Isotopic Discrimination Factors Research Articles

Tissue‐ and diet‐dependent stable carbon and nitrogen isotope discrimination: a calibration study in a captive shorebird species

In ecology, stable‐isotope ratios are widely used to determine diets of organisms and reconstruct food webs. This is usually done by analyzing the stable‐isotope ratios of nitrogen (δ15N), which increase with increasing trophic level, and those of carbon (δ13C), which correlate with the δ13C value of food source(s) and generally differ between terrestrial and marine food sources. Assimilation of food changes stable‐isotope ratios, resulting in different values between the food source and its consumer. These differences are known as isotope trophic discrimination factors and, if known, can be used to determine from the stable‐isotope ratios in the consumer's tissue what the consumer has been eating. What is often ignored is that discrimination factors can differ between consumer's food sources and also between tissue types. Therefore, we performed a controlled feeding study in red knots Calidris canutus to determine discrimination factors between different food sources and red knot tissues. We kept two groups of red knots in captivity on a stable diet, one group feeding on mudsnails and the other on Trouvit pellets, for several months, during which the birds molted their feathers. We analyzed δ13C and δ15N in both food sources and in five red knot tissues (blood cells, blood plasma and three feather types) and subsequently calculated the isotope discrimination factors. We confirmed that the discrimination factors differed between tissues, and also between diets. Our values deviated from general averages reported in reviews on a wide range of animals/birds, but were very similar to values from previous red knot and dunlin studies. We therefore think that our discrimination factors can be used in future stable isotope studies, not only on red knots, but also on other marine shorebird species and plea for careful consideration of using the right discrimination factors.Keywords: δ13C, δ15N, discrimination factor, red knot, shorebird, stable isotope

Sulfur isotopic discrimination factors differ among avian tissues and diets: Insights from a case study in Gentoo Penguins

Abstract The use of stable isotopes of sulfur (δ34S) to infer avian diets, foraging habitats, and movements is relatively uncommon, resulting in a lack of information on patterns of δ34S incorporation in avian tissue. In a controlled study of Gentoo Penguins (Pygoscelis papua), we found that diet-tissue isotopic discrimination factors (Δ34Sdiet-tissue) differed among egg components and feathers synthesized from a common diet, ranging from –0.4 to –1.7‰. We also found that methodical choices such as lipid extraction and prey tissue selection influenced calculated Δ34Sdiet-tissue values. Specifically, Δ34Sdiet-tissue values were lower (i.e., more negative) when calculated using whole fish relative to fish muscle and lipid-extraction biased egg yolk, but not fish tissue, δ34S values. The Δ34Sdiet-tissue values obtained for Gentoo Penguins fed a marine fish diet were generally lower than those reported for freshwater fish consumption by Double-crested Cormorants (Phalacrocorax auritus), the only other bird species in which Δ34Sdiet-tissue has been quantified. We found support for the hypothesis that tissue Δ34Sdiet-tissue values are inversely related to dietary δ34S values in birds, similar to what has been observed in mammals. Given this relationship, the discrimination factors reported here for Gentoo Penguins may be broadly applicable to other avian species with a similar marine diet. Finally, we provide recommendations for future studies seeking to quantify Δ34Sdiet-tissue in avian tissues and guidance to allow for greater application of sulfur stable isotope analysis in ornithological research.

Toxicity and Starvation Induce Major Trophic Isotope Variation in Daphnia Individuals: A Diet Switch Experiment Using Eight Phytoplankton Species of Differing Nutritional Quality.

Stable isotope values can express resource usage by organisms, but their precise interpretation is predicated using a controlled experiment-based validation process. Here, we develop a stable isotope tracking approach towards exploring resource shifts in a key primary consumer species Daphnia magna. We used a diet switch experiment and model fitting to quantify the stable carbon (δ13C) and nitrogen (δ15N) isotope turnover rates and discrimination factors for eight dietary sources of the plankton species that differ in their cellular organization (unicellular or filamentous), pigment and nutrient compositions (sterols and polyunsaturated fatty acids), and secondary metabolite production rates. We also conduct a starvation experiment. We evaluate nine tissue turnover models using Akaike's information criterion and estimate the repetitive trophic discrimination factors. Using the parameter estimates, we calculate the hourly stable isotope turnover rates. We report an exceedingly faster turnover value following dietary switching (72 to 96 h) and a measurable variation in trophic discrimination factors. The results show that toxic stress and the dietary quantity and quality induce trophic isotope variation in Daphnia individuals. This study provides insight into the physiological processes that underpin stable isotope patterns. We explicitly test multiple alternative dietary sources and fasting and discuss the parameters that are fundamental for field- and laboratory-based stable isotope studies.

