuals with relatively heavy livers for their  body mass had relatively heavy kidneys and intestines. This agrees with the findings o f Konarzewski and Diamond (1995) who found positive correlations among internal organs in mice. Surprisingly, Chappell et al. (1999), did not find a correlation among organs o f the abdominal cavity in sparrows. Kidney mass and intestine mass (wet) when combined account for about 5% o f an adult swallow's body mass. Kidney mass and dry intestine mass explain 21 % o f the variation in residual V O 2 . Konarzewski and Diamond (1994) found that kidney and heart when combined explained 12% o f the variation in R M R in mice. Both o f these estimates are considerably below the 52% o f variation in R M R explained by heart, liver, intestine, and kidney masses o f among inbred strains o f mice (Konarzewski and Diamond 1995). However, such mice strains have been selectively bred to maximize differences. Individual swallows with a relatively high resting V O 2 had a relatively high Het. Although avian red blood cells are nucleated and undergo oxidative metabolism, I feel the relationship between Het and V O 2 is not causal.  77  Costs and benefits of a high RMR Traditionally, physiologists have considered much o f the variation surrounding intraspecific regression lines as noise (Bennett 1987). Such variation can, however, be acted on by natural selection, and can potentially impact on fitness (Jayne and Bennett 1990).  For  example, in pied flycatchers (Ficedula hypoleuca), great tits (Parus major, Roskaft et al. 1986) and w i l l o w tits (P. montanus • ll0gstad 1987) the most dominant individuals also have x  the highest resting metabolic rates (but see Vezina and Thomas 2000). Similar relationships have also been demonstrated for B M R i n dippers (Cinclus cinclus; Bryant and Newton 1994). If a relatively high resting V 0 is associated with increased fitness (through for 2  example, increased dominance), it is unclear how such physiological variability is maintained in the population. One possibility is that it is energetically expensive to maintain the organs (e.g., kidney) associated with a high V O 2 (Konarzewski and Diamond 1994; 1995). Although individuals with relatively high R M R s may have potential fitness benefits, during extended food shortage high levels of maintenance metabolism likely carry associated costs.  Conclusion Within a single population of tree swallows I detected considerable inter-individual variation in resting V O 2 and in the size o f various internal organs and tissues. Inter-individual differences in the masses o f the kidney and small intestine explained 2 1 % o f the variation in resting V O 2 . Although individuals with relatively high V 0 s had relatively large, 2  metabolically active kidneys, they had relatively small intestines. Mechanistically, a negative relationship with intestine mass is difficult to interpret. Significant inter-annual differences were found for most tissues, although not for resting V O 2 . The cause o f inter-annual variation in organ size is unknown, although it is hypothesized that it is a response to variation in food availability.  78  CHAPTER 4 E F F E C T OF B R O O D SIZE M A N I P U L A T I O N O N OFFSPRING P H Y S I O L O G Y : A N E X P E R I M E N T W I T H PASSERINE BIRDS  PREFACE This chapter is adapted from a paper by G . P . Burness, G . B . M c C l e l l a n d , S.L. Wardrop, and P.W. Hochachka, which has been accepted for publication in the Journal of Experimental Biology. Measurements o f resting metabolic rate were performed with the assistance o f G . B . McClelland; S.L. Wardrop assisted with field work. I generated the research question, collected the majority o f the data, and was responsible for all aspects o f data analyses and presentation.  INTRODUCTION The environment experienced during avian and mammalian ontogeny can have important morphological, behavioural and life history consequences.  Individuals raised under  poor conditions often exhibit smaller structural size, are lighter in mass at independence, and have decreased over winter survival and recruitment rates (e.g., Perrins 1965, Boag 1987, Richner 1989, Dijkstra et al. 1990, Koskela, 1998). A s breeding adults, these individuals may have reduced fecundity (e.g., smaller clutches) or decreased attractiveness o f secondary sexual characters (Haywood and Perrins 1992, Schluter and Gustaffson 1993, Gustaffson et al. 1995, de Kogel and Prijs 1996). Implicit in the above studies is that variation in the quality o f the rearing environment has an effect on the "physiological condition" o f individuals reared in that environment. Differences in condition at fledging are then manifested through survival probabilities and variation in the adult phenotype (Perrins 1965, Haywood and Perrins 1992, Schluter and Gustaffson 1993, de Kogel and Prijs 1996). Despite the prominent role that the concept o f  79  condition plays in many evolutionary studies (e.g., M c N a m a r a and Houston 1992), the physiological and biochemical characters that define it remain relatively unexplored. I used the tree swallow (Tachycineta bicolor) to investigate whether the brood size experienced during ontogeny would affect the physiology and metabolism o f nestlings shortly before fledging. Brood size was experimentally manipulated and the resting metabolic rate o f individuals reared in each brood was determined. To look for potential trade-offs in energy allocation near fledging, total lipid mass, and the masses o f skeletal muscle and internal organs were measured. A s I hypothesized a priori that differences i n condition may also be linked to differences i n blood oxygen carrying capacity, I measured blood haemoglobin concentration and haematocrit. Finally, the activities o f the following key metabolic enzymes were measured i n various tissues: (i) citrate synthase (an index o f aerobic capacity), (ii) pyruvate kinase (an index o f glycolytic capacity), (iii) 3-hydroxyacyl C o A dehydrogenase (an index of capacity for fatty acid catabolism), (iv) lactate dehydrogenase (an index o f capacity for anaerobic glycolysis). Although some characters responded to brood manipulation, overall, the rearing environment appears to play a relatively minor role i n determining the physiological and biochemical phenotype of individuals near fledging.  