Oecologia https://doi.org/10.1007/s00442-020-04645-5

PHYSIOLOGICAL ECOLOGY – ORIGINAL RESEARCH

Fasting afects amino acid nitrogen isotope values: a new tool for identifying nitrogen balance of free‑ranging mammals

Nico Lübcker1 · John P. Whiteman2,4 · Robert P. Millar3 · P. J. Nico de Bruyn1 · Seth D. Newsome4

Received: 5 August 2019 / Accepted: 7 April 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract Changes in the nutritional status of free-ranging animals have a strong infuence on individual ftness, yet it remains challeng- ing to monitor longitudinally. Nitrogen (δ15N) and carbon (δ13C) isotope values measured chronologically along the length of metabolically inert keratinous tissues can be used as a nutritional biomarker to retrospectively reconstruct the foraging ecology and eco-physiology of consumers. We quantitatively describe the physiological efects of fasting on amino acid using sequentially measured bulk tissue and amino acid δ15N values along the length of whiskers sampled from free-ranging juvenile, subadults, adult female, and male southern elephant seals (SES; Mirounga leonina) on Marion Island in the Southern Ocean. For both juveniles and adult females, whisker segments representing fasting had signifcantly higher bulk tissue δ15N values of 0.6 ± 0.5‰ and 1.3–1.8‰, respectively, in comparison to segments unafected by fasting. We also found a large increase (2–6‰) in δ15N values for most glucogenic amino acids and a simultaneous depletion (2–3‰) of in segments refecting fasting, which enabled us to accurately predict (74%) the nutritional status of our model species. We hypothesize that the -alanine cycle is the mechanism driving the observed depletion of alanine δ15N val- ues during fasting. We demonstrated that keratinaceous tissues can be used as a longitudinal nutritional biomarker to detect changes in the nitrogen balance of an individual. Moreover, it is evident that physiological factors have an important infuence on tissue δ15N values and can lead to erroneous bulk tissue or amino acid isotope-based reconstructions of foraging habits.

Keywords Amino acid metabolism · Compound-specifc stable isotopes · Diet · Elephant seals · Nutritional biomarker

Introduction

Animals often experience prolonged fasts during which met- abolic processes regulate changes in the utilization of stored Communicated by Helene Marsh. endogenous substrates such as (proteins) and adipose tissue (lipids) (Raubenheimer et al. 2009; Fontana Electronic supplementary material The online version of this article (https​://doi.org/10.1007/s0044​2-020-04645​-5) contains and Partridge 2015; Secor and Carey 2011; Vidal et al. supplementary material, which is available to authorized users. 2019). Ultimately, the integrative physiological response to extended fasting bouts can have strong impacts on individual * Nico Lübcker ftness, and are thus intrinsically linked to their evolution [email protected] (Secor and Carey 2011). Yet, assessing the relative impor- 1 Department of Zoology and Entomology, Mammal tance of various biochemical and physiological mechanisms Research Institute, University of Pretoria, Private Bag X20, that enable organisms to maintain physiological homeostasis Hatfeld 0028, South Africa while solely relying on endogenous resources remains a con- 2 Department of Biological Sciences, Old Dominion tentious topic (Secor and Carey 2011; Lee et al. 2012). Fur- University, Norfolk, VA 23529, USA thermore, our understanding of such mechanisms that enable 3 Centre for Neuroendocrinology and Department free-ranging animals to withstand and recover from extended of Immunology, Faculty of Health Sciences, University fasts are often hampered by the challenge of building longi- of Pretoria, Pretoria 0001, South Africa tudinal (temporal) records of their nutritional status. 4 Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA

