Multiple trophic pathways support fish on floodplains of California's Central Valley.

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.

Isotopic and genetic methods reveal the role of the gut microbiome in mammalian host essential amino acid metabolism.

Intestinal microbiota perform many functions for their host, but among the most important is their role in metabolism, especially the conversion of recalcitrant biomass that the host is unable to digest into bioavailable compounds. Most studies have focused on the assistance gut microbiota provide in the metabolism of carbohydrates, however, their role in host amino acid metabolism is poorly understood. We conducted an experiment on Mus musculus using 16S rRNA gene sequencing and carbon isotope analysis of essential amino acids (AAESS) to quantify the community composition of gut microbiota and the contribution of carbohydrate carbon used by the gut microbiome to synthesize AAESS that are assimilated by mice to build skeletal muscle tissue. The relative abundances of Firmicutes and Bacteroidetes inversely varied as a function of dietary macromolecular content, with Firmicutes dominating when mice were fed low-protein diets that contained the highest proportions of simple carbohydrates (sucrose). Mixing models estimated that the microbial contribution of AAESS to mouse muscle varied from less than 5% (threonine, lysine, and phenylalanine) to approximately 60% (valine) across diet treatments, with the Firmicute-dominated microbiome associated with the greatest contribution. Our results show that intestinal microbes can provide a significant source of the AAESS their host uses to synthesize structural tissues. The role that gut microbiota play in the amino acid metabolism of animals that consume protein-deficient diets is likely a significant but under-recognized aspect of foraging ecology and physiology.

Compound-specific δ2H analysis highlights the relationship between direct assimilation and de novo synthesis of amino acids from food and water in a terrestrial mammalian omnivore.

Hydrogen isotope (δ2H) analysis has been routinely used as an ecological tracer for animal movement and migration, yet a biochemical understanding of how animals incorporate this element in the synthesis of tissues is poorly resolved. Here, we apply a new analytical tool, amino acid (AA) δ2H analysis, in a controlled setting to trace the influence of drinking water and dietary macromolecules on the hydrogen in muscle tissue. We varied the δ2H of drinking water and the proportions of dietary protein and carbohydrates with distinct hydrogen and carbon isotope compositions fed to house mice among nine treatments. Our results show that hydrogen in the non-essential (AANESS) and essential (AAESS) AAs of mouse muscle is not readily exchanged with body water, but rather patterns among these compounds can be described through consideration of the major biochemical pathway(s) used by organisms to synthesize or route them from available sources. Dietary carbohydrates contributed more hydrogen than drinking water to the synthesis of AANESS in muscle. While neither drinking water nor dietary carbohydrates directly contributed to muscle AAESS, we did find that a minor but measurable proportion (10-30%) of the AAESS in muscle was synthesized by the gut microbiome using hydrogen and carbon from dietary carbohydrates. δ2H patterns among individual AAs in mice muscle are similar to those we previously reported for bacteria, which provides additional support that this approach may allow for the simultaneous analysis of different AAs that are more influenced by drinking water (AANESS) versus dietary (AAESS) sources of hydrogen.

Hydrogen isotopes in individual amino acids reflect differentiated pools of hydrogen from food and water in Escherichia coli.

Hydrogen isotope (δ(2)H) analysis is widely used in animal ecology to study continental-scale movement because δ(2)H can trace precipitation and climate. To understand the biochemical underpinnings of how hydrogen is incorporated into biomolecules, we measured the δ(2)H of individual amino acids (AAs) in Escherichia coli cultured in glucose-based or complex tryptone-based media in waters with δ(2)H values ranging from -55‰ to +1,070‰. The δ(2)H values of AAs in tryptone spanned a range of ∼250‰. In E. coli grown on glucose, the range of δ(2)H among AAs was nearly 200‰. The relative distributions of δ(2)H of AAs were upheld in cultures grown in enriched waters. In E. coli grown on tryptone, the δ(2)H of nonessential AAs varied linearly with the δ(2)H of media water, whereas δ(2)H of essential AAs was nearly identical to δ(2)H in diet. Model calculations determined that as much as 46% of hydrogen in some nonessential AAs originated from water, whereas no more than 12% of hydrogen in essential AAs originated from water. These findings demonstrate that δ(2)H can route directly at the molecular level. We conclude that the patterns and distributions in δ(2)H of AAs are determined through biosynthetic reactions, suggesting that δ(2)H could become a new biosignature for studying novel microbial pathways. Our results also show that δ(2)H of AAs in an organism's tissues provides a dual tracer for food and environmental (e.g., drinking) water.

