FACTORS AFFECTING THE PHYSIOLOGICAL CONSEQUENCES OF STRESS …

The Pennsylvania State University

The Graduate School

Intercollege Graduate Degree Program in Ecology

FACTORS AFFECTING THE PHYSIOLOGICAL CONSEQUENCES OF STRESS IN

EASTERN FENCE LIZARDS (SCELOPORUS UNDULATUS)

A Dissertation in

Ecology

by

Gail Lindsey McCormick

© 2016 Gail Lindsey McCormick

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

May 2016

The dissertation of Gail Lindsey McCormick was reviewed and approved* by the following:

Tracy Langkilde Associate Professor of Biology Dissertation Advisor Chair of Committee

Victoria Braithwaite Professor of Fisheries and Biology

Katriona Shea Alumni Professor of Biology

Sonia Cavigelli Associate Professor of Biobehavioral Health

David Eissenstat Professor of Woody Plant Physiology Chair of the Intercollege Graduate Program in Ecology

*Signatures are on file in the Graduate School

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ABSTRACT

Understanding the consequences of stress is integral to predicting how organisms will

respond to global environmental change. The stress response is generally adaptive, promoting

physiological changes that allow an organism to deal with and recover from a threat. However,

the stress response can lead to negative fitness outcomes because it diverts energy away from

growth, reproduction, and immune function. My dissertation broadly investigates how traits of a

stressor and of an organism affect the physiological outcomes of stress. To do so, I utilized

populations of eastern fence lizards (Sceloporus undulatus) that co-evolved with different levels

of stress associated with presence of invasive predatory fire ants (Solenopsis invicta).

Stress is typically characterized by duration, with short-duration (“acute”) stress assumed

to be neutral or beneficial and long-duration (“chronic”) stress assumed to have negative

outcomes. However, the outcomes of stressors that result from repeated short-duration stress (e.g.

frequent predator attacks) are not well understood. I first investigated the immune outcomes of

these “repeated acute” stressors. Lizards from both high-and low-stress populations enhanced

immune function following exposure to repeated acute elevations of exogenous corticosterone

(CORT). This demonstrates that repeated acute stressors produce immune outcomes more typical

of those expected from short-duration stress (i.e. immune enhancement). I then investigated the

role of stressor duration, frequency, and intensity in determining the immune outcomes of stress.

My results reveal that stressor intensity is a major driver of immune outcomes of stress in this

system and suggest that the current duration-centric terminology is not adequate. Finally, I

explored how exposure to stress within a lifetime and across generations affects stress physiology

and immune function by raising offspring of lizards from high- and low-stress sites under high

and low-stress conditions in the lab and measuring physiological outcomes in adulthood. Early

life stress did not affect adult stress physiology, but offspring of lizards from high-stress sites had

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more robust stress responses than those from uninvaded sites. This suggests that, in this system,

stress experienced by an individual’s ancestors may be more important in shaping adult stress

physiology than stress that an individual faces within its lifetime. By contrast, within- and across-

generational factors interacted to affect adult immune function; CORT-elevation during early life

suppressed adult immune function in lizards from low-stress sites but enhanced immune function

in lizards from high-stress sites. Together, these results further our understanding of how traits of

a stressor and those of an organism influence physiological outcomes of stress. This insight

allows us to better predict how organisms will be affected by stress, including that imposed as a

result of global change, and to better allocate resources to manage and mitigate these fitness-

relevant effects.

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TABLE OF CONTENTS

LIST OF FIGURES .................................................................................................................. viii

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

ACKNOWLEDGEMENTS ..................................................................................................... xi

Chapter 1 Introduction ............................................................................................................. 1

The Vertebrate Stress Response and its Outcomes .......................................................... 1Selective Environment .............................................................................................. 3Stressor Characteristics ............................................................................................. 3History of Stress Exposure ....................................................................................... 5

Research Objectives and Study System ............................................................................ 6References ........................................................................................................................ 10

Chapter 2 Immune responses of eastern fence lizards (Sceloporus undulatus) to repeated acute elevations of corticosterone ..................................................................................... 20

Abstract ............................................................................................................................. 21Introduction ...................................................................................................................... 21Methods ............................................................................................................................ 24

Collection and Housing ............................................................................................ 24Hormone Manipulation and Blood Collection .......................................................... 25Hormone Assays ....................................................................................................... 26Hemagglutination Assay ........................................................................................... 27Bacterial Killing Ability ........................................................................................... 28Data Analysis ............................................................................................................ 28

Results .............................................................................................................................. 29Plasma Corticosterone .............................................................................................. 29Hemagglutination ...................................................................................................... 29Bacterial Killing Ability ........................................................................................... 30

Discussion ......................................................................................................................... 30Acknowledgements .......................................................................................................... 33Figures .............................................................................................................................. 35References ........................................................................................................................ 37

Chapter 3 How do duration, frequency, and intensity of exogenous CORT elevation affect immune outcomes of stress? ................................................................................... 45


Study System ............................................................................................................ 49Treatments ................................................................................................................ 49Blood Collection ....................................................................................................... 51

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Hemagglutination Assay ........................................................................................... 51Bacterial Killing Ability ........................................................................................... 52Data Analysis ............................................................................................................ 53

Results .............................................................................................................................. 53Hemagglutination ...................................................................................................... 53Bacterial Killing Ability ........................................................................................... 53

Discussion ......................................................................................................................... 54Total or Average Stress (CORT) .............................................................................. 54Duration .................................................................................................................... 55Frequency .................................................................................................................. 56Intensity .................................................................................................................... 57Conclusions ............................................................................................................... 59

Acknowledgements .......................................................................................................... 59Tables ................................................................................................................................ 60Figures .............................................................................................................................. 61References ........................................................................................................................ 63

Chapter 4 Ancestry trumps experience: Cross-generational but not early life stress affects the adult physiological stress response ............................................................................. 68


Study System and Animal Collection ....................................................................... 72Animal Husbandry .................................................................................................... 72Treatments ................................................................................................................ 73Blood Collection and Stress Assays ......................................................................... 74ACTH Challenge ...................................................................................................... 75Hormone Analysis .................................................................................................... 76Data Analysis ............................................................................................................ 76

Results .............................................................................................................................. 78Baseline Corticosterone ............................................................................................ 78Corticosterone Reactivity to Restraint ...................................................................... 78Corticosterone Response to Fire Ants ...................................................................... 78Corticosterone Response to ACTH Challenge ......................................................... 79

Discussion ......................................................................................................................... 79Early Life Stress ........................................................................................................ 80Cross-Generational Exposure to Stress ..................................................................... 81Baseline CORT ......................................................................................................... 83Conclusions ............................................................................................................... 83

Acknowledgements .......................................................................................................... 84Figures .............................................................................................................................. 85References ........................................................................................................................ 88

Chapter 5 Reaping the rewards: High-stressed populations up-regulate immune function in the face of stress ........................................................................................................... 95

Abstract ............................................................................................................................. 96

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Introduction ...................................................................................................................... 97Methods ............................................................................................................................ 98

Study System and Collection .................................................................................... 99Animal Husbandry .................................................................................................... 100Treatments ................................................................................................................ 100Blood Collection ....................................................................................................... 102Hemagglutination Assay ........................................................................................... 102Bacterial Killing Assay ............................................................................................. 103Data Analysis ............................................................................................................ 104

Results .............................................................................................................................. 104Hemagglutination ...................................................................................................... 104Bacterial Killing Ability ........................................................................................... 105

Discussion ......................................................................................................................... 105Conclusions ............................................................................................................... 110

Acknowledgements .......................................................................................................... 110Figures .............................................................................................................................. 112References ........................................................................................................................ 114

Chapter 6 Conclusions ............................................................................................................. 122

References ........................................................................................................................ 126

Appendix What Makes Stress Stressful? Extending the Acute-Chronic Stress Paradigm ...... 129

Tables ................................................................................................................................ 131 References ........................................................................................................................ 132

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LIST OF FIGURES

Figure 2-1: Plasma CORT concentrations of lizards from high-stress (fire ant invaded) and low-stress (uninvaded) populations after 23 days of treatment with CORT (shaded bars) or control (oil vehicle only; open bars). CORT-treated lizards had significantly higher plasma CORT concentrations than control lizards. This relationship was consistent across high- and low-stress populations (i.e. no significant effect of invasion status). Bars represent means ± one standard error. Sample size for each group is given within each bar. ....................................................... 35

Figure 2-2: A) Hemagglutination scores and B) percent bacterial killing by plasma of lizards from high-stress (fire ant invaded) and low-stress (uninvaded) populations after 23 days of treatment with CORT (shaded bars) or control (oil vehicle only; open bars). CORT-treated lizards had significantly higher hemagglutination scores than but similar bacterial killing ability to control lizards. These relationships were consistent across high- and low-stress populations (i.e. no significant effect of invasion status). Bars represent means ± one standard error. Sample size for each group is given within each bar. ......................................................................................... 36

Figure 3-1: A) The frequency, intensity, and duration of CORT application in each of the treatments used in this study, and the total amount of CORT received in each 3-day period. Text in parentheses indicates: for Frequency, how frequently a CORT-oil solution was applied (oil-vehicle only was applied on remaining days); for Intensity, the amount of CORT applied during each application; and for Duration, whether the period of CORT elevation was short or long. Italicized pairs in each column represents treatments that differ in only the parameter shown in that column. B) A graphical representation of the amount of CORT applied for each of the treatments used in this study (Control (Ctl) had no CORT applied). This is provided for illustrative purposes, to convey the expected duration of CORT release following application. ....................................................................................................................... 61

Figure 3-2: A) Hemagglutination scores and B) Percent bacterial killing by lizard plasma after 9 days of treatment (see Fig. 3-1). Lizards in the Low Acute (LA), Repeated Acute (RA), and Control (Ctl) treatments had significantly higher hemagglutination scores than did those in the High Acute (HA) treatment, and those in the Chronic (Ch) treatment had hemagglutination scores that were intermediate to these groups. Lines above the columns connect treatments that do not significantly differ from one another. Bacterial killing ability did not significantly differ across treatments. Error bars represent means ± one standard error. The sample size for each group is given within each bar. ................................................................................................................. 62

Figure 4-1: CORT reactivity to restraint is greater in offspring of lizards from fire ant-invaded sites. Adult concentrations of CORT at baseline (shaded bars) and following restraint in a bag (white bars) of lizards exposed weekly to fire ants (FA),

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exogenous CORT, or control treatment during early life. CORT reactivity (post-restraint stressor minus baseline) was assessed in the statistical model but stress-induced concentrations are plotted here for ease of comparisons between graphs. Bars represent means ± one standard error. The sample size for each group is given above each bar. ................................................................................................................. 85

Figure 4-2: CORT concentrations following adult fire ant exposure are greater in offspring of lizards from fire ant-invaded sites. CORT concentrations following exposure in adulthood to an empty arena (FA control; shaded bars) or attack by fire ants (FA; white bars) of lizards exposed weekly to fire ants (FA), exogenous CORT, or control treatment during early life. Bars represent means ± one standard error. The sample size for each group is given above each bar. ................................................. 86

Figure 4-3: ACTH-induced CORT concentrations are greater in offspring of lizards from fire ant-invaded sites. Adult CORT concentrations following injection with saline solution (shaded bars) or ACTH (white bars) of lizards exposed weekly to fire ants (FA), exogenous CORT, or control treatment during early life. Bars represent means ± one standard error. The sample size for each group is given above each bar. ... 87

Figure 5-1: Early life and cross-generational history of stress exposure interact to affect adult baseline, but not post-inoculation, hemagglutination scores. a) In offspring of lizards from fire ant-uninvaded populations, CORT exposure during early life suppressed adult baseline plasma hemagglutination compared to controls. The opposite effect was seen in offspring of lizards from fire ant-invaded populations: early life CORT exposure enhanced adult baseline hemagglutination compared to controls. b) Post-inoculation hemagglutination scores did not differ across early life stress treatment or fire ant-invasion status. Bars represent means ± one standard error and sample size for each group is shown above each set of bars. ...... 112

Figure 5-2: Bacterial killing by plasma of adult lizards is not related to early life or cross-generational history with stress. Offspring of lizards from fire ant invaded and uninvaded populations exposed weekly to fire ants (FA), CORT, or control treatment from hatching until maturity had similar percent bacterial killing ability of plasma as adults. Bars represent means ± one standard error and sample size for each group is shown above each bar. ........................................................................................ 113

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LIST OF TABLES

Table 3-1: This table shows the different immune outcomes (enhancement, no change, or suppression) to repeated exposure to stress (exogenous application of CORT, handling stress, or an ecologically-relevant stressor), indicating the study organism and immune component measured. Exogenous CORT was elevated using topical application or feeding. Handling includes handling or chasing, placement in a bag, or air exposure (fish). Ecologically relevant stressors include food deprivation, social isolation, social defeat, or exposure to predator scents. *Animals were simultaneously restrained. Abbreviations as follows: Hemag. = hemagglutination; BKA = bacterial killing ability; DTH = delayed-type hyper sensitivity; Antibody Resp. = antibody response. ............................................................................................... 60

Table A-1: Representative studies from the literature showing the full range of both duration of stress applications (rows) and consequences (columns) of stress, using definitions of “acute” and “chronic” stress from each source paper. Shaded boxes represent combinations expected according to existing theory. ....................................... 131

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ACKNOWLEDGEMENTS

Firstly, I thank my advisor, Tracy Langkilde, for her guidance and enthusiasm over the

last five years. Her encouragement, patience, and many many track changes greatly contributed to

my growth as a scientist, and her friendship made my journey through graduate school not only

possible, but also pleasurable. I am also grateful to my committee members for their advice and

thoughtful feedback and to my undergraduate mentors, Phil Myers and Catherine Badgey,

without whom I would not have considered attending grad school.

I am forever indebted to the past and present members of the Langkilde Lab. For their

friendship, manuscript edits, assistance bleeding lizards, movie nights, and all of the Cheez-Its

flavors ever, I thank: Renee Rosier, Lindsey Swierk, Brad Carlson, Jenny Tennessen, Chris

Thawley, Chris Howey, Sean Graham, Travis Robbins, Nicole Freidenfelds, Kelly Brossman,

Caty Tylan, Braulio Assis, Dustin Owen, and Cam Venable. I am also grateful to the horde of

undergraduates who made this work possible, especially Courtney Norjen, Melissa O’Brien,

Tommy Cerri, Mark Herr, and Mark Goldy-Brown for their patience and dedication.

My deepest thanks to my friends and family. Thank you to the State College theatre

community for making State College home; to Katie, Sean, Lia, Nitesh, Heather, and Katelyn for

their never-ending supply of support, understanding, and whimsy; to TL for sharing DPTL, which

translates to all facets of life; and to EJ for encouraging simultaneous pursuit of disparate

passions. I am grateful to my parents and brother for their patience and support of all my

endeavors. And finally, thank you to my boyfriend, Rich, for his unwavering support, constant

presence, and for smashing lizard poop on a Friday night. Thanks for saving the day on multiple

occasions and for the adventures, dinosaurs, and love.

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***

I am grateful to the National Science Foundation, the American Society for Ichthyologists and

Herpetologists, the Society for Integrative and Comparative Biology, the Ecological Society of

America Physiology Section, the Huck Institute of the Life Sciences, the Penn State Department

of Biology, and the Penn State Intercollege Graduate Degree Program in Ecology for providing

conference funding as well as financial and logistical support. The research presented here

adheres to the Guidelines for the Use of Animals in Research and the Institutional Guidelines of

Penn State University (IACUC #35780) and animal collection was permitted by the respective

states.

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Chapter 1

Introduction

The global environment is changing with increasing speed (Vitousek, 1997), exposing

populations to novel stressors (Tylianakis et al., 2008; Vitousek, 1997). Environmental changes

including habitat loss (Homan et al., 2003; Suorsa et al., 2004), pollution (Norris et al., 1999;

Tomei et al., 2003; Wikelski et al., 2001), urbanization (French et al., 2008), and the introduction

of novel predators (Berger et al., 2007; Graham et al., 2012), can elicit a physiological stress

response in vertebrates. Although the physiological stress response is critical in the short-term,

long-term or frequent activation can reduce fitness by altering behavior, reproduction, stress

physiology, and immune function (McEwen, 1998a). The magnitude and direction of these

outcomes may vary with the type of stressor (e.g. social defeat, metabolic stress, immune stress,

trauma; Segerstrom and Miller 2004; Koolhaas et al. 2011; Ariza Traslaviña et al. 2014), stressor

characteristics (e.g. duration, frequency, intensity; Busch et al., 2008; Dhabhar and McEwen,

1997; Martin, 2009; McEwen et al., 1997), or an individual’s or population’s previous exposure

to stress (Carpenter et al., 2007; Franklin et al., 2010; Spencer et al., 2009; Yehuda et al., 2000).

Understanding how such factors may influence the outcomes of stress (e.g. immune costs) will

enable predictions of how organisms respond to environmental change.

The Vertebrate Stress Response and its Outcomes

Stress can be defined as an external challenge to an organism’s normal functioning

(homeostasis; Burchfield, 1979; Dhabhar, 2007; Levine, 2005). A great number of stimuli

(stressors) can present such a challenge, and stress is thus unavoidable (Selye, 1978). When a

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stressor is encountered, the body undergoes a series of changes in order to survive and recover

from the threat. This physiological response to stress is highly conserved across vertebrates and

includes activation of both the sympathetic nervous system and the hypothalamic–pituitary–

adrenal (HPA) axis (Seaward, 2006). The sympathetic nervous system induces the fight-or-flight

response by rapid production of the catecholamines epinephrine and norepinephrine, whose

effects last only seconds (Seaward, 2006). Longer term effects (minutes to hours, or even weeks)

are mediated by the HPA axis (Seaward, 2006; Stratakis and Chrousos, 1995). Briefly, the

hypothalamus produces corticotropin-releasing factor (CRF), which acts on the pituitary. The

pituitary then secretes adrenocorticotropic hormone (ACTH), which stimulates the adrenal glands

to produce and release glucocorticoid hormones such as cortisol or corticosterone (CORT). The

effects of glucocorticoids are numerous and long-lasting (Sapolsky et al., 2000), and CORT is

frequently measured as a proxy for stress.

