STABLE DELTA 13 CARBON AND DELTA 15 NITROGEN ISOTOPE VALUES FROM NESTING LEATHERBACK SEA TUR TLES IN FLORIDA AND EFFECTS OF PRESERVATIVES ON STABLE DELTA 13 CARBON AND DELTA 15 NITROGEN ISOTOPE ANALYSES By LINDY MICHELLE BARROW A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006
Copyright 2006 by Lindy Michelle Barrow
To my parents and my husbandÂ—without them, the completion of this research would not have been possible. I will always be grat eful to them for thei r patience, words of encouragement, and unconditional love.
iv ACKNOWLEDGMENTS This thesis was completed thanks to th e financial support of Sigma Xi and the Riewald-Olowo Grant at the University of Florida. These studies were conducted in compliance with the Florida Fish and W ildlife Conservation Commission permit TP #016 and IACUC #D588 and #E025. I would like to thank Chris Johnson and his team, for colle cting leatherback epidermis and yolkless egg samples for both the 2004 and 2005 nesting seasons; Dr. Brian Stacy and Karrie Singel, for providing se a turtle epidermis and collection data on stranded sea turtles for my preservation st udy; Kim Reich, for preserving and preparing the sea turtle epidermis samples after th e preservation interval ; April Johnson, for providing the freshwater turtle epidermis for the preservati on study; Dr. Jason Curtis and the Stable Isotope Lab at the University of Florida, for helping to analyze my samples; and my undergraduate assistant Siobhan Proksell. I would also like to thank Dr . Mary Christman, for her invaluable assistance with my statistical analysis. Lastly, I would like to thank my advisor, Dr. Karen Bjorndal, and the rest of my masterÂ’s thesis committee, Dr s. David Evans and Louis Guillette, for their invaluable input and guidance in my research and thesis.
v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................vi LIST OF FIGURES..........................................................................................................vii ABSTRACT.....................................................................................................................vi ii CHAPTER 1 EXPERIMENT OVERVIEW.......................................................................................1 2 CAPITAL VERSUS INCOME: REPR ODUCTIVE STRATEGIES OF THE ATLANTIC LEATHERBACK SEA TURTLE...........................................................6 Introduction................................................................................................................... 6 Methods........................................................................................................................ 9 Sample Collection.................................................................................................9 Sample Analyses.................................................................................................10 Results........................................................................................................................ .11 Discussion...................................................................................................................12 3 EFFECTS OF PRESERVATION METHOD ON STABLE CARBON AND NITROGEN ISOTOPIC RATIOS OF TURTLE EPIDERMIS.................................20 Introduction.................................................................................................................20 Materials and Methods...............................................................................................21 Results and Discussion...............................................................................................23 4 SUMMARY OF STUDIES........................................................................................29 APPENDIX: SUMMARY OF RESULTS IN STABLE ISOTOPE PRESERVATION STUDIES....................................................................................................................33 LIST OF REFERENCES...................................................................................................45 BIOGRAPHICAL SKETCH.............................................................................................50
vi LIST OF TABLES Table page 3-1 Protocol for sampling of each tr eatment at each time interval.................................27 3-2 P values from DunnettÂ’s adjustment te sts evaluating preservation treatments on 15N and 13C in turtle epidermis samples...............................................................28 A-1 Summary of results in stable isotope preserva tion studies.......................................34
vii LIST OF FIGURES Figure page 2-1 Linear relationship between the 15N ratios of adult female Atlantic leatherback epidermis samples versus their body size given as CCL min (cm, p = 0.0004)......18 2-2 Linear relationship of 13C ratios of Atlantic leatherback sea turtle eggshell carbonate versus nesting da te (date given by Julian calendar beginning in April and ending in June, p = 0.0075)...............................................................................18 2-3 The 13C ratios of female adult Atlantic leatherback epidermis, albumen, and eggshell carbonate samples over the nesting season................................................19
viii Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science STABLE DELTA 13 CARBON AND DELTA 15 NITROGEN ISOTOPE VALUES FROM NESTING LEATHERBACK SEA TUR TLES IN FLORIDA AND EFFECTS OF PRESERVATIVES ON STABLE DELTA 13 CARBON AND DELTA 15 NITROGEN ISOTOPE ANALYSES By Lindy Michelle Barrow December 2006 Chair: Dr. Karen A. Bjorndal Major Department: Zoology The objective of the first porti on of my research was to determine if the Atlantic leatherbacks ( Dermochelys coriacea ) nesting at Juno Beach, Florida, were capital breeders, income breeders, or a combination of these two methods. Capital breeders use body stores to provide nutrient s for egg production while income breeders use recently ingested nutrients fo r egg production. In this study, I found that the female ep idermis did represent an oceanic foraging carbon stable isotopic ratio and the stable nitrogen ratio increased as curved carapace length increased. I also found th at the stable carbon and nitr ogen ratios of the albumen tissue sampled from yolkless eggs, collected at the time of their deposition, did not change significantly over th e nesting season. This sugge sts that albumen tissue is produced from body stores (capital breeding). However, the stable carbon ratio of the eggshell of the yolkless egg did change significantly over the nesting season. The
ix significant change in the eggshell stable carbon ratio over time suggests an income breeding strategy is being used to produce this tissue. Epidermis samples that were collected on Juno Beach from nesting female leatherbacks were preserved in a 70% ethano l solution. Because the effects of this preservative on the epidermis tissue of sea turtles were not known, the objective of the second portion of my research was to determin e the effects of four preservation methods on turtle epidermis tissue. In my study, epidermis tissue from two green sea turtle ( Chelonia mydas ), two loggerhead sea turtles ( Caretta caretta ), and two red-eared slider turtles ( Trachemys scripta elegans ) were subjected to 4 methods of preser vation: dried at 60Â°C for 24 h (the control), placed in 70% ethanol solution, pl aced in saturated NaCl (sodium chloride) aqueous solution, frozen at -10Â°C in a fros t-free freezer, and placed in DMSO (dimethyl sulfoxide) buffer (250 mM EDTA (ethylened iaminetetraacetic acid) pH 7.5; 20% DMSO). In this study, I found that tissues preserved in 70% ethanol and NaCl aqueous solution showed no significant difference from tissues dried at 60Â°C. Therefore, the stable isotope ratios obtained from the le atherback epidermis tissue preserved in 70% ethanol from the first portion of my thesis s hould be reliable. However, I also found that samples preserved in DMSO were significantly altered from the dried samples. Samples that have been preserved using DMSO are not ideal for use in stable isotope analysis. The freezing preservation only showed a signif icant change in isotopi c ratios at 60 days. I believe this difference was due to the use of a frost-free freezer in this experiment and that the effects seen in this study could be corrected by using a different type of freezer.
1 CHAPTER 1 EXPERIMENT OVERVIEW Atlantic leatherbacks ( Dermochelys coriacea ) are the largest extant species of sea turtle (Boulon, Dutton, and McDonald, 1996). Adult leatherbacks commonly forage on jellyfish in the open ocean, typically near ar eas of cool water convergence zones as far north as the subpolar regions of the Nort h Atlantic (Davenport and Balazs, 1991; Ferraroli, Gerges, Gaspar, and LeMaho, 2004; Hays, Houghton, and Myers, 2004). Atlantic leatherbacks migrate to nesting b eaches in Florida and the Greater Caribbean every 2Â–7 years with a mean of 3 years (Boulon et al., 1996). Atlantic leatherbacks nest from March to July with individual females us ually depositing 4 to 8 clutches of eggs in one season at about 9-day intervals (Boul on et al., 1996; Fretey and Girondot, 1998). These clutches often contain both viab le and non-viable (yolkless) eggs. The largest nesting population of leatherbacks in the continental United States is located from Martin and St. Lucie counties to Palm Beach County in South Florida, with an important rookery located at Juno Beach, Fl orida. Research is needed to understand the physiology and behavior of these turtles. Therefore, for the first portion of my masterÂ’s thesis I used stable isotope anal ysis to determine if females use body stores (capital) or recently ingested nutrients (income) to produce eggshell and albumen. Stable isotopes exist within all biologi cal systems and are being used increasingly to answer questions about beha viors and diets of organisms. Many elements have stable isotopes, meaning that besides thei r commonly known stable forms (i.e., 12C and 14N), another stable isotope naturally occurs (i.e., 13C and 15N). These heavier isotopes occur
2 in a significantly reduced propor tion to the lighter form (12C 98.89 % and 13C 1.11%; Boutton, 1991). Isotopes of an element do not function in the same manner for all chemical or physical processe s (Peterson and Fry, 1987). When a consumer assimilates nutrients from an organism that it has ingest ed, the distinct isotopic signature of that organism is incorporated into the tissues of the consumer. The lighter isotope of an element is excreted preferentially by the orga nism resulting in an increase of the heavier isotope (Macko, Fogel-Estep, Engel, and Hare , 1986). The lighter isotope is typically preferentially excreted by the organism because of its lighter weight and weaker molecular bonds. Most studies that use stable isotopes e xpress their results in terms of delta ( ) values. The value is a ratio of the stable isotope composition of the sample ( Rsample) with respect to a universal standard ( Rstandard) expressed in parts per mil (Â‰). These calculations are made using the equation: 310 * 1 ndard sta sampleR R X where X is the heavy isotope such as 13C or 15N, and R is the ratio of heavy isotope to light isotope (i.e., 13C/12C or 15N/14N) for the sample and the standard. Therefore, a higher value would indicate a pro portionally greater amount of heavy isotope present in the sample with respect to the standard. Th e standard for carbon used in this thesis was sucrose ANU (sucrose, 13C = -10.5Â‰) and NBS19 (limestone, 13C = 1.95Â‰). The standard used for 15N was IAEA-N1 (ammonium sulfate, 15N = 0.4Â‰). These standards were calibrated against Vienna PeeDee Belemnite (carbon) and atmospheric N2 (nitrogen).