Application of stable isotope analysis to evaluate the assimilation of protein sources in juvenile slipper lobsters (Thenus australiensis)

Stable carbon and nitrogen isotope analyses were used to evaluate the contribution of five protein sources to the growth of juvenile slipper lobster Thenus australiensis. Protein sources tested were fish meal, krill meal, lupin meal, soybean meal and a squid by-product meal. A commercial in confidence basal mix was utilised to produce five experimental feeds containing 30% of each protein source. Juvenile lobsters were fed daily at 5% of their body weight for 12 weeks. Lobster performance was assessed by survival, feed intake and growth performance, and the effects of protein source on nutrient assimilation and discrimination factor were determined. The squid by-product meal, soybean meal, and krill meal produced better growth performance and survival. The specific growth rate on lupin meal was significantly (p < 0.05) lower than all the other protein sources. Apparent feed intake was significantly (p < 0.001) higher in squid by-product meal than lupin meal but with no significant differences in feed efficiency ratio. Protein source assimilation proportion in lobster ranged from 7% for soybean meal to 32% for squid by-product meal, whereby squid by-product meal and fish meal (29%) were the only protein sources assimilated at approximately their dietary inclusion level (30%). Overall, squid by-product meal had the best growth performance, high survival, and the highest assimilation into growth. This study demonstrated the potential of stable isotopic analysis to screen protein sources, evaluate the nutritional performance of the experimental feeds, and provide a deeper insight into ingredient utilisation. This study also provides the first whole-body isotopic discrimination factors for juvenile lobster, which can determine ingredient contribution to growth in future studies.

Stable isotope measurements as analytical tools for the traceability of crocodile–derived products

In this preliminary study we examined the application of dual stable isotope analysis (δ13C and δ15N) to identify the origin of skins and meat derived from wild and farmed crocodiles. Traceability protocols can benefit from analytical techniques that are able to distinguish farmed or wild organisms. Scutes and muscle samples were obtained from wild and farmed crocodiles Crocodylus acutus (n = 14) and C. moreletii (n = 9). Isotopic values in scutes differed significantly between wild and farmed organisms, this difference being higher for δ15N than for δ13C values. When both isotopic values were integrated using a discriminant analysis, we observed a significant categorization. The isotopic values of muscle samples were very similar to those measured in scutes from the same individuals. In addition, two specimens of C. acutus were kept on a constant diet for 97 days to obtain reference isotopic values and tissues were compared. We also estimated the isotopic discrimination factors between tissues and the supplied diet.

Turnover Rates and Diet-Tissue Discrimination Factors of Nitrogen and Carbon Stable Isotopes in Seahorse Hippocampus reidi Juveniles Following a Laboratory Diet Shift.

Simple SummaryThe main aim of the present study was to ascertain the effect of two feeding schedules (including copepods and Artemia nauplii) on the early development and physiology of seahorse Hippocampus reidi juveniles. For that, we analyzed seahorse performance (growth and survival) and trophic patterns by means of stable isotopes. Our results highlight that the welfare and condition of juveniles were enhanced by extending the period of feeding on copepods up to day 10 after the male’s pouch release. The analysis of turnover rates for δ13C and δ15N revealed that switching copepods to Artemia nauplii at earlier developmental stages would reduce prey assimilation resulting in lower growth rates and survivals. The present study also provides for the diet–tissue discrimination factors for δ13C and δ15N in seahorse juveniles for the first time.The initial development of seahorse juveniles is characterized by low digestion capabilities. Stable isotope analysis is an effective tool in studies of trophic food webs and animal feeding patterns. The present study provides new insights for the understanding of growth and food assimilation in early developing seahorses following a laboratory diet switch. The study was performed in the early life stages of the seahorse Hippocampus reidi by assessing the influence of diet shift on changes and turnovers in carbon (δ13C) and nitrogen (δ15N) stable isotope in juveniles. Newborn seahorses were fed for 60 days following two feeding schedules (A6 and A11) based initially on copepods Acartia tonsa and subsequently on Artemia nauplii (since days 6 and 11, respectively). After the prey shift, we determined δ13C and δ15N turnover rates as functions of change in either body mass (fitting model G) and days of development (fitting model D), contributions of metabolism and growth to those turnover rates, and diet–tissue discrimination factors. Survival, final dry weight, and final standard length for diet A11 were higher compared to diet A6. The shift from copepods to Artemia led to fast initial enrichments in δ13C and δ15N. Afterwards, the enrichment was gradually reduced until the isotopic equilibrium with the diet was reached. In most cases, both fitting models performed similarly. The isotopic analysis revealed that 100% of tissue turnover was attributed to growth in diet A11, whereas 19–25% was linked to metabolism in diet A6. Diet–tissue discrimination factors were estimated for the first time in seahorse juveniles, resulting in higher estimates for diet A11 (2.9 ± 0.7‰ for δ13C; 2.5 ± 0.2‰ for δ15N) than in diet A6 (1.8 ± 0.1‰ for δ13C; 1.9 ± 0.1‰ for δ15N). This study highlights the relevance of feeding on copepods and their effect on isotopic patterns and discrimination factors in seahorse juveniles after a dietary shift. Regarding the application of the results achieved in relation to the feeding schedules in the rearing of H. reidi, a long period of feeding on copepods during the first days of development is highly recommended.

