Diet–consumer nitrogen isotope fractionation for prolonged fasting arthropods

Nitrogen acquisition for cellular metabolism during diapause is a primary concern for herbivorous arthropods. Analyses of naturally occurring stable isotopes of nitrogen help elucidate the mechanism. Relevant articles have cited (58 times up to mid-June 2011) anomalously elevated δ¹⁵N (per mil deviation of ¹⁵N/¹⁴N, relative to atmospheric nitrogen=0 ‰) values (diet–consumer nitrogen isotope fractionation; up to 12 ‰) for a prolonged fasting raspberry beetle (Byturus tomentosus Degeer (Coleoptera: Byturidae)), which feeds on red raspberries (Rubus idaeus: δ¹⁵N=～+2 ‰). Biologists have hypothesised that extensive recycling of amino acid nitrogen is responsible for the prolonged fasting. Since this hypothesis was proposed in 1995, scientists have integrated biochemical and molecular knowledge to support the mechanism of prolonged diapausing of animals. To test the validity of the recycling hypothesis, we analysed tissue nitrogen isotope ratios for four Japanese arthropods: the shield bug Parastrachia japonensis Scott (Hemiptera: Cydnidae), the burrower bug Canthophorus niveimarginatus Scott (Hemiptera: Cydnidae), leaf beetle Gastrophysa atrocyanea Motschulsky (Coleoptera: Chrysomelidae) and the Japanese oak silkworm Antheraea yamamai (Lepidoptera: Saturniidae), all of which fast for more than 6 months as part of their life-history strategy. Resulting diet–consumer nitrogen isotope discrimination during fasting ranged from 0 to 7‰, as in many commonly known terrestrial arthropods. We conclude that prolonged fasting of arthropods does not always result in anomalous diet–consumer nitrogen isotope fractionation, since the recycling process is closed or nearly closed with respect to nitrogen isotopes.     

Seasonal variation in natural abundance of δ13C and 15N in Salicornia brachiata Roxb. populations from a coastal area of India

High and fluctuating salinity is characteristic for coastal salt marshes, which strongly affect the physiology of halophytes consequently resulting in changes in stable isotope distribution. The natural abundance of stable isotopes (δ¹³C and δ¹⁵N) of the halophyte plant Salicornia brachiata and physico-chemical characteristics of soils were analysed in order to investigate the relationship of stable isotope distribution in different populations in a growing period in the coastal area of Gujarat, India. Aboveground and belowground biomass of S. brachiata was collected from six different populations at five times (September 2014, November 2014, January 2015, March 2015 and May 2015). The δ¹³C values in aboveground (?30.8 to ?23.6?‰, average: ?26.6?±?0.4?‰) and belowground biomass (?30.0 to ?23.1?‰, average: ?26.3?±?0.4?‰) were similar. The δ¹³C values were positively correlated with soil salinity and Na concentration, and negatively correlated with soil mineral nitrogen. The δ¹⁵N values of aboveground (6.7–16.1?‰, average: 9.6?±?0.4?‰) were comparatively higher than belowground biomass (5.4–13.2?‰, average: 7.8?±?0.3?‰). The δ¹⁵N values were negatively correlated with soil available P. We conclude that the variation in δ¹³C values of S. brachiata was possibly caused by soil salinity (associated Na content) and N limitation which demonstrates the potential of δ¹³C as an indicator of stress in plants.     

13C and 15N Abundances in the Sediment Core (VER 92/1-St-10-GC2) from Northern Lake Baikal

Abstract

Carbon and nitrogen stable isotope compositions of organic matter, TOC/TN ratio, and manganese concentration in a sediment core that was collected in northern part of Lake Baikal (VER92ST10-GC2, water depth at 922 m, about 3 m long) were investigated to elucidate the origin of the sedimentary organic matter and its associated environmental factors.

