Carbon Isotope Ratios of Plasma and RBC Fatty Acids Identify Meat Consumers in a 12-Week Inpatient Feeding Study of 32 Men.

BACKGROUND: Molecular stable isotope ratios are a novel type of dietary biomarker with high sensitivity and specificity for certain foods. Among these, fatty acid carbon isotope ratios (CIRs) have strong potential but have not been investigated as dietary biomarkers. OBJECTIVES: We evaluated whether fatty acid CIRs and mass proportions were associated with meat, fish, and sugar-sweetened beverage (SSB) intake. METHODS: Thirty-two men [aged 46.2 ± 10.5 y; BMI (kg/m2): 27.2 ± 4.0] underwent a 12-wk inpatient dietary intervention at the National Institute of Diabetes and Digestive and Kidney Diseases in Phoenix, Arizona. Men were randomly assigned to 1 of 8 dietary treatments varying the presence/absence of dietary meat, fish, and SSBs in all combinations. Fatty acid CIRs and mass proportions were measured in fasting blood samples and adipose tissue biopsies that were collected pre- and postintervention. Dietary effects were analyzed using multivariable regression and receiver operating characteristic AUCs were calculated using logistic regression. RESULTS: CIRs of the several abundant SFAs, MUFAs and arachidonic acid (20:4n-6) in plasma were strongly associated with meat, as were a subset of these fatty acids in RBCs. Effect sizes in plasma ranged from 1.01‰ to 1.93‰ and were similar but attenuated in RBCs. Mass proportions of those fatty acids were not associated with diet. CIRs of plasma dihomo-γ-linolenic acid (20:3n-6) and adipose palmitic acid (16:0) were weakly associated with SSBs. Mass proportions of plasma odd-chain fatty acids were associated with meat, and mass proportions of plasma EPA and DHA (20:5n-3 and 22:6n-3) were associated with fish. CONCLUSIONS: CIRs of plasma and RBC fatty acids show promise as sensitive and specific measures of dietary meat. These provide different information from that provided by fatty acid mass proportions, and are informative where mass proportion is not. This trial is registered at www.clinicaltrials.gov as NCT01237093.

Ecotoxicology of mercury concentrations in arctic lamprey (Lethenteron camtschaticum).

Arctic lamprey (Lethenteron camtschaticum) is an important dietary resource for rural and indigenous communities in parts of Alaska, with some commercial use. As with many fish species harvested for human consumption, there are concerns regarding mercury concentrations ([Hg]) in Arctic lamprey that may impact human health. To date, information regarding the life cycle and diet of Arctic lamprey is scarce, with no published studies examining [Hg] in Arctic lamprey tissues. Our goals were to investigate the feeding ecology of Arctic lamprey from the Bering Sea, determine how diet and potential dietary shifts might influence [Hg] in muscle, and determine if current [Hg] may pose a human health risk. The mean total [Hg] in Arctic lamprey muscle (n = 98) was 19 ng/g wet-weight. Log transformed total [Hg] were not associated with any measured biological variables including length, mass, δ13C values, or δ15N values. A stable isotope mixing model estimated that capelin (Mallotus villosus) accounted for 40.0 ± 4.0% of the Arctic lamprey diet, while Pacific sand lance (Ammodytes hexapterus) and Pacific herring (Clupea pallasii) accounted for 37.8 ± 3.1% and 22.2 ± 3.5% respectively. Finally, diet percentage compositions shifted based on size class (i.e., medium versus large). These results indicated that feeding location, bioaccumulation, and biomagnification are not important drivers of [Hg] in Arctic lamprey and current [Hg] do not pose a human health risk. Taken together, this research further expands our knowledge of Arctic lamprey trophic ecology in the eastern Bering Sea.

Amino Acid Nitrogen Isotope Ratios Respond to Fish and Meat Intake in a 12-Week Inpatient Feeding Study of Men.