Mercury concentrations, biomagnification and isotopic discrimination factors in two seabird species from the Humboldt Current ecosystem

Assessing mercury (Hg) biomagnification requires the description of prey-predator relationships, for each species and ecosystem, usually based on carbon and nitrogen isotope analyses. Here, we analyzed two seabirds from the Humboldt Current ecosystem, the Guanay cormorant (Phalacrocorax bougainvillii) and the Peruvian booby (Sula variegata), as well as their main prey, the Peruvian anchovy (Engraulis ringens). We reported Hg concentrations, Hg biomagnification (BMF) and isotopic discrimination factors (Δ13C and Δ15N) in seabird whole blood. BMFs and Δ13C in our study (on wild birds where diet was not controlled) were similar to other piscivorous seabirds previously studied in captive settings, but Δ15N were lower than most captive experiments. We observed lower Hg concentrations in Humboldt seabirds compared to other oligotrophic ecosystems, possibly due to Hg biodilution in the high biomass of the first trophic levels. This work calls for a better characterization of Hg trophic dynamics in productive upwelling ecosystems.

Four approaches for estimating isotope discrimination factors produce contrasting dietary estimates for bears

Isotope diet-tissue discrimination factors can significantly influence dietary proportions estimated with stable isotope mixing models. For bears, for which discrimination factors from controlled dietary experiments are currently lacking for most tissues and diets, literature reports 3 approaches have so far been used to compile carbon and nitrogen discrimination factors, and recently one additional approach has been proposed. We used these 4 approaches to compile carbon and nitrogen discrimination factors for the brown bear (Ursus arctos) population in Slovenia (southeastern Europe). By performing a sensitivity analysis of the stable isotope mixing models, we then tested how these different discrimination factors affected dietary estimates for bears. We found substantial differences in the dietary estimates among the applied approaches, with the approach that uses discrimination factors from different species producing most distinct results. Our results suggested that these differences were more pronounced among approaches with larger differences in discrimination factors and for isotopically less distinct food sources. We show that the 4 approaches can lead to contrasting conclusions about bear diets, which can have important ecological and management consequences. Therefore, when using stable isotope mixing models without experimentally obtained species-, diet-, and tissue-specific discrimination factors, the choice of the discrimination-factor compilation approach is vital. All 4 approaches have limitations that must be considered because they can result in erroneous discrimination factors when certain species, diets, and tissues are used. We review these limitations and provide recommendations about the use of these approaches in stable isotope dietary studies for bears.

Zinc isotopes from archaeological bones provide reliable trophic level information for marine mammals

In marine ecology, dietary interpretations of faunal assemblages often rely on nitrogen isotopes as the main or only applicable trophic level tracer. We investigate the geographic variability and trophic level isotopic discrimination factors of bone zinc 66Zn/64Zn ratios (δ66Zn value) and compared it to collagen nitrogen and carbon stable isotope (δ15N and δ13C) values. Focusing on ringed seals (Pusa hispida) and polar bears (Ursus maritimus) from multiple Arctic archaeological sites, we investigate trophic interactions between predator and prey over a broad geographic area. All proxies show variability among sites, influenced by the regional food web baselines. However, δ66Zn shows a significantly higher homogeneity among different sites. We observe a clear trophic spacing for δ15N and δ66Zn values in all locations, yet δ66Zn analysis allows a more direct dietary comparability between spatially and temporally distinct locations than what is possible by δ15N and δ13C analysis alone. When combining all three proxies, a more detailed and refined dietary analysis is possible.

