The metabolic rate of the biosphere and its components.

We assessed the relationship between rates of biological energy utilization and the biomass sustained by that energy utilization, at both the organism and biosphere level. We compiled a dataset comprising >10,000 basal, field, and maximum metabolic rate measurements made on >2,900 individual species, and, in parallel, we quantified rates of energy utilization, on a biomass-normalized basis, by the global biosphere and by its major marine and terrestrial components. The organism-level data, which are dominated by animal species, have a geometric mean among basal metabolic rates of 0.012 W (g C)-1 and an overall range of more than six orders of magnitude. The biosphere as a whole uses energy at an average rate of 0.005 W (g C)-1 but exhibits a five order of magnitude range among its components, from 0.00002 W (g C)-1 for global marine subsurface sediments to 2.3 W (g C)-1 for global marine primary producers. While the average is set primarily by plants and microorganisms, and by the impact of humanity upon those populations, the extremes reflect systems populated almost exclusively by microbes. Mass-normalized energy utilization rates correlate strongly with rates of biomass carbon turnover. Based on our estimates of energy utilization rates in the biosphere, this correlation predicts global mean biomass carbon turnover rates of ~2.3 y-1 for terrestrial soil biota, ~8.5 y-1 for marine water column biota, and ~1.0 y-1 and ~0.01 y-1 for marine sediment biota in the 0 to 0.1 m and >0.1 m depth intervals, respectively.

Proterozoic Acquisition of Archaeal Genes for Extracellular Electron Transfer: A Metabolic Adaptation of Aerobic Ammonia-Oxidizing Bacteria to Oxygen Limitation.

Many aerobic microbes can utilize alternative electron acceptors under oxygen-limited conditions. In some cases, this is mediated by extracellular electron transfer (or EET), wherein electrons are transferred to extracellular oxidants such as iron oxide and manganese oxide minerals. Here, we show that an ammonia-oxidizer previously known to be strictly aerobic, Nitrosomonas communis, may have been able to utilize a poised electrode to maintain metabolic activity in anoxic conditions. The presence and activity of multiheme cytochromes in N. communis further suggest a capacity for EET. Molecular clock analysis shows that the ancestors of ß-proteobacterial ammonia oxidizers appeared after Earth's atmospheric oxygenation when the oxygen levels were >10-4pO2 (present atmospheric level [PAL]), consistent with aerobic origins. Equally important, phylogenetic reconciliations of gene and species trees show that the multiheme c-type EET proteins in Nitrosomonas and Nitrosospira lineages were likely acquired by gene transfer from γ-proteobacteria when the oxygen levels were between 0.1 and 1 pO2 (PAL). These results suggest that ß-proteobacterial EET evolved during the Proterozoic when oxygen limitation was widespread, but oxidized minerals were abundant.

Carbonate-hosted microbial communities are prolific and pervasive methane oxidizers at geologically diverse marine methane seep sites.

At marine methane seeps, vast quantities of methane move through the shallow subseafloor, where it is largely consumed by microbial communities. This process plays an important role in global methane dynamics, but we have yet to identify all of the methane sinks in the deep sea. Here, we conducted a continental-scale survey of seven geologically diverse seafloor seeps and found that carbonate rocks from all sites host methane-oxidizing microbial communities with substantial methanotrophic potential. In laboratory-based mesocosm incubations, chimney-like carbonates from the newly described Point Dume seep off the coast of Southern California exhibited the highest rates of anaerobic methane oxidation measured to date. After a thorough analysis of physicochemical, electrical, and biological factors, we attribute this substantial metabolic activity largely to higher cell density, mineral composition, kinetic parameters including an elevated Vmax, and the presence of specific microbial lineages. Our data also suggest that other features, such as electrical conductance, rock particle size, and microbial community alpha diversity, may influence a sample's methanotrophic potential, but these factors did not demonstrate clear patterns with respect to methane oxidation rates. Based on the apparent pervasiveness within seep carbonates of microbial communities capable of performing anaerobic oxidation of methane, as well as the frequent occurrence of carbonates at seeps, we suggest that rock-hosted methanotrophy may be an important contributor to marine methane consumption.