Elevation of plasma CORT can help an organism appropriately respond to a stressor

(Munck et al., 1984; Sapolsky et al., 2000; Stratakis and Chrousos, 1995). For example, elevation

of CORT can trigger important behavior (including anti-predatory behavior, Remage-Healey and

Romero, 2001; Thaker et al., 2009), mobilize stored energy (Sapolsky et al., 2000), enhance

immune function in preparation for wounding or subsequent risk of infection (Dhabhar, 2009;

Martin, 2009), and alter metabolism to help an individual maintain homeostasis (Stratakis and

Chrousos, 1995). For these reasons, the stress response is generally considered adaptive in the

short term (McEwen, 2008). By contrast, persistent or long-duration elevation of CORT, such as

that experienced when an organism is continuously or repeatedly exposed to a threat, may

suppress reproduction (Moore and Jessop, 2003; Salvante and Williams, 2003), growth (Barton et

al., 1986; Bourgeon and Raclot, 2006; Morici et al., 1997), and immune function (Dhabhar, 2009;

Martin, 2009). This may result from the long-term diversion of energy away from these energy-

sensitive functions (McEwen and Wingfield, 2003; McEwen, 1998b; Romero et al., 2009).

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Despite these general trends, documented outcomes of both short- and long-duration stress vary

greatly (Table A-1). The factors underlying this variation are likely numerous and may include

the selective environment (French et al., 2008; Martin II et al., 2005), characteristics of the

stressor (Busch et al., 2008; Martin, 2009; McEwen et al., 1997), and an organism’s or

population’s history with stress (Harris and Seckl, 2011; McCormick and Green, 2013;

Veenema, 2009; Yehuda et al., 2000). The potential effects of these factors on the outcomes of

stress are addressed below.

Selective Environment

The nature of the costs of the stress response may vary depending on context. For

example, in populations where immune function is critical to survival, such as where the

prevalent stressor is likely to injure an individual or induce behavior that increases risk of

infection (Cox and John-Alder, 2007; Ezenwa et al., 2012), immune function may be maintained

by reallocating energy from growth or reproduction. Over time, one might expect to see selection

against immune suppression (when it exists), and maybe even selection for immune enhancement

within populations that are frequently exposed to immune-activating stressors (French et al.,

2008; Martin II et al., 2005). Understanding how populations vary in their response to stress may

have important management implications, as management strategies may need to be altered at a

population level according to the local selective environment.

Stressor Characteristics

Stress is typically characterized by the duration of the stressor: short-duration exposure to

a stressor lasting from minutes to a few days (Harbuz and Lightman, 1992; Martin, 2009) is

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typically termed “acute” (Burchfield, 1979; Romero, 2004), while long-duration continuous

exposure to a stressor over days or months (Dhabhar, 2009; Martin, 2009) is typically termed

“chronic” (Burchfield, 1979; Romero, 2004; Sapolsky et al., 2000). Repeated exposure to short-

duration stressors, such as frequent disturbances by humans or sub-lethal predator attacks, are

sometimes referred to as “repeated acute” (Burchfield, 1979; Busch et al., 2008) but usually

classified as “chronic” (Harbuz and Lightman, 1992; Romero, 2004). In spite of their frequent use

in the literature and in medical practices, the terms “acute” and “chronic” are inconsistently

applied (Appendix). It is also unclear when “acute” stress becomes “chronic” and where repeated

acute stressors fall on the acute-chronic spectrum. This distinct terminology can be problematic,

as beneficial or harmful physiological outcomes are typically associated with acute and chronic

stress, respectively (Martin, 2009; McEwen, 2008), but observed outcomes commonly vary from

these associations (Table A-1; e.g., Chester et al., 2010; Harris et al., 2002; Merrill et al., 2012;

Ottenweller et al., 1992).

Although prediction of stress outcomes is almost exclusively associated with stressor

duration, other aspects such as stressor intensity and frequency may play important roles. For

example, exposure to conspecifics and predators are stressors that may differ in intensity and may

be encountered at different rates. However, stressor intensity and frequency are largely ignored in

the literature (but see Busch et al., 2008; McCormick et al., 1998; McEwen et al., 1997;

Ottenweller et al., 1989). Predicting the independent and synergistic effects of stressor

characteristics is critical if we are to gain a complete understanding of how organisms respond to

stress.

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History of Stress Exposure

Stress experienced by an organism within its lifetime may affect its physiological

response to stress (Ladd et al., 1996; Spencer et al., 2009) and may affect HPA activity in future

generations (Jenkins et al., 2014; Yehuda et al., 2000). When persistently exposed to stress either

within a lifetime or across generations, an organism’s physiological stress response may change

to balance associated costs and benefits (Matthews, 2002; Meaney et al., 1994; Oitzl et al., 2010).

Down-regulation of HPA activity may reduce the costs associated with this stress response

(Martin, 2009; Romero, 2004; Romero et al., 2009). Alternatively, if the benefits of the stress

response outweigh the costs, organisms frequently exposed to stress may up-regulate HPA

activity to take advantage of these benefits (Romero, 2004; Romero et al., 2009; Sapolsky et al.,

2000). Altering the stress response to balance these costs and benefits may alter energy usage,

which may in turn affect other traits, such as growth or immune function.

Within an individual’s lifetime, stress exposure can have effects on behavior and

physiology that persist into adulthood. For example, early life stress can increase risk of

aggression, anxiety, and depressive behaviors in adult rodents and primates (reviewed in

McCormick and Green, 2013; Veenema, 2009), can affect adult HPA activity in a variety of

species (Carpenter et al., 2007; Ladd et al., 1996; Spencer et al., 2009), and has been linked to

increased risk of inflammatory, liver, lung, and ischemic heart disease in adult humans (Danese et

al., 2007; Dong et al., 2004; Felitti MD et al., 1998). The effects of early life stress are relatively

well studied in humans and rodents (McCormick and Green, 2013; Veenema, 2009). Expanding

this understanding to other taxa will inform the evolutionary pressures leading to the

consequences of early life stress.

A population’s experience with stress can affect stress physiology in the next generation

(Franklin et al., 2010; Harris and Seckl, 2011; Storm and Lima, 2010; Yehuda et al., 2000). For

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example, adult human offspring of Holocaust survivors have lower baseline CORT

concentrations compared to their peers (Yehuda et al., 2000). These cross-generational changes

could operate via a number of mechanisms, including selection favoring particular CORT profiles

(Jenkins et al., 2014; Lightman, 2008; Oitzl et al., 2010), maternal effects (e.g. mothers

differentially allocating hormones or nutrients to young or altering maternal behavior;

Champagne and Meaney, 2001; Liu et al., 1997; Love et al., 2013), or epigenetic processes (e.g.

altering gene expression within a lifetime at the level of the HPA axis that may be passed to

offspring; Anacker et al., 2014; Harper, 2005; Jablonka and Raz, 2009; Weaver et al., 2004).

Cross-generational exposure to perturbations following the introduction of novel threats, such as

predatory or competitive invasive species, may also drive changes in other traits that enhance

fitness, such as behavior (e.g. Griffiths et al., 1998; Langkilde, 2009) and morphology (e.g.

Langkilde, 2009; Phillips and Shine, 2004). However, the role of stress in driving these changes

is unknown.

Research Objectives and Study System

My dissertation broadly investigates how characteristics of a stressor (duration,

frequency, intensity) and those of an organism (history of stress exposure) affect the

physiological consequences of stress, including HPA and immune function. I utilized eastern

fence lizards (Sceloporus undulatus) as a study system, as populations of this species exhibit

documented variation in exposure to a known ecological stressor: red imported fire ants

(Solenopsis invicta). Fire ants are invasive across parts of the natural range of fence lizards

(Callcott and Collins, 1996), and these species occupy similar habitat and frequently interact

where their ranges overlap (Freidenfelds et al., 2012; Langkilde, 2009b). Fire ants prey upon

fence lizards, and attacks involve bites and stings that can break the skin of lizards, leaving

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lizards vulnerable to infection (Elkan and Cooper, 1980; Murphy, 2001). Sub-lethal encounters

elevate lizard plasma CORT concentrations (Langkilde, unpubl. data), and my early research

revealed that lizards from fire ant-invaded sites have higher baseline CORT concentrations and

post-stress CORT reactivity than lizards from uninvaded sites (Graham et al., 2012). This system

thus allows us to compare populations that differ in lifetime and cross-generational histories of

high versus low stress (sites with different histories of fire ant invasion).

I take advantage of elements of both field and lab studies by bringing field-caught

animals from populations with different histories of stress exposure into the lab. Field studies are

more realistic and ecologically relevant than laboratory manipulations, but stress exposure can be

challenging to manipulate in the field if not using long-term implants (which are not always

physiologically relevant). In contrast, short and long duration stress exposure can be easily

manipulated in the lab. By utilizing wild-caught lizards from high- and low-stress sites, I was able

to assess the effects of cross-generational history with stress and the effects of lifetime exposure

to stress by carefully manipulating animals reared in the lab from hatching.

In fence lizards, one can easily manipulate exposure to a variety of stress regimes,

including ecologically relevant fire ant stressors, restraint and handling (a common experimental

stressor), and topical application of exogenous CORT. In some cases, I chose to manipulate

CORT to replicate the CORT elevation that would occur in response to a stressor. Manipulation

of exogenous CORT allows one to target the role of this stress-relevant hormone without

potentially confounding effects of the stressor itself (e.g. wounding, venom). In some cases, I

used fire ant attack as a direct stressor to complement exogenous CORT treatments as it is more

realistic. Utilizing both CORT and fire ant exposure allows us to determine if any effects of fire

ants are due to CORT elevation or to something else, such as higher order HPA activity,

including sensitivity to other stress-relevant hormones (CRF, ACTH), or to other elements of the

attack (e.g. wounding).

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In my dissertation research, I measured HPA and immune function, both of which are

fitness-relevant and can be rapidly assessed. Many components of the HPA axis could be

measured to assess HPA activity, including concentrations of receptor numbers for CRF, ACTH,

and CORT. Identifying receptor density and location can increase our understanding of the

function of glucocorticoid production, but this can be challenging, particularly in non-model

organisms whose brain structures are not well mapped, and collection of the brain or tissue is

necessary, precluding serial sampling. By contrast, hormone concentrations or metabolites can be

determined using small amounts of blood, saliva, hair, feathers, urine, or feces, permitting non-

lethal and repeated sampling from the same individual. Hair and feathers provide a long-term

record of glucocorticoids due to their slow growth and replacement (Sheriff et al., 2011). Urine

and fecal metabolites reflect a shorter record of glucocorticoids (though metabolism is

complicated and species-specific; Sheriff et al., 2011). Saliva and plasma provide an immediate

snapshot of the circulating glucocorticoid concentrations (Sheriff et al., 2011). Following

extraction, glucocorticoid concentrations or metabolites can be easily measured using

commercially available kits and are repeatable within species (Tarlow and Blumstein, 2007). In

my research, I chose to measure circulating plasma concentrations of CORT due to its rapid

production, repeatability, and known methodological validity for this species (Trompeter and

Langkilde, 2011).

The immune system plays a critical role in maintaining health by protecting an organism

from pathogens and parasites, repairing damage, and responding to infection (Møller and Saino,

2004; Murphy, 2001). Fence lizards have high rates of wounding across their range (McCormick

and Langkilde, 2014), and immune function may be particularly important at sites where risk of

fire ant attack and associated wounding is high. Immune function can be assessed in a variety of

ways. For example, measuring rates of wound healing provides an integrative assessment of

immune function, but is challenging to measure in the field and does not indicate the specific

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immune mechanism responsible for any changes (Detillion et al., 2004; French et al., 2006;

Martin and Martin, 2014). By contrast, quantifying specific components of the immune system

(e.g. activity of natural antibodies, complement) can be carried out using relatively simple assays

with small amounts of blood, and can be used in the field (Matson et al., 2006, 2005). Lizards

rely primarily on innate rather than adaptive immune function (Zimmerman et al., 2010), and I

therefore utilized two measures of innate immune function that lizards may employ in response to

infection: specifically, the ability of antibodies to agglutinate (clump) foreign cells, providing

fewer targets to be engulfed by phagocytes (Matson et al., 2005; Millet et al., 2007; Sharon,

1998), and of complement to kill bacteria (bacterial killing; Graham et al., 2012; Matson et al.,

2006).

For my dissertation research, I used the above approaches to determine the effects of

stressor characteristics and history of stress exposure on stress physiology and immune function.

In Chapter 2, I investigate whether repeated acute elevations of CORT affect immune function in

lizards, and if these immune outcomes differ in lizards from high- and low-stress sites. In Chapter

3, I experimentally manipulate CORT in fence lizards to examine how stressor duration,

frequency, and intensity affect immune outcomes. In Chapters 4 and 5, I investigate the

consequences of early life stress on adult HPA activity and immune function, respectively, using

lizards from high-and low-stress sites raised under different stress regimes (fire ant exposure and

CORT elevation). Chapters 2, 3 and 5 describe how stressor characteristics and an organism’s

experience with stress affect immune outcomes of stress. Chapters 2 and 4 contribute to our

understanding of naturally occurring patterns of stress in this system and, taken together, allow us

to identify the cross-generational (genetic and maternal effects) and within-lifetime (plasticity)

mechanisms driving the ecology of this system. As a whole, my dissertation sheds light on factors

that influence the physiological outcomes of stress, guiding future research in this field.

10

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Chapter 2

Immune responses of eastern fence lizards (Sceloporus undulatus) to repeated acute elevations of corticosterone

Gail L. McCormicka and Tracy Langkildea

a Intercollege Graduate Degree Program in Ecology,

Department of Biology,

and The Center for Brain, Behavior and Cognition


Published in General and Comparative Endocrinology (2014) 204:135-140.

Author Contributions: GLM and TL designed the experiment and analyzed data. GLM collected

data and wrote the manuscript. TL contributed to the manuscript

21

Abstract

Prolonged elevations of glucocorticoids due to long-duration (chronic) stress can

suppress immune function. It is unclear, however, how natural stressors that result in repeated

short-duration (acute) stress, such as frequent agonistic social encounters or predator attacks, fit

into our current understanding of the immune consequences of stress. Because these types of

stressors may activate the immune system due to increased risk of injury, immune suppression

may be reduced at sites where individuals are repeatedly exposed to potentially damaging

stressors. We tested whether repeated acute elevations of corticosterone (CORT, a glucocorticoid)

suppress immune function in eastern fence lizards (Sceloporus undulatus), and whether this effect

varies between lizards from high-stress (high baseline CORT, invaded by predatory fire ants) and

low-stress (low baseline CORT, uninvaded) sites. Lizards treated daily with exogenous CORT

showed higher hemagglutination of novel proteins by their plasma (a test of constitutive humoral

immunity) than control lizards, a pattern that was consistent across sites. There was no significant

effect of CORT treatment on bacterial killing ability of plasma. These results suggest that

repeated elevations of CORT, which are common in nature, produce immune effects more typical

of those expected at the acute end of the acute-chronic spectrum and provide no evidence of

modulated consequences of elevated CORT in animals from high-stress sites.

Introduction

An organism’s physiological response to stress is generally adaptive, promoting

important behavioral and physiological changes to deal with the stressor (Sapolsky et al., 2000;

Stratakis and Chrousosa, 1995). The vertebrate stress response includes the release of

glucocorticoids, such as cortisol or corticosterone (CORT), by the hypothalamic–pituitary–

22

adrenal (HPA) axis. The production of CORT in response to short-duration (acute) stress, such as

that imposed by a predator encounter (Creel, 2001), may increase survival by triggering important

behavior (including anti-predatory behavior, Remage-Healey et al., 2006; Thaker et al., 2009),

enhancing immune responsiveness in preparation for wounding or subsequent infection that

might result (Deak et al., 1999; Dhabhar, 2009; Martin, 2009), and mobilizing stored energy

(Remage-Healey and Romero, 2001; Sapolsky et al., 2000).

While reallocation of resources to permit these changes is integral to dealing with acute

stressors, long-term reallocation of resources due to chronic stress may reduce fitness, by

suppressing reproduction (Moore and Miller, 1984; Moore and Jessop, 2003; Salvante and

Williams, 2003), growth (Barton et al., 1986; Bourgeon and Raclot, 2006; Davis et al., 1985;

Morici et al., 1997), and immune function (Dhabhar, 2009; Martin, 2009; Sapolsky et al., 2000;

Stratakis and Chrousosa, 1995). Immune effects of long-duration (chronic) stress can include a

reduced ability to heal wounds (Bourgeon and Raclot, 2006; French et al., 2010), suppressed

inflammatory (El-Lethey et al., 2003; Martin II et al., 2005) and antibody response to novel

foreign bodies (El-Lethey et al., 2003; Stier et al., 2009), reduced ability of plasma to kill bacteria

(French et al., 2010), and lymphocyte apoptosis (Sapolsky et al., 2000). There are examples,

however, in which long-term elevations of CORT do not affect immune function (Dabbert et al.,

1997; Klein et al., 1992; Martin II et al., 2005; Vegas et al., 2012). These conflicting findings

could result from differences in energy limitation during the study, as some species exhibit an

immune-suppressive effect of CORT only when resources are limited (food is limited or during

reproduction: French et al., 2010, 2007). Alternatively, it may be maladaptive to suppress

immune function in response to chronic or persistent stress in situations where the immune

system is particularly important for dealing with this stress, such as in response to agonistic social

interactions or predator attacks that can damage an organism or induce behavior that increases

risk of infection (Cox and John-Alder, 2007; Ezenwa et al., 2012). We may expect to see

23

selection against immune compromise (when it exists) and maybe even selection for immune

enhancement within populations chronically exposed to these types of immune-activating

stressors (French et al., 2008; Martin II et al., 2004).

Chronic stress, such as that induced by a storm or drought (Fitze et al., 2009; Wilson and

Wingfield, 1994; Wingfield and Kitaysky, 2002), is often studied by exposing animals to constant

levels of stress (e.g. implanting animals with silastic implants, gel, or pellets that provide a

continuous release of glucocorticoids for days to months; French et al., 2007; Martin II et al.,

2005; Morici et al., 1997). Acute stressors, such as predator attacks or disturbances by humans,

are much shorter in duration, and CORT concentrations following exposure to these stressors

typically return to baseline within a few hours (Malisch et al., 2010), which can be mimicked by

topical application or injection of CORT. However, acute stressors may occur at high frequency.

Such repeated exposure to an acute stressor is sometimes termed “chronic” (Burchfield, 1979;

Harbuz and Lightman, 1992; Romero, 2004), and studies investigating the consequences of

chronic stress often do so by exposing animals to repeated acute stress (Barton et al., 1986; Davis

et al., 1985; Fitze et al., 2009; Retana-Marquez et al., 1998). Repeated acute stress has been

shown to have consequences typical of chronic stress, including suppressed reproduction (Robert

et al., 2009) and growth (Busch et al., 2008a; McCormick et al., 1998; McGraw et al., 2011).