Methanogenesis in the Eocene Tharad lignite deposits of Sanchor Sub-Basin, Gujarat, India: Insights from gas molecular ratio and stable carbon isotopic compositions

The present study investigates the molecular ratio and stable carbon isotopic compositions of gas samples from the Lower to Middle Eocene lignite deposits of the Lower to Middle Tharad Formation in the Sanchor Sub-Basin, Gujarat, India, which were understudied, hitherto. The stable carbon isotopic composition of methane (−69.30 to −57.20‰ in BK-5 and from −67.40 to −64.50‰ in BK-7 well) may imply the generation of primary microbial gas through the hydrogenotrophic methanogenesis, which was mixed with the early mature thermogenic gas, and this is a novel finding from this study area. The relation between the gas molecular ratio [methane/(ethane + propane)] and the stable carbon isotopic composition of methane confirms the mixing trend (~90–~40%) of the primary microbial methane with the early mature thermogenic gas sourced from hydrogen-rich macerals either in situ or at a higher depth that subsequently migrated upwards. Further, the studied gas samples comprise a large amount of carbon dioxide (16.95–32.32 vol.% and from 18.91 to 28.60 vol.% in BK-5 and BK-7 wells, respectively). The relation between the carbon dioxide content and its stable carbon isotopic composition (−21.30 to −11.80‰ and from −11.20 to −9.40‰ in BK-5 and BK-7 wells, respectively) may suggest the origin of carbon dioxide from volcanic degassing from the Deccan Trap basaltic magma as well as from coalification through decarboxylation and decarbonylation reactions. So, the mixed origins of carbon dioxide (inorganic + organic) and methane (primary microbial and early mature thermogenic) would have influenced the carbon isotopic difference between carbon dioxide and methane as well as the isotopic discrimination factor, which is also reported for the first time from this investigation. Finally, the relation between the gas molecular ratio and the carbon isotopic difference between carbon dioxide and methane compliments the gas mixing effects. Hence, the novelty of this investigation lies in elucidating the influences of the early mature thermogenic gas on the gas molecular and stable carbon isotopic signatures of primary microbial gas originated through the hydrogenotrophic pathway as depicted by the gas molecular ratio and stable carbon isotopic compositions of methane and carbon dioxide.

Tissue-specific Isotopic Incorporation Turnover Rates and Trophic Discrimination Factors in the Freshwater Shrimp Macrobrachium borellii (Crustacea: Decapoda: Palaemonidae).

The interpretation of isotopic data in ecology requires knowledge about two factors: turnover rate and the trophic discrimination factor, which have not been well described in freshwater shrimps. We performed a 142-day diet shift experiment on 174 individuals of the omnivorous shrimp Macrobrachium borellii, measured their growth, and temporally serially sampled muscle and hepatopancreas tissue to quantify carbon and nitrogen incorporation rates and isotope discrimination factors. Shrimps were fed with artificial diets (δ13C = -26.1‰, δ15N= 2.1‰) for 45 days in attempt to standardize the shrimps' initial δ13C and δ15N values for subsequent experiments. Shrimps were then fed with another artificial diet (δ13C = -16.1‰, δ15N = 15.8‰) and the change in δ13C and δ15N was observed for a period of 97 days. The trophic discrimination factor (∆) for δ13C was significantly higher in hepatopancreas (0.7 ± 0.36‰) than in muscle (-0.1 ± 0.83‰); however, the opposite was the case for δ15N (1.7 ± 0.43‰ and 3.6 ± 0.42‰, respectively). In the hepatopancreas the mean residence time (τ) of 13C was 26.3 ± 4.3 days compared to a residence time of 16.6 ± 5.51 days for δ15N, whereas the τ in muscle was 75.8 ± 25 days for δ13C and 40 ± 25 days for δ15N. The rate of incorporation of carbon into muscle was higher than that predicted by allometric equations relating isotopic incorporation rate to body mass that was developed previously for invertebrates. Our results support ranges of traditional trophic discrimination factor values observed in muscles samples of different taxa (∆15N around 3‒3.5‰ and ∆13C around 0‒1‰), but our work provides evidence that these traditionally used values may vary in other tissues, as we found that in the hepatopancreas ∆15N is around 1.7‰.

