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Stable isotope fingerprinting: A novel method for identifying plant, fungal, or bacterial origins of amino acids
Ecology
Abstract
Amino acids play an important role in ecology as essential nutrients for animals and as currencies in symbiotic associations. Here we present a new approach to tracing the origins of amino acids by identifying unique patterns of carbon isotope signatures generated by amino acid synthesis in plants, fungi, and bacteria ("13C fingerprints"). We measured amino acid delta 13C from 10 C3 plants, 13 fungi, and 10 bacteria collected and isolated from a boreal forest in interior Alaska, USA, using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Microorganisms were cultured under amino-acid-free conditions and identified based on DNA sequences. Bacteria, fungi, and plants generated consistent, unique 13C fingerprints based on the more complex amino acids (five or more biosynthetic steps) that are classified as essential for animals. Linear discriminant analysis classified all samples correctly with >99% certainty and correctly classified nearly all insect samples from a previous study by diet. Our results suggest that 13C fingerprints of amino acids could provide a powerful in situ assay of the biosynthetic sources of amino acids and a potential new tool for understanding nutritional linkages in food webs.
... They can be used as markers of the basal resources that support soil food webs because metazoans lack the metabolic pathways to synthesize them (Wu et al., 2014). Since basal resources such as bacteria, fungi and plants each exhibit distinctive δ 13 C eAAs patterns, or fingerprints, it is possible to estimate the relative contribution of these three groups to the diet of consumers (Larsen et al., 2009). ...
... To predict the biosynthetic origin of eAAs in earthworms, we used the fingerprinting approach as described in Larsen et al. (2009). In brief, we conducted linear discriminant analysis (LDA) with δ 13 C values of the eAAs including Ile, Leu, Phe, Thr and Val. ...
... We excluded Met because the chromatography of this amino acid was not satisfactory in all samples. We used eAAs δ 13 C values of bacteria, fungi and plants obtained from Larsen et al. (2009Larsen et al. ( , 2016 and Pollierer et al. (2020) as classifier variables to identify the contribution of the basal resources to the diet of earthworms in the LDA. We then ran multivariate analyses of variance (MANOVAs) for the LDA classification to inspect the effects of earthworm species and plant treatments on the use of basal resources by earthworms. ...
... In this Article, we employ an emerging technique-essential amino acid (EAA) carbon isotope (δ 13 C) fingerprinting 30 -to quantify the impact of climate warming on energy flow and C fluxes between green and brown food webs in Arctic tundra and boreal forests in Alaska, USA. We use δ 13 C analysis of EAAs to trace the flow of C through food webs, quantify the proportional assimilation of green and brown C by above-ground consumers and estimate C fluxes between trophic compartments on the basis of food-web shifts [30][31][32] https://doi.org/10.1038/s41558-023-01893-0 and we predicted that the proportion of brown C (bacteria and fungi) assimilated by consumers would be higher under warming conditions due to the greater availability of microbial energy from increased decomposition of permafrost soils. ...
... We used δ 13 C fingerprinting 30,31 to quantify C assimilation by consumers, focusing on six EAAs: isoleucine, leucine, lysine, phenylalanine, threonine and valine. We combined measured values with published EAA δ 13 C data 31,32 for plants (n = 5), fungi (n = 13) and bacteria (n = 10) collected in boreal forest near Fairbanks, Alaska (Supplementary Appendix 1) 55 . ...
... We combined measured values with published EAA δ 13 C data 31,32 for plants (n = 5), fungi (n = 13) and bacteria (n = 10) collected in boreal forest near Fairbanks, Alaska (Supplementary Appendix 1) 55 . We characterized δ 13 C fingerprints using linear discriminant analysis (LDA) 30,31 and trained the LDA using EAA δ 13 C data for plant, fungal and bacterial sources. We also included filamentous green algae as a potential source of aquatic C (refs. ...
Rapid warming of high-latitude ecosystems is increasing microbial activity and accelerating the decomposition of permafrost soils. This proliferation of microbial energy could restructure high-latitude food webs and alter carbon cycling between above-ground and below-ground habitats. We used stable isotope analysis (δ¹³C) of amino acids to trace carbon flow through food webs exposed to warming and quantified changes in the assimilation of microbial carbon by Arctic tundra and boreal forest consumers. From 1990 to 2021, small mammals in boreal forests exhibited a significant reduction in the use of plant-based ‘green’ food webs and an increased use of microbially mediated ‘brown’ food webs, punctuated by a >30% rise in fungal carbon assimilation. Similarly, fungal carbon assimilation rose 27% in wolf spiders under experimental warming in Arctic tundra. These findings reveal a climate-mediated ‘browning’ of high-latitude food webs and point to an understudied pathway by which animals can impact carbon cycling under climate warming.
... These specific amino acids offer a way to disentangle potential change in the source identification of nitrogen from changes in the trophic position of the consumer. Furthermore, the δ 13 C AA value can help us distinguish between the origin of the amino acids among plant, fungal and bacterial sources and help identifying detritivores from herbivores (Larsen et al., 2009). Therefore, the use compound-specific isotopes of amino acid can contribute to improve our understanding of complex food webs with multiple energy sources. ...
... Differences in amino acid metabolisms between major prokaryotic and eukaryotic lineages generate unique amino acid carbon isotopic signature (Larsen et al., 2009(Larsen et al., , 2013(Larsen et al., , 2022. Plants and fungi have different pathway to synthesize Lys and their pathways for biosynthesizing Leu are compartmentalized differently (Kohlhaw, 2003;Hudson et al., 2006). ...
... Plants and fungi have different pathway to synthesize Lys and their pathways for biosynthesizing Leu are compartmentalized differently (Kohlhaw, 2003;Hudson et al., 2006). The difference between Ile and Leu was found to be valuable in distinguishing plants and fungi from bacteria while the difference between Ile and Lys can be used to separate plants from fungi (Larsen et al., 2009). The δ 13 C AA of Ile, Lys and Leu was therefore used to assess the biosynthetic origin of our sample. ...
Mangrove forests are characterised by a specialised fauna with low ecological redundancy. Their faunal diversity is also decreasing across the tropics, with cascading effects on the ecosystem services provided by these forests. Traditional tools often failed to assess the importance of different sources of nutrients and a reliable trophic position estimate in mangroves, although this information is crucial to better advise on effective conservation strategies. Here, we present nitrogen and carbon isotope data of individual amino acids measured in primary producers and fauna inhabiting a subtropical mangrove. We quantified the relative importance of vascular and non-vascular source of nitrogen in the mangrove food web. We found that most gastropods mostly exclusively rely on nitrogen originating from the marine environment (non-vascular) while most brachyuran crabs integrate between 8 and 50% of their nitrogen from vascular sources, most probably by processing decaying mangrove leaves. This highlights the unique role of crabs in processing the low-nutritional mangrove leaves into the food web. Moreover, we estimated the trophic position for 17 invertebrate species inhabiting the mangrove. We highlighted previously unreported difference in term of trophic position and source of food for several species, which were previously thought to have overlapping feeding preferences. Our data thus suggest that the inherent lack of ecological redundancy present in mangrove may even be more severe than predicted by recent estimates.
... Compound-specific analysis of carbon isotopes in EAAs has shown potential to characterize specific primary producers as well as microorganisms (Larsen et al. 2009(Larsen et al. , 2012(Larsen et al. , 2013. As a likely consequence of different carbon isotopic fractionations during potentially variable EAA biosynthetic pathways across basal resources, the δ 13 C values of individual EAAs (δ 13 C EAA values) can be used to identify specific basal resources (Larsen et al. 2009). ...
... Compound-specific analysis of carbon isotopes in EAAs has shown potential to characterize specific primary producers as well as microorganisms (Larsen et al. 2009(Larsen et al. , 2012(Larsen et al. , 2013. As a likely consequence of different carbon isotopic fractionations during potentially variable EAA biosynthetic pathways across basal resources, the δ 13 C values of individual EAAs (δ 13 C EAA values) can be used to identify specific basal resources (Larsen et al. 2009). Although underlying mechanisms are poorly understood (Besser et al. 2022), discrimination among major taxa, such as bacteria, plants, microalgae, and their clades, has been observed by centering individual δ 13 C EAA values to the mean δ 13 C EAA value within samples of basal resources that creates a suite of relative δ 13 C values of EAAs, that is, δ 13 C EAA patterns (Larsen et al. 2009(Larsen et al. , 2012. ...
... As a likely consequence of different carbon isotopic fractionations during potentially variable EAA biosynthetic pathways across basal resources, the δ 13 C values of individual EAAs (δ 13 C EAA values) can be used to identify specific basal resources (Larsen et al. 2009). Although underlying mechanisms are poorly understood (Besser et al. 2022), discrimination among major taxa, such as bacteria, plants, microalgae, and their clades, has been observed by centering individual δ 13 C EAA values to the mean δ 13 C EAA value within samples of basal resources that creates a suite of relative δ 13 C values of EAAs, that is, δ 13 C EAA patterns (Larsen et al. 2009(Larsen et al. , 2012. The δ 13 C EAA patterns in autotrophs remain consistent despite spatiotemporal changes in environmental factors, including δ 13 C values of dissolved inorganic carbon, pH, salinity, and temperature that can alter the baseline δ 13 C EAA values of basal resources (Larsen et al. 2013(Larsen et al. , 2015Lynch et al. 2016;Elliott Smith et al. 2022). ...
A rapidly warming Arctic Ocean and associated sea-ice decline is resulting in changing sea-ice protist communities , affecting productivity of under-ice, pelagic, and benthic fauna. Quantifying such effects is hampered by a lack of biomarkers suitable for tracing specific basal resources (primary producers and microorganisms) through food webs. We investigate the potential of δ 13 C values of essential amino acids (EAAs) (δ 13 C EAA values) to estimate the proportional use of diverse basal resources by organisms from the under-ice (Apherusa glacialis), pelagic (Calanus hyperboreus) and benthic habitats (sponges, sea cucumber), and the cryo-pelagic fish Boreogadus saida. Two approaches were used: baseline δ 13 C EAA values, that is, the basal resource specific δ 13 C EAA values, and δ 13 C EAA fingerprints, or mean-centred baseline δ 13 C EAA values. Substantial use of sub-ice algae Melosira arctica by all studied organisms suggests that its role within Arctic food webs is greater than previously recognized. In addition, δ 13 C EAA fingerprints from algae-associated bacteria were clearly traced to the sponges, with an individually variable kelp use by sea cucumbers. Although mean-centred δ 13 C EAA values in A. glacialis, C. hyperboreus, and B. saida tissues were aligned with microalgae resources, they were not fully represented by the filtered pelagic-and sea-ice particulate organic matter constituting the spring diatom-dominated algal community. Under-ice and pelagic microalgae use could only be differentiated with baseline δ 13 C EAA values as similar micro-algae clades occur in both habitats. We suggest that δ 13 C EAA fingerprints combined with microalgae baseline δ 13 C EAA values are an insightful tool to assess the effect of ongoing changes in Arctic basal resources on their use by organisms.
... All statistical analyses were performed in Program R (v4.0.3; (R Core Team, 2021)) with RStudio interface (v1.1.463). To characterize the basal energetic sources supporting the CA-SRI-85 fish community, we employed essential amino acid δ 13 C "fingerprinting," via linear discriminant analysis (Larsen et al., 2009(Larsen et al., , 2013. This statistical technique is well validated in coastal marine ecosystems of the Pacific (Elliott Smith, 2019; Elliott Smith et al., 2018Smith et al., , 2022Fox et al., 2019;Larsen et al., 2013;Rowe et al., 2019). ...
... Our essential amino acid δ 13 C measurements of CA-SRI-85 fish specimens allowed us to measure key attributes of historical Santa Rosa marine food webs and provide unique insights into the ecology and stability of these systems. Stable isotope analysis of individual amino acids has been used previously to study energy flow in a range of coastal Pacific ecosystems (Elliott Smith et al., 2018, 2021Fox et al., 2019;Larsen et al., 2009Larsen et al., , 2013Nye, 2019;Rowe et al., 2019;Vokhshoori et al., 2014), and the utility of this technique relies on three key observations. First, δ 13 C values of essential amino acids are minimally altered during trophic transfers (Fantle et al., 1999;McMahon et al., 2015), although de novo synthesis by gut microbial communities cannot be ruled out. ...
... First, δ 13 C values of essential amino acids are minimally altered during trophic transfers (Fantle et al., 1999;McMahon et al., 2015), although de novo synthesis by gut microbial communities cannot be ruled out. Second, some primary producer clades, such as kelps (Elliott Smith et al., 2022), impart distinct δ 13 C values on essential amino acids, which can be identified in consumers using multivariate statistics Elliott Smith et al., 2018Fox et al., 2019;Larsen et al., 2009Larsen et al., , 2013. Finally, these "fingerprints" appear to be conserved over broad geographics scales, and over multiple centuries (Elliott Smith et al., 2022), enabling the reconstruction of energy flow in historical marine communities. ...
How do human communities integrate within food webs? Studies characterizing the historical flow of energy among people and local environments can yield important insights into managing sustainable ecosystems. Here, we combine zooarchaeological, bulk tissue, and compound-specific stable isotope data from late Holocene Santa Rosa Island to investigate the ecological role of people within a southern California nearshore marine ecosystem. We show people foraged in diverse marine habitats, including kelp forests and pelagic waters, and consumed a wide range of species. However, zooarchaeological results reveal >50% of recovered fish remains came from presumed kelp forest carnivores of mid-trophic level (3.0–4.0) such as surfperches and rockfishes. Local fishing strategies thus did not involve the preferential removal of large-bodied, high trophic level species – a pattern known as “fishing down the food web” which has been documented in modern commercial fisheries and results in the collapse of marine ecosystems. Essential amino acid δ ¹³ C analysis revealed that the historical fish community relied on basal resources from kelps and phytoplankton. This coupling of benthic and pelagic energy sources suggests late Holocene coastal food webs in the region were in a relatively stable configuration immediately prior to first European arrival. This stability likely contributed to sustaining densely populated Chumash settlements, intensive fisheries, and high local faunal diversity. Our findings provide important pre-industrial data for marine ecosystems and document some of the mechanisms behind the intensive, yet sustainable long-term fisheries of the Island Chumash.
... Essential amino acids (EAAs) are ideal biomarkers to trace the flow of green and brown energy because EAAs can only be synthesized by bacteria, fungi, and photoautotrophs (e.g., plants), each via unique biosynthetic pathways that impart distinct δ 13 C patterns across EAAs (Besser et al., 2022). These multivariate patterns, or "fingerprints," are distinct between green (i.e., plant) and brown (i.e., bacterial and fungal) protein sources (Larsen et al., 2009). Because animals cannot synthesize EAAs de novo, they must assimilate these molecules from their diets, and EAA δ 13 C fingerprints can then be used to quantify the use of green and brown food webs by consumers (Manlick & Newsome, 2022). ...
... To quantify multichannel feeding, we used δ 13 C values from six EAAs: isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), and valine (Val). We combined measured values with published EAA δ 13 C data (Larsen et al., 2009; Appendix S1) and characterized δ 13 C fingerprints for sources using linear discriminant analysis (LDA), which maximizes separation of groups in multivariate space and can distinguish green from brown energy channels (Larsen et al., 2009;Manlick & Newsome, 2022). We trained the LDA using EAA δ 13 C values of the three source groups, used leave-one-out cross-validation to assess classification accuracy, and then used the model to predict group membership for animal consumers. ...
... To quantify multichannel feeding, we used δ 13 C values from six EAAs: isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), and valine (Val). We combined measured values with published EAA δ 13 C data (Larsen et al., 2009; Appendix S1) and characterized δ 13 C fingerprints for sources using linear discriminant analysis (LDA), which maximizes separation of groups in multivariate space and can distinguish green from brown energy channels (Larsen et al., 2009;Manlick & Newsome, 2022). We trained the LDA using EAA δ 13 C values of the three source groups, used leave-one-out cross-validation to assess classification accuracy, and then used the model to predict group membership for animal consumers. ...
Food web ecology has revolutionized our understanding of ecological processes, but the drivers of food web properties like trophic position (TP) and food chain length are notoriously enigmatic. In terrestrial ecosystems, above‐ and belowground systems were historically compartmentalized into “green” and “brown” food webs, but the coupling of these systems by animal consumers is increasingly recognized, with potential consequences for trophic structure. We used stable isotope analysis (δ¹³C, δ¹⁵N) of individual amino acids to trace the flow of essential biomolecules and jointly measure multichannel feeding, food web coupling, and TP in a guild of small mammals. We then tested the hypothesis that brown energy fluxes to aboveground consumers increase terrestrial food chain length via cryptic trophic transfers during microbial decomposition. We found that the average small mammal consumer acquired nearly 70% of their essential amino acids (69.0% ± 7.6%) from brown food webs, leading to significant increases in TP across species and functional groups. Fungi were the primary conduit of brown energy to aboveground consumers, providing nearly half the amino acid budget for small mammals on average (44.3% ± 12.0%). These findings illustrate the tightly coupled nature of green and brown food webs and show that microbially mediated energy flow ultimately regulates food web structure in aboveground consumers. Consequently, we propose that the integration of green and brown energy channels is a cryptic driver of food chain length in terrestrial ecosystems.
... Compound-specific isotope analysis (CSIA) of amino acids (AAs) has emerged as a powerful tool for tracing organic C and nitrogen (N) in marine food webs and sediments (Larsen et al., 2009;Larsen et al., 2013;McMahon et al., 2013;Batista et al., 2014;Ohkouchi et al., 2017). The traditional approach to interpret stable isotope values of bulk organic materials has been widely used to study organic matter sources and cycling in different ecosystems (Fry 1998). ...
... Essential amino acids (EAAs) can only be synthesized by autotrophs or resynthesized by bacteria de novo and utilized directly by consumers with minor alteration to their C skeletons (Larsen et al., 2013;McMahon et al., 2013). Diverse biosynthetic pathways and associated isotopic effects lead to distinct and consistent d 13 C of EAAs in different functional groups of primary producers (Hayes, 1993;Larsen et al., 2009), which are transferred to higher trophic levels with little or no trophic discrimination (McMahon et al., 2013). Therefore, d 13 C-EAA patterns of consumers reflect the isotopic signatures of primary producers at the base of the food web (McMahon et al., 2013). ...