There are mixed outcomes of repeated acute stress on immune function: reduced wound healing

in tree lizards (Urosaurus ornatus; French et al., 2006); suppressed hemagglutination and

inflammatory response to phytohemagglutinin (PHA) in chickens (El-Lethey et al., 2003); and no

change in wound healing in mice (Vegas et al., 2012). Further research examining the immune

consequences of repeated acute stress is necessary to better understand how these ecologically-

relevant repeated acute stressors fit into the existing acute-chronic gradient of stress.

We examined the immune consequences of repeated acute elevations of CORT in eastern

fence lizards, Sceloporus undulatus. Some populations of this species have had their habitat

24

invaded by red imported fire ants, Solenopsis invicta, which prey upon fence lizards (along with

several native predators including birds and snakes; Crowley, 1985). Eastern fence lizards and red

imported fire ants frequently encounter one another in nature (Langkilde, 2009a); lizards exhibit

elevated plasma CORT concentrations following fire ant attack (Langkilde, unpublished data),

and lizards from fire ant invaded sites have higher baseline plasma CORT concentrations than

those from uninvaded sites (Graham et al., 2012a). This provides an excellent opportunity for

understanding whether the immune costs of repeated acute exposure to CORT vary between

animals from high- and low-stress populations.

Methods

Collection and Housing

During April and May 2012, we captured a total of 46 adult male fence lizards (S.

undulatus) from six sites across the southern United States using a hand-held noose. These sites

are similar in habitat (Langkilde, 2009a; Langkilde, unpublished data), but differ in fire ant

invasion history (Callcott and Collins, 1996). Three of these sites have no previous history of fire

ant invasion and lizards at these sites have relatively low baseline CORT concentrations (referred

to as “low-stress” populations): (1) St Francis National Forest, Lee County, Arkansas; (2) Edgar

Evins State Park, DeKalb County, Tennessee; and (3) Standing Stone State Park, Overton

County, Tennessee. The remaining sites were first invaded by fire ants 55-70 years ago and

lizards at these sites have relatively high baseline CORT concentrations (referred to “high-stress”

populations): (4) Blackwater River State Forest, Santa Rosa County, Florida; (5) Geneva State

Forest, Geneva County, Alabama; and (6) Conecuh National Forest, Covington County, Alabama.

25

We measured all lizards for snout-vent length (SVL) upon capture and transported them to our

lab in Pennsylvania, in individual cloth bags inside coolers, for this study.

Lizards were housed individually in plastic enclosures (42 x 28 x 27 cm L x W x H).

Outer walls of the enclosures were wrapped with dark paper to prevent lizards from seeing each

other. The floor of each enclosure was lined with paper towel and furnished with a water dish and

plastic shelter for refuge and basking. The shelter was placed underneath a 60-W incandescent

light bulb that provided heat for 2 h each day. Overhead florescent lights provided additional heat

and were set to a 12:12 light:dark schedule. Each lizard was fed 3 crickets (Acheta domestica)

every other day. All cages were cleaned on the same days to standardize stress (once during the

experimental phase of the study).

Hormone Manipulation and Blood Collection

Lizards were allowed to acclimate for at least 1.5 months (47 - 77 days depending on date

of capture; lizards from fire ant invaded sites were collected approximately 2 weeks earlier than

those from invaded sites) prior to starting the study. This provided adequate time for lizards to

acclimate to laboratory conditions and CORT concentrations to return to baseline (CORT levels

are reduced and remain stable after 2 weeks in captivity; Trompeter and Langkilde, 2011).

Lizards from each site were randomly assigned to a treatment or control group. Those in the

treatment group (“+CORT”) received daily application of CORT (≥92%, Sigma C2505) dissolved

in commercial sesame oil (1 µg CORT / 1 µL sesame oil) and those in the control group received

the sesame oil vehicle only. Every day for 23 days, we applied 6 µL of either CORT solution or

sesame oil only (control) to the backs of lizards using a repeat pipette. Due to the lipophilic nature

of lizard skin, both the oil and hormone/oil mixture were quickly absorbed (Belliure and Clobert,

2004; Meylan et al., 2003; Trompeter and Langkilde, 2011). This CORT application elevates

26

plasma CORT to ecologically relevant concentrations approximate 30 min after application, and

these return to baseline within 4 h after application (Knapp and Moore, 1997; Trompeter and

Langkilde, 2011). We treated lizards in the evening (between 1545 and 1750) because animals

were less active at these hours, negating the need to handle lizards during hormone application

and thus minimizing elevations of CORT caused by this methodology.

Eighteen to 21 h after the final treatment was applied, we collected blood samples from

the post-orbital sinus using 70 µl heparinized microhematocrit tubes (VWR, San Francisco, CA).

This timing allowed us to measure baseline CORT concentrations, as CORT elevations resulting

from CORT application would no longer be evident. All samples for hormone assays were

collected within 2 min of capture (mean 95.7 ± 2.97 SE sec) to prevent handling stress from

influencing plasma corticosterone concentrations in our samples. For a subset of animals (n=28),

additional time was needed to collect blood for immune assays, and this was collected within 3.5

min of capture (mean 194.4 ± 9.0 SE seconds). Blood samples were centrifuged, and plasma was

drawn off and assigned to 3 tubes, one for each assay, and immediately frozen (-20°C) until

assays were performed. Sufficient blood samples were not always available to perform all assays

for each lizard, so sample sizes were reduced in some cases (provided for each assay, below).

Hormone Assays

For 39 individuals, we measured CORT by enzyme immunoassay (Corticosterone High

Sensitivity EIA Kits, Immunodiagnostic Systems Ltd., Fountain Hills, AZ, USA) following

directions provided in the kit. These kits have been validated for S. undulatus (Trompeter and

Langkilde, 2011). Plasma was stored for 49 or 64 days before the assay was performed. We

diluted plasma 1:9 with buffer (5 µl plasma : 45 µl buffer) to ensure that samples fell within the

range of detection of this assay’s standard curve. We ran all samples in duplicate. The mean

27

intraassay coefficient of variation within the two kits was 7.73%, and mean interassay coefficient

of variation between the two kits was 2.79%.

Hemagglutination Assay

For 40 individuals, we measured the ability of plasma to hold sheep red blood cells

(SRBC) in suspension (hemagglutination) in vitro. This assay has previously been used as a

measure of innate immunity in eastern fence lizards (Graham et al., 2012a) as well as birds

(Matson et al., 2005), toads (Graham et al., 2012b), and snakes (Sparkman and Palacios, 2009).

SRBC (Innovative Research, Novi, MI) was washed with phosphate-buffered saline (PBS) up to

three times by diluting 2mL SRBC with 4mL PBS, then gently vortexing and centrifuging for 5

min. We drew off excess PBS and lysed SRBC and repeated the process until the supernatant

became clear. We then brought the washed SRBC to a 2% solution with PBS. After 16 hours in

the freezer, plasma (25 µL) was thawed and diluted 1:1 with PBS (25 µL) and then serially

diluted to 1:64 in a 96-well plate using a multichannel pipette. Control wells contained PBS only

(25 µL). The 2% SRBC solution (25 µL) was then added to each well. Plates were gently mixed

by tapping and incubated at room temperature for 1 h, after which plates were scored for

agglutination (action of antibodies in plasma to hold SRBC in suspension). Scores were

calculated as the negative log2 of the highest dilution at which agglutination was attained – higher

scores indicate a higher concentration of SRBC-specific antibodies in the plasma (Matson et al.,

2005). Half scores were recorded when SRBC precipitated partially but not to the extent of

control wells. Lysis of erythrocytes was not scored since this process has been shown to be

subjective (Matson et al., 2005).

28

Bacterial Killing Ability

For 32 individuals, we measured the ability of plasma to lyse Escherichia coli bacteria.

Heat inactivation experiments completely inhibit the ability of S. undulatus plasma to lyse

bacteria, indicating that complement (e.g., a non-specific protein cascade involved in innate and

adaptive immune lysis) plays a major role in lysis of E. coli in this assay (Graham et al., 2012a).

We use this assay as an additional measure of innate immunity. Detailed methods of this assay

are provided in (Graham et al., 2012a). Briefly, after 4 days in the freezer, thawed lizard plasma

(14 µl) was combined with E. coli (10 µl of 200 CFU bacteria dilution) and allowed to react. This

solution was combined with a growth medium solution (126 µl of a CO2 L-glutamine solution,

containing 400 µl L-glutamine and 19.6 mL CO2 medium). 50µl of each sample was spread in

duplicate on agar plates and incubated at 37°C for 16 h. Colonies on each plate were counted,

averaged (across duplicates) and compared to mean colony counts of two replicated control plates

that contained no plasma (140 µl CO2 L-glutamine medium + 10 µl E. coli). Percent bacterial

killing was calculated as 100 - (mean plasma treatment colony count/mean control colony count)

x 100. Plates that ranged from 0 to negative 10% killing (n=4) were corrected to 0%. Plates with

less than negative 10% killing (n=4) were discarded.

Data Analysis

Plasma CORT concentrations, SVL, and time to obtain blood were log transformed, and

percent lysis data were angular transformed, prior to analysis to meet assumptions of parametric

tests. We compared mean CORT concentrations using ANCOVA with treatment, fire ant invasion

status, and source population (site) nested within invasion status as factors and SVL and time to

obtain blood sample as covariates. We analyzed hemagglutination and bacterial killing ability of

29

plasma separately using ANCOVA with treatment, invasion status, and source population nested

within invasion status as factors and SVL as a covariate. Statistical analyses were performed

using JMP (version 7.0, SAS Institute Inc., Cary NC) with α = 0.05.

Results

Plasma Corticosterone

After treatment, all lizards exhibited plasma CORT concentrations within the

physiological limits of this species (Trompeter and Langkilde, 2011). Lizards repeatedly treated

with CORT had significantly higher plasma CORT concentrations at the end of this study than

control-treated lizards (Fig. 2-1; F1,29=4.44, P=0.04). This relationship was consistent across high-

and low-stress populations (treatment*invasion status: F1,29=0.20, P=0.66), and concentration of

CORT did not significantly differ between lizards from high- and low-stress populations

(invasion status: F1,29=0.15, P=0.70; site within invasion status: F4,29=0.65, P=0.63; SVL:

F1,29=0.52, P=0.48; time to bleed: F1,29=0.28, P=0.60).

Hemagglutination

Hemagglutination scores of lizards repeatedly treated with CORT were significantly

greater than those of control-treated lizards (Figh. 2-2a; F1,31=5.07, P=0.03). This relationship was

consistent across lizards from high- and low-stress populations (treatment*invasion status:

F1,31=0.23, P=0.64), and hemagglutination scores did not significantly differ between lizards from

high- and low-stress populations (invasion status: F1,31=2.32, P=0.14; site within invasion status:

F4,31=0.93, P=0.46; SVL: F1,31=0.01, P=0.92).

30


Lizards repeatedly treated with CORT had similar bacterial killing ability to control-

treated lizards (Fig. 2-2b; F1,19 = 1.56, P = 0.23). This was consistent across lizards from high-

and low-stress populations (treatment*invasion status: F1,19 = 0.00, P = 0.97). Bacterial killing

also did not differ between high- and low-stress populations (invasion status: F1,19 = 0.43, P =

0.52; site within invasion status: F4,19 = 0.38, P = 0.82).

Discussion

Repeated exposure to acute stress is typically considered chronic (Burchfield, 1979;

Harbuz and Lightman, 1992; Romero, 2004) and is thus expected to have “chronic” consequences

including immune suppression. Although each application of CORT should have resulted in only

a short-duration (4 h) elevation in CORT (Trompeter and Langkilde, 2011), baseline CORT

levels of CORT-treated individuals became elevated by the end of the study. However, we found

no evidence that daily application of CORT suppressed immune function of eastern fence lizards.

In fact, our results reveal that repeated acute exposure to CORT produces immune effects more

typical of those expected from stress categorized at the acute end of the spectrum (enhanced;

Dhabhar, 2009; Romero, 2004; Sapolsky et al., 2000): lizards repeatedly treated with acute levels

of CORT exhibited enhanced immune function (hemagglutination) compared to control-treated

lizards. There may be a number of possible reasons for this:

1) We treated lizards at the same time each evening, resulting in elevated plasma CORT

concentrations each evening. Although CORT levels in eastern fence lizards do not vary

considerably during the activity period of this species (1000 - 1600; Trompeter and Langkilde,

2011), quantifying CORT levels outside of this period would be informative for understanding

31

how timing of CORT application may affect our results. If CORT is typically low in the evening,

for example, addition of exogenous CORT at this time may not increase CORT to concentrations

necessary to incite changes typical of the stress response (i.e. concentrations to induce allostatic

overload, McEwen and Wingfield, 2003; Romero, 2004). Although lizards may be more likely to

experience stress during the day, evening elevations of CORT are ecologically relevant, as

eastern fence lizards likely experience stressful fire ant attacks in the evening (Calcaterra et al.,

2008; Porter and Tschinkel, 1987). Studies examining how timing affects immune outcomes

would be valuable.

2) Lizards may have acclimated to these daily elevations in plasma CORT due to the

consistency in timing of application (Harbuz and Lightman, 1992), and some studies randomize

treatment time to prevent this (Boyd, 2007; Busch et al., 2008a, 2008b). Again, however, our

treatment is likely ecologically relevant, as animals commonly experience stressors at predictable

times.

3) Chronic stress is often defined by its long duration (Burchfield, 1979; Romero, 2004;

Wingfield and Kitaysky, 2002). It is possible that we would have detected an immune-

suppressive effect if we continued our CORT treatment for longer than 23 days; however,

physiological effects have been detected following similar durations of CORT application in

common lizards (Lacerta vivipara; Cote et al., 2006) and Gambel’s white-crowned sparrows

(Zonotrichia leucophrys gambelii; Boyd, 2007; Busch et al., 2008a). Further research is necessary

to better understand the frequency or duration necessary for repeated acute stressors to be

interpreted as chronic by an organism.

4) It is also possible that we would have seen immune-suppressive effects of our CORT

treatment under more food-limited conditions (French et al., 2010, 2007). Lizards in our

experiment were well fed and likely not energy limited. Because resources are likely more limited

in nature (body condition (mass/SVL) of field-caught adults = 1.43 +/- 0.03, lab reared adults =

32

1.50 +/- 0.04; P<0.01; TL unpublished data), we might expect to see immune-suppression from

chronic stress in the wild even when we do not see it in the lab. In some cases, however, immune

suppression has been observed even when resources were not limited (repeated acute stress:

Barton et al., 1986; Boyd, 2007; El-Lethey et al., 2003; chronic stress: Morici et al., 1997) and so

this may not have been a constraining factor in this study.

5) Some components of the immune system may trade-off differentially with the stress

response based on the costs and benefits of suppressing that component (Kurtz et al., 2000; Stier

et al., 2009). This may explain why differential effects of CORT on various immune components

have been observed within the same study (this study; Bourgeon and Raclot, 2006; Graham et al.,

2012b; Ilmonen et al., 2003; Stier et al., 2009) and could contribute to the varied effects of CORT

on immune function described in literature. We would benefit from a better understanding of

which components of the immune system trade off with other systems under stress.

Exposure of fence lizard populations to fire ants has driven evolutionary changes in

behavior and morphology within seven decades (35 lizard generations; Langkilde, 2009b;

Robbins, unpubl. data). Plasma CORT concentrations at the end of our study did not, however,

differ between lizards from high- and low-stress populations, suggesting lifetime and/or

evolutionary exposure to stress may not affect an animal’s response to exogenous CORT. Lizards

from high-stress (fire ant invaded) and low-stress (uninvaded) populations return to similar

baseline CORT concentrations in the lab (this study; Trompeter and Langkilde, 2011), despite

lizards from high-stress populations having higher baseline CORT concentrations than lizards

from low-stress populations in the field (Graham et al., 2012a). This supports the notion that

differences in CORT concentrations between invaded and uninvaded populations in the field are a

direct result of exposure to environmental stressors (likely attack by fire ants) rather than a result

of genetic or acquired sensitivity to CORT.

33

Similarly, immune function did not differ between high- and low-stress populations in

this study. The lack of immune-suppressive effects of CORT in high-stress fire ant invaded sites

may not be surprising given that the immune system may be important for dealing with these

predatory ants. Immune function may be important for these lizards in general - they exhibit high

rates of tail autotomy and other wounding (30% (100/337) of individuals surveyed; T.L. unpubl.

data). To maintain a high-functioning immune system, it is possible that less immediately

important systems, such as growth or reproduction, may be traded-off against CORT in this and

possibly other wild species. Additionally, the risk of injury from encounters with fire ants can be

high in this species—fire ants puncture the skin when they sting lizards—and suppressing

immune function when stressed would likely be maladaptive for this species. We may, in fact,

expect lizards from fire ant invaded populations to up-regulate immune function, but we have

found no evidence of this in the field (Graham et al., 2012a). Further research on a range of

organisms that have evolved under different stressors is necessary to assess the contexts under

which costs of CORT are incurred and the nature of these costs.

Acknowledgements

We thank T. Robbins, S. Graham, and J. Newman for help with lizard collection, S.

Graham, G. DeWitt, and S. McGinley for assistance with immune assays, the Cavener lab for use

of their plate reader, and B. Chitterling for valuable comments on this manuscript. We thank the

Landsdale family for access to their land and lizards and personnel at St Francis National Forest,

Edgar Evins State Park, Standing Stone State Park, Blackwater River State Forest, Geneva State

Forest, Conecuh National Forest, and especially the Solon Dixon Forestry Education Center for

logistical support. The research presented in this article adheres to the Guidelines for the Use of

Animals in Research and the Institutional Guidelines of Penn State University, and animal

34

collection was permitted by the respective states. Funding was provided in part by the American

Society of Ichthyologists and Herpetologists (Gaige Award to GLM) and the National Science

Foundation (DGE1255832 to GLM and IOS1051367 to TL).

35

Figures

Figure 2-1: Plasma CORT concentrations of lizards from high-stress (fire ant invaded) and low-stress (uninvaded) populations after 23 days of treatment with CORT (shaded bars) or control (oil vehicle only; open bars). CORT-treated lizards had significantly higher plasma CORT concentrations than control lizards. This relationship was consistent across high- and low-stress populations (i.e. no significant effect of invasion status). Bars represent means ± one standard error. Sample size for each group is given within each bar.

