Stable Isotopes and the Ecology and Physiology of Reptiles

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Andrew M. Durso Utah State University

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This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. STABLE ISOTOPES AND THE ECOLOGY AND PHYSIOLOGY OF REPTILES

Andrew M. Durso

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Biology

Approved:

______Susannah S. French Alan H. Savitzky Major Professor Committee Member

______Edmund D. Brodie Lise M. Aubry Committee Member Committee Member

______Daniel A. Warner Mark McLellan Committee Member Vice President for Research and Dean of the School of Graduate Studies

UTAH STATE UNIVERSITY Logan, Utah

2016

ABSTRACT

Stable Isotopes and the Ecology and Physiology of Reptiles

Andrew M. Durso, Doctor of Philosophy

Utah State University, 2016

Major Professor: Dr. Susannah S. French Department: Biology

Animals trade-off limited resources among competing demands. Trade-offs are difficult to quantify because it is challenging to measure investment into disparate physiological systems using a common scale. Additionally, biologists desire methods to more precisely measure energy status in wild animals. I used stable isotopes to help solve both of these problems. I examined natural spatial and temporal variation in stable isotope signatures of wild lizards and found significant variation. In the lab, I was able to demonstrate the utility of nitrogen stable isotope ratios of uric acid pellets for measuring nutritional stress. By tracing labeled amino acids through the bodies of gravid female lizards, I demonstrated that vitellogenesis and wound healing compete for amino acids and quantified the direction and magnitude of the trade-offs. I showed that reproductive-immune trade-offs vary based on reproductive stage and energy availability, have effects on metabolism and immune function, and are influenced by hormonal mechanisms.

My findings shed light on the interconnectedness of stable isotope endpoints and key physiological systems in animals. I showed that isotopic signatures of physiological stress can be reflected at a large scale in natural populations, and I made novel measurements of the size and direction of trade-offs, which were formerly limited to physiological and performance outcomes.

(160 pages) iv

PUBLIC ABSTRACT

Stable Isotopes and the Ecology and Physiology of Reptiles

Andrew M. Durso

When animals don’t have enough food, they have to “choose” between “spending” their limited energy on themselves or on their offspring. Biologists think that reptiles can make this choice quickly in response to different environments. But, it can be hard to study these choices because it is hard to convert between, for example, the number of eggs laid and the speed of healing a wound. By using stable isotope chemistry, we can collect more detailed and comparable information about how lizards and other animals spend their limited resources than with any other method. For example, lizards in the wild have similar stable isotope signatures to nearby plants and insects, because their bodies are built out of these foods. In the lab, the stable isotope signatures of lizard “pee” (uric acid) change in predictable ways when they are hungry. We could essentially measure how hungry a wild lizard is by looking at the stable isotope signatures of its uric acid. We can also use a stable isotope “spike” to track where resources go within a lizard’s body. Because protein from food is used for both healing wounds and creating egg yolk, we can measure which of these is a greater priority for lizards by examining the amount of our stable isotope spike that ends up in both places. I found that lizards make different choices under different circumstances; in particular, their stage of pregnancy had a strong effect on how much protein they put into their eggs. This technique could be used on any animal, isn’t very expensive, and could help us learn a lot more about exactly how our bodies, and those of other animals, work. v

ACKNOWLEDGMENTS

During the course of this dissertation, I received funding, directly or indirectly, from the

National Science Foundation, USDA Agricultural Research Service, USU College of Science,

USU Department of Biology, USU Ecology Center, and Utah Public Radio. Wild lizards were collected under the authority of Utah State Department of Wildlife Resources COR #1COLL8382 and handled in accordance with USU IACUC protocol #2068.

I received generous technical and logistical support with mass spectrometry from John

Stark, Kendal Morris, Katrina Slabaugh, and Tom Maddox. Michael Angilletta and Dale

DeNardo provided support to Geoff Smith as he collected respirometry data at Arizona State

University. Michael Piep and Carlee Coleman helped me with plant identification, and James

Pitts helped me with insect identification. Susan Durham advised me on statistics.

I had the privilege of working with several wonderful lab assistants, especially Sydney

Greenfield and Cheyne Warren, but also Sabrina Anderson, Tyler Hansen, Heather Jones, Kati

Mattinson, Sarah Mueller, and Taylor Pettit. I also enjoyed the company and help of numerous field assistants, including Alex Berryman, Jennica Blasi, Trevor Brown, Naomi Clements, Holly

Flann, Dacia Hunter, Jenny Kordosky, Georgia Kosmala, Nick Kiriazis, John Pettit, Austin

Spence, Marilize van der Walt, Richard Walker, Eleanor Watson, and Julianne Wood.

I wish to thank my advisor, Susannah French, and my committee members, Al Savitzky,

Butch Brodie, Lise Aubry, and Dan Warner, for their mentorship and support. Their graduate students and my friends, Lori Neuman-Lee, Geoff Smith, Alison Webb, Spencer Hudson, Shab

Mohammadi, and Gareth Hopkins, were indispensable companions in the field and lab. USU

Biology faculty Marty Crump and Nancy Huntly mentored me in numerous ways.

Finally, I wish to thank my partner, Kendal Morris, and my family, especially my mother

Sandy Durso, father Paul Durso, and brother Kevin Durso, for their love and support in all things. vi

CONTENTS

Page

ABSTRACT ...... iii

PUBLIC ABSTRACT...... iv

ACKNOWLEDGMENTS ...... v

LIST OF TABLES ...... viii

LIST OF FIGURES ...... ix

CHAPTER

1. INTRODUCTION ...... 1 A Stable Isotope Primer ...... 1 Stable Isotopes and Animal Physiology...... 2 Trade-offs in Animal Physiology ...... 4 Natural Abundance Variation in Stable Isotope Ratios ...... 5 Stable Isotope Tracer Experiments ...... 7 Reptiles...... 7 Dissertation Overview...... 9 References ...... 12

2. STOICHIOMETRIC AND STABLE ISOTOPE RATIOS SUPPORT STRESS PHYSIOLOGY OF WILD LIZARDS IN AN URBAN LANDSCAPE ...... 22 Introduction ...... 22 Methods ...... 25 Results ...... 28 Tables and Figures...... 29 Discussion...... 40 References ...... 44

3. STABLE ISOTOPE RATIOS OUTPERFORM OTHER PHYSIOLOGICAL ENDPOINTS OF NUTRITIONAL STRESS ...... 55 Introduction ...... 55 Methods ...... 58 Results ...... 61 Tables and Figures...... 65 Discussion...... 71 References ...... 75

4. STABLE ISOTOPE TRACERS REVEAL A TRADE-OFF BETWEEN REPRODUCTION AND IMMUNITY IN A REPTILE WITH COMPETING NEEDS ...... 81 Introduction ...... 81 Methods ...... 83 vii

Results ...... 86 Tables and Figures...... 88 Discussion...... 92 References ...... 95

5. ASSESSING THE PROTEIN AND METABOLIC COSTS OF A TRADE-OFF BETWEEN REPRODUCTION AND IMMUNITY ...... 100 Introduction ...... 100 Methods ...... 103 Results ...... 108 Tables and Figures...... 111 Discussion...... 120 References ...... 123

6. CONCLUSION ...... 131 References ...... 133

CURRICULUM VITAE ...... 134

viii

LIST OF TABLES

Table Page

2-1 Number of lizards captured at all sites in all years ...... 29

2-2 Type III Anova Table showing variation in δ13C, δ15N, and C:N of lizard toes among sites, years, and sexes ...... 30

2-3 Type III Anova Table showing three-way interaction among site, sex, and body condition on the C:N ratio of lizard toes...... 31

3-1 Linear models testing for differences between treatment groups on a variety of response variables ...... 65

3-2 Six linear mixed-effects models describing the relationship between the amount of food eaten and two interacting fixed effects representing initial and either final or average δ15N values of uric acid...... 66

4-1 Summary statistics for validation showing low inter-individual variation in APE N of lizard eggs, feces, and uric acid...... 88

5-1 Timeline of procedures ...... 111

LIST OF FIGURES

Figure Page

2-1 Stable carbon and nitrogen isotope ratios of toes of Side-blotched Lizards (Uta stansburiana), ants, and plants at three urban and three rural sites in southwestern Utah...... 32

2-2 Annual variation in stable carbon and nitrogen isotope ratios of toes of Side-blotched Lizards (Uta stansburiana) at three urban and three rural sites in southwestern Utah over four years...... 34

2-3 Co-variation of toe C:N ratio and body condition of male and female Side-blotched Lizards (Uta stansburiana) at three urban and three rural sites in southwestern Utah over four years...... 35

2-4 Co-variation of toe δ15N and body condition of male and female Side-blotched Lizards (Uta stansburiana) at three urban and three rural sites in southwestern Utah over four years...... 36

2-5 Co-variation of toe C:N ratio and bacterial killing ability of plasma of male and female Side-blotched Lizards (Uta stansburiana) at three urban and three rural sites in southwestern Utah over three years...... 37

2-6 Co-variation of toe δ13C ratio and clutch size of female Side-blotched Lizards (Uta stansburiana) at three urban and three rural sites in southwestern Utah over three years...... 38

2-7 Higher increase in plasma corticosterone after handling in female Side-blotched Lizards (Uta stansburiana) with higher toe C:N ratio in 2013 ...... 39

3-1 Relationships between the amount of food eaten and three measurements of uric acid nitrogen stable isotope ratios ...... 67

3-2 Scatterplots showing the relationship among the amount of food eaten, the average δ15N value of the first three uric acid pellets collected, and the average δ15N value of the last three uric acid pellets collected ...... 69

3-3 Output of piecewise-linear (‘segmented’) regressions ...... 70

3-4 Relationship between body condition at the end of the experiment and δ15N value of the last uric acid pellet collected...... 71

4-1 Relationship between quantity of food consumed (g) and amount of nitrogen-15 found in scabs (APE N) ...... 89 x

4-2 Relationship between size of follicles at the beginning of the experiment (g) and ratio of nitrogen-15 found in eggs to that found in scabs ...... 90

4-3 Relationship between A) amount of nitrogen-15 found in scabs and corticosterone, and B) ratio of nitrogen-15 found in eggs to that found in scabs and corticosterone ...... 91

4-4 Relationship between amount of nitrogen-15 found in scabs and bactericidal ability ...... 92

5-1 Effect of FSH and wounding treatments on A) cumulative follicular growth; B) Effect of FSH and wounding treatments on reactive oxygen metabolites; C) Effect of FSH and wounding treatments on body mass ...... 112

5-2 Effect of FSH and wounding treatments on metabolic rate over time ...... 113

5-3 Relationships among the change in metabolic rate and (A) wound healing and (B) the quantity of 15N in eggs...... 114

5-4 Effect of FSH treatment on 15N atom percent enrichment (APE) of egg tissue ...... 115

5-5 Effect of treatment on total quantity of 15N of A) eggs, B) scabs, C) hearts, D) livers, E) uric acid, and F) feces...... 116

5-6 15N atom percent enrichment (APE) of eggs and scabs with (A) and without (B) outliers ...... 117

5-7 Co-variance of egg mass and egg 15N atom percent enrichment (APE) ...... 118

5-8 Relationship between oxygen (O2) consumption (Kleiber mass-adjusted) and ratio of total 15N in eggs to that in livers in lizards that did not receive (top) and received (bottom) FSH ...... 119

CHAPTER 1

INTRODUCTION

A Stable Isotope Primer

Differences in the stable isotope ratios of organisms have become increasingly useful and popular tools in ecology (Gannes et al. 1997, Gannes et al. 1998, Karasov and Martínez del Rio

2007). Most elements have at least two stable isotopes, which differ in the number of neutrons in their nucleus, the mass of which is denoted by a superscript number to the upper left of the elemental symbol (e.g., 14N for the stable isotope of nitrogen containing 7 protons and 7 neutrons). Typically, one isotope is much more abundant than the other(s). For example, the average relative abundances of 14N and 15N atoms in nature are 99.635% and 0.365%, respectively. Unlike radioactive isotopes (e.g., 14C), stable isotopes never decay into one another

(hence the name “stable”).

Almost all substances contain a mixture of stable isotopes of any given element. Stable isotope analysis determines the ratio of two isotopes in a sample, which can be denoted as, for example, 15N:14N. To transform these very small values into numbers that are easier to understand and compare, ecologists use either delta notation (δ15N; units are parts per thousand [often written as ‰ or spoken as “per mil”]) or atom percent enrichment (APE, the percentage above or below a reference material), both of which are calculated using comparisons to consistent standard reference materials. For nitrogen, the universal standard is atmospheric air, although the inorganic salt ammonium sulfate [(NH₄)₂SO₄] is also used. Two commonly used terms in isotopic ecology are “enriched” and “depleted”. A sample is “enriched” when its δ value or APE is more positive than that of another sample; it is “depleted” when its δ value or APE is more negative instead. For nitrogen, “enriched” means that the rarer, heavier 15N isotope is relatively more abundant and

“depleted” means that the rarer, heavier isotope is relatively less abundant. Some confusion can arise when comparing carbon isotope values, which are always negative. For more detail, see 2

Ehleringer and Osmond (1989), Ehleringer and Rundel (1989), and Karasov and Martínez del Rio

(2007).

Because the structure of two stable isotopes of the same element differ only in their number of neutrons, their relative abundance can be measured and used to inform our knowledge of various ecological and physiological phenomena. In a broad sense, two different stable isotopes of the same element behave in very similar ways: they have the same number of protons and electrons, the same structure and electronegativity, can form the same chemical bonds, and become parts of the same chemical molecules, from which they participate in the same chemical reactions and physical transformations. However, tiny differences in the masses of two different stable isotopes of the same element caused by the different number of neutrons can and do cause slight changes in the reactivity of molecules into which they are incorporated (Steele and Daniel

1978). Differential reactivity of molecules containing heavier or lighter stable isotopes due to the effect of their differences in mass is called fractionation. Molecules containing heavier stable isotopes have higher mass, stronger bond strength, and different equilibrium constants than those containing lighter stable isotopes (Peterson and Fry 1987, Schimel 1993). As a result, one can think of heavy stable isotopes as tending to bio-accumulate in a manner similar to that of heavy metals and other environmental contaminants; that is, once a molecule containing a heavy stable isotope goes somewhere, it is more likely to stay there than the same molecule containing a light stable isotope. For example, animal waste products (e.g., nitrogenous excretory products, respired

CO2) are typically more depleted than the tissues of the animals that excrete them (Olive et al.

2003).

Stable Isotopes and Animal Physiology

Stable isotopes have been used in studies of animal physiology in two fundamentally different types of experiments. The first, natural abundance experiments, make use of the natural 3 variation in stable isotope ratios to understand more about how energy and nutrients move through the ecosystem and the bodies of animals. The most widely-used application of natural abundance stable isotope analysis is to examine natural abundance variation in a consumer and several of its potential sources of food to decide what mixture of these sources constitute its diet.

Predictable variation in isotope ratios of carbon and nitrogen exists in most food webs (Post

2002), because plants at the base of the food web create carbohydrates with different carbon isotope ratios depending on whether they are made using C3 or C4 photosynthesis, and heavy isotopes of nitrogen bio-accumulate up trophic levels. Because the isotopic composition of a sample contains both source and process information, studies of animal diet assuming that the former always dominates (asserted by Griffiths 1991) have been criticized as unrealistic in many cases (Pilgrim 2005), but continue to be popular because of their relative ease. Spatial variation in stable isotope signatures is also used to understand animal migration and other spatial patterns

(Rubenstein and Hobson 2004). Less common are natural abundance studies that focus on the physiological processing of nutrients within the bodies of animals (Hatch 2012). Metabolic pathways are subject to fractionation, which creates variation in the stable isotope ratios of different animal tissues, and this variation can be measured to understand more about how animals process nutrients, waste, and toxins (Karasov and Martínez del Rio 2007). A few studies have combined insights from ecology and physiology by focusing on the stable isotope signatures of essential dietary nutrients, which are less subject to fractionation within the animal’s body

(Iverson et al. 2004, Käkelä et al. 2007).

The second type of experiment uses artificially-produced molecules containing unnaturally high proportions of rare stable isotopes (e.g., 99.9% 15N). Because the natural abundance of these rare isotopes is so low, a pulse of “labeled” nutrients or other molecules can be used as a tracer (Schimel 1993, Stark 2000). The concept is similar to using a radioactive tracer, but without the associated health and safety concerns. A wide variety of molecules are 4 available for purchase and, although they are relatively expensive, a very small amount of recovered label can be accurately measured because of the very high precision of isotope-ratio mass spectrometry. Perhaps the most widely-known application of stable isotope tracer studies are doubly-labeled water experiments for measuring metabolic rates of wild animals in the field

(Nagy 1989). Very few tracer experiments using carbon or nitrogen labels have been performed on wild vertebrates (Gilbert et al. 2014, Scott et al. 2015). In the lab, methods for labeling and tracing amino acids using stable isotopes are developing rapidly (McCue 2011), but to date most applications have been limited to in vitro protein expression assays (e.g., Ong et al. 2002, Gruhler et al. 2005). Some in vivo studies have examined the effects of starvation on metabolism (McCue et al. 2013), but only a few have traced nutrients to different, competing ultimate fates within the bodies of living organisms.

Trade-offs in Animal Physiology

For most organisms, the primary life history trade-off is that between reproduction and self-maintenance (Stearns 1992). Investment into reproduction generally takes the form of more or larger offspring. Investment into self-maintenance is more variable, because it depends on the circumstances of each individual. Individuals that have been unlucky enough to become sick or wounded have to pay larger self-maintenance costs than those that have been able to maintain good health throughout their lives. Furthermore, individuals may trade-off between investment into somatic growth and that into other forms of self-maintenance, such as immunocompetence or fat storage.

Most studies of life history trade-offs are hindered because of the complexity of the physiological systems involved and the difficulty of interpreting measurements that lack a common unit. To date, all such studies have compared measurements of indirect outcomes of resource competition (e.g., hormone levels, immune function, oxidative stress, body mass or 5 condition change; Dickens and Romero 2013, Husak et al. 2016). Because the endpoints are so varied, no study has made direct measurements of resource competition within the bodies of living animals (Zera and Harshman 2001).

In heterotrophs, reproduction and self-maintenance compete for the same limited resources: dietary macromolecules (proteins, lipids, and carbohydrates), as well as certain vitamins and minerals. Unlike carbohydrates and most lipids, many animals must obtain certain amino acids for building proteins from their diets, as they cannot be synthesized de novo by most species. As such, essential amino acids represent a limiting resource for most animals, including vertebrates. Amino acids contain hydrogen, oxygen, carbon, and nitrogen (and some also contain other elements). Of these elements, nitrogen is present in the lowest abundance and has the fewest possible fates within the bodies of heterotrophic animals (i.e., it is not breathed out or lost through evaporation). As a result, both natural abundance stable isotope ratios and labeled tracer experiments focusing on nitrogen in essential amino acids have promise as techniques to directly measure allocation of resources to both reproduction and self-maintenance using a common currency.

Natural Abundance Variation in Stable Isotope Ratios

The δ15N values of an animal’s body tissues are influenced by their diet. However, more minor variation in isotope ratios is caused by a variety of physiological processes (Vanderklift and Ponsard 2003), including pregnancy (Fuller et al. 2004) and resource limitation (Fuller et al.

2005, Mekota et al. 2006). Our understanding of the relationship between these processes and isotope ratios is rapidly evolving and has been the subject of a recent review (Hatch 2012) and meta-analysis (Hertz et al. 2015).

Although results vary among systems and studies, one of the most consistent factors influencing δ15N is the nutritional state of an animal. Differences in nitrogen balance appear to 6 cause δ15N to decrease during anabolism and increase with catabolism (Gaebler et al. 1966, Hare et al. 1991, Gaye-Siessegger et al. 2004, Gaye-Siessegger et al. 2007), because of the principle reviewed above that proteins or other molecules containing slightly heavier 15N atoms are slightly less chemically reactive (Steele and Daniel 1978). The effect of fasting on δ15N has been measured in a variety of systems and has been found to increase with the length of the fast (e.g.,

Martinez del Rio and Wolf 2005, Hatch 2012). A recent meta-analysis (Hertz et al. 2015) determined that the average magnitude of the increase in δ15N is 0.5‰ and that the most significant moderator of this effect was the type of tissue examined. Specifically, nitrogenous waste is more sensitive to isotopic changes than other bodily tissues (Barboza and Parker 2006,

Gustine et al. 2011), making it a good candidate for a sensitive biomarker of nutritional stress in captive and wild animals.

Although the late stages of fasting and starvation are obvious, early detection of nutritional stress can be difficult, especially in wild animals, many of which can only be captured once. The most reliable method, direct measurement of body composition, is too destructive for most purposes (Speakman 2008). Nitrogen isotope ratios are promising because they appear to be sensitive to nutritional stress, collecting them is non-lethal and minimally invasive, serial measurements on a fine temporal scale are possible even from small animals, and they are relatively inexpensive (~$8/sample) to process (Hatch 2012). The technique integrates information over the preceding several weeks of the animal’s lifetime and can be used in both the lab and the field. This technique has special utility for reptiles, which are often very secretive in the wild and can be very difficult to monitor because of low and variable detection probability

(Durso and Seigel 2015, Lardner et al. 2015, Rodda et al. 2015).

Stable Isotope Tracer Experiments

Nutrients are allocated among organs and tissues within an animal’s body according to physiological mechanisms determined by environmental conditions and physiological status

(McCue 2011). Stable isotope tracers are emerging as powerful tools to address questions about the nutritional status and nutrient allocation of animals. The primary strength of tracing essential amino acids through the bodies of organisms is that they can be used to directly measure allocation of resources to fundamentally different processes (e.g., reproduction and self- maintenance) using a common currency.

Several authors have examined the effects of stress on carbon and nitrogen allocation to reproduction in insects (Hood‐Nowotny and Knols 2007, Boggs and Niitepõld 2014, McCue et al.

2015a). Very few such studies have examined vertebrate physiology, and almost none have done so in the context of life-history trade-offs. Winne (2008) used a 15N label to examine income breeding in Black Swampsnakes (Seminatrix pygaea), and Van Dyke and Beaupre (2012) provided evidence for placentation in viviparous snakes using the a similar technique. Another research group used 3H labels to examine placental protein (Itonaga et al. 2012) and hormone

(Itonaga et al. 2011) transfer in a viviparous lizard. McCue et al. (2013) measured a 13C label in breath CO2 to show that quail spared protein from oxidation during the day but not at night,

McCue and Pollock (2013) conducted a similar study in mice, and McCue et al. (2015b) used the same technique to describe how and when pythons use the proteins in their meals. Recently,

Brace et al. (2015) used a 13C label to trace the competing fates of fatty acids in Brown Anoles

(Anolis sagrei).

Reptiles

Reptiles are important components of biodiversity that are often overlooked. They can reach high densities (Rodda et al. 2001) and may represent large standing stocks of nitrogen and 8 other limiting nutrients in many ecosystems (Milanovich et al. 2015). Studies of their stoichiometry are in their infancy (Sterrett et al. 2015), and stable isotope studies on the ecology and physiology of reptiles have proliferated over only the past decade (Pilgrim 2007, Willson et al. 2010, Durso and Mullin 2016). Recent work has suggested that that stable isotope levels of ectotherms may be less sensitive to short and moderate periods of fasting than is expected of other taxa because of their metabolic efficiency (McCue 2007, Milanovich and Maerz 2013).

Reptiles likely play important roles in many ecosystems, but their ecology is harder to study than those of other vertebrates, because 1) many species are cryptic, 2) most individuals are only caught once, and 3) they feed relatively infrequently. Collecting ecological data from indirect sources such as stable isotopes has great potential to improve our knowledge of reptile ecology and physiology.

In this dissertation, I focus on Side-blotched Lizards (Uta stansburiana), a widespread and common species of sceloporine lizard that is found throughout southwestern North America

(Jones and Lovich 2009). For a reptile, much is known about the ecology of U. stansburiana

(Tinkle 1967, Zani 2005), and they are emerging as potential model organisms in the fledgling field of reptile eco-physiology. Reptiles are particularly good choices as model organisms in studies of physiological trade-offs, because their low energetic demands and metabolic flexibility

(Pough 1980, Pough 1983) allow them to modify their life history quickly and precisely to match their environment. Uta stansburiana is a good choice because they are abundant, not of conservation concern, relatively easy to detect, hold stable territories that permit repeated sampling (Scoular et al. 2011), and can be kept in the lab. In addition, several easily-sampled parts of their anatomy (e.g., their toes) are an ideal size for stable isotope analysis. Although isotopic variation among color morphs has been documented in male Urosaurus ornatus

(Lattanzio and Miles 2016), populations of U. stansburiana in southern Utah do not exhibit stable 9 color morphs as they do in other parts of their range (Sinervo and Lively 1996), so we did not focus on this widely-known aspect of their ecology.

Dissertation Overview

In chapter 1, I explore relationships among stoichiometric and isotopic ratios and physiological and morphological parameters of wild U. stansburiana living across an urban landscape. I examined a large data set on the stoichiometric (C:N) and stable isotope (δ13C and

δ15N) ratios of the toes of wild U. stansburiana from populations that vary in their exposure to urbanization and anthropogenic stressors. We compare these biochemical markers to other physiological measurements of stress (immunity, clutch size, corticosterone, oxidative stress) within each population, as well as to inter-annual survival, and assess the generality of these relationships. We found support for relationships predicted by theory, such as that between C:N and body condition, but these relationships varied with context. We suggest that co-variation between biochemical ratios and hematological measurements is likely underlain by variation in nutritional stress, but emphasize that “rules” about the meaning of isotopic and stoichiometric ratios do not apply in all situations.

Because the mechanisms underlying the variation examined in chapter 1 are difficult to assess in the wild, I focus on elucidating some of these mechanisms throughout the rest of the dissertation. In chapter 2, I manipulated food availability in the lab and measured the isotopic and physiological consequences for male U. stansburiana. I examined serial variation in nitrogen isotope ratios in several tissue types, including toes, tail tips, feces, and uric acid. Three studies have reported an increase in the enrichment of the nitrogenous waste of reptiles with starvation

(Castillo and Hatch 2007, McCue 2007, McCue and Pollock 2008), but these studies are limited in their ability to make comparisons to other physiological endpoints of nutritional stress, which we simultaneously measured. I demonstrated that δ15N of lizard uric acid is a sensitive biomarker 10 of nutritional stress, although it is not related to other measures of nutritional stress. We found that a single pellet of uric acid served as a metric for the nutritional state of lizards that had been food restricted, but that taking the average value of several pellets over time can describe even more subtle differences in past nutritional history.

In chapter 3, I used 15N labeled amino acids to directly compare the protein allocation of female U. stansburiana that varied in resource availability and reproductive stage with indirect physiological outcomes. We measured the amount of 15N deposited into eggs and compared it with that allocated to the scab that formed over a healing wound. We found that resource limitation and reproductive stage had important effects on the absolute and relative allocation of protein to self-maintenance, providing evidence for a trade-off between the reproductive and immune systems. Female lizards later in pregnancy allocated more protein to healing their wounds, whereas female lizards earlier in pregnancy had a higher egg:scab 15N ratio (i.e., they allocated more protein to growing their eggs relative to healing their wound). The effect of reproductive stage outweighed the effect of resource limitation in our study, but both were important, emphasizing the necessity of understanding individual variation in physiology when attempting to study life history trade-offs (Winne 2008).

Finally, in chapter 4 I manipulated an endocrine mechanism controlling allocation of resources to reproduction: follicle-stimulating hormone (FSH). Again, I used a stable isotope label to trace amino acids through the bodies of female U. stansburiana that were undergoing both reproductive and immune costs, and measured both the direct allocation of protein to tissues involved in these activities as well as the combined oxygen consumption costs of these demands.

I found that there was a significant increase in the 15N content of eggs when FSH was applied, and that the application of FSH reversed the direction of the relationship between egg and scab

15N content. I also found a significant interaction between the effect of FSH treatment and isotope 11 ratio on the metabolic rate of lizards, bolstering evidence for the role of FSH in mediating the costs and benefits of a trade-off between wound healing and vitellogenesis.

