Fall 08

UNDERSTANDING EXPERIMENTALLY-INDUCED AND NATURALLY-

OCCURRING δ2H AND δ13C VARIATION IN A MARINE PREDATOR, THE

BROWN BOOBY

A Thesis

Presented to

The Graduate Faculty of the University of Akron

In Partial Fulfillment

of the Requirements for the Degree

Master of Biology

Madelyn Jacobs

May, 2020 Fall 08

UNDERSTANDING EXPERIMENTALLY-INDUCED AND NATURALLY-

OCCURRING δ2H AND δ13C VARIATION IN A MARINE PREDATOR, THE

BROWN BOOBY

Madelyn Jacobs

Thesis

Approved: Accepted:

Advisor Department Chair Dr. Anne Wiley Dr. Steve Weeks

Committee Member Dean of the College Dr. Brian Bagatto Dr. Linda Subich

Committee Member Acting Dean of the Graduate School Dr. Randall Mitchell Dr. Marnie Saunders

Date

ii ABSTRACT

Refining our understanding of the sources of δ2H variation in terrestrial and marine organisms is a necessary step to expand the usefulness of stable hydrogen isotope values in ecological studies. Marine consumers in particular provide a simplified hydrogen isotope study system in which to test hypotheses concerning non-geographical sources of δ2H variation. The primary goal of our study was to test hypotheses about the source of hydrogen isotope variation in seabirds using manipulative experiments in brown booby chicks and naturally occurring variation in δ2H along a continuum of ages in a colony of brown booby (Sula leucogaster). First, we hypothesized that increased salt ingestion will increase salt gland activity and proportionately more protium will be expelled in salt gland excretions, causing the δ2H of seabird tissues to increase. Second, we hypothesized that increased lipid ingestion will result in proportionately more incorporations of dietary-lipid derived hydrogen into seabird tissues, resulting in lower

δ2H values. A third, related hypothesis regarding carbon isotope variation was tested simultaneously: increased lipid ingestion will result in proportionately more incorporation of dietary-lipid derived carbon into seabird tissues, resulting in lower δ13C values. To test these hypotheses, a population of brown booby (Sula leucogaster) chicks was tube-fed one of two treatments for 14 consecutive days: a high salt solution (3%

NaCl), or a high lipid solution (50% salmon oil). Two groups of chicks were tube-fed normal saline solution (0.9% NaCl) as control groups alongside the treatment groups. We then measured the hydrogen, carbon, and nitrogen isotopic composition (δ2H, δ13C, and

δ15N) of the plasma prior to and after the 14-day manipulation. The change in δ2H plasma

iii values was significantly larger for chicks in the high salt ingestion treatment group compared to the salt control group (0=0.036). The change in plasma δ2H of chicks receiving the high lipid solution treatment did not differ statistically from the change in plasma δ2H observed in the control group (p=0.0702). High salt or lipid ingestion, however, had no apparent effect on δ13C or δ15N plasma values compared to the chicks in the control groups. Our study further aimed to understand whether δ2H values change along a continuum of ages in a seabird species and if amino acid-specific isotope analysis could illuminate the mechanism behind widely reported age-related shifts in δ2H.

Therefore, we compared bulk plasma δ2H values from brown booby chicks to corresponding amino acid-specific δ2H analyses to understand whether some amino acids may dictate patterns in bulk δ2H. In addition, we explored how age affected bulk and amino acid-specific δ2H values within the brown booby colony. We found several lines of evidence that age is an important source of δ2H variation in the brown booby.

Importantly, none of the amino acid δ2H values correlated significantly with chick age in our study. We propose that changes in the amino acid composition of plasma and newly synthesized tissues may cause bulk δ2H values to change with age. To our knowledge, this is the first reported investigation of avian amino acid-specific δ2H values.

iv ACKNOWLEDGMENTS

I would like to acknowledge my advisor, Dr. Anne Wiley, for her unwavering support, optimism, and guidance throughout my master’s program. Her wisdom and thoughtfulness on experimental design and data interpretation were invaluable. In addition, I would like to acknowledge my collaborators that made sample collection and analysis possible. This study was completed in collaboration with Dr. Roxana Torres and

Gala Castro Mejias from the National Autonomous University of Mexico, and Dr.

Kaycee Morra and Dr. Marilyn Fogel from the University of California, Riverside.

Lastly, I would like to thank my lab mates at the University of Akron for their help in sample analysis and data interpretation: Nathan Michael and Ryan Trimbath (PhD candidates), Allison Carpenter and Erin Taylor (undergraduate students).

v TABLE OF CONTENTS

Page

LIST OF FIGURES...... viii

LIST OF TABLES...... ix

CHAPTER

I. UNDERSTANDING THE ISOTOPIC INFLUENCE OF SALT AND LIPID INGESTION IN BROWN BOOBY (SULA LEUCOGASTER) CHICKS ON ISLA MARIETAS, MEXICO...... 1

INTRODUCTION...... 1

MATERIALS AND METHODS

Experimental design...... 8

Sample collection and analysis...... 11

Statistical analyses...... 12

RESULTS

Change in chick plasma hydrogen isotope values...... 13

Change in chick plasma carbon and nitrogen isotope values...... 17

Change in chick body mass...... 17

DISCUSSION

The effect of salt ingestion on δ2H...... 18

The effect of lipid ingestion on δ2H...... 20

The effect of lipid ingestion on δ13C...... 21

SUMMARY...... 23

vi II. EFFECTS OF AGE AND PATTERNS OF AMINO ACID-SPECIFIC

δ2H VALUES IN A COLONY OF BROWN BOOBIES (SULA

LEUCOGASTER)...... 24

INTRODUCTION...... 24

METHODS

Plasma sample collection and preparation...... 27

Prey sample collection and preparation...... 28

Bulk sample analysis...... 29

Amino acid-specific analysis...... 30

Reporting δ2H...... 30

Statistical analyses...... 30

RESULTS

Chick plasma bulk δ2H and chick age...... 31

Prey muscle δ2H...... 34

Amino acid-specific chick plasma...... 34

DISCUSSION

δ2H and age...... 37

Amino acid-specific δ2H...... 43

LITERATURE CITED...... 45

APPENDIX...... 55

vii LIST OF FIGURES

FIGURE PAGE

CHAPTER 1

1. Tube-feeding 50% salmon oil solution to a chick in the lipid treatment

group...... 8

2. Mean change in isotopic composition of brown booby chick plasma post-

manipulation...... 15

CHAPTER 2

1. δ2H values of prey muscle and brown booby plasma and feather samples...... 33

2 2. Brown booby chick plasma amino acid δ H values...... 36

viii LIST OF TABLES

CHAPTER 1 Page

1. Description of tube-feeding treatment solutions and sample sizes...... 9

2. Summary statistics of brown booby chick plasma isotopic change post-

manipulation and supplementary sample δ2H values...... 16

CHAPTER 2

1. Mean and standard deviation of δ2H values for 13 amino acids from brown booby

chick plasma...... 35

ix CHAPTER 1

UNDERSTANDING THE ISOTOPIC INFLUENCE OF SALT AND LIPID INGESTION IN BROWN BOOBY (SULA LEUCOGASTER) CHICKS ON ISLA MARIETAS, MEXICO

INTRODUCTION

Changing climate and industrialized fishing are shifting marine food web dynamics and may be partially responsible for the alarming global decline of seabird populations (69% in 50 years, Paleczny et al. 2015). For example, Croxall et al. (2012) estimate 160,000 seabirds are killed incidentally by longline fisheries each year. In addition, Jones et al. (2018) showed a single marine heat wave event, which disrupted the food web in the Northeast Pacific Ocean, caused mass starvation and mortality in

Cassin’s Auklet populations in 2014 and 2015. Lastly, Bertrand et al. (2012) found significant and immediate negative effects on Peruvian booby (Sula variegata) foraging efficiency and breeding success following the opening of an anchovy fishery off the coast of Peru. These studies highlight the potential for mass, diet-linked seabird mortality during short spans of time. Ecologists further their understanding of seabird diet using seabird regurgitations and stomach contents, which can reflect seasonal and annual shifts to prey availability (see Barrett et al. 2007 review). However, anthropogenically-driven 1 climate change and industrialized fishing have wrought changes in marine ecosystems over the past century, and thoroughly understanding their effects on seabird populations will require studies that detail seabird diet on a decadal to millennial scales.

Stable isotope values of seabirds have become important tools to study long-term shifting marine conditions (Hobson 1999, Kelly 2000, Cherel & Hobson 2007). Stable isotope-based studies take advantage of naturally-occurring patterns in isotope ratios within the environment. For example, there is a stepwise increase in the ratio of nitrogen isotopes as you move up trophic levels; the heavy N isotope (15N) becomes more abundant relative to the light N isotope (14N) in consumers’ tissues with each trophic level increase (DeNiro & Epstein 1981, Fry 2006). Additionally, differential

13 incorporation of the heavy stable carbon isotope ( C) among C3 and C4 plants during photosynthesis produces distinct patterns in δ13C of consumer tissues, depending on which plants fuel the base of their food web (Post et al. 2002, Farquhar et al. 2012).