Assimilation and discrimination of hydrogen isotopes in a terrestrial mammal.

Stable isotope analysis has revolutionized the way ecologists study animal resource use from the individual to the community level. Recent interest has emerged in using hydrogen isotopes (2H/1H) as ecological tracers, because they integrate information from both abiotic and biotic processes. A better physiological understanding of how animals assimilate hydrogen and use it to synthesize tissues is needed to further refine this tool and broaden its use in animal ecology. We conducted a controlled-feeding experiment using laboratory mice (Mus musculus) in which we varied the hydrogen isotope (δ2H) values of water and the proportions of dietary protein and carbohydrates among nine experimental treatments. For each tissue, we calculated the percent of hydrogen derived from water and the percent hydrogen derived from dietary protein versus carbohydrates using linear relationships and isotope mixing models based on accompanying carbon isotope (δ13C) data. The net discrimination (∆2HNet) between mice tissues and potential water and dietary sources of hydrogen differed among tissues. ∆2HNet was positively correlated with dietary protein content in red blood cells (RBC) and muscle, but negatively correlated in liver and plasma. We also report the first estimates for hydrogen isotope discrimination factors (∆2H) for different sources of hydrogen (∆2HWater, ∆2HProtein, and ∆2HCarbs) available for tissue synthesis. This research provides a foundation for understanding how diet quality (e.g., protein content) influences hydrogen isotope assimilation and discrimination in different tissues of a terrestrial mammal, which is a first step towards using δ2H as a tracer of resource use in free-ranging mammals.

Variability in the routing of dietary proteins and lipids to consumer tissues influences tissue-specific isotopic discrimination.

RATIONALE: The eco-physiological mechanisms that govern the incorporation and routing of macronutrients from dietary sources into consumer tissues determine the efficacy of stable isotope analysis (SIA) for studying animal foraging ecology. We document how changes in the relative amounts of dietary proteins and lipids affect the metabolic routing of these macronutrients and the consequent effects on tissue-specific discrimination factors in domestic mice using SIA. We also examine the effects of dietary macromolecular content on a commonly used methodological approach: lipid extraction of potential food sources. METHODS: We used carbon ((13) C) and nitrogen ((15) N) isotopes to examine the routing of carbon from dietary proteins and lipids that were used by mice to biosynthesize hair, blood, muscle, and liver. Growing mice were fed one of four diet treatments in which the total dietary content of C4 -based lipids (δ(13) C = -14.5‰) and C(3) -based proteins (δ(13) C = -27‰) varied inversely between 5% and 40%. RESULTS: The δ(13) C values of mouse tissues increased by approximately 2-6‰ with increasing dietary lipid content. The difference in δ(13) C values between mouse tissues and bulk diet ranged from 0.1 ± 1.5‰ to 2.3 ± 0.6‰ for all diet treatments. The mean (±SD) difference between the δ(13) C values of mouse tissues and dietary protein varied systematically among tissues and ranged from 3.1 ± 0.1‰ to 4.5 ± 0.6‰ for low fat diets and from 5.4 ± 0.4‰ to 10.5 ± 7.3‰ for high fat diets. CONCLUSIONS: Mice used some fraction of their dietary lipid carbon to synthesize tissue proteins, suggesting flexibility in the routing of dietary macromolecules to consumer tissues based on dietary macromolecular availability. Consequently, all constituent dietary macromolecules, not just protein, should be considered when determining the relationship between diets and consumer tissues using SIA. In addition, in cases where animals consume diets with high lipid contents, non lipid-extracted prey samples should be analyzed to estimate diets using SIA.

Pleistocene to historic shifts in bald eagle diets on the Channel Islands, California.