... In the ocean, the d 13 C-EAA signatures of primary producers are passed, unaltered, to higher trophic levels and eventually preserved in paleo-archives such as proteinaceous deep-sea corals and marine sediments Larsen et al., 2015;McMahon et al., 2015). Such stable isotope "fingerprints" have been developed for primary producers from terrestrial and aquatic ecosystems (Larsen et al., 2009;Larsen et al., 2013;McMahon et al., 2015) and used in Bayesian mixing models to reconstruct plankton community structure from archived materials (McMahon et al., 2015). The changes in d 15 N in AAs during trophic transfers lead to different groupings of AAs that are independent of those based on d 13 C. ...
The on-going decline in Arctic sea ice represents a significant loss of habitat for sea ice algae, which are a major contributor to primary production in the Arctic. Data on sea ice algal production is limited due to difficulties in both accessing sea-ice and sampling under-ice algae. Compound-specific isotope analysis (CSIA) of amino acids (AAs) is emerging as a powerful tool to trace element origins and biogeochemical processes in marine food webs and may address the knowledge gaps in sea ice algal productivity dynamics. Here we measured δ¹³C-AA and δ¹⁵N-AA in natural communities of sea ice and pelagic algae collected from regions offshore Labrador and Nunavut, Canada. Significant difference in δ¹³C-AA patterns between sea ice and pelagic algae was observed in different size classes. This difference was further supported by multivariate analyses based on normalized δ¹³C of essential amino acids (EAAs), which demonstrated a clear separation between sea ice and pelagic algae. Beta (β) values and trophic position (TP) calculated from δ¹⁵N of Glutamic Acid and Phenylalanine (Phe) and ∑V parameter for microbial resynthesis indicated a slightly higher heterotrophic biomass in pelagic and sea ice samples as compared to cultured samples. This finding is consistent with the Phe-normalized δ¹⁵N of Alanine and Threonine, which provided better separations between sea ice/pelagic algae and other end-member groups. Overall, our study provides first insights into the potential differences in δ¹³C-AA and δ¹⁵N-AA patterns between sea ice and pelagic algae and suggests carbon of sea ice origins may be distinguished from pelagic sources using CSIA-AA approach. These observations highlight the potential of CSIA-AA to estimate proportional contributions of sea ice and pelagic algae to export production and efficiency of benthic-pelagic coupling in polar marine environments.
... Second, inferring TP from bulk stable isotope composition remains challenging, as the δ 15 N values of organisms are affected not only by trophic fractionation, but also by variations in stable isotope values of basal resources Potapov et al., 2019a). Recently, compound specific isotope analysis (CSIA) of amino acids (AAs) (McClelland and Montoya, 2002;Chikaraishi et al., 2014;Larsen et al., 2009Larsen et al., , 2013 has been introduced to study soil food webs and provides accurate estimates of TP and basal resources of soil organisms, such as microarthropods (Larsen et al., 2011;Pollierer et al., 2019), Annelida (Larsen et al., 2016a, b;Potapov et al., 2019) and Diptera . The TP estimated by CSIA of AAs (TP CSIA ) is based on the differential isotopic fractionation of N between the canonical 'trophic' and 'source' AAs glutamine/glutamic acid (Glx) and phenylalanine (Phe), respectively, during metabolic processes; the difference in δ 15 N values between glutamine/glutamic acid (Glx) and phenylalanine (Phe) increases with the trophic position of organisms. ...
... The TP estimated by CSIA of AAs (TP CSIA ) is based on the differential isotopic fractionation of N between the canonical 'trophic' and 'source' AAs glutamine/glutamic acid (Glx) and phenylalanine (Phe), respectively, during metabolic processes; the difference in δ 15 N values between glutamine/glutamic acid (Glx) and phenylalanine (Phe) increases with the trophic position of organisms. Furthermore, using δ 13 C values of essential AAs (eAAs), the AA fingerprinting approach allows to distinguish between saprotrophic fungi, mycorrhizal fungi, bacteria and plants as basal resources of soil organisms (Larsen et al., 2009;Pollierer et al., 2019Pollierer et al., , 2020. ...
... To compare the contribution of different resources to the diet of Collembola, linear discriminant function analysis (LDA) was performed with δ 13 C values of three eAAs, i.e. threonine (Thr), phenylalanine (Phe) and methionine (Met), using package 'MASS' (Venables and Ripley, 2002). To predict the biosynthetic origin of these eAAs in consumers, we used classifier variables (i.e., 'training' data) comprising the resources from previous studies (Larsen et al., 2009(Larsen et al., , 2013Pollierer et al., 2020). LDA finds the linear combination (linear classifier) of δ 13 C values of eAAs that most efficiently separates different groups of basal resources. ...
The vertical heterogeneity of trophic interactions in soil food webs in temperate forest systems and its response to deprivation of root energy supply are poorly understood. In the present study, community-level trophic niches of Collembola functional groups (epedaphic, hemiedaphic and euedaphic) were analyzed using compound specific isotope analysis (CSIA) of carbon (C) and nitrogen (N) in amino acids (AAs) in a root-trenching experiment. The δ¹⁵N values of phenylalanine, serving as proxy for N resources, were higher in euedaphic Collembola than in ep-/hemiedaphic Collembola, suggesting that euedaphic Collembola mainly utilized N from processed organic substrate in deeper soil, while leaf litter was the dominant N source for ep-/hemiedaphic Collembola. The trophic position calculated from CSIA (TPCSIA) of euedaphic Collembola was higher than that of epedaphic Collembola in most of the studied forest sites, reflecting a higher number of trophic transfers in the soil than in the litter layer. For the first time, we found evidence that the deprivation of root energy supply can decrease the TPCSIA of soil omnivores, suggesting that root-derived C may increase the number of trophic transfers and food chain length in soil food webs. However, effects varied among functional groups of Collembola in different forest stands, indicating that the association between root energy supply and trophic interactions is only partially in line with the ‘productivity hypothesis’ and strongly mediated by vertical resource heterogeneity, regional conditions and feeding strategies of consumers. Finally, our results are in line with recent studies, suggesting that saprotrophic microorganisms, especially saprotrophic fungi, are the predominant resources for Collembola, whereas the contribution of mycorrhizal fungi and plants is subordinate. Overall, by using CSIA of AAs our study provides novel information on trophic niches of microarthropods in forest soils, extending our understanding of the influences of vertical resource heterogeneity and root energy supply on trophic interactions in soil food webs.
... For at least 30 years, stable carbon and nitrogen isotope analyses of individual amino acids in living organisms have provided insights into microbe, plant and animal physiology, trophic dynamics, and ecology (Abelson and Hoering, 1961;Macko et al., 1987;Hayes, 2001;Scott et al., 2006;Ohkouchi et al., 2017). More recently, exploiting systematic differences in the carbon isotope composition of amino acids to tease apart the sources of amino acids in consumer tissues has been termed "isotope fingerprinting" (Larsen et al., 2009). Stable hydrogen isotope analyses of amino acids, on the other hand, have only been conducted recently and have yet to be fully exploited due to a lack of fundamental understanding of how these isotopes are distributed in amino acids Newsome et al., 2020;Morra et al., 2021). ...
... Central carbon fluxes of the mutant E. coli strains are documented to differ from the wild type (Hua et al., 2003;Kabir and Shimizu, 2003;Zhao et al., 2004a,b;Matsuoka and Shimizu, 2013) and growth rate has been shown to alter carbon flux (e.g., Ihssen and Egli, 2004;Kayser et al., 2005) as discussed throughout the text. Despite these known differences in flux, across the different growth rate regimes in continuous culture and in each of the strains grown in batch, there are consistent isotopic fractionation patterns in hydrogen and carbon isotope values and fractionations for the branched [Leu, Ileu, Lys, Val, and Thr] Scott et al., 2006;Larsen et al., 2009) and may or may not be directly comparable to continuous cultures. ...
... Isotopic compositions of amino acids are increasingly being used for determining the sources of matter and energy in marine and terrestrial food webs (Larsen et al., 2009;Wang et al., 2018). For fingerprinting applications, supervised machine learning (LDA) is often used in conjunction with some iterative model parameterization stage to determine the most effective combination of predictors (i.e., amino acids) to parse production sources. ...
Compound specific stable isotope analysis (CSIA) of amino acids from bacterial biomass is a newly emerging powerful tool for exploring central carbon metabolism pathways and fluxes. By comparing isotopic values and fractionations relative to water and growth substrate, the impact of variable flow path for metabolites through different central metabolic pathways, perturbations of these paths, and their resultant consequences on intracellular pools and resultant biomass may be elucidated. Here, we explore the effects that central carbon metabolism and growth rate can have on stable hydrogen (δ2H) and carbon (δ13C) compound specific isotopic values of amino acids, and whether diagnostic isotopic fingerprints are revealed by these paired analyses. We measured δ2H and δ13C in amino acids in the wild type Escherichia coli (MG1655) across a range of growth rates in chemostat cultures to address the unknown isotopic consequences as metabolic fluxes are shuffled between catabolic and anabolic metabolisms. Additionally, two E. coli knockout mutants, one with deficiency in glycolysis –pgi (LC1888) and another inhibiting the oxidative pentose phosphate pathway (OPPP) –zwf (LC1889), were grown on glucose and used as a comparison against the wild type E. coli (MG1655) to address the isotopic changes of amino acids produced in these perturbed metabolic pathways. Amino acid δ2H values, which collectively vary in composition by more than 400‰, are altered along with δ13C values demonstrating fundamental shifts in central metabolic pathways and/or fluxes. Within our linear discriminant analysis with a simple model organism to examine potential amino acid fingerprinting, our knockout strains and variable growth rate samples plot across a wider array of organism classification than merely within the boundaries of other bacterial data.
... SIR in compound level is more tightly associated with the exact biomolecules (e.g., AAs), whereas bulk SIR of whole tissue or total protein depends on inter-molecular isotope differences and the mo- Nonetheless, organisms that share similar biosynthetic/metabolic pathways tend to exhibit analogous patterns of isotope fractionation (e.g., Hayes, 2001;Larsen et al., 2009;Scott et al., 2006). ...
... The dashed lines help get approximate diet proportion composed of different basal members derived from each organic C resources 3.2 | Identifying multiple producers using AA isotope fingerprints AA-SIR has been applied for identifying trophic bases (nutritional origins) in local habitats (Table 1). This is due to several empirical evidences that naturally occurring AA δ 13 C variables are unique to distinguish phylogenetically different producers (Larsen et al., 2009(Larsen et al., , 2013Scott et al., 2006) (Figure 5a), which is known as "stable isotope fingerprints" (Larsen et al., 2009(Larsen et al., , 2013. The stable isotope fingerprints based on AAs are related to phylogenetically distinctive characters of producers, such as how to fix inorganic C substrate (e.g., HCO 3 − and CO 2 ) and how to synthesize C skeletons of AAs (Brett et al., 2017;Macko et al., 1987;Scott et al., 2006) in diverse routes (e.g., glycolysis, the citric acid cycle, and the pentose phosphate pathway). ...
... The dashed lines help get approximate diet proportion composed of different basal members derived from each organic C resources 3.2 | Identifying multiple producers using AA isotope fingerprints AA-SIR has been applied for identifying trophic bases (nutritional origins) in local habitats (Table 1). This is due to several empirical evidences that naturally occurring AA δ 13 C variables are unique to distinguish phylogenetically different producers (Larsen et al., 2009(Larsen et al., , 2013Scott et al., 2006) (Figure 5a), which is known as "stable isotope fingerprints" (Larsen et al., 2009(Larsen et al., , 2013. The stable isotope fingerprints based on AAs are related to phylogenetically distinctive characters of producers, such as how to fix inorganic C substrate (e.g., HCO 3 − and CO 2 ) and how to synthesize C skeletons of AAs (Brett et al., 2017;Macko et al., 1987;Scott et al., 2006) in diverse routes (e.g., glycolysis, the citric acid cycle, and the pentose phosphate pathway). ...
Abstract Explaining food web dynamics, stability, and functioning depend substantially on understanding of feeding relations within a community. Bulk stable isotope ratios (SIRs) in natural abundance are well‐established tools to express direct and indirect feeding relations as continuous variables across time and space. Along with bulk SIRs, the SIRs of individual amino acids (AAs) are now emerging as a promising and complementary method to characterize the flow and transformation of resources across a diversity of organisms, from microbial domains to macroscopic consumers. This significant AA‐SIR capacity is based on empirical evidence that a consumer's SIR, specific to an individual AA, reflects its diet SIR coupled with a certain degree of isotopic differences between the consumer and its diet. However, many empirical ecologists are still unfamiliar with the scope of applicability and the interpretative power of AA‐SIR. To fill these knowledge gaps, we here describe a comprehensive approach to both carbon and nitrogen AA‐SIR assessment focusing on two key topics: pattern in AA‐isotope composition across spatial and temporal scales, and a certain variability of AA‐specific isotope differences between the diet and the consumer. On this basis we review the versatile applicability of AA‐SIR to improve our understanding of physiological processes as well as food web functioning, allowing us to reconstruct dominant basal dietary sources and trace their trophic transfers at the specimen and community levels. Given the insightful and opportunities of AA‐SIR, we suggest future applications for the dual use of carbon and nitrogen AA‐SIR to study more realistic food web structures and robust consumer niches, which are often very difficult to explain in nature.
... bacteria, fungi). Importantly, each of these groups synthesize EAAs using unique biosynthetic pathways that lead to distinct, multivariate patterns in δ 13 C values-termed 'fingerprints'-that differ significantly between green and brown energy sources (Larsen et al., 2009(Larsen et al., , 2013. In contrast, animals cannot synthesize EAAs at rates sufficient to meet metabolic demands and therefore must acquire these molecules from their diets, potentially enabling the use of δ 13 C fingerprints to trace the flow of green versus brown carbon from primary synthesizers to consumers (Harada et al., 2022). ...
... Lastly, we used our controlled data to test the efficacy of two common practices: (1) the use of published δ 13 C source fingerprints (e.g. Larsen et al., 2009Larsen et al., , 2013 to estimate multichannel feeding in consumers and (2) statistical normalization of source data when estimating consumer diets. ...
... To identify δ 13 C fingerprints and predict the origins of EAAs in consumer tissues, we then used linear discriminant analysis (LDA) in the r package mass v7.3-54 (Ripley et al., 2013). LDA maximizes separation of groups in multivariate space and has proved a powerful approach for distinguishing energy channels based on δ 13 C values of EAAs (Larsen et al., 2009(Larsen et al., , 2013. We used the δ 13 C values of all EAAs from the plant, fungi and bacteria protein sources in an LDA to first train the model, and we used leave-one-out cross-validation to assess whether sources could be assigned to their respective groups with high (>80%) classification accuracy. ...
Animals often consume resources from multiple energy channels, thereby connecting food webs and driving trophic structure. Such ‘multichannel feeding’ can dictate ecosystem function and stability, but tools to quantify this process are lacking. Stable isotope ‘fingerprints’ are unique patterns in essential amino acid (EAA) δ ¹³ C values that vary consistently between energy channels like primary production and detritus, and they have emerged as a tool to trace energy flow in wild systems. Because animals cannot synthesize EAAs de novo and must acquire them from dietary proteins, ecologists often assume δ ¹³ C fingerprints travel through food webs unaltered. Numerous studies have used this approach to quantify energy flow and multichannel feeding in animals, but δ ¹³ C fingerprinting has never been experimentally tested in a vertebrate consumer.
We tested the efficacy of δ ¹³ C fingerprinting using captive deer mice Peromyscus maniculatus raised on diets containing bacterial, fungal and plant protein, as well as a combination of all three sources. We measured the transfer of δ ¹³ C fingerprints from diet to consumer liver, muscle and bone collagen, and we used linear discriminant analysis (LDA) and isotopic mixing models to estimate dietary proportions compared to known contributions. Lastly, we tested the use of published δ ¹³ C source fingerprints previously used to estimate energy flow and multichannel feeding by consumers.
We found that EAA δ ¹³ C values exhibit significant isotopic (i.e. trophic) fractionation between consumer tissues and diets. Nevertheless, LDA revealed that δ ¹³ C fingerprints are consistently routed and assimilated into consumer tissues, regardless of isotopic incorporation rate. Isotopic mixing models accurately estimated the proportional diets of consumers, but all models overestimated plant‐based protein contributions, likely due to the digestive efficiencies of protein sources. Lastly, we found that δ ¹³ C source fingerprints from published literature can lead to erroneous diet reconstruction.
We show that δ ¹³ C fingerprints accurately measure energy flow to vertebrate consumers across tissues with different isotopic incorporation rates, thereby enabling the estimation of multichannel feeding at various temporal scales. Our findings illustrate the power of δ ¹³ C fingerprinting for quantifying food web dynamics, but also reveal that careful selection of dietary source data is critical to the accuracy of this emerging technique.
... Furthermore, because some amino acids cannot be synthesised by animals, these essential amino acids are assimilated by consumers with little carbon isotopic enrichment (Howland et al. 2003;McMahon et al. 2010). In addition, δ 13 C values of these amino acids differ between producer groups, allowing identification of the source of carbon in amino acids within consumer tissue (Larsen et al. 2009;McMahon and Newsome 2019). Based on these principles, stable carbon isotope 'fingerprinting' of amino acids (Larsen et al. 2009(Larsen et al. , 2013, has been used by Arthur et al. (2014) to identify the source of carbon in the diet of green, olive ridley, and loggerhead turtles. ...
... In addition, δ 13 C values of these amino acids differ between producer groups, allowing identification of the source of carbon in amino acids within consumer tissue (Larsen et al. 2009;McMahon and Newsome 2019). Based on these principles, stable carbon isotope 'fingerprinting' of amino acids (Larsen et al. 2009(Larsen et al. , 2013, has been used by Arthur et al. (2014) to identify the source of carbon in the diet of green, olive ridley, and loggerhead turtles. ...
... To determine the source origins of essential amino acids in turtle whole blood tissue we use the amino acid stable isotope fingerprinting method (Larsen et al. 2009(Larsen et al. , 2013. A linear discriminant analysis (LDA) was performed using the R package MASS (Venables and Ripley 2002). ...