In conclusion, my findings shed light on the interconnectedness of stable isotope endpoints and key physiological systems. This basic research can lead to advances in physiology, ecology, conservation, and animal husbandry, and highlights the metabolic flexibility of reptiles and how much we have left to learn about their ecology and physiology. 12

References

Barboza, P.S. & Parker, K.L. (2006) Body protein stores and isotopic indicators of N balance in

female reindeer (Rangifer tarandus) during winter. Physiological and Biochemical

Zoology, 79, 628-644.

Boggs, C. & Niitepõld, K. (2014) Insights from stable isotopic tracers on reproductive allocation

under stress. Integrative and Comparative Biology, 54, 880-889.

Brace, A.J., Sheikali, S. & Martin, L.B. (2015) Highway to the danger zone: exposure-dependent

costs of immunity in a vertebrate ectotherm. Functional Ecology, 29, 924-930.

Castillo, L. & Hatch, K. (2007) Fasting increases d15N-values in the uric acid of Anolis

carolinensis and Uta stansburiana as measured by nondestructive sampling. Rapid

Communications in Mass Spectrometry, 21, 4125-4128.

Dickens, M.J. & Romero, L.M. (2013) A consensus endocrine profile for chronically stressed

wild animals does not exist. General and Comparative Endocrinology, 191, 177-189.

Durso, A.M. & Mullin, S.J. (2016) Ontogenetic shifts in the diets of Plains Hog-nosed Snakes

(Colubridae: Heterodon) revealed by stable isotope analysis. Zoology,

10.1016/j.zool.2016.07.004.

Durso, A.M. & Seigel, R.A. (2015) A snake in the hand is worth 10,000 in the bush. Journal of

Herpetology, 49, 503-506.

Ehleringer, J.R. & Osmond, C.B. (1989) Stable isotopes. Plant Physiological Ecology: Field

Methods & Instrumentation (eds R.W. Pearcy, J. Ehleringer, H.A. Mooney & P.W.

Rundel), pp. 281-300. Chapman & Hall Ltd., New York. 13

Ehleringer, J.R. & Rundel, P.W. (1989) Stable isotopes: history, units, and instrumentation.

Stable Isotopes in Ecological Research (eds P.W. Rundel, J.R. Ehleringer & K.A. Nagy),

pp. 1-16. Springer-Verlag, New York.

Fuller, B.T., Fuller, J.L., Sage, N.E., Harris, D.A., O'Connell, T.C. & Hedges, R.E. (2004)

Nitrogen balance and δ15N: why you're not what you eat during pregnancy. Rapid

Communications in Mass Spectrometry, 18, 2889-2896.

Fuller, B.T., Fuller, J.L., Sage, N.E., Harris, D.A., O'Connell, T.C. & Hedges, R.E. (2005)

Nitrogen balance and δ15N: why you're not what you eat during nutritional stress. Rapid

Communications in Mass Spectrometry, 19, 2497-2506.

Gaebler, O., Vitti, T.G. & Vukmirovich, R. (1966) Isotope effects in metabolism of 14N and 15N

from unlabeled dietary proteins. Canadian Journal of Biochemistry, 44, 1249-1257.

Gannes, L.Z., Martinez del Rio, C. & Koch, P. (1998) Natural abundance variations in stable

isotopes and their potential uses in animal physiological ecology. Comparative

Biochemistry and Physiology A Comparative Physiology, 119, 725-737.

Gannes, L.Z., O'Brien, D.M. & Martinez del Rio, C. (1997) Stable isotopes in animal ecology:

assumptions, caveats, and a call for more laboratory experiments. Ecology, 78, 1271-

1276.

Gaye-Siessegger, J., Focken, U., Abel, H. & Becker, K. (2007) Starvation and low feeding levels

result in an enrichment of 13C in lipids and 15N in protein of Nile tilapia Oreochromis

niloticus L. Journal of Fish Biology, 71, 90-100. 14

Gaye-Siessegger, J., Focken, U., Muetzel, S., Abel, H. & Becker, K. (2004) Feeding level and

individual metabolic rate affect d13C and d15N values in carp: implications for food web

studies. Oecologia, 138, 175-183.

Gilbert, K.J., Fahey, T.J., Maerz, J.C., Sherman, R.E., Bohlen, P., Dombroskie, J.J., . . . Yavitt,

J.B. (2014) Exploring carbon flow through the root channel in a temperate forest soil

food web. Soil Biology and Biochemistry, 76, 45-52.

Griffiths, H. (1991) Applications of stable isotope technology in physiological ecology.

Functional Ecology, 5, 254-269.

Gruhler, A., Schulze, W.X., Matthiesen, R., Mann, M. & Jensen, O.N. (2005) Stable isotope

labeling of Arabidopsis thaliana cells and quantitative proteomics by mass spectrometry.

Molecular & Cellular Proteomics, 4, 1697-1709.

Gustine, D.D., Barboza, P.S., Adams, L.G., Farnell, R.G. & Parker, K.L. (2011) An isotopic

approach to measuring nitrogen balance in caribou. The Journal of Wildlife Management,

75, 178-188.

Hare, P., Fogel, M., Stafford, T., Mitchell, A. & Hoering, T. (1991) The isotopic composition of

carbon and nitrogen in individual amino acids isolated from modern and fossil proteins.

Journal of Archaeological Science, 18, 277-292.

Hatch, K.A. (2012) The use and application of stable isotope analysis to the study of starvation,

fasting, and nutritional stress in animals. Comparative Physiology of Fasting, Starvation,

and Food Limitation (ed. M.D. McCue), pp. 337-364. Springer-Verlag, Berlin. 15

Hertz, E., Trudel, M., Cox, M.K. & Mazumder, A. (2015) Effects of fasting and nutritional

restriction on the isotopic ratios of nitrogen and carbon: a meta‐analysis. Ecology and

Evolution, 5, 4829-4839.

Hood‐Nowotny, R. & Knols, B.G. (2007) Stable isotope methods in biological and ecological

studies of arthropods. Entomologia Experimentalis et Applicata, 124, 3-16.

Husak, J.F., Ferguson, H.A. & Lovern, M.B. (2016) Trade-offs among locomotor performance,

reproduction, and immunity in lizards. Functional Ecology, 10.1111/1365-2435.12653.

Itonaga, K., Wapstra, E. & Jones, S.M. (2011) Evidence for placental transfer of maternal

corticosterone in a viviparous lizard. Comparative Biochemistry and Physiology-Part A:

Molecular & Integrative Physiology, 160, 184-189.

Itonaga, K., Wapstra, E. & Jones, S.M. (2012) A novel pattern of placental leucine transfer during

mid to late gestation in a highly placentotrophic viviparous lizard. Journal of

Experimental Zoology Part B: Molecular and Developmental Evolution, 318, 308-315.

Iverson, S.J., Field, C., Don Bowen, W. & Blanchard, W. (2004) Quantitative fatty acid signature

analysis: a new method of estimating predator diets. Ecological Monographs, 74, 211-

235.

Jones, L. & Lovich, R. (2009) Lizards of the American Southwest. Rio Nuevo Publishers, Tucson,

Arizona.

Käkelä, A., Furness, R.W., Kelly, A., Strandberg, U., Waldron, S. & Käkelä, R. (2007) Fatty acid

signatures and stable isotopes as dietary indicators in North Sea seabirds. Marine Ecology

Progress Series, 342, 291-301. 16

Karasov, W.H. & Martínez del Rio, C. (2007) Physiological Ecology: How Animals Process

Energy, Nutrients, and Toxins. Princeton University Press, Princeton, New Jersey, USA.

Lardner, B., Rodda, G.H., Yackel Adams, A.A., Savidge, J.A. & Reed, R.N. (2015) Detection

rates of geckos in visual surveys: turning confounding variables into useful knowledge.

Journal of Herpetology, 49, 522-532.

Lattanzio, M.S. & Miles, D.B. (2016) Trophic niche divergence among colour morphs that

exhibit alternative mating tactics. Royal Society Open Science, 3, 150531.

Martinez del Rio, C. & Wolf, B. (2005) Mass-balance models for animal isotopic ecology.

Physiological and Ecological Adaptations to Feeding in Vertebrates (eds J.M. Starck &

T. Wang), pp. 141-174. Science Publishers, Inc, Enfield, New Hampshire.

McCue, M., Guzman, R., Passement, C. & Davidowitz, G. (2015a) How and when do insects rely

on endogenous protein and lipid resources during lethal bouts of starvation? A new

application for 13C-breath testing. PLoS ONE, 10, e0140053.

McCue, M.D. (2007) Western diamondback rattlesnakes demonstrate physiological and

biochemical strategies for tolerating prolonged starvation. Physiological and Biochemical

Zoology, 80, 25-34.

McCue, M.D. (2011) Tracking the oxidative and nonoxidative fates of isotopically labeled

nutrients in animals. Bioscience, 61, 217–230.

McCue, M.D., Amaya, J.A., Yang, A.S., Erhardt, E.B., Wolf, B.O. & Hanson, D.T. (2013)

Targeted 13C enrichment of lipid and protein pools in the body reveals circadian changes

in oxidative fuel mixture during prolonged fasting: a case study using Japanese quail. 17

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology,

166, 546-554.

McCue, M.D., Guzman, R.M. & Passement, C.A. (2015b) Digesting pythons quickly oxidize the

proteins in their meals and save the lipids for later. The Journal of Experimental Biology,

215, 2089-2096.

McCue, M.D. & Pollock, E.D. (2008) Stable isotopes may provide evidence for starvation in

reptiles. Rapid Communications in Mass Spectrometry, 22, 2307-2314.

13 McCue, M.D. & Pollock, E.D. (2013) Measurements of substrate oxidation using CO2-breath

testing reveals shifts in fuel mix during starvation. Journal of Comparative Physiology B,

183, 1039-1052.

Mekota, A.M., Grupe, G., Ufer, S. & Cuntz, U. (2006) Serial analysis of stable nitrogen and

carbon isotopes in hair: monitoring starvation and recovery phases of patients suffering

from anorexia nervosa. Rapid Communications in Mass Spectrometry, 20, 1604-1610.

Milanovich, J.R. & Maerz, J.C. (2013) Realistic fasting does not affect stable isotope levels of a

metabolically efficient salamander. Journal of Herpetology, 47, 544-548.

Milanovich, J.R., Maerz, J.C. & Rosemond, A.D. (2015) Stoichiometry and estimates of nutrient

standing stocks of larval salamanders in Appalachian headwater streams. Freshwater

Biology, 10.1111/fwb.12572.

Nagy, K.A. (1989) Doubly-labeled water studies of vertebrate physiological ecology. Stable

Isotopes in Ecological Research (eds P.W. Rundel, J.R. Ehleringer & K.A. Nagy), pp.

270-287. Springer-Verlag, New York, NY, USA. 18

Olive, P.J., Pinnegar, J.K., Polunin, N.V.C., Richards, G. & Welch, R. (2003) Isotope trophic-

step fractionation: A dynamic equilibrium model. Journal of Animal Ecology, 72, 608-

617.

Ong, S.-E., Blagoev, B., Kratchmarova, I., Kristensen, D.B., Steen, H., Pandey, A. & Mann, M.

(2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and

accurate approach to expression proteomics. Molecular & Cellular Proteomics, 1, 376-

386.

Peterson, B.J. & Fry, B. (1987) Stable isotopes in ecosystem studies. Annual Review of Ecology

and Systematics, 18, 293-320.

Pilgrim, M. (2005) Linking Microgeographic Variation in Pigmy Rattlesnake (Sistrurus

miliarius) Life History and Demography with Diet Composition: A Stable Isotope

Approach. Ph. D. Ph. D. Dissertation, University of Arkansas.

Pilgrim, M. (2007) Expression of maternal isotopes in offspring: Implications for interpreting

ontogenetic shifts in isotopic composition of consumer tissues. Isotopes in Environmental

& Health Studies, 43, 155-163.

Post, D.M. (2002) Using stable isotopes to estimate trophic position: models, methods, and

assumptions. Ecology, 83, 703-718.

Pough, F.H. (1980) The advantages of ectothermy for tetrapods. The American Naturalist, 115,

92-112.

Pough, F.H. (1983) Amphibians and reptiles as low-energy systems. Behavioral Energetics: The

Cost of Survival in Vertebrates (eds W.P. Aspey & S.I. Lustick), pp. 141-188. Ohio State

University Press, Columbus, OH, U.S.A. 19

Rodda, G.H., Dean-Bradley, K., Campbell, E.W., Fritts, T.H., Lardner, B., Yackel Adams, A.A.

& Reed, R.N. (2015) Stability of detectability over 17 years at a single site and other

lizard detection comparisons from Guam. Journal of Herpetology, 49, 513-521.

Rodda, G.H., Perry, G.A.D., Rondeau, R.J. & Lazell, J. (2001) The densest terrestrial vertebrate.

Journal of Tropical Ecology, 17, 331-338.

Rubenstein, D.R. & Hobson, K.A. (2004) From birds to butterflies: animal movement patterns

and stable isotopes. Trends in Ecology & Evolution, 19, 256-263.

Schimel, D.S. (1993) Theory and Application of Tracers. Academic Press, Inc., London, UK.

Scott, D.E., Yanes, Y., Rothermel, B.B., Pilgrim, M. & Romanek, C.S. (2015) Efficacy of

Labeling Wetlands with Enriched 15N to Determine Amphibian Dispersal. Wetlands, 35,

349-356.

Scoular, K.M., Caffry, W.C., Tillman, J.L., Finan, E.S., Schwartz, S.K., Sinervo, B. & Zani, P.A.

(2011) Multiyear Home-Range Ecology of Common Side-blotched Lizards in Eastern

Oregon with Additional Analysis of Geographic Variation in Home-range Size.

Herpetological Monographs, 25, 52-75.

Sinervo, B. & Lively, C.M. (1996) The rock-paper-scissors game and the evolution of alternative

male strategies. Nature, 380, 240-243.

Speakman, J.R. (2008) Body Composition Analysis of Animals: A Handbook of Non-destructive

Methods. pp. 260. Cambridge University Press, Cambridge, UK.

Stark, J.M. (2000) Nutrient transformations. Methods in Ecosystem Science (eds O.E. Sala, R.B.

Jackson, H.A. Mooney & R.W. Howarth), pp. 214-234. Springer, New York, NY, USA.

Stearns, S. (1992) The Evolution of Life Histories. Oxford University Press, Oxford, UK. 20

Steele, K. & Daniel, R. (1978) Fractionation of nitrogen isotopes by animals: a further

complication to the use of variations in the natural abundance of 15N for tracer studies.

Journal of Agricultural Science, 90, 7-9.

Sterrett, S.C., Maerz, J.C. & Katz, R.A. (2015) What can turtles teach us about the theory of

ecological stoichiometry? Freshwater Biology, 60, 443-455.

Tinkle, D.W. (1967) The life and demography of the Side-blotched Lizard, Uta stansburiana.

Miscellaneous Publications of the Museum of Zoology University of Michigan, 132, 1-

182.

Van Dyke, J.U. & Beaupre, S.J. (2012) Stable isotope tracer reveals that viviparous snakes

transport amino acids to offspring during gestation. The Journal of Experimental Biology,

215, 760-765.

Vanderklift, M.A. & Ponsard, S. (2003) Sources of variation in consumer-diet δ15N enrichment: a

meta-analysis. Oecologia, 136, 169-182.

Willson, J.D., Winne, C.T., Pilgrim, M.A., Romanek, C.S. & Gibbons, J.W. (2010) Seasonal

variation in terrestrial resource subsidies influences trophic niche width and overlap in

two aquatic snake species: a stable isotope approach. Oikos, 119, 1161-1171.

Winne, C.T. (2008) Seminatrix pygaea, a Model of Ecological Resilience in Dynamic Habitats.

Ph. D. dissertation Ph. D. dissertation, University of Georgia.

Zani, P.A. (2005) Life-history strategies near the limits of persistence: winter survivorship and

spring reproduction in the Common Side-blotched Lizard (Uta stansburiana) in eastern

Oregon. Journal of Herpetology, 39, 166-169. 21

Zera, A.J. & Harshman, L.G. (2001) The physiology of life history trade-offs in animals. Annual

Review of Ecology and Systematics, 32, 95-126.

CHAPTER 2

STOICHIOMETRIC AND STABLE ISOTOPE RATIOS SUPPORT STRESS PHYSIOLOGY OF WILD LIZARDS IN AN URBAN LANDSCAPE

Urbanization modifies wildlife habitat and often has major impacts on animal ecology and physiology. Changes to feeding ecology and nutritional status resulting from urbanization have been documented, but studying the diet and nutrition of wild reptiles is challenging. Stable isotopes integrate dietary information over long periods of time and have the potential to provide ecologists with information about wildlife health and nutrition as well. We measured the stable isotope ratios of toe clips from 426 wild Uta stansburiana at three urban and three rural sites and compared them with stable isotope ratios of plants and ants from those same sites, as well as with data on other physiological indicators of nutritional status on a within-site basis. We found that, although substantial spatial variation in plant, ant, and lizard stable isotope signatures was present, the relative values of stable isotope ratios were more similar among rural sites than among urban sites. We also found support for relationships between δ13C and clutch size in urban areas, and between C:N and body condition, corticosterone reactivity, and immune function at some sites. We suggest that stoichiometric and isotopic ratios can provide important ecological information about the diet and nutritional status of wild reptiles.

Introduction

Nutritional stress is a common challenge faced by wild animals. In some wildlife populations, nutritional stress can compromise individual health and thereby decrease survivorship, with potential conservation implications (McNamara and Houston 1990, Wikelski and Romero 2003, Romero and Wikelski 2010). Starvation is a major source of mortality for 23 some wildlife (Stout and Cornwell 1976, Young 1994, Krebs et al. 2004), particularly when populations are subject to unpredictable temporal variation in resource availability (Romero and

Wikelski 2001, Monteith et al. 2014) or only have access to resources of poor quality (Knapp et al. 2013, Murray et al. 2015).

Landscape change through urbanization is a major factor influencing the distribution, conservation status, foraging behavior, and nutritional status of wildlife (Ditchkoff et al. 2006).

Our understanding of some of the mechanisms driving the negative effects of urbanization on wildlife is becoming more complete (e.g., Bradley and Altizer 2007, McKinney 2008, Hutton and

McGraw 2016). Nutritional stress due to habitat loss or alteration can contribute to wildlife declines (Naug 2009, Vangestel et al. 2010). However, most studies of how nutrition affects survival take place away from urban centers, and few studies of urban wildlife address nutrition.

Measuring nutritional stress in wild animals can be challenging. Biologists desire rapid, non-invasive measurement techniques for assessing nutritional stress in wild animals (Romero and Reed 2005, 2008, Speakman 2008). Although several such techniques are available

(reviewed in McCue 2010), including measuring stable isotope and stoichiometric composition, lipid profiles, hormones, and circulating metabolites, consensus about their meaning has not been reached (Sarre et al. 1994, Dickens and Romero 2013). Few studies have measured more than one of these endpoints simultaneously, although proximate hematological measurements underlying energy allocation among physiological demands might be expected to co-vary with isotope and stoichiometric ratios, both of which are ultimately underpinned by variation in nutritional stress.

Recently, studies of amphibians and reptiles in urban areas have proliferated (Mitchell et al. 2008). These ectothermic vertebrates have relatively limited capacity for dispersal but often persist in urban areas due to their remarkable plasticity in response to environmental change (e.g.,

Glanville and Seebacher 2006, Refsnider and Janzen 2012, Ackley et al. 2015). Although the role 24 of amphibians as sentinels in environmental change is becoming more widely recognized

(Hopkins 2007), studies of the urban ecology of reptiles still lag behind those of other vertebrates

(French et al. 2008, Lucas and French 2012). Variable resource availability can have major effects on reptile populations (Romero and Wikelski 2001), although their poikilothermic metabolism can allow individuals to persist through relatively long periods of resource scarcity

(Willson et al. 2006).

Because stable isotopes integrate ecological information over relatively long periods of time compared with other metrics, they can offer unique insights into the ecology of amphibians and reptiles (Willson et al. 2010), which can be very cryptic, often have low detection probabilities (Durso and Seigel 2015), and whose metabolic flexibility may limit the utility of plasma metabolites for predicting recent feeding history (Price 2016). Changes to elemental and isotopic ratios appear to result from mobilization, reorganization, and catabolism of stored lipid and protein reserves during fasting. In other taxa, empirical data support relationships between carbon-to-nitrogen ratio (C:N) and body condition (Graves et al. 2012), and between stable carbon (13C:12C or δ13C) and nitrogen (15N:14N or δ15N) isotope ratios and body condition or nutritional stress (Mekota et al. 2006, Hatch 2012). Controlled laboratory studies of reptiles report that nutritional stress causes isotopic enrichment (McCue and Pollock 2008, Martinez del Rio et al. 2009), but the utility of C:N and stable isotope ratios for monitoring the nutritional status of wild reptiles is still largely untested, and the relationships between these metrics and other physiological endpoints are unknown.

Here, we tested for relationships among stoichiometric and isotopic ratios and physiological and morphological parameters of wild lizards living across an urban landscape.

Although several studies have found relationships between stable isotope ratios of reptiles and aspects of their ecology (Barrett et al. 2005, Reddin et al. 2016), none have yet to focus on 25 changing landscape parameters or physiology of free-living vertebrates. We examined a large data set on the stoichiometric (C:N) and stable isotope (δ13C and δ15N) ratios of wild Side- blotched Lizards (Uta stansburiana) from populations that vary in their exposure to urbanization and anthropogenic stressors. We compared these biochemical markers to other commonly used measures of physiological stress (immunocompetence, clutch size, glucocorticoid concentrations, oxidative stress) within each population, and assessed the generality of these relationships. We predicted that higher C:N, δ13C, and δ15N ratios would be found in lizards with better body condition and immune function, and with lower corticosterone and oxidative stress.

Methods

We collected wild Side-blotched Lizards (Uta stansburiana) from six locations in and around St. George, Utah, USA, every May for four years (N = 426; see Table 2-1 for more details). We collected one to four toes from each lizard, dried them to a constant mass (0.5-2.0 mg) using a drying oven set to 60°C, and wrapped them in tin capsules (5x9mm; Costech

Analytical). The samples were analyzed using continuous-flow direct-combustion mass spectrometry on an isotope-ratio mass spectrometer (Europa Scientific ANCA-2020; PDZ,

Crewe, England) at the Utah State University Stable Isotope Lab. We measured δ13C and δ15N as well as the total mass of C and N, from which we calculated the C:N ratio. Standard reference materials were used for calibration. Standard deviations of replicate standards did not exceed 0.1 per mil. We removed seven outlying data points that might have been influenced by measurement error (δ15N > 20‰).

In addition to recording the sex and color morph of each lizard, we also measured their mass, snout-vent length, and the clutch size of the females using ultrasound. Upon capture, we 26 collected blood samples from the retro-orbital sinus within three minutes, from which we measured bacterial killing ability (BKA; French and Neuman-Lee 2012), corticosterone (CORT;

Neuman-Lee et al. 2015), and two components of oxidative stress: reactive oxygen metabolites

(dROM) and plasma oxidative barrier (OXY; Diacron International, Grosseto, Italy). In 2013, we collected a second blood sample 10 minutes after the first to measure the difference between baseline and post-stress corticosterone (CORT reactivity; Moore et al. 1991, French et al. 2008,

Lucas and French 2012). We individually marked and released lizards, and recorded recaptures.

We calculated a body condition index using the residuals of a regression of snout-vent length and mass.

Our choice of toes was motivated by the fact that we were already clipping them off for capture-mark-recapture. Additionally, toe-clipping is non-lethal and minimally-invasive (Perry et al. 2011), toes have been used in other stable isotope studies of small lizards (Lattanzio and Miles

2016a, Lattanzio and Miles 2016b), and the toes of U. stansburiana and other small (3-6 g total body mass) lizards weigh approximately 0.75 mg, a convenient size for measuring stable isotope ratios. However, the tissues that make up the bulk of a lizard toe (bone, scale and claw keratin) are relatively inert, so their stoichiometric and isotopic ratios may not reflect small variations in lipid or nitrogen loss over short time scales. Other tissues, such as splanchnic organs or excreta, may be better choices for detecting chemical signals of nutritional stress (Karasov and Pinshow

1998, Doucett et al. 1999, Martinez del Rio et al. 2009). Evidence in favor of using δ15N of uric acid of reptiles for this purpose is mounting (Castillo and Hatch 2007, McCue and Pollock 2008,

Hatch 2012, Durso et al. In review). Collecting uric acid pellets from wild animals is only marginally more work than collecting toes and is less invasive, although it would likely require holding individual animals in separate containers for several hours, which has other costs. 27

Because geographic variation in the isotopic signatures of soils, plants, and invertebrates propagates to higher trophic levels (Pilgrim 2005), we did not make comparisons of absolute values among sites, but rather analyzed within-site variation in isotopic signature as it related to other physiological endpoints of condition and stress. No site was larger than 1.8 ha in size, and we assumed that spatial variation in isotopic signature within a site was negligible. Sites were separated by at least 4.0 km (average distance among urban sites=5.4 km, average distance among rural sites=12.3 km, average distance among all sites=20.7 km). To test whether spatial variation in source was important among sites, we collected isotopic data on plants (N=41) and ants (N=9) collected at each site. We attempted to collect the same species of plants and insects across sites, but because the plant and insect communities varied so much from site to site, we were forced to use ecological equivalents in some cases. At each site we attempted to collect representatives of the dominant plant species present. We chose to collect harvester ants (genus

Pogonomyrmex) because they forage widely, thus accumulating their own food from many species of plants, and because they make up a large proportion of the diet of U. stansburiana at other sites (Tinkle 1967, Best and Gennaro 1984).

We modeled three response variables (C:N, δ13C, δ15N) individually against 10 continuous explanatory variables (SVL, mass, body condition, reproductive investment [females only], BKA, CORT, dROM, OXY, and the two other isotopic/stoichiometric ratios), with fixed block effects for sex (except in the reproductive investment model), year and site. We also tested whether the same three response variables differed among color morphs or between lizards that were later recaptured (known survivors) and those that were not using the ‘Anova’ function in the

‘car’ package (Fox and Weisberg 2011). We used the package ‘nlme’ in R (R Core Team 2015, version 3.2.2, Pinheiro et al. 2016) for modeling and the package ‘ggplot2’ to make figures

(Wickham 2009). 28

Results

We found that lizards from different sites differed from one another in both δ13C and

δ15N, but not C:N (Table 2-2; Fig. 2-1A,B). The variation in δ13C was much higher at urban sites than at rural sites (Fig. 2-1A,B). Annual variation was present but lesser in magnitude than spatial variation (Fig. 2-2). Male lizards in better body condition had higher C:N ratios at four of six sites

(Fig. 2-3). Female lizards in better body condition had higher C:N ratios at two of six sites, and lower C:N ratios at three of six sites (Fig. 2-3). There was no relationship between δ15N and body condition (Fig. 2-4). We found a significant three-way interaction among site, sex, and body condition on the C:N ratio (F5,269 = 5.59, p < 0.0001; Table 2-3).

At two urban sites, C:N ratio was related to bacterial killing ability (interaction F5,214 =

3.20, p = 0.008; Fig. 2-5). At urban sites, δ13C was related to the total clutch mass of females

(interaction F5,141 = 2.74, p = 0.02; Fig. 2-6). In 2013, C:N ratio was positively related to corticosterone reactivity of female lizards, but not males, at both urban and rural sites (F1,16 =

12.51, p = 0.002; Fig. 2-7). We did not find significant relationships between isotopic or stoichiometric ratios and other physiological parameters, or significant differences between lizards that were later recaptured and those that were not. Although isotopic variation among color morphs has been documented in male Urosaurus ornatus (Lattanzio and Miles 2016b), we did not find evidence for such differences in our populations of U. stansburiana, which do not exhibit stable color morphs in southern Utah as they do in other parts of their range (Sinervo and

Lively 1996).

Stable isotope signatures of plants and ants were closely and consistently paired with those of lizard toes at all three rural sites (Fig. 2-1B). Differences between plants and lizard toes were greater at rural sites (δ13C = 4.82-5.54; δ15N = 3.88-6.76) than at urban sites (δ13C = 2.93-

3.51; δ15N = 1.68-3.58). Stable isotope signatures of ants at rural sites were intermediate between 29 those of plants and lizard toes (difference between ant and lizard toe δ13C = 2.60-3.30; δ15N =

0.24-1.06). In contrast, at urban sites, ants were more enriched than lizard tows in both elements

(difference between ant and lizard toe δ13C = -4.90-0.11; δ15N = -4.08 to -1.20).