36

Figure 2-2: A) Hemagglutination scores and B) percent bacterial killing by plasma of lizards from high-stress (fire ant invaded) and low-stress (uninvaded) populations after 23 days of treatment with CORT (shaded bars) or control (oil vehicle only; open bars). CORT-treated lizards had significantly higher hemagglutination scores than but similar bacterial killing ability to control lizards. These relationships were consistent across high- and low-stress populations (i.e. no significant effect of invasion status). Bars represent means ± one standard error. Sample size for each group is given within each bar.

37

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doi:10.1093/icb/42.3.600

45

Chapter 3

How do duration, frequency, and intensity of exogenous CORT elevation affect immune outcomes of stress?

Gail L. McCormicka,b, Katriona Shea a, and Tracy Langkilde a,b

a Department of Biology, Intercollege Graduate Degree Program in Ecology

b The Center for Brain, Behavior and Cognition


Published in General and Comparative Endocrinology (2015) 222:81-87.

Author Contributions: GLM, KS, and TL designed the experiment. GLM and TL analyzed data.

GLM collected data and wrote the manuscript. TL and KS contributed to the manuscript.

46

Abstract

Stress is typically characterized as “acute” (lasting from minutes to hours) or “chronic”

(lasting from days to months). These terms are of limited use as they are inconsistently used and

only encompass one aspect of the stressor (duration). Short and long duration stress are generally

thought to produce specific outcomes (e.g. acute stress enhances while chronic stress suppresses

immune function). We propose that aspects of stress other than duration, such as frequency and

intensity, are important in determining its outcome. We experimentally manipulated duration,

frequency, and intensity of application of exogenous corticosterone, CORT, in Sceloporus

undulatus (Eastern fence lizards) and measured the immune outcomes. Our findings reveal that

immune outcomes of stress are not easily predicted from the average amount or duration of

CORT elevation, but that intensity plays an important role. Although three of our treatments

received the same average amount of CORT, they produced different effects on immune

outcomes (hemagglutination). As predicted by the literature, short-duration exposure to low-dose

CORT enhanced hemagglutination; however, short-duration exposure to high-dose CORT

suppressed hemagglutination, suggesting that stressor intensity affects immune outcomes of

stress. While both are traditionally termed “acute” based on duration, these treatments produced

different immune outcomes. Long-duration (“chronic”) exposure to CORT did not produce the

expected suppression of hemagglutination. Frequency of CORT application did not alter immune

outcomes at low intensities. These results highlight the need to quantify more than just the

duration of a stressor if we are to understand and manage the ecological consequences of stress.

Specifically, we should consider stressor frequency and intensity, as well as duration, for a more

complete characterization and understanding of stress.

47

Introduction

The influence of stressor duration on physiological outcomes is well recognized;

however, less is known about the role of other stress aspects. Stress is typically characterized by

its duration, as “acute” or “chronic”. Acute stress is characterized as “short” in duration

(Burchfield, 1979; Romero, 2004), lasting from minutes to hours (Boonstra, 2012; Harbuz and

Lightman, 1992; Martin, 2009), and the stressor is usually not repeated. The stress response is

generally considered to be adaptive in the short term as it can facilitate the response to and

recovery from a threat (Munck et al., 1984; Sapolsky et al., 2000). For example, the production of

glucocorticoids (such as cortisol or corticosterone, CORT) in response to a stressor can mobilize

energy to allow a fight or flight response (Sapolsky et al., 2000), enhance immune function to

deal with increased risk of injury and infection (Dhabhar, 2009; Martin, 2009), and alter

metabolism to help an individual maintain homeostasis (Stratakis and Chrousosa, 1995). Chronic

stress is characterized as “long” in duration (Burchfield, 1979; Romero, 2004; Sapolsky et al.,

2000), lasting from days to months (Boonstra, 2012; Dhabhar, 2009; Martin, 2009), and includes

persistent or frequent repeated stress (Harbuz and Lightman, 1992; Romero, 2004), such as

frequent tourist visits or sub-lethal predator attacks (“repeated acute;” Burchfield 1979; Busch et

al. 2008a, b). Persistent or long term activation of the physiological stress response can push

animals into allostatic overload, whereby more energy is required for allostasis than can be

obtained from the environment (McEwen and Wingfield, 2003). This can lead to energy being

diverted away from other energy-sensitive functions, which in turn may lead to suppressed

immune function (Dhabhar, 2009; French et al., 2007; Guillette Jr. et al., 1994; Martin, 2009),

growth rates (Chrousos and Gold, 1992; Laugero and Moberg, 2000), and reproductive output

(Greenberg and Wingfield, 1987; McGrady, 1984). However, long-duration exposure to stress

does not always have these suppressive effects (Chester et al., 2010; McCormick and Langkilde,

48

2014; Miles et al., 2007). It is also unclear at what point in time acute stress become chronic, and

where repeated acute stressors fall on the acute - chronic spectrum (Busch et al., 2008a, b). The

terms “acute” and “chronic” may not be entirely useful, as they are inconsistently used and only

encompass one aspect of the stressor (duration).

While it is clear that stressor duration influences the physiological outcome of stress

(citations above), other characteristics of a stressor may also play a role. Intensity and frequency

may have important influences on the outcomes of stress. These characteristics are largely

ignored in the literature (but see intensity: Ottenweller et al. 1989; McEwen et al. 1997;

frequency: McCormick et al. 1998; Busch et al. 2008), and are not considered simultaneously or

in combination with duration. We suspect that considering only duration of stress is hampering

progress towards understanding how stress can have different outcomes (Romero et al., 2009).

In this study, we systematically investigated the effects of duration, frequency, and

intensity of CORT elevation on immune outcomes using Sceloporus undulatus (Eastern fence

lizards). We experimentally elevated CORT, the primary glucocorticoid stress-relevant hormone

in reptiles, using various application regimes that differed in duration (length of time exposed),

intensity (concentration of CORT dosage), and frequency (how often exposed), and determined

how these stress aspects affected immune outcomes (Fig. 3-1). We then determined whether the

traditional classification of these treatments as “acute” or “chronic” matched the expected

immune outcomes (immune enhancement vs suppression, respectively). We chose to manipulate

CORT. Many other components of the HPA axis, including hypothalamic and pituitary

hormones, as well as the sympathetic nervous system are also important in the physiological

stress response, and may respond differently to various stress aspects. CORT plays an important

role in the stress response, its elevation is frequently linked to negative consequences (Dhabhar,

2009; French et al., 2007; Guillette Jr. et al., 1994; Martin, 2009), and our manipulation of CORT

will provide insight into how organisms respond to stressors with different characteristics.

49

Methods

Study System

Between May and July 2013, we collected 32 adult male S. undulatus (Eastern fence

lizards) using a hand-held noose. Lizards were collected from five sites across the Southern

United States: (1) Standing Stone State Park, Overton County, Tennessee; (2) Fall Creek Falls

State Park, Van Buren County, Tennessee; (3) Holly Springs National Forest, Marshall County,

Mississippi; (4) Conecuh National Forest, Covington County, Alabama; and (5) Blakeley State

Park, Baldwin County, Alabama. We measured lizards for snout-vent length (SVL) at capture and

placed lizards in individual cloth bags for transport.

Lizards were housed individually in plastic enclosures (42 x 28 x 27 cm L x W x H).

Outer walls of these enclosures were wrapped with dark paper to prevent lizards from visually

interacting, which could affect CORT concentrations. Each enclosure was lined with a paper

towel and furnished with a water dish and plastic shelter for refuge and basking. Overhead lights

were set to a 12:12 light:dark schedule, and a 60-W incandescent light bulb placed above the

shelter provided additional heat for 4 h each day to allow lizards to thermoregulate. Lizards were

fed crickets (Acheta domestica) every second day.

Treatments

Lizards were housed for at least 10 days (10-73 days) to allow lizards to acclimate and

CORT concentrations to return to baseline (i.e. CORT levels declined and then remained

relatively constant; Trompeter and Langkilde, 2011). Lizards from each site were assigned

randomly to one of 5 treatment groups, accounting for site of capture: 4 CORT-application

treatments and one control, all with appropriate handling controls (Fig. 3-1). These were selected

50

to allow us to test the contribution of differences in duration, frequency, and intensity of

elevations in CORT concentrations in determining immune outcomes, ensuring all lizards

received the same handling and exposure to CORT vehicle. Two treatments represent those

typically termed acute (by duration), but with differing intensities (different dosages applied):

high-dose acute (HA; high but ecologically relevant) and low-dose acute (LA). Lizards in these

treatments received a high or low dose of CORT, respectively, every third day and oil vehicle

only on the remaining days (described below). One treatment represented repeated acute (RA)

and involved daily applications of the low dosage of CORT. The final treatment represented a

chronic treatment (Ch), and lizards in this treatment received CORT via a slow-release patch.

Lizards in the HA, RA, and Ch treatment groups all received the same total (and average) amount

of CORT in each 3-day period (and thus over the entire treatment period).

Each day for 9 days, all lizards were given topical oil containing the appropriate amount

of CORT (high-dose, low-dose, or none), and every third day all lizards were fitted with slow-

release patches containing CORT or oil-vehicle only (Fig. 3-1). All lizards received topical doses

daily and a slow-release patch every third day, resulting in similar handling for all lizards (only

the presence of CORT in the oil solutions differed). The oil (and hormone, where relevant) was

quickly absorbed due to the lipophilic nature of lizard skin (Belliure and Clobert, 2004), resulting

in physiologically and ecologically-relevant increases in plasma CORT concentrations (Knapp

and Moore, 1997; Trompeter and Langkilde, 2011). The lower dosage was selected to mimic the

CORT response that occurs in this species in response to an attack by a fire ant predator. We

treated animals in the evening (between 1500 and 1700) because animals were less active at these

hours, negating the need to handle lizards during topical application.

We dissolved CORT (≥92%, Sigma C2505, Saint Louis MO) in commercial sesame oil at

two different physiologically relevant concentrations: 1 µg CORT / 1 µL sesame oil and 3 µg

CORT / 1 µL sesame oil. We applied 6µL of the appropriate dose to provide 6µg (“low” dose) or

51

18µg (“high,” but physiologically relevant, dose) of CORT (= ~ 0.66µg or 2µg CORT / g lizard

respectively; Fig. 3-1). Topical oil and oil-CORT solutions were applied using a repeat pipette,

resulting in elevated CORT levels 30 min after application (Trompeter and Langkilde, 2011).

Patches were constructed from the padded section of an adhesive bandage, which held the oil or

oil-CORT solution, and electrical tape and transparent surgical adhesive, which secure the patch

to the back of a lizard (Knapp and Moore, 1997). Patches were loaded with 6µL of either the high

CORT solution (18ug CORT) or oil-vehicle alone (Fig. 3-1) and provided a slow release of

hormone to the lizard over the 3-day period (Knapp and Moore, 1997). Patches were replaced on

Days 1, 4, and 7 of the treatment period at the usual time of treatment, and handling animals was

necessary to attach patches.

Blood Collection

After the 9 days of treatment, we collected blood samples from the lizards’ post-orbital

sinus using 70 µl heparinized microhematocrit tubes (VWR, San Francisco CA). Blood was

collected within 3 min of capture (51 – 198 s). Blood samples were centrifuged, and plasma was

drawn off and immediately frozen (-20°C) until assays were performed (24h for hemagglutination

assay; 4 days for bacterial killing assay; Graham et al. 2012).


As a measure of innate immunity, we measured the ability of lizard plasma to hold sheep

red blood cells (SRBC) in suspension in vitro. We washed SRBC (Innovative Research,

Novi, MI) with phosphate buffered saline (PBS) and brought it to a 2% solution with PBS. We

thawed 25µl of plasma, diluted it 1:1 with PBS (25 µL), and serially diluted this solution to 1:64

52

in a 96-well plate using a multichannel pipette. Control wells contained PBS only (25 µL). We

then added 25µL of 2% SRBC solution to each well and gently mixed plates by tapping. After

incubation at room temperature for 1 h, we scored plates for agglutination (ability of antibodies in

plasma to hold SRBC in suspension). Scores were calculated as the negative log2 of the last

dilution at which agglutination was attained – higher scores indicate higher concentration of

SRBC-specific antibodies in the plasma (Matson et al., 2005). Half scores were recorded when

SRBC precipitated partially but not to the extent of control wells. Lysis of erythrocytes was not

scored since this process has been shown to be subjective (Matson et al., 2005).


As an additional measure of innate immunity, we measured the ability of plasma to lyse

Escherichia coli bacteria. We mixed thawed plasma (14 µL) with E. coli (10 µl of 200 CFU

bacteria dilution). We allowed this solution to react and combined it with a growth medium

dilution (126 µl CO2 L-glutamine, containing 400 µL L-glutamine and19.6 mL CO2 medium).

We spread 50 µl of each sample in duplicate on agar plates, which were incubated at 37°C for 15

h. Colonies on each plate were then counted, averaged (across duplicates) and compared to mean

colony counts of two replicated control plates that contained no plasma (140 µl CO2 L-glutamine

medium + 10 µl E. coli). Percent bacterial killing was calculated as 100 - (mean plasma treatment

colony count/mean control colony count) x 100. For detailed methods, see Graham et al., 2012.

Plates that ranged from 0 to negative 10% killing (n=11) were corrected to 0%. Plates with less

than negative 10% killing (n=9) were discarded (see Fig. 3-2b for final sample sizes).

53

Data Analysis

SVL was log transformed and percent killing data were angular transformed prior to

analysis to meet assumptions of parametric tests. We analyzed hemagglutination of plasma and

percent killing separately using ANCOVA with treatment and source population as factors and

snout-vent length (SVL) and time in captivity as covariates. Time in captivity did not

significantly explain variation in the immune data (p > 0.78), suggesting that being held in

captivity for longer than 10 days does not affect these physiological measures. It was thus

removed from the final model. We used a post-hoc least square means student’s t-test to test for

differences in hemagglutination scores between treatments. All statistical analyses were

performed using JMP (version 7.0, SAS Institute Inc., Cary NC) with α = 0.05.

Results

Hemagglutination

Hemagglutination scores were significantly affected by treatment (Fig. 3-2a; F4,21=4.503,

p=0.009; SVL F1,21=1.747, p=0.200; source population F5,21=1.816, p=0.153). Post-hoc tests

revealed that lizards in the HA treatment had significantly lower hemagglutination scores than

those from LA, RA, and Control treatments; and lizards from the Chronic treatment had

intermediate hemagglutination scores.


Bacterial killing ability did not significantly differ among our treatment groups (Fig. 3-

2b; F4,17=0.865, p=0.510; SVL F1,17=0.099, p=0.757; source population F5,17=4.685, p=0.007).

54

Discussion

The four treatments in which we manipulated CORT (mimicking the physiological stress

response) and the control treatment produced different immune outcomes in this study: lizards in

the High Dose Acute (HA) treatment expressed lower concentrations of sheep red blood cell

(SRBC)-specific antibodies in their plasma (lower hemagglutination scores) than those from the

Low Dose Acute (LA), Repeated Acute (RA), and Control (Ctl) treatments, and lizards from the

Chronic (Ch) treatment expressed intermediate hemagglutination (Fig. 3-1 and 3-2). This raises a

number of concerns with considering duration alone when investigating the physiological

consequences of stress, as discussed below. Here we describe our results for the effects of total,

average, duration, frequency, and intensity of CORT application on immune function, and the

potential implications for furthering our understanding of the consequences of stress more

broadly. Future research examining how other aspects of the stress response, such as the

sympathetic nervous system or hypothalamic and pituitary hormones, might affect these general

conclusions is needed.

Total or Average Stress (CORT)

Three of our treatments (HA, RA, Ch) all received the same amount of corticosterone

(18µg CORT in each 3-day period, for 9 days total, Fig. 3-1). The immune outcomes of these

treatments, however, differed. Plasma hemagglutination of novel protein (sheep red blood cells)

was significantly lower for lizards receiving short infrequent elevations of high doses of CORT

(High Dose Acute, HA) than for lizards receiving short frequent elevations of low doses of CORT

(Repeated Acute, RA), and plasma of lizards receiving a slow release of high doses of CORT

(Chronic, Ch) had an intermediate level of hemagglutination. While hemagglutination of lizards

55

in the Ch group did not differ significantly from controls, this nonetheless supports the notion that

not all stress is the same: the average or total amount of CORT to which an individual is exposed

(or the associated stress it experiences) is not a good predictor of the immune outcome, and

specific details of the stress regime are needed to understand outcomes. The treatments in this

study were selected to reflect differences in duration, frequency, and intensity of exposure to

CORT (Fig. 3-1). Under these circumstances, intensity, but not frequency or duration of CORT

elevation, affected the immune outcome.

Duration

Standard terminology implies that duration of exposure to a stressor affects immune

outcome: repeated or continuous (days to months, “chronic”) exposure to a stressor should

suppress immune function (Dhabhar, 2009; Martin, 2009; Sapolsky et al., 2000), whereas short-

duration (minutes to hours, “acute”) exposure to a stressor should enhance or have no effect on

immune function (Deak et al., 1999; Dhabhar, 2009; Martin, 2009). We found no significant

difference in plasma hemagglutination of lizards exposed to continuous elevation of CORT

(Chronic, Ch) for 9 days versus that of lizards given short exposure to CORT (High Acute, HA)

at the same dosage, every 3 days, suggesting that duration of exposure to a single stress event

may not always have important consequences for immune function. However, we tested duration

of the discrete events (e.g. CORT was elevated for hours vs. days; Knapp and Moore, 1997) while

keeping exposure to the entire stress series consistent (all CORT treatments were applied over 9

days). It is therefore possible that we could find that duration affects immune outcomes if we alter

the total duration of exposure to CORT (e.g. 1 day vs. 30 days of exposure to continuously

elevated CORT).

