Factors that influence assimilation rates and fractionation of nitrogen and carbon stable isotopes in avian blood and feathers

By switching great skuas Catharacta skua from one isotopically distinct diet to another, we measured diet-tissue discrimination factors and tested the assumption that dietary nitrogen and carbon isotope signatures are incorporated into blood and feathers at similar rates. We also examined the effects of metabolic rate and looked for evidence of isotopic routing. We found that blood delta(15)N and delta(13)C signatures altered after the diet switch at similar rates (14.4 d and 15.7 d, respectively). Qualitative analyses imply that the same was true with feathers. Mass balance calculations suggest that only a small amount of lipid is likely to be involved in the synthesis of blood and feathers. Differences in diet-tissue discrimination factors before and after the diet switch may mean that toward the end of the experiment, some of the nutrients for blood synthesis had been coming from stores. Repeated measures mixed models provided evidence that increases in metabolic rate might accelerate fractional turnover rates in blood. There is a need for more laboratory-based experimental isotope studies in order to address further questions that this study has raised.     

Effects of elemental composition on the incorporation of dietary nitrogen and carbon isotopic signatures in an omnivorous songbird

The use of stable isotopes to infer diet requires quantifying the relationship between diet and tissues and, in particular, knowing of how quickly isotopes turnover in different tissues and how isotopic concentrations of different food components change (discriminate) when incorporated into consumer tissues. We used feeding trials with wild-caught yellow-rumped warblers (Dendroica coronata) to determine delta15N and delta13C turnover rates for blood, delta15N and delta13C diet-tissue discrimination factors, and diet-tissue relationships for blood and feathers. After 3 weeks on a common diet, 36 warblers were assigned to one of four diets differing in the relative proportion of fruit and insects. Plasma half-life estimates ranged from 0.4 to 0.7 days for delta13C and from 0.5 to 1.7 days for delta15N . Half-life did not differ among diets. Whole blood half-life for delta13C ranged from 3.9 to 6.1 days. Yellow-rumped warbler tissues were enriched relative to diet by 1.7-3.6% for nitrogen isotopes and by -1.2 to 4.3% for carbon isotopes, depending on tissue and diet. Consistent with previous studies, feathers were the most enriched and whole blood and plasma were the least enriched or, in the case of carbon, slightly depleted relative to diet. In general, tissues were more enriched relative to diet for birds on diets with high percentages of insects. For all tissues, carbon and nitrogen isotope discrimination factors increased with carbon and nitrogen concentrations of diets. The isotopic signature of plasma increased linearly with the sum of the isotopic signature of the diet and the discrimination factor. Because the isotopic signature of tissues depends on both elemental concentration and isotopic signature of the diet, attempts to reconstruct diet from stable isotope signatures require use of mixing models that incorporate elemental concentration.     

Stable‐hydrogen isotope turnover in red blood cells of two migratory thrushes: application to studies of connectivity and carry‐over effects

ABSTRACT Understanding turnover rates of stable isotopes in metabolically active tissues is critical for making spatial connections for migratory birds because samples provide information about pre‐migratory location only until the tissue turns over to reflect local values. We calculated stable‐hydrogen isotope (δ²H) turnover rate in the red blood cells of two long‐distance migratory songbirds, Bicknell's Thrushes (Catharus bicknelli) and Swainson's Thrushes (Catharus ustulatus), using samples collected at a breeding site in New Brunswick, Canada. Blood from both species captured early in the breeding site was more positive in δ²H than blood sampled later in the summer, but did not match blood values for wintering Bicknell's Thrushes. An asymptotic exponential model was used to estimate turnover of red blood cell δ²H and yielded a half‐life estimate of 21 days and 14 days for Bicknell's and Swainson's thrushes, respectively. Red blood cells of both species approached the local breeding site value one month after the first individuals were detected at the site. For Bicknell's Thrushes, estimated δ²H in blood at arrival (?72‰) was closer to blood collected at wintering sites (mean ?61‰) than to expected breeding site δ²H (?120‰). Discrimination values calculated for red blood cells collected at the breeding site for both species were greater than expected based on studies using keratin. Turnover during migration currently limits the use of blood sampled early in the breeding season for connectivity/carry‐over effect studies. However, direct tracking technology such as geolocators can provide information about migration duration, timing, and stopovers that can be used to improve isotopic turnover equations for metabolically active tissues.     