The sediment core was composed of mainly two parts: turbidite sections and other sections. Constant δ¹³C and δ¹⁵N values of the turbidite sections were observed (- 26.8 ±0.2 ‰ for δ¹³C and 3.2 ± 0.1 ‰ for δ¹⁵N) throughout the core. The higher δ¹³C in turbidite sections (about - 27 ‰) than that of the other sections (- 31 to - 29 ‰) was clearly observed, and δ¹⁵N was different between turbidite sections (about 3‰) and other sections (3 to 5 ‰). δ¹³C of other sections was close to that of pelagic phytoplankton, indicating that sediment other than turbidite sections is composed of autochthonous components. The variation of stable isotopes in other sections may be possibly caused by the changes in either phytoplankton growth rate or contribution ratios of terrestrial to aquatic plants for δ¹³C. Either denitrification or fluctuation of δ¹⁵N in pelagic phytoplankton can be the cause of variable δ¹⁵N in other sections.     

The Effect of Diet Quality on δ(13)C and δ(15)N in the Tissues of Locusts, Locusta migratoria L

Abstract Locust nymphs were raised from hatching to adult locusts on either seedling wheat (C(3)) or maize (C(4)), to determine whether relative enrichments/depletions of (15)N and (13)C within body tissues are influenced by diet. The maize contained less hexose sugars and protein per gram than wheat. The isotopic spacing between the food and the whole insect was found to differ between the two diets. The lower quality maize diet showed an overall +5.1‰ enrichment in δ(15)N compared to + 2.8‰ for wheat, possibly due to increased fractionation due to protein recycling. The maize diet resulted in increased depletion in lipid and trehalose and depletion in chitin relative to diet. The results for both δ(15)N and δ(13)C suggest that substrate recycling was occurring on the low quality maize diet. Therefore diet quality determines the enrichment/depletion in δ(15)N and δ(13)C within organisms.     

Estimates of dietary overlap for six species of Amazonian manakin birds using stable isotopes

We used stable isotope ratios to determine the metabolic routing fraction of carbon and nitrogen in feathers in addition to faecal analysis to estimate diet overlap of six sympatric species of manakins in the eastern lowland forest of Ecuador. Collectively, all species varied from?23.7 to?32.7 ‰ for δ¹³C, and from 6.0 to 9.9‰ for δ¹⁵N, with Machaeropterus regulus showing isotopic differences from the other species. We developed a mixing model that explicitly addresses the routing of carbon and nitrogen to feathers. Interestingly, these results suggest a higher proportion of nitrogen and carbon derived from insects than anticipated based on feeding observations and faecal analysis. A concentration-dependent mixing isotopic model was also used to look at dietary proportions. While larvae and arachnids had higher δ¹⁵N values, these two groups may also be preferred prey of manakins and may be more assimilated into tissues, leading to a potential overestimation of the contribution to diet. This study supports the finding that manakin species, previously thought be primarily frugivorous, contain a significant amount of arthropods in their diet.     

Feeding and Growth of Apple Snail Pomacea lineata in the Pantanal Wetland,Brazil—a Stable Isotope Approach

Abstract

Apple snails Pomacea lineata (SPIX 1827) are widespread in the tropical regions of Brazil as well as in the Pantanal wetland of Mato Grosso in the western part of the country. They have a key position in the Pantanal food web and serve as food for many animals e.g. fishes, birds, and caimans. However, little is known about their feeding preferences and growth rates. Stable isotopes have been used successfully on numerous studies as food source indicator. Therefore, the δ¹⁵N and δ¹³C values of snails from 0.45 to 3.03cm in length, which were collected in the rainy season from March through May, were analyzed. Snails signatures revealed ambiguous evidence for food preferences. δ¹⁵N and δ¹³C values ranged between ?2.8 and 12.4‰ and between ?24.2 and ?16.4‰, respectively. This range of values mirrors the highly variable isotope values of possible food sources comprising C₃ and C₄ macrophytes. To test whether all common food sources were similarly assimilated, feeding experiments with different diets were conducted. Snail eggs were reared in tanks and offered different but single plants. Snails fed different diets and δ¹³C values of the food were reflected in the animal tissue. Growth varied considerably in experiments with different diets indicating the preference for certain food sources. Also, the fractionation of nitrogen isotopes between food and animal varied from 0.1 to 17.0‰. The results are explained by different feeding habits, and it is supposed that animals fed either on the plant itself or on bacteria mats growing in the tanks. In an additional experiment juvenile snails were offered one single food with a distinctive C₄ grass signature. These snails did not grow detectably, but nevertheless isotope signatures approached to values of the diet.     