3 In marine environments, the ra tio of stable carbon isotopes (13C:12C) decreases from neritic to oceanic habitats (Ruben stein and Hobson, 2004). Because organisms reflect the carbon ratio of their diet animals that forage in neritic habitats will have a higher ratio of 13C:12C than those that forage in ocean ic habitats. The stable nitrogen isotopic ratio (15N:14N) can be used to determine the tr ophic level at which an animal is feeding and whether an animal is fasting. The nitrogen ratio will be higher if the organisms feed at a higher trophic level or are not foraging but using their body stores for survival (DeNiro and Epstein, 1981; Minagawa and Wada, 1984). To determine if Atlantic leatherbacks ar e capital or income breeders, I began by using the principle that sea turtles that fora ge in oceanic environments will have more depleted 13C and 15N ratios then turtles from coastal foraging grounds (Hatase et al., 2002; Bolten, unpublished). Because leatherbacks spend the majority of their time in the open ocean, isotopic ratios taken from body tissu es, such as epidermis, will reflect an oceanic ratio. Isotopic turnover of carbon and nitrogen in epidermis tissue of sea turtles has been shown to take several months in loggerhead turtles (R eich, unpubl. data); therefore it is believed that sa mples taken early in the nestin g season will reflect isotopic ratios from the foraging ground. I will also use the 13C ratios from eggshell carbonate and the 13C and 15N ratios of albumen samples collect ed from yolkless eggs to see if these tissues reflect foraging gr ound or nesting beach ratios. The carbonate that is used to construct the eggshell is believed to come from circulating levels of bicarbonate within th e blood stream of the female, generated from the metabolism of ingested nutrients (DeN iro and Epstein, 1978; Krueger and Sullivan, 1984; Hobson, 1995). Circulating levels of carbonate obtained from diet should be
4 incorporated into the eggshell within days of ingestion because new clutches of eggs are shelled prior to each nesting event (Wyneke n, 2001). Therefore, if nesting female leatherbacks are foraging in coastal habitats during their internesting intervals, a larger quantity of 13C will become incorporated into the stable carbon ratios, causing the 13C ratios obtained from the eggshell to beco me more enriched over the nesting season reflecting the change in nutrients. The use of recently ingested nutrients to produce eggs is known as income breeding. If the ratios do not change, it can be concluded that the females are using body stores or are returning to the oceanic Florida Current to forage between nesting events. Using body stores ac quired before the migration to the nesting beach is known as capital breeding. Albumen mainly consists of proteins that are believed to be synthesized directly from amino acids in the blood (Taylor, 1970). Similar to eggshell, if nesting females are income breeders, the 13C ratios should become more en riched over the nesting season. The 15N ratios of albumen should also become en riched if the leatherbacks are fasting. Fasting turtles would use body stores to pr oduce energy. The lighter nitrogen isotopes would be preferentially used and excreted from the orga nism, leaving an increasing number of the heavier nitrogen isotope. This would result in an enriched ratio. A study by Hobson (1995) on quail ( Coturnix japonica ) revealed that shell carbonate and albumen reflected diet integrated over 3Â–5 days. Acknowledging that the metabolism of reptiles is much slower than that of birds, I expect to see an enrichment of both stable carbon and nitrogen isotopic ratio s if leatherbacks are foraging coastally between nesting events.
5 Because leatherbacks spend the majority of their time in the open ocean, migrating to coastal areas for reproduction, stable isot opes can be used to determine their breeding strategy. However, in my study to addre ss this question epidermis samples were preserved in 70% ethanol solution, a treatm ent that could potentially influence the apparent isotopic ratios. Ther efore, I needed to determine the effect of preservatives on sea turtle epidermis stable isotope ratios. Thus, the second portion of my masterÂ’s thesis focused on evaluating the effects of different preservative treatments and the re moval of lipids on isotopic ratios in turtle epidermis samples. I tested epidermis sa mples taken from deceased green sea turtles ( Chelonia mydas ), loggerhead sea turtles ( Caretta caretta ), and red eared slider turtles ( Trachemys scripta elegans) . I used four preservatives common in field research: 70% ethanol, freezing, DMSO (dimethyl sulfoxide) buffer (250 mM EDTA (ethylenediaminetetraacetic acid) pH 7.5; 20% DMSO), and saturated NaCl (sodium chloride) aqueous solution. I compared samples treated in each preser vative at different time intervals to dried samples that were used as controls. The use of stable isotope ratios in behavior and diet research is a valuable technique that could potentially answer many questions.
6 CHAPTER 2 CAPITAL VERSUS INCOME: REPRODUCTI VE STRATEGIES OF THE ATLANTIC LEATHERBACK SEA TURTLE Introduction During reproduction, organisms must expend nu trients for egg development. This process can be accomplished using one or a combination of the following methods. First, eggs may receive nutrients from body stores accumulated before the reproductive season. This is known as Â“capitalÂ” breeding. Second, organisms can use recently ingested nutrients for reproduction. This method is known as Â“incomeÂ” breeding (Thomas, 1988; Stearns, 1989; JÃ¶nsson, 1997). Little information on capital versus income breeding is available for reptiles; most studies focus on endothermic animals (Bonnet, 1998; Meijer and Drent, 1999; Kullberg, Jakobsson, Kaby, and Lind, 2005). It has long been thought, howev er, that sea turtles are capital breeders (Hamann, Limpus, and Whittier, 2002; Hamann, Limpus, and Owens, 2003). This assumption has been supported by the great distances a nd habitat differences between foraging grounds and nesting beaches as well as the apparent lack of normal prey items at most nesting beaches. Female s delay their migration to nesting beaches until sufficient fat stores are acquired fo r reproduction (Hamann et al., 2002, 2003). Also, empty digestive tracts in nesting green turtles have been reported at Ascension Island and Costa Rica (Hays, Broderick, Glen, and Godley, 2002a; Bjorndal, pers. comm.) supporting the idea that no significant foraging is occurring at these nesting beaches.
7 Recent studies, however, have suggested that sea turtles may be, at least in part, income breeders (Hughes, Luschi, Mencacci, and Papi, 1998; Tucker and Read, 2001). Using data from time-depth recorders, Ha ys, Glen, Broderick, Godley, and Metcalfe (2002b) suggested that green turtles nesti ng on northern Cyprus may feed during the inter-nesting interval. Eckert, Eckert, Ponganis, and Kooyman (1989) documented nesting leatherbacks diving deeply during th e inter-nesting period, suggesting feeding. These observations alone do not re veal the extent to which sea turtles need to feed during their inter-nesting period s to produce egg tissues. To determine the type of breeding stra tegy that is being used by different organisms, scientists are increasingly using th e properties of stable isotopes. In marine environments, the ratio of stable carbon isotopes (13C:12C) decreases from neritic to oceanic habitats (Rubenstein and Hobson, 2004) . Because organisms reflect the carbon ratio of their diet in a predictable way, animals that forage in neritic habitats will have a higher, or more enriched, ratio of 13C:12C than those that forage in oceanic habitats. The stable nitrogen isotopic ratio can be used to determine trophic level. This is due to the increased ratio of 15N to 14N that occurs when an organism ingests food at higher trophic levels. The life stages and reproductive physiology of the Atlantic leatherback sea turtle ( Dermochelys coriacea ) facilitate the use of stable is otopes in determining their breeding strategy. Adult leatherbacks forage in the ope n ocean (Ferraroli et al., 2004; Hays et al., 2004) and migrate to nesting beaches in Fl orida and the Greate r Caribbean every 2Â–7 years, with a mean of 3 years (Boulon et al ., 1996). Individual females usually deposit 4 to 8 clutches of viable an d non-viable (yolkless) eggs in one season at about 9-day
8 intervals (Boulon et al., 1996; Fretey and Gi rondot, 1998). Because leatherbacks are believed to spend the majority of their time in the open ocean, isotopi c ratios taken from body tissues that have slow turnover times (i.e ., red blood cells, egg yolk) will reflect an oceanic ratio (Wallace, Seminoff, Kilham, Spotila, and Dutton, 2006). Isotopic turnover of carbon and nitrogen in epider mis tissue of sea turtles has been shown to take several months (Seminoff, Jones, Eguchi, Jones, and Dutton, 2006; Reich, unpubl. data). Therefore, if these turtles ar e feeding neritically after leav ing their foraging grounds for the nesting beach, the isotopic ratio of epid ermis will still reflect the oceanic foraging grounds. If body stores, such as fat acquired duri ng oceanic foraging before the nesting migration, are used to produce egg components, the isotopic ratio of e gg tissues will also reflect the oceanic isotopic ratio. The car bonate in eggshell come s from circulating bicarbonates in the blood of the female ge nerated from the metabolism of ingested nutrients or body stores (DeNiro and Epst ein, 1978; Krueger and Sullivan, 1984; Hobson, 1995). Circulating levels of carbonate obtained from diet should be incorporated into the eggshell within days of ingestion because new clutches of eggs are shelled prior to each nesting event (Wyneken, 2001). Therefore, if nesting leatherbacks forage in coastal habitats during their internesting intervals, the 13C ratios obtained from the eggshell should become more enriched over the nesting season reflecting this change in nutrients used to produce the eggshell. Albumen is also thought to be produced a few weeks prior to egg deposition (Wyneken, 2001). Therefor e, if leatherbacks are using recently ingested nutrients to produ ce their eggs, egg tissues should reflect a more enriched, coastal ratio acquired from the coastal food items.
