The Effect of Testosterone on the Spring Migratory Phenotype of a North American Songbird (Zonotrichia Albicollis)

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The Effect Of Testosterone On The Spring Migratory Phenotype Of A North American Songbird (Zonotrichia albicollis)

Caitlin L. Vandermeer The University of Western Ontario

Supervisor Dr. Chris G. Guglielmo The University of Western Ontario

Graduate Program in Biology A thesis submitted in partial fulfillment of the equirr ements for the degree in Master of Science © Caitlin L. Vandermeer 2013

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Recommended Citation Vandermeer, Caitlin L., "The Effect Of Testosterone On The Spring Migratory Phenotype Of A North American Songbird (Zonotrichia albicollis)" (2013). Electronic Thesis and Dissertation Repository. 1788. https://ir.lib.uwo.ca/etd/1788

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(Thesis format: Integrated Article)

Caitlin L. Vandermeer

Graduate Program in Biology

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science

The School of Graduate and Postdoctoral Studies The University of Western Ontario London, Ontario, Canada

Abstract

In passerines, the endocrine modulators responsible for seasonal changes in migratory behaviour and physiology are unclear. Spring photoperiods alter androgen levels, influencing muscle mass and fat deposition rates to power migration, as well as enhance nocturnal migratory restlessness activity (Zugunruhe). My study compared physiological indicators and migratory restlessness in castrated and intact white-throated sparrows (Zonotrichia albicollis) following photoperiod and hormone manipulation. Nocturnal restlessness activity was highest in migratory intact males or migratory castrated males that received testosterone replacement. Liver mass decreased in all photostimulated migratory groups regardless of testosterone treatment. Likewise, oxidative enzyme activity was unrelated to testosterone, but was higher in the migratory males relative to the non-migratory group. My results indicate that long day photoperiods in spring will enhance nocturnal restlessness through testosterone, but that testosterone does not appear to be responsible for changes in oxidative enzymes or digestive organ mass.

Keywords

Migration, Nocturnal Restlessness, Photoperiod, Testosterone, Zugunruhe, Body Composition, Phenotypic Flexibility, Oxidative Enzyme Activity.

Co-Authorship Statement

My advisors, Dr. Christopher G. Guglielmo and Dr. Scott A. MacDougall-Shackleton contributed to the experimental design used in chapters two and three. Both provided funding and access to equipment necessary for my experiments. My advisors both provided editorial comments and advise on the statistical analyses used in drafts of both data and bookend chapters. Crystalline testosterone was purchsed by Scott MacDougall-Shackleton. Androgen blocking and aromatase-inhibiting pharmaceuticals were purchased using funding from Chris Guglielmo. I was responsible for all bird care during the entire experiment.

A version of chapter 2 is currently in review in Hormones and Behaviour with Wayne Bezner-Kerr, Scott A. MacDougall-Shackleton and Christopher G. Guglielmo as co-authors. Wayne Bezner-Kerr designed the infrared sensors used to measure nocturnal restlessness. The infrared sensors were built and assembled by Wayne Bezner-Kerr and myself. Scott MacDougall-Shackleton performed the castration and laporotomy surgeries as well as implanted birds with silastic implants containing pharmaceuticals. Using enzyme immunoassay kits, provided by Scott MacDougall-Shackleton, I determined androgen levels of blood samples for my treatment groups. Data collection (nocturnal activity, body mass, and fat scores) and organization was all performed by myself. I was also responsible for the maintenance and repair of infrared sensors.

In chapter 3, muscle and liver samples as well as the brain of each of my experimental birds was taken at the initial point of euthanization. After I had euthanized each of the birds, Chris Guglielmo dissected each of the brains, whereas Morag Dick and Brendan McCabe took liver and muscle samples, respectively. Dissections and measurements of additional organs were performed by me, under the guidance of Alex MacMillan. Chris Guglielmo provided advice and equipment for running enzymes assays alongside Alex Gerson and Dr. Jim Staples. Enzyme assays were conducted by myself with the company of Morag Dick. In the future, a these results will be submitted as a manuscript; Scott MacDougall-Shackleton, Chris Guglielmo and myself will be co-authors.

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Acknowledgments

None of this work could have been completed without the great support of everyone at the Advanced Facility for Avian Research at Western. During my time here, I was fortunate to be a part of two great labs. I would like to thank both the Guglielmo and MacDougall-Shackleton labs (past and present) for all of their help during my degree: Tara Crewe, Morag Dick, Alex Gerson, Kristen Jonasson, Lisa Kennedy, Alex MacMillan, Ivan Maggini, Brendan McCabe, Liam McGuire, Greg Mitchell, Adriana Diez, Tara Farrell, Mélanie Guigueno, Micheal Hasstedt, Tosha Kelly, Pralle Kriengwatana, Shannon Mischler, Adam Piraino, Brian Robertson, Kim Schmidt, and Haruka Wada. The dedication and interest everyone has for their projects is inspiring and encouraging!

Each lab would not have been the same without my advisors Scott MacDougall- Shackleton or Chris Guglielmo. I was lucky to have two mentors who are hardworking, enthusiastic and extremely patient. Without the combination of Chris and Scott's expertise and insight, my project would not have been nearly as cool. I am very grateful I had the opportunity to work with both of them at this awesome facility.

The first chapter of my thesis would not have been possible without the help of Wayne Bezner-Kerr. Wayne designed and helped build the infrared sensors used to measure nocturnal activity. Having zero electrical engineering experience, I would not have had a hope in initiating, let alone finishing their construction in time for my experiment without his help.

Most importantly, I have to thank my family. My parents and sister have always supported me throughout my undergrad and masters. Biology is far from their forte, but they've never contested my interests. For times when I've questioned myself and my ability, I'd also like to thank Joshua K. Robertson, alongside my Mom and Dad for all their pep talks and words of encouragement.

Table of Contents

Abstract ...... ii

Co-Authorship Statement ...... iii

Acknowledgments ...... iv

List of Tables ...... vii

List of Figures ...... viii

List of Abbreviations ...... ix

List of Appendices ...... x

Chapter 1 : General Introduction ...... 1

1.1 Fuelling Migration ...... 3

1.2 Improving Endurance Exercise Machinery ...... 4

1.3 Migratory Behaviour ...... 6

1.4 Regulation of Migration by Endogenous Programs...... 7

1.5 Migration and Reproduction ...... 9

1.6 Thesis Objectives ...... 11

1.7 Literature Cited ...... 14

Chapter 2 : Effects of testosterone on spring nocturnal migratory restlessness in the white-throated sparrow (Zonotrichia albicollis)...... 29

2.1 Introduction ...... 29

2.2 Methods and Materials ...... 31

2.3 Results ...... 39

2.4 Discussion ...... 44

2.5 Conclusions ...... 46

2.6 Literature Cited ...... 49

Chapter 3 : Effects of testosterone on migration-related changes in body composition and muscle oxidative enzyme activity in the white-throated sparrow (Zonotrichia albicollis)...... 59

3.1 Introduction ...... 59

3.2 Methods and Materials ...... 63

3.3 Results ...... 69

3.4 Discussion ...... 83

3.5 Conclusions ...... 89

3.6 Literature Cited ...... 91

Chapter 4 : General Conclusions...... 102

4.1 Thesis Summary...... 103

4.2 Future Directions ...... 105

4.3 Conclusions ...... 109

4.4 Literature Cited ...... 111

Appendices ...... 118

Curriculum Vitae ...... 122

List of Tables

Table 2 - 1: Treatment groups containing either castrated or sham-operated intact male white- throated sparrows (Zonotrichia albicollis)...... 34

Table 3 - 1: Assay conditions used to measure activity of Carnitine Palmitoyl Transferase (CPT), 3-Hydroxyacyl-CoA-Dehydrogenase (HOAD), Citrate Synthase (CS), Lactate Dehydrogenase (LDH) ...... 67

Table 3 - 2: Eigenvalues of the first three principal components of log-transformed masses of dried organ components of male white-throated sparrows, Zonotrichia albicollis (n = 32) ...... 78

vii

List of Figures

Figure 2 - 1: Circulating androgen levels of male white-throated sparrows (Zonotrichia albicollis) before and after testosterone and photoperiod manipulations ...... 40

Figure 2 - 2: Behavior recorded for male white-throated sparrows (Zonotrichia albicollis) subject to testosterone and photoperiod manipulations ...... 43

Figure 3 - 1: Body mass (A) and furcular fat score (B) of male white-throated sparrows (Zonotrichia albicollis) following manipulations of photoperiod and testosterone...... 71

Figure 3 - 2: Dry mass of digestive organs of male white throated sparrows (Zonotrichia albicollis) following testosterone and photoperiod manipulations...... 73

Figure 3 - 3: The relationship between dry gut mass and body mass of male white-throated sparrows (Zonotrichia albicollis) after testosterone and photoperiod manipulation ...... 74

Figure 3 - 4: Hematocrit (percentage of packed cell volume) of male white-throated sparrows (Zonotrichia albicollis) after photoperiod and hormonal manipulation ...... 75

Figure 3 - 5: Dry mass of exercise organs of male white-throated sparrows (Zonotrichia albicollis) following hormone and photoperiod treatment...... 76

Figure 3 - 6: Principal component scores for male white-throated sparrows (Zonotrichia albicollis) after photopeiod and hormone manipulation...... 79

Figure 3 - 7: Oxidative enzyme activity of male white-throated sparrows (Zonotrichia albicollis) after hormone and photoperiod manipulation ...... 82

viii

List of Abbreviations

ATD - 1-4-6 androstatrien-3,17 dione

BMR - basal metabolic rate

CPT - carnitine palmitoyl transferase

CS - citrate synthase

EIA - enzyme immunoassay

FABPpm - plasma membrane fatty-acid binding protein

FAT/CD36 - fatty acid translocase

FLUT - fluoro-2-methyl-4'-nitro-m-propionotoluidide

GH - growth hormone

HFABP - heart-type fatty acid binding protein

HOAD - 3-hydroxyacyl-CoA-dehydrogenase

IGF-I - insulin-like-growth factor

IR - infrared i.d. - inside diameter o.d. - outside diameter

JH - juvenile hormone

LDH - lactate dehydrogenase

T3 - triiodothyronin

T4 - thyroxin

List of Appendices

Appendix A : Ethics Approval...... 118

Appendix B: Circuit Board Configuration and Electrical Diagrams for Infrared Sensors, Detectors and Data Acquisition Plates...... 119

Appendix C: Validation Curve for testosterone in white-throated sparrows (Zonotrichia albicollis)...... 121

Chapter 1 : General Introduction

Migratory movements, both obvious large-scale migration journeys and subtle inconspicuous passages, have amazed researchers for generations. Migration is an important component of the life history of many animals and during this time, migrants face unique physiological challenges (Bowlin et al, 2010; Dingle 1996; Dingle and Drake, 2007; Ramenofsky and Wingfield, 2007). The most familiar migrations involve round-trip, annual movements between breeding and wintering grounds, however many one-way and indirect return movements are also considered true migrations (Dingle, 1996; Rankin, 1991).

Migration occurs in a tremendous variety of animals, and migration strategies are diverse. For example, after several years at sea, adult Pacific salmon (Oncorhynchus spp.) travel hundreds or thousands of kilometers to their natal fresh water rivers to spawn and die (Groot and Margolis, 1991; Ramenofsky and Wingfield, 2007). In the late summer and fall monarch butterflies (Danaus plexippus) fly up to 4000 km from Canada and the northern United States to over-winter in central Mexico (Brower, 1996; Brower et al., 2006). The return migration in spring for monarchs may occur over multiple generations, with offspring from each generation continuing the journey north (Brower et al., 2006; Brower, 1996; Davis and Howard, 2005), although some individuals appear to make the return migration all the way from Mexico back to the northernmost breeding areas (Miller et al., 2011; Norris et al., 2010). Stimulated by monsoonal rains, red crabs (Gecarcoidea natalis) on Christmas Island migrate from rainforest habitats down coastal cliffs to breed along the shore (reviewed in Agnieszka et al., 2001). Migratory wildebeest (Connochaetes taurinus) predictably move to northern grazing areas at the end of the wet season in anticipation of months of drought in southern Africa (Dingle, 1980; Voeten et al., 2010). Thus, though migration is widespread, how animals migrate varies among species.

Some of the most impressive examples of migration occur in birds, and avian migration has been extensively studied in many species all over the world. In North 1

America alone, over 400 bird species migrate biannually, and approximately eighty percent of birds that breed in Canada migrate south each fall (Bartell and Moore, 2013). This tally includes species in all major avian taxa: waterfowl, shorebirds, birds of prey, and songbirds (Kaufman, 2001). Avian migration is periodically interrupted for refuelling. These periods of stopover prepare birds to travel sometimes incredible distances on the subsequent legs of their journey. Typically, songbirds (suborder Passeri) undergo nocturnal migrations, stopping over during the day (Morton, 1967; Wikelski et al., 2003). Some species however, must make multiday flights over large geographic barriers, such as oceans, mountains or deserts. For example, northern wheatears (Oenanthe oenanthe), breed in the Canadian Arctic and Alaska, and migrate to wintering grounds in Africa (Bairlein et al., 2012). Alaskan wheatear populations stopover periodically, passing through Asia, however songbirds leaving Iqaluit, Nunavut, must fly 3,500 km over the open Atlantic ocean on route to Europe (Bairlein et al., 2012). Many old-world species, such as garden warblers (Sylvia borin), stopover to store large amounts of fuel (mainly fat) prior to crossing the Mediterranean Sea and Sahara Desert (Bauchinger et al., 2005; Biebach, 1998; Hume and Biebach, 1996). Similarly, North American passerines wintering in Central and South America also must stopover to fuel up prior to crossing the Gulf of Mexico (Kuenzi et al., 1991; Moore and Kerlinger, 1987).

To cope with the demands imposed by migration, migratory species possess many physiological, morphological and behavioural traits, which together constitute a “migratory syndrome” (Dingle, 1996; Dingle and Drake, 2007; Ramenofsky and Wingfield, 2007). The migratory phenotype includes the following characteristics: fuel deposition in advance of movement, up-regulation of biochemical pathways required for fuel metabolism, and modifications to promote movement, and correct navigation (Alerstam and Lindstrӧm, 1990; Bairlein, 2002; Beauchamp, 2010; Berthold, 2001; Caraco et al., 1980; Driedzic et al., 1993; Guglielmo et al., 2002; Jenni and Jenni- Eiermann, 1998; Lundgren and Kiessling, 1985; Marsh, 1981; Piersma and Gill, 1998; Ramenofsky and Wingfield, 2007).

1.1 Fuelling Migration

Powered flight and swimming locomotion have lower costs of transport per unit distance than walking or running (Schmidt-Nielsen, 1972), consequently the majority of long-distance migratory species swim or fly (Alerstam et al., 2003; Bowlin et al., 2010). Regardless of the mode of locomotion, most animals use fat as the primary metabolic fuel for migration, and they often deposit large amounts of fat during premigratory seasons (Dingle 1996, Jenni and Jenni-Eiermann, 1998). For example, many insects store fat in the thorax and abdomen prior to migration (Blem 1980; Downer and Matthews, 1976). Migratory birds can attain body fat levels as high as 50 % of body mass or greater (Piersma and Gill, 1998). Bats also appear to increase fat stores and become hyperphagic prior to migration (Ewing et al., 1970; McGuire and Guglielmo, 2009; Welch et al., 2008; Widimaier et al., 1996), however, it has been difficult to discern whether these fat stores are required for hibernation rather than for migratory flights because there are species of bats that migrate to hibernaculum (Orr, 1954; Twente, 1955). Fat is also used by swimmers like migratory green turtles (Chelonia mydas), many anadromous and catadromous fish, and marine mammals (Blem, 1980; Carr, 1964; Carr and Goodman, 1970; Krueger et al., 1968; Young, 1976). Lipids are ideal for fueling migration because they are energetically dense, providing higher energy yields (37.6 kJ g -1 than carbohydrates (3.5 - 4.4 kJ g-1; Blem, 1980; Jenni and Jenni-Eirmann, 1998). Stored lipids are also lighter and require less space than glycogen or protein stores of the same weight because lipids are stored anhydrously (Ramenofsky, 1990; Weis-Fogh, 1952;).

Protein is also catabolised for fuel in migrants that maintain locomotion for long periods of time; the resulting amino acids are required as intermediates of the citric acid cycle, and gluconeogenesis (Dohm, 1986; Jenni and Jenni-Eiermann, 1998). Protein catabolism can serve to balance weight distribution for improved locomotive efficiency, and can act as a source of metabolic water (Gerson and Guglielmo, 2011; Jenni and Jenni-Eiermann, 1998; Piersma, 1990). This is particularly important for birds undergoing multiday flights and for insects that risk desiccation in dry climates (Weis- Fogh, 1967). Changes in visceral lean mass are well documented in birds; in particular, digestive organs are flexible in size and in their ability to assimilate nutrients

(McWilliams and Karasov, 2005). During migratory seasons, the sizes of digestive organs differ within and among species depending on the distance they travel and the likelihood of encountering geographic barriers during migration (Piersma, 1998). Long- distance migrants reduce non-essential digestive organs to maintain lower weights during flights: this follows the "guts don't fly" hypothesis (Piersma and Gill, 1998). This pattern has been seen in species prior to crossing oceans or deserts (Battley et al., 2000; Battley et al., 2001; Hume and Biebach, 1996; McWilliams and Karasov, 2000; Piersma,1998; Piersma et al., 1999; Piersma and Gill, 1998). Once landscape barriers are crossed, it is necessary for birds of these species to rebuild digestive organs before additional fuel can be deposited and migration can continue. Short distance migrants that make frequent stops along migration routes may maintain large digestive organs (McWilliams and Karasov, 2003). Migrants using this strategy (the "migration takes guts" pattern), are able to digest food and assimilate nutrients at a faster rate, and therefore leave stopover sites more quickly (McWilliams and Karasov, 2003). Dissections revealed that migratory bats had smaller intestines, kidneys and stomachs than non-migrants (McGuire et al., 2013b), however anecdotal evidence suggest that migrating bats may sometimes feed on the wing during migratory flights (McGuire and Guglielmo, 2009; Voigt et al., 2010; Voigt et al., 2012), and therefore would require large functional digestive organs. Like birds, this strategy would allow bats to reduce overall mass and minimize the cost of flight by maintaining high rates of nutrient absorption in the intestinal tract even with relatively small guts (Caviedes-Vidal et al., 2007, 2008).