56

Frequency

Frequent exposure to a short-duration high intensity stressor may result in continuous

elevation of CORT, if CORT concentrations do not have the opportunity to return to baseline

between events. Continuously elevated CORT could lead to outcomes typical of chronic stress

(Dhabhar, 2009). For example, exposure to CORT in our High Acute (HA) treatment (every 3

days) may have constituted a physiological response to persistent (chronic) exposure to a stressor,

which may explain why lizards in this treatment had similar immune outcomes to those

continuously exposed to CORT in the Chronic (Ch) treatment, despite the fact that lizards were

given three days between exposures to CORT in the HA treatment. Measuring circulating CORT

levels would inform whether frequent, high intensity stressors prevent CORT concentrations from

returning to baseline. These measurements would need to be non-invasive to avoid affecting

CORT levels and compromising the treatments. Our results, however, also suggest that repeated

exposure to acute stressors do not always produce “chronic” (immune) outcomes. Lizards

exposed to daily short-duration low dosage elevations of CORT (Repeated Acute, RA) produced

outcomes typical of acute stress: this treatment elevated plasma hemagglutination similarly to

lizards exposed to short duration low-dosage elevations of CORT every 3rd day (Low Dose Acute,

LA). Repeated exposure to low-intensity elevations of CORT (Repeated Acute, RA) may have

allowed CORT concentrations to return to baseline (though our previous study reveals that CORT

concentrations are somewhat elevated above controls after 21 days of exposure; McCormick &

Langkilde 2014). Therefore, frequency of CORT application did not appear to have important

consequences for immune outcomes at low intensities, but frequency may be important at high

intensities. Interactions between frequency and intensity of perturbations have also been shown to

result in complex effects on species diversity in community ecology (Miller et al., 2011).

Understanding how frequency and intensity of exposure to stress interact to affect the recovery of

57

CORT concentrations to baseline, and subsequent immune outcomes, would be valuable for

understanding the consequences of stress.

Our results suggest that repeated exposure to acute stressors may not always produce

“chronic” (immune) outcomes, despite the fact that repeated acute stressors are frequently

considered chronic (Harbuz and Lightman, 1992; Romero, 2004) and indeed are sometimes

referred to as “chronic intermittent stressors” (Burchfield, 1979; Martí et al., 1994). These results

support previous findings that repeated exposure to short-duration elevations in CORT enhanced

immune function in this species and had no effect on immune function in other species (Table 1).

However, many studies have found that repeated exposure to short-duration stress suppressed

immune function (Table 1) and/or had negative effects on other fitness relevant traits including

growth rate (Barton et al., 1986; McCormick et al., 1998) and condition (Busch et al., 2008b).

Together with these studies, our results demonstrate the range of physiological outcomes

associated with repeated acute stress, and that repeated exposure to short-duration stressors does

not always produce “chronic” effects (at least at low intensities). Delimiting the boundary at

which the outcomes of stress are reversed would be hugely informative.

Intensity

Our results suggest that intensity of CORT exposure can affect immune outcomes. Short-

duration exposure to stress is typically predicted to enhance or not affect immune function (Deak

et al., 1999; Dhabhar, 2009; Martin, 2009). However, our two short-durations treatments

produced very different immune outcomes: plasma hemagglutination of lizards exposed to short-

duration low-intensity CORT (Low Acute, LA) were significantly greater than those exposed to

short-duration high-intensity CORT (High Acute, HA); with hemagglutination of lizards in the

control treatment being intermediate to these two (Fig. 3-2a). This suggests that “chronic”

58

outcomes (e.g. immune suppression) could result even in response to short-duration stress if the

stressor is sufficiently intense. Immune suppression has been observed in response to short-

duration pharmacological levels of stress (Dhabhar, 2009, 2000), but our results suggest that

immune suppression in response to short-duration stress is possible even at physiologically

relevant levels. Such lasting consequences of high intensity stressors on immune function are also

observed in response to post-traumatic stress disorder (PTSD) in humans (reviewed in: Altemus

et al., 2006; Gill et al., 2009), suggesting the potentially broad importance of understanding how

exposure to stress can modulate immune outcomes.

It is possible that our results showing different immune outcomes to acute elevations of

CORT arise in part from our binary treatment design (we only applied two levels of each stress

aspect). For example, when three levels of stressor intensity were examined, both mild and

intense short-duration stressors suppressed while moderate stressor enhanced cellular immune

responses (in rats: McEwen et al. 1997; Dhabhar & McEwen 1997). It may be that there is a non-

linear, unimodal response of immune function to stress, where mild and intense short-duration

stressors enhance immune function, while moderate stressors suppress immune function. Our

study may have constituted moderate (LA, immune enhancement) and intense (HA, immune

suppression) levels of stress. Only a study that systematically examines outcomes for all possible

combinations of frequency, intensity and duration of stress can comprehensively delineate the

complete stress response surface (Miller, Roxburgh, & Shea, 2011). And these stress aspects will,

of course, need to be scaled to the relevant traits of the organism of interest (e.g. life cycle, HPA

function) in order to make them generally applicable (Roxburgh et al., 2004).

59

Conclusions

Our results reveal that average, total, or other single measures of stress (CORT) do not

satisfactorily encompass either how stress is experienced (aspects such as frequency, intensity,

and duration) or the physiological outcomes (immune function). Additionally, categorizing stress

as acute or chronic – by duration alone – and the lack of consistency in use of these terms may be

hindering progress in this field. Our results suggest that additional underlying aspects of stress,

such as stressor intensity and frequency, can affect the outcomes of stress and should also be

considered and reported if we are to adequately describe and assess the ecological and human

health consequences of stress.

Acknowledgements

We thank S. Graham, C. Thawley, M. Goldy-Brown, and M. Herr for help with lizard

collection, C. Thawley for assistance with blood collection, C. Norjen and D. McGregor for

assistance with immune assays, C. Thawley, C. Norjen, M. Goldy-Brown, and S. McGinley for

lizard care, the Cavener lab for use of their plate reader, and B. Chitterlings for valuable

comments on this manuscript. We thank personnel at Standing Stone State Park, Fall Creek Falls

State Park, Holly Spring National Forest, Blakeley State Park, Conecuh National Forest, and

especially the Solon Dixon Forestry Education Center for logistical support. The research

presented in this article adheres to the Guidelines for the Use of Animals in Research and the

Institutional Guidelines of Penn State University (IACUC #35780), and animal collection was

permitted by the respective states. Funding was provided in part by the National Science

Foundation (DGE1255832 to GLM, DEB0815373 to KS, and IOS1051367 to TL).

60

Tables

Table 3-1: This table shows the different immune outcomes (enhancement, no change, or suppression) to repeated exposure to stress

(exogenous application of CORT, handling stress, or an ecologically-relevant stressor), indicating the study organism and immune component measured. Exogenous CORT was elevated using topical application or feeding. Handling includes handling or chasing, placement in a bag, or air exposure (fish). Ecologically relevant stressors include food deprivation, social isolation, social defeat, or exposure to predator scents. *Animals were simultaneously restrained. Abbreviations as follows: Hemag. = hemagglutination; BKA = bacterial killing ability; DTH = delayed-type hyper sensitivity; Antibody Resp. = antibody response.

61

Figures

Figure 3-1: A) The frequency, intensity, and duration of CORT application in each of the treatments used in this study, and the total amount of CORT received in each 3-day period. Text in parentheses indicates: for Frequency, how frequently a CORT-oil solution was applied (oil-vehicle only was applied on remaining days); for Intensity, the amount of CORT applied during each application; and for Duration, whether the period of CORT elevation was short or long. Italicized pairs in each column represents treatments that differ in only the parameter shown in that column. B) A graphical representation of the amount of CORT applied for each of the treatments used in this study (Control (Ctl) had no CORT applied). This is provided for illustrative purposes, to convey the expected duration of CORT release following application.

62

Figure 3-2: A) Hemagglutination scores and B) Percent bacterial killing by lizard plasma after 9 days of treatment (see Fig. 3-1). Lizards in the Low Acute (LA), Repeated Acute (RA), and Control (Ctl) treatments had significantly higher hemagglutination scores than did those in the High Acute (HA) treatment, and those in the Chronic (Ch) treatment had hemagglutination scores that were intermediate to these groups. Lines above the columns connect treatments that do not significantly differ from one another. Bacterial killing ability did not significantly differ across treatments. Error bars represent means ± one standard error. The sample size for each group is given within each bar.

63

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Chapter 4

Ancestry trumps experience: Cross-generational but not early life stress affects the adult physiological stress response

Gail L. McCormicka,b, Travis R. Robbinsa,1, Sonia A. Cavigellib,c, Tracy Langkildea,b


b The Center for Brain, Behavior and Cognition, Huck Institute of Life Sciences,

c Department of Biobehavioral Health,

The Pennsylvania State University,

1Present address: Department of Biology, Northern New Mexico College

Submitted to Hormones and Behavior

Author Contributions: GLM, TRR, SAC, and TL designed the experiment. TRR performed early

life treatments. GLM collected data in adulthood and wrote the manuscript. GLM and TL

analyzed the data. All authors contributed to the manuscript.

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Abstract

Exposure to stress within a lifetime or in previous generations can have important lasting

effects on an organism’s physiology and behavior. Because prolonged exposure to stress-relevant

hormones (e.g. corticosterone, CORT) can have fitness-relevant costs, the stress response may be

up- or down-regulated to balance costs and benefits following stress exposure. We investigated

the effects of stress exposure during early life and across generations on the physiological stress

response in adulthood. We captured gravid female eastern fence lizards (Sceloporus undulatus)

from populations that naturally differ in their cross-generational exposure to stress associated

with the invasion of predatory fire ants (Solenopsis invicta). Offspring from high- and low-stress

populations were exposed weekly to either sub-lethal attack by fire ants, topical treatment of

CORT (mimicking the stress of fire ant attack), or a control treatment until maturity. Several

months after treatments ended, we quantified plasma CORT concentrations at baseline and

following restraint, exposure to fire ants, and injection with adrenocorticotrophic hormone

(ACTH). Exposure to stress (CORT or fire ants) during early life did not affect any of our

measures of the adult stress response. Offspring of lizards from fire ant-invaded populations that

have experienced high stress over multiple generations exhibited more robust CORT responses to

restraint, fire ant exposure, and ACTH-injection as adults than offspring from low-stress

uninvaded populations. Together, these results reveal that cross-generational history of stress may

be more important than early life exposure to stress in influencing adult stress responses.

Introduction

An organism’s environment in early life, including the availability of nutrients, maternal

behavior, and presence of predators and competitors, can have important consequences for

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physiology, morphology, and behavior that persist into or manifest during adulthood (Champagne

and Meaney, 2001; Kasumovic, 2013; Relyea, 2001). For example, stress exposure during early

life (i.e. prior to adulthood) can program physiology and behavior (Dalmaz et al., 2015; de Kloet

et al., 2005; Meaney et al., 1994), and lead to increased risk of aggression, anxiety, and

depressive behaviors in adult rodents and primates, including post-traumatic stress disorder

(PTSD) in humans (reviewed in Heim and Nemeroff, 2001; McCormick and Green, 2013;

Veenema, 2009). These effects of early life stress may be modulated by an individual’s genotype

(de Kloet et al., 2005; Lightman, 2008; Oitzl et al., 2010). Additionally, because stress

experienced in previous generations (by one’s ancestors) can affect an individual’s stress

physiology (Harris and Seckl, 2011; Storm and Lima, 2010; Yehuda et al., 2000), one may also

expect stress exposure in previous generations to alter how an organism responds to early life

stress (e.g. Sheriff and Love, 2013).

When persistently exposed to stressors either within a lifetime (e.g. during early life) or

across generations, an organism’s physiological stress response may change to balance associated

costs and benefits (Matthews, 2002; Meaney et al., 1994; Oitzl et al., 2010). This could take the

form of up- or down-regulation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in

increased or decreased baseline or post-stress concentrations of stress hormones (e.g.

corticosterone, CORT; adrenocorticotrophic hormone, ACTH; corticotropin-releasing hormone,

CRH) (e.g. Carpenter et al., 2007; Heim et al., 2000; Ladd et al., 1996; Plotsky and Meaney,

1993). Suppression of the stress response could reflect habituation to a specific stressor (Grissom

and Bhatnagar, 2009; Romero and Reed, 2005; Romero et al., 2009) or general down-regulation

of the response (Romero, 2004). This suppression may protect the organism from stress-related

costs (e.g. diversion of energy away from non-critical functions such as growth, reproduction, and

immune function; Chrousos and Gold, 1992; Greenberg and Wingfield, 1987; Martin, 2009) but

could reduce benefits associated with heightened stress reactivity (e.g. supporting behavioral and

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metabolic responses to stressors; Sapolsky, 2000; Thaker et al., 2009). Alternatively, up-

regulation of the stress response may allow an organism to more effectively mount responses to

current threats (Romero, 2004) while potentially incurring longer-term costs of elevated

corticosterone (Korte et al., 2005; Romero et al., 2009; Sapolsky et al., 2000). Both up- and

down-regulation of the adult stress response has been observed in response to early life stress

(Ariza Traslaviña et al., 2014; Carpenter et al., 2007; Caruso et al., 2014; Spencer et al., 2009)

and in some cases adult baseline CORT is not altered (e.g. Mirescu et al., 2004; Plotsky and

Meaney, 1993). The effects of early life stress are relatively well studied in humans and rodents

(McCormick and Green, 2013; Veenema, 2009); expanding this to other organisms with

documented differences in ancestral stress exposure will inform the evolutionary pressures

leading to the consequences of both early life and cross-generational stress.

Physiological stress has been documented in response to a variety of environmental

perturbations including habitat loss (Homan et al., 2003; Suorsa et al., 2004), urbanization

(French et al., 2008), and the introduction of invasive species (Berger et al., 2007; Graham et al.,

2012). We took advantage of naturally occurring high- and low-stress populations of native

lizards associated with cross-generational presence or absence, respectively, of predatory invasive

fire ants (Graham et al., 2012). We manipulated early life stress of offspring from these

populations and measured aspects of their stress physiology in adulthood. This allowed us to

experimentally investigate whether adult HPA activity is affected by early life and cross-

generational exposure to stress and whether these stress histories interact.

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Methods

Study System and Animal Collection

Red imported fire ants (Solenopsis invicta) are invasive predators of eastern fence lizards

(Sceloporus undulatus) (Langkilde, 2009a), and these species utilize similar habitat where their

ranges overlap (Langkilde, 2009b). Fire ants frequently attack fence lizards in nature

(Freidenfelds et al., 2012). These encounters induce anti-predator behavior (Langkilde, 2009a)

and trigger the release of CORT in lizards (Trompeter and Langkilde, 2011). Lizards in areas

invaded by fire ants have higher baseline concentrations of CORT than do those from uninvaded

sites (Graham et al., 2012).

During April and May 2012, we captured gravid female eastern fence lizards (n = 86)

from 6 sites across the southeastern United States: (1) Blackwater River State Forest, Santa Rosa

County, Florida; (2) Geneva State Forest, Geneva County, Alabama; (3) Conecuh National

Forest, Covington County, Alabama; (4) St Francis National Forest, Lee County, Arkansas; (5)

Edgar Evins State Park, DeKalb County, Tennessee; and (6) Standing Stone State Park, Overton

County, Tennessee. All sites have similar habitat (Langkilde, 2009, unpubl. data) but differ in fire

ant invasion history (Callcott and Collins, 1996): Sites 1, 2, and 3 were first invaded by fire ants

57 to 75 years ago (“invaded”), while sites 4, 5, and 6 have no previous history of fire ant

invasion (“uninvaded”).

Animal Husbandry

Gravid lizards were housed in pairs in plastic enclosures (56 x 40 x 30 cm, L x W x H)

furnished with a shelter for refuge and basking, a water bowl, and moist sand for nesting.

Overhead lights were set to a 12:12 hour light:dark schedule (light: 0800 – 2000 hours), and a 60-

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W incandescent light bulb was placed at one end of the enclosure to provide heat for 6 hours each

day to allow lizards to thermoregulate. Lizards were fed crickets (Acheta domestica) dusted with

calcium and vitamin supplements every second day, and water was available ad libitum.

We checked enclosures at least twice daily for eggs and immediately placed clutches in

plastic containers (11 x 11 x 7.5 cm) filled with moist vermiculite (-200 kpa), covered with plastic

wrap, and sealed with a rubber band (Langkilde and Freidenfelds, 2010). We placed containers in

an incubator (29o C ± 1oC) until eggs hatched (approximately 45 days), rotating the containers

every other day to avoid any within-incubator effects of position. We checked incubators twice

daily for hatchlings.

We toe-clipped hatchlings for unique identification and housed them in groups of six

based on age. Each enclosure contained two lizards from each of the three treatments (described

under Treatments) and no more than two lizards from each clutch, each from different treatments.

Lizards from fire ant-invaded sites never shared enclosures with those from uninvaded sites.

Hatchlings were housed under similar conditions as gravid females but without sand; the floor of

each enclosure was instead lined with paper towel. At 42 weeks of age, we measured all lizards

for mass and snout-vent length (SVL).

Treatments

To determine the effects of exposure to CORT or an ecologically-relevant stressor during

early life, hatchlings were assigned to one of 3 treatments using a split-clutch design. Starting at 2

weeks of age, lizards were exposed to topical application of CORT (CORT), fire ants (FA), or a

handling and oil-vehicle control (Ctl) once a week for 42 weeks, at which time lizards had

reached maturity. This regimen was selected to be ecologically-relevant, while avoiding

potentially lethal effects of frequent exposure to fire ant venom (Freidenfelds et al., 2012). To

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ensure all lizards received the same handling and topical application, each week all lizards were

individually placed in a sand-lined arena (with or without fire ants; 9 x 22 cm R x H) for 30

seconds, after which 3 µl sesame oil (with or without CORT) was applied to their backs with a

pipette. Lizards were returned to their home enclosure after treatment.

Lizards in the fire ant treatment were placed inside the testing arena with 15 - 20 fire ants.

Ants were allowed to encounter and sting the lizard, as they do in nature. A trial ended 30

seconds after the first ant contacted the lizard, and any attacking fire ants were removed from the

lizard. This provided a non-lethal exposure that induces CORT elevation (Trompeter and

Langkilde, 2011). They then had sesame oil applied to their backs.

Lizards in the CORT treatment topically received CORT (≥92%, Sigma C2505, Saint

Louis MO) dissolved in commercial sesame oil, after being removed from the sand-lined testing

arena. The oil and hormone were quickly absorbed due to the lipophilic nature of lizard skin

(Belliure and Clobert, 2004) and resulted in physiologically-relevant increases in plasma CORT

concentrations that simulated CORT responses to fire ant exposure (Knapp and Moore, 1997;

Trompeter and Langkilde, 2011). CORT doses were calculated based on the average growth of

this species in the laboratory to avoid stress associated with measuring size each week

(Freidenfelds et al., 2012) and ranged from 0.6 to 1 µg CORT/g body mass.

