Application of a porous graphitic carbon column to carbon and nitrogen isotope analysis of underivatized individual amino acids using high-performance liquid chromatography coupled with elemental analyzer/isotope ratio mass spectrometry.

RATIONALE: Isolation of underivatized amino acids (AAs) using high-performance liquid chromatography (HPLC) is becoming a popular method for carbon (δ13 C) and nitrogen isotope (δ15 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, δ13 C and δ15 N values of AAs were determined using elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). We demonstrated the application of this method by determining δ13 C and δ15 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 δ13 C and δ15 N values of AA standards remained unchanged during the whole experimental procedure. The δ13 C values of AAs in mackerel muscle are also in good agreement with the values obtained using another verified method for δ13 C analysis. CONCLUSIONS: The good separation performance of hydrophilic AAs and the reliability of δ13 C and δ15 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 δ13 C and δ15 N analyses of AAs in natural samples.

Tracking long-distance migration of marine fishes using compound-specific stable isotope analysis of amino acids.

The long-distance migrations by marine fishes are difficult to track by field observation. Here, we propose a new method to track such migrations using stable nitrogen isotopic composition at the base of the food web (δ15 NBase ), which can be estimated by using compound-specific isotope analysis. δ15 NBase exclusively reflects the δ15 N of nitrate in the ocean at a regional scale and is not affected by the trophic position of sampled organisms. In other words, δ15 NBase allows for direct comparison of isotope ratios between proxy organisms of the isoscape and the target migratory animal. We initially constructed a δ15 NBase isoscape in the northern North Pacific by bulk and compound-specific isotope analyses of copepods (n = 360 and 24, respectively), and then we determined retrospective δ15 NBase values of spawning chum salmon (Oncorhynchus keta) from their vertebral centra (10 sections from each of two salmon). We then estimated the migration routes of chum salmon during their skeletal growth by using a state-space model. Our isotope tracking method successfully reproduced a known chum salmon migration route between the Okhotsk and Bering seas, and our findings suggest the presence of a new migration route to the Bering Sea Shelf during a later growth stage.

A method for stable carbon isotope measurement of underivatized individual amino acids by multi-dimensional high-performance liquid chromatography and elemental analyzer/isotope ratio mass spectrometry.

RATIONALE: To achieve better precision and accuracy for δ13 C analysis of individual amino acids (AAs), we have developed a new analytical method based on multi-dimensional high-performance liquid chromatography (HPLC) and elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). Unlike conventional methods using gas chromatography, this approach omits pre-column chemical derivatization, thus reducing systematic errors associated with the isotopic measurement. METHODS: The separation and isolation of individual AAs in a standard mixture containing 15 AAs and a biological sample, spear squid (Heterololigo bleekeri) were performed. AAs were isolated using an HPLC system equipped with a reversed-phase column and a mixed-mode column and collected using a fraction collector. After the chromatographic separation and further post-HPLC purification, the δ13 C values of AAs were measured by EA/IRMS. RESULTS: The complete isolation of all 15 AAs in the standard mixture was achieved. The δ13 C values of these AAs before and after the experiment were in good agreement. Also, 15 AAs in the biological sample, H. bleekeri, were successfully measured. The δ13 C values of AAs in H. bleekeri varied by as much as 30‰ with glycine being most enriched in13 C. CONCLUSIONS: The consistency between the δ13 C values of reference and processed AAs demonstrates that the experimental procedure generates accurate δ13 C values unaffected by fractionation effects and contamination. This method is therefore suitable for δ13 C analysis of biological samples with higher precision than conventional approaches. We propose this new method as a tool to measure δ13 C values of AAs in biological, ecological and biogeochemical studies.

Using food network unfolding to evaluate food-web complexity in terms of biodiversity: theory and applications.

Food-web complexity often hinders disentangling functionally relevant aspects of food-web structure and its relationships to biodiversity. Here, we present a theoretical framework to evaluate food-web complexity in terms of biodiversity. Food network unfolding is a theoretical method to transform a complex food web into a linear food chain based on ecosystem processes. Based on this method, we can define three biodiversity indices, horizontal diversity (DH ), vertical diversity (DV ) and range diversity (DR ), which are associated with the species diversity within each trophic level, diversity of trophic levels, and diversity in resource use, respectively. These indices are related to Shannon's diversity index (H'), where H' = DH  + DV  - DR . Application of the framework to three riverine macroinvertebrate communities revealed that D indices, calculated from biomass and stable isotope features, captured well the anthropogenic, seasonal, or other within-site changes in food-web structures that could not be captured with H' alone.

Improved Method for Isolation and Purification of Underivatized Amino Acids for Radiocarbon Analysis.