MATERIALS AND METHODS Study site and species The field component o f this study was performed in May-June 1996 and 1998 at the Creston Valley Wildlife Area, near Creston, British Columbia, Canada. Beginning in early M a y checks o f nest boxes began in search for signs o f breeding by tree swallows. Females in this population lay between 1 and 8 eggs with a modal clutch o f 6 (Chapter 3). Clutch completion is followed by 12-14 days o f incubation. After hatching, nestlings follow a sigmoidal growth curve, reaching maximum mass at ca. day 12 (hatch day = day 1). This is followed by a weight recession which continues until fledging at 18-22 days o f age (for a  80  review, see Robertson et al. 1992). Nestlings cannot be handled beyond day 16 due to risk o f premature fledging (De Steven 1980).  Manipulation  of nestling  environment  In both years o f study, manipulations consisted o f either increasing or decreasing the number o f nestlings in a brood. One nestling was either added to or removed from a nest on day 4 (1996) or day 6 (1998). A l l nestlings were banded, and the growth o f members o f the brood was followed until day 16. I did not use a control group as I was interested only i n demonstrating an effect o f manipulation and not in predicting a directionality o f the response (i.e., an increase versus a decrease in a given character). In 1996 I used only nests in which females had laid 6 eggs; due to a shortage o f suitable nests i n 1998,1 used both 5- and 6-egg nests. It is unknown i f differences in protocol between years w i l l affect the measurements, consequently, the term "year" was included in all statistical analyses.  Morphometries A l l nestlings in experimental broods were weighed (± 0.5 g) on either day 4 (1996) or day 6 (1998) and then again on days 8, 12, and 16. A t day 16 the nestling with the mass closest to the average for a given brood had the following additional measurements taken: tarsus length, total body length, middle toe and keel lengths, bill length, depth and width. In addition, the length o f the ninth primary feather (plucked) was measured, as its length at 16 days o f age correlates with age of nest departure (De Steven 1980). To minimize interobserver variability, the same individual performed all measurements o f a given character.  Resting V02 and VCO2 In 1998, on day 6, 8, 12, and 16 the nestling o f average mass from each brood was transported to the field lab for determination o f resting metabolic rate ( R M R ) . A s the same individual was never used on two consecutive days, a repeated measures experimental design  81 was not possible. Rates o f oxygen consumption (VO2) and carbon dioxide production (VCO2) were obtained using a flow-through respirometry system (Sable Systems T R - 3 , Henderson, N V ) , consisting o f an Ametek S-3 A oxygen analyzer and a L i - C o r LI-6251 carbon dioxide analyzer. Within approximately 30 minutes o f being removed from the nest, nestlings were placed in a black Plexiglas metabolic chamber in the field lab. The volume o f the chamber was either 500 m L or 1000 m L depending on the size/age of the nestling. A i r inlet and outlet of the metabolic chamber consisted o f brass tubes, extending from the top to the bottom o f the chamber, and perforated along their length to maximize mixing o f air within the chamber. The chamber was placed in a temperature controlled cabinet. The temperature inside the chamber was maintained at 32.1-33.0°C, and was continuously monitored using a thermocouple placed in the air outlet o f the metabolism chamber. Water- and carbon dioxide-free air was drawn through the metabolic chamber at 200 500 m L / m i n . using a combination pump/mass flow meter (Sable Systems TR-SS1). A subsample o f out-flowing air was drawn through the analyzers at 150-200 m L / m i n after being dried with magnesium perchlorate (Mg(C104)2). Measurements were taken for 60 minutes and the lowest 5 minutes o f recording i n the last 30 minutes was used in calculations o f resting oxygen consumption rate. The system was found to be accurate to ± 1% (N=3) by burning methanol. Frequently, two nestlings were brought to the lab simultaneously. In these cases, to minimize metabolic variation due to differences in the degree o f post-absorptiveness, one individual was placed in a metabolic chamber for 60 minutes (as above), and the other was fed ca. 0.4 g o f moistened cat food (Vineland, Abottsford, B C ) . The fed individual was then placed in a duplicate chamber, and both chambers were then placed in the temperature controlled cabinet. For the fasted individual, the time elapsed between last possible parental feeding and first measurement o f V O 2 and V C 0  2  was 60 minutes. For the individual fed in  82  the lab, measurement was 90 minutes post feeding. There was no systematic bias between the two treatments. Following metabolic trials, birds were removed from the chamber, re-weighed and i f 6, 8, or 12 days old, returned to their nest. Nestlings that were 16 days old were retained for additional measurements.  Calculations  Values for  V C 0  V 0  2  2  V O 2  and  =  V E(  F E C 0  - FICO2)  (1)  =  V E CFICh - F E C M - V C O ( F I C M  (2)  V C O 2  2  were calculated by Datacan 5.1 using the following equations:  I-FIO2  Where V E is the flow rate o f air leaving the metabolic chamber corrected for standard temperature and pressure.  F I C 0  2  and F I O 2 are the fractional concentrations o f carbon  dioxide and oxygen entering the chamber. F E C O 2 and F E O 2 are the fractional concentrations of carbon dioxide and oxygen leaving the chamber.  Blood  parameters Following metabolism trials, a 100-200 u L blood sample was collected from each 16  day old nestling into heparinized microcapillary tubes. Haematocrit and haemoglobin were determined as reported previously (Chapter 2). The number o f replicates for each character was determined by the size o f the blood sample and ranged from one to three (which were averaged).  83  Carcass  analyses  I sacrificed Day 16 nestlings immediately after blood sampling (following the guidelines o f the Canadian Committee on A n i m a l Care). A sample (ca. 150 mg) o f the right pectoralis major and liver were removed from each bird (within 1-2 min. o f death) and immediately frozen in a liquid N2 charged dry shipper. These samples were later transferred to liquid N2 for 3 month storage. Nestling were dissected, and the organs and carcass were stored as described previously for adults (Chapter 2). Carcasses were weighed ( ± 0 . 0 0 0 l g ) upon removal from the freezer and plucked o f all feathers. A l l muscles on the tibiotarsus and femur were then removed from one side o f the bird, rinsed with 0.9% N a C l , blotted dry and weighed (± 0.000lg). To calculate total leg muscle mass, values were multiplied by 2. Wet weights were determined for other organs and tissues. The empty mass o f the small intestine and gizzard were determined as described previously (Chapter 2). In preparation for fat extraction, all organs and tissues (with the exception o f the heart, liver and kidney) were freeze dried to constant mass. Carcasses were dried to constant mass in a 70°C oven. A l l dried samples were then fat extracted as in Chapter 2.  