1.2 Improving Endurance Exercise Machinery

Muscle performance can increase at the whole muscle and molecular level in a number of ways that could be important for migration. Skeletal muscle mass can increase prior to migration to promote endurance exercise, to store extra amino acids that may be needed for key metabolic pathways, or to provide extra power to transport large fuel stores. In birds, muscle growth via hypertrophy increases the power output of primary flight muscles (Jenni and Jenni-Eiermann, 1998; Karasov and Pinshow, 1998; Lindstrӧm and Piersma, 1993). Christmas Island crabs also show adaptive modulation of muscles that improves aerobic exercise. Prior to migration, the expression of genes that regulate 4

muscular contraction (actin, troponin and tropomyosin) increase, resulting in crabs having improved fatigue resistance (Postel et al., 2010).

In order for active muscle groups to process large volumes of fuel required for migratory flight, many animals increase their capacity to transport and metabolize lipids. A variety of proteins bind to, and facilitate the transport of fatty acids through the circulation and within the cytosol of cells (Schaffer, 2002), such as albumin and lipoproteins in plasma or haemolymph and heart type fatty acid binding protein (H- FABP) in the cytosol (Schaffer, 2002; Storch and Thumser, 2000; Van der Horst et al., 1993). Membrane bound transporter proteins (e.g. fatty acid translocase: FAT/CD36, and plasma membrane fatty-acid binding protein: FABPpm) move fatty acids across the sarcrolemma of muscle cells (Bonen et al., 2007; Luiken et al., 1999). In desert locusts (Schistocerca gregaria), elevated levels of fatty acids cause the up-regulation of mRNA expression of H-FABP improving lipid transport during extended flights (Chen and Haunerland, 1994). In addition, the concentrations of muscle cell transporter proteins increase during migration in waterfowl, shorebirds and passerine bird species (Guglielmo et al., 2002; McFarlan et al., 2009; Pelsers et al., 1999; Zajac et al., 2011). Bats may also up regulate these transporter proteins to improve fuel transport during long-distance flights, however evidence so far suggests that increases in H-FABP may also be due to increased body mass, fat metabolism and longer migration distances in pregnant migrating females (McGuire et al., 2013a).

Changes also occur in the activities of oxidative mitochondrial enzymes during migration in many species, which help to increase aerobic capacity and the use of fatty acids as fuel for locomotion. Once inside mitochondria, fatty acids undergo β-oxidation and enter the Krebs cycle. In migrating bats and birds, enzymes such as carnitine palmitoyl transferase (CPT), 3-hydroxyacyl-CoA-dehydrogenase (HOAD), and citrate synthase (CS) increase prior to as well as during migration to increase fatty acid oxidation capacity (McFarlan et al., 2009; McGuire et al., 2013; Zajac et al., 2011). Exercised rainbow trout (Oncorhynchus mykiss) had higher maximal CPT, CS and HOAD activities than non-exercised fish (Farrell et al., 1991). Mitochondrial enzyme activity was higher in red muscle of anadromous cisco (Coregonus artedii) that swam 5

longer distances than populations who swam along shorter rivers to breeding grounds (Couture and Guderley, 1989). In addition, developing migratory locusts (Locusta migratoria) increase beta-oxidative enzyme activity as they mature into flying adults (Beenakkers et al., 1975; Kammer and Heinrich, 1978).

1.3 Migratory Behaviour

At the onset of migratory seasons, changes in physiology are paired with modifications of behaviour. As alluded to above, songbirds extend their daily activity into nocturnal periods to migrate at night while foraging during daylight hours (Morton, 1967). In captivity, when birds are in a migratory condition this nocturnal motivation to move, deemed migratory restlessness (Zugunruhe), causes extensive hopping and wing- whirling in their cages at night (Agatsuma and Ramenofsky, 2006; Berthold, 1973; Coverdill et al., 2011; Gwinner and Czschlik, 1978). The intensity and persistence of nocturnal restlessness of captive birds is correlated, both inter- and intra-specifically, with migratory distance (Wingfield et al., 1990). This suggests that this behaviour is regulated, in part, by an endogenous program (Wingfield et al., 1990). However, factors such as low resource availability, and poor weather conditions can also affect migration speed and can over-ride the endogenous program (reviewed in Wingfield et al., 1990). Light wavelength also influences the initiation of migratory behaviour in the black bean aphid (Aphis fabae). Once an asexual female has laid enough eggs on a host plant, she becomes attracted to the sky's blue-wavelengths and flies into the wind (reviewed in Dingle, 1980). After spending some time in the wind column, females then become attracted to yellow wavelengths of new host plants, and descend to lay more eggs (Dingle, 1980). Outside of the summer breeding season, aphids do not display an attraction to light wavelengths (Dingle, 1980).

Other behaviours that are associated with migration have been observed in many animal taxa and include formation of social groups and changes in dietary preferences. In some species, solitary individuals become gregarious and join groups for migration (Ramenofsky and Wingfield, 2007). Flocking is well documented in both non-migratory and migratory bird species, however this behavioural adaptation also occurs in migrating

sharks, ungulates and invertebrates (Geist, 1974; Jacoby et al., 2012; Rainey and Betts, 1979; Rose et al., 1985). Autumn migration of spiny lobsters (Panulirus argus) involves the formation of long queues as they travel south across the ocean floor to wintering grounds in the Gulf of Mexico (Kanciruk and Herrnkind, 1978). Similarly, locusts transform from a solitary to gregarious phenotype before joining the massive plague-like swarms that periodically move across sub-Saharan Africa (Pener and Yerushalmi, 1998). Forming herds and flocks provides added benefits for navigation and predator avoidance during migration, however the mechanisms for these changes in behaviour are unknown in many species (Ramenofsky and Wingfield, 2007; Simons, 2004).

Diet switches are often observed during migration, and are usually associated with a preference for more energetically dense or more abundant and reliable foods for fuel (Lee and McCraken, 2005; McWilliams and Karasov, 2005). Gregarious locusts are visually attracted to and preferentially swarm on tall, vertical crops (Kennedy, 1939; Mulkern, 1967). This pattern has not been correlated with nutrition quality, but diet selection does appear to affect the timing of locust molt and sexual maturation (Ellis et al., 1965; Mulkern, 1967). Depending on their nutrient requirements, Canada geese (Branta canadensis) choose to feed on a specific variety of vegetation (McLandress and Raveling, 1981). Migratory garden warblers (Sylvia borin) and chaffinches (Fringilla coelebs) are typically insectivores, however, both species include fruits and seeds, respectively, in their diet during migratory seasons (Bairlein, 1990; Bairlien, 2002). Fruits and insects are more available during these seasons, and the carbohydrates acquired from this diet could be readily converted to fat (Bairlein, 2002).

1.4 Regulation of Migration by Endogenous Programs

The onset of a migratory phenotype occurs through interactions of endogenous programs with external environmental cues that are often transduced as endocrine signals (Roenneberg and Merrow, 2005). Circadian rhythms regulate daily patterns, whereas, circannual rhythms are responsible for maintaining patterns on a yearly cycle (Gwinner, 1996; Merrow and Roenneberg, 2005, 2009). These rhythms are maintained in constant conditions without external stimuli with a period that deviates slightly from the external

environmental cycle (Collison and Sheridan, 2012; Gwinner, 1990). Some environmental cues, known as zeitgebers, such as photoperiod, temperature, lunar and tidal cycles reset endogenous rhythms through entrainment mechanisms (Collison and Sheridan, 2012; Gwinner, 1990; Merrow and Roenneberg, 2009). Over evolutionary time, organisms have presumably gained adaptive advantages from their biological clocks to keep them synchronized with seasonal cycles. Using signals from the environment to anticipate environmental changes, organisms can prepare for periods where resources are scarce, or when climatic conditions will deteriorate (Collison and Sheridan, 2012; Panda et al., 2002). Photoperiod is a critical zeitgeber for species residing in northern latitudes; shifts in light cycle represent the changing of seasons and maintain aspects of an organism's life history at appropriate times of the year (Stephan, 1982).

Seasonal migratory changes have been experimentally induced in captive birds exposed to natural rhythmic changes in light cycles (Gwinner, 1990). Light interacts with deep-brain opsin 5 photoreceptors in birds (located in the cerebrospinal-fluid-contacting neurons of the paraventricular organ), which results in a release of hormones from the hypothalamus and pituitary to mediate seasonal changes in physiology (Ikegami and Yoshimura, 2012). Increasing day lengths imitate vernal photoperiods and initiate spring migration and breeding, whereas decreasing day lengths signal the advancement of autumn and subsequently, fall migration. In addition to exhibiting migratory restlessness in response to the external cue of photoperiod, captive studies have demonstrated the contribution of an endogenous annual rhythm to migratory behaviour (reviewed in Gwinner, 1977, 1986, 1989, 1990, 1996). When kept in certain constant conditions, such as a constant 12:12 light:dark cycle, birds of some species still increase body mass and express migratory restlessness at night twice each year. Some bird species will also express the migratory phenotype during spring and fall even if their photoperiod does not change from short to long or from long to short day lengths, respectively (Gwinner, 1987, 1989). Thus migratory restlessness is controlled by an endogenous annual rhythm whose timing is influenced by changes in external environmental cues such as photoperiod.

1.5 Migration and Reproduction

Vernal migration is linked to the onset of reproduction, but the relationship between these life history events differs across taxa. In insects, migration and reproduction pose life history trade-offs. The hypothesis of an oogenesis-flight syndrome posits that migration and reproduction are separate physiological states and each life stage is partly inhibited by the other (Rankin and Burchsted, 1992). Flightless and short- winged species of waterstriders (for example: Gerris lacustris, Limnoporus canalicalatus) benefit from an earlier onset of reproduction and have an increased lifetime fecundity (Andersen, 1973; Zera, 1984), but migratory species (Prokelisia marginata and Horvathiolus gibbicollis) are capable of moving to better quality habitats and can produce high numbers of small eggs (Denno et al., 1985; Rankin and Burchsted, 1992; Solbreck, 1986). Reproduction is also stimulated by flight in several insect species (reviewed in Rankin and Burchsted, 1992). Both migratory flight and reproduction are inhibited and re-established when juvenile hormone (JH) are removed and replaced in several species (Rankin et al., 1986).

On the other hand, migration and preparations for reproduction overlap in birds. During spring, birds travel towards their breeding grounds. Following increasing day lengths associated with spring, the gonadal tissue grows over the course of migratory flights and secretes gonadal hormones (androgens and estrogens, Wingfield et al., 1990). This pattern in hormone secretion has been replicated in captive studies of photo- manipulated birds (Wingfield et al., 1990). Fall migration differs from movements occuring in spring in that it occurs after the breeding season, when gonadal hormones are mininal.

Because spring and fall migrations of birds occur during different life history stages, it was believed that separate neuroendocrine systems regulated the migratory phenotype during each season. From these observations, Rowan (1932) first postulated the gonadal hypothesis for spring migration where gonadal hormones facilitate the migratory phenotype of birds and accelerate the speed of migration. Males and females both produce androgens, however males produce greater amounts. In males, cholesterol is

converted into testosterone through several enzymatic reactions within the Leydig cells of the testes (Nieschlag et al., 1998). The metabolism of testosterone can then produce alternative steroids, such as 5α-dihydrotestosterone (Nieschlag et al., 1998). In males, estrogens are produced by the conversion of testosterone to estradiol via the enzyme aromatase (O'Connell and Hofmann, 2012): estrogens are important endocrine signals affecting behaviour (O'Connell and Hofmann, 2012). In females, androgens and estrogens are produced primarily by the ovary and adrenal gland (Snyder, 2003).

The role of androgens during migration in both sexes is unclear. Male and female birds both migrate during spring and fall, and require similar seasonal changes in behaviour and physiology. For many species, the overall speed of fall migration for males and females is similar (Morris and Glasgow, 2001; Morris et al., 1996), however, spring elevations in testosterone may provide an advantage to males who initiate nocturnal restlessness earlier in the spring and arrive earlier at breeding grounds (Coppack and Pulido, 2009; Maggini and Bairlein, 2012; Morris et al., 2003). Under the influence of testosterone, males rush to breeding grounds in order to establish and defend territories (Ketterson and Nolan, 1999; Wingfield et al., 2001).

In addition to promoting aggressive behaviours required for attracting mates and defending territories, androgens like testosterone have anabolic effects on skeletal muscle (Berthold, 2001). Elevated testosterone levels elicit hyperphagia, or increased feeding rates. Hyperphagia occurs in migrants during the fueling stages prior to endurance flights (Berthold, 2001), and allows birds to deposit large volumes of fat. Hematocrit is elevated in migratory birds, increasing the ability to transport oxygen to working muscles; studies manipulating testosterone have shown similar increases in hematocrit (Fair et al., 2007; Robinzon and Rodgers, 1979; Thapliyal et al., 1983).

Castration studies have been used to manipulate testosterone levels in migrant birds under spring photoperiods, however these methods were likely unsuccessful at completely inhibiting circulating androgens. Inhibiting androgens (via castration or pharmaceutical treatment) during wintering photoperiods appeared to inhibit migratory behaviour and fuel deposition when birds were photostimulated (Gupta and Kumar, 2013;

Tonra et al., 2011; Weise, 1967). However, these results were not replicated in studies where castration surgeries occurred after photostimulation (Lofts and Marshall, 1961; Millar, 1960; Morton and Mewaldt, 1962). In addition, castration studies did not determine if androgens alter body components associated with exercise (muscles, heart) or food processing (digestive organs) or the biochemical pathways for exercise in muscle. So far, previous work has only examined the effect of sex steroids on nocturnal restlessness, body weight and fat deposition.

1.6 Thesis Objectives

The primary objective of my thesis was to determine if androgens regulate physiological and behavioural changes associated with a migratory phenotype during spring in birds. I examined several indices of migratory condition in a migratory passerine species: nocturnal restlessness, body composition, and fat metabolism. To do this I manipulated both photoperiod and testosterone. Photostimulating captive birds with a switch from short-days to long-days mimicked the arrival of spring and elicited the specific migratory changes mentioned above. By using a combination of castration, androgen blockers and aromatase inhibitors I was able to completely inhibit circulating sex steroid hormones in male white-throated sparrows (Zonotrichia albicollis). I was able to reintroduce testosterone in castrated males by implanting sub-cutaneous testosterone- filled implants.

In chapter two, I measured the nocturnal activity of sparrows in each of my treatment groups. I quantified nocturnal restlessness of photostimulated male birds in comparison to males on short day photoperiods that remained in a non-migratory condition. The intensity of nocturnal restlessness behaviour increases as the bird's motivation to migrate strengthens, and therefore data were compared through time within treatment groups, as well as between treatment groups. In addition to measurements of testes volume, I used blood samples taken before and after the four-week experimental period to measure the levels of circulating androgens and validate the efficacy of the castration surgeries. Briefly, I found that spring photoperiods initiate migratory behaviour

but testosterone treatment intensifies the level of migratory restlessness exhibited by individuals.

In chapter three, I examined if differences in body composition and muscle biochemistry existed between treatment groups after birds underwent photoperiod and hormone manipulation. After behaviour was recorded for four weeks, all experimental birds were euthanized. At this time, samples of muscle were frozen to measure activities of oxidative enzymes: carnitine palmitoyl transferase (CPT), hydroxyacyl-CoA dehydrogenase (HOAD), citrate synthase (CS), and lactate dehydrogenase (LDH), an indicator of anaerobic metabolism. I dissected the experimental birds to measure sizes of organs and muscles. I compared organ masses and enzyme activities among treatment groups to test whether androgens affect body composition and the biochemical machinery of active muscle in photostimulated birds in contrast to non-migratory males. Spring photoperiods stimulated fat deposition, decreases in liver mass, and increases in mitochondrial enzyme activity. While increases in mitochondrial enzymes were primarily photoperiod driven, testosterone treatment or removal in castrated males appeared to dampen the increase in mitochondrial enzyme activity.

This thesis examines the long-standing gonadal hypothesis in new ways. I used a combination of castration surgeries and the implantation of pharmaceuticals to definitively block androgens produced in and outside the gonadal tissue. Unlike previous research, measuring nocturnal restlessness under these conditions would more clearly test whether androgens influence spring migratory behaviour. This work also examines the role of androgens in migratory preparations at the molecular and organ levels. In the past, migratory preparations and strategies were indicated by making inferences from organ mass and by quantifying the amount fat deposited. Androgens have anabolic effects, however testosterone levels have not previously been correlated with exercise organ size or a muscle's ability to transport or oxidize lipids for fuel. In addition, the endocrine mechanisms responsible for the seasonal upregulation in mitochondrial enzyme activity are relatively unknown, however this physiological change is vital for successful fuel metabolism and migratory flight. Ultimately, the results presented in this thesis will

identify if the gonadal hypothesis can be expanded to include physiological changes associated with migration in addition to behaviour and fuel deposition.

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Chapter 2 : Effects of testosterone on spring nocturnal migratory restlessness in the white-throated sparrow (Zonotrichia albicollis).

2.1 Introduction

Migration of songbirds involves movement to their breeding grounds in the spring and to their wintering grounds in the fall. The coarse timing of such migration is controlled through circannual rhythms (reviewed in Gwinner, 1977, 1989 and 1996). However, photoperiod is a critical cue that entrains and synchronizes these circannual rhythms (reviewed in Gwinner, 1977, 1989 and 1996). The timing of spring migration and the breeding season are thus both regulated in large part by changes in photoperiod (Wingfield and Kenagy, 1991). Photostimulation by exposure to longer day lengths initiates increases in muscle and fat stores in preparation for endurance flights as well as the growth and maturation of gonadal tissue (reviewed in Scott et al., 1994; Wingfield and Kenagy, 1991; Dawson et al., 2001).

Most migratory songbirds are nocturnal migrants, refuelling at stopover sites during the day (Morton, 1967). This nocturnal migratory behavior has been well characterized and is made up of several specific behaviors (Berthold, 1973; Gwinner and Czeschlik, 1978). Captive studies include altering photoperiod cycles to illicit migratory condition in captive birds (reviewed in Gwinner, 1996) that then exhibit "beak-up" and "beak-up flight" behaviors in their cages at night (Agatsuma and Ramenofsky, 2006; Coverdill et al., 2011). In comparison, non-migratory birds in captivity are not active at night, and spend most of the night motionless and asleep (Agatsuma and Ramenofsky, 2006; Coverdill et al., 2011). In addition, captive birds in migratory condition also show depressed activity during a quiescent phase around dusk (Agatsuma and Ramenofsky, 2006). This quiescent phase has been characterized as a 2 h period before night during vernal migration and as the first 2 h after lights go out during fall migration (Agatsuma and Ramenofsky, 2006). The quiescent phase and night migratory activity are

collectively referred to as Zugunruhe or migratory restlessness behavior (Ramenofsky et al., 2003; Landys et al., 2004).

