Changes in carbon and nitrogen stable isotopes of chironomid larvae during growth, starvation and metamorphosis

We conducted experiments to determine isotope changes in the deposit-feeding chironomid larvae Chironomus acerbiphilus during feeding, starvation and metamorphosis. Isotope changes in chironomid larvae occurred mainly during growth and rarely afterward. This finding indicates that chironomid isotope turnover mainly occurs in conjunction with growth and suggests that chironomid larvae only break down newly assimilated food for energy during periods of no growth. Chironomid delta(13)C values significantly increased throughout the starvation experiment, indicating that chironomids preferentially break down components with lower delta(13)C content during starvation. We found significant changes in chironomid isotope ratios ((15)N enrichment) during pupation. This evidence suggests that the physiological condition of animals (such as during an active growth phase or pre- or post-molting) is important to their stable isotope ratios.     

Metabolic turnover rates of carbon and nitrogen stable isotopes in captive juvenile snakes

Metabolic turnover rates (m) of δ¹⁵N and δ¹³C were assessed in different tissues of newly hatched captive‐raised corn snakes (Elaphe guttata guttata) fed maintenance diets consisting of earthworms (Eisenia foetida) that varied substantially in δ¹⁵N (by 644‰) and δ¹³C (by 5.0‰). Three treatments were used during this 144 day experiment that consisted of the same diet throughout (control), shifting from a depleted to an enriched stable isotope signature diet (uptake), and shifting from an enriched to depleted stable isotope signature diet (elimination). Values of δ¹³C in the liver, blood, and muscle of the control snakes reached equilibrium with and were, respectively, 1.73, 2.25 and 2.29 greater than in their diet, this increase is called an isotopic discrimination factor (Δδ¹³C = δ¹³C_snake ? δ¹³C_food). Values of δ¹⁵N in snake tissues did not achieve equilibrium with the diets in any of the exposures and thus Δ¹⁵N could not be estimated. Values of metabolic turnover rates (m) for δ¹³C and δ¹⁵N were greater in liver than in muscle and blood, which were similar, and relative results remained the same if the fraction of ¹⁵N and ¹³C were modeled. Although caution is warranted because equilibrium values of stable isotopes in the snakes were not achieved, values of m were greater for δ¹³C than δ¹⁵N, resulting in shorter times to dietary equilibrium for δ¹³C upon a diet shift, and for both stable isotopes in all tissues, greater during an elimination than in an uptake shift in diet stable isotope signature. Multiple explanations for the observed differences between uptake and elimination shifts raise new questions about the relationship between animal and diet stable isotope concentrations. Based on this study, interpretation of feeding ecology using stable isotopes is highly dependent on the kind of stable isotope, tissue, direction of diet switch (uptake versus elimination), and the growth rate of the animal. Copyright © 2009 John Wiley & Sons, Ltd.     

Fractionation and metabolic turnover of carbon and nitrogen stable isotopes in black fly larvae

Diet-tissue fractionation factors and metabolic turnover rates of delta15N and delta13C were assessed in laboratory-reared black fly (Simulium vittatum IS-7) larvae fed isotopically distinct diets. Five treatments consisted of using food with different delta15N signatures throughout the experiments (19-26 days), a sixth shifted from a low to high delta15N signature diet (uptake) on day 14, and the last shifted from a high to low delta15N signature diet (elimination) on day 14. In the larvae, diet-tissue fractionation factors for delta13C, which were in steady state with food, ranged from -0.61 to 2.0, with a median of 1.87. The delta15N diet-tissue fractionation factors were mostly negative, ranging from +2.85 to -24.96 per thousand, with a single positive value from the elimination treatment in which larval delta15N did not achieve steady state with the food. Diet-tissue fractionation factors also had a significant negative relationship (r2 = 0.98) with delta15N values in the food suggesting that nitrogen diet-tissue fractionation factors are 15N concentration-dependent. The delta15N of shed head capsules and feces were enriched in 15N and could be mechanisms for elimination of 15N by the larvae. For delta15N, metabolic turnover values based on the Hesslein model were highly consistent (0.40 to 0.43 delta15N*day(-1)) between uptake and elimination phases and across experiments and were an order of magnitude greater than growth rates. The rapid turnover of nitrogen in black fly larvae, which was orders of magnitude greater than measured in vertebrates, makes them an excellent indicator of short-term changes in nitrogen inputs to aquatic systems.     