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Biomarkers can refect changes in how animals catabolize catabolism during fasting and thus will show minimal to no or synthesize molecules during fasting (Petzke et al. 2010), change in δ15N because they are not closely associated with and chemical analysis of such compounds can provide valu- nitrogen cycling and cannot easily be deaminated without able insights on the relationship between an organism’s phys- catabolizing the entire compound. In summary, we predict iology, diet, and environmental conditions (Fuller et al. 2005; that the ofsets between the δ15N of trophic and source amino Mekota et al. 2009; Hatch 2012; Ohkouchi et al. 2017). For acids in fasting animals are expected to be larger than in ani- example, nitrogen (δ15N) and carbon (δ13C) isotope analysis mals that are consuming exogenous resources. of tissues provide information about resource and habitat use, Adult southern elephant seals (SES; Mirounga leonina) as well as physiological status (Jaeger et al. 2010; Newsome experience two distinct prolonged fasting periods during their et al. 2010; O’Brien 2015). While δ13C values are often used annual life cycle (Fig. 1S; Le Boeuf and Laws 1994). One to infer sources of primary production that fuel the food webs occurs during the breeding season (Oct.–Nov.) which ends that animals rely on, δ15N values are commonly used to esti- with a brief foraging bout, and the other during the annual mate trophic level (Peterson and Fry 1987). The pattern of obligatory molt (Dec.–Jan.). Tissues synthesized while fast- trophic enrichment in tissue δ15N is a result of fractionation ing provide a means to examine the efects of protein catabo- 15 among the amino radicals (–NH2) that are produced during lism on nitrogen balance and by extension on δ N values amino acid catabolism; the isotopically depleted nitrogen of the consumer. After birth, juvenile SES pups suckle for (14N) is preferentially excreted in the form of ammonia, , 22–23 days before undergoing a post-weaning fast that or uric acid (Peterson and Fry 1987) and the retained 15N is lasts ~ 40–60 days (Wilkinson and Bester 1990). During this then incorporated into body nitrogen pools and used to build fast, recently-weaned pups lose up to ~ 30% of their original and maintain tissues. Ofsets in δ15N values between the ani- weaning body mass (Wilkinson and Bester 1990; Carlini mals and their food are referred to as trophic discrimination et al. 2001) before departing on their frst foraging trip; the factors (TDF; Peterson and Fry 1987), which vary depending mass lost consists of 39% water, 47% fat, and 12% protein. on the type of nitrogenous waste compound produced (Van- Using estimates of whisker growth rates (Lubcker et al. 2016), derklift and Ponsard 2003), as well as the quantity and quality we showed that δ15N values of longitudinally sub-sampled of protein in their diets (Hughes et al. 2018). whisker segments from recently-weaned SES pups showed In addition to diet composition, physiological processes enrichment of ~ 1.0‰ while fasting (Lübcker et al. 2017). such as starvation or gestation can also afect the isotopic Here we used a combined bulk tissue and compound- composition of tissues (Hobson et al. 1993; Fuller et al. 2004, specifc isotope approach to advance our understanding of 2005). Recent meta-analyses demonstrated that fasting causes physiological changes in amino acid metabolism that occur a small yet measurable increase in bulk tissue mean (± SD) during fasting, and potentially establish amino acid δ15N δ15N values of + 0.5 ± 0.2‰ (McCue 2010; Hertz et al. 2015) analysis as a robust proxy for identifying the use of endoge- as the individual essentially ‘consumes’ itself by cataboliz- nous versus exogenous resources. We frst compared how the ing its protein- (skeletal muscle) and lipid-rich (adipose) tis- δ15N of amino acids changed during known catabolic and sues to fuel metabolism (Fuller et al. 2005). Theoretically, anabolic states that were identifed using bulk tissue δ15N isotopic analysis of individual compounds may provide data measured chronologically along the length of juvenile additional information about the biochemical mechanisms SES whiskers with known whisker growth histories. We then that enable animals to fast for prolonged periods (Ohkouchi used these data to determine if fasting infuenced the δ15N et al. 2017; Whiteman et al. 2019). For animals that do not values measured along the length of whiskers sampled from consume appreciable amounts of glucose (e.g., carnivores), free-ranging adult SES with unknown whisker growth his- amino acids are catabolized for glucose synthesis via gluco- tories. Although we have previously described the growth neogenesis to fuel activity, which results in the preferential rate and shedding phenology of SES whiskers (Lübcker et al. deamination and excretion of 14N and yields the measurable 2016), it is still challenging to ascertain when the whisker TDFs described above. During fasting, endogenous protein growth of free-ranging seals commences (McHuron et al. (i.e., skeletal muscle), which is 15N-enriched relative to 2019). Identifying the segment(s) of the whisker grown exogenous dietary protein, becomes the primary substrate while fasting on land could clarify the whisker growth his- for (Krebs 1964) and consequently enriches tories and refne the use of this tissue to provide longitudinal the δ15N of the remaining amino acids in the body pool that physiological and ecological information (McHuron et al. are used to synthesize tissues. Specifcally, “trophic” amino 2019). Lastly, by assessing how δ15N values of individual acids (e.g., ) that are closely linked to nitro- amino acids respond during periods of known fasting, we gen cycling in animals are subject to more deamination aimed to advance the use of this approach to assess the nitro- (O’Connell 2017), should show increases in δ15N values gen-balance of free-ranging animals and to provide insights in tissues synthesized during fasting. In contrast, we expect into the biochemical mechanisms that lead to δ15N enrich- that “source” amino acids may not be subject to extensive ment of tissues during fasting.

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Methods coupled to a Thermo Scientifc Delta V Plus isotope ratio mass spectrometer (EA-IRMS) in the Stable Isotope Labora- Sample collection and whisker subsampling tory at the University of Pretoria (Pretoria, South Africa). Isotope results are expressed as delta (δ) values in parts per Sample collection occurred at Marion Island (46.88° S, thousand (‰) relative to the international standard atmos- 2 37.87° E). We sampled a single whisker by cutting as close pheric ­N . Two internal standards (Merck gel and DL-Ala- to the skin as possible from SES pups < 2 days after they nine) were used to assess analytical precision (SD), which 15 were weaned (21–23 days after birth) during 2012 and 2013. was ± 0.3‰ for δ N. 15 At ~ 362 days after the initial sampling, we sampled the For amino acid δ N analysis, juvenile whisker regrowth regrown whisker from the returning juveniles (e.g., Fig. 1S). segments with a mean (± SD) sample mass of 8.2 ± 2.0 mg The mean (± SD) length of these regrown whiskers was (minimum: 5.6 mg; n = 10 samples) and adult female 68.6 ± 13.8 mm (n = 17; 6 females and 11 males). Thus, iso- whisker segments with a mass of 9.9 ± 2.9 mg (n = 30 sam- topic information in the regrown whiskers encompasses the ples), were hydrolyzed in 1 ml 6 N hydrochloric acid (HCl) post-weaning fast (catabolic state) followed by independent for 20 h at 110 °C. During the hydrolysis, asparagine was foraging at sea (anabolic state). Whisker regrowths of n = 5 of converted to aspartic acid and glutamine was converted to the individuals used for the bulk δ15N analysis were also used glutamic acid (Whiteman et al. 2019). Amino acids were for the amino acid δ15N analysis. Further details on how these subsequently derivatized with 2-isopropanol and N-TFAA 15 whiskers were sub-sampled for isotope analysis are provided (Fantle et al. 1999) and then analyzed for δ N after separa- in the Supplementary Material and in Lübcker et al. (2017). tion on a 60 m DB-5 column (SGE Analytical Science) in a We also collected multiple fully-grown whiskers with Thermo Scientifc Trace 1310 gas chromatographer coupled unknown growth histories from 23 individuals representing to a Isolink II and Thermo Scientifc Delta V Plus IRMS at subadult (2 males, 1 female), adult female (n = 17), and adult the University of New Mexico Center for Stable Isotopes male SES (n = 3) ranging from 2 to 13-years old (median (Albuquerque, NM, USA). All samples were analyzed in 5-years). Sampling occurred between 2009 and 2016 during duplicate and were bracketed by an internal stock standard satellite telemetry tag deployments. The mean (± SD) length consisting of pure (powdered) amino acids (Sigma-Aldrich of these whiskers was 116.4 ± 19.1 mm (range 79.0–165.0 mm Co.) that were derivatized alongside batches of unknown 15 long) and we noted cases where the whiskers were plucked samples; δ N values of the pure amino acids were meas- 15 instead of cut. Although we considered the growth history ured with EA-IRMS. This method provided δ N measure- as unknown, the growth of the sampled whiskers most likely ments of 13 amino acids: alanine (Ala), isoleucine (Iso), commenced during the molt (Lübcker et al. 2016; McHuron leucine (Leu), valine (Val), proline (Pro), glycine (Gly), ser- et al. 2019). A second whisker from adult females (n = 10) ine (Ser), phenylalanine (Phe), lysine (Lys), tyrosine (Tyr), used for the bulk δ15N analysis was also used for the amino threonine (Thr), glutamic acid (Glu) and aspartic acid (Asp). 15 acid δ15N analysis. We used the whiskers sampled from one Within-run precision of amino acid δ N measurements was adult male SES to demonstrate how the whisker subsampling estimated by calculating SD of duplicate samples analyzed for the amino acid δ15N analysis was achieved (Fig. 2S). Fur- with bracketing standards; the mean SD for all amino acids ther details on how these whiskers were sub-sampled for iso- was 0.4‰ and ranged from 0.3‰ for lysine and 0.5‰ for tope analysis are provided in the Supplementary Material. threonine.

