Metabolic origin of δ15N values in nitrogenous compounds from Brassica napus L. leaves

Nitrogen isotope composition (δ¹⁵N) in plant organic matter is currently used as a natural tracer of nitrogen acquisition efficiency. However, the δ¹⁵N value of whole leaf material does not properly reflect the way in which N is assimilated because isotope fractionations along metabolic reactions may cause substantial differences among leaf compounds. In other words, any change in metabolic composition or allocation pattern may cause undesirable variability in leaf δ¹⁵N. Here, we investigated the δ¹⁵N in different leaf fractions and individual metabolites from rapeseed (Brassica napus) leaves. We show that there were substantial differences in δ¹⁵N between nitrogenous compounds (up to 30‰) and the content in (¹⁵N enriched) nitrate had a clear influence on leaf δ¹⁵N. Using a simple steady‐state model of day metabolism, we suggest that the δ¹⁵N value in major amino acids was mostly explained by isotope fractionation associated with isotope effects on enzyme‐catalysed reactions in primary nitrogen metabolism. δ¹⁵N values were further influenced by light versus dark conditions and the probable occurrence of alternative biosynthetic pathways. We conclude that both biochemical pathways (that fractionate between isotopes) and nitrogen sources (used for amino acid production) should be considered when interpreting the δ¹⁵N value of leaf nitrogenous compounds.     

Comparative response of δ13C, δ18O and δ15N in durum wheat exposed to salinity at the vegetative and reproductive stages

This study compared the performance of the stable isotope composition of carbon (δ¹³C), oxygen (δ¹⁸O) and nitrogen (δ¹⁵N) by tracking plant response and genotypic variability of durum wheat to different salinity conditions. To that end, δ¹³C, δ¹⁸O and δ¹⁵N were analysed in dry matter (dm) and the water‐soluble fraction (wsf) of leaves from plants exposed to salinity, either soon after plant emergence or at anthesis. The δ¹³C and δ¹⁸O of the wsf recorded the recent growing conditions, including changes in evaporative conditions. Regardless of the plant part (dm or wsf), δ¹³C and δ¹⁸O increased and δ¹⁵N decreased in response to stress. When the stress conditions were established just after emergence, δ¹⁵N and δ¹³C correlated positively with genotypic differences in biomass, whereas δ¹⁸O correlated negatively in the most severe treatment. When the stress conditions were imposed at anthesis, relationships between the three isotope signatures and biomass were only significant and positive within the most severe treatments. The results show that nitrogen metabolism, together with stomatal limitation, is involved in the genotypic response to salinity, with the relative importance of each factor depending on the severity and duration of the stress as well as the phenological stage that the stress occurs.     

Decrypting stable‐isotope (δ13C and δ15N) variability in aquatic plants

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Intraskeletal isotopic compositions (δ13C, δ15N) of bone collagen: Nonpathological and pathological variation

Paleodiet research traditionally interprets differences in collagen isotopic compositions (δ¹³C, δ¹⁵N) as indicators of dietary distinction even though physiological processes likely play some role in creating variation. This research investigates the degree to which bone collagen δ¹³C and δ¹⁵N values normally vary within the skeleton and examines the influence of several diseases common to ancient populations on these isotopic compositions. The samples derive from two medieval German cemeteries and one Swiss reference collection and include examples of metabolic disease (rickets/osteomalacia), degenerative joint disease (osteoarthritis), trauma (fracture), infection (osteomyelitis), and inflammation (periostitis). A separate subset of visibly nonpathological skeletal elements from the German collections established normal intraindividual variation. For each disease type, tests compared bone lesion samples to those near and distant to the lesions sites. Results show that normal (nonpathological) skeletons exhibit limited intraskeletal variation in carbon‐ and nitrogen‐isotope ratios, suggesting that sampling of distinct elements is appropriate for paleodiet studies. In contrast, individuals with osteomyelitis, healed fractures, and osteoarthritis exhibit significant intraskeletal differences in isotope values, depending on whether one is comparing lesions to near or to distant sites. Skeletons with periostitis result in significant intraskeletal differences in nitrogen isotope values only, while those with rickets/osteomalacia do not exhibit significant intraskeletal differences. Based on these results, we suggest that paleodiet researchers avoid sampling collagen at or close to lesion sites because the isotope values may be reflecting both altered metabolic processes and differences in diet relative to others in the population. Am J Phys Anthropol 153:598–604, 2014. © 2013 Wiley Periodicals, Inc.     