Effects of acidification on carbon and nitrogen stable isotopes of benthic macrofauna from a tropical coral reef

Stable isotope analyses are widely used to determine trophic levels in ecological studies. We have investigated the effects of carbonate removal via acidification on the stable carbon and nitrogen isotopic composition of 33 species of tropical benthic macrofauna, and we report guidelines for standardizing this procedure for higher taxa in tropical coral reef ecosystems. Many tropical benthic invertebrates are small in size, and therefore body tissue isolation (separating organic carbon from inorganic structures) is difficult and time-consuming. Literature reviews of invertebrate studies show a lack of consistent procedures and guidelines for preparation techniques, especially for carbonate removal via acidification of whole individuals. We find that acidification decreases the delta(13)C values of samples containing carbonate, with shifts ranging from 0.21 to 3.20 per thousand, which can be related to CaCO(3) content (assessed by a carbonate proxy), justifying acid pre-treatment. Carbonate-containing taxa benefiting from acidification included Amphinomida, Terebellida (Annelida), Anomura, Brachyura, Caridea, Amphipoda, Tanaidacea (Arthropoda) and Edwardsiida (Cnidaria). The delta(13)C shifts of samples containing no carbonate varied up to 0.02 +/- 0.20 per thousand. As this induced delta(13)C shift was lower than the range of an average trophic level shift (0.5 to 1 per thousand), we conclude that acid pre-treatment is unnecessary. Carbonate-free taxa consisted of Eunicida, Phyllodocida (Annelida) and Mollusca. We note minimal impact of acidification on delta(15)N values except for Brachyura, which showed a shift of 0.83 +/- 0.46 per thousand, which is still lower than a single trophic level shift (2.9-3.8 per thousand). We conclude that for trophic level studies, both the delta(13)C and the delta(15)N of carbonate-rich macrofauna can be determined from the same acidified sample. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Effect of a controlled dietary change on carbon and nitrogen stable isotope ratios of human hair

The carbon (¹³C/¹²C) and nitrogen (¹⁵N/¹⁴N) stable isotope ratios of human hair can be used for the interpretation of dietary habits and nutritional status in contemporary or past populations. Although the results of bulk or segmental isotope ratio analysis of human hair have been used for the reconstruction of an individual's diet for years, only limited data of controlled dietary changes on the carbon and nitrogen isotopic composition of human hair are available. Hair of four individuals, two males and two females, who participated in a dietary change experiment for 28 days was segmentally analysed for δ¹³C and δ¹⁵N. The dietary change included a change from C3 to C4 plant enriched diets and a simultaneous replacement of terrestrial animal products by marine products. This resulted in an increase in δ¹³C_diet of +8.5 to +9.9‰ and in δ¹⁵N_diet of +1.5 to +2.2‰. All subjects showed significant increases in δ¹³C_hair and δ¹⁵N_hair during the dietary change period, although no subject reached a new steady state for either carbon or nitrogen. The change in δ¹⁵N_hair was faster than the change in δ¹³C_hair for all individuals. The magnitude of change of the isotopic composition during the dietary change period could be attributed to the degree of physical activity of the individuals, with a higher physical activity resulting in a faster change. Copyright © 2009 John Wiley & Sons, Ltd.     

Assessment of effects of the rising atmospheric nitrogen deposition on nitrogen uptake and long-term water-use efficiency of plants using nitrogen and carbon stable isotopes

This study assesses the effects of the atmospheric nitrogen (N) deposition on the N uptake and the long-term water-use efficiency of two C(3) plants (Agropyron cristatum and Leymus chinensis) and two C(4) plants (Amaranthus retroflexus and Setaria viridis) using N and C stable isotopes. In addition, this study explores the potential correlation between leaf N isotope (δ(15)N) values and leaf C isotope (δ(13)C) values. This experiment shows that the atmospheric N deposition has significant effects on the N uptake, δ(15)N and leaf N content (N(m)) of C(3) plants. As the atmospheric N deposition rises, the proportion and the amount of N absorbed from the simulated atmospheric deposition become higher, and the δ(15)N and N(m) of the two C(3) plants both also increase, suggesting that the rising atmospheric N deposition is beneficial for C(3) plants. However, C(4) plants display different patterns in their N uptake and in their variations of δ(15)N and N(m) from those of C(3) plants. C(4) plants absorb less N from the atmospheric deposition, and the leaf N(m) does not change with the elevated atmospheric N deposition. Photosynthetic pathways may account for the differences between C(3) and C(4) plants. This study also shows that atmospheric N deposition does not play a role in determining the δ(13)C and in the long-term water-use efficiency of C(3) and C(4) plants, suggesting that the long-term water-use pattern of the plants does not change with the atmospheric N input. In addition, this study does not observe any relationship between leaf δ(15)N and leaf δ(13)C in both C(3) and C(4) plants.     