Variable δ13C values among organisms have helped to delineate food web energetics and carbon cycling (Martinez del Rio et al. 2009). Naturally-occurring carbon and nitrogen isotope variation in seabirds have also allowed ecologists to trace foraging locations and trophic position of seabirds from modern and historic populations (Bearhop et al. 2006,

Votier et al. 2010, Blight et al. 2015, Meier et al. 2017, Ostrom et al. 2017). Importantly, retrospective isotope studies have shown that increased fishing pressures can induce dietary shifts and trophic declines in seabirds; shifts that are sometimes associated with population decline (Becker & Beissinger 2006, Norris et al. 2007, Morra et al. 2019). In wide-ranging, generalist seabirds, these same dietary shifts are interpreted as evidence of

2 large-scale, human-driven change to marine food webs (Wiley et al. 2013, Gagne et al.

2018, Morra et al. 2019).

Environmental patterns in isotope ratios ultimately derive from the chemical and physical differences between heavy and light isotopes of a given element. In general, heavy isotopes react slower and concentrate more readily in strong bonds than do light isotopes (Peterson & Fry 1987, Fry 2006). For example, when water evaporates from the ocean, the light H isotope (protium, 1H) evaporates more quickly than the heavy H isotope (deuterium, 2H). This results in deuterium-depleted water vapor and clouds.

Conversely, when a cloud releases precipitation, deuterium is more easily formed into rain, and as such the cloud becomes more and more deuterium-depleted as it moves inland and away from the equator. The northwest movement of clouds from the Gulf of

Mexico across continental North America generates a hydrogen isotope gradient in which precipitation becomes more and more depleted in deuterium as one moves north and west of the Gulf of Mexico (Kelly 2000, Fry 2006, Rundel et al. 2012). Importantly, the terrestrial gradients in hydrogen isotope ratios (reported as δ2H values) have fueled a large subfield of migration studies, in which songbird populations are linked to isotopically distinct breeding and wintering regions across North America (Chamberlain et al. 1996, Hobson & Wassenaar 1996, Wassenaar & Hobson 2000, Bowen et al. 2015,

Vander Zanden et al. 2016).

In contrast to the strong geographical variation in hydrogen isotope ratios found in terrestrial precipitation (e.g. >200 ‰ range from Mexico to Canada), seawater shows minimal spatial variation (<15 ‰ from Mexican to Canadian waters). Because of the isotopic homogeneity of seawater and marine fish and squid, isotope ecologists

3 previously assumed seabirds would show minimal δ2H variation (Hobson 1999).

Surprisingly, a large range (142 ‰) and distinct patterns of δ2H have recently been observed in seabird populations (Wiley et al. 2012, Quillfeldt et. al. 2017). A similarly large range of δ2H values is present among sympatric colonies of Hawaiian petrel chicks, which derive 100% of their hydrogen from isotopically-homogenous prey (Ostrom et. al.

2014). This finding suggests that seabirds do not simply transfer the hydrogen isotope values from their hydrogen sources into their tissues. Rather, it implies that hydrogen isotope ratios are physiologically altered within seabirds’ bodies. Indeed, if the patterned variation in seabird δ2H can be understood, hydrogen isotopes may provide a new tool for retrospective studies of seabird biology and marine communities. Understanding physiologically-derived δ2H variation among seabirds may also help us to refine hydrogen isotope-based studies of migration, which don’t account for physiological sources of δ2H variation.

The primary goal of our study was to test hypotheses about the source of hydrogen isotope variation in seabirds using manipulative experiments. We chose to investigate these hypotheses using a subpopulation of Brewster’s brown booby (Sula leucogaster brewsteri) chicks on the Long Island of Islas Marietas, Mexico. Brown boobies are a good model species in this study because they are widely distributed and common in tropical waters worldwide. In addition, many brown booby colonies are inhabited year-round, and nests are built on flat, sparsely vegetated terrain, making the exposed chicks relatively accessible. In the Marietas colony in particular, the chick- rearing season is relatively synchronized. This allowed us to work on a large number of chicks of similar age. Using chicks is advantageous over using adults for several reasons.

4 Primarily, all chicks are accessible every day and remain immobile at their natal site until they fledge. Conversely, adults forage at sea and return to the colony only briefly to rest and feed their chick. In addition, adults may incidentally ingest seawater as they forage.

Seawater is a source of hydrogen that may add to hydrogen isotope variation in consumer tissues. Chicks do not ingest seawater, and are limited to one source of hydrogen: the prey regurgitated by their parents. This simplified our study system and allowed us to assess the mechanisms influencing hydrogen isotope variation with fewer confounding variables. Lastly, the brown booby, along with several other seabird populations, are in decline around the Marietas Islands and Gulf of California, potentially due to unsustainable fishing pressure and warming sea surface temperatures (Rebon 1997,

BirdLife International 2016). Our focus on brown boobies on the Marietas Islands provides a starting point for constructing and interpreting decadal-scale isotope chronologies that could reflect local seabird diet and physiology – research that may help to uncover the factors driving local seabird population decline.

We used brown booby chicks to test two hypotheses concerning hydrogen isotope variation in seabirds as well as a third, related hypothesis concerning carbon isotopes values, which are commonly used in animal isotope ecology.

H1: Increased salt ingestion will increase salt gland activity and proportionately more protium will be expelled in salt gland excretions, causing the δ2H of seabird tissues to increase. Because squid and other marine invertebrates are isosmotic with seawater, while teleost marine fish regulate their ionic composition to remain hyposmotic with seawater, the amount of salt a seabird ingests is dependent on the proportion of marine invertebrates (e.g. squid) vs. teleost fish in its diet, and the intake of seawater (Goldstein,

5 2002). After a meal, seabirds rely predominantly on their salt glands to remove excess ingested salt (Goldstein 1993, Schmidt-Nielson 1997). Reabsorption of sodium and chloride ions in the renal system allows marine birds to minimize water lost as urine and to gain more free water by excreting a highly concentrated salt solution from glands in their supraorbital cavity (Peaker & Linzell 1975, Skadhauge 1981, Holmes & Phillips

1985). As this solution is formed and expelled, protium (the light isotope of hydrogen) may move more quickly across the epithelial cell walls and therefore be preferentially used in salt gland excrement. Wiley et al. (2012) has proposed this as the mechanism explaining elevated δ2H values in seabird tissues relative to seabird prey. Indeed, feather

δ2H comparisons among three sympatric seabird populations showed that the proportion of squid in their diets positively correlated with δ2H values (Ostrom et al. 2014).

H2: Increased lipid ingestion will result in proportionately more dietary-lipid derived hydrogen getting/being? incorporated into seabird tissues, resulting in lower δ2H values. In addition to salt ingestion, lipid catabolism may drive variation in seabird δ2H.

Lipids are known to have low δ2H values (Wolf et al. 2009), and lipid-derived hydrogen used to build proteinaceous tissues could similarly result in relatively low δ2H values.

While proteinaceous tissues such as feather and blood plasma are widely used in isotope- based studies, it is still unclear whether proportionately more dietary lipid-derived H is used to synthesize proteinaceous tissues in consumers with a lipid-rich diet. Routing dietary lipids directly to fat stores may be advantageous in adult consumers that ingest a naturally low-lipid diet. These organisms may instead route H found in dietary proteins and carbohydrates to synthesize proteinaceous tissues (Newsome et al. 2017, Curras et al.

2018). As a result, the amount of dietary lipid-derived hydrogen that is incorporated into

6 proteinaceous tissues of these organisms may be low. However, in growing seabird chicks, which have a lipid-heavy diet and a high demand for tissue synthesis, we expect the proportion of lipid-derived hydrogen in their plasma to vary based on the lipid content of their diet.

H3: Increased lipid ingestion will result in proportionately more dietary-lipid derived carbon getting incorporated into seabird tissues, resulting in lower δ13C values.

Just as lipid ingestion may alter hydrogen isotope values of proteins, an analogous effect has been proposed for carbon isotope values, as lipids are depleted in the heavy carbon isotope (i.e. they have very low δ13C). In fact, some isotope ecologists propose that as a field, we underestimate the influence of lipid metabolism on δ13C variation (Tieszen et al.

1993, Post et al. 2007). In many consumers, dietary carbohydrates are preferentially used to meet the demands of energy metabolism, dietary proteins are routed to tissue synthesis, and dietary lipids are either stored for future energy needs or used immediately to meet daily energy demands (Tieszen et al. 1993). Importantly, however, Whiteman et al.

(2012) and Ben-David et al. (2012) have shown that predators that consume little or no carbohydrates, such as seabirds, use lipids to help meet the demands of tissue synthesis.