Isotopic Discrimination in the Double-Crested Cormorant (Phalacrocorax auritus)

The diet-tissue discrimination factor is the amount by which a consumer’s tissue varies isotopically from its diet, and is therefore a key element in models that use stable isotopes to estimate diet composition. In this study we measured discrimination factors in blood (whole blood, red blood cells and plasma), liver, muscle and feathers of Double-crested Cormorants (Phalacrocorax auritus) for stable isotope ratios of carbon, nitrogen and sulfur. Cormorants exhibited discrimination factors that differed significantly among tissue types (for carbon and nitrogen), and differed substantially (in the context of the isotopic variation among relevant prey species) from those observed in congeneric species. The Double-crested Cormorant has undergone rapid population expansion throughout much of its historic range over the past three decades, leading to both real and perceived conflicts with fisheries throughout North America, and this study provides an essential link for the use of stable isotope analysis in researching foraging ecology, diet, and resource use of this widespread and controversial species.     

Tissue turnover and stable isotope clocks to quantify resource shifts in anadromous rainbow trout

Stable isotopes can illuminate resource usage by organisms, but effective interpretation is predicated on laboratory validation. Here we develop stable isotope clocks to track resource shifts in anadromous rainbow trout (Oncorhynchus mykiss). We used a diet-switch experiment and model fitting to quantify N stable isotope (δ¹⁵N) turnover rates and discrimination factors for seven tissues: plasma, liver, fin, mucus, red blood cells, muscle, and scales. Among tissues, diet-tissue δ¹⁵N discrimination factors ranged from 1.3 to 3.4 ‰. Model-supported tissue turnover half-lives ranged from 9.0 (fin) to 27.7 (scale) days. We evaluated six tissue turnover models using Akaike’s information criterion corrected for small sample sizes. The use of equilibrium tissue values was supported in all tissues and two-compartment models were supported in plasma, liver, and mucus. Using parameter estimates and their uncertainty we developed stable isotope clocks to estimate the time since resource shifts. Longer turnover tissues provided accurate estimates of time since resource switch for durations approximately twice their half-life. Faster turnover tissues provided even higher precision estimates, but only within their half-life post-switch. Averaging estimates of time since resource shift from multiple tissues provided the highest precision estimates of time since resource shift for the longest duration (up to 64 days). This study therefore provides insight into physiological processes that underpin stable isotope patterns, explicitly tests alternative models, and quantifies key parameters that are the foundation of field-based stable isotope analysis.     

同位素富集-稀释法研究食性转变对鱼类不同组织N同位素转化率的影响

稳定同位素技术广泛地用于描绘生态系统中食物网的食物来源和营养级关系,但是消费者不同组织转化率的研究相对较少。通过锦鲤摄食人工添加15N蓝藻的食性转化实验,研究不同组织N同位素转化率的差异,探讨组织生长和代谢对同位素转化的相对贡献,为不同时间尺度的稳定同位素研究取样奠定基础。结果表明,通过42d的加富蓝藻饲喂,各组织的N稳定同位素发生显著变化。肝的δ15N为(19.3±1.4)‰,显著高于其它组织,其次为鱼鳍((15.6±1.0)‰)和血液((12.6±0.4)‰),肌肉的δ15N‰最低,为(9.9±0.7)‰。在随后的同位素稀释实验中,锦鲤的体重增加,相对生长速率为0.011d-1,鳍肉的转化率最快,达到11.4%/d,半衰期仅为6.1d,其次是血液和肝,肌肉的转化率最低,仅有3.8%/d,半衰期最长,为18.4d。代谢衰减指数c和-1不存在显著差异,表明锦鲤各组织的N同位素转化主要由组织生长引起。结论显示,同位素富集-稀释法可以有效评价鱼类食性转变对不同组织同位素转化的差异,鳍肉和血液同位素分析可以作为锦鲤食性转变快速追踪的手段。     