Studies of current interactions among species, their prey, and environmental factors are essential for mitigating immediate threats to population viability, but the true range of behavioral and ecological flexibility can be determined only through research on deeper timescales. Ecological data spanning centuries to millennia provide important contextual information for long-term management strategies, especially for species that now are living in relict populations. Here we use a variety of methods to reconstruct bald eagle diets and local abundance of their potential prey on the Channel Islands from the late Pleistocene to the time when the last breeding pairs disappeared from the islands in the mid-20th century. Faunal and isotopic analysis of bald eagles shows that seabirds were important prey for immature/adult eagles for millennia before the eagles' local extirpation. In historic times (A.D. 1850-1950), however, isotopic and faunal data show that breeding bald eagles provisioned their chicks with introduced ungulates (e.g., sheep), which were locally present in high densities. Today, bald eagles are the focus of an extensive conservation program designed to restore a stable breeding population to the Channel Islands, but native and nonnative prey sources that were important for bald eagles in the past are either diminished (e.g., seabirds) or have been eradicated (e.g., introduced ungulates). In the absence of sufficient resources, a growing bald eagle population on the Channel Islands could expand its prey base to include carrion from local pinniped colonies, exert predation pressure on a recovering seabird population, and possibly prey on endangered island foxes.

Comparative metagenomics at Solfatara and Pisciarelli hydrothermal systems in Italy reveal that ecological differences across substrates are not ubiquitous.

Introduction: Continental hydrothermal systems (CHSs) are geochemically complex, and they support microbial communities that vary across substrates. However, our understanding of these variations across the complete range of substrates in CHS is limited because many previous studies have focused predominantly on aqueous settings. Methods: Here we used metagenomes in the context of their environmental geochemistry to investigate the ecology of different substrates (i.e., water, mud and fumarolic deposits) from Solfatara and Pisciarelli. Results and Discussion: Results indicate that both locations are lithologically similar with distinct fluid geochemistry. In particular, all substrates from Solfatara have similar chemistry whereas Pisciarelli substrates have varying chemistry; with water and mud from bubbling pools exhibiting high SO4 2- and NH4 + concentrations. Species alpha diversity was found to be different between locations but not across substrates, and pH was shown to be the most important driver of both diversity and microbial community composition. Based on cluster analysis, microbial community structure differed significantly between Pisciarelli substrates but not between Solfatara substrates. Pisciarelli mud pools, were dominated by (hyper)thermophilic archaea, and on average, bacteria dominated Pisciarelli fumarolic deposits and all investigated Solfatara environments. Carbon fixation and sulfur oxidation were the most important metabolic pathways fueled by volcanic outgassing at both locations. Together, results demonstrate that ecological differences across substrates are not a widespread phenomenon but specific to the system. Therefore, this study demonstrates the importance of analyzing different substrates of a CHS to understand the full range of microbial ecology to avoid biased ecological assessments.

Molecular exploration of fossil eggshell uncovers hidden lineage of giant extinct bird.

The systematics of Madagascar's extinct elephant birds remains controversial due to large gaps in the fossil record and poor biomolecular preservation of skeletal specimens. Here, a molecular analysis of 1000-year-old fossil eggshells provides the first description of elephant bird phylogeography and offers insight into the ecology and evolution of these flightless giants. Mitochondrial genomes from across Madagascar reveal genetic variation that is correlated with eggshell morphology, stable isotope composition, and geographic distribution. The elephant bird crown is dated to ca. 30 Mya, when Madagascar is estimated to have become less arid as it moved northward. High levels of between-clade genetic variation support reclassifying Mullerornis into a separate family. Low levels of within-clade genetic variation suggest there were only two elephant bird genera existing in southern Madagascar during the Holocene. However, we find an eggshell collection from Madagascar's far north that represents a unique lineage of Aepyornis. Furthermore, divergence within Aepyornis coincides with the aridification of Madagascar during the early Pleistocene ca. 1.5 Ma, and is consistent with the fragmentation of populations in the highlands driving diversification and the evolution of extreme gigantism over shorts timescales. We advocate for a revision of their taxonomy that integrates palaeogenomic and palaeoecological perspectives.

An experimental exploration of the incorporation of hydrogen isotopes from dietary sources into avian tissues.