In fact, Thompson et al. (2000) proposed that liver δ13C variation among several species of seabird was explained by differential lipid ingestion. Seabird species that consumed a more lipid-rich diet presumably synthesized their liver tissue using proportionately more lipid-derived carbon, resulting in significantly lower δ13C values. Therefore, if flexibility in nutrient routing exists in a growing seabird chick with high tissue synthesis demands, we expect proportionately more dietary lipids to be used for tissue synthesis in chicks that consume more lipids. This would be evidenced by proportionately more dietary lipid-

7 derived carbon, and therefore lower δ13C values present in the plasma of brown booby chicks ingesting a lipid-rich diet.

By simultaneously studying the influence of lipid-enriched diets on brown booby

(Sula leucogaster) plasma δ2H and δ13C, we aim to clarify how dietary lipids are incorporated into consumer tissues. Furthermore, in contrast to the previously mentioned studies that manipulate the diet of adult consumers, our study provides novel data regarding the flexibility of nutrient routing and carbon and hydrogen isotope incorporation in a growing organism.

MATERIALS AND METHODS

Experimental design

We used a subpopulation of brown booby chicks (n=28) on Islas

Marietas, Mexico from July 16th-

August 17th, 2018 to assess the isotopic effect of salt and lipid ingestion on blood plasma. Chicks ranged in estimated age from 29 days Figure 1: Tube-feeding 50% salmon oil solution to a old to 59 days old at the onset of chick in the lipid treatment group. Photograph taken by Ytzayanna Peñuñuri Ochoa. manipulation (see appendix for age estimations). The experimental chicks also ranged in

8 mass from 520g (nearly half of a typical adult’s mass) to 1480g (asymptotic chick mass).

Chicks are covered in a thick layer of down after ~21 days, begin growing flight feathers

~40 days, and fledge ~95-110 days of age (Schreiber & Norton 2002).

Table 1. Description of tube-feeding treatment solutions and sample sizes. Chicks were tube-fed their respective treatments once every 24 hours for fourteen consecutive days. The volume of solution given was determined by weighing each chick at least every four days.

Treatment Tube-feeding Total volume given Volume of aqueous Sample group solu4on daily solu4on given daily size (n) Salt 3% NaCl 3% chick body weight 3% chick body weight 9 Salt control 0.9% NaCl 3% chick body weight 3% chick body weight 6 Lipid 50% salmon oil 3% chick body weight 1.5% chick body weight 9 Lipid control 0.9% NaCl 1.5% chick body weight 1.5% chick body weight 4

Chicks were divided into one of four treatment groups (Table 1). To understand whether high salt ingestion elevates δ2H values in brown booby chick plasma, we tube-fed nine chicks an aqueous 3% NaCl solution. The volume tube-fed to each chick was equal to 3% of the chick’s body mass, which was measured at least every 4 days during the study period. To assess the influence of lipid ingestion on chick plasma δ13C and δ2H, we tube- fed nine chicks an aqueous solution of salmon oil (50% oil:50% water by volume, Figure

1). The total volume tube-fed was equal to 3% of each chick’s body mass. We also tube- fed chicks saline (0.9% NaCl) solution in order to control for the influence of water- derived hydrogen being provided to the chicks undergoing the salt and lipid treatments.

The volume of saline solution tube-fed to the salt control group (n=6) was equivalent to the volume of 3% NaCl solution being fed to the salt group chicks (i.e. 3% of the chick’s body mass). The volume of saline solution tube-fed to the lipid-control group (n=4) was

9 equivalent to the volume of water given to the lipid treatment group (1.5% of chick’s body mass). Unfortunately, the sample sizes for some treatment groups (primarily the lipid control group) were smaller than anticipated due to the limited number of chicks at the Marietas colony that were of appropriate age, several unexpected but natural chick deaths during the study period, and sample loss during international shipment to the

University of Akron.

Treatments were tube fed to each chick once every 24 hours for 14 consecutive days. Tube feedings were provisioned only at night (from 7pm to 5am) to minimize interference with diurnal parental care, and the chick was returned to its nest immediately after the treatment (handling time was limited to <1 minute for feeding and <5 minute for blood collection and morphometric measurement collection). The tube-feeding solutions were made using the same, homogenous fresh water source. Chick mass and overall body condition was assessed at least every 4 days to ensure our tube-feeding treatment and daily handling was not restricting normal development. The chicks were left in their natal nest site and we allowed their parents to feed them a normal diet because this study aimed to assess only whether elevated salt or lipid ingestion altered plasma δ2H. These methods could artificially induce increased salt and lipid ingestion for our experimental chicks without otherwise altering their normal diet, thereby minimizing any unintended effects of our manipulations on chick health, development, and isotope ratios. Compared to chicks that were being tube-fed salt or lipid solutions, chicks in the control groups, supplemented only with normal saline solution, likely experienced minimal variation in lipid or salt ingestion from the diet provided by their parents.

10 The experimental water used throughout the duration of the study (for all treatments) was stored in a 5-gallon cooler. Water samples were collected for δ2H analysis immediately before and after the study period to ensure any changes to chick plasma δ2H was not due to shifting experimental water δ2H. Pure NaCl weighed on a digital scale was used to mix 0.9% saline and 3% salt solutions. Salmon oil was mixed with the fresh experimental water to create the high-lipid treatment solution. A sample of salmon oil was also collected for hydrogen isotope analysis. 60ml catheter tip syringes and 40cm long flexible rubber feeding tubes were used to measure and deliver tube- feeding treatments.

Sample collection and analysis

A sample of whole blood (1.5ml) was collected from the brachial vein of each chick prior to and following the two-week tube-feeding treatment period using a 3ml syringe and 22G needle. Whole blood was stored in a vial coated with heparin to prevent coagulation for up to two hours prior to centrifugation. Plasma was then transferred to a vial containing ethanol for storage and remained in ethanol until being prepared for isotopic analysis at the University of Akron.

Once they arrived at the University of Akron, all plasma samples were dried under vacuum to remove ethanol, freeze-dried to remove water, and then continuously washed in chloroform-methanol (87:13) solvent via Soxhlet extraction for at least 6 hours to extract lipids. Lipid extraction of tissues prior to stable isotope analysis is widely used to remove the isotopic influence of tissue-specific lipid content (Post et al. 2007, Wolf et al. 2009, Vander Zanden et al. 2016). Lipid-extracted samples were then weighed into

11 silver capsules alongside reference materials and allowed to equilibrate with ambient air for a minimum of two weeks, enabling us to account for exchangeable hydrogen (Hobson and Wassenaar 2003). Experimental water samples (used to create tube feeding solutions throughout the duration of our study) were sent to the stable isotope ratio facility for environmental research (SIRFER) at the University of Utah (Salt Lake City, Utah) for analysis using a Picarro L2130i Analyzer. Plasma and salmon oil samples were analyzed at the University of Akron with a Vario PYRO Cube elemental analyzer (Elementar

Americas) coupled to an Isoprime100 stable isotope ratio mass spectrometer (Isoprime).

Stable isotope values are reported in delta notation: δX = ([RSample/RStandard] – 1) × 1,000, where X is 2H, 13C, or 15N, R is the corresponding ratio of heavy/light isotope, and

Rstandard is in reference to the internationally accepted standards Vienna Standard Mean

Ocean Water (VSMOW, δ2H), Vienna Pee Dee Belemnite (VPDB, δ13C ), and atmospheric nitrogen (δ15N). Analytical precision of reported δ2H values are within ±1.5

‰, while δ13C and δ15N values are within ±0.2 ‰

Statistical analyses

To assess the impact of diet treatments to individual birds, we focused on the magnitude of change in plasma δ2H (hereafter, ∆2H: δ2H of plasma sample taken after experimental treatment minus δ2H of plasma sample taken before initiation of experiment). We were interested in testing two different, directional hypotheses (the effect of elevated salt ingestion and the effect of elevated lipid ingestion on chick plasma

δ2H), therefore, we compared the mean ∆2H of each “pair” of treatments (salt vs. salt control chicks, and lipid vs. lipid control chicks) using one-tailed t-tests.

12 We performed a power analysis to understand whether our study’s small sample size limited our ability to determine whether our treatments caused significant effects.

We also compared the change in chick plasma δ13C and δ15N values after two weeks of tube feeding (∆13C and ∆15N) between our treatment groups using four separate one- tailed t-tests (change in δ13C for salt vs. salt control, change in δ13C for lipid vs. lipid control, change in δ15N for salt vs. salt control, and change in δ15N for lipid vs. lipid control).

Lastly, we tested the assumptions of all the t-tests performed. We used a Shapiro-

Wilk test to assess whether the residuals in each of our groups was normally distributed.

RESULTS

Change in chick plasma hydrogen isotope values

Prior to manipulation, the range in δ2H values for all chicks (n=28) was from -49 to -8 ‰). There were no significant differences in mean δ2H values between groups before tube feeding began (One-way ANOVA F(3, 24)=1.038 p=0.3738). After tube feeding all chicks their respective treatments for fourteen consecutive days, the range in

δ2H was 52 ‰ (from -54 to -2 ‰). A one-way ANOVA revealed that treatment was a significant source of variation to describe the Δ2H experienced by all groups post- manipulation (F(3, 24)=3.145, p=0.0437).