9 In this study, I will use stable carbon and nitrogen isotopes to study the breeding strategy of Atlantic le atherback sea turtles. Comparison of isotopic ratios from epidermis samples taken from nesting females and eggshell and albumen samples from yolkless eggs will be used to help determine if these turtles are using capital or income strategies for reproduction. Methods Sample Collection All leatherback samples were collected from nesting females on Juno Beach in South Florida (26.879Â°N, -80.053Â°W). The nesti ng season for these turtles on this beach begins in March and typically extends through June. The date of the nesting event and the minimum curved carapace length (CCL min) to the nearest 0.1 cm of each nesting females was recorded. Epidermis samples in this study were only collected from turtles nesting from April through June in 2004 (n =7) and in 2005 (n=19). Epidermis samples were collected from the shoulder region of the nesting females with sterile 6-mm biopsy punches. Alcohol swabs were used to cleanse the area before sampling. The sample was immediately placed in 70% ethanol for stor age. Blood clotting ointment (Traumaex, Emergency Medical Products, Inc. ) was applied to the sample site to reduce any bleeding. One or two non-viable, yolkless eggs (n=27) were also collected from each nesting female. Yolkless eggs were collected from nesting females during oviposition in the 2005 season. These eggs are easily identified by their small size. At time of collection, they were placed into bags and frozen in a freezer for transport to the Archie Carr Center for Sea Turtle Research (ACCSTR), where the samples were maintained at -10Â°C. Some yolkless eggs were collected from the same fema le at different nesting events (n=3). All
10 samples collected in the field were transporte d to ACCSTR, at the Un iversity of Florida, for analysis. Sample Analyses After fewer than 4 months, epidermis samples were removed from the 70% ethanol solution and rinsed in deionized water. C onnective tissue was removed from each sample using a scalpel blade. Samples were diced using the scalpel blade and half of each sample was placed in a cryovial and dried at 60Â°C for approximately 24 h. Yolkless eggs were removed from the freezer fewer than 5 months after transport back to ACCSTR and thoroughly rinsed in deio nized water. A scalpel blade was used to open the egg, and the albumen was poured into a plastic weighboat. Eggshells were then rinsed with deionized water, placed in plas tic cups, and dried at 60Â°C for 24 h. Albumen samples were frozen at -80Â°C for 24 h, lyophi lized, and ground into a fine powder with a mortar and pestle. Dried eggshells were ground into a fine powder with a mortar and pestle. A portion of the ground powder was placed in a cryovial and a 3.5% NaOCl (sodium hypochloride) solution was added to each cryovial under a fume hood. After 24 h, the NaOCl solution was decanted, and all samples were rinsed a minimum of six times with deionized water. Samples were th en returned to the drying oven. Stable carbon and stable nitrogen isotope ratios were obtained from the epidermis samples by loading 400Â–500 Âµg of dried ep idermis tissue and from the albumen by loading approximately 400 Âµg into tin capsules. These capsules were then combusted using a COSTECH ECS 4010 elemental anal yzer interfaced via a Finnigan-MAT ConFlow III device to a Finnigan-MAT Delta Plus XL isotope ratio mass spectrometer located in the Geological Sciences Department at the University of Florida. Stable
11 isotope ratios are expressed in delta ( ) notation relative to a standard for carbon and nitrogen using the following equation: 1000 1 standard sampleR R X where X is 13C or 15N and Rsample is the corresponding ratio 13C/12C or 15N/14N. The standard for 13C in this experiment was sucrose ANU (sucrose, 13C = -10.5Â‰) and for 15N was IAEA-N1 (ammonium sulfate, 15N = +0.4Â‰). These sta ndards were calibrated against Vienna PeeDee Belemnite (carbon) and atmospheric N2 (nitrogen). Stable carbon isotope ratios were obtained for the eggshell by loading approximately 400 Âµg of samples into tin capsu les. Samples were then placed in a VG PRISM (Series II) mass spectrometer (SIR MS) for analyses. The standard for 13C in these analyses was NBS19 (limestone, 13C = +1.25Â‰). A pooled t-test was used to determine if epidermis ratios from the 2004 season were significantly different from those of the 2005 season. Linear regressions were conducted for 13C and 15N ratios of adult epidermis and albumen versus body size (CCL min values) and nesting date. Li near regressions we re also conducted on 13C ratios of eggshell versus body size (ccl mi n values) and nesting date. A Pearsons correlation analysis was used to test for a relationship between nesting date and body size (CCL min values). All analyses we re performed using JMP v. 5, Duxbury. Results The minimum curved carapace length (CCL min) ranged from 142-167.5 cm (n = 7) for the 2004 season and for 138Â–159.5 cm (n = 18) for the 2005 season. The 13C mean Â± SD for the 2004 and 2005 seasons were as follows: -17.17 Â± 0.41, -17.14 Â± 0.59Â‰. The 15N mean Â± SD for the 2004 season was 12.91 Â± 1.82Â‰ and for the 2005
12 season 11.70 Â± 1.19Â‰. There was no statis tical difference between the mean 13C (t = 0.11, p = 0.91) and 15N ratios (t = 1.96, p = 0.06) between the two seasons. There was no significant relationship between 13C ratios of adult epidermis samples and body size (F = 0.05, p = 0.83); however, 15N ratios did increase significantly with body size (F = 16.92, p = 0.0004; Figure 2-1). No statistical relationship was found between 13C or 15N ratios and nesting date (F = 3.08, p = 0.09; F = 0.24, p = 0.63, respectively). No correlati on was found between nesting date and body size (p = 0.60, n = 26). The 13C mean ratio Â± SD for eggshell was found to be -13.18 Â± 0.51Â‰. No statistical relationshi p was found between the 13C ratios from the eggshells and body size (F = 0.04, p = 0.84). A significa nt relationship was found for the 13C ratios of the eggshells and nesting date (F = 8.50, p = 0.01; Figure 2-2). No correlation was found between nesting date an d body size (p = 0.18, n = 27). The 13C mean ratio Â± SD for albumen was found to be -19.03 Â± 0.88Â‰. The 15N mean Â± SD for albumen was found to be 8.63 Â± 1.42Â‰. There were no significant relationships between the 13C or 15N ratios of albumen and body size (F = 0.09, p = 0.77; F = 0.01, p = 0.94, respectively) or nes ting date (F = 0.51, p = 0.48; F = 0.01, p = 0.91 respectively). Discussion In this study, leatherback ep idermis stable carbon ratios were found to reflect the stable carbon isotopic ratios of the oceanic foraging grounds. This conclusion was supported by comparison of the leatherback epidermis stable carbon ratios with those obtained from another known oceanic dwelling tu rtle species, juvenile loggerhead turtles ( Caretta caretta; Bolten, 2003; Reich et al., unpub). The leatherback samples were
13 found to be more depleted than those from th e oceanic loggerheads. This conclusion was also supported by comparison of the epidermis ratios with ratios obt ained from red blood cells (RBCs) of leatherbacks nesting in St. Croi x. Wallace et al. (2006) stated that ratios from RBCs would reflect resources that were being consumed on foraging grounds before migration to the nesting beache s. The mean carbon ratios for RBCs of leatherbacks nesting on St. Croix ( 13C = -18.3 Â± 0.7Â‰) and epidermis from leatherbacks nesting on Juno Beach ( 13C2004 = -17.17 Â± 0.41Â‰, 13C2005 = -17.14 Â± 0.59Â‰) are similar. The epidermis values are also similar to those found by Godley, Thompson, Waldron, and Furness (1998) for bone collagen from stranded leatherb ack turtles in the Mediterranean Sea ( 13C = -19.0 Â± 5.43Â‰). Bone collagen is known to reflect the past several years of diet and would be reflective of foraging ground diet. Therefore, it can be assumed that the stable carbon isotope ratios of the epidermis tissue at the nesting beach continue to reflect the ratio of the foragi ng grounds. This finding was expected because of the slow turnover rate of the epidermis tissue in sea turtles. The mean stable nitrogen ratios of the leatherback epidermis samples were also similar to those found by Wallace et al. (2006, 15N = 10.2 Â± 1.3Â‰) and Godley et al. (1998, 15N = 14.1 Â± 0.52Â‰); however, these ratios va ried significantly with body size (Figure 2-1). Animal tissues tend to be enriched 3 to 4 Â‰ for 15N per trophic level (DeNiro and Epstein 1978, 1981). Based on th is, the significant increase in nitrogen ratios as body size increases suggests that sm aller females may possibly be foraging at a slightly lower trophic level th an larger females. A study by Hatase et al. (2002) found a correlation between 13C and 15N ratios of egg yolks and body size for loggerhead turtles. Using satellite tracki ng, they showed that turtles with larger body size and more
14 enriched 13C and 15N egg yolk ratios were utilizing fora ging areas closer to the coast as opposed to smaller turtles that were foraging in oceanic habitats. In my study, a correlation was only found between 15N epidermis ratios an d body size; however, because satellite tracking was not used in my study I can not conclusively discern a difference in feeding between females. Ho wever, different species of jellyfish, the leatherback primary food source, consume va rying food items from zooplankton to fish thereby making the 15N ratios of each species different (Brodeur, Sugisaki, and Hunt, 2002). The relationship between the nitrogen ratios of the epidermis and body size may be explained by a differential intake of sp ecific food items resulting in a less enriched nitrogen ratio for the smalle r females. Further studies should be conducted to support this idea. Eggshell obtained from yolkless eggs wa s found to become significantly enriched in stable carbon ratios over the nesting seas on (Figure 2-2). This finding would suggest that the female is foraging during the in ternesting period on neri tic food items whose carbon ratios are then becoming in corporated into the carbon ate of the eggshell (income breeding) or possibly that th e sea turtle diving behavior during the internesting period may be affecting carbon isotope ratios. Sea turtles produce the shells for each clutch during their internesting periods (Wyneken, 2001). Dissolved bicarbonates in the body fluids, derived primarily from recently ingested food items, are thought to be incorporated into the eggshell carbonate; theref ore, based on this reasoning, if turtles are ingesting nutrients during the internesting pe riod, it should be reflected in this tissue (Krueger and Sullivan, 1984; Hobson, 1995).