Temporal diet changes recorded by stable isotopes in Asiatic black bear (Ursus thibetanus) hair

Carbon and nitrogen stable isotope ratios were measured in hair samples of the Asiatic black bear (Ursus thibetanus) inhabiting the Northern Japanese Alps (NJA) (n?=?20) and the periphery of Nagano City (NC) (n?=?6), in Nagano Prefecture, Japan. The hair of NJA bears, which did not have access to anthropogenic foods, showed lower values of δ¹³C and δ¹⁵N than that of NC bears which had access to garbage and corn fields, especially during the summer. These results reflect somewhat differing diets between the NJA and NC bears. We attempted to assess the feeding history during the hair growth cycle using the growth section analysis method. Each hair sample had been cut into 3?mm lengths from root to tip, labeled, and analyzed along the hair growth. We measured the carbon and nitrogen stable isotope ratios of each 3?mm length of hair sample from one NC bear which had been killed while raiding a corn field. The sections showed wide ranges of isotope ratios, from ?23.2‰ to ?14.6‰ for δ¹³C, and from 0.3‰ to 4.6‰ for δ¹⁵N. It was shown that the diet of this bear shifted dramatically from principally C₃ plants to more C₄ plants and to foods of animal origin. An analysis of the whole hair reflects just the average feeding habit during hair growth, but the present method can trace its diet history. This method can contribute to obtain precise ecological information of wildlife.     

The Effect of Diet Quality on δ13C and δ15N in the Tissues of Locusts,Locusta migratoria L

Abstract

Locust nymphs were raised from hatching to adult locusts on either seedling wheat (C₃) or maize (C₄), to determine whether relative enrichments/depletions of ¹⁵N and ¹³C within body tissues are influenced by diet. The maize contained less hexose sugars and protein per gram than wheat.

The isotopic spacing between the food and the whole insect was found to differ between the two diets. The lower quality maize diet showed an overall +5.1‰ enrichment in δ¹⁵N compared to + 2.8‰ for wheat, possibly due to increased fractionation due to protein recycling.

The maize diet resulted in increased depletion in lipid and trehalose and depletion in chitin relative to diet. The results for both δ¹⁵N and δ¹³C suggest that substrate recycling was occurring on the low quality maize diet. Therefore diet quality determines the enrichment/depletion in δ¹⁵N and δ¹³C within organisms.     

Carbon-13 Kinetic Isotope Effects in the Decarbonylation of Liquid Formic Acid Assisted with Acetic Anhydride

Abstract

We evaluated the use of δ¹⁵N- and δ¹³C-values to monitor the development of food web complexity and biodiversity in a regenerating ecosystem. Therefore a model food chain was established feeding cultivated woodlice (Porcellio dilatatus) on a cellulolytic fungus (Chaetomium globosum) grown on cellulose paper. Two diets of different quality (C:N ratios of 54 vs. 200) with different δ¹⁵N- (1.3‰ vs. 3.1‰) but identical δ¹³C-values caused low and high dietary stress in animals of treatment A and B, respectively. After an incubation time of 7 weeks amount, elemental and isotopic composition of collected faeces and exuviae as well as woodlice and remaining food were determined.

The increase of δ¹⁵N-values of woodlice relative to the diet was 5.7‰ and 2.5‰ in treatments A and B, respectively, whereas δ¹³C-shifts were 1.0‰ and 1.6‰, showing a reverse relationship. Modelling of elemental and isotopic mass balances indicated that faeces recycling explains the unexpected high ¹⁵N-enrichments. Moreover, ¹³C-enrichments were positively correlated to the degree of starvation. Considering the effects of starvation and recycling of faeces, stable isotopes represent a useful tool to elucidate trophic interactions in regenerating food webs.     

