Amino Acid δ15N Research Articles

Amino acid isotope incorporation and enrichment factors in Pacific bluefin tuna, Thunnus orientalis.

Compound specific isotopic analysis (CSIA) of amino acids has received increasing attention in ecological studies in recent years due to its ability to evaluate trophic positions and elucidate baseline nutrient sources. However, the incorporation rates of individual amino acids into protein and specific trophic discrimination factors (TDFs) are largely unknown, limiting the application of CSIA to trophic studies. We determined nitrogen turnover rates of individual amino acids from a long-term (up to 1054 days) laboratory experiment using captive Pacific bluefin tuna, Thunnus orientalis (PBFT), a large endothermic pelagic fish fed a controlled diet. Small PBFT (white muscle δ15N∼11.5‰) were collected in San Diego, CA and transported to the Tuna Research and Conservation Center (TRCC) where they were fed a controlled diet with high δ15N values relative to PBFT white muscle (diet δ15N∼13.9‰). Half-lives of trophic and source amino acids ranged from 28.6 to 305.4 days and 67.5 to 136.2 days, respectively. The TDF for the weighted mean values of amino acids was 3.0 ‰, ranging from 2.2 to 15.8 ‰ for individual combinations of 6 trophic and 5 source amino acids. Changes in the δ15N values of amino acids across trophic levels are the underlying drivers of the trophic 15N enrichment. Nearly all amino acid δ15N values in this experiment changed exponentially and could be described by a single compartment model. Significant differences in the rate of 15N incorporation were found for source and trophic amino acids both within and between these groups. Varying half-lives of individual amino acids can be applied to migratory organisms as isotopic clocks, determining the length of time an individual has spent in a new environment. These results greatly enhance the ability to interpret compound specific isotope analyses in trophic studies.

Deep‐sea coral record of human impact on watershed quality in the Mississippi River Basin

AbstractOne of the greatest drivers of historical nutrient and sediment transport into the Gulf of Mexico is the unprecedented scale and intensity of land use change in the Mississippi River Basin. These landscape changes are linked to enhanced fluxes of carbon and nitrogen pollution from the Mississippi River, and persistent eutrophication and hypoxia in the northern Gulf of Mexico. Increased terrestrial runoff is one hypothesis for recent enrichment in bulk nitrogen isotope (δ15N) values, a tracer for nutrient source, observed in a Gulf of Mexico deep‐sea coral record. However, unambiguously linking anthropogenic land use change to whole scale shifts in downstream Gulf of Mexico biogeochemical cycles is difficult. Here we present a novel approach, coupling a new tracer of agro‐industrialization to a multiproxy record of nutrient loading in long‐lived deep‐sea corals collected in the Gulf of Mexico. We found that coral bulk δ15N values are enriched over the last 150–200 years relative to the last millennia, and compound‐specific amino acid δ15N data indicate a strong increase in baseline δ15N of nitrate as the primary cause. Coral rhenium (Re) values are also strongly elevated during this period, suggesting that 34% of Re is of anthropogenic origin, consistent with Re enrichment in major world rivers. However, there are no pre‐anthropogenic measurements of Re to confirm this observation. For the first time, an unprecedented record of natural and anthropogenic Re variability is documented through coral Re records. Taken together, these novel proxies link upstream changes in water quality to impacts on the deep‐sea coral ecosystem.

Preservation effects on the isotopic and elemental composition of skeletal structures in the deep-sea bamboo coral Lepidisis spp. (Isididae)

Trace elements and stable isotopes (δ13C and δ15N) in deep-sea coral have been used as proxies to reconstruct past climate, and to investigate food web structure. However, there is a paucity of information regarding the effect of preservation on the chemical integrity of archived coral. In this study a live-caught colony of Bamboo coral (southern Australia), genus (Lepidisis), was sectioned into three pieces and stored for approximately one year to investigate the influence of preservation in ethanol and preservation in seawater, that mimics the early stages of fossilization, against a dry preserved control. Storage and preservation have no significant effect on the isotopic signature of Δ14C, bulk δ15N, δ15N and δ13C of individual amino acids, or C:N in the fibrillar protein matrix, with the only offset (~0.2‰) being observed in part of the δ13C record. In the high-magnesium calcite lattice Ba/Ca appears to be significantly different after storage in ethanol, whereas Δ14C, B/Ca, Mg/Ca, Sr/Ca, and U/Ca remain largely unaltered. Possible mechanisms responsible for these observed differences center around the decomposition, or contamination, by organics and we recommend further investigation, and caution when comparing samples with differing preservation histories.