We have improved a method for isolation and purification of individual amino acids for compound-specific radiocarbon analysis (CSRA). To remove high-performance liquid chromatography (HPLC) eluent blanks from isolated amino acid fractions prior to the radiocarbon (Δ14C) measurement, each fraction was filtered through a membrane filter and then washed with diethyl ether twice. Radiocarbon measurements on standard amino acids processed and purified with the above method using elemental analyzer-accelerator mass spectrometry resulted in Δ14C values that were in strong agreement ( R2 = 0.998) with the original Δ14C value of each amino acid standard. From these measurements, we calculate dead and modern carbon contamination contributions as 1.2 ± 0.2 and 0.3 ± 0.1 µgC, respectively, which are consistent with direct assessments of HPLC procedural blanks of 1.0 ± 0.8 µgC per sample. These contamination constraints allow correction of measured Δ14C values for accurate and precise CSRA and are widely applicable to future archeological and biogeochemical studies.

Terrestrial-aquatic linkage in stream food webs along a forest chronosequence: multi-isotopic evidence.

Long-term monitoring of ecosystem succession provides baseline data for conservation and management, as well as for understanding the dynamics of underlying biogeochemical processes. We examined the effects of deforestation and subsequent afforestation of a riparian forest of Japanese cedar (Cryptomeria japonica) on stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹5N) and natural abundances of radiocarbon (Δ¹4C) in stream biota in the Mt. Gomadan Experimental Forest and the Wakayama Forest Research Station, Kyoto University, central Japan. Macroinvertebrates, periphytic algae attached to rock surfaces (periphyton), and leaf litter of terrestrial plants were collected from six headwater streams with similar climate, topography, and bedrock geology, except for the stand ages of riparian forests (from 3 to 49 yr old in five stands and > 90 yr old in one reference stand). Light intensity and δ¹³C values of both periphyton and macroinvertebrates decreased synchronously with forest age in winter. A Bayesian mixing model indicates that periphyton contributions to the stream food webs are maximized in 23-yr-old forests. Except for grazers, most macroinvertebrates showed Δ¹4C values similar to those of terrestrial leaf litter, reflecting the influence of modern atmospheric CO2 Δ¹4C values. On the other hand, the Δ¹4C values of both periphyton and grazers (i.e., aquatic primary consumers) were significantly lower than that of modern atmospheric CO2, and were lowest in 23-yr-old forest stands. Previous studies show that root biomass of C. japonica peaks at 15-30 yr after planting. These evidences suggest that soil CO2 released by root respiration and dispersed by groundwater weathers carbonate substrata, and that dissolved inorganic carbon (DIC) with low Δ¹4C is incorporated into stream periphyton and some macroinvertebrates. The ecological response in the studied streams to clear-cutting and replanting of Japanese cedar is much slower (~20 yr) than the chemical response (< 5 yr). More than 50 yr is required for the food web structure to completely recover from clear-cutting. The ecological delay is attributed to several biogeochemical factors, the understanding of which is critical to integrated management of forest-stream continuum and the prediction of ecosystem resilience in response to environmental change.

Carbon storage reservoirs in watersheds support stream food webs via periphyton production.

We measured the natural abundances of radiocarbon (delta14C) in macroinvertebrates, fishes, and their potential food sources, collected from the upper and lower reaches of six temperate streams in Lake Biwa basin (central Japan), three of which flow on limestone bedrock. Several carbon storage reservoirs in the watersheds show distinctive delta14C signatures (e.g., ancient carbonate rocks, -1000 per thousand; modern atmospheric CO2, +50 per thousand). Our analyses showed that the delta14C values for periphytic algae range from -361 per thousand to +21 per thousand, reflecting 14C-depleted signals from watershed storage reservoirs (carbonate rocks and/or soils). In contrast, the delta14C values for coarse particulate organic matter (CPOM) range from -6 per thousand to +62 per thousand, reflecting 14C-enriched signals from modern atmospheric CO2. The periphyton from streams on limestone bedrock was more 14C-depleted than that from streams in non-limestone areas, although the delta14C values for periphyton from the latter were less than modern atmospheric 14CO2 concentration. The delta14C values for most of the consumers were between those for periphyton and CPOM. Based on a delta14C two-source mixing models, the results suggested that the grazers rely on periphyton, while the carbon source for collectors and predators shifts from CPOM in the upper reaches of streams to periphyton in the lower reaches. The delta14C signature can trace carbon from watershed storage reservoirs to benthic production, which suggests that stream food webs are composed of mixtures of carbon originating from various sources of different ages.

Stable nitrogen isotopic composition of amino acids reveals food web structure in stream ecosystems.

The stable N isotopic composition of individual amino acids (SIAA) has recently been used to estimate trophic positions (TPs) of animals in several simple food chain systems. However, it is unknown whether the SIAA is applicable to more complex food web analysis. In this study we measured the SIAA of stream macroinvertebrates, fishes, and their potential food sources (periphyton and leaf litter of terrestrial C3 plants) collected from upper and lower sites in two streams having contrasting riparian landscapes. The stable N isotope ratios of glutamic acid and phenylalanine confirmed that for primary producers (periphyton and C3 litter) the TP was 1, and for primary consumers (e.g., mayfly and caddisfly larvae) it was 2. We built a two-source mixing model to estimate the relative contributions of aquatic and terrestrial sources to secondary and higher consumers (e.g., stonefly larva and fishes) prior to the TP calculation. The estimated TPs (2.3-3.5) roughly corresponded to their omnivorous and carnivorous feeding habits, respectively. We found that the SIAA method offers substantial advantages over traditional bulk method for food web analysis because it defines the food web structure based on the metabolic pathway of amino groups, and can be used to estimate food web structure under conditions where the bulk method cannot be used. Our result provides evidence that the SIAA method is applicable to the analysis of complex food webs, where heterogeneous resources are mixed.