While these behaviors have been well documented, the endocrine controls regulating these activities are unclear. Following the gonadal hypothesis (Morton and Mewaldt, 1962; Rowan, 1932), androgens have been suspected to play a role during spring migration because of their association with the breeding season. Testosterone levels begin to increase during spring migration (simultaneously during maturation of the reproductive system), and peak during the breeding season (Wingfield and Hahhn, 1994). In addition to stimulating testicular growth and sperm production (Balthazart, 1983; Ketterson and Nolan, 1999; Wingfield et al., 2001), testosterone and its metabolites regulate seasonal aggression required to defend and acquire territories, as well as induce courtship behaviors to attract mates (Blas et al., 2007; Beletsky et al., 1990; De Ridder et al., 2000; Hegner and Wingfield, 1987; Ketterson et al., 1992; Moore, 1984; Saino and Møller, 1995; Wingfield, 1984). Hyperphagia and increases in fat deposits are two additional effects attributed to the anabolic effects of testosterone, and may accelerate the timing of migration during spring (Berthold, 2001). Exercise muscle mass (flight muscles and heart) increases due to higher rates of foraging, induced by testosterone, ultimately aiding birds during endurance flights. Combined, these observations support the gonadal hypothesis and imply a role for testosterone in mediating vernal migration.

One limitation of prior studies, however, is that hormone manipulation experiments have likely not completely inhibited circulating testosterone. Generally these studies assumed that castration was sufficient to eliminate circulating androgens. However, even without gonads, testosterone can be produced de novo in the brain (Nomura et al., 1998; Roble and Baulieu, 1995; Tsutsui and Yamakazi,1995) and in the adrenal gland (Labrie et al., 1995; Schlinger et al., 1999). Thus, androgens may still have been present in past castration experiments. Despite this shortcoming, some studies found a decrease in migratory restlessness as well as a delay in the onset of migratory restlessness following castration (Gupta and Kumar, 2013; Weise, 1967). However, other studies did not find such an effect of castration (Lofts and Marshall, 1961; Morton and Mewaldt, 1962; Millar, 1960). It appears that the effects of castration were only observed 30

in birds that underwent surgery prior to photostimulation, suggesting that androgen removal must occur prior to the onset of neuroendocrine response to photostimulation in order to inhibit migratory restlessness (Wingfield et al., 1990). A more recent study examined the effect of androgens on migratory restlessness in male dark-eyed juncos (Junco hyemalis) using anti-androgen drugs (aromatase inhibitors and androgen receptor blockers) rather than castration, and found reduced nocturnal activity in testosterone- inhibited males (Tonra et al., 2011). However, because the birds in this study were not castrated it is possible that androgens were not completely suppressed. Combined, the above studies suggest that testosterone, in conjunction with photostimulation, initiates spring migratory restlessness. However, further work is required to clarify this relationship and to determine if complete elimination of testosterone abolishes migratory restlessness.

The present experiment sought to determine whether the complete inhibition of testosterone in migratory white-throated sparrows (Zonotrichia albicolis) would fully suppress the initiation of nightly migratory restlessness. My experiment involved the photostimulation of male sparrows following castration surgeries and the administration of implants. Hormone treatment involved implants containing either anti-androgen compounds or testosterone. We predicted that male white-throated sparrows that were completely testosterone inhibited (via castrations and implantation with anti-androgens) would not display nocturnal restlessness or a quiescent phase after photoperiod manipulation, and that testosterone treatment of castrated birds would rescue these behaviors.

2.2 Methods and Materials

Study Species

In comparison to long-distance migrants travelling up to 10000 km (example: bar-tailed godwits, Limosa lapponica bauero; Gill et al., 2009), white-throated sparrows (25-30 g) are short distance migrants, travelling 3000-5000 km between breeding grounds in northern Ontario and Quebec and wintering grounds in the southeastern United States

(Kaufman, 2001). Sparrows were caught using mist nets and Potter traps during their fall migration in October 2011 on undeveloped property belonging to the University of Western Ontario. Birds were individually housed in cages of identical size (39X34X42 cm) at the Advanced Facility of Avian Research at the University of Western Ontario. Birds were fed a mixture of white millet, parakeet seed and ground bird chow (Mazuri Small Bird Maintenance); food and water was replaced on a daily basis. Birds were kept at a constant temperature (21 °C) for the entire experiment. Birds were captured under a scientific collection permit from the Canadian Wildlife Service (CA 0256). All animal procedures were approved by The University of Western Ontario Institutional Animal Use Subcommittee (protocol # 2010-216; Appendix A).

The sex of the birds was initially determined in the field using wing chord (Kaufman, 2001) and confirmed genetically using PCR (method developed by Brendan McCabe: Griffiths et al., 1998; Bercovich et al., 1999; Tomasulo et al., 2002). Approximately 150 µL of blood was taken from the brachail vein from each bird. Whole blood samples were diluted to 1% packed erythrocytes using distilled water. Diluted blood samples (5 µL) were added to 18 µL of stock solution (containing 10 mM Tris-HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 200 µM of each dNTP, 1 µM each of P2 and P8 primer and water), and 1 Unit (2 µL) of Taq Polymerase (0.5 U/µL) before loaded into a thermocycler. Samples were exposed to three 3 minutes cycles at 94°C, and three cycles at 48°C followed by a four minute denaturing step at 94°C. Samples were then run for an additional 35 cycles (45 seconds at 94°C, 45 seconds at 48°C, 45 seconds at 72°C and 5 minutes at 72°C). The lid temperature of the thermocycler was set at a constant 94°C. Once these cycles were completed, 3 µl of 6x loading dye could be added to each sample; 20 ul of each PCR product was then added into wells of a 3% aragose gel alongside a 2 µL standard. A voltage of 8.5 V/cm (120 Volts) was applied to the gel for 60 - 80 minutes. The gel was then examined under a UV light, and the number of bands in each well's lane was recorded; one band was representative of male white throated sparrows and two bands indicated females. A total of 32 male birds were required for my experiment, and following PCR those identified as females were released.

Photoperiod Manipulation

Photoperiod followed the civil dawn and dusk for London, Ontario (Sunrise/Sunset Calculator, National Research Council Canada) until the winter solstice (21 December 2011). Following this, photoperiod was switched to an 8 h light:16 h dark (8L:16D) cycle for 80 d in order to dissipate photorefractoriness. By breaking photorefractoriness, birds could then become sensitive again to spring photoperiods. The day after implant surgeries (10 April 2012; see below), birds in the three long-day treatment groups were switched to a 16L:8D photoperiod for four weeks. During this experimental period, dim nightlights (< 1 lux) were added to each of the short-day and long-day treatment rooms to facilitate migratory restlessness behavior (Ramenofsky, et al., 2003; Landys et al., 2004 and Coverdill et al., 2008). Because previous work has shown that transferring birds from short to long days promotes migratory behaviour and changes in body composition (Gwinner, 1990), I will henceforth refer to photostimulated males in this study as 'migratory' as well as refer to the non-photostimulated group as being 'non-migratory'.

Hormone Manipulations

Surgical castration was used to eliminate the production of gonadal testosterone, and pharmacological treatments were used to block the effects of non-gonadal sources of testosterone. Birds were randomly assigned to one of four treatment groups: each containing eight birds (Table 2 - 1). Two treatment groups contained castrated sparrows. Birds were anesthetised with isoflurane (4% for induction, 2% for maintenance) in oxygen (2 L/min) before a lateral incision was made between the last two ribs. Once a blunt probe was able to deflect the intestines, testes were removed with arched tip forceps. Two treatment groups contained laparotomized sham-operated sparrows. Sham- operated birds were similarly anesthetised and opened using a lateral incision, however the testes remained intact. All birds were given an analgesic, Metacam (0.5 mg/kg in 0.5cc volume i.m.) immediately before and 24 h after their surgery. Castrations were performed when the birds were exposed to short days and in non-breeding condition (8 through 18 December 2011); this ensured the testes would be regressed, allowing for

complete removal and minimal bleeding due to the decrease in vascularisation (Farner, 1955; Wingfield and Farner, 1980).

Table 2 - 1: Treatment groups used to study effects of androgens on migration in white- throated sparrows (Zonotrichia albicollis). Groups contained either castrated or sham- operated intact males. Both castration groups and one intact group (groups 2, 3, and 4) were switched to long days (16 hours of light and eight hours of dark; 16L:8D), while group 1 intact males remained on short days (8 hours of light and 16 hours of dark; 8L:16D). n = 8 for all treatment groups.

Treatment Group Light Cycle Surgery Type and Quantity (Hours of Performed Light:Dark) of Implants per WTSP

Group 1: Intact Males 8L:16D Laporotomy 1 Blank silastic implant.

Group 2: Intact Males 16L:8D Laporotomy 1 Blank silastic implant.

Group 3: Hormone 16L:8D Castration 1 Silastic implant containing crystalline Replacement Males testosterone (Sigma-Aldrich, 2012).

Group 4: Castrated 16L:8D Castration 2 Silastic implants containing crystalline Males ATD (Steraliods Inc., 2012).

2 Silastic implants containing crystalline Flutamide (Sigma-Aldrich, 2011).

To block any androgens from non-gonadal tissue an androgen receptor blocker, tri-fluoro-2-methyl-4'-nitro-m-propionotoluidide was used (flutamide; Product No. F9397, Sigma-Aldrich). Flutamide has been used in several bird species (Adkins-Regan and Garcia 1986; Hegner, and Wingfield, 1987; Neri and Peets, 1975; Peets et al., 1974). In addition, to block any estrogenic signalling from non-gonadal sources of steroids a steroidal aromatase inhibitor, 1-4-6 androstatrien-3,17 dione was also used (ATD; Product No. A4100-000, Steraloids Inc. Newport, RI). ATD will irreversibly inactivate aromatase (Numazawa and Tachibana, 1997) and inhibit aromatase mRNA transcription (Foidart et al., 1995) and has been used in several bird species (Adkins and Nock, 1976; Adkins et al., 1980; Adkins-Regan et al., 1982; Archawaranon and Wiley, 1988; Beletsky et al., 1990; Johnson and Bottjer, 1995; Walters and Harding, 1988). Many studies in the past have used combinations of ATD and flutamide to eliminate androgenic and estrogenic steroid signalling (Archawaranon and Wiley, 1988; Johnson and Bottjer, 1995; Schlinger et al., 1992; Soma et al., 1999; Tonra et al., 2011). By castrating, blocking circulating testosterone with flutamide, and by preventing the activity of aromatase with ATD all effects of testosterone were thus inhibited.

Both castration groups and one intact group of males were switched to long days (16L:8D) on 11 April 2012. One sham-operated group of intact males remained on short days (8L:16D). One of the two castration groups was implanted with two silastic implants filled with ATD and two silastic implants containing flutamide (each implant was 10 mm long, i.d. 1.47 mm, o.d. 1.96 mm; n = 8). Past studies have used similar doses of ATD and flutamide in songbirds of similar size and have calculated that each implant type (both ATD and flutamide) would lose approximately 3 mg over 30 days (8 mg /kg /day; Johnson and Bottjer, 1995; Schlinger et al., 1992; Soma et al., 1999). The second castration group (n = 8) contained birds implanted with a single silastic implant containing testosterone (10 mm, i.d. 1.47 mm, o.d. 1.96 mm; Archawaranon and Wiley, 1988; Tonra et al., 2011). The two sham-operated groups were implanted with empty silastic tubes (both groups: n = 8). All silastic implants were sealed using Silastic Medical Adhesive (Dow Corning).

Recording Activity

Infrared (IR) sensors have been used in the past to record migratory restlessness behavior (Coverdill et al., 2008; Coverdill et al., 2011; Rani et al., 2006). Each bird’s cage was equipped with a single perch that had an IR emitter at one end and an IR sensor at the other end (Appendix B). There was thus an IR beam parallel to, and 10 cm above, the perch. Hopping or flight activity above the perch would break the beam and beam breaks were recorded by HOBO data loggers (HOBO 4-Channel Pulse Data Logger: UX120-017M, Onset Computer), with each logger monitoring a bank of 4 cages and recording beam breaks every 1 s.

Data were recorded over 24 h, across the 28 d during the four-week experimental period, and was downloaded on a daily basis using HOBOware Pro software (Version 3.3.0, Onset Computer). The 24 h activity profiles allowed us to quantify the quiescent phase in addition to night activity for each bird.

Plasma Androgen Levels

Blood samples (approximately 150 µL) were taken before and at the end of the experimental period (3 April and 2 May 2012, respectively). All blood samples were collected within 30 min of entering the birds’ rooms and were kept on ice until processed. Samples were processed immediately after each bleeding session. Microhematocrit capillary tubes were centrifuged for 10 min at 13,000 g to separate plasma from hematocrit. Hematocrit was measured, then the plasma was harvested and kept frozen at - 30 °C until a hormone assay was run (Expanded Range Salivary Testosterone Enzyme Immunoassay Kit 1-2402-5, Salimetrics PA, USA). This kit has been validated in a variety of bird species (Washburn et al., 2007). All instructions included with this kit were followed with the exception that we used 15 µL of plasma diluted in 60 µL of assay buffer. The order of plasma samples on each immunoassay plate was randomly assigned (2 plates were used to assay all samples). A standard consisting of a pooled sample of plasma from multiple birds was run on each plate alongside a standard curve and high and low testosterone standards supplied with the kit. To validate this kit for white- throated sparrows the pooled plasma sample was serially diluted to demonstrate 36

parallelism with the standard curve (Appendix C). The intra-assay coefficient of variation was 20.73% for the pooled plasma standard, 25.50% for high standard, and 1.54% for the low standard. The sensitivity of the assay, defined as 2 standard deviations above a blank control, was 3.56 ng/mL. Because this kit cross-reacts with dihydrotestosterone (according to the manufacturer) and other androgens (see below), we report hormone levels below as androgen levels. Because the steroid aromatase inhibitor used (ATD) has an extremely similar structure to testosterone, we conducted an assay using a dilution of ATD dissolved in saline. ATD had high cross-reactivity with the kit antibody, yielding concentration estimates similar to testosterone (data not shown).

Measurements of Testis Size

After the 28 d experimental period, all birds were euthanized under isoflurane overdose and decapitation. All carcasses were initially frozen and stored at -20 °C. Dissections were later performed on thawed specimens to confirm successful castration. For intact birds testis width and length (mm) were recorded to the nearest mm and averaged for each individual prior to statistical analysis. Length and width measurements for left and right testis were averaged for each individual prior to statistical analysis.

Testis volume for each bird was calculated using the equation , where V represents volume, a is the radius and b is half of the length of the testis (Perfito et al, 2008). Dissections of additional organs will be described further in Chapter 3.

Statistical Analyses

In total, 28 d of activity were recorded. For each 24 h activity profile recorded for each bird the data were first filtered to exclude anomalously high levels of activity. Although over 99% of activity fell below 100 beam breaks per minute there were occasional registers of much greater value. These high values were likely a result of birds fluttering their wings within the IR beam. To reduce positive skew in the data we thus set a maximum activity value at 100 beam-breaks per min, and then calculated the average number of beam breaks per minute for each 30-min time bin. Activity of each individual was then averaged over the night, day and quiescent phase periods. The mean activity of

birds within each treatment group was then averaged across four-day time bins. Over the entire experimental period, short-day intact white-throated sparrows did not express nocturnal activity above three beam breaks per min. Based on this criterion, photostimulated birds were considered to be displaying migratory restlessness if they were displaying nocturnal activity above this three beam breaks per minute threshold.

The quiescent phase was defined as activity that occurred during the last two hours prior to when lights were turned off (Agatsuma and Ramenofsky, 2006). Night activity was classified as activity that occurred during the dark phase, and day activity was classified as activity that occurred during the light phase. The diurnal phase included activity included during the quiescent phase activity.

Square root transformations were performed on all behavioral data to reduce positive skew prior to statistical analyses. Linear mixed models were used to determine if there were differences in activity between treatment groups: activity was classified as the dependent variable and treatment group, date interval (binned into seven four-day intervals) and time bin over the night or day were entered as independent variables (fixed factor). Bird ID was entered as a random factor to control for repeated measures from the same individuals. If no difference was found in activity over time bins, this factor was removed from the final model. Post-hoc LSD tests were used to assess pairwise comparisons across treatment groups.

Differences in testis volume between photostimulated and non-photostimulated sham operated males were determined using a one-way ANOVA. Testis volume was log transformed to reduce positive skewing of data. Separate ANOVAs were run with and without testicular volumes for testosterone inhibited castrated males. All statistical analyses were performed using SPSS version 20.0 software (SPSS Inc. Chicago IL, USA).

2.3 Results

Androgen Levels and Testis Size

Levels of circulating androgens in plasma and comparisons of testes size between groups validated that the hormone manipulations were successful. A two-way ANOVA revealed a main effect of photostimulation on circulating androgen levels (F1,27 = 47.02, P < 0.001; Figure 2 - 1), as well as a significant interaction between photostimulation and hormonal treatment groups (F3,27 = 12.80, P < 0.001). To assess this interaction we conducted separate one-way ANOVA for androgen levels for the samples collected before and after manipulation. There was no difference in androgen levels in samples taken during short day photoperiods prior to hormone manipulation (F3,28 = 0.55, P = 0.65), but there were significant differences between groups 20 days after photostimulation (F3,28 = 12.78, P > 0.001; Figure 2 - 1). Post-hoc tests indicated that castrates implanted with ATD and FLUT had androgen levels significantly greater than all other groups, likely reflecting high circulating ATD (see Methods above). Castrates implanted with testosterone had androgen levels higher than the intact groups, but lower than the ATD and FLUT treated birds. Interestingly, androgen level in photostimulated intact males had increased, but not statistically higher than those for short-day birds (P = 0.51).

We also used paired t-tests to compare androgens before and after manipulation for each group. There was a significant increase in androgens after photostimulation for all three photostimulated groups (migratory intact: P = 0.039; testosterone inhibited: P = 0.006; testsosterone treated castrates: P = 0.001). Non-migratory (short day) intact males also had significantly increased levels of androgens 28 days after the initial blood sample (P = 0.002) but continued to have low androgen levels during the entire experimental period (0.44 ng/mL).

Figure 2 - 1: Circulating androgen levels of male white-throated sparrows (Zonotrichia albicollis) before and after testosterone and photoperiod manipulations. Migratory intact males, migratory ATD+FLUT castrates and migratory testosterone-treated castrates were switched from short (8L:1D) to long days (16L:8D). Non migratory intact males remained on short days during the entire experiment. Mean (+ s.e.) androgen levels (ng/mL) of n = 8 for all groups except for migratory ATD+FLUT castrates after photostimulation (n = 7), are given. Post-manipulation groups indicated by the same lower-case letter (a,b,c) did not statistically differ from each other.