Cereal grain, rachis and pulse seed amino acid δ15N values as indicators of plant nitrogen metabolism.

Natural abundance δ(15)N values of plant tissue amino acids (AAs) reflect the cycling of N into and within plants, providing an opportunity to better understand environmental and anthropogenic effects on plant metabolism. In this study, the AA δ(15)N values of barley (Hordeum vulgare) and bread wheat (Triticum aestivum) grains and rachis and broad bean (Vicia faba) and pea (Pisum sativum) seeds, grown at the experimental farm stations of Rothamsted, UK and Bad Lauchstädt, Germany, were determined by GC-C-IRMS. It was found that the δ(15)N values of cereal grain and rachis AAs could be largely attributed to metabolic pathways involved in their biosynthesis and catabolism. The relative (15)N-enrichment of phenylalanine can be attributed to its involvement in the phenylpropanoid pathway and glutamate has a δ(15)N value which is an average of the other AAs due to its central role in AA-N cycling. The relative AA δ(15)N values of broad bean and pea seeds were very different from one another, providing evidence for differences in the metabolic routing of AAs to the developing seeds in these leguminous plants. This study has shown that AA δ(15)N values relate to known AA biosynthetic pathways in plants and thus have the potential to aid understanding of how various external factors, such as source of assimilated N, influence metabolic cycling of N within plants.

Compound-specific amino acid δ15N patterns in marine algae: Tracer potential for cyanobacterial vs. eukaryotic organic nitrogen sources in the ocean

Stable nitrogen isotopic analysis of individual amino acids (δ15N-AA) has unique potential to elucidate the complexities of food webs, track heterotrophic transformations, and understand diagenesis of organic nitrogen (ON). While δ15N-AA patterns of autotrophs have been shown to be generally similar, prior work has also suggested that differences may exist between cyanobacteria and eukaryotic algae. However, δ15N-AA patterns in differing oceanic algal groups have never been closely examined. The overarching goals of this study were first to establish a more quantitative understanding of algal δ15N-AA patterns, and second to examine whether δ15N-AA patterns have potential as a new tracer for distinguishing prokaryotic vs. eukaryotic N sources. We measured δ15N-AA from prokaryotic and eukaryotic phytoplankton cultures and used a complementary set of statistical approaches (simple normalization, regression-derived fractionation factors, and multivariate analyses) to test for variations. A generally similar δ15N-AA pattern was confirmed for all algae, however significant AA-specific variation was also consistently identified between the two groups. The relative δ15N fractionation of Glx (glutamine+glutamic acid combined) vs. total proteinaceous N appeared substantially different, which we hypothesize could be related to differing enzymatic forms. In addition, the several other AA (most notably glycine and leucine) appeared to have strong biomarker potential. Finally, we observed that overall patterns of δ15N values in algae correspond well with the Trophic vs. Source-AA division now commonly used to describe variable AA δ15N changes with trophic transfer, suggesting a common mechanistic basis. Overall, these results show that autotrophic δ15N-AA patterns can differ between major algal evolutionary groupings for many AA. The statistically significant multivariate results represent a first approach for testing ideas about relative eukaryotic vs. prokaryotic ON sources in the sea.