Diet is fundamental to an individual’s biology because energy acquired from food constrains growth and reproduction, which subsequently influences survival. It is, therefore, important to have a strong understanding of a population’s diet for species of conservation concern, such as the green turtle (Chelonia mydas). While the diet of adult green turtles is generally characterised as primarily herbivorous, growing evidence suggests variation in diet between and within populations is prevalent. We use complementary stable isotope analysis techniques to elucidate diet variation within a C. mydas population (ranging from small juveniles to adults) foraging at Ningaloo Reef in Western Australia. Analyses of multiple tissues and samples from ten individuals recaptured between 4 months and 4.5 years apart revealed that adults showed the highest levels of individual specialisation and consistency in diet over time. Analysis of red blood cell δ¹³C and δ¹⁵N values revealed macroalgae is likely the dominant food source for all size classes, and sub-adult and adults also ate animals (probably jellyfish). Compound-specific stable isotope analysis of amino acids indicated the main sources of essential amino acids for Ningaloo C. mydas were macroalgae or bacteria. Taken together, these results suggest C. mydas at Ningaloo conform to the general description of adult C. mydas diet as predominantly herbivorous, but diet varies with size and between adult individuals. Consideration of within-population diet variation will be important for predicting responses to stressors such as climate change, that directly affect foraging resources, as fitness consequences may vary for individuals with different diets.
... CSI-AA ecological proxies are based on the differences in fractionation of specific AAs between diet and consumer. For C, primary producers synthesize essential AAs (EAA) with a unique δ 13 C "fingerprint" based on evolutionary divergence (Larsen et al., 2009(Larsen et al., , 2013. Since animals must acquire EAAs from their diet, δ 13 C EAA fingerprints are passed up the food chain unaltered, and thus preserve the signature of the primary producer assemblage through trophic transfers (Hare et al., 1991;Howland et al., 2003;McMahon et al., 2010). ...
... Since animals must acquire EAAs from their diet, δ 13 C EAA fingerprints are passed up the food chain unaltered, and thus preserve the signature of the primary producer assemblage through trophic transfers (Hare et al., 1991;Howland et al., 2003;McMahon et al., 2010). Libraries of the EAA δ 13 C values for potential primary producer types for a given ecosystem (e.g., Larsen et al., 2009Larsen et al., , 2013Tipple et al., 2021) combined with Bayesian mixing models (e.g., MixSIAR; Stock and Semmens, 2016) can therefore be used for quantifying shifting baseline resource contribution to consumers within that system. For N, the "trophic AA" group (e.g., Glutamic Acid: Glu) undergo significant transamination/deamination which predictably increases the δ 15 N values with each trophic transfer. ...
... Normality (Q-Q plots) and homoscedasticity (Bartlett's test) of the data were verified before statistical analyses. From bulk isotopes, we calculated total area (TA) and standard ellipse areas (SEA; n > 10) for each site population using the stable isotope Bayesian ellipses in R package (SIBER; Jackson et al., 2011), and with normalized EAA δ 13 C values we used the Bayesian mixing model stable isotope analysis in R package (MixSIAR; Stock and Semmens, 2016) to estimate resource contribution to bivalve diet using a "training set" of potential endmember sources from Larsen et al., 2009and Tipple et al., 2021. ...
Ecological isotope proxies measured in ancient bivalve shell matrix protein have great promise for paleoecological reconstruction. Compound-specific isotopes of amino acids (CSI-AA) may be an ideal tool for developing paleoecological proxies, as initial work has indicated that CSI-AA is less subject to alteration under geologic conditions relative to bulk isotope values. While CSI-AA proxies have been applied in modern bivalve soft tissues and a few shell studies, they have yet to be systematically investigated in shell matrix protein. Here, we measured stable isotope values of carbon (δ¹³C) and nitrogen (δ¹⁵N) in both bulk and individual amino acids (AA), comparing soft tissue and shell organic fractions to test the fidelity of a suite of ecological isotope proxies in shell. We sampled three ubiquitous bivalve species in four seasons for one year from two common coastal environments: littoral rocky intertidal and estuarine delta ecosystems. Particulate organic matter (POM) was simultaneously collected to investigate relationships between tissue types and local POM isotope signatures. The ecological proxies tested include trophic niche breadth from bulk isotopes, and baseline δ¹³C and δ¹⁵N values, resource contribution, and trophic level from CSI-AA data. We found niche breadth corresponded well between tissue types, but that bulk isotope values were significantly higher in shell compared to soft tissue. In the CSI-AA record, there were no differences in baseline δ¹³C and δ¹⁵N proxies between tissue types. While no consistent seasonal trends were observed in the δ¹³C record, summer bulk and baseline δ¹⁵N values were lowest in both ecosystems in both tissues. Food resource contribution estimates from shell also closely matched soft tissue, however trophic level estimates were consistently higher in shell, attributed to a systematic offset in Glutamic acid δ¹⁵N values. We therefore propose here a new mollusk-specific trophic discrimination factor, and corresponding shell isotope enrichment factor to correct for isotopic routing, both required for accurate paleoecological reconstructions.
... Each EAA can be characterised by its δ 13 C, that is the ratio of the heavy but rare carbon-13 stable isotope compared to the lighter but common carbon-12. Another notable advancement lies in the capacity to differentiate primary producers from various phyla based on the relative difference between EAA δ 13 C, an approach commonly referred to as fingerprinting (Larsen et al., 2009). Based on the fingerprinting, studies have estimated the relative importance of producers as food source of several consumers (Arsenault et al., 2022;Liew et al., 2019;Manlick et al., 2024;Manlick & Newsome, 2022;Saboret et al., 2023). ...
... Different phyla of primary producers evolve different anabolism pathways, resulting in distinct δ 13 C differences between EAAs, referred to as fingerprinting (Larsen et al., 2009). A challenge when applying this approach to the wild is to sample the different producers (e.g. ...
Global change drives multiple facets of biodiversity including interaction diversity, which is fundamental for ecosystem functioning. However, studying trophic interactions is challenging in meta‐ecosystems, that is ecosystems connected by spatial flows of energy, materials and organisms across ecosystem boundaries. While analytical methods based on abundances of polyunsaturated fatty acids (PUFAs) and stable isotopes of amino acids (AAs) are being increasingly used, it has never been explored if both approaches could be: (i) combined in mixing models to enhance precision in dietary inference (ii) compared to disentangle transfers of various PUFAs and proteins in food webs in the wild.
We explore the utility of analytical approaches based on PUFA abundances and AA isotopes to resolve resource transfers in a natural riparian food web. We focus on spiders and their potential prey from blue, green and brown sources to address three important and persisting methodological issues in food‐web ecology, namely whether (i) essential AA carbon isotopes can resolve protein origin from blue, green and brown resources, (ii) PUFA relative abundance and AA isotopes can be combined in a mixing model to provide higher precision estimates (i.e. narrower intervals) and (iii) combining the two approaches can unveil the coupling of protein and PUFA transfers in food webs.
Our research demonstrates the power of AA isotopes and PUFAs to distinguish blue, green, and brown sources and their transfer up to consumers. We show that combining PUFA relative abundance and AA isotopes in a mixing model provides overall estimates similar to the individual estimates but significantly increases precision. In addition, we showcase how combining approaches unveil the coupling of protein and PUFA transfers. For instance, we show that most PUFAs are less concentrated from prey to predators, relative to proteins, highlighting uncoupling of PUFAs and protein transfer along food chains.
We show for the first time the effectiveness of combining AA isotopes and PUFA abundances, particularly relevant for complex trophic interactions in a meta‐ecosystem context. Our study illustrates the trophic uncoupling of proteins and PUFAs, highlighting the necessity in combining both approaches.
... Stable isotope ratios of 13 C/ 12 C of amino acids, on the other hand, allow identifying major basal resources of food chains, such as plants, bacteria, and fungi (Larsen et al., 2009;Pollierer et al., 2019), as these phylogenetically old groups feature some distinct or modified synthesis pathways for amino acids (Hagelstein et al., 1997;Hudson et al., 2005;Kohlhaw, 2003;Velasco et al., 2002). These distinct 13 C patterns can be traced in essential amino acids (δ 13 C eAA ) of consumers ("stable isotope fingerprinting"; Larsen et al., 2009) and thereby allow to quantify energy fluxes via the major energy channels of soil food webs. ...
... Stable isotope ratios of 13 C/ 12 C of amino acids, on the other hand, allow identifying major basal resources of food chains, such as plants, bacteria, and fungi (Larsen et al., 2009;Pollierer et al., 2019), as these phylogenetically old groups feature some distinct or modified synthesis pathways for amino acids (Hagelstein et al., 1997;Hudson et al., 2005;Kohlhaw, 2003;Velasco et al., 2002). These distinct 13 C patterns can be traced in essential amino acids (δ 13 C eAA ) of consumers ("stable isotope fingerprinting"; Larsen et al., 2009) and thereby allow to quantify energy fluxes via the major energy channels of soil food webs. ...
Mountain forests are at risk as the consequences of climate change will likely lead to altered tree species boundaries. Characterizing food webs along elevation gradients in primary forests may help to predict the potential consequences of such changes, for example with regard to the decomposition of dead organic matter. Here, for the first time, we studied trophic variations in two species‐rich microarthropod taxa, Collembola and Oribatida, along an elevation gradient of primary forest at Changbai Mountain, China. Samples were taken at seven elevations of 150‐m elevational difference between 800 and 1700 m. At each elevation, Collembola and Oribatida were extracted from litter samples of eight subplots. We applied three state‐of‐the‐art methods to elucidate trophic positions and basal resource use at community level: Bulk stable isotope analysis of nitrogen (Δ ¹⁵ N bulk ) and carbon (Δ ¹³ C bulk ), compound‐specific stable isotope analysis of amino acids (CSIA‐AA), and dietary routing of neutral lipid fatty acids (NLFAs). Trophic positions calculated using Δ ¹⁵ N bulk and CSIA‐AA (TP CSIA ) in both taxa increased similarly with elevation by about half and one third of a trophic position, respectively. Stable isotope mixing models and linear discriminant analysis bootstrapping using δ ¹³ C of essential amino acids indicated fungi as the most important resource at all elevations for both taxa. Also, proportions of marker NLFAs changed little across elevations in both taxa; overall high proportions of linoleic acid indicated high fungal contributions, but in Collembola the contribution of bacterial markers was generally higher than in Oribatida. Δ ¹³ C bulk did not respond linearly to the elevation gradient; however, changes in elevation differed between Collembola and Oribatida. A strong linear relationship between δ ¹⁵ N of phenylalanine and δ ¹⁵ N of litter indicated litter as the basis of energy channels in both taxa. Overall, food web functioning likely changes with changing forest types along elevation gradients, with microarthropods switching from feeding closer to the base of the food web at lower elevations to feeding at higher trophic levels at higher elevations, potentially compromising their role in litter decomposition and nutrient cycling.
... The δ 13 C values of both AA NESS and AA ESS have been used to study animal ecology (Phillips et al. 2020) and ecophysiology (O'Brien et al. 2002;Barreto-Curiel et al. 2017;Hesse et al. 2022) and to identify and quantify the sources of production fueling consumers at different levels in the food chain (Elliott Smith et al. 2018). One notable example is AA ESS δ 13 C "fingerprinting" (Scott et al. 2006;Larsen et al. 2009; which has been employed to study energy channel use by consumers in a variety of ecosystems (Larsen et al. 2013;Elliott Smith et al. 2018Fox et al. 2019;Pollierer and Scheu 2021). This technique relies on differences in patterns among AA ESS δ 13 C values, or "fingerprints", (Larsen et al. 2013) of different functional groups at or near the base of food webs such as terrestrial plants, phytoplankton, macroalgae, bacteria, and fungi (Scott et al. 2006;Larsen et al. 2009). ...
... One notable example is AA ESS δ 13 C "fingerprinting" (Scott et al. 2006;Larsen et al. 2009; which has been employed to study energy channel use by consumers in a variety of ecosystems (Larsen et al. 2013;Elliott Smith et al. 2018Fox et al. 2019;Pollierer and Scheu 2021). This technique relies on differences in patterns among AA ESS δ 13 C values, or "fingerprints", (Larsen et al. 2013) of different functional groups at or near the base of food webs such as terrestrial plants, phytoplankton, macroalgae, bacteria, and fungi (Scott et al. 2006;Larsen et al. 2009). These AA ESS δ 13 C fingerprints are assumed to be passed up food chains with little isotopic modification since eukaryotic consumers cannot synthesize these compounds de novo (McMahon et al. 2010;Whiteman et al. 2019). ...
The measurement of stable isotope values of individual compounds, such as amino acids (AAs), has become a powerful tool in animal ecology and ecophysiology. As with any emerging technique, questions remain regarding the capabilities and limitations of this approach, including how metabolism and tissue synthesis impact the isotopic values of individual AAs and subsequent multivariate patterns. We measured carbon isotope (δ¹³C) values of essential (AAESS) and nonessential (AANESS) AAs in bone collagen, whisker, muscle, and liver from ten southern sea otters (Enhydra lutris nereis) that stranded in Monterey Bay, California. Sea otters in this population exhibit high degrees of individual dietary specialization, making this an excellent dataset to explore differences in AA δ¹³C values among tissues in a wild population. We found the δ¹³C values of the AANESS glutamic acid, proline, serine, and glycine and the AAESS threonine differed significantly among tissues, indicating possible isotopic discrimination during tissue synthesis. Threonine δ¹³C values were higher in liver relative to bone collagen and muscle, which may indicate catabolism of threonine for gluconeogenesis, an interpretation further supported by correlations between the δ¹³C values of threonine and its gluconeogenic products glycine and serine in liver. This intraindividual isotopic variation yielded different ecological interpretations among tissues; for 6/10 of the sea otter individuals analyzed, at least one tissue indicated reliance on a different primary producer source than the other tissues. Our results highlight the importance of gluconeogenesis in a carnivorous marine mammal and indicate that metabolic processes influence AAESS and AANESSδ¹³C values and multivariate AA δ¹³C patterns.
... Following Scott et al. (2006), Larsen et al. (2009) found that patterns of δ 13 C in the essential amino acids in primary producers such as plants, bacteria, and fungi consistently differed among groups (see also Scott et al. 2006). By applying multivariate ordination techniques (PCA), these authors were able to show distinct clustering of isotopic data in bivariate space that separated these primary producers. ...
... Subsequent papers used linear discriminant analysis (LDA) to show how samples could be assigned to such groups as a form of essential amino acid δ 13 C "fingerprinting". Since that investigation of terrestrial primary production (Larsen et al. 2009), studies have confirmed the multivariate fingerprinting approach to identifying sources of primary production in terrestrial (Besser et al. 2022), and freshwater aquatic (Besser et al. 2022) and marine (Larsen et al. 2013;Elliott Smith et al. 2022) food webs. Because δ 13 C values in essential amino acids are conserved trophically, the CSIA approach offers considerable advantages over the bulk tissue isotope approach for the purpose of identifying sources of primary production. ...
The measurement of naturally occurring stable isotope ratios of the light elements (C, N, H, O, S) in animal tissues and associated organic and inorganic fractions of associated environments holds immense potential as a means of addressing effects of global change on animals. This paper provides a brief review of studies that have used the isotope approach to evaluate changes in diet, isotopic niche, contaminant burden, reproductive and nutritional investment, invasive species and shifts in migration origin or destination with clear links to evaluating effects of global change. This field has now reached a level of maturity that is impressive but generally underappreciated and involves technical as well as statistical advances and access to freely available R-based packages. There is a need for animal ecologists and conservationists to design tissue collection networks that will best answer current and anticipated questions related to the global change and the biodiversity crisis. These developments will move the field of stable isotope ecology toward a more hypothesis driven discipline related to rapidly changing global events.
... There is tremendous diversity in the enzymatic synthesis pathways, carbon concentration mechanisms, and carbon fixation pathways that evolutionarily distinct primary producers use to make their amino acids (Hayes 2001;Scott et al. 2006). Taxon-specific biosynthetic processes have significant variations in carbon isotope dynamics (Hayes 2001;Scott et al. 2006;Bianchi and Canuel 2011;Besser et al. 2022), yielding distinct patterns or "fingerprints" when the δ 13 C values of multiple amino acids (δ 13 C AA ) diagnostic of their primary producer sources are considered together (Scott et al. 2006;Larsen et al. 2009Larsen et al. , 2013. Furthermore, the complex side chains of essential amino acids (EAA) typically cannot be de novo biosynthesized by most metazoans and must be directly acquired from diet with minimal isotopic discrimination (McMahon et al. 2010). ...
... Generally, the CSIA-AA approach has used multivariate coordinate space of EAAs (e.g., Thr, Ile, Val, Phe, and Leu), termed δ 13 C EAA fingerprints, to distinguish among broad taxonomic groups of primary producers, for example, among bacteria, fungi, terrestrial plants, and marine algae (Larsen et al. 2009(Larsen et al. , 2013. These broad taxonomic fingerprints have been used to reconstruct microalgae production in a coastal upwelling zone (Vokhshoori et al. 2014), examine grazing vs. detritivory in mangrove food webs (Harada et al. 2022), examine nutritional sources for facultative hindgut fermenting green sea turtles (Arthur et al. 2014), and characterize trophic niches of fish functional groups (Larsen et al. 2020). ...