DIET-TISSUE FRACTIONATION OF STABLE CARBON AND NITROGEN ISOTOPES IN PHOCID SEALS

Diet-tissue isotopic fractionation of carbon (C) and nitrogen (N) isotopes in short- and longer-term diet integrators of diet ( i. e. , blood serum and red cells), that involve non-invasive sampling techniques was examined using three species of phocid seals (harbor seals, gray seals, and harp seals) fed a known diet. Variability in diet-tissue fractionation values within and between species was also scrutinized to determine the legitimacy of using values obtained from one species to explore trophic positions and diets of other related species. All captive seals raised on a constant diet had tissues enriched in ¹³C and ¹⁵N relative to their diet. Diet-tissue isotopic fractionation values were generally consistent among conspecifics and among phocid species for a given tissue. Trophic isotopic enrichment in ¹³C was significantly higher in red blood cells (+1.5%±) than in blood serum (+0.8%±), whereas the reverse was observed for nitrogen isotopes (+1.7%± in red cells vs . +3.1%± in serum). However, ¹³C-depleted lipids were not extracted from blood tissues in this study. This results in a downward bias in the diet-tissue fractionation factors for carbon for both red cells and blood serum, particularly the latter because of their significantly higher lipid contents ( ± SD = 14.6 ± 2.3%; n = 20; red blood cells 3.8 ± 0.9%±; n = 50, muscle 7.7 ± 2.0; n = 21) in marine mammals.     

Variability in isotope discrimination factors in coral reef fishes: implications for diet and food web reconstruction

Interpretation of stable isotope ratios of carbon and nitrogen (δ(13)C and δ(15)N) is generally based on the assumption that with each trophic level there is a constant enrichment in the heavier isotope, leading to diet-tissue discrimination factors of 3.4‰ for (15)N (ΔN) and ～0.5‰ for (13)C (ΔC). Diet-tissue discrimination factors determined from paired tissue and gut samples taken from 152 individuals from 26 fish species at Ningaloo Reef, Western Australia demonstrate a large amount of variability around constant values. While caution is necessary in using gut contents to represent diet due to the potential for high temporal variability, there were significant effects of trophic position and season that may also lead to variability in ΔN under natural conditions. Nitrogen enrichment increased significantly at higher trophic levels (higher tissue δ(15)N), with significantly higher ΔN in carnivorous species. Changes in diet led to significant changes in ΔN, but not tissue δ(15)N, between seasons for several species: Acanthurus triostegus, Chromis viridis, Parupeneus signatus and Pomacentrus moluccensis. These results confirm that the use of meta-analysis averages for ΔN is likely to be inappropriate for accurately determining diets and trophic relationships using tissue stable isotope ratios. Where feasible, discrimination factors should be directly quantified for each species and trophic link in question, acknowledging the potential for significant variation away from meta-analysis averages and, perhaps, controlled laboratory diets and conditions.     

Stable carbon and nitrogen isotopic discrimination in whole blood of red-throated ant tanagers Habia fuscicauda

Analysis of stable isotope ratios is increasingly used to reconstruct diets in passerine birds, but studies of diet–tissue isotopic discrimination for this avian group are scarce. We determined ¹⁵N and ¹³C diet–tissue discrimination factors on whole blood in the red-throated ant tanager (Habia fuscicauda), an insectivorous–frugivorous passerine. Birds were fed an isotopically uniform, semi-synthetic diet of dog puppy dry food, soy protein isolate, wheat germ, and other ingredients, during 92 days. Average (± SD) diet–tissue discrimination was 2.6 ± 0.2‰ for N and 2.2 ± 0.1‰ for C. Nitrogen diet-tissue discrimination was similar to the values found previously in other passerines fed animal protein and it can probably be used to accurately reconstruct protein dietary origin in passerines feeding on animal protein (e.g., insects). In the case of C, diet reconstruction might be affected by metabolic routing of dietary nutrients.     

