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Trophic enrichment patterns of δ 13C in organic matter of molluscan shell

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Title:
Trophic enrichment patterns of δ 13C in organic matter of molluscan shell implications for reconstructing ancient environments and food webs
Alternate title:
Trophic enrichment patterns of delta13C in organic matter of molluscan shell
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McKnight, Julie
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Amino acids
Stable isotopes
Food web
Trophic
Mollusk shell
Dissertations, Academic -- Geology -- Masters -- USF   ( lcsh )
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non-fiction   ( marcgt )

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ABSTRACT: Shell organic matrix proteins in fossils are valuable geochemical archives for studying ancient environments and food webs. Compound-specific studies of stable carbon isotope ratios offer particularly good resolution of trophic level of consumers and the identities of primary producers and can be used to detect diagenetic alteration of isotopic ratios. To interpret compound specific isotope data, however, controlled diet studies in the laboratory are needed to reveal trophic enrichment patterns of 13C in tissues and shell organic matter. This study examines the relationship between δ 13C of 11 amino acids in diet, soft tissues, and shell organic matter in laboratory-cultured Strombus alatus, an herbivorous marine gastropod. The δ 13C values of amino acids in this animal's foot and mantle tissues are consistently enriched in 13C relative to the diet.Phenylalanine (+1.8 ppm) and alanine (+3.8 ppm) showed the least fractionation between diet and tissues, while aspartic acid (+10.7 ppm) and glutamic acid (+14.6 ppm) showed the greatest enrichment. On average, nonessential amino acids exhibited greater enrichment than did essential amino acids (+7.1 ppm vs. + 4.1 ppm). Shell organic matter amino acids showed a very similar pattern, with aspartic and glutamic acids again showing the greatest enrichment (+7.2 ppm and +11.1 ppm respectively). Nonessential amino acids in shell (+4.9 ppm) were also more enriched than the essential amino acids (+3.5 ppm). Overall, the carbon isotopic compositions of amino acids in shell organic matrix appear to parallel those in animal tissue, validating the utility of employing this material as a surrogate for animal tissue in fossil samples.Interpreting trophic position information in consumers is difficult, however, as the variation in the magnitude of trophic enrichments for glutamic and aspartic acids between species, tissue types and diet is still poorly understood. As phenylalanine has the most consistent diet-consumer enrichments, the most suitable application for δ 13C isotope analysis at this time is the reconstruction of base food sources.
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Thesis (M.S.)--University of South Florida, 2009.
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by Julie McKnight.
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ABSTRACT: Shell organic matrix proteins in fossils are valuable geochemical archives for studying ancient environments and food webs. Compound-specific studies of stable carbon isotope ratios offer particularly good resolution of trophic level of consumers and the identities of primary producers and can be used to detect diagenetic alteration of isotopic ratios. To interpret compound specific isotope data, however, controlled diet studies in the laboratory are needed to reveal trophic enrichment patterns of 13C in tissues and shell organic matter. This study examines the relationship between 13C of 11 amino acids in diet, soft tissues, and shell organic matter in laboratory-cultured Strombus alatus, an herbivorous marine gastropod. The 13C values of amino acids in this animal's foot and mantle tissues are consistently enriched in 13C relative to the diet.Phenylalanine (+1.8 ppm) and alanine (+3.8 ppm) showed the least fractionation between diet and tissues, while aspartic acid (+10.7 ppm) and glutamic acid (+14.6 ppm) showed the greatest enrichment. On average, nonessential amino acids exhibited greater enrichment than did essential amino acids (+7.1 ppm vs. + 4.1 ppm). Shell organic matter amino acids showed a very similar pattern, with aspartic and glutamic acids again showing the greatest enrichment (+7.2 ppm and +11.1 ppm respectively). Nonessential amino acids in shell (+4.9 ppm) were also more enriched than the essential amino acids (+3.5 ppm). Overall, the carbon isotopic compositions of amino acids in shell organic matrix appear to parallel those in animal tissue, validating the utility of employing this material as a surrogate for animal tissue in fossil samples.Interpreting trophic position information in consumers is difficult, however, as the variation in the magnitude of trophic enrichments for glutamic and aspartic acids between species, tissue types and diet is still poorly understood. As phenylalanine has the most consistent diet-consumer enrichments, the most suitable application for 13C isotope analysis at this time is the reconstruction of base food sources.
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T 13 C in Organic Matter of Molluscan Shell: Implications for Reconstructing Anci ent Environments and Food Webs b y Julie McKnight A thesis submitted in partial fulfillment of the requirements for the degree of Mast er of Science Department of Geology College of Arts and Sciences University of South Florida Major Professor: Gregory Herbert, Ph.D. Peter Harries, Ph.D. Jonathan Wynn, Ph.D. Date of Approval: June 5, 2009 Keywords: Amino acids, Stable isotopes, Fo od web, Trophic, Mollusk shell Copyright 2009, Julie McKnight

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i Table of Contents List of Tables ii List of Figures iii Abstract iv Chapter One : Introduction 1 Chapter Two : Experimental Study 7 M e t h o d s 7 Controlled Feeding Experiment 7 Sample Selection 8 Preparation and Analysis 8 Bulk Analysis 9 Compound Specific Analysis 9 Data Processing 11 Results 12 Bulk vs. Compound Speci fic Carbon Isotopes 1 4 Compound 13 C Fractionations 1 8 Essential vs. Non Ess ential Amino Acids 2 0 D iscussion 2 2 Trophic Discrimination Factor in Mollusks 2 2 The Biomine ralization Effect 2 5 13 C Fractionation 2 7 Chapter Three : Conclusions 3 0 References 3 2 Appendices 3 6 Appendix A: Bulk 13 C Values for All Samples 3 7 Append ix B: Corrected 13 C Values for Individual AA for All Samples 3 8 Appendix C: Raw 13 C Values for Individua l AA for All Samples 4 2 Appendix D: Kinetic Isotope Effect Correctional Value s 4 6

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ii List of Tables Table 1 Relative Abundances of Individual Amino Acids 1 3 Table 2 13 C Values for Individual Amino Acids in Strombus spp. 1 6 Table 3 13 C Values for Diet Consumer Enrichments in Strombus spp. 1 9 Table 4 Comparison of Amino Acid 13 C R esu lts to Other Studies 2 8

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iii List of Figures Figure 1 13 C Values for Diet and Foot Relative to Amino Acid Synthesis 1 4 Figure 2 A 13 C Values for Essential Amino Acids 1 7 Figure 2B 13 C Values fo r Non Essential Amino Acids 1 8 Figure 3A 13 C Consumer Diet Enrichments for Essential Amino Acids 2 1 Figure 3B 13 C Consumer Diet Enrichments for Non Essential Amino Acids 2 2

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iv 13 C in O rganic Matter of Molluscan Shell: Implications for Reconstructing Ancient Environments and Food Webs Julie McKnight ABSTRACT Shell organic matrix proteins in fossils are valuable geochemical archives for studying ancient environments and food webs. Co mpound specific studies of stable carbon isotope ratios offer particularly good resolution of trophic level of consumers and the identities of primary producers and can be used to detect diagenetic alteration of isotop ic ratios. To interpret compound spec ific isotope data, however, controlled diet studies in the laboratory are needed to reveal trophic enrichment patterns of 13 C in tissues and shell organic matter. 13 C of 11 amino acids in diet, soft tissues, an d shell organic matter in laboratory cultured Strombus alatus an herbivorous 13 are consistently enriched in 13 nonessential amino acids exhibited greater enrichment than did essential amino acids (+7.1 vs.

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v were also more enriched than th compositions of amino acids in shell organic matrix appear to parallel those in animal tissue, validating the utility of employing this material as a surrogate for animal tissue in fossil sampl es. Interpreting trophic position information in consumers is difficult, however, as the v ariation in the magnitude of trophic enrichments for glutamic and aspartic acids between species, tissue types and diet is still poorly understood. As phenylalanine has the most consistent diet consumer enrichments the most suitable 13 C isotope analysis at this time is the reconstruction of base food sources.