Eukaryotic microalgae play critical roles in the structure and function of marine food webs. The contribution of microalgae to food webs can be tracked using compound‐specific isotope analysis of amino acids (CSIA‐AA). Previous CSIA‐AA studies have defined eukaryotic microalgae as a single functional group in food web mixing models, despite their vast taxonomic and ecological diversity. Using controlled cultures, this work characterizes the amino acid δ ¹³ C ( δ ¹³ C AA ) fingerprints—a multivariate metric of amino acid carbon isotope values—of four major groups of eukaryotic microalgae: diatoms, dinoflagellates, raphidophytes, and prasinophytes. We found excellent separation of essential amino acid δ ¹³ C ( δ ¹³ C EAA ) fingerprints among four microalgal groups (mean posterior probability reclassification of 99.2 ± 2.9%). We also quantified temperature effects, a primary driver of microalgal bulk carbon isotope variability, on the fidelity of δ ¹³ C AA fingerprints. A 10°C range in temperature conditions did not have significant impacts on variance in δ ¹³ C AA values or the diagnostic microalgal δ ¹³ C EAA fingerprints. These δ ¹³ C EAA fingerprints were used to identify primary producers at the base of food webs supporting consumers in two contrasting systems: (1) penguins feeding in a diatom‐based food web and (2) mixotrophic corals receiving amino acids directly from autotrophic endosymbiotic dinoflagellates and indirectly from water column diatoms, prasinophytes, and cyanobacteria, likely via heterotrophic feeding on zooplankton. The increased taxonomic specificity of CSIA‐AA fingerprints developed here will greatly improve future efforts to reconstruct the contribution of diverse eukaryotic microalgae to the sources and cycling of organic matter in food web dynamics and biogeochemical cycling studies.
... First, essential AAs (EAAs) are only synthetised at the basis of food webs by primary producers, meaning that the carbon skeleton and δ 13 C values EAAs remain largely unchanged up the food web (Takizawa et al., 2020). This can help alleviate some of the uncertainties in mixing models based on baseline variability of δ 13 C. Second, different phyla of primary producers leave consistent δ 13 C offsets between AAs (i.e., the δ 13 C difference between pairs of AAs is fixed) referred to as carbon fingerprinting (Larsen et al., 2009), which is believed to be mostly independent of source δ 13 C and metabolism Larsen et al., 2013). In recent studies, the C fingerprinting of algae and vascular plants revealed high levels of autochthony in large rivers (Thorp & Bowes, 2017) or cave fish (Liew et al., 2019), two ecosystems that were believed to depend mainly on OC Terr . ...
... Since the first description of the C fingerprinting method (Larsen et al., 2009;Scott et al., 2006), a decade of growing evidence supporting (2) and (3) Organic carbon in lakes could be derived from three sources of primary producers: vascular plants (allochthonous production); algae and cyanobacteria (autochthonous production); and bacteria (recycling of organic matter or chemoautotrophy) (Brett et al., 2017;Cole et al., 2006;Vlah et al., 2018). Based on a first dataset, Larsen et al. (2013) were able to distinguish among plants, bacteria and aquatic phototrophs, which comprised microalgae and cyanobacteria. ...
Lakes are recipients of allochthonous organic matter and nutrients. However, the importance of these subsidies for food webs and how they vary with lake trophic status remains unclear, especially for large lakes.
We assessed the source and fate of organic matter and nutrients in seven perialpine lakes across a gradient of trophic status. We measured carbon and nitrogen stable isotopes of amino acids of lake‐residing Atlantic trout, Salmo trutta , to determine the source of primary production (i.e., how carbon is fixed in the ecosystem) and how it is transferred through food webs, respectively. Based on essential amino acid carbon fingerprinting, we estimated the probability of organic carbon originating from autochthonous (algal), allochthonous (terrestrial plant), and recycled (bacterial) sources. In addition, we used amino acid δ ¹⁵ N to track how this primary production is transferred to consumers in general, and by using different trophic amino acids (glutamic acid and alanine), identify the trophic pathways involving either metazoan or protozoans.
We found a high likelihood of autochthonous origin of organic carbon (86 ± 9%) in trout that contrasted with allochthonous origins of particulate organic matter and some sediments. We showed that those estimates are good proxies of source reliance. Our results also highlighted the importance of bacterial origin of organic carbon in fish (12%). The likely autochthonous origin of this carbon was supported by trophic markers (Ala δ ¹⁵ N) that suggest the role of protists in transferring recycled organic carbon up the food web. While the sources of nitrogen sustaining food webs varied among lakes, we found a conserved carbon fingerprinting of fish. Overall, this suggests an uncoupling between the source of nutrients and organic carbon in large perialpine lakes.
Across a wide range of trophic status ( c. 2 orders of magnitude range of phosphorus concentration), several lines of evidence suggested that perialpine lake food webs shared a common reliance on autochthonous and bacterial production.
Our study is the first to quantify the dependence on allochthonous organic carbon in lake food webs based on new amino acid stable isotope markers (carbon fingerprinting and Ala δ ¹⁵ N) and shows promise for estimating the source of carbon fixation in ecosystems. Our results support previous suggestions that terrestrial organic carbon is a relatively minor source for aquatic consumers despite contributing to the pool of organic matter, and more importantly, its contribution does not vary substantially with trophic status in perialpine lakes.
... In what many consider to be the most rigorous application of SIA, CSIA-AA provides less ambiguous insights into the trophic biology of corals [73,74]. The analysis of both δ 15 N (trophic versus source amino acids) and δ 13 C (essential versus non-essential amino acids) values can be used effectively to unravel trophic sources and sinks with their associated metabolic processes and with better resolution than bulk tissue isotope analysis because the confounding influence of trophic fractionation is largely absent [75][76][77][78]. Algae, bacteria, and fungi have highly conserved modes of carbon acquisition and amino acid biosynthesis that produce unique patterns of carbon isotopic fractionation that can be used to "fingerprint" their biosynthetic origin [75,76]. ...
... The analysis of both δ 15 N (trophic versus source amino acids) and δ 13 C (essential versus non-essential amino acids) values can be used effectively to unravel trophic sources and sinks with their associated metabolic processes and with better resolution than bulk tissue isotope analysis because the confounding influence of trophic fractionation is largely absent [75][76][77][78]. Algae, bacteria, and fungi have highly conserved modes of carbon acquisition and amino acid biosynthesis that produce unique patterns of carbon isotopic fractionation that can be used to "fingerprint" their biosynthetic origin [75,76]. This "fingerprinting" approach and the isotopic fingerprints of different amino acid carbon source end members in CSIA have been shown to be faithfully maintained through a coral reef food web [77,78] and to study the trophic ecology of sponges from shallow to mesophotic depths [79]. ...
Studies on the trophic ecology of scleractinian corals often include stable isotope analyses of tissue and symbiont carbon and nitrogen. These approaches have provided critical insights into the trophic sources and sinks that are essential to understanding larger-scale carbon and nitrogen budgets on coral reefs. While stable isotopes have identified most shallow water (<30 m) corals as mixotrophic, with variable dependencies on autotrophic versus heterotrophic resources, corals in the mesophotic zone (~30–150 m) transition to heterotrophy with increasing depth because of decreased photosynthetic productivity. Recently, these interpretations of the stable isotope data to distinguish between autotrophy and heterotrophy have been criticized because they are confounded by increased nutrients, reverse translocation of photosynthate, and changes in irradiance that do not influence photosynthate translocation. Here we critically examine the studies that support these criticisms and show that they are contextually not relevant to interpreting the transition to heterotrophy in corals from shallow to mesophotic depths. Additionally, new data and a re-analysis of previously published data show that additional information (e.g., skeletal isotopic analysis) improves the interpretation of bulk stable isotope data in determining when a transition from primary dependence on autotrophy to heterotrophy occurs in scleractinian corals.
... In contrast, CSIA has gained momentum in food web studies only over the last 20 years focusing almost exclusively on SIs of nitrogen and carbon (McClelland and Montoya 2002;Chikaraishi et al. 2009;Larsen et al. 2009;McMahon and McCarthy 2016), with some first applications of hydrogen SI (Fogel et al. 2016;Pilecky et al. 2021). CSIA has a number of potential advantages over BSIA, including the possibility to determine consumer trophic levels without the need for external baseline samples, and an increased power to differentiate among basal resources or dietary sources. ...
... Similarly, the even lower number of BSIA studies focusing on d 2 H and d 18 O (Deutsch and Berth 2006;Bartels et al. 2018) contrasts with the usefulness of these isotopes to generate isoscapes and of d 2 H to contribute to trophic studies (Vander Zanden et al. 2016). Regarding CSIA, the late onset and low number of studies in the Baltic contrast with the rapidly growing number of studies globally (McMahon and McCarthy 2016), and are unfortunate given the demonstrated applicability of CSIA to elucidate organic matter sources at the base of food webs (Larsen et al. 2009) that are so essential for Baltic Sea food web understanding. Likely explanations for these patterns include the higher entry barriers compared to ''traditional'' BSIA, due to the technological and methodological challenges of d 2 H and d 18 O BSIA (Vander Zanden et al. 2016) and CSIA (Nielsen et al. 2018), confounded by the substantially higher cost of the latter. ...
Food web research provides essential insights into ecosystem functioning, but practical applications in ecosystem-based management are hampered by a current lack of knowledge synthesis. To address this gap, we provide the first systematic review of ecological studies applying stable isotope analysis, a pivotal method in food web research, in the heavily anthropogenically impacted Baltic Sea macro-region. We identified a thriving research field, with 164 publications advancing a broad range of fundamental and applied research topics, but also found structural shortcomings limiting ecosystem-level understanding. We argue that enhanced collaboration and integration, including the systematic submission of Baltic Sea primary datasets to stable isotope databases, would help to overcome many of the current shortcomings, unify the scattered knowledge base, and promote future food web research and science-based resource management. The effort undertaken here demonstrates the value of macro-regional synthesis, in enhancing access to existing data and supporting strategic planning of research agendas.
... Scott et al. (2006) found that carbon SIA-AA could elucidate variations in microbial metabolism when the carbon isotope composition of AAs in bacterial cultures was analysed with discriminant function analysis. Larsen et al. (2009) applied this concept in a natural system to differentiate basal sources of carbon to consumers (e.g., plant, fungal, bacterial), as well as corroborate known insect diets through a comparison of δ 13 C of AAs in dietary items with those in consumers. Since then, this methodology has been applied to a diverse array of questions involving a variety of carbon sources supporting consumers in freshwater (Besser et al., 2022;Bowes & Thorp, 2015; , terrestrial (Chua et al., 2021;Larsen et al., 2016), mangrove , and marine and coastal systems (Larsen et al., 2015;Smith et al., 2018;Smith, Fox, et al., 2022;Smith, Nakamoto, et al., 2022), as well as aquaculture authentication (Wang et al., 2018). ...
... We recoded the taxonomic information available in the original publication to four classes to facilitate interpretation in our system: algae, bacteria, fungi and plants. In total, we used previously published δ 13 C values for eight AAs (Ala, Gly, Glx, Asx, Phe, Val, Leu, Ile) (Larsen 2009(Larsen , 2013). ...
We used compound‐specific isotope analysis of carbon isotopes in amino acids (AAs) to determine the biosynthetic source of AAs in fish from major tributaries to California's Sacramento‐San Joaquin river delta (i.e., the Sacramento, Cosumnes and Mokelumne rivers). Using samples collected in winter and spring between 2016 and 2019, we confirmed that algae are a critical component of floodplain food webs in California's Central Valley. Results from bulk stable isotope analysis of carbon and nitrogen in producers and consumers were adequate to characterize a general trophic structure and identify potential upstream and downstream migration into our study site by American shad Alosa sapidissima and rainbow trout Oncorhynchus mykiss, respectively. However, owing to overlap and variability in source isotope compositions, our bulk data were unsuitable for conventional bulk isotope mixing models. Our results from compound‐specific carbon isotope analysis of AAs clearly indicate that algae are important sources of organic matter to fish of conservation concern, such as Chinook salmon Oncorhynchus tshawytscha in California's Central Valley. However, algae were not the exclusive source of energy to metazoan food webs. We also revealed that other sources of AAs, such as bacteria, fungi and higher plants, contributed to fish as well. While consistent with the well‐supported notion that algae are critical to aquatic food webs, our results highlight the possibility that detrital subsidies might intermittently support metazoan food webs.
... In contrast, CSIA has gained momentum in food web studies only over the last 20 years focusing almost exclusively on SIs of nitrogen and carbon (McClelland and Montoya 2002;Chikaraishi et al. 2009;Larsen et al. 2009;McMahon and McCarthy 2016), with some first applications of hydrogen SI (Fogel et al. 2016;Pilecky et al. 2021). CSIA has a number of potential advantages over BSIA, including the possibility to determine consumer trophic levels without the need for external baseline samples, and an increased power to differentiate among basal resources or dietary sources. ...
... Similarly, the even lower number of BSIA studies focusing on d 2 H and d 18 O (Deutsch and Berth 2006;Bartels et al. 2018) contrasts with the usefulness of these isotopes to generate isoscapes and of d 2 H to contribute to trophic studies (Vander Zanden et al. 2016). Regarding CSIA, the late onset and low number of studies in the Baltic contrast with the rapidly growing number of studies globally (McMahon and McCarthy 2016), and are unfortunate given the demonstrated applicability of CSIA to elucidate organic matter sources at the base of food webs (Larsen et al. 2009) that are so essential for Baltic Sea food web understanding. Likely explanations for these patterns include the higher entry barriers compared to ''traditional'' BSIA, due to the technological and methodological challenges of d 2 H and d 18 O BSIA (Vander Zanden et al. 2016) and CSIA (Nielsen et al. 2018), confounded by the substantially higher cost of the latter. ...
... A new approach to identify phytoplankton origins of organic matter in marine ecosystems is through the use of compound-specific carbon isotope analysis of amino acids (McCarthy et al. 2004;Close 2019). This technique is based on the characteristic carbon isotope fractionation that occurs during the biosynthesis of major groups of organisms (Hayes 2001;Larsen et al. 2009). Essential amino acids like leucine and isoleucine can be transferred across food chains with no fractionation, which is thought to be due to the absence of pathways to synthesize essential amino acids and to the resulting obligate uptake of these amino acids from the food source (O'Brien et al. 2002). ...
Identifying the phytoplankton origin of particulate organic matter (POM) in the deep ocean is challenging due to the changing phytoplankton composition and varied extent of decomposition by heterotrophic bacteria and zooplankton. Here, we report vertical distributions of amino acid stable carbon isotope values (δ¹³C) measured in particles in the South China Sea. Carbon‐weighted amino acid δ¹³C values generally parallel bulk POM δ¹³C profiles with a positive offset from 2.4‰ in the surface water to 6.0‰ in deep. Accordingly, a lability model is introduced to describe the relative distribution and isotopic linkage of labile and refractory particulate organic carbon. Temporal changes of amino acid δ¹³C were observed during the microbial decomposition of two phytoplankton strains, the prokaryote cyanobacterium Synechococcus and the eukaryote diatom Thalassiosira weissflogii. Principal component analysis of individual amino acid δ¹³C values from the decomposing cells separate samples into two components, reflecting the influence of microbial decomposition and taxonomic differences, respectively. These two components were further applied to particles. A major contribution from decomposing phytoplankton to particles below the euphotic zone was observed, with more prokaryote organic matter in the basin and a larger contribution from eukaryotes at stations near the productive northern shelf. Our findings show the applicability of amino acid δ¹³C values in tracing the lability and phytoplankton origins of POM in samples with varied extents of decomposition in marine environments.
... When this second caveat is not met, it indicates the presence of an alternate intrinsic source(s) of EAAs for the consumer. Following this, the source of alternative EAAs in insects can be inferred with source diagnostic d 13 C EAA patterns of bacteria, fungi, and plants (5). This approach has been used to determine alternate EAA contribution in a variety of macroinvertebrates (6)(7)(8)(9), as well as insects (10)(11)(12)(13). ...
Introduction
Building on our previous work, we investigate how dietary shifts affect gut microbial essential amino acid (EAA) provisioning in the lactating cockroach Diploptera punctata .
Method
To that end, we fed cockroaches three distinct diets: a plant-only Gari diet composed of starchy and granulated root tuber Yucca ( Manihot esculenta ), a dog food diet (DF), and a cellulose-amended dog food (CADF) diet. We anticipated that the high carbohydrate, low protein Gari would highlight increased microbial EAA supplementation to the host.
Results
By day 28, we observed distinct profiles of 14 bacterial families in the insect gut microbiomes of the three dietary groups. CADF-fed insects predominantly harbored cellulolytic and nitrogen-fixing bacteria families Streptococcaceae and Xanthomonadaceae . In contrast, Gari-fed insects were enriched in anaerobic lignocellulolytic bacteria families Paludibacteraceae and Dysgonomonadaceae , while DF-fed insects had a prevalence of proteolytic anaerobes Williamwhitmaniaceae and sulfate-reducing bacteria Desulfovibrionaceae . Furthermore, we confirmed significantly higher EAA supplementation in Gari-fed insects than in non-Gari-fed insects based on δ ¹³ C-EAA offsets between insect and their diets. The δ ¹³ C-EAA offsets between DF and CADF were nearly indistinguishable, highlighting the relevance of using the plant-based Gari in this experiment to unequivocally demonstrate this function in this insect. These results were underscored by lower standard metabolic rate (SMR) relative to the DF insect in Gari-fed (intermediate SMR and dietary quality) and CADF (least SMR and dietary quality) insects.
Discussion
The influence of diet on EAA provisioning and SMR responses in insects underscores the need for further exploration into the role of gut microbial functions in modulating metabolic responses
... An emerging alternative approach is measuring the 13 C values of essential amino acids. Plants and algae have distinct patterns in essential amino acid 13 C values ( 13 C EAA ) due to di erences in the way(s) each producer type synthesizes these compounds (Larsen et al., 2009(Larsen et al., , 2013Besser et al., 2022). Most eukaryotic consumers cannot synthesize essential amino acids de novo and must route these compounds directly from the protein in their food, leading to minimal isotope alteration of essential amino acids as they are passed up the food chain (McMahon et al., 2015;Whiteman et al., 2019;Manlick and Newsome, 2022). ...
Land-use conversion and resulting habitat fragmentation can affect the source(s) of primary productivity that fuels food webs and alter their structure in ways that leads to biodiversity loss. We investigated the effects of landscape modification on food webs in the Araguaia River floodplain in central Brazil using the top predator, and indicator species Caiman crocodilus (Crocodilia, Alligatoridae). We measured carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope values of three tissues with different isotopic incorporation rates to evaluate spatial and temporal changes in caiman isotopic niche width with hierarchical Bayesian models that accounted for habitat use, intraspecific trait variation (sex and body size), and landscape attributes (composition and configuration). We also measured δ¹³C values of essential amino acids to assess if different primary producers are fueling aquatic food webs in natural and anthropogenic areas. Spatial analysis showed that caiman in agricultural areas had larger isotopic niche widths, which likely reflects some use of terrestrial resources in environments dominated by C4 plants. Patterns in δ¹³C values among essential amino acids were clearly different between natural and anthropogenic habitats. Overall, our findings suggest that caimans can persist in heterogeneous landscapes fueled by natural and agricultural energy sources of energy, which has implications for effectively managing such landscapes to maximize biodiversity.