Dietary macronutrients influence 13C and 15N signatures of pinnipeds: captive feeding studies with harbor seals (Phoca vitulina)

Metabolic effects of dietary macronutrients on diet-tissue isotopic discrimination factors were investigated in harbor seals. Three seals were fed either high fat/low protein herring (H), or low fat/high protein pollock (P), and switched to the alternative every 4 months. This allowed each seal to be subjected to two dietary treatments in each of three metabolically defined seasons (breeding from May to September, molting from September to January, and late winter/early spring period from January to May) over a 2 year cycle, and function as its internal control regardless of physiological changes over season. One seal was fed a constant equal mix of H and P over the entire trial. Up to 1 per thousand differences in serum delta15N values of one seal fed alternatively on H and P were observed. Progressively more enriched serum delta15N values as diet switching from H to P might link to changes in seal digestive physiology and protein metabolism in response to very high protein intake on P diet. These findings demonstrate that dietary macronutrients of prey species and protein intake level of consumers also play important roles in shaping isotopic patterns of a consumer's tissues, and thus influence accurate data interpretation of stable isotope techniques in ecological applications.     

Fractionation and turnover of stable carbon isotopes in animal tissues: Implications for δ13C analysis of diet

The use of stable carbon isotopes as a means of studying energy flow is increasing in ecology and paleoecology. However, secondary fractionation and turnover of stable isotopes in animals are poorly understood processes. This study shows that tissues of the gerbil (Meriones unguienlatus) have different δ¹³C values when equilibrated on corn (C₄) or wheat (C₃) diets with constant ¹³C/¹²C contents. Lipids were depleted 3.0‰ and hair was enriched 1.0‰ relative to the C₄ diet. Tissue δ¹³C values were ranked hair>brain>muscle>liver>fat. After changing the gerbils to a wheat (C₃) diet, isotope ratios of the tissues shifted in the direction of the δ¹³C value of the new diet. The rate at which carbon derived from the corn diet was replaced by carbon derived from the wheat diet was adequately described by a negative exponential decay model for all tissues examined. More metabolically active tissues such as liver and fat had more rapid turnover rates than less metabolically active tissues such as hair. The half-life for carbon ranged from 6.4 days in liver to 47.5 days in hair. The results of this study have important implications for the use of δ¹³C values as indicators of animal diet. Both fractionation and turnover of stable carbon isotopes in animal tissues may obscure the relative contributions of isotopically distinct dietary components (such as C₃ vs. C₄, or marine vs. terrestrial) if an animal's diet varies through time. These complications deserve attention in any study using stable isotope ratios of animal tissue as dietary indicators and might be minimized by analysis of several tissues or products covering a range of turnover times.     

Variability in stable isotopes of snowy owl feathers and contribution of marine resources to their winter diet

The snowy owl is an elusive arctic predator known for its nomadic behaviour. Satellite tracking has revealed that some adult snowy owls could make an extensive use of the marine environment during the non‐breeding season. However, the relative contribution of marine resources to their diet is unknown. Stable isotope analyses can be useful to document the diet of mobile animals during periods of the year when individuals are less accessible. This study aimed to assess variation in isotopic values (δ¹³C and δ¹⁵N) of various feather types, and the usefulness of feathers to determine the contribution of the marine environment to the winter diet of snowy owls captured in summer. We sampled feathers coming from 6 body regions of 18 breeding females at two sites in the eastern Canadian Arctic in 2013 and 2014. Prior to analyses, diet‐tissue discrimination factors of snowy owl feathers were established in captivity. Variability in isotopic values among feather types was relatively low and pairwise correlations in isotopic values between feathers on the same individual were variable and often low, which suggests differences in the diet at the time when various feathers were synthesized. Diet reconstruction models detected a contribution of marine sources to snowy owl feathers ranging from 4 to 19% among feather types. However, the marine contribution was highly variable when single feathers were examined within individuals, ranging from 3 to 71%. This indicated that no single feather type could be used alone to reliably infer the contribution of marine resources to the winter diet of owls, possibly due to a high variability in the timing and sequence of molt. For asynchronous molters like snowy owls, we recommend sampling multiple feathers from various body regions, excluding wing feathers, to investigate winter diet or habitat use.     