Stable Isotope Dynamics of Herbivorous Reef Fishes and Their Ectoparasites

Acanthurids (surgeonfishes) are an abundant and diverse group of herbivorous fishes on coral reefs. While their contribution to trophic linkages and dynamics in coral reef systems has received considerable attention, the role of linkages involving their parasites has not. As both consumers of fish tissue and prey to microcarnivores, external parasites may play a significant role in trophic transfer between primary consumers (and hence their predominantly algae-based diet) and the broader coral reef community. Stable isotope analysis is a common tool for studying trophic linkages which can be used for studies involving parasites. We examined the stable isotope ecology (13C and 15N) of copepod (Caligus atromaculatus) and monogenean (Neobenedenia sp.) ectoparasites collected from two species of Caribbean acanthurids (Acanthurus coeruleus and Acanthurus bahianus). There were significant intraspecific differences in isotope discrimination factors between parasites collected from the two different host species as well as interspecific differences between parasites collected from the same host species. Discrimination factors for 15N were consistently positive but varied in magnitude depending on host and parasite species and were slightly lower than what would be expected for consumers. The 13C discrimination factors for both monogeneans and copepods collected from A. coeruleus were consistently positive but were negative for copepods collected from A. bahianus. These findings emphasize the complexity of the stable isotope trophic interactions occurring between parasites and their hosts, highlighting the value of these types of host-parasite isotopic studies.

Isotopic discrimination between carrion and elytra clippings of lab-reared American burying beetles (Nicrophorus americanus): Implications for conservation and evaluation of feeding relationships in the wild.

Differences in stable isotope composition between an animal and its diet are quantified by experimentally derived diet-tissue discrimination factors. Appropriate discrimination factors between consumers and prey are essential for interpreting stable isotope patterns in ecological studies. While available for many taxa, these values are rarely estimated for organisms within the carrion food web. We used a controlled-diet stable isotope feeding trial to quantify isotopic diet-tissue discrimination factors of carbon (δ13 C values) and nitrogen (δ15 N values) from laboratory-reared Nicrophorus americanus raised on carrion. We used exoskeleton samples of beetle elytra (wing covers) to determine diet-tissue discrimination factors using a continuous flow isotope ratio mass spectrometer equipped with an elemental analyzer. We also measured the isotopic compositions of five species of co-occurring, wild-caught burying beetles and evaluated feeding relationships. We found differences in stable carbon discrimination between carrion sources (mammalian and avian) and lab-reared beetles, but no difference in stable nitrogen discrimination. Values for δ13 C did not differ among wild-caught burying beetle species, but values for δ15 N were significantly different for the three species with overlapping breeding seasons. Furthermore, wild-caught burying beetles within our study area do not appear to use avian carrion resources to rear their young. This study informs future interpretation of stable isotope data for insects within the carrion food web. In addition, these results provide insight into carrion resources used by co-occurring burying beetle species in situ. We also demonstrated that independent of adult food type, the larval food source has a significant impact on the isotopic signatures of adult beetles, which can be estimated using a minimally invasive elytra clipping.

Lipid normalization and stable isotope discrimination in Pacific walrus tissues

Analysis of stable carbon and nitrogen isotope values (δ13C and δ15N) of animal tissues can provide important information about diet, physiology, and movements. Interpretation of δ13C and δ15N values, however, is influenced by factors such as sample lipid content, tissue-specific isotope discrimination, and tissue turnover rates, which are typically species- and tissue-specific. In this study, we generated lipid normalization models for δ13C and investigated the effects of chemical lipid extractions on δ13C and δ15N in Pacific walrus (Odobenus rosmarus divergens) muscle, liver, and skin. We also evaluated tissue-specific isotope discrimination in walrus muscle, liver, skin, and bone collagen. Mean δ13Clipid-free of skin and bone collagen were similar, as were mean δ15N of muscle and liver. All other tissues differed significantly for both isotopes. Differences in δ13Clipid-free and δ15N among tissues agreed with published estimates of marine mammal tissue-specific isotope discrimination factors, with the exception of skin. The results of this work will allow researchers to gain a clearer understanding of walrus diet and the structure of Arctic food webs, while also making it possible to directly compare the results of contemporary walrus isotope research with those of historic and paleoecological studies.