Tables and Figures

Table 2-1. Number of lizards captured at all sites in all years. All sites are rocky areas ≤1.8 ha in size near or along riparian corridors. No site is within 4 km of any other.

Urban Sites Rural Sites Big Year Bluff Dinosaur Manowar Browse Leeds Hollow Total 2013 20 30 8 18 20 19 115 2014 21 19 16 19 18 29 122 2015 12 15 19 13 22 13 94 2016 13 - 29 20 11 22 95 Total 66 64 72 70 71 83 426

Table 2-2. Type III Anova Table showing variation in δ13C, δ15N, and C:N of lizard toes among sites, years, and sexes.

δ13C δ15N C:N

Factor Fdf P Factor Fdf p Factor Fdf p

Site 18.35,257 1.5e-15 Site 9.725,257 1.6e-8 Site 0.805,257 0.55

Year 0.072,257 0.94 Year 0.272,257 0.76 Year 0.932,257 0.40

Sex 0.011,257 0.93 Sex 2.721,257 0.10 Sex 0.081,257 0.77

Site*Year 1.4410,257 0.16 Site*Year 0.7710,257 0.66 Site*Year 1.0910,257 0.37

Site*Sex 0.675,257 0.64 Site*Sex 0.905,257 0.48 Site*Sex 0.515,257 0.77

Year*Sex 0.012,257 0.99 Year*Sex 0.592,257 0.55 Year*Sex 0.292,257 0.75

Site*Year*Sex 0.4210,257 0.94 Site*Year*Sex 0.4410,257 0.93 Site*Year*Sex 1.1510,257 0.32

Table 2-3. Type III Anova Table showing three-way interaction among site, sex, and body condition on the C:N ratio of lizard toes.

Factor Fdf p

Site 2.265,267 0.048

Body condition 0.011,267 0.914

Sex 0.691,267 0.406

Year 8.232,267 0.0003

Site*Body condition 5.845,267 3.86e-5

Site*Sex 3.695,267 0.003

Body condition*Sex 0.331,267 0.565

Site*Sex*Body condition 3.935,267 0.002

Figure 2-1. A) Stable carbon and nitrogen isotope ratios of toes of Side-blotched Lizards (Uta stansburiana) at three urban and

32 three rural sites in southwestern Utah. Years and sites are pooled.

Figure 2-1. B) Stable carbon and nitrogen isotope ratios of toes of Side-blotched Lizards (Uta stansburiana), ants, and plants at

33 three urban and three rural sites in southwestern Utah. Years are pooled.

Figure 2-2. Annual variation in stable carbon and nitrogen isotope ratios of toes of Side-blotched Lizards (Uta stansburiana) at

34 three urban and three rural sites in southwestern Utah over four years.

Figure 2-3. Co-variation of toe C:N ratio and body condition of male and female Side-blotched Lizards (Uta stansburiana) at

three urban and three rural sites in southwestern Utah over four years.

Figure 2-4. Co-variation of toe δ15N and body condition of male and female Side-blotched Lizards (Uta stansburiana) at

three urban and three rural sites in southwestern Utah over four years.

Figure 2-5. Co-variation of toe C:N ratio and bacterial killing ability of plasma of male and female Side-blotched Lizards

(Uta stansburiana) at three urban and three rural sites in southwestern Utah over three years.

Figure 2-6. Co-variation of toe δ13C ratio and clutch size of female Side-blotched Lizards (Uta stansburiana) at three urban and

38 three rural sites in southwestern Utah over three years.

Figure 2-7. Higher increase in plasma corticosterone after handling in female Side-blotched Lizards (Uta stansburiana) with

39 higher toe C:N ratio in 2013. 40

Discussion

Across sites, we documented substantial variation in plant, ant, and lizard stable isotope signatures. However, the relative values of stable isotope ratios were more similar among rural sites than among urban sites. By examining lizard toe stable isotope ratios within sites, we found support for relationships predicted by theory, such as that between C:N and body condition, but these relationships varied greatly with context. Specifically, we found support for relationships among C:N ratio, body condition, CORT reactivity, and bacterial killing ability, but these relationships varied temporally and across sites. We also found that δ13C was related to clutch size in urban animals.

We present the first evidence that proximate physiological mechanisms underlying energy allocation (CORT) and non-reproductive physiological demands (immunity) sometimes also co-vary with stoichiometric ratios, possibly as a result of variation in nutritional stress. In cases of resource limitation, trade-offs among competing demands often emerge. We found that higher CORT reactivity and higher immune function were associated with higher C:N ratios in some cases, suggesting that animals in superior body condition that likely had access to ample lipid reserves may also be more capable of mobilizing stored energy and have more energy to allocate to immunity (French et al. 2007, Price 2016). This co-variation is likely underlain by variation in nutritional stress, and in animals whose nutritional status is above a certain threshold one might expect to see a predictable positive relationship between C:N ratio and immune function.

Sites differed from one another in both δ13C and δ15N (Fig. 2-1B); however, it is difficult to ascribe mechanisms to the variation in isotopic signatures across sites. The isotopic structure of food webs is highly variable in space and time (Gannes et al. 1997, Gannes et al. 1998, Pilgrim

2005), and the absolute differences we observed among lizards at different sites are likely driven 41 largely by geographic variation in climate, soil, plants, or invertebrates (Smiley et al. 2016; Fig.

2-1B). However, some authors have made the suggestion that inter-individual isotopic variation increases as preferred resources become scarce (Reddin et al. 2016). We found that variation in

δ13C at urban sites exceeded that at rural sites, which is consistent with the idea that resources at disturbed urban sites may be less optimal than those in relatively undisturbed rural areas. In contrast, other studies have suggested that there may be costs to specialization on high-quality forage (Darimont et al. 2007), such that individuals occupying more peripheral niches have higher fitness.

Lattanzio and Miles (2016a) estimated isotopic discrimination of Urosaurus ornatus claw tissue in the laboratory as δ13C = 1.2 ± 0.1 ‰ and δ15N = 0.7 ± 0.1 ‰. Differences in our wild populations were larger for δ13C but similar for δ15N at rural sites. Assuming that these discrimination values are approximately correct, our data suggest that ants make up a large proportion of the diet of our wild Uta in rural areas, which is consistent with results from more directed studies of diet (Tinkle 1967, Best and Gennaro 1984). The diet of Uta in urban areas has not been studied, but our data suggest that it may vary substantially from that in rural areas. In particular, we suggest that urban Uta diet is more varied, because the stable isotopes signatures of

Uta exhibited high inter-individual variation. Because ants at urban sites did not have stable isotope signatures that were depleted relative to Uta toes, we suggest that urban Uta are feeding on other invertebrates at these sites.

Surprisingly, even though the desert plant communities at our rural sites were dominated by C4 species, Uta at urban sites had carbon signatures that were more similar to those of C4 rather than C3 plants. Although our urban sites included a mixture of C3 and C4 plants, three elements associated with urbanization may lead to C4 food web dominance in urban areas: 1) cheatgrass (Bromus tectorum) or other grass invasion (Hooker et al. 2008, Bradford et al. 2010, 42

Schaeffer et al. 2012), 2) agriculture of C4 plants such as corn (Finucane et al. 2006), and 3) human detritus such as food (Schoeller et al. 1986, Jahren et al. 2014). These same three factors could also be contributors to the overall higher δ15N values seen in urban lizards. We suspect that the first and third mechanisms are more likely at our sites, considering that all of them are relatively distant from agriculture (and, the most commonly grown crop in Washington County is alfalfa, a C3 legume).

If lizards varied in the degree of nutritional stress across sites, this could explain why relationships between toe C:N and body condition were observed at some locations but not others. Animals with insufficient dietary energetic resources first metabolize lipids and only as a last resort catabolize protein for energy (McCue et al. 2013, McCue et al. 2015a, McCue et al.

2015b). Lipid catabolism without replenishment of lipid stores may cause changes in the δ13C and

C:N ratio of animal tissues, whereas protein catabolism may cause changes in the δ15N signature of animal tissues. Lab studies suggest that these two phases of nutritional stress rarely overlap; that is, starving animals spare protein until lipid reserves are exhausted (McCue 2010). We suggest that individuals with poor body condition at different sites were in different stages of nutritional stress, such that thresholds beyond which C:N begins to change were reached at some sites but not others, and or thresholds beyond which δ15N begins to change were sufficiently close to death that we did not capture many lizards in this stage of starvation. In contrast, we find that female lizards with larger clutches at urban sites increased in δ13C as a result of the larger nutritional (lipid) cost of vitellogenesis (Fuller et al. 2004, Hatch 2012). We observed this relationship only at urban sites, which could be a result of differences in lipid storage capacity of female U. stansburiana in urbanized habitats, potentially resulting from suboptimal diet. Because overwinter mortality is significant in this species (Tinkle 1967, Zani 2005), there could be strong fitness effects resulting from allocation of lipids to vitellogenesis (Price 2016). 43

The life history of U. stansburiana (Lucas and French 2012, Smith et al. 2013) and the occupancy (Smart et al. 2005, Ackley et al. 2015) and physiology (French et al. 2008, French et al. 2010, Knapp et al. 2013) of other lizards differs between urbanized and rural areas across the globe, a conservation concern that may be partly underpinned by both direct and indirect bottom- up effects of changes to resource quality and availability (Suarez and Case 2002, Barrett et al.

2005, Knapp et al. 2013, DeVore and Maerz 2014). The use of stable isotope and stoichiometric ratios to assess animal health is a technique that is becoming more widespread (Hatch 2012). It is a rapid, non-invasive measurement that integrates information over a relatively long time, and the cost is decreasing. However, our test of the generality of relationships between isotopic and stoichiometric ratios and other measures of nutrition and health in a wild reptile revealed that

“rules” about the meaning of isotopic and stoichiometric ratios do not apply in all situations

(Pilgrim 2005), and caution should be used when interpreting these data.

Amphibians and reptiles can reach high densities (Rodda et al. 2001) and may represent large standing stocks of nitrogen and other limiting nutrients in many ecosystems (Milanovich et al. 2015). Studies of their stoichiometry are in their infancy (Sterrett et al. 2015). In particular, the excreta of many tetrapods represent a contribution of organic forms of nitrogen (urea or uric acid) rather than inorganic ammonia (excreted by fishes), thereby providing both carbon and nitrogen to microbes and potentially representing an important and overlooked mechanism of nutrient recycling of limiting nutrients in many ecosystems (Milanovich et al. 2015, Milanovich and

Hopton 2016, Milanovich and Peterman 2016). As urbanization continues to impact populations of wild reptiles, we stand to lose both biodiversity and probably unknown and overlooked ecosystem functions (Gibbons et al. 2000, Willson and Winne 2016). 44

References

Ackley, J.W., Wu, J., Angilletta Jr, M.J., Myint, S.W. & Sullivan, B. (2015) Rich lizards: how

affluence and land cover influence the diversity and abundance of desert reptiles

persisting in an urban landscape. Biological Conservation, 182, 87-92.

Barrett, K., Anderson, W.B., Wait, D.A. & Grismer, L.L. (2005) Marine subsidies alter the diet

and abundance of insular and coastal lizard populations. Oikos, 109, 145-153.

Best, T.L. & Gennaro, A. (1984) Feeding ecology of the lizard, Uta stansburiana, in southeastern

New Mexico. Journal of Herpetology, 18, 291-301.

Bradford, M.A., DeVore, J.L., Maerz, J.C., McHugh, J.V., Smith, C.L. & Strickland, M.S. (2010)

Native, insect herbivore communities derive a significant proportion of their carbon from

a widespread invader of forest understories. Biological Invasions, 12, 721-724.

Bradley, C.A. & Altizer, S. (2007) Urbanization and the ecology of wildlife diseases. Trends in

Ecology & Evolution, 22, 95-102.

Castillo, L. & Hatch, K. (2007) Fasting increases δ15N-values in the uric acid of Anolis

carolinensis and Uta stansburiana as measured by nondestructive sampling. Rapid

Communications in Mass Spectrometry, 21, 4125-4128.

Darimont, C., Paquet, P. & Reimchen, T. (2007) Stable isotopic niche predicts fitness of prey in a

wolf–deer system. Biological Journal of the Linnean Society, 90, 125-137.

DeVore, J.L. & Maerz, J.C. (2014) Grass invasion increases top-down pressure on an amphibian

via structurally mediated effects on an intraguild predator. Ecology, 95, 1724-1730.

Dickens, M.J. & Romero, L.M. (2013) A consensus endocrine profile for chronically stressed

wild animals does not exist. General and Comparative Endocrinology, 191, 177-189. 45

Ditchkoff, S.S., Saalfeld, S.T. & Gibson, C.J. (2006) Animal behavior in urban ecosystems:

modifications due to human-induced stress. Urban Ecosystems, 9, 5-12.

Donihue, C.M. (2016) Aegean wall lizards switch foraging modes, diet, and morphology in a

human-built environment. Ecology and Evolution, 6, 7433-7442.

Doucett, R., Booth, R., Power, G. & McKinley, R. (1999) Effects of the spawning migration on

the nutritional status of anadromous Atlantic salmon (Salmo salar): insights from stable-

isotope analysis. Canadian Journal of Fisheries and Aquatic Sciences, 56, 2172-2180.

Durso, A.M. & Seigel, R.A. (2015) A snake in the hand is worth 10,000 in the bush. Journal of

Herpetology, 49, 503-506.

Durso, A.M., Smith, G.D. & French, S.S. (In review) Stable isotope ratios outperform other

physiological endpoints of nutritional stress. Journal of Experimental Biology.

Finucane, B., Agurto, P.M. & Isbell, W.H. (2006) Human and animal diet at Conchopata, Peru:

stable isotope evidence for maize agriculture and animal management practices during

the Middle Horizon. Journal of Archaeological Science, 33, 1766-1776.

Fox, J. & Weisberg, S. (2011) An {R} Companion to Applied Regression. Sage, Thousand Oaks,

CA, USA.

French, S., Fokidis, H. & Moore, M. (2008) Variation in stress and innate immunity in the tree

lizard (Urosaurus ornatus) across an urban–rural gradient. Journal of Comparative

Physiology B: Biochemical, Systemic, and Environmental Physiology, 178, 997-1005.

French, S.S., DeNardo, D.F., Greives, T.J., Strand, C.R. & Demas, G.E. (2010) Human

disturbance alters endocrine and immune responses in the Galapagos marine iguana

(Amblyrhynchus cristatus). Hormones and Behavior, 58, 792-799. 46

French, S.S., DeNardo, D.F. & Moore, M.C. (2007) Trade-offs between the reproductive and

immune systems: Facultative responses to resources or obligate responses to

reproduction? The American Naturalist, 170, 79-89.

French, S.S. & Neuman-Lee, L.A. (2012) Improved ex vivo method for microbiocidal activity

across vertebrate species. Biology Open, 1, 482-487.

Fuller, B.T., Fuller, J.L., Sage, N.E., Harris, D.A., O'Connell, T.C. & Hedges, R.E. (2004)

Nitrogen balance and δ15N: why you're not what you eat during pregnancy. Rapid

Communications in Mass Spectrometry, 18, 2889-2896.

Gannes, L.Z., Martinez del Rio, C. & Koch, P. (1998) Natural abundance variations in stable

isotopes and their potential uses in animal physiological ecology. Comparative

Biochemistry and Physiology A Comparative Physiology, 119, 725-737.

Gannes, L.Z., O'Brien, D.M. & Martinez del Rio, C. (1997) Stable isotopes in animal ecology:

assumptions, caveats, and a call for more laboratory experiments. Ecology, 78, 1271-

1276.

Gibbons, J.W., Scott, D.E., Ryan, T.J., Buhlmann, K.A., Tuberville, T.D., Metts, B.S., . . . Poppy,

S. (2000) The global decline of reptiles, déjà vu amphibians. Bioscience, 50, 653-666.

Glanville, E. & Seebacher, F. (2006) Compensation for environmental change by complementary

shifts of thermal sensitivity and thermoregulatory behaviour in an ectotherm. Journal of

Experimental Biology, 209, 4869-4877.

Graves, G.R., Newsome, S.D., Willard, D.E., Grosshuesch, D.A., Wurzel, W.W. & Fogel, M.L.

(2012) Nutritional stress and body condition in the Great Gray Owl (Strix nebulosa)

during winter irruptive migrations. Canadian Journal of Zoology, 90, 787-797. 47

Hatch, K.A. (2012) The use and application of stable isotope analysis to the study of starvation,

fasting, and nutritional stress in animals. Comparative Physiology of Fasting, Starvation,

and Food Limitation (ed. M.D. McCue), pp. 337-364. Springer-Verlag, Berlin.

Hooker, T.D., Stark, J.M., Norton, U., Leffler, A.J., Peek, M. & Ryel, R. (2008) Distribution of

ecosystem C and N within contrasting vegetation types in a semiarid rangeland in the

Great Basin, USA. Biogeochemistry, 90, 291-308.

Hopkins, W.A. (2007) Amphibians as models for studying environmental change. ILAR Journal,

48, 270-277.

Hutton, P. & McGraw, K.J. (2016) Urban impacts on oxidative balance and animal signals.

Frontiers in Ecology and Evolution, 4, 54.

Jahren, A.H., Bostic, J.N. & Davy, B.M. (2014) The potential for a carbon stable isotope

biomarker of dietary sugar intake. Journal of Analytical Atomic Spectrometry, 29, 795-

816.

Karasov, W.H. & Pinshow, B. (1998) Changes in lean mass and in organs of nutrient assimilation

in a long‐distance passerine migrant at a springtime stopover site. Physiological and

Biochemical Zoology, 71, 435-438.

Knapp, C.R., Hines, K.N., Zachariah, T.T., Perez-Heydrich, C., Iverson, J.B., Buckner, S.D., . . .

Romero, L.M. (2013) Physiological effects of tourism and associated food provisioning

in an endangered iguana. Conservation Physiology, 1, 1-12.

Krebs, J., Lofroth, E., Copeland, J., Banci, V., Cooley, D., Golden, H., . . . Shults, B. (2004)

Synthesis of survival rates and causes of mortality in North American wolverines.

Journal of Wildlife Management, 68, 493-502. 48

Lattanzio, M. & Miles, D. (2016a) Stable carbon and nitrogen isotope discrimination and

turnover in a small-bodied insectivorous lizard. Isotopes in Environmental and Health

Studies, 10.1080/10256016.2016.1154854.

Lattanzio, M.S. & Miles, D.B. (2016b) Trophic niche divergence among colour morphs that

exhibit alternative mating tactics. Royal Society Open Science, 3, 150531.

Lucas, L. & French, S.S. (2012) Stress-induced tradeoffs in a free-living lizard across a variable

landscape: Consequences for individuals and populations. PLoS ONE, 7, e49895.

Martinez del Rio, C., Wolf, N., Carleton, S.A. & Gannes, L.Z. (2009) Isotopic ecology ten years

after a call for more laboratory experiments. Biological Reviews, 84, 91-111.

McCue, M., Guzman, R., Passement, C. & Davidowitz, G. (2015a) How and when do insects rely

on endogenous protein and lipid resources during lethal bouts of starvation? A new

application for 13C-breath testing. PLoS ONE, 10, e0140053.

McCue, M.D. (2010) Starvation physiology: reviewing the different strategies animals use to

survive a common challenge. Comparative Biochemistry and Physiology-Part A:

Molecular & Integrative Physiology, 156, 1-18.

McCue, M.D., Amaya, J.A., Yang, A.S., Erhardt, E.B., Wolf, B.O. & Hanson, D.T. (2013)

Targeted 13C enrichment of lipid and protein pools in the body reveals circadian changes

in oxidative fuel mixture during prolonged fasting: a case study using Japanese quail.

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology,

166, 546-554. 49

McCue, M.D., Guzman, R.M. & Passement, C.A. (2015b) Digesting pythons quickly oxidize the

proteins in their meals and save the lipids for later. The Journal of Experimental Biology,

215, 2089-2096.

McCue, M.D. & Pollock, E.D. (2008) Stable isotopes may provide evidence for starvation in

reptiles. Rapid Communications in Mass Spectrometry, 22, 2307-2314.

McKinney, M.L. (2008) Effects of urbanization on species richness: a review of plants and

animals. Urban Ecosystems, 11, 161-176.

McNamara, J.M. & Houston, A.I. (1990) The value of fat reserves and the tradeoff between

starvation and predation. Acta Biotheoretica, 38, 37-61.

Mekota, A.M., Grupe, G., Ufer, S. & Cuntz, U. (2006) Serial analysis of stable nitrogen and

carbon isotopes in hair: monitoring starvation and recovery phases of patients suffering

from anorexia nervosa. Rapid Communications in Mass Spectrometry, 20, 1604-1610.

Milanovich, J.R. & Hopton, M.E. (2016) Stoichiometry of excreta and excretion rates of a

stream-dwelling plethodontid salamander. Copeia, 104, 26-34.

Milanovich, J.R., Maerz, J.C. & Rosemond, A.D. (2015) Stoichiometry and estimates of nutrient

standing stocks of larval salamanders in Appalachian headwater streams. Freshwater

Biology, 10.1111/fwb.12572.

Milanovich, J.R. & Peterman, W.E. (2016) Revisiting Burton and Likens (1975): nutrient

standing stock and biomass of a terrestrial salamander in the midwestern United States.

Copeia, 104, 165-171.

Mitchell, J.C., Jung, R.E. & Bartholomew, B. (2008) Urban Herpetology. Society for the Study

of Amphibians and Reptiles, Salt Lake City. 50

Monteith, K.L., Bleich, V.C., Stephenson, T.R., Pierce, B.M., Conner, M.M., Kie, J.G. &

Bowyer, R.T. (2014) Life‐history characteristics of mule deer: Effects of nutrition in a

variable environment. Wildlife Monographs, 186, 1-62.

Moore, M.C., Thompson, C.W. & Marler, C.A. (1991) Reciprocal changes in corticosterone and

testosterone levels following acute and chronic handling stress in the tree lizard,

Urosaurus ornatus. General and Comparative Endocrinology, 81, 217-226.

Murray, M., Cembrowski, A., Latham, A., Lukasik, V., Pruss, S. & St Clair, C. (2015) Greater

consumption of protein‐poor anthropogenic food by urban relative to rural coyotes

increases diet breadth and potential for human–wildlife conflict. Ecography, 38, 1235-

1242.

Naug, D. (2009) Nutritional stress due to habitat loss may explain recent honeybee colony

collapses. Biological Conservation, 142, 2369-2372.

Neuman-Lee, L.A., Stokes, A.N., Greenfield, S., Hopkins, G.R., Brodie, E.D. & French, S.S.

(2015) The role of corticosterone and toxicity in the antipredator behavior of the Rough-

skinned Newt (Taricha granulosa). General and Comparative Endocrinology, 213, 59-

64.

Perry, G., Wallace, M.C., Perry, D., Curzer, H. & Muhlberger, P. (2011) Toe clipping of

amphibians and reptiles: science, ethics, and the law. Journal of Herpetology, 45, 547-

555.

Pilgrim, M. (2005) Linking Microgeographic Variation in Pigmy Rattlesnake (Sistrurus

miliarius) Life History and Demography with Diet Composition: A Stable Isotope

Approach. Ph. D. Ph. D. Dissertation, University of Arkansas. 51

Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D. & R Core Team (2016) nlme: Linear and

Nonlinear Mixed Effects Models.

Price, E.R. (2016) The physiology of lipid storage and use in reptiles. Biological Reviews,

10.1111/brv.12288.

R Core Team (2015) R: A language and environment for statistical computing. R Foundation for

Statistical Computing, Vienna, Austria.

Reddin, C.J., O’Connor, N.E. & Harrod, C. (2016) Living to the range limit: consumer isotopic

variation increases with environmental stress. PeerJ, 4, e2034.

Refsnider, J.M. & Janzen, F.J. (2012) Behavioural plasticity may compensate for climate change

in a long-lived reptile with temperature-dependent sex determination. Biological

Conservation, 152, 90-95.

Rodda, G.H., Perry, G.A.D., Rondeau, R.J. & Lazell, J. (2001) The densest terrestrial vertebrate.

Journal of Tropical Ecology, 17, 331-338.

Romero, L.M. & Reed, J.M. (2005) Collecting baseline corticosterone samples in the field: is

under 3 min good enough? Comparative Biochemistry and Physiology-Part A: Molecular

& Integrative Physiology, 140, 73-79.

Romero, L.M. & Reed, J.M. (2008) Repeatability of baseline corticosterone concentrations.

General and Comparative Endocrinology, 156, 27-33.

Romero, L.M. & Wikelski, M. (2001) Corticosterone levels predict survival probabilities of

Galapagos marine iguanas during El Niño events. Proceedings of the National Academy

of Sciences, 98, 7366-7370. 52

Romero, L.M. & Wikelski, M. (2010) Stress physiology as a predictor of survival in Galapagos

marine iguanas. Proceedings of the Royal Society of London B: Biological Sciences, 277,

3157-3162.

Sarre, S., Dearn, J.M. & Georges, A. (1994) The application of fluctuating asymmetry in the

monitoring of animal populations. Pacific Conservation Biology, 1, 118-122.

Schaeffer, S.M., Ziegler, S.E., Belnap, J. & Evans, R. (2012) Effects of Bromus tectorum

invasion on microbial carbon and nitrogen cycling in two adjacent undisturbed arid

grassland communities. Biogeochemistry, 111, 427-441.

Schoeller, D.A., Minagawa, M., Slater, R. & Kaplan, I. (1986) Stable isotopes of carbon, nitrogen

and hydrogen in the contemporary North American human food web. Ecology of Food

and Nutrition, 18, 159-170.

Sinervo, B. & Lively, C.M. (1996) The rock-paper-scissors game and the evolution of alternative

male strategies. Nature, 380, 240-243.

Smart, R., Whiting, M.J. & Twine, W. (2005) Lizards and landscapes: integrating field surveys

and interviews to assess the impact of human disturbance on lizard assemblages and

selected reptiles in a savanna in South Africa. Biological Conservation, 122, 23-31.

Smiley, T.M., Cotton, J.M., Badgley, C. & Cerling, T.E. (2016) Small-mammal isotope ecology

tracks climate and vegetation gradients across western North America. Oikos, 125, 1100-

1109.

Smith, G.D., Lucas, L.D. & French, S.S. (2013) Town and country lizards: physiological ecology

of Side-blotched Lizards across a variable landscape. Reptiles in Research (ed. W.I.

Lutterschmidt), pp. 51-71. Nova Biomedical, New York, New York. 53

Speakman, J.R. (2008) Body Composition Analysis of Animals: A Handbook of Non-destructive

Methods. pp. 260. Cambridge University Press, Cambridge, UK.

Sterrett, S.C., Maerz, J.C. & Katz, R.A. (2015) What can turtles teach us about the theory of

ecological stoichiometry? Freshwater Biology, 60, 443-455.

Stout, I.J. & Cornwell, G.W. (1976) Nonhunting mortality of fledged North American waterfowl.

The Journal of Wildlife Management, 40, 681-693.

Suarez, A.V. & Case, T.J. (2002) Bottom‐up effects on persistence of a specialist predator: ant

invasions and horned lizards. Ecological Applications, 12, 291-298.

Tinkle, D.W. (1967) The life and demography of the Side-blotched Lizard, Uta stansburiana.

Miscellaneous Publications of the Museum of Zoology University of Michigan, 132, 1-

182.

Vangestel, C., Braeckman, B.P., Matheve, H. & Lens, L. (2010) Constraints on home range

behaviour affect nutritional condition in urban house sparrows (Passer domesticus).

Biological Journal of the Linnean Society, 101, 41-50.

Wickham, H. (2009) ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag, New York.

Wikelski, M. & Romero, L.M. (2003) Body size, performance and fitness in Galapagos marine

iguanas. Integrative and Comparative Biology, 43, 376-386.