Novel Insights on Obligate Symbiont Lifestyle and Adaptation to Chemosynthetic Environment as Revealed by the Giant Tubeworm Genome.

The mutualism between the giant tubeworm Riftia pachyptila and its endosymbiont Candidatus Endoriftia persephone has been extensively researched over the past 40 years. However, the lack of the host whole-genome information has impeded the full comprehension of the genotype/phenotype interface in Riftia. Here, we described the high-quality draft genome of Riftia, its complete mitogenome, and tissue-specific transcriptomic data. The Riftia genome presents signs of reductive evolution, with gene family contractions exceeding expansions. Expanded gene families are related to sulfur metabolism, detoxification, antioxidative stress, oxygen transport, immune system, and lysosomal digestion, reflecting evolutionary adaptations to the vent environment and endosymbiosis. Despite the derived body plan, the developmental gene repertoire in the gutless tubeworm is extremely conserved with the presence of a near intact and complete Hox cluster. Gene expression analyses establish that the trophosome is a multifunctional organ marked by intracellular digestion of endosymbionts, storage of excretory products, and hematopoietic functions. Overall, the plume and gonad tissues both in contact to the environment harbor highly expressed genes involved with cell cycle, programed cell death, and immunity indicating a high cell turnover and defense mechanisms against pathogens. We posit that the innate immune system plays a more prominent role into the establishment of the symbiosis during the infection in the larval stage, rather than maintaining the symbiostasis in the trophosome. This genome bridges four decades of physiological research in Riftia, whereas it simultaneously provides new insights into the development, whole organism functions, and evolution in the giant tubeworm.

Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

CRISPR/Cas9-induced disruption of Bodo saltans paraflagellar rod-2 gene reveals its importance for cell survival.

Developing transfection protocols for marine protists is an emerging field that will allow the functional characterization of protist genes and their roles in organism responses to the environment. We developed a CRISPR/Cas9 editing protocol for Bodo saltans, a free-living kinetoplastid with tolerance to both marine and freshwater conditions and a close non-parasitic relative of trypanosomatids. Our results show that SaCas9/single-guide RNA (sgRNA) ribonucleoprotein (RNP) complex-mediated disruption of the paraflagellar rod 2 gene (BsPFR2) was achieved using electroporation-mediated transfection. The use of CRISPR/Cas9 genome editing can increase the efficiency of targeted homologous recombination when a repair DNA template is provided. Our sequence analysis suggests two mechanisms for repairing double-strand breaks in B. saltans are active; homologous-directed repair (HDR) utilizing an exogenous DNA template that carries an antibiotic resistance gene and likley non-homologous end joining (NHEJ). However, HDR was only achieved when a single (vs. multiple) SaCas9 RNP complex was provided. Furthermore, the biallelic knockout of BsPFR2 was detrimental for the cell, highlighting its essential role for cell survival because it facilitates the movement of food particles into the cytostome. Our Cas9/sgRNA RNP complex protocol provides a new tool for assessing gene functions in B. saltans and perhaps similar protists with polycistronic transcription.

Spatially resolved correlative microscopy and microbial identification reveal dynamic depth- and mineral-dependent anabolic activity in salt marsh sediment.

Coastal salt marshes are key sites of biogeochemical cycling and ideal systems in which to investigate the community structure of complex microbial communities. Here, we clarify structural-functional relationships among microorganisms and their mineralogical environment, revealing previously undescribed metabolic activity patterns and precise spatial arrangements within salt marsh sediment. Following 3.7-day in situ incubations with a non-canonical amino acid that was incorporated into new biomass, samples were resin-embedded and analysed by correlative fluorescence and electron microscopy to map the microscale arrangements of anabolically active and inactive organisms alongside mineral grains. Parallel sediment samples were examined by fluorescence-activated cell sorting and 16S rRNA gene sequencing to link anabolic activity to taxonomic identity. Both approaches demonstrated a rapid decline in the proportion of anabolically active cells with depth into salt marsh sediment, from ~60% in the top centimetre to 9.4%-22.4% between 2 and 10 cm. From the top to the bottom, the most prominent active community members shifted from sulfur cycling phototrophic consortia, to putative sulfate-reducing bacteria likely oxidizing organic compounds, to fermentative lineages. Correlative microscopy revealed more abundant (and more anabolically active) organisms around non-quartz minerals including rutile, orthoclase and plagioclase. Microbe-mineral relationships appear to be dynamic and context-dependent arbiters of biogeochemical cycling.