Estimating stable isotope turnover rates of epidermal mucus and dorsal muscle for an omnivorous fish using a diet-switch experiment

Stable isotope (SI) analysis studies rely on knowledge of isotopic turnover rates and trophic-step discrimination factors. Epidermal mucus (‘mucus’) potentially provides an alternative SI ‘tissue’ to dorsal muscle that can be collected non-invasively and non-destructively. Here, a diet-switch experiment using the omnivorous fish Cyprinus carpio and plant- and fish-based formulated feeds compared SI data between mucus and muscle, including their isotopic discrimination factors and turnover rates (as functions of time T and mass G, at isotopic half-life (50) and equilibrium (95)). Mucus isotope data differed significantly and predictively from muscle data. The fastest δ13C turnover rate was for mucus in fish on the plant-based diet (T_{50}: 17 days, T_{95}: 74 days; G_{50}: 1.08(BM), G_{95}: 1.40(BM)). Muscle turnover rates were longer for the same fish (T_{50}: 44 days, T_{95}: 190 days; G_{50}: 1.13(BM), G_{95}: 1.68(BM)). Longer half-lives resulted in both tissues from the fish-based diet. δ13C discrimination factors varied by diet and tissue (plant-based: 3.11–3.28‰; fishmeal: 1.28–2.13‰). Mucus SI data did not differ between live and frozen fish. These results suggest that mucus SI half-lives provide comparable data to muscle, and can be used as a non-destructive alternative tissue in fish-based SI studies.

Amino acid isotope discrimination factors for a carnivore: physiological insightsfrom leopard sharks and their diet.

Stable isotopes are important ecological tools, because the carbon and nitrogen isotopic composition of consumer tissue reflects the diet. Measurements of isotopes of individual amino acids can disentangle the effects of consumer physiology from spatiotemporal variation in dietary isotopic values. However, this approach requires knowledge of assimilation patterns of dietary amino acids. We reared leopard sharks (Triakis semifasciata) on diets of squid (Loligo opalescens; 1250days; control sharks) or squid then tilapia (Oreochromis sp.; switched at 565days; experimental sharks) to evaluate consumer-diet discrimination factors for amino acids in muscle tissue. We found that control sharks exhibited lower nitrogen isotope discrimination factors (∆15N) than most previous consumer studies, potentially because of urea recycling. Control sharks also had large carbon isotope discrimination factors (∆13C) for three essential amino acids, suggesting microbial contributions or fractionation upon assimilation. Compared to controls, experimental sharks exhibited higher ∆13C values for four amino acids and ∆15N values for seven amino acids, corresponding with differences between diets in δ13C and δ15N values. This suggests that not all amino acids in experimental sharks had reached steady state, contrary to the conclusion of a bulk isotope study of these sharks. Our results imply that (1) the magnitude of a shift in dietary δ13C and δ15N values temporarily influences the appearance of discrimination factors; (2) slow turnover of amino acid isotopes in elasmobranch muscle precludes inferences about seasonal dietary changes; (3) elasmobranch discrimination factors for amino acids may be affected by urea recycling and microbial contributions of amino acids.

Assimilation and discrimination of hydrogen isotopes in a terrestrial mammal.

Stable isotope analysis has revolutionized the way ecologists study animal resource use from the individual to the community level. Recent interest has emerged in using hydrogen isotopes (2H/1H) as ecological tracers, because they integrate information from both abiotic and biotic processes. A better physiological understanding of how animals assimilate hydrogen and use it to synthesize tissues is needed to further refine this tool and broaden its use in animal ecology. We conducted a controlled-feeding experiment using laboratory mice (Mus musculus) in which we varied the hydrogen isotope (δ2H) values of water and the proportions of dietary protein and carbohydrates among nine experimental treatments. For each tissue, we calculated the percent of hydrogen derived from water and the percent hydrogen derived from dietary protein versus carbohydrates using linear relationships and isotope mixing models based on accompanying carbon isotope (δ13C) data. The net discrimination (∆2HNet) between mice tissues and potential water and dietary sources of hydrogen differed among tissues. ∆2HNet was positively correlated with dietary protein content in red blood cells (RBC) and muscle, but negatively correlated in liver and plasma. We also report the first estimates for hydrogen isotope discrimination factors (∆2H) for different sources of hydrogen (∆2HWater, ∆2HProtein, and ∆2HCarbs) available for tissue synthesis. This research provides a foundation for understanding how diet quality (e.g., protein content) influences hydrogen isotope assimilation and discrimination in different tissues of a terrestrial mammal, which is a first step towards using δ2H as a tracer of resource use in free-ranging mammals.