Nitrogen Isotope Ratios in Different Compartments of a Mixed Stand of Spruce,Larch and Beech Trees and of Understorey Vegetation Including Fungi

Abstract

Natural nitrogen isotope ratios were measured in different compartments (needles or leaves and twigs of different age classes and crown positions, roots and soil of different horizons) of spruce (Picea abies), larch (Larix decidua) and beech (Fagus sylvatica) trees in an 11-year-old mixed stand in the Fichtelgebirge, NE Bavaria, Germany. In addition, samples of understorey vegetation (mainly ericaceous shrubs and grass) and of ectomycorrhizal and saprophytic fungi were analyzed. The δ¹⁵N values found for all samples ranged between ?7.5 and + 4.5‰. No significant differences were found for the nitrogen isotope ratios of the three tree species despite of their evergreen versus deciduous foliage and despite of their different rooting depth. Ericaceous shrubs had the most negative and fungi and soil from the mineral horizon the most positive δ¹⁵N values. Positive δ¹⁵N values of the fungi indicate their ability to utilize organic soil nitrogen, but the data do not unequivocally show that plants forming mycorrhizas profit from this organic nitrogen source.     

Food webs in Mongolian grasslands: The analysis of 13C and 15N natural abundances

Overgrazing often lowers species richness and productivity of grassland communities. For Mongolian grassland ecosystems, a lack of detailed information about food-web structures makes it difficult to predict the effects of overgrazing on species diversity and community composition. We analysed the δ¹³C and δ¹⁵N signatures of herbaceous plants, arthropods (grouped by feeding habit), wild and domestic mammals, and humans in central Mongolia to understand the predominant food-web pathways in this grassland ecosystem. The δ¹³C and δ¹⁵N values of mammals showed little variation within species, but varied considerably with slope position for arthropods. The apparent isotopic discrimination between body tissue and hair of mammals was estimated as 2.0 ‰ for δ¹³C and 2.1 ‰ for δ¹⁵N, which was large enough to cause overestimation of the trophic level of mammals if not taken into account when using hair samples to measure isotopic enrichment.     

Biological fractionation of stable Ca isotopes in Göttingen minipigs as a physiological model for Ca homeostasis in humans

In order to investigate fractionation of calcium (Ca) isotopes in vertebrates as a diagnostic tool to detect Ca metabolism dysfunction we analyzed the Ca isotopic composition (δ^44/40Ca?=?[(⁴⁴Ca/⁴⁰Ca)_sample/(⁴⁴Ca/⁴⁰Ca)_reference]?1) of diet, faeces, blood, bones and urine from Göttingen minipigs, an animal model for human physiology. Samples of three groups were investigated: 1. control group (Con), 2. group with glucocorticosteroid induced osteoporosis (GIO) and 3. group with Ca and vitamin D deficiency induced osteomalacia (?CaD). In contrast to Con and GIO whose average δ^44/40Ca_faeces values (0.39?±?0.13‰ and 0.28?±?0.08‰, respectively) tend to be lower than their diet (0.47?±?0.02‰), δ^44/40Ca_faeces of ?CaD (?0.27?±?0.21‰) was significantly lower than their δ^44/40Ca_diet (0.37?±?0.03‰), but also lower than δ^44/40Ca_faeces of Con and GIO. We suggest that the low δ^44/40Ca_faeces of ?CaD might be due to the contribution of isotopically light Ca from gastrointestinal fluids during gut passage. Assuming that this endogenous Ca source is a common physiologic feature, a fractionation during Ca absorption is also required for explaining δ^44/40Ca_faeces of Con and GIO. The δ^44/40Ca_urine of all groups are high (>2.0‰) reflecting preferential renal reabsorption of light Ca isotopes. In Göttingen minipigs we found a Ca isotope fractionation between blood and bones (Δ^44/40Ca_blood-bone) of 0.68?±?0.15‰.     

Sample preparation techniques for the determination of natural 15N/14N variations in amino acids by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS)

In order to identify natural nitrogen isotope variations of biologically important amino acids four derivatization reactions (t-butylmethylsilylation, esterification with subsequent trifluoroacetylation, acetylation and pivaloylation) were tested with standard mixtures of 17 proteinogenic amino acids and plant (moss) samples using GC-C-IRMS. The possible fractionation of the nitrogen isotopes, caused for instance by the formation of multiple reaction products, was investigated. For biological samples, the esterification of the amino acids with subsequent trifluoroacetylation is recommended for nitrogen isotope ratio analysis. A sample preparation technique is described for the isotope ratio mass spectrometric analysis of amino acids from the non-protein (NPN) fraction of terrestrial moss. 14N/15N ratios from moss (Scleropodium spec.) samples from different anthropogenically polluted areas were studied with respect to ecotoxicologal bioindication.     