Cooccurring Activities of Two Autotrophic Pathways in Symbionts of the Hydrothermal Vent Tubeworm Riftia pachyptila.

Genome and proteome data predict the presence of both the reductive citric acid cycle (rCAC; also called the reductive tricarboxylic acid cycle) and the Calvin-Benson-Bassham cycle (CBB) in "Candidatus Endoriftia persephonae," the autotrophic sulfur-oxidizing bacterial endosymbiont from the giant hydrothermal vent tubeworm Riftia pachyptila. We tested whether these cycles were differentially induced by sulfide supply, since the synthesis of biosynthetic intermediates by the rCAC is less energetically expensive than that by the CBB. R. pachyptila was incubated under in situ conditions in high-pressure aquaria under low (28 to 40 µmol · h-1) or high (180 to 276 µmol · h-1) rates of sulfide supply. Symbiont-bearing trophosome samples excised from R. pachyptila maintained under the two conditions were capable of similar rates of CO2 fixation. Activities of the rCAC enzyme ATP-dependent citrate lyase (ACL) and the CBB enzyme 1,3-bisphosphate carboxylase/oxygenase (RubisCO) did not differ between the two conditions, although transcript abundances for ATP-dependent citrate lyase were 4- to 5-fold higher under low-sulfide conditions. δ13C values of internal dissolved inorganic carbon (DIC) pools were varied and did not correlate with sulfide supply rate. In samples taken from freshly collected R. pachyptila, δ13C values of lipids fell between those collected for organisms using either the rCAC or the CBB exclusively. These observations are consistent with cooccurring activities of the rCAC and the CBB in this symbiosis. IMPORTANCE Previous to this study, the activities of the rCAC and CBB in R. pachyptila had largely been inferred from "omics" studies of R. pachyptila without direct assessment of in situ conditions prior to collection. In this study, R. pachyptila was maintained and monitored in high-pressure aquaria prior to measuring its CO2 fixation parameters. Results suggest that ranges in sulfide concentrations similar to those experienced in situ do not exert a strong influence on the relative activities of the rCAC and the CBB. This observation highlights the importance of further study of this symbiosis and other organisms with multiple CO2-fixing pathways, which recent genomics and biochemical studies suggest are likely to be more prevalent than anticipated.

Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase.

Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa3 and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD(+))/NADH ratio via binding of NAD(+) to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration.

Hydrogen Does Not Appear To Be a Major Electron Donor for Symbiosis with the Deep-Sea Hydrothermal Vent Tubeworm Riftia pachyptila.

Use of hydrogen gas (H2) as an electron donor is common among free-living chemolithotrophic microorganisms. Given the presence of this dissolved gas at deep-sea hydrothermal vents, it has been suggested that it may also be a major electron donor for the free-living and symbiotic chemolithoautotrophic bacteria that are the primary producers at these sites. Giant Riftia pachyptila siboglinid tubeworms and their symbiotic bacteria ("Candidatus Endoriftia persephone") dominate many vents in the Eastern Pacific, and their use of sulfide as a major electron donor has been documented. Genes encoding hydrogenase are present in the "Ca Endoriftia persephone" genome, and proteome data suggest that these genes are expressed. In this study, high-pressure respirometry of intact R. pachyptila and incubations of trophosome homogenate were used to determine whether this symbiotic association could also use H2 as a major electron donor. Measured rates of H2 uptake by intact R. pachyptila in high-pressure respirometers were similar to rates measured in the absence of tubeworms. Oxygen uptake rates in the presence of H2 were always markedly lower than those measured in the presence of sulfide, as was the incorporation of 13C-labeled dissolved inorganic carbon. Carbon fixation by trophosome homogenate was not stimulated by H2, nor was hydrogenase activity detectable in these samples. Though genes encoding [NiFe] group 1e and [NiFe] group 3b hydrogenases are present in the genome and transcribed, it does not appear that H2 is a major electron donor for this system, and it may instead play a role in intracellular redox homeostasis.IMPORTANCE Despite the presence of hydrogenase genes, transcripts, and proteins in the "Ca Endoriftia persephone" genome, transcriptome, and proteome, it does not appear that R. pachyptila can use H2 as a major electron donor. For many uncultivable microorganisms, omic analyses are the basis for inferences about their activities in situ However, as is apparent from the study reported here, there are dangers in extrapolating from omics data to function, and it is essential, whenever possible, to verify functions predicted from omics data with physiological and biochemical measurements.