Effect of ecosystem retrogression on stable nitrogen and carbon isotopes of plants,soils and consumer organisms in boreal forest islands

In the prolonged absence of catastrophic disturbance, ecosystem retrogression occurs, and this involves increased nutrient limitation, and reduced aboveground and belowground ecosystem processes rates. Little is known about how the nitrogen and carbon stable isotope ratios (δ¹⁵N and δ¹³C) of plants, soils and consumer organisms respond to retrogression in boreal forests. We investigated a 5000 year chronosequence of forested islands in the boreal zone of northern Sweden, for which the time since lightning‐induced wildfire increases with decreasing island size, leading to ecosystem retrogression. For this system, tissue δ¹⁵N of three abundant plant species (Betula pubescens, Vaccinium myrtillus and Pleurozium schreberi) and humus all increased as retrogression proceeded. This is probably due to enhanced ecosystem inputs of N by biological fixation, and greater dependency of the plants on organic N during retrogression. The δ¹³C of B. pubescens and plant‐derived humus also increased during retrogression, probably through nutrient limitation increasing plant physiological stress. Unlike the plants, δ¹⁵N of invertebrates (lycosid spiders and ants) did not increase during retrogression, probably because of their partial dependence on aquatic‐derived prey that had a variable δ¹⁵N signature. The δ¹³C of the invertebrates increased as retrogression proceeded and converged towards that of an aquatic prey source (chironomid flies), suggesting increased dependence on aquatic‐derived prey during retrogression. These results show that measurement of δ¹⁵N and δ¹³C of plants, soils, and consumers across the same environmental gradient can provide insights into environmental factors that drive both the aboveground and belowground subsystems, as well as the linkages between them. Copyright © 2009 John Wiley & Sons, Ltd.     

Detection of royal jelly adulteration using carbon and nitrogen stable isotope ratio analysis

Stable isotope ratios ((13)C/(12)C and (15)N/(14)N) were measured in royal jelly (RJ) samples by isotope ratio mass spectrometry (IRMS) to evaluate authenticity and adulteration. Carbon and nitrogen isotope contents (given as delta values relative to a standard, delta(13)C, delta(15)N) of RJ samples from various European origins and samples from commercial sources were analyzed. Uniform delta(13)C values from -26.7 to -24.9 per thousand were observed for authentic RJ from European origins. Values of delta(15)N ranged from -1.1 to 5.8 per thousand depending on the plant sources of nectars and pollen. High delta(13)C values of several commercial RJ samples from -20.8 to -13.3 per thousand indicated adulteration with high fructose corn syrup (HFCS) as a sugar source. Use of biotechnologically produced yeast powder as protein source for the adulterated samples was assumed as delta(15)N values were lower, as described for C(4) or CAM plant sources. RJ samples from authentic and from adulterated production were distinguished. The rapid and reliable method is suitable for urgent actual requirements in food monitoring.     

An objective means of diagnosing anorexia nervosa and bulimia nervosa using 15N/14N and 13C/12C ratios in hair

An objective means based on the carbon and nitrogen stable isotope analysis of five hairs per individual is presented for distinguishing between individuals with anorexia nervosa and/or bulimia nervosa from non-clinical individuals (i.e. clinically normal controls). Using discriminant analysis, an algorithm has been developed that provides both sensitivity and specificity of 80% in making diagnoses of individuals with these eating disorders. With further refinements, the results suggest that it may be also possible to distinguish between individuals with anorexia or bulimia. Finally, the study shows the value of conducting blind tests and using larger sample sizes of both control and treatment groups. Both groups are needed to validate the diagnostic value of a method and to provide measures of sensitivity and specificity of any diagnostic test.     

Measure of carbon and nitrogen stable isotope ratios in cultured cells

We report the measurement of the natural isotope ratios of nitrogen and carbon in subcellular volumes of individual cells among a population of cultured cells using a multi-isotope imaging mass spectrometer (MIMS), [MIMS is the prototype of the NanoSIMS 50, Cameca, France.] We also measured the nitrogen and carbon isotope ratio in cells after they had been cultured in media enriched with the amino acid glycine labeled with either 13C or 15N. The results demonstrate that 13C/12C and 15N/14N isotope ratios can be measured directly on a subcellular scale. This opens the way for the use of stable isotopes, in particular 15N, as labels to measure the intracellular turnover of biomolecules. Such a capability should help resolve a wide range of biomedical problems.