Enzyme assays Sub-samples (ca. 150 mg) o f the pectoralis major and liver, and the ventricles o f the heart were prepared for enzyme assays following the protocol in Chapter 2. Homogenates were stored at -80°C until assays were conducted (maximum 1 month). A s an index o f capacity for flux through various metabolic pathways I measured the maximum catalytic activity ( V a x ) o f key metabolic enzymes under optimal conditions. A l l m  assays were performed on a 96-well Thermomax microplate reader (Molecular Devices Corp., Sunnyvale, C A ) .  In all assays, un-centrifuged homogenates were used to avoid potential loss  in the pellet. Each reaction was replicated i n 5 wells. The wells with the highest and lowest activity were omitted and the remaining 3 values were averaged. Preliminary experiments  84  confirmed that all substrates and cofactors were saturating but not inhibitory. Initially, control wells (containing no substrate) were run simultaneously with all reactions. The control rates for pyruvate kinase and lactate dehydrogenase represented <2% o f total activity and in subsequent assays were omitted. Control wells were included for all 3-hydroxyacylC o A dehydrogenase and citrate synthase assays. W i t h the exception o f citrate synthase, all assays were at p H 7.0 and 340 nm. Citrate synthase was assayed at p H 8.0, 412 nm. A l l reactions were at 40°C. Activities are expressed as international units (umoles substrate converted to product per minute) per gram wet weight o f tissue. Although the enzymes assayed were the same as described previously (Chapter 2), some o f the assay conditions differed. Assays were performed as follows. Citrate synthase ( E C 4.1.3.7; CS): 5 0 m M Tris buffer, 0.05% Triton X - 1 0 0 , 0 . 2 m M D T N B , 0.12mM acetyl C o A , 0 . 5 m M oxaloacetate (omitted from the control well). 3-hydroxyacyl C o A dehydrogenase ( E C 1.1.1.35; H O A D ) : 5 0 m M Imidazole, 0.15mM N A D H , l O m M B-mercaptoethanol, 1.0 m M N a C N , acetoacetyl C o A (0.1 m M for the pectoralis and ventricle, 0.05mM for the liver; omitted from the control well).  Pyruvate kinase ( E C 2.7.1.40; P K ) : 5 0 m M Imidazole, 0 . 1 5 m M N A D H , l O m M fi-  mercaptoethanol, L O m M N a C N , 5 . 0 m M A D P , lOOmM K C 1 , l O m M M g C l 2 , 5 m M P E P , l O u M fructose 1,6-bisphosphate, excess lactate dehydrogenase (5U/mL); assayed in the ventricles and pectoralis only. Lactate dehydrogenase ( E C 1.1.1.27; L D H ) : 2 0 m M Imidazole, 0 . 1 5 m M N A D H , 2 m M Pyruvate, l O m M B-mercaptoethanol, 1.0 m M N a C N ; assayed i n the ventricles and pectoralis only.  Statistical analyses Many physiological variables scale allometrically with the mass o f an animal. In my study, the effect o f brood manipulation on body mass was o f interest. Consequently, rather than controlling for body mass, in most analyses I instead controlled for structural size. To generate an index o f size, I performed a principal component analysis ( P C A ) on the  85  correlation matrix o f 7 external morphological variables (tarsus length, total body length, middle toe and keel lengths, bill length, depth and width). Loadings were positive for all variables and ranged from 0.24-0.47, with a corresponding eigenvalue o f 3.13.  The first  principal component ( P C I ) accounted for 44.7% of the total original variance. I used the scores along P C I as a measure o f body size (e.g., Alisaukas and Ankney 1987) with positive values representing individuals that were larger than average body size and negative values representing individuals that were smaller than average. The effects o f treatment (brood manipulation) and year on phenotypic variation o f 16 day old nestlings were explored using a 2-way analysis o f covariance ( A N C O V A ) with body size included as a covariate. Initially, all interaction terms were included as additional covariates and i f not significant were excluded. Further analyses were then performed including only the covariate and main effects (treatment and year). I used a liberal P<0.15 for inclusion o f interaction terms; for main effects significance was claimed at P<0.05. Unless otherwise noted, all means are least squares means ± 1 S . E . M . A l l analyses were performed using J M P statistical software ( S A S Institute Inc.).  RESULTS Growth and metabolism of nestlings To determine the impact o f brood manipulation on nestling growth I averaged the mass o f all individuals within a brood. O n the day o f manipulation (Day 4 or 6) there was no difference between treatments in the mass o f the average nestling (P>0.50, F i g 4.1). B y 12 days o f age, however, nestlings in the Reduced broods were significantly heavier than those in Enlarged broods (Fi 29=7.963, P=0.009); this difference was maintained at 16 days )  ( F i = 8 . 8 5 7 , P-0.006, Fig. 4.1). )29  86  m  ^  °  m  i u i—'—i—<—i—<—i—<—i—'—i—'—i—i—i—*— 2  4  6  8  10  12  14  16  18  Nestling age (days)  Fig. 4.1  (A) Body mass, and (B) body mass adjusted-resting oxygen consumption rate o f tree swallow nestlings, as a function o f age and treatment. Enlarged broods (o), Reduced broods (•). Sample sizes (Enlarged, Reduced), Panel A : Day 4 (9, 6), Day 6 (8, 8), Days 8, 12, and 16 (17, 15). Panel B : Day 6 (6, 4), Day 8, (8, 8), Day 12 (8, 8), Day 16 (7, 7). Values are least squares means ± 1 S . E . M . Asterisks indicate that treatments differed significantly from each other (P<0.01).  87  I measured the resting V O 2 and V C O 2 of the nestling o f average mass from each brood at 6, 8, 12 and 16 days of age. Different nestlings were used each day. There was no effect of manipulation on the R E R (P>0.15, Table 4.1). I could not generate an index o f structural size for nestlings less than 16 days old and instead adjusted resting V O 2 for body mass. Body mass-adjusted V O 2 at 6, 8, and 12 days o f age did not differ between treatments (P>0.10, Fig. 4.1). B y 16 days o f age, nestlings in reduced broods had marginally higher mass-adjusted V O 2 (F\ \i=3.548, P=0.086). I re-analyzed the V O 2 o f the 16 day old t  nestlings with P C I (rather than mass) included as a covariate. Individuals in Reduced broods had a 15% greater body-size adjusted V O 2 than those in Enlarged broods ( F j i i=6.108, 5  P=0.031).  Morphology and physiology of 16 day old nestlings Brood manipulation had no effect on nestling structural size ( P C I , P=0.500), nor on the length o f the ninth primary feather (P=0.681) at 16 days o f age.  Individuals from the  Reduced broods were heavier than those from Enlarged broods after controlling for size (F 8=8.450, P=0.007). 