Global meta-analysis for controlling factors on carbon stable isotope ratios of lotic periphyton.

Carbon stable isotope ratios (δ(13)C) are widely used to trace resource transfer pathways, yet δ(13)C variation in freshwater autotrophs is not yet fully understood. We have analyzed data from 42 published studies, supplemented with some unpublished data, to show the determinants of lotic periphyton δ(13)C. At large spatial scales, we observed broad differences in periphyton δ(13)C among biomes and consistent longitudinal variation related to watershed area. Longitudinal increases in δ(13)C indicate the importance of in-stream processes on lotic carbon cycles and autotroph δ(13)C variation. At local spatial scales, periphyton δ(13)C was negatively related to canopy cover and water current velocity and positively related to chlorophyll a density. Autotroph δ(13)C varied among taxonomic groups. Cyanobacteria and red algae had significantly higher and lower δ(13)C than other taxa, respectively. A hierarchical model across spatial scales showed that local controls for periphyton δ(13)C were nested by regional controls, which suggested that productivity and CO(2) availability determine δ(13)C. Overall, our results reveal general patterns of periphyton δ(13)C and provide improved information for study design and the use of δ(13)C in isotopic mixing models in lotic food web studies.

Trophic discrimination factor of nitrogen isotopes within amino acids in the dobsonfly Protohermes grandis (Megaloptera: Corydalidae) larvae in a controlled feeding experiment.

The trophic discrimination factor (TDF) of nitrogen isotopes (15N/14N) within amino acids, between a stream-dwelling dobsonfly larva (Protohermes grandis: Megaloptera; Corydalidae) and its diet (chironomid larvae), was determined in controlled feeding experiments. Last-instar larvae of P. grandis were collected from the Yozawa-gawa River, central Japan, and reared in the laboratory. After fed to satiation for 1 month, one group of larvae was each fed one living chironomid larva per day for 4 weeks, while a second group was starved for 8 weeks. The larvae were harvested at intervals and the nitrogen isotopic composition of glutamic acid (δ15NGlu) and phenylalanine (δ15NPhe) were determined to calculate TDF. The mean TDF of satiated and starved larvae were 7.1‰ ± 0.5‰ (n = 3) and 7.3‰ ± 0.5‰ (n = 5), respectively. Thus, the TDF for P. grandis larvae in this study was similar to that reported for other arthropods (approximately 7‰) and was independent of satiation or starvation. A previous study of wild P. grandis larvae, based on the δ15NGlu and δ15NPhe values, estimated its trophic position (TP) as approximately 2.0 ± 0.1 (n = 5), a low value close to that of algivores, although they are generally characterized as carnivores (usually accepted as TP ≥ 3). The TDF for P. grandis larvae suggests that their low TPs in nature were caused by incorporation of vascular plant-derived amino acids (with a different δ15N profile from that of algae) and not by an unusually low TDF or by the effects of the satiation/starvation on amino acid metabolism.

Integrated trophic position decreases in more diverse communities of stream food webs.

The relationship between biodiversity and ecosystem functioning is an important theme in environmental sciences. We propose a new index for configuration of the biomass pyramid in an ecosystem, named integrated trophic position (iTP). The iTP is defined as a sum of trophic positions (i.e. the average number of steps involved in biomass transfer) of all the animals in a food web integrated by their individual biomass. The observed iTP for stream macroinvertebrates ranged from 2.39 to 2.79 and was negatively correlated with the species density and the Shannon-Wiener diversity index of the local community. The results indicate a lower efficiency of biomass transfer in more diverse communities, which may be explained by the variance in edibility hypothesis and/or the trophic omnivory hypothesis. We found a negative effect of biodiversity on ecosystem functioning.

High-resolution food webs based on nitrogen isotopic composition of amino acids.

Food webs are known to have myriad trophic links between resource and consumer species. While herbivores have well-understood trophic tendencies, the difficulties associated with characterizing the trophic positions of higher-order consumers have remained a major problem in food web ecology. To better understand trophic linkages in food webs, analysis of the stable nitrogen isotopic composition of amino acids has been introduced as a potential means of providing accurate trophic position estimates. In the present study, we employ this method to estimate the trophic positions of 200 free-roaming organisms, representing 39 species in coastal marine (a stony shore) and 38 species in terrestrial (a fruit farm) environments. Based on the trophic positions from the isotopic composition of amino acids, we are able to resolve the trophic structure of these complex food webs. Our approach reveals a high degree of trophic omnivory (i.e., noninteger trophic positions) among carnivorous species such as marine fish and terrestrial hornets.This information not only clarifies the trophic tendencies of species within their respective communities, but also suggests that trophic omnivory may be common in these webs.