... tools for studying food web structures, trophic relationships, and biogeochemical processes in the ecosystem (see Ohkouchi et al 2017, 1 Whiteman et al 2019, 2 Yun et al 2022, 3 and Ohkouchi 2023 4 for general reviews). The δ 13 C values of AAs are "fingerprints" of the biosynthetic sources of AAs and are used to trace the carbon sources in food webs, [5][6][7] and δ 15 N values of AAs can be used to calculate the trophic positions of species and to reconstruct the food web structures in aquatic and terrestrial ecosystems. [8][9][10][11] For the isotope analyses of individual AAs, most studies have used gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), 12 which is convenient and applicable to the analyses of a variety of samples (see Silverman et al 13 for a general review on the analytical methods). ...
Rationale
Isolation of underivatized amino acids (AAs) using high‐performance liquid chromatography (HPLC) is becoming a popular method for carbon (δ ¹³ C) and nitrogen isotope (δ ¹⁵ N) analyses of AAs because of the high analytical precision and for performing dual‐isotope analysis. However, some AAs in natural samples, especially small, hydrophilic AAs, are not suitably separated using reversed‐phase columns (e.g., C18) and ion‐exchange columns (e.g., Primesep A).
Methods
We developed a new method for HPLC using a porous graphitic carbon column for the separation of nine hydrophilic AAs. After purification, δ ¹³ C and δ ¹⁵ N values of AAs were determined using elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). We demonstrated the application of this method by determining δ ¹³ C and δ ¹⁵ N values of individual hydrophilic AAs in a biological sample, the muscle of blue mackerel ( Scomber australasicus ).
Results
Chromatographically, the baseline separation of hydrophilic AAs was achieved in both the standard mixture and the biological sample. We confirmed that δ ¹³ C and δ ¹⁵ N values of AA standards remained unchanged during the whole experimental procedure. The δ ¹³ C values of AAs in mackerel muscle are also in good agreement with the values obtained using another verified method for δ ¹³ C analysis.
Conclusions
The good separation performance of hydrophilic AAs and the reliability of δ ¹³ C and δ ¹⁵ N analyses of individual AAs using the porous graphite column offer a significant advantage over conventional settings. We suggest that, in the future, the HPLC × EA/IRMS method can be used for reliable δ ¹³ C and δ ¹⁵ N analyses of AAs in natural samples.
... development of a variety of approaches (e.g., Phillips et al. 2005;Parnell et al. 2010) but challenges will persist in systems where sources share very similar δ 13 C and δ 15 N values and therefore cannot be distinguished as unique dietary items. With compound-specific analysis, sources can often be assigned unique isotopic fingerprints (Larsen et al. 2009), avoiding these issues and providing insight into complex ecosystems (see Whiteman et al. 2019 for a review). ...
The application of fatty acid (FA) isotopic analysis has great potential in elucidating food web structure, but it has not experienced the same wide-spread use as amino acid isotopic analyses. The failure to adopt FA isotopic methods is almost certainly linked to a lack of reliable information on trophic fractionation of FA, particularly in higher predators. In this work, we attempt to address this shortfall, through comparison of FA δ¹³C values in captive Atlantic pollock (Pollachius virens) liver and their known diets. Since catabolism is likely the main cause of fractionation and it may vary with dietary fat content, we investigated the impact of dietary fat concentration on isotopic discrimination in FA. We fed Atlantic pollock three formulated diets with similar FA isotopic compositions but different fat concentrations (5–9% of diet), representative of the range found in natural prey, for 20 weeks. At the conclusion of the study, δ¹³C values of liver FA were very similar to the FA within the corresponding diets, with most discrimination factors < 1. For all FA except 22:6n-3, dietary fat had no effect on discrimination factors. Only for 22:6n-3 did fish fed the highest fat diet have lower δ¹³C values than the diet consumed. Thus, these FA-specific discrimination factors can be applied to evaluate diets in marine fish consuming natural diets and will serve as additional and valuable biomarkers in fish feeding ecology.
... Animals cannot synthesise the carbon skeletons of essential AAs and must therefore route them directly from diet or the gut microbiome with minimal isotopic fractionation (McMahon et al., 2010;Newsome et al., 2020). Pathways of de novo essential AA synthesis differ among plants, algae, bacteria and fungi, leading to unique essential AA δ 13 C "fingerprints" among these potential energy channels (Besser et al., 2022;Elliott Smith et al., 2022;Larsen et al., 2009Larsen et al., , 2012. Recent work using δ 13 C analysis of essential AAs in consumer tissues has proven robust for tracing the contribution of different energy channels to individuals and communities (Elliott Smith et al., 2021;Manlick et al., 2023;Skinner et al., 2021). ...
1. Identifying potential links between food web structure and animal body condition is fundamental for predicting the long-term persistence of populations under rapidly changing environments. Northern lakes, and the cold water adapted species that inhabit them, are particularly vulnerable to a warming climate.
2. We explored relationships among body condition and energy channel use in lake trout (Salvelinus namaycush) and seasonal variation in food-chain length in the Lake of Two Rivers, Ontario, Canada. Bayesian mixing models using carbon isotope values (δ13C) of essential amino acids showed that individuals utilizing multiple energy channels had higher gonad mass, suggesting dietary diversity has reproductive fitness benefits.
3. Amino acid δ15N analysis revealed seasonal variation in food chain length, with extension of the food web during winter and spring and truncation of the food web during summer. These findings illustrate increased omnivory of lake trout during summer, however, there was no clear evidence that seasonal changes in food-chain length impacted trout condition.
4. These findings provide new insight into potential relationships between food web structure, energy flow, and consumer condition in freshwater ecosystems, thereby revealing potential responses of species to shifting ecosystem states under intensifying climate change at high latitudes.
... AA ESS δ 13 C 'fingerprinting' (Larsen et al., 2009(Larsen et al., , 2013Scott et al., 2006) is a promising approach for quantifying gut microbial AA ESS contributions to host tissues in wild animals. Organisms capable of AA ESS synthesis (e.g. ...
Herbivory is a dominant feeding strategy among animals, yet herbivores are often protein limited. The gut microbiome is hypothesized to help maintain host protein balance by provisioning essential macromolecules, but this has never been tested in wild consumers. Using amino acid carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope analysis, we estimated the proportional contributions of essential amino acids (AAESS) synthesized by gut microbes to five co‐occurring desert rodents representing herbivorous, omnivorous and insectivorous functional groups. We found that herbivorous rodents occupying lower trophic positions (Dipodomys spp.) routed a substantial proportion (~40%–50%) of their AAESS from gut microbes, while higher trophic level omnivores (Peromyscus spp.) and insectivores (Onychomys arenicola) obtained most of their AAESS (~58%) from plant‐based energy channels but still received ~20% of their AAESS from gut microbes. These findings empirically demonstrate that gut microbes play a key functional role in host protein metabolism in wild animals.
... Besides standard analyses of the bulk δ 13 C, the δ 13 C of amino acid (δ 13 C AA ) patterns can often transcend bulk isotope analyses. Research has shown that naturally occurring δ 13 C AA patterns contain information of both biosynthetic origin and mode of carbon acquisition (Scott et al. 2006), and that the essential AA group is particularly diagnostic of origin (Larsen et al. 2009(Larsen et al. , 2013. Therefore, these ''stable isotope fingerprints'' were proposed as a tool for tracing sources of organic matter in terrestrial ecosystems, and hence these techniques could additionally be used to trace sources of organic fertilizer. ...
The steadily growing demand for fertilizers and increasing interest for organic inputs result in rapid expansion and diversification of the solid nitrogen (N) fertilizer market. Fertilizer legislations distinct different fertilizers classes (i.e. organic, organo-mineral, inorganic), but standards and norms related to nutrient- and carbon origin remain dynamic and lag behind. This, together with poor analytical understanding of commercially available N sources leaves many open questions to industries and farmers, fostering increased prevalence of fertilizer adulteration and false claims on the organic fertilizer market. This work presents a thorough, science-based multivariate assessment on a wide sample set (n = 52) of the solid N fertilizer market, including multiple state-of-the-art analytical attributes, such as stable isotopes of nitrogen and carbon. Results present the possibility to correctly (94%) classify N fertilizers using multivariate fingerprinting with linear discriminant analysis. We extract analytical cut-off values for discriminants indicative for ingredient origin and conclude that, when a fertilizer has (i) a bulk δ¹⁵N below 2‰; and (ii) a relatively high total N content (> 15%), from which (iii) a high share (> 50%) is water soluble (i.e. in ammonium or nitrate form), it is extremely unlikely to be of pure biologic origin. We also present additional analyses (e.g. amino acids, peptide sequences, δ¹³C of specific compounds, and stable isotopes of boron) that can then be used to further trace down the N sources in novel fertilizer products. This work contributes to future debates, regulations, and further development of analytical standards for solid N fertilizers, possibly to be used in fraud detection.
Graphical abstract
... Dietary reconstruction through isotopic analysis is a powerful technique that elucidates the relative contributions of isotopically distinct food resources to individuals' diets, thus improving our knowledge of the nutrient flows within complex ecosystems. Patterns of stable carbon isotope (δ 13 C) values generated by different pathways of amino acid (AA) synthesis in aquatic and terrestrial plants, fungi, and bacteria have been termed " 13 C fingerprints" and are used to distinguish among different types of nutrient sources at the base of the food web and to identify their incorporation into consumer tissues (Larsen et al. 2009(Larsen et al. , 2013. This is premised on the fact that the C skeletons of essential AAs typically pass from dietary source to consumer without alteration and therefore with minimal isotopic fractionation (McMahon et al. 2010). ...
Recent studies have demonstrated that there exists significant variability in amino acid (AA) δ¹⁵N values of terrestrial plants, discriminating among plant types (i.e., legume seeds, grasses, tree leaves) as well as tissues of the same plant. For the first time, we investigate the potential of the spacing between the δ¹⁵N values of different AAs to differentiate between plant types and thus elucidate their relative importance in herbivore diet. Using principal component analysis, we show that it is possible to distinguish among five plant categories—cereal grains, rachis, legume seeds, herbaceous plants, and woody plants—whose consumption has different implications for understanding herbivore ecology and management practices. Furthermore, we were able to correctly classify the herbaceous plant diet of modern cattle using AA δ¹⁵N values of their tooth dentine adjusted for trophic enrichment. The AA δ¹⁵N patterns of wild and domestic herbivores from archaeological sites seem to be consistent with diets comprised predominantly of herbaceous plants, but there is variation in AA δ¹⁵N values among individuals that may reflect differing inputs of other plant types. The variation in AA δ¹⁵N values does not necessarily reflect the variation in herbivore bulk collagen δ¹³C and δ¹⁵N values, indicating that AA δ¹⁵N values have the potential to provide additional insights into plant dietary sources compared to bulk tissue isotope values alone. Future work should focus on defining trophic enrichment factors for a wider range of terrestrial herbivores and expanding libraries of primary producer AA δ¹⁵N values.
... However, substantial isotopic variability exists within both freshwater and marine ecosystems (Fuller et al., 2012;Sulak et al., 2012;Guiry, 2019), and studies employing essential amino acid δ 13 C analyses [e.g. (Larsen et al., 2009, Elliott Smith et al., 2022] are needed to confirm marine resource use by archaeological A. transmontanus. These studies could be paired with population genetic studies of both ancient and modern sturgeon to explore potential correlations among population structures and isotopic (e.g., dietary) niches (Cook et al., 2007;Brophy et al., 2020). ...
Understanding the habitats people were fishing in the past is central to evaluating the relationship between coastal environmental change and human behavior. Researchers often use zooarchaeological identification of fishes and modern ecological data to infer the habitats people fished in the past. However, these inferences assume stable environmental conditions through time and can be hindered by precision issues in identification of archaeological specimens (species vs. genus or family). Here, we integrate genetic and bulk tissue stable isotope data to investigate a late Holocene sturgeon fishery in northern Oregon. Ancient DNA analysis indicated that people were fishing for both green and white sturgeon (Acipenser medirostris and Acipenser transmontanus) in comparable numbers. Stable isotope analyses of these same bones documented distinct isotope values for each species, correlating with species-specific habitat preferences. These findings highlight the value of paired isotope and genetic data to elucidate human fishing strategies and environmental change and provide baseline ecological data for modern fisheries.
... Another method which might be employed when investigating intricate trophic strategies is compound-specific stable isotope analysis of amino acids, specifically the δ 13 C of essential amino acids termed "δ 13 C fingerprints" (Larsen et al., 2009), which has recently shown promise in differentiating the niches of marine consumers (Larsen et al., 2020). ...
Fish have one of the highest occurrences of individual specialization in trophic strategies among Eukaryotes. Yet, few studies characterize this variation during trophic niche analysis, limiting our understanding of aquatic food web dynamics. Stable isotope analysis (SIA) with advanced Bayesian statistics is one way to incorporate this individual trophic variation when quantifying niche size. However, studies using SIA to investigate trophodynamics have mostly focused on species-or guild-level (i.e., assumed similar trophic strategy) analyses in settings where source isotopes are well-resolved. These parameters are uncommon in an ecological context. Here, we use Stable Isotope Bayesian Ellipses in R (SIBER) to investigate cross-guild trophodynam-ics of 11 reef fish species within an oceanic atoll. We compared two-(δ15N and δ13C) versus three-dimensional (δ15N, δ13C, and δ34S) reconstructions of isotopic niche space for interpreting guild-, species-, and individual-level trophic strategies. Reef fish isotope compositions varied significantly among, but also within, guilds. Individuals of the same species did not cluster together based on their isotope values, suggesting within-species specializations. Furthermore, while two-dimensional isotopic niches helped differentiate reef fish resource use, niche overlap among species was exceptionally high. The addition of δ34S and the generation of three-dimensional isotopic niches were needed to further characterize their isotopic niches and better evaluate potential trophic strategies. These data suggest that δ34S may reveal fluctuations in resource availability, which are not detectable using only δ15N and δ13C. We recommend that researchers include δ34S in future aquatic food web studies
... Indeed, carbon CSIA of essential AAs has been tested in conjugation with nitrogen CSIA AAs to study trophic relationships in complex soil food webs (Pollierer et al., 2019), although to our knowledge true integration of two elements to quantify mixing ratio among different sources has not been accomplished yet. Nevertheless, carbon CSIA of essential AA seem to differentiate main basal energy sources (fungi, bacteria and plants) of soil food webs relatively well (Larsen et al., 2009;Pollierer et al., 2019) and possibly enables monitoring of arthropod food web interactions in different type of systems (Pollierer et al., 2019). While use of AA CSIA in arthropods monitoring has the greatest potential in estimation of trophic position of individuals, there are new emerging applications such as using AA δ 13 C, δ 15 N and δ 2 H in monitoring animal movements in high resolution (McMahon and Newsome, 2019). ...
Monitoring of arthropods focuses typically on changes in population and range size over time. Yet, there are a myriad of other aspects that could and should be monitored under the ongoing global and local environmental change. Stable isotope analysis, widely employed in short-term ecological studies, has potential in long-term monitoring of arthropods. Here we discuss the use of stable isotopes in monitoring terrestrial arthropods, provide some empirical examples of the use of bulk tissue samples in stable isotope analysis, and outline future directions in using compound-specific stable isotope analysis in monitoring. We performed a literature search for 2012–2021 to see if stable isotopes have been specifically used in monitoring of terrestrial arthropods. The literature shows that stable isotopes have been successfully used to reveal ecological phenomena (dispersal, trophic interactions, resource use) that would have been difficult or impossible to detect by other means. Yet, stable isotopes have been underused in arthropod monitoring programs, but the growing number of basic studies on stable isotope ecology and methodology provides crucial basis needed for developing monitoring programs. Stable isotopes provide technically, economically and ecologically feasible addition to the traditional monitoring methods of terrestrial arthropods.
... Due to the low availability of contemporary comparative paleontological samples from these contexts on Vancouver Island, it was not possible to employ the CSIA carbon isotope fingerprinting technique used by other researchers, which uses different species to reconstruct food-webs and diets (e.g. Corr et al., 2007;Larsen et al., 2009Larsen et al., , 2013Jarman et al., 2017). However, several trends in individual amino acid carbon isotope composition have been observed across a variety of ecosystems and periods, and syntheses of published CSIA carbon data (e.g. ...
Competition between taxa related to climate changes has been proposed as a possible factor in Pleistocene megafaunal extinctions, and here we present isotope evidence of the diets of three co‐existing bear species [black bear (Ursus americanus), brown bear (Ursus arctos), and the now extinct short‐faced bear (Arctodus simus)] from a locale in western North America dating to the Late (Terminal) Pleistocene (~14.5–11.7 ka). The three bear species were found at several sites on Vancouver Island, on the western coast of Canada. To examine the chronological overlap and niche partitioning between these species of bear, we used direct radiocarbon dating, stable isotope analysis and ZooMS proteomic identification methods. Here we present new radiocarbon evidence from Terminal Pleistocene U. americanus, U. arctos and A. simus from several sites on the island, along with both bulk collagen and compound‐specific isotope data for these species. Radiocarbon dates confirm the chronological overlap of Arctodus and both Ursus species in the montane regions of the island at the end of the Pleistocene. Stable isotope data reveal niche differentiation between these species, with U. americanus occupying a distinctly lower trophic position than the other two taxa.
... However, more controlled feeding studies on mammalian model species are needed to validate the extent to which δ 13 C essential amino acid values persist through multiple trophic transfers (Webb et al. 2017). Another key feature of the essential amino acids is that the primary organisms synthesizing them-algae, bacteria, fungi, and terrestrial plants-have distinct δ 13 C amino acid patterns in which the relative differences among amino acids are consistent, regardless of the actual baseline δ 13 C values (Scott et al. 2006, Larsen et al. 2009, Larsen et al. 2013). These patterns are termed fingerprints when they are unique and unequivocal for a given basal resource. ...