BACKGROUND: The natural abundance nitrogen stable isotope ratio (NIR) of whole tissue correlates with animal protein intakes, including meat and fish. Amino acid (AA) NIRs (NIRAAs) are more variable than the whole-tissue NIRs and may thus better differentiate among foods. OBJECTIVES: We evaluated whether NIRAAs were associated with intakes of fish and meat and whether these dietary associations were larger than with whole-tissue NIRs. METHODS: Men were recruited at the National Institute of Diabetes and Digestive and Kidney Diseases in Phoenix, Arizona, and randomly assigned to one of eight 12-wk inpatient dietary interventions, which varied the presence/absence of fish, meat, and sugar-sweetened beverages (SSBs) in all possible combinations. Fasting blood was drawn pre- and postintervention and plasma and RBC NIRAAs (free and protein-bound) were measured as secondary outcomes in 32 participants. Multivariable regression was used to determine responses of postintervention NIRAAs to dietary variables, and logistic regression was used to calculate receiver operating characteristic AUCs. RESULTS: Most plasma NIRAAs increased with fish and meat intakes, but to a greater extent with fish intake. The largest increase in response to fish intake was plasma NIRLeucine (ß = 2.19, SE = 0.26). The NIRThreonine decreased with both fish and meat intakes. Fewer RBC NIRAAs increased with fish intake, and only RBC NIRProline increased with meat intake. No plasma or RBC NIRAA responded to SSB intake. We identified fish intake with a high degree of accuracy using plasma NIRLeucine (corrected AUC, cAUC = 0.96) and NIRGlutamic acid/glutamine (cAUC = 0.93), and meat intake to a lower degree using plasma NIRProline (cAUC = 0.80) and RBC NIRProline (cAUC = 0.85). CONCLUSIONS: Plasma and RBC NIRAAs were associated with fish and meat intakes but were not superior to whole-tissue stable isotope biomarkers in identifying these intakes in a US diet. This trial is registered at www.clinicaltrials.gov as NCT01237093.

Ancient horse genomes reveal the timing and extent of dispersals across the Bering Land Bridge.

The Bering Land Bridge (BLB) last connected Eurasia and North America during the Late Pleistocene. Although the BLB would have enabled transfers of terrestrial biota in both directions, it also acted as an ecological filter whose permeability varied considerably over time. Here we explore the possible impacts of this ecological corridor on genetic diversity within, and connectivity among, populations of a once wide-ranging group, the caballine horses (Equus spp.). Using a panel of 187 mitochondrial and eight nuclear genomes recovered from present-day and extinct caballine horses sampled across the Holarctic, we found that Eurasian horse populations initially diverged from those in North America, their ancestral continent, around 1.0-0.8 million years ago. Subsequent to this split our mitochondrial DNA analysis identified two bidirectional long-range dispersals across the BLB ~875-625 and ~200-50 thousand years ago, during the Middle and Late Pleistocene. Whole genome analysis indicated low levels of gene flow between North American and Eurasian horse populations, which probably occurred as a result of these inferred dispersals. Nonetheless, mitochondrial and nuclear diversity of caballine horse populations retained strong phylogeographical structuring. Our results suggest that barriers to gene flow, currently unidentified but possibly related to habitat distribution across Beringia or ongoing evolutionary divergence, played an important role in shaping the early genetic history of caballine horses, including the ancestors of living horses within Equus ferus.

Validation of dried blood spot sampling for determining trophic positions of Arctic char using nitrogen stable isotope analyses of amino acids.