Stable isotope analysis Statistical analyses

Whiskers were pre-treated following the protocol reported in A piecewise linear regression model was used to character- Lübcker et al. (2017). Samples were scrubbed with deionized ize isotopic variation along the length of the whisker cor- (DI) water and rinsed with a 2:1 chloroform:methanol sol- responding to specifc life-history events (R package seg- vent solution to remove surface contaminants. Samples were mented; Muggeo 2008). This was done after estimating the then rinsed twice with the chloroform:methanol solution breakpoints by visual inspection of the plotted data and the and subsequently rinsed thoroughly with DI and dried. All ftted Loess smoothing polynomial regression. In all cases, whiskers were subsampled sequentially into ~ 2.1 ± 0.4 mm normality of the data and model residuals was assessed segments for bulk tissue isotope analysis. Because the distal using a Shapiro–Wilk normality test before applying appro- end (tip) of the fully-grown whiskers was not as thick as priate parametric or non-parametric statistical analyses. For 15 the base, segments of 6–16 mm were required to obtain the bulk tissue δ N results, we reported the mean and standard 15 desired 0.5–0.6 mg sample mass. Segments were weighed deviation (SD). For amino acid δ N results, we reported into tin capsules and δ15N values were measured with a medians and associated upper and lower 95% confdence Thermo Scientifc Flash 1112 Series elemental analyzer intervals. We used linear discriminant analysis (LDA) to

1 3 Oecologia classify the amino acid δ15N values as being synthesized Results during fasting or active foraging (MASS package; Venables and Ripley 2013); details are in the Supplementary Mate- Efects of known fasting on bulk tissue δ15N values rial. The amino acid δ15N values were divided for model training (n = 24 data points, 60% of data) and testing (n = 19 A total of 560 chronologically subsampled whisker regrowth data points, 40% of data) and multivariate normality was segments from juvenile SES (32.9 ± 6.6 segments per indi- confrmed using the ICS package in R (Nordhausen et al. vidual) were analyzed for bulk tissue δ15N. The mean δ15N 2008). Statistical computations were performed using R for values were higher (Kruskal–Wallis χ2 = 351.75, df = 4, statistical computing (R Development Core Team 2018, P < 0.001) for whisker segments grown during the cata- version 3.4.4) coupled with the RStudio interface (version bolic, post-weaning fast as compared to segments of the 1.0.153). whisker grown in utero and while nursing (Fig. 1). The

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Fig. 1 Bulk (whole sample) tissue δ15N values measured along the ments grown during lactation (darker gray; 6.6 ± 1.4 mm segment), length of whiskers sampled from a juvenile (n = 17), b adult female the post-weaning fast (darkest gray; 20.5 ± 1.1 mm), and independ- (n = 17), c adult males (n = 3), and d male and female subadult ent foraging (white). The green boxes on the whisker schematic (top) (n = 3) southern elephant seals (Mirounga leonina). The plot position indicate the position on the whisker where subsamples were collected (x-axis) was set to zero at the onset of data refecting independent for- for AA-CSIA. Samples representing (T1) ‘fasting’ was recorded aging and the red vertical lines indicate the predicted position along 35.6 mm from the tip of the whisker regrowths (13.6 ± 2.9 mm long the length of the whiskers where the isotope values are no longer segment), while samples representing (T2) ‘foraging’ were subsam- afected by preceding fast. The blue lines with gray bars (standard pled on average 7.2 mm from the whisker base. For panel b–d, the error) represent the Loess smoother, while the black line represents light gray boxes (far left) indicate the segment of whisker grown on the ftted Piecewise Linear Regression Models. For panel a, the light- land while fasting. The darker gray bar at the base of the whiskers in est gray area (far left) refects the segment of the whisker that was panel b refects the embedded segment of three whiskers that were grown in utero (10.0 ± 8.2 mm at the tip of whisker), followed by seg- plucked and excluded from further analyses

