Acquisition of epibiotic bacteria along the life cycle of the hydrothermal shrimp Rimicaris exoculata

The caridean shrimp Rimicaris exoculata dominates the fauna at several Mid-Atlantic Ridge hydrothermal vent sites. This shrimp has an enlarged gill chamber, harboring a dense ectosymbiotic community of chemoautotrophic bacteria associated with mineral oxide deposits. Until now, their acquisition is not fully understood. At three hydrothermal vent sites, we analyzed the epibionts diversity at different moult stages and also in the first stages of the shrimp life (eggs, hatched eggs (with larvae) and juveniles). Hatched eggs associated with young larvae were collected for the first time directly from gravid females at the Logachev vent site during the Serpentine cruise. An approach using 16S rRNA clone libraries, scanning and transmission electron microscopy, and fluorescent in situ hybridization was used. Molecular results and microscope observations indicated a switch in the composition of the bacterial community between early R. exoculata life cycle stage (egg libraries dominated by the Gammaproteobacteria) and later stages (juvenile/adult libraries dominated by the Epsilonproteobacteria). We hypothesized that the epibiotic phylotype composition could vary according to the life stage of the shrimp. Our results confirmed the occurrence of a symbiosis with Gammaproteobacteria and Epsilonproteobacteria, but more complex than previously assumed. We revealed the presence of active type-I methanotrophic bacteria colonizing the cephalothorax of shrimps from the Rainbow site. They were also present on the eggs from the Logachev site. This could be the first ‘epibiotic'' association between methanotrophic bacteria and hydrothermal vent crustacean. We discuss possible transmission pathways for epibionts linked to the shrimp life cycle.     

Screening and its potential application of lipolytic activity from a marine environment: characterization of a novel esterase from <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> CL180

To develop an enantioselective lipase/esterase hydrolyzing racemic ofloxacin ester to levofloxacin, samples were collected from a variety of marine environments such as cold sea, hydrothermal vent area, sediment, tidal flat area, arctic sea, marine organisms, and so on. Microorganisms were isolated by plating on an enrichment medium with simultaneous detection of lipolytic activities and screened for the hydrolysis of ofloxacin ester. Three candidates among isolates were selected, and one of them, identified as Yarrowia lipolytica CL180, hydrolyzed preferentially S-enantiomer of racemic ofloxacin ester. The lipase/esterase gene (yli180) was cloned by screening a genomic library. The sequence analysis revealed an open reading frame consisting of 1,431 bp that encoded a protein of 476 amino acids with a molecular mass of 53 kDa. The yli180 gene was expressed in Escherichia coli and purified to homogeneity. The optimum activity of the recombinant protein (rYli180) occurred at pH 7.5 and 35°C, respectively. rYli180 preferentially hydrolyzed p-nitrophenyl esters of fatty acids with short chain lengths of ≤10 carbon atoms. This study represents a novel esterase of type B1 carboxylesterase/lipase family from a marine isolate, showing a potential usage as a biocatalyst because of enantioselectivity toward racemic ofloxacin ester.     

Pathways of carbon and energy metabolism of the epibiotic community associated with the deep-sea hydrothermal vent shrimp Rimicaris exoculata

Background

The shrimp Rimicaris exoculata dominates the faunal biomass at many deep-sea hydrothermal vent sites at the Mid-Atlantic Ridge. In its enlarged gill chamber it harbors a specialized epibiotic bacterial community for which a nutritional role has been proposed.

Methodology/Principal Findings

We analyzed specimens from the Snake Pit hydrothermal vent field on the Mid-Atlantic Ridge by complementing a 16S rRNA gene survey with the analysis of genes involved in carbon, sulfur and hydrogen metabolism. In addition to Epsilon- and Gammaproteobacteria, the epibiotic community unexpectedly also consists of Deltaproteobacteria of a single phylotype, closely related to the genus Desulfocapsa. The association of these phylogenetic groups with the shrimp was confirmed by fluorescence in situ hybridization. Based on functional gene analyses, we hypothesize that the Gamma- and Epsilonproteobacteria are capable of autotrophic growth by oxidizing reduced sulfur compounds, and that the Deltaproteobacteria are also involved in sulfur metabolism. In addition, the detection of proteobacterial hydrogenases indicates the potential for hydrogen oxidation in these communities. Interestingly, the frequency of these phylotypes in 16S rRNA gene clone libraries from the mouthparts differ from that of the inner lining of the gill chamber, indicating potential functional compartmentalization.