RATIONALE: Dried blood spots (DBSs) are gaining popularity for biomarker analyses in ecological research due to their advantages for use in field-based research and in remote settings; however, many DBS biomarkers remain unvalidated. We validated the application of compound-specific stable nitrogen isotope analyses of amino acids (CSIA-AAs) to field-prepared DBSs for determining trophic positions of wild-caught Arctic char (Salvelinus alpinus). METHODS: Whole blood and muscle from Arctic char were collected, and DBSs were created in the field. We measured the stable nitrogen isotope ratios (expressed as δ15 N values) of the amino acids glutamic acid (Glu) and phenylalanine (Phe) isolated from Arctic char samples using CSIA-AAs. We then compared amino acid δ15 N values from DBSs and the other sample types (whole blood and muscle) from the same specimens. We calculated and compared trophic position estimates generated from whole blood, DBSs, and muscle. RESULTS: The δ15 N values of Glu and Phe, as well as trophic position estimates from DBSs, were highly correlated with δ15 N values and estimates from both whole blood and muscle. The DBS amino acid δ15 N values and trophic position estimates agreed well with those from whole blood. Although mean differences between amino acid δ15 N values from DBSs and muscle were noted, the offsets were small and resulted in a 0.2 mean difference between trophic position estimates for DBSs and muscle. CONCLUSIONS: We demonstrate that the application of CSIA-AAs to field-prepared DBSs of Arctic char generates similar trophic position estimates to those from whole blood and muscle. We suggest that DBSs could be developed as a minimally invasive sampling technique to study feeding ecology of wild fish and perhaps other organisms of interest.

Ecological drivers of mercury concentrations in fish species in subsistence harvests from Kotzebue Sound, Alaska.

The State of Alaska assesses human exposure to mercury (Hg) via fish consumption producing consumption guidelines for fish tailored for children and women of childbearing age. Under these guidelines, unrestricted consumption is suggested for many fish species, while limited consumption is recommended for others. Subsequent questions have arisen regarding ecological drivers influencing [Hg] in fishes consumed by Alaskans. This community-assisted public health study evaluates [Hg] in fishes from Kotzebue Sound to examine factors that may drive observed [Hg]. We examined eight species of subsistence harvested fish (least cisco, chum salmon, Pacific herring, humpback whitefish, sheefish, starry flounder, Pacific tomcod, and fourhorn sculpin) from Kotzebue Sound. We report total Hg concentrations ([THg]) and monomethyl Hg+ concentrations ([MeHg+]) in the context of various factors (such as species, fork length, carbon and nitrogen stable isotope (δ15N or δ13C)) values that may influence [Hg] and [MeHg+]. Across all 297 fish, [THg] ranged from 3.4 - 235.2 ng/g ww. [THg] was positively correlated with fork length in six of eight fish species, as well as with trophic level (indicated by δ15N values) in five species. [MeHg+] was positively correlated with fork length in four species, and with δ15N values over all specimens examined, and specifically for three individual species. In six of the seven species analyzed, %MeHg was >80% of [THg]. This value decreased with fork length in three species, with no relationship for δ15N values in any species. Among top ranked models based on Akaike Information Criterion correction (AICc), fork length was more frequently included as an explanatory factor for [Hg] than δ15N or δ13C values. The food web magnification factor for [THg] was 11.3, and 12.6 for [MeHg+]. Biomagnification is likely driving [THg] and [MeHg+] over the entire food web, while within species, bioaccumulation is likely a stronger driver of [THg] and [MeHg+] than feeding ecology or trophic position. The [THg] for all species fell within the established unrestricted consumption guideline of 200 ng/g weight wet as established by the State of Alaska's fish consumption guidelines for Hg.

Chemical profiling of ancient hearths reveals recurrent salmon use in Ice Age Beringia.

Current approaches to reconstruct subsistence and dietary trends in ancient hunter-gatherer societies include stable isotope analyses, but these have focused on human remains, cooking pottery, and food residues, which are relatively rare in the archaeological record. In contrast, short-term hearths are more ubiquitous worldwide, and these features can provide valuable evidence for ancient subsistence practices, particularly when faunal remains are not preserved. To test the suitability of hearths for this purpose, we conducted multiple chemical analyses: stable carbon and nitrogen isotope analyses of total organic matter (expressed as δ(13)C and δ(15)N values) and compound-specific carbon isotope analyses of individual fatty acids (δ(13)C16:0 and δ(13)C18:0) from 17 well-preserved hearths present in three occupations dating between ∼13,200-11,500 calibrated years B.P. at the Upward Sun River (USR) site in central Alaska. We combined δ(15)N and δ(13)CFA data in a Bayesian mixing model (stable isotope analysis in R) with concentration dependency to each hearth. Our model values were tested against faunal indices, indicating a strong positive relationship between marine proportional contributions to each hearth and salmon abundance. Results of the models show substantial anadromous salmon use in multiple USR components, indicating recurrent use of the site for salmon processing during the terminal Pleistocene. Our results demonstrate that salmonid and freshwater resources were more important for late Pleistocene hunter-gatherers than previously thought and highlight the potential of chemical profiling of hearth organic residues for providing greater geographic and temporal insights into resource use by prepottery societies.