Harnessing a methane-fueled, sediment-free mixed microbial community for utilization of distributed sources of natural gas.

Harnessing the metabolic potential of uncultured microbial communities is a compelling opportunity for the biotechnology industry, an approach that would vastly expand the portfolio of usable feedstocks. Methane is particularly promising because it is abundant and energy-rich, yet the most efficient methane-activating metabolic pathways involve mixed communities of anaerobic methanotrophic archaea and sulfate reducing bacteria. These communities oxidize methane at high catabolic efficiency and produce chemically reduced by-products at a comparable rate and in near-stoichiometric proportion to methane consumption. These reduced compounds can be used for feedstock and downstream chemical production, and at the production rates observed in situ they are an appealing, cost-effective prospect. Notably, the microbial constituents responsible for this bioconversion are most prominent in select deep-sea sediments, and while they can be kept active at surface pressures, they have not yet been cultured in the lab. In an industrial capacity, deep-sea sediments could be periodically recovered and replenished, but the associated technical challenges and substantial costs make this an untenable approach for full-scale operations. In this study, we present a novel method for incorporating methanotrophic communities into bioindustrial processes through abstraction onto low mass, easily transportable carbon cloth artificial substrates. Using Gulf of Mexico methane seep sediment as inoculum, optimal physicochemical parameters were established for methane-oxidizing, sulfide-generating mesocosm incubations. Metabolic activity required >∼40% seawater salinity, peaking at 100% salinity and 35 °C. Microbial communities were successfully transferred to a carbon cloth substrate, and rates of methane-dependent sulfide production increased more than threefold per unit volume. Phylogenetic analyses indicated that carbon cloth-based communities were substantially streamlined and were dominated by Desulfotomaculum geothermicum. Fluorescence in situ hybridization microscopy with carbon cloth fibers revealed a novel spatial arrangement of anaerobic methanotrophs and sulfate reducing bacteria suggestive of an electronic coupling enabled by the artificial substrate. This system: 1) enables a more targeted manipulation of methane-activating microbial communities using a low-mass and sediment-free substrate; 2) holds promise for the simultaneous consumption of a strong greenhouse gas and the generation of usable downstream products; and 3) furthers the broader adoption of uncultured, mixed microbial communities for biotechnological use.

Toward establishing model organisms for marine protists: Successful transfection protocols for Parabodo caudatus (Kinetoplastida: Excavata).

We developed protocols for, and demonstrated successful transfection of, the free-living kinetoplastid flagellate Parabodo caudatus with three plasmids carrying a fluorescence reporter gene (pEF-GFP with the EF1 alpha promoter, pUB-GFP with Ubiquitin C promoter, and pEYFP-Mitotrap with CMV promoter). We evaluated three electroporation approaches: (1) a square-wave electroporator designed for eukaryotes, (2) a novel microfluidic transfection system employing hydrodynamically-controlled electric field waveforms, and (3) a traditional exponential decay electroporator. We found the microfluidic device provides a simple and efficient platform to quickly test a wide range of electric field parameters to find the optimal set of conditions for electroporation of target species. It also allows for processing large sample volumes (>10 ml) within minutes, increasing throughput 100 times over cuvettes. Fluorescence signal from the reporter gene was detected a few hours after transfection and persisted for 3 days in cells transfected by pEF-GFP and pUB-GFP plasmids and for at least 5 days post-transfection for cells transfected with pEYFP-Mitotrap. Expression of the reporter genes (GFP and YFP) was also confirmed using reverse transcription-PCR (RT-PCR). This work opens the door for further efforts with this taxon and close relatives toward establishing model systems for genome editing.