15 This process is similar to what is th ought to occur for carbonate found in bone apatite. A study by Biasatti (2004) stated that the pr imary influence on the carbon isotopic composition of bone carbona te was diet, but that respir ation also influenced this ratio. For leatherbacks, Biasatti found that the accumulation of respired CO2 in the blood during extended breath-holding diving was causi ng the typically preferentially excreted, lighter isotope 12C to be incorporated in to the bone carbonate from circulating levels in the blood bicarbonate. This was causing the bone carbonate ratios to be less carbon enriched compared to diet values then thos e of shallower diving tu rtles. Biasatti found a carbon isotope ratios for bone carbonate of leatherback humeri to be -12.3Â‰, a value similar to the mean value that was found for eggshell in this study ( 13C = -13.18 Â± 0.51Â‰). It is possible that the turtles are making shallower dives as the season progresses. Therefore, as seen in bone carbonate, the eggshell carbonate is becoming increasingly enriched as le ss of the lighter isotope, 12C, is incorporated. Although the significant change in carbon ratios of e ggshells over the nesting season could be signifying income breeding, I do not believe the small sample size and limited turtle replication (sampling the same turtle multiple times) throughout the season allows for this finding to be conclusive. Closer evaluati on of Figure 2-3 reveal s an asymptotic line around the value of -12.50Â‰. This value is a pproached several times during the season. However because of the reduction in sample number at the end of the season, the significance of the carbon eggshell values ma y be influenced. Further sampling should be conducted to further validate this finding. The 13C and 15N ratios for albumen found in this study ( 13C = -19.03 Â± 0.88Â‰, 15N = 8.63 Â± 1.42Â‰) were similar to those found by Maros, Louveaux, Lelarge, and
16 Girondot (2006) for leatherbacks nesting in French Guiana ( 13C = -16.57 Â± 0.42Â‰, 15N = 10.10 Â± 0.25Â‰). The ratios for the albumen in my study did not significantly change over the nesting season and are more depleted in carbon then either epidermis or eggshell (Figure 2-3). This finding first suggests that the females are not foraging during the internesting period, but are using body stores to produce this tissue (capital breeding). However, because of the lack of enrichment of the nitrogen ratios , the use of body stores is not fully supported. A second possible explan ation for these results is that females are returning to the Florida Curre nt during their internesting pe riods to forage on food items found in these waters. The Florida Current is the beginning portion of the Gulf Stream and is located close to the South Florida coas tline. At Juno Beach the Florida Current is less than 14 kilometers away from shore. The Loggerhead Marine Life Center has conducted satellite tracks of females during th eir internesting periods and it appears the females are returning to this current during th ese periods. If females are foraging in this current, it would not be reflected in the isot opic ratios of the albumen because food items in the current would most likely also have 13C and 15N ratios reflective of the open ocean. Therefore, conclusively determini ng the breeding strategy, capital, income, or combination of both, which is being used by these organisms, is not possible. Although a conclusive result on the breed ing strategy of female leatherbacks could not be determined in this study, it doe s provide a first look at the stable isotope ratios of specific leatherback egg tissues and th eir relationship to the female. It also is one of the first studies to describe a proce dure by which sea turtle eggshell and albumen may be prepared for isotope analysis. Stable isotope research comb ined with satellite tracking and possibly direct obs ervations of internesting feed ing behavior would provide
17 the necessary information about the reproducti ve biology of these organisms to determine breeding strategy. Knowing the breeding stra tegy these organisms utilize may aid in the preservation of this species for future generations.
18 R2 = 0.4238 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 135140145150155160165170 CCL min (cm) 15N (Â‰) Figure 2-1. Linear relationship between the 15N ratios of adult female Atlantic leatherback epidermis samples versus their body size given as CCL min (cm, p = 0.0004). R2 = 0.2488 -15.00 -14.00 -13.00 -12.00 95105115125135145155 Nesting Date (Julian calendar) 13C (Â‰) Figure 2-2. Linear relationship of 13C ratios of Atlantic leatherback sea turtle eggshell carbonate versus nesting date (date given by Julian calendar beginning in April and ending in June, p = 0.0075). Samples collected from the same turtle at different nesting events ar e connected by a straight line.
19 -22.00 -20.00 -18.00 -16.00 -14.00 -12.00 -10.00 95105115125135145155165175 Nesting Date (Julian Calendar) Epidermis Albumen Eggshell 13C (Â‰) Figure 2-3. The 13C ratios of female adult Atlantic leatherback epidermis, albumen, and eggshell carbonate samples over the ne sting season (dates given by Julian calendar beginning in April and ending in June).
20 CHAPTER 3 EFFECTS OF PRESERVATION MET HOD ON STABLE CARBON AND NITROGEN ISOTOPIC RATIOS OF TURTLE EPIDERMIS Introduction Stable isotope analysis is being used in creasingly by field researchers to answer questions about habitat use, migration patte rns, and diets of various organisms (e.g., Braune, Donaldson, and Hobson, 2002; Kurle and Worthy, 2002; Hatase et al., 2002). However, samples collected in the field mu st be preserved for varying amounts of time before analyses can be conducted in the labora tory. If the preservation technique alters the isotopic values, improper inte rpretation of the results will ensue. Similarly, effects of preservatives are a concern in the use of archived samples in museum collections (Kiriluk, Whittle, Keir, Carswell, and Huestis, 1997; Hobbie, Weber, and Trappe, 2001). If preservation does not signifi cantly affect archived samples, these repositories have tremendous potential for reconstructing food webs of past ecosystems. Despite the possible severity of this problem, however, st udies on the effects of type and duration of preservation on stable isotopes in tissue samp les have been limited and are scattered in the literature. The goals of this study were (1) to summari ze the results from studies of preservation effects in the literature a nd (2) to test the e ffects of four common preservatives on the isotopic ratios in epidermis tissue of turtles to validate my studies in these species.
21 Materials and Methods Four sea turtles that had stranded alive on the southeas t coast of Florida were necropsied shortly after death at the School of Veterinary Medicine, University of Florida, during which I collected epider mal samples. The two green turtles, Chelonia mydas , had a curved carapace length [CCL] of 29.0 cm ( Chelonia 1) and 44.2 cm ( Chelonia 2) The two loggerhead turtles, Caretta caretta , had a CCL of 68.5 cm ( Caretta 1) and 58.0 cm ( Caretta 2). The two red-eared slider turtles, Trachemys scripta elegans , ( Trachemys 1, CCL = 18.1 cm; Trachemys 2, CCL = 18.8 cm) were wildÂ–caught adults from Louisiana, sacrificed as controls in an experiment at the UF School of Veterinary Medicine, and epidermal samples were collect ed shortly after death. No turtles were sacrificed for this study. The epidermis was cleaned with alco hol and then washed thoroughly with deionized water. Epidermis samples were co llected with 6-mm MiltexÂ® biopsy punches. Four preservation methods were used: dried at 60C for 24 h (the control), placed in 70% ethanol, placed in saturated NaCl (sodium ch loride) aqueous solution, frozen at -10Â°C in a frost-free freezer, and placed in DMSO (dimethyl sulfoxide) buffer (250 mM EDTA (ethylenediaminetetraacetic acid) pH 7.5; 20% DMSO). The samples that were frozen or placed in preservative solutions were held for different time intervals (Table 3-1). Three epidermis samples were collected from each turtle for each treatment (preservative x duration) and placed in 3 separate vials. For analysis, samples were removed from each treatment, washed in deionized water, cleaned of connective tissue and diced wi th a scalpel blade, placed into individual cryovials, and dried at 60Â°C for 24 h. Lipids were extracted from half of each sample using petroleum ether in a DionexÂ® Accelerat ed Solvent Extractor (ASEÂ®). Samples,
22 both with and without lipids, were analyzed for stable carbon and st able nitrogen ratios by loading 350Â–550 Âµg of dried epidermis tissue into tin capsules. These capsules were combusted using a COSTECH ECS 4010 elementa l analyzer interfac ed via a FinniganMAT ConFlow III device to a Finnigan-MAT DeltaPlus XL isotope ratio mass spectrometer. Stable isotope ratios are expressed in delta ( ) notation relative to a standard for carbon and nitrogen using the following equation: 1000 1 standard sampleR R X where X is 13C or 15N and Rsample is the corresponding ratio 13C/12C or 15N/14N in the sample. The standard for 13C in this experiment was sucrose ANU (sucrose, 13C = 10.5Â‰) and for 15N was IAEA-N1 ((NH4)2SO4, 15N = 0.4Â‰). These standards were calibrated monthly against Vienna PeeDee Be lemnite limestone formation international standard (carbon) and atmospheric N2 (nitrogen). Samples of standard materials were inserted at regular intervals in all analytical runs to calibra te the system and evaluate any drift over time. Data were analyzed according to a random ized complete block design with nesting within individual treatments. Turtles were th e random blocks and th e three factors were source (marine or freshwater species), presen ce/absence of lipids and a treatment factor for each combination of time and preservative . Data were collected for a total of 21 combinations of time and preservative, but not for all turtles because of different amounts of available epidermis with homogeneous appe arance (Table 3-1). The three replicate observations taken within each combination of tu rtle and the three factors were the nested samples. TukeyÂ’s adjustment was used to perform multiple comparisons, which included differences between treatments within one time period and differences within one
23 treatment between time intervals. DunnettÂ’s adjustment was used to determine any significant differences between treatment and th e control. All statis tical analyses were performed using SAS v. 9.1 software (SAS Institute, Cary, NC). Results and Discussion The effects of preservative type and dur ation on stable isotope values were not different for the 3 species and were the same for carbon and nitrogen isotopes (Table 3-2, Appendix A). Isotope values from samples pres erved in ethanol or saturated NaCl for all durations or frozen up through 30 days were not significantly different from those of the dried controls. Samples frozen for 60 days or preserved in DMSO buffer for 1 to 30 days had significant preservation e ffects. A surprising resu lt for both carbon and nitrogen isotopes was the loss or reversal of the preser vative effect in samples stored for 60 days in DMSO buffer. The effect of lipid ex traction was dependent on the preservative and time period being analyzed (F20,116 = 2.92, p = 0.0002). The only sample that demonstrated a significant difference between the isotopic carbon signature with lipids and lipid extracted was the DMSO treatment on day four. Therefore, within the analysis, the extraction of lipids did not ch ange the preservative effects (F1,116 = 2.73, p = 0.1011). Several studies that looked at the effect of preservation in 70 % ethanol over time also found no significant effect on carbon and nitrogen isotope ratios (Hobson, Gibbs, and Gloutney, 1997; Gloutney and Hobson, 1998; Appendix A). However, Kaehler and Pakhomov (2001) found a significant effect in 3 species of invertebrates preserved in 70% ethanol. In addition, studies that exam ined the effects of ethanol concentrations above 70% found that both car bon and nitrogen ratios could be significantly altered by the ethanol treatment (Ponsard and Am lou, 1999; Sarakinos, Johnson, and Vander Zanden, 2002; Appendix A). Therefore, 70% ethanol may not be an appropriate