Dissection following the experiment revealed two males (both in the ATD+FLUT group) had remnants of testicular tissue. These birds were excluded from further behavioral analyses (below). However, inclusion of these birds did not qualitatively alter the results, likely because the ATD and flutamide prevented any androgens secreted by the testicular remnants from binding to receptors.

Testes were located and measured in all migratory intact males. For three birds the testes were too small to accurately measure, so we assigned them a volume of 1 mm3.

Intact migratory males had an average testicular volume of 71.48 mm3, significantly 3 larger than intact non-migratory males (volume of 1.35 mm ; F1,14 = 59.42, P < 0.001).

Behavior

Nocturnal Migratory Restlessness

No interaction existed between nightly beam breaks and the day of the experiment

(F1,18 = 0.516, P = 0.95), so this interaction term was removed from the further models. Linear mixed models revealed a significant difference between treatment groups in the total number of beam breaks during the night (Figure 2 - 2 A; F1,3 = 7.13, P < 0.0001); however, there was no significant effect of date across the seven four-day intervals (F1,6 = 1.27, P = 0.28). The onset of migratory restlessness activity occurred in all three photostimulated/migratory treatment groups between days 5 and 8, and peak levels of activity were seen in migrants by days 17-21 (data not shown). Post-hoc tests revealed that castrated migrants implanted with testosterone had a higher number of beam breaks per night than the other three treatment groups (P < 0.05 for all comparisons). There was no difference in the total nightly beam breaks between castrates implanted with ATD and flutamide and migratory sham operates (P = 0.60). Over the entire experimental period, non-migratory (short day) intact birds displayed minimal nocturnal beam break activity, significantly lower than testosterone replaced castrates and migratory intact birds (P < 0.05 for both). However, there was no significant difference between the short day birds and castrated males treated with ATD and flutamide (P = 0.10). Ad hoc observations of the birds using an infrared camera confirmed that birds in all three photostimulated groups exhibited typical migratory restlessness behaviour with wing fluttering and beak- up postures. In summary, long-day testosterone-treated birds had the highest levels of nocturnal restlessness followed by intact long-day birds. Long-day birds that had androgens completely eliminated through castration, ATD and flutamide displayed nocturnal restlessness intermediate to intact long-day birds and intact short-day (non- migratory) birds.

Quiescent Phase and Diurnal Activity

Linear mixed models revealed significant differences in quiescent phase activity between the four treatment groups (F1,3 = 6.25, P < 0.001). As with the nocturnal activity, quiescent phase activity levels did not vary over the experimental period (F1,6 = 1.70, P = 0.12). When data were collapsed across date there remained a significant difference between treatment groups in total quiescent phase activity (Figure 2 - 2 B; F1,3 = 6.90, P < 0.001). Non-migratory (short day) intact males maintained higher levels of activity than the three long-day treatment groups (P < 0.005 for all comparisons), which did not differ from each other (all P > 0.05).

Similar results were observed for diurnal activity. Linear mixed models revealed significant differences in daytime activity between the four treatment groups (F1,3 =

16.72, P < 0.001), but there was no effect of date across the experimental period (F1.6 = 1.03, P = 0.41). When data were collapsed across date there remained a significant difference between treatment groups in daytime activity (Figure 2 - 2 C; F1,3 = 22.5, P < 0.001). Migratory intact birds displayed a lower number of beam breaks during the diurnal phase than the rest of the three treatment groups (all P < 0.05). Non-migratory sham operates had significantly higher levels of diurnal activity than the rest of the three treatment groups: P < 0.001, for all pairwise comparisons. There was no difference between the total beam break activity of castrates implanted with ATD and flutamide and testosterone (P = 0.098).

Figure 2 - 2: Behaviour recorded for male white-throated sparrows (Zonotrichia albicollis) subject to testosterone and photoperiod manipulations. Mean (± s.e.) infrared beam breaks per 30 minutes, over the entire experimental period for Migratory restlessness (A), Quiescent Phase (B) and Diurnal Activity (C). Migratory intact males, migratory ATD+FLUT castrates and migratory testosterone-treated castrates were switched to longer light cycles, 16L:8D, from short days, 8L:16D. Non-migratory intact males remained on 8L:16D. The vernal quiescent phase included activity recorded during the last 2 h before lights had gone out (7:30 pm for the three migratory groups and 3:30 pm for the single non-migratory group). Treatment groups with the same lower-case letters indicate groups that are not significantly different. For all treatment groups, n = 8. 43

2.4 Discussion

Overall, long days induced a migratory phenotype, decreasing diurnal and quiescent phase activity and increasing nocturnal restlessness. Both castrated groups were active at night regardless of hormone treatment; however, there was evidence that testosterone modulates this behaviour. Testosterone-replaced males had significantly elevated levels of activity in comparison to intact photostimulated males, whereas testosterone-inhibited males displayed reduced levels of nocturnal activity. Testosterone manipulations however, did not affect activity occurring during the diurnal or quiescent phase.

Androgen Levels and Testis Volume

The EIA results validated our hormone manipulation. Photostimulated castrates who underwent hormone replacement (via testosterone implants) had higher levels of circulating androgens than sham operated migratory males (19.15 ng/mL in comparison to 4.3 ng/mL). Although this level of circulating testosterone is high, it is not out of the range seen in wild free-living birds (Spinney et al., 2006). Testosterone-inhibited birds had the highest levels of androgens, but this is almost certainly due to cross-reactivity of the EIA antibody with ATD, as the only difference between testosterone and 1-4-6 androstatrien-3,17 dione is an extra double bond on a cyclohexene of ATD. Moreover, in pilot studies I verified that the Salimetrics kit used cross-reacts with ATD. I thus conclude that the ATD implants released the drug at a high dose and would have successfully inhibited endogenous aromatase.

Increases in plasma androgen levels compliments testes measurements acquired from sham operated males. Photostimulated intact males had much larger testes than non- migratory short day males; this finding is comparable to captive white-throated sparrows seen by Harris and Turek (1982). Both of these findings indicate that the effect of photoperiod was enough to bring males into breeding condition.

Behavior

Nocturnal Migratory Restlessness

The level of nocturnal restlessness reflected the amount of circulating androgens within each treatment group. Despite castration surgeries and the use of pharmaceutical administration, testosterone-inhibited males still exhibited migratory restlessness behavior when photostimulated. As seen in Gupta and Kumar's recent publication (2013), the behavior shown by this group was depressed in comparison to long-day sham operated and testosterone replacement groups. On the other hand, photostimulated castrated males given testosterone implants displayed elevated levels of nocturnal restlessness behavior in comparison to the three other treatment groups. These effects differ from those found by Tonra et al. (2011a), who found testosterone treated males displayed nocturnal activity similar to control males. Migratory restlessness of the testosterone-inhibited males may result from endogenous programs that are sex steroid- independent (Bartell and Gwinner, 2005; reviewed in Kumar et al., 2010; Rani et al., 2006). Birds were photostimulated mid April, around the normal spring migratory period of this species. Migration and nocturnal restlessness are highly regulated by cirannual and circadian rhythms. Thus, photostimulating males during this time period may induce migratory restlessness regardless any effects of gonadal hormones. That is, photostimulation in spring was sufficient to induce a migratory phenotype, even in the absence of sex-steroids.

Unlike results found by Tonra et al., (2011a) or Weise (1967), we did not see a difference in the onset or the timing in the peak of migratory restlessness for our photostimulated groups. Our statistical analyses did not indicate that there was an interaction between treatment group and day of experimental period. Regardless of hormonal treatment, testosterone inhibited and replacement groups initiated activity over the same time intervals. However, Tonra et al., (2011a) used a longer day length to stimulate birds with (18L:6D). The more extreme the photostimulation, the faster the onset of migratory behaviour and physiology.

Quiescent Phase and Diurnal Activity

Photostimulated white-throated sparrows exhibited similar decreases in quiescent phase activity as previously described (Agatsuma and Ramenofsky, 2006). Non- migratory males were more active than photostimulated males during the daytime and during the quiescent phase. Activity expressed by long-day males during both phases was similar: hormonal manipulations did not affect diurnal activity or activity occurring during the quiescent phase. Differences in diurnal activity may have been because non- migratory males had less time to forage than photostimulated males (8 hours of light instead of 16) and therefore needed to work harder to meet their dietary requirements. Hyperphagia is induced in migrants by elevations in testosterone during spring (reviewed by Wingfeild et al., 1990), so perhaps migratory birds spent more time feeding at stationary food cups than actively breaking the infrared beam. It is possible that another hormone system regulates day time activity independently since separate circadian oscillators control each day and night behaviours (Bartell and Gwinner, 2005; Rani et al., 2006).

An effect of captivity may have also contributed to differences in activity. Birds were fed ad lib, without the risk of predators or the need to actively forage, photostimulated males could minimize activity to save energy prior to nocturnal endurance flights. Migratory restlessness is elevated, and diurnal activity is suppressed in captive birds with high levels of stored fat (Fusani et al., 2009; Ramenofsky et al., 2008), and has also been documented in fatter birds at stopover sites (Bairlein 1985; Yong and Moore, 1993). Thus, the unlimited food supply combined with photostimulation could have contributed to reduced diurnal activity in photostimulated birds.

2.5 Conclusions

Unlike previous work, our findings suggest that eliminating androgens does not completely eliminate nocturnal migratory activity, at least for this species. Stimulation with spring photoperiods promoted migratory restlessness independently of androgens, but testosterone enhanced this photoperiod-induced behaviour, as intact and testosterone-

treated birds exhibited higher levels of migratory restlessness. Similar patterns are seen with the expression of hormone-mediated social behaviors. While highly influenced by hormones, aggression, parental and mating behaviors can all be evoked in the absence of hormones; hormones modulate the activity of neural circuits, but these neural circuits may still function in the absence of hormones (reviewed by Sternson, 2013). We conclude that, like other behaviors, migratory restlessness is modulated by androgens but is not androgen-dependent.

A modulatory role of testosterone for migratory restlessness is consistent with comparisons between spring and fall migration. Vernal migration occurs faster than autumnal migration, and the life history events and subsequent endocrine mechanisms at play during these periods of time are quite different (Maggini and Bairlein, 2010; reviewed in Wingfield et al., 1990). Driven by androgens, males rush to breeding grounds in spring to establish territories. Males who arrive earlier to breeding grounds have higher levels of testosterone than late-comers (Tonra et al., 2011b). Fall migration occurs when gonadal hormone levels are minimal (Mattocks, 1976). At this time, weather is more variable and migration is slower and includes the movement of juveniles alongside experienced adults (Wingfield et al., 1990). Thus, the migratory phenotype is expressed in both spring and fall, but the accelerated rate of spring migration, particularly by males, may be facilitated by androgens.

Nocturnal restlessness is likely regulated by a combination of many endocrine signals present during migratory seasons. Of particular interest are endocrine factors associated with regulating circadian oscillators. In birds, the pineal gland regulates daily rhythms by rhythmically producing melatonin (Takahashi et al., 1980); the highest levels are reached at night. Peak levels of melatonin are lower during migratory seasons in comparison to when birds are non-migratory (Gwinner et al., 1993). During spring and fall, there is also increased thyroid activity in migratory bird species (George and Naik, 1964; Oakeson and Lilley, 1960; Wilson and Farner, 1969) and inhibiting thyroid hormones depresses nocturnal restlessness (reviewed by Dawson, 2001 and 2003; Pathak and Chandola, 1982). Treatment with thyroid stimulating hormone (TSH; Wingfield et al., 1990) or injections of T3 and T4 (Pathak and Chandola, 1982) were effective in re- 47

instating this activity. Thus, hormones other than androgens are likely important, and may be necessary, for a migratory phenotype.

Our results add greater clarity to the gonadal hypothesis (Morton and Mewaldt, 1962; Rowan, 1932), in that we conclude that sex steroid hormones are not required for the expression of spring migratory restlessness activity. Testosterone appears to play a modulating role in rather than directly activating migratory restlessness. Although information regarding the endocrine modulation of nocturnal migratory restlessness has increased in recent years (Agatsuma and Ramenofsky, 2006; Coverdill et al., 2011; Gupta and Kumar, 2013; Ramenofsky et al., 2008; reviewed in Ramenofsky et al., 2003; Stuber et al., 2013), further investigations regarding interactions of internal mechanisms inducing this circadian behavior should be examined to broaden our understanding of important circannual patterns.

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Chapter 3 : Effects of testosterone on migration-related changes in body composition and muscle oxidative enzyme activity in the white-throated sparrow (Zonotrichia albicollis).

3.1 Introduction

Changes in body composition and energy metabolism are crucial for the successful migration of birds (Klassen et al., 2000; Jenni and Jenni-Eiermann, 1998; Jenni-Eiermann et al., 2002; Piersma, 1998). Migrating birds can fly for hours and sometimes days at a time, requiring very high aerobic and lipid oxidation capacities (Jenni and Jenni-Eiermann, 1998). These energetically expensive endurance flights are interrupted for refueling at stopovers, where the ability to digest and process food into fuel stores is paramount (Schmidt-Nielsen, 1972; Maina, 2000; Hederstrom and Alerstam, 1997). During migratory seasons, many bird species alter their physiology to enhance flight capability, fuel storage and fuel metabolism. Because these changes are reversible outside of migratory seasons they provide an important example of adult phenotypic flexibility (Piersma and Lindstrӧm, 1997; McWilliams and Karasov, 2001; Piersma and Drent, 2003).

At the individual level, changes in body composition involve shifts in the amounts of fat and lean mass. Fatty acids are the primary fuel source for avian migratory flight (Jenni and Jenni-Eiermann 1998; Guglielmo, 2010), and so in preparation for migratory seasons, birds deposit large amounts of subcutaneous and abdominal fat (Jenni and Jenni- Eiermann 1998). Fat stores can make up to over > 50 % of body mass in birds just prior to initiating migratory flights (Piersma and Gill, 1998). Fat is the primary fuel for migration because 1) fat can provide 8 - 10 times more energy per unit mass than protein or carbohydrates, 2) fat can be stored using far less water than protein or carbohydrates (in the form of glycogen), and 3) fat stores require as much as 10 times less energy to maintain than glycogen stores within the liver, or protein stores in lean tissues (Ramenofsky, 1990, Jenni and Jenni-Eiermann 1998).

Birds have a remarkable capacity to use fat during intensive exercise in comparison to other vertebrate animals. Mammals use fat primarily for low intensity exercise, and above about 40 % of maximal aerobic exercise, running mammals primarily use carbohydrates as fuel (Roberts et al., 1996; Weber et al., 1996). Mammals however, may be limited by their metabolic machinery required to process fat for energy. Flapping flight in birds on the other hand, is on average 2.3 times more energetically expensive than the maximal aerobic performance demonstrated by running mammals, but is fueled almost completely by fatty acids (Jenni and Jenni-Eiermann, 1998).

Lean mass increases in birds prior to migratory seasons in concert with changes in lipid stores (Lindstrӧm and Piersma, 1993; Karasov and Pinshow,1998; Jenni and Jenni- Eiermann, 1998; McWilliams and Karasov, 2001). During migratory periods, exercise organs, such as the major flight muscles (pectoralis, supracoracoideus and scapulohumeralis caudalis) and heart increase in size independently of body mass (Driedzic et al., 1993; Peirsma et al., 1999; Bauchinger et al., 2005). Despite hypertrophy of exercise muscles, there is no evidence for fibre type change in small passerines, as they have fast oxidative-glycolytic fibres (Lundgren and Kiessling, 1988). By having larger exercise muscles, migrants can adjust their power output and enhance their aerobic capacity (Hedenstrӧm and Alerstam, 1997; Pennycuick, 1998; Guglielmo and Williams, 2003; Bauchinger et al., 2005).

Migratory birds also change the size of digestive organs. Digestive organs will decrease in size during flight, as birds use stored protein as fuel or as a metabolic water sources (Piersma, 1990; Jenni and Jenni-Eiermann, 1998; Gerson and Guglielmo, 2011). Decreases in digestive organs are particularly evident in migrants that make multi-day flights over geographic barriers (Hume and Biebach, 1996; Piersma,1998; Piersma and Gill, 1998; Piersma et al., 1999; Battley et al., 2000; Battley et al., 2001; McWilliams and Karasov, 2001). However, birds can also make changes in digestive organ masses in anticipation of migration. In some cases, a decrease in digestive organs (gut, liver, kidneys and pancreas) may occur in order to carry less weight (Piersma and Gill, 1998; Karasov and Pinshow, 1998). By reducing the mass of such organs, birds can save energy during flights (Hedenstrӧm and Alerstam, 1997; Piersma and Gill, 1998). In other cases, 60

digestive organs increase in size during the migration season, presumably to increase refueling rate during stopover in short-hop migrants (McWilliams and Karasov, 2001, 2005; Guglielmo and Williams, 2003).

To meet the high energy demands of migratory flight, birds also have distinct metabolic adaptations in flight muscle. High rates of transport and oxidation of fatty acids are required for migratory flight, which flight muscles achieve through the upregulation of fatty acid transport proteins and oxidative enzymes (Marsh 1981; Lundgren and Kiessling, 1985; Driedzic et al., 1993; Guglielmo et al., 2002; McFarlan et al., 2009' McClelland, 2004).

Fatty acids are hydrophobic and transport must be facilitated by fatty acid transport proteins (FABP; Guglielmo, 2010; McClelland, 2004). Specifically, in muscle, fatty acid translocase (FAT/CD36) and plasma membrane fatty-acid binding protein (FABPpm) transport fatty acids from the circulation, across the muscle membrane and into the cytosol (Bonen et al., 2007; Luiken et al., 1999). Heart-type fatty acid binding protein (H-FABP) transports fatty acids within the cytosol of cells (McWilliams et al., 2004). These transporters increase in abundance/concentration during migration in birds (Guglielmo et al., 2002; McFarlan et al., 2009; Zajac et al., 2011).