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1 Chapter One Introduction Stable isotope analysis has been a vital resource in under standing the general structure of ecosystems In addition to providing insights into modern communities, isotope techniques have also been employed in the reconstruction of ancient ecosystems ( Fox Dobbs et al 2008). These reconstructions are essential to our understanding of how energy flow through ecosystems has evolved in response to long term environmental changes and how modern ecosystems have been affected by anthropogenic alteration. Bulk isotope analysis has been a key asset in trophic and food sou rce studies for the better part of four decades. Carbon and nitrogen are among the most common isotopes used in bulk analysis as consistent diet consumer enrichments 13 C 15 N (DeNiro and Epstein 1981) have been 15 N makes it more suitable for resolving consumer hierarchy, or trophic position, within a food web. Carbon, however, often used to determine what primary producers likely served as the base nutritional source in a system. These trends have led to the use of bulk isotope analysis in a variety of studies identifying food web interactions ( Corbisier et al 2006 Grall et al 2006 ), diet shifts

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2 (Paulet et al 2006), migrational patterns (MacNeil et al 2005) and general dietary trends across spatial and temporal boundaries (Phillips and Eldridge 2006). 15 13 C (Fry 1988), there are several shortcomings in the bulk nitrogen approach. For instance, variation in the isotopic signature of different tissues within the same organism has been demonstrated in various taxa (DeNiro and Epstein 1981 Hobson and Clark 1992, Lorrain et al 2002, Yokoyama et al 2008 ). Additionally, studies have also demonstrated interspecies variation in diet 15 N enrichments (Vanderklift and Ponsard 2003, D ubois et al 2007 ). O ne o f the most prominent issues, however, is that bulk nitrogen does not directly indicate baseline or source, values and can only be used to determine 15 N signature is largely based on both the trophi c position of the consumer and the primary producers at the base of the food web. In marine 15 N values of these baseline primary producers can vary both spatially and S hifts 15 N values in consumer tissues that appear to be indicative of diet switching may thus be a mere artifact of the natural variation in the primary nitrogen source. The isotopic values of individual organic components can elucidate valuable source and trophic information that would otherwise be confounded in the averaged bulk 15 N is significantly enriched in some amino acids (AA) while others undergo very little to no fractionation (M cClelland and Montoya 2002). If these trends in fractionation are well defined, in dividual amino acids from a single organism can be used to determine either the

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3 food web (trophic AA). Based on these principles, simultaneous shifts in source nitrogen and trophic position can be deconvolved (Popp et al 2007) with compound specific isotope analysis (CSIA). 15 N, there is great potential for the use of CSIA of carbon isotopes for food web research As carbon is more abundant in organic material than nitrogen, instrument sensitivity is less of a limiting f actor and thus smaller sample sizes can be used and still obtain reliable data. The ability to utilize smaller sample sizes is particularly useful when working with limited specimens such as fossils, which can lose large amounts of material due to diagene sis (Mitterer 1972, Hare and Hoering 1977, Hoering 1980 ). The fractionation of carbon isotopes is more complex however, due to the biological processes underlying amino acid synthesis in animals. Amino acids are commonly partitioned into what are known as essential amino acids (EAA) and non essential amino acids (NAA). EAA s which cannot be synthesized de novo The incorporation of EAAs directly from diet in to consumer tissues is not accompanied 13 C fractionation I n theory, the result i s similar EAA isotopic values in both diet and consumer. NAA s however, are generally isotopically enriched with respect to diet as they are synthesized de novo. ketoacids, are produced from the breakdown of proteins, carbohydrates and lipids during the citric acid cycle. As the ketoacids is cycled extensively during this process, there is 13 C during NAA synthesi s. Finally, NAA s are ketoacid via a transamination reaction. The effects of these processes on carbon fractionation have been

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4 supported by controlled feeding experiments which showed 13 C values of EAA s in consumer tissues remained very similar to dietary values whereas NAA s were significantly enriched (Fantle et al 1999). 13 C fractionation between individual amino acids can thus provide valuable molecular level insight into bulk isotopic signals. Amino acids are vital to our understanding of diagenetic processes that call into question the ind i geneity of organic material in fossil shell specimens. Apart from physical processes such as leaching and contamination chemical reactions such as hydrolysis and defunctionalization, result in the breakdown and loss of original organic material (Mitterer 1995). S ome amino acids are less stable than others due to biochemical features such as side chain acidity/basicity, side chain polarity, and hydrophilic/hydrophobic properties, which make them more susceptible to diagenetic processes. The result is a dominant suite of the more stable, i.e. neutral or nonpolar, amino acids in older shells (Vallentyne 1964, Hare and Mit terer 1967). 13 C values between a mino acid s can et al 200 7 ) thus large variation in the proportion of amino acids in diagenetically alter fossil shell would significant ly change the bulk 13 C value. Although a loss of up to 98 % of origin al organic material has been reported in a Miocene Mercencaria shell ( Hare and Mitterer 1967 ), experimentally aged shells show that the retention of certain amino acids during diagenesis is considerably high (Qian et al 1995) and in fact sufficient organ ic material 2005) and even Cretaceous (Ostrom et al 1993) fossil specimens for paleoecological reconstructions The same unstable characteristics that result in the los s of certain amino

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5 acids, however, also drive the incorporation of newer foreign material into the shell matrix These contaminating amino acids, such as serine, are often by products of metabolic processes (Hamilton 1965, Hare 1965 ) in surrounding organi c materials and thus bear their own distinct isotopic signatures The result is a fossil sample with an amino acid range resembling modern organisms or younger fossils Engel and Macko (1986 ) presented one solution to this in a study that showed that rac emization, the process by which an L amino acid becomes a mirror image of itself, a D amino acid, does not od for establishing the Although useful, this method does not provide a solution for the isotopic analysis of less stable amino acids. D uring the biomineralization of molluscan shell, calciu m carbonate crystals are formed in an organic matrix secreted by the mantle tissue. A mino acids are subsequently transferred from the mantle tissue into these hard crystalline structures where they can be preserved for up to 20 million years ( Hare and Hoe ring 1977, Hoering 1980 ). As a result, the intracrystalline fraction of fossil shell organics provides the best representation of material indigenous to the original organism (Towe 1980). In fact previous research shows that for amino acids the analysis of only intra crystalline proteins can reduce the inter shell variability in D/L AA values by up to 50% (Penkman et al 2008). In order to successfully apply stable isotope techniques to fossil shell material, however, a comprehensive understanding of the fractionation effects during the biomineralization process is necessary

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6 In this paper I 13 C in individual amino acids from diet to consumer tissues i.e. foot muscle and mantle tissue, and shell organic matter in a marine herbivorous gastropod reared on a controlled diet. My primary research objective wa s to de 13 C is reflected in the shell organic material of mollusks. It wa s also my aim to determine w hich amino acids, if any, in the shell organic material were best suited to serve as trophic or source indicators in reconstr uctions of modern and ancient systems.

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7 Chapter Two E x p e r i m e n t a l S t u d y M ethods Controlled Feeding Experiment Twenty five Strombus alatus were reared on a controlled diet under laboratory conditions at the Harbor Branch Oceanographic Institution in Ft. Pierce, Florida. Cultivation procedures follow those outlined by Davis (2005). Eggs were collected from captive breeding tanks and, once hatched, veliger stage larvae were cultured in 130 to 530 gallon conical tanks maintained at 28C and 30 ppt salinity for ~21 days. After metamorphosis, juveniles were held in a recirculating seawater system at a temperature of 27 29 C and fed a flocculated form of the diatom Chaetoceros gracilis for the first 2 3 weeks. Once juveniles reached a shell length of 3 4 mm they were fe d a gel based diet consisting of a homogenized mixture of ground catfish pellets, dried Ulva spp. seaweed, alginate, and seawater. A daily feeding regime of ~10 mg of feed per conch per day was maintained until they reached a shell length of 6 mm after wh ich they were fed approximately 2 to 7 percent of the wet meat weight. These prepared pellets, made in house, served as the sole food source for the juvenile conchs for approximately 15 months until they reached a size range of 30 37 mm in length.