24 preservative for all tissue types. Concentra tions of ethanol above 70% should be tested before being used as a preservative. Although ethanol has been found to be an acceptable preservative for several tissues, ethanol is flammable and difficult to transport because of safety regulations. Preservation in a saturated NaCl aqueous so lution or in salt is becoming more popular because of the ease of transport. Turtle ep idermis samples preserved in saturated NaCl solutions for up to 60 days in my study did not demonstrate significant differences in stable carbon and nitrogen ratios compared to the controls. While differences were not significant in this experiment, the characteri stics of samples being preserved (e.g., lipid content) should be considered before usi ng saturated salt preservation. Interaction of specific factors with the preservative may aff ect the stable isotopic ratios of the sample and should be tested before the preservative is used (Appendix A). Muscle tissues from four fish species preserved in salt had a signi ficant increase in the stable nitrogen ratio (Arrington and Winemiller, 2002; Appendix A). Again, th e effects of NaCl as a preservative may vary with species and tissue type. Freezing samples prior to analysis has always been considered a relatively safe method of preservation, although sometimes diff icult in the field. I found that samples preserved frozen at -10Â°C in a frost-fr ee freezer for up to 30 days did not differ significantly from the control; however, samp les preserved frozen up to 60 days were significantly depleted in both 13C and 15N compared to the control. Many other studies that evaluated the effects of freezing at this temperature and colder temperatures did not find significant changes in stable carbon and nitrogen si gnatures (Gloutney and Hobson, 1998; Kaehler and Pakhomov, 2001; Sweeting, Polunin, and Jennings, 2004; Appendix
25 A). However, one study by Feuchtmayr a nd Grey (2003) found both carbon and nitrogen ratios in zooplankton were significantly altered from the control in a freezing treatment. They attributed these differences in ratios to the loss of the lighter is otopes of carbon and nitrogen from the mechanical breakdown of cells and via leaching when the samples were thawed or filtered during their preparatory procedure. The fourth preservative, DMSO buffer, has been commonly used to preserve samples for genetic analyses. These archived samples could be used for studies based on stable isotopes, if the preservative has no effect. However, in this study I found that samples preserved in DMSO buffer for 1, 4, 15, and 30 days, but not 60 days, had significantly altered 13C and 15N values compared to those of the controls. A study by Hobson et al. (1997) also found that DMSO buffer si gnificantly affected tissue isotopic ratios, but a study by Todd, Ostrom, Lien, a nd Abrajano (1997) found that DMSO alone did not have a significant effect if lipid s were extracted from the samples after preservation. Todd et al. (1997) suggested that the EDTA in the buffer solution is responsible for the isotopic alterations. In this study, both samples with and without lipids were found to be significantly different from the control samples. We cannot offer an explanation for the lack of a preservative effect after 60 days in DMSO buffer; further evaluation is needed. In conclusion, 70% ethanol and saturate d NaCl aqueous solution are suitable methods of preserving turtle epidermis for st able carbon and nitrogen isotope analysis, as well as short-term freezing at -10Â°C. DMSO buffer unpredictably alters the isotopic ratios making results from these preserved samp les difficult if not impossible to interpret. The effects of all of these preservatives ove r durations greater than 60 days should be
26 evaluated. Given the different effects of preservatives on different species and tissue types, studies should attempt to develop pred ictable patterns of wh at preservatives are appropriate for what tissue. Finally, more samples preser ved in DMSO buffer should be analyzed to see if the apparent Â“recoveryÂ” of the isotopic ratios by 60 days is real and lasting. Twenty different preservative methods and their effects on different tissue samples are represented in appendix A. However, of these twenty methods, only seven have been examined by more than one research group. All of these seven methods had mixed results concerning their effect on the tissue tested. The samp les that were preserved by freezing typically showed no change, but there were instances of effects on both carbon and nitrogen. Shock-frozen samples only show ed changes in stable nitrogen ratios. Aqueous NaCl preserved samples either showed no change or a change in nitrogen ratios. Formalin/ethanol and DMSO resulted in significant alterations of both carbon and nitrogen ratios for almost every tissue examin ed. Formalin mainly affected either carbon or nitrogen ratios in every test. The 70% et hanol solutions that were tested usually showed no effect; however, one study did report a significant change in the carbon ratio. Ethanol solutions that were stronger than 70% demonstrat ed mixed results affecting mainly carbon ratios. The results found in appendix A, while not conclusive to determining the best preservative for ev ery tissue, do help in determining the preservatives that should not be used and th e areas where further research is needed.
27 Table 3-1. Protocol for sampling of each treat ment at each time interval. The symbol Â“XÂ” indicates 3 epidermis samples were collected for that organism. Blank cells indicate no samples were collected. TURTLE TREATMENT TIME (days) Chelonia 1 Chelonia 2 Caretta 1 Caretta 2 Trachemys 1 Trachemys 2 Dry 0 X X X X X X 1 X X X X 4 X X X X X X 15 X X X X X X 30 X X X X X X Ethanol 60 X X 1 X X 4 X X 15 X X 30 X X DMSO buffer 60 X X 1 X X X X 4 X X X X X X 15 X X X X X X 30 X X X X X X Frozen 60 X X 1 X X 4 X X 15 X X 30 X X Saturated NaCl 60 X X
28 Table 3-2. P values from DunnettÂ’s adjustme nt tests evaluating preservation treatments on 15N and 13C in turtle epidermis samples. Values that differed significantly from dried controls are signified with an asterisk (*). TREATMENT TIME (days) p for 15N p for 13C 11.00000.8958 40.99780.6003 150.92300.8680 300.99980.9964 Ethanol 600.99690.7099 10.94940.0832 40.99720.9432 150.08340.3784 300.98720.9998 Saturated NaCl 601.00000.5245 11.00001.0000 41.00000.9997 151.00001.0000 300.95701.0000 Frozen 600.0440*< 0.0001* 10.0001*< 0.0001* 4< 0.0001*< 0.0001* 150.0055*< 0.0001* 300.0096*< 0.0001* DMSO buffer 601.00000.9734
29 CHAPTER 4 SUMMARY OF STUDIES The objective of the first porti on of my research was to determine if the Atlantic leatherback ( Dermochelys coriacea ) population nesting at Ju no Beach, Florida, were capital breeders, income breeders, or a co mbination of these two methods. Capital breeders use body stores to provi de nutrients for egg production while income breeders use recently ingested nutrients for eg g production (Thomas, 1988; Stearns, 1989; JÃ¶nsson, 1997). The leatherbacks nesting at Juno Beach were thought to provide an opportunity to study this question using carbon and nitrogen stable isotope analysis, because of their life stages and reproduc tive physiology. Leatherbacks are believed to spend the majority of their time in the open ocean. Therefore, isotopic ratios taken from body tissues would reflect an oceanic rati o. If they are us ing body stores for reproduction, no significant cha nge in isotopic signatures shou ld be seen over the season in their eggs. If income breeding is being used, a change should be seen over the nesting season. In this study, I found that the female ep idermis did represent an oceanic foraging carbon stable isotopic ratio and the stable ni trogen ratio increased as CCL increased. I also found that the stable carbon and nitrogen ratios of the albumen tissue sampled from yolkless eggs, collected at the time of their deposition, did not change significantly over the nesting season. This suggests that album en tissue is produced from body stores (capital breeding). However, the stable ca rbon ratio of the eggshe ll of the yolkless egg did change significantly over the nesting s eason. This change suggests an income
30 breeding strategy is being used to produce this tissue or that the phys iological influences of the turtles internesting dives throughout the season ar e influencing the carbon ratio. Although these results are supported in my study, they are not conclusive. Sample size for yolkless eggs at the e nd of the nesting season is too small to produce definitive results and may be contributi ng to the significant stable ca rbon isotope effect found in this study. Another confounding factor ma y be the unknown effect of the Florida Current. Satellite tracking studies conducted by Chris J ohnson and his team at Juno Beach reveal that nesting females are retu rning to the Florida Current during their internesting intervals. Stable isotopic ratios incorporated from any foraging occurring in this current would most likely appear as oceanic in origin, making it impossible to delineate from body stores using only stable isotope analysis. Epidermis samples that were collected on Juno Beach from the nesting female leatherbacks were preserved in a 70% ethano l solution. Because the effects of this preservative on the epidermis tissue of s ea turtles was not known, the objective of the second portion of my research was to determin e the effects of four preservation methods on turtle epidermis tissue. In my study, epidermis tissue from two green sea turtle ( Chelonia mydas ), two loggerhead sea turtles ( Caretta caretta ), and two red eared-slider turtles ( Trachemys scripta elegans ) were subjected to preservation by being dried at 60Â°C for 24 h (the control), placed in 70% ethanol solution, pl aced in saturated NaCl (sodium chloride) aqueous solution, frozen at -10Â°C in a fros t-free freezer, and placed in DMSO (dimethyl sulfoxide) buffer (250 mM EDTA (ethylened iaminetetraacetic acid) pH 7.5; 20% DMSO).
31 In this study, I found that tissues preserved in 70% ethanol and NaCl aqueous solution showed no significant difference from tissues dried at 60Â°C. Therefore, the stable isotope ratios obtained from the le atherback epidermis tissue preserved in 70% ethanol from the first portion of my thesis s hould be reliable. However, I also found that DMSO preserved samples were significantly altered from the dried samples. Samples that have been preserved using DMSO are not ideal for use in stable isotope analysis. The freezing preservative only showed a signif icant change in isotopi c ratios at 60 days. I believe this difference was due to the use of a frost-free freezer in this experiment and that the effects seen in this study could be corrected by using a different type of freezer that does not cycle temperatur e (to melt accumulating ice). In summary, my research supports 1) Atla ntic leatherback sea turtles may use both the capital and income breeding strategy for reproduction, 2) turtle epidermis can be preserved in 70% ethanol and aqueous NaCl without significantly altering the stable carbon and nitrogen isotopic ratio s, 3) DMSO preserved sample s are not ideal for use in stable isotope analysis because of the unpredictable changes th is preservative can have on the isotopic ratios, and 4) samples that are fr ozen for preservation s hould not be kept in a frost-free freezer. Future studies in this area should focu s on (1) increasing the sample size of leatherback yolkless eggs for stable isotope analysis thr oughout the nesting season, (2) collection of both epidermis and yolkless egg samples from the same turtle at time of nesting for comparison using stab le isotopes, (3) collection of dead in nest hatchlings to compare isotopic ratios from mothers to offspr ing, (4) incorporation of satellite tracking studies with stable isotopic an alysis to help determine the effects of the Florida Current,
32 (5) creation of a way to capture expelled ca rbon dioxide from nesting females for stable carbon isotope analysis to help determine defi nitively if the income breeding strategy is being used, and (6) re-examination of the e ffects of freezing on tissue samples using a freezer that is not frost-free.