Mitochondrial enzymes such as carnitine palmitoyl transferase (CPT), 3- hydroxyacyl-CoA-dehydrogenase (HOAD) and citrate synthase (CS) also increase in exercise muscle during migration to increase fatty acid oxidation capacity (Marsh 1981; Lundgren and Kiessling, 1985; Driedzic et al., 1993; Guglielmo et al., 2002; McFarlan et al., 2009; Price et al., 2010; Zajac et al., 2011). CPT is a key enzyme involved in the translocation of fatty acids from the cytosol of muscle cells across the mitochondrial membranes, and into the mitochondrial matrix (Crabtree and Newsholme 1972,1975; Marsh, 1981). HOAD is involved in the catabolism of fatty acids by the β-oxidation pathway (Beenakkers et al., 1967; Bass et al. 1969; Beenakkers 1969; Studte and Pette, 1972; Marsh, 1981). CPT and HOAD are commonly used as indicators of fatty acid oxidation capacity, whereas CS, a Krebs cycle enzyme, is used as an indicator of aerobic capacity (Lundgren and Kiessling, 1985; Marsh, 1981).

In migrating birds, the endocrine signals responsible for the up-regulation of aerobic performance, fatty acid oxidation capacity, and for changes in organ and muscle mass largely are unknown. Seasonal variation in photoperiod triggers endocrine signalling that alters behaviour and physiology in ways that promote migration (Gwinner,1990). In captive studies, manipulating photoperiod can cause birds to deposit fat and alter lean mass (Gwinner, 1989). Manipulating the light cycle can also promote the up-regulation of oxidative enzymes and fatty acid transporters during spring (Zajac et al., 2011).

The long-standing gonadal hypothesis has been used to explain the regulation of the migratory phenotype in spring (Rowan, 1932). Spring (vernal) migration is linked to the onset of reproduction and increases in gonadal steroid hormones (androgens and estrogens; Wingfield and Hahhn, 1994; Balthazart, 1983; Ketterson and Nolan, 1999; Wingfield et al., 2001). The gonadal hypothesis suggests that in addition to promoting courtship behaviours and territory defence (Moore, 1984; Wingfield, 1984; Beletsky et al., 1990; Ketterson et al., 1992; De Ridder et al., 2000; Hegner and Wingfield, 1987; Saino and Møller, 1995; Blas et al., 2006), testosterone also increases muscle mass, induces hyperphagia, and elevates fat deposition rates (reviewed in Berthold, 2001), increasing fuel stores in preparation for migration. Testosterone is also thought to increase hematocrit in birds, increasing their ability to supply active tissues with oxygen, and ultimately increasing their capacity for intense aerobic exercise (reviewed in Wingfield et al., 1990).

Previous studies of the role of gonadal hormones on vernal migration have focussed primarily on the effect of testosterone on the expression of migratory behaviour (see chapter 2), with fat scores, body mass and hematocrit typically being used as the only physiological indicators of migratory preparedness. To my knowledge, there is no literature to support the hypothesis that testosterone is involved in the regulation of organ sizes, or the biochemical machinery involved in fuel metabolism. The objective of this study was to test the hypothesis that testosterone is responsible for the adjustments in body composition and muscle oxidative enzyme activities of white-throated sparrows (Zonotrichia allibicollis) during spring migration. I predicted that the removal of 62

testosterone (via castrations and the implantation of anti-androgen pharmaceuticals) in photostimulated males, would prevent the building of exercise organs as well as inhibit the upregulation of CS, CPT and HOAD required for migratory flights.

3.2 Methods and Materials

The same white-throated sparrows used in chapter 2 were also used in this study. In brief, thirty-two male white-throated sparrows were captured in London, Ontario in October 2011 and maintained as described previously. Birds were captured under a scientific collection permit from the Canadian Wildlife Service (CA-0256) and animal procedures were approved by the University of Western Ontario Institutional Animal Care and Use Sub-Committee (protocol number 2010-216; Appendix A). The photoperiod of my animal holding room was set initially to match local civil twilight for dawn and dusk until the winter solstice (21 December 2011). At this point, photoperiod was maintained at 8 h light:16 h dark (8L:16D) for 80 days. As mentioned in chapter 2, castration surgeries were performed before the winter solstice (15 - 21 December 2011) prior to implant surgeries (10 April 2012) to produce experimental groups of sham operated males, castrated males that were treated with an aromatase inhibitor 1-4-6 androstatrien-3,17 dione (ATD) and the androgen receptor blocker flutamide (tri-fluoro- 2-methyl-4'-nitro-m-propionotoluidide), and castrated males given testosterone filled implants. Immediately following implantation, eight of the sham operated males were kept on 8L:16D photoperiod (non-migratory sham group), while the remaining 24: sham operated (n = 8), ATD/flutamide treated (n = 8), and testosterone treated (n = 8) birds were switched to a 16L:8D photoperiod for a period of 28 days (from 11 April to 11 May, 2012) to stimulate migratory behaviour and physiology. Treatment groups will be referred to hence forth as: non-migratory intact, migratory intact, migratory testosterone inhibited castrates and migratory testosterone replaced castrates.

Body Composition and Tissue Collection

During the four-week experimental period, each bird was weighed and fat scored on a weekly basis (Tonra et al., 2011). Fat scores ranged from 0 to 6; 0 meaning zero fat visible through the skin, and 6 referring to birds with fat slightly bulging from the 63

furcular fossa. This method of fat scoring was first developed in 1960 by Helms and Drury; it is the most commonly used scheme used for assessing fat quantity in birds (Krementz and Pendleton, 1990).

After 28 days of long-day photoperiod treatment (16L:8D), birds in all groups were euthanized by decapitation under isoflurane anaesthesia. Samples of right pectoralis muscle and liver were collected, weighed and immediately snap frozen using a liquid nitrogen-cooled dry shipper and stored at -80° C until analysis. Likewise, carcasses were individually packaged in plastic and frozen at -20°C until dissections were performed on the remaining organs.

Plasma Testosterone and Hematocrit

Blood samples (~ 150 µL) were collected from each of the birds before the experimental period when all birds were on a short day photoperiod, and again on day 22 of the experimental period. (3 April and 2 May 2012). This was done to measure circulating testosterone levels. All blood samples were taken within 30 minutes of disturbing birds, and were processed immediately after each bleeding session (Chapter 2). The blood was centrifuged for 10 minutes at 13,000 x g (IEC Micro-MB Centrifuge, International Equipment Company, U.S.A), and hematocrit was only measured (only at the second blood sampling during the long day photoperiod (2 May 2012). This was done by dividing the length of packed cells by the total length of all contents in the capillary tube. Hematocrit was calculated as the mean of all of the capillary tubes taken from a bird (usually 3; Fair et al., 2007).

Dissections

Prior to the day of dissection, carcasses were moved to a refrigerator to allow them to thaw. Before each dissection took place, each carcass was weighed, and tarsus and keel lengths were measured with digital calipers (± 0.01 mm). I dissected and weighed (0.001 g) the heart, flight muscle (pectoralis and supracoracoideus combined), liver, kidney, gizzard, gut (small intestine, colon and cecae combined) and the pancreas. Mesenteric fat was removed from the organs before weights were recorded. In addition,

weights of the gizzard and gut were measured after the contents were removed. The gizzard was cut open in order to be emptied, while tissue making up the small and large intestines was gently squeezed to eliminate any undigested material. Flight muscle masses obtained from the left side of the sternum were doubled to estimate of total flight muscle mass. Likewise, the total wet mass of liver was calculated by adding the amount extracted during the dissection to the weight of the sample taken and frozen when the birds were euthanized. As mentioned in Chapter 2, measurements of testis volume (mm3) acquired during dissections as well as plasma androgen levels (with an Expanded Range Salivary Testosterone Enzyme Immunoassay Kit: 1-2402-5; Salimetrics PA, USA) validated that castration surgeries and hormone manipulations were successful.

After measuring wet masses, organs were placed in an oven at 60 °C for a period of 3 to 7 days in order to obtain dry masses. Organs were weighed daily and were considered completely dry if the mass of the organ remained identical (to the nearest 0.001 gram) over two consecutive days. Once completely dry, each bird's organs were packaged in plastic and stored in a cool and dry environment. Remaining carcasses were re-packaged and frozen at -80 °C. Because a portion of the liver remained frozen at -80 °C, an estimate of the total dry liver mass was calculated using a dry fraction of the subsamples. The fraction consisted of the dissected dry liver mass divided by dissected wet liver mass. The total wet mass of the liver (wet dissected liver and liver samples taken when birds were sacrificed) was then multiplied by the fraction to acquire an estimate of the total dry mass.

Enzyme Assays

A previous study found negligible differences in oxidative enzyme activity in cardiac tissue in non-migratory and migratory white-throated sparrows (Zajac et al., 2011), therefore, enzyme assays were only performed using pectoralis muscle. Pectoralis muscle (approximately 100 mg) was homogenized three times (10 s each with 30 s between) using a Polytron PT 10 - 35 homogenizer with a 7 mm generator (Kinetica

USA), in 20 mM Na2HPO4, 0.5 mM EDTA, 0.2 % defatted BSA, 50 % glycerol (Caledon Laboratories, Georgetown, ON), 0.1 % Triton x-100, and Aprotinin at 50µg/mL (Price et

al., 2010). To disrupt the membrane of muscle cells, the homogenized samples were then sonicated three times (again, waiting 30 s between each 10 s sonication; VirSonic Ultrasonic Cell Disrupter 100, VirTis, USA). The wattage of the sonicator was kept just below a level that caused foaming, homogenates were kept on ice at all times during the homogenization process, and stored at -80° C until the enzyme assays. Homogenates were used the next day in enzyme assays for lactate dehydrogenase (LDH). Assays concluded approximately three months after homogenates were made.

Enzyme assays were performed in duplicate at 39° C using a Cary 100 UV/Vis spectrophotometer (Varian, Palo Alto, CA) using disposable polystyrene cuvettes with a 1 mL reaction volume. Temperature was maintained using a Peltier temperature control module. Measurements were accepted if replicates were within 5 %, otherwise additional replicates were assayed until this criterion was met. CPT, HOAD, CS and LDH were assayed under the conditions shown in Table 3.1 following McFarlan et al., (2009), Price et al., (2010) and Zajac et al., (2011). Activities were calculated from ΔA412 for CS and

CPT and from ΔA340 for HOAD and LDH.

Table 3 - 1: Assay conditions used to measure activity of Carnitine Palmitoyl Transferase (CPT), 3-Hydroxyacyl-CoA-Dehydrogenase (HOAD), Citrate Synthase (CS), Lactate Dehydrogenase (LDH).

Enzyme Substrates 50 mM Tris Buffer (pH 8.0), 5 mM carnitine Carnitine Palmitoyl (C0283), 0.15 mM DTNB (D8130), 0.035 mM Transferase, CPT palmitoyl CoA (P9716), 10 µL of homogenate (EC2.3.1.21) diluted 1:5

3-Hydroxyacyl-CoA- 50 mM Imidazole buffer (pH 7.4), 0.2 mM NADH Dehydrogenase, HOAD (N9129), 1 mM EDTA (D0632), 0.1 mM acetoacetyl (EC1.1.1.35) CoA (A1625), 10 µL of homogenate diluted 1:10

Citrate Synthase, 50 mM Tris buffer (pH 8.0), 0.5 mM oxaloacetic acid CS (EC 2.3.3.1) (O4126), 0.15 mM DTNB (D8130), 0.3 mM acetyl

CoA (A2056), 10 µL of homogenate diluted 1:20

Lactate Dehydrogenase, 50 mM Imidazole buffer (ph 7.4), 8 mM pyruvate LDH (EC1.1.127) (P8574), 0.3 mM NADH (N9129), 5 mM DTT (D0632), 10 µL of homogenate diluted 1:100

**All reagents were purchased from Sigma-Aldrich, Oakville Ontario Canada. Product numbers are in brackets beside each compound.

Statistical Analyses

Statistical analyses were conducted using SPSS version 20.0 software (SPSS Inc. Chicago IL, USA). A principle components analysis (PCA) of tarsus and keel lengths was used to derive a measure of structural body size (Rising and Somers, 1989). PC1 explained 57.4 % of the variation, and the eigenvectors for the two variables were both positive (0.76 for both keel and tarsus loadings).

Hematocrit was compared among treatment groups by an ANOVA followed by a least significant difference (LSD) post hoc test. A repeated measures general linear model using PC1 as a covariate was used to compare differences in body masses and fat scores 67

within and among treatment groups across the 28 d experimental period. LSD post hoc tests were performed if the main effect of treatment was significant and there was no significant interaction between treatment and PC1.

Dry organ/muscle masses were compared among treatment groups by ANCOVA with log(body mass - wet organ mass) as a covariate. This covariate was used to eliminate part-whole correlations between organ/muscle mass and body mass (Christians, 1999). The covariate for body size (PC1) was also used as a covariate in analyses for flight muscle because flight muscle mass is dependent on the size of the rib cage. All mass variables were log transformed to account for allometric scaling. Comparisons were only made if treatment by body size or treatment by body mass interactions were not significant. Least significant difference (LSD) post hoc analyses were conducted if ANCOVA tests determined that there were significant differences in organ/muscle masses among treatment groups. A principle components analysis of log10 dry masses of organ/muscles was also used to examine relationships among body components for birds within all treatment groups (Guglielmo and Williams, 2003). Loadings on each axis from the principle component analyses were compared in all treatment groups using ANCOVA, with the body size covariate (PC1). If no interactions existed between loadings and body size and main effects were evident, LSD post hoc tests were performed. Organ masses were plotted as the least square mean log 10 dry mass (g). To maintain positive bars for each of the graphs, + 2 was added to each of the measurements in all organ component data sets.

Muscle enzyme activities (CPT, CS, HOAD and LDH) were compared among treatments using ANCOVA with log10body mass as a covariate. LSD post hoc tests were performed if there was no significant treatment by mass interaction and if there was evidence of a significant difference in enzyme activity among treatments.

Validation of Treatment Efficacy

As described in chapter 2, EIA concentrations of androgens alongside measurements of testis volume (mm3) validated that the castration surgeries and administration of pharmaceuticals (ATD and FLUT) were successful at inhibiting 68

circulating testosterone (Figure 2 - 1). Similar to free-living birds, testosterone levels increased after exposure to spring photoperiods (Wingfield et al., 1990). This finding agrees with testes measurements acquired from migratory intact males. Migratory intact males had much larger testes than non-migratory intact males who remained on short day light cycles; this finding is comparable to prior data from captive white-throated sparrows (Harris and Turek, 1982). Both of these results indicate that the effect of photoperiod was enough to bring males into breeding condition.

3.3 Results

Body Mass and Fat Scores

For body mass there was no three-way interaction between treatment group, experimental day and the covariate for body size (PC1; Figure 3 - 1: A; F3,24 = 0.439, P = 0.727). There was also no interaction between treatment group and the body size covariate once the three-way interaction term was removed (F12, 108 = 0.911, P = 0.46). After the body size covariate was removed, there was a significant difference in body mass among treatment groups (F4, 112 = 18.35, P < 0.001) as well as a significant effect of treatment on body mass over the experimental period (F12, 112 = 3.32, P < 0.001). LSD Post hoc tests indicated that over the experimental period, testosterone-inhibited castrates were on average 12% lower in body mass than intact migratory males (P = 0.049), but were not statistically different from the two other treatment groups. The body masses of intact non-migratory males, testosterone-treated castrates, and migratory intact males were similar over the 28 day period. Post hoc comparisons of body mass over the experimental period indicated that body mass decreased in all groups during the second week (day 7) of the photostimulation experiment (P < 0.001), and then stabilized in all treatment groups (including those that remained on the winter short light cycle), staying lower than prior to photostimulation for the rest of the experimental period (P < 0.001 for all comparisons between day 1 and subsequent experimental days). Body mass on day 7 was significantly lower than masses recorded on days 1, 14 and 21 (P < 0.005 for all comparisons), but these masses were identical to those recorded on day 28 (P = 0.13). Body mass between all treatment groups did not change over days 14, 21 and 28 (P > 0.1

for all). One way ANOVAs that separately analyzed body mass on each experimental day did not reveal a significant difference among treatment groups. (Day 1: F3,27 = 2.41, P =

0.089; Day 7: F3, 27= 1.80, P = 0.17; Day 14: F3,27 = 2.9, P = 0.053; Day 21: F3,27 = 1.11, P

= 0.36; Day 28: F3,27 = 1.37, P = 0.27).

For fat score there was no interaction between experimental day, treatment group and body size (PC1; Figure 3 - 1:B; F12,96 = 1.828, P = 0.054). Once the PC1 interaction term was removed there was a significant effect of experimental date on fat score over the 28 day experimental period (F4, 112 = 6.34, P < 0.001), as well as an effect of treatment on fat score over the 28 days (F12, 112 = 2.36, P = 0.010). Post hoc tests indicated that the average fat score in all treatment groups did not change during the first week after photostimulation (P = 0.917 between days 1 and 7). Over days 7 to 21, fat score increased in all treatment groups (P < 0.001 between days 7 and 14, P = 0.005 between days 14 and 21). Fat score remained stable over the last week of the experiment (P = 0.886 between days 21 and 28). There was no significant difference in fat score in all treatment groups over the 28 day period (P > 0.1 for all comparisons). Subsequent univariate ANOVAs analysing fat score among treatment groups separately on each experimental day also did not find any statistical difference (Day 1: F3,28 = 0.25, P = 0.86; Day 7:F3,28 = 1.5, P =

0.24; Day 14:F3,28 = 1.97, P = 0.14; Day 21: F3,28 = 1.23, P = 0.32; Day 28: F3,28 = 1.51, P = 0.23).

Figure 3 - 1: Body mass (A) and furcular fat score (B) of male white-throated sparrows (Zonotrichia albicollis) following manipulations of photoperiod and testosterone. Prior to day one, birds were exposed to short days (8L:16D). On day one photostimulated birds (16L:8D) included migratory intact males (filled circles with thick solid line), ATD+FLUT castrates (solid squares with dotted line) and testosterone-treated castrates (filled triangles with dashed line). Non-photostimulated males (represented by open circles and a thin solid line) remained on short light cycles throughout the experimental period. For all treatment groups, n = 8. 71

Variation in Digestive Organs

The only organ component where differences in mass were found among treatment groups was the liver (Figure 3 - 2). There was no interaction between body mass and dry liver mass among treatments (F3,24 = 1.387, P = 0.271), but after the interaction was removed, there was evidence of a significant difference between treatment groups (F3,27 = 14.78, P < 0.001). However the body mass covariate had no effect in the model (F1,27 = 3.782, P = 0.062). LSD post hoc tests determined that migratory groups had decreased liver masses in comparison to short-day non-migratory males (P < 0.001 for all pair wise comparisons). Migratory groups were not statistically different from each other (P > 0.05 for all comparisons).