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8 Samp le Selection The prepared pellet diet described previously was analyzed to establish the isotopic baseline for this system. Ten pellets (0.8 1.5 g each) were selected at random from the in house supply and prepared for analysis. Separate samples of foo t and mantle tissues and powdered shell samples were collected from thirteen juvenile Strombus spp. selected at random from the cultivated stock. Selected specimens were kept frozen at 60 C until preparation for analysis. Foot and mantle tissue samples were obtained via dissection with the tissues being rinsed twice in DI water to remove surface contaminants. The entire shell for an individual was homogenized and used as a single sample. Preparation and Analysis In order to isolate organic material from the shell matrix, dialysis was performed under weak acidic conditions. Whole shells were first dried in an oven at 60C for 48 hours then homogenized into a fine powder. Samples were mixed with deionized water and transferred into dialysis tubing wi th a nominal pore size of 3 kilodaltons. Samples were first dialyzed in a 20 liter volume of 0.05 M HCl for 72 hours to remove all carbonate and then dialyzed in a 20 liter volume of deionized water to remove salts and residual acid. The insoluble materi al was extracted via filtration using 0.6 micron Teflon filters and dried in an oven at 60C for 48 hours. Samples for diet, foot tissue and mantle

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9 tissue were dried in an oven at 60C and homogenized into a fine powder. All samples were stored in a desi ccation chamber until preparation for compound specific analysis. Bulk Analysis Bulk analysis was only performed for diet, foot tissue and mantle tissue samples. From the previously prepared samples 0.8 1.5 mg of dried material was put in 3.5 mm x 5 mm tin boats. Bulk 13 C values were measured on a Thermo Delta Plus XL mass spectrometer. Carbon isotope ratios were determined relative to the PDB standard in parts per mil. Results are expressed as: 13 C = [(R sample / R standard ) 1] X 1000 where R sample and R standard are the ratios of 13 C/ 12 C for the sample and standard respectively. Compound Specific Ana lysis Preparation methods for compound specific isotope analysis followed procedures outlined by McClelland and Montoya (2002). Samples were dried in an oven at 60C for 48 hours and then homogenized into a fine powder. Then 5 mg of dry powder was digested in 6 M HCl under a nitrogen atmosphere for 24 hours at 110 o C, evaporated to dryness and redissolved in 2 ml of 0.01 N HCl. Liquid c olumn chromatogr aphy was used to extract amino acids from sample digests. Columns were assembled in Pasteur pipettes using 5 cm Dowex 50WX8 400 ion exchange resin. Under the acidic conditions of the digests, amino acids adhered to the resin, were eluted using 4 ml of 2 M ammonium hydroxide and then dried under an N 2 stream prior to derivatization.

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10 In order to facilitate analysis on the gas chromatograph, a derivatization process was necessary prior to analysis Samples were derivatized to N acetyl i propyl ( NAIP ) esters in a two step process. First, samples were esterified by heating to 100 o C for 1 hour in 1 m L of acidified isopropanol (2.8 M with acetyl chloride). After quenching the reaction, by chilling to 0 o C, the residual reagents were evaporated under an N 2 stream rinsed twice with reagent grade dichloromethane to remove any remaining solvents or water and evaporated to dryness. In the acylation phase, samples were dissolved in a 1.0 ml mix of acetic anhydride, triethylamine, and acetone (1:2:5 by volume), heated to 60 o C for 10 minutes and again evaporated to dryness. Samples were then redissolved using 1. 0 ml of ethyl acetate and 1.0 mL of NaCl saturated water and vigorou sly mixed producing a two phase solution. The organic phase of this solution, containing th e derivatized products, was retained whereas the aqueous phased was discarded. The derivatized amino acids were dried under an N 2 stream, rinsed twice with reagent grade dichloromethane and again evaporated to dryness and stored in this state until analys is. For injection on the gas chromatograph s amples were redissolved in 1.0 m L of L of this solution was injected into an Agilent 6890 gas chromatograph with a split/splitless injector in splitless mode linked to a Thermo Delta Plus XL mass spectrometer via a GC Combustion III interface. Two columns, a DB 5 (30 m x 0.32 mm an Rtx chromatography. Combustion oven temperature was 950C and reduction oven temper ature was 650C.

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11 Data Processing 13 C values are affected by a kinetic isotope effect (KIE) during the acylation step of the derivatization process (Silfer et al 1991). As 13 C values for derivatized amino acids will not match those predicted by stoichometric mass balance equations using the carbon isotopic compositions of the amino acids and derivatizing reagents. Therefore, the isotopic composition of sample amino acids cannot be determined directly by me asurement of the values of reagents and derivatized products. To circum 13 C values can be determined by derivatizing and measuring amino acid standards of known isotopic composition (previously determined by EA 13 C d in the following equation (Corr et al 2007): n cd 13 C cd = n c 13 C c + n d 13 C d where n is the number of carbon atoms in each molecule, the subscript c is the amino acid under study, d is the derivative group, and cd is the derivatized product. Since the extent of the kinetic isotope in this reaction has been shown to be consistent, this value can then 13 C c (the original, underivatized isotopic composition) for sample amino acids (Silfer et al 1991).

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12 Results As an initial test of the soundness o f my methods and accuracy of my data, 13 C values for the diet and foot tissue for individual amino acids were compared to underlying metabolic processes driving their synthesis (Figure 1) Amino acids sharing common biosynthetic pathways should 13 C values. 13 C diet values and trophic enrichments for threonine and isoleucine, for example, were consistent with the fact that isoleucine is derived from a biosynthetic pathway which begins with threonine. Similarly, glycine i s synthesized directly from 13 C diet values in our study. 13 C values of foot and mantle tissues for alanine were closest to t 13 C values of bulk tissues. Alanine is a major component in muscle tissue and also one of the most abundant circulating amino acids. 13 C of bulk organic matter was, as expected, strongly influenced by the 13 C value of alanine. T he r elative abun dances of individual amino acids analyzed were determined for each organic component analyzed (Table 1) Values are based on the total area integrated under each peak in the chromatogram normalized to standards of known concentration. Alanine, glycine, an d glutamic acid we re of the most abundant amino acids in the diet and consumer mantle and foot tissues. The average d 13 C value for these three amino acids was similar to the bulk value indicating supporting the notion that relative AA abundance is influential in bulk isotopic measurements. Proline also contributed greatly to the pool of diet ary amino acids but was les s abundant in the tissues. Aspartic acid, however, was more abundant in soft tissues compared to diet and was the next largest

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13 contributing amino acid to alanine, glycine, and glutamic acid. Relative abundances of all amino acids were similar between the mantle and foot tissues. In shell organic matter the two most contributing amino acids were glutamic and aspartic acid, both of which have been shown to be important in the biominerlization process. Phenylalanine contributed little in all four organic m aterials and was the least abundant amino acid in foot tissue, mantle tissue and shell organic matter. Table 1 : Relative A bundances of I ndividual A mino A cids Amino Acid Diet Foot Mantle Shell alanine 10.2 13.6 13.2 8.6 aspartic acid 7.9 10.5 10.3 15.1 glutamic acid 21.7 14.2 12.1 16.9 glycine 16.2 23.8 21.4 9.8 isoleucine 2.7 5.0 4.5 5.4 leucine 9.6 8.5 7.8 10.1 phenylalanine 3.8 2.0 2.8 4.1 proline 14.6 5.7 7.7 7.1 serine 7.2 6.0 6.8 7.8 threonine 3.1 4.2 5.6 6.8 valine 3.0 6.5 7.7 8.5 Va lues for aspartic acid and glutamic acid are the sum of aspartic acid plus asparagines and glutamic acid plus glutamine respectively.

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14 Fig. 1. 13 C Values for Diet and Foot Relative to Amino Acid Synthesis: 13 C values (n=13) are shown for die t and foot tissue with error bars indicating the standard deviation. Essential and non essential amino acids are grouped with respect to common precursors involved in metabolic pathways resulting in their synthesis. Essential amino acids are divided into the aromatic, aspartate and pyruvate families. Non essential amino acids are divided into the TCA and glycolytic groups which are synthesized from precursors created during the Citric Acid Cycle and glycolysis, respectively. Proline is defined as an imi no acid as it contains both imino and carboxyl functional groups. Bulk versus Compound Specific Carbon Isotopes Measured bulk and compound 13 C values for all analyzed materials are listed in Table 2. Averaged bulk 13 C diet tissue enrichmen conch foot and mantle tissues. Although diet consumer fractionation was anticipated, 1978). CSIA of 13 C values varied from the In spite of the variation among individual components the averaged value of all amino acids in the diet,

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15 ino acids in both foot and mantle foot and mantle respectively. Essential ami no acids in both diet and tissues were generally depleted relative to 13 C values of diet and tissues with the exception of threonine and, in the case of foot tissue, isoleucine as well (Figure 2A). Fractionation trends in non essential amino acids w ere more variable. In the diet, only glycine and serine were significantly enriched with respect to bulk whereas for both foot and mantle tissues all NAA except alanine and proline were enriched (Figure 2B). Diet values for bulk and all NAA were depleted relative to both tissues and shell organic matter, s 13 C from diet 13 C values remained similar in most NAA however the mantle is depleted with respect to foot tissue in aspartic acid and glycine. Shell organic matter was enriched with respect to diet for all NAA but is depleted with respect to tissues in all NAA except alanine.