Latitudinal distribution of nitrogen isotopic composition in suspended particulate organic matter in tropical/subtropical seas

Natural nitrogen isotopic composition (δ(15)N) of suspended particulate organic matter (POM) and nitrogen fixation rates via (15)N(2) assay were measured in surface waters along 120° E from 30° N to 30° S in the Asian marginal seas (the East/South China Seas and the Sulu/Celebes/Java Seas) and the northeastern Indian Ocean in November-December 2005 and March 2006. The POM δ(15)N values ranged from-1.8 to 12.2‰ with an average of 3.6‰ and showed a decreasing trend towards the equator in both hemispheres. In parallel, the measured N(2) fixation rates showed an increase from the subtropical to the tropical seas. This implies that a higher contribution of (15)N-depleted POM was derived from enhanced N(2) fixation. Water temperature and the stability of water column were partly responsible for the observed variations in nitrogen fixation. The large-scale spatial variations in suspended POM δ(15)N and N(2) fixation rates suggest that the suspended POM δ(15)N may be a potential indicator of nitrogen fixation in surface waters in tropical/subtropical seas.     

Obituary: Professor Dr.-Ing. Justus Mühlenpfordt

Abstract

The dose-dependent isotope fractionation during decarboxylation (¹³C/¹²C) amplifies the radiation effect in the relatively low dose range. The fractionation of the nitrogen isotopes during deamination (¹⁵N/¹⁴N) at the dose of 17 Gy was found to be smaller than that of decarboxylation.     

Natural abundances of 15N and 13C in leaves of some N2-fixing and non-N2-fixing trees and shrubs in Syria

A survey study was conducted on man-made plantations located at two different areas in the arid region of Syria to determine the variations in natural abundances of the ¹⁵N and ¹³C isotopes in leaves of several woody legume and non-legume species, and to better understand the consequence of such variations on nitrogen fixation and carbon assimilation. In the first study area (non-saline soil), the δ¹⁵N values in four legume species (Acacia cyanophylla,?1.73 ‰ Acacia farnesiana,?0.55 ‰ Prosopis juliflora,?1.64 ‰; and Medicago arborea,+1.6 \textperthousand) and one actinorhizal plant (Elaeagnus angustifolia,?0.46 to?2.1 ‰) were found to be close to that of the atmospheric value pointing to a major contribution of N₂ fixing in these species; whereas, δ¹⁵N values of the non-fixing plant species were highly positive. δ¹³C ‰; in leaves of the C3 plants were found to be affected by plant species, ranging from a minimum of?28.67 ‰; to a maximum of?23 ‰. However, they were relatively similar within each plant species although they were grown at different sites. In the second study area (salt affected soil), a higher carbon discrimination value (Δ¹³C ‰) was exhibited by P. juliflora, indicating that the latter is a salt tolerant species; however, its δ¹⁵N was highly positive (+7.03 ‰) suggesting a negligible contribution of the fixed N₂. Hence, it was concluded that the enhancement of N₂ fixation might be achieved by selection of salt-tolerant Rhizobium strains.     

Maternal obesity does not influence human milk protein 15N natural isotope abundance

ABSTRACT

Obesity increases protein metabolism with a potential effect on nitrogen isotope fractionation. The aim of this study was to test the influence of obesity on human milk extracted protein ¹⁵N natural isotope abundance (NIA) at one month post-partum and to compare human milk extracted protein ¹⁵N NIA and bulk infant hair ¹⁵N NIA. This cross-sectional observational study involved 16 obese mothers (body mass index (BMI)?≥?30?kg?m^?2 before pregnancy) matched with 16 normal-weight mothers (18.5?kg?m^?2?≤?BMI?<?25?kg?m^?2) for age and pregnancy characteristics. Human milk extracted protein and bulk infant hair ¹⁵N NIA were determined by isotope ratio monitoring by mass spectrometry interfaced to an elemental analyser (IRM-EA/MS). No significant difference was found in human milk protein ¹⁵N NIA values between obese and normal-weight mothers (8.93?±?0.48?‰ vs. 8.95?±?0.27?‰). However, human milk protein ¹⁵N NIA was significantly lower than bulk infant hair ¹⁵N NIA: 8.94?±?0.38?‰ vs. 9.66?±?0.69?‰, respectively. On the basis of these results, it is concluded that human milk protein ¹⁵N NIA measured at one month post-partum is not influenced by maternal obesity. These findings suggest that ¹⁵N NIA may be exploited to study metabolism without considering maternal obesity as a confounder.     