Timing and causes of mid-Holocene mammoth extinction on St. Paul Island, Alaska.

Relict woolly mammoth (Mammuthus primigenius) populations survived on several small Beringian islands for thousands of years after mainland populations went extinct. Here we present multiproxy paleoenvironmental records to investigate the timing, causes, and consequences of mammoth disappearance from St. Paul Island, Alaska. Five independent indicators of extinction show that mammoths survived on St. Paul until 5,600 ± 100 y ago. Vegetation composition remained stable during the extinction window, and there is no evidence of human presence on the island before 1787 CE, suggesting that these factors were not extinction drivers. Instead, the extinction coincided with declining freshwater resources and drier climates between 7,850 and 5,600 y ago, as inferred from sedimentary magnetic susceptibility, oxygen isotopes, and diatom and cladoceran assemblages in a sediment core from a freshwater lake on the island, and stable nitrogen isotopes from mammoth remains. Contrary to other extinction models for the St. Paul mammoth population, this evidence indicates that this mammoth population died out because of the synergistic effects of shrinking island area and freshwater scarcity caused by rising sea levels and regional climate change. Degradation of water quality by intensified mammoth activity around the lake likely exacerbated the situation. The St. Paul mammoth demise is now one of the best-dated prehistoric extinctions, highlighting freshwater limitation as an overlooked extinction driver and underscoring the vulnerability of small island populations to environmental change, even in the absence of human influence.

Carbon sources and trophic relationships of ice seals during recent environmental shifts in the Bering Sea.

Dramatic multiyear fluctuations in water temperature and seasonal sea ice extent and duration across the Bering-Chukchi continental shelf have occurred in this century, raising a pressing ecological question: Do such environmental changes alter marine production processes linking primary producers to upper trophic-level predators? We examined this question by comparing the blubber fatty acid (FA) composition and stable carbon isotope ratios of individual FA (δ¹³CFA) of adult ringed seals (Pusa hispida), bearded seals (Erignathus barbatus), spotted seals (Phoca largha), and ribbon seals (Histriophoca fasciata), collectively known as "ice seals," sampled during an anomalously warm, low sea ice period in 2002-2005 in the Bering Sea and a subsequent cold, high sea ice period in 2007-2010. δ¹³C(FA) values, used to estimate the contribution to seals of carbon derived from sea ice algae (sympagic production) relative to that derived from water column phytoplankton (pelagic production), indicated that during the cold period, sympagic production accounted for 62-80% of the FA in the blubber of bearded seals, 51-62% in spotted seals, and 21-60% in ringed seals. Moreover, the δ¹³CFA values of bearded seals indicated a greater incorporation of sympagic FAs during the cold period than the warm period. This result provides the first empirical evidence of an ecosystem-scale effect of a putative change in sympagic production in the Western Arctic. The FA composition of ice seals showed clear evidence of resource partitioning among ringed, bearded, and spotted seals, and little niche separation between spotted and ribbon seals, which is consistent with previous studies. Despite interannual variability, the FA composition of ringed and bearded seals showed little evidence of differences in diet between the warm and cold periods. The findings that sympagic production contributes significantly to food webs supporting ice seals, and that the contribution apparently is less in warm years with low sea ice, raise an important concern: Will the projected warming and continuing loss of seasonal sea ice in the Arctic, and the associated decline of organic matter input from sympagic production, be compensated for by pelagic production to satisfy both pelagic and benthic carbon and energy needs?

Methane-derived carbon flow through microbial communities in arctic lake sediments.

