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.     

Dye-laser-induced separation of nitrogen and carbon isotopes

A unimolecular, laser-induced dissociation reaction of s-tetrazine (C₂H₂N₄), with a quantum yield of close to unity, was used to separate nitrogen $\text{14}\text{15}$ and carbon $\text{12}\text{13}$ isotopes. The sharp Q-branch of the 0-0 band of the 5515 Å absorption system of tetrazine was irradiated for 1 minute periods with a 10 mW cw tunable dye laser output that was monochromatic to about 0.05 Å. Each of the decomposition products, found here to be HCN and N₂, as well as the starting material, could be altered in isotopic composition, in either direction, by selection of an appropriate laser wavelength. The largest one-step enrichment ratio measured was 72. It makes possible mass separation at an efficiency of 17 moles per kWh of laser energy.     

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.     

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.     

Fractionation of nitrogen stable isotopes by ion exchange chromatography

The ion exchange chromatography displacement technique was used for the enrichment of ¹⁵N. The runs were conducted in laboratory and bench scales using two systems of columns filled with Wofatit KPS ion exchange resin (medium porosity type). Ammonia NH₄ ⁺/NH₃ aq. was chosen as the isotopic exchange system. The ammonium bands formed in the columns were eluted by means of sodium hydroxide solution. Hydrodynamic patterns in the column beds was evaluated in terms of dimensionless Reynolds number. The results show that separation process can be performed in the turbulent or laminar flow regime.     

Serial analysis of stable nitrogen and carbon isotopes in hair: monitoring starvation and recovery phases of patients suffering from anorexia nervosa

Stable nitrogen and carbon isotopic ratios of hair strands of six patients suffering from anorexia nervosa were measured to monitor a dietary change from near starvation to recovery. This paper presents the results of a first-time study of nitrogen and carbon balance of the patients prior to and after admittance to a hospital and therapy. Sequential analysis of the isotopic ratios of hair strands of all patients could be related to the respective body mass index (BMI) of each patient. Our hypothesis concerning the diachronic change in delta15N and delta13C during therapy was met: The delta15N values were inversely related to the BMI, indicating a slow-down in catabolism of bodily protein due to the process of gluconeogenesis during the starvation phase. In contrast, the delta13C values and BMI were in phase: an increase in BMI resulted in an increase in the delta13C values. This rise in delta13C ratios is best interpreted by an increased supply of protein in the diet. Furthermore, delta15N and delta13C were inversely related. We conclude that hair, which is easily and non-traumatically sampled, is an adequate monitor that reflects dietary change and nitrogen balance within days. This isotopic method may also be applied in forensic studies with regard to cases of deprivation, and starvation, and may be a method for investigating starvation in historic populations.     

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.     

The effect of acidification on the determination of organic carbon, total nitrogen and their stable isotopic composition in algae and marine sediment

We investigated the effects of sample acidification on the stable carbon and nitrogen isotopic composition (delta13C and delta15N), as well as the organic carbon (OC) and total nitrogen (TN) composition, of an algal culture and a marine sediment. Replicate measurements of untreated and acid-treated samples were made using 1 M, 2 M and 6 M HCl, 6% H2SO3 and 1 M H3PO4. For all treatments the precision of the analysis for the acid-treated sample was equal to or less than that in the non-acidified sample. For the algae, analysis of variance (ANOVA) indicated no significant differences in the mean OC and TN concentration, or delta13C and delta15N composition, between any acid treatment and non-acidified samples. For the sediment sample a comparison could only be made between the different acid treatments because the untreated contained significant amounts ( approximately 30%) of carbonate carbon. ANOVA indicated that the mean OC determined in sediment samples after the 1 M HCl treatment and the mean delta13C values after the 6% H2SO3 and 1 M H3PO4 treatments were significantly different (p < 0.013 and < .05, respectively) from all other treatments. Mass balance calculations indicate that in some instances delta13C values were biased due to a contribution from unreacted carbonate carbon. There were no significant differences in the mean TN between any acid-treated and non-acidified samples. The mean delta15N values after 6 M HCl, 6% H2SO3 and 1 M H3PO4 treatments were significantly different from the untreated sediment sample (p < 0.044). Based on the significant bias observed for the delta15N and delta13C values, a weak (1-2 M) HCl solution is confirmed as the most appropriate acid for the removal of inorganic carbon from natural materials requiring elemental and isotopic analysis.     

Effect of preparation and preservation procedures on carbon and nitrogen stable isotope determinations from zooplankton

A literature survey of zooplankton stable isotope studies revealed inconsistencies between authors concerning (a) fixation and (b) allowance for gut clearance of zooplankton prior to delta13C and delta15N determinations. To address whether commonly used preservation techniques induce changes in stable isotope values, fresh lake zooplankton (control) were compared with preserved (ethanol, methanol, formaldehyde, gluteraldehyde, frozen and shock frozen) material. Differences of up to 1.1 per thousand for carbon and 1.5 per thousand for nitrogen isotopic signatures were found. Even freezing, the most frequently used method identified from the literature, caused significant changes compared with the control. We advocate the use of fresh material prepared immediately whenever possible, or complementary testing of the preservative method to be used. Larger organisms are routinely eviscerated, or specific tissues are dissected, and analysed for stable isotopes to reduce errors introduced via the gut contents. Yet zooplankton gut clearance is rarely performed: the gut content assumed to be negligible relative to organism mass. Experimental determinations of relative gut mass, from both original and compiled data, range from 1-26% for different zooplankton species. Using reported isotopic values of basal resources from natural systems, we calculated that, when analysing bulk zooplankton, inclusion of the gut mass may introduce substantial errors of >3 per thousand. Thus it appears prudent to perform the simple procedure of gut clearance, especially for copepod species.     

Post-photosynthetic fractionation of stable carbon isotopes between plant organs--a widespread phenomenon

Discrimination against 13C during photosynthesis is a well-characterised phenomenon. It leaves behind distinct signatures in organic matter of plants and in the atmosphere. The former is depleted in 13C, the latter is enriched during periods of preponderant photosynthetic activity of terrestrial ecosystems. The intra-annual cycle and latitudinal gradient in atmospheric 13C resulting from photosynthetic and respiratory activities of terrestrial plants have been exploited for the reconstruction of sources and sinks through deconvolution by inverse modelling. Here, we compile evidence for widespread post-photosynthetic fractionation that further modifies the isotopic signatures of individual plant organs and consequently leads to consistent differences in delta13C between plant organs. Leaves were on average 0.96 per thousand and 1.91 per thousand more depleted than roots and woody stems, respectively. This phenomenon is relevant if the isotopic signature of CO2-exchange fluxes at the ecosystem level is used for the reconstruction of individual sources and sinks. It may also modify the parameterization of inverse modelling approaches if it leads to different isotopic signatures of organic matter with different residence times within the ecosystems and to a respiratory contribution to the average difference between the isotopic composition of plant organic matter and the atmosphere. We discuss the main hypotheses that can explain the observed inter-organ differences in delta13C.