Variations in stable isotope ratios of carbon (δ13C) and nitrogen (δ15N) in different body parts and eggs of adult stoneflies (Plecoptera)

Variations in δ¹³C and δ¹⁵N might arise from differences in nutrient allocation. Residence times of δ¹³C and δ¹⁵N vary among tissues depending on metabolic turnover rates. However, because of their small size, entire individual insects are generally used as single samples in isotope analyses. The present study aimed to determine the degree of isotope similarity among regions of the adult body and eggs in four species of Plecoptera (Amphinemura sp., Sweltsa sp., Kamimuria tibialis Pictet and Ostrovus sp.). Levels of δ¹³C and δ¹⁵N differ between the four species, being lowest in Amphinemura sp., and with δ¹⁵N being highest in Sweltsa sp. Egg masses contain consistently the lowest values of δ¹³C in the four species, with the δ¹⁵N value of eggs being highest in K. tibialis and Ostrovus sp., and lower in Amphinemura and Sweltsa spp. In Sweltsa sp., the δ¹⁵N levels of the dermal layers and cuticle are lowest, whereas the δ¹³C values of the dermal layers and cuticle are almost equal to those in other regions of the body, except egg masses. Oviposited individuals of Amphinemura and Sweltsa spp. have lower δ¹⁵N levels than individuals that have not oviposited. The rates of metabolism and incorporation of dietary metabolites will differ depending on the body regions and species. Differences in egg ecology such as egg developmental period and egg buoyancy among species are considered to impact on the values of δ¹³C and δ¹⁵N. These results will be useful for understanding the nutritional status of aquatic insects and their energy allocation.     

Changing gull diet in a changing world: A 150‐year stable isotope (δ13C, δ15N) record from feathers collected in the Pacific Northwest of North America

The world's oceans have undergone significant ecological changes following European colonial expansion and associated industrialization. Seabirds are useful indicators of marine food web structure and can be used to track multidecadal environmental change, potentially reflecting long‐term human impacts. We used stable isotope (δ¹³C, δ¹⁵N) analysis of feathers from glaucous‐winged gulls (Larus glaucescens) in a heavily disturbed region of the northeast Pacific to ask whether diets of this generalist forager changed in response to shifts in food availability over 150 years, and whether any detected change might explain long‐term trends in gull abundance. Sampled feathers came from birds collected between 1860 and 2009 at nesting colonies in the Salish Sea, a transboundary marine system adjacent to Washington, USA and British Columbia, Canada. To determine whether temporal trends in stable isotope ratios might simply reflect changes to baseline environmental values, we also analysed muscle tissue from forage fishes collected in the same region over a multidecadal timeframe. Values of δ¹³C and δ¹⁵N declined since 1860 in both subadult and adult gulls (δ¹³C, ~ 2–6‰; δ¹⁵N, ~4–5‰), indicating that their diet has become less marine over time, and that birds now feed at a lower trophic level than previously. Conversely, forage fish δ¹³C and δ¹⁵N values showed no trends, supporting our conclusion that gull feather values were indicative of declines in marine food availability rather than of baseline environmental change. Gradual declines in feather isotope values are consistent with trends predicted had gulls consumed less fish over time, but were equivocal with respect to whether gulls had switched to a more garbage‐based diet, or one comprising marine invertebrates. Nevertheless, our results suggest a long‐term decrease in diet quality linked to declining fish abundance or other anthropogenic influences, and may help to explain regional population declines in this species and other piscivores.     