Stable isotope analysis of teeth and bones is regularly applied by archeologists and paleoanthropologists seeking to reconstruct diets, ecologies, and environments of past hominin populations. Moving beyond the now prevalent study of stable isotope ratios from bulk materials, researchers are increasingly turning to stable isotope ratios of individual amino acids to obtain more detailed and robust insights into trophic level and resource use. In the present article, we provide a guide on how to best use amino acid stable isotope ratios to determine hominin dietary behaviors and ecologies, past and present. We highlight existing uncertainties of interpretation and the methodological developments required to ensure good practice. In doing so, we hope to make this promising approach more broadly accessible to researchers at a variety of career stages and from a variety of methodological and academic backgrounds who seek to delve into new depths in the study of dietary composition.
... These groups often rely on a heterogeneous mixture of mangrove litter, microphytobenthos and detrital organic compounds in sediments, and encrusting bacteria from mangrove roots (Agusto et al. 2020). A lower δ 15 N value in leaf-eating crabs and surface-grazing gastropods is attributed to nitrogen-fixing bacteria consolidating atmospheric N into their biomass, and generating δ 15 N values close to 0 (Ganguly et al. 2009;Larsen et al. 2009). However, more studies are needed to determine the type of symbiotic bacteria living on areal roots or the sediments responsible for nitrogen fixation in the mangrove ecosystem. ...
Stable isotope analysis was used to investigate the food sources of benthic macroinvertebrates in the arid mangrove of the Persian Gulf. Stable carbon (δ13C) and nitrogen (δ15N) isotope values were analyzed for the 23 species and their potential food sources to examine the seasonal variation in the contribution of different potential food sources to macroinvertebrates. Primary food sources, e.g., mangrove leaves, microphytobenthos, sediment organic matter, and particulate organic matter, showed a wide range of δ13C values ranging between −28.07 and −13.54‰ and δ15N from 1.44 to 10.72‰. The wide range of the isotopic composition of consumers indicated that different food sources are important in the mangrove ecosystem of the Persian Gulf. The grazers tended to be more 13C-depleted than other feeding groups at both sampling seasons. In contrast, predators had significantly high δ13C, reflecting their carnivorous feeding habit of consuming 13C-enriched foods. The higher δ13C values of suspension feeders reflected the significant role of re-suspended microphytobenthos in the benthic-pelagic coupling of the mangrove ecosystem. The Bayesian stable isotope mixing model outputs suggested that the benthic food webs are supported by multiple primary sources. Microphytobenthos contribution was greater (> 40%), indicating their significant ecological role in structuring the food web of the arid mangroves. While sediment and particulate organic matter sources are involved in sustaining deposit feeders and partially suspension feeders, respectively, the mangrove-derived carbon played a significant role in maintaining herbivorous crabs.
In a warming world, the input of glacier meltwater to inland water ecosystems is predicted to change, potentially affecting their productivity. Meta-ecosystem theory, which posits that the nutrient availability in the recipient ecosystem can determine the extent of cross-ecosystem boundary utilization, can be useful for studying landscape-scale influences of glacier meltwater on inland waters. Here, we investigate how the input of glacier meltwater in a river system in Southern Greenland influences the utilization of marine subsidies in freshwater fish. Our study system comprised four sites, with controls for glacial meltwater and marine subsidies, harboring a partially migrating population of arctic char, meaning that some individuals migrate to the ocean and others remain in freshwaters, and two fully resident populations as a freshwater reference. We assessed the incorporation of marine carbon in freshwater resident char using both bulk and amino acid stable isotope analysis of muscle tissue. In the population with partial migration, marine subsidies were a significant resource for resident char individuals, and estimates of trophic position suggest that egg cannibalism is an important mechanism underlying the assimilation of these marine subsidies. In proglacial streams, namely those with high glacial meltwater, the total dependence on marine subsidies increased and reached 83% because char become cannibals at smaller sizes. In the configuration of our focal meta-ecosystem, our results suggest that the importance of marine subsidies to freshwater fish strengthens within increasing meltwater flux from upstream glaciers.
The present study assessed and compared the diet and trophic positions (TP) of two carnivorous fish H. macrolepidota and C. ocellaris from Chenderoh Reservoir, Malaysia. The focal goal of the study was to understand the effects of invasive non-indigenous species (NIS), C. ocellaris, on the native indigenous (IS) fish species, H. macrolepidota. Data were acquired from September 2014 to February 2015 within the study area. The assessment was grounded in stomach content analysis (SCA) and stable isotope analysis (SIA), which collectively clarified the feeding habits and trophic positions (TP) of these selected fish. In total, 184 fish samples (comprising 64 individuals of H. macrolepidota and 120 individuals of C. ocellaris) underwent stomach content analysis (SCA). Additionally, 24 individuals (12 of H. macrolepidota and 12 of C. ocellaris), sampled from December 2014 to February 2015, were selected for stable isotope analysis (SIA). The mean RGL values for H. macrolepidota and C ocellaris were 0.98 ± 0.18 and 1.10 ± 0.15 (Mean ± SD), respectively, aligning with known ranges for carnivorous fish. These values also clarified that both species occupy higher TP in the food web as tertiary or quaternary consumers. SCA findings also revealed that fish and crustaceans were the predominant food categories for H. macrolepidota, while C. ocellaris predominantly fed on fish. The mean stomach fullness index (MSF) and the gastrosomatic index (GSI) corroborated the differences in the foraging performance of the fishes, with C. ocellaris having a higher MSF (2.03) compared to H. macrolepidota (0.65). These implied that C. ocellaris had plentiful of food and encountered fewer diet-related challenges in the ecosystem. From SIA, δ13C values indicated that the primary carbon sources for both species are C3 plants, particularly aquatic vegetation. Further, δ15N values further ensured that both H. macrolepidota and C. ocellaris are carnivorous in nature and occupy higher TP in the ecosystem.
Total mercury concentrations ([THg]) exceed thresholds of concern in some Steller sea lion (Eumetopias jubatus) tissues from certain portions of the Aleutian Islands, Alaska. We applied compound-specific stable isotope analyses of both carbon and nitrogen in amino acids from fish muscle tissue to quantify the proportional contributions of primary production sources and trophic positions of eight prey species (n = 474 total) that are part of Steller sea lion diets. Previous THg analyses of fish muscle, coupled with monomethylmercury analyses of a subset of samples, substantiated previous findings that fishes from the west of Amchitka Pass, a discrete oceanographic boundary of the Aleutian Archipelago, have higher muscle Hg concentrations relative to fishes from the east. The δ13C values of essential amino acids (EAAs) in fish muscle demonstrated that although most fishes obtained their EAAs primarily from algae, some species varied in the extent to which they relied on this EAA source. The δ15N values of phenylalanine (0.9 to 7.8 ‰), an indicator of the isotopic baseline of a food web, varied widely within and among fish species. Trophic position estimates, accounting for this baseline variation, were higher from the west relative to the east of the pass for some fish species. Trophic magnification slopes using baseline-corrected trophic position estimates indicated similar rates of Hg biomagnification to the east and west of Amchitka Pass. Multiple linear regression models revealed that trophic position was the most important driver of fish muscle [THg] with less variation explained by other parameters. Thus, higher trophic positions but not the rate of Hg biomagnification to the west of Amchitka Pass may play a role in the regional differences in both fish and Steller sea lion [THg]. Although, differences in Hg contamination and uptake at the base of the east and west food webs could not be excluded.
This chapter discusses the current state and future directions of the application of stable isotopes to archaeological materials. New technologies have facilitated massive growth in this field over the last twenty years. I briefly discuss some of these advancements and highlight some issues associated with maintaining quality control in the publication process. I highlight some interpretive techniques, such as mixing models, that have been used to make sense of isotopic data, highlighting the importance of generating excellent isotopic baselines to minimize inaccurate interpretations. The characterization of the isotopic composition of heavier elements and individual compounds is a burgeoning area of study that promises to produce important new insights. I close with my thoughts on the importance of mentoring the next generation of scholars in this field.KeywordsIsotope analysisMixing modelsIsotopic methodsCompound specific amino acids
The measurement of stable isotope values of individual compounds, such as amino acids (AAs), has quickly become a powerful tool in animal ecology and ecophysiology. As with any emerging technique, fundamental questions remain regarding the capability and limitations of this approach, including how metabolism and tissue synthesis impact AA isotope values and patterns. We measured carbon isotope ( d ¹³ C) values of essential (AA ESS ) and nonessential (AA NESS ) AAs in bone collagen, whisker, muscle, and liver from ten southern sea otters ( Enhydra lutris nereis ) stranded in Monterey Bay, California. Sea otters in this population exhibit high degrees of individual dietary specialization, making this an excellent opportunity to explore differences in AA d ¹³ C values among tissues in a wild population. We found the d ¹³ C values of glutamic acid, proline, serine, lysine, and threonine differed significantly among tissues, indicating possible isotopic discrimination during tissue synthesis. Threonine d ¹³ C values were higher in liver relative to bone collagen and muscle, which may indicate catabolism of threonine for gluconeogenesis, an interpretation further supported by statistically significant correlations between the d ¹³ C values of threonine and its gluconeogenic products glycine and serine. For 6/10 of the sea otters analyzed, tissues from the same individual sea otter classified with different primary producers in multivariate space. We thus recommend caution when using data from multiple tissues in an AA ESS d ¹³ C fingerprinting framework. Broadly, our results indicate that metabolism influences both AA ESS and AA NESS d ¹³ C values and highlight the importance of gluconeogenesis in driving compound-specific isotope patterns in this endangered apex marine consumer.
Quantifying the trophic basis of production for freshwater metazoa at broad spatial scales is key to understanding ecosystem function and has been a research priority for decades. However, previous lotic food web studies have been limited by geographic coverage or methodological constraints. We used compound‐specific stable carbon isotope analysis of amino acids (AAs) to estimate basal resource contributions to fish consumers in streams spanning grassland, montane and semi‐arid ecoregions of the temperate steppe biome on two continents. Across a range of stream sizes and light regimes, we found consistent trophic importance of aquatic resources. Essential AAs of heterotrophic microbial origin generally provided secondary support for fishes, while terrestrial carbon did not seem to provide significant, direct support. These findings provide strong evidence for the dominant contribution of carbon to higher‐order consumers by aquatic autochthonous resources (primarily) and heterotrophic microbial communities (secondarily) in temperate steppe streams.
Rationale:
Position-specific 13 C/12 C ratios within amino acids remain largely unexplored in environmental samples due to methodological limitations. We hypothesized that natural-abundance isotope patterns in serine may serve as a proxy for plant metabolic fluxes including photorespiration. Here we describe an Orbitrap method optimized for the position-specific carbon isotope analysis of serine to test our hypothesis and discuss the generalizability of this method to other amino acids.
Methods:
Position-specific carbon isotope ratios of serine were measured using a Thermo Scientific™ Q Exactive™ GC Orbitrap™. Amino acids were hydrolyzed from Arabidopsis biomass, purified from potential matrix interferences, and derivatized alongside standards. Derivatized serine (N,O-bis (trifluoroacetyl) methyl ester) was isolated by GC, trapped in a reservoir, and purged into the electron ionization source over tens of minutes, producing fragment ions containing different combinations of atoms from the serine-derivative molecule. The 13 C/12 C ratios of fragments with monoisotopic masses of 110.0217, 138.0166, and 165.0037 Da were monitored in the mass analyzer and used to calculate position-specific δ13 C values relative to a working standard.
Results:
This methodology constrains position-specific δ13 C values for nanomole amounts of serine isolated from chemically complex mixtures. The δ13 C values of fragment ions of serine were characterized with ≤ 1‰ precisions, leading to propagated standard errors of 0.7 - 5‰ for each carbon position. Position-specific δ13 C values differed by up to ~28‰ (± 5‰) between serine molecules hydrolyzed from plants grown under contrasting pCO2 , selected to promote different fluxes through photosynthesis and photorespiration. The method was validated using pure serine standards characterized offline.
Conclusions:
This study presents the first Orbitrap-based measurements of natural-abundance, position-specific carbon isotope variation in an amino acid isolated from a biological matrix. We present a method for the precise characterization of isotope ratios in serine and propose applications probing metabolism in plants. We discuss the potential for extending these approaches to other amino acids, paving the way for novel applications.
Nitrogen isotope (δ15N) analysis of bulk tissues and individual amino acids (AA) can be used to assess how consumers maintain nitrogen balance with broad implications for predicting individual fitness. For elasmobranchs, a ureotelic taxa thought to be constantly nitrogen limited, the isotopic effects associated with nitrogen-demanding events such as prolonged gestation remain unknown. Given the linkages between nitrogen isotope variation and consumer nitrogen balance, we used AA δ15N analysis of muscle and liver tissue collected from female bonnethead sharks (Sphyrna tiburo, n = 16) and their embryos (n = 14) to explore how nitrogen balance may vary across gestation. Gestational stage was a strong predictor of bulk tissue and AA δ15N values in pregnant shark tissues; decreasing as individuals neared parturition. This trend was observed in trophic (e.g., Glx, Ala, Val), source (e.g., Lys), and physiological (e.g., Gly) AAs. Several potential mechanisms may explain these results including nitrogen conservation, scavenging, and bacterially mediated breakdown of urea to free ammonia that is used to synthesize AAs. We observed contrasting patterns of isotopic discrimination in embryo tissues, which generally became enriched in 15N throughout development. This was attributed to greater excretion of nitrogenous waste in more developed embryos, and the role of physiologically sensitive AAs (i.e., Gly and Ser) to molecular processes such as nucleotide synthesis. These findings underscore how AA isotopes can quantify shifts in nitrogen balance, providing unequivocal evidence for the role of physiological condition in driving δ15N variation in both bulk tissues and individual AAs.
In soil a high number of species co-exist without extensive niche differentiation, which was assigned as ‘the enigma of soil animal species diversity’. In particular, the detritivores are regarded as food generalists. We have investigated nitrogen stable isotope ratios (15N/14N) of a major decomposer group, the Collembola, to evaluate trophic relationship and determine feeding guilds. Additionally, the δ15N values of potential food sources such as mosses, lichens and other plant derived material (bark, nuts, leaves) were analysed. The natural variation in nitrogen isotopes was assessed in 20 Collembola taxa from three deciduous forest stands. The δ15N signature formed a continuum from phycophages/herbivores to primary and secondary decomposers, reflecting a gradual shift from more detrital to more microbial diets. The δ15N gradient spanned over 9 δ units, which implies a wide range in food sources used. Assuming a shift in 15N of about 3 ‰ per trophic level, the results indicate a range of three trophic levels. These variations in 15N/14N ratios suggest that trophic niches of Collembola species differ and this likely contributes to Collembola species diversity.
Plant growth in the boreal forest, the largest terrestrial biome, is generally limited by the availability of nitrogen. The presumed cause of this limitation is slow mineralization of soil organic nitrogen. Here we demonstrate, to our knowledge for the first time, the uptake of organic nitrogen in the field by the trees Pinus
sylvestris and Picea abies, the dwarf shrub Vaccinium myrtillus and the grass Deschampsia flexuosa. These results show that these plants, irrespective of their different types of root–fungal associations (mycorrhiza), bypass nitrogen mineralization. A trace of the amino acid glycine, labelled with the stable isotopes 13C and 15N, was injected into the organic (mor) layer of an old successional boreal coniferous forest. Ratios of 13C:15Nin the roots showed that at least 91, 64 and 42% of the nitrogen from the absorbed glycine
was taken up in intact glycine by the dwarf shrub, the grass and the trees, respectively. Rates of glycine uptake were similar to those of 15N-ammonium. Our data indicate that organic nitrogen is important for these different plants, even when they are competing with each other and with non-symbiotic microorganisms. This has major implications for our understanding of the effects of nitrogen deposition, global warming and intensified forestry.
Two classes of procaryotic organisms were cultured on specific inorganic and organic nitrogenous substrates. The organisms fractionated these substrates in characteristic ways during the synthesis of their cellular biochemical compounds. Blue-green algae, Anabaena sp., were raised on molecular nitrogen, nitrate and ammonia in the presence of excess carbon dioxide. The difference between δ15N of either nitrate or ammonium and the δ15N of the algae grown on either source was 13%o. The δ15N of Anabaena that fixed N2 was 2% lighter than the nitrogen gas supplied to the algae. These fractionations are associated with enzymatic incorporation of the nitrogen into the cell. A heterotrophic bacterium, Vibrio harveyi was grown on a single amino acid as its source of both nitrogen and carbon. Cells grown on glutamic acid were enriched in 15N relative to substrate, whereas those grown on alanine were depleted in 15N compared to source nitrogen. The bacterial cultures were enriched in 13C relative to the substrate. These cultures were then hydrolyzed and individual amino acids isolated and isotopically analyzed. The isotopic compositions of the amino acids have a wide range of values; most appear to have isotope fractionations associated with the metabolic pathways in their synthesis. These results and the application of the coupled separation-isotopic analysis of amino acids yield a better understanding of comparative biochemistry for these organisms. Such analyses offer valuable information for the tracing of biosynthesis and early diagenesis to help explain the fossil record.
Progress towards understanding the extent to which mycorrhizal fungi are involved in the mobilization of nitrogen (N) and phosphorus (P) from natural substrates is reviewed here. While mycorrhiza research has emphasized the role of the symbiosis in facilitation of capture of these nutrients in ionic form, attention has shifted since the mid-1980s to analysing the mycorrhizal fungal abilities to release N and P from the detrital materials of microbial faunal and plant origins, which are the primary sources of these elements in terrestrial ecosystems. Ericoid, and some ectomycorrhizal fungi have the potential to be directly involved in attack both on structural polymers, which may render nutrients inaccessible, and in mobilization of N and P from the organic polymers in which they are sequestered. The advantages to the plant of achieving intervention in the microbial mobilization–immobilization cycles are stressed. While the new approaches may initially lack the precision achieved in studies of readily characterized ionic forms of N and P, they do provide insights of greater ecological relevance. The results support the hypothesis that selection has favoured ericoid and ectomycorrhizal systems with well developed saprotrophic capabilities in those ecosystems characterized by retention of N and P as organic complexes in the soil. The need for further investigation of the abilities of arbuscular mycorrhizal fungi to intervene in nutrient mobilization processes is stressed.