Wilder, S.M. & Jeyasingh, P.D. (2016) Merging elemental and macronutrient approaches for a

comprehensive study of energy and nutrient flows. Journal of Animal Ecology, 85, 1427-

1430. 54

Willson, J.D. & Winne, C.T. (2016) Evaluating the functional importance of secretive species: A

case study of aquatic snake predators in isolated wetlands. Journal of Zoology, 298, 266-

273.

Willson, J.D., Winne, C.T., Dorcas, M.E. & Gibbons, J.W. (2006) Post-drought responses of

semi-aquatic snakes inhabiting an isolated wetland: insights on different strategies for

persistence in a dynamic habitat. Wetlands, 26, 1071-1078.

Willson, J.D., Winne, C.T., Pilgrim, M.A., Romanek, C.S. & Gibbons, J.W. (2010) Seasonal

variation in terrestrial resource subsidies influences trophic niche width and overlap in

two aquatic snake species: a stable isotope approach. Oikos, 119, 1161-1171.

Young, T.P. (1994) Natural die‐offs of large mammals: implications for conservation.

Conservation Biology, 8, 410-418.

Zani, P.A. (2005) Life-history strategies near the limits of persistence: winter survivorship and

spring reproduction in the Common Side-blotched Lizard (Uta stansburiana) in eastern

Oregon. Journal of Herpetology, 39, 166-169.

Zhang, C., Jansen, M., De Meester, L. & Stoks, R. (2016) Energy storage and fecundity explain

deviations from ecological stoichiometry predictions under global warming and size-

selective predation. Journal of Animal Ecology, 85, 1431-1441.

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CHAPTER 3

STABLE ISOTOPE RATIOS OUTPERFORM OTHER PHYSIOLOGICAL

ENDPOINTS OF NUTRITIONAL STRESS

Resource limitation is a common challenge faced by animals, but precise, non-destructive methods for measuring resource availability often hinder experiments aimed at assessing the effects of nutritional stress. In particular, assessing the past nutritional state of wild animals is nearly impossible, especially for species with low recapture rates, including many reptiles. We examined how stable isotope ratios of nitrogen (δ15N) of a variety of tissues performed next to other physiological measures of nutritional stress, including immunological, endocrinological, and morphological metrics. In a lab experiment where the quantity of food available to Side- blotched Lizards (Uta stansburiana) was manipulated and their intake carefully measured over 49 days, we found that nitrogen stable isotope ratios of uric acid pellets outperform other physiological endpoints of nutritional stress. Average values of uric acid δ15N were capable of detecting smaller differences in food intake. Although this measure was not related to other physiological metrics, we suggest that it has great utility in future field and lab studies of reptile physiology and ecology.

INTRODUCTION

Nutritional stress is a common challenge faced by many wild animals. Limited nutritional resources underlie many life-history trade-offs and their fitness consequences (Stearns, 1992;

Zera and Harshman, 2001). Nitrogen, acquired as amino acids, is the limiting element in many systems (White, 2012), partly because, unlike lipids, animals cannot store proteins over long periods of time. When lipid reserves are exhausted and exogenous resources are not available, 56 animals rely on protein catabolism for energy (McCue et al., 2015; McCue et al., 2013), influencing their nitrogen balance.

Stable isotope ratios of nitrogen (δ15N) are often used to examine the diet of wild animals.

The δ15N values of an animal’s body tissues typically reflect the isotopic signatures of their diet, plus a predictable fractionation factor (normally ~3‰ for nitrogen; Post, 2002). However, more minor variation in isotope ratios is caused by a variety of physiological processes, including resource availability. Our understanding of the relationship between these processes and isotope ratios is rapidly evolving (Fuller et al., 2004; Fuller et al., 2005) and has been the subject of a recent review (Hatch, 2012) and meta-analysis (Hertz et al., 2015).

2015). The principle is that proteins or other molecules containing slightly heavier 15N atoms are slightly less chemically reactive (Steele and Daniel, 1978), meaning that they are both less likely to be incorporated into a growing animal’s tissues from its diet during anabolism, and less likely to leave during tissue catabolism. The effect of fasting on δ15N has been measured in a variety of systems and has been found to increase with the length of the fast (e.g., Hatch, 2012; Martinez del

Rio and Wolf, 2005). A recent meta-analysis (Hertz et al., 2015) determined that the average magnitude of the increase in δ15N is 0.5‰ and that the most significant moderator of this effect was the type of tissue examined.

Recent work has suggested that that stable isotope levels of reptiles and amphibians may be less sensitive to short and moderate periods of fasting than is expected of other taxa because of their metabolic efficiency (McCue, 2007; Milanovich and Maerz, 2013), but most studies have assessed only one or a few tissue types. Three studies have reported an increase in the enrichment 57 of the nitrogenous waste of reptiles with starvation (Castillo and Hatch, 2007; McCue, 2007;

McCue and Pollock, 2008), but are limited in their ability to make comparisons to other physiological endpoints of nutritional stress.

Nitrogenous waste is more sensitive to isotopic changes than other bodily tissues

(Barboza and Parker, 2006; Gustine et al., 2011). Uric acid makes up >92% of the nitrogen excreted by reptiles (Greenwald and Kanter, 1979; Khalil, 1951), which use a uricotelic pathway for its production. Although small insectivorous ectotherms may be particularly recalcitrant to changes in the stable isotope ratios of all tissues (Milanovich and Maerz, 2013), it seems that uric acid is emerging as the most promising place to look for effects of fasting on δ15N in reptiles.

This is significant because, if true, the δ15N signatures of uric acid could represent a sensitive biomarker of nutritional stress that can easily and non-lethally be collected from both captive and wild animals.

In this study, we set out to answer how the amount of food eaten by Side-blotched

Lizards (Uta stansburiana Baird and Girard, 1852) influences the δ15N signatures of different lizard tissues, including uric acid, feces, toes, tail tips, and red blood cells, as well as other physiological endpoints of nutritional stress, and whether any of these measures of nutritional stress were correlated. To accomplish these aims we subjected 45 male U. stansburiana to high and low food treatments for 32 days and measured the δ15N signatures of several of their tissues as well as other metrics of nutritional stress. We predicted that food restriction would increase the

δ15N of at least one of the tissues of interest, most likely uric acid, by the end of the experiment.

We also predicted that we would see an effect of nutritional stress on one or more of the other physiological endpoints (corticosterone, immune function, and oxidative stress). We did not have a specific prediction about the relationship between δ15N and other physiological endpoints of nutritional stress. 58

METHODS

Animal Collection

Lizards were collected under the authority of Utah State Department of Wildlife

Resources COR #1COLL8382 and handled in accordance with USU IACUC protocol #2068. We captured 45 adult male U. stansburiana from a single site near St. George, Utah (37.06220,-

113.59535; WGS 84), in the spring of 2014. Unlike in California (Sinervo and Lively, 1996), in this part of their range, U. stansburiana do not exhibit stable color morphs. An acclimation period of 18 days preceded the start of the experiment, during which time all lizards were offered 0.2-0.4 g of farm-raised crickets (Acheta domestica, Fluker’s®, Port Allen, Louisiana, USA) every other day. Half (22) of the lizards were then assigned to a food restriction treatment (0.2-0.4 g of crickets every eight days), and the remaining 23 continued to be fed ad libitum (0.2-0.4 g of crickets every other day) for a period of 32 days. At the beginning of the experiment, we measured the mass, snout-vent length, tail length, and the length of any tail regrowth of each lizard. We also collected tissue from the tip of each lizard’s tail, as well as a single toe from each lizard.

Laboratory Methods

All lizards were housed individually in 30x45x15 cm plastic terraria with a plastic hide, water bowl, and newspaper substrate. The racks of terraria were in a climate-controlled room

(25.2°C, 20% humidity) and each terrarium had an ultraviolet (UVA/UVB) light on a 12:12 hour cycle. We recorded how much food was offered to each lizard and calculated how much was consumed by subtracting the total mass of all crickets put into each cage from the total mass of all crickets removed from each cage. Every cage was checked for crickets every other day. At the 59 same time, any feces and uric acid pellets, which are normally deposited simultaneously

(Greenwald and Kanter, 1979) were collected, separated, dried, and weighed.

At the end of the experiment, we measured the mass, snout-vent length, tail length, and the length of any tail regrowth of each lizard. We again collected any regrown tissue from the tip of each lizard’s tail, as well as a single toe from each lizard. We also took a blood sample, which we separated into a red blood cell pellet and a plasma supernatant. We were unable to collect blood samples or accurate morphometric data from 14 lizards that were removed from the experiment because they did not feed in captivity and their physiological condition deteriorated; however, we did collect toe and tail clips from these lizards at the time when they were removed, and we considered data from uric acid and feces deposited prior to their removal valid.

We measured the nitrogen stable isotope ratios of the pre and post tails and toes, the red blood cell pellet, and all fecal and uric acid samples. Each sample was homogenized and between

0.05 and 2.0 mg of each sample were weighed into a tin capsule (5x9mm; Costech Analytical,

Valencia, California, USA), folded, and analyzed for δ15N by continuous-flow direct-combustion and isotope ratio mass spectrometry using a Europa Scientific ANCA-2020 (PDZ, Crewe,

England) at the Utah State University Stable Isotope Lab.

Additionally, we assessed immunological, endocrinological, and morphological endpoints to compare the utility of stable isotopes with that of other endpoints for measuring nutritional stress and induction of life-history trade-offs. Corticosterone was measured using a radioimmunoassay (Neuman-Lee et al., 2015). Plasma bactericidal capability was quantified using the method of French and Neuman-Lee (2012). We assayed markers of oxidative stress using d-ROMs and OXY-Adsorbent test kits from Diacron International (Grosseto, Italy). The length and mass of any tail regrowth, and the difference between the final and initial 60 measurements of body mass and body condition (residual of regression of mass and SVL) were also calculated.

Statistical Analysis

We analyzed the difference between the two treatment groups using one-way ANOVAs

('Anova' function in the 'car' package in R, with option type=3 for unbalanced designs; Fox and

Weisberg, 2011; R Core Team, 2015) if the variable in question was normally-distributed, or using non-parametric Kruskal-Wallace tests (function ‘kruskal.test’ in the ‘stats’ package in base

R) if the variable was not normally distributed. Although the amount of food eaten was not normally distributed and could not be transformed, the skewness (0.98) and kurtosis (2.60) were not abnormally distant from normal parameters, so we justified testing for differences between treatments in these data using parametric tests (Schmider et al., 2010). Results from parametric tests ultimately agreed with those from non-parametric tests, which we could not use universally because we wished to look at interactions and lacked a balanced design. We used a Bonferroni correction (α = 0.002) to correct for multiple analyses.

Even though the feeding treatments were randomly assigned, nearly half of the lizards assigned to the ad libitum feeding treatment consumed as little or less food than was being offered to the food-restricted lizards, with one individual consuming only 0.09 g of food throughout the entire experiment. As a result, we re-analyzed the data using general linear models

(function ‘lm’ in package ‘stats’ in R) to examine the effect of the amount of food consumed both before and during the experiment on physiological response variables. We incorporated a two- level categorical effect for treatment into this analysis to account for the fact that ad libitum lizards were offered more food, and tested for an interaction between treatment and quantity of 61 food eaten. We also used a piecewise-linear regression (package 'segmented'; Muggeo, 2008) to test for non-linear (i.e., threshold) effects of food eaten on δ15N of feces and uric acid.

Finally, to correct for individual variation in the starting isotopic values of repeatedly- sampled feces and nitrogenous waste, we incorporated the earliest values for these tissues as continuous covariates into our linear models, and we conducted a repeated measures analysis by building and comparing a set of random coefficient models (Bliese and Ployhart, 2002) in R using the package nlme (Pinheiro and Bates, 2000) to analyze the change in δ15N of feces and uric acid over the course of the experiment, with random effects for both the slope (pattern over time) and the intercept (initial value for each individual lizard). We used AIC (Akaike, 1973) to compare random coefficient models of the effect of the amount of food eaten both cumulatively or over the preceding three days on the δ15N of uric acid and feces. All figures were made using ggplot2

(Wickham, 2009).

RESULTS

The amount of food eaten by each lizard over the course of the experiment varied from

0.09 g (1.7% of initial body mass) to 7.41 g (158% of initial body mass). Most lizards lost mass in spite of eating, ranging from a 1% to a 43% decrease. Only four lizards gained mass throughout the experiment (maximum gain = 0.95 g; a 20% increase).

At the beginning of the food manipulation, none of the variables we measured differed between treatment groups (p ≥ 0.10; Table 3-1). At the end of the food manipulation, five variables (mass [F = 21.601,29, p < 0.0001], body condition [F1,29 = 23.82, p < 0.0001], total mass

2 of feces [Χ 1 = 7.29, p = 0.009], total mass of uric acid [F = 7.851,42, p = 0.008], and log-

15 transformed δ N of feces [F = 8.101,24, p < 0.0001]; Table 3-1) were significantly lower in food- 62

15 restricted lizards, and one variable, δ N of uric acid, was significantly higher (F = 13.701,29, p =

0.0009; Table 3-1) in food-restricted lizards.

Isotopic changes due to fasting

The δ15N values of uric acid samples collected at the end of the experiment were negatively related to the quantity of food eaten (slope = -1.22, p < 0.0001, R2 = 0.94; Fig. 3-1A).

Other physiological measures of nutritional stress that were strongly negatively related to quantity of food eaten included the average δ15N value of the last three uric acid samples deposited (slope

= -1.18, p < 0.0001, R2 = 0.85; Fig. 3-1B), and the average δ15N value of uric acid samples over the preceding month (slope = -1.22, p = 0.0004, R2 = 0.91; Fig. 3-1C). The total mass of uric acid

(slope = 0.02, p < 0.001, R2 = 0.92) and feces (slope = 0.05, p < 0.0001, R2 = 0.93) produced over the preceding month, and the average mass of uric acid (slope = 0.24, p < 0.001, R2 = 0.92) and feces (slope = 0.12, p < 0.001, R2 = 0.91) produced over the preceding month were positively related to quantity of food eaten. Weaker statistical support was observed between quantity of food eaten and the average δ15N value of fecal samples over the preceding month (p = 0.06, R2 =

0.90), body condition at the end of the experiment (p = 0.05, R2 = 0.90), and change in mass over the entire experiment (p = 0.07, R2 = 0.89). Other morphological endpoints (tail regrowth; p =

0.97, R2 = 0.88), and evidence from immunology (BKA; p = 0.86, R2 = 0.88), endocrinology

(CORT; p = 0.59, R2 = 0.90), and oxidative stress (dROM; p = 0.63, R2 = 0.89) were not significantly related to the quantity of food eaten, nor were stable isotope ratios of red blood cells

(p = 0.16, R2 = 0.87), toes (p = 0.69, R2 = 0.88), regrown tail tissue (p = 0.95, R2 = 0.90), or feces

(p = 0.35, R2 = 0.87) collected at the end of the experiment. 63

Overall, δ15N values of uric acid at the end of the experiment were negatively related to the quantity of food eaten (Fig. 3-1A; Table 3-2). The average of the δ15N values of the last three

(Fig. 3-1B) and all uric acid pellets collected (Fig. 3-1C) were also negatively related to the quantity of food eaten. We measured an average increase of 1.50‰ in the uric acid of food restricted lizards.

Comparing linear mixed models with continuous covariates for initial δ15N values of uric acid and treatment as the random effect showed that the model using the average δ15N value of all uric acid samples collected throughout the experiment to explain the variation in the amount of food eaten had the lowest AIC (Table 3-2). This model suggested that there was a significant

15 effect of the average δ N value of all uric acid pellets on the amount of food eaten (t1,38 = -4.43, p

15 = 0.0001), while correcting for less important effects of the initial δ N value of uric acid (t1,38 = -

15 1.87, p = 0.07) and a possible interaction (t1,38 = 2.00, p = 0.05). All models suggested that δ N values of uric acid from the end of the experiment were stronger predictors of the amount of food eaten than initial values, however many uric acid pellets were used to calculate the isotope ratios

(Fig. 3-2).

We found support for a threshold effect in our data (Fig. 3-3). At a threshold between

4.20 ± 0.75 and 3.12 ± 0.79 g of food (80-197% of initial body mass over 32 days), the slope of the relationship between amount of food eaten over the preceding month and the δ15N of uric acid pellets decreased from between 0.01 ± 0.32 and -0.26 ± 0.20 to between -0.86 ± 0.23 and -1.26 ±

0.29, depending on which uric acid pellets were included (Fig. 3-3).

Our random coefficient (mixed-effect) models revealed that the effect of the cumulative amount of food eaten over the course of the experiment on both the δ15N of uric acid (t = -4.47, p

< 0.0001) and feces (t = 22.11, p = 0.0003) was highly significant. We found support (lowest 64

AIC) for models with random effects for both the slope and the intercept for both response variables.

Other physiological endpoints

The δ15N values of uric acid samples collected at the end of the experiment were higher in lizards with poorer body condition (Fig. 3-4), but were not related to CORT, dROM, BKA, or tail regrowth. Lizards fed ad libitum had higher BKA than food-restricted lizards (F1,29 = 6.7, p =

0.015), an effect that was driven by four individuals with 100% killing in the ad libitum treatment.

TABLES AND FIGURES

Table 3-1. Linear models testing for differences between treatment groups on a variety of response variables. We used a Bonferroni correction (α = 0.002) to correct for multiple comparisons

At beginning of food manipulation

2 2 Response variable Normally distributed? Fdf or Χ df p R

Mass Yes 0.771,43 0.39 0.02

Body condition Yes 2.051,43 0.16 0.05

15 Δ N of tail tip Yes 2.881,42 0.10 0.06

15 Δ N of toe No 0.531 0.47 NA

15 Δ N of feces No 0.271 0.60 NA

15 Δ N of uric acid Yes 0.201,33 0.66 0.01 At end of food manipulation

2 2 Response variable Normally distributed? Fdf or Χ df p R

Mass Yes 21.601,29 < 0.0001 0.43

Body condition Yes 23.821,29 < 0.0001 0.45

15 Δ N of tail tip Yes 1.501,24 0.24 0.06

15 Δ N of toe No 0.051 0.81 NA

15 Δ N of feces Yes (log-transformed) 8.101,24 0.009 0.25

15 Δ N of uric acid Yes 13.701,30 0.0009 0.31

15 Δ N of red blood cells Yes 0.051,28 0.82 < 0.01

Total mass of feces No 7.291 0.007 NA

Total mass of uric acid Yes 7.851,42 0.008 0.16

Tail regrowth No 0.181 0.67 NA

CORT Yes (log-transformed) 1.551,30 0.22 0.05

dROM Yes 1.841,25 0.19 0.07

BKA No 0.851 0.36 NA 66

Table 3-2. Six linear mixed-effects models describing the relationship between the amount of food eaten (response variable) and two interacting fixed effects representing initial and either final or average δ15N values of uric acid. All models have treatment as a random effect

Model ID Term df t-value p-value AIC Δ15N last uric acid 38 -2.93 0.006 1 Δ15N first uric acid 38 -1.84 0.07 137 Interaction 38 1.00 0.33 Δ15N last uric acid 38 -1.86 0.07 Average of δ15N first week of 2 38 -1.33 0.19 137 uric acid Interaction 38 0.48 0.63 Average δ15N of last three uric 38 -2.57 0.01 acid pellets 3 139 Δ15N first uric acid 38 -1.25 0.22 Interaction 38 0.62 0.54 Average δ15N of last three uric 38 -2.14 0.04 acid pellets 4 Average of δ15N first week of 135 38 -1.60 0.12 uric acid Interaction 38 0.94 0.35 Average δ15N of all uric acid 38 -4.43 0.0001 pellets 5 131 Δ15N first uric acid 38 -1.87 0.07 Interaction 38 2.00 0.05 Average δ15N of all uric acid 38 -3.71 0.0007 pellets 6 Average of δ15N first week of 132 38 -0.81 0.42 uric acid Interaction 38 1.32 0.20

Fig. 3-1. Relationships between the amount of food eaten and three measurements of uric acid nitrogen stable isotope ratios. (A) Relationship between the amount of food eaten and the

δ15N value of the final uric acid collected, (B) the average of the δ15N value of the last three uric acid pellets collected, and (C) the average δ15N value of all uric acid pellets collected over the course of the experiment. Test and goodness of fit statistics: A) t = -7.234, p = 4.72e-08, R2 = 68

0.6235; B) t = -8.057, p = 5.54e-10, R2 = 0.6035; C) t = -9.339, p = 1.05e-11, R2 = 0.6724. A = ad libitum, R = restricted. 69

Fig. 3-2. Scatterplots showing the relationship among the amount of food eaten, the average

δ15N value of the first three uric acid pellets collected, and the average δ15N value of the last three uric acid pellets collected. 70

Fig. 3-3. Output of piecewise-linear (‘segmented’) regressions. These figures show the thresholds of change in the slopes of the relationships between amount of food eaten over the preceding month and A) the δ15N value of the final uric acid pellet collected, B) the average ± s.e.m. of the δ15N values of the last three uric acid pellets collected, and C) the average ± s.e.m.

δ15N value of all uric acid pellets collected over the course of the experiment. The change was estimated at 4.20 ± 0.75 g of food (80-146% of initial body mass; A), 4.15 ± 1.02 g of food (81-

148% of initial body mass; B), and 3.12 ± 0.79 g of food (109-197% of initial body mass; C). The slope decreased from 0.01 ± 0.32 to -1.26 ± 0.29 (A; t = -4.33, p = 0.0001, R2 = 0.67), -0.12 ±

0.26 to -0.86 ± 0.23 (B; t = -3.68, p = 0.0007, R2 = 0.63), and -0.26 ± 0.20 to -1.14 ± 0.27 (C; t = -

4.19, p = 0.0002, R2 = 0.70). 71

Fig. 3-4. Relationship between body condition at the end of the experiment and δ15N value of the last uric acid pellet collected.

DISCUSSION

Our study demonstrated that δ15N of lizard uric acid is a sensitive biomarker of nutritional stress, although it yields different results than other measures of nutritional stress. We found that a single pellet of uric acid served as a metric for the nutritional state of lizards that had been food restricted, but that taking the average value of several pellets over time can describe even more subtle differences in past nutritional history (Fig. 3-3). We showed that the magnitude of the effect of nutritional stress on δ15N of uric acid increases with the degree of nutritional stress. Our measured average increase was 1.50‰, three times the average increase of 0.50‰ calculated in a recent meta-analysis (Hertz et al., 2015), though much less than the maximum shift observed

(nearly 6.0‰; Gaye-Siessegger et al., 2004). 72

Unlike uric acid, the other tissues that we measured (toes, tail tips, red blood cells) did not appear to be useful markers of nutritional stress. Fecal nitrogen did track nutritional stress to some degree, but the association was not as strong as with uric acid. This is consistent with the findings of other studies of small insectivorous ectotherms conducted over shorter periods of time

(Castillo and Hatch, 2007; Milanovich and Maerz, 2013). We do not anticipate a residual effect of diet switching from the wild to captivity, because we acclimated our lizards to a cricket diet over

18 days, and 95% turnover of toe isotope signature occurs after ~15.5 days in a similar species,

Urosaurus ornatus (Lattanzio and Miles, 2016).

Although δ15N of lizard uric acid was related to nutritional stress, it was not related to other physiological endpoints that we measured. However, lizards fed ad libitum had higher plasma bacterial-killing ability than food-restricted lizards. Although nutritional stress is a risk factor for infection (e.g., Plowright et al., 2008), bacterial-killing ability 1) is just one component of immunity, 2) may be elevated for reasons other than nutritional stress, and 3) participates in trade-offs with other aspects of immunity as well as other demands for resources (Lochmiller and

Deerenberg, 2000; Zera and Harshman, 2001). We did not collect data on other endpoints of immunity, on infection status, or on reproductive investment in these lizards, which, taken together, might have presented a more complete picture of the relationships among nutritional stress, δ15N ratios, and immunity.

In studies of endothermic animals with significant lipid stores, no relationship between

δ15N and nutritional stress has been found (e.g., Ben-David et al., 1999; Gómez‐Campos et al.,

2011; Kempster et al., 2007). We were unable to quantify the lipid reserves of our lizards because no suitable non-destructive technique exists to do so (Labocha and Hayes, 2012), but it seems that the duration of our study was sufficient that more than half of our subjects exhausted any lipid stores they may have had. We found that a single δ15N value from uric acid is not a continuous 73 index of nutritional stress; rather, there is a threshold above which the relationship between δ15N of uric acid and amount of food consumed is neither positive nor negative. It is likely that the nitrogen balance (and therefore the δ15N) of an animal’s tissues and excreta is not influenced by nutritional stress when the animals have ample lipid reserves to catabolize and do not need to recycle protein (Bar and Volkoff, 2012; Hatch, 2012; Martinez del Rio and Wolf, 2005).

Variation in the amount of stored lipids likely causes variation in the strength of trade-offs among competing physiological processes, including immunity and reproduction (Doughty and Shine,

1997).

Measuring stable isotope ratios is relatively inexpensive (~$8/sample), can be accomplished fairly rapidly, and is non-lethal and non-invasive. Among the several strengths of using δ15N of uric acid as an index of nutritional stress include that the technique integrates information over the preceding several weeks of the animal’s lifetime, can be used in both the lab and the field, and is relatively inexpensive. Another strength is that serial measurements on a fine temporal scale are possible, unlike with variables measured from blood, which can only be taken so often from small animals. This technique has special utility for reptiles, which are often very secretive in the wild and can be very difficult to monitor because of low and variable detection probability (Lardner et al., 2015; Rodda et al., 2015). Weaknesses include the fact that variation in food sources among wild populations of animals usually equals or exceeds the magnitude of these effects (Melissa A. Pilgrim, Linking Microgeographic Variation in Pigmy Rattlesnake

(Sistrurus miliarius) Life History and Demography with Diet Composition: A Stable Isotope

Approach, PhD thesis, University of Arkansas-Fayetteville, 2005), so this technique should only be used within a wild population that can reasonably be expected to be feeding on isotopically- similar sources; otherwise the isotopic effects of nutritional stress will likely by swamped by the 74 noise coming from the different sources (which, ironically, is the signal of interest in most stable isotope studies of animal ecology).

This study builds upon knowledge collected by others (Castillo and Hatch, 2007) and integrates techniques for assessment of nutritional stress across four physiological systems

(digestive, reproductive, immune, and endocrine). It is the most physiologically comprehensive study of its kind to date (McCue, 2010). As we continue to refine our ability to measure nutritional stress, we will be better able to incorporate individual differences in physiology into studies of population dynamics, trade-offs, and their fitness costs (Demas et al., 2012; French et al., 2009).

References

Akaike, H. (1973). Information theory and an extension of the maximum likelihood principle. In

Second International Symposium on Information Theory (eds. B. Petrov and F. Csáki),

pp. 267-281. Tsahkadsor, Armenia, USSR: Akademiai Kidao, Budapest.

Baird, S.F. and Girard, C. (1852). Descriptions of new species of reptiles, collected by the U.S.

exploring expedition under the command of Capt. Charles Wilkes, U.S.N. First part. -

Including the species from the Western coast of America. Proc. Acad. Nat. Sci. Phila. 6,

174-177.

Bar, N. and Volkoff, H. (2012). Adaptation of the physiological, endocrine, and digestive system

functions to prolonged food deprivation in fish. In Comparative Physiology of Fasting,

Starvation, and Food Limitation (ed. M. D. McCue), pp. 69-89. Berlin: Springer-Verlag.

Barboza, P.S. and Parker, K.L. (2006). Body protein stores and isotopic indicators of N balance

in female reindeer (Rangifer tarandus) during winter. Physiol. Biochem. Zool. 79, 628-

644.

Ben-David, M., McColl, C., Boonstra, R. and Karels, T. (1999). 15N signatures do not reflect

body condition in Arctic ground squirrels. Can. J. Zool. 77, 1373-1378.

Bliese, P.D. and Ployhart, R.E. (2002). Growth modeling using random coefficient models:

model building, testing, and illustrations. Organizational Research Methods 5, 362-387.

Castillo, L. and Hatch, K. (2007). Fasting increases d15N-values in the uric acid of Anolis

carolinensis and Uta stansburiana as measured by nondestructive sampling. Rapid

Commun. Mass Spectrom. 21, 4125-4128. 76

Demas, G.E., T.J., G., Chester, E.M. and French, S.S. (2012). The energetics of immunity:

mechanisms mediating trade-offs in eco-immunology. In Ecoimmunology (eds. G. E.