The influence of lipid‐extraction and long‐term DMSO preservation on carbon (δ13C) and nitrogen (δ15N) isotope values in cetacean skin

Stable isotope analysis (SIA) has rapidly become a useful tool to study the ecology of wild animal populations, especially for elusive, wide‐ranging predators like marine mammals. The development of projectile biopsy techniques resulted in the collection of thousands of cetacean tissue samples that were archived in a dimethyl sulfoxide (DMSO) solution for long‐term, multidecadal preservation. Here we examine the influence of DMSO preservation on carbon (δ¹³C) and nitrogen (δ¹⁵N) values by comparing a set of paired delphinid skin samples stored frozen without preservative and in DMSO for up to 22 yr. Treatment of paired frozen and DMSO‐preserved skin in a 2:1 chloroform:methanol solution yielded similar δ¹³C and δ¹⁵N values, revealing that DMSO and lipid contamination have similar isotopic effects on skin, and that these effects can be removed using routine lipid‐extraction methods. Further, amino acid concentrations in DMSO‐preserved and frozen skin tissue were similar, providing independent evidence of minimal protein alteration due to preservation. Access to a rich archive of skin samples preserved in DMSO will expand our ability to examine temporal and spatial variability in the isotope values of cetaceans, which will aid our understanding of how their ecology has been influenced by historical changes in environmental conditions.     

Nonlethal sampling for stable isotope analysis of juvenile Chinese sturgeon (Acipenser sinensis): Comparing δ13C and δ15N signatures in muscle and fin tissues

We compared δ¹³C and δ¹⁵N values of muscle with fin from juvenile Chinese sturgeon (Acipenser sinensis), to evaluate the feasibility of using nonlethal (fin) as an alternative to lethal (muscle) sampling. Size and lipid effect on the relationship between fin and muscle were also investigated. Dorsal muscle (DM) and fin clip (FC) were collected from A. sinensis with different body length (120–373 mm) in the Yangtze Estuary for isotope analysis. The result showed that (1) muscle isotope values could estimated by the values of fin, from either use the regression model (δ¹³C_DM = 0.939 × FC ? 2.577; δ¹⁵N_DM = 0.737 × FC + 4.638) or constants factors (δ¹³C_DM = δ¹³C_FC ? 1.27; δ¹⁵N_DM = δ¹⁵N_FC + 0.59); (2) no size‐based relationships with δ¹³C and δ¹⁵N from either fin or muscle; (3) lipid extraction significantly improving the fin and muscle regression model fit for both δ¹³C and δ¹⁵N values. Therefore, this study support the use of nonlethal fin tissues for isotope analysis of juvenile A. sinensis, and will allow trophic studies to avoid the effect of lipid accumulation from muscle.     

Effects of decomposition and storage conditions on the δ13C and δ15N isotope values of killer whale (Orcinus orca) skin and blubber tissues

Several different factors in the collection and preservation of whale skin and blubber samples were examined to determine their effect on the results obtained by stable nitrogen and carbon isotope (δ¹⁵N and δ¹³C) analysis. Samples of wet killer whale skin retained their original stable isotope values for up to 14 d at 4°C or lower. However, decomposition significantly changed the δ¹⁵N value within 3 d at 20°C. Storage at ?20°C was as effective as ?80°C for the preservation of skin and blubber samples for stable isotope analysis for at least a year. By contrast, once a skin sample had been freeze‐dried and lipid extracted, the stable isotope values did not change significantly when it was stored dry at room temperature for at least 12 mo. Preservation of whale skin samples for a month in DMSO‐salt solution, frozen or at room temperature, did not significantly change the δ¹⁵N and δ¹³C values of lipid extracted tissues, although the slight changes seen could influence results of a study if only small changes are expected.     