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Table 1 Bulk tissue δ15N values refecting diferent life-history events adult female (n = 17) southern elephant seals [Mirounga leonina; captured along the length of whiskers sampled for juvenile (n = 17), mean ± SD, expressed as parts per mil (‰)] male and female subadult (n = 3), adult male (n = 3) and breeding

Age In utero Lactation Fasting (catabolic) Transition Foraging (anabolic)

Juvenile 10.9 ± 0.5 11.3 ± 0.8 11.9 ± 0.7 10.3 ± 1.3 8.7 ± 0.5 Subadult – – 11.7 ± 0.3 11.3 ± 0.6 10.9 ± 0.4 Adult ­femalea – – 11.6 ± 0.8 11.2 ± 0.8 10.0 ± 0.7 Adult male – – 12.9 ± 0.6 12.5 ± 0.8 11.6 ± 0.6 a Excludes plucked whisker segments

15 fasting-associated δ15N values were also higher than the Efects of fasting on amino acid δ N values transition from milk to independent foraging (Table 1), and higher than independent foraging (P < 0.001). A piecewise We observed both increases and decreases in the δ15N val- linear regression model (Adj. R2 = 0.79) was used to defne ues of individual amino acids measured along the length of the changes that occurred in the δ15N values captured along the whisker regrowth segments synthesized during a known the length of the whisker regrowths (Table 2). δ15N val- catabolic and anabolic period, respectively (n = 5 juveniles; ues increased by an average of 0.6 ± 0.5‰ during the post- n = 10 samples; Fig. 2). A paired Wilcoxon Signed-Rank weaning fast from post-lactation δ15N values; an increase of Test indicated that the rank sum of the δ15N of the trophic 1.2 ± 0.6‰ (max = 2.4‰) from pre-lactation δ15N values. amino acids, aspartic acid and proline (Z = 2.611, P < 0.01), The transition from fasting to independent foraging at sea were higher during the post-weaning fast as compared to (an anabolic state), was associated with a decrease of 3.7‰, the independent foraging period (Table 1S). The δ15N val- whereafter the mean (±SD) δ15N values equilibrated to the ues of the branched-chain amino acids, leucine, isoleucine, new diet and remained low (8.7 ± 0.5‰). and valine remained unchanged. The δ15N values of several source amino acids were > 2‰ higher during fasting, includ- 15 Identifying unknown fasting using bulk tissue δ N ing lysine (Z = 2.193, P < 0.05), phenylalanine (Z = 2.402, values P < 0.05), and tyrosine (Z = 2.402, P < 0.05; Table 1S). The δ15N of threonine was lower when fasting (Z = 2.611, To determine if we could detect fasting-related δ15N changes P < 0.01) by 5.0‰ than during active foraging. Glycine and along the length of the whiskers with unknown growth serine were both (Z = 2.611, P < 0.01) enriched by 6.3‰ histories, we analyzed a total of 1136 whisker segments during fasting as compared to foraging. Surprisingly, the (49.4 ± 10.6 segments per individual) collected from sub- δ15N value of the trophic amino acid alanine was 2.4‰ adult (n = 3) and adult male (n = 3) and adult female (n = 17) lower during the fast (Z = − 2.311, P < 0.05) than during SES. The distinctive slope (b) and positive change in δ15N foraging. values observed during the catabolic state of the post-wean- The δ15N of the amino acids measured along the length ing fast (b = 0.027; Table 2) did not difer signifcantly from of the whiskers of breeding adult females (n = 10 indi- the change in δ15N values observed at the tip of the whisk- viduals; n = 30 samples; Fig. 3) and the one adult male ers of subadult (b = 0.023), adult female (b = 0.035), and (Fig. 2S) showed a similar pattern of increases and adult male SES (b = 0.030) (Kruskal–Wallis χ2 = 3, df = 3, decreases as observed in juvenile SES (Fig. 2). The gly- P = 0.391), suggesting that the whisker tips were grown cine δ15N value in the tip of adult female whiskers, likely while fasting. The trends in the δ15N values measured along refecting fasting on land, was 2.4‰ higher than in the the length of whiskers sampled from male and female SES middle and basal segments (Kruskal–Wallis χ2 = 12.963, of various ages were comparable (Fig. 1b–d). The pre- df = 2, P < 0.001) that represented at-sea foraging. The sumed fasting-associated changes in δ15N of subadult and serine δ15N value was 2.1‰ higher (Kruskal–Wallis adult male SES included increases of 1.3‰ (Kruskal–Wal- χ2 = 4.560, df = 2, P = 0.099) during fasting compared to lis χ2 = 53.40, df = 2, P < 0.001) and 1.6‰ (Kruskal–Wallis when foraging (Table 1S). The δ15N value of alanine was χ2 = 65.12, df = 2, P < 0.001) from the start to end of the 2.1‰ lower (Kruskal–Wallis χ2 = 5.546, df = 2, P = 0.059) fast, respectively (Table 2). The largest predicted enrich- during fasting as compared to the middle of the whisker ment occurred in adult females (1.8‰). Thereafter, the bulk when at-sea foraging commenced (Table 1S). There were tissue δ15N of subadult, adult females and males declined no signifcant diferences between fasting and foraging by 1.4‰ by 1.6‰ respectively when whisker growth is measured for any of the source amino acids (phenyla- assumed to refect foraging at sea. lanine, tyrosine, lysine), branched-chain amino acids

