Brian Fry

Despite differences in their overall metabolism, eukaryotes share a common mitochondrial biochemistry. We investigated how this fundamental biochemistry supports overall metabolism using a high-resolution carbon isotope approach, position-specific isotope analysis. We measured carbon isotope ¹³ C/ ¹² C cycling in animals, focusing on amino acids that are formed in mitochondrial reactions and are most metabolically active. Carboxyl isotope determinations for amino acids showed strong signals related to common biochemical pathways. Contrasting isotope patterns were measured for metabolism associated with major life history patterns, including growth and reproduction. Turnover of proteins and lipids as well as gluoconeogensis dynamics could be estimated for these metabolic life histories. The high-resolution isotomics measurements fingerprinted metabolism and metabolic strategies across the eukaryotic animal kingdom, yielding results for humans, ungulates, whales, and diverse fish and invertebrates in a nearshore marine food web.

Metabolomic and genomic markers in plants have helped diagnose evolutionary pressures and resulting modern-day floristic diversification. Here we use a different set of metrics, 17 biochemical measures made at the whole tissue or bulk tissue level, to study diversification in resource use and productivity among Pacific mangroves. Three mangrove species Bruguiera gynmorhiza (BRGY), Rhizphora apiculata (RHAP), and Sonneratia alba (SOAL) were studied across 5 sites on the island of Kosrae, Federated States of Micronesia with measurements of the following chemical metrics: C, N, P, K, Na, Mg, Ca, B, S, Mn, Fe, Cu, Zn elements and isotope values δ²H, δ¹³C, δ¹⁵N, and δ³⁴S. Species were remarkably distinct in chemical profiles, showing significant differences across all metrics. This indicated long-term resource use partitioning and optimization, with metrics showing physiology and patch-related differences. The patch-related differences meant that metrics were not really fixed in species, but represented flexible traits (“flexitraits”) in fingerprinting mangrove ecology. Effects of tree harvesting could be fingerprinted with the metrics at one of the Kosrae sites. Modeling showed two results. (1) Conservation efforts to preserve low-nutrient specialists like BRGY probably should involve removal of competing SOAL and RHAP rather than nutrient reductions. (2) Although mangrove growth rates were most limited by P, water was a strongly co-limiting factor. This study introduces a new physiological parameter to plant ecology, a water-to-phosphorus ratio, “normalized δ¹³C/P” or “f_13C/P”, that should generally help diagnose how plant N and P nutrient use can be co-limited by water.

Metabolomic and genomic markers have helped diagnose evolutionary pressures and environmental responses in plants. Here we use a different set of metrics, 17 biochemical measures made at the whole tissue or bulk tissue level, to study resource use and productivity among Pacific mangroves. Three mangrove species Bruguiera gynmorhiza (BRGY), Rhizphora apiculate (RHAP), and Sonneratia albans (SOAL) were studied across 5 sites on the island of Kosrae, Federated States of Micronesia with measurements of the following chemical metrics: C, N, P, K, Na, Mg, Ca, B, S, Mn, Fe, Cu, Zn elements and isotope values d²H, d¹³C, d¹⁵N, and d³⁴S. Species were remarkably distinct in chemical profiles, showing significant differences across all metrics. This indicated long-term resource use partitioning and optimization, with metrics showing physiology and patch-related differences. The patch differences meant that metrics were not really fixed in species, but represented flexible traits (“flexitraits”) in fingerprinting mangrove ecology. Effects of tree harvesting could be fingerprinted with the metrics at one of the Kosrae sites. Modeling showed two results. 1) Conservation efforts aimed at enhancing abundance of conservative species like BRGY that have low N and P probably should involve removal of competing SOAL and RHAP rather than nutrient reductions. 2) While P most limited mangrove growth rates, water was a strongly co-limiting factor. This study introduces a new physiological parameter to plant ecology, a water-to-phosphorus ratio, “normalized d¹³C/P” or “f_13C/P”, that should generally help diagnose how plant N and P nutrient use can be co-limited by water.