1;2  In 1998, 16-day nestlings were structurally larger (Fi a=64.517, j2  P<0.001) and had longer primaries (Fi 29=9.018, P=0.006) than in 1996, but were no heavier )  (P=0.484). To explore the basis of body mass differences resulting from brood manipulation (above), I measured total lipid mass. P C I was included as a covariate, with year and treatment as main effects.  Nestlings from Reduced broods had 19% greater lipid mass at day  16 than those from Enlarged broods (Fi 28 5-623, P=0.025, F i g . 4.2). There was no =  j  difference between years (P=0.486).  88  Table 4.1  Rates o f resting oxygen consumption and carbon dioxide production o f tree swallow nestlings.  Age  N  Mass(g)  VC^fmLlr ) 1  VOMmLh- ) 1  RER  6 Days R  (4)  11.40 ± 0.32  26.89 ± 3.97  19.82 ± 2.31  0.75 ± 0.03  E  (5)  10.21 ± 0.34  20.39 ± 2.37  16.34 ±  1.24  0.80 ± 0.04  R  (8)  16.40 ± 0.40  41.46 ± 2.73  31.72 ± 2.19  0.77 ± 0.01  E  (8)  14.84 ± 1.10  38.19 ± 4.63  28.49 ±  3.30  0.76 ± 0.02  R  (8)  21.36 ± 0.31  65.61 ± 2.63  47.05 ± 2.12  0.72 ± 0.01  E  (8)  19.79 ± 0.34  52.64 ± 1.93  39.48 ±  1.88  0.75 ± 0.04  R  (7)  21.19 ± 0.61  65.81 ± 2.73  45.97 ±  1.56  0.70 ± 0.02  E  (7)  20.05 ± 0.31  56.52 ± 2.01  39.68 ±  1.27  0.71 ± 0.03  8 Days  12 Days  16 Days  Values are means ± 1 S . E . M . R, broods reduced by a single individual; E , broods enlarged by a single individual. R E R , respiratory exchange ratio  89  1.75 i  Fig. 4.2  L i p i d mass o f 16 day old tree swallow nestlings, adjusted for body size ( P C I ; see text), as a function of brood manipulation. Reduced broods (black bar), Enlarged broods (white bar). Least squares means + 1 S . E . M . Enlarged, N=17; Reduced N=15. Asterisks indicate that treatments differed significantly from each other (PO.05).  90  After controlling for structural size, individuals in the Reduced treatment had heavier organs than those in the Enlarged treatment. However, only the mass o f the gizzard showed a significant difference ( P O . 0 5 , Table 4.2). There was no effect o f year on the wet mass o f any organ except the intestine (P<0.01). There was no effect o f brood manipulation on the wet masses o f either the pectoral or leg muscles (P>0.05, Table 4.3). The water content o f a muscle (total water/lipid-free wet mass o f tissue) decreases with chronological age (Konarzewski 1988), and is a useful index of muscle maturation (Ricklefs and Webb 1985). There was no significant effect o f treatment on water fraction o f either the pectoral or leg muscles (P>0.10, Table 4.3), suggesting that at 16 days o f age individuals from the two treatments were o f similar degrees o f functional maturity. Significant year effects (Table 4.3) may be due to desiccation in the freezer, consequently I assign them no particular functional significance. A s an index o f blood oxygen carrying capacity I measured the Het and H b content o f the blood. The average Het was 41.7 % (S.D. = 4.50, N=32) and ranged from 27.3 - 51.0 %; the average H b concentration was 13.51 g d L " (S.D =2.063, N=31) and ranged from 6.7 -16.3 1  g d L " . There were no significant effect of treatment or year on either character (P>0.25). 1  Enzyme activities There was little effect o f brood manipulation on maximum enzyme activities. Individuals from Reduced broods had significantly higher H O A D activity in the heart ( P O . 0 1 , Table 4.4), suggesting an increased capacity for fatty acid oxidation. N o other enzyme changed significantly between treatments.  9 1  Table 4.2  Effect o f brood manipulation on organ masses o f 16 day old tree swallows.  Treatment Enlarged  P-value Reduced  Treatment  Year  Heart (g)  0.25 ±  0.008  0.26 ±  0.008  0.476  0.498  Liver (g)  0.90 ±  0.030  0.98 ±  0.032  0.074  0.990  Kidney (g)  0.25 ±  0.011  0.27 ±  0.012  0.149  0.927  Gizzard (g)  0.57 ±  0.017  0.62 ±  0.018  0.043  0.472  Intestine (g)  0.75 ±  0.028  0.83 ±  0.030  0.079  0.006  Values are least squares means ± 1 S . E . M . from two-way A N C O V A with body size as a covariate ( P C I ; see text), interaction terms were not significant (P>0.15). Samples sizes: Enlarged, N=17; Reduced, N=15. Significant P-values are underlined.  92  Table 4.3  Effect o f brood manipulation on muscle mass and water fraction o f 16 day old tree swallows.  P-value  Treatment Variable Pectoralis (g) Water (g/g)  Leg (g) Water (g/g)  Enlarged  Reduced  Treatment  Year  ±  0.065  2.15 ±  0.069  0.092  0.315  0.78 ±  0.003  0.78 ±  0.003  0.907  0.007  0.66 ±  0.020  0.62 ±  0.021  0.181  0.946  0.78 ±  0.006  0.77 ±  0.007  0.154  <0.001  1.98  Values for pectoral and leg muscle are least squares means ± 1 S . E . M . from two way A N C O V A with body size as a covariate ( P C I ; see text). Values for water fraction are least squares means ± 1 S . E . M . from two-way A N O V A . A l l interactions were non-significant (P>0.15). Sample sizes: Enlarged, N=17; Reduced, N=15. Significant P-values are underlined.  93  Table 4.4  M a x i m u m enzyme activities from tissues o f 16 day old tree swallows.  Treatment Enlarged  Statistic Reduced  F  P-value  Heart CS  132.9  ± 11.6  131.4  ±  11.6  0.008  0.930  PK  146.8  ± 13.6  173.9  ±  13.6  1.937  0.187  LDH  61.5  ±  5.4  74.9  ±  5.4  3.019  0.106  HOAD  14.3  ±  1.1  19.1  ±  1.1  9.502  0.009  CS  136.3  ±  12.6  137.6  ±  12.6  0.006  0.941  PK  405.7  ±  20.8  387.1  ±  20.8  0.394  0.541  LDH  406.7  ±  24.6  447.0  ±  24.6  1.304  0.274  HOAD  10.3  ±  1.0  9.1  ±  1.0  0.867  0.369  CS  11.3  ±  0.7  9.7  ±  0.7  2.757  0.121  HOAD  36.1  ±  1.3  35.5  ±  1.4  1.209  0.293  Pectoralis  Liver  £  Values are least squares means ± 1 S . E . M . from one-way A N C O V A with body size as a covariate ( P C I ; see text).For enzyme names, see text. Significant body size*treatment interaction (P<0.15). Enzyme activity is in U/gram tissue (umoles substrate converted to product per minute). N=8 for each treatment. Significant P-values are underlined. a  94  DISCUSSION The brood size o f tree swallows was manipulated to determine i f the environment experienced during ontogeny would affect the physiology and biochemistry of nestlings shortly before they were to fledge. A s skeletal characters are known to respond to environmental variation during development (Lindstrom, 1999, and references within), a minimal response at the physiological and biochemical level was surprising. A lack o f variation in basal measures suggests that physiological and biochemical development may be relatively invariant except perhaps, under extreme conditions (e.g., Schew and Ricklefs 1998). Nonetheless, variation in the rearing environment did affect some characters. A decrease in the number o f nestlings i n a brood resulted in increased body mass, total lipid mass, gizzard mass, body size-adjusted V O 2 , and the activity o f H O A D in the heart.  