There were no interactions between body mass and dry kidney, gizzard and pancreas mass (kidney: F3,24 = 1.297, P = 0.298; gizzard: F3,24 = 0.717, P = 0.552; pancreas: F3,24 = 0.011, P = 0.998). Once interaction terms were removed from each model, no significant main-effects were detected for kidney, gizzard and pancreas mass among treatment groups (kidney: F3,27 = 1.25, P = 0.31; gizzard: F3,27 = 2.043, P = 0.132; pancreas: F3,27 = 0.291, P = 0.832). Kidney and gizzard mass scaled positively with body mass (kidney: F1,27 = 12.1, P = 0.002; gizzard: F1,27 = 9.01, P = 0.006), but there was no effect of the body mass covariate in analyses for the pancreas (F1,27 = 0.815, P = 0.375).

Figure 3 - 2: Dry mass of digestive organs of male white throated sparrows ((Zonotrichia albicollis) following testosterone and photoperiod manipulations. Least square means of log10 +2 dried liver, kidney, gizzard and pancreas (g). Migratory intact males, migratory ATD+FLUT castrates and migratory testosterone-treated castrates were switched to longer light cycles (16L:8D), from short days (8L:16D). Treatment groups with letters (a, b, c) are statistically different, P < 0.05. For all treatment groups, n = 8.

A significant interaction existed between body mass and treatment groups for dry gut mass (F3,24 = 3.68, P = 0.026; Figure 3 - 3). When the ANCOVA was run after testosterone inhibited males were removed from the data set, this interaction was no longer significant (F2,18 = 2.10, P = 0.152). Once this interaction term was removed, there was no difference among the three remaining treatment groups (F2,20 = 0.36, P = 0.70), and dry gut mass scaled positively with the body mass covariate (F1,20 = 32.50, P <

0.001). No relationship existed between body mass and dry gut mass for testosterone inhibited males when analysed separately (r2 = 0.190, P = 0.28).

Figure 3 - 3: The relationship between dry gut mass and body mass of male white- throated sparrows (Zonotrichia albicollis) after testosterone and photoperiod manipulation. Migratory intact males, ATD+FLUT castrates and testosterone-treated castrates were switched from short (8L:16D) to long-days (16L:8D). Non migratory intact males remained on short days for the entire experiment. The following symbols represent treatment groups that have a positive linear relationship between dry gut mass and body mass signified by the solid line (P < 0.001, r2 = 0.613, y = 1.027x -2.62), filled circles: migratory intact males; open circles: non-migratory males; filled triangles: testosterone-treated castrated males. No relationship between dry gut mass and body mass existed in migratory ATD+FLUT castrates, represented by filled squares (following the dashed line: P = 0.28, r2 = 0.19, y = -1.63x + 1.22). For all treatment groups, n = 8.

Exercise Machinery

Hematocrit

Hematocrit differed in treatment groups (Figure 3 - 4; F1,3 = 3.91 P = 0.019). Post hoc tests indicated that non-migratory short-day males had significantly lower hematocrit than the three migratory long-day groups (by approximately 6%), with no significant difference among the three migratory groups.

Figure 3 - 4: Hematocrit (percentage of packed cell volume) of male white-throated sparrows (Zonotrichia albicollis) after photoperiod and hormonal manipulation. Prior to the experimental period, all birds were kept on short days (8L:16D), non-migratory males were maintained on this light cycle. Migratory intact males, migratory ATD+FLUT and testosterone-treated castrates were moved to long days (16L:8D) at the onset of the experiment. Letters (a, b) indicated treatment groups that are considered statistically similar (P > 0.05). Error bars represent one standard error. For all treatment groups, n = 8.

Variation in Flight Muscle and Heart

There was no evidence of a three-way interaction between body size, body mass and dry flight muscle (F4,16 = 0.35, P = 0.84), nor significant evidence of an interaction between body size (F3,20 = 2.27, P = 0.11) or body mass (F3,23 = 1.69, P = 0.198) and dry flight muscle. Once all interaction terms were removed from the model, no significant main-effects in dry flight muscle were present among treatment groups (Figure 3 - 5; F3,26 = 0.881, P = 0.464). Likewise, there was no interaction between body mass and dry heart mass (F3,24 = 0.15, P = 0.93). After this interaction term was removed, no significant differences were seen in dry heart mass in all treatment groups (F3,27 = 1.641, P = 0.203).

Figure 3 - 5: Dry mass of exercise organs of male white-throated sparrows (Zonotrichia albicollis) following hormone and photoperiod treatment. Least square means of log10 + 2 dry flight muscle (pectoralis and supracoracoideus) and heart mass (g). Non-migratory intact males remained on short photoperiods (8L:16D), and migratory intact, ATD+FLUT castrates and testosterone-treated castrates were transitioned to long day light cycles (16:8D). Error bars represent one standard error. For all treatment groups, n = 8.

Multivariate Analysis of Body Composition

The first two principal component axes explained 60.8 % of the variation in dry organ and muscle masses (Table 3 - 2, PC1: 40.2 %; PC2: 20.6 %). All variables loaded positively on the first principal component axis, which reflected absolute component mass. No interaction existed between the body size covariate and loadings of PC1 within each treatment group (F3,24 = 0.58, P = 0.63). Controlling for body size, there was a significant difference in the PC1 scores among treatment groups (F3,27 = 5.61, P = 0.004). Migratory intact males, testosterone inhibited and testosterone replaced castrates had significantly lower component scores than non-migratory males (P = 0.03, P < 0.01 and P = 0.02, respectively). The three migratory treatment groups had statistically similar PC1scores (P > 0.05 for all pair wise comparisons). The second principle component axis mainly reflected differences in residual variation related to the size of exercise and digestive organs. The flight muscle and heart loaded positively alongside the pancreas, whereas negative loadings were found in the liver, kidney, gut, and gizzard. Once insignificant interactions between body size and organ component among groups was removed (F3,24 = 2.1, P = 0.12), no difference existed in PC2 scores among treatment groups (F3,27 = 1.96, P = 0.14). To show differences in allocation and component size among treatment scores, PC1 scores were plotted against PC2 scores (Figure 3 - 6).

Table 3 - 2: Eigenvalues of the first three principal components of log-transformed masses of dried organ components of males white-throated sparrows, Zonotrichia albicollis (n = 32).

Organ Component PC1 PC2

Flight Muscle 0.220 0.447 Heart 0.154 0.365 Liver 0.292 -0.207 Kidney 0.292 -0.035

Gizzard 0.254 -0.177 Pancreas 0.129 0.320 Gut 0.190 -0.429

Variance Explained 40.2% 22.6%

Figure 3 - 6: Principal component scores for male white-throated sparrows (Zonotrichia albicollis) after photopeiod and hormone manipulation. Two axes were generated from a principal components analysis of body composition. Open circles represent non- migratory intact males kept on short days (8L:16D). Migratory groups were photostimulated from short days to long days (16L:8D). Closed circles represent migratory intact males. Filled triangles represent migratory ATD+FLUT castrates and filled squares represent migratory testosterone-treated castrates. PC1 reflected overall component masses and PC2 reflected exercise and digestion organ components (positive vs. negative loadings, respectively). Non-migratory males had significantly larger PC1 scores than migratory groups (F3,27 = 5.61, P = 0.004). For all treatment groups, n = 8.

Variation in Metabolic Enzyme Activity

There was no interaction between body mass and treatment group for LDH,

HOAD, or CPT activity (Figure 3 - 7: F3,56 = 0.58, P = 0.63; F3,56 = 2.17, P = 0.10; F3,56 = 1.76, P = 0.16, respectively). Once these interaction terms were removed in each model, there was a statistical difference among the treatment groups for each enzyme (F3,59 =

25.07, P < 0.001 for LDH; F3,59 = 8.13, P < 0.001 for HOAD and F3,59 = 16.08, P < 0.001 79

for CPT). LDH activity scaled positively with body mass (F1,59 = 14.20, P < 0.001), but the effect of this covariate was not significant in analyses for HOAD or CPT (F1,59 = 0.89,

P = 0.35 and F1,59 = 0.80, P = 0.38, respectively).

Activity of LDH decreased by 37 % in migratory treatments when compared to wintering intact males (Figure 3 - 7; P < 0.050 for all pair wise comparisons), indicating an overall affect of the photoperiod treatment. Differences within photostimulated groups existed as well. LDH activity was 28 % greater in castrated males treated with testosterone and 11 % lower in castrates implanted with ATD and flutamide in comparison to migratory intact males. Migratory testosterone treated castrates had significantly greater LDH activity levels than the other two migratory groups (P < 0.001 for migratory intact males and migratory testosterone-inhibited castrates). There was no difference between migratory intact males and testosterone inhibited castrates; (P = 0.58).

Activities of enzymes used for fatty acid oxidation were greater in the migratory groups compared to non-migratory groups (HOAD: + 84 %, CPT: + 259 % , Figure 3 - 7; P < 0.001 for all pair wise comparisons of treatment groups for HOAD and CPT). Within the three migratory treatment groups, there were differences in the levels of enzyme activity, primarily between the intact males and the two groups containing castrated males. For HOAD, testosterone replaced castrates were similar to testosterone inhibited males and non-migratory intact males (P = 0.23 and P = 0.082, respectively). Migratory intact males and testosterone inhibited castrated males were statistically similar to each other (P = 0.091). CPT activity of testosterone inhibited and testosterone treated castrated males were also statistically similar (P = 0.603). Migratory intact males displayed the greatest upregulation in CPT activity and were statistically different than the two migratory castrated groups (P = 0.012 for testosterone treated castrates, and P = 0.004 for testosterone inhibited castrates).

A significant interaction was found between body mass and treatment group for

CS activity (Figure 3 - 7; F3,56 = 5.97, P = 0.001). Linear regressions were significant for 2 both migratory castrated treatment groups (F1,14 = 8.30, P = 0.012, r = 0.37 for 2 testosterone treated males; F1,14 = 24.71, P < 0.001, r = 0.64 for testosterone inhibited

males), indicating a positive relationship between CS activity and body mass. There was no evidence of a relationship between body mass and CS activity for either migratory intact males (F1,14 = 0.007, P = 0.94) or for non-migratory intact males (F1,14 = 0.78, P = 0.39). Comparing the two groups, CS increased by 38 % in migratory intact birds from intact non-migrants. Even taking into account the different effects of body mass among treatment groups, both castrated treatment groups (testosterone inhibited and testosterone replaced males) had CS activities that were generally intermediate to migratory and non- migratory intact males.

Figure 3 - 7: Oxidative enzyme activity of male white-throated sparrows (Zonotrichia albicollis) after hormone and photoperiod manipulation. Mean maximal enzyme activity (µmol/min/g of wet weight in pectoralis muscle)  standard error of lactate dehydrogenase (A), hydroxyacyl-CoA dehydrogenase (B) and carnitine palmitoyl transferase (C). Migratory intact males, ATD+FLUT castrates and testosterone treated castrates were transitioned from short (8L:16D) to long days (16D:8L). Non migratory intact males remained on short days. Citrate synthase, CS activity taking into account the effects of body mass (D). Treatment groups in panel D are represented as follows: non- migratory intact males: open circles with thin solid line, migratory intact males: filled circles with thick solid line, migratory ATD+FLUT castrates: squares with dashed line (P < 0.001, r2 = 0.64, y = 795.6x - 994.76), migratory testosterone-treated castrates: triangles with dotted line (P = 0.012, r2 = 0.37, y = 300.21x - 317.28). Letters (a,b,c) indicate treatment groups that are statistically different, P < 0.05). For all treatment groups, n = 8. 82

3.4 Discussion

Photoperiod had a greater effect on body composition (fat and lean mass), organ component mass, enzyme activity, and hematocrit than testosterone manipulation. White- throated sparrows photostimulated with long days displayed several physiological changes in body composition that were consistent with preparation for vernal migration. Migratory males increased fat stores, hematocrit and decreased liver mass, despite hormonal treatment. Likewise, the upregulation of oxidative enzymes was primarily photoperiod-driven. However, without gonadal tissue, oxidative enzyme activity of castrated males did not reach levels of migratory intact males. These results suggest gonadal hormones do not play a major role in promoting seasonal phenotypic flexibility of physiology and biochemistry in migratory birds. Despite assumptions that the anabolic effects of testosterone build exercise organs as well as increase appetite (hyperphagia) and fuel deposition during spring, it appears other neuroendocrine signals are responsible for the upregulation of these systems. In addition, this study emphasises that biochemical indicators may be more reliable at signifying whether individuals are primed for migratory flights than comparisons of organ component mass.

Body Mass and Fat Stores

Stress from increased handling may have affected total body mass and fatness in all treatment groups, particularly in the first week following photomanipulation, where birds were handled and disturbed more frequently. After photostimulation, captive birds initially lose weight, but then become hyperphagic and rapidly gain mass and fat (Lindstrӧm et al., 2000; reviewed in Wingfield et al., 1990). The body masses I observed were similar to those recorded in free-living birds, where wintering white-throated sparrows had a similar mass to spring migrants (McFarlan et al., 2009).

Testosterone inhibited males were statistically smaller at the outset of the study than the other treatment groups. This was evident in analyses comparing body mass over the experimental period. Migratory treatment groups were also found to be statistically smaller than non-migrants in analyses of the first principal component score generated

from dry organ components. While I randomly assigned birds to treatment groups, castration surgeries may have favoured leaner birds. Weise (1967) had similar issues performing castrations on white-throated sparrows. If testes could not be located or safely removed, birds were assigned to a sham-operated treatment group. Testes are more easily detected when there is less fat in the body cavity, therefore, the final organization of treatment groups may have resulted in castrated groups containing overall smaller individuals than sham-operate groups.

Fat stores generally increased in migratory treatment groups over weeks two through four after photostimulation. As the primary fuel source for flight, fat is crucial for the success of migration, and both captive and free-living birds increase fat stores in preparation of endurance flights (Wingfield et al., 1990). While the statistical analyses seem to hide this trend, fat scores of non-migratory males did not appear to increase over the experimental period. These birds had substantial fat stores prior to manipulations, therefore fat scores would not be expected to increase further if short photo-cycles were maintained over the experiment. Fat deposition in migratory treatment groups did not occur until after week two. The delayed deposition of fat may also have been caused by induced stress and increased glucocorticoid hormone levels (corticosterone) after implantation surgeries. Corticosterone levels triple in captive starlings during handling; and have catabolic effects on fat stores (Remage-Healey and Romero, 2001). Glucocorticoids regulate the mobilization of stored lipids from adipose tissue, providing the stressed individual with energy (Gregoire et al., 1991; Ramenofsky, 1990).

At the end of the experimental period, males maintained lower weights than prior to photostimulation, however fat scores steadily increased. The density of fat is less than muscle, therefore fat could increase without dramatically increasing total body mass. Increased fat scores despite maintenance of total body mass appear to reflect decreases in organ mass. This indicates that the experimental treatment affected both fat and lean components.

Phenotypic Flexibility of Digestive Organs

My results are consistent with previous trends, where the liver follows a "digestive organ role" during migratory seasons (Hume and Biebach, 1996; Piersma and Gill, 1998; Battley et al., 2000; Landys-Ciannelli et al., 2003; Bauchinger et al., 2005; Karasov and Pinshow, 1998). Liver mass decreased in all migratory treatment groups regardless of hormone treatment. This suggests that testosterone is not responsible for altering the size of this organ. In addition, in a principal components analysis, the liver loaded negatively with the kidney, gizzard and gut; a pattern that is similar to several other studies (Piersma et al., 1996a; Piersma et al., 1999; Guglielmo and Williams, 2003). Unlike the liver, gut mass did not decrease in migrants. Dry gut mass was strongly positively related to body mass in non-migrant males, migratory males and testosterone treated castrates. When testosterone was removed gut mass was no longer positively correlated withbody mass, suggesting that androgens may be related in a complex or indirect manner to changes in gut size. A variety of hormones and enzymes regulate digestion (Denbow, 2000), however the endocrine signals that control digestive organ masses are poorly understood. Metabolic and growth regulating hormones may play a role in the seasonal shifts in digestive organ mass often documented in migratory birds. For example, thyroid hormones regulate body temperature, and stimulate thermogenesis and cell metabolism (Wrutniak-Cabello et al., 2001; Zaninovich et al., 2003).

Triiodothyronin, (T3) has been correlated with increased lean organ components in sparrows acclimated to cold temperatures (Zheng et al., 2008). The growth hormone and insulin-like-growth factor I axis (GH-IGF-I) is a complex system that regulates pre- and post-natal growth (reviewed in Roith et al., 2001). Growth hormone stimulates body growth. IGF-I is secreted from most tissues (including the liver, pancreas, intestines, kidneys and skeletal muscle) and mediates the effects of growth hormone secreted from the anterior pituitary on target tissues (Roith et al., 2001).

Exercise Machinery

Hematocrit

Hematocrit fell within normal levels typically seen in captivity (35 - 55 %), and therefore the birds were not anemic or polycythemic (Campbell, 1994). Major factors that influence hematocrit include age, reproductive status, energy metabolism, season, parasitism and nutritional state (Fair et al., 2007). While decreases in hematocrit have been reported in egg-laying female European starlings, Sturnus vulgaris (Williams et al., 2004), several studies document that increasing gonadal androgens increase hematocrit in birds during the breeding season (Robinzon and Rodgers, 1979; Thapliyal et al., 1983; Fair et al., 2007). Unlike these studies, testosterone removal and replacement did not affect hematocrit. Hematocrit increases during prior to migration and during stop-over in free living migrants (deGraw et al., 1979; Piersma et al., 1996b; Landys-Ciannelli et al., 2002); increased hematocrit would benefit migrants by increasing their aerobic capacity via improving their ability to transport oxygen to active muscle groups during energetically demanding aerobic flights (Bairlein and Totzke, 1992; Piersma et al., 1996b; Prats et al., 1996; Wingfield et al., 1990).

Increased energy expenditure could provide an alternative explanation for the increase in hematocrit in migratory groups (Fair et al., 2007). Migratory groups were exposed to longer day lengths (16 hours) and exhibited nocturnal restlessness (Chapter 2, Figure 2 - 1). In comparison, non-migratory males were only active for 8 hours and exhibited minimal movement at night. Prolonged increased activity in migratory males may have been sufficient to decrease plasma volume, elevate erythropoietin levels and increase hematocrit to levels higher than seen in non-migratory males (Birchard, 1997; Geyssant et al., 1981; Jelkmann, 2004). This could be tested further by including migratory groups kept in complete darkness at night, which have been shown to not express migratory restlessness (Ramenofsky et al., 2008).