Demas and R. J. Nelson), pp. 259-296. New York, New York, USA: Oxford University

Press.

Doughty, P. and Shine, R. (1997). Detecting life history tradeoffs: measuring energy stores in

‘capital’ breeders reveals costs of reproduction. Oecologia 110, 508–513.

Fox, J. and Weisberg, S. (2011). An {R} Companion to Applied Regression. Thousand Oaks,

CA, USA: Sage.

French, S.S., Moore, M.C. and Demas, G.E. (2009). Ecological immunology: the organism in

context. Integr. Comp. Biol. 49, 246-253.

French, S.S. and Neuman-Lee, L.A. (2012). Improved ex vivo method for microbiocidal activity

across vertebrate species. Biology Open 1, 482-487.

Fuller, B.T., Fuller, J.L., Sage, N.E., Harris, D.A., O'Connell, T.C. and Hedges, R.E. (2004).

Nitrogen balance and δ15N: why you're not what you eat during pregnancy. Rapid

Commun. Mass Spectrom. 18, 2889-2896.

Fuller, B.T., Fuller, J.L., Sage, N.E., Harris, D.A., O'Connell, T.C. and Hedges, R.E. (2005).

Nitrogen balance and δ15N: why you're not what you eat during nutritional stress. Rapid

Commun. Mass Spectrom. 19, 2497-2506.

Gaye-Siessegger, J., Focken, U., Muetzel, S., Abel, H. and Becker, K. (2004). Feeding level

and individual metabolic rate affect d13C and d15N values in carp: implications for food

web studies. Oecologia 138, 175-183. 77

Gómez‐Campos, E., Borrell, A. and Aguilar, A. (2011). Nitrogen and carbon stable isotopes do

not reflect nutritional condition in the striped dolphin. Rapid Commun. Mass Spectrom.

25, 1343-1347.

Greenwald, O. and Kanter, M.E. (1979). The effects of temperature and behavioral

thermoregulation on digestive efficiency and rate in corn snakes (Elaphe guttata guttata).

Physiol. Zool. 52, 398-408.

Gustine, D.D., Barboza, P.S., Adams, L.G., Farnell, R.G. and Parker, K.L. (2011). An

isotopic approach to measuring nitrogen balance in caribou. The Journal of Wildlife

Management 75, 178-188.

Hatch, K.A. (2012). The use and application of stable isotope analysis to the study of starvation,

fasting, and nutritional stress in animals. In Comparative Physiology of Fasting,

Starvation, and Food Limitation (ed. M. D. McCue), pp. 337-364. Berlin: Springer-

Verlag.

Hertz, E., Trudel, M., Cox, M.K. and Mazumder, A. (2015). Effects of fasting and nutritional

restriction on the isotopic ratios of nitrogen and carbon: a meta‐analysis. Ecology and

evolution 5, 4829-4839.

Kempster, B., Zanette, L., Longstaffe, F.J., MacDougall-Shackleton, S.A., Wingfield, J.C.

and Clinchy, M. (2007). Do stable isotopes reflect nutritional stress? Results from a

laboratory experiment on song sparrows. Oecologia 151, 365-371.

Khalil, F. (1951). Excretion in reptiles. 4. Nitrogenous constituents of the excreta of lizards. J.

Biol. Chem. 189, 443-445. 78

Labocha, M.K. and Hayes, J.P. (2012). Morphometric indices of body condition in birds: a

review. Journal of Ornithology 153, 1-22.

Lardner, B., Rodda, G.H., Yackel Adams, A.A., Savidge, J.A. and Reed, R.N. (2015).

Detection rates of geckos in visual surveys: turning confounding variables into useful

knowledge. J. Herpetol. 49, 522-532.

Lattanzio, M. and Miles, D. (2016). Stable carbon and nitrogen isotope discrimination and

turnover in a small-bodied insectivorous lizard. Isotopes Environ. Health Stud.

10.1080/10256016.2016.1154854.

Lochmiller, R.L. and Deerenberg, C. (2000). Trade‐offs in evolutionary immunology: just what

is the cost of immunity? Oikos 88, 87-98.

Martinez del Rio, C. and Wolf, B. (2005). Mass-balance models for animal isotopic ecology. In

Physiological and Ecological Adaptations to Feeding in Vertebrates (eds. J. M. Starck

and T. Wang), pp. 141-174. Enfield, New Hampshire: Science Publishers, Inc.

McCue, M., Guzman, R., Passement, C. and Davidowitz, G. (2015). How and when do insects

rely on endogenous protein and lipid resources during lethal bouts of starvation? A new

application for 13C-breath testing. PLoS ONE 10, e0140053.

McCue, M.D. (2007). Western diamondback rattlesnakes demonstrate physiological and

biochemical strategies for tolerating prolonged starvation. Physiol. Biochem. Zool. 80,

25-34.

McCue, M.D. (2010). Starvation physiology: reviewing the different strategies animals use to

survive a common challenge. Comparative Biochemistry and Physiology-Part A:

Molecular & Integrative Physiology 156, 1-18. 79

McCue, M.D., Amaya, J.A., Yang, A.S., Erhardt, E.B., Wolf, B.O. and Hanson, D.T. (2013).

Targeted 13C enrichment of lipid and protein pools in the body reveals circadian changes

in oxidative fuel mixture during prolonged fasting: a case study using Japanese quail.

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology

166, 546-554.

McCue, M.D. and Pollock, E.D. (2008). Stable isotopes may provide evidence for starvation in

reptiles. Rapid Commun. Mass Spectrom. 22, 2307-2314.

Milanovich, J.R. and Maerz, J.C. (2013). Realistic fasting does not affect stable isotope levels

of a metabolically efficient salamander. J. Herpetol. 47, 544-548.

Muggeo, V.M.R. (2008). Segmented: an R package to fit regression models with broken-line

relationships. R News 8, 20-25.

Neuman-Lee, L.A., Stokes, A.N., Greenfield, S., Hopkins, G.R., Brodie, E.D. and French,

S.S. (2015). The role of corticosterone and toxicity in the antipredator behavior of the

Rough-skinned Newt (Taricha granulosa). Gen. Comp. Endocrinol. 213, 59-64.

Pinheiro, J.C. and Bates, D.M. (2000). Mixed-effects models in S and S-PLUS. New York,

New York: Springer-Verlag.

Plowright, R.K., Field, H.E., Smith, C., Divljan, A., Palmer, C., Tabor, G., Daszak, P. and

Foley, J.E. (2008). Reproduction and nutritional stress are risk factors for Hendra virus

infection in little red flying foxes (Pteropus scapulatus). Proceedings of the Royal

Society of London B: Biological Sciences 275, 861-869.

Post, D.M. (2002). Using stable isotopes to estimate trophic position: models, methods, and

assumptions. Ecology 83, 703-718. 80

R Core Team. (2015). R: A language and environment for statistical computing. Vienna,

Austria: R Foundation for Statistical Computing.

Rodda, G.H., Dean-Bradley, K., Campbell, E.W., Fritts, T.H., Lardner, B., Yackel Adams,

A.A. and Reed, R.N. (2015). Stability of detectability over 17 years at a single site and

other lizard detection comparisons from Guam. J. Herpetol. 49, 513-521.

Schmider, E., Ziegler, M., Danay, E., Beyer, L. and Bühner, M. (2010). Is it really robust?

Reinvestigating the robustness of ANOVA against violations of the normal distribution

assumption. Methodology 6, 147-151.

Sinervo, B. and Lively, C.M. (1996). The rock-paper-scissors game and the evolution of

alternative male strategies. Nature 380, 240-243.

Stearns, S. (1992). The Evolution of Life Histories. Oxford, UK: Oxford University Press.

Steele, K. and Daniel, R. (1978). Fractionation of nitrogen isotopes by animals: a further

complication to the use of variations in the natural abundance of 15N for tracer studies. J.

Agric. Sci. 90, 7-9.

White, T.C. (2012). The inadequate environment: nitrogen and the abundance of animals. Berlin,

Germany: Springer-Verlag.

Wickham, H. (2009). ggplot2: Elegant Graphics for Data Analysis. New York: Springer-Verlag.

Zera, A.J. and Harshman, L.G. (2001). The physiology of life history trade-offs in animals.

Annu. Rev. Ecol. Syst. 32, 95-126.

CHAPTER 4

STABLE ISOTOPE TRACERS REVEAL A TRADE-OFF BETWEEN

REPRODUCTION AND IMMUNITY IN A REPTILE WITH COMPETING NEEDS

Trade-offs between the reproductive and immune systems are predicted when resources are limited, but are difficult to measure. We used 15N labeled amino acids to directly compare protein allocation by lizards to eggs and healing wounds. We showed that these two demands compete for the same resource, and that the ratio of protein allocation between them was related to resource availability, corticosterone, and immune function. We found that reproductive stage had important effects on the absolute and relative allocation of protein to self-maintenance.

Introduction

Energy allocation to competing life-history demands is highly plastic (Ford & Seigel

1989; Warne et al. 2012). In particular, trade-offs between the reproductive and immune systems are predicted to be quite strong when resources are limited (French, DeNardo & Moore 2007;

Stahlschmidt et al. 2013), although the magnitude and direction of the trade-off can be influenced by many factors (French & Moore 2007; Skibiel, Speakman & Hood 2013; Peck, Costa &

Crocker 2015). Studies attempting to measure these trade-offs are often hindered because of the complexity of the physiological systems involved and the difficulty of interpreting measurements that lack a common unit. To date, all such studies have compared measurements of indirect outcomes of resource competition (e.g., hormone levels, immune function, oxidative stress, body mass or condition change; Dickens & Romero 2013; Husak, Ferguson & Lovern 2016), but no study has made direct measurements of resource competition (Zera & Harshman 2001). 82

Amino acids are the building blocks of proteins. It is essential that many animals obtain certain amino acids from their diets, as they cannot be synthesized de novo by most species. As such, essential amino acids represent a limiting resource for most animals, including vertebrates.

Methods for labeling and tracing amino acids using stable isotopes are developing rapidly

(McCue 2011), but to date most applications have been limited to in vitro protein expression assays (e.g., Ong et al. 2002). A few in vivo studies have examined the effects of starvation on metabolism (McCue et al. 2013), but none have traced amino acids to different, competing ultimate fates within the bodies of living organisms. McCue et al. (2013) measured a 13C label in breath CO2 to show that quail spared protein from oxidation during the day but not at night, and

McCue, Guzman and Passement (2015) used the same technique to describe how and when pythons use the proteins in their meals. Recently, Brace, Sheikali and Martin (2015) used a 13C label to trace the competing fates of fatty acids in Brown Anoles (Anolis sagrei), but no study has traced amino acids and compared their ultimate fates. Unlike carbon, nitrogen is not lost in the breath of heterotrophic animals, so studying the fates of essential amino acids can help us understand trade-offs where protein is the limiting resource.

Here, we used 15N labeled amino acids to directly compare the protein allocation of lizards that varied in resource availability and reproductive stage with indirect physiological outcomes. During vitellogenesis, lizards allocate large amounts of protein to their eggs

(Thompson et al. 2001; Shawsuan, Sasuang & Thammasirirak 2007), and studies of other animals suggest that wound healing has significant protein costs (Zhang et al. 1998; Lochmiller &

Deerenberg 2000; MacKay & Miller 2003; Zhang, Chinkes & Wolfe 2004). Because these two competing demands share a common resource (amino acids), we used a stable isotope label to trace amino acids through the bodies of resource-limited lizards that were undergoing both reproductive and immune costs. We predicted that we would observe enrichment in both eggs and 83 scabs, that lizards with higher amounts of 15N in their eggs would have lower amounts in scabs, and that lizards that ate less would exhibit a stronger co-relationship between these two measurements of protein investment.

Methods

Experimental Design

We collected 56 female Side-blotched Lizards (Uta stansburiana) from a single site near

St. George, Utah (37.06220,-113.59535; WGS 84), during the second week of May 2013. We assumed that all lizards were approximately one year old, as U. stansburiana at this location rarely live longer than one and a half years (Lucas & French 2012). An acclimation period of 48 hours preceded the start of the experiment, during which time we measured the snout-vent length

(SVL, in mm), mass (in g), and all follicle lengths of each lizard using ultrasound (MicroMaxx,

SonoSite, Bothell, Washington, USA). We selected lizards that were as similar as possible to one another in size and reproductive stage from a larger population of captured individuals. Lizards were housed individually in 30x45x15 cm plastic terraria, each with a water bowl, plastic hide, and newspaper substrate. The racks of terraria were kept in a climate-controlled room (25.2°C,

20% humidity) and each terrarium had an ultraviolet (UVA/UVB) light on a 12:12 hour cycle.

We divided lizards into four groups in a 2x2 design. Half (28) of the lizards were randomly assigned to a food restriction treatment (<0.1 g of farm-raised crickets [Acheta domestica, Fluker’s®, Port Allen, Louisiana, USA] every other day), and the other half were fed ad libitum (0.2-0.7 g of crickets every other day) for a period of 7 days. We also included a handling stress treatment, wherein we removed half of the lizards in each food treatment group from their cages each day and placed them into a cloth bag for one hour. On the first day of the experiment, we used sterile 3.5 mm biopsy punches to remove a circle of skin from each lizard’s 84 back after anesthetizing the lizards using surface-induced hypothermia (French, Matt & Moore

2006) and collected the skin punch from each lizard biopsy to use as a baseline for stable isotope values. We also measured the C:N ratio of the skin biopsy in an attempt to obtain an index of lipid stores.

On the first day, we injected each lizard with a small amount (20 mg/kg) of 99% 15N stable isotope in the form of leucine, an essential amino acid. In doing so, we labeled the dietary protein source pool. We monitored flow out of the source pool into two sinks: the protein deposited in newly-formed tissue (scab, a measure of allocation to self-maintenance) and the protein deposited into eggs (a measure of allocation to reproduction). We also measured excreted nitrogen in uric acid and feces, which we considered sink pools that were not of particular interest, in order to account for variation in assimilation of our label. At the end of the experiment, we anesthetized each lizard and collected the scab that had formed over its biopsy, as well as a single egg via laparotomy. Ultimately, we removed from the analysis two lizards that died partway through the experiment and four lizards that laid eggs partway through the experiment.

We performed a pilot experiment to examine within-individual variation in egg 15N uptake and fecal and uric acid 15N loss. Because within-individual variation in eggs, feces, and

UA pellets was low (Table 1), we justified collecting just a single egg and combining feces and uric acid pellets across the whole experiment. This allowed us to reduce the number of samples we needed to run by almost an order of magnitude.

Stable Isotope Sample Preparation

We measured the nitrogen stable isotope ratios of all samples collected. Each sample was dried to a constant mass, homogenized, and between 0.5 and 2.0 mg of each sample were weighed into a tin capsule (5x9mm; Costech Analytical, Valencia, California, USA), folded, and analyzed for total N and δ15N by continuous-flow direct-combustion and isotope ratio mass spectrometry using a Carlo Erba CHN Elemental Analyzer [model NA1500] coupled to a Thermo

Finnigan Delta V Isotope Ratio Mass Spectrometer via Thermo Finnigan Conflo III Interface at the University of Georgia Analytical Chemistry Laboratory. The enrichment values were corrected for individual egg mass by dividing them by the dry mass of the entire egg.

Other Physiological Endpoints

The rate of wound healing was measured by serially photographing the biopsy site and analyzing the percent healed using ImageJ (Rasband 1997-2014; French, Matt & Moore 2006).

Using blood plasma collected just prior to euthanasia and within three minutes of capture and handling, we measured 1) plasma bactericidal capability using the method of French and

Neuman-Lee (2012), 2) corticosterone using a radioimmunoassay (Neuman-Lee et al. 2015), and

3) a marker of oxidative stress using a d-ROM test kit from Diacron International (Grosseto,

Italy).

Statistical Analyses

We used R (R Core Team 2015, version 3.2.2) to construct linear models (function ‘lm’) to examine the effects of nutrient limitation, handling stress, and stage of pregnancy on isotopic 86 enrichment values and other measures of physiological stress. We evaluated the effects on three response variables: the enrichment of the egg, the enrichment of the scab, and the ratio of the enrichment of the two. We used continuous covariates to control for differences in isotopic enrichment, assimilation efficiency, C:N ratio, and follicle size at the start of the experiment. We verified assumptions of least-squares regression where appropriate (Williams, Grajales &

Kurkiewicz 2013).

Results

The best-fitting model (R2 = 0.21) revealed significant positive effects of the quantity of food eaten (p = 0.0283) and the size of the follicles at the beginning of the experiment (p =

0.0193) on the allocation of protein to wound healing. A continuous covariate accounting for variation in assimilation efficiency (measured from the enrichment of excreted nitrogen over the course of the experiment) was marginally significant (p = 0.0787). The best-fitting model (R2 =

0.19) revealed significant positive effects of the size of the follicles at the beginning of the experiment (p = 0.00752) and the change in follicle size over the course of the experiment (p =

0.04355) on the egg to scab ratio. No model contained support for an effect of handling stress on any response variable.

The estimated mass of the follicles at the beginning of the experiment varied from 0.018 g to 0.626 g. The absolute mass-corrected enrichment of eggs was not related to treatments (p ≥

0.45), and was marginally related to the initial mass of the clutch (p = 0.064). Lizards whose follicles were larger at the beginning of the experiment allocated more protein to healing their wounds (Fig. 4-1), whereas lizards whose follicles were smaller at the beginning of the experiment had a higher egg:scab nitrogen isotope ratio (i.e., they allocated more protein to growing their eggs relative to healing their wound; Fig. 4-2). 87

There were no significant relationships between nutrient limitation, handling stress, or stage of pregnancy and any of the other physiological parameters we measured. Lizards that allocated more protein to healing their wounds had significantly higher concentrations of corticosterone in their blood, both in terms of the absolute quantity of protein allocated (p =

0.0305; Fig. 4-3A) and its ratio with protein allocated to eggs (p = 0.0377; Fig. 4-3B). Lizards that allocated more protein to healing their wounds had significantly higher plasma bactericidal ability (p = 0.0305; Fig. 4-4), an effect that was modulated by both the quantity of food consumed

(p = 0.0402) and the stage of pregnancy at the beginning of the experiment (p = 0.009). We did not detect any relationships between protein allocation and measures of oxidative stress or wound healing rate. The C:N ratio of the skin biopsy was not a significant predictor or modifier of any relationship.

Overall, model fit was low, suggesting that unmeasured variation influenced response variables. We attempted to measure individual variation in assimilation of the label, reproductive stage, food intake, and stored energy, and to control for other sources of individual variation by selecting lizards that were as similar as possible at the beginning of the experiment.

Tables and Figures

Table 4-1. Summary statistics for validation showing low inter-individual variation in APE N of lizard eggs, feces, and uric acid.

Eggs Feces Uric Acid N Mean Min Max N Mean Min Max N Mean Min Max Mean 0.3702 0.3632 0.4021 0.3795 0.3677 0.4175 0.4244 0.3675 0.5746 SD 0.0011 1.06e-5 0.0159 0.0110 0.0003 0.0325 0.0771 0.0003 0.3186 46 41 42 SE 0.0006 7.54e-6 0.0071 0.0056 0.0001 0.0195 0.0358 0.0002 0.1586 CV 0.0029 2.87e-5 0.0396 0.0283 0.0007 0.0799 0.1642 0.0009 0.5544

Figure 4-1. Relationship between quantity of food consumed (g) and amount of nitrogen-15 found in scabs (APE N). We used ultrasound data to classify lizards into two reproductive stages, early

(follicles <50 mm in diameter) and mid (follicles >50 mm in diameter). 90

Figure 4-2. Relationship between size of follicles at the beginning of the experiment (g) and ratio of nitrogen-15 found in eggs to that found in scabs. Higher values of the ratio indicate relatively more investment of protein into eggs. 91

Figure 4-3. A) Relationship between amount of nitrogen-15 found in scabs and corticosterone; B)

Relationship between ratio of nitrogen-15 found in eggs to that found in scabs and corticosterone. 92

Figure 4-4. Relationship between amount of nitrogen-15 found in scabs and bactericidal ability.

Discussion

We found that resource limitation and reproductive stage had important effects on the absolute and relative allocation of protein to self-maintenance, providing evidence for a trade-off between the reproductive and immune systems (French, DeNardo & Moore 2007). Both reproduction and self-maintenance are critical processes that require substantial resource investment. However, we found evidence that higher resource requirements during early vitellogenesis shifted protein into eggs and away from wound healing, as has been suggested in studies of other lizards using indirect measurements (French & Moore 2007). The effect of reproductive stage outweighed the effect of resource limitation in our study (Fig. 4-2).

Our direct measurements of protein allocation to wound healing were related to two indirect measures of self-maintenance (Fig. 4-3). First, higher plasma bactericidal ability was 93 observed in lizards that allocated more leucine to their scabs, which could be interpreted as immune prioritization (relative to reproduction or other needs) or as immune activation in response to the stressor of the wound itself. These are not mutually exclusive, and although evidence for immune activation in reptiles is mixed (Zimmerman, Vogel & Bowden 2010), we are beginning to understand that the reptile immune system can be as sophisticated as those of endothermic vertebrates under the right conditions (Neuman-Lee & French 2014). Additionally, higher circulating baseline concentrations of corticosterone in lizards that allocated more leucine to their scabs could be interpreted as a mediator of the mobilization of energy away from other physiological processes, a response to the stress of healing a wound, or both (Sapolsky, Romero

& Munck 2000; Wingfield & Romero 2011), although the meaning of glucocorticoid hormone elevation is not consistent (Dickens & Romero 2013). Strong effects of corticosterone on maternal and offspring phenotype suggest that maternal corticosterone alters energy allocation among reproduction and other physiological processes (Itonaga, Jones & Wapstra 2012a).

We have only a rough understanding of the relative cost of different stages of reproduction in lizards (De Stasio et al. 1999). However, in our experiment, early vitellogenesis appeared to be the most costly stage. Other studies have shown the importance of stored energy to fueling reproduction, suggesting that lizard egg protein is derived almost equally from both income and capital resources (Warner et al. 2008), something that we either could not adequately assess or did not influence our results. In U. stansburiana, yolk deposition takes about 38 days

(Tinkle 1967), so the majority of our lizards had not yet completed vitellogenesis by the time they began shifting resource allocation towards healing their wounds. Female lizards late in reproduction sometimes withhold resources from their eggs if their own resources are limited

(Itonaga, Jones & Wapstra 2012a). 94

Viviparous lizards (Itonaga, Jones & Wapstra 2012b; Itonaga, Jones & Wapstra 2012a) and snakes (Van Dyke & Beaupre 2012) are capable of making flexible reproductive decisions.

Here, we have shown that oviparous reproductive female lizards can also modulate their allocation of resources between self-maintenance and reproduction. Because the lizards in our study were collected early in the reproductive season and would likely have produced two or more additional clutches of eggs in their lives (Tinkle 1967), they may have allocated more resources to self-maintenance during mid-vitellogenesis than they would have if they had been closer to senescence. These trade-offs are likely influenced by future effects of maternal resource availability on offspring fitness (Mousseau & Fox 1998; Flores et al. 2013).

Trade-offs are difficult to measure (Dickens & Romero 2013), and no one parameter is likely to tell us everything we want to know about the state of an animal. The primary strength of our experiment is that tracing essential amino acids through the bodies of organisms can be used to directly measure allocation of resources to both reproduction and self-maintenance using a common currency (McCue 2011). Nitrogen has fewer sinks than carbon in animals, because it is not breathed out, and unlike fatty acids, many amino acids cannot be synthesized de novo in most animals, including lizards, suggesting that studies tracing essential amino acids may have great utility in understanding trade-offs. 95

References

Brace, A.J., Sheikali, S. & Martin, L.B. (2015) Highway to the danger zone: exposure-dependent

costs of immunity in a vertebrate ectotherm. Functional Ecology, 29, 924-930.

De Stasio, R., Borrelli, L., Kille, P., Parisi, E. & Filosa, S. (1999) Isolation, characterization and

molecular cloning of cathepsin D from lizard ovary: changes in enzyme activity and

mRNA expression throughout ovarian cycle. Molecular Reproduction and Development,

52, 126-134.

Dickens, M.J. & Romero, L.M. (2013) A consensus endocrine profile for chronically stressed

wild animals does not exist. General and Comparative Endocrinology, 191, 177-189.

Flores, E.E., Stevens, M., Moore, A.J. & Blount, J.D. (2013) Diet, development and the

optimization of warning signals in post‐metamorphic green and black poison frogs.

Functional Ecology, 27, 816-829.

Ford, N.B. & Seigel, R.A. (1989) Phenotypic plasticity in reproductive traits: evidence from a

viviparous snake. Ecology, 70, 1768-1774.

French, S.S., DeNardo, D.F. & Moore, M.C. (2007) Trade-offs between the reproductive and

immune systems: Facultative responses to resources or obligate responses to

reproduction? The American Naturalist, 170, 79-89.

French, S.S., Matt, K. & Moore, M.C. (2006) The effects of stress on wound healing in male tree

lizards (Urosaurus ornatus). General and Comparative Endocrinology, 145, 128-132.

French, S.S. & Moore, M.C. (2007) Immune function varies with reproductive stage and context

in female and male tree lizards, Urosaurus ornatus. General and Comparative

Endocrinology, 155, 148-156. 96

French, S.S. & Neuman-Lee, L.A. (2012) Improved ex vivo method for microbiocidal activity

across vertebrate species. Biology Open, 1, 482-487.

Husak, J.F., Ferguson, H.A. & Lovern, M.B. (2016) Trade-offs among locomotor performance,

reproduction, and immunity in lizards. Functional Ecology, 10.1111/1365-2435.12653.

Itonaga, K., Jones, S.M. & Wapstra, E. (2012a) Do gravid females become selfish? Female

allocation of energy during gestation. Physiological and Biochemical Zoology, 85, 231-

242.

Itonaga, K., Jones, S.M. & Wapstra, E. (2012b) Effects of maternal basking and food quantity

during gestation provide evidence for the selective advantage of matrotrophy in a

viviparous lizard. PLoS ONE, 7, e41835.

Lochmiller, R.L. & Deerenberg, C. (2000) Trade‐offs in evolutionary immunology: just what is

the cost of immunity? Oikos, 88, 87-98.

Lucas, L. & French, S.S. (2012) Stress-induced tradeoffs in a free-living lizard across a variable

landscape: Consequences for individuals and populations. PLoS ONE, 7, e49895.

MacKay, D.J. & Miller, A.L. (2003) Nutritional support for wound healing. Alternative Medicine

Review, 8, 359-378.

McCue, M.D. (2011) Tracking the oxidative and nonoxidative fates of isotopically labeled

nutrients in animals. Bioscience, 61, 217–230.

McCue, M.D., Amaya, J.A., Yang, A.S., Erhardt, E.B., Wolf, B.O. & Hanson, D.T. (2013)

Targeted 13C enrichment of lipid and protein pools in the body reveals circadian changes

in oxidative fuel mixture during prolonged fasting: a case study using Japanese quail. 97

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology,

166, 546-554.

McCue, M.D., Guzman, R.M. & Passement, C.A. (2015) Digesting pythons quickly oxidize the

proteins in their meals and save the lipids for later. The Journal of Experimental Biology,

215, 2089-2096.

Mousseau, T.A. & Fox, C.W. (1998) The adaptive significance of maternal effects. Trends in

Ecology & Evolution, 13, 403-407.

Neuman-Lee, L.A. & French, S.S. (2014) Wound healing reduces stress-induced immune

changes: evidence for immune prioritization in the side-blotched lizard. Journal of

Comparative Physiology B, 184, 623-629.

Neuman-Lee, L.A., Stokes, A.N., Greenfield, S., Hopkins, G.R., Brodie, E.D. & French, S.S.

(2015) The role of corticosterone and toxicity in the antipredator behavior of the Rough-

skinned Newt (Taricha granulosa). General and Comparative Endocrinology, 213, 59-

64.

Ong, S.-E., Blagoev, B., Kratchmarova, I., Kristensen, D.B., Steen, H., Pandey, A. & Mann, M.