Tissue carbon incorporation rates and diet-to-tissue discrimination in ectotherms: tortoises are really slow

Abstract Understanding carbon incorporation rates and diet-to-tissue discrimination (Δ(13)C(tissue-diet)) in animals is necessary to interpret stable isotope data collected from animals in the field. Our current understanding of the carbon dynamics in terrestrial ectotherms such as snakes, lizards, and turtles is poorly developed. Here we use a diet switch experiment to estimate carbon incorporation rates and diet-to-tissue discrimination factors in growing desert tortoises (Gopherus agassizii). Average carbon retention times for red blood cells (RBCs) and plasma were 126.7 ± 40.3 and 32.9 ± 14.5 days, respectively. Tissue carbon incorporation rates were affected by both growth and metabolism, with growth accounting for 50% of the carbon turnover in RBCs and 13% of carbon turnover in plasma. At equilibrium, scute keratin (0.8 ± 0.1) and plasma (1.0 ± 0.2) showed enriched discrimination values (Δ(13)C) compared to the test diet, but RBC Δ(13)C values were indistinguishable from diet (0.2 ± 0.3). We also found that new keratin continued to contribute significant material to previously grown keratin rings on the tortoise's shell. Changes in the δ(13)C of previously laid down growth rings indicated that the old rings closest to the region of new growth received about 73% of the carbon from the current diet; these data suggest that the interpretation of dietary history using growth rings must recognize that each ring may represent the weighted average of the diet over several seasons. These results continue to highlight the importance of laboratory experiments in interpreting isotopic data derived from field studies.     

Inherent variation in stable isotope values and discrimination factors in two life stages of green turtles

Abstract We examine inherent variation in carbon and nitrogen stable isotope values of multiple soft tissues from a population of captive green turtles Chelonia mydas to determine the extent of isotopic variation due to individual differences in physiology. We compare the measured inherent variation in the captive population with the isotopic variation observed in a wild population of juvenile green turtles. Additionally, we measure diet-tissue discrimination factors to determine the offset that occurs between isotope values of the food source and four green turtle tissues. Tissue samples (epidermis, dermis, serum, and red blood cells) were collected from captive green turtles in two life stages (40 large juveniles and 30 adults) at the Cayman Turtle Farm, Grand Cayman, and analyzed for carbon and nitrogen stable isotopes. Multivariate normal models were fit to the isotope data, and the Bayesian Information Criterion was used for model selection. Inherent variation and discrimination factors differed among tissues and life stages. Inherent variation was found to make up a small portion of the isotopic variation measured in a wild population. Discrimination factors not only are tissue and life stage dependent but also appear to vary with diet and sea turtle species, thus highlighting the need for appropriate discrimination factors in dietary reconstructions and trophic-level estimations. Our measures of inherent variation will also be informative in field studies employing stable isotope analysis so that differences in diet or habitat are more accurately identified.     

Turnover rates of nitrogen stable isotopes in the salt marsh mummichog, Fundulus heteroclitus, following a laboratory diet switch

Nitrogen stable isotopes are frequently used in ecological studies to estimate trophic position and determine movement patterns. Knowledge of tissue-specific turnover and nitrogen discrimination for the study organisms is important for accurate interpretation of isotopic data. We measured δ¹⁵ N turnover in liver and muscle tissue in juvenile mummichogs, Fundulus heteroclitus, following a laboratory diet switch. Liver tissue turned over significantly faster than muscle tissue suggesting the potential for a multiple tissue stable isotope approach to study movement and trophic position over different time scales; metabolism contributed significantly to isotopic turnover for both liver and muscle. Nitrogen diet-tissue discrimination was estimated at between 0.0 and 1.2‰ for liver and –1.0 and 0.2‰ for muscle. This is the first experiment to demonstrate a significant variation in δ¹⁵ N turnover between liver and muscle tissues in a fish species.     