Lizards in the control treatment were placed in a sand-lined testing arena for 30 seconds

and then had sesame oil applied to their backs.

Blood Collection and Stress Assays

We measured baseline and stress reactive concentrations of CORT following restraint for

a subset of lizards (n=32) on a single day eight to 14 weeks after treatments ended. Baseline

blood samples were obtained from the post-orbital sinus using 70 µl heparinized microhematocrit

75

tubes (VWR, San Francisco CA) within 3 minutes of capture from their home enclosures

(Romero and Reed, 2005). We then individually placed lizards in cloth bags for 30 minutes, after

which we collected a second blood sample to assess CORT reactivity to this standardized restraint

stressor (Romero and Reed, 2005).

We measured the CORT response to an ecologically-relevant stressor, attack by fire ants,

for a separate subset of lizards (n=31) on a single day eight to 16 weeks after treatments ended.

Lizards were placed inside a sand-lined arena containing 15-20 fire ants for 60 seconds after the

first ant attacked. The number of ants attacking lizards during these trials was similar across early

life treatments (ANOVA: F1,30=1.068, p=0.358) and invasion statuses of the source population

(F1,30=0.041, p=0.841). A separate subset of lizards (n=30) was placed in a sand-lined arena with

no ants for 70 seconds to serve as a control. The difference in duration of these fire ant versus

control trials (60 vs. 70 seconds) reflects the average time it took for fire ants to attack after

placing a lizard in the arena (Robbins, unpubl. data). Lizards in both the fire ant-exposure and

control groups were immediately returned to their home enclosures for 60 minutes, after which

we obtained a blood sample as described above. All blood samples were maintained on ice during

collection, then centrifuged, and plasma drawn off and immediately frozen (-20°C) until assays

were performed.

ACTH Challenge

To determine the ability of lizards' adrenal glands to mount a CORT response, we

conducted an ACTH challenge on a separate subset of lizards (n=65) over two days 11 to 18

weeks after treatments ended. Lizards were injected intraperitoneally with 70 or 100 µl of either

adrenocorticotrophic hormone (ACTH), a pituitary hormone that stimulates the adrenal glands to

excrete CORT in lizards, (n=34) or saline solution as a control (n=31). ACTH (Sigma A6303,

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Saint Louis MO) was dissolved in saline prior to injection with doses ranging from 0.56-0.80 IU

(Klukowski 2011). Dosages did not significantly affect CORT concentrations (see Data

Analysis). After injection, all lizards were placed in individual cloth bags for 60 minutes, after

which we obtained a blood sample. Blood samples were processed using the previously described

methods.

Hormone Analysis

We measured plasma CORT concentrations using Corticosterone High Sensitivity EIA

Kits (Immunodiagnostic Systems Ltd., Fountain Hills, AZ, USA) following directions provided

in the kit. These kits have been validated for Eastern fence lizards (Trompeter and Langkilde,

2011). We diluted plasma 1:9 with buffer (5 µl plasma : 45 µl buffer) to ensure that samples fell

within the range of detection of the assay’s standard curve. We ran all samples in duplicate. The

mean intrassay coefficient of variation within the six kits was 2.35% (1.53% to 2.91%), and the

mean interassay coefficient of variation was 5.11%.

Data Analysis

CORT concentrations at baseline, following ACTH- and saline-injection, and following

exposure to fire ants or the associated control were log transformed prior to analysis to meet

assumptions of parametric tests. One data point was omitted from analysis of baseline CORT and

one from the analysis of CORT reactivity to restraint, as their values were greater than 2 standard

deviations from the mean.

For restraint stress, we calculated CORT reactivity as the baseline CORT value

subtracted from the CORT value after the 30-minute stressor. Because baseline CORT

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concentrations were not taken for lizards before exposure to fire ants or the control arena or

before ACTH- or saline-injection, we analyzed the CORT response to these stressors (post-

stressor concentrations). We analyzed baseline CORT, CORT reactivity to restraint, and CORT

response to fire ant exposure, ACTH-, or saline-injection separately using mixed-model

ANCOVA with early life treatment, fire ant invasion status, source population (nested within

invasion status), and sex as factors, maternal ID as a random effect, and snout-vent length and age

as covariates. Time to bleed was included as a covariate in the model for baseline CORT, baseline

CORT was included as a covariate in the model for CORT reactivity to restraint, and fire ant

exposure assay (FA vs. control exposure) was included as a factor in the model of CORT

response to fire ant exposure.

Site, sex, and age did not significantly explain variation in any of the CORT

concentration data (p>0.100); time to bleed and SVL did not significantly explain variation in

baseline CORT concentrations (p>0.146); SVL did not significantly explain variation in CORT

reactivity to restraint (p=0.503); the amount of ACTH or saline injected did not significantly

explain variation in the CORT response (p>0.831); and age and SVL did not significantly explain

variation in CORT responses to injection (p>0.203). These variables were thus omitted from the

respective final models. In cases where interactions were non-significant, they were removed

from the final model. All statistical analyses were performed using JMP (version 12.1, SAS

Institute Inc., Cary NC) with α = 0.05.

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Results

Baseline Corticosterone

Neither early life treatment (Fig. 4-1; F2,20=1.535, p=0.240) nor invasion status of the

source population affected baseline CORT concentrations of adult lizards (F1,6=2.094, p=0.196;

early life treatment x invasion status F2,14=0.713, p=0.508).

Corticosterone Reactivity to Restraint

The change in CORT following restraint (CORT reactivity) was not significantly affected

by early life treatment (Fig. 4-1; F2,10=0.293, p=0.752). However, CORT reactivity was

significantly greater in offspring of lizards from high-stress fire ant-invaded populations than in

those from low-stress uninvaded populations (F1,9=5.239, p=0.048; baseline F1,15=2.454,

p=0.140). The effect of early life treatment on CORT reactivity did not differ with invasion status

(early life treatment x invasion status F2,10=0.031, p=0.970).

Corticosterone Response to Fire Ants

CORT concentrations were significantly greater in lizards following exposure to fire ants

in adulthood compared to those that were control-handled (Fig 4-2; F1,33=12.954, p=0.010). This

result was not affected by the invasion status of the source population or early life treatment

(invasion status x fire ant exposure assay F1,25=0.078, p=0.782; early life treatment x fire ant

exposure assay F2,34=1.831, p=0.176; early life treatment x invasion status x fire ant exposure

assay F2,34=0.546, p=0.584). Among lizards exposed to fire ants in adulthood, offspring of lizards

from high-stress fire ant-invaded populations had greater CORT concentrations than did those

79

from low-stress uninvaded populations (F1,22=4.825, p=0.039; early life treatment F2,51=0.937,

p=0.398; SVL F1,43=12.222, p=0.001). This may have been driven by the fact that CORT-treated

offspring of lizards from high-stress populations had elevated CORT concentrations following

fire ant exposure relative to controls, whereas this was not observed in offspring from uninvaded

source populations; a trend which approached significance (early life treatment x invasion status

F2,50=2.788, p=0.070).

Corticosterone Response to ACTH Challenge

Injection of saline had a similar effect on CORT concentrations of lizards regardless of

their early life treatment (Fig. 4-3; F2,13=0.246 p=0.786) or the invasion status of the source

population (F1,8=0.814, p=0.394). These factors did not interact to explain the CORT

concentrations following injection with saline solution (early life treatment x invasion status

F2,9=0.182, p=0.837).

CORT concentrations following ACTH-injection were not affected by early life treatment

(Fig. 4-3; F2,22=2.389, p=0.115) but were significantly greater in offspring from high-stress fire

ant-invaded source populations than in those from low-stress uninvaded populations (F1,17=7.604,

p=0.013). This result was not affected by early life treatment (invasion status x early life

treatment F2,17=1.405, p=0.272).

Discussion

We investigated the effects of early life and cross-generational exposure to stress, and the

interaction of these exposure histories, on adult HPA activity. We found no effect of early life

stress (weekly exposure to CORT or fire ant attack) on adult baseline concentrations of CORT,

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CORT reactivity to restraint, or the CORT response to fire ant exposure or ACTH injection.

Offspring of lizards from high-stress fire ant-invaded and low-stress uninvaded populations had

similar baseline concentrations of CORT. However, cross-generational exposure to stress did

influence our measures of HPA reactivity: offspring of lizards from high-stress invaded

populations had greater CORT responses to restraint, fire ant exposure, and ACTH injection than

did offspring of lizards from low-stress uninvaded populations. These results suggest cross-

generational history with stress has important effects on adult HPA activity, while early life stress

may play a lesser role in this system.

Early Life Stress

We did not observe an effect of early life exposure to CORT or fire ants on HPA activity.

Early life stress does not affect adult baseline concentrations of CORT in rats (Mirescu et al.,

2004; Plotsky and Meaney, 1993); however, adult CORT reactivity is altered by early life stress

in rats and humans (Carpenter et al., 2007; Gunnar et al., 2009; Liu et al., 1997; Matthews, 2002)

and there is some evidence of this in birds (Spencer et al., 2009). We also did not observe any

effect of early life stress on CORT following ACTH injection, suggesting that this stress exposure

had no effect on adrenal function. It is important to note that we measured HPA activity several

months after treatments had ended. It is thus possible that early life stress exposure affected HPA

activity during or immediately following the treatment period, but any potential effects did not

persist two months beyond the completion of treatments. This is counter to the literature

documenting persistent effects of early life stress on adult behavior and physiology (McCormick

and Green, 2013; Meaney et al., 1994; Veenema, 2009). Lizards may have habituated to the

regular exposure to fire ants in the laboratory environment (Cyr and Romero, 2009; Romero,

2004; Romero et al., 2009) or the fire ant colony may have become less venomous in captivity

81

(Tschinkel, 2006; Xian-Fu et al., 2015), although this does not explain why no effects of early life

CORT treatment were observed. Lizards exposed to fire ants in adulthood had greater CORT

concentrations compare to those exposed to a sand-lined enclosure, suggesting that our fire ants

still induced a CORT response. Future research on whether more frequent, intense, or varied

duration of early life stress treatments produce lasting effects into adulthood would be

informative (Busch et al., 2008; McCormick et al., 1998; McCormick et al., 2015; McEwen et al.,

1997). The absence of maternal care in this species may also play a role, as maternal care may

exacerbate the effects of early life stress in mammals (Champagne and Meaney, 2001; Meaney,

2001).

Cross-Generational Exposure to Stress

Cross-generational exposure to stress predicted adult HPA activity in the current study.

Offspring of lizards from high-stress fire ant-invaded populations had greater CORT reactivity to

restraint and higher CORT concentrations following fire ant exposure than did offspring from

low-stress uninvaded populations, irrespective of early life stress treatment. These results mirror

the differences in adult HPA activity of these populations in the wild (Graham et al., 2012).

Because these patterns were observed even in lizards from the control treatment, the current study

indicates that field patterns of elevated CORT responsiveness in fire ant-invaded populations

(Graham et al., 2012) are not driven by within-lifetime stress, but rather by cross-generational

mechanisms. This could take two forms:

a) Mothers from high-stress invaded populations may differentially provision their eggs

(with nutrients, CORT, etc.; Hayward and Wingfield, 2004; Seckl and Meaney, 2004) or

alter their behavior (e.g. feeding, thermoregulation, maternal care; Champagne and

Meaney, 2001; Shine and Harlow, 1993). These changes may affect the perinatal

82

environment and lead to epigenetic changes (Fish et al., 2006; Weaver et al., 2004),

which can affect the stress responsiveness of offspring (maternal effects; Champagne and

Meaney, 2001; Liu et al., 1997; Love et al., 2013, 2008). These changes may prepare

offspring for a stressful environment (maternal matching: Sheriff and Love, 2013). Prior

research in this species argues against a role for increased yolk CORT in explaining

elevated CORT reactivity, as 3 month-old hatchlings from eggs treated with CORT had

lower, not higher, CORT reactivity to restraint than those from eggs treated with oil

vehicle only (Norjen, unpubl. data). Future research should investigate how maternal

mechanisms may affect adult HPA activity.

b) At high-stress fire ant-invaded sites, selection may favor heightened CORT

responsiveness, as elevated concentrations of CORT trigger important survival behaviors

that allow these lizards to escape attack from fire ants (Trompeter and Langkilde, 2011;

Langkilde, unpubl. data). The adult CORT response to fire ant exposure appeared to be

heightened by early life exposure to CORT in offspring from fire ant-invaded sites (a

trend which approached statistical significance; Fig. 2). Cross-generational history with

stress may thus select for greater sensitivity to early life stressors. This combination of

genetic and early life environmental factors affecting adult stress reactivity has been

documented in other systems (Gariépy et al., 2002; Jenkins et al., 2014). Further research

on how these stress histories interact in natural populations will increase our

understanding of the ecological and evolutionary significance of these patterns.

The greater CORT reactivity and CORT response to fire ant exposure in offspring of

lizards from high-stress fire ant-invaded populations is mirrored in our ACTH results: offspring

of lizards from high-stress sites had greater CORT responses to ACTH compared to those from

low-stress uninvaded sites. This indicates a general up-regulation of CORT reactivity in offspring

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from high-stress populations, with differences occurring at the adrenal glands rather than at the

level of the hypothalamus or the pituitary.

Baseline CORT

Neither early life stress nor cross-generational history with stress affected baseline

CORT. Baseline CORT varies greatly within individuals as seasons and metabolic demands

change (Bonier et al., 2009; Landys et al., 2006). Because this flexibility occurs on short time

scales, variation in baseline CORT concentrations may be best explained by environmental rather

than genetic factors (Jenkins et al., 2014). The results of this study suggest that field patterns of

lizards having higher baseline CORT at fire ant-invaded than at uninvaded sites (Graham et al.,

2012) are likely not due to early life stress or cross-generational history with stress. Instead, these

field patterns may reflect CORT responses to recent and frequent (on the order of minutes) fire

ant attack (Freidenfelds et al., 2012; Langkilde unpubl. data) rather than true baseline CORT.

This is supported by the fact that lizards from both fire ant-invaded and uninvaded sites return to

similar baseline CORT concentrations within one week in captivity (Langkilde, unpubl. data).

Alternatively, these field differences may reflect increases in true baseline CORT concentrations

as a result of persistent increases in CORT due to frequent attack by fire ants over long time

periods (McCormick and Langkilde, 2014).

Conclusions

This study demonstrates that measuring early life stress alone may not adequately capture

the population level drivers of changes in adult physiology, which may be affected by both cross-

generational and early life exposure to stress. The exposure of an individual’s ancestors to stress

84

may be more important in regulating physiological stress responses than the individual’s own

lifetime stress experiences. This has important consequences for predicting and managing the

effects of stress and for establishing whether these effects can be reversed within a lifetime.

Determining the mechanisms that drive changes in the physiological stress response would aid

predictions of how species will respond to environmental perturbations.

Acknowledgements

We thank S. Graham and C. Thawley for assistance with planning, S. Graham, C.

Thawley, and J. Newman for help with lizard collection, C. Thawley, S. McGinley and E. Baron

for assistance with adult lizard trials, C. Thawley for assistance with blood collection, C.

Thawley, G. Dewitt, D. Fricken, M. Goldy-Brown, A. Hollowell, L. Horne, M. Hook, A. Jacobs,

C. Norjen, S. McGinley, and M. O’Brien for lizard care and assistance with early life lizard

treatments, the Cavener lab for use of their plate reader, and B. Chitterlings for valuable

comments on this manuscript. We thank the Landsdale family for access to their land and lizards

and personnel at St. Francis National Forest, Edgar Evins State Park, Standing Stone State Park,

Blakeley State Park, Blackwater River State Forest, Geneva State Forest, Conecuh National

Forest, and especially the Solon Dixon Forestry Education Center for logistical support. All

methods detailed here adhere to the Guidelines for the Use of Animals in Research and the



Foundation (DGE1255832 to GLM and IOS1051367 to TL and SAC).

85

Figures

Figure 4-1: CORT reactivity to restraint is greater in offspring of lizards from fire ant-invaded sites. Adult concentrations of CORT at baseline (shaded bars) and following restraint in a bag (white bars) of lizards exposed weekly to fire ants (FA), exogenous CORT, or control treatment during early life. CORT reactivity (post-restraint stressor minus baseline) was assessed in the statistical model but stress-induced concentrations are plotted here for ease of comparisons between graphs. Bars represent means ± one standard error. The sample size for each group is given above each bar.

86

Figure 4-2: CORT concentrations following adult fire ant exposure are greater in offspring of lizards from fire ant-invaded sites. CORT concentrations following exposure in adulthood to an empty arena (FA control; shaded bars) or attack by fire ants (FA; white bars) of lizards exposed weekly to fire ants (FA), exogenous CORT, or control treatment during early life. Bars represent means ± one standard error. The sample size for each group is given above each bar.

87

Figure 4-3: ACTH-induced CORT concentrations are greater in offspring of lizards from fire ant-invaded sites. Adult CORT concentrations following injection with saline solution (shaded bars) or ACTH (white bars) of lizards exposed weekly to fire ants (FA), exogenous CORT, or control treatment during early life. Bars represent means ± one standard error. The sample size for each group is given above each bar.

88

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Chapter 5

Population history with stress predicts innate immune function response to early life glucocorticoid exposure

Gail L. McCormicka,b, Travis R. Robbinsa,1, Sonia A. Cavigellib,c, Tracy Langkildea,b


b The Center for Brain, Behavior and Cognition, Huck Institute of Life Sciences,

c Department of Biobehavioral Health,

The Pennsylvania State University,

1Present address: Department of Biology, Northern New Mexico College

Submitted to Physiological and Biochemical Zoology

Author Contributions: GLM, TRR, SAC, and TL designed the experiment. TRR performed early

life treatments. GLM collected data in adulthood and wrote the manuscript. GLM and TL

analyzed the data. All authors contributed to the manuscript.

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Abstract

Persistent or long-term stress generally suppresses immune function, but it is unclear if

the immune consequences of early life stress are modulated by cross-generational stress exposure.

In populations where the immune system is frequently activated, such as where the predominant

stressor leads to injury, it may be maladaptive to suppress immune function. Thus, the

relationship between stress and immune function may vary with environment and population

history of stress. To test this hypothesis, we collected gravid female fence lizards (Sceloporus

undulatus) from populations that naturally differ in long-term exposure to stress associated with

invasion by predatory fire ants (Solenopsis invicta). We manipulated physiological stress levels in

the resulting offspring via weekly exposure to fire ant attack, topical application of the stress-

relevant hormone corticosterone (CORT), or control treatment. Adult immune function was

quantified with assays of bacterial killing and baseline and antigen-induced hemagglutination.