Conclusions

Our data show the specific association of autotrophic bacteria with Rimicaris exoculata from the Snake Pit hydrothermal vent field, and suggest that autotrophic carbon fixation is contributing to the productivity of the epibiotic community with the reductive tricarboxylic acid cycle as one important carbon fixation pathway. This has not been considered in previous studies of carbon fixation and stable carbon isotope composition of the shrimp and its epibionts. Furthermore, the co-occurrence of sulfur-oxidizing and sulfur-reducing epibionts raises the possibility that both may be involved in the syntrophic exchange of sulfur compounds, which could increase the overall efficiency of this epibiotic community.     

Diversity and characterization of bacterial communities of five co‐occurring species at a hydrothermal vent on the Tonga Arc

Host–symbiont relationships in hydrothermal vent ecosystems, supported by chemoautotrophic bacteria as primary producers, have been extensively studied. However, the process by which densely populated co‐occurring invertebrate hosts form symbiotic relationships with bacterial symbionts remains unclear. Here, we analyzed gill‐associated symbiotic bacteria (gill symbionts) of five co‐occurring hosts, three mollusks (“Bathymodiolus” manusensis, B. brevior, and Alviniconcha strummeri) and two crustaceans (Rimicaris variabilis and Austinograea alayseae), collected together at a single vent site in the Tonga Arc. We observed both different compositions of gill symbionts and the presence of unshared operational taxonomic units (OTUs). In addition, the total number of OTUs was greater for crustacean hosts than for mollusks. The phylogenetic relationship trees of gill symbionts suggest that γ‐proteobacterial gill symbionts have coevolved with their hosts toward reinforcement of host specificity, while campylobacterial Sulfurovum species found across various hosts and habitats are opportunistic associates. Our results confirm that gill symbiont communities differ among co‐occurring vent invertebrates and indicate that hosts are closely related with their gill symbiont communities. Considering the given resources available at a single site, differentiation of gill symbionts seems to be a useful strategy for obtaining nutrition and energy while avoiding competition among both hosts and gill symbionts.     

Stable-Carbon-Isotope Composition of Fatty Acids in Hydrothermal Vent Mussels Containing Methanotrophic and Thiotrophic Bacterial Endosymbionts

Fatty acid biomarker analysis coupled with gas chromatography-isotope ratio mass spectrometry was used to confirm the presence of methanotrophic and thiotrophic bacterial endosymbionts in the tissues of a hydrothermal vent mussel (Bathymodiolus sp.), collected from the Menez Gwen vent field on the mid-Atlantic ridge. Monounsaturated (n-8) fatty acids, which are diagnostic of methanotrophic bacteria, were detected in all three types of tissues examined (gill, posterior adductor, and mantle), although levels were highest in gill tissues where the bacteria were found. Stable-carbon-isotope compositions (δ-¹³C per mille relative to that of Peedee belemnite) of fatty acids for all three tissues ranged from −24.9 to −34.9‰, which encompasses the range predicted for both thiotroph- and methanotroph-based nutrition. The data suggest that these thio- and methanotrophic bacterial endosymbionts are equally important in the nutrition of the vent mussel at this particular vent site.     

Analysis of Comparative Sequence and Genomic Data to Verify Phylogenetic Relationship and Explore a New Subfamily of Bacterial Lipases

Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca²⁺-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter P_lac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65°C and retained ≥ 97% activity after incubation at 50°C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.     