The analysis of hydrogen stable isotopes (δD) is a potentially powerful tool for studying animal ecology. Unlike other stable isotopes used in ecological research, however, we are less familiar with the physiological processes that influence the incorporation of hydrogen isotopes from dietary resources to animal tissues. Here we present the results of a controlled feeding experiment utilizing Japanese quail (Cortunix japonica) that was designed to: (1) estimate the relative contributions of diet to the δD signatures of blood plasma, red blood cells, intestine, liver, muscle and feathers; (2) investigate possible differences among these same tissues in diet to tissue discrimination; and (3) explore the differences in incorporation dynamics between deuterium ((2)H) and a well-studied isotope, (13)C, for blood plasma solids and red blood cells. Tissues differed in both the relative contribution of diet to tissue δD and diet to tissue discrimination. The average residence time of both hydrogen and carbon was significantly lower in plasma solids than in red blood cells. The average residence time of hydrogen was significantly lower than that of carbon in plasma solids, but not in red blood cells. Although the average residence times of hydrogen and carbon were positively correlated, the correlation was weak. Hence the incorporation of carbon seems to be a poor predictor of that of hydrogen.

High-pressure tolerance in Halobacterium salinarum NRC-1 and other non-piezophilic prokaryotes.

In this study, we examined the high-pressure survival of a range of prokaryotes not found in high-pressure environments to determine the effects of adaptations to osmotic and oxidative stresses on piezo-resistance. The pressure survivals of Halobacterium salinarum NRC-1, Deinococcus radiodurans R1, and Chromohalobacter salexigens were compared to that of Escherichia coli MG1655. C. salexigens, which uses the compatible solute ectoine as an osmolyte, was as piezo-sensitive as E. coli MG1655, suggesting that ectoine is not a piezolyte. D. radiodurans R1 and H. salinarum NRC-1, both resistant to oxidative stress, were found to be highly piezo-resistant. H. salinarum NRC-1 showed nearly full survival after pressurization up to 400 MPa; a survival 3.5 log units higher than E. coli MG1655. This piezo-resistance was maintained in H. salinarum NRC-1 for pressurizations up to 1 h. We hypothesize that the high-pressure resistance of H. salinarum NRC-1 is due to a combination of factors including cell envelope structure and the presence of intracellular salts.

Can amino acid carbon isotope ratios distinguish primary producers in a mangrove ecosystem?

RATIONALE: The relative contribution of carbon from terrestrial vs. marine primary producers to mangrove-based food webs can be challenging to resolve with bulk carbon isotope ratios (δ(13)C). In this study we explore whether patterns of δ(13)C values among amino acids (AAs) can provide an additional tool for resolving terrestrial and marine origins of carbon. METHODS: Amino acid carbon isotope ratios (δ(13)C(AA)) were measured for several terrestrial and marine primary producers in a mangrove ecosystem at Spanish Lookout Caye (SLC), Belize, using gas chromatography-combustion-isotope ratio mass spectrometry. The δ(13)C values of essential amino acids (δ(13)C(EAA)) were measured to determine whether they could be used to differentiate terrestrial and marine producers using linear discriminant analysis. RESULTS: Marine and terrestrial producers had distinct patterns of δ(13)C(EAA) values in addition to their differences in bulk δ(13)C values. Microbial mat samples and consumers (Crassostrea rhizophorae, Aratus pisonii, Littoraria sp., Lutjanus griseus) were most similar to marine producers. Patterns of δ(13)C(EAA) values for terrestrial producers were very similar to those described for other terrestrial plants. CONCLUSIONS: The findings suggest that δ(13)C(EAA) values may provide another tool for estimating the contribution of terrestrial and marine sources to detrital foodwebs. Preliminary analyses of consumers indicate significant use of aquatic resources, consistent with other studies of mangrove foodwebs.

Central Metabolism and Growth Rate Impacts on Hydrogen and Carbon Isotope Fractionation During Amino Acid Synthesis in E. coli.

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.

Physiological and metabolic responses of chemolithoautotrophic NO 3 - reducers to high hydrostatic pressure.