13

A one-tailed t-test assuming unequal variance revealed a significantly larger magnitude of 2H enrichment in the salt treatment chick plasma (n=9) compared to the salt control chick plasma (n=6, t(13)= 1.97, p=0.0359). The mean Δ2H for the salt treatment chick plasma was 11 ‰, whereas the Δ2H for the salt control chick plasma was 2 ‰

(Figure 2A, Table 2). The Δ2H for both groups was normally distributed (Shapiro-Wilk test, p=0.2004).

We found a marginally significant difference (t(11)= -1.6, p=0.0702) in Δ2H between the lipid treatment chick plasma (mean change= 0.2 ‰, n=9) and lipid control chick plasma (mean change= 6.1 ‰, n=4, Figure 2A and Table 2). The Δ2H for both groups was normally distributed (Shapiro-Wilk test, p=0.9701). An a priori power analysis (with known effect size of 0.85 and power set to 0.8) revealed that a significant difference between these groups (p<0.05) may have been detected if we increased the sample size of the lipid treatment group to 13 chicks and increased the sample size of the control group to 29 chicks. Similarly, an a priori power analysis for the salt treatment and salt control group suggested sample sizes of 18 for the salt treatment group and 28 in the salt control group would have been needed to detect significant changes.

14 Salt control Salt Lipid control Lipid 20 A P=0.0359 P=0.0702 15 H (‰)

2 10 Δ 5

0 Plasma

-5

0.2 B P=0.230 P=0.260

0.0 C (‰) 13

Δ -0.2

-0.4 Plasma -0.6

0.7 C P=0.300 P=0.344

N (‰) 0.2 15 Δ

-0.3

Plasma -0.8 Figure 2. Mean change in isotopic composition of brown booby chick plasma post- manipulation. Panel A: Δ2H, B: Δ13C, and C: Δ15N; all mean values are from lipid- extracted plasma samples after two weeks of being tube-fed 3% saline solution (“Salt”), 50% salmon oil solution (“Lipid”), or 0.9% saline solution (“Salt control” and “Lipid control”). Error bars represent the standard error around each treatment group’s mean.

A post hoc power analysis to compare lipid treated and lipid control groups (alpha value set to 0.05) showed that, although we had a large effect size (0.85), we achieved power of only 37.5% due to our limited sample size.

15 Table 2. Summary statistics of brown booby chick plasma isotopic change post- manipulation and supplementary sample δ2H values A: Mean change, standard deviation, and standard error in hydrogen, carbon, and nitrogen isotope values in brown booby chick plasma after two weeks of tube-feeding treatments. Rows are separated by treatment group (see text for further detail). Table abbreviations are as followed: n= sample size, SD= standard deviation around the mean of each group, and SE= standard error around the mean of each group. B: δ2H values of the fresh experimental water and the salmon oil being tube fed during the study period, and δ2H of a sample of ocean water from the Marietas colony site. 2 Experimental water δ H did not change during the study period.

A Treatment n Mean (‰) SD (‰) SE (‰) p-value

Salt control 6 2 8.18 2.34 0.0359*

H Salt 9 11 9.76 3.25 2 Δ Lipid control 4 6 4.57 2.29 0.0702 Lipid 6 0 8.63 2.88

Salt control 6 -0.2 0.33 0.14 0.230

C Salt 9 -0.3 0.55 0.18 13

Δ Lipid control 4 0. 1 0.29 0.15 0.264 Lipid 6 -0.3 0.44 0.15

Salt control 6 -0.2 1.067 0.43 0.300

N Salt 9 0.1 0.295 0.98 15

Δ Lipid control 4 -0.1 0.285 0.14 0.344 Lipid 6 -0.1 0.284 0.95

B Sample type δ2H (‰) SD (‰)

Experimental water -46 1 Salmon oil -212 1 Ocean water at Marietas 0 0

The δ2H of the experimental water used to create tube-feeding solutions throughout the duration of our study changed by only 2‰ (δ2H values of experimental

water collected on: July 17th = -45‰, and August 5th = -47‰). Samples were measured in triplicate at the stable isotope ratio facility for environmental research (SIRFER) at the

University of Utah (USA). Standard deviations for the water δ2H values fell below analytical uncertainty (0.2‰). 16 Change in chick plasma carbon and nitrogen isotope values

We found no significant differences in the Δ13C or Δ15N between salt treated and salt control chicks (t(13)= 0.76, p=0.230 and t(13)= 0.60, p=0.300, respectively) (Figure

2B & C). The mean Δ13C for the salt treatment chick plasma was 0 ‰, while the mean

Δ13C for the salt control group plasma was -0.2 ‰ (Table 2). The plasma Δ15N for the salt treatment and the salt control groups was 0.1 ‰ and -0.2 ‰, respectively.

Similarly, there were no significant differences in the plasma Δ13C or Δ15N between the lipid treatment and lipid control chicks (δ13C t(11)= -0.66, p=0.264 and δ15N t(11)= 0.42, p=0.344)(Figure 2B & C, Table 2). The mean Δ13C for the lipid treatment chick plasma was -0.3 ‰, while the Δ13C for the lipid control chick plasma was -0.1 ‰.

The Δ15N for the lipid treatment and lipid control groups was equal (-0.1 ‰).

Change in chick body mass

The range in chick body mass prior to manipulation was 1260g (from 520g to

1780g), however, no significant differences existed in mean chick body mass between groups (one-way ANOVA F(3, 24)=0.222, p=0.880). In fact, the mean body mass of chicks in each treatment group prior to manipulation varied by less than 200g (mean mass in lipid group=1073g vs. mean mass in lipid control group=1200g). After two weeks of tube feeding, the total range in chick body mass was 480g (from 980g to 1460g). The mean body mass of the chicks did not differ significantly by group, post-manipulation

(one-way ANOVA F(3, 24)=1.033, p=0.397).

17 In addition, the mean change in mass did not differ by treatment group, post- manipulation (one-way ANOVA F(3, 24)=1.636, p=0.2086).

DISCUSSION

The effect of salt ingestion on δ2H

Chicks receiving the 3% salt solution treatment had significantly elevated plasma

δ2H values compared to chicks receiving the control (0.9% normal saline solution) treatment (p=0.0378). In addition, the magnitude of change in plasma δ2H values in the salt treatment chick group was 9 ‰ larger, on average, than in the salt control group

(p=0.0359, Figure 2A). These findings provide support for my high salt load hypothesis: increased salt ingestion did apparently elevate the δ2H of brown booby chick plasma.

Seabird salt load is determined by diet composition (the proportion of isosmotic squid and invertebrates consumed) and seawater ingestion. For example, fairy prions

(Pachyptila turtur) and common diving petrels (Pelecanoides urinatrix), which rely heavily on krill for prey, consume approximately 170mmol of sodium per kilogram of body mass per day (Green and Brothers 1989). Conversely, a species that consumes primarily teleost fish such as the Buller’s shearwater only consumes 9mmol of sodium per day (Wiley unpublished data, Gould et al. 1997). Brown boobies are opportunistic feeders that can rely on both fish and squid as prey (Schreiber & Norton 2002). However, we have observed minimal within-year variation in chick provisions over several years of

18 observation and prey regurgitation sampling at the Marietas colony. Overwhelmingly the most common prey regurgitated by adults on Marietas are flying fish species

(Exocoetidae) and a single squid was sampled from only one nest site, once, in 2016

(roughly 150 prey items were sampled opportunistically from regurgitations in 2016,

2017, and 2019). It is therefore likely that the salt load of adult brown boobies on

Marietas is consistently low (relative to other species of seabird). If we make a similar assumption about the natural diet of the chicks in our feeding study, chicks in the salt treatment (provided 3% NaCl solution in a volume equal to 3% of the chick’s body mass) only ingested an additional 17mmol Na, compared to the chicks in the salt control treatment. This difference in salt load is relatively small compared to the variation in salt ingestion found naturally between species, yet it produced a 9 ‰ increase in δ2H. To our knowledge, this is the first experimental study to show a direct effect of salt load on hydrogen isotope values.

The large effect size we achieved after two weeks of manipulation within a single study species may have broader implications for the use of hydrogen isotopes in seabird studies and terrestrial migration studies. For example, Farmer et al. (2004) aimed to delineate arctic breeding grounds for three species of sandpipers using feather samples collected along the coast of Argentina, where they overwinter. The study found hydrogen isotopes alone to be an unreliable predictor of origin for all three species, and noted significant inter-individual variability in δ2H. Similarly, Reudink et al. (2016) found more than 100 ‰ range in δ2H values within a colony of American white pelicans. In both studies, variation in salt load may explain inter-individual δ2H variation that is not linked to geographic location. Our experiment provides evidence that salt ingestion may impact

19 tissue δ2H values. Hydrogen isotope-based studies of migration in salt gland-bearing birds should recognize variable salt load as a potential source of inter-individual δ2H variation.