(2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and

accurate approach to expression proteomics. Molecular & Cellular Proteomics, 1, 376-

386.

Peck, H.E., Costa, D.P. & Crocker, D.E. (2015) Body reserves influence allocation to immune

responses in capital breeding female northern elephant seals. Functional Ecology.

R Core Team (2015) R: A language and environment for statistical computing. R Foundation for

Statistical Computing, Vienna, Austria. 98

Rasband, W.S. (1997-2014) ImageJ. U. S. National Institutes of Health, Bethesda, Maryland,

USA.

Sapolsky, R.M., Romero, L.M. & Munck, A.U. (2000) How do glucocorticoids influence stress

responses? Integrating permissive, suppressive, stimulatory, and preparative actions.

Endocrine Reviews, 21, 55-89.

Shawsuan, L., Sasuang, S. & Thammasirirak, S. (2007) Comparative study of reptile egg white

proteins by 2D-PAGE. KMITL Science and Technology Journal, 7, 127-136.

Skibiel, A.L., Speakman, J.R. & Hood, W.R. (2013) Testing the predictions of energy allocation

decisions in the evolution of life‐history trade‐offs. Functional Ecology, 27, 1382-1391.

Stahlschmidt, Z.R., Rollinson, N., Acker, M. & Adamo, S.A. (2013) Are all eggs created equal?

Food availability and the fitness trade‐off between reproduction and immunity.

Functional Ecology, 27, 800-806.

Thompson, M.B., Speake, B.K., Russell, K.J. & McCartney, R.J. (2001) Utilisation of lipids,

protein, ions and energy during embryonic development of Australian oviparous skinks in

the genus Lampropholis. Comparative Biochemistry and Physiology Part A: Molecular

& Integrative Physiology, 129, 313-326.

Tinkle, D.W. (1967) The life and demography of the Side-blotched Lizard, Uta stansburiana.

Miscellaneous Publications of the Museum of Zoology University of Michigan, 132, 1-

182.

Van Dyke, J.U. & Beaupre, S.J. (2012) Stable isotope tracer reveals that viviparous snakes

transport amino acids to offspring during gestation. The Journal of Experimental Biology,

215, 760-765. 99

Warne, R.W., Gilman, C.A., Garcia, D.A. & Wolf, B.O. (2012) Capital breeding and allocation to

life-history demands are highly plastic in lizards. The American Naturalist, 180, 130.

Warner, D.A., Bonnet, X., Hobson, K.A. & Shine, R. (2008) Lizards combine stored energy and

recently acquired nutrients flexibly to fuel reproduction. Journal of Animal Ecology, 77,

1242-1249.

Williams, M.N., Grajales, C.A.G. & Kurkiewicz, D. (2013) Assumptions of multiple regression:

correcting two misconceptions. Practical Assessment, Research & Evaluation, 18, 2.

Wingfield, J. & Romero, L. (2001) Adrenocortical responses to stress and their modulation in

free-living vertebrates. Handbook of Physiology; Section 7: The Endocrine System;

Volume IV: Coping with the Environment: Neural and Endocrine Mechanisms (eds B.S.

McEwen & H.M. Goodman), pp. 211-234. Oxford University Press, New York.

Zera, A.J. & Harshman, L.G. (2001) The physiology of life history trade-offs in animals. Annual

Review of Ecology and Systematics, 32, 95-126.

Zhang, X.-j., Chinkes, D.L., Doyle, D. & Wolfe, R.R. (1998) Metabolism of skin and muscle

protein is regulated differently in response to nutrition. American Journal of Physiology-

Endocrinology And Metabolism, 274, E484-E492.

Zhang, X.-j., Chinkes, D.L. & Wolfe, R.R. (2004) Leucine supplementation has an anabolic

effect on proteins in rabbit skin wound and muscle. The Journal of Nutrition, 134, 3313-

3318.

Zimmerman, L., Vogel, L. & Bowden, R. (2010) Understanding the vertebrate immune system:

insights from the reptilian perspective. The Journal of Experimental Biology, 213, 661-

671. 100

CHAPTER 5

ASSESSING THE PROTEIN AND METABOLIC COSTS OF A TRADE-OFF

BETWEEN REPRODUCTION AND IMMUNITY

Animals must allocate limited resources to competing demands. A central trade-off between reproductive and immune costs underlies much of life history theory. We wounded gravid female lizards and manipulated their levels of follicle-stimulating hormone (FSH), an endocrine mechanism controlling allocation of protein resources to reproduction. Using a stable isotope label, we traced amino acids to their ultimate fates within their bodies. We measured both the direct allocation of protein to tissues involved in these activities as well as the combined oxygen consumption costs of these demands. We found that there was a significant increase in the

15N content of eggs when FSH was applied, and that the application of FSH reversed the direction of the relationship between egg and scab 15N content. There was also a significant interaction between the effect of FSH treatment and isotope ratio on the metabolic rate of lizards, bolstering evidence for the role of FSH in mediating the costs and benefits of a trade-off as these lizards underwent both reproductive and immune costs.

Introduction

Differential investments between self-replicating and self-maintaining processes have been of interest to life history theorists for many years (Moreau 1944, Lack 1947, Skutch 1949), and this discipline has grown in scope and complexity with the advent of new mathematical models (Williams 1966, Pianka 1970, Mesquita et al. 2016). Similarly, as new techniques to assess physiological parameters have been developed, new relationships among hormones

(French et al. 2007c, Lancaster et al. 2008), the immune system (Lochmiller and Deerenberg 101

2000a, Norris and Evans 2000), and other factors, including oxidative stress (Monaghan et al.

2009), have emerged. However, the underlying mechanisms that drive trade-offs in one direction or another, often depending on internal as well as external cues, still represent critical gaps in our knowledge (Demas et al. 2012).

Reproduction has both metabolic (Angilletta and Sears 2000, Nilsson and Råberg 2001) and protein (Robbins 1981, Lourdais et al. 2004) costs. Increased investment into the reproductive system can suppress other competing processes, such as immunity (Nordling et al.

1998, Adamo et al. 2001, Harshman and Zera 2007), and reciprocally, immunological investment can decrease reproductive effort (Råberg et al. 2000), especially during periods of resource limitation (Zera and Harshman 2001, French et al. 2007a). Because immune function also has energetic (Demas et al. 2012) and protein (Lochmiller and Deerenberg 2000b, Moret and Schmid-

Hempel 2000, Lee et al. 2006) costs, but is important to survival (Hanssen et al. 2004), understanding these trade-offs is key to understanding how organisms maximize their lifetime fitness (Stearns 1992).

In addition to external pressures like energetic and nutritional resource availability, internal cues such as gonadotropins (McGlothlin et al. 2007) and glucocorticoid hormones

(French et al. 2007c, Romero and Wikelski 2010) can influence trade-offs. What is not well understood is how hormones mediate trade-offs between energy (metabolic rate) and matter (the macronutrients composing the organism’s body; Groothuis and Schwabl 2008). Gonadotropins such as follicle-stimulating hormone (FSH) are prime candidates for experimentation given their role in reproductive investment, which is to recruit follicles during early vitellogenesis (Licht et al. 1977, Hamilton and Armstrong 1991, Jones and Swain 2000) and stimulate the in vitro uptake of the protein vitellogenin into ovarian follicles, where it is incorporated into yolk (Ferguson

1966, Callard and Zeigler 1970, Ho et al. 1982, Limatola and Filosa 1989). Sinervo and Licht 102

(1991a) showed that experimentally increasing FSH levels increased clutch size in Side-blotched

Lizards (Uta stansburiana). Furthermore, French et al. (2007b) showed that FSH injections increased follicle size but decreased healing rates in Ornate Tree Lizards (Urosaurus ornatus).

What is still unknown are the material (nutritional) and energetic costs of this apparent trade-off.

Methods for measuring use of both energy and matter in living organisms are well- developed. Advances in high-precision respirometry have allowed us to measure metabolic rates in fine detail (Lighton 2008). Using amino acids labeled with stable isotopes in vivo to measure allocation of a limiting resource (e.g., a macronutrient) is an emerging technique that has several advantages over more indirect techniques for assessing the allocation of these resources (McCue

2011, McCue et al. 2013, Brace et al. 2015). Both wound healing and reproduction have significant protein costs that put them in competition for common resources such as essential amino acids. A few experiments have measured the effects of starvation on metabolism (McCue et al. 2013), but only one has traced amino acids to different, competing ultimate fates within the bodies of living organisms (Durso et al. In review). Because trade-offs impose limits on energy expenditure, combining respirometry with direct measurements of protein allocation has the potential to quantify resource trade-offs with greater clarity and precision than either technique alone.

In this experiment, we used a stable isotope label to trace amino acids through the bodies of female Side-blotched Lizards (Uta stansburiana Baird and Girard, 1852) that were undergoing both reproductive and immune challenges, and measured both the direct allocation of protein to tissues involved in these activities and the combined oxygen consumption costs of these demands.

We also manipulated the level of follicle-stimulating hormone (FSH), which initiates and controls follicular growth and vitellogenesis (Licht 1979, Ho et al. 1982, Ho 1987), in half the lizards in order to better understand one of the mechanisms controlling reproductive trade-offs. We 103 hypothesized that lizards with high reproductive demand would have diminished allocation of resources to immunity and vice-versa. We also expected to see FSH increase allocation of resources to reproduction relative to controls.

Methods

Animals and Overview

We measured oxygen consumption and stable isotope deposition in response to two treatments, an immune challenge (cutaneous biopsy) and vitellogenesis in response to an injection of follicle-stimulating hormone in female U. stansburiana. Forty adult lizards averaging 46.1 ±

0.4 mm in snout-vent length and 2.65 ± 0.08 g in mass were captured via noosing from

Washington County, Utah, USA in May 2014. The lizards were taken to Arizona State University where they were individually housed in 40x15x13 cm plastic terraria with paper substrate. The terraria were held at a 12L:12D photoperiod at 36° C inside environmental chambers. Lizards were fed crickets (Fluker Farms, Port Allen, Louisiana, USA) daily except for the days prior to and of metabolic measurements. We weighed and counted crickets offered to lizards and removed, counted, and weighed crickets the following day to quantify food intake. The internal walls of the terraria were sprayed daily to provide water for the lizards.

We quantified the initial number and length of follicles via ultrasound on Day 1 of the experiment and randomly assigned subjects to one of four treatments (FSH, biopsy, both, or control) with a random number generator. We ultrasounded the lizards again on Days 5 and 10 to quantify follicular growth during the study. On Day 3, we administered 20 µg ovine FSH (Sigma-

Aldrich) suspended in 20 µl saline to stimulate vitellogenesis in the FSH-treatment lizards, and 20

µl vehicle (saline only) to the control group following French et al. (2007b). We also injected 20

μl of a 50:50 mixture of 14N and 15N-leucine (dissolved in Ringer’s solution at a concentration of 104

937.5 mg/ml) into all subjects. These injections were repeated to ensure follicular investment and isotope enrichment on Days 5 and 7. On Day 4, we measured initial oxygen consumption via closed respirometry and repeated metabolic trials on Days 6 and 8 to quantify changes in metabolic rate in response to treatment. On Day 5, we gave half of the lizards an immune challenge (cutaneous biopsy) and took a digital image of the wound. On Day 10, we took another digital image to quantify wound healing, and blood was collected from the retro-orbital sinus of all animals. Blood samples were centrifuged to isolate plasma, which was then frozen and stored at -80° C until hormone and immune assays were performed. Lizards were then transported to

Utah State University where they were euthanized and dissected on Day 14. A timeline of procedures can be found in Table 1.

Cutaneous Biopsy and Measurements

We simulated wounding of lizards via a cutaneous biopsy. After lizards were anesthetized using isoflurane gas, a biopsy tool (Miltex Instrument Company, York,

Pennsylvania, USA) was gently twisted against the dorsal skin surface anterior to the tail, creating a 3.5 mm circular wound. The piece of skin was then removed with forceps. Immediately after, the wound was photographed next to a ruler for scale. The wounds were also photographed at the end of the study, four days later. The wound images were analyzed for wound area using ImageJ

(Rasband 1997-2014) such that the investigator was blind to the treatment of the animal. Lizards that did not receive a cutaneous wound (control animals) were handled and anesthetized as described above, except the plastic blunt end of the biopsy punch was gently pressed against the dorsal surface to simulate the experience of the cutaneous biopsy process without actually taking skin from those animals. Lizards were placed back into their individual terraria, which were placed on heating pads until the lizards were alert and responsive and then returned to the 105 environmental chambers. Healing was assessed as the change in wound area from the initial biopsy.

Metabolic Measurements

We measured metabolic rates of lizards using closed-system respirometry three times throughout the experiment. Lizards were fasted on the days before and during metabolic assessments to avoid gathering data during a post-prandial response. To promote a quiescent state during measurements, lights in the environmental chambers were turned off an hour before the animals were moved to the test chambers. Each animal was quickly removed from its housing and placed in a glass chamber (473 ml), inside an incubator set at 36°C. This temperature lies within the optimal range for Uta stansburiana (Waldschmidt and Tracy 1983). The inside of the incubator was dark, and the lizards were given an additional 2 h before metabolic measurements were taken. Once lizards were inside the glass chambers, these chambers were slowly filled with dry, CO2-free air. Then, an automated sampling regime lasting 2.3 hours commenced, following respirometry protocols of Kolbe et al. (2014). Air samples taken from the metabolic chambers were pushed through columns of Drierite® with a mass-flow regulator to remove water vapor, then passed through analyzers for oxygen (Oxzilla, Sable Systems, Las Vegas, Nevada, USA) and carbon dioxide (LI-6252, LI-COR, Lincoln, Nebraska, USA).

Radioimmunoassay

Circulating corticosterone concentrations were determined using a radioimmunoassay protocol modified from Lucas and French (2012). Briefly, samples were extracted using a solution of 30% ethyl acetate:isooctane and assayed in duplicate for CORT (MP Biomedicals, Lot 106

#3R3PB-19E). Final concentrations were calculated by averaging the duplicate samples and adjusted for accuracy using individual recoveries.

Bactericidal Ability

We performed the bactericidal assay on samples following the protocol outlined in

French and Neuman-Lee (2012). Briefly, we combined a 1:5 plasma dilution with CO2- independent media plus 4 nM L-glutamine, 104 colony producing units of Escherichia coli

(EPowerTM Microorganisms #483-237-1, ATCC 8739, MicroBioLogics, St. Cloud, MN, USA), and agar broth on a 96-well microplate. We also included positive (media and bacteria with no plasma) and negative (media alone) controls to account for total possible growth and ensure no contamination was present. We incubated the plate for 12 h and calculated absorbance using a microplate reader (300 nm, BioRad Benchmark, Hercules, CA, USA). Microbiocidal ability was calculated as 1-(absorbance of sample/absorbance of positive controls).

Oxidative Stress

We measured plasma levels of reactive oxygen metabolites using the d-ROMs Test kit

(Diacron, Grosseto, Italy). Briefly, we mixed the provided R1 and R2 reagents in a 1:100 dilution to create an acidic buffered solution with a chromogen. This resulting solution was kept in the dark until 5 µl of sample plasma was added into separate wells of a 96-well microplate and 100 µl of the R1/R2 solution was added to each well. Nanopure water was used as sample blanks and the provided serum was used as a calibrator solution. We followed the “end-point mode” from the manufacturer’s protocol and measured absorbance at 505 nm after a 90 m incubation at 37° C.

The resulting units are in mg H2O2/dl (1 CARR U = 0.08 mg H2O2/dl). 107

Stable Isotope Label

We injected each lizard with 20 μL of a 50:50 mixture of 14N and 15N-leucine (dissolved in Ringer’s solution at a concentration of 937.5 mg/ml) on Days 3, 5, and 7 to ensure enrichment during the study. We measured the flow of this label into two pools: the protein deposited in eggs, and protein deposited into newly-formed tissue (scab forming over the cutaneous wound). We also measured excreted nitrogen in uric acid and feces, which we considered sink pools that were not of particular interest, in order to account for variation in assimilation of our label. At the end of the experiment, we collected eggs, scabs, hearts, livers, and pooled uric acid and feces from each lizard, dried them to a constant mass, (0.5-2.0 mg) in a drying oven set at 60°C, and folded them into 5x9mm tin capsules (Costech Analytical, Valencia, California, USA). In addition to the forty animals described above, five additional animals were used as unlabeled controls and were given neither FSH nor 15N-leucine. We measured stable nitrogen isotope ratios (15N:14N or δ15N) using continuous-flow direct-combustion and isotope ratio mass spectrometry using a Europa

Scientific ANCA-2020 (PDZ, Crewe, England) at the Utah State University Stable Isotope Lab.

The enrichment values were corrected for individual organ mass by dividing them by the dry mass of the entire organ. Standard deviations of replicate standards were < 0.1‰.

Statistical Analysis

We used linear models (function ‘lm’) and ANOVA (function ‘Anova’, package 'car';

Fox and Weisberg 2011) in R (R Core Team 2015) to assess the relationships among treatments, hematological parameters, isotopic ratios, and metabolic rates. We conducted a repeated measures analysis using random coefficient models (Bliese and Ployhart 2002) in R using the package 108

‘nlme’ (Pinheiro et al. 2016) to analyze the change in metabolic rate over the course of the experiment, with a random effect for each individual lizard. The R package ‘ggplot2’ (Wickham

2009) was used to create figures.

Results

The amount of food eaten did not differ among treatments (F1,36 ≤ 1.02, p ≥ 0.31), although it was a significant predictor of body mass change (F1,36 = 37.52, p = 4.73e-7). The number of follicles laid by or dissected from each lizard varied from 1 to 8. Lizards given FSH ovulated more follicles (F1,14 = 5.32, p = 0.03) and there was a marginally significant interaction between the number of follicles and their initial size as measured by ultrasound (F1,14 = 4.13, p =

0.06). The cumulative length of all follicles of lizards given extra FSH increased by 0.29 ± 0.07 cm (F1,34 = 7.95, p = 0.007; Fig. 5-1A). There was no effect of cutaneous biopsy (F1,34 = 0.37, p =

0.55) on cumulative follicle length. Lizards given FSH had reactive oxygen metabolites 71% higher than controls at the end of the experiment with an average of 18.88 ± 1.8 mg H2O2/dl (F1,31

= 4.38, P = 0.04; Fig. 5-1B), but there was no effect of cutaneous biopsy (F1,31 = 0.08, p = 0.78).

All animals lost mass throughout the course of the experiment, with an average loss of 0.34 ±

0.04 g, but biopsied animals lost more with an average of 0.44 ± 0.06 g (F1,34 = 6.93, p = 0.01;

Fig. 5-1C). There was no significant effect of FSH on weight loss (F1,34 = 2.56, P = 0.12), and no interaction (F1,34 = 1.52, p = 0.23).

Metabolic rates decreased throughout the course of this experiment, with total oxygen consumption on Day 4 averaging 2.26 ± 0.15 ml O2/h, followed by 2.12 ± 0.12 and 1.91 ± 0.15 on Days 6 and 8, respectively (Fig. 5-2). Repeated measures analysis suggested that this decrease was marginally significant (t75 = -1.82, p = 0.07), but that there were not significant differences among treatments in the decrease in metabolic rate (p ≥ 0.24). When only wounded animals were analyzed, there was a significant decrease in metabolic rate over time (t37 = -2.28, p = 0.028). The 109 change in metabolic rate from Day 6 to Day 8 (following wounding) was significantly related to wound healing rate (p = 0.027) and the quantity of 15N in eggs (p = 3.62e-5). Lizards that invested larger quantities of protein into their eggs had larger increases in metabolic rate, whereas lizards that had faster wound healing had larger decreases in metabolic rate (Fig. 5-3). Although this model was based on data from only a subset of lizards, the R2 was 0.67.

Wound healing was not affected by FSH (F1,17 = 0.13, P = 0.72), and lizards healed an average of 31.44 ± 0.06% of the biopsied area during the course of this experiment. Circulating corticosterone concentration was 119.24 ± 22.80 ng/ml at the end of this experiment, and did not differ among the treatment groups (F1,20 = 1.06, P = 0.31). Microbiocidal ability was 40.00 ±

5.65% for all animals, and there was no treatment effect (F1,33 = 0.42, P = 0.52).

The median ± SD atom percent enrichment of egg tissue from females treated with FSH was 0.423 ± 0.027, in contrast to eggs of females not treated with FSH (0.379 ± 0.008) and unlabeled controls (0.369 ± 0.0002; Fig. 5-4). The differences among these three groups are highly significant (F2,66 = 52.90, p = 1.95e-14). There was a strong negative relationship between

15 the percent of N in a follicle and the mass (F1,63 = 12.44, p = 2.8e-5) and length (F2,34 = 4.4, p =

0.02) of that follicle, regardless of whether or not FSH was applied. The average total (mass- corrected) quantity of 15N in eggs of females treated with FSH was 150-200 µg, whereas females not given FSH incorporated an average of only 50 µg of 15N into their eggs (Figure-5-5).

Individual variation was high and replication was low, so these differences were not statistically significant. Variation in the atom percent enrichment and mass-corrected total quantity of 15N in other tissues was not significantly affected by FSH (all p ≥ 0.30).

We used the percent of 15N in healed wounds (scabs) as a direct measure of protein allocation to self-maintenance, whereas we interpreted the percent of 15N in eggs as a direct measure of protein allocation to reproduction. We found that egg 15N and scab 15N were strongly 110 negatively related in the absence of our FSH treatment, whereas they were strongly positively correlated in lizards that had been given supplemental FSH (F1,20 = 109.4, p = 1.47e-9; Fig. 5-6).

This relationship remained when the two lizards with the most outlying data were removed (F1,15

= 8.47, p = 0.01). There was no relationship between the within-lizard standard deviation in individual follicle mass and the within-lizard standard deviation in 15N without FSH, but when

FSH was applied there was a significant positive relationship between these two measures of intra-clutch variance (F1,13 = 10.69, p = 0.006; Fig. 5-7).

We also examined the ratios of egg and scab 15N to one another and to the quantity of 15N in lizard hearts (an organ that represents self-maintenance) and livers (an organ that largely, but not completely represents a systemic measure of immune activity). Because not all lizards received a wound, we considered our analysis of the egg-to-liver ratio to be a second test of the trade-off between reproductive and immune costs. We found that there was a significant interaction between the effect of FSH treatment and egg:liver isotope ratio on the metabolic rate of lizards at the third time point (t = -2.68, p = 0.018; Fig. 5-8), such that FSH treated animals with higher egg:liver ratios also had higher metabolic rates. We did not find evidence for an effect of FSH treatment on the egg-to-heart ratio.

111

Tables and Figures

Table 5-1. Timeline of procedures.

Day 1 Ultrasound

Day 3 Inject 15N and FSH

Day 4 Metabolic Trial

Day 5 Biopsy / Wound Image / Inject 15N and FSH / Ultrasound

Day 6 Metabolic Trial

Day 7 Inject 15N and FSH

Day 8 Metabolic Trial

Day 10 Blood Sample / Wound Image / Ultrasound

Day 14 Animals sacrificed and organs harvested for isotopic analysis

Figure 5-1. A) Effect of FSH and wounding treatments on cumulative follicular growth; B) Effect of FSH and wounding treatments on reactive oxygen metabolites; C) Effect of FSH and wounding treatments on body mass. 112

113

Figure 5-2. Effect of FSH and wounding treatments on metabolic rate over time. 114

Figure 5-3. Relationships among the change in metabolic rate and (A) wound healing and (B) the quantity of 15N in eggs. 115

15 Figure 5-4. Effect of FSH treatment on N atom percent enrichment (APE) of egg tissue (F2,66 =

52.90, p = 1.95e-14). Individual lizards ranked from left to right by median egg APE. 116

Figure 5-5. Effect of treatment on total quantity of 15N of A) eggs, B) scabs, C) hearts, D) livers,

E) uric acid, and F) feces.

117

Figure 5-6. 15N atom percent enrichment (APE) of eggs and scabs with (A) and without (B) outliers. 118

Figure 5-7. Co-variance of egg mass and egg 15N atom percent enrichment (APE). 119

Figure 5-8. Relationship between oxygen (O2) consumption (Kleiber mass-adjusted) and ratio of total 15N in eggs to that in livers in lizards that did not receive (top) and received (bottom) FSH.

2 The interaction term between FSH and isotope ratio is significant (F1,13 = 6.78, p = 0.022; R =

0.46). 120

Discussion

We found that experimental increases in FSH increased maternal allocation of protein into eggs and elevated the metabolic rates of female lizards that invested the most protein in their eggs relative to other demands. Wounding had a minimal effect on these processes, but did affect total body mass change. We interpret the change in direction of the relationship between scab and egg 15N with FSH as evidence of a trade-off between reproduction and self-maintenance. This trade-off is further supported by the relationship among wound healing, protein deposition in the eggs, and the wound-induced decrease in metabolic rate.

We interpret the absence of a relationship between metabolic rate and egg:liver 15N ratio in unmanipulated lizards as evidence that resources are limiting the ability of these lizards to invest protein into the competing demands of wound healing and vitellogenesis, whereas the elevated metabolic rate of lizards investing large amounts of protein into their eggs suggests that

FSH forced these lizards to invest in reproduction at a level disproportionate to their resource availability. The relative increase in metabolism could also have caused the increased levels of reactive oxygen metabolites seen in these animals. Brace et al. (2015) also found that increased self-maintenance costs of reproductive female lizards was only evident when leucine allocation ratios between lymphoid and reproductive organs were examined, whereas we found direct evidence for such a trade-off. Although some yolk proteins are produced in the liver, some immune cells also encounter their specific antigens there (Racanelli and Rehermann 2006).

Previous studies of the role of FSH have shown that artificially increasing levels of the hormone increases egg number and decreases egg size (Sinervo and Licht 1991a), reflecting the trade-off between egg size and number (Sinervo and Licht 1991b). By experimentally manipulating FSH in the presence of a 15N-labeled leucine tracer, we showed that FSH also increases the protein content of eggs (Fig. 4), but not at the expense of wound healing. Increases 121 in the variance in protein content among eggs in a clutch with FSH corresponded with the pre- existing variance in egg size (Fig. 6), suggesting that the ability of FSH to increase protein allocation to a particular follicle is dependent on the maturity of that follicle. Although others have shown that vitellogenesis is directly or indirectly regulated by FSH (Ferguson 1966, Licht

1970, 1979, Limatola and Filosa 1989), we provide the first data directly showing greater incorporation of a free amino acid into yolk protein in the presence of elevated FSH.

Because FSH can influence the fate of circulating amino acids, directing them into reproduction, it serves as one mechanism for modifying resource investment. However, apparently wound healing is not compromised and instead metabolic rate increases. The relationship among these three parameters is complex. In male lizards of this species, wounding decreased metabolic rate (Smith et al. In review). These results are contrary to the general consensus that animals facing an immune challenge upregulate their metabolism. This surprising pattern could represent an exacerbation of existing resource limitation, or a compensatory effect of immune prioritization (Neuman-Lee and French 2014). Because the metabolic rates of all animals were measured at rest, sickness behavior is probably not the cause of the decreased metabolic rate in wounded animals in this study. However, we did recover significant effects of both wound healing rate and protein deposition into eggs on the change in metabolic rate of wounded lizards, such that lizards investing more protein into their eggs had larger increases in metabolic rate, whereas lizards with faster wound healing had larger decreases in metabolic rate

(Fig. 3).

The lizards in our study, which were not food-restricted and were relatively early in their reproductive lives, apparently had sufficient stored resources to invest in both wound healing and vitellogenesis. We suggest that unmanipulated lizards spared these stored resources for later use, whereas artificially elevated FSH caused manipulated lizards to spend them. Although we were 122 unable to quantify stored resources in our lizards, other studies have shown the important influence of resource availability on the strength and direction of life-history trade-offs (Doughty and Shine 1997, French et al. 2007b). Surprisingly, we did not find evidence for a mediatory role of corticosterone in this process or an impact on bacterial killing ability.