We investigated the impact of pressure on thermophilic, chemolithoautotrophic NO 3 - reducing bacteria of the phyla Campylobacterota and Aquificota isolated from deep-sea hydrothermal vents. Batch incubations at 5 and 20 MPa resulted in decreased NO 3 - consumption, lower cell concentrations, and overall slower growth in Caminibacter mediatlanticus (Campylobacterota) and Thermovibrio ammonificans (Aquificota), relative to batch incubations near standard pressure (0.2 MPa) conditions. Nitrogen isotope fractionation effects from chemolithoautotrophic NO 3 - reduction by both microorganisms were, on the contrary, maintained under all pressure conditions. Comparable chemolithoautotrophic NO 3 - reducing activities between previously reported natural hydrothermal vent fluid microbial communities dominated by Campylobacterota at 25 MPa and Campylobacterota laboratory isolates at 0.2 MPa, suggest robust similarities in cell-specific NO 3 - reduction rates and doubling times between microbial populations and communities growing maximally under similar temperature conditions. Physiological and metabolic comparisons of our results with other studies of pressure effects on anaerobic chemolithoautotrophic processes (i.e., microbial S0 -oxidation coupled to Fe(III) reduction and hydrogenotrophic methanogenesis) suggest that anaerobic chemolithoautotrophs relying on oxidation-reduction (redox) reactions that yield higher Gibbs energies experience larger shifts in cell-specific respiration rates and doubling times at increased pressures. Overall, our results advance understanding of the role of pressure, its relationship with temperature and redox conditions, and their effects on seafloor chemolithoautotrophic NO 3 - reduction and other anaerobic chemolithoautotrophic processes.

Quaternary record of aridity and mean annual precipitation based on δ15N in ratite and dromornithid eggshells from Lake Eyre, Australia.

The cause(s) of the late Pleistocene megafauna extinction on the Australian continent remains largely unresolved. Unraveling climatic forcing mechanisms from direct or indirect human agents of ecosystem alteration has proven to be extremely difficult in Australia due to the lack of (1) well-dated vertebrate fossils and (2) paleo-environmental and -ecological records spanning the past approximately 100 ka when regional climatic conditions are known to have significantly varied. We have examined the nitrogen isotope composition (δ(15)N) of modern emu (Dromaius novaehollandiae) eggshells collected along a precipitation gradient in Australia, along with modern climatological data and dietary δ(15)N values. We then used modern patterns to interpret an approximately 130-ka record of δ(15)N values in extant Dromaius and extinct Genyornis newtoni eggshells from Lake Eyre to obtain a novel mean annual precipitation (MAP) record for central Australia spanning the extinction interval. Our data also provide the first detailed information on the trophic ecology and environmental preferences of two closely related taxa, one extant and one extinct. Dromaius eggshell δ(15)N values show a significant shift to higher values during the Last Glacial Maximum and Holocene, which we interpret to indicate more frequent arid conditions (<200 mm MAP), relative to δ(15)N from samples just prior to the megafauna extinction. Genyornis eggshells had δ(15)N values reflecting wetter nesting conditions overall relative to those of coeval Dromaius, perhaps indicating that Genyornis was more reliant on mesic conditions. Lastly, the Dromaius eggshell record shows a significant decrease in δ(13)C values prior to the extinction, whereas the Genyornis record does not. Neither species showed a concomitant change in δ(15)N prior to the extinction, which suggests that a significant change in vegetation surrounding Lake Eyre occurred prior to an increase in local aridity.

Variation in delta13C and delta15N diet-vibrissae trophic discrimination factors in a wild population of California sea otters.

The ability to quantify dietary inputs using stable isotope data depends on accurate estimates of isotopic differences between a consumer (c) and its diet (d), commonly referred to as trophic discrimination factors (TDFs) and denoted by delta(c-d). At present, TDFs are available for only a few mammals and are usually derived in captive settings. The magnitude of TDFs and the degree to which they vary in wild populations is unknown. We determined delta13C and delta15N TDFs for vibrissae (i.e., whiskers), a tissue that is rapidly becoming an informative isotopic substrate for ecologists, of a wild population of sea otters for which individual diet has been quantified through extensive observational study. This is one of the very few studies that report TDFs for free-living wild animals feeding on natural diets. Trophic discrimination factors of 2.2 per thousand +/- 0.7 per thousand for delta13C and 3.5 per thousand +/- 0.6 per thousand for delta15N (mean +/- SD) were similar to those reported for captive carnivores, and variation in individual delta13C TDFs was negatively but significantly related to sea urchin consumption. This pattern may relate to the lipid-rich diet consumed by most sea otters in this population and suggests that it may not be appropriate to lipid-extract prey samples when using the isotopic composition of keratinaceous tissues to examine diet in consumers that frequently consume lipid-rich foods, such as many marine mammals and seabirds. We suggest that inherent variation in TDFs should be included in isotopically based estimates of trophic level, food chain length, and mixing models used to quantify dietary inputs in wild populations; this practice will further define the capabilities and limitations of isotopic approaches in ecological studies.

