Metabolomics as a tool for in situ study of chronic metal exposure in estuarine invertebrates.

Estuaries are subject to intense human use globally, with impacts from multiple stressors, such as metal contaminants. A key challenge is extending beyond traditional monitoring approaches to understand effects to biota and system function. To explore the metabolic effects of complex metal contaminants to sediment dwelling (benthic) fauna, we apply a multiple-lines-of-evidence approach, coupling environmental monitoring, benthic sampling, total metals analysis and targeted metabolomics. We characterise metabolic signatures of metal exposure in three benthic invertebrate taxa, which differed in distribution across sites and severity of metal exposure: sipunculid (very high), amphipod (high), maldanid polychaete (moderate). We observed sediment and tissue metal loads far exceeding sediment guidelines where toxicity-related adverse effects may be expected, for metals including, As, Cd, Pb, Zn and Hg. Change in site- and taxa-specific metabolite profiles was highly correlated with natural environmental drivers (sediment total organic carbon and water temperature). At the most metal influenced sites, metabolite variation was also correlated with sediment metal loads. Using supervised multivariate regression, taxa-specific metabolic signatures of increased exposure and possibility of toxic effects were characterised against multiple reference sites. Metabolic signatures varied according to each taxon and degree of metal exposure, but primarily indicated altered cysteine and methionine metabolism, metal-binding and elimination (lysosomal) activity, coupled to change in complex biosynthesis pathways, responses to oxidative stress, and cellular damage. This novel multiple-lines-of-evidence approach combining metabolomics with traditional environmental monitoring, enabled detection and characterisation of chronic metal exposure effects in situ in multiple invertebrate taxa. With capacity for application to rapid and effective monitoring of non-model species in complex environments, these approaches are critical for improved assessment and management of systems that are increasingly subject to anthropogenic drivers of change.

Metabolic pathways inferred from a bacterial marker gene illuminate ecological changes across South Pacific frontal boundaries.

Global oceanographic monitoring initiatives originally measured abiotic essential ocean variables but are currently incorporating biological and metagenomic sampling programs. There is, however, a large knowledge gap on how to infer bacterial functions, the information sought by biogeochemists, ecologists, and modelers, from the bacterial taxonomic information (produced by bacterial marker gene surveys). Here, we provide a correlative understanding of how a bacterial marker gene (16S rRNA) can be used to infer latitudinal trends for metabolic pathways in global monitoring campaigns. From a transect spanning 7000 km in the South Pacific Ocean we infer ten metabolic pathways from 16S rRNA gene sequences and 11 corresponding metagenome samples, which relate to metabolic processes of primary productivity, temperature-regulated thermodynamic effects, coping strategies for nutrient limitation, energy metabolism, and organic matter degradation. This study demonstrates that low-cost, high-throughput bacterial marker gene data, can be used to infer shifts in the metabolic strategies at the community scale.

Can We Use Functional Genetics to Predict the Fate of Nitrogen in Estuaries?

Increasing nitrogen (N) loads present a threat to estuaries, which are among the most heavily populated and perturbed parts of the world. N removal is largely mediated by the sediment microbial process of denitrification, in direct competition to dissimilatory nitrate reduction to ammonium (DNRA), which recycles nitrate to ammonium. Molecular proxies for N pathways are increasingly measured and analyzed, a major question in microbial ecology, however, is whether these proxies can add predictive power around the fate of N. We analyzed the diversity and community composition of sediment nirS and nrfA genes in 11 temperate estuaries, covering four types of land use in Australia, and analyzed how these might be used to predict N removal. Our data suggest that sediment microbiomes play a central role in controlling the magnitude of the individual N removal rates in the 11 estuaries. Inclusion, however, of relative gene abundances of 16S, nirS, nrfA, including their ratios did not improve physicochemical measurement-based regression models to predict rates of denitrification or DNRA. Co-occurrence network analyses of nirS showed a greater modularity and a lower number of keystone OTUs in pristine sites compared to urban estuaries, suggesting a higher degree of niche partitioning in pristine estuaries. The distinctive differences between the urban and pristine network structures suggest that the nirS gene could be a likely gene candidate to understand the mechanisms by which these denitrifying communities form and respond to anthropogenic pressures.

Eukaryotic assimilatory nitrate reductase fractionates N and O isotopes with a ratio near unity.

In order to (i) establish the biological systematics necessary to interpret nitrogen (N) and oxygen (O) isotope ratios of nitrate ((15)N/(14)N and (18)O/(16)O) in the environment and (ii) investigate the potential for isotopes to elucidate the mechanism of a key N cycle enzyme, we measured the nitrate N and O isotope effects ((15)ε and (18)ε) for nitrate reduction by two assimilatory eukaryotic nitrate reductase (eukNR) enzymes. The (15)ε for purified extracts of NADPH eukNR from the fungus Aspergillus niger and the (15)ε for NADH eukNR from cell homogenates of the marine diatom Thalassiosira weissflogii were indistinguishable, yielding a mean (15)ε for the enzyme of 26.6 ± 0.2‰. Both forms of eukNR imparted near equivalent fractionation on N and O isotopes. The increase in (18)O/(16)O versus the increase in (15)N/(14)N (relative to their natural abundances) was 0.96 ± 0.01 for NADPH eukNR and 1.09 ± 0.03 for NADH eukNR. These results are the first reliable measurements of the coupled N and O isotope effects for any form of eukNR. They support the prevailing view that intracellular reduction by eukNR is the dominant step in isotope fractionation during nitrate assimilation and that it drives the (18)ε:(15)ε ≈ 1 observed in phytoplankton cultures, suggesting that this O-to-N isotope signature will apply broadly in the environment. Our measured (15)ε and (18)ε may represent the intrinsic isotope effects for eukNR-mediated N-O bond rupture, a potential constraint on the nature of the enzyme's transition state.