Morphological response Brood manipulation did not effect the structural size o f 16 day old tree swallows (PCI scores). This is consistent with previous studies o f this species (Wiggins 1990, Wheelwright et al. 1991). It could be argued that the addition or subtraction o f a single nestling may have been insufficient to elicit a response. This is unlikely for two reasons. First, addition o f two nestlings (rather than one) exceeds the provisioning capacities o f the parents i n this population and frequently results i n brood reduction ( G P B unpublished, data). Second, at 16 days o f age, individuals in Enlarged broods had smaller lipid stores and a lower body mass than those in Reduced broods, suggesting they were resource limited. Previous studies have shown that under food restriction, other altricial species can maintain skeletal growth through catabolism o f body tissues and lipid. Skeletal growth drops significantly, however, when energy reserves are depleted (Schew and Ricklefs 1998). Although I detected few differences in organ and muscle mass as a consequence o f treatment, individuals in Enlarged broods had significantly lower lipid stores than those in Reduced  95  broods. Rather than utilize stored energy to maintain growth, in the present study it is more likely that nestlings first met energetic requirements for growth and maturation and then stored the remaining energy as lipid. A t 16 days o f age nestlings were structurally larger in 1998 than in 1996. Systematic measurement error is unlikely as the same individuals (the same field assistant or myself) performed all measurements in both years.  Interannual differences in morphology were  more likely due to variation in weather conditions or food availability, as has been suggested previously for adults (Chapters 2 and 3).  Phenotypic variation and developmental  plasticity  During periods o f reduced nutrition, nestlings o f some species delay tissue maturation and feather growth, or increase the duration o f the nestling period (Schew and Ricklefs 1998). If this occurred in the present study, the phenotypic values o f characters at 16 days o f age may have little similarity to the values o f those same characters a few days later when individuals actually left the nest. I argue that nestlings probably did not exhibit an adaptive suspension o f maturation nor an extension o f the nestling period. There is a well established negative relationship between the hydration state o f a muscle and its mature function (e.g., Ricklefs and Webb 1985). Water content, normalized to lean dry mass (an index o f protein content), typically decreases with a nestling's chronological age (Konarzewski 1988). If nestlings in Enlarged broods were able to arrest their developmental program in response to unfavourable conditions, their muscles would likely have had an increased water content (less mature muscles) than nestlings o f similar age from Reduced broods. This was not observed (Table 4.3). Although exact dates o f nest departure were not determined, no difference in the duration o f the nestling period has been reported previously under similar brood manipulations (Wheelwright et al. 1991). In addition, the duration o f the nestling period is inversely related to the length of the ninth primary feather at 16 days o f age (De Steven  96  1980); the length o f the primaries did not differ between treatments. Taken together, these data suggest that nestlings from each o f the two treatments would have fledged at similar ages, with those from Enlarged broods being i n poorer condition for a given structural size.  Heat increment of feeding Following ingestion o f a meal there is an unavoidable increase in metabolic rate, the heat increment of feeding (HIF; also called specific dynamic action). Conclusions drawn from my measurements o f resting V O 2 rely on the assumption that when nestlings in the two treatments were measured, they were o f a similar absorptive state. T w o lines o f evidence indicate that this was the case: (1) A s the magnitude and duration o f the H I F increase linearly with increasing meal mass (Chappell et al 1997), I compared the mass o f food in the small intestine o f day 16 nestlings between treatments.  Individuals i n Reduced broods did not  have more food in their intestines (P>0.29): Enlarged broods = 0.15 g (SD=0.05, N=7), Reduced broods = 0.12 g (SD=0.06, N=7). There was also no increase in resting V O 2 with increasing mass o f intestine contents (P>0.69). Finally, I performed an A N C O V A with brood manipulation as a main effect and both intestine contents and P C I as covariates. Nestlings i n the Reduced broods still had a significantly higher V O 2 than individuals in the Enlarged broods (Fi io=9.146, P=0.013); neither P C I , nor intestine contents were significant 3  covariates (P>0.10). (2) In other passerines, as nestlings get older the duration o f the HIF decreases (Chappell et al 1997). If differences in V O 2 between treatments at 16 days o f age were due primarily to a H I F , such differences should have been detectable on day 8. Contrary to the pattern seen on day 16, day 8 nestlings in the Reduced treatment had on average a lower mass-adjusted V O 2 than individuals in the Enlarged broods (although not significantly, Fig. 4.1). Taken together these arguments indicate that i f my measurements were affected by a H I F , both treatments were affected equally.  97  Phenotypic flexibility of organ size The only organ that differed between brood sizes was the gizzard, being greater in individuals in the Reduced broods. A s individuals in the Reduced broods were presumably receiving more food, variation in size o f the muscular gizzard may be a result o f an increased work load, analogous to a training effect (Piersma et al. 1993). For example, the gizzard o f Japanese quail {Coturnix japonica) demonstrates repeated up- and down-regulation o f size coincident with the fibre content o f the diet (Stark 1999). Captive red knots (Calidris canutus) also display phenotypic flexibility, and decrease the size o f their gizzards by ca. 75% upon switching from small bivalves to soft food pellets (Dietz et al. 1999). Morphological responses to changes in diet are rapid, and measurable within 24-48 hours o f diet switching (Stark 1999). Interestingly, following diet switching experiments, Stark (1999) reported that the gizzards o f experimental quail never returned to the same size as unchallenged controls. Whether differences I observed between nestling swallows are fixed is unknown.  