The carbon isotopic compositions of amino acids are increasingly measured to characterize diets and metabolic response to diets. We report a new high-resolution system to measure the stable carbon isotopic composition of carboxyl atoms within amino acids. The automated system used HPLC to separate amino acids followed by addition of ninhydrin for decarboxylation and transfer of the evolved CO₂ to a stable isotope ratio mass spectrometer for δ¹³C_CARBOXYL measurement. The ninhydrin reaction was conducted at acidic pH (1.5) and elevated temperature (160 ^oC) giving yields close to 100% for most common amino acids. Eight mammalian keratin samples from herbivores (kudu and caribou), omnivores (humans) and carnivores (bowhead and humpback zooplanktivorous whales) were analysed with this new system. The data provide an initial calibration of reference materials to be used in studies of this type and is the first report of carboxyl carbon isotope distributions in mammals. Results showed widespread ¹³C enrichments in both essential and non-essential amino acid carboxyl groups, likely linked to decarboxylation of amino acids during normal metabolism. Analyses of non-essential amino acid isotope profiles showed (1) consistent and general taxon-level metabolic differences between the herbivore, human and whale samples, (2) marked differences among individual humans, ruminants and whales (3) evidence for gluconeogenesis in the wildlife samples, and (4) extensive ¹³C enrichment likely associated with fasting in the humpback whale sample. Future mammalian research related to the metabolism of growth, reproduction, aging and disease may benefit from using this technique. Values obtained for internationally available samples USGS42 and USGS43 (Tibetan and Indian human hair) provide a first characterization of reference materials for δ¹³C_CARBOXYL profiles.

Rationale: The fundamental level of stable isotopic knowledge lies at specific atomic positions within molecules but existing methods of analysis require lengthy off‐line preparation to reveal this information. An automated position‐specific isotope analysis (PSIA) method is presented to determine the stable carbon isotopic compositions of the carboxyl groups of amino acids ( δ ¹³ C _CARBOXYL values). This automation makes PSIA measurements easier and routine. Methods: An existing high‐performance liquid chromatography (HPLC) gas handling interface/stable isotope ratio mass spectrometry system was modified by the addition of a post‐column derivatisation unit between the HPLC system and the interface. The post‐column reaction was optimised to yield CO ₂ from the carboxyl groups of amino acids by reaction with ninhydrin. Results: The methodology described produced δ ¹³ C _CARBOXYL values with typical standard deviations below ±0.1 ‰ and consistent differences ( Δ ¹³ C _CARBOXYL values) between amino acids over a 1‐year period. First estimates are presented for the δ ¹³ C _CARBOXYL values of a number of internationally available amino acid reference materials. Conclusions: The PSIA methodology described provides a further dimension to the stable isotopic characterisation of amino acids at a more detailed level than the bulk or averaged whole‐molecule level. When combined with on‐line chromatographic separation or off‐line fraction collection of protein hydrolysates the technique will offer an automated and routine way to study position‐specific carboxyl carbon isotope information for amino acids, enabling more refined isotopic studies of carbon uptake and metabolism.

Stream food webs are changing in response to anthropogenic stressors, such as nutrient enrichment and water extraction. One way to monitor these changes is to examine shifts in the relative importance of autotrophic and detrital energetic pathways in food webs. This understanding is particularly pertinent in intermittent streams whose prevalence is projected to increase because of a changing climate and groundwater extraction. We used the stable-isotope values (δ¹³C and δ¹⁵N) of autotrophic and detrital basal resources and the tissue of consumers to examine the relative importance of these 2 energetic pathways during wet and dry phases in 5 groundwater-influenced intermittent streams. In addition, we used surface-water biogeochemical data to estimate the use of groundwater-derived dissolved inorganic C (DIC) in food webs. Autotrophic basal resources were the dominant C source for primary consumers during wet (>95%, on average) and dry (>77%, on average) hydrological phases. ¹³C-depleted autotrophs and consumers and negative correlations between the δ¹³C values of autotrophs and consumers with aqueous CO₂ concentrations, indicated that primary producers probably are assimilating microbial respired CO₂ from groundwater. Overall, we suggest that groundwater discharge is probably central to foodweb productivity in these streams by extending the duration of wetted habitat for autotrophic growth during dry phases and by delivering dissolved resources that potentially contribute to the energetic base of producers and consumers. Consequently, extended periods of reduced flow (and cease-to-flow events) caused by groundwater extraction or changed climatic conditions may reduce the overall productivity of groundwater-influenced intermittent streams.

To assess coastal ecosystem status and pollution baselines, prawns were collected from the commercial catches of eight Asia-Pacific countries (Australia, Bangladesh, Indonesia, Myanmar, Philippines, Pakistan, Sri Lanka and Thailand). Samples collected from 21 sites along regional coastlines were analysed for trace metal and stable isotopic compositions of H, C, N, O and S. A combination of simple averaging and multivariate analyses was used to evaluate the data. Sites could be assigned to easily recognise polluted and unpolluted groups based on the prawn results. Some filter-feeding clams were also collected and analysed together with the benthic-feeding prawns, and the prawns generally had lower trace metal burdens. Climate change effects were not strongly evident at this time, but altered ocean circulation and watershed run-off patterns accompanying future climate change are expected to change chemical patterns recorded by prawns along these and other coastlines. Stable isotopes, especially ¹⁵N, can help to distinguish between relatively polluted and unpolluted sites.