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Table 2 Piecewise linear Period Segments Slope (b) y-axis intercept Predicted regression models (y = a + bx) 15 (a) Δ NStart–End characterizing the changes (‰) observed in the bulk δ15N n Estimate SE (95% CI) Estimate SE values during diferent a life-history events measured Juvenile sequentially along the length of In utero growth 77 0.002 0.007 (− 0.023; 0.025) 10.9 0.025 < 0.1 whiskers sampled from juvenile Lactation 73 0.062 0.022 (0.019; 0.105) 12.9 0.023 + 0.7 (n = 560 whisker segments), male and female subadult Fasting 186 0.027 0.007 (0.012; 0.042) 12.2 0.019 + 0.6 (n = 141 segments), adult Transition 105 − 0.287 0.018 (− 0.322; − 0.252) 12.4 0.019 − 3.7 male (n = 174 segments) and Foraging 119 0.018 0.007 (0.004; 0.032) 8.3 0.186 + 0.9 n breeding adult female ( = 821 Subadultb segments) southern elephant seals (Mirounga leonina) Fasting 30 0.023 0.008 (0.008; 0.038) 12.0 0.014 + 1.3 Transition 29 − 0.073 0.012 (− 0.097; − 0.050) 11.8 0.013 − 1.4 Foraging 82 0.005 0.006 (− 0.006; 0.016) 10.5 0.009 + 0.2 Foraging† 0.025 0.007 (0.012; 0.038) 9.5 0.007 + 1.0 Adult ­femalec Fasting 170 0.035 0.003 (0.029; 0.042) 12.0 0.028 + 1.8 Transition 158 − 0.135 0.027 (− 0.187; − 0.082) 11.8 0.027 − 1.4 Foraging 493 − 0.012 0.001 (− 0.014; − 0.009) 10.6 0.22 − 1.3 Adult ­maled Fasting 23 0.030 0.010 (0.010; 0.049) 13.2 0.049 + 1.6 Transition 31 − 0.165 0.048 (− 0.259; − 0.070) 13.8 0.049 − 1.6 Foraging 120 − 0.018 0.010 (− 0.039; 0.002) 11.9 0.011 − 0.5 Foraging† 0.016 0.003 (0.010; 0.022) 10.5 0.003 + 1.3

We report the standard error (SE) with lower and upper 95% confdence intervals in brackets † Breakpoint during foraging a Piecewise linear regression model standard error = 0.656; P < 0.001; Adj. R2 = 0.79 (df = 550); x-axis estimated break-points on set plot position (± SE) = 13.5 ± 0.5 mm, 0.7 ± 0.5 mm, − 21.4 ± 4.8 mm, and − 32.6 ± 3.1 mm b Piecewise linear regression model standard error = 0.526; P < 0.001; Adj. R2 = 0.15 (df = 139); x-axis esti- mated break-points (± SE) = − 2.2 ± 1.8 mm, 16.9 ± 2.2 mm, and 48.2 ± 7.6 mm c Piecewise linear regression model standard error = 0.724; P < 0.001; Adj. R2 = 0.48 (df = 799); x-axis esti- mated break-points (± SE) = − 1.3 ± 1.1 mm, 9.4 ± 1.4 mm, and 115.5 ± 0.7 mm. Plucked whisker values excluded d Piecewise linear regression model standard error = 0.335; P < 0.001; Adj. R2 = 0.09 (df = 172); x-axis esti- mated break-points (± SE) = 3.1 ± 1.7 mm, 12.9 ± 2.2 mm, and 42.0 ± 6.0 mm

(isoleucine, leucine and valine), other trophic amino acids Discussion (proline, aspartic acid, glutamic acid), or threonine. Fasting caused an enrichment in whisker bulk tissue Identifying fasting based on amino acid δ15N values δ15N values

15 The basic LDA models parameterized with the δ N of Fasting-related enrichment of bulk tissue δ15N values has all the amino acids or with the baseline-corrected data been reported in a variety of tissues from various species 15 (Δ NTrophic AA–Phe) produced identical outputs. The training (Hobson et al. 1993; Fuller et al. 2004; Doi et al. 2017). dataset accurately predicted fasting in 96% of the cases, with Here, we observed bulk tissue δ15N enrichments of 0.8‰ a single misclassifcation of actual foraging as fasting. The and 1.8‰ in the tip of subadult and adult whiskers with accuracy of the model based on the testing dataset was 74%, unknown whisker growth histories, respectively (Fig. 1b–d), with fasting incorrectly classifed as foraging in 12% of the which is comparable to the fasting-related δ15N enrichment 15 data points. The δ N of glycine, serine, alanine and proline observed in the whiskers segments of juvenile SES that were were the most signifcant predictors (P < 0.001) for separating synthesized during a known catabolic period while fasting foraging and fasting (Table 2S). on land (Lübcker et al. 2016, 2017). The consistency of the location of the segments exhibiting the fasting-associated

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6 8 -2 -36 *** * ** ** ** ** * * * ** Bulk AlaAsp Glu IleLeu Val Pro GlySer Phe Lys Tyr Thr Trophic T/S Source

Fig. 2 Comparison of the mean (± SD) bulk tissue δ15N values (left bars refect the 25% and 75% confdence intervals (CI) around the panel) and median amino acid δ15N values of juvenile southern median δ15N values, while the dashed lines refect the 5% and 95% elephant seals (n = 5) whiskers refecting the post-weaning fast (red CI. *P < 0.05, **P < 0.01, ***P < 0.001. Amino acid abbreviations open symbols) and foraging at-sea (blue open symbols). Solid error are defned in “Methods”