Aerobic methane (CH4 ) oxidation mitigates CH4 release and is a significant pathway for carbon and energy flow into aquatic food webs. Arctic lakes are responsible for an increasing proportion of global CH4 emissions, but CH4 assimilation into the aquatic food web in arctic lakes is poorly understood. Using stable isotope probing (SIP) based on phospholipid fatty acids (PLFA-SIP) and DNA (DNA-SIP), we tracked carbon flow quantitatively from CH4 into sediment microorganisms from an arctic lake with an active CH4 seepage. When 0.025 mmol CH4 g(-1) wet sediment was oxidized, approximately 15.8-32.8% of the CH4 -derived carbon had been incorporated into microorganisms. This CH4 -derived carbon equated to up to 5.7% of total primary production estimates for Alaskan arctic lakes. Type I methanotrophs, including Methylomonas, Methylobacter and unclassified Methylococcaceae, were most active at CH4 oxidation in this arctic lake. With increasing distance from the active CH4 seepage, a greater diversity of bacteria incorporated CH4 -derived carbon. Actinomycetes were the most quantitatively important microorganisms involved in secondary feeding on CH4 -derived carbon. These results showed that CH4 flows through methanotrophs into the broader microbial community and that type I methanotrophs, methylotrophs and actinomycetes are important organisms involved in using CH4 -derived carbon in arctic freshwater ecosystems.

Fatty acid and stable isotope characteristics of sea ice and pelagic particulate organic matter in the Bering Sea: tools for estimating sea ice algal contribution to Arctic food web production.

We determined fatty acid (FA) profiles and carbon stable isotopic composition of individual FAs (δ(13)CFA values) from sea ice particulate organic matter (i-POM) and pelagic POM (p-POM) in the Bering Sea during maximum ice extent, ice melt, and ice-free conditions in 2010. Based on FA biomarkers, differences in relative composition of diatoms, dinoflagellates, and bacteria were inferred for i-POM versus p-POM and for seasonal succession stages in p-POM. Proportions of diatom markers were higher in i-POM (16:4n-1, 6.6-8.7%; 20:5n-3, 19.6-25.9%) than in p-POM (16:4n-1, 1.2-4.0%; 20:5n-3, 5.5-14.0%). The dinoflagellate marker 22:6n-3/20:5n-3 was highest in p-POM. Bacterial FA concentration was higher in the bottom 1 cm of sea ice (14-245 µg L(-1)) than in the water column (0.6-1.7 µg L(-1)). Many i-POM δ(13)C(FA) values were higher (up to ~10‰) than those of p-POM, and i-POM δ(13)C(FA) values increased with day length. The higher i-POM δ(13)C(FA) values are most likely related to the reduced dissolved inorganic carbon (DIC) availability within the semi-closed sea ice brine channel system. Based on a modified Rayleigh equation, the fraction of sea ice DIC fixed in i-POM ranged from 12 to 73%, implying that carbon was not limiting for primary productivity in the sympagic habitat. These differences in FA composition and δ(13)C(FA) values between i-POM and p-POM will aid efforts to track the proportional contribution of sea ice algal carbon to higher trophic levels in the Bering Sea and likely other Arctic seas.

A female woolly mammoth's lifetime movements end in an ancient Alaskan hunter-gatherer camp.

Woolly mammoths in mainland Alaska overlapped with the region's first people for at least a millennium. However, it is unclear how mammoths used the space shared with people. Here, we use detailed isotopic analyses of a female mammoth tusk found in a 14,000-year-old archaeological site to show that she moved ~1000 kilometers from northwestern Canada to inhabit an area with the highest density of early archaeological sites in interior Alaska until her death. DNA from the tusk and other local contemporaneous archaeological mammoth remains revealed that multiple mammoth herds congregated in this region. Early Alaskans seem to have structured their settlements partly based on mammoth prevalence and made use of mammoths for raw materials and likely food.

Acute Toxicity of Copper to Three Species of Pacific Salmon Fry in Water with Low Hardness and Low Dissolved Organic Carbon.