By tracing an essential amino acid through the bodies of lizards, we directly measured allocation of resources to fundamentally different processes (i.e., reproduction and self- maintenance) using a common currency (McCue 2011) and showed that a trade-off between these two competing demands takes place, of which FSH is a potent mediator. We found that experimentally-induced increases in reproductive investment impacted both overall energy and protein budgets, leading to trade-offs. Although we did not measure the ultimate fitness outcomes of the trade-offs we observed, future studies should focus on documenting the long-term effects of life-history trade-offs for individuals and populations. 123

References

Adamo, S.A., Jensen, M. & Younger, M. (2001) Changes in lifetime immunocompetence in male

and female Gryllus texensis (formerly G. integer): trade-offs between immunity and

reproduction. Animal Behaviour, 62, 417-425.

Angilletta, M. & Sears, M. (2000) The metabolic cost of reproduction in an oviparous lizard.

Functional Ecology, 14, 39-45.

Baird, S.F. & Girard, C. (1852) Descriptions of new species of reptiles, collected by the U.S.

exploring expedition under the command of Capt. Charles Wilkes, U.S.N. First part. -

Including the species from the Western coast of America. Proceedings of the Academy of

Natural Sciences of Philadelphia, 6, 174-177.

Bliese, P.D. & Ployhart, R.E. (2002) Growth modeling using random coefficient models: model

building, testing, and illustrations. Organizational Research Methods, 5, 362-387.

Brace, A.J., Sheikali, S. & Martin, L.B. (2015) Highway to the danger zone: exposure-dependent

costs of immunity in a vertebrate ectotherm. Functional Ecology, 29, 924-930.

Callard, I.P. & Zeigler, H. (1970) Inhibitory effects of prolactin upon gonadotrophin-stimulated

ovarian growth in the iguanid lizard, Dipsosaurus dorsalis. Journal of Endocrinology, 47,

131-132.

Demas, G.E., T.J., G., Chester, E.M. & French, S.S. (2012) The energetics of immunity:

mechanisms mediating trade-offs in eco-immunology. Ecoimmunology (eds G.E. Demas

& R.J. Nelson), pp. 259-296. Oxford University Press, New York, New York, USA.

Doughty, P. & Shine, R. (1997) Detecting life history tradeoffs: measuring energy stores in

‘capital’ breeders reveals costs of reproduction. Oecologia, 110, 508–513. 124

Durso, A.M., Smith, G.D. & French, S.S. (In review) Stable isotope ratios outperform other

physiological endpoints of nutritional stress. Journal of Experimental Biology.

Ferguson, G.W. (1966) Effect of follicle-stimulating hormone and testosterone propionate on the

reproduction of the Side Blotched Lizard, Uta stansburiana. Copeia, 1966, 495-498.

Fox, J. & Weisberg, S. (2011) An {R} Companion to Applied Regression. Sage, Thousand Oaks,

CA, USA.

French, S., Johnston, G. & Moore, M. (2007a) Immune activity suppresses reproduction in food

in food-limited female tree lizards Urosaurus ornatus. Functional Ecology, 21, 1115-

1122.

French, S.S., DeNardo, D.F. & Moore, M.C. (2007b) Trade-Offs between the Reproductive and

Immune Systems: Facultative Responses to Resources or Obligate Responses to

Reproduction? The American Naturalist, 170, 79-89.

French, S.S., McLemore, R., Vernon, B., Johnston, G.I.H. & Moore, M.C. (2007c) Corticosterone

modulation of reproductive and immune systems trade-offs in female tree lizards: long-

term corticosterone manipulations via injectable gelling material. Journal of

Experimental Biology, 210, 2859-2865.

French, S.S. & Neuman-Lee, L.A. (2012) Improved ex vivo method for microbiocidal activity

across vertebrate species. Biology Open, 1, 482-487.

Groothuis, T.G. & Schwabl, H. (2008) Hormone-mediated maternal effects in birds: mechanisms

matter but what do we know of them? Philosophical Transactions of the Royal Society B:

Biological Sciences, 363, 1647-1661. 125

Hamilton, G.S. & Armstrong, D.T. (1991) The superovulation of synchronous adult rats using

follicle-stimulating hormone delivered by continuous infusion. Biology of Reproduction,

44, 851-856.

Hanssen, S.A., Hasselquist, D., Folstad, I. & Erikstad, K.E. (2004) Costs of immunity: immune

responsiveness reduces survival in a vertebrate. Proceedings of the Royal Society of

London, Series B: Biological Sciences, 271, 925-930.

Harshman, L.G. & Zera, A.J. (2007) The cost of reproduction: the devil in the details. Trends in

Ecology & Evolution, 22, 80-86.

Ho, S.-m. (1987) Endocrinology of vitellogenesis. Hormones and Reproduction in Fishes,

Amphibians, and Reptiles (eds D.O. Norris & R.E. Jones), pp. 145-171. Springer Science

& Business Media, New York.

Ho, S.-m., Kleis, S., McPherson, R., Heisermann, G.J. & Callard, I.P. (1982) Regulation of

vitellogenesis in reptiles. Herpetologica, 38, 40-50.

Jones, S.M. & Swain, R. (2000) Effects of exogenous FSH on follicular recruitment in a

viviparous lizard Niveoscincus metallicus (Scincidae). Comparative Biochemistry and

Physiology Part A, 127, 487-493.

Kolbe, J.J., Ehrenberger, J.C., Moniz, H.A. & Angilletta Jr, M.J. (2014) Physiological variation

among invasive populations of the Brown Anole (Anolis sagrei). Physiological and

Biochemical Zoology, 87, 92-104.

Lack, D. (1947) The significance of clutch-size. Ibis, 89, 302-352. 126

Lancaster, L., Hazard, L., Clobert, J. & Sinervo, B. (2008) Corticosterone manipulation reveals

differences in hierarchical organization of multidimensional reproductive trade‐offs in r-

strategist and K-strategist females. Journal of Evolutionary Biology, 21, 556-565.

Lee, K., Cory, J., Wilson, K., Raubenheimer, D. & Simpson, S. (2006) Flexible diet choice

offsets protein costs of pathogen resistance in a caterpillar. Proceedings of the Royal

Society of London B: Biological Sciences, 273, 823-829.

Licht, P. (1970) Effects of mammalian gonadotropins (ovine FSH and LH) in female lizards.

General and Comparative Endocrinology, 14, 98-106.

Licht, P. (1979) Reproductive endocrinology of reptiles and amphibians: gonadotropins. Annual

Review of Physiology, 41, 337-351.

Licht, P., Gallo, A.B., Hartree, A.S. & Shownkeen, R.C. (1977) Physiological actions of human

follicle-stimulating hormone and its β-subunit in reptiles. Journal of Endocrinology, 74,

441-447.

Lighton, J. (2008) Measuring Metabolic Rates: A Manual for Scientists. Oxford University Press,

Oxford.

Limatola, E. & Filosa, S. (1989) Exogenous vitellogenesis and micropinocytosis in the lizard,

Podarcis sicula, treated with follicle-stimulating hormone. General and Comparative

Endocrinology, 75, 165-176.

Lochmiller, R.L. & Deerenberg, C. (2000a) Trade-offs in evolutionary immunology: just what is

the cost of immunity? Oikos, 88, 87-98.

Lochmiller, R.L. & Deerenberg, C. (2000b) Trade‐offs in evolutionary immunology: just what is

the cost of immunity? Oikos, 88, 87-98. 127

Lourdais, O., Brischoux, F., Denardo, D. & Shine, R. (2004) Protein catabolism in pregnant

snakes (Epicrates cenchria maurus Boidae) compromises musculature and performance

after reproduction. Journal of Comparative Physiology B, 174, 383-391.

Lucas, L.D. & French, S.S. (2012) Stress-Induced Tradeoffs in a Free-Living Lizard across a

Variable Landscape: Consequences for Individuals and Populations. PLoS One, 7,

e49895.

McCue, M.D. (2011) Tracking the oxidative and nonoxidative fates of isotopically labeled

nutrients in animals. Bioscience, 61, 217–230.

McCue, M.D., Amaya, J.A., Yang, A.S., Erhardt, E.B., Wolf, B.O. & Hanson, D.T. (2013)

Targeted 13C enrichment of lipid and protein pools in the body reveals circadian changes

in oxidative fuel mixture during prolonged fasting: a case study using Japanese quail.

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology,

166, 546-554.

McGlothlin, J.W., Jawor, J.M. & Ketterson, E.D. (2007) Natural variation in a testosterone‐

mediated trade‐off between mating effort and parental effort. The American Naturalist,

170, 864-875.

Mesquita, D.O., Costa, G.C., Colli, G.R., Costa, T.B., Shepard, D.B., Vitt, L.J. & Pianka, E.R.

(2016) Life-History Patterns of Lizards of the World. The American Naturalist, 187, 689-

705.

Monaghan, P., Metcalfe, N.B. & Torres, R. (2009) Oxidative stress as a mediator of life history

trade-offs: mechanisms, measurements and interpretation. Ecology Letters, 12, 75-92. 128

Moreau, R.E. (1944) Clutch-size: a comparative study, with special reference to African birds.

Ibis, 86, 286-347.

Moret, Y. & Schmid-Hempel, P. (2000) Survival for immunity: the price of immune system

activation for bumblebee workers. Science, 290, 1166-1168.

Neuman-Lee, L.A. & French, S.S. (2014) Wound healing reduces stress-induced immune

changes: evidence for immune prioritization in the side-blotched lizard. Journal of

Comparative Physiology B, 184, 623-629.

Nilsson, J.-Å. & Råberg, L. (2001) The resting metabolic cost of egg laying and nestling feeding

in great tits. Oecologia, 128, 187-192.

Nordling, D., Andersson, M., Zohari, S. & Lars, G. (1998) Reproductive effort reduces specific

immune response and parasite resistance. Proceedings of the Royal Society of London B:

Biological Sciences, 265, 1291-1298.

Norris, K. & Evans, M.R. (2000) Ecological immunology: life history trade-offs and immune

defense in birds. Behavioral Ecology, 11, 19-26.

Pianka, E.R. (1970) On r-and K-selection. The American Naturalist, 104, 592-597.

Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D. & R Core Team (2016) nlme: Linear and

Nonlinear Mixed Effects Models.

R Core Team (2015) R: A language and environment for statistical computing. R Foundation for

Statistical Computing, Vienna, Austria.

Råberg, L., Nilsson, J., Ilmonen, P., Stjernman, M. & Hasselquist, D. (2000) The cost of an

immune response: vaccination reduces parental effort. Ecology Letters, 3, 382-386. 129

Racanelli, V. & Rehermann, B. (2006) The liver as an immunological organ. Hepatology, 43.

Rasband, W.S. (1997-2014) ImageJ. U. S. National Institutes of Health, Bethesda, Maryland,

USA.

Robbins, C.T. (1981) Estimation of the relative protein cost of reproduction in birds. The Condor,

83, 177-179.

Romero, L.M. & Wikelski, M. (2010) Stress physiology as a predictor of survival in Galapagos

marine iguanas. Proceedings of the Royal Society of London B: Biological Sciences, 277,

3157-3162.

Sinervo, B. & Licht, P. (1991a) Hormonal and physiological control of clutch size, egg size, and

egg shape in side‐blotched lizards (Uta stansburiana): constraints on the evolution of

lizard life histories. Journal of Experimental Zoology, 257, 252-264.

Sinervo, B. & Licht, P. (1991b) Proximate constraints on the evolution of egg size, number, and

total clutch mass in lizards. Science, 252, 1300.

Skutch, A.F. (1949) Do tropical birds rear as many young as they can nourish? Ibis, 91, 430-458.

Smith, G.D., Neuman-Lee, L.A., Webb, A.C., Angilletta Jr, M.J., DeNardo, D. & French, S.S. (In

review) Metabolic responses to immune challenges: re-evaluating the energetics of

immunity. Journal of Experimental Biology.

Stearns, S. (1992) The Evolution of Life Histories. Oxford University Press, Oxford, UK.

Waldschmidt, S. & Tracy, C.R. (1983) Interactions between a lizard and its thermal environment:

implications for sprint performance and space utilization in the lizard Uta stansburiana.

Ecology, 64, 476-484. 130

Wickham, H. (2009) ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag, New York.

Williams, G.C. (1966) Natural selection, the costs of reproduction, and a refinement of Lack's

principle. The American Naturalist, 1966, 687-690.

Zera, A.J. & Harshman, L.G. (2001) The physiology of life history trade-offs in animals. Annual

Review of Ecology and Systematics, 32, 95-126.

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CHAPTER 6

CONCLUSION

The goals of my research were 1) to determine how we can more precisely measure energy status in wild animals, 2) to understand how animals trade-off limited resources among competing demands, and 3) to demonstrate how we can quantify these trade-offs using a common scale.

In chapter 1, I examined natural spatial and temporal variation in stable isotope signatures of wild lizards and found some support for hypothetical relationships among body condition, physiology, and stable isotope ratios, but also that “rules” about the meaning of isotopic and stoichiometric ratios do not apply in all situations. In chapter 2, by experimentally manipulating energy status in the lab, I was able to compare the utility of numerous physiological endpoints of nutritional stress. I determined that the nitrogen stable isotope ratios of uric acid pellets outperformed all other measures for precision and accuracy. In chapters 3 and 4, by tracing labeled amino acids through the bodies of gravid female lizards that were healing wounds, I demonstrated that vitellogenesis and wound healing compete for amino acids and quantified the direction and magnitude of the trade-offs. I showed that reproductive-immune trade-offs vary based on reproductive stage and energy availability, have effects on metabolism and immune function, and are influenced by hormonal mechanisms.

My findings shed light on the interconnectedness of stable isotope endpoints and key physiological systems in animals. I showed that stable isotope signatures of physiological stress can be reflected at a large scale in natural populations. I made novel measurements of the size and 132 direction of trade-offs, descriptions of which were formerly limited to their physiological and performance outcomes.

This basic research can lead to advances in physiology, ecology, conservation, and animal husbandry, and highlights the metabolic flexibility of reptiles and how much we have left to learn about their ecology and physiology. Studying life-history trade-offs that reptiles make under varying circumstances can allow us to predict how they will respond to environmental change (Hutton and McGraw 2016). Because reptiles are of increasing conservation concern

(Gibbons et al. 2000, Böhm et al. 2013), rapid, minimally-invasive techniques that allow us to collect information on their diet, nutritional status, and allocation of resources to competing demands are of increasing importance, particularly where their energy, nitrogen, or water budgets are near their physiological extremes (Nagy and Shoemaker 1975). As we currently use water labeled with stable isotopes of hydrogen and oxygen to study field metabolic rates (Nagy 1989), perhaps we will someday use stable isotopes of carbon and nitrogen to study the balance of these elements in wild reptiles and the roles that these animals play in nutrient cycling (e.g., Milanovich et al. 2015, Sterrett et al. 2015). It is my hope that by more fully understanding the importance of reptiles to ecosystems, we can strengthen arguments in favor of their conservation. 133

References

Böhm, M., Collen, B., Baillie, J.E.M., Bowles, P., Chanson, J., Cox, N., . . . Zug, G. (2013) The

conservation status of the world’s reptiles. Biological Conservation, 157, 372-385.

Gibbons, J.W., Scott, D.E., Ryan, T.J., Buhlmann, K.A., Tuberville, T.D., Metts, B.S., . . . Poppy,

S. (2000) The global decline of reptiles, déjà vu amphibians. Bioscience, 50, 653-666.

Hutton, P. & McGraw, K.J. (2016) Urban impacts on oxidative balance and animal signals.

Frontiers in Ecology and Evolution, 4, 54.

Milanovich, J.R., Maerz, J.C. & Rosemond, A.D. (2015) Stoichiometry and estimates of nutrient

standing stocks of larval salamanders in Appalachian headwater streams. Freshwater

Biology, 10.1111/fwb.12572.

Nagy, K.A. (1989) Doubly-labeled water studies of vertebrate physiological ecology. Stable

Isotopes in Ecological Research (eds P.W. Rundel, J.R. Ehleringer & K.A. Nagy), pp.

270-287. Springer-Verlag, New York, NY, USA.

Nagy, K.A. & Shoemaker, V.H. (1975) Energy and nitrogen budgets of the free-living desert

lizard Sauromalus obesus. Physiological Zoology, 48, 252-262.

Sterrett, S.C., Maerz, J.C. & Katz, R.A. (2015) What can turtles teach us about the theory of

ecological stoichiometry? Freshwater Biology, 60, 443-455. 134

Andrew Michael Durso curriculum vitae

Utah State University Phone: 001.919.349.7967 Department of Biology E-mail: [email protected] 5305 Old Main Hill Twitter: @am_durso Logan, UT 84322 ORCID: 0000-0002-3008-7763 Education

Ph.D. Ecology – Utah State University 2016 . Dissertation: Stable isotopes and the ecology and physiology of reptiles . Graduate Advisor: Susannah S. French

M.S. Biological Sciences – Eastern Illinois University 2011 . Thesis: Interactions of diet and behavior in a death-feigning snake (Heterodon nasicus) . Graduate Advisor: Stephen J. Mullin

B.S. Ecology – University of Georgia 2009 . Research Advisors: J. Whitfield Gibbons, John C. Maerz

Research Interests

. Physiological, population, community, evolutionary, and behavioral ecology of reptiles

. Using stable isotopes as tracers of nutrients and energy in field and lab systems

. Developing field and analytical methods for monitoring cryptic species

Peer-reviewed Publications (* denotes undergraduate or high school co-authors)

Durso, A.M. & S.J. Mullin. In press. Ontogenetic shifts in the diet of Plains Hog-nosed Snakes (Colubridae: Heterodon) revealed by stable isotope analysis. Zoology. DOI: 10.1016/j.zool.2016.07.004

Tingley, R., P.J. Mahoney, A.M. Durso, A.G. Tallian, A. Morán-Ordóñez, and K.H. Beard. 2016. Threatened and invasive reptiles are not two sides of the same coin. Global Ecology and Biogeography. 25(9):1050-1060.

Feldman, C.R., A.M. Durso, C.T. Hanifin, M.E. Pfrender, P.K. Ducey, A.N. Stokes, K.E. Barnett, E.D. Brodie III, & E.D. Brodie Jr. 2016. Is there more than one way to skin a newt? North American snakes with convergent feeding adaptations do not share a common genetic mechanism. Heredity. 116:84-91.

Durso, A.M. & R.A. Seigel. 2015. A snake in the hand is worth 10,000 in the bush. Journal of Herpetology. 49(4):503-506.

Mahoney, P.J., K.H. Beard, A.M. Durso, A.G. Tallian, A.L. Long, R.J. Kindermann, N.E. Nolan, D. Kinka, & H.E. Mohn. 2015. Introduction effort, climate matching, and species traits as 135 predictors of global establishment success in non-native reptiles. Diversity & Distributions. 21:64-74.

O’Donnell, R.P. & A.M. Durso. 2014. Harnessing the power of a global network of citizen herpetologists by improving citizen science databases. Herpetological Review. Letter to the Editor. 45(1):151-157.

Durso, A.M. & S.J. Mullin. 2014. Intrinsic and extrinsic factors influence expression of defensive behavior in Plains Hog-nosed Snakes (Heterodon nasicus). Ethology. 120(2):140-148.

Neuman-Lee, L.A., A.M. Durso, N.M. Kiriazis*, M.J. Olds, & S.J. Mullin. 2013. Differential habitat use by Northern Watersnakes (Nerodia sipedon). IRCF Reptiles & Amphibians. 20(4):166-171.

Durso, A.M., J.D. Willson, & C.T. Winne. 2013. Habitat influences diet overlap in aquatic snake assemblages. Journal of Zoology (London). 291(3):185-193.

Graham, S.P., E.K. Timpe, A.M. Durso, D.A. Steen, W.B. Sutton, K.T. Nelson, G.J. Brown, M.A. Connell, K.M. Gray, & J.C. Godwin. 2012. The second known contact zone between Plethodon websteri & P. ventralis, & other new county records for Bibb County, Alabama. Herpetological Review. 43(2):312-313.

Durso, A.M., J.D. Willson, & C.T. Winne. 2011. Needles in haystacks: estimating detection probability & occupancy of rare & cryptic snakes. Biological Conservation. 144(5):1506-1513.

Graham, S.P., A.M. Durso, et al. 2010. An additional bioblitz competition in northwest Georgia yields new county records. Herpetological Review. 41(3):383-384.

Graham, S.P., D.A. Steen, K.T. Nelson, A.M. Durso, & J.C. Maerz. 2010. An overlooked hotspot? Rapid biodiversity assessment reveals a region of exceptional herpetofaunal richness in the southeastern United States. Southeastern Naturalist. 9(1):19-34.

Davis, A.K. & A.M. Durso. 2009. White blood cell differentials of northern cricket frogs (Acris c. crepitans) with a compilation of published values from other amphibians. Herpetologica. 65(3):260-267.

In review:

Skinner, H.M.*, A.M. Durso, L.A. Neuman-Lee, S.L. Durham, S.D. Mueller*, & S.S. French. In review. Effects of energy manipulation and diet complexity on life history strategies in Side- blotched Lizards (Uta stansburiana). Journal of Experimental Zoology Part A.

Spence, A.R.*, A.M. Durso, G.D. Smith, H.M. Skinner*, & S.S. French. In review. Limited physiological consequences of parasitic infection in Side-blotched Lizards. Physiological and Biochemical Zoology.

Durso, A.M. & S.S. French. In review. Stable isotope tracers reveal a trade-off between reproduction and immunity in a reptile with competing needs. Functional Ecology.

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Durso, A.M. & J.C. Maerz. 2016. Behavior. In: Mader’s Reptile Medicine & Surgery. 3rd ed. (Divers, S., ed.) pp. TBD

Honors & Awards

College of Science Graduate Teacher of the Year 2014-2015 Utah State University

Department of Biology Graduate Teacher of the Year 2014 Utah State University

Graduate Research Assistantship 2014-2016 National Science Foundation & Utah State University

Claude E. Zobell Scholarship 2014 Utah State University ($1000)

Summer Research Assistantship 2013 U. S. Department of Agriculture & Utah State University

Graduate Enhancement Award 2013 Utah State University ($4000)

James A. and Patty MacMahon Ecology Graduate Student Research Award 2013 Utah State University ($1000)

Department of Biology Graduate Teacher of the Year 2012 Utah State University

Hamand Society Scholar 2011 Eastern Illinois University

Graduate Student Investigator Award 2011 Eastern Illinois University

Summer Research Assistantship 2011 Eastern Illinois University

Williams Travel Award 2011 Eastern Illinois University Graduate School ($500)

Distinguished Graduate Student in Biology 2011 Eastern Illinois University

Stephen J. Gould Award 2011 Eastern Illinois University Darwin Day essay contest ($500)

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Provost’s Research Assistantship 2010-2011 Eastern Illinois University

Student Travel Award 2010 Society for the Study of Amphibians & Reptiles ($400)

Williams Travel Award 2010 Eastern Illinois University Graduate School ($500)

Summer Research Assistantship 2010 Eastern Illinois University

Richardson-Golley Undergraduate Citizenship Award 2009 Odum School of Ecology

Joshua Laerm Outstanding Ecology Undergraduate Award 2008

Courts International Scholarship 2007 University of Georgia Honors Program

Research Experience for Undergraduates 2006 Savannah River Ecology Laboratory & National Science Foundation

Center for Undergraduate Research Opportunities Apprenticeship 2005-2007 University of Georgia Honors Program

North Carolina District Employees Association Scholarship 2005

National Merit Foundation Scholarship 2005

Grants

Awarded to me:

2015. Utah State University Student Association Research Grant – Comparing multiple indirect methods for assessing snake diets - $1000

2015. Utah State University Ecology Center Research Support Award – Comparing multiple indirect methods for assessing snake diets - $2500

2014. Utah State University Ecology Center Research Support Award – Assessing ecologists’ ability to distinguish isotopic signals of nutrient limitation from those caused by other common stressors – $3500

2013. Herpetologists’ League E.E. Williams Research Grant – The effects of isotopic routing, discrimination factors, and equilibration on stable isotope studies of lizards – $1000

138

2013. Utah State University Ecology Center Research Support Award – Using labeled nutrient tracers to reveal resource allocation in lizards with competing needs – $3500

2011. Utah State University Graduate Student Senate Research & Project Grant – Dietary sequestration of toxic compounds by North American Hog-nosed Snakes – $1000

Spring 2011. Eastern Illinois University Graduate School Research/Creative Activity Grant – Interactions of diet and behavior in death-feigning snakes – $850

Fall 2010. Eastern Illinois University Graduate School Research/Creative Activity Grant – Interactions of diet and behavior in death-feigning snakes – $750

Spring 2010. Eastern Illinois University Graduate School Research/Creative Activity Grant – Interactions of diet and behavior in death-feigning snakes – $1000

2009. North Carolina Herpetological Society Research Grant – The interaction of diet and behavior in toad-eating snakes that feign death – $1000

2006. Georgia Museum of Natural History – Joshua Laerm Academic Support Award – Effects of environmental factors on genetic diversity within populations of Ambystoma in the Whitehall Forest – A.M. Durso & J.F. Dean – $1000

Awarded to students I have mentored:

2016. Utah State University Undergraduate Research and Creative Opportunities Grant – Effects of dehydration on oxygen stable isotopes present in Uta stansburiana – Tyler Hansen (undergraduate) – $750

Professional Employment

Graduate Research Assistantship – National Science Foundation (IOS-1350070) 2015-2016 Physiological Trade-offs in Ecoimmunology: Costs for Individuals and Populations Utah State University Supervisor: Susannah French . Administered study to measure the effect of carrying out scientific research on science teachers

Graduate Research Assistantship – USDA Agricultural Experiment Station Utah State University Summers 2013, 2014, 2015

Contracted Software Developer – National Science Foundation (DRL-1114558) Herpetology Education in Rural Places and Spaces Project Spring 2012-2014 University of North Carolina-Greensboro http://theherpproject.uncg.edu/ Supervisors: Ann B. Somers & Bruce Kirchoff

Van Driver – Ornithology course Spring 2012 Utah State University, Department of Biology Supervisor: Ryan O’Donnell 139

Field Assistant – Diamondback Terrapin Project Summer 2008 University of Georgia & Georgia Department of Natural Resources http://jcmaerz.myweb.uga.edu/lab/Terrapins/index.htm Supervisors: Andrew Grosse & Dr. John C. Maerz

Herpetology Lab Technician 2008 University of Georgia, Warnell School of Forestry & Natural Resources http://maerzlab.uga.edu/Site/Home.html Supervisors: Kristen Cecala & Dr. John C. Maerz

Field Technician/Volunteer Summer 2007 Reptile & Amphibian Ecology International, Manabí/Pichincha, Ecuador www.reptilesandamphibians.org Supervisor: Dr. Paul S. Hamilton

Website Technician 2005-2006 University of Georgia, Odum School of Ecology, Discover Life www.discoverlife.org Supervisor: Dr. John Pickering . Built global taxonomic biodatabase checklists & scored keys for reptiles & amphibians, uploaded photos & locality records to worldwide server

Teaching Experience (* denotes volunteer experience; † denotes field course)

Department of Biology Instructor, Utah State University . Biodiversity of Utah† – Summer 2012

Department of Biology Teaching Assistantship, Utah State University . Biology I – Dr. James Pitts – Fall 2011, 2012 . Biology II – Dr. James Pitts – Spring 2012 . Evolutionary Biology – Dr. Frank Messina – Fall 2013, 2014 . Ornithology† – Dr. Kimberly Sullivan – Spring 2013, 2014

Department of Biological Sciences Teaching Assistantship, Eastern Illinois University . Herpetology† – Dr. Stephen J. Mullin – Spring 2011* . Animal Diversity – Dr. Ann Fritz – Spring 2010 . Functional Comparative Vertebrate Anatomy – Dr. Stephen J. Mullin – Spring 2010 . Vertebrate Natural History† – Dr. Stephen J. Mullin – Fall 2009 & Fall 2010* . General Biology – Dr. Zhiwei Liu – Fall 2009 . Animal Diversity – Dr. Jeffrey R. Laursen – Fall 2009

Undergraduate Teaching Assistant, University of Georgia . Population Ecology - Dr. John M. Drake - Fall 2008 . Ichthyology† - Drs. Byron J. Freeman & Brett Albanese - Spring 2009 . Herpetology† - Drs. John C. Maerz & Kurt A. Buhlmann - Spring 2007 & 2008

Guest Instructor, Herpetology of the Southwest, Southwestern Research Station 140

. 2015

Guest Lectures Utah State University . Reptile Anatomy and Physiology – Anatomy and Physiology of Animals . Microbes & the Immune System – General Microbiology (2 years) . Human Immunology – Human Physiology (2 years) . Introduction to Immunology – Biology and the Citizen . Biodiversity of Birds – Biology and the Citizen . Comparative Immunology of Animals – Comparative Animal Physiology . Nutrition, Feeding, & Metabolism – Comparative Animal Physiology (2 years) . Avian Evolution – Ornithology (2 years) . Breeding Biology of Birds – Ornithology . Inventory & Monitoring of Amphibians & Reptiles – Wildlife Ecology & Identification . Living with Snakes – Living with Wildlife (4 years + 2 summer semesters) . Introduction to eBird – EcoLunch . The Only Good Snake is a Dead Snake – Honors Seminar (2 years) . Amphibians & Reptiles of Northern Utah – EcoLunch/Biology Grad. Student Assoc. . Snakes of Logan Canyon – Bridgerland Audubon Society field trip . Graduate School in Biology (panel discussion) – Women in Science (2 years) . EndNote Basics – EcoLunch & Biology Graduate Student Association workshop . Evolution & Diversity of Snakes – Herpetology (3 lectures) . Snake Identification – Herpetology Lab . Graduate School – Preparing for Scholarships (honors seminar; 3 years)

Eastern Illinois University . Lizard Diversity – Herpetology Lab . Tetrapod Evolution – Vertebrate Natural History . Reptiles – Vertebrate Natural History Lab

University of Georgia (as undergraduate) . Evolution & Diversity of Snakes – Herpetology (2 lectures x 2 consecutive years) . Amphibians & Reptiles as Companion Animals – Companion Animal Care (2 years) . Biodiversity & Conservation – Roosevelt Institution panel discussion . Research & the Critical Voice – Undergraduate Research Seminar . Biodiversity – Population & Community Ecology . Internships & Undergraduate Research Opportunities – Ecology Junior Seminar . Aquatic Snakes – Herpetology

I also tutor high school and college students in biology, chemistry, and statistics.