New insigths on the metabolic diversity among the epibiotic microbial communitiy of the hydrothermal shrimp Rimicaris exoculata

The Rimicaris exoculata dominates the megafauna of some of the Mid Atlantic ridge hydrothermal vent sites. This species harbors a rich community of bacterial epibionts inside its gill chamber. Literature data indicate that a single 16S rRNA phylotype dominates this epibiotic community, and is assumed to be a sulfide-oxidizing bacteria. However attempts of cultivation were not successful and did not allow to confirm it. The aim of our study was to test the hypothesis of sulfide oxidation in the gill chamber, by a multidisciplinary approach, using in vivo experiments at in situ pressure in the presence of sulfide, microscopic observations and a molecular survey. Morphology of microorganisms, before and after treatment, was analyzed to test the effect of sulfide depletion and re-exposure. Our observations, as well as molecular data indicate a wider diversity than previously described for this shrimp's epibiotic community. We observed occurrence of bacterial intracellular sulfur- and iron-enriched granules and some methanotrophic-like bacteria cells for the first time. Genes that are characteristic of methane-oxidizing (pmoA) and sulfide-oxidizing (APS) bacteria were identified. These results suggest that three metabolic types (iron, sulfide and methane oxidation) may co-occur within the epibiont community associated with Rimicaris exoculata. As this shrimp colonizes chemically contrasted environments, the relative abundance of each metabolic type could vary according to the local availability of reduced compounds.     

Growth and Phylogenetic Properties of Novel Bacteria Belonging to the Epsilon Subdivision of the Proteobacteria Enriched from Alvinella pompejana and Deep-Sea Hydrothermal Vents

Recent molecular characterizations of microbial communities from deep-sea hydrothermal sites indicate the predominance of bacteria belonging to the epsilon subdivision of Proteobacteria (epsilon Proteobacteria). Here, we report the first enrichments and characterizations of four epsilon Proteobacteria that are directly associated with Alvinella pompejana, a deep sea hydrothermal vent polychete, or with hydrothermal vent chimney samples. These novel bacteria were moderately thermophilic sulfur-reducing heterotrophs growing on formate as the energy and carbon source. In addition, two of them (Am-H and Ex-18.2) could grow on sulfur lithoautrotrophically using hydrogen as the electron donor. Optimal growth temperatures of the bacteria ranged from 41 to 45°C. Phylogenetic analysis of the small-subunit ribosomal gene of the two heterotrophic bacteria demonstrated 95% similarity to Sulfurospirillum arcachonense, an epsilon Proteobacteria isolated from an oxidized marine surface sediment. The autotrophic bacteria grouped within a deeply branching clade of the epsilon Proteobacteria, to date composed only of uncultured bacteria detected in a sample from a hydrothermal vent along the mid-Atlantic ridge. A molecular survey of various hydrothermal vent environments demonstrated the presence of two of these bacteria (Am-N and Am-H) in more than one geographic location and habitat. These results suggest that certain epsilon Proteobacteria likely fill important niches in the environmental habitats of deep-sea hydrothermal vents, where they contribute to overall carbon and sulfur cycling at moderate thermophilic temperatures.     

Experimentally induced endosymbiont loss and re-acquirement in the hydrothermal vent bivalve Bathymodiolus azoricus