15N/14N ratios of amino acids as a tool for studying terrestrial food webs: a case study of terrestrial insects (bees, wasps, and hornets)

AbstractCompound‐specific stable isotope analysis (CSIA) of amino acids is a new method that enables estimates of trophic position for consumers in food webs. We examined the nitrogen isotopic composition (δ15N) of amino acids of Japanese social insects (three bee, three wasp, and four hornet species) to evaluate the potential of CSIA of amino acids in studies of terrestrial food webs. For wasps, we also examined samples at different growth stages (ranging from egg to adult) to assess the effect of metamorphosis on CSIA estimates of trophic position. The δ15N values of bulk tissues for Japanese social insects are only weakly correlated with the biologically expected trophic positions. In contrast, the trophic positions estimated from the δ15N values of amino acids (yielding values of between 2.0 and 2.3 for bees, between 2.8 and 3.3 for wasps, and between 3.5 and 4.1 for hornets) are consistent with the biologically expected trophic positions for these insects (i.e., 2.0 for bees, 3.0 for wasps, and 3.0–4.0 for hornets). Although large variability is observed among the δ15N values of individual amino acids (e.g., ranging from 3.0 to 14.9‰ for phenylalanine), no significant change is observed in the trophic position during wasp metamorphosis. Thus, the CSIA of amino acids is a powerful tool for investigating not only aquatic food webs but also terrestrial food webs with predatory insects.

Quantitative evaluation of marine protein contribution in ancient diets based on nitrogen isotope ratios of individual amino acids in bone collagen: An investigation at the Kitakogane Jomon site

Nitrogen stable isotopes analysis of individual bone collagen amino acids was applied to archeological samples as a new tool for assessing the composition of ancient human diets and calibrating radiocarbon dates. We used this technique to investigate human and faunal samples from the Kitakogane shell midden in Hokkaido, Japan (5,300-6,000 cal BP). Using compound-specific nitrogen isotope analysis of individual amino acids, we aimed to estimate i) the quantitative contribution of marine and terrestrial protein to the human diet, and ii) the mean trophic level (TL) from which dietary protein was derived from marine ecosystems. Data were interpreted with reference to the amino acid trophic level (TL(AA)) model, which uses empirical amino acid delta(15)N from modern marine fauna to construct mathematical equations that predict the trophic position of organisms. The TL(AA) model produced realistic TL estimates for the Kitakogane marine animals. However, this model was not appropriate for the interpretation of human amino acid delta(15)N, as dietary protein is derived from both marine and terrestrial environments. Hence, we developed a series of relevant equations that considered the consumption of dietary resources from both ecosystems. Using these equations, the mean percentage of marine protein in the Kitakogane human diet was estimated to be 74%. Although this study is one of the first systematic investigations of amino acid delta(15)N in archeological bone collagen, we believe that this technique is extremely useful for TL reconstruction, palaeodietary interpretation, and the correction of marine reservoir effects for radiocarbon dating.

Determination of aquatic food‐web structure based on compound‐specific nitrogen isotopic composition of amino acids

The nitrogen isotopic composition (δ15N) of amino acids is potentially useful as an alternative method for estimating the trophic levels of organisms in food webs. However, because this “amino acid method” has been constructed from the observations of only a few case studies of food‐consumer combinations in previous studies, the universality of the approach remains unclear. In this study, we investigated the δ15N signatures of amino acids in 17 photoautotrophs and the trophic relationships during four controlled feeding experiments using green algae, zooplankton, and fish. The results are consistent with those reported in previous studies, implying that the amino acid method can be applied to a variety of organisms. From these and previously published data, we estimate the two factors (β, isotope differences among amino acids in primary producers; Δ, the 15N‐enrichment factor for each trophic level) required to calculate the trophic level. Based on the lowest error (1σ = 0.12) in the estimated trophic level, we conclude that a comparison of the δ15N values for glutamic acid and phenylalanine is most useful in calculating precise estimates of the trophic level, using the following equation: trophic level (TLGlu/Phe) = (δ15NGlu − δ15NPhe − 3.4)/7.6 + 1.

Transformation of plant biochemicals to geological macromolecules during early diagenesis.