Spatio‐temporal isotopic signatures (δ13C and δ15N) reveal that two sympatric West African mullet species do not feed on the same basal production sources

Potential trophic competition between two sympatric mullet species, Mugil cephalus and Mugil curema, was explored in the hypersaline estuary of the Saloum Delta (Senegal) using δ¹³C and δ¹⁵N composition of muscle tissues. Between species, δ¹⁵N compositions were similar, suggesting a similar trophic level, while the difference in δ¹³C compositions indicated that these species did not feed from exactly the same basal production sources or at least not in the same proportions. This result provides the first evidence of isotopic niche segregation between two limno‐benthophageous species belonging to the geographically widespread, and often locally abundant, Mugilidae family.     

Nitrogen and carbon contents and δ15N and δ13C signatures in six bryophyte species: assessment of long‐term deposition changes (1980–2010) in Spanish beech forests

In this study we used recent (2010) and herbarium material (1980) of six bryophyte species to assess long‐term atmospheric deposition in natural forested areas in northern Spain. For this purpose, tissue nitrogen and carbon content, as well as δ¹³C and δ¹⁵N signatures of samples of Hypnum cupressiforme, Polytrichastrum formosum, Leucobryum juniperoideum, Rhytidiadelphus loreus, Homalothecium lutescens and Diplophyllum albicans were analysed and comparisons made between years and species. In addition, the usefulness of each of the six species was evaluated. The range of values observed was similar to that in other studies carried out in rural areas. Significantly lower values were found in 2010 for N (H. cupressiforme), δ¹⁵N (R. loreus and D. albicans), C (R. loreus) and δ¹³C (all except L. juniperoideum). Our natural areas are thus now less influenced by atmospheric pollutants than they were, most probably due to changes in some traditional local activities. Differences were observed between species for all the four parameters studied, so different species must not be analysed together. Finally, R. loreus and H. lutescens seem to be good bioindicators, sensitive even with a few samples, although further studies are needed to corroborate their usefulness.     

Accumulation,selection and covariation of amino acids in sieve tube sap of tansy (Tanacetum vulgare) and castor bean (Ricinus communis): evidence for the function of a basic amino acid transporter and the absence of a γ‐amino butyric acid transporter

Sieve tube sap was obtained from Tanacetum by aphid stylectomy and from Ricinus after apical bud decapitation. The amino acids in sieve tube sap were analyzed and compared with those from leaves. Arginine and lysine accumulated in the sieve tube sap of Tanacetum more than 10‐fold compared to the leaf extracts and they were, together with asparagine and serine, preferably selected into the sieve tube sap, whereas glycine, methionine/tryptophan and γ‐amino butyric acid were partially or completely excluded. The two basic amino acids also showed a close covariation in sieve tube sap. The acidic amino acids also grouped together, but antagonistic to the other amino acids. The accumulation ratios between sieve tube sap and leaf extracts were smaller in Ricinus than in Tanacetum. Arginine, histidine, lysine and glutamine were enriched and preferentially loaded into the phloem, together with isoleucine and valine. In contrast, glycine and methionine/tryptophan were partially and γ‐amino butyric acid almost completely excluded from sieve tube sap. The covariation analysis grouped arginine together with several neutral amino acids. The acidic amino acids were loaded under competition with neutral amino acids. It is concluded from comparison with the substrate specificities of already characterized plant amino acid transporters, that an AtCAT1‐like transporter functions in phloem loading of basic amino acids, whereas a transporter like AtGAT1 is absent in phloem. Although Tanacetum and Ricinus have different minor vein architecture, their phloem loading specificities for amino acids are relatively similar.     

Mother–embryo isotope (δ15N, δ13C) fractionation and mercury (Hg) transfer in aplacental deep‐sea sharks

Stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotopic values and total mercury (Hg) concentrations were analysed in muscle and liver of mothers and embryos of two aplacental shark species, Squalus megalops and Centrophorus moluccensis. Embryos of the two species had similar or lower isotopic values than their respective mothers, the only exception being for δ¹³C, which was higher in the liver of C. moluccensis embryos than in their mothers. Hg concentrations were systematically lower in embryos compared with their mothers suggesting a low transfer of this element in muscle and liver.     