The effect of lipid ingestion on δ2H

Surprisingly, the change in plasma δ2H of chicks receiving the 50% lipid solution treatment (Δ2H= 0.23 ‰) did not differ statistically from the Δ2H of the lipid control chicks receiving the 0.9% saline solution treatment (6.11 ‰, p=0.0702, Figure 2A).

However, our ability to detect a significant difference between the lipid treatment and lipid control groups was hampered. Unfortunately, obtaining low statistical power is fairly common in behavioral studies with limited sample sizes. Jennions and Moller

(2003) surveyed 697 papers from 10 ecology and behavioral biology journals and conducted power analyses for the statistics reported within each paper. For studies reporting non-significant findings alongside large effect size (n=219), less than half of the papers obtained statistical power of 80%. In addition, only 10-20% of all the papers reviewed (n=697) achieved statistical power above 80% in their study. In our study, we achieved an isotopic effect that was near-significant, characterized by a large effect size

(nearly 6 ‰ offset between treatment groups), and the lack of significance is potentially a result of limited statistical power.

Newsome et al. (2017) and Curras et al. (2018) recently explored the fate of dietary lipid-derived hydrogen in fresh water tilapia and mice using controlled feeding experiments. Both studies determined that dietary lipids have minimal (<2%) influence on the bulk hydrogen isotope values of proteinaceous tissues when consumers were

20 provided either a high protein or high carbohydrate diet. Importantly, both focal species in these studies have a diet primarily comprised of proteins and carbohydrates. The lipid content of the diets in both studies remained constant and was only 2-8% of the total diet.

Neither study manipulated the lipid component of diet to ascertain whether the proportion of lipid-derived hydrogen being incorporated into proteinaceous tissues would change.

The brown booby chicks in our study naturally consume almost no carbohydrates, and therefore must rely on some dietary lipids to meet tissue synthesis demands

(Schreiber and Norton 2002, Whiteman et al. 2012). Though our results aren’t conclusive, we argue that a 6 ‰ effect size in this small dataset, alongside a near- significant result achieved with limited statistical power suggests that dietary lipid ingestion may well have an impact on lipid-derived hydrogen isotope incorporation in brown booby chick plasma. We suggest another manipulation study with larger sample sizes and higher statistical power to better understand whether incorporation of dietary lipid-derived hydrogen is plastic in brown booby chicks and whether that plasticity may be reflected in variable δ2H values. Further study will additionally allow us to determine whether flexible dietary lipid incorporation is unique to growing seabird chicks, or a more widespread phenomenon.

The effect of lipid ingestion on δ13C

The plasma δ13C of chicks receiving the lipid treatment was not significantly different from the plasma δ13C of chicks in the lipid control group after the 2-week study period. In fact, the difference in mean post-manipulation δ13C between lipid treated and the lipid control groups fell within our analytical error. In addition, the Δ13C for both

21 groups was similar and the difference between the treatments was negligible. Though our statistical power to detect significant differences between these groups is low, the nearly identical carbon isotope values in these groups suggests the proportion of dietary lipid- derived carbon incorporated into brown booby chick plasma was not affected by additional lipid ingestion.

Newsome et al. (2014) and Wolf et al. (2015) showed that altering the lipid and protein content of diet in mice affected how macronutrients in the diet were used and concurrently altered carbon isotope values of proteinaceous tissues. Specifically, mice given a low protein/high lipid diet routed proportionately more dietary lipid-derived carbon to synthesize hair, blood, liver, and muscle tissues. Importantly, both studies utilized amino acid-specific carbon isotope analysis to understand how nutrient routing and isotopic incorporation differed in each diet treatment. Ben-David et al.’s (2012) diet manipulation study using captive mink was unable to show increased lipid-derived carbon incorporation into red blood cells using lipid-rich diet treatments and bulk tissue carbon isotope analysis. Ben-David et al. (2012) argued that amino acid-specific carbon isotope analysis is necessary to understand the complex nature of nutrient routing and isotope incorporation in consumers with variable diets. Our limited data set, which shows no marked differences in bulk plasma δ13C, suggests the additional lipids we supplied to the chicks in the lipid treatment may have been used immediately or stored as endogenous reserves, and not used to synthesize proteinaceous tissues. However, to determine the true nature of macronutrient routing and isotopic incorporation in brown booby chicks, we agree that a higher level of resolution provided in amino acid-specific isotope analysis is warranted.

22 SUMMARY

Refining our understanding of the sources of δ2H variation in terrestrial and marine organisms is a necessary step to expand the usefulness of stable hydrogen isotope values in ecological studies. Marine consumers in particular provide a simplified hydrogen isotope study system in which to test hypotheses concerning non-geographical sources of δ2H variation. In this study, we show a significant effect of salt ingestion and a marginally significant effect of lipid ingestion on plasma δ2H values in juvenile brown boobies. Further exploration of how salt and lipid ingestion impact seabird tissue δ2H is a necessary component of advancing stable hydrogen isotope ecology in marine systems.

Understanding patterns of stable hydrogen isotope values alongside carbon and nitrogen isotope values in marine consumers may provide a powerful new perspective on marine food web dynamics. In addition, refining our understanding of how lipid and salt ingestion affects seabird δ13C and δ2H values through compound-specific isotope analysis may illuminate previously uncharacterized patterns of nutrient routing and may help explain unresolved isotopic variation in bulk tissues. By improving our ability to study the biology of seabirds using stable isotopes, ecologists can gain further insight into how seabird diets have changed over time and identify human-caused impacts to local and large-scale shifts in marine food web energetics.

23 CHAPTER II

EFFECTS OF AGE AND PATTERNS OF AMINO ACID-SPECIFIC δ2H VALUES IN

A COLONY OF BROWN BOOBIES (SULA LEUCOGASTER)

INTRODUCTION

Hydrogen isotope ratios of terrestrial animal tissues (reported as δ2H values) can act as intrinsic biomarkers of geographic location and have fueled a large subfield of studies on animal migration (e.g.Wolf et al. 2009, Vander Zanden et al. 2016, Hobson and Wassenaar 2018). This subfield is founded on the observation that the δ2H of precipitation declines at higher latitudes and elevations. For example, the δ2H of precipitation in North America ranges from -19 ‰ in coastal Texas to -219 ‰ in Arctic

Canada (Meehan et al. 2004). This location-specific hydrogen isotope value is transferred to the tissues of animals that consume local water and food. Analyzing δ2H values in consumer tissues can indicate where “source” hydrogen originated, and therefore where that consumer resided while synthesizing its tissues. Although hydrogen isotope values in consumer tissues have been widely adopted as tools to study migration patterns,

24 unexplained variation within and between individuals of the same animal population belies our still limited understanding of hydrogen isotope systematics. Recently, ecologists have begun to study δ2H in marine systems. Unlike water in terrestrial systems, ocean water δ2H is largely invariable. The 200 ‰ range in δ2H found across terrestrial North America (ca. 27° to 55°N) is accompanied by <15 ‰ range in seawater

δ2H over the same latitudinal range (Meehan et al. 2004, Xu et al. 2012). In addition, marine top predators, such as seabirds, consume prey that is relatively homogenous with regard to δ2H (Ostrom et al. 2014). In the present study, we used a colony of brown boobies (Sula leucogaster) on Long Island, Islas Marietas, Mexico to investigate potential sources of δ2H variation in a top predator with negligible geographic and dietary

δ2H variation.

Isotope biogeochemists and ecologists have recently expanded the use of compound-specific stable isotope analysis to refine stable isotope-based studies of animal ecology (Close et al. 2019, Whiteman et al. 2019). Traditional “bulk” stable isotope analyses, used in most isotope ecology studies, report a singe δ value from a homogenized tissue sample. In reality, these tissues are a complex mixture of compounds, each with a unique biosynthetic pathway and a potentially unique isotope ratio. Analysis of compound-specific δ values increases our ability to understand the complex nature of tissue synthesis, resource use/nutrient routing, and sources of bulk- tissue isotope variation. Amino acid-specific isotope analysis has become particularly useful for distinguishing between sources of nitrogen isotope ratio variation in consumer tissues. For example, the bulk δ15N of consumer tissues is commonly used to understand food web dynamics because δ15N becomes consistently elevated at each trophic level. 25 However, spatiotemporal variation in nitrogen cycling adds a second source of δ15N variation. Because some amino acids show dramatic increases in δ15N with trophic level, while others reliably reflect the δ15N at the base of the local food web, amino acid- specific δ15N analysis has allowed ecologists to distinguish between trophic level and geographical impacts on δ15N (Nielsen et al. 2015, McMahon and McCarthy 2016). For example, Ostrom et al. (2017) and Morra et al. (2019) were able to provide strong evidence of trophic declines in the North Pacific seabirds over the past century based on amino-acid-specific δ15N, discounting a shift in nitrogen cycling dynamics as a potential driver of seabird nitrogen isotope ratios through time.

As a field, isotope ecologists are beginning to recognize the usefulness of amino acid-specific carbon and nitrogen analyses (Ohkouchi et al. 2017, Thorp et al. 2017, Won et al. 2018, McMahon and Newsome 2019). However, amino acid-specific hydrogen analysis in isotope ecology studies remains relatively rare. In fact, Fogel et al.’s 2016 study on E. coli was the first published record of individual amino acid δ2H values.