The effects of early life stress on baseline hemagglutination differed with population-level history

of stress exposure. Early life CORT exposure suppressed baseline hemagglutination in offspring

of lizards from low-stress fire ant-uninvaded populations but enhanced hemagglutination in those

from high-stress fire ant-invaded populations, which may help prepare lizards for high rates of

wounding and infection associated with fire ant attack. Adult bacterial killing ability and antigen-

induced hemagglutination were not affected by early life or cross-generational stress exposure.

These results suggest that a population’s history of exposure to stress may alter specific immune

consequences of early life stress, providing insight into factors regulating costs of stress in natural

populations.

97

Introduction

The immune system plays a critical role in maintaining health by protecting an organism

from pathogens and parasites, repairing damage, and responding to infection (Murphy 2001;

Møller and Saino 2004). Various components of immune function are influenced by stress, often

through the action of glucocorticoid hormones that are produced in response to stress. Mild or

short-duration glucocorticoid elevation can enhance immune function in preparation for

wounding or subsequent infection that may occur (Deak et al. 1999; Dhabhar 2009; Martin 2009).

Intense or long-duration glucocorticoid elevation can, by contrast, suppress immune function

(Stratakis and Chrousos 1995; Sapolsky et al. 2000; Dhabhar 2009; Martin 2009); a result of

energy being diverted away from immune function and toward stress-relevant responses

(Lochmiller and Deerenberg 2000; McEwen and Wingfield 2003).

Immune suppression as a result of stress experienced early in life (i.e. before maturity)

can persist into adulthood (Michaut et al. 1981; Avitsur et al. 2006; Schmidt et al. 2015). The

immune-suppressive effects of early life stress have primarily been studied in humans (e.g. Felitti

MD et al., 1998; Dong et al., 2004; Danese et al., 2007; Miller et al., 2009) and rodents (e.g.

Michaut et al., 1981; Avitsur et al., 2006), with less known about lasting effects of early life stress

on immune function in other species (but see birds De Coster et al., 2011; Kriengwatana et al.,

2013; Schmidt et al., 2015). Ectotherms, for example, are unable to internally regulate

temperature and may allocate resources toward the immune system in a different manner than do

mammals (Zimmerman et al. 2010); thus ectotherms may experience different immune

consequences of early life stress. Understanding the scope of the effects of stress on immune

outcomes is critical to comprehending how species respond to environmental challenges.

The immune outcomes of stress can vary between populations (e.g. in response to

tourism and urbanization; Martin II et al. 2005; French et al. 2008, 2010). It is possible that

98

differences in outcomes reflect a population’s history of stress exposure (e.g. HPA function and

behavior; Yehuda et al. 2000; Storm and Lima 2010; Harris and Seckl 2011), or how limited

energy is allocated during duress (i.e. either growth, reproduction, and/or immunity; Martin et al.

2004; Pörtner et al. 2005; Fischer and Thatje 2008; Du et al. 2012). For example, in populations

where the prevalent stressor (e.g. predators, aggressive competitors, pathogens) is likely to injure

an individual or increase infection risk (Cox and John-Alder 2007; Ezenwa et al. 2012), it may be

maladaptive to compromise immune function in response to stress.

We experimentally investigated the independent and interactive effects of early life and

cross-generational stress exposure on adult immune function. We took advantage of lizard

populations that vary in exposure to an environmental stressor that can lead to wounding— i.e.

the presence or absence of predatory invasive fire ants (Langkilde 2009a; Graham et al. 2012). To

test the prediction that lizards from predator-exposed populations up-regulate, rather than

suppress, immune function in response to stress, we manipulated early life stress exposure then

measured adult immune function in lab-reared offspring of lizards from fire ant-invaded and

uninvaded populations. Previous research in this system has demonstrated effects of cross-

generational predator-exposure, but not early life stress exposure, on adult HPA activity (Chapter

4). Here we investigate long-term effects of cross-generational and early life stress exposure on

immune function in this system.

Methods

Lizards were collected, housed, handled, and treated following procedures outlined in

Chapter 4. An overview of these methods is provided below.

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Study System and Collection

Eastern fence lizards (Sceloporus undulatus) and red imported fire ants (Solenopsis

invicta) occupy similar habitat where their ranges overlap (Langkilde 2009b). These invasive fire

ants are predators of fence lizards (Langkilde 2009a) and frequently bite and sting lizards in the

wild (Freidenfelds et al. 2012). These attacks break the skin, leaving lizards vulnerable to

infection, and envenomation can be lethal (Langkilde 2009a). Fire ant attacks trigger the release

of CORT in lizards (Trompeter and Langkilde 2011), and lizard populations in fire ant invaded

areas have higher baseline CORT concentrations (“high-stress” populations) than those from fire

ant uninvaded sites (“low-stress” populations) (Graham et al. 2012). All methods used in this

study adhere to the Guidelines for the Use of Animals in Research and the Institutional

Guidelines of Penn State University (IACUC #35780), and animal collection was permitted by

the respective states.

During April and May 2012, we collected 86 gravid female eastern fence lizards from 6

sites with similar habitats (Langkilde, 2009, unpubl. data) across the southeastern United States:

(1) Blackwater River State Forest, Santa Rosa County, Florida; (2) Geneva State Forest, Geneva

County, Alabama; (3) Conecuh National Forest, Covington County, Alabama; (4) St Francis

National Forest, Lee County, Arkansas; (5) Edgar Evins State Park, DeKalb County, Tennessee;

and (6) Standing Stone State Park, Overton County, Tennessee. The first three sites were invaded

by fire ants 57 to 76 years ago (“invaded”), while the last three sites have no history of fire ant

invasion (“uninvaded”) (Callcott and Collins 1996).

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Animal Husbandry

Gravid females were returned to the Pennsylvania State University and housed in pairs in

plastic enclosures (56 x 40 x 30 cm, L x W x H) until oviposition. Each enclosure was furnished

with moist sand, a water dish, and a shelter at one end for refuge and basking. Overhead lights

were set to a 12:12 light:dark schedule (800 – 2000 hrs), and a 60-W incandescent light bulb

placed at one end of the enclosure provided a thermal gradient for 6 hrs each day to allow lizards

to thermoregulate. Lizards were fed crickets (Acheta domestica) dusted with calcium and vitamin

supplements every second day, and water was available ad libitum.

Eggs were placed by clutch in plastic containers (11 x 11 x 7.5 cm L x W x H) filled with

moist vermiculite (-200 kpa), covered with plastic wrap and sealed with a rubber band (Langkilde

and Freidenfelds 2010). Egg containers were placed in the incubator (29 ± 1oC) until hatching

(approx. 45 days) and were rotated every other day to avoid any within-incubator effects of

position. Incubators were checked twice daily for hatchlings.

Once lizards hatched, they were toe-clipped for unique identification and housed in

groups of six based on age. Each enclosure contained two lizards from each of the three

treatments and no more than two lizards from each clutch. Lizards from fire ant invaded sites

never shared enclosures with those from uninvaded sites. Hatchlings were housed under similar

conditions as gravid females but without sand; the floor of each enclosure was instead lined with

paper towel. Mass and snout-vent length (SVL) of all lizards were measured at 42 weeks of age.

Treatments

To determine the effects of exposure to CORT or an ecologically-relevant stressor during

early life, hatchlings were assigned to one of three treatments using a split-clutch design. Lizards

101

were exposed to fire ants (FA), topical application of CORT (CORT), or a handling and oil-

vehicle control (Ctl) once a week between 2 and 43 weeks of age, at which time lizards had

reached maturity. Each week, lizards were individually placed in a sand-lined arena (with or

without fire ants) for 30 seconds, after which 3 µl sesame oil (with or without CORT) was applied

to their backs with a pipette. This allowed us to control for effects of handling and topical

application across all treatments.

Lizards in the fire ant exposure treatment were exposed to 15 to 20 fire ants, which were

allowed to bite and sting the lizard as they do in nature. The trial was ended 30 seconds after the

first ant contacted the lizard, providing a non-lethal level of exposure known to induce CORT

elevation (Trompeter and Langkilde 2011). They then received a topical application of sesame

oil.

Lizards in the CORT treatment were placed in an empty testing arena for 30 seconds and

then received a topical dose of CORT (≥92%, Sigma C2505, Saint Louis MO) dissolved in

commercial sesame oil, which was quickly absorbed due to the lipophilic nature of lizard skin

(Belliure and Clobert 2004). This type of application results in physiologically-relevant increases

in plasma CORT concentrations that simulate CORT responses to fire ant exposure (Knapp and

Moore 1997; Trompeter and Langkilde 2011). CORT doses were calculated based on the average

growth for this species in the laboratory to avoid stress associated with measuring size each week

(Freidenfelds et al. 2012). Doses ranged from 0.6 to 1 µg CORT / g body mass.

Lizards in the control treatment were placed in an empty testing arena for 30 seconds and

then received a topical dose of sesame oil.

102

Blood Collection

Eight to 14 weeks after treatments ended, we collected blood from the post-orbital sinus

of 32 lizards (5 to 6 per treatment) using 70 µl heparinized microhematocrit tubes (VWR, San

Francisco CA). Blood samples were collected within 4.3 min of capture (116 ± 50 seconds). Time

to catch and bleed did not influence the immune parameters that we measured (ANOVA;

p>0.661). We maintained blood samples on ice until blood collection was completed. Blood

samples were centrifuged, and plasma was drawn off and immediately frozen (-20°C) until

immune assays were performed (24hr for hemagglutination assay; 4 days for bacterial killing

assay; Graham et al., 2012).


We measured baseline and acquired hemagglutination of lizard plasma to assess innate

and adaptive humoral immune function, respectively (Matson et al. 2005). This assay measures

ability of lizard plasma to hold sheep red blood cells (SRBC) in suspension in vitro. We first

measured baseline hemagglutination, which is primarily driven by natural antibodies. SRBC

(Innovative Research, Novi, MI) were washed with phosphate buffered saline (PBS) and brought

to a 2% solution with PBS. Plasma (25 µL) was diluted 1:1 with PBS, then serially-diluted to

1:64 in a 96-well plate using a multichannel pipette. Control wells contained PBS only (25 µL).

Twenty-five µl of 2% SRBC solution was then added to each well, and the plate contents were

mixed by gentle tapping. After incubation at room temperature for 1 hour, plates were scored for

agglutination (ability of antibodies in plasma to hold SRBC in suspension). Scores were

calculated as the negative log2 of the last dilution at which agglutination was attained—higher

scores are associated with a stronger immune response (Matson et al. 2005). Half scores were

103

recorded when SRBC precipitated partially but not to the extent of control wells. Lysis of

erythrocytes was not scored since this process has been shown to be subjective (Matson et al.

2005).

To measure acquired SRBC-specific antibody responses, after blood was collected for

baseline assays, lizards were injected intraperitoneally with 50 µL of 25% SRBC solution using

an insulin syringe and a 26 g needle. Lizards were then returned to their home enclosures for 15

days to ensure time for an appropriate antibody response (Graham et al. 2012). We re-sampled

lizards and calculated hemagglutination scores using the previously described methods.

Bacterial Killing Assay

We measured the ability of plasma to lyse Escherichia coli bacteria (American Type

Culture Collection 8739; Epower Microorganisms, catalog no. 0483E7, MicroBiologics, St.

Cloud, MN) as a coarse measure of innate immunity. Thawed plasma (14 µL) was mixed with E.

coli (10 µl of 200 CFU bacteria dilution), and this solution was allowed to react. This solution

was then combined with a growth medium dilution (126 µl CO2 L-glutamine, containing 400 µL

L-glutamine and19.6 mL CO2 medium). Each sample (50 µl) was spread on agar plates in

duplicate and incubated at 37°C for 15 hr. Control plates contained no plasma and an additional

14 µL CO2 L-glutamine to create an equal volume (140 µl CO2 L-glutamine medium + 10 µl E.

coli). Colonies on each plate were then counted, averaged (across duplicates) and compared to

mean colony counts of four replicated control plates. Percent bacterial killing was calculated as

100 - (mean plasma treatment colony count/mean control colony count) x 100. For detailed

methods, see (Graham et al., 2012). Plates that ranged from 0 to negative 10% killing (n=4) were

corrected to 0%. Plates with less than negative 10% killing (n=1) were discarded.

104

Data Analysis

We analyzed hemagglutination of plasma and percent killing separately using ANCOVA

with treatment, fire ant invasion status, source population (nested within invasion status), and sex

as factors, maternal ID as a random effect, and SVL and time in captivity as covariates. For

SRBC-inoculated lizards, baseline hemagglutination score was also included as a covariate to

account for initial variation in humoral activity. Percent killing data were angular transformed

prior to analysis to meet assumptions of parametric tests. Sex, SVL, and source population did not

significantly explain variation (p>0.169) and were omitted from the final models. All statistical

analyses were performed using JMP (version 12.1, SAS Institute Inc., Cary NC) with α = 0.05.

Results

Hemagglutination

The effect of early life stress on adult baseline hemagglutination was dependent upon the

fire ant invasion status of the source population (Fig. 1; Treatment x Invasion F2,12=4.170,

p=0.041; Treatment F2,13=0.127, p=0.882; Invasion F1,8=0.042, p=0.843). In offspring of lizards

from low-stress uninvaded populations, early life CORT exposure suppressed baseline

hemagglutination scores in adulthood relative to controls, but in offspring from invaded

populations, early CORT exposure enhanced baseline hemagglutination scores.

Adult post-inoculation hemagglutination scores were not related to early life treatment

(Fig. 1; F2,11=0.717, p=0.509), invasion status (F1,4=4.840, p=0.095) or an interaction of the two

(F2,14=0.222, p=0.804; baseline hemagglutination covariate F1,24=6.65, p=0.017).

105


Adult percent killing of E. coli by plasma was not related to early life stress exposure

(Fig. 2; F2,19=0.014, p=0.986), invasion status of the source population (F1,15=1.875, p=0.192), or

an interaction of the two (F2,19=2.045, p=0.157).

Discussion

In offspring of lizards from fire ant-uninvaded sites, early life CORT (but not fire ant)

exposure suppressed adult baseline hemagglutination compared to controls, but in offspring from

invaded sites, early life CORT exposure enhanced baseline hemagglutination. Neither early life

nor cross-generational exposure to stress affected adult bacterial killing ability or post-inoculation

hemagglutination scores.

Suppression of adult baseline hemagglutination by early life CORT exposure in lab-

reared offspring of lizards from low-stress fire ant-uninvaded sites is consistent with the

suppressive effects of long-term and early life stress on innate immune function of rodents, birds,

and humans (Michaut et al. 1981; Avitsur et al. 2006; De Coster et al. 2011; Kriengwatana et al.

2013; Schmidt et al. 2015). Because development of the immune system is energetically costly

(Sheldon and Verhulst 1996; Lochmiller and Deerenberg 2000; Norris and Evans 2000; Klasing

2004), immune function development may not be prioritized if energy is limited as a result of

early life stress. The opposite pattern, however, was observed in lab-reared offspring of lizards

from high-stress fire ant-invaded sites; when exposed to CORT during early life, these offspring

had higher baseline hemagglutination scores in adulthood compared to controls. Enhancing

immune function in response to early life stress may be beneficial for lizards from these

106

populations. Fire ants are the predominant stressor facing lizards in these populations (Langkilde

2009a; Graham et al. 2012), and fire ant attacks are frequent (on the order of every few minutes;

Freidenfelds et al., 2012). The bites and stings of fire ants break the skin, increasing risk of

infection (Elkan and Cooper 1980; Murphy 2001), and lizards from fire ant-invaded sites have

higher rates of tail autonomy and other wounding (Thawley unpubl. data), possibly due to

behavioral responses of lizards to fire ants attracting the attention of visual predators

(Freidenfelds et al. 2012). This frequent wounding may lead to selection against stress-induced

innate immune suppression at these sites, and may even favor enhancement of innate immunity

(including hemagglutination). Innate immune function is essential for a rapid response to frequent

low-grade wounding and infection (Murphy 2001), and may be favored over the slower adaptive

immune function in this context (McDade et al. 2001). Other energy-sensitive traits (e.g.

behavior, growth, reproduction) may also be traded-off in order to maintain a high-functioning

innate immune system at these sites (Svensson et al. 1998; Norris and Evans 2000; Van Der Most

et al. 2011; Rauw 2012), and should be further investigated.

The mechanism behind the documented interaction between early-life and cross-

generational stress exposure are unclear, but could be induced by epigenetic processes

(Mostoslavsky and Bergman 1997; Fitzpatrick and Wilson 2003; Teitell and Richardson 2003),

and/or maternal effects, such as the transfer of maternal immunological memory (Hasselquist et

al. 2012; Ismail et al. 2015). These processes could affect the immune system directly or could

affect other systems, such as the HPA axis, that have cascading effects on the immune system.

We have found that lizards from fire ant-invaded populations have greater stress (CORT)

responsiveness as adults in the lab regardless of stress exposure in early life (McCormick, unpubl.

data), but it is unclear if underlying mechanisms affect HPA and immune function in a similar

manner. Additional research on the mechanisms behind these early life and cross-generational

changes would be useful.

107

In contrast to the effects of CORT exposure in early life, exposure to fire ants in early life

did not affect baseline hemagglutination scores. Whereas CORT exposure remained consistent

throughout the treatment period, fire ant exposure may have become less stressful over time if

lizards habituated to this exposure (Romero 2004; Cyr and Romero 2009; Romero et al. 2009) or

if the fire ant colony became less venomous in captivity (Tschinkel 2006; Xian-Fu et al. 2015),

rendering this treatment similar to the control. Alternatively, it may be that the immune enhancing

effect of CORT elevation due to fire ant attack is counteracted by potentially immune suppressive

effects of venom (Yi et al. 2003; Tankersley 2008) or frequent wounding (Plaistow et al. 2003).