Transgenic Arabidopsis thaliana plants expressing a β‐1,3‐glucanase from sweet sorghum (Sorghum bicolor L.) show reduced callose deposition and increased tolerance to aluminium toxicity

Seventy‐one cultivars of sweet sorghum (Sorghum bicolor L.) were screened for aluminium (Al) tolerance by measuring relative root growth (RRG). Two contrasting cultivars, ROMA (Al tolerant) and POTCHETSTRM (Al sensitive), were selected to study shorter term responses to Al stress. POTCHETSTRM had higher callose synthase activity, lower β‐1,3‐glucanase activity and more callose deposition in the root apices during Al treatment compared with ROMA. We monitored the expression of 12 genes involved in callose synthesis and degradation and found that one of these, SbGlu1 (Sb03g045630.1), which encodes a β‐1,3‐glucanase enzyme, best explained the contrasting deposition of callose in ROMA and POTCHETSTRM during Al treatment. Full‐length cDNAs of SbGlu1 was prepared from ROMA and POTCHETSTRM and expressed in Arabidopsis thaliana using the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Independent transgenic lines displayed significantly greater Al tolerance than wild‐type plants and vector‐only controls. This phenotype was associated with greater total β‐1,3‐glucanase activity, less Al accumulation and reduced callose deposition in the roots. These results suggest that callose production is not just an early indicator of Al stress in plants but likely to be part of the toxicity pathway that leads to the inhibition of root growth.     

Is there a relationship between N‐acetyl‐β‐d‐glucosaminidase activity of Metarhizium anisopliae (Metschn.) Sorokin (Hyphomycetes) isolates from peridomestic areas in Central Brazil and larvicidal effect on Aedes aegypti (L.) (Diptera,Culicidae)?

Abstract:  The larvicidal effect of Metarhizium anisopliae (Metschn.) Sorokin (Hyphomycetes) isolated in peridomestic areas in Central Brazil was tested in Aedes aegypti (L.) (Dipt., Culicidae), which is worldwide the primary vector for the viruses that cause human dengue and yellow fever. Highest susceptibility of larvae was found after application of suspended ungerminated conidia. However, conidia, which were found on the larval cuticle and in the gut did not germinate in live or dead larvae. Mortality dropped when testing germinating conidia or supernatants, which originated from chitin-amended minimal medium (MM) inoculated conidia and cultures up to 72 h. Paralysis of larval movement was observed a few hours after application, especially of untreated conidia. Isolates showed a high variability of total protein production and N -acetyl- β - d -glucosaminidase activity after 48 and 72 h incubation in MM. No relationship between enzyme levels and insecticidal activity could be detected. The results indicate that toxic compounds emitted by ungerminated conidia on the cuticle or in the gut are involved in the activity of M. anisopliae against A. aegypti larvae.     

Carbon isotope compositions (δ13C) of leaf,wood and holocellulose differ among genotypes of poplar and between previous land uses in a short‐rotation biomass plantation

The efficiency of water use to produce biomass is a key trait in designing sustainable bioenergy‐devoted systems. We characterized variations in the carbon isotope composition (δ¹³C) of leaves, current year wood and holocellulose (as proxies for water use efficiency, WUE) among six poplar genotypes in a short‐rotation plantation. Values of δ¹³C_wood and δ¹³C_{holocellulose} were tightly and positively correlated, but the offset varied significantly among genotypes (0.79–1.01‰). Leaf phenology was strongly correlated with δ¹³C, and genotypes with a longer growing season showed a higher WUE. In contrast, traits related to growth and carbon uptake were poorly linked to δ¹³C. Trees growing on former pasture with higher N‐availability displayed higher δ¹³C as compared with trees growing on former cropland. The positive relationships between δ¹³C_leaf and leaf N suggested that spatial variations in WUE over the plantation were mainly driven by an N‐related effect on photosynthetic capacities. The very coherent genotype ranking obtained with δ¹³C in the different tree compartments has some practical outreach. Because WUE remains largely uncoupled from growth in poplar plantations, there is potential to identify genotypes with satisfactory growth and higher WUE.