Flight Muscle and Heart Mass

Flight muscle and heart mass were not affected by photoperiod manipulation or hormonal treatment. Negligible differences in these organs among intact migratory and non-migratory controls may have been due to unlimited food abundance while in captivity. Free-living garden warblers migrating over food-rich landscapes also did not experience changes in flight muscle mass (Bauchinger et al., 2005). Body mass may have already reached a "maximum level" given the structural frame size in non-migrants, inhibiting their capacity for flight (Hedenstrӧm and Alerstam, 1992). Perhaps further increases in muscle and heart mass may not have been achievable. Hypertrophy of flight muscle and heart mass may also be dependent on exercise: heart mass of western sandpipers (Calidris mauri) increased 10 – 25 % during migration (Guglielmo and Williams, 2003). Testosterone has anabolic effects on skeletal muscle by binding to androgen receptors in myocytes that activate genes regulating protein synthesis (Griggs et al., 1989; Bhasin et al., 2012). However, myostatin and insulin-like growth factor can also independently increase lean mass (Bhasin et al., 2012; Lee, 2004; Velloso, 2008). Testosterone-inhibited males had the smallest flight muscle and heart however, analyses of body mass over the experimental period indicated that this group was inherently smaller in total body mass. In previous studies, flight muscle and heart mass track body mass (Marsh, 1984; Lindstrӧm et al., 2000), therefore, the small mass of testosterone- inhibited individuals may have been an artifact of body size. However, positive eigenvalues for the heart and flight muscle are similar to patterns seen in previous studies (Piersma et al., 1996a; Piersma et al., 1999; Guglielmo and Williams, 2003).

Enzyme Activities in Flight Muscle

My results confirm those of Zajac et al., (2011), who found that vernal photostimulation of white-throated sparrows led to upregulation of oxidative enzymes (CS, CPT, HOAD) and down-regulation of LDH in flight muscles. The negative relationship between LDH and CS activity in photostimulated birds suggests that tradeoffs exist between anaerobic and aerobic metabolism in muscle during migratory seasons (McFarlan et al., 2009; Zajac et al., 2011). Premigratory changes anticipate the

demand for increased rates of lipid metabolism and enhanced oxidative capacity during endurance flights.

Migration related changes in oxidative enzymes and LDH were not fully expressed in castrated sparrows, regardless of testosterone replacement. This suggests that endocrine factors secreted by gonadal tissue, other than testosterone may influence metabolic enzyme activity. Similar conclusions can be made concerning the interactions present in analyses for CS. Strong positive relationships existed between CS activity and body mass for both castrated treatment groups, while no such relationship was present in either sham-operated group. Enzyme activity may be dependent on estrogens rather than androgens. Aromatase-inhibited male mice (via disruption of the gene Crp19) had suppressed activity of β-oxidation enzymes in the liver (Toda et al., 2001), which was restored when mice were treated with 17β-estradiol (Toda et al., 2001). However, aromatase was not inhibited in testosterone treated castrated males, so theoretically enzyme activity should have been rescued to levels seen in migratory intact males.

Phenotypic Flexibility and Migration Strategy

Changes in organ mass (maintenance of flight muscle and heart and reductions of liver, kidney and gizzard) in migrants are typical of seasonal phenotypic flexibility (reviewed in Piersma and Linstrӧm, 1997; McWilliams and Karasov, 2001). Flight performance is selectively improved by metabolizing digestive organs in order to maintain a lighter weight during endurance flights while increasing power output with larger exercise organs (Driedzic et al., 1993; Piersma et al., 1996a; Piersma et al., 1999; Guglielmo and Williams, 2003; Landys-Ciannelli et al., 2003; Bauchinger et al., 2005). However, this re-arrangement of lean components is typical of long-distance migrants that cross geographic barriers (Piersma and Gill, 1998; Bauchinger et al., 2005). Short distance migrants, stop to refuel often, and therefore retain large digestive organs to maintain increased levels of digestive assimilation (McWilliams and Karasov, 2005; Guglielmo and Williams, 2003; Karasov, 1990). This strategy allows these species to refuel at a faster rate since they do not have to spend extra time rebuilding digestive organs prior to depositing fat such as long-distance migrants (McWilliams and Karasov,

2005). White-throated sparrows are short-hop migrants making frequent stop-overs, however, in my study they appear to follow a long-distance migrant strategy described previously in shorebirds and garden warblers (Hume and Biebach, 1996; Piersma, 1998; Piersma and Gill, 1998; Piersma et al., 1999; Battley et al., 2000; Battley et al., 2001).

The physiological strategy for migration by white-throated sparrows may be related to the environmental conditions on wintering grounds (Dawson et al., 1983). Some populations of white-throated sparrows spend the winter in more northern latitudes, and endure cold climates (Dawson et al., 1983; Falls and Kopachena, 2010). Surviving colder climates requires increases in digestive capacity and an increased production of metabolic heat (Dawson et al., 1983). Birds in northern latitudes have higher basal metabolic rates (BMR) than birds found in lower latitudes (Kersten and Piersma, 1987; Piersma et al., 1995). Higher metabolic rates are achieved by maintaining larger proportions of metabolically active lean tissues, which increase total heat production (Block, 1994, Daan et al., 1990; Marsh and Dawson, 1989; Weber and Piersma, 1996; Zheung et al., 2008). Cold acclimated captive red knots (C. islandica), hoopoe larks (Alaemon alaudipes) and Eurasian tree sparrows (Passer montanus) had increased body masses, food intake, metabolic rates as well as increases in lean mass (Vezina et al., 2006,Williams and Tielman, 2000; Zheng et al., 2008). Larger digestive components can assimilate more energy and nutrients from food per unit time, and therefore reduce time and energy spent foraging (reviewed by Karasov, 1990; McWilliams and Karasov, 2003). In white-throated sparrows, large digestive components necessary for winter survival may be atrophied in spring in anticipation of endurance flights and migration.

3.5 Conclusions

My study indicates that separate neuroendocrine factors may regulate the various physiological modifications that are typical of the migratory phenotype. Effects of photoperiod on liver mass and hematocrit appear to occur independently of androgens. Changes in light availability may regulate alternate hormone signalling pathways released by deep brain photoreceptors in birds mediating seasonal changes in physiology (Ikegami and Yoshimura, 2012). Intermediate increases of oxidative enzyme activity in castrated

males suggest gonadal hormones other than sex steroids may partially control this seasonal upregulation. Although testosterone appears to modulate nocturnal migratory restlessness (Chapter 2), there was little evidence that it is involved in regulating physiological and biochemical adaptations important for improving and fuelling flight performance.

These findings further emphasize the effect of environmental cues on life history strategies. External stimuli, particularly photoperiod, are crucial for the organization of circadian and circannual rhythms regulating seasonal events (reviewed in Gwinner,1990, 1996). In addition, temperature and climate influence metabolic strategies and the distribution of organ component mass. Our findings also show that changes in organ component size are not indicative of up regulated oxidative enzyme activity required to metabolize lipids. Future migration studies should examine biochemical indicators as well as compare masses of digestive and exercise organs, in addition to photostimulating birds to fully interpret the life history strategy of a species. There are likely many neuroendocrine signals working to produce physiological adaptations in response to environmental signals, primarily photoperiod. More research should be conducted to indentify which hormones are linked to the selective catabolism of digestive organs and the upregulation of oxidative enzymes in migratory birds.

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Roith, D.L., Scavo, L. and Butler, A. (2001). What is the role of circulating IGF-I? J Endocrinol Metab, 12, 48-52.

Schmidt-Nielsen, K. (1972). Locomotion: energy cost of swimming, flying, and running. Science, 177, 222-228.

Soma, K. K., Sullivan, K., and Wingfield, J. (1999). Combined aromatase inhibitor and antiandrogen treatment decreases Territorial aggression in a wild songbird during the nonbreeding season. Gen Comp Endocrinol, 115, 442-453. doi:10.1006/gcen.1999.7334

Staudte, H.W., and Pette, D. (1972). Correlations between enzymes of energy-supplying metabolism as a basic pattern of organization in muscle. Comp Biochem Physiol, 41, 533-540.

Tonra, C. M., Marra, P. P., and Holberton, R. L. (2011). Migration phenology and winter habitat quality are related to circulating androgen in a long-distance migratory bird. J Avian Biol, 42, 397-404.

Toda, K., Takeda, K., Akira, S., Saibara, T., Okada, T., Onishi, S., and Shitzuta, Y. (2001). Alterations in hepatic expression of fatty-acid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17β-estradiol or peroxisome proliferator. J Steroid Biochem Mol Biol, 79, 11-17.

Velloso, C. P. (2008). Regulation of muscle mass by growth hormone and IGF-I. Br J Pharmacol, 154, 557-568. doi:10.1038/bjp.2008.153

Vezina, F., Jalvingh, K.M., Dekinga, A., and Piersma, T. (2006). Acclimation to different thermal conditions in a northerly wintering shorebird is driven by body mass-related changes in organ size. J Exp Biol, 209, 3141-3154

Weber, J., Brichon, G., Zwingelstein, G., McClelland, G., Saucedo, C., Weibel, E. R., and Taylor, C. R. (1996). Design of the oxygen and substrate pathways. IV. Partitioning energy provision from fatty acids. J Exp Biol, 199, 1667-1674.

Weber, T.P. and Piersma, T. (1996). Basal metabolic rate and the mass of tissues differing in metabolic scope: migration-related covariation between individual Knots Calidris canutus. J Avian Biol, 27, 215-224

Weise, C. M. (1967). Castration and spring migration in the white-throated sparrow. Condor, 69, 49-68.

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Williams, J.B., and Tielman, B.I. (2000). Flexibility in basal metabolic rate and evaporative water loss among hoope larks exposed to different environmental temperatures. J Exp Biol, 203, 3153-3159.

Williams, T.D., Challenger, W.O., Christians, J.K., Evanson, M., Love, O., and Vezina, F. (2004). What causes the decrease in haematocrit during egg production? Funct Ecol, 18, 330-336.

Wingfield, J.C. and Hahn, T.P. (1994). Testosterone and territorial behaviour in sedentary and migratory sparrows. Anim Behav, 47: 77-89.

Wingfield, J.C., Lynn, S.E., Soma, K.K. (2001). Avoiding the costs of testosterone: ecological bases of hormone-behavior interactions. Brain, Behav Evol, 57, 239-251

Wingfield, J. C. (1984). Environmental and endocrine control of reproduction in the song sparrow, Melospiza melodia: I. Temporal organization of the breeding cycle. Gen Comp Endocrinol, 56, 406-416.

Zheng, W.H., Li, M.,Liu, J.S., Shao, S.L. (2008). Seasonal acclimatization of metabolism in Eurasian tree sparrows (Passer montanus). Comp Biochem Physiol, 151, 519- 525.

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Chapter 4 : General Conclusions

The research presented in this thesis represents the most comprehensive manipulation of gonadal hormones in a migratory bird, where surgical castrations were combined with pharmaceutical blockers of androgens and with testosterone replacement under different photoperiods. This study is also the first to investigate the potential role of androgens in the changes in body composition (muscle and organ sizes) and muscle biochemistry (enhanced aerobic capacity and fatty acid oxidation) that are crucial to migration in birds. In general, my results do not support the gonadal hypothesis, and suggest that transitions to a migratory state in both spring and fall are controlled by non- gonadal hormones and neural mechanisms, with gonadal hormones modifying certain aspects of migration, such as speed, in spring.

Since the 1930's, the gonadal hypothesis has dominated the way scientists understand endocrine regulation of vernal migratory behaviour and physiology in birds (Rowan, 1932). This hypothesis postulated that vernal migration was driven primarily by circulating sex steroid hormones; during spring, androgen levels increase simultaneously with the growth of gonadal tissue (Wingfield and Hahn, 1994). In addition to promoting territory defence and mate attracting behaviour required for the breeding season (Blas et al., 2006; Beletsky et al., 1990; De Ridder et al., 2000; Hegner and Wingfield, 1987; Ketterson et al., 1992; Moore, 1984; Saino and Møller, 1995; Wingfield, 1984), testosterone increases muscle mass and induces hyperphagia, which would also provide benefits to migrants during endurance flights (Berthold, 2001; Holberton and Dufty, 2005). While there has been support for this idea over the years, not one of the early studies addressing this hypothesis performed manipulations that completely inhibited circulating androgens; therefore the effects of testosterone on the avian migratory phenotype could not be confirmed. Previous studies testing the gonadal hypothesis have also focused primarily on the effects of testosterone on behaviour, and have ignored additional physiological indicators outside of seasonal increases in body mass and fat deposition. By limiting the parameters used to assess the migratory state of individual 102

birds, these studies over-simplified the avian migratory phenotype. In birds, preparing for migration requires modifying both behaviour and physiology, however, to my knowledge there have been no studies that have examined these seasonal changes together in detail. A major weakness of the gonadal hypothesis has always been that it requires a different mechanism to prepare birds for migration in fall than in spring. A simpler explanation is that the same basic, and as yet undescribed, neuroendocrine mechanism operates in spring and fall, with gonadal hormones acting to enhance or speed-up migratory movements in the spring (see below).

4.1 Thesis Summary

My studies examined the effect of testosterone on seasonal changes in both migratory behaviour and physiology. I experimentally manipulated photoperiod and androgen levels in castrated male white-throated sparrows (Zonotrichia albicollis). Transitioning the light cycle from short to long photoperiods simulated the approach of spring, and initiated the expression of the migratory phenotype in white-throated sparrows. To block the action of androgens produced by non-gonadal tissues such as the adrenal gland and the brain, I used implants containing anti-androgen pharmaceuticals. I also treated a second group of castrated males with implants containing testosterone. Testosterone-inhibited and testosterone-replaced castrated groups of males were photostimulated to long days and brought into a migratory condition alongside a single group of sham-operated males, whose testes remained intact. The behaviour and physiology of migratory males was compared to an additional group of non-migratory sham-operates who remained on a short photoperiod for the duration of the experiment.

In chapter two, I found that nocturnal restlessness was a testosterone-mediated behaviour, but the initiation of this migratory activity occurred independently of androgens. After photostimulation, all migratory treatment groups were active at night; testosterone-treated castrates were more active than the two other groups of migratory males. Testosterone-inhibited castrated males displayed low levels of nocturnal activity not unlike migratory intact males, however this level of behaviour was also statistically similar to activity recorded in non-migrants.

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In chapter three, I found that spring photoperiods increased fat stores and hematocrit, as well as decreased liver mass in migratory white-throated sparrows. These physiological changes are typical of the migratory phenotype and occurred despite testosterone manipulation, indicating that other factors are responsible for fuel deposition and building of exercise organs. The decreases in liver mass that were induced by photoperiod were attributed to the migratory strategy of some white-throated sparrows that winter in northern latitudes and experience cooler temperatures (Dawson et al., 1983; Falls and Kopachena, 1994). Larger visceral organs may be required to support thermogenesis in cold climates (Block, 1994, Daan et al., 1990; Marsh and Dawson, 1989; Weber and Piersma, 1996; Zheung et al., 2008), however these organs would need to be catabolised prior to migration to save energy in flight by reducing body weight (Piersma and Gill, 1998).

Exposure to long day lengths also increased oxidative enzyme activity and decreased anaerobic enzyme activity in flight muscle, but there was evidence that the removal of gonadal tissue dampened this up-regulation. Castrated males in both hormone-manipulated groups had intermediate levels of enzyme activity in comparison to non-migratory and migratory sham-operated groups. The gonads produce other hormones (gonadotropin-releasing hormone, inhibin, activin, androgen-binding protein) in addition to sex steroids, and several other endocrine systems were likely disrupted with the surgical removal of this tissue. Because the body composition of all migratory groups was similar despite hormone treatment, this difference in oxidative enzyme activity suggests that biochemical indicators within exercise machinery may be more dependable as indicators of the ability of individuals to successfully migrate than changes in body composition.

Testosterone manipulation did not affect any of the physiological indicators measured in my experiment. In measurements of nocturnal behaviour, testosterone treatment only modulated the intensity of activity, but the onset of this activity was primarily photoperiod driven. As described above, these results do not support the gonadal hypothesis, and instead seem to fit well with what is known about spring and fall migrations of free-living birds. Migrations during both seasons require the same seasonal 104

changes in behaviour and physiology, however fall migration occurs when gonadal hormone levels are minimal (Wingfield and Farner, 1978; Wingfield 1990). The gonadal hypothesis suggested that because each migration involved different life history events, two separate endocrine systems regulated the migratory phenotype during spring and fall (Wingfield et al., 1990). I propose a more parsimonious hypothesis where both migrations are regulated by a single underlying system, but that testosterone regulates migration speed during spring. Spring migration is faster than fall migration (Fransson, 1995). In spring, elevations of gonadal androgens may induce birds to rush to breeding grounds (Tonra et al., 2011; Wingfield et al., 1990). Gonadal hormones are minimal during fall migration, however migratory birds still display nocturnal restlessness and exhibit changes in body composition (Wingfield et al., 1990). The pace of fall migration may be slower than that measured during spring because birds utilise energy-minimizing strategies to cope with variable weather patterns during autumn (Alerstam and Hedenstrӧm, 1998; Lack, 1960; Wingfield et al., 1990). If testosterone modulates nocturnal restlessness and eagerness to migrate, then increases in gonadal hormones in spring would significantly increase total spring migration. With this in mind, the slowed fall migration may be comparable to the spring migration of females who arrive at breeding grounds after males have arrived and established territories (Cooper et al., 2009; Morris and Glasgow, 2001; Morris et al., 2003).

While this alternative hypothesis appears to be congruent with the ecology and life history of some migratory bird species, more research will be needed before the gonadal hypothesis can be completely discounted. In particular, further experiments involving hormone manipulations in both field and captive studies would broaden our understanding of the endogenous systems regulating changes in seasonal behaviour and physiology.

4.2 Future Directions

Alternative Treatment Groups

My results suggest adding some additional experimental treatments. To determine if testosterone has an effect on behaviour and physiology in non-migratory seasons, I 105

would treat castrated males with crystalline testosterone filled implants while maintaining a short-day photoperiod. If wintering birds in this treatment increase exercise muscle mass, rates of feeding (hyperphagia), and hematocrit it would indicate that testosterone can over-ride the effect of exogenous photoperiod cues. To my knowledge, no studies have examined the effect of testosterone on non-migratory biochemical physiology or phenotypic flexibility, however some work has looked at aggressive behaviour (Soma et al., 1999; Hau et al., 2000). Non-breeding aggression was inhibited in song sparrows (Melospiza melodia morphna) given anti-androgens and anti-estrogens (Soma et al., 1999). Additionally, androgen-treated tropical spotted antbirds (Hylophylax naevioides) had enhanced song production, and were more aggressive than controls outside of the breeding season (Hau et al., 2000). Since evidence exists that testosterone treatment and inhibition can over-ride aggressive behaviour regulated by circannual rhythms, perhaps hormone-manipulation could also induce nocturnal restlessness and migratory physiology during wintering periods.

