Yoshito Chikaraishi

Compound-specific δD–δ13C analyses of n-alkanes extracted from terrestrial and aquatic plants

Abstract Stable hydrogen and carbon isotopic compositions of individual n -alkanes were determined for various terrestrial plants (33 samples including 27 species) and aquatic plants (six species) in natural environments from Japan and Thailand. In C3 plants, n- alkanes extracted from angiosperms have a δ D value of −152±26‰ (relative to Standard Mean Ocean Water [SMOW]) and δ 13 C value of −36.1±2.7‰ (relative to Peedde Belemnite [PDB]), and those from gymnosperms have a δ D value of −149±16‰ and δ 13 C value of −31.6±1.7‰. Angiosperms have n -alkanes depleted in 13 C relative to gymnosperms. n- Alkanes from C4 plants have a δ D value of −171±12‰ and δ 13 C value of −20.5±2.1‰, being a little depleted in D and much enriched in 13 C compared to C3 plants. n- Alkanes of CAM plants are a little depleted in D and vary widely in δ 13 C relative to those of C3 and C4 plants. In aquatic plants, n- alkanes from freshwater plants have a δ D value of −187±16‰ and δ 13 C value of −25.3±1.9‰, and those from seaweeds have a δ D value of −155±34‰ and δ 13 C value of −22.8±1.0‰. All n -alkanes from various plant classes are more depleted in D and 13 C relative to environmental water and bulk tissue, respectively. In addition, the hydrogen and carbon isotopic fractionations during n -alkane synthesis are distinctive for these various plant classes. While C3 plants have smaller isotopic fractionations in both D and 13 C, seaweed has larger isotopic fractionations.

Geographical origin of polished rice based on multiple element and stable isotope analyses

We determined carbon and nitrogen contents (C and N contents) and stable carbon, nitrogen, and oxygen isotopic compositions (δ(13)C, δ(15)N, and δ(18)O) of polished rice in order to develop a simple method to discriminate its geographical origin. As a first attempt, we examined a single cultivar, Koshihikari rice, from 14 different cultivation areas including Australia (n=1), Japan (n=12), and USA (n=1). For all rice samples, C and N contents and the isotopic compositions are consistent with those of general plant materials, being 37.2-40.0% (C content), 0.8-1.4% (N content), -27.1 to -25.4% (δ(13)C), +0.4 to +9.0% (δ(15)N), and +18.8 to +22.9% (δ(18)O). However, its cultivated area is clearly distinguished by a pentagonal radar plot based on the elemental and isotopic compositions. Thus, the comparison of C and N contents and δ(13)C, δ(15)N, and δ(18)O values would potentially be useful for rapid and routine discrimination of geographical origin of the polished rice.

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

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

Quantitative Evaluation of Marine Protein Contribution in Ancient Diets Based on Nitrogen Isotope Ratios of Individual Amino Acids in Bone Collagen: An Investigation at the Kitakogane Jomon Site

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

Stable carbon, nitrogen, and oxygen isotope analysis as a potential tool for verifying geographical origin of beef.

Stable isotope analysis of organic elements such as carbon and nitrogen has been employed as a powerful tool for provenance determination of food materials, because isotopic compositions of the materials reflect many factors in natural environment. In this study, we examined carbon, nitrogen, and oxygen isotope signatures of beef from Australia, Japan, and USA, in order to confirm the method as a potential tool for verifying geographical origin of beef commercially distributed in Japan. Defatted dry matter of beef from USA was characterized by higher carbon isotopic composition (-13.6 per thousand to -11.1 per thousand) than that from Japan (-19.6 per thousand to -17.0 per thousand) and Australia (-23.6 per thousand to -18.7 per thousand). That from Australia was characterized by higher oxygen isotopic composition (+15.0 per thousand to +19.4 per thousand) than that from Japan (+7.3 per thousand to +13.6 per thousand) and USA (+9.5 per thousand to +11.7 per thousand). The oxygen isotopic composition in Japanese beef showed a positive correlation with the isotopic composition of cattle drinking water, the difference in which is clearly latitude dependent. These results suggest that a comparison of carbon, nitrogen, and oxygen isotopic compositions is applicable as a potential tool to discriminate the provenance of beef not only between different countries (i.e. Australia, Japan, and USA) but also among different regions within Japan.

A low trophic position of Japanese eel larvae indicates feeding on marine snow.

What eel larvae feed on in the surface layer of the ocean has remained mysterious. Gut contents and bulk nitrogen stable isotope studies suggested that these unusual larvae, called leptocephali, feed at a low level in the oceanic food web, whereas other types of evidence have suggested that small zooplankton are eaten. In this study, we determined the nitrogen isotopic composition of amino acids of both natural larvae and laboratory-reared larvae of the Japanese eel to estimate the trophic position (TP) of leptocephali. We observed a mean TP of 2.4 for natural leptocephali, which is consistent with feeding on particulate organic matter (POM) such as marine snow and discarded appendicularian houses containing bacteria, protozoans and other biological materials. The nitrogen isotope enrichment values of the reared larvae confirm that the primary food source of natural larvae is consistent only with POM. This shows that leptocephali feed on readily available particulate material originating from various sources closely linked to ocean primary production and that leptocephali are a previously unrecognized part of oceanic POM cycling.