Understanding how δ2H varies in amino acids and what amino acids drive the patterns of bulk δ2H variation may provide substantial gains in the field of hydrogen isotope ecology.

In addition to understanding amino acid-specific δ2H variation and its relationship to bulk δ2H, we also investigated the influence of the organism’s age on the δ2H composition of two commonly-sampled consumer tissues: blood plasma and feather.

Many studies have reported an offset in juvenile and adult δ2H values in terrestrial and freshwater organisms. Though widely reported, the finding that juvenile tissues typically have a lower δ2H value compared to adults is rarely a primary focus of studies and 26 therefore, the mechanism(s) that drives this offset is largely unexplored. In addition, nearly all studies that report juvenile and adult δ2H values only report the age class of individuals. Understanding patterns of age-related δ2H variation in marine organisms could improve our ability to interpret patterns of δ2H in both marine and terrestrial organisms. Our study aimed to understand whether δ2H values change along a continuum of ages and if amino acid-specific isotope analysis could illuminate the mechanism behind any age-related shifts in δ2H. Therefore, we first aimed to compare bulk plasma

δ2H values from brown booby chicks to corresponding amino acid-specific δ2H analyses to understand whether some amino acids may dictate patterns in bulk δ2H. In addition, we explored how age affected bulk and amino acid-specific δ2H values within the brown booby colony.

METHODS

Plasma sample collection and preparation

I collected plasma samples from a subpopulation of brown booby chicks (n=27), immatures (n=9), and adults (n=22) on Long Island, Islas Marietas, Mexico from June through August in 2018. 30 additional chick plasma samples were collected from the same colony in July and August of 2016 by my collaborators, and were included in this study’s analyses. Chicks ranged in age from an estimated 20 to 52 days old (appendix).

Immatures were classified as a distinct age class based on several morphological features

27 that are present only after 75 days of age (e.g. fully grown flight feathers, minimal post- natal down present, mobility outside nest site).

1.5ml of whole blood was collected from the brachial vein of each individual using a 3ml syringe and 22G needle. Whole blood was stored in a vial coated with heparin to prevent coagulation for up to two hours prior to centrifugation. Plasma was then transferred to a vial containing ethanol for storage and remained in ethanol until being prepared for isotopic analysis at the University of Akron.

Prey sample collection and preparation

Regurgitated brown booby prey were collected opportunistically throughout the colony and analyzed for bulk δ2H to test the assumption that this colony of brown boobies consumed prey with relatively invariant δ2H values. Muscle biopsies were collected from whole prey regurgitations and were stored in ethanol until arrival at the

University of Akron. For the purposes of this study, prey type classification was assigned based on morphological features identified in the field. However, collaborators A. Welch and E. Bonillas-Monge (University of Durham) are currently working to provide taxonomic identification of the prey morphotypes using next-generation sequencing.

Once they arrived at the University of Akron, all plasma and prey samples were dried under vacuum to remove ethanol, freeze-dried to remove water, and then continuously washed in chloroform-methanol (87:13) solvent for at least 6 hours to extract lipids via Soxhlet extraction. Prey muscle samples were then homogenized by grinding to a fine powder.

28 Feather sample collection and preparation

I collected barbs from various locations along the third tail feather (R3) of 10-13 week old immature brown boobies (n=15). Tail feathers of brown booby chicks erupt at

~40 days of age and continue to grow until ~70 days old, reaching a length of ~120mm.

Approximately five barbs were sampled from four locations along the tail feather: tip of the rachis (0mm, grown at 40 days of age), tip-middle (20mm from the tip of the rachis, grown at 45 days of age), middle (60mm from the tip of the rachis, grown at 55 days of age), and base (100-120mm from the tip of the rachis, grown at age 65-70 days). Though

15 individual immatures were captured for sampling, not every barb sample collected was analyzed for δ2H values.

Once they arrived at the University of Akron, feather barbs were washed in chloroform-methanol (87:13) solution to remove contaminants, rinsed with ultrapure distilled water (E-pure, Barnstead) and allowed to dry.

Bulk sample analysis

Following preparation, samples were weighed into silver capsules alongside reference materials and allowed to equilibrate with ambient air for a minimum of two weeks, enabling us to account for exchangeable hydrogen (Hobson and Wassenaar 2003).

All samples were analyzed at the University of Akron with a Vario PYRO Cube elemental analyzer (Elementar Americas) coupled to an Isoprime100 stable isotope ratio mass spectrometer (Isoprime).

29 Amino acid-specific analysis

Following lipid extraction, a subset of chick plasma samples (n=10) was sent to the University of California, Riverside for amino-acid specific analysis. Sample preparation, analysis, and δ2H corrections closely followed methods described in Fogel et al. (2016) and O’Brien et al. (2002). Briefly, dry plasma samples were hydrolyzed to amino acids in 6N hydrochloric acid, and then derivatized to N-trifluoroacetic acid isopropyl esters. The δ2H values of thirteen amino acids were measured in triplicate alongside reference material using a Thermo-fisher Trace Gas Chromatograph.

Reporting δ2H

2 Stable isotope values are reported in delta notation: δ H = ([RSample/RStandard] – 1) ×

2 1 1,000, where R is the ratio of H to H, and Rstandard is in reference to the internationally accepted standard Vienna Standard Mean Ocean Water (VSMOW). Analytical precision of reported δ2H values are within ±1.5 ‰ for bulk tissue samples and within 4 ‰ for amino acids.

Statistical analyses

I conducted multiple bivariate least squares linear regressions to assess the influence of age (predictor variable) on bulk and amino acid-specific δ2H values

(response variable). I tested the residuals of the bulk and amino acid δ2H linear regression for normal distribution. Leucine and glutamine δ2H values followed a non-normal

30 distribution, and therefore were transformed before analysis (Johnson SI transformation).

Bulk and all remaining amino acid-specific δ2H values followed a normal distribution.

I also performed several least square linear regressions to understand how much of the variation in bulk δ2H values could be explained by the variation among individual amino acid δ2H values. In all cases, Bonferroni corrections were used to account for increased type I error associated with running multiple linear regressions. In addition, I used a one-way ANOVA to determine whether immature feather barb mean δ2H values differed by feather barb location (tip, tip-middle, middle, and base). Immature feather

δ2H data was tested for homogeneity of variance and were transformed to correct for non- normal distribution (SHASH transformation). All statistical analyses were performed using JMP®Pro version 14.0. Mean δ2H values are reported in the text with their associated standard deviations (SD).

RESULTS

Chick plasma bulk δ2H and chick age

Age was a significant predictor of brown booby plasma δ2H. Among brown booby chicks, estimated age was positively correlated to bulk plasma δ2H values (Figure 1A, linear regression R2=0.599; p<0.0001; n=57) I combined 2016 and 2018 chick plasma samples in order to perform one linear regression because there was no significant effect

31 of sampling year (t-test: df =1; p=0.32). Residuals of the regression were normally distributed (Shapiro Wilk test, p=0.645).

Age class was also a significant predictor of bulk plasma δ2H values. Mean adult and immature plasma δ2H values were significantly higher than mean chick plasma δ2H values (one-way ANOVA F(2,87)=173.96, whole model p<0.0001; mean δ2H plasma values ± SD of chicks = -43±10 ‰, n=57; immatures = -7± 6 ‰, n=9; adults = -5± 7 ‰, n=23; Tukey’s post-hoc comparison of means: p<0.0001 for chick-immature and chick- adult comparisons, p=0.806 for immature-adult comparison).

Lastly, I found a similar pattern of increasing δ2H values with increasing age when analyzing SHASH transformed immature feather barb δ2H values. Feather barb location (“tip”, “tip-middle”, “middle”, and “base”), which correlates to the age at which the feather barbs were grown, was a significant predictor of mean δ2H value (Figure 1B,

ANOVA F(3, 39)=35.13, p<0.0001). Barbs that were collected from the tip of the tail feather (n=5), grown ~40 days post-hatch, had a significantly lower mean δ2H value

(mean δ2H± standard deviation= -25 ±9 ‰) than feather barbs grown at any other age (tip vs. tip middle δ2H p=0.002; tip vs. middle δ2H p<0.0001; tip vs. base δ2H p<0.0001).

Additionally, the mean δ2H of feather barbs grown ~45 days post-hatch (“tip-middle”, n=13, mean ± SD= -1 ±6 ‰) were significantly lower than the mean δ2H of feather barbs grown ~55 days post-hatch (“middle”, n=13, mean ± standard deviation= 6 ±6 ‰, p=0.0027). Feather barbs grown ~70 days post-hatch (“base”, n=12, mean δ2H ± SD= 4

±5 ‰) did not differ from either middle feather barb δ2H locations analyzed (base vs. middle p=0.5391, base vs. tip-middle p=0.0980).