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16 Table 2 : 13 C Values for Individual Amino Acids in Strombus spp. Diet SD Foot SD Mantle SD Shell SD Bulk 23.4 0.2 20.9 0.3 21.2 0.7 Essential Isoleucine 24.3 1.2 19.3 3.1 21.9 2.1 22.3 2.9 Leucine 34.1 0.6 28.6 1.0 29.8 1.6 22.3 1.5 Phenylalanine 28.1 0.4 27.2 1.0 27.1 1.2 27.4 1.7 Threonine 22.8 4.5 15.6 3.3 14.9 2.1 16.6 2.9 Valine 28.0 1.1 24.3 1.2 24.7 1.1 25.2 1.8 Non Esssential Alanine 24.2 1.9 21.6 1.5 22.0 1.3 22.0 2.1 Aspartic Acid 25.5 3.3 14.8 1.5 16.4 2.1 17.4 1.6 Glutamic Acid 27.9 0.7 14.7 2.2 14.6 1.5 16.9 2.8 Glycine 15.0 1.0 7.6 2.3 10.1 3.3 13.3 2.7 Prol ine 23.2 0.3 19.6 1.4 19.4 1.8 20.7 1.3 Serine 10.0 1.2 5.4 3.0 6.4 4.2 9.1 2.3 SD=Standard Deviation

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17 Fig.2A 13 C Values for Essential Amino Acids: Mean (n=13) 13 C values of essential amino acids for diet (diamond), foot (square) and mantle (triangle) tissues, 13 C of each component is also shown in conjunction with compound specific measurements. The standard deviation for each mean value is represented by error bars on each data point. T 13 C value for the diet indicated by the dotted line, is included as a point of reference for data comparison.

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18 Fig.2B 13 C Values for Non Essential Amino Acids: Mean (n=13) 13 C values of non essential amino acids for diet (diamond), foot (square) and mantle 13 C of each component is also shown in conjunction with compound specific measurements. The standard deviation for each mean value is represented by error bars on each 13 C value for the diet indicated by the dotted line, is included as a point of reference for data comparison. Compound 13 C Fractionations Diet 13 C enrichments 13 C) for bulk and all analyzed amino acids ar e listed in Table 3 In both foot and mantle tissues, amino acids were enriched relative enrichments t representative of the dietary source. Interestingly, apart from phenylalanine the least enriched amino acid was the NAA alanine with enrichments

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19 mantle. Glutamic acid (NAA) was the most enriched amino acid with respect to die t, with enrichments enrichment in aspartic acid is very high ose of glutamic acid. Diet consumer fractionations for almost all other amino acids ranged from +3.6 to enrichments remained similar in over however, the mantle was more depleted than the foot with respect to diet ( Figures 3A and 3B) Mantle tissue values for serine, aspartic acid and leucine were depleted by ~1 compared to foot values with the greatest foot or both glycine and isoleucine. Table 3: 13 C Values for Diet Consumer Enrichments in Strombus spp. Foot Diet SD Mantle Diet SD Shell Diet SD Bulk 2.6 0.3 2.3 0.4 NA NA Essential Isoleucine 5.0 2.1 2.5 1.7 2.0 2.0 Leucine 5.6 0.8 4.3 1.1 4.8 1.1 Phenylalanine 1.0 0.7 1. 0 0.8 0.8 1.0 Threonine 7.3 4.0 7.9 3.3 6.2 3.7 Valine 3.7 1.2 3.4 1.1 2.8 1.5 Non Essential Alanine 2.6 1.7 2.2 1.6 2.2 2.0 Aspartic Acid 10.7 2.4 9.2 2.7 8.1 2.4 Glutamic Acid 13.2 1.4 13.3 1.1 11.0 1.8 Glycine 7.4 1.6 4.8 2.1 1 .7 1.8 Proline 3.6 0.9 3.9 1.0 2.5 0.8 Serine 4.7 2.1 3.7 2.7 1.0 1.8 SD=Standard Deviation

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20 In general diet shell enrichments for individual amino acids were less than their respective diet tissue enrichments with the exception of leucine and alanine which had shell values that fell between those of the foot and mantle. Phenylalanine and serine were the least enriched amino acids in the shell organic material with enrichments to diet were glutamic shell enrichments for other amino acids Essential vs. Non Essentia l Amino Acids The most distinctive difference between the essential and non essential amino acids groups was the relationship between diet tissues and diet shell enrichments. With the exception of isoleucine, diet consumer enrichments for EAA varied by le 13 C values for shell organic matter were similar to both tissue values. While alanine (NAA) followed this trend, all other NAA exhibited two very distinct fractionation relations hips between the three organic materials. As is seen in proline and glutamic acid, enrichments for the foot shell values were Aspartic acid, glycine and 13 C diet consumer values from foot to mantle to shell. For instance, serine had enrichments of +4.7 and +3.7 for foot and mantle respectively, but was only enriched relative to di

PAGE 27

21 decreases in the dietary values were more consistent for aspartic acid and glycine. These step 13 C enrichment relative material). Fig. 3A. 13 C Consumer Diet Enrichments for Essential Amino Acids: Mean (n=13) 13 C diet consumer enrichments for essential amino acids in foot tissue (square), mantle tissue (triangle), and shell o rganic matter (circle) are presented. Error bars indicate the standard deviation for each data point. Enrichments measured at the compound specific level are compared to the averaged bulk measurements for the foot and mantle tissues. The dotted line, re 13 C enrichment, is included as a point of reference for the comparison of data.

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22 Fig.3B 13 C Consumer Diet Enrichments for Non Essential Amino Acids: 13 C diet consumer enrichments for non essential amino acids in foot tissue (square), mantle tissue (triangle), and shell organic matter (circle) are presented. Error bars indicate the standard deviation for each data point. Enrichments measured at the compound specific level are compared to the averaged bul k measurements for the 13 C enrichment, is included as a point of reference for the comparison of data. Discussion T r ophic Discrimination Factor in Mollusks Essential amino acids (EAA) are generally thought to be derived directly from diet, thus avoiding any de novo synthesis reactions that in non essential amino acids 13 C ( Reeds 2000 ). Thus, our initial prediction was that EAAs would show no diet 13 C

PAGE 29

23 enrichment, whereas NAAs would be slightly to gre atly enriched depending on the specific metabolic pathways involved in their formation Enrichment trends in EAAs did not match our expectations. In the foo t tissue, all and was thus the only EAA that followed the EAA prediction Extensive trophic enrichment for EAAs, however, has been reported in at least one other study. H owland et al. (2003) found that among EAAs in consumer bone collagen, only phenylalanine and 13 C value of the diet. Although Wolf et al. (2009) 13 lt with no combination with the results of Howland et al. (2003) suggest that our expectations of EAAs and NAAs need clarification. synthesized by the animal organism out of material ordinarily available to the cells at a speed commensurate with the demands for normal growth emphasis in original). The requirements for an EAA are, thus, provisional in that animals may actuall y synthesize their own EAAs, in part, under certain conditions. Animals are not able to produce, de novo the base structural features required for EAA synthesis. However, intermediary molecules obtained from dietary sources may serve as precursors from which some EAAs can be synthesized by an animal. Based on these principles, Reeds (2000) suggests that EAAs leucine, isoleucine, valine, pheynylalanine and methionine can be synthesized by animals from available precursors, while only threonine and lysine 13 C trophic

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24 enrichment from multipl e studies of a wide range of organisms, phenylalanine, rather than threonine or lysine, is the most consistent representative of the dietary signal and behaves most like what is expected of EAAs. 13 C. The least enriched NAA in the foot tissue, alanine, was enriched with respect to 13 traditional trophic level indicator) reported widely in the literature (DeNiro and Epstein 1981, Fry 1988). The greatest enrichments between diet and consumer for NAAs, 13 C trophic enrichments reported thus far in amino acid s (see Jim et al 2006, Fantle et al 1999) and even exceed trophic enrichments 15 N of individual amino acids (McClelland and Montoya 2002, Popp et al 2007). Thus, at least for simple food webs (see CONCLUSIONS below), the 13 C of individual amino acids has potential for use in high resolution estimates of trophic position for secondary consumers. Although often grouped with NAAs, proline and glycine are actually Reeds 2000 ). This ter m is meant to convey that the rate at which synthesis for these amino acids can occur within an animal is limited by the availability of precursor molecules (Womack and Rose 1947, reviewed in Reeds 2000). Once that limit is reached, the amino acid becomes 13 C values for foot and mantle tissues