Patterns of immune function in the field mirror the lack of immune modulation in

response to early life fire ant exposure in this study. In the field, baseline hemaggglutination

scores are similar for lizards at fire ant-invaded and -uninvaded sites (Graham et al. 2012) despite

the presence of both early life and cross-generational exposure to fire ants at invaded sites. The

field patterns do not, however, reflect the up-regulation of baseline hemagglutination following

lifetime CORT exposure in the present study. Several possibilities could explain these conflicting

findings: 1) CORT doses in the lab may have been greater than those elicited by stress in the

field. However, we selected the laboratory CORT dosage to reflect CORT elevations that occur in

response to natural encounters with fire ants (Trompeter and Langkilde 2011). 2) Adult lizards

used in this study were smaller and likely younger than those surveyed in the field (SVL;

McCormick unpubl. data). Younger lizards may up-regulate immune function in response to early

life CORT, as seen in this study, but older lizards may prioritize other traits, such as reproduction

(reviewed in Forslund and Pärt, 1995). 3) It is possible that intrinsic immune function is up-

regulated in lizards at fire ant-invaded sites in the field, but suppressed by extrinsic environmental

factors (e.g. frequent wounding), resulting in baseline levels that are similar to lizards at

uninvaded sites (Martin et al. 2011; Du et al. 2012). 4) Energy limitations in the field may prevent

lizards at high-stress fire ant-invaded sites from up-regulating immune function. This is supported

108

by the fact that immune effects of early life stress in adult birds are only observed under

energetically favorable conditions, and are not observed when energy demands are high (De

Coster et al. 2011). 5) Our application of CORT does not mimic the full spectrum of the HPA

response to an environmental stressor, as would be experienced in the field, including higher-

order effects (e.g. norepinephrine response) that may offset CORT effects. Further research into

how these factors affect the interaction of within- and across-generation stress exposure are

needed.

In the current study, adult acquired hemagglutination scores 2-weeks after inoculation

with sheep red blood cells (SRBC) were not affected by either early life or cross-generational

history with stress. This mirrors work in birds, in which early life nutritional stress did not affect

post-inoculation hemagglutination scores (Kriengwatana et al. 2013). It may not be surprising that

we observed modulation of innate (baseline hemagglutination) but not adaptive (post-inoculation)

immunity following exposure to stress. In reptiles, the adaptive immune system generally takes

longer to respond than in mammals and birds, in part because the innate immune system produces

a stronger response than in mammals and birds (Zimmerman et al. 2010). It is important to note

that hemagglutination scores did not increase overall in response to SRBC-injection, as would be

expected if an adaptive response had occurred (Ochsenbein and Zinkernagel 2000; Zimmerman et

al. 2010; Graham et al. 2012). The incubation period of fifteen days should have allowed an

appropriate adaptive response of SRBC-specific antibodies in this species, as demonstrated in

wild-caught lizards (Graham et al. 2012). However, in some cases the maximal response of

humoral immunity in reptiles takes six to eight weeks (Zimmerman et al. 2010) and our younger

lizards may have taken longer to respond than the older field lizards (Palacios et al. 2009;

Hopkins and Durant 2011).

In contrast to the effects of stress on baseline hemagglutination, early life stress exposure

(CORT or fire ants) did not affect bacterial killing ability of adult lizard plasma. Previous work

109

on this species has revealed similar short-term effects of stress on baseline hemagglutination but

not bacterial killing of E. coli (McCormick and Langkilde 2014; McCormick et al. 2015). In

birds, the opposite pattern is found—early life stress does not affect baseline hemagglutination

but does affect bacterial killing (De Coster et al. 2011; Kriengwatana et al. 2013; Schmidt et al.

2015). This varied sensitivity to early life stress may be a result of differential resource allocation

to specific immune responses (i.e. immune components, including complement, antibodies,

cellular activity) in ectotherms versus endotherms (Zimmerman et al. 2010), or due to species-

specific effects of early life stress on immune function (Schmidt et al. 2015). However, the

immune consequences of stress are known to vary depending on the immune component

measured (in response to short term stress: Matson et al., 2006; Stier et al., 2009; Brooks and

Mateo, 2013; McCormick and Langkilde, 2014; in response to early life stress: von Hoersten et

al., 1993; De Coster et al., 2011; Kriengwatana et al., 2013; Schmidt et al., 2015). Even within the

same branch of the immune system (e.g. innate), there is modulation of some components, such

as activity of phagocytes, but not others, such as complement (Schmidt et al. 2015) in response to

stress. It has been suggested that up-regulating a specific immune component may allow animals

to avoid an energetically-costly generalized immune response (Wegner et al. 2007). Bacterial

killing of E. coli 8739 (Millet et al. 2007; Graham et al. 2012) and hemagglutination (Matson et

al. 2005; Graham et al. 2012) both involve complement and natural antibody immune responses,

and we are unable to tease apart the effects of early life stress on these components. However,

early life and cross-generational history with stress may have different effects on other

components of the immune system.

110

Conclusions

The results of this study demonstrate that exposure to stress within a lifetime and across

generations interact to affect adult immune function. This is in contrast to our prior findings on

adult lizard HPA axis regulation, where cross-generational, but not early life, stress exposure was

shown to affect adult HPA activity in this species (Chapter 4). Cross-generational history with

stress, but not early life stress, also influenced survival and morphology of this species

(Langkilde, unpubl. data). Together, these results suggest that cross-generational and early life

stress affect different traits in different manners, and caution that cross-generational history with

stress likely plays an important role in determining adult phenotype. Thus, the interaction

between early life and cross-generational stress exposure should be considered.

Many organisms will be exposed to novel stressors as a result of global environmental

change, and it is critical that we understand how immune function and other fitness-relevant traits

trade off to balance the energetic costs of responding to these stressors. Assessing how these

responses may vary across taxa (including endotherms versus ectotherms), traits, and

environmental gradients is necessary to allow generalizations that permit the prediction and

management of the effects of stress on wild populations.

Acknowledgements

We thank S. Graham and C. Thawley for assistance with planning, S. Graham, C.

Thawley, and J. Newman for help with lizard collection; C. Thawley, S. McGinley and E. Baron

for assistance with lizard trials; C. Thawley for assistance with blood collection, D. McGregor for

assistance with immune assays; C. Thawley, G. Dewitt, D. Fricken, M. Goldy-Brown, A.

Hollowell, M. Hook, C. Norjen, S. McGinley, L. Horne, A. Jacobs and M. O’Brien for lizard care

111

and assistance with developmental lizard treatments; the Cavener lab for use of their plate reader;

and B. Chitterlings for valuable comments on this manuscript. We thank the Lansdale family for

access to their land and lizards and personnel at St. Francis National Forest, Edgar Evins State

Park, Standing Stone State Park, Blackwater River State Forest, Geneva State Forest, Conecuh

National Forest, and especially the Solon Dixon Forestry Education Center for logistical support.

All methods detailed here adhere to the Guidelines for the Use of Animals in Research and the



Foundation (DGE1255832 to GLM and IOS1051367 to TL and SAC).

112

Figures

Figure 5-1: Early life and cross-generational history of stress exposure interact to affect adult baseline, but not post-inoculation, hemagglutination scores. a) In offspring of lizards from fire ant-uninvaded populations, CORT exposure during early life suppressed adult baseline plasma hemagglutination compared to controls. The opposite effect was seen in offspring of lizards from fire ant-invaded populations: early life CORT exposure enhanced adult baseline hemagglutination compared to controls. b) Post-inoculation hemagglutination scores did not differ across early life stress treatment or fire ant-invasion status. Bars represent means ± one standard error and sample size for each group is shown above each set of bars.

113

Figure 5-2: Bacterial killing by plasma of adult lizards is not related to early life or cross-generational history with stress. Offspring of lizards from fire ant invaded and uninvaded populations exposed weekly to fire ants (FA), CORT, or control treatment from hatching until maturity had similar percent bacterial killing ability of plasma as adults. Bars represent means ± one standard error and sample size for each group is shown above each bar.

114

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Chapter 6

Conclusions

The environment is changing rapidly (Vitousek 1997), and these changes can be stressful

for native organisms (e.g. Homan et al. 2003; Tomei et al. 2003; Berger et al. 2007; French et al.

2008). My dissertation research reveals that, in order to understand the consequences of this

stress, a new stress framework is necessary that considers stressor characteristics and an

individual and population’s history with stress. Using populations with differing histories of

stress exposure in manipulative lab experiments, I provide important insight into the

physiological consequences of stress. Although repeated acute stressors are typically considered

chronic, they do not produce typical “chronic” outcomes (immune suppression) in lizards

(Chapter 2). The current duration-centric stress terminology does not adequately describe stress

or its outcomes, as the outcomes of stress depend upon not only stressor duration, but also on

intensity and perhaps frequency (Chapter 3). Stress experienced by previous generations is more

influential than stress experienced during an individual’s lifetime (i.e. early life) in determining

adult lizard stress physiology (Chapter 4), and these stress histories interact to affect immune

function (Chapter 5), demonstrating that the drivers of stress outcomes can differ among traits.

These insights provided by the study system of fence lizards and fire ants reveal a general need to

consider both stressor characteristics and stress history when investigating the outcomes of stress

in other systems.

This work suggests further avenues of research addressing additional factors that may

affect the outcomes of stress:

123

1) The stress framework of allostatic overload indicates that stress may cause pathology

only when the energetic costs of normal maintenance and the stressor become a burden (McEwen

and Wingfield 2003). Lizards held in the lab for my research were fed ad libitum and thus were

likely not constrained by energy. Further research examining whether animals experience more

pronounced outcomes of stress when energy is limited, as it is in the field, would be informative.

2) The invasion gradient of fire ants corresponds to latitude so that fire ant-invaded sites

generally occur at lower latitudes than uninvaded sites. Although all sites used in my dissertation

research are similar in habitat, observed differences between lizards from invaded and uninvaded

sites may be confounded by latitude and corresponding variables such as temperature (De Frenne

et al. 2013). Investigating the existence of latitudinal gradients in lizard stress physiology at fire

ant-free locations could inform whether existing gradients or fire ant presence play a greater role

in the documented patterns of stress physiology.

3) Lizards used in the lab studies presented here were limited in their capacity to

behaviorally mitigate stress (Schreck et al. 1997; Trompeter and Langkilde 2011). Studies that

allow animals to behave naturally would provide more ecologically relevant results.

4) My dissertation research reveals that stress can have differing effects across

physiological traits; early life and cross-generational history with stress affected lizard HPA

activity and immune function in different ways. We may thus expect stress to have varied effects

on other traits (Greenberg and Wingfield 1987; Breuner et al. 1998; Meylan and Clobert 2005;

Schmidt et al. 2012). Future research investigating the effects of stressor characteristics and stress

history on traits including behavior, reproduction, morphology, and survival would allow a

broader understanding of the ecological consequences of stress.

5) Just as traits may differentially respond to stress, so too may branches (i.e. innate vs.

adaptive) or components (e.g. cellular, antibodies, complement) of the immune system (Ilmonen

et al. 2003; Stier et al. 2009; De Coster et al. 2011; Schmidt et al. 2015). Depending on the type

124

of stressor or the predominant challenges faced by a population, either innate or adaptive

immunity may be favored. Similarly, certain immune components may be favored, perhaps at a

cost to other components (Norris and Evans 2000). It may be that up-regulating a specific

immune component allows animals to avoid an energetically-costly generalized immune response

(Wegner et al. 2007). Research that systematically employs a suite of complementary immune

assays could pinpoint which components of the immune system are affected by stress, to what

extent they are affected, and the corresponding fitness consequences.

6) Similarly, observed immune outcomes may vary with timing of the stressor and

anatomical focus of the assay. CORT and other stress-associated hormones (i.e. norepinephrine,

epinephrine) initiate redistribution of immune cells within the body (Dhabhar et al. 2012).

Immediately after an organism is exposed to a short-term stressor, immune cells increase in

concentration in the bloodstream, followed by a decrease in immune cell concentration as cells

are redistributed toward the skin (Dhabhar et al. 2012). As such, observed immune suppression

within the bloodstream may in fact represent immune redistribution of immune cells toward the

skin and out of the bloodstream (Dhabhar and McEwen 1997; Dhabhar et al. 2012). Because this

redistribution occurs over time, immune measures may appear to be affected differently by stress

if measured immediately after the stressor occurs versus a few hours or a few days later.

Similarly, changes in adaptive immunity may not be observable immediately after the stressor is

induced, because adaptive immune responses act more slowly than innate responses (Murphy

2001; Zimmerman et al. 2010). Future studies investigating the effects of stress on immune

function over time and in different locations of the body (e.g. blood vs. skin) would provide a

clearer picture of how overall immune function is affected by stress, and relevant fitness

consequences.

My dissertation research provides the foundation for future work by addressing a number

of important gaps in our understanding of the physiological consequences of stress. I considered

125

limitations of the duration-centric terminology used to define stress and revealed that other

stressor characteristics— specifically intensity and frequency—may influence the outcomes of

stress. Additionally, my work indicates that stress exposure both within a lifetime and across

generations can influence an organism’s physiology. These insights into how stressor

characteristics and stress history affect the outcomes of stress will allow us to better predict how

organisms will be affected by the increasing stress imposed by global environmental change, and

to better allocate resources to manage these fitness-relevant consequences.

126

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Appendix

What Makes Stress Stressful? Extending the Acute-Chronic Stress Paradigm

The ecological causes and consequences of stress have received increasing attention in

the last decade and have been the focus of many special features and symposia (e.g. Functional

Ecology Special Feature (2013): The Ecology of Stress; Society of Integrative and Comparative

Biology Symposium (2013): Coping with Uncertainty; Journal of Experimental Biology Special

Feature (2014): Stress, Challenging Homeostasis).

Stress is typically characterized by the duration of the stressor. Acute stress is

characterized as “short” in duration (Burchfield, 1979; Romero, 2004), lasting from minutes to a

few days (Boonstra, 2012; Martin, 2009), and is usually not repeated. Chronic stress is

characterized as “long” in duration (Burchfield, 1979; Romero, 2004; Sapolsky et al., 2000),

lasting from days to months (Boonstra, 2012; Dhabhar, 2009; Martin, 2009): a stimulus to which

the organism is continuously or repeatedly exposed. This terminology is frequently used in the

animal stress literature (Table A-1), as well as in human medical practice (A.D.A.M. Inc., 2013;

Merriam-Webster Incorporated, n.d.). These terms are also used widely in popular culture.

Despite the fact that some studies have independently addressed stressor intensity

(McEwen et al., 1997; Ottenweller et al., 1989), or frequency (Busch et al., 2008; McCormick et

al., 1998) we know of only one study that examines more than one aspect simultaneously

(McCormick et al., 2015). Furthermore, while stress is usually characterized by stressor duration,

it is also characterized by the consequence of the stressor (e.g. typically acute stress enhances,

while chronic stress suppresses, immune function).

130

Unfortunately, common usage of these terms for both the cause and the consequence

of stress, as well as the acute-chronic paradigm’s focus on duration, results in confusion.

Inconsistent use of terminology contributes to circular definitions and conflicting results on the

consequences of stress with different durations (Table A-1), and hinders our ability to predict

how an organism will be affected by stress. Relying on “acute” and “chronic” to encompass a

plethora of meanings (different stressor aspects; cause vs. consequence), and limiting our

definitions to duration of stress, may be hampering progress towards understanding how stress

can have different consequences (Romero et al., 2009). A more complete stress framework, that

includes other aspects of stress that are likely critically important in determining the

consequences of stress, will allow more rapid progress in an increasingly important field that is

currently hampered by a limited terminological framework.

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Tables

Table A-1: Representative studies from the literature showing the full range of both duration of stress applications (rows) and consequences (columns) of stress, using definitions of “acute” and “chronic” stress from each source paper. Shaded boxes represent combinations expected according to existing theory.

132

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VITA — GAIL LINDSEY MCCORMICK

EDUCATION Ph.D. Ecology Intercollege Graduate Degree Program. Penn State University. May 2016. B.S. with high distinction. (Ecology & Evolutionary Biology) University of Michigan. 2010. B.T.A. with highest honors. (Theatre Arts) University of Michigan. Ann Arbor, MI. 2010.

PUBLICATIONS McCormick, GL, T Robbins, S Cavigelli, T Langkilde. Under review. Ancestry

trumps experience: Cross-generational but not developmental stress affects the physiological stress response. Horm Behav.

McCormick, GL, K Shea, T Langkilde. 2015. How do duration, frequency, and intensity of exogenous CORT elevation affect immune outcomes of stress? Gen Comp Endocrinol 222: 81-87.

McCormick, GL, T Langkilde. 2014. Immune responses of Eastern fence lizards (Sceloporus undulatus) to repeated acute elevation of corticosterone. Gen Comp Endocrinol 204: 135-140.

Stuble, KL, MA Rodriguez-Cabal, GL McCormick, RR Dunn, NJ Sanders. 2013 Tradeoffs, competition, and coexistence in eastern deciduous forest ant communities. Oecologia 171(4): 981-992.

Graham, SP, NA Freidenfelds, GL McCormick, T Langkilde. 2012. The impacts of invaders: Basal and acute stress glucocorticoid profiles and immune function in native lizards threatened by invasive ants. Gen Comp Endocrinol 176(3): 400-408.

GRANTS, AWARDS & FELLOWSHIPS 2016 Alumni Association Dissertation Award. Penn State Alumni Association. $5,000 2015 Ecology Research Assistantship. PSU Ecology Program. $11,800 (declined) ESA Travel Award. Ecological Society of America Physiology Section. $500 2014 Ecology Travel Award. PSU Ecology Intercollege Graduate Degree Program. $200 Biology Travel Grant. PSU Department of Biology. $250 2013 Charlotte Mangum Student Support. Society for Integrative and Comparative Biol. $300 2012 Graduate Research Fellowship. National Science Foundation. $126,000 Gaige Award. American Society of Ichthyologists and Herpetologists. $500 2011 University Graduate Fellowship. Penn State University. $41,730 Arthur J. Schmitt Presidential Fellowship. University of Notre Dame. $284,800 (declined)

TEACHING AND LEADERSHIP 2015 Teaching Assistant. Populations and Communities. BIO 220. Penn State University. 2014 Guest lecturer. BIOL 429. Animal Behavior. Penn State University. 2014 Guest lecturer. WFS 462. Amphibians & Reptiles Penn State 2010 Teaching Assistant. Field Mammalogy. University of Michigan Biological Station 2010 Teaching Assistant. Inside the Dramatic Process. University of Michigan

LEADERSHIP AND OUTREACH EXPERIENCE 2014 Ecology Graduate Student Association. President. Penn State University. 2013–2015 Science Café. Committee Chair. Penn State University Ecology Program. 2014 Science Café. Presenter. Penn State University Ecology Program. 2012–2013 Ecology Graduate Student Association. Webmaster. Penn State University. 2012–2013 Ecology Spring Seminar Series. Co-Coordinator. Penn State University

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