Carbon isotope fractionation of amino acids in fish muscle reflects biosynthesis and isotopic routing from dietary protein.

1. Analysis of stable carbon isotopes is a valuable tool for studies of diet, habitat use and migration. However, significant variability in the degree of trophic fractionation (Delta(13)C(C-D)) between consumer (C) and diet (D) has highlighted our lack of understanding of the biochemical and physiological underpinnings of stable isotope ratios in tissues. 2. An opportunity now exists to increase the specificity of dietary studies by analyzing the delta(13)C values of amino acids (AAs). Common mummichogs (Fundulus heteroclitus, Linnaeus 1766) were reared on four isotopically distinct diets to examine individual AA Delta(13)C(C-D) variability in fish muscle. 3. Modest bulk tissue Delta(13)C(C-D) values reflected relatively large trophic fractionation for many non-essential AAs and little to no fractionation for all essential AAs. 4. Essential AA delta(13)C values were not significantly different between diet and consumer (Delta(13)C(C-D) = 0.0 +/- 0.4 per thousand), making them ideal tracers of carbon sources at the base of the food web. Stable isotope analysis of muscle essential AAs provides a promising tool for dietary reconstruction and identifying baseline delta(13)C values to track animal movement through isotopically distinct food webs. 5. Non-essential AA Delta(13)C(C-D) values showed evidence of both de novo biosynthesis and direct isotopic routing from dietary protein. We attributed patterns in Delta(13)C(C-D) to variability in protein content and AA composition of the diet as well as differential utilization of dietary constituents contributing to the bulk carbon pool. This variability illustrates the complicated nature of metabolism and suggests caution must be taken with the assumptions used to interpret bulk stable isotope data in dietary studies. 6. Our study is the first to investigate the expression of AA Delta(13)C(C-D) values for a marine vertebrate and should provide for significant refinements in studies of diet, habitat use and migration using stable isotopes.

Using stable isotopes to investigate individual diet specialization in California sea otters (Enhydra lutris nereis).

Differences in diet composition among conspecifics (dietary specialization) have been documented across a broad range of taxonomic groups and habitats, and such variation at the individual level is increasingly recognized as an important component of diversity in trophic interactions. Accurate identification of individual dietary specialization, however, requires longitudinal dietary records that are labor-intensive and cost-prohibitive to obtain for many species. Here we explore the use of stable isotopes (delta13C and delta15N) as a promising technique for detecting and quantifying patterns of individual dietary specialization. Southern sea otters (Enhydra lutris nereis) offer a unique opportunity for testing this approach because (1) they consume a wide variety of prey that span multiple trophic levels, habitats, and ecologically defined functional groups; and (2) individual diet specialization can be validated with existing observational data. We analyzed the isotopic composition of sea otter vibrissae (n = 31) in order to characterize inter- and intra-individual variation in sea otter diets at Monterey Bay, California, USA. At the population level, sea otters showed substantial variation in both delta13C and delta15N values, occupying nearly all of the "isotopic space" created by the diversity of isotopic signatures of potential prey taxa. Most of the variation in sea otter vibrissae was accounted for by differences between individuals, with much less contributed by within-individual variation. A majority of sea otters (approximately 80%) showed relatively little temporal variability in isotopic composition, suggesting that the proportional composition of most individuals' diets is relatively constant over time; a few individuals (approximately 20%) exhibited a high degree of intra-vibrissa isotopic variability, suggesting seasonal shifts in diet composition. These results and our interpretation of them were supported by long-term observational data on the diets of radio-tagged sea otters from the same population (n = 23). Our results demonstrate that stable isotopes can provide an efficient tool for measuring individual- and population-level dietary breadth and may be useful for studying populations where longitudinal data on individuals would otherwise be impossible to acquire. This will be critical for examining the causes and consequences of dietary variation within and among consumer populations, thereby improving our understanding of these important ecological and evolutionary processes at the community level.