Diet: tissue stable isotope fractionation of carbon and nitrogen in blood plasma and whole blood of male reindeer Rangifer tarandus

Estimates of diet derived from stable isotope analyses are sensitive to the accuracy of corrections made for diet-tissue fractionation. In particular, diet-tissue fractionation in reindeer Rangifer tarandus may be expected to differ significantly from the generic values often used in stable isotope dietary calculations, given the known values obtained from other ungulates and the complex digestive system and nutrient recycling characteristic of the species. We fed domestic reindeer a homogenous artificial diet of known isotopic value in order to directly determine diet-tissue isotopic fractionation of carbon and nitrogen, the main elements used in stable isotope dietary analyses. Diet-tissue fractionation values for blood plasma were +3.5 ± 0.1‰ (δ¹³C) and +4.2 ± 0.3‰ (δ¹⁵N). Diet-tissue fractionation values for whole blood were +3.7 ± 0.2‰ (δ¹³C) and +2.5 ± 0.3‰ (δ¹⁵N). Metabolic turnover rates were clearly sufficient for complete tissue replacement over the period of artificial feeding for blood plasma, but may not have been so for whole blood, especially for δ¹⁵N. Our values, except for whole blood δ¹⁵N, differ considerably from the generic values often used in dietary studies and interspecific comparisons of trophic niche. The results underline the importance of obtaining as specific as possible fractionation values for the species, tissue, and in some cases sex and physiological status of animals under examination, and the potential problems associated with assuming ‘generic’ fractionation values when comparing species, especially where digestive processes are dissimilar.     

Carotenoid pigments and the selectivity of psittacofulvin-based coloration systems in parrots

Carotenoid pigments are commonly used as colorants of feathers and bare parts by birds. However, parrots (Aves: Psittaciformes) use a novel class of plumage pigments (called psittacofulvins) that, like carotenoids, are lipid-soluble and red, orange, or yellow in color. To begin to understand how and why parrots use these pigments and not carotenoids in their feathers, we must first describe the distribution of these two types of pigments in the diet, tissues, and fluids of these birds. Here, we studied the carotenoid content of blood in five species of parrots with red in their plumage to see if they show the physiological ability to accumulate carotenoids in the body. Although Scarlet (Ara macao) and Greenwing Macaws (Ara chloroptera) and Eclectus (Eclectus roratus), African Gray (Psittacus erithacus) and Blue-fronted Amazon (Amazona aestiva) Parrots all use psittacofulvins to color their feathers red, we found that they also circulated high concentrations of both dietary (lutein, zeaxanthin, beta-cryptoxanthin) and metabolically derived (anhydrolutein, dehydrolutein) carotenoids through blood at the time of feather growth, at levels comparable to those found in many other carotenoid-colored birds. These results suggest that parrots have the potential to use carotenoids for plumage pigmentation, but preferentially avoid depositing them in feathers, which is likely under the control of the maturing feather follicle. As there is no evidence of psittacofulvins in parrot blood at the tune of feather growth, we presume that these pigments are locally synthesized by growing feathers within the follicular tissue.     

Feather isotope analysis discriminates age-classes of Western, Least, and Semipalmated sandpipers when plumage methods are unreliable