Proposed development of a mine within Alaska's Bristol Bay watershed (USA) has raised concerns about the potential impact of copper (Cu) on Pacific salmon (Oncorhynchus spp.). We conducted 96-h flow-through bioassays using low-hardness and low dissolved organic carbon water to determine the acute lethal toxicity of Cu to sockeye (Oncorhynchus nerka), Chinook (Oncorhynchus tshawytscha), and coho salmon (Oncorhynchus kisutch) fry. We aimed to determine Cu toxicity under field-relevant water quality conditions and to assess three methods of calculating ambient Cu criteria: the biotic ligand model (BLM), a multiple linear regression model endorsed by the US Environmental Protection Agency, and the hardness-based model currently used by the State of Alaska. The criteria generated by all models were below 20% lethal Cu concentrations by factors ranging from 2.2 to 54.3, indicating that all criteria would be protective against mortality. The multiple linear regression-based criteria were the most conservative and were comparable to BLM-based criteria. The median lethal concentrations (LC50s) for sockeye, Chinook, and coho were 35.2, 23.9, and 6.3 µg Cu/L, respectively. We also used the BLM to predict LC50s for each species. Model predictions differed from empirical LC50s by factors of 0.7 for sockeye and Chinook salmon, and 1.1 for coho salmon. These differences fell within the acceptable range of ±2, indicating the model's accuracy. We calculated critical lethal Cu accumulation values for each species to account for differing water chemistry in each bioassay; the present study revealed that coho salmon were most sensitive to Cu, followed by sockeye and Chinook salmon. Our findings underscore the importance of considering site- and species-specific factors when modeling Cu toxicity. The empirical data we present may enhance Cu risk assessments for Pacific salmon. Environ Toxicol Chem 2023;42:2440-2452. © 2023 SETAC.

Identification of functionally active aerobic methanotrophs in sediments from an arctic lake using stable isotope probing.

Arctic lakes are a significant source of the greenhouse gas methane (CH(4) ), but the role that methane oxidizing bacteria (methanotrophs) play in limiting the overall CH(4) flux is poorly understood. Here, we used stable isotope probing (SIP) techniques to identify the metabolically active aerobic methanotrophs in upper sediments (0-1 cm) from an arctic lake in northern Alaska sampled during ice-free summer conditions. The highest CH(4) oxidation potential was observed in the upper sediment (0-1 cm depth) with 1.59 µmol g wet weight(-1) day(-1) compared with the deeper sediment samples (1-3 cm, 3-5 cm and 5-10 cm), which exhibited CH(4) oxidation potentials below 0.4 µmol g wet weight(-1) day(-1) . Both type I and type II methanotrophs were directly detected in the upper sediment total communities using targeted primer sets based on 16S rRNA genes. Sequencing of 16S rRNA genes and functional genes (pmoA and mxaF) in the (13) C-DNA from the upper sediment indicated that type I methanotrophs, mainly Methylobacter, Methylosoma, Methylomonas and Methylovulum miyakonense, dominated the assimilation of CH(4) . Methylotrophs, including the genera Methylophilus and/or Methylotenera, were also abundant in the (13) C-DNA. Our results show that a diverse microbial consortium acquired carbon from CH(4) in the sediments of this arctic lake.

Shifts in identity and activity of methanotrophs in arctic lake sediments in response to temperature changes.

Methane (CH(4)) flux to the atmosphere is mitigated via microbial CH(4) oxidation in sediments and water. As arctic temperatures increase, understanding the effects of temperature on the activity and identity of methanotrophs in arctic lake sediments is important to predicting future CH(4) emissions. We used DNA-based stable-isotope probing (SIP), quantitative PCR (Q-PCR), and pyrosequencing analyses to identify and characterize methanotrophic communities active at a range of temperatures (4°C, 10°C, and 21°C) in sediments (to a depth of 25 cm) sampled from Lake Qalluuraq on the North Slope of Alaska. CH(4) oxidation activity was measured in microcosm incubations containing sediments at all temperatures, with the highest CH(4) oxidation potential of 37.5 µmol g(-1) day(-1) in the uppermost (depth, 0 to 1 cm) sediment at 21°C after 2 to 5 days of incubation. Q-PCR of pmoA and of the 16S rRNA genes of type I and type II methanotrophs, and pyrosequencing of 16S rRNA genes in (13)C-labeled DNA obtained by SIP demonstrated that the type I methanotrophs Methylobacter, Methylomonas, and Methylosoma dominated carbon acquisition from CH(4) in the sediments. The identity and relative abundance of active methanotrophs differed with the incubation temperature. Methylotrophs were also abundant in the microbial community that derived carbon from CH(4), especially in the deeper sediments (depth, 15 to 20 cm) at low temperatures (4°C and 10°C), and showed a good linear relationship (R = 0.82) with the relative abundances of methanotrophs in pyrosequencing reads. This study describes for the first time how methanotrophic communities in arctic lake sediments respond to temperature variations.