15N-enrichment in the whiskers of SES adults supports that deaminated during catabolism (O’Connell 2017). Specif- elephant seals shed their whiskers synchronously during the cally, glycine, serine, proline, and aspartic acid had signif- annual pelage molt (Lübcker et al. 2016; Aurioles-Gamboa cantly higher δ15N values in whisker segments grown during et al. 2019; McHuron et al. 2019). Thus, the longitudinal the post-weaning fast in juveniles and molting period in adult record captured in the subadult and adult SES whiskers females (Figs. 2, 3). These four amino acids serve as primary essentially represents the ~ 8-month period of the foraging substrates for gluconeogenesis, a crucial biochemical path- trip that commences at the end of the molt and continues way through which carnivorous mammals convert exogenous until the onset of the breeding season when the whiskers (dietary) or endogenous (body) protein into glucose (Krebs were collected (Fig. 1S). This enables the identifcation of 1964). Our fndings suggest that after muscle peptides are segments of the whiskers representing the phase when SES hydrolyzed into amino acids, those amino acids containing are solely relying on exogenous energy sources, and thus isotopically light amine groups (14N) are then preferentially accurate reconstructions of habitat use and diet for phocids deaminated in the frst step of gluconeogenesis, resulting in that can function as sentinels of ocean health (Fedak 2004). the isotopic enrichment of the remaining free amino acids in plasma that is subsequently used to synthesize whiskers dur- Fasting caused an enrichment in glucogenic amino ing fasting (Fig. 4). In addition, splanchnic de novo synthesis acid δ15N values of glycine, serine, and proline (Felig et al. 1969; Kalhan and Hanson 2012; Wang et al. 2013) from endogenous pools of 15 Amino acid nitrogen isotope data show that the fasting- nitrogen that are N-enriched relative to exogenous sources 15 induced 15N-enrichment in bulk whisker tissue is driven by can also contribute to the observed increase in δ N values increases in the δ15N pool of amino acids that are readily of these amino acids in whiskers (Fig. 5); this process is

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8 12 2 -34

10 0

6 8 -2 -36 *** ** Bulk Ala Asp Glu IleLeu Val Pro Gly Ser Phe Lys Tyr Thr Trophic T/S Source

Fig. 3 Mean (± SD) bulk tissue δ15N values (left panel) and median fdence intervals (CI) around the median δ15N values and the dashed amino acid δ15N values measured in the tip (red), middle (green) and lines refect the 5% and 95% CI. **P < 0.01. Amino acid abbrevia- basal (blue) whisker segments from breeding adult female (n = 10) tions are defned in the “Methods” southern elephant seals. Solid error bars refect the 25% and 75% con- known as the anabolic model of nitrogen fux (Lee et al. would further enrich the remaining aspartic acid used to 2012). Data from humans show support for the anabolic synthesize tissues (McMahon and McCarthy 2016). model, where plasma glycine, serine, and proline concen- We observed a decrease in threonine δ15N values during trations increase during fasting (Felig et al. 1969). Thus, fasting relative to foraging, which may result from selec- our fndings suggest that the δ15N enrichments (0.4–6.3‰; tive (enzymatic or microbial) recycling of threonine sourced Table 1S) of these three amino acids could result from from intestinal mucin during fasting to maintain amino acid their de novo synthesis from recycled 15N-enriched nitro- homeostasis (Wallace and Hedges 2016). Although the gen pools. Concerning proline, its ring structure prevents molecular mechanism responsible for the often-observed transamination of its nitrogen (O’Connell 2017), suggesting negative fractionation of threonine δ15N between consumer that the observed increase in δ15N of proline during fast- and diet is unclear (Whiteman et al. 2019) it nonetheless may ing occurs when 15N-enriched glutamic acid (sourced from be a useful ecological (e.g., trophic level) or physiological 15N-enriched endogenous protein or synthesized de novo), (e.g., fasting) biomarker. is converted into proline (O’Connell 2017). Lastly, aspartic 15 acid is synthesized when the nitrogen of glutamic acid is Depletion of alanine δ N values during fasting transaminated to oxaloacetate in the tricarboxylic acid cycle (Macko et al. 1986). The amide transferred from glutamic The signifcant decrease in alanine δ15N values of ~ 2–3‰ acid is likely 15N-enriched as a result of gluconeogenesis in both juvenile and adult female SES during fasting was when fasting. Furthermore, deamination of 14N-containing unexpected. We suggest that this pattern is driven by isotopic aspartic acid during the and purine synthesis fractionation associated with the glucose-alanine (Cahill) cycle (Fig. 5), which is the dominant metabolic pathway

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Glucose Threonine 3-phoshoglycerate Serine Glycine (cytosol)

Phosphoenolpyruvate

Pyruvate Mitochondria Lys, Phe, Tyr Acetyl-CoA (ketogenic) Endogenous + (enriched) (skeletal muscle) Citric Acid Cycle Catabolic

Oxaloacetate α-KG Ala, Ser, Gly, Glu, Asp, Pro, Phe, Tyr δ¹ N (glucogenic) Fumarate Succinyl-CoA Anabolic Exogenous – (depleted) (dietary protein)

Fig. 4 Gluconeogenesis (gray dashed lines) represents a key meta- via conversion to acetyl CoA can contribute to gluconeogenesis. bolic pathway that results in nitrogen isotope discrimination when Catabolism involves preferential deamination of amino acids contain- either exogenous (dietary) or endogenous (muscle) protein is catab- ing 14N, which leads to isotopic enrichment of the remaining amino olized to produce glucose. Both glucogenic amino acids (green) via acid pool used to synthesize new tissue (e.g., whiskers) Citric Acid Cycle intermediaries and ketogenic (blue) amino acids