Intracellular associations between bacteria and insects are widespread in nature (Baumann and Moran 1997; Buchner 1965; Dasch et al. 1984; Douglas 1989; Houk and Griffiths 1980). Extensive studies of the natural history of such associations have led to the conclusion that they are commonly found in insects that utilize diets containing an excess of one class of compounds but a deficiency of some essential nutrients (Buchner 1965; Dadd 1985). It was thought that the function of the endosymbionts was to rectify this imbalance by the synthesis of these essential nutrients for the host. Extensive compilations of the occurrence of endosymbionts in different groups of insects are found in Buchner (1965) and Dasch et al. (1984). Because most of the prokaryotes involved in such associations are not cultivable on common laboratory media, their characterization had to await the development of recombinant DNA methodology. The past 10 years have witnessed the initiation of studies on the intracellular association of prokaryotes with a variety of insect hosts. In this chapter, we will provide an overview of the evolution and, where possible, genetics and physiology of such recently studied associations. A summary of some of their features is presented in Table 19.1, and the phylogeny of the endosymbionts based on 16S rDNA is presented in Fig. 19.1.
We measured in situ uptake of amino acids and ammonium across deciduous and coniferous taiga forest ecosystems in interior Alaska to examine the idea that late successional (coniferous) forests rely more heavily on dissolved organic nitrogen (DON), than do early successional (deciduous) ecosystems. We traced 15N-NH
4+ and 13C-15N-amino acids from the soil solution into plant roots and soil pools over a 24 h period in stands of early successional willow and late successional black spruce. Late successional soils have much higher concentrations of amino acid in soil solution and a greater ratio of DON to dissolved inorganic N (DIN) (ammonium plus nitrate) than do early successional soils. Moreover, late successional coniferous forests exhibit higher rates of soil proteolytic activity, but lower rates of inorganic N turnover. Differences in ammonium and amino acid uptake by early successional willow stands were insignificant. By contrast, the in situ uptake of amino acid by late successional black spruce forests were approximately 4-fold greater than ammonium uptake. The relative difference in uptake of ammonium and amino acids in these forests was approximately proportional to the relative difference of these N forms in the soil solution. Thus, we suggest that differences in uptake of different N forms across succession in these boreal forests largely reflect edaphic variation in available soil N (composition), rather than any apparent physiological specialization to absorb particular forms of N. These finding are relevant to our understanding of how taiga ecosystems may respond to increases in temperature, fire frequency, N deposition, and other potential consequences of global change.
It has been demonstrated that plant roots can take up small amounts of low-molecular weight (LMW) compounds from the surrounding
soil. Root uptake of LMW compounds have been investigated by applying isotopically labelled sugars or amino acids but not
labelled organic matter. We tested whether wheat roots took up LMW compounds released from dual-labelled (13C and 15N) green manure by analysing for excess 13C in roots. To estimate the fraction of green manure C that potentially was available for root uptake, excess 13C and 15N in the primary decomposers was estimated by analysing soil dwelling Collembola that primarily feed on fungi or microfauna.
The experimental setup consisted of soil microcosm with wheat and dual-labelled green manure additions. Plant growth, plant
N and recoveries of 13C and 15N in soil, roots, shoots and Collembola were measured at 27, 56 and 84days. We found a small (<1%) but significant uptake
of green manure derived 13C in roots at the first but not the two last samplings. About 50% of green manure C was not recovered from the soil-plant
system at 27days and additional 8% was not recovered at 84days. Up to 23% of C in collembolans derived from the green manure
at 56days (the 27days sampling was lost). Using a linear mixing model we estimated that roots or root effluxes provided
the main C source for collembolans (54−79%). We conclude that there is no solid support for claiming that roots assimilated
green manure derived C due to very small or no recoveries of excess 13C in wheat roots. During the incubation the pool of green manure derived C available for root uptake decreased due to decomposition.
However, the isotopic composition in Collembola indicated that there was a considerable fraction of green manure derived C
in the decomposer system at 56days thus supporting the premise that LMW compounds containing C from the green manure was
released throughout the incubation.
The stable isotope compositions (C and N) of plants and animals of a marsh dominated by Spartina alterniflora in the Delaware Estuary were determined. The study focused on the juvenile stage of the Atlantic blue crab, Callinectes sapidus, and the importance of marsh-derived diets in supporting growth during this stage. Laboratory growth experiments and field
data indicated that early juvenile blue crabs living in the Delaware Bay habitat fed primarily on zooplankton, while marsh-dwelling
crabs, which were enriched in 13C relative to bay juveniles, utilized marsh-derived carbon for growth. In laboratory experiments, the degree to which juvenile
blue crabs isotopically fractionated dietary nitrogen, as well as the growth rate, depended on the protein quality of the
diet. The range of δ13C of amino acids in laboratory-reared crabs and their diets was almost 20‰, similar to the isotopic range of amino acids of
other organisms. In laboratory studies, the δ13C of nonessential and essential amino acids in the diet were compared to those in juvenile crabs. Isotopic fractionation at
the molecular level depended on diet quality and the crabs' physiological requirements. Comparison of whole-animal isotope
data with individual amino acid C isotope measurements of wild juvenile blue crabs from the bay and marsh suggested a different
source of total dietary carbon, yet a shared protein component, such as zooplankton.
The transfer of dissolved organic carbon (DOC) and nitrogen (DON) out of the surface ocean where it is produced to storage in the ocean's interior creates one of the largest reservoirs of reduced carbon and organic nitrogen on earth. In nutrient-depleted surface waters of the oligotrophic ocean, dissolved nitrogenous material is of key importance as a source of fixed nitrogen for heterotrophic organisms. Recent work has increasingly indicated that, contrary to previous ideas, recalcitrant chemical structure is not the central factor underlying the preservation of DOC and DON, leaving the major preservation mechanisms largely unknown. We employ here a stable isotopic approach to examine the metabolic source and transformation signatures imprinted in carbon isotopic fractionation patterns of amino acids, which are the major components of both particulate and dissolved organic nitrogen that can be identified at the molecular level. Compound-specific isotopic signatures from central Pacific particulate and dissolved organic matter indicate a profound difference in processing histories between these two material pools. Sinking particles show a clear imprint of heterotrophic resynthesis and alteration, while the much larger and older dissolved pool retains an unaltered signature of photoautotrophic synthesis, even in samples from the abyssal ocean. In addition, δ13C signatures of enantiomers of alanine (d vs. l) in dissolved materials are indistinguishable. This isotopic data, in light of previously observed abundant d-amino acids in oceanic DOM, suggests that autotrophic prokaryotes may be a main source for dissolved nitrogenous material preserved over long time scales in the sea. Taken together, our results suggest that dissolved organic nitrogen preservation is not predominantly linked to heterotrophic reworking and resynthesis, but instead there exists a non-discriminating and rapid shunt, effectively removing recently formed autotrophic biomolecules from further recycling.
To understand how enzymatic pathways respond to changing external conditions, the fluxes through the tricarboxylic acid cycle and ancillary reactions were determined under three different growth conditions in Escherichia coli. The velocities through the major steps in each pathway were measured (a) for growth on acetate alone, (b) for growth on acetate plus glucose, and (c) during the transition caused by addition of glucose to cells growing on acetate. During the transition, the carbon flow through the Krebs cycle decreased by a factor of 5 despite an increase in the growth rate of the culture. Under these conditions, the dephosphorylation of isocitrate dehydrogenase caused a 4-fold increase in its activity. This, together with the decreased rate of substrate production and the kinetic parameters of the branch point enzymes, led to a cessation of the flux through the glyoxylate shunt. The decreased rate of acetyl-CoA turnover, not an inhibition of acetate transport, caused a slower rate of acetate uptake in the presence of glucose. The modulation of protein phosphorylation and metabolite levels is one of the regulatory mechanisms which enables the bacterium to make dramatic shifts between metabolic pathways within a fraction of a doubling time.
Differences in the natural-abundance carbon stable isotopic compositions between products from aerobic cultures of Escherichia coli K-12 were measured. Respired CO2 was 3.4% depleted in 13C relative to the glucose used as the carbon source, whereas the acetate was 12.3% enriched in 13C. The acetate 13C enrichment was solely in the carboxyl group. Even though the total cellular carbon was only 0.6% depleted in 13C, intracellular components exhibited a significant isotopic heterogeneity. The protein and lipid fractions were -1.1 and -2.7%, respectively. Aspartic and glutamic acids were -1.6 and +2.7%, respectively, yet citrate was isotopically identical to the glucose. Probable sites of carbon isotopic fractionation include the enzyme, phosphotransacetylase, and the Krebs cycle.
A method has been developed for the positional 13C isotope analysis of pyruvate and acetate by stepwise quantitative degradation. On its base, the kinetic isotope effects on the pyruvate dehydrogenase reaction (enzymes from Escherichia coli and Saccharomyces cerevisiae) for both of the carbon atoms involved in the bond scission (double isotope effect determination) and on C-3 of pyruvate have been determined. The experimental k12/k13 values with the enzyme from E. coli on C-1 and C-2 of pyruvate are 1.0093 +/- 0.0007 and 1.0213 +/- 0.0017, respectively, and, with the enzyme from S. cerevisiae, the values are 1.0238 +/- 0.0013 and 1.0254 +/- 0.0016, respectively. A secondary isotope effect of 1.0031 +/- 0.0009 on C-3 (CH3-group) was found with both enzymes. The size of the isotope on C-1 indicates that decarboxylation is more rate-determining with the yeast enzyme than with the enzyme from E. coli, although it is not the entirely rate-limiting step in the overall reaction sequence. Assuming appropriate values for the intrinsic isotope effect on the decarboxylation step (k3) and the equilibrium isotope effect on the reversible substrate binding (k1, k2), one can calculate values for the partitioning factor R (k3/k2: E. coli enzyme 4.67, S. cerevisiae enzyme 1.14) and the intrinsic isotope effects related to the carbonyl-C (k1/k'1 = 1.019; k3/k'3 = 1.033). The isotope fractionation at C-2 of pyruvate gives strong evidence that the well known relative carbon-13 depletion in lipids from biological material is mainly caused by the isotope effect on the pyruvate dehydrogenase reaction. In addition, our results indicate an alternating 13C abundance in fatty acids, that has already been verified in some cases.
The allocation of nutritional resources to reproduction in animals is a complex process of great evolutionary significance. We use compound-specific stable isotope analysis of carbon (GC/combustion/isotope ratio MS) to investigate the dietary sources of egg amino acids in a nectar-feeding hawkmoth. Previous work suggests that the nutrients used in egg manufacture fall into two classes: those that are increasingly synthesized from adult dietary sugar over a female's lifetime (renewable resources), and those that remain exclusively larval in origin (nonrenewable resources). We predict that nonessential and essential amino acids correspond to these nutrient classes and test this prediction by analyzing egg amino acids from females fed isotopically distinct diets as larvae and as adults. The results demonstrate that essential egg amino acids originate entirely from the larval diet. In contrast, nonessential egg amino acids were increasingly synthesized from adult dietary sugars, following a turnover pattern across a female's lifetime. This study demonstrates that female Lepidoptera can synthesize a large fraction of egg amino acids from nectar sugars, using endogenous sources of nitrogen. However, essential amino acids derive only from the larval diet, placing an upper limit on the use of adult dietary resources to enhance reproductive success.
Heliconius and Laparus butterflies exhibit a unique pollen-collecting behaviour that enhances lifespan and fecundity. The specific nutritional contribution of pollen, however, had not been previously demonstrated. We used stable isotope variation to trace the carbon flow into eggs from corn pollen provided experimentally to ovipositing female Heliconius charitonia, and to evaluate the use of isotopically contrasting nectar sugars in egg amino acids. The delta(13)C of individual amino acids from pollen, larval host plant and the eggs from experimental butterflies was measured with gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), to evaluate amino acid transfer. The delta(13)C of egg essential amino acids indicated a transfer of essential amino acids from pollen to butterfly eggs. However, the delta(13)C of non-essential amino acids reflected the isotopic composition of the artificial nectar, indicating that H. charitonia synthesizes non-essential amino acids from dietary sugars. This, to our knowledge, is the first direct demonstration of amino acid transfer from pollen to butterfly eggs, and suggests that essential amino acids in particular are a key resource for extending lifespan and fecundity in Heliconius butterflies.
Most mycorrhizas are 'balanced' mutualistic associations in which the fungus and plant exchange commodities required for their growth and survival. Myco-heterotrophic plants have 'exploitative' mycorrhizas where transfer processes apparently benefit only plants. Exploitative associations are symbiotic (in the broad sense), but are not mutualistic. A new definition of mycorrhizas that encompasses all types of these associations while excluding other plant-fungus interactions is provided. This definition recognises the importance of nutrient transfer at an interface resulting from synchronised plant-fungus development. The diversity of interactions between mycorrhizal fungi and plants is considered. Mycorrhizal fungi also function as endophytes, necrotrophs and antagonists of host or non-host plants, with roles that vary during the lifespan of their associations. It is recommended that mycorrhizal associations are defined and classified primarily by anatomical criteria regulated by the host plant. A revised classification scheme for types and categories of mycorrhizal associations defined by these criteria is proposed. The main categories of vesicular-arbuscular mycorrhizal associations (VAM) are 'linear' or 'coiling', and of ectomycorrhizal associations (ECM) are 'epidermal' or 'cortical'. Subcategories of coiling VAM and epidermal ECM occur in certain host plants. Fungus-controlled features result in 'morphotypes' within categories of VAM and ECM. Arbutoid and monotropoid associations should be considered subcategories of epidermal ECM and ectendomycorrhizas should be relegated to an ECM morphotype. Both arbuscules and vesicles define mycorrhizas formed by glomeromycotan fungi. A new classification scheme for categories, subcategories and morphotypes of mycorrhizal associations is provided.
The EcoCyc database (http://EcoCyc.org/) is a comprehensive source of information on the biology of the prototypical model organism Escherichia coli K12. The mission for EcoCyc is to contain both computable descriptions of, and detailed comments describing, all genes, proteins,
pathways and molecular interactions in E.coli. Through ongoing manual curation, extensive information such as summary comments, regulatory information, literature citations
and evidence types has been extracted from 8862 publications and added to Version 8.5 of the EcoCyc database. The EcoCyc database
can be accessed through a World Wide Web interface, while the downloadable Pathway Tools software and data files enable computational
exploration of the data and provide enhanced querying capabilities that web interfaces cannot support. For example, EcoCyc
contains carefully curated information that can be used as training sets for bioinformatics prediction of entities such as
promoters, operons, genetic networks, transcription factor binding sites, metabolic pathways, functionally related genes,
protein complexes and protein–ligand interactions.
With the aim of elucidating how plants synthesize lysine, extracts prepared from corn, tobacco, Chlamydomonas and soybean were tested and found to lack detectable amounts of N-alpha-acyl-L,L-diaminopimelate deacylase or N-succinyl-alpha-amino-epsilon-ketopimelate-glutamate aminotransaminase, two key enzymes in the central part of the bacterial pathway for lysine biosynthesis. Corn extracts missing two key enzymes still carried out the overall synthesis of lysine when provided with dihydrodipicolinate. An analysis of available plant DNA sequences was performed to test the veracity of the negative biochemical findings. Orthologs of dihydrodipicolinate reductase and diaminopimelate epimerase (enzymes on each side of the central pathway) were readily found in the Arabidopsis thaliana genome. Orthologs of the known enzymes needed to convert tetrahydrodipicolinate to diaminopimelic acid (DAP) were not detected in Arabidopsis or in the plant DNA sequence databases. The biochemical and reinforcing bioinformatics results provide evidence that plants may use a novel variant of the bacterial pathways for lysine biosynthesis.
It is a nutritional challenge for nectar-feeding insects to meet the amino acid requirements of oviposition. Here we investigate whether egg amino acids derive from larval diet or are synthesized from nectar sugar in four species of butterfly: Colias eurytheme, Speyeria mormonia, Euphydryas chalcedona, and Heliconius charitonia. These species exhibit a range of life history and differ in degree of shared phylogeny. We use 13C differences among plants to identify dietary sources of amino acid carbon, and we measure amino acid 13C using compound-specific stable isotope analysis. Egg essential amino acids derived solely from the larval diet, with no evidence for metabolic carbon remodeling. Carbon in nonessential amino acids from eggs derived primarily from nectar sugars, with consistent variation in amino acid turnover. There was no relationship between the nonessential amino acids of eggs and host plants, demonstrating extensive metabolic remodeling. Differences between species in carbon turnover were reflected at the molecular level, particularly by glutamate and aspartate. Essential amino acid 13C varied in a highly consistent pattern among larval host plants, reflecting a common isotopic "fingerprint" associated with plant biosynthesis. These data demonstrate conservative patterns of amino acid metabolism among Lepidoptera and the power of molecular stable isotope analyses for evaluating nutrient metabolism in situ.
A guide to using S environments to perform statistical analyses providing both an introduction to the use of S and a course in modern statistical methods. The emphasis is on presenting practical problems and full analyses of real data sets.
Recent finding that a prokaryote synthesizes lysine through the alpha-aminoadipate pathway demonstrates that the lysine synthesis through the alpha-aminoadipate pathway is not typical of fungi. However, the fungal lysine biosynthesis is not completely the same as the prokaryotic one. We point out that alpha-aminoadipate reductase is a key enzyme to the evolution of fungal lysine synthesis. In addition, fungi have two different saccharopine dehydrogenases, which is also characteristic of fungi.