Evidence of Global Chlorophyll d

Although analyses of chlorophyll d (Chl d)‐dominated oxygenic photosystems have been conducted since their discovery 12 years ago, Chl d distribution in the environment and quantitative importance for aquatic photosynthesis remain to be investigated. We analyzed the pigment compositions of surface sediments and detected Chl d and its derivatives from diverse aquatic environments. Our data show that the viable habitat for Chl d‐producing phototrophs extends across salinities of 0 to 50 practical salinity units and temperatures of 1� to 40�C, suggesting that Chl d production can be ubiquitously observed in aquatic environments that receive near-infrared light. The relative abundances of Chl d derivatives over that of Chl a derivatives in the studied samples are up to 4%, further suggesting that Chl d‐based photosynthesis plays a quantitatively important role in the aquatic photosynthesis.

Biochemical and physiological bases for the use of carbon and nitrogen isotopes in environmental and ecological studies

We review the biochemical and physiological bases of the use of carbon and nitrogen isotopic compositions as an approach for environmental and ecological studies. Biochemical processes commonly observed in the biosphere, including the decarboxylation and deamination of amino acids, are the key factors in this isotopic approach. The principles drawn from the isotopic distributions disentangle the complex dynamics of the biosphere and allow the interactions between the geosphere and biosphere to be analyzed in detail. We also summarize two recently examined topics with new datasets: the isotopic compositions of individual biosynthetic products (chlorophylls and amino acids) and those of animal organs for further pursuing the basis of the methodology. As a tool for investigating complex systems, compound-specific isotopic analysis compensates the intrinsic disadvantages of bulk isotopic signatures. Chlorophylls provide information about the particular processes of various photoautotrophs, whereas amino acids provide a precise measure of the trophic positions of heterotrophs. The isotopic distributions of carbon and nitrogen in a single organism as well as in the whole biosphere are strongly regulated, so that their major components such as amino acids are coordinated appropriately rather than controlled separately.

Trophic hierarchies illuminated via amino acid isotopic analysis.

Food web ecologists have long sought to characterize the trophic niches of animals using stable isotopic analysis. However, distilling trophic position from isotopic composition has been difficult, largely because of the variability associated with trophic discrimination factors (inter-trophic isotopic fractionation and routing). We circumvented much of this variability using compound-specific isotopic analysis (CSIA). We examined the 15N signatures of amino acids extracted from organisms reared in pure culture at four discrete trophic levels, across two model communities. We calculated the degree of enrichment at each trophic level and found there was a consistent trophic discrimination factor (~7.6‰). The constancy of the CSIA-derived discrimination factor permitted unprecedented accuracy in the measurement of animal trophic position. Conversely, trophic position estimates generated via bulk-15N analysis significantly underestimated trophic position, particularly among higher-order consumers. We then examined the trophic hierarchy of a free-roaming arthropod community, revealing the highest trophic position (5.07) and longest food chain ever reported using CSIA. High accuracy in trophic position estimation brings trophic function into sharper focus, providing greater resolution to the analysis of food webs.

Microbes are trophic analogs of animals

Significance We report evidence that microbes are trophically equivalent to animals. When bacteria or fungi are fed the same diets as animals, the microbes register the same trophic position as animals. This discovery reframes how microbes can be viewed within food chains and facilitates the inclusion of the microbiome in functional diversity studies. To demonstrate the broad applicability of our approach, we investigated the ancient symbioses represented by leaf-cutter ant fungus gardens, revealing four discrete trophic levels within this community and providing evidence that fungi, not ants, are the dominant herbivores of the Neotropics. Altogether, we show that microbes can be integrated with plants and animals in a food chain, thereby unifying the macro- and microbiome in studies of trophic ecology. In most ecosystems, microbes are the dominant consumers, commandeering much of the heterotrophic biomass circulating through food webs. Characterizing functional diversity within the microbiome, therefore, is critical to understanding ecosystem functioning, particularly in an era of global biodiversity loss. Using isotopic fingerprinting, we investigated the trophic positions of a broad diversity of heterotrophic organisms. Specifically, we examined the naturally occurring stable isotopes of nitrogen (15N:14N) within amino acids extracted from proteobacteria, actinomycetes, ascomycetes, and basidiomycetes, as well as from vertebrate and invertebrate macrofauna (crustaceans, fish, insects, and mammals). Here, we report that patterns of intertrophic 15N-discrimination were remarkably similar among bacteria, fungi, and animals, which permitted unambiguous measurement of consumer trophic position, independent of phylogeny or ecosystem type. The observed similarities among bacterial, fungal, and animal consumers suggest that within a trophic hierarchy, microbiota are equivalent to, and can be interdigitated with, macrobiota. To further test the universality of this finding, we examined Neotropical fungus gardens, communities in which bacteria, fungi, and animals are entwined in an ancient, quadripartite symbiosis. We reveal that this symbiosis is a discrete four-level food chain, wherein bacteria function as the apex carnivores, animals and fungi are meso-consumers, and the sole herbivores are fungi. Together, our findings demonstrate that bacteria, fungi, and animals can be integrated within a food chain, effectively uniting the macro- and microbiome in food web ecology and facilitating greater inclusion of the microbiome in studies of functional diversity.