Muscle Plasma LiverKidneys Protein 14 14 N 14 14 N Alanine N N Amino Acids α-KG2Alanine 2Alanine α-KG 14NH4+ Urea Cycle Alanine Alanine Transamination Transamination aminotransferase aminotransferase α-Keto Acid Glu 2Pyruvate 2Pyruvate Glu Glycine 14N 14N Glycolysis Gluconeogenesis Amino Acid 14N Glucose Glucose Catabolism 15N

Amino Acid 15 Synthesis N

Tissues (Alanine, Serine, Glycine) 14N 15N 15N

Fig. 5 Schematic of the glucose-alanine (Cahill) cycle portraying the vate during gluconeogenesis. The pathway by which 14N are delivered mechanism responsible for isotopic depletion of alanine during fast- from the muscle to the urea cycle is indicated in red. The blue dashed ing. Alanine aminotransferase catalyzes the synthesis of alanine from lines depict the pathway by which the 14N-alanine and 15N-enriched pyruvate, in which isotopically light nitrogen (14N) is transaminated gluconic amino acids (glycine and serine) are incorporated into newly from glutamate. Alanine is then transferred to the and deami- synthesized tissues (e.g., whiskers) during fasting nated to produce urea while the carbon skeleton is recycled into pyru-

1 3 Oecologia used by vertebrates to (a) transport nitrogen from the mus- expectations, whisker phenylalanine, lysine, and tyrosine cle to the liver (Felig et al. 1969), and (b) cycle carbon in δ15N values signifcantly increased by ~ 2–4‰ during fast- the form of glucose between the liver and muscle during ing in both pups (Fig. 2) and adult females (Fig. 3), sug- fasting (Felig et al. 1970). The observed isotopic depletion gesting that these amino acids were catabolized to fuel glu- is likely caused by the transamination of isotopically light coneogenesis; tyrosine is considered to be a conditionally (14N) nitrogen atoms from glutamate to alanine that is trans- essential amino acid because it can only be synthesized ported via blood plasma to the liver where it is deaminated from phenylalanine. Importantly, phenylalanine and lysine to produce glucose (Krebs 1964; Felig et al. 1970). While in are irreversibly lost when catabolized during fasting because blood circulation, this 14N-alanine can also be used to main- they are essential amino acids that cannot be synthesized de 15 15 tain actively growing tissues like whiskers during fasting. novo. The δ N diference (δ NGlu–Phe) between glutamic acid (trophic) and phenylalanine (source) has been widely 15 Lack of change in δ N values of branched‑chain used to infer the trophic level (TL) of marine consumers amino acids and glutamic acid (Chikaraishi et al. 2009; Germain et al. 2013; McMahon and McCarthy 2016). Because δ15N values of source amino acids While the branched-chained amino acids valine, leucine, and are often assumed to be insensitive to TL and physiology isoleucine can be catabolized to fuel for gluconeogenesis, the (Chikaraishi et al. 2009), the enrichment of phenylalanine lack of change in their δ15N value suggests that these amino has a strong infuence of TL estimates for SES. For example, 15 acids are being spared during fasting. These three amino acids the δ NGlu–Phe of foraging adult female SES during gestation are considered essential for most eukaryotes and cannot be was 16.0‰ which, produces a TL estimate of ~ 3.5. If we synthesized de novo, thus it may be advantageous to spare assume that nursing pups are feeding approximately one TL them and keep their catabolism to a minimum during peri- above adult females (Jenkins et al. 2001), we would expect 15 ods of fasting, a strategy that has been previously shown to to observe δ NGlu–Phe of ~ 23.6‰ and an associated TL occur in nutritionally stressed captive mammals (Adibi 1980; estimate of ~ 4.5. In contrast, the unexpectedly stable δ15N Tom and Nair 2006). In addition, the interchangeability of values of glutamic acid and higher phenylalanine observed 15 the amine group between branched-chain amino acids and during fasting resulted in these pups having a δ NGlu–Phe glutamic acid during amino acid catabolism, anabolism, and of ~ 12.5‰ and a calculated TP of ~ 3.1. In other words, urea (14N) nitrogen recycling may reduce isotopic fractiona- the trophic-source ofset in nitrogen isotope values placed tion leading to 15N-enrichment (O’Connell 2017). the pups at roughly half of a trophic level lower than their We also observed no diference in whisker glutamic acid mothers, rather than at a trophic level higher. This discrep- δ15N values between fasting and foraging for juvenile and ancy, combined with the observed increase in phenylala- adult SES. We suggest that the lack of change is likely the nine, lysine, and tyrosine δ15N values during fasting suggests result of the large degree of nitrogen exchange between glu- that physiological status must be taken into account when tamic acid and other amino acids (O’Connell 2017), com- interpreting ofsets in amino acid δ15N values as proxies for bined with the fact that glutamate and glutamine represent trophic level (Chikaraishi et al. 2009). the largest nitrogen pool in the body. The insensitivity of glutamic acid nitrogen isotope values to fasting is benef- Accuracy of identifcation of fasting events 15 cial for studies of trophic ecology, which commonly use the with amino acid δ N values δ15N value of this amino acid to estimate the trophic position occupied by consumers (Chikaraishi et al. 2009). Yet, when The various patterns in δ15N enrichment enabled us to combined with observed patterns in source amino acid δ15N identify fasting events with an accuracy of 74%. The small values (see below), this fnding contradicts our expectation sample size used for model testing and the possible inclu- that the ofset between the δ15N values of trophic and source sion of whisker segments that refected a mixture of fasting amino acids of animals should increase during fasting rela- and foraging potentially reduced the predictive power of the tive to when their energy demands are satisfed by exogenous models (Fig. 2S). Nevertheless, the correlation coefcients (dietary) resources. (R2 values) met the required criteria for biomarker evalua- tion (Yun et al. 2018), even though our study was focused on free-ranging animals. The bulk whisker tissue δ15N enrich- 15 Fasting leads to increases in δ N values of source ments associated with fasting in juveniles (1.2‰) was simi- amino acids lar to that observed in adult females (1.3‰) (Table 2). The mass of protein lost as a percentage of body weight for fast- Source amino acids like phenylalanine and lysine are ing adult female SES during the molt (14%) is comparable expected to have unchanged δ15N values during fasting, to that lost by juveniles (12–32%) during the post-weaning similar to branched-chained amino acids. Contrary to fast (Boyd et al. 1993; Carlini et al. 2001). The higher body