Chemical and isotopic changes in plant biochemicals that were transformed into organic geochemicals have been measured in anaerobic, freshwater marsh environments. In two litter bag studies, plant biochemicals decayed extensively in the first year, as recorded by dry weight, C:N ratios, δ15N of bulk tissue and amino acids, and δ13C of individual amino acids. Molecular analyses of Rubisco revealed that the high-molecular-weight enzyme subunit could be recognized antigenically for at least 12 months, but concentrations and amounts declined. Geochemical compounds, advanced glycation endproducts, were not found in fresh plants, but formed gradually with first indications documented at 3 months. The organic remains of plants were reworked or replaced by microbial products from decomposition, as indicated by a shift in the isotopic composition of individual amino acids in total plant protein. In experiments with Rubisco, isotopic changes over time in the individual amino acids in the 50-60 kDa molecular weight range were substantial. These high-molecular-weight substances were no longer pristine molecules. Biochemical and isotopic tools for studying living processes have been demonstrated to be effective and novel approaches to identify and quantify altered geochemical remnants.

Vascular plant 15N natural abundance in heath and forest tundra ecosystems is closely correlated with presence and type of mycorrhizal fungi in roots.

In this study we show that the natural abundance of the nitrogen isotope 15, δ15N, of plants in heath tundra and at the tundra-forest ecocline is closely correlated with the presence and type of mycorrhizal association in the plant roots. A total of 56 vascular plant species, 7 moss species, 2 lichens and 6 species of fungi from four heath and forest tundra sites in Greenland, Siberia and Sweden were analysed for δ15N and N concentration. Roots of vascular plants were examined for mycorrhizal colonization, and the soil organic matter was analysed for δ15N, N concentration and soil inorganic, dissolved organic and microbial N. No arbuscular mycorrhizal (AM) colonizations were found although potential host plants were present in all sites. The dominant species were either ectomycorrhizal (ECM) or ericoid mycorrhizal (ERI). The δ15N of ECM or ERI plants was 3.5-7.7‰ lower than that of non-mycorrhizal (NON) species in three of the four sites. This corresponds to the results in our earlier study of mycorrhiza and plant δ15N which was limited to one heath and one fellfield in N Sweden. Hence, our data suggest that the δ15N pattern: NON/AM plants > ECM plants ≥ ERI plants is a general phenomenon in ecosystems with nutrient-deficient organogenic soils. In the fourth site, a␣birch forest with a lush herb/shrub understorey, the differences between functional groups were considerably smaller, and only the ERI species differed (by 1.1‰) from the NON species. Plants of all functional groups from this site had nearly twice the leaf N concentration as that found in the same species at the other three sites. It is likely that low inorganic N availability is a prerequisite for strong δ15N separation among functional groups. Both ECM roots and fruitbodies were 15N enriched compared to leaves which suggests that the difference in δ15N between plants with different kinds of mycorrhiza could be due to isotopic fractionation at the␣fungal-plant interface. However, differences in δ15N between soil N forms absorbed by the plants could also contribute to the wide differences in plant δ15N found in most heath and forest tundra ecosystems. We hypothesize that during microbial immobilization of soil ammonium the microbial N pool could become 15N-depleted and the remaining, plant-available soil ammonium 15N-enriched. The latter could be a main source of N for NON/AM plants which usually have high δ15N. In contrast, amino acids and other soil organic N compounds presumably are 15N-depleted, similar to plant litter, and ECM and ERI plants with high uptake of these N forms hence have low leaf δ15N. Further indications come from the δ15N of mosses and lichens which was similar to that of ECM plants. Tundra cryptogams (and ECM and ERI plants) have previously been shown to have higher uptake of amino acid than ammonium N; their low δ15N might therefore reflect the δ15N of free amino acids in the soil. The concentration of dissolved organic N was 3-16 times higher than that of inorganic N in the sites. Organic nitrogen could be an important N source for ECM and, in particular, ERI plants in heath and forest tundra ecosystems with low release rate of inorganic N from the soil organic matter.