Implications for post-fledging  survival  There is a well established positive relationship between body mass at fledging and the probability o f subsequent recruitment (e.g., Perrins 1965, Tinbergen and Boerlijst 1990, Magrath 1991, Both et al. 1999). In my study, brood manipulation resulted in significant variation in body mass just prior to fledging which likely had fitness consequences. The mechanism underlying the relationship between mass at fledging and recruitment is unclear. Body mass may represent a general indicator of health; for example, lipid stores may allow for survival during periods o f adverse weather or reduced food intake. Although heavier individuals have greater lipid stores, they are often structurally larger. Garnett (1981) hypothesized that this may enhance survival by allowing large individuals to dominate smaller ones i n competitive interactions. Recent work on great tits has shown that  9 8  individuals that are heavy when compared to others i n the population have an increased probability o f subsequent recruitment (Both et al. 1999). Apart from structural size and large lipid stores, how physiological factors may influence the probability o f survival and recruitment is unclear. A n elevated size (or mass) adjusted V O 2 , although i n itself presumably detrimental, may be linked to an elevated V02max  (aerobic capacity model, Bennett and Ruben 1979). Support for a correlation  between resting V O 2 and V 0  2 m a  x is, however, equivocal (Hayes and Garland 1995). For  example, i n house sparrows (Passer domesticus) resting V O 2 is correlated with V 0 2 m a x i  n  juveniles but not adults (Chappell et al. 1999). A n elevated resting V O 2 has also been linked with dominance status (Rj0skaft et al. 1986, Bryant and Newton, 1994), although not in all species (Hammond et al. 2000, Vezina and Thomas 2000). E v e n with an estimate o f aerobic capacity or dominance, the consequence o f an elevated resting V O 2 in tree swallow fledglings would be speculative without estimates o f differential survivorship (e.g., Hayes and O'Connor 1999). The only enzyme showing a clear response to brood manipulation was H O A D . Individuals i n the Reduced broods had significantly higher cardiac H O A D activities, suggesting an increased capacity for oxidation o f fatty acids. Mechanistically this may be simply coupled to their elevated fat stores. Marsh (1981) found a correlation between H O A D activity i n the pectoral muscle and carcass fat levels in birds preparing for migration. However, during pre-migratory fattening, semipalmated sandpipers (Calidris pusUla) increase their capacity for fatty acid oxidation i n skeletal muscle, but not the heart (Driedzic et al. 1993). I could not detect a correlation between carcass fat levels and cardiac H O A D activity (P>0.50). Although the amount o f fat in the diet can affect enzyme activities i n both skeletal (Fisher et al. 1983) and cardiac muscle (Power and Newsholme 1997) nestlings presumably received diets o f similar fat content. A t present the mechanisms underlying elevated heart H O A D activities are unclear.  99  Conclusion Manipulation o f brood size early in ontogeny had minimal affects on the physiology and biochemistry o f tree swallows shortly before fledging. A lack o f response suggests that these characters may be relatively insensitive to environmental variation. Consequently, the early rearing environment may play a relatively small role in determining variation in the adult physiological or biochemical phenotype. Some characters did respond to environmental variation, including lipid levels, cardiac H O A D activity, and resting V O 2 . which variation in these characters influences survivorship is unknown.  The mechanism by  100  CHAPTER 5 GENERAL CONCLUSIONS  This is the first study to identify both physiological causes and ecological consequences of inter-individual variation in the aerobic metabolism of free-living birds. In this final chapter, I shall summarize the main findings of each o f the 3 research chapters, present some general conclusions, and suggest areas of future research.  Research summary Chapter 2. I used the doubly labelled water technique to estimate the energy expenditure of male and female tree swallows provisioning various natural brood sizes. I addressed two primary questions: (i) how does sustained metabolic rate affect Darwinian fitness, and (ii) what are the physiological and biochemical correlates of S u s M R ? Although there was no correlation between natural brood size and parental S u s M R , nestlings in large natural broods grew at the same rate as those in smaller broods. One interpretation o f this result is that adults with large natural broods were energetically more efficient than those rearing smaller natural broods.  After statistically controlling for brood size, male and female parental  S u s M R increased with increasing nestling mass, and in females, with nestling growth over the previous 4 days (in one year only). Thus S u s M R was positively related to a surrogate of Darwinian fitness. In one of two years, individuals with relatively high S u s M R had relatively large intestines. This suggests that a high digestive capacity was necessary to attain a high SusMR. This is the first demonstration of such a relationship in the field. It also suggests that capacity to maintain a high S u s M R likely entails a cost in terms of increased resting metabolism.  101  Chapter 3. I measured the resting oxygen consumption rate and body composition o f breeding tree swallows in two breeding seasons. I asked two primary questions: (i) what is the relationship between inter-individual variation in organ size and resting oxygen consumption rate ( V O 2 ) , and (ii) do V O 2 and body composition display inter-annual variation? Individuals with relatively high resting oxygen consumption rates for their body mass had relatively heavy kidneys. This was the first intra-specific study o f birds to demonstrate such a relationship (Burness et al. 1998). Contrary to previous (Konarzewski and Diamond 1995) and subsequent studies (Bech and 0stnes 1999, Chappell et al. 1999) o f other species, the size o f the small intestine did not correlate with resting V O 2 . Although phenotypic flexibility in organ size is increasingly well recognized (reviewed in Piersma and Lindstrom 1997), this study was the first to demonstrate large scale inter-annual variation. Despite inter-annual variation in the sizes of organs o f the abdominal cavity, resting V O 2 did not vary between years.  