Invertebrates harbouring endosymbiotic chemoautotroph bacteria are widely distributed in a variety of reducing marine habitats, including deep-sea hydrothermal vents. In these species mechanisms of symbiont transmission are likely to be key elements of dispersal strategies that remained partially unresolved because the early life stages are not available for developmental studies. To study cessation and re-establishment of symbiosis in the host gill a laboratory experiment was conducted over 45 days in a controlled set-up (LabHorta) that endeavour re-creation of the hydrothermal vent chemical environment. Our animal model was the vent bivalve Bathymodiolus azoricus from the Menez Gwen vent site of the Mid Atlantic Ridge (MAR). Animals were exposed to conditions lacking inorganic S supply for 30 days, which is vital for their symbionts, and then re-acclimatized in sulphide-supplied seawater for an additional 15 days.Gradual disappearance of bacteria from the symbiont-bearing gill cells was observed in animals kept in seawater free of dissolved sulphide for up to 30 days, and was evidenced by histological, ultrastructural observations and Polymerase Chain Reaction tests. Following re-acclimatisation in S-supplied seawater, proliferation of sulphur-bacteria in the gill bacteriocytes confirms the functionality of our sulfide-feeding system in supporting chemoautotrophic symbionts. It may also indicate a horizontal endosymbiont acquisition, i.e. from the environment to the host by means of phagocytosis-like mechanism involving special “pit-like” structures on the apical cell membrane.The present work reports the first laboratory set-up successfully used to maintain the hydrothermal vent bivalve B. azoricus for prolonged periods of time by supplying inorganic sulphur as an energy source for its bacterial endosymbionts. Survival of symbiont bacteria is a critical factor influencing the host physiology and thus the methods reported here represent great potential for future studies of host-symbiont dynamics and for post-capture experimental investigations.     

Microdistribution of Faunal Assemblages at Deep-Sea Hydrothermal Vents in the Southern Ocean

Chemosynthetic primary production by microbes supports abundant faunal assemblages at deep-sea hydrothermal vents, with zonation of invertebrate species typically occurring along physico-chemical gradients. Recently discovered vent fields on the East Scotia Ridge (ESR) in the Southern Ocean represent a new province of vent biogeography, but the spatial dynamics of their distinct fauna have yet to be elucidated. This study determines patterns of faunal zonation, species associations, and relationships between faunal microdistribution and hydrothermal activity in a vent field at a depth of 2,400 m on the ESR. Remotely operated vehicle (ROV) dives obtained high-definition imagery of three chimney structures with varying levels of hydrothermal activity, and a mosaic image of >250 m² of seafloor co-registered with temperature measurements. Analysis of faunal microdistribution within the mosaiced seafloor reveals a consistent pattern of faunal zonation with increasing distance from vent sources and peak temperatures. Assemblages closest to vent sources are visibly dominated by a new species of anomuran crab, Kiwa n. sp. (abundance >700 individuals m⁻²), followed by a peltospiroid gastropod (>1,500 individuals m⁻²), eolepadid barnacle (>1,500 individuals m⁻²), and carnivorous actinostolid anemone (>30 individuals m⁻²). Peripheral fauna are not dominated by a single taxon, but include predatory and scavenger taxa such as stichasterid seastars, pycnogonids and octopus. Variation in faunal microdistribution on chimneys with differing levels of activity suggests a possible successional sequence for vent fauna in this new biogeographic province. An increase in δ³⁴S values of primary consumers with distance from vent sources, and variation in their δ¹³C values also indicate possible zonation of nutritional modes of the vent fauna. By using ROV videography to obtain a high-resolution representation of a vent environment over a greater extent than previous studies, these results provide a baseline for determining temporal change and investigations of processes structuring faunal assemblages at Southern Ocean vents.     

Inorganic carbon fixation by chemosynthetic ectosymbionts and nutritional transfers to the hydrothermal vent host-shrimp Rimicaris exoculata

The shrimp Rimicaris exoculata dominates several hydrothermal vent ecosystems of the Mid-Atlantic Ridge and is thought to be a primary consumer harbouring a chemoautotrophic bacterial community in its gill chamber. The aim of the present study was to test current hypotheses concerning the epibiont''s chemoautotrophy, and the mutualistic character of this association. In-vivo experiments were carried out in a pressurised aquarium with isotope-labelled inorganic carbon (NaH¹³CO₃ and NaH¹⁴CO₃) in the presence of two different electron donors (Na₂S₂O₃ and Fe²⁺) and with radiolabelled organic compounds (¹⁴C-acetate and ³H-lysine) chosen as potential bacterial substrates and/or metabolic by-products in experiments mimicking transfer of small biomolecules from epibionts to host. The bacterial epibionts were found to assimilate inorganic carbon by chemoautotrophy, but many of them (thick filaments of epsilonproteobacteria) appeared versatile and able to switch between electron donors, including organic compounds (heterotrophic acetate and lysine uptake). At least some of them (thin filamentous gammaproteobacteria) also seem capable of internal energy storage that could supply chemosynthetic metabolism for hours under conditions of electron donor deprivation. As direct nutritional transfer from bacteria to host was detected, the association appears as true mutualism. Import of soluble bacterial products occurs by permeation across the gill chamber integument, rather than via the digestive tract. This first demonstration of such capabilities in a decapod crustacean supports the previously discarded hypothesis of transtegumental absorption of dissolved organic matter or carbon as a common nutritional pathway.     