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.

Metabolic flexibility of aerobic methanotrophs under anoxic conditions in Arctic lake sediments.

Methane (CH4) emissions from Arctic lakes are a large and growing source of greenhouse gas to the atmosphere with critical implications for global climate. Because Arctic lakes are ice covered for much of the year, understanding the metabolic flexibility of methanotrophs under anoxic conditions would aid in characterizing the mechanisms responsible for limiting CH4 emissions from high-latitude regions. Using sediments from an active CH4 seep in Lake Qalluuraq, Alaska, we conducted DNA-based stable isotope probing (SIP) in anoxic mesocosms and found that aerobic Gammaproteobacterial methanotrophs dominated in assimilating CH4. Aerobic methanotrophs were also detected down to 70 cm deep in sediments at the seep site, where anoxic conditions persist. Metagenomic analyses of the heavy DNA from 13CH4-SIP incubations showed that these aerobic methanotrophs had the capacity to generate intermediates such as methanol, formaldehyde, and formate from CH4 oxidation and to oxidize formaldehyde in the tetrahydromethanopterin (H4MPT)-dependent pathway under anoxic conditions. The high levels of Fe present in sediments, combined with Fe and CH4 profiles in the persistent CH4 seep site, suggested that oxidation of CH4, or, more specifically, its intermediates such as methanol and formaldehyde might be coupled to iron reduction. Aerobic methanotrophs also possessed genes associated with nitrogen and hydrogen metabolism, which might provide potentially alternative energy conservation options under anoxic conditions. These results expand the known metabolic spectrum of aerobic methanotrophs under anoxic conditions and necessitate the re-assessment of the mechanisms underlying CH4 oxidation in the Arctic, especially under lakes that experience extended O2 limitations during ice cover.

Carbon isotopic fractionation in eider adipose tissue varies with fatty acid structure: implications for trophic studies.

Carbon isotopic fractionation was investigated in fatty acids (FA) of adipose tissue and blood serum of threatened Steller's eiders (Polysticta stelleri) and spectacled eiders (Somateria fischeri) relative to the FA in their diets. Captive eiders were fed a known diet for 180 days with serum sampled at 60, 120 and 180 days immediately after a 12 fast; adipose was collected at 180 days. Essential FA (EFA) in the adipose showed varying degrees of isotope fractionation (0-4‰), depending on FA structure. The δ(13)C values of long-chain FA 20:5n-3 and 22:6n-3 did not differ from those in the diet, while those of 18:2n-6 and 18:3n-3 were ∼2‰ greater than in the diet. The δ(13)C values of free FA (FFA) in serum were not consistent within individuals or sampling dates; fractionation varied randomly, suggesting that FFA were arising from diet, rather than mobilization from adipose tissue. Discrimination factors were used in combination with a mixing model incorporating FA and lipid concentrations to estimate the diet of eiders fed a binary mixture with contrasting isotopic signatures. Diet estimates varied with FA but mean values closely approximated the actual proportions consumed. By tracking EFA, this study avoided the complications in interpretation arising from isotopic routing of carbon in bulk isotope analyses and serves as a basis for the development of compound-specific isotopic methods to trace dietary input in wild eiders. However, our understanding of the processes contributing to the variation in isotopic signatures of FA in nature is currently limited, and we recommend that future research directions focus on elucidating these mechanisms.