1 3 Oecologia masses and slower tissue turnover rates of adult female SES fndings show that continuously growing, but metabolically may be a factor in explaining why the fasting-associated inert keratinaceous tissues like whiskers and baleen, can be increases in δ15N values for some amino acids were smaller used to construct a longitudinal dietary and physiological in magnitude (Fig. 3) in comparison to those observed in record of an individual that can span months (phocids) to juveniles (Fig. 2). years (mysticetes) depending on the species. Importantly, Source and trophic amino acids can have diferent turno- the isotopic variation associated with physiological chal- ver rates (Bradley et al. 2014; Downs et al. 2014) that should lenges like fasting must be taken into consideration when also be considered when using this approach to identify interpreting isotope-based reconstructions of diet. This is when the isotope values measured in the sampled tissue especially important for species with complex annual life matrix are afected by fasting. The half-lives of trophic histories (e.g., marine mammals) that include extended peri- and source amino acids measured in the muscle of Pacifc ods of fasting associated with migration and/or reproduction. bluefn tuna (Thunnus orientalis) for example, ranged from 28.6–305.4 days and 67.5–36.2 days, respectively (Brad- Acknowledgements The Animal Use and Care Committee (AUCC) of the Faculty of Veterinary Science, University of Pretoria, provided ley et al. 2014). We emphasized that apart from consider- ethical clearance for the research under AUCC 040827-022, AUCC ing the turnover rate of diferent amino acids, amino acids 040827-023, EC030602-016, and EC077-15. We thank Prof Dan Costa that are readily deaminated during catabolic states were (University of California, Santa Cruz, USA) for his vital role with more likely to show a response in fasting SES, while the the sample handling [National Marine Fisheries Service (NMFS), National Oceanic and Atmospheric Administration (NOAA) Permit large glutamate and glutamine pool and the interchangeabil- number 19439]. The National Research Foundation (NRF) (Grant no. ity of their amide–nitrogen could mask expected changes. SFH14070373490) funded the research and supported N. Lübcker More research is required to determine the rate at which through an Innovation Scholarship. The ‘Society for Marine Mam- amino acids reach a steady-state when the nitrogen balance malogy (SMM) Small Grant in Aid of Research’ provided additional support for the sample analyses. The conclusions drawn are attributed of organisms is disturbed. Still, diferential fasting-induced 15 to the authors and not necessarily to the NRF and SMM. Logistical sup- changes in the δ N values of trophic and source amino acids port was provided by the Department of Environmental Afairs under can lead to erroneous TP estimates and are best excluded the South African National Antarctic Program (SANAP). We express from trophic studies. our sincerest gratitude towards the staf and colleagues at the University of New Mexico Center for Stable Isotopes (Albuquerque, NM, USA) for their support, with a special mention for Dr. Viorel Atudorei and Emma Elliot Smith, as well as to Dr. Grant Hall, Nicolas Prinsloo, Conclusions Michael Mole, and André van Tonder and feld personne from the University of Pretoria, South Africa. Lastly, we thank the reviewers for their time and valuable contributions. Our study illustrates the utility of using both bulk tissue and amino acid isotope analysis as nutritional biomarkers. Our Author contribution statement NL, SDN, RPM, JPW, and PJNdB con- sampling methodology and analytical approach provide a ceived and designed the experiments. PJNdB, JPW, SDN, and RPM longitudinal record of nutrient inputs (diet), trophic posi- enabled sample collection and sample analyses. NL and JW analysed tion, and physiological status (e.g., nitrogen-balance) for the data. NL wrote the manuscript; other authors provided editorial advice. consumers (Mekota et al. 2009; McCue 2010). Non-inva- sively obtaining such information from a single tissue sam- Compliance with ethical standards ple is currently unattainable with other techniques (Reitsema 2013; Ohkouchi et al. 2017; Yun et al. 2018). Conflict of interest The authors declare that they have no confict of Our study establishes that it is feasible to identify periods interest. of fasting by bulk tissue δ15N analysis, while also provid- ing a new compound-specifc amino acid framework for Ethical approval All procedures performed in the studies involving human participants were in accordance with the ethical standards of assessing nitrogen balance. Specifcally, substantial iso- the institutional and/or national research committee and with the 1964 topic enrichments (~ 2–6‰) in glucogenic and even some Helsinki declaration and its later amendments or comparable ethical ketogenic amino acids are likely driven by a switch in the standards. catabolism of exogenous (prey) amino acids during foraging to endogenous (skeletal muscle) amino acids during fasting for gluconeogenesis. The signifcant depletion in alanine δ15N during fasting is likely associated with the glucose- References alanine cycle that shuttles nitrogen from muscle to liver and fuels amino acid metabolism. Adibi SA (1980) Roles of branched-chain amino acids in metabolic Moreover, we can also accurately identify the segment regulation. J Lab Clin Med 95(4):475–484 of the whisker grown while free-ranging SES are on land Aurioles-Gamboa D, Newsome SD, Hassrick JL, Acosta-Pachón T, Aurioles-Rodríguez F, Costa DP (2019) Use of 15N-enriched without prior knowledge of whisker growth histories. These

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