Hydrogen is an energy source for hydrothermal vent symbioses.

The discovery of deep-sea hydrothermal vents in 1977 revolutionized our understanding of the energy sources that fuel primary productivity on Earth. Hydrothermal vent ecosystems are dominated by animals that live in symbiosis with chemosynthetic bacteria. So far, only two energy sources have been shown to power chemosynthetic symbioses: reduced sulphur compounds and methane. Using metagenome sequencing, single-gene fluorescence in situ hybridization, immunohistochemistry, shipboard incubations and in situ mass spectrometry, we show here that the symbionts of the hydrothermal vent mussel Bathymodiolus from the Mid-Atlantic Ridge use hydrogen to power primary production. In addition, we show that the symbionts of Bathymodiolus mussels from Pacific vents have hupL, the key gene for hydrogen oxidation. Furthermore, the symbionts of other vent animals such as the tubeworm Riftia pachyptila and the shrimp Rimicaris exoculata also have hupL. We propose that the ability to use hydrogen as an energy source is widespread in hydrothermal vent symbioses, particularly at sites where hydrogen is abundant.

Heterotrophic Proteobacteria in the vicinity of diffuse hydrothermal venting.

Deep-sea hydrothermal vents are highly dynamic habitats characterized by steep temperature and chemical gradients. The oxidation of reduced compounds dissolved in the venting fluids fuels primary production providing the basis for extensive life. Until recently studies of microbial vent communities have focused primarily on chemolithoautotrophic organisms. In our study, we targeted the change of microbial community compositions along mixing gradients, focusing on distribution and capabilities of heterotrophic microorganisms. Samples were retrieved from different venting areas within the Menez Gwen hydrothermal field, taken along mixing gradients, including diffuse fluid discharge points, their immediate surroundings and the buoyant parts of hydrothermal plumes. High throughput 16S rRNA gene amplicon sequencing, fluorescence in situ hybridization, and targeted metagenome analysis were combined with geochemical analyses. Close to diffuse venting orifices dominated by chemolithoautotrophic Epsilonproteobacteria, in areas where environmental conditions still supported chemolithoautotrophic processes, we detected microbial communities enriched for versatile heterotrophic Alpha- and Gammaproteobacteria. The potential for alkane degradation could be shown for several genera and yet uncultured clades. We propose that hotspots of chemolithoautotrophic life support a 'belt' of heterotrophic bacteria significantly different from the dominating oligotrophic microbiota of the deep sea.

Oxygen, ecology, and the Cambrian radiation of animals.

The Proterozoic-Cambrian transition records the appearance of essentially all animal body plans (phyla), yet to date no single hypothesis adequately explains both the timing of the event and the evident increase in diversity and disparity. Ecological triggers focused on escalatory predator-prey "arms races" can explain the evolutionary pattern but not its timing, whereas environmental triggers, particularly ocean/atmosphere oxygenation, do the reverse. Using modern oxygen minimum zones as an analog for Proterozoic oceans, we explore the effect of low oxygen levels on the feeding ecology of polychaetes, the dominant macrofaunal animals in deep-sea sediments. Here we show that low oxygen is clearly linked to low proportions of carnivores in a community and low diversity of carnivorous taxa, whereas higher oxygen levels support more complex food webs. The recognition of a physiological control on carnivory therefore links environmental triggers and ecological drivers, providing an integrated explanation for both the pattern and timing of Cambrian animal radiation.

Evidence for the role of endosymbionts in regional-scale habitat partitioning by hydrothermal vent symbioses.