APPENDIX A SUMMARY OF RESULTS IN STABLE ISOTOPE PRESERVA TION STUDIES
34Table A-1. Summary of results in stable isotope preservation st udies. Controls against which pr eservative treatments were com pared in each study are given in the footnotes. Preservative Tissue Species LEa 13Cb (Â‰) 15Nb (Â‰) Nc n = indiv Preservation Time 13C Signif 15N Signif Ref.d Oven Dried 50Â°C Muscle Argyrosomus hololepidotus No n/v n/v 3 per interval 1 fillet 1, 4, 12 wk No No 2 50Â°C Arm Octopus vulgaris No n/v n/v 3 per interval 1 arm 1, 4, 12 wk No No 2 50Â°C Frond Ecklonia radiate No n/v n/v 3 per interval 1 frond 1, 4, 12 wk No No 2 Air Dried 20Â–24 C Blood Coturnix coturnix japonica No -0.04 0 5 total 25 8 wk No No 4 20Â–24 C Blood Ovis aries No 0.02 -0.08 5 total 5 8 wk No No 4 Frozen -10Â°C Epidermis Chelonia mydas No 0.07 -0.6 3 per interval 2 1 d, 4 d, 15 d, 30 d, 1 individual 60 d 1 -10Â°C Epidermis Caretta caretta No -0.16 -0.03 3 per interval 2 1 d, 4 d, 15 d, 30 d, 1 individual 60 d 1 -10Â°C Epidermis Trachemys scripta elegans No -0.05 0.22 3 per interval 2 4 d, 15 d, 30 d 1 -10Â°C Epidermis Chelonia mydas Yes -0.09 -0.76 3 per interval 2 1 d, 4 d, 15 d, 30 d, 1 individual 60 d 1 -10Â°C Epidermis Caretta caretta Yes -0.15 0.17 3 per interval 2 1 d, 4 d, 15 d, 30 d, 1 individual 60 d 1 -10Â°C Epidermis Trachemys scripta elegans Yes 0.26 -0.21 3 per interval 2 4 d, 15 d, 30 d Yes, 60 d only Yes, 60 d only 1 -20Â°C Entire organisms Drosophila melanogaster No 0.23 -0.08 10 4 per sample 12 wk No No 5
35Table A-1. Continued Preservative Tissue Species LEa 13Cb (Â‰) 15Nb (Â‰) Nc n = indiv Preservation Time 13C Signif 15N Signif Ref.d -20Â°C egg, yolk lipids Coturnix japonica NA -0.02 n/a 5 5 eggs 50 d No n/a 7 -20Â°C Egg, yolk Coturnix japonica Yes 0.08 0.05 5 5 eggs 50 d No No 7 -20Â°C egg, albumin Coturnix japonica Yes 0.03 0.02 5 5 eggs 50 d No No 7 -10Â°C muscle Gadus morhua No -0.13 0.06 3 per interval 1 mature female 1 d, 8 d, 2 wk, 1 mo, 3 mo, 7 mo, 11 mo, 15 mo, 19 mo, 21 mo No No 6 -10Â°C Roe Gadus morhua No -0.21 0.14 3 per interval 1 mature female 1 d, 8 d, 2 wk, 1 mo, 3 mo, 7 mo, 11 mo, 15 mo, 19 mo, 21 mo No No 6 -10Â°C liver Gadus morhua No 0.16 0.17 3 per interval 1 mature female 1 d, 8 d, 2 wk, 1 mo, 3 mo, 7 mo, 11 mo, 15 mo, 19 mo, 21 mo No No 6 -18Â°C muscle Argyrosomus hololepidotus No n/v n/v 3 per interval 1 fillet 1, 4, 12 wk No No 2 -18Â°C Arm Octopus vulgaris No n/v n/v 3 per interval 1 arm 1, 4, 12 wk No No 2 -18Â°C frond Ecklonia radiata No n/v n/v 3 per interval 1 frond 1, 4, 12 wk No No 2 -20Â°C Entire organisms Bulk zooplankton No -0.83 0.61 5 n/a 4 d Yes Yes 3 Shock-frozen Immersed in N2 Entire organisms Bulk zooplankton No 0.11 1.5 5 n/a 4 d No Yes 3 Drowned in liquid N2 Entire organisms Drosophila melanogaster No -0.34 0.07 10 4 per sample 12 hr No Yes 5
36Table A-1. Continued Preservative Tissue Species LEa 13Cb (Â‰) 15Nb (Â‰) Nc n = indiv Preservation Time 13C Signif 15N Signif Ref.d Ethanol 70% Epidermis Chelonia mydas No 0.29 0.1 3 per interval 2 1 d, 4 d, 15 d, 30 d, 1 individual 60d 1 70% Epidermis Caretta caretta No 0.11 0.14 3/interval 2 1 d, 4 d, 15 d, 30 d, 1 individual 60 d 1 70% Epidermis Trachemys scripta elegans No 0.16 0.22 3 per interval 2 4 d, 15 d, 30 d 1 70% Epidermis Chelonia mydas Yes 0.11 -0.04 3 per interval 2 1 d, 4 d, 15 d, 30 d, 1 individual 60 d 1 70% Epidermis Caretta caretta Yes 0 0.41 3 per interval 2 1 d, 4 d, 15 d, 30 d, 1 individual 60 d 1 70% Epidermis Trachemys scripta elegans Yes 0.47 -0.26 3 per interval 2 4 d, 15 d, 30 d No No 1 70% Egg, yolk lipids Coturnix japonica n/a 0.27 n/a 5 5 eggs 50 d No n/a 7 70% Egg, yolk Coturnix japonica Yes 0.08 -0.1 5 5 eggs 50 d No No 7 70% Egg, albumen Coturnix japonica Yes 0 -0.07 5 5 eggs 50 d No No 7 95% Entire organisms Drosophila melanogaster No -1.38 0.17 10 4 per sample 10 d Yes No 5 95% Entire organisms Drosophila melanogaster No -1.17 0.12 10 4 per sample 6 wk Yes No 5 75% Muscle Catostomus occidentalis No 0.21 0.37 n/v n/v 3 d, 3 wk, 3 mo, 6 mo No Yes 9 75% Tissue removed from shell Corbicula fluminea No 2.18 -0.39 n/v n/v 3 d, 3 wk, 3 mo, 6 mo Yes Yes 9
37Table A-1. Continued Preservative Tissue Species LEa 13Cb (Â‰) 15Nb (Â‰) Nc n = indiv Preservation Time 13C Signif 15N Signif Ref.d 75% Entire organism Hydropsyche sp. No 0.04 -0.21 n/v n/v 3 d, 3 wk, 3 mo, 6 mo No No 9 70% Blood Coturnix coturnix japonica No -0.12 -0.26 5 total 25 8 wk No No 4 70% Blood Ovis aries No 0.02 -0.06 5 total 5 8 wk No No 4 70% Muscle Coturnix coturnix japonica No -0.44 0.4 5 total 5 8 wk No No 4 70% Muscle Argyrosomus hololepidotus No 0.7 to 1.5 n/v 3 per interval 1 1, 4, 12 wk Yes No 2 70% Arm Octopus vulgaris No 0.7 to 1.5 n/v 3 per interval 1 1, 4, 12 wk Yes No 2 70% Frond Ecklonia radiate No 0.7 to 1.5 n/v 3 per interval 1 1, 4, 12 wk Yes No 2 96%/ total of 30% Entire organisms Bulk zooplankton No 0.24 0.77 5 n/a 4 d No Yes 3 Industrial Ethanol (95% EtOH, 5% methanol) 80% Muscle Gadus morhua No 0.54 1.05 3 per interval 1 1 d, 8 d, 2 wk, 1 mo, 3 mo, 7 mo, 11 mo, 15 mo, 19 mo, 21 mo Yes Yes 6 80% Roe Gadus morhua No 0.81 0.44 3 per interval 1 1 d, 8 d, 2 wk, 1 mo, 3 mo, 7 mo, 11 mo, 15 mo, 19 mo, 21 mo No Yes 6 80% Liver Gadus morhua No 1.57 0.5 3 per interval 1 1 d, 8 d, 2 wk, 1 mo, 3 mo, 7 mo, 11 mo, 15 mo, 19 mo, 21 mo Yes No 6
38Table A-1. Continued Preservative Tissue Species LEa 13Cb (Â‰) 15Nb (Â‰) Nc n = indiv Preservation Time 13C Signif 15N Signif Ref.d 100% muscle Gadus morhua No 0.42 0.95 3 per interval 1 1 d, 8 d, 2 wk, 1 mo, 3 mo, 7 mo, 11 mo, 15 mo, 19 mo, 21 mo Yes Yes 6 Formalin/ Ethanol 10%/90% Muscle Arius felis No -1.12 0.62 1 1 2 wk formalin /4 wk EtOH Yes Yes 10 10%/90% Muscle Cynoscion nebulosus No -1.12 0.62 1 1 2 wk formalin /4 wk EtOH Yes Yes 10 10%/90% Muscle Dorosoma cepedianum No -1.12 0.62 2 2 2 wk formalin /4 wk EtOH Yes Yes 10 10%/90% Muscle Mugil cephalus No -1.12 0.62 12 12 2 wk formalin /4 wk EtOH Yes Yes 10 10%/90% Tail Crangon septemspinosa No -2.15 1.1 6 3 2 mo formalin /2 mo EtOH Yes Yes 8 10%/90% Muscle Pleuronectes americanus No -2.17 1.41 3 3 2 mo formalin /2 mo EtOH Yes Yes 8 Formalin 10% Egg, yolk lipids Coturnix japonica n/a -0.16 n/a 5 5 eggs 50 d No n/a 7 10% Egg, yolk Coturnix japonica Yes -2.44 0.03 5 5 eggs 50 d Yes No 7 10% Egg, albumen Coturnix japonica Yes -2.39 0.24 5 5 eggs 50 d Yes No 7 37% (10-15% methanol) Entire organisms Drosophila melanogaster No -2.92 0.34 10 4 per sample 10 d Yes Yes 5 37% (10-15% methanol) Entire organisms Drosophila melanogaster No -2.69 0.08 10 4 per sample 6 wk Yes No 5 10% Muscle Catostomus occidentalis No -1.33 0.16 n/v n/v 3 d, 3 wk, 3 mo, 6 mo Yes No 9 10% Tissue removed from shell Corbicula fluminea No 0.67 -0.48 n/v n/v 4 d, 3 wk, 3 mo, 6 mo No Yes 9