32 A 20

0 R2=0.59897 P<0.0001 -20

-40 H (‰) 2

δ -60 20 30 40 50 60 -80 Age in days -100 Prey Chick Immat Adult (25) (57) (9) (23) B 20 C 10 B,C B 0 -10 H (‰) 2

δ -20 A -30 -40 Tip Tip-Mid Middle Base (5) (13) (13) (12)

Increasing age

Figure 1. δ2H values of prey muscle and brown booby plasma and feather samples A: δ2H values of lipid-extracted prey muscle and brown booby chick, immature, and adult plasma (from left to right). Sample sizes are described in parentheses next to data label along the x-axis. Chick plasma δ2H values are plotted against estimated chick age and a line of best fit was applied. Reported R2 and p-values derive from a linear least square regression analysis. B: Mean (+/- std err) δ2H feather values from immature brown booby tail feathers. Feather barbs were collected from various locations along the feather, from the tip of the rachis (left) to the base (right). Sample sizes are represented in parentheses next to the label along the x-axis. Unique letters next to data points indicate significant differences between groups (ANOVA and Tukey’s post hoc analysis, p<0.05).

33 Prey muscle δ2H

As anticipated, analysis of 25 prey muscle samples representing nine different fish morphotypes showed minimal variation in δ2H (range of δ2H values <30 ‰, average

±SD= -88.56 ±7 ‰). Mean prey δ2H values were significantly lower than mean chick, immature, and adult bulk plasma δ2H (one-way ANOVA F(3,111) = 438.8, p<0.0001).

Amino acid-specific chick plasma

Mean proline δ2H was elevated by at least 350 ‰ relative to all other amino acids and also showed the largest variation among individual brown boobies (Figure 2, Table 1;

δ2H ranged from 628 to 1,214 ‰). Following proline, threonine had the highest δ2H values but was much less variable, ranging from 200 to 271 ‰. Excluding threonine, all essential amino acids had relatively low δ2H (mean δ2H: -100 to -170 ‰) and were relatively invariable (SD ≤30 ‰; Table 1).

No amino acid δ2H values showed a significant relationship with chick age. The highest correlation coefficients were found for leucine (R2=0.313), glycine (R2=0.294), and proline (R2=0.271). However, none of these correlations were significant once a

Bonferroni-adjusted alpha value of 0.0038 was considered (all p values > 0.091).

34 Though the variation in proline δ2H did not correlate with chick age, proline δ2H very tightly correlated with bulk plasma δ2H (R2=0.842, p=0.0002, below the adjusted alpha value of 0.0038, Figure 2B). The only other amino acid that showed any potential relationship to bulk plasma δ2H was glycine ((R2=0.3833, Figure 2C), however, this relationship was not significant (p=0.0561).

Table 1. Mean and standard deviation of δ2H values for 13 amino acids from brown booby chick plasma. n=10 for each amino acid, except for argenine (n=9). Standard deviation is abbreviated as SD in table.

Abbr Amino Acid Mean δ2H (‰) SD (‰) Ileu Isoleucine -189 15 Leu Leucine -141 10 Lys Lysine -101 7 Phe Phenylalanine -171 24

Essen8al Thr Threonine 241 25 Val Valine -122 14 Arg Argenine -101 28 Pro Proline 902 168 Gly Glycine -32 21 Ser Serine -107 29 Asp AsparCc acid -21 19

Non-Essen8al Glu Glutamine -7 11 Ala Alanine 74 19

35 A 1400 Essential Non-Essential 1200

1000 H (‰)

2 800

600

400

200

Amino acid δ 0

-200

-400 Ileu Leu Lys Phe Thr Val Arg Pro Gly Ser Asp Glu Ala B -20 C R² = 0.84186 R² = 0.3833 -25 p=0.0002 p=0.0561

H (‰) -30 2 -35

-40

Bulk δ -45

-50 600 800 1000 1200 -80 -60 -40 -20 0 Proline δ2H (‰) Glycine δ2H (‰)

Figure 2. Brown booby chick plasma amino acid δ2H values A: δ2H values of thirteen amino acids from brown booby chick plasma (n=10, n=9 for Arg). B: Univariate linear least square regression and line of best fit for brown booby chick plasma proline δ2H vs. bulk plasma δ2H values (Bonferoni-corrected alpha value=0.0038). C: Univariate least square regression and line of best fit for brown booby chick plasma glycine δ2H and bulk plasma δ2H (correlation is not statistically significant; p>adjusted alpha of 0.0038).

36 DISCUSSION

δ2H and age

We found several lines of evidence that age is an important source of δ2H variation in the brown booby. First, by comparing three distinct age classes (chick, immature, and adult), we found that the mean bulk δ2H plasma values of chicks were significantly lower than those of immature and adult brown boobies. This observation aligns with other studies that have shown δ2H values are lower in juvenile bird tissues compared to adult tissues (e.g. Meehan et al. 2003, Smith and Duffy 2005, Langin et al.

2007, Hatche et al. 2012, Studds et al. 2012, and Ostrom et al. 2014). Notably, in our study immature plasma δ2H (from chicks that were over 75 days of age) values were not significantly different from adult δ2H values. Second, the δ2H values of bulk chick plasma increased linearly with chick age (for chicks aged 20 to 60 days). Lastly, feather barb δ2H values from the tip of immature brown boobies were significantly lower than the δ2H of more proximal and more recently grown feather barbs. Meanwhile, the δ2H values of feather barbs from the base of the feather (grown last) did not differ from feather barbs sampled from the middle of the feather. Therefore, the positive relationship between δ2H values and age appears to be most pronounced for younger individuals undergoing rapid growth and dissipates during the latter stages of development.

Importantly, none of the amino acid δ2H values correlated significantly with chick age in our study. This finding suggests that the pattern of increasing δ2H with age 37 may be independent of amino acid isotopic values. We discuss three non-exclusive hypotheses to explain these results: 1) an increase in evaporative loss with age, 2) a shift in how nutrients are routed from diet to tissues, and 3) a shift in the amino acid composition of tissues with age.

The pattern of δ2H increasing with age may be explained by increased evaporative water loss. As chicks grow, the amount of water lost via evaporation increases (Nagy

1988, Withers 1992). Water that evaporates due to respiration has lower δ2H value than water that remains in an animal’s body (McKechnie et al. 2004). This body water δ2H may be transferred to the δ2H values of various tissues as they are synthesized. Several previous studies have proposed increased evaporative water loss as an explanation for observed offsets between juvenile and adult δ2H (Smith and Duffy 2005, Powell and

Hobson 2006, Hatche et al. 2012). Indeed, experimental manipulation by McKechnie et al. (2004) showed rock doves placed in an unnaturally hot environment and experiencing four times more evaporative water loss than doves kept in thermoneutral conditions had significantly elevated whole blood δ2H values.

Ostrom et al. (2014), however, report δ2H values of seabird feathers that do not align with the evaporative water loss hypothesis. They show that Hawaiian petrel chicks from two different colonies had very different δ2H values (86 ‰ offset in mean δ2H), despite residing in similar environmental conditions and presumably experiencing similar rates of evaporative water loss. Furthermore, our brown booby data fail to fully support this hypothesis. Though our brown booby chicks did have significantly lower δ2H values than adults, and likely experienced less evaporative water loss due to inactivity (sitting in a nest compared to flying and foraging adults), our immature brown booby plasma was

38 not significantly different from adult plasma. If evaporative water losses related to activity levels dictate patterns of δ2H variation, we would expect the plasma δ2H of pre- fledging immatures to be lower than full-fledged adult δ2H. Lastly, if increased evaporative water loss increases δ2H values, we would expect to see increasing δ2H values of amino acids as chicks’ age.

A second potential explanation for the age-δ2H relationship observed in bulk chick plasma and feathers may lie in the physiological and morphological changes that occur throughout a chick’s development. Specifically, shifts in dietary resource use during chick development may explain the observed effect of age on bulk δ2H values. A review by Vander Zanden et al. (2016) argues that the flexible nature of dietary nutrient routing may be an important factor in understanding the unexplained variability of δ2H values among consumer tissues in terrestrial studies. Seabird chick livers, kidneys, and gut increase in size and functional ability during early development in order to help chicks digest prey, synthesize structural tissues, and meet growing metabolic demands

(Ricklefs and Schew 1994, and Ricklefs 1979). For example, a study on northern fulmar seabird chicks revealed chick liver lean mass increased from 2g to over 10g in only 20 days during the chicks’ most rapid growth phase (Phillips and Hammer 2000). As these digestive organs grow, the ability for chicks to utilize dietary protein may simultaneously increase. In addition, seabird chicks deposit proportionately more dietary lipids as fat reserves as they near fledging (Ricklefs 1994, Phillips and Hammer 2000, Navarro et al.

2015). Routing dietary lipids directly to adipose tissue minimizes the potential for dietary lipid-derived hydrogen to be incorporated into proteinaceous tissues.