Deep-sea hydrothermal vents are populated by dense communities of animals that form symbiotic associations with chemolithoautotrophic bacteria. To date, our understanding of which factors govern the distribution of host/symbiont associations (or holobionts) in nature is limited, although host physiology often is invoked. In general, the role that symbionts play in habitat utilization by vent holobionts has not been thoroughly addressed. Here we present evidence for symbiont-influenced, regional-scale niche partitioning among symbiotic gastropods (genus Alviniconcha) in the Lau Basin. We extensively surveyed Alviniconcha holobionts from four vent fields using quantitative molecular approaches, coupled to characterization of high-temperature and diffuse vent-fluid composition using gastight samplers and in situ electrochemical analyses, respectively. Phylogenetic analyses exposed cryptic host and symbiont diversity, revealing three distinct host types and three different symbiont phylotypes (one ε-proteobacteria and two γ-proteobacteria) that formed specific associations with one another. Strikingly, we observed that holobionts with ε-proteobacterial symbionts were dominant at the northern fields, whereas holobionts with γ-proteobacterial symbionts were dominant in the southern fields. This pattern of distribution corresponds to differences in the vent geochemistry that result from deep subsurface geological and geothermal processes. We posit that the symbionts, likely through differences in chemolithoautotrophic metabolism, influence niche utilization among these holobionts. The data presented here represent evidence linking symbiont type to habitat partitioning among the chemosynthetic symbioses at hydrothermal vents and illustrate the coupling between subsurface geothermal processes and niche availability.

Biological capacitance studies of anodes in microbial fuel cells using electrochemical impedance spectroscopy.

It is known that cell potential increases while anode resistance decreases during the start-up of microbial fuel cells (MFCs). Biological capacitance, defined as the apparent capacitance attributed to biological activity including biofilm production, plays a role in this phenomenon. In this research, electrochemical impedance spectroscopy was employed to study anode capacitance and resistance during the start-up period of MFCs so that the role of biological capacitance was revealed in electricity generation by MFCs. It was observed that the anode capacitance ranged from 3.29 to 120 mF which increased by 16.8% to 18-20 times over 10-12 days. Notably, lowering the temperature and arresting biological activity via fixation by 4% para formaldehyde resulted in the decrease of biological capacitance by 16.9 and 62.6%, indicating a negative correlation between anode capacitance and anode resistance of MFCs. Thus, biological capacitance of anode should play an important role in power generation by MFCs. We suggest that MFCs are not only biological reactors and/or electrochemical cells, but also biological capacitors, extending the vision on mechanism exploration of electron transfer, reactor structure design and electrode materials development of MFCs.

Characterizing the plasticity of nitrogen metabolism by the host and symbionts of the hydrothermal vent chemoautotrophic symbioses Ridgeia piscesae.

Chemoautotrophic symbionts of deep sea hydrothermal vent tubeworms are known to provide their hosts with all their primary nutrition. While studies have examined how chemoautotrophic symbionts provide the association with nitrogen, fewer have examined if symbiont nitrogen metabolism varies as a function of environmental conditions. Ridgeia piscesae tubeworms flourish at Northeastern Pacific vents, occupy a range of microhabitats, and exhibit a high degree of morphological plasticity [e.g. long-skinny (LS) and short-fat (SF) phenotypes] that may relate to environmental conditions. This plasticity affords an opportunity to examine whether symbiont nitrogen metabolism varies among host phenotypes. LS and SF R. piscesae were recovered from the Axial and Main Endeavour Field hydrothermal vents. Nitrate and ammonium were quantified in Ridgeia blood, and the expression of key nitrogen metabolism genes, as well as stable nitrogen isotope ratios, was quantified in host branchial plume and symbiont-containing tissues. Nitrate and ammonium were abundant in the blood of both phenotypes though environmental ammonium concentrations were, paradoxically, lowest among individuals with the highest blood ammonium. Assimilatory nitrate reductase transcripts were always below detection, though in both LS and SF R. piscesae symbionts, we observed elevated expression of dissimilatory nitrate reductase genes, as well as symbiont and host ammonium assimilation genes. Site-specific differences in expression, along with tissue stable isotope analyses, suggest that LS and SF Ridgeia symbionts are engaged in both dissimilatory nitrate reduction and ammonia assimilation to varying degrees. As such, it appears that environmental conditions -not host phenotype-primarily dictates symbiont nitrogen metabolism.