39Table A-1. Continued Preservative Tissue Species LEa 13Cb (Â‰) 15Nb (Â‰) Nc n = indiv Preservation Time 13C Signif 15N Signif Ref.d 10% Entire organism Hydropsyche sp. No -0.75 -0.121 n/v n/v 5 d, 3 wk, 3 mo, 6 mo Yes No 9 10% Tail Crangon septemspinosa No -2.05 0.35 6 3 n/v No No 8 10% Muscle Pleuronectes americanus No -0.74 1.21 3 3 n/v No Yes 8 5% Entire organisms Neomysis intermedia No n/a 0.04 n/a n/v 8.5 mo n/a No 11 10% Blood Coturnix coturnix japonica No -0.94 -0.34 5 total 25 8 wk No Yes 4 10% Blood Ovis aries No -1.32 -0.44 5 total 5 8 wk No Yes 4 10% Muscle Coturnix coturnix japonica No -1.78 0.04 5 total 5 8 wk Yes No 4 4% sodium phosphate Muscle Gadus morhua No -1.96 0.89 3 per interval 1 1 d, 8 d, 2 wk, 1 mo, 3 mo, 7 mo, 11 mo, 15 mo, 19 mo, 21 mo Yes Yes 6 4%/ 3gL-1 Na acetate trihydrate Muscle Gadus morhua No -1.49 0.71 3 per interval 1 1 d, 8 d, 2 wk, 1 mo, 3 mo, 7 mo, 11 mo, 15 mo, 19 mo, 21 mo Yes Yes 6 4%/ 3gL-1 Na acetate trihydrate Roe Gadus morhua No -1.22 0.28 3 per interval 1 1 d, 8 d, 2 wk, 1 mo, 3 mo, 7 mo, 11 mo, 15 mo, 19 mo, 21 mo Yes No 6 4%/ 3gL-1 Na acetate trihydrate Liver Gadus morhua No -1.06 -0.14 3 per interval 1 1 d, 8 d, 2 wk, 1 mo, 3 mo, 7 mo, 11 mo, 15 mo, 19 mo, 21 mo Yes No 6
40Table A-1. Continued Preservative Tissue Species LEa 13Cb (Â‰) 15Nb (Â‰) Nc n = indiv Preservation Time 13C Signif 15N Signif Ref.d Hexamine buffered 4% saline formalin Muscle Argyrosomus hololepidotus No -0.6 -0.28 to -3 3 per interval 1 1, 4, 12 wk Yes No 2 Hexamine buffered 4% saline formalin Arm Octopus vulgaris No -0.6 -0.28 to -3 3 per interval 1 1, 4, 12 wk Yes No 2 Hexamine buffered 4% saline formalin frond Ecklonia radiata No -1.5 -0.28 to -3 3 per interval 1 1, 4, 12 wk Yes No 2 37% formaldehyde / total 10% Entire organisms Bulk zooplankton No 1.09 0.8 5 n/a 4 d Yes Yes 3 Unbuffered formalinseawater (3% v/v) Entire organisms Marine zooplankton No -2.5 -1 n/v n/a 5Â–8 years No No 12 DMSO (250 mM EDTA pH 7.5; 20% DMSO) Epidermis Chelonia mydas No -2.49 -1.18 3/interval 1 1 d, 4 d, 15 d, 30 d, 1 individual 60 d 1 Epidermis Caretta caretta No -1.9 -0.47 3/interval 1 1 d, 4 d, 15 d, 30 d, 1 individual 60 d 1 Epidermis Chelonia mydas Yes -2.02 -0.8 3/interval 1 1 d, 4 d, 15 d, 30 d, 1 individual 60 d 1 Epidermis Caretta caretta Yes -1.49 -0.24 3/interval 1 1 d, 4 d, 15 d, 30 d, 1 individual 60 d 1d, 4d, 15d, and 30d only 1d, 4d, 15d, and 30d only 1
41Table A-1. Continued Preservative Tissue Species LEa 13Cb (Â‰) 15Nb (Â‰) Nc n = indiv Preservation Time 13C Signif 15N Signif Ref.d Muscle Coturnix coturnix japonica No -4.74 -0.74 5 total 5 8 wk Yes Yes 4 NaCl (>99.1%) muscle Arius felis No 0.13 0.72 16 1 6 wk No Yes 10 muscle Cynoscion nebulosus No 0.13 0.72 16 1 6 wk No Yes 10 muscle Dorosoma cepedianum No 0.13 0.72 16 2 6 wk No Yes 10 muscle Mugil cephalus No 0.13 0.72 16 12 6 wk No Yes 10 Aqueous NaCl Epidermis Chelonia mydas No 0.25 -0.07 3/interval 1 1 d, 4 d, 15 d, 30 d, 1 individual 60 d 1 Epidermis Caretta caretta No 0.38 -0.25 3/interval 1 1 d, 4 d, 15 d, 30 d, 1 individual 60 d 1 Epidermis Chelonia mydas Yes 0.09 0.4 3/interval 1 1 d, 4 d, 15 d, 30 d, 1 individual 60 d 1 Epidermis Caretta caretta Yes 0.25 0.46 3/interval 1 1 d, 4 d, 15 d, 30 d, 1 individual 60 d No No 1 33 g/L Entire organisms Drosophila melanogaster No -0.63 0.21 10 4 per sample 10 d No No 5 Seawater/Hg Cl muscle Pleuronectes americanus No -0.62 0.69 3 3 n/v No Yes 8 ABI Buffer Blood Coturnix coturnix japonica No -18.76 -5.16 5 total 25 8 wk Yes Yes 4 Blood Ovis aries No -17.2 -6.58 5 total 5 8 wk Yes Yes 4
42Table A-1. Continued Preservative Tissue Species LEa 13Cb (Â‰) 15Nb (Â‰) Nc n = indiv Preservation Time 13C Signif 15N Signif Ref.d QueenÂ’s Buffer Blood Coturnix coturnix japonica No -4.56 -1.32 5 total 25 8 wk Yes Yes 4 Blood Ovis aries No -4.36 -1.92 5 total 5 8 wk Yes Yes 4 Rotting 10 days in 5 mL of H2O Entire organisms Drosophila melanogaster No -0.81 0.42 10 4 per sample 10 d Yes Yes 5 Museum Preservation Muscle Rhinichthys cataractae No -2 0.4 3 3 per interval 10 d formalin n/v n/v 13 Muscle Rhinichthys cataractae No -2 0.4 3 3 per interval 40 d formalin n/v n/v 13 Muscle Rhinichthys cataractae No -2 0.4 3 3 per interval 70 d formalin n/v n/v 13 Muscle Rhinichthys cataractae No -2 0.4 2 3 per interval 100 d formalin n/v n/v 13 Muscle Rhinichthys cataractae No -2 0.4 2 3 per interval 130 d formalin n/v n/v 13 Muscle Rhinichthys cataractae No -2 0.4 3 3 per interval 160 d formalin n/v n/v 13 Muscle Rhinichthys cataractae No -2 0.4 3 3 per interval 190 d formalin n/v n/v 13 Muscle Percina caprodes No -0.8 0.5 3 3 12Â–15 yr n/v n/v 13 Muscle Percina roanoka No -0.8 0.5 3 3 12Â–15 yr n/v n/v 13 Muscle Etheostoma tippecanoe No -0.8 0.5 3 3 12Â–15 yr n/v n/v 13 10% formalinwater Distilled water-3Â–5 days 35% EtOH-2 wk 70% EtOHlong term Muscle Rhinichthys cataractae No -1.5 n/a 10 10 n/v Yes n/a 13
43Table A-1. Continued Preservative Tissue Species LEa 13Cb (Â‰) 15Nb (Â‰) Nc n = indiv Preservation Time 13C Signif 15N Signif Ref.d Ethylene Glycol Entire organisms Drosophila melanogaster No -1.52 0.15 10 4 per sample 10 d Yes No 5 Petroleum ether treated Muscle Rhinichthys cataractae Yes -0.058 n/a 10 10 4 hours No n/a 13 Methanol 30% Entire organisms Bulk zooplankton No 0.48 0.68 5 n/a 4 d Yes Yes 3 Gluteraldehyde 4% Entire organisms Bulk zooplankton No 0.65 0.04 5 n/a 4 d Yes Yes 3 Boiled Egg, yolk Lipids Coturnix japonica NA 0.14 n/a 5 5 eggs frozen 50 d No n/a 7 Egg, yolk Coturnix japonica Yes -0.03 0.22 5 5 eggs frozen 50 d No No 7 Egg, albumin Coturnix japonica Yes -0.11 0.14 5 5 eggs frozen 50 d/ 7 d room temp No No 7 Egg, yolk lipids Coturnix japonica NA 0.41 n/a 5 5 eggs frozen 50 d/ 7 d room temp No No 7 Egg, yolk Coturnix japonica Yes 0.03 0.3 5 5 eggs frozen 50 d/ 7 d room temp No No 7 Egg, albumin Coturnix japonica Yes -0.21 -0.2 5 5 eggs frozen 50 d/ 7 d @ 6Â°C No No 7 Egg, yolk lipids Coturnix japonica NA 0.13 n/a 5 5 eggs frozen 50 d/ 7 d @ 6Â°C No n/a 7 Egg, yolk Coturnix japonica Yes -0.55 -0.36 5 5 eggs frozen 50 d/ 7 d @ 6Â°C Yes No 7 Egg, albumin Coturnix japonica Yes -0.2 0.21 5 5 eggs frozen 50 d No No 7
44Table A-1. Continued Preservative Tissue Species LEa 13Cb (Â‰) 15Nb (Â‰) Nc n = indiv Preservation Time 13C Signif 15N Signif Ref.d Egg, yolk lipids Coturnix japonica n/a 0.03 n/a 5 5 eggs frozen 50 d @ 6Â°C No n/a 7 Egg, yolk Coturnix japonica Yes -0.13 0.13 5 5 eggs frozen 50 d @ 6Â°C No No 7 Egg, albumin Coturnix japonica Yes 0 0.41 5 5 eggs frozen 50 d @ 6Â°C No Yes 7 aLipid Extracted bDifference between treatment and control values. A positive value reflects an enrichment from the control and a negative value reflects a depletion. cN = within sample replicates dReferences identified by numb er and control used for eval uation of preservative effect efresh = control samples were collected at a di fferent time then the experimental samples 1. Present Study; dried at 60Â°C 2. Kaehler and Pakhomov, 2001; dried at 50Â°C 3. Feuchtmayr and Grey , 2003; dried at 60Â°C 4. Hobson et al., 1997; freeze-dried 5. Ponsard and Amlou, 1999; frozen at -80Â°C, then freeze-dried 6. Sweeting et al., 2004; freeze-dried 7. Gloutney and Hobson, 1998; separated in to eg g components, dried at 60Â°C, preserved in a 2:1 chloroform to methanol solution for 50 days, then dried 8. Bosley and Wainright, 1999; frozen at -80Â°C 9. Sarakinos et al., 2002; frozen at -25Â°C 10. Arrington and Winemiller, 2001; no control was used 11. Toda and Wada, 1990; freshe dried samples 12. Mullin, Rau, and Eppley, 1984; freshe samples dried at 60Â°C for several days 13. Edwards, Turner, and Sharp, 2002; frozen at -80Â°C n/v = denotes no value or information is available for that study n/a = denotes that the column does not apply to the study