Profile of the GSK Published Protein Kinase Inhibitor Set Across ATP-Dependent and-Independent Luciferases: Implications for Reporter-Gene Assays

A library of 367 protein kinase inhibitors, the GSK Published Kinase Inhibitor Set (PKIS), which has been annotated for protein kinase family activity and is available for public screening efforts, was assayed against the commonly used luciferase reporter enzymes from the firefly, Photinus pyralis (FLuc) and marine sea pansy, Renilla reniformis (RLuc). A total of 22 compounds (∼6% of the library) were found to inhibit FLuc with 10 compounds showing potencies ≤1 µM. Only two compounds were found to inhibit RLuc, and these showed relatively weak potency values (∼10 µM). An inhibitor series of the VEGFR2/TIE2 protein kinase family containing either an aryl oxazole or benzimidazole-urea core illustrate the different structure activity relationship profiles FLuc inhibitors can display for kinase inhibitor chemotypes. Several FLuc inhibitors were broadly active toward the tyrosine kinase and CDK families. These data should aid in interpreting the results derived from screens employing the GSK PKIS in cell-based assays using the FLuc reporter. The study also underscores the general need for strategies such as the use of orthogonal reporters to identify kinase or non-kinase mediated cellular responses.     

Molecular cloning and characterization of thermostable esterase and lipase from Geobacillus thermoleovorans YN isolated from desert soil in Egypt

Genes encoding an esterase (EstA) and lipase (LipA) from Geobacillus thermoleovorans YN, a strain isolated from Egyptian desert soil, were cloned and the respective proteins were expressed in Escherichia coli and characterized. Whereas LipA was cloned directly by PCR amplification from genomic DNA, a genomic library composed of 3000 clones was screened on tributyrin agar plates to find EstA. An open reading frame of 744 bps encoding a polypeptide of 247 amino acid residues was identified as esterase due to its conserved GXSXG motif and its high similarity toward other carboxyl esterases. LipA (416 aa residues) is encoded by an ORF of 1251 bps and constitutes a pre-protein with a calculated molecular mass of 46 kDa including a signal sequence of 28 aa resulting in a mature lipase of 43 kDa. Both, LipA and EstA were sub-cloned and expressed under control of the temperature-inducible λ-promoter and purified by IMAC and gel filtration. The molecular mass of the purified EstA was 29 kDa. Both enzymes were most active at pH ∼9.5 and remarkably stable at pH 5 and 10.5. Temperature optima and stabilities (up to 70 °C) of both enzymes as well as their reaction kinetics and substrate spectra were determined.     

Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with α-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 × 10⁴-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 × 10⁴-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H₂O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.     

Functional Metagenomic Investigations of Microbial Communities in a Shallow-Sea Hydrothermal System

Little is known about the functional capability of microbial communities in shallow-sea hydrothermal systems (water depth of <200 m). This study analyzed two high-throughput pyrosequencing metagenomic datasets from the vent and the surface water in the shallow-sea hydrothermal system offshore NE Taiwan. This system exhibited distinct geochemical parameters. Metagenomic data revealed that the vent and the surface water were predominated by Epsilonproteobacteria (Nautiliales-like organisms) and Gammaproteobacteria ( Thiomicrospira -like organisms), respectively. A significant difference in microbial carbon fixation and sulfur metabolism was found between the vent and the surface water. The chemoautotrophic microorganisms in the vent and in the surface water might possess the reverse tricarboxylic acid cycle and the Calvin−Bassham−Benson cycle for carbon fixation in response to carbon dioxide highly enriched in the environment, which is possibly fueled by geochemical energy with sulfur and hydrogen. Comparative analyses of metagenomes showed that the shallow-sea metagenomes contained some genes similar to those present in other extreme environments. This study may serve as a basis for deeply understanding the genetic network and functional capability of the microbial members of shallow-sea hydrothermal systems.     