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25 show less trophic enrichment than for glycine, although it is unclear that proline synthesis is more limited by the availabili ty of precursors than glycine because metabolic fractionations for these amino acids derived de novo is not yet known. The Biomineralization Effect 13 C values of amino acids in biominerals refle 13 C values of the diet and have potential to be developed as geochemical archives of ancient environments and food webs. Initially, I hypothesized that trophic fractionation might occur as amino acids pass through the mantle, the tissue directly i nvolved in biomineralization, to the site of shell deposition. Trophic discrimination between bulk tissue and the bulk organic component in shell of the landsnail Helix was reported to range between proportion of C 3 and C 4 pla 13 C and bulk proteins of shell of the marine clam Mercenaria mercenaria caught specimens does n ot allow for methodological control over potential variation in isotopic turnover rates between tissue and shell components. Soft tissues, which generally have turnover times of under two months (Tieszen et al. 1987, McIntyre and Flecker 2006), more close growth is accretionary and can reflect, if shell growth is continuous, the entire ontogenetic dietary history of that animal. In the present study, this problem was circumvented by way of a lab oratory experiment with juvenile snails reared from larvae on a controlled and known diet. Shell

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26 and tissues were thus equilibrated with respect to diet. Our results indicate that the incorporation of proteins into shell does, indeed, involve an additio n al layer of isotopic discrimina tion. Amino acids in shell were found to be consistently depleted in 13 C 13 C values of the same compounds in both mantle tissue and foot muscle. The biomineralization process thus appears to suppress or reverse trophic enrichment 13 C of shell amino acids, however, does ge 13 C of amino acids in tissue. Diet to shell trophic 13 C of shell organics. The largest diet shell enr ichments are again seen in glutamic acid and aspartic acid. With enrichment s of of trophic position based on analyses of shell. 13 C of amino acids in mantle as this tissue is directly involved in biomineralization and, at least during rapid juvenile growth, should exhibit trophic enrichment factors similar to those of shell. With the exception of threonine and proline, 13 C values of amino acid s in mantle tissue are closer to those of the shell and generally depleted in 13 C relative to foot muscle. Trophic enrichment of mantle amino elative to diet. As in foot muscle, glutamic and aspartic acids in the mantle show the greatest trophic enrichment with respect to diet with enrichment

PAGE 33

27 13 C Fractionation 13 C values of phenylalanine hold promise as a marker of diet, although we hesitate to recommend glutamic and aspartic acids for estimating trophic position. The trophic level (TL) of a consumer is typically estimated by the following equation: 13 C consumer 13 C base n 13 C consumer 13 C base is the isotopic n 13 C per trophic level (Post 2002). However, large errors in estimated trophic level can result if trophic enrichment factors are highly variable between species in an ecosystem. This variability appears to be the case based on a comparison of amino acid level enrichment patterns in mollusks (this study) with those of vertebrates and arthropods (Table 4 ).

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2 8 Table 4 13 C Results to Other Studies: Only data based on controlled diet experiments were included. The diet source used and organic material (with taxa specified) sampled for analysis is indicated for comparison between the given studies. Amino acids are abbreviated as fo llows: Glu glutamic acid, Asp aspartic acid, Gly glycine, Thr threonine, Leu leucine, Ile isoleucine, Ser serine, Val valine, Pro proline, Ala alanine, and Phe phenylalanine. relative to diet, whereas trophic enrichment for the same amino acid in pigs and rats fed C 3 ver, these enrichment factors were found to fluctuate with changes in diet. Pigs and rats fed C 4 while rats that obtained protein from C 3 plants but carbohydrates from C 4 p lants had an enrichment factor of of dietary carbon, protein content of the diet, and perhaps even phylogeny confound any trophic signal that might otherwise be present in this system. Our Data Fantle et al (1999) Hare et al (1991) Jim et al (2006) Diet Algae pell et Algae pellet Zoo plankton Detritus C3 C4 C3 C3(p)/ C4(e)* C4 C4(p)/ C3(e)* Organic Material Mollusk Foot Muscle Mollusk Shell Organic Matter Crab Whole Body Crab Whole body Pig Collagen Pig Collagen Rat Collagen Rat Collagen Rat Collagen Rat Collagen Bulk 2.5 0.1 0.1 1.4 3.2 5.2 2.2 4.4 9.9 Glu 13.2 10.9 1.0 0.7 6 7.4 1.8 12.5 0.9 6.8 Asp 10.7 8.1 1.0 1.7 2.6 3.5 0.4 11.3 1.7 2.4 Gly 7.3 1.6 2.4 2.4 0.4 0.9 1.3 10.5 1.9 5.5 Thr 7.2 6.2 0.7 0 Leu 5.5 4.7 1.1 2.5 1.4 0. 1 2.1 4.9 Ile 5 1.9 1.5 0.7 2.7 0.7 0.9 0.5 Ser 4.6 0.9 1.7 1.2 Val 3.7 2.8 0.6 0.9 1.3 Pro 3.6 2.5 0.8 2.4 2 2.9 4.8 3.4 4.6 Ala 2.6 2.2 0.1 1.8 1.4 3.4 1.5 17 5.5 19.7 Phe 0.9 0.7 0.5 4.0 1.2 0.1 0.6 2.6

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29 Trop 13 C of phenylalanine is consumer enrichment for phenylalanine reported for juvenile blue crabs raised on a controlled diet of zooplankton (Fantle et al. 1999). This suggests that phen ylalanine is a stable indicator of diet across taxa and across diets. In the same study, Fantle et al. (1999) also reported trophic depletion in consumer phenylalanine of c omparable. Unlike other potential dietary sources, detritus is subject to an array of microbial processes that break down and can potential alter the isotopic signature of the original material. Additionally, as detrital material is comprised of various components and little is known about the feeding selectivity of detrivore crabs, it is difficult to ascertain which components of the detrital material the crabs are actually ingesting and the specific 13 C s ignatures of those components.

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30 Chapter Th ree Conclusions Based on the results of this study, the following conclusions may 13 C analyses of amino acids in molluscan shell organics: 1. 13 C for foot muscle, mantle tissue, and shell proteins. This EAA is therefore appropriate for identifying primary producers based on isotopic analyses of molluscan organic carbon. Potential questions that could be addressed with molluscan phenylalanine in future work could include the impo rtance of terrestrial productivity for coastal ecosystems, the influence of past climate change on the proportions of C 3 vs. C 4 plants, migrational patterns of dispersing larval mollusks, and the nature of anthropogenic alteration of basal food web ecology 2. Although phenylalanine is the most effective source indicator of the amino acids examined in this study, it is also the least abundant in shell organic material (Table 1). With the loss of free amino acids in diagenetically altered specimens, the amou nt of phenylalanine in a sample may thus be so diminished that reliable isotope data is unattainable. For the application to fossil shell, f uture work is necessary to address this issue and determine the full potential of this technique as it may be const rained by the low abundances of phenylalanine.

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31 3. Other EAAs do not reflect closely the carbon isotopic composition of the diet but instead are capable of substantial trophic enrichment. The present notion of the essentiality of amino acids needs further cla rification as there can be instances where amino acids are conditionally essential to the diet or where essential amino acids can be synthesized in part by the animal. As a result, the use of the NAA and EAA partitioning should not be used to predict the 13 C diet consumer enrichments for individual amino acids. 4. Glutamic and aspartic acids in molluscan tissues and shell are significantly er, comparison of compound specific carbon isotope data reported in the literature reveals that trophic enrichment patterns of NAAs vary considerably between species, organic components (tissues and amino acids), protein content, and dietary sources ( e.g., C 3 vs. C 4 plants). This variation could lead to large errors in estimates of trophic position for a given consumer, 13 C of shell organics should not be used to resolve trophic positions of secondary consumers. 5. Biomineralization in mollusks influence s the magnitude of trophic enrichment between diet and consumer. However, the overall pattern of enrichment, with low trophic fractionation for phenylalanine and large fractionation for glutamic and aspartic acids, was maintained from mollusk tissue to sh ell. Carbon isotopes of amino acids (particularly phenylalanine) obtained from shell organic matter are a potentially valuable geochemical archive of past environments and food webs.