Science Communication & Outreach

Science Reporting Internship – Utah Public Radio 2016 http://upr.org/term/andrew-durso KUSU FM 91.5 Logan Supervisor: Kerry Bringhurst

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Blog author – Life is Short, but Snakes are Long 2012-present http://www.snakesarelong.blogspot.com . I write a blog in English and Spanish about snake ecology which has received >800,000 hits from >450,000 unique users representing nearly every country since April 2012 . My blog material has been covered or republished by: . National Geographic . The Sierra Club . Scientific American . HerpDigest . National Public Radio . The Herpetologists’ League . The Conversation . Biodiversity Heritage Library . Huffington Post . HerpNation Media . BBC Earth . The Other Herpcast . Discover Magazine . Center for Snake Conservation . Small Pond Science . Partners in Amphibian & Reptile Conservation . KVRX 91.7 UT-Austin . North Carolina Herpetological Society . Logan Herald-Journal . US Amphibian and Reptile Keepers . Salt Lake City Tribune . One of my posts was nominated for the ‘Best Science Writing Online 2013’ contest . Additionally, I have consulted on reptile biology with fiction novelists, Bones on FOX TV, The Blacklist on NBC, and Cherry Lake Publishing, all of which I have connected with through my blog.

In-person outreach programs Invited Lectures: . Life is Short but Snakes are Long – USU Science Unwrapped; Cache Naturalists; Logan Summer Citizens (x2) . Why Birds are Dinosaurs – Venture Academy Charter High School, Ogden, UT . Amphibians & Reptiles of Northern Utah – Bear River Migratory Bird Refuge Volunteer Training Workshop . Research in Herpetology – Slip Slidin’ Away (high school herpetology field camp) . Estimating Detection Probabilities of Aquatic Snakes – NCSU Herpetology Club . Effect of Roads & Habitat Fragmentation on Snakes – NC Museum of Natural Sciences . Aquatic Snakes of the Southeast – NC Museum of Natural Sciences Reptile & Amphibian Day . Invited National Issues Forum moderator on water resources, CURO International Symposium, Costa Rica (May 2008)

As opportunities arise, I often accept invitations to do educational public outreach programs using live animals to teach groups of various ages, including public and private K-12 classes, clubs, and teams, and groups at zoos, libraries, community events, and amateur societies. Since 1999 I have done 15-20 programs a year on average; in some years up to 50. Annual average participants ~2,000.

Science fair judging and coaching 2007-present . High School Environmental Science, Bridgerland Science & Engineering Fair (February 2014) . Elementary School Behavioral Science, Cache County Regional Science Fair (February 2013) . Middle School Plant Science, Cache County Regional Science Fair (February 2012) 142

. Behavioral Science, Regional Illinois Junior Academy of Science Fair (March 2011) . Georgia Science & Engineering Fair, Environmental Sciences, High School (2009) . Georgia Science & Engineering Fair, Behavioral Sciences, Middle School (2008) . Coach, Georgia Regional & State Science Olympiad, Divisions B & C (Fall 2007 & 2008)

Other outreach Field trip coordinator, Bridgerland Audubon Society (2015-2016)

Ask an Expert volunteer, USDA Cooperative Extension (2011-present; ~30 questions/year)

Administrator for Facebook Snake Identification (>25,000 members) and Wild Snakes: Education & Discussion (>12,000 members) groups

Citizen scientist data contributor for eBird (>4450 checklists entered), HerpMapper, iNaturalist, Project BudBurst, Butterflies & Moths of North America, & Mushroom Observer

Professional Service

Board member, Bridgerland Audubon Society (2015-2016)

Associate Editor, Herpetological Review, Snake Natural History Notes (2015-present)

Associate Editor, Journal of Herpetology, special section on detectability (2014, published 2015), and chair, Herpetologists’ League sponsored symposium: Detectability and studying rare species: when cryptic natural histories defy both conventional and progressive statistics. Joint Meetings of Ichthyologists & Herpetologists (2013), Albuquerque, NM, including organizing workshop through Southwest PARC and editing special issue of contributed papers

USU Biology Department Seminar Committee Member & Host (2013-2016)

Chair, USU Ecology Center Seminar Committee (2014-2015)

USU Ecology Center Seminar Committee Member & Host (2012-2013)

Outreach Chair, Biology Graduate Student Association, Utah State University (2013-2014)

Vice President, Biology Graduate Student Association, Utah State University (2012-2013)

Society for Study of Amphibians & Reptiles meeting mentorship committee (Fall 2011-present)

Updater & editor, Wildlife Profiles, North Carolina Wildlife Resources Commission (2011)

Grants Chair, North Carolina Herpetological Society (2010-present)

Committee Member, SSAR/HL/ASIH Meritorious Teaching Award in Herpetology (2010-2012)

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Vice President, Biological Sciences Graduate Student Association, Eastern Illinois University (2010-2011) . Organized departmental seminar series

Chair, SE Partners in Amphibian & Reptile Conservation Development Task Team (2008-10)

Undergraduate representative, Odum School of Ecology, Undergraduate Curriculum Committee (2008-09)

President, University of Georgia Herpetological Society (2006-2007)

Vice President of Outreach, University of Georgia Herpetological Society (2005-2006)

Membership Chair, North Carolina Herpetological Society (2004-2006)

Reviewer for Biological Conservation (2015), Conservation Biology (2015), Copeia (2015), Journal of North American Herpetology (2015), Ethology (2014), Biology of the Rattlesnakes II (book; 2014), Herpetologica (2013-present; 2 papers), Animal Conservation (2013-present),

Professional Memberships Herpetological Conservation and Biology (2013-present; 2 papers), Journal of Herpetology (2012-present; 3 papers), Herpetological Review (2012-present; 3 papers), Journal of African Ecology (2010-present; 2 papers), Contemporary Herpetology (2008-present)

Natural History Network (2013-present)

American Association for the Advancement of Science (2012-2015)

Society for Integrative & Comparative Biology (2010-2011, 2013-2015)

Snake Conservation & Leadership Education Society (2010-present)

Sigma Xi (2009-2015)

The Herpetologists’ League (2009-present)

Association of Southeastern Biologists (2006-2008)

Southeast & North Carolina Partners in Amphibian & Reptile Conservation (2005-present)

Society for the Study of Amphibians & Reptiles (2005-present)

North Carolina Herpetological Society (2002-present)

Oral Presentations (* denotes undergraduate or high school co-authors) 144

Invited seminars:

Durso, A.M. 2016. Behavioral ecology of hog-nosed snakes. Junior Curator Program, North Carolina Museum of Natural Sciences. Raleigh, NC.

Durso, A.M. 2016. Use of nitrogen stable isotope ratios and labels in the physiological ecology of reptiles. Max Planck Institute for Chemical Ecology. Jena, DE.

Durso, A.M. 2016. Amphibians and reptiles of the Majadas Region and their contributions to nutrient cycling. Max Planck Institute for Biogeochemistry. Jena, DE.

Durso, A.M. 2015. Life is Short, but Snakes are Long. International Herpetological Symposium. San Antonio, TX. (Science Café)

Durso, A.M. 2012. Snakes as models in community ecology. Northern Illinois University Department of Biological Sciences. Dekalb, IL.

Durso, A.M. 2006. Effects of expanding road systems and habitat fragmentation on snake populations. Junior Curator Program, North Carolina Museum of Natural Sciences. Raleigh, NC.

Conference presentations:

Durso, A.M. & R.P. O’Donnell. 2015. Realizing the potential of herpetological citizen science databases. Joint Meeting of Ichthyologists & Herpetologists. Reno, NV.

Smith, G.D., A.M. Durso, L.A. Neuman-Lee, & S.S. French. 2015. The town lizard and the country lizard: The physiological ecology of urbanization in Uta stansburiana. Society for Integrative and Comparative Biology. West Palm Beach, FL.

Mahoney, P.J., K.H. Beard, A.M. Durso, A.G. Tallian, A.L. Long, R.J. Kindermann, N.E. Nolan, D. Kinka, & H.E. Mohn. 2014. Introduction effort, climate matching, and species traits as predictors of global establishment success in non-native reptiles. Joint Meeting of Ichthyologists & Herpetologists. Chattanooga, TN.

Vogrinc, P.N., J.D. Willson, and A.M. Durso. 2014. Landscape-scale responses of semi-aquatic snakes to drought. Joint Meeting of Ichthyologists & Herpetologists. Chattanooga, TN.

Mahoney, P.J., K.H. Beard, A.G. Tallian, A.L. Long, A.M. Durso, R.J. Kindermann, N.E. Nolan, D. Kinka, & H.E. Mohn. 2014. Getting to the bottom of non-native reptile establishment success. Society for Conservation Biology. Missoula, MT.

Durso, A.M., G. D. Smith, L. A. Neuman-Lee, & S.S. French. 2014. Using labeled nutrient tracers to reveal resource allocation in lizards with competing needs. Society for Integrative and Comparative Biology. Austin, TX.

Smith, G.D., L.A. Neuman-Lee, A.M. Durso, & S.S. French. 2014. Investment between reproductive and immune systems varies with latitude and time in Uta stansburiana. Society for Integrative and Comparative Biology. Austin, TX. 145

Durso, A.M. & R.A. Seigel. 2013. Which snakes cross the road and when do they cross it? Use of a detectability analysis on a long-term road survey data set. Joint Meeting of Ichthyologists & Herpetologists. Albuquerque, NM. Invited symposium talk.

Gross, I.P.,* A.M. Durso, C.P. Lennon & S.J. Mullin. August 2012. Why did the Brownsnake cross the road? Understanding how snake populations are impacted by vehicle access within a state park. World Congress of Herpetology. Vancouver, BC, Canada.

Durso, A.M. & S.J. Mullin. August 2012. Dietary ecology of a sand prairie snake community. World Congress of Herpetology. Vancouver, BC, Canada.

Durso, A.M. & S.J. Mullin. July 2011. Interactions of sex, age & behavior in death-feigning snakes (Heterodon). Joint Meeting of Ichthyologists & Herpetologists. Minneapolis, MN. . Honorable Mention, Henri Seibert Award in Ecology, Society for the Study of Amphibians and Reptiles

Durso, A.M. & S.J. Mullin. April 2011. Influence of diet, sex & age on defensive behavior of western hog-nosed snakes (Heterodon nasicus). Midwest Ecology & Evolution Conference. Carbondale, IL.

Durso, A.M., K.T. Nelson & J.M. Pahlas. February 2008. Bringing Reptiles into the Classroom. Georgia Science Teachers Association Conference. Athens, GA.

Durso, A.M. & J.C. Maerz. April 2007. Environmental & ontogenetic changes in detection probability of pond-breeding salamanders in the Georgia piedmont. Center for Undergraduate Research Opportunities Symposium. Athens, GA. . First place, Sigma Xi Undergraduate poster/presentation award,

Durso, A.M. July 2006. Needle in a Haystack: Estimating Detection Probabilities of Aquatic Snakes. Research Experience for Undergraduates Symposium. Savannah River Ecology Lab, Aiken, SC.

Poster Presentations (* denotes undergraduate or high school co-authors)

Keehn, J.E., A.M. Durso, S.S. French, C.R. Feldman. 2016. Chronic stress response of side- blotched lizards (Uta stansburiana) at noisy California wind farms. Joint Meeting of Ichthyologists & Herpetologists. New Orleans, LA.

Durso, A.M., G.D. Smith, L.A. Neuman-Lee, & S.S. French. August 2014. Using labeled nutrient tracers to reveal resource allocation in lizards with competing needs. Joint Meeting of Ichthyologists & Herpetologists. Chattanooga, TN. 146

Gross, I.P.,* C.P. Lennon, M.A. Thomas, A.M. Durso, & S.J. Mullin. July 2013. Patterns of snake (Colubridae: Storeria) movement and mortality during seasonal migrations between habitats. Joint Meeting of Ichthyologists & Herpetologists. Albuquerque, NM.

Durso, A.M., A.B. Somers, & B.K. Kirchoff. August 2012. Development of visual learning tools for identifying herpetofauna. World Congress of Herpetology. Vancouver, BC, Canada.

Piñon, M.,* J. Maciel,* C. Hinsley,* A. Reedy, A. Durso, T. Mitchell, D. Warner & F. Janzen. July 2011. Microhabitat selection by Western Hognose Snakes. Joint Meeting of Ichthyologists & Herpetologists. Minneapolis, MN.

Gross, I.P.,* L.A. Neuman-Lee, A.M. Durso, & S.J. Mullin. July 2011. Assessing the relationship between parasite loads & limb deformities in Small-mouthed Salamanders (Caudata: Ambystomatidae). Joint Meeting of Ichthyologists & Herpetologists. Minneapolis, MN.

Durso, A.M. & J.D. Willson. July 2011. Dietary plasticity of southeastern aquatic snakes. Joint Meeting of Ichthyologists & Herpetologists. Minneapolis, MN.

Ginder, D.J., A.M. Durso, B.M. Daugherty, Z. Liu & G.C. Tucker. April 2011. Morphological & molecular systematics of the genus Triosteum (Caprifoliaceae). Illinois State Academy of Sciences. Charleston, IL.

Gross, I.P.,* D.P. Fecarotta* & A.M. Durso. April 2011. Getting beaten on the path: migrating Midland Brownsnakes (Colubridae, Storeria) experience high mortality within a state park. Midwest Ecology & Evolution Conference. Carbondale, IL.

Durso, A.M. & S.J. Mullin. July 2010. Interactions of diet & behavior in death-feigning snakes (Heterodon). Joint Meeting of Ichthyologists & Herpetologists. Providence, RI.

Durso, A.M. & S.J. Mullin. March 2010. Interactions between diet & behavior in the death- feigning snakes Heterodon nasicus & H. platirhinos. Midwest Ecology & Evolution Conference. Ames, IA.

Durso, A.M. March 2010. The Junior Curator Program at the North Carolina Museum of Natural Sciences. North Carolina Partners in Amphibian & Reptile Conservation. Haw River State Park, NC.

Durso, A.M., J.D. Willson & C.T. Winne. July 2009. Modeling Detectability of Aquatic Snake Communities. Joint Meeting of Ichthyologists & Herpetologists. Portland, OR.

Durso, A.M., J.D. Willson & C.T. Winne. July 2009. Modeling Detectability of Aquatic Snake Communities. Snake Ecology Group V. Donnelly, ID.

Durso, A.M., J.D. Willson & C.T. Winne. February 2009. Needle in a Haystack: Estimating Detection Probabilities for Aquatic Snakes. SE/NCPARC. Montreat, NC.

Durso, A.M., J.D. Willson & C.T. Winne. January 2009. Needle in a Haystack: Estimating Detection Probabilities for Aquatic Snakes. Odum School of Ecology Graduate Student Symposium. Athens, GA. 147

. Best Undergraduate Poster

Durso, A.M., J.M. Pahlas & P.S. Hamilton. February 2008. Cataloging biodiversity: Filling in the gaps for coastal Ecuadorian herpetofauna. Odum School of Ecology Graduate Student Symposium. Athens, GA. . Best Undergraduate Poster

Durso, A.M., J.M. Pahlas & P.S. Hamilton. February 2008. Cataloging biodiversity: Filling in the gaps for coastal Ecuadorian herpetofauna. Southeast Partners in Amphibian & Reptile Conservation. Athens, GA.

Durso, A.M., J.D. Willson & C.T. Winne. February 2007. Estimating Detection Probabilities for Aquatic Snakes. 2007 Southeast Partners in Amphibian & Reptile Conservation meeting. Chattanooga, TN.

Durso, A.M., J.D. Willson & C.T. Winne. January 2007. Estimating Detection Probabilities for Aquatic Snakes. Institute of Ecology Graduate Student Symposium. Athens, GA.

Durso, A.M., J.D. Willson & C.T. Winne. October 2006. Estimating Detection Probabilities for Aquatic Snakes. Academy of the Environment Symposium. Athens, GA.

Natural History & Geographic Distribution Notes (* denotes undergraduate or high school co-authors)

Durso, A.M. E. Nixon, and K.T. Nelson. In prep. Eurycea longicauda (Long-tailed Salamander). Chattooga County, Georgia. Geographic Distribution. Herpetological Review.

Durso, A.M. In review. Agkistrodon contortrix (Copperhead). Diet. Natural History Note. Herpetological Review.

Durso, A.M. 2016. New developments in telemetry, in: Somers, A.B., Matthews, C.E. (Eds.), The Box Turtle Connection, Greensboro, North Carolina.

Durso, A.M. and B. Rosenthal. 2016. Seminatrix pygaea (Black Swampsnake). Predation. Natural History Note. Herpetological Review. 47(3):484-485.

Durso, A.M. & P. Norberg. 2016. Coniophanes piceivittis. Nicaragua: Región Autónoma Atlántico Sur. Mesoamerican Herpetology. 3(1):194-197.

Golla, J.M. and A.M. Durso. 2015. Crotalus oreganus helleri (Southern Pacific Rattlesnake). Diet/Scavenging. Natural History Note. Herpetological Review. 46(4):641-642.

Durso, A.M. and K.A. Morris. 2015. Agkistrodon contortrix laticinctus (Broad-banded Copperhead). Diet. Natural History Note. Herpetological Review. 46(4):636.

Durso, A. M., E. Lugo, & M. Rodriguez. 2015. Hydrophis (=Pelamis) platura (Yellow-bellied Seasnake). Interaction with dolphins. Natural History Note. Herpetological Review. 46(1):104 (with addendum printed 46(3):455).

148

Durso, A. M., C.M. Carter, T.W. Pierson, & N. Bayona. 2014. Plethodon chattahoochee (Chattahoochee Slimy Salamander). Habitat Use. Natural History Note. Herpetological Review. 45(4):676-677.

Mebert, K., & A.M. Durso. 2014. When predation and defense intermingle - an intercepted predation attempt by a Flying Snake on a Tokay Gecko. Sauria. 36(3):41-46.

Durso, A.M. 2014. Scincella lateralis (Little Brown Skink). Predation. Natural History Note. Herpetological Review. 45(3):508.

Cates, C.D., D.M. Delaney, A.M. Buckelew*, A.M. Durso, S.S. French, A.M. Reedy, & D.A. Warner. 2014. Anolis sagrei (Brown Anole). Egg Predation. Natural History Note. Herpetological Review. 45(3):491-492.

Cates, C.D., D.M. Delaney, A.M. Buckelew*, A.M. Durso, S.S. French, A.M. Reedy, & D.A. Warner. 2014. Anolis sagrei (Brown Anole). Cannibalism. Natural History Note. Herpetological Review. 45(3):491.

Durso, A.M., H.M. Heinz, D. Lockwood, & S. Durso. 2014. Boaedon (Lamprophis) fuliginosus (African House Snake). Longevity. Herpetoculture Note. Herpetological Review. 45(3):455.

Delaney, D.M., C.D. Cates, A.M. Buckelew*, A.M. Durso, S.S. French, A.M. Reedy, & D.A. Warner. 2014. Anolis sagrei (Brown Anole). Prey Stealing Behavior. Natural History Note. Herpetological Review. 45(2):324-325.

Gross, I.P.*, M.A. Thomas, C.M. Carter, & A.M. Durso. 2014. Coluber (Masticophis) flagellum (Coachwhip). Nocturnal Activity. Natural History Note. Herpetological Review. 45(2):336-337.

Vogrinc, P., J.D. Willson, A.M. Durso, L.A. Bryan, Z. Ross, J. Holbrook, & D. Filipiak. 2013. Nerodia floridana (Florida Green Watersnake). Diet. Natural History Note. Herpetological Review. 44(4):695.

Delaney, D.M.*, A.M. Reedy, T.S. Mitchell, A.M. Durso, K.P. Durso*, A.J. Morrison* and D.A. Warner. 2013. Anolis sagrei (Brown Anole) Nest-Site Choice. Natural History Note. Herpetological Review. 44(2): 314.

Durso, A.M., T.S. Mitchell, A.M. Reedy, & D.A. Warner. 2013. Ophisaurus compressus (Island Glass Lizard) Swimming. Natural History Note. Herpetological Review. 44(1):146.

Durso, A.M. & D.G. Mulcahy. 2012. Hypsiglena chlorophaea deserticola (Desert Nightsnake) Cache County, Utah. Geographic Distribution. Herpetological Review. 43(1):106.

Durso, A.M. & K.P. Durso*. 2012. Coluber (=Masticophis) flagellum (Coachwhip) Lincoln County, Kansas. Geographic Distribution. Herpetological Review. 43(1):105.

Durso, A.M., D.A. Warner, T.S. Mitchell, & A.M. Reedy. 2011. Heterodon nasicus (Western Hog-nosed Snake) Diet. Natural History Note. Herpetological Review. 42(3):439-440.

149

Durso, A.M. 2011. Thamnophis radix (Plains Gartersnake) Carroll County, Illinois. Geographic Distribution. Herpetological Review. 42(3):396.

Durso, A.M. & N.M. Kiriazis*. 2011. Coluber constrictor (North American Racer) Prey Size. Natural History Note. Herpetological Review. 42(2):285.

Neuman-Lee, L.A. & A.M. Durso. 2011. Graptemys pseudogeographica (False Map Turtle) Carroll & Whiteside Counties, Illinois. Geographic Distribution. Herpetological Review. 42(1):110.

Neuman-Lee, L.A. & A.M. Durso. 2011. Graptemys geographica (Northern Map Turtle) Whiteside County, Illinois. Geographic Distribution. Herpetological Review. 42(1):110.

Durso, A.M. & L.A. Neuman-Lee. 2011. Chelydra serpentina (Snapping Turtle) Whiteside County, Illinois. Geographic Distribution. Herpetological Review. 42(1):110.

N.M. Kiriazis* & A.M. Durso. 2011. Lithobates palustris (Pickerel Frog) Edgar County, Illinois. Geographic Distribution. Herpetological Review. 42(1):107.

Durso, A.M., G.J. Brown,* & T.W. Pierson*. 2010. Plethodon petraeus (Pigeon Mountain Salamander) Depredation. Natural History Note. Herpetological Review. 41(4):469-470.

Durso, A.M., P.S. Hamilton, & K.M. Donithan. 2010. Trachycephalus jordani (Jordan’s Casque- Headed Treefrog) Predation. Natural History Note. Herpetological Review. 41(3):343-344.

Durso, A.M. 2010. Alligator mississippiensis (American Alligator) Miller County, Georgia. Geographic Distribution. Herpetological Review. 41(2):242.

Durso, A.M. & K.P. Durso. 2010. A Defensive Display by a Smooth Earth Snake (Virginia valeriae). IRCF Reptiles & Amphibians (Henry Fitch Memorial Issue). 17(1):41.

Durso, A.M. & K.T. Nelson. 2010. Lampropeltis triangulum elapsoides (Scarlet Kingsnake) Miller & Calhoun Counties, Georgia. Geographic Distribution. Herpetological Review. 41(1):110.

Durso, A.M. & K.P. Durso. 2010. Opheodrys aestivus (Rough Green Snake) Foraging. Natural History Note. Herpetological Review. 41(1):95-96.

Durso, A.M., E.P. Hill, & K.J. Sash. 2010. Lampropeltis getula getula (Eastern Kingsnake) Scavenging & Diet. Natural History Note. Herpetological Review. 41(1):94.

Nelson, K.T., A.M. Durso, & R.V. Horan. 2010. Diadophis punctatus punctatus (Southern ring- necked snake) Diet. Natural History Note. Herpetological Review. 41(1):90-91.

Durso, A.M., S. Jarrett, C. Jarrett, & J.T. Oguni. 2009. Lampropeltis getula (Common Kingsnake) Madison County, Georgia. Geographic Distribution. Herpetological Review. 40(4):456.

150

Durso, A.M., J. Moree, & C. Stoudenmire. 2009. Diadophis punctatus (Ring-necked snake) Madison County, Georgia. Geographic Distribution. Herpetological Review. 40(4):455.

Durso, A.M., J. Moree, & C. Stoudenmire. 2009. Plestiodon fasciatus (Five-lined skink) Madison County, Georgia. Geographic Distribution. Herpetological Review. 40(4):453.

Durso, A.M. 209. Sternotherus odoratus (Eastern Musk Turtle) Madison County, Georgia. Geographic Distribution. Herpetological Review. 40(4):449-450.

Durso, A.M. 2009. Kinosternon subrubrum (Eastern Mud Turtle) Madison County, Georgia. Geographic Distribution. Herpetological Review. 40(4):449.

Durso, A.M. & M.O. Milby. 2009. Eurycea guttolineata (Three-lined Salamander) Madison County, Georgia. Geographic Distribution. Herpetological Review. 40(4):444.

Nelson, K.T. & A.M. Durso. 2009. Coluber (=Masticophis) flagellum (Coachwhip) Emanuel County, Georgia. Geographic Distribution. Herpetological Review. 40(3):364-365.

Durso, A.M., K.T. Nelson, & E.D. Osburn. 2009. Virginia valeriae (Smooth Earth Snake) Elbert County, Georgia. Geographic Distribution. Herpetological Review. 40(2):239-240.

Durso, A.M., K.L. Holcomb, & P.G. Barnett. 2009. Carphophis amoenus (Eastern Worm Snake) Madison County, Georgia. Geographic Distribution. Herpetological Review. 40(2):237.

Holcomb, K.L., A.M. Durso, & P.G. Barnett. 2009. Hemidactylium scutatum (Four-toed Salamander) Madison County, Georgia. Geographic Distribution. Herpetological Review. 40(2):233.

Technical Skills

. Programming (R, SAS, some experience with SPSS & Python) . GIS (ArcMap) . Microsoft Access . EndNote . Mark-recapture and occupancy estimation field and modeling techniques (packages rmark and unmarked in R; Program PRESENCE; Program MARK) . Reptile and amphibian inventory & monitoring . Field blood draw methods for snakes, turtles, lizards, and salamanders . Venomous snake handling safety protocol . General lab safety, radiation safety, IACUC, IRB, and NSF-RCR (ethics) training . Radioimmunoassay . Oxidative stress assays . Bacterial killing microplate assay . Design and interpretation of stable isotope studies . Design and implementation of radio telemetry studies . University science library reference training (2 years) . PADI open water SCUBA Certified . Excellent naturalist with experience throughout the US and in the Neotropics