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47 Hays, G.C., Glen, F., Broderick, A.C., Godl ey, B.J., Metcalfe, J.D., 2002b. Behavioral plasticity in a large marine herbivore: Contrasting patterns of depth utilisation between two green turtle ( Chelonia mydas ) populations. Marine Biology 141, 985Â– 990. Hays, G.C., Houghton, J.D.R., Myers, A.E ., 2004. Pan-Atlantic leatherback turtle movements. Nature 429, 522. Hobbie, E.A., Weber, N.S., Trappe, J.M., 2001. Mycorrhizal vs sapr otrophic status of fungi: The isotopic evidence. New Phytologist 150, 601Â–610. Hobson, K.A., 1995. Reconstructing avian diets using stable-carbon a nd nitrogen isotope analysis of egg components: patters of isotopic fractionation and turnover. Condor 97, 752Â–762. Hobson, K.A., Gibbs, H.L., Gloutney, M.L ., 1997. Preservation of blood and tissue samples for stable-carbon and stable-nitrogen isotope anal ysis. Canadian Journal of Zoology 75, 1720Â–1723. Hughes, G.R., Luschi, P., Mencacci, R., Pa pi, F., 1998. The 7000-km oceanic journey of a leatherback turtle tracked by satellite. Journal of Experimental Marine Biology and Ecology 229, 209Â–217. JÃ¶nsson, K.I., 1997. Capital and income breeding as alternative tactic s of resource use in reproduction. Oikos 78, 57Â–66. Kaehler, S., Pakhomov, E.A., 2001. Effects of storage and preservation on the 13C and 15N signatures of selected marine organi sms. Marine Ecology Progress Series 219, 299Â–304. Kiriluk, R.M., Whittle, D.M., Keir, M.J., Ca rswell, A.A., Huestis, S.Y., 1997. The great lakes fisheries specimen bank: A Canadian perspective in environmental specimen banking. Chemosphere 34, 1921Â–1932. Krueger, H.W., Sullivan, C.H., 1984. Models of carbon isotope fractionation between diet and bone. In: Turnland, J.R., Johnson, P. E. (Eds.), Stable Isotopes in Nutrition. ACS Symp. Ser. 258. American Chem ical Society, Washington DC. pp. 205Â–220. Kullberg, C., Jakobsson, S., Kaby, U., Lind, J ., 2005. Impaired flight ability to egglaying: a cost of being a capital breeder. Functional Ecology 19, 98Â–101. Kurle, C.M., Worthy, G.A.J., 2002. Stable nitr ogen and carbon isotope ratios in multiple tissues of the northern fur seal Callorhinus ursinus : implications for dietary and migratory reconstructions. Marine Ecology Progress Series 236, 289Â–300. Macko, S.A., Fogel-Estep, M.L., Engel, M.H ., Hare, P.E., 1986. Kinetic fractionation of nitrogen isotopes during amino acid tran samination. Geochimica et Cosmochimica Acta 50, 2143Â–2146.
48 Maros, A., Louveaux, A., Lelarge, C., Gi rondot, M., 2006. Evidence of the exploitation of marine resource by th e terrestrial insect Scapteriscus didactylus through stable isotope analyzes of its cuticle. BMC Ecology 6, 6. Meijer, T., Drent, R., 1999. Re-examination of the capital and income dichotomy in breeding birds. Ibis 141, 399Â–414. Minagawa, M., Wada, E., 1984. Stepwise enrichment of 15N along food chains: Further evidence and the relationship between 15N and animal age. Geochimica et Cosmochimica Acta 48, 1135Â–1140. Mullin, M.M., Rau, G.H., Eppley, R.W., 1984. Stab le nitrogen isotopes in zooplankton: some geographic and temporal variations in the north paci fic. Limnology and Oceanography 29, 1267Â–1273. Peterson, B.J., Fry, B., 1987. Stable isotopes in ecosystem studies. Annual Review of Ecology and Systematics 18, 293Â–320. Ponsard, S., Amlou, M., 1999. Effects of seve ral preservation methods on the isotopic content of Drosophila samples. Comptes Rendus de lÂ’Academie des Sciences, Sciences de la vie 322, 35Â–41. Rubenstein, D.R., Hobson, K. A., 2004. From birds to butterflies: Animal movement patterns and stable isotopes. Tre nds in Ecology and Evolution 19, 256Â–263. Sarakinos, H.C., Johnson, M.L., Vander Za nden, M.J., 2002. A synthesis of tissuepreservation effects on carbon and nitrogen stable isotope signatures. Canadian Journal of Zoology 80, 381Â–387. Seminoff, J.A., Jones, T.T., Eguchi, T., J ones, D.R., Dutton, P.H., 2006. Stable isotope discrimination ( 13C and 15N) between soft tissues of the green sea turtle Chelonia mydas and its diet. Marine Ecology Progress Series 308, 271Â–278. Stearns, S.C., 1989. Trade-offs in life hi story evolution. Functional Ecology 3, 259Â–268. Sweeting, C.J., Polunin, N.V.C., Jennings, S ., 2004. Tissue and fixative dependent shifts of 13C and 15N in preserved ecological materi al. Rapid Communications in Mass Spectrometry 18, 2587Â–2592. Taylor, T.G., 1970. How an eggshell is made. Scientific American 222, 88Â–95. Thomas, V.G., 1988. Body composition, ovarian hi erarchies, and their relation to egg formation in anseriform and galliform sp ecies. In: Ouellet, H., (Ed.), Acta XIX Congressus Internationalis Ornithologici. University of Ottawa Press, Ottawa, pp. 353Â–363.
49 Toda, H., Wada, E., 1990. Use of 15N/14N rations to evaluate the food source of the mysid, Neomysis intermedia Czerniawsky, in a eutrophic lake in Japan. Hydrobiologia 194, 85Â–90. Todd, S., Ostrom, P., Lien, J., Abrajano, J., 1997. Use of biopsy samples of humpback whales (Megaptera novaeangliae) skin for stable isotope ( 13C) determination. Journal of Northwest Atlantic Fishery Science 22, 71Â–76. Tucker, A.D., Read, M.A., 2001. Frequency of foraging by gravid green turtles ( Chelonia mydas ) at Raine Island, Great Barrier R eef. Journal of Herpetology 35, 500Â–503. Wallace, B.P., Seminoff, J.A., Kilham, S.S., Spotila, J.R., Dutton, P.H., 2006. Leatherback turtles as oceanographic indica tors: stable isotope analyses reveal a trophic dichotomy between ocean basins. Marine Biology 149, 953Â–960. Wyneken, J., 2001. The Anatomy of Sea Turtle s. U.S. Department of Commerce NOAA Technical Memorandum NMRS-SEFSC-470. pp. 1Â–172.
50 BIOGRAPHICAL SKETCH Lindy Michelle Barrow graduated from Pace High School in Pace, Florida in 1999. She then moved to Gainesville, Florida, where she pursued a bachelorÂ’s degree in zoology and chemistry. In May of 2003, Lindy graduated summa cum laude in zoology and cum laude in chemistry. A few months after graduation, Lindy traveled to Playa Grande, Costa Rica to be the Volunteer Coor dinator for the Earthw atch Leatherback Sea Turtle Program being conducted on that beach. After five and a half months on the project, Lindy returned to Ga inesville, Florida to pursue he r masterÂ’s degree in zoology, specifically to conduct sea tur tle research. After completi ng both her course work and her laboratory research, Li ndy was married and moved to Boston, Massachusetts. While in Boston, Lindy completed writing her thesis , returning to Gainesville to defend and graduate from UF.