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32 References Borman, A., Wood, T.R., Balck, H.C., Anderson, E.G., Oes terling, M.J., Womack, M., Rose, W.C., 1946. The role of arginine in growth with some observations of the effects of argininic acid. Journal of Biological Chemistry 166, 585 594. Corbisier, T., Soares, L., Petti, M., Muto, E., Silva, M., McClelland, J ., Valiela, I., 2006. U se of isotopic signatures to assess the food web in a tropical shallow marine ecosystem of Southeastern Brazil. Aquatic Ecology 40, 381 390. Corr, L.T., Berstan, R., Evershed, R.P., 2007. Optimisation of derivatisation procedures 13 C values of amino acids by gas chromatography/ combustion/isotope ratio mass spectrometry. Rapid Communications in Mass Spectrometry 21, 3759 3771. Davis, M., 2005. Species Profile Queen Conch, Strombus gigas Southern Regi onal Aquaculture Center. SRAC Publication No. 7203. DeNiro, M., Epstein, S., 1978. Influence of diet on the distribution of carbon isotopes in animals. Geochimica et Cosmochimica Acta 42, 495 506. DeNiro, M., Epstein, S., 1981. Influence of diet on th e distribution of nitrogen isotopes in animals. Geochimica et Cosmochimica Acta 45, 341 351. Dubois, S., Jean Louis, B., Bertrand, B., Lefebvre, S., 2007. Isotope trophic step fractionation of suspension feeding species: Implications for food partition ing in coastal ecosystems. Journal of Experimental Marine Biology and Ecology 351, 121 128. Engel, M.H., Macko, S.A., 1986. Stable isotope evaluation of the origins of amino acids in fossils. Nature 323, 531 533. Fantle, M.S., Dittel, A.I., Schwalm, S. M., Epifanio, C.E., Fogel, M.L., 1999. A food web analysis of the juvenile blue crab, Callinectes sapidus using stable isotopes in whole animals and individual amino acids. Oecologia 120, 416 426. Fox Dobbs, K., Leonard, J., Koch, P., 2008. Pleistocen e megafauna from eastern Beringia: Paleoecological and paleoenvironmental interpretations of stable carbon and nitrogen isotope and radiocarbon records. Paleogeography, Paleoclimatology, Paleoecology 261, 30 46.

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33 Fry, B., 1988. Food web structure on Geor ges Bank from stable C, N, and S isotopic compositions. Limnology and Oceanography 33, 1182 1190. 15 13 C) analysis of a North Eastern Atlantic maerl bed. Journal of Experimental Marine Biology and Ecology 338, 1 15. Hamilton, P.B., 1965. Amino acids on hands. Nature 205, 284 285. Hare, P.E., 1965. Amino acid artifacts in organic geochemistry. Carnegie Inst. Washington Yearb. 64, 23 2 235. Hare, P.E., Mitterer, R.M., 1967. Nonprotein amino acids in fossil shells. Carnegie Inst. Washington Yearb 65 362 364 Hare, P.E., Hoering, T.C., 1977. The organic constituents of fossil mollusc shells. Carnegie Inst.Washington Yearb. 76, 625 631. Hare, P.E., Fogel, M.L., Stafford Jr., T.W., Mitchell, A.D., Hoering, T.C., 1991. The isotopic composition of carbon and nitrogen in individual amino acids isolated from modern and fossil proteins. Journal of Archaeological Science 18, 277 292. Ho bson, K., Clark, R., 1992. Assessing avian diets using stable isotopes II: Factors influencing diet tissue fractionation. The Condor 94, 189 197. Hoering, T.C., 1980. The organic constituents of fossil mollusc shells. In: Hare, P.E., Hoering, T.C., King Jr., K. (Ed.), Biogeochemistry of Amino Acids. John Wiley & Sons, New York, pp.193 201. Howland, M.R., Corr, L.T., Young, S.M.M., Jones, V., Jim, S., Van Der Merwe, N.J., Mitchell, A.D., Evershed, R.P., 2003. Expression of the dietary isotope signal i n the compound 13 C values of pig bone lipids and amino acids. International Journal of Osteoarchaeology 13, 54 65. Jim, S., Jones, V., Ambrose, S.H., Evershed, R.P., 2006. Quantifying dietary macronutrient sources of carbon for bone collagen biosynthesis using natural abundance stable carbon isotope analysis. British Journal of Nutrition 95, 1055 1062. Lorrain, A., Paulet, Y., Chauvard, L., Savoye, N., Donval, A., Saout, C., 2002. 13 15 N signatures among scallop tissues : implications for ecology and physiology. Journal of Experimental Marine Biology and Ecology 275, 47 61.

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34 MacNeil, M., Skomal, G., Fisk, A., 2005. Stable isotopes from multiple tissues reveal diet switching in sharks. Marine Ecology Progress Series 302 199 206. McClelland, J., Montoya, J., 2002. Trophic relationships and the nitrogen isotopic composition of amino acids in plankton. Ecology 83, 2173 2180. McIntyre, P.B., Flecker, A.S., 2006. Rapid turnover of tissue nitrogen of primary consumers in tropical freshwaters. Oecologia 148, 12 21. Mitterer, R.M., 1972. Biogeochemistry of aragonite mud and oolites. Geochemica et Cosmochimica Acta 36, 1407 1422. Mitterer, R.M., 1993. The diagenesis of proteins and amino acids in fossil shells. In: Engel, M.H., Macko, S.A. (Ed.), Organic Geochemistry. Plenum Press, New York, pp.739 753. Hartten, K.L., Wehmiller, J.F., 2003. 13 15 34 S in organic matter from the biominerals of modern and fossil Mercenaria spp. Organic Geochemistry 34, 165 183. e hmiller, J.F., 2006. Stable carbon isotope composition of amino acids in modern and fossil Mercenaria Organic Geochemistry 38, 485 498. Ostrom, P.H., Macko, S.A ., Engel, M.H., Russell, D.A., 1993. Assessment of trophic structure of Cretaceous communities based on stable nitrogen isotope analysis. Geology 21, 491 494. 13 C: A potential tool for ecophysiological studies in marine bivalves. Organic Geochemistry 37, 1359 1370. Penkman, K.E.H., Kaufman, D.S., Maddy, D., Collins, M.J., 2008. Closed system behavior of the intra crystalline fraction of amino acids in mollusc shell. Quaternary Geochronology 3, 2 25. Phillips, D., Eldridge, P., 2006. Estimating the timing of diet shifts using stable isotopes. Oecologia 147, 195 203. Popp, B., Graham, B., Olson, R., Hanndies, C., Lott, M., Lpez, Ibarra, G., Galv n, Magaa, F., Fry B., 2007. Stable Isotope as Indicators of Ecological Change, 173 190. Post, D.M., 2002. Using stable isotopes to estimate trophic position: Models, methods, and assumptions. Ecology 83, 703 718.

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35 Reeds, P.J., 2000. Dispensable and indispensable amino acids for humans. Journal of Nutrition 130, 1835S 1840S. Schweizer, M.K., Wooller, M.J., Toporski, J., Fogel, M.L., Steele, A., 2005. Examination of an Oligocene lacustrine ecosystem using C and N stable isotopes. Palaeogeography, Palaeoclimatology, Palaeoecology, 1 17. Silfer, J.A., Engel, M.H., Macko, S.A., Jumeau, E.J., 1991. Stable carbon isotope analysis of amino acid enantiomers by conventional isotope ratio mass spectrometry and combined gas chromatography/isotope ratio mass spectrometry. Analytical Chemistry 63, 370 374. 13 C of shell carbon in the pulmonate snail Helix aspersa Earth and Planetary Science Letters 195, 249 259. Towe, K.M., 1980. Preserved organic ultrastructure: An unreli able indicator for Paleozoic amino acid biogeochemistry. In: Hare, P.E., Hoering, T.C., King, K. Jr. (Ed.), The Biogeochemistry of Amino Acids. Wiley, NewYork, pp.65 74. Tamelander, T., Kivime, C., Bellerby, R., Renaud, P., Kristiansen, S., 2009. Base line variations in stable isotope values in an Arctic marine system: effects of carbon and nitrogen uptake by phytoplankton. Hyrdobiologi a 630, 63 73. Tieszen, L.L., Boutton, T.W., Tesdahl, K.G., Slade, N.A., 1983. Fractionation and turnover of stabl e carbon isotopes in animal tissues: Implications 13 C analysis of diet. Oecologia 57, 32 37. Vallentyne, J.R., 1964. Biogeochemistry of organic matter II Thermal reaction kinetics and transformation products of amino compounds. Geochemica et Cosm ochimica Acta 28, 157 188. Vanderklift, M., Ponsard, S., 2003. Sources of variation in consumer 15 N enrichment: a meta analysis. Oecologia 136, 169 182. Wolf, N., Carleton, S.A., Martnez del Rio, C., 2009. Ten years of experimental animal isot opic ecology. Functional Ecology 23, 17 26. Womack, M., Rose, W.C., 1947. The role of proline, hydroxyproline and glutamic acid in growth. Journal of Biological Chemistry 171, 37 50. Yokoyama, H., Ishihi, Y., Yamamoto, S., 2008. Diet tissue isotopic fra ctionation of the Pacific oyster Crassostrea gigas Marine Ecology Progress Series 358, 173