ABSTRACT Avian age‐class discrimination is typically based on the completeness of the first prebasic molt. In several calidrid sandpiper species, juvenal flight feathers grown on Arctic breeding grounds are retained through the first three migrations. Thereafter, flight feathers are grown annually at temperate migratory stopover sites during the fall or on the subtropical wintering grounds. Standard methods for distinguishing age classes of sandpipers rely on a combination of traits, including body plumage, coloration of protected inner median covert edges, and extent of flight feather wear. We tested the ability of stable hydrogen isotope ratios in flight feathers (δD_f) to distinguish young birds in their first winter through second fall from older adults in three calidrid sandpiper species, Western (Calidris mauri), Least (C. minutilla), and Semipalmated (C. pusilla) sandpipers. We compared the apparent reliability of the isotope approach to that of plumage‐based aging. The large expected differences in δD_f values of flight feathers grown at Arctic versus non‐Arctic latitudes enabled use of this technique to discriminate between age‐classes. We determined δD_f values of known Arctic‐grown feathers from juveniles that grew their flight feathers on the breeding grounds. Flight feather δD_f values of southward‐migrating adults showed bimodal distributions for all three species. Negative values overlapped with species‐specific juvenile values, identifying putative second fall birds with high‐latitude grown juvenal feathers retained from the previous year. The more positive values identified older adults who grew their feathers at mid‐ and low latitudes. Importantly, δD_f analysis successfully identified first‐winter and second‐fall birds not detected by plumage‐based aging. Flight feather wear alone was a poor basis for age classification because scores overlapped extensively between putative second fall birds and older adults. Flight feather hydrogen isotope analysis enables more definitive assignment of age classes when standard plumage methods are unreliable.     

Reply to Hussey et al.: The requirement for accurate diet-tissue discrimination factors for interpreting stable isotopes in sharks

Carbon and nitrogen stable isotope analyses have improved our understanding of food webs and movement patterns of aquatic organisms. These techniques have recently been applied to diet studies of elasmobranch fishes, but isotope turnover rates and isotope diet–tissue discrimination are still poorly understood for this group. We performed a diet switch experiment on captive sandbar sharks (Carcharhinus plumbeus) as a model shark species to determine tissue turnover rates for liver, whole blood, and white muscle. In a second experiment, we subjected captive coastal skates (Leucoraja spp.) to serial salinity reductions to measure possible impacts of tissue urea content on nitrogen stable isotope values. We extracted urea from spiny dogfish (Squalus acanthias) white muscle to test for effects on nitrogen stable isotopes. Isotope turnover was slow for shark tissues and similar to previously published estimates for stingrays and teleost fishes with low growth rates. Muscle isotope data would likely fail to capture seasonal migrations or diet switches in sharks, while liver and whole blood would more closely reflect shorter term movement or shifts in diet. Nitrogen stable isotope values of skate blood and skate and dogfish white muscle were not affected by tissue urea content, suggesting that available diet–tissue discrimination estimates for teleost fishes with similar physiologies would provide accurate estimates for elasmobranchs.     

Using hydrogen isotopes of freshwater fish tissue as a tracer of provenance

Hydrogen isotope (δ²H) measurements of consumer tissues in aquatic food webs are useful tracers of diet and provenance and may be combined with δ¹³C and δ¹⁵N analyses to evaluate complex trophic relationships in aquatic systems. However, δ²H measurements of organic tissues are complicated by analytical issues (e.g., H exchangeability, lack of matrix‐equivalent calibration standards, and lipid effects) and physiological mechanisms, such as H isotopic exchange with ambient water during protein synthesis and the influence of metabolic water. In this study, δ²H (and δ¹⁵N) values were obtained from fish muscle samples from Lake Winnipeg, Canada, 2007–2010, and were assessed for the effects of species, feeding habits, and ambient water δ²H values. After lipid removal, we used comparative equilibration to calibrate muscle δ²H values to nonexchangeable δ²H equivalents and controlled for H isotopic exchange between sample and laboratory ambient water vapor. We then examined the data for evidence of trophic δ²H enrichment by comparing δ²H values with δ¹⁵N values. Our results showed a significant logarithmic correlation between fork length and δ²H values, and no strong relationships between δ¹⁵N and δ²H. This suggests the so‐called apparent trophic compounding effect and the influence of metabolic water into tissue H were the potential mechanisms for δ²H enrichment. We evaluated the importance of water in controlling δ²H values of fish tissues and, consequently, the potential of H isotopes as a tracer of provenance by taking account of confounding variables such as body size and trophic effects. The δ²H values of fish appear to be a good tracer for tracking provenance, and we present a protocol for the use of H isotopes in aquatic ecosystems, which should be applicable to a broad range of marine and freshwater fish species. We advise assessing size effects or working with fish of relatively similar mass when inferring fish movements using δ²H measurements.