This work demonstrates that the complete synthesis of leucine is localized in chloroplasts. Isolated, purified spinach chloroplasts, supplied with 2-oxoisovalerate (3 mmol L−1), incorporated [2−14C] acetate (2 mmol L−1) into leucine as the only amino acid labelled. A leucine/isoleucine aminotransferase and a valine aminotransferase as terminal enzymes of the branched-chain amino acid synthesis (both enzymes belonging to the EC 2.6.1 class) were isolated from chloroplast stroma and purified about 110-fold. The enzymes differed strongly in their substrate specificities. The apparent Km-values for 2-oxoisocaproate and 2-oxo-3-methylvalerate of the leucine/isoleucine aminotransferase and for 2-oxoisovalerate of the valine aminotransferase were 0.12; 0.089 and 1.13 mmol L−1, respectively. The optimum pH of the aminotransferase reaction was 8. 9. Both enzymes were competitively inhibited by higher concentrations of leucine, isoleucine and valine. However, the efficient regulation of leucine synthesis takes place by a sensitive feedback control of 2-isopropylmalate synthase by leucine at a micromolar level (Hagelstein and Schultz, 1993). Concerning the integration of leucine synthesis into chloroplast metabolism, the problem of pathways competing for the same introductory substrates, acetyl-CoA and 2-oxoisovalerate, is discussed.
This review is concerned with the isotopic relationships between organic compounds produced by a single organism, specifically their enrichments or depletions in 13C relative to total-biomass carbon. These relationships are biogeochemically significant because
1. An understanding of biosynthetically controlled, between-compound isotopic contrasts is required in order to judge whether plausibly related carbon skeletons found in a natural mixture might come from a single source or instead require multiple sources.
2. An understanding of compound-to-biomass differences must underlie the interpretation of isotopic differences between individual compounds and total organic matter in a natural mixture.
My approach is pedagogic. The coverage is meant to be thorough, but the emphases and presentation have been chosen for readers approaching this subject as students rather than as research specialists. In common with the geochemists in my classes, many readers of this paper may not be very familiar with biochemistry and microbiology. I have not tried to explain every concept from those subjects and I have not inserted references for points that appear in standard texts in biochemistry or microbiology. Among such books, I particularly recommend the biochemistry text by Garrett and Grisham (1999) and the microbiology text by Madigan et al. (2000). The biochemistry text edited by Zubay (1998) is also particularly elegant and detailed. White (1999) has written a superb but condensed text on the physiology and biochemistry of prokaryotes.
A schematic overview of the relevant processes is shown in Figure 1⇓. Plants and other autotrophs fix CO2. Animals and other heterotrophs utilize organic compounds. If the assimilated carbon is a small molecule (like CO2, CH4, or acetate), significant isotopic fractionation is likely to accompany the fixation or assimilation of C. Such fractionations establish the isotopic relationship between an organism and its carbon source. Those associated …
The application of a combined gas chromatography/isotope ratio mass spectrometry (GC/IRMS) method for stable carbon isotope analysis of amino acid enantiomers is presented. This method eliminates the numerous preparative steps integral to the isolation of amino acids and amino acid enantiomers from protein hydrolyzates that precede delta-C-13 analysis by conventional isotope ratio mass spectrometry. Unlike hydrocarbons, amino acids require derivatization prior to GC/IRMS analysis. Replicate delta-C-13 analyses of trifluoroacetyl (TFA) isopropyl ester derivatives of 22 amino acids by IRMS revealed that the derivatization process is reproducible, with an average error (1 standard deviation) of 0.10% +/- 0.09%. The average analytical error for analysis of amino acid derivatives by GC/IRMS was 0.26% +/- 0.09%. In general, absolute differences between IRMS and GC/IRMS analyses were less than 0.5%. The derivatization process introduces a distinct, reproducible isotopic fractionation that is constant for each amino acid type. The observed fractionations preclude direct calculation of underivatized amino acid delta-C-13 values from their respective TFA isopropyl ester delta-C-13 compositions through mass balance relationships. Derivatization of amino acid standards of known stable carbon isotope compositions in conjunction with natural samples, however, permits computation of the original, underivatized amino acid delta-C-13 values through use of an empirical correction for the carbon introduced during the derivatization process.
The carbon isotopic composition of any naturally synthesized organic compound depends on (1) the carbon source utilized, (2) isotope effects associated with assimilation of carbon by the producing organism, (3) isotope effects associated with metabolism and biosynthesis, and (4) cellular carbon budgets. These factors are reviewed and quantitative considerations summarized, particularly with regard to active and passive modes of carbon assimilation by phytoplankton and the existence of discernible regularities in isotope effects associated with lipid biosynthesis. It is concluded that n-alkyl lipids are in general depleted in 13C by about 1.5‰ relative to polyisoprenoid lipids produced by the same organism. The effects of biological reworking on isotopic compositions of organic carbon are examined and it is suggested that enrichment of 13C in sedimentary organic carbon may result from loss of CH4 from zooplanktonic gut communities. Isotopic methods for estimation of ancient CO2 levels are considered and a hyperbolic form favored for the relationship between concentrations of dissolved CO2 and isotopic fractionation.
Dihydrodipicolinate synthase (DHDPS; EC4.2.1.52) catalyses the first reaction of lysine biosynthesis in plants and bacteria. Plant DHDPS enzymes are strongly inhibited by lysine (I0.5≈ 10 μM), whereas the bacterial enzymes are less (50-fold) or insensitive to lysine inhibition. We found that plant dhdps sequences expressing lysine-sensitive DHDPS enzymes are unable to complement a bacterial auxotroph, although a functional plant DHDPS enzyme is formed. As a consequence of this, plant dhdps cDNA clones which have been isolated through functional complementation using the DHDPS-deficient Escherichia coli strain encode mutated DHDPS enzymes impaired in lysine inhibition. The experiments outlined in this article emphasize that heterologous complementation can select for mutant clones when altered protein properties are requisite for functional rescue. In addition, the mutants rescued by heterologous complementation revealed a new critical amino acid substitution which renders lysine insensitivity to the plant DHDPS enzyme. An interpretation is given for the impaired inhibition mechanism of the mutant DHDPS enzyme by integrating the identified amino acid substitution in the DHDPS protein structure.
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.
Pigs were reared in laboratory pens on controlled diets that consisted of either 100% C3 plants or 100% C4 plants. Carbon and nitrogen isotopic compositions of the diets, and the resulting pig products, purified collagen and muscle tissue, were measured to determine isotopic fractionation during growth and metabolism. Total collagen from pigs grown on C3 diets was enriched in 13C by 3·2‰ and in 15N by 2·2‰, whereas that from pigs reared on C4 diets was enriched in 13C by 1·4‰ and in 15N by 2·3‰. In addition, fractionation between pigs and their diets was determined at the molecular level on individual amino acids separated by ion exchange chromatography. The carbon isotopic compositions of separated amino acids from the C3 and C4 diets were transferred to amino acids in bone collagen. For nitrogen, the isotopic compositions of all nonessential amino acids were enriched in 15N relative to those amino acids in the diet. Threonine, an essential amino acid, behaved oppositely, in that its isotope ratio (δ15N) was depleted by an average of 6‰ from the δ15N of the whole collagen. Similar isotopic patterns were analysed in collagenous amino acids extracted from field specimens that included both herbivores and carnivores; marine animals and terrestrial animals; and C3 and C4 feeders. Amino acids from two fossil bones, a bison (4500 years old) and a whale (70,000 years old), recorded the same isotopic signals as modern collagen. The ubiquity of these isotopic patterns at the molecular level suggests that distinct biochemical mechanisms control the metabolism of amino acids in animals rather than random synthesis.
To evaluate the importance of reactions within the central metabolism under different flux burdens the fluxes within the pentose phosphate pathway (PPP), as well as the other reactions of the central metabolism, were intensively analyzed and quantitated. For this purpose, Corynebacterium glutamicum was grown with [1-(13)C]glucose to metabolic and isotopic steady state and the fractional enrichments in precursor metabolites (e.g., pentose 5-phosphate) were quantified. Matrix calculus was used to express these data together with metabolite mass data. The detailed analysis of the dependence of (13)C enrichments on exchange fluxes enabled the transketolase-catalyzed exchange rate (2 pentose 5-phosphate <--> sedoheptulose 7-phosphate + glyceraldehyde 3-phosphate) to be quantified as 74.3% (molar metabolite flux) at a net flux of 10.3% and the exchange rate (pentose 5-phosphate + erythrose 4-phosphate <--> fructose 6-phosphate + glyceraldehyde 3-phosphate) to be quantified as 5.6% at a net flux of 8.1%. The flux entering the tricarboxylic acid cycle was 93.3%. The same comprehensive flux analysis as performed for the nonexcreting condition was done with the identical strain that had been forced to excrete L-glutamate. Because we had already quantified the fluxes for L-lysine excretion with an isogenic strain, three directly comparable flux situations are thus available. Consequently, this comparison permits a direct cause-and-effect relationship to be specified. In response to the different flux burdens of the cell, the PPP flux decreased from a maximum of 67% to 26%, with the glycolytic flux increasing accordingly. The carbon flux through isocitrate dehydrogenase increased from 20% to 36%. The bidirectional carbon flux between pyruvate and oxaloacetate decreased from 36% to 9%. Since the cause of the three different flux states was the allelic exchange in the final L-lysine assembling pathway or the glutamate export activity, respectively, the flexible response is the effect. This shows conclusively the enormous flexibility within the central metabolism of C. glutamicum to supply precursors upon their withdrawal for the synthesis of amino acids. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 168-180, 1997.
High throughput sequencing methods are widely used in analyses of microbial diversity, but are generally applied to small numbers of samples, which precludes characterization of patterns of microbial diversity across space and time. We have designed a primer-tagging approach that allows pooling and subsequent sorting of numerous samples, which is directed to amplification of a region spanning the nuclear ribosomal internal transcribed spacers and partial large subunit from fungi in environmental samples. To test the method for phylogenetic biases, we constructed a controlled mixture of four taxa representing the Chytridiomycota, Zygomycota, Ascomycota and Basidiomycota. Following cloning and colony restriction fragment length polymorphism analysis, we found no significant difference in representation in 19 of the 23 tested primers. We also generated a clone library from two soil DNA extracts using two primers for each extract and compared 456 clone sequences. Community diversity statistics and contingency table tests applied to counts of operational taxonomic units revealed that the two DNA extracts differed significantly, while the pairs of tagged primers from each extract were indistinguishable. Similar results were obtained using UniFrac phylogenetic comparisons. Together, these results suggest that the pig-tagged primers can be used to increase ecological inference in high throughput sequencing projects on fungi.
Abstract The isotope fractionation of carbon from substrates possessing different isotope ratios into fatty acids of polar lipids and amino acids was determined for four different fungi (Rhizopus arrhizus, Mortierella isabellina, Fusarium solani, Aspergillus niger). Carbon isotope ratios of fungi closely followed that of the substrates. Palmitic acid (C16:0), derived from phospholipids, did not display a large carbon isotope fractionation against the substrate. Stearic acid (C18:0), however, was depleted in (13)C against C16:0 in all strains. The desaturation of C18:0 to oleic acid (C18:1omega9) had little effect on the carbon isotope ratio. The subsequent desaturation of C18:1omega9 to linolic acid (C18:2omega6,9) enriched the resulting C18:2omega6,9 by +3.9 per thousand and varied little among strains. This result is important because C18:2omega6,9 is often used as a biomarker in environmental studies. Most amino acids were enriched in (13)C compared to the substrates, but isoleucine and lysine were close to the isotope ratio of the substrate and phenylalanine and leucine were depleted. Interestingly, the carbon isotope ratios of many amino acids differed significantly among different species. A discriminant analysis based on the isotope ratio of four amino acids (Thr, Ile, Phe, Val) resolved the two phyla in the first discriminant function and all four strains in the first two discriminant functions and confirmed a taxon-specific manner of isotope fractionation. The derived rules provide the basis for the use of stable isotopes in environmental studies to elucidate the role of fungi in the carbon flow in the environment.
The genetic basis for resistance to a number of antibiotics was examined in Rhizobium japonicum. Resistance to penicillin G, neomycin, and chloramphenicol appears to be mediated by an extrachromosomal element similar
to that found in the Enterobacteriaceae. Resistance to these antibiotics was eliminated from cells by treatment with acridine orange, and resistance to all three
antibiotics could be transferred en bloc to Agrobacterium tumefaciens under conditions excluding transformation or transduction as possible genetic mechanisms.
Compound-specific isotope analysis (CSIA) by isotope ratio mass spectrometry (IRMS) following on-line combustion (C) of compounds separated by gas chromatography (GC) is a relatively young analytical method. Due to its ability to measure isotope distribution at natural abundance level with great accuracy and high precision, GC-C-IRMS has increasingly become the method of choice in authenticity control of foodstuffs and determination of origin in archaeology, geochemistry, and environmental chemistry. In combination with stable isotope labelled compounds, GC-C-IRMS is also used more and more in biochemical and biomedical application as it offers a reliable and risk-free alternative to the use of radioactive tracers. The literature on these topics is reviewed from the advent of commercial GC-C-IRMS systems in 1990 up to the beginning of 1998. Demands on sample preparation and quality of GC separation for GC-C-IRMS are discussed also.
Dihydrodipicolinate synthase (DHDPS; EC4.2.1.52) catalyses the first reaction of lysine biosynthesis in plants and bacteria. Plant DHDPS enzymes are strongly inhibited by lysine (I0.5 approximately 10 microM), whereas the bacterial enzymes are less (50-fold) or insensitive to lysine inhibition. We found that plant dhdps sequences expressing lysine-sensitive DHDPS enzymes are unable to complement a bacterial auxotroph, although a functional plant DHDPS enzyme is formed. As a consequence of this, plant dhdps cDNA clones which have been isolated through functional complementation using the DHDPS-deficient Escherichia coli strain encode mutated DHDPS enzymes impaired in lysine inhibition. The experiments outlined in this article emphasize that heterologous complementation can select for mutant clones when altered protein properties are requisite for functional rescue. In addition, the mutants rescued by heterologous complementation revealed a new critical amino acid substitution which renders lysine insensitivity to the plant DHDPS enzyme. An interpretation is given for the impaired inhibition mechanism of the mutant DHDPS enzyme by integrating the identified amino acid substitution in the DHDPS protein structure.
Molecular phylogenetic studies reveal that many endosymbioses between bacteria and invertebrate hosts result from ancient infections followed by strict vertical transmission within host lineages. Endosymbionts display a distinctive constellation of genetic properties including AT-biased base composition, accelerated sequence evolution, and, at least sometimes, small genome size; these features suggest increased genetic drift. Molecular genetic characterization also has revealed adaptive, host-beneficial traits such as amplification of genes underlying nutrient provision.
Recent finding that a prokaryote synthesizes lysine through the alpha-aminoadipate pathway demonstrates that the lysine synthesis through the alpha-aminoadipate pathway is not typical of fungi. However, the fungal lysine biosynthesis is not completely the same as the prokaryotic one. We point out that alpha-aminoadipate reductase is a key enzyme to the evolution of fungal lysine synthesis. In addition, fungi have two different saccharopine dehydrogenases, which is also characteristic of fungi.
Many insect species rely on intracellular bacterial symbionts for their viability and fecundity. Large-scale DNA-sequence analyses are revealing the forces that shape the evolution of these bacterial associates and the genetic basis of their specialization to an intracellular lifestyle. The full genome sequences of two obligate mutualists, Buchnera aphidicola of aphids and Wigglesworthia glossinidia of tsetse flies, reveal substantial gene loss and an integration of host and symbiont metabolic functions. Further genomic comparisons should reveal the generality of these features among bacterial mutualists and the extent to which they are shared with other intracellular bacteria, including obligate pathogens.
After exploring evolutionary aspects of branched-chain amino acid biosynthesis, the review focuses on the extended leucine biosynthetic pathway as it operates in Saccharomyces cerevisiae. First, the genes and enzymes specific for the leucine pathway are considered: LEU4 and LEU9 (encoding the alpha-isopropylmalate synthase isoenzymes), LEU1 (isopropylmalate isomerase), and LEU2 (beta-isopropylmalate dehydrogenase). Emphasis is given to the unusual distribution of the branched-chain amino acid pathway enzymes between mitochondrial matrix and cytosol, on the newly defined role of Leu5p, and on regulatory mechanisms governing gene expression and enzyme activity, including new evidence for the metabolic importance of the regulation of alpha-isopropylmalate synthase by coenzyme A. Next, structure-function relationships of the transcriptional regulator Leu3p are addressed, defining its dual role as activator and repressor and discussing evidence in support of the self-masking model. Recent data pointing at a more extended Leu3p regulon are discussed. An overview of the layered controls of the extended leucine pathway is provided that includes a description of the newly recognized roles of Ilv5p and Bat1p in maintaining mitochondrial integrity. Finally, branched-chain amino acid biosynthesis and its regulation in other fungi are summarized, the question of leucine as metabolic signal is addressed, and possible directions of future research in this area are outlined.
Stable isotopes are increasingly being used as tracers in environmental studies. One application is to use isotopic ratios to quantitatively determine the proportional contribution of several sources to a mixture, such as the proportion of various pollution sources in a waste stream. In general, the proportional contributions of n+1 different sources can be uniquely determined by the use of n different isotope system tracers (e.g., delta13C, delta15N, delta18O) with linear mixing models based on mass balance equations. Often, however, the number of potential sources exceeds n+1, which prevents finding a unique solution of source proportions. What can be done in these situations? While no definitive solution exists, we propose a method that is informative in determining bounds for the contributions of each source. In this method, all possible combinations of each source contribution (0-100%) are examined in small increments (e.g., 1%). Combinations that sum to the observed mixture isotopic signatures within a small tolerance (e.g., +/-0.1 per thousand ) are considered to be feasible solutions, from which the frequency and range of potential source contributions can be determined. To avoid misrepresenting the results, users of this procedure should report the distribution of feasible solutions rather than focusing on a single value such as the mean. We applied this method to a variety of environmental studies in which stable isotope tracers were used to quantify the relative magnitude of multiple sources, including (1) plant water use, (2) geochemistry, (3) air pollution, and (4) dietary analysis. This method gives the range of isotopically determined source contributions; additional non-isotopic constraints specific to each study may be used to further restrict this range. The breadth of the isotopically determined ranges depends on the geometry of the mixing space and the similarity of source and mixture isotopic signatures. A sensitivity analysis indicated that the estimated ranges vary only modestly with different choices of source increment and mass balance tolerance parameter values. A computer program (IsoSource) to perform these calculations for user-specified data is available at http://www.epa.gov/wed/pages/models.htm.