Chapter 4: Numerous studies have investigated the role o f the rearing environment in morphological development (structural size or body mass, Lindstrom 1999). In chapter 4,1 performed the first study to ask how the environment during early development affects individual variation in physiology and biochemistry. To mimic "good" and "bad" environments, I either reduced or enlarged broods by one nestling. Within a few days o f fledging, nestlings in "good" environments were significantly heavier, had greater mass of lipid, increased cardiac H O A D levels, heavier gizzards and higher size-specific resting V O 2 than individuals raised under "poor" conditions. Perhaps more important than characters that differed as a consequence o f treatment, are the number that did not. A lack o f difference between treatments in the size o f most organs suggests that development o f many physiological and biochemical traits is relatively insensitive to this form o f environmental variation.  102  Conclusions Tree swallows rearing different sized broods did not differ in their energy expenditures. This suggests that within a single population o f aerial insectivores there likely exists variation in foraging efficiency. Such variation has also been demonstrated in Eurasian kestrels; males with large natural broods had higher hunting yields per unit time, but similar energy expenditures than did males rearing smaller broods. In kestrels, however, differences in hunting yield were due to differences in territory quality (Masman et al. 1989). In contrast to kestrels, tree swallows are aerial insectivores and do not hold feeding territories. A s individual swallows rearing different sized broods did not differ in the physiological or biochemical traits that I measured, I propose that inter-individual differences in efficiency resulted from variation in foraging strategies. Although a positive relationship between S u s M R and frequency o f nest site visitations (an index o f feeding frequency) has been shown previously in tree swallows (Williams 1988), feeding frequency is an incomplete measure o f parental provisioning. In addition to feeding frequency, aerial insectivores may vary bolus size or prey composition (e.g., Wright et al. 1998). These behavioural strategies are not detectable through studies o f energy expenditure or frequency o f nest site visitations. Studies o f foraging behaviour in this same population suggest that individuals with different natural brood sizes vary i n their foraging decisions and capacity to respond to energetic challenges (R.C. Ydenberg, pers. comm.). These behavioural studies complement my physiological ones, and support the contention that individuals rearing different sized broods differ in quality, independent of their energy expenditure. Despite a lack o f relationship between S u s M R and the number o f nestlings an individual was rearing, among individuals o f the same brood size, S u s M R was related to nestling mass. This suggests that increased parental effort resulted in an increase in nestling growth. Because relatively heavy fledglings have greater chances o f over-winter survival and recruitment (Both et al. 1999) variation in growth rates or nestling body mass probably have  103  fitness consequences. Operationally, natural selection is defined as the correlation between the variation in a phenotypic trait and variation in Darwinian fitness (Garland and Carter 1994). Under this definition, S u s M R may have been under selection in one o f two years. Although measures such as nestling mass or growth rates are incomplete measures o f fitness, they provide a useful first step in analyzing evolutionary patterns o f phenotypic variation (Garland and Carter 1994). Although I consider parental energy expenditure to be a performance trait, it can also be viewed as an environmental effect that influences the nestling phenotype. The resemblance among offspring in a nest is a consequence o f the interaction between genetics and the common nutritional environment provided by parental care. Consequently, selection on the environmental component o f offspring size (e.g., Alatalo et al 1990) may result in selection on parental S u s M R as a correlated response (Moreno et al. 1997). Recent evidence indicates that measurements o f parental S u s M R are repeatable between breeding seasons, suggesting that an individual's S u s M R may retain a genetic component o f variation (Potti et al 1999). I f this is the case, S u s M R may evolve under natural selection. Additional studies are required in order to estimate the seasonal stability and heritability o f parental SusMR. Individual tree swallows with relatively high SusMRs had relatively large intestines. Although this relationship has been shown in the lab (Konarzewski and Diamond 1994), this is the first study to demonstrate such a relationship i n the field. It is well recognized that individuals w i l l undergo intestinal hypertrophy under conditions o f chronically high energy expenditure (Dykstra and Karasov 1992, Hammond and Diamond 1992, 1994, Hammond et al. 1994, Konarzewski and Diamond 1994). In my study, whether inter-individual variation in intestine mass was due to differential hypertrophy remains unknown. One o f the central theorems o f life-history theory is that there exists a trade-off between current and future reproduction (Williams 1966, Charnov and Krebs 1974). This leads to the question: i f among individuals with the same brood size a high energy expenditure is reproductively beneficial, what are the associated costs? One argument is that there is a  104  trade-off between the benefits o f attaining a high S u s M R and the energetic costs o f maintaining large internal organs (Hammond and Diamond 1997). I f such a trade-off exists I would predict that individuals with relatively large intestines for their body mass would have relatively high R M R s . Data presented in Chapter 3 failed to support this hypothesis; the only significant predictor o f an individual's R M R (estimated by measuring resting V O 2 ) was the mass o f its kidney. A lack o f correlation between R M R and intestine mass was surprising given that such correlations exist in laboratory mice (Konarzewski and Diamond 1994, 1995) and w i l d birds (Bech and 0stnes 1999, Chappell et al. 1999). I f I were to assume that a relationship between R M R and intestine mass did exist (and I simply failed to detect it), could the resultant increase in R M R reduce survival? In their study o f dippers, Cinclus cinclus, Bryant and Newton (1994) calculated that the difference i n R M R between dominant an subordinate individuals was small, constituting ~ 3 % o f the daily energy budget. Whether such differences would still be considered small under conditions o f reduced food availability is unknown. Although there is an implicit trade-off between the costs and benefits o f maintaining large internal organs (Hammond