An alternative experiment would include measuring physiology of photoperiod and androgen manipulated treatment groups while preventing nocturnal migratory behaviour. In complete darkness (< 1 lux of light; Ramenofsky et al., 2008), migratory birds do not express nocturnal restlessness. This reduction in activity would lower each individual's daily energy expenditure (Gwinner, 1974, 1977). It would be interesting to see if testosterone treatment stimulates fuel deposition and weight gain in activity- reduced birds who were anticipating migratory flights. As briefly mentioned in Chapter 3, this experiment may also clarify if testosterone, rather than increased activity, exercise and energy expenditure after exposure to spring photoperiods, increases hematocrit in white-throated sparrows (Fair et al., 2007).

Endocrine Mechanisms

A complex system of neuroendocrine signals probably interact to regulate the expression of spring and fall migratory behaviour and physiology. Examining hormones that influence growth as well as regulate circannual and circadian rhythms would be crucial for understanding seasonal migratory changes. Melatonin is secreted by the pineal

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gland and is a powerful endocrine signal involved in the generation of circadian rhythms. Melatonin levels peak at night (Takahashi et al., 1980), and in birds, the melatonin peak is depressed during migratory seasons in comparison to other times of the year (Gwinner et al., 1997). Reductions in the nocturnal melatonin peak have been linked to migratory nocturnal restlessness. The causal link between this activity and melatonin is still unknown (Fusani and Gwinner, 2005), however food availability seems to mediate this relationship and appears to correspond to refueling patterns that birds experience at stopover (Fusani and Gwinner, 2004). On a similar note, thyroid hormones regulate metabolism and protein synthesis, and could benefit birds that are refueling for migratory flights (Wingfield et al., 1990; Zhang and Lazar, 2000). Thyroid hormone secreation increases during both spring and fall migratory seasons in free-living birds (George and Naik, 1964; Oakeson and Lilley, 1960; Wilson and Farner, 1969). In addition, nocturnal restlessness can be inhibited and re-instated by removing and replacing thyroid hormones

(triiodothyronin, T3 and thyroxin, T4), respectively (reviewed by Dawson, 2001; Dawson et al., 2003; Pathak and Chandola, 1982).

Despite the breadth of knowledge of digestive hormones (Denbow, 2000), little is known about the mechanisms affecting visceral organ mass. Migratory phenotypic flexibility involves shifts in digestive organ mass (liver, gut, gizzard, pancreas and kidney), but in my study androgens did not affect these organs. While there is a lack of evidence regarding adults, studies on developing animals indicates growth hormone

(GH), insulin-like-growth factor I (IGF-I) and thyroid hormones (specifically T3) may be involved in changes in digestive organ mass (Roith et al., 2001; Zheng et al., 2008). Future manipulations should look at the effects of these hormones on body composition of adult migrating birds.

Field Studies

One of the most important general results of my study is that photoperiod has a dominant effect on behaviour and physiology, regardless of androgen hormone treatment. Natural photoperiods entrain endogenous programmes to environmental cues. While there are still gaps in our knowledge as to what induces migratory changes,

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environmental and internal factors are likely highly integrated to synchronize these life history events (reviewed in Kumar et al., 2010). Understanding the neuroendocrine mechanisms that influence seasonal changes is important, however an individual's ability to interpret abiotic and biotic factors are just as crucial for successful migration. Integrating information on environmental conditions, such as food availability, temperature, weather, and social context, (Åkesson and Hedenstrӧm, 2000; Richardson, 1978) could prevent migrants from undergoing endurance flights during suboptimal conditions (Ramenofsky et al., 2008; Ramenofsky, 2012). This would ultimately reduce the proximate (e.g. energy) and ultimate (fitness) costs of migration by improving a migrant's chances of arriving at their breeding or wintering grounds. With these implications in mind, new experiments should attempt to integrate endogenous and exogenous factors to achieve a more realistic interpretation of the migratory strategies birds use. One way to acquire this kind of information would be to perform experimental manipulations of androgens in the field.

If the technology were available, the next step for this experiment would be to validate lab experiment results in a free-living study. Castration and laporotomy surgeries as well as the administration of implants could be performed in the field on non- migratory birds residing on wintering grounds. At the same time, tracking devices could be attached to individual birds: allowing researchers to track the speed, length of stopover and directional movements during spring migration. While these techniques are still relatively new, migratory movements of birds have been documented this way in several other studies (Bairlein et al., 2012; Fudickar et al., 2012; Gill et al., 2009; Stanley et al., 2012; Stutchbury et al., 2009). Driven by androgens, castrated males given testosterone implants would be expected to travel faster than testosterone inhibited males. Arrival date and testosterone levels are correlated in American redstarts (Setophaga ruticilla), suggesting that migration speed may be affected by androgens (Tonra et al., 2011). If the recapture of tracked individuals is possible, body composition and organ mass can be assessed upon arrival of birds to their breeding grounds.

In addition, the role of androgens on both sexes could be further examined by documenting arrival dates during spring and fall field studies. As mentioned previously, 108

females in several avian species arrive at breeding grounds later than males (Cooper et al., 2009; Morris and Glasgow, 2001; Morris et al., 2003); circulating levels of androgens are much higher in males than in females during spring migration (Wingfield and Farner, 1978). Perhaps the administration of testosterone to migrating females would increase their migration speed to that of a migrating male during spring. Treating fall migrants with testosterone implants would theoretically increase migration speed of both males and females in comparison to control birds.

Field studies would provide the most accurate representation of migration, as they would take into account weather conditions as well as resource availability during stopover. Changes in physiology may also be more prominent in a free-living study, since birds would actually experience exercise imposed by endurance flights.

4.3 Conclusions

By providing evidence against the gonadal hypothesis, this thesis has opened many new areas of research regarding both the life-history and migration strategies used by migrants in spring and fall. In particular, my conclusions stressed the importance of modifications to metabolic machinery in migrants. In light of this, new experiments should definitely examine biochemical indicators as well as seasonal changes in body composition and behaviour in order to assess the migratory condition of individuals. Future captive studies could also examine other endocrine systems that regulate the migratory phenotype during both spring and fall: primarily focussing on hormones that regulate seasonal shifts in body composition in adults as well as nocturnal behaviour. On the other hand, future field studies could focus on the effect of androgen manipulations during true flight, as well as differences in arrival date between sexes (with and without hormone manipulation). These experiments would provide researchers with the opportunity to manipulate endocrine systems while allowing migrants to be exposed to all exogenous cues from the surrounding environment. Future work should attempt to link ecology and physiology of migratory birds. Ultimately, by gaining a thorough understanding of the interacting factors affecting the timing of migration, researchers can

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make inferences on how populations of migrants will be affected by changing weather patterns and anthropological influences.

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Beletsky, L. D., Orians, G. H., and Wingfield, J. C. (1990). Effects of exogenous androgen and antiandrogen on territorial and nonterritorial red‐winged blackbirds (Aves: Icterinae). Ethology, 85, 58-72.

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Blas, J., Pérez-Rodríguez, L., Bortolotti, G. R., Viñuela, J., & Marchant, T. A. (2006). Testosterone increases bioavailability of carotenoids: insights into the honesty of sexual signaling. Proceed Natl Acad Sci, 103, 18633-18637.

Block, B. (1994). Thermogenesis in muscle. Annu Rev Physiol, 56, 535-577

Cooper, N. W., Murphy, M. T., and Redmond, L. J. (2009). Age‐and sex‐dependent spring arrival dates of Eastern Kingbirds. J Field Ornithol, 80, 35-41.

Daan, S., D. Masman, and A. Groenewold. 1990. Avian basal metabolic rates: their association with body composition and energy expenditure in nature. Am J Physiol, 259, R333–R340

Dawson, A. (2003). Photoperiodic control of the annual cycle in birds and comparison with mammals. Ardea, 90, 355-367.

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Dawson, A., King, V. M., Bentley, G. E., and Ball, G. F. (2001). Photoperiodic control of seasonality in birds. J Biol Rhythms, 16, 365-380.

Dawson, W. R., Marsh, R. L., and Yacoe, M. E. (1983). Metabolic adjustments of small passerine birds for migration and cold. Am J Physiol, 245, 755-767.

Fair, J., Whitaker, S., and Pearson, B. (2007). Sources of variation in haematocrit in birds. Ibis, 149, 535-552.

Falls, J. B. and J. G. Kopachena. (1994). White-throated Sparrow (Zonotrichia albicollis). In The Birds of North America, No. 128 (A. Poole, Ed.). The Birds of North America Online, Ithaca, New York.

Fransson, T. (1995). Timing and speed of migration in North and West European populations of Sylvia warblers. J Avian Biol, 26, 39-48.

Fudickar, A. M., Wikelski, M., and Partecke, J. (2012). Tracking migratory songbirds: accuracy of light‐level loggers (geolocators) in forest habitats. Methods Ecol Evol, 3, 47-52.

Fusani, L., and Gwinner, E. (2004). Simulation of migratory flight and stopover affects night levels of melatonin in a nocturnal migrant. Proc Biol Sci, 271, 205-211. doi:10.1098/rspb.2003.2561

Fusani, L., and Gwinner, E. (2005). Melatonin and nocturnal migration. Ann N Y Acad Sci, 1046, 264-270.

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George, J. C., and Naik, C. V. (1964). Cyclic changes in the thyroid of the migratory starling (Sturnus roseus). Linnaeus. Pavo, 2, 37-47.

Gill, R. E., Tibbitts, T. L., Douglas, D. C., Handel, C. M., Mulcahy, D. M., Gottschalck, J. C., and Piersma, T. (2009). Extreme endurance flights by landbirds crossing the Pacific Ocean: ecological corridor rather than barrier? Proc R Soc B Biol Sci, 276, 447-457.

Gwinner, E., Hau, M., and Heigl, S. (1997). Melatonin: generation and modulation of avian circadian rhythms. Brain Res Bull, 44, 439-444.

Gwinner, E. (1974). Endogenous temporal control of migratory restlessness in warblers. Naturwissenschaften, 61, 405-405. doi:10.1007/BF00622629

Gwinner, E. (1977). Circannual rhythms in bird migration. Ann Rev Ecol Syst, 8, 381- 405.

Hau, M., Wikelski, M., Soma, K. K., and Wingfield, J. C. (2000). Testosterone and year- round territorial aggression in a tropical bird. Gen Comp Endocrinol, 117, 20-33. doi:10.1006/gcen.1999.7390

Hedenström, A., and Alerstam, T. (1997). Optimum fuel loads in migratory birds: distinguishing between time and energy minimization. J Theor Biol, 189, 227-234. doi:10.1006/jtbi.1997.0505

Hegner, R. E., and Wingfield, J. C. (1987). Effects of experimental manipulation of testosterone levels on parental investment and breeding success in male house sparrows. Auk, 104, 462-469.

Holberton, R. L., and Dufty, A. M. (2005). Hormones and variation in life history strategies of migratory and non-migratory birds. In J. A. Greenberg, and P. P. Marra (Eds.), Birds of Two Worlds: The Ecology and Evolution of Migration (pp. 290-302). Baltimore, MD: John Hopkins University Press.

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Lack, D. (1960). The influence of weather on passerine migration, A review. Auk, 77, 171-209.

Morris, S. R., and Glasgow, J. L. (2001). Comparison of spring and fall migration of American Redstarts on Appledore Island, Maine. Wilson Bull, 113, 202-210.

Morris, S. R., Pusateri, C. R., and Battaglia, K. A. (2003). Spring migration and stopover ecology of Common Yellowthroats on Appledore Island, Maine. Wilson Bull, 115, 64-72

Oakeson, B. B., and Lilley, B. R. (1960). Annual cycle of thyroid histology in two races of white-crowned sparrow. Anat Rec Suppl, 136, 41-57.

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Piersma, T., and Gill Jr, R. E. (1998). Guts don't fly: small digestive organs in obese Bar- tailed Godwits. Auk, 115, 196-203.

Ramenofsky, M. (2012). Reconsidering the role of photoperiod in relation to effects of precipitation and food availability on spring departure of a migratory bird. Pro Biol Sci, 279, 15-16. doi:10.1098/rspb.2011.1591 ER

Ramenofsky, M., Agatsuma, R., and Ramfar, T. (2008). Environmental conditions affect the behavior of captive, migratory white-crowned sparrows. Condor, 110, 658-671. doi:10.1525/cond.2008.8523

Richardson, W. J. (1978). Timing and amount of bird migration in relation to weather: a review. Oikos, 30, 224-272.

Roith, D.L., Scavo, L. and Butler, A. (2001). What is the role of circulating IGF-I? Endocrinol Metab, 12, 48-52.

Rowan, W. (1932). Experiments in bird migration: III. The effects of artificial light, castration and certain extracts on the autumn movements of the American crow (Corvus brachyrhynchis). Proc Natl Acad Sci U S A, 18, 639.

Stanley, C. Q., MacPherson, M., Fraser, K. C., McKinnon, E. A., & Stutchbury, B. J. (2012). Repeat tracking of individual songbirds reveals consistent migration timing but flexibility in route. PloS One, 7, e40688.

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Stutchbury, B. J., Tarof, S. A., Done, T., Gow, E., Kramer, P. M., Tautin, J., and Afanasyev, V. (2009). Tracking long-distance songbird migration by using geolocators. Science, 323, 896-896.

Takahashi, J. S., Hamm, H., and Menaker, M. (1980). Circadian rhythms of melatonin release from individual superfused chicken pineal glands in vitro. Proc Natl Acad Sci, 77, 2319-2322.

Tonra, C. M., Marra, P. P., and Holberton, R. L. (2011). Migration phenology and winter habitat quality are related to circulating androgen in a long‐distance migratory bird. J Avian Biol, 42, 397-404.

Wilson, A. C., and Farner, D. S. (1960). The annual cycle of thyroid activity in white- crowned sparrows of eastern Washington. Condor, 62, 414-425.

Wingfield, J.C., and Farner, D.S. (1978). The annual cycle of plasma irLH and steroid hormones in feral populations of the white-crowned sparrow, Zonotrichia leucophrys gambelii. Biol Reprod, 19, 1046-1056.

Wingfield, J. C., and Hahn, T. P. (1994). Testosterone and territorial behaviour in sedentary and migratory sparrows. Animal Behav, 47, 77-89. doi:ISSN 0003-3472

Wingfield, J. C. (1984). Environmental and endocrine control of reproduction in the song sparrow, Melospiza melodia: I. Temporal organization of the breeding cycle. Gen Comp Endocrinol, 56, 406-416

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Zhang, J., and Lazar, M. A. (2000). The mechanism of action of thyroid hormones. Annu Rev Physiol, 62, 439-466.

Zheng, W.H., Li, M.,Liu, J.S., Shao, S.L. (2008). Seasonal acclimatization of metabolism in Eurasian tree sparrows (Passer montanus). Comp Biochem Physiol, 151, 519- 525.

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Appendices

Appendix A: Ethics Approval

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Appendix B: Circuit Board Configuration and Electrical Diagrams for Infrared Sensors, Detectors and Data Acquisition Plates.

B - 1 Circuit Board and Electrical Diagrams for Infrared Beam Emmitor and Detector.

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B - 2 Circuit Board and Electrical Diagrams for Data Acquisition Plates.

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Appendix C: Validation Curve for testosterone in white-throated sparrows (Zonotrichia albicollis). A serial dilution was performed using a pooled standard of plasma (10 µL of plasma taken from 15 samples; 150 µL total) and testosterone assay dilutent. This serial dilution was compared to a standard curve using a 600pg/ml standard and testosterone assay diluents supplied in an Expanded Range Salivary Testosterone Enzyme Immunoassay Kit (Catalog No. 1-2402-5, Salimetrics PA, USA).

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Curriculum Vitae

Caitlin L. Vandermeer

Academic Training

2011-2013 M.Sc. Biology The University of Western Ontario Advisors: Dr. Christopher G. Guglielmo and Dr. Scott A. MacDougall-Shackleton Thesis: The Effect Of Testosterone On The Spring Migratory Phenotype Of A North American Songbird (Zonotrichia albicollis)

2007 - 2011 B.Sc. Zoology, Honors University of Guelph Guelph, Ontario, Canada

Honours and Awards

2013 Graduate Student Thesis Award, The University of Western Ontario

2011 - 2013 Western Graduate Research Scholarship (Institutional)

Related Work Experience

Teaching Assistant The University of Western Ontario 2011 - 2012 Population Ecology BIOL 2483A 2012 - 2013 Environmental Biology BIOL 2485B

2011 Research Assistant The University of Guelph, Department of Biology Advisor: Dr. M. Alex Smith

2010-2011 Lab Technician & Field Assistant The Biodiversity Institute of Ontario, The University of Guelph Advisor: Dr. Sarah Adamowics 122

Presentations and Posters

Vandermeer, C.L., Bezner-Kerr, W., MacDougall-Shackleton, S.A., Guglielmo, C.G. (2013). The effect of testosterone on migratory restlessness, body composition and oxidative enzyme activity in white-throated sparrows (Zonotrichia albicollis) during spring migration. 52nd Annual Meeting of the Canadian Society of Zoologists. Guelph, ON. (presentation)

Vandermeer, C.L., Bezner-Kerr, W., MacDougall-Shackleton, S.A., Guglielmo, C.G. (2013). Effects of testosterone on spring nocturnal migratory restlessness and body composition in Zonotrichia albicollis. The Annual Meeting for the Society of Integrative Biology. San Francisco, CA. (presentation)

** represented at the 2012 Biology Graduate Student Research Forum, The University of Western Ontario, London ON. (presentation)

Vandermeer, C.L. McCubbin, S., Fruetel, C., Hall, J., and Smith, M.A. (2011). Insect Diversity in the University of Guelph Dairy Bush: Preliminary case study of ants (Formicidae) in the Dairy Bush and the Adjacent Field. CBS Undergraduate Summer Student Poster Symposium, University of Guelph, Guelph ON. (poster).

Publications

Vandermeer, C.L., Bezner-Kerr, W., Guglielmo, C.G., and MacDougall-Shackleton, S.A. (2013). Effects of testosterone on spring nocturnal migratory restlessness in white-throated sparrows (Zonotrichia albicollis). Horm Behav. In revision.

Robertson, B.D., Hasstedt, M.R., Vandermeer, C.L., and MacDougall-Shackleton, S.A. (2013). Sex steroid-independent effects of photostimulation on the song-control system of white-throated sparrows (Zonotrichia albicollis). Gen Comp Endocrinol. In revision.

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