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36 Appendices

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37 Appendix A: Bulk 13 C Values for All Samples Bulk isotope values for all analyzed samples of diet, foot tissue and mantle ti ssue. Individual specimens used for analysis are represented by their sample ID and shell lengths are provided for each consumer specimen. Values presented here are the mean representative of duplicate analyses for each specimen. Diet ID 15 N ) 13 C D AP 1 2.72 23.59 D AP 6 3.74 23.30 D AP 7 2.87 23.49 D AP 8 3.85 23.49 D AP 9 3.57 23.41 D AP 10 3.79 23.24 Specimen ID Shell Length (mm) Foot Avg 15 Foot Avg 13 Mantle Avg 15 Mantle Avg 13 S2 30. 57 6.78 21.25 5.13 20.78 S3 31.02 7.21 20.85 4.22 21.35 S5 35.13 8.32 20.57 5.69 20.07 S7 31.53 6.98 21.30 5.27 21.29 S12 34.53 6.19 21.31 4.11 21.70 S19 36.79 7.56 20.37 5.56 21.76 S20 33.48 6.91 20.99 5.09 21.87 S23 32.14 7.46 20.57 5.57 21.64 S25 33.43 6.45 21.26 4.86 21.88 S26 30.75 7.04 21.04 4.98 20.74 S29 35.77 7.79 20.65 5.99 20.44 S30 35.66 6.73 21.15 5.37 20.03 S33 34.5 7.70 20.72 5.73 21.44

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38 Appendix B: Corrected 13 C Values for Individual AA for All Sampl es L ist of corrected 13 C values for individual amino acids for each sample analyzed. Values have been corrected for a kinetic isotope effect using a mass balance equation. Each of the four lists represents the organic materials analyzed including: algae diet pellet (I) consumer mantle tissue ( II ), consumer foot muscle tissue ( III ), and shell organic material ( IV ). I. Sample ID (Diet) Amino Acid D AP 1 D AP 6 D AP 7 D AP 8 D AP 9 D AP 10 alanine 23.46 23.64 24.02 24.20 22.05 27.75 aspartic acid 24.55 28.57 26.28 24.18 29.24 20.40 glutamic acid 27.01 28.22 28.94 27.99 27.87 27.26 glycine 14.78 14.48 16.37 15.67 13.66 14.80 isoleucine 22.75 N/A N/A 24.87 24.28 25.42 leucine 33.47 34.49 35.03 34.53 33.62 33.72 pheny lalanine 28.25 28.49 27.64 28.61 28.12 27.93 proline 22.74 23.38 23.02 23.32 23.30 23.57 serine 11.15 10.73 8.51 9.50 11.47 8.88 threonine 20.30 28.41 28.79 19.62 20.88 18.89 valine 26.89 27.95 29.34 29.47 27.17 27.33 II Sample ID (Mantle Tissue) Amino Acid S2M S3M S5M S7M S12M S19M S20M alanine 20.39 22.40 21.48 22.03 23.70 22.71 20.90 aspartic acid 18.07 18.16 15.23 17.82 19.01 17.25 16.75 glutamic acid 13.30 14.15 12.92 15.59 16.58 15.28 15.30 glycine 9.86 14.60 6.71 10.59 13.64 14.04 13.29 isoleucine 21.38 23.77 18.48 21.51 24.26 25.31 N/A leucine 28.10 30.21 26.86 28.13 31.07 31.76 32.34 phenylalanine 25.95 26.67 24.81 27.16 27.83 27.00 26.96 proline 18.89 19 .33 14.11 19.25 19.41 19.84 20.50 serine 4.98 3.88 3.44 7.26 6.78 10.97 16.38 threonine 13.46 14.49 12.13 14.70 14.16 19.89 16.16 valine 24.68 25.40 23.11 25.53 26.37 24.73 25.12

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39 Appendix B : (Continued) II. (Continued) Sam ple ID (Mantle Tissue) Amino Acid S23M S25M S26M S29M S30M S33M alanine 22.65 20.38 24.09 20.15 22.81 22.77 aspartic acid 18.95 12.65 14.62 13.31 14.42 16.74 glutamic acid 15.25 14.14 17.35 12.50 12.47 15.39 glycine 11.24 6.27 11. 56 5.37 5.81 8.90 isoleucine 21.86 21.09 21.02 18.67 24.15 20.98 leucine 29.29 30.32 31.05 28.53 30.86 28.92 phenylalanine 27.70 25.69 29.03 27.85 28.00 28.13 proline 19.26 20.49 21.67 19.13 20.66 19.03 serine 2.53 11.34 4.73 3.64 5.63 1.53 threonine 15.79 15.39 15.66 11.54 15.75 15.04 valine 25.58 22.10 24.85 23.56 24.99 24.38 III. Sample ID (Foot Tissue) Amino Acid S2F S3F S5F S7F S12F S19F S20F alanine 20.52 19.99 18.97 22.75 22.47 20.76 21.8 4 aspartic acid 15.29 13.63 15.16 18.46 14.68 14.42 16.28 glutamic acid 12.71 12.09 15.38 15.26 14.61 13.16 12.69 glycine 7.80 4.65 6.15 10.41 9.52 5.42 10.19 isoleucine 19.86 18.34 18.67 10.68 22.42 22.98 21.08 leucine 29 .17 28.21 27.40 28.36 29.02 29.10 28.52 phenylalanine 26.63 26.44 25.56 27.46 27.68 27.17 28.00 proline 19.69 18.87 19.72 17.37 19.01 18.90 19.33 serine 6.40 2.03 4.33 5.26 7.80 7.05 10.46 threonine 21.63 20.05 14.45 N/A 19.57 16.80 13.79 valine 24.40 22.23 21.92 24.96 25.02 23.72 25.22

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40 Appendix B : (Continued) III. (Continued) Sample ID (Foot Tissue) Amino Acid S23F S25F S26F S29F S30F S33F alanine 22.91 21.99 22.27 19.19 23.31 23.31 aspartic acid 16.26 13.34 13.84 12.53 13.98 14.54 glutamic acid 12.79 14.71 15.04 14.42 18.82 18.97 glycine 6.98 5.24 6.03 6.20 11.55 8.78 isoleucine 20.51 19.38 19.26 17.66 22.16 18.32 leucine 27.75 28.42 28.61 27.34 31.33 28.01 phenyl alanine 26.86 25.54 27.12 28.13 29.23 27.97 proline 16.95 21.45 20.52 20.87 21.56 20.85 serine 6.53 0.84 4.08 1.99 9.78 3.33 threonine 14.11 14.50 14.38 9.59 14.00 13.88 valine 24.32 23.95 24.54 24.43 26.21 25.24 IV. Sam ple ID (Shell Organic Matter) Amino Acid S2S S3S S5S S7S S12S S19S S20S alanine 24.07 19.70 22.31 25.12 19.80 19.14 19.22 aspartic acid 17.97 17.57 14.91 18.79 18.98 18.35 18.31 glutamic acid 14.80 15.87 13.15 21.14 15.32 18.42 17. 37 glycine 18.37 12.40 11.08 14.86 11.80 12.35 8.26 isoleucine 25.42 24.23 19.92 23.91 22.42 22.52 23.99 leucine 32.09 25.75 28.88 30.65 29.83 28.83 28.51 phenylalanine 28.03 22.21 27.68 28.06 27.87 26.90 27.05 proline 20. 59 18.86 19.40 23.68 21.98 21.18 19.53 serine 8.28 6.25 8.72 12.95 12.63 10.49 6.85 threonine 16.96 17.63 20.38 23.97 17.28 15.60 15.89 valine 25.52 23.48 23.14 28.28 27.01 27.32 24.59

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41 Appendix B : (Continued) IV. (Continu ed) Sample ID (Shell Organic Matter) Amino Acid S23S S25S S26S S29S S30S S33S alanine 22.14 21.72 20.79 23.04 24.26 24.22 aspartic acid 17.70 15.06 15.71 17.42 15.90 20.03 glutamic acid 16.72 13.32 21.57 19.71 N/A 15.65 glycine 14.36 11.84 11.11 17.19 14.14 14.73 isoleucine 24.71 27.01 17.04 19.17 20.88 19.16 leucine 28.39 29.77 29.32 30.36 28.65 30.76 phenylalanine 27.96 27.94 28.51 28.50 28.77 27.00 proline 21.25 19.74 21.29 19.19 21.35 20.84 serine 8.91 8.30 11.11 5.39 7.61 10.22 threonine 16.45 15.08 14.95 13.84 15.39 12.49 valine 26.19 22.97 22.86 24.61 26.19 25.73