and Diamond 1997), future research should explicitly test this hypothesis. Variation in parental care affects nestling survival, condition at fledging and the probability o f subsequent recruitment (Tinbergen and Boerlijst 1990, Magrath 1991, Both et al. 1999). Few studies, however, have investigated the potential state variables that may link nestling condition with survivorship (McNamara and Houston 1992, M e r i l a and Svensson 1997). To date, the only physiological characters that have been studied with respect to variation in parental care are the size o f subcutaneous fat reserves and the immunocompetence o f nestlings (e.g., Merila 1996, Saino et al. 1999). In the present study, although a number o f physiological characters responded to brood manipulation (including, not surprisingly, lipid mass), it is perhaps more striking how few i n fact showed variation. A lack o f difference i n body composition between treatments suggests that development o f many physiological and biochemical traits is relatively insensitive to environmental variation.  105  One trait that did respond to variation was resting oxygen consumption rate. In some species R M R is related to dominance (Bryant and Newton 1994, R0skaft et al. 1986, but see Hammond et al 2000, Vezina and Thomas 2000), suggesting that a high R M R may be an indicator o f 'quality'. In blue tits, Parus caeruleus, an individual's lipid level established prior to 15 days o f age is a good predictor o f body condition up to four months later during migration (Merila and Svensson 1997). H o w long inter-individual differences established during the nestling phase are maintained is unknown but it is tempting to speculate that the rearing environment may have contributed to variation o f adult traits such as S u s M R .  Future directions: I conclude by identifying areas for future research: Inter-individual variation in capacity to elevate SusMR. I have shown that individuals rearing different sized broods have similar energy expenditures. Whether under times of increased energetic demand (e.g., adverse weather conditions) all individuals have the same capacity to elevate their S u s M R is unknown. Flexibility i n parental effort would be best explored by manipulating work load directly without changing brood demand. This could be accomplished through the clipping o f primary feathers o f adults or through the addition o f weights. Inter-annual costs of high SusMR. N o study has demonstrated an effect o f variation i n S u s M R on fecundity the following year. This could be investigated through studying clutch size variation in the year following manipulation (above). Repeatability and heritability of SusMR. To date, only a single study has shown significant statistical repeatability o f S u s M R (Potti et al. 1999). Additional studies should be establish how stable S u s M R is for other avian species, and assess its components o f variation.  106  Repeatability of body composition. Although an individual's physiology and biochemistry are phenotypically flexible, the significant repeatability o f R M R measurements (Bech et al. 1999) suggests that individuals may show consistent differences i n body composition. The increasing use o f non-invasive technologies (e.g., ultrasonography, Dietz et al. 1999) may make a repeatability study o f body composition during different life stages possible. E n v i r o n m e n t a l components of adult physiology. H o w important is the rearing environment in determining adult physiology? 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Measurement o f V O 2 , V C O 2 , and evaporative water loss with a flow through mask. J. A p p l . Physiol. 42: 120-123. Wright, J., Both, C , Cotton, P. A . and Bryant, D . 1998. Quality vs. quantity: energetics and nutritional trade-offs in parental provisioning strategies. J. A n i m . E c o l . 67: 620-634. Zimmitti, S. J. 1999. Individual variation in morphological physiological, and biochemical features associated with calling i n spring peepers (Pseudacris crucifer). Physiol. Biochem. Zool. 72: 666-676.  121  APPENDIX 1 Rates o f C O 2 production and estimated sustained metabolic rates o f male and female tree swallows rearing natural sized broods.  Nest  Year  Sex  Brood  Mass  size  (a)  a  Mass change  rC0  b 2  SusM  ta d - h  tmLd- ^ 1  (kJd-i  2A-41  1996  M  5  19.38  -0.23  2179.7  57.1  D2-31  1996  M  5  18.63  -0.27  4614.6  120.9  D2-01  1997  M  5  20.63  +0.22  3907.7  102.4  2A-16  1997  M  5  18.63  -0.26  3830.5  100.4  2A-57  1997  M  5  19.50  0.00  4185.9  109.7  2A-63  1997  M  5  18.25  -0.48  3696.4  96.9  D2-20  1997  M  6  21.13  -0.24  4201.2  110.1  D2-32  1997  M  6  19.50  d  4676.6  122.5  2A-14  1997  M  6  18.13  -0.25  3931.1  103.0  2A-56  1997  M  6  19.88  +0.25  4567.9  119.7  2A-76  1997  M  6  17.38  +0.25  4556.4  119.4  2A-77  1996  M  7  21.64  -0.62  3635.9  95.3  D2-12  1997  M  7  20.00  -0.61  3883.5  101.8  D2-44  1997  M  7  18.88  +0.74  3306.2  86.6  2A-17  1997  M  7  18.75  +0.46  4260.2  111.6  2A-21  1997  M  7  19.75  +0.46  4579.7  120.0  2A-78  1997  M  7  18.88  +0.25  5203.0  136.3  2A-59  1996  F  5  19.88  -1.24  4487.9  118.5  2A-41  1996  F  5  21.50  0.00  3804.0  99.7  D2-25  1996  F  5  18.75  2515.3  65.9  D3-15  1996  F  5  18.00  3622.3  94.9  0.00  122  2A-16  1997  F  5  17.50  0.00  4209.0  110.3  2A-57  1997  F  5  18.25  -0.51  3908.9  102.4  2A-63  1997  F  5  16.50  -0.49  4177.1  109.4  D2-01  1997  F  5  19.00  +0.47  4578.8  120.0  D2-11  1997  F  5  16.75  -0.48  3691.3  96.7  D2-21  1997  F  5  19.00  -0.46  3716.8  97.4  D2-29  1997  F  5  17.25  -0.54  4147.9  108.7  2A-37  1996  F  6  17.25  +0.42  3406.7  89.3  2A-85  1996  F  6  19.38  -0.75  4772.4  125.0  D3-09  1996  F  6  19.25  2141.5  56.1  2A-07  1997  F  6  18.00  0.00  4310.9  113.0  2A-25  1997  F  6  19.63  -0.23  4092.4  107.2  2A-33  1997  F  6  18.00  -1.06  3218.6  85.1  2A-73  1997  F  6  18.63  +0.78  3754.3  98.4  2A-40  1996  F  7  17.38  -0.24  3352.5  87.8  2A-77  1996  F  7  19.10  4551.0  119.2  D2-24  1996  F  7  16.63  -0.70  2205.6  58.3  D2-16  1996  F  7  17.50  0.00  2449.3  64.2  2A-17  1997  F  7  17.13  -1.25  3583.7  94.8  2A-21  1997  F  7  17.13  +0.23  3713.2  97.3  2A-78  1997  F  7  18.25  -1.00  4683.6  123.4  2A-80  1997  F  7  20.13  +0.25  4616.1  120.9  D3-01  1997  F  7  17.75  +0.46  3397.7  89.0  D3-16  1997  F  7  19.25  0.00  4641.4  121.6  D2-06  1997  F  7  17.25  +0.42  3759.4  98.5  83.9 -1.21 3169.5 18.50 F 7 D2-12 1997 Average o f initial and final mass, Rate o f carbon dioxide production, Sustained metabolic a  b  rate. Either the initial or final mass was missed. d  

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