Distribution and Diversity of Sulfur-Oxidizing Thiomicrospira spp. at a Shallow-Water Hydrothermal Vent in the Aegean Sea (Milos, Greece)

A shallow-water hydrothermal vent system in the Aegean Sea close to the island of Milos (Greece) was chosen to study the diversity and distribution of the chemolithoautotrophic sulfur-oxidizing bacterium Thiomicrospira. Cell numbers in samples from different regions around a solitary vent decreased toward the center of the vent (horizontal distribution), as well as with depth (vertical distribution), corresponding to an increase in temperature (from ca. 25 to 60°C) and a decrease in pH (from ca. pH 7 to 5). Thiomicrospira was one of the most abundant culturable sulfur oxidizers and was even dominant in one region. Phylogenetic analysis of Thiomicrospira spp. present in the highest most-probable-number (MPN) dilutions revealed that most of the obtained sequences grouped in two new closely related clusters within the Thiomicrospira branch. Two different new isolates, i.e., Milos-T1 and Milos-T2, were obtained from high-dilution (10⁻⁵) enrichments. Phylogenetic analysis indicated that isolate Milos-T1 is related to the recently described Thiomicrospira kuenenii and Hydrogenovibrio marinus, whereas isolate Milos-T2 grouped with the MPN sequences of cluster 2. The predominance of strain Milos-T2 was indicated by its identification in several environmental samples by hybridization analysis of denaturing gradient gel electrophoresis (DGGE) patterns and by sequencing of one of the corresponding bands, i.e., ML-1, from the DGGE gel. The results shown in this paper support earlier indications that Thiomicrospira species are important members of hydrothermal vent communities.     

Spatial Differences in East Scotia Ridge Hydrothermal Vent Food Webs: Influences of Chemistry,Microbiology and Predation on Trophodynamics

The hydrothermal vents on the East Scotia Ridge are the first to be explored in the Antarctic and are dominated by large peltospiroid gastropods, stalked barnacles (Vulcanolepas sp.) and anomuran crabs (Kiwa sp.) but their food webs are unknown. Vent fluid and macroconsumer samples were collected at three vent sites (E2, E9N and E9S) at distances of tens of metres to hundreds of kilometres apart with contrasting vent fluid chemistries to describe trophic interactions and identify potential carbon fixation pathways using stable isotopes. δ¹³C of dissolved inorganic carbon from vent fluids ranged from −4.6‰ to 0.8‰ at E2 and from −4.4‰ to 1.5‰ at E9. The lowest macroconsumer δ¹³C was observed in peltospiroid gastropods (−30.0‰ to −31.1‰) and indicated carbon fixation via the Calvin-Benson-Bassham (CBB) cycle by endosymbiotic gamma-Proteobacteria. Highest δ¹³C occurred in Kiwa sp. (−19.0‰ to −10.5‰), similar to that of the epibionts sampled from their ventral setae. Kiwa sp. δ¹³C differed among sites, which were attributed to spatial differences in the epibiont community and the relative contribution of carbon fixed via the reductive tricarboxylic acid (rTCA) and CBB cycles assimilated by Kiwa sp. Site differences in carbon fixation pathways were traced into higher trophic levels e.g. a stichasterid asteroid that predates on Kiwa sp. Sponges and anemones at the periphery of E2 assimilated a proportion of epipelagic photosynthetic primary production but this was not observed at E9N. Differences in the δ¹³C and δ³⁴S values of vent macroconsumers between E2 and E9 sites suggest the relative contributions of photosynthetic and chemoautotrophic carbon fixation (rTCA v CBB) entering the hydrothermal vent food webs vary between the sites.