39 If young brown booby chicks transition from synthesizing proteinaceous tissues using dietary lipids (which have consistently low δ2H) to utilizing proportionately more dietary proteins (which have higher δ2H values) as they age, we would expect the δ2H values of the chick’s tissues to increase. This does explain the relationship we observed between bulk plasma δ2H values and age; bulk plasma δ2H values increased linearly with age in chicks undergoing rapid growth, while plasma δ2H values of immature brown boobies that approximated adult body size did not differ from adult plasma δ2H values.

Similarly, Soto et al. (2011) found a significant positive relationship between catfish

(Siluris glanis) length and muscle δ2H values. Fish are indeterminate growers; therefore, length can be used as a proxy for age. However, the rate of growth declines as total fish length increases. Soto et al. (2011) report significant increase in δ2H values of catfish between 50 and 1,000mm in length (during rapid growth phase), and a plateau in δ2H values in catfish between 1,000 and 2,000mm long (during slower growth phase). They argue that this shift in δ2H values during fish growth signifies a shift in dietary resource use. Young fish that grow very rapidly rely more heavily on dietary lipids to meet tissue synthesis demands. Meanwhile, older, slower-growing fish can meet all tissue synthesis demands by utilizing only dietary protein, saving dietary lipids for immediate metabolic or future energy demands.

Unlike fish body length increases, feather growth in immature brown boobies occurs mostly during the rapid, linear chick growth phase. Chick growth only slows towards the end of feather growth (i.e. feather barbs near the base of the feather rachis are grown when a chick has reached its’ fledgling weight). If growth rate influences dietary nutrient routing and δ2H values in the brown booby in the same way as Soto et al. argue 40 for fish, we would expect to have seen a linear increase in feather δ2H values from tip to base, signifying a shift to proportionately more dietary protein utilization as a chick grows. Instead, only the tip of the feather had significantly lower δ2H values from all other feather sections, and changes in feather δ2H values appeared to plateau (i.e. basal feather barb δ2H did not differ from the middle portions of the feather). In addition, our amino acid-specific δ2H data fails to support the nutrient routing hypothesis. If variation in δ2H values primarily reflects a shift in dietary resource use, we should see a shift in amino acid δ2H values similar to bulk δ2H values. We instead found no correlation between amino acid δ2H values and chick age.

Shifting amino acid composition of tissues is the last hypothesis that may explain all observed patterns of δ2H variation in our dataset. The amino acid composition of bird plasma is highly variable, even at the scale of days (Klasing and Austic 1984, Klasing

1998) and may shift in predictable directions as a chick develops. Changes in the amino acid composition of plasma and newly synthesized tissues (e.g. feather barbs) may cause bulk δ2H values to change. The δ2H values of proline are significantly higher than those of other amino acids (Table 1, Figure 2; δ2H of proline ≥370 ‰ higher than any other amino acid). Notably, proline is a major component of collagen and other structural proteins (e.g. keratin). These proteins are being synthesized quickly during rapid growth and therefore, the demand for proline is high in the growing chicks we sampled. As a result, we expect the proline concentration of plasma in growing chicks to be lower than in a non-molting adult (Hebert et al. 2002). Older chicks and juveniles nearing the end of their rapid growth phase may possess higher concentrations of proline in their plasma

41 compared to younger chicks, as the demand for proline to synthesize new tissues declines with age.

Indeed, early work on tissue amino acid composition has shown changing amino acid content of feathers in young chickens and geese (Fisher et al. 1981, Nitsan et al.

1981, Stilborn et al. 1997). Markedly, Stilborn et al. (1997) reported significantly higher chicken feather proline content in 28-day-old chicks compared to 14-day-old chicks.

Although Fisher et al. (1981) did not measure proline content, they reported an increase in threonine, isoleucine, and valine content in chicken feathers from 1-7 weeks of age.

The age- δ2H relationship observed in our chick plasma and along immature feather vanes, in addition to the offset in δ2H values between chicks and adults, may therefore be a consequence of changing amino acid composition. Overall, we find that a shift in amino acid composition of plasma and feather to be the most parsimonious explanation for our brown booby dataset. However, further work detailing the amino acid composition of tissues in rapidly growing birds is needed to test this new hypothesis.

While many hydrogen isotope studies report juveniles to have lower δ2H values than adults, there are several inconsistencies in this trend. Most importantly, several studies have found the magnitude of the juvenile-adult δ2H offset to vary between consecutive years in the same study system (American redstarts (Setophaga ruticilla) in

Ontario, Canada, Langin et al. 2007; Wood thrush (Hylocichla mustelina) in Crawford county, Pennsylvania, Gow et al. 2012; and Ovenbirds (Seirus aurocapilla) in New

Brunswick, Canada, Hatche et al. 2012). In addition, some authors report no difference in mean δ2H values between juvenile and adult tissues (Powell and Hobson 2006, Studds et al. 2012). Though age appears to be a potentially large source of δ2H variation, it is clear

42 from our data and the data of others that δ2H can be impacted by variables other than age and the δ2H of local water.

Amino acid-specific δ2H

To our knowledge, this is the first reported observation of avian amino acid- specific δ2H values. Understanding what drives bulk tissue δ2H variation in a system with negligible geographic hydrogen isotope influence is an important first step that may illuminate sources of δ2H variation not yet understood in terrestrial systems. Hydrogen isotope values of consumer tissues are broadly applied as a tool to study resource use and migration in terrestrial organisms. However, some ecologists argue the unexplained δ2H variation commonly found in these studies limit the usefulness of hydrogen isotopes as a tool. An analogous limitation in interpreting nitrogen isotope variation among consumer tissues was recently resolved by implementing amino-acid specific analyses. Amino acid- specific nitrogen isotope analysis allowed ecologists to understand whether trends in bulk

δ15N values derived from trophic level variation versus variation of δ15N at the base of food webs (McMahon and McCarthy 2016, Nielsen et al. 2015). Substantially more insight can now be gleamed from patterns in nitrogen isotope variation because amino acid-specific nitrogen analysis was developed.

Though our amino acid-specific δ2H data set is limited, we can draw several important conclusions. First, proline appears to be uniquely enriched in deuterium compared to all other amino acids (that is, it has a substantially higher δ2H value). To our knowledge, our study’s brown booby plasma proline δ2H values are the highest of any

43 δ2H measured for any animal tissue or animal-derived compound (Wiley, Morra, and

Fogel personal communication). Proline δ2H was also highly variable among our brown booby chicks, but did not correlate with chick age. Determining what drives variation in proline δ2H and understanding what causes its high δ2H values may provide useful insight into bulk δ2H analysis. Importantly, the variation in proline δ2H was tightly associated with bulk δ2H values in our study (R2=0.84, p=0.0002). Limited variation in

δ2H values of amino acids such as glutamine, valine, and leucine, may be similarly useful in clarifying interpretations of bulk tissue δ2H. If we can eliminate unexplained δ2H variation caused by non-geographic sources by studying invariable amino acid δ2H, we may be able to refine the use of hydrogen isotopes as a tool to study migration patterns.

For example, the δ2H of glutamine, valine, and leucine may track geographic location more closely than the δ2H of proline or bulk δ2H, which (based on our current dataset) can be highly variable in a population of birds from a single location. Lastly, though variation in δ2H between amino acids may be informative, we believe it is just as essential to describe the amino acid composition of tissues. Unlike amino acid-specific

δ2H, bulk tissue δ2H is a widespread and relatively inexpensive analysis. Understanding which amino acids comprise different tissues and how the proportions of amino acids may change as a result of physiological processes (such as development in juvenile organisms) will further our understanding of how bulk hydrogen isotopes behave within a consumer.

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APPENDIX

Chapter 1 and 2 brown booby chick age estimate calculations

In 2016, 31 chicks were monitored from hatch to 40 days of age. Chick mass, beak length, and ulna length were measured every 5 days. Mean mass, beak length, and ulna length was calculated from this population of chicks at each age step (1, 5, 10, 15, 20, 25, 30, 25, and 40 days post hatch). Growth rates for each metric were then calculated by plotting known age against mean mass or lengths. (graphs 1-3, below). Because each growth metric was highly predictive of known age (R2>0.98 for each), age was calculated based on each growth metric, and then averaged to produce the estimated age of chicks (table 1, below). The average age calculated by using each metric was more accurate at assigning chick age compared to any individual growth curve (e.g. using only ulna length as a way to back-calculate age from 2016 chicks produced larger error than averaging the three ages calculated for each metric). Error in back-calculating chick ages for each metric (weight, beak length, and ulna length) for the 2016 chicks with known age was always less than 3 days. This process was applied to all 2016 and 2018 chicks used in our study (chapter 1 and 2) to estimate chick age. 1. 2. Weight Beak y = 0.0448x + 0.4524 y = 0.5868x - 8.7815 40 40 R² = 0.99291 R² = 0.99724 30 30 20 20 10 10 Known Age Known Age 0 0 0 200 400 600 800 0 50 100 Chick weight (g) Beak length (mm)

Ulna y = 0.2873x - 2.2343 R² = 0.98898 40 30 20 10 0 Known Age 0 100 200 Ulna length (mm) 3.

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