Differential response of two Pinus spp. to avian nitrogen input as revealed by nitrogen isotope analysis for tree rings

Temporal variations in N concentration and δ(15)N value of annual tree rings (1 year of time resolution) of two Japanese Black Pine (Pinus thunbergii) and three Japanese Red Pine (Pinus densiflora) trees under current breeding activity of the Great Cormorant (Pharacrocorax carbo) and the Black-tailed Gull (Larus crassirostris), respectively, in central and northeastern Japan were studied. Both species from control sites where no avian input occurs show negative values (δ(15)N = around -4 ‰ to -2 ‰) which are common among higher plants growing under high rainfall regimes. The δ(15)N values of P. densiflora show uniformly positive values several years before and after the breeding event, indicating N translocation that moved the absorbed N of a given growth year to tree rings of the previous year while a clear historical value of soil N dynamics was kept intact in the annual rings of P. thunbergii. Long-term N trends inferred from tree rings must take into account tree species with limited translocation rates that can retain actual N annual acquisition.     

Measuring δ(13)C values of atmospheric acetaldehyde via sodium bisulfite adsorption and cysteamine derivatisation

δ(13)C values of gaseous acetaldehyde were measured by gas chromatograph-combustion-isotope ratio mass spectrometer (GC-C-IRMS) via sodium bisulfite (NaHSO(3)) adsorption and cysteamine derivatisation. Gaseous acetaldehyde was collected via NaHSO(3)-coated Sep-Pak(?) silica gel cartridge, then derivatised with cysteamine, and then the δ(13)C value of the acetaldehyde-cysteamine derivative was measured by GC-C-IRMS. Using two acetaldehydes with different δ(13)C values, derivatisation experiments were carried out to cover concentrations between 0.009×10(-3) and 1.96×10(-3)?mg·l(-1)) of atmospheric acetaldehyde, and then δ(13)C fractionation was evaluated in the derivatisation of acetaldehyde based on stoichiometric mass balance after measuring the δ(13)C values of acetaldehyde, cysteamine and the acetaldehyde-cysteamine derivative. δ(13)C measurements in the derivertisation process showed good reproducibility (<0.5?‰) for gaseous acetaldehyde. The differences between predicted and measured δ(13)C values were 0.04-0.31?‰ for acetaldehyde-cysteamine derivative, indicating that the derivatisation introduces no isotope fractionation for gaseous acetaldehyde, and obtained δ(13)C values of acetaldehyde in ambient air at the two sites were distinct (-34.00?‰ at an urban site versus-31.00?‰ at a forest site), implying potential application of the method to study atmospheric acetaldehyde.     

Sample preparation techniques for the determination of natural 15N/14N variations in amino acids by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS)

In order to identify natural nitrogen isotope variations of biologically important amino acids four derivatization reactions (t-butylmethylsilylation, esterification with subsequent trifluoroacetylation, acetylation and pivaloylation) were tested with standard mixtures of 17 proteinogenic amino acids and plant (moss) samples using GC-C-IRMS. The possible fractionation of the nitrogen isotopes, caused for instance by the formation of multiple reaction products, was investigated. For biological samples, the esterification of the amino acids with subsequent trifluoroacetylation is recommended for nitrogen isotope ratio analysis. A sample preparation technique is described for the isotope ratio mass spectrometric analysis of amino acids from the non-protein (NPN) fraction of terrestrial moss. ¹⁴N/¹⁵N ratios from moss (Scleropodium spec.) samples from different anthropogenically polluted areas were studied with respect to ecotoxicologal bioindication.