Stable hydrogen isotope variability within and among plumage tracts (δ2HF) of a migratory wood warbler.

Hydrogen isotope analysis of feather keratin (δ2HF) has become an essential tool for tracking movements between breeding and wintering populations of migratory birds. In particular, δ2HF has been used to create δ2HF isoscapes that can be used to assign the geographic origins of molt. The majority of past studies have sampled a portion of a single feather as an isotopic proxy for the entire plumage although surprisingly little is known about variation of stable isotopes within and between feather tracts of individuals in local populations. Here we examine δ2HF variation in 24 pterylographic variables (9 primaries, 6 secondaries, 6 rectrices, and 3 patches of ventral contour feathers) in individual specimens of black-throated blue warbler (Setophaga caerulescens) breeding in the Big Santeetlah Creek watershed (5350 ha), southern Appalachian Mountains. By restricting our study to territorial ASY males (after second year) inhabiting a small watershed, we could focus on δ2HF variation generated during the complete prebasic annual molt in a circumscribed population while factoring out age and sexual differences in foraging behavior, isotopic incorporation, and post-breeding dispersal. Summed within-individual variation (δ2HF) across 24 pterylographic variables ranged from 12 to 60‰ (= 21.8 ± 9.4‰), with 81% of the individuals exhibiting variation ≥ 16‰ (reproducibility of analyses was ≤ 4 ‰). Adjacent feathers in feather tracts tend to have more similar δ2HF values than feathers grown weeks apart, consistent with the stepwise replacement of flight feathers. The pooled population sample exhibited significant δ2HF variation in primaries (-78 to -21‰), secondaries (-80 to -17‰), rectrices (-78 to -23‰), and ventral contour feathers (-92 to -32‰). Strong year effects in δ2HF variation were observed in each of the 24 pterylographic variables. Altitudinal effects were observed only in ventral contour feathers. The current findings demonstrate that within-individual variation (δ2HF) may be much greater than previously thought in migratory species that molt on or near breeding territories. Our study also highlights the need for greater pterylographic precision in research design of isotope-based studies of avian movement. Within-individual and within-population δ2HF variation should be incorporated in geographic assignment models. In a broader context, the staggered Staffelmauser pattern of molt in wood warblers provides an exceptional view of the seasonal variation of hydrogen isotopes circulating in blood plasma during the six-week period of annual molt.

Climate change promotes parasitism in a coral symbiosis.

Coastal oceans are increasingly eutrophic, warm and acidic through the addition of anthropogenic nitrogen and carbon, respectively. Among the most sensitive taxa to these changes are scleractinian corals, which engineer the most biodiverse ecosystems on Earth. Corals' sensitivity is a consequence of their evolutionary investment in symbiosis with the dinoflagellate alga, Symbiodinium. Together, the coral holobiont has dominated oligotrophic tropical marine habitats. However, warming destabilizes this association and reduces coral fitness. It has been theorized that, when reefs become warm and eutrophic, mutualistic Symbiodinium sequester more resources for their own growth, thus parasitizing their hosts of nutrition. Here, we tested the hypothesis that sub-bleaching temperature and excess nitrogen promotes symbiont parasitism by measuring respiration (costs) and the assimilation and translocation of both carbon (energy) and nitrogen (growth; both benefits) within Orbicella faveolata hosting one of two Symbiodinium phylotypes using a dual stable isotope tracer incubation at ambient (26 °C) and sub-bleaching (31 °C) temperatures under elevated nitrate. Warming to 31 °C reduced holobiont net primary productivity (NPP) by 60% due to increased respiration which decreased host %carbon by 15% with no apparent cost to the symbiont. Concurrently, Symbiodinium carbon and nitrogen assimilation increased by 14 and 32%, respectively while increasing their mitotic index by 15%, whereas hosts did not gain a proportional increase in translocated photosynthates. We conclude that the disparity in benefits and costs to both partners is evidence of symbiont parasitism in the coral symbiosis and has major implications for the resilience of coral reefs under threat of global change.