Use of isotopic enrichment to assess the relationship among dietary protein levels, growth and nitrogen retention in juvenile Totoaba macdonaldi

Stable isotope analysis was used to investigate the relationships among dietary protein levels, growth, protein utilization efficiency, and isotopic enrichment per amino acid for juvenile Totoaba macdonaldi. Four isocaloric diets were formulated to contain different protein levels (400, 430, 460 and 490 g kg−1 DM). After 60 days, growth increased proportionally as dietary protein content increased. The isotopic discrimination factor in bulk samples, revealed increases of 0.83 to 1.24‰ and 3.31 to 3.55‰ for ∆δ15N values of liver and muscle tissues, respectively, of fish fed diets containing 40% to 49% dietary protein. Compound-specific isotope analysis (CSIA) revealed that Δδ15N for Glu in the liver and Ala in the muscle tissues were enriched by up to 9.37% and 9.0%, respectively, for fish in the 49% dietary treatment, possibly due to transamination processes. Using the traditional parameters such as protein efficiency ratio (PER) failed to show significant differences. Growth of totoaba increased in conjunction with a lower retention of nitrogen according to bulk isotopic values, suggesting that proportionately more protein was being used as an energy source. Relationships among protein productivity values (PPV) corresponded well to those among bulk isotopic values. Considering the low isotopic enrichment in bulk from treatment D40, a diet containing 400 g kg−1 crude protein is recommended for use in future investigations, through the addition of each particular essential amino acids to reach the amount found in a diet contained with 49% protein. And thereafter to determine whether dietary levels of non-essential amino acids might be limiting using the CSIA for amino acids.

Incorporation of carbon and nitrogen isotopes in age-0 walleye (Sander vitreus) tissues following a laboratory diet switch experiment

Trophic dynamics are often described by following the exchange of naturally occurring isotopes through aquatic communities. However, without experimentally derived isotopic turnover rates and discrimination factors for each species, tissue, and life stage, these trophic models can be misleading. We conducted a laboratory diet shift experiment to describe isotopic turnover and discrimination in age-0 walleye (Sander vitreus) dorsal muscle and gutted carcass samples. Although turnover of dietary δ13C (half-life: 10–12 days) and δ15N (half-life: ∼13 days) signatures was relatively rapid, the diet change was undetected in both tissues during a short transitional period (up to 1.2 times shorter in muscle). Our discrimination estimates generally conform to those of other fishes (ΔCarbon= 0.91, ΔNitrogen= 1.6), but were 30%–50% higher in muscle tissues than in gutted carcass samples. The assumption that young walleye tissues are in equilibrium with their diet is untrue for weeks following a diet shift, and when incorporated, discrimination factors differ between tissues. We provide tissue-specific parameters that remove uncertainty associated with the analysis of field collected isotopic age-0 walleye data.

Stable isotope discrimination factors of omnivorous fishes: influence of tissue type, temperature, diet composition and formulated feeds

Application of stable isotope data to trophic studies requires understanding of factors influencing the isotopic discrimination factor (Δ) between consumers and their prey resources. This is missing for many omnivorous species, despite their diet and environment potentially impacting Δ. The effects of temperature, diet (including formulated feeds) and tissue type on Δ13C and Δ15N were thus tested experimentally. A temperature experiment exposed three species to identical diets at 18 and 23°C, whereas a diet experiment exposed one species to four diets at 18°C. At 23°C, C:N ratios, Δ13C and Δ15N were generally elevated versus 18°C. After lipid correction, tissue/species-specific differences at 23°C in Δ13C and Δ15N were up to 0.73 and 0.54‰ higher, respectively. Across the four diets, there were also significant differences in Δ13C and Δ15N between a natural diet and diets based on formulated feeds. Δ13C and Δ15N of muscle were 1.51 to 2.76‰ and 3.13 to 5.44‰, respectively. Highest Δ for both isotopes was from a formulated feed based on plant material that resulted in lower dietary protein content and quality. Thus, diet and environment fundamentally affected the isotopic discrimination factors and these factors require consideration within trophic studies based on stable isotopes.