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42 Appendix C: Raw 13 C Values for Individual AA for All Samples L ist of raw 13 C values for individual amino ac ids for each sample analyzed. Values are an average of duplicate isotope measurements for each sample. Each of the four lists represents the organic materials analyzed including: algae diet pellet (I) consumer mantle tissue ( II ), consumer foot muscle tis sue ( III ), and shell organic material ( IV ). I. Sample ID (Diet) Amino Acid D AP 1 D AP 6 D AP 7 D AP 8 D AP 9 D AP 10 alanine 29.8 29.9 30.1 30.1 29.3 31.5 aspartic acid 31.1 32.4 31.7 31.0 32.7 29.7 glutamic acid 30.4 30.8 31.0 30.7 30.7 30.5 glycine 28.3 28.2 28.8 28.6 28.0 28.3 isoleucine 30.6 N/A N/A 31.7 31.4 32.1 leucine 34.1 34.7 35.0 34.7 34.2 34.3 phenylalanine 32.3 32.5 31.9 32.6 32.2 32.1 proline 29.5 29.8 29.7 29.8 29.8 29.9 serine 29.2 29.1 28.4 28.7 29.3 28.5 threonine 31.3 34.2 34.4 31.0 31.5 30.8 valine 32.3 32.8 33.5 33.6 32.5 32.5 II. Sample ID (Mantle Tissue) Amino Acid S2M S3M S5M S7M S12M S19M S20M alanine 27.7 28.4 28.1 28.3 28.9 28.6 27.9 aspartic acid 28.6 28.7 27.7 28.6 29.0 28.4 28.2 glutamic acid 26.5 26.8 26.3 27.2 27.6 27.1 27.1 glycine 26.1 27.5 25.2 26.3 27.2 27.3 27.1 isoleucine 29.9 31.2 28.3 29.9 31.4 32.0 34.2 leucine 31.2 32.3 30.5 31.2 32.8 33.2 33.5 phenylalanine 30.5 30.9 29.7 31.2 31.7 31.1 31.1 proline 28.3 28.5 25.9 28.5 28.6 28.8 29.1 serine 27.2 26.8 26.7 27.8 27.7 29.0 30.6 threonine 27.7 28.1 27.2 28.1 27.9 30.0 28.7 valine 31.2 31.6 30.5 31.7 32.1 3 1.3 31.5

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43 Appendix C : (Continued) II. (Continued) Sample ID (Mantle Tissue) Amino Acid S23M S25M S26M S29M S30M S33M alanine 28.5 26.6 27.9 26.5 27.5 28.6 aspartic acid 28.9 27.0 27.6 27.2 27.6 28.2 glutamic acid 27.1 25.3 26.4 24. 8 24.8 26.6 glycine 26.5 23.9 25.4 23.6 23.8 25.2 isoleucine 30.1 29.8 29.8 28.5 31.5 29.9 leucine 31.8 32.5 32.9 31.5 32.8 31.6 phenylalanine 31.6 29.3 31.5 30.7 30.8 31.2 proline 28.5 26.9 27.5 26.2 27.0 26.1 serine 26.4 28.0 26.0 25.7 26.3 25.8 threonine 28.5 29.0 29.1 27.6 29.2 29.2 valine 31.7 29.8 31.1 30.5 31.2 30.9 III. Sample ID (Foot Tissue) Amino Acid S2F S3F S5F S7F S12F S19F S20F alanine 27.7 28.4 28.1 28.6 28.5 27.8 28.2 asp artic acid 28.6 28.7 27.7 28.8 27.5 27.4 28.0 glutamic acid 26.5 26.8 26.3 27.1 26.9 26.4 26.3 glycine 26.1 27.5 25.2 26.3 26.0 24.8 26.2 isoleucine 29.9 31.2 28.3 24.0 30.4 30.7 29.7 leucine 31.2 32.3 30.5 31.3 31.7 3 1.7 31.4 phenylalanine 30.5 30.9 29.7 31.4 31.6 31.3 31.8 proline 28.3 28.5 25.9 27.5 28.4 28.3 28.5 serine 27.2 26.8 26.7 27.2 28.0 27.8 28.8 threonine 27.7 28.1 27.2 42.9 29.9 28.9 27.8 valine 31.2 31.6 30.5 31.4 31 .4 30.8 31.5

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44 Appendix C : (Continued) III. (Continued) Sample ID (Foot Tissue) Amino Acid S23F S25F S26F S29F S30F S33F alanine 28.6 27.2 27.3 26.1 28.8 27.7 aspartic acid 28.0 27.2 27.4 26.9 27.3 27.6 glutamic acid 26.3 25.5 25.6 25.4 27.8 26.9 glycine 25.3 23.6 23.8 23.9 25.9 24.6 isoleucine 29.4 28.9 28.8 27.9 30.5 28.3 leucine 31.0 31.5 31.6 30.9 32.9 31.2 phenylalanine 31.0 29.2 30.2 30.9 32.0 30.8 proline 27.3 27.4 26.9 27.1 27.4 27.1 ser ine 27.6 24.8 25.8 25.2 28.3 25.6 threonine 27.9 28.7 28.7 26.9 28.8 28.5 valine 31.1 30.7 31.0 30.9 31.8 31.3 IV. Sample ID (Shell Organic Matter) Amino Acid S2S S3S S5S S7S S12S S19S S20S alanine 27.9 26.9 27.3 26.1 26.9 26. 6 26.7 aspartic acid 28.7 27.9 27.7 26.8 28.3 28.1 28.1 glutamic acid 25.5 25.6 25.0 23.4 25.4 26.4 26.1 glycine 27.4 23.9 25.3 23.1 23.7 23.8 22.7 isoleucine 32.2 33.0 29.2 32.6 32.0 32.1 32.9 leucine 33.5 29.5 31.7 2 7.3 31.7 31.2 31.0 phenylalanine 30.8 28.1 30.6 19.3 31.7 31.1 31.2 proline 26.9 24.5 26.3 23.3 26.1 25.7 24.8 serine 27.1 24.1 27.2 23.8 26.0 25.4 24.3 threonine 29.6 30.9 30.9 29.0 30.8 30.2 30.3 valine 31.5 29.6 30 .3 29.1 31.4 31.6 30.2

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45 Appendix C : (Continued) IV. (Continued) Sample ID (Shell Organic Matter) Amino Acid S23S S25S S26S S29S S30S S33S alanine 28.4 27.1 26.7 28.7 28.0 29.2 aspartic acid 28.5 27.8 28.0 28.4 28.1 29.3 glutamic aci d 27.1 25.0 27.8 28.1 44.7 26.7 glycine 26.7 25.5 25.3 27.6 26.2 26.8 isoleucine 31.9 33.0 27.6 28.9 29.7 28.9 leucine 31.3 32.2 32.0 32.3 31.6 32.6 phenylalanine 31.1 30.8 31.1 31.5 31.3 30.5 proline 27.2 26.5 27.3 2 6.2 27.3 27.0 serine 28.0 27.1 27.9 26.9 26.9 28.4 threonine 29.7 28.9 28.9 28.8 29.1 28.3 valine 31.8 30.2 30.1 31.0 31.8 31.6

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46 Appendix D: Kinetic Isotope Effect Correctional Values List of values used to determine the isotopi c influence of the deriviatizing reagent as a 13 C of laboratory amino acid standards with known isotopic values (std) was determined independently via the GC/C/IRMS (GC) p rocedure used in this study. Using these values, in conjunction with the percentage of carbon atoms inherent to the native amino 13 C of the derivatizing reagent was determined. A mino Acid 13 C AA (GC) 13 C AA (std) % native % derivative 13 C reagent alanine 29.50 25.6 0.38 0.62 31.9 aspartic acid 30.41 23.5 0.33 0.67 33.9 glutamic acid 25.21 12.1 0.33 0.67 31.7 glycine 35.12 45.1 0.29 0.71 31.1 isoleucine 24.97 11.7 0.55 0.45 40.9 leucine 31.59 29.0 0.55 0.45 34.7 phenylalanine 34.76 33.0 0.64 0.36 37.9 proline 26.55 19.0 0.50 0.50 34.1 serine 28.08 11.2 0.30 0.70 35.3 threonine 33.31 26.5 0.36 0.64 37.2 valine 23.83 10.4 0.50 0.50 37.3