Tracing the origin of nutrients is a fundamental goal of food web research but methodological issues associated with current research techniques such as using stable isotope ratios of bulk tissue can lead to confounding results. We investigated whether naturally occurring δ¹³C patterns among amino acids (δ¹³C_AA) could distinguish between multiple aquatic and terrestrial primary production sources. We found that δ¹³C_AA patterns in contrast to bulk δ¹³C values distinguished between carbon derived from algae, seagrass, terrestrial plants, bacteria and fungi. Furthermore, we showed for two aquatic producers that their δ¹³C_AA patterns were largely unaffected by different environmental conditions despite substantial shifts in bulk δ¹³C values. The potential of assessing the major carbon sources at the base of the food web was demonstrated for freshwater, pelagic, and estuarine consumers; consumer δ¹³C patterns of essential amino acids largely matched those of the dominant primary producers in each system. Since amino acids make up about half of organismal carbon, source diagnostic isotope fingerprints can be used as a new complementary approach to overcome some of the limitations of variable source bulk isotope values commonly encountered in estuarine areas and other complex environments with mixed aquatic and terrestrial inputs.

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.

Diet is a leading modifiable risk factor for chronic disease, but it remains difficult to measure accurately due to the error and bias inherent in self-reported methods of diet assessment. Consequently, there is a pressing need for more objective biomarkers of diet for use in health research. The stable isotope ratios of light elements are a promising set of candidate biomarkers because they vary naturally and reproducibly among foods, and those variations are captured in molecules and tissues with high fidelity. Recent studies have identified valid isotopic measures of short- and long-term sugar intake, meat intake, and fish intake in specific populations. These studies provide a strong foundation for validating stable isotopic biomarkers in the general US population. Approaches to improve specificity for specific foods are needed; for example, by modeling intake using multiple stable isotope ratios or by isolating and measuring specific molecules linked to foods of interest.

While conventional nutrition research has yielded biomarkers such as doubly labeled water for energy metabolism and 24-h urinary nitrogen for protein intake, a critical need exists for additional, equally robust biomarkers that allow for objective assessment of specific food intake and dietary exposure. Recent advances in high-throughput MS combined with improved metabolomics techniques and bioinformatic tools provide new opportunities for dietary biomarker development. In September 2018, the NIH organized a 2-d workshop to engage nutrition and omics researchers and explore the potential of multiomics approaches in nutritional biomarker research. The current Perspective summarizes key gaps and challenges identified, as well as the recommendations from the workshop that could serve as a guide for scientists interested in dietary biomarkers research. Topics addressed included study designs for biomarker development, analytical and bioinformatic considerations, and integration of dietary biomarkers with other omics techniques. Several clear needs were identified, including larger controlled feeding studies, testing a variety of foods and dietary patterns across diverse populations, improved reporting standards to support study replication, more chemical standards covering a broader range of food constituents and human metabolites, standardized approaches for biomarker validation, comprehensive and accessible food composition databases, a common ontology for dietary biomarker literature, and methodologic work on statistical procedures for intake biomarker discovery. Multidisciplinary research teams with appropriate expertise are critical to moving forward the field of dietary biomarkers and producing robust, reproducible biomarkers that can be used in public health and clinical research.

SUMMARY: The widely accepted idea that bees fuel flight through the oxidation of carbohydrate is based on studies of only a few species. We tested this hypothesis as part of our research program to investigate the size-dependence of flight energetics in Panamanian orchid bees. We succeeded in measuring rates of O2 consumption and CO2 production in vivo during hovering flight, as well as maximal activities(Vmax values) in vitro of key enzymes in flight muscle energy metabolism in nine species belonging to four genera. Respiratory quotients (ratios of rates of CO2 production to O2consumption) in all nine species are close to 1.0. This indicates that carbohydrate is the main fuel used for flight. Trehalase, glycogen phosphorylase and hexokinase activities are sufficient to account for the glycolytic flux rates estimated from rates of CO2 production. High activities of other glycolytic enzymes, as well as high activities of mitochondrial oxidative enzymes, are consistent with the estimated rates of carbohydrate-fueled oxidative metabolism. In contrast, hydroxyacylCoA dehydrogenase, an enzyme involved in fatty acid oxidation, was not detectable in any species. Thoracic homogenates displayed ADP-stimulated oxidition of pyruvate + proline, but did not oxidize palmitoyl l-carnitine +proline as substrates. A metabolic map, based on data reported herein and information from the literature, is presented. The evidence available supports the hypothesis that carbohydrate serves as the main fuel for flight in bees.