The carbon isotope ratios of nonessential amino acids identify sugar-sweetened beverage (SSB) consumers in a 12-wk inpatient feeding study of 32 men with varying SSB and meat exposures.

BACKGROUND: The carbon isotope ratios (CIRs) of individual amino acids (AAs) may provide more sensitive and specific biomarkers of sugar-sweetened beverages (SSBs) than total tissue CIR. Because CIRs turn over slowly, long-term controlled-feeding studies are needed in their evaluation. OBJECTIVE: We assessed the responses of plasma and RBC CIRAA's to SSB and meat intake in a 12-wk inpatient feeding study. METHODS: Thirty-two men (aged 46.2 ± 10.5 y) completed the feeding study at the National Institute of Diabetes and Digestive and Kidney Diseases in Phoenix, Arizona. The effects of SSB, meat, and fish intake on plasma and RBC CIRAA's were evaluated in a balanced factorial design with each dietary variable either present or absent in a common weight-maintaining, macronutrient-balanced diet. Fasting blood samples were collected biweekly from baseline. Dietary effects on the postfeeding CIR of 5 nonessential AAs (CIRNEAA's) and 4 essential AAs (CIREAA's) were analyzed using multivariable regression. RESULTS: In plasma, 4 of 5 CIRNEAA's increased with SSB intake. Of these, the CIRAla was the most sensitive (ß = 2.81, SE = 0.38) to SSB intake and was not affected by meat or fish intake. In RBCs, all 5 CIRNEAA's increased with SSBs but had smaller effect sizes than in plasma. All plasma CIREAA's increased with meat intake (but not SSB or fish intake), and the CIRLeu was the most sensitive (ß = 1.26, SE = 0.23). CIRs of leucine and valine also increased with meat intake in RBCs. Estimates of turnover suggest that CIRAA's in plasma, but not RBCs, were in equilibrium with the diets by the end of the study. CONCLUSIONS: The results of this study in men support CIRNEAA's as potential biomarkers of SSB intake and suggest CIREAA's as potential biomarkers of meat intake in US diets. This trial was registered at clinicaltrials.gov/ct2/show/NCT01237093 as NCT01237093.

Whiskers as a novel tissue for tracking reproductive and stress-related hormones in North Pacific otariid pinnipeds.

Keratinized tissues, including whiskers, are ideal for acquiring a record of physiological parameters. Most tissues provide a snapshot of physiological status; however, whiskers may support longitudinal sampling for reproductive and stress-related hormones, if hormones are incorporated as whiskers grow and concentrations change with physiological state. Whiskers from female Steller sea lions (Eumetopias jubatus) and northern fur seals (Callorhinus ursinus) were serially sectioned and pulverized and steroid hormones were extracted. Standard methods were used to validate enzyme immunoassay kits for cortisol, progesterone, 17ß-estradiol and testosterone. All hormones were measurable in whisker segments from both species with progesterone concentrations showing cyclical patterns, which appear to signify previous pregnancies or luteal phases. Yearly progesterone concentrations were greater in years a pup was produced compared with years when no pup was observed. Free-ranging female Steller sea lions had reproductive rates between 0 and 1.0 (0.53 ± 0.33, n = 12) using a yearly progesterone concentration of 30 pg/mg or greater to classify a reproductive year as producing a pup and below 30 pg/mg as non-reproductive. Cortisol concentrations were greater near the root and rapidly declined, lacking any obvious patterns, throughout the rest of the whisker. Progesterone and testosterone concentrations were able to help determine sex of unknown individuals. Immunohistochemistry revealed that steroid hormones most likely do not leach out of whiskers based on the deposition patterns of progesterone and cortisol being present throughout the whisker length. Overall, measuring steroid hormones in whiskers can reveal individual reproductive histories over multiple years in sea lions and fur seals. Cyclical patterns of δ15N were useful for identifying periods of up to ~10 years of growth within whiskers, and measuring both stable isotopes and hormones may be useful for differentiating periods of active gestation from diapause and potentially track multi-year reproductive histories of female otariids.